Ionizable lipid molecule containing arginine structure, lipid nanoparticles comprising same, and use thereof

Abstract

The present invention relates to an ionizable lipid molecule containing an arginine structure, lipid nanoparticles comprising same, and use thereof. Specifically, provided are an ionizable lipid molecule represented by formula (1) containing a structure of arginine and a derivative thereof, lipid nanoparticles comprising same, a preparation method therefor, and use thereof. Compared with ionizable lipid molecules conventionally used in the art, the lipid nanoparticles prepared from the ionizable lipid molecule represented by formula (1) of the present invention can achieve highly efficient delivery and expression of nucleic acids.

Description

Ionizable lipid molecules containing arginine, lipid nanoparticles containing arginine, and their applications. Technical Field

[0001] This disclosure pertains to the field of pharmaceutical biotechnology, specifically relating to ionizable lipid molecules that can deliver nucleic acids and contain an arginine structure, lipid nanoparticles containing such molecules, and their uses. Background Technology

[0002] In recent years, nucleic acid therapy has achieved several breakthroughs. The global COVID-19 pandemic in 2020 saw the emergence of mRNA nucleic acid vaccines, which became a dark horse in the field of nucleic acid therapy. Simultaneously, it propelled nucleic acid therapy to unprecedented heights. Nucleic acid drugs are a type of therapy that regulates the post-transcriptional, pre-translational stages of protein synthesis, acting upstream of protein synthesis. They possess advantages such as simple design, short development cycles, strong targeting specificity, broad therapeutic applications, and long-lasting effects. Currently, they are widely used in the treatment of genetic diseases, tumors, and viral infections, and are expected to become the third largest class of drugs after small molecule drugs and antibody drugs. However, delivery systems remain a crucial and unavoidable issue for nucleic acid drugs. How to stably and efficiently deliver nucleic acid molecules to target cells through suitable delivery systems, while preventing their degradation, is one of the major challenges currently facing the field of nucleic acid therapy.

[0003] An ideal delivery vector should possess the following characteristics: safety, stability, and efficiency. Lipid nanoparticles (LNPs) are lipid nanoparticles composed of phospholipids that encapsulate mRNA. This protects the mRNA from enzymatic degradation and clearance by the immune system during delivery, promotes its transmembrane transport, and releases it into the cytoplasm for protein translation. LNPs also neutralize antibodies, thus achieving immunity. They are currently the most advanced non-viral nucleic acid vectors used clinically. The advent of LNPs is a milestone in the development of nucleic acid therapy, successfully solving the challenges of protecting and delivering RNA. Furthermore, due to their superior flexibility, safety, and relatively easy and scalable production methods, LNPs are now used in cutting-edge mRNA vaccine candidates and widely used novel coronavirus vaccines.

[0004] Arginine, an amino acid compound, is widely distributed in various tissues and organs of the human body and is an indispensable nutrient for normal life activities. It exists in nature in multiple forms. As an essential amino acid, it plays a crucial role in protein synthesis and is essential for muscle growth and repair, making it an ideal supplement for athletes, fitness enthusiasts, and physically active individuals. Furthermore, arginine participates in regulating nitrogen balance in the body, converting excess amino nitrogen into urea, which is excreted through urine, helping to maintain normal metabolic levels. In addition, arginine has the function of enhancing immunity. It can stimulate the activity of lymphocytes, thereby enhancing the function of the immune system and improving the body's ability to fight disease. At the same time, arginine also has a certain antioxidant effect, reducing the damage of oxygen free radicals to the body and delaying cell aging. Arginine also has significant benefits for the circulatory system and liver function. It helps dilate blood vessels, improve blood circulation, lower blood pressure, and prevent cardiovascular disease. Simultaneously, arginine can promote the decomposition and excretion of toxins in the liver, helping to protect liver cells and maintain liver health.

[0005] Therefore, arginine, as an important amino acid, not only plays a crucial role in protein synthesis but also offers numerous benefits to immunity, nitrogen metabolism, antioxidation, the circulatory system, and liver function. Its multiple functions make it an important nutritional supplement for improving human health and athletic performance. Further research and application of its unique effects are expected to bring even more benefits to the health field.

[0006] Based on the properties of arginine and its derivatives, we developed a class of ionizable lipid molecules containing arginine and its derivatives and applied them to the preparation of nucleic acid (including DNA and RNA) delivery vectors. We found that using these lipid molecules for nucleic acid delivery can effectively improve the delivery efficiency of nucleic acids in vivo, achieving efficient nucleic acid expression.

[0007] Summary of the Invention

[0008] This invention relates to lipid molecules containing arginine, lipid nanoparticles containing arginine, and their uses. Specifically, it provides an ionizable lipid molecule containing arginine and its derivatives as shown in formula (1), lipid nanoparticles containing arginine, a method for preparing the ionizable lipid molecule, and its uses. Compared with conventionally used ionizable lipid molecules in the art, the lipid nanoparticles prepared from the ionizable lipid molecule shown in formula (1) of this invention can achieve highly efficient delivery and expression of nucleic acids, especially targeting the pancreas for nucleic acid delivery.

[0009] [Ionizable lipid molecules - compounds of formula (1) or pharmaceutically acceptable salts thereof]

[0010] This invention provides a compound of formula (1) or a pharmaceutically acceptable salt thereof.

[0011] wherein,

[0012] X is selected from N or CH;

[0013] P 1 selected from monovalent radicals derived from arginine or a derivative thereof, and monovalent radicals derived from dipeptides, tripeptides, tetrapeptides and pentapeptides of arginine with amino acids selected from arginine, histidine, glycine, alanine, valine, leucine, isoleucine, methionine, proline, tryptophan, serine, tyrosine, cysteine, phenylalanine, asparagine, glutamine, threonine, aspartic acid, glutamic acid and lysine;

[0014] R 1 and R 2 are each independently selected from H, C 1-30 alkyl, C 2-30 alkenyl or C 2-30 alkynyl; said alkyl, alkenyl or alkynyl is unsubstituted or substituted by one or more OH, NH2, halogen, -OC 1-20 alkyl, -OC(=O)C 1-20 alkyl, -C(=O)OC 1-20 alkyl, -SC 1-20 alkyl, -NHC(=O)C 1-20 alkyl, -C(=O)NHC 1-20 alkyl, -OC 2-20 alkenyl, -OC(=O)C 2-20 alkenyl, -C(=O)OC 2-20 alkenyl, -SC 2-20 alkenyl, -NHC(=O)C 2-20 alkenyl, -C(=O)NHC 2-20 alkenyl, C 3-8 cycloalkyl, 5-8 membered heterocyclyl, C 6-10 aryl or 5-10 membered heteroaryl;

[0015] L 1 , L 2 and L 3 are independently selected from a single bond, C 1-10 alkylene or C 2-10 alkenylene, said alkylene or alkenylene is unsubstituted or substituted by one or more OH, NH2or halogen;

[0016] L 4 , L 5 and L 6 are independently selected from a single bond, C 1-20 alkylene, C 2-10Alkenyl group, -(OCH2CH2) m -or-C 1-10 Alkylene-(OCH2CH2) m - The alkylene or alkenyl group is unsubstituted or is converted by one or more OH, NH2, halogen or C 6-10 Aryl substitution; the C 6-10 The aryl group is either unsubstituted or substituted with one or more OH groups, NH2 groups, or halogens; m is selected from integers from 1 to 10;

[0017] G 1 G 2 and G 3 Independently selected from single bonds, -NR 3 -、-O-、-NR 3 C(=O)O-、-OC(=O)NR 3 -, -C(=O)-, -OC(=O)-, -C(=O)O-, -OC(=O)O-, -C(=O)C(=O)O-, -OC(=O)C(=O)-, -OC(=O)C(=O)O-, -OC(=O)-L a -C(=O)O-、-NR 3 C(=O)NR 3 -、-C(=O)NR 3 -、-NR 3 C(=O)- or -C(=O)NH-L a -C(=O)O-;

[0018] G 4 G 5 and G 6 Independently selected from single bonds, -NR 3 -、-S-、-O-、-NR 3 C(=O)O-、-OC(=O)NR 3 -, -C(=O)-, -OC(=O)-, -C(=O)O-, -SC(=O)-, -C(=O)S-, -OC(=O)O-, -C(=O)C(=O)O-, -OC(=O)C(=O)-, -OC(=O)C(=O)O-, -OC(=O)-L a -C(=O)O-、-OC(=O)-L a -C(=O)-、-C(=O)-L a -C(=O)-、-C(=O)-L a -C(=O)NR 3 -、-NR 3 C(=O)NR 3 -、-C(=O)NR 3 -、-NR3 C(=O)-, -OC(=O)-L a -S-L a -C(=O)O- or -OC(=O)-L a -S-S-L a -C(=O)O-;

[0019] each R 3 is independently selected from H, C 1-10 alkyl, C 2-10 alkenyl, C 3-8 cycloalkyl, 5-8 membered heterocyclyl, C 6-10 aryl or 5-10 membered heteroaryl, which alkyl, alkenyl, cycloalkyl, heterocyclyl, aryl or heteroaryl is unsubstituted or substituted by one or more OH, NH2or halogen;

[0020] each L a is independently selected from C 1-10 alkylene or C 2-10 alkenylene, which alkylene or alkenylene is unsubstituted or substituted by one or more OH, NH2or halogen.

[0021] In some embodiments, P 1 is selected from the group consisting of a monovalent radical derived from L-arginine, D-arginine or a derivative thereof, and a monovalent radical derived from a dipeptide, tripeptide, tetrapeptide and pentapeptide formed from L-arginine or D-arginine and an amino acid selected from the group consisting of L-glycine, L-alanine, L-valine, L-leucine, L-isoleucine, L-methionine, L-proline, L-tryptophan, L-serine, L-tyrosine, L-cysteine, L-phenylalanine, L- asparagine, L-glutamine, L-threonine, L-aspartic acid, L-glutamic acid, L-lysine, L-arginine, L-histidine, D-glycine, D-alanine, D-valine, D-leucine, D-isoleucine, D-methionine, D-proline, D-tryptophan, D-serine, D-tyrosine, D-cysteine, D-phenylalanine, D-asparagine, D-glutamine, D-threonine, D-aspartic acid, D-glutamic acid, D-lysine, D-arginine and D-histidine.

[0022] In some embodiments, P 1 is selected from the group consisting of a monovalent radical derived from arginine or a derivative thereof, and a monovalent radical derived from a dipeptide, tripeptide, tetrapeptide and pentapeptide formed from arginine and said amino acid, which monovalent radical means a monovalent radical obtained by removing a hydrogen atom from an amino group and / or a hydroxyl group from a carboxyl group and / or a hydrogen atom from a thiol group of said arginine or derivative thereof, dipeptide, tripeptide, tetrapeptide or pentapeptide.

[0023] In some embodiments, the dipeptide containing arginine has the following connection mode: arginine is at the outermost end and connects another amino acid from the N-terminus, or arginine is at the outermost end and connects the second amino acid with the C-terminus of arginine; or arginine is at the inner end (the position close to X in formula (1) is the inner end, and the position away from X in formula (1) is the outer end), and the second amino acid is at the outermost end, connected with the N-terminus or the C-terminus of arginine.

[0024] In some embodiments, the tripeptide containing arginine has the following connection mode: arginine is at the outermost end and connects two other amino acids from the N-terminus, or arginine is at the outermost end and connects the second amino acid with the C-terminus of arginine, and the third amino acid can be freely connected to the first two amino acids; or arginine is at the inner end, and the second amino acid is at the outermost end, connected with the N-terminus of arginine, and the third amino acid can be freely connected to the first two amino acids.

[0025] In some embodiments, in the dipeptide, tripeptide, tetrapeptide and pentapeptide formed by arginine and the amino acid, the chirality of the amino acid can be randomly combined.

[0026] In some embodiments, P 1 is selected from

[0027] In some embodiments, R 1 and R 2 are each independently selected from C 5-30 alkyl or C 5-30 alkenyl; the alkyl or alkenyl is unsubstituted or substituted with one or more OH, NH2, halogen, -OC 1-20 alkyl, -OC(=O)C 1-20 alkyl, -C(=O)OC 1-20 alkyl, -NHC(=O)C 2-20 alkenyl, -C(=O)NHC 2-20 alkenyl, -NHC(=O)C 1-20 alkyl or -C(=O)NHC 1-20 alkyl.

[0028] In some embodiments, R 1 and R 2 are each independently selected from C 5-24 alkyl or C 5-24 alkenyl; the alkyl or alkenyl is unsubstituted or substituted with one or more OH, NH2, halogen, -OC 6-20 alkyl, -OC(=O)C 6-20 alkyl, -C(=O)OC 6-20 alkyl, -NHC(=O)C6-20 alkenyl, -C(=O)NHC 6-20 alkenyl, -NHC(=O)C 6-20 alkyl or -C(=O)NHC 6-20 alkyl substituted.

[0029] In some embodiments, R 1 and R 2 are each independently selected from C 5-20 alkyl or C 5-20 alkenyl; said alkyl or alkenyl is unsubstituted or substituted with one or more OH, NH2, halo, -NHC(=O)C 10-20 alkenyl or -C(=O)NHC 10-20 alkenyl.

[0030] In some embodiments, R 1 and R 2 are each independently selected from H,

[0031] In some embodiments, L 1 , L 2 and L 3 are independently selected from a single bond or C 1-10 alkylene, said alkylene is unsubstituted or substituted with one or more OH, NH2or halo.

[0032] In some embodiments, L 1 , L 2 and L 3 are independently selected from a single bond or C 1-6 alkylene, said alkylene is unsubstituted or substituted with one or more OH, NH2or halo.

[0033] In some embodiments, L 1 , L 2 and L 3 are independently selected from a single bond, -CH2-, -CH2CH2-,

[0034] In some embodiments, L 4 , L 5 and L 6 are independently selected from a single bond, C 1-10 alkylene, -(OCH2CH2) m - or -C 1-6 alkylene-(OCH2CH2) m -, said alkylene is unsubstituted or substituted with one or more OH, NH2or halo; m is selected from 1, 2, 3, 4, 5, 6, 7 or 8.

[0035] In some embodiments, L 4 , L 5 , and L 6 are independently selected from a single bond, C 1-8 alkylene, -(OCH2CH2) m -, or -C 1-4 alkylene-(OCH2CH2) m -, said alkylene being unsubstituted or substituted with one or more OH, NH2, halo, or phenyl; and m is selected from 1, 2, 3, 4, 5, or 6.

[0036] In some embodiments, L 4 , L 5 , and L 6 are independently selected from a single bond, -CH2-, -CH2CH2-, -CH(CH3)-,

[0037] In some embodiments, each R 3 is independently selected from H, C 1-10 alkyl, C 2-10 alkenyl, C 3-8 cycloalkyl, or 5-8 membered heterocyclyl, said alkyl, alkenyl, cycloalkyl, or heterocyclyl being unsubstituted or substituted with one or more OH, NH2, or halo.

[0038] In some embodiments, each R 3 is independently selected from H, C 1-10 alkyl, or C 2-10 alkenyl, said alkyl or alkenyl being unsubstituted or substituted with one or more OH, NH2, or halo.

[0039] In some embodiments, each R 3 is independently selected from H, C 1-6 alkyl, or C 2-6 alkenyl, said alkyl or alkenyl being unsubstituted or substituted with one or more OH, NH2, or halo.

[0040] In some embodiments, each R 3 is independently selected from H, C 1-4 alkyl, or C 2-4 alkenyl, said alkyl or alkenyl being unsubstituted or substituted with one or more OH, NH2, F, Cl, or Br.

[0041] In some embodiments, each R 3 is independently H.

[0042] In some embodiments, L ais selected from C 1-10 alkylene, which alkylene is unsubstituted or substituted by one or more OH, NH2, or halogen.

[0043] In some embodiments, L a is selected from C 1-6 alkylene, which alkylene is unsubstituted or substituted by one or more OH, NH2, or halogen.

[0044] In some embodiments, L a is selected from C 1-4 alkylene, which alkylene is unsubstituted or substituted by one or more OH, NH2, or halogen.

[0045] In some embodiments, L a is selected from -CH2-, -CH2CH2-, or -CH2CH2CH2-.

[0046] In some embodiments, G 1 , G 2 , and G 3 are independently selected from a single bond, -NH-, -O-, -C(=O)-, -NHC(=O)O-, -OC(=O)NH-, -OC(=O)-, -C(=O)O-, -NHC(=O)-, -C(=O)NH-, or 3 -, 3 C(=O)O-, -OC(=O)NR 3 -, 3 -C(=O)-, -C(=O)NR 3 -, a -C(=O)O-.

[0047] In some embodiments, G 1 , G 2 , and G 3 are independently selected from a single bond, -NH-, -O-, -C(=O)-, -NHC(=O)O-, -OC(=O)NH-, -OC(=O)-, -C(=O)O-, -NHC(=O)-, -C(=O)NH-, or

[0048] In some embodiments, G 4 , G 5 , and G 6 are independently selected from a single bond, -NH-, -O-, -C(=O)-, -NHC(=O)O-, -OC(=O)NH-, -OC(=O)-, -C(=O)O-, -NHC(=O)-, -C(=O)NH-, or 3 -, 3 C(=O)O-, -OC(=O)NR 3 -, a -C(=O)-, -C(=O)NR3 -, -NR 3 C(=O)-, -OC(=O)-L a -S-L a -C(=O)O- or -OC(=O)-L a -S-S-L a -C(=O)O-.

[0049] In some embodiments, G 4 , G 5 and G 6 are independently selected from a single bond, -NH-, -O-, -OC(=O)-, -C(=O)O-, -C(=O)-, -NHC(=O)-, -C(=O)NH-, -NHC(=O)O-, -OC(=O)NH- or

[0050] In some embodiments, the present application provides a compound or a pharmaceutically acceptable salt thereof, which is selected from the following:

[0051] [lipid carrier]

[0052] The present application provides a lipid carrier comprising an ionizable lipid molecule, a polyethylene glycol lipid molecule, a steroidal lipid molecule and a helper lipid molecule, the ionizable lipid molecule comprising a compound represented by the above formula (1) or a pharmaceutically acceptable salt thereof.

[0053] In some embodiments, the ionizable lipid molecule contains one or more ionizable sites, including pyridine, imidazole, primary amine, secondary amine and tertiary amine, etc.

[0054] In some embodiments, the ionizable lipid molecule is a compound represented by the above formula (1) or a pharmaceutically acceptable salt thereof.

[0055] In some embodiments, the ionizable lipid molecule further comprises at least one of: (1-octyl nonyl 8-[(2-hydroxyethyl)[6-O-6-(undecyloxy)hexyl]amino]-octanoate) SM-102, ((4-hydroxybutyl)azanediyl)bis(hexan-6,1-diyl)bis(2-hexyldecanoate) ALC-0315, 1,2-dioleoyl-3-dimethylammonium-propane DODAP, 1,2-dioleoyloxy-3-dimethylamino-propane DODMA, N-(4-carboxybenzyl)-N,N-dimethyl-2,3-bis(oleoyloxy)propane DOBAQ, YSK05, Dlin-DMA, N,N-dimethyl-2,2-di-(9Z,12Z)-9,12-octadecadien-1-yl-1,3-dioxolane-4- ethanamine Dlin-KC2-DMA, 4-(N,N-dimethylamino)butanoic acid (dilinoleyl) methyl ester Dlin-MC3-DMA, and the like.

[0056] In some embodiments, the ionizable lipid molecule further comprises at least one of: SM-102, ALC-0315, DODAP, DODMA, DOBAQ, YSK05, Dlin-DMA, Dlin-KC2-DMA, and Dlin-MC3-DMA.

[0057] In some embodiments, the ionizable lipid molecule further comprises at least one of: SM-102, ALC-0315, DODAP, and Dlin-DMA.

[0058] In some embodiments, the ionizable lipid molecule further comprises at least one of: SM-102 and ALC-0315.

[0059] In some embodiments, the polyethylene glycol lipid molecule is selected from at least one of: 2-[(polyethylene glycol)-2000]-N,N-tetracosanylacetamide (ALC-0159), 1,2-dimyristoyl-sn-glycero-methoxypolyethylene glycol (PEG-DMG), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N- [amino(polyethylene glycol)] (PEG-DSPE), PEG-disteraryl glycerol (PEG-DSG), PEG-dipalmitoyl, PEG-dioleyl, PEG-distearoyl, PEG-digalactosylceramide (PEG-DAG), PEG-dipalmitoyl phosphatidyl ethanolamine (PEG-DPPE), and PEG-1,2-dimyristoyloxypropyl-3-amine (PEG-c-DMA).

[0060] In some embodiments, the sterol lipid molecule is selected from at least one of: avenasterol, beta-sitosterol, brassicasterol, ergocalciferol, campesterol, cholestanol, cholesterol, coprostanol, dehydrocholesterol, desmosterol, dihydroergocalciferol, dihydrocholesterol, dihydroergosterol, dinosterol, epicholesterol, ergosterol, fucosterol, hexahydroergosterol, hydroxycholesterol, lanosterol, lumisterol, mycocerotic acid, sitostanol, sitosterol, stigmasterol, cholic acid, glycocholic acid, taurocholic acid, deoxycholic acid, and lithocholic acid.

[0061] In some embodiments, the sterol lipid molecule is selected from at least one of: cholesterol, cholestanol, ergocalciferol, dihydrocholesterol, fucosterol, taurocholic acid, and deoxycholic acid.

[0062] In some embodiments, the sterol lipid molecule is selected from at least one of: cholesterol, cholestanol, dihydrocholesterol, fucosterol, and deoxycholic acid.

[0063] In some embodiments, the sterol lipid molecule is selected from at least one of: cholesterol and dihydrocholesterol.

[0064] In some embodiments, the helper lipid molecule is selected from at least one of: 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dipalmitoyl-sn-glycero-3- phosphocholine (DPPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2- dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), 1,2-dimyristoyl-sn-glycero-3- phosphoethanolamine (DMPE), 2-dioleoyl-sn-glycero-3-phospho-(1 '-rac-glycerol) (DOPG), oleoyl phosphatidylcholine (POPC), and 1-palmitoyl-2-oleoyl phosphatidyl ethanolamine (POPE).

[0065] In some embodiments, the helper lipid molecule is selected from at least one of: 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dipalmitoyl-sn-glycero-3- phosphocholine (DPPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2- dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE).

[0066] In some embodiments, the helper lipid molecule is selected from at least one of: 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and 1,2-dioleoyl-sn-glycero-3- phosphoethanolamine (DOPE).

[0067] In some embodiments, the lipid carrier comprises 10-70% of the ionizable lipid molecule (the compound represented by formula (1) above or a pharmaceutically acceptable salt thereof), 5-60% of the steroidal lipid molecule, 1-60% of the polyethylene glycol lipid molecule, and 1-30% of the helper lipid molecule, in terms of molar percentage.

[0068] In some embodiments, the molar percentage of the compound represented by formula (1) or a pharmaceutically acceptable salt thereof is 15-65%.

[0069] In some embodiments, the molar percentage of the compound represented by formula (1) or a pharmaceutically acceptable salt thereof is 30-60%.

[0070] In some embodiments, the molar percentage of the compound represented by formula (1) or a pharmaceutically acceptable salt thereof is 45-55%.

[0071] In some embodiments, the molar percentage of the steroidal lipid molecule is 10-50%.

[0072] In some embodiments, the molar percentage of the steroidal lipid molecule is 25-45%.

[0073] In some embodiments, the molar percentage of the steroidal lipid molecule is 30-40%.

[0074] In some embodiments, the molar percentage of the polyethylene glycol lipid molecule is 1-30%.

[0075] In some embodiments, the molar percentage of the polyethylene glycol lipid molecule is 1-10%.

[0076] In some embodiments, the molar percentage of the polyethylene glycol lipid molecule is 1-5%.

[0077] In some embodiments, the molar percentage of the helper lipid molecule is 1-25%.

[0078] In some embodiments, the molar percentage of the helper lipid molecule is 5-15%.

[0079] In some embodiments, the molar percentage of the helper lipid molecule is 8-12%.

[0080] In some embodiments, the molar ratio of the ionizable lipid molecule, the helper lipid molecule, the steroidal lipid molecule, and the ionizable lipid molecule in the lipid carrier is 50:10:38:2, 45:10:42:3, 30:25:30:10, 46:15:40:3, 50:10:38.5:1.5, 50:10:37:3, 50:9:38:3, or the like.

[0081] [NUCLEIC ACID LIPID NANOPARTICLE COMPOSITION]

[0082] The present application provides a nucleic acid lipid nanoparticle composition comprising the above-described lipid carrier and at least one ingredient selected from a nucleic acid, a small molecule drug, and a protein polypeptide.

[0083] The present application provides a nucleic acid lipid nanoparticle composition comprising the above-described lipid carrier and a nucleic acid.

[0084] In some embodiments, the nucleic acid is selected from at least one of DNA, mRNA, rRNA, siRNA, tRNA, microRNA, antisense nucleic acid, self-replicating RNA, and circular RNA.

[0085] In some embodiments, the nucleic acid is mRNA.

[0086] In some embodiments, the nucleic acid is Firefly Luciferase mRNA, Green Fluorescent Protein (GFP) mRNA, Chicken Ovalbumin (OVA) mRNA, IL-2 protein mRNA, or H1N1 influenza virus mRNA.

[0087] In some embodiments, the above-described small molecule drug is selected from at least one of small molecule chemotherapeutic drugs commonly used in the art (such as doxorubicin, cisplatin, oxaliplatin, amphotericin B, paclitaxel, KRAS inhibitors, etc.).

[0088] In some embodiments, the above-described protein polypeptide is selected from at least one of polypeptide and protein drugs commonly used in the art, such as insulin, calcitonin, oxytocin, octreotide, leuprolide, desmopressin, etc.

[0089] In some embodiments, the mass ratio of the lipid carrier to the nucleic acid in the nucleic acid lipid nanoparticle composition is 5:1-50:1.

[0090] In some embodiments, the mass ratio of the lipid carrier to the nucleic acid in the nucleic acid lipid nanoparticle composition is 10:1-30:1.

[0091] In some embodiments, the nucleic acid is mRNA, and the mass ratio of the lipid carrier to the nucleic acid in the nucleic acid lipid nanoparticle composition is 20:1-30:1.

[0092] In some embodiments, the nucleic acid is mRNA, and the mass ratio of the lipid carrier to the nucleic acid in the nucleic acid lipid nanoparticle composition is 20:1, 25:1, or 30:1.

[0093] In some embodiments, the particle size of the nucleic acid lipid nanoparticle composition is 30-500 nm.

[0094] In some embodiments, the particle size of the nucleic acid lipid nanoparticle composition is 30-200 nm.

[0095] In some embodiments, the particle size can be 30 nm, 40 nm, 50 nm, 60 nm, 70 nm, 80 nm, 90 nm, 100 nm, 110 nm, 120 nm, 130 nm, 140 nm, 150 nm, 160 nm, 170 nm, 180 nm, 190 nm, 200 nm, etc.

[0096] In some embodiments, the encapsulation efficiency of the nucleic acid in the nucleic acid lipid nanoparticle composition is greater than 50%. Illustratively, the encapsulation efficiency can be 55%, 60%, 65%, 70%, 75%, 79%, 80%, 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, etc.

[0097] [Nucleic acid lipid nanoparticle preparation, use, and treatment method]

[0098] The present application provides a nucleic acid lipid nanoparticle preparation comprising the above-mentioned nucleic acid lipid nanoparticle composition, and a pharmaceutically acceptable excipient.

[0099] The present application also provides the use of the above-mentioned compound represented by formula (1) or a pharmaceutically acceptable salt thereof, the above-mentioned lipid carrier, or the above-mentioned nucleic acid lipid nanoparticle composition in the preparation of a nucleic acid drug, a vaccine, a polypeptide drug, and a small molecule drug.

[0100] The present application also provides the use of the above-mentioned compound represented by formula (1) or a pharmaceutically acceptable salt thereof, the above-mentioned lipid carrier, or the above-mentioned nucleic acid lipid nanoparticle composition in the preparation of a nucleic acid drug or a vaccine targeting the pancreas.

[0101] The present application also provides the above-mentioned compound represented by formula (1) or a pharmaceutically acceptable salt thereof, the above-mentioned lipid carrier, or the above-mentioned nucleic acid lipid nanoparticle composition for use in the in vivo delivery of a nucleic acid drug or a vaccine, in particular, the delivery of a nucleic acid drug or a vaccine targeting the pancreas.

[0102] The present disclosure also provides methods for in vivo delivery of a nucleic acid drug or vaccine, the methods comprising administering to a subject in need thereof the above nucleic acid lipid nanoparticle composition or the above nucleic acid lipid nanoparticle formulation.

[0103] The present disclosure also provides methods for targeted pancreatic delivery of a nucleic acid drug or vaccine, the methods comprising administering to a subject in need thereof the above nucleic acid lipid nanoparticle composition or the above nucleic acid lipid nanoparticle formulation.

[0104] The present disclosure also provides methods for treating or preventing a disease (e.g., an inflammatory disease, a viral infection, and a cancer) or a condition in a subject by delivering a nucleic acid, the methods comprising administering to a subject in need thereof the above nucleic acid lipid nanoparticle composition or the above nucleic acid lipid nanoparticle formulation.

[0105] The term “inflammatory disease” includes autoimmune disorders, allergic disorders, and inflammatory disorders, such as selected from the group consisting of acute pancreatitis, alcoholic chronic pancreatitis, biliary obstructive chronic pancreatitis, idiopathic chronic pancreatitis, arthritis, ankylosing spondylitis, inflammatory bowel disease, ulcerative colitis, gastritis, pancreatitis, Crohn’s disease, celiac disease, multiple sclerosis, systemic lupus erythematosus, rheumatoid arthritis, rheumatic fever, gout, organ or transplant rejection, acute or chronic graft-versus-host disease, chronic allograft rejection, Behcet’s disease, uveitis, psoriasis, dermatitis, atopic dermatitis, dermatomyositis, myasthenia gravis, Grave’s disease, Hashimoto’s thyroiditis, Sjogren’s syndrome, and blistering disorders (e.g., pemphigus vulgaris), antibody-mediated vasculitis syndromes, including ANCA-associated vasculitis, purpura, and immune complex vasculitis (cancer or infection stage one or two). The allergic disorder can be selected from the group consisting of contact dermatitis, celiac disease, asthma, hypersensitivity to house dust mites, pollen and related allergens, berylliosis.

[0106] The term “viral infection” includes, but is not limited to, retroviral infection, hepatitis viral infection, COVID-19 coronavirus infection, Zika virus infection, dengue virus infection, and the like.

[0107] The term “cancer” includes, but is not limited to, primary pancreatic cancer (including ductal adenocarcinoma, endocrine tumors, cystic papillary adenocarcinoma, papillary pancreatic cancer, mucinous cystadenoma), primary lung cancer (including non-small cell lung cancer and small cell lung cancer), metastatic lung cancer (liver cancer lung metastasis, breast cancer lung metastasis, colon cancer lung metastasis, melanoma lung metastasis, and the like), and cancer in other parts of the body.

[0108] In some embodiments, the above nucleic acid lipid nanoparticle composition or the above nucleic acid lipid nanoparticle formulation is administered by one of the following routes of administration: intraperitoneal, oral, intranasal, intravenous, intraperitoneal, intramuscular, intraarticular, intralesional, intratracheal, subcutaneous, and intradermal. In some embodiments, the above nucleic acid lipid nanoparticle composition or the above nucleic acid lipid nanoparticle formulation is administered, for example, via enteral or parenteral routes of administration. In some embodiments, a dose of about 0.001 mg / kg to about 10 mg / kg of the nucleic acid lipid nanoparticle composition or nucleic acid lipid nanoparticle formulation is administered to the subject.

[0109] [Method for producing nucleic acid-encapsulating lipid nanoparticles]

[0110] The present application provides a method for producing nucleic acid-encapsulating lipid nanoparticles, comprising the following steps:

[0111] (A1) mixing ionizable lipid molecules (the compound represented by the formula (1) or a pharmaceutically acceptable salt thereof), polyethylene glycol lipid molecules, helper lipid molecules, and steroidal lipid molecules in the above-described proportions, dissolving with a solvent to obtain an organic phase liposome solution;

[0112] (A2) dissolving a nucleic acid with a buffer solution of an appropriate pH to obtain an aqueous phase nucleic acid solution;

[0113] (A3) uniformly mixing the organic phase liposome solution and the aqueous phase nucleic acid solution in the above-described mass ratio and a certain volume ratio using a microfluidic device to produce a nucleic acid-encapsulating lipid nanoparticle solution;

[0114] In some embodiments, the solvent used to dissolve the lipid molecules in step (A1) is methanol, ethanol, tetrahydrofuran, acetone, dimethyl sulfoxide, or N,N-dimethylformamide.

[0115] In some embodiments, the solvent in step (A1) is ethanol, tetrahydrofuran, or acetone.

[0116] In some embodiments, the solvent in step (A1) is ethanol.

[0117] In some embodiments, the buffer solution in step (A2) is an acetic acid / sodium acetate solution or a citric acid / sodium citrate solution.

[0118] In some embodiments, the buffer solution in step (A2) is a citric acid / sodium citrate solution.

[0119] In some embodiments, the pH of the buffer solution in step (A2) is 3-9.

[0120] In some embodiments, the pH of the buffer solution in step (A2) is 4-6.

[0121] In some embodiments, the pH of the buffer solution in step (A2) is 5.

[0122] In some embodiments, the concentration of the buffer solution in step (A2) is 1 mM-1 M.

[0123] In some embodiments, the concentration of the buffer solution in step (A2) is 20 mM-500 mM.

[0124] In some embodiments, the concentration of the buffer solution in step (A2) is 100 mM.

[0125] In some embodiments, the volume ratio of the organic phase liposome solution to the aqueous phase nucleic acid solution in step (A3) is 1:1-1:10.

[0126] In some embodiments, the volume ratio of the organic phase liposome solution to the aqueous phase nucleic acid solution in step (A3) is 1:1-1:5.

[0127] In some embodiments, the volume ratio of the organic phase liposome solution to the aqueous phase nucleic acid solution in step (A3) is 1:3.

[0128] In some embodiments, the microfluidic device in step (A3) can be a microfluidic device commonly used in the art. For example, INano TM L / L+, Miaena or BT, Precision NanoSystems.

[0129] [Liposomes and liposome compositions]

[0130] The present application also provides a liposome comprising the compound represented by the above formula (1) or a pharmaceutically acceptable salt thereof.

[0131] In some embodiments, the liposome comprises the compound represented by the above formula (1) or a pharmaceutically acceptable salt thereof, the above steroidal lipid molecule, and the above helper lipid molecule.

[0132] In some embodiments, the liposome comprises the compound represented by the above formula (1) or a pharmaceutically acceptable salt thereof, the above steroidal lipid molecule, the above polyethylene glycol lipid molecule, and the above helper lipid molecule.

[0133] The present application also provides a liposome composition comprising the above liposome and at least one component selected from the group consisting of nucleic acids, small molecule drugs, and protein polypeptides.

[0134] In some embodiments, the above-mentioned nucleic acid is selected from at least one of DNA, mRNA, rRNA, siRNA, tRNA, microRNA, antisense nucleic acid, self-replicating RNA, and circular RNA.

[0135] In some embodiments, the above-mentioned small molecule drug is selected from at least one of small molecule chemotherapy drugs commonly used in the art (such as doxorubicin, cisplatin, oxaliplatin, amphotericin B, paclitaxel, KRAS inhibitor, etc.).

[0136] In some embodiments, the above-mentioned protein polypeptide is selected from at least one of polypeptide and protein drugs commonly used in the art, such as insulin, calcitonin, oxytocin, octreotide, leuprolide, desmopressin, etc.

[0137] In some embodiments, the liposome composition comprises 1% to 100% of the ionizable lipid molecule (the compound of formula (1) or a pharmaceutically acceptable salt thereof), 0% to 80% of the steroidal lipid molecule, 0% to 80% of the polyethylene glycol lipid molecule, and 0% to 80% of the helper lipid molecule, in terms of molar percentage.

[0138] In some embodiments, in the above-mentioned liposome composition, the mass ratio of the lipid carrier to the nucleic acid is 5:1 to 50:1; or, the mass ratio of the lipid carrier to the nucleic acid is 10:1 to 30:1.

[0139] In some embodiments, in the above-mentioned liposome composition, the mass ratio of the lipid carrier to the small molecule drug or protein polypeptide is 2:1 to 100:1; or, the mass ratio is 5:1 to 50:1; or, the mass ratio is 10:1 to 30:1; or, the mass ratio is 20.5:1.

[0140] [Method for preparing liposome composition]

[0141] The present application provides a method for preparing a liposome composition, which comprises the following steps:

[0142] (A1) Mix the ionizable lipid molecule (the compound of formula (1) or a pharmaceutically acceptable salt thereof), the polyethylene glycol lipid molecule, the helper lipid molecule, and the steroidal lipid molecule in the above-mentioned proportions, and dissolve them with a solvent to obtain an organic phase lipid solution;

[0143] (A2) In a round-bottom flask, add the above-mentioned organic phase lipid solution, and slowly evaporate the solvent with a rotary evaporator to form a uniform organic lipid film.

[0144] (A3) Disperse the small molecule drug or protein polypeptide in a solvent, add the obtained solution to the lipid film, and gently shake until a uniform liposome suspension is formed.

[0145] (A4) pour the liposome suspension into an ultrasonic processor for processing, and further control the size of the liposome by using an aperture sieve.

[0146] In some embodiments, the solvent used for dissolving the lipid molecules in step (A1) is chloroform, dichloromethane, methanol, ethanol, tetrahydrofuran, acetone, dimethyl sulfoxide, N,N-dimethylformamide or a mixture of the above solvents.

[0147] The solvent in step (A3) is selected from distilled water, PBS buffer, citrate buffer, acetate buffer or phosphate buffer.

[0148] The frequency of the ultrasonic treatment in step (A4) is 5-100 Hz.

[0149] The present application has the following beneficial effects:

[0150] 1. The lipid carrier comprising the compound represented by formula (1) or a pharmaceutically acceptable salt thereof can achieve pancreatic targeting delivery.

[0151] 2. The lipid carrier comprising the compound represented by formula (1) or a pharmaceutically acceptable salt thereof is tightly combined with nucleic acid, and can achieve high encapsulation efficiency and stable protection of nucleic acid.

[0152] 3. The formed LNP has good biocompatibility and is more stable; can improve the efficiency of LNP in delivering nucleic acid in vivo, and achieve efficient expression of nucleic acid.

[0153] 4. The lipid nanoparticle is suitable for nucleic acid delivery of different nucleic acid molecular weights, lengths and sequences, and has universality.

[0154] 5. The present application has simple synthesis, low raw material price and is suitable for large-scale production. BRIEF DESCRIPTION OF DRAWINGS

[0155] Figure 1 shows the particle size distribution of Basic LNP@mRNA Luc and I-1 LNP@mRNA Luc , I-2 LNP@mRNA Luc , I-3 LNP@mRNA Luc of the present application.

[0156] Figure 2 shows the particle size distribution of Basic LNP@mRNA OVA and I-1 LNP@mRNA OVA , I-2 LNP@mRNA OVA , I-3 LNP@mRNA OVA , I-4 LNP@mRNA OVAI-5 LNP@mRNA OVA VII-1 LNP@mRNA OVA Antigen presentation efficiency achieved by transfecting DC2.4 cells.

[0157] Figure 3 shows mouse in vivo / ex vivo fluorescent protein expression experiment of I-2 LNP@mRNA Luc of the present application.

[0158] Figure 4 shows mouse in vivo / ex vivo fluorescent protein expression experiment of Basic LNP@mRNA Luc .

[0159] Figure 5 shows pig ex vivo fluorescent protein expression experiment of Basic LNP@mRNA Luc and I-2 LNP@mRNA Luc of the present application.

[0160] Figure 6 shows mouse pancreas fluorescent protein expression results of I-2 LNP@mRNA Luc+GFP of the present application.

[0161] Figure 7 shows mouse anti-tumor results of Basic LNP@mRNA IL-2 and I-3 LNP@mRNA IL-2 of the present application.

[0162] Figure 8 shows mouse pancreas T cell infiltration results of Basic LNP@mRNA IL-2 and I-1 LNP@mRNA IL-2 of the present application.

[0163] Figure 9 shows mouse inflammatory factor IL-6 levels after injection with Basic LNP@mRNA GFP and I-1 LNP@mRNA GFP , II-1 LNP@mRNA GFP , III-1 LNP@mRNA GFP , IV-1 LNP@mRNA GFP and V-1 LNP@mRNA GFP of the present application.

[0164] Figure 10 shows mouse inflammatory factor IL-6 levels after injection with Basic LNP@mRNA GFP and I-1 LNP@mRNA GFP , II-1 LNP@mRNA GFP , III-1 LNP@mRNA GFP , IV-1 LNP@mRNA GFP and V-1 LNP@mRNAGFP Inflammatory factor TNF-a level in mice after injection.

[0165] Figure 11 shows the particle size distribution of liposomes obtained using compound V-1.

[0166] Figure 12 shows the luciferase organ biodistribution results in mice of I-2 LNP@mRNA Luc+GFP of the present application. DETAILED DESCRIPTION

[0167] I. DEFINITIONS

[0168] In the present disclosure, unless otherwise indicated, the scientific and technical terms used herein have the meanings that would be generally understood by one of ordinary skill in the art. Also, the relevant terms and laboratory procedures steps used herein are those that are conventional in the relevant art. Also, for better understanding of the present disclosure, the definitions and explanations of the relevant terms are provided below.

[0169] Unless otherwise indicated, the terms “comprise(s),” “contain(s),” “include(s),” “have(s),” “hold(s),” and “possess(s)” as used throughout this description and in the claims, are each construed in the sense that they allow for items not excluded by those terms to be present.

[0170] The term “pharmaceutically acceptable” is used in reference to those compounds, materials, compositions, and / or dosage forms that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0171] The term “pharmaceutically acceptable salt” refers to a salt of a compound of the present application that is prepared from a compound of the present application having a particular substituent with a relatively nontoxic acid or base. Alkali addition salts can be obtained by contacting the compound of the present application in pure solution or in a suitable inert solvent with a sufficient amount of a base to produce the desired salt. Acid addition salts can be obtained by contacting the compound of the present application in pure solution or in a suitable inert solvent with a sufficient amount of an acid to produce the desired salt. Certain specific compounds of the present application contain both basic and acidic functionalities that allow them to be converted into either alkali or acid addition salts.

[0172] The pharmaceutically acceptable salts of the present application can be synthesized from the parent compound that contains an acidic or basic moiety by conventional chemical methods. Generally, the salt will be prepared by reacting the free acid or base forms of these compounds with a stoichiometric amount of the appropriate base or acid in water or in an organic solvent or in a mixture of both.

[0173] The compounds of the present disclosure can be asymmetric, e.g., having one or more stereocenters. Unless otherwise indicated, all stereoisomers are included, e.g., enantiomers and diastereomers. Compounds of the present disclosure containing asymmetric carbon atoms can be isolated in optically active form or as racemic mixtures. Optically active forms can be separated from racemic mixtures or synthesized from chiral starting materials or chiral reagents. The racemates, diastereomers, enantiomers are all included within the scope of the present disclosure.

[0174] In the present disclosure, denotes the position at which the substituent is bonded.

[0175] The term "optionally" or "optional" means that the subsequently described event or circumstance can or can not occur, and this description includes instances where the event or circumstance occurs and instances where it does not.

[0176] When a numerical range is disclosed, any number or any sub-range within the range is specifically disclosed. In particular, each and every stated numerical range and subrange has its own specifically disclosed meaning. For example, a disclosure of a range of "a to b" is specifically disclosed as a range of "a to b," and also as each and every number and sub-range within the range of "a to b." For example, "C 1-4 " is specifically disclosed as C 2-4 , C 3-4 , C 1-2 , C 1-3 , C 1-4 , etc., as well as C1, C2, C3, C4, etc. Also, for example, "5-10 membered" is specifically disclosed as any sub-range and each and every point value within the range of 5-10 membered, e.g., 5-6 membered, 5-7 membered, 5-8 membered, 5-9 membered, 6-7 membered, 6-8 membered, etc., as well as 5, 6, 7, 8, 9, 10 membered, etc.

[0177] When any variable (e.g., R n ) occurs more than one time in a constituent or

[0178] The term "substituted" or "substitution" means that any one or more hydrogen atoms on a specified atom or group is / are replaced with a substituent, as long as the valence of the specified atom or group is normal and the resulting compound is stable. When the substituent is oxo (i.e., =0), it means that two hydrogen atoms are replaced. Unless otherwise specified, the kind and number of substituents can be any that are chemically possible, on the basis of chemistry. The substituents can be selected from one, two or more of deuterium, halogen group, cyano group, nitro group, -C(=0)R, -C(=0)OR', -OC(=0)R", imido group, amido group, hydroxyl group, substituted or unsubstituted amine group, substituted or unsubstituted alkyl group, substituted or unsubstituted cycloalkyl group, substituted or unsubstituted haloalkyl group, substituted or unsubstituted alkoxy group, substituted or unsubstituted alkenyl group, substituted or unsubstituted alkynyl group, substituted or unsubstituted aryl group, substituted or unsubstituted aryloxy group, substituted or unsubstituted heteroaryl group, and the like, but are not limited thereto.

[0179] The term "independently" means that at least two groups (or ring systems) present in a structure, which have the same or similar range of values, can have the same or different meaning in a particular case. For example, substituent X and substituent Y are independently hydrogen, halogen, hydroxyl, cyano, alkyl or aryl, then when substituent X is hydrogen, substituent Y can be hydrogen, halogen, hydroxyl, cyano, alkyl or aryl; similarly, when substituent Y is hydrogen, substituent X can be hydrogen, halogen, hydroxyl, cyano, alkyl or aryl.

[0180] The term "halogen" means fluorine (F), chlorine (Cl), bromine (Br) and iodine (I).

[0181] The term "alkyl" refers to a saturated aliphatic hydrocarbon group, including straight-chain or branched-chain saturated hydrocarbon groups having the number of carbon atoms indicated. As the term "C 1-10 The term "alkyl" refers to a saturated aliphatic hydrocarbon group, including straight-chain or branched-chain saturated hydrocarbon groups having the number of carbon atoms indicated. As the term "C 10 The term "alkyl" refers to a saturated aliphatic hydrocarbon group, including straight-chain or branched-chain saturated hydrocarbon groups having the number of carbon atoms indicated. As the term "C

[0182] The term "alkylene" refers to a straight-chain or branched-chain divalent saturated aliphatic hydrocarbon group, which can be connected to two groups (or fragments) of the same carbon atom or different carbon atoms. For example, the term "C 1-10"Alkylene" refers to an alkylene group having 1-10 carbon atoms (such as methylene, 1,1-ethylene, 1,2-ethylene, 1,2-propylene, 1,3-butylene, etc.). Alkylenes can be optionally substituted or unsubstituted.

[0183] The term "alkenyl" refers to a monovalent, straight-chain or branched alkane group consisting only of carbon and hydrogen atoms, containing at least one double bond, and connected to other segments via a single bond. This includes (but is not limited to) vinyl, propenyl, allyl, isopropenyl, butenyl, and isobutenyl groups. For example, "C..." 2-30 "Alkenyl" refers to a monovalent straight-chain or branched hydrocarbon group containing 2 to 30 carbon atoms and having at least one carbon-carbon double bond. Alkenyl groups can be optionally substituted or unsubstituted.

[0184] The term "alkenyl" refers to a divalent, straight-chain or branched alkane group consisting only of carbon and hydrogen atoms, containing at least one double bond, and connected to other segments via two single bonds, including (but not limited to) vinylenes. For example, "C 2-10 "Alkenyl" refers to a divalent straight-chain or branched hydrocarbon group containing 2 to 10 carbon atoms and having at least one carbon-carbon double bond. Alkenyl groups can be optionally substituted or unsubstituted.

[0185] The term "alkynyl" refers to a monovalent, straight-chain or branched alkane group composed only of carbon and hydrogen atoms, containing at least one carbon-carbon triple bond, and connected to other segments by a single bond. This includes (but is not limited to) ethynyl, propynyl, butynyl, and pentyynyl groups. For example, "C..." 2-30 "Alynyl" refers to a monovalent straight-chain or branched hydrocarbon group containing 2 to 30 carbon atoms and having at least one carbon-carbon triple bond. The alkynyl group can be optionally substituted or unsubstituted.

[0186] The term "cycloalkyl" refers to a non-aromatic hydrocarbon group that is saturated or partially saturated, monocyclic or polycyclic (such as bicyclic, e.g., fused, bridged, or spirocyclic) in nature. For example, the term "C 3-8 "Cycloalkyl" refers to a cycloalkyl group having 3 to 8 carbon atoms. Examples include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl. Cycloalkyl groups can be optionally substituted or unsubstituted.

[0187] The term "heterocyclic group" refers to a saturated or partially saturated, monocyclic or polycyclic (such as bicyclic, e.g., fused, bridged, or spirocyclic) non-aromatic group whose ring atoms consist of a carbon atom and at least one heteroatom selected from N, O, and S, wherein the S atom is optionally substituted to form S(=O), S(=O)2, or S(=O)(=NR). x ), R x Independently selected from H or C 1-4alkyl. If valence requirements are met, the heterocyclyl group can be attached to the remainder of the molecule through any one of the ring atoms. For example, the term "5-8 membered heterocyclyl" refers to a heterocyclyl group having 5 to 8 ring atoms. Common heterocyclyl groups include, but are not limited to, oxiranyl, aziridinyl, azetidinyl, oxetanyl, tetrahydrofuranyl, dioxolanyl, pyrrolidinyl, pyrrolidonyl, imidazolidinyl, pyrazolidinyl, tetrahydropyranyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl, dithianyl, or trithianyl. The heterocyclyl groups in the present application are optionally substituted with one or more substituents described in the present application.

[0188] The term "aryl" refers to a monocyclic or fused polycyclic aromatic hydrocarbon group having a conjugated pi-electron system. For example, the term "C 6-10 The term "aryl" refers to a monocyclic or fused polycyclic aromatic hydrocarbon group having a conjugated pi-electron system. For example, the term "C

[0189] The term "heteroaryl" refers to a monocyclic or fused polycyclic aromatic group having a conjugated pi-electron system, the ring atoms of which are composed of carbon atoms and at least one heteroatom selected from N, O, and S. If valence requirements are met, the heteroaryl group can be attached to the remainder of the molecule through any one of the ring atoms. For example, the term "5-10 membered heteroaryl" refers to a heteroaryl group having 5 to 10 ring atoms. Common heteroaryl groups include, but are not limited to, thienyl, furanyl, pyrrolyl, oxazolyl, thiazolyl, imidazolyl, pyrazolyl, isoxazolyl, isothiazolyl, oxadiazolyl, triazolyl, thiadiazolyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl, and benzo derivatives thereof, pyrrolopyridyl, pyrrolopyrazinyl, pyrazolopyridyl, imidazopyridyl, pyrrolopyrimidinyl, pyrazolopyrimidinyl, purinyl, and the like. The heteroaryl groups in the present application are optionally substituted with one or more substituents described in the present application, such as halo, C 1-6 The term "heteroaryl" refers to a monocyclic or fused polycyclic aromatic group having a conjugated pi-electron system, the ring atoms of which are composed of carbon atoms and at least one heteroatom selected from N, O, and S. If valence requirements are met, the heteroaryl group can be attached to the remainder of the molecule through any one of the ring atoms. For example, the term "5-10 membered heteroaryl" refers to a heteroaryl group having 5 to 10 ring atoms. Common heteroaryl groups include, but are not limited to, thienyl, furanyl, pyrrolyl, oxazolyl, thiazolyl, imidazolyl, pyrazolyl, isoxazolyl, isothiazolyl, oxadiazolyl, triazolyl, thiadiazolyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl, and benzo derivatives thereof, pyrrolopyridyl, pyrrolopyrazinyl, pyrazolopyridyl, imidazopyridyl, pyrrolopyrimidinyl, pyrazolopyrimidyl, purinyl, and the like. The heteroaryl groups in the present application are optionally substituted with one or more substituents described in the present application, such as halo, C

[0190] The term "pharmaceutically acceptable excipient" means an excipient that is compatible with the nucleic acid lipid nanoparticle composition described above, and that is not deleterious to the recipient thereof at dosages to be employed. Examples include, but are not limited to, carriers, diluents, binders, absorbents, colorants, adjuvants, excipients, preservatives, fillers, disintegrants, wetting agents, emulsifiers, suspending agents, sweetening agents, flavoring agents, flavorants, antibacterial agents, antifungal agents, lubricants, dispersing agents, temperature-sensitive materials, temperature-regulating agents, adhesion agents, stabilizing agents, suspending agents, and the like.

[0191] II. Specific Embodiments

[0192] The application will be described in further detail by way of examples. The examples are only used to further illustrate the present application and should not be construed as limiting the scope of the present application. Some non-essential improvements and adjustments made by those skilled in the art based on the content of the present application are within the scope of the present application.

[0193] The reagents or instruments used in the examples are all commercially available conventional products. If the specific conditions are not specified, the conventional conditions or the conditions recommended by the manufacturer are used. In the present application, the term "room temperature" refers to 20°C ± 5°C. When used to modify a numerical value or a range of numerical values, the term "about" used in the present application refers to the numerical value or range of numerical values and the acceptable error range of the numerical value or range of numerical values by those skilled in the art, for example, the error range is ± 10%, ± 5%, ± 4%, ± 3%, ± 2%, ± 1%, ± 0.5%, etc.

[0194] In the following examples, the experimental methods are described, and if not specifically stated, they are all conventional methods; the reagents and materials are commercially available, unless otherwise specified.

[0195] The abbreviations used herein have the following meanings:

[0196] Example 1: Synthesis method of compound I-1

[0197] Compound 1 (1.0 eq) and compound 2 (1.0 eq) were dissolved in DCM, EDC (1.0 eq) and DMAP (0.1 eq) were added, stirred at room temperature for 12 h, after the reaction was completed, purified by column chromatography (DCM:MeOH=10:1) to obtain compound 3 (yield: 59%). Compound 3 (1.0 eq) and compound 4 (2.0 eq) were dissolved in DCM, EDC (2.0 eq) and DMAP (0.2 eq) were added, stirred at room temperature for 24 h, after the reaction was completed, purified by column chromatography (DCM:MeOH=20:1) to obtain compound 5 (yield: 76%). Compound 5 (1.0 eq) was dissolved in DCM, 10% TFA was slowly added at 0°C, stirred at room temperature for 48 h, after the reaction was completed, precipitated with ether to obtain compound 6, i.e. compound I-1 (yield: 87%). 1H NMR (400 MHz, CDC13, 293 K) δ (ppm): 5.40-5.35 (tt, J = 3.1, 1.4 Hz, 4H), 4.32-4.15 (dt, J = 12.9, 6.2 Hz, 6H), 3.93-3.74 (p, J = 6.5 Hz, 1H), 3.33-3.21 (td, J = 5.4, 3.6 Hz, 2H), 3.06-2.94 (dt, J = 9.6, 6.2 Hz, 6H), 2.32-2.24 (t, J = 8.5 Hz, 4H), 2.02-1.94 (dddd, J = 7.9, 4.4, 3.3, 1.7 Hz, 8H), 1.91-1.59 (m, 8H), 1.40-1.25 (m, 42H), 0.94-0.82 (m, 6H). HRMS m / z: calcd: 833.6969 (M+H + ), found: 833.6978.

[0198] Example 2: Synthesis method of compound I-2

[0199] Compound 1 (1.0 eq) and compound 7 (1.0 eq) were dissolved in DCM, and NHS (1.0 eq), EDC (1.0 eq) and DMAP (0.1 eq) were added, stirred at room temperature for 12 h, and after the reaction was completed, compound 8 was obtained by column chromatography purification (DCM:MeOH:CH3COOH=100:10:1) (yield: 78%). Compound 8 (1.0 eq) and compound 2 (2.0 eq) were dissolved in DCM, and EDC (1.0 eq) and DMAP (0.1 eq) were added, stirred at room temperature for 12 h, and after the reaction was completed, compound 9 was obtained by column chromatography purification (DCM:MeOH=10:1) (yield: 56%). Compound 9 (1.0 eq) and compound 4 (2.0 eq) were dissolved in DCM, and EDC (2.0 eq) and DMAP (0.2 eq) were added, stirred at room temperature for 12 h, and after the reaction was completed, compound 10 was obtained by column chromatography purification (DCM:MeOH=20:1) (yield: 68%). Compound 10 (1.0 eq) was dissolved in DCM, and 10% TFA was slowly added at 0°C, stirred at room temperature for 48 h, and after the reaction was completed, compound 11, i.e., compound I-2, was obtained by precipitation with ether (yield: 81%). 1H NMR (400 MHz, CDC13, 293 K) δ (ppm): 7.60-7.54 (dd, J = 5.9, 1.6 Hz, 1H), 6.85-6.81 (dd, J = 4.7, 1.6 Hz, 1H), 5.33-5.24 (tt, J = 3.1, 1.4 Hz, 4H), 4.79-4.68 (dt, J = 9.9, 6.8 Hz, 1H), 4.29-4.25 (m, 6H), 3.78-3.69 (m, 1H), 3.13-2.82 (m, 10H), 2.44-2.37 (t, J = 8.5 Hz, 4H), 2.05-1.97 (dddd, J = 7.9, 4.4, 3.3, 1.7 Hz, 8H), 1.72-1.56 (m, 8H), 1.45-1.28 (m, 42H), 0.99-0.86 (m, 6H). HRMS m / z: calcd: 970.7558 (M+H + ), found: 970.7563.

[0200] Example 3: Method for synthesis of compound I-3

[0201] Compound 12 (1.0 eq) and compound 13 (1.0 eq) were dissolved in DCM, and NHS (1.0 eq), EDC (1.0 eq) and DMAP (0.1 eq) were added, stirred at room temperature for 12 h, after the reaction was completed, purified by column chromatography (DCM:MeOH:CH3COOH = 100:10:1) to obtain compound 14 (yield: 72%). Compound 14 (1.0 eq) and compound 2 (2.0 eq) were dissolved in DCM, and EDC (1.0 eq) and DMAP (0.1 eq) were added, stirred at room temperature for 12 h, after the reaction was completed, purified by column chromatography (DCM:MeOH = 10:1) to obtain compound 15 (yield: 46%). Compound 15 (1.0 eq) and compound 4 (2.0 eq) were dissolved in DCM, and EDC (2.0 eq) and DMAP (0.2 eq) were added, stirred at room temperature for 12 h, after the reaction was completed, purified by column chromatography (DCM:MeOH = 20:1) to obtain compound 16 (yield: 65%). Compound 16 (1.0 eq) was dissolved in DCM, 10% TFA was slowly added at 0°C, stirred at room temperature for 48 h, after the reaction was completed, precipitated by ether to obtain compound 17, i.e. compound I-3 (yield: 83%). 1H NMR (400 MHz, CDC13, 293 K) δ (ppm): 7.60-7.54 (dd, J = 5.9, 1.6 Hz, 1H), 6.85-6.81 (dd, J = 4.7, 1.6 Hz, 1H), 5.33-5.24 (tt, J = 3.1, 1.4 Hz, 4H), 4.79-4.68 (dt, J = 9.9, 6.8 Hz, 1H), 4.29-4.25 (m, 6H), 3.78-3.69 (m, 1H), 3.13-2.82 (m, 10H), 2.44-2.37 (t, J = 8.5 Hz, 4H), 2.05-1.97 (dddd, J = 7.9, 4.4, 3.3, 1.7 Hz, 8H), 1.72-1.56 (m, 8H), 1.45-1.28 (m, 42H), 0.99-0.86 (m, 6H). HRMS m / z: calcd: 970.7558 (M+H + ), found: 970.7563.

[0202] Example 4: Method of synthesis of compound I-4

[0203] Compound 18 (1.0 eq) and compound 4 (2.0 eq) were dissolved in DCM, and NHS (2.0 eq), EDC (2.0 eq), and DMAP (0.2 eq) were added, and stirred at room temperature for 12 h. After the reaction was completed, purification was performed by column chromatography (DCM:MeOH:CH3COOH=100:10:1) to obtain compound 19 (yield: 68%). Compound 19 (2.0 eq) and compound 2 (1.0 eq) were dissolved in DCM, and EDC (1.0 eq) and DMAP (0.1 eq) were added, and stirred at room temperature for 12 h. After the reaction was completed, purification was performed by column chromatography (DCM:MeOH=10:1) to obtain compound 20 (yield: 51%). Compound 20 (1.0 eq) and compound 21 (1.0 eq) were dissolved in DCM, and stirred at room temperature for 12 h. After the reaction was completed, water was washed three times, and the solvent was evaporated under reduced pressure to obtain compound 22 (yield: 88%). Compound 22 (1.0 eq) and compound 23 (2.0 eq) were dissolved in DCM, and stirred at room temperature for 12 h. After the reaction was completed, water was washed three times, and the solvent was evaporated under reduced pressure to obtain compound 24 (yield: 88%). Compound 1 (1.0 eq) was dissolved in DCM, and 10% TFA was slowly added at 0°C, and stirred at room temperature for 3 h. After the reaction was completed, water was washed three times, and the solvent was evaporated under reduced pressure to obtain compound 25 (yield: 79%). Compound 25 (1.0 eq) and compound 24 (1.0 eq) were dissolved in DCM, and NHS (1.0 eq), EDC (1.0 eq), and DMAP (0.1 eq) were added, and stirred at room temperature for 12 h. After the reaction was completed, purification was performed by column chromatography (DCM:MeOH:CH3COOH=100:10:1) to obtain compound 26 (yield: 68%). Compound 26 (1.0 eq) was dissolved in DCM, and 10% TFA was slowly added at 0°C, and stirred at room temperature for 48 h. After the reaction was completed, precipitation was performed with ether to obtain compound 27, which is compound I-4 (yield: 83%). 1H NMR (400 MHz, CDC13, 293 K) δ (ppm): 5.46-5.33 (td, J = 3.1, 1.6 Hz, 8H), 4.35-4.20 (dt, J = 24.2, 6.3 Hz, 6H), 4.17-4.04 (ddt, J = 30.4, 8.8, 6.5 Hz, 3H), 3.34-3.28 (q, J = 5.6 Hz, 2H), 3.16-3.09 (td, J = 5.0, 3.6 Hz, 2H), 3.08-3.03 (q, J = 5.1 Hz, 4H), 3.02-2.90 (q, J = 6.0 Hz, 6H), 2.31-2.23 (td, J = 8.6, 2.9 Hz, 6H), 2.18-2.15 (t, J = 8.4 Hz, 4H), 2.05-1.89 (m, 16H), 1.81-1.45 (m, 22H), 1.40-1.24 (m, 85H), 0.98-0.80 (m, 12H). HRMS m / z: calc. 1812.5486 (M+H + ), found: 1812.5489.

[0204] Example 5: Method of synthesis of compound I-5

[0205] Compound 2 (1.0 eq) and compound 28 (1.0 eq) were dissolved in DCM, EDC (2.0 eq) and DMAP (0.2 eq) were added, stirred at room temperature for 12 h, after the reaction was completed, purified by column chromatography (DCM:MeOH=10:1) to obtain compound 29 (yield: 68%). Compound 29 (2.0 eq) and compound 30 (1.0 eq) were dissolved in DCM, EDC (1.0 eq) and DMAP (0.1 eq) were added, stirred at room temperature for 12 h, after the reaction was completed, purified by column chromatography (DCM:MeOH=15:1) to obtain compound 31 (yield: 51%). Compound 31 (1.0 eq) and compound 32 (1.0 eq) were dissolved in DCM, EDC (2.0 eq) and DMAP (0.2 eq) were added, stirred at room temperature for 12 h, after the reaction was completed, purified by column chromatography (DCM:MeOH=10:1) to obtain compound 33 (yield: 68%). Compound 33 (1.0 eq) and compound 34 (1.0 eq) were dissolved in DCM, EDC (2.0 eq) and DMAP (0.2 eq) were added, stirred at room temperature for 12 h, after the reaction was completed, purified by column chromatography (DCM:MeOH=10:1) to obtain compound 35 (yield: 68%). Compound 35 (1.0 eq) and compound 36 (1.0 eq) were dissolved in DCM, NHS (1.0 eq), EDC (1.0 eq) and DMAP (0.2 eq) were added, stirred at room temperature for 12 h, after the reaction was completed, purified by column chromatography (DCM:MeOH=10:1) to obtain compound 37 (yield: 68%). Compound 37 (1.0 eq) and compound 25 (1.0 eq) were dissolved in DCM, NHS (1.0 eq), EDC (1.0 eq) and DMAP (0.1 eq) were added, stirred at room temperature for 12 h, after the reaction was completed, purified by column chromatography (DCM:MeOH=8:1) to obtain compound 38 (yield: 68%). Compound 38 (1.0 eq) was dissolved in DCM, 10% TFA was slowly added at 0°C, stirred at room temperature for 48 h, after the reaction was completed, washed with water three times, the solvent was evaporated under reduced pressure to obtain compound 39, which was compound I-5 (yield: 79%). 1H NMR (400 MHz, CDC13, 293 K) δ (ppm): 5.56-5.46 (m, 8H), 4.65-4.48 (dt, J = 8.2, 7.2 Hz, 1H), 4.36-4.28 (td, J = 6.1, 1.0 Hz, 6H), 4.20-4.13 (dt, J = 8.9, 6.2 Hz, 1H), 3.90-3.80 (dd, J = 6.0, 1.1 Hz, 2H), 3.81-3.65 (p, J = 5.6 Hz, 1H), 3.43-3.29 (td, J = 5.0, 3.6 Hz, 2H), 3.16-2.96 (m, 8H), 2.76-2.62 (m, 6H), 2.44-2.35 (t, J = 8.5 Hz, 2H), 2.30-2.16 (td, J = 8.3, 2.0 Hz, 2H), 2.13-1.98 (m, 4H), 1.90-1.72 (m, 2H), 1.69-1.59 (m, 11H), 1.57-1.40 (pd, J = 7.9, 1.0 Hz, 2H), 1.33-1.18 (m, 21H), 0.90-0.78 (td, J = 6.7, 2.2 Hz, 6H). HRMS m / z: calc. 1062.6763 (M+H + ), found: 1062.6760.

[0206] Example 6: Synthesis method of compound II-1

[0207] Compound 40 (1.0 eq) and compound 41 (1.0 eq) were dissolved in DCM, EDC (2.0 eq) and DMAP (0.2 eq) were added, stirred at room temperature for 12 h, after the reaction was completed, purified by column chromatography (DCM:MeOH:CH3COOH = 100:10:1) to obtain compound 42 (yield: 68%). Compound 42 (2.0 eq) and compound 43 (1.0 eq) were dissolved in DCM, EDC (1.0 eq) and DMAP (0.1 eq) were added, stirred at room temperature for 12 h, after the reaction was completed, purified by column chromatography (DCM:MeOH = 15:1) to obtain compound 44 (yield: 51%). Compound 44 (1.0 eq) and compound 1 (1.0 eq) were dissolved in DCM, EDC (2.0 eq) and DMAP (0.2 eq) were added, stirred at room temperature for 12 h, after the reaction was completed, purified by column chromatography (DCM:MeOH = 20:1) to obtain compound 45 (yield: 68%). Compound 45 (1.0 eq) was dissolved in DCM, 10% TFA was slowly added at 0°C, stirred at room temperature for 48 h, after the reaction was completed, washed with water three times, the solvent was evaporated under reduced pressure to obtain compound 46, which is compound II-1 (yield: 81%). 1H NMR (400 MHz, CDC13, 293 K) δ (ppm): 4.38-4.19 (m, 10H), 3.98-3.85 (m, 1H), 3.65-3.56 (d, J = 1.1 Hz, 8H), 3.36-3.23 (td, J = 5.4, 3.6 Hz, 2H), 2.98-2.76 (m, 1H), 1.95-1.87 (m, 2H), 1.78-1.69 (m, 6H), 1.55-1.34 (m, 42H), 0.99-0.80 (m, 6H). HRMS m / z: calcd: 890.5472 (M+H + ), found: 890.5472.

[0208] Example 7: Synthesis method of compound II-2

[0209] Compound 28 (2.0 eq) and compound 43 (1.0 eq) were dissolved in DCM, EDC (2.0 eq) and DMAP (0.2 eq) were added, stirred at room temperature for 12 h, after the reaction was completed, purified by column chromatography (DCM:MeOH=20:1) to obtain compound 47 (yield: 68%). Compound 47 (2.0 eq) and compound 48 (1.0 eq) were dissolved in DCM, EDC (1.0 eq) and DMAP (0.1 eq) were added, stirred at room temperature for 12 h, after the reaction was completed, purified by column chromatography (DCM:MeOH=5:1) to obtain compound 49 (yield: 51%). Compound 49 (1.0 eq) and compound 1 (1.0 eq) were dissolved in DCM, NHS ((1.0 eq), EDC (1.0 eq) and DMAP (0.1 eq) were added, stirred at room temperature for 12 h, after the reaction was completed, purified by column chromatography (DCM:MeOH:CH3COOH=100:10:1) to obtain compound 50 (yield: 68%). Compound 50 (1.0 eq) was dissolved in DCM, 10% TFA was slowly added at 0°C, stirred at room temperature for 48 h, after the reaction was completed, washed with water three times, the solvent was evaporated under reduced pressure to obtain compound 51, which is compound II-2 (yield: 74%). 1H NMR (400 MHz, CDC13, 293 K) δ (ppm): 5.66-5.38 (m, 16H), 4.89-4.75 (dt, J = 9.2, 7.3 Hz, 1H), 4.34-4.18 (m, 6H), 3.89-3.75 (m, 1H), 3.45-3.32 (td, J = 5.2, 3.6 Hz, 2H), 2.98-2.85 (m, 14H), 2.76-2.69 (p, J = 7.1 Hz, 1H), 2.54-2.48 (t, J = 8.0 Hz, 4H), 2.23-1.92 (m, 8H), 1.88-1.66 (m, 8H), 1.60-1.49 (pd, J = 7.8, 1.0 Hz, 4H), 1.38-1.18 (m, 12H), 0.90-0.78 (t, J = 6.3 Hz, 6H). HRMS m / z: calcd: 977.6817 (M+H + ), found: 977.6815.

[0210] Example 8: Synthesis method of compound II-3

[0211] Compound 47 (2.0 eq) and compound 52 (1.0 eq) were dissolved in DCM, EDC (1.0 eq) and DMAP (0.1 eq) were added, stirred at room temperature for 12 h, after the reaction was completed, purified by column chromatography (DCM:MeOH=5:1) to obtain compound 53 (yield: 51%). Compound 53 (1.0 eq) and compound 14 (1.0 eq) were dissolved in DCM, NHS (1.0 eq), EDC (1.0 eq) and DMAP (0.1 eq) were added, stirred at room temperature for 12 h, after the reaction was completed, purified by column chromatography (DCM:MeOH:CH3COOH=100:10:1) to obtain compound 54 (yield: 68%). Compound 54 (1.0 eq) was dissolved in DCM, 10% TFA was slowly added at 0°C, stirred at room temperature for 48 h, after the reaction was completed, washed with water three times, the solvent was evaporated under reduced pressure to obtain compound 55, i.e. compound II-3 (yield: 79%). 1H NMR (400 MHz, CDC13, 293 K) δ (ppm): 5.66-5.38 (m, 16H), 4.89-4.75 (dt, J = 9.2, 7.3 Hz, 1H), 4.34-4.18 (m, 6H), 3.89-3.75 (m, 1H), 3.45-3.32 (td, J = 5.2, 3.6 Hz, 2H), 2.98-2.85 (m, 14H), 2.76-2.69 (p, J = 7.1 Hz, 1H), 2.54-2.48 (t, J = 8.0 Hz, 4H), 2.23-1.92 (m, 8H), 1.88-1.66 (m, 8H), 1.60-1.49 (pd, J = 7.8, 1.0 Hz, 4H), 1.38-1.18 (m, 12H), 0.90-0.78 (t, J = 6.3 Hz, 6H). HRMS m / z: calcd: 977.6817 (M+H + ), found: 977.6815.

[0212] Example 9: Method for synthesis of compound II-4

[0213] Compound 56 (1.0 eq) and compound 43 (1.0 eq) were dissolved in DCM, EDC (2.0 eq) and DMAP (0.2 eq) were added, stirred at room temperature for 12 h, after the reaction was completed, purified by column chromatography (DCM:MeOH:CH3COOH=80:10:1) to obtain compound 57 (yield: 68%). Compound 57 (2.0 eq) and compound 58 (1.0 eq) were dissolved in DCM, EDC (1.0 eq) and DMAP (0.1 eq) were added, stirred at room temperature for 12 h, after the reaction was completed, purified by column chromatography (DCM:MeOH=8:1) to obtain compound 59 (yield: 51%). Compound 59 (1.0 eq) and compound 60 (1.0 eq) were dissolved in DCM, EDC (2.0 eq) and DMAP (0.2 eq) were added, stirred at room temperature for 12 h, after the reaction was completed, purified by column chromatography (DCM:MeOH=15:1) to obtain compound 61 (yield: 68%). Compound 61 (1.0 eq) and compound 62 (1.0 eq) were dissolved in DCM, EDC (2.0 eq) and DMAP (0.2 eq) were added, stirred at room temperature for 12 h, after the reaction was completed, purified by column chromatography (DCM:MeOH:CH3COOH=150:10:1) to obtain compound 63 (yield: 68%). Compound 63 (1.0 eq) and compound 25 (1.0 eq) were dissolved in DCM, NHS (1.0 eq), EDC (1.0 eq) and DMAP (0.2 eq) were added, stirred at room temperature for 12 h, after the reaction was completed, purified by column chromatography (DCM:MeOH:CH3COOH=100:10:1) to obtain compound 64 (yield: 58%). Compound 64 (1.0 eq) was dissolved in DCM, 10% TFA was slowly added at 0°C, stirred at room temperature for 48 h, after the reaction was completed, washed with water three times, the solvent was evaporated under reduced pressure to obtain compound 65, which was compound II-4 (yield: 80%). 1H NMR (400 MHz, CDC13, 293 K) δ (ppm): 5.50-5.43 (tt, J = 3.0, 1.6 Hz, 2H), 4.45-4.31 (m, 9H), 3.99-3.80 (t, J = 6.7 Hz, 4H), 3.75-3.61 (s, 4H), 3.30-3.13 (td, J = 5.0, 3.6 Hz, 2H), 2.89-2.78 (m, 8H), 2.67-2.54 (hept, J = 7.1 Hz, 1H), 2.45-2.32 (m, 2H), 2.14-2.04 (tdd, J = 8.1, 3.1, 1.5 Hz, 4H), 1.91-1.72 (m, 2H), 1.54-1.49 (m, 4H), 1.42-1.23 (m, 43H), 0.98-0.80 (m, 6H). HRMS m / z: calcd: 1028.7600 (M+H + ), found: 1028.7605.

[0214] Example 10: Method of synthesis of compound II-5

[0215] Compound 66 (1.0 eq) and compound 43 (1.0 eq) were dissolved in DCM, EDC (2.0 eq) and DMAP (0.2 eq) were added, stirred at room temperature for 12 h, after the reaction was completed, purified by column chromatography (DCM:MeOH=8:1) to obtain compound 67 (yield: 68%). Compound 67 (2.0 eq) and compound 60 (1.0 eq) were dissolved in DCM, EDC (1.0 eq) and DMAP (0.1 eq) were added, stirred at room temperature for 12 h, after the reaction was completed, purified by column chromatography (DCM:MeOH=15:1) to obtain compound 68 (yield: 51%). Compound 68 (1.0 eq) and compound 21 (2.0 eq) were dissolved in DCM, stirred at room temperature for 12 h, after the reaction was completed, washed with water three times, and the solvent was evaporated under reduced pressure to obtain compound 69 (yield: 79%). Compound 69 (1.0 eq) and compound 70 (1.0 eq) were dissolved in DCM, stirred at room temperature for 12 h, after the reaction was completed, washed with water three times, and the solvent was evaporated under reduced pressure to obtain compound 71 (yield: 79%). Compound 71 (1.0 eq) and compound 72 (1.0 eq) were dissolved in DCM, EDC (1.0 eq) and DMAP (0.1 eq) were added, stirred at room temperature for 12 h, after the reaction was completed, purified by column chromatography (DCM:MeOH:NEt3=1500:100:1) to obtain compound 73 (yield: 70%). Compound 73 (1.0 eq) and compound 14 (1.0 eq) were dissolved in DCM, NHS (1.0 eq), EDC (1.0 eq) and DMAP (0.1 eq) were added, stirred at room temperature for 12 h, after the reaction was completed, purified by column chromatography (DCM:MeOH=30:1) to obtain compound 74 (yield: 68%). Compound 74 (1.0 eq) was dissolved in DCM, 10% TFA was slowly added at 0°C, stirred at room temperature for 48 h, after the reaction was completed, washed with water three times, and the solvent was evaporated under reduced pressure to obtain compound 75, which is compound II-5 (yield: 69%). 1 H NMR (400 MHz, CDC13, 293 K) δ (ppm): 5.64-5.51 (tt, J = 3.0, 1.6 Hz, 2H), 4.38-4.26 (m, 8H), 4.22-3.99 (dd, J = 9.0, 7.8 Hz, 1H), 3.86-3.71 (m, 1H), 3.35-3.19 (m, 4H), 2.64-2.48 (m, J = 7.1 Hz, 1H), 2.36-2.26 (t, J = 8.5 Hz, 4H), 2.13-1.98 (m, 5H), 1.86-1.23 (m, 47H), 0.95-0.81 (td, J = 7.0, 1.6 Hz, 12H). HRMS m / z: calcd: 908.6926 (M+H +), found: 908.6923.

[0216] Example 11: Method for synthesis of compound III-1

[0217] Compound 76 (2.0 eq) and compound 77 (1.0 eq) were dissolved in DCM, EDC (2.0 eq) and DMAP (0.2 eq) were added, stirred at room temperature for 12 h, after the reaction was completed, purified by column chromatography (DCM:MeOH:NEt3=1000:100:1) to obtain compound 78 (yield: 68%). Compound 78 (1.0 eq) and compound 79 (2.0 eq) were dissolved in DMF, CuSO4·5H2O (1.0 eq) and ascorbic acid (2.0 eq) were added, stirred at room temperature for 12 h, after the reaction was completed, purified by column chromatography (DCM:MeOH=15:1) to obtain compound 80 (yield: 51%). Compound 80 (1.0 eq) and compound 1 (2.0 eq) were dissolved in DCM, NHS (1.0 eq), EDC (1.0 eq) and DMAP (0.1 eq) were added, stirred at room temperature for 12 h, after the reaction was completed, purified by column chromatography (DCM:MeOH:NEt3=1500:100:1) to obtain compound 81 (yield: 59%). Compound 81 (1.0 eq) was dissolved in DCM, 10% TFA was slowly added at 0°C, stirred at room temperature for 48 h, after the reaction was completed, washed with water three times, the solvent was evaporated under reduced pressure to obtain compound 82, i.e. compound III-1 (yield: 86%). 1 H NMR (400 MHz, CDC13, 293 K) δ (ppm): 7.53-7.44 (s, 2H), 4.52-4.44 (t, J = 6.0 Hz, 4H), 4.42-4.35 (t, J = 6.4 Hz, 4H), 3.88-3.75 (m, 1H), 3.42-3.25 (m, 4H), 3.15-3.05 (t, J = 6.1 Hz, 4H), 2.91-2.83 (t, J = 8.0 Hz, 4H), 2.76-2.64 (t, J = 6.0 Hz, 2H), 2.57-2.45 (t, J = 8.3 Hz, 4H), 2.39-2.29 (p, J = 6.6 Hz, 4H), 2.06-1.94 (m, 4H), 1.88-1.77 (m, 10H), 1.67-1.56 (m, 4H), 1.35-1.28 (m, 12H), 0.92-0.70 (m, 6H). HRMS m / z: calculated: 816.6062 (M+H + ), found: 816.6060.

[0218] Example 12: Method for synthesis of compound III-2

[0219] Compound 83 (1.0 eq) and compound 77 (1.0 eq) were dissolved in DCM, EDC (2.0 eq) and DMAP (0.2 eq) were added, stirred at room temperature for 12 h, after the reaction was completed, purified by column chromatography (DCM:MeOH:NEt3=1000:100:1) to obtain compound 84 (yield: 63%). Compound 84 (2.0 eq) and compound 19 (1.0 eq) were dissolved in DCM, EDC (1.0 eq) and DMAP (0.1 eq) were added, stirred at room temperature for 12 h, after the reaction was completed, purified by column chromatography (DCM:MeOH:NEt3=1500:100:1) to obtain compound 85 (yield: 51%). Compound 85 (1.0 eq) and compound 86 (2.0 eq) were dissolved in DCM, NHS (1.0 eq), EDC (1.0 eq) and DMAP (0.1 eq) were added, stirred at room temperature for 12 h, after the reaction was completed, purified by column chromatography (DCM:MeOH=30:1) to obtain compound 87 (yield: 65%). Compound 87 (1.0 eq) and compound 14 (1.0 eq) were dissolved in DCM, NHS (1.0 eq), EDC (1.0 eq) and DMAP (0.1 eq) were added, stirred at room temperature for 12 h, after the reaction was completed, purified by column chromatography (DCM:MeOH=30:1) to obtain compound 88 (yield: 78%). Compound 88 (1.0 eq) was dissolved in DCM, 10% TFA was slowly added at 0°C, stirred at room temperature for 48 h, after the reaction was completed, washed with water three times, the solvent was evaporated under reduced pressure to obtain compound 89, which was compound III-2 (yield: 88%). 1H NMR (400 MHz, CDC13, 293 K) δ (ppm): 7.71-7.68 (m, 1H), 7.45-7.39 (dd, J = 7.6, 1.7 Hz, 1H), 6.99-6.95 (d, J = 6.8 Hz, 1H), 5.65-5.43 (td, J = 3.1, 1.6 Hz, 4H), 4.64-4.53 (dt, J = 8.4, 6.2 Hz, 1H), 4.31-4.24 (td, J = 6.2, 2.2 Hz, 4H), 4.08-4.06 (dt, J = 9.2, 6.6 Hz, 1H), 3.70-3.51 (m, 1H), 3.24-3.12 (m, 6H), 3.10-3.01 (q, J = 5.1 Hz, 2H), 2.94-2.86 (t, J = 6.0 Hz, 4H), 2.71-2.68 (t, J = 6.0 Hz, 2H), 2.53-2.34 (dt, J = 31.6, 8.6 Hz, 4H), 2.21-2.16 (t, J = 8.4 Hz, 2H), 2.12-1.88 (tdt, J = 5.6, 3.9, 1.6 Hz, 8H), 1.70-1.63 (m, 14H), 1.32-1.25 (tt, J = 7.5, 5.1 Hz, 2H), 1.20-1.08 (m, 69H), 0.90-0.78 (m, 9H). HRMS m / z: calc. 1443.1948 (M+H + ), found: 1443.1951.

[0220] Example 13: Method for synthesis of compound III-3

[0221] Compound 85 (1.0 eq) and compound 90 (2.0 eq) were dissolved in DCM, and NHS (1.0 eq), EDC (1.0 eq), and DMAP (0.1 eq) were added, and stirred at room temperature for 12 h. After the reaction was completed, column chromatography purification (DCM:MeOH = 30:1) was performed to obtain compound 91 (yield: 61%). Compound 91 (1.0 eq) and compound 1 (1.0 eq) were dissolved in DCM, and NHS (1.0 eq), EDC (1.0 eq), and DMAP (0.1 eq) were added, and stirred at room temperature for 12 h. After the reaction was completed, column chromatography purification (DCM:MeOH = 30:1) was performed to obtain compound 92 (yield: 60%). Compound 92 (1.0 eq) was dissolved in DCM, and 10% TFA was slowly added at 0°C, and stirred at room temperature for 48 h. After the reaction was completed, water was washed three times, and the solvent was evaporated under reduced pressure to obtain compound 93, which is compound III-3 (yield: 77%). 1H NMR (400 MHz, CDC13, 293 K) δ (ppm): 7.71-7.68 (m, 1H), 7.45-7.39 (dd, J = 7.6, 1.7 Hz, 1H), 6.99-6.95 (d, J = 6.8 Hz, 1H), 5.65-5.43 (td, J = 3.1, 1.6 Hz, 4H), 4.64-4.53 (dt, J = 8.4, 6.2 Hz, 1H), 4.31-4.24 (td, J = 6.2, 2.2 Hz, 4H), 4.08-4.06 (dt, J = 9.2, 6.6 Hz, 1H), 3.70-3.51 (m, 1H), 3.24-3.12 (m, 6H), 3.10-3.01 (q, J = 5.1 Hz, 2H), 2.94-2.86 (t, J = 6.0 Hz, 4H), 2.71-2.68 (t, J = 6.0 Hz, 2H), 2.53-2.34 (dt, J = 31.6, 8.6 Hz, 4H), 2.21-2.16 (t, J = 8.4 Hz, 2H), 2.12-1.88 (tdt, J = 5.6, 3.9, 1.6 Hz, 8H), 1.70-1.63 (m, 14H), 1.32-1.25 (tt, J = 7.5, 5.1 Hz, 2H), 1.20-1.08 (m, 69H), 0.90-0.78 (m, 9H). HRMS m / z: calc. 1443.1948 (M+H + ), found: 1443.1957.

[0222] Example 14: Method of synthesis of compound III-4

[0223] Compound 94 (1.0 eq) and compound 77 (1.0 eq) were dissolved in DCM, EDC (2.0 eq) and DMAP (0.2 eq) were added, stirred at room temperature for 12 h, after the reaction was completed, purified by column chromatography (DCM:MeOH: = 5:1) to obtain compound 95 (yield: 80%). Compound 95 (2.0 eq) and compound 96 (1.0 eq) were dissolved in DCM, EDC (1.0 eq) and DMAP (0.1 eq) were added, stirred at room temperature for 12 h, after the reaction was completed, purified by column chromatography (DCM:MeOH:NEt3= 1500:100:1) to obtain compound 97 (yield: 51%). Compound 97 (1.0 eq) and compound 98 (1.0 eq) were dissolved in DCM, EDC (1.0 eq) and DMAP (0.1 eq) were added, stirred at room temperature for 12 h, after the reaction was completed, purified by column chromatography (DCM:MeOH:NEt3= 1000:100:1) to obtain compound 99 (yield: 68%). Compound 99 (1.0 eq) and compound 100 (1.0 eq) were dissolved in DCM, NHS (1.0 eq), EDC (1.0 eq) and DMAP (0.1 eq) were added, stirred at room temperature for 12 h, after the reaction was completed, purified by column chromatography (DCM:MeOH:NEt3= 800:100:1) to obtain compound 101 (yield: 63%). Compound 101 (1.0 eq) and compound 1 (1.0 eq) were dissolved in DCM, NHS (1.0 eq), EDC (1.0 eq) and DMAP (0.1 eq) were added, stirred at room temperature for 12 h, after the reaction was completed, purified by column chromatography (DCM:MeOH:NEt3= 1000:100:1) to obtain compound 102 (yield: 71%). Compound 102 (1.0 eq) was dissolved in DCM, 10% TFA was slowly added at 0°C, stirred at room temperature for 48 h, after the reaction was completed, washed with water three times, the solvent was evaporated under reduced pressure to obtain compound 103, which was compound III-4 (yield: 80%). 1 H NMR (400 MHz, CDC13, 293 K) δ (ppm): 4.34-4.20 (m, 5H), 3.97-3.87 (t, J = 6.1 Hz, 2H), 3.71-3.59 (m, 1H), 3.22-3.11 (m, 4H), 2.99-2.88 (t, J = 6.1 Hz, 4H), 2.69-2.54 (m, 4H), 2.36-2.21 (t, J = 8.5 Hz, 6H), 1.78-1.52 (m, 18H), 1.49-1.19 (m, 42H), 1.02-0.72 (m, 6H). 1H NMR (400 MHz, CDC13, 293 K) δ (ppm): 4.97-4.92 (m, 1H), 4.50-4.41 (m, 4H), 3.73-3.53 (m, 180H), 3.38, (s, 3H), 2.85-2.61 (m, 8H), 2.57 (s, 3H), 2.48 (s, 3H), 2.23 (s, 3H), 2.19 (s, 3H), 2.15 (s, 3H), 2.13 (s, 3H), 1.94-1.84 (m, 4H), 1.59-1.01 (m, 48H), 0.88-0.76 (m, 24H). HRMS m / z: calcd: 952.7664 (M+H + ), found: 952.7668.

[0224] Example 15: Method for synthesis of compound III-5

[0225] Compound 98 (1.0 eq) and compound 77 (1.0 eq) were dissolved in DCM, EDC (2.0 eq) and DMAP (0.2 eq) were added, stirred at room temperature for 12 h, after the reaction was completed, purified by column chromatography (DCM:MeOH: = 5:1) to obtain compound 104 (yield: 68%). Compound 104 (2.0 eq) and compound 4 (1.0 eq) were dissolved in DCM, EDC (1.0 eq) and DMAP (0.1 eq) were added, stirred at room temperature for 12 h, after the reaction was completed, purified by column chromatography (DCM:MeOH:NEt3= 1500:100:1) to obtain compound 105 (yield: 51%). Compound 105 (1.0 eq) and compound 106 (1.0 eq) were dissolved in DCM, stirred at room temperature for 8 h, then compound 106 (1.0 eq) dissolved in DCM was added, after the reaction was completed, purified by column chromatography (DCM:MeOH:NEt3= 15:1) to obtain compound 107 (yield: 68%). Compound 107 (1.0 eq) and compound 1 (1.0 eq) were dissolved in DCM, EDC (1.0 eq) and DMAP (0.1 eq) were added, stirred at room temperature for 12 h, after the reaction was completed, purified by column chromatography (DCM:MeOH:NEt3= 20:1) to obtain compound 108 (yield: 68%). Compound 108 (1.0 eq) was dissolved in DCM, 10% TFA was slowly added at 0°C, stirred at room temperature for 48 h, after the reaction was completed, washed with water three times, the solvent was evaporated under reduced pressure to obtain compound 109, i.e. compound III-5 (yield: 82%). 1H NMR (400 MHz, CDC13, 293 K) δ (ppm): 5.39-5.27 (tt, J = 3.1, 1.4 Hz, 2H), 5.24-5.11 (m, 3H), 4.31-4.10 (m, 8H), 3.83-3.69 (m, 3H), 3.69-3.59 (d, J = 5.9 Hz, 6H), 3.25-3.08 (m, 4H), 3.00-2.89 (t, J = 6.1 Hz, 4H), 2.64-2.52 (t, J = 6.0 Hz, 2H), 2.36-2.24 (t, J = 8.5 Hz, 4H), 2.08-1.97 (dddd, J = 7.9, 4.4, 3.2, 1.3 Hz, 4H), 1.81-1.69 (m, 4H), 1.69-1.51 (m, 6H), 1.39-1.18 (m, 37H), 0.95-0.81 (m, 6H). HRMS m / z: calc. 940.7188 (M+H + ), found: 940.7193.

[0226] Example 16: Method for synthesis of compound IV-1

[0227] Compound 4 (2.0 eq) and compound 110 (1.0 eq) were dissolved in DCM, EDC (2.0 eq) and DMAP (0.2 eq) were added, stirred at room temperature for 12 h, after the reaction was completed, purified by column chromatography (DCM:MeOH = 15:1) to obtain compound 111 (yield: 68%). Compound 111 (1.0 eq) and compound 1 (1.0 eq) were dissolved in DCM, EDC (1.0 eq) and DMAP (0.1 eq) were added, stirred at room temperature for 12 h, after the reaction was completed, purified by column chromatography (DCM:MeOH = 20:1) to obtain compound 112 (yield: 51%). Compound 112 (1.0 eq) was dissolved in DCM, 10% TFA was slowly added at 0°C, stirred at room temperature for 48 h, after the reaction was completed, washed with water three times, the solvent was evaporated under reduced pressure to obtain compound 113, i.e. compound IV-1 (yield: 79%). 1H NMR (400 MHz, CDC13, 293 K) δ (ppm): 5.53 - 5.16 (m, 6H), 4.99 - 4.79 (p, J = 5.8 Hz, 1H), 4.36 - 4.10 (m, 4H), 3.94 - 3.68 (p, J = 6.5 Hz, 1H), 3.34 - 3.08 (td, J = 5.4, 3.6 Hz, 2H), 2.42 - 2.18 (td, J = 9.3, 8.9, 5.8 Hz, 4H), 2.14 - 1.94 (m, 8H), 1.88 - 1.41 (m, 8H), 1.39 - 1.16 (m, 40H), 1.01 - 0.74 (m, 6H). HRMS m / z: calcd: 776.6391 (M+H + ), found: 776.6396.

[0228] Example 17: Method for synthesis of compound IV-2

[0229] Compound 111 (1.0 eq) and compound 114 (1.0 eq) were dissolved in DCM, EDC (2.0 eq) and DMAP (0.2 eq) were added, stirred at room temperature for 12 h, after the reaction was completed, purified by column chromatography (DCM:MeOH:NEt3=1500:100:1) to obtain compound 115 (yield: 65%). Compound 115 (1.0 eq) and compound 1 (1.0 eq) were dissolved in DCM, NHS (1.0 eq), EDC (1.0 eq) and DMAP (0.1 eq) were added, stirred at room temperature for 12 h, after the reaction was completed, purified by column chromatography (DCM:MeOH=20:1) to obtain compound 116 (yield: 54%). Compound 116 (1.0 eq) was dissolved in DCM, 10% TFA was slowly added at 0°C, stirred at room temperature for 48 h, after the reaction was completed, washed with water three times, the solvent was evaporated under reduced pressure to obtain compound 117, which is compound IV-2 (yield: 79%). 1H NMR (400 MHz, CDC13, 293 K) δ (ppm): 7.37-7.23 (m, 5H), 5.49-5.28 (ddd, J = 4.6, 3.1, 1.4 Hz, 4H), 5.05-4.71 (dd, J = 5.8, 3.0 Hz, 3H), 4.54-4.38 (dt, J = 9.3, 7.1 Hz, 1H), 4.29-3.98 (m, 4H), 3.72-3.49 (m, 1H), 3.27-3.14 (td, J = 5.2, 3.6 Hz, 2H), 3.12-2.88 (m, 2H), 2.47-2.23 (td, J = 9.3, 8.9, 5.8 Hz, 4H), 2.12-1.93 (m, 8H), 1.80-1.45 (m, 8H), 1.41-1.17 (m, 40H), 1.02-0.77 (m, 6H). HRMS m / z: calc. 923.7075 (M+H + ), found: 923.7071.

[0230] Example 18: Method for synthesis of compound IV-3

[0231] Compound 111 (1.0 eq) and compound 118 (1.0 eq) were dissolved in DCM, EDC (2.0 eq) and DMAP (0.2 eq) were added, stirred at room temperature for 12 h, after the reaction was completed, purified by column chromatography (DCM:MeOH:NEt3=1500:100:1) to obtain compound 119 (yield: 61%). Compound 119 (1.0 eq) and compound 1 (1.0 eq) were dissolved in DCM, NHS (1.0 eq), EDC (1.0 eq) and DMAP (0.1 eq) were added, stirred at room temperature for 12 h, after the reaction was completed, purified by column chromatography (DCM:MeOH=20:1) to obtain compound 120 (yield: 51%). Compound 120 (1.0 eq) was dissolved in DCM, 10% TFA was slowly added at 0°C, stirred at room temperature for 48 h, after the reaction was completed, washed with water three times, the solvent was evaporated under reduced pressure to obtain compound 121, i.e. compound IV-3 (yield: 87%). 1H NMR (400 MHz, CDC13, 293 K) δ (ppm): 7.39-7.20 (m, 5H), 5.54-5.38 (ddd, J = 4.6, 3.1, 1.4 Hz, 4H), 5.15-4.71 (dd, J = 5.8, 3.0 Hz, 3H), 4.54-4.38 (dt, J = 9.3, 7.1 Hz, 1H), 4.29-3.98 (m, 4H), 3.72-3.49 (m, 1H), 3.27-3.14 (td, J = 5.2, 3.6 Hz, 2H), 3.12-2.88 (m, 2H), 2.47-2.23 (td, J = 9.3, 8.9, 5.8 Hz, 4H), 2.12-1.93 (m, 8H), 1.83-1.45 (m, 8H), 1.44-1.17 (m, 40H), 1.02-0.77 (m, 6H). HRMS m / z: calc. 923.7075 (M+H + ), found: 923.7068.

[0232] Example 19: Method for synthesis of compound IV-4

[0233] Compound 122 (1.0 eq) and compound 110 (1.0 eq) were dissolved in DCM, EDC (2.0 eq) and DMAP (0.2 eq) were added, stirred at room temperature for 12 h, after the reaction was completed, purified by column chromatography (DCM:MeOH: = 10:1) to obtain compound 123 (yield: 60%). Compound 123 (2.0 eq) and compound 124 (1.0 eq) were dissolved in DCM, EDC (1.0 eq) and DMAP (0.1 eq) were added, stirred at room temperature for 12 h, after the reaction was completed, purified by column chromatography (DCM:MeOH: = 15:1) to obtain compound 125 (yield: 51%). Compound 125 (1.0 eq) and compound 126 (1.0 eq) were dissolved in DCM, EDC (1.0 eq) and DMAP (0.1 eq) were added, stirred at room temperature for 12 h, after the reaction was completed, purified by column chromatography (DCM:MeOH:NEt3 = 1500:100:1) to obtain compound 127 (yield: 51%). Compound 127 (1.0 eq) and compound 1 (1.0 eq) were dissolved in DCM, EDC (1.0 eq) and DMAP (0.1 eq) were added, stirred at room temperature for 12 h, after the reaction was completed, purified by column chromatography (DCM:MeOH: = 10:1) to obtain compound 128 (yield: 68%). Compound 128 (1.0 eq) was dissolved in DCM, 10% TFA was slowly added at 0°C, stirred at room temperature for 48 h, after the reaction was completed, washed with water three times, the solvent was evaporated under reduced pressure to obtain compound 129, i.e. compound IV-4 (yield: 82%).1 HNMR (400 MHz, CDC13, 293 K) δ (ppm): 5.48-5.29 (pd, J = 3.9, 2.3 Hz, 6H), 4.98-4.77 (p, J = 5.8 Hz, 1H), 4.55-4.01 (m, 7H), 3.86-3.54 (m, 1H), 3.24-3.13 (td, J = 5.4, 3.6 Hz, 2H), 2.76-2.63 (hd, J = 2.1, 1.4 Hz, 4H), 2.41-2.26 (t, J = 8.7 Hz, 4H), 2.08-1.91 (m, 4H), 1.82-1.70 (m, 2H), 1.67-1.55 (m, 4H), 1.53-1.12 (m, 18H), 0.97-0.74 (td, J = 6.7, 2.2 Hz, 6H). HRMS m / z: calcd: 721.4990 (M+H + ), found: 721.7996.

[0234] Example 20: Method for synthesis of compound IV-5

[0235] Compounds 130 (1.0 eq) and 110 (1.0 eq) were dissolved in DCM, and EDC (2.0 eq) and DMAP (0.2 eq) were added. The mixture was stirred at room temperature for 12 h. After the reaction was complete, the mixture was purified by column chromatography (DCM:MeOH:CH3COOH = 1000:100:1) to give compound 131 (yield: 59%). Compounds 131 (2.0 eq) and 132 (1.0 eq) were dissolved in DCM, and EDC (1.0 eq) and DMAP (0.1 eq) were added. The mixture was stirred at room temperature for 12 h. After the reaction was complete, the mixture was purified by column chromatography (DCM:MeOH = 10:1) to give compound 133 (yield: 51%). Compounds 133 (1.0 eq) and 134 (1.0 eq) were dissolved in DCM, and EDC (1.0 eq) and DMAP (0.1 eq) were added. The mixture was stirred at room temperature for 12 h. After the reaction was complete, the mixture was purified by column chromatography (DCM:MeOH = 15:1) to give compound 135 (yield: 70%). Compounds 135 (1.0 eq) and 136 (1.0 eq) were dissolved in DCM, and EDC (1.0 eq) and DMAP (0.1 eq) were added. The mixture was stirred at room temperature for 12 h. After the reaction was complete, the mixture was purified by column chromatography (DCM:MeOH = 15:1) to give compound 137 (yield: 68%). Compounds 137 (1.0 eq) and 138 (1.0 eq) were dissolved in DCM, and EDC (1.0 eq) and DMAP (0.1 eq) were added. The mixture was stirred at room temperature for 12 h. After the reaction was complete, the mixture was purified by column chromatography (DCM:MeOH:CH3COOH = 1000:100:1) to give compound 139 (yield: 58%). Compounds 139 (1.0 eq) and 25 (1.0 eq) were dissolved in DCM, and NHS (1.0 eq), EDC (1.0 eq), and DMAP (0.1 eq) were added. The mixture was stirred at room temperature for 12 h. After the reaction was complete, the mixture was purified by column chromatography (DCM:MeOH:CH3COOH = 1000:100:1) to give compound 140 (yield: 68%). Compound 140 (1.0 eq) was dissolved in DCM, and 10% TFA was slowly added at 0 °C. The mixture was stirred at room temperature for 48 h. After the reaction was completed, the mixture was washed three times with water, and the solvent was evaporated under reduced pressure to obtain compound 141, namely compound IV-5 (yield: 82%). 1H NMR (400 MHz, CDC13, 293 K) δ (ppm): 4.91-4.81 (p, J = 5.7 Hz, 1H), 4.57-4.40 (m, 4H), 4.38-4.29 (dt, J = 8.9, 6.5 Hz, 1H), 3.97-3.89 (t, J = 6.1 Hz, 4H), 3.27-2.98 (td, J = 5.0, 3.6 Hz, 2H), 2.30-2.20 (m, 12H), 1.97-1.65 (m, 6H), 1.35-1.17 (m, 39H), 0.94-0.83 (m, 6H). HRMS m / z: calcd: 898.5878 (M+H + ), found: 898.5884.

[0236] Example 21: Method for synthesis of compound V-1

[0237] Compound 142 (2.0 eq) and compound 143 (1.0 eq) were dissolved in DCM, EDC (2.0 eq) and DMAP (0.2 eq) were added, stirred at room temperature for 12 h, after the reaction was completed, purified by column chromatography (DCM:MeOH:CH3COOH = 1000:100:1) to obtain compound 144 (yield: 56%). Compound 144 (2.0 eq) and compound 14 (1.0 eq) were dissolved in DCM, EDC (1.0 eq) and DMAP (0.1 eq) were added, stirred at room temperature for 12 h, after the reaction was completed, purified by column chromatography (DCM:MeOH = 20:1) to obtain compound 145 (yield: 51%). Compound 145 (1.0 eq) was dissolved in DCM, 10% TFA was slowly added at 0°C, stirred at room temperature for 48 h, after the reaction was completed, washed with water three times, and the solvent was evaporated under reduced pressure to obtain compound 146, i.e. compound V-1 (yield: 83%). 1 H NMR (400 MHz, CDC13, 293 K) δ (ppm): 4.62-4.53 (m, 6H), 4.12-3.99 (p, J = 6.4 Hz, 1H), 3.45-3.31 (td, J = 5.4, 3.6 Hz, 2H), 2.36-2.25 (p, J = 7.7 Hz, 2H), 1.98-1.61 (m, 19H), 1.39-1.17 (m, 40H), 0.99-0.80 (m, 12H). HRMS m / z: calcd: 780.6704 (M+H + ), found: 780.6704.

[0238] Example 22: Method for synthesis of compound V-2

[0239] Compound 4 (2.0 eq) and compound 143 (1.0 eq) were dissolved in DCM, EDC (2.0 eq) and DMAP (0.2 eq) were added, stirred at room temperature for 12 h, after the reaction was completed, purified by column chromatography (DCM:MeOH=15:1) to obtain compound 147 (yield: 68%). Compound 25 (2.0 eq) was dissolved in DCM, NHS (1.0 eq), EDC (1.0 eq) and DMAP (0.1 eq) were added, stirred at room temperature for 12 h, after the reaction was completed, purified by column chromatography (DCM:MeOH:NEt3=1000:100:1) to obtain compound 148 (yield: 51%). Compound 148 (1.0 eq) and compound 147 (1.0 eq) were dissolved in DCM, EDC (1.0 eq) and DMAP (0.1 eq) were added, stirred at room temperature for 12 h, after the reaction was completed, purified by column chromatography (DCM:MeOH:NEt3=1000:100:1) to obtain compound 149 (yield: 77%). Compound 149 (1.0 eq) was dissolved in DCM, 10% TFA was slowly added at 0°C, stirred at room temperature for 48 h, after the reaction was completed, washed with water three times, the solvent was evaporated under reduced pressure to obtain compound 150, i.e. compound V-2 (yield: 80%). 1 H NMR (400 MHz, CDC13, 293 K) δ (ppm): 5.75-5.65 (tt, J = 3.1, 1.4 Hz, 4H), 4.76-4.64 (m, 7H), 3.81-3.74 (p, J = 5.5 Hz, 1H), 3.58-3.28 (dtd, J = 24.5, 5.1, 3.6 Hz, 4H), 2.35-2.25 (t, J = 8.5 Hz, 4H), 2.19-1.97 (dddd, J = 7.9, 4.4, 3.3, 1.7 Hz, 8H), 1.89-1.78 (m, 19H), 1.48-1.31 (m, 42H), 0.95-0.80 (m, 6H). HRMS m / z: calcd: 788.8028 (M+H + ), found: 788.8023.

[0240] Example 23: Method for synthesizing compound V-3

[0241] Compound 14 (1.0 eq) was dissolved in DCM, 10% TFA was added slowly at 0 °C, stirred at room temperature for 3 h, washed with water for three times after the reaction was completed, and the solvent was evaporated under reduced pressure to obtain compound 151 (yield: 79%). Compound 151 (1.0 eq) and compound 25 (1.0 eq) were dissolved in DCM, and NHS (1.0 eq), EDC (1.0 eq) and DMAP (0.2 eq) were added, stirred at room temperature for 12 h, and compound 152 (yield: 60%) was obtained after the reaction was completed and purified by column chromatography (DCM:MeOH=5:1). Compound 152 (1.0 eq) and compound 147 (1.0 eq) were dissolved in DCM, and EDC (1.0 eq) and DMAP (0.1 eq) were added, stirred at room temperature for 12 h, and compound 153 (yield: 51%) was obtained after the reaction was completed and purified by column chromatography (DCM:MeOH:NEt3=1000:100:1). Compound 153 (1.0 eq) was dissolved in DCM, 10% TFA was added slowly at 0 °C, stirred at room temperature for 48 h, washed with water for three times after the reaction was completed, and the solvent was evaporated under reduced pressure to obtain compound 154, i.e. compound V-3 (yield: 88%). 1 H NMR (400 MHz, CDC13, 293 K) δ (ppm): 5.79-5.61 (tt, J = 3.1, 1.4 Hz, 4H), 4.86-4.77 (m, 7H), 3.81-3.74 (p, J = 5.5 Hz, 1H), 3.58-3.28 (dtd, J = 24.5, 5.1, 3.6 Hz, 4H), 2.55-2.45 (t, J = 8.5 Hz, 4H), 2.19-1.97 (dddd, J = 7.9, 4.4, 3.3, 1.7 Hz, 8H), 1.89-1.78 (m, 19H), 1.48-1.31 (m, 42H), 0.95-0.80 (m, 6H). HRMS m / z: calcd: 788.8028 (M+H + ), found: 788.8023.

[0242] Example 24: Synthesis method of compound V-4

[0243] Compound 155 (1.0 eq) and compound 21 (1.0 eq) were dissolved in DCM, stirred at room temperature for 12 h, washed with water three times after the reaction was completed, and the solvent was evaporated under reduced pressure to obtain compound 156 (yield: 61%). Compound 32 (1.0 eq) and compound 143 (1.0 eq) were dissolved in DCM, EDC (2.0 eq) and DMAP (0.2 eq) were added, stirred at room temperature for 12 h, and compound 157 (yield: 68%) was obtained by column chromatography purification (DCM:MeOH=10:1) after the reaction was completed. Compound 157 (1.0 eq) and compound 156 (1.0 eq) were dissolved in DCM, stirred at room temperature for 12 h, washed with water three times after the reaction was completed, and compound 158 (yield: 79%) was obtained by evaporating the solvent under reduced pressure. Compound 158 (1.0 eq) and compound 28 (1.0 eq) were dissolved in DCM, EDC (1.0 eq) and DMAP (0.1 eq) were added, stirred at room temperature for 12 h, and compound 159 (yield: 51%) was obtained by column chromatography purification (DCM:MeOH=15:1) after the reaction was completed. Compound 159 (1.0 eq) and compound 160 (1.0 eq) were dissolved in DCM, EDC (1.0 eq) and DMAP (0.1 eq) were added, stirred at room temperature for 12 h, and compound 161 (yield: 68%) was obtained by column chromatography purification (DCM:MeOH:NEt3=1500:100:1) after the reaction was completed. Compound 161 (1.0 eq) and compound 12 (1.0 eq) were dissolved in DCM, NHS (1.0 eq), EDC (1.0 eq), and DMAP (0.1 eq) were added, stirred at room temperature for 12 h, and compound 162 (yield: 72%) was obtained by column chromatography purification (DCM:MeOH=30:1) after the reaction was completed. Compound 162 (1.0 eq) was dissolved in DCM, 10% TFA was slowly added at 0°C, stirred at room temperature for 48 h, washed with water three times after the reaction was completed, and compound 163, which is compound V-4 (yield: 80%), was obtained by evaporating the solvent under reduced pressure. 1H NMR (400 MHz, CDC13, 293 K) δ (ppm): 5.73-5.61 (m, 8H), 4.53-4.43 (dt, J = 8.8, 7.3 Hz, 1H), 4.29-4.24 (m, 6H), 3.94-3.85 (d, J = 6.1 Hz, 2H), 3.73-3.58 (m, 1H), 3.25-3.14 (td, J = 5.2, 3.7 Hz, 2H), 2.88-2.65 (tdd, J = 5.6, 3.0, 1.0 Hz, 6H), 2.41-2.32 (t, J = 8.5 Hz, 2H), 2.25-2.21 (t, J = 8.5 Hz, 2H), 2.18-2.11 (m, 2H), 2.08-2.00 (m, 2H), 1.87-1.61 (m, 18H), 1.40-1.19 (m, 26H), 0.95-0.85 (td, J = 6.8, 1.8 Hz, 12H). HRMS m / z: calcd: 970.7446 (M+H + ), found: 970.7442.

[0244] Example 25: Method for synthesis of compound V-5

[0245] Compound 159 (1.0 eq) and compound 32 (1.0 eq) were dissolved in DCM, EDC (1.0 eq) and DMAP (0.1 eq) were added, stirred at room temperature for 12 h, after the reaction was completed, purified by column chromatography (DCM:MeOH=10:1) to obtain compound 164 (yield: 55%). Compound 164 (1.0 eq) and compound 165 (1.0 eq) were dissolved in DCM, NHS (1.0 eq), EDC (1.0 eq) and DMAP (0.1 eq) were added, stirred at room temperature for 12 h, after the reaction was completed, purified by column chromatography (DCM:MeOH=30:1) to obtain compound 166 (yield: 45%). Compound 166 (1.0 eq) and compound 12 (1.0 eq) were dissolved in DCM, EDC (1.0 eq) and DMAP (0.1 eq) were added, stirred at room temperature for 12 h, after the reaction was completed, purified by column chromatography (DCM:MeOH=20:1) to obtain compound 167 (yield: 51%). Compound 167 (1.0 eq) was dissolved in DCM, 10% TFA was slowly added at 0°C, stirred at room temperature for 48 h, after the reaction was completed, washed with water three times, the solvent was evaporated under reduced pressure to obtain compound 168, i.e. compound V-5 (yield: 89%). 1H NMR (400 MHz, CDC13, 293 K) δ (ppm): 5.87-5.71 (m, 8H), 4.56-4.48 (m, 2H), 4.36-4.24 (m, 8H), 4.12-3.98 (d, J = 6.6 Hz, 2H), 3.93-3.87 (d, J = 6.0 Hz, 2H), 3.82-3.59 (m, 13H), 3.35-3.10 (td, J = 5.4, 3.6 Hz, 2H), 2.86-2.79 (m, 6H), 2.54-2.47 (t, J = 6.0 Hz, 2H), 2.41-2.30 (t, J = 8.5 Hz, 2H), 2.20-2.10 (m, 2H), 2.08-1.99 (m, 2H), 1.89-1.61 (m, 15H), 1.44-1.18 (m, 25H), 0.97-0.79 (td, J = 7.1, 6.7, 2.2 Hz, 6H). HRMS m / z: calc. 1120.7611 (M+H + ), found: 1120.7615.

[0246] Example 26: Synthesis method of compound VI-1

[0247] Compound 83 (2.0 eq) and compound 169 (1.0 eq) were dissolved in DCM, EDC (1.0 eq) and DMAP (0.1 eq) were added, stirred at room temperature for 12 h, after the reaction was completed, purified by column chromatography (DCM:MeOH=10:1) to obtain compound 170 (yield: 63%). Compound 170 (1.0 eq) and compound 1 (1.0 eq) were dissolved in DCM, NHS (1.0 eq), EDC (1.0 eq) and DMAP (0.1 eq) were added, stirred at room temperature for 12 h, after the reaction was completed, purified by column chromatography (DCM:MeOH:CH3COOH=1500:100:1) to obtain compound 171 (yield: 72%). Compound 171 (1.0 eq) was dissolved in DCM, 10% TFA was slowly added at 0°C, stirred at room temperature for 48 h, after the reaction was completed, washed with water three times, the solvent was evaporated under reduced pressure to obtain compound 172, which was compound VI-1 (yield: 83%). 1H NMR (400 MHz, CDC13, 293 K) δ (ppm): 4.26-4.10 (m, 4H), 4.03-3.89 (dt, J = 9.7, 5.3 Hz, 1H), 3.72-3.61 (m, 1H), 3.28-3.06 (td, J = 5.3, 3.7 Hz, 2H), 2.36-2.29 (m, 4H), 1.74-1.54 (m, 8H), 1.35-1.21 (m, 51H), 0.94-0.86 (m, 6H). HRMS m / z: calcd: 779.6864 (M+H + ), found: 779.6871.

[0248] Example 27: Method for synthesis of compound VI-2

[0249] Compound 173 (2.0 eq) and compound 169 (1.0 eq) were dissolved in DCM, EDC (2.0 eq) and DMAP (0.2 eq) were added, stirred at room temperature for 12 h, after the reaction was completed, purified by column chromatography (DCM:MeOH=10:1) to obtain compound 174 (yield: 66%). Compound 174 (1.0 eq) and compound 175 (2.0 eq) were dissolved in DCM, EDC (2.0 eq) and DMAP (0.2 eq) were added, stirred at room temperature for 12 h, after the reaction was completed, purified by column chromatography (DCM:MeOH=5:1) to obtain compound 176 (yield: 78%). Compound 176 (1.0 eq) and compound 1 (1.0 eq) were dissolved in DCM, NHS (1.0 eq), EDC (1.0 eq) and DMAP (0.1 eq) were added, stirred at room temperature for 12 h, after the reaction was completed, purified by column chromatography (DCM:MeOH=20:1) to obtain compound 177 (yield: 51%). Compound 177 (1.0 eq) was dissolved in DCM, 10% TFA was slowly added at 0°C, stirred at room temperature for 48 h, after the reaction was completed, washed with water three times, the solvent was evaporated under reduced pressure to obtain compound 178 (yield: 77%). Compound 178 (1.0 eq) and compound 179 (1.0 eq) were dissolved in DCM, NHS (1.0 eq), EDC (1.0 eq) and DMAP (0.1 eq) were added, stirred at room temperature for 12 h, after the reaction was completed, purified by column chromatography (DCM:MeOH:NEt3=1500:100:1) to obtain compound 180, i.e. compound VI-2 (yield: 68%). 1H NMR (400 MHz, CDC13, 293 K) δ (ppm): 4.45-4.36 (m, 9H), 3.98-3.88 (m, 1H), 3.73-3.57 (m, 9H), 3.28-3.13 (tdd, J = 5.7, 3.9, 1.0 Hz, 2H), 1.73-1.54 (m, 8H), 1.44-1.23 (m, 19H), 0.93-0.86 (m, 6H). HRMS m / z: calcd: 842.3410 (M+H + ), found: 842.3413.

[0250] Example 28: Method for synthesis of compound VI-3

[0251] Compound 176 (1.0 eq) and compound 12 (1.0 eq) were dissolved in DCM, and NHS (1.0 eq), EDC (1.0 eq), and DMAP (0.1 eq) were added, and stirred at room temperature for 12 h. After the reaction was completed, purification by column chromatography (DCM:MeOH: = 20:1) gave compound 181 (yield: 51%). Compound 181 (1.0 eq) was dissolved in DCM, and 10% TFA was slowly added at 0°C, and stirred at room temperature for 48 h. After the reaction was completed, water was washed three times, and the solvent was evaporated under reduced pressure to obtain compound 182 (yield: 79%). Compound 182 (1.0 eq) and compound 183 (1.0 eq) were dissolved in DCM, and NHS (1.0 eq), EDC (1.0 eq), and DMAP (0.1 eq) were added, and stirred at room temperature for 12 h. After the reaction was completed, purification by column chromatography (DCM:MeOH:NEt3 = 1500:100:1) gave compound 184, which is compound VI-3 (yield: 68%). 1 H NMR (400 MHz, CDC13, 293 K) δ (ppm): 4.45-4.36 (m, 9H), 3.98-3.88 (m, 1H), 3.73-3.57 (m, 9H), 3.28-3.13 (tdd, J = 5.7, 3.9, 1.0 Hz, 2H), 1.73-1.54 (m, 8H), 1.44-1.23 (m, 19H), 0.93-0.86 (m, 6H). HRMS m / z: calcd: 842.3410 (M+H + ), found: 842.3413.

[0252] Example 29: Method for synthesis of compound VI-4

[0253] Compound 185 (1.0 eq) and compound 169 (1.0 eq) were dissolved in DCM, compound 21 (1.0 eq) was added, stirred at room temperature for 12 h, after the reaction was completed, purified by column chromatography (DCM:MeOH=5:1) to obtain compound 186 (yield: 68%). Compound 186 (2.0 eq) and compound 96 (1.0 eq) were dissolved in DCM, EDC (1.0 eq) and DMAP (0.1 eq) were added, stirred at room temperature for 12 h, after the reaction was completed, purified by column chromatography (DCM:MeOH=10:1) to obtain compound 187 (yield: 51%). Compound 187 (1.0 eq) and compound 188 (1.0 eq) were dissolved in DCM, EDC (1.0 eq) and DMAP (0.1 eq) were added, stirred at room temperature for 12 h, after the reaction was completed, purified by column chromatography (DCM:MeOH=15:1) to obtain compound 189 (yield: 55%). Compound 189 (1.0 eq) and compound 190 (1.0 eq) were dissolved in DCM, NHS (1.0 eq), EDC (1.0 eq) and DMAP (0.1 eq) were added, stirred at room temperature for 12 h, after the reaction was completed, purified by column chromatography (DCM:MeOH=15:1) to obtain compound 191 (yield: 63%). Compound 191 (1.0 eq) and compound 21 (1.0 eq) were dissolved in DCM, EDC (1.0 eq) and DMAP (0.1 eq) were added, stirred at room temperature for 12 h, after the reaction was completed, washed with water three times, and the solvent was evaporated under reduced pressure to obtain compound 192 (yield: 54%). Compound 12 (1.0 eq) and compound 185 (1.0 eq) were dissolved in DCM, NHS (1.0 eq), EDC (1.0 eq) and DMAP (0.1 eq) were added, stirred at room temperature for 12 h, after the reaction was completed, purified by column chromatography (DCM:MeOH=15:1) to obtain compound 193 (yield: 69%). Compound 193 (1.0 eq) was dissolved in DCM, 10% TFA was slowly added at 0°C, stirred at room temperature for 3 h, after the reaction was completed, washed with water three times, and the solvent was evaporated under reduced pressure to obtain compound 194 (yield: 79%). Compound 194 (2.0 eq) and compound 192 (1.0 eq) were dissolved in DCM, stirred at room temperature for 12 h, after the reaction was completed, washed with water three times, and the solvent was evaporated under reduced pressure to obtain compound 195 (yield: 63%). Compound 195 (1.0 eq) was dissolved in DCM, 10% TFA was slowly added at 0°C, stirred at room temperature for 48 h, after the reaction was completed, washed with water three times, and the solvent was evaporated under reduced pressure to obtain compound 196 (yield: 79%).Compound 196 (1.0 eq) and compound 197 (1.0 eq) were dissolved in DCM, and NHS (1.0 eq), EDC (1.0 eq) and DMAP (0.1 eq) were added, stirred at room temperature for 12 h, after the reaction was completed, purified by column chromatography (DCM:MeOH = 15:1) to obtain compound 198, i.e. compound VI-4 (yield: 82%). 1 H NMR (400 MHz, CDC13, 293 K) δ (ppm): 5.56-5.48 (tt, J = 3.1, 1.6 Hz, 2H), 4.79-4.35 (m, 13H), 3.98-3.86 (ddd, J = 5.3, 4.0, 2.3 Hz, 1H), 3.63-3.44 (tdd, J = 5.3, 3.8, 1.7 Hz, 2H), 3.27-2.94 (m, 2H), 2.37-2.29 (m, 2H), 2.25-2.18 (t, J = 8.7 Hz, 2H), 2.04-1.92 (m, 5H), 1.89-1.51 (m, 15H), 1.50-1.41 (m, 2H), 1.39-1.27 (m, 48H), 0.94-0.84 (m, 6H). HRMS: m / z calcd for C68H110N10O10P2, 1134.7879 (M+H + ), found 1134.7872.

[0254] Example 30: Method for synthesizing compound VI-5

[0255] Compound 199 (1.0 eq) and compound 200 (1.0 eq) were dissolved in DCM, stirred at room temperature for 12 h, washed with water three times after the reaction was completed, and the solvent was evaporated under reduced pressure to obtain compound 201 (yield: 47%). Compound 188 (2.0 eq) and compound 169 (1.0 eq) were dissolved in DCM, EDC (1.0 eq) and DMAP (0.1 eq) were added, stirred at room temperature for 12 h, and compound 202 (yield: 68%) was obtained by column chromatography purification (DCM:MeOH:=10:1) after the reaction was completed. Compound 202 (1.0 eq) and compound 32 (1.0 eq) were dissolved in DCM, NHS (1.0 eq), EDC (1.0 eq), and DMAP (0.1 eq) were added, stirred at room temperature for 12 h, and compound 203 (yield: 63%) was obtained by column chromatography purification (DCM:MeOH:=15:1) after the reaction was completed. Compound 203 (1.0 eq) and compound 185 (1.0 eq) were dissolved in DCM, NHS (1.0 eq), EDC (1.0 eq), and DMAP (0.1 eq) were added, stirred at room temperature for 12 h, and compound 204 (yield: 62%) was obtained by column chromatography purification (DCM:MeOH:=15:1) after the reaction was completed. Compound 204 (1.0 eq) and compound 201 (1.0 eq) were dissolved in DCM, NHS (1.0 eq), EDC (1.0 eq), and DMAP (0.1 eq) were added, stirred at room temperature for 12 h, and compound 205 (yield: 50%) was obtained by column chromatography purification (DCM:MeOH:=15:1) after the reaction was completed. Compound 205 (1.0 eq) was dissolved in DCM, 10% TFA was slowly added at 0°C, stirred at room temperature for 48 h, washed with water three times after the reaction was completed, and compound 206 (yield: 55%) was obtained by evaporating the solvent under reduced pressure. Compound 206 (1.0 eq) and compound 12 (1.0 eq) were dissolved in DCM, NHS (1.0 eq), EDC (1.0 eq), and DMAP (0.1 eq) were added, stirred at room temperature for 12 h, and compound 207 (yield: 68%) was obtained by column chromatography purification (DCM:MeOH:=15:1) after the reaction was completed. Compound 207 (1.0 eq) was dissolved in DCM, 10% TFA was slowly added at 0°C, stirred at room temperature for 48 h, washed with water three times after the reaction was completed, and compound 208, which is compound VI-5, was obtained by evaporating the solvent under reduced pressure (yield: 89%). 1H NMR (400 MHz, CDC13, 293 K) δ (ppm): 5.44-5.29 (tt, J = 3.1, 1.6 Hz, 4H), 4.35-4.09 (m, 7H), 4.06-3.95 (dp, J = 9.7, 5.5 Hz, 1H), 3.89-3.82 (d, J = 5.9 Hz, 2H), 3.73-3.58 (m, 1H), 3.28-3.11 (td, J = 5.3, 3.7 Hz, 2H), 2.64-2.57 (td, J = 6.0, 1.0 Hz, 2H), 2.35-2.27 (m, 4H), 2.03-1.81 (m, 10H), 1.74-1.54 (m, 8H), 1.46-1.39 (d, J = 5.6 Hz, 3H), 1.32-1.19 (m, 41H), 1.10-1.04 (d, J = 5.8 Hz, 3H), 0.94-0.85 (m, 6H). HRMS: m / z calcd for C64H110N2O2, 1105.7912 (M+H + ), found 1105.7918.

[0256] Example 31: Method for synthesis of compound VII-1

[0257] Compound 209 (1.0 eq) and compound 130 (1.0 eq) were dissolved in DCM, stirred at room temperature for 12 h, after the reaction was completed, purified by column chromatography (DCM:MeOH:CH3COOH=800:100:1) to obtain compound 210 (yield: 54%). Compound 210 (2.0 eq) and compound 211 (1.0 eq) were dissolved in DCM, EDC (1.0 eq) and DMAP (0.1 eq) were added, stirred at room temperature for 12 h, after the reaction was completed, purified by column chromatography (DCM:MeOH:CH3COOH=1000:100:1) to obtain compound 212 (yield: 66%). Compound 212 (1.0 eq) and compound 32 (1.0 eq) were dissolved in DCM, NHS (1.0 eq), EDC (1.0 eq) and DMAP (0.1 eq) were added, stirred at room temperature for 12 h, after the reaction was completed, purified by column chromatography (DCM:MeOH=15:1) to obtain compound 213 (yield: 72%). Compound 213 (1.0 eq) and compound 25 (1.0 eq) were dissolved in DCM, NHS (1.0 eq), EDC (1.0 eq) and DMAP (0.1 eq) were added, stirred at room temperature for 12 h, after the reaction was completed, purified by column chromatography (DCM:MeOH=15:1) to obtain compound 214 (yield: 77%). Compound 214 (1.0 eq) was dissolved in DCM, 10% TFA was slowly added at 0°C, stirred at room temperature for 48 h, after the reaction was completed, washed with water three times, the solvent was evaporated under reduced pressure to obtain compound 215, which was compound VII-1 (yield: 79%). 1 H NMR (400 MHz, CDC13, 293 K) δ (ppm): 4.12-4.01 (m, 5H), 3.98-3.89 (m, 2H), 3.82-3.77 (d, J = 6.2 Hz, 2H), 3.19-3.10 (td, J = 5.0, 3.6 Hz, 2H), 2.86-2.76 (q, J = 5.2 Hz, 4H), 2.51-2.31 (m, 8H), 2.06-1.98 (p, J = 4.9 Hz, 1H), 1.95-1.52 (m, 17H), 1.45-1.20 (m, 24H), 0.95-0.84 (m, 6H). HRMS: m / z calcd for C52H84N4O8, 826.5779 (M+H + ), found 826.5783.

[0258] Example 32: Synthesis method of compound VII-2

[0259] Compound 213 (1.0 eq) and compound 161 (1.0 eq) were dissolved in DCM, and NHS (1.0 eq), EDC (1.0 eq), and DMAP (0.1 eq) were added, and stirred at room temperature for 12 h. After the reaction was completed, purification by column chromatography (DCM:MeOH = 15:1) gave compound 216 (yield: 59%). Compound 216 (1.0 eq) was dissolved in DCM, and 10% TFA was slowly added at 0°C, and stirred at room temperature for 48 h. After the reaction was completed, water was washed three times, and the solvent was evaporated under reduced pressure to obtain compound 217, which is compound VII-2 (yield: 81%). 1 H NMR (400 MHz, CDC13, 293 K) δ (ppm): 4.22-4.16 (m, 5H), 3.88-3.81 (m, 2H), 3.82-3.77 (d, J = 6.2 Hz, 2H), 3.19-3.10 (td, J = 5.0, 3.6 Hz, 2H), 2.86-2.76 (q, J = 5.2 Hz, 4H), 2.56-2.38 (m, 8H), 2.06-1.98 (p, J = 4.9 Hz, 1H), 1.95-1.52 (m, 17H), 1.47-1.24 (m, 24H), 0.95-0.83 (m, 6H). HRMS: m / z calcd for C46H73N5O7, 826.5779 (M+H + ), found 826.5783.

[0260] Example 33: Synthesis method of compound VII-3

[0261] Compound 1 (1.0 eq) and compound 218 (1.0 eq) were dissolved in DCM, and EDC (1.0 eq) and DMAP (0.1 eq) were added, and stirred at room temperature for 12 h. After the reaction was completed, purification by column chromatography (DCM:MeOH = 20:1) gave compound 219 (yield: 60%). Compound 219 (1.0 eq) was dissolved in DCM, and 10% TFA was slowly added at 0°C, and stirred at room temperature for 48 h. After the reaction was completed, water was washed three times, and the solvent was evaporated under reduced pressure to obtain compound 220, which is compound VII-3 (yield: 70%). 1 H NMR (400 MHz, CDC13, 293 K) δ (ppm): 4.58-4.46 (t, J = 6.5 Hz, 2H), 3.91-3.74 (p, J = 6.4 Hz, 1H), 3.29-3.08 (td, J = 5.4, 3.6 Hz, 2H), 1.72-1.47 (m, 6H), 1.41-1.20 (m, 32H), 0.95-0.81 (m, 3H). HRMS: m / z calcd for C42H71N5O7, 454.4247 (M+H +), found 454.4242.

[0262] Example 34: Method for synthesis of compound VII-4

[0263] Compound 28 (1.0 eq) and compound 165 (1.0 eq) were dissolved in DCM, stirred at room temperature for 12 h, after the reaction was completed, purified by column chromatography (DCM:MeOH:CH3COOH=1000:100:1) to obtain compound 221 (yield: 63%). Compound 221 (2.0 eq) and compound 25 (1.0 eq) were dissolved in DCM, added with NHS (1.0 eq), EDC (1.0 eq) and DMAP (0.1 eq), stirred at room temperature for 12 h, after the reaction was completed, purified by column chromatography (DCM:MeOH:CH3COOH=1000:100:1) to obtain compound 222 (yield: 72%). Compound 222 (1.0 eq) and compound 214 (1.0 eq) were dissolved in DCM, slowly added with 10% TFA at 0°C, stirred at room temperature for 48 h, after the reaction was completed, washed with water three times, and the solvent was evaporated under reduced pressure to obtain compound 223 (yield: 79%). Compound 223 (2.0 eq) and compound 23 (1.0 eq) were dissolved in DCM, added with NHS (1.0 eq), EDC (1.0 eq) and DMAP (0.1 eq), stirred at room temperature for 12 h, after the reaction was completed, purified by column chromatography (DCM:MeOH=5:1) to obtain compound 224, i.e. compound VII-4 (yield: 68%). 1 H NMR (400 MHz, CDC13, 293 K) δ (ppm): 5.53-5.29 (m, 8H), 4.39-4.29 (t, J = 5.3 Hz, 2H), 4.11-4.01 (dt, J = 8.9, 6.5 Hz, 1H), 3.77-3.58 (m, 12H), 3.31-3.14 (tdd, J = 5.4, 3.8, 1.7 Hz, 2H), 2.86-2.77 (tt, J = 6.4, 5.3 Hz, 2H), 2.74-2.64 (m, 6H), 2.61-2.56 (t, J = 6.5 Hz, 2H), 2.53-2.48 (t, J = 5.7 Hz, 2H), 2.32-2.24 (t, J = 8.1 Hz, 2H), 2.11-2.01 (dtdd, J = 14.4, 7.5, 3.9, 1.3 Hz, 4H), 1.86-1.55 (m, 10H), 1.54-1.44 (pd, J = 7.9, 1.0 Hz, 2H), 1.39-1.20 (m, 6H), 0.93-0.85 (t, J = 6.3 Hz, 3H). HRMS: m / z calcd for C46H64N4O8, 779.5408 (M+H +), found 779.5412.

[0264] Example 35: Method for synthesis of compound VII-5

[0265] Compound 40 (1.0 eq) and compound 25 (1.0 eq) were dissolved in DCM, and NHS (1.0 eq), EDC (1.0 eq) and DMAP (0.1 eq) were added, stirred at room temperature for 12 h, after the reaction was completed, purified by column chromatography (DCM:MeOH=10:1) to obtain compound 225 (yield: 68%). Compound 225 (1.0 eq) was dissolved in DCM, 10% TFA was slowly added at 0°C, stirred at room temperature for 48 h, washed with water three times after the reaction was completed, and the solvent was evaporated under reduced pressure to obtain compound 226 (yield: 43%). Compound 226 (2.0 eq) and compound 23 (1.0 eq) were dissolved in DCM, and NHS (1.0 eq), EDC (1.0 eq) and DMAP (0.1 eq) were added, stirred at room temperature for 12 h, after the reaction was completed, purified by column chromatography (DCM:MeOH:NEt3=1000:100:1) to obtain compound 227 (yield: 52%). Compound 227 (1.0 eq) and compound 4 (1.0 eq) were dissolved in DCM, and NHS (1.0 eq), EDC (1.0 eq) and DMAP (0.1 eq) were added, stirred at room temperature for 12 h, after the reaction was completed, purified by column chromatography (DCM:MeOH=10:1) to obtain compound 228 (yield: 82%). 1 H NMR (400 MHz, CDC13, 293 K) δ (ppm): 5.53-5.41 (tt, J = 3.1, 1.4 Hz, 2H), 5.16-4.92 (t, J = 6.7 Hz, 1H), 4.29-4.20 (dt, J = 8.4, 6.5 Hz, 1H), 3.55-3.48 (d, J = 5.3 Hz, 2H), 3.36-3.32 (s, 2H), 3.28-3.19 (m, 4H), 3.17-3.06 (td, J = 5.0, 3.6 Hz, 2H), 2.30-2.24 (t, J = 8.3 Hz, 2H), 2.19-2.16 (t, J = 8.4 Hz, 2H), 2.07-1.76 (m, 16H), 1.56-1.43 (m, 21H), 0.93-0.81 (m, 3H). HRMS: m / z calcd for C52H84N10O8, 828.5143 (M+H + ), found 828.5138.

[0266] Example 36: Preparation and characterization of mRNA-encapsulating base LNP and LNP of the application

[0267] Ionizable lipid molecules (ALC-0315), DSPC, cholesterol, and PEG-DSPE were dissolved in ethanol at a molar ratio of 50:10:38:2. Firefly luciferase mRNA was dissolved in a 100mM sodium citrate buffer solution at pH 5.0. The volume ratio of the organic phase to the aqueous phase was 1:3, and the lipid carrier was mixed with mRNA at a mass ratio of 25:1 to obtain a slightly white solution. Ethanol was then removed by ultrafiltration to obtain basic lipid nanoparticles encapsulating mRNA (represented as Basic LNP@mRNA). Luc ).

[0268] The compounds (I-1 to VII-5) synthesized in Examples 1-35, DSPC, cholesterol, and PEG-DSPE were dissolved in ethanol at a molar ratio of 50:10:38:2. The mRNA, firefly luciferase mRNA, was dissolved in a 100 mM sodium citrate buffer solution at pH 5.0. The organic phase solution and aqueous phase solution were mixed at a volume ratio of 1:3, and the lipid carrier and mRNA were mixed at a mass ratio of 25:1 to obtain a slightly white solution. The ethanol was then removed by ultrafiltration to obtain the lipid nanoparticles of the present invention encapsulating mRNA (named according to the compounds synthesized in Examples 1-35. For example, if compound I-1 is used, the lipid nanoparticles can be represented as I-1 LNP@mRNA). Luc ).

[0269] The particle size distribution of the obtained basic lipid nanoparticles and the lipid nanoparticles of this invention was characterized using dynamic light scattering (DLS). Specifically, Basic LNP@mRNA... Luc I-1 LNP@mRNA Luc I-2 LNP@mRNA Luc and I-3 LNP@mRNA Luc The particle size distribution is shown in Figure 1.

[0270] DLS results (Table 1) show that Basic LNP@mRNA Luc Compared with the LNP@mRNA of the present invention prepared based on I-1 to VII-5 Luc In comparison, there was no significant difference in hydrated particle size, and both met the usable standards.

[0271] LNP@mRNA Luc Encapsulation efficiency determination

[0272] The resulting slightly white solution was dialyzed against a suitable volume of PBS for 4 hours. The filtrate was collected, and the mRNA content in the filtrate was determined using Nanodrop. The encapsulation efficiency was calculated using the following formula: Encapsulation efficiency = mRNA总量 - mRNA 滤液 / mRNA 总量 .

[0273] The results show (Table 1) that the prepared LNP@mRNA of the application Luc all have good mRNA encapsulation rates.

[0274] Table 1

[0275] Example 37: In vitro transfection efficiency experiment of LNP@mRNA of the application Luc

[0276] The compounds (I-1 to VII-5) synthesized in Examples 1-35, DSPC, cholesterol and PEG-DSPE were respectively dissolved in ethanol according to a molar ratio of 50:10:38:2. The mRNA was firefly luciferase mRNA, which was dissolved with a sodium citrate (100 mM) buffer solution with a pH of 5.0. The volume ratio of the organic phase solution to the aqueous phase solution was 1:3, and the lipid carrier and the mRNA were mixed in a mass ratio of 25:1 to obtain a slightly white solution. Subsequently, ethanol was removed by ultrafiltration. The mRNA-encapsulated I-1 LNP@mRNA of the application Luc to VII-5 LNP@mRNA of the application were obtained. Luc Basic LNP@mRNA encapsulating mRNA was obtained in the same way using ALC-0315 instead of the compounds synthesized in Examples 1-35. Luc .

[0277] DC2.4 cells were seeded in a 24-well plate at a density of 5x10 5 cells were incubated in DMEM medium (10% fetal bovine serum and 1% penicillin-streptomycin double antibody) at 37°C in an atmosphere containing 5% CO2. After 24 hours of cell incubation, fresh medium was replaced. PBS, Basic LNP@mRNA Luc and LNP@mRNA of the application Luc were added to the cells (the mRNA dose per well of cells was 1 μg). After 24 hours of cell incubation, the cells were lysed, and a firefly luciferase detection reagent (Luciferase Reporter Gene Assay Kit, Yeasen) was added, mixed well, and the RLU (Relative light unit) was measured by using the Luminescence mode of a multifunctional enzyme label instrument, thereby reflecting the in vitro transfection efficiency of each group of LNP@mRNA Luc .

[0278] The results show (Table 2) that the Basic LNP@mRNA​Luc The transfection efficiency in vitro is low, while the LNP@mRNA of the present application Luc The transfection efficiency in vitro is low, while the LNP@mRNA of the present application Luc There is a significant improvement, and the transfection effect is excellent.

[0279] Table 2

[0280] Example 38: LNP@mRNA of the present application OVA Antigen presentation efficiency experiment

[0281] The example compounds (I-1, I-2, I-3, I-4, I-5 or VII-1), DSPC, cholesterol and PEG-DSPE are respectively dissolved in ethanol according to the molar ratio of 50:10:38:2. The mRNA is chicken ovalbumin mRNA (mRNA OVA ) dissolved in a sodium citrate (100 mM) buffer solution with pH of 5.0. The volume ratio of the organic phase solution to the aqueous phase solution is 1:3, and the lipid carrier and the mRNA are mixed in a mass ratio of 25:1 to obtain a slightly white solution. Then ethanol is removed by ultrafiltration to obtain mRNA-loaded I-1 LNP@mRNA OVA , I-2 LNP@mRNA OVA , I-3 LNP@mRNA OVA , I-4 LNP@mRNA OVA , I-5 LNP@mRNA OVA and VII-1 LNP@mRNA OVA . ALC-0315 is used instead of the above example compound to obtain mRNA-loaded Basic LNP@mRNA OVA in the same way.

[0282] DC2.4 cells are seeded in a 24-well plate at a density of 5×10 5 in DMEM medium (10% fetal bovine serum and 1% penicillin-streptomycin double antibody) at 37°C in an atmosphere containing 5% CO2. After 24 hours of cell incubation, fresh medium is replaced. PBS, Basic LNP@mRNA OVA and LNP@mRNA of the present application OVA are added to the cells respectively (the mRNA dose per well of cells is 1 μg). After 24 hours of cell incubation, flow cytometry analysis is performed to detect the proportion of OVA-H-2Kb positive cell population, and the antigen presentation efficiency of the two LNP@mRNA is compared.

[0283] The results show (Figure 2) that the antigen presentation efficiency of the Basic LNP@mRNA OVAAfter in vitro transfection, the proportion of OVA-H-2Kb positive cells was low, which proved that the DC2.4 cells transfected by Basic LNP@mRNA OVA After transfection of OVA mRNA, the antigen presentation efficiency was very limited. However, when the LNP@mRNA OVA After in vitro transfection of OVA mRNA, the proportion of OVA-H-2Kb positive cells was significantly increased, which proved that the LNP@mRNA of the application could significantly improve the antigen presentation efficiency. Among them, *P<0.05 indicates that the results have significant difference; **P<0.01 indicates that the results have obvious significant difference; ***P<0.001 indicates that the results have extremely significant difference.

[0284] Example 39: In vivo expression experiment of LNP@mRNA Luc of the application in mice

[0285] The example compound I-2, DSPC, cholesterol and PEG-DSPE were dissolved in ethanol according to the molar ratio of 50:10:38:2. The mRNA was firefly luciferase mRNA, which was dissolved with sodium citrate (100 mM) buffer solution with pH of 5.0. The volume ratio of organic phase solution to aqueous phase solution was 1:3, and the lipid carrier and mRNA were mixed in a mass ratio of 25:1 to obtain a slightly white solution. Then, ethanol was removed by ultrafiltration to obtain I-2 LNP@mRNA Luc containing mRNA. ALC-0315 was used instead of the above example compound to obtain Basic LNP@mRNA Luc containing mRNA in the same way.

[0286] Subsequently, the prepared two kinds of LNP@mRNA Luc were intraperitoneally injected into C57BL / 6J mice (4-6 weeks old, male, body weight about 18-20 g) at a dose of 2 μg mRNA per mouse. After 6 hours, the mice were intraperitoneally injected with a substrate (fluorescent sodium salt, D-Luciferin, 150 mg / kg, Yeasen), and then bioluminescence imaging was performed using a small animal live fluorescence imaging system (IVIS Spectrum, PerkinElmer).

[0287] The results showed that the LNP@mRNA Luc of the application (Figure 3) could significantly improve the luciferase expression level compared with the Basic LNP@mRNA Luc (Figure 4), and the luciferase of the application was only expressed in the pancreas. The LNP@mRNA Luc of the application could achieve targeted pancreatic delivery and high expression of nucleic acids in vivo.

[0288] ​Example 40: LNP@mRNA of the application Luc Large animal in vivo expression experiment

[0289] Example compound I-2, DSPC, cholesterol and PEG-DSPE were dissolved in ethanol according to the molar ratio of 50:10:38:2. The mRNA was firefly luciferase mRNA, which was dissolved in sodium citrate (100 mM) buffer solution with pH of 5.0. The volume ratio of organic phase solution to aqueous phase solution was 1:3, and the lipid carrier and mRNA were mixed in a mass ratio of 25:1 to obtain a slightly white solution. Then, ethanol was removed by ultrafiltration to obtain mRNA-loaded I-2 LNP@mRNA Luc . ALC-0315 was used instead of the above example compound to obtain mRNA-loaded Basic LNP@mRNA Luc in the same way.

[0290] Subsequently, the prepared two LNP@mRNA Luc were intraperitoneally injected into domestic pigs (7 weeks old, male, body weight about 15 kg) at a dose of 100 μg mRNA per pig. Six hours later, the pigs were intraperitoneally injected with a substrate (sodium salt of luciferin, D-Luciferin, 150 mg / kg, Yeasen). Then the pigs were euthanized, and the pig organs (heart, liver, spleen, lung, kidney, pancreas) were subjected to bioluminescence imaging using a live fluorescence imaging system (IVIS® Spectrum, PerkinElmer).

[0291] The results show (Figure 5) that the luciferase expression level of the LNP@mRNA of the application Luc is significantly higher than that of the Basic LNP@mRNA Luc , and the luciferase of the application is only expressed in the pancreas. The LNP@mRNA of the application Luc can achieve targeted pancreas delivery and high expression of nucleic acids in vivo.

[0292] Example 41: LNP of the application can simultaneously deliver multiple mRNA

[0293] Example compound I-2, DSPC, cholesterol and PEG-DSPE were dissolved in ethanol according to the molar ratio of 50:10:38:2. The mRNA was firefly luciferase mRNA and green fluorescent protein mRNA, which was dissolved in sodium citrate (100 mM) buffer solution with pH of 5.0. The volume ratio of organic phase solution to aqueous phase solution was 1:3, and the lipid carrier and mRNA were mixed in a mass ratio of 25:1 to obtain a slightly white solution. Then, ethanol was removed by ultrafiltration to obtain mRNA-loaded LNP@mRNA Luc+GFP . ​

[0294] Subsequently, the prepared two kinds of LNP@mRNA Luc+GFP C57BL / 6J mice (4-6 weeks old, male, body weight about 18-20 g) were intraperitoneally injected at a dose of 2 μg mRNA per mouse. Six hours later, the mice were intraperitoneally injected with a substrate (fluorescent sodium salt, D-Luciferin, 150 mg / kg, Yeasen), and then bioluminescence imaging was performed using a small animal live fluorescence imaging system (IVIS® Spectrum, PerkinElmer). The mice were then euthanized, and the pancreas of the mice was sectioned.

[0295] The results showed that the mouse pancreas had the highest protein firefly luciferase content (Figure 12), and the pancreas fluorescent protein expression section results based on the example compound I-2 LNP showed that the GFP protein was successfully expressed (Figure 6). Therefore, this proves that the LNP of the application can simultaneously deliver multiple mRNAs.

[0296] Example 42: LNP@mRNA IL-2 of the application

[0297] Example compounds I-1 to VII-5, DSPC, cholesterol and PEG-DSPE were dissolved in ethanol at a molar ratio of 50:10:38:2. The mRNA was IL-2 protein mRNA (IL-2 mRNA), which was dissolved with a sodium citrate (100 mM) buffer solution at pH 5.0. The volume ratio of the organic phase solution to the aqueous phase solution was 1:3, and the lipid carrier and the mRNA were mixed at a mass ratio of 25:1 to obtain a slightly white solution. Subsequently, ethanol was removed by ultrafiltration to obtain mRNA-loaded LNP@mRNA IL-2 (I-1 LNP@mRNA IL-2 to VII-5 LNP@mRNA IL-2 ) of the application. Basic LNP@mRNA IL-2 was obtained by using ALC-0315 instead of the above example compounds I-1 to VII-5 in the same way.

[0298] C57BL / 6J mice (4-6 weeks old, male, body weight about 18-20 g) were implanted with PANC02 cells (1 × 10 5 ) with luciferase in the pancreas of the mice. The day of tumor bearing was recorded as day 0, and PBS, Basic LNP@mRNA IL-2 and LNP@mRNA IL-2 ​, mRNA injection dose was 2 μg, and the tumor volume of mice was measured on day 0 and day 21, respectively. As shown in Figure 7, the LNP@mRNA IL-2 of the present application had a significant anti-tumor effect.

[0299] The tumor growth inhibition rate was calculated according to the formula TGI (Tumor Growth Inhibition) = (1-treatment group tumor volume / control group tumor volume) * 100%.

[0300] The results show (Table 3) that the Basic LNP@mRNA IL-2 has a tumor growth inhibition rate of about 10%, while the LNP@mRNA IL-2 of the present application has a tumor growth inhibition rate of about 80%, which is significantly improved compared with the Basic LNP@mRNA IL-2 , indicating that the LNP@mRNA IL-2 of the present application can significantly enhance the in vivo expression efficiency of mRNA.

[0301] Table 3

[0302] Example 43: T cell pancreatic enrichment experiment of LNP@mRNA IL-2 of the present application

[0303] The example compounds I-1 to VII-5, DSPC, cholesterol and PEG-DSPE were dissolved in ethanol according to the molar ratio of 50:10:38:2. The mRNA was IL-2 protein mRNA (IL-2 mRNA) dissolved with sodium citrate (100 mM) buffer solution with pH 5.0. The volume ratio of organic phase solution to aqueous phase solution was 1:3, and the lipid carrier and mRNA were mixed in a mass ratio of 25:1 to obtain a slightly white solution. Then ethanol was removed by ultrafiltration to obtain mRNA-loaded LNP@mRNA IL-2 (I-1 LNP@mRNA IL-2 to VII-5 LNP@mRNA IL-2 ) of the present application. ALC-0315 was used instead of the above example compounds, and mRNA-loaded Basic LNP@mRNA IL-2 was obtained by the same method.

[0304] C57BL / 6J mice (4-6 weeks old, male, body weight about 18-20 g) were used. The day of injection of LNP was recorded as day 0, and on day 12, PBS, Basic LNP@mRNA IL-2 and LNP@mRNA IL-2 of the present application were injected intraperitoneally, respectively., mRNA injection dose was 2 μg.

[0305] After 15 days, the mice were euthanized, the pancreas tissue was quickly removed and immediately placed in ice-cold PBS. The pancreas tissue was then cut into small pieces (1-2 mm 3 ) under sterile conditions. The cut pancreas tissue was placed in a solution of digestive enzymes (collagenase D, 1 mg / ml; DNase I, 100 U / ml) and incubated at 37°C on a shaker for 30-60 minutes, with gentle shaking every 10 minutes. After digestion was complete, an equal volume of ice-cold flow cytometry buffer was added to stop the digestion reaction. The digested tissue was then filtered through a 70 μm cell strainer to obtain a single cell suspension. The cells were washed twice with flow cytometry buffer (centrifugation for 5 minutes at 300 g at 4°C). The cells were counted using an automatic cell counter, and the total number was controlled at 1 x 10 6 -2 x 10 6 cells / sample. The fluorescently labeled antibody cocktail (anti-CD3-FITC) was added according to the experimental design and incubated on ice in the dark for 30 minutes. The labeled cells were washed twice with flow cytometry buffer (centrifugation for 5 minutes at 300 g at 4°C). The labeled cell suspension was resuspended in an appropriate amount of flow cytometry buffer and run on a flow cytometer. Depending on the fluorescent antibody, the appropriate excitation and detection parameters were set. The data were analyzed using flow cytometry software, with the gating strategy: total T cells: CD3-positive cells. As shown in Figure 8, the LNP@mRNA IL-2 of the application was able to significantly enhance T cell infiltration in the pancreas.

[0306] The results show (Table 4) that the Basic LNP@mRNA IL-2 had an immune cell proportion in the pancreas of 3.2%, while the LNP@mRNA IL-2 of the application all had an immune cell proportion in the pancreas of about 20%, which was significantly higher than the Basic LNP@mRNA IL-2 , indicating that the LNP@mRNA IL-2 of the application was able to enhance the in vivo expression efficiency of mRNA.

[0307] Table 4

[0308] Example 44: CAR-T cell pancreas enrichment experiment of LNP@mRNA IL-2 of the application

[0309] Example compounds I-1 to VII-5, DSPC, cholesterol and PEG-DSPE were dissolved in ethanol according to the molar ratio of 50:10:38:2. The mRNA was IL-2 protein mRNA (IL-2 mRNA) dissolved with sodium citrate (100 mM) buffer solution with pH 5.0. The volume ratio of organic phase solution to aqueous phase solution was 1:3, and the lipid carrier and mRNA were mixed at a mass ratio of 25:1 to obtain a slightly white solution. Subsequently, ethanol was removed by ultrafiltration to obtain the mRNA-loaded LNP@mRNA of the application IL-2 (I-1 LNP@mRNA IL-2 to VII-5 LNP@mRNA IL-2 ). ALC-0315 was used instead of the above example compounds to obtain the mRNA-loaded Basic LNP@mRNA IL-2 .

[0310] C57BL / 6J mice (4-6 weeks old, male, body weight about 18-20 g) were used. Green fluorescent label-mesothelin (MSLN)-related CAR-T GFP-MSLN 10 6 cells were injected intraperitoneally with PBS, Basic LNP@mRNA IL-2 and the LNP@mRNA of the application IL-2 of the application on day 3, respectively, with the day recorded as day 0. The mRNA injection dose was 2 μg.

[0311] After 6 days, the mice were euthanized, and the pancreas tissue was quickly removed and immediately placed in ice-cold PBS. The pancreas tissue was then cut into small pieces (1-2 mm 3 ) under sterile conditions. The cut pancreas tissue was placed in a digestion enzyme solution (collagenase D, 1 mg / ml; DNase I, 100 U / ml) and incubated at 37°C on a shaking table for 30-60 minutes, with gentle shaking every 10 minutes. After digestion was complete, an equal volume of ice-cold flow cytometry buffer was added to terminate the digestion reaction. The digested tissue was then filtered through a 70 μm cell strainer to obtain a single cell suspension. The cells were washed twice with flow cytometry buffer (centrifugation for 5 minutes at 300 g, 4°C). The cells were counted using an automatic cell counter, with the total number controlled at 1×10 6 -2×10 6 cells / sample. The cell suspension was resuspended in an appropriate amount of flow cytometry buffer and run on a flow cytometer. According to the CAR-T fluorescence channel, the corresponding excitation and detection parameters were set. The data were analyzed using flow cytometry software, with the gating strategy: CAR-T GFP-MSLN : FITC positive cells. The output was the infiltration ratio of CAR-T cells in the pancreas tissue.

[0312] The results (Table 5) show that Basic LNP@mRNA IL-2 The proportion of CAR-T cells in the pancreas is 0.7%, while the LNP@mRNA of this invention... IL-2 The proportion of CAR-T cells in the pancreas reached approximately 4%, compared to Basic LNP@mRNA. IL-2 The significant improvement demonstrates the effectiveness of the LNP@mRNA of this invention. IL-2 It can significantly enhance CAR-T infiltration in the pancreas.

[0313] Table 5

[0314] Example 45: The LNP@mRNA of the present invention has no obvious side effects.

[0315] The compounds of the examples (I-1, II-1, III-1, IV-1, or V-1), DSPC, cholesterol, and PEG-DSPE were dissolved in ethanol at a molar ratio of 50:10:38:2. The mRNA was GFP protein mRNA (GFP mRNA), dissolved in a 100 mM sodium citrate buffer solution at pH 5.0. The volume ratio of the organic phase solution to the aqueous phase solution was 1:3, and the lipid carrier was mixed with the mRNA at a mass ratio of 25:1 to obtain a slightly white solution. The ethanol was then removed by ultrafiltration to obtain the LNP@mRNA of the present invention encapsulating the mRNA. GFP (I-1 LNP@mRNA GFP II-1LNP@mRNA GFP III-1 LNP@mRNA GFP IV-1 LNP@mRNA GFP V-1 LNP@mRNA GFP Using ALC-0315 instead of the compounds in the above examples, Basic LNP@mRNA encapsulating mRNA was obtained in the same manner. GFP .

[0316] C57BL / 6J mice (4-6 weeks old, male, weighing approximately 18-20g) were used. The LNP@mRNA of the present invention was injected in the same manner as in Example 44. GFP (I-1 LNP@mRNA GFP To VII-5 LNP@mRNA GFP ) and Basic LNP@mRNA GFP .

[0317] The levels of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) were measured. We found that after injection of the LNP@mRNA of this invention... GFPWithin 48 hours after treatment, serum IL-6 levels in the mice increased slightly, but the increase was insignificant and transient (Figure 9), while serum TNF-α levels did not increase (Figure 10). This indicates that the LNP@mRNA of the present invention... GFP It will not trigger a strong immune response in the system.

[0318] Example 46: The compounds of the present invention can be used to construct liposomes.

[0319] 100 mg of compound V-1, 80 mg of DSPC, 20 mg of cholesterol, and 5 mg of PEG-DSPE were dissolved in an equal volume of chloroform (5 mL) and methanol (5 mL). The solvent was evaporated at 45 °C using a rotary evaporator until a homogeneous lipid film was formed. 10 mL of distilled water was added to resuspend the film, and it was gently agitated until a homogeneous emulsion was formed. The emulsion was placed in an ultrasonic processor and treated at 60 Hz for 1 minute to form liposomes. The resulting liposome solution was frozen at -20 °C for 24 hours and then thawed at room temperature. This process was repeated 3 times to improve the stability of the liposomes. The liposomes were then extruded through a 100 nm polycarbonate membrane using an extruder to ensure a particle size of approximately 100 nm. The particle size distribution of the obtained V-1 Lipsome is shown in Figure 11.

[0320] Example 47: The liposomes of the present invention can be used to encapsulate molecular drugs (taking cisplatin (Cis) as an example).

[0321] 100 mg of the compound from the examples (I-1, II-1, III-1, IV-1, or V-1), 80 mg of DSPC, 20 mg of cholesterol, and 5 mg of PEG-DSPE were dissolved in a mixed solvent of 5 mL chloroform and 5 mL methanol. The solvent was evaporated at 45 °C using a rotary evaporator until a homogeneous lipid film was formed. 10 mg of cisplatin was then dispersed in 10 mL of distilled water. The cisplatin solution was added to the lipid film and gently agitated until a homogeneous emulsion was formed. The mixture was sonicated at 60 Hz for 1 minute to allow the lipid molecules to form primary liposomes encapsulating cisplatin. The resulting liposome solution was frozen at -20 °C for 24 hours and then thawed at room temperature. This freeze-thaw process was repeated three times to improve the stability of the liposomes and the encapsulation efficiency of cisplatin. The liposomes were extruded through a 200 nm polycarbonate membrane using an extrusion device, ensuring an average diameter of approximately 200 nm. Unencapsulated cisplatin was separated by centrifugation at 10,000 rpm for 30 minutes. The concentration of cisplatin in the supernatant was determined, and the encapsulation efficiency was calculated.

[0322] Table 6

[0323] Example 48: The liposomes of the present invention can be used for targeted delivery of molecular drugs (using cisplatin as an example).

[0324] Dissolve 100 mg of the compound of Example (V-1), 80 mg of DSPC, 20 mg of cholesterol and 5 mg of PEG-DSPE in a mixed solvent of 5 mL of chloroform and 5 mL of methanol. Evaporate the solvent using a rotary evaporator at 45°C until a uniform lipid film is formed. Dissolve 10 mg of cisplatin in 10 mL of distilled water. Add the cisplatin solution to the lipid film and shake gently until a uniform emulsion is formed. Subject the emulsion to ultrasonic treatment in an ultrasonic processor at a frequency of 60 Hz for 1 minute to form primary liposomes encapsulating cisplatin. Freeze the resulting liposome solution at -20°C for 24 hours and then thaw at room temperature. Repeat the freeze-thaw process 3 times to improve the stability of the liposomes and the encapsulation rate of cisplatin. Extrude the liposomes through a 200 nm polycarbonate membrane using an extrusion device to ensure that the average diameter of the liposomes is about 200 nm.

[0325] After intraperitoneal injection of the mice (C57BL / 6J mice (4-6 weeks old, male, body weight about 18-20 g)) for 6 hours, collect the blood (0.5 mL), heart, liver, spleen, kidney, lung and pancreas tissue samples of the mice, 100-200 mg of tissue sample for each organ. Strict aseptic operation is required during collection, and the samples are immediately frozen for storage to avoid sample deterioration. Blood sample: centrifugal separation of plasma, and addition of an appropriate amount of nitric acid or other acid for digestion. Take 100 mg of tissue and add 2 mL of 1M nitric acid for digestion, and fully dissolve the tissue. Take 0.1 mL of serum and add 2 mL of 1M nitric acid for digestion, and fully dissolve the tissue. Introduce the pretreated sample solution into the ICP-MS instrument for detection. The instrument detects the concentration of cisplatin. Calculate the total content of cisplatin in each organ: Total amount of cisplatin in blood (μg) = cisplatin concentration in plasma (μg / L) x plasma volume (L) Total amount of cisplatin in liver, kidney, lung (μg) = cisplatin concentration in tissue (μg / g) x tissue mass (g)

[0326] The distribution percentage of cisplatin in each organ is analyzed in comparison with the injection dose (mg), and Table 7 is obtained.

[0327] Table 7

[0328] The foregoing description of specific exemplary embodiments of the disclosure has been presented for the purposes of illustration and explanation. It is not intended to be exhaustive or to limit the disclosure to the precise forms disclosed, and obviously many modifications and variations are possible in light of the above teaching. The examples were chosen and described in order to explain the principles of the disclosure and its practical application and to thereby enable others skilled in the art to best utilize the disclosure and various embodiments with various modifications as are suited to the particular use contemplated.

Claims

1. A compound represented by the formula (1) or a pharmaceutically acceptable salt thereof, wherein X is selected from N or CH; P 1 monovalent radical derived from arginine or a derivative thereof, and monovalent radicals derived from dipeptides, tripeptides, tetrapeptides and pentapeptides formed from arginine and an amino acid selected from the group consisting of arginine, histidine, glycine, alanine, valine, leucine, isoleucine, methionine, proline, tryptophan, serine, tyrosine, cysteine, phenylalanine, asparagine, glutamine, threonine, aspartic acid, glutamic acid and lysine; R 1 and R 2 are each independently selected from H, C 1-30 alkyl, C 2-30 alkenyl or C 2-30 alkynyl; which alkyl, alkenyl or alkynyl is unsubstituted or substituted by one or more OH, NH2, halogen, -OC 1-20 alkyl, -OC(=O)C 1-20 alkyl, -C(=O)OC 1-20 alkyl, -SC 1-20 alkyl, -NHC(=O)C 1-20 alkyl, -C(=O)NHC 1-20 alkyl, -OC 2-20 alkenyl, -OC(=O)C 2-20 alkenyl, -C(=O)OC 2-20 alkenyl, -SC 2-20 alkenyl, -NHC(=O)C 2-20 alkenyl, -C(=O)NHC 2-20 alkenyl, C 3-8 cycloalkyl, 5-8 membered heterocyclyl, C 6-10 aryl or 5-10 membered heteroaryl; L 1 , L 2 and L 3 are independently selected from a single bond, C 1-10 alkylene or C 2-10 alkenylene, which alkylene or alkenylene is unsubstituted or substituted by one or more OH, NH2or halogen; L 4 , L 5 and L 6 are independently selected from a single bond, C 1-20 alkylene, C 2-10 alkenylene, -(OCH2CH2) m - or -C 1-10 alkylene-(OCH2CH2) m -, said alkylene or alkenylene being unsubstituted or substituted by one or more OH, NH2, halogen or C 6-10 aryl; said C 6-10 aryl being unsubstituted or substituted by one or more OH, NH2or halogen; m is selected from an integer between 1 and 10; G 1 , G 2 and G 3 are independently selected from a single bond, -NR 3 -, -O-, -NR 3 C(=O)O-, -OC(=O)NR 3 -, -C(=O)-, -OC(=O)-, -C(=O)O-, -OC(=O)O-, -C(=O)C(=O)O-, -OC(=O)C(=O)-, -OC(=O)C(=O)O-, -OC(=O)-L a -C(=O)O-, -NR 3 C(=O)NR 3 -, -C(=O)NR 3 -, -NR 3 C(=O)- or -C(=O)NH-L a -C(=O)O-; G 4 , G 5 and G 6 are independently selected from a single bond, -NR 3 -, -S-, -O-, -NR 3 C(=O)O-, -OC(=O)NR 3 -, -C(=O)-, -OC(=O)-, -C(=O)O-, -SC(=O)-, -C(=O)S-, -OC(=O)O-, -C(=O)C(=O)O-, -OC(=O)C(=O)-, -OC(=O)C(=O)O-, -OC(=O)-L a -C(=O)O-, -OC(=O)-L a -C(=O)-, -C(=O)-L a -C(=O)-, -C(=O)-L a -C(=O)NR 3 -, -NR 3 C(=O)NR 3 -, -C(=O)NR 3 -, -NR 3 C(=O)-, -OC(=O)-L a -S-L a -C(=O)O- or -OC(=O)-L a -S-S-L a -C(=O)O-; Each R 3 Independently selected from H and C 1-10 Alkyl, C 2-10 alkenyl, C 3-8 cycloalkyl, 5-8 membered heterocyclic, C 6-10 Aryl or 5-10-membered heteroaryl, wherein the alkyl, alkenyl, cycloalkyl, heterocyclic, aryl or heteroaryl group is unsubstituted or substituted by one or more OH, NH2 or halogens; each L is independently selected from C a is independently selected from C 1-10 alkylene or C 2-10 alkylene or C 2. The compound of claim 1 or a pharmaceutically acceptable salt thereof, wherein, P 1 monovalent radical derived from L-arginine, D-arginine or a derivative thereof, and monovalent radicals derived from dipeptides, tripeptides, tetrapeptides and pentapeptides formed from L-arginine or D-arginine and an amino acid selected from the group consisting of L-glycine, L-alanine, L-valine, L-leucine, L-isoleucine, L-methionine, L-proline, L-tryptophan, L-serine, L-tyrosine, L-cysteine, L-phenylalanine, L- asparagine, L-glutamine, L-threonine, L-aspartic acid, L-glutamic acid, L-lysine, L-arginine, L-histidine, D-glycine, D-alanine, D-valine, D-leucine, D-isoleucine, D-methionine, D-proline, D-tryptophan, D-serine, D-tyrosine, D-cysteine, D-phenylalanine, D-asparagine, D-glutamine, D-threonine, D-aspartic acid, D-glutamic acid, D-lysine, D-arginine and D-histidine; or P 1 selected from 3. The compound of claim 1 or 2 or a pharmaceutically acceptable salt thereof, wherein, R 1 and R 2 are each independently selected from C 5-30 alkyl or C 5-30 alkenyl; which alkyl or alkenyl is unsubstituted or substituted by one or more OH, NH2, halogen, -OC(=O)R 1-20 alkyl, -OC(=O)C 1-20 alkyl, -C(=O)OC 1-20 alkyl, -NHC(=O)C 2-20 alkenyl, -C(=O)NHC 2-20 alkenyl, -NHC(=O)C 1-20 alkyl or -C(=O)NHC 1-20 alkyl; or R 1 and R 2 are each independently selected from C 5-20 alkyl or C 5-20 alkenyl; which alkyl or alkenyl is unsubstituted or substituted by one or more OH, NH2, halogen, -NHC(=O)C 10-20 alkenyl or -C(=O)NHC 10-20 alkenyl; or R 1 and R 2 are each independently selected from H, 4. The compound of any one of claims 1-3 or a pharmaceutically acceptable salt thereof, wherein, L 1 , L 2 and L 3 are independently selected from a single bond or C 1-10 alkylene, which alkylene is unsubstituted or substituted by one or more OH, NH2or halogen; or L 1 , L 2 and L 3 are independently selected from a single bond or C 1-6 alkylene, which alkylene is unsubstituted or substituted by one or more OH, NH2or halogen; or L 1 , L 2 and L 3 are independently selected from a single bond, -CH2-, -CH2CH2-, 5. The compound of any one of claims 1-4 or a pharmaceutically acceptable salt thereof, wherein, L 4 , L 5 and L 6 are independently selected from a single bond, C 1-10 alkylene, -(OCH2CH2) m - or -C 1-6 alkylene-(OCH2CH2) m -, said alkylene being unsubstituted or substituted by one or more OH, NH2or halogen; m is selected from 1, 2, 3, 4, 5, 6, 7 or 8; or L 4 , L 5 and L 6 are independently selected from a single bond, C 1-8 alkylene, -(OCH2CH2) m - or -C 1-4 alkylene-(OCH2CH2) m -, said alkylene being unsubstituted or substituted by one or more OH, NH2, halogen or phenyl; m is selected from 1, 2, 3, 4, 5 or 6; or L 4 , L 5 and L 6 are independently selected from a single bond, -CH2-, -CH2CH2-, -CH(CH3)-, 6. The compound of any one of claims 1-5 or a pharmaceutically acceptable salt thereof, wherein, Each R 3 Independently selected from H and C 1-10 Alkyl, C 2-10 alkenyl, C 3-8 Cycloalkyl or 5-8 membered heterocyclic group, wherein the alkyl, alkenyl, cycloalkyl or heterocyclic group is unsubstituted or substituted by one or more OH, NH2 or halogen; or each R is independently selected from the group consisting of H, C 3 alkyl or C 1-6 alkyl or C 2-6 alkyl or C Or, each R 3 H stands for H independently.

7. The compound of any one of claims 1-6 or a pharmaceutically acceptable salt thereof, wherein, L a selected from C 1-6 alkylene, which alkylene is unsubstituted or substituted by one or more OH, NH2or halogen; Or, L a Selected from C 1-4 Alkylene, wherein the alkylene is unsubstituted or substituted by one or more OH, NH2 or halogens; or L a is selected from -CH2-, -CH2CH2- or -CH2CH2CH2-.

8. The compound of any one of claims 1-7 or a pharmaceutically acceptable salt thereof, wherein, G 1 , G 2 and G 3 are independently selected from a single bond, -NR 3 -, -O-, -NR 3 C(=O)O-, -OC(=O)NR 3 -, -C(=O)-, -OC(=O)-, -C(=O)O-, -OC(=O)O-, -C(=O)NR 3 -, -NR 3 C(=O)- or -C(=O)NH-L a -C(=O)O-; or G 1 , G 2 and G 3 is independently selected from a single bond, -NH-, -0-, -C(=0)-, -NHC(=0)0-, -OC(=0)NH-, -OC(=0)-, -C(=O)O-, -NHC(=O)-, -C(=O)NH- or 9. The compound of any one of claims 1-8 or a pharmaceutically acceptable salt thereof, wherein, G 4 , G 5 and G 6 are independently selected from a single bond, -NR 3 -, -O-, -NR 3 C(=O)O-, -OC(=O)NR 3 -, -C(=O)-, -OC(=O)-, -C(=O)O-, -OC(=O)-L a -C(=O)-, -C(=O)NR 3 -, -NR 3 C(=O)-, -OC(=O)-L a -S-L a -C(=O)O- or -OC(=O)-L a -S-S-L a -C(=O)O-; Or, G 4 G 5 and G 6 Independently selected from single bonds, -NH-, -O-, -OC(=O)-, -C(=O)O-, -C(=O)-, -NHC(=O)-, -C(=O)NH-, -NHC(=O)O-, -OC(=O)NH- or 10. A compound selected from the following: ###0007### or a pharmaceutically acceptable salt thereof.

11. A lipid carrier comprising an ionizable lipid molecule, a polyethylene glycol lipid molecule, a steroidal lipid molecule, and a helper lipid molecule, wherein the ionizable lipid molecule comprises a compound of any one of claims 1-10 or a pharmaceutically acceptable salt thereof.

12. The lipid carrier of claim 11, wherein, the polyethylene glycol lipid molecule is selected from at least one of 2-[(polyethylene glycol)-2000]-N,N-tetracosanoylacetamide (ALC-0159), 1,2-dimyristoyl-sn-glycero- methoxypolyethylene glycol (PEG-DMG), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[ammonium(polyethylene glycol)] (PEG-DSPE), PEG- disteraridylglycerol (PEG-DSG), PEG-dipalmitoyl, PEG-dioleoyl, PEG- distearoyl, PEG-diaclylglycerol amide (PEG-DAG), PEG-dipalmitoyl phosphatidyl ethanolamine (PEG-DPPE), and PEG-1,2-dimyristoyloxypropyl-3- amine (PEG-c-DMA); the steroidal lipid molecule is selected from at least one of avenasterol, beta-sitosterol, campesterol, ergocalciferol, brassicasterol, cholestanol, cholesterol, coprostanol, desmosterol, chalinosterol, dihydroergocalciferol, dihydrocholesterol, dihydroergosterol, febrifugasterol, epicholesterol, ergosterol, fucosterol, hexahydrophotosterol, hydroxycholesterol, lanosterol, photosterol, phytosterol, sitostanol, sitosterol, stigmaterol, stigmastanol, stigmasterol, cholic acid, glycocholic acid, taurocholic acid, deoxycholic acid, and lithocholic acid; the steroidal lipid molecule is selected from at least one of avenasterol, beta-sitosterol, campesterol, ergocalciferol, brassicasterol, cholestanol, cholesterol, coprostanol, desmosterol, chalinosterol, dihydroergocalciferol, dihydrocholesterol, dihydroergosterol, febrifugasterol, epicholesterol, ergosterol, fucosterol, hexahydrophotosterol, hydroxycholesterol, lanosterol, photosterol, phytosterol, sitostanol, sitosterol, stigmaterol, stigmastanol, stigmasterol, cholic acid, glycocholic acid, taurocholic acid, deoxycholic acid, and lithocholic acid; The helper lipid molecule is selected from at least one of 1,2-distearoyl-sn-glycero-3-phosphocholine DSPC, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine DPPC, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine DOPE, 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine DPPE, 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine DMPE, 2-dioleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) DOPG, oleoyl phosphatidylcholine POPC, and 1-palmitoyl-2-oleoyl phosphatidylethanolamine POPE.

13. The lipid carrier according to claim 11 or 12, wherein, The lipid carrier comprises 10-70% of the ionizable lipid molecule, 5-60% of the steroidal lipid molecule, 1-60% of the polyethylene glycol lipid molecule, and 1-30% of the helper lipid molecule, in terms of molar percentage.

14. A lipid nanoparticle composition comprising the lipid carrier according to any one of claims 11-13 and at least one ingredient selected from nucleic acid, small molecule drug, and protein polypeptide.

15. A liposome composition comprising the lipid carrier according to any one of claims 11-13 and at least one ingredient selected from nucleic acid, small molecule drug, and protein polypeptide.

16. The lipid nanoparticle composition of claim 14 or the liposome composition of claim 15, wherein, The nucleic acid is selected from at least one of DNA, mRNA, rRNA, siRNA, tRNA, microRNA, antisense nucleic acid, self-replicating RNA, and circular RNA.

17. The lipid nanoparticle composition of claim 14 or the liposome composition of claim 15, wherein, The mass ratio of the lipid carrier to the nucleic acid is 5:1 to 50:1; or the mass ratio of the lipid carrier to the nucleic acid is 10:1 to 30:

1.

18. Use of the compound or pharmaceutically acceptable salt thereof according to any one of claims 1-10, the lipid carrier according to any one of claims 11-13, the lipid nanoparticle composition according to claim 14, or the liposome composition according to claim 15 in the preparation of a nucleic acid drug or vaccine.

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