Lipid composition and preparation method therefor and use thereof

Abstract

A lipid composition and a preparation method therefor and the use thereof. The lipid composition comprises the following components: an ionizable lipid and an auxiliary lipid, and does not contain a component having a preventive or therapeutic effect. The lipid composition can be used for delivering nucleic acids, macromolecular substances and small molecular substances, has a good stability, can be stored at room temperature for a long time, and can be used as a carrier in multiple fields such as cell and disease treatment and prevention.

Description

A lipid composition and its preparation method and application

[0001] Related applications

[0002] This application claims priority to Chinese patent application No. 202410080713.7 filed on January 19, 2024, the entire contents of which are incorporated by reference into this application. Technical Field

[0003] The present application belongs to the field of biomedicine technology, and specifically relates to a lipid composition, a preparation method and an application thereof. Background Art

[0004] From the perspective of cell biology, drug delivery is a process of introducing active substances into cells to change the function of host cells. The above active substances include deoxyribonucleic acid (DNA), ribonucleic acid (RNA) and small non-coding RNA, such as siRNA, shRNA and miRNA, as well as active macromolecules and small molecules.

[0005] To date, a variety of biologically derived and chemically derived materials have been developed for the delivery of the above-mentioned active substances, some of which have shown good performance. Biologically derived carriers, including exosomes, bacterial outer membrane vesicles, and virus-like particles (such as PEG10 VLPs), have good applications in certain scenarios. Among chemical-based delivery systems, polymers and lipid nanoparticles have been the most widely used due to their high efficiency and diversity. Polymers include polyamine compounds (such as polyethyleneimine), copoly(amine esters) (PACE), poly(glycidyl butylamine) (PGBA), poly(ornithine) (PLO), poly(phosphine-amino esters) (PBAEs), poly(trimethylolpropane allyl ether-poly(vinyl chloride)), and charge-altered release transporters (CARTs). Due to their controllable synthesis routes and ease of scale-up, they have been well developed. In terms of lipid carriers, lipid nanoparticles (LNPs) are considered the first choice for delivering the above-mentioned bioactive substances, such as the COVID-19 mRNA vaccine (Comirnaty and Spikevax). Currently, more than a dozen nucleic acid drugs are using LNPs as delivery vehicles in clinical trials. These examples span infectious disease and cancer vaccines, as well as genetic disease treatments. Some of these drugs have already made significant progress, demonstrating the promising potential of this platform. Recently released data showed that a Phase 3 trial of a respiratory syncytial virus (RSV) vaccine (Moderna's mRNA-1345) met its primary efficacy endpoint in adults 60 years and older, achieving an 83.7% vaccine efficacy against RSV lower respiratory tract disease. In another example, a personalized nucleic acid cancer vaccine (Moderna and Merck's mRNA-4157 / V940) demonstrated positive results as adjuvant therapy in patients with completely resected high-risk melanoma. Compared to pembrolizumab alone, the combination therapy reduced the risk of recurrence or death by 44%. The FDA recently granted "breakthrough therapy" designation to the two aforementioned mRNA-LNP vaccines, demonstrating the promising prospects of this field.

[0006] Currently, commonly used lipid nanotechnology consists of four components: ionizable cationic lipids, phospholipids, cholesterol, and PEGylated lipids. To form nucleic acid LNPs, different lipids and nucleic acids are typically dissolved in ethanol and an acidic aqueous phase (such as a pH 4.0 citric acid buffer), respectively. The ethanol and aqueous phases are then mixed in a 1:3 volume ratio using a microfluidic device to complete the self-assembly process. During the formation process, the ionizable cationic lipids become protonated, becoming positively charged. They then electrostatically bind to the negatively charged nucleic acid, encapsulating the nucleic acid within the lipid nanoparticle. Simultaneously, other helper lipids (including phospholipids, cholesterol, and polymeric lipids) self-assemble on top to stabilize the resulting nucleic acid LNPs. Subsequently, the mRNA-LNP solution is adjusted to a neutral pH through buffer exchange, during which the ionizable lipids become uncharged, rendering them stable at physiological pH and less toxic. This preparation method is cumbersome and requires expensive equipment, such as microfluidics. Encapsulation of the bioactive substance must be performed simultaneously with nanoparticle preparation. Due to material and formulation reasons, lipid nanoparticles can only be stored for a long time at extremely low temperatures, which limits the widespread use of lipid nanoparticle technology.

[0007] Based on this, the present application provides a lipid composition composed of ionizable lipids and helper lipids, free of cationic polymers and components with preventive or therapeutic effects, and suitable for use as a drug carrier. The lipid composition provided herein has excellent stability, a simple preparation method, and can be stored for long periods at room or low temperatures. It can be used as a carrier in a variety of fields, including cell therapy and disease treatment and prevention. Summary of the Invention

[0008] The present application addresses the problems existing in the prior art and provides a lipid composition, a preparation method thereof, and an application thereof. The lipid composition provided in the present application comprises the following components: ionizable lipids and auxiliary lipids, and does not contain ingredients with preventive or therapeutic effects. The lipid composition provided in the present application can be used to deliver nucleic acids, small molecules, or macromolecules. The lipid composition provided in the present application has good stability and can be stored for a long time at room temperature. Moreover, by simply mixing with the active ingredient, good encapsulation of the active ingredient can be achieved, and it can be used as a carrier in multiple fields such as cell and disease treatment and prevention.

[0009] To achieve the above objectives, in a first aspect, the present application provides a lipid composition comprising the following components: an ionizable lipid and a helper lipid; the lipid composition does not contain an ingredient with a preventive or therapeutic effect; the ionizable lipid comprises a first ionizable lipid, which is selected from a compound of formula (1), or a salt, stereoisomer, or tautomer thereof:

[0010] Wherein, R1, R2, and R3 are independently H, C5-40 Straight or branched alkyl, C 5-40 Straight-chain or branched alkenyl, C 5-40 A straight or branched alkynyl group, a 3-6 membered saturated or partially unsaturated cyclic hydrocarbon group containing 1-3 side chains, or a 6-10 membered aromatic group containing 1-3 side chains; the side chains are independently selected from C 10-30 Straight-chain or branched alkyl, C 10-30 Straight-chain or branched alkenyl, C 10-30 Straight or branched alkynyl; provided that at most one of R1, R2, and R3 is H;

[0011] M is selected from -NR4R5, a saturated or partially unsaturated 3-6 membered heterocyclic group containing at least one nitrogen atom, a 6-10 membered heteroaryl group containing at least one nitrogen atom, wherein the heterocyclic group and the heteroaryl group are unsubstituted or substituted with one or more -OH, carboxyl, amino, oxo or halogen;

[0012] R4 and R5 are independently H, C 1-6 Straight-chain or branched alkyl, C 2-6 Straight chain or branched alkenyl or C 2-6 Straight chain or branched chain alkynyl, the C 1-6 Straight-chain or branched alkyl, C 2-6 Straight chain or branched alkenyl or C 2-6 The straight-chain or branched alkynyl group is unsubstituted or substituted with one or more -OH, carboxyl, aminoamide, amidino, guanidino or halogen;

[0013] G1, G2, and G3 are independently -O-, -S-, -NR6-, -SS-, -C(=O)-, -C(=S)-, -C(=O)O-, -CH(OH)-, -OC(=O)-, -C(=O)NR6-, -NR6C(=O)-, -OC(=O)O-, -NR6C(=O)O-, -OC(=O)NR6-, -NR6C(=O)NR 13 -, -C(=O)S-, -C(=S)S-, -SC(=S)-, -SC(=O)-, -OC(=O)S-, -SC(=O)O-, -SC(=O)S-, -OS(=O)2O-, -S(=O)2O-, -OS (=O)2-, -S(=O)2-, -S(=O)2-NR6-, -NR6-S(=O)2-, -P(=O)(OR6)O-, -OP(=O)(OR6)- or -OP(=O)(OR6)O-; where each R6, R 13 independently selected from H, hydroxyl, C 1-30 Straight-chain or branched alkyl or cycloalkyl, C 2-30 straight-chain or branched alkenyl;

[0014] L1 is selected from -X1- or -(CR7R8) m -X1-, wherein each X1 is independently selected from -O-, -S-, -NR 14 -, -SS-, -C(=O)-, -C(=S)-, -C(=O)O-, -OC(=O)-, -C(=O)NR 14 -、-NR 14 C(=O)-, -OC(=O)O-, -NR 14 C(=O)O-, -OC(=O)NR 14 -、-NR 14 C(=O)NR 15 -, -C(=O)S-, -C(=S)S-, -SC(=S)-, -SC(=O)-, -OC(=O)S-, -SC(=O)O-, -SC(=O)S-, -OS(=O)2O-, -S(=O)2O-, -OS(=O)2-, -S(=O)2-, -S(=O)2-NR 14 -、-NR 14 -S(=O)2-、-P(=O)(OR 14 )O-、-OP(=O)(OR 14 )-or-OP(=O)(OR 14 )O-; wherein m is an integer from 2 to 6, R 7、 R8 are independently H, hydroxy, halogen, C 1-6 Straight or branched alkyl or cycloalkyl, C 2-6 A straight-chain or branched alkenyl group, each R 14 、R 15 independently selected from H, C 1-30 Straight-chain or branched alkyl or cycloalkyl, C 2-30 straight-chain or branched alkenyl;

[0015] L2 is -(CR9R 10 ) n -or-(CR9R 10 ) n -X2-(CR 11 R 12 ) k -, wherein X2 is selected from -O-, -S-, -NR 16 -, -SS-, -C(=O)-, -C(=S)-, -C(=O)O-, -OC(=O)-, -C(=O)NR 16 -、-NR 16 C(=O)-, -OC(=O)O-, -NR 16 C(=O)O-, -OC(=O)NR 16 -、-NR16 C(=O)NR 17 -, -C(=O)S-, -C(=S)S-, -SC(=S)-, -SC(=O)-, -OC(=O)S-, -SC(=O)O-, -SC(=O)S-, -OS(=O)2O-, -S(=O)2O-, -OS(=O)2-, -S(=O)2-, -S(=O)2-NR 16 -、NR 16 -S(=O)2-、-P(=O)(OR 16 )O-、-OP(=O)(OR 16 )-or-OP(=O)(OR 16 )O-; n is an integer from 1 to 6; k is an integer from 1 to 6; R 9、 R 10、 R 11、 R 12 are independently H, hydroxy, halogen, C 1-6 Straight or branched alkyl or cycloalkyl, C 2-6 A straight-chain or branched alkenyl group, each R 16 、R 17 independently selected from H, C 1-30 Straight-chain or branched alkyl or cycloalkyl, C 2-30 straight-chain or branched alkenyl;

[0016] Where R4 to R 17 The alkyl, cycloalkyl, and alkenyl groups are unsubstituted or substituted by one or more groups selected from hydroxyl, thiol, amino, substituted amino, and halogen;

[0017] The salts mentioned do not include quaternary ammonium salts.

[0018] In one embodiment, the preventive or therapeutic ingredient is a pharmaceutically active ingredient.

[0019] In one embodiment, R1, R2, and R3 are independently the following groups:

[0020] Where Y does not exist or is C 1-30 Straight chain or branched chain alkyl or cycloalkyl, C 2-20 Straight-chain or branched alkenyl, C 2-20 Straight or branched alkynyl; R1', R2' are independently H, C 1-30 Straight-chain or branched alkyl, C 2-30 Straight-chain or branched alkenyl, C 2-30 The total carbon chain length of Y, R1' and R2' is 8-40.

[0021] In one embodiment, R1, R2, and R3 are independently selected from the following groups:

[0022] Wherein, R1' and R2' are independently H, C 1-30 Straight-chain or branched alkyl, C 2-30 Straight-chain or branched alkenyl, C 2-30 The total carbon chain length of R1' and R2' is 8-30.

[0023] In one embodiment, R1, R2, and R3 are independently selected from any one of the following groups:

[0024] In one embodiment, G1, G2, and G3 are independently -O-, -S-, -NR6-, -SS-, -C(=O)-, -C(=O)O-, -CH(OH)-, -OC(=O)-, -C(=O)NR6-, -NR6C(=O)-, -OC(=O)O-, -NR6C(=O)O-, -OC(=O)NR6-, -NR6C(=O)NR 13 -, -P(=O)(OR6)O-, -OP(=O)(OR6)- or -OP(=O)(OR6)O-.

[0025] In one embodiment, L1 is selected from -(CR7R8) m -X1-, wherein X1 is selected from -O-, -S-, -NR 14 -, -SS-, -C(=O)-, -C(=O)O-, -OC(=O)-, -C(=O)NR 14 -、-NR 14 C(=O)-, -OC(=O)O-, -NR 14 C(=O)O-, -OC(=O)NR 14 -、-NR 14 C(=O)NR 15 -、-P(=O)(OR 14 )O-、-OP(=O)(OR 14 )-or-OP(=O)(OR 14 )O-.

[0026] In one embodiment, L2 is -(CR9R 10 ) n -X2-(CR 11 R 12 ) k -, wherein X2 is selected from -O-, -S-, -NR 16-, -SS-, -C(=O)-, -C(=O)O-, -OC(=O)-, -C(=O)NR 16 -、-NR 16 C(=O)-, -OC(=O)O-, -NR 16 C(=O)O-, -OC(=O)NR 16 -、-NR 16 C(=O)NR 17 -、-P(=O)(OR 16 )O-、-OP(=O)(OR 16 )-or-OP(=O)(OR 16 )O-.

[0027] In one embodiment, M is selected from the following structures:

[0028] wherein m' and n' are independently integers of 0-6, R1" and R2" are independently H, C 1-6 Alkyl, C 2-6 The alkenyl, guanidino, amidino, amide, fatty amine, 3-10 membered nitrogen-containing heterocycle; the nitrogen-containing heterocycle is selected from pyrrole, imidazole, pyridine, pyrazole, triazole, oxazole, isoxazole, thiophene, isothiazole, pyridazine, pyrazine, piperazine, indole, benzimidazole, carbazole, quinoline, isoquinoline, purine and pyrimidine and tautomeric forms thereof, which are unsubstituted or optionally substituted with one or more selected from hydroxyl, thiol, amino, substituted amino, halogen, C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 6-14 The aromatic group is substituted with an organic group.

[0029] In one embodiment, the compound of formula (1) is selected from the compound represented by formula (1A):

[0030] In one embodiment, the compound of formula (1) is selected from the compound represented by formula (1B):

[0031] In one embodiment, the compound of formula (1) is selected from compounds of formula (1C):

[0032] In one embodiment, the compound of formula (1) is selected from the group consisting of compounds represented by formula (1D):

[0033] In one embodiment, the compound of formula (1) is selected from the compound represented by formula (1E)

[0034] In one embodiment, the compound of formula (1) is selected from the compound represented by formula (1F):

[0035] In one embodiment, the compound of formula (1) is selected from the compound represented by formula (1G):

[0036] In one embodiment, the compound of formula (1) is selected from the compound represented by formula (1H):

[0037] In one embodiment, M is selected from any one of the following groups:

[0038] In one embodiment, the compound of formula (1) is selected from the compound represented by formula (II):

[0039] In one embodiment, the compound of formula (1) is selected from the compound represented by formula (1J):

[0040] In one embodiment, the compound of formula (1) is selected from the compound represented by formula (1K):

[0041] In one embodiment, Y is absent and the compound of formula (1) is selected from the compounds represented by formula (1L):

[0042] Wherein R1', R2' are independently selected from H, C 1-30 Straight-chain or branched alkyl, C 2-30 Straight-chain or branched alkenyl, C 2-30 The total carbon chain length of R1' and R2' is 8-40.

[0043] In one embodiment, the compound of formula (1) is selected from the compound represented by formula (1M):

[0044] In one embodiment, the compound of formula (1) is selected from:

[0045] In one embodiment, the method for preparing the first ionizable lipid comprises the step of reacting a compound of formula (II) with a compound of formula (III):

[0046] Wherein, Xa and Xb are groups containing a leaving group or a nucleophilic group, and Xa and Xb form L1 through a nucleophilic reaction or a condensation reaction.

[0047] In one embodiment, the method for preparing the first ionizable lipid comprises the step of reacting a compound of formula (IV) with a compound of formula (V):

[0048] wherein Xc and Xd are groups containing a leaving group or a nucleophilic group, and Xc and Xd form L2 through a nucleophilic reaction or a condensation reaction.

[0049] In one embodiment, the method for preparing the first ionizable lipid comprises the step of reacting a compound of formula (VI):

[0050] wherein Xe is a group containing a leaving group or a nucleophilic group, Xf is a compound containing a leaving group or a nucleophilic group, and Xe and Xf form M through a nucleophilic reaction or a condensation reaction.

[0051] In one embodiment, the method for preparing the first ionizable lipid comprises the steps of reacting a compound of formula (VII) with a compound of formula (VIII), a compound of formula (IX), and a compound of formula (X) in sequence:

[0052] wherein Xg, Xh, Xi, Xj, Xk, and Xl are groups containing a leaving group or a nucleophilic group, and Xg and Xj form G1 through a nucleophilic reaction or a condensation reaction, Xh and Xk form G2 through a nucleophilic reaction or a condensation reaction, and Xi and Xl form G3 through a nucleophilic reaction or a condensation reaction.

[0053] In one embodiment, the method for preparing the first ionizable lipid comprises the step of reacting a compound of formula (XI) with a compound of formula (XII):

[0054] or

[0055] Steps for reacting the compound of formula (XIII) with the compound of formula (XII):

[0056] wherein Xm is a group containing a nucleophilic group, and Xm and C=CL of the compound of formula (XII) 2a X1-L2 is formed by addition reaction.

[0057] In one embodiment, R1, R2, and R3 are independently of one another:

[0058] Wherein, Y, R1', and R2' have the same meanings as above;

[0059] It also includes the steps of forming tail chains R1, R2, and R3:

[0060] Wherein X is a leaving group.

[0061] In one embodiment, the helper lipid is a mixture of a phospholipid and a steroid or a derivative thereof.

[0062] In one embodiment, the phospholipids include 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-diondecanoyl-sn-glycero-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-0-octadecenyl-sn-glycero-3-phosphocholine (18:0Diether PC), 1-oleoyl-2-cholesteryl hemisuccinyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), 1,2-dialinolenoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME 16.0PE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-bisdocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG), dipalmitoylphosphatidylglycerol (DPPG), palmitoyloleoyl at least one of 1-stearoyl-2-oleoyl-stearoylethanolamine (SOPE), 1-stearoyl-2-oleoyl-phosphatidylcholine (SOPC), sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoylphosphatidylcholine, lysophosphatidylcholine, and lysophosphatidylethanolamine (LPE).

[0063] In one embodiment, the steroid or its derivative comprises at least one of cholesterol, cholesterol stearate, coprosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, salinarate, tomatine, ursolic acid, and α-tocopherol.

[0064] In one embodiment, in the lipid composition, the amount of the ionizable lipid is 5-90 mol% of the total lipids in the formulation; and the amount of the auxiliary lipid is 10-95 mol% of the total lipids in the formulation.

[0065] In one embodiment, the molar ratio of phospholipid to steroid or its derivative in the helper lipid is (5-80):(5-80).

[0066] In one embodiment, the starting material of the lipid composition further comprises PEG-conjugated lipids in an amount of 0.1-15 mol% of the total lipids in the formulation.

[0067] In one embodiment, the PEG-conjugated lipid comprises at least one of PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, and PEG-modified dialkylglycerol.

[0068] In one embodiment, the PEG conjugated lipid includes at least one of PEG-distearyloxypropyl (PEG-DSA), PEG-c-DOMG, PEG-DPPC, polyethylene glycol dimethacrylate (PEG-DMA), 1,2-dimethylstyrene-rac-glycero-3-methoxypolyethylene glycol (PEG-DMG), dipalmitoylglycerol-polyethylene glycol (DPG-PEG), 1,2-distearoyl-rac-glycero-3-methoxypolyethylene glycol (DSG-PEG), 2-[(polyethylene glycol)-2000]-N,N-tetracosylacetamide (ALC-0159), dipalmitoylphosphatidylethanolamine-polyethylene glycol (DPPE-PEG), distearoylphosphatidylethanolamine-polyethylene glycol (PEG-DSPE), dilauroylphosphatidylethanolamine-polyethylene glycol (PEG-DLPE), and dimyristoylphosphatidylethanolamine-polyethylene glycol (PEG-DMPE) lipids.

[0069] In one embodiment, in the lipid composition, the amount of the ionizable lipid is 5-80 mol% of the total lipids in the formulation; the amount of the auxiliary lipid is 10-90 mol% of the total lipids in the formulation; and the amount of the PEG-conjugated lipid is 0.1-15 mol% of the total lipids in the formulation.

[0070] In one embodiment, the ionizable lipid further comprises at least one of a second ionizable lipid and a third ionizable lipid;

[0071] The second ionizable lipid is a compound of formula (2), or a salt, stereoisomer, or tautomer thereof:

[0072] Wherein N1 is NH or O;

[0073] R a Selected from C6-C 24 Alkyl, C6-C 24 Alkenyl, C6-C 24 Cycloalkyl, C6-C 24 Alcohol, C6-C 24 Short chain polyethylene oxide; the C6-C 24 Alkyl, C6-C 24 Alkenyl, C6-C 24 Cycloalkyl, C6-C 24 Alcohol, C6-C 24 Short-chain polyoxyethylene is a straight chain or branched chain structure; R b and R c Each independently selected from C1-C 12 Alkyl, C2-C 12 Alkenyl, C1-C 12 Alkynyl, C3-C 12 Cycloalkane, C6-C 12 Aromatic hydrocarbon groups, C1-C 12 Alkyl alcohol, C1-C 12 Heterocyclic groups, alkylamines;

[0074] The alkylamine is Among them, R a ' is C1-C 12 Alkyl, the R b ' and R b " are each independently selected from H, C1-C6 alkylamine, R c " is selected from C1-C6 alkyl groups which are unsubstituted or substituted with amino groups, R c ”' is H, or -R c '-A1'-R c ”-NH2;

[0075] Provided that, when A1' is -CO-NH-, -NH-CO- or -CO-O-, R c ' is a C1-C6 alkyl group; when A1' is -CO-, R c 'does not exist;

[0076] The third ionizable lipid is selected from at least one of the following compounds:

[0077] In one embodiment, R b and R c Each independently selected from C1-C12 Alkyl alcohols, alkylamines.

[0078] In one embodiment, provided that: when R a C6-C 24 In the case of alkenyl, A1 is NH, R b and R c Each independently selected from C1-C 12 Alkyl alcohols;

[0079] When R a C6-C 24 When alkyl, A1 is NH, R b and R c Each independently selected from C1-C 12 Alkyl alcohols, alkylamines;

[0080] R a For straight chain C6-C 24 When alkyl, A1 is NH or O, R b and R c each independently selected from alkylamines;

[0081] When R a For straight chain C6-C 24 When it is alkyl, A1 is O, R b and R c Each independently selected from C1-C 12 Alkyl alcohols;

[0082] When R a C6-C 24 In the case of short-chain polyoxyethylene, A1 is NH or O, R b and R c each independently selected from alkylamines;

[0083] Or when R a C6-C 24 In the case of alkyl alcohol, A1 is NH or O, R b and R c Each is independently selected from alkylamines.

[0084] In one embodiment, R a Selected from the following compound structures:

[0085] In one embodiment, R b and R c Selected from the following compound structures:

[0086] R d Selected from C1-C6 alkanes or cycloalkanes.

[0087] In one embodiment, the compound of formula (2) is selected from at least one of the following compounds:

[0088] In one embodiment, the method for preparing the second ionizable lipid comprises the following reaction steps:

[0089] R a -NH2① reacts with α,β-unsaturated carbonyl compound② to form ionizable lipid compound③:

[0090] Among them, R a -NH2 is R a -NH2 or

[0091] R b ' and R b "At the same time H, or R b ' and R b " is also a C1-C6 amine, or R b ' and R b "At the same time -R c '-A1'-R c ”-NH2.

[0092] In one embodiment, the method for preparing the second ionizable lipid comprises:

[0093] 1) R a -NH2① reacts with α,β-unsaturated carbonyl compound④ to form compound⑤;

[0094] 2) Compound ⑤ reacts with a nucleophile ⑥ to generate an ionizable lipid compound ③;

[0095] Among them, the nucleophile ⑥ is R b -NH2 or R b -OH; R a -NH2 is selected from R a -NH2 or

[0096] R b ' and R b "At the same time H, or R b ' and R b " is also a C1-C6 amine, or R b ' and R b "At the same time -R c '-A1'-R c ”-NH2; Z2 is a leaving group, and Z2 reacts with NH2 to obtain A1.

[0097] In one embodiment, the method for preparing the second ionizable lipid comprises:

[0098] 1) R a -NH2① reacts with α,β-unsaturated carbonyl compound② to form compound⑦;

[0099] 2) Compound ⑦ reacts with an α,β-unsaturated carbonyl compound ⑧ to form compound ⑨;

[0100] 3) Compound ⑨ reacts with nucleophile ⑩ to form an ionizable lipid compound

[0101] Among them, R a -NH2 is R a -NH2, Z3 is a leaving group, A3 reacts with Z3 to obtain A1.

[0102] In one embodiment, in Formula 2a-Formula 2c, at least one nucleophile undergoes a Michael addition reaction with the β-carbon atom of at least one α,β-unsaturated carbonyl compound to generate the ionizable lipid compound with two forks containing a carbon-carbon bond, a carbon-oxygen bond, a carbon-nitrogen bond, a carbon-sulfur bond or a carbon-selenium bond.

[0103] In one embodiment, Formula 2a-Formula 2b further comprises the step of using the compound of Formula (2) with a terminal amino group obtained by the reaction of Formula 2a-Formula 2b as a starting material, and performing an iterative reaction according to Step 1 in Formula 2a or Steps 1-2 in Formula 2b;

[0104] The compound of formula (2) with a terminal amino group is

[0105] Among them, R b ' and R b "At the same time H, or R b ' and R b " is also a C1-C6 amine, or R b ' and R b "At the same time -R c '-A1'-R c ”-NH2.

[0106] In one embodiment, the reaction formula 2a-2c further comprises using the compound of formula (2) with a terminal amino group obtained by the reaction formula 2a-2c as a raw material, and The steps of the reaction;

[0107] The compound of formula (2) with a terminal amino group is

[0108] Among them, R b ' and R b "At the same time H, or R b ' and R b " is also a C1-C6 amine, or R b ' and R b "At the same time -R c '-A1'-R c ”-NH2.

[0109] The leaving group mentioned above refers to the leaving part in the nucleophilic reaction or condensation reaction, including but not limited to: H,

[0110] OH, H2O, halogen (such as F, Cl, Br and I), cyanate anion, inorganic acid (such as nitric acid, sulfuric acid, phosphoric acid), carboxylic acid (such as acetic acid, trifluoroacetic acid and benzoic acid, etc.), sulfonic acid (such as methanesulfonic acid, trifluoromethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid and p-nitrobenzenesulfonic acid, etc.), carbon dioxide (CO2), nitrogen (N2), imidazole, alkoxy (RO-), amino (-NHR, wherein R is an alkyl or aryl group with H removed), phenoxy, tertiary carbon cation (such as tert-butyl cation), carbon cation stabilized by an unsaturated system or a heteroatom, or the various protecting groups mentioned above.

[0111] The nucleophilic groups mentioned above refer to molecules or ions that can provide electron pairs to form new chemical bonds in chemical reactions. Common nucleophilic groups include: hydroxide (HO - ), ammonia (NH3), hydroxylamine (NH2OH), hydrazine (NH2-NH2), substituted hydrazines, nucleophilic halogens (such as Cl - Br - or I - ), hydrogen ions (H - ), azide anion (N3 - ), cyanate anion (CN - ), alcohol or alkoxy anion (such as alcohol with hydroxyl hydrogen removed), amine group (including primary amine, secondary amine and tertiary amine) or amine anion, carbon anion (such as carbon anion in organometallic reagents such as Grignard reagent, organic lithium reagent, Gilman reagent), sulfhydryl or sulfhydryl anion, sulfide, enol or enol anion, alkenyl ether, enamine, carboxylic acid or carboxylic acid anion, alkyl or aryl phosphine (such as triphenylphosphine), aromatic heterocycle with lone pair electrons (such as pyridine), etc.

[0112] In one embodiment, the starting materials used in the reaction further contain a protecting group, and the reaction steps include protection and / or deprotection steps.

[0113] Those skilled in the art will recognize that it may be necessary to use protecting groups to protect certain groups from reaction conditions. Protecting groups can also be used to distinguish similar functional groups in molecules. A list of protecting groups and methods for introducing and removing these groups can be found in Wuts, PGM, Greene, TW, Greene's Protective Groups in Organic Synthesis, 4th edition, John Wiley & Sons: New Jersey, 2007.

[0114] Protecting groups include, but are not limited to, protecting groups for hydroxyl groups, protecting groups for amino or amine groups, protecting groups for carboxyl groups, protecting groups for aldehydes or ketones, protecting groups for thiol groups, and protecting groups for any combination of functional groups such as hydroxyl groups, amino or amine groups, carboxyl groups, aldehydes or ketones, and thiol groups.

[0115] In one embodiment, the synthesis route of the first ionizable lipid is as follows:

[0116] Wherein, M' is M or M containing a protecting group.

[0117] In one embodiment, the synthesis route of the first ionizable lipid is as follows:

[0118] Wherein M' is M or M containing a protecting group; A is O, NH or S.

[0119] In one embodiment, the synthesis route of the first ionizable lipid is as follows:

[0120] Wherein, X is halogen; R4' is R4 or R4 containing a protecting group; R5' is R5 or R5 containing a protecting group.

[0121] In one embodiment, the synthesis route of the first ionizable lipid is as follows:

[0122] Wherein, X is halogen; R4' is R4 or R4 containing a protecting group; R5' is R5 or R5 containing a protecting group.

[0123] In one embodiment, the synthesis route of the first ionizable lipid is as follows:

[0124] Among them, M pro is M or M containing a protecting group.

[0125] In a second aspect, the present application provides a method for preparing the aforementioned lipid composition, comprising the following steps:

[0126] 1) dissolving the lipid compound in an organic solvent as an organic phase;

[0127] 2) using an aqueous solvent as the aqueous phase;

[0128] 3) The organic phase and the aqueous phase are mixed and ultrafiltered to obtain the lipid composition.

[0129] In one embodiment, after the lipid compound is dissolved in the organic phase in step 1), the total lipid concentration is 0.1 mg / mL-30 mg / mL; in step 3), the volume ratio of the organic phase to the aqueous phase is 1:1-6, more preferably 1:2-5.

[0130] There are many types of organic solvents, which can be divided into 10 categories according to their chemical structure: ① Aromatic hydrocarbons: benzene, toluene, xylene, etc.; ② Aliphatic hydrocarbons: pentane, hexane, octane, etc.; ③ Alicyclic hydrocarbons: cyclohexane, cyclohexanone, toluene, cyclohexanone, etc.; ④ Halogenated hydrocarbons: chlorobenzene, dichlorobenzene, dichloromethane, etc.; ⑤ Alcohols: methanol, ethanol, isopropanol, etc.; ⑥ Ethers: ethyl ether, propylene oxide, etc.; ⑦ Esters: methyl acetate, ethyl acetate, propyl acetate, etc.; ⑧ Ketones: acetone, methyl butyl ketone, methyl isobutyl ketone, etc.; ⑨ Diol derivatives: ethylene glycol monomethyl ether, ethylene glycol monoethyl ether, ethylene glycol monobutyl ether, etc.; ⑩ Others: acetonitrile, pyridine, phenol, etc.

[0131] The aqueous solvent mentioned in this application refers to a solution using water as a solvent, including but not limited to water, sugar solution, salt solution, etc.

[0132] In one embodiment, the aqueous solvent comprises at least one of water, sucrose solution, and buffered saline solution.

[0133] In one embodiment, the pH value of the buffered salt solution is 1-10, and the concentration of the buffered salt is 1-200 mM.

[0134] In one embodiment, the organic solvent comprises an alcohol, preferably ethanol.

[0135] In a third aspect, the present application provides a method for delivering a pharmaceutically active ingredient, wherein the method uses the aforementioned lipid composition as a carrier; the pharmaceutically active ingredient comprises at least one of a macromolecular substance and a small molecule substance.

[0136] In one embodiment, the macromolecular substance includes nucleic acid, protein, polypeptide, recombinant human insulin, interferon, human growth hormone, insulin analog, and staphylokinase.

[0137] In a fourth aspect, the present application provides the use of the aforementioned lipid composition in preparing a carrier for delivering pharmaceutically active ingredients.

[0138] Compared with the prior art, this application has the following beneficial effects:

[0139] 1. The present application provides a lipid composition, which comprises degradable components such as ionizable lipids, auxiliary lipids and PEG-conjugated lipids, and does not contain components with preventive or therapeutic effects.

[0140] 2. The lipid composition provided in this application has nanoparticle characteristics, a particle size of 2-400 nm, a PDI < 0.5, and good stability.

[0141] 3. The lipid composition provided in this application has a simple preparation process and high process reproducibility. It can achieve good encapsulation of the active ingredients by simply mixing with the active ingredients. No organic reagents need to be added during the combination with nucleic acids, small molecules or macromolecules, which is safer.

[0142] 4. The lipid composition provided in this application has a wide range of applications, and its use method is simple and has good reproducibility.

[0143] 5. The lipid composition provided in this application can deliver a variety of active ingredients such as nucleic acids, macromolecules and small molecules, meeting the delivery requirements of different active ingredients. As shown in the examples, it can effectively encapsulate nucleic acid substances such as mRNA, pDNA, and siRNA. DETAILED DESCRIPTION

[0144] The synthetic process of the present application can tolerate multiple functional groups, so various substituted starting materials can be used. These processes generally provide the desired final compound at the end or near the end of the entire process, but in some cases it may be necessary to further convert the compound into its pharmaceutically acceptable salt. The compounds of the present application can use commercially available starting materials, compounds known in the literature, or intermediates that are easy to prepare, by using standard synthetic methods and procedures known to those skilled in the art or obvious to the skilled person based on the teachings of this article, prepared in a variety of ways. Standard synthetic methods and procedures for preparing organic molecules and functional group transformations and operations can be obtained from relevant scientific literature or from standard textbooks in this area. The following description of the synthetic method is designed to illustrate, but not limit, the general procedures for preparing the compounds of the present application.

[0145] The compounds of the present invention having the various formulas described herein can be prepared from commercially available starting materials or starting materials that can be prepared using literature processes according to the processes described in the corresponding general synthetic routes. The variables in each general synthetic route (e.g., R1, R2, and R3, etc.) are as defined herein. One of ordinary skill in the art will note that in the reaction procedures and synthetic schemes described herein, the order of certain steps may be varied, such as the introduction and removal of protecting groups.

[0146] In the reaction schemes described herein, a variety of stereoisomers can be produced. When a specific stereoisomer is not indicated, this is understood to include all possible stereoisomers produced by the reaction. One of ordinary skill in the art will recognize that the reaction can be optimized to preferentially obtain a single isomer, or a new scheme can be designed to produce a single isomer. If a mixture is produced, the isomers can be separated using techniques such as preparative thin layer chromatography, preparative HPLC, preparative chiral HPLC, or preparative SFC.

[0147] (1) Synthesis of the first ionizable lipid in Example 1

[0148] The general synthetic routes for the first ionizable lipids are as shown in general synthetic routes 1 to 5.

[0149] General synthetic route 1

[0150] Wherein, M' is M or M containing a protecting group.

[0151] As described in the general synthetic route 1 above, Boc-aminotris(hydroxymethyl)methane reacts with an acid (Compound 2) to form Compound 3. Step 1 can be carried out in an organic solvent (e.g., dichloromethane (DCM)) in the presence of, for example, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCl) and 4-dimethylaminopyridine (DMAP). Step 1 can be carried out at room temperature for 24 hours.

[0152] Next, the Boc protecting group of compound 3 is removed to generate compound 4. Step 2 can be carried out in an organic solvent (eg, DCM) under the catalysis of an acid (eg, trifluoroacetic acid).

[0153] Next, compound 4 and compound 5 undergo condensation reaction to obtain compound 6. Step 3 can be carried out in an organic solvent (such as DCM or DMF) using EDCl and DMAP or dicyclohexylcarbodiimide (DCC) as a catalyst.

[0154] If the M' group of compound 6 contains the aforementioned protecting group, the protecting group is removed to obtain the target lipid compound. Step 4 is carried out under the selected protecting group removal reaction conditions.

[0155] General synthetic route 2

[0156] Wherein M' is M or M containing a protecting group; A is O, NH or S.

[0157] As described in the above general synthetic route 2, compound 1 and compound 2 undergo condensation reaction to give compound 3. Step 1 can be carried out in an organic solvent (eg, DCM) in the presence of EDCl and DMAP.

[0158] Next, the tert-butyloxy protecting group of compound 3 is removed to obtain compound 4. Step 2 can be carried out in an organic solvent (eg, DCM) in the presence of an acid (eg, trifluoroacetic acid) and a positive ion scavenger (eg, triisopropylsilane (TiPS)).

[0159] Next, compound 4 undergoes a condensation reaction with a compound 5 to obtain compound 6. Step 3 can be carried out in an organic solvent (such as DCM or DMF) in the presence of EDCl and DMAP or DCC.

[0160] If the M' group of compound 6 contains the above-mentioned protecting group, the protecting group is removed to obtain the target lipid compound. Step 4 is carried out under the deprotection reaction conditions of the selected protecting group.

[0161] General synthetic route 3

[0162] Wherein, X is a halogen, such as Cl, Br or I; R4' is R4 or R4 containing a protecting group; R5' is R5 or R5 containing a protecting group.

[0163] As described in the general synthetic route 3 above, Boc-aminotris(hydroxymethyl)methane undergoes a condensation reaction with compound 2 to produce compound 3. Step 1 can be carried out in an organic solvent (e.g., DCM) in the presence of, for example, EDCl and DMAP. Step 1 can be carried out at room temperature for 24 hours.

[0164] Next, the Boc protecting group of compound 3 is removed to generate compound 4. Step 2 can be carried out in an organic solvent (eg, DCM) under the catalysis of an acid (eg, trifluoroacetic acid).

[0165] Next, compound 4 is reacted with a halogen-substituted aldehyde (compound 5) through condensation and reduction to obtain compound 6. Step 3 can be carried out in an organic solvent (such as dichloroethane (DCE)) in the presence of a reducing agent (such as sodium triacetylborohydride (NaBH(OAc)3)).

[0166] Next, compound 6 reacts with an amine (compound 7) to produce compound 8. Step 4 can be carried out in an organic solvent (such as DMF) in the presence of a base (such as a non-nucleophilic organic base (e.g., triethylamine, iPr2EtN) or an inorganic base (e.g., K2CO3)) and a catalyst (KI or NaI).

[0167] If the R4' and / or R5' groups of compound 8 contain the aforementioned protecting groups, the protecting groups are removed to obtain the target lipid compound. Step 5 is carried out under the deprotection reaction conditions of the selected protecting groups.

[0168] General synthetic route 4

[0169] Wherein, X is a halogen, such as Cl, Br or I; R4' is R4 or R4 containing a protecting group; R5' is R5 or R5 containing a protecting group.

[0170] As described in the general synthetic route 4 above, Boc-aminotris(hydroxymethyl)methane undergoes a condensation reaction with compound 2 to produce compound 3. Step 1 can be carried out in an organic solvent (e.g., DCM) in the presence of, for example, EDCl and DMAP. Step 1 can be carried out at room temperature for 24 hours.

[0171] Next, the Boc protecting group of compound 3 is removed to generate compound 4. Step 2 can be carried out in an organic solvent (eg, DCM) under the catalysis of an acid (eg, trifluoroacetic acid).

[0172] Next, compound 4 undergoes a condensation reaction with a halogen-substituted compound 5 to obtain compound 6. Step 3 can be carried out in an organic solvent such as DCM or DMF using EDCl and DMAP or DCC as catalyst.

[0173] Next, compound 6 reacts with an amine (compound 7) to form compound 8. Step 4 can be carried out in an organic solvent (e.g., DMF) in the presence of a base (e.g., a non-nucleophilic organic base (e.g., triethylamine, iPr2EtN) or an inorganic base (e.g., K2CO3)) and a catalyst (KI or NaI).

[0174] If the R4' and / or R5' groups of compound 8 contain the aforementioned protecting groups, the protecting groups are removed to obtain the target lipid compound. Step 5 is carried out under the deprotection reaction conditions of the selected protecting groups.

[0175] General synthetic route 5

[0176] Among them, M pro is M or M containing a protecting group.

[0177] As described in the general synthetic route 5 above, Boc-aminotris(hydroxymethyl)methane undergoes a condensation reaction with compound 2 to produce compound 3. Step 1 can be carried out in an organic solvent (e.g., DCM) in the presence of, for example, EDCl and DMAP. Step 1 can be carried out at room temperature for 24 hours.

[0178] Next, the Boc protecting group of compound 3 is removed to generate compound 4. Step 2 can be carried out in an organic solvent (eg, DCM) under the catalysis of an acid (eg, trifluoroacetic acid).

[0179] Next, compound 4 and compound 5 are subjected to condensation and reduction reaction to obtain compound 6. Step 3 can be carried out in an organic solvent (such as dichloroethane (DCE)) in the presence of a reducing agent (such as sodium triacetylborohydride (NaBH(OAc)3)).

[0180] If the M of compound 6 pro If the group contains the above-mentioned protecting group, the protecting group is removed to obtain the target lipid compound. Step 4 is carried out under the deprotection reaction conditions of the selected protecting group.

[0181] Furthermore, it should be understood that any particular embodiment of the present application that is within the prior art may be explicitly excluded from any one or more claims. Because such embodiments are deemed to be known to one of ordinary skill in the art, they may be excluded even if such exclusion is not explicitly stated herein.

[0182] All cited sources, such as references, publications, databases, database entries, and techniques cited herein, are incorporated herein by reference, even if not explicitly stated in the citation. In the event of a conflict between a cited source and the present application, the present application shall prevail.

[0183] (1) Synthesis of a compound according to formula (1), (1A), (1B), (1C), (1D), (1E), (1F), (1G), (1H), (1I), (1J), (1K), (1L) or (1M)

[0184] A. General considerations

[0185] It is worth noting that the raw materials used in this application are all common commercially available products, and their sources are not specifically limited.

[0186] The process routes described below can be used to synthesize compounds 1001-3422 of this application.

[0187] The following abbreviations are used herein: THF: tetrahydrofuran MeCN: acetonitrile MeOH: methanol PE: petroleum ether EA: ethyl acetate DMF: N,N-dimethylformamide EDCl: 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride LAH: lithium aluminum hydride DCM: dichloromethane DMAP: 4-dimethylaminopyridine LDA: lithium diisopropylamide rt: room temperature DCE: 1,2-dichloroethane n-BuLi: n-butyllithium i-Pr2EtN: N,N-diisopropylethylamine

[0188] B. Intermediate Synthesis

[0189] Intermediate A:

[0190] Intermediate A is obtained by the following synthetic process:

[0191] Tris(hydroxymethyl)aminomethane (50.0 g) and di-tert-butyl carbonic anhydride (Boc2O) (99.1 g) were dissolved in a methanol (300 mL) / H2O (30 mL) mixed solvent and reacted at room temperature for 72 h. The mixture was purified by silica gel column chromatography (DCM:MeOH = 20:1-10:1) to obtain a white solid compound intermediate A (N-Boc-aminotrihydroxymethylmethane) (75.0 g). LCMS (ESI) calculation for C9H 19 NO 5， [M+H] + m / z 222.13, found 222.25.

[0192] Intermediate B:

[0193] Intermediate B is obtained by the following synthetic process:

[0194] To a single-necked flask, diethyl 2-ethyl-1,3-propanedioate (29.0 g), THF (90 mL), and DMF (30 mL) were added, and NaH (3.7 g) was added under ice bath. The mixture was stirred at room temperature for 30 min under nitrogen protection; pentadecane bromide (15.0 g) was added, and the mixture was reacted at 80° C. for 2 h under nitrogen protection; THF was concentrated under reduced pressure to remove THF, and the reaction solution was added dropwise to ice water. Ethyl acetate was added and stirred, and the mixture was filtered through diatomaceous earth. The organic phase was collected and then separated. The organic phase was washed with saturated brine, separated, and purified by silica gel column chromatography (PE:EA=100:1-50:1) to obtain the compound diethyl 2-ethyl-2-pentadecylamealate (16.5 g). To diethyl 2-ethyl-2-pentadecylmaleate (8.0 g) was added EtOH (50 mL), H2O (50 mL), and KOH (11.3 g), and the mixture was reacted at 90°C for 12 h; the EtOH was removed by concentration under reduced pressure, and the reaction system was adjusted to pH 4-5 by adding dilute hydrochloric acid. Water and ethyl acetate were added, stirred, and separated, and the organic phase was washed with saturated brine, separated, and purified by column chromatography (PE:EA=5:1-1:1) to obtain 2-ethyl-2-pentadecylmaleic acid (5.2 g). 2-ethyl-2-pentadecylmaleic acid (10.0 g) was reacted at 170°C for 6 h under open conditions, cooled to room temperature, and water and ethyl acetate were added, stirred, separated, and the organic phase was washed with saturated brine, separated, concentrated, and purified by column chromatography (DCM:MeOH=20:1-10:1) to obtain intermediate B (2-ethylheptadecanoic acid) (8.2 g). 1HNMR (400MHz, CDCl3) δ2.31 (tt, J = 8.6, 5.3Hz, 1H), 1.71-1.46 (m, 4H), 1.28 (s, 26H), 0.96 (t, J = 7.4Hz, 3H), 0.90 (t, J = 6.7Hz, 3H).

[0195] Intermediate C:

[0196] Intermediate C is obtained by the following synthetic process:

[0197] To a three-necked flask, add decanoic acid (50 g) and THF (500 mL). After cooling the system to 0°C, slowly add NaH (23.22 g). After stirring at 0°C under nitrogen for 1 hour, slowly add LDA (62.19 g) dropwise. Stirring at 0°C under nitrogen for another 1 hour is then continued. Finally, add iodonane (88.52 g) dropwise, warm to room temperature, stir overnight, then dilute with 1 L of DCM and wash with saturated NH4Cl solution and water, respectively. The organic layer is dried over anhydrous Na2SO4, filtered, and concentrated. Purification by silica gel chromatography (PE:EA = 50:1) yields intermediate C (2-octylundecanoic acid) (11 g). 1H NMR (400MHz, Chloroform-d) δ2.37 (tt, J = 8.7, 5.1 Hz, 1H), 1.71-1.58 (m, 2H), 1.48 (dt, J = 13.3, 6.7 Hz, 2H), 1.29 (d, J = 9.4 Hz, 25H), 0.90 (t, J = 6.8 Hz, 6H).

[0198] Intermediate D:

[0199] Intermediate D is obtained by the following synthetic route:

[0200] Tridecanoic acid (79.5 g) and THF (800 mL) were added to a three-necked flask. The temperature was lowered to 0°C, and NaH (22.25 g) was slowly added. The reaction mixture was stirred at 0°C under nitrogen for 1 hour. LDA (317.87 g) was then slowly added dropwise to the reaction mixture. The reaction mixture was stirred at 0°C under nitrogen for another 1 hour. After adding n-hexyl iodide (94.3 g) dropwise, the mixture was warmed to room temperature and stirred overnight. The reaction mixture was then diluted with 1 L of DCM and washed with saturated NH4Cl solution and then water. The organic layer was dried over anhydrous Na2SO4, filtered, concentrated, and purified by silica gel column chromatography (PE:EA = 50:1) to yield intermediate D (2-hexyltridecanoic acid) (26 g). 1H NMR(400MHz,Chloroform-d)δ2.36(tt,J=8.7,5.4Hz,1H),1.63(ddd,J=14.3,8.7,5.5 Hz, 2H), 1.49 (dq, J = 13.5, 6.6 Hz, 2H), 1.29 (d, J = 10.1 Hz, 26H), 0.90 (t, J = 6.6 Hz, 6H).

[0201] Intermediate E:

[0202] Intermediate E is obtained by the following synthetic process:

[0203] Tris(hydroxymethyl)aminomethane (10.0 g) and tert-butyl acrylate (21.1 1) were dissolved in EtOH (150 mL) and reacted at 45°C under nitrogen for 30 h. The mixture was then concentrated under reduced pressure to remove the EtOH. Three 100 mL portions of (PE:EA=20:1) were added for slurrying, and the mixture was filtered to obtain Intermediate E (11.5 g) as a white solid. 1H NMR (400 MHz, CDCl3) δ 3.60 (s, 6H), 2.84 (t, J=5.9 Hz, 2H), 2.47 (t, J=5.8 Hz, 2H), 1.48 (s, 9H).

[0204] Intermediate F:

[0205]

[0206] Intermediate F is obtained by the following synthetic route:

[0207] To a single-necked flask, tert-butyl (2-aminoethyl)carbamate (2.7 g), MeCN (90 mL), benzyl 2-bromoethyl ether (7.99 g), and K2CO3 (11.65 g) were added. After reacting at 80°C overnight, water and ethyl acetate were added and stirred for separation. The organic phase was washed with saturated brine, separated, concentrated, and purified by silica gel column chromatography (PE:EA=10:1-5:1) to obtain compound 2 (5.5 g). Dioxane (30 mL) and dioxane hydrochloride solution (30 mL) were added to compound 2, stirred at room temperature for 3 h, and then concentrated under reduced pressure to obtain compound 3, intermediate F (6 g). LCMS (ESI) calculated for C 20 H 28 N2O2,[M+H] + m / z 329.22, found 329.24.

[0208] Intermediate G:

[0209] Intermediate G is obtained by the following synthetic process:

[0210] Compound 1 (3.0 g), MeCN (90 mL), 3-benzyloxypropane bromide (9.45 g), and K2CO3 (12.94 g) were added to a single-necked flask and reacted overnight at 80°C. Water and ethyl acetate were then added for separation by stirring. The organic phase was washed with saturated brine, separated, concentrated, and purified by column chromatography (PE:EA=10:1-5:1) to obtain compound 2 (6.9 g). Dioxane (30 mL) and dioxane hydrochloride solution (30 mL) were added to compound 2 (6.9 g). After stirring at room temperature for 3 h, the mixture was concentrated under reduced pressure to obtain intermediate G (7.5 g). LCMS (ESI) calculated for C 22 H 32 N2O2,[M+H] + m / z 357.25, found 357.51.

[0211] Intermediate H:

[0212] Intermediate H is obtained by the following synthetic process:

[0213] Compound 1 (1.5 g), MeCN (90 mL), 4-bromobutyl benzyl ether (5.02 g), and K2CO3 (6.47 g) were added to a single-necked flask and reacted overnight at 80°C. Water and ethyl acetate were then added and stirred for separation. The organic phase was washed with saturated brine, separated, concentrated, and purified by column chromatography (PE:EA = 20:1-10:1) to obtain compound 2 (4.1 g). Dioxane (30 mL) and dioxane hydrochloride solution (30 mL) were added to compound 2 (4.1 g). After stirring at room temperature for 3 h, the mixture was concentrated under reduced pressure to obtain intermediate H (4.9 g). LCMS (ESI) calculated for C 24 H 36 N2O2, [M+H] + m / z 385.28, found 385.56.

[0214] Intermediate I:

[0215] Intermediate I is obtained by the following synthetic route:

[0216] Compound 1 (3.9 g), MeCN (90 mL), benzyl 2-bromoethyl ether (5.78 g), and K2CO3 (15.46 g) were added to a single-necked flask and reacted overnight at 80°C. Water and ethyl acetate were then added for separation by stirring. The organic phase was washed with saturated brine, separated, concentrated, and purified by column chromatography (PE:EA=20:1-10:1) to obtain compound 2 (6.8 g). Dioxane (30 mL) and dioxane hydrochloride solution (30 mL) were added to compound 2 (6.8 g). After stirring at room temperature for 3 h, the mixture was concentrated under reduced pressure to obtain intermediate I (6.5 g). LCMS (ESI) calculated for C 12 H 20 N2O, [M+H] + m / z 209.16, found 209.31.

[0217] Intermediate J:

[0218] Intermediate J is obtained using the following synthetic route:

[0219] To a single-necked flask, butyric acid (5.0 g) and THF (100 mL) were added. NaH (2.73 g) was added at 0°C, and LDA (56.8 mL) was slowly added dropwise. The mixture was reacted at room temperature for 30 min. 1-Bromotridecane was added and the reaction was continued at room temperature overnight. Ice water and ethyl acetate were added and stirred for separation. The organic phase was washed with saturated brine, separated, concentrated, and purified by column chromatography (PE:EA = 10:1-5:1) to obtain intermediate J (5.0 g). LCMS (ESI) calculated for C 17 H 34 O2, [M+H] + m / z 271.26, found 271.46.

[0220] Intermediate K:

[0221] Intermediate K is obtained by the following synthetic process:

[0222] To a three-necked flask, reactant 1 (24 g), imidazole (19.00 g), and DCM (200 mL) were added. The temperature was lowered to 0°C, and then TBDMSCI (38.57 g) was slowly added. After stirring at 25°C for 4 h, the mixture was diluted with 300 mL of DCM. The organic phase was washed twice with 1 L of water and dried over anhydrous Na2SO4. After filtration and concentration, silica gel was added and column chromatography with 1:20 DCM:MeOH was performed. The sample was collected and concentrated to yield intermediate K (28.00 g). 1H NMR (400 MHz, CDCl3) δ 4.89 (s, 1H), 3.58 (t, J = 6.1 Hz, 2H), 2.66 (t, J = 7.3 Hz, 2H), 2.45 (s, 3H), 1.67-1.40 (m, 4H), 0.84 (s, 9H).

[0223] Intermediate L:

[0224] Intermediate L is obtained by the following synthetic process:

[0225] Compound DMSO (3.58 g) and anhydrous DCM (30 mL) were added to a three-necked flask. The system was cooled to -78°C and oxalyl chloride (2.91 g) was slowly added. The reaction solution was stirred at -78°C under N2 protection for 10 minutes, and compound 1 (3 g) was slowly added dropwise. Stirring was continued at -78°C under N2 protection for 1 hour. Finally, TEA (9.28 g) was added dropwise. Stirring was continued at -78°C under N2 protection for 0.5 hour, and the mixture was diluted with 100 mL DCM and washed with saturated NH4Cl solution and water, respectively. The organic layer was dried over anhydrous Na2SO4, filtered, and concentrated to obtain intermediate L (3.0 g). LCMS (ESI) calculated for C 11 H 14 O3, [M+H] + m / z 195.09, found 195.23.

[0226] Intermediate M:

[0227] Intermediate M is obtained by the following synthetic process:

[0228] To a sealed tube, intermediate K (2.0 g), compound 1 (1.79 g), and ACN (20 mL) were added. The reactants, KCO (3.81 g) and KI (1.52 g), were then added. The mixture was stirred overnight at 70°C under N2 protection, then diluted with 100 mL of EA and washed with saturated NH4Cl solution and water. The organic layer was dried over anhydrous Na2SO4, filtered, and concentrated. The mixture was then added to a silica gel column and filtered with 15:1 DCM:MeOH. The sample was collected and concentrated to yield compound 2 (2.0 g). Reactant 2 (1.80 g) and solvent DCM (20 mL) were added to a three-necked flask. The system was purged with nitrogen and the temperature was lowered to -78°C. DIBAL-H (1.00 g) was then added dropwise. After stirring at -78°C for 4 h, methanol and sodium carbonate solution were added dropwise to the reaction solution to quench the mixture. The mixture was then extracted with DCM (100 mL). The organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated to obtain intermediate M (1.36 g). LCMS (ESI) calculated for C 15 H 33 NO2Si,[M+H] + m / z 288.52, found 288.23.

[0229] C. Compound 1003 was synthesized according to general synthetic route 1

[0230] Structural formula:

[0231] Chemical formula: C 63 H 122 N2O7

[0232] Molecular weight: 1019.68

[0233] Step 1: Synthesis of compound 3 in general synthetic route 1

[0234] Compound 1 (intermediate A) (1.0 g), DCM (20 mL), DMAP (2.2 g), and EDCI (3.4 g) were added to compound 2 (intermediate B) (4.3 g). The mixture was stirred at room temperature for 12 h under nitrogen protection, and then water and dichloromethane were added for stirring and separation. The organic phase was washed with saturated brine and separated. The organic phase was dried over anhydrous sodium sulfate and concentrated. The organic phase was purified by silica gel column chromatography (PE:EA=40:1-30:1) to give compound 3 (2.6 g).

[0235] Step 2: Synthesis of compound 4 in general synthetic route 1

[0236] DCM (15 mL) and TFA (5 mL) were added to compound 3 (2.6 g), and the mixture was stirred at room temperature for 3 h, concentrated, and purified by silica gel column chromatography (PE:EA=10:1-5:1) to give compound 4 (3.2 g).

[0237] Step 3: Synthesis of compound 6 in general synthetic route 1

[0238] To compound 4 (3.2 g) were added DCM (20 mL), Boc-glycine (compound 5, 578 mg), and DCC (6.8 g). The mixture was stirred at room temperature under nitrogen for 12 h, and then water and ethyl acetate were added for stirring and separation. The organic phase was washed with saturated brine, separated, dried over anhydrous sodium sulfate, concentrated, and purified by column chromatography (PE:EA=5:1-2:1) to give compound 6 (2.2 g).

[0239] Step 4: Compound 1003

[0240] To compound 6 (2.2 g) were added DCM (15 mL) and TFA (5 mL), and the mixture was stirred at room temperature for 12 h and concentrated. The mixture was purified by column chromatography (DCM:MeOH=30:1-20:1) to give compound 1003 (1.3 g). 1 H NMR (400MHz, CDCl3) δ4.49 (d, J=2.6Hz, 6H), 3.28 (s, 2H), 2.32 (tt, J=8.6, 5. 5Hz, 3H), 1.62 (q, J = 7.1Hz, 12H), 1.27 (d, J = 3.4Hz, 78H), 0.94-0.86 (m, 18H).

[0241] D. Compound 1002 was synthesized according to general synthetic route 1

[0242] Structural formula:

[0243] Chemical formula: C 60 H 116 N2O7

[0244] Molecular weight: 977.60

[0245] The compound was synthesized according to the general synthetic route 1, which is similar to the synthetic process of compound 1003, except that intermediate C was used as compound 2 in the general synthetic route 1. 1H NMR (400MHz, CDCl3) δ4.48 (s, 6H), 3.27 (s, 2H), 2.37 (tt, J = 8.5, 5.5Hz, 3H), 1 .64(d,J=6.9Hz,6H),1.50-1.42(m,6H),1.27(s,72H),0.90(t,J=6.7Hz,18H).

[0246] E. Compound 1004 was synthesized according to general synthetic route 1

[0247] Structural formula:

[0248] Chemical formula: C 54 H 104 N2O7

[0249] Molecular weight: 893.43

[0250] The compound was synthesized according to the general synthetic route 1, which is similar to the synthetic process of compound 1003, except that palmitic acid was used as compound 2 in the general synthetic route 1. 1 HNMR (400MHz, CDCl3) δ7.55(s,1H),4.45(s,6H),3.74(s,2H),2.35(t,J=7.6Hz,6H),1.60(p,J=6.9Hz,6H),1.27(s,72H),0.90(t,J=6.7Hz,9H).

[0251] F. Compound 1001 was synthesized according to general synthetic route 1

[0252] Structural formula:

[0253] Chemical formula: C 59 H 108 N2O7

[0254] Molecular weight: 957.52

[0255] The product was synthesized according to the general synthetic route 1, which is similar to the synthetic process of compound 1003, except that (9Z)-9-hexadecenoic acid was used as compound 2 in the general synthetic route 1, and 5-(N,N-dimethylamino)pentanoic acid was used as compound 5 in the general synthetic route 1. 1H NMR (400MHz, CDCl3) δ5.42-5.30(m,6H),4.51-4.38(m,6H),3.19(dt,J=11.3,5 .6Hz,2H),2.97(t,J=5.9Hz,6H),2.46(t,J=6.6Hz,1H),2.35(td,J=7.6,3.1Hz, 5H),2.20(t,J=7.6Hz,2H),2.03(d,J=6.2Hz,12H),1.89-1.81(m,3H),1.74(q, J=7.8Hz,2H),1.67-1.51(m,6H),1.31(d,J=7.8Hz,48H),0.90(t,J=6.7Hz,9H).

[0256] G. Compound 1014 was synthesized according to general synthetic route 2

[0257] Structural formula:

[0258] Chemical formula: C 70 H 137 N3O9

[0259] Molecular weight: 1164.88

[0260] Step 1: Synthesis of Intermediate 3 in General Synthesis Route 2

[0261] Intermediate 3 was synthesized according to the method shown in step 1 of general synthetic route 1, except that intermediate E was used as compound 1 in general synthetic route 1.

[0262] Step 2: Synthesis of compound 4 of general synthetic route 2

[0263] DCM (15 mL), TFA (12 mL), and TiPS (3 mL) were added to compound 3 (4.8 g). After reacting at room temperature for 12 h, water and dichloromethane were added for stirring and separation. The organic phase was washed with saturated brine, separated, concentrated, and purified by silica gel column chromatography (DCM:MeOH=20:1-10:1) to give compound 4 (3.3 g).

[0264] Step 3: Synthesis of compound 6 of general synthetic route 2

[0265] Compound 4 (4.0 g), DCM (80 mL), EDCI (1.1 g), 1-hydroxybenzotriazole (HOBt) (0.78 g), and DIEA (2.5 g) were added to compound 5 (1.9 g). After stirring at room temperature for 12 h, water and dichloromethane were added, stirred and separated, and the organic phase was washed with saturated brine. The organic phase was separated and concentrated, and purified by silica gel column chromatography (DCM:MeOH=30:1-20:1) to give compound 6 (4.0 g).

[0266] Step 4: Compound 1014

[0267] To compound 6 (4.0 g) were added MeOH (30 mL), DCM (10 mL), and Pd / C (4.73 g (10%)). The mixture was stirred at room temperature overnight under hydrogen atmosphere, filtered through celite, concentrated, and purified by silica gel column chromatography (DCM:MeOH 30:1-15:1) to give compound 1014 (2 g). 1 H(400MHz, CDCl3)δ7.38(t,J=5.8Hz,1H),4.14-4.05(m,6H),3.58(t,J=4.9Hz,4H),3.31(q,J=5.7Hz,2H),2.89(t,J=6.3Hz, 2H), 2.64 (q, J=5.2Hz, 6H), 2.29 (dq, J=10.8, 4.4Hz, 6H), 1.66-1.37 (m, 12H), 1.24 (d, J=4.3Hz, 78H), 0.87 (t, J=7.1Hz, 18H).

[0268] H. Compound 1015 was synthesized according to general synthetic route 2

[0269] Structural formula:

[0270] Chemical formula: C 72 H 141 N3O9

[0271] Molecular weight: 1192.93

[0272] The compound was synthesized according to general synthetic route 2, which is similar to the synthetic process of compound 1014, except that intermediate G was used as compound 5 in general synthetic route 2. 1H(400MHz, CDCl3)δ7.58(t,J=5.7Hz,1H),4.15-4.01(m,6H),3.70(t,J=5.4Hz,4H),3.39(q,J=5.7Hz,2H),2.86(t,J=6.2Hz,2H),2 .57(dt,J=20.5,5.8Hz,6H),2.36-2.22(m,5H),1.76-1.67(m,4H),1.62-1.35(m,13H),1.23(d,J=4.6Hz,78H),0.92-0.80(m,18H).

[0273] I. Compound 1020, synthesized according to general synthetic route 2

[0274] Structural formula:

[0275] Chemical formula: C 74 H 145 N3O9

[0276] Molecular weight: 1220.99

[0277] The compound was synthesized according to the general synthetic route 2, which is similar to the synthetic process of compound 1014, except that intermediate H was used as compound 5 in the general synthetic route 2.

[0278] J. Compound 1025 was synthesized according to general synthetic route 2

[0279] Structural formula:

[0280] Chemical formula: C 69 H 135 N3O8

[0281] Molecular weight: 1134.85

[0282] The compound was synthesized according to the general synthetic route 2, which is similar to the synthetic process of compound 1014, except that intermediate I was used as compound 5 in the general synthetic route 2. 1 H(400MHz, CDCl3)δ6.98(t,J=5.7Hz,1H),4.10(m,J=3.8Hz,6H),3.63(t,J=5.2Hz,2H),3.35(q,J=5.8Hz,2H),2.89(t,J=6.1Hz,2 H), 2.57 (q, J = 5.3Hz, 4H), 2.32 (s, 3H), 2.28 (q, J = 4.1Hz, 5H), 1.65-1.41 (m, 13H), 1.24 (d, J = 4.4Hz, 78H), 0.87 (t, J = 7.1Hz, 18H).

[0283] K. Compound 1005, synthesized according to general synthetic route 2

[0284] Structural formula:

[0285] Chemical formula: C 68 H 133 N3O7

[0286] Molecular weight: 1104.83

[0287] The compound 5 was synthesized according to the general synthetic route 2, which is similar to the synthetic process of compound 1014, except that N,N-dimethylethylenediamine was used as compound 5 in the general synthetic route 2. 1 H NMR (400MHz, CDCl3) δ4.19-4.07(m,6H),3.40(d,J=5.7Hz,2H),2.95-2.87(m,2H),2.82(s,4H),2.64-2.55(m,2H),2.38 (s, 6H), 2.33-2.29 (m, 3H), 1.63-1.55 (m, 6H), 1.49-1.44 (m, 2H), 1.26 (d, J = 5.4Hz, 78H), 0.89 (td, J = 7.0, 2.8Hz, 18H).

[0288] L. Compound 1006 was synthesized according to general synthetic route 2

[0289] Structural formula:

[0290] Chemical formula: C 53 H 103 N3O7

[0291] Molecular weight: 894.42

[0292] The compound was synthesized according to general synthetic route 2, which is similar to the synthetic process of compound 1014, except that tetradecanoic acid was used as compound 2 in general synthetic route 2 and N,N-dimethylethylenediamine was used as compound 5 in general synthetic route 2. 1 H NMR (400MHz, CDCl3) δ7.99(t,J=5.8Hz,1H),4.13(s,6H),3.60(q,J=5.3Hz,2H),3.38-3.34(m,2H),3.00(s,6H),2.94(t, J=5.8Hz,2H),2.45(t,J=5.8Hz,2H),2.35(t,J=7.6Hz,6H),1.66-1.56(m,6H),1.33-1.23(m,60H),0.90(t,J=6.8Hz,9H).

[0293] M. Compound 1007, synthesized according to general synthetic route 2

[0294] Structural formula:

[0295] Chemical formula: C 68 H 133 N3O7

[0296] Molecular weight: 1104.83

[0297] The compound was synthesized according to general synthetic route 2, which is similar to the synthetic process of compound 1014, except that intermediate D was used as compound 2 in general synthetic route 2 and N,N-dimethylethylenediamine was used as compound 5 in general synthetic route 2. 1 H NMR (400MHz, Chloroform-d) δ7.13(t,J=5.0Hz,1H),4.13(s,6H),3.33(q,J=5.6Hz,2H),2.92(t,J=6.0Hz,2H),2.47-2.28(m,7H ), 2.24 (s, 6H), 1.60 (dq, J = 14.7, 7.2Hz, 6H), 1.47 (dd, J = 14.2, 6.7Hz, 6H), 1.27 (d, J = 3.4Hz, 78H), 0.89 (td, J = 6.7, 2.5Hz, 18H).

[0298] N. Compound 1008, synthesized according to general synthetic route 2

[0299] Structural formula:

[0300] Chemical formula: C 68 H 133 N3O7

[0301] Molecular weight: 1104.01

[0302] The compound was synthesized according to general synthetic route 2, which is similar to the synthetic process of compound 1014, except that intermediate C was used as compound 2 in general synthetic route 2 and N,N-dimethylethylenediamine was used as compound 5 in general synthetic route 2. 1H NMR(400MHz,Chloroform-d)δ4.13(s,6H),3.33(q,J=5.6Hz,2H),2.92(t,J=5.9Hz,2H),2.51-2.26(m,7H),2.24 (s, 6H), 1.59 (ddt, J = 14.8, 10.9, 6.4Hz, 6H), 1.47 (tq, J = 11.0, 5.4Hz, 6H), 1.26 (s, 77H), 0.89 (t, J = 6.7Hz, 18H).

[0303] O. Compound 1009, synthesized according to general synthetic route 2

[0304] Structural formula:

[0305] Chemical formula: C 62 H 121 N3O7

[0306] Molecular weight: 1020.66

[0307] The compound was synthesized according to general synthetic route 2, which is similar to the synthetic process of compound 1014, except that intermediate J was used as compound 2 in general synthetic route 2 and N,N-dimethylethylenediamine was used as compound 5 in general synthetic route 2. 1 H(400MHz, CDCl3)δ7.04(t,J=5.0Hz,1H),4.12(m,J=3.3Hz,6H),3.30(q,J=5.6Hz,2H),2.91(t,J=6.0Hz,2H),2.39 (t,J=6.0Hz,2H),2.33-2.25(m,5H),2.22(s,6H),1.76-1.54(m,12H),1.24(m,J=3.7Hz,66H),0.90-0.84(m,18H).

[0308] P. Compound 1011 was synthesized according to general synthetic route 2

[0309] Structural formula:

[0310] Chemical formula: C 70 H 137 N3O7

[0311] Molecular weight: 1132.88

[0312] The compound 5 was synthesized according to the general synthetic route 2, which is similar to the synthetic process of compound 1014, except that N,N-diethylethylenediamine was used as compound 5 in the general synthetic route 2. 1H NMR (400MHz, CDCl3) δ4.19-4.08(m,6H),3.31(q,J=5.8Hz,2H),2.92(t,2H),2.57(t,J=6.9Hz,6H),2.37-2.26( m,5H),1.62(ddd,J=18.3,9.0,4.3Hz,12H),1.27(d,J=4.7Hz,78H),1.04(t,J=7.1Hz,6H),0.94-0.86(m,18H).

[0313] Q. Compound 1012, synthesized according to general synthetic route 2

[0314] Structural formula:

[0315] Chemical formula: C 72 H 141 N3O7

[0316] Molecular weight: 1160.93

[0317] The compound 5 was synthesized according to the general synthetic route 2, which is similar to the synthetic process of compound 1014, except that N,N-diethylethylenediamine was used as compound 5 in the general synthetic route 2. 1 H NMR (400MHz, CDCl3) δ6.59 (s, 1H), 4.18-4.07 (m, 6H), 3.29 (q, J = 5.7Hz, 2H), 2.91 (t, J = 6.2Hz, 2H), 2.54 (t, J = 6.1 Hz, 2H), 2.40 (t, J = 7.5Hz, 4H), 2.35-2.27 (m, 5H), 1.63-1.42 (m, 16H), 1.27 (m, J = 4.5Hz, 78H), 0.93-0.85 (m, 24H).

[0318] R. Compound 1013, synthesized according to general synthetic route 2

[0319] Structural formula:

[0320] Chemical formula: C 74 H 145 N3O7

[0321] Molecular weight: 1188.99

[0322] The compound 5 was synthesized according to the general synthetic route 2, which is similar to the synthetic process of compound 1014, except that N,N-diethylethylenediamine was used as compound 5 in the general synthetic route 2. 1HNMR (400MHz, CDCl3) δ4.10(d,J=4.1Hz,6H),3.26(q,J=5.8Hz,2H),2.89(t,J=6.2Hz,2H),2.52(d,J=6.6Hz,2H) ,2.41(t,J=7.4Hz,4H),2.33-2.25(m,5H),1.60(d,J=11.8Hz,16H),1.24(d,J=4.5Hz,82H),0.96-0.79(m,24H).

[0323] S. Compound 1029, synthesized according to general synthetic route 2

[0324] Structural formula:

[0325] Chemical formula: C 68 H 132 N2O8

[0326] Molecular weight: 1105.81

[0327] The compound 5 was synthesized according to the general synthetic route 2, which is similar to the synthetic process of compound 1014, except that N,N-diethyl-2-hydroxyethylamine was used as compound 5 in the general synthetic route 2. 1 H NMR (400MHz, CDCl3) δ4.16(t,J=5.8Hz,2H),4.13-4.05(m,6H),2.86(t,J=6.3Hz,2H),2.55(t,J=5.8Hz,2H) ,2.44(t,J=6.3Hz,2H),2.35-2.20(m,9H),1.60-1.39(m,11H),1.24(d,J=4.5Hz,80H),1.02-0.79(m,18H).

[0328] T. Compound 1111, synthesized according to general synthetic route 2

[0329] Structural formula:

[0330] Chemical formula: C 70 H 136 N4O7

[0331] Molecular weight: 1145.88

[0332] The product was synthesized according to general synthetic route 2, which was similar to the synthetic process of compound 1014, except that intermediate C was used as compound 2 in general synthetic route 2 and tert-butyl 4-(2-aminoethyl)piperazine-1-carboxylate was used as compound 5 in general synthetic route 2. 1H NMR (400 MHz, Chloroform-d) δ 4.28 (s, 6H), 3.41 (s, 2H), 3.31 (s, 4H), 3.22 (s, 3H), 2.97-2.80 (m, 5H), 2.70 (s, 5H), 2.46-2.32 (m, 4H), 1.60 (t, J = 7.1 Hz, 6H), 1.54-1.40 (m, 6H), 1.27 (s, 78H), 0.89 (t, J = 6.7 Hz, 18H).

[0333] U. Compound 1118, synthesized according to general synthetic route 2

[0334] Structural formula:

[0335] Chemical formula: C 71 H 139 N3O8

[0336] Molecular weight: 1162.91

[0337] The compound was synthesized according to general synthetic route 2, which is similar to the synthetic process of compound 1014, except that intermediate C was used as compound 2 in general synthetic route 2 and intermediate K was used as compound 5 in general synthetic route 2. 1 H NMR(400MHz,Chloroform-d)δ4.11(s,6H),3.61(s,2H),3.42(d,J=33.5Hz,2H),2.91(t,J=6.2Hz,3H ), 2.36 (dd, J = 9.2, 3.6Hz, 6H), 1.47 (dd, J = 13.9, 6.2Hz, 14H), 1.27 (s, 71H), 0.90 (t, J = 6.7Hz, 18H).

[0338] V. Compound 1010, synthesized according to general synthetic route 3

[0339] Structural formula:

[0340] Chemical formula: C 67 H 133 N3O6

[0341] Molecular weight: 1076.82

[0342] Step 1 and Step 2: Synthesis of Compound 3 and Compound 4 of General Synthesis Route 3

[0343] According to steps 1 and 2 of general synthetic route 1, the synthesis process is the same as that of compound 1003.

[0344] Step 3: Synthesis of compound 6 of general synthetic route 3

[0345] Compound 4 (600 mg), chloroacetaldehyde (compound 5, 242 mg (40%)), and NaBH(OAc)3 (394 mg) were added to DCE (60 mL). After reacting at room temperature overnight, water and dichloromethane were added, stirred, and the liquids were separated. The organic phase was washed with saturated brine, separated, and concentrated. Compound 6 (270 mg) was obtained by silica gel column chromatography (DCM:MeOH=30:1-15:1).

[0346] Step 4: Synthesis of Compound 1010

[0347] MeCN (20 mL), N,N-dimethylethylenediamine (compound 7, 229 mg), KI (43 mg), and K2CO3 (179 mg) were added to compound 6 (270 mg). The mixture was reacted at 70°C under nitrogen protection overnight, and then silica gel column chromatography was used to obtain compound 1010 (16 mg). 1 H(400MHz, CDCl3)δ4.10(m,J=2.9Hz,6H),2.76-2.64(m,6H),2.40(t,J=6.2Hz,2H),2.29(ddd,J=8.5,5.6 ,2.9Hz,3H),2.22(s,6H),1.97(s,6H),1.50-1.41(m,6H),1.24(d,J=4.2Hz,78H),0.87(t,J=7.1Hz,18H).

[0348] W. Compound 1059, synthesized according to general synthetic route 3

[0349] Structural formula:

[0350] Chemical formula: C 67 H 132 N2O7

[0351] Molecular weight: 1077.80

[0352] The synthesis was carried out according to the general synthetic route 3, which is similar to the synthesis process of compound 1010, except that intermediate C and intermediate L were used as compound 2 and compound 5 in the general synthetic route 3, respectively. In addition, compound 6 in the general synthetic route 3 was subjected to the following process steps for functional group transformation:

[0353] To intermediate 6a (1.5 g) was added MeOH (5 mL) and THF (5 mL), and the reactants Pd(OH)2 / C (10%) (0.46 g) and Pd / C (10%) (0.35 g) were added. The mixture was reacted under H2 at 25°C overnight, filtered through celite, and the filtrate was concentrated and chromatographed on a silica gel column (PE:EA 30:1-10:1) to afford intermediate 6b (520 mg). To intermediate 6b (470 mg) was added DCM (5 mL), and SOCl2 (1064.26 mg, 8.946 mmol) was added dropwise at 0°C. The mixture was allowed to react at room temperature for 3 hours, and then the reaction mixture was extracted with water and DCM. The organic phase was concentrated and chromatographed on a silica gel column (PE:EA 20:1) to afford intermediate 6c (181 mg). To intermediate 6c (170 mg) were added dimethylamine (143.39 mg) and DMF (2 mL), potassium carbonate (43.96 mg) and potassium iodide (26.39 mg). The reaction solution was stirred at 70°C, filtered, concentrated, and purified by silica gel column chromatography (DCM:MeOH 20:1) to give compound 1059 (91 mg, 53.22% yield). 1 H NMR(400MH z,Chloroform-d)δ4.11(s,6H),3.51(dt,J=18.3,5.6Hz,4H),2.81(dd,J=10.3,5.1Hz,2H),2.50(t,J=6.0Hz,2 H),2.42-2.32(m,3H),2.28(s,6H),2.08-1.98(m,1H),1.53-1.38(m,7H),1.27(s,77H),0.90(t,J=6.7Hz,18H).

[0354] X. Compound 1112, synthesized according to general synthetic route 3

[0355] Structural formula:

[0356] Chemical formula: C 69 H 136 N2O6

[0357] Molecular weight: 1089.85

[0358] The compound was synthesized according to the general synthetic route 3, which is similar to the synthetic process of compound 1010, except that intermediate C and 6-bromohexanal were used to synthesize compound 2 and compound 5 in the general synthetic route 3, respectively. 1HNMR(400MHz,Chloroform-d)δ4.11(s,6H),3.11(s,1H),2.57(t,J=6.8Hz,2H),2.44-2.19 (m,12H),2.04(q,J=6.5Hz,2H),1.53-1.38(m,13H),1.27(s,92H),0.90(t,J=6.7Hz,20H).

[0359] Y. Compound 1113, synthesized according to general synthetic route 4

[0360] Structural formula:

[0361] Chemical formula: C 71 H 138 N2O8

[0362] Molecular weight: 1147.89

[0363] Step 1 and Step 2: Synthesis of Compound 3 and Compound 4 of General Synthesis Route 4

[0364] According to steps 1 and 2 of general synthetic route 1, the synthesis process is the same as that of compound 1003.

[0365] Step 3: Synthesis of compound 6 of general synthetic route 4

[0366] DIEA (0.30 g) and DCM (20 mL) were added to compound 4. Under nitrogen, the temperature was cooled to 0°C, and 5-bromovaleryl chloride (compound 5, 0.27 g) was added. The mixture was stirred at 25°C for 2 h. After dilution with 100 mL of DCM, the organic phase was washed twice with 100 mL of water. The organic phase was dried over anhydrous NaSO, filtered, concentrated, and then purified by silica gel column chromatography (PE:EA 20:1) to obtain compound 6 (800 mg).

[0367] Step 4: Synthesis of compound 8 of general synthetic route 4

[0368] Compound 6 (750.00 mg), intermediate K (compound 7, 217.31 mg), and DMF (20 mL) were added to a three-necked flask. KCO (184.16 mg) and KI (110.60 mg) were then added and stirred at 25°C for 16 h. The temperature was then raised to 100°C and stirred for 3 h. The reaction solution was diluted with 200 mL of EA, and the organic phase was washed twice with 300 mL of saturated brine. The organic phase was dried over anhydrous NaSO, filtered, and concentrated. Silica gel was added and the mixture was filtered through a column using 20:1 DCM:MeOH. The sample was collected and concentrated to yield compound 8 (700.00 mg).

[0369] Step 5: Synthesis of Compound 1113

[0370] Compound 8 (700.00 mg) and THF (5 mL) were added to a three-necked flask, followed by a 1 M dioxane hydrochloride solution (5.55 mL). The reaction mixture was stirred at 25°C for 2 h, diluted with 50 mL of EA, and the organic phase was washed with a saturated NaHCO solution and water. The organic phase was dried over anhydrous NaSO, filtered, concentrated, and then purified by silica gel column chromatography (MeOH:DCM 1:20) to afford compound 1113 (201.1 mg, 31.59% yield). 1 H NMR (400MHz, CDCl3) δ4.50-4.39(m,6H),3.59(t,J=4.6Hz,2H),2.49(s,3H),2.35-2.26(m,6H),2.14 (t,J=7.5Hz,2H),1.75-1.69(m,4H),1.66-1.43(m,17H),1.27(d,J=3.7Hz,78H),0.94-0.87(m,18H).

[0371] Z. Compound 1115, synthesized according to general synthetic route 4

[0372] Structural formula:

[0373] Chemical formula: C 67 H 131 N3O7

[0374] Molecular weight: 1090.80

[0375] The compound was synthesized according to general synthetic route 4, which is similar to the synthetic process of compound 1010, except that 2-bromoacetyl chloride and N,N-dimethylethylenediamine were used to synthesize compound 5 and compound 7 in general synthetic route 4, respectively. 1H NMR(400MHz, CDCl3)δ7.42(s,1H),4.48(tt,J=11.9,5.7Hz,6H),3.27(s,2H),3.13(s,2H),3.01(s,2H),2.83(s,6 H), 2.38-2.25 (m, 3H), 1.55 (dtt, J = 36.3, 14.1, 7.3Hz, 12H), 1.27 (d, J = 3.3Hz, 78H), 0.90 (td, J = 7.2, 3.4Hz, 18H).

[0376] AA. Compound 1114, synthesized according to general synthetic route 5

[0377] Structural formula:

[0378] Chemical formula: C 70 H 138 N2O7

[0379] Molecular weight: 1119.88

[0380] Step 1 and Step 2: Synthesis of Compound 3 and Compound 4 of General Synthesis Route 5

[0381] According to steps 1 and 2 of general synthetic route 1, the synthesis process is the same as that of compound 1003.

[0382] Step 3: Synthesis of compound 6 of general synthetic route 5

[0383] Compound 4 (500 mg), intermediate M (compound 5, 298.68 mg), reactant STAB (328.68 mg) and DCE (10 mL) were added to a three-necked flask. The reaction solution was stirred under N2 at 25°C for 16 h, then 100 mL of water was added to the reaction solution. The mixture was extracted twice with EA (100 mL). The organic phases were combined and concentrated. Silica gel was added and the mixture was passed through a column with 1:20 DCM:MeOH. The sample was collected and concentrated to give compound 6 (500 mg).

[0384] Step 4: Synthesis of Compound 1114

[0385] Compound 6 (450 mg) and THF (5 mL) were added to a three-necked flask, followed by a 1 M dioxane hydrochloride solution (3.65 mL). After stirring at 25°C for 2 h, the mixture was diluted with 50 mL of EA, and the organic phase was washed with 50 mL of saturated NaHCO solution and water. The organic layer was dried over anhydrous NaSO, filtered, concentrated, and then purified by silica gel column chromatography (MeOH:DCM 1:20) to afford compound 1114 (107 mg, 24.84% yield). 1 HNMR (400MHz, CDCl3) δ4.16-4.07(m,6H),3.60(t,J=4.8Hz,2H),2.62(t,J=7.0Hz,2H),2.49(s,3H),2.31(tt, J=8.4,5.5Hz,6H),1.70(d,J=4.6Hz,3H),1.64-1.39(m,18H),1.27(d,J=4.0Hz,78H),0.90(t,J=7.1Hz,18H).

[0386] Z. Compound 1120, synthesized according to general synthetic route 5

[0387] Structural formula:

[0388] Chemical formula: C 72 H 142 N2O7

[0389] Molecular weight: 1147.90

[0390] The product was synthesized according to general synthetic route 5, which was similar to the synthetic process of compound 1114, except that intermediate C and 6-bromohexanal were used to synthesize compound 2 and compound 5 in general synthetic route 5, respectively. 1H NMR (400 MHz, Chloroform-d) δ 4.11 (s, 6H), 3.63 (s, 2H), 2.57 (t, J = 6.8 Hz, 4H), 2.37 (ddd, J = 8.5, 7.1, 4.3 Hz, 5H), 1.67-1.41 (m, 24H), 1.27 (s, 79H), 0.90 (t, J = 6.7 Hz, 18H).

[0391] (2) Synthesis of the second ionizable lipid in Preparation Example 2

[0392] 1. Preparation of Compound 6001

[0393] Structural formula:

[0394] Molecular weight: 499.43

[0395] 0.8 g of oleylamine and 0.8 g of hydroxyethyl acrylamide were added to 6.4 mL of ethanol, stirred and heated to 70°C. After 3.5 h, 0.8 g of hydroxyethyl acrylamide was added. The reaction was continued for 16 h and then passed through a reverse column to obtain compound 6002.

[0396] 0.7 g of compound 6002, 0.4 g of palladium on carbon and 28 mL of tetrahydrofuran were added to a reaction flask and stirred. The gas was replaced with hydrogen and the pressure was maintained with a hydrogen balloon. After 16 h, the mixture was filtered and passed through a column (methanol-DCM system) in the forward direction to obtain 200 mg of compound 6001. 1 H NMR (400MHz, CDCl3) δ7.54(t,J=5.5Hz,2H),3.79-3.68(m,4H),3.42(dd,J=10.0,5.4Hz,4H),2.89(t,J =6.0Hz, 4H), 2.55 (dt, J = 11.8, 6.8Hz, 6H), 1.54 (s, 2H), 1.27 (d, J = 11.2Hz, 32H), 0.91 (t, J = 6.8Hz, 3H).

[0397] 2. Preparation of Compound 6002

[0398] Structural formula:

[0399] Molecular weight: 497.42

[0400] 0.8 g of oleylamine and 0.8 g of hydroxyethyl acrylamide were added to 6.4 mL of ethanol, stirred and heated to 70°C. After 3.5 h, 0.8 g of hydroxyethyl acrylamide was added. The reaction was continued for 16 h, and the organic solvent was concentrated. The mixture was passed through a reverse phase column and freeze-dried to obtain 400 mg of compound 6002. 1 H NMR(400MHz, CDCl3) δ7.31(s,2H),5.38(dd,J=13.0,7.4Hz,2H),3.79-3.66(m,4H),3.41(dd,J=10.1,5.4Hz,4H),2. 82-2.72(m,4H),2.44(dd,J=15.1,9.2Hz,6H),2.10-1.99(m,4H),1.46(s,2H),1.29(s,24H),0.90(t,J=6.8Hz,3H).

[0401] 3. Preparation of Compound 6003

[0402] Structural formula:

[0403] Molecular weight: 445.34

[0404] 4.00 g of tetradecylamine, 6.53 g of hydroxyethyl acrylate and 80 mL of tert-butanol were added to a reaction flask, stirred and heated to 70°C. After 29 h, the organic solvent was concentrated, passed through a reverse phase column and freeze-dried to obtain 1.5 g of compound 6003. 1 H NMR(400MHz, CDCl3)δ4.29(t,J=7.5Hz,4H),3.89-3.75(m,4H),2.81(t,J=6.2Hz,4H), 2.50(ddd,J=23.2,14.1,7.1Hz,6H),1.46(s,2H),1.28(s,24H),0.91(t,J=6.3Hz,3H).

[0405] 4. Preparation of Compound 6004

[0406] Structural formula:

[0407] Molecular weight: 553.48

[0408] 0.8 g of oleylamine, 1.5 g of n-butyl acrylate, and 5.6 mL of n-butanol were added to a reaction flask, stirred, and heated to 100°C. After 4 h, 1 mL of n-butyl acrylate was added, and the reaction was continued for 1 h. The mixture was passed through a normal phase column (petroleum ether-ethyl acetate system), and 0.4 g of sodium hydroxide, 1 mL of water, and 10 mL of methanol were added, stirred and hydrolyzed for 30 min. 1 mL of concentrated hydrochloric acid and 10 mL of methanol were added to obtain a mixed solution, and the pH was adjusted to neutral. The solution was spin-dried, dissolved in DCM, dried over anhydrous magnesium sulfate, filtered, and the solvent was spin-dried. 10 mL of DCM, 1.06 g of 4-amino-1-butanol, 0.81 g of HOBT, and 2.3 g of EDCI were added, and the reaction was allowed to react at room temperature for 18 h. After concentrating the organic solvent, the solution was passed through a reverse phase column and freeze-dried to obtain 240 mg of compound 6004. 1 HNMR (400MHz, CDCl3) δ7.58(d,J=16.6Hz,2H),5.44-5.31(m,2H),3.72(d,J=16.5Hz,4H),3.31(d,J=5.6Hz,4H),3.17( s,4H),2.84(s,2H),2.69(s,4H),2.09-2.01(m,4H),1.67(d,J=2.7Hz,10H),1.37-1.26(m,24H),0.91(t,J=6.8Hz,3H).

[0409] 5. Preparation of Compound 6005

[0410] Structural formula:

[0411] Molecular weight: 597.54

[0412] 25.00 g of 11-henicosone, 62.05 g of ammonium acetate, and 500 mL of methanol were added to a reaction flask and stirred. 6.55 g of sodium cyanoborohydride was added and reacted for 16 h. 250 mL of water and 250 mL of DCM were added, and the layers were separated. The aqueous phase was extracted with 50 mL of DCM, and the combined organic phases were passed through a normal phase column (methanol-DCM system) to obtain 21.00 g of compound 6005-A.

[0413] 21.00 g of compound 6005-A, 58 mL of n-butyl acrylate and 100 mL of n-butanol latex were stirred in a reaction flask and heated to 100° C. 10 mL of n-butyl acrylate was added. After 16 h, the mixture was passed through a column (petroleum ether-ethyl acetate system) to obtain 31.00 g of compound 6005-B.

[0414] A solution prepared by mixing 31.00 g of compound 6005-B with 6.55 g of sodium hydroxide, 310 mL of methanol, and 31 mL of water was added to a reaction flask and stirred. After 20 minutes, 150 mL of THF was added, and the temperature was raised to 50°C with stirring. After 30 minutes, 16.13 g of concentrated hydrochloric acid and 160 mL of methanol were added and stirred. After 30 minutes, the solvent was dried by spin-drying, and 300 mL of DCM was added to dissolve the mixture. The mixture was dried over anhydrous magnesium sulfate, filtered, and dried to obtain 27.00 g of compound 6005-C as an oil.

[0415] 27.00 g of compound 6005-C, 25.00 g of 4-amino-1-butanol, 41.85 g of EDCI, 14.74 g of HOBT, and 310 mL of DCM were added to a reaction flask and stirred. After 18 h, the organic solvent was concentrated and filtered through a column (methanol-DCM system) to remove excess 4-amino-1-butanol, yielding 6.80 g of compound 6005. 1 H NMR (400MHz, CDCl3) δ3.70(t,J=5.6Hz,4H),3.28(t,J=5.6Hz,4H),2.74(t,J=6.0Hz,4H),2.40-2 .45(m,1H),2.34(t,J=6.4Hz,4H),1.63-1.65(m,8H),1.25-1.35(m,36H),0.91(t,J=6.4Hz,6H).

[0416] 6. Preparation of Compound 6006

[0417] Structural formula:

[0418] Molecular weight: 553.48

[0419] 1.00g of oleylamine, 0.47g of hydroxyethyl acrylamide, and 8mL of ethanol were added to a reaction flask, stirred, and heated to 70°C. After 13 hours, the solvent was dried and passed through a methanol-DCM column to obtain 800mg of intermediate 1. 1.5mL of n-butyl acrylate and 5mL of n-butanol were added, stirred, and heated to 100°C. After 3 hours, the solvent was dried and passed through a methanol-DCM column to obtain 800mg of intermediate 1. A solution of 0.4g of sodium hydroxide, 10mL of methanol, and 1mL of water was added, stirred for 1h, and a solution of 1mL of concentrated hydrochloric acid and 8mL of methanol was added. The mixture was stirred for 30min, the solvent was dried and dissolved in 100mL of DCM, dried over anhydrous magnesium sulfate, filtered, and dried. 0.45g of 6-amino-1-hexanol, 0.27g of HOBT, 0.78g of EDCI, and 10mL of DCM were added, stirred. After 14 hours, the organic solvent was concentrated and passed through a methanol-DCM column to obtain 300mg of compound 6006. 1 H NMR (400MHz, CDCl3) δ7.83(s,1H),6.95(s,1H),5.43-5.28(m,2H),3.74-3. 67(m,2H),3.64(t,J=6.3Hz,2H),3.39(dd,J=10.0,5.3Hz,2H),3.24(dd,J= 12.9,6.7Hz,2H),2.74(t,J=5.8Hz,4H),2.48-2.42(m,2H),2.41-2.33(m,4 H), 2.01 (dd, J = 13.6, 6.8 Hz, 4H), 1.66-1.17 (m, 34H), 0.89 (t, J = 6.7 Hz, 3H).

[0420] 7. Compound 6007

[0421] Structural formula:

[0422] Molecular weight: 953.78

[0423] At room temperature, octadecylamine (50 g) was added to a three-necked flask, followed by MeOH (200 mL) and stirring. The mixture was then cooled to 5°C in an ice-water bath, and methyl acrylate (31.5 g) was slowly added dropwise. The mixture was stirred and returned to room temperature. The reaction mixture was allowed to react at room temperature for 4 h. The mixture was concentrated under reduced pressure and purified by column chromatography (PE:EA = 20:1-10:1) to obtain product 2 (68 g). At room temperature, product 2 (15.0 g), MeOH (100 mL), and ethylenediamine (1.0 g) were added to a single-necked flask, heated to 60°C, and maintained at this temperature for overnight reaction. The mixture was concentrated under reduced pressure, and 30 mL of toluene was added. The mixture was heated to 60°C to dissolve, then slowly cooled to room temperature and evaporated to dryness under reduced pressure to obtain compound 3 (15 g). Compound 3 (2 g) was added to a single-necked flask at room temperature. MeOH (20 mL) was added and stirred to dissolve. Methyl acrylate (2.5 g) was then slowly added and stirred to mix. The mixture was heated to 60°C overnight. The reaction was stopped, and the reaction solution was concentrated under reduced pressure and purified by column chromatography (DCM:MeOH = 50:1-20:1) to obtain compound 4 (2.5 g). Compound 4 (2.3 g) and ethylenediamine (20 mL) were added to the single-necked flask at room temperature and reacted overnight at room temperature. The reaction was stopped and the mixture was directly concentrated under reduced pressure to obtain compound 6007 (24 mg). 1H NMR (400MHz, Methanol-d4) δ3.87(t,J=6.6Hz,2H),3.55-3.36(m,8H),3.33(s,8H),3.27-3.16(m,2H),3.06-2.90(m,4H),2.77(t,J=6.5Hz,4H) ,1.90(dtdd,J=21.2,14.0,9.5,6.9Hz,4H),1.73(p,J=7.7Hz,4H),1.49(tdd,J=9.2,7.9,7.1,4.0Hz,4H),1.31(s,28H),0.92(t,J=6.8Hz,3H).

[0424] 8. Compounds 6008-6018

[0425] Synthesis of compound 6009

[0426] Structural formula:

[0427] At room temperature, a solution of freshly recrystallized hexadecylamine (0.03 mol) in methanol (20 mL) was added dropwise to a solution of stirred methyl methacrylate (6 mL) in methanol (20 mL) under a nitrogen atmosphere and the reaction was continued overnight. The reactant was spin-dried at room temperature, and the residue was dissolved in chloroform and washed twice with 0.1 M NaOH solution. The chloroform solution was collected and dried over anhydrous calcium chloride. The resulting product was then separated by column chromatography to obtain a colorless oil. 1H-NMR (300 MHz, CDCl 3 ): 0.78 (t, 3H), 1.16 (s, 30H), 2.38 (m, 6H), 2.71 (t, 4H), 3.57 (m, 6H).

[0428] Then, a solution of the above colorless oil (11.05 g) in methanol (20 ml) was added to a solution of 1,2-diaminoethane (75 g) in methanol (100 ml) that was vigorously stirred at room temperature. After complete addition, the mixture was stirred at room temperature for another 24 hours. The solvent was removed under reduced pressure, maintaining the temperature not higher than 40°C. Excess 1,2-diaminoethane was removed with an azeotropic mixture of toluene and methanol (9:1). The remaining toluene was removed by azeotropic distillation with methanol. Finally, a white powder (10.5 g) was obtained, which was repeatedly recrystallized from chloroform and cyclohexane to obtain a white solid. 1H-NMR (300MHz, CDCl3): 0.88(t,3H), 1.25(s,30H), 1.84(s,4H), 2.38(m,6H), 2.73(m,4H), 2.82(m,4H), 3.29(m,4H), 7.47(s,2H).

[0429] The preparation methods of 6008 and 6009 differ only in that an equal molar amount of tetradecylamine is used instead of hexadecylamine. 6010-6017 refer to the preparation method of 6009 and use an equal molar amount of the corresponding R a -NH2 is used to replace hexadecylamine.

[0430] 6018 was prepared by referring to the method of 6007, using an equimolar amount of the corresponding R a -NH2 replaces octadecylamine to prepare.

[0431] 9. Compound 6019

[0432] Structural formula:

[0433] Molecular weight: 753.66

[0434] At room temperature, compound 2483-46-7 (3 g) was added to a single-necked flask, followed by DCM (50 mL) and stirring. 18807-71-1 (2.5 g), DCC (2.7 g), and DMAP (1.6 g) were then added and stirred. The mixture was allowed to react overnight at room temperature. The mixture was concentrated under reduced pressure and purified by column chromatography (DCM:MeOH = 5:1) to afford product 3 (4 g). At room temperature, product 3 (5.0 g), MeOH (50 mL), and Pd / C (1.0 g) were added to a three-necked flask. The hydrogen atmosphere was replaced, the temperature was raised to 50°C, and the mixture was stirred for 1 h. The reaction was stopped, the reaction solution was cooled to room temperature, filtered, and the filtrate was dried to afford product 4 (3.5 g). At room temperature, raw material 5 (1.2 g) was added to a single-necked flask. DMF (50 mL) was added and stirred to dissolve. Then, product 4 (3.3 g), HATU (3.2 g), and TEA (0.9 g) were added and stirred. The mixture was allowed to react overnight at room temperature. Water (500 mL) and EA (500 mL) were added, and the mixture was separated and extracted. The organic phase was dried over Na2SO4 and concentrated under reduced pressure. The crude product was purified by column chromatography (DCM:MeOH = 20:1-10:1) to obtain solid 6 (380 mg). Solid 6 (380 mg) and HCl / Dioxane (4 M) (1.6 mL) were added to the single-necked flask at room temperature and reacted at room temperature for 2 h. The mixture was concentrated under reduced pressure and purified to obtain compound 6019 (220 mg). 1H NMR (400MHz, Methanol-d4) δ3.87(t,J=6.6Hz,2H),3.55-3.36(m,8H),3.33(s,8H),3.27-3.16(m,2H),3.06-2.90(m,4H),2.77(t,J=6.5Hz,4H) ,1.90(dtdd,J=21.2,14.0,9.5,6.9Hz,4H),1.73(p,J=7.7Hz,4H),1.49(tdd,J=9.2,7.9,7.1,4.0Hz,4H),1.31(s,28H),0.92(t,J=6.8Hz,3H).

[0435] 10. Compound 6020

[0436] Structural formula:

[0437] Molecular weight: 810.19

[0438] At room temperature, octadecylamine (50 g) was added to a three-necked flask, followed by MeOH (200 mL) and stirring. The mixture was then cooled to 5°C in an ice-water bath, and methyl acrylate (31.5 g) was added dropwise. The mixture was stirred and returned to room temperature for 4 h. The reaction was stopped, and the mixture was directly concentrated under reduced pressure. Purification by column chromatography (PE:EA = 20:1-10:1) afforded product 2 (68 g). At room temperature, product 2 (15.0 g), MeOH (100 mL), and ethylenediamine (1.0 g) were added to a single-necked flask, heated to 60°C, and maintained at this temperature for overnight reaction. The reaction was stopped, the reaction solution was concentrated under reduced pressure, 30 mL of toluene was added, and the mixture was heated to 60°C for dissolution. The mixture was slowly cooled to room temperature, filtered, and evaporated to dryness under reduced pressure to afford compound 3 (15 g). At room temperature, compound 3 (1.3 g) was added to a single-necked flask, and DMF (20 mL) was added and stirred. Then, 35897-34-8 (3.6 g), EDCI (3.0 g), HOBt (2.1 g), and DIEA (2.0 g) were added, stirred, and reacted at room temperature overnight. Water (200 mL) was added and the mixture was directly freeze-dried under reduced pressure to obtain compound 4 (4 g). Compound 4 (100 mg) and HCl / Dioxane (4 M) (2 mL) were added to a single-necked flask at room temperature and reacted for 1 h. The reaction was stopped and the reaction solution was concentrated under reduced pressure to obtain compound 6020. 1H NMR (400 MHz, Methanol-d4) δ 3.90 (t, J = 6.4 Hz, 2H), 3.57-3.34 (m, 10H), 3.24 (dt, J = 16.1, 8.0 Hz, 8H), 2.77 (t, J = 6.5 Hz, 4H), 2.03-1.58 (m, 10H), 1.31 (s, 30H), 1.01-0.82 (m, 3H).

[0439] 11. Compound 6021

[0440] Structural formula:

[0441] Molecular weight: 1182.79

[0442] At room temperature, compound 2 (3.5 g, the synthesis process is the same as that of product 2 in compound 6020) was added to a single-necked flask, MeOH (20 mL) was added and stirred, and then TREN (23.0 g) was added, stirred, and the temperature was raised to 60°C, stirred overnight, and concentrated under reduced pressure. The crude product was lyophilized to obtain product 3 (25 g). At room temperature, product 3 (22.0 g) and DCM (200 mL) were added to a single-necked flask, the temperature was lowered to 0°C, and (Boc)2O (71.7 g) was slowly added dropwise. After the temperature was restored to room temperature, the reaction was carried out for 3 h, water (200 mL) was added for extraction, and DCM (100 mL) was added for separation and extraction. The organic phase was dried over sodium sulfate, concentrated under reduced pressure, and purified by column chromatography to obtain a yellow oil 4 (5.8 g). At room temperature, yellow oil 4 (1.4 g) was added to a single-necked flask. HCl / Dioxane (4 M) (15 mL) was slowly added under an ice-water bath and stirred. After addition, the mixture was returned to room temperature and reacted for 0.5 h. The mixture was then concentrated under reduced pressure to obtain compound 5 (800 mg). Compound 5 (1.0 g), (S)-2,6-di-tert-butyloxycarbonylaminohexanoic acid (2.6 g), EDCI (1.4 g), HOBt (1.0 g), DIEA (1.0 g), and DMF (10 mL) were added to a single-necked flask at room temperature. The mixture was reacted overnight at room temperature. 100 mL of water and 100 mL of EA were added, and the mixture was separated and extracted. The EA phase was washed once with 100 mL of saturated brine, concentrated under reduced pressure, and purified by column chromatography (DCM:MeOH = 10:1) to obtain compound 6 (1.1 g). To a one-necked flask at room temperature, compound 6 (1.2 g), HCl / Dioxane (4 M) (10 mL) and solvent DCM (10 mL) were added. The reaction was allowed to proceed overnight at room temperature. The mixture was concentrated under reduced pressure and purified to obtain compound 6021 (220 mg). 1H NMR(400MHz, Methanol-d4)δ3.96(t,J=6.6Hz,4H),3.75(dt,J=14.0,6.6Hz,4H),3.62-3.45(m,12H),3.36-3.33(m,14H),3.25-3.17(m,2H),3.04 -2.90(m,8H),2.83(t,J=6.9Hz,4H),2.05-1.82(m,8H),1.73(p,J=7.7Hz ,10H),1.53(qd,J=8.3,7.7,4.3Hz,8H),1.30(s,28H),0.97-0.87(m,3H).

[0443] 12. Compound 6023

[0444] Structural formula:

[0445] Molecular weight: 670

[0446] At room temperature, compound dodecylamine (10 g) was added to a three-necked flask, followed by MeOH (100 mL) and stirring. The mixture was then cooled to 5°C in an ice-water bath, and methyl acrylate (10.2 g) was slowly added dropwise. The mixture was stirred and returned to room temperature. The reaction mixture was allowed to react at room temperature for 4 h, then stopped, concentrated under reduced pressure, and purified by column chromatography (PE:EA=20:1-10:1) to obtain product 2 (19 g). At room temperature, product 2 (19.0 g), MeOH (200 mL), and ethylenediamine (127.7 g) were added to a single-necked flask, heated to 60°C, maintained at this temperature for overnight, and concentrated under reduced pressure to obtain compound 3 (20 g). 500 mg of the product was purified and lyophilized to obtain compound 6023-1 (218 mg). At room temperature, raw material 6023-1 (1.4 g) was added to a single-necked flask. DCM (20 mL) was added and stirred to dissolve. (S)-2,6-di-tert-butyloxycarbonylaminocaproic acid (3.4 g), EDCI (1.9 g), and DMAP (1.2 g) were then added and stirred. The mixture was allowed to react overnight at room temperature. The mixture was separated, extracted, dried, and concentrated under reduced pressure to obtain compound 4 (3.0 g). Compound 4 (2.8 g) and HCl / 1,4-Dioxane (4 M) (20 mL) were added to the single-necked flask at room temperature and reacted for 2 h. The mixture was then concentrated under reduced pressure, purified, and lyophilized to obtain compound 6023 (123 mg). 1H NMR(400MHz, Methanol-d4)δ3.88(t,J=6.6Hz,2H),3.55-3.36(m,8H),3.36-3.33(m,2H),3.31-3.14(m,4H),3.04-2.93(m,4H),2. 77(t,J=6.5Hz,4H),2.00-1.82(m,4H),1.82-1.66(m,6H),1.50(qd,J=8.2,7.8,4.2Hz,4H),1.45-1.25(m,18H),0.96-0.87(m,3H).

[0447] 13. Compound 6024

[0448] Structural formula:

[0449] Molecular weight: 870.24

[0450] Product 6023-1 (1.4 g, synthesized by the same method as 6023-1 in compound 6023) was added to a single-necked flask at room temperature. MeOH (20 mL) was added and stirred to dissolve, and then methyl acrylate (2.8 g) was slowly added and stirred to mix. The temperature was raised to 60°C, and the reaction was allowed to proceed overnight at this temperature. The mixture was concentrated under reduced pressure to obtain compound 4 (2.0 g). Compound 4 (2.4 g) and ethylenediamine (20 mL) were added to a single-necked flask at room temperature, and the reaction was allowed to proceed overnight at room temperature. The mixture was concentrated under reduced pressure and purified to obtain compound 6024 (191 mg). 1HNMR (400MHz, Methanol-d4) δ3.69(t,J=6.0Hz,4H),3.60-3.48(m,20H),3.42(t,J=6.1Hz,4H),3.25-3.19(m,2H),3 .12(t,J=5.8Hz,8H),2.83(t,J=6.5Hz,12H),1.78(tt,J=11.0,6.4Hz,2H),1.51-1.18(m,20H),0.91(t,J=6.7Hz,3H).

[0451] 14. Compound 6026

[0452] Structural formula:

[0453] Molecular weight: 443.67

[0454] Compound 6026 was synthesized by referring to the synthetic route of compound 6019, with the following differences: an equal molar amount of compound 1 was used instead of starting material 5 of compound 6019, and an equal molar amount of N-(tert-butyloxycarbonyl)ethanolamine was used instead of intermediate 4 of compound 6019. 1H NMR (300 MHz, DMSO) δ: 4.51 (t, J = 7.3 Hz, 4H), 3.76 (t, J = 6.1 Hz, 4H), 3.18 (t, J = 6.7 Hz, 4H), 3.01 (t, J = 5.8 Hz, 2H), 2.49 (t, J = 7.1 Hz, 4H), 1.36-1.26 (m, 24H), 0.89 (t, J = 6.2 Hz, 3H).

[0455] Compounds 6025-6034 were obtained by referring to the synthetic route of the aforementioned compounds, except that hydroxyethylamine was used instead of the corresponding ethylenediamine, and the amino group of the hydroxyethylamine was first protected with a protecting group such as Fmoc or Boc. After the reaction was completed, the protecting group was removed according to conventional methods.

[0456] (3) First Ionizable Lipid Example Compounds and Their Properties

[0457] The exemplary compounds of the present application and their properties are listed in Table 1, which were synthesized according to the process routes in Preparation Example 1.

[0458] The calculated c-pKa (molnetwork), LogP (cLogP driver) of the exemplary compounds of this application, c-pKa and cLogP values ​​were generated by the ChemDraw module of Chemoffice.

[0459] Table 1 Properties of exemplary compounds of the first ionizable lipid

[0460] The above properties of the first ionizable lipid exemplary compounds of the present application indicate that they can serve as excellent surfactants and are particularly suitable for preparing lipid nanoparticles for use as drug delivery carriers.

[0461] (4) Preparation of lipid composition

[0462] The lipid compositions were prepared according to the above molar ratios, and the specific methods are as follows:

[0463] The lipid raw material was dissolved in ethanol to obtain a lipid ethanol solution, and the total concentration of all lipid raw materials in ethanol was 8 mg / mL. The lipid ethanol solution was mixed with 50 mM citrate buffer saline (pH 4.0) solution at a volume ratio of 1:3 in a nanopreparation device, and then ultrafiltration was performed to collect the sample to obtain a lipid composition, and the lipid concentration of the composition was 2 mg / mL.

[0464] (5) Determination of nanoparticle size and zeta potential

[0465] 1. Particle Size and Polydispersity Index (PDI) Determination: The average particle size and PDI of the nanoparticle sample solutions in the examples were determined by dynamic light scattering using a Malvern ZetaSizerNano ZS90. The measurement angle was 90°, the refractive index of the dispersant was 1.330, and the test temperature was 25°C.

[0466] 2. Zeta potential: The zeta potential of the nanoparticle sample solution in the examples was measured using a Malvern ZetaSizer Nano ZS90 based on electrophoretic light scattering (ELS) technology. The refractive index of the dispersant was 1.330 and the test temperature was 25°C.

[0467] The average particle size, PDI, and Zeta potential data of the lipid compositions prepared from each first ionizable lipid exemplary compound and the second ionizable lipid are shown in Table 2.

[0468] Table 2 Summary of particle size, PDI, and Zeta potential of lipid compositions

[0469] As can be seen from Table 2, the lipid composition provided in the present application has a small and uniform particle size and is neutral or weakly electrically charged.

[0470] Physical stability data summary table

[0471] As shown in Table 3, the lipid nanoparticles provided in the present application were placed under conditions of 25° C. and RH 60% for one month, and there was no significant change in particle size, PDI, and potential, indicating good stability.

[0472] (7) Investigation of the encapsulation effect of lipid compositions on nucleic acid substances

[0473] Using nucleic acid as the active ingredient, the nucleic acid was prepared into a 40 ng / μl solution using nuclease-free water as solution 1; the lipid composition was diluted 1-fold using nuclease-free water as solution 2; equal volumes of solution 1 and solution 2 were mixed and vortexed for 2-3 seconds to obtain a lipid composition-nucleic acid complex, and the encapsulation efficiency was tested.

[0474] The encapsulation efficiency test method is as follows:

[0475] The mRNA encapsulation efficiency in lipid nanoparticles was determined using the Quant-it Ribogreen RNA quantification kit (ThermoFisher Scientific, UK) according to the manufacturer's instructions.

[0476] The encapsulation efficiency of pDNA in lipid nanoparticles was determined using a dsDNA HS kit (Novozymes / EQ121) according to the manufacturer's instructions.

[0477] Follow the manufacturer's instructions for use. microRNA Reagent Kit, used to determine the encapsulation efficiency of siRNA in lipid nanoparticles.

[0478] The test results are shown in Table 4.

[0479] Table 4 Encapsulation efficiency of lipid composition-nucleic acid complex

[0480] (8) Investigation of the encapsulation effect of lipid compositions on small molecules

[0481] Small molecules were used as active ingredients, and the active ingredients were prepared into a 300 ng / μl solution using nuclease-free water as solution 1; the lipid compositions prepared in Examples 2, 4-5, 8, 11 and 23 of Part (4) were used as solution 2; equal volumes of solution 1 and solution 2 were mixed and vortexed for 2-3 seconds to obtain a lipid composition-small molecule complex, and the encapsulation efficiency was tested.

[0482] Encapsulation efficiency test method:

[0483] The encapsulated LNPs of each component were ultrafiltrated and centrifuged using ultrafiltration centrifuge tubes (Amicon Ultra-4 Centrifugal Filters, molecular cutoff 30 kD). The amount of free drug in the centrifuge tube and the amount of encapsulated drug retained on the filter membrane were determined by high performance liquid chromatography (encapsulation efficiency calculation formula: encapsulation efficiency (EE%) = retained drug amount / (retained drug amount + free drug amount) × 100%). The test results are shown in Table 5.

[0484] Table 5 Encapsulation efficiency of lipid composition-small molecule substance complex

[0485] (9) Investigation of the encapsulation effect of lipid compositions on macromolecular substances

[0486] A macromolecular substance was used as the active ingredient, and the active ingredient was prepared into a 200 ng / μl solution using nuclease-free water as solution 1; the lipid composition prepared in Examples 3, 5, 7, 14 and 22 of Part (4) was used as solution 2; equal volumes of solution 1 and solution 2 were mixed and vortexed for 2-3 seconds to obtain a lipid composition-macromolecular substance complex, and the encapsulation efficiency was tested.

[0487] Encapsulation efficiency test method: add nanoparticles to retain relative molecular mass of 100×10 3 The free drug was separated by centrifugation in the ultrafiltration tube, and the amount of free drug in the centrifuge tube and the amount of encapsulated drug retained on the filter membrane were determined by RP-HPLC. The encapsulation efficiency was calculated as follows: Encapsulation efficiency (EE%) = retained drug amount / (retained drug amount + free drug amount) × 100%.

[0488] The test results are shown in Table 6.

[0489] Table 6 Encapsulation efficiency of lipid composition-macromolecule complex

[0490] The above studies show that the lipid composition provided in this application has a good encapsulation effect on nucleic acids, small molecules or macromolecules, with an encapsulation rate of >80%, and can be used as a delivery vehicle for active ingredients.

[0491] The present application is not limited to the technical means disclosed by the above technical means, but also includes technical solutions composed of any combination of the above technical features. The above is a specific embodiment of the present application. It should be pointed out that for ordinary technicians in the field of this application, several improvements and modifications can be made without departing from the principles of this application. These improvements and modifications are also considered to be within the scope of protection of this application.

Claims

1. A lipid composition, wherein, The lipid composition comprises the following components: an ionizable lipid and a helper lipid; the lipid composition does not contain ingredients having a prophylactic or therapeutic effect, the ionizable lipid comprises a first ionizable lipid, and the first ionizable lipid is selected from the compounds of formula (1), or salts, stereoisomers, tautomers thereof: wherein, R1, R2, and R3 are each independently H, C 5-40 a linear or branched alkyl group, C 5-40 a linear or branched alkenyl group, C 5-40 a linear or branched alkynyl group, a 3- to 6-membered saturated or partially unsaturated cycloalkyl group having 1 to 3 side chains, or a 6- to 10-membered aromatic group having 1 to 3 side chains; the side chains are independently selected from C 10-30 a linear or branched alkyl group, C 10-30 a linear or branched alkenyl group, C 10-30 a linear or branched alkynyl group; provided that at most one of R1, R2, and R3 is H; M is selected from -NR4R5, a saturated or partially unsaturated 3-6 membered heterocyclic group containing at least one nitrogen atom, a 6-10 membered heteroaryl group containing at least one nitrogen atom, and the heterocyclic group and heteroaryl group are unsubstituted or substituted by one or more -OH, carboxyl, amino, oxo or halogen; R4 and R5 are each independently H, C 1-6 a straight-chain or branched-chain alkyl group, C 2-6 a straight-chain or branched-chain alkenyl group or C 2-6 a straight-chain or branched-chain alkynyl group, and the C 1-6 a straight-chain or branched-chain alkyl group, C 2-6 a straight-chain or branched-chain alkenyl group or C 2-6 a straight-chain or branched-chain alkynyl group is unsubstituted or substituted by one or more -OH, carboxyl, aminoamide, amidino, guanidine or halogen; G1, G2, G3 are each independently -O-, -S-, -NR6-, -S-S-, -C(=O)-, -C(=S)-, -C(=O)O-, -CH(OH)-, -OC(=O)-, -C(=O)NR6-, -NR6C(=O)-, -OC(=O)O-, -NR6C(=O)O-, -OC(=O)NR6-, -NR6C(=O)NR 13 -, -C(=O)S-, -C(=S)S-, -SC(=S)-, -SC(=O)-, -OC(=O)S-, -SC(=O)O-, -SC(=O)S-, -OS(=O)2O-, -S(=O)2O-, -OS(=O)2-, -S(=O)2-, -S(=O)2-NR6-, -NR6-S(=O)2-, -P(=O)(OR6)O-, -OP(=O)(OR6)- or -OP(=O)(OR6)O-; where each R6, R 13 is independently selected from H, hydroxy, C 1-30 a straight-chain or branched alkyl or cycloalkyl, C 2-30 a straight-chain or branched alkenyl; L1 is selected from -X1- or -(CR7R8) m -X1-, wherein each X1 is independently selected from -O-, -S-, -NR 14 -, -S-S-, -C(=O)-, -C(=S)-, -C(=O)O-, -OC(=O)-, -C(=O)NR 14 -, -NR 14 C(=O)-, -OC(=O)O-, -NR 14 C(=O)O-, -OC(=O)NR 14 -, -NR 14 C(=O)NR 15 -, -C(=O)S-, -C(=S)S-, -SC(=S)-, -SC(=O)-, -OC(=O)S-, -SC(=O)O-, -SC(=O)S-, -OS(=O)2O-, -S(=O)2O-, -OS(=O)2-, -S(=O)2-, -S(=O)2-NR 14 -, -NR 14 -S(=O)2-, -P(=O)(OR 14 )O-, -OP(=O)(OR 14 )-, or -OP(=O)(OR 14 )O-; wherein, m is an integer from 2 to 6, and R7 and R8 are independently of each other H, hydroxy, halogen, C 1-6 of straight-chain or branched-chain alkyl or cycloalkyl, C 2-6 of straight-chain or branched-chain alkenyl, and each R 14 , R 15 are independently selected from H, C 1-30 of straight-chain or branched-chain alkyl or cycloalkyl, C 2-30 of straight-chain or branched-chain alkenyl; L2 is -(CR9R 10 ) n - or -(CR9R 10 ) n -X2-(CR 11 R 12 ) k -, where X2 is selected from -O-, -S-, -NR 16 -, -S-S-, -C(=O)-, -C(=S)-, -C(=O)O-, -OC(=O)-, -C(=O)NR 16 (-), -NR 16 C(=O)-, -OC(=O)O-, -NR 16 C(=O)O-, -OC(=O)NR 16 (-), -NR 16 C(=O)NR 17 (-), -C(=O)S-, -C(=S)S-, -SC(=S)-, -SC(=O)-, -OC(=O)S-, -SC(=O)O-, -SC(=O)S-, -OS(=O)2O-, -S(=O)2O-, -OS(=O)2-, -S(=O)2-, -S(=O)2-NR 16 (-), NR 16 -S(=O)2-, -P(=O)(OR 16 )O-, -OP(=O)(OR 16 )-, or -OP(=O)(OR 16 )O-; n is an integer from 1 to 6; k is an integer from 1 to 6; R9, R 10 , R 11 , R 12 are independently of each other H, hydroxy, halogen, a straight-chain or branched-chain alkyl or cycloalkyl of C 1-6 , a straight-chain or branched-chain alkenyl of C 2-6 ; each R 16 , R 17 is independently selected from H, a straight-chain or branched-chain alkyl or cycloalkyl of C 1-30 , a straight-chain or branched-chain alkenyl of C 2-30 ; wherein R4 to R 17 the alkyl, cycloalkyl, alkenyl groups described therein are unsubstituted or substituted by one or more groups selected from hydroxyl, mercapto, amino, substituted amino, halogen; The salt is not a quaternary ammonium salt.

2. The lipid composition according to claim 1, wherein R1, R2, and R3 are, independently of one another, groups as follows: wherein Y does not exist or is C 1-30 a linear or branched alkyl or cycloalkyl group, C 2-20 a linear or branched alkenyl group, C 2-20 a linear or branched alkynyl group; R1’ and R2’ are each independently H, C 1-30 a linear or branched alkyl group, C 2-30 a linear or branched alkenyl group, C 2-30 a linear or branched alkynyl group, and the total carbon chain length of Y, R1’ and R2’ is 8 - 40.

3. The lipid composition according to claim 1, wherein R1, R2, and R3 are each independently selected from the following groups: wherein, R1’ and R2’ are each independently H, C 1-30 a linear or branched alkyl group, C 2-30 a linear or branched alkenyl group, C 2-30 a linear or branched alkynyl group, and the total carbon chain length of R1’ and R2’ is 8 - 30.

4. The lipid composition according to claim 1, wherein, R1, R2, and R3 are each independently selected from any one of the following groups:

5. The lipid composition according to claim 1, wherein, G1, G2, G3 are each independently -O-, -S-, -NR6-, -S-S-, -C(=O)-, -C(=O)O-, -CH(OH)-, -OC(=O)-, -C(=O)NR6-, -NR6C(=O)-, -OC(=O)O-, -NR6C(=O)O-, -OC(=O)NR6-, - NR6C(=O)NR 13 —, —P(=O)(OR6)O—, —OP(=O)(OR6)— or —OP(=O)(OR6)O—.

6. The lipid composition according to claim 1, wherein, L1 is selected from -(CR7R8) m -X1-, where X1 is selected from -O-, -S-, -NR 14 -, -S-S-, -C(=O)-, -C(=O)O-, -OC(=O)-, -C(=O)NR 14 -, -NR 14 C(=O)-, -OC(=O)O-, -NR 14 C(=O)O-, -OC(=O)NR 14 -, -NR 14 C(=O)NR 15 -, -P(=O)(OR 14 )O-, -OP(=O)(OR 14 )-, or -OP(=O)(OR 14 )O-.

7. The lipid composition according to claim 1, wherein, L2 is -(CR9R 10 ) n -X2-(CR 11 R 12 ) k -, where X2 is selected from -O-, -S-, -NR 16 -, -S-S-, -C(=O)-, -C(=O)O-, -OC(=O)-, -C(=O)NR 16 -, -NR 16 C(=O)-, -OC(=O)O-, -NR 16 C(=O)O-, -OC(=O)NR 16 -, -NR 16 C(=O)NR 17 -, -P(=O)(OR 16 )O-, -OP(=O)(OR 16 )-, or -OP(=O)(OR 16 )O-.

8. The lipid composition according to claim 1, wherein, M is selected from the following structures: wherein, m' and n' are independently integers from 0 to 6, and R1'' and R2'' are independently H, C 1-6 alkyl, C 2-6 alkenyl, guanidyl, amidino, amido, aliphatic amine group, 3- to 10-membered nitrogen-containing heterocycle; the nitrogen-containing heterocycle is selected from pyrrole, imidazole, pyridine, pyrazole, triazole, oxazole, isoxazole, thiophene, isothiazole, pyridazine, pyrazine, piperazine, indole, benzimidazole, carbazole, quinoline, isoquinoline, purine, and pyrimidine and their tautomeric forms, which are unsubstituted or optionally substituted by one or more organic groups selected from hydroxy, mercapto, amino, substituted amino, halogen, C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, C 6-14 aryl.

9. The lipid composition according to claim 1, wherein, The compound of formula (1) is selected from the compounds represented by formula (1A):

10. The lipid composition according to claim 1, wherein, The compound of formula (1) is selected from the compounds shown in formula (1B):

11. The lipid composition according to claim 1, wherein, The compound of formula (1) is selected from the compounds represented by formula (1C):

12. The lipid composition according to claim 10, wherein, The compound of formula (1) is selected from the compounds represented by formula (1D):

13. The lipid composition according to claim 12, wherein, The compound of formula (1) is selected from the compounds represented by formula (1E) 14. The lipid composition according to claim 13, wherein, The compound of formula (1) is selected from the compounds represented by formula (1F):

15. The lipid composition according to claim 14, wherein, The compound of formula (1) is selected from the compounds represented by formula (1G):

16. The lipid composition according to claim 14, wherein, The compound of formula (1) is selected from the compounds represented by formula (1H):

17. The lipid composition according to claim 1, wherein, The lipid nanoparticle structure is a compound represented by formula (1I):

18. The lipid composition according to claim 10, wherein, The compound of formula (1) is selected from the compounds represented by formula (1J):

19. The lipid composition according to claim 17, wherein, The compound of formula (1) is selected from the compounds represented by formula (1K):

20. The lipid composition according to claim 2, wherein, Y does not exist, and the compound of formula (1) is selected from the compounds represented by formula (1L): wherein R1’ and R2’ are independently selected from H, C 1-30 a linear or branched alkyl group, C 2-30 a linear or branched alkenyl group, C 2-30 a linear or branched alkynyl group, and the total carbon chain length of R1’ and R2’ is 8-40.

21. The lipid composition according to claim 2, wherein, The compound of formula (1) is selected from the compounds represented by formula (1M):

22. The lipid composition according to claim 1, wherein, The compound of formula (1) is selected from:

23. The lipid composition according to claim 1, wherein, The auxiliary lipid is a mixture of phospholipids and steroids or their derivatives.

24. The lipid composition according to claim 23, wherein, The phospholipids include at least one of 1,2-dilinoleoyl-sn-glycero-3-phosphocholine, 1,2-dimyristoyl-sn-glycero-phosphocholine, 1,2-dioleoyl-sn-glycero-3-phosphocholine, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine, 1,2-distearoyl-sn-glycero-3-phosphocholine, 1,2-dielaidoyl-sn-glycero-phosphocholine, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, 1,2-di-0-octadecenoyl-sn-glycero-3-phosphocholine, 1-oleoyl-2-cholesteryl succinyl-sn-glycero-3-phosphocholine, 1-hexadecyl-sn-glycero-3-phosphocholine, 1,2-dilinolenoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt, dipalmitoyl phosphatidylglycerol, palmitoyl oleoyl phosphatidylethanolamine, distearoyl-phosphatidylethanolamine, dipalmitoyl phosphatidylethanolamine, dimyristoyl phosphoethanolamine, 1-stearoyl-2-oleoyl-stearoylethanolamine, 1-stearoyl-2-oleoyl-phosphatidylcholine, sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyl oleoyl phosphatidylcholine, lysophosphatidylcholine, lysophosphatidylethanolamine.

25. The lipid composition according to claim 23, wherein, The steroid or its derivative includes at least one of cholesterol, cholesteryl stearate, coprosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, dunaliol, tomatine, ursolic acid, and α-tocopherol.

26. The lipid composition according to claim 1, wherein, In the lipid composition, the amount of the ionizable lipid is 5-90 mol% of the total lipids in the formulation; the amount of the helper lipid is 10-95 mol% of the total lipids in the formulation.

27. The lipid composition according to claim 26, wherein, The molar ratio of phospholipid to steroid or its derivative in the helper lipid is (5-80):(5-80).

28. The lipid composition according to any one of claims 1 to 27, wherein, The lipid composition further comprises a PEG-conjugated lipid.

29. The lipid composition according to claim 28, wherein, The PEG-conjugated lipid includes at least one of PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, and PEG-modified dialkylglycerol.

30. The lipid composition according to claim 29, wherein, The PEG-conjugated lipid includes at least one of PEG-distearyloxypropyl, PEG-c-DOMG, PEG-DPPC, polyethylene glycol dimethacrylate, 1,2-dimethylstyrene-rac-glycerol-3-methoxypolyethylene glycol, glyceryl dipalmitate-polyethylene glycol, 1,2-distearoyl-rac-glycerol-3-methoxypolyethylene glycol, 2-[(polyethylene glycol)-2000]-N,N-tetracosanoylacetamide, dipalmitoylphosphatidylethanolamine-polyethylene glycol, distearoylphosphatidylethanolamine-polyethylene glycol, dilauroylphosphatidylethanolamine-polyethylene glycol, and dimyristoylphosphatidylethanolamine-polyethylene glycol lipid.

31. The lipid composition according to claim 28, wherein, In the lipid composition, the amount of the ionizable lipid is 5-80 mol% of the total lipids in the formulation; the amount of the helper lipid is 10-90 mol% of the total lipids in the formulation; the amount of the PEG-conjugated lipid is 0.1-15 mol% of the total lipids in the formulation.

32. The lipid composition according to any one of claims 1-27, wherein, The ionizable lipid further includes at least one of a second ionizable lipid and a third ionizable lipid; The second ionizable lipid is selected from the compounds of formula (2), or salts, stereoisomers, tautomers thereof: wherein A1 is NH or O; R a selected from C6-C 24 alkyl, C6-C 24 alkenyl, C6-C 24 cycloalkyl, C6-C 24 alcohol, C6-C 24 short-chain polyoxyethylene; the C6-C 24 alkyl, C6-C 24 alkenyl, C6-C 24 cycloalkyl, C6-C 24 alcohol, C6-C 24 short-chain polyoxyethylene has a straight-chain or branched-chain structure; R b and R c are each independently selected from C1-C 12 alkyl, C2-C 12 alkenyl, C1-C 12 alkynyl, C3-C 12 cycloalkane group, C6-C 12 aryl group, C1-C 12 alkyl alcohol, C1-C 12 heterocyclic group, alkylamine; The alkylamine is Among them, R a ’ is C1-C 12 alkyl, and said R b ’ and R b ” are each independently selected from H, C1-C6 alkylamine, R c ” selected from unsubstituted or amino-substituted C1-C6 alkyl, R c ”’ is H, or -R c '-A1'-R c '-NH2; Provided that when A1' is -CO-NH-, -NH-CO- or -CO-O-, R c ' is a C1-C6 alkyl group; when A1' is -CO-, R c ' does not exist; The third ionizable lipid is selected from at least one of the following compounds:

33. The lipid composition according to claim 32, wherein, The compound of formula (2) is selected from at least one of the following compounds:

34. The lipid composition according to claim 32, wherein, In the ionizable lipid, the molar ratio of the first ionizable lipid to the second ionizable lipid and / or the third ionizable lipid is (5-90):(1-80).

35. The lipid composition according to any one of claims 1 to 27, wherein, The lipid composition is a nanoparticle.

36. The lipid composition according to claim 35, wherein, The nanoparticle has a particle size of 20-400 nm and a polydispersity index <0.

5.

37. The method for preparing a lipid composition according to any one of claims 1 to 36, wherein, It includes the following steps: 1) Dissolve the lipid compound in an organic solvent to obtain an organic phase; 2) Use an aqueous solvent as the aqueous phase; 3) Mix the organic phase and the aqueous phase, and perform ultrafiltration to obtain the lipid composition.

38. A method for delivering a pharmaceutically active ingredient, wherein, The method uses the lipid composition according to any one of claims 1 to 36 as a carrier; the medicinally active ingredient includes at least one of a macromolecular substance and a small molecule substance.

Images