Auxiliary lipid molecule containing tocopherol structure, lipid nanoparticle containing same and application thereof

Abstract

The invention relates to an auxiliary lipid molecule containing a tocopherol structure, a lipid nanoparticle containing the auxiliary lipid molecule and application of the auxiliary lipid molecule. Specifically, the invention provides an auxiliary lipid molecule containing tocopherol and a derivative structure thereof as shown in a formula (1), a lipid nanoparticle containing the auxiliary lipid molecule, and a preparation method and application of the auxiliary lipid molecule. Compared with auxiliary lipid molecules conventionally used in the field, the lipid nanoparticles prepared from the auxiliary lipid molecules 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 auxiliary lipid molecules capable of delivering nucleic acids and containing a tocopherol structure, lipid nanoparticles containing the auxiliary lipid molecules, and uses thereof. Background Art

[0002] Nucleic acids, as a new generation of biopharmaceuticals, not only treat diseases at their root, but also possess significant platform-based characteristics in both technology and production, thus holding broad application prospects in the medical field. However, the poor in vivo and in vitro stability and low delivery efficiency of nucleic acids significantly limit their druggability. In recent years, with the rapid development of materials science and biology, new gene vectors have emerged one after another, and nucleic acid synthesis and modification technologies have achieved significant breakthroughs, ushering in a new era for the clinical application of nucleic acid drugs. The FDA's emergency authorization of the COVID-19 mRNA vaccine has also made nucleic acid vaccines one of the hottest research areas. Nucleic acid drugs are therapeutics that regulate genes at the post-transcriptional and pre-protein translation stages, acting upstream of protein synthesis. They offer advantages such as simple design, short development cycles, strong target specificity, broad therapeutic areas, and long-lasting efficacy. They are widely used in the treatment of various diseases, including genetic disorders, tumors, and infections. However, the delivery system plays a crucial role in the development of nucleic acid drugs. The delivery vehicle not only ensures the intact delivery of the RNA component to the target, but also ensures timely release under appropriate timing and environmental conditions. Therefore, how to stably and efficiently deliver nucleic acid molecules to target cells through a suitable delivery system and ensure the most efficient nucleic acid expression is one of the major challenges facing the field of nucleic acid therapy today.

[0003] Lipid nanoparticles (LNPs) are one of the most advanced and widely used delivery systems in clinical practice. LNPs are composed of phospholipids, which act like cell membranes to encapsulate and protect fragile RNA, allowing it to enter cells and function. LNPs can also be metabolized by the body. LNPs successfully address the challenges of RNA protection and delivery. Due to their improved flexibility, safety, ease of preparation, and scalable production, LNPs are now being used in cutting-edge mRNA vaccine candidates and the widely used COVID-19 vaccine.

[0004] Tocopherol, also known as vitamin E, is the collective name for all tocopherols, tocotrienols, and their derivatives. It is an important fat-soluble antioxidant and an essential nutrient for normal life. Tocopherols are isoprene derivatives of the 6-hydroxybenzodihydropyran ring and include eight compounds, namely α-, β-, γ-, and δ-tocopherols and α-, β-, γ-, and δ-tocotrienols. Although the eight tocopherols have highly similar chemical structures, their biological activities vary significantly. α-Tocopherol is the most widely distributed, abundant, and active form of tocopherol in nature. The activities of β-tocopherol, γ-tocopherol, and δ-tocopherol are 50%, 10%, and 2% of those of α-tocopherol, respectively. Tocopherols and their derivatives are widely used in various industries, including pharmaceuticals, food, and cosmetics. As antioxidants, they effectively prevent and mitigate oxidative damage to cellular DNA and lipids caused by free radicals or reactive oxygen species, thereby protecting lipid membranes. In addition to its antioxidant properties, tocopherol and its derivatives also have excellent anti-inflammatory, lipid-regulating, and immunomodulatory effects. Furthermore, studies have found that tocopherol and its derivatives have good therapeutic effects on cardiovascular diseases, neurological diseases, reproductive system diseases, and other diseases, and also have excellent anti-tumor potential.

[0005] Based on the aforementioned properties of tocopherol and its derivatives, we have developed a class of helper lipid molecules containing tocopherol and its derivatives 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 helper lipid molecule containing the structure of tocopherol and its derivatives represented by formula (1), lipid nanoparticles containing the same, and methods for preparing and using the same. Compared with conventional helper lipid molecules used in the art, the lipid nanoparticles prepared from the helper lipid molecule represented by formula (1) of the present invention can achieve high-efficiency delivery and expression of nucleic acids.

[0007] [Helper lipid molecule - a 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] in,

[0010] X is selected from N or CH;

[0011] represents a single bond or a double bond;

[0012] R a 、Rb and R c independently selected from H or methyl;

[0013] L 1 Selected from single bond, C 1-10 Alkylene or C 2-10 Alkenylene, said alkylene or alkenylene being unsubstituted or substituted with one or more OH;

[0014] 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;

[0015] G 1 , G 2 , G 3 and G 4 are independently selected from a single bond, -NR 3 -, -O-, -S-, -OC(=O)NR 3 -、-NR 3 C(=O)-, -C(=O)-, -OC(=O)-, -SC(=O)O-, -OC(=O)O-, -C(=O)-L a -C(=O)-, -C(=O)-L a -C(=O)O-, -OC(=O)-L a -C(=O)O-, -C(=O)-L a -C(=O)NR 3 -、-NR 3 C(=O)NR 3 -, or a combination of these groups and amino acid residues;

[0016] The amino acid residue is selected from a divalent group derived 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 3-8 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;

[0018] Each L a 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;

[0019] L 4 and L 5 are independently selected from a single bond, C 1-20 Alkylene, C 2-10 Alkenylene or -(OCH2CH2) n -, the alkylene or alkenylene is unsubstituted or substituted by one or more OH, NH2 or halogen;

[0020] P 1 Selected from or -NH2;

[0021] R 1 Selected from C 1-30 Alkyl, C 2-30 Alkenyl or C 2-30 Alkynyl, said alkyl, alkenyl or alkynyl being unsubstituted or substituted by one or more OH, NH2, halogen, -OC 1-10 Alkyl, -SC 1-10 Alkyl, C 3-8 Cycloalkyl, 5-8 membered heterocyclic group, C 6-10 substituted with aryl or 5-10 membered heteroaryl;

[0022] n is an integer of 1-10; m is an integer of 2-10.

[0023] In some embodiments, L 1 Selected from single bond or C 1-10 Alkylene, which is unsubstituted or substituted with one or more OH groups.

[0024] In some embodiments, L 1 Selected from single bond or C 1-6 Alkylene, which is unsubstituted or substituted with one or more OH groups.

[0025] In some embodiments, L 1 Selected from single bond, -CH2-, -CH2CH2-,

[0026] In some embodiments, 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 2 and L 3 are independently selected from a single bond, -CH2-, -CH2CH2-,

[0028] In some embodiments, G 1 , G 2 , G 3 and G 4 are independently selected from a single bond, -NR 3 -, -O-, -S-, -OC(=O)NR 3 -、-NR 3 C(=O)-, -C(=O)-, -OC(=O)-, -SC(=O)O-, -OC(=O)O-, -C(=O)-L a -C(=O)-, -C(=O)-L a -C(=O)O-, -C(=O)-L a -C(=O)NR 3 -, or a combination of these groups and amino acid residues;

[0029] 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.

[0030] In some embodiments, G 1 , G 2 , G 3 and G 4 are independently selected from a single bond, -NR 3 -, -O-, -S-, -OC(=O)NR 3 -、-NR 3 C(=O)-, -C(=O)-, -OC(=O)-, -SC(=O)O-, -OC(=O)O-, -C(=O)-L a -C(=O)-, -C(=O)-L a -C(=O)O-, -C(=O)-L a -C(=O)NR 3 -, or a combination of these groups and amino acid residues;

[0031] The amino acid residue is selected from the divalent groups obtained by removing H and / or OH from leucine, threonine, glutamic acid, phenylalanine, tryptophan, serine, histidine, alanine or glycine.

[0032] 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.

[0033] In some embodiments, each R 3 Independently selected from H, C 1-10 Alkyl or C 2-10 Alkenyl, said alkyl or alkenyl is unsubstituted or substituted with one or more OH, NH2 or halogen.

[0034] In some embodiments, each R 3 Independently selected from H, C 1-6 Alkyl or C 2-6 Alkenyl, said alkyl or alkenyl is unsubstituted or substituted with one or more OH, NH2 or halogen.

[0035] In some embodiments, each R 3 Independently selected from H, C 1-4 Alkyl or C 2-4 Alkenyl, the alkyl or alkenyl is unsubstituted or substituted with one or more OH, NH2, F, Cl or Br.

[0036] In some embodiments, each R 3 are independently H.

[0037] In some embodiments, L a Selected from single bond or C 1-10 Alkylene, which is unsubstituted or substituted with one or more OH, NH2 or halogen.

[0038] In some embodiments, L a Selected from single bond or C 1-6 Alkylene, which is unsubstituted or substituted with one or more OH, NH2 or halogen.

[0039] In some embodiments, L a Selected from single bond or C 1-4 Alkylene, which is unsubstituted or substituted with one or more OH, NH2 or halogen.

[0040] In some embodiments, La Selected from single bond, -CH2-, -CH2CH2-,

[0041] In some embodiments, G 1 , G 2 , G 3 and G 4 are independently selected from a single bond, -NH-, -O-, -S-, -C(=O)-, -OC(=O)-, -SC(=O)O-, -NHC(=O)-, -OC(=O)NH-,

[0042] In some embodiments, L 4 and L 5 are independently selected from a single bond, C 1-20 Alkylene or -(OCH2CH2) n -, the alkylene group is unsubstituted or substituted by one or more OH, NH2 or halogen.

[0043] In some embodiments, L 4 and L 5 are independently selected from a single bond, C 1-16 Alkylene or -(OCH2CH2) n -, the alkylene group is unsubstituted or substituted by one or more OH, NH2 or halogen.

[0044] In some embodiments, L 4 and L 5 are independently selected from a single bond, C 1-14 Alkylene or -(OCH2CH2) n -, the alkylene group is unsubstituted or substituted by one or more OH, NH2 or halogen.

[0045] In some embodiments, L 4 and L 5 are independently selected from a single bond, C 1-11 Alkylene or -(OCH2CH2) n -, the alkylene group is unsubstituted or substituted by one or more OH, NH2 or halogen.

[0046] In some embodiments, n is an integer from 1 to 8; alternatively, n is an integer from 1 to 6; alternatively, n is an integer from 1 to 3.

[0047] In some embodiments, L 4 and L 5 are independently selected from a single bond, -CH2-, -CH2CH2-,

[0048] In some embodiments, m is an integer from 2 to 8; alternatively, m is an integer from 2 to 6.

[0049] In some embodiments, Selected from

[0050] In some embodiments, Selected from

[0051] In some embodiments, R 1 A monovalent group selected from vitamin E and its derivatives including but not limited to α-tocopherol, β-tocopherol, γ-tocopherol, δ-tocopherol, α-tocotrienol, β-tocotrienol, γ-tocotrienol, δ-tocotrienol, C 1-30 Alkyl, C 2-30 Alkenyl or C 2-30 Alkynyl, said alkyl, alkenyl or alkynyl being unsubstituted or substituted by one or more OH, NH2, halogen, -OC 1-10 Alkyl, -SC 1-10 Alkyl, C 3-8 Cycloalkyl, 5-8 membered heterocyclic group, C 6-10 aryl or 5-10 membered heteroaryl.

[0052] In some embodiments, R 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 1-10 Alkyl or -SC 1-10 Alkyl substitution.

[0053] In some embodiments, R 1 Selected from C 6-24 Alkyl or C 6-24 Alkenyl, the alkyl or alkenyl is unsubstituted or substituted with one or more OH, NH2, halogen, -OC 1-10 Alkyl or -SC 1-10 Alkyl substitution.

[0054] In some embodiments, R 1 Selected from C 8-20 Alkyl or C 8-20 Alkenyl, the alkyl or alkenyl is unsubstituted or substituted with one or more OH, NH2, halogen, -OC 1-6 Alkyl or -SC 1-6Alkyl substitution.

[0055] In some embodiments, R 1 Selected from

[0056] C 8-20 Alkyl or C 8-20 Alkenyl, the alkyl or alkenyl is unsubstituted or substituted with one or more OH, NH2, halogen, -OC 1-6 Alkyl or -SC 1-6 Alkyl substitution.

[0057] In some embodiments, R 1 Selected from

[0058] C 8-20 Alkyl or C 8-20 Alkenyl, the alkyl or alkenyl is unsubstituted or substituted with one or more OH, NH2, halogen, -OC 1-6 Alkyl or -SC 1-6 Alkyl substitution.

[0059] In some embodiments, R 1 Selected from

[0060] 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:

[0061]

[0062]

[0063]

[0064]

[0065] [Lipid carrier]

[0066] The present invention provides a lipid carrier comprising an ionizable lipid molecule, a polyethylene glycol lipid molecule, a steroid lipid molecule and an auxiliary lipid molecule, wherein the auxiliary lipid molecule comprises the compound represented by the above formula (1) or a pharmaceutically acceptable salt thereof.

[0067] In some embodiments, the ionizable lipid molecule contains one or more ionizable sites, including pyridine, imidazole, primary amine, secondary amine, and tertiary amine.

[0068] 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.

[0069] 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.

[0070] In some embodiments, the ionizable lipid molecule is selected from at least one of SM-102, ALC-0315, DODAP, and Dlin-DMA.

[0071] In some embodiments, the ionizable lipid molecule is selected from at least one of SM-102 and ALC-0315.

[0072] 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.

[0073] 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.

[0074] In some embodiments, the steroidal lipid molecule is selected from at least one of cholesterol, cholestanol, dihydrocholesterol, alginosterol, and deoxycholic acid.

[0075] In some embodiments, the steroidal lipid molecule is selected from at least one of cholesterol and dihydrocholesterol.

[0076] In some embodiments, the polyethylene glycol lipid molecule is selected from 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) or PEG-1,2-dimyristoyloxypropyl-3-amine (PEG-c-DMA).

[0077] In some embodiments, the polyethylene glycol lipid molecule is selected from at least one of the following: 2-[(polyethylene glycol)-2000]-N,N-tetracosylacetamide (ALC-0159), 1,2-dimyristoyl-sn-glyceromethoxypolyethylene glycol (PEG-DMG), and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)] (PEG-DSPE).

[0078] In some embodiments, the polyethylene glycol lipid molecule is selected from at least one of the following: 2-[(polyethylene glycol)-2000]-N,N-tetracosylacetamide (ALC-0159) or 1,2-dimyristoyl-sn-glyceromethoxypolyethylene glycol (PEG-DMG).

[0079] In some embodiments, the helper lipid molecule is a compound represented by the above formula (1) or a pharmaceutically acceptable salt thereof.

[0080] In some embodiments, the helper lipid molecule further includes 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).

[0081] 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 (auxiliary lipid molecule) and 1%-30% of polyethylene glycol lipid molecules.

[0082] In some embodiments, the molar percentage of the ionizable lipid molecules is 15%-65%.

[0083] In some embodiments, the molar percentage of the ionizable lipid molecules is 30%-60%.

[0084] In some embodiments, the molar percentage of the ionizable lipid molecules is 45%-55%.

[0085] In some embodiments, the molar percentage of the steroidal lipid molecules is 10%-50%.

[0086] In some embodiments, the molar percentage of the steroidal lipid molecules is 25%-45%.

[0087] In some embodiments, the molar percentage of the steroidal lipid molecules is 30%-40%.

[0088] In some embodiments, the molar percentage of the compound represented by formula (1) or a pharmaceutically acceptable salt thereof is 1%-50%.

[0089] In some embodiments, the molar percentage of the compound represented by formula (1) or a pharmaceutically acceptable salt thereof is 5%-40%.

[0090] In some embodiments, the molar percentage of the compound represented by formula (1) or a pharmaceutically acceptable salt thereof is 10%-20%.

[0091] In some embodiments, the molar percentage of the polyethylene glycol lipid molecules is 1%-20%.

[0092] In some embodiments, the molar percentage of the polyethylene glycol lipid molecules is 1%-10%.

[0093] In some embodiments, the molar percentage of the polyethylene glycol lipid molecules is 1%-5%.

[0094] 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.

[0095] [Nucleic acid lipid nanoparticle composition]

[0096] The present invention provides a nucleic acid lipid nanoparticle composition, which comprises the lipid carrier and nucleic acid.

[0097] 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.

[0098] In some embodiments, the nucleic acid is mRNA.

[0099] In some embodiments, the nucleic acid is firefly luciferase mRNA, green fluorescent protein (GFP) mRNA, chicken ovalbumin (OVA) mRNA, or H1N1 influenza virus mRNA.

[0100] In some embodiments, the mass ratio of lipid carrier to nucleic acid in the nucleic acid lipid nanoparticle composition is 5:1-50:1.

[0101] In some embodiments, the mass ratio of lipid carrier to nucleic acid in the nucleic acid lipid nanoparticle composition is 10:1-30:1.

[0102] 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.

[0103] 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.

[0104] In some embodiments, the nucleic acid lipid nanoparticle composition has a particle size of 30 to 500 nm.

[0105] In some embodiments, the nucleic acid lipid nanoparticle composition has a particle size of 30 to 200 nm.

[0106] 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.

[0107] 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.

[0108] [Nucleic acid lipid nanoparticle preparations, uses and treatment methods]

[0109] The present invention provides a nucleic acid lipid nanoparticle preparation, which comprises the nucleic acid lipid nanoparticle composition and pharmaceutically acceptable excipients.

[0110] 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.

[0111] 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.

[0112] The present invention also provides the compound represented by the above formula (1) or a pharmaceutically acceptable salt thereof, the above lipid carrier or the above nucleic acid lipid nanoparticle composition, which is used for delivering nucleic acid drugs or gene vaccines.

[0113] 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.

[0114] 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.

[0115] 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.

[0116] 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.

[0117] 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.

[0118] [Method for preparing lipid nanoparticles containing nucleic acids]

[0119] The present invention provides a method for preparing nucleic acid-encapsulated lipid nanoparticles, comprising the following steps:

[0120] (A1) mixing ionizable lipid molecules, the compound represented by formula (1) or a pharmaceutically acceptable salt thereof, polyethylene glycol lipid molecules, and steroid lipid molecules in the proportions described above, and dissolving them in a solvent to obtain an organic phase liposome solution;

[0121] (A2) dissolving the nucleic acid in a buffer solution of appropriate pH to obtain an aqueous nucleic acid solution;

[0122] (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;

[0123] 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.

[0124] In some embodiments, the solvent in step (A1) is ethanol, tetrahydrofuran or acetone.

[0125] In some embodiments, the solvent in step (A1) is ethanol.

[0126] In some embodiments, the buffer solution in step (A2) is an acetic acid / sodium acetate solution or a citric acid / sodium citrate solution.

[0127] In some embodiments, the buffer solution in step (A2) is a citric acid / sodium citrate solution.

[0128] In some embodiments, the pH of the buffer solution in step (A2) is 3-9.

[0129] In some embodiments, the pH of the buffer solution in step (A2) is 4-6.

[0130] In some embodiments, the pH of the buffer solution in step (A2) is 5.

[0131] In some embodiments, the concentration of the buffer solution in step (A2) is 1 mM-1 M.

[0132] In some embodiments, the concentration of the buffer solution in step (A2) is 20 mM-500 mM.

[0133] In some embodiments, the concentration of the buffer solution in step (A2) is 100 mM.

[0134] 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.

[0135] 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.

[0136] 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.

[0137] 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.

[0138] The present invention has the following beneficial effects:

[0139] 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.

[0140] 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.

[0141] 3. The lipid nanoparticles are suitable for the delivery of nucleic acids with different molecular weights and sequences and are universal.

[0142] 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

[0143] Figure 1 Basic LNP@mRNA with DSPC as the auxiliary lipid component is shown Luc and I-1LNP@mRNA of the present invention with compounds I-1, III-1, IV-1, and V-11 as auxiliary lipid components Luc 、III-1LNP@mRNA Luc , IV-1LNP@mRNA Luc and V-11LNP@mRNA Luc particle size distribution.

[0144] Figure 2 Basic LNP@mRNA with DSPC as the auxiliary lipid component is shown Luc and I-1LNP@mRNA of the present invention with compounds I-1, III-1, IV-1, and V-11 as auxiliary lipid components Luc 、III-1LNP@mRNA Luc , IV-1LNP@mRNA Luc and V-11LNP@mRNA Luc TEM photo of .

[0145] Figure 3 Basic LNP@mRNA with DSPC as the auxiliary lipid component is shown GFPand I-1LNP@mRNA of the present invention with compounds I-1, III-1, IV-1, and V-11 as auxiliary lipid components GFP 、III-1LNP@mRNA GFP , IV-1LNP@mRNA GFP and V-11LNP@mRNA GFP The expression of GFP protein in DC2.4 cells after in vitro transfection.

[0146] Figure 4 Basic LNP@mRNA with DSPC as the auxiliary lipid component is shown OVA and I-1LNP@mRNA of the present invention with compounds I-1 and I-2 as auxiliary lipid components OVA and I-2LNP@mRNA OVA Comparison of tumor growth curves of mice after injection treatment.

[0147] Figure 5 Basic LNP@mRNA with DSPC as the auxiliary lipid component is shown OVA and I-1LNP@mRNA of the present invention with compounds I-1 and I-2 as auxiliary lipid components OVA and I-2LNP@mRNA OVA Comparison of the survival curves of mice after injection treatment.

[0148] Figure 6 Basic LNP@mRNA with DSPC as the auxiliary lipid component is shown OVA and I-1LNP@mRNA of the present invention with compounds I-1 and I-2 as auxiliary lipid components OVA and I-2LNP@mRNA OVA Comparison of the expression of antigen-specific cytotoxic T lymphocytes in mice after injection treatment.

[0149] Figure 7 Basic LNP@mRNA with DSPC as the auxiliary lipid component is shown OVA and I-1LNP@mRNA of the present invention with compounds I-1 and I-2 as auxiliary lipid components OVA and I-2LNP@mRNA OVA Comparison of the infiltration of cytotoxic T lymphocytes in the tumor tissues of mice after injection treatment.

[0150] Figure 8 Basic LNP@mRNA with DSPC as the auxiliary lipid component is shown H1N1and V-10LNP@mRNA of the present invention with compounds V-10, V-11, V-12, and V-13 as auxiliary lipid components H1N1 、V-11LNP@mRNA H1N1 、V-12LNP@mRNA H1N1 and V-13LNP@mRNA H1N1 The production of humoral immune antibodies in vaccine-immunized mice. DETAILED DESCRIPTION

[0151] I. Definition

[0152] 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.

[0153] 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.

[0154] 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.

[0155] 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.

[0156] 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 preparation method of such salts is: in water or an organic solvent or a mixture of the two, the compounds in the form of free acid or base are reacted with a stoichiometric amount of a suitable base or acid to prepare.

[0157] 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.

[0158] In this disclosure, It refers to the position where a substituent is bonded.

[0159] 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.

[0160] 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.

[0161] 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.

[0162] 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.

[0163] 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.

[0164] The term "halogen" refers to fluorine (F), chlorine (Cl), bromine (Br) and iodine (I).

[0165] 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.

[0166] 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.

[0167] 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.

[0168] 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 can be optionally substituted or unsubstituted.

[0169] 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.

[0170] The term "cycloalkyl" refers to a saturated or partially saturated, monocyclic or polycyclic (such as bicyclic, for example, cyclic, bridged or spiro) non-aromatic hydrocarbon group. For example, the term "C 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.

[0171] 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.

[0172] 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.

[0173] 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.

[0174] 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.

[0175] The tocopherols and tocotrienols described herein are all natural tocopherols and tocotrienols, and the amino acids described herein are all natural amino acids.

[0176] II. Specific Examples

[0177] 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.

[0178] 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.

[0179] The experimental methods described in the following examples are conventional methods unless otherwise specified; the reagents and materials are commercially available unless otherwise specified.

[0180] The abbreviations used in this document have the following meanings:

[0181] abbreviation meaning abbreviation meaning EDC 1-Ethyl-(3-dimethylaminopropyl)carbodiimide DMAP 4-Dimethylaminopyridine TEA Triethylamine DCM dichloromethane NHS N-Hydroxysuccinimide MeOH Methanol <![CDATA[CDCl3]]> Deuterated chloroform <![CDATA[SOCl2]]> Thionyl chloride THF Tetrahydrofuran

[0182] Example 1: Synthesis of Compound I-1

[0183]

[0184] Compound 1 (1.0 eq) and compound 2 (1.0 eq) were dissolved in DCM, TEA (1.0 eq) was added, and the mixture was stirred at room temperature for 12 h. After completion of the reaction, the mixture was separated by column chromatography (DCM:MeOH = 10:1) to obtain compound 3 (yield: 56%). Compound 4 (1.0 eq) and compound 5 (1.0 eq) were dissolved in DCM, stirred at room temperature for 12 h, and after completion of the reaction, the excess solvent was removed in vacuo to obtain compound 6 (yield: 86%). Compound 3 (1.0 eq) and compound 6 (2.0 eq) were dissolved in DCM, TEA (1.0 eq) was added, and the mixture was stirred at room temperature for 12 h. After completion of the reaction, the mixture was separated by column chromatography (DCM:MeOH = 20:1) to obtain compound I-1 (yield: 42%). 1H NMR (400 MHz, CDCl3, 293 K) δ (ppm): 4.97-4.93 (m, 1H), 4.37-4.22 (m, 6H), 3.67-3.66 (m, 2H), 3.25 (s, 1H), 2.88-2.82 (m, 4H), 2.75-2.64 (m, 8H), 2.21 (s, 6H), 2.19 (s, 6H), 2.17 (s, 6H), 1.91-1.88 (m, 2H), 1.60-1.14 (m, 46H), 1.06-0.98 (m, 4H), 0.85-0.80 (m, 24H). HRMS m / z: calcd: 1283.7802 (M+H + ), measured value: 1283.7800.

[0185] Example 2: Synthesis of Compound I-2

[0186]

[0187] Compound 6 (1.0 eq) and compound 2 (1.0 eq) were dissolved in DCM, TEA (1.0 eq) was added, and the mixture was stirred at room temperature for 12 h. After completion of the reaction, the mixture was separated by column chromatography (DCM:MeOH = 15:1) to obtain compound 8 (yield: 54%). Compound 8 (1.0 eq) and compound 9 (POCl3, 1.5 eq) were dissolved in THF, TEA (1.5 eq) was added, and the mixture was stirred at 0°C for 12 h. After completion of the reaction, the mixture was separated by column chromatography (DCM:MeOH = 15:1) to obtain compound 10 (yield: 67%). Compound 10 (1.0 eq) and compound 11 (1.2 eq) were dissolved in THF, TEA (1.5 eq) was added, and the mixture was stirred at room temperature for 12 h. After completion of the reaction, the mixture was separated by column chromatography (DCM:MeOH = 15:1) to obtain compound 12 (yield: 64%). Compound 12 (1.0 eq) was dissolved in DCM, and TEA (1.5 eq) was added. The mixture was stirred at room temperature for 12 h. After the reaction was completed, the mixture was separated by column chromatography (DCM:MeOH=20:1) to obtain compound I-2 (yield: 46%). 1 H NMR (400 MHz, CDCl3, 293 K) δ (ppm): 5.06-4.97 (m, 1H), 4.44-4.21 (m, 4H), 4.10-4.04 (m, 2H), 3.18-3.16 (m, 2H), 2.89-2.81 (m, 4H), 2.76-2.64 (m, 8H), 2.21 (s, 6H), 2.19 (s, 6H), 2,16 (s, 6H), 1.98-1,88 (m, 4H), 1.74-0.98 (m, 50H), 0.87-0.80 (m, 24H). HRMS m / z: calcd: 1240.6918 (M+H+ ), measured value: 1240.6905.

[0188] Example 3: Synthesis of Compound I-3

[0189]

[0190] Compound 14 (1.0 eq) and compound 15 (1.0 eq) were dissolved in DCM and stirred at room temperature for 6 h. After completion of the reaction, the excess solvent was removed in vacuo to obtain compound 16 (yield: 84%). Compound 3 (1.0 eq) and compound 16 (2.0 eq) were dissolved in DCM, TEA (1.0 eq) was added, and stirred at room temperature for 12 h. After completion of the reaction, the mixture was separated by column chromatography (DCM:MeOH = 15:1) to obtain compound I-3 (yield: 63%). 1 H NMR (400 MHz, CDCl3, 293 K) δ (ppm): 5.16-5.07 (m, 1H), 4.53-4.45 (m, 2H), 4.38-4.25 (m, 4H), 3.70-3.66 (m, 2H), 3.23 (s, 9H), 2.75-2.64 (m, 4H), 2.21 (s, 6H), 2.18 (s, 6H), 2.15 (s, 6H), 1.95-1.88 (m, 2H), 1.73-0.99 (m, 50H), 0.88-0.80 (m, 24H). HRMS m / z: calcd: 1227.6722 (M+H + ), measured value: 1227.6718.

[0191] Example 4: Synthesis of Compound I-4

[0192]

[0193] Compound 2 (1.0 eq) and compound 16 (2.0 eq) were dissolved in DCM, TEA (1 eq) was added, and the mixture was stirred at room temperature for 12 h. After completion of the reaction, the mixture was separated by column chromatography (DCM:MeOH = 30:1) to obtain compound 18 (yield: 75%). Compound 18 (1.0 eq) and compound 9 (POCl3, 1.5 eq) were dissolved in THF, TEA (1.5 eq) was added, and the mixture was stirred at 0°C for 12 h. After completion of the reaction, the mixture was separated by column chromatography (DCM:MeOH = 15:1) to obtain compound 19 (yield: 57%). Compound 19 (1.0 eq) and compound 11 (1.2 eq) were dissolved in THF, TEA (1.5 eq) was added, and the mixture was stirred at room temperature for 12 h. After completion of the reaction, the mixture was separated by column chromatography (DCM:MeOH = 20:1) to obtain compound 20 (yield: 58%). Compound 20 (1.0 eq) was dissolved in DCM, and TEA (1.5 eq) was added. The mixture was stirred at room temperature for 12 h. After the reaction was completed, the mixture was separated by column chromatography (DCM:MeOH=10:1) to obtain compound I-4 (yield: 65%). 1 H NMR (400 MHz, CDCl3, 293 K) δ (ppm): 5.17-5.06 (m, 1H), 4.64-4.29 (m, 4H), 4.15-4.05 (m, 2H), 3.29-3.13 (m, 2H), 2.75-2.61 (m, 4H), 2.21 (s, 6H), 2.18 (s, 6H), 2.15 (s, 6H), 1.96-1.88 (m, 4H), 1.69-0.96 (m, 50H), 0.86-0.75 (m, 24H). HRMS: m / z calcd: 1183.7664 (M+H + ), measured value: 1184.7658.

[0194] Example 5: Synthesis of Compound I-5

[0195]

[0196] Compound 22 (1.0 eq) and compound 23 (1.0 eq) were dissolved in DCM, and NHS (1.00 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 separated by column chromatography (DCM:MeOH = 15:1) to obtain compound 24 (yield: 35%). Compound 24 (1.0 eq) and compound 3 (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 separated by column chromatography (DCM:MeOH = 10:1) to obtain compound 25 (yield: 53%). Compound 25 (1.0 eq) and compound 6 (2.0 eq) were dissolved in DCM, stirred at room temperature for 12 h. After completion of the reaction, the mixture was purified by column chromatography to obtain compound I-5 (yield: 42%). 1 H NMR (400 MHz, CDCl3, 293 K) δ (ppm): 5.25-4.90 (m, 4H), 4.44-4.21 (m, 7H), 3.70-3.66 (m, 2H), 3.26 (s, 9H), 2.87-2.64 (m, 8H), 2.25-1.82 (m, 32H), 1.77-1.13 (m, 42H), 1.06-0.98 (m, 2H), 0.89-0.78 (m, 18H). HRMS: m / z calcd: 1334.8282 (M+H + ), measured value: 1334.8272.

[0197] Example 6: Synthesis of Compound I-6

[0198]

[0199] Compound 3 (1.0 eq) and compound 16 (1.0 eq) were dissolved in DCM and 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 27 (yield: 54%). Compound 27 (1.0 eq) and compound 6 (1.0 eq) were dissolved in DCM and 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 I-6 (yield: 79%). 1H NMR (400 MHz, CDCl3, 293 K) δ (ppm): 5.12-5.09 (m, 1H), 4.38-4.24 (m, 6H), 3.73-3.66 (m, 2H), 3.25 (s, 9H), 2.87-2.64 (m, 8H), 2.21 (s, 6H), 2.19 (s, 6H), 2.17 (s, 6H), 1.95-1.88 (m, 2H), 1.71-0.98 (m, 50H), 0.89-0.80 (m, 24H). HRMS: m / z calcd: 1255.7262 (M+H + ), measured value: 1255.7257.

[0200] Example 7: Synthesis of Compound I-7

[0201]

[0202] Compound 2 (1.0 eq) and compound 16 (1.0 eq) were dissolved in DCM. After the reaction, the mixture was washed with water and purified by column chromatography (DCM:MeOH = 10:1) to give compound 29 (yield: 64%). Compound 29 (1.0 eq) and compound 30 (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 the reaction, the mixture was separated by column chromatography (DCM:MeOH = 15:1) to give compound 31 (yield: 55%). Compound 31 (1.0 eq) and compound 9 (POCl3, 1.5 eq) were dissolved in THF, TEA (1.5 eq) was added, and the mixture was stirred at 0°C for 12 h. After the reaction, the mixture was separated by column chromatography (DCM:MeOH = 15:1) to give compound 32 (yield: 59%). Compound 32 (1.0 eq) and compound 11 (1.2 eq) were dissolved in THF, TEA (1.5 eq) was added, and the mixture was stirred at room temperature for 12 h. After completion of the reaction, the mixture was separated by column chromatography (DCM:MeOH = 20:1) to obtain compound 33 (yield: 58%). Compound 33 (1.0 eq) was dissolved in DCM, TEA (1.5 eq) was added, and the mixture was stirred at room temperature for 12 h. After completion of the reaction, the mixture was separated by column chromatography (DCM:MeOH = 10:1) to obtain compound I-7 (yield: 45%). 1H NMR (400 MHz, CDCl3, 293 K) δ (ppm): 5.17-5.06 (m, 1H), 4.56-4.15 (m, 4H), 4.07-4.03 (m, 2H), 3.26-3.11 (m, 2H), 2.78-2.62 (m, 2H), 2.35-2.29 (m, 2H), 2.21 (s, 3H), 2.19 (s, 3H), 2.17 (s, 3H), 1.98-1.88 (m, 3H), 1.71-0.98 (m, 54H), 0.92-0.78 (m, 15H). HRMS: m / z calcd: 980.3588 (M+H + ), measured value: 980.3578.

[0203] Example 8: Synthesis of Compound I-8

[0204]

[0205] Compound 4 (1.0 eq) and compound 35 (1.0 eq) were dissolved in DCM, and NHS (1.00 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 washed with water and purified by column chromatography (DCM:MeOH=15:1) to give compound 36 (yield: 46%). Compound 2 (1.0 eq) and compound 36 (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 washed with water and purified by column chromatography (DCM:MeOH=10:1) to give compound 37 (yield: 63%). Compound 38 (1.0 eq) and compound 37 (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 washed with water and purified by column chromatography (DCM:MeOH = 20:1) to give compound 39 (yield: 63%). Compound 39 (1.0 eq) and compound 9 (POCl3, 1.5 eq) were dissolved in THF, TEA (1.5 eq) was added, and the mixture was stirred at 0°C for 12 h. After completion of the reaction, the mixture was separated by column chromatography (DCM:MeOH = 15:1) to give compound 40 (yield: 69%). Compound 40 (1.0 eq) and compound 11 (1.2 eq) were dissolved in THF, TEA (1.5 eq) was added, and the mixture was stirred at room temperature for 12 h. After completion of the reaction, the mixture was separated by column chromatography (DCM:MeOH = 20:1) to give compound 41 (yield: 68%). Compound 41 (1.0 eq) was dissolved in DCM, and TEA (1.5 eq) was added. The mixture was stirred at room temperature for 12 h. After the reaction was completed, the mixture was separated by column chromatography (DCM:MeOH=10:1) to obtain compound I-8 (yield: 62%).1 H NMR (400MHz, CDCl3, 293K) δ (ppm): 5.45-5.34 (m, 6H), 5.05-4.96 (m, 1H), 4.37- 4.20(m,4H),4.10-4.04(m,2H),3.21-3.04(m,4H),2.82-2.57(m,10H),2.34-2. 24(m,4H),2.21(s,3H),2.19(s,3H),2.18(s,3H),2.06-1.86(m,7H),1.71-1.1 2(m,41H),1.06-0.98(m,2H),0.92-0.76(m,15H).HRMS:m / z calculated value:1115.5248(M+H + ), measured value: 1115.5254.

[0206] Example 9: Synthesis of Compound I-9

[0207]

[0208] Compound 2 (1.0 eq) and compound 6 (1.0 eq) were dissolved in DCM and 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 = 15:1) to give compound 43 (yield: 46%). Compound 43 (1.0 eq) and compound 30 (1.0 eq) were dissolved in DCM, EDC (1.0 eq) and DMAP (0.1 eq) were added, and 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 44 (yield: 66%). Compound 44 (1.0 eq) and compound 9 (POCl3, 1.5 eq) were dissolved in THF, TEA (1.5 eq) was added, and stirred at 0°C for 12 h. After completion of the reaction, the mixture was separated by column chromatography (DCM:MeOH = 15:1) to give compound 45 (yield: 78%). Compound 45 (1.0 eq) and compound 11 (1.2 eq) were dissolved in THF, TEA (1.5 eq) was added, and the mixture was stirred at room temperature for 12 h. After completion of the reaction, the mixture was separated by column chromatography (DCM:MeOH = 20:1) to obtain compound 46 (yield: 68%). Compound 46 (1.0 eq) was dissolved in DCM, TEA (1.5 eq) was added, and the mixture was stirred at room temperature for 12 h. After completion of the reaction, the mixture was separated by column chromatography (DCM:MeOH = 10:1) to obtain compound I-9 (yield: 64%). 1H NMR (400 MHz, CDCl3, 293 K) δ (ppm): 5.05-4.97 (m, 1H), 4.37-4.17 (m, 4H), 4.10-4.05 (m, 2H), 3.26-3.16 (m, 2H), 2.87-2.81 (m, 2H), 2.75-2.64 (m, 4H), 2.32-2.29 (m, 2H), 2.21 (s, 3H), 2.19 (s, 3H), 2.17 (s, 3H), 1.96-1.88 (m, 3H), 1.68-0.98 (m, 55H), 0.91-0.74 (m, 15H). HRMS: m / z calcd: 1008.4128 (M+H + ), measured value: 1008.4124.

[0209] Example 10: Synthesis of Compound I-10

[0210]

[0211] Compound 3 (1.0 eq) and compound 6 (1.0 eq) were dissolved in DCM and stirred at room temperature for 12 h. After the reaction, the mixture was washed with water and purified by column chromatography (DCM:MeOH = 10:1) to give compound 48 (yield: 45%). Compound 49 (1.0 eq) and compound 50 (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 the reaction, the mixture was washed with water and purified by column chromatography (DCM:MeOH = 15:1) to give compound 51 (yield: 62%). Compound 48 (1.0 eq) and compound 51 (1.0 eq) were dissolved in DCM, NHS (1.0 eq), EDC (1.0 eq) and DMAP (0.1 eq) were added, and the mixture was stirred at room temperature for 12 h. After the reaction, the mixture was washed with water and purified by column chromatography (DCM:MeOH = 20:1) to give compound I-10 (yield: 46%). 1 H NMR (400 MHz, CDCl3, 293 K) δ (ppm): 5.01-4.89 (m, 1H), 4.37-4.07 (m, 9H), 3.70-3.63 (m, 2H), 3.23 (s, 9H), 2.87-2.64 (m, 6H), 2.21 (s, 3H), 2.19 (s, 3H), 2.17 (s, 3H), 1.95-1.88 (m, 1H), 1.71-1.11 (m, 49H), 1.06-0.98 (m, 2H), 0.90-0.75 (m, 15H). HRMS: m / z calcd: 1068.4442 (M+H + ), measured value: 1068.4448.

[0212] Example 11: Synthesis of Compound II-1

[0213]

[0214] Compound 6 (1.0 eq) and compound 53 (1.0 eq) were dissolved in DCM, TEA (1.5 eq) was added, and the mixture was stirred at room temperature for 12 h. The mixture was purified by column chromatography (DCM:MeOH = 15:1) to give compound 54 (yield: 53%). Compound 54 (1.0 eq) and compound 9 (POCl3, 1.5 eq) were dissolved in THF, TEA (1.5 eq) was added, and the mixture was stirred at 0°C for 12 h. After completion of the reaction, the mixture was separated by column chromatography (DCM:MeOH = 15:1) to give compound 55 (yield: 80%). Compound 55 (1.0 eq) and compound 11 (1.2 eq) were dissolved in THF, TEA (1.5 eq) was added, and the mixture was stirred at room temperature for 12 h. After completion of the reaction, the mixture was separated by column chromatography (DCM:MeOH = 20:1) to give compound 56 (yield: 78%). Compound 56 (1.0 eq) was dissolved in DCM, and TEA (1.5 eq) was added. The mixture was stirred at room temperature for 12 h. After the reaction was completed, the mixture was separated by column chromatography (DCM:MeOH=10:1) to obtain compound II-1 (yield: 63%). 1 H NMR (400 MHz, CDCl3, 293 K) δ (ppm): 4.20-3.95 (m, 7H), 3.26-3.17 (m, 2H), 2.86-2.64 (m, 10H), 2.54-2.50 (m, 2H), 2.21 (s, 6H), 2.19 (s, 6H), 2.17 (s, 6H), 1.96-1.86 (m, 4H), 1.70-1.10 (m, 46H), 1.06-0.98 (m, 4H), 0.90-0.80 (m, 24H). HRMS: m / z calcd: 1239.7078 (M+H + ), measured value: 1239.7069.

[0215] Example 12: Synthesis of Compound II-2

[0216]

[0217] Compound 53 (1.0 eq) and compound 58 (1.0 eq) were dissolved in DCM, TEA (1.5 eq) was added, and the mixture was stirred at room temperature for 12 h. After the reaction was complete, the mixture was washed with water and the organic solvent was removed in vacuo to obtain compound 59 (yield: 87%). Compound 59 (1.0 eq) and compound 1 (1.0 eq) were dissolved in DCM, TEA (1.5 eq) was added, and the mixture was stirred at room temperature for 12 h. After the reaction was complete, the mixture was washed with water and purified by column chromatography (DCM:MeOH=10:1) to obtain compound 60 (yield: 67%). Compound 60 (1.0 eq) was dissolved in THF, 15% TFA was added, and the mixture was stirred at room temperature for 3 h. After the reaction was complete, the mixture was washed with water and the organic solvent was removed in vacuo to obtain compound 61 (yield: 78%). Compound 61 (1.0 eq) and compound 6 (1.0 eq) were dissolved in DCM, TEA (1.5 eq) was added, and the mixture was stirred at room temperature for 12 h. After the reaction was completed, column chromatography purification (DCM:MeOH=15:1) was performed to obtain compound II-2 (yield: 63%). 1 H NMR (400 MHz, CDCl3, 293 K) δ (ppm): 4.33-4.28 (m, 2H), 4.18-4.09 (m, 2H), 4.04-3.96 (m, 3H), 3.66-3.63 (m, 2H), 3.25 (s, 9H), 2.86-2.63 (m, 10H), 2.56-2.50 (m, 2H), 2.21 (s, 6H), 2.19 (s, 6H), 2.16 (s, 6H), 1.95-1.88 (m, 2H), 1.70-0.98 (m, 50H), 0.85-0.78 (m, 24H). HRMS: m / z calcd: 1282.7962 (M+H + ), measured value: 1282.7960.

[0218] Example 13: Synthesis of Compound II-3

[0219]

[0220] Compound 53 (1.0 eq) was dissolved in DCM, and compound 16 (2.0 eq) was added. 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 give compound 63 (yield: 65%). Compound 63 (1.0 eq) and compound 9 (POCl3, 1.5 eq) were dissolved in THF, and TEA (1.5 eq) was added. The mixture was stirred at 0°C for 12 h. After completion of the reaction, the mixture was separated by column chromatography (DCM:MeOH = 15:1) to give compound 64 (yield: 78%). Compound 64 (1.0 eq) and compound 11 (1.2 eq) were dissolved in THF, and TEA (1.5 eq) was added. The mixture was stirred at room temperature for 12 h. After completion of the reaction, the mixture was separated by column chromatography (DCM:MeOH = 20:1) to give compound 65 (yield: 73%). Compound 65 (1.0 eq) was dissolved in DCM, TEA (1.5 eq) was added, and the mixture was stirred at room temperature for 12 h. After the reaction was completed, the mixture was separated by column chromatography (DCM:MeOH=10:1) to obtain compound II-3 (yield: 74%). 1 H NMR (400 MHz, CDCl3, 293 K) δ (ppm): 4.36-4.25 (m, 2H), 4.16-3.94 (m, 5H), 3.30-3.16 (m, 2H), 2.79-2.61 (m, 4H), 2.21 (s, 6H), 2.19 (s, 6H), 2.17 (s, 6H), 1.98-1.88 (m, 4H), 1.73-1.13 (m, 46H), 1.07-1.00 (m, 4H), 0.88-0.80 (m, 24H). HRMS: m / z calculated: 1183.5998 (M+H + ), measured value: 1183.5991.

[0221] Example 14: Synthesis of Compound II-4

[0222]

[0223] Compound 61 (1.0 eq) was dissolved in DCM, and compound 16 (2.0 eq) was added, and stirred at room temperature for 6 h. After the reaction was completed, the mixture was washed with water and purified by column chromatography (DCM:MeOH=10:1) to obtain compound II-4 (yield: 85%). 1H NMR (400 MHz, CDCl3, 293 K) δ (ppm): 4.33-4.22 (m, 4H), 4.17-4.11 (m, 1H), 4.03-3.90 (m, 2H), 3.71-3.66 (m, 2H), 3.28 (s, 9H), 2.78-2.55 (m, 4H), 2.21 (s, 6H), 2.19 (s, 6H), 2.17 (s, 6H), 1.98-1.88 (m, 2H), 1.70-0.98 (m, 50H), 0.87-0.80 (m, 24H). HRMS: m / z calcd: 1226.6882 (M+H + ), measured value: 1226.6878.

[0224] Example 15: Synthesis of Compound II-5

[0225]

[0226] Compound 53 (1.0 eq) was dissolved in DCM, and compound 16 (2.0 eq) was added. 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 give compound 68 (yield: 63%). Compound 68 (1.0 eq) was dissolved in DCM, and compound 69 (1.2 eq) was added. EDC (2.0 eq) and DMAP (0.1 eq) were added, and the mixture was stirred at room temperature for 12 h. The mixture was purified by column chromatography (DCM:MeOH = 20:1) to give compound 70 (yield: 53%). Compound 70 (1.0 eq) and compound 9 (POCl3, 1.5 eq) were dissolved in THF, and TEA (1.5 eq) was added. The mixture was stirred at 0°C for 12 h. After completion of the reaction, the mixture was separated by column chromatography (DCM:MeOH = 15:1) to give compound 71 (yield: 68%). Compound 71 (1.0 eq) and compound 11 (1.2 eq) were dissolved in THF, TEA (1.5 eq) was added, and the mixture was stirred at room temperature for 12 h. After completion of the reaction, the mixture was separated by column chromatography (DCM:MeOH = 20:1) to obtain compound 72 (yield: 69%). Compound 72 (1.0 eq) was dissolved in DCM, TEA (1.5 eq) was added, and the mixture was stirred at room temperature for 12 h. After completion of the reaction, the mixture was separated by column chromatography (DCM:MeOH = 10:1) to obtain compound II-5 (yield: 55%). 1H NMR (400 MHz, CDCl3, 293 K) δ (ppm): 5.17-5.06 (m, 3H), 4.21-3.92 (m, 7H), 3.24-3.16 (m, 2H), 2.89-2.64 (m, 8H), 2.21 (s, 6H), 2.19 (s, 6H), 2.17 (s, 6H), 2.15-0.95 (m, 57H), 0.85-0.80 (m, 12H). HRMS: m / z calcd: 1205.6058 (M+H + ), measured value: 1205.6049.

[0227] Example 16: Synthesis of Compound II-6

[0228]

[0229] Compound 4 (1.0 eq) and compound 74 (1.0 eq) were dissolved in DCM, EDC (2.0 eq) and DMAP (0.1 eq) were added, the mixture was stirred at room temperature for 12 h, and purified by column chromatography (DCM:MeOH = 10:1) to give compound 75 (yield: 63%). Compound 75 (1.0 eq) and compound 53 (1.0 eq) were dissolved in DCM, EDC (2.0 eq) and DMAP (0.1 eq) were added, the mixture was stirred at room temperature for 12 h, and after completion of the reaction, the mixture was washed with water and purified by column chromatography (DCM:MeOH = 15:1) to give compound 76 (yield: 87%). Compound 76 (1.0 eq) and compound 69 (1.0 eq) were dissolved in DCM, EDC (2.0 eq) and DMAP (0.1 eq) were added, the mixture was stirred at room temperature for 12 h, and purified by column chromatography (DCM:MeOH = 15:1) to give compound 77 (yield: 73%). Compound 77 (1.0 eq) and compound 9 (POCl3, 1.5 eq) were dissolved in THF, TEA (1.5 eq) was added, and the mixture was stirred at 0°C for 12 h. After completion of the reaction, the mixture was separated by column chromatography (DCM:MeOH = 15:1) to obtain compound 78 (yield: 78%). Compound 78 (1.0 eq) and compound 11 (1.2 eq) were dissolved in THF, TEA (1.5 eq) was added, and the mixture was stirred at room temperature for 12 h. After completion of the reaction, the mixture was separated by column chromatography (DCM:MeOH = 20:1) to obtain compound 79 (yield: 73%). Compound 79 (1.0 eq) was dissolved in DCM, TEA (1.5 eq) was added, and the mixture was stirred at room temperature for 12 h. After completion of the reaction, the mixture was separated by column chromatography (DCM:MeOH = 10:1) to obtain compound II-6 (yield: 83%). 1H NMR (400MHz, CDCl3, 293K) δ (ppm): 5.19-5.04 (m, 4H), 4.37-3.94 (m, 11H), 3.77-3.72 (m, 4H), 3.67 (s, 4H), 3.26-3.16 (m, 2H), 2. 89-2.64(m,12H),2.21(s,6H),2.19(s,6H),2.17(s,6H),2.15-0.98(m,57H),0.89-0.80(m,12H).HRMS: m / z calculated value: 1409.8278(M+H + ), measured value: 1409.8285.

[0230] Example 17: Synthesis of Compound II-7

[0231]

[0232] Compound 14 (1.0 eq) and compound 81 (1.0 eq) were dissolved in DCM, EDC (2.0 eq) and DMAP (0.1 eq) were added, the mixture was stirred at room temperature for 12 h, and the mixture was purified by column chromatography (DCM:MeOH = 10:1) to give compound 82 (yield: 72%). Compound 82 (1.0 eq) and compound 53 (1.0 eq) were dissolved in DCM, EDC (2.0 eq) and DMAP (0.1 eq) were added, the mixture was stirred at room temperature for 12 h, and the mixture was purified by column chromatography (DCM:MeOH = 15:1) to give compound 83 (yield: 67%). Compound 83 (1.0 eq) and compound 84 (1.0 eq) were dissolved in DCM, 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, and the mixture was purified by column chromatography (DCM:MeOH = 18:1) to give compound 85 (yield: 72%). Compound 85 (1.0 eq) and compound 9 (POCl3, 1.5 eq) were dissolved in THF, TEA (1.5 eq) was added, and the mixture was stirred at 0°C for 12 h. After completion of the reaction, the mixture was separated by column chromatography (DCM:MeOH = 15:1) to obtain compound 86 (yield: 78%). Compound 86 (1.0 eq) and compound 11 (1.2 eq) were dissolved in THF, TEA (1.5 eq) was added, and the mixture was stirred at room temperature for 12 h. After completion of the reaction, the mixture was separated by column chromatography (DCM:MeOH = 20:1) to obtain compound 87 (yield: 81%). Compound 87 (1.0 eq) was dissolved in DCM, TEA (1.5 eq) was added, and the mixture was stirred at room temperature for 12 h. After completion of the reaction, the mixture was separated by column chromatography (DCM:MeOH = 10:1) to obtain compound II-7 (yield: 82%). 1H NMR (400 MHz, CDCl3, 293 K) δ (ppm): 5.40-5.35 (m, 2H), 4.25-3.96 (m, 7H), 3.25-3.17 (m, 2H), 2.75-2.60 (m, 4H), 2.38-2.28 (m, 2H), 2.25-2.25 (m, 11H), 2.02-1.88 (m, 7H), 1.69-0.98 (m, 57H), 0.90-0.80 (m, 15H). HRMS: m / z calcd: 1061.5208 (M+H + ), measured value: 1061.5201.

[0233] Example 18: Synthesis of Compound II-8

[0234]

[0235] Compound 60 (1.0 eq) was dissolved in DCM, and compound 16 (1.2 eq) was added. The mixture was stirred at room temperature for 6 h. After the reaction was complete, the mixture was washed with water and purified by column chromatography (DCM:MeOH = 10:1) to give compound 89 (yield: 45%). Compound 89 (1.0 eq) was dissolved in THF, 15% TFA was added, and the mixture was stirred at room temperature for 3 h. After the reaction was complete, the mixture was washed with water, and the organic solvent was removed in vacuo to give compound 90 (yield: 78%). Compound 90 (1.0 eq) and compound 91 (1.0 eq) were dissolved in DCM, and NHS (1.0 eq), EDC (1.0 eq), and DMAP (0.1 eq) were added. The mixture was stirred at room temperature for 12 h. After the reaction was complete, the mixture was washed with water and purified by column chromatography (DCM:MeOH = 15:1) to give compound II-8 (yield: 74%). 1 H NMR (400 MHz, CDCl3, 293 K) δ (ppm): 4.33-4.21 (m, 4H), 4.06-3.95 (m, 3H), 3.67-3.65 (m, 2H), 3.27 (s, 9H), 2.75-2.62 (m, 2H), 2.27-2.14 (m, 11H), 1.93-1.86 (m, 1H), 1.71-1.10 (m, 42H), 1.06-0.98 (m, 2H), 0.93-0.80 (m, 15H). HRMS: m / z calcd: 938.3012 (M+H + ), measured value: 938.3008.

[0236] Example 19: Synthesis of Compound II-9

[0237]

[0238] Compound 4 (1.0 eq) and compound 53 (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 the reaction was completed, the mixture was washed with water and purified by column chromatography (DCM:MeOH=15:1) to give compound 93 (yield: 52%). Compound 93 (1.0 eq) and compound 94 (1.0 eq) were dissolved in DCM, NHS (1.0 eq), EDC (1.0 eq) and DMAP (0.1 eq) were added, and the mixture was stirred at room temperature for 12 h. After the reaction was completed, the mixture was washed with water and purified by column chromatography (DCM:MeOH=15:1) to give compound 95 (yield: 52%). Compound 95 (1.0 eq) and compound 9 (POCl3, 1.5 eq) were dissolved in THF, TEA (1.5 eq) was added, and the mixture was stirred at 0°C for 12 h. After completion of the reaction, the mixture was separated by column chromatography (DCM:MeOH = 15:1) to obtain compound 96 (yield: 77%). Compound 96 (1.0 eq) and compound 11 (1.2 eq) were dissolved in THF, TEA (1.5 eq) was added, and the mixture was stirred at room temperature for 12 h. After completion of the reaction, the mixture was separated by column chromatography (DCM:MeOH = 20:1) to obtain compound 97 (yield: 73%). Compound 97 (1.0 eq) was dissolved in DCM, TEA (1.5 eq) was added, and the mixture was stirred at room temperature for 12 h. After completion of the reaction, the mixture was separated by column chromatography (DCM:MeOH = 10:1) to obtain compound II-9 (yield: 46%). 1 H NMR (400 MHz, CDCl3, 293 K) δ (ppm): 5.45-5.34 (m, 8H), 4.28-3.96 (m, 7H), 3.23-3.16 (m, 2H), 2.86-2.64 (m, 12H), 2.27-2.15 (m, 11H), 2.09-1.88 (m, 7H), 1.70-0.96 (35H), 0.89-0.72 (m, 15H). HRMS: m / z calculated: 1027.4188 (M+H + ), measured value: 1027.4179.

[0239] Example 20: Synthesis of Compound II-10

[0240]

[0241] Compound 61 (1.0 eq) was dissolved in DCM, and compound 4 (1.2 eq) was added. 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=10:1) to give compound 99 (yield: 63%). Compound 99 (1.0 eq) and compound 30 (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 washed with water and purified by column chromatography (DCM:MeOH=20:1) to give compound II-10 (yield: 73%). 1 H NMR (400 MHz, CDCl3, 293 K) δ (ppm): 4.31-4.27 (m, 2H), 4.16-4.10 (m, 2H), 4.02-3.84 (m, 3H), 3.71-3.66 (m, 2H), 3.25 (s, 9H), 2.92-2.82 (m, m, 2H), 2.75-2.63 (m, 4H), 2.27-2.23 (m, 2H), 2.21 (s, 3H), 2.19 (s, 3H), 2.17 (s, 3H), 1.93-1.88 (m, 1H), 1.69-0.98 (m, 54H), 0.92-0.76 (m, 15H). HRMS: m / z calcd: 1050.5172 (M+H + ), measured value: 1050.5168.

[0242] Example 21: Synthesis of Compound III-1

[0243]

[0244] Compound 16 (1.0 eq) and compound 101 (1.0 eq) were dissolved in DCM, TEA (1.5 eq) was added, and the mixture was stirred at room temperature for 12 h. The mixture was purified by column chromatography (DCM:MeOH = 10:1) to give compound 102 (yield: 64%). Compound 102 (1.0 eq) and compound 9 (POCl3, 1.5 eq) were dissolved in THF, TEA (1.5 eq) was added, and the mixture was stirred at 0°C for 12 h. After completion of the reaction, the mixture was separated by column chromatography (DCM:MeOH = 15:1) to give compound 103 (yield: 75%). Compound 103 (1.0 eq) and compound 11 (1.2 eq) were dissolved in THF, TEA (1.5 eq) was added, and the mixture was stirred at room temperature for 12 h. After completion of the reaction, the mixture was separated by column chromatography (DCM:MeOH = 20:1) to give compound 104 (yield: 70%). Compound 104 (1.0 eq) was dissolved in DCM, and TEA (1.5 eq) was added. The mixture was stirred at room temperature for 12 h. After the reaction was completed, the mixture was separated by column chromatography (DCM:MeOH=10:1) to obtain compound III-1 (yield: 57%). 1 H NMR (400 MHz, CDCl 3 , 293 K) δ (ppm): 4.20-4.04 (m, 8H), 3.23-3.16 (m, 2H), 2.79-2.67 (m, 4H), 2.56-2.47 (m, 1H), 2.21 (s, 6H), 2.19 (s, 6H), 2.17 (s, 6H), 1.98-1.88 (m, 4H), 1.74-0.96 (m, 50H), 0.90-0.81 (m, 24H). HRMS: m / z calculated: 1198.6108 (M+H + ), measured value: 1198.6103.

[0245] Example 22: Synthesis of Compound III-2

[0246]

[0247] Compound 1 (1.0 eq) and compound 101 (1.0 eq) were dissolved in DCM, TEA (1.5 eq) was added, and the mixture was stirred at room temperature for 12 h. The mixture was purified by column chromatography (DCM:MeOH=10:1) to give compound 106 (yield: 53%). Compound 106 (1.0 eq) was dissolved in DCM, compound 16 (1.2 eq) was added, TEA (1.5 eq) was added, and the mixture was stirred at room temperature for 6 h. After the reaction was completed, the mixture was washed with water and purified by column chromatography (DCM:MeOH=15:1) to give compound III-2 (yield: 47%). 1H NMR (400 MHz, CDCl3, 293 K) δ (ppm): 4.35-4.26 (m, 2H), 4.19-3.95 (m, 6H), 3.67-3.63 (m, 2H), 3.25 (s, 9H), 2.79-2.64 (m, 4H), 2.57-2.48 (m, 1H), 2.21 (s, 6H), 2.19 (s, 6H), 2.17 (s, 6H), 1.98-1.89 (m, 2H), 1.69-1.01 (m, 50H), 0.87-0.80 (m, 24H). HRMS: m / z calcd: 1241.6992 (M+H + ), measured value: 1241.6999.

[0248] Example 23: Synthesis of Compound III-3

[0249]

[0250] Compound 101 (1.0 eq) was dissolved in DCM, and compound 6 (2.0 eq) was added. 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 give compound 108 (yield: 78%). Compound 108 (1.0 eq) and compound 9 (POCl3, 1.5 eq) were dissolved in THF, and TEA (1.5 eq) was added. The mixture was stirred at 0°C for 12 h. After completion of the reaction, the mixture was separated by column chromatography (DCM:MeOH = 15:1) to give compound 109 (yield: 65%). Compound 109 (1.0 eq) and compound 11 (1.2 eq) were dissolved in THF, and TEA (1.5 eq) was added. The mixture was stirred at room temperature for 12 h. After completion of the reaction, the mixture was separated by column chromatography (DCM:MeOH = 20:1) to give compound 110 (yield: 46%). Compound 110 (1.0 eq) was dissolved in DCM, and TEA (1.5 eq) was added. The mixture was stirred at room temperature for 12 h. After the reaction was completed, the mixture was separated by column chromatography (DCM:MeOH=10:1) to obtain compound III-3 (yield: 64%). 1 H NMR (400 MHz, CDCl3, 293 K) δ (ppm): 4.19-3.96 (m, 8H), 3.23-3.17 (m, 2H), 2.86-2.81 (m, 4H), 2.75-2.63 (m, 8H), 2.54-2.47 (m, 1H), 2.21 (s, 6H), 2.19 (s, 6H), 2.17 (s, 6H), 2.00-1.88 (m, 4H), 1.71-0.98 (m, 50H), 0.89-0.77 (m, 24H). HRMS: m / z calcd: 1254.7188 (M+H +), measured value: 1254.7180.

[0251] Example 24: Synthesis of Compound III-4

[0252]

[0253] Compound 106 (1.0 eq) was dissolved in DCM, and compound 6 (2.0 eq) was added, and stirred at room temperature for 6 h. After the reaction was completed, the mixture was washed with water and purified by column chromatography (DCM:MeOH=20:1) to obtain compound III-4 (yield: 53%). 1 H NMR (400 MHz, CDCl3, 293 K) δ (ppm): 4.32-4.28 (m, 2H), 4.16-4.01 (m, 6H), 3.66-3.63 (m, 2H), 3.27 (s, 9H), 2.89-2.64 (m, 12H), 2.58-2.49 (m, 1H), 2.21 (s, 6H), 2.19 (s, 6H), 2.17 (s, 6H), 1.95-1.88 (m, 2H), 1.71-0.98 (m, 50H), 0.85-0.80 (m, 24H). HRMS: m / z calcd: 1297.8072 (M+H + ), measured value: 1297.8067.

[0254] Example 25: Synthesis of Compound III-5

[0255]

[0256] Compound 14 (1.0 eq) and compound 113 (1.0 eq) were dissolved in DCM, EDC (2.0 eq) and DMAP (0.1 eq) were added, the mixture was stirred at room temperature for 12 h, and the mixture was purified by column chromatography (DCM:MeOH = 10:1) to give compound 114 (yield: 46%). Compound 114 (1.0 eq) and compound 101 (1.0 eq) were dissolved in DCM, EDC (2.0 eq) and DMAP (0.1 eq) were added, the mixture was stirred at room temperature for 12 h, and the mixture was purified by column chromatography (DCM:MeOH = 15:1) to give compound 115 (yield: 64%). Compound 115 (1.0 eq) and compound 4 (1.0 eq) were dissolved in DCM, EDC (2.0 eq) and DMAP (0.1 eq) were added, the mixture was stirred at room temperature for 12 h, and the mixture was purified by column chromatography (DCM:MeOH = 18:1) to give compound 116 (yield: 78%). Compound 116 (1.0 eq) and compound 9 (POCl3, 1.5 eq) were dissolved in THF, TEA (1.5 eq) was added, and the mixture was stirred at 0°C for 12 h. After completion of the reaction, the mixture was separated by column chromatography (DCM:MeOH = 15:1) to obtain compound 117 (yield: 74%). Compound 117 (1.0 eq) and compound 11 (1.2 eq) were dissolved in THF, TEA (1.5 eq) was added, and the mixture was stirred at room temperature for 12 h. After completion of the reaction, the mixture was separated by column chromatography (DCM:MeOH = 20:1) to obtain compound 118 (yield: 76%). Compound 118 (1.0 eq) was dissolved in DCM, TEA (1.5 eq) was added, and the mixture was stirred at room temperature for 12 h. After completion of the reaction, the mixture was separated by column chromatography (DCM:MeOH = 10:1) to obtain compound III-5 (yield: 74%). 1 H NMR (400 MHz, CDCl3, 293 K) δ (ppm): 4.19-4.02 (m, 8H), 3.21-3.16 (m, 2H), 2.87-2.81 (m, 2H), 2.79-2.59 (m, 8H), 2.54-2.47 (m, 1H), 2.34-2.30 (m, 2H), 2.21 (s, 6H), 2.19 (s, 9H), 2.17 (s, 9H), 1.98-1.86 (m, 4H), 1.71-0.98 (m, 56H), 0.85-0.78 (m, 24H). HRMS: m / z calcd: 1296.7998 (M+H + ), measured value: 1296.7991.

[0257] Example 26: Synthesis of Compound III-6

[0258]

[0259] Compound 106 (1.0 eq) was dissolved in DCM, and compound 16 (1.2 eq) was added, 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=10:1) to give compound 120 (yield: 64%). Compound 120 (1.0 eq) was dissolved in DCM, and compound 6 (1.2 eq) was added, 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 give compound III-6 (yield: 74%). 1 H NMR (400 MHz, CDCl3, 293 K) δ (ppm): 4.33-4.02 (m, 8H), 3.67-3.63 (m, 2H), 3.25 (s, 9H), 2.89-2.82 (m, 2H), 2.76-2.62 (m, 6H), 2.55-2.45 (m, 1H), 2.21 (s, 6H), 2.19 (s, 6H), 2.17 (s, 6H), 1.97-1.86 (m, 2H), 1.71-0.98 (m, 50H), 0.89-0.80 (m, 24H). HRMS: m / z calcd: 1269.7532 (M+H + ), measured value: 1269.7532.

[0260] Example 27: Synthesis of Compound III-7

[0261]

[0262] Compound 122 (1.0 eq) and compound 123 (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 washed with water and purified by column chromatography (DCM:MeOH=10:1) to give compound 124 (yield: 56%). Compound 124 (1.0 eq) and compound 125 (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 washed with water and purified by column chromatography (DCM:MeOH=10:1) to give compound 126 (yield: 74%). Compound 126 (1.0 eq) and compound 101 (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 washed with water and purified by column chromatography (DCM:MeOH = 15:1) to give compound 127 (yield: 76%). Compound 127 (1.0 eq) and compound 16 (1.0 eq) were dissolved in DCM, TEA (1.5 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 and purified by column chromatography (DCM:MeOH = 20:1) to give compound 128 (yield: 56%). Compound 128 (1.0 eq) and compound 9 (POCl3, 1.5 eq) were dissolved in THF, TEA (1.5 eq) was added, and the mixture was stirred at 0°C for 12 h. After completion of the reaction, the mixture was separated by column chromatography (DCM:MeOH = 15:1) to give compound 129 (yield: 75%). Compound 129 (1.0 eq) and compound 11 (1.2 eq) were dissolved in THF, TEA (1.5 eq) was added, and the mixture was stirred at room temperature for 12 h. After completion of the reaction, the mixture was separated by column chromatography (DCM:MeOH = 20:1) to obtain compound 130 (yield: 73%). Compound 130 (1.0 eq) was dissolved in DCM, TEA (1.5 eq) was added, and the mixture was stirred at room temperature for 12 h. After completion of the reaction, the mixture was separated by column chromatography (DCM:MeOH = 10:1) to obtain compound III-7 (yield: 46%). 1H NMR (400MHz, CDCl3, 293K) δ (ppm): 7.53-7.49 (m, 2H), 6.83-6.78 (s, 1H), 4.77-4.64 (m, 1 H),4.30-3.94(m,10H),3.27-3.16(m,2H),3.09-2.96(m,2H),2.75-2.64(m,3H),2.57-2. 48 (m, 1H), 2.34-2.23 (m, 4H), 2.21 (s, 3H), 2.19 (s, 3H), 2.17 (s, 3H), 1.98-1.88 (m, 3H), 1.71-1.14 (m, 61H), 1.06-0.98 (m, 2H), 0.91-0.80 (m, 15H). HRMS: m / z calculated value: 1244.6238 (M+H + ), measured value: 1244.6230.

[0263] Example 28: Synthesis of Compound III-8

[0264]

[0265] Compound 132 (1.0 eq) and compound 133 (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 washed with water and purified by column chromatography (DCM:MeOH=10:1) to give compound 134 (yield: 64%). Compound 120 (1.0 eq) and compound 134 (1.0 eq) were dissolved in DCM, and EDC (1.0 eq) and DMAP (0.1 eq) were added. The mixture was stirred at room temperature for 12 h. After completion of the reaction, the mixture was washed with water and purified by column chromatography (DCM:MeOH=16:1) to give compound III-8 (yield: 54%). 1 H NMR (400 MHz, CDCl3, 293 K) δ (ppm): 4.36-4.02 (m, 8H), 3.92-3.86 (m, 2H), 3.69-3.66 (m, 2H), 3.27 (m, 9H), 2.80-2.59 (m, 2H), 2.53-2.47 (m, 1H), 2.30-2.24 (m, 2H), 2.21 (s, 3H), 2.19 (s, 3H), 2.17 (s, 3H), 1.95-1.86 (m, 1H), 1.71-0.98 (m, 46H), 0.92-0.70 (m, 15H). HRMS: m / z calcd: 1024.3912 (M+H + ), measured value: 1024.3906.

[0266] Example 29: Synthesis of Compound III-9

[0267]

[0268] Compound 136 (1.0 eq) and compound 137 (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 the reaction, the mixture was washed with water and purified by column chromatography (DCM:MeOH = 10:1) to give compound 138 (yield: 75%). Compound 138 (1.0 eq) and compound 101 (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 the reaction, the mixture was washed with water and purified by column chromatography (DCM:MeOH = 10:1) to give compound 139 (yield: 55%). Compound 139 (1.0 eq) was dissolved in DCM, compound 6 (1.2 eq) was added, and the mixture was stirred at room temperature for 6 h. After the reaction, the mixture was washed with water and purified by column chromatography (DCM:MeOH = 20:1) to give compound 140 (yield: 56%). Compound 140 (1.0 eq) and compound 9 (POCl3, 1.5 eq) were dissolved in THF, TEA (1.5 eq) was added, and the mixture was stirred at 0°C for 12 h. After completion of the reaction, the mixture was separated by column chromatography (DCM:MeOH = 15:1) to obtain compound 141 (yield: 75%). Compound 141 (1.0 eq) and compound 11 (1.2 eq) were dissolved in THF, TEA (1.5 eq) was added, and the mixture was stirred at room temperature for 12 h. After completion of the reaction, the mixture was separated by column chromatography (DCM:MeOH = 20:1) to obtain compound 142 (yield: 73%). Compound 142 (1.0 eq) was dissolved in DCM, TEA (1.5 eq) was added, and the mixture was stirred at room temperature for 12 h. After completion of the reaction, the mixture was separated by column chromatography (DCM:MeOH = 10:1) to obtain compound III-9 (yield: 75%). 1 H NMR (400MHz, CDCl3, 293K) δ (ppm): 4.37-4.31 (m, 2H), 4.18-4.01 (m, 8H), 3.81-3.73 (m,4H),3.68-3.58(m,4H),3.21-3.15(m,2H),2.88-2.82(m,2H),2.75-2.64(m,4H) ,2.57-2.47(m,3H),2.34-2.29(m,2H),2.21(s,3H),2.19(s,3H),2.17(s,3H),1.98-1.86(m,3H),1.73-0.98(m,38H),0.93-0.77(m,15H).HRMS: m / z calculated value: 1056.3658(M+H +), measured value: 1056.3651.

[0269] Example 30: Synthesis of Compound III-10

[0270]

[0271] Compound 106 (1.0 eq) was dissolved in DCM, compound 6 (1.2 eq) was 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 = 10:1) to give compound 144 (yield: 64%). Compound 145 (1.0 eq) and compound 146 (1.0 eq) were dissolved in DCM, stirred at room temperature for 6 h, and washed with water to give compound 147 (yield: 87%). Compound 147 (1.0 eq) was dissolved in DCM, SOCl2 (1.2 eq) was added, and the mixture was stirred at room temperature for 6 h. After completion of the reaction, the solvent was removed in vacuo to give compound 148 (yield: 65%). Compound 144 (1.0 eq) was dissolved in DCM, compound 148 (1.2 eq) was 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 give compound III-10 (yield: 57%). 1 H NMR (400 MHz, CDCl3, 293 K) δ (ppm): 4.36-4.28 (m, 2H), 4.18-3.91 (m, 8H), 3.67-3.64 (m, 2H), 3.25 (s, 9H), 2.89-2.82 (m, 2H), 2.75-2.63 (m, 8H), 2.55-2.47 (m, 1H), 2.21 (s, 3H), 2.19 (s, 3H), 2.17 (s, 3H), 1.95-1.86 (m, 1H), 1.74-0.98 (m, 49H), 0.94-0.80 (m, 18H). HRMS: m / z calcd: 1109.5372 (M+H + ), measured value: 1109.5367.

[0272] Example 31: Synthesis of Compound IV-1

[0273]

[0274] Compound 16 (1.0 eq) and compound 150 (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. The mixture was purified by column chromatography (DCM:MeOH = 10:1) to give compound 151 (yield: 46%). Compound 151 (1.0 eq) and compound 9 (POCl3, 1.5 eq) were dissolved in THF, TEA (1.5 eq) was added, and the mixture was stirred at 0°C for 12 h. After completion of the reaction, the mixture was separated by column chromatography (DCM:MeOH = 15:1) to give compound 152 (yield: 75%). Compound 152 (1.0 eq) and compound 11 (1.2 eq) were dissolved in THF, TEA (1.5 eq) was added, and the mixture was stirred at room temperature for 12 h. After completion of the reaction, the mixture was separated by column chromatography (DCM:MeOH = 20:1) to give compound 153 (yield: 73%). Compound 153 (1.0 eq) was dissolved in DCM, and TEA (1.5 eq) was added. The mixture was stirred at room temperature for 12 h. After the reaction was completed, the mixture was separated by column chromatography (DCM:MeOH=10:1) to obtain compound IV-1 (yield: 74%). 1 H NMR (400MHz, CDCl3, 293K) δ (ppm): 4.43-4.36 (m, 4H), 4.17-4.00 (m, 4H), 3.20-3.16 (m, 2H), 3.00-2.88 (m, 6H), 2.81-2.64 (m, 4H) ,2.21(s,6H),2.19(s,6H),2.15(s,6H),1.96-1.88(s,4H),1.69-0.98(m,50H),0.87-0.78(m,24H).HRMS: m / z calculated value: 1241.6798(M+H + ), measured value: 1241.6790.

[0275] Example 32: Synthesis of Compound IV-2

[0276]

[0277] Compound 1 (1.0 eq) and compound 150 (1.0 eq) were dissolved in DCM, TEA (1.5 eq) was added, and the mixture was stirred at room temperature for 12 h. The mixture was purified by column chromatography (DCM:MeOH=10:1) to give compound 155 (yield: 57%). Compound 155 (1.0 eq) was dissolved in DCM, compound 16 (1.2 eq) was added, TEA (1.5 eq) was added, and the mixture was stirred at room temperature for 6 h. After the reaction was completed, the mixture was washed with water and purified by column chromatography (DCM:MeOH=20:1) to give compound IV-2 (yield: 75%). 1H NMR (400MHz, CDCl3, 293K) δ (ppm): 4.43-4.32 (m, 4H), 4.28-4.18 (m, 2H), 4.17-4.14 (m, 2H), 3.70-3.66 (m, 2H), 3.25 (s, 9H), 3.02-2.93 (m, 6H), 2. 75-2.60(m,4H),2.21(s,6H),2.19(s,6H),2.16(s,6H),1.96-1.86(m,2H ),1.71-0.98(m,50H),0.89-0.80(m,24H).HRMS:m / z calculated value:1284.7682(M+H + ), measured value: 1284.7677.

[0278] Example 33: Synthesis of Compound IV-3

[0279]

[0280] Compound 150 (1.0 eq) was dissolved in DCM, and compound 6 (2.0 eq) was added. 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 give compound 157 (yield: 63%). Compound 157 (1.0 eq) and compound 9 (POCl3, 1.5 eq) were dissolved in THF, and TEA (1.5 eq) was added. The mixture was stirred at 0°C for 12 h. After completion of the reaction, the mixture was separated by column chromatography (DCM:MeOH = 15:1) to give compound 158 (yield: 75%). Compound 158 (1.0 eq) and compound 11 (1.2 eq) were dissolved in THF, and TEA (1.5 eq) was added. The mixture was stirred at room temperature for 12 h. After completion of the reaction, the mixture was separated by column chromatography (DCM:MeOH = 20:1) to give compound 159 (yield: 73%). Compound 159 (1.0 eq) was dissolved in DCM, and TEA (1.5 eq) was added. The mixture was stirred at room temperature for 12 h. After the reaction was completed, the mixture was separated by column chromatography (DCM:MeOH=10:1) to obtain compound IV-3 (yield: 73%). 1H NMR (400MHz, CDCl3, 293K) δ (ppm): 4.33-4.27 (m, 4H), 4.18-4.02 (m, 4H), 3.24-3.16 (m, 2H), 3.03-2.96 (m, 6H), 2.85-2.82 (m, 4H), 2.75-2. 61(m,8H),2.21(s,6H),2.19(s,6H),2.16(s,6H),1.98-1.88(m,4H),1.71-0.98(m,50H),0.88-0.80(m,24H).HRMS: m / z calculated value: 1297.7878(M+H + ), measured value: 1297.7871.

[0281] Example 34: Synthesis of Compound IV-4

[0282]

[0283] Compound 155 (1.0 eq) was dissolved in DCM, and compound 6 (2.0 eq) was added, and stirred at room temperature for 6 h. After the reaction was completed, the mixture was washed with water and purified by column chromatography (DCM:MeOH=15:1) to obtain compound IV-4 (yield: 71%). 1 H NMR (400 MHz, CDCl3, 293 K) δ (ppm): 4.31-4.15 (m, 6H), 4.18-4.12 (m, 2H), 3.66-3.64 (m, 2H), 3.29 (s, 9H0), 3.04-2.94 (m, 6H), 2.88-2.82 (m, 4H), 2.74-2.62 (m, 8H), 2.21 (s, 6H), 2.19 (s, 6H), 2.17 (s, 6H), 1.95-1.86 (m, 2H), 1.69-0.95 (m, 50H), 0.89-0.78 (m, 24H). HRMS: m / z calcd: 1340.8762 (M+H + ), measured value: 1340.8762.

[0284] Example 35: Synthesis of Compound IV-5

[0285]

[0286] Compound 150 (1.0 eq) was dissolved in DCM, and compound 6 (1.2 eq) was added. 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 = 10:1) to give compound 162 (yield: 82%). Compound 162 (1.0 eq) was dissolved in DCM, and compound 16 (1.2 eq) was added. 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 = 16:1) to give compound 163 (yield: 63%). Compound 163 (1.0 eq) and compound 9 (POCl3, 1.5 eq) were dissolved in THF, and TEA (1.5 eq) was added. The mixture was stirred at 0°C for 12 h. After completion of the reaction, the mixture was separated by column chromatography (DCM:MeOH = 15:1) to give compound 164 (yield: 75%). Compound 164 (1.0 eq) and compound 11 (1.2 eq) were dissolved in THF, TEA (1.5 eq) was added, and the mixture was stirred at room temperature for 12 h. After completion of the reaction, the mixture was separated by column chromatography (DCM:MeOH = 20:1) to obtain compound 165 (yield: 73%). Compound 165 (1.0 eq) was dissolved in DCM, TEA (1.5 eq) was added, and the mixture was stirred at room temperature for 12 h. After completion of the reaction, the mixture was separated by column chromatography (DCM:MeOH = 10:1) to obtain compound IV-5 (yield: 63%). 1 H NMR (400MHz, CDCl3, 293K) δ (ppm): 4.44-4.36 (m, 2H), 4.31-4.24 (m, 2H), 4.18-4.04 (m, 4H), 3.21-3.16 (m, 2H), 3.02-2.96 (m, 6H), 2.8 6-2.82(m,2H),2.75-2.65(m,6H),2.21(s,6H),2.19(s,6H),2.15(s,6H),1.98-1.88(m,4H),1.70-0.98(m,50H),0.87-0.78(m,24H). HRMS:m / z calculated value:1269.7338(M+H + ), measured value: 1269.7335.

[0287] Example 36: Synthesis of Compound IV-6

[0288]

[0289] Compound 4 (1.0 eq) and compound 123 (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 washed with water and purified by column chromatography (DCM:MeOH=10:1) to give compound 167 (yield: 74%). Compound 167 (1.0 eq) and compound 155 (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 washed with water and purified by column chromatography (DCM:MeOH=15:1) to give compound 168 (yield: 46%). Compound 168 (1.0 eq) and compound 4 (1.0 eq) were dissolved in DCM, and EDC (1.0 eq) and DMAP (0.1 eq) were added. The mixture was stirred at room temperature for 12 h. After the reaction was completed, the mixture was washed with water and purified by column chromatography (DCM:MeOH=20:1) to obtain compound IV-6 (yield: 84%). 1 H NMR (400 MHz, CDCl3, 293 K) δ (ppm): 7.51-7.49 (m, 1H), 6.80-6.78 (m, 1H), 4.69-4.62 (m, 1H), 4.32-4.27 (m, 6H), 4.18-4.15 (m, 2H), 3.67-3.63 (m, 2H), 3.25 (s, 9H), 3.11-2.56 (m, 21H), 2.21 (s, 6H), 2.19 (s, 6H), 2.17 (s, 6H), 1.95-1.87 (m, 2H), 1.69-0.96 (m, 50H), 0.87-0.78 (m, 24H). HRMS: m / z calcd: 1478.0182 (M+H + ), measured value: 1478.0182.

[0290] Example 37: Synthesis of Compound IV-7

[0291]

[0292] Compound 150 (1.0 eq) was dissolved in DCM, and compound 16 (1.0 eq) was added. 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 give compound 170 (yield: 62%). Compound 170 (1.0 eq) and compound 30 (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 washed with water and purified by column chromatography (DCM:MeOH = 20:1) to give compound 171 (yield: 65%). Compound 171 (1.0 eq) and compound 9 (POCl3, 1.5 eq) were dissolved in THF, and TEA (1.5 eq) was added. The mixture was stirred at 0°C for 12 h. After completion of the reaction, the mixture was separated by column chromatography (DCM:MeOH = 15:1) to give compound 172 (yield: 75%). Compound 172 (1.0 eq) and compound 11 (1.2 eq) were dissolved in THF, TEA (1.5 eq) was added, and the mixture was stirred at room temperature for 12 h. After completion of the reaction, the mixture was separated by column chromatography (DCM:MeOH = 20:1) to obtain compound 173 (yield: 73%). Compound 173 (1.0 eq) was dissolved in DCM, TEA (1.5 eq) was added, and the mixture was stirred at room temperature for 12 h. After completion of the reaction, the mixture was separated by column chromatography (DCM:MeOH = 10:1) to obtain compound IV-7 (yield: 73%). 1 H NMR (400 MHz, CDCl3, 293 K) δ (ppm): 4.43-4.36 (m, 2H), 4.29-4.26 (m, 2H), 4.18-4.14 (m, 2H), 4.08-4.02 (m, 2H), 3.25-3.16 (m, 2H), 3.02-2.96 (m, 6H), 2.75-2.62 (m, 2H), 2.33-2.29 (m, 2H), 2.21 (s, 3H), 2.19 (s, 3H), 2.15 (s, 3H), 1.98-1.88 (m, 3H), 1.68-0.98 (m, 55H), 0.91-0.80 (m, 15H). HRMS: m / z calcd: 1037.4548 (M+H + ), measured value: 1037.4540.

[0293] Example 38: Synthesis of Compound IV-8

[0294]

[0295] Compound 4 (1.0 eq) and compound 136 (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 the reaction was complete, the mixture was washed with water and purified by column chromatography (DCM:MeOH=10:1) to give compound 175 (yield: 75%). Compound 175 (1.0 eq) and compound 155 (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 the reaction was complete, the mixture was washed with water and purified by column chromatography (DCM:MeOH=15:1) to give compound 176 (yield: 63%). Compound 176 (1.0 eq) and compound 30 (1.0 eq) were dissolved in DCM, and EDC (1.0 eq) and DMAP (0.1 eq) were added. The mixture was stirred at room temperature for 12 h. After the reaction was completed, the mixture was washed with water and purified by column chromatography (DCM:MeOH=20:1) to give compound IV-8 (yield: 75%). 1 H NMR (400 MHz, CDCl3, 293 K) δ (ppm): 4.40-4.27 (m, 8H), 4.18-4.15 (m, 2H), 3.80-3.63 (m, 10H), 3.27 (m, 9H), 3.19-3.17 (m, 2H), 3.00-2.93 (m, 6H), 2.86-2.64 (m, 6H), 2.33-2.29 (m, 2H), 2.21 (s, 3H), 2.19 (s, 3H), 2.17 (s, 3H), 1.93-1.86 (m, 1H), 1.71-1.00 (m, 54H), 0.92-0.80 (m, 15H). HRMS: m / z calcd: 1316.8692 (M+H + ), measured value: 1316.8687.

[0296] Example 39: Synthesis of Compound IV-9

[0297]

[0298] Compound 162 (1.0 eq) and compound 178 (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 washed with water and purified by column chromatography (DCM:MeOH = 15:1) to give compound 179 (yield: 85%). Compound 179 (1.0 eq) and compound 9 (POCl3, 1.5 eq) were dissolved in THF, TEA (1.5 eq) was added, and the mixture was stirred at 0°C for 12 h. After completion of the reaction, the mixture was separated by column chromatography (DCM:MeOH = 15:1) to give compound 180 (yield: 75%). Compound 180 (1.0 eq) and compound 11 (1.2 eq) were dissolved in THF, TEA (1.5 eq) was added, and the mixture was stirred at room temperature for 12 h. After completion of the reaction, the mixture was separated by column chromatography (DCM:MeOH = 20:1) to give compound 181 (yield: 73%). Compound 181 (1.0 eq) was dissolved in DCM, and TEA (1.5 eq) was added. The mixture was stirred at room temperature for 12 h. After the reaction was completed, the mixture was separated by column chromatography (DCM:MeOH=10:1) to obtain compound IV-9 (yield: 85%). 1 H NMR (400 MHz, CDCl3, 293 K) δ (ppm): 4.36-4.26 (m, 4H), 4.18-4.14 (m, 2H), 4.08-4.04 (m, 2H), 3.26-3.16 (m, 2H), 3.00-2.90 (m, 6H), 2.86-2.65 (m, 6H), 2.33-2.29 (m, 2H), 2.21 (s, 3H), 2.19 (s, 3H), 2.16 (s, 3H), 1.98-1.88 (m, 3H), 1.69-0.98 (m, 51H), 0.90-0.80 (m, 15H). HRMS: m / z calcd: 1037.4548 (M+H + ), measured value: 1037.4545.

[0299] Example 40: Synthesis of Compound IV-10

[0300]

[0301] Compound 155 (1.0 eq) and compound 4 (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 the reaction was completed, the mixture was washed with water and purified by column chromatography (DCM:MeOH=15:1) to give compound 183 (yield: 46%). Compound 183 (1.0 eq) and compound 184 (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 the reaction was completed, the mixture was washed with water and purified by column chromatography (DCM:MeOH=25:1) to give compound IV-10 (yield: 64%). 1 H NMR (400 MHz, CDCl3, 293 K) δ (ppm): 5.48-5.34 (m, 2H), 4.37-4.26 (m, 6H), 4.18-4.15 (m, 2H), 3.67-3.63 (m, 2H), 3.28 (s, 9H), 3.03-2.96 (m, 6H), 2.86-2.65 (m, 6H), 2.32-2.27 (m, 2H), 2.21 (s, 3H), 2.19 (s, 3H), 2.16 (m, 3H), 2.03-1.87 (m, 5H), 1.69-0.98 (m, 45H), 0.91-0.80 (m, 15H). HRMS: m / z calcd: 1078.5272 (M+H + ), measured value: 1078.5277.

[0302] Example 41: Synthesis of Compound V-1

[0303]

[0304] Compound 16 (1.0 eq) and compound 186 (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 and purified by column chromatography (DCM:MeOH = 15:1) to give compound 187 (yield: 63%). Compound 187 (1.0 eq) and compound 9 (POCl3, 1.5 eq) were dissolved in THF, and TEA (1.5 eq) was added. The mixture was stirred at 0°C for 12 h. After completion of the reaction, the mixture was separated by column chromatography (DCM:MeOH = 15:1) to give compound 188 (yield: 75%). Compound 188 (1.0 eq) and compound 11 (1.2 eq) were dissolved in THF, and TEA (1.5 eq) was added. The mixture was stirred at room temperature for 12 h. After completion of the reaction, the mixture was separated by column chromatography (DCM:MeOH = 20:1) to give compound 189 (yield: 73%). Compound 189 (1.0 eq) was dissolved in DCM, and TEA (1.5 eq) was added. The mixture was stirred at room temperature for 12 h. After the reaction was completed, the mixture was separated by column chromatography (DCM:MeOH=10:1) to obtain compound V-1 (yield: 69%). 1 H NMR (400MHz, CDCl3, 293K) δ (ppm): 4.39-4.36 (m, 2H), 4.22-4.15 (m, 2H), 4.08-4.02 (m, 2H), 3.24-3.16 (m, 4H), 3.01-2.93 (m, 4H), 2.79-2. 53(m,6H),2.21(s,6H),2.19(s,6H),2.16(s,6H),1.95-1.88(m,4H),1.77-0.95(m,52H),0.88-0.80(m,24H).HRMS: m / z calculated value: 1254.7228(M+H + ), measured value: 1254.7226.

[0305] Example 42: Synthesis of Compound V-2

[0306]

[0307] Compound 186 (1.0 eq) and compound 58 (1.0 eq) were dissolved in DCM, TEA (1.5 eq) was added, and the mixture was stirred at room temperature for 12 h. After the reaction was complete, the mixture was washed with water and the organic solvent was removed in vacuo to obtain compound 191 (yield: 87%). Compound 191 (1.0 eq) and compound 1 (1.0 eq) were dissolved in DCM, TEA (1.5 eq) was added, and the mixture was stirred at room temperature for 12 h. After the reaction was complete, the mixture was washed with water and purified by column chromatography (DCM:MeOH=30:1) to obtain compound 192 (yield: 67%). Compound 192 (1.0 eq) was dissolved in THF, 15% TFA was added, and the mixture was stirred at room temperature for 3 h. After the reaction was complete, the mixture was washed with water and the organic solvent was removed in vacuo to obtain compound 193 (yield: 78%). Compound 16 (1.0 eq) and compound 193 (1.0 eq) were dissolved in DCM, TEA (1.5 eq) was added, and the mixture was stirred at room temperature for 12 h. The mixture was purified by column chromatography (DCM:MeOH=20:1) to give compound V-2 (yield: 75%). 1 H NMR (400MHz, CDCl3, 293K) δ (ppm): 4.39-4.36 (m, 4H), 4.29-4.25 (m, 2H), 3.67-3.65 (m, 2H), 3.28 (s, 9H), 3.21-3.13 (m, 2H), 2.99-2.95 (m, 4H), 2. 78-2.57(m,6H),2.21(s,6H),2.19(s,6H),2.15(s,6H),1.93-1.88(m,2H ),1.77-0.95(m,52H),0.88-0.80(m,24H).HRMS: m / z calculated value: 1297.8112(M+H + ), measured value: 1297.8108.

[0308] Example 43: Synthesis of Compound V-3

[0309]

[0310] Compound 186 (1.0 eq) was dissolved in DCM, and compound 6 (2.0 eq) was added. 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 = 25:1) to give compound 195 (yield: 87%). Compound 195 (1.0 eq) and compound 9 (POCl3, 1.5 eq) were dissolved in THF, and TEA (1.5 eq) was added. The mixture was stirred at 0°C for 12 h. After completion of the reaction, the mixture was separated by column chromatography (DCM:MeOH = 15:1) to give compound 196 (yield: 62%). Compound 196 (1.0 eq) and compound 11 (1.2 eq) were dissolved in THF, and TEA (1.5 eq) was added. The mixture was stirred at room temperature for 12 h. After completion of the reaction, the mixture was separated by column chromatography (DCM:MeOH = 20:1) to give compound 197 (yield: 72%). Compound 197 (1.0 eq) was dissolved in DCM, and TEA (1.5 eq) was added. The mixture was stirred at room temperature for 12 h. After the reaction was completed, the mixture was separated by column chromatography (DCM:MeOH=10:1) to obtain compound V-3 (yield: 87%). 1 H NMR (400MHz, CDCl3, 293K) δ (ppm): 4.29-4.26 (m, 2H), 4.19-4.15 (m, 2H), 4.08-4.02 (m, 2H), 3.27 (s, 9H), 3.23-3.13 (m, 4H), 2.96-2.92 (m, 4H), 2. 86-2.57(m,14H),2.21(s,6H),2.19(s,6H),2.17(s,6H),1.96-1.86(m,4 H),1.71-0.98(m,52H),0.88-0.78(m,24H).HRMS:m / z calculated value:1310.8308(M+H + ), measured value: 1310.8301.

[0311] Example 44: Synthesis of Compound V-4

[0312]

[0313] Compound 193 (1.0 eq) was dissolved in DCM, and compound 6 (2.0 eq) was added, and stirred at room temperature for 6 h. After the reaction was completed, the mixture was washed with water and purified by column chromatography (DCM:MeOH=20:1) to obtain compound V-4 (yield: 82%). 1H NMR (400MHz, CDCl3, 293K) δ (ppm): 4.31-4.26 (m, 2H), 4.19-4.15 (m, 2H), 4.08-4.01 (m, 2H), 3.21-3.15 (m, 4H), 2.97-2.94 (m, 4H), 2.86-2.5 7(m,14H),2.21(s,6H),2.19(s,6H),2.16(s,6H),1.96-1.86(m,4H),1.71-0.98(m,52H),0.86-0.78(m,24H).HRMS: m / z calculated value: 1353.9192(M+H + ), measured value: 1353.9188.

[0314] Example 45: Synthesis of Compound V-5

[0315]

[0316] Compound 186 (1.0 eq) and compound 16 (1.0 eq) were dissolved in DCM, TEA (1.5 eq) was added, and the mixture was stirred at room temperature for 12 h. The mixture was purified by column chromatography (DCM:MeOH = 10:1) to give compound 200 (yield: 63%). Compound 200 (1.0 eq) was dissolved in DCM, compound 6 (1.2 eq) was added, TEA (1.5 eq) was 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 give compound 201 (yield: 52%). Compound 201 (1.0 eq) and compound 9 (POCl3, 1.5 eq) were dissolved in THF, TEA (1.5 eq) was added, and the mixture was stirred at 0°C for 12 h. After completion of the reaction, the mixture was separated by column chromatography (DCM:MeOH = 15:1) to give compound 202 (yield: 62%). Compound 202 (1.0 eq) and compound 11 (1.2 eq) were dissolved in THF, TEA (1.5 eq) was added, and the mixture was stirred at room temperature for 12 h. After completion of the reaction, the mixture was separated by column chromatography (DCM:MeOH = 20:1) to obtain compound 203 (yield: 72%). Compound 203 (1.0 eq) was dissolved in DCM, TEA (1.5 eq) was added, and the mixture was stirred at room temperature for 12 h. After completion of the reaction, the mixture was separated by column chromatography (DCM:MeOH = 10:1) to obtain compound V-5 (yield: 73%). 1H NMR (400 MHz, CDCl3, 293 K) δ (ppm): 4.31-4.24 (m, 2H), 4.22-4.15 (m, 2H), 4.03-3.93 (m, 2H), 3.23-3.17 (m, 2H), 3.00-2.94 (m, 4H), 2.86-2.65 (m, 10H), 2.59-2.26 (m, 2H), 2.21 (s, 6H), 2.19 (s, 6H), 2.15 (s, 6H), 1.95-1.88 (m, 2H), 1.80-1.00 (m, 58H), 0.85-0.80 (m, 24H). HRMS: m / z calcd: 1310.8308 (M+H + ), measured value: 1310.8300.

[0317] Example 46: Synthesis of Compound V-6

[0318]

[0319] Compound 14 (1.0 eq) was dissolved in DCM, compound 205 (1.2 eq) was added, and the mixture was stirred at room temperature for 6 h. After completion of the reaction, the mixture was washed with water to give compound 206 (yield: 63%). Compound 206 (1.0 eq) was dissolved in DCM, compound 193 (1.2 eq) was added, and the mixture was stirred at room temperature for 6 h. After completion of the reaction, the mixture was washed with water to give compound 207 (yield: 67%). Compound 207 (1.0 eq) was dissolved in DCM, compound 6 (1.2 eq) was 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 give compound V-6 (yield: 52%). 1 H NMR (400MHz, CDCl3, 293K) δ (ppm): 4.31-4.24 (m, 2H), 4.22-4.15 (m, 2H), 4.03 -3.93(m,2H),3.25(s,9H),3.23-3.17(m,2H),3.00-2.94(m,4H),2.86-2.65( m,10H),2.59-2.26(m,2H),2.21(s,6H),2.19(s,6H),2.15(s,6H),1.95-1.88 (m,2H),1.80-1.00(m,58H),0.85-0.80(m,24H).HRMS:m / z calculated value:1297.8552(M+H + ), measured value: 1297.8549.

[0320] Example 47: Synthesis of Compound V-7

[0321]

[0322] Compound 209 (1.0 eq) and compound 186 (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 and purified by column chromatography (DCM:MeOH = 15:1) to give compound 210 (yield: 57%). Compound 210 (1.0 eq) was dissolved in DCM, and compound 16 (1.0 eq) was added. 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 211 (yield: 52%). Compound 211 (1.0 eq) and compound 9 (POCl3, 1.5 eq) were dissolved in THF, and TEA (1.5 eq) was added. The mixture was stirred at 0°C for 12 h. After completion of the reaction, the mixture was separated by column chromatography (DCM:MeOH = 15:1) to give compound 212 (yield: 72%). Compound 212 (1.0 eq) and compound 11 (1.2 eq) were dissolved in THF, TEA (1.5 eq) was added, and the mixture was stirred at room temperature for 12 h. After completion of the reaction, the mixture was separated by column chromatography (DCM:MeOH = 20:1) to obtain compound 213 (yield: 74%). Compound 213 (1.0 eq) was dissolved in DCM, TEA (1.5 eq) was added, and the mixture was stirred at room temperature for 12 h. After completion of the reaction, the mixture was separated by column chromatography (DCM:MeOH = 10:1) to obtain compound V-7 (yield: 74%). 1 H NMR (400 MHz, CDCl3, 293 K) δ (ppm): 4.41-4.34 (m, 2H), 4.19-4.15 (m, 2H), 4.08-4.04 (m, 2H), 3.21-3.13 (m, 4H), 3.01-2.93 (m, 4H), 2.75-2.54 (m, 4H), 2.21-2.15 (m, 11H), 2.00-1.88 (m, 3H), 1.74-0.98 (m, 39H), 0.91-0.80 (m, 15H). HRMS: m / z calcd: 910.2278 (M+H + ), measured value: 910.2273.

[0323] Example 48: Synthesis of Compound V-8

[0324]

[0325] Compound 193 (1.0 eq) and compound 94 (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 and purified by column chromatography (DCM:MeOH = 15:1) to give compound 215 (yield: 52%). Compound 215 (1.0 eq) was dissolved in DCM, and compound 16 (1.2 eq) was added. The mixture was stirred at room temperature for 6 h. After the reaction was completed, the mixture was washed with water and purified by column chromatography (DCM:MeOH = 20:1) to give compound V-8 (yield: 67%). 1 H NMR (400 MHz, CDCl3, 293 K) δ (ppm): 5.45-5.29 (m, 8H), 4.43-4.13 (m, 6H), 3.67-3.58 (m, 2H), 3.27 (m, 9H), 3.19-3.10 (m, 2H), 2.99-2.92 (m, 4H), 2.75-2.65 (m, 8H), 2.59-2.52 (m, 2H), 2.21-2.15 (m, 11H), 2.10-2.00 (m, 4H), 1.93-1.86 (m, 1H), 1.71-0.98 (m, 37H), 0.92-0.73 (m, 15H). HRMS: m / z calcd: 1113.5762 (M+H + ), measured value: 1113.5760.

[0326] Example 49: Synthesis of Compound V-9

[0327]

[0328] Compound 30 (1.0 eq) and compound 186 (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 washed with water and purified by column chromatography (DCM:MeOH = 10:1) to give compound 217 (yield: 45%). Compound 217 (1.0 eq) was dissolved in DCM, compound 6 (1.2 eq) was 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 give compound 218 (yield: 62%). Compound 218 (1.0 eq) and compound 9 (POCl3, 1.5 eq) were dissolved in THF, and TEA (1.5 eq) was added. The mixture was stirred at 0°C for 12 h. After completion of the reaction, the mixture was separated by column chromatography (DCM:MeOH = 15:1) to give compound 219 (yield: 72%). Compound 219 (1.0 eq) and compound 11 (1.2 eq) were dissolved in THF, TEA (1.5 eq) was added, and the mixture was stirred at room temperature for 12 h. After completion of the reaction, the mixture was separated by column chromatography (DCM:MeOH = 20:1) to obtain compound 220 (yield: 74%). Compound 220 (1.0 eq) was dissolved in DCM, TEA (1.5 eq) was added, and the mixture was stirred at room temperature for 12 h. After completion of the reaction, the mixture was separated by column chromatography (DCM:MeOH = 10:1) to obtain compound V-9 (yield: 72%). 1 H NMR (400 MHz, CDCl3, 293 K) δ (ppm): 4.31-4.24 (m, 2H), 4.19-4.15 (m, 2H), 4.01-3.97 (m, 2H), 3.19-3.15 (m, 2H), 2.97-2.94 (m, 4H), 2.86-2.82 (m, 2H), 2.76-2.64 (m, 6H), 2.59-2.57 (m, 2H), 2.22-2.11 (m, 11H), 1.95-1.86 (m, 1H), 1.71-0.98 (m, 66H), 0.89-0.78 (m, 15H). HRMS: m / z calcd: 1134.6598 (M+H + ), measured value: 1134.6590.

[0329] Example 50: Synthesis of Compounds V-10 to V-13

[0330]

[0331] Compound 192 (1.0 eq) was dissolved in DCM, and compound 6 (1.2 eq) was added. 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 = 10:1) to give compound 222 (yield: 56%). Compound 222 (1.0 eq) was dissolved in THF, 15% TFA was added, and the mixture was stirred at room temperature for 3 h. After completion of the reaction, the mixture was washed with water, and the organic solvent was removed in vacuo to give compound 223 (yield: 78%). Compound 223 (1.0 eq) and compound 141 (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 washed with water and purified by column chromatography (DCM:MeOH = 20:1) to give compound V-10 (yield: 52%). 1 H NMR (400MHz, CDCl3, 293K) δ (ppm): 4.40-4.28 (m, 6H), 4.19-4.08 (m, 2H), 3.82-3.6 1(m,10H),3.25(s,9H),3.18-3.12(m,2H),2.96-2.56(m,12H),2.48-2.43(m,2H),2 .36-2.29 (m, 2H), 2.21 (s, 3H), 2.19 (s, 3H), 2.16 (s, 3H), 1.95-1.86 (m, 1H), 1.74-1.13 (m, 38H), 1.07-0.98 (m, 2H), 0.92-0.78 (m, 15H). HRMS: m / z calculated value: 1155.5662 (M+H + ), measured value: 1155.5656.

[0332] Compounds V-11, V-12, and V-13 were obtained using β-tocopherol, γ-tocopherol, and δ-tocopherol as raw materials, respectively, using the same synthesis method as in the previous examples (yields: 54%, 41%, and 56%, respectively).

[0333]

[0334] Compound V-11: 1H NMR (400MHz, CDCl3, 293K) δ (ppm): 6.80 (s, 1H), 4.40-4.28 (m, 6H), 4.19-4.08 (m, 2 H),3.82-3.61(m,10H),3.25(s,9H),3.18-3.12(m,2H),2.96-2.56(m,12H),2.48-2 .43 (m, 2H), 2.36-2.29 (m, 2H), 2.22 (s, 3H), 2.19 (s, 3H), 1.95-1.86 (m, 1H), 1.74-1.13 (m, 38H), 1.07-0.98 (m, 2H), 0.92-0.78 (m, 15H). HRMS: m / z calculated value: 1141.5392 (M+H + ), measured value: 1141.5388.

[0335] Compound V-12: 1 H NMR (400MHz, CDCl3, 293K) δ (ppm): 6.69 (s, 1H), 4.40-4.28 (m, 6H), 4.19-4.08 (m, 2 H),3.82-3.61(m,10H),3.25(s,9H),3.18-3.12(m,2H),2.96-2.56(m,12H),2.48-2 .43 (m, 2H), 2.36-2.29 (m, 2H), 2.22 (s, 3H), 2.19 (s, 3H), 1.95-1.86 (m, 1H), 1.74-1.13 (m, 38H), 1.07-0.98 (m, 2H), 0.92-0.78 (m, 15H). HRMS: m / z calculated value: 1141.5392 (M+H + ), measured value: 1141.5391.

[0336] Compound V-13: 1 H NMR (400MHz, CDCl3, 293K) δ (ppm): 6.85 (m, 1H), 6.67 (m, 1H), 4.40-4.28 (m, 6H), 4. 19-4.08(m,2H),3.82-3.61(m,10H),3.25(s,9H),3.18-3.12(m,2H),2.96-2.56(m, 12H), 2.48-2.43 (m, 2H), 2.36-2.29 (m, 2H), 2.17 (s, 3H), 1.95-1.86 (m, 1H), 1.74-1.13 (m, 38H), 1.07-0.98 (m, 2H), 0.92-0.78 (m, 15H). HRMS: m / z calculated value: 1127.5122 (M+H+ ), measured value: 1127.5129.

[0337] Example 51: Preparation and Characterization of Basic LNPs and LNPs of the Present Invention Encapsulating mRNA

[0338] Ionizable lipid molecules (ALC-0315), DSPC, cholesterol and polyethylene glycol lipids (ALC-0159) 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. The basic lipid nanoparticles encapsulating mRNA (expressed as BasicLNP@mRNA) were obtained. Luc ).

[0339] Ionizable lipid molecules (ALC-0315), polyethylene glycol lipids (ALC-0159), cholesterol, and the compounds synthesized in Examples 1-50 (I-1 to V-13) were dissolved in ethanol at a molar ratio of 50:2:38:10. The mRNA was firefly luciferase mRNA, which 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. The ethanol was then removed by ultrafiltration. The lipid nanoparticles of the present invention (named according to the compounds synthesized in Examples 1-50) containing mRNA were obtained. For example, it can be expressed as I-1LNP@mRNA Luc or V-13LNP@mRNA Luc ).

[0340] 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 the representative I-1LNP@mRNA Luc 、III-1LNP@mRNA Luc , IV-1LNP@mRNA Luc and V-11LNP@mRNA Luc The particle size distribution of Figure 1 shown.

[0341] DLS results showed (Table 1) that Basic LNP@mRNA LucLNP@mRNA of the present invention prepared based on I-1 to V-13 Luc In comparison, there is no significant difference in the hydrated particle size, and both meet the applicable standards.

[0342] Basic LNP@mRNA was observed using transmission electron microscopy. Luc , I-1 LNP@mRNA Luc 、III-1 LNP@mRNA Luc , IV-1LNP@mRNA Luc and V-11 LNP@mRNA Luc The morphology of five mRNA-encapsulated lipid nanoparticles. Electron microscopy images show ( Figure 2 ), the five mRNA-loaded lipid nanoparticles had good morphology, were all quasi-spherical, and had a particle size of about 50 nm.

[0343] LNP@mRNA Luc Determination of encapsulation efficiency

[0344] The white solution obtained above was dialyzed with an appropriate volume of PBS solution for 4 hours, 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: Encapsulation efficiency = mRNA 总量 -mRNA 滤液 / mRNA 总量 .

[0345] The results show (Table 1) that the prepared LNP@mRNA of the present invention Luc All of them have good mRNA encapsulation efficiency.

[0346] Table 1

[0347]

[0348] Example 52: LNP@mRNA of the present invention GFP In vitro transfection efficiency experiment

[0349] The ionizable lipid molecules (ALC-0315), polyethylene glycol lipids (ALC-0159), cholesterol, and compounds I-1, III-1, IV-1, and V-11 synthesized in Examples 1, 21, 31, and 50 were dissolved in ethanol at a molar ratio of 50:2:38:10. The mRNA was green fluorescent protein mRNA (GFP 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 was mixed with the mRNA at a mass ratio of 25:1 to obtain a slightly white solution. The ethanol was then removed by ultrafiltration. The I-1 LNP@mRNA of the present invention containing mRNA was obtained. GFP、III-1 LNP@mRNA GFP IV-1 LNP@mRNA GFP and V-11LNP@mRNA GFP DSPC was used instead of the compound synthesized in the above example to obtain BasicLNP@mRNA loaded with mRNA in the same way. GFP .

[0350] 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. GFP And the I-1 LNP@mRNA of the present invention GFP 、III-1LNP@mRNA GFP , IV-1LNP@mRNA GFP 、V-11 LNP@mRNA GFP (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 GFP-positive cell populations and compare the transfection efficiency of the two LNP@mRNAs.

[0351] The results show that ( Figure 3 ), using Basic LNP@mRNA GFP After in vitro transfection, the proportion of GFP-positive cells was low, proving that DC2.4 cells were transfected with Basic LNP@mRNA GFP After transfection of GFP mRNA, the transfection efficiency is very limited. GFP 、III-1LNP@mRNA GFP , IV-1LNP@mRNA GFP 、V-11LNP@mRNA GFP After in vitro transfection with GFP mRNA, the proportion of GFP-positive cells increased significantly, demonstrating that the LNP@mRNA of the present invention can significantly improve antigen presentation efficiency. ***P<0.001 indicates a highly significant difference.

[0352] Example 53: LNP@mRNA of the present invention Luc In vitro expression experiments

[0353] The ionizable lipid molecule (ALC-0315), polyethylene glycol lipid (ALC-0159), cholesterol and the compound synthesized in Example 1-50 (I-1 to V-13) were dissolved in ethanol at a molar ratio of 50:2:38:10. 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. Ethanol was then removed by ultrafiltration. The I-1LNP@mRNA of the present invention containing mRNA was obtained. Luc to V-13LNP@mRNA Luc DSPC was used instead of the compound synthesized in the above example to obtain Basic LNP@mRNA loaded with mRNA in the same way. Luc .

[0354] 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. 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.

[0355] 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.

[0356] Table 2

[0357]

[0358] Example 54: Evaluation of the anti-tumor properties of lipid nanoparticles of the present invention containing the compound of formula (1)

[0359] The ionizable lipid molecule (ALC-0315), polyethylene glycol lipid (ALC-0159), cholesterol, and the compounds synthesized in Examples 1 and 2 (I-1 and I-2) were dissolved in ethanol at a molar ratio of 50:2:38:10. The mRNA was ovalbumin mRNA (OVA 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. The ethanol was then removed by ultrafiltration. The I-1LNP@mRNA of the present invention containing mRNA was obtained. OVA and I-2LNP@mRNA OVA DSPC was used instead of the compound synthesized in the above example to obtain Basic LNP@mRNA loaded with mRNA in the same way. OVA .

[0360] 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, and PBS and BasicLNP@mRNA were injected into the thigh muscle twice on days 9 and 15, respectively. OVA , I-1LNP@mRNA OVA and I-2LNP@mRNA OVA The mRNA injection dose was 5 μg each time, and the changes in tumor volume of mice were recorded within 25 days.

[0361] The results show that ( Figure 4 ), receiving I-1LNP@mRNA OVA and I-2LNP@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 and has excellent anti-tumor effects.

[0362] Example 55: LNP@mRNA of the present invention OVA Tumor model mouse survival rate experiment

[0363] The ionizable lipid molecule (ALC-0315), polyethylene glycol lipid (ALC-0159), cholesterol, and the compounds synthesized in Examples 1 and 2 (I-1 and I-2) were dissolved in ethanol at a molar ratio of 50:2:38:10. The mRNA was ovalbumin mRNA (OVA 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. The ethanol was then removed by ultrafiltration. The I-1LNP@mRNA of the present invention containing mRNA was obtained. OVA and I-2LNP@mRNA OVA DSPC was used instead of the compound synthesized in the above example to obtain Basic LNP@mRNA loaded with mRNA in the same way. OVA .

[0364] 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, and PBS and BasicLNP@mRNA were injected into the thigh muscle twice on days 9 and 15, respectively. OVA , I-1LNP@mRNA OVA and I-2LNP@mRNA OVA The mRNA injection dose was 5 μg each time, and the survival of mice was recorded for 50 days.

[0365] The results show that ( Figure 5 ), the median survival time of mice in the PBS group was 22 days, and those receiving Basic LNP@mRNA OVA The median survival time of the treated tumor model mice was 35 days. OVA and I-2LNP@mRNA OVA The survival of the treated tumor model mice was significantly prolonged. At the end of the 50-day observation period, the survival rates of the two groups of mice were 80% and 100%, respectively.

[0366] Example 56: LNP@mRNA of the present invention OVA Anti-tumor immune response experiments

[0367] The ionizable lipid molecule (ALC-0315), polyethylene glycol lipid (ALC-0159), cholesterol, and the compounds synthesized in Examples 1 and 2 (I-1 and I-2) were dissolved in ethanol at a molar ratio of 50:2:38:10. The mRNA was ovalbumin mRNA (OVA 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. The ethanol was then removed by ultrafiltration. The I-1LNP@mRNA of the present invention containing mRNA was obtained. OVA and I-2LNP@mRNA OVA DSPC was used instead of the compound synthesized in the above example to obtain Basic LNP@mRNA loaded with mRNA in the same way. OVA .

[0368] 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, and PBS and BasicLNP@mRNA were injected into the thigh muscle twice on days 9 and 15, respectively. OVA , I-1LNP@mRNA OVA and I-2LNP@mRNA OVA Each mRNA injection dose was 5 μg. After 30 days, the mice were sacrificed, and the spleen and tumor tissue were completely removed and ground. The mixed lymphocyte suspension obtained from the spleen was inoculated into a 24-well plate and stimulated with OVA antigen peptide in vitro. Golgi inhibitor intervention was also given. After 6 hours of culture, the cell suspension was collected and stained with flow cytometry antibodies together with the mixed cell suspension obtained from the tumor. Flow cytometry analysis was performed to detect the levels of antigen-specific cytotoxic T lymphocytes and compare the anti-tumor immune response capabilities of BasicLNP@mRNA and the two LNP@mRNAs of the present invention.

[0369] The results showed that I-1LNP@mRNA OVA and I-2LNP@mRNA OVA The proportion of antigen-specific reactive T cells in the spleen cells of treated mice ( Figure 6 ) and IFN-γ in tumor tissue + CD8 + T cells ( 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 OVAIt can significantly enhance the in vivo expression efficiency of mRNA, thereby significantly enhancing the anti-tumor immune response. *P<0.05 indicates a significant difference; **P<0.01 indicates a significantly significant difference; ***P<0.001 indicates an extremely significant difference.

[0370] Example 57: LNP@mRNA constructed with β-tocopherol, γ-tocopherol and δ-tocopherol instead of α-tocopherol H1N1 Humoral immunity test

[0371] The ionizable lipid molecule (ALC-0315), polyethylene glycol lipid (ALC-0159), cholesterol and the compound synthesized in Example 50 (V-10, V-11, V-12 and V-13) were dissolved in ethanol at a molar ratio of 50:2:38:10. The mRNA was H1N1 influenza virus mRNA (H1N1 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. Ethanol was then removed by ultrafiltration. The V-10LNP@mRNA of the present invention containing mRNA was obtained. H1N1 、V-11LNP@mRNA H1N1 、V-12LNP@mRNA H1N1 、V-13LNP@mRNA H1N1 DSPC was used instead of the compound synthesized in the above example to obtain BasicLNP@mRNA loaded with mRNA in the same way. H1N1 .

[0372] The vaccine was administered to C57BL / 6J mice at a dose of 5 μg mRNA per mouse via intramuscular injection twice, one week apart. Peripheral blood was collected on the 7th and 21st days after the last immunization, and the levels of anti-IgM and IgG antibodies 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 8 shown.

[0373] 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 H1N1The vaccine significantly improved humoral immunity, with excellent results. *P<0.05 indicates a significant difference; **P<0.01 indicates a significantly significant difference; ***P<0.001 indicates an extremely significant difference; and NS indicates no statistically significant difference.

[0374] 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; represents a single bond or a double bond; R a 、R b and R c independently selected from H or methyl; L 1 Selected from single bond, C 1-10 Alkylene or C 2-10 Alkenylene, said alkylene or alkenylene being unsubstituted or substituted with one or more OH; 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 , G 3 and G 4 are independently selected from a single bond, -NR 3 -, -O-, -S-, -OC(=O)NR 3 -、-NR 3 C(=O)-, -C(=O)-, -OC(=O)-, -SC(=O)O-, -OC(=O)O-, -C(=O)-L a -C(=O)-, -C(=O)-L a -C(=O)O-, -OC(=O)-L a -C(=O)O-, -C(=O)-L a -C(=O)NR 3 -、-NR 3 C(=O)NR 3 -, or a combination of these groups and amino acid residues; The amino acid residue is selected from a divalent group derived 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 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; L 4 and L 5 are independently selected from a single bond, C 1-20 Alkylene, C 2-10 Alkenylene or -(OCH2CH2) n -, the alkylene or alkenylene is unsubstituted or substituted by one or more OH, NH2 or halogen; P 1 Selected from or -NH2; R 1 Selected from C 1-30 Alkyl, C 2-30 Alkenyl or C 2-30 Alkynyl, said alkyl, alkenyl or alkynyl being unsubstituted or substituted by one or more OH, NH2, halogen, -OC 1-10 Alkyl, -SC 1-10 Alkyl, C 3-8 Cycloalkyl, 5-8 membered heterocyclic group, C 6-10 substituted with aryl or 5-10 membered heteroaryl; n is an integer of 1-10; m is an integer of 2-10.

2. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, wherein L 1 Selected from single bond or C 1-10 Alkylene, which is unsubstituted or substituted with one or more OH groups; Or, L 1 Selected from single bond or C 1-6 Alkylene, which is unsubstituted or substituted with one or more OH groups; Or, L 1 Selected from single bond, -CH2-, -CH2CH2-, 3. The compound according to claim 1 or 2 or a pharmaceutically acceptable salt thereof, wherein 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 2 and L 3 are independently selected from a single bond, -CH2-, -CH2CH2-, 4. The compound according to any one of claims 1 to 3 or a pharmaceutically acceptable salt thereof, wherein G 1 , G 2 , G 3 and G 4 are independently selected from a single bond, -NR 3 -, -O-, -S-, -OC(=O)NR 3 -、-NR 3 C(=O)-, -C(=O)-, -OC(=O)-, -SC(=O)O-, -OC(=O)O-, -C(=O)-L a -C(=O)-, -C(=O)-L a -C(=O)O-, -C(=O)-L a -C(=O)NR 3 -, or a combination of these groups and an amino acid residue; the amino acid residue is selected from glycine, alanine, valine, leucine, isoleucine, methionine, proline, tryptophan, serine, tyrosine, cysteine, phenylalanine, asparagine, glutamine, threonine, aspartic acid, glutamic acid, lysine, arginine or histidine by removing H and / or OH divalent groups, or a combination thereof; Or, G 1 , G 2 , G 3 and G 4 are independently selected from a single bond, -NR 3 -, -O-, -S-, -OC(=O)NR 3 -、-NR 3 C(=O)-, -C(=O)-, -OC(=O)-, -SC(=O)O-, -OC(=O)O-, -C(=O)-L a -C(=O)-, -C(=O)-L a -C(=O)O-, -C(=O)-L a -C(=O)NR 3 -, 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, histidine, alanine or glycine; Or, G 1 , G 2 , G 3 and G 4 are independently selected from a single bond, -NH-, -O-, -S-, -C(=O)-, -OC(=O)-, -SC(=O)O-, -NHC(=O)-, -OC(=O)NH-, 5. The compound according to any one of claims 1 to 4 or a pharmaceutically acceptable salt thereof, wherein Each R 3 Independently selected from H, C 1-10 Alkyl or C 2-10 alkenyl, said alkyl or alkenyl being unsubstituted or substituted with one or more OH, NH2 or halogen; Or, each R 3 Independently selected from H, C 1-6 Alkyl or C 2-6 alkenyl, said alkyl or alkenyl being unsubstituted or substituted with one or more OH, NH2 or halogen; Or, each R 3 are independently H.

6. The compound according to any one of claims 1 to 5 or a pharmaceutically acceptable salt thereof, wherein: L a Selected from single bond or C 1-10 Alkylene, which is unsubstituted or substituted with one or more OH, NH2 or halogen; Or, L a Selected from single bond or C 1-6 Alkylene, which is unsubstituted or substituted with one or more OH, NH2 or halogen; Or, L a Selected from single bond, -CH2-, -CH2CH2-, 7. The compound according to any one of claims 1 to 6 or a pharmaceutically acceptable salt thereof, wherein: L 4 and L 5 are independently selected from a single bond, C 1-20 Alkylene or -(OCH2CH2) n -, the alkylene group is unsubstituted or substituted by one or more OH, NH2 or halogen; Or, L 4 and L 5 are independently selected from a single bond, C 1-16 Alkylene or -(OCH2CH2) n -, the alkylene group is unsubstituted or substituted by one or more OH, NH2 or halogen; Or, L 4 and L 5 are independently selected from a single bond, -CH2-, -CH2CH2-, 8. The compound according to any one of claims 1 to 7 or a pharmaceutically acceptable salt thereof, wherein n is an integer of 1-8; or, n is an integer of 1-6; or, n is an integer of 1-3.

9. The compound according to any one of claims 1 to 8 or a pharmaceutically acceptable salt thereof, wherein m is an integer of 2-8; alternatively, m is an integer of 2-6.

10. The compound according to any one of claims 1 to 9 or a pharmaceutically acceptable salt thereof, wherein Selected from 11. The compound according to any one of claims 1 to 10 or a pharmaceutically acceptable salt thereof, wherein: R 1 Selected from C 6-24 Alkyl or C 6-24 Alkenyl, the alkyl or alkenyl is unsubstituted or substituted with one or more OH, NH2, halogen, -OC 1-10 Alkyl or -SC 1-10 Alkyl substitution; Or, R 1 Selected from C 8-20 Alkyl or C 8-20 Alkenyl, the alkyl or alkenyl is unsubstituted or substituted with one or more OH, NH2, halogen, -OC 1-6 Alkyl or -SC 1-6 Alkyl substitution; Or, R 1 Selected from 12. The following compound or a pharmaceutically acceptable salt thereof:

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 helper lipid molecule comprises the 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 polyethylene glycol lipid molecule is selected from 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 or PEG-1,2-dimyristoyloxypropyl-3-amine PEG-c-DMA.

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 auxiliary lipid molecules and 1%-30% of polyethylene glycol 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.

Images