Ionizable 2-amino-1,3-propanediol-based cationic lipid compounds, compositions comprising the same, and uses thereof

Abstract

The application discloses an ionizable 2-amino-1,3-propanediol cationic lipid compound, a composition containing the same and application. Specifically, the application discloses an ionizable 2-amino-1,3-propanediol cationic lipid compound shown in formula (I-A), (I-B), (II-A) or (II-B), or a pharmaceutically acceptable salt or stereoisomer thereof, the ionizable 2-amino-1,3-propanediol cationic lipid compound has good biocompatibility and high delivery efficiency, provides more choices for delivery of nucleic acid drugs, small molecule drugs, polypeptide and protein drugs and the like, and has important significance for development and application of nucleic acid therapeutic drugs and vaccines and the like preventive agents.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to an ionizable 2-amino-1,3-propanediol cationic lipid compound, compositions containing the same, and their applications. Background Technology

[0002] Gene therapy is a novel treatment method that transfers exogenous genetic material into target cells to treat specific diseases, aiming to achieve a "root cause cure" and possessing enormous potential. It offers new options and directions for diseases that are currently untreatable by traditional therapies. Within gene therapy, nucleic acid drug therapy has become a hot topic in innovative medical and pharmaceutical research. Nucleic acid drugs and vaccines include four main classes: antisense oligonucleotides (ASO), small interfering RNA (siRNA), RNA aptamers, and microRNAs (miRNA), as well as nucleic acid mRNA vaccines. In 1998, the world's first nucleic acid drug, ASO fomivirsen sodium, was approved. In 2004, the first RNA aptamer drug, pegaptanib, was approved. In 2018, the first siRNA drug, Patisiran, was approved for marketing. In 2020, Biotech and Moderna launched two mRNA vaccines, Tozinameran and Elasomeran, respectively. In addition, many drugs and vaccines are in various stages of preclinical and clinical trials.

[0003] Nucleic acid drugs must enter the cytoplasm or nucleus to exert their effects. However, nucleic acid molecules are large, highly hydrophilic, and contain a large number of phosphate groups in their molecular chains. Under normal physiological pH conditions, they carry a negative charge, making it difficult for them to penetrate the cell membrane and enter the cell. Therefore, the development of nucleic acid drug delivery carriers and related delivery technologies is a crucial foundation for the clinical application of nucleic acid drugs.

[0004] An ideal nucleic acid drug carrier should possess characteristics such as high nucleic acid drug loading efficiency, protection of nucleic acid stability in blood circulation, tissue-targeted accumulation ability, and efficient cellular uptake, transfection, and drug release; the manufacturing process for mass production of the carrier is also a key factor. The development and improvement of carrier materials and delivery systems are directly related to the progress of nucleic acid drug development. Summary of the Invention

[0005] Based on the material basis of biological membrane structure, the purpose of this invention is to provide an ionizable 2-amino-1,3-propanediol cationic lipid compound for delivering therapeutic drugs or preventive agents, thereby enriching the types of ionizable lipid compounds and providing more options for the efficient and safe delivery of nucleic acid drugs, small molecule drugs, gene vaccines, peptide or protein drugs, which is of great significance for the development and application of nucleic acid drugs.

[0006] To solve the above-mentioned technical problems, the present invention adopts the following technical solution:

[0007] A first aspect of the invention provides ionizable lipid compounds of formula (IA), (IB), (II-A), or (II-B), or pharmaceutically available salts or stereoisomers thereof.

[0008]

[0009] in:

[0010] L1 is selected from: optionally substituted linear C1-C 20 Alkylene, optionally substituted C2-C 20 alkenyl, optionally substituted C2-C 20 Ethyne group;

[0011] L2 is selected from: -CH2CH2-, -CH=CH-;

[0012] X1 and X2 do not exist independently, or they are each independently selected from -CH2-, O, S, and NH;

[0013] Y is selected from N or CH;

[0014] R1 and R2 are each independently selected from H, or optionally substituted with the following groups: C1-C 12 Straight-chain or branched alkyl groups, C2-C 12 alkenyl, C2-C 12 alkynyl group, C3-C 10 cycloalkyl, -CH2-C3-C 10 Cycloalkyl, -(CH2) n -OH, C3-C 10 Cycloalkenyl, C6-C 12 Aryl or 5-12-membered heteroaryl, where n is an integer selected from 1-10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10); or R1, R2 together with the Y or N atom to which they are attached form an optionally substituted 3-10-membered heterocycle or an optionally substituted C3-C. 10 The carbon ring; the heterocycle or heteroaryl group contains 1-4 heteroatoms selected from N, O, and S (e.g., 2 or 3 types);

[0015] R3 is selected from: C3-C 30 Straight-chain or branched alkyl groups, C3-C 30 alkenyl or C3-C 30 alkynyl group;

[0016] R4 is selected from: C3-C 30 Straight-chain or branched alkyl groups, C3-C 30 alkenyl or C3-C 30 alkynyl group;

[0017] R5 is selected from H or

[0018] The substitution refers to being replaced by one or more substituents selected from the following: hydroxyl, mercapto, amino, halogen, cyano, C6-C. 10 Aryl, 5-10 heteroaryl, C1-C6 alkyl (e.g., C1-C4 alkyl), C1-C6 alkoxy (e.g., C1-C4 alkyl), hydroxy C1-C6 alkyl (e.g., C1-C4 alkyl), C2-C6 alkenyl (e.g., C2-C4 alkenyl), C2-C6 ynyl (e.g., C2-C4 ynyl), C3-C8 cycloalkyl (e.g., C3-C7 cycloalkyl), C3-C8 cycloalkenyl (e.g., C3-C7 cycloalkenyl).

[0019] In some embodiments, in formulas (IA), (IB), (II-A), (II-B): L1 is selected from: optionally substituted C1-C 12 Straight-chain or branched alkylene, optionally substituted C2-C 12 alkenyl, optionally substituted C2-C 12 Alynyl group; preferably, L1 is selected from: optionally substituted C1-C 10 Straight-chain or branched alkylene, optionally substituted C2-C 10 alkenyl, optionally substituted C2-C 10 Alynyl group; the definition of the substitution is as described above; more preferably, L1 is C1-C 10 Straight-chain alkylene compounds, such as C2-C8 straight-chain alkylene compounds and C3 straight-chain alkylene compounds.

[0020] In some embodiments, in formulas (IA), (IB), (II-A), (II-B): X1 and X2 are independently absent or are independently selected from -CH2-, O, and NH; preferably, X1 is absent or is selected from -CH2-, O, and NH, and X2 is absent or is -CH2-.

[0021] In some implementations, in formulas (IA), (IB), (II-A), (II-B): Y is selected from N or CH;

[0022] R1 and R2 are each independently selected from H, or optionally substituted with the following groups: straight-chain or branched C1-C8 alkyl (e.g., straight-chain or branched C1-C6 alkyl), C2-C8 alkenyl (e.g., C2-C6 alkenyl), C2-C8 ynyl (e.g., C2-C6 ynyl), C3-C8 cycloalkyl, -CH2-(C3-C8 cycloalkyl), -(CH2) n -OH, C3-C8 cycloalkenyl, C6-C 10 The aryl or 5-6 membered heteroaryl, or R1, R2 together with the Y or N atom to which they are attached, form an optionally substituted 3-8 membered N-containing heterocycle; wherein, n, the definition of the substitution is as described above.

[0023] Furthermore, Y is selected from N or CH;

[0024] R1 and R2 are each independently selected from H, methyl, ethyl, isobutyl, n-hexyl, 3-methylpentyl, or R1 and R2 together with the Y or N atom they are attached to form a structure selected from the following:

[0025] In some implementations, in formulas (IA), (IB), (II-A), (II-B): R3 is selected from: C3-C 24 Straight-chain or branched alkyl groups, C3-C 24 alkenyl or C3-C 24 Alkyne group; preferably, R3 is selected from: C6-C 15 Straight-chain or branched alkyl groups, C6-C 15 alkenyl or C6-C 15 Alkyne group; preferably, R3 is C6-C 15 Straight-chain alkyl groups.

[0026] In some implementations, in formulas (IA), (IB), (II-A), (II-B): R4 is selected from: C3-C 24 Straight-chain or branched alkyl groups, C3-C 24 alkenyl or C3-C 24 Alkyne group; preferably, R4 is selected from: straight-chain or branched C6-C 20 Alkyl, C6-C 20 alkenyl or C6-C 20 Alkyne group; preferably, R4 is C6-C 20 Straight-chain alkyl groups.

[0027] In some implementations, in formulas (IA), (IB), (II-A), (II-B): R5 is selected from H, or R4 and X1 are defined as described above.

[0028] In some embodiments, the compounds represented by formulas (IA), (IB), (II-A), and (II-B) are selected from the following structures:

[0029]

[0030] The definitions of L1, L2, X1, X2, Y, R1, R2, R3, R4, and R5 are as described above.

[0031] In some embodiments, the compound represented by formula (IA) is selected from the following structures:

[0032]

[0033] The definitions of R1, R2, R4, and L1 are as described above;

[0034] In some implementations, in formula (IA-1) or formula (IA-2):

[0035] R1 and R2 are each independently selected from H and optionally substituted C1-C 12 Straight-chain or branched alkyl; preferably, R1 and R2 are each independently selected from H, or optionally substituted C1-C8 straight-chain or branched alkyl; more preferably, R1 and R2 are each independently selected from H, or optionally substituted C1-C6 straight-chain or branched alkyl.

[0036] R4 is an optional substituted C3-C 24 Straight-chain alkyl or optionally substituted C3-C 24 Alkenyl; preferably optionally substituted C6-C 20 Straight-chain alkyl or optionally substituted C6-C 20 Alkenyl group, more preferably C6-C 20 Straight-chain alkyl;

[0037] L1 is an optional substituted C1-C 12 Straight-chain alkylene or optionally substituted C2-C 12 Alkenyl group; preferably C2-C8 straight-chain alkylene group or optionally substituted C2-C8 alkenyl group; more preferably C2-C8 straight-chain alkylene group;

[0038] The substitution refers to being replaced by one or more groups selected from the following groups: C1-C4 alkoxy, halogen, C1-C4 alkyl, C2-C4 alkenyl, hydroxyl, and C3-C7 cycloalkyl.

[0039] The term "optional substitution" refers to the possibility of substitution or non-substitution; for example, optional substituted alkyl groups include substituted alkyl groups and unsubstituted alkyl groups.

[0040] Specifically, when the groups are “substituted”, they can be substituted by any suitable one or more (e.g., 1, 2, 3 or 4) substituents.

[0041] In this invention, "halogen" refers to fluorine, chlorine, bromine, and iodine.

[0042] In this invention, "the salt in which the drug is available" refers to an acid addition salt or a base addition salt.

[0043] The acid addition salt refers to a salt prepared by adding an acid to a free base compound. The acid includes inorganic and organic acids, examples of which include, but are not limited to, hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, acetic acid, hydrobromic acid, adipic acid, alginic acid, ascorbic acid, aspartic acid, benzenesulfonic acid, benzoic acid, decanoic acid, hexanoic acid, caprylic acid, carbonic acid, cinnamic acid, citric acid, cycloamic acid, dodecyl sulfate, formic acid, fumaric acid, galactobionic acid, gluconic acid, glucuronic acid, glutamic acid, glutamate, glycerophosphate, glycolic acid, hippuric acid, isobutyric acid, lactic acid, lactobionic acid, lauric acid, maleic acid, malic acid, malonic acid, methanesulfonic acid, nicotinic acid, oleic acid, orotic acid, oxalic acid, palmitic acid, palmitic acid, propionic acid, pyroglutamic acid, salicylic acid, stearic acid, succinic acid, tartaric acid, p-toluenesulfonic acid, and trifluoroacetic acid.

[0044] The base addition salt refers to a salt prepared by the addition of an inorganic or organic base. Salts derived from inorganic bases include, but are not limited to, sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, and aluminum salts. The organic base includes, but is not limited to, ammonia, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, diethanolamine, ethanolamine, dealcoholized, lysine, arginine, histidine, caffeine, procaine, hydrazine, choline, betaine, ethylenediamine, glucosamine, methylglucosamine, theobromine, triethanolamine, purine, piperazine, piperidine, and N-ethylpiperidine. Preferably, the organic base is isopropylamine, diethylamine, ethanolamine, or trimethylamine.

[0045] In this invention, "stereoisomer" refers to isomers produced by different spatial arrangements of atoms in a molecule, including cis-trans isomers, enantiomers, and diastereomers.

[0046] In this invention, all features or conditions defined in the form of numerical ranges are for simplicity and convenience only. Accordingly, the description of numerical ranges should be considered as covering and specifically disclosing all possible secondary ranges and individual values ​​within those ranges, particularly integer values. For example, the description of the range "1-6" should be considered as specifically disclosing all secondary ranges such as 1 to 6, 2 to 6, 2 to 5, 3 to 5, 4 to 6, 3 to 6, etc., particularly secondary ranges defined by all integer values, and should be considered as specifically disclosing individual values ​​within those ranges such as 1, 2, 3, 4, 5, 6, etc. Unless otherwise specified, the foregoing interpretation applies to all content throughout this invention, regardless of its scope.

[0047] According to some embodiments, the ionizable lipid compound is one or more selected from compounds with the following structures:

[0048]

[0049]

[0050]

[0051]

[0052] A second aspect of the present invention provides a composition comprising:

[0053] Treatment drugs and / or preventative agents, and

[0054] Carrier material used to deliver the therapeutic or preventative agent;

[0055] The carrier material includes ionizable lipids, which include one or more of the ionizable lipid compounds of formula (IA), (IB), (II-A), or (II-B) or their pharmaceutically available salts or stereoisomers.

[0056] In some embodiments, the therapeutic agent and / or preventive agent are encapsulated within or associated with a carrier material.

[0057] In some embodiments, the therapeutic drugs and preventive agents have therapeutic and preventive effects on diseases, respectively, and include one or more of nucleic acids, small molecule compounds, polypeptides, and proteins.

[0058] In some embodiments, the nucleic acid includes any form of nucleic acid molecule, including but not limited to single-stranded DNA, double-stranded DNA, short isomers, agomir, antagomir, antisense molecules, small interfering RNA (siRNA), asymmetric interfering RNA (aiRNA), microRNA (miRNA), Dicer-substrate RNA (dsiRNA), small hairpin RNA (shRNA), transfer RNA (tRNA), messenger RNA (mRNA), and other forms of RNA molecules known in the art.

[0059] In some embodiments, the ionizable lipids also include one or more other ionizable lipid compounds.

[0060] Specifically, the other ionizable lipid compounds refer to other charged lipid compounds, whether disclosed or not.

[0061] Specifically, the charged lipid compound refers to any lipid molecule that exists in a positively or negatively charged form within a selected pH value or range. The selected pH value or range corresponds to the pH conditions of the intended use environment of the lipid, such as physiological pH.

[0062] More specifically, the charged lipid compounds include, but are not limited to: 1,2-dilinoleoyloxy-N,N-dimethylaminopropane (DLinDMA), 2,2-dilinoleoyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), heptatriaconta-6,9,28,31-tetraen-19-yl-4-(dimethylamino)butyrate (DLin-MC3-DMA), 2,2-dilinoleoyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA), 1,2-dioloxy-N,N-dimethyl... Aminopropane (DODMA), N-[1-(2,3-diolenoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA), N-[1-(2,3-diolenoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTAP), 1,2-dioleoyl-sm-glycero-3-ethylcholine phosphate (DOEPC), 1,2-dilauroyl-sn-glycero-3-ethylcholine phosphate (DLEPC), 1,2-dimyristoyl-sn-glycero-3-ethylcholine phosphate (DMEPC), 1,2-dimyristoyl-sn-glycero-3-ethylcholine phosphate (14) ∶1(9Z)), N1-[2-((1S)-1-[(3-aminopropyl)amino]-4-[di(3-aminopropyl)amino]butylcarbamoyl)ethyl]-3,4-di[oleyloxy]-benzamide (MVL5), bis(octadecylamino)glycylspermine (DOGS), 3b-[N-(N′,N′-dimethylaminoethyl)carbamoyl]cholesterol (DC-Chol), bis(octadecyl)dimethylammonium bromide (DDAB), SAINT-2, N-methyl-4-(dioleyl)methylpyridine, 1,2-dimyristyloxypropyl-3-dimethylhydroxyethylammonium bromide (DMRIE) 1,2-Dioleoyl-3-dimethyl-hydroxyethyl ammonium bromide (DORIE), 1,2-dioleoyloxypropyl-3-dimethyl-hydroxyethyl ammonium chloride (DORI), di-alkylated amino acid (DILA2), diolenoyl dimethyl ammonium chloride (DODAC), 3-(docoacylamino)-N1,N1,4-tridodecyl-1-piperazine ethylamine (KL10), N1-[2-(docoacylamino)ethyl]-N1,N4,N4-tridodecyl-1,4-piperazine diene amide (KL22), 14,25-docoacyl-15,18,21,24-tetraazaoctaporane (KL25).

[0063] According to some embodiments, the carrier material also includes neutral lipids.

[0064] Specifically, the neutral lipid compound is any lipid molecule, whether disclosed or undisclosed, present in an uncharged or neutral zwitterionic form within a selected pH value or range. The selected useful pH value or range corresponds to the pH conditions of the environment in which the lipid is intended to be used, such as physiological pH.

[0065] More specifically, the neutral lipid is selected from one or more of phosphatidylcholine, phosphatidylethanolamine, ceramide, sphingomyelin and their derivatives.

[0066] More specifically, the neutral lipids include, but are not limited to, 1,2-distearyl-sn-glycerol-3-phosphate choline (DSPC), 1,2-dipalmitoyl-sn-glycerol-3-phosphate choline (DPPC), 1,2-dimyristoyl-sn-glycerol-3-phosphate choline (DMPC), 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphate choline (POPC), 1,2-dioleoyl-sn-glycerol-3-phosphate choline (DOPC), 1,2-dioleoyl-sn-glycerol-3-phosphate ethanolamine (DOPE), 2-(((2,3-bis(oleoyloxy)propyl))dimethylammonium phosphate)ethylhydrogen (DOCP), sphingosine (SP), ceramides and their derivatives, preferably 1,2-distearyl-sm-glycerol-3-phosphate choline (DSPC).

[0067] In some embodiments, the molar ratio of the ionizable lipid to the neutral lipid is 1 to 15:1, more preferably 2 to 10:1, and even more preferably 3 to 8:1, for example 5:1.

[0068] According to some embodiments, the carrier material further includes structural lipids.

[0069] Structural lipids can stabilize the amphiphilic structure of the delivery system. Specifically, the structural lipid is selected from one or more of cholesterol, sphingomyelin, sphingosine, sitosterol, α-tocopherol, and corticosteroids, preferably cholesterol.

[0070] In some embodiments, the molar ratio of the ionizable lipid to the structural lipid is 1 to 3:1, more preferably 1 to 2:1, for example 1.3:1.

[0071] According to some embodiments, the carrier material further includes polyethylene glycol conjugated lipids.

[0072] Specifically, the polyethylene glycol conjugated lipids mainly include publicly disclosed or undisclosed PEG-modified lipid compounds, which can improve the stability of liposomes and reduce protein absorption of liposomes, such as one or more of PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, and PEG-modified dialkylglycerol; wherein the molecular weight of the PEG used for modification is 600-12000 Da, preferably 2000-5000 Da.

[0073] More specifically, the polyethylene glycol conjugated lipid can be R-3-[(ω-methoxy-poly(ethylene glycol))carbamoyl]-1,2-dimyristyloxypropyl-3-amine (PEG-c-DOMG), 1-(monomethoxy-polyethylene glycol)-2,3-dimyristylglycerol (DMG-PEG), polyethylene glycol-dilauroylphosphatidylethanolamine (PEG-DLPE), polyethylene glycol-dimyristoylphosphatidylethanolamine (PEG-DMPE), polyethylene glycol-dipalmitoylphosphatidylcholine (PEG-DPPC), polyethylene glycol-distearateylphosphatidylethanolamine (PEG-DSPE), etc. The preferred formulations are amide-polyethylene glycol, cholesterol-polyethylene glycol (Chol-PEG), PEG-PE, 4-O-(2′,3′-di(tetradecanoyloxy)propyl-1-O-(ω-methoxy(polyethoxy)ethyl)succinate (PEG-S-DMG), PEG-ceramide, ω-methoxy(polyethoxy)ethyl-N-(2,3-di(tetradecanoyloxy)propyl)carbamate or 2,3-di(tetradecanoyloxy)propyl-N-(ω-methoxy)(polyethoxy)ethyl)carbamate, and DMG-PEG2000.

[0074] In some embodiments, the molar ratio of the ionizable lipid to the polyethylene glycol conjugated lipid is 30–80:1, preferably 30–60:1, more preferably 30–50:1, and even more preferably 30–40:1, for example 33:1.

[0075] In some embodiments, the mass ratio of the carrier material to the therapeutic drug and / or preventive agent is 8 to 40:1, preferably 10 to 35:1, more preferably 10 to 30:1, and even more preferably 12 to 18:1.

[0076] In some embodiments, the carrier material also includes neutral lipids, structural lipids, and polyethylene glycol conjugated lipids.

[0077] In some embodiments, the composition is a lipid nanoparticle formulation comprising:

[0078] Treatment drugs and / or preventative agents, and

[0079] Carrier materials for delivering the therapeutic drugs and / or preventive agents; preferably, the carrier materials include ionizable lipids, neutral lipids, structured lipids, and polyethylene glycol conjugated lipids;

[0080] Preferably, the ionizable lipid is an ionizable lipid compound of formula (IA), (IB), (II-A) or (II-B) or a salt or stereoisomer available for use with a drug thereof, the neutral lipid is 1,2-distearate-sn-glycerol-3-phosphocholine, the structural lipid is cholesterol, and the polyethylene glycol conjugated lipid is DMG-PEG2000;

[0081] Preferably, the molar ratio of the ionizable lipids, neutral lipids, structural lipids, and polyethylene glycol conjugated lipids is (30-60):(1-15):(20-40):1, more preferably (30-55):(1-12):(20-35):1, more preferably (30-40):(5-10):(20-30):1, for example 33.3:6.7:25.7:1;

[0082] The therapeutic drug or preventive agent is a nucleic acid molecule, preferably small interfering RNA; the mass ratio of the carrier material to the therapeutic drug or preventive agent is 8-40:1, preferably 10-35:1, more preferably 10-30:1, and even more preferably 12-18:1.

[0083] The average size of the nanoparticle formulation is 30nm to 300nm, preferably 50nm to 250nm, more preferably 80nm to 200nm, and even more preferably 80nm to 180nm. Preferably, the polydispersity index of the nanoparticle formulation is ≤0.4, more preferably ≤0.3, and even more preferably ≤0.25.

[0084] A third aspect of the present invention provides a method for preparing the above-described composition, wherein the composition is a lipid nanoparticle formulation, and the method comprises the following steps:

[0085] (1) Dissolve the ionizable lipids, neutral lipids, structural lipids and polyethylene glycol conjugated lipids in an organic solvent to prepare a lipid solution; dissolve the therapeutic drug or preventive agent (preferably a nucleic acid molecule, more preferably a small interfering RNA) in a buffer solution to obtain a therapeutic drug or preventive agent solution;

[0086] (2) The lipid solution and the therapeutic or preventive agent solution are mixed at a certain flow rate ratio using a microfluidic device to prepare lipid nanoparticles, and the resulting product is dialyzed in a buffer solution.

[0087] (3) Filter the product after dialysis in step (2) to obtain lipid nanoparticle formulation.

[0088] In some embodiments, in step (1), the organic solvent is one or more combinations selected from ethanol, diethyl ether, and acetone, preferably ethanol; the concentration of lipid in the lipid solution is 2 to 25 mg / mL.

[0089] In some embodiments, in step (1), the buffer solution is a citrate buffer with a pH of 4.0; and the concentration of the therapeutic drug or preventive agent in the therapeutic drug or preventive agent solution is 5 to 1000 μg / mL.

[0090] In some embodiments, in step (2), the flow rate ratio of the lipid solution to the therapeutic or preventative agent solution is 1:2 to 5, preferably 1:3.

[0091] In some embodiments, in step (2), dialysis is used to remove organic solvents. The dialysis method can be a molecular weight cutoff (MWCO) tube or dialysis bag with a molecular weight cutoff of 3-100 kDa and static dialysis. The buffer solution is selected from Tris buffer, citrate buffer, and phosphate buffer solution. The static dialysis time is 12-24 h.

[0092] In some embodiments, in step (3), the product is filtered through a sterile filter with a membrane pore size of 0.2 μm.

[0093] In a fourth aspect, the present invention provides an ionizable lipid compound of formula (IA), (IB), (II-A), or (II-B), or a pharmaceutically usable salt or stereoisomer thereof, or the use of the composition thereof in the preparation of a medicament; preferably, the medicament comprises a nucleic acid drug, a small molecule drug, a polypeptide or a protein drug; preferably, the nucleic acid drug comprises a gene vaccine or a gene editing drug.

[0094] Compared with the prior art, the present invention has the following advantages:

[0095] Based on the material basis of biological membrane structure, this invention provides a novel 2-amino-1,3-propanediol-based cationic lipid compound with a chemical structure resembling that of a cell membrane and exhibiting good biocompatibility. This invention offers more delivery options for nucleic acid drugs, small molecule drugs (peptides), and protein drugs, and is of great significance for the development and application of nucleic acid therapeutics and preventative agents such as vaccines. Detailed Implementation

[0096] The present invention will be further described in detail below through embodiments. However, this should not be construed as limiting the scope of the above-described subject matter of the present invention to the following embodiments. Various substitutions or modifications made based on ordinary technical knowledge and conventional methods in the art without departing from the above-described technical concept of the present invention should be included within the scope of the present invention.

[0097] In the specific embodiments of this invention, all raw materials used are commercially available.

[0098] Abbreviation Explanation

[0099]

[0100]

[0101] Example 1

[0102] The synthetic routes for compounds WO1 and WO2 are as follows:

[0103]

[0104] Step 1: Synthesis of compound W01-1

[0105] D-Sphingosine (2.0 g, 6.68 mmol) was added to DMF (10 mL) and THF (30 mL), stirred until dissolved, and then octanoic acid (0.96 g, 6.68 mmol) and HATU (2.79 g, 7.35 mmol) were added. The mixture was cooled to 0–10 °C. A solution of triethylamine (2.02 g, 20.04 mmol) in THF (5 mL) was slowly added dropwise, and the mixture was stirred overnight at room temperature. EA (60 mL) and water (60 mL) were added, and the mixture was separated. The organic phase was washed with saturated sodium chloride solution (60 mL). The solution was dried over anhydrous sodium sulfate, concentrated, and purified by column chromatography (PE:EA = 5:1–1:3) to give a white to off-white waxy solid compound (2 g, 72% yield). 1 H NMR (400MHz, CDCl3) δ: 5.89(1H), 5.57(1H), 5.52(1H), 5.38(2H), 4.47(1H), 4.36(1H), 3.74 (2H), 2.19(2H), 2.01(2H), 1.57(2H), 1.36(2H), 1.28(4H), 1.27(4H), 1.26(20H), 0.90(6H).

[0106] Step 2: Synthesis of compound W01

[0107] 4-Dimethylaminobutyrate (158 mg, 0.941 mmol), EDCI (362 mg, 1.882 mmol), and DMAP (253 g, 2.07 mmol) were dissolved in DCM (4 mL) and stirred at room temperature for half an hour. In another reaction flask, WO1-1 (400 mg, 0.941 mmol) was dissolved in DCM (4 mL). The 4-dimethylaminobutyrate solution was added dropwise to the WO1-1 solution while cooling in an ice-water bath (0–10 °C). The reaction was stirred for 1 hour while maintaining the ice-water bath (0–10 °C). Water (20 mL) was added, and the mixture was separated. The aqueous phase was extracted twice with DCM. The organic phases were combined, dried over anhydrous sodium sulfate, concentrated, and purified by column chromatography (DCM:MeOH = 50:1–7:1). The purified compound was concentrated to give 130 mg of a white waxy solid. 1 H NMR (400MHz, CDCl3) δ: 5.89(1H), 5.58(1H), 5.52(1H), 5.38(1H), 4.48(1H), 4.36(1H), 3.52(2H), 2.72(2H), 2.31 (2H), 2.26(2H), 2.18(2H), 2.05(2H), 2.01(2H), 1.57(2H), 1.36(2H), 1.28(4H), 1.27(4H), 1.26(20H), 0.89(6H).

[0108] Step 3: Synthesis of compound WO2

[0109] WO1 (130 mg, 0.941 mmol) was dissolved in methanol (2 mL) and THF (2 mL), and palladium on carbon (50 mg) was added. The mixture was reacted overnight under a hydrogen balloon, filtered, concentrated, and purified by column chromatography (DCM:MeOH = 50:1 to 7:1). The purified product was concentrated to give 88 mg of a pale yellow waxy solid compound. 1 H NMR (400MHz, CDCl3) δ6.04 (d, J=8.0Hz, 1H), 4.43 (dd, J=11.6, 5.7Hz, 1H), 4.26-4.06 (m, 2H), 3.67-3.59 (m, 1H), 2.38 (t, J=7 .2Hz, 2H), 2.32(s, 2H), 2.24-2.18(m, 8H), 1.83-1.78(m, 2H), 1.63(s, 2H), 1.49(s, 2H), 1.26(s, 34H), 0.89(d, J=6.6Hz, 6H).

[0110] ESI-MS m / z = 541.4 [M+1] + .

[0111] Example 2

[0112] The synthetic route for compound WO3 is as follows:

[0113]

[0114] Step 1: Synthesis of compound WO3

[0115] Add WO1-1 (500 mg, 1.18 mmol) to DCM (5 mL), add triethylamine (238 mg, 2.35 mmol), and DMAP (72 mg, 0.59 mmol), and cool to 0–10 °C; slowly add a DCM (2 mL) solution of p-nitrobenzene chloroformate (239 mg, 1.18 mmol), and stir for 10 minutes; add N,N-dimethylpropanol (364 mg, 3.53 mmol), raise to room temperature and stir overnight, concentrate, add water (20 mL), add ethyl acetate (20 mL), add 0.5N hydrochloric acid (5 mL), separate, extract with ethyl acetate in aqueous phase (10 mL x 4), wash with saturated sodium bicarbonate solution in organic phase, dry with anhydrous sodium sulfate, concentrate, and purify by column chromatography (DCM:MeOH = 50:1–10:1) with a little ammonia to give 270 mg of yellow oily compound.

[0116] 1 H NMR (400MHz, MeOD) δ5.72 (d, J=15.3Hz, 1H), 5.45 (dd, J=15.2, 7.1Hz, 1H), 4.35 (dd, J=11.0, 3.2Hz, 1H), 4.28-3.96 (m, 4H), 3.63 (dd, J=18.2, 12.1Hz, 1H), 2.65 -2.51 (m, 3H), 2.39 (d, J=5.1Hz, 3H), 2.18 (dd, J=16.5, 9.0Hz, 2H), 2.04 (d, J=7 .3Hz, 2H), 1.96-1.77 (m, 2H), 1.58 (s, 2H), 1.29 (s, 32H), 0.90 (t, J=6.5Hz, 6H).

[0117] ESI-MS m / z = 555.5 [M+1] + .

[0118] Example 3

[0119] The synthetic route for compound WO4 is as follows:

[0120]

[0121] Step 1: Synthesis of compound WO4

[0122] Add WO1-1 (500 mg, 1.18 mmol) to DCM (5 mL), add triethylamine (238 mg, 2.35 mmol), and DMAP (72 mg, 0.59 mmol), and cool to 0–10 °C; slowly add a DCM (2 mL) solution of p-nitrobenzene chloroformate (239 mg, 1.18 mmol), and stir for 10 minutes; add N,N-dimethylpropylamine (364 mg, 3.53 mmol), raise to room temperature and stir overnight, concentrate, add water (20 mL), add ethyl acetate (20 mL), add 0.5N hydrochloric acid (5 mL), separate, extract with ethyl acetate in aqueous phase (10 mL x 4), wash with saturated sodium bicarbonate solution in organic phase, dry with anhydrous sodium sulfate, concentrate, and purify by column chromatography (DCM:MeOH = 50:1–10:1) with a little ammonia to give 330 mg of yellow oily compound.

[0123] 1 H NMR (400MHz, MeOD) 65.82-5.65 (m, 1H), 5.46 (dd, J=15.3, 7.1Hz, 1H), 4.28 (d, J=7.9Hz, 1H), 4.20-3.90 (m, 3H), 3.14 (t, J=5.7Hz, 2H), 2.75 (d, J=7.3H z, 2H), 2.57 (s, 6H), 2.18 (dd, J=16.4, 8.9Hz, 2H), 2.12-1.93 (m, 2H), 1.78 (s, 2H), 1.58 (s, 2H), 1.34 (d, J=39.2Hz, 30H), 0.90 (dd, J=6.6, 4.8Hz, 6H).

[0124] ESI-MS m / z = 554.5 [M+1] + .

[0125] Example 4

[0126] The synthetic route for compound W50 is as follows:

[0127]

[0128] Step 1: Synthesis of compound W50-1

[0129] WO1-0 (1 g, 3.34 mmol) was added to methanol (20 mL), 0.2 M borate buffer (20 mL), sodium cyanoborohydride (2.7 g, 43.54 mmol), and formaldehyde aqueous solution (12 mL) under ice-water bath. The mixture was stirred overnight at room temperature, water was added, and the mixture was extracted with dichloromethane. The product was concentrated and column filtered to obtain 1.03 g of product.

[0130] 1H NMR (400MHz, CDCl3) δ5.84-5.68 (m, 1H), 5.55 (d, J=6.4Hz, 1H), 4.72 (s, 1H), 4.36 (t, J=5.6Hz, 1H), 3.75 (d, J=5.4 Hz, 2H), 2.60-2.12 (m, 10H), 2.06 (dd, J=14.1, 7.0Hz, 2H), 1.45-1.35 (m, 2H), 1.26 (s, 18H), 0.88 (t, J=6.7Hz, 3H).

[0131] ESI-MS m / z = 328.3 [M+1] + .

[0132] Step 2: Synthesis of compound W50

[0133] Add W50-1 (100 mg, 0.306 mmol) to dichloromethane (2 mL), triethylamine (62 mg, 0.612 mmol), and DMAP (18 mg, 0.153 mmol), and cool to 0–10 °C. Slowly add a solution of myristoyl chloride (80 mg, 0.321 mmol) in dichloromethane (0.5 mL), and stir for 10 minutes. Add water (10 mL), extract with dichloromethane, wash with saturated sodium bicarbonate solution, concentrate, add ethyl acetate (10 mL), add 0.5 N hydrochloric acid solution, separate, extract with ethyl acetate (10 mL x 4), wash with saturated sodium bicarbonate solution, dry with anhydrous sodium sulfate, concentrate, and purify by column chromatography (DCM:MeOH = 50:1–10:1) to obtain 100 mg of white waxy substance.

[0134] 1 H NMR (400MHz, CDCl3) δ5.71 (dt, J=13.8, 6.7Hz, 1H), 5.46 (dd, J=15.6, 6.5Hz, 1H), 5.05-3.40 (m, 3H), 3.08-2.62 (m, 3H), 2.40 (d, J=22.6Hz, 6H), 2.29 (dt, J=15.8, 7.6Hz, 2H), 2.21-1.97 (m, 2H), 1.70-1.56 (m, 2H), 1.26 (s, 40H), 0.88 (t, J=6.7Hz, 6H).

[0135] ESI-MS m / z = 538.5 [M+1] + .

[0136] Example 5

[0137] The synthetic route for compound W51 is as follows:

[0138]

[0139] Step 1: Synthesis of compound W51

[0140] W50 (270 mg, 0.504 mmol) was dissolved in methanol (4 mL) and tetrahydrofuran (4 mL), and palladium on carbon (50 mg) was added. The mixture was stirred overnight at room temperature under a hydrogen balloon. The mixture was filtered, concentrated, and purified by column chromatography (DCM:MeOH = 50:1 to 10:1). The concentrate yielded 120 mg of a white waxy substance.

[0141] 1 H NMR (400MHz, CDCl3) δ4.16 (dd, J=15.1, 5.1Hz, 3H), 2.72 (dd, J=24.9, 7.6Hz, 1H), 2. 57-2.12 (m, 8H), 1.68-1.44 (m, 4H), 1.27 (d, J=10.9Hz, 46H), 0.88 (t, J=6.8Hz, 6H).

[0142] ESI-MS m / z = 540.5 [M+1] + .

[0143] Example 6

[0144] The synthetic routes for compounds W52 and W54 are as follows:

[0145]

[0146] Step 1: Synthesis of compound W52

[0147] Add W50-1 (100 mg, 0.306 mmol) to dichloromethane (2 mL), triethylamine (124 mg, 1.224 mmol), and DMAP (36 mg, 0.306 mmol), and cool to 0–10 °C. Slowly add a solution of myristoyl chloride (160 mg, 0.642 mmol) in dichloromethane (0.5 mL), and stir for 10 minutes. Increase the temperature to room temperature and react for 2 hours. Add water (10 mL), extract with dichloromethane, wash with saturated sodium bicarbonate solution, concentrate, add ethyl acetate (10 mL), add 0.5 N hydrochloric acid solution, separate the layers, extract with ethyl acetate (10 mL x 4), wash with saturated sodium bicarbonate solution, dry with anhydrous sodium sulfate, concentrate, and purify by column chromatography (DCM:MeOH = 500:1–100:1) to obtain 150 mg of white waxy substance.

[0148] 1H NMR (400MHz, CDCl3) δ5.68 (d, J=6.8Hz, 1H), 5.46 (d, J=13.1Hz, 2H), 4.24 (t, J=4.8Hz, 2H), 2.85 (d, J=4.4Hz, 1H ), 2.40-2.19 (m, 10H), 2.02 (q, J=6.9Hz, 2H), 1.60 (dd, J=13.4, 5.9Hz, 6H), 1.25 (s, 60H), 0.88 (t, J=6.6Hz, 9H).

[0149] ESI-MS m / z = 748.7 [M+1] + .

[0150] Step 2: Synthesis of compound W54

[0151] W52 (300 mg, 0.401 mmol) was dissolved in methanol (4 mL) and tetrahydrofuran (4 mL), and palladium on carbon (50 mg) was added. The mixture was stirred overnight at room temperature under a hydrogen balloon. The mixture was filtered, concentrated, and purified by column chromatography (DCM:MeOH = 50:1 to 10:1). The purified product was concentrated to give 150 mg of a white waxy substance.

[0152] 1 H NMR (400MHz, CDCl3) δ4.37 (dd, J=22.6, 5.3Hz, 2H), 3.86 (s, 1H), 2.66 (s, 1H), 2.48 (s, 6H), 2.33 (d, J=7.7Hz, 2H), 1.65-1.58 (m, 2H), 1.50 (s, 2H), 1.26 (s, 46H), 0.88 (t, J=6.8Hz, 6H).

[0153] ESI-MS m / z = 750.7 [M+1] + .

[0154] Example 7

[0155] The synthetic routes for compounds W33 and W34 are as follows:

[0156]

[0157] Step 1: Synthesis of compound W33-1

[0158] WO1-1 (300 mg, 0.706 mmol) was added to dichloromethane (6 mL), triethylamine (143 mg, 1.412 mmol), and DMAP (9 mg, 0.0706 mmol), and cooled to 0–10 °C. A solution of 4-bromobutyryl chloride (131 mg, 0.706 mmol) in dichloromethane (0.5 mL) was slowly added dropwise, and the mixture was stirred and kept warm for 10 minutes. Water (10 mL) was added, and the mixture was extracted with dichloromethane, washed with saturated sodium bicarbonate solution, concentrated, and purified by column chromatography (PE:EA = 10:1–2:1) to obtain 160 mg of white solid.

[0159] 1 H NMR (400MHz, CDCl3) 65.92-5.67 (m, 2H), 5.48 (dd, J=15.5, 6.5Hz, 1H), 4.46-4.08 (m, 4H), 3.47 (t, J=6.4Hz, 2H), 2.60 (d, J=4.8Hz, 1H), 2.52 ( t, J=7.1Hz, 2H), 2.18 (dd, J=15.2, 7.6Hz, 4H), 2.04 (dd, J=14.1, 7.1Hz, 2H), 1.62 (d, J=7.0Hz, 2H), 1.45-1.10 (m, 32H), 0.88 (t, J=6.6Hz, 6H).

[0160] ESI-MS m / z = 574.3 [M+1] + .

[0161] Step 2: Synthesis of compound W33

[0162] W33-1 (100 mg, 0.17 mmol) was added to ethanol (6 mL) and pyrrole (61 mg, 0.85 mmol), heated to 88 °C, stirred for 3 h, water (10 mL) was added, extracted with dichloromethane, washed with saturated sodium bicarbonate aqueous solution, concentrated, and purified by column chromatography (DCM:MeOH = 100:1 to 10:1) to obtain 50 mg of colorless oil.

[0163] 1H NMR (400MHz, CDCl3) 66.21 (d, J = 36.7Hz, 1H), 5.85-5.65 (m, 1H), 5.50 (d, J = 5.9Hz, 1H ), 4.39 (dd, J=10.9, 4.6Hz, 1H), 4.15 (dt, J=14.1, 6.1Hz, 4H), 2.62 (s, 4H), 2.57 (d, J =7.5Hz, 2H), 2.43 (s, 2H), 2.38 (d, J = 7.3Hz, 2H), 2.26-2.19 (m, 2H), 2.03 (d, J = 7.2Hz , 2H), 1.83 (s, 4H), 1.61 (d, J=7.2Hz, 2H), 1.36-1.21 (m, 30H), 0.88 (t, J=6.6Hz, 6H).

[0164] ESI-MS m / z = 565.5 [M+1] + .

[0165] Step 3: Synthesis of compound W34

[0166] W33 (100 mg, 0.177 mmol) was dissolved in methanol (4 mL) and tetrahydrofuran (4 mL), and palladium on carbon (50 mg) was added. The mixture was stirred overnight at room temperature under a hydrogen balloon, filtered, and concentrated to obtain 80 mg of the product.

[0167] Example 8

[0168] The synthetic routes for compounds W43 and W44 are as follows:

[0169]

[0170] Step 1: Synthesis of compound W43

[0171] W33-1 (100 mg, 0.17 mmol) was added to ethanol (6 mL) and 1-(2-hydroxyethylpiperazine) (110 mg, 0.85 mmol), heated to 88 °C, stirred for 3 h, water (10 mL) was added, extracted with dichloromethane, washed with saturated sodium bicarbonate aqueous solution, concentrated, and purified by column chromatography (DCM∶MeOH=100∶1-10∶1) to obtain 100 mg of colorless oil.

[0172] 1H NMR (400MHz, CDCl3) 65.90 (d, J=7.9Hz, 1H), 5.72 (s, 1H), 5.47 (dd, J=15.5, 6.5Hz, 1H), 4.41-3.99 (m, 4H), 3.62 (t, J=5.3Hz, 2H), 2.46 (ddd, J=21.9, 12.4, 6.2Hz, 15H), 2.19 (t, J=7.6Hz, 2H), 2.04 (d, J=7.1Hz, 2H), 1.85-1.7 8 (m, 2H), 1.60 (d, J=6.6Hz, 2H), 1.40-1.16 (m, 30H), 0.88 (t, J=6.5Hz, 6H).

[0173] ESI-MS m / z = 624.5 [M+1] + .

[0174] Step 2: Synthesis of compound W44

[0175] W43 (200 mg, 0.321 mmol) was dissolved in methanol (4 mL) and tetrahydrofuran (4 mL), and palladium on carbon (50 mg) was added. The mixture was stirred overnight at room temperature under a hydrogen balloon, and then filtered and concentrated to obtain 140 mg of the product.

[0176] 1 H NMR (400MHz, CDCl3) δ6.02 (d, J=8.2Hz, 1H), 4.43-4.32 (m, 1H), 4.18 (dd, J=21.8, 10.2Hz, 2H), 3.62 (t, J=5.3Hz, 3H) , 2.67-2.09(m, 19H), 1.89-1.75(m, 2H), 1.67-1.58(m, 2H), 1.49(s, 2H), 1.34-1.22(m, 32H), 0.88(t, J=6.7Hz, 6H).

[0177] ESI-MS m / z = 626.5 [M+1] + .

[0178] Example 9

[0179] The synthetic route for compound W71 is as follows:

[0180]

[0181] Step 1: Synthesis of compound W71-1

[0182] D-Sphingosine (2.0 g, 6.68 mmol, 1.0 eq) was added to 15 mL of DMF and 45 mL of THF, stirred until dissolved, and then icosanoic acid (2.18 g, 6.68 mmol, 1.0 eq) and HATU (2.79 g, 7.35 mmol, 1.1 eq) were added. The solution was cooled to 0–10 °C. Triethylamine (2.02 g, 20.04 mmol, 3.0 eq) was dissolved in 5 mL of THF and then slowly added dropwise to the above solution. The reaction mixture was stirred overnight at room temperature, and TLC showed that D-sphingosine had completely disappeared. The reaction mixture was diluted with 100 mL of EA and washed successively with water (150 mL) and saturated sodium chloride solution (150 mL). The organic phases were combined, dried over anhydrous sodium sulfate, and the solvent was removed under vacuum to obtain a crude product. The crude product was purified by column chromatography (silica gel column, eluent PE:EA = 3:1 to 1:3 (volume ratio)). The product fraction was evaporated to obtain a colorless oily compound W71-1 (2.87 g, 70.8% yield). 1 H NMR (400MHz, CDCl3) δ: 5.87(1H), 5.58(1H), 5.52(1H), 5.37(2H), 4.48(1H), 4.37(1H), 3.74 (2H), 2.18(2H), 2.02(2H), 1.57(2H), 1.37(2H), 1.28(4H), 1.27(4H), 1.26(46H), 0.89(6H).

[0183] Step 2: Synthesis of compound W71

[0184] 7-Dimethylaminoheptanoate (0.72 g, 2.47 mmol, 1.0 eq), EDCI (1.42 g, 7.41 mmol, 3.0 eq), and DMAP (0.66 g, 5.43 mmol, 2.2 eq) were dissolved in 15 mL of DCM and stirred at room temperature for half an hour. Compound W71-1 (1.5 g, 2.47 mmol, 1.0 eq) was dissolved in 25 mL of DCM and stirred in an ice-water bath (0–10 °C) for half an hour. The above 7-dimethylaminoheptanoate solution was added dropwise to the above compound 2-1 solution, and the reaction was maintained in an ice-water bath (0–10 °C) with stirring for 1 hour. The reaction was observed by TLC. If there was still compound 2-1 remaining, 0.5 eq of 7-dimethylaminoheptanoate was added, and the reaction was continued in an ice-water bath for half an hour. The extent of the reaction was observed by TLC until compound 2-1 disappeared on TLC. The mixture was washed with water (80 mL) and saturated sodium chloride solution (80 mL). The aqueous phase was extracted twice with DCM, the organic phases were combined, dried over anhydrous sodium sulfate, and the solvent was removed under vacuum to obtain the crude product. The crude product was purified by column chromatography (silica gel column, eluent DCM:MeOH = 30:1 to 7:1 (volume ratio)), and the product fraction was evaporated to obtain a colorless oily compound W71 (0.79 g, 42.1% yield). 1 H NMR (400MHz, CDCl3) δ: 5.89(1H), 5.59(1H), 5.52(1H), 5.38(2H), 4.48(1H), 4.38(1H), 3.53(2H), 2.71(2H), 2.33(2H), 2.27 (6H), 2.19(2H), 2.02(2H), 1.63(2H), 1.58(2H), 1.56(2H), 1.37(2H), 1.29(2H), 1.28(4H), 1.27(4H), 1.26(48H), 0.90(6H).

[0185] Example 10

[0186] The synthetic route for compound W73 is as follows:

[0187]

[0188] Step 1: Synthesis of compound W73-1

[0189] D-Sphingosine (2.0 g, 6.68 mmol, 1.0 eq) was added to 10 mL of DMF and 30 mL of THF, stirred until dissolved, and then octanoic acid (0.96 g, 6.68 mmol, 1.0 eq) and HATU (2.79 g, 7.35 mmol, 1.1 eq) were added. The solution was cooled to 0–10 °C. Triethylamine (2.02 g, 20.04 mmol, 3.0 eq) was dissolved in THF (5 mL) and slowly added dropwise to the above solution. The reaction mixture was stirred overnight at room temperature, and then TLC showed that D-sphingosine had completely disappeared. The reaction mixture was diluted with EA (60 mL) and washed successively with water (150 mL) and saturated sodium chloride solution (150 mL). The organic phases were combined, dried over anhydrous sodium sulfate, and the solvent was removed under vacuum to obtain a crude product. The crude product was purified by column chromatography (silica gel column, eluent PE:EA = 5:1 to 1:3 (volume ratio)). The product fraction was evaporated to obtain a white to off-white waxy solid compound 3-1 (2.2 g, 75.7% yield). 1 H NMR (400MHz, CDCl3) δ: 5.89(1H), 5.57(1H), 5.52(1H), 5.38(2H), 4.47(1H), 4.36(1H), 3.74 (2H), 2.19(2H), 2.01(2H), 1.57(2H), 1.36(2H), 1.28(4H), 1.27(4H), 1.26(20H), 0.90(6H).

[0190] Step 2: Synthesis of compound W73-2

[0191] Compound W73-1 (2.2 g, 5.18 mmol, 1.0 eq) was dissolved in DMF (20 mL), and imidazole (528 mg, 7.16 mmol, 1.5 eq) was added. Under nitrogen protection, the mixture was cooled to 0–10 °C, and TBDPSCl (1.7 g, 6.22 mmol, 1.2 eq) was added. The reaction mixture was stirred at room temperature for 12 hours. The reaction mixture was diluted with EA (100 mL) and washed with water (50 x 3 mL). The organic layer was washed with brine (50 mL), dried over anhydrous sodium sulfate, concentrated, and the crude product was purified by silica gel chromatography to give W73-2 (1.5 g, 44% yield). 1H NMR (400MHz, CDCl3) δ: 7.41(4H), 7.37(4H), 7.35(2H), 5.89(1H), 5.57(1H), 5.52(1H), 5.38(1H), 4.47(1H), 4.36 (1H), 4.12(2H), 2.01(2H), 1.91(2H), 1.57(2H), 1.38(2H), 1.36(9H), 1.28(4H), 1.27(4H), 1.26(20H), 0.89(6H).

[0192] Step 3: Synthesis of compound W73-3

[0193] 6-Dimethylaminohexanoate (442 mg, 2.26 mmol, 1.0 eq), EDCI (1.30 g, 6.78 mmol, 3.0 eq), and DMAP (610 mg, 4.97 mmol, 2.2 eq) were dissolved in 20 ml of DCM and stirred at room temperature for half an hour. Compound W73-2 (1.5 g, 2.26 mmol, 1.0 eq) was dissolved in 30 ml of DCM and stirred in an ice-water bath (0–10 °C) for half an hour. The above 6-dimethylaminohexanoate solution was added dropwise to the above compound 3-2 solution, and the reaction was maintained in an ice-water bath (0–10 °C) with stirring for 1 hour. The reaction was observed by TLC. If there was still compound 3-2 remaining, 0.5 eq of 6-dimethylaminohexanoate was added, and the reaction was continued in an ice-water bath for half an hour. The extent of the reaction was observed by TLC until compound 3-2 disappeared on TLC. The mixture was washed with water (100 ml) and saturated sodium chloride solution (100 ml). The aqueous phase was extracted twice with DCM, the organic phases were combined, dried over anhydrous sodium sulfate, and the solvent was removed under vacuum to obtain the crude product. The crude product was purified by column chromatography to obtain compound W73-3 (1.2 g, 67.0% yield). 1 H NMR (400MHz, CDCl3) δ: 7.40(4H), 7.37(4H), 7.35(2H), 5.88(1H), 5.58(1H), 5.53(1H), 4.46(1H), 4.37(1H), 4.13(2H), 2.71(2H), 2.32(2H), 2.27(6H), 2.01(2H), 1.90(2H), 1.62(2H), 1.57(2H), 1.56(2H), 1.37( 2H), 1.36(9H), 1.30(2H), 1.29(4H), 1.27(4H), 1.26(18H), 0.89(6H).

[0194] Step 4: Synthesis of compound W73

[0195] Compound W73-3 (1.2 g, 1.51 mmol, 1 eq) was dissolved in anhydrous THF (20 mL), and TBAF (2.4 mL, 2.27 mmol, 1.5 eq, 1 M in THF) was added. The reaction was carried out at room temperature for 2 h, quenched with water, extracted with ethyl acetate, washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, and the solvent was removed under vacuum to obtain the crude product. The crude product was purified by column chromatography to obtain compound W73 (500 mg, 60.0% yield). 1 H NMR (400MHz, CDCl3) δ: 5.89(1H), 5.59(1H), 5.53(1H), 5.36(1H), 4.46(1H), 4.37(1H), 3.74(2H), 2.71(2H), 2.32(2H), 2.27 (6H), 2.19(2H), 2.01(2H), 1.62(2H), 1.57(2H), 1.56(2H), 1.37(2H), 1.30(2H), 1.29(4H), 1.27(4H), 1.26(18H), 0.89(6H).

[0196] Example 11

[0197] The synthetic route for compound W74 is as follows:

[0198]

[0199] Step 1: Synthesis of compound W74-1

[0200] D-Sphingosine (2.0 g, 6.68 mmol, 1.0 eq) was added to 15 mL of DMF and 45 mL of THF, stirred until dissolved, and then undecanoic acid (1.24 g, 6.68 mmol, 1.0 eq) and HATU (2.79 g, 7.35 mmol, 1.1 eq) were added. The solution was cooled to 0–10 °C. Triethylamine (2.02 g, 20.04 mmol, 3.0 eq) was dissolved in 5 mL of THF and then slowly added dropwise to the above solution. The reaction mixture was stirred overnight at room temperature, and TLC showed that D-sphingosine had completely disappeared. The reaction mixture was diluted with 100 mL of EA and washed successively with water (150 mL) and saturated sodium chloride solution (150 mL). The organic phases were combined, dried over anhydrous sodium sulfate, and the solvent was removed under vacuum to obtain a crude product. The crude product was purified by column chromatography (silica gel column, eluent PE:EA = 3:1 to 1:3 (volume ratio)). The product fraction was evaporated to obtain a colorless oily compound W74-1 (2.15 g, 68.9% yield). 1H NMR (400MHz, CDCl3) δ: 5.89(1H), 5.59(1H), 5.52(1H), 5.36(2H), 4.48(1H), 4.37(1H), 3.74 (2H), 2.19(2H), 2.01(2H), 1.57(2H), 1.37(2H), 1.30(4H), 1.28(4H), 1.26(26H), 0.89(6H).

[0201] Step 2: Synthesis of compound W74-2

[0202] Compound W74-1 (2.0 g, 4.27 mmol, 1.0 eq) was dissolved in DMF (20 mL), and imidazole (473 mg, 6.41 mmol, 1.5 eq) was added. Under nitrogen protection, the mixture was cooled to 0–10 °C, and TBDPSCl (1.4 g, 5.13 mmol, 1.2 eq) was added. The reaction mixture was stirred at room temperature for 12 hours. The reaction mixture was diluted with EA (100 mL) and washed with water (50 x 3 mL). The organic layer was washed with brine (50 mL), dried over anhydrous sodium sulfate, concentrated, and the crude product was purified by silica gel chromatography to give W74-2 (1.4 g, 46.4% yield). 1 H NMR (400MHz, CDCl3) δ: 7.40(4H), 7.37(4H), 7.35(2H), 5.88(1H), 5.58(1H), 5.53(1H), 5.37(1H), 4.49(1H), 4.37 (1H), 4.13(2H), 2.01(2H), 1.91(2H), 1.57(2H), 1.38(2H), 1.36(9H), 1.30(4H), 1.27(4H), 1.26(26H), 0.89(6H).

[0203] Step 3: Synthesis of compound W74-3

[0204] 7-Dimethylaminoheptanoate (357 mg, 1.70 mmol, 1.0 eq), EDCI (978 mg, 5.1 mmol, 3.0 eq), and DMAP (457 mg, 3.74 mmol, 2.2 eq) were dissolved in 20 ml of DCM and stirred at room temperature for half an hour. Compound W74-2 (1.2 g, 1.70 mmol, 1.0 eq) was dissolved in 30 ml of DCM and stirred in an ice-water bath (0–10 °C) for half an hour. The solution of 7-dimethylaminoheptanoate was added dropwise to the solution of compound 3-2, and the reaction was maintained in an ice-water bath (0–10 °C) with stirring for 1 hour. The reaction was observed by TLC. If compound 3-2 remained, 0.5 eq of 7-dimethylaminoheptanoate was added, and the reaction was continued in an ice-water bath for half an hour. The extent of the reaction was observed by TLC until compound 3-2 disappeared. The sample was washed successively with water (100 ml) and saturated sodium chloride solution (100 ml). The aqueous phase was extracted twice with DCM, and the combined organic phases were dried over anhydrous sodium sulfate. The solvent was removed under vacuum to obtain the crude product. The crude product was purified by column chromatography to obtain compound W74-3 (980 mg, 67.1% yield). 1 HNMR (400MHz, CDCl3) δ: 7.4l(4H), 7.38(4H), 7.36(2H), 5.88(1H), 5.58(1H), 5.53(1H), 4.46(1H), 4.37(1H), 4.13(2H), 2.71(2H), 2.32(2H) ,2.27(6H),2.01(2H),1.91(2H),1.62(2H),1.58(2H),1.56(2H),1.37 (2H), 1.36(9H), 1.30(2H), 1.29(4H), 1.27(4H), 1.26(28H), 0.89(6H).

[0205] Step 4: Synthesis of compound W74

[0206] Compound W74-3 (900 mg, 1.04 mmol, 1 eq) was dissolved in anhydrous THF (20 mL), and TBAF (1.7 mL, 1.57 mmol, 1.5 eq, 1 M in THF) was added. The reaction was carried out at room temperature for 2 h, quenched with water, extracted with ethyl acetate, washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, and the solvent was removed under vacuum to obtain the crude product. The crude product was purified by column chromatography to obtain compound W74 (400 mg, 61.7% yield). 1H NMR (400MHz, CDCl3) δ: 5.89(1H), 5.57(1H), 5.53(1H), 5.36(1H), 4.46(1H), 4.37(1H), 3.74(2H), 2.71(2H), 2.32(2H), 2.27 (6H), 2.19(2H), 2.00(2H), 1.62(2H), 1.57(2H), 1.56(2H), 1.38(2H), 1.30(2H), 1.29(4H), 1.27(4H), 1.26(28H), 0.90(6H).

[0207] It should be noted that: fluctuations of 50% in the reaction conditions of the above synthetic routes and steps are applicable to the present invention. The preparation methods of the ionizable lipid compounds included in general formulas (I) and (II) can be obtained by referring to the routes and synthetic steps of the above embodiments, and are all within the protection scope of the present invention.

[0208] Other compounds in this invention can be prepared by a method similar to that in Examples 1-11 (the appropriate acyl chloride or acid is used as a raw material depending on the structure of the compound).

[0209] Example 12 Preparation and characterization of lipid nanoparticles (LNPs)

[0210] The ionizable lipid compounds prepared in Examples 1 to 8, and the Dlin-MC3-DMA ionizable lipid molecule (CAS No. 1224606-06-7, the main component of the marketed LNP product (Onpattro)) were dissolved in ethanol at a molar ratio of 50:10:38.5:1.5 to prepare ethanol lipid solutions (lipid concentration 5 mg / mL); double-stranded siRNA targeting SNCA and double-stranded siRNA targeting Dgat2 (siRNA sequences are shown in Table 1) were diluted in 50 mM citrate buffer (pH = 4.0) to obtain siRNA aqueous solutions (concentration 0.4 mg / mL).

[0211] Lipid nanoparticles were prepared by mixing an ethanol lipid solution and an siRNA aqueous solution at a flow rate ratio of 1:3 using a microfluidic device (PG-MFC-8CH model, PreciGenome), with a total lipid to siRNA weight ratio of 12:1. The ethanol was then removed by dialysis in DPBS for 12–24 h in a dialysis bag with a rejection capacity of 3500 D.

[0212] Finally, the lipid nanoparticles were filtered through a 0.2 μm sterile filter to obtain LNP formulations 1–24 encapsulated with different siRNA model drugs using ionizable lipids / DSPC / cholesterol / DMG-PEG2000 (50 / 10 / 38.5 / 1.5 mol%).

[0213] The particle size, polydispersity index (PDI), and potential of lipid nanoparticles were determined by dynamic light scattering using a nanoparticle size and Zeta potential meter (BeNano 90Zeta model, Dandong Better Instruments Co., Ltd.). The test results are shown in Tables 2 and 3.

[0214] Example 13 Preparation and characterization of lipid nanoparticles (LNPs)

[0215] Ionizable lipid compounds (W01, W71, W73, W74) and Dlin-MC3-DMA ionizable lipid molecules (CAS No. 1224606-06-7, the main component of the marketed LNP product (Onpattro)) were dissolved in ethanol at a molar ratio of 50:10:38.5:1.5 to prepare ethanol lipid solutions (lipid concentration 15 mg / mL); double-stranded DNA (XH04) or FAM probe fluorescently labeled double-stranded DNA (FAM-XH04) (XH04 sequence shown in Table 1) were diluted in 50 mM citrate buffer (pH = 4.0) to obtain DNA aqueous solution (concentration 0.5 mg / mL).

[0216] Lipid nanoparticles were prepared by mixing an ethanol lipid solution and a DNA aqueous solution at a flow rate ratio of 1:3 using a microfluidic device (PG-MFC-8CH, PreciGenome) to achieve a total lipid to DNA weight ratio of 12:1 or 18:1 (12:1 for total lipids containing ionizable lipid compounds W01, W71, or Dlin-MC3-DMA, and 18:1 for total lipids containing ionizable lipid compounds W73 and W74). Ethanol was then removed by dialysis in DPBS for 12–24 h in a dialysis bag with a rejection capacity of 3500 D.

[0217] Finally, the lipid nanoparticles were filtered through a 0.2 μm sterile filter to obtain LNP formulations 25-29 encapsulated with XH04 using ionizable lipids / DSPC / cholesterol / DMG-PEG2000 (50 / 10 / 38.5 / 1.5 mol%) and LNP formulations 30-34 encapsulated with FAM-XH04.

[0218] The particle size and polydispersity index (PDI) of lipid nanoparticles were determined by dynamic light scattering using a Malvern Zetasizer Nano ZS90. The test results are shown in Table 4.

[0219] The encapsulation efficiency of lipid nanoparticles was determined using RiboGreen fluorescence staining and the Quant-it RiboGreen RNA quantification kit (Thermo Fisher Scientific). The test results are shown in Table 4.

[0220] Example 14 HEK-293T cell uptake assay

[0221] Cell culture: HEK-293T cells (Wuhan Pronosai Life Science Technology Co., Ltd.) were resuscitated and cultured with RPMI 1640 + 10% FBS.

[0222] Plating: Take normally growing cells, digest them with trypsin cell digestion solution, centrifuge, count, and plate at 2 x 10⁻⁶. 5 Cells were seeded at a density of 100 μL per well in 13 wells of a 24-well plate. The wells were sealed with an appropriate amount of PBS to prevent water evaporation from the edge wells.

[0223] Drug administration: 24 h after cell plating, add drug (LNP formulation 30-34 prepared according to Example 13), 100 μL per well, and set concentration gradients of 10 nM, 100 nM and 1000 nM for each group according to the encapsulation efficiency.

[0224] Detection: After 48 hours of drug treatment, the culture medium was carefully removed, 500 μL of PBS was added, and FAM fluorescence was detected using a microplate reader. Parameter settings: Ex = 485 nm, Em = 528 nm. Results are shown in Table 4.

[0225] Example 15 AML-12 Cell (Normal Mouse Hepatocytes) Toxicity Test

[0226] Preparation of blank lipid nanoparticles (LNPs): Blank LNP samples were prepared according to the preparation process in Example 12, wherein the siRNA aqueous phase solution was replaced with blank 50mM citrate buffer (pH=4.0) to obtain blank LNP formulations 35-46, which were used for cytotoxicity tests.

[0227] Cell resuscitation and passage: AML-12 cells (normal mouse hepatocytes, Wuhan Pronosai Life Science Technology Co., Ltd.) were resuscitated and passaged in culture flasks to the required number of cells.

[0228] Cell plating and transfection: Cells in logarithmic growth phase were digested with 0.25% trypsin and resuspended in fresh complete culture medium. AML-12 cells were adjusted to 2.2*102 5 / ml density. Add 10 μl of blank LNP diluted to different concentrations (0.01–400 μM different cationic lipid concentrations) to a 96-well plate, followed by 90 μl of cell suspension (at which point the cell density is 2*10^6 cells / ml). 4 Cells / well), cultured in a 37℃ / 5% CO2 cell culture incubator for 24 hours.

[0229] Assay: After culturing for 24 hours, 10 μl of enhanced CCK-8 reagent (CCK-8 cell proliferation and virulence assay kit, Beyotime) was added to each well, and incubated for an appropriate time (OD value around 1). The absorbance at 450 nm was measured using a microplate reader (SpectraMax iD5, Molecular MD). The results are shown in Table 5.

[0230] Example 16: Gene silencing efficiency assay in NCI-H460 cells (human large cell lung cancer cells)

[0231] Cell resuscitation and passage: NCI-H460 cells (Wuhan Pronosai Life Science Technology Co., Ltd.) were resuscitated and passaged in culture flasks to the required number of cells.

[0232] Cell plating and transfection: Cells in logarithmic growth phase were digested with 0.25% trypsin and resuspended in fresh complete culture medium. NCI-H460 cells were adjusted to 2.2*102 5 / ml density. Add 10 μl of diluted LNP formulation containing SNCA-siRNA (concentration: siRNA 500 nM, ionizable cationic lipid 83 μM) to a 96-well plate, followed by 90 μl of cell suspension (cell density at this point is 2*10^6 cells / ml). 4 Cells / well; final LNP formulation concentration after dilution: siRNA 50 nM, ionizable cationic lipids 8.3 μM), cultured at 37℃ / 5% CO2 cell culture incubator for 24 h.

[0233] Cell lysis, reverse transcription, and qPCR: LNP preparations containing SNCA-siRNA were co-cultured with NCI-H460 cells for 24 h, and then subjected to (96-well) QuickEasy PCR using Fuji Biotechnology. TM Cell Direct RT-qPCR Kit (One Step) - Taqman kit: Follow the instructions to perform cell lysis, reverse transcription, and qPCR (fluorescent probe method, housekeeping gene selected GAPDH); Instrument: The data were collected and analyzed using a Roche 96 real-time fluorescence qPCR instrument, and the results are shown in Table 2.

[0234] Example 17: Gene silencing efficiency test in HepG2 cells (human liver cancer cells)

[0235] Cell resuscitation and passage: HepG2 cells (Wuhan Pronosai Life Science Technology Co., Ltd.) were resuscitated and passaged in culture flasks to the required number of cells.

[0236] Cell plating and transfection: Cells in logarithmic growth phase were digested with 0.25% trypsin and resuspended in fresh complete culture medium. HepG2 cells were adjusted to 5.5*102 5 / ml density. Add 10 μl of diluted LNP formulation containing SNCA-siRNA (concentration: siRNA 500 nM, ionizable cationic lipid 83 μM) to a 96-well plate, followed by 90 μl of cell suspension (cell density at this point is 5*10^6 cells / ml). 4 Cells / well; final LNP formulation concentration after dilution: siRNA 50 nM, ionizable cationic lipids 8.3 μM), cultured at 37℃ / 5% CO2 cell culture incubator for 24 h.

[0237] Cell lysis, reverse transcription, qPCR: HepG2 cells were co-cultured with an LNP formulation containing SNCA-siRNA for 24 h and then subjected to qPCR using Fuji Bio's (96-well) QuickEasy PCR. TM Cell Direct RT-qPCR Kit (One Step) - Taqman kit: Follow the instructions to perform cell lysis, reverse transcription, and qPCR (fluorescent probe method, housekeeping gene selected GAPDH); Instrument: The data were collected and analyzed using a Roche 96 real-time fluorescence qPCR instrument, and the results are shown in Table 3.

[0238] Table 1 Nucleic Acid Sequences

[0239]

[0240] Note: m indicates 2′-methoxy modification, f indicates 2′-fluorine modification, g indicates glycol nucleic acid (GNA) modification, and * indicates thiodiol reagent DDTT.

[0241] Table 2 Detection results of SNCA target siRNA-LNP samples

[0242]

[0243]

[0244] Table 3. Detection results of Dgat2 target siRNA-LNP samples

[0245]

[0246] Table 4. Detection results of XH04-LNP samples

[0247]

[0248] Table 5. Cytotoxicity results of AML-12 in blank LNP samples.

[0249] Lipid nanoparticles (LNP) Ionizable lipids <![CDATA[IC 50 (μM)]]> 35 Dlin-MC3-DMA 161.10 36 W01 201.2 37 W02 150.3 38 W03 181.0 39 W04 63.7 40 W33 57.4 41 W43 148.8 42 W44 142.9 43 W50 784.5 44 W51 188.0 45 W52 176.1 46 W54 263.9

[0250] Analysis of experimental results:

[0251] 1) LNP samples prepared using the ionizable lipid compounds of the present invention showed superior cellular safety at the cellular level compared to Dlin-MC3-DMA lipids (IC50, W51, W52, and W54). 50 The value was greater than that of the Dlin-MC3-DMA control group. At commonly used lipid concentrations for cell transfection (<50 μM), the cell viability of all LNP samples prepared with lipids was above 90%, and this lipid concentration had no adverse effect on cell viability in cell transfection experiments.

[0252] 2) The LNP prepared by using the ionizable lipid compound of the present invention to deliver nucleic acid molecules has a uniform particle size distribution, good PDI stability, and a potential close to electroneutrality.

[0253] 3) In both nucleic acid model drugs, the gene silencing efficiency of W50 lipid samples was non-inferior to that of commercially available Dlin-MC3-DMA.

[0254] 4) The uptake efficiency of W01, W71, W73, and W74 cells at all concentrations was not inferior to that of commercially available Dlin-MC3-DMA. The ionizable lipid compounds of this invention can be used to deliver nucleic acid molecules, small molecule compounds such as peptides or proteins.

[0255] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the above embodiments do not limit the present invention in any way, and all technical solutions obtained by equivalent substitution or equivalent transformation fall within the protection scope of the present invention.

Claims

1. The compounds represented by formulas (IA), (IB), (II-A), (II-B), or their pharmaceutically available salts or stereoisomers, in: L1 is selected from: optionally substituted linear C1-C 20 Alkylene, optionally substituted C2-C 20 alkenyl, optionally substituted C2-C 20 Ethyne group; L2 is selected from: -CH2CH2-, -CH=CH-; X1 and X2 do not exist independently, or they are each independently selected from -CH2-, O, S, and NH; Y is selected from N or CH; R1 and R2 are each independently selected from H, or optionally substituted with the following groups: C1-C 12 Straight-chain or branched alkyl groups, C2-C 12 alkenyl, C2-C 12 alkynyl group, C3-C 10 cycloalkyl, -CH2-C3-C 10 Cycloalkyl, -(CH2) n -OH, C3-C 10 Cycloalkenyl, C6-C 12 Aryl or 5-12-membered heteroaryl, where n is an integer selected from 1 to 10; or R1, R2 together with the Y or N atom to which they are attached to form an optionally substituted 3-10-membered heterocycle or an optionally substituted C3-C. 10 The carbon ring; the heterocycle or heteroaryl group contains 1-4 heteroatoms selected from N, O, and S; R3 is selected from: C3-C 30 Straight-chain or branched alkyl groups, C3-C 30 alkenyl or C3-C 30 alkynyl group; R4 is selected from: C3-C 30 Straight-chain or branched alkyl groups, C3-C 30 alkenyl or C3-C 30 alkynyl group; R5 is selected from H or The substitution refers to being replaced by one or more substituents selected from the following: hydroxyl, mercapto, amino, halogen, cyano, C6-C. 10 Aryl, 5-10 heteroaryl, C1-C6 alkyl, C1-C6 alkoxy, hydroxy C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C8 cycloalkyl, C3-C8 cycloalkenyl.

2. The compound according to claim 1, or a salt or stereoisomer thereof suitable for pharmaceutical use, characterized in that, In formula (IA), (IB), (II-A), (II-B): L1 is selected from: optional substituted C1-C 12 Straight-chain or branched alkylene, optionally substituted C2-C 12 alkenyl, optionally substituted C2-C 12 Alynyl group; preferably, L1 is selected from: optionally substituted C1-C 10 Straight-chain or branched alkylene, optionally substituted C2-C 10 alkenyl, optionally substituted C2-C 10 The alkynyl group; the definition of the substitution is as described in claim 1; more preferably, L1 is C1-C 10 Straight-chain alkylene compounds, such as C2-C8 straight-chain alkylene compounds, C3 straight-chain alkylene compounds; and / or X1 and X2 are either independently absent or independently selected from -CH2-, O, and NH; preferably, X1 is absent or selected from -CH2-, O, and NH, and X2 is absent or is -CH2-; and / or Y is selected from N or CH; R1 and R2 are each independently selected from H, or optionally substituted with the following groups: straight-chain or branched C1-C8 alkyl (e.g., straight-chain or branched C1-C6 alkyl), C2-C8 alkenyl (e.g., C2-C6 alkenyl), C2-C8 ynyl (e.g., C2-C6 ynyl), C3-C8 cycloalkyl, -CH2-(C3-C8 cycloalkyl), -(CH2) n -OH, C3-C8 cycloalkenyl, C6-C 10 The aryl or 5-6-membered heteroaryl group, or R1 and R2 together with the Y or N atom to which they are attached, form an optionally substituted 3-8-membered N-containing heterocycle; wherein, n, the definition of the substitution is as described in claim 1; preferably, R1 and R2 are each independently selected from H, methyl, ethyl, isobutyl, n-hexyl, 3-methylpentyl, or R1 and R2 together with the Y or N atom to which they are attached form a structure selected from the following: and / or R3 is selected from: C3-C 24 Straight-chain or branched alkyl groups, C3-C 24 alkenyl or C3-C 24 Alkyne group; preferably, R3 is selected from: C6-C 15 Straight-chain or branched alkyl groups, C6-C 15 alkenyl or C6-C 15 Alkyne group; preferably, R3 is C6-C 15 Straight-chain alkyl groups; and / or R4 is selected from: C3-C 24 Straight-chain or branched alkyl groups, C3-C 24 alkenyl or C3-C 24 Alkyne group; preferably, R4 is selected from: straight-chain or branched C6-C 20 Alkyl, C6-C 20 alkenyl or C6-C 20 Alkyne group; preferably, R4 is C6-C 20 Straight-chain alkyl groups; and / or R5 is selected from H, or R4 and X1 are defined as described in claim 1.

3. The compound according to claim 1 or 2, or a salt or stereoisomer thereof suitable for pharmaceutical purposes, characterized in that, The compounds represented by formulas (IA), (IB), (II-A), and (II-B) are selected from the following structures: Wherein, L1, L2, X1, X2, Y, R1, R2, R3, R4, and R5 are defined as described in claim 1 or 2.

4. The compound according to claim 1 or 2, or a salt or stereoisomer thereof suitable for pharmaceutical purposes, characterized in that, The compound represented by formula (IA) is selected from the following structures: The definitions of R1, R2, R4, and L1 are as described in claim 1 or 2; Preferably, in formula (IA-1) or formula (IA-2): R1 and R2 are each independently selected from H and optionally substituted C1-C 12 Straight-chain or branched alkyl; preferably, R1 and R2 are each independently selected from H, or optionally substituted C1-C8 straight-chain or branched alkyl; more preferably, R1 and R2 are each independently selected from H, or optionally substituted C1-C6 straight-chain or branched alkyl. R4 is an optional substituted C3-C 24 Straight-chain alkyl or optionally substituted C3-C 24 Alkenyl; preferably optionally substituted C6-C 20 Straight-chain alkyl or optionally substituted C6-C 20 Alkenyl group, more preferably C6-C 20 Straight-chain alkyl; L1 is an optional substituted C1-C 12 Straight-chain alkylene or optionally substituted C2-C 12 Alkenyl group; preferably C2-C8 straight-chain alkylene group or optionally substituted C2-C8 alkenyl group; more preferably C2-C8 straight-chain alkylene group; The substitution refers to being replaced by one or more groups selected from the following groups: C1-C4 alkoxy, halogen, C1-C4 alkyl, C2-C4 alkenyl, hydroxyl, and C3-C7 cycloalkyl.

5. The compound according to any one of claims 1-4, or a salt or stereoisomer thereof suitable for use as a pharmaceutical preparation, characterized in that, The compound is selected from:

6. A composition comprising: Treatment drugs and / or preventative agents, and Carrier material used for delivering the therapeutic drugs and / or preventive agents; The carrier material includes ionizable lipids, which include one or more of the compounds of any one of claims 1-5 or their pharmaceutical salts or stereoisomers. Preferably, the therapeutic agent or preventative agent is selected from one or more of nucleic acid molecules, small molecule compounds, peptides, and proteins; more preferably, the nucleic acid molecule is selected from one or more of single-stranded DNA, double-stranded DNA, short isomers, agomir, antagomir, antisense molecules, small interfering RNA, asymmetric interfering RNA, microRNA, Dicer-substrate RNA, small hairpin RNA, transfer RNA, and messenger RNA; and / or The therapeutic and / or preventative agents are encapsulated within or associated with a carrier material.

7. The composition according to claim 6, characterized in that, The ionizable lipid further includes one or more other ionizable lipid compounds; preferably, the other ionizable lipid compounds are selected from 1,2-dilinoleoyloxy-N,N-dimethylaminopropane, 2,2-dilinoleoyl-4-dimethylaminomethyl-[1,3]-dioxolane, heptatriaconta-6,9,28,31-tetraen-19-yl-4-(dimethylamino)butyrate, 2,2-dilinoleoyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane, 1, 2-Dioloxy-N,N-Dimethylaminopropane, N-[1-(2,3-diolenoyloxy)propyl]-N,N,N-trimethylammonium chloride, N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride, 1,2-dioleoyl-sn-glycero-3-ethylcholine phosphate, 1,2-dilauroyl-sn-glycero-3-ethylcholine phosphate, 1,2-dimyristoyl-sn-glycero-3-ethylcholine phosphate, 1,2-dimyristoleoyl-Sn-glycero-3-ethylcholine phosphate Choline phosphate, N1-[2-((1S)-1-[(3-aminopropyl)amino]-4-[di(3-aminopropyl)amino]butylcarbamoyl)ethyl]-3,4-di[oleylyloxy]-benzamide, bis(octadecylamino)glycylspermine, 3b-[N-(N′,N′-dimethylaminoethyl)carbamoyl]cholesterol, bis(octadecyl)dimethylammonium bromide, SAINT-2,N-methyl-4-(dioleylyl)methylpyridine, 1,2-dimyristyloxypropyl-3-dimethylhydroxyethylammonium bromide One or more of the following: 1,2-dioleoyl-3-dimethyl-hydroxyethyl ammonium bromide, 1,2-dioleoyloxypropyl-3-dimethyl-hydroxyethyl ammonium chloride, dialkylated amino acid, dioleenyldimethyl ammonium chloride, 3-(docomonasylamino)-N1,N1,4-tridodecyl-1-piperazinethylamine, N1-[2-(docomonasylamino)ethyl]-N1,N4,N4-tridodecyl-1,4-piperazindienamide, 14,25-docomonasyl-15,18,21,24-tetraazaoctaporane; and / or The carrier material further includes neutral lipids; preferably, the neutral lipids are selected from one or more of phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, ceramides and their derivatives; preferably, the neutral lipids are selected from 1,2-distearyl-sn-glycerol-3-phosphate choline, 1,2-dipalmitoyl-sn-glycerol-3-phosphate choline, 1,2-dimyristoyl-sn-glycerol-3-phosphate choline, 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphate choline, 1,2 -Dioleoyl-sn-glycerol-3-phosphate choline, 1,2-dioleoyl-sn-glycerol-3-phosphate ethanolamine, 2-(((2,3-bis(oleoyloxy)propyl))dimethylammonium phosphate)ethylhydrogen, sphingosine, ceramide and their derivatives, more preferably 1,2-distearate-sn-glycerol-3-phosphate choline; preferably, the molar ratio of the ionizable lipid to the neutral lipid is 1-15:1, more preferably 2-10:1, more preferably 3-8:1; and / or The carrier material further includes structural lipids, preferably selected from one or more of cholesterol, sphingomyelin, sphingosine, sitosterol, α-tocopherol, and corticosteroids, with cholesterol being the most preferred; preferably, the molar ratio of the ionizable lipid to the structural lipid is 1–3:1, more preferably 1–2:1; and / or The carrier material further includes polyethylene glycol conjugated lipids. Preferably, the polyethylene glycol conjugated lipids are selected from one or more of PEG-modified phosphatidic acid, PEG-modified phosphatidylethanolamine, PEG-modified ceramide, PEG-modified dialkylamine, and PEG-modified diacylglycerol. Preferably, the polyethylene glycol conjugated lipids are selected from R-3-[(ω-methoxy-poly(ethylene glycol))carbamoyl)]-1,2-dimyristyloxypropyl-3-amine, 1-(monomethoxy-polyethylene glycol)-2,3-dimyristylglycerol, polyethylene glycol-dilauroylphosphatidylethanolamine, polyethylene glycol-dimyristoylphosphatidylethanolamine, polyethylene glycol-dipalmitoylphosphatidylcholine, polyethylene glycol-distearateylphosphatidylethanolamine, and ceramide. Amine-polyethylene glycol, cholesterol-polyethylene glycol, PEGylated phosphatidylethanolamine, 4-O-(2′,3′-di(tetradecanoyloxy)propyl-1-O-(ω-methoxy(polyethoxy)ethyl)succinate, PEGylated ceramide, ω-methoxy(polyethoxy)ethyl-N-(2,3-di(tetradecanoyloxy)propyl)carbamate or 2,3-di(tetradecanoyloxy)propyl-N-(ω-methoxy)(polyethoxy)ethyl)carbamate, more preferably DMG-PEG2000; preferably, the molar ratio of the ionizable lipid to the polyethylene glycol conjugated lipid is 30-80:1, preferably 30-60:1, more preferably 30-50:1, further preferably 30-40:1; and / or The mass ratio of the carrier material to the therapeutic drug or preventive agent is 8-40:1, preferably 10-35:1, more preferably 10-30:1, and even more preferably 12-18:

1.

8. The composition according to claim 6 or 7, characterized in that, The composition is a lipid nanoparticle formulation, comprising: Treatment drugs and / or preventative agents, and Carrier materials for delivering the therapeutic drugs and / or preventive agents; preferably, the carrier materials include ionizable lipids, neutral lipids, structured lipids, and polyethylene glycol conjugated lipids; Preferably, the ionizable lipid is a compound as described in any one of claims 1-5 (IA), (IB), (II-A) or (II-B) or a salt or stereoisomer available for use with a drug thereof, the neutral lipid is 1,2-distearate-sn-glycerol-3-phosphocholine, the structural lipid is cholesterol, and the polyethylene glycol conjugated lipid is DMG-PEG2000; Preferably, the molar ratio of the ionizable lipid, the neutral lipid, the structural lipid, and the polyethylene glycol conjugated lipid is (30-60):(1-15):(20-40):1, more preferably (30-55):(1-12):(20-35):1, and even more preferably (30-40):(5-10):(20-30):1; Preferably, the therapeutic drug or preventive agent is a nucleic acid molecule, preferably small interfering RNA; the mass ratio of the carrier material to the therapeutic drug or preventive agent is 10-35:1, more preferably 10-30:1, and even more preferably 12-18:1; Preferably, the average size of the nanoparticle formulation is 30nm to 300nm, more preferably 50nm to 250nm, further preferably 80nm to 200nm, and even more preferably 80nm to 180nm; the polydispersity index of the nanoparticle formulation is ≤0.4, preferably ≤0.3, and even more preferably ≤0.

25.

9. The method for preparing the composition according to claim 8, characterized in that, The composition is a lipid nanoparticle formulation, and its preparation method includes the following steps: (1) Dissolve the ionizable lipids, neutral lipids, structural lipids and polyethylene glycol conjugated lipids in an organic solvent to prepare a lipid solution; dissolve the therapeutic drug or preventive agent (preferably a nucleic acid molecule, more preferably a small interfering RNA) in a buffer solution to obtain a therapeutic drug or preventive agent solution; (2) The lipid solution and the therapeutic or preventive agent solution were mixed using a microfluidic device to prepare lipid nanoparticles, and the resulting product was dialyzed in a buffer solution; (3) Filter the product after dialysis in step (2) to obtain lipid nanoparticle formulation.

10. The method for preparing the composition according to claim 9, characterized in that, In step (1), the organic solvent is one or more combinations selected from ethanol, diethyl ether, and acetone, preferably ethanol; the concentration of lipid in the lipid solution is 2-25 mg / mL; Preferably, in step (1), the buffer solution is a citrate buffer with a pH of 4.0; the concentration of the therapeutic drug or preventative agent in the therapeutic drug or preventative agent solution is 5–1000 μg / mL; and / or In step (2), the flow rate ratio of the lipid solution to the therapeutic drug or preventive agent solution is 1:2 to 5, preferably 1:3; Preferably, in step (2), the dialysis is performed using a molecular weight cutoff tube or dialysis bag with a molecular weight cutoff of 3-100 kDa; the buffer solution is selected from Tris buffer, citrate buffer, and phosphate buffer solution; the dialysis time is 12-24 hours; and / or In step (3), the product is filtered through a sterile filter with a membrane pore size of 0.2 μm.

11. The use of the compound of any one of claims 1-5, or a salt or stereoisomer thereof that is usable as a drug, or the composition of any one of claims 6-8, or the composition prepared by the method of claim 9 or 10, in the preparation of a drug; Preferably, the drug includes nucleic acid drugs, small molecule drugs, polypeptide or protein drugs; preferably, the nucleic acid drugs include gene vaccines and gene editing drugs.

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