WO2025113656A1 - Ionizable cationic lipid compound and use thereof - Google Patents

Abstract

An ionizable cationic lipid compound having a structure represented by formula (I), which can be used to prepare lipid nanoparticles (LNP) for the delivery of a therapeutic and/or prophylactic agent. The LNP prepared using the ionizable cationic lipid compound has good stability and transfection efficiency, and can efficiently and stably deliver bioactive substances (including nucleic acids, such as mRNA) to target cells or organs, thereby eliciting a high-specificity antibody response in vivo.

Description

An ionizable cationic lipid compound and its application Technical Field

The invention belongs to the field of biomedicine, and in particular relates to a steroid-cationic lipid compound and its application in the delivery of bioactive substances.

Background Art

Nucleic acid drugs mainly refer to compounds containing nucleotide or deoxynucleotide structures with genetic characteristics and pharmacological activity. They can be used to treat tumors, tissue regeneration, wound healing, pulmonary fibrosis, inflammatory diseases, microbial infections, etc. After nucleic acid drugs are injected into the human body, an efficient and safe drug delivery system is required to deliver them to the lesion site. The drug delivery system needs to remain for a sufficient time to accurately target the lesion site while avoiding damage to normal cells.

The current delivery systems can be divided into viral vectors and non-viral vectors. Viral vectors are less used in nucleic acid drugs due to their immunogenicity, tumorigenicity, and limited drug loading; non-viral vectors, such as polymers and lipids (liposomes or LNPs), can bind nucleic acid drugs to specific ligands to enable them to target specific cells, and are widely used in current nucleic acid drugs. LNP is one of the most widely used delivery systems for nucleic acid drugs. The LNP delivery system can safely and effectively deliver nucleic acids. It has the advantages of high nucleic acid encapsulation rate, effective cell transfection, strong tissue penetration, low cytotoxicity and immunogenicity, etc., which are conducive to drug delivery. Compared with other delivery systems, it has great advantages. Therefore, the LNP delivery system has broad development and application prospects.

In the prior art, LNP delivery systems are often composed of ionizable lipids (cationic lipids), steroids, neutral lipids, PEG-lipids, nucleic acid drugs and other components. For example: Patent document AU2020325221A1 discloses a composition for delivering LNPs to target cells, including (i) ionizable lipids; (ii) sterols or other structural lipids; (iii) non-cationic auxiliary lipids or phospholipids; (iv) PEG lipids and (v) agents encapsulated in and/or associated with LNPs (such as nucleic acid molecules). These four components enhance the delivery efficiency of target cells in a specific ratio. Patent document WO 2021/055849A1 discloses a lipid with the following structure: This structure can improve its safety, effectiveness and specificity. Patent WO2010021865A1 publicly reports a class of ionizable cholesterol-modified amino lipid compounds This type of compound can be combined with DOPE, DOPC, DOPS and DMG-PEG to form lipid nanoparticles to deliver siRNA. Studies have shown that this asymmetric cholesterol amino lipid compound can improve the order of lipid nanoparticles and can effectively promote the transformation of lipid nanoparticles from multilayer structures to hexagonal phase structures in vivo, thereby improving the endosomal escape ability of liposomes. Patent CN112424214A also discloses cationic lipid compounds formed by cholesterol and linear olefins (3) The compound is combined with cholesterol, DPPC, DOPE and DMG-PEG200 to construct LNP to deliver nucleic acids. The above compounds all need to be combined with 3 or even more than 4 different lipid excipients to form a nucleic acid drug delivery carrier preparation, and the construction process is relatively complicated. The prior art has an urgent need to optimize the structure of each component in LNP, especially the structure of ionizable cationic lipids, in order to further obtain a safe, effective, stable, simple to construct LNP delivery system that can be simultaneously applied to different routes of administration.

Summary of the invention

In one aspect, the present invention provides a lipid compound represented by formula (I),

or its stereoisomers, tautomers, and pharmaceutically acceptable salts; wherein,

L ₁ , L ₂ and L ₃ are each independently selected at each occurrence from a bond, an optionally substituted C ₁ -C ₂₀ alkylene group, an optionally substituted C ₂ -C ₂₀ alkenylene group, an optionally substituted C ₂ -C ₂₀ alkynylene group, an optionally substituted C ₁ -C ₂₀ acyl group;

_G1 , _G2 , and _G3 are each independently selected at each occurrence from a bond, -O-, -S-, -C(=O)-, -C(=O)O-, -OC(=O)-, -OC(=O)O-, -C(=O)N(R _a )-, -NR _a C(=O)-, -S(=O)O-, -OS(=O) _O- , -S(=O)2O-, -OS(=O) _2O- , -C(=O)S-, -C(=S)S-, -OP(=O)(OR _a )O-, -N(R _a )C(=O)O-, -OC(=O)N(R _a )-, -SS-, -OC(=O)S-, -SC(=O)O-, -N(R _a )C(=O)N(R _b )-;

One of R ₁ , R ₂ and R ₃ is selected from optionally substituted C ₁ -C ₂₀ alkyl, optionally substituted C ₂ -C ₂₀ alkenyl, optionally substituted C ₂ -C ₂₀ alkynyl; wherein one or more -CH ₂ - in the C ₁ -C ₂₀ alkyl, C ₂ -C ₂₀ alkenyl, C ₂ -C ₂₀ alkynyl may be optionally replaced by -O-, -S-, -NR _a -, carbocyclyl, aryl, heteroaryl, and/or heterocyclyl;

Meanwhile, the other of R ₁ , R ₂ and R ₃ is selected from a steroidal compound group;

At the same time, the third of R ₁ , R ₂ and R ₃ is selected from hydrogen, C ₁ -C ₂₀ alkyl, -(R ₄ ) _q -NR _a R _b , -(R ₄ ) _q -nitrogen-containing heteroaryl, -(R ₄ ) _q -nitrogen-containing heterocyclic group, -(R ₄ ) _q -guanidinyl; wherein the nitrogen-containing heteroaryl, nitrogen-containing heterocyclic group and guanidinyl are optionally substituted by one or more groups selected from the following: C ₁ -C ₂₀ alkyl, C ₂ -C ₂₀ alkenyl, C ₂ -C ₂₀ alkynyl, C ₁ -C ₂₀ alkoxy, halogen, hydroxyl, thiol, cyano, nitro, amino, carboxyl, C ₁ -C ₂₀ acyl, C ₁ -C ₂₀ acyloxy;

R ₄ is selected from C ₁ -C ₂₀ alkylene, C ₂ -C ₂₀ alkenylene, C ₂ -C ₂₀ alkynylene;

_Ra and _Rb are each independently selected from H, optionally substituted _C1 - _C20 alkyl, optionally substituted _C2 - _C20 alkenyl, optionally substituted _C2 - _C20 alkynyl, optionally substituted carbocyclyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted heterocyclyl;

m, n and p are each independently selected from 1, 2 or 3;

q is selected from 0 or 1.

On the other hand, the present invention also provides a lipid nanoparticle, comprising a lipid compound represented by formula (I) or a stereoisomer, a tautomer, or a pharmaceutically acceptable salt thereof.

In another aspect, the present invention also provides a pharmaceutical composition comprising the lipid nanoparticles described herein and a pharmaceutically acceptable carrier.

In another aspect, the present invention also provides a method of delivering a therapeutic agent and/or a prophylactic agent, comprising administering the pharmaceutical composition described herein to an individual in need thereof.

On the other hand, the present invention also provides use of the lipid compound represented by formula (I) described herein or its stereoisomers, tautomers, and pharmaceutically acceptable salts in the preparation of a therapeutic and/or preventive agent delivery system.

Beneficial effects:

The three-component lipid nanoparticles prepared by using the lipid compound of the present invention or its stereoisomers, tautomers, and pharmaceutically acceptable salts have simple processes and good stability and transfection efficiency. The lipid nanoparticles are used to deliver nucleic acids (such as mRNA), which can be efficiently and stably delivered to target cells or organs, and induce high specific antibody responses and cellular immune responses in experimental animals, and have good safety.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 shows the detection of eGFP-mRNA expression in HEKT cells by fluorescence microscopy.

Figure 2 shows the ELISA serum antibody titer after mice were immunized with SARS-CoV-2 antigen mRNA-LNP.

Figure 3 shows the level of antigen-specific IFN-γ cell activation in PBMC detected by ELSIPOT after mice were immunized with SARS-CoV-2 antigen mRNA-LNP.

FIG. 4 shows the proliferation toxicity effect of mRNA-LNP on HEK293T cells.

DETAILED DESCRIPTION

definition

As used in this specification, the following words and phrases are generally intended to have the meanings set forth below, unless the context in which they are used indicates otherwise.

As used herein, the term "lipid nanoparticle," or "LNP," refers to a particle having a nanometer scale, for example, 1 nm to 1,000 nm, which comprises one or more types of lipid molecules.

As used herein, the term "gene medicine" generally consists of a vector or delivery system containing an engineered gene construct, the active ingredient of which may be DNA, RNA, genetically modified viruses, bacteria or cells. It is used to replace, compensate, block or correct specific genes by introducing exogenous genes into target cells or tissues to achieve the purpose of treating and preventing diseases.

As used herein, the term "nucleic acid" refers to a polymer containing at least two deoxyribonucleotides or ribonucleotides in a single-stranded or double-stranded form, and includes DNA, RNA, and hybrids thereof.

As used herein, the term "lipid compound" refers to a group of organic compounds, which include but are not limited to esters of fatty acids, and are generally characterized by being poorly soluble in water but soluble in many organic solvents. The organic solvents of the present invention include but are not limited to: benzene, toluene, pentane, hexane, methanol, ethanol, isopropanol, ether, ethyl acetate, acetone, carbon tetrachloride.

As used herein, the term "alkyl" refers to a monovalent group of a straight or branched saturated hydrocarbon chain having 1 to 20 carbon atoms, more typically 1 to 10 carbon atoms, 1 to 8 carbon atoms, or 1 to 6 carbon atoms. This term is exemplified by groups such as methyl, ethyl, 1-propyl (n-propyl), 2-propyl (isopropyl), 1-butyl (n-butyl), 2-methyl-1-propyl (isobutyl), 2-butyl (sec-butyl), 2-methyl-2-propyl (tert-butyl), 1-pentyl (n-pentyl), 2-pentyl, 3-pentyl, 2-methyl-2-butyl, 3-methyl-2-butyl, 3-methyl-1-butyl, 2-methyl-1-butyl, 1-hexyl, 2-hexyl, 3-hexyl, 2-methyl-2-pentyl, 3-methyl-2-pentyl, 4-methyl-2-pentyl, 3-methyl-3-pentyl, 2-methyl-3-pentyl, 2,3-dimethyl-2-butyl, 3,3-dimethyl-2-butyl, 1-heptyl, 1-octyl, 1-nonyl, 1-decyl, and the like.

As used herein, the term "alkylene" refers to a divalent group of a straight or branched saturated hydrocarbon chain having 1 to 20 carbon atoms (more typically 1 to 10 carbon atoms, 1 to 8 carbon atoms, or 1 to 6 carbon atoms). The term is exemplified by groups such as methylene, ethylene, propylene, butylene, pentylene, hexylene, and the like.

As used herein, the term "alkenyl" refers to a linear or branched unsaturated hydrocarbon chain monovalent group having 2 to 20 carbon atoms (more typically 2 to 10 carbon atoms, 2 to 8 carbon atoms, or 2 to 6 carbon atoms) and having carbon-carbon double bonds (e.g., 1, 2 or 3 carbon-carbon double bonds). The unsaturated carbon-carbon double bonds may be present at any stable point along the chain. The term is exemplified by groups such as vinyl (i.e., -CH=CH ₂ ), propen-1-yl (i.e., -CH=CHCH ₃ ), propen-3-yl (or allyl, i.e., -CH ₂ CH=CH ₂ ), propen-2-yl (i.e., -C(CH ₃ )=CH ₂ ), butadienyl (including 1,2-butadienyl and 1,3-butadienyl), and the like.

As used herein, the term "alkenylene" refers to a divalent group of a straight or branched unsaturated hydrocarbon chain having 2 to 20 carbon atoms (more typically 1 to 10 carbon atoms, 1 to 8 carbon atoms, or 1 to 6 carbon atoms) and having carbon-carbon double bonds (e.g., 1, 2 or 3 carbon-carbon double bonds). The unsaturated carbon-carbon double bonds may be present at any stable point along the chain. The term is exemplified by groups such as vinylene, propenylene, butenylene, pentenylene, hexenylene, etc.

As used herein, the term "alkynyl" refers to a linear or branched unsaturated hydrocarbon chain monovalent group having 2 to 20 carbon atoms (more typically 2 to 10 carbon atoms, 2 to 8 carbon atoms, or 2 to 6 carbon atoms) and having carbon-carbon triple bonds (e.g., 1, 2 or 3 carbon-carbon triple bonds). The term is exemplified by groups such as ethynyl (i.e., -C≡CH), propargyl (i.e., -CH ₂ C≡CH), propynyl (i.e., -C≡CCH ₃ ), and the like.

As used herein, the term "alkynylene" refers to a divalent group of a straight or branched unsaturated hydrocarbon chain having 2 to 20 carbon atoms (more typically 1 to 10 carbon atoms, 1 to 8 carbon atoms, or 1 to 6 carbon atoms) and having carbon-carbon triple bonds (e.g., 1, 2 or 3 carbon-carbon triple bonds). The unsaturated carbon-carbon triple bonds may be present at any stable point along the chain. The term is exemplified by groups such as ethynylene, propynylene, butynylene, pentynylene, hexynylene, and the like.

As used herein, the term "halogen" refers to fluorine, chlorine, bromine and iodine.

As used herein, the term "alkoxy" refers to an "alkyl-O-" group, wherein alkyl is as defined herein. This term is exemplified by groups such as methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, tert-butoxy, and the like.

As used herein, the term "acyl" refers to "alkyl-C(=O)-", "alkenyl-C(=O)-", "alkynyl-C(=O)-", "aryl-C(=O)-", "heteroaryl-C(=O)-", "carbocyclyl-C(=O)-", "heterocyclyl-C(=O)-" groups, wherein alkyl, alkenyl, alkynyl, aryl, heteroaryl, carbocyclyl, heterocyclyl are as defined herein. The term is exemplified by groups such as formyl, acetyl, propionyl, n-butyryl, isobutyryl, n-valeryl, n-hexanoyl, acryloyloxy, benzoyl, cyclopropylacyl, and the like.

As used herein, the term "acyloxy" refers to "alkyl-C(=O)O-", "alkenyl-C(=O)O-", "alkynyl-C(=O)O-", "aryl-C(=O)O-", "heteroaryl-C(=O)O-", "carbocyclyl-C(=O)O-", "heterocyclyl-C(=O)-" groups, wherein alkyl, alkenyl, alkynyl, aryl, heteroaryl, carbocyclyl, heterocyclyl are as defined herein. The term is exemplified by groups such as formyloxy, acetyloxy, propionyloxy, n-butyryloxy, isobutyryloxy, n-valeryloxy, n-hexanoyloxy, and the like.

As used herein, the term "aryl" refers to an aromatic carbocyclic group of 6 to 14 carbon atoms (more typically 6 to 10 carbon atoms, or 6 carbon atoms) having a single ring (e.g., phenyl) or multiple rings (e.g., biphenyl) or multiple condensed (fused) rings (e.g., naphthyl, fluorenyl, and anthracenyl). The term is exemplified by groups such as phenyl, fluorenyl, naphthyl, anthracenyl, 1,2,3,4-tetrahydronaphthalene (if the point of attachment is through the aryl group), and the like.

As used herein, the term "carbocyclic radical" refers to a monocyclic or monoradical saturated or partially unsaturated group having 3 to 14 carbon atoms (more typically 3 to 8 carbon atoms, or 3 to 6 carbon atoms) as ring atoms or a plurality of thick (fused) rings or bridged rings or spirocyclic rings.Carbocyclic ring or carbocyclic radical can be saturated or partially unsaturated, and can be fused with another saturated, partially unsaturated or aromatic ring, provided that the ring atoms connected with the target molecule are not aromatic carbon.The example of carbocyclic ring or carbocyclic radical includes, but is not limited to cyclopropane, cyclobutane, cyclopentane, cyclohexane, cyclobutene, cyclopentene, cyclohexene, cycloheptene, cyclopentadiene etc.

As used herein, the term "heteroaryl" refers to an aromatic ring group containing 5 to 14 ring atoms (more typically 5 to 10 ring atoms, or 5 to 6 ring atoms) in the ring or multiple condensed (fused) rings (e.g., containing 2 or 3 rings), wherein in addition to carbon atoms, the ring atoms also contain at least one heteroatom selected from oxygen, nitrogen and/or sulfur. If the ring is aromatic, sulfur and nitrogen atoms can also exist in oxidized form. Multiple condensed (fused) ring heteroaryl is a monocyclic heteroaryl as defined above fused with one or more rings selected from the following to form a multiple condensed ring system: heteroaryl (to form, for example, naphthyridinyl, such as 1,8-naphthyridinyl), heterocyclic (for example, 1,2,3,4-tetrahydronaphthyridinyl, such as 1,2,3,4-tetrahydro-1,8-naphthyridinyl), carbocyclic (to form, for example, 5,6,7,8-tetrahydroquinolinyl) and aryl (to form, for example, indazolyl). It will be appreciated that the point of attachment of the heteroaryl group may be at any suitable atom of the heteroaryl group, including carbon atoms and heteroatoms (eg, nitrogen). Exemplary heteroaryl groups include, but are not limited to, pyridinyl, pyrrolyl, pyrazinyl, pyrimidinyl, pyridazinyl, pyrazolyl, thienyl, indolyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl, furanyl, oxadiazolyl, thiadiazolyl, quinolinyl, isoquinolinyl, benzothiazolyl, benzoxazolyl, indazolyl, quinoxalinyl, quinazolinyl, 5,6,7,8-tetrahydroisoquinolinyl, benzofuranyl, benzimidazolyl, thiaindenyl, pyrrolo[2,3-b]pyridinyl, quinazolinyl-4(3H)-one, triazolyl, 4,5,6,7-tetrahydro-1H-indazolyl, and 3b,4,4a,5-tetrahydro-1H-cyclopropane[3,4]cyclopenta[1,2-c]pyrazolyl.

As used herein, the term "heterocyclic radical" refers to a monocyclic or multiple condensed (condensed) rings or bridged rings or spirocyclic monovalent or divalent saturated or partially unsaturated group having 3 to 14 ring atoms (more typically 3 to 10 ring atoms, or 3 to 6 ring atoms) in the ring, wherein in addition to carbon atoms, the ring atoms also include at least one or more nitrogen atoms. Examples of heterocyclic radical groups include, but are not limited to, aziridine rings, azetidine rings, tetrahydropyrrole rings, piperidine rings, azepane rings, azocyclooctane rings, tetrahydroimidazole rings, tetrahydropyrazole rings, tetrahydrooxazole rings, tetrahydroisoxazole rings, tetrahydrothiazole rings, tetrahydroisothiazole rings, piperazine rings, morpholine rings, dihydropyridyl, 4,5,6,7-tetrahydro-1H-benzo [d] imidazole, 4,5,6,7-tetrahydro-1H-imidazo [4,5-c] pyridine, etc. The nitrogen heterocyclic group in the present invention is a heterocyclic group containing a nitrogen atom in the structure, including but not limited to substituted or unsubstituted: aziridinyl, azetidine, β-propiolactam, pyrrolyl, piperidinyl, pyrazolyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl, pyridinyl, caprolactam, pyranyl, pyridazinyl, pyrimidinyl, pyrazinyl, piperazinyl, piperazinyl, indolyl, benzimidazolyl, carbazolyl, quinolyl, isoquinolyl, pteridinyl, acridinyl, 7H-purinyl, phenazinyl, phenothiazinyl or 1H-azepinyl.

As used herein, the term "optionally substituted" means unsubstituted or substituted with one or more groups selected from C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, halogen, hydroxy, cyano, nitro, amino, C ₃ -C ₆ cycloalkyl, oxo.

As used herein, the term "steroid" is an organic compound having a four-ring carbon skeleton structure as shown below.

Steroids include naturally occurring or synthetic steroids and their analogs. Steroids or their analogs include sterols or their analogs derived from plants and/or animals. Examples of steroids described herein include, but are not limited to, avenasterol, β-sitosterol, brassicasterol, ergocalciferol, campesterol, cholestanol, coprostanol, dehydrocholesterol, streptosterol, dihydroergocalciferol, cholesterol, dihydrocholesterol, dihydroergosterol, black seasterol, epicholesterol, ergosterol, fucosterol, hexahydroluminosterol, hydroxycholesterol, luminosterol, alginosterol, sitostanol, stigmasterol, stigmasterol, cholic acid, glycocholic acid, taurocholic acid, deoxycholic acid, lithocholic acid, ent-cholesterol, epicholesterol, demosterol, cholestanol, cholestanone, choletenone, 3p-[N-(N'N'-dimethylaminoethyl)carbamoylcholesterol (DC-Ch ol), 24(S)-hydroxycholesterol, 25-hydroxycholesterol, 25(R)-27-hydroxycholesterol, 22-oxa-cholesterol, 23-oxa-cholesterol, 24-oxa-cholesterol, cyclohexyl alcohol, 22-ketosterol, 20-hydroxysterol, 7-hydroxycholesterol, 19-hydroxycholesterol, 22-hydroxycholesterol, 25-hydroxycholesterol, 7-dehydrocholesterol, dehydroergosterol, dehydroepiandrosterone, lanosterol, dihydrolanosterol, lumiesterol, cetocalciferol, calcipotriol, coprostol, cholecalciferol, lupeol, ergocalciferol, 22-dihydroautocalciferol, tomatine, ursolic acid, chenodeoxycholic acid, yeast sterol, diosgenin, etc.

As used herein, the term "therapeutically effective amount" refers to an amount sufficient to affect treatment as defined below when administered to a mammal in need of such treatment. The therapeutically effective amount will vary with the subject and disease condition being treated, the weight and age of the subject, the severity of the disease condition, the mode of administration, etc., and can be readily determined by one of ordinary skill in the art.

As used herein, the term "stereoisomer" refers to a compound having the same chemical composition and connectivity, but whose atoms have different orientations in space that cannot be interchanged by single bond rotation. "Stereoisomer" includes "diastereomers" and "enantiomers". "Diastereomers" refers to stereoisomers with two or more chiral centers and whose molecules are not mirror images of each other. Diastereomers have different physical properties, such as melting points, boiling points, spectral characteristics and reactivity. Diastereomeric mixtures can be separated under high-resolution analytical procedures (such as crystallization, electrophoresis and chromatography). "Enantiomers" refers to two stereoisomers that are non-overlapping mirror images of each other.

As used herein, the term "tautomer" refers to the coexistence of two (or more) compounds that differ only in the position and electron distribution of one (or more) mobile atoms, such as keto-enol tautomers.

As used herein, the term "pharmaceutically acceptable salt" refers to salts that retain the biological effectiveness and properties of a given compound and are not biologically or otherwise undesirable. Pharmaceutically acceptable salts can be acid addition salts and/or base addition salts. Acid addition salts can be prepared from inorganic acids and organic acids. Salts derived from inorganic acids include hydrochlorides, hydrobromides, sulfates, nitrates, phosphates, carbonates, bisulfates, hydrogenphosphates, dihydrogenphosphates, bicarbonates, etc.; salts derived from organic acids include formates, acetates, propionates, glycolates, pyruvates, oxalates, malates, malonates, succinates, maleates, fumarates, tartrates, citrates, benzoates, cinnamates, mandelates, methanesulfonates, ethanesulfonates, p-toluenesulfonates, salicylates, lactates, nicotinates, lauryl sulfates, naphthylsulfonates, camphorsulfonates, gluconates, glucuronates, oleates, palmitates, stearates, pamoates, trifluoroacetates, etc. Base addition salts can be formed with inorganic or organic bases. Salts derived from inorganic bases include sodium, potassium, ammonium, calcium, magnesium, iron, zinc, copper, lithium, barium, aluminum salts and the like; salts derived from organic bases include salts formed with various primary, secondary and tertiary amines, for example, ethylamine, diethylamine, n-propylamine, isopropylamine, diethanolamine, meglumine, lysine, piperazine, piperidine, morpholine, tromethamine, choline and the like.

As used herein, the term "pharmaceutically acceptable" means that the substance or composition must be chemically and/or toxicologically compatible with the other ingredients comprising the formulation and/or the mammal to be treated therewith.

As used herein, the term "delivery system" refers to a formulation or composition that regulates the spatial, temporal and dosage distribution of a biologically active ingredient in an organism.

Compound

In some embodiments, the present invention provides a lipid compound represented by formula (I)

or stereoisomers, tautomers, and pharmaceutically acceptable salts thereof; wherein L ₁ , _{L 2} , L ₃ , G 1 , G ₂ , G ₃ , R ₁ , R ₂ , R ₃ , m, n, and p are as defined herein.

In some embodiments, in the lipid compound represented by formula (I) provided by the present invention or its stereoisomers, tautomers, and pharmaceutically acceptable salts,

L ₁ is independently selected at each occurrence from a bond, an optionally substituted C ₁ -C ₂₀ alkylene group, an optionally substituted C ₂ -C ₂₀ alkenylene group, an optionally substituted C ₂ -C ₂₀ alkynylene group, an optionally substituted C ₁ -C ₂₀ acyl group;

G1 is independently selected at _each occurrence from -C(＝O)O-, -OC(＝O)-, -OC(＝O)O-, -C(＝O)N(R _a )-, -NR _a C(＝O)-, -S(＝O)O-, -OS(＝O)O-, -S(＝O) ₂ O-, -OS(＝O) ₂ O-, -C(＝O)S-, -C(＝S)S-, -OP(＝O)(OR _a )O-, -N(R _a )C(＝O)O-, -OC(＝O)N(R _a )-, -SS-, -OC(＝O)S-, -SC(＝O)O-, -N(R _a )C(＝O)N(R _b )-;

R ₁ is selected from optionally substituted C ₁ -C ₂₀ alkyl, optionally substituted C ₂ -C ₂₀ alkenyl, optionally substituted C ₂ -C ₂₀ alkynyl; wherein one or more -CH ₂ - in the C ₁ -C ₂₀ alkyl, C ₂ -C ₂₀ alkenyl, C ₂ -C ₂₀ alkynyl may be optionally replaced by O, S, -NR _a -, carbocyclyl, aryl, heteroaryl, and/or heterocyclyl;

_L2 and _L3 are each independently selected at each occurrence from a bond, an optionally substituted _C1 - _C20 alkylene group, an optionally substituted _C2 - _C20 alkenylene group, an optionally substituted _C2 - _C20 alkynylene group, an optionally substituted _C1 - _C20 acyl group;

_G2 and _G3 are each independently selected at each occurrence from a bond, -O-, -S-, -C(=O)-, -C(=O)O-, -OC(=O)-, -OC(=O)O-, -C(=O) _NRa- , -NRaC(=O)-, _-S (=O)O-, -OS(=O)O-, -S(=O) _2O- , -OS(=O) _2O- , -C(=O)S-, -C(=S)S-, -OP(= _O )(ORa)O-, -N( _Ra )C(=O)O-, -OC(=O) _NRa- , -SS-, -OC(=O)S-, -SC(=O)O-, _-NRaC (=O) _NRb- ;

One of _R2 and _R3 is selected from a steroidal compound group, and the other is selected from hydrogen, _C1 - _C20 alkyl, -( _R4 ) _q - _NRaRb , _- ( _R4 ) _q -nitrogen-containing heteroaryl, -( _R4 ) _q -nitrogen-containing heterocyclic group, -( _R4 ) _q -guanidinyl; wherein the nitrogen-containing heteroaryl, nitrogen-containing heterocyclic group and guanidinyl are optionally substituted by one or more groups selected from the following: _C1 - _C20 alkyl, _C2 - _C20 alkenyl, _C2 - _C20 alkynyl, _C1 - _C20 alkoxy, halogen, hydroxyl, thiol, cyano, nitro, amino, carboxyl, _C1 - _C20 acyl, _C1 - _C20 acyloxy;

R ₄ is selected from C ₁ -C ₂₀ alkylene, C ₂ -C ₂₀ alkenylene, C ₂ -C ₂₀ alkynylene;

_Ra and _Rb are each independently selected from H, optionally substituted _C1 - _C20 alkyl, optionally substituted _C2 - _C20 alkenyl, optionally substituted _C2 - _C20 alkynyl, optionally substituted carbocyclyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted heterocyclyl;

m, n and p are each independently selected from 1, 2 or 3;

q is selected from 0 or 1.

In some embodiments, in the lipid compound represented by formula (I) provided by the present invention or its stereoisomers, tautomers, and pharmaceutically acceptable salts,

L ₁ is independently selected at each occurrence from a bond, an optionally substituted C ₁ -C ₂₀ alkylene group, an optionally substituted C ₂ -C ₂₀ alkenylene group, an optionally substituted C ₂ -C ₂₀ alkynylene group, an optionally substituted C ₁ -C ₂₀ acyl group;

G1 is independently selected at _each occurrence from -C(＝O)O-, -OC(＝O)-, -OC(＝O)O-, -C(＝O)N(R _a )-, -NR _a C(＝O)-, -S(＝O)O-, -OS(＝O)O-, -S(＝O) ₂ O-, -OS(＝O) ₂ O-, -C(＝O)S-, -C(＝S)S-, -OP(＝O)(OR _a )O-, -N(R _a )C(＝O)O-, -OC(＝O)N(R _a )-, -SS-, -OC(＝O)S-, -SC(＝O)O-, -N(R _a )C(＝O)N(R _b )-;

R ₁ is selected from optionally substituted C ₁ -C ₂₀ alkyl, optionally substituted C ₂ -C ₂₀ alkenyl, optionally substituted C ₂ -C ₂₀ alkynyl; wherein one or more -CH ₂ - in the C ₁ -C ₂₀ alkyl, C ₂ -C ₂₀ alkenyl, C ₂ -C ₂₀ alkynyl may be optionally replaced by O, S, -NR _a -, carbocyclyl, aryl, heteroaryl, and/or heterocyclyl;

_L2 is independently selected at each occurrence from a bond, an optionally substituted C ₁ -C ₂₀ alkylene group, an optionally substituted C ₂ -C ₂₀ alkenylene group, an optionally substituted C ₂ -C ₂₀ alkynylene group, an optionally substituted C ₁ -C ₂₀ acyl group;

G2 is independently selected at _each occurrence from -O-, -S-, -C(＝O)-, -C(＝O)O-, -OC(＝O)-, -OC(＝O)O-, -C(＝O) _NRa- , _-NRaC (＝O)-, -S(＝O)O-, -OS(＝O)O-, -S(＝O) _2O- , -OS(＝O) _2O- , -C(＝O)S-, -C(＝S)S-, -OP(＝ _O )(ORa)O-, -N( _Ra )C(＝O) _O- , -OC(＝O)NRa-, -SS-, -OC(＝O)S-, -SC(＝O)O-, _-NRaC (＝O) _NRb- ;

_R2 is selected from a steroid group;

_L3 is independently selected at each occurrence from a bond, an optionally substituted _C1 - _C20 alkylene group, an optionally substituted _C2 - _C20 alkenylene group, an optionally substituted _C2 - _C20 alkynylene group, an optionally substituted _C1 - _C20 acyl group;

_G3 is independently selected at each occurrence from a bond, -O-, -S-, -C(=O)-, -C(=O)O-, -OC(=O)-, -OC(=O)O-, -C(=O) _NRa- , _-NRaC (=O)-, -S(=O)O-, -OS(=O)O-, -S(=O) _2O- , -OS(=O) _2O- , -C(=O)S-, -C(=S)S-, -OP(= _O )(ORa)O-, -N( _Ra )C(=O)O-, -OC(=O) _NRa- , -SS-, -OC(=O)S-, -SC(=O)O-, _-NRaC (=O) _NRb- ;

R ₃ is selected from hydrogen, C ₁ -C ₂₀ alkyl, -(R ₄ ) _q -NR _a R _b , -(R ₄ ) _q -nitrogen-containing heteroaryl, -(R ₄ ) _q -nitrogen-containing heterocyclic group, -(R ₄ ) _q -guanidinyl; wherein the nitrogen-containing heteroaryl, nitrogen-containing heterocyclic group and guanidinyl are optionally substituted by one or more groups selected from the following groups: C ₁ -C ₂₀ alkyl, C ₂ -C ₂₀ alkenyl, C ₂ -C ₂₀ alkynyl, C ₁ -C ₂₀ alkoxy, halogen, hydroxyl, thiol, cyano, nitro, amino, carboxyl, C ₁ -C ₂₀ acyl, C ₁ -C ₂₀ acyloxy.

In some embodiments, in the lipid compound represented by formula (I) provided by the present invention or its stereoisomers, tautomers, and pharmaceutically acceptable salts,

L ₁ is independently selected at each occurrence from a bond, an optionally substituted C ₁ -C ₂₀ alkylene group, an optionally substituted C ₂ -C ₂₀ alkenylene group, an optionally substituted C ₂ -C ₂₀ alkynylene group, an optionally substituted C ₁ -C ₂₀ acyl group;

G1 is independently selected at _each occurrence from -C(＝O)O-, -OC(＝O)-, -OC(＝O)O-, -C(＝O)N(R _a )-, -NR _a C(＝O)-, -S(＝O)O-, -OS(＝O)O-, -S(＝O) ₂ O-, -OS(＝O) ₂ O-, -C(＝O)S-, -C(＝S)S-, -OP(＝O)(OR _a )O-, -N(R _a )C(＝O)O-, -OC(＝O)N(R _a )-, -SS-, -OC(＝O)S-, -SC(＝O)O-, -N(R _a )C(＝O)N(R _b )-;

R ₁ is selected from optionally substituted C ₁ -C ₂₀ alkyl, optionally substituted C ₂ -C ₂₀ alkenyl, optionally substituted C ₂ -C ₂₀ alkynyl; wherein one or more -CH ₂ - in the C ₁ -C ₂₀ alkyl, C ₂ -C ₂₀ alkenyl, C ₂ -C ₂₀ alkynyl may be optionally replaced by O, S, -NR _a -, carbocyclyl, aryl, heteroaryl, and/or heterocyclyl;

_L2 is independently selected at each occurrence from a bond, an optionally substituted C ₁ -C ₂₀ alkylene group, an optionally substituted C ₂ -C ₂₀ alkenylene group, an optionally substituted C ₂ -C ₂₀ alkynylene group, an optionally substituted C ₁ -C ₂₀ acyl group;

_G2 is independently selected at each occurrence from a bond, -O-, -S-, -C(=O)-, -C(=O)O-, -OC(=O)-, -OC(=O)O-, -C(=O) _NRa- , _-NRaC (=O)-, -S(=O)O-, -OS(=O)O-, -S(=O) _2O- , -OS(=O) _2O- , -C(=O)S-, -C(=S)S-, -OP(= _O )(ORa)O-, -N( _Ra )C(=O)O-, -OC(=O) _NRa- , -SS-, -OC(=O)S-, -SC(=O)O-, _-NRaC (=O) _NRb- ;

_R2 is selected from hydrogen, _C1 - _C20 alkyl, -( _R4 ) _q - _NRaRb , -( _R4 ) _q - _nitrogen -containing heteroaryl, -( _R4 ) _q -nitrogen-containing heterocyclic group, -( _R4 ) _q -guanidinyl; wherein the nitrogen-containing heteroaryl, nitrogen-containing heterocyclic group and guanidinyl are optionally substituted by one or more groups selected from the following: _C1 - _C20 alkyl, _C2 - _C20 alkenyl, _C2 - _C20 alkynyl, _C1 - _C20 alkoxy, halogen, hydroxyl, thiol, cyano, nitro, amino, carboxyl, _C1 - _C20 acyl, _C1 - _C20 acyloxy;

_L3 is independently selected at each occurrence from a bond, an optionally substituted _C1 - _C20 alkylene group, an optionally substituted _C2 - _C20 alkenylene group, an optionally substituted _C2 - _C20 alkynylene group, an optionally substituted _C1 - _C20 acyl group;

G3 is independently selected at _each occurrence from -O-, -S-, -C(＝O)-, -C(＝O)O-, -OC(＝O)-, -OC(＝O)O-, -C(＝O) _NRa- , _-NRaC (＝O)-, -S(＝O)O-, -OS(＝O)O-, -S(＝O) _2O- , -OS(＝O) _2O- , -C(＝O)S-, -C(＝S)S-, -OP(＝ _O )(ORa)O-, -N( _Ra )C(＝O) _O- , -OC(＝O)NRa-, -SS-, -OC(＝O)S-, -SC(＝O)O-, _-NRaC (＝O) _NRb- ;

_R3 is selected from the group consisting of steroidal compounds;

R ₄ is selected from C ₁ -C ₂₀ alkylene, C ₂ -C ₂₀ alkenylene, C ₂ -C ₂₀ alkynylene;

_Ra and _Rb are each independently selected from H, optionally substituted _C1 - _C20 alkyl, optionally substituted _C2 - _C20 alkenyl, optionally substituted _C2 - _C20 alkynyl, optionally substituted carbocyclyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted heterocyclyl;

m, n and p are each independently selected from 1, 2 or 3;

q is selected from 0 or 1.

In some embodiments, the present invention provides a lipid compound represented by formula (I) or a stereoisomer, a tautomer, and a pharmaceutically acceptable salt thereof;

Wherein, the steroidal compounds in the steroidal compound group are selected from naturally occurring steroidal compounds or their analogs; preferably, they include plant sterols and animal sterols, or their analogs; more preferably, they are selected from avenasterol, β-sitosterol, brassicasterol, ergocalciferol, campesterol, cholestanol, coprostanol, dehydrocholesterol, streptosterol, dihydroergocalciferol, cholesterol, dihydrocholesterol, dihydroergosterol, black sea sterol, epicholesterol, ergosterol, fucosterol, hexahydroluminosterol, hydroxycholesterol, luminosterol, alginosterol, sitostanol, stigmasterol, stigmasterol, bile acid, glycocholic acid, taurocholic acid, deoxycholic acid, lithocholic acid, ent-cholesterol, epicholesterol, demosterol, cholestanol, cholestanone, cholenone, 3p-[N-(N'N'-dimethylaminoethyl)carbamoylcholesterol (DC-Chol), 24(S)-hydroxycholesterol, 25-hydroxycholesterol, 25(R)-27-hydroxycholesterol, 22-oxa-cholesterol, 23-oxa-cholesterol, 24-oxa-cholesterol, cyclohexyl alcohol, 22-ketosterol, 20-hydroxysterol, 7-hydroxycholesterol, 19-hydroxycholesterol, 22-hydroxycholesterol, 25-hydroxycholesterol, 7-dehydrocholesterol, dehydroergosterol, dehydroepiandrosterone, lanosterol, dihydrolanosterol, lumiesterol, cetocalciferol, calcipotriol, coprostol, cholecalciferol, lupeol, ergocalciferol, 22-dihydroautocalciferol, tomatine, ursolic acid, chenodeoxycholic acid, yeast sterol, diosgenin, etc.

In some embodiments, the present invention provides a lipid compound represented by formula (I) or a stereoisomer, a tautomer, and a pharmaceutically acceptable salt thereof;

Wherein, the steroid compound in the steroid compound group is selected from cholesterol and cholesterol derivatives.

In some embodiments, the present invention provides a lipid compound represented by formula (I) or a stereoisomer, a tautomer, and a pharmaceutically acceptable salt thereof;

Wherein, the steroidal compound group has the following structure:

_R5 is selected from hydrogen, _C1 - _C20 alkyl, _C2 - _C20 alkenyl, _C2 - _C20 alkynyl, _C1 - _C20 alkoxycarbonyl- _C1 - _C20 alkyl-;

_R6 is selected from hydrogen, halogen, cyano, hydroxy, amino, oxo, _C1 - _C20 alkyl, _C2 - _C20 alkenyl, _C2 - _C20 alkynyl;

m is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

In some embodiments, the steroid group is selected from:

wherein R' is a C _{1 -} C ₂₀ alkyl group.

In some embodiments, the lipid compound provided by the present invention is a lipid compound represented by formula (II-a) or formula (II-b) or formula (II-c):

or its stereoisomers, tautomers, and pharmaceutically acceptable salts, wherein

_L1 , _L2 , _L3 , _G1 , _G2 , _G3 , _R1 , _R2 , _R3 , m, n and p are as defined herein.

In some embodiments, the lipid compound provided by the present invention is a lipid compound represented by formula (III-a) or formula (III-b) or formula (III-c):

or its stereoisomers, tautomers, and pharmaceutically acceptable salts, wherein

X is selected from O, S, NH; L ₁ , L ₂ , L ₃ , G ₁ , G ₂ , G ₃ , R ₁ , R ₂ , R ₃ are as defined herein.

In some embodiments, the lipid compound provided by the present invention is a lipid compound represented by formula (IV-a) or formula (IV-b) or formula (IV-c):

or its stereoisomers, tautomers, and pharmaceutically acceptable salts, wherein

X is selected from O, S, NH; L ₁ , L ₂ , L ₃ , G ₁ , G ₂ , G ₃ , R ₁ , R ₂ , R ₃ are as defined herein.

In some embodiments, the lipid compound provided by the present invention is a compound represented by formula (Va), formula (Vb), formula (Vc), formula (Vd), formula (Ve) or formula (Vd):

or its stereoisomers, tautomers, and pharmaceutically acceptable salts, wherein

X is selected from O, S, and NH; L ₁ , L ₂ , L ₃ , G ₁ , G ₂ , G ₃ , R ₁ , R ₂ , and R ₃ are as defined above.

In some embodiments, each occurrence of L ₁ is independently selected from optionally substituted C ₁ -C ₂₀ alkylene, optionally substituted C ₂ -C ₂₀ acyl; preferably, L ₁ is selected from optionally substituted C ₁ -C ₆ alkylene, optionally substituted C ₂ -C ₆ acyl;

G1 is independently selected at _each occurrence from -C(＝O)O-, -OC(＝O)-, -OC(＝O)O-, -C(＝O)N(R a )-, -NR a C(＝ _O )- _, -S(＝O)O-, -OS(＝O)O-, -S(＝O) 2 O-, -OS(＝O) 2 O-, -C(＝O)S-, -C(＝S)S-, -OP(＝O)(OR _a )O-, -N(R a )C(＝O)O-, -OC(＝O)N(R _a )-, -SS-, -OC(＝O)S-, -SC(＝O)O-, -N(R _{a )} C(＝O)N(R b )-; preferably, G1 is selected from -C(＝O)O-, -OC(＝ _O )-, -OC(＝O)O-, -C(＝O)N(R a )-, -NR a C(＝ _O )-, -S(＝O)O-, -OS(＝O)O-, -S(＝O) 2 O-, -C(＝O)S-, -C(＝ _S )S-, -OP(＝O)(OR a )O-, -N(R _a ) _C (＝O)O-, -OC(＝O)N(R a )-, -SS-, -OC(＝O)S-, -SC(＝O)O-, -N(R a )C(＝O)N(R b ) _-. )-, -NR _a C(═O)-, -N(R _a )C(═O)O-, -OC(═O)N(R _a )-; more preferably, G ₁ is selected from -C(═O)O-, -OC(═O)-;

_Ra and _Rb are each independently selected from H, _C1 - _C20 alkyl, _C1 - _C20 alkenyl, _C1 - _C20 alkynyl, _C3 - _C14 carbocyclyl, _C6 - _C14 aryl, heteroaryl, heterocyclyl; preferably, _Ra and _Rb are each independently selected from H, _C1 - _C20 alkyl, C1- _C20 alkenyl, _C1 - _C20 alkynyl; more preferably, _Ra and _Rb are _{each independently selected from H, C1-C20 alkyl ;}

R ₁ is selected from optionally substituted C ₁ -C ₂₀ alkyl groups; wherein one or more -CH ₂ - in the C ₁ -C ₂₀ alkyl groups may be optionally replaced by O, S, or C ₃ -C ₆ carbocyclic groups;.

In some embodiments, each occurrence of L ₁ is independently selected from optionally substituted methylene, ethylene, propylene, butylene, pentylene, hexylene, acetyl, propionyl, butyryl, pentanoyl, hexanoyl;

G1 is independently selected at _each occurrence from -C(＝O)O-, -OC(＝O)-, -OC(＝O)O-, -C(＝O)N(R _a )-, -NR _a C(＝O)-, -S(＝O)O-, -OS(＝O)O-, -S(＝O) ₂ O-, -OS(＝O) ₂ O-, -C(＝O)S-, -C(＝S)S-, -OP(＝O)(OR _a )O-, -N(R _a )C(＝O)O-, -OC(＝O)N(R _a )-, -SS-, -OC(＝O)S-, -SC(＝O)O-, -N(R _a )C(＝O)N(R _b )-;

R _a and R _b are each independently selected from H, C ₁ -C ₆ alkyl;

R ₁ is selected from

In some embodiments, L ₂ and L ₃ are each independently selected at each occurrence from a bond, an optionally substituted C ₁ -C ₂₀ alkylene, an optionally substituted C ₂ -C ₂₀ acyl;

_G2 and _G3 are each independently selected at each occurrence from a bond, -O-, -S-, -C(=O)-, -C(=O)O-, -OC(=O)-, -OC(=O)O-, -C(=O) _NRa- , -NRaC(=O)-, _-S (=O)O-, -OS(=O)O-, -S(=O) _2O- , -OS(=O) _2O- , -C(=O)S-, -C(=S)S-, -OP(= _O )(ORa)O-, -N( _Ra )C(=O)O-, -OC(=O) _NRa- , -SS-, -OC(=O)S-, -SC(=O)O-, _-NRaC (=O) _NRb- ;

One of _R2 and _R3 is selected from a cholesterol group, and the other is selected from hydrogen, _C1 - _C20 alkyl, -( _R4 ) _q - _NRaRb , _- ( _R4 ) _q -5 or 6-membered nitrogen-containing heteroaryl, -( _R4 ) _q -5 or 6-membered nitrogen-containing heterocyclic group; wherein the 5- or 6-membered nitrogen-containing heteroaryl and the 5- or 6 _{-membered nitrogen-containing heterocyclic group are optionally substituted by a group selected from the following: C1-C6 alkyl} , _C2 - _C6 alkenyl, _C2 - _C6 alkynyl, _C1 - _C6 alkoxy, halogen, hydroxyl, thiol, cyano, nitro, amino, _C1 - _C6 acyl, C1- _C6 acyloxy _;

R ₄ is selected from C ₁ -C ₆ alkylene;

R _a and R _b are each independently selected from H, C ₁ -C ₆ alkyl;

q is selected from 0 or 1.

In some embodiments, L ₂ and L ₃ are each independently selected at each occurrence from a bond, an optionally substituted C ₁ -C ₆ alkylene, an optionally substituted C ₂ -C ₆ acyl;

_G2 and _G3 are each independently selected at each occurrence from a bond, -O-, -S-, -C(=O)-, -C(=O)O-, -OC(=O)-, -OC(=O)O-, -C(=O) _NRa- , -NRaC(=O)-, _-S (=O)O-, -OS(=O)O-, -S(=O) _2O- , -OS(=O) _2O- , -C(=O)S-, -C(=S)S-, -OP(= _O )(ORa)O-, -N( _Ra )C(=O)O-, -OC(=O) _NRa- , -SS-, -OC(=O)S-, -SC(=O)O-, _-NRaC (=O) _NRb- ;

One of R ₂ and R ₃ is selected from a cholesterol group, and the other is selected from hydrogen, a C ₁ -C ₆ alkyl group, -(R ₄ ) _q -NR _a R _b , -(R ₄ ) _q -5 or 6-membered nitrogen-containing heteroaryl, -(R ₄ ) _q -5 or 6-membered nitrogen-containing heterocyclic group; wherein the 5- or 6-membered nitrogen-containing heteroaryl group and the 5- or 6-membered nitrogen-containing heterocyclic group are optionally substituted by a C ₁ -C ₆ alkyl group;

R ₄ is selected from C ₁ -C ₆ alkylene;

R _a and R _b are each independently selected from H, C ₁ -C ₆ alkyl;

q is selected from 0 or 1.

In some embodiments, the present invention provides a compound selected from the group consisting of compounds shown in Table 1 or a pharmaceutically acceptable salt thereof.

Table 1

Lipid Nanoparticles (LNP)

In some embodiments, the present invention provides a lipid nanoparticle as a delivery carrier of a therapeutic and/or prophylactic agent (e.g., nucleic acids including DNA, RNA, etc.), comprising a lipid compound as described herein or its stereoisomers, tautomers, and pharmaceutically acceptable salts thereof. In some embodiments, the lipid nanoparticles described herein further comprise one or more phospholipids. In some embodiments, the lipid nanoparticles described herein further comprise one or more PEG lipids. In some embodiments, the lipid nanoparticles described herein further comprise a combination of phospholipids and PEG lipids. The lipid nanoparticles described herein can deliver therapeutic and/or prophylactic agents to target sites of interest (e.g., cells, tissues, organs, etc.). Therefore, the lipid nanoparticles described herein further comprise one or more therapeutic or prophylactic agents (e.g., nucleic acids, particularly therapeutic nucleic acids (TNA)).

In some embodiments, the molar ratio of lipid compound described herein: phospholipid: PEG lipid in lipid nanoparticles is 40-90:10-60:0.5-20; preferably, the molar ratio of lipid compound described herein: phospholipid: PEG lipid is 30-80:30-80:0.5-20; more preferably, the molar ratio of lipid compound described herein: phospholipid: PEG-lipid is 40-60:40-60:0.5-5; most preferably, the molar ratio of lipid compound described herein: phospholipid: PEG-lipid is 49.25:49.25:1.5.

Phospholipids

In some embodiments, the lipid nanoparticles described herein further comprise phospholipids. Examples of phospholipids include, but are not limited to, distearoyl-sn-glycero-phosphoethanolamine, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE), dioleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE), dioleoylphosphatidylcholine (DOPC), dioleoylphosphatidylcholine (DP ... Phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoylphosphatidylethanolamine (DSPE), monomethylphosphatidylethanolamine (e.g. 16-O-monomethyl PE), dimethylphosphatidylethanolamine (e.g. 16-O-dimethyl PE), 18-1-trans PE, 1-stearoyl-2-oleoylphosphatidylethanolamine (DPPE), Ethanolamine (SOPE), hydrogenated soy phosphatidylcholine (HSPC), egg phosphatidylcholine (EPC), dioleoylphosphatidylserine (DOPS), sphingomyelin (SM), dimyristoylphosphatidylcholine (DMPC), dimyristoylphosphatidylglycerol (DMPG), distearoylphosphatidylglycerol (DSPG), rutoylphosphatidylcholine (DEPC), palmitoyloleoylphosphatidylglycerol (POPG), dioleoyl-phosphatidylethanolamine (DEPE) , 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine (DLPE), 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (DPHyPE), lecithin, phosphatidylethanolamine, lysolecithin, lysophosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, egg sphingomyelin (ESM), cephalin, cardiolipin, phosphatidic acid, cerebroside, hexacosyl phosphate, lysophosphatidylcholine, dilinoleoylphosphatidylcholine or its mixture. It should be understood that other diacylphosphatidylcholine and diacylphosphatidylethanolamine phospholipids can also be used. The acyl group in these lipids is preferably an acyl group derived from a fatty acid with a C10-C24 carbon chain, such as lauroyl, myristoyl, palmitoyl, stearyl or oleoyl.

In some embodiments, the molar percentage of phospholipids in the total lipid of the lipid nanoparticles is about 15% to about 65%, for example, about 20% to about 65%, about 25% to about 65%, about 30% to about 65%, about 35% to about 65%, about 40% to about 65%, about 45% to about 65%, about 50% to about 65%, about 55% to about 65%, about 60% to about 65%, about 15% to about 20%, about 20% to about 25%, about 25% to about 30%, about 30% to about 35%, about 35% to about 40%, about 40% to about 45%, about 45% to about 50%, about 50% to about 55%, about 55% to about 60%, about 60% to about 65%.

PEG lipids

In some embodiments, PEG lipids are incorporated into lipid nanoparticles described herein to inhibit the aggregation of particles, thereby improving the stability of lipid nanoparticles. In some embodiments, PEG lipids described herein are lipids that are covalently or non-covalently connected to one or more polyethylene glycol (PEG) chains. In some embodiments, PEG lipids described herein are lipids that are covalently connected to one or more polyethylene glycol (PEG) chains.

In some embodiments, the molecular weight of the PEG molecules suitable for use in the PEG lipids described herein is about 500 to about 10,000, about 1,000 to about 10,000, about 1,000 to about 5,000, about 1,000 to about 4,000, about 1,000 to about 3,000, about 1,000 to about 2,000, for example, PEG2000, PEG2500, PEG3000, etc.

Examples of PEG lipids include, but are not limited to, PEG-diacylglycerol (DAG) (e.g., 1-(monomethoxy-polyethylene glycol)-2,3-dimyristoylglycerol (PEG-DMG)), PEG-dialkoxypropyl (DAA), PEG-phospholipids, PEG-ceramide (Cer), PEGylated phosphatidylethanolamine (PEG-PE), PEG succinate diacylglycerol (PEGS-DAG) (e.g., 4-0-(2',3'-di(tetradecanoyloxy) )propyl-1-0-(w-methoxy(polyethoxy)ethyl)succinate (PEG-S-DMG)), PEG dialkoxypropylaminoformamide, N-(carbonyl-methoxypolyethylene glycol 2000)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine sodium, PEG-dilauroyloxypropyl, PEG-dimyristoyloxypropyl, PEG-dipalmitoyloxypropyl, PEG-distearoyloxypropyl, 1-(monomethoxy-polyethylene glycol)-2,3 -Dimyristoylglycerol-PEG (DMG-PEG), distearoyl-rac-glycerol-PEG (DSG-PEG), PEG-dilauroylglycerol, PEG-dipalmitoylglycerol, PEG-distearoylglycerol, PEG-dilauroylglyceramide, PEG-dimyristoylglyceramide, PEG-dipalmitoylglyceramide, PEG-distearoylglyceramide, (1-[8'-(cholest-5-ene-3β-oxy)formamido-3', 6'-dioxaoctyl]carbamoyl-ω-methyl-poly(ethylene glycol) (PEG-cholesterol), 3,4-ditetradecyloxybenzyl-ω-methyl-poly(ethylene glycol) ether (PEG-DMB), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)] (DSPE-PEG), and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-poly(ethylene glycol)-hydroxy (DSPE-PEG-OH).

In some embodiments, the molar percentage of PEG lipid in the total lipid of the lipid nanoparticle is about 0.1% to about 10%, for example, about 1.0% to about 10%, about 1.5% to about 10%, about 2.0% to about 10%, about 2.5% to about 10%, about 3.0% to about 10%, about 3.5% to about 10%, about 4.0% to about 10%, about 4.5% to about 10%, about 5.0% to about 10%, about 5.5% to about 10%, about 6.0% to about 10%, about 6.5% to about 10%, about 7.0% to about 10%, about 7.5% to about 10%, about 8.0% to about 10%, about 8.5% to about 10%, about 9.0% to about 10%, about 9.5% to about 10%, about 1.0% to about 1.5%, about 1.5% to about 2.0%, about 2.0% to about 2.5%, about 2.5% to about 3.0%, about 3.0% to about 3.5%, about 3.5% to about 4.0%, about 4.0% to about 4.5%, about 4.5% to about 5.0%, about 5.0% to about 5.5%, about 5.5% to about 6.0%, about 6.0% to about 6.5%, about 6.5% to about 7.0%, about 7.0% to about 7.5%, about 7.5% to about 8.0%, about 8.0% to about 8.5%, about 8.5% to about 9.0%, about 9.0% to about 10%.

Particle size of lipid nanoparticles

In some embodiments, the particle size range of the lipid nanoparticles described herein is from about 40 nm to about 150 nm, for example, from about 45 nm to about 150 nm, from about 50 nm to about 150 nm, from about 55 nm to about 150 nm, from about 60 nm to about 150 nm, from about 65 nm to about 150 nm, from about 70 nm to about 150 nm, from about 75 nm to about 150 nm, from about 80 nm to about 150 nm, from about 85 nm to about 150 nm, from about 90 nm to about 150 nm, from about 95 nm to about 150 nm, from about 100 nm to about 150 nm, from about 105 nm to about 150 nm, from about 110 nm to about 150 nm, from about 115 nm to about 150 nm, from about 120 nm to about 150 nm, from about 125 nm to about 150 nm, from about 130 nm to about 150 nm, from about 135 nm to about 150 nm, from about 140 nm to about 150 nm, from about 145 nm to about 150 nm. In some embodiments, the particle size range of the lipid nanoparticles described herein is about 40 nm to about 120 nm, for example, about 45 nm to about 120 nm, about 50 nm to about 120 nm, about 55 nm to about 120 nm, about 60 nm to about 120 nm, about 65 nm to about 120 nm, about 70 nm to about 120 nm, about 75 nm to about 120 nm, about 80 nm to about 120 nm, about 85 nm to about 120 nm, about 90 nm to about 120 nm, about 95 nm to about 120 nm, about 100 nm to about 120 nm, about 105 nm to about 120 nm, 110 nm to about 120 nm, 115 nm to about 120 nm. In some embodiments, the particle size range of the lipid nanoparticles described herein is about 40 nm to about 110 nm, for example, about 45 nm to about 110 nm, about 50 nm to about 110 nm, about 55 nm to about 110 nm, about 60 nm to about 110 nm, about 65 nm to about 110 nm, about 70 nm to about 110 nm, about 75 nm to about 110 nm, about 80 nm to about 110 nm, about 85 nm to about 110 nm, about 90 nm to about 110 nm, about 95 nm to about 110 nm, about 100 nm to about 110 nm, about 105 nm to about 110 nm. In some embodiments, the particle size range of the lipid nanoparticles described herein is from about 40nm to about 100nm, for example, from about 45nm to about 100nm, from about 50nm to about 100nm, from about 55nm to about 100nm, from about 60nm to about 100nm, from about 65nm to about 100nm, from about 70nm to about 100nm, from about 75nm to about 100nm, from about 80nm to about 100nm, from about 85nm to about 100nm, from about 90nm to about 100nm, from about 95nm to about 100nm. In some embodiments, the particle size range of the lipid nanoparticles described herein is from about 40nm to about 90nm, for example, from about 45nm to about 90nm, from about 50nm to about 90nm, from about 55nm to about 90nm, from about 60nm to about 90nm, from about 65nm to about 90nm, from about 70nm to about 90nm, from about 75nm to about 90nm, from about 80nm to about 90nm, from about 85nm to about 90nm. In some embodiments, the particle size range of the lipid nanoparticles described herein is from about 40nm to about 85nm, for example, from about 45nm to about 85nm, from about 50nm to about 85nm, from about 55nm to about 85nm, from about 60nm to about 85nm, from about 65nm to about 85nm, from about 70nm to about 85nm, from about 75nm to about 85nm, from about 80nm to about 85nm. In some embodiments, the particle size range of the lipid nanoparticles described herein is from about 40nm to about 80nm, for example, from about 45nm to about 80nm, from about 50nm to about 80nm, from about 55nm to about 80nm, from about 60nm to about 80nm, from about 65nm to about 80nm, from about 70nm to about 80nm, from about 75nm to about 80nm. In some embodiments, the particle size range of the lipid nanoparticles described herein is from about 40nm to about 70nm, such as from about 45nm to about 70nm, from about 50nm to about 70nm, from about 55nm to about 70nm, from about 60nm to about 70nm, from about 65nm to about 70nm. In some embodiments, the particle size range of the lipid nanoparticles described herein is from about 40nm to about 60nm, such as from about 45nm to about 60nm, from about 50nm to about 60nm, from about 55nm to about 60nm.

Lipid/nucleic acid ratio

In some embodiments, the weight or molar ratio of lipid to nucleic acid of the lipid nanoparticle is from about 10:1 to about 100:1, e.g., from about 10:1 to about 95:1, from about 10:1 to about 90:1, from about 10:1 to about 85:1, from about 10:1 to about 80:1, from about 10:1 to about 75:1, from about 10:1 to about 70:1, from about 10:1 to about 65:1, from about 10:1 to about 60:1, from about 10:1 to about 55:1, from about 10:1 to about 50:1, from about 10:1 to about 45:1, from about 10:1 to about 40:1, from about 10:1 to about 35:1, from about 10:1 to about 30:1, from about 10:1 to about 25:1, from about 10:1 to about 20:1, from about 10:1 to about 15:1.

In some embodiments, the N/P ratio of the lipid nanoparticles (i.e., the ratio of positively charged lipid amine groups to negatively charged nucleic acid phosphate groups) is about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more.

Therapeutic/preventive agents

In some embodiments, the lipid nanoparticles of the present invention also include therapeutic and/or preventive agents. In some embodiments, the therapeutic and/or preventive agents described herein include organic molecules, inorganic molecules, proteins, polypeptides, nucleic acids, vaccines, immunotherapeutics, etc. In some embodiments, the therapeutic and/or preventive agents described herein include nucleic acids. In some embodiments, the therapeutic and/or preventive agents described herein include DNA. In some embodiments, the therapeutic and/or preventive agents described herein include single-stranded DNA (ssDNA), double-stranded DNA (dsDNA), genomic DNA (gDNA), complement DNA (cDNA), antisense DNA, chloroplast DNA (ctDNA or cpDNA), microsatellite DNA, mitochondrial DNA (mtDNA or mDNA), kinetoplastid DNA (kDNA), provirus, lysogen, repeat DNA, satellite DNA, or viral DNA. In some embodiments, the therapeutic and/or preventive agents described herein include RNA. In some embodiments, the therapeutic and/or preventive agents described herein include small interfering RNA (siRNA). In some embodiments, the therapeutic and/or preventive agents described herein include messenger RNA (mRNA). In some embodiments, the therapeutic and/or prophylactic agents described herein include single-stranded RNA (ssRNA), double-stranded RNA (dsRNA), precursor messenger RNA (pre-mRNA), small hairpin RNA or short hairpin RNA (shRNA), microRNA (miRNA), guide RNA (gRNA), transfer RNA (tRNA), nuclear heterogeneous RNA (hnRNA), coding RNA, non-coding RNA (ncRNA), long non-coding RNA (long ncRNA or lncRNA), satellite RNA, viral satellite RNA, signal recognition particle RNA, small cytoplasmic RNA, small nuclear RNA (snRNA), ribosomal RNA (rRNA), Piwi-interacting RNA (piRNA), polyinosinic acid, and ribozyme.

Pharmaceutical compositions and preparations

In some embodiments, the present invention provides a pharmaceutical composition comprising the lipid nanoparticles described herein and a pharmaceutically acceptable carrier. In some embodiments, the present invention provides a therapeutic and/or preventive agent (e.g., nucleic acid including DNA and RNA, etc.) vaccine comprising the lipid nanoparticles described herein and a pharmaceutically acceptable carrier.

In some embodiments, the pharmaceutically acceptable carriers described herein include diluents, buffers, stabilizers, and the like.

In some embodiments, the diluent includes ethylene glycol, glycerol, polyethylene glycol, sucrose, trehalose, or a combination thereof, etc. In some embodiments, the content of the diluent is 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19% or 20%.

In some embodiments, the buffer comprises phosphate, citrate, imidazole, histidine, Tris, HEPES, or a combination thereof, etc. In some embodiments, the concentration of the buffer in the pharmaceutical composition is about 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, 100 mM, or more than 100 mM.

In some embodiments, stabilizers include salts, including inorganic metal salts such as sodium chloride, potassium chloride, calcium chloride, etc. In some embodiments, the concentration of the stabilizer in the pharmaceutical composition is about 10mM, 20mM, 30mM, 40mM, 50mM, 60mM, 70mM, 80mM, 90mM, 100mM, 110mM, 120mM, 130mM, 140mM, 150mM, 160mM, 170mM, 180mM, 190mM, 200mM, or more than 200mM.

In some embodiments, depending on the different routes of drug administration, the pharmaceutical composition and/or vaccine of the present invention can be prepared into oral preparations, intramuscular injection preparations, subcutaneous injection preparations, intravenous injection preparations, nebulized inhalation preparations, nasal spray inhalation preparations or dry powder inhalation preparations, and ocular administration preparations.

Indications

The lipid nanoparticles and/or pharmaceutical compositions provided by the present invention can be used to prevent and/or treat cancer, inflammation, fibrotic diseases, autoimmune diseases, infections, mental disorders, blood diseases, chromosomal diseases, genetic diseases, connective tissue diseases, digestive diseases, ear, nose and throat diseases, endocrine diseases, eye diseases, reproductive diseases, heart diseases, kidney diseases, lung diseases, metabolic diseases, oral diseases, musculoskeletal diseases, neonatal screening, nutritional diseases, parasitic diseases, skin diseases, etc.

In some embodiments, the lipid nanoparticles and/or pharmaceutical compositions provided by the present invention are mRNA vaccines, which can be used to prevent cancer, viral infection, bacterial infection, fungal infection, etc. The viruses include but are not limited to: norovirus, Ebola virus, coronavirus (including the new coronavirus SARS CoV2), cytomegalovirus, dengue virus, Zika virus, coxsackie virus, enterovirus, hepatitis virus, herpes simplex virus, human papillomavirus, influenza virus, Marburg virus, measles virus, polio virus, rabies virus, rotavirus, measles virus, etc.

Treatment

The present invention provides a method for in vivo delivery of lipid nanoparticles as described herein, comprising administering the lipid nanoparticles or pharmaceutical compositions described herein to an individual in need. In some embodiments, the present invention provides a method for in vivo delivery of lipid nanoparticles as described herein, comprising administering the lipid nanoparticles or pharmaceutical compositions described herein to an individual in need by pulmonary delivery. In some embodiments, the present invention provides a method for in vivo delivery of lipid nanoparticles as described herein, comprising administering the lipid nanoparticles or pharmaceutical compositions described herein to an individual in need by intranasal delivery. In some embodiments, the present invention provides a method for in vivo delivery of lipid nanoparticles as described herein, comprising administering the lipid nanoparticles or pharmaceutical compositions described herein to an individual in need by inhalation. In some embodiments, the present invention provides a method for in vivo delivery of lipid nanoparticles as described herein, comprising administering the lipid nanoparticles or pharmaceutical compositions described herein to an individual in need by nebulized inhalation.

Preparation of lipid nanoparticles

The lipid nanoparticles of encapsulated therapeutic agents and/or preventive agents can be prepared using a variety of methods known in the art. Typically, a solution comprising various lipid mixtures is first prepared, and the solution is mixed with a solution of a therapeutic agent and/or preventive agent before forming lipid nanoparticles, and the therapeutic agent and/or preventive agent is encapsulated in lipid nanoparticles formed by a mixture of various lipids (e.g., described in WO2016004318, US20160038432). Alternatively, a solution comprising various lipid mixtures is first prepared to form lipid nanoparticles, and then the obtained lipid nanoparticles are mixed with a therapeutic agent and/or preventive agent to encapsulate the therapeutic agent and/or preventive agent in lipid nanoparticles formed by a mixture of various lipids (e.g., described in WO2018089801, US20180153822). These methods can effectively encapsulate the therapeutic agent and/or prophylactic agent in the lipid nanoparticles, and the encapsulation efficiency is generally not less than about 80%, not less than about 85%, not less than about 90%, not less than about 95%, not less than about 96%, not less than about 97%, not less than about 98%, and not less than about 99%.

The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

Example

Example 1 Synthesis of Compound CP101

Step 1: Synthesis of 6-bromohexyl 2-hexyldecanoate (Compound 2)

Dissolve 2-hexyldecanoic acid (2.12 g, 5.0 mmol) in 30 mL of dichloromethane, add 6-bromohexanol (0.93 g, 5.0 mmol), DMAP (0.21 g, 2.0 mmol), triethylamine (0.62 g, 6.0 mmol), and stir to dissolve. Add a dichloromethane solution of EDC.HCL (1.10 g, 6.0 mmol) dropwise, and stir at room temperature for 16 hours after the addition is complete. Quench with water, add dilute hydrochloric acid, adjust the pH value to 1-3, and separate the liquids. Wash the organic phase with saturated brine, dry over anhydrous sodium sulfate, filter and concentrate to obtain compound 2 (1.45 g, light yellow oil), with a yield of 70%.

MS m/z(ESI):419.2[M+1];

¹ H NMR: (400MHz, CDCl ₃ -d) δ4.08(t,J＝6.6Hz,2H),3.41(t,J＝6.8Hz,2H),2.35-2.27(m,1H),1.92-1.75(m,2H),1.70-1.54(m,5H),1.51-1.35(m,7H),1.26(br s,20H),0.88(t,J＝6.7Hz,7H)

Step 2: Synthesis of compound 4

Compound 3 (23.0 g, 112 mmol), compound 2 (47.0 g, 112 mmol) and potassium carbonate (23.2 g, 168 mmol) were added to 230 mL of DMF and reacted at 80° C. overnight. After the reaction was completed, 100 mL of water was added to quench, and methyl tert-butyl ether was used for extraction. The organic phases were combined, concentrated, and separated by column chromatography to obtain compound 4 (32 g, yellow oil) with a yield of 52.5%.

¹ HNMR: (400MHz, CDCl ₃ -d) δ5.44 (br s,1H),4.27-4.04(m,5H),4.01-3.88(m,2H),2.41-2.24(m,2H),1.64-1.64(m,1H) ,1.70-1.56(m,6H),1.50-1.38(m,16H),1.32-1.21(m,22H),0.88(t,J＝6.7Hz,6H)

Step 3: Synthesis of compound 6

Compound 4 (9.0 g, 16.6 mmol) and compound 5 (7.43 g, 16.6 mmol) were added to 90 mL of dichloromethane, and triethylamine (2.51 g, 24.8 mmol) was added, and stirred at room temperature overnight. After the reaction was completed, the solvent was removed by rotary evaporation, and compound 6 (9.0 g, yellow oil) was directly separated by column chromatography, with a yield of 56.9%.

¹ H NMR: (400MHz, CDCl ₃ -d) δ5.40-5.34(m,1H),5.31-5.30(m,2H),4.57-4.35(m,4H),4.18(t,J＝6.7Hz,2H),4.07(t,J＝6.6Hz,2H),2.42-2.28(m,3H),2 .08-1.78(m,5H),1.70-1.55(m,10H),1.53-1.43(m,1H),1.33-1.19(m,24H),1.14-1.05(m,5H),0.99-0.85(m,17H),0.73(s,3H)

Step 4: Synthesis of compound CP101

Compound 6 (7.50 g, 7.84 mmol) was added to 75 mL of dichloromethane, and then 75 mL of 4N hydrochloric acid/1,4-dioxane solution was added. The mixture was stirred at room temperature for 3 hours, and the solvent was removed by rotary evaporation to obtain compound CP101.

¹ H NMR: (400MHz, CD ₃ OD-d4) δ5.32 (br d,J＝4.9Hz,1H),4.64-4.53(m,1H),4.50-4.26(m,4H),4.17-4.06(m,1H),3.99(t,J＝6.5Hz, 2H),2.37-2.17(m,3H),1.99-1.74(m,5H),1.67-1.42(m,13H),1.39-1.27(m,10H),1.19(br s,20H),1.11-0.98(m,7H),0.98-0.88(m,6H),0.88-0.76(m,15H),0.63(s,3H)

Example 2 Synthesis of Compound CP102

Compound CP101 (2.00 g, 2.24 mmol), N,N-dimethylaminoacetic acid (347 mg, 3.36 mmol), EDCI (664 mg, 3.36 mmol) and DMAP (411 mg, 3.36 mmol) were added to 15 mL of dichloromethane and stirred overnight at room temperature. The reaction solution was washed with brine, the organic phase was concentrated, and column chromatography analysis gave compound CP102 (260 mg, yellow viscous solid) with a yield of 12.3%.

¹ H NMR: (400MHz, CDCl ₃ -d)δ5.48-5.35(m,2H),4.68-4.47(m,3H),4.44-4.36(m,1H),4.23-4.13(m,2H),4.07(t,J＝6.6Hz,2H),3.18(d,J＝1.9Hz,2H),2.43-2.26( m,9H),2.09-1.79(m,8H),1.72-1.53(m,12H),1.50-1.27(m,30H),1.18-1.08(m,6H),1.05-0.99(m,5H),0.95-0.85(m,15H),0.68(s,3H).

The compounds of Examples 3-6 were prepared using a method similar to that of Examples 1HE 2 using corresponding starting materials.

Example 3 Synthesis of Compound CP103

¹ H NMR (400 MHz, CDCl ₃ -d)δ7.91-7.81(m,1H),5.45-5.29(m,1H),4.92-4.80(m,1H),4.56(s,3H),4 .27-4.13(m,2H),4.10-3.96(m,2H),3.20-3.00(m,2H),2.85-2.56(m,7H),2. 54-2.18(m,7H),2.10-1.72(m,6H),1.70-1.52(m,10H),1.51-1.35(m,12H),1 .34-1.22(m,22H),1.21-1.03(m,8H),1.01-0.82(m,18H),0.72-0.61(m,3H).

Example 4 Synthesis of Compound CP104

¹ H NMR (400MHz, CDCl ₃ -d) δ7.88(br s,1H),7.25-7.19(m,1H),7.15-6.99(m,1H),6.89-6.60(m,1H),5.42(br d,J＝4.0Hz,1H),5.36(br d,J＝5.0Hz,1H),4.89-4.70(m,2H),4.51-4.40(m,2H),4.17(t,J＝6.6Hz,1H),4.09-4.06(m,1H),3.6 1-3.46(m,1H),2.46-2.18(m,5H),2.08-1.78(m,8H),1.72-1.44(m,15H),1.43-1.33(m,8H),1.26(br s,13H),1.19-1.05(m,10H),1.04-1.00(m,6H),0.92(d,J=6.4Hz,4H),0.90-0.86(m,12H),0.69(s,3H).

Example 5 Synthesis of Compound CP105

¹ H NMR (400MHz, CDCl ₃ -d) δ12.3 (br s, 1H), 6.40 (d, J = 7.5Hz, 1H), 6.55 (br d,J＝7.5Hz,1H),5.35-5.27(m,1H),4.76-4.66(m,1H),4.49-4.31(m,3H),4.21-4.05(m, 2H),4.05-3.94(m,2H),3.70-3.50(m,1H),3.40-3.14(m,3H),2.78-2.66(m,3H),2.61(br s,1H),2.35-2.17(m,6H),1.74(br s,6H),1.65-1.45(m,10H),1.44-1.18(m,29H),1.10-0.98(m,7H),0.96-0.91(m,5H),0.87-0.72(m,18H),0.61(s,3H).

Example 6 Synthesis of Compound CP106

¹ H NMR (400MHz, CDCl ₃ -d) δ7.36-7.23(m,1H),5.47-5.27(m,1H),4.76-4.70(m,1H),4.42-4.35(m,2H),4.09(t,J＝6.6Hz,2H),4.01-3.99(m,2H),3.58(t,J＝6 .6Hz,2H),3.21-3.13(m,1H),3.01-2.93(m,1H),2.83-2.75(m,3H),2.49-2.20(m,6H),2.10-1.67(m,8H),1.43-1.29(m,14H),1.18(br s,30H),1.11-1.00(m,6H),0.96-0.89(m,5H),0.86-0.73(m,20H),0.70-0.54(m,3H).

Example 7 Synthesis of Compound CP201

Step 1: Synthesis of compound 7

Compound 4 (20.0 g, 36.8 mmol), N,N-dimethylaminoacetic acid (4.55 g, 44.1 mmol), DMAP (5.39 g, 44.1 mmol), and EDCI (8.46 g, 44.1 mmol) were added to 200 mL of dichloromethane and reacted at room temperature for 15 hours. Water was added to stop the reaction, and the organic phase was separated. The organic phase was washed with saturated brine and dried over anhydrous sodium sulfate, filtered and concentrated, and separated by column chromatography to obtain compound 7 (16.0 g, yellow oil) with a yield of 69%.

¹ H NMR: ET67677-100-P1A2 (400MHz, CDCl ₃ -d) δ5.35-5.25(m,1H),4.60-4.48(m,2H),4.44-4.32(m,1H),4.17-4.11(m,2H),4.09-4.03(m,2H),3. 21-3.13(m,2H),2.35-2.33(m,7H),1.70-1.57(m,7H),1.47-1.43(m,10H),1.41-1.36(m,5H),1.25(br s,20H),0.87(t,J=6.6Hz,6H).

Step 2: Synthesis of compound 8

Compound 7 (8.00 g, 12.7 mmol) was added to 80 mL of dichloromethane, and then 80 mL of 4N hydrochloric acid/1,4-dioxane solution was added. The mixture was stirred at room temperature for 3 hours, and the solvent was removed by rotary evaporation to obtain compound 8 (6.73 g, yellow oil).

Step 3: Synthesis of compound CP201

Compound 8 (500 mg, 946 μmol), compound 9 (460 mg, 946 μmol), EDCI (272 mg, 1.42 mmol), and DMAP (139 mg, 1.13 mmol) were added to 5 mL of dichloromethane solution and stirred overnight at room temperature. Water was added to stop the reaction, and the organic phase was separated. The organic phase was washed with saturated brine and dried over anhydrous sodium sulfate, filtered and concentrated, and separated by column chromatography to obtain compound CP201 (222 mg, yellow oil) with a yield of 24%.

¹ H NMR (400MHz, CDCl ₃ -d) δ7.81 (br d, J=8.5Hz, 1H), 5.37 (br d,J＝4.0Hz,1H),4.94-4.84(m,1H),4.69-4.56(m,1H),4.53-4.37(m,2 H),4.23-4.13(m,2H),4.07(t,J＝6.6Hz,2H),4.00-3.90(m,1H),3.00(q ,J＝16.3Hz,2H),2.70-2.54(m,4H),2.44-2.24(m,9H),2.05-1.94(m,3 H),1.91-1.79(m,4H),1.70-1.53(m,10H),1.51-1.37(m,11H),1.26(br s,21H),1.17-1.07(m,6H),1.02(s,5H),0.94-0.84(m,15H),0.68(s,3H).

Example 8 Synthesis of Compound CP202

Step 1: Synthesis of compound 11

Compound 4 (10 g), compound 10 (4.36 g), EDCI (4.23 g), and DMAP (2.70 g) were added to 100 mL of dichloromethane solution and stirred at room temperature overnight. Water was added to stop the reaction, and the organic phase was separated. The organic phase was washed with saturated brine and dried over anhydrous sodium sulfate, filtered and concentrated, and separated by column chromatography to obtain compound 11 (5.77 g, yellow oil) with a yield of 46%.

¹ H NMR (400MHz, CDCl ₃ -d) δ5.36-5.21(m,1H),4.60-4.52(m,1H),4.52-4.45(m,1H),4.43-4.35(m,1H),4.21-4.10(m,2H),4.06(t,J＝6.6Hz,2H),3.21(d,J ＝2.3Hz,2H),2.71-2.40(m,8H),2.36-2.27(m,4H),1.70-1.55(m,6H),1.50-1.33(m,15H),1.31-1.19(m,20H),0.88(t,J＝6.7Hz,6H).

Step 2: Synthesis of compound 12

Compound 11 (5 g) was added to 50 mL of dichloromethane, followed by 50 mL of 4N hydrochloric acid/1,4-dioxane solution. The mixture was stirred at room temperature for 3 hours, and the solvent was removed by rotary evaporation to obtain compound 12 (4.27 g, yellow oil).

Step 3: Synthesis of compound CP202

Compound 12 (500 mg), compound 9 (417 mg), EDCI (246 mg), and DMAP (126 mg) were added to 20 mL of dichloromethane solution and stirred at room temperature overnight. Water was added to stop the reaction, and the organic phase was separated. The organic phase was washed with saturated brine and dried over anhydrous sodium sulfate, filtered and concentrated, and separated by column chromatography to obtain compound CP202 (154 mg, yellow oil) with a yield of 46%.

¹ H NMR (400MHz, CDCl ₃ -d) δ7.88 (d, J = 8.5Hz, 1H), 5.37 (br d,J＝4.3Hz,1H),4.92-4.82(m,1H),4.68-4.58(m,1H),4.54-4.40(m,2H),4.23-4.13(m,2H),4.11-4.03(m,2H),4.02-3.90(m,1H),3.16-2 .97(m,2H),2.68-2.46(m,11H),2.38-2.27(m,6H),2.09-1.93(m,3H),1.91-1.78(m,4H),1.71-1.54(m,10H),1.52-1.37(m,12H),1.26(br s,21H),1.18-1.08(m,6H),1.02(s,4H),0.96-0.85(m,16H),0.68(s,3H).

The compounds of Examples 9 to 10 were prepared in a similar manner to Example 8 using the corresponding starting materials.

Example 9 Synthesis of Compound CP203

¹ H NMR (400MHz, CDCl ₃ -d) δ7.88 (d, J = 8.4Hz, 1H), 5.38 (br d,J＝4.1Hz,1H),4.93-4.81(m,1H),4.69-4.56(m,1H),4.54-4.46(m,1H),4.44-4.33(m,1H),4.21-4.12(m,2H),4.07(t,J＝6.6Hz,2H ),3.15-2.93(m,2H),2.70-2.47(m,6H),2.42-2.28(m,10H),2.06-1.78(m,9H),1.72-1.21(m,44H),1.19-0.83(m,28H),0.68(s,3H).

Example 10 Synthesis of Compound CP204

¹ H NMR (400MHz, CDCl ₃ -d) δ7.84 (br d, J = 8.5Hz, 1H), 5.73-5.48 (m, 1H), 5.38 (br d,J＝4.0Hz,1H),4.94-4.78(m,1H),4.69-4.56(m,1H),4.54-4.44(m,1H),4.43-4.34(m,1H),4.22-4.02(m,3H),4.01-3.92(m,1H) ),3.68(s,1H),3.13-2.85(m,2H),2.44-2.24(m,11H),2.12-1.75(m,11H),1.73-1.20(m,38H),1.20-0.80(m,29H),0.68(s,3H).

Example 11 Synthesis of Compound CP302

Step 1: Synthesis of compound 13

Compound 4 (12.3 g, 22.6 mol), compound 9 (11.0 g, 22.6 mmol), EDCI (6.5 g, 33.9 mmol), and DMAP (3.31 g, 27.1 mmol) were added to 100 mL of dichloromethane solution and stirred overnight at room temperature. Water was added to stop the reaction, and the organic phase was separated. The organic phase was washed with saturated brine and dried over anhydrous sodium sulfate, filtered and concentrated, and separated by column chromatography to obtain compound 13 (15.5 g, yellow oil) with a yield of 67.7%.

¹ H NMR (400MHz, CDCl ₃ -d)δ5.41-5.33(m,2H),4.68-4.52(m,2H),4.43(dq,J＝3.5,11.1Hz,2H ),4.26-4.13(m,2H),4.11-4.03(m,2H),2.72-2.49(m,4H),2.42-2.22 (m,3H),2.10-1.79(m,5H),1.72-1.52(m,11H),1.52-1.35(m,23H),1. 34-1.20(m,26H),1.18-0.98(m,12H),0.95-0.83(m,17H),0.68(s,3H).

Step 2: Synthesis of compound CP301

Compound 13 (20.5 g, 20.3 mmol) was added to 125 mL of dichloromethane, and then 125 mL of 4N hydrochloric acid/1,4-dioxane solution was added. The mixture was stirred at room temperature for 3 hours, and the solvent was removed by rotary evaporation. The mixture was redissolved in 200 mL of dichloromethane, and saturated aqueous sodium carbonate solution was added to adjust the pH to neutral. The mixture was extracted with dichloromethane, and the organic phases were combined, dried over anhydrous sodium sulfate, concentrated, filtered, and separated by column chromatography to obtain compound CP301 (10.5 g, yellow oil) in a yield of 54.3%.

¹ H NMR (400MHz, CDCl ₃ -d)δ6.64-6.55(m,1H),5.47-5.33(m,1H),4.70-4.59(m,2H),4.24-3.92(m,5H),2 .85-2.46(m,5H),2.39-2.26(m,3H),2.09-1.92(m,2H),1.92-1.75(m,3H),1.54(br s,10H),1.51-1.33(m,12H),1.33-1.20(m,21H),1.20-1.05(m,7H),1.04-0.98(m,5H),0.96-0.85(m,15H),0.68(s,3H).

Step 3: Synthesis of compound CP302

Compound CP301 (0.60 g, 658 μmol), compound 14 (156 mg, 986 μmol), EDCI (151. mg, 789 μmol), and DMAP (96.4 mg, 789 μmol) were added to 6 mL of dichloromethane solution and stirred overnight at room temperature. Water was added to stop the reaction, and the organic phase was separated. The organic phase was washed with saturated brine and dried over anhydrous sodium sulfate, filtered and concentrated, and separated by column chromatography to obtain compound CP302 (312 mg, yellow oil) with a yield of 45.1%.

1H NMR (400MHz, CDCl ₃ -d) δ6.55-6.37(m,1H),5.43-5.32(m,1H),4.94-4.81(m,1H),4.69-4.35(m,3H),4.2 1-4.12(m,2H),4.10-4.01(m,2H),3.23(s,2H),2.68-2.46(m,11H),2.36-2.25(m,6H) ,2.04-1.93(m,2H),1.89-1.80(m,3H),1.69-1.54(m,9H),1.51-1.36(m,12H),1.33- 1.22(m,23H),1.18-1.08(m,6H),1.05-0.97(m,6H),0.94-0.85(m,16H),0.68(s,3H).

The compounds of Examples 12 to 16 were prepared in a similar manner to Example 11 using the corresponding starting materials.

Example 12 Synthesis of Compound CP303

¹ H NMR (400MHz, CDCl ₃ -d)δ6.72-6.37(m,1H),5.46-5.34(m,1H),4.94-4.82(m,1H),4.66-4.52(m,2H),4.48-4.36( m,1H),4.23-4.13(m,2H),4.09-4.00(m,2H),3.30-3.10(m,2H),2.71-2.52(m,4H),2.45-2.2 6(m,9H),2.06-1.96(m,2H),1.90-1.77(m,3H),1.71-1.53(m,11H),1.51-1.36(m,11H),1.35 -1.21(m,26H),1.15-1.06(m,5H),1.05-0.94(m,5H),0.92-0.85(m,13H),0.73-0.62(m,3H).

Example 13 Synthesis of Compound CP304

¹ H NMR (400MHz, CDCl ₃ -d) δ6.52-6.39(m,1H),5.37(br d,J＝4.0Hz,1H),4.91-4.77(m,1H),4.68-4.57(m,1H),4.48(dd,J＝3.8,11.3Hz ,1H),4.36(dd,J＝3.3,11.3Hz,1H),4.22-4.03(m,4H),2.71-2.52(m,6H),2.40- 2.27(m,11H),2.07-1.76(m,8H),1.69-1.54(m,10H),1.42-1.35(m,6H),1.34- 1.21(m,29H),1.18-1.08(m,6H),1.02(s,5H),0.93-0.83(m,18H),0.68(s,3H).

Example 14 Synthesis of Compound CP305

¹ H NMR (400MHz, CDCl ₃ -d) δ6.52(br d,J＝8.0Hz,1H),5.37(br d,J＝3.8Hz,1H),4.89-4.81(m,1H),4.61(br s,1H),4.40(br s,2H),4.23-4.02(m,4H),2.76-2.44(m,14H),2.39-2.26(m,6H),2.06-1.92(m,2H),1.89-1.77(m,3H),1.72-1.53(m, 10H),1.52-1.35(m,12H),1.34-1.20(m,23H),1.20-1.06(m,7H),1.05-0.98(m,5H),0.97-0.82(m,16H),0.68(s,3H).

Example 15 Synthesis of Compound CP306

¹ H NMR (400MHz, CDCl ₃ -d) δ6.47 (d, J = 7.6Hz, 1H), 5.38 (br d,J＝3.9Hz,1H),4.88-4.80(m,1H),4.69-4.56(m,1H),4.50(dd,J＝3.7,1 1.3Hz,1H),4.41-4.33(m,1H),4.20-4.11(m,2H),4.10-4.04(m,2H),2.91 -2.75(m,2H),2.69-2.51(m,4H),2.38-2.24(m,6H),2.08-1.92(m,4H),1 .92-1.80(m,5H),1.66-1.55(m,8H),1.38(td,J＝3.5,7.2Hz,7H),1.26(br s,28H),1.18-1.08(m,8H),1.02(s,4H),0.93-0.84(m,18H),0.68(s,3H).

Example 16 Synthesis of Compound CP307

¹ H NMR (400MHz, CDCl ₃ -d) δ6.46 (br d, J=7.5Hz, 1H), 5.37 (br d,J＝4.1Hz,1H),4.87-4.80(m,1H),4.68-4.55(m,1H),4.47(dd,J＝3.8,11.4Hz, 1H),4.36(dd,J＝3.3,11.4Hz,1H),4.20-4.11(m,2H),4.10-4.04(m,2H),2.88(br d,J＝4.1Hz,1H),2.70-2.52(m,4H),2.38-2.24(m,6H),2.07-1.93(m,4H),1.92-1.78(m,6H),1.67-1.54(m,9H),1.26(br s,35H),1.02(s,5H),0.96-0.81(m,24H),0.68(s,3H).

Example 17 Synthesis of Compound CP401

Step 1: Synthesis of compound 4

Compound 2 (1.0 g, 2.38 mmol), cysteine (433.2 mg, 3.57 mmol), tetrabutylammonium iodide (866.5 mg, 2.4 mmol), and NaOH (190.4 mg, 4.76 mmol) were added to 50 mL of ethanol, stirred at room temperature for 48 h, and then di-tert-butyl dicarbonate (780.0 mg, 3.58 mmol) was added and stirred at room temperature for 15 hours. After the reaction was completed, 100 mL of water was added to quench, and the mixture was extracted with ethyl acetate. The organic phases were combined, concentrated, and separated by column chromatography to obtain compound 4 (731 mg, yellow oil) with a yield of 54.8%. ESI-MS m/z: 582.4 [M+Na ⁺ ].

Step 2: Synthesis of compound 5

Compound 4 (5.0 g, 8.93 mmol) was dissolved in 100 mL of dichloromethane, and compound 5 (2.6 g, 6.0 mmol), DMAP (1.1 g, 9.0 mmol), and EDCI (1.8 g, 9.0 mmol) were added and stirred at room temperature for 16 hours. Water was added to quench the mixture, and the organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered and concentrated to obtain compound 5 (4.1 g, light yellow oil) with a yield of 70%. ESI-MS m/z: 972.8 [M+H ⁺ ].

Step 3: Synthesis of compound 6

Compound 5 (4 g, 4.1 mmol) was added to 40 mL of dichloromethane, and then 40 mL of 4N hydrochloric acid/1,4-dioxane solution was added, stirred at room temperature for 3 hours, and the solvent was removed by rotary evaporation to obtain compound 6 (3.4 g, light yellow oil), with a yield of 95%. ESI-MS m/z: 872.7 [M+H ⁺ ].

Step 4: Synthesis of compound CP401

Compound 6 (1.00 g, 1.15 mmol), N,N-dimethylaminoacetic acid (179 mg, 1.73 mmol), EDCI (342 mg, 1.73 mmol) and DMAP (311 mg, 1.73 mmol) were added to 25 mL of dichloromethane and stirred overnight at room temperature. The reaction solution was washed with brine, the organic phase was concentrated, and column chromatography analysis gave compound CP401 (385 mg, yellow viscous solid) with a yield of 35%. ESI-MS m/z: 957.8 [M+H ⁺ ].

¹ HNMR (400MHz, CDCl ₃ -d)δ5.25(1H,dd,J＝7.2,6.9Hz),4.37-4.51(3H,t,J＝7.4Hz),4.20-4.32(2H,t,J＝7.1Hz),3.44-3.74(5H,m),2.93-3.05(2H,d,J＝7.1Hz),2 .56-2.68(2H,m),2.31-2.51(3H,m),2.02-2.19(7H,m),1.17-1.96(5 6H,m), 1.04(3H,d,J＝6.8Hz), 0.73-0.93(17H,m), δ0.60-0.72(2H,m).

Example 18 Synthesis of Compound CP402

Step 1: Synthesis of compound 3

Compound 1 (2.0 g, 4.11 mmol), N-hydroxysuccinimide (708 mg, 6.16 mmol), DMAP (752.5 g, 6.16 mmol), and EDCI (1.2 g, 6.16 mmol) were added to 200 mL of dichloromethane and reacted at room temperature for 15 hours. Cysteine (982 mg, 8.22 mmol) and triethylamine (1.2 mL, 8.22 mmol) were added and the reaction was continued at room temperature for 15 hours. Water was added to stop the reaction, and the organic phase was separated. The organic phase was washed with saturated brine and dried over anhydrous sodium sulfate, filtered, concentrated, and separated by column chromatography to obtain compound 3 (16.0 g, yellow oil) with a yield of 69%. ESI-MS m/z: 590.4 [M+H ⁺ ].

Step 2: Synthesis of compound 4

Compound 3 (20.0 g, 33.4 mmol), compound A1 (14.0 g, 33.4 mmol), tetrabutylammonium iodide (12.3 g, 33.4 mmol) and NaOH (2.7 g, 67.5 mmol) were added to 100 mL of ethanol and stirred at room temperature for 48 h. After the reaction was completed, 100 mL of water was added to quench the reaction, and the mixture was extracted with dichloromethane. The organic phases were combined, concentrated and separated by column chromatography to obtain compound 4 (20.2 g, yellow oil) with a yield of 65%.

Step 3: Synthesis of compound CP402

Compound 6 (1.00 g, 1.08 mmol), N,N-dimethylethanolamine (167 mg, 1.62 mmol), EDCI (320 mg, 1.62 mmol) and DMAP (198 mg, 1.62 mmol) were added to 15 mL of dichloromethane and stirred overnight at room temperature. After the reaction, the reaction solution was washed with brine, dried over anhydrous sodium sulfate, filtered, concentrated, and separated by silica gel column chromatography to obtain compound CP402 (272 mg, yellow viscous solid) with a yield of 25.2%. ESI-MS m/z: 999.8 [M+H ⁺ ], 1006.7 [M+Na ⁺ ].

Example 19 Synthesis of Compound CP501

Step 1: Synthesis of compound 3

Compound 1 (20.0 g, 41.1 mmol), N-hydroxysuccinimide (7.1 g, 61.6 mmol), DMAP (6.02 g, 49.3 mmol), EDCI (9.74 g, 49.3 mmol) were added to 200 mL of dichloromethane and reacted at room temperature for 15 hours. 1-tert-butyl L-glutamic acid (8.35 g, 41.1 mmol) and triethylamine (17 mL, 123 mmol) were added and the reaction was continued at room temperature for 15 hours. Water was added to stop the reaction, and the organic phase was separated. The organic phase was washed with saturated brine and dried over anhydrous sodium sulfate, filtered, concentrated, and separated by column chromatography to obtain compound 3 (22.6 g, yellow oil) with a yield of 82%. ESI-MS m/z: 672.5 [M+H ⁺ ].

Step 2: Synthesis of compound 4

Compound 3 (10.0 g, 14.9 mmol), compound A2 (8.0 g, 22.4 mmol), EDCI (4.4 g, 22.4 mmol) and DMAP (2.7 g, 22.4 mmol) were added to 200 mL of dichloromethane and stirred overnight at room temperature. The reaction solution was washed with brine, the organic phase was concentrated, and column chromatography analysis gave compound 4 (7.5 g, yellow viscous liquid) with a yield of 51%. ESI-MS m/z: 980.7 [M+H ⁺ ].

Step 3: Synthesis of compound 5

Compound 5 (7.00 g, 7.84 mmol) was added to 75 mL of dichloromethane, and then 75 mL of 4N hydrochloric acid/1,4-dioxane solution was added. The mixture was stirred at room temperature for 3 hours, and the solvent was removed by rotary evaporation to obtain Compound 5.

Step 4: Synthesis of compound CP501

Compound 5 (2.00 g, 3.93 mmol), N,N-dimethylethanolamine (609 mg, 5.90 mmol), EDCI (1.17 g, 5.9 mmol) and DMAP (7.21 mg, 5.9 mmol) were added to 15 mL of dichloromethane and stirred overnight at room temperature. The reaction solution was washed with brine, the organic phase was concentrated, and column chromatography analysis gave compound CP501 (860 mg, yellow viscous solid) with a yield of 21.5%. ESI-MS m/z: 1025.8 [M+H ⁺ ].

Example 20: Preparation and Characterization of mRNA-LNP

1. Materials and Instruments

Table 1 Main experimental consumables

Table 2 Main experimental equipment

Table 3 Other main reagents

2. Experimental Plan

Preparation of mRNA-LNPs

The lipid compound, phospholipid (DOPE) and PEG lipid (DMG-PEG) of the present invention are mixed in an ethanol solution at a molar ratio of 49.25:49.25:1.5, and the mRNA is diluted into a 25mM pH 4.0 sodium acetate buffer with a final concentration of 135ng/uL. The aqueous phase and the ethanol phase are mixed through a microfluidic device, and the mixed flow rate of the aqueous phase is 9mL/min and the ethanol phase is 3mL/min. The prepared encapsulation solution is diluted 40 times into a pH 7.525mM Tris 25mM sodium acetate buffer solution, and after ultrafiltration with a 30KDa ultrafiltration tube, 25% of the final volume of 435mg/mL sucrose 20mM Tris 10.7mM sodium acetate buffer solution is added, and the experimental sample is obtained after sterile filtration.

Characterization of mRNA-LNPs

The prepared mRNA-LNP experimental sample was diluted 50 times with buffer (final concentration was 2-100ng/μL), and the average particle size, PDI and ζ potential of the nanoparticles were detected using a Malvern particle size potential instrument; the average particle size and PDI used a ZEN0040 DLS sample cell with a sample volume of 200μL; the ζ potential was detected using a DTS1070 potential cell with a sample volume of 800μL. The mRNA content and encapsulation efficiency were detected using the Quant-iT ^TM RiboGreen RNA detection kit, the free mRNA content _Cfree was detected with TE buffer, and the total mRNA content _Ctotal was detected with 2% Triton buffer. The encapsulation efficiency was calculated according to the formula EE = (1- _Cfree / _Ctotal ) × 100%. The experimental results are shown in Table 4.

Table 4 Physicochemical parameters of GFPmRNA-LNP


*Note: For comparison with lipid compounds, the synthesis of each compound was performed according to the method provided in the corresponding patent examples, and the structural characterization showed that the compound structure was correctly synthesized.

The results show that the composition formed by the cationic lipid compound of the present invention, phospholipid (DOPE), PEG lipid (DMG-PEG) and mRNA has good physical and chemical parameters. The average particle size is in the range of 60-105nm, the PDI is less than 0.2, the zeta potential is between -10mV-10mV, and the LNP encapsulation rate of mRNA is higher than 88%.

Example 21: mRNA-LNP in vitro cell transfection activity

Fluorescence microscopy was used to detect the expression level of green fluorescent protein eGFP to evaluate the transfection activity of mRNA-LNP for HEK293T cells. 6.5×10 ⁵ cells/mL of HEK293T cell solution was inoculated into a 24-well cell culture plate at a volume of 1 mL/well. After 24 hours, each well was transfected with 500 ng of eGFP mRNA-LNP and the cell culture plate was placed in a 37°C, 5% CO ₂ cell culture incubator for culture. The negative control group was transfected with an equal volume of saline. After 24 hours, the microscope was used to take pictures and the results are shown in Figure 1. The results show that the three-component LNP composition formed by the cationic lipid compound of the present invention can achieve high expression of eGFP-mRNA in cells, and the expression level is better than that of the control group, and the expression level is better than that of other patented compounds (compound (3), CLinDMA, HGT4001, ICE).

Example 22: Animal Activity Evaluation of LNP Compositions

We used the new coronavirus S protein mRNA to evaluate the immune activity of mRNA-LNP in mice. The LNP formula used was an ionizable cationic lipid compound: DOPE: DMG-PEG2K = 49.25: 49.25: 1.5 molar ratio for mRNA encapsulation. The specific formula is shown in Table 4. Female BALB/c mice aged 6-8 weeks were randomly divided into 6/group and immunized by hind leg intramuscular injection. Immunization was performed on days 0 and 14, and the immunization dose was 5μg mRNA-LNP. Blood was collected and serum was separated on day 28. The specific antibody titer against the SARS-CoV2 virus S protein antigen was detected by ELISA. PBMC cells were collected and S protein-specific IFNγ-ELISPOT was performed. The antibody titer detection value GMT (95% CI) is shown in Figure 2. The results show that the mRNA vaccine composition formed by lipid nanoparticles provided by the present invention has higher immunogenicity than the control group. The ELISPOT data are shown in Figure 3. The mRNA vaccine composition formed by lipid nanoparticles provided by the present invention can induce higher cellular immunity.

Example 23: Safety Evaluation of mRNA-LNP

The effect of mRNA-LNP on the growth state of HEK293T cells was evaluated using the CCK-8 method. HEK293T cells were plated in a 96-well plate, 10 ⁴ cells were inoculated in each well, and 2 μg of mRNA-LNP was transfected after 24 hours (transfection volume 20 μL, culture medium volume 10%, final concentration of cationic lipid compound about 180 μM), 10% DMSO was selected as a positive control, PBS as a negative control, three wells were paralleled, and cultured for 24 hours at 37 ° C and 5% CO _2. After adding CCK-8 substrate and incubating for 2 hours, the absorbance value was detected by a microplate reader, and the relative cell survival rate was calculated. The experimental results are shown in Figure 4, and the results show that the compounds provided by the present invention have no effect on the proliferation of cells and have good safety.

Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims (20)

The lipid compound represented by formula (I):
or its stereoisomers, tautomers, and pharmaceutically acceptable salts; wherein, L ₁ , L ₂ and L ₃ are each independently selected at each occurrence from a bond, an optionally substituted C ₁ -C ₂₀ alkylene group, an optionally substituted C ₂ -C ₂₀ alkenylene group, an optionally substituted C ₂ -C ₂₀ alkynylene group, an optionally substituted C ₁ -C ₂₀ acyl group; _G1 , _G2 , and _G3 are each independently selected at each occurrence from a bond, -O-, -S-, -C(=O)-, -C(=O)O-, -OC(=O)-, -OC(=O)O-, -C(=O)N(R _a )-, -NR _a C(=O)-, -S(=O)O-, -OS(=O) _O- , -S(=O)2O-, -OS(=O) _2O- , -C(=O)S-, -C(=S)S-, -OP(=O)(OR _a )O-, -N(R _a )C(=O)O-, -OC(=O)N(R _a )-, -SS-, -OC(=O)S-, -SC(=O)O-, -N(R _a )C(=O)N(R _b )-; One of R ₁ , R ₂ and R ₃ is selected from optionally substituted C ₁ -C ₂₀ alkyl, optionally substituted C ₂ -C ₂₀ alkenyl, optionally substituted C ₂ -C ₂₀ alkynyl; wherein one or more -CH ₂ - in the C ₁ -C ₂₀ alkyl, C ₂ -C ₂₀ alkenyl, C ₂ -C ₂₀ alkynyl may be optionally replaced by -O-, -S-, -NR _a -, carbocyclyl, aryl, heteroaryl, and/or heterocyclyl; Meanwhile, the other of R ₁ , R ₂ and R ₃ is selected from a steroidal compound group; At the same time, the third of R ₁ , R ₂ and R ₃ is selected from hydrogen, C ₁ -C ₂₀ alkyl, -(R ₄ ) _q -NR _a R _b , -(R ₄ ) _q -nitrogen-containing heteroaryl, -(R ₄ ) _q -nitrogen-containing heterocyclic group, -(R ₄ ) _q -guanidinyl; wherein the nitrogen-containing heteroaryl, nitrogen-containing heterocyclic group and guanidinyl are optionally substituted by one or more groups selected from the following: C ₁ -C ₂₀ alkyl, C ₂ -C ₂₀ alkenyl, C ₂ -C ₂₀ alkynyl, C ₁ -C ₂₀ alkoxy, halogen, hydroxyl, thiol, cyano, nitro, amino, carboxyl, C ₁ -C ₂₀ acyl, C ₁ -C ₂₀ acyloxy; R ₄ is selected from C ₁ -C ₂₀ alkylene, C ₂ -C ₂₀ alkenylene, C ₂ -C ₂₀ alkynylene; _Ra and _Rb are each independently selected from H, optionally substituted _C1 - _C20 alkyl, optionally substituted _C2 - _C20 alkenyl, optionally substituted _C2 - _C20 alkynyl, optionally substituted carbocyclyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted heterocyclyl; m, n and p are each independently selected from 1, 2 or 3; q is selected from 0 or 1. The lipid compound according to claim 1 or its stereoisomers, tautomers, and pharmaceutically acceptable salts; wherein L ₁ is independently selected at each occurrence from a bond, an optionally substituted C ₁ -C ₂₀ alkylene group, an optionally substituted C ₂ -C ₂₀ alkenylene group, an optionally substituted C ₂ -C ₂₀ alkynylene group, an optionally substituted C ₁ -C ₂₀ acyl group; G1 is independently selected at _each occurrence from -C(＝O)O-, -OC(＝O)-, -OC(＝O)O-, -C(＝O)N(R _a )-, -NR _a C(＝O)-, -S(＝O)O-, -OS(＝O)O-, -S(＝O) ₂ O-, -OS(＝O) ₂ O-, -C(＝O)S-, -C(＝S)S-, -OP(＝O)(OR _a )O-, -N(R _a )C(＝O)O-, -OC(＝O)N(R _a )-, -SS-, -OC(＝O)S-, -SC(＝O)O-, -N(R _a )C(＝O)N(R _b )-; R ₁ is selected from optionally substituted C ₁ -C ₂₀ alkyl, optionally substituted C ₂ -C ₂₀ alkenyl, optionally substituted C ₂ -C ₂₀ alkynyl; wherein one or more -CH ₂ - in the C ₁ -C ₂₀ alkyl, C ₂ -C ₂₀ alkenyl, C ₂ -C ₂₀ alkynyl may be optionally replaced by O, S, -NR _a -, carbocyclyl, aryl, heteroaryl, and/or heterocyclyl; _L2 and _L3 are each independently selected at each occurrence from a bond, an optionally substituted _C1 - _C20 alkylene group, an optionally substituted _C2 - _C20 alkenylene group, an optionally substituted _C2 - _C20 alkynylene group, an optionally substituted _C1 - _C20 acyl group; _G2 and _G3 are each independently selected at each occurrence from a bond, -O-, -S-, -C(=O)-, -C(=O)O-, -OC(=O)-, -OC(=O)O-, -C(=O) _NRa- , -NRaC(=O)-, _-S (=O)O-, -OS(=O)O-, -S(=O) _2O- , -OS(=O) _2O- , -C(=O)S-, -C(=S)S-, -OP(= _O )(ORa)O-, -N( _Ra )C(=O)O-, -OC(=O) _NRa- , -SS-, -OC(=O)S-, -SC(=O)O-, _-NRaC (=O) _NRb- ; One of _R2 and _R3 is selected from a steroidal compound group, and the other is selected from hydrogen, _C1 - _C20 alkyl, -( _R4 ) _q - _NRaRb , _- ( _R4 ) _q -nitrogen-containing heteroaryl, -( _R4 ) _q -nitrogen-containing heterocyclic group, -( _R4 ) _q -guanidinyl; wherein the nitrogen-containing heteroaryl, nitrogen-containing heterocyclic group and guanidinyl are optionally substituted by one or more groups selected from the following: _C1 - _C20 alkyl, _C2 - _C20 alkenyl, _C2 - _C20 alkynyl, _C1 - _C20 alkoxy, halogen, hydroxyl, thiol, cyano, nitro, amino, carboxyl, _C1 - _C20 acyl, _C1 - _C20 acyloxy; R ₄ is selected from C ₁ -C ₂₀ alkylene, C ₂ -C ₂₀ alkenylene, C ₂ -C ₂₀ alkynylene; _Ra and _Rb are each independently selected from H, optionally substituted _C1 - _C20 alkyl, optionally substituted _C2 - _C20 alkenyl, optionally substituted _C2 - _C20 alkynyl, optionally substituted carbocyclyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted heterocyclyl; m, n and p are each independently selected from 1, 2 or 3; q is selected from 0 or 1. The lipid compound according to claim 2 or its stereoisomers, tautomers, and pharmaceutically acceptable salts; wherein _L2 is independently selected at each occurrence from a bond, an optionally substituted C ₁ -C ₂₀ alkylene group, an optionally substituted C ₂ -C ₂₀ alkenylene group, an optionally substituted C ₂ -C ₂₀ alkynylene group, an optionally substituted C ₁ -C ₂₀ acyl group; G2 is independently selected at _each occurrence from -O-, -S-, -C(＝O)-, -C(＝O)O-, -OC(＝O)-, -OC(＝O)O-, -C(＝O) _NRa- , _-NRaC (＝O)-, -S(＝O)O-, -OS(＝O)O-, -S(＝O) _2O- , -OS(＝O) _2O- , -C(＝O)S-, -C(＝S)S-, -OP(＝ _O )(ORa)O-, -N( _Ra )C(＝O) _O- , -OC(＝O)NRa-, -SS-, -OC(＝O)S-, -SC(＝O)O-, _-NRaC (＝O) _NRb- ; _R2 is selected from the group consisting of steroidal compounds; _L3 is independently selected at each occurrence from a bond, an optionally substituted _C1 - _C20 alkylene group, an optionally substituted _C2 - _C20 alkenylene group, an optionally substituted _C2 - _C20 alkynylene group, an optionally substituted _C1 - _C20 acyl group; _G3 at each occurrence is independently selected from a bond, -O-, -S-, -C(＝O)-, -C(＝O)O-, -OC(＝O)-, -OC(＝O)O-, -C(＝O) _NRa- , _-NRaC (＝O)-, -S(＝O)O-, -OS(＝O)O-, -S(＝O) _2O- , -OS(＝O) _2O- , -C(＝O)S-, -C(＝S)S-, -OP(＝ _O )(ORa)O-, -N( _Ra )C(＝O)O-, -OC(＝O) _NRa- , -SS-, -OC(＝O)S-, -SC(＝O)O-, _-NRaC (＝O) _NRb- ; _R3 is selected from hydrogen, _C1 - _C20 alkyl, -( _R4 ) _q - _NRaRb , -( _R4 ) _q - _nitrogen -containing heteroaryl, -( _R4 ) _q -nitrogen-containing heterocyclic group, -( _R4 ) _q -guanidinyl; wherein the nitrogen-containing heteroaryl, nitrogen-containing heterocyclic group and guanidinyl are optionally substituted by one or more groups selected from the following: _C1 - _C20 alkyl, _C2 - _C20 alkenyl, _C2 - _C20 alkynyl, _C1 - _C20 alkoxy, halogen, hydroxyl, thiol, cyano, nitro, amino, carboxyl, _C1 - _C20 acyl, _C1 - _C20 acyloxy; or _L2 is independently selected at each occurrence from a bond, an optionally substituted C ₁ -C ₂₀ alkylene group, an optionally substituted C ₂ -C ₂₀ alkenylene group, an optionally substituted C ₂ -C ₂₀ alkynylene group, an optionally substituted C ₁ -C ₂₀ acyl group; _G2 is independently selected at each occurrence from a bond, -O-, -S-, -C(=O)-, -C(=O)O-, -OC(=O)-, -OC(=O)O-, -C(=O) _NRa- , _-NRaC (=O)-, -S(=O)O-, -OS(=O)O-, -S(=O) _2O- , -OS(=O) _2O- , -C(=O)S-, -C(=S)S-, -OP(= _O )(ORa)O-, -N( _Ra )C(=O)O-, -OC(=O) _NRa- , -SS-, -OC(=O)S-, -SC(=O)O-, _-NRaC (=O) _NRb- ; _R2 is selected from hydrogen, _C1 - _C20 alkyl, -( _R4 ) _q - _NRaRb , -( _R4 ) _q - _nitrogen -containing heteroaryl, -( _R4 ) _q -nitrogen-containing heterocyclic group, -( _R4 ) _q -guanidinyl; wherein the nitrogen-containing heteroaryl, nitrogen-containing heterocyclic group and guanidinyl are optionally substituted by one or more groups selected from the following: _C1 - _C20 alkyl, _C2 - _C20 alkenyl, _C2 - _C20 alkynyl, _C1 - _C20 alkoxy, halogen, hydroxyl, thiol, cyano, nitro, amino, carboxyl, _C1 - _C20 acyl, _C1 - _C20 acyloxy; _L3 is independently selected at each occurrence from a bond, an optionally substituted _C1 - _C20 alkylene group, an optionally substituted _C2 - _C20 alkenylene group, an optionally substituted _C2 - _C20 alkynylene group, an optionally substituted _C1 - _C20 acyl group; G3 is independently selected at _each occurrence from -O-, -S-, -C(＝O)-, -C(＝O)O-, -OC(＝O)-, -OC(＝O)O-, -C(＝O) _NRa- , _-NRaC (＝O)-, -S(＝O)O-, -OS(＝O)O-, -S(＝O) _2O- , -OS(＝O) _2O- , -C(＝O)S-, -C(＝S)S-, -OP(＝ _O )(ORa)O-, -N( _Ra )C(＝O) _O- , -OC(＝O)NRa-, -SS-, -OC(＝O)S-, -SC(＝O)O-, _-NRaC (＝O) _NRb- ; _R3 is selected from the group consisting of steroidal compounds; R ₄ is selected from C ₁ -C ₂₀ alkylene, C ₂ -C ₂₀ alkenylene, C ₂ -C ₂₀ alkynylene; _Ra and _Rb are each independently selected from H, optionally substituted _C1 - _C20 alkyl, optionally substituted _C2 - _C20 alkenyl, optionally substituted _C2 - _C20 alkynyl, optionally substituted carbocyclyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted heterocyclyl; m, n and p are each independently selected from 1, 2 or 3; q is selected from 0 or 1. A compound according to any one of the preceding claims or a stereoisomer, a tautomer, and a pharmaceutically acceptable salt thereof; Wherein, the steroidal compound in the steroidal compound group is selected from naturally occurring steroidal compounds or their analogs; preferably, it includes plant sterols and animal sterols, or their analogs; more preferably, it is selected from cholesterol and its derivatives. A compound according to any one of the preceding claims or a stereoisomer, a tautomer, and a pharmaceutically acceptable salt thereof; Wherein, the steroidal compound group has the following structure:
_R5 is selected from hydrogen, _C1 - _C20 alkyl, _C2 - _C20 alkenyl, _C2 - _C20 alkynyl, _C1 - _C20 alkoxycarbonyl- _C1 - _C20 alkyl-; _R6 is selected from hydrogen, halogen, cyano, hydroxy, amino, oxo, _C1 - _C20 alkyl, _C2 - _C20 alkenyl, _C2 - _C20 alkynyl; m is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; Preferably, the steroid group is selected from:

wherein R' is a C _1-20 alkyl group. A compound according to any one of the preceding claims, selected from
or its stereoisomers, tautomers, and pharmaceutically acceptable salts, wherein _L1 , _L2 , _L3 , _G1 , _G2 , _G3 , _R1 , _R2 , _R3 , m, n, p are as defined in any of the preceding claims. A compound according to any one of the preceding claims, selected from:
or its stereoisomers, tautomers, and pharmaceutically acceptable salts, wherein X is selected from O, S, NH; L ₁ , L ₂ , L ₃ , G ₁ , G ₂ , G ₃ , R ₁ , R ₂ , R ₃ are as defined in the preceding claims. A compound according to any one of the preceding claims, selected from:
or its stereoisomers, tautomers, and pharmaceutically acceptable salts, wherein X is selected from O, S, NH; L ₁ , L ₂ , L ₃ , G ₁ , G ₂ , G ₃ , R ₁ , R ₂ , R ₃ are as defined in the preceding claims. A compound according to any one of the preceding claims or a stereoisomer, a tautomer, and a pharmaceutically acceptable salt thereof; wherein Each occurrence of _L1 is independently selected from optionally substituted _C1 - _C20 alkylene, optionally substituted _C2 - _C20 acyl; preferably, _L1 is selected from optionally substituted _C1 - _C6 alkylene, optionally substituted _C2 - _C6 acyl; G1 is independently selected at _each occurrence from -C(＝O)O-, -OC(＝O)-, -OC(＝O)O-, -C(＝O)N(R a )-, -NR a C(＝ _O )- _, -S(＝O)O-, -OS(＝O)O-, -S(＝O) 2 O-, -OS(＝O) 2 O-, -C(＝O)S-, -C(＝S)S-, -OP(＝O)(OR _a )O-, -N(R a )C(＝O)O-, -OC(＝O)N(R _a )-, -SS-, -OC(＝O)S-, -SC(＝O)O-, -N(R _{a )} C(＝O)N(R b )-; preferably, G1 is selected from -C(＝O)O-, -OC(＝ _O )-, -OC(＝O)O-, -C(＝O)N(R a )-, -NR a C(＝ _O )-, -S(＝O)O-, -OS(＝O)O-, -S(＝O) 2 O-, -C(＝O)S-, -C(＝ _S )S-, -OP(＝O)(OR a )O-, -N(R _a ) _C (＝O)O-, -OC(＝O)N(R a )-, -SS-, -OC(＝O)S-, -SC(＝O)O-, -N(R a )C(＝O)N(R b ) _-. )-, -NR _a C(═O)-, -N(R _a )C(═O)O-, -OC(═O)N(R _a )-; more preferably, G ₁ is selected from -C(═O)O-, -OC(═O)-; _Ra and _Rb are each independently selected from H, _C1 - _C20 alkyl, _C2 - _C20 alkenyl, _C2 - _C20 alkynyl, carbocyclyl, aryl, heteroaryl, heterocyclyl; preferably, Ra and _Rb are each independently selected from H, _C1 - _C20 alkyl, _C2 - _C20 alkenyl, _C2 - _C20 alkynyl; more preferably, _Ra and _{Rb are} each independently selected from H, _C1 - _C20 alkyl; R ₁ is selected from optionally substituted C ₁ -C ₂₀ alkyl groups; wherein one or more -CH ₂ - in the C ₁ -C ₂₀ alkyl groups may be optionally replaced by O, S, or a carbocyclic group. A compound according to any one of the preceding claims or a stereoisomer, a tautomer, and a pharmaceutically acceptable salt thereof; wherein _L1 is independently selected at each occurrence from optionally substituted methylene, ethylene, propylene, butylene, pentylene, hexylene, acetyl, propionyl, butyryl, pentanoyl, hexanoyl; G1 is independently selected at _each occurrence from -C(＝O)O-, -OC(＝O)-, -OC(＝O)O-, -C(＝O)N(R _a )-, -NR _a C(＝O)-, -S(＝O)O-, -OS(＝O)O-, -S(＝O) ₂ O-, -OS(＝O) ₂ O-, -C(＝O)S-, -C(＝S)S-, -OP(＝O)(OR _a )O-, -N(R _a )C(＝O)O-, -OC(＝O)N(R _a )-, -SS-, -OC(＝O)S-, -SC(＝O)O-, -N(R _a )C(＝O)N(R _b )-; R _a and R _b are each independently selected from H, C ₁ -C ₆ alkyl; R ₁ is selected from A compound according to any one of the preceding claims or a stereoisomer, a tautomer, and a pharmaceutically acceptable salt thereof; wherein _L2 and _L3 are each independently selected from a bond, an optionally substituted _C1 - _C20 alkylene group, an optionally substituted _C2 - _C20 acyl group at each occurrence; _G2 and _G3 are each independently selected at each occurrence from a bond, -O-, -S-, -C(=O)-, -C(=O)O-, -OC(=O)-, -OC(=O)O-, -C(=O) _NRa- , -NRaC(=O)-, _-S (=O)O-, -OS(=O)O-, -S(=O) _2O- , -OS(=O) _2O- , -C(=O)S-, -C(=S)S-, -OP(= _O )(ORa)O-, -N( _Ra )C(=O)O-, -OC(=O) _NRa- , -SS-, -OC(=O)S-, -SC(=O)O-, _-NRaC (=O) _NRb- ; One of R ₂ and R ₃ is selected from a cholesterol group, and the other is selected from hydrogen, a C ₁ -C ₂₀ alkyl group, -(R ₄ ) _q -NR _a R _b , -(R ₄ ) _q -5 or 6-membered nitrogen-containing heteroaryl group, -(R ₄ ) _q -5 or 6-membered nitrogen-containing heterocyclic group, -(R ₄ ) _q -guanidinyl group; wherein the 5- or 6-membered nitrogen-containing heteroaryl group, the 5- or 6-membered nitrogen-containing heterocyclic group and the guanidinyl group are optionally substituted by one or more groups selected from the following: C ₁ -C ₆ alkyl group, C ₂ -C ₆ alkenyl group, C ₂ -C ₆ alkynyl group, C ₁ -C ₆ alkoxy group, halogen, hydroxyl group, thiol group, cyano group, nitro group, amino group, C ₁ -C ₆ acyl group, C ₁ -C ₆ acyloxy group; R ₄ is selected from C ₁ -C ₆ alkylene; R _a and R _b are each independently selected from H, C ₁ -C ₆ alkyl; q is selected from 0 or 1. A compound according to any one of the preceding claims or a stereoisomer, a tautomer, and a pharmaceutically acceptable salt thereof; wherein _L2 and _L3 are each independently selected from a bond, an optionally substituted _C1 - _C6 alkylene group, an optionally substituted _C2 - _C6 acyl group; _G2 and _G3 are each independently selected at each occurrence from a bond, -O-, -S-, -C(=O)-, -C(=O)O-, -OC(=O)-, -OC(=O)O-, -C(=O) _NRa- , -NRaC(=O)-, _-S (=O)O-, -OS(=O)O-, -S(=O) _2O- , -OS(=O) _2O- , -C(=O)S-, -C(=S)S-, -OP(= _O )(ORa)O-, -N( _Ra )C(=O)O-, -OC(=O) _NRa- , -SS-, -OC(=O)S-, -SC(=O)O-, _-NRaC (=O) _NRb- ; One of R ₂ and R ₃ is selected from a cholesterol group, and the other is selected from hydrogen, a C ₁ -C ₂₀ alkyl group, -(R ₄ ) _q -NR _a R _b , -(R ₄ ) _q -5 or 6-membered nitrogen-containing heteroaryl group, -(R ₄ ) _q -5 or 6-membered nitrogen-containing heterocyclic group, -(R ₄ ) _q -guanidinyl group; wherein the 5- or 6-membered nitrogen-containing heteroaryl group, the 5- or 6-membered nitrogen-containing heterocyclic group and the guanidinyl group are optionally substituted by a C ₁ -C ₆ alkyl group; R ₄ is selected from C ₁ -C ₆ alkylene; R _a and R _b are each independently selected from H, C ₁ -C ₆ alkyl; q is selected from 0 or 1. The compound according to claim 1, selected from:


or its stereoisomers, tautomers, and pharmaceutically acceptable salts. A lipid nanoparticle comprising the lipid compound according to any one of claims 1 to 13 or its stereoisomers, tautomers, and pharmaceutically acceptable salts thereof. The lipid nanoparticle according to claim 14, further comprising phospholipids and/or polyethylene glycol lipids. The lipid nanoparticle according to claim 15, further comprising a therapeutic agent and/or a preventive agent. According to the lipid nanoparticles according to claim 16, the therapeutic agent and/or preventive agent comprises one or more nucleic acids, such as DNA, RNA, etc. A pharmaceutical composition comprising the lipid nanoparticles according to any one of claims 14 to 17 and a pharmaceutically acceptable carrier. A method for treating and/or preventing a disease, comprising administering a therapeutically effective amount of the lipid nanoparticle according to any one of claims 14-147 or the pharmaceutical composition according to claim 18 to an individual in need thereof. Use of the compound according to any one of claims 1 to 13 and/or the lipid nanoparticles according to claims 14 to 17 in the preparation of a therapeutic and/or preventive agent delivery system.