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WO2025134071A1 - Malic and glutaric acid based ionizable lipids - Google Patents

Malic and glutaric acid based ionizable lipids Download PDF

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Publication number
WO2025134071A1
WO2025134071A1 PCT/IB2024/063068 IB2024063068W WO2025134071A1 WO 2025134071 A1 WO2025134071 A1 WO 2025134071A1 IB 2024063068 W IB2024063068 W IB 2024063068W WO 2025134071 A1 WO2025134071 A1 WO 2025134071A1
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Prior art keywords
alkyl
pharmaceutically acceptable
acceptable salt
lipid
formula
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French (fr)
Inventor
Hongfeng Deng
Shrirang KARVE
Neha KAUSHAL
Younan MA
Peter Justin Mikochik
Trent Northen
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Sanofi SA
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Sanofi SA
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C229/00—Compounds containing amino and carboxyl groups bound to the same carbon skeleton
    • C07C229/02—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton
    • C07C229/04—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated
    • C07C229/06—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having only one amino and one carboxyl group bound to the carbon skeleton
    • C07C229/10—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having only one amino and one carboxyl group bound to the carbon skeleton the nitrogen atom of the amino group being further bound to acyclic carbon atoms or to carbon atoms of rings other than six-membered aromatic rings
    • C07C229/12—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having only one amino and one carboxyl group bound to the carbon skeleton the nitrogen atom of the amino group being further bound to acyclic carbon atoms or to carbon atoms of rings other than six-membered aromatic rings to carbon atoms of acyclic carbon skeletons
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C219/00—Compounds containing amino and esterified hydroxy groups bound to the same carbon skeleton
    • C07C219/02—Compounds containing amino and esterified hydroxy groups bound to the same carbon skeleton having esterified hydroxy groups and amino groups bound to acyclic carbon atoms of the same carbon skeleton
    • C07C219/04—Compounds containing amino and esterified hydroxy groups bound to the same carbon skeleton having esterified hydroxy groups and amino groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated
    • C07C219/06—Compounds containing amino and esterified hydroxy groups bound to the same carbon skeleton having esterified hydroxy groups and amino groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having the hydroxy groups esterified by carboxylic acids having the esterifying carboxyl groups bound to hydrogen atoms or to acyclic carbon atoms of an acyclic saturated carbon skeleton
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C271/00—Derivatives of carbamic acids, i.e. compounds containing any of the groups, the nitrogen atom not being part of nitro or nitroso groups
    • C07C271/06—Esters of carbamic acids
    • C07C271/08—Esters of carbamic acids having oxygen atoms of carbamate groups bound to acyclic carbon atoms
    • C07C271/10—Esters of carbamic acids having oxygen atoms of carbamate groups bound to acyclic carbon atoms with the nitrogen atoms of the carbamate groups bound to hydrogen atoms or to acyclic carbon atoms
    • C07C271/20—Esters of carbamic acids having oxygen atoms of carbamate groups bound to acyclic carbon atoms with the nitrogen atoms of the carbamate groups bound to hydrogen atoms or to acyclic carbon atoms to carbon atoms of hydrocarbon radicals substituted by nitrogen atoms not being part of nitro or nitroso groups
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C271/00—Derivatives of carbamic acids, i.e. compounds containing any of the groups, the nitrogen atom not being part of nitro or nitroso groups
    • C07C271/06—Esters of carbamic acids
    • C07C271/08—Esters of carbamic acids having oxygen atoms of carbamate groups bound to acyclic carbon atoms
    • C07C271/10—Esters of carbamic acids having oxygen atoms of carbamate groups bound to acyclic carbon atoms with the nitrogen atoms of the carbamate groups bound to hydrogen atoms or to acyclic carbon atoms
    • C07C271/22—Esters of carbamic acids having oxygen atoms of carbamate groups bound to acyclic carbon atoms with the nitrogen atoms of the carbamate groups bound to hydrogen atoms or to acyclic carbon atoms to carbon atoms of hydrocarbon radicals substituted by carboxyl groups
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0043—Nose
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5107—Excipients; Inactive ingredients
    • A61K9/5123—Organic compounds, e.g. fats, sugars

Definitions

  • lipid of Formula (I): (I), or a pharmaceutically acceptable salt thereof wherein: two of R 1 , R 2 , and R 3 are independently selected from -C (6-24) alkyl, -C (6-24) alkenyl, - C (6-24) alkynyl, , , and , wherein the -C (6-24) alkyl, - C (6-24) alkenyl, and -C (6-24) alkynyl are each optionally substituted with one to six groups independently selected from halo, -OC (1-6) alkyl, -OC (2-6) alkenyl, -SC (1-6) alkyl, -SC (2-6) alkenyl, and -C(O)OC (1-6) alkyl; one of R 1 , R 2 , and R 3 is or X 1 independently for each occurrence is -C (3-12) alkyl, -C (3-12) alkenyl, or -C
  • lipid of Formula (I) (I), or a pharmaceutically acceptable salt thereof, wherein R 1 , R 2 , R 3 , X, and n are as defined herein.
  • a lipid of Formula (I) is provided: (I), or a pharmaceutically acceptable salt thereof, wherein: two of R 1 , R 2 , and R 3 are independently selected from -C (6-24) alkyl, -C (6-24) alkenyl, - C (6-24) alkynyl, , , and , wherein the -C (6-24) alkyl, - C (6-24) alkenyl, and -C (6-24) alkynyl are each optionally substituted with one to six groups independently selected from halo, -OC (1-6) alkyl, -OC (2-6) alkenyl, -SC (1-6) alkyl, -SC (2-6) alkenyl, and -C(O)OC (1-6) alkyl; one of R
  • two of R 1 , R 2 , and R 3 are independently selected from -C (6- 24) alkyl, -C (6-24) alkenyl, and ;
  • X 1 independently for each occurrence is -C (3-12) alkyl or -C (3-12) alkenyl;
  • X 2 independently for each occurrence is -C(O)O-, -OC(O)-, or -OC(O)O-;
  • X 3 independently for each occurrence is -C (5-24) alkyl or -C (5-24) alkenyl.
  • R 1 , R 2 , and R 3 are independently selected from -C (6-24) alkyl, -C (6- 24) alkenyl, or ; R 3 is or ; X 1 independently for each occurrence is -C (3-12) alkyl or -C (3-12) alkenyl; X 2 independently for each occurrence is -C(O)O- , -OC(O)-, or -OC(O)O-; X 3 independently for each occurrence is -C (5-24) alkyl or -C(5- 2 4) alkenyl; X 4 is -C (1-6) alkyl; X 5 is -C (1-6) alkyl; X 6 is -C(O)O-, -OC(O)-, or -OC(O)O
  • A is ; and R A1 and R A2 are each independently -C (1-6) alkyl that is optionally substituted with - OH; or R A1 and R A2 are taken together to form a 3- to 6-membered heteroaryl that is optionally substituted with one to three -C (1-3) alkyl groups.
  • A is -N(CH 3 ) 2 .
  • the lipid has a structure selected from the group consisting of: or a pharmaceutically acceptable salt thereof.
  • the lipid has a structure selected from the group consisting of: or a pharmaceutically acceptable salt thereof.
  • the lipid has a structure selected from the group consisting of: or a pharmaceutically acceptable salt thereof. In some embodiments, the lipid has a structure selected from the group consisting of: or a pharmaceutically acceptable salt thereof. In some embodiments, R X is -H or -CH 3 . In some embodiments, n is 1 or 2. In some embodiments, n is 1. In some embodiments, n is 2. In some embodiments, the lipid has a structure selected from the group consisting of:
  • the present disclosure provides a composition comprising a lipid nanoparticle (LNP), wherein the LNP comprises a lipid of the present disclosure (e.g., a lipid of Formula (I)).
  • the LNP comprises: (I) a lipid of the present disclosure; (II) a stealth lipid; (III) a structural lipid; and (IV) a helper lipid.
  • the stealth lipid is a polyethylene glycol-conjugated (PEGylated) lipid, a polyoxazoline polymer-conjugated lipid, or a polysarcosine-conjugated (pSar) lipid.
  • PEGylated polyethylene glycol-conjugated
  • pSar polysarcosine-conjugated
  • the stealth lipid is a PEGylated lipid selected from 1,2- dimyristoyl-rac-glycero-3-methoxypolyethylene glycol (DMG-PEG), 1,2-distearoyl-sn- glycero-3-phosphoethanolamine-polyethylene glycol (DSPE-PEG), 1,2-dilauroyl-sn-glycero- 3-phosphoethanolamine-polyethylene glycol (DLPE-PEG), and 1,2-distearoyl-rac-glycero- polyethelene glycol (DSG-PEG).
  • DMG-PEG 1,2- dimyristoyl-rac-glycero-3-methoxypolyethylene glycol
  • DSPE-PEG 1,2-distearoyl-sn- glycero-3-phosphoethanolamine-polyethylene glycol
  • DLPE-PEG 1,2-dilauroyl-sn-glycero- 3-phosphoethanolamine-polyethylene glycol
  • DSG-PEG
  • the helper lipid is 1,2-dioleoyl-SN-glycero-3- phosphoethanolamine (DOPE).
  • DOPE 1,2-dioleoyl-SN-glycero-3- phosphoethanolamine
  • the LNP comprises: the lipid of the present disclosure at a molar ratio between 35% and 45%, the stealth lipid at a molar ratio between 0.5% and 7%, the structural lipid at a molar ratio between 20% and 35%, and the helper lipid at a molar ratio of between 20% and 30%.
  • the LNP comprises: the lipid of any one of claims 1-23 at a molar ratio of about 40%, the stealth lipid at a molar ratio of about 1.5%, the structural lipid at a molar ratio of about 30%, and the helper lipid at a molar ratio of about 28.5%. In some embodiments, the LNP comprises: the lipid of any one of claims 1-23 at a molar ratio of about 40%, the stealth lipid at a molar ratio of about 5%, the structural lipid at a molar ratio of about 30%, and the helper lipid at a molar ratio of about 25%.
  • the composition comprising a lipid nanoparticle (LNP) further comprises a nucleic acid molecule, wherein the nucleic acid molecule is encapsulated in the LNP.
  • the LNP comprises 1-20, optionally 5-10 or 6-8, nucleic acid molecules.
  • the nucleic acid molecule is an mRNA molecule.
  • the mRNA molecule encodes an antigen, optionally a viral antigen or a bacterial antigen.
  • the LNP encapsulates two or more mRNA molecules, wherein each mRNA molecule encodes a different antigen, optionally wherein the different antigens are from the same pathogen or from different pathogens.
  • the present disclosure provides a kit comprising a container comprising a single-use or multi-use dosage of a composition of the present disclosure, optionally wherein the container is a vial or a pre-filled nasal spray device.
  • a kit comprising a container comprising a single-use or multi-use dosage of a composition of the present disclosure, optionally wherein the container is a vial or a pre-filled nasal spray device.
  • LNP lipid nanoparticle
  • LNPs comprising the ionizable or cationic lipids of the present disclosure exhibit enhanced expression of proteins encoded by cargo nucleic acid molecules.
  • compositions or processes as “consisting of” and “consisting essentially of” the enumerated ingredients/steps, which allows the presence of only the named ingredients/steps and excludes other ingredients/steps.
  • the term “approximately” or “about,” as applied to one or more values of interest refers to a value that is similar to a stated reference value.
  • the term “approximately” or “about” refers to a range of values that fall within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than or less than) of the stated reference value unless otherwise stated or otherwise evident from the context (except where such number would exceed 100% of a possible value).
  • delivery encompasses both local and systemic delivery.
  • delivery of mRNA encompasses situations in which an mRNA is delivered to a target tissue and the encoded protein is expressed and retained within the target tissue (also referred to as “local distribution” or “local delivery”), and situations in which an mRNA is delivered to a target tissue and the encoded protein is expressed and secreted into patient's circulation system (e.g., serum) and systematically distributed and taken up by other tissues (also referred to as “systemic distribution” or “systemic delivery).
  • patient's circulation system e.g., serum
  • expression of a nucleic acid sequence refers to translation of an mRNA into a polypeptide, assembly of multiple polypeptides (e.g., heavy chain or light chain of antibody) into an intact protein (e.g., antibody), and/or post-translational modification of a polypeptide or fully assembled protein (e.g., antibody).
  • expression and production are used inter-changeably.
  • half-life is the time required for a quantity such as nucleic acid or protein concentration or activity to fall to half of its value as measured at the beginning of a time period.
  • lipid refers to any lamellar, multilamellar, or solid nanoparticle vesicle.
  • a liposome as used herein can be formed by mixing one or more lipids or by mixing one or more lipids and polymer(s).
  • a liposome suitable for the present disclosure contains an ionizable or cationic lipid(s) and optionally non-cationic lipid(s), optionally cholesterol-based lipid(s), and/or optionally PEG- modified lipid(s).
  • mRNA messenger RNA
  • mRNA refers to a polynucleotide that encodes at least one polypeptide.
  • mRNA as used herein may encompass both modified and unmodified RNA.
  • mRNA may contain one or more coding and non-coding regions.
  • mRNA can be purified from natural sources, produced using recombinant expression systems and optionally purified, chemically synthesized, etc. Where appropriate, e.g., in the case of chemically synthesized molecules, mRNA can comprise nucleoside analogs such as analogs having chemically modified bases or sugars, backbone modifications, etc.
  • An mRNA sequence is presented in the 5′ to 3′ direction unless otherwise indicated.
  • an mRNA is or comprises natural nucleosides (e.g., adenosine, guanosine, cytidine, uridine); nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyl adenosine, 5-methylcytidine, C-5 propynyl-cytidine, C-5 propynyl-uridine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5-propynyl-cytidine, C5-methylcytidine, 2-aminoadenosine, 7- deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, O(6)-methyl
  • nucleic acid in its broadest sense, refers to any compound and/or substance that is or can be incorporated into a polynucleotide chain.
  • a nucleic acid is a compound and/or substance that is or can be incorporated into a polynucleotide chain via a phosphodiester linkage.
  • nucleic acid refers to individual nucleic acid residues (e.g., nucleotides and/or nucleosides).
  • nucleic acid refers to a polynucleotide chain comprising individual nucleic acid residues.
  • the term “subject” refers to a human or any non-human animal (e.g., mouse, rat, rabbit, dog, cat, cattle, swine, sheep, horse or primate).
  • a human includes pre- and post-natal forms.
  • a subject is a human being.
  • a subject can be a patient, which refers to a human presenting to a medical provider for diagnosis or treatment of a disease.
  • the term “subject” is used herein interchangeably with “individual” or “patient.”
  • a subject can be afflicted with or is susceptible to a disease or disorder but may or may not display symptoms of the disease or disorder.
  • target tissues refers to any tissue that is affected by a disease to be treated. In some embodiments, target tissues include those tissues that display disease-associated pathology, symptom, or feature.
  • treatment is defined as the application or administration of a therapeutic agent to a patient, or application or administration of a therapeutic agent to an isolated tissue or cell line from a patient (e.g., for diagnosis or ex vivo applications), who has a disorder or disease as described herein, a symptom thereof; or the potential to develop such disorder or disease, where the purpose of the application or administration is to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve or affect the disorder or disease, or its symptoms.
  • Such treatments may be specifically tailored or modified, based on knowledge obtained from the field of pharmacogenomics.
  • Isomers can be isolated from mixtures by methods known to those skilled in the art, including chiral high performance liquid chromatography (HPLC) and the formation and crystallization of chiral salts; or preferred isomers can be prepared by asymmetric syntheses. See, for example, Jacques et al., Enantiomers. Racemates and Resolutions (Wiley Interscience, New York, 1981); Wilen et al., Tetrahedron 33:2725 (1977); Eliel, E. L. Stereochemistry of Carbon Compounds (McGraw-Hill, NY, 1962); and Wilen, S. H. Tables of Resolving Agents and Optical Resolutions p.268 (E. L. Eliel, Ed., Univ.
  • alkyl refers to a straight or branched saturated hydrocarbon.
  • an alkyl group can have 1 to 30 carbon atoms (i.e., (C 1 -C 30 )alkyl), 1 to 20 carbon atoms (i.e., (C 1 -C 20 )alkyl), 1 to 12 carbon atoms (i.e., (C 1 -C 12 )alkyl), 1 to 6 carbon atoms (i.e., (C 1 -C 6 )alkyl), or 1 to 3 carbon atoms (i.e., (C 1 -C 3 )alkyl).
  • alkyl groups include, but are not limited to, methyl (Me, -CH 3 ), ethyl (Et, -CH 2 CH 3 ), 1- propyl (n-Pr, n-propyl, -CH 2 CH 2 CH 3 ), isopropyl (i-Pr, i-propyl, -CH(CH 3 )2), 1-butyl (n-bu, n-butyl, -CH 2 CH 2 CH 2 CH 3 ), 2-butyl (s-bu, s-butyl, -CH(CH 3 )CH 2 CH 3 ), tert-butyl (t-bu, t- butyl, -CH(CH 3 ) 3 ), 1-pentyl (n-pentyl, -CH 2 CH 2 CH 2 CH 2 CH 3 ), 2-pentyl (-CH(CH 3 ) CH 2 CH 2 CH 3 ), neopentyl (CH 2 C(CH 3 )3), 1-hex
  • alkyl When a bivalent variable is defined as “alkyl,” it is to be understood that such a group is a bivalent alkylene group.
  • alkenyl refers to a straight or branched saturated hydrocarbon having at least one site of carbon-carbon double bond unsaturation.
  • an alkenyl group can have 2 to 30 carbon atoms (i.e., (C2-C30)alkenyl), 2 to 20 carbon atoms (i.e., (C2-C20)alkenyl), 2 to 12 carbon atoms (i.e., (C2-C12)alkenyl) or 2 to 6 carbon atoms (i.e., (C 2 -C 6 )alkenyl), and the alkenyl group can contain 1, 2, 3, or 4 carbon- carbon double bonds.
  • the one or more carbon-carbon double bonds can be internal (such as in 2-butenyl) or terminal (such as in 1-butenyl). Included within this term are the cis and trans isomers or mixtures of these isomers.
  • alkenyl groups include prop- 2-enyl, but-2-enyl, but-3-enyl, 2-methylprop-2-enyl, hex-2-enyl, hex- 5-enyl, 2,3- dimethylbut-2-enyl, and the like.
  • alkenyl it is to be understood that such a group is a bivalent alkenylene group.
  • alkynyl refers to a straight or branched saturated hydrocarbon having at least one site of carbon-carbon triple bond unsaturation occurring at any stable point along the chain.
  • an alkynyl group can have 2 to 30 carbon atoms (i.e., (C 2 -C 30 )alkynyl), 2 to 20 carbon atoms (i.e., (C 2 -C 20 )alkynyl), 2 to 12 carbon atoms (i.e., (C 2 -C 12 )alkynyl) or 2 to 6 carbon atoms (i.e., (C 2 -C 6 )alkynyl), and the alkynyl group can contain 1, 2, 3, or 4 carbon-carbon triple bonds.
  • the one or more carbon-carbon triple bonds can be internal or terminal.
  • the alkynyl group may include one or more double bonds (e.g., 1, 2, 3, or 4 double bonds).
  • a “counteranion” is a negatively charged group associated with a positively charged quarternary amine in order to maintain electronic neutrality.
  • exemplary counteranions include halide ions (e.g., F—, Cl—, Br—, I—), NO3-, ClO4-, OH—, H2PO4-, HSO4-, sulfonate ions (e.g., methansulfonate, trifluoromethanesulfonate, p-toluenesulfonate, benzenesulfonate, 10-camphor sulfonate, naphthalene-2-sulfonate, naphthalene-1-sulfonic acid-5-sulfonate, ethan-1-sulfonic acid-2-sulfonate, and the like), and carboxylate ions (e.g., acetate, ethanoate, propanoate, benzoate, glycerate, lactate,
  • R 1 and R 2 are independently selected from -C (6-24) alkyl, -C (6- 24) alkenyl, -C (6-24) alkynyl, , , and , wherein the -C (6- 24) alkyl, -C (6-24) alkenyl, and -C (6-24) alkynyl are each optionally substituted with one to six groups independently selected from halo, -OC (1-6) alkyl, -OC (2-6) alkenyl, -SC (1-6) alkyl, -SC (2- 6) alkenyl, and -C(O)OC (1-6) alkyl.
  • R 1 and R 3 are independently . In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, R 1 and R 3 are independently . In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, R 1 and R 3 are independently . In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, R 1 and R 3 are independently . In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, R 1 and R 3 are independently . In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, R 2 and R 3 are independently -C (6-24) alkyl.
  • X 2 is -OC(O)NH-. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, X 2 is -NHC(O)O-. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, X 2 is -NHC(O)NH-. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, X 3 is -C (5-24) alkyl or -C (5-24) alkenyl. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, X 3 is -C (5-24) alkyl.
  • X 3 is -C(5-20)alkyl. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, X 3 is -C(10-18)alkyl. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, X 3 is . In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, X 3 is -C (5-24) alkenyl. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, X 3 is -C (5-15) alkenyl.
  • two of R 1 , R 2 , and R 3 are independently selected from the group consisting of formulas A1-A21: In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, two of R 1 , R 2 , and R 3 are independently selected from the group consisting of formulas A1, A3, and A7-A18. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, two of R 1 , R 2 , and R 3 are independently selected from the group consisting of formulas A13, A14, and A15.
  • R 1 , R 2 , and R 3 are independently selected from the group consisting of formulas A13 and A14:
  • R 1 and R 2 are independently selected from the group consisting of formulas A1, A3, and A7-A18.
  • R 1 and R 2 are independently selected from the group consisting of formulas A1-A21.
  • R 1 and R 2 are independently selected from the group consisting of formulas A13, A14, and A15.
  • R 1 and R 3 are independently selected from the group consisting of formulas A13 and A14.
  • R 2 and R 3 are independently selected from the group consisting of formulas A1, A3, and A7-A18.
  • R 2 and R 3 are independently selected from the group consisting of formulas A1-A21.
  • R 2 and R 3 are independently selected from the group consisting of formulas A13, A14, and A15.
  • R 2 and R 3 are independently selected from the group consisting of formulas A13 and A14. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, two of R 1 , R 2 , and R 3 are independently selected from the group consisting of formulas B1-58: In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, two of R 1 , R 2 , and R 3 are independently selected from the group consisting of formulas B1-56.
  • two of R 1 , R 2 , and R 3 are independently selected from the group consisting of formulas B1, B2, B3, B4, B5, B7, B10, B12, B15, B16, B18, B21, B24, B28, B31, B33, B36, B37, B40, B41, B43, B44, B46, B47, B48, B49, B50, and B51.
  • two of R 1 , R 2 , and R 3 are independently selected from the group consisting of formulas B1, B2, B5, B33-B53, B57, and B58.
  • two of R 1 , R 2 , and R 3 are independently selected from the group consisting of formulas B33-B51. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, two of R 1 , R 2 , and R 3 are independently selected from the group consisting of formulas B36- B44. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, R 1 and R 2 are independently selected from the group consisting of formulas B1-B58. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, R 1 and R 2 are independently selected from the group consisting of formulas B1-B56.
  • R 1 and R 2 are independently selected from the group consisting of formulas B1, B2, B3, B4, B5, B7, B10, B12, B15, B16, B18, B21, B24, B28, B31, B33, B36, B37, B40, B41, B43, B44, B46, B47, B48, B49, B50, and B51.
  • R 1 and R 2 are independently selected from the group consisting of formulas B1, B2, B5, B33- B53, B57, and B58.
  • R 1 and R 2 are independently selected from the group consisting of formulas B33-B51. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, R 1 and R 2 are independently selected from the group consisting of formulas B36-B44. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, R 1 and R 3 are independently selected from the group consisting of formulas B1-B58. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, R 1 and R 3 are independently selected from the group consisting of formulas B1-B56.
  • R 1 and R 3 are independently selected from the group consisting of formulas B1, B2, B3, B4, B5, B7, B10, B12, B15, B16, B18, B21, B24, B28, B31, B33, B36, B37, B40, B41, B43, B44, B46, B47, B48, B49, B50, and B51.
  • R 1 and R 3 are independently selected from the group consisting of formulas B1, B2, B5, B33- B53, B57, and B58.
  • R 1 and R 3 are independently selected from the group consisting of formulas B33-B51. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, R 1 and R 3 are independently selected from the group consisting of formulas B36-B44. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, R 2 and R 3 are independently selected from the group consisting of formulas B1-B58. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, R 2 and R 3 are independently selected from the group consisting of formulas B1-B56.
  • R 1 is . In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, R 1 is . In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, R 2 is or . In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, R 2 is . In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, R 2 is . In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, R 3 is or . In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, R 3 is .
  • A is ; and R A1 and R A2 are each independently -C (1-6) alkyl that is optionally substituted with - OH; or R A1 and R A2 are taken together to form a 3- to 6-membered heteroaryl that is optionally substituted with one to three -C (1-3) alkyl groups.
  • R A1 and R A2 are each independently -C (1-4) alkyl that is optionally substituted with hydroxyl; or R A1 and R A2 are taken together to form a 5- to 6-membered heteroaryl that is optionally substituted with methyl.
  • A is . In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, A is . In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, A is . In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, A is . In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, is selected from the group consisting of Formulas C1-C12: In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, is selected from the group consisting of Formulas C1-C10. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, has the structure of formula C1.
  • the compound of Formula (I), or a pharmaceutically acceptable salt thereof has the structure of formula C8. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, has the structure of formula C9. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, has the structure of formula C10. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, has the structure of formula C11. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, has the structure of formula C12.
  • the compound of Formula (I), or a pharmaceutically acceptable salt thereof is selected from the group consisting of Formulas D1-D48: In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, is selected from the group consisting of Formulas D1-D40. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, is selected from the group consisting of formulas D1-D4. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, is selected from the group consisting of formulas E1-E12:
  • the compound of Formula (I), or a pharmaceutically acceptable salt thereof is selected from the group consisting of formulas E1-E10. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, is selected from the group consisting of formulas E1 and E3: In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, is selected from the group consisting of formulas F1- F2: In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, R 1 is selected from the group consisting of formulas C1-C12 and E1- E12.
  • R 1 is selected from the group consisting of formulas E1-E12. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, R 1 is selected from the group consisting of formulas E1-E10. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, R 1 is selected from the group consisting of formulas E1 and E3. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, R 1 is selected from the group consisting of formulas F1 and F2.
  • R 2 is selected from the group consisting of formulas C1-C12 and E1- E12. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, R 2 is selected from the group consisting of formulas C1-C10 and E1-E10. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, R 2 is selected from the group consisting of formulas C1-C12. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, R 2 is selected from the group consisting of formulas C1-C10.
  • R 2 has the structure of formula C1. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, R 2 is selected from the group consisting of formulas D1-D4. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, R 2 is selected from the group consisting of formulas E1-E12. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, R 2 is selected from the group consisting of formulas E1-E10. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, R 2 is selected from the group consisting of formulas E1 and E3.
  • R 2 is selected from the group consisting of formulas F1 and F2.
  • R 3 is selected from the group consisting of formulas C1-C12 and E1- E12.
  • R 3 is selected from the group consisting of formulas C1-C10 and E1-E10.
  • R 3 is selected from the group consisting of formulas C1-C12.
  • R 3 is selected from the group consisting of formulas C1-C10. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, R 3 has the structure of formula C1. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, R 3 is selected from the group consisting of formulas D1-D4. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, R 3 is selected from the group consisting of formulas E1-E12. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, R 3 is selected from the group consisting of formulas E1-E10.
  • R 3 is selected from the group consisting of formulas E1 and E3. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, R 3 is selected from the group consisting of formulas F1 and F2. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, X is selected from the group consisting of: , In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, X is selected from the group consisting of: In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, X is .
  • X is . In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, X is . In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, X is . In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, X is . In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, X is . In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, X is . In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, X is . In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, X is . In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, X is . In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, X is . In some embodiments of the compound of Formula (I), or a
  • X is . In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, X is . In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, X is . In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, X is .
  • the compound of Formula (I) has a structure selected from the group consisting of Formulas (I-a1), (I-a2), (I-a3), and (I-a4), or a pharmaceutically acceptable salt thereof: In some embodiments, the compound of Formula (I) has a structure according to Formula (I-a1), or a pharmaceutically acceptable salt thereof. In some embodiments, the compound of Formula (I) has a structure according to Formula (I-a2), or a pharmaceutically acceptable salt thereof. In some embodiments, the compound of Formula (I) has a structure according to Formula (I-a3), or a pharmaceutically acceptable salt thereof.
  • the compound of Formula (I) has a structure according to Formula (I-a4), or a pharmaceutically acceptable salt thereof.
  • the compound of Formula (I) has a structure selected from the group consisting of Formulas (I-b1), (I-b2), (I-b3), and (I-b4), or a pharmaceutically acceptable salt thereof:
  • the compound of Formula (I) has a structure according to Formula (I-b1), or a pharmaceutically acceptable salt thereof.
  • the compound of Formula (I) has a structure according to Formula (I-b2), or a pharmaceutically acceptable salt thereof.
  • the compound of Formula (I) has a structure according to Formula (I-b3), or a pharmaceutically acceptable salt thereof. In some embodiments, the compound of Formula (I) has a structure according to Formula (I-b4), or a pharmaceutically acceptable salt thereof. In some embodiments, the compound of Formula (I) has a structure selected from the group consisting of Formulas (I-c1), (I-c2), (I-c3), and (I-c4), or a pharmaceutically acceptable salt thereof: In some embodiments, the compound of Formula (I) has a structure according to Formula (I-c1), or a pharmaceutically acceptable salt thereof.
  • the compound of Formula (I) has a structure according to Formula (I-c2), or a pharmaceutically acceptable salt thereof. In some embodiments, the compound of Formula (I) has a structure according to Formula (I-c3), or a pharmaceutically acceptable salt thereof. In some embodiments, the compound of Formula (I) has a structure according to Formula (I-c4), or a pharmaceutically acceptable salt thereof.
  • the compound of Formula (I) has a structure selected from the group consisting of Formulas (I-d1), (I-d2), (I-d3), and (I-d4), or a pharmaceutically acceptable salt thereof: In some embodiments, the compound of Formula (I) has a structure according to Formula (I-d1), or a pharmaceutically acceptable salt thereof. In some embodiments, the compound of Formula (I) has a structure according to Formula (I-d2), or a pharmaceutically acceptable salt thereof. In some embodiments, the compound of Formula (I) has a structure according to Formula (I-d3), or a pharmaceutically acceptable salt thereof.
  • the compound of Formula (I) has a structure according to Formula (I-d4), or a pharmaceutically acceptable salt thereof.
  • the compound of Formula (I) has a structure selected from the group consisting of Formulas (I-e1), (I-e2), (I-e3), and (I-e4), or a pharmaceutically acceptable salt thereof:
  • the compound of Formula (I) has a structure according to Formula (I-e1), or a pharmaceutically acceptable salt thereof.
  • the compound of Formula (I) has a structure according to Formula (I-e2), or a pharmaceutically acceptable salt thereof.
  • the compound of Formula (I) has a structure according to Formula (I-e3), or a pharmaceutically acceptable salt thereof. In some embodiments, the compound of Formula (I) has a structure according to Formula (I-e4), or a pharmaceutically acceptable salt thereof. In some embodiments, the compound of Formula (I) has a structure selected from the group consisting of Formulas (I-f1), (I-f2), (I-f3), and (I-f4), or a pharmaceutically acceptable salt thereof: In some embodiments, the compound of Formula (I) has a structure according to Formula (I-f1), or a pharmaceutically acceptable salt thereof.
  • the compound of Formula (I) has a structure according to Formula (I-f2), or a pharmaceutically acceptable salt thereof. In some embodiments, the compound of Formula (I) has a structure according to Formula (I-f3), or a pharmaceutically acceptable salt thereof. In some embodiments, the compound of Formula (I) has a structure according to Formula (I-f4), or a pharmaceutically acceptable salt thereof.
  • the compound of Formula (I) has a structure selected from the group consisting of Formulas (I-g1), (I-g2), (I-g3), and (I-g4), or a pharmaceutically acceptable salt thereof: In some embodiments, the compound of Formula (I) has a structure according to Formula (I-g1), or a pharmaceutically acceptable salt thereof. In some embodiments, the compound of Formula (I) has a structure according to Formula (I-g2), or a pharmaceutically acceptable salt thereof. In some embodiments, the compound of Formula (I) has a structure according to Formula (I-g3), or a pharmaceutically acceptable salt thereof.
  • the compound of Formula (I) has a structure according to Formula (I-g4), or a pharmaceutically acceptable salt thereof.
  • the compound of Formula (I) has a structure selected from the group consisting of Formulas (I-h1), (I-h2), (I-h3), and (I-h4), or a pharmaceutically acceptable salt thereof:
  • the compound of Formula (I) has a structure according to Formula (I-h1), or a pharmaceutically acceptable salt thereof. In some embodiments, the compound of Formula (I) has a structure according to Formula (I-h2), or a pharmaceutically acceptable salt thereof. In some embodiments, the compound of Formula (I) has a structure according to Formula (I-h3), or a pharmaceutically acceptable salt thereof. In some embodiments, the compound of Formula (I) has a structure according to Formula (I-h4), or a pharmaceutically acceptable salt thereof.
  • the compound of Formula (I) has a structure selected from the group consisting of Formulas (I-i1), (I-i2), (I-i3), and (I-i4), or a pharmaceutically acceptable salt thereof: In some embodiments, the compound of Formula (I) has a structure according to Formula (I-i1), or a pharmaceutically acceptable salt thereof. In some embodiments, the compound of Formula (I) has a structure according to Formula (I-i2), or a pharmaceutically acceptable salt thereof. In some embodiments, the compound of Formula (I) has a structure according to Formula (I-i3), or a pharmaceutically acceptable salt thereof.
  • the compound of Formula (I) has a structure according to Formula (I-i4), or a pharmaceutically acceptable salt thereof.
  • the compound of Formula (I) has a structure selected from the group consisting of Formulas (I-j1), (I-j2), (I-j3), and (I-j4), or a pharmaceutically acceptable salt thereof:
  • the compound of Formula (I) has a structure according to Formula (I-j1), or a pharmaceutically acceptable salt thereof.
  • the compound of Formula (I) has a structure according to Formula (I-j2), or a pharmaceutically acceptable salt thereof.
  • the compound of Formula (I) has a structure according to Formula (I-j3), or a pharmaceutically acceptable salt thereof. In some embodiments, the compound of Formula (I) has a structure according to Formula (I-j4), or a pharmaceutically acceptable salt thereof. In some embodiments, the compound of Formula (I) has a structure selected from the group consisting of Formulas (I-k1), (I-k2), (I-k3), and (I-k4), or a pharmaceutically acceptable salt thereof: In some embodiments, the compound of Formula (I) has a structure according to Formula (I-k1), or a pharmaceutically acceptable salt thereof.
  • the compound of Formula (I) has a structure according to Formula (I-k2), or a pharmaceutically acceptable salt thereof. In some embodiments, the compound of Formula (I) has a structure according to Formula (I-k3), or a pharmaceutically acceptable salt thereof. In some embodiments, the compound of Formula (I) has a structure according to Formula (I-k4), or a pharmaceutically acceptable salt thereof.
  • the compound of Formula (I) has a structure selected from the group consisting of Formulas (I-l1), (I-l2), (I-l3), and (I-l4), or a pharmaceutically acceptable salt thereof: In some embodiments, the compound of Formula (I) has a structure according to Formula (I-l1), or a pharmaceutically acceptable salt thereof. In some embodiments, the compound of Formula (I) has a structure according to Formula (I-l2), or a pharmaceutically acceptable salt thereof. In some embodiments, the compound of Formula (I) has a structure according to Formula (I-l3), or a pharmaceutically acceptable salt thereof.
  • the compound of Formula (I) has a structure according to Formula (I-l4), or a pharmaceutically acceptable salt thereof.
  • R X is -H or -CH 3.
  • R X is -H.
  • R X is -CH 3 .
  • n is 1 or 2. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, n is 1.
  • the compound of Formula (I) has a structure according to Formula (I-a1), (I-b1), (I-c1), (I-d1), (I-e1), (I-f1), (I-g1), (I-h1), (I-i1), (I-j1), (I-k1), or (I-l1).
  • the compound of Formula (I) has a structure according to Formula (I-a1), (I-b1), (I-c1), (I-d1), (I-e1), (I-f1), (I-g1), (I-h1), or (I-i1).
  • n is 2.
  • the compound of Formula (I) has a structure according to Formula (I-a2), (I-b2), (I-c2), (I-d2), (I-e2), (I-f2), (I-g2), (I-h2), (I-i2), (I-j2), (I-k2), or (I-l2).
  • the compound of Formula (I) has a structure according to Formula (I-a2), (I-b2), (I-c2), (I-d2), (I-e2), (I-f2), (I-g2), (I-h2), or (I-i2).
  • n is 3.
  • the compound of Formula (I) has a structure according to Formula (I-a3), (I-b3), (I-c3), (I-d3), (I-e3), (I-f3), (I-g3), (I-h3), (I-i3), (I-j3), (I-k3), or (I-l3).
  • the compound of Formula (I) has a structure according to Formula (I-a3), (I-b3), (I-c3), (I-d3), (I-e3), (I-f3), (I-g3), (I-h3), or (I-i3).
  • n is 4.
  • the compound of Formula (I) has a structure according to Formula (I-a4), (I-b4), (I-c4), (I-d4), (I-e4), (I-f4), (I-g4), (I-h4), (I-i4), (I-j4), (I-k4), or (I-l4).
  • the compound of Formula (I) has a structure according to Formula (I-a4), (I-b4), (I-c4), (I-d4), (I-e4), (I-f4), (I-g4), (I-h4), or (I-i4).
  • R 1 and R 2 are independently selected from the group consisting of formulas A1-A21; and R 3 is selected from the group consisting of formulas C1-C12 and E1- E12. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, R 1 and R 2 are independently selected from the group consisting of formulas A1- A21; and R 3 is selected from the group consisting of formulas C1-C10 and E1-E10.
  • R 1 and R 2 are independently selected from the group consisting of formulas A1-A21; and R 3 is selected from the group consisting of formulas C1-C12.
  • R 1 and R 2 are independently selected from the group consisting of formulas A1-A21; and R 3 is selected from the group consisting of formulas C1-C10.
  • R 1 and R 2 are independently selected from the group consisting of formulas A1, A3, and A7-A18; and R 3 is selected from the group consisting of formulas C1-C12.
  • R 1 and R 2 are independently selected from the group consisting of formulas A13-A15; and R 3 has the structure of formula C1.
  • R 1 and R 2 are independently selected from the group consisting of formulas B1-B58; and R 3 is selected from the group consisting of formulas D1-D48, F1, and F2.
  • R 1 and R 2 are independently selected from the group consisting of formulas B1- B56; and R 3 is selected from the group consisting of formulas D1-D40, F1, and F2.
  • R 1 and R 2 are independently selected from the group consisting of formulas B1, B2, B3, B4, B5, B7, B10, B12, B15, B16, B18, B21, B24, B28, B31, B33, B36, B37, B40, B41, B43, B44, B46, B47, B48, B49, B50, and B51; and R 3 is selected from the group consisting of formulas D1-D40.
  • R 1 and R 2 are independently selected from the group consisting of formulas B1, B2, B3, B4, B5, B7, B10, B12, B15, B16, B18, B21, B24, B28, B31, B33, B36, B37, B40, B41, B43, B44, B46, B47, B48, B49, B50, and B51; and R 3 is selected from the group consisting of formulas D1-D4.
  • R 1 and R 2 are independently selected from the group consisting of formulas B1, B2, B5, B33-B53, B57, and B58; and R 3 is selected from the group consisting of formulas D1-D48.
  • R 1 and R 2 are independently selected from the group consisting of formulas B33-B51; and R 3 is selected from the group consisting of formulas D1-D48.
  • R 1 and R 2 are independently selected from the group consisting of formulas B33-B51; and R 3 is selected from the group consisting of formulas D1-D40.
  • R 1 and R 2 are independently selected from the group consisting of formulas B36-B44; and R 3 is selected from the group consisting of formulas D1-D48.
  • R 1 and R 2 are independently selected from the group consisting of formulas B36-B44; and R 3 is selected from the group consisting of formulas D1-D4.
  • R 1 and R 3 are independently selected from the group consisting of formulas A1-A21; and R 2 is selected from the group consisting of formulas C1-C12 and E1- E12. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, R 1 and R 3 are independently selected from the group consisting of formulas A1- A21; and R 2 is selected from the group consisting of formulas C1-C10 and E1-E10.
  • R 1 and R 3 are independently selected from the group consisting of formulas A1-A21; and R 2 is selected from the group consisting of formulas C1-C12. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, R 1 and R 3 are independently selected from the group consisting of formulas A1-A21; and R 2 is selected from the group consisting of formulas C1-C10. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, R 1 and R 3 are independently selected from the group consisting of formulas A1, A3, and A7-A18; and R 2 is selected from the group consisting of formulas C1-C12.
  • R 1 and R 3 are independently selected from the group consisting of formulas A13-A15; and R 2 has the structure of formula C1.
  • R 1 and R 3 are independently selected from the group consisting of formulas B1-B58; and R 2 is selected from the group consisting of formulas D1-D48, F1, and F2.
  • R 1 and R 3 are independently selected from the group consisting of formulas B1- B56; and R 2 is selected from the group consisting of formulas D1-D40, F1, and F2.
  • R 1 and R 3 are independently selected from the group consisting of formulas B1, B2, B3, B4, B5, B7, B10, B12, B15, B16, B18, B21, B24, B28, B31, B33, B36, B37, B40, B41, B43, B44, B46, B47, B48, B49, B50, and B51; and R 2 is selected from the group consisting of formulas D1-D40.
  • R 1 and R 3 are independently selected from the group consisting of formulas B1, B2, B3, B4, B5, B7, B10, B12, B15, B16, B18, B21, B24, B28, B31, B33, B36, B37, B40, B41, B43, B44, B46, B47, B48, B49, B50, and B51; and R 2 is selected from the group consisting of formulas D1-D4.
  • R 1 and R 3 are independently selected from the group consisting of formulas B1, B2, B5, B33-B53, B57, and B58; and R 2 is selected from the group consisting of formulas D1-D48.
  • R 1 and R 3 are independently selected from the group consisting of formulas B33-B51; and R 2 is selected from the group consisting of formulas D1-D48.
  • R 1 and R 3 are independently selected from the group consisting of formulas B33-B51; and R 2 is selected from the group consisting of formulas D1-D40.
  • R 1 and R 3 are independently selected from the group consisting of formulas B36-B44; and R 2 is selected from the group consisting of formulas D1-D48.
  • R 1 and R 3 are independently selected from the group consisting of formulas B36-B44; and R 2 is selected from the group consisting of formulas D1-D4.
  • R 2 and R 3 are independently selected from the group consisting of formulas A1-A21; and R 1 is selected from the group consisting of formulas C1-C12 and E1- E12. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, R 2 and R 3 are independently selected from the group consisting of formulas A1- A21; and R 1 is selected from the group consisting of formulas C1-C10 and E1-E10.
  • R 2 and R 3 are independently selected from the group consisting of formulas A1-A21; and R 1 is selected from the group consisting of formulas C1-C12.
  • R 2 and R 3 are independently selected from the group consisting of formulas A1-A21; and R 1 is selected from the group consisting of formulas C1-C10.
  • R 2 and R 3 are independently selected from the group consisting of formulas A1, A3, and A7-A18; and R 1 is selected from the group consisting of formulas C1-C12.
  • R 2 and R 3 are independently selected from the group consisting of formulas A13-A15; and R 1 has the structure of formula C1.
  • R 2 and R 3 are independently selected from the group consisting of formulas B1-B58; and R 1 is selected from the group consisting of formulas D1-D48, F1, and F2.
  • R 2 and R 3 are independently selected from the group consisting of formulas B1- B56; and R 1 is selected from the group consisting of formulas D1-D40, F1, and F2.
  • R 2 and R 3 are independently selected from the group consisting of formulas B1, B2, B3, B4, B5, B7, B10, B12, B15, B16, B18, B21, B24, B28, B31, B33, B36, B37, B40, B41, B43, B44, B46, B47, B48, B49, B50, and B51; and R 1 is selected from the group consisting of formulas D1-D48.
  • R 2 and R 3 are independently selected from the group consisting of formulas B1, B2, B3, B4, B5, B7, B10, B12, B15, B16, B18, B21, B24, B28, B31, B33, B36, B37, B40, B41, B43, B44, B46, B47, B48, B49, B50, and B51; and R 1 is selected from the group consisting of formulas D1-D40.
  • R 2 and R 3 are independently selected from the group consisting of formulas B1, B2, B3, B4, B5, B7, B10, B12, B15, B16, B18, B21, B24, B28, B31, B33, B36, B37, B40, B41, B43, B44, B46, B47, B48, B49, B50, and B51; and R 1 is selected from the group consisting of formulas D1-D4.
  • R 2 and R 3 are independently selected from the group consisting of formulas B1, B2, B5, B33-B53, B57, and B58; and R 1 is selected from the group consisting of formulas D1-D48.
  • R 2 and R 3 are independently selected from the group consisting of formulas B33-B51; and R 1 is selected from the group consisting of formulas D1-D48.
  • R 2 and R 3 are independently selected from the group consisting of formulas B33-B51; and R 1 is selected from the group consisting of formulas D1-D40.
  • R 2 and R 3 are independently selected from the group consisting of formulas B36-B44; and R 1 is selected from the group consisting of formulas D1-D48.
  • R 2 and R 3 are independently selected from the group consisting of formulas B36-B44; and R 1 is selected from the group consisting of formulas D1-D4.
  • the compound of Formula (I) has a structure selected from the group consisting of: or a pharmaceutically acceptable salt thereof.
  • the compound of Formula (I) has a structure selected from the group consisting of:
  • ionizable/Cationic Lipid Synthesis The ionizable or cationic lipids of the present disclosure (e.g., compounds of Formula (I)) can be prepared according to methods known in the art. Nonlimiting and exemplary synthetic methods are disclosed herein. For example, Scheme A provides exemplary synthetic routes for preparing certain ionizable lipids of the present disclosure. Malic acid (1) can be esterified with methanol under acidic conditions to provide dimethyl malate (2), which can then be reacted with an alcohol (3) to provide intermediate (4). Intermediate (4) can be reacted with a carboxylic acid (5) in the presence of a coupling reagent, such as EDCI, to yield the ester product (8). Alternately, intermediate (4) can be reacted with a carbonate compound (6) to afford the carbonate product (9). Intermediate (4) can also be reacted with a carbamate compound (7), to afford the carbamate product (10).
  • a coupling reagent such as EDCI
  • Scheme A (a is 1-7; R is -C (6-24) alkyl, -C (6-24) alkenyl, -C (6-24) alkynyl, , and wherein the -C (6-24) alkyl, -C (6-24) alkenyl, and -C (6-24) alkynyl are each optionally substituted with one to six groups independently selected from halo, -OC (1-6) alkyl, -OC (2- 6 )alkenyl, -SC (1-6) alkyl, -SC (2-6) alkenyl, and -C(O)OC (1-6) alkyl)
  • Scheme B provides an exemplary synthetic route for preparing certain ionizable lipids of the present disclosure.
  • N-Boc-(L)-aspartic acid (11) can be reacted with an alcohol (3) in the presence of a coupling reagent, such as EDCI, to provide intermediate (12), which can be deprotected with TFA to afford amine (13).
  • a coupling reagent such as EDCI
  • Amine (13) can then be reacted with 4- nitrophenylchloroformate (14) to yield intermediate (15), which can be reacted with an alcohol (16) to afford the carbamate product (17).
  • Scheme B (a is 1-7; R is -C (6-24) alkyl, -C (6-24) alkenyl, -C (6-24) alkynyl, , and wherein the -C (6-24) alkyl, -C (6-24) alkenyl, and -C (6-24) alkynyl are each optionally substituted with one to six groups independently selected from halo, -OC (1-6) alkyl, -OC (2- 6 )alkenyl, -SC (1-6) alkyl, -SC (2-6) alkenyl, and -C(O)OC (1-6) alkyl)
  • Scheme C provides exemplary synthetic routes for preparing certain ionizable lipids of the present disclosure.
  • ⁇ -Ketoglutaric acid (18) can be reacted with alcohol (3) under acidic conditions to afford diester (19), which can be reduced with a reducing agent, such as NaBH 4 , to provide intermediate (20).
  • Intermediate (20) can be reacted with a carboxylic acid (5) and a coupling reagent like EDCI to yield the ester product (22).
  • intermediate (20) can be reacted sequentially with 4-nitrophenylchloroformate (14) and an alcohol (16) to afford the carbonate product (23).
  • Intermediate (20) can also be reacted sequentially with 4- nitrophenylchloroformate (14) and an amine (21) to afford the carbonate product (24).
  • Scheme C (a is 1-7; R is -C (6-24) alkyl, -C (6-24) alkenyl, -C (6-24) alkynyl, , and wherein the -C (6-24) alkyl, -C (6-24) alkenyl, and -C (6-24) alkynyl are each optionally substituted with one to six groups independently selected from halo, -OC (1-6) alkyl, -OC (2- 6) alkenyl, -SC (1-6) alkyl, -SC (2-6) alkenyl, and -C(O)OC (1-6) alkyl)
  • Scheme D provides an exemplary synthetic route for preparing certain ionizable lipids of the present disclosure.
  • (tert-Butoxycarbonyl)-L-glutamic acid (25) can be reacted with an alcohol (3) in the presence of a coupling reagent, such as EDCI, to provide intermediate (26), which can be deprotected with TFA to afford amine (27).
  • a coupling reagent such as EDCI
  • Amine (27) can be reacted with 4- nitrophenylchloroformate (14) to yield intermediate (28), which can then be reacted with an alcohol (16) to afford the carbamate product (29).
  • Scheme D (a is 1-7; R is -C (6-24) alkyl, -C (6-24) alkenyl, -C (6-24) alkynyl, , and wherein the -C (6-24) alkyl, -C (6-24) alkenyl, and -C (6-24) alkynyl are each optionally substituted with one to six groups independently selected from halo, -OC (1-6) alkyl, -OC (2- 6 )alkenyl, -SC (1-6) alkyl, -SC (2-6) alkenyl, and -C(O)OC (1-6) alkyl)
  • Scheme E provides an exemplary synthetic route for preparing certain ionizable lipids of the present disclosure.
  • Dimethyl malate (2) can be reacted with a benzyl protected diol such as compound (30) under acidic conditions to afford intermediate (31).
  • Intermediate (31) can be protected with a hydroxyl protecting group, such as TBDPS, to afford intermediate (32) before deprotecting the benzyl groups (e.g., under hydrolytic conditions) to provide intermediate (33).
  • Intermediate (33) can be oxidized to afford diacid (34), which can then be reacted with an alcohol (35) in the presence of a coupling reagent, such as EDCI, to provide intermediate (36).
  • the hydroxyl group of intermediate (36) can be deprotected to provide intermediate (37), which can then be reacted with a carboxylic acid (5) to afford the product (38').
  • intermediate (37) can be reacted with carbamate (5’) in the presence of a base, such as DIPEA, to afford the product (38’).
  • Scheme E (a is 1-7; R’ is -C (5-24) alkyl, -C (5-24) alkenyl, or -C (5-24) alkynyl, each of which is optionally substituted by one to six groups independently selected from halo, -OC (1-6) alkyl, - OC (2-6) alkenyl, -SC (1-6) alkyl, -SC (2-6) alkenyl, and -C(O)OC (1-6) alkyl)
  • Scheme F provides an exemplary synthetic route for preparing certain ionizable lipids of the present disclosure.
  • (S)-2-(2,2-Dimethyl-5-oxo-1,3-dioxolan-4-yl)acetic acid (39) can be protected with a benzyl group to afford intermediate (40), the acetal moiety of which can then be cleaved under acidic conditions to provide intermediate (41).
  • Intermediate (41) can be reacted with an alcohol (3) under acidic conditions to provide intermediate (42), the benzyl group of which can then be deprotected (e.g., under hydrolytic conditions) to afford intermediate (43).
  • Intermediate (43) can be reacted sequentially with an alcohol (16) and a carboxylic acid (45) in the presence of a coupling reagent such as EDCI to provide the product (46).
  • Scheme F (a is 1-7; R is -C (6-24) alkyl, -C (6-24) alkenyl, -C (6-24) alkynyl, , and , wherein the -C (6-24) alkyl, -C (6-24) alkenyl, and -C (6-24) alkynyl are each optionally substituted with one to six groups independently selected from halo, -OC (1-6) alkyl, -OC (2- 6) alkenyl, -SC (1-6) alkyl, -SC (2-6) alkenyl, and -C(O)OC (1-6) alkyl)
  • Scheme G provides an exemplary synthetic route for preparing certain ionizable lipids of the present disclosure.
  • An alcohol (3) can be reacted with nitrophenylchloroformate (14) to yield intermediate (47), which can then be reacted with intermediate (44) to afford the carbamate product (48).
  • Scheme G (a is 1-7; R is -C (6-24) alkyl, -C (6-24) alkenyl, -C (6-24) alkynyl, , and wherein the -C (6-24) alkyl, -C (6-24) alkenyl, and -C (6-24) alkynyl are each optionally substituted with one to six groups independently selected from halo, -OC (1-6) alkyl, -OC (2- 6 )alkenyl, -SC (1-6) alkyl, -SC (2-6) alkenyl, and -C(O)OC (1-6) alkyl)
  • Scheme H provides an exemplary synthetic route for preparing certain ionizable lipids of the present disclosure.
  • An alcohol (3) can be reacted with (S)-2-(2,2-dimethyl-5-oxo-1,3- dioxolan-4-yl)acetic acid (39) in the presence of a coupling reagent to provide intermediate (49), the acetal moiety of which can be cleaved under acidic conditions to afford intermediate (50).
  • Intermediate (50) can then be reacted sequentially with an alcohol (16) and a carboxylic acid (45) in the presence of a coupling reagent such as EDCI to provide the product (52).
  • Scheme H (a is 1-7; R is -C (6-24) alkyl, -C (6-24) alkenyl, -C (6-24) alkynyl, , and wherein the -C (6-24) alkyl, -C (6-24) alkenyl, and -C (6-24) alkynyl are each optionally substituted with one to six groups independently selected from halo, -OC (1-6) alkyl, -OC (2- 6 )alkenyl, -SC (1-6) alkyl, -SC (2-6) alkenyl, and -C(O)OC (1-6) alkyl)
  • Scheme I provides an exemplary synthetic route for preparing certain ionizable lipids of the present disclosure.
  • An alcohol (3) can be reacted with nitrophenylchloroformate (14) to yield intermediate (47), which can then be reacted with intermediate (51) to afford the carbamate product (53).
  • Scheme I (a is 1-7; R is -C (6-24) alkyl, -C (6-24) alkenyl, -C (6-24) alkynyl, , and wherein the -C (6-24) alkyl, -C (6-24) alkenyl, and -C (6-24) alkynyl are each optionally substituted with one to six groups independently selected from halo, -OC (1-6) alkyl, -OC (2- 6) alkenyl, -SC (1-6) alkyl, -SC (2-6) alkenyl, and -C(O)OC (1-6) alkyl)
  • Scheme J provides an exemplary synthetic route for preparing certain ionizable lipids of the present disclosure.
  • a diol (54) and 2-mercaptosuccinic acid (55) can be reacted in the presence of zinc chloride to provide intermediate (56).
  • Intermediate (56) can then be reacted with dipyridyl disulfide to provide disulfide (57), which can then be reacted with an acyl chloride (58) to provide intermediate (59).
  • intermediate (59) can be reacted with a thiol (60) to provide the disulfide product (61).
  • Scheme J (a is 1-7; R’ is -C (5-24) alkyl, -C (5-24) alkenyl, or -C (5-24) alkynyl, each of which is optionally substituted by one to six groups independently selected from halo, -OC (1-6) alkyl, - OC (2-6) alkenyl, -SC (1-6) alkyl, -SC (2-6) alkenyl, and -C(O)OC (1-6) alkyl) IV.
  • compositions comprising a lipid nanoparticle (LNP), wherein the LNP comprises an ionizable or cationic lipid of the present disclosure (e.g., a lipid of Formula (I)).
  • LNP lipid nanoparticle
  • the LNP further comprises at least one of a structural lipid, a helper lipid, or a stealth lipid.
  • the LNP comprises an ionizable or cationic lipid of the present disclosure and a structural lipid. In some embodiments, the LNP comprises an ionizable or cationic lipid of the present disclosure and a helper lipid. In some embodiments, the LNP comprises an ionizable or cationic lipid of the present disclosure and a stealth lipid. In some embodiments, the LNP comprises an ionizable or cationic lipid of the present disclosure, a structural lipid, a helper lipid, and a stealth lipid.
  • A. Structural Lipids A structural lipid component provides stability to the lipid bilayer structure within the lipid nanoparticle. In some embodiments, the LNP comprises one or more structural lipid.
  • the structural lipid is a cholesterol-based lipid.
  • Suitable cholesterol-based lipids include, for example: DC-Choi (N,N-dimethyl-N-ethylcarboxamidocholesterol), l,4- bis(3-N-oleylamino-propyl)piperazine (Gao et al., Biochem Biophys Res Comm. (1991) 179:280; Wolf et al., BioTechniques (1997) 23:139; U.S.
  • the structural lipid is cholesterol.
  • B. Stealth Lipids A stealth lipid component provides control over particle size and stability of the nanoparticle. The addition of such components may prevent complex aggregation and provide a means for increasing circulation lifetime and increasing the delivery of a lipid- nucleic acid pharmaceutical composition to target tissues.
  • the stealth lipid is a polyethylene glycol-conjugated (PEGylated) lipid. These components may be selected to rapidly exchange out of the pharmaceutical composition in vivo (see, e.g., U.S. Pat.5,885,613).
  • Contemplated PEGylated lipids include, but are not limited to, a polyethylene glycol (PEG) chain of up to 5 kDa in length covalently attached to a lipid with alkyl chain(s) of C 6- C 20 (e.g., C 8 , C 10 , C 12 , C 14 , C 16 , or C 18 ) length, such as a derivatized ceramide (e.g., N- octanoyl-sphingosine-1-[succinyl(methoxypolyethylene glycol)] (C8 PEG ceramide)).
  • PEG polyethylene glycol
  • the PEGylated lipid is 1,2-dimyristoyl-rac-glycero-3- methoxypolyethylene glycol (DMG-PEG); 1,2-distearoyl-sn-glycero-3- phosphoethanolamine-polyethylene glycol (DSPE- PEG); 1,2-dilauroyl-sn-glycero-3- phosphoethanolamine-polyethylene glycol (DLPE-PEG); or 1,2-distearoyl-rac-glycero- polyethelene glycol (DSG-PEG).
  • DMG-PEG 1,2-dimyristoyl-rac-glycero-3- methoxypolyethylene glycol
  • DSPE- PEG 1,2-distearoyl-sn-glycero-3- phosphoethanolamine-polyethylene glycol
  • DLPE-PEG 1,2-dilauroyl-sn-glycero-3- phosphoethanolamine-polyethylene glycol
  • DSG-PEG 1,2-dist
  • the PEG is PEG2000 (or PEG-2K). In some embodiments, the PEGylated lipid is DMG-PEG2000, DSPE-PEG2000, DLPE-PEG2000, DSG-PEG2000, or C8 PEG2000. In some embodiments, the PEGylated lipid is dimyristoyl-PEG2000 (DMG- PEG2000). In some embodiments, the stealth lipid is a polyoxazoline polymer-conjugated lipid. Polyoxazoline polymer-conjugated lipids suitable for the LNP compositions of the present disclosure are described, for example, in WO2022/173667 and WO2023/031394.
  • the stealth lipid is a polysarcosine-conjugated (pSar) lipid.
  • the polysarcosine comprises 25-45 sarcosine units.
  • the polysarcosine comprises 25 sarcosine units.
  • the polysarcosine comprises 35 sarcosine units.
  • the polysarcosine comprises 45 sarcosine units.
  • Nonlimiting examples of pSar lipids include N-tetradecyl-pSar25, N-hexadecyl- pSar25, N-octadecyl-pSar25, N-dodecyl-pSar25, 1,2-dimyristoyl-sn-glycero-3-succinyl-N- polysarcosine-25 (DMG-pSar25), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N- polysarcosine-25 (18:1 PE (DOPE) pSar25), N,N-ditetradecylamine-N- succinyl[methyl(polysarcosine)45], N,N-ditetradecylamine-N- succinyl[methyl(polysarcosine)35], and N,N-ditetradecyl-polysarcosine-25.
  • DMG-pSar25 1,2-dimy
  • helper lipids suitable for the LNP compositions of the present disclosure are described in WO2020/070040.
  • a helper lipid enhances the structural stability of the LNP and helps the LNP in endosomal escape.
  • a helper lipid may improve uptake and release of an mRNA drug payload encapsulated in the LNP.
  • the helper lipid is a zwitterionic lipid.
  • the helper lipid can have fusogenic properties for enhancing uptake and release of the drug payload.
  • helper lipids are 1,2-dioleoyl- SN-glycero-3-phosphoethanolamine (DOPE); 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC); 1,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS); 1,2-dielaidoyl-sn-glycero-3- phosphoethanolamine (DEPE); and 1,2-dioleoyl-sn-glycero-3-phosphocholine (DPOC), dipalmitoylphosphatidylcholine (DPPC), 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC), 1,2-Distearoylphosphatidylethanolamine (DSPE), and 1,2-dilauroyl-sn-glycero-3- phosphoethanolamine (DLPE).
  • DOPE 1,2-dioleoyl- SN-glycero-3-phosphoethanolamine
  • helper lipids are dioleoylphosphatidylcholine (DOPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (POPE), dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-l- carboxylate (DOPE-mal), dipalmitoyl phosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), phosphatidylserine, sphingolipids, cerebrosides, gangliosides, 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, l-stearoyl-2-oleoyl- phosphatidyethanol
  • the helper lipid is 1,2-dioleoyl-SN-glycero-3- phosphoethanolamine (DOPE).
  • DOPE 1,2-dioleoyl-SN-glycero-3- phosphoethanolamine
  • the LNP comprises a lipid of Formula (I) and a structural lipid.
  • the LNP comprises a lipid of Formula (I) and a stealth lipid.
  • the LNP comprises a lipid of Formula (I) and a helper lipid.
  • the LNP comprises a lipid of Formula (I), a structural lipid, a stealth lipid, and a helper lipid.
  • the LNP comprises a lipid of Formula (I-a1), (I-b1), (I-c1), (I- d1), (I-e1), (I-f1), (I-g1), (I-h1), or (I-i1) and a structural lipid.
  • the LNP comprises a lipid of Formula (I-a1), (I-b1), (I-c1), (I-d1), (I-e1), (I-f1), (I-g1), (I-h1), or (I-i1) and a stealth lipid.
  • the LNP comprises a lipid of Formula (I-a1), (I-b1), (I-c1), (I-d1), (I-e1), (I-f1), (I-g1), (I-h1), or (I-i1) and a helper lipid.
  • the LNP comprises a lipid of Formula (I-a1), (I-b1), (I-c1), (I-d1), (I-e1), (I-f1), (I-g1), (I-h1), or (I-i1); a structural lipid; a stealth lipid; and a helper lipid.
  • the LNP comprises a lipid of Formula (I-a2), (I-b2), (I-c2), (I- d2), (I-e2), (I-f2), (I-g2), (I-h2), or (I-i2) and a structural lipid.
  • the LNP comprises a lipid of Formula (I-a2), (I-b2), (I-c2), (I-d2), (I-e2), (I-f2), (I-g2), (I-h2), or (I-i2) and a stealth lipid.
  • the LNP comprises a lipid of Formula (I-a2), (I-b2), (I-c2), (I-d2), (I-e2), (I-f2), (I-g2), (I-h2), or (I-i2) and a helper lipid.
  • the LNP comprises a lipid of Formula (I-a2), (I-b2), (I-c2), (I-d2), (I-e2), (I- f2), (I-g2), (I-h2), or (I-i2); a structural lipid; a stealth lipid; and a helper lipid.
  • the LNP comprises a lipid of Formula (I-a3), (I-b3), (I-c3), (I- d3), (I-e3), (I-f3), (I-g3), (I-h3), or (I-i3) and a structural lipid.
  • the LNP comprises a lipid of Formula (I-a3), (I-b3), (I-c3), (I-d3), (I-e3), (I-f3), (I-g3), (I-h3), or (I-i3) and a stealth lipid.
  • the LNP comprises a lipid of Formula (I-a3), (I-b3), (I-c3), (I-d3), (I-e3), (I-f3), (I-g3), (I-h3), or (I-i3) and a helper lipid.
  • the LNP comprises a lipid of Formula (I-a3), (I-b3), (I-c3), (I-d3), (I-e3), (I-f3), (I-g3), (I-h3), or (I-i3); a structural lipid; a stealth lipid; and a helper lipid.
  • the LNP comprises a lipid of Formula (I-a4), (I-b4), (I-c4), (I- d4), (I-e4), (I-f4), (I-g4), (I-h4), or (I-i4) and a structural lipid.
  • the LNP comprises a lipid of Formula (I-a4), (I-b4), (I-c4), (I-d4), (I-e4), (I-f4), (I-g4), (I-h4), or (I-i4) and a stealth lipid.
  • the LNP comprises a lipid of Formula (I-a4), (I-b4), (I-c4), (I-d4), (I-e4), (I-f4), (I-g4), (I-h4), or (I-i4) and a helper lipid.
  • the LNP comprises a lipid of Formula (I-a4), (I-b4), (I-c4), (I-d4), (I-e4), (I- f4), (I-g4), (I-h4), or (I-i4); a structural lipid; a stealth lipid; and a helper lipid.
  • the ionizable or cationic lipid may comprise a molar ratio from about 35% to about 45% of the total lipid present in the lipid nanoparticle. In some embodiments, the ionizable or cationic lipid may comprise a molar ratio of about 40% of the total lipid present in the lipid nanoparticle. In some embodiments, the stealth (e.g., PEGylated) lipid may comprise a molar ratio from about 0.5% to about 7% of the total lipid present in the lipid nanoparticle. In some embodiments, the stealth (e.g., PEGylated) lipid may comprise a molar ratio of about 1.5% of the total lipid present in the lipid nanoparticle.
  • the stealth (e.g., PEGylated) lipid may comprise a molar ratio of about 5% of the total lipid present in the lipid nanoparticle.
  • the structural lipid may comprise a molar ratio from about 20% to about 35% of the total lipid present in the lipid nanoparticle.
  • the structural lipid may comprise a molar ratio of about 30% of the total lipid present in the lipid nanoparticle.
  • the helper lipid may comprise a molar ratio from about 20% to about 30% of the total lipid present in the lipid nanoparticle.
  • the helper lipid may comprise a molar ratio of about 25% of the total lipid present in the lipid nanoparticle. In some embodiments, the helper lipid may comprise a molar ratio of about 28.5% of the total lipid present in the lipid nanoparticle. In some embodiments, the LNP comprises the ionizable or cationic lipid at a molar ratio between 35% and 45%; the structural lipid at a molar ratio between 20% and 35%, the stealth lipid at a molar ratio between 0.5% and 7%, and the helper lipid at a molar ratio between 20% and 30%.
  • the LNP comprises the ionizable or cationic lipid at a molar ratio of about 40%; the structural lipid at a molar ratio of about 30%; the stealth lipid at a molar ratio of about 1.5%, and the helper lipid at a molar ratio of about 28.5%.
  • the LNP comprises the ionizable or cationic lipid at a molar ratio of about 40%; the structural lipid at a molar ratio of about 30%; the stealth lipid at a molar ratio of about 5%, and the helper lipid at a molar ratio of about 25%.
  • the molar amount of the cationic or ionizable lipid is first determined based on a desired N/P ratio, where N is the number of nitrogen atoms in the cationic lipid and P is the number of phosphate groups in the mRNA to be transported by the LNP.
  • N is the number of nitrogen atoms in the cationic lipid
  • P is the number of phosphate groups in the mRNA to be transported by the LNP.
  • the molar amount of each of the other lipids is calculated based on the molar amount of the cationic lipid and the molar ratio selected. These molar amounts are then converted to weights using the molecular weight of each lipid.
  • Active Ingredients of the LNPs The active ingredient of the present LNP composition may be an mRNA that encodes a polypeptide of interest.
  • the polypeptide is an antigen. In certain embodiments, the polypeptide is a therapeutic polypeptide.
  • the therapeutic polypeptide may be an antibody (e.g., an antibody heavy chain or an antibody light chain.
  • the therapeutic polypeptide may be an enzyme.
  • the mRNA molecule encapsulated by the present disclosure LNPs may comprise at least one ribonucleic acid (RNA) comprising an ORF encoding a polypeptide of interest.
  • the mRNA further comprises at least one 5’ UTR, 3’ UTR, a poly(A) tail, and/or a 5’ cap.
  • i.5’ Cap An mRNA 5’ cap can provide resistance to nucleases found in most eukaryotic cells and promote translation efficiency.
  • a 7- methylguanosine cap (also referred to as “m 7 G” or “Cap-0”), comprises a guanosine that is linked through a 5’ – 5’ - triphosphate bond to the first transcribed nucleotide.
  • a 5' cap is typically added as follows: first, an RNA terminal phosphatase removes one of the terminal phosphate groups from the 5’ nucleotide, leaving two terminal phosphates; guanosine triphosphate (GTP) is then added to the terminal phosphates via a guanylyl transferase, producing a 5 ‘5 ‘5 triphosphate linkage; and the 7-nitrogen of guanine is then methylated by a methyltransferase.
  • GTP guanosine triphosphate
  • cap structures include, but are not limited to, m7G(5’)ppp, (5’(A,G(5’)ppp(5’)A, and G(5’)ppp(5’)G. Additional cap structures are described in U.S.
  • 5’-capping of polynucleotides may be completed concomitantly during the in vitro- transcription reaction using the following chemical RNA cap analogs to generate the 5’- guanosine cap structure according to manufacturer protocols: 3’-O-Me-m7G(5’)ppp(5’)G (the ARCA cap); G(5’)ppp(5’)A; G(5’)ppp(5’)G; m7G(5’)ppp(5’)A; m7G(5’)ppp(5’)G; m7G(5')ppp(5')(2'OMeA)pG; m7G(5')ppp(5')(2'OMeA)pU; m7G(5')ppp(5')(2'OMeG)pG (New England BioLab
  • Cap 1 structure may be generated using both vaccinia virus capping enzyme and a 2’-O methyl-transferase to generate: m7G(5’)ppp(5’)G- 2’-O-methyl.
  • Cap 2 structure may be generated from the Cap 1 structure followed by the 2’- O-methylation of the 5’-antepenultimate nucleotide using a 2’-O methyl-transferase.
  • Cap 3 structure may be generated from the Cap 2 structure followed by the 2’-O-methylation of the 5’-preantepenultimate nucleotide using a 2’-O methyl-transferase.
  • the mRNA of the disclosure comprises a 5’ cap selected from the group consisting of 3’-O-Me-m7G(5’)ppp(5’)G (the ARCA cap), G(5’)ppp(5’)A, G(5’)ppp(5’)G, m7G(5’)ppp(5’)A, m7G(5’)ppp(5’)G, m7G(5')ppp(5')(2'OMeA)pG, m7G(5')ppp(5')(2'OMeA)pU, and m7G(5')ppp(5')(2'OMeG)pG.
  • a 5’ cap selected from the group consisting of 3’-O-Me-m7G(5’)ppp(5’)G (the ARCA cap), G(5’)ppp(5’)A, G(5’)ppp(5’)G, m7G(5’
  • the mRNA of the disclosure comprises a 5’ cap of: . ii. Untranslated Region (UTR)
  • the mRNA of the disclosure includes a 5’ and/or 3’ untranslated region (UTR).
  • the 5’ UTR starts at the transcription start site and continues to the start codon but does not include the start codon.
  • the 3’ UTR starts immediately following the stop codon and continues until the transcriptional termination signal.
  • the mRNA disclosed herein may comprise a 5’ UTR that includes one or more elements that affect an mRNA’s stability or translation.
  • a 5’ UTR may be about 10 to 5,000 nucleotides in length.
  • a 5’ UTR may be about 50 to 500 nucleotides in length.
  • the 5’ UTR is at least about 10 nucleotides in length, about 20 nucleotides in length, about 30 nucleotides in length, about 40 nucleotides in length, about 50 nucleotides in length, about 100 nucleotides in length, about 150 nucleotides in length, about 200 nucleotides in length, about 250 nucleotides in length, about 300 nucleotides in length, about 350 nucleotides in length, about 400 nucleotides in length, about 450 nucleotides in length, about 500 nucleotides in length, about 550 nucleotides in length, about 600 nucleotides in length, about 650 nucleotides in length, about 700 nucleotides in length, about 750 nucleotides in length, about 800 nucleotides in length, about 850 nucleotides in length, about
  • the mRNA disclosed herein may comprise a 3’ UTR comprising one or more of a polyadenylation signal, a binding site for proteins that affect an mRNA’s stability of location in a cell, or one or more binding sites for miRNAs.
  • a 3’ UTR may be 50 to 5,000 nucleotides in length or longer. In some embodiments, a 3’ UTR may be 50 to 1,000 nucleotides in length or longer.
  • the 3’ UTR is at least about 50 nucleotides in length, about 100 nucleotides in length, about 150 nucleotides in length, about 200 nucleotides in length, about 250 nucleotides in length, about 300 nucleotides in length, about 350 nucleotides in length, about 400 nucleotides in length, about 450 nucleotides in length, about 500 nucleotides in length, about 550 nucleotides in length, about 600 nucleotides in length, about 650 nucleotides in length, about 700 nucleotides in length, about 750 nucleotides in length, about 800 nucleotides in length, about 850 nucleotides in length, about 900 nucleotides in length, about 950 nucleotides in length, about 1,000 nucleotides in length, about 1,500 nucleotides in length, about 2,000 nucleotides in length, about 2,500 nucleotides in length, about
  • the mRNA disclosed herein may comprise a 5’ or 3’ UTR that is derived from a gene distinct from the one encoded by the mRNA transcript (i.e., the UTR is a heterologous UTR).
  • the 5’ and/or 3’ UTR sequences can be derived from mRNA which are stable (e.g., globin, actin, GAPDH, tubulin, histone, or citric acid cycle enzymes) to increase the stability of the mRNA.
  • a 5’ UTR sequence may include a partial sequence of a CMV immediate-early 1 (IE1) gene, or a fragment thereof, to improve the nuclease resistance and/or improve the half-life of the mRNA.
  • IE1 immediate-early 1
  • hGH human growth hormone
  • these modifications improve the stability and/or pharmacokinetic properties (e.g., half-life) of the mRNA relative to their unmodified counterparts, and include, for example, modifications made to improve such mRNA resistance to in vivo nuclease digestion.
  • exemplary 5’ UTRs include a sequence derived from a CMV immediate-early 1 (IE1) gene (U.S.
  • the 5’ UTR may be derived from the 5’ UTR of a TOP gene.
  • TOP genes are typically characterized by the presence of a 5’-terminal oligopyrimidine (TOP) tract.
  • TOP genes are characterized by growth-associated translational regulation.
  • TOP genes with a tissue specific translational regulation are also known.
  • the 5’ UTR derived from the 5’ UTR of a TOP gene lacks the 5’ TOP motif (the oligopyrimidine tract) (e.g., U.S. Publication Nos.2017/0029847, 2016/0304883, 2016/0235864, and 2016/0166710, each of which is incorporated herein by reference).
  • the 5’ UTR is derived from a ribosomal protein Large 32 (L32) gene (U.S. Publication No.2017/0029847, supra).
  • the 5’ UTR is derived from the 5’ UTR of an hydroxysteroid (17-b) dehydrogenase 4 gene (HSD17B4) (U.S.
  • the 5’ UTR is derived from the 5’ UTR of an ATP5A1 gene (U.S. Publication No.2016/0166710, supra).
  • an internal ribosome entry site IVS
  • the 5’UTR comprises a nucleic acid sequence reproduced below: GGACAGAUCGCCUGGAGACGCCAUCCACGCUGUUUUGACCUCCAUAGAA GACACCGGGACCGAUCCAGCCUCCGCGGCCGGGAACGGUGCAUUGGAACGCGG AUUCCCCGUGCCAAGAGUGACUCACCGUCCUUGACACG(SEQ ID NO:2).
  • the 3’UTR comprises a nucleic acid sequence reproduced below: CGGGUGGCAUCCCUGUGACCCCUCCCCAGUGCCUCUCCUGGCCCUGGAA GUUGCCACUCCAGUGCCCACCAGCCUUGUCCUAAUAAAAUUAAGUUGCAUC (SEQ ID NO:3).
  • the 5’ UTR and 3’UTR are described in further detail in WO2012/075040, incorporated herein by reference.
  • iii. Polyadenylated Tail As used herein, the terms “poly(A) sequence,” “poly(A) tail,” and “poly(A) region” refer to a sequence of adenosine nucleotides at the 3’ end of the mRNA molecule.
  • the poly(A) tail may confer stability to the mRNA and protect it from exonuclease degradation.
  • the poly(A) tail may enhance translation.
  • the poly(A) tail is essentially homopolymeric.
  • a poly(A) tail of 100 adenosine nucleotides may have essentially a length of 100 nucleotides.
  • the poly(A) tail may be interrupted by at least one nucleotide different from an adenosine nucleotide (e.g., a nucleotide that is not an adenosine nucleotide).
  • a poly(A) tail of 100 adenosine nucleotides may have a length of more than 100 nucleotides (comprising 100 adenosine nucleotides and at least one nucleotide, or a stretch of nucleotides, that are different from an adenosine nucleotide).
  • the poly(A) tail comprises the sequence AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA
  • the term likewise relates to corresponding sequences in a DNA molecule (e.g., a “poly(T) sequence”).
  • the poly(A) tail(SEQ ID NO:5) may comprise about 10 to about 500 adenosine nucleotides, about 10 to about 200 adenosine nucleotides, about 40 to about 200 adenosine nucleotides, or about 40 to about 150 adenosine nucleotides.
  • the length of the poly(A) tail may be at least about 10, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, or 500 adenosine nucleotides.
  • the poly(A) (SEQ ID NO:5)tail of the nucleic acid is obtained from a DNA template during RNA in vitro transcription.
  • the poly(A) tail is obtained in vitro by common methods of chemical synthesis without being transcribed from a DNA template.
  • poly(A) tails are generated by enzymatic polyadenylation of the RNA (after RNA in vitro transcription) using commercially available polyadenylation kits and corresponding protocols, or alternatively, by using immobilized poly(A)polymerases, e.g., using methods and means as described in WO2016/174271.
  • the nucleic acid may comprise a poly(A) tail obtained by enzymatic polyadenylation, wherein the majority of nucleic acid molecules comprise about 100 (+/-20) to about 500 (+/- 50) or about 250 (+/-20) adenosine nucleotides.
  • the nucleic acid may comprise a poly(A) tail derived from a template DNA and may additionally comprise at least one additional poly(A) tail generated by enzymatic polyadenylation, e.g., as described in WO2016/091391.
  • the nucleic acid comprises at least one polyadenylation signal.
  • the nucleic acid may comprise at least one poly(C) sequence.
  • poly(C) SEQ ID NO:6sequence
  • the poly(C) sequence comprises about 10 to about 200 cytosine nucleotides, about 10 to about 100 cytosine nucleotides, about 20 to about 70 cytosine nucleotides, about 20 to about 60 cytosine nucleotides, or about 10 to about 40 cytosine nucleotides.
  • the poly(C) sequence comprises about 30 cytosine nucleotides.
  • the mRNA may comprise at least one chemical modification.
  • the mRNA disclosed herein may contain one or more modifications that typically enhance RNA stability. Exemplary modifications can include backbone modifications, sugar modifications, or base modifications.
  • the disclosed mRNA may be synthesized from naturally occurring nucleotides and/or nucleotide analogues (modified nucleotides) including, but not limited to, purines (adenine (A) and guanine (G)) or pyrimidines (thymine (T), cytosine (C), and uracil (U)).
  • the disclosed mRNA may be synthesized from modified nucleotide analogues or derivatives of purines and pyrimidines, such as, e.g., 1-methyl-adenine, 2-methyl-adenine, 2-methylthio-N-6-isopentenyl-adenine, N6-methyl-adenine, N6-isopentenyl-adenine, 2-thio-cytosine, 3-methyl-cytosine, 4-acetyl- cytosine, 5-methyl-cytosine, 2,6-diaminopurine, 1-methyl-guanine, 2-methyl-guanine, 2,2- dimethyl-guanine, 7-methyl-guanine, inosine, 1-methyl-inosine, pseudouracil (5-uracil), dihydro-uracil, 2-thio-uracil, 4-thio-uracil, 5-carboxymethylaminomethyl-2-thio-uracil, 5- (carboxyhydroxymethyl)-uracil, 5-
  • the disclosed mRNA may comprise at least one chemical modification including, but not limited to, pseudouridine, N1-methylpseudouridine, 2- thiouridine, 4’-thiouridine, 5-methylcytosine, 2-thio-l-methyl-1-deaza-pseudouridine, 2-thio- l-methyl-pseudouridine, 2-thio-5-aza-uridine, 2-thio-dihydropseudouridine, 2-thio- dihydrouridine, 2-thio-pseudouridine, 4-methoxy-2-thio-pseudouridine, 4-methoxy- pseudouridine, 4-thio-l-methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methyluridine, 5-methyluridine, 5-methoxyuridine, and 2’-O-methyl uridine.
  • pseudouridine N1-methylpseudouridine
  • the chemical modification is selected from the group consisting of pseudouridine, N1-methylpseudouridine, 5-methylcytosine, 5-methoxyuridine, and a combination thereof. In some embodiments, the chemical modification comprises N1-methylpseudouridine. In some embodiments, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% of the uracil nucleotides in the mRNA are chemically modified.
  • At least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% of the uracil nucleotides in the ORF are chemically modified.
  • the preparation of such analogues is described, e.g., in U.S. Pat. No.4,373,071, U.S. Pat. No.4,401,796, U.S. Pat. No.4,415,732, U.S. Pat. No.4,458,066, U.S. Pat. No. 4,500,707, U.S. Pat. No.4,668,777, U.S. Pat. No.4,973,679, U.S. Pat.
  • IVT is typically performed with a linear or circular DNA template containing a promoter, a pool of ribonucleotide triphosphates, a buffer system that may include DTT and magnesium ions, an appropriate RNA polymerase (e.g., T3, T7, or SP6 RNA polymerase), DNase I, pyrophosphatase, and/or RNase inhibitor.
  • RNA polymerase e.g., T3, T7, or SP6 RNA polymerase
  • DNase I e.g., pyrophosphatase
  • RNase inhibitor e.g., RNase inhibitor.
  • the exact conditions may vary according to the specific application.
  • the presence of these reagents is generally undesirable in a final mRNA product and these reagents can be considered impurities or contaminants which can be purified or removed to provide a clean and/or homogeneous mRNA that is suitable for therapeutic use.
  • the LNP or the LNP formulation may be multi-valent.
  • the LNP may carry mRNAs that encode more than one polypeptide (e.g., antigen), such as two, three, four, five, six, seven, eight, nine, ten, or more polypeptides.
  • the LNP may carry multiple mRNA molecules, each encoding a different polypeptide; or carry a polycistronic mRNA that can be translated into more than one polypeptide (e.g., each polypeptide-coding sequence is separated by a nucleotide linker encoding a self-cleaving peptide such as a 2A peptide).
  • An LNP carrying different mRNA molecules typically comprises (encapsulate) multiple copies of each mRNA molecule.
  • an LNP carrying or encapsulating two different mRNA molecules typically carries multiple copies of each of the two different mRNA molecules.
  • a single LNP formulation may comprise multiple kinds (e.g., two, three, four, five, six, seven, eight, nine, ten, or more) of LNPs, each kind carrying a different mRNA.
  • F. Buffer and Other Components To stabilize the nucleic acid and/or LNPs (e.g., to prolong the shelf-life of the vaccine product), to facilitate administration of the LNP pharmaceutical composition, and/or to enhance in vivo expression of the nucleic acid, the nucleic acid and/or LNP can be formulated in combination with one or more carriers, targeting ligands, stabilizing reagents (e.g., preservatives and antioxidants), and/or other pharmaceutically acceptable excipients.
  • stabilizing reagents e.g., preservatives and antioxidants
  • the LNP compositions of the present disclosure can be provided as a frozen liquid form or a lyophilized form.
  • cryoprotectants may be used, including, without limitations, sucrose, trehalose, glucose, mannitol, mannose, dextrose, and the like.
  • the cryoprotectant may constitute 5-30% (w/v) of the LNP composition.
  • the LNP composition comprises trehalose, e.g., at 5-30% (e.g., 10%) (w/v).
  • the LNP compositions may be frozen (or lyophilized and cryopreserved) at -20 o C to -80 o C.
  • the LNP compositions may be provided to a patient in an aqueous buffered solution – thawed if previously frozen, or if previously lyophilized, reconstituted in an aqueous buffered solution at bedside.
  • the buffered solution may be isotonic and suitable for e.g., intramuscular or intradermal injection.
  • the buffered solution is a phosphate-buffered saline (PBS).
  • multilamellar vesicles may be prepared according to conventional techniques, such as by depositing a selected lipid on the inside wall of a suitable container or vessel by dissolving the lipid in an appropriate solvent, and then evaporating the solvent to leave a thin film on the inside of the vessel or by spray drying. An aqueous phase may then be added to the vessel with a vortexing motion that results in the formation of MLVs.
  • Unilamellar vesicles (ULV) can then be formed by homogenization, sonication or extrusion of the multilamellar vesicles.
  • unilamellar vesicles can be formed by detergent removal techniques.
  • Various methods are described in US 2011/0244026, US 2016/0038432, US 2018/0153822, US 2018/0125989, and PCT/US2020/043223 (filed July 23, 2020) and can be used to practice the present disclosure.
  • One exemplary process entails encapsulating mRNA by mixing it with a mixture of lipids, without first pre-forming the lipids into lipid nanoparticles, as described in US 2016/0038432.
  • Another exemplary process entails encapsulating mRNA by mixing pre-formed LNPs with mRNA, as described in US 2018/0153822.
  • the process of preparing mRNA-loaded LNPs includes a step of heating one or more of the solutions to a temperature greater than ambient temperature, the one or more solutions being the solution comprising the pre-formed lipid nanoparticles, the solution comprising the mRNA and the mixed solution comprising the LNP-encapsulated mRNA.
  • the process includes the step of heating one or both of the mRNA solution and the pre-formed LNP solution, prior to the mixing step.
  • the process includes heating one or more of the solutions comprising the pre- formed LNPs, the solution comprising the mRNA and the solution comprising the LNP- encapsulated mRNA, during the mixing step.
  • the process includes the step of heating the LNP- encapsulated mRNA, after the mixing step.
  • the temperature to which one or more of the solutions is heated is or is greater than about 30°C, 37°C, 40°C, 45°C, 50°C, 55°C, 60°C, 65°C, or 70°C.
  • the temperature to which one or more of the solutions is heated ranges from about 25-70°C, about 30-70°C, about 35-70°C, about 40-70°C, about 45-70°C, about 50-70°C, or about 60- 70°C.
  • the temperature is about 65°C.
  • Various methods may be used to prepare an mRNA solution suitable for the present disclosure.
  • mRNA may be directly dissolved in a buffer solution described herein.
  • an mRNA solution may be generated by mixing an mRNA stock solution with a buffer solution prior to mixing with a lipid solution for encapsulation.
  • an mRNA solution may be generated by mixing an mRNA stock solution with a buffer solution immediately before mixing with a lipid solution for encapsulation.
  • a suitable mRNA stock solution may contain mRNA in water or a buffer at a concentration at or greater than about 0.2 mg/ml, 0.4 mg/ml, 0.5 mg/ml, 0.6 mg/ml, 0.8 mg/ml, 1.0 mg/ml, 1.2 mg/ml, 1.4 mg/ml, 1.5 mg/ml, or 1.6 mg/ml, 2.0 mg/ml, 2.5 mg/ml, 3.0 mg/ml, 3.5 mg/ml, 4.0 mg/ml, 4.5 mg/ml, or 5.0 mg/ml.
  • an mRNA stock solution is mixed with a buffer solution using a pump.
  • Exemplary pumps include but are not limited to gear pumps, peristaltic pumps and centrifugal pumps.
  • the buffer solution is mixed at a rate greater than that of the mRNA stock solution.
  • the buffer solution may be mixed at a rate at least 1x, 2x, 3x, 4x, 5x, 6x, 7x, 8x, 9x, 10x, 15x, or 20x greater than the rate of the mRNA stock solution.
  • a buffer solution is mixed at a flow rate ranging between about 100-6000 ml/minute (e.g., about 100-300 ml/minute, 300-600 ml/minute, 600-1200 ml/minute, 1200-2400 ml/minute, 2400-3600 ml/minute, 3600-4800 ml/minute, 4800-6000 ml/minute, or 60-420 ml/minute).
  • a buffer solution is mixed at a flow rate of, or greater than, about 60 ml/minute, 100 ml/minute, 140 ml/minute, 180 ml/minute, 220 ml/minute, 260 ml/minute, 300 ml/minute, 340 ml/minute, 380 ml/minute, 420 ml/minute, 480 ml/minute, 540 ml/minute, 600 ml/minute, 1200 ml/minute, 2400 ml/minute, 3600 ml/minute, 4800 ml/minute, or 6000 ml/minute.
  • an mRNA stock solution is mixed at a flow rate ranging between about 10-600 ml/minute (e.g., about 5-50 ml/minute, about 10-30 ml/minute, about 30-60 ml/minute, about 60-120 ml/minute, about 120-240 ml/minute, about 240-360 ml/minute, about 360-480 ml/minute, or about 480-600 ml/minute).
  • a flow rate ranging between about 10-600 ml/minute (e.g., about 5-50 ml/minute, about 10-30 ml/minute, about 30-60 ml/minute, about 60-120 ml/minute, about 120-240 ml/minute, about 240-360 ml/minute, about 360-480 ml/minute, or about 480-600 ml/minute).
  • an mRNA stock solution is mixed at a flow rate of or greater than about 5 ml/minute, 10 ml/minute, 15 ml/minute, 20 ml/minute, 25 ml/minute, 30 ml/minute, 35 ml/minute, 40 ml/minute, 45 ml/minute, 50 ml/minute, 60 ml/minute, 80 ml/minute, 100 ml/minute, 200 ml/minute, 300 ml/minute, 400 ml/minute, 500 ml/minute, or 600 ml/minute.
  • the process of incorporation of a desired mRNA into a lipid nanoparticle is referred to as “loading.” Exemplary methods are described in Lasic et al., FEBS Lett. (1992) 312:255-8.
  • the LNP-incorporated nucleic acids may be completely or partially located in the interior space of the lipid nanoparticle, within the bilayer membrane of the lipid nanoparticle, or associated with the exterior surface of the lipid nanoparticle membrane.
  • the incorporation of an mRNA into lipid nanoparticles is also referred to herein as “encapsulation” wherein the nucleic acid is entirely or substantially contained within the interior space of the lipid nanoparticle. Suitable LNPs may be made in various sizes.
  • decreased size of lipid nanoparticles is associated with more efficient delivery of an mRNA.
  • Selection of an appropriate LNP size may take into consideration the site of the target cell or tissue and to some extent the application for which the lipid nanoparticle is being made.
  • a variety of methods known in the art are available for sizing of a population of lipid nanoparticles. Preferred methods herein utilize Zetasizer Nano ZS (Malvern Panalytical) to measure LNP particle size. In one protocol, 10 ⁇ l of an LNP sample are mixed with 990 ⁇ l of 10% trehalose. This solution is loaded into a cuvette and then put into the Zetasizer machine.
  • the z-average diameter (nm), or cumulants mean, is regarded as the average size for the LNPs in the sample.
  • the Zetasizer machine can also be used to measure the polydispersity index (PDI) by using dynamic light scattering (DLS) and cumulant analysis of the autocorrelation function.
  • PDI polydispersity index
  • DLS dynamic light scattering
  • Average LNP diameter may be reduced by sonication of formed LNP. Intermittent sonication cycles may be alternated with quasi-elastic light scattering (QELS) assessment to guide efficient lipid nanoparticle synthesis.
  • QELS quasi-elastic light scattering
  • the majority of purified LNPs i.e., greater than about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the LNPs, have a size of about 70-150 nm (e.g., about 145 nm, about 140 nm, about 135 nm, about 130 nm, about 125 nm, about 120 nm, about 115 nm, about 110 nm, about 105 nm, about 100 nm, about 95 nm, about 90 nm, about 85 nm, or about 80 nm).
  • nm e.g., about 145 nm, about 140 nm, about 135 nm, about 130 nm, about 125 nm, about 120 nm, about 115 nm, about 110 nm, about 105 nm, about 100 nm, about 95 nm, about 90
  • substantially all (e.g., greater than 80 or 90%) of the purified lipid nanoparticles have a size of about 70-150 nm (e.g., about 145 nm, about 140 nm, about 135 nm, about 130 nm, about 125 nm, about 120 nm, about 115 nm, about 110 nm, about 105 nm, about 100 nm, about 95 nm, about 90 nm, about 85 nm, or about 80 nm).
  • about 70-150 nm e.g., about 145 nm, about 140 nm, about 135 nm, about 130 nm, about 125 nm, about 120 nm, about 115 nm, about 110 nm, about 105 nm, about 100 nm, about 95 nm, about 90 nm, about 85 nm, or about 80 nm.
  • the LNPs in the present composition have an average size of less than 150 nm, less than 120 nm, less than 100 nm, less than 90 nm, less than 80 nm, less than 70 nm, less than 60 nm, less than 50 nm, less than 30 nm, or less than 20 nm.
  • greater than about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% of the LNPs in the present composition have a size ranging from about 40-90 nm (e.g., about 45-85 nm, about 50-80 nm, about 55-75 nm, about 60-70 nm), about 40-90 nm (e.g., about 45-85 nm, about 50-80 nm, about 55-75 nm, about 60-70 nm), or about 50-70 nm (e.g., 55-65 nm) are particular suitable for pulmonary delivery via nebulization.
  • about 40-90 nm e.g., about 45-85 nm, about 50-80 nm, about 55-75 nm, about 60-70 nm
  • about 50-70 nm e.g., 55-65 nm
  • the dispersity, or measure of heterogeneity in size of molecules (PDI), of LNPs in a pharmaceutical composition provided by the present disclosure is less than about 0.5.
  • an LNP has a PDI of less than about 0.5, less than about 0.4, less than about 0.3, less than about 0.28, less than about 0.25, less than about 0.23, less than about 0.20, less than about 0.18, less than about 0.16, less than about 0.14, less than about 0.12, less than about 0.10, or less than about 0.08.
  • the PDI may be measured by a Zetasizer machine as described above.
  • a lipid nanoparticle has an encapsulation efficiency of between 50% and 99%; or greater than about 60, 65, 70, 75, 80, 85, 90, 92, 95, 98, or 99%.
  • lipid nanoparticles for use herein have an encapsulation efficiency of at least 90% (e.g., at least 91, 92, 93, 94, or 95%).
  • an LNP has a N/P ratio of between 1 and 10.
  • a lipid nanoparticle has a N/P ratio above 1, about 1, about 2, about 3, about 4, about 5, about 6, about 7, or about 8.
  • a typical LNP herein has an N/P ratio of 4.
  • a pharmaceutical composition according to the present disclosure contains at least about 0.5 ⁇ g, 1 ⁇ g, 5 ⁇ g, 10 ⁇ g, 100 ⁇ g, 500 ⁇ g, or 1000 ⁇ g of encapsulated mRNA.
  • a pharmaceutical composition contains about 0.1 ⁇ g to 1000 ⁇ g, at least about 0.5 ⁇ g, at least about 0.8 ⁇ g, at least about 1 ⁇ g, at least about 5 ⁇ g, at least about 8 ⁇ g, at least about 10 ⁇ g, at least about 50 ⁇ g, at least about 100 ⁇ g, at least about 500 ⁇ g, or at least about 1000 ⁇ g of encapsulated mRNA.
  • the mRNA-LNP can be packaged for parenteral (e.g., intramuscular, intradermal, subcutaneous, or intravenous) administration, nasopharyngeal (e.g., intranasal) administration, or mucosal (e.g., intranasal, oral, rectal) administration.
  • parenteral e.g., intramuscular, intradermal, subcutaneous, or intravenous
  • nasopharyngeal e.g., intranasal
  • mucosal e.g., intranasal, oral, rectal
  • the compositions may be in the form of an extemporaneous formulation, where the LNP composition is lyophilized and reconstituted with a physiological buffer (e.g., PBS) just before use.
  • PBS physiological buffer
  • compositions also may be shipped and provided in the form of an aqueous solution or a frozen aqueous solution and can be directly administered to subjects without reconstitution (after thawing, if previously frozen).
  • the present disclosure provides an article of manufacture, such as a kit, that provides the mRNA-LNP in a single container, or provides the mRNA-LNP in one container and a physiological buffer for reconstitution in another container.
  • the container(s) may contain a single-use dosage or multi-use dosage.
  • the containers may be pre-treated glass vials or ampules.
  • the article of manufacture may include instructions for use as well.
  • the present disclosure provides methods of preventing or treating a disease or disorder by administering the composition of the disclosure to a subject in need thereof.
  • the subject is suffering from or susceptible to an infection.
  • the present disclosure provides methods of eliciting an immune response in a subject in need thereof, comprising administering to the subject a prophylactically effective amount of a composition described herein.
  • the present disclosure provides methods of preventing an infection or reducing one or more symptoms of an infection in a subject in need thereof, comprising administering to the subject a prophylactically effective amount of the composition.
  • the composition is administered to the subject mucosally, intramuscularly, intranasally, intravenously, subcutaneously, or intradermally. In some embodiments, the composition is administered mucosally.
  • the composition is administered intranasally.
  • Intranasal administration includes administration via the nose, either with or without concomitant inhalation during administration. Such administration is typically through contact by the composition with the nasal mucosa, nasal turbinates or sinus cavity.
  • the pharmaceutical compositions for administration may be applied in a single administration or in multiple administrations. For example, one dose can be placed in each nostril during administration.
  • bi-dose delivery can be used with the compositions according to the invention.
  • Bi-dose devices contain two sub-doses of a single dose, one sub-dose for administration to each nostril. Generally, the two sub-doses are present in a single chamber and the construction of the device allows the efficient delivery of a single sub-dose at a time.
  • a mono-dose device may be used for administering the compositions according to the invention.
  • the composition can be given in one, two, three, four, or more doses, so that the subject is given a first dose (which can be a bi-dose or mono-dose, as described above), and then a second dose is administered within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,27, 28, 29, or 30 days, or 4 ,5, 6, 7, 8, 9, 10, 11, or 12 weeks, or 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months, or 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more years apart.
  • Exemplary devices for intranasal administration of the compositions according to the invention are spray devices.
  • Suitable commercially available nasal spray devices include AccusprayTM (Becton Dickinson). Nebulizers produce a very fine spray (such as a mist) which can be easily inhaled and are also contemplated herein.
  • Exemplary spray devices for intranasal use are devices for which the performance of the device is not dependent upon the pressure applied by the user. These devices are known as pressure threshold devices. Liquid is released from the nozzle only when a threshold pressure is applied. These devices make it easier to achieve a spray with a regular droplet size. Pressure threshold devices suitable for use with the present invention are known in the art.
  • the invention provides in a further aspect a pharmaceutical kit comprising an intranasal administration device as described herein containing a formulation according to the invention.
  • compositions according to the invention may be administered in other forms e.g. as a powder.
  • the subject is administered one or more doses of the composition, wherein each dose comprises 1 ug – 25 mg of mRNA.
  • each dose comprises 2.5-135 ⁇ g of mRNA.
  • each dose comprises 250 ug – 13.5 mg of mRNA.
  • each dose comprises 2.5., 5, 15, 30, 45, 50, 135, 250, or 500 ⁇ g or 1, 1.5, 2.5, 3, 4, 5, 7.5 or 10 mg of mRNA.
  • the subject is administered two doses of the composition.
  • the two doses of the composition are administered with an interval of 2-6 weeks. In some embodiments, the two doses of the composition are administered with an interval of 2, 3, 4, 5, or 6 weeks. In some embodiments, the two doses of the composition are administered with an interval of 4 weeks.
  • the present disclosure also provides the use of a composition described herein for the manufacture of a medicament for use in any of the methods described herein.
  • the present disclosure further provides a kit comprising a composition of the present disclosure.
  • the kit comprises a containing comprising a single-use or multi-use dosage of the composition.
  • the containing is a vial.
  • the container is a pre-filled nasal spray device. V.
  • R 1 , R 2 , and R 3 are independently selected from -C (6-24) alkyl, -C (6-24) alkenyl, and ;
  • X 1 independently for each occurrence is -C (3-12) alkyl or -C (3-12) alkenyl;
  • X 2 independently for each occurrence is -C(O)O-, -OC(O)-, -C(O)S-, -SC(O)-, or - OC(O)O-;
  • X 3 independently for each occurrence is -C (5-24) alkyl or -C (5-24) alkenyl. 4.
  • R 1 and R 2 are independently selected from -C (6-24) alkyl, -C (6-24) alkenyl, or R 3 is X 1 independently for each occurrence is -C (3-12) alkyl or -C (3-12) alkenyl; X 2 independently for each occurrence is -C(O)O-, -OC(O)-, -C(O)S-, -SC(O)-, or - OC(O)O-; X 3 independently for each occurrence is -C (5-24) alkyl or -C (5-24) alkenyl; X 4 is -C (1-6) alkyl; X 5 is -C (1-6) alkyl; X 6 is -C(O)O-, -OC(O)-, or -OC(O)O-; X 7 is -C (1-6) alkyl; and A is an ionizable or
  • R 1 and R 2 are independently selected from -C (6-24) alkyl, -C (6-24) alkenyl, or R 3 is or X 1 independently for each occurrence is -C (3-12) alkyl or -C (3-12) alkenyl; X 2 independently for each occurrence is -C(O)O-, -OC(O)-, or -OC(O)O-; X 3 independently for each occurrence is -C (5-24) alkyl or -C (5-24) alkenyl; X 4 is -C (1-6) alkyl; X 5 is -C (1-6) alkyl; X 6 is -C(O)O-, -OC(O)-, or -OC(O)O-; X 7 is -C (1-6) alkyl; and A is an ionizable or cationic nitrogen-containing group.
  • A is selected from the group consisting of:
  • the lipid of any one of embodiments 1-16 having a structure selected from the group consisting of: or a pharmaceutically acceptable salt thereof.
  • 22. The lipid of any one of embodiments 1-16, having a structure selected from the group consisting of: or a pharmaceutically acceptable salt thereof.
  • 23. The lipid of any one of embodiments 1-16, having a structure selected from the group consisting of: or a pharmaceutically acceptable salt thereof.
  • 24. The lipid of any one of embodiments 1-16, having a structure selected from the group consisting of: or a pharmaceutically acceptable salt thereof.
  • 25. The lipid of any one of embodiments 1-16, having a structure selected from the group consisting of: or a pharmaceutically acceptable salt thereof. 26.
  • the lipid of any one of embodiments 1-16 having a structure selected from the group consisting of: or a pharmaceutically acceptable salt thereof.
  • the lipid of any one of embodiments 1-16 having a structure selected from the group consisting of: or a pharmaceutically acceptable salt thereof.
  • 28. The lipid of any one of embodiments 1-16, having a structure selected from the group consisting of: or a pharmaceutically acceptable salt thereof.
  • 29. The lipid of any one of embodiments 1-16, 18, 20-22, and 26, or a pharmaceutically acceptable salt thereof, wherein R X is -H or -CH 3 .
  • 30. The lipid of any one of embodiments 1-29, or a pharmaceutically acceptable salt thereof, wherein n is 1 or 2. 31.
  • LNP lipid nanoparticle
  • composition of embodiment 36, wherein the stealth lipid is a polyethylene glycol- conjugated (PEGylated) lipid, a polyoxazoline polymer-conjugated lipid, or a polysarcosine- conjugated (pSar) lipid. 38.
  • PEGylated polyethylene glycol- conjugated
  • pSar polysarcosine- conjugated
  • the composition of embodiment 37, wherein the stealth lipid is a PEGylated lipid selected from 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol (DMG-PEG), 1,2- distearoyl-sn-glycero-3-phosphoethanolamine-polyethylene glycol (DSPE-PEG), 1,2- dilauroyl-sn-glycero-3-phosphoethanolamine-polyethylene glycol (DLPE-PEG), and 1,2- distearoyl-rac-glycero-polyethelene glycol (DSG-PEG).
  • DMG-PEG 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol
  • DSPE-PEG 1,2- distearoyl-sn-glycero-3-phosphoethanolamine-polyethylene glycol
  • DLPE-PEG 1,2- dilauroyl-sn-glycero-3-phosphoethanolamine-polyethylene glyco
  • composition of any one of embodiments 36-39, wherein the structural lipid is a sterol. 41. The composition of embodiment 40, wherein the sterol is cholesterol. 42. The composition of any one of embodiments 36-41, wherein the helper lipid is 1,2- dioleoyl-SN-glycero-3-phosphoethanolamine (DOPE); 1,2-distearoyl-sn-glycero-3- phosphocholine (DSPC); 1,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS); 1,2- dielaidoyl-sn-glycero-3-phosphoethanolamine (DEPE); and 1,2-dioleoyl-sn-glycero-3- phosphocholine (DPOC), dipalmitoylphosphatidylcholine (DPPC), 1,2-dilauroyl-sn-glycero- 3-phosphocholine (DLPC), 1,2-Distearoylphosphatidylethanolamine (DS
  • composition of embodiment 42, wherein the helper lipid is 1,2-dioleoyl-SN- glycero-3-phosphoethanolamine (DOPE).
  • DOPE 1,2-dioleoyl-SN- glycero-3-phosphoethanolamine
  • the LNP comprises: the lipid of any one of claims 1-34 at a molar ratio between 35% and 45%, the stealth lipid at a molar ratio between 0.5% and 7%, the structural lipid at a molar ratio between 20% and 35%, and the helper lipid at a molar ratio of between 20% and 30%. 45.
  • the composition of claim 44, wherein the LNP comprises: the lipid of any one of claims 1-23 at a molar ratio of about 40%, the stealth lipid at a molar ratio of about 1.5%, the structural lipid at a molar ratio of about 30%, and the helper lipid at a molar ratio of about 28.5%.
  • the LNP comprises: the lipid of any one of claims 1-23 at a molar ratio of about 40%, the stealth lipid at a molar ratio of about 5%, the structural lipid at a molar ratio of about 30%, and the helper lipid at a molar ratio of about 25%. 47.
  • composition of embodiment 49 or embodiment 50 wherein the LNP encapsulates two or more mRNA molecules, wherein each mRNA molecule encodes a different antigen, optionally wherein the different antigens are from the same pathogen or from different pathogens.
  • the composition comprises two or more LNPs, wherein each LNP encapsulates an mRNA encoding a different antigen, optionally wherein the different antigens are from the same pathogen or from different pathogens.
  • 53 The composition of any one of embodiments 35-52, wherein the composition is formulated for intranasal administration. 54.
  • 55. The composition of any one of embodiments 35-54, wherein the composition comprises trehalose, optionally at 10% (w/v) of the composition.
  • 56. A method of eliciting an immune response in a subject in need thereof, comprising administering to the subject, optionally mucosally, intramuscularly, intranasally, intravenously, subcutaneously, or intradermally, a prophylactically effective amount of the composition of any one of embodiments 47-55. 57.
  • a method of preventing an infection or reducing one or more symptoms of an infection in a subject in need thereof comprising administering to the subject, optionally mucosally, intramuscularly, intranasally, intravenously, subcutaneously, or intradermally, a prophylactically effective amount of the composition of any one of embodiments 47-55. 58. The method of embodiment 56 or embodiment 57, wherein the composition is administered intranasally. 59. The method of any one of embodiments 56-58, comprising administering to the subject one or more doses of the composition, each dose comprising 1-250, optionally 2.5., 5, 15, 45, or 135, ⁇ g of mRNA. 60.
  • any one of embodiments 56-59 comprising administering to the subject two doses of the composition with an interval of 2-6, optionally 4, weeks.
  • 61. Use of the composition of any one of embodiments 47-55 for the manufacture of a medicament for use in treating a subject in need thereof, optionally in a method of any one of embodiments 56-60.
  • 62. The composition of any one of embodiments 47-55 for use in treating a subject in need thereof, optionally in a method of any one of embodiments 56-60.
  • a kit comprising a container comprising a single-use or multi-use dosage of the composition of any one of embodiments 47-55, optionally wherein the container is a vial or a pre-filled nasal spray device.
  • HPLC Analytical Methods Method 1 HPLC: Agilent 1100 Column: Agela C18 column, 4.6 x 50 mm, 3 ⁇ m Column temperature: 60°C Flow Rate: 1.0 mL/min Detector: ELSD Eluents: A, acetonitrile with 0.1% TFA; B, water with 0.1% TFA. Gradient: Method 2: HPLC: Agilent 1100 Column: Agela C18 column, 4.6 x 50 mm, 3 ⁇ m Column temperature: 60°C Flow Rate: 0.5 mL/min Detector: ELSD Eluents: A, Isopropanol; B, water with 0.1% TFA.
  • Step 2 Synthesis of Dioctadecyl (S)-2-hydroxysuccinate (2) A mixture of dimethyl malate 1 (4.35 g, 26.8 mmol), octadecan-1-ol (14.51 g, 53.6 mmol), p-toluene sulfonic acid (800 mg, 4.6 mmol) in 650 mL toluene, was refluxed vigorously with Dean-stark apparatus.
  • Step 3 Synthesis of Dioctadecyl (S)-2-((3-(dimethylamino)propanoyl)oxy)succinate (Example 1)
  • Example 3 Dihexadecyl (S)-2-((3-(dimethylamino)propanoyl)oxy)succinate
  • the derivative compound was prepared in a manner analogous to Example 1.
  • 1 H NMR 300 MHz, CDCl3) ⁇ 5.48 (t, 1H), 4.14 (t, 2H), 4.10 (t, 2H), 2.87 (d, 2H), 2.68-2.52 (m, 4H), 2.23 (s, 6H), 1.61 (s, br., 4H), 1.25 (m, 52H), 0.87 (t, 6H).
  • Example 4 Dihexadecyl (S)-2-((3-(dimethylamino)butanoyl)oxy)succinate
  • the derivative compound was prepared in a manner analogous to Example 1.
  • 1 H NMR 300 MHz, CDCl3) ⁇ 5.46 (t, 1H), 4.14 (t, 2H), 4.10 (t, 2H), 2.86 (d, 2H), 2.43 (m, 2H), 2.31 (t, 2H), 2.21 (s, 6H), 1.81 (quint, 2H), 1.62 (s, br., 4H), 1.24 (m, 52H), 0.87 (t, 6H).
  • Example 6 Di((9Z,12Z)-octadeca-9,12-dien-1-yl) (S)-2-((3- (dimethylamino)butanoyl)oxy)succinate
  • the derivative compound was prepared in a manner analogous to Example 1.
  • Example 7 Bis(7-(nonanoyloxy)heptyl) (S)-2-((3-(dimethylamino)propanoyl)oxy)succinate
  • Step 1 Synthesis of 7-hydroxyheptyl nonanoate (3) A mixture of 1,7-heptanediol (10.6 g, 80 mmol), nonanoic acid (3.12 g, 20 mmol), EDCI-HCl (4.22 g, 22 mmol) and DMAP (2.7 g, 22 mmol) in 60 mL dichloromethane was stirred at room temperature for 17 h.
  • the dichloromethane was removed on rotavapor, the residue was diluted with ethyl acetate (200 mL), and the solution was washed with aqueous saturated ammonium chloride, followed by brine and dried over sodium sulfate and concentrated to give an oil.
  • the crude product was purified by column chromatography using 15-10% ethyl acetate in hexanes to yield the desired product as colorless oil (4.5 g, 82%).
  • Step 2 Bis(7-(nonanoyloxy)heptyl) (S)-2-hydroxysuccinate (4)
  • the reaction mixture was cooled to room temperature and washed with saturated NaHCO3 solution, and the aqueous layer was extracted with EtOAc.
  • Step 3 Synthesis of bis(7-(nonanoyloxy)heptyl) (S)-2-((3- (dimethylamino)propanoyl)oxy)succinate (Example 7)
  • Example 8 Bis(7-(nonanoyloxy)heptyl) (S)-2-((3-(dimethylamino)butanoyl)oxy)succinate
  • the derivative compound was prepared in a manner analogous to Example 7.
  • Example 11 Dihexadecyl (S)-2-(((2-(dimethylamino)ethoxy)carbonyl)oxy)succinate
  • the derivative compound was prepared in a manner analogous to Example 9.
  • Example 12 Dihexadecyl (S)-2-(((3-(dimethylamino)propoxy)carbonyl)oxy)succinate
  • the derivative compound was prepared in a manner analogous to Example 9.
  • 1 H NMR 300 MHz, CDCl 3 ) ⁇ 5.37 (t, 1H), 4.25 (t, 2H), 4.16 (dt, 2H), 4.10 (t, 2H), 2.90 (d, 2H), 2.54 (s, br., 2H), 2.36 (s, 6H), 1.94 (quint, 2H), 1.66-1.59 (m, 4H), 1.25 (m, 52H), 0.87 (t, 6H).
  • Example 14 Di((9Z,12Z)-octadeca-9,12-dien-1-yl) (S)-2-(((3- (dimethylamino)propoxy)carbonyl)oxy)-succinate
  • the derivative compound was prepared in a manner analogous to Example 9.
  • Example 15 Bis(7-(nonanoyloxy)heptyl) (S)-2-(((2- (dimethylamino)ethoxy)carbonyl)oxy)succinate
  • the derivative compound was prepared in a manner analogous to Example 9.
  • Example 17 Dioctadecyl (S)-2-(((2-(dimethylamino)ethyl)(methyl)carbamoyl)oxy)succinate
  • N 1 ,N 1 ,N 2 -trimethylethane-1,2-diamine 408 mg, 4 mmol
  • 4-nitrophenylchloroformate 804 mg, 4 mmol
  • Example 18 Dioctadecyl (S)-2-(((3- (dimethylamino)propyl)(methyl)carbamoyl)oxy)succinate
  • the derivative compound was prepared in a manner analogous to Example 17.
  • 1 H NMR (300 MHz, CDCl3) ⁇ 5.42-5.35 (m, 1H), 4.11 (t, 2H), 4.09 (t, 2H), 3.27 (t, 2H), 2.90 (s, 3H), 2.84 (d, 2H), 2.21 (t, 2H), 2.18 (s, 6H), 1.68 (quint, 2H), 1.61-1.54 (m, 4H), 1.23 (m, 60H), 0.85 (t, 6H).
  • Example 20 Dihexadecyl (S)-2-(((3- (dimethylamino)propyl)(methyl)carbamoyl)oxy)succinate
  • the derivative compound was prepared in a manner analogous to Example 17.
  • Example 22 Di((9Z,12Z)-octadeca-9,12-dien-1-yl) (S)-2-(((3- (dimethylamino)propyl)(methyl)-carbamoyl)oxy)succinate
  • the derivative compound was prepared in a manner analogous to Example 17.
  • Example 23 Dioctadecyl ((2-(dimethylamino)ethoxy)carbonyl)-L-aspartate Step 1: Synthesis of Dioctadecyl (tert-butoxycarbonyl)-L-aspartate (5) A mixture of N-Boc-(L)-aspartic acid (2.33g, 10 mmol), octadecan-1-ol (5.4 g, 20 mmol), EDCI-HCl (4.8 g, 25 mmol) and DMAP (3.05 g, 25 mmol) in a mixture of 25 mL dichloromethane and 20 mL DMF was stirred at room temperature for 17 h.
  • reaction mixture was diluted with saturated ammonium chloride solution (100 mL) and extracted with dichloromethane (100 mL x 3). The combined organic layers were washed with brine, dried (sodium sulfate) and concentrated, and the crude was purified by column chromatography using 10-35% ethyl acetate in hexanes to get the desired product as white solid (5.2 g, 71%).
  • Step 2 Synthesis of Dioctadecyl L-aspartate (6)
  • dioctadecyl (tert-butoxycarbonyl)-L-aspartate 5 1.2 g, 1.63 mmol
  • dichloromethane 3 mL trifluoroacetic acid
  • the volatiles were concentrated under vacuum, and the residue was dissolved in dichloromethane, and washed with saturated sodium bicarbonate solution.
  • the organic layer was dried over sodium sulfate and concentrated to give the desired product (1.0 g, 99%), which was used without further purification.
  • Step 3 Synthesis of Dioctadecyl ((2-(dimethylamino)ethoxy)carbonyl)-L-aspartate (Example 23) To a solution of 2-(dimethylamino)ethan-1-ol (267 mg, 3 mmol) in a mixture of 1 mL pyridine and 8 mL dichloromethane, was added 4-nitrophenylchloroformate (603 mg, 3 mmol), and the resulting mixture was stirred at room temperature for 3 h.
  • Example 24 Dioctadecyl ((3-(dimethylamino)propoxy)carbonyl)-L-aspartate
  • the derivative compound was prepared in a manner analogous to Example 23.
  • 1 H NMR 300 MHz, CDCl3) ⁇ 5.66 (d, 1H), 4.59 (m, 1H), 4.20-4.03 (m, 6H), 3.02 (dd, 1H), 2.82 (dd, 1H), 2.36 (t, 2H), 2.24 (s, 6H), 1.88-1.77 (m, 2H), 1.67-1.53 (m, 4H), 1.25 (m, 60H), 0.87 (t, 6H).
  • Example 26 Dihexadecyl ((3-(dimethylamino)propoxy)carbonyl)-L-aspartate
  • the derivative compound was prepared in a manner analogous to Example 23.
  • 1 H NMR 300 MHz, CDCl 3 ) ⁇ 5.65 (d, 1H), 4.62-4.53 (m, 1H), 4.18-4.04 (m, 6H), 3.02 (dd, 1H), 2.82 (dd, 1H), 2.33 (t, 2H), 2.22 (s, 6H), 1.79 (quint, 2H), 1.67-1.56 (m, 4H), 1.25 (m, 52H), 0.87 (t, 6H).
  • Example 28 Di((9Z,12Z)-octadeca-9,12-dien-1-yl) ((3-(dimethylamino)propoxy)carbonyl)- L-aspartate
  • the derivative compound was prepared in a manner analogous to Example 23.
  • Example 29 Dioctadecyl 2-((3-(dimethylamino)propanoyl)oxy)pentanedioate
  • Step 1 Synthesis of Dioctadecyl 2-oxopentanedioate (7)
  • Step 2 Synthesis of Dioctadecyl 2-hydroxypentanedioate (8) To a solution of dioctadecyl 2-oxopentanedioate 7 (14 g, 20.5 mmol) in 100 mL THF, was added sodium borohydride (1.2 g, 32.2 mmol), and the reaction mixture was stirred at room temperature for 2 hours.
  • Step 3 Synthesis of Dioctadecyl 2-((3-(dimethylamino)propanoyl)oxy)pentanedioate (Example 29)
  • Example 31 Dihexadecyl 2-((3-(dimethylamino)propanoyl)oxy)pentanedioate
  • the derivative compound was prepared in a manner analogous to Example 29.
  • 1 H NMR 300 MHz, CDCl 3 ) ⁇ 5.07 (dd, 1H), 4.12 (t, 2H), 4.06 (t, 2H), 2.68-2.53 (m, 4H), 2.43 (m, 2H), 2.24 (s, 6H), 2.22-2.08 (m, 2H), 1.66-1.55 (m, 4H), 1.24 (m, 52H), 0.87 (t, 6H).
  • Example 33 Di((9Z,12Z)-octadeca-9,12-dien-1-yl) 2-((3- (dimethylamino)propanoyl)oxy)pentanedioate
  • the derivative compound was prepared in a manner analogous to Example 29.
  • Example 34 Di((9Z,12Z)-octadeca-9,12-dien-1-yl) 2-((4- (dimethylamino)butanoyl)oxy)pentanedioate
  • the derivative compound was prepared in a manner analogous to Example 29.
  • Example 35 Bis(7-(nonanoyloxy)heptyl) 2-((3- (dimethylamino)propanoyl)oxy)pentanedioate
  • the derivative compound was prepared in a manner analogous to Example 29 and was obtained by column purification chromatography eluted with hexane/acetone.
  • Example 36 Bis(7-(nonanoyloxy)heptyl) 2-((4-(dimethylamino)butanoyl)oxy)pentanedioate
  • the derivative compound was prepared in a manner analogous to Example 29 and was obtained by column purification chromatography eluted with hexane/acetone.
  • Example 37 Dioctadecyl 2-(((2-(dimethylamino)ethoxy)carbonyl)oxy)pentanedioate
  • a solution of dioctadecyl 2-hydroxypentanedioate 8 (150 mg, 0.23 mmol) and pyridine (75 ⁇ L, 0.92 mmol) in 10 mL dichloromethane was added p-nitrophenyl chloroformate (138 mg, 0.69 mmol), and the resulting mixture was stirred for 2 hours.
  • 2-dimethylaminoethanol 73 mg, 0.92 mmol was added, and the reaction mixture was stirred at room temperature overnight. MS showed the formation of desired product.
  • Example 38 Dioctadecyl 2-(((3-(dimethylamino)propoxy)carbonyl)oxy)pentanedioate
  • the derivative compound was prepared in a manner analogous to Example 37.
  • Example 40 Dihexadecyl 2-(((3-(dimethylamino)propoxy)carbonyl)oxy)pentanedioate
  • the derivative compound was prepared in a manner analogous to Example 37.
  • 1 H NMR 300 MHz, CDCl3) ⁇ 4.95 (dd, 1H), 4.21 (t, 2H), 4.15 (t, 2H), 4.06 (t, 2H), 2.50- 2.42 (m, 2H), 2.36 (t, 2H), 2.22 (s, 6H), 2.21-2.10 (m, 2H), 1.85 (quint, 2H), 1.66-1.56 (m, 4H), 1.25 (m, 52H), 0.87 (t, 6H).
  • Example 42 Di((9Z,12Z)-octadeca-9,12-dien-1-yl) 2-(((3- (dimethylamino)propoxy)carbonyl)oxy)-pentanedioate
  • the derivative compound was prepared in a manner analogous to Example 37.
  • Example 43 Bis(7-(nonanoyloxy)heptyl) 2-(((2- (dimethylamino)ethoxy)carbonyl)oxy)pentanedioate
  • the derivative compound was prepared in a manner analogous to Example 37.
  • Example 44 Bis(7-(nonanoyloxy)heptyl) 2-(((3- (dimethylamino)propoxy)carbonyl)oxy)pentanedioate
  • the derivative compound was prepared in a manner analogous to Example 37.
  • Example 45 Dioctadecyl 2-(((2-(dimethylamino)ethyl)(methyl)carbamoyl)oxy)- pentanedioate
  • dioctadecyl 2-hydroxypentanedioate 8 150 mg, 0.23 mmol
  • pyridine 75 ⁇ L, 0.92 mmol
  • p-nitrophenyl chloroformate 138 mg, 0.69 mmol
  • N 1 ,N 1 ,N 2 -trimethylethane-1,2-diamine (94 mg, 0.92 mmol) was added, and the reaction mixture was stirred at room temperature overnight. MS showed the formation of desired product.
  • the reaction mixture was concentrated, and the residue was partitioned between hexane and acetonitrile. The hexane layer was concentrated, and the crude was purified by column chromatography eluted with dichloromethane/acetone to afford dioctadecyl 2-(((2-(dimethylamino)ethyl)(methyl)carbamoyl)oxy)-pentanedioate as pale yellow wax (142 mg, 79%).
  • Example 46 Dioctadecyl 2-(((3- (dimethylamino)propyl)(methyl)carbamoyl)oxy)pentanedioate
  • the derivative compound was prepared in a manner analogous to Example 45.
  • Example 47 Dihexadecyl 2-(((2- (dimethylamino)ethyl)(methyl)carbamoyl)oxy)pentanedioate
  • the derivative compound was prepared in a manner analogous to Example 45.
  • Example 49 Di((9Z,12Z)-octadeca-9,12-dien-1-yl) 2-(((2- (dimethylamino)ethyl)(methyl)carbamoyl)oxy)-pentanedioate
  • the derivative compound was prepared in a manner analogous to Example 45.
  • Example 50 Di((9Z,12Z)-octadeca-9,12-dien-1-yl) 2-(((3- (dimethylamino)propyl)(methyl)carbamoyl)-oxy)pentanedioate
  • the derivative compound was prepared in a manner analogous to Example 45.
  • Step 2 Synthesis of Dioctadecyl L-glutamate trifluoroacetate (10) To a solution of dioctadecyl (tert-butoxycarbonyl)-L-glutamate 9 (4.0 g, 5.3 mmol) in 10 mL dichloromethane, was added 9 mL trifluoroacetic acid slowly, and the reaction mixture was stirred at room temperature for 3 hours.
  • Step 3 Synthesis of Dioctadecyl ((2-(dimethylamino)ethoxy)carbonyl)-L-glutamate (Example 51) To a solution of dioctadecyl L-glutamate trifluoroacetate 10 (500 mg, 0.65 mmol) and pyridine (0.21 mL, 2.6 mmol) in 10 mL dichloromethane, was added p-nitrophenyl chloroformate (393 mg, 2 mmol), and the resulting mixture was stirred for 2 hours.
  • Example 52 Dioctadecyl ((3-(dimethylamino)propoxy)carbonyl)-L-glutamate
  • the derivative compound was prepared in a manner analogous to Example 51.
  • Example 54 Dihexadecyl ((3-(dimethylamino)propoxy)carbonyl)-L-glutamate
  • the derivative compound was prepared in a manner analogous to Example 51.
  • Example 56 Di((9Z,12Z)-octadeca-9,12-dien-1-yl) ((3-(dimethylamino)propoxy)carbonyl)- L-glutamate
  • the derivative compound was prepared in a manner analogous to Example 51.
  • Example 57 Bis(7-(nonanoyloxy)heptyl) ((2-(dimethylamino)ethoxy)carbonyl)-L-glutamate
  • the derivative compound was prepared in a manner analogous to Example 51.
  • 1 H NMR 300 MHz, CDCl3) ⁇ 5.43 (d, 1H), 4.40-4.32 (m, 1H), 4.18-4.08 (m, 2H), 4.04 (t, 8H), 2.56 (t, 2H), 2.38 (dd, 2H), 2.28 (s, 6H), 2.28 (t, 4H), 2.25-2.13 (m, 1H), 2.02-1.88 (m, 1H), 1.68-1.50 (m, 12H), 1.38-1.18 (m, 32H), 0.86 (t, 6H).
  • Step 2 Synthesis of 5-Hydroxypentyl octanoate (12) A mixture of 1,5-pentanediol (15.7 mL, 0.15 mole), octanoic acid (7.92 mL, 50 mmol), EDCI-HCl (9.58 g, 50 mmol) and DMAP (1.52 g, 12.5 mmol) in 100 mL dichloromethane was stirred at room temperature for 17 h. The dichloromethane was removed, the residue was diluted with 200 mL ethyl acetate, and the solution was washed with aqueous saturated ammonium chloride.
  • Step 3 Synthesis of Bis(5-(octanoyloxy)pentyl) 2-hydroxysuccinate (13)
  • Step 4 Synthesis of Bis(5-(octanoyloxy)pentyl) 2-((3-(dimethylamino)propanoyl)oxy)- succinate (Example 58)
  • Step 2 Synthesis of Bis(5-((2-hexyldecanoyl)oxy)pentyl) 2-hydroxysuccinate (15) A mixture of dimethyl malate 11 (2.11 g, 13 mmol), 5-hydroxypentyl 2- hexyldecanoate 14 (8.89 g, 26 mmol) and p-toluenesulfonic acid monohydrate (200 mg) in 200 mL toluene was heated to reflux vigorously with Dean-stark apparatus. After 150 mL toluene was distilled, more toluene was added. This process was repeated one more time.
  • reaction mixture was cooled to room temperature and washed with saturated sodium bicarbonate solution, and the aqueous layer was extracted with EtOAc. The combined organic layers were dried over sodium sulfate and concentrated to give the crude which was purified by column chromatography using 0-10 % ethyl acetate in hexane as eluent to give the desired product as colorless oil (5.0 g, 50%).
  • Step 3 Synthesis of Bis(5-((2-hexyldecanoyl)oxy)pentyl) 2-((3-(dimethylamino)propanoyl)- oxy)succinate
  • Example 59 A mixture of bis(5-((2-hexyldecanoyl)oxy)pentyl) 2-hydroxysuccinate 15 (265 mg, 0.34 mmol), 3-(dimethylamino)propanoic acid hydrochloride (88 mg, 0.57 mmol), EDC-HCl (109 mg, 0.57 mmol), DMAP (70 mg, 0.57 mmol) in 10 mL dichloromethane was stirred at room temperature for 17 h.
  • Example 61 Bis(6-oxo-6-(pentadecan-7-yloxy)hexyl) 2-((3-(dimethylamino)- propanoyl)oxy)succinate
  • Step 1 Synthesis of Pentadecan-7-ol (16) At 0 °C, to a solution of octylmagnesium bromide (2 M in ether, 52.5 mL, 0.105 mole) in 100 mL ether, was slowly added a solution of heptaldehyde (12.6 mL, 90 mmol) in 40 mL ether, and the mixture was warmed up to room temperature and stirred overnight.
  • Step 2 Synthesis of Bis(6-(benzyloxy)hexyl) 2-hydroxysuccinate (17) A mixture of dimethyl malate 11 (2.1 g, 13 mmol), 6-benzyoxyhexanol (5.4 g, 26 mmol) and p-toluenesulfonic acid monohydrate (300 mg) in 200 mL toluene was refluxed vigorously with Dean-stark apparatus.
  • Step 3 Synthesis of Bis(6-(benzyloxy)hexyl) 2-((tert-butyldiphenylsilyl)oxy)succinate (18)
  • the reaction mixture was partitioned with ethyl acetate and saturated ammonium chloride solution, and the combined organic layer was dried over sodium sulfate.
  • Step 4 Synthesis of Bis(6-hydroxyhexyl) 2-((tert-butyldiphenylsilyl)oxy)succinate (19) A mixture of bis(6-(benzyloxy)hexyl) 2-((tert-butyldiphenylsilyl)oxy)succinate 18 (1.6 g, 2,12 mmol) and 5% palladium on carbon (500 mg) in 100 mL ethyl acetate was subjected to hydrogenolysis for 15 h.
  • Step 5 Synthesis of 6,6'-((2-((tert-Butyldiphenylsilyl)oxy)succinyl)bis(oxy))dihexanoic acid (20)
  • Step 6 Synthesis of Bis(6-oxo-6-(pentadecan-7-yloxy)hexyl) 2-((tert-butyldiphenylsilyl)oxy)- succinate (21)
  • a mixture of 6,6'-((2-((tert-butyldiphenylsilyl)oxy)succinyl)bis(oxy))dihexanoic acid 20 800 mg, 1.33 mmol
  • 7-pentadecanol 914 mg, 4 mmol
  • EDCI-HCl (768 mg, 4 mmol
  • DMAP 488 mg, 4 mmol
  • Step 7 Synthesis of Bis(6-oxo-6-(pentadecan-7-yloxy)hexyl) 2-hydroxysuccinate (22)
  • pyridine 2 mL
  • HF-pyridine complex 70%, 1.5 mL
  • Step 8 Synthesis of Bis(6-oxo-6-(pentadecan-7-yloxy)hexyl) 2-((3-(dimethylamino)- propanoyl)oxy)succinate (Example 61)
  • Example 62 Bis(6-oxo-6-(pentadecan-7-yloxy)hexyl) 2-((3-(dimethylamino)- butanoyl)oxy)succinate
  • the derivative compound was prepared in a manner analogous to Example 61.
  • Example 63 Bis(6-((2-butylnonyl)oxy)-6-oxohexyl) 2-hydroxysuccinate
  • Step 1 Synthesis of 2-Butylnonanoic acid (23) To a solution of nonanoic acid (17.7 mL, 0.114 mole) in 200 mL THF at 0 °C, was added NaH (60%, 5.0 g, 0.125 mole) portionwise, followed by adding a solution of LDA (2 M in THF/heptane/ethylbenzene, 62.5 mL, 0.125 mole) dropwise, and the mixture was stirred at room temperature for 30 min.
  • LDA 2 M in THF/heptane/ethylbenzene
  • Step 2 Synthesis of 2-Butylnonan-1-ol (24)
  • a solution of impure 2-butylnonanoic acid 23 1.0 g, 4.7 mmol
  • a solution of LiAlH4 2 M in THF, 23.5 mL, 47 mmol
  • EtOAc 2 M in THF
  • the organic layer was dried over sodium sulfate.
  • the solvent was removed under vacuum, and the crude was purified by flash column chromatography eluted with 0-40% EtOAc in hexane to give the desired product as colorless oil (600 mg, 64%).
  • Step 3 Synthesis of Bis(6-((2-butylnonyl)oxy)-6-oxohexyl) 2-((tert- butyldiphenylsilyl)oxy)succinate (25)
  • Step 4 Synthesis of Bis(6-((2-butylnonyl)oxy)-6-oxohexyl) 2-hydroxysuccinate (26)
  • pyridine 2 mL
  • HF-pyridine complex 70%, 1.5 mL
  • the reaction was quenched by adding saturated sodium bicarbonate to pH 7, and then extracted with ethyl acetate (3 x 60 mL).
  • Step 5 Synthesis of Bis(6-((2-butylnonyl)oxy)-6-oxohexyl) 2-hydroxysuccinate (Example 63) A mixture of bis(6-((2-butylnonyl)oxy)-6-oxohexyl) 2-hydroxysuccinate 26 (73 mg, 0.10 mmol), 3-(dimethylamino)propanoic acid hydrochloride (76 mg, 0.49 mmol), EDC-HCl (95 mg, 0.49 mmol) and DMAP (60 mg, 0.49 mmol) in 10 mL dichloromethane was stirred at room temperature for 17 h.
  • Example 64 4-(2-(Dimethylamino)ethyl) 1-(4-((2-hexyldecanoyl)oxy)butyl) (2S)-2-((6-((2- hexyldecanoyl)oxy)hexanoyl)oxy)succinate
  • Step 1 Synthesis of Benzyl (S)-2-(2,2-dimethyl-5-oxo-1,3-dioxolan-4-yl)acetate (27)
  • Compound 27 was prepared according the procedure described in CN111039919A.
  • Step 2 Synthesis of (S)-4-(Benzyloxy)-2-hydroxy-4-oxobutanoic acid (28)
  • Compound 28 was prepared according the procedure described in CN111039919A. A solution of benzyl (S)-2-(2,2-dimethyl-5-oxo-1,3-dioxolan-4-yl)acetate 27 (6.5 g, 24.6 mmol) in 30 mL THF, 30 mL acetic acid and 30 mL water was heated to 40 °C overnight.
  • Step 3 Synthesis of 4-Benzyl 1-(4-((2-hexyldecanoyl)oxy)butyl) (2S)-2-hydroxysuccinate (29)
  • Step 4 Synthesis of (3S)-4-(4-((2-Hexyldecanoyl)oxy)butoxy)-3-hydroxy-4-oxobutanoic acid (30) A mixture of 4-benzyl 1-(4-((2-hexyldecanoyl)oxy)butyl) (2S)-2-hydroxysuccinate 29 (155 mg, 0.29 mmol) and palladium on carbon (5%, 200 mg) in 15 mL hexane was subjected to hydrogenolysis for 30 h.
  • Step 5 Synthesis of 4-(2-(Dimethylamino)ethyl) 1-(4-((2-hexyldecanoyl)oxy)butyl) (2S)-2- hydroxysuccinate (31)
  • a solution of (3S)-4-(4-((2-hexyldecanoyl)oxy)butoxy)-3-hydroxy-4-oxobutanoic acid 30 130 mg, 0.29 mmol
  • 2-(dimethylamino)ethan-1-ol 260 mg, 2.9 mmol
  • EDCI-HCl 192 mg, 1 mmol
  • DMAP 122 mg, 1 mmol
  • reaction mixture was diluted with 100 mL dichloromethane, washed with saturated ammonium chloride solution (50 mL x 3), and followed by brine. The organic layer was dried over sodium sulfate and concentrated to give the crude product which was carried to the next step without further purification.
  • Step 6 Synthesis of 4-(2-(Dimethylamino)ethyl) 1-(4-((2-hexyldecanoyl)oxy)butyl) (2S)-2-((6- ((2-hexyldecanoyl)oxy)hexanoyl)oxy)succinate (Example 64)
  • Example 65 4-(2-(Dimethylamino)propyl) 1-(4-((2-hexyldecanoyl)oxy)butyl) (2S)-2-((6-((2- hexyldecanoyl)oxy)hexanoyl)oxy)succinate
  • the derivative compound was prepared in a manner analogous to Example 64.
  • Example 66 4-(2-(Dimethylamino)ethyl) 1-(5-((2-hexyldecanoyl)oxy)-pentyl) 2-((7-((2- hexyldecanoyl)oxy)heptanoyl)oxy)succinate
  • the derivative compound was prepared in a manner analogous to Example 64.
  • Example 67 4-(3-(Dimethylamino)propyl) 1-(5-((2-hexyldecanoyl)oxy)pentyl) (2S)-2-((7- ((2-hexyldecanoyl)oxy)heptanoyl)oxy)succinate
  • the derivative compound was prepared in a manner analogous to Example 64.
  • Example 68 4-(2-(Dimethylamino)ethyl) 1-(5-((2-hexyldecanoyl)oxy)pentyl) (2S)-2-((((5- ((2-hexyldecanoyl)oxy)pentyl)oxy)carbonyl)oxy)succinate
  • Step 1 Synthesis of 5-Hydroxypentyl 2-hexyldecanoate (32) A mixture of 1,5-pentanediol (10.4 g, 0.10 mole), 2-hexyldecanoic acid (7.92 mL, 50 mmol), EDCI-HCl (9.58 g, 50 mmol) and DMAP (1.52 g, 12.5 mmol) in 100 mL dichloromethane was stirred at room temperature for 16 h.
  • Step 2 Synthesis of 4-(2-(Dimethylamino)ethyl) 1-(5-((2-hexyldecanoyl)oxy)pentyl) (2S)-2- ((((5-((2-hexyldecanoyl)oxy)pentyl)oxy)carbonyl)oxy)succinate (Example 68)
  • p-nitrophenyl chloroformate 404 mg, 2.0 mmol
  • Example 69 4-(3-(Dimethylamino)propyl) 1-(5-((2-hexyldecanoyl)oxy)pentyl) (2S)-2-((((5- ((2-hexyldecanoyl)oxy)pentyl)oxy)carbonyl)oxy)succinate
  • the derivative compound was prepared in a manner analogous to Example 68.
  • Example 70 1-(2-(Dimethylamino)ethyl) 4-(5-((2-hexyldecanoyl)oxy)pentyl) (2S)-2-((7-((2- hexyldecanoyl)oxy)heptanoyl)oxy)succinate
  • Step 1 7-Hydroxyheptyl 2-hexyldecanoate (33)
  • 1,7-heptanediol 4 g, 35 mmol
  • 2-hexyldecanoic acid 4.5 g, 17.5 mmol
  • EDCI-HCl 4.0 g, 21 mmol
  • DMAP 427 mg, 3.5 mmol
  • Step 2 7-((2-Hexyldecanoyl)oxy)heptanoic acid (34) To a solution of 7-hydroxyheptyl 2-hexyldecanoate 33 (4.9 g, 13.2 mmol) in 50 mL acetone, was added Jones reagent until the orange color persisted and the resulting mixture was stirred for 1 h.
  • Step 3 Synthesis of 5-(2-((S)-2,2-Dimethyl-5-oxo-1,3-dioxolan-4-yl)acetoxy)pentyl 2- hexyldecanoate (35)
  • a mixture of (S)-2-(2,2-dimethyl-5-oxo-1,3-dioxolan-4-yl)acetic acid (174 mg, 1 mmol)
  • 5-hydroxypentyl 2-hexyldecanoate 32 (342 mg, 1 mmol)
  • triethylamine 0.5 mL
  • EDCI-HCl 192 mg, 1 mmol
  • DMAP 122 mg, 1 mmol
  • Step 4 Synthesis of (2S)-4-((5-((2-Hexyldecanoyl)oxy)pentyl)oxy)-2-hydroxy-4-oxobutanoic acid (36)
  • Step 5 Synthesis of 1-(2-(Dimethylamino)ethyl) 4-(5-((2-hexyldecanoyl)oxy)pentyl) (2S)-2- hydroxysuccinate (37) A mixture of (2S)-4-((5-((2-hexyldecanoyl)oxy)pentyl)oxy)-2-hydroxy-4-oxobutanoic acid 36 (200 mg, 0.44 mmol), 2-(dimethylamino)ethan-1-ol (390 mg, 4.4 mmol), EDCI-HCl (192 mg, 1 mmol) and DMAP (122 mg, 1 mmol) in 7 mL dichloromethane was stirred at room temperature for 18 h.
  • reaction mixture was diluted with dichloromethane (100 mL) and washed with saturated ammonium chloride solution (50 mL x 3), followed by brine (50 mL x 1).
  • the organic layer was dried over sodium sulfate and concentrated to get the desired product as brown oil (250 mg) which was used for the next step without further purification.
  • Step 6 Synthesis of 1-(2-(Dimethylamino)ethyl) 4-(5-((2-hexyldecanoyl)oxy)pentyl) (2S)-2- ((7-((2-hexyldecanoyl)oxy)heptanoyl)oxy)succinate (Example 70)
  • Example 71 1-(3-(Dimethylamino)propyl) 4-(5-((2-hexyldecanoyl)oxy)pentyl) (2S)-2-((7- ((2-hexyldecanoyl)oxy)heptanoyl)oxy)succinate
  • the derivative compound was prepared in a manner analogous to Example 70.
  • Example 72 1-(2-(Dimethylamino)ethyl) 4-(4-((2-hexyldecanoyl)oxy)butyl) (2S)-2-((6-((2- hexyldecanoyl)oxy)hexanoyl)oxy)succinate
  • the derivative compound was prepared in a manner analogous to Example 70.
  • Example 73 1-(18-hexyl-2-methyl-6,9,16-trioxo-5,10,17-trioxa-2-azahexacosan-7-yl) 8- (pentadecan-7-yl) octanedioate
  • the derivative compound was prepared in a manner analogous to Example 70.
  • Example 74 1-(2-(Dimethylamino)ethyl) 4-(5-((2-hexyldecanoyl)oxy) pentyl) 2-((6-((2- hexyldecanoyl)oxy)hexanoyl)oxy)succinate
  • the derivative compound was prepared in a manner analogous to Example 70.
  • Example 75 1-(2-(Dimethylamino)ethyl) 4-(5-((2-hexyldecanoyl)oxy)pentyl) (2S)-2-((((5- ((2-hexyldecanoyl)oxy)pentyl)oxy)carbonyl)oxy)succinate
  • 5-hydroxypentyl 2-hexyldecanoate 32 513 mg, 1.5 mmol
  • pyridine (1 mL)
  • p-nitrophenyl chloroformate 303 mg, 1.5 mmol
  • reaction mixture was diluted with dichloromethane (100 mL) and washed with saturated ammonium chloride solution (50 mL x 3), followed by brine (50 mL x 1).
  • the organic layer was dried over sodium sulfate and concentrated to get the desired product as brown oil (260 mg) which was used for the next step without further purification.
  • Step 2 Synthesis of 1-(3-(dimethylamino)propyl) 4-(5-((2-hexyldecanoyl)oxy)pentyl) (2S)-2- ((((5-((2-hexyldecanoyl)oxy)pentyl)oxy)carbonyl)oxy)succinate (Example 76)
  • 5-hydroxypentyl 2-hexyldecanoate 32 513 mg, 1.5 mmol
  • pyridine (1 mL)
  • dichloromethane p-nitrophenyl chloroformate
  • Step 2 Synthesis of Bis(8-(benzyloxy)octyl) 2-((tert-butyldiphenylsilyl)oxy)succinate (40)
  • the reaction mixture was partitioned with ethyl acetate and saturated ammonium chloride solution, and the combined organic layer was dried over sodium sulfate.
  • Step 3 Synthesis of Bis(8-hydroxyoctyl) 2-((tert-butyldiphenylsilyl)oxy)succinate (41)
  • Step 4 Synthesis of 8,8'-((2-((tert-Butyldiphenylsilyl)oxy)succinyl)bis(oxy))dioctanoic acid (42)
  • Step 5 Synthesis of Bis(8-oxo-8-(pentadecan-7-yloxy)octyl) 2-((tert- butyldiphenylsilyl)oxy)succinate (43) A mixture of 8,8'-((2-((tert-butyldiphenylsilyl)oxy)succinyl)bis(oxy))dioctanoic acid 42 (400 mg, 0.61 mmol), 7-pentadecanol (310 mg, 1.28 mmol), EDCI-HCl (384 mg, 2 mmol) and DMAP (244 mg, 2 mmol) in 10 mL dichloromethane was stirred at room temperature for 16 h.
  • Step 6 Synthesis of Bis(8-oxo-8-(pentadecan-7-yloxy)octyl) 2-hydroxysuccinate (44)
  • pyridine 2 mL
  • HF-pyridine complex 70%, 1.5 mL
  • Step 7 Synthesis of Bis(8-oxo-8-(pentadecan-7-yloxy)octyl) 2-(((2- (dimethylamino)ethoxy)carbonyl)oxy)succinate (Example 77) To a solution of 2,2-dimethylaminoethanol (534 mg, 6 mmol) in a mixture of 3mL pyridine and 8 mL dichloromethane, was added 4-nitrophenylchloroformate (603 mg, 6 mmol), and the reaction mixture was stirred at room temperature for 3 h.
  • Step 2 Synthesis of Bis(8-(((Z)-non-2-en-1-yl)oxy)-8-oxooctyl) 2-hydroxysuccinate (46)
  • pyridine 2 mL
  • HF-pyridine complex 70%, 0.5 mL
  • reaction was quenched by adding saturated sodium bicarbonate to pH 7, and then extracted with ethyl acetate (3 x 60 mL). The combined organic layers were dried and concentrated, and the crude was purified by column chromatography using 0-30% ethyl acetate in hexane to yield the desired product (270 mg, 89%).
  • Step 3 Synthesis of Bis(8-(((Z)-non-2-en-1-yl)oxy)-8-oxooctyl) 2-(((2- (dimethylamino)ethoxy)carbonyl)oxy)succinate (Example 78)
  • 2-(dimethylamino)ethan-1-ol 534 mg, 6 mmol
  • 4-nitrophenylchloroformate 1.20 g, 6 mmol
  • Step 2 Synthesis of 7-(Benzyloxy)heptyl 8-methylnonanoate (48)
  • Step 3 Synthesis of 7-Hydroxyheptyl 8-methylnonanoate (49) A mixture of 7-(benzyloxy)heptyl 8-methylnonanoate 48 (13.8 g, 48.2 mmol) and 20% palladium hydroxide on carbon (1.5 g) in 100 mL ethyl acetate was subjected to hydrogenolysis in a Parr apparatus (40 psi) for 16 h.
  • Step 4 Synthesis of Bis(7-((8-methylnonanoyl)oxy)heptyl) 2-hydroxysuccinate (50)
  • Step 5 Synthesis of Bis(7-((8-methylnonanoyl)oxy)heptyl) 2-(((2- (dimethylamino)ethoxy)carbonyl)oxy)succinate (Example 79)
  • 2-(dimethylamino)ethan-1-ol 534 mg, 6 mmol
  • 4-nitrophenylchloroformate 1.20 g, 6 mmol
  • Example 80 Bis(8-oxo-8-((2-propylnonyl)oxy)octyl) 2-(((2- (dimethylamino)ethoxy)carbonyl)oxy)succinate
  • Step 1 Synthesis of 2-Propylnonanoic acid (51) To a solution of nonanoic acid (10.5 mL, 60 mmol) in 100 mL THF at 0°C, was added NaH (60%, 2.4 g, 60 mmol) in portions, followed by adding a solution of LDA (2 M in THF/heptane/ethylbenzene, 60 mL, 0.12 mole) dropwise, and the mixture was stirred at room temperature for 30 min.
  • LDA 2 M in THF/heptane/ethylbenzene
  • Step 2 Synthesis of 2-propylnonan-1-ol (52) To a solution of impure 2-propylnonanoic acid 51 (5.5 g, 30 mmol) in 30 mL ether, was added a solution of LiAlH 4 (2 M in THF, 15 mL, 30 mmol) dropwise, and the resulting mixture was stirred at room temperature overnight. After quenched by EtOAc, the solution was washed with water, and the organic layer was dried over sodium sulfate. The solvent was removed under vacuum, and the crude was purified by flash column chromatography eluted with 0-40% EtOAc in hexane to give the desired product as colorless oil (3.5 g, 69%).
  • Step 3 Synthesis of Bis(8-oxo-8-((2-propylnonyl)oxy)octyl) 2-((tert- butyldiphenylsilyl)oxy)succinate (53)
  • a mixture of 8,8'-((2-((tert-butyldiphenylsilyl)oxy)succinyl)bis(oxy))dioctanoic acid 42 700 mg, 1.06 mmol
  • 2-propylnonan-1-ol 52 510 mg, 2.74 mmol
  • EDCI-HCl 576 mg, 3 mmol
  • DMAP 366 mg, 3 mmol
  • Step 4 Synthesis of Bis(8-oxo-8-((2-propylnonyl)oxy)octyl) 2-hydroxysuccinate (54) To a solution of crude bis(8-oxo-8-((2-propylnonyl)oxy)octyl) 2-((tert- butyldiphenylsilyl)oxy)succinate 53 (900 mg) in 10 mL THF, was added pyridine (2 mL) followed by HF-pyridine complex (70%, 1.5 mL), and the mixture was stirred at room temperature overnight.
  • reaction was quenched by adding saturated sodium bicarbonate to pH 7, and then extracted with ethyl acetate (3 x 60 mL). The combined organic layers were dried and concentrated, and the crude was purified by column chromatography using 0-30% ethyl acetate in hexane to yield the desired product (460 mg, 57%).
  • Step 5 Synthesis of Bis(8-oxo-8-((2-propylnonyl)oxy)octyl) 2-(((2- (dimethylamino)ethoxy)carbonyl)oxy)succinate (Example 80)
  • 2-(dimethylamino)ethan-1-ol 534 mg, 6 mmol
  • 4-nitrophenylchloroformate 1.20 g, 6 mmol
  • Example 81 Bis(6-(decanoyloxy)hexyl) 2-(((2- (dimethylamino)ethoxy)carbonyl)oxy)succinate
  • Step 1 Synthesis of Bis(6-(decanoyloxy)hexyl) 2-((tert-butyldiphenylsilyl)oxy)succinate (55)
  • a mixture of bis(6-hydroxyhexyl) 2-((tert-butyldiphenylsilyl)oxy)succinate 19 370 mg, 0.65 mmol
  • decanoic acid (223 mg, 1.3 mmol
  • EDCI-HCl 288 mg, 1.5 mmol
  • DMAP 183 mg, 1.5 mmol
  • Step 2 Synthesis of Bis(6-(decanoyloxy)hexyl) 2-hydroxysuccinate (56)
  • pyridine 2 mL
  • HF-pyridine complex 70%, 1 mL
  • the reaction was quenched by adding saturated sodium bicarbonate to pH 7, and then extracted with ethyl acetate (3 x 60 mL).
  • Step 3 Synthesis of Bis(6-(decanoyloxy)hexyl) 2-(((2- (dimethylamino)ethoxy)carbonyl)oxy)succinate (Example 81) To a solution of 2-(dimethylamino)ethan-1-ol (534 mg, 6 mmol) in a mixture of 1 mL pyridine and 8 mL dichloromethane, was added 4-nitrophenylchloroformate (1.20 g, 6 mmol), and the reaction mixture was stirred at room temperature for 1 h.
  • Example 86 Bis(6-(((Z)-non-2-en-1-yl)oxy)-6-oxohexyl) 2-(((2- (dimethylamino)ethoxy)carbonyl)oxy)succinate
  • Step 1 Synthesis of Bis(6-(((Z)-non-2-en-1-yl)oxy)-6-oxohexyl) 2-((tert- butyldiphenylsilyl)oxy)succinate (57)
  • Step 2 Synthesis of Bis(6-(((Z)-non-2-en-1-yl)oxy)-6-oxohexyl) 2-hydroxysuccinate (58)
  • pyridine 2 mL
  • HF-pyridine complex 70%, 1.5 mL
  • reaction was quenched by adding saturated sodium bicarbonate to pH 7, and then extracted with ethyl acetate (3 x 60 mL). The combined organic layers were dried and concentrated, and the crude was purified by column chromatography using 0-40% ethyl acetate in hexane to yield the desired product (300 mg, 82%).
  • Step 3 Synthesis of Bis(6-(((Z)-non-2-en-1-yl)oxy)-6-oxohexyl) 2-(((2- (dimethylamino)ethoxy)carbonyl)oxy)succinate (Example 86)
  • 2-(dimethylamino)ethan-1-ol 445 mg, 5 mmol
  • 4-nitrophenylchloroformate 1.0 g, 5 mmol
  • Step 2 Synthesis of Bis(7-((2-hexyldecanoyl)oxy)heptyl) 2-(((2- (dimethylamino)ethoxy)carbonyl)oxy)succinate (Example 87)
  • 2-(dimethylamino)ethan-1-ol 445 mg, 5 mmol
  • 4-nitrophenylchloroformate 1.0 g, 5 mmol
  • Example 88 Bis(5-(decanoyloxy)pentyl) 2-(((2- (dimethylamino)ethoxy)carbonyl)oxy)succinate
  • Step 1 Synthesis of 5-Hydroxypentyl decanoate (60) A mixture of 1,5-pentanediol (10 g, 0.10 mole), decanoic acid (5.67 g, 33 mmol), EDCI-HCl (8.85 g, 46.2 mmol) and DMAP (805 mg, 6.6 mmol) in 100 mL dichloromethane was stirred at room temperature for 17 h.
  • Step 2 Synthesis of Bis(5-(decanoyloxy)pentyl) 2-hydroxysuccinate (61)
  • a mixture of dimethyl malate (1.22 g, 7.5 mmol), 5-hydroxypentyl decanoate 60(4.0 g, 15 mmol) and p-toluenesulfonic acid monohydrate (250 mg) in 150 mL toluene was heated to reflux vigorously with Dean-stark apparatus overnight.
  • the reaction mixture was cooled to room temperature and washed with saturated sodium bicarbonate solution, and the aqueous layer was extracted with EtOAc.
  • Step 3 Synthesis of Bis(5-(decanoyloxy)pentyl) 2-(((2- (dimethylamino)ethoxy)carbonyl)oxy)succinate (Example 88)
  • 2-(dimethylamino)ethan-1-ol 445 mg, 5 mmol
  • 4-nitrophenylchloroformate 1.0 g, 5 mmol
  • Step 2 Synthesis of Bis(5-hydroxypentyl) 2-(pyridin-2-yldisulfaneyl)succinate (63)
  • a solution of bis(5-hydroxypentyl) 2-mercaptosuccinate 62 (2.1 g, 6.52 mmol) and dipyridyl disulfide (2.86 g, 13 mmol) in 50 mL dichloromethane was purged with nitrogen three times, and then the mixture was stirred at room temperature overnight. After concentration, the crude was purified by column chromatography eluted with 0-80% ethyl acetate in hexanes to give the desired product (2.15 g, 77%).
  • Step 3 Synthesis of Bis(5-((2-hexyldecanoyl)oxy)pentyl) 2-(pyridin-2-yldisulfaneyl)succinate (64)
  • a solution of 2-hexyldecanoic acid (1.54 g, 6 mmol) in 20 mL dichloromethane was cooled to 0°C, then oxalyl chloride (1.1 mL, 12 mmol) and 5 drops of DMF were added, and the mixture was stirred at room temperature for 2 h.
  • Step 4 Synthesis of Bis(5-((2-hexyldecanoyl)oxy)pentyl) 2-((3- (dimethylamino)propyl)disulfaneyl)succinate
  • Example 89 A solution of bis(5-((2-hexyldecanoyl)oxy)pentyl) 2-(pyridin-2- yldisulfaneyl)succinate 64 (350 mg, 0.38 mmol) and 3-(dimethylamino)propane-1-thiol hydrochloride (156 mg, 1 mmol) in a mixture of 2 mL methanol and 10 mL dichloromethane was purged with nitrogen three times, and then stirred at room temperature for 5 h.
  • Example 90 Bis(6-(nonyloxy)-6-oxohexyl) 2-(((2- (dimethylamino)ethoxy)carbonyl)oxy)pentanedioate
  • Step 1 Synthesis of 6-(Benzyloxy)hexanoic acid (65)
  • 6-(benzyloxy)hexan-1-ol 9.7 g, 46.5 mmol
  • 60 mL acetone was added Jones reagent until the orange color persisted, and the resulting mixture was stirred for 90 min.
  • Step 2 Synthesis of Nonyl 6-(benzyloxy)hexanoate (66)
  • 6-(benzyloxy)hexanoic acid 65 (9.0 g, 40.5 mmol), nonan-1-ol (7.3 g, 42 mmol), EDCI-HCl (9.6 g, 50 mmol) and DMAP (1.22 g, 10 mmol) in 200 mL dichloromethane was stirred at room temperature for 16 h. After concentration, the crude was partitioned between EtOAc and saturated ammonium chloride solution, and the combined organic layer was dried over sodium sulfate.
  • Step 3 Synthesis of Nonyl 6-hydroxyhexanoate (67) A mixture of nonyl 6-(benzyloxy)hexanoate 66 (11.2 g, 32.2 mmol) and 5% palladium on carbon (1 g) in 100 mL ethyl acetate was subjected to hydrogenolysis for 15 h.
  • Step 4 Synthesis of Bis(6-(nonyloxy)-6-oxohexyl) 2-oxopentanedioate (68) A mixture of ⁇ -ketoglutaric acid (514 mg, 3.5 mmol), nonyl 6-hydroxyhexanoate 67 (2.0 g, 7.74 mmol) and p-toluenesulfonic acid monohydrate (100 mg) in 80 mL toluene was heated to reflux for 30 min.
  • Step 5 Synthesis of Bis(6-(nonyloxy)-6-oxohexyl) 2-hydroxypentanedioate (69) To a solution of bis(6-(nonyloxy)-6-oxohexyl) 2-oxopentanedioate 68 (600 mg, 0.95 mmol) in 20 mL THF, was added sodium borohydride (53 mg, 1.4 mmol), and the reaction mixture was stirred at room temperature for 2 h.
  • Step 6 Synthesis of Bis(6-(nonyloxy)-6-oxohexyl) 2-(((2- (dimethylamino)ethoxy)carbonyl)oxy)pentanedioate (Example 90)
  • p-nitrophenyl chloroformate 382 mg, 1.9 mmol
  • Example 91 Bis(6-(((Z)-non-2-en-1-yl)oxy)-6-oxohexyl) 2-(((2- (dimethylamino)ethoxy)carbonyl)oxy)pentanedioate
  • Step 1 Synthesis of 6-((tert-Butyldiphenylsilyl)oxy)hexan-1-ol (70)
  • reaction mixture was partitioned with ethyl acetate and saturated ammonium chloride solution, and the combined organic layer was dried over sodium sulfate. After concentration, the crude was purified by column chromatography using 0-25% ethyl acetate in hexane to give the desired product (13 g, 48%).
  • Step 2 Synthesis of 6-((tert-Butyldiphenylsilyl)oxy)hexanoic acid (71)
  • 6-((tert-butyldiphenylsilyl)oxy)hexan-1-ol 70 9.5 g, 26.6 mmol
  • Jones reagent was consumed by adding few drops of 2-propanol, then the blue solution was diluted with water (100 mL) and extracted with ethyl acetate (3 x 100 mL).
  • Step 3 Synthesis of (Z)-Non-2-en-1-yl 6-((tert-butyldiphenylsilyl)oxy)hexanoate (72) A mixture of 6-((tert-butyldiphenylsilyl)oxy)hexanoic acid 71 (8.9 g, 24 mmol), (Z)- non-2-en-1-ol (3.0 g, 21.6 mmol), EDCI-HCl (8.3 g, 43.2 mmol) and DMAP (658 mg, 5.4 mmol) in 150 mL dichloromethane was stirred at room temperature for 16 h.
  • the crude was triturated with hexanes three times, and the solvent was removed under vacuum, then the crude was purified by column chromatography using 0-10% ethyl acetate in hexane to give the desired product (10.2 g, 99%).
  • Step 4 Synthesis of (Z)-Non-2-en-1-yl 6-hydroxyhexanoate (73) To a solution of (Z)-non-2-en-1-yl 6-((tert-butyldiphenylsilyl)oxy)hexanoate 72 (10.1 g, 20.4 mmol) in 80 mL THF, was added pyridine (8 mL) followed by HF-pyridine complex (70%, 5 mL), and the mixture was stirred at room temperature for 20 h. The reaction was quenched by adding saturated sodium bicarbonate to pH 7, and then extracted with ethyl acetate (3 x 60 mL).
  • Step 5 Synthesis of Bis(6-(((Z)-non-2-en-1-yl)oxy)-6-oxohexyl) 2-oxopentanedioate (74)
  • Step 6 Synthesis of Bis(6-(((Z)-non-2-en-1-yl)oxy)-6-oxohexyl) 2-hydroxypentanedioate (75)
  • Step 7 Synthesis of Bis(6-(((Z)-non-2-en-1-yl)oxy)-6-oxohexyl) 2-(((2- (dimethylamino)ethoxy)carbonyl)oxy)pentanedioate (Example 91)
  • p- nitrophenyl chloroformate 322 mg, 1.6 mmol
  • Example 92 Bis(6-(decanoyloxy)hexyl) 2-(((2- (dimethylamino)ethoxy)carbonyl)oxy)pentanedioate
  • Step 1 Synthesis of Bis(6-(decanoyloxy)hexyl) 2-oxopentanedioate (76)
  • ⁇ -ketoglutaric acid 536 mg, 3.7 mmol
  • 6-hydroxyhexyl decanoate 2.0 g, 7.3 mmol
  • p-toluenesulfonic acid monohydrate 10 mg
  • Step 2 Synthesis of Bis(6-(decanoyloxy)hexyl) 2-hydroxypentanedioate (77)
  • Step 3 Synthesis of Bis(6-(decanoyloxy)hexyl) 2-(((2- (dimethylamino)ethoxy)carbonyl)oxy)pentanedioate (Example 92)
  • p-nitrophenyl chloroformate 367 mg, 1.83 mmol
  • Example 93 Bis(8-oxo-8-((2-propylnonyl)oxy)octyl) 2-(((2- (dimethylamino)ethoxy)carbonyl)oxy)-pentanedioate
  • Step 1 Synthesis of 8-(Benzyloxy)octanoic acid (78) To a solution of 8-(benzyloxy)octan-1-ol (4.8 g, 20.3 mmol) in 60 mL acetone, was added Jones reagent until the orange color persisted, and the resulting mixture was stirred for 90 min.
  • Step 2 Synthesis of 2-Propyloctyl 8-(benzyloxy)octanoate (79)
  • Step 3 Synthesis of 2-Propyloctyl 8-hydroxyoctanoate (80) A mixture of 2-propyloctyl 8-(benzyloxy)octanoate 79 (4.3 g, 10.3 mmol) and 20% palladium hydroxide on carbon (0.2 g) in 60 mL ethyl acetate was subjected to hydrogenolysis for 15 h.
  • Step 4 Synthesis of Bis(8-oxo-8-((2-propylnonyl)oxy)octyl) 2-oxopentanedioate (81)
  • Step 5 Synthesis of Bis(8-oxo-8-((2-propylnonyl)oxy)octyl) 2-hydroxypentanedioate (82) To a solution of bis(8-oxo-8-((2-propylnonyl)oxy)octyl) 2-oxopentanedioate 81 (800 mg, 1.04 mmol) in 20 mL THF, was added sodium borohydride (63.5 mg, 1.67 mmol), and the reaction mixture was stirred at room temperature for 2 h.
  • Step 6 Synthesis of Bis(8-oxo-8-((2-propylnonyl)oxy)octyl) 2-(((2- (dimethylamino)ethoxy)carbonyl)oxy)-pentanedioate (Example 93)
  • p-nitrophenyl chloroformate 366 mg, 1.82 mmol
  • Example 94 Bis(8-oxo-8-(pentadecan-7-yloxy)octyl) 2-(((2- (dimethylamino)ethoxy)carbonyl)oxy)pentanedioate
  • Step 1 Synthesis of Pentadecan-7-yl 8-(benzyloxy)octanoate (83)
  • pentadecan-7-ol 16 (1.8 g, 8 mmol)
  • EDCI-HCl 1.9 g, 10 mmol
  • DMAP 1.2 g, 14 mmol
  • Step 2 Synthesis of Pentadecan-7-yl 8-hydroxyoctanoate (84) A mixture of pentadecan-7-yl 8-(benzyloxy)octanoate 83 (2.3 g, 5 mmol) and 5% palladium on carbon (0.6 g) in 50 mL ethyl acetate was subjected to hydrogenolysis for 15 h.
  • Step 3 Synthesis of bis(8-oxo-8-(pentadecan-7-yloxy)octyl) 2-oxopentanedioate (85)
  • Step 4 Synthesis of Bis(8-oxo-8-(pentadecan-7-yloxy)octyl) 2-hydroxypentanedioate (86) To a solution of bis(8-oxo-8-(pentadecan-7-yloxy)octyl) 2-oxopentanedioate 85 (1.2 g, 1.4 mmol) in 40 mL THF, was added sodium borohydride (80 mg, 2.1 mmol), and the reaction mixture was stirred at room temperature for 2 h.
  • Step 5 Synthesis of Bis(8-oxo-8-(pentadecan-7-yloxy)octyl) 2-(((2- (dimethylamino)ethoxy)carbonyl)oxy)pentanedioate (Example 94)
  • a solution of bis(8-oxo-8-(pentadecan-7-yloxy)octyl) 2-hydroxypentanedioate 86 800 mg, 0.9 mmol
  • p-nitrophenyl chloroformate 377 mg, 1.87 mmol
  • Example 95 Bis(6-oxo-6-(pentadecan-7-yloxy)hexyl) 2-((3- (dimethylamino)propanoyl)oxy)pentanedioate
  • Step 1 Synthesis of Pentadecan-7-yl 6-(benzyloxy)hexanoate (87) A mixture of 6-(benzyloxy)hexanoic acid 65 (8.4 g, 37.8 mmol), pentadecan-7-ol 16 (12.9 g, 56.7 mmol), EDCI-HCl (10.88 g, 56.7 mmol) and DMAP (1.22 g, 10 mmol) in 200 mL dichloromethane was stirred at room temperature for 16 h.
  • Step 2 Synthesis of Pentadecan-7-yl 6-hydroxyhexanoate (88) A mixture of pentadecan-7-yl 6-(benzyloxy)hexanoate 87 (12.5 g, 36.5 mmol) and 5% palladium on carbon (1.2 g) in 100 mL ethyl acetate was subjected to hydrogenolysis for 15 h.
  • Step 3 Synthesis of Bis(6-oxo-6-(pentadecan-7-yloxy)hexyl) 2-oxopentanedioate (89)
  • Step 4 Synthesis of Bis(6-oxo-6-(pentadecan-7-yloxy)hexyl) 2-hydroxypentanedioate (90)
  • Step 5 Synthesis of Bis(6-oxo-6-(pentadecan-7-yloxy)hexyl) 2-((3- (dimethylamino)propanoyl)oxy)pentanedioate (Example 95)
  • a mixture of bis(6-oxo-6-(pentadecan-7-yloxy)hexyl) 2-hydroxypentanedioate 90 (170 mg, 0.21 mmol), 3-(dimethylamino)propanoic acid hydrochloride (65 mg, 0.42 mmol), EDCI (82 mg, 0.42 mmol) and 4-dimethylaminopyridine (26 mg, 0.21 mmol) in 10 mL dichloromethane was stirred at room temperature overnight.
  • the reaction mixture was diluted with dichloromethane and washed with water. The combined organic layer was dried over sodium sulfate. After filtration and concentration, the crude was purified by column chromatography with 0-10% methanol in dichloromethane to afford the desired product (170 mg, 90%).
  • Example 96 Bis(6-oxo-6-(pentadecan-7-yloxy)hexyl) 2-((4- (dimethylamino)butanoyl)oxy)pentanedioate
  • reaction mixture was diluted with dichloromethane and washed with water. The combined organic layer was dried over sodium sulfate. After filtration and concentration, the crude was purified by column chromatography with 0-10% methanol in dichloromethane to afford the desired product (239 mg, 71%).
  • Example 97 Bis(5-((2-hexyldecanoyl)oxy)pentyl) 2-(((2- (dimethylamino)ethoxy)carbonyl)oxy)pentanedioate
  • Step 1 Synthesis of Bis(5-((2-hexyldecanoyl)oxy)pentyl) 2-oxopentanedioate (91)
  • 5-hydroxypentyl 2- hexyldecanoate 32 3.6 g, 10.5 mmol
  • p-toluenesulfonic acid monohydrate 100 mg
  • Step 2 Synthesis of Bis(5-((2-hexyldecanoyl)oxy)pentyl) 2-hydroxypentanedioate (92) To a solution of bis(5-((2-hexyldecanoyl)oxy)pentyl) 2-oxopentanedioate 91 (3.1 g, 3.9 mmol) in 60 mL THF, was added sodium borohydride (222 mg, 5.8 mmol), and the reaction mixture was stirred at room temperature for 2 h.
  • Step 3 Synthesis of Bis(5-((2-hexyldecanoyl)oxy)pentyl) 2-(((2- (dimethylamino)ethoxy)carbonyl)oxy)pentanedioate (Example 97)
  • a solution of bis(5-((2-hexyldecanoyl)oxy)pentyl) 2-hydroxypentanedioate 92 (1.5 g, 1.9 mmol) in 5 mL pyridine and 10 mL dichloromethane was added p-nitrophenyl chloroformate (1.13 g, 5.6 mmol), and the resulting mixture was stirred for 2 h.
  • Example 98 Bis(7-((8-methylnonanoyl)oxy)heptyl) 2-(((2- (dimethylamino)ethoxy)carbonyl)oxy)pentanedioate
  • Step 1 Synthesis of Bis(7-((8-methylnonanoyl)oxy)heptyl) 2-oxopentanedioate (93)
  • a mixture of ⁇ -ketoglutaric acid (607 mg, 4.15 mmol), 7-hydroxyheptyl 8- methylnonanoate 49 (2.5 g, 8.73 mmol) and p-toluenesulfonic acid monohydrate (10 mg) in 100 mL toluene was heated to reflux for 2 h.
  • Step 2 Synthesis of Bis(7-((8-methylnonanoyl)oxy)heptyl) 2-hydroxypentanedioate (94) To a solution of bis(7-((8-methylnonanoyl)oxy)heptyl) 2-oxopentanedioate 93 (2.3 g, 3.3 mmol) in 60 mL THF, was added sodium borohydride (188 mg, 4.9 mmol), and the reaction mixture was stirred at room temperature for 2 h.
  • Step 3 Synthesis of Bis(7-((8-methylnonanoyl)oxy)heptyl) 2-(((2- (dimethylamino)ethoxy)carbonyl)oxy)pentanedioate (Example 98)
  • a solution of bis(7-((8-methylnonanoyl)oxy)heptyl) 2-hydroxypentanedioate 94 500 mg, 0.73 mmol
  • p-nitrophenyl chloroformate 293 mg, 1.46 mmol
  • Example 99 Bis(8-(((Z)-non-2-en-1-yl)oxy)-8-oxooctyl) 2-(((2- (dimethylamino)ethoxy)carbonyl)oxy)pentanedioate
  • Step 1 Synthesis of 8-((tert-Butyldiphenylsilyl)oxy)octan-1-ol (95)
  • reaction mixture was partitioned with ethyl acetate and saturated ammonium chloride solution, and the combined organic layer was dried over sodium sulfate. After concentration, the crude was purified by column chromatography using 0-25% ethyl acetate in hexane to give the desired product (10.0 g, 52%).
  • Step 2 Synthesis of 8-((tert-Butyldiphenylsilyl)oxy)octanoic acid (96) To a solution of 8-((tert-butyldiphenylsilyl)oxy)octan-1-ol 95 (5 g, 13 mmol) in 100 mL acetone, was added Jones reagent until the orange color persisted, and the resulting mixture was stirred for 30 min. The excess Jones reagent was consumed by adding few drops of 2-propanol, then the blue solution was diluted with water (100 mL) and extracted with ethyl acetate (3 x 100 mL).
  • Step 3 Synthesis of (Z)-Non-2-en-1-yl 8-((tert-butyldiphenylsilyl)oxy)octanoate (97) A mixture of 8-((tert-butyldiphenylsilyl)oxy)octanoic acid 96 (5 g, 12.6 mmol), (Z)- non-2-en-1-ol (1.69 g, 11.3 mmol), EDCI-HCl (3.62 g, 18.9 mmol) and DMAP (307 mg, 2.5 mmol) in 100 mL dichloromethane was stirred at room temperature for 16 h.
  • Step 4 Synthesis of (Z)-Non-2-en-1-yl 8-hydroxyoctanoate (98)
  • pyridine 5 mL
  • HF-pyridine complex 70%, 3 mL
  • the reaction was quenched by adding saturated sodium bicarbonate to pH 7, and then extracted with ethyl acetate (3 x 60 mL).
  • Step 5 Synthesis of Bis(8-(((Z)-non-2-en-1-yl)oxy)-8-oxooctyl) 2-oxopentanedioate (99)
  • Example 104 Bis(7-((2-hexyldecanoyl)oxy)heptyl) 2-(((2- (dimethylamino)ethoxy)carbonyl)oxy)pentanedioate
  • Step 1 Synthesis of Bis(7-((2-hexyldecanoyl)oxy)heptyl) 2-oxopentanedioate (103)
  • Step 2 Synthesis of Bis(7-((2-hexyldecanoyl)oxy)heptyl) 2-hydroxypentanedioate (104) To a solution of bis(7-((2-hexyldecanoyl)oxy)heptyl) 2-oxopentanedioate 103 (1.8 g, 2.1 mmol) in 20 mL THF, was added sodium borohydride (120 mg, 3.2 mmol), and the reaction mixture was stirred at room temperature for 2 h.
  • Step 3 Synthesis of Bis(7-((2-hexyldecanoyl)oxy)heptyl) 2-(((2- (dimethylamino)ethoxy)carbonyl)oxy)pentanedioate (Example 104)
  • p-nitrophenyl chloroformate 422 mg, 2.1 mmol
  • Example 105 bis(8-oxo-8-(pentadecan-7-yloxy)octyl) 2-(((2-((2- hydroxyethyl)(methyl)amino)ethoxy)carbonyl)oxy)pentanedioate
  • Step 1 Synthesis of 8-(Benzyloxy)octan-1-ol (105)
  • NaH 60% in mineral oil, 12 g, 0.3 mole
  • Step 2 Synthesis of Bis(8-(benzyloxy)octyl) 2-oxopentanedioate (106)
  • Step 3 Synthesis of Bis(8-(benzyloxy)octyl) 2-hydroxypentanedioate (107)
  • a solution of bis(8-(benzyloxy)octyl) 2-oxopentanedioate 106 (10.8 g, 18.5 mmol) in 30 mL THF was cooled to 0°C, then sodium borohydride (701 mg, 18.5 mmol) was added in portions in 5 min, and the reaction mixture was stirred at room temperature for 1 h. Saturated ammonium chloride solution was added slowly, and the resulting mixture was extracted with ethyl acetate. The combined organic layer was washed with brine and dried over sodium sulfate.
  • Step 4 Synthesis of Bis(8-(benzyloxy)octyl) 2-((tert-butyldiphenylsilyl)oxy)pentanedioate (108)
  • tert-butylchlorodiphenylsilane 5.08 g, 18.5 mmol
  • reaction mixture was quenched by saturated sodium bicarbonate solution and extracted with dichloromethane.
  • the combined organic layer was dried and concentrated, and the crude was purified by column chromatography using 0-20% ethyl acetate in hexanes to give bis(8-(benzyloxy)octyl) 2-((tert- butyldiphenylsilyl)oxy)pentanedioate as colorless oil (9.8 g, 64%).
  • Step 5 Synthesis of Bis(8-hydroxyoctyl) 2-((tert-butyldiphenylsilyl)oxy)pentanedioate (109)
  • Step 6 Synthesis of 8,8'-((2-((tert-Butyldiphenylsilyl)oxy)pentanedioyl)bis(oxy))dioctanoic acid (110)
  • the excess Jones reagent was consumed by adding 2-propanol, then the blue solution was diluted with water and extracted with ethyl acetate.
  • Step 7 Synthesis of Bis(8-oxo-8-(pentadecan-7-yloxy)octyl) 2-((tert- butyldiphenylsilyl)oxy)pentanedioate (111)
  • a mixture of 8,8'-((2-((tert-butyldiphenylsilyl)oxy)pentanedioyl)bis(oxy))dioctanoic acid 110 (4.2 g, 6.3 mmol)
  • pentadecan-7-ol 16 2.9 g, 13 mmol
  • EDCI 6.0 g, 31 mmol
  • DMAP 0.76 g, 6.3 mmol
  • Step 8 Synthesis of Bis(8-oxo-8-(pentadecan-7-yloxy)octyl) 2-hydroxypentanedioate (112)
  • a solution of bis(8-oxo-8-(pentadecan-7-yloxy)octyl) 2-((tert- butyldiphenylsilyl)oxy)pentanedioate 111 (4.2 g, 3.8 mmol) in a mixture of THF (15 mL) and pyridine (6.0 mL) in a Teflon flask, was added HF-pyridine complex (70wt%, 2.5 mL) at 0°C, and the resulting mixture was stirred at room temperature for 18 h.
  • Step 9 Synthesis of Bis(8-oxo-8-(pentadecan-7-yloxy)octyl) 2-(((2-((2- hydroxyethyl)(methyl)amino)ethoxy)carbonyl)oxy)pentanedioate (Example 105)
  • p-nitrophenyl chloroformate 157 mg, 0.78 mmol
  • Step 2 Synthesis of bis(8-oxo-8-(pentadecan-7-yloxy)octyl) 2-((5,10,10,11,11-pentamethyl- 2,9-dioxa-5-aza-10-siladodecanoyl)oxy)pentanedioate (114)
  • a solution of bis(8-oxo-8-(pentadecan-7-yloxy)octyl) 2-hydroxypentanedioate 112 (280 mg, 0.33 mmol) in 2 mL pyridine and 8 mL dichloromethane, was added p-nitrophenyl chloroformate (198 mg, 0.98 mmol), and the resulting mixture was stirred for 30 min.
  • Step 3 Synthesis of bis(8-oxo-8-(pentadecan-7-yloxy)octyl) 2-(((2-((3- hydroxypropyl)(methyl)amino)ethoxy)carbonyl)oxy)pentanedioate (Example 106)
  • Step 2 Synthesis of bis(8-oxo-8-(pentadecan-7-yloxy)octyl) 2-(((2-((4- (benzyloxy)butyl)(methyl)amino)ethoxy)carbonyl)oxy)pentanedioate (116)
  • p-nitrophenyl chloroformate 77 mg, 0.38 mmol
  • Step 3 Synthesis of bis(8-oxo-8-(pentadecan-7-yloxy)octyl) 2-(((2-((4- hydroxybutyl)(methyl)amino)ethoxy)carbonyl)oxy)pentanedioate (Example 107)
  • reaction mixture was filtered through a pad of silica gel and eluted with 10% methanol in ethyl acetate to give bis(8-oxo-8-(pentadecan-7-yloxy)octyl) 2-(((2-((4- hydroxybutyl)-(methyl)amino)ethoxy)-carbonyl)oxy)pentanedioate as pale yellow oil (86 mg, 80%).
  • Step 2 Synthesis of Bis(8-oxo-8-(pentadecan-7-ylthio)octyl) 2-((tert- butyldiphenylsilyl)oxy)pentanedioate (118)
  • a mixture of 8,8'-((2-((tert-butyldiphenylsilyl)oxy)pentanedioyl)bis(oxy))dioctanoic acid 68 mg, 0.10 mmol
  • pentadecane-7-thiol 117 100 mg, 0.41mmol
  • EDCI (0.19 g, 1.0 mmol
  • DMAP (12 mg, 0.10 mmol) in 8.0 mL dichloromethane was stirred at room temperature overnight.
  • Step 3 Synthesis of Bis(8-oxo-8-(pentadecan-7-ylthio)octyl) 2-hydroxypentanedioate (119)
  • a Teflon flask To a Teflon flask, to a solution of bis(8-oxo-8-(pentadecan-7-ylthio)octyl) 2-((tert- butyldiphenylsilyl)oxy)pentanedioate 118 (70 mg, 0.06 mmol) in 8 mL THF and 1 mL pyridine, was added HF-pyridine complex (70wt%, 0.3 mL) slowly, and the mixture was stirred at room temperature overnight.
  • HF-pyridine complex 70wt%, 0.3 mL
  • Step 4 Synthesis of Bis(8-oxo-8-(pentadecan-7-ylthio)octyl) 2-(((2- (dimethylamino)ethoxy)carbonyl)oxy)pentanedioate (Example 120)
  • To a solution of bis(8-oxo-8-(pentadecan-7-ylthio)octyl) 2-hydroxypentanedioate 119 (55 mg, 0.062 mmol) in 6.0 mL dichloromethane and 0.5 mL pyridine (0.5 ml) was added 4- nitrophenyl carbonochloridate (63 mg, 0.31 mmol), and the mixture was stirred for 90 min.
  • Step 2 Synthesis of Bis(6-(benzyloxy)hexyl) 2-hydroxypentanedioate (121)
  • a solution of bis(6-(benzyloxy)hexyl) 2-oxopentanedioate 120 (3.0 g, 5.7 mmol) in 20 mL THF was cooled to 0°C, then sodium borohydride (200 mg, 5 mmol) was added by portions in 10 min, and the reaction mixture was stirred at room temperature for 2 h. Saturated ammonium chloride solution was added slowly, and the resulting mixture was extracted with ethyl acetate. The combined organic layer was washed with brine and dried over sodium sulfate.
  • Step 3 Synthesis of Bis(6-(benzyloxy)hexyl) 2-((tert-butyldiphenylsilyl)oxy)pentanedioate (122)
  • tert-butylchlorodiphenylsilane 2.04 g, 7.4 mmol
  • reaction mixture was quenched by saturated sodium bicarbonate solution and extracted with dichloromethane.
  • the combined organic layer was dried and concentrated, and the crude was purified by column chromatography using 0-20% ethyl acetate in hexanes to give bis(6-(benzyloxy)hexyl) 2-((tert- butyldiphenylsilyl)oxy)pentanedioate as colorless oil (3.1 g, 70%).
  • Step 4 Synthesis of Bis(6-hydroxyhexyl) 2-((tert-butyldiphenylsilyl)oxy)pentanedioate (123)
  • Step 5 Synthesis of Bis(6-((2-hexyldecanoyl)oxy)hexyl) 2-((tert- butyldiphenylsilyl)oxy)pentanedioate (124)
  • Step 6 Synthesis of Bis(6-((2-hexyldecanoyl)oxy)hexyl) 2-hydroxypentanedioate (125)
  • a solution of bis(6-((2-hexyldecanoyl)oxy)hexyl) 2-((tert- butyldiphenylsilyl)oxy)pentanedioate 124 (2.6 g, 2.4 mmol) in a mixture of THF (20 mL) and Pyridine (6.0 mL) in a Teflon flask was added HF-pyridine complex (70wt%, 3 mL) at 0°C, and the resulting mixture was stirred at room temperature for 18 h.
  • Step 7 Synthesis of bis(6-((2-hexyldecanoyl)oxy)hexyl) 2-(((2- (dimethylamino)ethoxy)carbonyl)oxy)pentanedioate (Example 121)
  • p-nitrophenyl chloroformate 629 mg, 3.12 mmol
  • Step 2 Synthesis of Dibenzyl 2-hydroxypentanedioate (134)
  • a solution of dibenzyl 2-oxopentanedioate 133 (1.8 g, 5.5 mmol) in 25 mL THF was cooled to 0°C, then sodium borohydride (210 mg, 5.5 mmol) was added by portions in 5 min, and the reaction mixture was stirred at same temperature for 1 h. Saturated ammonium chloride solution was added slowly, and the resulting mixture was extracted with ethyl acetate. The combined organic layer was washed with brine and dried over sodium sulfate. After concentration, 1.7 g crude was used for the next step without purification.
  • Step 4 Synthesis of 5-(Benzyloxy)-4-((tert-butyldiphenylsilyl)oxy)-5-oxopentanoic acid (136)
  • Step 5 Synthesis of 1-Benzyl 5-(4-((2-hexyldecanoyl)oxy)butyl) 2-((tert- butyldiphenylsilyl)oxy)pentanedioate (137)
  • a mixture of 5-(benzyloxy)-4-((tert-butyldiphenylsilyl)oxy)-5-oxopentanoic acid 136 (contains diacid impurity, 105 mg, 0.22 mmol), 4-hydroxybutyl 2-hexyldecanoate (109 mg, 0.33 mmol), DMAP (40 mg, 0.33 mmol) and EDCI (169 mg, 0.88 mmol) in 8 mL dichloromethane was stirred at room temperature overnight.
  • Step 6 Synthesis of 2-((tert-butyldiphenylsilyl)oxy)-5-(4-((2-hexyldecanoyl)oxy)butoxy)-5- oxopentanoic acid (138)
  • Step 7 Synthesis of Bis(4-((2-hexyldecanoyl)oxy)butyl) 2-((tert- butyldiphenylsilyl)oxy)pentanedioate (139)
  • a mixture of 2-((tert-butyldiphenylsilyl)oxy)-5-(4-((2-hexyldecanoyl)oxy)butoxy)-5- oxopentanoic acid 8 75 mg, 0.11 mmol
  • 4-hydroxybutyl 2-hexyldecanoate 138 35 mg, 0.11 mmol
  • DMAP 13 mg, 0.11 mmol
  • EDCI 83 mg, 0.43 mmol
  • Step 8 Synthesis of Bis(4-((2-hexyldecanoyl)oxy)butyl) 2-hydroxypentanedioate (140)
  • a solution of bis(4-((2-hexyldecanoyl)oxy)butyl) 2-((tert- butyldiphenylsilyl)oxy)pentanedioate 139 80 mg, 0.079 mmol
  • a mixture of THF (8 mL) and pyridine (1 mL) in a Teflon flask was added HF-pyridine solution (70wt%, 0.3 mL) at 0°C, and the resulting mixture was stirred at room temperature for 18 h.
  • Step 9 Synthesis of bis(4-((2-hexyldecanoyl)oxy)butyl) 2-(((2- (dimethylamino)ethoxy)carbonyl)oxy)pentanedioate (Example 124)
  • p-nitrophenyl chloroformate 59 mg, 0.29 mmol
  • Step 2 Synthesis of (((8,8-dimethoxyoctyl)oxy)methyl)benzene (142)
  • a mixture of 8-(benzyloxy)octanal 141 (3.1 g, 13 mmol), 4-methylbenzenesulfonic acid hydrate (2.5 g, 13 mmol) and trimethyl orthoformate (11 g, 0.11 mole) was stirred at room temperature for 2 h.
  • reaction mixture was diluted with dichloromethane and washed with saturated sodium bicarbonate solution and brine. After dried and concentrated, the residue was azeotropic evaporated with toluene to get (((8,8- dimethoxyoctyl)oxy)methyl)benzene as colorless oil (3.65 g, 98%), which was used for the next step without any further purification.
  • Step 3 Synthesis of (((8,8-Bis(octyloxy)octyl)oxy)methyl)benzene (143)
  • a mixture of (((8,8-dimethoxyoctyl)oxy)methyl)benzene 142 (2.8 g, 10 mmol), 1- octanol (2.6 g, 20 mmol) and pyridine 4-methylbenzenesulfonate (0.25 g, 1.0 mmol) was heated at 120°C for 4 h. After cooled to room temperature, the reaction mixture was diluted with hexanes and washed with saturated sodium bicarbonate.
  • Step 4 Synthesis of 8,8-bis(octyloxy)octan-1-ol (144) A mixture of (((8,8-bis(octyloxy)octyl)oxy)methyl)benzene 143 (800 mg, 1.68 mmol) and 20% palladium hydroxide on carbon (47 mg) in 15 mL hexane was subjected to hydrogenation at room temperature overnight.
  • reaction mixture was carefully quenched by saturated sodium bicarbonate and extracted with ethyl acetate.
  • the combined organic layer was dried and concentrated, and the crude was purified by column chromatography using 0-25% ethyl acetate in hexanes to 8,8-bis(octyloxy)octan-1-ol as colorless oil (280 mg, 90%).
  • Step 5 Synthesis of 2-((tert-Butyldiphenylsilyl)oxy)pentanedioic acid (145)
  • Step 6 Synthesis of Bis(8,8-bis(octyloxy)octyl) 2-((tert-butyldiphenylsilyl)oxy)pentanedioate (146)
  • Step 7 Synthesis of Bis(8,8-bis(octyloxy)octyl) 2-hydroxypentanedioate (147)
  • a solution of bis(8,8-bis(octyloxy)octyl) 2-((tert- butyldiphenylsilyl)oxy)pentanedioate 146 150 mg, 0.133 mmol
  • a mixture of THF (8 mL) and pyridine (3 mL) in a Teflon flask was added HF-pyridine solution (70wt%, 0.2 mL) at 0°C, and the resulting mixture was stirred at room temperature overnight.
  • Step 8 Synthesis of bis(8,8-bis(octyloxy)octyl) 2-(((2- (dimethylamino)ethoxy)carbonyl)oxy)pentanedioate (Example 125)
  • p-nitrophenyl chloroformate 68 mg, 0.34 mmol
  • Step 2 Synthesis of N,N-Dimethyl-4-(tritylthio)butan-1-amine (149)
  • a seal tube To a mixture of (4-bromobutyl)(trityl)sulfane 148 (3.0 g, 7.3 mmol) in 20 mL acetonitrile, was added a solution of dimethylamine in THF (2M, 11 mL, 22 mmol), and the mixture was heated at 80°C overnight.
  • Step 3 Synthesis of 4-(Dimethylamino)butane-1-thiol trifluoroacetate (150) To a solution of N,N-dimethyl-4-(tritylthio)butan-1-amine 149 (1.1 g, 2.9 mmol) and triethylsilane (1.4 mL, 8.8 mmol) in 5 mL dichloromethane, wad added trifluoroacetic acid (3.4 mL, 44 mmol), and the mixture was stirred at room temperature for 4 h.
  • Step 4 Synthesis of Dimethyl 2-(acetylthio)pentanedioate (151) To a solution of potassium ethanethioate (2.5 g, 22 mmol) in 10 mL acetonitrile and 20 mL DMF, was added dimethyl 2-bromopentanedioate (4.3 g, 18 mmol) dropwise, and the resulting mixture was allowed to stir for 2 h.
  • Step 5 Synthesis of 2-Mercaptopentanedioic acid (152) To a solution of dimethyl 2-(acetylthio)pentanedioate 151 (950 mg, 4.06 mmol) in 20 mL THF, was added 10 mL 2 M sodium hydroxide solution, and the mixture was stirred at room temperature overnight.
  • reaction mixture was partitioned with water and ethyl acetate, and the aqueous layer was acidified using 6M HCl solution to pH 2. After extracted with ethyl acetate, the combined organic layer was dried and concentrated to give 2- mercaptopentanedioic acid as pale yellow solid (600 mg, 90%), which was used for the next step without purification.
  • Step 6 Synthesis of Bis(5-hydroxypentyl) 2-mercaptopentanedioate (153)
  • a mixture of 2-mercaptopentanedioic acid 152 (450 mg, 2.74 mmol), pentane-1,5-diol (4.28 g, 41.1 mmol) and zinc chloride (93.4 mg, 0.685 mmol) was heated under nitrogen atomsphere at 130°C for 4 h. After cooled to room temperature, the mixture was diluted with water and extracted with dichloromethane, the combined organic layer was dried and concentrated to give bis(5-hydroxypentyl) 2-mercaptopentanedioate as colorless oil (750 mg, 73%), which was used for the next step without further purification.
  • Step 7 Synthesis of Bis(5-hydroxypentyl) 2-(pyridin-2-yldisulfaneyl)pentanedioate (154)
  • the mixture was purified by column purification using 0-5% methanol in ethyl acetate to give bis(5-hydroxypentyl) 2-(pyridin-2- yldisulfaneyl)pentanedioate as pale yellow oil (600 mg, 67%).
  • Step 8 Synthesis of Bis(5-((2-hexyldecanoyl)oxy)pentyl) 2-(pyridin-2- yldisulfaneyl)pentanedioate (155)
  • 2-hexyldecanoic acid (1.53 g, 5.97 mmol) in 10 mL dichloromethane
  • oxalyl chloride (1.57 mL, 17.9 mmol) at 0°C followed by few drops of DMF, and the mixture was warmed up to room temperature for 90 min. After concentration, the residue was azeotropic evaporated with toluene several times.
  • the crude 2-hexyldecanoyl chloride was dissolved in 4 mL pyridine and cooled to 0°C, then a solution of bis(5-hydroxypentyl) 2- (pyridin-2-yldisulfaneyl)pentanedioate 154 (0.62 g, 1.4 mmol) in 4 mL dichloromethane was slowly added, and the resulting mixture was stirred at room temperature for 30 min. TLC showed complete reaction. The reaction mixture was quenched with saturated ammonium chloride solution and extracted with dichloromethane.
  • Step 9 Synthesis of bis(5-((2-hexyldecanoyl)oxy)pentyl) 2-((4- (dimethylamino)butyl)disulfaneyl)pentanedioate (Example 126)
  • Example 127 dioctadecyl 2-((3-(dimethylamino)propyl)disulfaneyl)succinate
  • Step 1 Synthesis of Dioctadecyl 2-mercaptosuccinate (156)
  • mercaptosuccinic acid (15.0 g, 0.1 mole)
  • 1-octadecanol 59.49 g, 0.22 mole
  • 0.25 mL concentrated sulfuric acid was added to reflux with Dean-Stark apparatus overnight. After cooled to room temperature, the reaction mixture was washed with saturated bicarbonate solution and water.
  • Step 2 Synthesis of Dioctadecyl 2-(pyridin-2-yldisulfaneyl)succinate (157) A solution of dioctadecyl 2-mercaptosuccinate 156 (10.0 g, 15.2 mmol) in 150 mL dichloromethane was purged with nitrogen, then 2,2-dipyridyl disulfide (4.53 g, 16.8 mmol) was added, and the resulting solution was stirred at room temperature overnight under nitrogen.
  • Step 3 Synthesis of Dioctadecyl 2-((3-(dimethylamino)propyl)disulfaneyl)succinate (Example 127)
  • a solution of dioctadecyl 2-(pyridin-2-yldisulfaneyl)succinate 157 (1.0 g, 1.3 mmol) in 30 mL chloroform was purged with nitrogen, then 3-(dimethylamino)propane-1-thiol hydrochloride (425 mg, 2.75 mmol) was added, and the resulting solution was stirred at room temperature for 3 h.
  • Example 128 dihexadecyl 2-((3-(dimethylamino)propyl)disulfaneyl)succinate
  • Step 1 Synthesis of Dihexadecyl 2-mercaptosuccinate (158) To a mixture of mercaptosuccinic acid (10.0 g, 66.6 mmol) and 1-hexadecanol (35.5 g, 0.146 mole) in 100 mL benzene, was added 0.25 mL concentrated sulfuric acid, and the mixture was heated to reflux with Dean-Stark apparatus overnight. After cooled to room temperature, the reaction mixture was washed with saturated bicarbonate solution and water.
  • Step 2 Synthesis of Dihexadecyl 2-(pyridin-2-yldisulfaneyl)succinate (159) A solution of dihexadecyl 2-mercaptosuccinate 158 (5.0 g, 8.3 mmol) in 150 mL dichloromethane was purged with nitrogen, then 2,2-dipyridyl disulfide (2.0 g, 9.2 mmol) was added, and the resulting solution was stirred at room temperature overnight under nitrogen.
  • Step 3 Synthesis of dihexadecyl 2-((3-(dimethylamino)propyl)disulfaneyl)succinate (Example 128)
  • a solution of dihexadecyl 2-(pyridin-2-yldisulfaneyl)succinate 159 (2.0 g, 2.8 mmol) in 60 mL chloroform was purged with nitrogen, then 3-(dimethylamino)propane-1-thiol hydrochloride (872 mg, 5.6 mmol) was added, and the resulting solution was stirred at room temperature for 3 h.
  • Example 129 di((9Z,12Z)-octadeca-9,12-dien-1-yl) 2-((3- (dimethylamino)propyl)disulfaneyl)succinate
  • Step 1 Synthesis of Di((9Z,12Z)-octadeca-9,12-dien-1-yl) 2-mercaptosuccinate (160)
  • a mixture of mercaptosuccinic acid (5.0 g, 33.3 mmol) and linoleyl alcohol (17.7 g, 66.6 mmol) in 60 mL benzene was added 0.15 mL concentrated sulfuric acid, and the mixture was heated to reflux with Dean-Stark apparatus overnight.
  • Step 2 Synthesis of Di((9Z,12Z)-octadeca-9,12-dien-1-yl) 2-(pyridin-2- yldisulfaneyl)succinate (161)
  • Step 3 Synthesis of di((9Z,12Z)-octadeca-9,12-dien-1-yl) 2-((3- (dimethylamino)propyl)disulfaneyl)succinate (Example 129)
  • Example 130 dihexadecyl 2-(N-(3-(dimethylamino)propyl)sulfamoyl)succinate
  • Step 1 Synthesis of Dihexadecyl maleate (162)
  • Step 2 Synthesis of Sodium 1,4-bis(hexadecyloxy)-1,4-dioxobutane-2-sulfonate (163)
  • aqueous sodium bisulfite solution 7.2 g in 30 mL water, 69.2 mmol
  • the residue was partitioned between water and EtOAc, and the combined organic layer was washed with brine.
  • Step 2 Synthesis of Dihexadecyl 2-(N-(3-(dimethylamino)propyl)sulfamoyl)succinate (Example 130)
  • oxalyl chloride 40 ⁇ L, 0.51 mmol
  • Step 2 Synthesis of 2-(3-((tert-Butyldimethylsilyl)oxy)propyl)propane-1,3-diol (165)
  • a solution of diethyl 2-(3-((tert-butyldimethylsilyl)oxy)propyl)malonate 164 (1.4 g, 4.2 mmol) in 20 mL ether, was slowly added a solution of lithium aluminum hydride in THF (2 M, 6.5 mL, 13 mmol), and the mixture was stirred at room temperature overnight.
  • Step 3 Synthesis of 2-((Benzyloxy)methyl)-5-((tert-butyldimethylsilyl)oxy)pentan-1-ol (166)
  • a suspension of sodium hydride (60% in mineral oil, 270 mg, 11.3 mmol) in 8.0 mL DMF was cooled to 0°C, then a solution of 2-(3-((tert- butyldimethylsilyl)oxy)propyl)propane-1,3-diol 165 (700 mg, 2.82 mmol) in 2 mL THF was slowly added, and the mixture was stirred for 15 min at this temperature.
  • Step 4 Synthesis of 2-((Benzyloxy)methyl)pentane-1,5-diol (167)
  • 2-((benzyloxy)methyl)-5-((tert-butyldimethylsilyl)oxy)pentan-1-ol 166 500 mg, 1.48 mmol
  • dichloromethane 6.0 mL dichloromethane
  • five drops of diluted hydrochloride solution in methanol was stirred at room temperature for 1 h. TLC showed complete reaction.
  • the reaction was quenched by saturated sodium bicarbonate solution and extracted with dichloromethane.
  • Step 5 Synthesis of 2-((Benzyloxy)methyl)pentanedioic acid (168) To a solution of 2-((benzyloxy)methyl)pentane-1,5-diol 167 (320 mg, 1.43 mmol) in 10 mL acetone, was added Jones reagent until orange color persisted. The mixture was stirred at room temperature for 2 h.
  • Step 7 Synthesis of Bis(8-oxo-8-(pentadecan-7-yloxy)octyl) 2-(hydroxymethyl)pentanedioate (170)
  • Step 8 Synthesis of Bis(8-oxo-8-(pentadecan-7-yloxy)octyl) 2-((((2- (dimethylamino)ethoxy)carbonyl)oxy)methyl)pentanedioate (Example 131)
  • pyridine 1.0 mL
  • 4-nitrophenyl chloroformate 74 mg, 0.37 mmol
  • Example 132 1,4-bis(hexadecyloxy)-1,4-dioxobutane-2-sulfonic 3- (dimethylamino)propanoic anhydride
  • Example 133 1,4-bis(octadecyloxy)-1,4-dioxobutane-2-sulfonic 3- (dimethylamino)propanoic anhydride
  • Example 134 3-(dimethylamino)propanoic 9,20,23,34-tetraoxo-10,19,24,33- tetraoxadotetracontane-21-sulfonic anhydride
  • Example 135 1,4-bis(((9Z,12Z)-octadeca-9,12-dien-1-yl)oxy)-1,4-dioxobutane-2-sulfonic 3- (dimethylamino)propanoic anhydr
  • Lipid nanoparticle formulation Ionizable lipids described herein can be used in the preparation of lipid nanoparticles according to methods known in the art.
  • suitable methods include methods described in International Publication No. WO 2018/089801, which is hereby incorporated by reference in its entirety.
  • the lipid nanoparticles in the examples of the present invention were formulated using Process A of WO 2018/089801 (see, e.g., Example 1 and Figure 1 of WO 2018/089801).
  • Process A (“A”) relates to a method of encapsulating mRNA by mixing mRNA with a mixture of lipids, without first pre-forming the lipids into lipid nanoparticles.
  • an ethanolic solution of a mixture of lipids (cationic lipid, phosphatidylethanolamine, cholesterol, and polyethylene glycol-lipid) at a fixed lipid to mRNA ratio were combined with an aqueous buffered solution of target mRNA at an acidic pH under controlled conditions to yield a suspension of uniform LNPs.
  • the resulting nanoparticle suspensions were diluted to final concentration, filtered, and stored frozen at ⁇ 80°C until use.
  • Lipid nanoparticle formulations of Table 1 using certain cationic lipids as described herein were prepared by Process A.
  • lipid nanoparticle formulations comprised firefly luciferase (FFL) mRNA and the different lipids (Cationic Lipid: DMG-PEG2000: Cholesterol: DOPE) in the mol % ratios specified in Table 1.
  • Table 1 Exemplary Lipid Nanoparticle Formulations * The N/P ratio is defined as the ratio of the number of nitrogen in cationic lipid to the number of phosphate in nucleic acid.
  • Example 106 The N/P ratio is defined as the ratio of the number of nitrogen in cationic lipid to the number of phosphate in nucleic acid.
  • Lipid nanoparticle formulation 1 listed in Table 1 comprising FFL mRNA, cationic lipid, DMG-PEG2000, cholesterol and DOPE was administered in mice via pipetting the formulations at 10 ⁇ g/Animal and 15 ⁇ l per nostril.
  • FFL Firefly Luciferase
  • DMG-PEG2000 cationic lipid
  • DOPE DOPE
  • mice were pipetting the formulations at 10 ⁇ g/Animal and 15 ⁇ l per nostril.
  • IVIS IVIS with separate ROIs on the nose and lungs.
  • Whole body imaging was performed 10-15 minutes following D-Luciferin administration. All animals were dosed with 0.2 mL of 15 mg/mL D-luciferin solution via intraperitoneal (IP) injection.
  • IP intraperitoneal
  • Anesthesia was performed by isoflurane during the procedure and animals were placed sternal recumbency (face-down).
  • An intranasal vaccine drug product may be administered via nasal spray.
  • Exemplary data are provided in Table 2, which describes the average radiance in p/s/cm2/sr (the number of photons per second that leave a square centimeter of tissue and radiate into a solid angle of one steradian (sr)).
  • cKK-E12 having the following structure, was used in a control formulation: Table 2. Exemplary in vivo protein expression following intranasal administration

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Abstract

Provided herein are ionizable or cationic lipids of Formula (I): (I), or pharmaceutically acceptable salts thereof. The ionizable or cationic lipids provided herein can be useful for delivery and expression of mRNA and encoded protein, e.g., as a component of liposomal delivery vehicle, and accordingly can be useful for treating various diseases, disorders and conditions, such as those associated with deficiency of one or more proteins.

Description

Malic and Glutaric Acid Based Ionizable Lipids CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to European Patent Application No.23307350.1, filed December 22, 2023, the disclosure of which is incorporated herein by reference in its entirety. BACKGROUND Effective targeted delivery of biologically active substances such as nucleic acid molecules (e.g., mRNA) represents a continuing medical challenge. In particular, the delivery of nucleic acids to cells is made difficult by their low in vivo stability, propensity toward rapid degradation, and low cell permeability. Lipid-containing nanoparticle compositions have proven effective as transport vehicles into cells and/or intracellular compartments for biologically active substances such as small molecule drugs, proteins, and nucleic acids. Such compositions generally include one or more ionizable or cationic lipid, phospholipids including polyunsaturated lipids, cholesterol-based lipids, and/or lipids containing polyethylene glycol (PEGylated lipids). While liposomal-based vehicles that comprise an ionizable or cationic lipid have shown promising results with regards to encapsulation, stability, and site localization, there remains a great need for improvement of liposomal-based delivery systems. In particular, there remains a need for improved ionizable or cationic lipids that demonstrate improved pharmacokinetic properties and which are capable of delivering macromolecules, such as nucleic acids, to a wide variety of cell types and tissues with enhanced efficiency. Importantly, there also remains a particular need for novel ionizable and cationic lipids that are characterized as having reduced toxicity and are capable of efficiently delivering encapsulated nucleic acids and polynucleotides to cells, tissues, and organs by various routes of administration. SEQUENCE LISTING The instant application contains a Sequence Listing which has been submitted herewith and is hereby incorporated by reference in its entirety. Said .xml copy, created on December 19, 2024 is named 758925_SA9-394PC_SL, and is 7,409 bytes in size. SUMMARY The present disclosure provides, inter alia, a lipid of Formula (I): (I), or a pharmaceutically acceptable salt thereof, wherein: two of R1, R2, and R3 are independently selected from -C(6-24)alkyl, -C(6-24)alkenyl, - C(6-24)alkynyl, , , and , wherein the -C(6-24)alkyl, - C(6-24)alkenyl, and -C(6-24)alkynyl are each optionally substituted with one to six groups independently selected from halo, -OC(1-6)alkyl, -OC(2-6)alkenyl, -SC(1-6)alkyl, -SC(2-6)alkenyl, and -C(O)OC(1-6)alkyl; one of R1, R2, and R3 is or X1 independently for each occurrence is -C(3-12)alkyl, -C(3-12)alkenyl, or -C(3-12)alkynyl, each of which is optionally substituted by one to six groups independently selected from halo, -OC(1-6)alkyl, -OC(2-6)alkenyl, -SC(1-6)alkyl, -SC(2-6)alkenyl, and -C(O)OC(1-6)alkyl; X2 independently for each occurrence is -C(O)O-, -OC(O)-, -C(O)NH-, -C(O)N(X3)-, -NHC(O)-, -N(X3)C(O)-, -C(O)S-, -SC(O)-, -OC(O)O-, -OC(O)NH-, -NHC(O)O-, or - NHC(O)NH-; X3 independently for each occurrence is -C(5-24)alkyl, -C(5-24)alkenyl, or -C(5-24)alkynyl, each of which is optionally substituted by one to six groups independently selected from halo, -OC(1-6)alkyl, -OC(2-6)alkenyl, -SC(1-6)alkyl, -SC(2-6)alkenyl, and -C(O)OC(1-6)alkyl; X4 is -C(1-8)alkyl; X5 is -C(1-8)alkyl; X6 is -C(O)O-, -OC(O)-, -C(O)NH-, -NHC(O)-, -C(O)S-, -SC(O)-, -OC(O)O-, - OC(O)NH-, -NHC(O)O-, or -NHC(O)NH-; X7 is -C(1-8)alkyl; A is an ionizable or cationic nitrogen-containing group; X is selected from the group consisting of: , , , , , , , , , , , and ; wherein * indicates the point of attachment to R3; RX independently for each occurrence is -H or -C(1-3)alkyl; and n is 1, 2, 3, or 4. The present disclosure further provides a lipid of Formula (I): (I), or a pharmaceutically acceptable salt thereof, wherein R1, R2, R3, X, and n are as defined herein. In one aspect, a lipid of Formula (I) is provided: (I), or a pharmaceutically acceptable salt thereof, wherein: two of R1, R2, and R3 are independently selected from -C(6-24)alkyl, -C(6-24)alkenyl, - C(6-24)alkynyl, , , and , wherein the -C(6-24)alkyl, - C(6-24)alkenyl, and -C(6-24)alkynyl are each optionally substituted with one to six groups independently selected from halo, -OC(1-6)alkyl, -OC(2-6)alkenyl, -SC(1-6)alkyl, -SC(2-6)alkenyl, and -C(O)OC(1-6)alkyl; one of R1, R2, and R3 is or ; X1 independently for each occurrence is -C(3-12)alkyl, -C(3-12)alkenyl, or -C(3-12)alkynyl, each of which is optionally substituted by one to six groups independently selected from halo, -OC(1-6)alkyl, -OC(2-6)alkenyl, -SC(1-6)alkyl, -SC(2-6)alkenyl, and -C(O)OC(1-6)alkyl; X2 independently for each occurrence is -C(O)O-, -OC(O)-, -C(O)NH-, -C(O)N(X3)-, -NHC(O)-, -N(X3)C(O)-, -C(O)S-, -SC(O)-, -OC(O)O-, -OC(O)NH-, -NHC(O)O-, or - NHC(O)NH-; X3 independently for each occurrence is -C(5-24)alkyl, -C(5-24)alkenyl, or -C(5-24)alkynyl, each of which is optionally substituted by one to six groups independently selected from halo, -OC(1-6)alkyl, -OC(2-6)alkenyl, -SC(1-6)alkyl, -SC(2-6)alkenyl, and -C(O)OC(1-6)alkyl; X4 is -C(1-8)alkyl; X5 is -C(1-8)alkyl; X6 is -C(O)O-, -OC(O)-, -C(O)NH-, -NHC(O)-, -C(O)S-, -SC(O)-, -OC(O)O-, - OC(O)NH-, -NHC(O)O-, or -NHC(O)NH-; X7 is -C(1-8)alkyl; A is an ionizable or cationic nitrogen-containing group; X is selected from the group consisting of: , , , , , , , , and ; wherein * indicates the point of attachment to R3; RX independently for each occurrence is -H or -C(1-3)alkyl; and n is 1, 2, 3, or 4. In some embodiments, two of R1, R2, and R3 are independently selected from -C(6- 24)alkyl, -C(6-24)alkenyl, and ; X1 independently for each occurrence is -C(3-12)alkyl or -C(3-12)alkenyl; X2 independently for each occurrence is -C(O)O-, -OC(O)-, or -OC(O)O-; and X3 independently for each occurrence is -C(5-24)alkyl or -C(5-24)alkenyl. In some embodiments, two of R1, R2, and R3 are independently selected from the group consisting of:
Figure imgf000006_0001
Figure imgf000007_0001
Figure imgf000008_0001
Figure imgf000009_0001
In some embodiments, one of R1, R2, and R3 is or ; X4 is - C(1-6)alkyl; X5 is -C(1-6)alkyl; X6 is -C(O)O-, -OC(O)-, or -OC(O)O-; X7 is -C(1-6)alkyl; and A is an ionizable or cationic nitrogen-containing group. In some embodiments, one of R1, R2, and R3 is ; X4 is -C(1-6)alkyl; and A is an ionizable or cationic nitrogen-containing group. In some embodiments, R1 and R2 are independently selected from -C(6-24)alkyl, -C(6- 24)alkenyl, or ; R3 is or ; X1 independently for each occurrence is -C(3-12)alkyl or -C(3-12)alkenyl; X2 independently for each occurrence is -C(O)O- , -OC(O)-, or -OC(O)O-; X3 independently for each occurrence is -C(5-24)alkyl or -C(5- 24)alkenyl; X4 is -C(1-6)alkyl; X5 is -C(1-6)alkyl; X6 is -C(O)O-, -OC(O)-, or -OC(O)O-; X7 is - C(1-6)alkyl; and A is an ionizable or cationic nitrogen-containing group. In some embodiments, A is ; and RA1 and RA2 are each independently -C(1-6)alkyl that is optionally substituted with - OH; or RA1 and RA2 are taken together to form a 3- to 6-membered heteroaryl that is optionally substituted with one to three -C(1-3)alkyl groups. In some embodiments, A is -N(CH3)2. In some embodiments, is selected from the group consisting of: , , , and . In some embodiments, the lipid has a structure selected from the group consisting of:
Figure imgf000010_0001
or a pharmaceutically acceptable salt thereof. In some embodiments, the lipid has a structure selected from the group consisting of:
Figure imgf000011_0001
or a pharmaceutically acceptable salt thereof. In some embodiments, the lipid has a structure selected from the group consisting of:
Figure imgf000011_0002
or a pharmaceutically acceptable salt thereof. In some embodiments, the lipid has a structure selected from the group consisting of:
Figure imgf000011_0003
Figure imgf000012_0001
or a pharmaceutically acceptable salt thereof. In some embodiments, the lipid has a structure selected from the group consisting of:
Figure imgf000012_0002
or a pharmaceutically acceptable salt thereof. In some embodiments, the lipid has a structure selected from the group consisting of:
Figure imgf000012_0003
or a pharmaceutically acceptable salt thereof. In some embodiments, the lipid has a structure selected from the group consisting of:
Figure imgf000013_0001
or a pharmaceutically acceptable salt thereof. In some embodiments, the lipid has a structure selected from the group consisting of:
Figure imgf000013_0002
or a pharmaceutically acceptable salt thereof. In some embodiments, the lipid has a structure selected from the group consisting of:
Figure imgf000013_0003
Figure imgf000014_0001
or a pharmaceutically acceptable salt thereof. In some embodiments, RX is -H or -CH3. In some embodiments, n is 1 or 2. In some embodiments, n is 1. In some embodiments, n is 2. In some embodiments, the lipid has a structure selected from the group consisting of:
Figure imgf000014_0002
Figure imgf000015_0001
Figure imgf000016_0001
Figure imgf000017_0001
Figure imgf000018_0001
Figure imgf000019_0001
Figure imgf000020_0001
Figure imgf000021_0001
Figure imgf000022_0001
Figure imgf000023_0001
Figure imgf000024_0001
Figure imgf000025_0001
Figure imgf000026_0001
Figure imgf000027_0001
Figure imgf000028_0001
Figure imgf000029_0001
Figure imgf000030_0001
Figure imgf000031_0001
Figure imgf000032_0001
Figure imgf000033_0001
Figure imgf000034_0001
Figure imgf000035_0001
Figure imgf000036_0001
or a pharmaceutically acceptable salt thereof. In some embodiments, the lipid has a structure selected from the group consisting of:
Figure imgf000036_0002
Figure imgf000037_0001
Figure imgf000038_0001
Figure imgf000039_0001
Figure imgf000040_0001
Figure imgf000041_0001
or a pharmaceutically acceptable salt thereof. In another aspect, the present disclosure provides a composition comprising a lipid nanoparticle (LNP), wherein the LNP comprises a lipid of the present disclosure (e.g., a lipid of Formula (I)). In some embodiments, the LNP comprises: (I) a lipid of the present disclosure; (II) a stealth lipid; (III) a structural lipid; and (IV) a helper lipid. In some embodiments, the stealth lipid is a polyethylene glycol-conjugated (PEGylated) lipid, a polyoxazoline polymer-conjugated lipid, or a polysarcosine-conjugated (pSar) lipid. In some embodiments, the stealth lipid is a PEGylated lipid selected from 1,2- dimyristoyl-rac-glycero-3-methoxypolyethylene glycol (DMG-PEG), 1,2-distearoyl-sn- glycero-3-phosphoethanolamine-polyethylene glycol (DSPE-PEG), 1,2-dilauroyl-sn-glycero- 3-phosphoethanolamine-polyethylene glycol (DLPE-PEG), and 1,2-distearoyl-rac-glycero- polyethelene glycol (DSG-PEG). In some embodiments, the PEGylated lipid is dimyristoyl-PEG2000 (DMG- PEG2000). In some embodiments, the structural lipid is a sterol. In some embodiments, the sterol is cholesterol. In some embodiments, the helper lipid is 1,2-dioleoyl-SN-glycero-3- phosphoethanolamine (DOPE); 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC); 1,2- dioleoyl-sn-glycero-3-phospho-L-serine (DOPS); 1,2-dielaidoyl-sn-glycero-3- phosphoethanolamine (DEPE); and 1,2-dioleoyl-sn-glycero-3-phosphocholine (DPOC), dipalmitoylphosphatidylcholine (DPPC), 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC), 1,2-Distearoylphosphatidylethanolamine (DSPE), or 1,2-dilauroyl-sn-glycero-3- phosphoethanolamine (DLPE). In some embodiments, the helper lipid is 1,2-dioleoyl-SN-glycero-3- phosphoethanolamine (DOPE). In some embodiments, the LNP comprises: the lipid of the present disclosure at a molar ratio between 35% and 45%, the stealth lipid at a molar ratio between 0.5% and 7%, the structural lipid at a molar ratio between 20% and 35%, and the helper lipid at a molar ratio of between 20% and 30%. In some embodiments, the LNP comprises: the lipid of any one of claims 1-23 at a molar ratio of about 40%, the stealth lipid at a molar ratio of about 1.5%, the structural lipid at a molar ratio of about 30%, and the helper lipid at a molar ratio of about 28.5%. In some embodiments, the LNP comprises: the lipid of any one of claims 1-23 at a molar ratio of about 40%, the stealth lipid at a molar ratio of about 5%, the structural lipid at a molar ratio of about 30%, and the helper lipid at a molar ratio of about 25%. In some embodiments, the composition comprising a lipid nanoparticle (LNP) further comprises a nucleic acid molecule, wherein the nucleic acid molecule is encapsulated in the LNP. In some embodiments, the LNP comprises 1-20, optionally 5-10 or 6-8, nucleic acid molecules. In some embodiments, the nucleic acid molecule is an mRNA molecule. In some embodiments, the mRNA molecule encodes an antigen, optionally a viral antigen or a bacterial antigen. In some embodiments, the LNP encapsulates two or more mRNA molecules, wherein each mRNA molecule encodes a different antigen, optionally wherein the different antigens are from the same pathogen or from different pathogens. In some embodiments, the composition comprises two or more LNPs, wherein each LNP encapsulates an mRNA encoding a different antigen, optionally wherein the different antigens are from the same pathogen or from different pathogens. In some embodiments, the composition is formulated for intranasal administration. In some embodiments, the composition comprises a phosphate-buffer saline. In some embodiments, the composition comprises trehalose, optionally at 10% (w/v) of the composition. In another aspect, the present disclosure provides a method of eliciting an immune response in a subject in need thereof, comprising administering to the subject, optionally mucosally, intramuscularly, intranasally, intravenously, subcutaneously, or intradermally, a prophylactically effective amount of a composition of the present disclosure. In yet another aspect, the present disclosure provides a method of preventing an infection or reducing one or more symptoms of an infection in a subject in need thereof, comprising administering to the subject, optionally mucosally, intramuscularly, intranasally, intravenously, subcutaneously, or intradermally, a prophylactically effective amount of a composition of the present disclosure. In some embodiments, the composition is administered intranasally. In some embodiments, the methods of the present disclosure comprise administering to the subject one or more doses of the composition, each dose comprising 1-250, optionally 2.5., 5, 15, 45, or 135, μg of mRNA. In some embodiments, the methods of the present disclosure comprise administering to the subject two doses of the composition with an interval of 2-6, optionally 4, weeks. In another aspect, the present disclosure provides the use of a composition of the present disclosure for the manufacture of a medicament for use in treating a subject in need thereof. In yet another aspect, the present disclosure provides a composition, as described herein, for use in treating a subject in need thereof. In still another aspect, the present disclosure provides a kit comprising a container comprising a single-use or multi-use dosage of a composition of the present disclosure, optionally wherein the container is a vial or a pre-filled nasal spray device. DETAILED DESCRIPTION The present disclosure provides ionizable or cationic lipids that may be incorporated into lipid nanoparticle (LNP) formulations for delivering cargo, such as a nucleic acid molecule (e.g., mRNA), to a target cell. LNPs comprising the ionizable or cationic lipids of the present disclosure exhibit enhanced expression of proteins encoded by cargo nucleic acid molecules. I. Definitions Unless otherwise defined herein, scientific and technical terms used in this application shall have the meanings that are commonly understood by those of ordinary skill in the art. As used in the specification and in the claims, the term “comprising” can include the embodiments “consisting of” and “consisting essentially of.” The terms “comprise(s),” “include(s),” “having,” “has,” “can,” “contain(s),” and variants thereof, as used herein, are intended to be open-ended transitional phrases, terms, or words that require the presence of the named ingredients/steps and permit the presence of other ingredients/steps. However, such description should be construed as also describing compositions or processes as “consisting of” and “consisting essentially of” the enumerated ingredients/steps, which allows the presence of only the named ingredients/steps and excludes other ingredients/steps. As used herein, the term “approximately” or “about,” as applied to one or more values of interest, refers to a value that is similar to a stated reference value. In certain embodiments, the term “approximately” or “about” refers to a range of values that fall within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than or less than) of the stated reference value unless otherwise stated or otherwise evident from the context (except where such number would exceed 100% of a possible value). As used herein, the term “delivery” encompasses both local and systemic delivery. For example, delivery of mRNA encompasses situations in which an mRNA is delivered to a target tissue and the encoded protein is expressed and retained within the target tissue (also referred to as “local distribution” or “local delivery”), and situations in which an mRNA is delivered to a target tissue and the encoded protein is expressed and secreted into patient's circulation system (e.g., serum) and systematically distributed and taken up by other tissues (also referred to as “systemic distribution” or “systemic delivery). As used herein, “expression” of a nucleic acid sequence refers to translation of an mRNA into a polypeptide, assembly of multiple polypeptides (e.g., heavy chain or light chain of antibody) into an intact protein (e.g., antibody), and/or post-translational modification of a polypeptide or fully assembled protein (e.g., antibody). In this application, the terms “expression” and “production,” and grammatical equivalent, are used inter-changeably. As used herein, the term “half-life” is the time required for a quantity such as nucleic acid or protein concentration or activity to fall to half of its value as measured at the beginning of a time period. As used herein, the terms “improve,” “increase” or “reduce,” or grammatical equivalents, indicate values that are relative to a baseline measurement, such as a measurement in the same individual prior to initiation of the treatment described herein, or a measurement in a control subject (or multiple control subject) in the absence of the treatment described herein. As used herein, the term “in vitro” refers to events that occur in an artificial environment, e.g., in a test tube or reaction vessel, in cell culture, etc., rather than within a multi-cellular organism. As used herein, the term “in vivo” refers to events that occur within a multi-cellular organism, such as a human and a non-human animal. In the context of cell-based systems, the term may be used to refer to events that occur within a living cell (as opposed to, for example, in vitro systems). As used herein, the term "liposome" refers to any lamellar, multilamellar, or solid nanoparticle vesicle. Typically, a liposome as used herein can be formed by mixing one or more lipids or by mixing one or more lipids and polymer(s). In some embodiments, a liposome suitable for the present disclosure contains an ionizable or cationic lipid(s) and optionally non-cationic lipid(s), optionally cholesterol-based lipid(s), and/or optionally PEG- modified lipid(s). As used herein, the term “messenger RNA (mRNA)” refers to a polynucleotide that encodes at least one polypeptide. mRNA as used herein may encompass both modified and unmodified RNA. mRNA may contain one or more coding and non-coding regions. mRNA can be purified from natural sources, produced using recombinant expression systems and optionally purified, chemically synthesized, etc. Where appropriate, e.g., in the case of chemically synthesized molecules, mRNA can comprise nucleoside analogs such as analogs having chemically modified bases or sugars, backbone modifications, etc. An mRNA sequence is presented in the 5′ to 3′ direction unless otherwise indicated. In some embodiments, an mRNA is or comprises natural nucleosides (e.g., adenosine, guanosine, cytidine, uridine); nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyl adenosine, 5-methylcytidine, C-5 propynyl-cytidine, C-5 propynyl-uridine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5-propynyl-cytidine, C5-methylcytidine, 2-aminoadenosine, 7- deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, O(6)-methylguanine, and 2-thiocytidine); chemically modified bases; biologically modified bases (e.g., methylated bases); intercalated bases; modified sugars (e.g., 2′-fluororibose, ribose, 2′-deoxyribose, arabinose, and hexose); and/or modified phosphate groups (e.g., phosphorothioates and 5′-N- phosphoramidite linkages). As used herein, the term “nucleic acid,” in its broadest sense, refers to any compound and/or substance that is or can be incorporated into a polynucleotide chain. In some embodiments, a nucleic acid is a compound and/or substance that is or can be incorporated into a polynucleotide chain via a phosphodiester linkage. In some embodiments, “nucleic acid” refers to individual nucleic acid residues (e.g., nucleotides and/or nucleosides). In some embodiments, “nucleic acid” refers to a polynucleotide chain comprising individual nucleic acid residues. In some embodiments, “nucleic acid” encompasses RNA as well as single and/or double-stranded DNA and/or cDNA. The term “pharmaceutically acceptable” as used herein, refers to substances that, within the scope of sound medical judgment, are suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio. Pharmaceutically acceptable salts are well known in the art. For example, S. M. Berge et al., describes pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences (1977) 66:1-19. Pharmaceutically acceptable salts of the compounds of this disclosure include those derived from suitable inorganic and organic acids and bases. As used herein, the term “subject” refers to a human or any non-human animal (e.g., mouse, rat, rabbit, dog, cat, cattle, swine, sheep, horse or primate). A human includes pre- and post-natal forms. In many embodiments, a subject is a human being. A subject can be a patient, which refers to a human presenting to a medical provider for diagnosis or treatment of a disease. The term “subject” is used herein interchangeably with “individual” or “patient.” A subject can be afflicted with or is susceptible to a disease or disorder but may or may not display symptoms of the disease or disorder. As used herein, the term “substantially” refers to the qualitative condition of exhibiting total or near-total extent or degree of a characteristic or property of interest. One of ordinary skill in the biological arts will understand that biological and chemical phenomena rarely, if ever, go to completion and/or proceed to completeness or achieve or avoid an absolute result. The term “substantially” is therefore used herein to capture the potential lack of completeness inherent in many biological and chemical phenomena. As used herein, the term “target tissues” refers to any tissue that is affected by a disease to be treated. In some embodiments, target tissues include those tissues that display disease-associated pathology, symptom, or feature. As used herein, the term “therapeutically effective amount” of a therapeutic agent means an amount that is sufficient, when administered to a subject suffering from or susceptible to a disease, disorder, and/or condition, to treat, diagnose, prevent, and/or delay the onset of the symptom(s) of the disease, disorder, and/or condition. It will be appreciated by those of ordinary skill in the art that a therapeutically effective amount is typically administered via a dosing regimen comprising at least one unit dose. As used herein, the term “treatment” or “treating,” is defined as the application or administration of a therapeutic agent to a patient, or application or administration of a therapeutic agent to an isolated tissue or cell line from a patient (e.g., for diagnosis or ex vivo applications), who has a disorder or disease as described herein, a symptom thereof; or the potential to develop such disorder or disease, where the purpose of the application or administration is to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve or affect the disorder or disease, or its symptoms. Such treatments may be specifically tailored or modified, based on knowledge obtained from the field of pharmacogenomics. As used herein, the term “prevent” or “prevention” means no disorder or disease development if none had occurred, or no further disorder or disease development if there had already been development of the disorder or disease. Also considered is the ability of one to prevent some or all of the symptoms associated with the disorder or disease. Compounds described herein can comprise one or more asymmetric centers, and thus can exist in various isomeric forms, e.g., enantiomers and/or diastereomers. For example, the compounds described herein can be in the form of an individual enantiomer, diastereomer or geometric isomer, or can be in the form of a mixture of stereoisomers, including racemic mixtures and mixtures enriched in one or more stereoisomer. Isomers can be isolated from mixtures by methods known to those skilled in the art, including chiral high performance liquid chromatography (HPLC) and the formation and crystallization of chiral salts; or preferred isomers can be prepared by asymmetric syntheses. See, for example, Jacques et al., Enantiomers. Racemates and Resolutions (Wiley Interscience, New York, 1981); Wilen et al., Tetrahedron 33:2725 (1977); Eliel, E. L. Stereochemistry of Carbon Compounds (McGraw-Hill, NY, 1962); and Wilen, S. H. Tables of Resolving Agents and Optical Resolutions p.268 (E. L. Eliel, Ed., Univ. of Notre Dame Press, Notre Dame, Ind. 1972). The present disclosure additionally contemplates compounds as individual isomers substantially free of other isomers, and alternatively, as mixtures of various isomers. When a range of values is listed, it is intended to encompass each value and sub-range within the range. For example “ C1-6 alkyl” is intended to encompass, C1, C2, C3, C4, C5, C6, C1-6, C1-5, C1-4, C1-3, C1-2, C2-6, C2-5, C2-4, C2-3, C3-6, C3-5, C3-4, C4-6, C4-5, and C5-6 alkyl. As used herein, the term “alkyl” refers to a straight or branched saturated hydrocarbon. For example, an alkyl group can have 1 to 30 carbon atoms (i.e., (C1-C30)alkyl), 1 to 20 carbon atoms (i.e., (C1-C20)alkyl), 1 to 12 carbon atoms (i.e., (C1-C12)alkyl), 1 to 6 carbon atoms (i.e., (C1-C6)alkyl), or 1 to 3 carbon atoms (i.e., (C1-C3)alkyl). Examples of alkyl groups include, but are not limited to, methyl (Me, -CH3), ethyl (Et, -CH2CH3), 1- propyl (n-Pr, n-propyl, -CH2CH2CH3), isopropyl (i-Pr, i-propyl, -CH(CH3)2), 1-butyl (n-bu, n-butyl, -CH2CH2CH2CH3), 2-butyl (s-bu, s-butyl, -CH(CH3)CH2CH3), tert-butyl (t-bu, t- butyl, -CH(CH3)3), 1-pentyl (n-pentyl, -CH2CH2CH2CH2CH3), 2-pentyl (-CH(CH3) CH2CH2CH3), neopentyl (CH2C(CH3)3), 1-hexyl (-CH2CH2CH2CH2CH2CH3), 2-hexyl (- CH(CH3)CH2CH2CH2CH3), heptyl (-(CH2)6CH3), octyl (-(CH2)7CH3), 2,2,4-trimethylpentyl (-CH2C(CH3)2CH2CH(CH3)2), nonyl (-(CH2)8CH3), decyl (-(CH2)9CH3), undecyl (- (CH2)10CH3), and dodecyl (-(CH2)11CH3). When a bivalent variable is defined as “alkyl,” it is to be understood that such a group is a bivalent alkylene group. As used herein, the term “alkenyl” refers to a straight or branched saturated hydrocarbon having at least one site of carbon-carbon double bond unsaturation. For example, an alkenyl group can have 2 to 30 carbon atoms (i.e., (C2-C30)alkenyl), 2 to 20 carbon atoms (i.e., (C2-C20)alkenyl), 2 to 12 carbon atoms (i.e., (C2-C12)alkenyl) or 2 to 6 carbon atoms (i.e., (C2-C6)alkenyl), and the alkenyl group can contain 1, 2, 3, or 4 carbon- carbon double bonds. The one or more carbon-carbon double bonds can be internal (such as in 2-butenyl) or terminal (such as in 1-butenyl). Included within this term are the cis and trans isomers or mixtures of these isomers. Nonlimiting examples of alkenyl groups include prop- 2-enyl, but-2-enyl, but-3-enyl, 2-methylprop-2-enyl, hex-2-enyl, hex- 5-enyl, 2,3- dimethylbut-2-enyl, and the like. When a bivalent variable is defined as “alkenyl,” it is to be understood that such a group is a bivalent alkenylene group. As used herein, the term “alkynyl” refers to a straight or branched saturated hydrocarbon having at least one site of carbon-carbon triple bond unsaturation occurring at any stable point along the chain. For example, an alkynyl group can have 2 to 30 carbon atoms (i.e., (C2-C30)alkynyl), 2 to 20 carbon atoms (i.e., (C2-C20)alkynyl), 2 to 12 carbon atoms (i.e., (C2-C12)alkynyl) or 2 to 6 carbon atoms (i.e., (C2-C6)alkynyl), and the alkynyl group can contain 1, 2, 3, or 4 carbon-carbon triple bonds. The one or more carbon-carbon triple bonds can be internal or terminal. Optionally, the alkynyl group may include one or more double bonds (e.g., 1, 2, 3, or 4 double bonds). For purposes of the present disclosure, a hydrocarbon group having one or more triple bonds and one or more double bonds (e.g., an “ene-yne”) is categorized as an alkynyl moiety. Nonlimiting examples of an alkynyl groups include prop-2-ynyl, but-2-ynyl, but-3-ynyl, pent-2-ynyl, 3-methylpent-4-ynyl, hex-2-ynyl, hex-5-ynyl, etc. When a bivalent variable is defined as “alkynyl,” it is to be understood that such a group is a bivalent alkynylene group. The term “heterocycle” or “heterocyclyl” refers to a saturated or partially unsaturated ring system that has at least one atom other than carbon in the ring system, wherein the atom is selected from the group consisting of oxygen, nitrogen and sulfur. The heterocyclyl group may, for example, consist of a single ring or multiple rings (e.g., in the form of a spirocyclic or bicyclic ring system). Exemplary heterocycles include, but are not limited to oxetanyl, aziridinyl, azetidinyl, pyrrolidinyl, piperidinyl, piperazinyl, morpholinyl, tetrahydropyranyl, tetrahydrofuranyl, and thiomorpholinyl. The term “heteroaryl” refers to a single aromatic ring that has at least one atom other than carbon in the ring, wherein the atom is selected from the group consisting of oxygen, nitrogen and sulfur. The term “heteroaryl” includes single aromatic rings of from 1 to 6 carbon atoms and 1 to 4 heteroatoms selected from the group consisting of oxygen, nitrogen and sulfur. Exemplary heteroaryl ring systems include but are not limited to pyridinyl, pyrimidinyl, pyrazinyl, pyridazinyl, pyrimidinyl, pyrazolyl, oxazolyl, oxadiazolyl, isoxazolyl, triazolyl, imidazolyl, tetrazolyl, thienyl, thiazolyl, isothiazolyl, thiadiazolyl, or furyl. The term “halogen” or “halo” refers to bromo (-Br), chloro (-Cl), fluoro (-F) or iodo (- I). As used herein, a “counteranion” is a negatively charged group associated with a positively charged quarternary amine in order to maintain electronic neutrality. Exemplary counteranions include halide ions (e.g., F—, Cl—, Br—, I—), NO3-, ClO4-, OH—, H2PO4-, HSO4-, sulfonate ions (e.g., methansulfonate, trifluoromethanesulfonate, p-toluenesulfonate, benzenesulfonate, 10-camphor sulfonate, naphthalene-2-sulfonate, naphthalene-1-sulfonic acid-5-sulfonate, ethan-1-sulfonic acid-2-sulfonate, and the like), and carboxylate ions (e.g., acetate, ethanoate, propanoate, benzoate, glycerate, lactate, tartrate, glycolate, and the like). Nitrogen atoms can be substituted or unsubstituted as valency permits, and include primary, secondary, tertiary, and quarternary nitrogen atoms. Exemplary methods and materials are described below, although methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure. In case of conflict, the present specification, including definitions, will control. Generally, nomenclature used in connection with, and techniques of, cell and tissue culture, molecular biology, virology, immunology, microbiology, genetics, analytical chemistry, synthetic organic chemistry, medicinal and pharmaceutical chemistry, and protein and nucleic acid chemistry and hybridization described herein are those well- known and commonly used in the art. Enzymatic reactions and purification techniques are performed according to manufacturer’s specifications, as commonly accomplished in the art or as described herein. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. Although a number of documents are cited herein, this citation does not constitute an admission that any of these documents forms part of the common general knowledge in the art. II. Ionizable/Cationic Lipids The present disclosure provides a lipid of Formula (I): (I), or a pharmaceutically acceptable salt thereof, wherein: two of R1, R2, and R3 are independently selected from -C(6-24)alkyl, -C(6-24)alkenyl, - C(6-24)alkynyl, , , and , wherein the -C(6-24)alkyl, - C(6-24)alkenyl, and -C(6-24)alkynyl are each optionally substituted with one to six groups independently selected from halo, -OC(1-6)alkyl, -OC(2-6)alkenyl, -SC(1-6)alkyl, -SC(2-6)alkenyl, and -C(O)OC(1-6)alkyl; one of R1, R2, and R3 is or ; X1 independently for each occurrence is -C(3-12)alkyl, -C(3-12)alkenyl, or -C(3-12)alkynyl, each of which is optionally substituted by one to six groups independently selected from halo, -OC(1-6)alkyl, -OC(2-6)alkenyl, -SC(1-6)alkyl, -SC(2-6)alkenyl, and -C(O)OC(1-6)alkyl; X2 independently for each occurrence is -C(O)O-, -OC(O)-, -C(O)NH-, -C(O)N(X3)-, -NHC(O)-, -N(X3)C(O)-, -C(O)S-, -SC(O)-, -OC(O)O-, -OC(O)NH-, -NHC(O)O-, or - NHC(O)NH-; X3 independently for each occurrence is -C(5-24)alkyl, -C(5-24)alkenyl, or -C(5-24)alkynyl, each of which is optionally substituted by one to six groups independently selected from halo, -OC(1-6)alkyl, -OC(2-6)alkenyl, -SC(1-6)alkyl, -SC(2-6)alkenyl, and -C(O)OC(1-6)alkyl; X4 is -C(1-8)alkyl; X5 is -C(1-8)alkyl; X6 is -C(O)O-, -OC(O)-, -C(O)NH-, -NHC(O)-, -C(O)S-, -SC(O)-, -OC(O)O-, - OC(O)NH-, -NHC(O)O-, or -NHC(O)NH-; X7 is -C(1-8)alkyl; A is an ionizable or cationic nitrogen-containing group; X is selected from the group consisting of:
Figure imgf000051_0001
wherein * indicates the point of attachment to R3; RX independently for each occurrence is -H or -C(1-3)alkyl; and n is 1, 2, 3, or 4. In some embodiments, the present disclosure provides a lipid of Formula (I): (I), or a pharmaceutically acceptable salt thereof, wherein: two of R1, R2, and R3 are independently selected from -C(6-24)alkyl, -C(6-24)alkenyl, - C(6-24)alkynyl, , , and , wherein the -C(6-24)alkyl, - C(6-24)alkenyl, and -C(6-24)alkynyl are each optionally substituted with one to six groups independently selected from halo, -OC(1-6)alkyl, -OC(2-6)alkenyl, -SC(1-6)alkyl, -SC(2-6)alkenyl, and -C(O)OC(1-6)alkyl; one of R1, R2, and R3 is or ; X1 independently for each occurrence is -C(3-12)alkyl, -C(3-12)alkenyl, or -C(3-12)alkynyl, each of which is optionally substituted by one to six groups independently selected from halo, -OC(1-6)alkyl, -OC(2-6)alkenyl, -SC(1-6)alkyl, -SC(2-6)alkenyl, and -C(O)OC(1-6)alkyl; X2 independently for each occurrence is -C(O)O-, -OC(O)-, -C(O)NH-, -C(O)N(X3)-, -NHC(O)-, -N(X3)C(O)-, -C(O)S-, -SC(O)-, -OC(O)O-, -OC(O)NH-, -NHC(O)O-, or - NHC(O)NH-; X3 independently for each occurrence is -C(5-24)alkyl, -C(5-24)alkenyl, or -C(5-24)alkynyl, each of which is optionally substituted by one to six groups independently selected from halo, -OC(1-6)alkyl, -OC(2-6)alkenyl, -SC(1-6)alkyl, -SC(2-6)alkenyl, and -C(O)OC(1-6)alkyl; X4 is -C(1-8)alkyl; X5 is -C(1-8)alkyl; X6 is -C(O)O-, -OC(O)-, -C(O)NH-, -NHC(O)-, -C(O)S-, -SC(O)-, -OC(O)O-, - OC(O)NH-, -NHC(O)O-, or -NHC(O)NH-; X7 is -C(1-8)alkyl; A is an ionizable or cationic nitrogen-containing group; X is selected from the group consisting of:
Figure imgf000052_0001
wherein * indicates the point of attachment to R3; RX independently for each occurrence is -H or -C(1-3)alkyl; and n is 1, 2, 3, or 4. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof: two of R1, R2, and R3 are independently selected from -C(6-24)alkyl, -C(6-24)alkenyl, or ; one of R1, R2, and R3 is or ; X1 independently for each occurrence is -C(3-12)alkyl or -C(3-12)alkenyl; X2 independently for each occurrence is -C(O)O-, -OC(O)-, -C(O)S-, -SC(O)-, or - OC(O)O-; X3 independently for each occurrence is -C(5-24)alkyl or -C(5-24)alkenyl; X4 is -C(1-6)alkyl; X5 is -C(1-6)alkyl; X6 is -C(O)O-, -OC(O)-, or -OC(O)O-; X7 is -C(1-6)alkyl; A is an ionizable or cationic nitrogen-containing group; X is selected from the group consisting of:
Figure imgf000053_0001
wherein * indicates the point of attachment to R3; RX is -H or -C(1-3)alkyl; and n is 1, 2, 3, or 4. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof: two of R1, R2, and R3 are independently selected from -C(6-24)alkyl, -C(6-24)alkenyl, or ; one of R1, R2, and R3 is or ; X1 independently for each occurrence is -C(3-12)alkyl or -C(3-12)alkenyl; X2 independently for each occurrence is -C(O)O-, -OC(O)-, or -OC(O)O-; X3 independently for each occurrence is -C(5-24)alkyl or -C(5-24)alkenyl; X4 is -C(1-6)alkyl; X5 is -C(1-6)alkyl; X6 is -C(O)O-, -OC(O)-, or -OC(O)O-; X7 is -C(1-6)alkyl; A is an ionizable or cationic nitrogen-containing group; X is selected from the group consisting of:
Figure imgf000054_0001
wherein * indicates the point of attachment to R3; RX is -H or -C(1-3)alkyl; and n is 1, 2, 3, or 4. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, R1 and R2 are independently selected from -C(6-24)alkyl, -C(6- 24)alkenyl, -C(6-24)alkynyl, , , and , wherein the -C(6- 24)alkyl, -C(6-24)alkenyl, and -C(6-24)alkynyl are each optionally substituted with one to six groups independently selected from halo, -OC(1-6)alkyl, -OC(2-6)alkenyl, -SC(1-6)alkyl, -SC(2- 6)alkenyl, and -C(O)OC(1-6)alkyl. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, R1 and R3 are independently selected from -C(6-24)alkyl, -C(6- 24)alkenyl, -C(6-24)alkynyl, , , and , wherein the -C(6- 24)alkyl, -C(6-24)alkenyl, and -C(6-24)alkynyl are each optionally substituted with one to six groups independently selected from halo, -OC(1-6)alkyl, -OC(2-6)alkenyl, -SC(1-6)alkyl, -SC(2- 6)alkenyl, and -C(O)OC(1-6)alkyl. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, R2 and R3 are independently selected from -C(6-24)alkyl, -C(6- 24)alkenyl, -C(6-24)alkynyl, , , and , wherein the -C(6- 24)alkyl, -C(6-24)alkenyl, and -C(6-24)alkynyl are each optionally substituted with one to six groups independently selected from halo, -OC(1-6)alkyl, -OC(2-6)alkenyl, -SC(1-6)alkyl, -SC(2- 6)alkenyl, and -C(O)OC(1-6)alkyl. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, two of R1, R2, and R3 are independently selected from -C(6-24)alkyl, - C(6-24)alkenyl, , , and . In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, two of R1, R2, and R3 are independently -C(6-24)alkyl. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, two of R1, R2, and R3 are independently -C(6- 24)alkenyl. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, two of R1, R2, and R3 are independently . In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, two of R1, R2, and R3 are independently . In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, two of R1, R2, and R3 are independently . In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, two of R1, R2, and R3 are independently . In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, two of R1, R2, and R3 are independently . In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, R1 and R2 are independently -C(6-24)alkyl. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, R1 and R2 are independently -C(6-24)alkenyl. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, R1 and R2 are independently . In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, R1 and R2 are independently . In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, R1 and R2 are independently . In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, R1 and R2 are independently . In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, R1 and R2 are independently . In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, R1 and R3 are independently -C(6-24)alkyl. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, R1 and R3 are independently -C(6-24)alkenyl. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, R1 and R3 are independently . In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, R1 and R3 are independently . In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, R1 and R3 are independently . In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, R1 and R3 are independently . In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, R1 and R3 are independently . In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, R2 and R3 are independently -C(6-24)alkyl. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, R2 and R3 are independently -C(6-24)alkenyl. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, R2 and R3 are independently . In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, R2 and R3 are independently . In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, R2 and R3 are independently . In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, R2 and R3 are independently . In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, R2 and R3 are independently . In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, X1 is -C(3-12)alkyl or -C(3-12)alkenyl. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, X1 is -C(3-12)alkyl. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, X1 is -C(4-8)alkyl. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, X2 is -C(O)O-, -OC(O)-, -C(O)S-, -SC(O)-, or -OC(O)O-. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, X2 is -C(O)O-, -OC(O)-, or -OC(O)O-. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, X2 is -C(O)O-. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, X2 is -OC(O)-. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, X2 is -C(O)NH-. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, X2 is -C(O)N(X3)-. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, X2 is -NHC(O)-. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, X2 is -N(X3)C(O)-. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, X2 is -C(O)S-. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, X2 is -SC(O)-. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, X2 is -OC(O)O-. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, X2 is -OC(O)NH-. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, X2 is -NHC(O)O-. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, X2 is -NHC(O)NH-. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, X3 is -C(5-24)alkyl or -C(5-24)alkenyl. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, X3 is -C(5-24)alkyl. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, X3 is -C(5-20)alkyl. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, X3 is -C(10-18)alkyl. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, X3 is . In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, X3 is -C(5-24)alkenyl. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, X3 is -C(5-15)alkenyl. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, two of R1, R2, and R3 are independently selected from the group consisting of formulas A1-A21:
Figure imgf000058_0001
Figure imgf000059_0001
Figure imgf000060_0001
In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, two of R1, R2, and R3 are independently selected from the group consisting of formulas A1, A3, and A7-A18. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, two of R1, R2, and R3 are independently selected from the group consisting of formulas A13, A14, and A15. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, two of R1, R2, and R3 are independently selected from the group consisting of formulas A13 and A14: In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, R1 and R2 are independently selected from the group consisting of formulas A1, A3, and A7-A18. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, R1 and R2 are independently selected from the group consisting of formulas A1-A21. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, R1 and R2 are independently selected from the group consisting of formulas A13, A14, and A15. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, R1 and R2 are independently selected from the group consisting of formulas A13 and A14. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, R1 and R3 are independently selected from the group consisting of formulas A1, A3, and A7-A18. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, R1 and R3 are independently selected from the group consisting of formulas A1-A21. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, R1 and R3 are independently selected from the group consisting of formulas A13, A14, and A15. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, R1 and R3 are independently selected from the group consisting of formulas A13 and A14. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, R2 and R3 are independently selected from the group consisting of formulas A1, A3, and A7-A18. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, R2 and R3 are independently selected from the group consisting of formulas A1-A21. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, R2 and R3 are independently selected from the group consisting of formulas A13, A14, and A15. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, R2 and R3 are independently selected from the group consisting of formulas A13 and A14. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, two of R1, R2, and R3 are independently selected from the group consisting of formulas B1-58:
Figure imgf000061_0001
Figure imgf000062_0001
Figure imgf000063_0001
Figure imgf000064_0001
Figure imgf000065_0001
In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, two of R1, R2, and R3 are independently selected from the group consisting of formulas B1-56. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, two of R1, R2, and R3 are independently selected from the group consisting of formulas B1, B2, B3, B4, B5, B7, B10, B12, B15, B16, B18, B21, B24, B28, B31, B33, B36, B37, B40, B41, B43, B44, B46, B47, B48, B49, B50, and B51. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, two of R1, R2, and R3 are independently selected from the group consisting of formulas B1, B2, B5, B33-B53, B57, and B58. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, two of R1, R2, and R3 are independently selected from the group consisting of formulas B33-B51. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, two of R1, R2, and R3 are independently selected from the group consisting of formulas B36- B44. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, R1 and R2 are independently selected from the group consisting of formulas B1-B58. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, R1 and R2 are independently selected from the group consisting of formulas B1-B56. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, R1 and R2 are independently selected from the group consisting of formulas B1, B2, B3, B4, B5, B7, B10, B12, B15, B16, B18, B21, B24, B28, B31, B33, B36, B37, B40, B41, B43, B44, B46, B47, B48, B49, B50, and B51. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, R1 and R2 are independently selected from the group consisting of formulas B1, B2, B5, B33- B53, B57, and B58. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, R1 and R2 are independently selected from the group consisting of formulas B33-B51. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, R1 and R2 are independently selected from the group consisting of formulas B36-B44. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, R1 and R3 are independently selected from the group consisting of formulas B1-B58. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, R1 and R3 are independently selected from the group consisting of formulas B1-B56. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, R1 and R3 are independently selected from the group consisting of formulas B1, B2, B3, B4, B5, B7, B10, B12, B15, B16, B18, B21, B24, B28, B31, B33, B36, B37, B40, B41, B43, B44, B46, B47, B48, B49, B50, and B51. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, R1 and R3 are independently selected from the group consisting of formulas B1, B2, B5, B33- B53, B57, and B58. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, R1 and R3 are independently selected from the group consisting of formulas B33-B51. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, R1 and R3 are independently selected from the group consisting of formulas B36-B44. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, R2 and R3 are independently selected from the group consisting of formulas B1-B58. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, R2 and R3 are independently selected from the group consisting of formulas B1-B56. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, R2 and R3 are independently selected from the group consisting of formulas B1, B2, B3, B4, B5, B7, B10, B12, B15, B16, B18, B21, B24, B28, B31, B33, B36, B37, B40, B41, B43, B44, B46, B47, B48, B49, B50, and B51. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, R2 and R3 are independently selected from the group consisting of formulas B1, B2, B5, B33- B53, B57, and B58. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, R2 and R3 are independently selected from the group consisting of formulas B33-B51. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, R2 and R3 are independently selected from the group consisting of formulas B36-B44. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, one of R1, R2, and R3 is . In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, one of R1, R2, and R3 is . In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, R1 is or . In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, R1 is . In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, R1 is . In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, R2 is or . In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, R2 is . In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, R2 is . In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, R3 is or . In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, R3 is . In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, R3 is . In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, X4 is -C(1-6)alkyl. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, X4 is -C(2-8)alkyl. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, X4 is -C(2-4)alkyl. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, X4 is -CH2CH2-. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, X4 is -CH2CH2CH2-. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, X4 is -CH2CH2CH2CH2-. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, X4 is -CH2CH2CH2CH2CH2-. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, X4 is - CH2CH2CH2CH2CH2CH2-. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, X5 is -C(1-6)alkyl. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, X5 is -C(2-8)alkyl. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, X5 is -C(2-4)alkyl. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, X5 is -CH2CH2-. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, X5 is -CH2CH2CH2-. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, X5 is -CH2CH2CH2CH2-. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, X6 is -C(O)O-, -OC(O)-, or -OC(O)O-. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, X6 is -C(O)O-. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, X6 is -OC(O)-. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, X6 is -C(O)NH-. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, X6 is -NHC(O)-. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, X6 is -C(O)S-. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, X6 is -SC(O)-. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, X6 is -OC(O)O-. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, X6 is -OC(O)NH-. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, X6 is -NHC(O)O-. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, X6 is -NHC(O)NH-. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, X7 is -C(1-6)alkyl. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, X7 is -C(2-8)alkyl. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, X7 is -C(2-4)alkyl. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, X7 is -CH2CH2-. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, X7 is -CH2CH2CH2-. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, X7 is -CH2CH2CH2CH2-. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, A is an ionizable nitrogen-containing group. For example, A may be a group comprising a basic, ionizable nitrogen atom that can be converted to a charged group by protonation of the nitrogen with an acid. Nonlimiting examples of ionizable nitrogen- containing groups include NH2, guanidine, amidine, monoalkylamine, dialkylamine, 5- to 6- membered heterocycloalkyl, or 5- to 6-membered nitrogen-containing heteroaryl. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, A is a cationic nitrogen-containing group, i.e., a group comprising a nitrogen atom having a positive charge. Nonlimiting examples of cationic nitrogen- containing groups include ammonium and 5- to 6-membered nitrogen-containing heteroaryl. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof: A is ; and RA1 and RA2 are each independently -C(1-6)alkyl that is optionally substituted with - OH; or RA1 and RA2 are taken together to form a 3- to 6-membered heteroaryl that is optionally substituted with one to three -C(1-3)alkyl groups. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, RA1 and RA2 are each independently -C(1-4)alkyl that is optionally substituted with hydroxyl; or RA1 and RA2 are taken together to form a 5- to 6-membered heteroaryl that is optionally substituted with methyl. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof: A is ; and RA1 and RA2 are each independently -C(1-6)alkyl that is optionally substituted with - OH; or RA1 and RA2 are taken together to form a 3- to 6-membered heterocyclyl that is optionally substituted with one to three -C(1-3)alkyl groups. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, RA1 and RA2 are each independently -C(1-4)alkyl that is optionally substituted with hydroxyl; or RA1 and RA2 are taken together to form a 4- to 6-membered heterocyclyl that is optionally substituted with methyl. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, A is selected from the group consisting of:
Figure imgf000070_0001
wherein RA3 is -C(1-6)alkyl, and An- is a counteranion (e.g., a halide). In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, A is selected from the group consisting of:
Figure imgf000070_0002
Figure imgf000071_0004
wherein RA3 is -C(1-6)alkyl, and An- is a counteranion (e.g., a halide). In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, A is selected from the group consisting of:
Figure imgf000071_0001
In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, A is selected from the group consisting of:
Figure imgf000071_0003
In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, A is selected from the group consisting of:
Figure imgf000071_0002
In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, A is . In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, A is . In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, A is . In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, A is . In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, A is . In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, A is . In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, A is . In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, A is . In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, A is . In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, A is . In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, A is . In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, A is . In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, is selected from the group consisting of Formulas C1-C12:
Figure imgf000073_0001
Figure imgf000074_0001
In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, is selected from the group consisting of Formulas C1-C10. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, has the structure of formula C1. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, has the structure of formula C2. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, has the structure of formula C3. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, has the structure of formula C4. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, has the structure of formula C5. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, has the structure of formula C6. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, has the structure of formula C7. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, has the structure of formula C8. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, has the structure of formula C9. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, has the structure of formula C10. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, has the structure of formula C11. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, has the structure of formula C12. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, is selected from the group consisting of Formulas D1-D48:
Figure imgf000075_0001
Figure imgf000076_0001
Figure imgf000077_0001
Figure imgf000078_0001
In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, is selected from the group consisting of Formulas D1-D40. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, is selected from the group consisting of formulas D1-D4. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, is selected from the group consisting of formulas E1-E12:
Figure imgf000078_0002
Figure imgf000079_0001
In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof,
Figure imgf000079_0003
is selected from the group consisting of formulas E1-E10. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof,
Figure imgf000079_0004
is selected from the group consisting of formulas E1 and E3: In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof,
Figure imgf000079_0005
is selected from the group consisting of formulas F1- F2:
Figure imgf000079_0002
In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, R1 is selected from the group consisting of formulas C1-C12 and E1- E12. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, R1 is selected from the group consisting of formulas C1-C10 and E1-E10. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, R1 is selected from the group consisting of formulas C1-C12. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, R1 is selected from the group consisting of formulas C1-C10. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, R1 has the structure of formula C1. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, R1 is selected from the group consisting of formulas D1-D4. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, R1 is selected from the group consisting of formulas E1-E12. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, R1 is selected from the group consisting of formulas E1-E10. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, R1 is selected from the group consisting of formulas E1 and E3. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, R1 is selected from the group consisting of formulas F1 and F2. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, R2 is selected from the group consisting of formulas C1-C12 and E1- E12. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, R2 is selected from the group consisting of formulas C1-C10 and E1-E10. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, R2 is selected from the group consisting of formulas C1-C12. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, R2 is selected from the group consisting of formulas C1-C10. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, R2 has the structure of formula C1. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, R2 is selected from the group consisting of formulas D1-D4. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, R2 is selected from the group consisting of formulas E1-E12. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, R2 is selected from the group consisting of formulas E1-E10. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, R2 is selected from the group consisting of formulas E1 and E3. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, R2 is selected from the group consisting of formulas F1 and F2. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, R3 is selected from the group consisting of formulas C1-C12 and E1- E12. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, R3 is selected from the group consisting of formulas C1-C10 and E1-E10. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, R3 is selected from the group consisting of formulas C1-C12. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, R3 is selected from the group consisting of formulas C1-C10. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, R3 has the structure of formula C1. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, R3 is selected from the group consisting of formulas D1-D4. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, R3 is selected from the group consisting of formulas E1-E12. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, R3 is selected from the group consisting of formulas E1-E10. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, R3 is selected from the group consisting of formulas E1 and E3. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, R3 is selected from the group consisting of formulas F1 and F2. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, X is selected from the group consisting of:
Figure imgf000081_0001
, In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, X is selected from the group consisting of:
Figure imgf000081_0002
In some embodiments of the compound of Formula (I), or a pharmaceutically
Figure imgf000081_0003
acceptable salt thereof, X is . In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, X is . In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, X is . In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, X is . In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, X is . In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, X is . In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, X is . In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, X is . In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, X is . In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, X is . In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, X is . In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, X is . In some embodiments, the compound of Formula (I) has a structure selected from the group consisting of Formulas (I-a1), (I-a2), (I-a3), and (I-a4), or a pharmaceutically acceptable salt thereof:
Figure imgf000083_0001
In some embodiments, the compound of Formula (I) has a structure according to Formula (I-a1), or a pharmaceutically acceptable salt thereof. In some embodiments, the compound of Formula (I) has a structure according to Formula (I-a2), or a pharmaceutically acceptable salt thereof. In some embodiments, the compound of Formula (I) has a structure according to Formula (I-a3), or a pharmaceutically acceptable salt thereof. In some embodiments, the compound of Formula (I) has a structure according to Formula (I-a4), or a pharmaceutically acceptable salt thereof. In some embodiments, the compound of Formula (I) has a structure selected from the group consisting of Formulas (I-b1), (I-b2), (I-b3), and (I-b4), or a pharmaceutically acceptable salt thereof:
Figure imgf000084_0001
In some embodiments, the compound of Formula (I) has a structure according to Formula (I-b1), or a pharmaceutically acceptable salt thereof. In some embodiments, the compound of Formula (I) has a structure according to Formula (I-b2), or a pharmaceutically acceptable salt thereof. In some embodiments, the compound of Formula (I) has a structure according to Formula (I-b3), or a pharmaceutically acceptable salt thereof. In some embodiments, the compound of Formula (I) has a structure according to Formula (I-b4), or a pharmaceutically acceptable salt thereof. In some embodiments, the compound of Formula (I) has a structure selected from the group consisting of Formulas (I-c1), (I-c2), (I-c3), and (I-c4), or a pharmaceutically acceptable salt thereof:
Figure imgf000084_0002
Figure imgf000085_0002
In some embodiments, the compound of Formula (I) has a structure according to Formula (I-c1), or a pharmaceutically acceptable salt thereof. In some embodiments, the compound of Formula (I) has a structure according to Formula (I-c2), or a pharmaceutically acceptable salt thereof. In some embodiments, the compound of Formula (I) has a structure according to Formula (I-c3), or a pharmaceutically acceptable salt thereof. In some embodiments, the compound of Formula (I) has a structure according to Formula (I-c4), or a pharmaceutically acceptable salt thereof. In some embodiments, the compound of Formula (I) has a structure selected from the group consisting of Formulas (I-d1), (I-d2), (I-d3), and (I-d4), or a pharmaceutically acceptable salt thereof:
Figure imgf000085_0001
In some embodiments, the compound of Formula (I) has a structure according to Formula (I-d1), or a pharmaceutically acceptable salt thereof. In some embodiments, the compound of Formula (I) has a structure according to Formula (I-d2), or a pharmaceutically acceptable salt thereof. In some embodiments, the compound of Formula (I) has a structure according to Formula (I-d3), or a pharmaceutically acceptable salt thereof. In some embodiments, the compound of Formula (I) has a structure according to Formula (I-d4), or a pharmaceutically acceptable salt thereof. In some embodiments, the compound of Formula (I) has a structure selected from the group consisting of Formulas (I-e1), (I-e2), (I-e3), and (I-e4), or a pharmaceutically acceptable salt thereof:
Figure imgf000086_0001
In some embodiments, the compound of Formula (I) has a structure according to Formula (I-e1), or a pharmaceutically acceptable salt thereof. In some embodiments, the compound of Formula (I) has a structure according to Formula (I-e2), or a pharmaceutically acceptable salt thereof. In some embodiments, the compound of Formula (I) has a structure according to Formula (I-e3), or a pharmaceutically acceptable salt thereof. In some embodiments, the compound of Formula (I) has a structure according to Formula (I-e4), or a pharmaceutically acceptable salt thereof. In some embodiments, the compound of Formula (I) has a structure selected from the group consisting of Formulas (I-f1), (I-f2), (I-f3), and (I-f4), or a pharmaceutically acceptable salt thereof:
Figure imgf000086_0002
Figure imgf000087_0001
In some embodiments, the compound of Formula (I) has a structure according to Formula (I-f1), or a pharmaceutically acceptable salt thereof. In some embodiments, the compound of Formula (I) has a structure according to Formula (I-f2), or a pharmaceutically acceptable salt thereof. In some embodiments, the compound of Formula (I) has a structure according to Formula (I-f3), or a pharmaceutically acceptable salt thereof. In some embodiments, the compound of Formula (I) has a structure according to Formula (I-f4), or a pharmaceutically acceptable salt thereof. In some embodiments, the compound of Formula (I) has a structure selected from the group consisting of Formulas (I-g1), (I-g2), (I-g3), and (I-g4), or a pharmaceutically acceptable salt thereof:
Figure imgf000087_0002
In some embodiments, the compound of Formula (I) has a structure according to Formula (I-g1), or a pharmaceutically acceptable salt thereof. In some embodiments, the compound of Formula (I) has a structure according to Formula (I-g2), or a pharmaceutically acceptable salt thereof. In some embodiments, the compound of Formula (I) has a structure according to Formula (I-g3), or a pharmaceutically acceptable salt thereof. In some embodiments, the compound of Formula (I) has a structure according to Formula (I-g4), or a pharmaceutically acceptable salt thereof. In some embodiments, the compound of Formula (I) has a structure selected from the group consisting of Formulas (I-h1), (I-h2), (I-h3), and (I-h4), or a pharmaceutically acceptable salt thereof:
Figure imgf000088_0001
In some embodiments, the compound of Formula (I) has a structure according to Formula (I-h1), or a pharmaceutically acceptable salt thereof. In some embodiments, the compound of Formula (I) has a structure according to Formula (I-h2), or a pharmaceutically acceptable salt thereof. In some embodiments, the compound of Formula (I) has a structure according to Formula (I-h3), or a pharmaceutically acceptable salt thereof. In some embodiments, the compound of Formula (I) has a structure according to Formula (I-h4), or a pharmaceutically acceptable salt thereof. In some embodiments, the compound of Formula (I) has a structure selected from the group consisting of Formulas (I-i1), (I-i2), (I-i3), and (I-i4), or a pharmaceutically acceptable salt thereof:
Figure imgf000088_0002
In some embodiments, the compound of Formula (I) has a structure according to Formula (I-i1), or a pharmaceutically acceptable salt thereof. In some embodiments, the compound of Formula (I) has a structure according to Formula (I-i2), or a pharmaceutically acceptable salt thereof. In some embodiments, the compound of Formula (I) has a structure according to Formula (I-i3), or a pharmaceutically acceptable salt thereof. In some embodiments, the compound of Formula (I) has a structure according to Formula (I-i4), or a pharmaceutically acceptable salt thereof. In some embodiments, the compound of Formula (I) has a structure selected from the group consisting of Formulas (I-j1), (I-j2), (I-j3), and (I-j4), or a pharmaceutically acceptable salt thereof:
Figure imgf000089_0001
In some embodiments, the compound of Formula (I) has a structure according to Formula (I-j1), or a pharmaceutically acceptable salt thereof. In some embodiments, the compound of Formula (I) has a structure according to Formula (I-j2), or a pharmaceutically acceptable salt thereof. In some embodiments, the compound of Formula (I) has a structure according to Formula (I-j3), or a pharmaceutically acceptable salt thereof. In some embodiments, the compound of Formula (I) has a structure according to Formula (I-j4), or a pharmaceutically acceptable salt thereof. In some embodiments, the compound of Formula (I) has a structure selected from the group consisting of Formulas (I-k1), (I-k2), (I-k3), and (I-k4), or a pharmaceutically acceptable salt thereof:
Figure imgf000089_0002
Figure imgf000090_0001
In some embodiments, the compound of Formula (I) has a structure according to Formula (I-k1), or a pharmaceutically acceptable salt thereof. In some embodiments, the compound of Formula (I) has a structure according to Formula (I-k2), or a pharmaceutically acceptable salt thereof. In some embodiments, the compound of Formula (I) has a structure according to Formula (I-k3), or a pharmaceutically acceptable salt thereof. In some embodiments, the compound of Formula (I) has a structure according to Formula (I-k4), or a pharmaceutically acceptable salt thereof. In some embodiments, the compound of Formula (I) has a structure selected from the group consisting of Formulas (I-l1), (I-l2), (I-l3), and (I-l4), or a pharmaceutically acceptable salt thereof:
Figure imgf000090_0002
In some embodiments, the compound of Formula (I) has a structure according to Formula (I-l1), or a pharmaceutically acceptable salt thereof. In some embodiments, the compound of Formula (I) has a structure according to Formula (I-l2), or a pharmaceutically acceptable salt thereof. In some embodiments, the compound of Formula (I) has a structure according to Formula (I-l3), or a pharmaceutically acceptable salt thereof. In some embodiments, the compound of Formula (I) has a structure according to Formula (I-l4), or a pharmaceutically acceptable salt thereof. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, RX is -H or -CH3. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, RX is -H. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, RX is -CH3. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, n is 1 or 2. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, n is 1. In some embodiments, the compound of Formula (I) has a structure according to Formula (I-a1), (I-b1), (I-c1), (I-d1), (I-e1), (I-f1), (I-g1), (I-h1), (I-i1), (I-j1), (I-k1), or (I-l1). In some embodiments, the compound of Formula (I) has a structure according to Formula (I-a1), (I-b1), (I-c1), (I-d1), (I-e1), (I-f1), (I-g1), (I-h1), or (I-i1). In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, n is 2. In some embodiments, the compound of Formula (I) has a structure according to Formula (I-a2), (I-b2), (I-c2), (I-d2), (I-e2), (I-f2), (I-g2), (I-h2), (I-i2), (I-j2), (I-k2), or (I-l2). In some embodiments, the compound of Formula (I) has a structure according to Formula (I-a2), (I-b2), (I-c2), (I-d2), (I-e2), (I-f2), (I-g2), (I-h2), or (I-i2). In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, n is 3. In some embodiments, the compound of Formula (I) has a structure according to Formula (I-a3), (I-b3), (I-c3), (I-d3), (I-e3), (I-f3), (I-g3), (I-h3), (I-i3), (I-j3), (I-k3), or (I-l3). In some embodiments, the compound of Formula (I) has a structure according to Formula (I-a3), (I-b3), (I-c3), (I-d3), (I-e3), (I-f3), (I-g3), (I-h3), or (I-i3). In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, n is 4. In some embodiments, the compound of Formula (I) has a structure according to Formula (I-a4), (I-b4), (I-c4), (I-d4), (I-e4), (I-f4), (I-g4), (I-h4), (I-i4), (I-j4), (I-k4), or (I-l4). In some embodiments, the compound of Formula (I) has a structure according to Formula (I-a4), (I-b4), (I-c4), (I-d4), (I-e4), (I-f4), (I-g4), (I-h4), or (I-i4). In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, R1 and R2 are independently selected from the group consisting of formulas A1-A21; and R3 is selected from the group consisting of formulas C1-C12 and E1- E12. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, R1 and R2 are independently selected from the group consisting of formulas A1- A21; and R3 is selected from the group consisting of formulas C1-C10 and E1-E10. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, R1 and R2 are independently selected from the group consisting of formulas A1-A21; and R3 is selected from the group consisting of formulas C1-C12. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, R1 and R2 are independently selected from the group consisting of formulas A1-A21; and R3 is selected from the group consisting of formulas C1-C10. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, R1 and R2 are independently selected from the group consisting of formulas A1, A3, and A7-A18; and R3 is selected from the group consisting of formulas C1-C12. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, R1 and R2 are independently selected from the group consisting of formulas A13-A15; and R3 has the structure of formula C1. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, R1 and R2 are independently selected from the group consisting of formulas B1-B58; and R3 is selected from the group consisting of formulas D1-D48, F1, and F2. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, R1 and R2 are independently selected from the group consisting of formulas B1- B56; and R3 is selected from the group consisting of formulas D1-D40, F1, and F2. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, R1 and R2 are independently selected from the group consisting of formulas B1, B2, B3, B4, B5, B7, B10, B12, B15, B16, B18, B21, B24, B28, B31, B33, B36, B37, B40, B41, B43, B44, B46, B47, B48, B49, B50, and B51; and R3 is selected from the group consisting of formulas D1-D48. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, R1 and R2 are independently selected from the group consisting of formulas B1, B2, B3, B4, B5, B7, B10, B12, B15, B16, B18, B21, B24, B28, B31, B33, B36, B37, B40, B41, B43, B44, B46, B47, B48, B49, B50, and B51; and R3 is selected from the group consisting of formulas D1-D40. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, R1 and R2 are independently selected from the group consisting of formulas B1, B2, B3, B4, B5, B7, B10, B12, B15, B16, B18, B21, B24, B28, B31, B33, B36, B37, B40, B41, B43, B44, B46, B47, B48, B49, B50, and B51; and R3 is selected from the group consisting of formulas D1-D4. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, R1 and R2 are independently selected from the group consisting of formulas B1, B2, B5, B33-B53, B57, and B58; and R3 is selected from the group consisting of formulas D1-D48. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, R1 and R2 are independently selected from the group consisting of formulas B33-B51; and R3 is selected from the group consisting of formulas D1-D48. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, R1 and R2 are independently selected from the group consisting of formulas B33-B51; and R3 is selected from the group consisting of formulas D1-D40. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, R1 and R2 are independently selected from the group consisting of formulas B36-B44; and R3 is selected from the group consisting of formulas D1-D48. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, R1 and R2 are independently selected from the group consisting of formulas B36-B44; and R3 is selected from the group consisting of formulas D1-D4. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, R1 and R3 are independently selected from the group consisting of formulas A1-A21; and R2 is selected from the group consisting of formulas C1-C12 and E1- E12. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, R1 and R3 are independently selected from the group consisting of formulas A1- A21; and R2 is selected from the group consisting of formulas C1-C10 and E1-E10. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, R1 and R3 are independently selected from the group consisting of formulas A1-A21; and R2 is selected from the group consisting of formulas C1-C12. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, R1 and R3 are independently selected from the group consisting of formulas A1-A21; and R2 is selected from the group consisting of formulas C1-C10. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, R1 and R3 are independently selected from the group consisting of formulas A1, A3, and A7-A18; and R2 is selected from the group consisting of formulas C1-C12. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, R1 and R3 are independently selected from the group consisting of formulas A13-A15; and R2 has the structure of formula C1. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, R1 and R3 are independently selected from the group consisting of formulas B1-B58; and R2 is selected from the group consisting of formulas D1-D48, F1, and F2. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, R1 and R3 are independently selected from the group consisting of formulas B1- B56; and R2 is selected from the group consisting of formulas D1-D40, F1, and F2. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, R1 and R3 are independently selected from the group consisting of formulas B1, B2, B3, B4, B5, B7, B10, B12, B15, B16, B18, B21, B24, B28, B31, B33, B36, B37, B40, B41, B43, B44, B46, B47, B48, B49, B50, and B51; and R2 is selected from the group consisting of formulas D1-D48. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, R1 and R3 are independently selected from the group consisting of formulas B1, B2, B3, B4, B5, B7, B10, B12, B15, B16, B18, B21, B24, B28, B31, B33, B36, B37, B40, B41, B43, B44, B46, B47, B48, B49, B50, and B51; and R2 is selected from the group consisting of formulas D1-D40. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, R1 and R3 are independently selected from the group consisting of formulas B1, B2, B3, B4, B5, B7, B10, B12, B15, B16, B18, B21, B24, B28, B31, B33, B36, B37, B40, B41, B43, B44, B46, B47, B48, B49, B50, and B51; and R2 is selected from the group consisting of formulas D1-D4. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, R1 and R3 are independently selected from the group consisting of formulas B1, B2, B5, B33-B53, B57, and B58; and R2 is selected from the group consisting of formulas D1-D48. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, R1 and R3 are independently selected from the group consisting of formulas B33-B51; and R2 is selected from the group consisting of formulas D1-D48. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, R1 and R3 are independently selected from the group consisting of formulas B33-B51; and R2 is selected from the group consisting of formulas D1-D40. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, R1 and R3 are independently selected from the group consisting of formulas B36-B44; and R2 is selected from the group consisting of formulas D1-D48. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, R1 and R3 are independently selected from the group consisting of formulas B36-B44; and R2 is selected from the group consisting of formulas D1-D4. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, R2 and R3 are independently selected from the group consisting of formulas A1-A21; and R1 is selected from the group consisting of formulas C1-C12 and E1- E12. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, R2 and R3 are independently selected from the group consisting of formulas A1- A21; and R1 is selected from the group consisting of formulas C1-C10 and E1-E10. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, R2 and R3 are independently selected from the group consisting of formulas A1-A21; and R1 is selected from the group consisting of formulas C1-C12. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, R2 and R3 are independently selected from the group consisting of formulas A1-A21; and R1 is selected from the group consisting of formulas C1-C10. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, R2 and R3 are independently selected from the group consisting of formulas A1, A3, and A7-A18; and R1 is selected from the group consisting of formulas C1-C12. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, R2 and R3 are independently selected from the group consisting of formulas A13-A15; and R1 has the structure of formula C1. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, R2 and R3 are independently selected from the group consisting of formulas B1-B58; and R1 is selected from the group consisting of formulas D1-D48, F1, and F2. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, R2 and R3 are independently selected from the group consisting of formulas B1- B56; and R1 is selected from the group consisting of formulas D1-D40, F1, and F2. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, R2 and R3 are independently selected from the group consisting of formulas B1, B2, B3, B4, B5, B7, B10, B12, B15, B16, B18, B21, B24, B28, B31, B33, B36, B37, B40, B41, B43, B44, B46, B47, B48, B49, B50, and B51; and R1 is selected from the group consisting of formulas D1-D48. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, R2 and R3 are independently selected from the group consisting of formulas B1, B2, B3, B4, B5, B7, B10, B12, B15, B16, B18, B21, B24, B28, B31, B33, B36, B37, B40, B41, B43, B44, B46, B47, B48, B49, B50, and B51; and R1 is selected from the group consisting of formulas D1-D40. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, R2 and R3 are independently selected from the group consisting of formulas B1, B2, B3, B4, B5, B7, B10, B12, B15, B16, B18, B21, B24, B28, B31, B33, B36, B37, B40, B41, B43, B44, B46, B47, B48, B49, B50, and B51; and R1 is selected from the group consisting of formulas D1-D4. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, R2 and R3 are independently selected from the group consisting of formulas B1, B2, B5, B33-B53, B57, and B58; and R1 is selected from the group consisting of formulas D1-D48. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, R2 and R3 are independently selected from the group consisting of formulas B33-B51; and R1 is selected from the group consisting of formulas D1-D48. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, R2 and R3 are independently selected from the group consisting of formulas B33-B51; and R1 is selected from the group consisting of formulas D1-D40. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, R2 and R3 are independently selected from the group consisting of formulas B36-B44; and R1 is selected from the group consisting of formulas D1-D48. In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, R2 and R3 are independently selected from the group consisting of formulas B36-B44; and R1 is selected from the group consisting of formulas D1-D4. In some embodiments, the compound of Formula (I) has a structure selected from the group consisting of:
Figure imgf000096_0001
Figure imgf000097_0001
Figure imgf000098_0001
Figure imgf000099_0001
Figure imgf000100_0001
Figure imgf000101_0001
Figure imgf000102_0001
Figure imgf000103_0001
Figure imgf000104_0001
Figure imgf000105_0001
Figure imgf000106_0001
Figure imgf000107_0001
Figure imgf000108_0001
Figure imgf000109_0001
Figure imgf000110_0001
Figure imgf000111_0001
Figure imgf000112_0001
Figure imgf000113_0001
Figure imgf000114_0001
Figure imgf000115_0001
Figure imgf000116_0001
Figure imgf000117_0001
Figure imgf000118_0001
or a pharmaceutically acceptable salt thereof. In some embodiments, the compound of Formula (I) has a structure selected from the group consisting of:
Figure imgf000118_0002
Figure imgf000119_0001
Figure imgf000120_0001
Figure imgf000121_0001
Figure imgf000122_0001
or a pharmaceutically acceptable salt thereof. III. Ionizable/Cationic Lipid Synthesis The ionizable or cationic lipids of the present disclosure (e.g., compounds of Formula (I)) can be prepared according to methods known in the art. Nonlimiting and exemplary synthetic methods are disclosed herein. For example, Scheme A provides exemplary synthetic routes for preparing certain ionizable lipids of the present disclosure. Malic acid (1) can be esterified with methanol under acidic conditions to provide dimethyl malate (2), which can then be reacted with an alcohol (3) to provide intermediate (4). Intermediate (4) can be reacted with a carboxylic acid (5) in the presence of a coupling reagent, such as EDCI, to yield the ester product (8). Alternately, intermediate (4) can be reacted with a carbonate compound (6) to afford the carbonate product (9). Intermediate (4) can also be reacted with a carbamate compound (7), to afford the carbamate product (10).
Scheme A (a is 1-7; R is -C(6-24)alkyl, -C(6-24)alkenyl, -C(6-24)alkynyl, , and
Figure imgf000124_0001
wherein the -C(6-24)alkyl, -C(6-24)alkenyl, and -C(6-24)alkynyl are each optionally substituted with one to six groups independently selected from halo, -OC(1-6)alkyl, -OC(2- 6)alkenyl, -SC(1-6)alkyl, -SC(2-6)alkenyl, and -C(O)OC(1-6)alkyl) Scheme B provides an exemplary synthetic route for preparing certain ionizable lipids of the present disclosure. N-Boc-(L)-aspartic acid (11) can be reacted with an alcohol (3) in the presence of a coupling reagent, such as EDCI, to provide intermediate (12), which can be deprotected with TFA to afford amine (13). Amine (13) can then be reacted with 4- nitrophenylchloroformate (14) to yield intermediate (15), which can be reacted with an alcohol (16) to afford the carbamate product (17).
Figure imgf000124_0002
Scheme B (a is 1-7; R is -C(6-24)alkyl, -C(6-24)alkenyl, -C(6-24)alkynyl, , and
Figure imgf000124_0003
wherein the -C(6-24)alkyl, -C(6-24)alkenyl, and -C(6-24)alkynyl are each optionally substituted with one to six groups independently selected from halo, -OC(1-6)alkyl, -OC(2- 6)alkenyl, -SC(1-6)alkyl, -SC(2-6)alkenyl, and -C(O)OC(1-6)alkyl) Scheme C provides exemplary synthetic routes for preparing certain ionizable lipids of the present disclosure. α-Ketoglutaric acid (18) can be reacted with alcohol (3) under acidic conditions to afford diester (19), which can be reduced with a reducing agent, such as NaBH4, to provide intermediate (20). Intermediate (20) can be reacted with a carboxylic acid (5) and a coupling reagent like EDCI to yield the ester product (22). Alternately, intermediate (20) can be reacted sequentially with 4-nitrophenylchloroformate (14) and an alcohol (16) to afford the carbonate product (23). Intermediate (20) can also be reacted sequentially with 4- nitrophenylchloroformate (14) and an amine (21) to afford the carbonate product (24). Scheme C (a is 1-7; R is -C(6-24)alkyl, -C(6-24)alkenyl, -C(6-24)alkynyl, , and
Figure imgf000125_0001
wherein the -C(6-24)alkyl, -C(6-24)alkenyl, and -C(6-24)alkynyl are each optionally substituted with one to six groups independently selected from halo, -OC(1-6)alkyl, -OC(2- 6)alkenyl, -SC(1-6)alkyl, -SC(2-6)alkenyl, and -C(O)OC(1-6)alkyl) Scheme D provides an exemplary synthetic route for preparing certain ionizable lipids of the present disclosure. (tert-Butoxycarbonyl)-L-glutamic acid (25) can be reacted with an alcohol (3) in the presence of a coupling reagent, such as EDCI, to provide intermediate (26), which can be deprotected with TFA to afford amine (27). Amine (27) can be reacted with 4- nitrophenylchloroformate (14) to yield intermediate (28), which can then be reacted with an alcohol (16) to afford the carbamate product (29). Scheme D (a is 1-7; R is -C(6-24)alkyl, -C(6-24)alkenyl, -C(6-24)alkynyl, , and
Figure imgf000126_0001
wherein the -C(6-24)alkyl, -C(6-24)alkenyl, and -C(6-24)alkynyl are each optionally substituted with one to six groups independently selected from halo, -OC(1-6)alkyl, -OC(2- 6)alkenyl, -SC(1-6)alkyl, -SC(2-6)alkenyl, and -C(O)OC(1-6)alkyl) Scheme E provides an exemplary synthetic route for preparing certain ionizable lipids of the present disclosure. Dimethyl malate (2) can be reacted with a benzyl protected diol such as compound (30) under acidic conditions to afford intermediate (31). Intermediate (31) can be protected with a hydroxyl protecting group, such as TBDPS, to afford intermediate (32) before deprotecting the benzyl groups (e.g., under hydrolytic conditions) to provide intermediate (33). Intermediate (33) can be oxidized to afford diacid (34), which can then be reacted with an alcohol (35) in the presence of a coupling reagent, such as EDCI, to provide intermediate (36). The hydroxyl group of intermediate (36) can be deprotected to provide intermediate (37), which can then be reacted with a carboxylic acid (5) to afford the product (38'). Alternately, intermediate (37) can be reacted with carbamate (5’) in the presence of a base, such as DIPEA, to afford the product (38’).
Scheme E (a is 1-7; R’ is -C(5-24)alkyl, -C(5-24)alkenyl, or -C(5-24)alkynyl, each of which is optionally substituted by one to six groups independently selected from halo, -OC(1-6)alkyl, - OC(2-6)alkenyl, -SC(1-6)alkyl, -SC(2-6)alkenyl, and -C(O)OC(1-6)alkyl) Scheme F provides an exemplary synthetic route for preparing certain ionizable lipids of the present disclosure. (S)-2-(2,2-Dimethyl-5-oxo-1,3-dioxolan-4-yl)acetic acid (39) can be protected with a benzyl group to afford intermediate (40), the acetal moiety of which can then be cleaved under acidic conditions to provide intermediate (41). Intermediate (41) can be reacted with an alcohol (3) under acidic conditions to provide intermediate (42), the benzyl group of which can then be deprotected (e.g., under hydrolytic conditions) to afford intermediate (43). Intermediate (43) can be reacted sequentially with an alcohol (16) and a carboxylic acid (45) in the presence of a coupling reagent such as EDCI to provide the product (46). Scheme F (a is 1-7; R is -C(6-24)alkyl, -C(6-24)alkenyl, -C(6-24)alkynyl, , and
Figure imgf000128_0001
, wherein the -C(6-24)alkyl, -C(6-24)alkenyl, and -C(6-24)alkynyl are each optionally substituted with one to six groups independently selected from halo, -OC(1-6)alkyl, -OC(2- 6)alkenyl, -SC(1-6)alkyl, -SC(2-6)alkenyl, and -C(O)OC(1-6)alkyl) Scheme G provides an exemplary synthetic route for preparing certain ionizable lipids of the present disclosure. An alcohol (3) can be reacted with nitrophenylchloroformate (14) to yield intermediate (47), which can then be reacted with intermediate (44) to afford the carbamate product (48).
Scheme G (a is 1-7; R is -C(6-24)alkyl, -C(6-24)alkenyl, -C(6-24)alkynyl, , and
Figure imgf000129_0001
wherein the -C(6-24)alkyl, -C(6-24)alkenyl, and -C(6-24)alkynyl are each optionally substituted with one to six groups independently selected from halo, -OC(1-6)alkyl, -OC(2- 6)alkenyl, -SC(1-6)alkyl, -SC(2-6)alkenyl, and -C(O)OC(1-6)alkyl) Scheme H provides an exemplary synthetic route for preparing certain ionizable lipids of the present disclosure. An alcohol (3) can be reacted with (S)-2-(2,2-dimethyl-5-oxo-1,3- dioxolan-4-yl)acetic acid (39) in the presence of a coupling reagent to provide intermediate (49), the acetal moiety of which can be cleaved under acidic conditions to afford intermediate (50). Intermediate (50) can then be reacted sequentially with an alcohol (16) and a carboxylic acid (45) in the presence of a coupling reagent such as EDCI to provide the product (52). Scheme H (a is 1-7; R is -C(6-24)alkyl, -C(6-24)alkenyl, -C(6-24)alkynyl, , and
Figure imgf000129_0002
wherein the -C(6-24)alkyl, -C(6-24)alkenyl, and -C(6-24)alkynyl are each optionally substituted with one to six groups independently selected from halo, -OC(1-6)alkyl, -OC(2- 6)alkenyl, -SC(1-6)alkyl, -SC(2-6)alkenyl, and -C(O)OC(1-6)alkyl) Scheme I provides an exemplary synthetic route for preparing certain ionizable lipids of the present disclosure. An alcohol (3) can be reacted with nitrophenylchloroformate (14) to yield intermediate (47), which can then be reacted with intermediate (51) to afford the carbamate product (53).
Scheme I (a is 1-7; R is -C(6-24)alkyl, -C(6-24)alkenyl, -C(6-24)alkynyl, , and
Figure imgf000130_0001
wherein the -C(6-24)alkyl, -C(6-24)alkenyl, and -C(6-24)alkynyl are each optionally substituted with one to six groups independently selected from halo, -OC(1-6)alkyl, -OC(2- 6)alkenyl, -SC(1-6)alkyl, -SC(2-6)alkenyl, and -C(O)OC(1-6)alkyl) Scheme J provides an exemplary synthetic route for preparing certain ionizable lipids of the present disclosure. A diol (54) and 2-mercaptosuccinic acid (55) can be reacted in the presence of zinc chloride to provide intermediate (56). Intermediate (56) can then be reacted with dipyridyl disulfide to provide disulfide (57), which can then be reacted with an acyl chloride (58) to provide intermediate (59). Finally, intermediate (59) can be reacted with a thiol (60) to provide the disulfide product (61).
Figure imgf000131_0001
Scheme J (a is 1-7; R’ is -C(5-24)alkyl, -C(5-24)alkenyl, or -C(5-24)alkynyl, each of which is optionally substituted by one to six groups independently selected from halo, -OC(1-6)alkyl, - OC(2-6)alkenyl, -SC(1-6)alkyl, -SC(2-6)alkenyl, and -C(O)OC(1-6)alkyl) IV. Lipid Nanoparticles Compositions The present disclosure also provides a composition comprising a lipid nanoparticle (LNP), wherein the LNP comprises an ionizable or cationic lipid of the present disclosure (e.g., a lipid of Formula (I)). An ionizable or cationic lipid, such as those described herein, affords a positively charged environment at low pH and facilitates efficient encapsulation of a negatively charged drug substance (e.g., mRNA) in the LNP. In some embodiments, the LNP further comprises at least one of a structural lipid, a helper lipid, or a stealth lipid. In some embodiments, the LNP comprises an ionizable or cationic lipid of the present disclosure and a structural lipid. In some embodiments, the LNP comprises an ionizable or cationic lipid of the present disclosure and a helper lipid. In some embodiments, the LNP comprises an ionizable or cationic lipid of the present disclosure and a stealth lipid. In some embodiments, the LNP comprises an ionizable or cationic lipid of the present disclosure, a structural lipid, a helper lipid, and a stealth lipid. A. Structural Lipids A structural lipid component provides stability to the lipid bilayer structure within the lipid nanoparticle. In some embodiments, the LNP comprises one or more structural lipid. In some embodiments, the structural lipid is a cholesterol-based lipid. Suitable cholesterol-based lipids include, for example: DC-Choi (N,N-dimethyl-N-ethylcarboxamidocholesterol), l,4- bis(3-N-oleylamino-propyl)piperazine (Gao et al., Biochem Biophys Res Comm. (1991) 179:280; Wolf et al., BioTechniques (1997) 23:139; U.S. Pat.5,744,335), imidazole cholesterol ester (“ICE”; WO2011/068810), sitosterol (22,23-dihydrostigmasterol), β- sitosterol, sitostanol, fucosterol, stigmasterol (stigmasta-5,22-dien-3-ol), ergosterol, desmosterol (3ß-hydroxy-5,24-cholestadiene), lanosterol (8,24-lanostadien-3b-ol), 7- dehydrocholesterol (Δ5,7-cholesterol), dihydrolanosterol (24,25-dihydrolanosterol), zymosterol (5α-cholesta-8,24-dien-3ß-ol), lathosterol (5α-cholest-7-en-3ß-ol), diosgenin ((3β,25R)-spirost-5-en-3-ol), campesterol (campest-5-en-3ß-ol), campestanol (5a-campestan- 3b-ol), 24-methylene cholesterol (5,24(28)-cholestadien-24-methylen-3ß-ol), cholesteryl margarate (cholest-5-en-3ß-yl heptadecanoate), cholesteryl oleate, cholesteryl stearate and other modified forms of cholesterol. In some embodiments, the structural lipid is cholesterol. B. Stealth Lipids A stealth lipid component provides control over particle size and stability of the nanoparticle. The addition of such components may prevent complex aggregation and provide a means for increasing circulation lifetime and increasing the delivery of a lipid- nucleic acid pharmaceutical composition to target tissues. In some embodiments, the stealth lipid is a polyethylene glycol-conjugated (PEGylated) lipid. These components may be selected to rapidly exchange out of the pharmaceutical composition in vivo (see, e.g., U.S. Pat.5,885,613). Contemplated PEGylated lipids include, but are not limited to, a polyethylene glycol (PEG) chain of up to 5 kDa in length covalently attached to a lipid with alkyl chain(s) of C6- C20 (e.g., C8, C10, C12, C14, C16, or C18) length, such as a derivatized ceramide (e.g., N- octanoyl-sphingosine-1-[succinyl(methoxypolyethylene glycol)] (C8 PEG ceramide)). In some embodiments, the PEGylated lipid is 1,2-dimyristoyl-rac-glycero-3- methoxypolyethylene glycol (DMG-PEG); 1,2-distearoyl-sn-glycero-3- phosphoethanolamine-polyethylene glycol (DSPE- PEG); 1,2-dilauroyl-sn-glycero-3- phosphoethanolamine-polyethylene glycol (DLPE-PEG); or 1,2-distearoyl-rac-glycero- polyethelene glycol (DSG-PEG). In some embodiments, the PEG has a high molecular weight, e.g., 2000-2400 g/mol. In some embodiments, the PEG is PEG2000 (or PEG-2K). In some embodiments, the PEGylated lipid is DMG-PEG2000, DSPE-PEG2000, DLPE-PEG2000, DSG-PEG2000, or C8 PEG2000. In some embodiments, the PEGylated lipid is dimyristoyl-PEG2000 (DMG- PEG2000). In some embodiments, the stealth lipid is a polyoxazoline polymer-conjugated lipid. Polyoxazoline polymer-conjugated lipids suitable for the LNP compositions of the present disclosure are described, for example, in WO2022/173667 and WO2023/031394. In some embodiments, the stealth lipid is a polysarcosine-conjugated (pSar) lipid. In some embodiment, the polysarcosine comprises 25-45 sarcosine units. In some embodiment, the polysarcosine comprises 25 sarcosine units. In some embodiment, the polysarcosine comprises 35 sarcosine units. In some embodiment, the polysarcosine comprises 45 sarcosine units. Nonlimiting examples of pSar lipids include N-tetradecyl-pSar25, N-hexadecyl- pSar25, N-octadecyl-pSar25, N-dodecyl-pSar25, 1,2-dimyristoyl-sn-glycero-3-succinyl-N- polysarcosine-25 (DMG-pSar25), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N- polysarcosine-25 (18:1 PE (DOPE) pSar25), N,N-ditetradecylamine-N- succinyl[methyl(polysarcosine)45], N,N-ditetradecylamine-N- succinyl[methyl(polysarcosine)35], and N,N-ditetradecyl-polysarcosine-25. Further examples of pSar lipids suitable for the LNP compositions of the present disclosure are described in WO2020/070040. C. Helper Lipids A helper lipid enhances the structural stability of the LNP and helps the LNP in endosomal escape. A helper lipid may improve uptake and release of an mRNA drug payload encapsulated in the LNP. In some embodiments, the helper lipid is a zwitterionic lipid. Without wishing to be bound by theory, the helper lipid can have fusogenic properties for enhancing uptake and release of the drug payload. Examples of helper lipids are 1,2-dioleoyl- SN-glycero-3-phosphoethanolamine (DOPE); 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC); 1,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS); 1,2-dielaidoyl-sn-glycero-3- phosphoethanolamine (DEPE); and 1,2-dioleoyl-sn-glycero-3-phosphocholine (DPOC), dipalmitoylphosphatidylcholine (DPPC), 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC), 1,2-Distearoylphosphatidylethanolamine (DSPE), and 1,2-dilauroyl-sn-glycero-3- phosphoethanolamine (DLPE). Other exemplary helper lipids are dioleoylphosphatidylcholine (DOPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (POPE), dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-l- carboxylate (DOPE-mal), dipalmitoyl phosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), phosphatidylserine, sphingolipids, cerebrosides, gangliosides, 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, l-stearoyl-2-oleoyl- phosphatidyethanolamine (SOPE), or a combination thereof. In particular embodiments, the helper lipid is 1,2-dioleoyl-SN-glycero-3- phosphoethanolamine (DOPE). D. Combinations of Lipid Components and Molar Ratios In some embodiments, the LNP comprises a lipid of Formula (I) and a structural lipid. In some embodiments, the LNP comprises a lipid of Formula (I) and a stealth lipid. In some embodiments, the LNP comprises a lipid of Formula (I) and a helper lipid. In some embodiments, the LNP comprises a lipid of Formula (I), a structural lipid, a stealth lipid, and a helper lipid. In some embodiments, the LNP comprises a lipid of Formula (I-a1), (I-b1), (I-c1), (I- d1), (I-e1), (I-f1), (I-g1), (I-h1), or (I-i1) and a structural lipid. In some embodiments, the LNP comprises a lipid of Formula (I-a1), (I-b1), (I-c1), (I-d1), (I-e1), (I-f1), (I-g1), (I-h1), or (I-i1) and a stealth lipid. In some embodiments, the LNP comprises a lipid of Formula (I-a1), (I-b1), (I-c1), (I-d1), (I-e1), (I-f1), (I-g1), (I-h1), or (I-i1) and a helper lipid. In some embodiments, the LNP comprises a lipid of Formula (I-a1), (I-b1), (I-c1), (I-d1), (I-e1), (I- f1), (I-g1), (I-h1), or (I-i1); a structural lipid; a stealth lipid; and a helper lipid. In some embodiments, the LNP comprises a lipid of Formula (I-a2), (I-b2), (I-c2), (I- d2), (I-e2), (I-f2), (I-g2), (I-h2), or (I-i2) and a structural lipid. In some embodiments, the LNP comprises a lipid of Formula (I-a2), (I-b2), (I-c2), (I-d2), (I-e2), (I-f2), (I-g2), (I-h2), or (I-i2) and a stealth lipid. In some embodiments, the LNP comprises a lipid of Formula (I-a2), (I-b2), (I-c2), (I-d2), (I-e2), (I-f2), (I-g2), (I-h2), or (I-i2) and a helper lipid. In some embodiments, the LNP comprises a lipid of Formula (I-a2), (I-b2), (I-c2), (I-d2), (I-e2), (I- f2), (I-g2), (I-h2), or (I-i2); a structural lipid; a stealth lipid; and a helper lipid. In some embodiments, the LNP comprises a lipid of Formula (I-a3), (I-b3), (I-c3), (I- d3), (I-e3), (I-f3), (I-g3), (I-h3), or (I-i3) and a structural lipid. In some embodiments, the LNP comprises a lipid of Formula (I-a3), (I-b3), (I-c3), (I-d3), (I-e3), (I-f3), (I-g3), (I-h3), or (I-i3) and a stealth lipid. In some embodiments, the LNP comprises a lipid of Formula (I-a3), (I-b3), (I-c3), (I-d3), (I-e3), (I-f3), (I-g3), (I-h3), or (I-i3) and a helper lipid. In some embodiments, the LNP comprises a lipid of Formula (I-a3), (I-b3), (I-c3), (I-d3), (I-e3), (I- f3), (I-g3), (I-h3), or (I-i3); a structural lipid; a stealth lipid; and a helper lipid. In some embodiments, the LNP comprises a lipid of Formula (I-a4), (I-b4), (I-c4), (I- d4), (I-e4), (I-f4), (I-g4), (I-h4), or (I-i4) and a structural lipid. In some embodiments, the LNP comprises a lipid of Formula (I-a4), (I-b4), (I-c4), (I-d4), (I-e4), (I-f4), (I-g4), (I-h4), or (I-i4) and a stealth lipid. In some embodiments, the LNP comprises a lipid of Formula (I-a4), (I-b4), (I-c4), (I-d4), (I-e4), (I-f4), (I-g4), (I-h4), or (I-i4) and a helper lipid. In some embodiments, the LNP comprises a lipid of Formula (I-a4), (I-b4), (I-c4), (I-d4), (I-e4), (I- f4), (I-g4), (I-h4), or (I-i4); a structural lipid; a stealth lipid; and a helper lipid. In some embodiments, the ionizable or cationic lipid may comprise a molar ratio from about 35% to about 45% of the total lipid present in the lipid nanoparticle. In some embodiments, the ionizable or cationic lipid may comprise a molar ratio of about 40% of the total lipid present in the lipid nanoparticle. In some embodiments, the stealth (e.g., PEGylated) lipid may comprise a molar ratio from about 0.5% to about 7% of the total lipid present in the lipid nanoparticle. In some embodiments, the stealth (e.g., PEGylated) lipid may comprise a molar ratio of about 1.5% of the total lipid present in the lipid nanoparticle. In some embodiments, the stealth (e.g., PEGylated) lipid may comprise a molar ratio of about 5% of the total lipid present in the lipid nanoparticle. In some embodiments, the structural lipid may comprise a molar ratio from about 20% to about 35% of the total lipid present in the lipid nanoparticle. In some embodiments, the structural lipid may comprise a molar ratio of about 30% of the total lipid present in the lipid nanoparticle. In some embodiments, the helper lipid may comprise a molar ratio from about 20% to about 30% of the total lipid present in the lipid nanoparticle. In some embodiments, the helper lipid may comprise a molar ratio of about 25% of the total lipid present in the lipid nanoparticle. In some embodiments, the helper lipid may comprise a molar ratio of about 28.5% of the total lipid present in the lipid nanoparticle. In some embodiments, the LNP comprises the ionizable or cationic lipid at a molar ratio between 35% and 45%; the structural lipid at a molar ratio between 20% and 35%, the stealth lipid at a molar ratio between 0.5% and 7%, and the helper lipid at a molar ratio between 20% and 30%. In some embodiments, the LNP comprises the ionizable or cationic lipid at a molar ratio of about 40%; the structural lipid at a molar ratio of about 30%; the stealth lipid at a molar ratio of about 1.5%, and the helper lipid at a molar ratio of about 28.5%. In some embodiments, the LNP comprises the ionizable or cationic lipid at a molar ratio of about 40%; the structural lipid at a molar ratio of about 30%; the stealth lipid at a molar ratio of about 5%, and the helper lipid at a molar ratio of about 25%. To calculate the actual amount of each lipid to be put into an LNP formulation, the molar amount of the cationic or ionizable lipid is first determined based on a desired N/P ratio, where N is the number of nitrogen atoms in the cationic lipid and P is the number of phosphate groups in the mRNA to be transported by the LNP. Next, the molar amount of each of the other lipids is calculated based on the molar amount of the cationic lipid and the molar ratio selected. These molar amounts are then converted to weights using the molecular weight of each lipid. E. Active Ingredients of the LNPs The active ingredient of the present LNP composition may be an mRNA that encodes a polypeptide of interest. In certain embodiments, the polypeptide is an antigen. In certain embodiments, the polypeptide is a therapeutic polypeptide. The therapeutic polypeptide may be an antibody (e.g., an antibody heavy chain or an antibody light chain. The therapeutic polypeptide may be an enzyme. The mRNA molecule encapsulated by the present disclosure LNPs may comprise at least one ribonucleic acid (RNA) comprising an ORF encoding a polypeptide of interest. In certain embodiments, the mRNA further comprises at least one 5’ UTR, 3’ UTR, a poly(A) tail, and/or a 5’ cap. i.5’ Cap An mRNA 5’ cap can provide resistance to nucleases found in most eukaryotic cells and promote translation efficiency. Several types of 5’ caps are known. A 7- methylguanosine cap (also referred to as “m7G” or “Cap-0”), comprises a guanosine that is linked through a 5’ – 5’ - triphosphate bond to the first transcribed nucleotide. A 5' cap is typically added as follows: first, an RNA terminal phosphatase removes one of the terminal phosphate groups from the 5’ nucleotide, leaving two terminal phosphates; guanosine triphosphate (GTP) is then added to the terminal phosphates via a guanylyl transferase, producing a 5 ‘5 ‘5 triphosphate linkage; and the 7-nitrogen of guanine is then methylated by a methyltransferase. Examples of cap structures include, but are not limited to, m7G(5’)ppp, (5’(A,G(5’)ppp(5’)A, and G(5’)ppp(5’)G. Additional cap structures are described in U.S. Publication No. US 2016/0032356 and U.S. Publication No. US 2018/0125989, which are incorporated herein by reference. 5’-capping of polynucleotides may be completed concomitantly during the in vitro- transcription reaction using the following chemical RNA cap analogs to generate the 5’- guanosine cap structure according to manufacturer protocols: 3’-O-Me-m7G(5’)ppp(5’)G (the ARCA cap); G(5’)ppp(5’)A; G(5’)ppp(5’)G; m7G(5’)ppp(5’)A; m7G(5’)ppp(5’)G; m7G(5')ppp(5')(2'OMeA)pG; m7G(5')ppp(5')(2'OMeA)pU; m7G(5')ppp(5')(2'OMeG)pG (New England BioLabs, Ipswich, MA; TriLink Biotechnologies).5’-capping of modified RNA may be completed post-transcriptionally using a vaccinia virus capping enzyme to generate the Cap 0 structure: m7G(5’)ppp(5’)G. Cap 1 structure may be generated using both vaccinia virus capping enzyme and a 2’-O methyl-transferase to generate: m7G(5’)ppp(5’)G- 2’-O-methyl. Cap 2 structure may be generated from the Cap 1 structure followed by the 2’- O-methylation of the 5’-antepenultimate nucleotide using a 2’-O methyl-transferase. Cap 3 structure may be generated from the Cap 2 structure followed by the 2’-O-methylation of the 5’-preantepenultimate nucleotide using a 2’-O methyl-transferase. In certain embodiments, the mRNA of the disclosure comprises a 5’ cap selected from the group consisting of 3’-O-Me-m7G(5’)ppp(5’)G (the ARCA cap), G(5’)ppp(5’)A, G(5’)ppp(5’)G, m7G(5’)ppp(5’)A, m7G(5’)ppp(5’)G, m7G(5')ppp(5')(2'OMeA)pG, m7G(5')ppp(5')(2'OMeA)pU, and m7G(5')ppp(5')(2'OMeG)pG. In certain embodiments, the mRNA of the disclosure comprises a 5’ cap of: . ii. Untranslated Region (UTR) In some embodiments, the mRNA of the disclosure includes a 5’ and/or 3’ untranslated region (UTR). In mRNA, the 5’ UTR starts at the transcription start site and continues to the start codon but does not include the start codon. The 3’ UTR starts immediately following the stop codon and continues until the transcriptional termination signal. In some embodiments, the mRNA disclosed herein may comprise a 5’ UTR that includes one or more elements that affect an mRNA’s stability or translation. In some embodiments, a 5’ UTR may be about 10 to 5,000 nucleotides in length. In some embodiments, a 5’ UTR may be about 50 to 500 nucleotides in length. In some embodiments, the 5’ UTR is at least about 10 nucleotides in length, about 20 nucleotides in length, about 30 nucleotides in length, about 40 nucleotides in length, about 50 nucleotides in length, about 100 nucleotides in length, about 150 nucleotides in length, about 200 nucleotides in length, about 250 nucleotides in length, about 300 nucleotides in length, about 350 nucleotides in length, about 400 nucleotides in length, about 450 nucleotides in length, about 500 nucleotides in length, about 550 nucleotides in length, about 600 nucleotides in length, about 650 nucleotides in length, about 700 nucleotides in length, about 750 nucleotides in length, about 800 nucleotides in length, about 850 nucleotides in length, about 900 nucleotides in length, about 950 nucleotides in length, about 1,000 nucleotides in length, about 1,500 nucleotides in length, about 2,000 nucleotides in length, about 2,500 nucleotides in length, about 3,000 nucleotides in length, about 3,500 nucleotides in length, about 4,000 nucleotides in length, about 4,500 nucleotides in length or about 5,000 nucleotides in length. In some embodiments, the mRNA disclosed herein may comprise a 3’ UTR comprising one or more of a polyadenylation signal, a binding site for proteins that affect an mRNA’s stability of location in a cell, or one or more binding sites for miRNAs. In some embodiments, a 3’ UTR may be 50 to 5,000 nucleotides in length or longer. In some embodiments, a 3’ UTR may be 50 to 1,000 nucleotides in length or longer. In some embodiments, the 3’ UTR is at least about 50 nucleotides in length, about 100 nucleotides in length, about 150 nucleotides in length, about 200 nucleotides in length, about 250 nucleotides in length, about 300 nucleotides in length, about 350 nucleotides in length, about 400 nucleotides in length, about 450 nucleotides in length, about 500 nucleotides in length, about 550 nucleotides in length, about 600 nucleotides in length, about 650 nucleotides in length, about 700 nucleotides in length, about 750 nucleotides in length, about 800 nucleotides in length, about 850 nucleotides in length, about 900 nucleotides in length, about 950 nucleotides in length, about 1,000 nucleotides in length, about 1,500 nucleotides in length, about 2,000 nucleotides in length, about 2,500 nucleotides in length, about 3,000 nucleotides in length, about 3,500 nucleotides in length, about 4,000 nucleotides in length, about 4,500 nucleotides in length, or about 5,000 nucleotides in length. In some embodiments, the mRNA disclosed herein may comprise a 5’ or 3’ UTR that is derived from a gene distinct from the one encoded by the mRNA transcript (i.e., the UTR is a heterologous UTR). In certain embodiments, the 5’ and/or 3’ UTR sequences can be derived from mRNA which are stable (e.g., globin, actin, GAPDH, tubulin, histone, or citric acid cycle enzymes) to increase the stability of the mRNA. For example, a 5’ UTR sequence may include a partial sequence of a CMV immediate-early 1 (IE1) gene, or a fragment thereof, to improve the nuclease resistance and/or improve the half-life of the mRNA. Also contemplated is the inclusion of a sequence encoding human growth hormone (hGH), or a fragment thereof, to the 3’ end or untranslated region of the mRNA. Generally, these modifications improve the stability and/or pharmacokinetic properties (e.g., half-life) of the mRNA relative to their unmodified counterparts, and include, for example, modifications made to improve such mRNA resistance to in vivo nuclease digestion. Exemplary 5’ UTRs include a sequence derived from a CMV immediate-early 1 (IE1) gene (U.S. Publication Nos.2014/0206753 and 2015/0157565, each of which is incorporated herein by reference), or the sequence GGGAUCCUACC(SEQ ID NO:1) (U.S. Publication No.2016/0151409, incorporated herein by reference). In various embodiments, the 5’ UTR may be derived from the 5’ UTR of a TOP gene. TOP genes are typically characterized by the presence of a 5’-terminal oligopyrimidine (TOP) tract. Furthermore, most TOP genes are characterized by growth-associated translational regulation. However, TOP genes with a tissue specific translational regulation are also known. In certain embodiments, the 5’ UTR derived from the 5’ UTR of a TOP gene lacks the 5’ TOP motif (the oligopyrimidine tract) (e.g., U.S. Publication Nos.2017/0029847, 2016/0304883, 2016/0235864, and 2016/0166710, each of which is incorporated herein by reference). In certain embodiments, the 5’ UTR is derived from a ribosomal protein Large 32 (L32) gene (U.S. Publication No.2017/0029847, supra). In certain embodiments, the 5’ UTR is derived from the 5’ UTR of an hydroxysteroid (17-b) dehydrogenase 4 gene (HSD17B4) (U.S. Publication No.2016/0166710, supra). In certain embodiments, the 5’ UTR is derived from the 5’ UTR of an ATP5A1 gene (U.S. Publication No.2016/0166710, supra). In some embodiments, an internal ribosome entry site (IRES) is used instead of a 5’ UTR. In some embodiments, the 5’UTR comprises a nucleic acid sequence reproduced below: GGACAGAUCGCCUGGAGACGCCAUCCACGCUGUUUUGACCUCCAUAGAA GACACCGGGACCGAUCCAGCCUCCGCGGCCGGGAACGGUGCAUUGGAACGCGG AUUCCCCGUGCCAAGAGUGACUCACCGUCCUUGACACG(SEQ ID NO:2). In some embodiments, the 3’UTR comprises a nucleic acid sequence reproduced below: CGGGUGGCAUCCCUGUGACCCCUCCCCAGUGCCUCUCCUGGCCCUGGAA GUUGCCACUCCAGUGCCCACCAGCCUUGUCCUAAUAAAAUUAAGUUGCAUC (SEQ ID NO:3). The 5’ UTR and 3’UTR are described in further detail in WO2012/075040, incorporated herein by reference. iii. Polyadenylated Tail As used herein, the terms “poly(A) sequence,” “poly(A) tail,” and “poly(A) region” refer to a sequence of adenosine nucleotides at the 3’ end of the mRNA molecule. The poly(A) tail may confer stability to the mRNA and protect it from exonuclease degradation. The poly(A) tail may enhance translation. In some embodiments, the poly(A) tail is essentially homopolymeric. For example, a poly(A) tail of 100 adenosine nucleotides may have essentially a length of 100 nucleotides. In certain embodiments, the poly(A) tail may be interrupted by at least one nucleotide different from an adenosine nucleotide (e.g., a nucleotide that is not an adenosine nucleotide). For example, a poly(A) tail of 100 adenosine nucleotides may have a length of more than 100 nucleotides (comprising 100 adenosine nucleotides and at least one nucleotide, or a stretch of nucleotides, that are different from an adenosine nucleotide). In certain embodiments, the poly(A) tail comprises the sequence AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGCAUAUGACUAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAA(SEQ ID NO:4). The “poly(A) tail,” as used herein, typically relates to RNA. However, in the context of the disclosure, the term likewise relates to corresponding sequences in a DNA molecule (e.g., a “poly(T) sequence”). The poly(A) tail(SEQ ID NO:5) may comprise about 10 to about 500 adenosine nucleotides, about 10 to about 200 adenosine nucleotides, about 40 to about 200 adenosine nucleotides, or about 40 to about 150 adenosine nucleotides. The length of the poly(A) tail may be at least about 10, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, or 500 adenosine nucleotides. In some embodiments where the nucleic acid is an RNA, the poly(A) (SEQ ID NO:5)tail of the nucleic acid is obtained from a DNA template during RNA in vitro transcription. In certain embodiments, the poly(A) tail is obtained in vitro by common methods of chemical synthesis without being transcribed from a DNA template. In various embodiments, poly(A) tails are generated by enzymatic polyadenylation of the RNA (after RNA in vitro transcription) using commercially available polyadenylation kits and corresponding protocols, or alternatively, by using immobilized poly(A)polymerases, e.g., using methods and means as described in WO2016/174271. The nucleic acid may comprise a poly(A) tail obtained by enzymatic polyadenylation, wherein the majority of nucleic acid molecules comprise about 100 (+/-20) to about 500 (+/- 50) or about 250 (+/-20) adenosine nucleotides. In some embodiments, the nucleic acid may comprise a poly(A) tail derived from a template DNA and may additionally comprise at least one additional poly(A) tail generated by enzymatic polyadenylation, e.g., as described in WO2016/091391. In certain embodiments, the nucleic acid comprises at least one polyadenylation signal. In various embodiments, the nucleic acid may comprise at least one poly(C) sequence. The term ‘‘poly(C) (SEQ ID NO:6)sequence,” as used herein, is intended to be a sequence of cytosine nucleotides of up to about 200 cytosine nucleotides. In some embodiments, the poly(C) sequence comprises about 10 to about 200 cytosine nucleotides, about 10 to about 100 cytosine nucleotides, about 20 to about 70 cytosine nucleotides, about 20 to about 60 cytosine nucleotides, or about 10 to about 40 cytosine nucleotides. In some embodiments, the poly(C) sequence comprises about 30 cytosine nucleotides. iv. Chemical Modification The mRNA disclosed herein may be modified or unmodified. In some embodiments, the mRNA may comprise at least one chemical modification. In some embodiments, the mRNA disclosed herein may contain one or more modifications that typically enhance RNA stability. Exemplary modifications can include backbone modifications, sugar modifications, or base modifications. In some embodiments, the disclosed mRNA may be synthesized from naturally occurring nucleotides and/or nucleotide analogues (modified nucleotides) including, but not limited to, purines (adenine (A) and guanine (G)) or pyrimidines (thymine (T), cytosine (C), and uracil (U)). In certain embodiments, the disclosed mRNA may be synthesized from modified nucleotide analogues or derivatives of purines and pyrimidines, such as, e.g., 1-methyl-adenine, 2-methyl-adenine, 2-methylthio-N-6-isopentenyl-adenine, N6-methyl-adenine, N6-isopentenyl-adenine, 2-thio-cytosine, 3-methyl-cytosine, 4-acetyl- cytosine, 5-methyl-cytosine, 2,6-diaminopurine, 1-methyl-guanine, 2-methyl-guanine, 2,2- dimethyl-guanine, 7-methyl-guanine, inosine, 1-methyl-inosine, pseudouracil (5-uracil), dihydro-uracil, 2-thio-uracil, 4-thio-uracil, 5-carboxymethylaminomethyl-2-thio-uracil, 5- (carboxyhydroxymethyl)-uracil, 5-fluoro-uracil, 5-bromo-uracil, 5- carboxymethylaminomethyl-uracil, 5-methyl-2-thio-uracil, 5-methyl-uracil, N-uracil-5-oxy acetic acid methyl ester, 5-methylaminomethyl-uracil, 5-methoxyaminomethyl-2-thio-uracil, 5’-methoxycarbonylmethyl-uracil, 5-methoxy-uracil, uracil-5-oxyacetic acid methyl ester, uracil-5-oxyacetic acid (v), 1-methyl-pseudouracil, queosine, β-D-mannosyl-queosine, phosphoramidates, phosphorothioates, peptide nucleotides, methylphosphonates, 7- deazaguanosine, 5-methylcytosine, and inosine. In some embodiments, the disclosed mRNA may comprise at least one chemical modification including, but not limited to, pseudouridine, N1-methylpseudouridine, 2- thiouridine, 4’-thiouridine, 5-methylcytosine, 2-thio-l-methyl-1-deaza-pseudouridine, 2-thio- l-methyl-pseudouridine, 2-thio-5-aza-uridine, 2-thio-dihydropseudouridine, 2-thio- dihydrouridine, 2-thio-pseudouridine, 4-methoxy-2-thio-pseudouridine, 4-methoxy- pseudouridine, 4-thio-l-methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methyluridine, 5-methyluridine, 5-methoxyuridine, and 2’-O-methyl uridine. In some embodiments, the chemical modification is selected from the group consisting of pseudouridine, N1-methylpseudouridine, 5-methylcytosine, 5-methoxyuridine, and a combination thereof. In some embodiments, the chemical modification comprises N1-methylpseudouridine. In some embodiments, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% of the uracil nucleotides in the mRNA are chemically modified. In some embodiments, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% of the uracil nucleotides in the ORF are chemically modified. The preparation of such analogues is described, e.g., in U.S. Pat. No.4,373,071, U.S. Pat. No.4,401,796, U.S. Pat. No.4,415,732, U.S. Pat. No.4,458,066, U.S. Pat. No. 4,500,707, U.S. Pat. No.4,668,777, U.S. Pat. No.4,973,679, U.S. Pat. No.5,047,524, U.S. Pat. No.5,132,418, U.S. Pat. No.5,153,319, U.S. Pat. No.5,262,530, and U.S. Pat. No. 5,700,642. v. mRNA Synthesis The mRNAs disclosed herein may be synthesized according to any of a variety of methods. For example, mRNAs according to the present disclosure may be synthesized via in vitro transcription (IVT). Some methods for in vitro transcription are described, e.g., in Geall et al. (2013) Semin. Immunol.25(2): 152-159; Brunelle et al. (2013) Methods Enzymol.530:101-14. Briefly, IVT is typically performed with a linear or circular DNA template containing a promoter, a pool of ribonucleotide triphosphates, a buffer system that may include DTT and magnesium ions, an appropriate RNA polymerase (e.g., T3, T7, or SP6 RNA polymerase), DNase I, pyrophosphatase, and/or RNase inhibitor. The exact conditions may vary according to the specific application. The presence of these reagents is generally undesirable in a final mRNA product and these reagents can be considered impurities or contaminants which can be purified or removed to provide a clean and/or homogeneous mRNA that is suitable for therapeutic use. While mRNA provided from in vitro transcription reactions may be desirable in some embodiments, other sources of mRNA can be used according to the instant disclosure including wild-type mRNA produced from bacteria, fungi, plants, and/or animals. Where desired, the LNP or the LNP formulation may be multi-valent. In some embodiments, the LNP may carry mRNAs that encode more than one polypeptide (e.g., antigen), such as two, three, four, five, six, seven, eight, nine, ten, or more polypeptides. For example, the LNP may carry multiple mRNA molecules, each encoding a different polypeptide; or carry a polycistronic mRNA that can be translated into more than one polypeptide (e.g., each polypeptide-coding sequence is separated by a nucleotide linker encoding a self-cleaving peptide such as a 2A peptide). An LNP carrying different mRNA molecules typically comprises (encapsulate) multiple copies of each mRNA molecule. For example, an LNP carrying or encapsulating two different mRNA molecules typically carries multiple copies of each of the two different mRNA molecules. In some embodiments, a single LNP formulation may comprise multiple kinds (e.g., two, three, four, five, six, seven, eight, nine, ten, or more) of LNPs, each kind carrying a different mRNA. F. Buffer and Other Components To stabilize the nucleic acid and/or LNPs (e.g., to prolong the shelf-life of the vaccine product), to facilitate administration of the LNP pharmaceutical composition, and/or to enhance in vivo expression of the nucleic acid, the nucleic acid and/or LNP can be formulated in combination with one or more carriers, targeting ligands, stabilizing reagents (e.g., preservatives and antioxidants), and/or other pharmaceutically acceptable excipients. Examples of such excipients are parabens, thimerosal, thiomersal, chlorobutanol, bezalkonium chloride, and chelators (e.g., EDTA). The LNP compositions of the present disclosure can be provided as a frozen liquid form or a lyophilized form. A variety of cryoprotectants may be used, including, without limitations, sucrose, trehalose, glucose, mannitol, mannose, dextrose, and the like. The cryoprotectant may constitute 5-30% (w/v) of the LNP composition. In some embodiments, the LNP composition comprises trehalose, e.g., at 5-30% (e.g., 10%) (w/v). Once formulated with the cryoprotectant, the LNP compositions may be frozen (or lyophilized and cryopreserved) at -20oC to -80oC. The LNP compositions may be provided to a patient in an aqueous buffered solution – thawed if previously frozen, or if previously lyophilized, reconstituted in an aqueous buffered solution at bedside. The buffered solution may be isotonic and suitable for e.g., intramuscular or intradermal injection. In some embodiments, the buffered solution is a phosphate-buffered saline (PBS). G. Processes for Making the Present LNP Formulations The present LNPs can be prepared by various techniques presently known in the art. For example, multilamellar vesicles (MLV) may be prepared according to conventional techniques, such as by depositing a selected lipid on the inside wall of a suitable container or vessel by dissolving the lipid in an appropriate solvent, and then evaporating the solvent to leave a thin film on the inside of the vessel or by spray drying. An aqueous phase may then be added to the vessel with a vortexing motion that results in the formation of MLVs. Unilamellar vesicles (ULV) can then be formed by homogenization, sonication or extrusion of the multilamellar vesicles. In addition, unilamellar vesicles can be formed by detergent removal techniques. Various methods are described in US 2011/0244026, US 2016/0038432, US 2018/0153822, US 2018/0125989, and PCT/US2020/043223 (filed July 23, 2020) and can be used to practice the present disclosure. One exemplary process entails encapsulating mRNA by mixing it with a mixture of lipids, without first pre-forming the lipids into lipid nanoparticles, as described in US 2016/0038432. Another exemplary process entails encapsulating mRNA by mixing pre-formed LNPs with mRNA, as described in US 2018/0153822. In some embodiments, the process of preparing mRNA-loaded LNPs includes a step of heating one or more of the solutions to a temperature greater than ambient temperature, the one or more solutions being the solution comprising the pre-formed lipid nanoparticles, the solution comprising the mRNA and the mixed solution comprising the LNP-encapsulated mRNA. In some embodiments, the process includes the step of heating one or both of the mRNA solution and the pre-formed LNP solution, prior to the mixing step. In some embodiments, the process includes heating one or more of the solutions comprising the pre- formed LNPs, the solution comprising the mRNA and the solution comprising the LNP- encapsulated mRNA, during the mixing step. In some embodiments, the process includes the step of heating the LNP- encapsulated mRNA, after the mixing step. In some embodiments, the temperature to which one or more of the solutions is heated is or is greater than about 30°C, 37°C, 40°C, 45°C, 50°C, 55°C, 60°C, 65°C, or 70°C. In some embodiments, the temperature to which one or more of the solutions is heated ranges from about 25-70°C, about 30-70°C, about 35-70°C, about 40-70°C, about 45-70°C, about 50-70°C, or about 60- 70°C. In some embodiments, the temperature is about 65°C. Various methods may be used to prepare an mRNA solution suitable for the present disclosure. In some embodiments, mRNA may be directly dissolved in a buffer solution described herein. In some embodiments, an mRNA solution may be generated by mixing an mRNA stock solution with a buffer solution prior to mixing with a lipid solution for encapsulation. In some embodiments, an mRNA solution may be generated by mixing an mRNA stock solution with a buffer solution immediately before mixing with a lipid solution for encapsulation. In some embodiments, a suitable mRNA stock solution may contain mRNA in water or a buffer at a concentration at or greater than about 0.2 mg/ml, 0.4 mg/ml, 0.5 mg/ml, 0.6 mg/ml, 0.8 mg/ml, 1.0 mg/ml, 1.2 mg/ml, 1.4 mg/ml, 1.5 mg/ml, or 1.6 mg/ml, 2.0 mg/ml, 2.5 mg/ml, 3.0 mg/ml, 3.5 mg/ml, 4.0 mg/ml, 4.5 mg/ml, or 5.0 mg/ml. In some embodiments, an mRNA stock solution is mixed with a buffer solution using a pump. Exemplary pumps include but are not limited to gear pumps, peristaltic pumps and centrifugal pumps. Typically, the buffer solution is mixed at a rate greater than that of the mRNA stock solution. For example, the buffer solution may be mixed at a rate at least 1x, 2x, 3x, 4x, 5x, 6x, 7x, 8x, 9x, 10x, 15x, or 20x greater than the rate of the mRNA stock solution. In some embodiments, a buffer solution is mixed at a flow rate ranging between about 100-6000 ml/minute (e.g., about 100-300 ml/minute, 300-600 ml/minute, 600-1200 ml/minute, 1200-2400 ml/minute, 2400-3600 ml/minute, 3600-4800 ml/minute, 4800-6000 ml/minute, or 60-420 ml/minute). In some embodiments, a buffer solution is mixed at a flow rate of, or greater than, about 60 ml/minute, 100 ml/minute, 140 ml/minute, 180 ml/minute, 220 ml/minute, 260 ml/minute, 300 ml/minute, 340 ml/minute, 380 ml/minute, 420 ml/minute, 480 ml/minute, 540 ml/minute, 600 ml/minute, 1200 ml/minute, 2400 ml/minute, 3600 ml/minute, 4800 ml/minute, or 6000 ml/minute. In some embodiments, an mRNA stock solution is mixed at a flow rate ranging between about 10-600 ml/minute (e.g., about 5-50 ml/minute, about 10-30 ml/minute, about 30-60 ml/minute, about 60-120 ml/minute, about 120-240 ml/minute, about 240-360 ml/minute, about 360-480 ml/minute, or about 480-600 ml/minute). In some embodiments, an mRNA stock solution is mixed at a flow rate of or greater than about 5 ml/minute, 10 ml/minute, 15 ml/minute, 20 ml/minute, 25 ml/minute, 30 ml/minute, 35 ml/minute, 40 ml/minute, 45 ml/minute, 50 ml/minute, 60 ml/minute, 80 ml/minute, 100 ml/minute, 200 ml/minute, 300 ml/minute, 400 ml/minute, 500 ml/minute, or 600 ml/minute. The process of incorporation of a desired mRNA into a lipid nanoparticle is referred to as “loading.” Exemplary methods are described in Lasic et al., FEBS Lett. (1992) 312:255-8. The LNP-incorporated nucleic acids may be completely or partially located in the interior space of the lipid nanoparticle, within the bilayer membrane of the lipid nanoparticle, or associated with the exterior surface of the lipid nanoparticle membrane. The incorporation of an mRNA into lipid nanoparticles is also referred to herein as “encapsulation” wherein the nucleic acid is entirely or substantially contained within the interior space of the lipid nanoparticle. Suitable LNPs may be made in various sizes. In some embodiments, decreased size of lipid nanoparticles is associated with more efficient delivery of an mRNA. Selection of an appropriate LNP size may take into consideration the site of the target cell or tissue and to some extent the application for which the lipid nanoparticle is being made. A variety of methods known in the art are available for sizing of a population of lipid nanoparticles. Preferred methods herein utilize Zetasizer Nano ZS (Malvern Panalytical) to measure LNP particle size. In one protocol, 10 μl of an LNP sample are mixed with 990 μl of 10% trehalose. This solution is loaded into a cuvette and then put into the Zetasizer machine. The z-average diameter (nm), or cumulants mean, is regarded as the average size for the LNPs in the sample. The Zetasizer machine can also be used to measure the polydispersity index (PDI) by using dynamic light scattering (DLS) and cumulant analysis of the autocorrelation function. Average LNP diameter may be reduced by sonication of formed LNP. Intermittent sonication cycles may be alternated with quasi-elastic light scattering (QELS) assessment to guide efficient lipid nanoparticle synthesis. In some embodiments, the majority of purified LNPs, i.e., greater than about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the LNPs, have a size of about 70-150 nm (e.g., about 145 nm, about 140 nm, about 135 nm, about 130 nm, about 125 nm, about 120 nm, about 115 nm, about 110 nm, about 105 nm, about 100 nm, about 95 nm, about 90 nm, about 85 nm, or about 80 nm). In some embodiments, substantially all (e.g., greater than 80 or 90%) of the purified lipid nanoparticles have a size of about 70-150 nm (e.g., about 145 nm, about 140 nm, about 135 nm, about 130 nm, about 125 nm, about 120 nm, about 115 nm, about 110 nm, about 105 nm, about 100 nm, about 95 nm, about 90 nm, about 85 nm, or about 80 nm). In some embodiments, the LNPs in the present composition have an average size of less than 150 nm, less than 120 nm, less than 100 nm, less than 90 nm, less than 80 nm, less than 70 nm, less than 60 nm, less than 50 nm, less than 30 nm, or less than 20 nm. In some embodiments, greater than about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% of the LNPs in the present composition have a size ranging from about 40-90 nm (e.g., about 45-85 nm, about 50-80 nm, about 55-75 nm, about 60-70 nm), about 40-90 nm (e.g., about 45-85 nm, about 50-80 nm, about 55-75 nm, about 60-70 nm), or about 50-70 nm (e.g., 55-65 nm) are particular suitable for pulmonary delivery via nebulization. In some embodiments, the dispersity, or measure of heterogeneity in size of molecules (PDI), of LNPs in a pharmaceutical composition provided by the present disclosure is less than about 0.5. In some embodiments, an LNP has a PDI of less than about 0.5, less than about 0.4, less than about 0.3, less than about 0.28, less than about 0.25, less than about 0.23, less than about 0.20, less than about 0.18, less than about 0.16, less than about 0.14, less than about 0.12, less than about 0.10, or less than about 0.08. The PDI may be measured by a Zetasizer machine as described above. In some embodiments, greater than about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the purified LNPs in a pharmaceutical composition provided herein encapsulate an mRNA within each individual particle. In some embodiments, substantially all (e.g., greater than 80% or 90%) of the purified lipid nanoparticles in a pharmaceutical composition encapsulate an mRNA within each individual particle. In some embodiments, a lipid nanoparticle has an encapsulation efficiency of between 50% and 99%; or greater than about 60, 65, 70, 75, 80, 85, 90, 92, 95, 98, or 99%. Typically, lipid nanoparticles for use herein have an encapsulation efficiency of at least 90% (e.g., at least 91, 92, 93, 94, or 95%). In some embodiments, an LNP has a N/P ratio of between 1 and 10. In some embodiments, a lipid nanoparticle has a N/P ratio above 1, about 1, about 2, about 3, about 4, about 5, about 6, about 7, or about 8. In further embodiments, a typical LNP herein has an N/P ratio of 4. In some embodiments, a pharmaceutical composition according to the present disclosure contains at least about 0.5 μg, 1 μg, 5 μg, 10 μg, 100 μg, 500 μg, or 1000 μg of encapsulated mRNA. In some embodiments, a pharmaceutical composition contains about 0.1 μg to 1000 μg, at least about 0.5 μg, at least about 0.8 μg, at least about 1 μg, at least about 5 μg, at least about 8 μg, at least about 10 μg, at least about 50 μg, at least about 100 μg, at least about 500 μg, or at least about 1000 μg of encapsulated mRNA. Packaging and Use of the mRNA-LNP The mRNA-LNP can be packaged for parenteral (e.g., intramuscular, intradermal, subcutaneous, or intravenous) administration, nasopharyngeal (e.g., intranasal) administration, or mucosal (e.g., intranasal, oral, rectal) administration. The compositions may be in the form of an extemporaneous formulation, where the LNP composition is lyophilized and reconstituted with a physiological buffer (e.g., PBS) just before use. The compositions also may be shipped and provided in the form of an aqueous solution or a frozen aqueous solution and can be directly administered to subjects without reconstitution (after thawing, if previously frozen). Accordingly, the present disclosure provides an article of manufacture, such as a kit, that provides the mRNA-LNP in a single container, or provides the mRNA-LNP in one container and a physiological buffer for reconstitution in another container. The container(s) may contain a single-use dosage or multi-use dosage. The containers may be pre-treated glass vials or ampules. The article of manufacture may include instructions for use as well. In some embodiments, the present disclosure provides methods of preventing or treating a disease or disorder by administering the composition of the disclosure to a subject in need thereof. In some embodiments, the subject is suffering from or susceptible to an infection. In some embodiments, the present disclosure provides methods of eliciting an immune response in a subject in need thereof, comprising administering to the subject a prophylactically effective amount of a composition described herein. In some embodiments, the present disclosure provides methods of preventing an infection or reducing one or more symptoms of an infection in a subject in need thereof, comprising administering to the subject a prophylactically effective amount of the composition. In some embodiments of the methods described herein, the composition is administered to the subject mucosally, intramuscularly, intranasally, intravenously, subcutaneously, or intradermally. In some embodiments, the composition is administered mucosally. In some embodiments, the composition is administered intranasally. Intranasal administration includes administration via the nose, either with or without concomitant inhalation during administration. Such administration is typically through contact by the composition with the nasal mucosa, nasal turbinates or sinus cavity. The pharmaceutical compositions for administration may be applied in a single administration or in multiple administrations. For example, one dose can be placed in each nostril during administration. For example, bi-dose delivery can be used with the compositions according to the invention. Bi-dose devices contain two sub-doses of a single dose, one sub-dose for administration to each nostril. Generally, the two sub-doses are present in a single chamber and the construction of the device allows the efficient delivery of a single sub-dose at a time. Alternatively, a mono-dose device may be used for administering the compositions according to the invention. The composition can be given in one, two, three, four, or more doses, so that the subject is given a first dose (which can be a bi-dose or mono-dose, as described above), and then a second dose is administered within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,27, 28, 29, or 30 days, or 4 ,5, 6, 7, 8, 9, 10, 11, or 12 weeks, or 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months, or 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more years apart. Exemplary devices for intranasal administration of the compositions according to the invention are spray devices. Suitable commercially available nasal spray devices include Accuspray™ (Becton Dickinson). Nebulizers produce a very fine spray (such as a mist) which can be easily inhaled and are also contemplated herein. Exemplary spray devices for intranasal use are devices for which the performance of the device is not dependent upon the pressure applied by the user. These devices are known as pressure threshold devices. Liquid is released from the nozzle only when a threshold pressure is applied. These devices make it easier to achieve a spray with a regular droplet size. Pressure threshold devices suitable for use with the present invention are known in the art. The invention provides in a further aspect a pharmaceutical kit comprising an intranasal administration device as described herein containing a formulation according to the invention. The invention is not necessarily limited to spray delivery of liquid formulations. Compositions according to the invention may be administered in other forms e.g. as a powder. In some embodiments of the methods described herein, the subject is administered one or more doses of the composition, wherein each dose comprises 1 ug – 25 mg of mRNA. In some embodiments, each dose comprises 2.5-135 μg of mRNA. In some embodiments, each dose comprises 250 ug – 13.5 mg of mRNA. In some embodiments, each dose comprises 2.5., 5, 15, 30, 45, 50, 135, 250, or 500 μg or 1, 1.5, 2.5, 3, 4, 5, 7.5 or 10 mg of mRNA. In some embodiments of the methods described herein, the subject is administered two doses of the composition. In some embodiments, the two doses of the composition are administered with an interval of 2-6 weeks. In some embodiments, the two doses of the composition are administered with an interval of 2, 3, 4, 5, or 6 weeks. In some embodiments, the two doses of the composition are administered with an interval of 4 weeks. The present disclosure also provides the use of a composition described herein for the manufacture of a medicament for use in any of the methods described herein. The present disclosure further provides a kit comprising a composition of the present disclosure. In some embodiments, the kit comprises a containing comprising a single-use or multi-use dosage of the composition. In some embodiments, the containing is a vial. In some embodiments, the container is a pre-filled nasal spray device. V. Particular Embodiments The present disclosure provides the following particular embodiments of the lipids, compositions, and uses disclosed herein: 1. A lipid of Formula (I):
Figure imgf000151_0001
or a pharmaceutically acceptable salt thereof, wherein: two of R1, R2, and R3 are independently selected from -C(6-24)alkyl, -C(6-24)alkenyl, - C(6-24)alkynyl, , , and , wherein the -C(6-24)alkyl, - C(6-24)alkenyl, and -C(6-24)alkynyl are each optionally substituted with one to six groups independently selected from halo, -OC(1-6)alkyl, -OC(2-6)alkenyl, -SC(1-6)alkyl, -SC(2-6)alkenyl, and -C(O)OC(1-6)alkyl; one of R1, R2, and R3 is or ; X1 independently for each occurrence is -C(3-12)alkyl, -C(3-12)alkenyl, or -C(3-12)alkynyl, each of which is optionally substituted by one to six groups independently selected from halo, -OC(1-6)alkyl, -OC(2-6)alkenyl, -SC(1-6)alkyl, -SC(2-6)alkenyl, and -C(O)OC(1-6)alkyl; X2 independently for each occurrence is -C(O)O-, -OC(O)-, -C(O)NH-, -C(O)N(X3)-, -NHC(O)-, -N(X3)C(O)-, -C(O)S-, -SC(O)-, -OC(O)O-, -OC(O)NH-, -NHC(O)O-, or - NHC(O)NH-; X3 independently for each occurrence is -C(5-24)alkyl, -C(5-24)alkenyl, or -C(5-24)alkynyl, each of which is optionally substituted by one to six groups independently selected from halo, -OC(1-6)alkyl, -OC(2-6)alkenyl, -SC(1-6)alkyl, -SC(2-6)alkenyl, and -C(O)OC(1-6)alkyl; X4 is -C(1-8)alkyl; X5 is -C(1-8)alkyl; X6 is -C(O)O-, -OC(O)-, -C(O)NH-, -NHC(O)-, -C(O)S-, -SC(O)-, -OC(O)O-, - OC(O)NH-, -NHC(O)O-, or -NHC(O)NH-; X7 is -C(1-8)alkyl; A is an ionizable or cationic nitrogen-containing group; X is selected from the group consisting of:
Figure imgf000152_0001
wherein * indicates the point of attachment to R3; RX independently for each occurrence is -H or -C(1-3)alkyl; and n is 1, 2, 3, or 4. 2. A lipid of Formula (I): (I), or a pharmaceutically acceptable salt thereof, wherein: two of R1, R2, and R3 are independently selected from -C(6-24)alkyl, -C(6-24)alkenyl, - C(6-24)alkynyl, , , and , wherein the -C(6-24)alkyl, - C(6-24)alkenyl, and -C(6-24)alkynyl are each optionally substituted with one to six groups independently selected from halo, -OC(1-6)alkyl, -OC(2-6)alkenyl, -SC(1-6)alkyl, -SC(2-6)alkenyl, and -C(O)OC(1-6)alkyl; one of R1, R2, and R3 is or ; X1 independently for each occurrence is -C(3-12)alkyl, -C(3-12)alkenyl, or -C(3-12)alkynyl, each of which is optionally substituted by one to six groups independently selected from halo, -OC(1-6)alkyl, -OC(2-6)alkenyl, -SC(1-6)alkyl, -SC(2-6)alkenyl, and -C(O)OC(1-6)alkyl; X2 independently for each occurrence is -C(O)O-, -OC(O)-, -C(O)NH-, -C(O)N(X3)-, -NHC(O)-, -N(X3)C(O)-, -C(O)S-, -SC(O)-, -OC(O)O-, -OC(O)NH-, -NHC(O)O-, or - NHC(O)NH-; X3 independently for each occurrence is -C(5-24)alkyl, -C(5-24)alkenyl, or -C(5-24)alkynyl, each of which is optionally substituted by one to six groups independently selected from halo, -OC(1-6)alkyl, -OC(2-6)alkenyl, -SC(1-6)alkyl, -SC(2-6)alkenyl, and -C(O)OC(1-6)alkyl; X4 is -C(1-8)alkyl; X5 is -C(1-8)alkyl; X6 is -C(O)O-, -OC(O)-, -C(O)NH-, -NHC(O)-, -C(O)S-, -SC(O)-, -OC(O)O-, - OC(O)NH-, -NHC(O)O-, or -NHC(O)NH-; X7 is -C(1-8)alkyl; A is an ionizable or cationic nitrogen-containing group; X is selected from the group consisting of:
Figure imgf000153_0001
wherein * indicates the point of attachment to R3; RX independently for each occurrence is -H or -C(1-3)alkyl; and n is 1, 2, 3, or 4. 3. The lipid of embodiment 1 or embodiment 2, or a pharmaceutically acceptable salt thereof, wherein: two of R1, R2, and R3 are independently selected from -C(6-24)alkyl, -C(6-24)alkenyl, and ; X1 independently for each occurrence is -C(3-12)alkyl or -C(3-12)alkenyl; X2 independently for each occurrence is -C(O)O-, -OC(O)-, -C(O)S-, -SC(O)-, or - OC(O)O-; and X3 independently for each occurrence is -C(5-24)alkyl or -C(5-24)alkenyl. 4. The lipid of any one of embodiments 1-3, or a pharmaceutically acceptable salt thereof, wherein: two of R1, R2, and R3 are independently selected from -C(6-24)alkyl, -C(6-24)alkenyl, and ; X1 independently for each occurrence is -C(3-12)alkyl or -C(3-12)alkenyl; X2 independently for each occurrence is -C(O)O-, -OC(O)-, or -OC(O)O-; and X3 independently for each occurrence is -C(5-24)alkyl or -C(5-24)alkenyl. 5. The lipid of embodiment 1 or embodiment 2, or a pharmaceutically acceptable salt thereof, wherein two of R1, R2, and R3 are independently selected from the group consisting of:
Figure imgf000154_0001
Figure imgf000155_0001
6. The lipid of embodiment 1 or embodiment 2, or a pharmaceutically acceptable salt thereof, wherein two of R1, R2, and R3 are independently selected from the group consisting of:
Figure imgf000155_0002
Figure imgf000156_0001
Figure imgf000157_0001
Figure imgf000158_0001
Figure imgf000159_0001
7. The lipid of any one of embodiments 1-6, or a pharmaceutically acceptable salt thereof, wherein: one of R1, R2, and R3 is
Figure imgf000159_0002
X4 is -C(1-6)alkyl; X5 is -C(1-6)alkyl; X6 is -C(O)O-, -OC(O)-, or -OC(O)O-; X7 is -C(1-6)alkyl; and A is an ionizable or cationic nitrogen-containing group. 8. The lipid of any one of embodiments 1-7, or a pharmaceutically acceptable salt thereof, wherein: one of R1, R2, and R3 is ; X4 is -C(1-6)alkyl; and A is an ionizable or cationic nitrogen-containing group. 9. The lipid of any one of embodiments 1-3, or a pharmaceutically acceptable salt thereof, wherein: R1 and R2 are independently selected from -C(6-24)alkyl, -C(6-24)alkenyl, or
Figure imgf000160_0002
R3 is
Figure imgf000160_0001
X1 independently for each occurrence is -C(3-12)alkyl or -C(3-12)alkenyl; X2 independently for each occurrence is -C(O)O-, -OC(O)-, -C(O)S-, -SC(O)-, or - OC(O)O-; X3 independently for each occurrence is -C(5-24)alkyl or -C(5-24)alkenyl; X4 is -C(1-6)alkyl; X5 is -C(1-6)alkyl; X6 is -C(O)O-, -OC(O)-, or -OC(O)O-; X7 is -C(1-6)alkyl; and A is an ionizable or cationic nitrogen-containing group. 10. The lipid of any one of embodiments 1-4, or a pharmaceutically acceptable salt thereof, wherein: R1 and R2 are independently selected from -C(6-24)alkyl, -C(6-24)alkenyl, or R3 is or X1 independently for each occurrence is -C(3-12)alkyl or -C(3-12)alkenyl; X2 independently for each occurrence is -C(O)O-, -OC(O)-, or -OC(O)O-; X3 independently for each occurrence is -C(5-24)alkyl or -C(5-24)alkenyl; X4 is -C(1-6)alkyl; X5 is -C(1-6)alkyl; X6 is -C(O)O-, -OC(O)-, or -OC(O)O-; X7 is -C(1-6)alkyl; and A is an ionizable or cationic nitrogen-containing group. 11. The lipid of any one of embodiments 1-10, or a pharmaceutically acceptable salt thereof, wherein A is ; and RA1 and RA2 are each independently -C(1-6)alkyl that is optionally substituted with - OH; or RA1 and RA2 are taken together to form a 3- to 6-membered heteroaryl that is optionally substituted with one to three -C(1-3)alkyl groups. 12. The lipid of any one of embodiments 1-10, or a pharmaceutically acceptable salt thereof, wherein A is ; and RA1 and RA2 are each independently -C(1-6)alkyl that is optionally substituted with - OH; or RA1 and RA2 are taken together to form a 3- to 6-membered heterocyclyl that is optionally substituted with one to three -C(1-3)alkyl groups. 13. The lipid of any one of embodiments 1-10 or 12, or a pharmaceutically acceptable salt thereof, wherein A is selected from the group consisting of:
Figure imgf000162_0001
14. The lipid of any one of embodiments 1-13, or a pharmaceutically acceptable salt thereof, wherein A is -N(CH3)2. 15. The lipid of any one of embodiments 1-10, 12, and 13, or a pharmaceutically acceptable salt thereof, wherein is selected from the group consisting of:
Figure imgf000162_0002
Figure imgf000163_0001
16. The lipid of any one of embodiments 1-15, or a pharmaceutically acceptable salt thereof, wherein is selected from the group consisting of:
Figure imgf000163_0002
17. The lipid of any one of embodiments 1-16, having a structure selected from the group consisting of:
Figure imgf000163_0003
Figure imgf000164_0001
or a pharmaceutically acceptable salt thereof. 18. The lipid of any one of embodiments 1-16, having a structure selected from the group consisting of:
Figure imgf000164_0002
or a pharmaceutically acceptable salt thereof. 19. The lipid of any one of embodiments 1-16, having a structure selected from the group consisting of:
Figure imgf000164_0003
or a pharmaceutically acceptable salt thereof. 20. The lipid of any one of embodiments 1-16, having a structure selected from the group consisting of:
Figure imgf000165_0001
or a pharmaceutically acceptable salt thereof. 21. The lipid of any one of embodiments 1-16, having a structure selected from the group consisting of:
Figure imgf000165_0002
or a pharmaceutically acceptable salt thereof. 22. The lipid of any one of embodiments 1-16, having a structure selected from the group consisting of:
Figure imgf000165_0003
Figure imgf000166_0001
or a pharmaceutically acceptable salt thereof. 23. The lipid of any one of embodiments 1-16, having a structure selected from the group consisting of:
Figure imgf000166_0002
or a pharmaceutically acceptable salt thereof. 24. The lipid of any one of embodiments 1-16, having a structure selected from the group consisting of:
Figure imgf000166_0003
Figure imgf000167_0001
or a pharmaceutically acceptable salt thereof. 25. The lipid of any one of embodiments 1-16, having a structure selected from the group consisting of:
Figure imgf000167_0002
or a pharmaceutically acceptable salt thereof. 26. The lipid of any one of embodiments 1-16, having a structure selected from the group consisting of:
Figure imgf000167_0003
or a pharmaceutically acceptable salt thereof. 27. The lipid of any one of embodiments 1-16, having a structure selected from the group consisting of:
Figure imgf000168_0001
or a pharmaceutically acceptable salt thereof. 28. The lipid of any one of embodiments 1-16, having a structure selected from the group consisting of:
Figure imgf000168_0002
or a pharmaceutically acceptable salt thereof. 29. The lipid of any one of embodiments 1-16, 18, 20-22, and 26, or a pharmaceutically acceptable salt thereof, wherein RX is -H or -CH3. 30. The lipid of any one of embodiments 1-29, or a pharmaceutically acceptable salt thereof, wherein n is 1 or 2. 31. The lipid of any one of embodiments 1-30, or a pharmaceutically acceptable salt thereof, wherein n is 1. 32. The lipid of any one of embodiments 1-30, or a pharmaceutically acceptable salt thereof, wherein n is 2. 33. A lipid having a structure selected from the group consisting of:
Figure imgf000169_0001
Figure imgf000170_0001
Figure imgf000171_0001
Figure imgf000172_0001
Figure imgf000173_0001
Figure imgf000174_0001
Figure imgf000175_0001
Figure imgf000176_0001
Figure imgf000177_0001
Figure imgf000178_0001
Figure imgf000179_0001
Figure imgf000180_0001
Figure imgf000181_0001
Figure imgf000182_0001
Figure imgf000183_0001
Figure imgf000184_0001
Figure imgf000185_0001
Figure imgf000186_0001
Figure imgf000187_0001
Figure imgf000188_0001
Figure imgf000189_0001
Figure imgf000190_0001
Figure imgf000191_0001
or a pharmaceutically acceptable salt thereof. 34. A lipid having a structure selected from the group consisting of:
Figure imgf000191_0002
Figure imgf000192_0001
Figure imgf000193_0001
Figure imgf000194_0001
Figure imgf000195_0001
Figure imgf000196_0001
or a pharmaceutically acceptable salt thereof. 35. A composition comprising a lipid nanoparticle (LNP), wherein the LNP comprises the lipid of any one of embodiments 1-34. 36. The composition of embodiment 35, wherein the LNP comprises: (I) the lipid of any one of embodiments 1-34; (II) a stealth lipid; (III) a structural lipid; and (IV) a helper lipid. 37. The composition of embodiment 36, wherein the stealth lipid is a polyethylene glycol- conjugated (PEGylated) lipid, a polyoxazoline polymer-conjugated lipid, or a polysarcosine- conjugated (pSar) lipid. 38. The composition of embodiment 37, wherein the stealth lipid is a PEGylated lipid selected from 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol (DMG-PEG), 1,2- distearoyl-sn-glycero-3-phosphoethanolamine-polyethylene glycol (DSPE-PEG), 1,2- dilauroyl-sn-glycero-3-phosphoethanolamine-polyethylene glycol (DLPE-PEG), and 1,2- distearoyl-rac-glycero-polyethelene glycol (DSG-PEG). 39. The composition of embodiment 38, wherein the PEGylated lipid is dimyristoyl- PEG2000 (DMG-PEG2000). 40. The composition of any one of embodiments 36-39, wherein the structural lipid is a sterol. 41. The composition of embodiment 40, wherein the sterol is cholesterol. 42. The composition of any one of embodiments 36-41, wherein the helper lipid is 1,2- dioleoyl-SN-glycero-3-phosphoethanolamine (DOPE); 1,2-distearoyl-sn-glycero-3- phosphocholine (DSPC); 1,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS); 1,2- dielaidoyl-sn-glycero-3-phosphoethanolamine (DEPE); and 1,2-dioleoyl-sn-glycero-3- phosphocholine (DPOC), dipalmitoylphosphatidylcholine (DPPC), 1,2-dilauroyl-sn-glycero- 3-phosphocholine (DLPC), 1,2-Distearoylphosphatidylethanolamine (DSPE), or 1,2- dilauroyl-sn-glycero-3-phosphoethanolamine (DLPE). 43. The composition of embodiment 42, wherein the helper lipid is 1,2-dioleoyl-SN- glycero-3-phosphoethanolamine (DOPE). 44. The composition of any one of embodiments 36-43, wherein the LNP comprises: the lipid of any one of claims 1-34 at a molar ratio between 35% and 45%, the stealth lipid at a molar ratio between 0.5% and 7%, the structural lipid at a molar ratio between 20% and 35%, and the helper lipid at a molar ratio of between 20% and 30%. 45. The composition of claim 44, wherein the LNP comprises: the lipid of any one of claims 1-23 at a molar ratio of about 40%, the stealth lipid at a molar ratio of about 1.5%, the structural lipid at a molar ratio of about 30%, and the helper lipid at a molar ratio of about 28.5%. 46. The composition of claim 44, wherein the LNP comprises: the lipid of any one of claims 1-23 at a molar ratio of about 40%, the stealth lipid at a molar ratio of about 5%, the structural lipid at a molar ratio of about 30%, and the helper lipid at a molar ratio of about 25%. 47. The composition of any one of embodiments 35-46, further comprising a nucleic acid molecule, wherein the nucleic acid molecule is encapsulated in the LNP. 48. The composition of embodiment 47, wherein the LNP comprises 1-20, optionally 5- 10 or 6-8, nucleic acid molecules. 49. The composition of embodiment 47 or embodiment 48, wherein the nucleic acid molecule is an mRNA molecule. 50. The composition of embodiment 49, wherein the mRNA molecule encodes an antigen, optionally a viral antigen or a bacterial antigen. 51. The composition of embodiment 49 or embodiment 50, wherein the LNP encapsulates two or more mRNA molecules, wherein each mRNA molecule encodes a different antigen, optionally wherein the different antigens are from the same pathogen or from different pathogens. 52. The composition of embodiment 49 or embodiment 50, wherein the composition comprises two or more LNPs, wherein each LNP encapsulates an mRNA encoding a different antigen, optionally wherein the different antigens are from the same pathogen or from different pathogens. 53. The composition of any one of embodiments 35-52, wherein the composition is formulated for intranasal administration. 54. The composition of any one of claims embodiments 35-53, wherein the composition comprises a phosphate-buffer saline. 55. The composition of any one of embodiments 35-54, wherein the composition comprises trehalose, optionally at 10% (w/v) of the composition. 56. A method of eliciting an immune response in a subject in need thereof, comprising administering to the subject, optionally mucosally, intramuscularly, intranasally, intravenously, subcutaneously, or intradermally, a prophylactically effective amount of the composition of any one of embodiments 47-55. 57. A method of preventing an infection or reducing one or more symptoms of an infection in a subject in need thereof, comprising administering to the subject, optionally mucosally, intramuscularly, intranasally, intravenously, subcutaneously, or intradermally, a prophylactically effective amount of the composition of any one of embodiments 47-55. 58. The method of embodiment 56 or embodiment 57, wherein the composition is administered intranasally. 59. The method of any one of embodiments 56-58, comprising administering to the subject one or more doses of the composition, each dose comprising 1-250, optionally 2.5., 5, 15, 45, or 135, μg of mRNA. 60. The method of any one of embodiments 56-59, comprising administering to the subject two doses of the composition with an interval of 2-6, optionally 4, weeks. 61. Use of the composition of any one of embodiments 47-55 for the manufacture of a medicament for use in treating a subject in need thereof, optionally in a method of any one of embodiments 56-60. 62. The composition of any one of embodiments 47-55 for use in treating a subject in need thereof, optionally in a method of any one of embodiments 56-60. 63. A kit comprising a container comprising a single-use or multi-use dosage of the composition of any one of embodiments 47-55, optionally wherein the container is a vial or a pre-filled nasal spray device. EXAMPLES The compounds and methods disclosed herein are further illustrated by the following examples, which should not be construed as further limiting. The practice of the present disclosure will employ, unless otherwise indicated, conventional techniques of organic synthesis, cell biology, cell culture, and molecular biology, which are within the skill of the art. The following examples further illustrate aspects of the present disclosure. However, they are in no way a limitation of the teachings of the present disclosure as set forth. HPLC Analytical Methods Method 1: HPLC: Agilent 1100 Column: Agela C18 column, 4.6 x 50 mm, 3 µm Column temperature: 60°C Flow Rate: 1.0 mL/min Detector: ELSD Eluents: A, acetonitrile with 0.1% TFA; B, water with 0.1% TFA. Gradient:
Figure imgf000200_0001
Method 2: HPLC: Agilent 1100 Column: Agela C18 column, 4.6 x 50 mm, 3 µm Column temperature: 60°C Flow Rate: 0.5 mL/min Detector: ELSD Eluents: A, Isopropanol; B, water with 0.1% TFA. Gradient:
Figure imgf000200_0002
Figure imgf000201_0001
Method 3: HPLC: Agilent 1100 Column: Agela C18 column, 4.6 x 50 mm, 3 µm Column temperature: 60°C Flow Rate: 0.5 mL/min Detector: ELSD Eluents: A, Isopropanol; B, water. Gradient:
Figure imgf000201_0002
Example 1: Dioctadecyl (S)-2-((3-(dimethylamino)propanoyl)oxy)succinate Step 1: Synthesis of Dimethyl (S)-2-hydroxysuccinate (1) To a solution of L-Malic acid (6 g, 44.7 mmol) in 30 mL methanol, was added 4 M hydrogen chloride in dioxane (5 mL) and the mixture was stirred at room temperature for 17 h. The reaction was carefully basified by adding saturated sodium bicarbonate solution, and the solution was extracted with dichloromethane (3 x 100 mL). The combined organic extracts were washed with brine, dried (anhydrous sodium sulfate) and concentrated to give dimethyl L-malate as an oil (4.35 g, 60%). The crude dimethyl malate was used for the next step without further purification. Step 2: Synthesis of Dioctadecyl (S)-2-hydroxysuccinate (2) A mixture of dimethyl malate 1 (4.35 g, 26.8 mmol), octadecan-1-ol (14.51 g, 53.6 mmol), p-toluene sulfonic acid (800 mg, 4.6 mmol) in 650 mL toluene, was refluxed vigorously with Dean-stark apparatus. After 500 mL of solvent was distilled off, another 500- mL of toluene was added, and the distillation and removal of distillate was continued. This process was repeated one more time. The reaction mixture was cooled to room temperature and washed with saturated NaHCO3 (100 mL), and the aqueous layer was extracted with ethyl acetate (150 mL x 3). The combined organic extracts were dried (anhydrous sodium sulfate) and concentrated, and the crude was purified by silica gel column chromatography using 5-25% ethyl acetate in hexane as eluent to give the desired product as thick oil (5.7 g, 33%). Step 3: Synthesis of Dioctadecyl (S)-2-((3-(dimethylamino)propanoyl)oxy)succinate (Example 1) A mixture of dioctadecyl (S)-2-hydroxysuccinate 2 (400 mg, 0.63 mmol), N,N- dimethylaminobutyric acid hydrochloride (128 mg, 0.76 mmol), EDC-HCl (192 mg, 1 mmol) and DMAP (122 mg, 1 mmol) in 10 mL dichloromethane was stirred at room temperature for 17 h. The volatiles were removed under vacuum, and the residue was dissolved in hexanes (100 mL). The hexane solution was washed with acetonitrile (40 mL x 3), and then the hexane layer was dried over sodium sulfate, filtered, and concentrated to yield the desired product as white solid (401 mg, 85%). 1H NMR (300 MHz, CDCl3) δ 5.48 (t, 1H), 4.14 (t, 2H), 4.11 (t, 2H), 2.87 (d, 2H), 2.68-2.52 (m, 4H), 2.23 (s, 6H), 1.62 (s, br., 4H), 1.25 (m, 60H), 0.87 (t, 6H). APCI-MS analysis: Calculated C45H87NO6 [M+H] = 738.2, Observed = 738.6. Example 2: Dioctadecyl (S)-2-((3-(dimethylamino)butanoyl)oxy)succinate The titular compound was prepared in a manner analogous to Example 1. 1H NMR (300 MHz, CDCl3) δ 5.46 (t, 1H), 4.13 (t, 2H), 4.09 (t, 2H), 2.86 (d, 2H), 2.42 (m, 2H), 2.29 (t, 2H), 2.26 (s, 6H), 1.80 (quint, 2H), 1.62 (s, br., 4H), 1.24 (m, 60H), 0.87 (t, 6H). APCI-MS analysis: Calculated C46H89NO6 [M+H] = 752.2, Observed = 752.6. Example 3: Dihexadecyl (S)-2-((3-(dimethylamino)propanoyl)oxy)succinate The titular compound was prepared in a manner analogous to Example 1. 1H NMR (300 MHz, CDCl3) δ 5.48 (t, 1H), 4.14 (t, 2H), 4.10 (t, 2H), 2.87 (d, 2H), 2.68-2.52 (m, 4H), 2.23 (s, 6H), 1.61 (s, br., 4H), 1.25 (m, 52H), 0.87 (t, 6H). APCI-MS analysis: Calculated C41H79NO6 [M+H] = 682.6, Observed = 682.5. Example 4: Dihexadecyl (S)-2-((3-(dimethylamino)butanoyl)oxy)succinate The titular compound was prepared in a manner analogous to Example 1. 1H NMR (300 MHz, CDCl3) δ 5.46 (t, 1H), 4.14 (t, 2H), 4.10 (t, 2H), 2.86 (d, 2H), 2.43 (m, 2H), 2.31 (t, 2H), 2.21 (s, 6H), 1.81 (quint, 2H), 1.62 (s, br., 4H), 1.24 (m, 52H), 0.87 (t, 6H). APCI-MS analysis: Calculated C42H81NO6 [M+H] = 696.6, Observed = 696.6. Example 5: Di((9Z,12Z)-octadeca-9,12-dien-1-yl) (S)-2-((3- (dimethylamino)propanoyl)oxy)succinate The titular compound was prepared in a manner analogous to Example 1. 1H NMR (300 MHz, CDCl3) δ 5.48 (t, 1H), 5.35 (m, 8H), 4.14 (t, 2H), 4.09 (t, 2H), 2.87 (d, 2H), 2.77 (t, 4H), 2.67-2.52 (m, 4H), 2.23 (s, 6H), 2.03 (m, 8H), 1.61 (s, br., 4H), 1.29 (m, 32H), 0.88 (t, 6H). APCI-MS analysis: Calculated C45H79NO6 [M+H] = 730.6, Observed = 730.5. Example 6: Di((9Z,12Z)-octadeca-9,12-dien-1-yl) (S)-2-((3- (dimethylamino)butanoyl)oxy)succinate The titular compound was prepared in a manner analogous to Example 1. 1H NMR (300 MHz, CDCl3) δ 5.48 (t, 1H), 5.34 (m, 8H), 4.13 (t, 2H), 4.09 (t, 2H), 2.86 (d, 2H), 2.76 (t, 4H), 2.42 (m, 2H), 2.28 (t, 2H), 2.20 (s, 6H), 2.04 (m, 8H), 1.81 (quint, 2H), 1.62 (s, br., 4H), 1.29 (m, 32H), 0.88 (t, 6H). APCI-MS analysis: Calculated C46H81NO6 [M+H] = 744.6, Observed = 744.6. Example 7: Bis(7-(nonanoyloxy)heptyl) (S)-2-((3-(dimethylamino)propanoyl)oxy)succinate Step 1: Synthesis of 7-hydroxyheptyl nonanoate (3) A mixture of 1,7-heptanediol (10.6 g, 80 mmol), nonanoic acid (3.12 g, 20 mmol), EDCI-HCl (4.22 g, 22 mmol) and DMAP (2.7 g, 22 mmol) in 60 mL dichloromethane was stirred at room temperature for 17 h. The dichloromethane was removed on rotavapor, the residue was diluted with ethyl acetate (200 mL), and the solution was washed with aqueous saturated ammonium chloride, followed by brine and dried over sodium sulfate and concentrated to give an oil. The crude product was purified by column chromatography using 15-10% ethyl acetate in hexanes to yield the desired product as colorless oil (4.5 g, 82%). Step 2: Bis(7-(nonanoyloxy)heptyl) (S)-2-hydroxysuccinate (4) A mixture of dimethyl malate 1 (648 mg, 4 mmol), 7-hydroxyheptyl nonanoate 3 (2.16 g, 8 mmol) and p-toluenesulfonic acid (200 mg) in 200 mL toluene was heated to reflux vigorously with Dean-stark apparatus. After 150 mL toluene was distilled, more toluene was added. This process was repeated one more time. The reaction mixture was cooled to room temperature and washed with saturated NaHCO3 solution, and the aqueous layer was extracted with EtOAc. The combined organic layers were dried over sodium sulfate and concentrated to give a viscous oil. The crude product was purified by silica gel column chromatography using 0-10 % ethyl acetate in hexane as eluent to give the desired product as colorless oil (260 mg, 10.1 %) Step 3: Synthesis of bis(7-(nonanoyloxy)heptyl) (S)-2-((3- (dimethylamino)propanoyl)oxy)succinate (Example 7) A mixture of bis(7-(nonanoyloxy)heptyl) (S)-2-hydroxysuccinate 4(130 mg, 0.2 mmol), N,N-dimethylaminobutyric acid hydrochloride (35 mg, 0.3 mmol), EDC-HCl (65 mg, 0.3 mmol) and DMAP (40 mg, 0.3 mmol) in 10 mL dichloromethane was stirred at room temperature for 17 h. The volatiles were removed under vacuum, and the residue was dissolved in EtOAc. The solution was washed with water, and the organic layer was dried over sodium sulfate, filtered, and concentrated, and the crude was purified by silica gel column chromatography using 0-10% methanol in dichloromethane to yield the desired product as colorless oil (120 mg, 81%). 1H NMR (300 MHz, CDCl3) δ 5.45 (t, 1H), 4.11 (t, 2H), 4.06 (t, 2H), 4.01 (t, 4H), 2.84 (d, 2H), 2.65-2.49 (m, 4H), 2.25 (t, 4H), 2.20 (s, 6H), 1.60 (m, 12H), 1.31-1.20 (m, 32H), 0.84 (t, 6H). APCI-MS analysis: Calculated C41H75NO10 [M+H] = 742.5, Observed = 742.5. Example 8: Bis(7-(nonanoyloxy)heptyl) (S)-2-((3-(dimethylamino)butanoyl)oxy)succinate The titular compound was prepared in a manner analogous to Example 7. 1H NMR (300 MHz, CDCl3) δ 5.44 (t, 1H), 4.12 (t, 2H), 4.09 (t, 2H), 4.02 (t, 4H), 2.84 (d, 2H), 2.42 (m, 2H), 2.29-2.24 (m, 6H), 2.19 (s, 6H), 1.78 (quint, 2H), 1.60 (m, 12H), 1.32- 1.25 (m, 32H), 0.85 (t, 6H). APCI-MS analysis: Calculated C42H77NO10 [M+H] = 756.5, Observed = 756.5. Example 9: Dioctadecyl (S)-2-(((2-(dimethylamino)ethoxy)carbonyl)oxy)succinate To a solution of N,N-dimethyl ethanol (356 mg, 4 mmol) in a mixture of 1 mL pyridine and 8 mL dichloromethane, was added 4-nitrophenylchloroformate (804 mg, 4 mmol), and the reaction mixture was stirred at room temperature for 3 h. TLC showed complete reaction. A solution of dioctadecyl (S)-2-hydroxysuccinate 2 (350 mg, 0.55 mmol) in 3 mL dichloromethane was added, then triethylamine (1 mL) was added, and the resulting mixture was stirred at room temperature for another 15 h. After concentration, the residue was dissolved in hexanes, and the solution was washed by acetonitrile several times until the acetonitrile layer contains no 4-nitrophenol. The hexane layer was concentrated, and the crude was purified by column chromatography using 50% ethyl acetate in hexanes, followed by ethyl acetate and acetone in ethyl acetate to give desired product (133 mg, 32%). 1H NMR (300 MHz, CDCl3) δ 5.37 (t, 1H), 4.26 (t, 2H), 4.15 (dt, 2H), 4.09 (t, 2H), 2.89 (d, 2H), 2.61 (dd, 2H), 2.28 (s, 6H), 1.64-1.57 (m, 4H), 1.24 (m, 60H), 0.87 (t, 6H). APCI-MS analysis: Calculated C45H87NO7 [M+H] = 754.6, Observed = 754.6. Example 10: Dioctadecyl (S)-2-(((3-(dimethylamino)propoxy)carbonyl)oxy)succinate The titular compound was prepared in a manner analogous to Example 9. 1H NMR (300 MHz, CDCl3) δ 5.37 (t, 1H), 4.23 (t, 2H), 4.16 (dt, 2H), 4.10 (t, 2H), 2.89 (d, 2H), 2.35 (t, 2H), 2.21 (s, 6H), 1.84 (quint, 2H), 1.64-1.57 (m, 4H), 1.24 (m, 60H), 0.87 (t, 6H). APCI-MS analysis: Calculated C46H89NO7 [M+H] = 768.7, Observed = 768.6. Example 11: Dihexadecyl (S)-2-(((2-(dimethylamino)ethoxy)carbonyl)oxy)succinate The titular compound was prepared in a manner analogous to Example 9. 1H NMR (300 MHz, CDCl3) δ 5.37 (t, 1H), 4.25 (t, 2H), 4.15 (dt, 2H), 4.09 (t, 2H), 2.89 (d, 2H), 2.61 (dd, 2H), 2.28 (s, 6H), 1.64-1.57 (m, 4H), 1.24 (m, 52H), 0.87 (t, 6H). APCI-MS analysis: Calculated C41H79NO7 [M+H] = 698.5, Observed = 698.5. Example 12: Dihexadecyl (S)-2-(((3-(dimethylamino)propoxy)carbonyl)oxy)succinate The titular compound was prepared in a manner analogous to Example 9. 1H NMR (300 MHz, CDCl3) δ 5.37 (t, 1H), 4.25 (t, 2H), 4.16 (dt, 2H), 4.10 (t, 2H), 2.90 (d, 2H), 2.54 (s, br., 2H), 2.36 (s, 6H), 1.94 (quint, 2H), 1.66-1.59 (m, 4H), 1.25 (m, 52H), 0.87 (t, 6H). APCI-MS analysis: Calculated C42H81NO7 [M+H] = 712.6, Observed = 712.5. Example 13: Di((9Z,12Z)-octadeca-9,12-dien-1-yl) (S)-2-(((2- (dimethylamino)ethoxy)carbonyl)oxy)-succinate The titular compound was prepared in a manner analogous to Example 9. 1H NMR (300 MHz, CDCl3) δ 5.42-5.25 (m, 9H), 4.24 (t, 2H), 4.15 (dt, 2H), 4.09 (t, 2H), 2.88 (d, 2H), 2.75 (t, 4H), 2.61 (dd, 2H), 2.26 (s, 6H), 2.06-2.00 (m, 8H), 1.64-1.57 (m, 4H), 1.28 (m, 32H), 0.87 (t, 6H). APCI-MS analysis: Calculated C45H79NO7 [M+H] = 746.6, Observed = 746.5. Example 14: Di((9Z,12Z)-octadeca-9,12-dien-1-yl) (S)-2-(((3- (dimethylamino)propoxy)carbonyl)oxy)-succinate The titular compound was prepared in a manner analogous to Example 9. 1H NMR (300 MHz, CDCl3) δ 5.42-5.25 (m, 9H), 4.20 (t, 2H), 4.15 (dt, 2H), 4.09 (t, 2H), 2.88 (d, 2H), 2.75 (t, 4H), 2.33 (t, 2H), 2.20 (s, 6H), 2.06-2.00 (m, 8H), 1.85 (quint, 2H), 1.64-1.57 (m, 4H), 1.28 (m, 32H), 0.87 (t, 6H). APCI-MS analysis: Calculated C46H81NO7 [M+H] = 760.6, Observed = 760.5. Example 15: Bis(7-(nonanoyloxy)heptyl) (S)-2-(((2- (dimethylamino)ethoxy)carbonyl)oxy)succinate The titular compound was prepared in a manner analogous to Example 9. 1H NMR (300 MHz, CDCl3) δ 5.36 (t, 1H), 4.24 (t, 2H), 4.15 (t, 2H), 4.08 (t, 2H), 4.03 (t, 4H), 2.87 (d, 2H), 2.59 (dd, 2H), 2.26 (s, 6H), 2.26 (t, 4H), 1.59 (m, 12H), 1.38-1.17 (m, 32H), 0.85 (t, 6H). APCI-MS analysis: Calculated C41H75NO11 [M+H] = 758.5, Observed = 758.5. Example 16: Bis(7-(nonanoyloxy)heptyl) (S)-2-(((3- (dimethylamino)propoxy)carbonyl)oxy)succinate The titular compound was prepared in a manner analogous to Example 9. 1H NMR (300 MHz, CDCl3) δ 5.35 (t, 1H), 4.23 (dt, 2H), 4.14 (t, 2H), 4.08 (t, 2H), 4.02 (t, 4H), 2.87 (d, 2H), 2.33 (t, 2H), 2.26 (t, 4H), 2.19 (s, 6H), 1.82 (quint, 2H), 1.64-1.57 (m, 12H), 1.38-1.17 (m, 32H), 0.85 (t, 6H). APCI-MS analysis: Calculated C42H77NO11 [M+H] = 772.5, Observed = 772.5. Example 17: Dioctadecyl (S)-2-(((2-(dimethylamino)ethyl)(methyl)carbamoyl)oxy)succinate To a solution of N1,N1,N2-trimethylethane-1,2-diamine (408 mg, 4 mmol) in a mixture of 1 mL pyridine and 8 mL dichloromethane, was added 4-nitrophenylchloroformate (804 mg, 4 mmol), and the resulting mixture was stirred at room temperature for 2 h. TLC showed complete reaction. A solution of dioctadecyl (S)-2-hydroxysuccinate 2 (320 mg, 0.5 mmol) in 3 mL dichloromethane, then 1 mL triethylamine was added, and the reaction was stirred at room temperature for 15 h. After concentrating under reduced pressure, the residue was dissolved in hexanes and washed with acetonitrile several times until the acetonitrile layer becomes colorless. The hexane layer was concentrated, and the crude was purified by column chromatography using 50% ethyl acetate in hexanes, followed by ethyl acetate and 50% acetone in ethyl acetate to give desired product (240 mg, 63 %). 1H NMR (300 MHz, CDCl3) δ 5.43-5.37 (m, 1H), 4.13 (t, 2H), 4.11 (t, 2H), 3.45-3.24 (m, 2H), 2.94 (s, 3H), 2.86 (d, 2H), 2.48-2.40 (m, 2H), 2.25 (d, 6H), 1.66-1.53 (m, 4H), 1.24 (m, 60H), 0.87 (t, 6H). APCI-MS analysis: Calculated C46H90N2O6 [M+H] = 767.7, Observed = 767.6. Example 18: Dioctadecyl (S)-2-(((3- (dimethylamino)propyl)(methyl)carbamoyl)oxy)succinate The titular compound was prepared in a manner analogous to Example 17. 1H NMR (300 MHz, CDCl3) δ 5.42-5.35 (m, 1H), 4.11 (t, 2H), 4.09 (t, 2H), 3.27 (t, 2H), 2.90 (s, 3H), 2.84 (d, 2H), 2.21 (t, 2H), 2.18 (s, 6H), 1.68 (quint, 2H), 1.61-1.54 (m, 4H), 1.23 (m, 60H), 0.85 (t, 6H). APCI-MS analysis: Calculated C47H92N2O6 [M+H] = 781.7, Observed = 781.6. Example 19: Dihexadecyl (S)-2-(((2-(dimethylamino)ethyl)(methyl)carbamoyl)oxy)succinate The titular compound was prepared in a manner analogous to Example 17. 1H NMR (300 MHz, CDCl3) δ 5.42-5.35 (m, 1H), 4.14 (t, 2H), 4.09 (t, 2H), 3.45-3.24 (m, 2H), 2.94 (s, 3H), 2.86 (d, 2H), 2.48-2.40 (m, 2H), 2.24 (d, 6H), 1.68-1.53 (m, 4H), 1.25 (m, 52H), 0.87 (t, 6H). APCI-MS analysis: Calculated C42H82N2O6 [M+H] = 711.6, Observed = 711.5. Example 20: Dihexadecyl (S)-2-(((3- (dimethylamino)propyl)(methyl)carbamoyl)oxy)succinate The titular compound was prepared in a manner analogous to Example 17. 1H NMR (300 MHz, CDCl3) δ 5.42-5.35 (m, 1H), 2.18-4.08 (m, 4H), 3.29 (t, 2H), 2.92 (s, 3H), 2.86 (d, 2H), 2.26 (t, 2H), 2.21 (s, 6H), 1.65-1.57 (m, 6H), 1.25 (m, 52H), 0.85 (t, 6H). APCI-MS analysis: Calculated C43H84N2O6 [M+H] = 725.6, Observed = 725.5. Example 21: Di((9Z,12Z)-octadeca-9,12-dien-1-yl) (S)-2-(((2- (dimethylamino)ethyl)(methyl)carbamoyl)-oxy)succinate The titular compound was prepared in a manner analogous to Example 17. 1H NMR (300 MHz, CDCl3) δ 5.44-5.28 (m, 9H), 4.14 (t, 2H), 4.09 (t, 2H), 3.45-3.24 (m, 2H), 2.94 (s, 3H), 2.86 (d, 2H), 2.76 (t, 4H), 2.48-2.40 (m, 2H), 2.24 (d, 6H), 2.10-1.97 (m, 8H), 1.68-1.53 (m, 4H), 1.29 (m, 32H), 0.88 (t, 6H). APCI-MS analysis: Calculated C46H82N2O6 [M+H] = 759.6, Observed = 759.6. Example 22: Di((9Z,12Z)-octadeca-9,12-dien-1-yl) (S)-2-(((3- (dimethylamino)propyl)(methyl)-carbamoyl)oxy)succinate The titular compound was prepared in a manner analogous to Example 17. 1H NMR (300 MHz, CDCl3) δ 5.44-5.28 (m, 9H), 4.13 (t, 2H), 4.09 (t, 2H), 3.29 (t, 2H), 2.92 (s, 3H), 2.86 (d, 2H), 2.76 (t, 4H), 2.24 (t, 2H), 2.21 (d, 6H), 2.10-1.96 (m, 8H), 1.70 (quint, 2H), 1.60 (m, 4H), 1.29 (m, 32H), 0.88 (t, 6H). APCI-MS analysis: Calculated C47H84N2O6 [M+H] = 773.6, Observed = 773.6. Example 23: Dioctadecyl ((2-(dimethylamino)ethoxy)carbonyl)-L-aspartate Step 1: Synthesis of Dioctadecyl (tert-butoxycarbonyl)-L-aspartate (5) A mixture of N-Boc-(L)-aspartic acid (2.33g, 10 mmol), octadecan-1-ol (5.4 g, 20 mmol), EDCI-HCl (4.8 g, 25 mmol) and DMAP (3.05 g, 25 mmol) in a mixture of 25 mL dichloromethane and 20 mL DMF was stirred at room temperature for 17 h. The reaction mixture was diluted with saturated ammonium chloride solution (100 mL) and extracted with dichloromethane (100 mL x 3). The combined organic layers were washed with brine, dried (sodium sulfate) and concentrated, and the crude was purified by column chromatography using 10-35% ethyl acetate in hexanes to get the desired product as white solid (5.2 g, 71%). Step 2: Synthesis of Dioctadecyl L-aspartate (6) To a solution of dioctadecyl (tert-butoxycarbonyl)-L-aspartate 5 (1.2 g, 1.63 mmol) in 10 mL dichloromethane, was added 3 mL trifluoroacetic acid, and the mixture was stirred at room temperature for 6 h. The volatiles were concentrated under vacuum, and the residue was dissolved in dichloromethane, and washed with saturated sodium bicarbonate solution. The organic layer was dried over sodium sulfate and concentrated to give the desired product (1.0 g, 99%), which was used without further purification. Step 3: Synthesis of Dioctadecyl ((2-(dimethylamino)ethoxy)carbonyl)-L-aspartate (Example 23) To a solution of 2-(dimethylamino)ethan-1-ol (267 mg, 3 mmol) in a mixture of 1 mL pyridine and 8 mL dichloromethane, was added 4-nitrophenylchloroformate (603 mg, 3 mmol), and the resulting mixture was stirred at room temperature for 3 h. A solution of dioctadecyl L-aspartate 6 (400 mg, 0.61 mmol) in 3 mL dichloromethane was added, then 1 mL triethylamine was added, and the reaction was stirred at room temperature for 15 h. After concentration under vacuum, the residue was dissolved in hexanes, and the solution was washed by acetonitrile several times until the acetonitrile layer becomes colorless. The hexane layer was concentrated to give the desired product (327 mg, 69%). 1H NMR (300 MHz, CDCl3) δ 5.75 (d, 1H), 4.62-4.54 (m, 1H), 4.17 (t, 2H), 4.13 (t, 2H), 4.05 (t, 2H), 3.02 (dd, 1H), 2.81 (dd, 1H), 2.56 (t, 2H), 2.28 (s, 6H), 1.66-1.53 (m, 4H), 1.24 (m, 60H), 0.87 (t, 6H). APCI-MS analysis: Calculated C46H90N2O6 [M+H] = 767.7, Observed = 767.6. Example 24: Dioctadecyl ((3-(dimethylamino)propoxy)carbonyl)-L-aspartate The titular compound was prepared in a manner analogous to Example 23. 1H NMR (300 MHz, CDCl3) δ 5.66 (d, 1H), 4.59 (m, 1H), 4.20-4.03 (m, 6H), 3.02 (dd, 1H), 2.82 (dd, 1H), 2.36 (t, 2H), 2.24 (s, 6H), 1.88-1.77 (m, 2H), 1.67-1.53 (m, 4H), 1.25 (m, 60H), 0.87 (t, 6H). APCI-MS analysis: Calculated C46H90N2O6 [M+H] = 767.7, Observed = 767.6. Example 25: Dihexadecyl ((2-(dimethylamino)ethoxy)carbonyl)-L-aspartate The titular compound was prepared in a manner analogous to Example 23. 1H NMR (300 MHz, CDCl3) δ 5.75 (d, 1H), 4.62-4.54 (m, 1H), 4.15 (t, 2H), 4.13 (t, 2H), 4.06 (t, 2H), 3.02 (dd, 1H), 2.81 (dd, 1H), 2.55 (t, 2H), 2.28 (s, 6H), 1.66-1.55 (m, 4H), 1.25 (m, 52H), 0.87 (t, 6H). APCI-MS analysis: Calculated C41H80N2O6 [M+H] = 697.6, Observed = 697.5. Example 26: Dihexadecyl ((3-(dimethylamino)propoxy)carbonyl)-L-aspartate The titular compound was prepared in a manner analogous to Example 23. 1H NMR (300 MHz, CDCl3) δ 5.65 (d, 1H), 4.62-4.53 (m, 1H), 4.18-4.04 (m, 6H), 3.02 (dd, 1H), 2.82 (dd, 1H), 2.33 (t, 2H), 2.22 (s, 6H), 1.79 (quint, 2H), 1.67-1.56 (m, 4H), 1.25 (m, 52H), 0.87 (t, 6H). APCI-MS analysis: Calculated C42H82N2O6 [M+H] = 711.6, Observed = 711.5. Example 27: Di((9Z,12Z)-octadeca-9,12-dien-1-yl) ((2-(dimethylamino)ethoxy)carbonyl)-L- aspartate The titular compound was prepared in a manner analogous to Example 23. 1H NMR (300 MHz, CDCl3) δ 5.76 (d, 1H), 5.45-5.25 (m, 8H), 4.62-4.54 (m, 1H), 4.17 (t, 2H), 4.12 (t, 2H), 4.05 (t, 2H), 3.02 (dd, 1H), 2.81 (dd, 1H), 2.76 (t, 4H), 2.57 (t, 2H), 2.28 (s, 6H), 2.08-1.96 (m, 8H), 1.66-1.52 (m, 4H), 1.29 (m, 32H), 0.87 (t, 6H). APCI-MS analysis: Calculated C45H80N2O6 [M+H] = 745.6, Observed = 745.6. Example 28: Di((9Z,12Z)-octadeca-9,12-dien-1-yl) ((3-(dimethylamino)propoxy)carbonyl)- L-aspartate The titular compound was prepared in a manner analogous to Example 23. 1H NMR (300 MHz, CDCl3) δ 5.65 (d, 1H), 5.45-5.25 (m, 8H), 4.62-4.54 (m, 1H), 4.19-4.04 (m, 6H), 3.02 (dd, 1H), 2.81 (dd, 1H), 2.76 (t, 4H), 2.35 (t, 2H), 2.23 (s, 6H), 2.08-1.96 (m, 8H), 1.78 (quint, 2H), 1.66-1.52 (m, 4H), 1.29 (m, 32H), 0.88 (t, 6H). APCI-MS analysis: Calculated C46H82N2O6 [M+H] = 759.6, Observed = 759.6. Example 29: Dioctadecyl 2-((3-(dimethylamino)propanoyl)oxy)pentanedioate Step 1: Synthesis of Dioctadecyl 2-oxopentanedioate (7) A mixture of α-ketoglutaric acid (3.0 g, 20.5 mmol), 1-octadecanol (11.0 g, 41 mmol) and p-toluenesulfonic acid monohydrate (0.5 g) in 80 mL toluene was heated to reflux for 5 hours. The reaction mixture was concentrated under vacuum, and the residue was crystallized in acetonitrile to give dioctadecyl 2-oxopentanedioate was obtained as brown solid (14.0 g, quant.) Step 2: Synthesis of Dioctadecyl 2-hydroxypentanedioate (8) To a solution of dioctadecyl 2-oxopentanedioate 7 (14 g, 20.5 mmol) in 100 mL THF, was added sodium borohydride (1.2 g, 32.2 mmol), and the reaction mixture was stirred at room temperature for 2 hours. After cooled to 0 °C, saturated ammonium chloride solution was added slowly, and the resulting mixture was extracted with ethyl acetate. The combined organic layer was washed with brine and dried over sodium sulfate. After concentration, the crude was purified by flash column chromatography eluted with hexane/ethyl acetate to get dioctadecyl 2-hydroxypentanedioate as white solid (2.0 g, 15%). The byproducts include 1- octadecanol and octadecyl 5-oxotetrahydrofuran-2-carboxylate. Step 3: Synthesis of Dioctadecyl 2-((3-(dimethylamino)propanoyl)oxy)pentanedioate (Example 29) A mixture of dioctadecyl 2-hydroxypentanedioate 8 (100 mg, 0.15 mmol), 2,2- dimethylaminopropanoic acid (27 mg, 0.23 mmol), EDCI (57.5 mg, 0.3 mmol) and 4- dimethylaminopyridine (37 mg, 0.3 mmol) in 10 mL dichloromethane was stirred at room temperature overnight. The reaction mixture was concentrated, and the residue was partitioned between hexane and acetonitrile. After separation, the hexane layer was washed with acetonitrile, and then concentrated to afford dioctadecyl 2-((3- (dimethylamino)propanoyl)oxy)pentanedioate as pale yellow wax (100 mg, 91%). 1H NMR (300 MHz, CDCl3) δ 5.05 (dd, 1H), 4.12 (t, 2H), 4.06 (t, 2H), 2.68-2.53 (m, 4H), 2.43 (m, 2H), 2.24 (s, 6H), 2.21-2.08 (m, 2H), 1.68-1.55 (m, 4H), 1.24 (m, 60H), 0.87 (t, 6H). APCI-MS analysis: Calculated C46H89NO6 [M+H] = 752.7, Observed = 752.6. Example 30: Dioctadecyl 2-((4-(dimethylamino)butanoyl)oxy)pentanedioate The titular compound was prepared in a manner analogous to Example 29. 1H NMR (300 MHz, CDCl3) δ 5.02 (dd, 1H), 4.12 (t, 2H), 4.06 (t, 2H), 2.42 (m, 4H), 2.29 (t, 2H), 2.21 (s, 6H), 2.21-2.08 (m, 2H), 1.81 (quint, 2H), 1.66-1.56 (m, 4H), 1.24 (m, 60H), 0.87 (t, 6H). APCI-MS analysis: Calculated C47H91NO6 [M+H] = 766.7, Observed = 766.6. Example 31: Dihexadecyl 2-((3-(dimethylamino)propanoyl)oxy)pentanedioate The titular compound was prepared in a manner analogous to Example 29. 1H NMR (300 MHz, CDCl3) δ 5.07 (dd, 1H), 4.12 (t, 2H), 4.06 (t, 2H), 2.68-2.53 (m, 4H), 2.43 (m, 2H), 2.24 (s, 6H), 2.22-2.08 (m, 2H), 1.66-1.55 (m, 4H), 1.24 (m, 52H), 0.87 (t, 6H). APCI-MS analysis: Calculated C42H81NO6 [M+H] = 696.6, Observed = 695.9. Example 32: Dihexadecyl 2-((4-(dimethylamino)butanoyl)oxy)pentanedioate The titular compound was prepared in a manner analogous to Example 29. 1H NMR (300 MHz, CDCl3) δ 5.02 (dd, 1H), 4.12 (t, 2H), 4.06 (t, 2H), 2.43 (m, 4H), 2.34 (t, 2H), 2.25 (s, 6H), 2.23-2.09 (m, 2H), 1.85 (quint, 2H), 1.68-1.56 (m, 4H), 1.24 (m, 52H), 0.87 (t, 6H). APCI-MS analysis: Calculated C43H83NO6 [M+H] = 710.6, Observed = 709.8. Example 33: Di((9Z,12Z)-octadeca-9,12-dien-1-yl) 2-((3- (dimethylamino)propanoyl)oxy)pentanedioate The titular compound was prepared in a manner analogous to Example 29. 1H NMR (300 MHz, CDCl3) δ 5.42-5.27 (m, 8H), 5.07 (dd, 1H), 4.12 (t, 2H), 4.06 (t, 2H), 2.76 (t, 2H), 2.67-2.55 (m, 4H), 2.44 (m, 2H), 2.24 (s, 6H), 2.22-2.08 (m, 2H), 2.07-2.01 (m, 8H), 1.66-1.55 (m, 6H), 1.29 (m, 32H), 0.88 (t, 6H). APCI-MS analysis: Calculated C46H81NO6 [M+H] = 744.6, Observed = 743.7. Example 34: Di((9Z,12Z)-octadeca-9,12-dien-1-yl) 2-((4- (dimethylamino)butanoyl)oxy)pentanedioate The titular compound was prepared in a manner analogous to Example 29. 1H NMR (300 MHz, CDCl3) δ 5.42-5.27 (m, 8H), 5.02 (dd, 1H), 4.12 (t, 2H), 4.06 (t, 2H), 2.76 (t, 4H), 2.46-2.40 (m, 4H), 2.30 (t, 2H), 2.22 (s, 6H), 2.20-2.09 (m, 2H), 2.08-2.01 (m, 8H), 1.81 (quint, 2H), 1.66-1.55 (m, 4H), 1.29 (m, 32H), 0.88 (t, 6H). APCI-MS analysis: Calculated C47H83NO6 [M+H] = 758.6, Observed = 757.7. Example 35: Bis(7-(nonanoyloxy)heptyl) 2-((3- (dimethylamino)propanoyl)oxy)pentanedioate The titular compound was prepared in a manner analogous to Example 29 and was obtained by column purification chromatography eluted with hexane/acetone. 1H NMR (300 MHz, CDCl3) δ 5.05 (dd, 1H), 4.12 (t, 2H), 4.06 (t, 2H), 4.04 (t, 4H), 2.67- 2.53 (m, 4H), 2.44 (m, 2H), 2.28 (t, 4H), 2.24 (s, 6H), 2.22-2.08 (m, 2H), 1.66-1.53 (m, 12H), 1.38-1.18 (m, 32H), 0.87 (t, 6H). APCI-MS analysis: Calculated C42H77NO10 [M+H] = 756.5, Observed = 756.5. Example 36: Bis(7-(nonanoyloxy)heptyl) 2-((4-(dimethylamino)butanoyl)oxy)pentanedioate The titular compound was prepared in a manner analogous to Example 29 and was obtained by column purification chromatography eluted with hexane/acetone. 1H NMR (300 MHz, CDCl3) δ 5.01 (dd, 1H), 4.12 (t, 2H), 4.06 (t, 2H), 4.04 (t, 4H), 2.49- 2.42 (m, 4H), 2.28 (t, 6H), 2.21 (s, 6H), 2.20-2.07 (m, 2H), 1.80 (quint, 2H), 1.68-1.55 (m, 12H), 1.36-1.19 (m, 32H), 0.87 (t, 6H). APCI-MS analysis: Calculated C43H79NO10 [M+H] = 770.6, Observed = 770.5. Example 37: Dioctadecyl 2-(((2-(dimethylamino)ethoxy)carbonyl)oxy)pentanedioate To a solution of dioctadecyl 2-hydroxypentanedioate 8 (150 mg, 0.23 mmol) and pyridine (75 μL, 0.92 mmol) in 10 mL dichloromethane, was added p-nitrophenyl chloroformate (138 mg, 0.69 mmol), and the resulting mixture was stirred for 2 hours. After TLC showed the complete reaction, 2-dimethylaminoethanol (73 mg, 0.92 mmol) was added, and the reaction mixture was stirred at room temperature overnight. MS showed the formation of desired product. The reaction mixture was concentrated, and the residue was partitioned between hexane and acetonitrile. The hexane layer was concentrated, and the crude was purified by column chromatography eluted with dichloromethane/acetone to afford dioctadecyl 2-(((2-(dimethylamino)ethoxy)carbonyl)oxy)pentanedioate as pale yellow wax (141 mg, 80%). 1H NMR (300 MHz, CDCl3) δ 4.94 (dd, 1H), 4.23 (dt, 2H), 4.13 (t, 2H), 4.04 (t, 2H), 2.68- 2.53 (m, 2H), 2.48-2.41 (m, 2H), 2.28 (s, 6H), 2.21-2.04 (m, 2H), 1.68-1.53 (m, 4H), 1.24 (m, 60H), 0.86 (t, 6H). APCI-MS analysis: Calculated C46H89NO7 [M+H] = 768.6, Observed = 768.6. Example 38: Dioctadecyl 2-(((3-(dimethylamino)propoxy)carbonyl)oxy)pentanedioate The titular compound was prepared in a manner analogous to Example 37. 1H NMR (300 MHz, CDCl3) δ 4.94 (dd, 1H), 4.20 (t, 2H), 4.12 (t, 2H), 4.05 (t, 2H), 2.48- 2.41 (m, 2H), 2.35 (t, 2H), 2.21 (s, 6H), 2.21-2.09 (m, 2H), 1.84 (quint, 2H), 1.66-1.56 (m, 4H), 1.24 (m, 60H), 0.86 (t, 6H). APCI-MS analysis: Calculated C47H91NO7 [M+H] = 782.7, Observed = 782.6. Example 39: Dihexadecyl 2-(((2-(dimethylamino)ethoxy)carbonyl)oxy)pentanedioate The titular compound was prepared in a manner analogous to Example 37. 1H NMR (300 MHz, CDCl3) δ 4.95 (dd, 1H), 4.25 (dt, 2H), 4.15 (t, 2H), 4.06 (t, 2H), 2.68- 2.52 (m, 2H), 2.50-2.44 (m, 2H), 2.28 (s, 6H), 2.25-2.10 (m, 2H), 1.68-1.54 (m, 4H), 1.25 (m, 52H), 0.87 (t, 6H). APCI-MS analysis: Calculated C42H81NO7 [M+H] = 712.6, Observed = 712.5. Example 40: Dihexadecyl 2-(((3-(dimethylamino)propoxy)carbonyl)oxy)pentanedioate The titular compound was prepared in a manner analogous to Example 37. 1H NMR (300 MHz, CDCl3) δ 4.95 (dd, 1H), 4.21 (t, 2H), 4.15 (t, 2H), 4.06 (t, 2H), 2.50- 2.42 (m, 2H), 2.36 (t, 2H), 2.22 (s, 6H), 2.21-2.10 (m, 2H), 1.85 (quint, 2H), 1.66-1.56 (m, 4H), 1.25 (m, 52H), 0.87 (t, 6H). APCI-MS analysis: Calculated C43H83NO7 [M+H] = 726.7, Observed = 726.5. Example 41: Di((9Z,12Z)-octadeca-9,12-dien-1-yl) 2-(((2- (dimethylamino)ethoxy)carbonyl)oxy)-pentanedioate The titular compound was prepared in a manner analogous to Example 37. 1H NMR (300 MHz, CDCl3) δ 5.44-5.27 (m, 8H), 4.95 (dd, 1H), 4.25 (dt, 2H), 4.15 (t, 2H), 4.06 (t, 2H), 2.76 (t, 4H), 2.68-2.55 (m, 2H), 2.51-2.43 (m, 2H), 2.28 (s, 6H), 2.25-2.12 (m, 2H), 2.14-2.01 (m, 8H), 1.66-1.55 (m, 4H), 1.29 (m, 32H), 0.88 (t, 6H). APCI-MS analysis: Calculated C46H81NO7 [M+H] = 760.6, Observed = 759.6. Example 42: Di((9Z,12Z)-octadeca-9,12-dien-1-yl) 2-(((3- (dimethylamino)propoxy)carbonyl)oxy)-pentanedioate The titular compound was prepared in a manner analogous to Example 37. 1H NMR (300 MHz, CDCl3) δ 5.43-5.27 (m, 8H), 4.95 (dd, 1H), 4.21 (dt, 2H), 4.15 (t, 2H), 4.06 (t, 2H), 2.76 (t, 4H), 2.51-2.42 (m, 2H), 2.39-2.34 (m, 2H), 2.22 (s, 6H), 2.20-2.12 (m, 2H), 2.07-2.01 (m, 8H), 1.85 (quint, 2H), 1.66-1.55 (m, 4H), 1.29 (m, 32H), 0.88 (t, 6H). APCI-MS analysis: Calculated C47H83NO7 [M+H] = 774.6, Observed = 773.7. Example 43: Bis(7-(nonanoyloxy)heptyl) 2-(((2- (dimethylamino)ethoxy)carbonyl)oxy)pentanedioate The titular compound was prepared in a manner analogous to Example 37. 1H NMR (300 MHz, CDCl3) δ 4.94 (dd, 1H), 4.27-4.21 (m, 2H), 4.14 (t, 2H), 4.07-4.01 (m, 6H), 2.68-2.56 (m, 2H), 2.50-2.42 (m, 2H), 2.27 (s, 6H), 2.27 (t, 4H), 2.24-2.04 (m, 2H), 1.66-1.53 (m, 12H), 1.38-1.18 (m, 32H), 0.86 (t, 6H). APCI-MS analysis: Calculated C42H77NO11 [M+H] = 772.5, Observed = 772.5. Example 44: Bis(7-(nonanoyloxy)heptyl) 2-(((3- (dimethylamino)propoxy)carbonyl)oxy)pentanedioate The titular compound was prepared in a manner analogous to Example 37. 1H NMR (300 MHz, CDCl3) δ 4.96 (dd, 1H), 4.27-4.21 (m, 2H), 4.15 (t, 2H), 4.08-4.02 (m, 8H), 2.50-2.42 (m, 2H), 2.39-2.33 (m, 2H), 2.28 (t, 4H), 2.22 (s, 6H), 2.20-2.08 (m, 2H), 1.85 (quint, 2H), 1.66-1.53 (m, 12H), 1.38-1.18 (m, 32H), 0.87 (t, 6H). APCI-MS analysis: Calculated C43H79NO11 [M+H] = 786.5, Observed = 785.5. Example 45: Dioctadecyl 2-(((2-(dimethylamino)ethyl)(methyl)carbamoyl)oxy)- pentanedioate To a solution of dioctadecyl 2-hydroxypentanedioate 8 (150 mg, 0.23 mmol) and pyridine (75 μL, 0.92 mmol) in 10 mL dichloromethane, was added p-nitrophenyl chloroformate (138 mg, 0.69 mmol), and the resulting mixture was stirred for 2 hours. After TLC showed the complete reaction, N1,N1,N2-trimethylethane-1,2-diamine (94 mg, 0.92 mmol) was added, and the reaction mixture was stirred at room temperature overnight. MS showed the formation of desired product. The reaction mixture was concentrated, and the residue was partitioned between hexane and acetonitrile. The hexane layer was concentrated, and the crude was purified by column chromatography eluted with dichloromethane/acetone to afford dioctadecyl 2-(((2-(dimethylamino)ethyl)(methyl)carbamoyl)oxy)-pentanedioate as pale yellow wax (142 mg, 79%). 1H NMR (300 MHz, CDCl3) δ 5.03-4.93 (m, 1H), 4.12 (dt, 2H), 4.05 (t, 2H), 3.50-3.25 (m, 2H), 2.95 (d, 3H), 2.52-2.39 (m, 4H), 2.25 (s, 6H), 2.21-2.08 (m, 2H), 1.68-1.53 (m, 4H), 1.24 (m, 60H), 0.86 (t, 6H). APCI-MS analysis: Calculated C47H92N2O6 [M+H] = 781.7, Observed = 781.6. Example 46: Dioctadecyl 2-(((3- (dimethylamino)propyl)(methyl)carbamoyl)oxy)pentanedioate The titular compound was prepared in a manner analogous to Example 45. 1H NMR (300 MHz, CDCl3) δ 5.03-4.93 (m, 1H), 4.11 (dt, 2H), 4.04 (t, 2H), 3.35-3.25 (m, 2H), 2.91 (d, 3H), 2.44-2.37 (m, 2H), 2.29-2.23 (m, 2H), 2.20 (s, 6H), 2.19-2.08 (m, 2H), 1.76-1.53 (m, 6H), 1.23 (m, 60H), 0.85 (t, 6H). APCI-MS analysis: Calculated C48H94N2O6 [M+H] = 795.7, Observed = 795.6. Example 47: Dihexadecyl 2-(((2- (dimethylamino)ethyl)(methyl)carbamoyl)oxy)pentanedioate The titular compound was prepared in a manner analogous to Example 45. 1H NMR (300 MHz, CDCl3) δ 5.03-4.93 (m, 1H), 4.11 (dt, 2H), 4.04 (t, 2H), 3.50-3.25 (m, 2H), 2.94 (d, 3H), 2.52-2.39 (m, 4H), 2.25 (s, 6H), 2.21-2.06 (m, 2H), 1.68-1.53 (m, 4H), 1.23 (m, 52H), 0.85 (t, 6H). APCI-MS analysis: Calculated C43H84N2O6 [M+H] = 725.6, Observed = 725.5. Example 48: Dihexadecyl 2-(((3- (dimethylamino)propyl)(methyl)carbamoyl)oxy)pentanedioate The titular compound was prepared in a manner analogous to Example 45. 1H NMR (300 MHz, CDCl3) δ 5.03-4.93 (m, 1H), 4.11 (dt, 2H), 4.05 (t, 2H), 3.38-3.25 (m, 2H), 2.93 (d, 3H), 2.48-2.39 (m, 2H), 2.24 (s, 6H), 2.35-2.13 (m, 4H), 1.74 (quint, 2H), 1.66- 1.53 (m, 4H), 1.24 (m, 52H), 0.87 (t, 6H). APCI-MS analysis: Calculated C44H86N2O6 [M+H] = 739.6, Observed = 739.6. Example 49: Di((9Z,12Z)-octadeca-9,12-dien-1-yl) 2-(((2- (dimethylamino)ethyl)(methyl)carbamoyl)oxy)-pentanedioate
Figure imgf000223_0001
The titular compound was prepared in a manner analogous to Example 45. 1H NMR (300 MHz, CDCl3) δ 5.44-5.27 (m, 8H), 5.03-4.95 (m, 1H), 4.12 (t, 2H), 4.06 (t, 2H), 3.53-3.24 (m, 2H), 2.96 (d, 3H), 2.76 (t, 4H), 2.52-2.41 (m, 4H), 2.26 (s, 6H), 2.24-2.12 (m, 2H), 2.09-2.01 (m, 8H), 1.66-1.55 (m, 4H), 1.29 (m, 32H), 0.88 (t, 6H). APCI-MS analysis: Calculated C47H84N2O6 [M+H] = 773.6, Observed = 772.7. Example 50: Di((9Z,12Z)-octadeca-9,12-dien-1-yl) 2-(((3- (dimethylamino)propyl)(methyl)carbamoyl)-oxy)pentanedioate The titular compound was prepared in a manner analogous to Example 45. 1H NMR (300 MHz, CDCl3) δ 5.43-5.27 (m, 8H), 5.01-4.94 (m, 1H), 4.12 (dt, 2H), 4.05 (t, 2H), 3.38-3.27 (m, 2H), 2.93 (d, 3H), 2.76 (t, 4H), 2.47-2.41 (m, 2H), 2.32-2.22 (m, 2H), 2.21 (s, 6H), 2.20-2.12 (m, 2H), 2.07-2.01 (m, 8H), 1.75 (quint, 2H), 1.66-1.55 (m, 4H), 1.29 (m, 32H), 0.88 (t, 6H). APCI-MS analysis: Calculated C48H86N2O6 [M+H] = 787.6, Observed = 786.7. Example 51: Dioctadecyl ((2-(dimethylamino)ethoxy)carbonyl)-L-glutamate Step 1: Synthesis of Dioctadecyl (tert-butoxycarbonyl)-L-glutamate (9) A solution of (tert-butoxycarbonyl)-L-glutamic acid (2.0 g, 8 mmol), 1-octadecanol (5.41 g, 20 mmol), EDCI (4.6 g, 24 mmol) and 4-dimethylaminopyridine (1.0 g, 8 mmol) in 80 mL dichloromethane was stirred at room temperature overnight. After concentration, the residue was partition between hexane and acetonitrile, and the hexane layer was washed by acetonitrile. After concentration, dioctadecyl (tert-butoxycarbonyl)-L-glutamate was obtained as white wax (4.4 g, 73%). Step 2: Synthesis of Dioctadecyl L-glutamate trifluoroacetate (10) To a solution of dioctadecyl (tert-butoxycarbonyl)-L-glutamate 9 (4.0 g, 5.3 mmol) in 10 mL dichloromethane, was added 9 mL trifluoroacetic acid slowly, and the reaction mixture was stirred at room temperature for 3 hours. MS showed complete reaction. After concentration, the residue was coevaporated with heptane three times, and the crude was used for the next step without further purification. Step 3: Synthesis of Dioctadecyl ((2-(dimethylamino)ethoxy)carbonyl)-L-glutamate (Example 51) To a solution of dioctadecyl L-glutamate trifluoroacetate 10 (500 mg, 0.65 mmol) and pyridine (0.21 mL, 2.6 mmol) in 10 mL dichloromethane, was added p-nitrophenyl chloroformate (393 mg, 2 mmol), and the resulting mixture was stirred for 2 hours. After TLC showed the complete reaction, 2-dimethylaminoethanol (231 mg, 2.6 mmol) was added, and the reaction mixture was stirred at room temperature overnight. MS showed the formation of desired product. The reaction mixture was concentrated, and the residue was partitioned between hexane and acetonitrile. The hexane layer was concentrated, and the crude was purified by column chromatography eluted with dichloromethane/acetone to afford dioctadecyl ((2-(dimethylamino)ethoxy)carbonyl)-L-glutamate as pale yellow wax (50 mg, 10%). 1H NMR (300 MHz, CDCl3) δ 5.42 (d, 1H), 4.42-4.32 (m, 1H), 4.15 (t, 2H), 4.12 (t, 2H), 4.07 (t, 2H), 2.54 (t, 2H), 2.38 (dd, 2H), 2.27 (s, 6H), 2.26-2.13 (m, 1H), 2.05-1.88 (m, 1H), 1.68-1.53 (m, 4H), 1.24 (m, 60H), 0.87 (t, 6H). APCI-MS analysis: Calculated C46H90N2O6 [M+H] = 767.7, Observed = 767.6. Example 52: Dioctadecyl ((3-(dimethylamino)propoxy)carbonyl)-L-glutamate
Figure imgf000225_0001
The titular compound was prepared in a manner analogous to Example 51. 1H NMR (300 MHz, CDCl3) δ 5.33 (d, 1H), 4.42-4.32 (m, 1H), 4.16-4.02 (m, 6H), 2.38 (t, 2H), 2.26 (s, 6H), 2.26-2.13 (m, 1H), 2.05-1.88 (m, 3H), 1.80 (quint, 2H), 1.68-1.55 (m, 4H), 1.24 (m, 60H), 0.87 (t, 6H). APCI-MS analysis: Calculated C47H92N2O6 [M+H] = 781.7, Observed = 781.6. Example 53: Dihexadecyl ((2-(dimethylamino)ethoxy)carbonyl)-L-glutamate The titular compound was prepared in a manner analogous to Example 51. 1H NMR (300 MHz, CDCl3) δ 5.42 (d, 1H), 4.42-4.32 (m, 1H), 4.15 (t, 2H), 4.12 (t, 2H), 4.07 (t, 2H), 2.54 (t, 2H), 2.38 (dd, 2H), 2.27 (s, 6H), 2.26-2.13 (m, 1H), 2.05-1.88 (m, 1H), 1.68-1.53 (m, 4H), 1.24 (m, 52H), 0.86 (t, 6H). APCI-MS analysis: Calculated C42H82N2O6 [M+H] = 711.6, Observed = 711.5. Example 54: Dihexadecyl ((3-(dimethylamino)propoxy)carbonyl)-L-glutamate
Figure imgf000226_0001
The titular compound was prepared in a manner analogous to Example 51. 1H NMR (300 MHz, CDCl3) δ 5.37 (m, 1H), 4.43-4.34 (m, 1H), 4.10 (t, 2H), 4.04 (t, 2H), 3.28-3.17 (m, 2H), 2.46-2.28 (m, 2H), 2.22 (s, 6H), 2.18-2.09 (m, 1H), 2.01-1.84 (m, 3H), 1.68-1.55 (m, 6H), 1.24 (m, 52H), 0.87 (t, 6H). APCI-MS analysis: Calculated C48H84N2O6 [M+H] = 725.6, Observed = 725.0. Example 55: Di((9Z,12Z)-octadeca-9,12-dien-1-yl) ((2-(dimethylamino)ethoxy)carbonyl)-L- glutamate The titular compound was prepared in a manner analogous to Example 51. 1H NMR (300 MHz, CDCl3) δ 5.45-5.27 (m, 9H), 4.42-4.32 (m, 1H), 4.15 (t, 2H), 4.12 (t, 2H), 4.04 (t, 2H), 2.76 (t, 4H), 2.54 (t, 2H), 2.38 (dd, 2H), 2.27 (s, 6H), 2.26-2.13 (m, 1H), 2.07-1.88 (m, 9H), 1.68-1.53 (m, 4H), 1.24 (m, 32H), 0.88 (t, 6H). APCI-MS analysis: Calculated C46H82N2O6 [M+H] = 759.6, Observed = 759.6. Example 56: Di((9Z,12Z)-octadeca-9,12-dien-1-yl) ((3-(dimethylamino)propoxy)carbonyl)- L-glutamate The titular compound was prepared in a manner analogous to Example 51. 1H NMR (300 MHz, CDCl3) δ 5.43-5.25 (m, 9H), 4.42-4.32 (m, 1H), 4.14-4.02 (m, 6H), 2.76 (t, 4H), 2.46-2.30 (m, 4H), 2.22 (s, 6H), 2.20-2.13 (m, 1H), 2.07-1.90 (m, 9H), 1.78 (quint, 2H), 1.68-1.53 (m, 4H), 1.24 (m, 32H), 0.88 (t, 6H). APCI-MS analysis: Calculated C47H84N2O6 [M+H] = 773.6, Observed = 773.6. Example 57: Bis(7-(nonanoyloxy)heptyl) ((2-(dimethylamino)ethoxy)carbonyl)-L-glutamate The titular compound was prepared in a manner analogous to Example 51. 1H NMR (300 MHz, CDCl3) δ 5.43 (d, 1H), 4.40-4.32 (m, 1H), 4.18-4.08 (m, 2H), 4.04 (t, 8H), 2.56 (t, 2H), 2.38 (dd, 2H), 2.28 (s, 6H), 2.28 (t, 4H), 2.25-2.13 (m, 1H), 2.02-1.88 (m, 1H), 1.68-1.50 (m, 12H), 1.38-1.18 (m, 32H), 0.86 (t, 6H). APCI-MS analysis: Calculated C42H78N2O10 [M+H] = 771.6, Observed = 771.5. Example 58: Bis(5-(octanoyloxy)pentyl) 2-((3-(dimethylamino)propanoyl)oxy)-succinate Step 1: Synthesis of Dimethyl 2-hydroxysuccinate (11) To a solution of racemic malic acid (10 g, 74.6 mmol) in 100 mL methanol, was added a solution of hydrogen chloride (4 M in dioxane, 6 mL, 24 mmol), and the mixture was stirred at room temperature for 17 h. The reaction was carefully basified by adding saturated sodium bicarbonate solution, and the solution was extracted with dichloromethane (3 x 100 mL). The combined organic extracts were washed with brine, dried over sodium sulfate, and concentrated to give dimethyl malate as colorless oil (10.0 g, 83%). The crude was used for the next step without further purification. Step 2: Synthesis of 5-Hydroxypentyl octanoate (12) A mixture of 1,5-pentanediol (15.7 mL, 0.15 mole), octanoic acid (7.92 mL, 50 mmol), EDCI-HCl (9.58 g, 50 mmol) and DMAP (1.52 g, 12.5 mmol) in 100 mL dichloromethane was stirred at room temperature for 17 h. The dichloromethane was removed, the residue was diluted with 200 mL ethyl acetate, and the solution was washed with aqueous saturated ammonium chloride. After dried over sodium sulfate and concentrated, the crude was purified by column chromatography using 15-10% ethyl acetate in hexane to yield the desired product as colorless oil (10.79 g, 85%). Step 3: Synthesis of Bis(5-(octanoyloxy)pentyl) 2-hydroxysuccinate (13) A mixture of dimethyl malate 11(2.43 g, 15 mmol), 5-hydroxypentyl octanoate 12 (6.94 g, 30 mmol) and p-toluenesulfonic acid monohydrate (200 mg) in 200 mL toluene was heated to reflux vigorously with Dean-stark apparatus. After 150 mL toluene was distilled, more toluene was added. This process was repeated one more time. The reaction mixture was cooled to room temperature and washed with saturated sodium bicarbonate solution, and the aqueous layer was extracted with EtOAc. The combined organic layers were dried over sodium sulfate and concentrated to give the crude which was purified by column chromatography eluted with 0-10 % ethyl acetate in hexane to give the desired product as colorless oil (1.0 g, 12%). Step 4: Synthesis of Bis(5-(octanoyloxy)pentyl) 2-((3-(dimethylamino)propanoyl)oxy)- succinate (Example 58) A mixture of bis(5-(octanoyloxy)pentyl) 2-hydroxysuccinate 13 (265 mg, 0.47 mmol), 3-(dimethylamino)propanoic acid hydrochloride (88 mg, 0.57 mmol), EDC-HCl (109 mg, 0.57 mmol) and DMAP (70 mg, 0.57 mmol) in 10 mL dichloromethane was stirred at room temperature for 17 h. The volatiles were removed under vacuum, and the residue was dissolved in EtOAc. The solution was washed with water, and the organic layer was dried over sodium sulfate. After concentration, the crude was purified by column chromatography using 0-10% methanol in dichloromethane to yield the desired product as colorless oil (170 mg, 55%). 1H NMR (300 MHz, CDCl3) δ 5.47 (t, 1H), 4.15 (t, 2H), 4.10 (t, 2H), 4.05 (m, 4H), 2.87 (d, 2H), 2.66-2.52 (m, 4H), 2.28 (t, 4H), 2.22 (s, 6H), 1.72-1.55 (m, 12H), 1.46-1.36 (m, 4H), 1.27 (m, 16H), 0.87 (t, 6H). APCI-MS analysis: Calculated C35H63NO10 [M+H] = 658.4, Observed = 658.4. Example 59: Bis(5-((2-hexyldecanoyl)oxy)pentyl) 2-((3-(dimethylamino)propanoyl)- oxy)succinate Step 1: Synthesis of 5-Hydroxypentyl 2-hexyldecanoate (14) A mixture of 1,5-pentanediol (12.6 mL, 0.12 mole), 2-hexyldecanoic acid (11.7 mL, 40 mmol), EDCI-HCl (7.66 g, 40 mmol) and DMAP (0.97 g, 8 mmol) in 80 mL dichloromethane was stirred at room temperature for 17 h. The solvent was removed, the residue was diluted with 200 mL ethyl acetate, and the solution was washed with aqueous saturated ammonium chloride. After dried over sodium sulfate and concentrated, the crude was purified by column chromatography using 15-10% ethyl acetate in hexane to yield the desired product as colorless oil (10.0 g, 75%). Step 2: Synthesis of Bis(5-((2-hexyldecanoyl)oxy)pentyl) 2-hydroxysuccinate (15) A mixture of dimethyl malate 11 (2.11 g, 13 mmol), 5-hydroxypentyl 2- hexyldecanoate 14 (8.89 g, 26 mmol) and p-toluenesulfonic acid monohydrate (200 mg) in 200 mL toluene was heated to reflux vigorously with Dean-stark apparatus. After 150 mL toluene was distilled, more toluene was added. This process was repeated one more time. The reaction mixture was cooled to room temperature and washed with saturated sodium bicarbonate solution, and the aqueous layer was extracted with EtOAc. The combined organic layers were dried over sodium sulfate and concentrated to give the crude which was purified by column chromatography using 0-10 % ethyl acetate in hexane as eluent to give the desired product as colorless oil (5.0 g, 50%). Step 3: Synthesis of Bis(5-((2-hexyldecanoyl)oxy)pentyl) 2-((3-(dimethylamino)propanoyl)- oxy)succinate (Example 59) A mixture of bis(5-((2-hexyldecanoyl)oxy)pentyl) 2-hydroxysuccinate 15 (265 mg, 0.34 mmol), 3-(dimethylamino)propanoic acid hydrochloride (88 mg, 0.57 mmol), EDC-HCl (109 mg, 0.57 mmol), DMAP (70 mg, 0.57 mmol) in 10 mL dichloromethane was stirred at room temperature for 17 h. After concentration, the residue was dissolved in EtOAc and washed with water. The organic layer was dried over sodium sulfate and concentrated, and the crude was purified by column chromatography using 0-10% methanol in dichloromethane to yield the desired product as colorless oil (240 mg, 71%). 1H NMR (300 MHz, CDCl3) δ 5.47 (t, 1H), 4.15 (t, 2H), 4.11 (t, 2H), 4.06 (m, 4H), 2.87 (d, 2H), 2.73-2.54 (m, 4H), 2.35-2.27 (m, 8H), 1.72-1.50 (m, 14H), 1.42 (m, 6H), 1.24 (m, 40H), 0.86 (t, 12H). APCI-MS analysis: Calculated C51H95NO10 [M+H] = 882.7, Observed = 882.6. Example 60: Bis(5-((2-hexyldecanoyl)oxy)pentyl) 2-((4- (dimethylamino)butanoyl)oxy)succinate The titular compound was prepared in a manner analogous to Example 59. 1H NMR (300 MHz, CDCl3) δ 5.45 (t, 1H), 4.15 (t, 2H), 4.11 (t, 2H), 4.06 (m, 4H), 2.86 (d, 2H), 2.42 (m, 2H), 2.35-2.25 (m, 4H), 2.20 (s, 6H), 1.79 (m, 2H), 1.69-1.50 (m, 14H), 1.35- 1.48 (m, 6H), 1.24 (m, 40H), 0.86 (t, 12H). APCI-MS analysis: Calculated C52H97NO10 [M+H] = 896.7, Observed = 896.6. Example 61: Bis(6-oxo-6-(pentadecan-7-yloxy)hexyl) 2-((3-(dimethylamino)- propanoyl)oxy)succinate Step 1: Synthesis of Pentadecan-7-ol (16) At 0 °C, to a solution of octylmagnesium bromide (2 M in ether, 52.5 mL, 0.105 mole) in 100 mL ether, was slowly added a solution of heptaldehyde (12.6 mL, 90 mmol) in 40 mL ether, and the mixture was warmed up to room temperature and stirred overnight. The reaction was quenched by saturated ammonium chloride and extracted with ether, the combined organic layers were dried over sodium sulfate. After concentration, the crude was recrystallized in acetonitrile to afford the desired product as white wax (10.0 g, 50%). Step 2: Synthesis of Bis(6-(benzyloxy)hexyl) 2-hydroxysuccinate (17) A mixture of dimethyl malate 11 (2.1 g, 13 mmol), 6-benzyoxyhexanol (5.4 g, 26 mmol) and p-toluenesulfonic acid monohydrate (300 mg) in 200 mL toluene was refluxed vigorously with Dean-stark apparatus. After 150 mL solvent was distilled off, more toluene was added and refluxed. This process was repeated one more time. After cooled to room temperature, the mixture was diluted with saturated sodium bicarbonate, and the resulting mixture was extracted with ethyl acetate. The combined organic layers were dried over sodium sulfate and concentrated to give oil which was purified by column chromatography using 0-20% ethyl acetate in hexane to give the desired product as colorless oil (2.7 g, 41%). Step 3: Synthesis of Bis(6-(benzyloxy)hexyl) 2-((tert-butyldiphenylsilyl)oxy)succinate (18) A mixture of bis(6-(benzyloxy)hexyl) 2-hydroxysuccinate 17 (1.4 g, 2.72 mmol), tert- butyldiphenylsilyl chloride (825 mg, 3 mmol) and imidazole (340 mg, 5 mmol) in 12 mL DMF was stirred room temperature for 17 h. The reaction mixture was partitioned with ethyl acetate and saturated ammonium chloride solution, and the combined organic layer was dried over sodium sulfate. After concentration, the crude was purified by column chromatography using 5-20% ethyl acetate in hexane to give the desired product (1.65 g, 83%). Step 4: Synthesis of Bis(6-hydroxyhexyl) 2-((tert-butyldiphenylsilyl)oxy)succinate (19) A mixture of bis(6-(benzyloxy)hexyl) 2-((tert-butyldiphenylsilyl)oxy)succinate 18 (1.6 g, 2,12 mmol) and 5% palladium on carbon (500 mg) in 100 mL ethyl acetate was subjected to hydrogenolysis for 15 h. After filtration, the filtrate was concentrated, and the crude was purified by column chromatography using 0-100% ethyl acetate in hexane to obtain the desired product (800 mg, 66%) along with partially deprotected intermediate (400 mg, 30%). Step 5: Synthesis of 6,6'-((2-((tert-Butyldiphenylsilyl)oxy)succinyl)bis(oxy))dihexanoic acid (20) To a solution of bis(6-hydroxyhexyl) 2-((tert-butyldiphenylsilyl)oxy)succinate 19 (700 mg, 1.22 mmol) in 15 mL acetone, was added Jones reagent until the orange color persisted, and the resulting mixture was stirred for 30 min. The excess Jones reagent was consumed by adding few drops of 2-propanol, then the blue solution was diluted with water (100 mL) and extracted with ethyl acetate (3 x 70 mL). The combined organic layers were washed with brine, dried and concentrated to give the desired product (700 mg, 95%), which was used for the next step without further purification. Step 6: Synthesis of Bis(6-oxo-6-(pentadecan-7-yloxy)hexyl) 2-((tert-butyldiphenylsilyl)oxy)- succinate (21) A mixture of 6,6'-((2-((tert-butyldiphenylsilyl)oxy)succinyl)bis(oxy))dihexanoic acid 20 (800 mg, 1.33 mmol), 7-pentadecanol (914 mg, 4 mmol), EDCI-HCl (768 mg, 4 mmol) and DMAP (488 mg, 4 mmol) in 10 mL dichloromethane was stirred at room temperature for 16 h. After concentration, the crude was partitioned between EtOAc and saturated ammonium chloride solution, and the combined organic layer was dried over sodium sulfate. The solvent was removed under vacuum, and the crude was purified by column chromatography using 2- 10% ethyl acetate in hexane to give the desired product (900 mg, 66%). Step 7: Synthesis of Bis(6-oxo-6-(pentadecan-7-yloxy)hexyl) 2-hydroxysuccinate (22) To a solution of bis(6-oxo-6-(pentadecan-7-yloxy)hexyl) 2-((tert- butyldiphenylsilyl)oxy)-succinate 21 (500 mg, 0.49 mmol) in 10 mL THF, was added pyridine (2 mL) followed by HF-pyridine complex (70%, 1.5 mL), and the mixture was stirred at room temperature for 4 h. The reaction was quenched by adding saturated sodium bicarbonate to pH 7, and then extracted with ethyl acetate (3 x 60 mL). The combined organic layers were dried and concentrated, and the crude was purified by column chromatography using 10-30% ethyl acetate in hexane to yield the desired product (280 mg, 73%). Step 8: Synthesis of Bis(6-oxo-6-(pentadecan-7-yloxy)hexyl) 2-((3-(dimethylamino)- propanoyl)oxy)succinate (Example 61) A mixture of bis(6-oxo-6-(pentadecan-7-yloxy)hexyl) 2-hydroxysuccinate 22 (125 mg, 0.16 mmol), 3-(dimethylamino)propanoic acid hydrochloride (76 mg, 0.49 mmol), EDC- HCl (95 mg, 0.49 mmol) and DMAP (60 mg, 0.49 mmol) in 10 mL dichloromethane was stirred at room temperature for 17 h. The volatiles were removed under vacuum, and the residue was dissolved in EtOAc. The solution was washed with water, and the organic layer was dried over sodium sulfate. After concentration, the crude was purified by column chromatography using 0-10% methanol in dichloromethane to yield the desired product as colorless oil (140 mg, 98%). 1H NMR (300 MHz, CDCl3) δ 5.46 (t, 1H), 4.84 (quint., 2H), 4.13 (t, 2H), 4.09 (t, 2H), 2.86 (d, 2H), 2.68-2.52 (m, 4H), 2.27 (t, 4H), 2.22 (s, 6H), 1.68-1.57 (m, 8H), 1.53-1.30 (m, 12H), 1.24 (m, 40H), 0.86 (t, 12H). APCI-MS analysis: Calculated C51H95NO10 [M+H] = 882.7, Observed = 882.6. Example 62: Bis(6-oxo-6-(pentadecan-7-yloxy)hexyl) 2-((3-(dimethylamino)- butanoyl)oxy)succinate The titular compound was prepared in a manner analogous to Example 61. 1H NMR (300 MHz, CDCl3) δ 5.44 (t, 1H), 4.85 (quint., 2H), 4.13 (t, 2H), 4.09 (t, 2H), 2.85 (d, 2H), 2.42 (m, 2H), 2.28 (t, 6H), 2.20 (s, 6H), 1.79 (quint., 2H), 1.68-1.57 (m, 8H), 1.53- 1.30 (m, 12H), 1.24 (m, 40H), 0.86 (t, 12H). APCI-MS analysis: Calculated C52H97NO10 [M+H] = 896.7, Observed = 896.6. Example 63: Bis(6-((2-butylnonyl)oxy)-6-oxohexyl) 2-hydroxysuccinate Step 1: Synthesis of 2-Butylnonanoic acid (23) To a solution of nonanoic acid (17.7 mL, 0.114 mole) in 200 mL THF at 0 °C, was added NaH (60%, 5.0 g, 0.125 mole) portionwise, followed by adding a solution of LDA (2 M in THF/heptane/ethylbenzene, 62.5 mL, 0.125 mole) dropwise, and the mixture was stirred at room temperature for 30 min. After addition of n-butyl iodide (15.5 mL, 0.137 mole), the resulting mixture was heated to 45 °C overnight. The suspension was cooled to room temperature and quenched with 1 N HCl to pH 2, the organic layer was separated and dried over sodium sulfate. After concentration, the crude was purified by flash column chromatography eluted with 0-20% EtOAc in hexane to give impure product (20 g), and the impurity is nonanoic acid. The impure product was used for the next step without further purification. Step 2: Synthesis of 2-Butylnonan-1-ol (24) To a solution of impure 2-butylnonanoic acid 23 (1.0 g, 4.7 mmol) in 30 mL ether, was added a solution of LiAlH4 (2 M in THF, 23.5 mL, 47 mmol) dropwise, and the resulting mixture was stirred at room temperature overnight. After quenched by EtOAc, the solution was washed with water, and the organic layer was dried over sodium sulfate. The solvent was removed under vacuum, and the crude was purified by flash column chromatography eluted with 0-40% EtOAc in hexane to give the desired product as colorless oil (600 mg, 64%). Step 3: Synthesis of Bis(6-((2-butylnonyl)oxy)-6-oxohexyl) 2-((tert- butyldiphenylsilyl)oxy)succinate (25) A mixture of 6,6'-((2-((tert-butyldiphenylsilyl)oxy)succinyl)bis(oxy))dihexanoic acid 20 (300 mg, 0.5 mmol), 2-butylnonan-1-ol 24 (300 mg, 1.5 mmol), EDCI-HCl (143 mg, 0.75 mmol) and DMAP (91 mg, 0.75 mmol) in 10 mL dichloromethane was stirred at room temperature for 16 h. After concentration, the crude was partitioned between EtOAc and saturated ammonium chloride, and the combined organic layer was dried over sodium sulfate. The solvent was removed under vacuum, and the crude was purified by column chromatography using 2-10% ethyl acetate in hexane to give the impure product (200 mg), which was used for the next step without further purification. Step 4: Synthesis of Bis(6-((2-butylnonyl)oxy)-6-oxohexyl) 2-hydroxysuccinate (26) To a solution of bis(6-((2-butylnonyl)oxy)-6-oxohexyl) 2-((tert- butyldiphenylsilyl)oxy)succinate 25 (200 mg, impure) in 10 mL THF, was added pyridine (2 mL) followed by HF-pyridine complex (70%, 1.5 mL), and the mixture was stirred at room temperature for 4 h. The reaction was quenched by adding saturated sodium bicarbonate to pH 7, and then extracted with ethyl acetate (3 x 60 mL). The combined organic layers were dried and concentrated, and the crude was purified by column chromatography using 10-30% ethyl acetate in hexane to yield the desired product as oil (73 mg, 20% in two steps). Step 5: Synthesis of Bis(6-((2-butylnonyl)oxy)-6-oxohexyl) 2-hydroxysuccinate (Example 63) A mixture of bis(6-((2-butylnonyl)oxy)-6-oxohexyl) 2-hydroxysuccinate 26 (73 mg, 0.10 mmol), 3-(dimethylamino)propanoic acid hydrochloride (76 mg, 0.49 mmol), EDC-HCl (95 mg, 0.49 mmol) and DMAP (60 mg, 0.49 mmol) in 10 mL dichloromethane was stirred at room temperature for 17 h. The volatiles were removed under vacuum, and the residue was partitioned in EtOAc and water, and the organic layer was dried over sodium sulfate. After concentration, the crude was purified by column chromatography using 0-10% methanol in dichloromethane to yield the desired product as colorless oil (63 mg, 76%). 1H NMR (300 MHz, CDCl3) δ 5.46 (t, 1H), 4.84 (quint., 2H), 4.13 (t, 2H), 4.09 (t, 2H), 2.86 (d, 2H), 2.68-2.52 (m, 4H), 2.27 (t, 4H), 2.22 (s, 6H), 1.68-1.57 (m, 8H), 1.53-1.30 (m, 12H), 1.24 (m, 40H), 0.86 (t, 12H). APCI-MS analysis: Calculated C47H87NO10 [M+H] = 826.6, Observed = 826.6. Example 64: 4-(2-(Dimethylamino)ethyl) 1-(4-((2-hexyldecanoyl)oxy)butyl) (2S)-2-((6-((2- hexyldecanoyl)oxy)hexanoyl)oxy)succinate
Figure imgf000237_0001
Step 1: Synthesis of Benzyl (S)-2-(2,2-dimethyl-5-oxo-1,3-dioxolan-4-yl)acetate (27)
Figure imgf000237_0002
Compound 27 was prepared according the procedure described in CN111039919A. To a solution of (S)-2-(2,2-dimethyl-5-oxo-1,3-dioxolan-4-yl)acetic acid (12 g, 69 mmol) and triethylamine (28.9 mL, 0.207 mole) in 200 mL acetone, was added benzyl bromide (24.6 mL, 0.207 mole) slowly, and then the resulting mixture was heated to reflux for 6 h. After cooled to room temperature, the suspension was filtered and washed with acetone, and the filtrate was concentrated. The residue was dissolved in EtOAc and washed with water, the organic layer was dried over sodium sulfate and concentrated. The residue was triturated with 25 mL ether to get the desired product as white solid (18.5 g, quant.). Step 2: Synthesis of (S)-4-(Benzyloxy)-2-hydroxy-4-oxobutanoic acid (28) Compound 28 was prepared according the procedure described in CN111039919A. A solution of benzyl (S)-2-(2,2-dimethyl-5-oxo-1,3-dioxolan-4-yl)acetate 27 (6.5 g, 24.6 mmol) in 30 mL THF, 30 mL acetic acid and 30 mL water was heated to 40 °C overnight. The solvent was evaporated, and the residue was coevaporated with toluene three times to give the desired product as pale yellow oil (6.0 g, quant.). Step 3: Synthesis of 4-Benzyl 1-(4-((2-hexyldecanoyl)oxy)butyl) (2S)-2-hydroxysuccinate (29) A solution of 4-hydroxybutyl 2-hexyldecanoate (800 mg, 2.35 mmol), (S)-4- (benzyloxy)-2-hydroxy-4-oxobutanoic acid 28 (800 mg, 3.57 mmol) and p-toluenesulfonic acid monohydrate (80 mg) in 60 mL toluene was heated to reflux overnight. The solvent was evaporated, and the residue was purified by column chromatography eluting with 0-20% EtOAc in hexane to give the desired product as pale yellow oil (650 mg, 47%). Step 4: Synthesis of (3S)-4-(4-((2-Hexyldecanoyl)oxy)butoxy)-3-hydroxy-4-oxobutanoic acid (30) A mixture of 4-benzyl 1-(4-((2-hexyldecanoyl)oxy)butyl) (2S)-2-hydroxysuccinate 29 (155 mg, 0.29 mmol) and palladium on carbon (5%, 200 mg) in 15 mL hexane was subjected to hydrogenolysis for 30 h. After filtration and concentration, the crude was purified by column chromatography eluted with 10-100% ethyl acetate in hexane to give the desired acid as colorless oil (116 mg, 90%). Step 5: Synthesis of 4-(2-(Dimethylamino)ethyl) 1-(4-((2-hexyldecanoyl)oxy)butyl) (2S)-2- hydroxysuccinate (31) A solution of (3S)-4-(4-((2-hexyldecanoyl)oxy)butoxy)-3-hydroxy-4-oxobutanoic acid 30 (130 mg, 0.29 mmol), 2-(dimethylamino)ethan-1-ol (260 mg, 2.9 mmol), EDCI-HCl (192 mg, 1 mmol) and DMAP (122 mg, 1 mmol) in 7 mL dichloromethane was stirred at room temperature for 18 h. The reaction mixture was diluted with 100 mL dichloromethane, washed with saturated ammonium chloride solution (50 mL x 3), and followed by brine. The organic layer was dried over sodium sulfate and concentrated to give the crude product which was carried to the next step without further purification. Step 6: Synthesis of 4-(2-(Dimethylamino)ethyl) 1-(4-((2-hexyldecanoyl)oxy)butyl) (2S)-2-((6- ((2-hexyldecanoyl)oxy)hexanoyl)oxy)succinate (Example 64) A solution of 4-(2-(dimethylamino)ethyl) 1-(4-((2-hexyldecanoyl)oxy)butyl) (2S)-2- hydroxysuccinate 31 (150 mg, 0.29 mmol), 6-((2-hexyldecanoyl)oxy)hexanoic acid (507 mg, 1.32 mmol), EDCI-HCl (253 mg, 1.32 mmol) and DMAP (122 mg, 1 mmol) in 8 mL dichloromethane was stirred at room temperature for 18 h. After the solvent was removed under vacuum, the residue was dissolved in ethyl acetate (80 mL), which was washed with saturated aqueous ammonium chloride (40 mL x 2), followed by brine. The organic layer was dried over sodium sulfate and concentrated, and the crude was purified by column chromatography eluting with 20-100% ethyl acetate in hexane to give the desired product as colorless oil (154 mg, 61%). 1H NMR (300 MHz, CDCl3) δ 5.46 (t, 1H), 4.23-4.16 (m, 4H), 4.09-4.01 (m, 4H), 2.90 (d, 2H), 2.55 (t, 2H), 2.38 (dt, 2H), 2.34-2.27 (m, 2H), 2.26 (s, 6H), 1.74-1.50 (m, 12H), 1.47- 1.36 (m, 6H), 1.24 (m, 40H), 0.86 (t, 12H). APCI-MS analysis: Calculated C50H93NO10 [M+H] = 868.6, Observed = 868.0. Example 65: 4-(2-(Dimethylamino)propyl) 1-(4-((2-hexyldecanoyl)oxy)butyl) (2S)-2-((6-((2- hexyldecanoyl)oxy)hexanoyl)oxy)succinate The titular compound was prepared in a manner analogous to Example 64. 1H NMR (300 MHz, CDCl3) δ 5.44 (t, 1H), 4.21-4.14 (m, 4H), 4.09-3.99 (m, 4H), 2.86 (d, 2H), 2.42-2.24 (m, 6H), 2.20 (s, 6H), 1.83-1.36 (m, 20H), 1.23 (m, 40H), 0.86 (t, 12H). APCI-MS analysis: Calculated C51H95NO10 [M+H] = 882.7, Observed = 882.0. Example 66: 4-(2-(Dimethylamino)ethyl) 1-(5-((2-hexyldecanoyl)oxy)-pentyl) 2-((7-((2- hexyldecanoyl)oxy)heptanoyl)oxy)succinate The titular compound was prepared in a manner analogous to Example 64. 1H NMR (300 MHz, CDCl3) δ 5.45 (t, 1H), 4.20 (t, 2H), 4.15 (t, 2H), 4.05 (m, 4H), 2.90 (d, 2H), 2.55 (t, 2H), 2.40-2.29 (m, 4H), 2.27 (s, 6H), 1.73-1.51 (m, 10H), 1.48-1.33 (m, 4H), 1.24 (m, 48H), 0.87 (t, 12H). APCI-MS analysis: Calculated C52H97NO10 [M+H] = 896.7, Observed = 896.9. Example 67: 4-(3-(Dimethylamino)propyl) 1-(5-((2-hexyldecanoyl)oxy)pentyl) (2S)-2-((7- ((2-hexyldecanoyl)oxy)heptanoyl)oxy)succinate The titular compound was prepared in a manner analogous to Example 64. 1H NMR (300 MHz, CDCl3) δ 5.45 (t, 1H), 4.20-4.13 (m, 4H), 4.08-4.02 (m, 4H), 2.86 (d, 2H), 2.42-2.28 (m, 6H), 2.24 (s, 6H), 1.82 (quint, 2H), 1.73-1.51 (m, 10H), 1.48-1.30 (m, 4H), 1.24 (m, 48H), 0.87 (t, 12H). APCI-MS analysis: Calculated C53H99NO10 [M+H] = 910.7, Observed = 910.8. Example 68: 4-(2-(Dimethylamino)ethyl) 1-(5-((2-hexyldecanoyl)oxy)pentyl) (2S)-2-((((5- ((2-hexyldecanoyl)oxy)pentyl)oxy)carbonyl)oxy)succinate Step 1: Synthesis of 5-Hydroxypentyl 2-hexyldecanoate (32) A mixture of 1,5-pentanediol (10.4 g, 0.10 mole), 2-hexyldecanoic acid (7.92 mL, 50 mmol), EDCI-HCl (9.58 g, 50 mmol) and DMAP (1.52 g, 12.5 mmol) in 100 mL dichloromethane was stirred at room temperature for 16 h. The mixture was diluted with 100 mL water, the organic layer was separated and washed by brine. After dried over sodium sulfate, the solvent was removed under vacuum, and the residue was purified by column chromatography using 0-10% ethyl acetate in hexane to yield the desired product as colorless oil (13.9 g, 81%). Step 2: Synthesis of 4-(2-(Dimethylamino)ethyl) 1-(5-((2-hexyldecanoyl)oxy)pentyl) (2S)-2- ((((5-((2-hexyldecanoyl)oxy)pentyl)oxy)carbonyl)oxy)succinate (Example 68) To a mixture of 5-hydroxypentyl 2-hexyldecanoate 32 (684 mg, 2.0 mmol) and pyridine (1.5 mL) in 6 mL dichloromethane, was added p-nitrophenyl chloroformate (404 mg, 2.0 mmol), and the resulting mixture was stirred at room temperature for 12 h. Then a solution of 4-(2-(dimethylamino)ethyl) 1-(5-((2-hexyldecanoyl)oxy)pentyl) (2S)-2- hydroxysuccinate (150 mg, 0.28 mmol) in 4 mL dichloromethane was added and followed by triethylamine (150 µL, 1 mmol), and the mixture was stirred at room temperature for 18 h. After evaporation, the residue was dissolved in hexane, and the solution was washed with 2% Na2CO3 solution several times until no p-nitrophenol present. After concentration, the crude was purified by column chromatography using 20% -100% ethyl acetate in hexane to give the desired product as colorless oil (60 mg, 24%). Example 69: 4-(3-(Dimethylamino)propyl) 1-(5-((2-hexyldecanoyl)oxy)pentyl) (2S)-2-((((5- ((2-hexyldecanoyl)oxy)pentyl)oxy)carbonyl)oxy)succinate The titular compound was prepared in a manner analogous to Example 68. 1H NMR (300 MHz, CDCl3) δ 5.36 (t, 1H), 4.23-4.12 (m, 6H), 4.05 (t, 4H), 2.89 (d, 2H), 2.54 (t, 2H), 2.37-2.26 (m, 4H), 2.21 (s, 6H), 1.79 (quint, 2H), 1.75-1.50 (m, 6H), 1.48-1.33 (m, 4H), 1.24 (m, 48H), 0.86 (t, 12H). APCI-MS analysis: Calculated C52H97NO11 [M+H] = 912.3, Observed = 912.0. Example 70: 1-(2-(Dimethylamino)ethyl) 4-(5-((2-hexyldecanoyl)oxy)pentyl) (2S)-2-((7-((2- hexyldecanoyl)oxy)heptanoyl)oxy)succinate Step 1: 7-Hydroxyheptyl 2-hexyldecanoate (33) A mixture of 1,7-heptanediol (4.6 g, 35 mmol), 2-hexyldecanoic acid (4.5 g, 17.5 mmol), EDCI-HCl (4.0 g, 21 mmol) and DMAP (427 mg, 3.5 mmol) in 50 mL dichloromethane was stirred at room temperature for 16 h. The mixture was diluted with 50 mL water, the organic layer was separated and washed by brine. After dried over sodium sulfate, the solvent was removed under vacuum, and the residue was purified by column chromatography using 0-10% ethyl acetate in hexane to yield the desired product as colorless oil (5.0 g, 77%). Step 2: 7-((2-Hexyldecanoyl)oxy)heptanoic acid (34) To a solution of 7-hydroxyheptyl 2-hexyldecanoate 33 (4.9 g, 13.2 mmol) in 50 mL acetone, was added Jones reagent until the orange color persisted and the resulting mixture was stirred for 1 h. The excess Jones reagent was consumed by adding few drops of 2- propanol. Then the blue solution was diluted with water (100 mL) and extracted with ethyl acetate (3 x 100 mL). The combined organic layers were washed with brine, dried and concentrated to give the desired product (5.0 g, 98%), which was used for the next step without further purification. Step 3: Synthesis of 5-(2-((S)-2,2-Dimethyl-5-oxo-1,3-dioxolan-4-yl)acetoxy)pentyl 2- hexyldecanoate (35) A mixture of (S)-2-(2,2-dimethyl-5-oxo-1,3-dioxolan-4-yl)acetic acid (174 mg, 1 mmol), 5-hydroxypentyl 2-hexyldecanoate 32 (342 mg, 1 mmol), triethylamine (0.5 mL), EDCI-HCl (192 mg, 1 mmol) and DMAP (122 mg, 1 mmol) in 8 mL dichloromethane was stirred at room temperature for 17 h. The solvent was removed under vacuum, the residue was dissolved in ethyl acetate (80 mL), and then the solution was washed with saturated ammonium chloride solution (25 mL x 2), followed by brine (25 mL x 2). After dried over sodium sulfate and concentration, the crude was purified by column chromatography using 5- 30% ethyl acetate in hexane to give the desired ester as pale yellow oil (433 mg, 87%). Step 4: Synthesis of (2S)-4-((5-((2-Hexyldecanoyl)oxy)pentyl)oxy)-2-hydroxy-4-oxobutanoic acid (36) A solution of 5-(2-((S)-2,2-dimethyl-5-oxo-1,3-dioxolan-4-yl)acetoxy)pentyl 2- hexyldecanoate 35 (433 mg, 0.87 mmol) in a mixture of acetic acid (5 mL), water (5 mL) and THF (5 mL) was heated at 40-50 °C for 18 h. The volatiles were removed, and the residue was coevaporated with toluene three times to get the crude product as brown oil (400 mg, quant.), which was carried over to the next step without further purification. Step 5: Synthesis of 1-(2-(Dimethylamino)ethyl) 4-(5-((2-hexyldecanoyl)oxy)pentyl) (2S)-2- hydroxysuccinate (37) A mixture of (2S)-4-((5-((2-hexyldecanoyl)oxy)pentyl)oxy)-2-hydroxy-4-oxobutanoic acid 36 (200 mg, 0.44 mmol), 2-(dimethylamino)ethan-1-ol (390 mg, 4.4 mmol), EDCI-HCl (192 mg, 1 mmol) and DMAP (122 mg, 1 mmol) in 7 mL dichloromethane was stirred at room temperature for 18 h. The reaction mixture was diluted with dichloromethane (100 mL) and washed with saturated ammonium chloride solution (50 mL x 3), followed by brine (50 mL x 1). The organic layer was dried over sodium sulfate and concentrated to get the desired product as brown oil (250 mg) which was used for the next step without further purification. Step 6: Synthesis of 1-(2-(Dimethylamino)ethyl) 4-(5-((2-hexyldecanoyl)oxy)pentyl) (2S)-2- ((7-((2-hexyldecanoyl)oxy)heptanoyl)oxy)succinate (Example 70) A mixture of 1-(2-(dimethylamino)ethyl) 4-(5-((2-hexyldecanoyl)oxy)pentyl) (2S)-2- hydroxysuccinate 37 (250 mg, 0.44 mmol), 7-((2-hexyldecanoyl)oxy)heptanoic acid 34 (507 mg, 1.32 mmol), EDCI-HCl (253 mg, 1.32 mmol) and DMAP (122 mg, 1 mmol) in 8 mL dichloromethane was stirred at room temperature for 18 h. After concentration, the residue was dissolved in ethyl acetate (80 mL), washed with saturated ammonium chloride solution (40 mL x 2), and followed by brine. The organic layer was dried over sodium sulfate and concentrated, and the crude was purified by column chromatography using 20-100% ethyl acetate in hexane to get the desired product as colorless oil (65 mg, 17%). 1H NMR (300 MHz, CDCl3) δ 5.45 (t, 1H), 4.24 (t, 2H), 4.09 (t, 2H), 4.05 (m, 4H), 2.86 (d, 2H), 2.54 (t, 2H), 2.40-2.26 (m, 4H), 2.24 (s, 6H), 1.70-1.48 (m, 10H), 1.46-1.30 (m, 4H), 1.23 (m, 48H), 0.85 (t, 12H). APCI-MS analysis: Calculated C52H97NO10 [M+H] = 896.3, Observed = 896.7. Example 71: 1-(3-(Dimethylamino)propyl) 4-(5-((2-hexyldecanoyl)oxy)pentyl) (2S)-2-((7- ((2-hexyldecanoyl)oxy)heptanoyl)oxy)succinate The titular compound was prepared in a manner analogous to Example 70. 1H NMR (300 MHz, CDCl3) δ 5.45 (t, 1H), 4.20 (t, 2H), 4.10 (t, 2H), 4.06 (m, 4H), 2.86 (d, 2H), 2.54 (t, 2H), 2.42-2.24 (m, 6H), 2.21 (s, 6H), 1.79 (quint, 2H), 1.70-1.48 (m, 8H), 1.46- 1.32 (m, 4H), 1.24 (m, 48H), 0.86 (t, 12H). APCI-MS analysis: Calculated C53H99NO10 [M+H] = 910.3, Observed = 910.6. Example 72: 1-(2-(Dimethylamino)ethyl) 4-(4-((2-hexyldecanoyl)oxy)butyl) (2S)-2-((6-((2- hexyldecanoyl)oxy)hexanoyl)oxy)succinate The titular compound was prepared in a manner analogous to Example 70. 1H NMR (300 MHz, CDCl3) δ 5.47 (t, 1H), 4.25 (t, 2H), 4.17-4.03 (m, 6H), 2.88 (d, 2H), 2.56 (t, 2H), 2.42-2.26 (m, 4H), 2.25 (s, 6H), 1.75-1.50 (m, 10H), 1.48-1.35 (m, 4H), 1.24 (m, 44H), 0.86 (t, 12H). APCI-MS analysis: Calculated C50H93NO10 [M+H] = 868.3, Observed = 868.0. Example 73: 1-(18-hexyl-2-methyl-6,9,16-trioxo-5,10,17-trioxa-2-azahexacosan-7-yl) 8- (pentadecan-7-yl) octanedioate The titular compound was prepared in a manner analogous to Example 70. 1H NMR (300 MHz, CDCl3) δ 5.46 (t, 1H), 4.85 (m, 2H), 4.25 (t, 2H), 4.09 (t, 2H), 2.86 (d, 2H), 2.56 (t, 2H), 2.42-2.26 (m, 6H), 2.24 (s, 6H), 1.76-1.55 (m, 10H), 1.55-1.43 (m, 4H), 1.24 (m, 48H), 0.86 (t, 12H). APCI-MS analysis: Calculated C52H97NO10 [M+H] = 896.3, Observed = 896.0. Example 74: 1-(2-(Dimethylamino)ethyl) 4-(5-((2-hexyldecanoyl)oxy) pentyl) 2-((6-((2- hexyldecanoyl)oxy)hexanoyl)oxy)succinate The titular compound was prepared in a manner analogous to Example 70. 1H NMR (300 MHz, CDCl3) δ 5.46 (t, 1H), 4.25 (t, 2H), 4.10 (t, 2H), 4.05 (m, 4H), 2.87 (d, 2H), 2.55 (t, 2H), 2.43-2.26 (m, 4H), 2.25 (s, 6H), 1.74-1.48 (m, 8H), 1.46-1.34 (m, 4H), 1.24 (m, 48H), 0.86 (t, 12H). APCI-MS analysis: Calculated C51H95NO10 [M+H] = 882.3, Observed = 882.6. Example 75: 1-(2-(Dimethylamino)ethyl) 4-(5-((2-hexyldecanoyl)oxy)pentyl) (2S)-2-((((5- ((2-hexyldecanoyl)oxy)pentyl)oxy)carbonyl)oxy)succinate To a mixture of 5-hydroxypentyl 2-hexyldecanoate 32 (513 mg, 1.5 mmol) and pyridine (1 mL) in 6 mL dichloromethane, was added p-nitrophenyl chloroformate (303 mg, 1.5 mmol), and the resulting mixture was stirred at room temperature for 12 h. Then a solution of 1-(2-(dimethylamino)ethyl) 4-(5-((2-hexyldecanoyl)oxy)pentyl) (2S)-2- hydroxysuccinate 37 (150 mg, 0.28 mmol) in 4 mL dichloromethane was added and followed by triethylamine (150 µL, 1 mmol), and the mixture was stirred at room temperature for 18 h. After evaporation, the residue was dissolved in hexane, and the solution was washed with 2% Na2CO3 solution several times until no p-nitrophenol was present. The solvent was evaporated, and the crude was purified by column chromatography using 0-100% ethyl acetate in hexane to give the desired product as colorless oil (84 mg, 35%). 1H NMR (300 MHz, CDCl3) δ 5.40-5.36 (m, 1H), 4.29-4.25 (m, 2H), 4.19-4.03 (m, 8H), 2.91-2.88 (m, 2H), 2.56 (t, 2H), 2.32-2.25 (m, 8H), 1.73-1.37 (m, 20H), 1.23 (m, 40H), 0.85 (t, 12H). APCI-MS analysis: Calculated C51H95NO11 [M+H] = 898.7, Observed = 898.6. Example 76: 1-(3-(Dimethylamino)propyl) 4-(5-((2-hexyldecanoyl)oxy)pentyl) (2S)-2-((((5- ((2-hexyldecanoyl)oxy)pentyl)oxy)carbonyl)oxy)succinate Step 1: Synthesis of 1-(3-(Dimethylamino)propyl) 4-(5-((2-hexyldecanoyl)oxy)pentyl) (2S)-2- hydroxysuccinate (38) A mixture of (2S)-4-((5-((2-hexyldecanoyl)oxy)pentyl)oxy)-2-hydroxy-4-oxobutanoic acid 36 (800 mg, 0.44 mmol), 3-(dimethylamino)propan-1-ol (453 mg, 4.4 mmol), EDCI-HCl (192 mg, 1 mmol) and DMAP (122 mg, 1 mmol) in 7 mL dichloromethane was stirred at room temperature for 18 h. The reaction mixture was diluted with dichloromethane (100 mL) and washed with saturated ammonium chloride solution (50 mL x 3), followed by brine (50 mL x 1). The organic layer was dried over sodium sulfate and concentrated to get the desired product as brown oil (260 mg) which was used for the next step without further purification. Step 2: Synthesis of 1-(3-(dimethylamino)propyl) 4-(5-((2-hexyldecanoyl)oxy)pentyl) (2S)-2- ((((5-((2-hexyldecanoyl)oxy)pentyl)oxy)carbonyl)oxy)succinate (Example 76) To a mixture of 5-hydroxypentyl 2-hexyldecanoate 32 (513 mg, 1.5 mmol) and pyridine (1 mL) in 6 mL dichloromethane, was added p-nitrophenyl chloroformate (303 mg, 1.5 mmol), and the resulting mixture was stirred at room temperature for 12 h. Then a solution of 1-(2-(dimethylamino)propyl) 4-(5-((2-hexyldecanoyl)oxy)pentyl) (2S)-2- hydroxysuccinate 38 (150 mg, 0.28 mmol) in 4 mL dichloromethane was added and followed by triethylamine (50 µL, 0.33 mmol), and the mixture was stirred at room temperature for 18 h. After evaporation, the residue was dissolved in hexane, and the solution was washed with 2% Na2CO3 solution several times until no p-nitrophenol was present. The solvent was evaporated, and the crude was purified by column chromatography using 0 -100% ethyl acetate in hexane and then 0-30% acetone in ethyl acetate to give the desired product as colorless oil (47 mg, 14%). 1H NMR (300 MHz, CDCl3) δ 5.32-5.41 (1H), 4.10-4.03 (m, 10H), 2.90 (m, 2H), 2.31-2.28 (m, 4H), 2.20 (s, 6H), 1.73-1.87 (m, 2H), 1.66-1.39 (m, 20H), 1.23 (m, 40H), 0.85 (t, 12H). APCI-MS analysis: Calculated C52H97NO11 [M+H] = 912.7, Observed = 912.0. Example 77: Bis(8-oxo-8-(pentadecan-7-yloxy)octyl) 2-(((2- (dimethylamino)ethoxy)carbonyl)oxy)succinate Step 1: Synthesis of Bis(8-(benzyloxy)octyl) 2-hydroxysuccinate (39) A mixture of dimethyl malate (4.05 g, 25 mmol), 8-benzoxyoctanol (11.8 g, 50 mmol) and p-toluenesulfonic acid monohydrate (600 mg) in 200 mL toluene was refluxed vigorously with Dean-stark apparatus. After 150 mL solvent was distilled off, more toluene was added and refluxed. This process was repeated one more time. After cooling to room temperature, the mixture was diluted with saturated sodium bicarbonate, and the resulting mixture was extracted with ethyl acetate. The combined organic layers were dried over sodium sulfate and concentrated, and the crude was purified by column chromatography using 0-20% ethyl acetate in hexane to give the desired product as colorless oil (5.7 g, 40%). Step 2: Synthesis of Bis(8-(benzyloxy)octyl) 2-((tert-butyldiphenylsilyl)oxy)succinate (40) A mixture of bis(8-(benzyloxy)octyl) 2-hydroxysuccinate 39 (5.0 g, 8.77 mmol), tert- butyldiphenylsilyl chloride (2.65 g, 9.65 mmol) and imidazole (657 mg, 9.65 mmol) in 50 mL DMF was stirred at room temperature for 17 h. The reaction mixture was partitioned with ethyl acetate and saturated ammonium chloride solution, and the combined organic layer was dried over sodium sulfate. After concentration, the crude was purified by column chromatography using 0-20% ethyl acetate in hexane to give the desired product as clear oil (6.4 g, quant.). Step 3: Synthesis of Bis(8-hydroxyoctyl) 2-((tert-butyldiphenylsilyl)oxy)succinate (41) A mixture of bis(8-(benzyloxy)octyl) 2-((tert-butyldiphenylsilyl)oxy)succinate 40 (6.4 g, 8.77 mmol) and 5% palladium on carbon (1 g) in 100 mL ethyl acetate was subjected to hydrogenolysis for 15 h. After filtration, the filtrate was concentrated, and the crude was purified by column chromatography using 0-100% ethyl acetate in hexane to obtain the desired product (800 mg) along with a mixture of partially deprotected intermediate and starting material (4 g), which was further hydrogenated with 800 mg 20% palladium hydroxide on carbon in a Parr apparatus (40 psi) for 16 h. After workup and column purification, 3.2 g pure product was obtained (total: 4.0 g, 72%). Step 4: Synthesis of 8,8'-((2-((tert-Butyldiphenylsilyl)oxy)succinyl)bis(oxy))dioctanoic acid (42) To a solution of bis(8-hydroxyoctyl) 2-((tert-butyldiphenylsilyl)oxy)succinate 41 (800 mg, 1.37 mmol) in 20 mL acetone, was added Jones reagent until the orange color persisted, and the resulting mixture was stirred for 90 min. The excess Jones reagent was consumed by adding few drops of 2-propanol, then the blue solution was diluted with water (100 mL) and extracted with ethyl acetate (3 x 70 mL). The combined organic layers were washed with brine, dried and concentrated to give the desired product (800 mg, 95%), which was used for the next step without further purification. Step 5: Synthesis of Bis(8-oxo-8-(pentadecan-7-yloxy)octyl) 2-((tert- butyldiphenylsilyl)oxy)succinate (43) A mixture of 8,8'-((2-((tert-butyldiphenylsilyl)oxy)succinyl)bis(oxy))dioctanoic acid 42 (400 mg, 0.61 mmol), 7-pentadecanol (310 mg, 1.28 mmol), EDCI-HCl (384 mg, 2 mmol) and DMAP (244 mg, 2 mmol) in 10 mL dichloromethane was stirred at room temperature for 16 h. After concentration, the crude was partitioned between EtOAc and saturated ammonium chloride solution, and the combined organic layer was dried over sodium sulfate. The solvent was removed under vacuum, and the crude was purified by column chromatography using 0- 10% ethyl acetate in hexane to give the desired product (590 mg, 90%). Step 6: Synthesis of Bis(8-oxo-8-(pentadecan-7-yloxy)octyl) 2-hydroxysuccinate (44) To a solution of bis(8-oxo-8-(pentadecan-7-yloxy)octyl) 2-((tert- butyldiphenylsilyl)oxy)succinate 43 (590 mg, 0.55 mmol) in 10 mL THF, was added pyridine (2 mL) followed by HF-pyridine complex (70%, 1.5 mL), and the mixture was stirred at room temperature overnight. The reaction was quenched by adding saturated sodium bicarbonate to pH 7, and then extracted with ethyl acetate (3 x 60 mL). The combined organic layers were dried and concentrated, and the crude was purified by column chromatography using 0-30% ethyl acetate in hexane to yield the desired product (440 mg, 95%). Step 7: Synthesis of Bis(8-oxo-8-(pentadecan-7-yloxy)octyl) 2-(((2- (dimethylamino)ethoxy)carbonyl)oxy)succinate (Example 77) To a solution of 2,2-dimethylaminoethanol (534 mg, 6 mmol) in a mixture of 3mL pyridine and 8 mL dichloromethane, was added 4-nitrophenylchloroformate (603 mg, 6 mmol), and the reaction mixture was stirred at room temperature for 3 h. TLC showed complete reaction. A solution of bis(8-oxo-8-(pentadecan-7-yloxy)octyl) 2-hydroxysuccinate 44 (440 mg, 0.53 mmol) in 3 mL dichloromethane was added, then diisopropylethylamine (1 mL) was added, and the resulting mixture was stirred at room temperature for another 15 h. After concentration, the residue was dissolved in hexanes, and the solution was washed by acetonitrile several times until the acetonitrile layer contains no 4-nitrophenol. The hexane layer was concentrated, and the crude was purified by column chromatography using 0-100% ethyl acetate in hexanes, followed by 0-30% acetone in ethyl acetate to give desired product (178 mg, 35%). 1H NMR (300 MHz, CDCl3) δ 5.36 (t, 1H), 4.86 (quint, 2H), 4.25 (t, 2H), 4.15 (t, 2H), 4.09 (t, 2H), 2.89-2.87 (m, 2H), 2.60 (m, 2H), 2.29-2.25 (m, 10H), 1.65-1.49 (m, 18H), 1.28 (m, 50H), 0.87 (t, 12H). APCI-MS analysis: Calculated C55H103NO11 [M+H] = 954.7, Observed = 954.7. Example 78: Bis(8-(((Z)-non-2-en-1-yl)oxy)-8-oxooctyl) 2-(((2- (dimethylamino)ethoxy)carbonyl)oxy)succinate Step 1: Synthesis of Bis(8-(((Z)-non-2-en-1-yl)oxy)-8-oxooctyl) 2-((tert- butyldiphenylsilyl)oxy)succinate (45) A mixture of 8,8'-((2-((tert-butyldiphenylsilyl)oxy)succinyl)bis(oxy))dioctanoic acid 42 (400 mg, 0.61 mmol), (Z)-non-2-en-1-ol (182 mg, 1.28 mmol), EDCI-HCl (384 mg, 2 mmol) and DMAP (244 mg, 2 mmol) in 10 mL dichloromethane was stirred at room temperature for 16 h. After concentration, the crude was partitioned between EtOAc and saturated ammonium chloride solution, and the combined organic layer was dried over sodium sulfate. The solvent was removed under vacuum, and the crude was purified by column chromatography using 0-10% ethyl acetate in hexane to give the desired product (410 mg, 74%). Step 2: Synthesis of Bis(8-(((Z)-non-2-en-1-yl)oxy)-8-oxooctyl) 2-hydroxysuccinate (46) To a solution of bis(8-(((Z)-non-2-en-1-yl)oxy)-8-oxooctyl) 2-((tert- butyldiphenylsilyl)oxy)succinate 45 (410 mg, 0.45 mmol) in 10 mL THF, was added pyridine (2 mL) followed by HF-pyridine complex (70%, 0.5 mL), and the mixture was stirred at room temperature for 20 h. The reaction was quenched by adding saturated sodium bicarbonate to pH 7, and then extracted with ethyl acetate (3 x 60 mL). The combined organic layers were dried and concentrated, and the crude was purified by column chromatography using 0-30% ethyl acetate in hexane to yield the desired product (270 mg, 89%). Step 3: Synthesis of Bis(8-(((Z)-non-2-en-1-yl)oxy)-8-oxooctyl) 2-(((2- (dimethylamino)ethoxy)carbonyl)oxy)succinate (Example 78) To a solution of 2-(dimethylamino)ethan-1-ol (534 mg, 6 mmol) in a mixture of 1 mL pyridine and 8 mL dichloromethane, was added 4-nitrophenylchloroformate (1.20 g, 6 mmol), and the reaction mixture was stirred at room temperature for 1 h. TLC showed complete reaction. A solution of bis(8-(((Z)-non-2-en-1-yl)oxy)-8-oxooctyl) 2- hydroxysuccinate 46 (270 mg, 0.4 mmol) in 3 mL dichloromethane was added, then diisopropylethylamine (0.52 mL, 3 mmol) was added, and the resulting mixture was stirred at room temperature for two days. After concentration, the residue was dissolved in hexanes, and the solution was washed by acetonitrile several times until the acetonitrile layer contains no 4-nitrophenol. The hexane layer was concentrated, and the crude was purified by column chromatography using 0-100% ethyl acetate in hexanes, followed by 0-30% acetone in ethyl acetate to give desired product as colorless oil (183 mg, 58%). 1H NMR (300 MHz, CDCl3) δ 5.68-5.47 (m, 4H), 5.37 (t, 1H), 4.61 (d, 4H), 4.25 (t, 2H), 4.16 (t, 2H), 4.09 (t, 2H), 2.90-2.87 (m, 2H), 2.63-2.58 (m, 2H), 2.32-2.23 (m, 10H), 2.09 (q, 4H), 1.61 (m, 6H), 1.32-1.25 (m, 30H), 0.87 (t, 6H). APCI-MS analysis: Calculated C43H75NO11 [M+H] = 782.5, Observed = 782.5. Example 79: Bis(7-((8-methylnonanoyl)oxy)heptyl) 2-(((2- (dimethylamino)ethoxy)carbonyl)oxy)succinate Step 1: Synthesis of 7-(Benzyloxy)heptan-1-ol (47) To a solution of 1,7-heptanediol (10 g, 75.7mmol) in 60 mL DMF, was added NaH (3.33 g, 83 mmol) at 0°C. After stirring for 30 min, a solution of benzylbromide (20.2 g, 91 mmol) in 30 mL DMF was added dropwise, and then reaction mixture was stirred at room temperature overnight. Water was added to quench the reaction, the mixture was extracted with EtOAc (2 x 200 mL). The combined organic layer was washed with water and brine, and then dried over sodium sulfate. After filtration and concentration, the crude was purified by column chromatography using 0-60% ethyl acetate in hexane to give the desired product (9.4 g, 59%). Step 2: Synthesis of 7-(Benzyloxy)heptyl 8-methylnonanoate (48) A mixture of 7-(benzyloxy)heptan-1-ol 47 (9.4 g, 42.3 mmol), 8-methylnonanoic acid (7.28 g, 42.3 mmol), EDCI-HCl (11.4 g, 59.3 mmol) and DMAP (1.03 g, 8.4 mmol) in 100 mL dichloromethane was stirred at room temperature for 16 h. After concentration, the crude was partitioned between dichloromethane and saturated ammonium chloride solution, and the combined organic layer was dried over sodium sulfate. The solvent was removed under vacuum, and the crude was purified by column chromatography using 0-10% ethyl acetate in hexane to give the desired product (14.47 g, 91%). Step 3: Synthesis of 7-Hydroxyheptyl 8-methylnonanoate (49) A mixture of 7-(benzyloxy)heptyl 8-methylnonanoate 48 (13.8 g, 48.2 mmol) and 20% palladium hydroxide on carbon (1.5 g) in 100 mL ethyl acetate was subjected to hydrogenolysis in a Parr apparatus (40 psi) for 16 h. After filtration, the filtrate was concentrated, and the crude was purified by column chromatography using 0-100% ethyl acetate in hexane to obtain the desired product (10.5 g, quant.). Step 4: Synthesis of Bis(7-((8-methylnonanoyl)oxy)heptyl) 2-hydroxysuccinate (50) A mixture of dimethyl malate (991 mg, 6.11 mmol), 7-hydroxyheptyl 8- methylnonanoate 49 (3.5 g, 12.3 mmol) and p-toluenesulfonic acid monohydrate (600 mg) in 200 mL toluene was refluxed vigorously with Dean-stark apparatus. After 150 mL solvent was distilled off, more toluene was added and refluxed. This process was repeated one more time. After cooled to room temperature, the mixture was diluted with saturated sodium bicarbonate, and the resulting mixture was extracted with ethyl acetate. The combined organic layers were dried over sodium sulfate and concentrated to give oil which was purified by column chromatography using 0-20% ethyl acetate in hexane to give the desired product as colorless oil (190 mg, 4.6%). The major product is heptane-1,7-diyl bis(8- methylnonanoate). Step 5: Synthesis of Bis(7-((8-methylnonanoyl)oxy)heptyl) 2-(((2- (dimethylamino)ethoxy)carbonyl)oxy)succinate (Example 79) To a solution of 2-(dimethylamino)ethan-1-ol (534 mg, 6 mmol) in a mixture of 1 mL pyridine and 8 mL dichloromethane, was added 4-nitrophenylchloroformate (1.20 g, 6 mmol), and the reaction mixture was stirred at room temperature for 1 h. TLC showed complete reaction. A solution of bis(7-((8-methylnonanoyl)oxy)heptyl) 2-hydroxysuccinate 50 (190 mg, 0.28 mmol) in 3 mL dichloromethane was added, then diisopropylethylamine (0.52 mL, 3 mmol) was added, and the resulting mixture was stirred at room temperature for two days. After concentration, the residue was dissolved in hexanes, and the solution was washed by acetonitrile several times until the acetonitrile layer contains no 4-nitrophenol. The hexane layer was concentrated, and the crude was purified by column chromatography using 0-100% ethyl acetate in hexanes, followed by 0-30% acetone in ethyl acetate to give desired product as colorless oil (98 mg, 43%). 1H NMR (300 MHz, CDCl3) δ 5.37 (t, 1H), 4.25 (t, 2H), 4.16 (t, 2H), 4.09 (t, 2H), 4.04 (t, 4H), 2.90-2.88 (m, 2H), 2.63-2.58 (m, 2H), 2.31-2.26 (m, 10H), 1.66-1.58 (m, 12H), 1.55- 1.46 (m, 2H), 1.33-1.10 (m, 28H), 0.86 (d, 12H). APCI-MS analysis: Calculated C43H79NO11 [M+H] = 786.5, Observed = 786.5. Example 80: Bis(8-oxo-8-((2-propylnonyl)oxy)octyl) 2-(((2- (dimethylamino)ethoxy)carbonyl)oxy)succinate
Figure imgf000255_0001
Step 1: Synthesis of 2-Propylnonanoic acid (51) To a solution of nonanoic acid (10.5 mL, 60 mmol) in 100 mL THF at 0°C, was added NaH (60%, 2.4 g, 60 mmol) in portions, followed by adding a solution of LDA (2 M in THF/heptane/ethylbenzene, 60 mL, 0.12 mole) dropwise, and the mixture was stirred at room temperature for 30 min. After addition of n-propyl iodide (11.22 g, 66 mmol), the resulting mixture was heated to 45°C overnight. The suspension was cooled to room temperature and quenched with 1 N HCl to pH 2, the organic layer was separated and dried over sodium sulfate. After concentration, the crude was purified by flash column chromatography eluted with 0-20% EtOAc in hexane to give impure product (10 g), and the impurity is nonanoic acid. The impure product was used for the next step without further purification. Step 2: Synthesis of 2-propylnonan-1-ol (52) To a solution of impure 2-propylnonanoic acid 51 (5.5 g, 30 mmol) in 30 mL ether, was added a solution of LiAlH4 (2 M in THF, 15 mL, 30 mmol) dropwise, and the resulting mixture was stirred at room temperature overnight. After quenched by EtOAc, the solution was washed with water, and the organic layer was dried over sodium sulfate. The solvent was removed under vacuum, and the crude was purified by flash column chromatography eluted with 0-40% EtOAc in hexane to give the desired product as colorless oil (3.5 g, 69%). Step 3: Synthesis of Bis(8-oxo-8-((2-propylnonyl)oxy)octyl) 2-((tert- butyldiphenylsilyl)oxy)succinate (53) A mixture of 8,8'-((2-((tert-butyldiphenylsilyl)oxy)succinyl)bis(oxy))dioctanoic acid 42 (700 mg, 1.06 mmol), 2-propylnonan-1-ol 52 (510 mg, 2.74 mmol), EDCI-HCl (576 mg, 3 mmol) and DMAP (366 mg, 3 mmol) in 12 mL dichloromethane was stirred at room temperature for 16 h. After concentration, the crude was partitioned between EtOAc and saturated ammonium chloride solution, and the combined organic layer was dried over sodium sulfate. After concentration, the crude (900 mg) was used for the next step without purification. Step 4: Synthesis of Bis(8-oxo-8-((2-propylnonyl)oxy)octyl) 2-hydroxysuccinate (54) To a solution of crude bis(8-oxo-8-((2-propylnonyl)oxy)octyl) 2-((tert- butyldiphenylsilyl)oxy)succinate 53 (900 mg) in 10 mL THF, was added pyridine (2 mL) followed by HF-pyridine complex (70%, 1.5 mL), and the mixture was stirred at room temperature overnight. The reaction was quenched by adding saturated sodium bicarbonate to pH 7, and then extracted with ethyl acetate (3 x 60 mL). The combined organic layers were dried and concentrated, and the crude was purified by column chromatography using 0-30% ethyl acetate in hexane to yield the desired product (460 mg, 57%). Step 5: Synthesis of Bis(8-oxo-8-((2-propylnonyl)oxy)octyl) 2-(((2- (dimethylamino)ethoxy)carbonyl)oxy)succinate (Example 80) To a solution of 2-(dimethylamino)ethan-1-ol (534 mg, 6 mmol) in a mixture of 1 mL pyridine and 8 mL dichloromethane, was added 4-nitrophenylchloroformate (1.20 g, 6 mmol), and the reaction mixture was stirred at room temperature for 1 h. TLC showed complete reaction. A solution of bis(8-oxo-8-((2-propylnonyl)oxy)octyl) 2-hydroxysuccinate 54 (350 mg, 0.46 mmol) in 3 mL dichloromethane was added, then diisopropylethylamine (0.52 mL, 3 mmol) was added, and the resulting mixture was stirred at room temperature for two days. After concentration, the residue was dissolved in hexanes, and the solution was washed by acetonitrile several times until the acetonitrile layer contains no 4-nitrophenol. The hexane layer was concentrated, and the crude was purified by column chromatography using 0-100% ethyl acetate in hexanes, followed by 0-30% acetone in ethyl acetate to give desired product (220 mg, 54%). 1H NMR (300 MHz, CDCl3) δ 5.37 (t, 1H), 4.25 (t, 2H), 4.15 (t, 2H), 4.09 (t, 2H), 3.96 (d, 4H), 2.89-2.87 (m, 2H), 2.63-2.57 (m, 2H), 2.31-2.27 (m, 10H), 1.63-1.59 (m, 10H), 1.32- 1.25 (m, 44H), 0.90-0.85 (m, 12H). APCI-MS analysis: Calculated C49H91NO11 [M+H] = 870.6, Observed = 870.6. Example 81: Bis(6-(decanoyloxy)hexyl) 2-(((2- (dimethylamino)ethoxy)carbonyl)oxy)succinate Step 1: Synthesis of Bis(6-(decanoyloxy)hexyl) 2-((tert-butyldiphenylsilyl)oxy)succinate (55) A mixture of bis(6-hydroxyhexyl) 2-((tert-butyldiphenylsilyl)oxy)succinate 19 (370 mg, 0.65 mmol), decanoic acid (223 mg, 1.3 mmol), EDCI-HCl (288 mg, 1.5 mmol) and DMAP (183 mg, 1.5 mmol) in 8 mL dichloromethane was stirred at room temperature for 16 h. After concentration, the crude was partitioned between EtOAc and saturated ammonium chloride solution, and the combined organic layer was dried over sodium sulfate. The solvent was removed under vacuum, and the crude was purified by column chromatography using 0- 10% ethyl acetate in hexane to give the desired product (550 mg, 88%). Step 2: Synthesis of Bis(6-(decanoyloxy)hexyl) 2-hydroxysuccinate (56) To a solution of bis(6-(decanoyloxy)hexyl) 2-((tert-butyldiphenylsilyl)oxy)succinate 55 (550 mg, 0.54 mmol) in 10 mL THF, was added pyridine (2 mL) followed by HF-pyridine complex (70%, 1 mL), and the mixture was stirred at room temperature for 16 h. The reaction was quenched by adding saturated sodium bicarbonate to pH 7, and then extracted with ethyl acetate (3 x 60 mL). The combined organic layers were dried and concentrated, and the crude was purified by column chromatography using 10-30% ethyl acetate in hexane to yield the desired product (330 mg, 95%). Step 3: Synthesis of Bis(6-(decanoyloxy)hexyl) 2-(((2- (dimethylamino)ethoxy)carbonyl)oxy)succinate (Example 81) To a solution of 2-(dimethylamino)ethan-1-ol (534 mg, 6 mmol) in a mixture of 1 mL pyridine and 8 mL dichloromethane, was added 4-nitrophenylchloroformate (1.20 g, 6 mmol), and the reaction mixture was stirred at room temperature for 1 h. TLC showed complete reaction. A solution of bis(6-(decanoyloxy)hexyl) 2-hydroxysuccinate 56 (350 mg, 0.46 mmol) in 3 mL dichloromethane was added, then diisopropylethylamine (0.52 mL, 3 mmol) was added, and the resulting mixture was stirred at room temperature for two days. After concentration, the residue was dissolved in hexanes, and the solution was washed by acetonitrile several times until the acetonitrile layer contains no 4-nitrophenol. The hexane layer was concentrated, and the crude was purified by column chromatography using 0-100% ethyl acetate in hexanes, followed by 0-30% acetone in ethyl acetate to give desired product (110 mg, 28%). 1H NMR (300 MHz, CDCl3) δ 5.37 (t, 1H), 4.26 (t, 2H), 4.17 (t, 2H), 4.12 (t, 2H), 4.05 (t, 4H), 2.89 (m, 2H), 2.63-2.58 (m, 2H), 2.31-2.26 (m, 10H), 1.63-1.61 (m, 12H), 1.37-1.26 (m, 32H), 0.87 (t, 6H). APCI-MS analysis: Calculated C41H75NO11 [M+H] = 758.5, Observed = 758.0. Example 82: Bis(6-oxo-6-(pentadecan-7-yloxy)hexyl) 2-((5- (dimethylamino)pentanoyl)oxy)succinate A mixture of bis(6-oxo-6-(pentadecan-7-yloxy)hexyl) 2-hydroxysuccinate 22 (120 mg, 0.15 mmol), 5-(dimethylamino)pentanoic acid hydrochloride (54 mg, 0.3 mmol), EDC- HCl (95 mg, 0.49 mmol) and DMAP (60 mg, 0.49 mmol) in 10 mL dichloromethane was stirred at room temperature for 17 h. The volatiles were removed under vacuum, and the residue was dissolved in EtOAc. The solution was washed with water, and the organic layer was dried over sodium sulfate. After concentration, the crude was purified by column chromatography using 0-10% methanol in dichloromethane to yield the desired product as colorless oil (196 mg, 71%). 1H NMR (300 MHz, CDCl3) δ 5.43 (t, 1H), 4.84 (quint, 2H), 4.15-4.06 (m, 4H), 2.84 (d, 2H), 2.42-2.15 (m, 12H), 1.67-1.47 (m, 18H), 1.41-1.23 (m, 48H), 0.90-0.83 (m, 12H). APCI-MS analysis: Calculated C53H99NO10 [M+H] = 910.7, Observed = 910.0. Example 83: Bis(6-oxo-6-(pentadecan-7-yloxy)hexyl) 2-(((3- (dimethylamino)propoxy)carbonyl)oxy)succinate
Figure imgf000260_0001
To a solution of 3-(dimethylamino)propan-1-ol (267 mg, 3 mmol) in a mixture of 1 mL pyridine and 8 mL dichloromethane, was added 4-nitrophenylchloroformate (603 mg, 3 mmol), and the reaction mixture was stirred at room temperature for 3 h. TLC showed complete reaction. A solution of bis(6-oxo-6-(pentadecan-7-yloxy)hexyl) 2-hydroxysuccinate 22 (300 mg, 0.29 mmol) in 3 mL dichloromethane was added, then diisopropylethylamine (1 mL) was added, and the resulting mixture was stirred at room temperature for another 15 h. After concentration, the residue was dissolved in hexanes, and the solution was washed by acetonitrile several times until the acetonitrile layer contains no 4-nitrophenol. The hexane layer was concentrated, and the crude was purified by column chromatography using 0-100% ethyl acetate in hexanes, followed by 0-30% acetone in ethyl acetate to give desired product (118 mg, 42%). 1H NMR (300 MHz, CDCl3) δ 5.36 (t, 1H), 4.85 (quint, 2H), 4.25-4.05 (m, 6H), 2.89 (d, 2H), 2.38-2.18 (m, 12H), 1.90-1.80 (m, 2H), 1.69-1.42 (m, 20H), 1.29 (m, 40H), 0.95-0.84 (m, 12H). APCI-MS analysis: Calculated C52H97NO11 [M+H] = 912.7, Observed = 912.1. Example 84: Bis(6-oxo-6-(pentadecan-7-yloxy)hexyl) 2-(((2- (dimethylamino)ethoxy)carbonyl)oxy)succinate To a solution of 2-(dimethylamino)ethan-1-ol (267 mg, 3 mmol) in a mixture of 1 mL pyridine and 8 mL dichloromethane, was added 4-nitrophenylchloroformate (603 mg, 3 mmol), and the reaction mixture was stirred at room temperature for 3 h. TLC showed complete reaction. A solution of bis(6-oxo-6-(pentadecan-7-yloxy)hexyl) 2-hydroxysuccinate 22 (140 mg, 0.18 mmol) in 3 mL dichloromethane was added, then diisopropylethylamine (1 mL) was added, and the resulting mixture was stirred at room temperature for another 15 h. After concentration, the residue was dissolved in hexanes, and the solution was washed by acetonitrile several times until the acetonitrile layer contains no 4-nitrophenol. The hexane layer was concentrated, and the crude was purified by column chromatography using 0-100% ethyl acetate in hexanes, followed by 0-30% acetone in ethyl acetate to give desired product (40 mg, 24%). 1H NMR (300 MHz, CDCl3) δ 5.36 (m, 1H), 4.86 (t, 2H), 4.26 (t, 2H), 4.17 (t, 2H), 4.10 (t, 2H), 2.89 (m, 2H), 2.61 (m, 2H), 2.31-2.26 (m, 12H), 1.70-1.49 (m, 18H), 1.42-1.25 (m, 40H), 0.87 (t, 12H). APCI-MS analysis: Calculated C51H95NO11 [M+H] = 898.7, Observed = 898.0. Example 85: Bis(5-((2-hexyldecanoyl)oxy)pentyl) 2-(((2- (dimethylamino)ethoxy)carbonyl)oxy)succinate To a solution of 2-(dimethylamino)ethan-1-ol (445 mg, 5 mmol) in a mixture of 3 mL pyridine and 10 mL dichloromethane, was added 4-nitrophenylchloroformate (1.0 g, 5 mmol), and the reaction mixture was stirred at room temperature for 3 h. TLC showed complete reaction. A solution of bis(5-((2-hexyldecanoyl)oxy)pentyl) 2-hydroxysuccinate 15 (500 mg, 0.66 mmol) in 3 mL dichloromethane was added, then diisopropylethylamine (1 mL) was added, and the resulting mixture was stirred at room temperature for two days. After concentration, the residue was dissolved in hexanes, and the solution was washed by acetonitrile several times until the acetonitrile layer contains no 4-nitrophenol. The hexane layer was concentrated, and the crude was purified by column chromatography using 0-100% ethyl acetate in hexanes to give desired product (360 mg, 61%). 1H NMR (300 MHz, CDCl3) δ 5.36 (t, 1H), 4.24 (t, 2H), 4.17 (t, 2H), 4.10 (t, 2H), 4.05 (t, 4H), 2.88 (d, 2H), 2.59 (m, 2H), 2.32-2.24 (m, 8H), 1.70-1.53 (m, 12H), 1.44-1.34 (m, 6H), 1.23 (m, 42H), 0.85 (t, 12H). APCI-MS analysis: Calculated C51H95NO11 [M+H] = 898.7, Observed = 898.0. Example 86: Bis(6-(((Z)-non-2-en-1-yl)oxy)-6-oxohexyl) 2-(((2- (dimethylamino)ethoxy)carbonyl)oxy)succinate Step 1: Synthesis of Bis(6-(((Z)-non-2-en-1-yl)oxy)-6-oxohexyl) 2-((tert- butyldiphenylsilyl)oxy)succinate (57) A mixture of 6,6'-((2-((tert-butyldiphenylsilyl)oxy)succinyl)bis(oxy))dihexanoic acid 20 (400 mg, 0.67 mmol), (Z)-non-2-en-1-ol (238 mg, 1.67 mmol), EDCI-HCl (321 mg, 1.67 mmol) and DMAP (204 mg, 1.67 mmol) in 10 mL dichloromethane was stirred at room temperature for 16 h. After concentration, the crude was partitioned between EtOAc and saturated ammonium chloride solution, and the combined organic layer was dried over sodium sulfate. The solvent was removed under vacuum, and the crude was purified by column chromatography using 0-10% ethyl acetate in dichloromethane to give the desired product (500 mg, 88%). Step 2: Synthesis of Bis(6-(((Z)-non-2-en-1-yl)oxy)-6-oxohexyl) 2-hydroxysuccinate (58) To a solution of bis(6-(((Z)-non-2-en-1-yl)oxy)-6-oxohexyl) 2-((tert- butyldiphenylsilyl)oxy)succinate 57 (500 mg, 0.49 mmol) in 10 mL THF, was added pyridine (2 mL) followed by HF-pyridine complex (70%, 1.5 mL), and the mixture was stirred at room temperature overnight. The reaction was quenched by adding saturated sodium bicarbonate to pH 7, and then extracted with ethyl acetate (3 x 60 mL). The combined organic layers were dried and concentrated, and the crude was purified by column chromatography using 0-40% ethyl acetate in hexane to yield the desired product (300 mg, 82%). Step 3: Synthesis of Bis(6-(((Z)-non-2-en-1-yl)oxy)-6-oxohexyl) 2-(((2- (dimethylamino)ethoxy)carbonyl)oxy)succinate (Example 86) To a solution of 2-(dimethylamino)ethan-1-ol (445 mg, 5 mmol) in a mixture of 3 mL pyridine and 10 mL dichloromethane, was added 4-nitrophenylchloroformate (1.0 g, 5 mmol), and the reaction mixture was stirred at room temperature for 3 h. TLC showed complete reaction. A solution of bis(6-(((Z)-non-2-en-1-yl)oxy)-6-oxohexyl) 2- hydroxysuccinate 58 (500 mg, 0.66 mmol) in 3 mL dichloromethane was added, then diisopropylethylamine (1 mL) was added, and the resulting mixture was stirred at room temperature for two days. After concentration, the residue was dissolved in hexanes, and the solution was washed by acetonitrile several times until the acetonitrile layer contains no 4- nitrophenol. The hexane layer was concentrated, and the crude was purified by column chromatography using 0-100% ethyl acetate in hexanes to give desired product (165 mg, 46%). 1H NMR (300 MHz, CDCl3) δ 5.68-5.46 (m, 4H), 5.36 (t, 1H), 4.61 (d, 4H), 4.25 (t, 2H), 4.16 (t, 2H), 4.10 (t, 2H), 2.88 (d, 2H), 2.63-2.57 (m, 2H), 2.34-2.27 (m, 10H), 2.09 (m, 4H), 1.75-1.59 (m, 8H), 1.42-1.25 (m, 20H), 0.87 (t, 6H). APCI-MS analysis: Calculated C39H67NO11 [M+H] = 726.4, Observed = 725.9. Example 87: Bis(7-((2-hexyldecanoyl)oxy)heptyl) 2-(((2- (dimethylamino)ethoxy)carbonyl)oxy)succinate Step 1: Synthesis of Bis(7-((2-hexyldecanoyl)oxy)heptyl) 2-hydroxysuccinate (59) A mixture of dimethyl malate (870 mg, 6 mmol), 7-hydroxyheptyl 2-hexyldecanoate 33 (4.4 g, 12 mmol) and p-toluenesulfonic acid monohydrate (300 mg) in 200 mL toluene was refluxed vigorously with Dean-stark apparatus overnight. After cooled to room temperature, the mixture was diluted with saturated sodium bicarbonate, and the resulting mixture was extracted with ethyl acetate. The combined organic layers were dried over sodium sulfate and concentrated to give oil which was purified by column chromatography using 0-20% ethyl acetate in hexane to give the desired product as colorless oil (3.0 g, 60%). Step 2: Synthesis of Bis(7-((2-hexyldecanoyl)oxy)heptyl) 2-(((2- (dimethylamino)ethoxy)carbonyl)oxy)succinate (Example 87) To a solution of 2-(dimethylamino)ethan-1-ol (445 mg, 5 mmol) in a mixture of 3 mL pyridine and 10 mL dichloromethane, was added 4-nitrophenylchloroformate (1.0 g, 5 mmol), and the reaction mixture was stirred at room temperature for 3 h. TLC showed complete reaction. A solution of bis(7-((2-hexyldecanoyl)oxy)heptyl) 2-hydroxysuccinate 59 (500 mg, 0.66 mmol) in 3 mL dichloromethane was added, then diisopropylethylamine (1 mL) was added, and the resulting mixture was stirred at room temperature for two days. After concentration, the residue was dissolved in hexanes, and the solution was washed by acetonitrile several times until the acetonitrile layer contains no 4-nitrophenol. The hexane layer was concentrated, and the crude was purified by column chromatography using 0-100% ethyl acetate in hexanes to give desired product (310 mg, 54%). 1H NMR (300 MHz, CDCl3) δ 5.36 (t, 1H), 4.24 (t, 2H), 4.15 (t, 2H), 4.08 (t, 2H), 4.04 (t, 4H), 2.88 (d, 2H), 2.60 (m, 2H), 2.32-2.22 (m, 8H), 1.63-1.46 (m, 12H), 1.46-1.23 (m, 56H), 0.85 (t, 12H). APCI-MS analysis: Calculated C55H103NO11 [M+H] = 954.7, Observed = 954.0. Example 88: Bis(5-(decanoyloxy)pentyl) 2-(((2- (dimethylamino)ethoxy)carbonyl)oxy)succinate Step 1: Synthesis of 5-Hydroxypentyl decanoate (60) A mixture of 1,5-pentanediol (10 g, 0.10 mole), decanoic acid (5.67 g, 33 mmol), EDCI-HCl (8.85 g, 46.2 mmol) and DMAP (805 mg, 6.6 mmol) in 100 mL dichloromethane was stirred at room temperature for 17 h. The dichloromethane was removed, the residue was diluted with 200 mL ethyl acetate, and the solution was washed with aqueous saturated ammonium chloride. After dried over sodium sulfate and concentrated, the crude was purified by column chromatography using 0-10% ethyl acetate in hexane to yield the desired product as colorless oil (13.9 g, 88%). Step 2: Synthesis of Bis(5-(decanoyloxy)pentyl) 2-hydroxysuccinate (61) A mixture of dimethyl malate (1.22 g, 7.5 mmol), 5-hydroxypentyl decanoate 60(4.0 g, 15 mmol) and p-toluenesulfonic acid monohydrate (250 mg) in 150 mL toluene was heated to reflux vigorously with Dean-stark apparatus overnight. The reaction mixture was cooled to room temperature and washed with saturated sodium bicarbonate solution, and the aqueous layer was extracted with EtOAc. The combined organic layers were dried over sodium sulfate and concentrated to give the crude which was purified by column chromatography eluted with 0-10% ethyl acetate in dichloromethane to give the desired product as colorless oil (350 mg, 8%). Step 3: Synthesis of Bis(5-(decanoyloxy)pentyl) 2-(((2- (dimethylamino)ethoxy)carbonyl)oxy)succinate (Example 88) To a solution of 2-(dimethylamino)ethan-1-ol (445 mg, 5 mmol) in a mixture of 3 mL pyridine and 10 mL dichloromethane, was added 4-nitrophenylchloroformate (1.0 g, 5 mmol), and the reaction mixture was stirred at room temperature for 3 h. TLC showed complete reaction. A solution of bis(5-(decanoyloxy)pentyl) 2-hydroxysuccinate 61 (500 mg, 0.66 mmol) in 3 mL dichloromethane was added, then diisopropylethylamine (0.52 mL) was added, and the resulting mixture was stirred at room temperature for two days. After concentration, the residue was dissolved in hexanes, and the solution was washed by acetonitrile several times until the acetonitrile layer contains no 4-nitrophenol. The hexane layer was concentrated, and the crude was purified by column chromatography using 0-100% ethyl acetate in hexanes to give desired product (180 mg, 46%). 1H NMR (300 MHz, CDCl3) δ 5.37 (t, 1H), 4.25 (t, 2H), 4.18 (t, 2H), 4.11 (t, 2H), 4.05 (td, 4H), 2.89 (d, 2H), 2.60 (m, 2H), 2.31-2.26 (m, 10H), 1.71-1.58 (m, 12H), 1.45-1.35 (m, 4H), 1.30 (m, 24H), 0.87 (t, 6H). APCI-MS analysis: Calculated C39H71NO11 [M+H] = 730.5, Observed = 730.5 Example 89: Bis(5-((2-hexyldecanoyl)oxy)pentyl) 2-((3- (dimethylamino)propyl)disulfaneyl)succinate Step 1: Synthesis of Bis(5-hydroxypentyl) 2-mercaptosuccinate (62) To a mixture of 2-mercaptosuccinic acid (1.10 g, 7.3 mmol) in 5 mL 1,5-pentanediol, was added 100 mg zinc chloride, and then the mixture was heated to 150°C for 5 h. After cooled to room temperature, 100 mL dichloromethane was added to dissolve the mixture. The solution was washed with water and brine. After dried over sodium sulfate, the solvent was removed under vacuum to give crude product (2.1 g), which was used for the next step without purification. Step 2: Synthesis of Bis(5-hydroxypentyl) 2-(pyridin-2-yldisulfaneyl)succinate (63) A solution of bis(5-hydroxypentyl) 2-mercaptosuccinate 62 (2.1 g, 6.52 mmol) and dipyridyl disulfide (2.86 g, 13 mmol) in 50 mL dichloromethane was purged with nitrogen three times, and then the mixture was stirred at room temperature overnight. After concentration, the crude was purified by column chromatography eluted with 0-80% ethyl acetate in hexanes to give the desired product (2.15 g, 77%). Step 3: Synthesis of Bis(5-((2-hexyldecanoyl)oxy)pentyl) 2-(pyridin-2-yldisulfaneyl)succinate (64) A solution of 2-hexyldecanoic acid (1.54 g, 6 mmol) in 20 mL dichloromethane was cooled to 0°C, then oxalyl chloride (1.1 mL, 12 mmol) and 5 drops of DMF were added, and the mixture was stirred at room temperature for 2 h. After concentration, the crude was dissolved in 20 mL dichloromethane and 3 mL pyridine, then bis(5-hydroxypentyl) 2- (pyridin-2-yldisulfaneyl)succinate 63 (900 mg, 2.1 mmol) was added dropwise at 0°C. The reaction mixture was stirred at room temperature overnight. TLC showed complete reaction. The mixture was partitioned between dichloromethane and saturated ammonium chloride solution, and the combined organic layer was dried over sodium sulfate. The solvent was removed under vacuum, and the crude was purified by column chromatography using 0- 100% ethyl acetate in hexanes to give the desired product (817 mg, 45%). Step 4: Synthesis of Bis(5-((2-hexyldecanoyl)oxy)pentyl) 2-((3- (dimethylamino)propyl)disulfaneyl)succinate (Example 89) A solution of bis(5-((2-hexyldecanoyl)oxy)pentyl) 2-(pyridin-2- yldisulfaneyl)succinate 64 (350 mg, 0.38 mmol) and 3-(dimethylamino)propane-1-thiol hydrochloride (156 mg, 1 mmol) in a mixture of 2 mL methanol and 10 mL dichloromethane was purged with nitrogen three times, and then stirred at room temperature for 5 h. MS showed complete reaction. After concentration, the residue was dissolved in dichloromethane, and the solution was washed by saturated sodium bicarbonate solution. The combined organic layer was dried over sodium sulfate. After filtration and concentration, the crude was purified by column chromatography using 0-100% ethyl acetate in dichloromethane to give desired product as pale yellow oil (200 mg, 57%). 1H NMR (300 MHz, CDCl3) δ 4.16-4.04 (m, 8H), 3.78-3.75 (m, 1H), 3.11 (dd, 1H), 2.83- 2.71 (m, 3H), 2.34-2.27 (m, 4H), 2.21 (s, 6H), 1.80 (m, 2H), 1.72-1.51 (m, 12H), 1.48-1.34 (m, 8H), 1.24 (m, 40H), 0.91-0.81 (m, 12H). APCI-MS analysis: Calculated C51H97NO8S2 [M+H] = 916.6, Observed = 916.6. Example 90: Bis(6-(nonyloxy)-6-oxohexyl) 2-(((2- (dimethylamino)ethoxy)carbonyl)oxy)pentanedioate Step 1: Synthesis of 6-(Benzyloxy)hexanoic acid (65) To a solution of 6-(benzyloxy)hexan-1-ol (9.7 g, 46.5 mmol) in 60 mL acetone, was added Jones reagent until the orange color persisted, and the resulting mixture was stirred for 90 min. The excess Jones reagent was consumed by adding few drops of 2-propanol, then the blue solution was diluted with water (100 mL) and extracted with ethyl acetate (3 x 100 mL). The combined organic layers were washed with brine, dried and concentrated to give the desired product (9.0 g), which was used for the next step without further purification. Step 2: Synthesis of Nonyl 6-(benzyloxy)hexanoate (66) A mixture of 6-(benzyloxy)hexanoic acid 65 (9.0 g, 40.5 mmol), nonan-1-ol (7.3 g, 42 mmol), EDCI-HCl (9.6 g, 50 mmol) and DMAP (1.22 g, 10 mmol) in 200 mL dichloromethane was stirred at room temperature for 16 h. After concentration, the crude was partitioned between EtOAc and saturated ammonium chloride solution, and the combined organic layer was dried over sodium sulfate. The solvent was removed under vacuum, and the crude was purified by column chromatography using 0-10% ethyl acetate in hexane to give the desired product (11.2 g, 79%). Step 3: Synthesis of Nonyl 6-hydroxyhexanoate (67) A mixture of nonyl 6-(benzyloxy)hexanoate 66 (11.2 g, 32.2 mmol) and 5% palladium on carbon (1 g) in 100 mL ethyl acetate was subjected to hydrogenolysis for 15 h. After filtration, the filtrate was concentrated, and the crude was purified by column chromatography using 0-100% ethyl acetate in hexane to afford the desired product (8.1 g, 97%). Step 4: Synthesis of Bis(6-(nonyloxy)-6-oxohexyl) 2-oxopentanedioate (68) A mixture of α-ketoglutaric acid (514 mg, 3.5 mmol), nonyl 6-hydroxyhexanoate 67 (2.0 g, 7.74 mmol) and p-toluenesulfonic acid monohydrate (100 mg) in 80 mL toluene was heated to reflux for 30 min. The reaction mixture was concentrated under vacuum, and the crude was purified by column chromatography with 0-20% ethyl acetate in hexanes to give the desired product (400 mg, 18%) Step 5: Synthesis of Bis(6-(nonyloxy)-6-oxohexyl) 2-hydroxypentanedioate (69) To a solution of bis(6-(nonyloxy)-6-oxohexyl) 2-oxopentanedioate 68 (600 mg, 0.95 mmol) in 20 mL THF, was added sodium borohydride (53 mg, 1.4 mmol), and the reaction mixture was stirred at room temperature for 2 h. After cooled to 0°C, saturated ammonium chloride solution was added slowly, and the resulting mixture was extracted with ethyl acetate. The combined organic layer was washed with brine and dried over sodium sulfate. After concentration, the crude (600 mg) was used for the next step without purification. Step 6: Synthesis of Bis(6-(nonyloxy)-6-oxohexyl) 2-(((2- (dimethylamino)ethoxy)carbonyl)oxy)pentanedioate (Example 90)
Figure imgf000270_0001
To a solution of bis(6-(nonyloxy)-6-oxohexyl) 2-hydroxypentanedioate 69 (600 mg, 0.95 mmol) in 3 mL pyridine and 10 mL dichloromethane, was added p-nitrophenyl chloroformate (382 mg, 1.9 mmol), and the resulting mixture was stirred for 2 h. After TLC showed the complete reaction, 2-dimethylaminoethanol (253 mg, 2.85 mmol) was added, and the reaction mixture was stirred at room temperature overnight. MS showed the formation of desired product. The reaction mixture was washed by saturated sodium bicarbonate solution, and the combined organic layer was dried over sodium sulfate. After filtration and concentration, the crude was purified by column chromatography eluted with 0-10% methanol in dichloromethane to afford the desired product as clear oil (250 mg, 35%). 1H NMR (300 MHz, CDCl3) δ 4.89-5.02 (1H), 4.28-4.21 (m, 2H), 4.16 (t, 2H), 4.09-4.03 (m, 6H), 2.66-2.54 (m, 2H), 2.42-2.52 (m, 2H), 2.33-2.23 (m, 12H), 1.74-1.52 (m, 12H), 1.42- 1.26 (m, 28H), 0.89-0.85 (m, 6H). APCI-MS analysis: Calculated C40H73NO11 [M+H] = 744.5, Observed = 744.5. Example 91: Bis(6-(((Z)-non-2-en-1-yl)oxy)-6-oxohexyl) 2-(((2- (dimethylamino)ethoxy)carbonyl)oxy)pentanedioate Step 1: Synthesis of 6-((tert-Butyldiphenylsilyl)oxy)hexan-1-ol (70) A mixture of hexane-1,6-diol (8.8 g, 75 mmol), tert-butyldiphenylsilyl chloride (22.7 g, 82.5 mmol) and imidazole (6.1 g, 90 mmol) in 20 mL DMF and 50 mL dichloromethane was stirred at room temperature for 17 h. The reaction mixture was partitioned with ethyl acetate and saturated ammonium chloride solution, and the combined organic layer was dried over sodium sulfate. After concentration, the crude was purified by column chromatography using 0-25% ethyl acetate in hexane to give the desired product (13 g, 48%). Step 2: Synthesis of 6-((tert-Butyldiphenylsilyl)oxy)hexanoic acid (71) To a solution of 6-((tert-butyldiphenylsilyl)oxy)hexan-1-ol 70 (9.5 g, 26.6 mmol) in 150 mL acetone, was added Jones reagent until the orange color persisted, and the resulting mixture was stirred for 30 min. The excess Jones reagent was consumed by adding few drops of 2-propanol, then the blue solution was diluted with water (100 mL) and extracted with ethyl acetate (3 x 100 mL). The combined organic layers were washed with brine, dried and concentrated to give the desired product (8.9 g, 90%), which was used for the next step without further purification. Step 3: Synthesis of (Z)-Non-2-en-1-yl 6-((tert-butyldiphenylsilyl)oxy)hexanoate (72) A mixture of 6-((tert-butyldiphenylsilyl)oxy)hexanoic acid 71 (8.9 g, 24 mmol), (Z)- non-2-en-1-ol (3.0 g, 21.6 mmol), EDCI-HCl (8.3 g, 43.2 mmol) and DMAP (658 mg, 5.4 mmol) in 150 mL dichloromethane was stirred at room temperature for 16 h. After concentration, the crude was triturated with hexanes three times, and the solvent was removed under vacuum, then the crude was purified by column chromatography using 0-10% ethyl acetate in hexane to give the desired product (10.2 g, 99%). Step 4: Synthesis of (Z)-Non-2-en-1-yl 6-hydroxyhexanoate (73) To a solution of (Z)-non-2-en-1-yl 6-((tert-butyldiphenylsilyl)oxy)hexanoate 72 (10.1 g, 20.4 mmol) in 80 mL THF, was added pyridine (8 mL) followed by HF-pyridine complex (70%, 5 mL), and the mixture was stirred at room temperature for 20 h. The reaction was quenched by adding saturated sodium bicarbonate to pH 7, and then extracted with ethyl acetate (3 x 60 mL). The combined organic layers were dried and concentrated, and the crude was purified by column chromatography using 0-30% ethyl acetate in hexane to yield the desired product (4.7 g, 90%). Step 5: Synthesis of Bis(6-(((Z)-non-2-en-1-yl)oxy)-6-oxohexyl) 2-oxopentanedioate (74) A mixture of α-ketoglutaric acid (700 mg, 4.8 mmol), (Z)-non-2-en-1-yl 6-hydroxyhexanoate 73 (2.7 g, 10.5 mmol) and p-toluenesulfonic acid monohydrate (100 mg) in 80 mL toluene was heated to reflux for 2 h. The reaction mixture was concentrated under vacuum, and the crude was purified by column chromatography with 0-20% ethyl acetate in hexanes to give the desired product (300 mg, 10%) Step 6: Synthesis of Bis(6-(((Z)-non-2-en-1-yl)oxy)-6-oxohexyl) 2-hydroxypentanedioate (75) To a solution of bis(6-(((Z)-non-2-en-1-yl)oxy)-6-oxohexyl) 2-oxopentanedioate 74 (500 mg, 0.8 mmol) in 20 mL THF, was added sodium borohydride (46 mg, 1.2 mmol), and the reaction mixture was stirred at room temperature for 2 h. After cooled to 0°C, saturated ammonium chloride solution was added slowly, and the resulting mixture was extracted with ethyl acetate. The combined organic layer was washed with brine and dried over sodium sulfate. After concentration, the crude (500 mg) was used for the next step without purification. Step 7: Synthesis of Bis(6-(((Z)-non-2-en-1-yl)oxy)-6-oxohexyl) 2-(((2- (dimethylamino)ethoxy)carbonyl)oxy)pentanedioate (Example 91) To a solution of bis(6-(((Z)-non-2-en-1-yl)oxy)-6-oxohexyl) 2-hydroxypentanedioate 75 (500 mg, 0.8 mmol) in 3 mL pyridine and 10 mL dichloromethane, was added p- nitrophenyl chloroformate (322 mg, 1.6 mmol), and the resulting mixture was stirred for 2 h. After TLC showed the complete reaction, 2-dimethylaminoethanol (213 mg, 2.4 mmol) was added, and the reaction mixture was stirred at room temperature overnight. MS showed the formation of desired product. The reaction mixture was washed by saturated sodium bicarbonate solution, and the combined organic layer was dried over sodium sulfate. After filtration and concentration, the crude was purified by column chromatography eluted with 0- 10% methanol in dichloromethane to afford the desired product as clear oil (200 mg, 33%). 1H NMR (300 MHz, CDCl3) δ 5.68-5.49 (m, 4H), 4.96-4.92 (m, 1H), 4.61 (d, 4H), 4.27-4.21 (m, 2H), 4.19-4.12 (m, 2H), 4.09-4.03 (m, 2H), 2.63-2.55 (m, 2H), 2.49-2.42 (m, 2H), 2.34- 2.16 (m, 12H), 2.17-2.05 (m, 4H), 1.70-1.59 (m, 8H), 1.42-1.25 (m, 20H), 0.87 (t, 6H). APCI-MS analysis: Calculated C40H69NO11 [M+H] = 740.5, Observed = 740.4. Example 92: Bis(6-(decanoyloxy)hexyl) 2-(((2- (dimethylamino)ethoxy)carbonyl)oxy)pentanedioate Step 1: Synthesis of Bis(6-(decanoyloxy)hexyl) 2-oxopentanedioate (76) A mixture of α-ketoglutaric acid (536 mg, 3.7 mmol), 6-hydroxyhexyl decanoate (2.0 g, 7.3 mmol) and p-toluenesulfonic acid monohydrate (10 mg) in 80 mL toluene was heated to reflux for 2 h. The reaction mixture was concentrated under vacuum, and the crude was purified by column chromatography with 0-20% ethyl acetate in hexanes to give the desired product (1.3 g, 53%) Step 2: Synthesis of Bis(6-(decanoyloxy)hexyl) 2-hydroxypentanedioate (77) To a solution of bis(6-(decanoyloxy)hexyl) 2-oxopentanedioate 76 (1.3 g, 2 mmol) in 20 mL THF, was added sodium borohydride (115 mg, 3 mmol), and the reaction mixture was stirred at room temperature for 1 h. TLC showed complete reaction. After diluted with 100 mL dichloromethane, the solution was washed by water and brine. The combined organic layer was dried over sodium sulfate. After concentration, the crude was purified by column chromatography with 0-10% methanol in dichloromethane to afford the desired product as clear oil (800 mg, 60%). Step 3: Synthesis of Bis(6-(decanoyloxy)hexyl) 2-(((2- (dimethylamino)ethoxy)carbonyl)oxy)pentanedioate (Example 92) To a solution of bis(6-(decanoyloxy)hexyl) 2-hydroxypentanedioate 77 (400 mg, 0.61 mmol) in 2 mL pyridine and 10 mL dichloromethane, was added p-nitrophenyl chloroformate (367 mg, 1.83 mmol), and the resulting mixture was stirred for 3 h. After TLC showed the complete reaction, 2-dimethylaminoethanol (213 mg, 2.44 mmol) was added, and the reaction mixture was stirred at room temperature overnight. MS showed the formation of desired product. The reaction mixture was washed by saturated sodium bicarbonate solution, and the combined organic layer was dried over sodium sulfate. After filtration and concentration, the crude was purified by column chromatography eluted with 0-10% methanol in dichloromethane to afford the desired product as clear oil (130 mg, 27%). 1H NMR (300 MHz, CDCl3) δ 4.95 (dd, 1H), 4.27-4.21 (m, 2H), 4.19-4.12 (m, 2H), 4.10- 4.02 (m, 6H), 2.65-2.57 (m, 2H), 2.50-2.44 (m, 2H), 2.31-2.12 (m, 12H), 1.72-1.54 (m, 12H), 1.43-1.25 (m, 32H), 0.87 (t, 6H). APCI-MS analysis: Calculated C42H77NO11 [M+H] = 772.5, Observed = 772.5. Example 93: Bis(8-oxo-8-((2-propylnonyl)oxy)octyl) 2-(((2- (dimethylamino)ethoxy)carbonyl)oxy)-pentanedioate Step 1: Synthesis of 8-(Benzyloxy)octanoic acid (78) To a solution of 8-(benzyloxy)octan-1-ol (4.8 g, 20.3 mmol) in 60 mL acetone, was added Jones reagent until the orange color persisted, and the resulting mixture was stirred for 90 min. The excess Jones reagent was consumed by adding few drops of 2-propanol, then the blue solution was diluted with water (100 mL) and extracted with ethyl acetate (3 x 100 mL). The combined organic layers were washed with brine, dried and concentrated to give the desired product (4.8 g, quant.), which was used for the next step without further purification. Step 2: Synthesis of 2-Propyloctyl 8-(benzyloxy)octanoate (79) A mixture of 8-(benzyloxy)octanoic acid 78 (3.2 g, 12.8 mmol), 2-propylnonan-1-ol 52 (3.0 g, 16.1 mmol), EDCI-HCl (4.9 g, 25.6 mmol) and DMAP (1.56 g, 12.8 mmol) in 20 mL dichloromethane was stirred at room temperature for 16 h. After concentration, the crude was partitioned between EtOAc and saturated ammonium chloride solution, and the combined organic layer was dried over sodium sulfate. The solvent was removed under vacuum, and the crude was purified by column chromatography using 0-10% ethyl acetate in hexane to give the desired product (4.3 g, 80%). Step 3: Synthesis of 2-Propyloctyl 8-hydroxyoctanoate (80) A mixture of 2-propyloctyl 8-(benzyloxy)octanoate 79 (4.3 g, 10.3 mmol) and 20% palladium hydroxide on carbon (0.2 g) in 60 mL ethyl acetate was subjected to hydrogenolysis for 15 h. After filtration, the filtrate was concentrated, and the crude was purified by column chromatography using 0-100% ethyl acetate in hexane to afford the desired product (3.6 g, quant.). Step 4: Synthesis of Bis(8-oxo-8-((2-propylnonyl)oxy)octyl) 2-oxopentanedioate (81) A mixture of α-ketoglutaric acid (398 mg, 2.7 mmol), 2-propyloctyl 8- hydroxyoctanoate 80 (2.0 g, 6 mmol) and p-toluenesulfonic acid monohydrate (100 mg) in 80 mL toluene was heated to reflux for 30 min. The reaction mixture was concentrated under vacuum, and the crude was purified by column chromatography with 0-20% ethyl acetate in hexanes to give the desired product (800 mg, 38%) Step 5: Synthesis of Bis(8-oxo-8-((2-propylnonyl)oxy)octyl) 2-hydroxypentanedioate (82) To a solution of bis(8-oxo-8-((2-propylnonyl)oxy)octyl) 2-oxopentanedioate 81 (800 mg, 1.04 mmol) in 20 mL THF, was added sodium borohydride (63.5 mg, 1.67 mmol), and the reaction mixture was stirred at room temperature for 2 h. After cooled to 0°C, saturated ammonium chloride solution was added slowly, and the resulting mixture was extracted with ethyl acetate. The combined organic layer was washed with brine and dried over sodium sulfate. After concentration, the crude (700 mg) was used for the next step without purification. Step 6: Synthesis of Bis(8-oxo-8-((2-propylnonyl)oxy)octyl) 2-(((2- (dimethylamino)ethoxy)carbonyl)oxy)-pentanedioate (Example 93) To a solution of bis(8-oxo-8-((2-propylnonyl)oxy)octyl) 2-hydroxypentanedioate 82 (700 mg, 0.91 mmol) in 3 mL pyridine and 10 mL dichloromethane, was added p-nitrophenyl chloroformate (366 mg, 1.82 mmol), and the resulting mixture was stirred for 2 h. After TLC showed the complete reaction, 2-dimethylaminoethanol (243 mg, 2.73 mmol) was added, and the reaction mixture was stirred at room temperature overnight. MS showed the formation of desired product. The reaction mixture was washed by saturated sodium bicarbonate solution, and the combined organic layer was dried over sodium sulfate. After filtration and concentration, the crude was purified by column chromatography eluted with 0-10% methanol in dichloromethane to afford the desired product as clear oil (325 mg, 40%). 1H NMR (300 MHz, CDCl3) δ 4.94 (dd, 1H), 4.27-4.19 (m, 2H), 4.17-4.11 (m, 2H), 4.08- 4.03 (m, 2H), 3.96 (d, 4H), 2.66-2.53 (m, 2H), 2.49-2.43 (m, 2H), 2.33-2.14 (m, 10H), 1.62 (m, 8H), 1.43-1.25 (m, 48H), 0.91-0.85 (m, 12H). APCI-MS analysis: Calculated C50H93NO11 [M+H] = 884.6, Observed = 884.7. Example 94: Bis(8-oxo-8-(pentadecan-7-yloxy)octyl) 2-(((2- (dimethylamino)ethoxy)carbonyl)oxy)pentanedioate Step 1: Synthesis of Pentadecan-7-yl 8-(benzyloxy)octanoate (83) A mixture of 8-(benzyloxy)octanoic acid 78 (1.6 g, 6.4 mmol), pentadecan-7-ol 16 (1.8 g, 8 mmol), EDCI-HCl (1.9 g, 10 mmol) and DMAP (1.2 g, 14 mmol) in 20 mL dichloromethane was stirred at room temperature for 16 h. After concentration, the crude was partitioned between EtOAc and saturated ammonium chloride solution, and the combined organic layer was dried over sodium sulfate. The solvent was removed under vacuum, and the crude was purified by column chromatography using 0-10% ethyl acetate in hexane to give the desired product (2.3 g, 78%). Step 2: Synthesis of Pentadecan-7-yl 8-hydroxyoctanoate (84) A mixture of pentadecan-7-yl 8-(benzyloxy)octanoate 83 (2.3 g, 5 mmol) and 5% palladium on carbon (0.6 g) in 50 mL ethyl acetate was subjected to hydrogenolysis for 15 h. After filtration, the filtrate was concentrated, and the crude was purified by column chromatography using 0-100% ethyl acetate in hexane to afford the desired product (1.9 g, quant.). Step 3: Synthesis of bis(8-oxo-8-(pentadecan-7-yloxy)octyl) 2-oxopentanedioate (85) A mixture of α-ketoglutaric acid (350 mg, 2.4 mmol), pentadecan-7-yl 8- hydroxyoctanoate 84 (1.9 g, 5.1 mmol) and p-toluenesulfonic acid monohydrate (100 mg) in 80 mL toluene was heated to reflux for 30 min. The reaction mixture was concentrated under vacuum, and the crude was purified by column chromatography with 0-20% ethyl acetate in hexanes to give the desired product (1.2 g, 38%) Step 4: Synthesis of Bis(8-oxo-8-(pentadecan-7-yloxy)octyl) 2-hydroxypentanedioate (86) To a solution of bis(8-oxo-8-(pentadecan-7-yloxy)octyl) 2-oxopentanedioate 85 (1.2 g, 1.4 mmol) in 40 mL THF, was added sodium borohydride (80 mg, 2.1 mmol), and the reaction mixture was stirred at room temperature for 2 h. After cooled to 0°C, saturated ammonium chloride solution was added slowly, and the resulting mixture was extracted with ethyl acetate. The combined organic layer was washed with brine and dried over sodium sulfate. After concentration, the crude (800 mg) was used for the next step without purification. Step 5: Synthesis of Bis(8-oxo-8-(pentadecan-7-yloxy)octyl) 2-(((2- (dimethylamino)ethoxy)carbonyl)oxy)pentanedioate (Example 94) To a solution of bis(8-oxo-8-(pentadecan-7-yloxy)octyl) 2-hydroxypentanedioate 86 (800 mg, 0.9 mmol) in 3 mL pyridine and 10 mL dichloromethane, was added p-nitrophenyl chloroformate (377 mg, 1.87 mmol), and the resulting mixture was stirred for 2 h. After TLC showed the complete reaction, 2-dimethylaminoethanol (240 mg, 2.7 mmol) was added, and the reaction mixture was stirred at room temperature overnight. MS showed the formation of desired product. The reaction mixture was washed by saturated sodium bicarbonate solution, and the combined organic layer was dried over sodium sulfate. After filtration and concentration, the crude was purified by column chromatography eluted with 0-10% methanol in dichloromethane to afford the desired product as clear oil (275 mg, 31%). 1H NMR (300 MHz, CDCl3) δ 4.94 (dd, 1H), 4.86 (quint, 2H), 4.24 (m, 2H), 4.14 (t, 2H), 4.05 (t, 2H), 2.64-2.58 (m, 2H), 2.49-2.43 (m, 2H), 2.30-2.11 (m, 12H), 1.63-1.49 (m, 12H), 1.39-1.17 (m, 56H), 0.89-0.81 (m, 12H). APCI-MS analysis: Calculated C56H105NO11 [M+H] = 968.7, Observed = 968.7. Example 95: Bis(6-oxo-6-(pentadecan-7-yloxy)hexyl) 2-((3- (dimethylamino)propanoyl)oxy)pentanedioate Step 1: Synthesis of Pentadecan-7-yl 6-(benzyloxy)hexanoate (87) A mixture of 6-(benzyloxy)hexanoic acid 65 (8.4 g, 37.8 mmol), pentadecan-7-ol 16 (12.9 g, 56.7 mmol), EDCI-HCl (10.88 g, 56.7 mmol) and DMAP (1.22 g, 10 mmol) in 200 mL dichloromethane was stirred at room temperature for 16 h. After concentration, the crude was partitioned between EtOAc and saturated ammonium chloride solution, and the combined organic layer was dried over sodium sulfate. The solvent was removed under vacuum, and the crude was purified by column chromatography using 0-10% ethyl acetate in hexane to give the desired product (12.5 g, 76%). Step 2: Synthesis of Pentadecan-7-yl 6-hydroxyhexanoate (88) A mixture of pentadecan-7-yl 6-(benzyloxy)hexanoate 87 (12.5 g, 36.5 mmol) and 5% palladium on carbon (1.2 g) in 100 mL ethyl acetate was subjected to hydrogenolysis for 15 h. After filtration, the filtrate was concentrated, and the crude was purified by column chromatography using 0-100% ethyl acetate in hexane to afford the desired product (10.3 g, quant.). Step 3: Synthesis of Bis(6-oxo-6-(pentadecan-7-yloxy)hexyl) 2-oxopentanedioate (89) A mixture of α-ketoglutaric acid (1.74 g, 11.9 mmol), pentadecan-7-yl 6- hydroxyhexanoate 88 (10.3 g, 29.8 mmol) and p-toluenesulfonic acid monohydrate (500 mg) in 150 mL toluene was heated to reflux overnight. The reaction mixture was concentrated under vacuum, and the crude was purified by column chromatography with 0-20% ethyl acetate in hexanes to give the desired product (2.3 g, 24%) Step 4: Synthesis of Bis(6-oxo-6-(pentadecan-7-yloxy)hexyl) 2-hydroxypentanedioate (90) To a solution of bis(6-oxo-6-(pentadecan-7-yloxy)hexyl) 2-oxopentanedioate 89 (250 mg, 0.31 mmol) in 40 mL THF, was added sodium borohydride (18 mg, 0.47 mmol), and the reaction mixture was stirred at room temperature for 2 h. After cooled to 0°C, saturated ammonium chloride solution was added slowly, and the resulting mixture was extracted with ethyl acetate. The combined organic layer was washed with brine and dried over sodium sulfate. After concentration, the crude (170 mg) was used for the next step without purification. Step 5: Synthesis of Bis(6-oxo-6-(pentadecan-7-yloxy)hexyl) 2-((3- (dimethylamino)propanoyl)oxy)pentanedioate (Example 95) A mixture of bis(6-oxo-6-(pentadecan-7-yloxy)hexyl) 2-hydroxypentanedioate 90 (170 mg, 0.21 mmol), 3-(dimethylamino)propanoic acid hydrochloride (65 mg, 0.42 mmol), EDCI (82 mg, 0.42 mmol) and 4-dimethylaminopyridine (26 mg, 0.21 mmol) in 10 mL dichloromethane was stirred at room temperature overnight. The reaction mixture was diluted with dichloromethane and washed with water. The combined organic layer was dried over sodium sulfate. After filtration and concentration, the crude was purified by column chromatography with 0-10% methanol in dichloromethane to afford the desired product (170 mg, 90%). 1H NMR (300 MHz, CDCl3) δ 5.04 (dd, 1H), 4.86 (quint, 2H), 4.15-4.04 (m, 4H), 2.65-2.53 (m, 4H), 2.46-2.41 (m, 2H), 2.31-2.10 (m, 12H), 1.72-1.58 (m, 10H), 1.55-1.45 (m, 6H), 1.42-1.25 (m, 44H), 0.87 (t, 12H). APCI-MS analysis: Calculated C52H97NO10 [M+H] = 896.7, Observed = 896.1. Example 96: Bis(6-oxo-6-(pentadecan-7-yloxy)hexyl) 2-((4- (dimethylamino)butanoyl)oxy)pentanedioate A mixture of bis(6-oxo-6-(pentadecan-7-yloxy)hexyl) 2-hydroxypentanedioate 90 (300 mg, 0.37 mmol), 4-(dimethylamino)butanoic acid hydrochloride (126 mg, 0.75 mmol), EDCI (144 mg, 0.75 mmol) and 4-dimethylaminopyridine (46 mg, 0.37 mmol) in 10 mL dichloromethane was stirred at room temperature overnight. The reaction mixture was diluted with dichloromethane and washed with water. The combined organic layer was dried over sodium sulfate. After filtration and concentration, the crude was purified by column chromatography with 0-10% methanol in dichloromethane to afford the desired product (239 mg, 71%). 1H NMR (300 MHz, CDCl3) δ 5.01 (dd, 1H), 4.90-4.82 (m, 2H), 4.15-4.03 (m, 4H), 2.45- 2.40 (m, 4H), 2.32-2.26 (m, 6H), 2.21-2.08 (m, 6H), 1.85-1.76 (m, 2H), 1.69-1.60 (m, 8H), 1.56-1.45 (m, 6H), 1.41-1.25 (m, 48H), 0.87 (t, 12H). APCI-MS analysis: Calculated C53H99NO10 [M+H] = 910.7, Observed = 910.0. Example 97: Bis(5-((2-hexyldecanoyl)oxy)pentyl) 2-(((2- (dimethylamino)ethoxy)carbonyl)oxy)pentanedioate
Figure imgf000283_0001
Step 1: Synthesis of Bis(5-((2-hexyldecanoyl)oxy)pentyl) 2-oxopentanedioate (91) A mixture of α-ketoglutaric acid (730 mg, 5 mmol), 5-hydroxypentyl 2- hexyldecanoate 32 (3.6 g, 10.5 mmol) and p-toluenesulfonic acid monohydrate (100 mg) in 120 mL toluene was heated to reflux for 2 h. The reaction mixture was concentrated under vacuum, and the crude was purified by column chromatography with 0-20% ethyl acetate in hexanes to give the desired product (3.1 g, 78%). Step 2: Synthesis of Bis(5-((2-hexyldecanoyl)oxy)pentyl) 2-hydroxypentanedioate (92) To a solution of bis(5-((2-hexyldecanoyl)oxy)pentyl) 2-oxopentanedioate 91 (3.1 g, 3.9 mmol) in 60 mL THF, was added sodium borohydride (222 mg, 5.8 mmol), and the reaction mixture was stirred at room temperature for 2 h. After cooled to 0°C, saturated ammonium chloride solution was added slowly, and the resulting mixture was extracted with ethyl acetate. The combined organic layer was washed with brine and dried over sodium sulfate. After concentration, the crude was purified by column chromatography using 0-100% ethyl acetate in hexanes to give the desired product as clear oil (1.5 g, 48%). Step 3: Synthesis of Bis(5-((2-hexyldecanoyl)oxy)pentyl) 2-(((2- (dimethylamino)ethoxy)carbonyl)oxy)pentanedioate (Example 97)
Figure imgf000284_0001
To a solution of bis(5-((2-hexyldecanoyl)oxy)pentyl) 2-hydroxypentanedioate 92 (1.5 g, 1.9 mmol) in 5 mL pyridine and 10 mL dichloromethane, was added p-nitrophenyl chloroformate (1.13 g, 5.6 mmol), and the resulting mixture was stirred for 2 h. After TLC showed the complete reaction, 2-dimethylaminoethanol (677 mg, 7.6 mmol) was added, and the reaction mixture was stirred at room temperature overnight. MS showed the formation of desired product. The reaction mixture was washed by saturated sodium bicarbonate solution, and the combined organic layer was dried over sodium sulfate. After filtration and concentration, the crude was purified by column chromatography eluted with 0-10% methanol in dichloromethane to afford the desired product as clear oil (530 mg, 30%). 1H NMR (300 MHz, CDCl3) δ 4.95 (dd, 1H), 4.27-4.22 (m, 2H), 4.20-4.14 (m, 2H), 4.09- 4.04 (m, 6H), 2.68-2.55 (m, 2H), 2.50-2.44 (m, 2H), 2.33-2.11 (m, 8H), 1.74-1.54 (m, 8H), 1.43-1.38 (m, 4H), 1.24 (m, 40H), 0.86 (t, 12H). APCI-MS analysis: Calculated C52H87NO11 [M+H] = 912.7, Observed = 912.7. Example 98: Bis(7-((8-methylnonanoyl)oxy)heptyl) 2-(((2- (dimethylamino)ethoxy)carbonyl)oxy)pentanedioate Step 1: Synthesis of Bis(7-((8-methylnonanoyl)oxy)heptyl) 2-oxopentanedioate (93) A mixture of α-ketoglutaric acid (607 mg, 4.15 mmol), 7-hydroxyheptyl 8- methylnonanoate 49 (2.5 g, 8.73 mmol) and p-toluenesulfonic acid monohydrate (10 mg) in 100 mL toluene was heated to reflux for 2 h. The reaction mixture was concentrated under vacuum, and the crude was purified by column chromatography with 0-20% ethyl acetate in hexanes to give the desired product (2.3 g, 81%). Step 2: Synthesis of Bis(7-((8-methylnonanoyl)oxy)heptyl) 2-hydroxypentanedioate (94)
Figure imgf000285_0001
To a solution of bis(7-((8-methylnonanoyl)oxy)heptyl) 2-oxopentanedioate 93 (2.3 g, 3.3 mmol) in 60 mL THF, was added sodium borohydride (188 mg, 4.9 mmol), and the reaction mixture was stirred at room temperature for 2 h. After cooled to 0°C, saturated ammonium chloride solution was added slowly, and the resulting mixture was extracted with ethyl acetate. The combined organic layer was washed with brine and dried over sodium sulfate. After concentration, the crude was purified by column chromatography using 0-100% ethyl acetate in hexanes to give the desired product as clear oil (500 mg, 22%). Step 3: Synthesis of Bis(7-((8-methylnonanoyl)oxy)heptyl) 2-(((2- (dimethylamino)ethoxy)carbonyl)oxy)pentanedioate (Example 98) To a solution of bis(7-((8-methylnonanoyl)oxy)heptyl) 2-hydroxypentanedioate 94 (500 mg, 0.73 mmol) in 3 mL pyridine and 10 mL dichloromethane, was added p-nitrophenyl chloroformate (293 mg, 1.46 mmol), and the resulting mixture was stirred for 2 h. After TLC showed the complete reaction, 2-dimethylaminoethanol (195 mg, 2.19 mmol) was added, and the reaction mixture was stirred at room temperature overnight. MS showed the formation of desired product. The reaction mixture was washed by saturated sodium bicarbonate solution, and the combined organic layer was dried over sodium sulfate. After filtration and concentration, the crude was purified by column chromatography eluted with 0-10% methanol in dichloromethane to afford the desired product as clear oil (396 mg, 67%). 1H NMR (300 MHz, CDCl3) δ 4.95 (dd, 1H), 4.29-4.20 (m, 2H), 4.15 (t, 2H), 4.08-4.00 (m, 6H), 2.63-2.55 (m, 2H), 2.50-2.43 (m, 2H), 2.31-2.11 (m, 12H), 1.64-1.44 (m, 16H), 1.44- 1.27 (m, 24H), 1.17-1.10 (m, 2H), 0.85 (d, 12H). APCI-MS analysis: Calculated C44H81NO11 [M+H] = 800.5, Observed = 800.5. Example 99: Bis(8-(((Z)-non-2-en-1-yl)oxy)-8-oxooctyl) 2-(((2- (dimethylamino)ethoxy)carbonyl)oxy)pentanedioate Step 1: Synthesis of 8-((tert-Butyldiphenylsilyl)oxy)octan-1-ol (95) A mixture of octane-1,8-diol (7.3 g, 50 mmol), tert-butyldiphenylsilyl chloride (15.1 g, 55 mmol) and imidazole (4.08 g, 60 mmol) in 20 mL DMF and 50 mL dichloromethane was stirred at room temperature for 17 h. The reaction mixture was partitioned with ethyl acetate and saturated ammonium chloride solution, and the combined organic layer was dried over sodium sulfate. After concentration, the crude was purified by column chromatography using 0-25% ethyl acetate in hexane to give the desired product (10.0 g, 52%). Step 2: Synthesis of 8-((tert-Butyldiphenylsilyl)oxy)octanoic acid (96) To a solution of 8-((tert-butyldiphenylsilyl)oxy)octan-1-ol 95 (5 g, 13 mmol) in 100 mL acetone, was added Jones reagent until the orange color persisted, and the resulting mixture was stirred for 30 min. The excess Jones reagent was consumed by adding few drops of 2-propanol, then the blue solution was diluted with water (100 mL) and extracted with ethyl acetate (3 x 100 mL). The combined organic layers were washed with brine, dried and concentrated to give the desired product (5 g, quant.), which was used for the next step without further purification. Step 3: Synthesis of (Z)-Non-2-en-1-yl 8-((tert-butyldiphenylsilyl)oxy)octanoate (97) A mixture of 8-((tert-butyldiphenylsilyl)oxy)octanoic acid 96 (5 g, 12.6 mmol), (Z)- non-2-en-1-ol (1.69 g, 11.3 mmol), EDCI-HCl (3.62 g, 18.9 mmol) and DMAP (307 mg, 2.5 mmol) in 100 mL dichloromethane was stirred at room temperature for 16 h. After concentration, the crude was triturated with hexanes three times, and the solvent was removed under vacuum, then the crude was purified by column chromatography using 0-10% ethyl acetate in hexane to give the desired product (5.2 g, 88%). Step 4: Synthesis of (Z)-Non-2-en-1-yl 8-hydroxyoctanoate (98) To a solution of (Z)-non-2-en-1-yl 8-((tert-butyldiphenylsilyl)oxy)octanoate 97 (5.2 g, 10 mmol) in 20 mL THF, was added pyridine (5 mL) followed by HF-pyridine complex (70%, 3 mL), and the mixture was stirred at room temperature for 20 h. The reaction was quenched by adding saturated sodium bicarbonate to pH 7, and then extracted with ethyl acetate (3 x 60 mL). The combined organic layers were dried and concentrated, and the crude was purified by column chromatography using 0-30% ethyl acetate in hexane to yield the desired product (2.7 g, 95%). Step 5: Synthesis of Bis(8-(((Z)-non-2-en-1-yl)oxy)-8-oxooctyl) 2-oxopentanedioate (99) A mixture of α-ketoglutaric acid (642 mg, 4.4 mmol), (Z)-non-2-en-1-yl 8- hydroxyoctanoate 98 (2.7 g, 9.4 mmol) and p-toluenesulfonic acid monohydrate (100 mg) in 80 mL toluene was heated to reflux for 2 h. The reaction mixture was concentrated under vacuum, and the crude was purified by column chromatography with 0-20% ethyl acetate in hexanes to give the desired product (1.3 g, 43%) Step 6: Synthesis of Bis(8-(((Z)-non-2-en-1-yl)oxy)-8-oxooctyl) 2-hydroxypentanedioate (100) To a solution of bis(8-(((Z)-non-2-en-1-yl)oxy)-8-oxooctyl) 2-oxopentanedioate 99 (1.3 g, 1.9 mmol) in 40 mL THF, was added sodium borohydride (109 mg, 2.8 mmol), and the reaction mixture was stirred at room temperature for 2 h. After cooled to 0°C, saturated ammonium chloride solution was added slowly, and the resulting mixture was extracted with ethyl acetate. The combined organic layer was washed with brine and dried over sodium sulfate. After concentration, the crude (1.3 g) was used for the next step without purification. Step 7: Synthesis of Bis(8-(((Z)-non-2-en-1-yl)oxy)-8-oxooctyl) 2-(((2- (dimethylamino)ethoxy)carbonyl)oxy)pentanedioate (Example 99) To a solution of bis(6-(((Z)-non-2-en-1-yl)oxy)-6-oxohexyl) 2-hydroxypentanedioate 100 (3.52 g, 1.76 mmol) in 3 mL pyridine and 10 mL dichloromethane, was added p- nitrophenyl chloroformate (708 mg, 3.52 mmol), and the resulting mixture was stirred for 2 h. After TLC showed the complete reaction, 2-dimethylaminoethanol 10 (470 mg, 5.28 mmol) was added, and the reaction mixture was stirred at room temperature overnight. MS showed the formation of desired product. The reaction mixture was washed by saturated sodium bicarbonate solution, and the combined organic layer was dried over sodium sulfate. After filtration and concentration, the crude was purified by column chromatography eluted with 0- 10% methanol in dichloromethane to afford the desired product as clear oil (605 mg, 43%). 1H NMR (300 MHz, CDCl3) δ 5.68-5.60 (m, 2H), 5.55-5.47 (m, 2H), 4.94 (dd, 1H), 4.61 (d, 4H), 4.27-4.03 (m, 6H), 2.63-2.55 (m, 2H), 2.49-2.43 (m, 2H), 2.32-2.16 (m, 10H), 2.16-2.05 (m, 4H), 1.76-1.61 (m, 6H), 1.32-1.25 (m, 32H), 0.93-0.82 (m, 6H). APCI-MS analysis: Calculated C44H77NO11 [M+H] = 796.5, Observed = 796.6. Example 100: Bis(6-oxo-6-(pentadecan-7-yloxy)hexyl) 2-(((2- (dimethylamino)ethoxy)carbonyl)oxy)pentanedioate To a solution of bis(6-oxo-6-(pentadecan-7-yloxy)hexyl) 2-hydroxypentanedioate 90 (280 mg, 0.35 mmol) in 3 mL pyridine and 10 mL dichloromethane, was added p-nitrophenyl chloroformate (140 mg, 0.7 mmol), and the resulting mixture was stirred for 2 h. After TLC showed the complete reaction, 2-dimethylaminoethanol (95 mg, 1.05 mmol) was added, and the reaction mixture was stirred at room temperature overnight. MS showed the formation of desired product. The reaction mixture was washed by saturated sodium bicarbonate solution, and the combined organic layer was dried over sodium sulfate. After filtration and concentration, the crude was purified by column chromatography eluted with 0-10% methanol in dichloromethane to afford the desired product as clear oil (205 mg, 64%). 1H NMR (300 MHz, CDCl3) δ 4.94 (dd, 1H), 4.86 (quint, 2H), 4.24 (m, 2H), 4.15 (t, 2H), 4.09-4.04 (m, 2H), 2.60 (m, 2H), 2.49-2.43 (m, 2H), 2.32-2.11 (m, 10H), 1.70-1.59 (m, 12H), 1.50 (m, 6H), 1.43-1.25 (m, 44H), 0.87 (t, 12H). APCI-MS analysis: Calculated C52H97NO11 [M+H] = 912.7, Observed = 912.0. Example 101: Bis(6-oxo-6-(pentadecan-7-yloxy)hexyl) 2-(((3- (dimethylamino)propoxy)carbonyl)oxy)pentanedioate To a solution of bis(6-oxo-6-(pentadecan-7-yloxy)hexyl) 2-hydroxypentanedioate 90 (250 mg, 0.31 mmol) in 3 mL pyridine and 10 mL dichloromethane, was added p-nitrophenyl chloroformate (124 mg, 0.62 mmol), and the resulting mixture was stirred for 2 h. After TLC showed the complete reaction, 3-dimethylaminopropanol (96 mg, 0.93 mmol) was added, and the reaction mixture was stirred at room temperature overnight. MS showed the formation of desired product. The reaction mixture was washed by saturated sodium bicarbonate solution, and the combined organic layer was dried over sodium sulfate. After filtration and concentration, the crude was purified by column chromatography eluted with 0-10% methanol in dichloromethane to afford the desired product as clear oil (100 mg, 34%). 1H NMR (300 MHz, CDCl3) δ 4.94 (dd, 1H), 4.86 (t, 2H), 4.23-4.19 (m, 2H), 4.19-4.13 (m, 2H), 4.07 (t, 2H), 2.49-2.12 (m, 16H), 1.86 (quint, 2H), 1.70-1.59 (m, 8H), 1.50 (m, 4H), 1.42-1.25 (m, 48H), 0.89-0.79 (m, 12H). APCI-MS analysis: Calculated C53H99NO11 [M+H] = 926.7, Observed = 926.0. Example 102: Bis(6-oxo-6-(pentadecan-7-yloxy)hexyl) 2-((5- (dimethylamino)pentanoyl)oxy)pentanedioate A mixture of bis(6-oxo-6-(pentadecan-7-yloxy)hexyl) 2-hydroxypentanedioate 90 (300 mg, 0.37 mmol), 5-(dimethylamino)pentanoic acid hydrochloride (136 mg, 0.75 mmol), EDCI (144 mg, 0.75 mmol) and 4-dimethylaminopyridine (46 mg, 0.37 mmol) in 10 mL dichloromethane was stirred at room temperature overnight. The reaction mixture was diluted with dichloromethane and washed with water. The combined organic layer was dried over sodium sulfate. After filtration and concentration, the crude was purified by column chromatography with 0-10% methanol in dichloromethane to afford the desired product (255 mg, 74%). 1H NMR (300 MHz, CDCl3) δ 5.01 (dd, 1H), 4.87 (quint, 2H), 4.14-4.04 (m, 4H), 2.45-2.38 (m, 4H), 2.32-2.24 (m, 4H), 2.21-2.10 (m, 8H), 1.72-1.60 (m, 10H), 1.56-1.49 (m, 8H), 1.43- 1.25 (m, 48H), 0.87 (t, 12H). APCI-MS analysis: Calculated C54H101NO10 [M+H] = 924.7, Observed = 924.0. Example 103: Bis(5-(decanoyloxy)pentyl) 2-(((2- (dimethylamino)ethoxy)carbonyl)oxy)pentanedioate Step 1: Synthesis of Bis(5-(decanoyloxy)pentyl) 2-oxopentanedioate (101) A mixture of α-ketoglutaric acid (514 mg, 3.5 mmol), 5-hydroxypentyl decanoate 60 (1.82 g, 7.05 mmol) and p-toluenesulfonic acid monohydrate (100 mg) in 80 mL toluene was heated to reflux for 2 h. The reaction mixture was concentrated under vacuum, and the crude was purified by column chromatography with 0-20% ethyl acetate in hexanes to give the desired product (1.0 g, 45%). Step 2: Synthesis of Bis(5-(decanoyloxy)pentyl) 2-hydroxypentanedioate (102) To a solution of bis(5-(decanoyloxy)pentyl) 2-oxopentanedioate 101 (1.0 g, 1.59 mmol) in 20 mL THF, was added sodium borohydride (91 mg, 2.4 mmol), and the reaction mixture was stirred at room temperature for 2 h. After cooled to 0°C, saturated ammonium chloride solution was added slowly, and the resulting mixture was extracted with ethyl acetate. The combined organic layer was washed with brine and dried over sodium sulfate. After concentration, the crude was purified by column chromatography using 0-10% methanol in dichloromethane to give the desired product (500 mg, 50%). Step 3: Synthesis of Bis(5-(decanoyloxy)pentyl) 2-(((2- (dimethylamino)ethoxy)carbonyl)oxy)pentanedioate (Example 103) To a solution of bis(5-(decanoyloxy)pentyl) 2-hydroxypentanedioate 102 (500 mg, 0.8 mmol) in 2 mL pyridine and 10 mL dichloromethane, was added p-nitrophenyl chloroformate (480 mg, 2.4 mmol), and the resulting mixture was stirred for 2 h. After TLC showed the complete reaction, 2-dimethylaminoethanol (284 mg, 3.2 mmol) was added, and the reaction mixture was stirred at room temperature overnight. MS showed the formation of desired product. The reaction mixture was washed by saturated sodium bicarbonate solution, and the combined organic layer was dried over sodium sulfate. After filtration and concentration, the crude was purified by column chromatography eluted with 0-10% methanol in dichloromethane to afford the desired product as clear oil (300 mg, 50%). 1H NMR (300 MHz, CDCl3) δ 4.95 (dd, 1H), 4.24 (td, 2H), 4.20-4.13 (m, 2H), 4.10-4.03 (m, 6H), 2.68-2.55 (m, 2H), 2.50-2.44 (m, 2H), 2.31-2.11 (m, 10H), 1.71-1.58 (m, 12H), 1.46- 1.25 (m, 32H), 0.87 (t, 6H). APCI-MS analysis: Calculated C40H73NO11 [M+H] = 744.5, Observed = 744.5. Example 104: Bis(7-((2-hexyldecanoyl)oxy)heptyl) 2-(((2- (dimethylamino)ethoxy)carbonyl)oxy)pentanedioate
Figure imgf000292_0001
Step 1: Synthesis of Bis(7-((2-hexyldecanoyl)oxy)heptyl) 2-oxopentanedioate (103) A mixture of α-ketoglutaric acid (708 mg, 4.85 mmol), 7-Hydroxyheptyl 2- hexyldecanoate 33 (3.6 g, 9.7 mmol) and p-toluenesulfonic acid monohydrate (10 mg) in 80 mL toluene was heated to reflux overnight. The reaction mixture was concentrated under vacuum, and the crude was purified by column chromatography with 0-20% ethyl acetate in hexanes to give the desired product (1.8 g, 43%). Step 2: Synthesis of Bis(7-((2-hexyldecanoyl)oxy)heptyl) 2-hydroxypentanedioate (104) To a solution of bis(7-((2-hexyldecanoyl)oxy)heptyl) 2-oxopentanedioate 103 (1.8 g, 2.1 mmol) in 20 mL THF, was added sodium borohydride (120 mg, 3.2 mmol), and the reaction mixture was stirred at room temperature for 2 h. After cooled to 0°C, saturated ammonium chloride solution was added slowly, and the resulting mixture was extracted with ethyl acetate. The combined organic layer was washed with brine and dried over sodium sulfate. After concentration, the crude was purified by column chromatography using 0-10% methanol in dichloromethane to give the desired product (600 mg, 33%). Step 3: Synthesis of Bis(7-((2-hexyldecanoyl)oxy)heptyl) 2-(((2- (dimethylamino)ethoxy)carbonyl)oxy)pentanedioate (Example 104) To a solution of bis(7-((2-hexyldecanoyl)oxy)heptyl) 2-hydroxypentanedioate 104 (600 mg, 0.7 mmol) in 2 mL pyridine and 10 mL dichloromethane, was added p-nitrophenyl chloroformate (422 mg, 2.1 mmol), and the resulting mixture was stirred for 2 h. After TLC showed the complete reaction, 2-dimethylaminoethanol (249 mg, 2.8 mmol) was added, and the reaction mixture was stirred at room temperature overnight. MS showed the formation of desired product. The reaction mixture was washed by saturated sodium bicarbonate solution, and the combined organic layer was dried over sodium sulfate. After filtration and concentration, the crude was purified by column chromatography eluted with 0-10% methanol in dichloromethane to afford the desired product as clear oil (115 mg, 17%). 1H NMR (300 MHz, CDCl3) δ 4.94 (dd, 1H), 4.24 (td, 2H), 4.15 (t, 2H), 4.08-4.03 (m, 6H), 2.60 (m, 2H), 2.50-2.43 (m, 2H), 2.35-2.11 (m, 10H), 1.70-1.54 (m, 12H), 1.47-1.24 (m, 56H), 0.86 (t, 12H). APCI-MS analysis: Calculated C56H105NO11 [M+H] = 968.7, Observed = 968.7. Example 105: bis(8-oxo-8-(pentadecan-7-yloxy)octyl) 2-(((2-((2- hydroxyethyl)(methyl)amino)ethoxy)carbonyl)oxy)pentanedioate Step 1: Synthesis of 8-(Benzyloxy)octan-1-ol (105) To a solution of 1,8-octanediol (43.8 g, 0.3 mole) in 100 mL DMF, was added NaH (60% in mineral oil, 12 g, 0.3 mole) at 0°C. After stirring for 30 min, a solution of benzylbromide (35 mL, 0.3 mole) in 20 mL DMF was added dropwise, and then reaction mixture was stirred at room temperature overnight. Water was added to quench the reaction, the mixture was extracted with EtOAc. The combined organic layer was washed with water and brine, and then dried over sodium sulfate. After filtration and concentration, the crude was purified by column chromatography using 0-60% ethyl acetate in hexane to give the desired product (35.5 g, 50%). Step 2: Synthesis of Bis(8-(benzyloxy)octyl) 2-oxopentanedioate (106) A mixture of α-ketoglutaric acid (5.4 g, 37 mmol), 8-(benzyloxy)octan-1-ol 105 (17 g, 74 mmol) and p-toluenesulfonic acid monohydrate (340 mg, 1.8 mmol) in 25 mL toluene was heated to reflux with Dean-Stark for 1 h. After cooled to room temperature, the mixture was diluted with hexanes and washed by saturated sodium bicarbonate solution. The aqueous layer was extracted with ethyl acetate, and the combined organic layer was dried over sodium sulfate. After filtration and concentration, the crude was purified by column chromatography with 0-25% ethyl acetate in hexanes to give bis(8-(benzyloxy)octyl) 2-oxopentanedioate as colorless oil (14.0 g, 65%). Step 3: Synthesis of Bis(8-(benzyloxy)octyl) 2-hydroxypentanedioate (107) A solution of bis(8-(benzyloxy)octyl) 2-oxopentanedioate 106 (10.8 g, 18.5 mmol) in 30 mL THF was cooled to 0°C, then sodium borohydride (701 mg, 18.5 mmol) was added in portions in 5 min, and the reaction mixture was stirred at room temperature for 1 h. Saturated ammonium chloride solution was added slowly, and the resulting mixture was extracted with ethyl acetate. The combined organic layer was washed with brine and dried over sodium sulfate. After concentration, the crude was used for the next step without purification. Step 4: Synthesis of Bis(8-(benzyloxy)octyl) 2-((tert-butyldiphenylsilyl)oxy)pentanedioate (108) To a stirred solution of crude bis(8-(benzyloxy)octyl) 2-hydroxypentanedioate 106 (10.8 g, 18.5 mmol) and imidazole (1.64 g, 24.1 mmol) in 30 mL dichloromethane, was added tert-butylchlorodiphenylsilane (5.08 g, 18.5 mmol), and the mixture was stirred room temperature for 18 h. The reaction mixture was quenched by saturated sodium bicarbonate solution and extracted with dichloromethane. The combined organic layer was dried and concentrated, and the crude was purified by column chromatography using 0-20% ethyl acetate in hexanes to give bis(8-(benzyloxy)octyl) 2-((tert- butyldiphenylsilyl)oxy)pentanedioate as colorless oil (9.8 g, 64%). Step 5: Synthesis of Bis(8-hydroxyoctyl) 2-((tert-butyldiphenylsilyl)oxy)pentanedioate (109) A mixture of bis(8-(benzyloxy)octyl) 2-((tert-butyldiphenylsilyl)oxy)pentanedioate 108 (3.8 g, 4.8 mmol) and 20% palladium hydroxide on carbon (300 mg) in 40 mL ethyl acetate was subjected to hydrogenolysis at 35 psi for 15 h. After filtration and concentration, the crude was purified by column chromatography using 0-100% ethyl acetate in hexane to afford bis(8-hydroxyoctyl) 2-((tert-butyldiphenylsilyl)oxy)pentanedioate as pale yellow oil (2.1 g, 71%). Step 6: Synthesis of 8,8'-((2-((tert-Butyldiphenylsilyl)oxy)pentanedioyl)bis(oxy))dioctanoic acid (110) To a solution of bis(8-hydroxyoctyl) 2-((tert-butyldiphenylsilyl)oxy)pentanedioate 109 (4.2 g, 6.5 mmol) in 20 mL acetone, was added Jones reagent until the orange color persisted, and the resulting mixture was stirred for 3 h. The excess Jones reagent was consumed by adding 2-propanol, then the blue solution was diluted with water and extracted with ethyl acetate. The combined organic layers were washed with brine, dried and concentrated to give the desired product (4.2 g, 96%), which was used for the next step without further purification. Step 7: Synthesis of Bis(8-oxo-8-(pentadecan-7-yloxy)octyl) 2-((tert- butyldiphenylsilyl)oxy)pentanedioate (111) A mixture of 8,8'-((2-((tert-butyldiphenylsilyl)oxy)pentanedioyl)bis(oxy))dioctanoic acid 110 (4.2 g, 6.3 mmol), pentadecan-7-ol 16 (2.9 g, 13 mmol), EDCI (6.0 g, 31 mmol) and DMAP (0.76 g, 6.3 mmol) in 30 mL dichloromethane was stirred at room temperature for 48 h. After concentration, the crude was partitioned between EtOAc and saturated ammonium chloride solution, and the combined organic layer was dried over sodium sulfate. The solvent was removed under vacuum, and the crude was purified by column chromatography using 0- 10% ethyl acetate in hexane to give bis(8-oxo-8-(pentadecan-7-yloxy)octyl) 2-((tert- butyldiphenylsilyl)oxy)pentanedioate mixed with pentadecan-7-ol (4.2 g, 61%). Step 8: Synthesis of Bis(8-oxo-8-(pentadecan-7-yloxy)octyl) 2-hydroxypentanedioate (112) To a solution of bis(8-oxo-8-(pentadecan-7-yloxy)octyl) 2-((tert- butyldiphenylsilyl)oxy)pentanedioate 111 (4.2 g, 3.8 mmol) in a mixture of THF (15 mL) and pyridine (6.0 mL) in a Teflon flask, was added HF-pyridine complex (70wt%, 2.5 mL) at 0°C, and the resulting mixture was stirred at room temperature for 18 h. The reaction mixture was carefully quenched with saturated sodium bicarbonate solution and extracted with ethyl acetate. The combined organic layer was dried and concentrated, and the crude was purified by column chromatography using 0-30% ethyl acetate in hexanes to give bis(8-oxo-8- (pentadecan-7-yloxy)octyl) 2-hydroxypentanedioate as colorless oil (3.2 g, 97%). Step 9: Synthesis of Bis(8-oxo-8-(pentadecan-7-yloxy)octyl) 2-(((2-((2- hydroxyethyl)(methyl)amino)ethoxy)carbonyl)oxy)pentanedioate (Example 105) To a solution of bis(8-oxo-8-(pentadecan-7-yloxy)octyl) 2-hydroxypentanedioate 112 (220 mg, 0.26 mmol) in 5 mL pyridine and 10 mL dichloromethane, was added p-nitrophenyl chloroformate (157 mg, 0.78 mmol), and the resulting mixture was stirred for 2 h. After TLC showed the complete reaction, 2,2'-(methylazanediyl)bis(ethan-1-ol) (1.6 g, 13.6 mmol) and Diisopropylethylamine(1 mL) were added, and the reaction mixture was stirred at room temperature overnight. MS showed the formation of desired product. The reaction mixture was washed by saturated sodium bicarbonate solution, and the combined organic layer was dried over sodium sulfate. After filtration and concentration, the crude was purified by column chromatography eluted with 0-10% methanol in dichloromethane to afford bis(8-oxo- 8-(pentadecan-7-yloxy)octyl) 2-(((2-((2- hydroxyethyl)(methyl)amino)ethoxy)carbonyl)oxy)pentanedioate as clear oil (206 mg, 68%). 1H NMR (400 MHz, CDCl3) δ 4.97-4.90 (m, 1H), 4.85 (quint, 2H), 4.33-4.18 (m, 2H), 4.14 (t, 2H), 4.05 (t, 2H), 3.62-3.53 (m, 2H), 2.74 (t, 2H), 2.59 (t, 2H), 2.52-2.37 (m, 2H), 2.32 (s, 3H), 2.30-2.20 (m, 4H), 2.20-2.05 (m, 1H), 1.71-1.54 (m, 8H), 1.53-1.43 (m, 8H), 1.39-1.13 (m, 52H), 0.86 (t, 12H). APCI-MS analysis: Calculated C57H107NO12 [M+H] = 998.7, Observed = 998.8. HPLC-ELSD: tR = 8.389 min (method 2). Example 106: bis(8-oxo-8-(pentadecan-7-yloxy)octyl) 2-(((2-((3- hydroxypropyl)(methyl)amino)ethoxy)carbonyl)oxy)pentanedioate Step 1: Synthesis of 2-((3-((tert-butyldimethylsilyl)oxy)propyl)(methyl)amino)ethan-1-ol (113) In a seal tube, a mixture of (3-bromopropoxy)(tert-butyl)dimethylsilane (8.36 g, 33.0 mmol), 2-(methylamino)ethan-1-ol (2.25 g, 30.0 mmol), diisopropylethylamine (13 mL, 75 mmol) and potassium iodide (498 mg, 3 mmol) in 20 mL ethanol was heated at 80°C overnight. After cooled to room temperature, the mixture was filtered, and the filtrate was concentrated under vacuum. The residue was triturated with hexanes, and the solution was washed with water to get the desired product (4.7 g, 63%), which was used for the next step without further purification. Step 2: Synthesis of bis(8-oxo-8-(pentadecan-7-yloxy)octyl) 2-((5,10,10,11,11-pentamethyl- 2,9-dioxa-5-aza-10-siladodecanoyl)oxy)pentanedioate (114) To a solution of bis(8-oxo-8-(pentadecan-7-yloxy)octyl) 2-hydroxypentanedioate 112 (280 mg, 0.33 mmol) in 2 mL pyridine and 8 mL dichloromethane, was added p-nitrophenyl chloroformate (198 mg, 0.98 mmol), and the resulting mixture was stirred for 30 min. After TLC showed the complete reaction, 2-((3-((tert- butyldimethylsilyl)oxy)propyl)(methyl)amino)ethan-1-ol 113 (1.35 g, 5.46 mmol) and Diisopropylethylamine(1.5 mL) were added, and the reaction mixture was stirred at 50°C in a seal tube for 48 h. MS showed the formation of desired product. The reaction mixture was concentrated and partitioned between hexanes and water, and the combined organic layer was washed with sodium carbonate solution. After concentration, the crude (370 mg) was used for the next step without further purification. Step 3: Synthesis of bis(8-oxo-8-(pentadecan-7-yloxy)octyl) 2-(((2-((3- hydroxypropyl)(methyl)amino)ethoxy)carbonyl)oxy)pentanedioate (Example 106) To a solution of bis(8-oxo-8-(pentadecan-7-yloxy)octyl) 2-((5,10,10,11,11- pentamethyl-2,9-dioxa-5-aza-10-siladodecanoyl)oxy)pentanedioate 114 (370 mg, 0.33 mmol) and pyridine (2 mL) in 8 mL THF, was slowly added HF-pyridine complex (70wt%, 1 mL), and the resulting mixture was stirred at room temperature overnight. The mixture was carefully quenched with NaHCO3 solution and extracted with ethyl acetate. The combined organic layer was dried and concentrated, and the crude was purified by column chromatography using 0-100% ethyl acetate in hexanes followed by 5% methanol in ethyl acetate to give bis(8-oxo-8-(pentadecan-7-yloxy)octyl) 2-(((2-((3- hydroxypropyl)(methyl)amino)ethoxy)carbonyl)oxy)pentanedioate as pale yellow oil (130 mg, 39%). 1H NMR (400 MHz, CDCl3) δ 4.95 (dd, 1H), 4.85 (quint, 2H), 4.34-4.19 (m, 2H), 4.14 (t, 2H), 4.05 (t, 2H), 3.76 (t, 2H), 2.78-2.67 (m, 2H), 2.67-2.58 (m, 2H), 2.55-2.39 (m, 2H), 2.32 (s, 3H), 2.26 (t, 4H), 2.23-2.19 (m, 1H), 2.19-2.06 (m, 1H), 1.69 (quint, 2H), 1.66-1.55 (m, 8H), 1.54-1.45 (m, 8H), 1.38-1.13 (m, 52H), 0.86 (t, 12H). APCI-MS analysis: Calculated C58H109NO12 [M+H] = 1012.7, Observed = 1012.7. HPLC-ELSD: tR = 7.235 min (method 2). Example 107: bis(8-oxo-8-(pentadecan-7-yloxy)octyl) 2-(((2-((4- hydroxybutyl)(methyl)amino)ethoxy)carbonyl)oxy)pentanedioate Step 1: Synthesis of 2-((4-(benzyloxy)butyl)(methyl)amino)ethan-1-ol (115) In a seal tube, a mixture of ((4-bromobutoxy)methyl)benzene (1.5 g, 20 mmol), 2- (methylamino)ethan-1-ol (5.3 g, 22 mmol), diisopropylethylamine (8.7 mL, 50 mmol) and potassium iodide (166 mg, 1 mmol) in 15 mL ethanol was heated at 80°C for two days. After cooled to room temperature, the mixture was filtered, and the filtrate was concentrated under vacuum to give the crude product (3.0 g, 63%), which was used for the next step without further purification. Step 2: Synthesis of bis(8-oxo-8-(pentadecan-7-yloxy)octyl) 2-(((2-((4- (benzyloxy)butyl)(methyl)amino)ethoxy)carbonyl)oxy)pentanedioate (116) To a solution of bis(8-oxo-8-(pentadecan-7-yloxy)octyl) 2-hydroxypentanedioate 112 (250 mg, 0.29 mmol) in 2 mL pyridine and 5 mL dichloromethane, was added p-nitrophenyl chloroformate (77 mg, 0.38 mmol), and the resulting mixture was stirred for 30 min. After TLC showed the complete reaction, 2-((4-(benzyloxy)butyl)(methyl)amino)ethan-1-ol 115 (1.47 g, 5.86 mmol) and diisopropylethylamine (2 mL) were added, and the reaction mixture was stirred at 45°C in a seal tube for four days. MS showed the formation of desired product. The reaction mixture was concentrated and partitioned between hexanes and water, and the combined organic layer was washed with sodium carbonate solution. After concentration, the crude was purified by column chromatography using 0-75% ethyl acetate in hexane to afford bis(8-oxo-8-(pentadecan-7-yloxy)octyl) 2-(((2-((4- (benzyloxy)butyl)(methyl)amino)ethoxy)carbonyl)oxy)pentanedioate as clear oil (117 mg, 36%). Step 3: Synthesis of bis(8-oxo-8-(pentadecan-7-yloxy)octyl) 2-(((2-((4- hydroxybutyl)(methyl)amino)ethoxy)carbonyl)oxy)pentanedioate (Example 107) A mixture of bis(8-oxo-8-(pentadecan-7-yloxy)octyl) 2-(((2-((4- (benzyloxy)butyl)(methyl)amino)ethoxy)carbonyl)oxy)pentanedioate 116 (117 mg, 0.10 mmol) and palladium hydroxide on carbon (20%, 20 mg) in 10 mL ethyl acetate was purged with nitrogen and filled with hydrogen, and then kept under balloon at room temperature overnight. The reaction mixture was filtered through a pad of silica gel and eluted with 10% methanol in ethyl acetate to give bis(8-oxo-8-(pentadecan-7-yloxy)octyl) 2-(((2-((4- hydroxybutyl)-(methyl)amino)ethoxy)-carbonyl)oxy)pentanedioate as pale yellow oil (86 mg, 80%). 1H NMR (400 MHz, CDCl3) δ 4.94 (dd, 1H), 4.85 (quint, 2H), 4.37-4.20 (m, 2H), 4.13 (t, 2H), 4.04 (t, 2H), 3.60-3.51 (m, 2H), 2.81-2.62 (m, 2H), 2.53-2.38 (m, 4H), 2.34-2.20 (m, 8H), 2.19-2.06 (m, 1H), 1.71-1.42 (m, 20H), 1.40-1.12 (m, 52H), 0.86 (t, 12H). APCI-MS analysis: Calculated C59H111NO12 [M+H] = 1026.8, Observed = 1026.5. HPLC-ELSD: tR = 7.181 min (method 2). Example 108: bis(8-oxo-8-(pentadecan-7-yloxy)octyl) 2-(((2-(bis(2- hydroxyethyl)amino)ethoxy)carbonyl)oxy)pentanedioate To a solution of bis(8-oxo-8-(pentadecan-7-yloxy)octyl) 2-hydroxypentanedioate 112 (220 mg, 0.26 mmol) in 2 mL pyridine and 7 mL dichloromethane, was added p-nitrophenyl chloroformate (157 mg, 0.78 mmol), and the resulting mixture was stirred for 1 h. After TLC showed the complete reaction, triethanolamine (2.6 g, 17 mmol) and diisopropylethylamine (1 mL) were added, and the reaction mixture was stirred at room temperature for two days. MS showed the formation of desired product. The reaction mixture was washed by saturated sodium bicarbonate solution, and the combined organic layer was dried over sodium sulfate. After filtration and concentration, the crude was purified by column chromatography eluted with 0-10% methanol in dichloromethane to afford bis(8-oxo-8-(pentadecan-7-yloxy)octyl) 2-(((2-((2-hydroxyethyl)(methyl)amino)ethoxy)-carbonyl)oxy)pentanedioate as clear oil (120 mg, 45%). 1H NMR (400 MHz, CDCl3) δ 4.94 (dd, 1H), 4.85 (quint, 2H), 4.45-4.34 (m, 1H), 4.23-4.10 (m, 4H), 4.05 (t, 2H), 3.68-3.53 (m, 4H), 2.91-2.60 (m, 8H), 2.53-2.37 (m, 2H), 2.30-2.19 (m, 5H), 2.18-2.07 (m, 1H), 1.69-1.42 (m, 16H), 1.37-1.14 (m, 52H), 0.86 (t, 12H). APCI-MS analysis: Calculated C58H109NO13 [M+H] = 1028.7, Observed = 1028.7. HPLC-ELSD: tR = 8.369 min (method 2) Example 109: bis(8-oxo-8-(pentadecan-7-yloxy)octyl) 2-(((2-(piperidin-1- yl)ethoxy)carbonyl)oxy)pentanedioate To a solution of bis(8-oxo-8-(pentadecan-7-yloxy)octyl) 2-hydroxypentanedioate 112 (150 mg, 0.17 mmol) in 2 mL pyridine and 8 mL dichloromethane, was added p-nitrophenyl chloroformate (103 mg, 0.51 mmol), and the resulting mixture was stirred for 2 h. After TLC showed the complete reaction, 2-(piperidin-1-yl)ethan-1-ol (220 mg, 1.7 mmol) and diisopropylethylamine (0.5 mL) were added, and the reaction mixture was stirred at room temperature overnight. MS showed the formation of desired product. The reaction mixture was washed by saturated sodium bicarbonate solution, and the combined organic layer was dried over sodium sulfate. After filtration and concentration, the crude was purified by column chromatography eluted with 0-10% methanol in dichloromethane to afford bis(8-oxo- 8-(pentadecan-7-yloxy)octyl) 2-(((2-(piperidin-1-yl)ethoxy)carbonyl)oxy)pentanedioate as clear oil (105 mg, 61%). 1H NMR (400 MHz, CDCl3) δ 4.93 (dd, 1H), 4.85 (quint, 2H), 4.26 (t, 2H), 4.13 (t, 2H), 4.04 (t, 2H), 2.70-2.56 (m, 2H), 2.51-2.35 (m, 6H), 2.31-2.18 (m, 5H), 2.18-2.06 (m, 1H), 1.67- 1.52 (m, 12H), 1.52-1.45 (m, 8H), 1.44-1.37 (m, 2H), 1.36-1.14 (m, 52H), 0.86 (t, 12H). APCI-MS analysis: Calculated C59H109NO11 [M+H] = 1008.8, Observed = 1008.8. HPLC-ELSD: tR = 8.405 min (method 1). Example 110: bis(8-oxo-8-(pentadecan-7-yloxy)octyl) 2-(((2- (diethylamino)ethoxy)carbonyl)oxy)pentanedioate To a solution of bis(8-oxo-8-(pentadecan-7-yloxy)octyl) 2-hydroxypentanedioate 112 (150 mg, 0.17 mmol) in 2 mL pyridine and 8 mL dichloromethane, was added p-nitrophenyl chloroformate (103 mg, 0.51 mmol), and the resulting mixture was stirred for 2 h. After TLC showed the complete reaction, 2-(diethylamino)ethan-1-ol (199 mg, 1.7 mmol) and diisopropylethylamine (0.5 mL) were added, and the reaction mixture was stirred at room temperature overnight. MS showed the formation of desired product. The reaction mixture was washed by saturated sodium bicarbonate solution, and the combined organic layer was dried over sodium sulfate. After filtration and concentration, the crude was purified by column chromatography eluted with 0-10% methanol in dichloromethane to afford bis(8-oxo- 8-(pentadecan-7-yloxy)octyl) 2-(((2-(diethylamino)ethoxy)carbonyl)oxy) pentanedioate as clear oil (136 mg, 80%). 1H NMR (400 MHz, CDCl3) δ 4.93 (dd, 1H), 4.85 (quint, 2H), 4.20 (t, 2H), 4.14 (t, 2H), 4.05 (t, 2H), 2.77-2.68 (m, 2H), 2.56 (q, 4H), 2.50-2.38 (m, 2H), 2.33-2.19 (m, 5H), 2.18-2.07 (m, 1H), 1.69-1.55 (m, 10H), 1.54-1.44 (m, 8H), 1.38-1.17 (m, 50H), 1.02 (t, 6H), 0.86 (t, 12H). APCI-MS analysis: Calculated C58H109NO11 [M+H] = 996.8, Observed = 996.8. HPLC-ELSD: tR = 8.395 min (method 1). Example 111: bis(8-oxo-8-(pentadecan-7-yloxy)octyl) 2-(((2-(pyrrolidin-1- yl)ethoxy)carbonyl)oxy)pentanedioate To a solution of bis(8-oxo-8-(pentadecan-7-yloxy)octyl) 2-hydroxypentanedioate 112 (200 mg, 0.23 mmol) in 2 mL pyridine and 8 mL dichloromethane, was added p-nitrophenyl chloroformate (139 mg, 0.69 mmol), and the resulting mixture was stirred for 2 h. After TLC showed the complete reaction, 2-(pyrrolidin-1-yl)ethan-1-ol (265 mg, 2.3 mmol) and diisopropylethylamine (0.5 mL) were added, and the reaction mixture was stirred at room temperature overnight. MS showed the formation of desired product. The reaction mixture was washed by saturated sodium bicarbonate solution, and the combined organic layer was dried over sodium sulfate. After filtration and concentration, the crude was purified by column chromatography eluted with 0-10% methanol in dichloromethane to afford bis(8-oxo- 8-(pentadecan-7-yloxy)octyl) 2-(((2-(pyrrolidin-1-yl)ethoxy)carbonyl)oxy)pentanedioate as clear oil (135 mg, 59%). 1H NMR (400 MHz, CDCl3) δ 4.92 (dd, 1H), 4.83 (quint, 2H), 4.25 (t, 2H), 4.12 (t, 2H), 4.03 (t, 2H), 2.80-2.69 (m, 2H), 2.58-2.49 (m, 4H), 2.48-2.37 (m, 2H), 2.30-2.16 (m, 5H), 2.15- 2.04 (m, 1H), 1.81-1.70 (m, 4H), 1.67-1.53 (m, 8H), 1.52-1.41 (m, 8H), 1.37-1.13 (m, 52H), 0.84 (t, 12H). APCI-MS analysis: Calculated C58H107NO11 [M+H] = 994.8, Observed = 994.8. HPLC-ELSD: tR = 8.439 min (method 1). Example 112: bis(8-oxo-8-(pentadecan-7-yloxy)octyl) 2-(((2- morpholinoethoxy)carbonyl)oxy)pentanedioate To a solution of bis(8-oxo-8-(pentadecan-7-yloxy)octyl) 2-hydroxypentanedioate 112 (200 mg, 0.23 mmol) in 2 mL pyridine and 8 mL dichloromethane, was added p-nitrophenyl chloroformate (139 mg, 0.69 mmol), and the resulting mixture was stirred for 2 h. After TLC showed the complete reaction, 2-morpholinoethan-1-ol (301 mg, 2.3 mmol) and diisopropylethylamine (0.5 mL) were added, and the reaction mixture was stirred at room temperature for four days. MS showed the formation of desired product. The reaction mixture was washed by saturated sodium bicarbonate solution, and the combined organic layer was dried over sodium sulfate. After filtration and concentration, the crude was purified by column chromatography eluted with 0-10% methanol in dichloromethane to afford bis(8-oxo- 8-(pentadecan-7-yloxy)octyl) 2-(((2-morpholinoethoxy)carbonyl)oxy)pentanedioate as clear oil (120 mg, 52%). 1H NMR (400 MHz, CDCl3) δ 4.94 (dd, 1H), 4.85 (quint, 2H), 4.27 (t, 2H), 4.14 (t, 2H), 4.05 (t, 2H), 3.70 (t, 4H), 2.72-2.61 (m, 2H), 2.54-2.41 (m, 6H), 2.31-2.20 (m, 5H), 2.19-2.07 (m, 1H), 1.69-1.54 (m, 8H), 1.53-1.42 (m, 8H), 1.39-1.14 (m, 52H), 0.86 (t, 12H). APCI-MS analysis: Calculated C58H107NO12 [M+H] = 1010.8, Observed = 1010.8. HPLC-ELSD: tR = 8.521 min (method 1). Example 113: bis(8-oxo-8-(pentadecan-7-yloxy)octyl) 2-(((2-(4-methylpiperazin-1- yl)ethoxy)carbonyl)oxy)pentanedioate To a solution of bis(8-oxo-8-(pentadecan-7-yloxy)octyl) 2-hydroxypentanedioate 112 (200 mg, 0.23 mmol) in 2 mL pyridine and 8 mL dichloromethane, was added p-nitrophenyl chloroformate (139 mg, 0.69 mmol), and the resulting mixture was stirred for 2 h. After TLC showed the complete reaction, 2-(4-methylpiperazin-1-yl)ethan-1-ol (332 mg, 2.3 mmol) and diisopropylethylamine (0.5 mL) were added, and the reaction mixture was stirred at room temperature for four days. MS showed the formation of desired product. The reaction mixture was washed by saturated sodium bicarbonate solution, and the combined organic layer was dried over sodium sulfate. After filtration and concentration, the crude was purified by column chromatography eluted with 0-10% methanol in dichloromethane to afford bis(8-oxo- 8-(pentadecan-7-yloxy)octyl) 2-(((2-(4-methylpiperazin-1- yl)ethoxy)carbonyl)oxy)pentanedioate as clear oil (138 mg, 80%). 1H NMR (400 MHz, CDCl3) δ 4.93 (dd, 1H), 4.85 (quint, 2H), 4.31-4.21 (m, 2H), 4.13 (t, 2H), 4.05 (t, 2H), 2.71-2.63 (m, 2H), 2.61-2.34 (m, 9H), 2.31-2.19 (m, 9H), 2.17-2.07 (m, 1H), 1.69-1.55 (m, 8H), 1.53-1.43 (m, 8H), 1.39-1.15 (m, 52H), 0.86 (t, 12H). APCI-MS analysis: Calculated C59H110N2O11 [M+H] = 1023.8, Observed = 1023.8. HPLC-ELSD: tR = 8.458 min (method 1). Example 114: bis(8-oxo-8-(pentadecan-7-yloxy)octyl) 2-(((3- (dimethylamino)propoxy)carbonyl)oxy)pentanedioate To a solution of bis(8-oxo-8-(pentadecan-7-yloxy)octyl) 2-hydroxypentanedioate 112 (100 mg, 0.12 mmol) in 2 mL pyridine and 8 mL dichloromethane, was added p-nitrophenyl chloroformate (100 mg, 0.49 mmol), and the resulting mixture was stirred for 30 min. After TLC showed the complete reaction, 3-(dimethylamino)propan-1-ol (1.2 g, 11.6 mmol) and diisopropylethylamine (2 mL) were added, and the reaction mixture was stirred at room temperature for two days. MS showed the formation of desired product. The reaction mixture was diluted with saturated ammonium chloride and extracted with dichloromethane. After concentration, the residue was dissolved in hexane and washed by sodium bicarbonate solution, and the combined organic layer was dried over sodium sulfate. After filtration and concentration, the crude was purified by column chromatography eluted with 0-10% methanol in dichloromethane to afford bis(8-oxo-8-(pentadecan-7-yloxy)octyl) 2-(((3- (dimethylamino)propoxy)carbonyl)oxy)pentanedioate as clear oil (97 mg, 84%). 1H NMR (400 MHz, CDCl3) δ 4.93 (dd, 1H), 4.84 (quint, 2H), 4.24-4.16 (m, 2H), 4.13 (t, 2H), 4.04 (t, 2H), 2.51-2.39 (m, 2H), 2.38-2.30 (m, 2H), 2.29-2.21 (m, 5H), 2.20 (s, 6H), 2.17-2.06 (m, 1H), 1.83 (quint, 2H), 1.67-1.54 (m, 8H), 1.53-1.41 (m, 8H), 1.38-1.13 (m, 54H), 0.85 (t, 12H). APCI-MS analysis: Calculated C57H107NO11 [M+H] = 982.7, Observed = 982.5. HPLC-ELSD: tR = 7.368 min (method 2). Example 115: bis(8-oxo-8-(pentadecan-7-yloxy)octyl) 2-(((4- (dimethylamino)butoxy)carbonyl)oxy)pentanedioate To a solution of bis(8-oxo-8-(pentadecan-7-yloxy)octyl) 2-hydroxypentanedioate 112 (250 mg, 0.29 mmol) in 2 mL pyridine and 8 mL dichloromethane, was added p-nitrophenyl chloroformate (177 mg, 0.88 mmol), and the resulting mixture was stirred for 45 min. After TLC showed the complete reaction, 4-(dimethylamino)butan-1-ol (858 mg, 7.3 mmol) and diisopropylethylamine (1.3 mL) were added, and the reaction mixture was stirred at room temperature overnight. MS showed the formation of desired product. After concentration, the residue was dissolved in hexane, washed by water and sodium bicarbonate solution, and the combined organic layer was dried over sodium sulfate. After filtration and concentration, the crude was purified by column chromatography eluted with 0-5% methanol in ethyl acetate to afford bis(8-oxo-8-(pentadecan-7-yloxy)octyl) 2-(((4- (dimethylamino)butoxy)carbonyl)oxy)pentanedioate as clear oil (230 mg, 78%). Due to the stability of the free base, the compound was converted into trifluoroacetate salt. 1H NMR of free base (400 MHz, CDCl3) δ 4.93 (dd, 1H), 4.84 (quint, 2H), 4.21-4.08 (m, 4H), 4.04 (t, 2H), 2.53-2.36 (m, 2H), 2.36-2.18 (m, 13H), 2.18-2.05 (m, 1H), 1.77-.41 (m, 20H), 1.37- 1.12 (m, 52H), 0.85 (t, 12H). 1H NMR of TFA salt (400 MHz, CDCl3) δ 10.58 (s, 1H), 5.50 (m, 8H), 4.94 (dd, 1H), 4.85 (quint, 2H), 4.34-4.25 (m, 1H), 4.19-4.10 (m, 3H), 4.06 (t, 2H), 3.20-3.08 (m, 2H), 2.89 (d, 6H), 2.54-2.38 (m, 2H), 2.33-2.19 (m, 5H), 2.18-2.05 (m, 1H), 1.93-1.72 (m, 4H), 1.69-1.43 (m, 16H), 1.37-1.12 (m, 52H), 0.86 (t, 12H). APCI-MS analysis: Calculated C58H109NO11 [M+H] = 996.8, Observed = 996.9. HPLC-ELSD: tR = 8.752 min (method 2). Example 116: bis(8-oxo-8-(pentadecan-7-yloxy)octyl) 2-(((2-(azetidin-1- yl)ethoxy)carbonyl)oxy)pentanedioate To a solution of bis(8-oxo-8-(pentadecan-7-yloxy)octyl) 2-hydroxypentanedioate 112 (250 mg, 0.29 mmol) in 2 mL pyridine and 8 mL dichloromethane, was added p-nitrophenyl chloroformate (118 mg, 0.59 mmol), and the resulting mixture was stirred for 30 min. After TLC showed the complete reaction, 2-(azetidin-1-yl)ethan-1-ol (741 mg, 7.3 mmol) and diisopropylethylamine (1.5 mL) were added, and the reaction mixture was stirred at room temperature overnight. MS showed the formation of desired product. After concentration, the residue was dissolved in hexane, washed by water and sodium bicarbonate solution, and the combined organic layer was dried over sodium sulfate. After filtration and concentration, the crude was purified by column chromatography eluted with 0-5% methanol in ethyl acetate to afford bis(8-oxo-8-(pentadecan-7-yloxy)octyl) 2-(((2-(azetidin-1- yl)ethoxy)carbonyl)oxy)pentanedioate as clear oil (270 mg, 94%). 1H NMR (400 MHz, CDCl3) δ 4.96-4.88 (m, 1H), 4.88-4.79 (m, 2H), 4.16-4.07 (m, 4H), 4.07-4.00 (m, 2H), 3.23 (t, 4H), 2.71-2.59 (m, 2H), 2.49-2.38 (m, 2H), 2.30-2.15 (m, 5H), 2.14-1.99 (m, 3H), 1.66-1.53 (m, 8H), 1.52-1.42 (m, 8H), 1.37-1.14 (m, 52H), 0.89-0.78 (m, 12H). APCI-MS analysis: Calculated C57H105NO11 [M+H] = 980.8, Observed = 980.1. HPLC-ELSD: tR = 7.318 min (method 2). Example 117: bis(8-oxo-8-(pentadecan-7-yloxy)octyl) 2-(((2- (isopropyl(methyl)amino)ethoxy)carbonyl)oxy)pentanedioate
Figure imgf000308_0001
To a solution of bis(8-oxo-8-(pentadecan-7-yloxy)octyl) 2-hydroxypentanedioate 112 (260 mg, 0.30 mmol) in 2 mL pyridine and 8 mL dichloromethane, was added p-nitrophenyl chloroformate (184 mg, 0.91 mmol), and the resulting mixture was stirred for 30 min. After TLC showed the complete reaction, 2-(isopropyl(methyl)amino)ethan-1-ol (714 mg, 6.1 mmol) and diisopropylethylamine (1 mL) were added, and the reaction mixture was stirred at room temperature overnight. MS showed the formation of desired product. After concentration, the residue was dissolved in hexane, washed by water and sodium bicarbonate solution, and the combined organic layer was dried over sodium sulfate. After filtration and concentration, the crude was purified by column chromatography eluted with 0-50 % ethyl acetate in hexane to afford bis(8-oxo-8-(pentadecan-7-yloxy)octyl) 2-(((2- (isopropyl(methyl)amino)ethoxy)carbonyl)oxy)pentanedioate as clear oil (175 mg, 57%). 1H NMR (400 MHz, CDCl3) δ 4.93 (dd, 1H), 4.85 (quint, 2H), 4.24-4.17 (m, 2H), 4.14 (t, 2H), 4.05 (t, 2H), 2.82 (quint, 1H), 2.72-2.58 (m, 2H), 2.51-2.39 (m, 2H), 2.31-2.18 (m, 8H), 2.18-2.05 (m, 1H), 1.70-1.42 (m, 16H), 1.38-1.14 (m, 52H), 0.99 (dd, 6H), 0.86 (t, 12H). APCI-MS analysis: Calculated C58H109NO11 [M+H] = 996.8, Observed = 996.1. HPLC-ELSD: tR = 8.64 min (method 1). Example 118: bis(8-oxo-8-(pentadecan-7-yloxy)octyl) 2-(((3-(pyrrolidin-1- yl)propoxy)carbonyl)oxy)pentanedioate To a solution of bis(8-oxo-8-(pentadecan-7-yloxy)octyl) 2-hydroxypentanedioate 112 (250 mg, 0.29 mmol) in 2 mL pyridine and 8 mL dichloromethane, was added p-nitrophenyl chloroformate (177 mg, 0.88 mmol), and the resulting mixture was stirred for 30 min. After TLC showed the complete reaction, 3-(pyrrolidin-1-yl)propan-1-ol (1.51 g, 11.7 mmol) and diisopropylethylamine (1.5 mL) were added, and the reaction mixture was stirred at room temperature overnight. MS showed the formation of desired product. After concentration, the residue was dissolved in hexane, washed by water and sodium bicarbonate solution, and the combined organic layer was dried over sodium sulfate. After filtration and concentration, the crude was purified by column chromatography eluted with 0-75 % ethyl acetate in hexane to afford bis(8-oxo-8-(pentadecan-7-yloxy)octyl) 2-(((3-(pyrrolidin-1- yl)propoxy)carbonyl)oxy)pentanedioate as clear oil (270 mg, 91%). 1H NMR (400 MHz, CDCl3) δ 4.94 (dd, 1H), 4.85 (quint, 2H), 4.22 (t, 2H), 4.14 (t, 2H), 4.05 (t, 2H), 2.59-2.37 (m, 8H), 2.32-2.19 (m, 5H), 2.18-2.06 (m, 1H), 1.89 (quint, 2H), 1.82-1.71 (m, 4H), 1.69-1.42 (m, 16H), 1.38-1.16 (m, 52H), 0.86 (t, 12H). APCI-MS analysis: Calculated C59H109NO11 [M+H] = 1008.8, Observed = 1008.7. HPLC-ELSD: tR = 7.496 min (method 2). Example 119: bis(8-oxo-8-(pentadecan-7-yloxy)octyl) 2-(((4-(pyrrolidin-1- yl)butoxy)carbonyl)oxy)pentanedioate To a solution of bis(8-oxo-8-(pentadecan-7-yloxy)octyl) 2-hydroxypentanedioate 112 (250 mg, 0.29 mmol) in 2 mL pyridine and 8 mL dichloromethane, was added p-nitrophenyl chloroformate (177 mg, 0.88 mmol), and the resulting mixture was stirred for 45 min. After TLC showed the complete reaction, 4-(pyrrolidin-1-yl)butan-1-ol (820 mg, 5.7 mmol) and diisopropylethylamine (1.5 mL) were added, and the reaction mixture was stirred at room temperature overnight. MS showed the formation of desired product. After concentration, the residue was dissolved in hexane, washed by water and sodium bicarbonate solution, and the combined organic layer was dried over sodium sulfate. After filtration and concentration, the crude was purified by column chromatography eluted with 0-5% methanol in ethyl acetate to afford bis(8-oxo-8-(pentadecan-7-yloxy)octyl) 2-(((4-(pyrrolidin-1- yl)butoxy)carbonyl)oxy)pentanedioate as clear oil (140 mg, 46%). Due to the stability of the free base, the compound was converted into trifluoroacetate salt. 1H NMR of free base (400 MHz, CDCl3) δ 4.93 (dd, 1H), 4.85 (quint, 2H), 4.22-4.15 (m, 4H), 4.05 (t, 2H), 2.55-2.37 (m, 8H), 2.32-2.18 (m, 5H), 2.18-1.08 (m, 1H), 1.82-1.68 (m, 6H), 1.66-1.41 (m, 16H), 1.41-1.15 (m, 54H), 0.86 (t, 12H). 1H NMR of TFA salt (400 MHz, CDCl3) δ 10.92 (s, 1H), 4.94 (dd, 1H), 4.85 (quint, 2H), 4.32-4.22 (m, 2H), 4.18-4.09 (m, 4H), 4.05 (t, 2H), 3.94-3.72 (m, 6H), 3.22-3.08 (m, 2H), 2.92-2.80 (m, 2H), 2.54-2.35 (m, 2H), 2.33-2.19 (m, 5H), 2.18-2.00 (m, 5H), 1.93-1.70 (m, 4H), 1.68-1.42 (m, 16H), 1.39-1.11 (m, 52H), 0.86 (t, 12H). APCI-MS analysis: Calculated C60H111NO11 [M+H] = 1022.8, Observed = 1022.7. HPLC-ELSD: tR = 8.911 min (method 2). Example 120: bis(8-oxo-8-(pentadecan-7-ylthio)octyl) 2-(((2- (dimethylamino)ethoxy)carbonyl)oxy)pentanedioate Step 1: Synthesis of Pentadecane-7-thiol (117) To a solution of pentadecan-7-ol (600 mg, 2.63 mmol) in pyridine (5 mL), was added 4-methylbenzenesulfonyl chloride (601 mg, 3.15 mmol), and the mixture was stirred at room temperature overnight. TLC showed completed reaction. The reaction mixture was diluted with dichloromethane and washed with sodium bicarbonate solution. The combined organic layer was dried and concentrated, and the crude was purified by column chromatography using 0-15% ethyl acetate in hexanes to get the pentadecan-7-yl 4-methylbenzenesulfonate (590 mg, 58%). A mixture of pentadecan-7-yl 4-methylbenzenesulfonate (580 mg, 1.52 mmol) and thiourea (173 mg, 2.27 mmol) in ethanol (8 mL) and pyridine (1.5 mL) was heated at 100°C overnight. After cooled to room temperature, the mixture was partitioned between water and dichloromethane, and the combined organic layer was dried and concentrated to give crude product which was used for the next step without purification. A mixture of crude pentadecan-7-yl carbamimidothioate (500 mg, 1.75 mmol) and sodium hydroxide (349 mg, 8.73 mmol) in ethanol (25 mL) and water (5.0 mL) was heated at 100°C overnight. The reaction mixture was poured into ice-water and acidified by acetic acid and then extracted with ethyl acetate. The combined organic layer was dried and concentrated, and the crude was purified by column chromatography eluting with 0-5% ethyl acetate in hexanes to obtain a mixture of thiol and disulfide (100 mg), which was dissolved in 12 mL diethyl ether and treated with lithium aluminum hydride (2 M solution in THF, 0.5 mL, 1 mmol) at room temperature for 4 h. The reaction was carefully quenched with water and acidified with dilute 1 M HCl, and then extracted with ether. The organic layer was dried and concentrated to give pentadecane-7-thiol as colorless oil (70 mg, 19%). Step 2: Synthesis of Bis(8-oxo-8-(pentadecan-7-ylthio)octyl) 2-((tert- butyldiphenylsilyl)oxy)pentanedioate (118) A mixture of 8,8'-((2-((tert-butyldiphenylsilyl)oxy)pentanedioyl)bis(oxy))dioctanoic acid (68 mg, 0.10 mmol), pentadecane-7-thiol 117 (100 mg, 0.41mmol), EDCI (0.19 g, 1.0 mmol) and DMAP (12 mg, 0.10 mmol) in 8.0 mL dichloromethane was stirred at room temperature overnight. After concentration, the residue was triturated with hexanes, and then the solvent was removed under vacuum. The crude was purified by column chromatography using 0-20% ethyl acetate in hexanes to give bis(8-oxo-8-(pentadecan-7-ylthio)octyl) 2-((tert- butyldiphenylsilyl)oxy)pentanedioate (70 mg, 61%). Step 3: Synthesis of Bis(8-oxo-8-(pentadecan-7-ylthio)octyl) 2-hydroxypentanedioate (119) In a Teflon flask, to a solution of bis(8-oxo-8-(pentadecan-7-ylthio)octyl) 2-((tert- butyldiphenylsilyl)oxy)pentanedioate 118 (70 mg, 0.06 mmol) in 8 mL THF and 1 mL pyridine, was added HF-pyridine complex (70wt%, 0.3 mL) slowly, and the mixture was stirred at room temperature overnight. The reaction was quenched by careful addition of sodium bicarbonate solution, and then extracted with ethyl acetate. The combined organic layer was dried and concentrated, and the crude was purified by column chromatography using 0-25% ethyl acetate in hexanes to give bis(8-oxo-8-(pentadecan-7-ylthio)octyl) 2- hydroxypentanedioate as colorless oil (50 mg, 91%). Step 4: Synthesis of Bis(8-oxo-8-(pentadecan-7-ylthio)octyl) 2-(((2- (dimethylamino)ethoxy)carbonyl)oxy)pentanedioate (Example 120) To a solution of bis(8-oxo-8-(pentadecan-7-ylthio)octyl) 2-hydroxypentanedioate 119 (55 mg, 0.062 mmol) in 6.0 mL dichloromethane and 0.5 mL pyridine (0.5 ml), was added 4- nitrophenyl carbonochloridate (63 mg, 0.31 mmol), and the mixture was stirred for 90 min. TLC-analysis confirmed the complete conversion of alcohol. A solution of 2- (dimethylamino)ethan-1-ol (0.28 g, 3.1 mmol) and diisopropylethylamine (0.2 mL) was added, and the mixture was stirred at room temperature overnight. MS showed the formation of desired product. After concentration, the residue was dissolved in hexane, washed by water and sodium bicarbonate solution, and the combined organic layer was dried over sodium sulfate. After filtration and concentration, the crude was purified by column chromatography eluted with 0-70% ethyl acetate in hexanes to afford bis(8-oxo-8-(pentadecan-7-ylthio)octyl) 2-(((2-(dimethylamino)ethoxy)carbonyl)oxy)pentanedioate as clear oil (42 mg, 68%) 1H NMR (400 MHz, CDCl3) δ 4.93 (dd, 1H), 4.30-4.17 (m, 2H), 4.13 (t, 2H), 4.04 (t, 2H), 3.54-3.42 (m, 2H), 2.67-2.54 (m, 2H), 2.54-2.39 (m, 6H), 2.27 (s, 6H), 2.24-2.05 (m, 2H), 1.76-1.42 (m, 16H), 1.41-1.13 (m, 52H), 0.86 (t, 12H). APCI-MS analysis: Calculated C56H105NO9S2 [M+H] = 1000.7, Observed = 1000.8. HPLC-ELSD: tR = 8.694 min (method 1). Example 121: bis(6-((2-hexyldecanoyl)oxy)hexyl) 2-(((2- (dimethylamino)ethoxy)carbonyl)oxy)pentanedioate Step 1: Synthesis of Bis(6-(benzyloxy)hexyl) 2-oxopentanedioate (120) A mixture of α-ketoglutaric acid (1.46 g, 10 mmol), 6-(benzyloxy)hexan-1-ol (4.16 g, 20 mmol) and p-toluenesulfonic acid monohydrate (100 mg, 0.52 mmol) in 25 mL toluene was heated to reflux with Dean-Stark for 1 h. After cooled to room temperature, the mixture was diluted with hexanes and washed by saturated sodium bicarbonate solution. The aqueous layer was extracted with ethyl acetate, and the combined organic layer was dried over sodium sulfate. After filtration and concentration, the crude was purified by column chromatography with 0-25% ethyl acetate in hexanes to give bis(6-(benzyloxy)hexyl) 2-oxopentanedioate as colorless oil (3.0 g, 57%). Step 2: Synthesis of Bis(6-(benzyloxy)hexyl) 2-hydroxypentanedioate (121) A solution of bis(6-(benzyloxy)hexyl) 2-oxopentanedioate 120 (3.0 g, 5.7 mmol) in 20 mL THF was cooled to 0°C, then sodium borohydride (200 mg, 5 mmol) was added by portions in 10 min, and the reaction mixture was stirred at room temperature for 2 h. Saturated ammonium chloride solution was added slowly, and the resulting mixture was extracted with ethyl acetate. The combined organic layer was washed with brine and dried over sodium sulfate. After concentration, the crude was used for the next step without purification. Step 3: Synthesis of Bis(6-(benzyloxy)hexyl) 2-((tert-butyldiphenylsilyl)oxy)pentanedioate (122)
Figure imgf000313_0001
To a stirred solution of crude bis(6-(benzyloxy)hexyl) 2-hydroxypentanedioate 121 (3.0 g, 5.7 mmol) and imidazole (503 mg, 7.4 mmol) in 25 mL dichloromethane, was added tert-butylchlorodiphenylsilane (2.04 g, 7.4 mmol), and the mixture was stirred room temperature for 18 h. The reaction mixture was quenched by saturated sodium bicarbonate solution and extracted with dichloromethane. The combined organic layer was dried and concentrated, and the crude was purified by column chromatography using 0-20% ethyl acetate in hexanes to give bis(6-(benzyloxy)hexyl) 2-((tert- butyldiphenylsilyl)oxy)pentanedioate as colorless oil (3.1 g, 70%). Step 4: Synthesis of Bis(6-hydroxyhexyl) 2-((tert-butyldiphenylsilyl)oxy)pentanedioate (123) A mixture of bis(6-(benzyloxy)hexyl) 2-((tert-butyldiphenylsilyl)oxy)pentanedioate 122 (3.1 g, 4 mmol) and 20% palladium hydroxide on carbon (1.2 g) in 20 mL ethyl acetate was subjected to hydrogenation at room temperature for 18 h. After filtration, the filtrate was concentrated, and the crude was purified by column chromatography using 0-100% ethyl acetate in hexane to afford bis(6-hydroxyhexyl) 2-((tert-butyldiphenylsilyl)oxy)pentanedioate as pale yellow oil (1.6 g, 70%). Step 5: Synthesis of Bis(6-((2-hexyldecanoyl)oxy)hexyl) 2-((tert- butyldiphenylsilyl)oxy)pentanedioate (124) A mixture of bis(6-hydroxyhexyl) 2-((tert-butyldiphenylsilyl)oxy)pentanedioate 123 (1.6 g, 3.16 mmol), 2-hexyldecanoic acid (1.79 g, 7 mmol), EDCI (1.34 g, 7 mmol) and DMAP (386 mg, 3.16 mmol) in 30 mL dichloromethane was stirred at room temperature overnight. After concentration, the crude was partitioned between EtOAc and saturated ammonium chloride solution, and the combined organic layer was dried over sodium sulfate. The solvent was removed under vacuum, and the crude was purified by column chromatography using 0-10% ethyl acetate in hexane to give bis(6-((2- hexyldecanoyl)oxy)hexyl) 2-((tert-butyldiphenylsilyl)oxy)pentanedioate as pale yellow oil (2.6 g, 79%). Step 6: Synthesis of Bis(6-((2-hexyldecanoyl)oxy)hexyl) 2-hydroxypentanedioate (125) To a solution of bis(6-((2-hexyldecanoyl)oxy)hexyl) 2-((tert- butyldiphenylsilyl)oxy)pentanedioate 124 (2.6 g, 2.4 mmol) in a mixture of THF (20 mL) and Pyridine (6.0 mL) in a Teflon flask, was added HF-pyridine complex (70wt%, 3 mL) at 0°C, and the resulting mixture was stirred at room temperature for 18 h. The reaction mixture was carefully quenched with saturated sodium bicarbonate solution and extracted with ethyl acetate. The combined organic layer was dried and concentrated, and the crude was purified by column chromatography using 0-30% ethyl acetate in hexanes to give bis(6-((2- hexyldecanoyl)oxy)hexyl) 2-hydroxypentanedioate as colorless oil (1.9 g, 96%). Step 7: Synthesis of bis(6-((2-hexyldecanoyl)oxy)hexyl) 2-(((2- (dimethylamino)ethoxy)carbonyl)oxy)pentanedioate (Example 121) To a solution of bis(6-((2-hexyldecanoyl)oxy)hexyl) 2-hydroxypentanedioate 125 (870 mg, 1.04 mmol) in 5 mL pyridine and 10 mL dichloromethane, was added p-nitrophenyl chloroformate (629 mg, 3.12 mmol), and the resulting mixture was stirred for 2 h. After TLC showed the complete reaction, 2-(dimethylamino)ethan-1-ol (0.93 g, 10.4 mmol) and diisopropylethylamine (0.5 mL) were added, and the reaction mixture was stirred at room temperature overnight. MS showed the formation of desired product. The reaction mixture was washed by saturated sodium bicarbonate solution, and the combined organic layer was dried over sodium sulfate. After filtration and concentration, the crude was purified by column chromatography eluted with 0-70% ethyl acetate in hexane to afford bis(6-((2- hexyldecanoyl)oxy)hexyl) 2-(((2-(dimethylamino)ethoxy)carbonyl)oxy)-pentanedioate as clear oil (700 mg, 70%). 1H NMR (400 MHz, CDCl3) δ 4.94 (dd, 1H), 4.29-4.18 (m, 2H), 4.17-4.11 (m, 2H), 4.09- 4.00 (m, 6H), 2.67-2.52 (m, 2H), 2.52-2.37 (m, 2H), 2.35-2.04 (m, 10H), 1.73-1.50 (m, 12H), 1.47-1.32 (m, 12H), 1.32-1.13 (m, 40H), 0.86 (t, 12H). APCI-MS analysis: Calculated C54H101NO11 [M+H] = 940.7, Observed = 940.4. HPLC-ELSD: tR = 8.512 min (method 1). Example 122: bis(6-((2-hexyldecanoyl)oxy)hexyl) 2-(((2-(bis(2- hydroxyethyl)amino)ethoxy)carbonyl)oxy)pentanedioate To a solution of bis(6-((2-hexyldecanoyl)oxy)hexyl) 2-hydroxypentanedioate 125 (100 mg, 0.12 mmol) in 5 mL pyridine and 10 mL dichloromethane, was added p-nitrophenyl chloroformate (73 mg, 0.36 mmol), and the resulting mixture was stirred for 2 h. After TLC showed the complete reaction, triethanolamine (179 mg, 1.2 mmol) and diisopropylethylamine (0.5 mL) were added, and the reaction mixture was stirred at room temperature overnight. MS showed the formation of desired product. The reaction mixture was washed by saturated sodium bicarbonate solution, and the combined organic layer was dried over sodium sulfate. After filtration and concentration, the crude was purified by column chromatography eluted with 0-5% methanol in dichloromethane to afford bis(6-((2- hexyldecanoyl)oxy)hexyl) 2-(((2-(bis(2- hydroxyethyl)amino)ethoxy)carbonyl)oxy)pentanedioateas clear oil (40 mg, 33%). 1H NMR (400 MHz, CDCl3) δ 4.94 (dd, 1H), 4.45-4.34 (m, 1H), 4.24-4.10 (m, 4H), 4.09- 3.98 (m, 6H), 3.68-3.49 (m, 4H), 2.99-2.57 (m, 6H), 2.55-2.37 (m, 2H), 2.35-2.19 (m, 4H), 2.19-2.07 (m, 1H), 1.72-1.48 (m, 10H), 1.47-1.06 (m, 52H), 0.85 (t, 12H). APCI-MS analysis: Calculated C56H105NO13 [M+H] = 1000.7, Observed = 1000.7. HPLC-ELSD: tR = 8.145 min (method 1). Example 123: bis(6-((3-hexylundecanoyl)oxy)hexyl) 2-(((2- (dimethylamino)ethoxy)carbonyl)oxy)pentanedioate Step 1: Synthesis of Pentadecan-7-one (126) To a stirred solution of pentadecan-7-ol (5.0 g, 22 mmol) in 20 mL acetone, was added Jones reagent dropwise at room temperature until the orange color persisted, and then the mixture was stirred for 30 min. After quenching by isopropanol, the resulting mixture was diluted with water and extracted with ethyl acetate, and the combined organic layer was dried over anhydrous sodium sulfate. After filtration and concentration, the crude was purified by column chromatography using 0-10% ethyl acetate in hexanes to give impure pentadecan-7- one (2.6 g, 52%), which was used for the next step without further purification. Step 2: Synthesis of 7-Vinylpentadecan-7-ol (127) To a solution of pentadecan-7-one 126 (2.5 g, 11 mmol) in 20 mL THF, was added vinylmagnesium bromide (1 M in THF, 22 mL, 22 mmol) dropwise at room temperature, and the resulting solution was stirred at room temperature overnight. The reaction was quenched with saturated ammonium chloride solution and extracted with ethyl acetate. The combined organic layer was washed with brine and dried over sodium sulfate. After filtration and concentration, 2.5 g crude was carried over to the next step without further purification. Step 3: Synthesis of 3-Hexylundec-2-enal (128) To a solution of 7-vinylpentadecan-7-ol 127 (1.5 g, 6 mmol) in 15 mL dichloromethane, was added pyridinium chlorochromate (2.6 g, 12 mmol), and the mixture was stirred room temperature overnight. TLC suggested complete conversion of starting material. The mixture was diluted with hexanes and filtered through Celite, and the filtrate was concentrated, and the crude was purified by silica gel column chromatography using 5- 20% ethyl acetate in hexanes to give 3-hexylundec-2-enal (1.2 g, 80%). Step 4: Synthesis of 3-Hexylundecan-1-ol (129) A suspension of 3-hexylundec-2-enal 128 (1.20 g, 4.75 mmol) and 10% palladium on carbon (500 mg) in 40 mL ethyl acetate was subjected to hydrogenation using a Parr hydrogenator at 35 psi for three days. After filtration and concentration, the crude was purified by column chromatography using 10-35% ethyl acetate in hexanes to give 3- hexylundecan-1-ol (1.02 g, 83%) as clear oil. Step 5: Synthesis of 3-Hexylundecanoic acid (130) To a solution of 3-hexylundecan-1-ol 129 (1.0 g, 3.9 mmol) in 15 mL acetone, was added Jones reagent until the orange color persisted, and then the mixture was stirred for 1.5 h. After quenching with isopropanol, the resulting solution was diluted with water and extracted with ethyl acetate, the combined organic layer was dried and concentrated to give 3-hexylundecanoic acid (0.8 g, 80%) as dark oil, which was used for next step without further purification. Step 6: Synthesis of Bis(6-((3-hexylundecanoyl)oxy)hexyl) 2-((tert- butyldiphenylsilyl)oxy)pentanedioate (131) A mixture of bis(6-hydroxyhexyl) 2-((tert-butyldiphenylsilyl)oxy)pentanedioate 123 (814 mg, 1.39 mmol), 3-hexylundecanoic acid 130 (750 mg, 2.77 mmol), EDCI (2.12 g, 11 mmol) and DMAP (488 mg, 4 mmol) in 20 mL dichloromethane was stirred at room temperature for four days. After concentration, the crude was partitioned between EtOAc and saturated ammonium chloride solution, and the combined organic layer was dried over sodium sulfate. The solvent was removed under vacuum, and the crude was purified by column chromatography using 0-20% ethyl acetate in hexane to give bis(6-((3- hexylundecanoyl)oxy)hexyl) 2-((tert-butyldiphenylsilyl)oxy)pentanedioate as pale yellow oil (1.21 g, 80%). Step 7: Synthesis of Bis(6-((2-hexyldecanoyl)oxy)hexyl) 2-hydroxypentanedioate (132) To a solution of bis(6-((3-hexylundecanoyl)oxy)hexyl) 2-((tert- butyldiphenylsilyl)oxy)pentanedioate 131 (1.2 g, 1.1 mmol) in a mixture of THF (10 mL) and Pyridine (4 mL) in a Teflon flask, was added HF-pyridine complex (70wt%, 2 mL) at 0°C, and the resulting mixture was stirred at room temperature for 18 h. The reaction mixture was carefully quenched with saturated sodium bicarbonate solution and extracted with ethyl acetate. The combined organic layer was dried and concentrated, and the crude was purified by column chromatography using 0-30% ethyl acetate in hexanes to give bis(6-((3- hexylundecanoyl)oxy)hexyl) 2-hydroxypentanedioate as colorless oil (800 mg, 85%). Step 8: Synthesis of bis(6-((3-hexylundecanoyl)oxy)hexyl) 2-(((2- (dimethylamino)ethoxy)carbonyl)oxy)pentanedioate (Example 123) To a solution of bis(6-((3-hexylundecanoyl)oxy)hexyl) 2-hydroxypentanedioate 132 (250 mg, 0.29 mmol) in 2 mL pyridine and 8 mL dichloromethane, was added p-nitrophenyl chloroformate (200 mg, 0.99 mmol), and the resulting mixture was stirred for 30 min. After TLC showed the complete reaction, 2-(dimethylamino)ethan-1-ol (1.3 g, 15 mmol) and diisopropylethylamine (2 mL) were added, and the reaction mixture was stirred at room temperature overnight. MS showed the formation of desired product. The reaction mixture was washed by saturated sodium bicarbonate solution, and the combined organic layer was dried over sodium sulfate. After filtration and concentration, the crude was purified by column chromatography eluted with 0-70% ethyl acetate in hexane to afford bis(6-((3- hexylundecanoyl)oxy)hexyl) 2-(((2-(dimethylamino)ethoxy)carbonyl)oxy)pentanedioate as clear oil (210 mg, 74%). 1H NMR (400 MHz, CDCl3) δ 4.94 (dd, 1H), 4.29-4.18 (m, 2H), 4.18-4.11 (m, 2H), 4.10- 3.99 (m, 6H), 2.68-2.52 (m, 2H), 2.51-2.37 (m, 2H), 2.27 (s, 6H), 2.21 (d, 4H), 2.25-2.06 (m, 2H), 1.89-1.75 (m, 2H), 1.72-1.52 (m, 8H), 1.45-1.15 (m, 56H), 0.86 (t, 12H). APCI-MS analysis: Calculated C56H105NO11 [M+H] = 968.7, Observed = 968.6. HPLC-ELSD: tR = 7.299 min (method 2). Example 124: bis(4-((2-hexyldecanoyl)oxy)butyl) 2-(((2- (dimethylamino)ethoxy)carbonyl)oxy)pentanedioate Step 1: Synthesis of dibenzyl 2-oxopentanedioate (133) A mixture of α-ketoglutaric acid (3.7 g, 25 mmol), benzyl alcohol (5.4 g, 50 mmol) and p-toluenesulfonic acid monohydrate (240 mg, 1.3 mmol) in 10 mL toluene was heated to reflux with Dean-Stark for 2 h. After cooled to room temperature, the mixture was diluted with hexanes and washed by saturated sodium bicarbonate solution. The aqueous layer was extracted with ethyl acetate, and the combined organic layer was dried over sodium sulfate. After filtration and concentration, the crude was purified by column chromatography with 0- 25% ethyl acetate in hexanes to give dibenzyl 2-oxopentanedioate as colorless oil (7.0 g, 69%). Step 2: Synthesis of Dibenzyl 2-hydroxypentanedioate (134) A solution of dibenzyl 2-oxopentanedioate 133 (1.8 g, 5.5 mmol) in 25 mL THF was cooled to 0°C, then sodium borohydride (210 mg, 5.5 mmol) was added by portions in 5 min, and the reaction mixture was stirred at same temperature for 1 h. Saturated ammonium chloride solution was added slowly, and the resulting mixture was extracted with ethyl acetate. The combined organic layer was washed with brine and dried over sodium sulfate. After concentration, 1.7 g crude was used for the next step without purification. Step 3: Synthesis of Dibenzyl 2-((tert-butyldiphenylsilyl)oxy)pentanedioate (135) To a stirred solution of crude dibenzyl 2-hydroxypentanedioate 134 (1.7 g, 5.2 mmol) and imidazole (420 mg, 6.2 mmol) in 15 mL dichloromethane, was added tert- butylchlorodiphenylsilane (1.4 g, 5.2 mmol), and the mixture was stirred room temperature overnight. The reaction mixture was quenched by saturated ammonium chloride solution and extracted with dichloromethane. The combined organic layer was dried and concentrated, and the crude was purified by column chromatography using 0-20% ethyl acetate in hexanes to give dibenzyl 2-((tert-butyldiphenylsilyl)oxy)pentanedioate as colorless oil (550 mg, 19%). Step 4: Synthesis of 5-(Benzyloxy)-4-((tert-butyldiphenylsilyl)oxy)-5-oxopentanoic acid (136) A mixture of dibenzyl 2-((tert-butyldiphenylsilyl)oxy)pentanedioate 135 (250 mg, 0.44 mmol) and 20% palladium hydroxide on carbon (47 mg) in 10 mL ethyl acetate was subjected to hydrogenation at room temperature for 5 h. After filtration, the filtrate was concentrated, and the crude was purified by column chromatography using 0-100% ethyl acetate in hexane to afford a mixture of 5-(benzyloxy)-4-((tert-butyldiphenylsilyl)oxy)-5- oxopentanoic acid and 2-((tert-butyldiphenylsilyl)oxy)pentanedioic acid as colorless oil (105 mg, 50%). Step 5: Synthesis of 1-Benzyl 5-(4-((2-hexyldecanoyl)oxy)butyl) 2-((tert- butyldiphenylsilyl)oxy)pentanedioate (137) A mixture of 5-(benzyloxy)-4-((tert-butyldiphenylsilyl)oxy)-5-oxopentanoic acid 136 (contains diacid impurity, 105 mg, 0.22 mmol), 4-hydroxybutyl 2-hexyldecanoate (109 mg, 0.33 mmol), DMAP (40 mg, 0.33 mmol) and EDCI (169 mg, 0.88 mmol) in 8 mL dichloromethane was stirred at room temperature overnight. After concentration, the residue was dissolved in ethyl acetate and washed with saturated ammonium chloride solution. The organic layer was dried and concentrated, and the crude was purified by column chromatography to give 1-benzyl 5-(4-((2-hexyldecanoyl)oxy)butyl) 2-((tert- butyldiphenylsilyl)oxy)pentanedioate as colorless oil (110 mg, 63%). Step 6: Synthesis of 2-((tert-butyldiphenylsilyl)oxy)-5-(4-((2-hexyldecanoyl)oxy)butoxy)-5- oxopentanoic acid (138) A mixture of 1-benzyl 5-(4-((2-hexyldecanoyl)oxy)butyl) 2-((tert- butyldiphenylsilyl)oxy)pentanedioate 137 (110 mg, 0.14 mmol) and 10% palladium on carbon (15 mg) in 10 mL ethyl acetate was subjected to hydrogenation at room temperature for 48 h. After filtration, the filtrate was concentrated, and the crude was purified by column chromatography using 0-100% ethyl acetate in hexane to afford a mixture of 2-((tert- butyldiphenylsilyl)oxy)-5-(4-((2-hexyldecanoyl)oxy)butoxy)-5-oxopentanoic acid as colorless oil (75 mg, 77%). Step 7: Synthesis of Bis(4-((2-hexyldecanoyl)oxy)butyl) 2-((tert- butyldiphenylsilyl)oxy)pentanedioate (139)
Figure imgf000322_0001
A mixture of 2-((tert-butyldiphenylsilyl)oxy)-5-(4-((2-hexyldecanoyl)oxy)butoxy)-5- oxopentanoic acid 8 (75 mg, 0.11 mmol), 4-hydroxybutyl 2-hexyldecanoate 138 (35 mg, 0.11 mmol), DMAP (13 mg, 0.11 mmol) and EDCI (83 mg, 0.43 mmol) in 8 mL dichloromethane was stirred at room temperature for three days. After concentration, the residue was dissolved in ethyl acetate and washed with saturated ammonium chloride solution. The organic layer was dried and concentrated, and the crude was purified by column chromatography eluted with 0-25% ethyl acetate in hexane to give bis(4-((2-hexyldecanoyl)oxy)butyl) 2-((tert- butyldiphenylsilyl)oxy)pentanedioate as colorless oil (90 mg, 83%). Step 8: Synthesis of Bis(4-((2-hexyldecanoyl)oxy)butyl) 2-hydroxypentanedioate (140) To a solution of bis(4-((2-hexyldecanoyl)oxy)butyl) 2-((tert- butyldiphenylsilyl)oxy)pentanedioate 139 (80 mg, 0.079 mmol) in a mixture of THF (8 mL) and pyridine (1 mL) in a Teflon flask, was added HF-pyridine solution (70wt%, 0.3 mL) at 0°C, and the resulting mixture was stirred at room temperature for 18 h. The reaction mixture was carefully quenched with saturated sodium bicarbonate solution and extracted with ethyl acetate. The combined organic layer was dried and concentrated, and the crude was purified by column chromatography using 0-25% ethyl acetate in hexanes to give bis(4-((2- hexyldecanoyl)oxy)butyl) 2-hydroxypentanedioate as colorless oil (45 mg, 74%). Step 9: Synthesis of bis(4-((2-hexyldecanoyl)oxy)butyl) 2-(((2- (dimethylamino)ethoxy)carbonyl)oxy)pentanedioate (Example 124) To a solution of bis(4-((2-hexyldecanoyl)oxy)butyl) 2-hydroxypentanedioate 140 (45 mg, 0.06 mmol) in 0.5 mL pyridine and 8 mL dichloromethane, was added p-nitrophenyl chloroformate (59 mg, 0.29 mmol), and the resulting mixture was stirred for 90 min. After TLC showed the complete reaction, 2-(dimethylamino)ethan-1-ol (0.26 g, 2.9 mmol) and diisopropylethylamine (0.5 mL) were added, and the reaction mixture was stirred at room temperature overnight. MS showed the formation of desired product. The reaction mixture was washed by saturated sodium bicarbonate solution, and the combined organic layer was dried over sodium sulfate. After filtration and concentration, the crude was purified by column chromatography eluted with 0-10% methanol in dichloromethane to afford bis(4-((2- hexyldecanoyl)oxy)butyl) 2-(((2-(dimethylamino)ethoxy)carbonyl)oxy)pentanedioate as clear oil (23 mg, 44%). 1H NMR (400 MHz, CDCl3) δ 4.99-4.89 (m, 1H), 4.31-4.13 (m, 4H), 4.13-3.99 (m, 6H), 2.67-2.52 (m, 2H), 2.52-2.37 (m, 2H), 2.35-2.18 (m, 9H), 2.18-2.07 (m, 1H), 1.85-1.62 (m, 8H), 1.62-1.48 (m, 4H), 1.48-1.34 (m, 4H), 1.34-1.05 (m, 40H), 0.86 (t, 12H). APCI-MS analysis: Calculated C50H93NO11 [M+H] = 884.6, Observed = 884.6. HPLC-ELSD: tR = 8.226 min (method 1). Example 125: bis(8,8-bis(octyloxy)octyl) 2-(((2- (dimethylamino)ethoxy)carbonyl)oxy)pentanedioate Step 1: Synthesis of 8-(Benzyloxy)octanal (141) To a solution of 8-(benzyloxy)octan-1-ol (4.0 g, 17 mmol) in 35 mL dichloromethane, was added pyridinium chlorochromate (4.7 g, 22 mmol), and the mixture was stirred at room temperature for 3 h. TLC monitoring confirmed the completion of reaction. The reaction mixture was triturated with hexanes, and the decanted solution was concentrated. The residue was purified by column chromatography using 0-20% ethyl acetate in hexanes to give 8- (benzyloxy)octanal as colorless oil (3.1 g, 78%). Step 2: Synthesis of (((8,8-dimethoxyoctyl)oxy)methyl)benzene (142) A mixture of 8-(benzyloxy)octanal 141 (3.1 g, 13 mmol), 4-methylbenzenesulfonic acid hydrate (2.5 g, 13 mmol) and trimethyl orthoformate (11 g, 0.11 mole) was stirred at room temperature for 2 h. The reaction mixture was diluted with dichloromethane and washed with saturated sodium bicarbonate solution and brine. After dried and concentrated, the residue was azeotropic evaporated with toluene to get (((8,8- dimethoxyoctyl)oxy)methyl)benzene as colorless oil (3.65 g, 98%), which was used for the next step without any further purification. Step 3: Synthesis of (((8,8-Bis(octyloxy)octyl)oxy)methyl)benzene (143) A mixture of (((8,8-dimethoxyoctyl)oxy)methyl)benzene 142 (2.8 g, 10 mmol), 1- octanol (2.6 g, 20 mmol) and pyridine 4-methylbenzenesulfonate (0.25 g, 1.0 mmol) was heated at 120°C for 4 h. After cooled to room temperature, the reaction mixture was diluted with hexanes and washed with saturated sodium bicarbonate. The combined organic layer was dried and concentrated, and the crude was purified by column chromatography using 0- 20% ethyl acetate in hexanes to give (((8,8-bis(octyloxy)octyl)oxy)methyl)benzene as colorless oil (2.5 g, 53%). Step 4: Synthesis of 8,8-bis(octyloxy)octan-1-ol (144) A mixture of (((8,8-bis(octyloxy)octyl)oxy)methyl)benzene 143 (800 mg, 1.68 mmol) and 20% palladium hydroxide on carbon (47 mg) in 15 mL hexane was subjected to hydrogenation at room temperature overnight. After filtration and concentration, the crude was purified by column chromatography using 0-50% ethyl acetate in hexanes to get 600 mg mixture of desired product with 1-octanol. The desired product was unstable in hydrogenation condition. The 600 mg mixture of 8,8-bis(octyloxy)octan-1-ol and 1-octanol was dissolved in 8 mL dichloromethane, then imidazole (251 mg, 3.69 mmol) and tert-butyldiphenylsilyl chloride (1.01 g, 3.69 mmol) were added, and the resulting mixture was stirred at room temperature overnight. After concentration, the residue was purified by column chromatography using hexane to get tert-butyl(octyloxy)diphenylsilane as colorless oil (600 mg, 78%). To a solution of ((8,8-bis(octyloxy)octyl)oxy)(tert-butyl)diphenylsilane (500 mg, 0.8 mmol) in THF (10 mL) and pyridine (3 mL) in a Teflon flask, was added hydrogen fluoride- pyridine complex (70wt%, 79 mg, 0.8 mmol), and the mixture was stirred at room temperature overnight. The reaction mixture was carefully quenched by saturated sodium bicarbonate and extracted with ethyl acetate. The combined organic layer was dried and concentrated, and the crude was purified by column chromatography using 0-25% ethyl acetate in hexanes to 8,8-bis(octyloxy)octan-1-ol as colorless oil (280 mg, 90%). Step 5: Synthesis of 2-((tert-Butyldiphenylsilyl)oxy)pentanedioic acid (145) A mixture of dibenzyl 2-((tert-butyldiphenylsilyl)oxy)pentanedioate 135 (2.9 g, 5.1 mmol) and 10% palladium on carbon (540 mg) in 20 mL ethyl acetate was subjected to hydrogenation at room temperature overnight. After filtration, the filtrate was concentrated, and the crude was purified by column chromatography using 0-100% ethyl acetate in hexane to afford 2-((tert-butyldiphenylsilyl)oxy)pentanedioic acid as colorless oil (2.0 g, 61%). Step 6: Synthesis of Bis(8,8-bis(octyloxy)octyl) 2-((tert-butyldiphenylsilyl)oxy)pentanedioate (146) A mixture of 8,8-bis(octyloxy)octan-1-ol 144 (0.25 g, 0.64 mmol), 2-((tert- butyldiphenylsilyl)oxy)pentanedioic acid 145 (125 mg, 323 μmol), DMAP (39 mg, 0.32 mmol) and EDCI (61 mg, 0.32 mmol) in 8 mL dichloromethane was stirred at room temperature for two days. After concentration, the residue was triturated with hexanes. The solution was concentrated and purified by column chromatography using 0-35% ethyl acetate in hexanes to get bis(8,8-bis(octyloxy)octyl) 2-((tert-butyldiphenylsilyl)oxy)pentanedioate as colorless oil (150 mg, 42%). Step 7: Synthesis of Bis(8,8-bis(octyloxy)octyl) 2-hydroxypentanedioate (147) To a solution of bis(8,8-bis(octyloxy)octyl) 2-((tert- butyldiphenylsilyl)oxy)pentanedioate 146 (150 mg, 0.133 mmol) in a mixture of THF (8 mL) and pyridine (3 mL) in a Teflon flask, was added HF-pyridine solution (70wt%, 0.2 mL) at 0°C, and the resulting mixture was stirred at room temperature overnight. The reaction mixture was carefully quenched with saturated sodium bicarbonate solution and extracted with ethyl acetate. The combined organic layer was dried and concentrated, and the crude was purified by column chromatography using 0-30% ethyl acetate in hexanes to give bis(8,8-bis(octyloxy)octyl) 2-hydroxypentanedioate as colorless oil (75 mg, 63%). Step 8: Synthesis of bis(8,8-bis(octyloxy)octyl) 2-(((2- (dimethylamino)ethoxy)carbonyl)oxy)pentanedioate (Example 125) To a solution of bis(8,8-bis(octyloxy)octyl) 2-hydroxypentanedioate 147 (75 mg, 0.085 mmol) in 1 mL pyridine and 8 mL dichloromethane, was added p-nitrophenyl chloroformate (68 mg, 0.34 mmol), and the resulting mixture was stirred for 90 min. After TLC showed the complete reaction, 2-(dimethylamino)ethan-1-ol (0.38 g, 4.2 mmol) and diisopropylethylamine (0.5 mL) were added, and the reaction mixture was stirred at room temperature overnight. MS showed the formation of desired product. The reaction mixture was washed by saturated sodium bicarbonate solution, and the combined organic layer was dried over sodium sulfate. After filtration and concentration, the crude was purified by column chromatography eluted with 0-10% methanol in dichloromethane to afford bis(8,8- bis(octyloxy)octyl) 2-(((2-(dimethylamino)ethoxy)carbonyl)oxy)pentanedioate as pale yellow oil (56 mg, 66%). 1H NMR (400 MHz, CDCl3) δ 4.93 (dd, 1H), 4.43 (t, 2H), 4.29-4.18 (m, 2H), 4.13 (t, 2H), 4.04 (t, 2H), 3.60-3.47 (m, 4H), 3.42-3.32 (m, 4H), 2.66-2.52 (m, 2H), 2.51-2.36 (m, 2H), 2.26 (s, 6H), 2.24-2.18 (m, 1H), 2.18-2.06 (m, 1H), 1.67-1.47 (m, 16H), 1.42-1.15 (m, 56H), 0.86 (t, 12H). APCI-MS analysis: Calculated C58H113NO11 [M+H] = 1000.8, Observed = 1000.9. HPLC-ELSD: tR = 8.650 min (method 3). Example 126: bis(5-((2-hexyldecanoyl)oxy)pentyl) 2-((4- (dimethylamino)butyl)disulfaneyl)pentanedioate Step 1: Synthesis of (4-Bromobutyl)(trityl)sulfane (148) To a suspension of triphenylmethanethiol (5.0 g, 18 mmol) in 40 mL ethanol and 40 mL water, was added a solution of sodium hydroxid (1.44 g, 36 mmol) in 40 mL water, and the mixture was stirred at room temperature for 10 min. Then a solution of 1,4- dibromobutane (3.6 g, 17 mmol) in 40 mL ethanol was added slowly, and the mixture was stirred vigorously for 4 h. The reaction mixture was diluted with dichloromethane and washed with saturated sodium bicarbonate solution. The combined organic layer was dried and concentrated, and the residue was triturated with methanol to give (4- bromobutyl)(trityl)sulfane as orange solid (5.5 g, 67%), which was used for the next step without further purification. Step 2: Synthesis of N,N-Dimethyl-4-(tritylthio)butan-1-amine (149) In a seal tube, to a mixture of (4-bromobutyl)(trityl)sulfane 148 (3.0 g, 7.3 mmol) in 20 mL acetonitrile, was added a solution of dimethylamine in THF (2M, 11 mL, 22 mmol), and the mixture was heated at 80°C overnight. After cooled to room temperature, the mixture was concentrated and purified by column chromatography using 0-25% methanol in ethyl acetate to get N,N-dimethyl-4-(tritylthio)butan-1-amine as brown oil (2.0 g, 73%). Step 3: Synthesis of 4-(Dimethylamino)butane-1-thiol trifluoroacetate (150) To a solution of N,N-dimethyl-4-(tritylthio)butan-1-amine 149 (1.1 g, 2.9 mmol) and triethylsilane (1.4 mL, 8.8 mmol) in 5 mL dichloromethane, wad added trifluoroacetic acid (3.4 mL, 44 mmol), and the mixture was stirred at room temperature for 4 h. After evaporation, the residue was triturated with hexanes to give 4-(dimethylamino)butane-1-thiol trifluoroacetate as brown semi-solid (700 mg, 93%), which was used for the next step without further purification. Step 4: Synthesis of Dimethyl 2-(acetylthio)pentanedioate (151) To a solution of potassium ethanethioate (2.5 g, 22 mmol) in 10 mL acetonitrile and 20 mL DMF, was added dimethyl 2-bromopentanedioate (4.3 g, 18 mmol) dropwise, and the resulting mixture was allowed to stir for 2 h. The reaction mixture was diluted with water and extracted with ethyl acetate, and the combined organic layer was washed with brine. After dried over sodium sulfate, the solvent was removed under vacuum to give dimethyl 2- (acetylthio)pentanedioate as light brown oil (4.0 g, 95%). Step 5: Synthesis of 2-Mercaptopentanedioic acid (152) To a solution of dimethyl 2-(acetylthio)pentanedioate 151 (950 mg, 4.06 mmol) in 20 mL THF, was added 10 mL 2 M sodium hydroxide solution, and the mixture was stirred at room temperature overnight. The reaction mixture was partitioned with water and ethyl acetate, and the aqueous layer was acidified using 6M HCl solution to pH 2. After extracted with ethyl acetate, the combined organic layer was dried and concentrated to give 2- mercaptopentanedioic acid as pale yellow solid (600 mg, 90%), which was used for the next step without purification. Step 6: Synthesis of Bis(5-hydroxypentyl) 2-mercaptopentanedioate (153) A mixture of 2-mercaptopentanedioic acid 152 (450 mg, 2.74 mmol), pentane-1,5-diol (4.28 g, 41.1 mmol) and zinc chloride (93.4 mg, 0.685 mmol) was heated under nitrogen atomsphere at 130°C for 4 h. After cooled to room temperature, the mixture was diluted with water and extracted with dichloromethane, the combined organic layer was dried and concentrated to give bis(5-hydroxypentyl) 2-mercaptopentanedioate as colorless oil (750 mg, 73%), which was used for the next step without further purification. Step 7: Synthesis of Bis(5-hydroxypentyl) 2-(pyridin-2-yldisulfaneyl)pentanedioate (154) A mixture of bis(5-hydroxypentyl) 2-mercaptopentanedioate 153 (0.67 g, 2.0 mmol) and 1,2-di(pyridin-2-yl)disulfane (0.44 g, 2.0 mmol) in 10 mL dichloromethane was stirred under nitrogen atmosphere at room temperature for 5 h. The mixture was purified by column purification using 0-5% methanol in ethyl acetate to give bis(5-hydroxypentyl) 2-(pyridin-2- yldisulfaneyl)pentanedioate as pale yellow oil (600 mg, 67%). Step 8: Synthesis of Bis(5-((2-hexyldecanoyl)oxy)pentyl) 2-(pyridin-2- yldisulfaneyl)pentanedioate (155) To a solution of 2-hexyldecanoic acid (1.53 g, 5.97 mmol) in 10 mL dichloromethane, was added oxalyl chloride (1.57 mL, 17.9 mmol) at 0°C followed by few drops of DMF, and the mixture was warmed up to room temperature for 90 min. After concentration, the residue was azeotropic evaporated with toluene several times. The crude 2-hexyldecanoyl chloride was dissolved in 4 mL pyridine and cooled to 0°C, then a solution of bis(5-hydroxypentyl) 2- (pyridin-2-yldisulfaneyl)pentanedioate 154 (0.62 g, 1.4 mmol) in 4 mL dichloromethane was slowly added, and the resulting mixture was stirred at room temperature for 30 min. TLC showed complete reaction. The reaction mixture was quenched with saturated ammonium chloride solution and extracted with dichloromethane. The combined organic layer was concentrated, and the residue was purified by column chromatography using 0-25% ethyl acetate in hexanes to get bis(5-((2-hexyldecanoyl)oxy)pentyl) 2-(pyridin-2- yldisulfaneyl)pentanedioate as pale yellow oil (750 mg, 58%). Step 9: Synthesis of bis(5-((2-hexyldecanoyl)oxy)pentyl) 2-((4- (dimethylamino)butyl)disulfaneyl)pentanedioate (Example 126) A mixture of bis(5-((2-hexyldecanoyl)oxy)pentyl) 2-(pyridin-2- yldisulfaneyl)pentanedioate 155 (350 mg, 0.379 mmol) and 4-(dimethylamino)butane-1-thiol trifluoroacetate 150 (176 mg, 0.759 mmol) in 8 mL dichloromethane and 3 mL methanol was stirred under nitrogen atmosphere for 5 h. TLC showed compete reaction. The reaction mixture was partitioned with sodium bicarbonate solution and dichloromethane, and the combined organic layer was concentrated. The crude was purified by column chromatography using 0-10% methanol in ethyl acetate to afford bis(5-((2- hexyldecanoyl)oxy)pentyl) 2-((4-(dimethylamino)butyl)disulfaneyl)pentanedioate as pale yellow oil (260 mg, 72 %). 1H NMR (400 MHz, CDCl3) δ 4.12 (t, 2H), 4.06 (t, 6H), 3.42 (t, 1H), 2.70 (t, 2H), 2.43 (t, 2H), 2.34-2.25 (m, 4H), 2.22 (s, 6H), 2.21-2.07 (m, 2H), 1.81-1.49 (m, 16H), 1.48-1.35 (m, 8H), 1.33-1.15 (m, 40H), 0.86 (t, 12H). APCI-MS analysis: Calculated C53H101NO8S2 [M+H] = 944.6, Observed = 943.9. HPLC-ELSD: tR = 10.334 min (method 1). Example 127: dioctadecyl 2-((3-(dimethylamino)propyl)disulfaneyl)succinate Step 1: Synthesis of Dioctadecyl 2-mercaptosuccinate (156) To a mixture of mercaptosuccinic acid (15.0 g, 0.1 mole) and 1-octadecanol (59.49 g, 0.22 mole) in 100 mL benzene, was added 0.25 mL concentrated sulfuric acid, and the mixture was heated to reflux with Dean-Stark apparatus overnight. After cooled to room temperature, the reaction mixture was washed with saturated bicarbonate solution and water. The organic layer was dried over sodium sulfate and concentrated to get dioctadecyl 2- mercaptosuccinate as white solid (65.4 g, quant.), which was used for the next step without purification. Step 2: Synthesis of Dioctadecyl 2-(pyridin-2-yldisulfaneyl)succinate (157) A solution of dioctadecyl 2-mercaptosuccinate 156 (10.0 g, 15.2 mmol) in 150 mL dichloromethane was purged with nitrogen, then 2,2-dipyridyl disulfide (4.53 g, 16.8 mmol) was added, and the resulting solution was stirred at room temperature overnight under nitrogen. After concentration, the crude was purified by flash column chromatography (SiO2: 0-50% EtOAc in hexane) to get dioctadecyl 2-(pyridin-2-yldisulfaneyl)succinate as white solid (6.2 g, 53%). Step 3: Synthesis of Dioctadecyl 2-((3-(dimethylamino)propyl)disulfaneyl)succinate (Example 127) A solution of dioctadecyl 2-(pyridin-2-yldisulfaneyl)succinate 157 (1.0 g, 1.3 mmol) in 30 mL chloroform was purged with nitrogen, then 3-(dimethylamino)propane-1-thiol hydrochloride (425 mg, 2.75 mmol) was added, and the resulting solution was stirred at room temperature for 3 h. After concentration, the crude was purified by flash column chromatography (SiO2: 0-10% methanol in dichloromethane) to get dioctadecyl 2-((3- (dimethylamino)propyl)disulfaneyl)succinate as white solid (85 mg, 8%). 1H NMR (300 MHz, CDCl3) δ 4.12 (t, 2H), 4.07 (t, 2H), 3.76 (dd, 1H), 3.11 (dd, 1H), 2.79 (dd, 1H), 2.74 (t, 2H), 2.32 (t, 2H), 2.20 (s, 6H), 1.80 (quint, 2H), 1.68-1.56 (m, 4H), 1.25 (m, 60H), 0.87 (t, 6H). APCI-MS analysis: Calculated C45H89NO4S2 [M+H] = 772.6, Observed = 772.6. Example 128: dihexadecyl 2-((3-(dimethylamino)propyl)disulfaneyl)succinate Step 1: Synthesis of Dihexadecyl 2-mercaptosuccinate (158) To a mixture of mercaptosuccinic acid (10.0 g, 66.6 mmol) and 1-hexadecanol (35.5 g, 0.146 mole) in 100 mL benzene, was added 0.25 mL concentrated sulfuric acid, and the mixture was heated to reflux with Dean-Stark apparatus overnight. After cooled to room temperature, the reaction mixture was washed with saturated bicarbonate solution and water. The organic layer was dried over sodium sulfate and concentrated to get dihexadecyl 2- mercaptosuccinate as white solid (24.5 g, 93%), which was used for the next step without purification. Step 2: Synthesis of Dihexadecyl 2-(pyridin-2-yldisulfaneyl)succinate (159) A solution of dihexadecyl 2-mercaptosuccinate 158 (5.0 g, 8.3 mmol) in 150 mL dichloromethane was purged with nitrogen, then 2,2-dipyridyl disulfide (2.0 g, 9.2 mmol) was added, and the resulting solution was stirred at room temperature overnight under nitrogen. After concentration, the crude was purified by flash column chromatography (SiO2: 0-50% EtOAc in hexane) to get dihexadecyl 2-(pyridin-2-yldisulfaneyl)succinate as white solid (3.6 g, 62%). Step 3: Synthesis of dihexadecyl 2-((3-(dimethylamino)propyl)disulfaneyl)succinate (Example 128) A solution of dihexadecyl 2-(pyridin-2-yldisulfaneyl)succinate 159 (2.0 g, 2.8 mmol) in 60 mL chloroform was purged with nitrogen, then 3-(dimethylamino)propane-1-thiol hydrochloride (872 mg, 5.6 mmol) was added, and the resulting solution was stirred at room temperature for 3 h. After concentration, the crude was purified by flash column chromatography (SiO2: 0-10% methanol in dichloromethane) to get dihexadecyl 2-((3- (dimethylamino)propyl)disulfaneyl)succinate as white solid (700 mg, 36%). 1H NMR (300 MHz, CDCl3) δ 4.12 (t, 2H), 4.07 (t, 2H), 3.75 (dd, 1H), 3.11 (dd, 1H), 2.78 (dd, 1H), 2.73 (t, 2H), 2.31 (t, 2H), 2.20 (s, 6H), 1.80 (quint, 2H), 1.68-1.55 (m, 4H), 1.24 (m, 52H), 0.87 (t, 6H). APCI-MS analysis: Calculated C41H81NO4S2 [M+H] = 716.5, Observed = 716.5. Example 129: di((9Z,12Z)-octadeca-9,12-dien-1-yl) 2-((3- (dimethylamino)propyl)disulfaneyl)succinate Step 1: Synthesis of Di((9Z,12Z)-octadeca-9,12-dien-1-yl) 2-mercaptosuccinate (160) To a mixture of mercaptosuccinic acid (5.0 g, 33.3 mmol) and linoleyl alcohol (17.7 g, 66.6 mmol) in 60 mL benzene, was added 0.15 mL concentrated sulfuric acid, and the mixture was heated to reflux with Dean-Stark apparatus overnight. After cooled to room temperature, the reaction mixture was washed with saturated bicarbonate solution and water. The organic layer was dried over sodium sulfate and concentrated, the crude was purified by column chromatography (SiO2: 0-30% EtOAc in hexane) to get di((9Z,12Z)-octadeca-9,12- dien-1-yl) 2-mercaptosuccinate as clear oil (14.2 g, 65%). Step 2: Synthesis of Di((9Z,12Z)-octadeca-9,12-dien-1-yl) 2-(pyridin-2- yldisulfaneyl)succinate (161) A solution of di((9Z,12Z)-octadeca-9,12-dien-1-yl) 2-mercaptosuccinate 160 (5.0 g, 7.7 mmol) in 100 mL dichloromethane was purged with nitrogen, then 2,2-dipyridyl disulfide (3.4 g, 15.4 mmol) was added, and the resulting solution was stirred at room temperature overnight under nitrogen. After concentration, the crude was purified by flash column chromatography (SiO2: 0-50% EtOAc in hexane) to get di((9Z,12Z)-octadeca-9,12-dien-1-yl) 2-(pyridin-2-yldisulfaneyl)succinate as pale yellow oil (2.5 g, 43%). Step 3: Synthesis of di((9Z,12Z)-octadeca-9,12-dien-1-yl) 2-((3- (dimethylamino)propyl)disulfaneyl)succinate (Example 129) A solution of di((9Z,12Z)-octadeca-9,12-dien-1-yl) 2-(pyridin-2- yldisulfaneyl)succinate 161 (700 mg, 0.92 mmol) in 30 mL chloroform was purged with nitrogen, then 3-(dimethylamino)propane-1-thiol hydrochloride (288 mg, 1.85 mmol) was added, and the resulting solution was stirred at room temperature for 3 h. After concentration, the crude was purified by flash column chromatography (SiO2: 0-10% methanol in dichloromethane) to get di((9Z,12Z)-octadeca-9,12-dien-1-yl) 2-((3- (dimethylamino)propyl)disulfaneyl)succinate as colorless oil (130 mg, 18%). 1H NMR (300 MHz, CDCl3) δ 5.45-5.27 (m, 8H), 4.12 (t, 2H), 4.07 (t, 2H), 3.76 (dd, 1H), 3.11 (dd, 1H), 2.85-2.68 (m, 7H), 2.31 (t, 2H), 2.21 (d, 6H), 2.07-1.96 (m, 8H), 1.80 (quint, 2H), 1.70-1.54 (m, 4H), 1.29 (m, 32H), 0.88 (t, 6H). APCI-MS analysis: Calculated C45H81NO4S2 [M+H] = 764.5, Observed = 764.5. Example 130: dihexadecyl 2-(N-(3-(dimethylamino)propyl)sulfamoyl)succinate Step 1: Synthesis of Dihexadecyl maleate (162) A mixture of maleic anhydride (1.74 g, 17.8 mmol), 1-hexadecanol (12.9 g, 53.3 mmol) and p-toluenesulfonic acid monohydrate (3.04 g, 17.8 mmol) in 80 mL toluene was heated with Dean-Stark apparatus to 130°C for 6 h. After concentration, the residue was recrystallized in methanol to give dihexadecyl maleate as waxy solid (7.0 g, 60%). Step 2: Synthesis of Sodium 1,4-bis(hexadecyloxy)-1,4-dioxobutane-2-sulfonate (163) To a solution of dihexadecyl maleate 162 (3.6 g, 6.37 mmol) in 60 mL dioxane, was added aqueous sodium bisulfite solution (7.2 g in 30 mL water, 69.2 mmol), and the resulting mixture was heated to reflux for 48 h. After concentration, the residue was partitioned between water and EtOAc, and the combined organic layer was washed with brine. The solvent was removed under vacuum to afford sodium 1,4-bis(hexadecyloxy)-1,4- dioxobutane-2-sulfonate as waxy solid (3.2 g, 75%), which was used for the next step without further purification. Step 2: Synthesis of Dihexadecyl 2-(N-(3-(dimethylamino)propyl)sulfamoyl)succinate (Example 130) To an ice-cold solution of sodium 1,4-bis(hexadecyloxy)-1,4-dioxobutane-2-sulfonate 163 (110 mg, 0.17 mmol) in 3 mL dichloromethane, was added oxalyl chloride (40 µL, 0.51 mmol) dropwise, then 2 drops DMF was added, and the mixture was warmed up to room temperature for 3 h. The volatiles were removed under vacuum, and the residue was dissolved in 5 mL dichloromethane. After cooled to 0°C, a solution of N1,N1- dimethylpropane-1,3-diamine 6 (197 mg, 1.93 mmol) in 1 mL dichloromethane was added slowly, and the resulting mixture was stirred at room temperature for 2 h. MS showed the formation of the desired product. The reaction mixture was diluted with water and extracted with dichloromethane, and the combined organic layer was washed with brine. After drying and concentration, the residue was purified by column chromatography (SiO2: 0-30% methanol in dichloromethane) to give dhexadecyl 2-(N-(3- (dimethylamino)propyl)sulfamoyl)succinate as pale yellow solid (25 mg, 20%) 1H NMR (400 MHz, CD3OD) δ 4.32-4.01 (m, 4H), 3.23-3.07 (m, 3H), 2.99-2.90 (m, 1H), 2.69-2.56 (m, 2H), 2.43 (s, 6H), 1.85-1.55 (m, 6H), 1.47-1.15 (m, 53H), 0.97-0.82 (m, 6H). APCI-MS analysis: Calculated C41H82N2O6S [M+H] = 731.6, Observed = 731.5. Example 131: bis(8-oxo-8-(pentadecan-7-yloxy)octyl) 2-((((2- (dimethylamino)ethoxy)carbonyl)oxy)methyl)pentanedioate Step 1: Synthesis of Diethyl 2-(3-((tert-butyldimethylsilyl)oxy)propyl)malonate (164) A mixture of diethyl malonate (1.6 g, 10 mmol), (3-bromopropoxy)(tert- butyl)dimethylsilane (2.0 g, 8.0 mmol) and potassium carbonate (1.1 g, 8.0 mmol) in THF (2.0 mL) and DMF (10 mL) was heated at 100°C overnight. The reaction was cooled to room temperature and diluted with saturated ammonium chloride. After extraction with hexanes, the organic layer was washed with brined and dried over sodium sulfate. The solvent was removed under vacuum, and the residue was purified by column chromatography using 0-6% ethyl acetate in hexanes to give diethyl 2-(3-((tert-butyldimethylsilyl)oxy)propyl)malonate as colorless oil (1.8 g, 68%). Step 2: Synthesis of 2-(3-((tert-Butyldimethylsilyl)oxy)propyl)propane-1,3-diol (165) To a solution of diethyl 2-(3-((tert-butyldimethylsilyl)oxy)propyl)malonate 164 (1.4 g, 4.2 mmol) in 20 mL ether, was slowly added a solution of lithium aluminum hydride in THF (2 M, 6.5 mL, 13 mmol), and the mixture was stirred at room temperature overnight. After workup, the solution was concentrated to give 2-(3-((tert- butyldimethylsilyl)oxy)propyl)propane-1,3-diol as colorless oil (710 mg, 68%), which was used for the next step without purification. Step 3: Synthesis of 2-((Benzyloxy)methyl)-5-((tert-butyldimethylsilyl)oxy)pentan-1-ol (166) A suspension of sodium hydride (60% in mineral oil, 270 mg, 11.3 mmol) in 8.0 mL DMF was cooled to 0°C, then a solution of 2-(3-((tert- butyldimethylsilyl)oxy)propyl)propane-1,3-diol 165 (700 mg, 2.82 mmol) in 2 mL THF was slowly added, and the mixture was stirred for 15 min at this temperature. A solution of benzyl bromide (482 mg, 2.82 mmol) in 1 mL THF was added dropwise in 10 min, and the resulting mixture was stirred at this temperature for 90 min. The reaction mixture was carefully quenched with ice followed by saturated ammonium chloride solution, and then extracted with ethyl acetate. The combined organic layer was washed with brine, dried and concentrated, and the residue was purified by column chromatography using 0-20% ethyl acetate in hexanes to afford 2-((benzyloxy)methyl)-5-((tert-butyldimethylsilyl)oxy)pentan-1- ol as colorless oil (750 mg, 78%). Step 4: Synthesis of 2-((Benzyloxy)methyl)pentane-1,5-diol (167) To a solution of 2-((benzyloxy)methyl)-5-((tert-butyldimethylsilyl)oxy)pentan-1-ol 166 (500 mg, 1.48 mmol) in 6.0 mL dichloromethane, was added five drops of diluted hydrochloride solution in methanol, and the mixture was stirred at room temperature for 1 h. TLC showed complete reaction. The reaction was quenched by saturated sodium bicarbonate solution and extracted with dichloromethane. The organic layers was washed with brine, dried and concentrated to give 2-((benzyloxy)methyl)pentane-1,5-diol as colorless oil (320 mg, 82%), which was used for the next step without purification. Step 5: Synthesis of 2-((Benzyloxy)methyl)pentanedioic acid (168)
Figure imgf000338_0001
To a solution of 2-((benzyloxy)methyl)pentane-1,5-diol 167 (320 mg, 1.43 mmol) in 10 mL acetone, was added Jones reagent until orange color persisted. The mixture was stirred at room temperature for 2 h. After quenched by isopropanol, the blue solution was diluted with water, extracted with ethyl acetate. The organic layer was washed with brine, dried and concentrated to give 2-((benzyloxy)methyl)pentanedioic acid as colorless viscous oil (280 mg, 70%), which was used for the next step without purification Step 6: Synthesis of Bis(8-oxo-8-(pentadecan-7-yloxy)octyl) 2- ((benzyloxy)methyl)pentanedioate (169)
Figure imgf000338_0002
A mixture of 2-((benzyloxy)methyl)pentanedioic acid 168 (200 mg, 0.793 mmol), pentadecan-7-yl 8-hydroxyoctanoate 84 (588 mg, 1.59 mmol), EDCI (152 mg, 0.793 mmol) and DMAP (96.9 mg, 0.793 mmol) in 10 mL dichloromethane was stirred at room temperature for 3 days. The reaction was quenched with saturated ammonium chloride solution and extracted with dichloromethane. The organic layer was concentrated, and the residue was purified by column chromatography using 0-25% ethyl acetate in hexanes to give bis(8-oxo-8-(pentadecan-7-yloxy)octyl) 2-((benzyloxy)methyl)pentanedioate as colorless oil (620 mg, 53%). Step 7: Synthesis of Bis(8-oxo-8-(pentadecan-7-yloxy)octyl) 2-(hydroxymethyl)pentanedioate (170) A suspension of bis(8-oxo-8-(pentadecan-7-yloxy)octyl) 2- ((benzyloxy)methyl)pentanedioate 169 (416 mg, 0.434 mmol) and 10% palladium on carbon (300 mg) in 30 mL hexanes was subjected to hydrogenation at room temperature overnight. TLC suggested completion of the reaction. After filtration, the solution was purified by column chromatography 0-40% ethyl acetate in hexanes to give bis(8-oxo-8-(pentadecan-7- yloxy)octyl) 2-(hydroxymethyl)pentanedioate as colorless oil (350 mg, 93%). Step 8: Synthesis of Bis(8-oxo-8-(pentadecan-7-yloxy)octyl) 2-((((2- (dimethylamino)ethoxy)carbonyl)oxy)methyl)pentanedioate (Example 131) To a solution of bis(8-oxo-8-(pentadecan-7-yloxy)octyl) 2- (hydroxymethyl)pentanedioate 170 (80 mg, 0.092 mmol) in 5.0 mL dichloromethane, was added pyridine (1.0 mL) followed by 4-nitrophenyl chloroformate (74 mg, 0.37 mmol), and the mixture was stirred at room temperature for 1 h. TLC showed disappearance of starting material.2-(Dimethylamino)ethan-1-ol (0.16 g, 1.8 mmol) was slowly added, and the mixture was stirred at room temperature overnight. The solvents were removed under reduced pressure, and the residue was purified by column chromatography using 0-100% ethyl acetate hexanes to give bis(8-oxo-8-(pentadecan-7-yloxy)octyl) 2-((((2- (dimethylamino)ethoxy)carbonyl)oxy)methyl)pentanedioate as colorless oil (68 mg, 75%). 1H NMR (300 MHz, CDCl3) δ 4.84 (quint, 2H), 4.35-4.27 (m, 1H), 4.26-4.16 (m, 3H), 4.13- 3.96 (m, 4H), 2.81-2.71 (m, 1H), 2.62-2.52 (m, 2H), 2.44-2.31 (m, 2H), 2.30-2.19 (m, 10H), 2.01-1.82 (m, 2H), 1.70-1.40 (m, 16H), 1.38-1.13 (m, 52H), 0.92-0.79 (m, 12H). APCI-MS analysis: Calculated C57H107NO11 [M+H] = 982.7, Observed = 982.7. HPLC-ELSD: tR = 8.645 min (method 1). Example 132: 1,4-bis(hexadecyloxy)-1,4-dioxobutane-2-sulfonic 3- (dimethylamino)propanoic anhydride Example 133: 1,4-bis(octadecyloxy)-1,4-dioxobutane-2-sulfonic 3- (dimethylamino)propanoic anhydride Example 134: 3-(dimethylamino)propanoic 9,20,23,34-tetraoxo-10,19,24,33- tetraoxadotetracontane-21-sulfonic anhydride Example 135: 1,4-bis(((9Z,12Z)-octadeca-9,12-dien-1-yl)oxy)-1,4-dioxobutane-2-sulfonic 3- (dimethylamino)propanoic anhydride Example 136: 1,4-bis(octadecyloxy)-1,4-dioxobutane-2-sulfonic 4-(dimethylamino)butanoic anhydride Example 137: 1,4-bis(((9Z,12Z)-octadeca-9,12-dien-1-yl)oxy)-1,4-dioxobutane-2-sulfonic 4- (dimethylamino)butanoic anhydride Example 105. Lipid nanoparticle formulation Ionizable lipids described herein can be used in the preparation of lipid nanoparticles according to methods known in the art. For example, suitable methods include methods described in International Publication No. WO 2018/089801, which is hereby incorporated by reference in its entirety. The lipid nanoparticles in the examples of the present invention were formulated using Process A of WO 2018/089801 (see, e.g., Example 1 and Figure 1 of WO 2018/089801). Process A (“A”) relates to a method of encapsulating mRNA by mixing mRNA with a mixture of lipids, without first pre-forming the lipids into lipid nanoparticles. In an exemplary process, an ethanolic solution of a mixture of lipids (cationic lipid, phosphatidylethanolamine, cholesterol, and polyethylene glycol-lipid) at a fixed lipid to mRNA ratio were combined with an aqueous buffered solution of target mRNA at an acidic pH under controlled conditions to yield a suspension of uniform LNPs. After ultrafiltration and diafiltration into a suitable diluent system, the resulting nanoparticle suspensions were diluted to final concentration, filtered, and stored frozen at −80°C until use. Lipid nanoparticle formulations of Table 1 using certain cationic lipids as described herein were prepared by Process A. All of the lipid nanoparticle formulations comprised firefly luciferase (FFL) mRNA and the different lipids (Cationic Lipid: DMG-PEG2000: Cholesterol: DOPE) in the mol % ratios specified in Table 1. Table 1. Exemplary Lipid Nanoparticle Formulations
Figure imgf000342_0001
* The N/P ratio is defined as the ratio of the number of nitrogen in cationic lipid to the number of phosphate in nucleic acid. Example 106. Delivery of Firefly Luciferase (FFL) mRNA by intranasal administration Lipid nanoparticle formulation 1 listed in Table 1 comprising FFL mRNA, cationic lipid, DMG-PEG2000, cholesterol and DOPE was administered in mice via pipetting the formulations at 10µg/Animal and 15µl per nostril. On Day 2, 24 hours post dose (±5%), all animals underwent a luminescent imaging session using IVIS with separate ROIs on the nose and lungs. Whole body imaging was performed 10-15 minutes following D-Luciferin administration. All animals were dosed with 0.2 mL of 15 mg/mL D-luciferin solution via intraperitoneal (IP) injection. Anesthesia was performed by isoflurane during the procedure and animals were placed sternal recumbency (face-down). An intranasal vaccine drug product may be administered via nasal spray. Exemplary data are provided in Table 2, which describes the average radiance in p/s/cm2/sr (the number of photons per second that leave a square centimeter of tissue and radiate into a solid angle of one steradian (sr)). cKK-E12, having the following structure, was used in a control formulation:
Figure imgf000342_0003
Table 2. Exemplary in vivo protein expression following intranasal administration
Figure imgf000342_0002
Figure imgf000343_0001
Figure imgf000344_0001

Claims

CLAIMS 1. A lipid of Formula (I):
Figure imgf000345_0001
or a pharmaceutically acceptable salt thereof, wherein: two of R1, R2, and R3 are independently selected from -C(6-24)alkyl, -C(6-24)alkenyl, - C(6-24)alkynyl, , , and , wherein the -C(6-24)alkyl, - C(6-24)alkenyl, and -C(6-24)alkynyl are each optionally substituted with one to six groups independently selected from halo, -OC(1-6)alkyl, -OC(2-6)alkenyl, -SC(1-6)alkyl, -SC(2-6)alkenyl, and -C(O)OC(1-6)alkyl; one of R1, R2, and R3 is
Figure imgf000345_0002
X1 independently for each occurrence is -C(3-12)alkyl, -C(3-12)alkenyl, or -C(3-12)alkynyl, each of which is optionally substituted by one to six groups independently selected from halo, -OC(1-6)alkyl, -OC(2-6)alkenyl, -SC(1-6)alkyl, -SC(2-6)alkenyl, and -C(O)OC(1-6)alkyl; X2 independently for each occurrence is -C(O)O-, -OC(O)-, -C(O)NH-, -C(O)N(X3)-, -NHC(O)-, -N(X3)C(O)-, -C(O)S-, -SC(O)-, -OC(O)O-, -OC(O)NH-, -NHC(O)O-, or - NHC(O)NH-; X3 independently for each occurrence is -C(5-24)alkyl, -C(5-24)alkenyl, or -C(5-24)alkynyl, each of which is optionally substituted by one to six groups independently selected from halo, -OC(1-6)alkyl, -OC(2-6)alkenyl, -SC(1-6)alkyl, -SC(2-6)alkenyl, and -C(O)OC(1-6)alkyl; X4 is -C(1-8)alkyl; X5 is -C(1-8)alkyl; X6 is -C(O)O-, -OC(O)-, -C(O)NH-, -NHC(O)-, -C(O)S-, -SC(O)-, -OC(O)O-, - OC(O)NH-, -NHC(O)O-, or -NHC(O)NH-; X7 is -C(1-8)alkyl; A is an ionizable or cationic nitrogen-containing group; X is selected from the group consisting of:
Figure imgf000346_0001
wherein * indicates the point of attachment to R3; RX independently for each occurrence is -H or -C(1-3)alkyl; and n is 1, 2, 3, or 4. 2. A lipid of Formula (I):
Figure imgf000346_0002
or a pharmaceutically acceptable salt thereof, wherein: two of R1, R2, and R3 are independently selected from -C(6-24)alkyl, -C(6-24)alkenyl, - C(6-24)alkynyl and wherein the -C(6-24)alkyl, -
Figure imgf000346_0004
Figure imgf000346_0005
C(6-24)alkenyl, and -C(6-24)alkynyl are each optionally substituted with one to six groups independently selected from halo, -OC(1-6)alkyl, -OC(2-6)alkenyl, -SC(1-6)alkyl, -SC(2-6)alkenyl, and -C(O)OC(1-6)alkyl; one of R1, R2, and R3 is
Figure imgf000346_0003
X1 independently for each occurrence is -C(3-12)alkyl, -C(3-12)alkenyl, or -C(3-12)alkynyl, each of which is optionally substituted by one to six groups independently selected from halo, -OC(1-6)alkyl, -OC(2-6)alkenyl, -SC(1-6)alkyl, -SC(2-6)alkenyl, and -C(O)OC(1-6)alkyl; X2 independently for each occurrence is -C(O)O-, -OC(O)-, -C(O)NH-, -C(O)N(X3)-, -NHC(O)-, -N(X3)C(O)-, -C(O)S-, -SC(O)-, -OC(O)O-, -OC(O)NH-, -NHC(O)O-, or - NHC(O)NH-; X3 independently for each occurrence is -C(5-24)alkyl, -C(5-24)alkenyl, or -C(5-24)alkynyl, each of which is optionally substituted by one to six groups independently selected from halo, -OC(1-6)alkyl, -OC(2-6)alkenyl, -SC(1-6)alkyl, -SC(2-6)alkenyl, and -C(O)OC(1-6)alkyl; X4 is -C(1-8)alkyl; X5 is -C(1-8)alkyl; X6 is -C(O)O-, -OC(O)-, -C(O)NH-, -NHC(O)-, -C(O)S-, -SC(O)-, -OC(O)O-, - OC(O)NH-, -NHC(O)O-, or -NHC(O)NH-; X7 is -C(1-8)alkyl; A is an ionizable or cationic nitrogen-containing group; X is selected from the group consisting of:
Figure imgf000347_0001
wherein * indicates the point of attachment to R3; RX independently for each occurrence is -H or -C(1-3)alkyl; and n is 1,
2, 3, or 4.
3. The lipid of claim 1 or claim 2, or a pharmaceutically acceptable salt thereof, wherein: two of R1, R2, and R3 are independently selected from -C(6-24)alkyl, -C(6-24)alkenyl, and
Figure imgf000347_0003
X1 independently for each occurrence is -C(3-12)alkyl or -C(3-12)alkenyl; X2 independently for each occurrence is -C(O)O-, -OC(O)-, or -OC(O)O-; and X3 independently for each occurrence is -C(5-24)alkyl or -C(5-24)alkenyl.
4. The lipid of claim 1 or claim 2, or a pharmaceutically acceptable salt thereof, wherein two of R1, R2, and R3 are independently selected from the group consisting of:
Figure imgf000347_0002
Figure imgf000348_0001
Figure imgf000349_0001
5. The lipid of claim 1 or claim 2, or a pharmaceutically acceptable salt thereof, wherein two of R1, R2, and R3 are independently selected from the group consisting of:
Figure imgf000349_0002
Figure imgf000350_0001
Figure imgf000351_0001
Figure imgf000352_0001
Figure imgf000353_0001
6. The lipid of claim 1 or claim 2, or a pharmaceutically acceptable salt thereof, wherein: R1 and R2 are independently selected from -C(6-24)alkyl, -C(6-24)alkenyl, or
Figure imgf000353_0002
; R3 is
Figure imgf000353_0003
X1 independently for each occurrence is -C(3-12)alkyl or -C(3-12)alkenyl; X2 independently for each occurrence is -C(O)O-, -OC(O)-, or -OC(O)O-; X3 independently for each occurrence is -C(5-24)alkyl or -C(5-24)alkenyl. X4 is -C(1-6)alkyl; X5 is -C(1-6)alkyl; X6 is -C(O)O-, -OC(O)-, or -OC(O)O-; X7 is -C(1-6)alkyl; and A is an ionizable or cationic nitrogen-containing group.
7. The lipid of any one of claims 1-6, or a pharmaceutically acceptable salt thereof, wherein A is ; and RA1 and RA2 are each independently -C(1-6)alkyl that is optionally substituted with - OH; or RA1 and RA2 are taken together to form a 3- to 6-membered heterocyclyl that is optionally substituted with one to three -C(1-3)alkyl groups.
8. The lipid of any one of claims 1-7, or a pharmaceutically acceptable salt thereof, wherein A is selected from the group consisting of:
Figure imgf000354_0002
9. The lipid of any one of claims 1-8, or a pharmaceutically acceptable salt thereof, wherein is selected from the group consisting of:
Figure imgf000354_0001
10. The lipid of any one of claims 1-9, having a structure selected from the group consisting of:
Figure imgf000355_0001
Figure imgf000356_0001
or a pharmaceutically acceptable salt thereof.
11. The lipid of any one of claims 1-9, having a structure selected from the group consisting of:
Figure imgf000357_0001
or a pharmaceutically acceptable salt thereof.
12. The lipid of any one of claims 1-9, having a structure selected from the group consisting of:
Figure imgf000358_0001
Figure imgf000359_0001
or a pharmaceutically acceptable salt thereof.
13. The lipid of any one of claims 1-12, or a pharmaceutically acceptable salt thereof, wherein n is 1 or 2.
14. A lipid having a structure selected from the group consisting of:
Figure imgf000359_0002
Figure imgf000360_0001
Figure imgf000361_0001
Figure imgf000362_0001
Figure imgf000363_0001
Figure imgf000364_0001
Figure imgf000365_0001
Figure imgf000366_0001
Figure imgf000367_0001
Figure imgf000368_0001
Figure imgf000369_0001
Figure imgf000370_0001
Figure imgf000371_0001
Figure imgf000372_0001
Figure imgf000373_0001
Figure imgf000374_0001
Figure imgf000375_0001
Figure imgf000376_0001
Figure imgf000377_0001
Figure imgf000378_0001
Figure imgf000379_0001
Figure imgf000380_0001
Figure imgf000381_0001
or a pharmaceutically acceptable salt thereof.
15. A lipid having a structure selected from the group consisting of:
Figure imgf000381_0002
Figure imgf000382_0001
Figure imgf000383_0001
Figure imgf000384_0001
Figure imgf000385_0001
Figure imgf000386_0001
or a pharmaceutically acceptable salt thereof.
16. A composition comprising a lipid nanoparticle (LNP), wherein the LNP comprises: (I) the lipid of any one of claims 1-15; (II) a stealth lipid; (III) a structural lipid; and (IV) a helper lipid.
17. The composition of claim 16, wherein: the stealth lipid is a polyethylene glycol-conjugated (PEGylated) lipid, a polyoxazoline polymer-conjugated lipid, or a polysarcosine-conjugated (pSar) lipid, optionally wherein the stealth lipid is a PEGylated lipid selected from 1,2-dimyristoyl-rac- glycero-3-methoxypolyethylene glycol (DMG-PEG), 1,2-distearoyl-sn-glycero-3- phosphoethanolamine-polyethylene glycol (DSPE-PEG), 1,2-dilauroyl-sn-glycero-3- phosphoethanolamine-polyethylene glycol (DLPE-PEG), and 1,2-distearoyl-rac-glycero- polyethelene glycol (DSG-PEG); the structural lipid is a sterol, optionally wherein the sterol is cholesterol; and the helper lipid is 1,2-dioleoyl-SN-glycero-3-phosphoethanolamine (DOPE); 1,2- distearoyl-sn-glycero-3-phosphocholine (DSPC); 1,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS); 1,2-dielaidoyl-sn-glycero-3-phosphoethanolamine (DEPE); and 1,2-dioleoyl-sn- glycero-3-phosphocholine (DPOC), dipalmitoylphosphatidylcholine (DPPC), 1,2-dilauroyl- sn-glycero-3-phosphocholine (DLPC), 1,2-Distearoylphosphatidylethanolamine (DSPE), or 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine (DLPE).
18. The composition of claim 16 or claim 17, further comprising a nucleic acid molecule, wherein the nucleic acid molecule is encapsulated in the LNP, and wherein the nucleic acid molecule is an mRNA molecule.
19. The composition of claim 18, wherein the mRNA molecule encodes an antigen, optionally a viral antigen or a bacterial antigen.
20. The composition of claim 18 or claim 19 for use in eliciting an immune response in a subject in need thereof.
21. The composition of claim 18 or claim 19 for use in preventing an infection or reducing one or more symptoms of an infection in a subject in need thereof.
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