Polyethylene glycol lipid molecule containing tocopherol structure, lipid nanoparticle containing same and application thereof

Abstract

The invention relates to a polyethylene glycol lipid molecule containing a tocopherol structure, lipid nanoparticles containing the polyethylene glycol lipid molecule and application of the polyethylene glycol lipid molecule. Specifically, the invention provides a polyethylene glycol lipid molecule containing tocopherol and a derivative structure thereof as shown in a formula (1), a lipid nanoparticle containing the polyethylene glycol lipid molecule, and a preparation method and application of the polyethylene glycol lipid molecule. Compared with a polyethylene glycol lipid molecule conventionally used in the field, the lipid nanoparticles prepared from the polyethylene glycol lipid molecule shown in the formula (1) can significantly improve the delivery efficiency and expression of nucleic acid. # imgabs0 #

Description

Technical Field

[0001] The present disclosure belongs to the field of medicine and biology, and particularly relates to polyethylene glycol lipid molecules capable of delivering nucleic acids and containing a tocopherol structure, lipid nanoparticles containing the same, and uses thereof. Background Art

[0002] In recent years, nucleic acid therapy has achieved numerous breakthroughs. Nucleic acid drugs are therapeutics that regulate genes after transcription and before protein translation, acting upstream of protein synthesis. They offer advantages such as ease of design, short development cycles, strong target specificity, a wide range of therapeutic applications, and long-lasting efficacy. Currently, they are widely used in the treatment of genetic diseases, tumors, viral infections, and other diseases, and are expected to become the third largest class of drugs after small molecule drugs and antibody drugs. However, delivery systems remain a key issue for nucleic acid drugs. One of the major challenges facing the field of nucleic acid therapy today is how to stably and efficiently deliver nucleic acid molecules to target cells through appropriate delivery systems while protecting them from degradation.

[0003] An ideal delivery vehicle should have the following characteristics: safety, stability, and high efficiency. Lipid nanoparticles (LNPs) are lipid nanoparticles composed of phospholipids. They encapsulate mRNA, protect mRNA from enzymatic degradation during the delivery process and clearance by the immune system, promote its transmembrane transport, and release mRNA in the cytoplasm for protein translation, neutralizing antibodies to achieve body immunity, and are currently the most advanced non-viral nucleic acid vectors in clinical practice. The emergence of LNPs is a milestone in the development of nucleic acid therapy, successfully solving the problem of protecting and delivering RNA. And because LNPs exhibit better flexibility, safety, and relatively easy and scalable production methods, they are now being used in cutting-edge mRNA vaccine candidates and widely used new coronavirus vaccines.

[0004] Tocopherol, also known as vitamin E, is a fat-soluble vitamin widely present in various tissues and organs in the human body and is an essential nutrient for normal life activities. It exists in various forms in nature, of which α-tocopherol is the most widely distributed, abundant, and active form of tocopherol in nature. In recent years, tocopherol and its derivatives have been widely used in various industries, including pharmaceuticals, food, and cosmetics. As antioxidants, they can effectively prevent and reduce non-enzymatic oxidative damage to cellular DNA and lipids caused by free radicals or reactive oxygen species (ROS), and play a protective role on biological membranes. In addition to antioxidant effects, tocopherol and its derivatives also have excellent anti-inflammatory, lipid-regulating, and radioprotective effects. In addition, studies have found that tocopherol and its derivatives have good therapeutic effects on cardiovascular diseases, Alzheimer's disease, Parkinson's disease, reproductive system diseases, and even have excellent anti-tumor capabilities.

[0005] Based on the aforementioned properties of tocopherol and its derivatives, we have developed a class of polyethylene glycol lipid molecules containing tocopherol and its derivative structures and applied them in the preparation of nucleic acid (including DNA and RNA) delivery vectors. We have found that the use of these lipid molecules for nucleic acid delivery can effectively improve the delivery efficiency of nucleic acids in vivo and achieve efficient nucleic acid expression. Summary of the Invention

[0006] The main purpose of the present invention is to provide a polyethylene glycol lipid molecule containing the structure of tocopherol and its derivatives represented by formula (1), a lipid nanoparticle containing the same, and a preparation method and use thereof. Compared with polyethylene glycol lipid molecules conventionally used in the art, the lipid nanoparticles prepared from the polyethylene glycol lipid molecule represented by formula (1) of the present invention can achieve high-efficiency delivery and expression of nucleic acids.

[0007] [Polyethylene glycol lipid molecule - compound represented by formula (1) or a pharmaceutically acceptable salt thereof]

[0008] The present invention provides a compound represented by formula (1) or a pharmaceutically acceptable salt thereof,

[0009]

[0010] in,

[0011] X is selected from N or CH;

[0012] 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 being unsubstituted or substituted with one or more OH, NH2 or halogen;

[0013] 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

[0014] L 4 , L 5 and L 6 are independently selected from a single bond, C 1-20 Alkylene, C 2-10 Alkenylene or -(OCH2CH2) m -, the alkylene or alkenylene is unsubstituted or substituted by one or more OH, NH2, halogen or C 6-10 Aryl substituted; the C 6-10 Aryl is unsubstituted or substituted with one or more OH, NH2 or halogen;

[0015] 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)-, -OP(=O)(OH)O-, or combinations of these groups with amino acid residues;

[0016] The amino acid residue is selected from a divalent group obtained by removing H and / or OH from glycine, alanine, valine, leucine, isoleucine, methionine, proline, tryptophan, serine, tyrosine, cysteine, phenylalanine, asparagine, glutamine, threonine, aspartic acid, glutamic acid, lysine, arginine or histidine, or a combination thereof;

[0017] Each R 3 Independently selected from H, C 1-10 Alkyl, C 2-10 Alkenyl, C 3-8Cycloalkyl, 5-8 membered heterocyclic group, C 6-10 Aryl or 5-10 membered heteroaryl, wherein the alkyl, alkenyl, cycloalkyl, heterocyclyl, aryl or heteroaryl is unsubstituted or substituted with one or more OH, NH2 or halogen;

[0018] Each L a Independently selected from C 1-10 Alkylene or C 2-10 Alkenylene, said alkylene or alkenylene being unsubstituted or substituted with one or more OH, NH2 or halogen;

[0019] R 1 selected from monovalent groups derived from tocopherol and its derivatives;

[0020] R 2 Selected from monovalent groups derived from tocopherol and its derivatives, C 1-30 Alkyl, C 2-30 Alkenyl or C 2-30 Alkynyl; the alkyl, alkenyl or alkynyl group 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 heterocyclic group, C 6-10 substituted with aryl or 5-10 membered heteroaryl;

[0021] P 1 Selected from C 1-30 Alkyl or C 2-30 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, -SC 1-20 Alkyl, -NHC(=O)C 1-20 Alkyl, -C(=O)NHC 1-20 Alkyl, -OC 2-20Alkenyl, -OC(=O)C 2-20 Alkenyl, -C(=O)OC 2-20 Alkenyl, -SC 2-20 Alkenyl, -NHC(=O)C 2-20 Alkenyl or -C(=O)NHC 2-20 Alkenyl substitution, provided that P 1 and G 3 At least one of them contains -(OCH2CH2) n -;

[0022] R 4 Selected from -OCH3, -NH2, -COOH, -SH or -N3;

[0023] n is an integer from 20 to 250;

[0024] m is an integer from 1 to 10.

[0025] In some embodiments, L 1 、L 2 and L 3 Independently selected from a single bond or C 1-10 Alkylene, which is unsubstituted or substituted with one or more OH, NH2 or halogen.

[0026] In some embodiments, L 1 、L 2 and L 3 Independently selected from a single bond or C 1-6 Alkylene, which is unsubstituted or substituted with one or more OH, NH2 or halogen.

[0027] In some embodiments, L 1 、L 2 and L 3 are independently selected from a single bond, -CH2-, -CH2CH2-,

[0028] In some embodiments, 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 -, -OC(=O)-, -C(=O)O-, -OC(=O)O-, -C(=O)C(=O)O-, -OC(=O)C(=O)O-, -OC(=O)-L a -C(=O)O-, -NR 3 C(=O)NR3 -, -C(=O)NR 3 -、-NR 3 C(=O)- or

[0029] In some embodiments, G 1 , G 2 and G 3 are independently selected from a single bond, -NR 3 -、-OC(=O)NR 3 -, -OC(=O)-, -OC(=O)O-, -C(=O)C(=O)O-, -OC(=O)C(=O)O-, -OC(=O)-L a -C(=O)O-, -NR 3 C(=O)- or

[0030] In some embodiments, each R 3 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 by one or more OH, NH2 or halogen.

[0031] In some embodiments, each R 3 Independently selected from H, C 1-10 Alkyl or C 2-10 Alkenyl, the alkyl, alkenyl is unsubstituted or substituted by one or more OH, NH2 or halogen.

[0032] In some embodiments, each R 3 Independently selected from H, C 1-6 Alkyl or C 2-6 Alkenyl, the alkyl, alkenyl is unsubstituted or substituted by one or more OH, NH2 or halogen.

[0033] In some embodiments, each R 3 Independently selected from H, C 1-4 Alkyl or C 2-4 Alkenyl, said alkyl, alkenyl is unsubstituted or substituted with one or more OH, NH2, F, Cl or Br.

[0034] In some embodiments, each R 3 are independently H.

[0035] In some embodiments, L a Selected from C 1-10Alkylene, which is unsubstituted or substituted with one or more OH, NH2 or halogen.

[0036] In some embodiments, L a Selected from C 1-6 Alkylene, which is unsubstituted or substituted with one or more OH, NH2 or halogen.

[0037] In some embodiments, L a Selected from C 1-4 Alkylene, which is unsubstituted or substituted with one or more OH, NH2 or halogen.

[0038] In some embodiments, L a is selected from -CH2-, -CH2CH2- or -CH2CH2CH2-.

[0039] In some embodiments, G 1 , G 2 and G 3 independently selected from a single bond, -NH-, -O-, -NHC(=O)O-, -OC(=O)NH-, -OC(=O)-, -C(=O)O-, -OC(=O)O-, -C(=O)C(=O)O-, -OC(=O)C(=O)-, -OC(=O)C(=O)O-, -OC(=O)CH2CH2C(=O)O-, -OC(=O)CH2CH2CH2C(=O)O-, -NHC(=O)NH-, -C(=O)NH-, -NHC(=O)-, or

[0040] In some embodiments, G 1 , G 2 and G 3 Independently selected from a single bond, -NH-, -O-, -NHC(=O)O-, -OC(=O)NH-, -OC(=O)-, -OC(=O)O-, -OC(=O)C(=O)O-, -OC(=O)CH2CH2C(=O)O-, -OC(=O)CH2CH2CH2C(=O)O-, -NHC(=O)NH- or -NHC(=O)-.

[0041] In some embodiments, L 4 、L 5 and L 6 are independently selected from a single bond, C 1-14 Alkylene, C 2-8 Alkenylene or -(OCH2CH2) m-, the alkylene or alkenylene is unsubstituted or substituted by one or more OH, NH2, halogen or phenyl; the phenyl is unsubstituted or substituted by one or more OH, NH2 or halogen; m is selected from 1, 2, 3, 4, 5, 6, 7 or 8.

[0042] In some embodiments, L 4 、L 5 and L 6 are independently selected from a single bond, C 1-11 Alkylene, C 2-7 Alkenylene or -(OCH2CH2) m -, the alkylene group is unsubstituted or substituted by one or more OH, NH2, halogen or phenyl; the phenyl group is unsubstituted or substituted by one or more OH, NH2 or halogen; m is selected from 1, 2, 3, 4, 5 or 6.

[0043] In some embodiments, L 4 、L 5 and L 6 are independently selected from a single bond, -CH2-, -CH2CH2-, -CH(CH3)-,

[0044] In some embodiments, 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)-, -OP(=O)(OH)O-, Or a combination of these groups and an amino acid residue; the amino acid residue is selected from a divalent group obtained by removing H and / or OH from valine, isoleucine, proline, tyrosine, cysteine, lysine, leucine, phenylalanine, threonine, serine, methionine, alanine, glutamic acid, glycine or tryptophan.

[0045] In some embodiments, 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-, -C(=O)C(=O)O-, -OC(=O)C(=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 -、-NR 3 C(=O)-, -OP(=O)(OH)O-, or a combination of these groups and an amino acid residue; the amino acid residue is selected from a divalent group obtained by removing H and / or OH from leucine, threonine, glutamic acid, phenylalanine, tryptophan, serine, methionine, alanine or glycine.

[0046] In some embodiments, G 4 , G 5 and G 6 Independently selected from single bonds, -NH-, -S-, -O-, -NHC(=O)O-, -OC(=O)NH-, -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)CH2CH2C(=O)O-, -OC(=O)CH2CH2CH2C(=O)O-, -NHC(=O)-, -C(=O)NH-, -NHC(=O)NH-, -OP(=O)(OH)O-,

[0047]

[0048] In some embodiments, R 1 The monovalent group selected from tocopherol and its derivatives includes but is not limited to α-tocopherol, β-tocopherol, γ-tocopherol, δ-tocopherol, α-tocotrienol, β-tocotrienol, γ-tocotrienol or δ-tocotrienol.

[0049] In some embodiments, R 1 A monovalent group selected from α-tocopherol, β-tocopherol, γ-tocopherol, δ-tocopherol, α-tocotrienol, β-tocotrienol, γ-tocotrienol or δ-tocotrienol obtained by removing H from the phenolic hydroxyl group.

[0050] In some embodiments, R 1 Selected from

[0051] In some embodiments, R 1 Selected from

[0052] In some embodiments, R 2 A monovalent group selected from tocopherol and its derivatives including but not limited to α-tocopherol, β-tocopherol, γ-tocopherol, δ-tocopherol, α-tocotrienol, β-tocotrienol, γ-tocotrienol or δ-tocotrienol, C 1-30 Alkyl, C 2-30 Alkenyl or C 2-30 Alkynyl; the alkyl, alkenyl or alkynyl group 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 heterocyclic group, C 6-10 aryl or 5-10 membered heteroaryl.

[0053] In some embodiments, R 2 A monovalent group selected from α-tocopherol, β-tocopherol, γ-tocopherol, δ-tocopherol, α-tocotrienol, β-tocotrienol, γ-tocotrienol or δ-tocotrienol obtained by removing H from the phenolic hydroxyl group, C 1-30 Alkyl, C 2-30 Alkenyl or C 2-30 Alkynyl; the alkyl, alkenyl or alkynyl group 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 heterocyclic group, C 6-10 aryl or 5-10 membered heteroaryl.

[0054] In some embodiments, R 2 Selected from

[0055] C 2-20 Alkyl or C 2-20 Alkenyl; the alkyl or alkenyl is unsubstituted or substituted by one or more OH, NH2, halogen, -OC(=O)C 1-20 Alkyl, -C(=O)OC 1-20 Alkyl, -NHC(=O)C 1-20 Alkyl, -C(=O)NHC 1-20 Alkyl, -OC(=O)C 2-20 Alkenyl, -C(=O)OC 2-20 Alkenyl, -NHC(=O)C 2-20 Alkenyl, -C(=O)NHC 2-20 The alkylene group is substituted with an alkenyl or 5-8 membered heteroaryl group.

[0056] In some embodiments, R 2 Selected from

[0057] C 2-20 Alkyl or C 2-20 Alkenyl; the alkyl or alkenyl is unsubstituted or substituted by one or more OH, NH2, halogen, -OC(=O)C 1-20 Alkyl, -C(=O)OC 1-20 Alkyl, -NHC(=O)C 2-20 The alkylene group is substituted with an alkenyl or 5-6 membered heteroaryl group.

[0058] In some embodiments, R 2 Selected from

[0059] C 2-20 Alkyl or C 2-20 Alkenyl; the alkyl or alkenyl is unsubstituted or substituted by one or more OH, NH2, halogen, -OC(=O)C 10-20 Alkyl, -C(=O)OC 10-20 Alkyl, -NHC(=O)C 10-20 The alkylene group is substituted with an alkenyl or 5-6 membered heteroaryl group.

[0060] In some embodiments, R 2 Selected from

[0061] C 2-20 Alkyl or C 2-20 Alkenyl; the alkyl or alkenyl is unsubstituted or substituted by one or more OH, NH2, halogen, -OC(=O)C 10-20 Alkyl, -C(=O)OC 10-20 Alkyl, -NHC(=O)C 10-20 The alkylene group is substituted with an alkenyl or 5-6 membered heteroaryl group.

[0062] In some embodiments, R 2 Selected from

[0063] In some embodiments, P 1 Selected from or C 1-30 Alkyl, said alkyl being unsubstituted or substituted by one or more OH, NH2, halogen, -OC 1-20 Alkyl, -OC(=O)C 1-20 Alkyl, -C(=O)OC1-20 Alkyl, -NHC(=O)C 1-20 Alkyl or -C(=O)NHC 1-20 Alkyl substitution.

[0064] In some embodiments, P 1 Selected from or C 1-20 Alkyl, said alkyl being unsubstituted or substituted by one or more OH, NH2, halogen, -OC 6-20 Alkyl, -OC(=O)C 6-20 Alkyl, -C(=O)OC 6-20 Alkyl, -NHC(=O)C 6-20 Alkyl or -C(=O)NHC 6-20 Alkyl substitution.

[0065] In some embodiments, P 1 Selected from or C 1-10 Alkyl, said alkyl being unsubstituted or substituted by one or more OH, NH2, halogen, -OC 10-20 Alkyl, -OC(=O)C 10-20 Alkyl, -C(=O)OC 10-20 Alkyl, -NHC(=O)C 10-20 Alkyl or -C(=O)NHC 10-20 Alkyl substitution.

[0066] In some embodiments, P 1 Selected from or C 1-6 Alkyl, said alkyl being unsubstituted or substituted by one or more OH, NH2, halogen or -OC(=O)C 10-20 Alkyl substitution.

[0067] In some embodiments, P 1 and G 3 One of them contains -(OCH2CH2) n -, n is an integer from 20 to 250.

[0068] In some embodiments, when P 1 Selected from C 1-30 Alkyl or C 2-30 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, -SC 1-20 Alkyl, -NHC(=O)C 1-20 Alkyl, -C(=O)NHC1-20 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 or -C(=O)NHC 2-20 When alkenyl is substituted, G 3 for n is an integer from 20 to 250.

[0069] In some embodiments, when P 1 Selected from C 1-10 Alkyl, said alkyl being unsubstituted or substituted by one or more OH, NH2, halogen, -OC 10-20 Alkyl, -OC(=O)C 10-20 Alkyl, -C(=O)OC 10-20 Alkyl, -NHC(=O)C 10-20 Alkyl or -C(=O)NHC 10-20 When alkyl is substituted, G 3 for n is an integer from 20 to 250.

[0070] In some embodiments, P 1 Selected from

[0071] In some embodiments, when P 1 for When G 3 for n is an integer from 20 to 250.

[0072] In some embodiments, P 1 The monovalent group is selected from polyethylene glycol and its derivatives including but not limited to methoxy polyethylene glycol, amino polyethylene glycol, carboxyl polyethylene glycol, mercapto polyethylene glycol, and azido polyethylene glycol.

[0073] In some embodiments, P 1 Selected from a monovalent group derived from methoxypolyethylene glycol, aminopolyethylene glycol, carboxylpolyethylene glycol, mercaptopolyethylene glycol or azidopolyethylene glycol.

[0074] In some embodiments, P 1 Selected from a monovalent group derived from methoxypolyethylene glycol, aminopolyethylene glycol, mercaptopolyethylene glycol or azidopolyethylene glycol.

[0075] In some embodiments, P 1Selected from monovalent groups derived from methoxypolyethylene glycol or aminopolyethylene glycol.

[0076] In some embodiments, P 1 The molecular weight range is 44-100,000 Daltons.

[0077] In some embodiments, P 1 The molecular weight range is 220-10,000 Daltons.

[0078] In some embodiments, P 1 The molecular weight range is 2000-5000 Daltons.

[0079] In some embodiments, n is an integer from 20 to 250; alternatively, n is an integer from 5 to 227; alternatively, n is an integer from 20 to 227; alternatively, n is an integer from 23 to 227; alternatively, n is an integer from 45 to 227; alternatively, n is an integer from 45 to 114; alternatively, n is an integer from 23 to 114.

[0080] In some embodiments, the present invention provides a compound represented by formula (1) or a pharmaceutically acceptable salt thereof, wherein the compound is selected from the following:

[0081]

[0082]

[0083]

[0084]

[0085]

[0086] Wherein n is an integer from 20 to 250.

[0087] In some embodiments, the present invention provides a compound represented by formula (1) or a pharmaceutically acceptable salt thereof, wherein the compound is selected from the following:

[0088]

[0089]

[0090]

[0091]

[0092]

[0093] [Lipid carrier]

[0094] The present invention provides a lipid carrier comprising an ionizable lipid molecule, a polyethylene glycol lipid (Lipid-PEG) molecule, a steroid lipid molecule and an auxiliary lipid molecule, wherein the polyethylene glycol lipid molecule comprises a compound represented by the above formula (1) or a pharmaceutically acceptable salt thereof.

[0095] In some embodiments, the ionizable lipid molecule contains one or more ionizable sites, including pyridine, imidazole, primary amine, secondary amine, and tertiary amine.

[0096] In some embodiments, the ionizable lipid molecule is selected from at least one of the following: (1-octylnonyl 8-[(2-hydroxyethyl)[6-O-6-(undecyloxy)hexyl]amino]-octanoate) SM-102, ((4-hydroxybutyl)azadialkyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate) ALC-0315, 1,2-dioleoyl-3-dimethylammonium-propane DODAP, 1,2-dioleoxy-3-dimethylammonium 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-octadecen-1-yl-1,3-dioxolane-4-ethylamine Dlin-KC2-DMA, 4-(N,N-dimethylamino)butyric acid (dilinoleyl) methyl ester Dlin-MC3-DMA, etc.

[0097] In some embodiments, the ionizable lipid molecule is selected from at least one of the following: SM-102, ALC-0315, DODAP, DODMA, DOBAQ, YSK05, Dlin-DMA, Dlin-KC2-DMA, and Dlin-MC3-DMA.

[0098] In some embodiments, the ionizable lipid molecule is selected from at least one of SM-102, ALC-0315, DODAP, and Dlin-DMA.

[0099] In some embodiments, the ionizable lipid molecule is selected from at least one of SM-102 and ALC-0315.

[0100] In some embodiments, the polyethylene glycol lipid molecule is a compound represented by the above formula (1) or a pharmaceutically acceptable salt thereof.

[0101] In some embodiments, the polyethylene glycol lipid molecules further include at least one of the following: 2-[(polyethylene glycol)-2000]-N,N-tetracosylacetamide (ALC-0159), 1,2-dimyristoyl-sn-glyceromethoxypolyethylene glycol (PEG-DMG), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)] (PEG-DSPE), PEG-disterylglycerol (PEG-DSG), PEG-dipalmitoleyl, PEG-dioleyl, PEG-distearyl, PEG-diacylglyceramide (PEG-DAG), PEG-dipalmitoylphosphatidylethanolamine (PEG-DPPE), and PEG-1,2-dimyristoyloxypropyl-3-amine (PEG-c-DMA).

[0102] In some embodiments, the steroidal lipid molecule is selected from at least one of the following: avenasterol, β-sitosterol, brassicasterol, ergocalciferol, campesterol, cholestanol, cholesterol, coprosterol, dehydrocholesterol, streptosterol, dihydroergocalciferol, dihydrocholesterol, dihydroergosterol, melanosterol, epicholesterol, ergosterol, fuccasterol, hexahydroluminosterol, hydroxycholesterol, lanosterol, luminosterol, alginosterol, sitostanol, sitosterol, stigmasterol, stigmasterol, cholic acid, glycocholic acid, taurocholic acid, deoxycholic acid, and lithocholic acid.

[0103] In some embodiments, the steroidal lipid molecule is selected from at least one of the following: cholesterol, cholestanol, ergocalciferol, dihydrocholesterol, alginosterol, taurocholic acid, and deoxycholic acid.

[0104] In some embodiments, the steroidal lipid molecule is selected from at least one of cholesterol, cholestanol, dihydrocholesterol, alginosterol, and deoxycholic acid.

[0105] In some embodiments, the steroidal lipid molecule is selected from at least one of cholesterol and dihydrocholesterol.

[0106] In some embodiments, the helper lipid molecule is selected from at least one of the following: 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), oleoylphosphatidylcholine (POPC) and 1-palmitoyl-2-oleoylphosphatidylethanolamine (POPE).

[0107] In some embodiments, the auxiliary lipid molecule is selected from at least one of the following: 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), and 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE).

[0108] 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).

[0109] In some embodiments, the lipid carrier comprises, in molar percentage, 10%-70% of ionizable lipid molecules, 5%-60% of steroidal lipid molecules, 1%-60% of the compound represented by the above formula (1) or a pharmaceutically acceptable salt thereof (polyethylene glycol lipid molecules) and 1%-30% of auxiliary lipid molecules.

[0110] In some embodiments, the molar percentage of the ionizable lipid molecules is 15%-65%.

[0111] In some embodiments, the molar percentage of the ionizable lipid molecules is 30%-60%.

[0112] In some embodiments, the molar percentage of the ionizable lipid molecules is 45%-55%.

[0113] In some embodiments, the molar percentage of the steroidal lipid molecules is 10%-50%.

[0114] In some embodiments, the molar percentage of the steroidal lipid molecules is 25%-45%.

[0115] In some embodiments, the molar percentage of the steroidal lipid molecules is 30%-40%.

[0116] In some embodiments, the molar percentage of the compound represented by formula (1) or a pharmaceutically acceptable salt thereof is 1%-30%.

[0117] In some embodiments, the molar percentage of the compound represented by formula (1) or a pharmaceutically acceptable salt thereof is 1%-10%.

[0118] In some embodiments, the molar percentage of the compound represented by formula (1) or a pharmaceutically acceptable salt thereof is 1%-5%.

[0119] In some embodiments, the molar percentage of the helper lipid molecules is 1%-25%.

[0120] In some embodiments, the molar percentage of the helper lipid molecules is 5%-15%.

[0121] In some embodiments, the molar percentage of the helper lipid molecules is 8%-12%.

[0122] In some embodiments, in the lipid carrier, the molar ratio of ionizable lipid molecules, helper lipid molecules, steroidal lipid molecules and polyethylene glycol lipid molecules 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, etc.

[0123] [Nucleic acid lipid nanoparticle composition]

[0124] The present invention provides a nucleic acid lipid nanoparticle composition, which comprises the lipid carrier and nucleic acid.

[0125] In some embodiments, the nucleic acid is selected from at least one of DNA, mRNA, rRNA, siRNA, tRNA, microRNA, antisense nucleic acid, and circular RNA.

[0126] In some embodiments, the nucleic acid is mRNA.

[0127] In some embodiments, the nucleic acid is firefly luciferase mRNA, green fluorescent protein (GFP) mRNA, chicken ovalbumin (OVA) mRNA, or H1N1 influenza virus mRNA.

[0128] In some embodiments, the mass ratio of lipid carrier to nucleic acid in the nucleic acid lipid nanoparticle composition is 5:1-50:1.

[0129] In some embodiments, the mass ratio of lipid carrier to nucleic acid in the nucleic acid lipid nanoparticle composition is 10:1-30:1.

[0130] 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.

[0131] In some embodiments, the nucleic acid is mRNA, and the mass ratio of lipid carrier to nucleic acid in the nucleic acid lipid nanoparticle composition is 20:1, 25:1 or 30:1.

[0132] In some embodiments, the nucleic acid lipid nanoparticle composition has a particle size of 30 to 500 nm.

[0133] In some embodiments, the nucleic acid lipid nanoparticle composition has a particle size of 30 to 200 nm.

[0134] 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.

[0135] In some embodiments, the encapsulation efficiency of nucleic acids in the nucleic acid lipid nanoparticle composition is greater than 50%. Exemplarily, 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.

[0136] [Nucleic acid lipid nanoparticle preparations, uses and treatment methods]

[0137] The present invention provides a nucleic acid lipid nanoparticle preparation, which comprises the nucleic acid lipid nanoparticle composition and pharmaceutically acceptable excipients.

[0138] The present invention also provides the use of the compound represented by formula (1) or a pharmaceutically acceptable salt thereof, the lipid carrier or the nucleic acid lipid nanoparticle composition in the preparation of nucleic acid drugs or gene vaccines.

[0139] The present invention also provides a method for in vivo delivery of nucleic acid drugs or gene vaccines, comprising administering the nucleic acid lipid nanoparticle composition or the nucleic acid lipid nanoparticle preparation to a subject in need thereof.

[0140] The present invention also provides a method for treating or preventing a disease (e.g., inflammatory disease, viral infection, and cancer) or condition in a subject by delivering a nucleic acid, the method comprising administering the above-mentioned nucleic acid lipid nanoparticle composition or the above-mentioned nucleic acid lipid nanoparticle formulation to a subject in need thereof.

[0141] The term "inflammatory disease" includes autoimmune disorders, allergic disorders and inflammatory disorders, for example, selected from 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 primary or secondary). The allergic disorder may be selected from contact dermatitis, celiac disease, asthma, hypersensitivity to house dust mites, pollen and related allergens, berylliosis.

[0142] The term "viral infection" includes but is not limited to retroviral infection, hepatitis virus infection, COVID-19 novel coronavirus infection, Zika virus infection, dengue virus infection, etc.

[0143] The term "cancer" includes but is not limited to primary lung cancer (including non-small cell lung cancer and small cell lung cancer), metastatic lung cancer (lung metastasis of liver cancer, lung metastasis of breast cancer, lung metastasis of colon cancer, lung metastasis of melanoma, etc.) and cancers in other parts of the body.

[0144] In some embodiments, the nucleic acid lipid nanoparticle composition or the nucleic acid lipid nanoparticle formulation is administered by one of the following routes of administration: oral, intranasal, intravenous, intraperitoneal, intramuscular, intraarticular, intralesional, intratracheal, subcutaneous, and intradermal. In some embodiments, the nucleic acid lipid nanoparticle composition or the nucleic acid lipid nanoparticle formulation is administered, for example, via an enteral or parenteral route of administration. In some embodiments, the nucleic acid lipid nanoparticle composition or the nucleic acid lipid nanoparticle formulation is administered to the subject at a dose of about 0.001 mg / kg to about 10 mg / kg.

[0145] [Method for preparing lipid nanoparticles containing nucleic acids]

[0146] The present invention provides a method for preparing nucleic acid-encapsulated lipid nanoparticles, comprising the following steps:

[0147] (A1) mixing an ionizable lipid molecule, the compound represented by formula (1) or a pharmaceutically acceptable salt thereof, an auxiliary lipid molecule, and a steroidal lipid molecule in the above-described proportions, and dissolving them in a solvent to obtain an organic phase liposome solution;

[0148] (A2) dissolving the nucleic acid in a buffer solution of appropriate pH to obtain an aqueous nucleic acid solution;

[0149] (A3) uniformly mixing the organic phase liposome solution and the aqueous phase nucleic acid solution according to the mass ratio and a certain volume ratio described above using a microfluidic device to prepare a nucleic acid-encapsulated lipid nanoparticle solution;

[0150] 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.

[0151] In some embodiments, the solvent in step (A1) is ethanol, tetrahydrofuran or acetone.

[0152] In some embodiments, the solvent in step (A1) is ethanol.

[0153] In some embodiments, the buffer solution in step (A2) is an acetic acid / sodium acetate solution or a citric acid / sodium citrate solution.

[0154] In some embodiments, the buffer solution in step (A2) is a citric acid / sodium citrate solution.

[0155] In some embodiments, the pH of the buffer solution in step (A2) is 3-9.

[0156] In some embodiments, the pH of the buffer solution in step (A2) is 4-6.

[0157] In some embodiments, the pH of the buffer solution in step (A2) is 5.

[0158] In some embodiments, the concentration of the buffer solution in step (A2) is 1 mM-1 M.

[0159] In some embodiments, the concentration of the buffer solution in step (A2) is 20 mM-500 mM.

[0160] In some embodiments, the concentration of the buffer solution in step (A2) is 100 mM.

[0161] 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.

[0162] 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.

[0163] 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.

[0164] In some embodiments, the microfluidic device in step (A3) can be a microfluidic device conventionally used in the art, such as INano TM L / L+, Myanna or BT, Precision NanoSystems.

[0165] The present invention has the following beneficial effects:

[0166] 1. The lipid carrier comprising the compound represented by formula (1) or a pharmaceutically acceptable salt thereof is tightly bound to the nucleic acid, and can achieve a high encapsulation rate and stable protection of the nucleic acid.

[0167] 2. The formed LNP has good biocompatibility and is more stable; it can improve the efficiency of LNP in delivering nucleic acids in the body and achieve efficient expression of nucleic acids.

[0168] 3. The lipid nanoparticles are suitable for the delivery of nucleic acids with different molecular weights and sequences and are universal.

[0169] 4. The technology of the present invention is simple to synthesize, the raw materials are cheap, and it is suitable for large-scale production. BRIEF DESCRIPTION OF THE DRAWINGS

[0170] Figure 1 The Basic LNP@mRNA prepared in Example 37 with PEG-DSPE as the polyethylene glycol lipid component is shown. Luc and the I-1LNP@mRNA of the present invention using compound I-1 as a polyethylene glycol lipid component Luc particle size distribution.

[0171] Figure 2 The Basic LNP@mRNA prepared in Example 37 with PEG-DSPE as the polyethylene glycol lipid component is shown. Luc , I-1LNP@mRNA with compound I-1 as the polyethylene glycol lipid component Luc , II-1LNP@mRNA with compound II-1 as the polyethylene glycol lipid component Luc and VI-4LNP@mRNA with compound VI-4 as the polyethylene glycol lipid component Luc TEM photo of.

[0172] Figure 3 The Basic LNP@mRNA prepared in Example 39 with PEG-DSPE as the polyethylene glycol lipid component is shown. OVA and III-2LNP@mRNA with compound III-2 as the polyethylene glycol lipid component OVA The proportion of OVA-H-2Kb-positive cell population after in vitro transfection of DC2.4 cells.

[0173] Figure 4 The I-1LNP@mRNA prepared in Example 40 with compound I-1 as the polyethylene glycol lipid component is shown. Luc , II-1LNP@mRNA with compound II-1 as the polyethylene glycol lipid component Luc , IV-4LNP@mRNA with compound IV-4 as the polyethylene glycol lipid component Luc and V-4LNP@mRNA with compound V-4 as the polyethylene glycol lipid component Luc Luciferase expression levels after intramuscular injection in the mouse leg.

[0174] Figure 5 The Basic LNP@mRNA prepared in Example 42 with PEG-DSPE as the polyethylene glycol lipid component is shown. OVA and V-3LNP@mRNA with compound V-3 as the polyethylene glycol lipid component OVA Tumor growth curve of mice after injection of treatment.

[0175] Figure 6 The Basic LNP@mRNA prepared in Example 43 with PEG-DSPE as the polyethylene glycol lipid component is shown. OVA and VI-4LNP@mRNA with compound VI-4 as the polyethylene glycol lipid component OVA The expression of IFN-γ in the tumor tissue of mice after injection treatment.

[0176] Figure 7 The Basic LNP@mRNA prepared in Example 43 with PEG-DSPE as the polyethylene glycol lipid component is shown. OVA and VI-4LNP@mRNA with compound VI-4 as the polyethylene glycol lipid component OVA The expression of TNF-α in the tumor tissue of mice after injection treatment.

[0177] Figure 8 The Basic LNP@mRNA prepared in Example 44 with PEG-DSPE as the polyethylene glycol lipid component is shown. Luc VI-6LNP@mRNA with compound VI-6 as the polyethylene glycol lipid component Luc VI-7LNP@mRNA with compound VI-7 as the polyethylene glycol lipid component Luc VI-8LNP@mRNA with compound VI-8 as the polyethylene glycol lipid component Luc , and VI-9LNP@mRNA with compound VI-9 as the polyethylene glycol lipid component Lucluciferase expression levels.

[0178] Figure 9 The Basic LNP@mRNA prepared in Example 45 with PEG-DSPE as the polyethylene glycol lipid component is shown. H1N1 VI-7LNP@mRNA with compound VI-7 as the polyethylene glycol lipid component H1N1 VI-10LNP@mRNA with compound VI-10 as the polyethylene glycol lipid component H1N1 VI-11LNP@mRNA with compound VI-11 as the polyethylene glycol lipid component H1N1 , and VI-12LNP@mRNA with compound VI-12 as the polyethylene glycol lipid component H1N1 Levels of humoral immune response in mice. DETAILED DESCRIPTION

[0179] I. Definition

[0180] In this disclosure, unless otherwise indicated, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the relevant terms and laboratory procedures used herein are those widely used in the relevant fields and routine procedures. To facilitate a better understanding of this disclosure, definitions and explanations of relevant terms are provided below.

[0181] Unless expressly stated otherwise, throughout the specification and claims, the term “comprise” or variations such as “include” or “comprising” will be understood to include the stated elements or components but not to exclude other elements or components.

[0182] The term "pharmaceutically acceptable" refers 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 problems or complications, commensurate with a reasonable benefit / risk ratio.

[0183] The term "pharmaceutically acceptable salt" refers to salts of the compounds of the present application, which are prepared by reacting the compounds with specific substituents discovered herein with relatively non-toxic acids or bases. When the compounds of the present application contain relatively acidic functional groups, base addition salts can be obtained by contacting the compounds with a sufficient amount of base in a pure solution or a suitable inert solvent. When the compounds of the present application contain relatively basic functional groups, acid addition salts can be obtained by contacting the compounds with a sufficient amount of acid in a pure solution or a suitable inert solvent. Certain specific compounds of the present application contain both basic and acidic functional groups and can be converted into either base or acid addition salts.

[0184] The pharmaceutically acceptable salts of the present application can be synthesized by conventional chemical methods from parent compounds containing acid radicals or bases. Generally, the salts are prepared by reacting the free acid or base form of the compound with a stoichiometric amount of an appropriate base or acid in water or an organic solvent or a mixture of the two.

[0185] The compounds of the present disclosure may be asymmetric, for example, having one or more stereoisomers. Unless otherwise indicated, all stereoisomers are included, such as enantiomers and diastereomers. The compounds of the present disclosure containing asymmetric carbon atoms can be isolated in optically pure forms or racemic forms. Optically pure forms can be resolved from racemic mixtures or synthesized by using chiral starting materials or chiral reagents. Racemates, diastereomers, and enantiomers are all included within the scope of the present disclosure.

[0186] In this disclosure, It refers to the position where a substituent is bonded.

[0187] The term "optional" or "optionally" means that the subsequently described event or circumstance may or may not occur, and that the description includes both occurring and not occurring.

[0188] When the lower and upper limits of a numerical range are disclosed, any value or sub-range falling within the range is specifically disclosed. In particular, each numerical range of a parameter disclosed herein (e.g., in the form of "about a to b," or equivalently "approximately a to b," or equivalently "about a b") should be understood to encompass every value and sub-range therein. For example, "C 1-4 " should be understood to include any sub-ranges and every point value therein, such 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. For another example, "5-10 yuan" should be understood to cover any sub-range and every point value therein, such as 5-6 yuan, 5-7 yuan, 5-8 yuan, 5-9 yuan, 6-7 yuan, 6-8 yuan, etc., as well as 5, 6, 7, 8, 9, 10 yuan, etc.

[0189] When any variable (such as R n ) occurs more than once in a compound's composition or structure, its definition at each occurrence is independent. Thus, for example, if a group is substituted with 1-5 R, the group may be optionally substituted with up to 5 R, with each occurrence of R being an independent choice. Furthermore, combinations of substituents and / or variants thereof are permissible only if such combinations result in stable compounds.

[0190] The term "substituted" or "substituted" means that any one or more hydrogen atoms on a particular atom or group are replaced by a substituent, as long as the valence state of the particular atom or group is normal and the compound after substitution is stable. When the substituent is an oxo group (i.e., =O), it means that two hydrogen atoms are replaced. Unless otherwise specified, the type and number of substituents can be any on the basis of chemical practicability. The substituent can be selected from one, two or more of the following substituents: deuterium, halogen group, cyano group, nitro group, -C(=O)R, -C(=O)OR', -OC(=O)R", imide group, amide 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, etc., but is not limited thereto.

[0191] The term "independently" means that at least two groups (or ring systems) present in a structure with the same or similar range of values may have the same or different meanings in specific circumstances. For example, if substituent X and substituent Y are independently hydrogen, halogen, hydroxyl, cyano, alkyl, or aryl, then when substituent X is hydrogen, substituent Y may be either hydrogen, or halogen, hydroxyl, cyano, alkyl, or aryl. Similarly, when substituent Y is hydrogen, substituent X may be either hydrogen, or halogen, hydroxyl, cyano, alkyl, or aryl.

[0192] The term "halogen" refers to fluorine (F), chlorine (Cl), bromine (Br) and iodine (I).

[0193] The term "alkyl" refers to a saturated aliphatic hydrocarbon group, including straight-chain or branched saturated hydrocarbon groups, having the indicated number of carbon atoms. 1-10 "Alkyl" refers to an alkyl group having 1 to 10 carbon atoms, including C1 alkyl, C2 alkyl, C3 alkyl, C4 alkyl, C5 alkyl, C6 alkyl, C7 alkyl, C8 alkyl, C9 alkyl, C 10 Alkyl groups, examples include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-pentyl, 2-pentyl, 3-pentyl, n-hexyl, 2-hexyl, 3-hexyl, etc. Alkyl groups can be optionally substituted or unsubstituted.

[0194] The term "alkylene" refers to a straight or branched divalent saturated aliphatic hydrocarbon group, and the two groups (or fragments) connected thereto may be connected to the same carbon atom or to different carbon atoms. For example, the term "C 1-10The term "alkylene" refers to an alkylene group having 1 to 10 carbon atoms (eg, methylene, 1,1-ethylene, 1,2-ethylene, 1,2-propylene, 1,3-butylene, etc.). The alkylene group may be optionally substituted or unsubstituted.

[0195] The term "alkenyl" refers to a monovalent straight or branched alkane group consisting only of carbon atoms and hydrogen atoms, containing at least one double bond, and connected to other fragments by a single bond, including (but not limited to) vinyl, propenyl, allyl, isopropenyl, butenyl and isobutenyl groups. For example, "C 2-30 "Alkenyl" refers to a monovalent straight or branched chain hydrocarbon radical containing from 2 to 30 carbon atoms and having at least one carbon-carbon double bond. Alkenyl groups may be optionally substituted or unsubstituted.

[0196] The term "alkenylene" refers to a divalent straight or branched alkane group consisting only of carbon atoms and hydrogen atoms, containing at least one double bond, and connected to other fragments by two single bonds, including (but not limited to) vinylene, etc. For example, "C 2-10 "Alkenylene" refers to a divalent straight or branched chain hydrocarbon radical containing from 2 to 10 carbon atoms and having at least one carbon-carbon double bond. Alkenylene may be optionally substituted or unsubstituted.

[0197] The term "alkynyl" refers to a monovalent straight or branched alkane group consisting only of carbon atoms and hydrogen atoms, containing at least one carbon-carbon triple bond, and connected to other fragments by a single bond, including (but not limited to) ethynyl, propynyl, butynyl and pentynyl groups. For example, "C 2-30 "Alkynyl" refers to a monovalent straight or branched chain hydrocarbon radical containing from 2 to 30 carbon atoms and having at least one carbon-carbon triple bond. Alkynyl groups may be optionally substituted or unsubstituted.

[0198] The term "cycloalkyl" refers to a saturated or partially saturated, monocyclic or polycyclic (such as bicyclic, for example, fused, bridged or spiro) non-aromatic hydrocarbon group. 3-8 "Cycloalkyl" refers to a cyclic alkyl group having 3 to 8 carbon atoms. Examples include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl, etc. Cycloalkyl groups may be optionally substituted or unsubstituted.

[0199] The term "heterocyclyl" refers to a saturated or partially saturated, monocyclic or polycyclic (such as bicyclic, for example, fused, bridged or spiro) non-aromatic group, the ring atoms of which are composed of carbon atoms 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 the valence bond requirements are met, the heterocyclic group can be attached to the rest of the molecule through any one of the ring atoms. For example, the term "5-8 membered heterocyclic group" refers to a heterocyclic group having 5 to 8 ring atoms. Common heterocyclic groups include, but are not limited to, oxiranyl, aziridine, azetidinyl, oxetanyl, tetrahydrofuranyl, dioxolyl, pyrrolidinyl, pyrrolidonyl, imidazolidinyl, pyrazolidinyl, tetrahydropyranyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl, dithianyl, or trithianyl. The heterocyclic groups in the present invention are optionally substituted with one or more substituents described herein.

[0200] The term "aryl" refers to a monocyclic or fused polycyclic aromatic hydrocarbon group having a conjugated π electron system. For example, the term "C 6-10 "Aryl" refers to an aromatic group having 6 to 10 carbon atoms. Common aromatic groups include (but are not limited to) phenyl, naphthyl, anthracenyl, phenanthrenyl, acenaphthenyl, azulenyl, fluorenyl, indenyl, pyrenyl, etc. The aromatic group in the present invention is optionally substituted by one or more substituents described in the present invention.

[0201] The term "heteroaryl" refers to a monocyclic or fused polycyclic aromatic group having a conjugated π electron system, wherein the ring atoms consist of carbon atoms and at least one heteroatom selected from N, O, and S. A heteroaryl group may be attached to the rest of the molecule via any ring atom if valence requirements are met. 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, pyridinyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl and their benzo derivatives, pyrrolopyridinyl, pyrrolopyrazinyl, pyrazolopyridinyl, imidazopyridinyl, pyrrolopyrimidinyl, pyrazolopyrimidinyl, purinyl, and the like. The heteroaryl groups of the present invention are optionally substituted with one or more substituents as described herein (e.g., halogen, C 1-6 alkyl, etc.) substituted.

[0202] The term "pharmaceutically acceptable excipient" refers to an excipient that is administered with the nucleic acid-lipid nanoparticle composition and is suitable, within the scope of sound medical judgment, for contact with human and / or other animal tissues without excessive toxicity, irritation, allergic reaction, or other problems or complications commensurate with a reasonable benefit / risk ratio. Examples include, but are not limited to, carriers, diluents, binders, absorbents, colorants, adjuvants, excipients, preservatives, fillers, disintegrants, wetting agents, emulsifiers, suspending agents, sweeteners, flavoring agents, fragrances, antibacterial agents, antifungal agents, lubricants, dispersants, temperature-sensitive materials, temperature regulators, adhesives, stabilizers, suspending agents, and the like.

[0203] The tocopherols and tocotrienols described herein are all natural tocopherols and tocotrienols, and the amino acids described herein are all natural amino acids.

[0204] II. Specific Examples

[0205] The present invention is described in detail below through examples. These examples are only used to further illustrate the present invention and cannot be understood as limiting the scope of protection of the present invention. Some non-essential improvements and adjustments made by those skilled in the art based on the contents of the above invention are all within the scope of protection of the present invention.

[0206] The reagents and instruments used in the examples are all commercially available conventional products. If no specific conditions are specified, the experiments were carried out under conventional conditions or the conditions recommended by the manufacturer. The term "room temperature" as used in the present invention refers to 20°C ± 5°C. When used to modify a certain numerical value or numerical range, the term "about" as used in the present invention refers to the numerical value or numerical range and the acceptable error range for those skilled in the art for the numerical value or numerical range, for example, the error range is ±10%, ±5%, ±4%, ±3%, ±2%, ±1%, ±0.5%, etc.

[0207] The experimental methods described in the following examples are conventional methods unless otherwise specified; the reagents and materials are commercially available unless otherwise specified.

[0208] The abbreviations used in this document have the following meanings:

[0209]

[0210] Example 1: Synthesis of Compound I-1

[0211]

[0212] Compound 1 (α-tocopherol, 1.0 eq) and compound 2 (1.0 eq) were dissolved in DCM, and compound 3 (2.0 eq) was slowly added at 0 ° C. and stirred at room temperature for 12 h. After the reaction was completed, compound 4 was precipitated with ether to obtain compound 4 (yield: 69%). Compound 5 (n = 45, purchased from Anaiji) (1.0 eq) was dissolved in DMF, DMAP and compound 6 (1.2 eq) were added, and heated and stirred at 40 ° C for 12 h. After the reaction was completed, the mixture was washed with water and precipitated with ether to obtain compound 7 (yield: 90%). Compound 4 (1.0 eq) and compound 7 (1.0 eq) were dissolved in DCM, EDC (2.0 eq) and DMAP (0.1 eq) were added, and stirred at room temperature for 12 h. After the reaction was completed, the mixture was washed with water and precipitated with ether to obtain compound I-1 (yield: 88%). 1H NMR (400MHz, CDCl3, 293K) 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 HRMS m / z: Calculated value: 3200.9988(M+H + ), measured value: 3200.9982.

[0213] Example 2: Synthesis of Compound I-2

[0214]

[0215] Compound 1 (α-tocopherol, 1.0 eq) and compound 2 (1.0 eq) were dissolved in DCM, compound 9 (1.0 eq) was added, and the mixture was stirred at room temperature for 12 h. After completion of the reaction, the mixture was purified by column chromatography (DCM:MeOH = 15:1) to obtain compound 10 (yield: 75%). Compound 11 (α-tocotrienol, 1.0 eq) was dissolved in DMF, DMAP and compound 6 (1.2 eq) were added, and the mixture was heated and stirred at 40°C for 12 h. After completion of the reaction, the mixture was washed with water and precipitated with diethyl ether to obtain compound 12 (yield: 90%). Compound 10 (1.0 eq) and compound 12 (1.0 eq) were dissolved in DCM, followed by the addition of EDC (2.0 eq) and DMAP (0.1 eq), and the mixture was stirred at room temperature for 12 h. After completion of the reaction, the mixture was purified by column chromatography (DCM:MeOH = 20:1) to obtain compound 13 (yield: 56%). Compound 13 (1.0 eq) and compound 7 (n=45) (1.0 eq) were dissolved in DCM, followed by the addition of EDC (2.0 eq) and DMAP (0.1 eq), and stirred at room temperature for 12 h. After the reaction was completed, the mixture was washed with water and precipitated with ether to obtain compound I-2 (yield: 77%). 1H NMR (400 MHz, CDCl3, 293 K) δ (ppm): 5.21-5.13 (m, 3H), 4.98-4.91 (m, 1H), 4.53-4.42 (m, 2H), 4.27-4.13 (m, 2H), 3.76-3.55 (m, 180H), 3.36 (s, 3H), 2.88-2.58 (m, 12H), 2.25-2.23 (s, 6H), 2.21-2.18 (s, 6H), 2.15-2.13 (s, 6H), 2.11-0.98 (m, 55H), 0.86-0.80 (m, 12H). HRMS: m / z calcd: 3293.9849 (M+H + ), measured value: 3293.9852.

[0216] Example 3: Synthesis of Compound I-3

[0217]

[0218] Compound 1 (α-tocopherol, 1.0 eq) was dissolved in DMF, and DMAP and compound 6 (1.2 eq) were added. The mixture was heated and stirred at 40°C for 12 h. After completion of the reaction, the mixture was washed with water and precipitated with diethyl ether to obtain compound 15 (yield: 90%). Compound 15 (1.0 eq) and compound 16 (1.0 eq) were dissolved in DCM, followed by the addition of EDC (2.0 eq) and DMAP (0.1 eq). The mixture was stirred at room temperature for 12 h. After completion of the reaction, the mixture was purified by column chromatography (DCM:MeOH = 15:1) to obtain compound 17 (yield: 64%). Compound 17 (1.0 eq) was dissolved in DCM, and compound 3 (1.0 eq) was slowly added dropwise. Compound 2 (1.0 eq) and TEA (1.2 eq) were then added. The mixture was stirred at room temperature for 12 h. After completion of the reaction, the mixture was purified by column chromatography (DCM:MeOH = 10:1) to obtain compound 18 (yield: 48%). Compound 18 (1.0 eq) and compound 19 (n = 45, purchased from Aladdin) (1.0 eq) were dissolved in DCM, followed by the addition of EDC (2.0 eq) and DMAP (0.1 eq), stirred at room temperature for 12 h, washed with water after completion of the reaction, and precipitated with diethyl ether to obtain compound 20 (yield: 77%). Compound 20 (1.0 eq) and compound 21 (1.0 eq) were dissolved in DCM, followed by the addition of EDC (2.0 eq) and DMAP (0.1 eq), stirred at room temperature for 12 h, washed with water after completion of the reaction, and precipitated with diethyl ether to obtain compound 22 (yield: 89%). Compound 22 (1.0 eq) and compound 23 (1.0 eq) were dissolved in DCM, followed by the addition of EDC (2.0 eq) and DMAP (0.1 eq), stirred at room temperature for 12 h, washed with water after completion of the reaction, and precipitated with diethyl ether to obtain compound I-3 (yield: 86%). 1 H NMR (400 MHz, CDCl3, 293 K) δ (ppm): 5.08-5.05 (m, 1H), 4.61-4.33 (m, 6H), 4.12-4.10 (m, 2H), 3.99-3.95 (m, 2H), 3.85-3.51 (m, 180H), 3.21-3.17 (m, 2H), 2.95-2.64 (m, 8H), 2.45-2.37 (m, 4H), 2.23 (s, 3H), 2.20 (s, 3H), 2.16 (s, 3H), 1.98-1.93 (m, 2H), 1.81-0.99 (m, 61H), 0.93-0.83 (m, 15H). HRMS: m / z calcd: 3289.9264 (M+H + ), measured value: 3289.9255.

[0219] Example 4: Synthesis of Compound I-4

[0220]

[0221] Compound 11 (α-tocotrienol, 1.0 eq) and compound 9 (1.0 eq) were dissolved in DCM, TEA (1.2 eq) was added, and the mixture was stirred at room temperature for 3 h. Compound 25 (1.0 eq) was then added, and the reaction was washed with water to give compound 26 (yield: 91%). Compound 26 (1.0 eq) and compound 2 (1.0 eq) were dissolved in DCM, EDC (2.0 eq) and DMAP (0.1 eq) were added, and the mixture was stirred at room temperature for 8 h. After the reaction was completed, compound 27 (yield: 53%) was obtained by column chromatography purification (DCM: MeOH = 10: 1). Compound 27 (1.0 eq) and compound 28 (1.0 eq) were dissolved in DCM, reacted at room temperature for 12 h, and compound 29 (n = 45, purchased from Anaiji) (1.0 eq) was subsequently added. The reaction was continued for 12 h. After the reaction was completed, the mixture was washed with water to give compound 30 (yield: 67%). Compound 30 (1.0 eq) and compound 31 (1.0 eq) were dissolved in DMF, followed by the addition of EDC (2.0 eq) and DMAP (0.1 eq). The mixture was stirred at room temperature for 8 h. After the reaction was completed, the mixture was washed with water to obtain compound I-4 (yield: 82%). 1 H NMR (400MHz, CDCl3, 293K) δ (ppm): 5.53-5.45 (m, 6H), 5.21-4.98 (m, 5H), 4.46-4.23 (m, 11H), 3.85-3.71 (m, 180H), 3.63-3.48 (m, 4H), 2.87-2.75 ( m,6H),2.43-2.30(m,6H),2.23(s,3H),2.17(s,3H),2.13(s,3H),2.11-1 .81(m,18H),1.70-1.52(m,21H),1.35-1.27(m,64H),0.96-0.81(m,15H). HRMS: m / z calculated value: 3714.4014 (M+H + ), measured value: 3714.4010.

[0222] Example 5: Synthesis of Compound I-5

[0223]

[0224]

[0225] Compound 1 (α-tocopherol, 1.0 eq) and compound 33 (1.0 eq) were dissolved in DCM, and compound 3 (1.0 eq) was slowly added at 0°C. The mixture was stirred at room temperature for 12 h. After completion of the reaction, the mixture was washed with water and precipitated with diethyl ether to obtain compound 34 (yield: 87%). Compound 34 (1.0 eq) and compound 2 (1.0 eq) were dissolved in DCM, and compound 3 (1.0 eq) was slowly added at 0°C. The mixture was stirred at room temperature for 12 h. After completion of the reaction, the mixture was washed with water and purified by column chromatography (DCM:MeOH = 10:1) to obtain compound 35 (yield: 45%). Compound 35 (1.0 eq) and compound 36 (1.0 eq) were dissolved in DCM, and EDC (2.0 eq) and DMAP (0.1 eq) were added. The mixture was stirred at room temperature for 12 h. After completion of the reaction, the mixture was washed with water and precipitated with diethyl ether to obtain compound 37 (yield: 61%). Compound 37 (1.0 eq) was dissolved in DMSO, sodium hydride (3.0 eq) was added in an ice bath and stirred for 10 min. The mixture was heated to room temperature and compound 38 (1.0 eq) was added. The mixture was stirred at room temperature for 6 h. After the reaction was completed, the mixture was purified by column chromatography (DCM:MeOH=15:1) to obtain compound 39 (yield: 67%). Compound 39 (1.0 eq) and compound 40 (n=45, purchased from Anaiji) (1.0 eq) were dissolved in acetonitrile, CuSO4·5H2O (0.01 eq) and sodium ascorbate (0.1 eq) were added, and the mixture was stirred at room temperature for 12 h. After the reaction was completed, the mixture was purified by column chromatography (DCM:MeOH=10:1) and precipitated with diethyl ether to obtain compound 41 (yield: 54%). Compound 41 (1.0 eq) and compound 42 (1.0 eq) were dissolved in DCM, and EDC (2.0 eq) and DMAP (0.1 eq) were added. The mixture was stirred at room temperature for 12 h. After the reaction was completed, the mixture was washed with water and precipitated with ether to obtain compound I-5 (yield: 81%). 1 H NMR (400 MHz, CDCl3, 293 K) δ (ppm): 7.83 (s, 1H), 5.02-4.90 (m, 2H), 4.64-4.37 (m, 8H), 4.28-4.19 (m, 5H), 4.15-4.08 (m, 1H), 3.94-3.78 (m, 190H), 2.80-2.71 (m, 2H), 2.63-2.54 (t, 2H), 2.31 (s, 3H), 2.20 (s, 3H), 2.18 (s, 3H), 1.88-1.01 (m, 46H), 0.95-0.81 (m, 15H). HRMS: m / z calcd: 3198.9142 (M+H + ), measured value: 3198.9146.

[0226] Example 6: Synthesis of Compound II-1

[0227]

[0228] Compound 44 (1.0 eq) and compound 15 (2.0 eq) were dissolved in DCM, EDC (4.0 eq) and DMAP (0.2 eq) were added, and the mixture was stirred at room temperature for 12 h. After completion of the reaction, the mixture was washed with water and purified by column chromatography (DCM:ethyl acetate = 5:1) to give compound 45 (yield: 79%). Compound 7 (n = 45, purchased from TCI) (1.0 eq) and compound 45 (1.0 eq) were dissolved in DCM, EDC (2.0 eq) and DMAP (0.1 eq) were added, and the mixture was stirred at room temperature for 12 h. After completion of the reaction, the mixture was washed with water and precipitated with diethyl ether to give compound II-1 (yield: 88%). 1 H NMR (400 MHz, CDCl3, 293 K) δ (ppm): 5.23-5.14 (m, 6H), 4.41-4.33 (t, 2H), 4.24-4.15 (m, 4H), 4.08-3.99 (m, 1H), 3.85-3.52 (m, 180H), 3.45 (s, 3H), 2.25 (s, 3H), 2.19 (s, 3H), 2.16 (s, 3H), 2.14-1.78 (m, 28H), 1.59-1.48 (m, 24H), 1.35 (s, 6H). HRMS: m / z calcd: 3258.1980 (M+H + ), measured value: 3258.1977.

[0229] Example 7: Synthesis of Compound II-2

[0230]

[0231]

[0232] Compound 15 (1.0 eq) was dissolved in DCM, and NHS (1.0 eq), EDC (2.0 eq), and DMAP (0.1 eq) were added. The mixture was stirred at room temperature for 8 h, followed by the addition of TEA (1.2 eq) and compound 45 (1.0 eq). Stirring at room temperature continued for 12 h. After completion of the reaction, the mixture was purified by column chromatography (DCM:MeOH = 10:1) to obtain compound 48 (yield: 47%). Compound 12 (1.0 eq) and compound 49 (1.0 eq) were dissolved in DCM, and EDC (2.0 eq) and DMAP (0.1 eq) were added. The mixture was stirred at room temperature for 8 h. After completion of the reaction, the mixture was washed with water to obtain compound 50 (yield: 69%). Compound 50 (1.0 eq) and compound 3 (1.0 eq) were dissolved in DCM, TEA (1.2 eq) was added, and the mixture was stirred at room temperature for 3 h. Compound 48 (1.0 eq) and potassium carbonate (1.0 eq) were added, and stirring continued for 6 h. After completion of the reaction, the mixture was purified by column chromatography (DCM:MeOH = 15:1) to obtain compound 51 (yield: 71%). Compound 51 (1.0 eq) and compound 6 (1.0 eq) were dissolved in DCM, DMAP (0.1 eq) was added, and the mixture was stirred at room temperature for 12 h. After completion of the reaction, the mixture was washed with water to obtain compound 52 (yield: 90%). Compound 52 (1.0 eq) and compound 53 (1.0 eq) were dissolved in DCM, EDC (2.0 eq) and DMAP (0.1 eq) were added, and the mixture was stirred at room temperature for 8 h. After completion of the reaction, the mixture was washed with water and purified by column chromatography (DCM:MeOH = 15:1) to obtain compound 54 (yield: 87%). Compound 54 (1.0 eq) was dissolved in DCM, and NHS (1.0 eq), EDC (2.0 eq) and DMAP (0.1 eq) were added, and the mixture was stirred at room temperature for 8 h. Subsequently, TEA (1.2 eq) and compound 55 (n=45, purchased from Myrel) (1.0 eq) were added, and stirring was continued at room temperature for 24 h. After the reaction was completed, the mixture was precipitated with diethyl ether to obtain compound II-2 (yield: 82%). 1 H NMR (400 MHz, CDCl3, 293 K) δ (ppm): 6.08-5.76 (m, 4H), 5.24-5.15 (m, 3H), 4.57-3.99 (m, 9H), 3.93-3.54 (m, 188H), 3.38-3.34 (m, 2H), 3.17-3.14 (m, 2H), 2.98-2.64 (m, 16H), 2.57-2.38 (m, 4H), 2.25 (s, 3H), 2.22 (s, 3H), 2.16 (s, 3H), 2.13-1.19 (m, 60H), 0.89-0.78 (m, 12H). HRMS: m / z calcd: 3673.1679 (M+Na + ), measured value: 3673.1683.

[0233] Example 8: Synthesis of Compound II-3

[0234]

[0235]

[0236] Compound 12 (1.0 eq) was dissolved in DMF, and NHS (1.0 eq), EDC (2.0 eq), and DMAP (0.1 eq) were added. The mixture was stirred at room temperature for 8 h, followed by the addition of TEA (1.2 eq) and compound 57 (1.0 eq). Stirring at room temperature was continued for 12 h. After completion of the reaction, the mixture was purified by column chromatography (DCM:MeOH=20:1) to obtain compound 58 (yield: 85%). Compound 58 (1.0 eq) and compound 44 (1.0 eq) were dissolved in DCM, and EDC (2.0 eq) and DMAP (0.1 eq) were added. The mixture was stirred at room temperature for 8 h. After completion of the reaction, the mixture was purified by column chromatography (DCM:MeOH=20:1) to obtain compound 59 (yield: 60%). Compound 60 (1.0 eq) and compound 28 (1.0 eq) were dissolved in DCM and stirred at room temperature for 8 h. Compound 59 (1.0 eq) was then added and stirred for another 8 h. After completion of the reaction, the mixture was washed with water and precipitated with diethyl ether to obtain compound 61 (yield: 91%). Compound 7 (n=45) (1.0 eq) was dissolved in DMF, EDC (2.0 eq) and DMAP (0.1 eq) were added, and stirred at room temperature for 8 h. Compound 61 (1.0 eq) was then added and stirred for another 12 h. After completion of the reaction, the mixture was washed with water and precipitated with diethyl ether to obtain compound II-3 (yield: 83%). 1 H NMR (400MHz, CDCl3, 293K) δ (ppm): 5.28-4.99 (m, 4H), 4.55-4.08 (m, 14H), 3 .98-3.63(m,180H),3.42-3.38(s,3H),2.95-2.65(m,10H),2.48-2.37(m,4H ),2.21(s,3H),2.19(s,3H),2.16(s,3H),2.14-1.80(m,12H),1.68-1.53(m, 16H),1.38-1.27(m,60H),0.98-0.86(m,6H).HRMS:m / z calculated value:3611.2247(M+Na + ), measured value: 3611.2239.

[0237] Example 9: Synthesis of Compound II-4

[0238]

[0239]

[0240] Compound 63 (1.0 eq) was dissolved in DCM, and NHS (1.0 eq), EDC (2.0 eq), and DMAP (0.1 eq) were added. The mixture was stirred at room temperature for 8 h, followed by the addition of TEA (1.2 eq) and compound 44 (1.0 eq). Stirring at room temperature continued for 12 h. After completion of the reaction, the mixture was purified by column chromatography (DCM:MeOH = 15:1) to obtain compound 64 (yield: 42%). Compound 11 (α-tocotrienol, 1.0 eq) and compound 65 (1.0 eq) were dissolved in DCM, and EDC (2.0 eq) and DMAP (0.1 eq) were added. The mixture was stirred at room temperature for 8 h. After completion of the reaction, the mixture was washed with water and purified by column chromatography (DCM:MeOH = 15:1) to obtain compound 66 (yield: 87%). Compound 66 (1.0 eq) and compound 9 (1.0 eq) were dissolved in DCM and stirred at room temperature for 8 h. TEA (1.2 eq) and compound 64 (1.0 eq) were then added, and the mixture was heated at 40°C and stirred for 12 h. After completion of the reaction, the mixture was purified by column chromatography (DCM:MeOH = 20:1) to obtain compound 67 (yield: 69%). Compound 68 (1.0 eq) was dissolved in DMSO, and sodium hydride (3.0 eq) was added in an ice bath and stirred for 10 min. The mixture was then heated to room temperature and compound 37 (1.0 eq) was added. The mixture was stirred at room temperature for 6 h. After completion of the reaction, the mixture was purified by column chromatography (DCM:MeOH = 20:1) to obtain compound 69 (yield: 83%). Compound 69 (1.0 eq) and compound 70 (n = 45, purchased from Anaiji) (1.0 eq) were dissolved in acetonitrile, and CuSO4 5H2O (0.01 eq) and sodium ascorbate (0.1 eq) were added. The mixture was stirred at room temperature for 12 h. After completion of the reaction, it was purified by column chromatography (DCM: MeOH = 20: 1) and precipitated with diethyl ether to obtain compound 71 (yield: 67%). Compound 71 (1.0 eq) and compound 67 (1.0 eq) were dissolved in DCM, EDC (2.0 eq) and DMAP (0.1 eq) were added, and the mixture was stirred at room temperature for 8 h. After completion of the reaction, it was precipitated with diethyl ether to obtain compound II-4 (yield: 81%). 1 H NMR (400 MHz, CDCl3, 293 K) δ (ppm): 7.78 (s, 1H), 5.18-5.15 (m, 3H), 4.71-4.61 (m, 2H), 4.48-4.44 (m, 2H), 4.23-3.98 (m, 7H), 3.95-3.55 (m, 180H), 3.37 (s, 3H), 2.77-2.59 (m, 4H), 2.24-1.53 (m, 43H), 1.43-1.23 (m, 17H), 0.91-0.86 (m, 3H). HRMS: m / z calcd: 3039.8568 (M+Na+ ), measured value: 3039.8563.

[0241] Example 10: Synthesis of Compound II-5

[0242]

[0243]

[0244] Compound 57 (1.0 eq) was dissolved in DMF, and NHS (1.0 eq), EDC (2.0 eq), and DMAP (0.1 eq) were added. The mixture was stirred at room temperature for 8 h, followed by the addition of TEA (1.2 eq) and compound 73 (1.0 eq). Stirring at room temperature was continued for 12 h. After completion of the reaction, the mixture was purified by column chromatography (DCM:MeOH=20:1) to obtain compound 74 (yield: 77%). Compound 74 (1.0 eq) was dissolved in DMF, and NHS (1.0 eq), EDC (2.0 eq), and DMAP (0.1 eq) were added. Stirring at room temperature for 8 h, followed by the addition of TEA (1.2 eq) and compound 44 (1.0 eq). Stirring at room temperature was continued for 12 h. After completion of the reaction, the mixture was purified by column chromatography (DCM:MeOH=15:1) to obtain compound 75 (yield: 43%). Compound 76 (1.0 eq) was dissolved in DMF, and NHS (1.0 eq), EDC (2.0 eq), and DMAP (0.1 eq) were added. The mixture was stirred at room temperature for 8 h, followed by the addition of TEA (1.2 eq) and compound 1 (α-tocopherol, 1.0 eq). Stirring was continued at room temperature for 12 h. After completion of the reaction, the mixture was purified by column chromatography (DCM:MeOH=20:1) to obtain compound 77 (yield: 89%). Compound 77 (1.0 eq) and compound 9 (1.0 eq) were dissolved in DCM and stirred at room temperature for 12 h. Compound 75 (1.0 eq) was then added. After completion of the reaction, the mixture was purified by column chromatography (DCM:MeOH=20:1) to obtain compound 78 (yield: 61%). Compound 79 (1.0 eq) and compound 9 (1.0 eq) were dissolved in DCM and stirred at room temperature for 12 h, followed by the addition of compound 80 (n=45, purchased from Aladdin) (1.0 eq). After the reaction was complete, the mixture was precipitated with diethyl ether to obtain compound 81 (yield: 76%). Compound 78 (1.0 eq) and compound 9 (1.0 eq) were dissolved in DCM and stirred at room temperature for 12 h, followed by the addition of compound 81 (1.0 eq). After the reaction was complete, the mixture was precipitated with diethyl ether to obtain compound II-5 (yield: 80%). 1H NMR (400MHz, CDCl3, 293K) δ (ppm): 5.88-5.75 (m, 4H), 5.61-5.53 (m, 4H), 4.43-4.38 (s, 2H ),4.35-4.12(m,7H),4.08-4.01(m,1H),3.75-3.24(m,186H),3.19-3.16(m,2H),2.99-2. 85 (m, 2H), 2.79-2.67 (m, 4H), 2.34-2.23 (m, 11H), 2.09-2.12 (m, 6H), 1.98-1.89 (m, 1H), 1.80-1.50 (m, 13H), 1.46-1.32 (m, 40H), 0.95-0.73 (m, 15H). HRMS: m / z calculated value: 3228.0345 (M+Na + ), measured value: 3228.0341.

[0245] Example 11: Synthesis of Compound III-1

[0246]

[0247]

[0248] Compound 79 (1.0 eq) and compound 15 (2.0 eq) were dissolved in DCM, EDC (4.0 eq) and DMAP (0.2 eq) were added, and the mixture was stirred at room temperature for 12 h. After completion of the reaction, the mixture was washed with water and purified by column chromatography (DCM:MeOH = 20:1) to give compound 80 (yield: 69%). Compound 7 (n = 45) (1.0 eq) and compound 80 (1.0 eq) were dissolved in DCM, EDC (2.0 eq) and DMAP (0.1 eq) were added, and the mixture was stirred at room temperature for 12 h. After completion of the reaction, the mixture was washed with water and precipitated with diethyl ether to give compound III-1 (yield: 82%). 1 H NMR (400 MHz, CDCl3, 293 K) δ (ppm): 4.32-4.27 (m, 2H), 4.15-4.02 (m, 6H), 3.85-3.48 (m, 180H), 3.38 (s, 3H), 2.87-2.39 (m, 17H), 2.21 (s, 3H), 2.19 (s, 3H), 2.15 (s, 3H), 1.87-0.99 (m, 51H), 0.88-0.77 (m, 24H). HRMS: m / z calcd: 3293.0637 (M+Na + ), measured value: 3293.0633.

[0249] Example 12: Synthesis of Compound III-2

[0250]

[0251] Compound 11 (α-tocotrienol, 1.0 eq) and compound 82 (1.0 eq) were dissolved in DCM, TEA (1.2 eq) was added, and the mixture was stirred at room temperature for 3 h. Compound 83 (1.0 eq) and TEA (1.2 eq) were then added, and the mixture was stirred at room temperature for 6 h. After completion of the reaction, the mixture was washed with water and purified by column chromatography (DCM:MeOH=20:1) to obtain compound 84 (yield: 57%). Compound 84 (2.0 eq) and compound 82 (2.0 eq) were dissolved in DCM, TEA (2.4 eq) was added, and the mixture was stirred at room temperature for 3 h. Compound 79 (1.0 eq) and TEA (2.4 eq) were then added, and the mixture was stirred at room temperature for 6 h. After completion of the reaction, the mixture was washed with water and purified by column chromatography (DCM:MeOH=15:1) to obtain compound 85 (yield: 68%). Compound 85 (1.0 eq) was dissolved in DMF, and NHS (1.0 eq), EDC (2.0 eq) and DMAP (0.1 eq) were added, and the mixture was stirred at room temperature for 8 h. Subsequently, TEA (1.2 eq) and compound 86 (1.0 eq) were added, and stirring was continued at room temperature for 12 h. Finally, EDC (2.0 eq), DMAP (0.1 eq) and compound 87 (n=45, purchased from Anaiji) (1.0 eq) were added, and stirring was continued at room temperature for 12 h. After the reaction was completed, the mixture was precipitated with ether to obtain compound III-2 (yield: 63%). 1 H NMR (400 MHz, CDCl3, 293 K) δ (ppm): 5.17-5.04 (m, 8H), 4.13-3.87 (m, 14H), 3.78-3.50 (m, 180H), 3.37 (s, 3H), 3.35-3.21 (m, 2H), 2.79-2.57 (m, 8H), 2.51-2.32 (m, 15H), 2.20 (s, 3H), 2.18 (s, 3H), 2.14 (s, 3H), 2.15-1.72 (m, 38H), 1.67-1.59 (m, 24H), 1.48-1.28 (m, 26H). HRMS: m / z calcd: 3771.3259 (M+H + ), measured value: 3771.3255.

[0252] Example 13: Synthesis of Compound III-3

[0253]

[0254] Compound 15 (1.0 eq) was dissolved in DMF, and NHS (1.0 eq), EDC (2.0 eq), and DMAP (0.1 eq) were added. The mixture was stirred at room temperature for 8 h, followed by the addition of TEA (1.2 eq) and compound 79 (1.0 eq). Stirring at room temperature was continued for 12 h. After completion of the reaction, the mixture was purified by column chromatography (DCM:MeOH=10:1) to obtain compound 89 (yield: 43%). Compound 90 (n=45, purchased from Myrel) (1.0 eq) was dissolved in DMF, and NHS (1.0 eq), EDC (2.0 eq), and DMAP (0.1 eq) were added. The mixture was stirred at room temperature for 8 h, followed by the addition of TEA (1.2 eq) and compound 91 (1.0 eq). Stirring at room temperature was continued for 12 h. After completion of the reaction, the mixture was precipitated with diethyl ether to obtain compound 92 (yield: 80%). Compound 92 (1.0 eq) was dissolved in DMF, and NHS (1.0 eq), EDC (2.0 eq), and DMAP (0.1 eq) were added. The mixture was stirred at room temperature for 8 h, followed by the addition of TEA (1.2 eq) and compound 89 (1.0 eq). The mixture was stirred at room temperature for 12 h. After completion of the reaction, the mixture was precipitated with diethyl ether to obtain compound 93 (yield: 63%). Compound 94 (1.0 eq) was dissolved in DCM, and NHS (1.0 eq), EDC (2.0 eq), and DMAP (0.1 eq) were added. The mixture was stirred at room temperature for 8 h, followed by the addition of TEA (1.2 eq) and compound 93 (1.0 eq). The mixture was stirred at room temperature for 12 h. After completion of the reaction, the mixture was precipitated with diethyl ether to obtain compound 95 (yield: 83%). Compound 95 (1.0 eq) and compound 96 (1.0 eq) were dissolved in DCM, EDC (2.0 eq) and DMAP (0.1 eq) were added, and the mixture was stirred at room temperature for 8 h. After completion of the reaction, the mixture was precipitated with diethyl ether to obtain compound 97 (yield: 67%). Compound 97 (1.0 eq) and compound 98 (1.0 eq) were dissolved in DCM, EDC (2.0 eq) and DMAP (0.1 eq) were added, and the mixture was stirred at room temperature for 12 h. After completion of the reaction, the mixture was precipitated with diethyl ether to obtain compound III-3 (yield: 78%). 1H NMR (400 MHz, CDCl3, 293 K) δ (ppm): 7.58-7.47 (m, 1H), 6.95-6.83 (m, 1H), 4.85-4.76 (m, 1H), 4.40-4.31 (m, 2H), 4.19-3.92 (m, 8H), 3.85-3.42 (m, 192H), 3.18-3.02 (m, 2H), 2.91-2.24 (m, 18H), 2.20 (s, 3H), 2.18 (s, 3H), 2.16 (s, 3H), 1.97-1.88 (s, 1H), 1.80-1.01 (m, 62H), 0.93-0.80 (m, 15H). HRMS: m / z calcd: 3210.9090 (M+H + ), measured value: 3210.9088.

[0255] Example 14: Synthesis of Compound III-4

[0256]

[0257] Compound 11 (α-tocotrienol, 1.0 eq) and compound 100 (1.0 eq) were dissolved in DCM, and EDC (2.0 eq) and DMAP (0.1 eq) were added. The mixture was stirred at room temperature for 6 h. After completion, the reaction was purified by column chromatography (DCM:MeOH = 20:1) to obtain compound 101 (yield: 85%). Compound 101 (1.0 eq) and compound 9 (1.0 eq) were dissolved in DCM and stirred at room temperature for 8 h. Subsequently, TEA (1.2 eq) and compound 79 (1.0 eq) were added. The mixture was heated at 40°C and stirred for 12 h. After completion, the reaction was purified by column chromatography (DCM:MeOH = 10:1) to obtain compound 102 (yield: 52%). Compound 103 (1.0 eq) and compound 104 (1.0 eq) were dissolved in DMF, TEA (1.2 eq) was added, and the mixture was stirred at room temperature for 3 h. Compound 105 (n=45, purchased from TCI) (1.0 eq) and TEA (1.2 eq) were then added. Stirring at room temperature continued for 6 h. After completion of the reaction, the mixture was precipitated with diethyl ether to obtain compound 106 (yield: 78%). Compound 102 (1.0 eq) and compound 106 (1.0 eq) were dissolved in DCM, EDC (2.0 eq) and DMAP (0.1 eq) were added, and the mixture was stirred at room temperature for 8 h. After completion of the reaction, the mixture was washed with water and precipitated with diethyl ether to obtain compound 107 (yield: 60%). Compound 107 (1.0 eq) and compound 103 (1.0 eq) were dissolved in DMF, EDC (2.0 eq) and DMAP (0.1 eq) were added, and the mixture was stirred at room temperature for 8 h. After completion of the reaction, the mixture was precipitated with diethyl ether to obtain compound 108 (yield: 81%). Compound 109 (1.0 eq) was dissolved in DCM, and NHS (1.0 eq), EDC (2.0 eq) and DMAP (0.1 eq) were added, and the mixture was stirred at room temperature for 8 h. Subsequently, TEA (1.2 eq) and compound 108 (1.0 eq) were added, and stirring was continued at room temperature for 12 h. After the reaction was completed, the mixture was precipitated with diethyl ether to obtain compound III-4 (yield: 83%). 1 H NMR (400 MHz, CDCl3, 293 K) δ (ppm): 5.37-5.25 (m, 3H), 4.40-4.15 (m, 8H), 4.05-3.98 (m, 4H), 3.78-3.28 (m, 180H), 3.12 (s, 2H), 2.85-2.53 (m, 7H), 2.37-2.29 (t, 2H), 2.21 (s, 3H), 2.19 (s, 3H), 2.16 (s, 3H), 2.14-1.89 (m, 12H), 1.80-1.56 (m, 20H), 1.49-1.19 (m, 19H), 0.90-0.82 (m, 3H). HRMS: m / z calcd: 2925.7025 (M+H +), measured value: 2925.7020.

[0258] Example 15: Synthesis of Compound III-5

[0259]

[0260]

[0261] Compound 111 (1.0 eq) was dissolved in DMF, and NHS (1.0 eq), EDC (2.0 eq), and DMAP (0.1 eq) were added. The mixture was stirred at room temperature for 8 h, followed by the addition of TEA (1.2 eq) and compound 112 (1.0 eq). Stirring at room temperature continued for 12 h. After completion of the reaction, the mixture was purified by column chromatography (DCM:MeOH = 20:1) to obtain compound 113 (yield: 55%). Compound 79 (1.0 eq) and compound 113 (1.0 eq) were dissolved in DCM, and EDC (2.0 eq) and DMAP (0.1 eq) were added. The mixture was stirred at room temperature for 8 h. After completion of the reaction, the mixture was washed with water to obtain compound 114 (yield: 41%). Compound 1 (α-tocopherol, 1.0 eq) and compound 9 (1.0 eq) were dissolved in DCM and stirred at room temperature for 8 h. TEA (1.2 eq) and compound 33 (1.0 eq) were then added and stirred at 40°C for 12 h. After completion of the reaction, the mixture was purified by column chromatography (DCM:MeOH = 15:1) to obtain compound 115 (yield: 55%). Compound 115 (1.0 eq) and compound 3 (1.0 eq) were dissolved in DCM, TEA (1.2 eq) was added, and stirred at room temperature for 3 h. Compound 114 (1.0 eq) and TEA (1.2 eq) were then added and stirred at room temperature for 6 h. After completion of the reaction, the mixture was washed with water to obtain compound 116 (yield: 45%). Compound 116 (1.0 eq) and compound 117 (1.0 eq) were dissolved in DMF, EDC (2.0 eq) and DMAP (0.1 eq) were added, and the mixture was stirred at room temperature for 8 h. After completion of the reaction, the mixture was purified by column chromatography (DCM:PE = 10:1) to obtain compound 118 (yield: 75%). Compound 118 (1.0 eq) was dissolved in DMSO, sodium hydride (1.0 eq) was added in an ice bath, and the mixture was stirred for 10 min. The mixture was then heated to room temperature and compound 38 (1.0 eq) was added. The mixture was stirred at room temperature for 6 h. After completion of the reaction, the mixture was purified by column chromatography (DCM:PE = 8:1) to obtain compound 119 (yield: 63%). Compound 119 (1.0 eq) and compound 120 (n = 45, purchased from Aladdin) (1.0 eq) were dissolved in acetonitrile, and CuSO4·5H2O (0.01 eq) and sodium ascorbate (0.1 eq) were added. The mixture was stirred at room temperature for 12 h. After the reaction was completed, the mixture was precipitated with ether to obtain compound III-5 (yield: 75%). 1H NMR (400MHz, CDCl3, 293K) δ (ppm): 7.59 (s, 1H), 5.37-5.30 (m, 4H), 4.73 (s, 2H), 4.42-4.41 (m,4H),4.26-4.23(t,2H),4.20-4.07(m,7H),3.88-3.44(m,188H),3.14-3.10(m,2H),2.8 9-2.81 (m, 2H), 2.79-2.65 (m, 2H), 2.57-2.53 (m, 1H), 2.35-2.11 (m, 15H), 2.08-1.97 (m, 8H), 1.96-1.87 (m, 1H), 1.78-1.16 (m, 94H), 0.93-0.80 (m, 18H). HRMS: m / z calculated value: 3729.5045 (M+H + ), measured value: 3729.5022.

[0262] Example 16: Synthesis of Compound IV-1

[0263]

[0264] Compound 15 (1.0 eq) and compound 122 (1.0 eq) were dissolved in DCM, EDC (2.0 eq) and DMAP (0.1 eq) were added, and the mixture was stirred at room temperature for 12 h. After completion of the reaction, the mixture was washed with water and precipitated with diethyl ether to obtain compound 123 (yield: 55%). Compound 123 (1.0 eq) and compound 7 (n=45) (1.0 eq) were dissolved in DCM, EDC (2.0 eq) and DMAP (0.1 eq) were added, and the mixture was stirred at room temperature for 12 h. After completion of the reaction, the mixture was washed with water and precipitated with diethyl ether to obtain compound IV-1 (yield: 72%). 1 H NMR (400 MHz, CDCl3, 293 K) δ (ppm): 4.44-4.35 (m, 8H), 3.80-3.53 (m, 180H), 3.46 (s, 3H), 3.11-2.99 (t, 6H), 2.88-2.80 (t, 4H), 2.76-2.60 (m, 12H), 1.73-1.00 (m, 50H), 0.87-0.78 (m, 24H). HRMS: m / z calculated: 3314.1159 (M+H + ), measured value: 3314.1148.

[0265] Example 17: Synthesis of Compound IV-2

[0266]

[0267]

[0268] Compound 122 (1.0 eq) and compound 9 (1.0 eq) were dissolved in DCM, followed by the addition of TEA (1.0 eq), stirred at room temperature for 12 h, followed by the addition of compound 103 (1.0 eq), stirred for 12 h, and washed with water after the reaction was complete to give compound 125 (yield: 42%). Compound 12 (1.0 eq) was dissolved in DCM, NHS (1.0 eq), EDC (2.0 eq) and DMAP (0.1 eq) were added, stirred at room temperature for 8 h, followed by the addition of TEA (1.2 eq) and compound 125 (1.0 eq), stirred at room temperature for 12 h, and precipitated with ether after the reaction was complete. Compound 126 (n=45, purchased from Anaiji) (1.0 eq) was then added, dissolved in DCM, EDC (2.0 eq) and DMAP (0.1 eq) were added, stirred at room temperature for 8 h, washed with water after the reaction was complete, and precipitated with ether to give compound 127 (yield: 63%). Compound 1 (α-tocopherol, 1.0 eq) and compound 3 (1.0 eq) were dissolved in DCM and stirred at room temperature for 8 h. Subsequently, TEA (1.2 eq) and compound 127 (1.0 eq) were added, and the mixture was heated at 40°C and stirred for 12 h. After the reaction was completed, the mixture was precipitated with diethyl ether to obtain compound IV-2 (yield: 75%). 1 H NMR (400MHz, CDCl3, 293K) δ (ppm): 5.18-5.07 (m, 3H), 4.42-4.36 (t, 2H), 4.29-4.2 3(4H),4.12-4.11(d,2H),3.78-3.49(m,180H),3.40(s,3H),3.17-3.07(t,2H),3. 01-2.96 (m, 6H), 2.85-2.82 (m, 2H), 2.75-2.64 (m, 6H), 2.21 (s, 3H), 2.18 (s, 3H), 2.15 (s, 3H), 2.12-0.95 (m, 41H), 0.88-0.79 (m, 12H). HRMS: m / z calculated value: 3297.0100 (M+H + ), measured value: 3297.0098.

[0269] Example 18: Synthesis of Compound IV-3

[0270]

[0271] Compound 7 (n = 45) (1.0 eq) and compound 122 (1.0 eq) were dissolved in DCM, EDC (2.0 eq) and DMAP (0.1 eq) were added, and the mixture was stirred at room temperature for 8 h. After completion of the reaction, the mixture was washed with water to obtain compound 129 (yield: 42%). Compound 15 (1.0 eq) was dissolved in DCM, EDC (2.0 eq) and DMAP (0.1 eq) were added, and the mixture was stirred at room temperature for 8 h. Subsequently, TEA (1.2 eq) and compound 129 (1.0 eq) were added, and the mixture was stirred at room temperature for another 12 h. After completion of the reaction, the mixture was washed with water to obtain compound 130 (yield: 55%). Compound 111 (1.0 eq) was dissolved in DMF, EDC (2.0 eq) and DMAP (0.1 eq) were added, and the mixture was stirred at room temperature for 8 h. Subsequently, compound 130 (1.0 eq) was added, and the mixture was stirred at room temperature for another 12 h. After completion of the reaction, the mixture was washed with water and precipitated with diethyl ether to obtain compound IV-3 (yield: 78%). 1 H NMR (400 MHz, CDCl3, 293 K) δ (ppm): 5.39-5.35 (m, 2H), 4.31-4.25 (m, 8H), 3.87-3.50 (m, 180H), 3.38 (s, 3H), 3.06-3.01 (t, 6H), 2.89-2.63 (m, 10H), 2.33-2.27 (t, 2H), 2.21 (s, 3H), 2.19 (s, 3H), 2.12 (s, 3H), 2.01-1.86 (m, 5H), 1.71-0.96 (m, 47H), 0.91-0.91 (m, 15H). HRMS: m / z calcd: 3065.9746 (M+H + ), measured value: 3065.9755.

[0272] Example 19: Synthesis of Compound IV-4

[0273]

[0274] Compound 34 (1.0 eq) and compound 9 (1.0 eq) were dissolved in DCM, TEA (1.2 eq) was added, and the mixture was stirred at room temperature for 3 h. Compound 122 (1.0 eq) and TEA (1.2 eq) were then added, and stirring continued at room temperature for 6 h. After completion of the reaction, the mixture was purified by column chromatography (DCM:MeOH = 10:1) to obtain compound 132 (yield: 38%). Compound 65 (1.0 eq) was dissolved in DMSO, sodium hydride (1.0 eq) was added in an ice bath, and the mixture was stirred for 10 min. The mixture was heated to room temperature and compound 38 (1.0 eq) was added. The mixture was stirred at room temperature for 6 h. After completion of the reaction, the mixture was purified by column chromatography (DCM:MeOH = 20:1) to obtain compound 133 (yield: 69%). Compound 133 (1.0 eq) and compound 69 (n=45) (1.0 eq) were dissolved in acetonitrile, and CuSO4·5H2O (0.01 eq) and sodium ascorbate (0.1 eq) were added. The mixture was stirred at room temperature for 12 h. After completion of the reaction, the mixture was precipitated with diethyl ether to obtain compound 134 (yield: 68%). Compound 134 (1.0 eq) and compound 132 (1.0 eq) were dissolved in DCM, and EDC (2.0 eq) and DMAP (0.1 eq) were added. The mixture was stirred at room temperature for 8 h. After completion of the reaction, the mixture was washed with water to obtain compound 135 (yield: 46%). Compound 135 (1.0 eq) and compound 136 (1.0 eq) were dissolved in DCM, and EDC (2.0 eq) and DMAP (0.1 eq) were added. The mixture was stirred at room temperature for 8 h. After completion of the reaction, the mixture was washed with water to obtain compound IV-4 (yield: 78%). 1 H NMR (400MHz, CDCl3, 293K) δ (ppm): 7.73 (s, 1H), 4.65 (s, 2H), 4.53-4.47 (t, 2H), 4 .42-4.28(m,10H),3.91-3.90(t,2H),3.80-3.38(m,196H),3.05-2.97(m,6H),2.8 0-2.66 (m, 2H), 2.36-2.31 (t, 4H), 2.21 (s, 3H), 2.18 (s, 3H), 2.15 (s, 3H), 1.95-1.89 (m, 1H), 1.71-0.98 (m, 62H), 0.91-0.80 (m, 15H). HRMS: m / z calculated value: 3369.1541 (M+H + ), measured value: 3369.1534.

[0275] Example 20: Synthesis of Compound IV-5

[0276]

[0277] Compound 11 (α-tocotrienol, 1.0 eq) and compound 9 (1.0 eq) were dissolved in DCM and stirred at room temperature for 8 h. TEA (1.2 eq) and compound 138 (1.0 eq) were then added and stirred at room temperature for 12 h. After completion of the reaction, the mixture was purified by column chromatography (DCM:MeOH = 20:1) to obtain compound 139 (yield: 86%). Compound 139 (1.0 eq) and compound 3 (1.0 eq) were dissolved in DCM, TEA (1.2 eq) was added, and stirred at room temperature for 6 h. Compound 122 (1.0 eq) and TEA (1.2 eq) were then added and stirred at room temperature for 8 h. After completion of the reaction, the mixture was purified by column chromatography (DCM:MeOH = 10:1) to obtain compound 140 (yield: 43%). Compound 33 (1.0 eq) and compound 9 (1.0 eq) were dissolved in DCM and stirred at room temperature for 8 h. TEA (1.2 eq) and compound 87 (n=45) (1.0 eq) were then added. The mixture was heated at 40°C and stirred for 12 h. After completion, the mixture was precipitated with diethyl ether to yield compound 141 (yield: 92%). Compound 140 (1.0 eq) and compound 9 (1.0 eq) were dissolved in DCM and stirred at room temperature for 8 h. TEA (1.2 eq) and compound 141 (1.0 eq) were then added. The mixture was heated at 40°C and stirred for 12 h. After completion, the mixture was precipitated with diethyl ether to yield compound 142 (yield: 56%). Compound 142 (1.0 eq) was dissolved in DMSO, sodium hydride (1.0 eq) was added in an ice bath, and stirred for 10 min. The mixture was then heated to room temperature and compound 143 (1.0 eq) was added. The mixture was stirred at room temperature for 6 h. After completion, the mixture was extracted with DCM and precipitated with diethyl ether to yield compound IV-5 (yield: 77%). 1 H NMR (400 MHz, CDCl3, 293 K) δ (ppm): 5.18-5.07 (m, 3H), 4.44-4.32 (m, 6H), 4.27-4.20 (m, 4H), 3.79-3.3.22 (m, 192H), 3.04-2.93 (m, 8H), 2.79-2.65 (m, 2H), 2.23 (s, 3H), 2.21 (s, 3H), 2.15 (s, 3H), 2.14-1.48 (m, 32H), 1.41-1.17 (m, 47H), 0.92-0.86 (m, 3H). HRMS: m / z calcd: 3369.1251 (M+H + ), measured value: 3369.1260.

[0278] Example 21: Synthesis of Compound V-1

[0279]

[0280] Compound 1 (α-tocopherol, 2.0 eq) and compound 9 (2.0 eq) were dissolved in DCM and stirred at room temperature for 8 h. TEA (2.4 eq) and compound 145 (1.0 eq) were then added, and stirring was continued at 40°C for 12 h. After completion of the reaction, the mixture was purified by column chromatography (DCM:MeOH = 20:1) to obtain compound 146 (yield: 56%). Compound 146 (1.0 eq) and compound 7 (n = 45) (1.0 eq) were dissolved in DCM, and EDC (2.0 eq) and DMAP (0.1 eq) were added. The mixture was stirred at room temperature for 12 h. After completion of the reaction, the mixture was washed with water and precipitated with diethyl ether to obtain compound V-1 (yield: 77%). 1 H NMR (400 MHz, CDCl3, 293 K) δ (ppm): 4.35-4.30 (t, 2H), 3.82-3.50 (m, 180H), 3.38 (s, 3H), 3.35-3.28 (m, 6H), 2.83-2.61 (m, 14H), 2.21 (s, 6H), 2.17 (s, 6H), 2.12 (s, 6H), 1.95-1.88 (m, 2H), 1.69-0.98 (m, 50H), 0.89-0.80 (m, 24H). HRMS: m / z calculated: 3199.1114 (M+H + ), measured value: 3199.1109.

[0281] Example 22: Synthesis of Compound V-2

[0282]

[0283]

[0284] Compound 11 (α-tocotrienol, 1.0 eq) and compound 3 (1.0 eq) were dissolved in DCM, TEA (1.2 eq) was added, and the mixture was stirred at room temperature for 3 h. Compound 100 (1.0 eq) and TEA (1.2 eq) were then added, and stirring continued at room temperature for 6 h. After completion of the reaction, the mixture was washed with water and purified by column chromatography (DCM:MeOH=20:1) to obtain compound 148 (yield: 65%). Compound 148 (1.0 eq) was dissolved in DCM, NHS (1.0 eq), EDC (2.0 eq), and DMAP (0.1 eq) were added, and stirring continued at room temperature for 8 h. TEA (1.2 eq) and compound 145 (1.0 eq) were then added, and stirring continued at room temperature for 12 h. After completion of the reaction, the mixture was purified by column chromatography (DCM:MeOH=20:1) to obtain compound 149 (yield: 36%).

[0285] Compound 100 (1.0 eq) was dissolved in DCM, EDC (2.0 eq) and DMAP (0.1 eq) were added, and the mixture was stirred at room temperature for 8 h. Subsequently, TEA (1.2 eq) and compound 150 (n=45) (1.0 eq) were added, and stirring at room temperature was continued for 12 h. After completion of the reaction, the mixture was precipitated with diethyl ether to obtain compound 151 (yield: 85%). Compound 151 (1.0 eq) and compound 11 (1.0 eq) were dissolved in DCM, NHS (1.0 eq), EDC (2.0 eq) and DMAP (0.1 eq) were added, and stirring was continued at room temperature for 8 h. Subsequently, TEA (1.2 eq) and compound 149 (1.0 eq) were added, and stirring was continued at room temperature for 12 h. After completion of the reaction, the mixture was precipitated with diethyl ether to obtain compound 152 (yield: 56%). Compound 100 (1.0 eq) was dissolved in DCM, and NHS (1.0 eq), EDC (2.0 eq), and DMAP (0.1 eq) were added. The mixture was stirred at room temperature for 8 h, followed by the addition of TEA (1.2 eq) and compound 152 (1.0 eq). Stirring at room temperature was continued for 12 h. After completion of the reaction, the mixture was precipitated with diethyl ether to obtain compound 153 (yield: 60%). Compound 15 (1.0 eq) was dissolved in DCM, and EDC (2.0 eq) and DMAP (0.1 eq) were added. Stirring at room temperature for 8 h, followed by the addition of TEA (1.2 eq) and compound 153 (1.0 eq). Stirring at room temperature was continued for 12 h. After completion of the reaction, the mixture was precipitated with diethyl ether to obtain compound V-2 (yield: 78%). 1 H NMR (400 MHz, CDCl3, 293 K) δ (ppm): 5.21-5.09 (m, 3H), 4.26-4.22 (m, 2H), 4.04-3.98 (m, 4H), 3.82-3.04 (m, 180H), 2.85-2.58 (m, 16H), 2.25 (s, 6H), 2.17-1.19 (m, 99H), 0.87-0.78 (m, 12H). HRMS: m / z calcd: 3133.9076 (M+H + ), measured value: 3133.9068.

[0286] Example 23: Synthesis of Compound V-3

[0287]

[0288]

[0289]

[0290] Compound 1 (α-tocopherol, 1.0 eq) and compound 155 (1.0 eq) were dissolved in DCM, EDC (2.0 eq) and DMAP (0.1 eq) were added, and the mixture was stirred at room temperature for 8 h. After completion of the reaction, the mixture was washed with water to obtain compound 156 (yield: 65%). Compound 156 (1.0 eq) was dissolved in DCM, NHS (1.0 eq), EDC (2.0 eq), and DMAP (0.1 eq) were added, and the mixture was stirred at room temperature for 8 h. Subsequently, TEA (1.2 eq) and compound 145 (1.0 eq) were added, and the mixture was stirred at room temperature for a further 12 h. After completion of the reaction, the mixture was purified by column chromatography (DCM:MeOH = 10:1) to obtain compound 157 (yield: 35%). Compound 158 (1.0 eq) was dissolved in DMF, and NHS (1.0 eq), EDC (2.0 eq) and DMAP (0.1 eq) were added. The mixture was stirred at room temperature for 8 h. Subsequently, TEA (1.2 eq) and compound 157 (1.0 eq) were added, and stirring was continued at room temperature for 12 h. After completion of the reaction, the mixture was purified by column chromatography (DCM:MeOH=20:1) to obtain compound 159 (yield: 59%).

[0291] Compound 160 (1.0 eq) was dissolved in DMF, and NHS (1.0 eq), EDC (2.0 eq), and DMAP (0.1 eq) were added. The mixture was stirred at room temperature for 8 h, followed by the addition of compound 159 (1.0 eq). Stirring at room temperature was continued for 12 h. After completion of the reaction, the mixture was purified by column chromatography (DCM:MeOH = 20:1) to obtain compound 161 (yield: 65%). Compound 161 (1.0 eq) was dissolved in DCM, and NHS (1.0 eq), EDC (2.0 eq), and DMAP (0.1 eq) were added. Stirring at room temperature for 8 h, followed by the addition of compound 162 (n = 45) (1.0 eq). Stirring at room temperature was continued for 12 h. After completion of the reaction, the mixture was precipitated with diethyl ether to obtain compound 163 (yield: 65%). Compound 100 (1.0 eq) was dissolved in DCM, and NHS (1.0 eq), EDC (2.0 eq), and DMAP (0.1 eq) were added. The mixture was stirred at room temperature for 8 h, followed by the addition of compound 163 (1.0 eq). Stirring at room temperature was continued for 12 h. After completion of the reaction, the mixture was precipitated with diethyl ether to obtain compound 164 (yield: 65%). Compound 164 (1.0 eq) and compound 111 (1.0 eq) were dissolved in DCM, and EDC (2.0 eq) and DMAP (0.1 eq) were added. The mixture was stirred at room temperature for 12 h. After completion of the reaction, the mixture was washed with water and precipitated with diethyl ether to obtain compound V-3 (yield: 65%). 1H NMR (400MHz, CDCl3, 293K) δ (ppm): 7.64-7.60 (m, 1H), 7.51-7.48 (m, 1H), 7.36-7.35 (m, 1H), 7.22-7.1 3(m,3H),6.88-6.82(m,3H),5.38-5.27(m,2H),4.98-4.86(m,3H),4.01-3.91(t,2H),3.82-3.70(m,1H ),3.67-3.08(m,180H),2.80-2.55(m,12H),2.33-2.29(t,2H),2.21(s,3H),2.19-2.08(m,15H),2.05-1.99(m,4H),1.95-1.85(m,3H),1.71-1.17(m,70H),0.94-0.73(m,15H).HRMS: m / z calculated value: 3243.9994(M+H + ), measured value: 3243.9988.

[0292] Example 24: Synthesis of Compound V-4

[0293]

[0294]

[0295] Compound 15 (1.0 eq) and compound 33 (1.0 eq) were dissolved in DCM, EDC (2.0 eq) and DMAP (0.1 eq) were added, and the mixture was stirred at room temperature for 8 h. After completion of the reaction, the mixture was washed with water to obtain compound 166 (yield: 35%). Compound 166 (1.0 eq) and compound 9 (1.0 eq) were dissolved in DCM and stirred at room temperature for 8 h. Subsequently, TEA (1.2 eq) and compound 145 (1.0 eq) were added, and the mixture was heated at 40°C and stirred for 12 h. After completion of the reaction, the mixture was purified by column chromatography (DCM:MeOH = 10:1) to obtain compound 167 (yield: 65%). Compound 155 (1.0 eq) was dissolved in DCM, and NHS (1.0 eq), EDC (2.0 eq) and DMAP (0.1 eq) were added. The mixture was stirred at room temperature for 8 h. Subsequently, TEA (1.2 eq) and compound 167 (1.0 eq) were added, and stirring was continued at room temperature for 12 h. After completion of the reaction, the mixture was purified by column chromatography (DCM:MeOH=15:1) to obtain compound 168 (yield: 62%).

[0296] Compound 168 (1.0 eq) was dissolved in DCM, and NHS (1.0 eq), EDC (2.0 eq), and DMAP (0.1 eq) were added. The mixture was stirred at room temperature for 8 h, followed by the addition of TEA (1.2 eq) and compound 105 (n=45) (1.0 eq). Stirring at room temperature was continued for 12 h. After completion of the reaction, the mixture was precipitated with diethyl ether to obtain compound 169 (yield: 75%). Compound 170 (1.0 eq) was dissolved in DMF, and NHS (1.0 eq), EDC (2.0 eq), and DMAP (0.1 eq) were added. The mixture was stirred at room temperature for 8 h. TEA (1.2 eq) and compound 169 (1.0 eq) were then added. Stirring at room temperature was continued for 12 h. After completion of the reaction, the mixture was precipitated with diethyl ether to obtain compound 171 (yield: 74%). Compound 109 (1.0 eq) was dissolved in DCM, and NHS (1.0 eq), EDC (2.0 eq) and DMAP (0.1 eq) were added, and the mixture was stirred at room temperature for 8 h. Subsequently, TEA (1.2 eq) and compound 171 (1.0 eq) were added, and stirring was continued at room temperature for 12 h. After the reaction was completed, the mixture was precipitated with diethyl ether to obtain compound V-4 (yield: 78%). 1 H NMR (400 MHz, CDCl3, 293 K) δ (ppm): 7.14-7.05 (m, 2H), 6.91-6.70 (m, 5H), 4.58-4.44 (m, 1H), 4.35-4.25 (t, 2H), 4.22-4.17 (t, 2H), 3.79-3.50 (m, 180H), 3.43-3.26 (m, 8H), 3.04-2.62 (m, 14H), 2.25-2.14 (m, 15H), 1.93-1.86 (m, 1H), 1.71-1.13 (m, 50H), 1.07-0.98 (m, 2H), 0.91-0.80 (m, 15H). HRMS: m / z calcd: 3377.1162 (M+H + ), measured value: 3377.1170.

[0297] Example 25: Synthesis of Compound V-5

[0298]

[0299] Compound 11 (α-tocotrienol, 1.0 eq) and compound 173 (1.0 eq) were dissolved in DMF, and EDC (2.0 eq) and DMAP (0.1 eq) were added. The mixture was stirred at room temperature for 8 h. After completion of the reaction, it was purified by column chromatography (DCM:MeOH = 20:1) to obtain compound 174 (yield: 75%). Compound 174 (1.0 eq) and compound 9 (1.0 eq) were dissolved in DCM and stirred at room temperature for 8 h. Subsequently, TEA (1.2 eq) and compound 145 (1.0 eq) were added. The mixture was heated at 40°C and stirred for 12 h. After completion of the reaction, it was purified by column chromatography (DCM:MeOH:NEt3 = 150:10:1) to obtain compound 175 (yield: 33%). Compound 176 (1.0 eq) and compound 28 (1.0 eq) were dissolved in DCM and stirred at room temperature for 8 h. TEA (1.2 eq) and compound 175 (1.0 eq) were then added, and the mixture was heated at 40°C and stirred for 12 h. After completion of the reaction, the mixture was purified by column chromatography (DCM:MeOH:NEt3=150:10:1) to obtain compound 177 (yield: 39%). Compound 177 (1.0 eq) and compound 90 (n=45, purchased from Myrel) (1.0 eq) were dissolved in DCM, and EDC (2.0 eq) and DMAP (0.1 eq) were added. The mixture was stirred at room temperature for 8 h. After completion of the reaction, the mixture was washed with water and precipitated with diethyl ether to obtain compound 178 (yield: 46%). Compound 179 (1.0 eq) was dissolved in DCM, and NHS (1.0 eq), EDC (2.0 eq) and DMAP (0.1 eq) were added, and the mixture was stirred at room temperature for 8 h. Subsequently, TEA (1.2 eq) and compound 178 (1.0 eq) were added, and stirring was continued at room temperature for 12 h. After the reaction was completed, the mixture was precipitated with diethyl ether to obtain compound V-5 (yield: 63%). 1 H NMR (400 MHz, CDCl3, 293 K) δ (ppm): 5.49-5.39 (m, 9H), 5.14-5.03 (m, 3H), 4.41-4.33 (3H), 4.22-4.20 (m, 2H), 3.78-3.63 (m, 180H), 3.35-3.23 (m, 6H), 2.78-2.55 (m, 24H), 2.27-1.86 (m, 34H), 1.79-1.52 (m, 16H), 1.40-1.19 (m, 9H), 0.92-0.86 (m, 3H). HRMS: m / z calcd: 3215.9569 (M+H + ), measured value: 3215.9550.

[0300] Example 26: Synthesis of Compound VI-1

[0301]

[0302] Compound 5 (n = 45, purchased from Anaiji) (2.0 eq) and compound 28 (2.0 eq) were dissolved in DCM and stirred at room temperature for 6 h, followed by the addition of compound 181 (1.0 eq), and continued stirring at room temperature for 8 h. After the reaction was complete, compound 182 was precipitated with diethyl ether (yield: 93%). Compound 1 (α-tocopherol, 1.0 eq) and compound 3 (1.0 eq) were dissolved in DCM and stirred at room temperature for 8 h. Subsequently, TEA (1.2 eq) and compound 182 (1.0 eq) were added, and continued stirring at room temperature for 12 h. After the reaction was complete, compound VI-1 was precipitated with diethyl ether (yield: 77%). 1 H NMR (400MHz, CDCl3, 293K) δ (ppm): 4.43-4.36 (t, 4H), 4.25-4.21 (t, 2H), 3.81-3.5 4(m,180H),3.40(s,3H),3.15-3.12(m,2H),3.01-2.97(m,4H),2.75-2.64(m,4H),2 .59-2.57 (m, 2H), 2.21 (s, 3H), 2.18 (s, 3H), 2.15 (s, 3H), 1.93-1.86 (m, 2H), 1.78-1.74 (m, 2H), 1.69-0.96 (m, 50H), 0.89-0.76 (m, 24H). HRMS: m / z calculated value: 3215.0587 (M+H + ), measured value: 3215.0569.

[0303] Example 27: Synthesis of Compound VI-2

[0304]

[0305] Compound 166 (1.0 eq) and compound 9 (1.0 eq) were dissolved in DCM and stirred at room temperature for 8 h. TEA (1.2 eq) and compound 181 (1.0 eq) were then added, and the mixture was heated at 40°C and stirred for 12 h. After completion of the reaction, the mixture was precipitated with diethyl ether to obtain compound 184 (yield: 62%). Compound 15 (1.0 eq) was dissolved in DCM, and NHS (1.0 eq), EDC (2.0 eq), and DMAP (0.1 eq) were added. The mixture was stirred at room temperature for 8 h. TEA (1.2 eq) and compound 185 (1.0 eq) were then added, and the mixture was stirred at room temperature for 12 h. After completion of the reaction, the mixture was purified by column chromatography (DCM:MeOH = 20:1) to obtain compound 186 (yield: 33%).

[0306] Compound 186 (1.0 eq) and compound 184 (1.0 eq) were dissolved in DCM, EDC (2.0 eq) and DMAP (0.1 eq) were added, and the mixture was stirred at room temperature for 10 h. After completion of the reaction, the mixture was purified by column chromatography (DCM:MeOH=20:1) to obtain compound 187 (yield: 33%). Compound 66 (1.0 eq) was dissolved in DCM, NHS (1.0 eq), EDC (2.0 eq) and DMAP (0.1 eq) were added, and the mixture was stirred at room temperature for 8 h. Subsequently, compound 120 (n=45, purchased from Aladdin) (1.0 eq) was added, and the mixture was stirred at room temperature for a further 12 h. After completion of the reaction, the mixture was precipitated with diethyl ether to obtain compound 188 (yield: 61%). Compound 187 (1.0 eq) and compound 9 (1.0 eq) were dissolved in DCM and stirred at room temperature for 8 h. Subsequently, TEA (1.2 eq) and compound 188 (1.0 eq) were added, and the mixture was heated at 40°C and stirred for 12 h. After the reaction was completed, the mixture was precipitated with diethyl ether to obtain compound VI-2 (yield: 80%). 1 H NMR (400 MHz, CDCl3, 293 K) δ (ppm): 5.19-5.07 (m, 3H), 4.42-4.17 (m, 10H), 3.92-3.61 (m, 180H), 3.49-3.40 (m, 4H), 3.18-3.04 (m, 4H), 2.86-2.56 (m, 18H), 2.36-2.27 (m, 2H), 2.23 (s, 6H), 2.19-1.14 (m, 97H), 0.88-0.78 (m, 12H). HRMS: m / z calcd: 3776.4113 (M+H + ), measured value: 3776.4108.

[0307] Example 28: Synthesis of Compound VI-3

[0308]

[0309]

[0310] Compound 15 (1.0 eq) was dissolved in DMF, and NHS (1.0 eq), EDC (2.0 eq), and DMAP (0.1 eq) were added. The mixture was stirred at room temperature for 8 h, followed by the addition of TEA (1.2 eq) and compound 190 (1.0 eq). Stirring at room temperature continued for 12 h. After completion of the reaction, the mixture was purified by column chromatography (DCM:ethyl acetate = 5:1) to obtain compound 191 (yield: 72%). Compound 191 (1.0 eq) and compound 192 (1.0 eq) were dissolved in DCM, and EDC (2.0 eq) and DMAP (0.1 eq) were added. The mixture was stirred at room temperature for 8 h. After completion of the reaction, the mixture was purified by column chromatography (DCM:MeOH = 20:1) to obtain compound 193 (yield: 62%).

[0311] Compound 193 (1.0 eq) and compound 28 (1.0 eq) were dissolved in DCM and stirred at room temperature for 8 h. Compound 181 (1.0 eq) was then added. After completion of the reaction, the mixture was purified by column chromatography (DCM:MeOH = 10:1) to give compound 194 (yield: 38%). Compound 194 (1.0 eq) and compound 91 (1.0 eq) were dissolved in DCM, EDC (2.0 eq) and DMAP (0.1 eq) were added, and the mixture was stirred at room temperature for 8 h. After completion of the reaction, the mixture was purified by column chromatography (DCM:MeOH = 15:1) to give compound 195 (yield: 55%). Compound 109 (1.0 eq) and compound 195 (1.0 eq) were dissolved in DCM, EDC (2.0 eq) and DMAP (0.1 eq) were added, and the mixture was stirred at room temperature for 10 h. After completion of the reaction, the mixture was washed with water and purified by column chromatography (DCM:MeOH = 20:1) to give compound 196 (yield: 42%). Compound 196 (1.0 eq) and compound 19 (n = 45, purchased from Aladdin) (1.0 eq) were dissolved in DCM, EDC (2.0 eq) and DMAP (0.1 eq) were added, and stirred at room temperature for 12 h. After the reaction was completed, the mixture was washed with water and precipitated with ether to obtain compound VI-3 (yield: 72%). 1H NMR (400MHz, CDCl3, 293K) δ (ppm): 7.32-7.19 (m, 5H), 4.63-4.55 (m, 1H), 4.37-4.28 (m, 4 H),4.19-4.02(m,8H),3.79-3.46(m,180H),3.18-3.12(m,2H),3.06-2.94(m,6H),2.86-2 .64(m,6H),2.59-2.55(m,6H),2.31-2.29(m,2H),2.21(s,3H),2.19(s,3H),2.15(s,3H),1.95-1.88(m,1H),1.78-1.10(m,50H),0.92-0.75(m,15H). HRMS: m / z calculated value: 3385.0680(M+H + ), measured value: 3385.0671.

[0312] Example 29: Synthesis of Compound VI-4

[0313]

[0314]

[0315] Compound 15 (1.0 eq) and compound 173 (1.0 eq) were dissolved in DCM, and NHS (1.0 eq), EDC (2.0 eq), and DMAP (0.1 eq) were added. The mixture was stirred at room temperature for 6 h. After completion of the reaction, the mixture was purified by column chromatography (DCM:ethyl acetate = 10:1) to obtain compound 198 (yield: 56%). Compound 198 (1.0 eq) and compound 48 (1.0 eq) were dissolved in DCM, and NHS (1.0 eq), EDC (2.0 eq), and DMAP (0.1 eq) were added. The mixture was stirred at room temperature for 6 h. After completion of the reaction, the mixture was purified by column chromatography (DCM:MeOH = 20:1) to obtain compound 199 (yield: 65%).

[0316] Compound 199 (1.0 eq) and compound 28 (1.0 eq) were dissolved in DCM and stirred at room temperature for 8 h, followed by the addition of compound 181 (1.0 eq), followed by heating at 40°C and continued stirring for 12 h. After completion of the reaction, the mixture was purified by column chromatography (DCM:MeOH=10:1) to obtain compound 200 (yield: 32%). Compound 28 (1.0 eq) and compound 200 (1.0 eq) were dissolved in DCM and stirred at room temperature for 8 h, followed by the addition of compound 29 (n=45, purchased from Anaiji) (1.0 eq) and TEA (1.2 eq), and continued stirring at room temperature for 6 h. After completion of the reaction, the mixture was precipitated with diethyl ether to obtain compound 201 (yield: 57%). Compound 201 (1.0 eq) and compound 202 (1.0 eq) were dissolved in DCM, EDC (2.0 eq) and DMAP (0.1 eq) were added, and stirred at room temperature for 8 h. After the reaction was completed, the mixture was washed with water and precipitated with ether to obtain compound VI-4 (yield: 79%). 1 H NMR (400MHz, CDCl3, 293K) δ (ppm): 5.03-4.96 (m, 1H), 4.42-4.17 (m, 17H), 3.90-3.6 4(m,180H),3.52-3.39(m,4H),3.15-3.12(m,2H),3.02-2.94(m,4H),2.86-2.58(m, 10H), 2.39-2.30 (m, 6H), 2.21 (s, 3H), 2.19 (s, 3H), 2.16 (s, 3H), 2.05 (s, 3H), 2.02-1.86 (m, 3H), 1.81-1.07 (m, 106H), 0.97-0.75 (m, 21H). HRMS: m / z calculated value: 4034.5995 (M+H + ), measured value: 4034.6003.

[0317] Example 30: Synthesis of Compound VI-5

[0318]

[0319] Compound 5 (n=45) (2.0 eq) and compound 28 (2.0 eq) were dissolved in DCM and stirred at room temperature for 6 h. Compound 181 (1.0 eq) was then added and stirred at room temperature for 8 h. After completion of the reaction, the mixture was precipitated with diethyl ether to obtain compound 182 (yield: 93%). Compound 182 (1.0 eq) was dissolved in DCM, SOCl2 (1.0 eq) was added, and stirred at room temperature for 6 h. Compound 15 (1.0 eq) was added and stirred at room temperature for 8 h. After completion of the reaction, the mixture was precipitated with diethyl ether to obtain compound VI-5 (yield: 80%). 1H NMR (400 MHz, CDCl3, 293 K) δ (ppm): 4.33-4.21 (m, 6H), 3.83-3.56 (m, 180H), 3.39 (s, 3H), 3.19-3.13 (m, 2H), 3.00-2.95 (t, 4H), 2.87-2.80 (t, 3H), 2.79-2.62 (m, 8H), 2.60-2.57 (t, 2H), 2.22 (s, 3H), 2.19 (s, 3H), 2.16 (s, 3H), 1.96-1.17 (m, 55H), 0.90-0.81 (m, 24H). HRMS: m / z calcd: 3271.1213 (M+H + ), the measured value is 3271.1202.

[0320] Example 31: Synthesis of Compounds VI-6 to VI-12

[0321]

[0322] Compound 182 (1.0 eq) was dissolved in DCM, SOCl2 (1.0 eq) was added, and the mixture was stirred at room temperature for 6 h. Compound 15 (1.0 eq) was added, and the mixture was further stirred at room temperature for 8 h. After completion of the reaction, the mixture was precipitated with diethyl ether to obtain Compound 205 (yield: 45%). Compound 205 (1.0 eq) was dissolved in DCM, and Compound 28 (1.0 eq) was added, and the mixture was stirred at room temperature for 12 h. Compound 58 (alanine) was then added, and the mixture was further stirred for 12 h. After completion of the reaction, the mixture was precipitated with diethyl ether to obtain Compound 206 (yield: 56%). Compound 206 (1.0 eq) was dissolved in DCM, EDC (2.0 eq) and DMAP (0.1 eq) were added, and then compound 23 (1.0 eq) was added. The mixture was stirred at room temperature for 12 h. After the reaction was completed, the mixture was precipitated with ether to obtain compound VI-6 (n = 23, yield: 78%), compound VI-7 (n = 45, yield: 70%), compound VI-8 (n = 114, yield: 65%) and compound VI-9 (n = 227, yield: 71%).

[0323] Compound VI-6: 1H NMR (400 MHz, CDCl3, 293 K) δ (ppm): 4.39-4.13 (m, 9H), 3.83-3.56 (m, 92H), 3.40 (s, 3H), 3.21-3.13 (m, 2H), 3.00-2.94 (t, 4H), 2.86-2.50 (m, 6H), 2.21 (s, 3H), 2.19 (s, 3H), 2.15 (s, 3H), 1.89-1.18 (m, 55H), 0.93-0.77 (m, 15H). HRMS: m / z calcd: 2086.3884 (M+H + ), the measured value is 2087.3876.

[0324] Compound VI-7: 1 H NMR (400 MHz, CDCl3, 293 K) δ (ppm): 4.39-4.13 (m, 9H), 3.83-3.56 (m, 180H), 3.40 (s, 3H), 3.21-3.13 (m, 2H), 3.00-2.94 (t, 4H), 2.86-2.50 (m, 6H), 2.21 (s, 3H), 2.19 (s, 3H), 2.15 (s, 3H), 1.89-1.18 (m, 55H), 0.93-0.77 (m, 15H). HRMS: m / z calcd: 3054.9601 (M+H + ), the measured value is 3055.9655.

[0325] Compound VI-8: 1 H NMR (400 MHz, CDCl3, 293 K) δ (ppm): 4.39-4.13 (m, 9H), 3.83-3.56 (m, 456H), 3.40 (s, 3H), 3.21-3.13 (m, 2H), 3.00-2.94 (t, 4H), 2.86-2.50 (m, 6H), 2.21 (s, 3H), 2.19 (s, 3H), 2.15 (s, 3H), 1.89-1.18 (m, 55H), 0.93-0.77 (m, 15H). HRMS: m / z calcd: 6092.7740 (M+H + ), the measured value is 6093.7749.

[0326] Compound VI-9: 1H NMR (400 MHz, CDCl3, 293 K) δ (ppm): 4.39-4.13 (m, 9H), 3.83-3.56 (m, 910H), 3.40 (s, 3H), 3.21-3.13 (m, 2H), 3.00-2.94 (t, 4H), 2.86-2.50 (m, 6H), 2.21 (s, 3H), 2.19 (s, 3H), 2.15 (s, 3H), 1.89-1.18 (m, 55H), 0.93-0.77 (m, 15H). HRMS: m / z calcd: 11067.7362 (M+H + ), the measured value is 11068.7367.

[0327] Using β-tocopherol, γ-tocopherol, and δ-tocopherol as raw materials, compounds VI-10, VI-11, and VI-12 (n=45, yields: 62%, 71%, and 68%, respectively) were obtained using the same synthesis method as in the previous examples.

[0328]

[0329] Compound VI-10: 1 H NMR (400 MHz, CDCl3, 293 K) δ (ppm): 6.88 (s, 1H), 4.39-4.13 (m, 9H), 3.83-3.56 (m, 180H), 3.40 (s, 3H), 3.21-3.13 (m, 2H), 3.00-2.94 (t, 4H), 2.86-2.50 (m, 6H), 2.21 (s, 3H), 2.15 (s, 3H), 1.89-1.18 (m, 55H), 0.93-0.77 (m, 15H). HRMS: m / z calcd: 3040.9495 (M+H + ), the measured value is 3040.9489.

[0330] Compound VI-11: 1 H NMR (400 MHz, CDCl3, 293 K) δ (ppm): 6.65 (s, 1H), 4.39-4.13 (m, 9H), 3.83-3.56 (m, 180H), 3.40 (s, 3H), 3.21-3.13 (m, 2H), 3.00-2.94 (t, 4H), 2.86-2.50 (m, 6H), 2.19 (s, 3H), 2.15 (s, 3H), 1.89-1.18 (m, 55H), 0.93-0.77 (m, 15H). HRMS: m / z calcd: 3040.9495 (M+H + ), the measured value is 3040.9490.

[0331] Compound VI-12: 1 H NMR (400 MHz, CDCl3, 293 K) δ (ppm): 6.87 (s, 1H), 6.63 (s, 1H), 4.39-4.13 (m, 9H), 3.83-3.56 (m, 180H), 3.40 (s, 3H), 3.21-3.13 (m, 2H), 3.00-2.94 (t, 4H), 2.86-2.50 (m, 6H), 2.19 (s, 3H), 1.89-1.18 (m, 55H), 0.93-0.77 (m, 15H). HRMS: m / z calcd: 3026.9338 (M+H + ), the measured value is 3026.9330.

[0332] Example 32: Synthesis of Compound VII-1

[0333]

[0334] Compound 7 (n=45) (1.0 eq) and compound 213 (1.0 eq) were dissolved in DCM, and NHS (1.0 eq), EDC (2.0 eq), and DMAP (0.1 eq) were added. The mixture was stirred at room temperature for 12 h. After completion of the reaction, the mixture was precipitated with diethyl ether to obtain compound 214 (yield: 59%). Compound 214 (1.0 eq) and compound 15 (2.0 eq) were dissolved in DCM, and EDC (2.0 eq) and DMAP (0.1 eq) were added. The mixture was stirred at room temperature for 12 h. After completion of the reaction, the mixture was precipitated with diethyl ether to obtain compound VII-1 (yield: 61%). 1 H NMR (400 MHz, CDCl3, 293 K) δ (ppm): 5.12-5.02 (m, 1H), 4.40-4.29 (m, 4H), 3.80-3.51 (m, 180H), 3.36 (s, 3H), 2.91-2.55 (m, 16H), 2.21 (s, 6H), 2.19 (s, 6H), 2.15 (s, 6H), 1.98-1.88 (m, 2H), 1.71-1.15 (m, 46H), 1.06-0.98 (m, 4H), 0.85-0.80 (m, 24H). HRMS: m / z calcd: 3357.1980 (M+H + ), the measured value is 3257.1976.

[0335] Example 33: Synthesis of Compound VII-2

[0336]

[0337] Compound 1 (2.0 eq) and compound 3 (2.0 eq) were dissolved in DCM and stirred at room temperature for 6 h. Compound 214 (n=45) (1.0 eq) was then added and stirred at room temperature for 12 h. After the reaction was completed, compound VII-2 was obtained by precipitation with ether (yield: 70%). 1 H NMR (400 MHz, CDCl3, 293 K) δ (ppm): 5.33-5.14 (m, 1H), 4.53-4.27 (m, 4H), 3.87-3.53 (m, 186H), 3.34 (s, 1H), 2.87-2.56 (m, 8H), 2.21 (s, 6H), 2.18 (s, 6H), 2.15 (s, 6H), 1.98-1.88 (m, 2H), 1.71-0.98 (m, 50H), 0.89-0.80 (m, 24H). HRMS: m / z calcd: 3201.0900 (M+H + ), the measured value is 3201.0908.

[0338] Example 34: Synthesis of Compound VII-3

[0339]

[0340] Compound 214 (n=45) (1.0 eq) and compound 15 (1.0 eq) were dissolved in DCM, EDC (1.0 eq) and DMAP (0.1 eq) were added, and the mixture was stirred at room temperature for 12 h. After completion of the reaction, the mixture was precipitated with diethyl ether to obtain compound 217 (yield: 51%). Compound 218 (1.0 eq) and compound 6 (2.0 eq) were dissolved in DCM, DMAP (0.1 eq) was added, and the mixture was stirred at room temperature for 12 h. After completion of the reaction, the mixture was washed twice with water to obtain compound 219 (yield: 91%). Compound 217 (1.0 eq) and compound 219 (1.0 eq) were dissolved in DCM, EDC (1.0 eq) and DMAP (0.1 eq) were added, and the mixture was stirred at room temperature for 12 h. After completion of the reaction, the mixture was precipitated with diethyl ether to obtain compound VII-3 (yield: 54%). 1H NMR (400 MHz, CDCl3, 293 K) δ (ppm): 5.09-5.01 (m, 1H), 4.44-4.29 (m, 4H), 4.00-3.93 (m, 1H), 3.88-3.45 (m, 180H), 3.34 (s, 3H), 2.88-2.60 (m, 14H), 2.21 (s, 6H), 2.19 (s, 6H), 2.15 (s, 6H), 1.95-1.86 (m, 1H), 1.71-1.12 (m, 48H), 1.06-0.98 (m, 2H), 0.91-0.76 (m, 18H). HRMS: m / z calcd: 3068.9280 (M+H + ), the measured value is 3068.9278.

[0341] Example 35: Synthesis of Compound VII-4

[0342]

[0343]

[0344] Compound 15 (1.0 eq) and compound 103 (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 completion of the reaction, the mixture was purified by column chromatography (DCM:MeOH = 15:1) to obtain compound 221 (yield: 45%). Compound 221 (1.0 eq) and compound 213 (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 completion of the reaction, the mixture was purified by column chromatography (DCM:MeOH = 10:1) to obtain compound 222 (yield: 43%). Compound 222 (1.0 eq) and compound 90 (n=45) (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 completion of the reaction, the mixture was precipitated with diethyl ether to obtain compound 223 (yield: 51%). Compound 223 (1.0 eq) and compound 202 (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 completion of the reaction, the mixture was precipitated with diethyl ether to obtain compound VII-4 (yield: 48%). 1H NMR (400 MHz, CDCl3, 293 K) δ (ppm): 6.30-6.18 (m, 1H), 4.42-4.29 (m, 2H), 3.83-3.50 (m, 182H), 3.10-3.07 (m, 2H), 2.83-2.46 (m, 10H), 2.33-2.30 (m, 4H), 2.21 (s, 3H), 2.19 (s, 3H), 2.15 (s, 3H), 1.95-1.88 (m, 1H), 1.71-0.98 (m, 52H), 0.90-0.74 (m, 15H). HRMS: m / z calcd: 2997.9150 (M+H + ), the measured value is 2997.9144.

[0345] Example 36: Synthesis of Compound VII-5

[0346]

[0347] Compound 41 (n=45) (1.0 eq) and compound 213 (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 completion of the reaction, the mixture was precipitated with diethyl ether to obtain compound 225 (yield: 55%). Compound 225 (1.0 eq) and compound 15 (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 completion of the reaction, the mixture was precipitated with diethyl ether to obtain compound 226 (yield: 46%). Compound 226 (1.0 eq) and compound 179 (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 completion of the reaction, the mixture was precipitated with diethyl ether to obtain compound VII-5 (yield: 52%). 1 H NMR (400 MHz, CDCl3, 293 K) δ (ppm): 6.25-6.21 (s, 1H), 5.48-5.34 (m, 8H), 4.38-4.15 (m, 2H), 3.82-3.28 (m, 180H), 2.87-2.62 (m, 12H), 2.52-2.31 (m, 4H), 2.25 (s, 3H), 2.16 (s, 3H), 2.15 (s, 3H), 2.15-2.11 (m, 2H), 2.07-1.89 (m, 3H), 1.77-1.11 (m, 31H), 1.04-0.95 (m, 2H), 0.91-0.80 (m, 15H). HRMS: m / z calcd: 2969.8060 (M+H + ), the measured value is 2969.8071.

[0348] Example 37: Preparation and Characterization of Basic LNPs and LNPs of the Present Invention Encapsulating mRNA

[0349] The ionizable lipid molecule (ALC-0315), DSPC, cholesterol and PEG-DSPE were dissolved in ethanol at a molar ratio of 50:10:38:2. The mRNA was firefly luciferase mRNA, which was dissolved in a sodium citrate (100mM) 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 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 basic lipid nanoparticles encapsulating mRNA (expressed as Basic LNP@mRNA Luc ).

[0350] Ionizable lipid molecules (ALC-0315), DSPC, cholesterol and the compounds synthesized by Example 1-36 (I-1 to VII-5) were dissolved in ethanol according to a molar ratio of 50:10:38:2. The mRNA was firefly luciferase mRNA, dissolved in a sodium citrate (100mM) 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 was mixed with the mRNA in a mass ratio of 25:1 to obtain a slightly white solution. The ethanol was then removed by ultrafiltration to obtain lipid nanoparticles of the present invention (named according to the compounds synthesized by Example 1-36) that encapsulate mRNA. For example, if compound I-1 is used, the lipid nanoparticles can be represented as I-1LNP@mRNA Luc ).

[0351] Dynamic light scattering (DLS) was used to characterize the particle size distribution of the obtained basic lipid nanoparticles and the lipid nanoparticles of the present invention. Luc and I-1LNP@mRNA Luc The particle size distribution of Figure 1 shown.

[0352] DLS results showed (Table 1) that Basic LNP@mRNA Luc LNP@mRNA of the present invention prepared based on I-1 to VII-5 Luc In comparison, there is no significant difference in the hydrated particle size, and both meet the applicable standards.

[0353] Basic LNP@mRNA was observed using transmission electron microscopy. Luc , I-1LNP@mRNA Luc , II-1LNP@mRNA Luc VI-4LNP@mRNALuc The morphology of four lipid nanoparticles loaded with mRNA. Electron microscopy images show ( Figure 2 ), the four mRNA-loaded lipid nanoparticles had good morphology, were all quasi-spherical, and had a particle size of about 50 nm.

[0354] LNP@mRNA Luc Determination of encapsulation efficiency

[0355] The slightly white solution obtained above was dialyzed with an appropriate volume of PBS solution for 4 h, and the filtrate was collected. The mRNA content in the filtrate was determined using Nanodrop, and the encapsulation efficiency was calculated using the following formula:

[0356] Encapsulation efficiency = mRNA 总量 -mRNA 滤液 / mRNA 总量 .

[0357] The results show (Table 1) that the prepared LNP@mRNA of the present invention Luc All of them have good mRNA encapsulation efficiency.

[0358] Table 1

[0359]

[0360]

[0361] Example 38: LNP@mRNA of the present invention Luc In vitro transfection efficiency experiments

[0362] The ionizable lipid molecule (ALC-0315), DSPC, cholesterol, and the compounds synthesized in Examples 1-36 (I-1 to VII-5) were dissolved in ethanol at a molar ratio of 50:10:38:2. The mRNA was firefly luciferase mRNA, dissolved in a sodium citrate (100 mM) buffer solution at 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 was mixed with the mRNA at a mass ratio of 25:1 to obtain a slightly white solution. Ethanol was then removed by ultrafiltration. The I-1LNP@mRNA of the present invention containing mRNA was obtained. Luc To VII-5LNP@mRNA Luc PEG-DSPE was used instead of the compound synthesized in Example 1-36, and the same method was used to obtain Basic LNP@mRNA loaded with mRNA. Luc .

[0363] DC2.4 cells were cultured at a rate of 5 × 10 5Cells were seeded at a high density in 24-well plates and incubated in DMEM medium (10% fetal bovine serum and 1% penicillin-streptomycin) at 37°C in an atmosphere containing 5% CO2. Fresh medium was replaced after 24 hours of incubation. PBS, Basic LNP@mRNA, and PBS were added to the cells. Luc And the LNP@mRNA of the present invention Luc (The mRNA dose for each well was 1 μg). After incubating the cells for 24 hours, the cells were lysed and the firefly luciferase detection reagent (Luciferase Reporter Gene Assay Kit, Yeasen) was added. After thorough mixing, the RLU (Relative light unit) was measured using the Luminescence mode of a multifunctional microplate reader to reflect the LNP@mRNA expression in each group. Luc in vitro transfection efficiency.

[0364] The results showed (Table 2) that Basic LNP@mRNA Luc The in vitro transfection efficiency is low, while the LNP@mRNA of the present invention Luc The in vitro transfection efficiency was higher than that of Basic LNP@mRNA Luc There has been a significant improvement, and the transfection effect is excellent.

[0365] Table 2

[0366]

[0367]

[0368] Example 39: LNP@mRNA of the present invention OVA Antigen presentation efficiency experiment

[0369] The ionizable lipid molecule (ALC-0315), DSPC, cholesterol and compound III-2 synthesized in Example 12 were dissolved in ethanol at a molar ratio of 50:10:38:2. The mRNA was chicken ovalbumin mRNA (OVA mRNA) and was dissolved in a sodium citrate (100mM) buffer solution at 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 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 III-2LNP@mRNA loaded with mRNA. OVA PEG-DSPE was used instead of compound III-2 synthesized in Example 12, and the same method was used to obtain BasicLNP@mRNA loaded with mRNA. OVA .

[0370] DC2.4 cells were cultured at a rate of 5 × 105 Cells were seeded at a high density in 24-well plates and incubated in DMEM medium (10% fetal bovine serum and 1% penicillin-streptomycin) at 37°C in an atmosphere containing 5% CO2. Fresh medium was replaced after 24 hours of incubation. PBS, Basic LNP@mRNA, and PBS were added to the cells. OVA and III-2LNP@mRNA OVA (The mRNA dose per well was 1 μg.) After incubating the cells for 24 hours, flow cytometry analysis was performed to detect the proportion of OVA-H-2Kb-positive cell populations and compare the antigen presentation efficiency of the two LNP@mRNAs.

[0371] The results show that ( Figure 3 ), using Basic LNP@mRNA OVA After in vitro transfection, the proportion of OVA-H-2Kb positive cells was low, proving that DC2.4 cells were transfected with Basic LNP@mRNA OVA After transfection of OVA mRNA, the antigen presentation efficiency is very limited. OVA After in vitro transfection with OVA mRNA, the proportion of OVA-H-2Kb-positive cells increased significantly, demonstrating that the LNP@mRNA of the present invention can significantly enhance antigen presentation efficiency. *P<0.05 indicates a significant difference; **P<0.01 indicates a significantly significant difference; ***P<0.001 indicates a highly significant difference.

[0372] Example 40: LNP@mRNA of the present invention Luc In vivo expression experiments

[0373] The ionizable lipid molecules (ALC-0315), DSPC, cholesterol and the compound I-1 synthesized in Example 1, the compound II-1 synthesized in Example 6, the compound IV-4 synthesized in Example 19 or the compound V-4 synthesized in Example 24 were dissolved in ethanol according to a molar ratio of 50:10:38:2. The mRNA is firefly luciferase mRNA, which is dissolved in a sodium citrate (100mM) buffer solution with a pH of 5.0. The volume ratio of the organic phase solution to the aqueous phase solution is 1:3, and the lipid carrier is mixed with the mRNA in a mass ratio of 25:1 to obtain a slightly white solution. Ethanol is then removed by ultrafiltration to obtain I-1LNP@mRNA encapsulated mRNA Luc 、II-1LNP@mRNA Luc IV-4LNP@mRNA Luc and V-4LNP@mRNA LucPEG-DSPE was used instead of the compound synthesized in the example, and the same method was used to obtain Basic LNP@mRNA encapsulated with mRNA. Luc .

[0374] Subsequently, the five prepared LNP@mRNA Luc C57BL / 6J mice (4-6 weeks old, male, weighing approximately 18-20 g) were injected intramuscularly in the thigh at a dose of 5 μg mRNA per mouse. Six hours later, the mice were intraperitoneally injected with substrate (fluorescein sodium salt, D-Luciferin, 150 mg / kg, Yeasen), and then the small animal in vivo fluorescence imaging system ( Spectrum, PerkinElmer) for bioluminescence imaging.

[0375] The results show that ( Figure 4 ), LNP@mRNA of the present invention Luc Basic LNP@mRNA Luc Compared with the LNP@mRNA, the expression level of luciferase was significantly improved. Luc Both achieved successful in vivo nucleic acid delivery and efficient expression. *P<0.05 indicates a significant difference; **P<0.01 indicates a significantly different result.

[0376] Example 41: LNP@mRNA of the present invention OVA Tumor growth inhibition rate experiment

[0377] The ionizable lipid molecule (ALC-0315), DSPC, cholesterol and the compound (I-1 to VII-5) synthesized by Example 1-36 were dissolved in ethanol according to a molar ratio of 50:10:38:2. The mRNA was chicken ovalbumin mRNA (OVA mRNA) and was dissolved in a sodium citrate (100mM) 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 was mixed with the mRNA in 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 containing the mRNA. OVA (I-1LNP@mRNA OVA To VII-5LNP@mRNA OVA PEG-DSPE was used instead of the compound synthesized in Example 1-36 to obtain Basic LNP@mRNA loaded with mRNA in the same manner. OVA .

[0378] C57BL / 6J mice (4-6 weeks old, male, weighing approximately 18-20 g) were cultured with B16-OVA cells (1×10 6 ) were injected subcutaneously to establish a tumor model. The day of tumor loading was recorded as day 0. On day 9, PBS and Basic LNP@mRNA were injected into the thigh muscle respectively. OVA And the LNP@mRNA of the present invention OVA The mRNA injection dose was 5 μg. The tumor volumes of mice were measured on day 0 and day 21, and the tumor growth inhibition rate was calculated according to the formula TGI (Tumor Growth Inhibition) = (1-tumor volume of treatment group / tumor volume of control group) * 100%.

[0379] The results showed (Table 3) that Basic LNP@mRNA OVA The tumor growth inhibition rate is about 50%, while the LNP@mRNA of the present invention OVA The tumor growth inhibition rate reached about 80%, which was significantly higher than that of Basic LNP@mRNA. OVA There is a significant improvement, indicating that the LNP@mRNA of the present invention OVA It can significantly enhance the in vivo expression efficiency of mRNA.

[0380] Table 3

[0381]

[0382]

[0383] Example 42: Injection of LNP@mRNA of the present invention OVA Tumor growth curve after

[0384] The ionizable lipid molecule (ALC-0315), DSPC, cholesterol and the compound V-3 synthesized in Example 23 were dissolved in ethanol at a molar ratio of 50:10:38:2. The mRNA was chicken ovalbumin mRNA (OVA mRNA) and was dissolved in a sodium citrate (100mM) 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 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 V-3LNP@mRNA encapsulated with mRNA. OVA PEG-DSPE was used instead of compound V-3 and the same method was used to obtain Basic LNP@mRNA loaded with mRNA. OVA .

[0385] C57BL / 6J mice (4-6 weeks old, male, weighing approximately 18-20 g) were cultured with B16-OVA cells (1×10 6 ) were injected subcutaneously to establish a tumor model. The day of tumor loading was recorded as day 0. On day 9, PBS and Basic LNP@mRNA were injected into the thigh muscle respectively. OVA and V-3LNP@mRNA OVA The mRNA injection dose was 5 μg, and the changes in tumor volume of mice were recorded within 23 days.

[0386] The results show that ( Figure 5 ), receiving V-3LNP@mRNA OVA The tumor volume of treated mice grew slowly, and the therapeutic effect was significantly better than that of mice receiving Basic LNP@mRNA. OVA Treated mice, indicating that the LNP@mRNA of the present invention OVA It can significantly enhance the in vivo expression efficiency of mRNA.

[0387] Example 43: LNP@mRNA of the present invention OVA Anti-tumor immune response experiments

[0388] The ionizable lipid molecule (ALC-0315), DSPC, cholesterol and compound VI-4 synthesized in Example 29 were dissolved in ethanol at a molar ratio of 50:10:38:2. The mRNA was chicken ovalbumin mRNA (OVA mRNA) and was dissolved in a sodium citrate (100mM) 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 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 VI-4LNP@mRNA loaded with mRNA. OVA PEG-DSPE was used instead of compound VI-4, and the same method was used to obtain Basic LNP@mRNA loaded with mRNA. OVA .

[0389] C57BL / 6J mice (4-6 weeks old, male, weighing approximately 18-20 g) were cultured with B16-OVA cells (1×10 6 ) were injected subcutaneously to establish a tumor model. The day of tumor loading was recorded as day 0. On day 9, PBS and Basic LNP@mRNA were injected into the thigh muscle respectively. OVA and VI-4LNP@mRNA OVA The mRNA injection dose was 5 μg. After 30 days, the mice were sacrificed, and the tumor tissues were completely removed and ground. ELISA assays were performed to detect the expression levels of IFN-γ and TNF-α in the tumor tissues and compare the anti-tumor immune response capabilities of the two LNP@mRNAs.

[0390] The results showed that VI-4LNP@mRNA OVA IFN-γ( Figure 6 ) and TNF-α( Figure 7 ) expression level was higher, and the therapeutic effect was significantly better than that of Basic LNP@mRNA OVA Treated mice, indicating that the LNP@mRNA of the present invention OVA It can significantly enhance the in vivo expression efficiency of mRNA. *P<0.05 indicates a significant difference; **P<0.01 indicates a significantly different result; ***P<0.001 indicates an extremely significant difference.

[0391] Example 44: LNP@mRNA constructed from compounds of formula (1) with different chain lengths (n different) Luc In vitro transfection efficiency experiments

[0392] The ionizable lipid molecule (ALC-0315), DSPC, cholesterol and the compound synthesized in Example 31 (Compound VI-6, n=23; Compound VI-7, n=45; Compound VI-8, n=114; Compound VI-9, n=227) were dissolved in ethanol at a molar ratio of 50:10:38:2. The mRNA was firefly luciferase mRNA and was dissolved in a sodium citrate (100mM) 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 mRNA were mixed at a mass ratio of 25:1 to obtain a slightly white solution. The ethanol was then removed by ultrafiltration. The VI-6LNP@mRNA of the present invention containing mRNA was obtained. Luc to VI-9LNP@mRNA Luc PEG-DSPE was used instead of the compound synthesized in Example 31, and the same method was used to obtain Basic LNP@mRNA loaded with mRNA. Luc .

[0393] DC2.4 cells were cultured at a rate of 5 × 10 5 Cells were seeded at a high density in 24-well plates and incubated in DMEM medium (10% fetal bovine serum and 1% penicillin-streptomycin) at 37°C in an atmosphere containing 5% CO2. Fresh medium was replaced after 24 hours of incubation. PBS, Basic LNP@mRNA, and PBS were added to the cells. Luc And the LNP@mRNA of the present invention Luc(The mRNA dose for each well was 1 μg). After incubating the cells for 24 hours, the cells were lysed and the firefly luciferase detection reagent (Luciferase Reporter Gene Assay Kit, Yeasen) was added and mixed thoroughly. The RLU (Relative light unit) was measured using the luciferase luminescence detection mode of a multifunctional microplate reader to reflect the LNP@mRNA expression in each group. Luc Among them, Basic LNP@mRNA Luc and VI-6 to VI-9 LNP@mRNA Luc The results of Luc mRNA transfection in vitro were as follows Figure 8 shown.

[0394] The results showed that Basic LNP@mRNA Luc The in vitro transfection efficiency of LNP@mRNA is low. Luc The in vitro transfection efficiency of LNP@mRNA was higher than that of Basic LNP@mRNA. Luc There was a significant improvement, and the transfection effect was excellent. *P<0.05 indicates a significant difference; **P<0.01 indicates a significantly significant difference; ***P<0.001 indicates a highly significant difference.

[0395] Example 45: LNP@mRNA constructed with β-tocopherol, γ-tocopherol and δ-tocopherol instead of α-tocopherol H1N1 Humoral immunity test

[0396] The ionizable lipid molecule (ALC-0315), DSPC, cholesterol, and the compounds synthesized in Example 31 (VI-7, VI-10, VI-11, and VI-12) were dissolved in ethanol at a molar ratio of 50:10:38:2. The mRNA was H1N1 influenza virus mRNA (H1N1 mRNA) and was dissolved in a sodium citrate (100 mM) buffer solution at 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 mRNA were mixed at a mass ratio of 25:1 to obtain a slightly white solution. Ethanol was then removed by ultrafiltration. The VI-7LNP@mRNA of the present invention containing mRNA was obtained. H1N1 VI-10LNP@mRNA H1N1 VI-11LNP@mRNA H1N1 and VI-12LNP@mRNA H1N1 Vaccine. Using PEG-DSPE instead of the compound synthesized in Example 31, the same method was used to obtain Basic LNP@mRNA encapsulated with mRNA. H1N1 .

[0397] The vaccine was administered to Balb / c mice twice by intramuscular injection at a dose of 10 μg mRNA per mouse, with an interval of one week. Peripheral blood was collected on the 7th and 21st days after the last immunization, and the anti-IgM and IgG antibody levels in the serum were detected by enzyme-linked immunosorbent assay to reflect the efficacy of each group of LNP@mRNA. H1N1 The level of humoral immune response after vaccination. Among them, Basic LNP@mRNA H1N1 and the LNP@mRNA of the present invention H1N1 Antibody production in mice immunized with the vaccine Figure 9 shown.

[0398] The results showed that Basic LNP@mRNA H1N1 Vaccines can induce a certain degree of humoral immune response, and the LNP@mRNA of the present invention H1N1 The antibody titer levels in mice induced by immunization were higher than those of Basic LNP@mRNA H1N1 The vaccine significantly improved humoral immunity, demonstrating excellent results. *P<0.05 indicates a significant difference; **P<0.01 indicates a significantly significant difference; NS indicates no statistically significant difference.

[0399] The foregoing descriptions of specific exemplary embodiments of the present disclosure are for purposes of illustration and description. These descriptions are not intended to limit the present disclosure to the precise form disclosed, and it is apparent that many modifications and variations are possible in light of the foregoing teachings. The exemplary embodiments have been selected and described for the purpose of explaining the specific principles of the present disclosure and their practical application, thereby enabling those skilled in the art to make and utilize the various exemplary embodiments and various options of the present disclosure.

Claims

1. A compound represented by formula (1) or a pharmaceutically acceptable salt thereof, in, X is selected from N or CH; 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 being unsubstituted or substituted with one or more OH, NH2 or halogen; 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 L 4 、L 5 and L 6 are independently selected from a single bond, C 1-20 Alkylene, C 2-10 Alkenylene or -(OCH2CH2) m -, the alkylene or alkenylene is unsubstituted or substituted by one or more OH, NH2, halogen or C 6-10 Aryl substituted; the C 6-10 Aryl is unsubstituted or substituted with one or more OH, NH2 or halogen; 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)-, -OP(=O)(OH)O-, or combinations of these groups with amino acid residues; The amino acid residue is selected from a divalent group obtained by removing H and / or OH from glycine, alanine, valine, leucine, isoleucine, methionine, proline, tryptophan, serine, tyrosine, cysteine, phenylalanine, asparagine, glutamine, threonine, aspartic acid, glutamic acid, lysine, arginine or histidine, or a combination thereof; Each R 3 Independently selected from H, C 1-10 Alkyl, C 2-10 Alkenyl, C 3-8 Cycloalkyl, 5-8 membered heterocyclic group, C 6-10 Aryl or 5-10 membered heteroaryl, wherein the alkyl, alkenyl, cycloalkyl, heterocyclyl, aryl or heteroaryl is unsubstituted or substituted with one or more OH, NH2 or halogen; Each L a Independently selected from C 1-10 Alkylene or C 2-10 Alkenylene, said alkylene or alkenylene being unsubstituted or substituted with one or more OH, NH2 or halogen; R 1 selected from monovalent groups derived from tocopherol and its derivatives; R 2 Selected from monovalent groups derived from tocopherol and its derivatives, C 1-30 Alkyl, C 2-30 Alkenyl or C 2-30 Alkynyl; the alkyl, alkenyl or alkynyl group 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 heterocyclic group, C 6-10 substituted with aryl or 5-10 membered heteroaryl; P 1 Selected from C 1-30 Alkyl or C 2-30 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, -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 or -C(=O)NHC 2-20 Alkenyl substitution, provided that P 1 and G 3 At least one of them contains -(OCH2CH2) n -; R 4 Selected from -OCH3, -NH2, -COOH, -SH or -N3; n is an integer from 20 to 250; m is an integer of 1-10.

2. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, wherein L 1 、L 2 and L 3 Independently selected from a single bond or C 1-10 Alkylene, which is unsubstituted or substituted with one or more OH, NH2 or halogen; Or, L 1 、L 2 and L 3 Independently selected from a single bond or C 1-6 Alkylene, which is unsubstituted or substituted with one or more OH, NH2 or halogen; Or, L 1 、L 2 and L 3 are independently selected from a single bond, -CH2-, -CH2CH2-, 3. The compound according to claim 1 or 2 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 -, -OC(=O)-, -C(=O)O-, -OC(=O)O-, -C(=O)C(=O)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 Or, G 1 , G 2 and G 3 are independently selected from a single bond, -NR 3 -、-OC(=O)NR 3 -, -OC(=O)-, -OC(=O)O-, -C(=O)C(=O)O-, -OC(=O)C(=O)O-, -OC(=O)-L a -C(=O)O-, -NR 3 C(=O)- or Or, G 1 , G 2 and G 3 independently selected from a single bond, -NH-, -O-, -NHC(=O)O-, -OC(=O)NH-, -OC(=O)-, -C(=O)O-, -OC(=O)O-, -C(=O)C(=O)O-, -OC(=O)C(=O)-, -OC(=O)C(=O)O-, -OC(=O)CH2CH2C(=O)O-, -OC(=O)CH2CH2CH2C(=O)O-, -NHC(=O)NH-, -C(=O)NH-, -NHC(=O)-, or 4. The compound according to any one of claims 1 to 3 or a pharmaceutically acceptable salt thereof, wherein L 4 、L 5 and L 6 are independently selected from a single bond, C 1-14 Alkylene, C 2-8 Alkenylene or -(OCH2CH2) m -, the alkylene or alkenylene is unsubstituted or substituted by one or more OH, NH2, halogen or phenyl; the phenyl is unsubstituted or substituted by one or more OH, NH2 or 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 1-11 Alkylene, C 2-7 Alkenylene or -(OCH2CH2) m -, the alkylene group is unsubstituted or substituted by one or more OH, NH2, halogen or phenyl; the phenyl group is unsubstituted or substituted by one or more OH, NH2 or halogen; 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)-, 5. The compound according to any one of claims 1 to 4 or a pharmaceutically acceptable salt thereof, wherein 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)-, -OP(=O)(OH)O-, or a combination of these groups and an amino acid residue; the amino acid residue is selected from a divalent group obtained by removing H and / or OH from valine, isoleucine, proline, tyrosine, cysteine, lysine, leucine, phenylalanine, threonine, serine, methionine, alanine, glutamic acid, glycine or tryptophan; Or, 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-, -C(=O)C(=O)O-, -OC(=O)C(=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 -、-NR 3 C(=O)-, -OP(=O)(OH)O-, or a combination of these groups and an amino acid residue; the amino acid residue is selected from a divalent group obtained by removing H and / or OH from leucine, threonine, glutamic acid, phenylalanine, tryptophan, serine, methionine, alanine or glycine; Or, G 4 , G 5 and G 6 Independently selected from single bonds, -NH-, -S-, -O-, -NHC(=O)O-, -OC(=O)NH-, -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)CH2CH2C(=O)O-, -OC(=O)CH2CH2CH2C(=O)O-, -NHC(=O)-, -C(=O)NH-, -NHC(=O)NH-, -OP(=O)(OH)O-, 6. The compound according to any one of claims 1 to 5 or a pharmaceutically acceptable salt thereof, wherein: Each R 3 Independently selected from H, C 1-10 Alkyl, C 2-10 Alkenyl, C 3-8 Cycloalkyl or 5-8 membered heterocyclyl, wherein the alkyl, alkenyl, cycloalkyl or heterocyclyl is unsubstituted or substituted with one or more OH, NH2 or halogen; Or, each R 3 Independently selected from H, C 1-6 Alkyl or C 2-6 Alkenyl, wherein the alkyl, alkenyl is unsubstituted or substituted with one or more OH, NH2 or halogen; Or, each R 3 are independently H.

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

8. The compound according to any one of claims 1 to 7 or a pharmaceutically acceptable salt thereof, wherein R 1 a monovalent group selected from α-tocopherol, β-tocopherol, γ-tocopherol, δ-tocopherol, α-tocotrienol, β-tocotrienol, γ-tocotrienol or δ-tocotrienol; Or, R 1 Selected from 9. The compound according to any one of claims 1 to 8 or a pharmaceutically acceptable salt thereof, wherein R 2 is selected from a monovalent group derived from α-tocopherol, β-tocopherol, γ-tocopherol, δ-tocopherol, α-tocotrienol, β-tocotrienol, γ-tocotrienol or δ-tocotrienol, C 1-30 Alkyl, C 2-30 Alkenyl or C 2-30 Alkynyl; the alkyl, alkenyl or alkynyl group 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 heterocyclic group, C 6-10 substituted with aryl or 5-10 membered heteroaryl; Or, R 2 Selected from C 2-20 Alkyl or C 2-20 Alkenyl; the alkyl or alkenyl is unsubstituted or substituted by one or more OH, NH2, halogen, -OC(=O)C 1-20 Alkyl, -C(=O)OC 1-20 Alkyl, -NHC(=O)C 2-20 Alkenyl or 5-6 membered heteroaryl substitution; Or, R 2 Selected from 10. The compound according to any one of claims 1 to 9 or a pharmaceutically acceptable salt thereof, wherein P 1 Selected from or C 1-20 Alkyl, said alkyl being unsubstituted or substituted by one or more OH, NH2, halogen, -OC 6-20 Alkyl, -OC(=O)C 6-20 Alkyl, -C(=O)OC 6-20 Alkyl, -NHC(=O)C 6-20 Alkyl or -C(=O)NHC 6-20 Alkyl substitution; Or, P 1 Selected from or C 1-6 Alkyl, said alkyl being unsubstituted or substituted by one or more OH, NH2, halogen or -OC(=O)C 10-20 Alkyl substitution; Or, P 1 Selected from 11. The compound according to any one of claims 1 to 10 or a pharmaceutically acceptable salt thereof, wherein: n is an integer of 20-227; alternatively, n is an integer of 23-227; alternatively, n is an integer of 45-227; alternatively, n is an integer of 45-114; alternatively, n is an integer of 23-114.

12. A compound represented by formula (1) or a pharmaceutically acceptable salt thereof, wherein the compound is selected from the following: Wherein n is an integer from 20 to 250.

13. A lipid carrier comprising an ionizable lipid molecule, a polyethylene glycol lipid molecule, a steroid lipid molecule and a helper lipid molecule, wherein the polyethylene glycol lipid molecule comprises a compound according to any one of claims 1 to 12 or a pharmaceutically acceptable salt thereof.

14. The lipid carrier according to claim 13, wherein The ionizable lipid molecule is selected from at least one of the following: (1-octylnonyl 8-[(2-hydroxyethyl)[6-O-6-(undecyloxy)hexyl]amino]-octanoate) SM-102, ((4-hydroxybutyl)azadialkyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate) ALC-0315, 1,2-dioleoyl-3-dimethylammonium-propane DODAP, 1,2-dioleoxy-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-octadecen-1-yl-1,3-dioxolane-4-ethylamine Dlin-KC2-DMA, 4-(N,N-dimethylamino)butyric acid (dilinoleyl)methyl ester Dlin-MC3-DMA; The steroidal lipid molecule is selected from at least one of the following: avenasterol, β-sitosterol, brassicasterol, ergocalciferol, campesterol, cholestanol, cholesterol, coprosterol, dehydrocholesterol, streptosterol, dihydroergocalciferol, dihydrocholesterol, dihydroergosterol, melanosterol, epicholesterol, ergosterol, fuccasterol, hexahydroluminosterol, hydroxycholesterol, lanosterol, luminosterol, alginosterol, sitostanol, sitosterol, stigmasterol, stigmasterol, cholic acid, glycocholic acid, taurocholic acid, deoxycholic acid and lithocholic acid; The auxiliary lipid molecule is selected from at least one of the following: 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, oleoylphosphatidylcholine POPC and 1-palmitoyl-2-oleoylphosphatidylethanolamine POPE.

15. The lipid carrier according to claim 13 or 14, wherein Calculated by molar percentage, the lipid carrier comprises 10%-70% of ionizable lipid molecules, 5%-60% of steroid lipid molecules, 1%-60% of polyethylene glycol lipid molecules and 1%-30% of auxiliary lipid molecules.

16. A nucleic acid lipid nanoparticle composition comprising the lipid carrier according to any one of claims 13 to 15 and a nucleic acid.

17. The nucleic acid lipid nanoparticle composition according to claim 16, wherein The nucleic acid is selected from at least one of DNA, mRNA, rRNA, siRNA, tRNA, microRNA, antisense nucleic acid and circular RNA.

18. The nucleic acid lipid nanoparticle composition according to claim 16 or 17, wherein The mass ratio of the lipid carrier to the nucleic acid is 5:1-50:1; or the mass ratio of the lipid carrier to the nucleic acid is 10:1-30:

1.

19. Use of the compound according to any one of claims 1 to 12 or a pharmaceutically acceptable salt thereof, the lipid carrier according to any one of claims 13 to 15, or the nucleic acid lipid nanoparticle composition according to any one of claims 16 to 18 in the preparation of a nucleic acid drug or a gene vaccine.

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