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WO2025088570A1 - Lipides ionisables et nanoparticules lipidiques ionisables - Google Patents

Lipides ionisables et nanoparticules lipidiques ionisables Download PDF

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Publication number
WO2025088570A1
WO2025088570A1 PCT/IB2024/060531 IB2024060531W WO2025088570A1 WO 2025088570 A1 WO2025088570 A1 WO 2025088570A1 IB 2024060531 W IB2024060531 W IB 2024060531W WO 2025088570 A1 WO2025088570 A1 WO 2025088570A1
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compound
bis
crisp
attorney docket
diyl
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Qisheng Xin
Lorenzo AULISA
Nicholas Louis RIZZI
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CRISPR Therapeutics AG
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CRISPR Therapeutics AG
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D251/00—Heterocyclic compounds containing 1,3,5-triazine rings
    • C07D251/02—Heterocyclic compounds containing 1,3,5-triazine rings not condensed with other rings
    • C07D251/12—Heterocyclic compounds containing 1,3,5-triazine rings not condensed with other rings having three double bonds between ring members or between ring members and non-ring members
    • C07D251/26—Heterocyclic compounds containing 1,3,5-triazine rings not condensed with other rings having three double bonds between ring members or between ring members and non-ring members with only hetero atoms directly attached to ring carbon atoms
    • C07D251/40—Nitrogen atoms
    • C07D251/54—Three nitrogen atoms
    • C07D251/70—Other substituted melamines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D403/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00
    • C07D403/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings
    • C07D403/12—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings linked by a chain containing hetero atoms as chain links
    • 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

  • the disclosure further provides lipid-nanoparticle compositions comprising such lipids towards delivery of therapeutic molecules, particularly therapeutic nucleic acids.
  • Nucleic acids-based therapies have attracted attention in the recent years as there is an enormous potential to treat diseases by targeting their genetic blueprints in vivo.
  • Nucleic acids-based therapeutics can achieve long-lasting or even curative effects via gene inhibition, addition, replacement or editing.
  • a lipid-based delivery system such as, but not limited to lipid nanoparticles (LNP) may provide an approach to stabilize and deliver nucleic acids and other therapeutic molecules, and there remains a significant need towards improving this technology.
  • LNP lipid nanoparticles
  • Design features such as optimal particle size, encapsulation efficiencies, robust manufacturing process, different lipophilicity and neutral surface charge, can be further advanced to provide efficient lipid-based delivery systems for nucleic acids and other therapeutic molecules.
  • BRIEF SUMMARY the present disclosure provides compounds, including stereoisomers, tautomers, or pharmaceutically acceptable salts thereof, which can be used alone or in combination with other therapeutic agents.
  • a compound having a structure of Formula (I) is provided: Attorney Docket No.: CRISP-43327.601 or a stereoisomer of the compound, tautomer of the compound, pharmaceutically acceptable salt thereof, wherein A 1 , A 2 , A 3 , R 1a , R 1b , R 2a , R 2b , m 1 , m 2 , m 3 , and X are as defined herein.
  • compositions comprising one or more of the foregoing compounds of Formula (I), (IA), (IB), (IA-a), (IA-b), (IB-a), (IB-b), (IA-a1), (IA-b1), (IB-a1), or (IB-b1) and an additional therapeutic agent are also provided.
  • methods of treatment by administering the foregoing compounds of Formula (I), (IA), (IB), (IA-a), (IA-b), (IB-a), (IB-b), (IA-a1), (IA-b1), (IB- a1), or (IB-b1) or the pharmaceutical compositions comprising a compound of Formula (I), (IA), (IB), (IA-a), (IA-b), (IB-a), (IB-b), (IA-a1), (IA-b1), (IB-a1), or (IB-b1), to a subject in need thereof to treat a disease are provided.
  • the compounds have the following structure of Formula (I): or a tautomer of the salt thereof, wherein: 1 2 A , A , and A 3 are, or X is -NR 3a R 3b or –OR 4 ; R 1a , R 1b , R 2a , and R 2b are, each independently, C4-C18 alkyl, C4-C18 alkenyl, or , wherein R 5 is C4-C18 alkyl or C4-C18 alkenyl, and n is an integer between 1 R 3a and R 3b are, each independently, C1-C18 alkyl, C2-C18 , or R 3a and R 3b , together with the nitrogen to which they are attached, heterocycle or heteroaryl; , wherein R 4a and R 4b are, each independently, H, C1-C8 alkyl, with the nitrogen they are attached to, form a 5-8 membered heterocycle, and p is an integer between 1 and 6; and
  • a 1 , A 2 , and A 3 are each O. In some embodiments, A 1 , A 2 , and A 3 are each NH. In some embodiments, the compound has the following structures of Formula (IA): Formula (IB): In some structures of Formula (IA-a)-(IA-b) or (IB-a)-(IB-b): , , or Attorney Docket No.: CRISP-43327.601 . In some embodiments, R 1a , R 1b , R 2a , R 2b , R 3a , and R 3b are, each independently, C 6 -C 16 alkyl, C 6 -C 16 .
  • alkyl of R 1a , R 1b , R 2a , R 2b , R 3a , or R 3b is, each independently, - - - -C 10 H 21 , -C 11 H 23 , -C 12 H 25 , -C 13 H 27 , -C 14 H 29 , or - C16H33.
  • the C 6 -C 16 alkenyl of R 1a , R 1b , R 2a , R 2b , R 3a , or R 3b has one, two, or three alkene moieties.
  • the C 6 -C 16 alkenyl of R 1a , R 1b , R 2a , R 2b , R 3a , or R 3b is, each independently, C10 alkenyl, C11 alkenyl, or C12 alkenyl. In some embodiments, the C 6 -C 16 alkenyl of R 1a , R 1b , R 2a , R 2b , R 3a , or R 3b is, each independently, . In alkyl or C7-C14 alkenyl.
  • the C 7 -C 14 alkyl of R 5 is -C 8 H 17 , -C 9 H 19 , -C 10 H 21 , -C 11 H 23 , - C12H25, -C13H27, or -C14H29.
  • the C 7 -C 14 alkenyl of R 5 has one, two, or three alkene moieties.
  • the C 7 -C 14 alkenyl of R 5 is C 10 alkenyl, C 11 alkenyl, or C 12 alkenyl.
  • the C7-C14 alkenyl of R 5 is .
  • n In some embodiments, n is an integer of 1, 2, or 3.
  • R 4 wherein R 4a and R 4b are, each independently, H, C1-C8 alkyl, 1 and 4. In some embodiments, R 4 , wherein R 4a and R 4b are, each independently, H, C1-C6 alkyl, 1 and 4. In some embodiments, the 5-8 membered heterocycle formed with R 4a and R 4b is further substituted with C1-C4 alkyl, C1-C4 heteroalkyl, -OH, or halo.
  • the compositions further comprise one or more additional lipids selected from the group consisting of helper lipids, cholesterol, and polymer conjugated lipids.
  • the nucleic acid comprises RNA, DNA, or mixtures thereof.
  • the RNA comprises mRNA, gRNA, or mixtures thereof.
  • the composition comprises a gene editing system.
  • the gene editing system comprises one or more components selected from the group consisting of a gRNA or sgRNA, a nucleic acid encoding an RNA-directed nuclease, and a donor polynucleotide.
  • the helper lipid is DSPC or DOPE. In some embodiments, the molar ratio of the compound to the helper lipid ranges from about 7:1 to about 1:7. In some embodiments, the molar ratio of the compound to the helper lipid ranges from about 5:1 to about 1:5. In some embodiments, the molar ratio of the compound to the helper lipid ranges from about 3:1 to about 1:3. In some embodiments, the composition further comprises cholesterol and the molar ratio of the compound to cholesterol ranges from 3:1 to 1:3.
  • compositions of the present disclosure can comprise, consist essentially of, or consist of, the components disclosed. All percentages, parts and ratios are based upon the total weight of the compositions and all measurements made are at about 25 oC., unless otherwise specified.
  • Amino refers to the -NH2, -NHR, or -NR2 radical
  • Cyano refers to the -CN radical
  • Hydroxyl refers to the -OH radical
  • Niro refers to the -NO2 radical
  • Trifluoromethyl refers to the -CF 3 radical
  • Hydrazido or hydrazino refers to N-N substituent, wherein each R of “amino” or “imino” is a compatible substituent as described in this disclosure and wherein an R group is chiral, isomers are contemplated and included herein.
  • Alkyl refers to a linear, saturated, acyclic, monovalent hydrocarbon radical or branched, saturated, acyclic, monovalent hydrocarbon radical, having from one to 30 carbon atoms, for example one to 24 carbon atoms, for example one to twelve carbon atoms, preferably one to eight carbon atoms or one to six carbon atoms, and which is attached to the Attorney Docket No.: CRISP-43327.601 rest of the molecule by a single bond, e.g., methyl, ethyl, n-propyl, 1-methylethyl (isopropyl), n-butyl, n-pentyl, 1,1-dimethylethyl (t-butyl), 3-methylhexyl, 2-methylhexyl and the like.
  • An optionally substituted alkyl radical is an alkyl radical that is optionally substituted, valence permitting, by one, two, three, four, or five substituents independently selected from the group consisting of halo, cyano, nitro, aryl, cycloalkyl, heterocyclyl, heteroaryl, oxo, trimethylsilyl, -OR ′ , -OC(O)R ′ , -N(R ′ )2, C(O)R′′, -C(O)OR ′ , -C(O)N(R ′ )2, - N(R ′ )C(O)OR′′′, N(R ′ )C(O)R′′′, -N(R ′ )S(O) t R′′′ (where t is 1 or 2), -S(O) t OR′′′ (where t is 1 or 2), -S(O)pR′′′ (where p is 0, 1, or 2) and -S(
  • Alkoxy refers to a radical of the formula -OR a where R a is an alkyl radical as defined above containing one to twelve carbon atoms. The alkyl part of the optionally substituted alkoxy radical is optionally substituted as defined above for an alkyl radical.
  • Alkylene refers to a bivalent saturated aliphatic radical. The alkylene is formed from an alkyl group by removal of one hydrogen.
  • Alkenyl refers to a linear, saturated, acyclic, monovalent hydrocarbon radical or branched, saturated, acyclic, monovalent hydrocarbon radical having at least one carbon- carbon double bond and no triple bonds. The double bond(s) may be located at any position(s) with the hydrocarbon chain.
  • the alkenyl group can be optionally substituted as defined above for an alkyl radical.
  • an alkenyl group refers to a radical of the formula -CnH2n-1, where n is an integer between 1 and 30.
  • the alkenyl is a monovalent group formed from an alkene by removal of one hydrogen atoms from any carbon atom.
  • Alkoxyalkyl refers to a radical of the formula -R a -O-R b where R a is alkylene and R b is alkyl as defined above. Alkyl and alkylene parts of the optionally substituted alkoxyalkyl radical are optionally substituted as defined above for an alkyl radical and alkylene chain, respectively.
  • Alkyl refers to a radical of the formula -R a -R b , where R a is alkylene and R b is aryl as described herein. Alkylene and aryl portions of optionally substituted aralkyl are optionally substituted as described herein for alkylene and aryl, respectively.
  • Aryl refers to an aromatic monocyclic or multicyclic hydrocarbon ring system radical containing from 6 to 18 carbon atoms, where the multicyclic aryl ring system is a bicyclic, tricyclic, or tetracyclic ring system.
  • Aryl radicals include, but are not limited to, Attorney Docket No.: CRISP-43327.601 groups such as fluorenyl, phenyl and naphthyl.
  • An optionally substituted aryl is an aryl radical that is optionally substituted by one, two, three, four, or five substituents independently selected from the group consisting of alkyl, alkenyl, alkynyl, halo, haloalkyl, haloalkenyl, cyano, nitro, aryl, heteroaryl, heteroarylalkyl, -R′′-OR ′ , -R′′-OC(O)-R ′ , - R′′-N(R ′ ) 2 , -R′′-C(O)R ′ , -R′′-C(O)OR ′ , -R′′-C(O)N(R ′ ) 2 , -R′′-N(R ′ )C(O)OR′′′
  • Arylalkoxy refers to a group of formula –O-R, where R is aralkyl.
  • An optionally substituted arylalkoxy is an arylalkoxy that is optionally substituted as described herein for aralkyl.
  • arylalkoxy is benzyloxy.
  • Cycloalkyl refers to a stable non-aromatic monocyclic or polycyclic hydrocarbon radical having from three to fifteen carbon atoms, preferably having from three to ten carbon atoms, and which is saturated or unsaturated, and which attaches to the rest of the molecule by a single bond.
  • a polycyclic hydrocarbon radical is bicyclic, tricyclic, or tetracyclic ring system.
  • An unsaturated cycloalkyl contains one, two, or three carbon-carbon double bonds and/or one carbon-carbon triple bond.
  • Monocyclic cycloalkyl radicals include, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl.
  • Polycyclic cycloalkyl radicals include, for example, adamantyl, norbornyl, decalinyl, and the like.
  • An optionally substituted cycloalkyl is a cycloalkyl radical that is optionally substituted by one, two, three, four, or five substituents independently selected from the group consisting of alkyl, alkenyl, halo, haloalkyl, haloalkenyl, cyano, nitro, oxo, aryl, aralkyl, cycloalkyl, heterocyclyl, heteroaryl, -R′′-OR ′ , -R′′-OC(O)-R ′ , -R′′-N(R ′ ) 2 , -R′′-C(O)R ′ , - R′′-C(O)OR ′ , -R′′-C(O)N(R ′ )2, -R′′-N(R ′ )C(O)OR′′′, -R′′-N(R ′ )C(O)R′′′, -R′′-N(R ′ )S(
  • Deuterated compounds are compounds where one or more hydrogen atoms have been replaced with a deuterium atom.
  • Deuterated drugs may be derivatives of an active compound.
  • Deuterated drugs may be prodrugs. Deuteration may alter the physical Attorney Docket No.: CRISP-43327.601 properties, metabolic properties, activity or safety of a drug.
  • Derivatives are related chemical species that can be derived from a similar compound via chemical reactions. They may encompass slight chemical modifications, substitution of atoms with deuterated atoms, substitution of atoms with stable or radioactive isotopes or other modifications that imbue a compound with desirable properties. "Fused” refers to any ring system described herein which is fused to an existing ring structure in the compounds of the invention.
  • any carbon atom on the existing ring structure which becomes part of the fused ring system may be replaced with a nitrogen atom.
  • Halo refers to the halogen substituents: bromo, chloro, fluoro, and iodo.
  • Haloalkyl refers to an alkyl radical, as defined above, that is further substituted by one or more halogen substituents. The number of halo substituents included in haloalkyl is from one and up to the total number of the hydrogen atoms available for replacement with the halo substituents (e.g., perfluoroalkyl).
  • Non-limiting examples of haloalkyl include trifluoromethyl, difluoromethyl, trichloromethyl, 2,2,2-trifluoroethyl, 1-fluoromethyl, 2- fluoroethyl, 3-bromo 2-fluoropropyl, 1-bromomethyl, 2-bromoethyl and the like.
  • the hydrogen atoms bonded to the carbon atoms of the alkyl part of the haloalkyl radical may be optionally replaced with substituents as defined above for an optionally substituted alkyl.
  • Haloalkenyl refers to an alkenyl radical, as defined above, that is further substituted by one or more halo substituents.
  • haloalkenyl is from one and up to the total number of the hydrogen atoms available for replacement with the halo substituents (e.g., perfluoroalkenyl).
  • haloalkenyl include 2,2-difluoroethenyl, 3-chloroprop-1-enyl, and the like.
  • the hydrogen atoms bonded to the carbon atoms of the alkenyl part of the haloalkenyl radical may be optionally replaced with substituents as defined above for an optionally substituted alkenyl group.
  • Haloalkynyl refers to an alkynyl radical, as defined above, that is further substituted by one or more halo substituents.
  • the number of halo substituents included in haloalkynyl is from one and up to the total number of the hydrogen atoms available for replacement with the halo substituents (e.g., perfluoroalkynyl).
  • Non-limiting examples of haloalkynyl include 3-chloroprop-1-ynyl and the like.
  • the alkynyl part of the haloalkynyl radical may be additionally optionally substituted as defined above for an alkynyl group.
  • Heteroarylalkyl refers to a radical of the formula -Ra-Rb, where Ra is alkylene and R b is heteroaryl as described herein. Alkylene and heteroaryl portions of optionally Attorney Docket No.: CRISP-43327.601 substituted heteroarylalkyl are optionally substituted as described herein for alkylene and heteroaryl, respectively.
  • Heterocyclyl refers to a stable 3- to 18--membered nonaromatic ring system radical having the carbon count of two to twelve and containing a total of one to six heteroatoms independently selected from the group consisting of nitrogen, oxygen, phosphorus, and sulfur.
  • a heterocyclyl radical is a monocyclic, bicyclic, tricyclic, or tetracyclic ring system.
  • a bicyclic, tricyclic, or tetracyclic heterocyclyl is a fused, spiro, and/or bridged ring system.
  • the heterocyclyl radical may be saturated or unsaturated.
  • An unsaturated heterocyclyl contains one, two, or three carbon-carbon double bonds and/or one carbon-carbon triple bond.
  • An optionally substituted heterocyclyl is a heterocyclyl radical that is optionally substituted by one, two, three, four, or five substituents independently selected from the group consisting of alkyl, alkenyl, halo, haloalkyl, haloalkenyl, cyano, oxo, thioxo, nitro, aryl, aralkyl, cycloalkyl, heterocyclyl-, heteroaryl, -R′′-OR ′ , -R′′-OC(O)-R ′ , -R′′-N(R ′ ) 2 , - R′′-C(O)R ′ , -R′′-C(O)OR ′ , -R′′-C(O)N(R ′ )2, -R′′-N(R ′ )C(O)OR′′′, -R′′-N(R ′ )C(O)R′′′, - R′′-N(R ′
  • the nitrogen, carbon, or sulfur atoms in the heterocyclyl radical may be optionally oxidized (when the substituent is oxo and is present on the heteroatom); the nitrogen atom may be optionally quaternized (when the substituent is alkyl, alkenyl, aryl, aralkyl, cycloalkyl, heterocyclyl, heteroaryl, -R′′-OR ′ , -R′′-OC(O)-R ′ , -R′′-N(R ′ )2, -R′′-C(O)R ′ , -R′′-C(O)OR ′ , - R′′-C(O)N(R ′ ) 2 , -R′′-N(R ′ )C(O)OR′′′, -R′′-N(R ′ )C(O)R′′′, -R′′-N(R ′ )S(O) t R′′′ (where t is 1 or 2), -R′′
  • optionally substituted heterocyclyl radicals include, but are not limited to, azetidinyl, dioxolanyl, thienyl[1,3]dithianyl, decahydroisoquinolyl, imidazolinyl, imidazolidinyl, isothiazolidinyl, isoxazolidinyl, morpholinyl, octahydroindolyl, octahydroisoindolyl, 2-oxopiperazinyl, 2-oxopiperidinyl, 2oxopyrrolidinyl, oxazolidinyl-, piperidinyl, piperazinyl, 4piperidonyl, pyrrolidinyl, pyrazolidinyl, thiazolidinyl, tetrahydrofuryl, trithianyl, tetrahydropyranyl, thiomorpholinyl, thiamorpholinyl, 1---
  • Heterocyclylene refers to a heterocyclyl in which one hydrogen atom is replaced with a valency. An optionally substituted heterocyclylene is optionally substituted as Attorney Docket No.: CRISP-43327.601 described herein for heterocyclyl.
  • Heteroaryl refers to a 5- to 18-membered ring system radical containing at least one aromatic ring, having the carbon count of one to seventeen carbon atoms, and containing a total of one to ten heteroatoms independently selected from the group consisting of nitrogen, oxygen, and sulfur.
  • the heteroaryl radical is a monocyclic, bicyclic, tricyclic, or tetracyclic ring system.
  • optionally substituted heteroaryl radicals include, but are not limited to, azepinyl, acridinyl, benzimidazolyl, benzthiazolyl, benzindolyl, benzodioxolyl, benzofuranyl, benzooxazolyl, benzothiazolyl, benzothiadiazolyl, benzo[b][1,4]dioxepinyl, 1,4benzodioxanyl, benzonaphthofuranyl, benzoxazolyl, benzodioxolyl, benzodioxinyl, benzopyranyl, benzopyranonyl, benzofuranyl, benzofuranonyl, benzothienyl (benzothiophenyl), benzotriazolyl, benzo[4,6]imidazo-[1,2a]pyridinyl, carbazolyl, cinnolinyl, dibenzofuranyl, dibenzo
  • phrases "pharmaceutically acceptable” is employed herein to refer to those compounds, salts, compositions, dosage forms, etc., which are--within the scope of sound medical judgment--suitable for use in contact with the tissues of human beings and/or other mammals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
  • pharmaceutically acceptable means approved by a regulatory agency of the federal or a state government, or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals (e.g., mammals), and more particularly, in humans.
  • “Prodrugs” are compounds that after administration are metabolized or otherwise chemically transformed into an active moiety.
  • the embodiments disclosed herein encompass all pharmaceutically acceptable compounds of the compound of (I), (IA), (IB), (IA-a), (IA-b), (IB-a), (IB-b), (IA-a1), (IA- b1), (IB-a1), or (IB-b1) being isotopically-labeled by having one or more atoms replaced by an atom having a different atomic mass or mass number.
  • the radioactive isotopes tritium, i.e., 3 H, and carbon-14, i.e., 14 C, are particularly useful for this purpose in view of their ease of incorporation and ready means of detection.
  • substitution with heavier isotopes such as deuterium, i.e., 2 H, may afford certain therapeutic advantages resulting from greater metabolic stability, for example, increased in vivo half-life or reduced dosage requirements, and hence may be preferred in some circumstances.
  • Substitution with positron emitting isotopes such as 11 C, 18 F, 15 O and 13 N, can be useful in Positron Emission Topography (PET) studies for examining substrate receptor occupancy.
  • PET Positron Emission Topography
  • the disclosure includes compounds produced by a process comprising administering a compound of this disclosure to a mammal for a period of time sufficient to yield a metabolic product thereof.
  • Such products are typically identified by administering a radiolabeled compound of the disclosure in a detectable dose to an animal, such as rat, mouse, guinea pig, monkey, or to human, allowing sufficient time for metabolism to occur, and isolating its conversion products from the urine, blood or other biological samples.
  • “Pharmaceutically acceptable salt” includes both acid and base addition salts.
  • “Pharmaceutically acceptable base addition salt” refers to those salts which retain the biological effectiveness and properties of the free acids, which are not biologically or otherwise undesirable. These salts are prepared from addition of an inorganic base or an organic base to the free acid. Salts derived from inorganic bases include, but are not limited to, the sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum salts and the like. Preferred inorganic salts are the ammonium, sodium, potassium, calcium, and magnesium salts.
  • Salts derived from organic bases include, but are not limited to, salts of primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines and basic ion exchange resins, such as ammonia, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, diethanolamine, ethanolamine, deanol, 2-dimethylaminoethanol, 2- diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline, betaine, benethamine, benzathine, ethylenediamine, glucosamine, methylglucamine, theobromine, triethanolamine, tromethamine, purines, piperazine, piperidine, N ethylpiperidine, polyamine resins and the like.
  • basic ion exchange resins such as
  • organic bases are isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline and caffeine.
  • a “pharmaceutical composition” refers to a formulation of a compound of the disclosure and a medium generally accepted in the art for the delivery of the biologically active compound to mammals, e.g., humans. Such a medium includes all pharmaceutically acceptable carriers, diluents and excipients therefor.
  • Effective amount or “therapeutically effective amount” refers to that amount of a compound of the disclosure which, when administered to a mammal, preferably a human, is sufficient to effect treatment in the mammal, preferably a human.
  • the amount of a compound which constitutes a “therapeutically effective amount” will vary depending on the compound, the condition and its severity, the manner of administration, and the age of the Attorney Docket No.: CRISP-43327.601 mammal to be treated, but can be determined routinely by one of ordinary skill in the art having regard to his own knowledge and to this disclosure.
  • a "stereoisomer” refers to a compound made up of the same atoms bonded by the same bonds but having different three-dimensional structures, which are not interchangeable.
  • the present disclosure contemplates various stereoisomers and mixtures thereof and includes “enantiomers”, which refers to two stereoisomers whose molecules are nonsuperimposeable mirror images of one another.
  • the present disclosure also contemplates "diastereomers”, which refers to non-mirror image of non-identical stereoisomers. Diastereomers occur when two or more stereoisomers of a compound have different configurations at one or more of the equivalent stereocenters and are not mirror images of each other.
  • a "tautomer” refers to a proton shift from one atom of a molecule to another atom of the same molecule. The present disclosure includes tautomers of any said compounds.
  • neutral lipid refers to any of a number of lipid species that exist either in an uncharged or neutral zwitterionic form at a selected pH.
  • lipids include, but are not limited to, phosphotidylcholines such as 1,2-Distearoyl-sn-glycero-3- phosphocholine (DSPC), 1,2-Dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2- Dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1-Palmitoyl-2-oleoyl-sn-glycero-3- phosphocholine (POPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), phophatidylethanolamines such as 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), sphingomyelins (SM),
  • DOPE 1,2-Di
  • Neutral lipids may be synthetic or naturally derived.
  • the term “lipid nanoparticle” refers to particles having at least one dimension on the order of nanometers (e.g., 1-1,000 nm) which include one or more of the compounds of structure (I) or other specified cationic lipids.
  • lipid nanoparticles are included in a formulation that can be used to deliver an active agent or therapeutic agent, such as a nucleic acid (e.g., mRNA) to a target site of interest (e.g., cell, tissue, organ, tumor, and the like).
  • the lipid nanoparticles of the invention comprise a nucleic acid.
  • Such lipid nanoparticles typically comprise a compound of structure Formula (I), (IA), (IB), (IA-a), (IA-b), (IB-a), (IB-b), (IA-a1), (IA-b1), (IB-a1), or (IB-b1) and one or more excipient selected from neutral lipids, charged lipids, steroids and polymer conjugated lipids.
  • the lipid nanoparticles have a mean diameter of from about 30 nm to about 150 nm, from about 40 nm to about 150 nm, from about 50 nm to about 150 nm, from about 60 nm to about 130 nm, from about 70 nm to about 110 nm, from about 70 nm to about 100 nm, from about 80 nm to about 100 nm, from about 90 nm to about 100 nm, from about 70 to about 90 nm, from about 80 nm to about 90 nm, from about 70 nm to about 80 nm, or about 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 n
  • nucleic acids when present in the lipid nanoparticles, are resistant in aqueous solution to degradation with a nuclease.
  • Lipid nanoparticles comprising nucleic acids and their method of preparation are disclosed in, e.g., U.S. Patent Publication Nos. 2004/0142025, 2007/0042031 and PCT Pub. Nos. WO 2013/016058 and WO 2013/086373, the full disclosures of which are herein incorporated by reference in their entirety for all purposes.
  • the term “polymer conjugated lipid” refers to a molecule comprising both a lipid portion and a polymer portion.
  • An example of a polymer conjugated lipid is a pegylated lipid.
  • pegylated lipid refers to a molecule comprising both a lipid portion and a polyethylene glycol portion. Pegylated lipids are known in the art and include 1- (monomethoxy-polyethyleneglycol)-2,3-dimyristoylglycerol (PEG-DMG) and the like.
  • PEG-DMG 1- (monomethoxy-polyethyleneglycol)-2,3-dimyristoylglycerol
  • lipid compound
  • lipid compound may be used interchangeably to refer to the ionizable lipids disclosed herein.
  • a compound i.e., a lipid compound having a structure of Formula (I), or a stereoisomer, tautomer of the compound, or a pharmaceutically acceptable salt thereof is provided: wherein A 1 , A 2 , O; X is -NR 3a R 3b or –OR 4 ; 1 a 1b 2 R , R , R a , and R 2b are, C18 alkenyl, or , wherein R 5 is C4-C18 alkyl or C4-C18 alkenyl and n is an integer between 1 and 7; R 3a and R 3b are, each independently, C1-C18 alkyl, C2-C18 alkenyl, or , or R 3a and R 3b , together with the nitrogen to which they are attached, heterocycle or heteroaryl; R 4 is H , wherein R 4a and R 4b are, each independently, H, C 1 -C 8 alkyl, or with the nitrogen they are attached to form a
  • a 1 , A 2 , and A 3 are, each independently, NH or O;
  • X is - NR 3a R 3b or –OR 4 ;
  • R 1a , R 1b , R 2a , R 2b , R 3a , and R 3b are, each independently, C 4 -C 18 alkyl, C 4 - C 18 , wherein R 5 is C 4 -C 18 alkyl or C 4 -C 18 alkenyl and n is an integer
  • R 4a and R 4b are, each independently, with the nitrogen they are attached to Attorney Docket No.: CRISP-43327.601 form a 5-8 membered heterocycle and p is an integer between 1 and 6; and
  • m 1 , m 2 , and m 3 are, each independently, an integer between 1 and 6.
  • a 1 , A 2 , and A 3 are each NH. In some embodiments, one of A 1 , A 2 , or A 3 is NH. In some other embodiments, two of A 1 , A 2 , or A 3 are NH. When A 1 , A 2 , and A 3 are each NH, the compound has the following structure of Formula (IA): . In one embodiments, one of A 1 , A 2 , or A 3 is O. In some are O. When A 1 , A 2 , and A 3 are each O, the compound has the following structure of Formula (IB): . In one X is –OR 4 .
  • X is - the following Formula (IA-a)- (IA-b): or .
  • the compound has the structure of Formula (IA-b).
  • the compound has one of the following Formula (IB-a)-(IB- b): Attorney Docket No.: CRISP-43327.601 or .
  • R 1a , R 1b , R 2a , R 2b , R 3a , and R 3b are, each independently, C4-C18 alkyl, C4-C18 alkenyl, or .
  • the C4-C18 alkyl of R 1a , R 1b , R 2a , R 2b , R 3a , or R 3b includes a C4-C18 alkyl.
  • the C4-C18 alkenyl of R 1a , R 1b , R 2a , R 2b , R 3a , or R 3b includes a linear or branched C 4 -C 18 alkenyl.
  • the C4-C18 alkenyl of R 1a , R 1b , R 2a , R 2b , R 3a , or R 3b includes one, two, or three alkene moieties.
  • R 1a , R 1b , R 2a , R 2b , R 3a , and R 3b are, each independently, C 6 -C 16 alkyl, C 6 -C 16 alkenyl, or .
  • the C 6 -C 16 alkyl of R 1a , R 1b , R 2a , R 2b , R 3a , or R 3b is, each -C8H17, -C9H19, -C10H21, -C11H23, -C12H25, - C 13 H 27 , -C 14 H 29 , or -C 16 H 33 .
  • the C 6 -C 16 alkyl of R 1a , R 1b , R 2a , and R 2b are, each independently -C6H13, -C8H17, -C9H19, -C10H21, -C11H23, -C12H25, -C13H27, - C 14 H 29 , or -C 16 H 33 .
  • the C 6 -C 16 alkyl of R 1a , R 1b , R 2a , and R 2b are - C6H13.
  • the C6-C16 alkyl of R 1a , R 1b , R 2a , and R 2b are -C8H17. In some embodiments, the C 6 -C 16 alkyl of R 1a , R 1b , R 2a , and R 2b , are -C 9 H 19 . In some embodiments, the C6-C16 alkyl of R 1a , R 1b , R 2a , and R 2b , are -C10H21. In some embodiments, the C6-C16 alkyl of R 1a , R 1b , R 2a , and R 2b , are -C11H23.
  • the C6-C16 alkyl of R 1a , R 1b , R 2a , and R 2b are -C12H25. In some embodiments, the C6-C16 alkyl of R 1a , R 1b , R 2a , and R 2b , are -C13H27. In some embodiments, the C6-C16 alkyl of R 1a , R 1b , R 2a , and R 2b , are - C 14 H 29 . In some embodiments, the C 6 -C 16 alkyl of R 1a , R 1b , R 2a , and R 2b , are -C 16 H 33 .
  • the C6-C16 alkenyl of R 1a , R 1b , R 2a , R 2b , R 3a , or R 3b has one, two, or three double bonds. In some embodiments, the C 6 -C 16 alkenyl of R 1a , R 1b , R 2a , R 2b , R 3a , or R 3b has one double bond. In some embodiments, the C6-C16 alkenyl of R 1a , R 1b , R 2a , Attorney Docket No.: CRISP-43327.601 R 2b , R 3a , or R 3b has two double bonds.
  • the C 6 -C 16 alkenyl of R 1a , R 1b , R 2a , R 2b , R 3a , or R 3b has three double bonds.
  • double bonds are present at any positions on the alkenyl group.
  • a double bond is present at a terminal position.
  • a double bond is present in the middle section.
  • the numbers and locations of double bonds present in the alkenyl group may vary.
  • the C6-C16 alkenyl of R 1a , R 1b , R 2a , R 2b , R 3a , or R 3b is, each independently, C 10 alkenyl, C 11 alkenyl, or C 12 alkenyl.
  • the C 6 -C 16 alkenyl of R 1a , R 1b , R 2a , R 2b , R 3a , and R 3b are, each independently, C10 alkenyl, C11 alkenyl, or C 12 alkenyl.
  • the C 6 -C 16 alkenyl of R 1a , R 1b , R 2a , R 2b , R 3a , and R 3b are C10 alkenyl. In some embodiments, the C6-C16 alkenyl of R 1a , R 1b , R 2a , R 2b , R 3a , and R 3b are C 11 alkenyl. In some embodiments, the C 6 -C 16 alkenyl of R 1a , R 1b , R 2a , R 2b , R 3a , and R 3b are C12 alkenyl.
  • the C6-C16 alkenyl of R 1a , R 1b , R 2a , and R 2b are C10 alkenyl. In some embodiments, the C 6 -C 16 alkenyl of R 1a , R 1b , R 2a , and R 2b , are C 11 alkenyl. In some embodiments, the C6-C16 alkenyl of R 1a , R 1b , R 2a , and R 2b , are C12 alkenyl. In some embodiments, the C6-C16 alkenyl of R 1a , R 1b , R 2a , R 2b , R 3a , or R 3b is, each independently, .
  • the C7-C14 alkyl of R 5 is -C7H15, -C8H17, -C9H19, -C10H21, -C11H23, -C12H25, -C13H27, or -C14H29.
  • C7-C14 alkyl of R 5 is -C8H17, -C9H19, -C10H21, -C11H23, -C12H25, -C13H27, or -C14H29.
  • R 5 is -C 8 H 17 .
  • R 5 is -C 9 H 19 .
  • R 5 is -C10H21.
  • R 5 is -C11H23. In some embodiments, R 5 is -C12H25. In some embodiments, R 5 is -C 13 H 27 . In some embodiments, R 5 is -C 14 H 29 . In some is of Attorney Docket No.: CRISP-43327.601 R 1a , R 1b , R 2a , R 2b , R 3a , or R 3b is . In some embodiments, moieties. In some particular embodiments, the C 7 -C 14 alkenyl of R 5 has one alkene moiety.
  • the C7-C14 alkenyl of R 5 is C7 alkenyl, C8 alkenyl, C9 alkenyl, C 10 alkenyl, C 11 alkenyl, C 12 alkenyl, C 13 alkenyl, or C 14 alkenyl.
  • the C7-C14 alkenyl of R 5 is C10 alkenyl, C11 alkenyl, or C12 alkenyl.
  • the C 7 -C 14 alkenyl of R 5 is .
  • n is an integer between 1 and 7.
  • n in is an integer between 1 and 4.
  • n in is an integer of 1, 2, or 3. In some embodiments, n in is an integer of 1. In some embodiments, n in is an integer of 2. In some integer of 3.
  • R 3b are, each independently, C1-C3 alkyl, each of which is substituted with one hydroxy group. In some embodiments, R 3a and R 3b are each methyl. In some embodiments, R 3a and R 3b are each ethyl. In some embodiments, R 3a is ethyl and R 3b is 2-hydroxyethyl. In some embodiments, R 3a is methyl and R 3b is 2-hydroxyethyl.
  • R 3a and R 3b together with the nitrogen to which they are attached, form a 5-8 membered heterocycle or heteroaryl.
  • R 3a and R 3b together with the nitrogen to which they are attached, form a 5-8 membered heterocycle or heteroaryl selected from imidazolyl, piperidinyl, morpholinyl, pyrrolidinyl, and piperazinyl, each of which is independently unsubstituted or substituted with 1 or 2 substituents independently selected from methyl and hydroxy.
  • R 3a and R 3b together with the nitrogen to which they are attached, form an imidazolyl group.
  • R 3a and R 3b together with the nitrogen to which they are attached, form a piperidinyl group, which is unsubstituted or substituted with a hydroxy group. In some embodiments, R 3a and R 3b , together with the nitrogen to which they are attached, form a morpholinyl group. In some embodiments, R 3a and R 3b , together with the nitrogen to which they are attached, form a pyrrolidinyl group, which is unsubstituted or substituted with a methyl group. In some embodiments, R 3a and R 3b , together with the nitrogen to which they are attached, form a piperazinyl group, which is unsubstituted or substituted with a methyl group.
  • the compound has one of the following structures of Formula (IA-a), (IA-b), (IB-a), or (IB-b): , Attorney Docket No.: CRISP-43327.601 (IA-a1) , . In some (IA-a1): . In some (IA-b1): . In some (IB-a1): Attorney Docket No.: CRISP-43327.601 . In some (IB-b1): . In one embodiment, R 4 is H . In some embodiments, R 4 is H.
  • R 4 wherein R 4a and R 4b are, each independently, H, C1-C8 alkyl, or R 4a and the nitrogen they are attached to form a 5-8 membered heterocycle. In some embodiments, R 4 , wherein R 4a and R 4b are, each independently, H, C 1 -C 8 alkyl, and p is 1 and 4. In some embodiments, R 4 is , wherein R 4a and R 4b are, each independently, H, C1-C6 alkyl, and p is an integer between 1 and 4.
  • the C 1 -C 6 alkyl of R 4a or R 4b is methyl, ethyl, n-propyl, iso-propyl, n-butyl, sec-butyl, tert-butyl, n-pentyl, or n- hexane.
  • the 5-8 membered heterocycle formed with R 4a and R 4b is further substituted with C 1 -C 4 alkyl, C 1 -C 4 heteroalkyl, -OH, or halo.
  • the 5-8 membered heterocycle formed with R 4a and R 4b is mono-, di-, or tri-substituted with C 1 -C 4 alkyl, C 1 -C 4 heteroalkyl, -OH, or halo. In some embodiments, the 5-8 membered heterocycle formed with R 4a and R 4b is mono-substituted with C1-C4 alkyl. In some embodiments, the 5-8 membered heterocycle formed with R 4a and R 4b is mono-substituted with C1-C4 heteroalkyl.
  • the 5-8 membered heterocycle formed with Attorney Docket No.: CRISP-43327.601 R 4a and R 4b is mono--substituted with -OH. In some embodiments, the 5-8 membered heterocycle formed with R 4a and R 4b is mono-substituted with halo. In one embodiment, R 4a and R 4b together with the nitrogen they are attached to form a 5-8 membered heterocycle. In some embodiments, the 5-8 membered heterocycle formed by R 4a and R 4b together with the nitrogen they are attached to includes one, two , or three heteroatoms.
  • the 5-8 membered heterocycle formed by R 4a and R 4b together with the nitrogen they are attached to includes one, two, or three nitrogen (N), oxygen (O), or sulfur (O) atoms.
  • the 5-8 membered heterocycle formed by R 4a and R 4b together with the nitrogen they are attached to is pyrrolidine, pyrrole, tetrahydrofuran, furan, tetrahydrothiophene, thiophene, imidazolidine, pyrazolidine, imidazole, pyrazole, oxazoline, isoxazoline, oxazole, isoxazole, thiazolidine, thiazole, isothiazole, dioxolane, dithiolane, piperidine, pyridine, oxane, pyran, thiane, thiopyran, diazinane, diazine, morpholine, oxazine, dio
  • the 5-8 membered heterocycle formed by R 4a and R 4b together with the nitrogen they are attached to is pyrrolidine. In some embodiments, the 5-8 membered heterocycle formed by R 4a and R 4b together with the nitrogen they are attached to is pyrrole. In some embodiments, the 5-8 membered heterocycle formed by R 4a and R 4b together with the nitrogen they are attached to is tetrahydrofuran. In some embodiments, the 5-8 membered heterocycle formed by R 4a and R 4b together with the nitrogen they are attached to is furan. In some embodiments, the 5-8 membered heterocycle formed by R 4a and R 4b together with the nitrogen they are attached to is imidazolidine.
  • the 5-8 membered heterocycle formed by R 4a and R 4b together with the nitrogen they are attached to is pyrazolidine. In some embodiments, the 5-8 membered heterocycle formed by R 4a and R 4b together with the nitrogen they are attached to is imidazole. In some embodiments, the 5-8 membered heterocycle formed by R 4a and R 4b together with the nitrogen they are attached to is pyrazole. In some embodiments, the 5-8 membered heterocycle formed by R 4a and R 4b together with the nitrogen they are attached to is oxazoline. In some embodiments, the 5-8 membered heterocycle formed by R 4a and R 4b together with the nitrogen they are attached to is isooxazoline.
  • the 5-8 membered heterocycle formed by R 4a and R 4b together with the nitrogen they are attached to is oxazole. In some embodiments, the 5-8 membered heterocycle formed by R 4a and R 4b together with the nitrogen they are attached to is isoxazole. In some embodiments, the 5-8 membered heterocycle formed by R 4a and R 4b together with the nitrogen they are attached to is dioxolane. In some embodiments, the 5-8 Attorney Docket No.: CRISP-43327.601 membered heterocycle formed by R 4a and R 4b together with the nitrogen they are attached to is piperidine.
  • the 5-8 membered heterocycle formed by R 4a and R 4b together with the nitrogen they are attached to is pyridine. In some embodiments, the 5-8 membered heterocycle formed by R 4a and R 4b together with the nitrogen they are attached to is oxane. In some embodiments, the 5-8 membered heterocycle formed by R 4a and R 4b together with the nitrogen they are attached to is pyran. In some embodiments, the 5-8 membered heterocycle formed by R 4a and R 4b together with the nitrogen they are attached to is diazinane. In some embodiments, the 5-8 membered heterocycle formed by R 4a and R 4b together with the nitrogen they are attached to is diazine.
  • the 5-8 membered heterocycle formed by R 4a and R 4b together with the nitrogen they are attached to is morpholine. In some embodiments, the 5-8 membered heterocycle formed by R 4a and R 4b together with the nitrogen they are attached to is oxazine. In some embodiments, the 5-8 membered heterocycle formed by R 4a and R 4b together with the nitrogen they are attached to is dioxane. In some embodiments, the 5-8 membered heterocycle formed by R 4a and R 4b together with the nitrogen they are attached to is dioxine. In some embodiments, the 5-8 membered heterocycle formed by R 4a and R 4b together with the nitrogen they are attached to is triazinane.
  • the 5-8 membered heterocycle formed by R 4a and R 4b together with the nitrogen they are attached to is triazine. In some embodiments, the 5-8 membered heterocycle formed by R 4a and R 4b together with the nitrogen they are attached to is trioxane. In some embodiments, the 5-8 membered heterocycle formed by R 4a and R 4b together with the nitrogen they are attached to is azepane. In some embodiments, the 5-8 membered heterocycle formed by R 4a and R 4b together with the nitrogen they are attached to is azepine. In some embodiments, the 5-8 membered heterocycle formed by R 4a and R 4b together with the nitrogen they are attached to is oxepane.
  • the 5-8 membered heterocycle formed by R 4a and R 4b together with the nitrogen they are attached to is oxepine. In some embodiments, the 5-8 membered heterocycle formed by R 4a and R 4b together with the nitrogen they are attached to is azocane. In some embodiments, the 5-8 membered heterocycle formed by R 4a and R 4b together with the nitrogen they are attached to is azocine. In some embodiments, R 4a and R 4b together with the nitrogen they are attached to form a 5-6 membered heterocycle. In some embodiments, the 5-8 membered heterocycle formed by R 4a and R 4b together with the nitrogen they are attached to is tetrahydrothiophene.
  • the 5-8 membered heterocycle formed by R 4a and R 4b together with the nitrogen they are attached to is thiophene. In some embodiments, the 5-8 membered heterocycle formed by R 4a and R 4b together with the nitrogen they are attached to is thiazolidine. In some embodiments, the 5-8 membered heterocycle formed by R 4a and R 4b Attorney Docket No.: CRISP-43327.601 together with the nitrogen they are attached to is thiazole. In some embodiments, the 5-8 membered heterocycle formed by R 4a and R 4b together with the nitrogen they are attached to is isothiazole.
  • the 5-8 membered heterocycle formed by R 4a and R 4b together with the nitrogen they are attached to is dithiolane. In some embodiments, the 5-8 membered heterocycle formed by R 4a and R 4b together with the nitrogen they are attached to is thiane. In some embodiments, the 5-8 membered heterocycle formed by R 4a and R 4b together with the nitrogen they are attached to is thiopyran. In some embodiments, the 5-8 membered heterocycle formed by R 4a and R 4b together with the nitrogen they are attached to is thiomorpholine. In some embodiments, the 5-8 membered heterocycle formed by R 4a and R 4b together with the nitrogen they are attached to is thiazine.
  • the 5-8 membered heterocycle formed by R 4a and R 4b together with the nitrogen they are attached to is oxathiane. In some embodiments, the 5-8 membered heterocycle formed by R 4a and R 4b together with the nitrogen they are attached to is dithiane. In some embodiments, the 5-8 membered heterocycle formed by R 4a and R 4b together with the nitrogen they are attached to is dithiin. In some embodiments, the 5-8 membered heterocycle formed by R 4a and R 4b together with the nitrogen they are attached to is trithiane. In some embodiments, the 5-8 membered heterocycle formed by R 4a and R 4b together with the nitrogen they are attached to is thiepane.
  • the 5-8 membered heterocycle formed by R 4a and R 4b together with the nitrogen they are attached to is thiepine.
  • R 4 wherein R 4a and R 4b together with the nitrogen they are attached to form heterocycle and p is an integer between 1 and 4.
  • R 4 wherein R 4a and R 4b together with the nitrogen they are attached to form a and p is an integer of 1.
  • the 6 membered heterocycle formed with R 4a and R 4b is substituted with C 1 alkyl.
  • the 6 membered heterocycle formed with R 4a and R 4b is diazinane.
  • the 6 membered heterocycle formed with R 4a and R 4b is 1,4-diazinane. In some embodiments, the 6 membered heterocycle formed with R 4a and R 4b is substituted with a methyl (-CH3). In some embodiments, the 6 membered heterocycle formed with R 4a and R 4b substituted with C 1 alkyl . In some Attorney Docket No.: CRISP-43327.601 embodiments, R 4 . In some embodiments, R 4 . In one between 1 and 6. In is an integer between 1 between 1 and 4. p is an 3. In some embodiments, p is an integer of 1. In some embodiments, p is an integer of 2. In some embodiments, p is an integer of 3.
  • p is an integer of 4. In some embodiments, p is an integer of 5. In some embodiments, p is an integer of 6. In one embodiment, m 1 , m 2 , and m 3 are, each independently, an integer between 1 and 6. In some embodiments, m 1 , m 2 , and m 3 are, each independently, an integer between 1 and 4. In some embodiments, m 1 , m 2 , and m 3 are, each independently, an integer between 1 and 3. In some embodiments, m 1 , m 2 , and m 3 are, each independently, 1, 2, or 3.
  • the compound of formula (I), (IA), (IB), (IA-a), (IA-b), (IB-a), (IB-b), (IA-a1), (IA-b1), (IB-a1), or (IB-b1) has one of the following structures shown in Table 1 below.
  • the exemplary compounds are designated Lipids 1-54 and 56-65. Lipids 1-54 and 56-65 were prepared according to the procedures described in Examples 6-88 as designated in Table 1. Table 1: A list of Lipids.
  • compositions Other embodiments are directed to pharmaceutical compositions.
  • the pharmaceutical composition comprises any one (or more) of the foregoing compounds and a pharmaceutically acceptable carrier.
  • the pharmaceutical composition is formulated for oral administration.
  • the pharmaceutical composition is formulated for injection.
  • the pharmaceutical compositions comprise a compound as disclosed herein and an additional therapeutic agent (e.g., anticancer agent).
  • additional therapeutic agents are described herein below.
  • a compound as described herein is administered in a local rather than systemic manner, for example, via injection of the compound directly into an organ, often in a depot preparation or sustained release formulation.
  • long acting formulations are administered by implantation (for example subcutaneously or intramuscularly) or by intramuscular injection.
  • the compound is delivered in a targeted drug delivery system, for example, in a liposome coated with an organ specific antibody.
  • the liposomes are targeted to and taken up selectively by the organ.
  • the compound as described herein is provided in the form of a rapid release formulation, in the form of an extended release formulation, or in the form of an intermediate release formulation.
  • the compound described herein is administered topically.
  • Effective amounts or doses may be ascertained by methods such as modeling, dose escalation studies or clinical trials, e.g., the mode or route of administration or drug delivery, the pharmacokinetics of the agent, the severity and course of the disease, disorder, or condition, the subject's previous or ongoing therapy, the subject's health status and response to drugs, and the judgment of the treating physician.
  • the compounds according to the disclosure are effective over a wide dosage range.
  • dosages from about 0.001 to 0.1 mg, 0.01 to 0.1 mg, 0.5 to 5 mg, 0.5 to 10 mg, 0.01 to 10 mg, 0.1 to 10 mg, 10 to 5000 mg, 100 to 5000 mg, 1000 mg to 4000 mg per day, or 1000 to 3000 mg per day are examples of dosages that are used in some embodiments.
  • the exact dosage will depend upon the route of Attorney Docket No.: CRISP-43327.601 administration, the form in which the compound is administered, the subject to be treated, the body weight of the subject to be treated, and the preference and experience of the attending physician.
  • compounds of the disclosure are administered in a single dose. In an embodiment, the single dose is administered orally.
  • the single dose is administered by injection.
  • other routes are used as appropriate.
  • compounds of the disclosure are administered in multiple doses.
  • dosing is about once, twice, three times, four times, five times, six times, or more than six times per day.
  • dosing is about once a month, once every two weeks, once a week, or once every other day.
  • compounds of the disclosure and another agent e.g., an additional anti-cancer agent
  • the administration of compounds of the disclosure and an agent continues for less than about 7 days.
  • the administration continues for more than about 6, 10, 14, 28 days, two months, six months, or one year.
  • continuous dosing is achieved and maintained as long as necessary.
  • Administration of compounds of the disclosure may continue as long as necessary.
  • compounds of the disclosure are administered for more than 1, 2, 3, 4, 5, 6, 7, 14, or 28 days.
  • compounds of the disclosure are administered for less than 28, 14, 7, 6, 5, 4, 3, 2, or 1 day.
  • compounds of the disclosure are administered chronically on an ongoing basis, e.g., for the treatment of chronic effects.
  • the compounds of the disclosure are administered in individual dosage forms. It is known in the art that due to intersubject variability in compound pharmacokinetics, individualization of dosing regimen is necessary for optimal therapy.
  • the compounds described herein are formulated into pharmaceutical compositions.
  • compositions are formulated in a conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries which facilitate processing of the disclosed compounds into preparations which can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen. Any pharmaceutically acceptable techniques, carriers, and excipients are used as suitable to formulate the pharmaceutical compositions described herein: Remington: The Science and Practice of Pharmacy, Nineteenth Ed (Easton, Pa.: Mack Publishing Company, 1995); Hoover, John E., Remington’s Pharmaceutical Sciences, Mack Publishing Co., Easton, Pennsylvania 1975; Liberman, H.A.
  • compositions comprising one or more compounds of Formula (I), (IA), (IB), (IA-a), (IA-b), (IB-a), (IB-b), (IA-a1), (IA-b1), (IB- a1), or (IB-b1), and a pharmaceutically acceptable carrier.
  • compositions comprising one or more compounds selected from compounds of Formula (I), (IA), (IB), (IA-a), (IA-b), (IB-a), (IB-b), (IA-a1), (IA-b1), (IB-a1), or (IB-b1) and pharmaceutically acceptable diluent(s), excipient(s), and carrier(s).
  • the compounds described are administered as pharmaceutical compositions in which one or more compounds selected from compounds of Formula (I), (IA), (IB), (IA-a), (IA-b), (IB-a), (IB-b), (IA-a1), (IA-b1), (IB-a1), or (IB-b1) are mixed with other active ingredients, as in combination therapy.
  • compounds of Formula (I), (IA), (IB), (IA-a), (IA-b), (IB-a), (IB-b), (IA-a1), (IA-b1), (IB-a1), or (IB-b1) are mixed with other active ingredients, as in combination therapy.
  • the pharmaceutical compositions include one or more compounds of Formula (I), (IA), (IB), (IA-a), (IA-b), (IB-a), (IB-b), (IA-a1), (IA-b1), (IB-a1), or (IB-b1).
  • a pharmaceutical composition refers to a mixture of one or more compounds selected from compounds of Formula (I), (IA), (IB), (IA-a), (IA-b), (IB-a), (IB- b), (IA-a1), (IA-b1), (IB-a1), or (IB-b1) with other chemical components, such as carriers, stabilizers, diluents, dispersing agents, suspending agents, thickening agents, and/or excipients.
  • the pharmaceutical composition facilitates administration of the compound to an organism.
  • therapeutically effective amounts of one or more compounds selected from compounds of Formula (I), (IA), (IB), (IA-a), (IA-b), (IB-a), (IB-b), (IA-a1), (IA-b1), (IB-a1), or (IB-b1) provided herein are administered in a pharmaceutical composition to a mammal having a disease, disorder or medical condition to be treated.
  • the mammal is a human.
  • therapeutically effective amounts vary depending on the severity of the disease, the age and relative health of the subject, the potency of the compound used and other factors.
  • the compounds described herein are used singly or in combination with one or more therapeutic agents as components of mixtures.
  • one or more compounds selected from compounds of Formula (I), (IA), (IB), (IA-a), (IA-b), (IB-a), (IB-b), (IA-a1), (IA-b1), (IB-a1), or (IB-b1) are formulated in aqueous solutions.
  • the aqueous solution is selected from, by way of example only, a physiologically compatible buffer, such as Hank’s solution, Ringer’s solution, or physiological saline buffer.
  • one or more Attorney Docket No.: CRISP-43327.601 compounds selected from compounds of Formula (I), (IA), (IB), (IA-a), (IA-b), (IB-a), (IB- b), (IA-a1), (IA-b1), (IB-a1), or (IB-b1) are formulated for transmucosal administration.
  • transmucosal formulations include penetrants that are appropriate to the barrier to be permeated.
  • appropriate formulations include aqueous or non-aqueous solutions.
  • such solutions include physiologically compatible buffers and/or excipients.
  • compounds described herein are formulated for oral administration. Compounds described herein are formulated by combining the active compounds with, e.g., pharmaceutically acceptable carriers or excipients. In various embodiments, the compounds described herein are formulated in oral dosage forms that include, by way of example only, tablets, powders, pills, dragees, capsules, liquids, gels, syrups, elixirs, slurries, suspensions and the like.
  • pharmaceutical preparations for oral use are obtained by mixing one or more solid excipient with one or more of the compounds described herein, optionally grinding the resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores.
  • Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as: for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methylcellulose, microcrystalline cellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose; or others such as: polyvinylpyrrolidone (PVP or povidone) or calcium phosphate.
  • disintegrating agents are optionally added.
  • Disintegrating agents include, by way of example only, cross linked croscarmellose sodium, polyvinylpyrrolidone, agar, or alginic acid or a pharmaceutically acceptable salt thereof such as sodium alginate.
  • the oral dosage forms such as a pill, capsule or tablet, comprises one or more suitable layers or coatings.
  • concentrated sugar solutions are used for coating the dosage form.
  • the sugar solutions optionally contain additional components, such as by way of example only, gum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures.
  • Dyestuffs and/or pigments are also optionally added to the coatings for identification purposes.
  • the dyestuffs and/or pigments are optionally utilized to characterize different combinations of active compound doses.
  • therapeutically effective amounts of at least one of the Attorney Docket No.: CRISP-43327.601 compounds described herein are formulated into other oral dosage forms.
  • Oral dosage forms include push fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol.
  • push fit capsules contain the active ingredients in admixture with one or more filler.
  • Fillers include, by way of example only, lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate and, optionally, stabilizers.
  • soft capsules contain one or more active compound that is dissolved or suspended in a suitable liquid.
  • suitable liquids include, by way of example only, one or more fatty oil, liquid paraffin, or liquid polyethylene glycol.
  • stabilizers are optionally added.
  • the compounds described herein are formulated for parental injection, including formulations suitable for bolus injection or continuous infusion.
  • formulations for injection are presented in unit dosage form (e.g., in ampoules) or in multi dose containers.
  • Preservatives are, optionally, added to the injection formulations.
  • the pharmaceutical compositions are formulated in a form suitable for parenteral injection as sterile suspensions, solutions or emulsions in oily or aqueous vehicles.
  • Parenteral injection formulations optionally contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
  • pharmaceutical formulations for parenteral administration include aqueous solutions of the active compounds in water soluble form.
  • suspensions of one or more compounds selected from compounds of Formula (I), (IA), (IB), (IA-a), (IA-b), (IB-a), (IB-b), (IA-a1), (IA-b1), (IB-a1), or (IB-b1) are prepared as appropriate oily injection suspensions.
  • Suitable lipophilic solvents or vehicles for use in the pharmaceutical compositions described herein include, by way of example only, fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes.
  • aqueous injection suspensions contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran.
  • the suspension contains suitable stabilizers or agents which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions.
  • the active ingredient is in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.
  • compositions include at least one pharmaceutically acceptable carrier, diluent or excipient, and one or more compounds selected from compounds of Formula (I), (IA), (IB), (IA-a), (IA-b), (IB-a), (IB-b), (IA-a1), (IA-b1), (IB-a1), or (IB-b1) as an active ingredient.
  • the active ingredient is in free-acid or free-base form, or in a pharmaceutically acceptable salt form.
  • the methods and pharmaceutical Attorney Docket No.: CRISP-43327.601 compositions described herein include the use of N-oxides, crystalline forms (also known as polymorphs), as well as active metabolites of these compounds having the same type of activity.
  • compositions optionally include other medicinal or pharmaceutical agents, carriers, adjuvants, such as preserving, stabilizing, wetting or emulsifying agents, solution promoters, pharmaceutically acceptable salts for regulating the osmotic pressure, buffers, and/or other therapeutically valuable substances.
  • compositions comprising the compounds described herein include formulating the compound(s) with one or more inert, pharmaceutically acceptable excipients or carriers to form a solid, semi-solid or liquid composition.
  • Solid compositions include, but are not limited to, powders, tablets, dispersible granules, capsules, cachets, and suppositories.
  • Liquid compositions include solutions in which a compound is dissolved, emulsions comprising a compound, or a solution containing liposomes, micelles, or nanoparticles comprising a compound as disclosed herein.
  • Semi-solid compositions include, but are not limited to, gels, ointments, suspensions and creams.
  • compositions described herein include liquid solutions or suspensions, solid forms suitable for solution or suspension in a liquid prior to use, or as emulsions. These compositions also optionally contain minor amounts of nontoxic, auxiliary substances, such as wetting or emulsifying agents, pH buffering agents, and so forth.
  • pharmaceutical compositions comprising one or more compounds selected from compounds of Formula (I), (IA), (IB), (IA-a), (IA-b), (IB-a), (IB- b), (IA-a1), (IA-b1), (IB-a1), or (IB-b1) illustratively takes the form of a liquid where the agents are present in solution, in suspension or both.
  • a liquid composition typically when the composition is administered as a suspension, a first portion of the agent is present in solution and a second portion of the agent is present in particulate form, in suspension in a liquid matrix.
  • a liquid composition includes a gel formulation.
  • the liquid composition is aqueous.
  • aqueous suspensions contain one or more polymers as suspending agents. Polymers include water-soluble polymers such as cellulosic polymers, e.g., hydroxypropyl methylcellulose, and water-insoluble polymers such as cross-linked carboxyl-containing polymers.
  • compositions described herein Attorney Docket No.: CRISP-43327.601 comprise a mucoadhesive polymer, selected for example from carboxymethylcellulose, carbomer (acrylic acid polymer), poly(methylmethacrylate), polyacrylamide, polycarbophil, acrylic acid/butyl acrylate copolymer, sodium alginate and dextran.
  • Pharmaceutical compositions also, optionally, include solubilizing agents to aid in the solubility of one or more compounds selected from compounds of Formula (I), (IA), (IB), (IA-a), (IA-b), (IB-a), (IB-b), (IA-a1), (IA-b1), (IB-a1), or (IB-b1).
  • solubilizing agent generally includes agents that result in formation of a micellar solution or a true solution of the agent.
  • Certain acceptable nonionic surfactants for example polysorbate 80, are useful as solubilizing agents, as can ophthalmically acceptable glycols, polyglycols, e.g., polyethylene glycol 400, and glycol ethers.
  • compositions optionally include one or more pH adjusting agents or buffering agents, including acids such as acetic, boric, citric, lactic, phosphoric and hydrochloric acids; bases such as sodium hydroxide, sodium phosphate, sodium borate, sodium citrate, sodium acetate, sodium lactate and tris- hydroxymethylaminomethane; and buffers such as citrate/dextrose, sodium bicarbonate and ammonium chloride.
  • acids such as acetic, boric, citric, lactic, phosphoric and hydrochloric acids
  • bases such as sodium hydroxide, sodium phosphate, sodium borate, sodium citrate, sodium acetate, sodium lactate and tris- hydroxymethylaminomethane
  • buffers such as citrate/dextrose, sodium bicarbonate and ammonium chloride.
  • Such acids, bases and buffers are included in an amount required to maintain pH of the composition in an acceptable range.
  • Compositions also, optionally, include one or more pharmaceutically acceptable salts in an amount required to bring
  • Such pharmaceutically acceptable salts include those having sodium, potassium or ammonium cations and chloride, citrate, ascorbate, borate, phosphate, bicarbonate, sulfate, thiosulfate or bisulfite anions; suitable pharmaceutically acceptable salts include sodium chloride, potassium chloride, sodium thiosulfate, sodium bisulfite and ammonium sulfate.
  • Other pharmaceutical compositions optionally include one or more preservatives to inhibit microbial activity.
  • Suitable preservatives include mercury-containing substances such as merfen and thiomersal; stabilized chlorine dioxide; and quaternary ammonium compounds such as benzalkonium chloride, cetyltrimethylammonium bromide and cetylpyridinium chloride.
  • Compositions may include one or more surfactants to enhance physical stability or for other purposes.
  • Suitable nonionic surfactants include polyoxyethylene fatty acid glycerides and vegetable oils, e.g., polyoxyethylene (60) hydrogenated castor oil; and polyoxyethylene alkylethers and alkylphenyl ethers, e.g., octoxynol 10, octoxynol 40.
  • Compositions may include one or more antioxidants to enhance chemical stability where required. Suitable antioxidants include, by way of example only, ascorbic acid and Attorney Docket No.: CRISP-43327.601 sodium metabisulfite.
  • aqueous suspension compositions are packaged in single- dose non-reclosable containers. Alternatively, multiple-dose reclosable containers are used, in which case it is typical to include a preservative in the composition.
  • other delivery systems for hydrophobic pharmaceutical compounds are employed. Liposomes and emulsions are examples of delivery vehicles or carriers useful herein.
  • organic solvents such as N- methylpyrrolidone are also employed.
  • the compounds described herein are delivered using a sustained release system, such as semipermeable matrices of solid hydrophobic polymers containing the therapeutic agent.
  • sustained release materials are useful herein.
  • sustained release capsules release the compounds for a few weeks up to over 100 days.
  • additional strategies for protein stabilization are employed.
  • the formulations described herein comprise one or more antioxidants, metal chelating agents, thiol containing compounds and/or other general stabilizing agents.
  • stabilizing agents include, but are not limited to: (a) about 0.5% to about 2% w/v glycerol, (b) about 0.1% to about 1% w/v methionine, (c) about 0.1% to about 2% w/v monothioglycerol, (d) about 1 mM to about 10 mM EDTA, (e) about 0.01% to about 2% w/v ascorbic acid, (f) 0.003% to about 0.02% w/v polysorbate 80, (g) 0.001% to about 0.05% w/v.
  • polysorbate 20 (h) arginine, (i) heparin, (j) dextran sulfate, (k) cyclodextrins, (l) pentosan polysulfate and other heparinoids, (m) divalent cations such as magnesium and zinc; or (n) combinations thereof.
  • the concentration of one or more compounds selected from compounds of Formula (I), (IA), (IB), (IA-a), (IA-b), (IB-a), (IB-b), (IA-a1), (IA-b1), (IB- a1), or (IB-b1) provided in the pharmaceutical compositions is greater than 90%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 40%, 30%, 20%, 19.75%, 19.50%, 19.25% 19%, 18.75%, 18.50%, 18.25% 18%, 17.75%, 17.50%, 17.25% 17%, 16.75%, 16.50%, 16.25% 16%, 15.75%, 15.50%, 15.25% 15%, 14.75%, 14.50%, 14.25% 14%, 13.75%, 13.50%, 13.25% 13%, 12.75%, 12.50%, 12.25% 12%, 11.75%, 11.50%, 11.25% 11%, 10.75%, 10.50%, 10.25% 10%, 9.
  • the amount of a compound selected from compounds of (I), (IA), (IB), (IA-a), (IA-b), (IB-a), (IB-b), (IA-a1), (IA-b1), (IB-a1), or (IB- b1) in the pharmaceutical compositions is an amount between about any two of the values recited in the preceding sentence, for example, between about 2-70 w/w%, 3.5-80 w/w%, 1- 30 w/w%, etc.
  • the concentration of one or more compounds selected from compounds of Formula (I), (IA), (IB), (IA-a), (IA-b), (IB-a), (IB-b), (IA-a1), (IA-b1), (IB- a1), or (IB-b1) provided in the pharmaceutical compositions of the present disclosure is in the range from approximately 0.0001% to approximately 50%, approximately 0.001% to approximately 40 %, approximately 0.01% to approximately 30%, approximately 0.02% to approximately 29%, approximately 0.03% to approximately 28%, approximately 0.04% to approximately 27%, approximately 0.05% to approximately 26%, approximately 0.06% to approximately 25%, approximately 0.07% to approximately 24%, approximately 0.08% to approximately 23%, approximately 0.09% to approximately 22%, approximately 0.1% to approximately 21%, approximately 0.2% to approximately 20%, approximately 0.3% to approximately 19%, approximately 0.4% to approximately 18%, approximately 0.5% to approximately 17%, approximately 0.6% to approximately 16%, approximately 0.7% to approximately 15%, approximately 0.8% to approximately 14%,
  • the amount the one or more compounds selected from compounds of Formula (I), (IA), (IB), (IA-a), (IA-b), (IB-a), (IB-b), (IA-a1), (IA-b1), (IB- a1), or (IB-b1) provided in the pharmaceutical compositions of the present disclosure is equal to or less than 10 g, 9.5 g, 9.0 g, 8.5 g, 8.0 g, 7.5 g, 7.0 g, 6.5 g, 6.0 g, 5.5 g, 5.0 g, 4.5 g, 4.0 g, 3.5 g, 3.0 g, 2.5 g, 2.0 g, 1.5 g, 1.0 g, 0.95 g, 0.9 g, 0.85 g, 0.8 g, 0.75 g, 0.7 g, 0.65 g, 0.6 g, 0.55 g, 0.5 g, 0.45 g, 0.4 g, 0.35 g, 0.3
  • the amount of the one or more compounds selected from compounds of Formula (I), (IA), (IB), (IA-a), (IA-b), (IB-a), (IB-b), (IA-a1), (IA-b1), (IB- a1), or (IB-b1) provided in the pharmaceutical compositions of the present disclosure is in the range of 0.0001-10 g, 0.0005-9 g, 0.001-8 g, 0.005-7 g, 0.01-6 g, 0.05-5 g, 0.1-4 g, 0.5-4 g, or 1-3 g.
  • Packaging materials for use in packaging pharmaceutical compositions described Attorney Docket No.: CRISP-43327.601 herein include those found in, e.g., U.S. Pat. Nos.5,323,907, 5,052,558 and 5,033,252.
  • Examples of pharmaceutical packaging materials include, but are not limited to, blister packs, bottles, tubes, inhalers, pumps, bags, vials, containers, syringes, bottles, and any packaging material suitable for a selected formulation and intended mode of administration and treatment.
  • the container(s) includes one or more compounds described herein, optionally in a composition or in combination with another agent as disclosed herein.
  • kits optionally have a sterile access port (for example the container is an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle).
  • kits optionally comprise a compound with an identifying description or label or instructions relating to its use in the methods described herein.
  • a kit typically includes one or more additional containers, each with one or more of various materials (such as reagents, optionally in concentrated form, and/or devices) desirable from a commercial and user standpoint for use of a compound described herein.
  • Non-limiting examples of such materials include, but not limited to, buffers, diluents, filters, needles, syringes; carrier, package, container, vial and/or tube labels listing contents and/or instructions for use, and package inserts with instructions for use.
  • a set of instructions will also typically be included.
  • a label is optionally on or associated with the container. For example, a label is on a container when letters, numbers or other characters forming the label are attached, molded or etched into the container itself, a label is associated with a container when it is present within a receptacle or carrier that also holds the container, e.g., as a package insert. In addition, a label is used to indicate that the contents are to be used for a specific therapeutic application.
  • the label indicates directions for use of the contents, such as in the methods described herein.
  • the pharmaceutical compositions are presented in a pack or dispenser device which contains one or more unit dosage forms containing a compound provided herein.
  • the pack for example contains metal or plastic foil, such as a blister pack.
  • the pack or dispenser device is accompanied by instructions for administration, or the pack or dispenser is accompanied with a notice associated with the container in form prescribed by a governmental agency regulating the manufacture, use, or sale of pharmaceuticals, which notice is reflective of approval by the agency of the form of the drug for human or veterinary administration.
  • a notice associated with the container in form prescribed by a governmental agency regulating the manufacture, use, or sale of pharmaceuticals which notice is reflective of approval by the agency of the form of the drug for human or veterinary administration.
  • Such notice for example, is the labeling approved by the U.S. Food and Drug Administration for prescription drugs, or the approved product insert.
  • compositions containing a compound provided herein formulated in a compatible pharmaceutical carrier are prepared, placed in an appropriate container, and Attorney Docket No.: CRISP-43327.601 labeled for treatment of an indicated condition.
  • a pharmaceutical composition has a compound described above and a pharmaceutically acceptable carrier including, for example, any adjuvant, carrier, excipient, glidant, sweetening agent, diluent, preservative, dye/colorant, flavor enhancer, surfactant, wetting agent, dispersing agent, suspending agent, stabilizer, isotonic agent, solvent, or emulsifier which has been approved by the United States Food and Drug Administration as being acceptable for use in humans or domestic animals.
  • a pharmaceutical composition comprising the compound of Formula (I), (IA), (IB), (IA-a), (IA-b), (IB-a), (IB-b), (IA-a1), (IA-b1), (IB-a1), or (IB-b1) and an additional therapeutic agent is disclosed.
  • LNP compositions for delivering biologically active agents, such as nucleic acids, e.g., DNA, mRNAs and gRNAs, including CRISPR/Cas cargoes.
  • Such LNP compositions include an “ionizable amine lipid”, along with, for example, cholesterol, a polymer conjugated lipid, and a helper lipid.
  • LNP Lip nanoparticle
  • the LNP composition comprises a compound of Formula (I), (IA), (IB), (IA-a), (IA-b), (IB-a), (IB-b), (IA-a1), (IA-b1), (IB-a1), or (IB-b1) or a combination of one or more of the compounds and one or more additional lipid components selected from the group consisting of neutral lipids and/or helper lipids (collectively helper lipids), polymer conjugated lipids, and cholesterol or a derivative thereof.
  • the composition further comprises a helper lipid and cholesterol or a derivative thereof. In some embodiments, the composition further comprises a helper lipid and a polymer conjugated lipid. In some embodiments, the composition further comprises a helper lipid, cholesterol or a derivative thereof, and a polymer conjugated lipid.
  • the helper lipid is selected from the group consisting of 1,2- dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero- phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2- dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-distearoyl-sn-glycero-3- phosphocholine (DSPC), 1,2-diundecanoyl-sn-glycero-phosphocholine (DUPC), l-palmitoyl- Attorney Docket No.: CRISP-43327.601 2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3- phosphocholine (18:0
  • the helper lipid is DOPE or DSPC. In some embodiments, the helper lipid is a lipid other than cholesterol.
  • Embodiments of the present disclosure provide lipid compositions described according to the respective molar ratios of the component lipids in the composition. All mol-% numbers are given as a fraction of the lipid component of the lipid composition or, more specifically, the LNP compositions.
  • the mol-% of the compound of Formula (I), (IA), (IB), (IA-a), (IA-b), (IB-a), (IB-b), (IA-a1), (IA-b1), (IB-a1), or (IB-b1) may be from about 10 mol-% to about 70 mol-%. In certain embodiments, the mol-% of the compound of Formula (I), (IA), (IB), (IA-a), (IA-b), (IB-a), (IB-b), (IA-a1), (IA-b1), (IB-a1), or (IB-b1) may be from about 20 mol-% to about 70 mol-%.
  • the mol-% of the compound of Formula (I), (IA), (IB), (IA-a), (IA-b), (IB-a), (IB-b), (IA-a1), (IA-b1), (IB-a1), or (IB-b1) may be at least 10 mol-%, at least 20 mol-%, at least 30 mol-%, at least 40 mol-%, or at least 50 mol-%.
  • the mol-% of the helper lipid may be from about 0 mol-% to about 60 mol-%. In certain embodiments, the mol-% of the helper lipid may be from about 20 mol-% to about 60 mol-%.
  • the mol-% of the helper lipid may be from about 20 mol-% to about 50 mol-%. In certain embodiments, the mol-% of the helper lipid may be about 40 mol-%. In certain embodiments, the mol-% of the helper lipid may be about 30 mol-%. In certain embodiments, the mol-% of cholesterol or a derivative thereof may be from about 0 mol-% to about 80 mol-%. In certain embodiments, the mol-% of cholesterol or a derivative thereof may be from about 20 mol-% to about 60 mol-%. In certain embodiments, the mol-% of cholesterol or a derivative thereof may be from about 30 mol-% to about 50 mol-%.
  • the mol-% of cholesterol or a derivative thereof may be Attorney Docket No.: CRISP-43327.601 from 30 mol-% to about 40 mol-% or from about 35% mol-% to about 45 mol-%.
  • the mol-% of cholesterol or a derivative thereof is adjusted based on compound of Formula (I), (IA), (IB), (IA-a), (IA-b), (IB-a), (IB-b), (IA-a1), (IA-b1), (IB-a1), or (IB-b1), helper lipid and/or PEG lipid concentrations to bring the lipid component to 100 mol-%.
  • the mol-% of the PEG lipid may be from about 0.5 mol-% to about 10 mol-%. In certain embodiments, the mol-% of the PEG lipid may be from about 0.5 mol-% to about 4 mol-%. In certain embodiments, the mol-% of the PEG lipid may be about 0.5 mol-% to about 2 mol-%. In certain embodiments, the mol-% of the PEG lipid may be about 1.5 mol-%. In certain embodiments, the mol-% of the PEG lipid may be about 1.0 mol-%. In certain embodiments, the mol-% of the PEG lipid may be about 0.5 mol-%.
  • the molar ratio of the compound of Formula (I), (IA), (IB), (IA- a), (IA-b), (IB-a), (IB-b), (IA-a1), (IA-b1), (IB-a1), or (IB-b1) to the helper lipid ranges from about 1:7 to about 7:1. In another embodiment, the molar ratio of the compound of Formula (I), (IA), (IB), (IA-a), (IA-b), (IB-a), (IB-b), (IA-a1), (IA-b1), (IB-a1), or (IB-b1) to the helper lipid ranges from about 1:5 to about 5:1.
  • the molar ratio of the compound of Formula (I), (IA), (IB), (IA-a), (IA-b), (IB-a), (IB-b), (IA-a1), (IA-b1), (IB- a1), or (IB-b1) to the helper lipid ranges from about 1:4 to about 4:1. In another embodiment, the molar ratio of the compound of Formula (I), (IA), (IB), (IA-a), (IA-b), (IB- a), (IB-b), (IA-a1), (IA-b1), (IB-a1), or (IB-b1) to the helper lipid ranges from about 1:3 to about 3:1.
  • the molar ratio of the compound of Formula (I), (IA), (IB), (IA-a), (IA-b), (IB-a), (IB-b), (IA-a1), (IA-b1), (IB-a1), or (IB-b1) to the helper lipid ranges from about 1:2.5 to about 2.5:1. In another embodiment, the molar ratio of the compound of Formula (I), (IA), (IB), (IA-a), (IA-b), (IB-a), (IB-b), (IA-a1), (IA-b1), (IB-a1), or (IB-b1) to the helper lipid ranges from about 1:2 to about 2:1.
  • the molar ratio of the compound of Formula (I), (IA), (IB), (IA-a), (IA-b), (IB-a), (IB-b), (IA- a1), (IA-b1), (IB-a1), or (IB-b1) to the helper lipid ranges from about 1:1 to about 1:3. In another embodiment, the molar ratio of the compound of Formula (I), (IA), (IB), (IA-a), (IA- b), (IB-a), (IB-b), (IA-a1), (IA-b1), (IB-a1), or (IB-b1) to the helper lipid ranges from about 1:1 to about 1:2.
  • the molar ratio of the compound of Formula (I), (IA), (IB), (IA- a), (IA-b), (IB-a), (IB-b), (IA-a1), (IA-b1), (IB-a1), or (IB-b1) to cholesterol ranges from 3:1 to 1:3.
  • the molar ratio of the compound of Formula (I), (IA), (IB), (IA-a), (IA-b), (IB-a), (IB-b), (IA-a1), (IA-b1), (IB-a1), or (IB-b1) to cholesterol ranges from 2:1 to 1:2.
  • the molar ratio of the compound of Formula (I), (IA), Attorney Docket No.: CRISP-43327.601 (IB), (IA-a), (IA-b), (IB-a), (IB-b), (IA-a1), (IA-b1), (IB-a1), or (IB-b1) to cholesterol ranges from 1:1 to 1:3.
  • the molar ratio of the compound of Formula (I), (IA), (IB), (IA-a), (IA-b), (IB-a), (IB-b), (IA-a1), (IA-b1), (IB-a1), or (IB-b1) to cholesterol ranges from 1:1 to 1: 2.
  • a composition comprising the compound of Formula (I), (IA), (IB), (IA-a), (IA-b), (IB-a), (IB-b), (IA-a1), (IA-b1), (IB-a1), or (IB-b1) and a nucleic acid is disclosed.
  • the composition is an LNP composition as described above.
  • the nucleic acid comprises RNA, DNA, or mixtures thereof.
  • the RNA comprises messenger RNA (mRNA), guide RNA (gRNA), interfering RNA (RNAi), such as small interfering RNA (siRNA), small activating RNA (saRNA), circular RNA (circRNA), or mixtures thereof.
  • the nucleic acid is messenger RNA (mRNA).
  • the nucleic acid is DNA.
  • the nucleic acid is a guide RNA (gRNA).
  • the RNA encodes a nuclease.
  • the nuclease is an RNA-directed nuclease or site-directed nuclease such as a Cas nuclease.
  • composition comprising the compound of Formula (I), (IA), (IB), (IA-a), (IA-b), (IB-a), (IB-b), (IA-a1), (IA-b1), (IB-a1), or (IB-b1) and a gene editing system is disclosed.
  • the composition is an LNP composition as described above.
  • the components of the gene editing system are nucleic acids or encoded by nucleic acids as is known in the art.
  • CRISPR clustered regularly interspaced short palindromic repeats
  • RT reverse transcriptase
  • TALEN transcription activator-like effector nuclease
  • ZFN zinc finger nuclease
  • the base editing, gene editing, and RT editing systems described herein comprise site-directed polypeptides that associate and/or complex with a genome-targeting nucleic acid to edit a genome of a cell in vitro, ex vivo, or in vivo (e.g., in a human subject).
  • the site-directed polypeptide can bind to a gRNA that, in turn, specifies the site in the target DNA to which the polypeptide is directed.
  • the site-directed polypeptide can bind to a template armed guide RNA (tagRNA) that, in turn, specifies the site in the target DNA to which the polypeptide is directed.
  • a site-directed polypeptide of a base editing system, gene editing system, and/or RT editing system disclosed herein can comprise one or more nuclease domains (e.g., a domain having endonuclease activity).
  • the site-directed polypeptide can be engineered to have one or more domains having endonuclease activity or a wild-type polypeptide with one or more domains having endonuclease activity (e.g., Cas9 from S. pyogenes).
  • the site-directed polypeptide can be an endonuclease, such as a DNA endonuclease.
  • Cas proteins include, without limitation, Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas5d, Cas5t, Cas5h, Cas5a, Cas6, Cas7, Cas8, Cas8a, Cas8b, Cas8c, Cas9 (also known as Csn1 and Csx12), Cas10, Cas11, Casl2a/Cpfl, Casl2b/C2cl, Casl2c/C2c3, Casl2d/CasY, Casl2e/CasX, Cas 12g, Casl2h, Casl2i, Csy1, Csy2, Csy3, Csy4, Cse1 , Cse2, Cse3, Cse4, Cse5e, Csc1 , Csc2, Csa5, Csnl, Csn2, Csml, Csm2, Cs
  • the site-directed polypeptide of a base editing system, gene editing system, and/or RT editing system is encoded by an RNA.
  • the base editing, gene editing and RT editing systems described herein use a Attorney Docket No.: CRISP-43327.601 genome-targeting nucleic acid that can direct a site-directed polypeptide to a target nucleic acid sequence to edit a target gene in the genome of a cell, tissue, or organism (e.g., human subject).
  • the disclosure is not limited to any particular type of genome-targeting nucleic acid.
  • the skilled artisan will appreciate that the genome-targeting nucleic acid can depend on the type of genomic editing used.
  • the genome-targeting nucleic acid can be an RNA.
  • a guide RNA can comprise at least a spacer sequence that hybridizes to a target nucleic acid sequence of interest, and a CRISPR repeat sequence.
  • the gRNA also comprises a second RNA called the trans-activating RNA (tracrRNA) sequence.
  • tracrRNA trans-activating RNA
  • the CRISPR repeat sequence and tracrRNA sequence hybridize to each other to form a duplex.
  • the crRNA forms a duplex.
  • the duplex can bind a site-directed polypeptide, such that the guide RNA and site-direct polypeptide form a complex.
  • the genome-targeting nucleic acid can provide target specificity to the complex by virtue of its association with the site-directed polypeptide.
  • the genome-targeting nucleic acid thus can direct the activity of the site-directed polypeptide.
  • the genome-targeting nucleic acid can be a single-molecule guide RNA (sgRNA).
  • a single-molecule guide RNA (sgRNA) in a Type II system can comprise, in the 5′ to 3′ direction, an optional spacer extension sequence, a spacer sequence, a minimum CRISPR repeat sequence, a single-molecule guide linker, a minimum tracrRNA sequence, a 3′ tracrRNA sequence and an optional tracrRNA extension sequence.
  • the optional tracrRNA extension can comprise elements that contribute additional functionality (e.g., stability) to the guide RNA.
  • the single-molecule guide linker can link the minimum CRISPR repeat and the minimum tracrRNA sequence to form a hairpin structure.
  • the optional tracrRNA extension can comprise one or more hairpins.
  • a single-molecule guide RNA (sgRNA) in a Type V system can comprise, in the 5′ to 3′ direction, a minimum CRISPR repeat sequence and a spacer sequence.
  • the genome-targeting nucleic acid can be an RT editing guide RNA (tagRNA) molecule.
  • a tagRNA can comprise an RNA scaffold, a guide sequence (spacer), a flap binding site (FBS) and an editing template (ET).
  • the tagRNA uses the spacer to direct a site- directed RT-editor polypeptide to a target nucleic acid sequence in a genome (protospacer) and initiates a nuclease- or nickase-mediated strand nick at a nick site, resulting in 3’ end hybridization to the FBS and initiation of reverse transcription by the ET to form a pair of redundant single-stranded DNA flaps.
  • Equilibrium between the edited 3’ flap and unedited 5’ flap mediates cleavage, ligation, DNA repair, and the incorporation of the desired edit Attorney Docket No.: CRISP-43327.601 encoded in the ET into the genome of a cell.
  • the gene editing system may further comprise a donor polynucleotide or sequence.
  • the donor template is DNA molecule that has homology to a target sequence.
  • the donor template is an exogenous polynucleotide sequence that can be inserted into a target nucleic acid cleavage site.
  • the donor polynucleotide, a portion of the donor polynucleotide, a copy of the donor polynucleotide, or a portion of a copy of the donor polynucleotide can be inserted into the target nucleic acid cleavage site.
  • the gene editing systems described herein include using site- directed nucleases to cut deoxyribonucleic acid (DNA) at precise target locations in the genome, thereby creating single-strand or double-strand DNA breaks at particular locations within the genome.
  • DNA deoxyribonucleic acid
  • Such breaks can be and regularly are repaired by natural, endogenous cellular processes, such as homology-directed repair (HDR) and NHEJ, as reviewed in Cox et al., Nature Medicine 21(2), 121-31 (2015).
  • HDR homology-directed repair
  • NHEJ directly joins the DNA ends resulting from a double-strand break, sometimes with the loss or addition of nucleotide sequence, which may disrupt or enhance gene expression.
  • HDR utilizes a homologous sequence, or donor sequence, as a template for inserting a defined DNA sequence at the break point.
  • the homologous sequence can be in the endogenous genome, such as a sister chromatid.
  • the donor can be an exogenous nucleic acid, such as a plasmid, a single-strand oligonucleotide, a double-stranded oligonucleotide, a duplex oligonucleotide or a virus, that has regions of high homology with the nuclease-cleaved locus, but which can also contain additional sequence or sequence changes including deletions that can be incorporated into the cleaved target locus.
  • a third repair mechanism can be microhomology-mediated end joining (MMEJ), also referred to as “Alternative NHEJ,” in which the genetic outcome is similar to NHEJ in that small deletions and insertions can occur at the cleavage site.
  • MMEJ can make use of homologous sequences of a few base pairs flanking the DNA break site to drive a more favored DNA end joining repair outcome, and recent reports have further elucidated the molecular mechanism of this process; see, e.g., Cho and Greenberg, Nature 518, 174-76 (2015); Kent et al., Nature Structural and Molecular Biology, Adv.
  • a lipid formulation preferably an LNP comprising a compound of Formula (I), (IA), (IB), (IA-a), (IA-b), (IB-a), (IB-b), (IA-a1), (IA-b1), (IB-a1), or (IB-b1), in association with a nucleic acid or gene editing system, is administered to the subject or the cells of the subject.
  • the subject is an animal.
  • the subject is a human.
  • Suitable protecting groups include hydroxy, amino, mercapto and carboxylic acid.
  • Suitable protecting groups for hydroxy include trialkylsilyl or diarylalkylsilyl (for example, t-butyldimethylsilyl, t-butyldiphenylsilyl or trimethylsilyl), tetrahydropyranyl, benzyl, and the like.
  • Suitable protecting groups for amino, amidino and guanidino include t- butoxycarbonyl, benzyloxycarbonyl, and the like.
  • Suitable protecting groups for mercapto include —C(O)—R′′ (where R′′ is alkyl, aryl or arylalkyl), p-methoxybenzyl, trityl and the like.
  • Suitable protecting groups for carboxylic acid include alkyl, aryl or arylalkyl esters.
  • Protecting groups may be added or removed in accordance with standard techniques, which are known to one skilled in the art and as described herein. The use of protecting groups is described in detail in Green, T. W. and P. G. M. Wutz, Protective Groups in Organic Synthesis (1999), 3rd Ed., Wiley.
  • the protecting group may also be a polymer resin such as a Wang resin, Rink resin or a 2-chlorotrityl- chloride resin.
  • a polymer resin such as a Wang resin, Rink resin or a 2-chlorotrityl- chloride resin.
  • a phosphate bearing compound of (I), (IA), (IB), (IA-a), (IA-b), (IB-a), (IB-b), (IA-a1), (IA-b1), (IB-a1), or (IB-b1) is a prodrug and the structure of such example is described in the disclosure.
  • all compounds of the invention which exist in free base or acid form can be converted to their pharmaceutically acceptable salts by treatment with the appropriate inorganic or organic base or acid by methods known to one skilled in the art. Salts of the Attorney Docket No.: CRISP-43327.601 compounds of the invention can be converted to their free base or acid form by standard techniques.
  • Example 6 2-((4,6-bis((3-(diundecylamino)propyl)amino)-1,3,5-triazin-2-yl)amino)ethan-1-ol (Lipid 1) (2-((4,6-bis((3- ethan-1-ol) can be prepared by performing a reductive amination with Intermediate B (di-tert-butyl (((6-((2- hydroxyethyl)amino)-1,3,5-triazine-2,4-diyl)bis(azanediyl))bis(propane-3,1- diyl))dicarbamate).150 mg (1 eq) of intermediate B (di-tert-butyl (((6-((2- hydroxyethyl)amino)-1,3,5-triazine-2,4-diyl)bis(azanediyl))bis(propane-3,1- diyl))dicarba
  • Example 7 2-((4,6-bis((3-(didodecylamino)propyl)amino)-1,3,5-triazin-2-yl)amino)ethan-1-ol (Lipid 2)
  • (2-((4,6-bis((3- ethan-1-ol) can be prepared in a similar was replaced with 6 eq of dodecanal yielding 12.5 mg (4.2% yield) of product.
  • M+1 957.91.
  • Example 9 3-((4,6-bis((3-(diundecylamino)propyl)amino)-1,3,5-triazin-2-yl)amino)propan-1-ol (Lipid 4) OH 2 3 (3-((4,6-bis((3- propan-1-ol) can be prepared by performing a reductive amination with Intermediate C (di-tert-butyl (((6-((3- hydroxypropyl)amino)-1,3,5-triazine-2,4-diyl)bis(azanediyl))bis(propane-3,1- diyl))dicarbamate).200 mg (1 eq, 0.4 mM) of intermediate C (di-tert-butyl (((6-((3- Attorney Docket No.: CRISP-43327.601 hydroxypropyl)amino)-1,3,5-triazine-2,4-diyl)
  • Example 10 3-((4,6-bis((3-(didodecylamino)propyl)amino)-1,3,5-triazin-2-yl)amino)propan-1-ol (Lipid 5) (3-((4,6-bis((3- propan-1-ol) can be prepared in a similar manner as Example 9.
  • Example 11 3-((4,6-bis((3-(di(undec-10-en-1-yl)amino)propyl)amino)-1,3,5-triazin-2-yl)amino)propan-1- ol (Lipid 6) 1-ol) can be prepared in a similar manner as Example 9.
  • the undecanal was Attorney Docket No.: CRISP-43327.601 replaced with 7 eq of undecylenic aldehyde yielding 67 mg (24.52% yield) of product.
  • M+1 907.85.
  • Example 12 4-((4,6-bis((3-(dioctylamino)propyl)amino)-1,3,5-triazin-2-yl)amino)butan-1-ol (Lipid 7) (4-((4,6-bis((3- butan-1-ol) can be prepared by (di-tert-butyl (((6-((4- hydroxybutyl)amino)-1,3,5-triazine-2,4-diyl)bis(azanediyl))bis(propane-3,1- diyl))dicarbamate).150 mg (1 eq, 0.3 mM) of intermediate D (di-tert-butyl (((6-((4- hydroxybutyl)amino)-1,3,5-triazine-2,4-diyl)bis(azanediyl))bis(propane-3,1- diyl))dicarbamate) was first deprotect
  • Example 13 4-((4,6-bis((3-(dinonylamino)propyl)amino)-1,3,5-triazin-2-yl)amino)butan-1-ol (Lipid 8) (4-((4,6-bis((3- butan-1-ol) can be prepared in a similar manner as Example 12.
  • Example 17 4-((4,6-bis((3-(ditetradecylamino)propyl)amino)-1,3,5-triazin-2-yl)amino)butan-1-ol (Lipid 12)
  • CRISP-43327.601 (4-((4,6-bis((3- amino)butan-1-ol) can be prepared in a was replaced with 6 eq of tetradecanal yielding 203 mg (63.11% yield) of product.
  • M+1 1098.17.
  • N2,N4,N6-tris(3-(dinonylamino)propyl)-1,3,5-triazine-2,4,6-triamine (Lipid 13) N2,N4,N6-tris(3- can be prepared by performing a reductive amination with Intermediate E (tri-tert-butyl (((1,3,5-triazine-2,4,6- triyl)tris(azanediyl))tris(propane-3,1-diyl))tricarbamate).150 mg (1 eq, 0.25 mM) of intermediate C (tri-tert-butyl (((1,3,5-triazine-2,4,6-triyl)tris(azanediyl))tris(propane-3,1- diyl))tricarbamate) was first deprotected overnight using 5 mL of DCM and 1mL of 4N HCl in 1,4 dioxane in a 40 mL vial
  • Example 21 N2,N4,N6-tris(3-(ditridecylamino)propyl)-1,3,5-triazine-2,4,6-triamine (Lipid 16) (N2,N4,N6-tris(3- can be prepared in a similar manner as Example 18.
  • Example 23 tetrahexyl 3,3',3'',3''-((((6-((2-hydroxyethyl)amino)-1,3,5-triazine-2,4- diyl)bis(azanediyl))bis(propane-3,1-diyl))bis(azanetriyl))tetrapropionate (Lipid 18) (tetrahexyl diyl)bis(azanediyl))bis(propane-3,1-diyl))bis(azanetriyl))tetrapropionate) can be prepared by a Michaels Addition of Intermediate B (di-tert-butyl (((6-((2-hydroxyethyl)amino)-1,3,5- triazine-2,4 diyl)bis(azanediyl))bis(propane-3,1-diyl))dicarbamate) with hexyl acrylate
  • Example 24 tetraoctyl 3,3',3'',3'''-((((6-((2-hydroxyethyl)amino)-1,3,5-triazine-2,4- diyl)bis(azanediyl))bis(propane-3,1-diyl))bis(azanetriyl))tetrapropionate (Lipid 19)
  • Example 30 tetradodecyl 3,3',3'',3''-((((6-((3-hydroxypropyl)amino)-1,3,5-triazine-2,4- diyl)bis(azanediyl))bis(propane-3,1-diyl))bis(azanetriyl))tetrapropionate (Lipid 25) (tetradodecyl diyl)bis(azanediyl))bis(propane-3,1-diyl))bis(azanetriyl))tetrapropionate) can be prepared in a similar manner as example 28.
  • Example 34 tetraoctyl 3,3',3'',3''-((((6-((4-hydroxybutyl)amino)-1,3,5-triazine-2,4- diyl)bis(azanediyl))bis(propane-3,1-diyl))bis(azanetriyl))tetrapropionate (Lipid 29) (tetraoctyl diyl)bis(azanediyl))bis(propane-3,1-diyl))bis(azanetriyl))tetrapropionate) can be prepared in a similar manner as example 33.
  • Example 36 tetrakis(decyl) 3,3',3'',3''-((((6-((4-hydroxybutyl)amino)-1,3,5-triazine-2,4- diyl)bis(azanediyl))bis(propane-3,1-diyl))bis(azanetriyl))tetrapropionate (Lipid 31) (tetrakis diyl)bis(azanediyl))bis(propane-3,1-diyl))bis(azanetriyl))tetrapropionate) can be prepared in a similar manner as example 33.
  • Example 41 4-((4,6-bis((3-(didodecylamino)propyl)amino)-1,3,5-triazin-2-yl)amino)butyl 3-(4- methylpiperazin-1-yl)propanoate (Lipid 36) N (4-((4,6-bis((3- butyl 3-(4- methylpiperazin-1-yl)propanoate) can be prepared via an EDC coupling between Example 16 (4-((4,6-bis((3-(didodecylamino)propyl)amino)-1,3,5-triazin-2-yl)amino)butan-1-ol) and 3-(4-methylpiperazin-1-yl)propanoic acid.100 mg (0.1 mM, 1 eq) of 4-((4,6-bis((3- (didodecylamino)propyl)amino)-1,3,5-triazin-2-yl)amin
  • Example 42 tetraoctyl 3,3',3'',3''-((((6-((4-((3-(4-methylpiperazin-1-yl)propanoyl)oxy)butyl)amino)- 1,3,5-triazine-2,4-diyl)bis(azanediyl))bis(propane-3,1-diyl))bis(azanetriyl))tetrapropionate (Lipid 37) N O N (tetraoctyl 3,3',3'',3''''-(( oxy)butyl)amino)- 1,3,5-triazine-2,4-diyl)bis(azanediyl))bis(propane-3,1-diyl))bis(azanetriyl))tetrapropionate) can be prepared in a similar manner as example 41.
  • Example 45 4-((4,6-bis((3-(bis((2E,4E,6E,8E)-3,7-dimethyl-9-(2,6,6-trimethylcyclohex-1-en-1-yl)nona- 2,4,6,8-tetraen-1-yl)amino)propyl)amino)-1,3,5-triazin-2-yl)amino)butan-1-ol (Lipid 40)
  • Example 46 Intermediate F (di-tert-butyl (((6-chloro-1,3,5-triazine-2,4-diyl)bis(oxy))bis(propane-3,1- diyl))dicarbamate)
  • Intermediate F (di- )bis(propane-3,1- diyl))dicarbamate) was prepared by mixing cyanuric chloride (1eq, 5000mg) with N-boc- 1,3-aminopropanol (2.4eq, 11413mg) in 150ml of chloroform and 10mL of DIPEA. The reaction was performed in a pressure vessel starting at 0C and after 30 minutes was heated to 50C before being left to run overnight.
  • intermediate F (1eq) was returned to the pressure vessel.
  • the material was dissolved in 1,4 dioxane and 4-hydroxybutylamine (2eq) was added. DIPEA was added last before the reaction vessel was closed and heated to 80C for 4 days.
  • the reaction was concentrated and purified via flash chromatography (DCM/Methanol, 0-20% over 30 minutes). The product eluted at around 10% methanol yielding 2g (45%) of tert- butyl (3-((4-(3-(3,3-dimethylbutanamido)propoxy)-6-((4-hydroxybutyl)amino)-1,3,5-triazin- 2-yl)oxy)propyl)carbamate.
  • Example 72 tetradodecyl 3,3',3'',3'''-((((6-((2-(pyrrolidin-1-yl)ethyl)amino)-1,3,5-triazine-2,4- diyl)bis(azanediyl))bis(propane-3,1-diyl))bis(
  • Example 74 tetradodecyl 3,3',3'',3'''-((((6-((2-(4-hydroxypiperidin-1-yl)ethyl)amino)-1,3,
  • Example 86 Tetradodecyl 3,3',3'',3'''-((((6-((2-(diethylamino)ethyl)amino)-1,3,5-triazine-2,4- diyl)bis(oxy))bis(propane-3,1-diyl))bis(azanetriyl))tetrapropionate (Lipid 64)
  • Example 88 LNP preparation method One volume of lipid mixtures (ionizable lipid, cholesterol, DOPE or DSPC, and DMG-PEG at 22/33/43/2 or 49/38.5/10/2.5 or 42.5/40/15/2.5 -mole ratio) in ethanol and three volumes of biologically active agents (DNA, mRNA, guideRNA) containing 50mM acetate buffer solutions were mixed through the micromixer at a combined flow rate of 12 mL /min. The resultant mixture underwent buffer exchange via amicon or dialysis to remove ethanol. Post dialysis the collected LNP were sterilely filter and mixed with a cryoprotectant.
  • lipid mixtures ionizable lipid, cholesterol, DOPE or DSPC, and DMG-PEG at 22/33/43/2 or 49/38.5/10/2.5 or 42.5/40/15/2.5 -mole ratio
  • biologically active agents DNA, mRNA, guideRNA
  • Example 89 In vitro editing of human Hep3 Cells Several of the ionizable lipids described above were selected for use in experiments examining in vitro editing in Hep3B cells. The selected lipid and their structures are provided in Table 2. Table 2. Lipid identifier Compound number, see Table 1 CC10-L2-T14 44 CC14-L2-T14 48 CC16-L2-T14 50 CC17-L2-T14 51 CC3-L5-T3 63 LNPs incorporating the ionizable lipids were produced as described in Example 88 above.
  • FIGs.1A and 1B provide editing efficiency (TIDE analysis) and toxicity data for editing in Hep3B cell at different LNP dosages.
  • FIGs.2A and 2B provide data for editing efficiency (TIDE analysis) and toxicity for LNPs comprising selected ionizable lipids at different dosages, pH and buffers.
  • FIGs.3A and 3B provide bar graphs that summarize the editing efficiency from FIGs.1A and 2A at the 0.0156 ng/ul dosage.
  • FIGs 4A and 4B provide data for editing efficiency (TIDE analysis) and toxicity for LNP formulations using selected ionizable lipids as indicated.
  • Lipid 48 is one of the top performing ionizable lipids in terms of in vitro gene editing efficiency.
  • Example 90 In vitro editing of mice AML12 Cells Several of the ionizable lipids described above were selected for use in experiments examining in vitro editing in mice AML-12 cells. Briefly, the LNPs were formulated to deliver Cas9 mRNA and gRNA targeting mice PCSK9 gene.
  • FIGs.5A and 5B provide Attorney Docket No.: CRISP-43327.601 editing efficiency (TIDE analysis) and toxicity data for editing in AML-12 cells cell at different LNP dosages. These data show LNP formulated with Lipid 63, Lipid 48 and Lipid 50 are also able to edit the target genes in mice AML12 cells in a dose-dependent manner.
  • Example 91 In vivo editing of liver cells Several of the ionizable lipids described above were selected for use in experiments examining in vivo editing of liver cells in a wildtype mouse model.
  • the LNPs were formulated to deliver CRISPR Cas9 mRNA and mice PCSK9 guide RNA at a ratio of 1:3 and intravenously injected into wildtype mice at a dose of 1mg/kg.
  • One week post dosing the mice were sacrificed, and the livers of the treated mice were collected.
  • Genomic DNA was extracted from the collected livers using Quick Extract TM DNA Extraction Solution (QE0905T) and following the manufacturer’s protocol. Editing efficiency in the extracted genomic DNA was determined by TIDE analysis.
  • FIG.6 provides editing efficiency (TIDE analysis) data.
  • Example 92 In Vitro Editing of Human HEP3 Cells LNPs incorporating the ionizable lipids were produced as described in Example 88 above. Briefly, the LNPs were formulated to deliver Cas9 mRNA and gRNA targeting the human ANGPTL3 gene.
  • FIGs.7A and B and 8A and B provide editing efficiency (TIDE analysis) and toxicity data for editing in Hep3B cells at different LNP dosages with LNPs comprising the specified lipids.

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Abstract

L'invention concerne un composé ayant la structure suivante de formule (I) ou un stéréoisomère du composé, un tautomère du composé, un sel pharmaceutiquement acceptable de celui-ci, A1, A2, A3, R1a, R1b, R2a, R2b, m1, m2, m3 et X étant tels que définis dans la description. L'invention concerne également une composition pharmaceutique comprenant les composés, et leur utilisation dans des méthodes de traitement de maladies.
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WO2013016058A1 (fr) 2011-07-22 2013-01-31 Merck Sharp & Dohme Corp. Nouveaux lipides cationiques contenant du bis-azote pour administration d'oligonucléotide
WO2013086373A1 (fr) 2011-12-07 2013-06-13 Alnylam Pharmaceuticals, Inc. Lipides pour l'administration d'agents actifs
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