Double-stranded RNA conjugate as well as preparation method and application thereof

Abstract

The invention relates to a double-stranded RNA conjugate and a preparation method and application thereof, the double-stranded RNA conjugate comprises a first nucleic acid molecule, a second nucleic acid molecule and a linker, the first nucleic acid molecule is connected with the second nucleic acid molecule through the linker, the linker is connected with the first nucleic acid molecule and the second nucleic acid molecule through phosphate (p) or thiophosphate (s), and the linker is connected with the first nucleic acid molecule and the second nucleic acid molecule through phosphate (p) or thiophosphate (s). The joint is selected from a structure as shown in a formula (I);

Description

TECHNICAL FIELD

[0001] The present application relates to the field of medicine, and more particularly, to a double-stranded RNA conjugate and a preparation method and use thereof. BACKGROUND

[0002] At present, the field needs a molecule capable of targeting multiple targets, and how to connect multiple target molecules, and how to break multiple target molecules in the body is a problem to be solved at present.

[0003] Although patent publication numbers WO2017015109A1 and WO2018136620 disclose some in vivo cleavable linkers, there are still various problems after the multi-target molecule enters the body, and there is no product on the market that can act on multiple targets at the same time, so it is urgent to research linkers suitable for in vivo cleavage and multi-target molecules. SUMMARY

[0004] In view of the problems existing in the prior art, the present application provides a double-stranded RNA conjugate and a preparation method and use thereof.

[0005] In a first aspect, the present application provides a double-stranded RNA conjugate, comprising: a first nucleic acid molecule, a second nucleic acid molecule and a linker, the first nucleic acid molecule is connected to the second nucleic acid molecule through the linker, the linker connects the first nucleic acid molecule and the second nucleic acid molecule through a phosphate (p) or a phosphorothioate (ps), and the linker is selected from the structure shown in formula (I):

[0006]

[0007] The T is selected from -NH-C(O)- or -C(O)-NH-;

[0008] The R 1 is selected from wherein y1, y2 and y3 are R 1 the connecting position;

[0009] The R 2 is selected from wherein y4 is R 2 the connecting position;

[0010] wherein the x, p, q, r are independently selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, and m, n are selected from positive integers from 1 to 10.

[0011] As a preferred technical solution of the present application, the linker is selected from:

[0012]

[0013]

[0014] The linker comprises a first connection site and a second connection site, the first connection site can be connected to the first nucleic acid molecule or the second nucleic acid molecule, and the second connection site can be connected to the first nucleic acid molecule or the second nucleic acid molecule, for example, when the first connection site is connected to the first nucleic acid molecule, the second connection site is connected to the second nucleic acid molecule, or when the first connection site is connected to the second nucleic acid molecule, the second connection site is connected to the first nucleic acid molecule.

[0015] As a preferred technical solution of the present application, the carboxylic acid of the linker is connected to the ligand of the asialoglycoprotein receptor ASGPR through an amide bond.

[0016] As a preferred technical solution of the present application, the ligand is selected from the following:

[0017]

[0018] As a preferred technical solution of the present application, the compound in which the carboxylic acid of the linker is connected to the ligand of the asialoglycoprotein receptor ASGPR through an amide bond is selected from the structures shown in Table 2.

[0019] As a preferred technical solution of the present application, the first nucleic acid molecule is used to inhibit the expression of a first target gene, the second nucleic acid molecule is used to inhibit the expression of a second target gene, and the first target gene and the second target gene are the same or different.

[0020] The first target gene and the second target gene can be independently selected from PCSK9, ANGPTL3, LPA, apo3, AGT, Factor B, Factor C3, MASP2 factor, PNP factor, HSD17B13, NR1H3, SOD1, HCV, HBV, TTR, FVII, etc.

[0021] In a second aspect, the present application provides a double-stranded RNA conjugate for inhibiting gene expression, comprising:

[0022] a linker,

[0023] a first nucleic acid molecule for inhibiting the expression of a first target gene, and

[0024] a second nucleic acid molecule for inhibiting the expression of a second target gene, the first nucleic acid molecule and the second nucleic acid molecule being connected by the linker, the linker being connected by a phosphate (p) or a phosphorothioate (s), and the linker being as described above;

[0025] The first target gene and the second target gene are independently selected from PCSK9, ANGPTL3, LPA, apo3, AGT, Factor B, Factor C3, MASP2, PNP, HSD17B13, NR1H3, SOD1, HCV, HBV, TTR, FVII.

[0026] As a preferred technical solution of the present application, the linker is selected from the structure shown in formula (I), preferably the linker of B1'-B9'.

[0027] As a preferred technical solution of the present application, the linker is selected from the structure shown in Table 2.

[0028] In a third aspect, the present application provides a double-stranded RNA conjugate for inhibiting the expression of ANGPTL3 and PCSK9, comprising:

[0029] a first nucleic acid molecule for inhibiting the expression of PCSK9; and

[0030] a second nucleic acid molecule for inhibiting the expression of ANGPTL3;

[0031] The first nucleic acid molecule and the second nucleic acid molecule are connected by the linker, the linker connects the first nucleic acid molecule and the second nucleic acid molecule by phosphate (p) or phosphorothioate (ps), the linker is selected from the structure shown in formula (I), preferably the linker of B1'-B9'.

[0032] As a preferred technical solution of the present application, the carboxylic acid of the linker is connected to the ligand of asialoglycoprotein receptor ASGPR by an amide bond.

[0033] As a preferred technical solution of the present application, the ligand is selected from:

[0034]

[0035] As a preferred technical solution of the present application, the compound in which the carboxylic acid of the linker is connected to the ligand of asialoglycoprotein receptor ASGPR by an amide bond is selected from the structure shown in Table 2.

[0036] As a preferred technical solution of the present application, the first nucleic acid molecule comprises a sense strand and an antisense strand, the second nucleic acid molecule comprises a sense strand and an antisense strand, and the sense strand of the first nucleic acid molecule is connected to the sense strand of the second nucleic acid molecule by the nucleic acid linker or covalent bond. For example, the nucleic acid linker or covalent bond is connected to the nucleotide at the 5' end or 3' end of the sense strand.

[0037] As a preferred technical solution of the present invention, the first nucleic acid molecule includes a sense strand and an antisense strand, the second nucleic acid molecule includes a sense strand and an antisense strand, and the antisense strand of the first nucleic acid molecule is connected to the antisense strand of the second nucleic acid molecule via the nucleic acid linker or covalent bond. For example, the nucleic acid linker or covalent bond is connected to the nucleotide at the 5' end or the 3' end of the antisense strand.

[0038] As a preferred technical solution of the present invention, the first nucleic acid molecule is selected from:

[0039] The sense strand comprises the nucleotide sequence of SEQ ID NO: 1 and the antisense strand comprises the nucleotide sequence of SEQ ID NO: 2.

[0040] The sense strand comprises the nucleotide sequence of SEQ ID NO: 3 and the antisense strand comprises the nucleotide sequence of SEQ ID NO: 4.

[0041] The sense strand comprises the nucleotide sequence of SEQ ID NO: 5 and the antisense strand comprises the nucleotide sequence of SEQ ID NO: 6.

[0042] As a preferred technical solution of the present invention, the second nucleic acid molecule is selected from:

[0043] The sense strand comprises the nucleotide sequence of SEQ ID NO: 1 and the antisense strand comprises the nucleotide sequence of SEQ ID NO: 2.

[0044] The sense strand comprises the nucleotide sequence of SEQ ID NO: 3 and the antisense strand comprises the nucleotide sequence of SEQ ID NO: 4.

[0045] The sense strand comprises the nucleotide sequence of SEQ ID NO: 5 and the antisense strand comprises the nucleotide sequence of SEQ ID NO: 6.

[0046] As a preferred technical solution of the present invention, the sense strand of the first nucleic acid molecule and / or the second nucleic acid molecule is linked to a targeting ligand. For example, the nucleotides at the 5' end or 3' end of the sense strand of the first nucleic acid molecule are linked to the targeting ligand, or the nucleotides at the 5' end or 3' end of the sense strand of the second nucleic acid molecule are linked to the targeting ligand.

[0047] As a preferred technical solution of the present invention, at least one nucleotide in the double-stranded RNA conjugate is modified, and the modification is selected from the group consisting of: alkyl nucleotides, methoxy nucleotides, ethoxy nucleotides, methoxyethyl nucleotides, amino nucleotides, fluoro nucleotides, deoxy nucleotides, 5′-methyl phosphate nucleotides, 5′-C-methyl phosphate nucleotides, 2′-deoxy-2′-fluoro nucleotides, vinyl phosphonate deoxyribonucleotides (VP), 5′-phosphate nucleotides (P), phosphorothioate nucleotides, phosphorodithioate nucleotides, locked nucleic acids (LNA), morpholino oligonucleotides (PMO), glycol nucleic acid modifications (GNA), hypoxanthine modifications (I), inverted abasic deoxyribose residues (invAb), and 5′-methyldeoxycytosine nucleotides (m5dC).

[0048] As a preferred technical solution of the present invention, the siRNA conjugate is selected from the nucleotide sequences described in Table 4.

[0049] In a fourth aspect, the present invention further provides intermediates and compounds of double-stranded RNA conjugates, selected from the structures shown in Table 1 and Table 3.

[0050] In a fifth aspect, the present invention further provides a pharmaceutical composition for inhibiting the expression of ANGPTL3 and PCSK9 genes, TTR and ANGPTL3 genes, TTR and FVII genes, wherein the pharmaceutical composition comprises the double-stranded RNA conjugate as described above.

[0051] As a preferred technical solution of the present invention, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient.

[0052] In a sixth aspect, the present invention also provides use of the double-stranded RNA conjugate described above in the preparation of a method for treating a disease, disorder or symptom mediated at least in part by the expression of ANGPTL3 and / or PCSK9 genes, TTR and / or ANGPTL3 genes, TTR and / or FVII genes.

[0053] The double-stranded RNA conjugate for inhibiting the expression of ANGPTL3 and PCSK9 provided by the present invention has good inhibitory activity against both ANGPTL3 and PCSK9, and can be used to prevent and / or treat related diseases mediated by the expression of ANGPTL3 and / or PCSK9 genes; the double-stranded RNA conjugate for inhibiting the expression of TTR and ANGPTL3 provided by the present invention has good inhibitory activity against both TTR and ANGPTL3, and can be used to prevent and / or treat related diseases mediated by the expression of TTR and / or ANGPTL3 genes; the double-stranded RNA conjugate for inhibiting the expression of TTR and FVII genes provided by the present invention has good inhibitory activity against both TTR and FVII genes, and can be used to prevent and / or treat related diseases mediated by the expression of TTR and / or FVII genes. DETAILED DESCRIPTION

[0054] The present invention will be further described in detail below with reference to examples, but the embodiments of the invention are not limited thereto.

[0055] As used herein, the term "comprising" is used to mean, and is used interchangeably with, the phrase "including, but not limited to," unless the context clearly dictates otherwise.

[0056] The term "or" is used herein to mean, and is used interchangeably with, the term "and / or," unless the context clearly dictates otherwise.

[0057] As used herein, the terms "sequence" and "nucleotide sequence" mean the order or sequence of nucleobases or nucleotides, described in alphabetical order using standard nomenclature.

[0058] As used herein, the term "PCSK9" refers to the proprotein convertase subtilisin / kexin9 gene or protein. PCSK9 is also known as FH3, HCHOLA3, NARC-1, or NARC1. The term PCSK9 includes human PCSK9, whose amino acid and nucleotide sequences can be found, for example, in GenBank Accession No. GI:299523249; mouse PCSK9, whose amino acid and nucleotide sequences can be found, for example, in GenBank Accession No. GI:163644257; and rat PCSK9, whose amino acid and nucleotide sequences can be found, for example, in GenBank Accession No. GI:77020249. Additional examples of PCSK9 mRNA sequences are readily available, for example, using GenBank.

[0059] Among them, the nucleic acid molecules used to inhibit PCSK9 gene expression in the present invention can be selected from patent application numbers or publication numbers CN201380063930.5, CN202080007281.7, CN202110643257.9, WO2022089486A1, CN201980041119.4, CN202210479298.3, WO2 The nucleic acid sequences disclosed in the patents 022266753A1, CN201880019823.5, CN201480036240.5, PCT / US2021 / 062831, CN201711429565.1, CN202210707102.1, WO2023049294A2, and PCT / US2022 / 034063.

[0060] The term "ANGPTL3" refers to angiopoietin-like protein 3 (NM_014495,4), a secreted protein primarily expressed in hepatocytes. Studies have shown that angiopoietin-like protein 3 (ANGPTL3) is a key regulator of LDL-C, HDL-C, and triglyceride metabolism and a key factor in lipid metabolism disorders, with multiple potential pathways of action. Loss-of-function mutations in ANGPTL3 can lead to lower LDL-C, VLDL-C, HDL-C, and triglycerides (TG), thereby reducing cardiovascular disease risk based on GWAS data, without the adverse phenotypes associated with known genetic defects. Therefore, inhibiting ANGPTL3 activity could be an effective way to prevent or treat dyslipidemia.

[0061] Among them, the nucleic acid molecules for inhibiting ANGPTL3 gene expression in the present invention can be selected from patent application numbers or publication numbers: WO2021188795A1, CN201480068063.9, CN201280041047.1, CN201680034444.4, CN201880048600.1, CN202180007277.5, CN201 The nucleic acid sequences disclosed in patents 880073901.X, WO2020099482A2, WO2022068923A1, CN201480068063.9, WO2022187435A1, WO2022268054A1, CN202210705962.1, WO2023045994A1, and WO2023134705A1.

[0062] The phrase "inhibiting the expression of ANGPTL3" in the present invention includes inhibiting the expression of any ANGPTL3 gene (e.g., mouse ANGPTL3 gene, rat ANGPTL3 gene, monkey ANGPTL3 gene, or human ANGPTL3 gene) as well as variants or mutants of ANGPTL3 genes encoding ANGPTL3 proteins.

[0063] The phrase "inhibiting the expression of PCSK9" includes inhibiting the expression of any PCSK9 gene (such as, for example, a mouse PCSK9 gene, a rat PCSK9 gene, a monkey PCSK9 gene, or a human PCSK9 gene) as well as variants (e.g., naturally occurring variants) or mutants of the PCSK9 gene. Thus, the PCSK9 gene can be a wild-type PCSK9 gene, a mutant PCSK9 gene, or a transgenic PCSK9 gene in the context of a genetically manipulated cell, cell group, or organism.

[0064] "Inhibiting the expression of ANGPTL3 and PCSK9" includes any level of inhibition of the ANGPTL3 and PCSK9 genes, such as at least partially suppressing the expression of the ANGPTL3 and PCSK9 genes, such as inhibiting at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%.

[0065] In the present invention, nucleic acid can refer to double-stranded siRNA or single-stranded ASO. In a double-stranded nucleic acid molecule, the bases of one chain are paired with the bases on the other chain in a complementary manner. The purine base adenine (A) always pairs with the pyrimidine base uracil (U); the purine base guanine (C) always pairs with the pyrimidine base cytosine (G). Each base pair includes a purine and a pyrimidine. When adenine on one chain always pairs with uracil on the other chain, and guanine always pairs with cytosine, the two chains are considered to be complementary to each other, and the sequence of the chain can be inferred from the sequence of its complementary chain.

[0066] In the present invention, modified nucleotides include but are not limited to: alkyl nucleotides, methoxy nucleotides, ethoxy nucleotides, methoxyethyl nucleotides, amino nucleotides, fluoro nucleotides, deoxy nucleotides, 5'-methyl phosphate nucleotides, 5'-C-methyl phosphate nucleotides, 2'-deoxy-2'-fluoro nucleotides, vinylphosphonate deoxyribonucleotides (VP), phosphorothioate nucleotides, phosphorodithioate nucleotides, locked nucleic acids (LNA), morpholino oligonucleotides (PMO), and inverted abasic deoxyribose residues (invAb).

[0067] Among them, alkyl-modified nucleotides, such as 2'-methyl nucleotides, 2'-ethyl nucleotides, 2'-methoxy modified nucleotides, e.g. 2'-methoxyethyl nucleotides, e.g. 2'-methoxyethoxy nucleotides, e.g. 2'-fluoronucleotides, e.g. 5'-C-methylphosphononucleotides, e.g.

[0068] (E)-vinylphosphonate deoxyribonucleotides (VP), e.g., phosphorothioate nucleotides (S), e.g., phosphorothioate nucleotides (S), e.g., 2'-deoxyribonucleotides, e.g., inverted abasic deoxyribose residues (invAb), e.g., glycol nucleic acids (GNA), including (S)-glycol nucleic acids ((S)-GNA), e.g., and (R)-glycol nucleic acids ((R)-GNA), e.g., inosine nucleotides (I), e.g., 5-methyldeoxypyrimidine nucleotides (m5dC).

[0069] wherein Base represents a base, and Me represents a methyl group, and Et represents an ethyl group.

[0070] The term "locked nucleic acid" is a nucleotide having a modified ribose moiety, wherein the ribose moiety includes an extra bridge connecting the 2' carbon and the 4' carbon. This structure effectively "locks" the ribose in the 3'-endo conformational state. The addition of locked nucleic acids to siRNA has been shown to increase siRNA stability in serum and to reduce off-target effects (Elmen, J. et al. (2005) Nucleic Acids Research 33(1): 439-447; Mook, OR. et al. (2007) Mol Cancer Ther 6(3): 833-843; Grunweller, A. et al. (2003) Nucleic Acids Research 31(12): 3185-3193).

[0071] Representative U.S. patents that teach the preparation of locked nucleic acid nucleotides include, but are not limited to, the following: U.S. Patent Nos. 6,268,490; 6,670,461; 6,794,499; 6,998,484; 7,053,207; 7,084,125; and 7,399,845, the entire contents of each of which are incorporated herein by reference.

[0072] Locked nucleic acid structures are as follows: In certain embodiments, the sugar surrogate comprises a ring having more than 5 atoms and more than 1 heteroatom. For example, nucleosides comprising morpholino sugar moieties have been reported and their use in oligomeric compounds (see, e.g., Braasch et al., Biochemistry, 2002, 41, 4503-4510; and U.S. Patents 5,698,685; 5,166,315; 5,185,444; and 5,034,506).

[0073] The term "morpholino" means a sugar surrogate having the formula:

[0074] In certain embodiments, the morpholino group can be modified, for example, by adding or changing various substituents according to the above morpholino structure. Such sugar surrogates are referred to herein as "modified morpholinos."

[0075] In the present invention, unless otherwise specified, capital letters C, G, U, and A represent the base composition of nucleotides. Lowercase letter m indicates that the nucleotide adjacent to the left of letter m is a methoxy-modified nucleotide; lowercase letter f indicates that the nucleotide adjacent to the left of letter f is a fluorine-modified nucleotide; LNA indicates that the nucleotide adjacent to the right of it is a locked nucleic acid (LNA) modified; lowercase letter s indicates that the two nucleotides on the left and right of the letter are connected by a thiophosphate group; VP indicates that the nucleotide to the right of the letter VP is a (E)-vinyl phosphate-modified nucleotide. invAb represents an inverted abasic deoxyribonucleotide; dN represents any deoxyribonucleotide; dA represents a deoxyadenine nucleotide; dT represents a deoxythymine nucleotide; dU represents a deoxyuridine nucleotide; dC represents a deoxycytosine nucleotide; and dG represents a deoxyguanine nucleotide.

[0076] It should be emphasized that the "modification" of nucleotides in the present disclosure includes but is not limited to the above examples, and nucleotides can also be replaced with other nucleotides, such as (S)-glycerol nucleic acid.

[0077] The term "targeting ligand" can include naturally occurring substances, such as proteins (e.g., human serum albumin (HAS), low-density lipoprotein (LDL) or globulin); carbohydrates (e.g., dextran, pullulan, chitin, chitosan, inulin, cyclodextrin, N-acetylglucosamine, N-acetylgalactosamine or hyaluronic acid); or lipids. The ligand can also be a recombinant or synthetic molecule, such as a synthetic polymer, for example, a synthetic polyamino acid. Examples of polyamino acids include the following polyamino acids: polylysine (PLL), poly-L-aspartic acid, poly-L-glutamic acid, styrene acid-maleic anhydride copolymer, poly-(L-lactide-co-glycolide) copolymer, divinyl ether-maleic anhydride copolymer, N-(2-hydroxypropyl) methacrylamide copolymer (HMPA), polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane, poly-(2-ethyl acrylic acid), N-isopropylacrylamide polymer or polyphosphazene. Examples of polyamines include polyethyleneimine, polylysine (PLL), spermine, spermidine, polyamines, pseudopeptide-polyamines, peptidomimetic polyamines, dendrimer polyamines, arginine, amidine, protamine, cationic lipids, cationic porphyrins, quaternary salts of polyamines, or alpha helical peptides.

[0078] The targeting ligand can also be a cell or tissue targeting agent that binds to a specified cell type, e.g., a lectin, glycoprotein, lipid, or protein, e.g., an antibody. The targeting group can be a thyrotropin, melanotropin, lectin, glycoprotein, surfactant protein A, mucin carbohydrate, multivalent lactose, multivalent galactose, N-acetyl-galactosamine, N-acetyl-glucosamine multivalent mannose, multivalent fucose, glycosylated polyamino acid, multivalent galactose, transferrin, bisphosphonate, polyglutamic acid, polyaspartic acid, lipid, cholesterol, steroid, bile acid, folate, vitamin B12, vitamin A, biotin, or an RGD peptide or RGD peptide mimetic.

[0079] The targeting ligand can also be a protein, e.g., a glycoprotein, or a peptide, e.g., a molecule with specific affinity for a co-ligand, or an antibody, e.g., an antibody that binds to a specified cell type, e.g., a hepatocyte. The ligand can also include a hormone and a hormone receptor. They can also include non-peptide species, e.g., a lipid, lectin, sugar, vitamin, co-factor, multivalent lactose, multivalent galactose, N-acetyl-galactosamine, N-acetyl-glucosamine multivalent mannose, or multivalent fucose. The ligand can be, e.g., a lipopolysaccharide, an activator of p38 MAP kinase, or an activator of NF-KB.

[0080] The targeting ligand can be a substance, e.g., a drug, that can increase cellular uptake of the iRNA agent into a cell, e.g., by perturbing the cytoskeleton of the cell, e.g., by perturbing the microtubules, microfilaments, and / or intermediate filaments of the cell. The drug can be, e.g., taxon, vincristine, vinblastine, cytochalasin, nocodazole, iaplakinolide, halichondrin A, phalloidin, swinholide A, indanotine, or myoservin.

[0081] The term "pharmaceutically acceptable excipient" is a substance that is intentionally included in a drug delivery system other than the active pharmaceutical ingredient (API, therapeutic product, e.g., ANGPTL3). The excipient does not exert or is not intended to exert a therapeutic effect at the intended dose. The excipient can serve the following purposes: a) aid in the handling of the drug delivery system during manufacture, b) protect, support, or enhance the stability, bioavailability, or patient acceptability of the API; c) aid in product identification; and / or d) enhance any other property of the API with respect to overall safety, effectiveness, or delivery during storage or use.

[0082] Among excipients include, but are not limited to, absorption enhancers, anti-adherents, antifoaming agents, antioxidants, binders, buffering agents, carriers, coatings, colorants, delivery enhancers, delivery polymers, detergents, dextran, dextrose, diluents, disintegrants, emulsifiers, effervescent agents, fillers, flavorants, glidants, humectants, oils, polymers, preservatives, saline, salts, solvents, sugars, surfactants, suspending agents, sustained release matrices, sweeteners, thickening agents, tonicity agents, vehicles, water repellents, wetting agents, lubricants such as sodium lauryl sulfate and magnesium stearate, flavorings, and aromatics.

[0083] Pharmaceutical compositions of the present disclosure include those suitable for oral, nasal, topical, buccal, sublingual, rectal, and / or parenteral administration. The formulations can conveniently be presented in unit dosage form and can be prepared by any methods well known in the art of pharmacy. The amount of active ingredient that can be combined with a carrier material to produce a single dosage form will generally be that amount of a compound that produces a therapeutic effect. Generally, this amount will range from about 1% to about 99% of the dosage, preferably from about 5% to about 70%, and most preferably from about 10% to about 30% by weight of the active ingredient.

[0084] The term "lipids" refers to any major lipid present in the blood. Lipids can exist in the blood either in free form or as part of a protein complex, such as a lipoprotein complex. Non-limiting examples of lipids can include triglycerides and cholesterol, such as total cholesterol (GT), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), very low-density lipoprotein cholesterol (VLDL-C), and intermediate-density lipoprotein cholesterol (IDLC).

[0085] The term "disorder of lipid metabolism" refers to any disorder associated with or caused by a perturbation in lipid metabolism. For example, the term includes any disorder, disease or condition that can result in hyperlipidemia, or a condition characterized by an abnormally elevated level of any or all lipids and / or lipoproteins in the blood. The term refers to both inherited disorders, such as familial hypertriglyceridemia, or acquired disorders, such as those acquired because of diet or ingestion of certain drugs. Exemplary disorders of lipid metabolism include, but are not limited to, atherosclerosis, dyslipidemia, hypertriglyceridemia (including drug-induced hypertriglyceridemia, diuretic-induced hypertriglyceridemia, alcohol-induced hypertriglyceridemia, beta-adrenergic blocker-induced hypertriglyceridemia, estrogen-induced hypertriglyceridemia, glucocorticoid-induced hypertriglyceridemia, retinoid-induced hypertriglyceridemia, cimetidine-induced hypertriglyceridemia, and familial hypertriglyceridemia), acute pancreatitis associated with hypertriglyceridemia, chylomicron syndrome, familial chylomicronemia, Apo-E deficiency or resistance, LPL deficiency or activity attenuation, hyperlipidemia (including familial combined hyperlipidemia), hypercholesterolemia, gout associated with hypercholesterolemia, xanthomatosis (subcutaneous cholesterol deposition).

[0086] The term "treatment" is used to mean obtaining a desired pharmacologic and / or physiologic effect. The effect can be prophylactic in terms of completely or partially preventing a disease or symptom thereof and / or can be therapeutic in terms of a partial or complete cure for a disease and / or adverse effect attributable to the disease. "Treatment" as used herein covers the treatment of a disease in a mammal, particularly in a human, and includes: (a) preventing the disease or condition from occurring in an individual which can be predisposed to the disease but has not yet developed the disease; (b) inhibiting the disease, i.e., arresting its development; or (c) relieving the disease, i.e., causing the partial or complete regression of the disease. "Treatment" as used herein covers any use of a drug or compound to treat, cure, relieve, alleviate, alter, improve, or inhibit a disease in an individual, including that of administering a drug containing a compound described herein to an individual in need thereof.

[0087] The term "inhibit" is used interchangeably with "reduce," "silence," "down-regulate," "arrest," and other similar terms and includes inhibition at any level.

[0088] Expression of the ANGPTL3 gene can be assessed based on the level of any variable associated with ANGPTL3 gene expression, such as ANGPTL3 mRNA levels or ANGPTL3 protein levels. Expression of ANGPTL3 can also be assessed indirectly based on the level of blood lipids, triglycerides, cholesterol (including LDL-C, HDL-C, VLDL-C, IDL-C, and total cholesterol), or free fatty acids. Inhibition can be assessed by a decrease in the absolute or relative level of one or more of these variables compared to a control level. The control level can be any type of control level utilized in the art, such as a pre-dose baseline level or a level determined from a similar subject, cell, or sample that has not been treated or that has been treated with a control (e.g., such as a buffer control or a non-active agent control only).

[0089] A "PCSK9-related disease" is intended to include any disease associated with the PCSK9 gene or protein. Such a disease can be caused, for example, by overproduction of the PCSK9 protein, by mutation of the PCSK9 gene, by abnormal cleavage of the PCSK9 protein, by abnormal interactions between PCSK9 and other proteins or other endogenous or exogenous agents. Exemplary PCSK9-related diseases include lipemia, such as hyperlipidemia, and other forms of lipid imbalance, such as hypercholesterolemia, hypertriglyceridemia, and pathological conditions associated with these imbalances, such as heart and circulatory diseases.

[0090] Example 1 Synthesis of Compound 1

[0091]

[0092] A specific synthesis route is as follows:

[0093] Step A: Synthesis of (S)-8-(2-hydroxymethyl)pyrrolidin-1-yl)-8-oxooctanoic acid methyl ester

[0094] (S)-8-(2-(Bis(4-methoxyphenyl)(phenyl)methoxy)methyl)pyrrolidin-1-yl)-8- oxooctanoic acid methyl ester

[0095] Step B: Synthesis of (S)-8-(2-(Bis(4-methoxyphenyl)(phenyl)methoxy)methyl)pyrrolidin-1-yl)-8- oxooctanoic acid methyl ester

[0096] (S)-8-(2-(Bis(4-methoxyphenyl)(phenyl)methoxy)methyl)pyrrolidin-1-yl)-8- oxooctanoic acid methyl ester

[0097] Step C: Synthesis of (S)-8-(2-(bis(4-methoxyphenyl)(phenyl)methoxy)methyl)pyrrolidin-1-yl)-8-oxooctanoic acid

[0098] At 15-25° C., (S)-8-(2-(bis(4-methoxyphenyl)(phenyl)methoxy)methyl)pyrrolidin-1-yl)-8-oxooctanoic acid methyl ester (100.00 g, 174.30 mmol) was dissolved in 750 ml of tetrahydrofuran and 250 ml of water, and lithium hydroxide dihydrate (209.16 g, 209.16 mmol) was added; after the system was reacted at 15-25° C. overnight, TLC showed that (S)-8-( Methyl 2-(bis(4-methoxyphenyl)(phenyl)methoxy)methyl)pyrrolidin-1-yl)-8-oxooctanoate completely disappeared; the system was concentrated to dryness at T≤35°C and then purified by column chromatography using a water / tetrahydrofuran system. The qualified fractions were concentrated until no fractions remained. After lyophilization, 73.20 g of (S)-8-(2-(bis(4-methoxyphenyl)(phenyl)methoxy)methyl)pyrrolidin-1-yl)-8-oxooctanoic acid was obtained (yield: 99.48%). (Exact Mass: 559.29, Found: (MH)=558.30).

[0099] Step D: Synthesis of N2-((9H-fluoren-9-yl)methoxy)carbonyl)-N6-((1s,4R)-4-hydroxycyclohexane-1-carbonyl)-L-lysine methyl ester: At 15-25°C, ((9H-fluoren-9-yl)methoxycarbonyl)-L-lysine methyl ester hydrochloride (20.00 g, 47.74 mmol) was dissolved in 200 ml of dichloromethane, and (1s,4s)-4-hydroxycyclohexane-1-carboxylic acid (7.64 g, 52.51 mmol) and N,N-diisopropylethylamine (9.31 g, 71.61 mmol) were added; the system was cooled to 0-5°C, and 1-(3-dimethylaminopropyl)-3-(4-nitropropoxy)-1 ... 1-Ethylcarbodiimide hydrochloride (11.89 g, 62.06 mmol) and HOPO (1.07 g, 9.55 mmol) were added and stirred for 10 minutes. The reaction was allowed to proceed overnight after the system temperature was returned to 15-25°C. TLC indicated the complete disappearance of ((9H-fluoren-9-yl)methoxycarbonyl)-L-lysine methyl ester hydrochloride, accompanied by the precipitation of a large amount of solid. Filtration afforded a white solid, which was then rinsed with 200 ml of dichloromethane and dried to afford N2-((9H-fluoren-9-yl)methoxy)carbonyl)-N6-((1s,4R)-4-hydroxycyclohexane-1-carbonyl)-L-lysine methyl ester (yield: 99.48%) (Exact Mass: 508.26, Found: (M+H)+ = 509.4).

[0100] Step E: Synthesis of N6-((1s,4R)-4-hydroxycyclohexane-1-carbonyl)-L-lysine methyl ester

[0101] At 15-25° C., N2-((9H-fluoren-9-yl)methoxy)carbonyl)-N6-((1s, 4R)-4-hydroxycyclohexane-1-carbonyl)-L-lysine methyl ester (16.00 g, 31.46 mmol) was dissolved in 160 ml of tetrahydrofuran and diethylamine (11.34 g, 157.29 mmol); the system was reacted at 15-25° C. overnight, and the N2-(( The reaction mixture (9H-fluoren-9-yl)-((1s,4R)-4-hydroxycyclohexane-1-carbonyl)-L-lysine methyl ester) completely disappeared, while a large amount of solid precipitated. Filtration afforded a white solid, which was then rinsed with 32 ml of dichloromethane and dried to yield 9 g of N-((1s,4R)-4-hydroxycyclohexane-1-carbonyl)-L-lysine methyl ester (yield: 99.9%). (Exact Mass: 286.19, Found: (M+H)+ = 287.3).

[0102] Step F: Synthesis of N2-(8-((S)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)pyrrolidin-1-yl)-8-oxooctanoyl)-N6-((1s,4R)-4-hydroxycyclohexane-1-carbonyl)-L-lysine methyl ester

[0103] At 15-25° C., (S)-8-(2-(bis(4-methoxyphenyl)(phenyl)methoxy)methyl)pyrrolidin-1-yl)-8-oxooctanoic acid (7.29 g, 13.02 mmol) was dissolved in 73 ml of dichloromethane, and N,N-diisopropylethylamine (4.21 g, 32.54 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (2.75 g, 14.32 mmol), and 1-hydroxybenzotriazole (1.94 g, 14.32 mmol) were added and stirred for 5 minutes; N6-((1s,4R)-4-hydroxycyclohexane-1-carbonyl)-L-lysine methyl ester (4.10 g, 14.32 mmol) was added under temperature control of 0-5° C. After the addition, the reaction system temperature was raised to 15-2 The reaction system was allowed to react at 15-25°C overnight, and the disappearance of (S)-8-(2-(bis(4-methoxyphenyl)(phenyl)methoxy)methyl)pyrrolidin-1-yl)-8-oxooctanoic acid was monitored by HPLC. The reaction system was washed twice with 35 ml of a saturated aqueous sodium bicarbonate solution; the organic phase was further washed once with 35 ml of a saturated aqueous sodium chloride solution; the organic phase was dried over anhydrous sodium sulfate and filtered; the filtrate was concentrated under a temperature control of T≤35°C until no fraction was obtained. The filtrate was subjected to dichloromethane / methanol column chromatography to obtain 9.12 g of N2-(8-((S)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)pyrrolidin-1-yl)-8-oxooctanoyl)-N6-((1s,4R)-4-hydroxycyclohexane-1-carbonyl)-L-lysine methyl ester (yield: 84.6%). (ExactMass: 827.47, Found: (MH)—=826.6).

[0104] Step G: Synthesis of N2-(8-((S)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)pyrrolidin-1-yl)-8-oxooctanoyl)-N6-((1s,4R)-4-hydroxycyclohexane-1-carbonyl)-L-lysine (B1)

[0105] N2-(8-((S)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)pyrrolidin-l-yl)-8- oxooctanoyl)-N6-((ls,4R)-4-hydroxycyclohexane-l-carbonyl)-L-lysine methyl ester (9 g, 10.87 mmol) was dissolved in 68 mL of tetrahydrofuran and 22 mL of water at 15-25 °C, and lithium hydroxide dihydrate (0.55 g, 13.04 mmol) was added; the system was reacted at 35 °C for 2 hours, and HPLC tracking showed that N2-(8-((S)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)pyrrolidin-l-yl)-8-oxooctanoyl)-N6-((ls,4R)-4-hydroxycyclohexane-l-carbonyl)-L-lysine methyl ester disappeared; the system was concentrated to no fraction, and the product N2-(8-((S)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)pyrrolidin-l-yl)-8-oxooctanoyl)-N6-((ls,4R)-4-hydroxycyclohexane-l-carbonyl)-L-lysine was obtained by lyophilization (yield: 88.1 %). (Exact Mass: 813.46, Found: (M-H) = 812.6).

[0106] Step H: synthesis of intermediate Cl

[0107] SM1 (14.06 g, 7.37 mmol) and N,N-diisopropylethylamine (3.32 g, 24.56 mmol) were dissolved in 100 ml of dichloromethane and stirred; the system was cooled to a target temperature of -5 to 5°C; the temperature was controlled at -5 to 5°C, and benzotriazole-N,N,N′,N′-tetramethyluronium hexafluorophosphate (2.94 g, 15.35 mmol) and N2-(8-((S)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)pyrrolidin-1-yl)-8-oxooctanoyl)-N6-((1s,4R)-4-hydroxycyclohexane-1-carbonyl)-L-lysine (4.21 g, 32.54 mmol) were added to the system; the reaction was returned to room temperature and stirred at room temperature for 2 hours; the reaction system temperature was raised to 400°C. 35 ° C, stirred overnight; HPLC showed that N2-(8-((S)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)pyrrolidin-1-yl)-8-oxooctanoyl)-N6-((1s,4R)-4-hydroxycyclohexane-1-carbonyl)-L-lysine completely disappeared; the reaction system was cooled to -5 to 5 ° C, 50 ml of buffer solution (pH = 7.0, K2HPO4 / KH2PO4,, temperature controlled at -5 to 5 ° C, and stirred for 5 minutes; after separation, the organic phase was washed once with 25 ml of water and twice with 25 ml of saturated NaCl solution; the organic phase was dried over sodium sulfate, filtered, and concentrated to obtain a crude product; the crude product was purified by reverse phase prep-HPLC (column: Daisogel The qualified fractions were concentrated at a temperature of T≤30°C and lyophilized to obtain 7 g of intermediate C1 (yield: 44.0%) (MW: 2589.95, (MW-DMTr+H+H)2+ / 2=1144.28, Found: 1144.5).

[0108] Step I: Synthesis of Compound 1

[0109] Under nitrogen protection, intermediate C1 (6.00 g, 2.32 mmol) was dissolved in 60 ml of dichloromethane, bis(diisopropylamino)(2-cyanoethoxy)phosphine (1.40 g, 4.64 mmol) was added, stirring was started, and the reaction system was cooled to 0-10°C; the temperature was maintained at 0-10°C, and tetrazole (0.16 g, 2.32 mmol) was added to the system; the system was returned to room temperature and stirred for 2-3 hours; HPLC showed that intermediate C1 had completely disappeared; the reaction system was washed twice with 18 ml of saturated sodium bicarbonate aqueous solution; the organic phase was washed once with 18 ml of saturated sodium chloride aqueous solution; and the reaction was dried over anhydrous sodium sulfate. The organic phase was dried and filtered to obtain a filtrate; the filtrate was concentrated to no fraction under the temperature of T≤30°C; the crude product was crystallized from 120 mL of DCM / MTBE (V / V=1 / 4) to obtain a white solid. The filter cake was rinsed with 12 mL of MTBE and nitrogen was purged for 14 h to obtain compound 1 (MW: 2790.17, (MW–DMTr–Diisopropylamine+H+H)2+ / 2=1194.3, Found: 1194.4; (MW–Propiononitrile–HH)2- / 2=1367.0, Found: 1367.2).

[0110] Example 2 Synthesis of Compound 2

[0111]

[0112] The specific synthetic route is as follows:

[0113] Step A: Synthesis of (S)-(6-(2-(hydroxymethyl)pyrrolidin-1-yl)-6-oxohexyl)carbamic acid

[0114] At 15-25°C, 6-((benzyloxy)carbonyl)amino)hexanoic acid (50.00 g, 188.45 mmol) was dissolved in 250 ml of dichloromethane and cooled to -5°C to 5°C; N,N-diisopropylethylamine (82.67 g, 637.55 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (43.35 g, 226.15 mmol), and 1-hydroxybenzotriazole (30.55 g, 226.15 mmol) were added, and the mixture was stirred for 20 minutes; (S)-pyrrolidin-2-ylmethanol (22.9 g, 510.04 mmol) in 250 ml of dichloromethane was added dropwise, and the mixture was stirred for 20 minutes. The solution was clarified by ethyl chloride. After the addition was complete, the temperature was raised to 15-25°C. The system was allowed to react overnight at 15-25°C, and TLC revealed the complete disappearance of 6-((benzyloxy)carbonyl)amino)hexanoic acid. The reaction system was washed twice with 125 mL of 10% citric acid solution. The organic phase was then washed once with 125 mL of saturated sodium bicarbonate solution and once with 125 mL of saturated sodium chloride solution. The organic phase was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated to dryness at T≤30°C and then purified by column chromatography using a dichloromethane / methanol system to obtain 50.50 g of benzyl (S)-(6-(2-(hydroxymethyl)pyrrolidin-1-yl)-6-oxohexyl)carbamate (yield: 84.6%) (Exact Mass: 348.20, Found: (M+H)+ = 349.2).

[0115] Step B: Synthesis of benzyl (S)-(6-(2-(bis(4-methoxyphenyl)(phenyl)methoxy)methyl)pyrrolidin-1-yl)-6-oxohexyl)carbamate

[0116] At 15-25° C., benzyl S)-(6-(2-(hydroxymethyl)pyrrolidin-1-yl)-6-oxohexyl)carbamate (47 g, 134.89 mmol), 4,4'-bismethoxytrityl chloride (54.84 g, 161.86 mmol), and 4-dimethylaminopyridine (1.65 g, 13.49 mmol) were dissolved in 470 ml of dichloromethane, and pyridine (53.35 g, 674.43 mmol) was added; the system was reacted at 15-25° C. overnight, and the reaction was monitored by TLC. The reaction mixture was washed twice with 50 ml of saturated sodium bicarbonate solution; the organic phase was washed once with 50 ml of saturated sodium chloride solution; the organic phase was dried over anhydrous sodium sulfate and filtered; the reaction mixture was concentrated to dryness at T≤35°C and then purified by column chromatography using n-heptane / ethyl acetate to obtain 90.5 g of benzyl (S)-(6-(2-(bis(4-methoxyphenyl)(phenyl)methoxy)methyl)pyrrolidin-1-yl)-6-oxohexyl)carbamate (yield: 98.0%). Exact Mass: 650.34, Found: (MH)=649.3.

[0117] Step C: Synthesis of (S)-6-amino-1-(2-(bis(4-methoxyphenyl)(phenyl)methoxy)methyl)pyrrolidin-1-yl)hexan-1-one

[0118] At 15-25° C., benzyl (54.84 g, 161.86 mmol) of S-(6-(2-(bis(4-methoxyphenyl)(phenyl)methoxy)methyl)pyrrolidin-1-yl)-6-oxohexyl)carbamate was dissolved in 500 ml of tetrahydrofuran in a 1 L four-necked flask. Palladium on carbon (10.00 g) and N,N-diisopropylethylamine (19.86 g, 153.65 mmol) were added. The system was reacted at 15-25° C. overnight under a hydrogen atmosphere. T LC tracking of benzyl (S)-(6-(2-(bis(4-methoxyphenyl)(phenyl)methoxy)methyl)pyrrolidin-1-yl)-6-oxohexyl)carbamate (disappearance; the system was filtered, and the filter cake was washed with 200 ml of tetrahydrofuran; the filtrate was concentrated to dryness, and then column chromatography using a dichloromethane / methanol system was used to obtain 35.0 g of (S)-6-amino-1-(2-(bis(4-methoxyphenyl)(phenyl)methoxy)methyl)pyrrolidin-1-yl)hexan-1-one (yield: 88%). Exact Mass: 516.30, Found: (M+Na)+=539.4).

[0119] Step D: Synthesis of 1-(tert-butyl)5-methyl((1s,4R)-4-hydroxycyclohexane-1-carbonyl)-L-glutamic acid

[0120] At 15-25°C, 1-(tert-butyl) 5-methyl L-glutamic acid (30.70 g, 141.47 mmol) was dissolved in 500 ml of dichloromethane and cooled to -5°C to 5°C; N,N-diisopropylethylamine (24.93 g, 192.92 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (29.59 g, 154.33 mmol), and 1-hydroxybenzotriazole (20.85 g, 154.33 mmol) were added in three batches while maintaining the temperature at -5°C to 5°C. The system was reacted overnight at 15-25°C. After completion of the reaction, post-treatment was initiated by TLC monitoring. The system was washed twice with 125 mL of 10% citric acid solution. The organic phase was washed once with 125 mL of saturated sodium bicarbonate solution and once with 125 mL of saturated sodium chloride solution. The organic phase was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated to dryness at T≤30°C and then purified by column chromatography using a dichloromethane / methanol system to obtain 37.0 g of 1-(tert-butyl)-5-methyl((1s,4R)-4-hydroxycyclohexane-1-carbonyl)-L-glutamic acid (yield: 83.9%). (Exact Mass: 343.20, Found: (2M+Na)+ = 709.3).

[0121] Step E: Synthesis of (S)-2-((1s,4R)-4-hydroxycyclohexane-1-carboxamido)-5-methoxy-5-oxopentanoic acid

[0122] 1-(tert-Butyl)-5-methyl((1s,4R)-4-hydroxycyclohexane-1-carbonyl)-L-glutamic acid (37.0 g, 108 mmol) was dissolved in 148 ml of dichloromethane at 15-25°C, and 74 ml of trifluoroacetic acid was added. After the system was reacted at 15-25°C overnight, TLC revealed the complete disappearance of 1-(tert-Butyl)-5-methyl((1s,4R)-4-hydroxycyclohexane-1-carbonyl)-L-glutamic acid. After concentration to dryness, column chromatography using dichloromethane / methanol was performed to obtain 15 g of (S)-2-((1s,4R)-4-hydroxycyclohexane-1-carboxamido)-5-methoxy-5-oxopentanoic acid (yield: 48.5%) (Exact Mass: 287.14, Found: (M+H)+=288.2).

[0123] Step F: Synthesis of (S)-methyl 5-((6-((S)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)pyrrolidin-1-yl)-6-oxohexyl)amino)-4-((1s,4R)-4-hydroxycyclohexane-1-carboxamido)-5-oxopentanoate

[0124] At 15-25° C., (S)-6-amino-1-(2-(bis(4-methoxyphenyl)(phenyl)methoxy)methyl)pyrrolidin-1-yl)hexan-1-one (11.00 g, 38.29 mmol) was dissolved in 33 ml of dichloromethane, and the system was cooled to -5 to 5° C.; N,N-diisopropylethylamine (4.95 g, 38.29 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (8.81 g, 4 mmol) were added to the system while maintaining the temperature at -5 to 5° C. g, 45.95 mmol), 1-hydroxybenzotriazole (6.225 g, 45.95 mmol), 22 ml of dichloromethane rinse the bottle mouth; at 15-25 ° C, weigh (S)-2-((1s, 4R)-4-hydroxycyclohexane-1-formamido)-5-methoxy-5-oxopentanoic acid in a 100 mL tetrafluoroethylene bottle, add N, N-diisopropylethylamine (9.89 g, 76.58 mmol) and 55 ml of dichloromethane, mix evenly, and dissolve clearly; control the temperature at -5 to 5 ° C, and add the mixture. A solution of (S)-2-((1s,4R)-4-hydroxycyclohexane-1-carboxamido)-5-methoxy-5-oxopentanoic acid was added dropwise to the reaction system; after the addition was complete, the system was naturally warmed to 15-25° C. overnight, and TLC showed that (S)-2-((1s,4R)-4-hydroxycyclohexane-1-carboxamido)-5-methoxy-5-oxopentanoic acid completely disappeared; the system was concentrated to dryness, and 110 ml of a mixed solvent of ethyl acetate / dichloromethane (V / V=10 / 1) was added to dissolve the solution; The organic phase was washed twice with 33 ml of saturated sodium bicarbonate solution; the organic phase was dried over anhydrous sodium sulfate and filtered; the filtrate was concentrated to dryness and then purified by column chromatography using a dichloromethane / methanol system to obtain 15.15 g of (S)-methyl 5-((6-((S)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)pyrrolidin-1-yl)-6-oxohexyl)amino)-4-((1s,4R)-4-hydroxycyclohexane-1-carboxamido)-5-oxopentanoate (yield 50.7%). (Exact Mass: 785.43, Found: (M+Na)+ = 808.6).

[0125] Step G: Synthesis of Compound B2

[0126] Methyl (S)-5-((6-((S)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)pyrrolidin-1-yl)-6-oxohexyl)amino)-4-((1s,4R)-4-hydroxycyclohexane-1-carboxamido)-5-oxopentanoate (11.00 g, 14.0 mmol) was dissolved in 83 ml of tetrahydrofuran and 28 ml of water at 15-25° C., and lithium hydroxide was added. The mixture was stirred and heated to 30°C, with stirring for 3 hours. HPLC analysis revealed the complete disappearance of (S)-methyl 5-((6-((S)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)pyrrolidin-1-yl)-6-oxohexyl)amino)-4-((1s,4R)-4-hydroxycyclohexane-1-carboxamido)-5-oxopentanoate. The system was concentrated to a temperature of T≤40°C until no more fractions were generated. The remaining mixture was lyophilized to obtain 8.3 g of Compound B2 (yield: 76.8%).

[0127] Step H: Synthesis of intermediate C2: SM1 (17.16 g, 8.55 mmol) was dissolved in 180 ml of dichloromethane and stirred to dissolve. The system was cooled to a target temperature of -5 to 5°C. The temperature was controlled at -5 to 5°C, and N,N-diisopropylethylamine (4.00 g, 31.12 mmol) and benzotriazole-N,N,N′,N′-tetramethyluronium hexafluorophosphate (4.40 g, 11.67 mmol) were added; (S)-5-((6-((S)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)pyrrolidin-1-yl)-6-oxohexyl)amino)-4-( (1s, 4R)-4-hydroxycyclohexane-1-carboxamido)-5-oxopentanoic acid (3.32 g, 24.56 mmol) was transferred to a constant drop; the temperature was controlled at -5 to 5°C, and the prepared (S)-5-((6-((S)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)pyrrolidin-1-yl)-6-oxohexyl)amino)-4-((1s, 4R)-4-hydroxycyclohexane-1-carboxamido) The dichloromethane solution of (S)-5-oxopentanoic acid was slowly added dropwise to the reaction system; the temperature was controlled at -5 to 5°C, and after reacting for 12 hours, HPLC monitoring was performed; HPLC showed that (S)-5-((6-((S)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)pyrrolidin-1-yl)-6-oxohexyl)amino)-4-((1s,4R)-4-hydroxycyclohexane-1-carboxamido)-5-oxopentanoic acid completely disappeared; the temperature was controlled at -5 to 5°C, 60 ml of buffer solution (pH = 7.0, K2HPO4 / KH2PO4) was added to the reaction system, the temperature was controlled at -5 to 5°C, and stirring was carried out for 5 minutes; after separation, the organic phase was washed once with 30 ml of water and twice with 30 ml of saturated NaCl solution; the organic phase was dried over sodium sulfate, filtered, and concentrated to obtain a crude product; the crude product was prepared and purified by reverse phase prep-HPLC (column: Daisogel SP-100-8-ODS-P, C1850*280mm#8um; mobile phase: [50mMNH4OAC / ACN]; B%: 15%-85%, 60min); qualified fractions were concentrated at a temperature of T≤30°C and lyophilized to obtain 12.3g of intermediate C2 (yield: 62.1%). (MW: 2547.86, (MW-DMTr+H+H) 2+ / 2=1123.2,Found:1123.6).

[0128] Step I: Synthesis of Compound 2: Under nitrogen protection, the intermediate C2 (5.50 g, 2.16 mmol) was dissolved in 55 ml of dichloromethane, bis(diisopropylamino)(2-cyanoethoxy)phosphine (1.30 g, 4.32 mmol) was added, stirring was started, and the reaction system was cooled to 0-10°C; the temperature was controlled at 0-10°C, tetrazole (0.37 g, 2.16 mmol) was added to the system; the system was heated to 15-25°C and stirred for 2-3 hours; HPLC showed that the intermediate C2 completely disappeared; the reaction system was washed twice with 15 ml of saturated sodium bicarbonate aqueous solution; the organic phase was washed once with 15 ml of saturated sodium chloride aqueous solution; anhydrous sulfur The organic phase was dried over sodium bicarbonate and filtered to obtain a filtrate; the filtrate was concentrated to a fraction free state with the temperature controlled at T≤30°C; the crude product was crystallized from 110 ml of DCM / MTBE (V / V=1 / 4) to obtain a white solid; the filter cake was rinsed with 11 ml of MTBE and nitrogen was purged for 14 hours to obtain compound 2 (MW: 2748.09, (MW-DMTr-Diisopropylamine+H+H)2+ / 2=1173.6, Found: 1173.4; (MW–Propiononitrile–HH)2- / 2=1346.0, Found: 1346.0 & 1346.1).

[0129] Example 3 Synthesis of Compound 3

[0130]

[0131] The specific synthetic route is as follows:

[0132] Step A: Synthesis of (S)-3-(((Benzyloxy)carbonyl)amino)amino)-4-(((6-((S)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)pyrrolidin-1-yl)-6-oxohexyl)amino)-4-oxobutanoic acid methyl ester. At room temperature, (S)-6-amino-1-(2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)pyrrolidin-1-yl) Hexane-1-one (2.1 g, 4.07 mmol) and (S)-2-(((benzyloxy)carbonyl)amino)-4-methoxy-4-oxobutanoic acid (1.09 g, 3.88 mmol) were dissolved in dichloromethane (18 ml). The system was cooled to 0°C and DIPEA (1.5 g, 11.64 mmol) and HATU (1.77 g, 4.66 mmol) were added with stirring, and the mixture was stirred at room temperature for 1 hour.

[0133] After completion of the reaction, the residue was purified by silica gel column chromatography (eluent: dichloromethane / methanol = 10 / 1) to give 2.84 g of methyl (S)-3-(((benzyloxy)carbonyl)amino)amino)-4-(((6-((S)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)pyrrolidin-1-yl)-6-oxohexyl)amino)-4-oxobutanoate as a yellow foamy solid (yield: 94%).

[0134] Step B: Synthesis of (S)-3-amino-4-((6-((S)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)pyrrolidin-1-yl)-6-oxohexyl)amino)-4-oxobutanoic acid methyl ester

[0135] Methyl (S)-3-(((benzyloxy)carbonyl)amino)amino)-4-(((6-((S)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)pyrrolidin-1-yl)-6-oxohexyl)amino)-4-oxobutanoate (1.78 g, 2.28 mmol) was dissolved in tetrahydrofuran (9 ml) at room temperature, and DIPEA (590 mg, 4.56 mmol) and 10% Pd(OH)2 / C (780 mg) were added with stirring, and hydrogenation was performed at room temperature and normal pressure for 4 hours.

[0136] After the reaction, the mixture was filtered and concentrated to give 1.46 g of methyl (S)-3-amino-4-((6-((S)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)pyrrolidin-1-yl)-6-oxohexyl)amino)-4-oxobutanoate as an off-white foamy solid (yield: 99%).

[0137] Step C: Synthesis of (S)-4-((6-((S)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)pyrrolidin-1-yl)-6-oxohexyl)amino)-3-((1S,4R)-4-hydroxycyclohexane-1-carboxamido)-4-oxobutanoic acid methyl ester

[0138] At room temperature, (S)-methyl 3-amino-4-((6-((S)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)pyrrolidin-1-yl)-6-oxohexyl)amino)-4-oxobutanoate (1.68 g, 2.6 mmol) and (1S,4S)-4-hydroxycyclohexane-1-carboxylic acid (410 mg, 2.86 mmol) were dissolved in dichloromethane (100 ml). The system was cooled to 0°C and DIPEA (1.01 g, 7.8 mmol) and HATU (1.19 g, 3.12 mmol) were added with stirring, and the mixture was stirred at room temperature for 1 hour.

[0139] After completion of the reaction, the residue was purified by silica gel column chromatography (eluent: dichloromethane / methanol = 10 / 1) to give 1.33 g of methyl (S)-4-((6-((S)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)pyrrolidin-1-yl)-6-oxohexyl)amino)-3-((1S,4R)-4-hydroxycyclohexane-1-carboxamido)-4-oxobutanoate as a yellow foamy solid (yield: 66%).

[0140] Step D: Synthesis of (S)-4-((6-((S)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)pyrrolidin-1-yl)-6-oxohexyl)amino)-3-((1S,4R)-4-hydroxycyclohexane-1-carboxamido)-4-oxobutanoic acid

[0141] At room temperature, (S)-methyl 4-((6-((S)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)pyrrolidin-1-yl)-6-oxohexyl)amino)-3-((1S,4R)-4-hydroxycyclohexane-1-carboxamido)-4-oxobutanoate (1.33 g, 1.73 mmol) was dissolved in a mixed solution of tetrahydrofuran (3.3 ml) and water (1.1 ml). Lithium hydroxide monohydrate (170 mg, 4.15 mmol) was added, and the system was heated to 30°C and stirred for 3 hours.

[0142] After the reaction, the system temperature was maintained below 40 degrees Celsius, tetrahydrofuran was concentrated until no fraction flowed out, and the remaining system was directly freeze-dried to obtain 1.23 grams of white solid (S)-4-((6-((S)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)pyrrolidin-1-yl)-6-oxohexyl)amino)-3-((1S,4R)-4-hydroxycyclohexane-1-carboxamido)-4-oxobutanoic acid (yield: 94%).

[0143] Step E: Synthesis of intermediate C3

[0144] 4-(5-chloro-2-(4-chloro-1H-1,2,3-triazol-1-yl)phenyl)pyridin-2(1H)-one (1.23 g, 0.44 mmol) was dissolved in dichloromethane (37 ml) at room temperature. The system was cooled to 0°C, and DIPEA (840 mg, 6.48 mmol) and HBTU (920 mg, 2.43 mmol) were added. Compound 1 (2.91 g, 1.62 mmol) was dissolved in dichloromethane (12 ml) and slowly added dropwise to the reaction system at 0°C. The reaction was allowed to proceed at 0°C for 12 hours.

[0145] After the reaction, the reaction system was washed twice with a saturated aqueous sodium bicarbonate solution (50 ml), and the organic phase was washed once with a saturated aqueous sodium chloride solution (50 ml). The organic phase was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated to dryness and purified by silica gel column chromatography (eluent: dichloromethane / methanol = 10 / 1) to obtain 457 mg of intermediate C3 as a yellow foamy solid (yield: 12%).

[0146] Step F: Synthesis of compound 3

[0147] At 0°C under nitrogen protection, intermediate C3 (377 mg, 0.15 mmol), 4A molecular sieves (400 mg) and tetrazole (130 mg, 0.75 mmol) were dissolved in dichloromethane (12 ml), and bis(diisopropylamino)(2-cyanoethoxy)phosphine (90 mg, 0.3 mmol) was added. The system was warmed to room temperature and stirred for 1 hour.

[0148] After the reaction, the reaction system was washed twice with a saturated aqueous sodium bicarbonate solution (12 ml), and the organic phase was washed once with a saturated aqueous sodium chloride solution (12 ml). The organic phase was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated to dryness and purified by silica gel column chromatography (eluent: dichloromethane / methanol = 10 / 1) to obtain 160 mg of compound 3 (yield: 39%). 1 HNMR (400MHz, DMSO-d6) δ7.91–7.82(m,5H),7.77(t,J=5.7Hz,2H),7.33(dd,J=11.6,7.1Hz,2H),7.23(t,J=7.9Hz,4H),6.90(t,J=6.6H z,2H),5.23(d,J=3.3Hz,2H),4.98(dd,J=11.1,3.3Hz,1H),4.50(d,J=8.5Hz,2H),4.04(s,11H),3.73(d,J=18.5Hz,11H),3.59–3.50(m, 15H),3.05(p,J=6.4,6.0Hz,13H),2.47(t,J=7.1Hz,14H),2.29(t,J=6.4Hz,5H),2.12(s,9H),2.06(t,J=7.1Hz,5H),2.01(s,11H),1.9 1(s,9H),1.79(s,9H),1.50(dt,J=16.6,8.1Hz,21H),1.25(s,6H),1.19(t,J=7.1Hz,3H),1.14(d,J=6.8Hz,9H),0.96(t,J=7.2Hz,25H). 31PNMR(162MHz,DMSO)δ144.96.LCMS:RT=2.08min,[M-DMTr+H+H] 2+ =1166.6.

[0149] Example 4 Synthesis of Compound 4

[0150]

[0151] Step A: Synthesis of Intermediate C4

[0152] SM2 (1 g, 0.67 mmol) was dissolved in dichloromethane (20 ml) at room temperature, and N,N-diisopropylethylamine (680 mg, 6.7 mol) and HATU (380 mg, 1.01 mmol) were added with stirring. Intermediate B4 (600 mg, 0.78 mmol) was added at 0°C, and the mixture was stirred at 0°C for 12 hours.

[0153] After the reaction, the mixture was quenched with sodium bicarbonate and extracted with dichloromethane (30 ml x 2). The organic phases were combined, dried, and concentrated to obtain an oil, which was purified by silica gel column chromatography (eluent: dichloromethane / methanol = 10 / 1) to obtain 360 mg of a white solid 0017-2 (yield: 24%). LCMS: RT = 1.94 min, [M-DMTr+H] + =1950.

[0154] Step B: Synthesis of Compound 4

[0155] At room temperature, intermediate C4 (360 mg, 0.16 mmol) and diisopropyl tetrazolyl ammonium salt (110 mg, 0.64 mmol) were dissolved in dichloromethane (10 ml), an appropriate amount of activated molecular sieves were added, and the atmosphere was replaced with nitrogen. Bis(diisopropylamino)(2-cyanoethoxy)phosphine (190 mg, 0.64 mmol) was added with stirring, and the mixture was stirred at room temperature for 8 hours.

[0156] After the reaction, the reaction mixture was added dropwise with stirring to a saturated sodium bicarbonate / saturated brine solution (6 ml / 6 ml) pre-cooled in an ice-water bath. The organic phase was then washed with saturated sodium bicarbonate / saturated brine (6 ml / 6 ml), dried over sodium sulfate, filtered, and concentrated to dryness to obtain an oil. This was purified by silica gel column chromatography (basified with triethylamine) (eluent: dichloromethane / methanol = 20:1) to obtain 177 mg of compound 4 (yield: 45%).

[0157] LCMS: RT=1.98min, (MW-Propiononitrile-HH)2- / 2=1198.1H NMR(400MHz,Chloroform-d)δ7.88(s,1H),7.42(d,J=7.6Hz,2H),7.36-7.29(m,7H),6.85(t,J=8.9Hz,5H),5.38(s,3H) ,5.34(d,J=3.5Hz,3H),5.26(d,J=10.6Hz,2H),4.78(d,J=9.0Hz,2H),4.34(s,3H),4.13(dt,J=30.4,10.6Hz,13H),3.98 (d,J=11.8Hz,6H),3.88-3.71(m,12H),3.55(t,J=38.6Hz,25H),3.26(d,J=13.9Hz,6H),2.67(s,5H),2.18(d,J=3.7Hz, 9H),2.11-2.06(m,12H),2.06-1.96(m,24H),1.89(d,J=12.2Hz,7H),1.62(d,J=49.4Hz,8H),1.21(d,J=5.8Hz,12H).31P NMR (400MHz, CDCl3): ppmδ145.84.

[0158] Example 5 Synthesis of Compound 5

[0159]

[0160] The specific synthetic route is as follows:

[0161] Step A: Synthesis of C5

[0162] To a solution of SM1 (2 g, 1.11 mmol) and B4 (686 mg, 0.86 mmol) in dichloromethane (45 mL) were added HBTU (631 mg, 1.67 mmol) and N,N-diisopropylethylamine (0.92 mL, 5.55 mmol) at room temperature. After stirring at 45°C for 16 hours, the mixture was concentrated under reduced pressure to obtain a residue, which was purified using a C18 column (eluent: acetonitrile / water = 1 / 1). The resulting product was further purified by silica gel column chromatography (eluent: dichloromethane / methanol = 10 / 1, containing 0.5% triethylamine) to obtain 1.69 g of C5 as a white solid (yield: 76%).

[0163] Step B: Synthesis of Compound 5

[0164] At room temperature, a mixture containing C5 (500 mg, 0.19 mmol) and To a solution of molecular sieves in dichloromethane (8 mL) was added diisopropylammonium tetrazolium salt (166 mg, 0.97 mmol) and bis(diisopropylamino)(2-cyanoethoxy)phosphine (0.6 mL, 1.9 mmol) and allowed to react at room temperature for 1 hour. After completion, the reaction was quenched with a saturated sodium bicarbonate / saturated sodium chloride solution (1:1, 5 mL) at -20°C and diluted with dichloromethane. The organic phase was backwashed three times with the above solution, 20 mL each time, then dried over anhydrous sodium sulfate and concentrated under reduced pressure to 1 mL. 20 mL of methyl tert-butyl ether was added dropwise to the solution at 0°C. The mixture was stirred for 5 minutes and then centrifuged. The supernatant was separated and the solid at the bottom was dissolved by sonication in 1 mL of dichloromethane. The above procedure was repeated four times and concentrated to yield 240 mg of compound 5 (yield: 45%). LCMS: RT = 2.036 min, [M-DMTr-Diisopropylamine+H+H] 2+ / 2=1187.0. 31 PNMR(162MHz,DMSO-d6)δ145.01(d,J=8.0Hz).

[0165] Example 6 Synthesis of Compound 6

[0166]

[0167] The specific synthetic route is as follows:

[0168] Step A: Synthesis of intermediate C6

[0169] To a solution of intermediate SM1 (1.3 g, 0.72 mmol) in dichloromethane (15.0 mL) at room temperature were added N,N-diisopropylethylamine (478.0 μL, 2.75 mmol), benzotriazole-N,N,N' 2 , N'-tetramethyluronium hexafluorophosphate (312.7 mg, 0.825 mmol) and (S)-5-((2-(3-((S)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)pyrrolidin-1-yl)-3-oxopropoxy)ethyl)amino)-4-((1S,4R)-4-hydroxycyclohexane-1-carboxamido)-5-oxopentanoic acid (428.7 mg, 0.55 mmol) were reacted at 45 °C overnight.

[0170] After the reaction was complete, the residue was concentrated under reduced pressure and purified by silica gel column chromatography (eluent: dichloromethane / methanol = 10 / 1, containing 0.5% triethylamine) to obtain 500.0 mg of intermediate C6 as a white foamy solid (yield: 35.7%). LCMS: RT = 1.870 min, [M-DMTr+H+H] 2+ / 2=1124.4.

[0171] Step B: Synthesis of compound 6

[0172] To a three-necked flask containing 4A molecular sieves and N,N-diisopropylammonium tetrazolide (171.2 mg, 1.0 mmol) at 0 °C was added intermediate C6 (500.0 mg, 0.20 mmol) in dichloromethane (10.0 mL), followed by bis(diisopropylamino)(2-cyanoethoxy)phosphine (190.0 μL, 0.60 mmol) and the reaction was allowed to proceed at room temperature for 1 h.

[0173] The reaction was quenched with saturated aqueous sodium bicarbonate solution of ice, diluted with dichloromethane, washed once with saturated aqueous sodium bicarbonate solution, once with saturated brine, then dried over anhydrous sodium sulfate and concentrated under reduced pressure to give a residue which was purified by column chromatography on silica gel (eluent: dichloromethane / methanol = 20 / 1 containing 0.5% triethylamine) to give 150.0 mg of compound 6 (yield: 27.3%). LCMS: RT = 2.155 min, [M-cyanoethyl]" = 1347.4. 31 PNMR (162 MHz, DMSO-d6) δ 141.54.

[0174] Example 7 Synthesis of compound 7

[0175]

[0176] The specific synthesis route is as follows:

[0177] Step A: Synthesis of intermediate compound C7

[0178] To a three-necked flask containing intermediate SM3 (1.1 g, 0.71 mmol) in dichloromethane (10.0 mL) at 0 °C was added N,N-diisopropylethylamine (494.0 μL, 2.84 mmol), benzotriazol-1-yl-oxy- tris-(dimethylamino-phosphonium hexafluorophosphate (404.0 mg, 1.07 mmol) and (S)-5-((6-((S)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)pyrrolidin-1-yl)-6- oxohexyl)amino)-4-((1S,4R)-4-hydroxycyclohexane-1-carbonylamino)-5-oxopentanoic acid (543.0 mg, 0.71 mmol) and the reaction was allowed to proceed at room temperature overnight.

[0179] The reaction was quenched with ice-cold saturated aqueous sodium bicarbonate solution, diluted with dichloromethane, washed once with saturated aqueous sodium bicarbonate solution, once with saturated aqueous sodium chloride solution, then dried over anhydrous sodium sulfate and concentrated under reduced pressure to give a residue which was purified by silica gel column chromatography (eluent: dichloromethane / methanol = 20 / 1 containing 0.5% triethylamine) to give 150.0 mg of compound 7 (yield: 50.4%). LCMS: RT = 2.052 min, [M-DMTr-Diisopropylamine + H + H]2+ / 2 = 1039.0.31P NMR (162 MHz, DMSO-d6) δ 144.96 (d, J = 9.9 Hz).

[0180] Step B: Synthesis of compound 7

[0181] To a three-necked flask containing 4A molecular sieves and N,N-diisopropylammonium tetrazolide (101.4 mg, 0.60 mmol) at 0 °C was added intermediate C7 (270.0 mg, 0.12 mmol) in dichloromethane (5.0 mL), followed by bis(diisopropylamino)(2-cyanoethoxy)phosphine (75.0 μL, 0.24 mmol) and the reaction was allowed to proceed at room temperature for 1 h.

[0182] The reaction was quenched with ice-cold saturated aqueous sodium bicarbonate solution, diluted with dichloromethane, washed once with saturated aqueous sodium bicarbonate solution, once with saturated aqueous sodium chloride solution, then dried over anhydrous sodium sulfate and concentrated under reduced pressure to give a residue which was purified by silica gel column chromatography (eluent: dichloromethane / methanol = 20 / 1 containing 0.5% triethylamine) to give 150.0 mg of compound 7 (yield: 50.4%). LCMS: RT = 2.052 min, [M-DMTr-Diisopropylamine + H + H]2+ / 2 = 1039.0.31P NMR (162 MHz, DMSO-d6) δ 144.96 (d, J = 9.9 Hz).

[0183] Example 8 Synthesis of compound 8

[0184]

[0185] The specific synthesis route is as follows:

[0186] Step A: Synthesis of C8

[0187] To SM1 (2 g, 1.11 mmol) and B6 (640 mg, 0.86 mmol) in dichloromethane (45 mL) at room temperature was added HBTU (631 mg, 1.67 mmol) and N,N-diisopropylethylamine (0.92 mL, 5.55 mmol). After stirring at 45 °C for 16 h, the residue was purified by C18 column (eluent: acetonitrile / water = 1 / 1) and the obtained product was further purified by silica gel column chromatography (eluent: dichloromethane / methanol = 10 / 1 containing 0.5% triethylamine) to give 1.8 g of C8 as a light yellow solid (yield: 83%).

[0188] Step B: Synthesis of compound 8

[0189] To a solution of C8 (600 mg, 0.24 mmol) and molecular sieves in dichloromethane (10 mL) was added diisopropylammonium salt tetrazole (203 mg, 1.19 mmol) and bis(diisopropylamino)(2-cyanoethoxy) phosphine (0.75 mL, 2.38 mmol) and the reaction was allowed to proceed at room temperature for 1 h. The reaction was quenched with saturated aqueous sodium bicarbonate / saturated aqueous sodium chloride (1 : 1, 5 mL) at -20 °C and the organic phase was washed with the above aqueous solution three times, 20 mL each time, then dried over anhydrous sodium sulfate and concentrated under reduced pressure to 1 mL. To 20 mL of methyl tert-butyl ether at 0 °C was added the above organic phase dropwise. After stirring for 5 min, the supernatant was separated by centrifugation. The bottom solid was dissolved in 1 mL of dichloromethane and the above operation was repeated four times. After concentration, 300 mg of compound 8 was obtained (yield: 46%). LCMS: RT = 1.981 min, [M-DMTr-Diisopropylamine + H+H] 2+ / 2 = 1160.2. 31 PNMR (162 MHz, DMSO-d6) δ 145.06.

[0190] Example 9 Synthesis of compound 9

[0191]

[0192] The specific synthesis route is as follows:

[0193] Step A: Synthesis of intermediate compound C9

[0194] To a solution of intermediate SM3 (1.0 g, 0.66 mmol) in dichloromethane (10.0 mL) at 0 °C was added N,N-diisopropylethylamine (459.0 μL, 2.64 mmol), benzotriazol- N,N,N',N'-tetramethyluronium hexafluorophosphate (375.5 mg, 0.99 mmol) and N 2 -(8-((S)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)pyrrolidin-1-yl)-8- oxooctanoyl)-N 6 -((1S,4R)-4-hydroxycyclohexane-1-carbonyl)-L-lysine (590.2 mg, 0.73 mmol) and the reaction was allowed to proceed at room temperature overnight.

[0195] After the reaction was complete, the residue was concentrated under reduced pressure and purified by silica gel column chromatography (eluent: dichloromethane / methanol = 10 / 1, containing 0.5% triethylamine) to obtain 750.0 mg of Intermediate C9 as a white foamy solid (yield: 49.0%). LCMS: RT = 4.259 min, [M-DMTr+H+H] 2+ / 2=1010.2.

[0196] Step B: Synthesis of compound 9

[0197] To a three-necked flask containing 4A molecular sieves and N,N-diisopropylammonium tetrazolium salt (140.0 mg, 0.82 mmol) was added intermediate C9 (380.0 mg, 0.16 mmol) in dichloromethane (5.0 mL) at 0°C, followed by the addition of bis(diisopropylamino)(2-cyanoethoxy)phosphine (104.0 μL, 0.32 mmol) and the reaction was carried out at room temperature for 1 hour.

[0198] After the reaction was complete, the reaction was quenched with an ice-cold saturated aqueous sodium bicarbonate solution, diluted with dichloromethane, washed once with a saturated aqueous sodium bicarbonate solution and once with saturated brine, then dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: dichloromethane / methanol = 20 / 1, containing 0.5% triethylamine) to obtain 190.0 mg of compound 9 (yield: 47.1%). LCMS: RT = 4.987 min, [M-DMTr-Diisopropylamine+H+H]2+ / 2 = 1060.0. 31 PNMR(162MHz,Chloroform-d)δ149.74.

[0199] Example 10 Synthesis of Compound 10

[0200]

[0201] The specific synthetic route is as follows:

[0202] Step A: Synthesis of Compound C10

[0203] SM2 (2 g, 1.34 mmol) and B8 (2.18 g, 2.68 mmol) were dissolved in acetonitrile (100 ml) at room temperature, and NMI (440 mg, 5.36 mol) and TCFH (750 mg, 2.68 mmol) were added with stirring, and the mixture was stirred at 60°C for 48 hours.

[0204] After the reaction, the mixture was quenched with sodium bicarbonate and extracted with dichloromethane (50 ml x 2). The organic phases were combined, dried, and concentrated to obtain an oil, which was purified by silica gel column chromatography (eluent: dichloromethane / methanol = 10 / 1) to obtain 600 mg of white solid C10 (yield: 20%). LCMS: RT = 2.03 min, [M-DMTr+H] + =1991.81.

[0205] Step B: Synthesis of Compound 10

[0206] At room temperature, C10 (360 mg, 0.16 mmol) and diisopropyltetrazolyl ammonium salt (110 mg, 0.64 mmol) were dissolved in dichloromethane (10 ml), an appropriate amount of activated molecular sieves were added, and the atmosphere was replaced with nitrogen. Bis(diisopropylamino)(2-cyanoethoxy)phosphine (190 mg, 0.64 mmol) was added with stirring, and the mixture was stirred at room temperature for 8 hours.

[0207] After the reaction, the reaction mixture was added dropwise with stirring to a saturated sodium bicarbonate / saturated brine solution (6 ml / 6 ml) pre-cooled in an ice-water bath. The organic phase was then washed with saturated sodium bicarbonate / saturated brine (6 ml / 6 ml), dried over sodium sulfate, filtered, and concentrated to dryness to obtain an oil. This was purified by silica gel column chromatography (basified with triethylamine) (eluent: dichloromethane / methanol = 20:1) to obtain 177 mg of compound 10 (yield: 45%).

[0208] LCMS:RT=1.98min,(MW-Propiononitrile-HH) 2 - / 2=1198. 1 H NMR(400MHz,DMSO-d6)δ8.07(d,J=5.5

[0209] Hz,2H),7.85(d,J=9.2Hz,2H),7.37-7.31(m,3H),7.23(td,J=9.3,2.7Hz,5H),6.99-6.79(m,3H),5.24(d,J=3. 4Hz,3H),5.01(dt,J=11.3,2.9Hz,2H),4.57(d,J=8.5Hz,3H),4.04(d,J=5.5Hz,10H),3.90(dt,J=11.0,8.7Hz,3 H),3.75(s,11H),3.64-3.47(m,11H),3.39(d,J=22.7Hz,26H),3.26(d,J=5.6Hz,7H),3.13-2.89(m,7H),2.12(d ,J=1.1Hz,11H),2.01(d,J=1.3Hz,9H),1.91(s,10H),1.80(s,10H),1.47(s,11H),1.15(dd,J=6.8,2.8Hz,13H). 31 P NMR (400MHz, DMSO-d6): ppmδ145.00.

[0210] Example 11 Synthesis of Compound 11

[0211]

[0212] The specific synthetic route is as follows:

[0213] Step A: Synthesis of Intermediate C11: To a solution of intermediate SM1 (1.3 g, 0.72 mmol) in dichloromethane (15.0 mL) was added N,N-diisopropylethylamine (478.0 μL, 2.75 mmol), benzotriazole-N,N,N'-diisopropylethylamine (478.0 μL, 2.75 mmol), and benzotriazole-N,N,N'-diisopropylethylamine (478.0 μL, 2.75 mmol). 2 , N'-tetramethyluronium hexafluorophosphate (312.7 mg, 0.825 mmol) and N 2 -(8-(4-(bis(4-methoxyphenyl)(phenyl)methoxy)piperidin-1-yl)-8-oxooctanoyl)-N 6 -((1S,4R)-4-hydroxycyclohexane-1-carbonyl)-L-lysine (447.2 mg, 0.55 mmol) was reacted at 45 °C overnight.

[0214] After the reaction was complete, the residue was concentrated under reduced pressure and purified by silica gel column chromatography (eluent: dichloromethane / methanol = 10 / 1, containing 0.5% triethylamine) to obtain 600.0 mg of Intermediate C11 as a white foamy solid (yield: 42.1%). LCMS: RT = 1.938 min, [M-DMTr+H+H] 2+ / 2=1144.4.

[0215] Step B: Synthesis of Compound 11

[0216] To a three-necked flask containing 4A molecular sieves and N,N-diisopropylammonium tetrazolium salt (198.3 mg, 1.12 mmol) was added intermediate C11 (600.0 mg, 0.23 mmol) in dichloromethane (10.0 mL) at 0°C. Bis(diisopropylamino)(2-cyanoethoxy)phosphine (291.8 μL, 0.92 mmol) was then added and the reaction was carried out at room temperature for 1 hour.

[0217] After the reaction was complete, the reaction was quenched with an ice-cold saturated aqueous sodium bicarbonate solution, diluted with dichloromethane, washed once with a saturated aqueous sodium bicarbonate solution and once with saturated brine, then dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: dichloromethane / methanol = 20 / 1, containing 0.5% triethylamine) to obtain 250.0 mg of compound 11 (yield: 42.0%). LCMS: RT = 2.132 min, [M-cyanoethyl]- = 1367.4. 31 P NMR (162 MHz, DMSO-d6) δ 144.94.

[0218] Example 12 Synthesis of Compound A12

[0219]

[0220] The specific synthetic route is as follows:

[0221] Step A: Synthesis of intermediate C12

[0222] To a solution of intermediate SM1 (1.3 g, 0.72 mmol) in dichloromethane (15.0 mL) at room temperature were added N,N-diisopropylethylamine (478.0 μL, 2.75 mmol), benzotriazole-N,N,N' 2 , N'-tetramethyluronium hexafluorophosphate (312.7 mg, 0.825 mmol) and N 2 -(6-((S)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)pyrrolidin-1-yl)-6-oxohexanoyl)-N 6 -((1S,4R)-4-hydroxycyclohexane-1-carbonyl)-L-lysine (432.3 mg, 0.55 mmol) was reacted at 45°C overnight.

[0223] After the reaction was complete, the residue was concentrated under reduced pressure and purified by silica gel column chromatography (eluent: dichloromethane / methanol = 10 / 1, containing 0.5% triethylamine) to obtain 650.0 mg of Intermediate C12 as a white foamy solid (yield: 46.2%). LCMS: RT = 1.893 min, [M-DMTr+H+H] 2+ / 2=1130.3.

[0224] Step B: Synthesis of compound 12

[0225] To a three-necked flask containing 4A molecular sieves and N,N-diisopropylammonium tetrazolium salt (214.1 mg, 1.25 mmol) was added intermediate 50-2 (650.0 mg, 0.25 mmol) in dichloromethane (10.0 mL) at 0°C, followed by the addition of bis(diisopropylamino)(2-cyanoethoxy)phosphine (317.2 μL, 1.0 mmol) and the reaction was carried out at room temperature for 1 hour.

[0226] After the reaction was complete, the reaction was quenched with an ice-cold saturated aqueous sodium bicarbonate solution, diluted with dichloromethane, washed once with a saturated aqueous sodium bicarbonate solution and once with saturated brine, then dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: dichloromethane / methanol = 20 / 1, containing 0.5% triethylamine) to obtain 180.0 mg of compound 12 (yield: 26.1%). LCMS: RT = 2.132 min, [M-cyanoethyl]- = 1353.2. 31 P NMR (162 MHz, DMSO-d6) δ 144.80.

[0227] Example 13 Synthesis of Compound 13

[0228]

[0229] The specific synthetic route is as follows:

[0230] Step A: Synthesis of 0013-2

[0231] At room temperature, 0013-1 (4.3 g, 9.46 mmol) was dissolved in methanol (43 ml), stirred in an ice bath and cooled, and thionyl chloride (5.6 g, 47.3 mmol) was added dropwise. After the addition, the mixture was stirred at room temperature for 16 h.

[0232] After the reaction was completed, the mixture was concentrated under reduced pressure to obtain 3.8 g of off-white solid 0013-2 (yield: 99.2%).

[0233] Step B: Synthesis of 0013-3

[0234] At room temperature, 0013-2 (3.8 g, 9.39 mmol) was dissolved in dichloromethane (75 ml), and cis-4-hydroxycyclohexanecarboxylic acid (1.5 g, 10.33 mmol) was added with stirring. The mixture was cooled in an ice bath, and DIPEA (1.8 g, 14.09 mmol), EDCI (2.3 g, 12.21 mmol), and HOPO (0.2 g, 1.88 mmol) were added. After the addition, the mixture was reacted at room temperature for 16 hours.

[0235] After the reaction, the mixture was washed with water (100 ml), the organic phase was dried over sodium sulfate, filtered, and concentrated to obtain a crude product, which was purified by silica gel column chromatography (eluent: dichloromethane / methanol = 10 / 1) to obtain 4.6 g of an off-white foamy solid 0013-3 (yield: 99.1%).

[0236] Step C: Synthesis of 0013-4

[0237] At room temperature, 0013-3 (4.6 g, 6.3 mmol) was dissolved in tetrahydrofuran (80 ml), and diethylamine (3.4 g, 46.5 mmol) was added with stirring, and the mixture was reacted at room temperature for 16 hours.

[0238] After the reaction was completed, the residue was concentrated to dryness to obtain 4.8 g of light yellow solid 0013-4 (yield: 100%).

[0239] Step D: Synthesis of 0013-6

[0240] At room temperature, 0013-4 (1.24 g, 4.55 mmol) was dissolved in dichloromethane (40 ml), and DIPEA (1.5 g, 11.38 mmol) and 0015-5 (2.6 g, 4.55 mmol) were added with stirring. The mixture was cooled in an ice bath, and HOBT (0.68 g, 5.0 mmol) and EDCI (0.96 g, 5.0 mmol) were added. The mixture was reacted at room temperature for 16 hours.

[0241] After the reaction, the mixture was washed with water (60 ml * 2), the organic phase was dried over sodium sulfate, filtered, and concentrated to obtain a crude product, which was purified by silica gel column chromatography (eluent: dichloromethane / methanol = 10 / 1) to obtain 3.1 g of an off-white foamy solid 0013-6 (yield: 83.6%).

[0242] Step E: Synthesis of 0013-7

[0243] At room temperature, 0013-6 (3.1 g, 3.81 mmol) was dissolved in tetrahydrofuran (24 ml), and water (6 ml) and lithium hydroxide (0.1 g, 4.19 mmol) were added with stirring, and the mixture was reacted at room temperature for 2 hours.

[0244] After the reaction was completed, water (20 ml) was added, tetrahydrofuran was concentrated, and lyophilization was carried out to obtain 2.75 g of off-white solid 0013-7 (yield: 89.6%).

[0245] Step F: Synthesis of Intermediate C13

[0246] At room temperature, SM1 (2.0 g, 1.0 mmol) was dissolved in dichloromethane (40 ml), and 0013-7 (0.81 g, 1.0 mmol), DIPEA (0.657 g, 5.0 mmol), HBTU (0.57 g, 1.5 mmol) were added with stirring.

[0247] The reaction was carried out at room temperature for 16 hours.

[0248] After the reaction, the crude product was concentrated and purified by silica gel column chromatography (eluent: dichloromethane / methanol = 7 / 1) to obtain 1.27 g of off-white solid C13 (yield: 49.2%).

[0249] Step G: Synthesis of compound 13

[0250] At room temperature, C13 (400 mg, 0.16 mmol) and diisopropyltetraisopropylammonium salt (30 mg, 0.18 mmol) were dissolved in dichloromethane (8 ml), an appropriate amount of activated molecular sieves were added, the atmosphere was replaced with nitrogen, and bis(diisopropylamino)(2-cyanoethoxy)phosphine (193 mg, 0.64 mmol) was added with stirring, and the mixture was stirred at room temperature for 1 hour.

[0251] After the reaction, the reaction mixture was added dropwise with stirring to a saturated sodium bicarbonate / saturated sodium chloride solution (6 ml / 6 ml) pre-cooled in an ice-water bath. The organic phase was then washed with saturated sodium bicarbonate / saturated sodium chloride solution (6 ml / 6 ml), dried over sodium sulfate, filtered, and concentrated to dryness to obtain an oil. Purification by silica gel column chromatography (basified with triethylamine) (eluent: dichloromethane / methanol = 10:1) afforded 378 mg of compound 13 (yield: 87.7%). LCMS: RT = 2.007 min, [M-DMTr-Diisopropylamine+H+H]+ / 2 = 1187.4. 31 PNMR (400MHz, DMSO-d6): ppmδ144.99.

[0252] Example 14 Synthesis of Compound 14

[0253]

[0254] The specific synthetic route is as follows:

[0255] Step A: Synthesis of intermediate C14

[0256] SM3A (0.6 g, 0.39 mmol) and B2 (0.30 g, 0.39 mmol) were dissolved in dry anhydrous dichloromethane (20 ml), and then DIEA (0.22 g, 1.56 mmol) was added, and finally HBTU (0.3 g, 0.78 mmol) was added and reacted at 45 °C for 12 hours.

[0257] After completion of the reaction, the mixture was quenched with aqueous sodium bicarbonate solution and extracted with dichloromethane (20 ml x 2). After drying, the mixture was concentrated to obtain an oil, which was purified by silica gel column chromatography (eluent: dichloromethane / methanol = 15 / 1) to obtain 236 mg of C14 (yield: 26%). LCMS: RT = 1.91 min, [M-DMTr+H] + =1979.47.

[0258] Step B: Synthesis of compound 14

[0259] At room temperature, C14 (500 mg, 0.22 mmol) and diisopropyltetrazolyl ammonium salt (188 mg, 1.10 mmol) were dissolved in dichloromethane (15 ml), an appropriate amount of activated molecular sieves were added, the atmosphere was replaced with nitrogen, and bis(diisopropylamino)(2-cyanoethoxy)phosphine (133 mg, 0.44 mmol) was added with stirring, and the mixture was stirred at room temperature for 2 hours.

[0260] After the reaction, the reaction mixture was added dropwise with stirring to a saturated sodium bicarbonate / saturated brine solution (6 ml / 6 ml) pre-cooled in an ice-water bath. The organic phase was then washed with saturated sodium bicarbonate / saturated brine (10 ml / 10 ml), dried over sodium sulfate, filtered, and concentrated to dryness to obtain an oil. This was purified by silica gel column chromatography (basified with triethylamine) (eluent: dichloromethane / methanol = 20:1) to obtain 351 mg of compound 14 (yield: 64%).

[0261] LCMS:RT=1.98min,(MW-Propiononitrile-HH) 2 - / 2=1212. 1 H NMR (400MHz, DMSO-d6) δ7.97 (d, J=7.0

[0262] Hz,4H),7.87(dd,J=20.8,7.7Hz,5H),7.42-7.27(m,4H),7.23(td,J=9.0,7.9,3.1Hz,5H),6.95-6.85(m,4H) ,5.23(d,J=3.4Hz,3H),5.00(dd,J=11.2,3.4Hz,3H),4.63-4.55(m,3H),4.19(d,J=6.9Hz,4H),4.04(d,J=4. 3Hz,10H),3.75(s,11H),3.65-3.48(m,14H),3.45-3.39(m,15H),3.20(s,8H),3.09(s,7H),2.12(s,14H),2. 01(d,J=1.3Hz,9H),1.91(s,15H),1.79(s,9H),1.72(s,5H),1.47(d,J=31.4Hz,3H),1.15(d,J=6.8Hz,12H). 31 PNMR (400MHz, DMSO-d6): ppmδ144.99,144.95.

[0263] Example 15 Synthesis of Compound 15

[0264] The specific synthetic route is as follows:

[0265]

[0266] Step A: Synthesis of Intermediate C15

[0267] SM3B (1.88 g, 1.23 mmol) was dissolved in dry anhydrous dichloromethane (30 ml), and then DIEA (0.48 g, 3.69 mmol), HATU (0.70 g, 1.84 mmol), and B1 (1.0 g, 1.23 mmol) were added in sequence, and then reacted at room temperature for 16 hours.

[0268] After completion of the reaction, the mixture was quenched with saturated aqueous sodium bicarbonate solution and extracted with dichloromethane (30 ml x 2). After drying, the mixture was concentrated to obtain an oil, which was purified by silica gel column chromatography (eluent: dichloromethane / methanol = 15 / 1) to afford 1 g of C15 (yield: 45%). LCMS: RT = 1.95 min, 0.5*[MH]- = 1159.6.

[0269] Step B: Synthesis of Compound 15

[0270] At room temperature, C15 (1400 mg, 0.37 mmol) and diisopropyltetrazolyl ammonium salt (513 mg, 1.85 mmol) were dissolved in dichloromethane (15 ml). An appropriate amount of activated molecular sieves was added and the atmosphere was replaced with nitrogen. Bis(diisopropylamino)(2-cyanoethoxy)phosphine (0.58 ml, 0.74 mmol) was added dropwise at zero degree and stirred at room temperature for 2 hours.

[0271] After the reaction, the reaction mixture was cooled to -20°C and added dropwise to a saturated sodium bicarbonate / saturated sodium chloride solution (6 ml / 6 ml) with stirring. The mixture was stirred for 2 minutes and then at room temperature for 5 minutes. The layers were separated, and the organic phase was dried over sodium sulfate, filtered, and concentrated to dryness to obtain an oil. The product was purified by silica gel column chromatography (basified with triethylamine) (eluent: dichloromethane / methanol = 20:1) to afford 1100 mg of compound 15 (yield: 82%). LCMS: RT = 4.748 min, [M-cyanoethyl-H]- = 1527.8. 31 P NMR (400MHz, DMSO-d6): ppmδ146.65 (dd, J=140Hz).

[0272] Example 16 Synthesis of Compound 16

[0273]

[0274] Step A: Synthesis of intermediate C16

[0275] SM2 (2 g, 1.34 mmol) and B7 (1.09 g, 1.34 mmol) were dissolved in tetrahydrofuran (100 ml) at room temperature, and NMI (440 mg, 5.36 mol) and TCFH (750 mg, 2.68 mmol) were added with stirring, and the mixture was stirred at 60°C for 48 hours.

[0276] After the reaction, the mixture was quenched with sodium bicarbonate and extracted with dichloromethane (50 ml x 2). The organic phases were combined, dried, and concentrated to obtain an oil, which was purified by silica gel column chromatography (eluent: dichloromethane / methanol = 10 / 1) to obtain 500 mg of C16 (yield: 16%). LCMS: RT = 2.02 min, [M-DMTr+H] + =1991.81.

[0277] Step B: Synthesis of Compound 16

[0278] At room temperature, C16 (1.2 g, 0.52 mmol) and diisopropyltetrazolyl ammonium salt (180 mg, 1.04 mmol) were dissolved in dichloromethane (10 ml), an appropriate amount of activated molecular sieves were added, the atmosphere was replaced with nitrogen, and bis(diisopropylamino)(2-cyanoethoxy)phosphine (310 mg, 1.04 mmol) was added with stirring, and the mixture was stirred at room temperature for 1 hour.

[0279] After the reaction, the reaction mixture was added dropwise with stirring to a saturated sodium bicarbonate / saturated brine solution (6 ml / 6 ml) pre-cooled in an ice-water bath. The organic phase was then washed with saturated sodium bicarbonate / saturated brine (6 ml / 6 ml), dried over sodium sulfate, filtered, and concentrated to dryness to obtain an oil. Purification by silica gel column chromatography (basified with triethylamine) (eluent: dichloromethane / methanol = 20:1) afforded 1.1 g of compound 16 (yield: 84%). LCMS: RT = 1.98 min, (MW-Propiononitrile-HH) 2 - / 2=1198. 1 HNMR (400MHz, DMSO-d6) δ8.06(d,J=5.3Hz,2H),7.84(d,J=9.3Hz,3H),7.45(d,J=7.7Hz,1H),7.33(d,J=8.0Hz,5H),6.90 (d,J=8.5Hz,3H),5.24(d,J=3.4Hz,3H),5.00(dt,J=11.2,2.9Hz,3H),4.57(dd,J=8.5,3.9Hz,3H),4.05(s,10H),3.90(q ,J=9.5Hz,3H),3.75(s,8H),3.71(dd,J=10.6,7.1Hz,3H),3.63-3.47(m,11H),3.41(d,J=7.9Hz,7H),3.26(s,7H),3.09( s,12H),2.12(d,J=2.5Hz,10H),2.01(d,J=3.1Hz,9H),1.91(s,9H),1.80(d,J=1.7Hz,10H),1.15(dd,J=6.8,2.8Hz,12H). 31 PNMR (400MHz, DMSO-d6): ppmδ144.97.

[0280] Table 1 shows the structures of the compounds of the present invention.

[0281]

[0282]

[0283]

[0284] Among them, the structures of compounds 1-16 after conjugation with siRNA sequences are shown in Table 2:

[0285] Table 2 shows the structure of A1-A16

[0286]

[0287]

[0288]

[0289] Among them, the intermediate structures used in the synthesis of compounds A1-A16 are B1, B2, B3, B4, B5, B6, B7, B8, and C1-C16 intermediate structures, respectively. The specific structures are shown in Table 3:

[0290] Table 3 shows the intermediate structures

[0291]

[0292]

[0293]

[0294] Among them, when A1-A16 is linked to nucleotides via phosphate or thiophosphate, the structure is shown in Table 4:

[0295] Table 4 shows the structures of A1-A16 connected with phosphate

[0296]

[0297]

[0298]

[0299] Example 17 Synthesis of nucleic acid molecules

[0300] Nucleic acid molecules, including a first nucleic acid molecule and a second nucleic acid molecule, were prepared using a Dr. 48 model DNA / RNA automatic synthesizer. The specific synthesis route can be found in Example 19, and the nucleic acid molecule sequences are shown in Table 5.

[0301] Table 5 is a nucleic acid sequence table

[0302]

[0303] Example 18 Synthesis of Modified Sequences of Nucleic Acid Molecules

[0304] The modified nucleic acid molecules were prepared using a Dr. 48 model DNA / RNA automatic synthesizer. The specific synthesis route can be found in Example 19. The sequences of the modified nucleic acid molecules are shown in Table 6:

[0305] Table 6 is a modified nucleic acid molecule sequence table

[0306]

[0307] Wherein, A = adenosine-3′-phosphate; U = uridine-3′-phosphate; C = cytidine-3′-phosphate; G = guanosine-3′-phosphate; Am = 2′-O-methyladenosine-3′-phosphate; Um = 2′-O-methyluridine-3′-phosphate; Cm = 2′-O-methylcytidine-3′-phosphate; Gm = 2′-O-methylguanosine-3′-phosphate; Gs = guanosine-3′-phosphorothioate; Ams = 2′-O-methyladenosine-3′-phosphorothioate; Ums = 2′-O-methyluridine-3′-phosphorothioate; Cms = 2′-O-methylcytidine-3′-phosphorothioate; Gms = 2′-O-methylguanosine-3′-phosphorothioate; Af = 2′-fluoroadenosine-3′-phosphate; Uf = 2′-fluorouridine-3′-phosphate; Cf = 2′-fluorocytidine-3′-phosphate; Gf = 2′-fluoroguanosine-3′-phosphate; Afs = 2′-fluoroadenosine-3′-phosphorothioate; Ufs = 2′-fluorouridine-3′-phosphorothioate; Cfs = 2′-fluorocytidine-3′-phosphorothioate; Gfs = 2′-fluoroguanosine-3′-phosphorothioate; dT = 2′-deoxythymidine-3′-phosphate; m = 2′-O-methyl; f = 2′-fluoro; s = phosphorothioate linkage.

[0308] Example 19 Preparation of double-stranded RNA conjugate

[0309] This preparation example synthesized a double-stranded RNA conjugate. The conjugated RNA in the double-stranded RNA conjugate had the sense and antisense strand sequences corresponding to Table 7.

[0310] Synthesize the sense strand of double-stranded RNA conjugates

[0311] By solid phase phosphoramidite method, nucleoside monomers are connected one by one from 3'-5' direction according to the arrangement order of the corresponding sense chain nucleotides in Table 7. Each connection of a nucleoside monomer includes four steps of deprotection, coupling, capping, oxidation or sulfurization. Among them, when phosphate is used to connect two nucleotides, the four steps of deprotection, coupling, capping and oxidation are included when connecting the next nucleoside monomer. When thiophosphate is used to connect two nucleotides, the four steps of deprotection, coupling, capping and sulfurization are included when connecting the next nucleoside monomer. The synthesis conditions are given as follows:

[0312] Commercial nucleoside monomers and compounds (A1-A16) prepared by the above steps were dissolved in ultra-dry anhydrous acetonitrile to prepare 0.05-0.1 M solutions, and 4A molecular sieves were added. The deprotection reaction conditions in each step were the same, namely, a temperature of 25°C, a reaction time of 70 seconds, and a deprotection reagent of trichloroacetic acid in dichloromethane (3% v / v). The molar ratio of trichloroacetic acid to the 4,4′-dimethoxytrityl protecting group on the solid support was 150:1.

[0313] The coupling reaction conditions were the same for each step, including a temperature of 25°C, a molar ratio of the nucleic acid sequence attached to the solid support to the nucleoside monomer of 1:20, a molar ratio of the nucleic acid sequence attached to the solid support to the coupling reagent of 1:120, a reaction time of 300 seconds, and a coupling reagent of 0.3 M acetonitrile solution of 5-benzylthio-1H-tetrazole.

[0314] The capping conditions were identical for each step, including a temperature of 25°C and a reaction time of 30 seconds. The capping reagent solution consisted of a 1:1 molar mixture of CapA and CapB. The molar ratio of capping reagent to the nucleic acid sequence attached to the solid support was 100:100:1 (acetic anhydride:N-methylimidazole:nucleic acid sequence attached to the solid support).

[0315] The oxidation reaction conditions for each step were identical, including a temperature of 25°C, a reaction time of 30 seconds, and 0.05 M iodine-water as the oxidizing agent. The molar ratio of iodine to the nucleic acid sequence attached to the solid support during the coupling step was 30:1. The reactions were performed in a solvent mixture of tetrahydrofuran:water:pyridine (7:2:1).

[0316] The sulfurization reaction conditions for each step were identical, including a temperature of 25°C, a reaction time of 300 seconds, and a sulfurization reagent of 0.2 M hydrogenated xanthan gum in pyridine. The molar ratio of the sulfurization reagent to the nucleic acid sequence attached to the solid support during the coupling step was 120:1.

[0317] After the last nucleoside monomer is attached, 250 μL of a 20% diethylamine / acetonitrile solution is added to each column and allowed to drain over 5 minutes, followed by complete flow. This wash is repeated two more times, for a total of 7500 μL and 15 minutes. The oligonucleotide / solid support is then washed with 3 x 300 μL of acetonitrile and dried by vacuum evacuation. The nucleic acid sample is purified using a preparative C18 column using reversed-phase ion-pair chromatography. Specifically, eluent A: 100 mM TEAA in water, eluent B: 100 mM TEAA in acetonitrile, with an elution gradient of 90:10 to 80:20 eluent A:Eluent B. The product eluate is collected and tested for purity. Nucleic acid samples meeting the required purity are pooled. Salt exchange is then performed using precipitation. After the sample is concentrated to dryness, sodium acetate and ethanol are added, and the precipitate is collected and washed with ethanol.

[0318] Synthesis of the antisense strand of double-stranded RNA conjugates

[0319] The antisense strand was synthesized using the solid-phase phosphoramidite method, using a universal solid support as the initiation cycle, according to the antisense strand nucleotide arrangement in Table 7. The deprotection, coupling, capping, oxidation or sulfurization reaction conditions, cleavage and deprotection, purification, and desalting conditions used in the solid-phase synthesis method were the same as those used for the sense strand.

[0320] 3. Deprotection

[0321] The synthesized nucleotide sequence connected to the vector was added to 25 wt% ammonia water (1 ml / μmol), reacted at 35° C. for 16 hours, filtered to remove the remaining vector, and the supernatant was vacuum concentrated to dryness.

[0322] 4. Purification and desalting

[0323] Nucleic acids were purified using a preparative ion chromatography column (Source 15Q) by gradient elution with NaBr.

[0324] 5. Annealing and freeze-drying

[0325] Heat equimolar amounts of the sense and antisense strands at 95°C for 2 minutes, then slowly cool to room temperature. Aliquot according to the pharmacology department's requirements, lyophilize to a solid powder, and store at -20°C until ready for use.

[0326] The sequences of the sense and antisense strands of the double-stranded RNA conjugates are shown in Tables 7, 8 and 9.

[0327] Table 7 shows the RNA sequences of PCSK9 and ANGPTL3

[0328]

[0329]

[0330] Table 8 shows the RNA sequences of TTR and ANGPTL3

[0331]

[0332]

[0333] Table 9 shows the RNA sequences of TTR and FVII

[0334]

[0335]

[0336] Wherein, A = adenosine-3′-phosphate; U = uridine-3′-phosphate; C = cytidine-3′-phosphate; G = guanosine-3′-phosphate; Am = 2′-O-methyladenosine-3′-phosphate; Um = 2′-O-methyluridine-3′-phosphate; Cm = 2′-O-methylcytidine-3′-phosphate; Gm = 2′-O-methylguanosine-3′-phosphate; Gs = guanosine-3′-phosphorothioate; Ams = 2′-O-methyladenosine-3′-phosphorothioate; Ums = 2′-O-methyluridine-3′-phosphorothioate; Cms = 2′-O-methylcytidine-3′-phosphorothioate; Gms = 2′-O-methylguanosine-3′-phosphorothioate; Af = 2′-fluoroadenosine-3′-phosphate; Uf = 2′-fluorouridine-3′ -phosphate; Cf = 2′-fluorocytidine-3′-phosphate; Gf = 2′-fluoroguanosine-3′-phosphate; Afs = 2′-fluoroadenosine-3′-phosphorothioate; Ufs = 2′-fluorouridine-3′-phosphorothioate; Cfs = 2′-fluorocytidine-3′-phosphorothioate; Gfs = 2′-fluoroguanosine-3′-phosphorothioate; dT = 2′-deoxythymidine-3′-phosphate; m = 2′-O methyl; f = 2′-fluoro; s = phosphorothioate, invAb = inverted abasic deoxyribose residue, wherein the structures of D1-D16 are shown in Table 4, D1-D16 are linked to RNA via phosphate (p) or phosphorothioate (s), wherein the phosphate-bearing end of the D1-D16 structure is linked to the 5′ end of the RNA, and the other end is linked to the 3′ end of the RNA.

[0337] Example 20 In vivo efficacy of double-stranded RNA conjugates

[0338] To evaluate the in vivo efficacy of the double-stranded RNA conjugates in Table 5, 6-8 week old PCSK9 humanized mice (purchased from Jicui Yaokang) were fed a high-fat diet for at least 2 weeks in advance and grouped according to the baseline low-density lipoprotein cholesterol levels of the mice, with 4 mice in each group. The group design was as follows:

[0339] A, vehicle group (3 mg / kg);

[0340] B, ANGPTL3 single-target siRNA compound (3 mg / kg) test group;

[0341] C, PCSK9 single-target (Inclisiran) siRNA compound (3 mg / kg) test group;

[0342] D. Test group of the double-stranded RNA conjugate in Table 7 (6 mg / kg).

[0343] On day -1 (the day before dosing), after a 4-hour fast, pre-dose plasma samples were obtained from mice to measure baseline target protein and lipid levels. Each group of siRNA compounds was diluted with saline and then subcutaneously injected on day 1 according to the experimental group design. The doses were 1 mg / kg, 2 mg / kg, 3 mg / kg, or 6 mg / kg, at concentrations of 0.1 mg / mL, 0.2 mg / mL, 0.3 mg / mL, or 0.6 mg / mL, in a volume of 10 mL / kg. Plasma samples were obtained after a 4-hour fast on days 7, 14, 21, 28, 35, and 42. Plasma ANGPTL3 and PCSK9 protein levels were measured by ELISA according to the protocol provided by the supplier (R&D Systems), and the inhibition rate was calculated according to the following formula. The results are shown in Table 10.

[0344] Target protein inhibition rate relative to the vehicle group (%) = (1-target protein expression level of a specific mouse / average target protein expression level of mice in the vehicle group) * 100

[0345] Inhibition rate of a specific mouse target protein relative to the baseline (%) = (1-expression level of a specific mouse target protein / baseline expression level of the corresponding target protein of a specific mouse) * 100

[0346] Table 10 shows the in vivo test results of RNA conjugates administered at 6 mg / kg

[0347]

[0348] Table 11 shows the in vivo test results of RNA conjugates administered at 2 mg / kg

[0349]

[0350] Among them, the inhibition rate B≥50%.

[0351] As shown in Tables 10 and 11, the double-stranded RNA conjugates of the present invention have good inhibitory activity against both ANGPTL3 and PCSK9.

[0352] The levels of four blood lipids in mouse plasma, namely total triglycerides (TG), total cholesterol (TC), high-density lipoprotein (HDL-C) and low-density lipoprotein cholesterol (LDL-C), were detected using a biochemical analyzer (Mindray) or a kit provided by the supplier (Nanjing Jiancheng). The test results showed that the double-stranded RNA conjugate of the present invention has a good inhibitory activity on the four blood lipids in mice.

[0353] Example 21 Drug Efficacy in Mice

[0354] In order to evaluate the in vivo efficacy of the RNA conjugates in Table 8, 6-8 week old male C57BL / 6 mice (purchased from Guangdong Weitong Lihua) were used for dosing and index detection. On the day before dosing, i.e., day -1, mouse plasma samples were obtained before dosing, and the baseline expression levels of mouse TTR (mTTR) and mouse Angptl3 (mAngptl3) proteins were detected using the kits provided by the suppliers (ALPCO and R&D). The mice were randomly divided into groups according to the baseline level of mAngptl3 protein, with 4-5 mice per group. The siRNA compound of each group was diluted with normal saline and then subcutaneously injected on day 0 according to the experimental group design. The dosage was 2 mg / kg, and the solvent group was directly injected with an equal amount of normal saline. Mouse plasma was collected on days 7, 14, 21, 28, and 35, and the expression levels of mTTR and mAngptl3 proteins were detected. The inhibition rate at a specific time point was then calculated according to the following formula:

[0355] mTTR inhibition rate (%) of a specific mouse relative to the baseline = (1-mTTR expression level of a specific mouse / mTTR baseline expression level of the mouse)*100

[0356] The mAngptl3 inhibition rate (%) of a specific mouse relative to the vehicle group = (1-mAngptl3 expression level of a specific mouse / average of the mAngptl3 baseline expression level of the vehicle group) * 100. The inhibition level of each compound on the target protein was taken as the average of the target protein inhibition rates of each group. The results are shown in Table 12.

[0357] Table 12

[0358]

[0359] As shown in Table 12, the RNA conjugates of the present invention have good inhibitory activity against both mTTR and mAngptl3.

[0360] Example 22 Drug efficacy in mice

[0361] In order to evaluate the in vivo efficacy of the RNA conjugates in Table 9, 6-8 week male C57BL / 6 mice (purchased from Guangdong Weitong Lihua) were used to carry out drug administration and index detection. Mice were randomly divided into 12 groups, 4 in each group. On the day of administration (day 0), the compound was dissolved in sterile saline in advance, and then injected subcutaneously, RNA conjugate solution or solvent (sterile saline) was applied to mice at a dosage of 3mg / kg or 6mg / kg. 2 weeks after administration (day 14), human F7 and Ttr gene expression in mouse liver was detected by RT-PCR. The expression level of the gene was evaluated by the relative expression level of F7 or Ttr mRNA of Gapdh mRNA of normalized corresponding mice, and the F7 or Ttr gene expression inhibition rate (relative to vehicle group) of specific mice was calculated by the following formula:

[0362] Inhibition rate of specific mouse F7 gene expression (%) = (1-relative expression level of F7 mRNA in the mouse / average relative expression level of F7 mRNA in the vehicle group) × 100

[0363] Inhibition rate of Ttr gene expression in a specific mouse (%) = (1-relative expression level of Ttr mRNA in the mouse / average relative expression level of Ttr mRNA in the vehicle group) × 100

[0364] Then the average inhibition rate of F7 and Ttr genes in each group of mice was calculated, and the results are shown in Table 13.

[0365] Table 13

[0366]

[0367] Among them, A≥60%.

[0368] As shown in Table 13, the RNA conjugates of the present invention have good inhibitory activity against both TTR and FVII.

[0369] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. A double-stranded RNA conjugate, characterized in that include: a first nucleic acid molecule, a second nucleic acid molecule, and a linker, wherein the first nucleic acid molecule is connected to the second nucleic acid molecule via the linker, the linker connects the first nucleic acid molecule and the second nucleic acid molecule via phosphate (P) or phosphorothioate (S), and the linker is selected from the structure represented by formula (I): Said T is selected from -NH-C(O)-, or -C(O)-NH-; The R 1 Selected from Among them, y1, y2 and y3 are R 1 Connection location; The R 2 Selected from Among them, y4 is R 2 Connection location; Wherein, x, p, q, and r are independently selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10, and m and n are selected from positive integers of 1-10.

2. The double-stranded RNA conjugate according to claim 1, characterized in that The linker is selected from:

3. The double-stranded RNA conjugate according to claim 1 or 2, characterized in that The carboxylic acid of the linker is linked to the ligand of the asialoglycoprotein receptor ASGPR through an amide bond.

4. The double-stranded RNA conjugate according to claim 3, characterized in that The ligand is selected from:

5. The double-stranded RNA conjugate according to claim 3, characterized in that The compound in which the carboxylic acid of the linker is connected to the ligand of the asialoglycoprotein receptor ASGPR via an amide bond is selected from the structures shown in Table 2.

6. The double-stranded RNA conjugate according to any one of claims 1 to 5, characterized in that The first nucleic acid molecule is used to inhibit the expression of a first target gene, and the second nucleic acid molecule is used to inhibit the expression of the second target gene. The first target gene and the second target gene are the same or different.

7. The double-stranded RNA conjugate according to any one of claims 1 to 5, characterized in that The first nucleic acid molecule includes a sense strand and an antisense strand, the second nucleic acid molecule includes a sense strand and an antisense strand, the sense strand of the first nucleic acid molecule is connected to the sense strand of the second nucleic acid molecule through the connector, or the antisense strand of the first nucleic acid molecule is connected to the antisense strand of the second nucleic acid molecule through the connector.

8. The double-stranded RNA conjugate according to any one of claims 1 to 5, characterized in that At least one nucleotide in the first nucleic acid molecule and the second nucleic acid molecule is modified, and the modification is selected from the group consisting of: alkyl nucleotides, methoxy nucleotides, ethoxy nucleotides, methoxyethyl nucleotides, amino nucleotides, fluoro nucleotides, deoxy nucleotides, 5′-methyl phosphate nucleotides, 5′-C-methyl phosphate nucleotides, 2′-deoxy-2′-fluoro nucleotides, vinylphosphonate deoxyribonucleotides (VP), 5′-phosphate nucleotides (P), phosphorothioate nucleotides, phosphorodithioate nucleotides, locked nucleic acids (LNA), morpholino oligonucleotides (PMO), glycol nucleic acid modifications (GNA), hypoxanthine modifications (I), inverted abasic deoxyribose residues (invAb), and 5′-methyldeoxycytosine nucleotides (m5dC).

9. A double-stranded RNA conjugate for inhibiting gene expression, characterized in that include: connector, a first nucleic acid molecule for inhibiting the expression of a first target gene, and a second nucleic acid molecule, wherein the second nucleic acid molecule is used to inhibit the expression of a second target gene, wherein the first nucleic acid molecule and the second nucleic acid molecule are connected by the linker, wherein the linker is connected by phosphate (p) or phosphorothioate (s), and the linker is as described in any one of claims 1 to 8; Wherein, the first target gene and the second target gene are independently selected from PCSK9, ANGPTL3, LPA, apo3, AGT, B factor, C3 factor, MASP2 factor, PNP factor, HSD17B13, NR1H3, SOD1, HCV, HBV, TTR, and FVII.

10. A compound, characterized in that Selected from the structures shown in Table 1 and Table 3.

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