Double-stranded RNA conjugate, preparation method therefor, and use thereof

Abstract

The present invention relates to a double-stranded RNA conjugate, a preparation method therefor, and the use 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 to the second nucleic acid molecule by means of the linker; the linker is connected to the first nucleic acid molecule and the second nucleic acid molecule by means of a phosphate ester (p) or a thiophosphate ester (s), and the linker is selected from a structure represented by formula (I).

Description

Double-stranded RNA conjugates, their preparation methods and applications Technical Field

[0001] This invention relates to the field of medicine, and more specifically, to a double-stranded RNA conjugate, its preparation method, and its uses. Background Technology

[0002] Currently, there is a need in this field for molecules that can target multiple targets. However, how to link multiple target molecules together and how to disconnect them once they are in the body are problems that need to be solved.

[0003] Although patent publication numbers WO2017015109A1 and WO2018136620 disclose some in vivo cleavable linkers, various problems still exist after multi-target molecules enter the body. Furthermore, there are currently no products on the market that can act on multiple targets simultaneously. Therefore, it is urgent to study linkers suitable for in vivo cleavage and multi-target molecules. Summary of the Invention

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

[0005] In a first aspect, the present invention provides a double-stranded RNA conjugate, comprising: 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, and the linker connects the first nucleic acid molecule and the second nucleic acid molecule via a phosphate ester (p) or a thiophosphate ester (s), wherein the linker is selected from the structure shown in formula (I):

[0006] The T is selected from -NH-C(O)- or -C(O)-NH-;

[0007] The R 1 Selected from Where y1, y2, and y3 are R 1 Connection location, the R 3 R 4 Together, cyclization into substitution The substitution is selected from a and b are the connection positions with the first or second nucleic acid molecule;

[0008] The R 2 Selected from Wherein, the R 5 Selected from the replaced C 3-8 Metacyclic alkyl groups and heterocyclic alkyl groups, wherein the substitution is selected from...

[0009] y4 is R 2 connecting position, c, d, e are the connecting position with the first nucleic acid molecule or the second nucleic acid molecule;

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

[0011] In a second aspect, the present application further provides a double-stranded RNA conjugate for inhibiting gene expression, comprising: a linker; a first nucleic acid molecule for inhibiting the expression of a first target gene; and a second nucleic acid molecule for inhibiting the expression of a second target gene;

[0012] wherein, 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 thiophosphate (s), and the linker is as described above.

[0013] wherein, the first target gene and the second target gene are independently selected from PCSK9, ANGPTL3, LPA, apo3, AGT, Factor B, Factor C3, MASP2 factor, PNP factor, HSD17B13, NR1H3, SOD1, HCV, HBV, TTR, FVII.

[0014] In a third aspect, the present application provides a double-stranded RNA conjugate for inhibiting the expression of ANGPTL3 and PCSK9, comprising:

[0015] a first nucleic acid molecule for inhibiting the expression of PCSK9; and a second nucleic acid molecule for inhibiting the expression of ANGPTL3; wherein, 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 thiophosphate (s), and the linker is selected from the structure shown in formula (I):

[0016] wherein, x, T, R 1 , R 2 are as described above.

[0017] As a preferred technical solution of the present application, m, n are independently selected from 2, 3, 4, and p is independently selected from 3, 4, 5, 6, 7.

[0018] As a preferred technical solution of the present application, R 3 , R 4 are cyclized together to substituted The substitution position can be located at the ortho position, the meta position, or the para position, and preferably the substitution position is the ortho position.

[0019] As a preferred technical solution of the present application, the C 3-8 The heterocycloalkyl refers to that the carbon atoms of the cycloalkyl are substituted by heteroatoms, and the heteroatoms are selected from nitrogen, oxygen or sulfur.

[0020] As a preferred technical solution of the present application, the R 5 Further selected from substituted The substitution position can be located at the ortho position, the meta position, or the para position, and preferably the substitution position is the para position.

[0021] As a preferred technical solution of the present application, the linker is selected from:

[0022] The linker includes 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.

[0023] 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.

[0024] As a preferred technical solution of the present application, the ligand is selected from:

[0025] 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 1 and Table 3.

[0026] As a preferred technical solution of the present application, the first nucleic acid molecule is used to inhibit the expression of the first target gene, and the second nucleic acid molecule is used to inhibit the expression of the second target gene, and the first target gene and the second target gene are the same or different.

[0027] 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.

[0028] 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, the sense strand of the first nucleic acid molecule is connected with the sense strand of the second nucleic acid molecule through the linker, or the antisense strand of the first nucleic acid molecule is connected with the antisense strand of the second nucleic acid molecule through the linker.

[0029] As a preferred technical solution of the present application, 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 nucleotide, methoxy nucleotide, ethoxy nucleotide, methoxy ethyl nucleotide, amino nucleotide, fluorinated nucleotide, deoxy nucleotide, 5'-methyl phosphate nucleotide, 5'-C-methyl phosphate nucleotide, 2'-deoxy-2'-fluoro nucleotide, vinyl phosphonate nucleotide (VP), 5'-phosphate nucleotide (P), phosphorothioate nucleotide, phosphorodithioate nucleotide, locked nucleic acid (LNA), morpholino oligonucleotide (PMO), glycol nucleic acid modification (GNA), hypoxanthine modification (I), inverted abasic deoxyribose residue (invAb), 5'-methyl deoxycytidine nucleotide (m5dC).

[0030] As a preferred technical solution of the present application, the first nucleic acid molecule is selected from the sequences shown in Table 4.

[0031] Preferably, 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, or

[0032] the sense strand comprises the nucleotide sequence of SEQ ID NO: 76 and the antisense strand comprises the nucleotide sequence of SEQ ID NO: 77.

[0033] As a preferred technical solution of the present application, the second nucleic acid molecule is selected from the sequences shown in Table 4.

[0034] Preferably, 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, or

[0035] 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; or

[0036] the sense strand comprises the nucleotide sequence of SEQ ID NO: 86 and the antisense strand comprises the nucleotide sequence of SEQ ID NO: 87.

[0037] As a preferred technical solution of the present application, the sense strand of the first nucleic acid molecule and / or the second nucleic acid molecule is connected with a targeting ligand. For example, the nucleotide at the 5' end or 3' end of the sense strand of the first nucleic acid molecule is connected with a targeting ligand, or the nucleotide at the 5' end or 3' end of the sense strand of the second nucleic acid molecule is connected with a targeting ligand.

[0038] As a preferred technical solution of the present application, 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, fluorinated nucleotides, deoxy nucleotides, 5'-methylphosphate nucleotides, 5'-C-methylphosphonate nucleotides, 2'-deoxy-2'-fluoro nucleotides, vinyl phosphonate nucleotides (VP), 5'-phosphate nucleotides (P), phosphorothioate nucleotides, phosphorodithioate nucleotides, locked nucleic acid (LNA), morpholino oligonucleotide (PMO), glycol nucleic acid modification (GNA), hypoxanthine modification (I), inverted abasic deoxyribose residue (invAb), 5'-methyldeoxycytosine nucleotide (m5dC).

[0039] As a preferred technical solution of the present application, the siRNA conjugate is selected from the nucleotide sequences described in Table 5.

[0040] The present application further provides a compound selected from the structures shown in Table 2 and Compounds 1-22. Among them, the structures of Compounds 1-22 are shown in Examples 1-22 of the specification.

[0041] In a fourth aspect, the present application also provides a pharmaceutical composition for inhibiting the expression of ANGPTL3 and PCSK9 genes, wherein the pharmaceutical composition comprises the double-stranded RNA conjugate as described above.

[0042] As a preferred technical solution of the present application, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient.

[0043] In a fifth aspect, the present application also provides the use of the above-mentioned double-stranded RNA conjugate in the preparation of a medicament for treating a disease, disorder or symptom mediated at least in part by the expression of ANGPTL3 and / or PCSK9 genes.

[0044] The double-stranded RNA conjugate provided by the present application for inhibiting the expression of ANGPTL3 and PCSK9, TTR and ANGPTL3, TTR and FVII has good inhibitory activity on ANGPTL3 and PCSK9, TTR and ANGPTL3, TTR and FVII, respectively, and can be used for preventing and / or treating diseases related to the expression of ANGPTL3 and / or PCSK9 genes, TTR and / or ANGPTL3 genes, TTR and / or FVII genes. Detailed Implementation

[0045] The present invention will be further described in detail below with reference to embodiments, but the implementation of the invention is not limited thereto.

[0046] The term “including” as used herein refers to the phrase “including (but not limited to)” and is used interchangeably with that phrase unless the context clearly indicates otherwise.

[0047] The term “or” is used herein to mean the term “and / or” and may be used interchangeably with that term unless the context clearly indicates otherwise.

[0048] The terms “sequence” and “nucleotide sequence” used in this article refer to the order or sequence of nucleobases or nucleotides, described in alphabetical order using standard nomenclature.

[0049] In this invention, the term "PCSK9" refers to the gene or protein of the preprotein convertase subtilisin Kexin9. PCSK9 is also known as FH3, HCHOLA3, NARC-1, or NARC1. The term PCSK9 includes human PCSK9, whose amino acid and nucleotide sequences are available, for example, in GenBank accession number GI:299523249; mouse PCSK9, whose amino acid and nucleotide sequences are available, for example, in GenBank accession number GI:163644257; and rat PCSK9, whose amino acid and nucleotide sequences are available, for example, in GenBank accession number GI:77020249. Other examples of PCSK9 mRNA sequences are readily available using, for example, GenBank.

[0050] The nucleic acid molecule used to inhibit PCSK9 gene expression in this 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.

[0051] The term "ANGPTL3" refers to Angiopoietin-like protein 3 (NM_014495, 4), a secreted protein that is primarily expressed in liver cells. Studies have shown that Angiopoietin-like protein 3 (ANGPTL3) is a key regulator of LDL-C, HDL-C, and triglyceride metabolism, and is an important factor in the dysregulation of lipid metabolism. ANGPTL3 has multiple potential nodes of action, and loss-of-function mutations in ANGPTL3 can result in reduced LDL-C, VLDL-C, HDL-C, and triglycerides (TG), thereby reducing the risk of cardiovascular disease based on GWAS, and no known adverse phenotypes of genetic deficiency. Therefore, inhibiting the activity of ANGPTL3 can effectively prevent or treat dyslipidemia.

[0052] In the present application, the nucleic acid molecule for inhibiting the expression of ANGPTL3 gene can be selected from the nucleic acid sequences disclosed in the patents with patent application number or publication number: WO2021188795A1, CN201480068063.9, CN201280041047.1, CN201680034444.4, CN201880048600.1, CN202180007277.5, CN201880073901.X, WO2020099482A2, WO2022068923A1, WO2022187435A1, WO2022268054A1, CN202210705962.1, WO2023045994A1, WO2023134705A1.

[0053] The phrase "inhibiting the expression of ANGPTL3" includes inhibiting the expression of any ANGPTL3 gene (e.g., a mouse ANGPTL3 gene, a rat ANGPTL3 gene, a monkey ANGPTL3 gene, or a human ANGPTL3 gene) as well as variants or mutants of the ANGPTL3 gene that encode an ANGPTL3 protein.

[0054] The phrase "inhibiting the expression of PCSK9" includes inhibiting the expression of any PCSK9 gene (e.g., 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, group of cells, or organism.

[0055] “Inhibiting expression of ANGPTL3 and PCSK9” includes any level of inhibition of the ANGPTL3 and PCSK9 genes, such as at least partially preventing 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%. In the present invention, the nucleic acid can refer to a double-stranded siRNA, or a single-stranded ASO. In a double-stranded nucleic acid molecule, the bases of one strand pair with the bases of the other strand 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 one purine and one pyrimidine. When the adenine on one strand always pairs with the uracil on the other strand, and the guanine always pairs with the cytosine, the two strands are said to be complementary to each other, and the sequence of one strand can be inferred from the sequence of its complementary strand.

[0056] 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'-methylphosphonate nucleotides, 5'-C-methylphosphonate nucleotides, 2'-deoxy-2'-fluoro nucleotides, vinylphosphonate nucleotides (VP), phosphorothioate nucleotides, phosphorodithioate nucleotides, locked nucleic acids (LNA), morpholino oligonucleotides (PMO), inverted abasic deoxyribose residues (invAb).

[0057] wherein alkyl modified nucleotides, such as 2'-methyl nucleotides, 2'-ethyl nucleotides, 2'-methoxy modified nucleotides, with the structure: 2'-methoxyethyl nucleotides, with the structure: 2'-fluoro nucleotides, with the structure: 5'-C-methylphosphonate nucleotides, with the structure: vinylphosphonate nucleotides (VP), with the structure: phosphorothioate nucleotides (S), with the structure: phosphorodithioate nucleotides, with the structure: 2'-deoxyribose nucleotides, with the structure: An inverted abasic deoxyribose residue (invAb), having the structure: A glycol nucleic acid (GNA), including (S)-glycol nucleic acid ((S)-GNA), having the structure: and (R)-glycol nucleic acid ((R)-GNA), having the structure: A hypoxanthine nucleotide (I), having the structure: A 5-methyl deoxycytosine nucleotide (m5dC), m5dC is 2'-deoxy-5-methylcytidine-3'-phosphate.

[0058] wherein Base represents a base, R represents an alkyl or alkoxy group, and Me represents a methyl group, and Et represents an ethyl group.

[0059] 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).

[0060] 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.

[0061] Locked nucleic acid and morpholino structures are as follows, respectively:

[0062] In certain embodiments, sugar surrogates include rings having more than 5 atoms and more than 1 heteroatom. For example, nucleosides comprising morpholino sugar moieties and their use in oligomeric compounds have been reported (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).

[0063] In certain embodiments, the morpholino group can be modified, e.g., by adding or changing various substituents according to the morpholino structures above. Such sugar substitutes are referred to herein as "modified morpholino groups."

[0064] In the present disclosure, capital letters C, G, U, A represent the base composition of nucleotides, unless otherwise specified. Lowercase letter m represents that the nucleotide adjacent to the left of the letter m is a methoxy-modified nucleotide; lowercase letter f represents that the nucleotide adjacent to the left of the letter f is a fluorinated-modified nucleotide; LNA represents that the nucleotide adjacent to the right of the letter is a locked nucleic acid (LNA)-modified; lowercase letter s represents that the linkage between the two nucleotides adjacent to the left and right of the letter is a phosphorothioate linkage; VP represents that the nucleotide adjacent to the right of the letter VP is a vinylphosphonate-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 deoxyuracil nucleotide; dC represents a deoxycytosine nucleotide; and dG represents a deoxyguanine nucleotide.

[0065] It is emphasized that the "modification" of the nucleotides described in the present disclosure includes, but is not limited to, the examples described above, and the nucleotides can also be replaced by other nucleotides, such as (S)-glycerol nucleic acid, etc.

[0066] The term "targeting ligand" can include a naturally occurring substance, such as a protein (e.g., human serum albumin (HAS), low-density lipoprotein (LDL), or globulin); a carbohydrate (e.g., dextran, pullulan, chitin, chitosan, inulin, cyclodextrin, N-acetylglucosamine, N-acetylgalactosamine, or hyaluronic acid); or a lipid. The ligand can also be a recombinant or synthetic molecule, such as a synthetic polymer, e.g., 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-ethylacrylic acid), N-isopropylacrylamide polymer, or polyphosphazene. Examples of polyamines include: polyethyleneimine, polylysine (PLL), spermine, spermidine, polyamine, pseudopeptide-polyamine, peptidomimetic polyamine, dendrimer polyamine, arginine, amidine, protamine, cationic lipid, cationic porphyrin, quaternary salt of polyamine, or alpha-helical peptide.

[0067] 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.

[0068] 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.

[0069] 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 cellular microtubules, microfilaments, and / or intermediate filaments. The drug can be, e.g., taxon, vincristine, vinblastine, cytochalasin, nocodazole, iaplakinolide, halichondrin A, phalloidin, swinholide A, indanocine, or myoservin.

[0070] 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.

[0071] 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.

[0072] 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.

[0073] 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).

[0074] 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 syndrom, 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).

[0075] 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.

[0076] The term "inhibit" is used interchangeably with "reduce," "silence," "down-regulate," "arrest," and other similar terms and includes inhibition at any level.

[0077] 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).

[0078] 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 substances. 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.

[0079] Example 1 Synthesis of Compound 1

[0080] A specific synthesis route is as follows:

[0081] Step A: Synthesis of (S)-8-(2-hydroxymethyl)pyrrolidin-1-yl)-8-oxooctanoic acid methyl ester

[0082] (S)-8-(2-(Bis(4-methoxyphenyl)(phenyl)methoxy)methyl)pyrrolidin-1-yl)-8- oxooctanoic acid methyl ester

[0083] Step B: Synthesis of (S)-8-(2-(Bis(4-methoxyphenyl)(phenyl)methoxy)methyl)pyrrolidin-1-yl)-8- oxooctanoic acid methyl ester

[0084] (S)-8-(2-(Bis(4-methoxyphenyl)(phenyl)methoxy)methyl)pyrrolidin-1-yl)-8- oxooctanoic acid methyl ester

[0085] Step C: Synthesis of (S)-8-(2-(bis(4-methoxyphenyl)(phenyl)methoxy)methyl)pyrrolidin-1-yl)-8-oxooctanoic acid methyl ester

[0086] (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 at 15-25 °C, and lithium hydroxide dihydrate (209.16 g, 209.16 mmol) was added; after the system was reacted overnight at 15-25 °C, TLC showed that (S)-8-(2-(bis(4-methoxyphenyl)(phenyl)methoxy)methyl)pyrrolidin-1-yl)-8-oxooctanoic acid methyl ester completely disappeared; after the system was concentrated to dryness at T < 35 °C, column chromatography using a water / tetrahydrofuran system was performed, and the qualified fractions were concentrated to remove the solvent; after lyophilization, (S)-8-(2-(bis(4-methoxyphenyl)(phenyl)methoxy)methyl)pyrrolidin-1-yl)-8-oxooctanoic acid (73.20 g, yield: 99.48%) was obtained. (Exact Mass: 559.29, Found: (M-H)- = 558.30).

[0087] Step D: Synthesis of N2-((9H-fluoren-9-yl)methoxy)carbonyl)-N6-((1s,4R)-4- hydroxycyclohexane-1-carbonyl)-L-lysine methyl ester

[0088] ((9H-fluoren-9-yl)methoxy carbonyl)-L-lysine methyl ester hydrochloride (20.00 g, 47.74 mmol) was dissolved in 200 mL of dichloromethane at 15-25 °C, 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; after the system was cooled to 0-5 °C, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (11.89 g, 62.06 mmol) and HOPO (1.07 g, 9.55 mmol) were added, and the system was stirred for 10 minutes; the temperature of the system was restored to 15-25 °C, and the system was reacted overnight, TLC showed that ((9H-fluoren-9-yl)methoxy carbonyl)-L-lysine methyl ester hydrochloride completely disappeared, and a large amount of solid precipitated in the system; the system was filtered to obtain a white solid, the solid was washed with 200 mL of dichloromethane, and dried to obtain 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).

[0089] Step E: Synthesis of N6-((1s,4R)-4-hydroxycyclohexane-1-carbonyl)-L- lysine methyl ester

[0090] N2-((9H-fluoren-9-yl)methyloxy)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 at 15-25 °C and diethylamine (11.34 g, 157.29 mmol) was added. The reaction mixture was stirred at 15-25 °C overnight. TLC tracking showed that N2-((9H-fluoren-9-yl)methyloxy)carbonyl)-N6-((1s,4R)-4- hydroxycyclohexane-1-carbonyl)-L-lysine methyl ester was completely consumed and a large amount of solid was precipitated. The solid was filtered and washed with 32 mL of dichloromethane. After drying, 9 g of N6-((1s,4R)-4-hydroxycyclohexane-1-carbonyl)-L-lysine methyl ester was obtained (yield: 99.9 %). (Exact Mass: 286.19, Found: (M+H)+= 287.3).

[0091] 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

[0092] (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 at 15-25 °C, N,N-diisopropylethylamine (4.21 g, 32.54 mmol), 1-(3- dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (2.75 g, 14.32 mmol), 1- hydroxybenzotriazole (1.94 g, 14.32 mmol) were added, stirred for 5 minutes; N6- ((1s,4R)-4-hydroxycyclohexane-1-carbonyl)-L-lysine methyl ester (4.10 g, 14.32 mmol) was added at 0-5 °C, the temperature of the reaction system was increased to 15-25 °C; the system was reacted at 15-25 °C overnight, HPLC monitoring (S)-8-(2-(bis(4- methoxyphenyl)(phenyl)methoxy)methyl)pyrrolidin-1-yl)-8-oxooctanoic acid disappeared; the reaction system was washed with 35 mL of saturated aqueous sodium bicarbonate solution twice; the organic phase was washed with 35 mL of saturated aqueous sodium chloride solution once; the organic phase was dried over anhydrous sodium sulfate, filtered; the filtrate was concentrated at T < 35 °C without fraction, and column chromatography with dichloromethane / methanol gave 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%). (Exact Mass: 827.47, Found: (M-H)"= 826.6).

[0093] 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)

[0094] N2-(8-((S)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)pyrrolidin-l-yl)-8- oxooctanoyl)-N6-((l s,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-((l s,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-((l s,4R)-4-hydroxycyclohexane-l-carbonyl)-L-lysine was obtained by lyophilization (yield: 88.1 %). (Exact Mass: 813.46, Found: (M-H) - = 812.6).

[0095] Step H: Synthesis of intermediate Cl

[0096] SM1 (14.06 g, 7.37 mmol), N, N-diisopropylethylamine (3.32 g, 24.56 mmol) were dissolved in 100 mL of dichloromethane, and stirred under the condition of -5-5 °C. Then, benzotriazole-N, N, N', N'-tetramethyluronium hexafluorophosphate (2.94 g, 15.35 mmol), N2-(8-((S)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)pyrrolidin-l-yl)-8-oxooctanoyl)-N6-((ls,4R)-4-hydroxycyclohexane-l-carbonyl)-L-lysine (4.21 g, 32.54 mmol) were added into the reaction system under the condition of -5-5 °C. After the reaction was stirred at room temperature for 2 hours, the temperature of the reaction system was increased to 35 °C and stirred overnight. HPLC 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 was completely disappeared. The reaction system was cooled to -5-5 °C, and 50 mL of buffer solution (pH = 7.0, K2HPO4 / KH2PO4) was added into the reaction system under the condition of -5-5 °C. After stirring for 5 minutes, the organic phase was washed with 25 mL of water once and 25 mL of saturated NaCl solution twice. After the organic phase was dried with sodium sulfate, it was filtered and concentrated to obtain a crude product. The crude product was purified by reverse phase prep-HPLC. After the qualified components were concentrated under the condition of T ≤ 30 °C, 7 g of intermediate Cl (yield: 44.0%) was obtained by freeze-drying (M.W.: 2589.95, (M.W.-DMTr+H+H)2+ / 2 = 1144.28, Found: 1144.5).

[0097] Step I: synthesis of compound 1

[0098] Intermediate CI (6.00 g, 2.32 mmol) was dissolved in 60 mL of dichloromethane under nitrogen protection, and bis(diisopropylamino)(2-cyanoethoxy)phosphine (1.40 g, 4.64 mmol) was added. The reaction system was stirred and cooled to 0-10 °C. Tetrazole (0.16 g, 2.32 mmol) was added to the system while the temperature was controlled at 0-10 °C. The system was allowed to return to room temperature and stirred for 2-3 hours. HPLC showed that intermediate CI was completely consumed. The reaction system was washed with 18 mL of saturated sodium bicarbonate aqueous solution twice. The organic phase was washed with 18 mL of saturated sodium chloride aqueous solution once. The organic phase was dried over anhydrous sodium sulfate, and the filtrate was obtained by filtration. The filtrate was concentrated to no distillate while the temperature was controlled at T≤30 °C. The crude product was crystallized from 120 mL of DCM / MTBE (V / V = 1 / 4), and 12 mL of MTBE was used to rinse the filter cake. After blowing nitrogen for 14 h, compound 1 (M.W.: 2790.17, (M.W.-DMTr-Diisopropylamine+H+H)2+ / 2 = 1194.3, Found: 1194.4; (M.W.-Propiononitrile-H-H)2- / 2 = 1367.0, Found: 1367.2) was obtained.

[0099] Example 2A synthesis of compound 2A

[0100] The specific synthesis route is as follows:

[0101] Step A: synthesis of (S)-(6-(2-(hydroxymethyl)pyrrolidin-1-yl)-6-oxohexyl) benzylcarbamate

[0102] (S)-Pyrrolidin-2-ylmethyl 6-((benzyloxy)carbonyl)aminohexanoate (50.00 g, 188.45 mmol) was dissolved in 250 mL of dichloromethane and cooled to -5 to 5 °C. N,N- diisopropylethylamine (82.67 g, 637.55 mmol), l-(3-dimethylaminopropyl)-3- ethylcarbodiimide hydrochloride (43.35 g, 226.15 mmol), and 1-hydroxybenzotriazole (30.55 g, 226.15 mmol) were added at -5 to 5 °C. The reaction mixture was stirred for 20 min. at -5 to 5 °C. A clear solution of (S)-pyrrolidin-2-ylmethanol (22.9 g, 510.04 mmol) in 250 mL of dichloroethane was added dropwise at -5 to 5 °C. The temperature was raised to 15 to 25 °C after the addition was complete. The reaction mixture was stirred at 15 to 25 °C overnight. TLC indicated that the 6-((benzyloxy)carbonyl)aminohexanoate was consumed. The reaction mixture was washed twice with 125 mL of 10% citric acid solution and once with 125 mL of saturated sodium bicarbonate solution. The organic phase was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated to dryness at T < 30 °C. The residue was purified by column chromatography using dichloromethane / methanol to give 50.50 g of (S)-(6-(2-(hydroxymethyl)pyrrolidin-l-yl)-6-oxohexyl)aminocarbonyl benzyl ester (84.6% yield). (Exact Mass: 348.20, Found: (M+H) = 349.2). +

[0103] Step B: Synthesis of (S)-(6-(2-(bis(4-methoxyphenyl)(phenyl)methoxy)methyl)pyrrolidin-l-yl)-6-oxohexyl)aminocarbonyl benzyl ester

[0104] ​Benzyl (S)-(6-(2-(hydroxymethyl)pyrrolidin-1-yl)-6-oxohexyl)carbamate (47 g, 134.89 mmol), 4,4'-dimethoxytrityl chloride (54.84 g, 161.86 mmol), 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 left to react at 15-25°C overnight, TLC tracking of the complete disappearance of benzyl (S)-(6-(2-(hydroxymethyl)pyrrolidin-1-yl)-6-oxohexyl)carbamate; the reaction system 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 under suction; the system was concentrated at T < 35°C until dryness and column chromatography was performed using a n-heptane / ethyl acetate system 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: (M-H) - = 649.3).

[0105] Step C: Synthesis of (S)-6-amino-1-(2-(bis(4-methoxyphenyl)(phenyl)methoxy)methyl)pyrrolidin-1-yl)hexan-1-one

[0106] To a 1 L four-necked flask, at 15-25°C, benzyl (S)-(6-(2-(bis(4-methoxyphenyl)(phenyl)methoxy)methyl)pyrrolidin-1-yl)-6-oxohexyl)carbamate (54.84 g, 161.86 mmol) was dissolved in 500 mL of tetrahydrofuran, palladium on carbon (10.00 g) was added, N,N-diisopropylethylamine (19.86 g, 153.65 mmol); under a hydrogen atmosphere, the system was left to react at 15-25°C overnight, TLC tracking of the disappearance of benzyl (S)-(6-(2-(bis(4-methoxyphenyl)(phenyl)methoxy)methyl)pyrrolidin-1-yl)-6-oxohexyl)carbamate; the system was filtered, the filter cake was washed with 200 mL of tetrahydrofuran; the filtrate was concentrated until dryness and column chromatography was performed using a dichloromethane / methanol system 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).

[0107] Step D: Synthesis of 1-(tert-butyl) 5-methyl ((1s,4R)-4-hydroxycyclohexane-1-carbonyl)-L-glutamate

[0108] To a solution of 1-(tert-butyl) 5-methyl ((1s,4R)-4-hydroxycyclohexane-1- carbonyl)-L-glutamate (37.0 g, 108 mmol) in 148 mL of dichloromethane was added 74 mL of trifluoroacetic acid at 15-25 °C. The reaction mixture was stirred at 15-25 °C overnight. TLC indicated that 1-(tert-butyl) 5-methyl ((1s,4R)-4-hydroxycyclohexane-1- carbonyl)-L-glutamate was consumed completely. The reaction mixture was concentrated to dryness. The residue was purified by column chromatography on silica gel using dichloromethane / methanol as eluent to give (S)-2-((1s,4R)-4-hydroxycyclohexane-1- carboxamido)-5-methoxy-5-oxopentanoic acid (15 g, 48.5% yield). (Exact Mass: 287.14, Found: (M+H) = 288.2).

[0109] Step E: Synthesis of (S)-2-((1s,4R)-4-hydroxycyclohexane-1-carboxamido)-5- methoxy-5-oxopentanoic acid

[0110] To a solution of 1-(tert-butyl) 5-methyl ((1s,4R)-4-hydroxycyclohexane-1- carbonyl)-L-glutamate (37.0 g, 108 mmol) in 148 mL of dichloromethane was added 74 mL of trifluoroacetic acid at 15-25 °C. The reaction mixture was stirred at 15-25 °C overnight. TLC indicated that 1-(tert-butyl) 5-methyl ((1s,4R)-4-hydroxycyclohexane-1- carbonyl)-L-glutamate was consumed completely. The reaction mixture was concentrated to dryness. The residue was purified by column chromatography on silica gel using dichloromethane / methanol as eluent to give (S)-2-((1s,4R)-4-hydroxycyclohexane-1- carboxamido)-5-methoxy-5-oxopentanoic acid (15 g, 48.5% yield). (Exact Mass: 287.14, Found: (M+H) = 288.2). +

[0111] Step F: Synthesis of (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 methyl ester

[0112] ​(S)-6-amino-l-(2-(bis(4-methoxyphenyl)(phenyl)methoxy)methyl)pyrrolidin-l- yl)hexan-l-one (11.00 g, 38.29 mmol) was dissolved in 33 mL of dichloromethane at 15-25 °C, and the system was cooled to -5-5 °C; N,N-diisopropylethylamine (4.95 g, 38.29 mmol) was added to the system at -5-5 °C, and 1-(3-dimethylaminopropyl)-3- ethylcarbodiimide hydrochloride (8.81 g, 45.95 mmol), 1-hydroxybenzotriazole (6.225 g, 45.95 mmol), and 22 mL of dichloromethane were added to the bottle mouth while washing; (S)-2-((ls,4R)-4-hydroxycyclohexane-l-carboxamido)-5-methoxy-5- oxopentanoic acid was weighed into a 100 mL tetrafluoro bottle at 15-25 °C, N,N- diisopropylethylamine (9.89 g, 76.58 mmol) and 55 mL of dichloromethane were mixed uniformly, and the solution was dissolved; the prepared (S)-2-((ls,4R)-4- hydroxycyclohexane-l-carboxamido)-5-methoxy-5-oxopentanoic acid solution was added dropwise to the reaction system at -5-5 °C; after the dropwise addition was completed, the system was naturally warmed to 15-25 °C overnight, TLC tracking was performed to confirm that (S)-2-((ls,4R)-4-hydroxycyclohexane-l-carboxamido)-5-methoxy-5- oxopentanoic acid was completely consumed; after the system was concentrated to dryness, 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 with 33 mL of saturated sodium bicarbonate solution twice; the organic phase was dried over anhydrous sodium sulfate and filtered; the filtrate was concentrated to dryness, and column chromatography was performed using a dichloromethane / methanol system to obtain 15.15 g of (S)-5-((6-((S)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)pyrrolidin-l-yl)-6-oxohexyl)amino)-4-((ls,4R)-4-hydroxycyclohexane-l-carboxamido)-5- oxopentanoic acid methyl ester (yield 50.7%). (Exact Mass: 785.43, Found: (M+Na) = 808.6). +

[0113] Step G: Synthesis of (S)-5-((6-((S)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)pyrrolidin-l-yl)-6-oxohexyl)amino)-4-((ls,4R)-4-hydroxycyclohexane-l-carboxamido)-5- oxopentanoic acid (B2A)

[0114] ​(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 methyl ester (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 dihydrate (0.71 g, 16.79 mmol) was added; stirred; the system was warmed to 30 °C, and stirred for 3 h. HPLC detection 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 methyl ester was completely consumed. The system was concentrated to no distillate at T < 40 °C; the remaining system was directly freeze-dried to obtain 8.3 g of (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. (Yield: 76.8%).

[0115] Step H: synthesis of intermediate C2A

[0116] 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 -5-5 °C. N, N-diisopropylethylamine (4.00 g, 31.12 mmol) and benzotriazol- N, N, N', N'-tetramethyluronium hexafluorophosphate (4.40 g, 11.67 mmol) were added while maintaining the temperature at -5-5 °C. (S)-5-((6-((S)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)pyrrolidin-l-yl)-6- oxohexyl)amino)-4-((ls,4R)-4-hydroxycyclohexane-l-carboxamido)-5-oxopentanoic acid (3.32 g, 24.56 mmol) was transferred to a constant drop feeder. The prepared (S)-5-((6-((S)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)pyrrolidin-l-yl)-6-oxohexyl)amino)-4-((ls,4R)-4-hydroxycyclohexane-l-carboxamido)-5-oxopentanoic acid solution in dichloromethane was slowly added to the reaction system while maintaining the temperature at -5-5 °C. After 12 h of reaction, the system was monitored by HPLC. HPLC showed that (S)-5-((6-((S)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)pyrrolidin-l-yl)-6-oxohexyl)amino)-4-((ls,4R)-4-hydroxycyclohexane-l-carboxamido)-5-oxopentanoic acid was completely consumed. A 60 mL buffer solution (pH = 7.0, K2HPO4 / KH2PO4) was added to the reaction system while maintaining the temperature at -5-5 °C. After stirring for 5 min, the system was allowed to separate. 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 with sodium sulfate, filtered, and concentrated to obtain a crude product. The crude product was purified by reverse-phase prep-HPLC. The qualified fractions were concentrated while maintaining the temperature at T < 30 °C, and then lyophilized to obtain 12.3 g of intermediate C2A (yield: 62.1 %). (M.W.: 2547.86, (M.W. - DMTr + H + H) 2+ / 2 = 1123.2, Found: 1123.6).

[0117] Step I: Synthesis of compound 2A

[0118] Intermediate C2A (5.50 g, 2.16 mmol) was dissolved in 55 mL of dichloromethane under nitrogen protection, and bis(diisopropylamino)(2-cyanoethoxy) phosphine (1.30 g, 4.32 mmol) was added. The reaction system was stirred and cooled to 0-10 °C. Tetrazole (0.37 g, 2.16 mmol) was added to the system while the temperature was controlled at 0-10 °C. The system was warmed to 15-25 °C and stirred for 2-3 hours. HPLC showed that intermediate C2A completely disappeared. The reaction system was washed with 15 mL of saturated sodium bicarbonate aqueous solution twice. The organic phase was washed with 15 mL of saturated sodium chloride aqueous solution once. The organic phase was dried over anhydrous sodium sulfate, and the filtrate was obtained by filtration. The filtrate was concentrated to no distillate while the temperature was controlled at T≤30 °C. The crude product was crystallized from 110 mL of DCM / MTBE (V / V=1 / 4), and 11 mL of MTBE was used to rinse the filter cake. After blowing for 14 hours, compound 2A ((M.W.: 2748.09, (M.W.-DMTr-Diisopropylamine+H+H)2+ / 2=1173.6, Found: 1173.4; (M.W.-Propiononitrile-H-H)2- / 2=1346.0, Found: 1346.0 & 1346.1) was obtained.

[0119] Example 2B synthesis of compound 2B

[0120] Intermediate B2B and B2A of compound 2B are isomers of each other, and compound 2B can be prepared according to the synthetic route of Example 2A. Compound 2B is characterized by LCMS: RT=1.883 min, [M-DMTr+H-Diisopropylamine+OH+2H]+ / 2=1181.6, 31P NMR (400 MHz, DMSO-d6): ppm δ 144.95.

[0121] Example 3 synthesis of compound 3

[0122] The specific synthetic route is as follows:

[0123] 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- oxobutanoate

[0124] (S)-6-amino-l-(2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)pyrrolidin-l- yl)hexan-l-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) at room temperature, and the system was cooled to 0°C. DIPEA (1.5 g, 11.64 mmol), HATU (1.77 g, 4.66 mmol) were added with stirring, and stirring was performed at room temperature for 1 hour.

[0125] After the reaction was completed, purification was performed by silica gel column chromatography (eluent: dichloromethane / methanol = 10 / 1) to obtain 2.84 g of yellow foam solid (S)-3-(((benzyloxy)carbonyl)amino)amino)-4-(((6-((S)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)pyrrolidin-l-yl)-6-oxohexyl)amino)-4-oxobutanoic acid methyl ester (yield: 94%).

[0126] Step B: Synthesis of (S)-3-amino-4-((6-((S)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)pyrrolidin-l-yl)-6-oxohexyl)amino)-4-oxobutanoic acid methyl ester

[0127] (S)-3-(((benzyloxy)carbonyl)amino)amino)-4-(((6-((S)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)pyrrolidin-l-yl)-6-oxohexyl)amino)-4-oxobutanoic acid methyl ester (1.78 g, 2.28 mmol) was dissolved in tetrahydrofuran (9 mL) at room temperature, and DIPEA (590 mg, 4.56 mmol), 10% Pd(OH)2 / C (780 mg) were added with stirring, and hydrogenation was performed at room temperature under normal pressure for 4 hours.

[0128] After the reaction was completed, filtration was performed, and concentration was performed to obtain 1.46 g of white foam solid (S)-3-amino-4-((6-((S)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)pyrrolidin-l-yl)-6-oxohexyl)amino)-4-oxobutanoic acid methyl ester (yield: 99%).

[0129] Step C: Synthesis of (S)-4-((6-((S)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)pyrrolidin-l-yl)-6-oxohexyl)amino)-3-((lS,4R)-4-hydroxycyclohexane-l-carbonylamino)-4-oxobutanoic acid methyl ester

[0130] (S)-3-amino-4-((6-((S)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)pyrrolidin-l- yl)-6-oxohexyl)amino)-4-oxobutanoic acid methyl ester (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) at room temperature, the system was cooled to 0 °C, DIPEA (1.01 g, 7.8 mmol), HATU (1.19 g, 3.12 mmol) were added with stirring, and the system was stirred at room temperature for 1 h.

[0131] After the reaction was completed, the system was purified by silica gel column chromatography (eluent: dichloromethane / methanol = 10 / 1) to obtain 1.33 g of yellow foam (S)-4-((6-((S)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)pyrrolidin-l-yl)-6-oxohexyl)amino)-3-((1S,4R)-4-hydroxycyclohexane-l-carboxamido)-4-oxobutanoic acid methyl ester (yield: 66%).

[0132] Step D: Synthesis of (S)-4-((6-((S)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)pyrrolidin-l- yl)-6-oxohexyl)amino)-3-((1S,4R)-4-hydroxycyclohexane-l-carboxamido)-4-oxobutanoic acid

[0133] (S)-4-((6-((S)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)pyrrolidin-l-yl)-6-oxohexyl)amino)-3-((1S,4R)-4-hydroxycyclohexane-l-carboxamido)-4-oxobutanoic acid methyl ester (1.33 g, 1.73 mmol) was dissolved in a mixed solution of tetrahydrofuran (3.3 mL) and water (1.1 mL) at room temperature, lithium hydroxide monohydrate (170 mg, 4.15 mmol) was added, and the system was stirred at 30 °C for 3 h.

[0134] After the reaction was completed, the system was kept below 40 °C, tetrahydrofuran was concentrated to no fraction was distilled, and the remaining system was directly freeze-dried to obtain 1.23 g of white solid (S)-4-((6-((S)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)pyrrolidin-l-yl)-6-oxohexyl)amino)-3-((1S,4R)-4-hydroxycyclohexane-l-carboxamido)-4-oxobutanoic acid (yield: 94%).

[0135] Step E: Synthesis of intermediate C3

[0136] 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) at 0°C under nitrogen atmosphere. Bis(diisopropylamino)(2-cyanoethoxy)phosphine (90 mg, 0.3 mmol) was added and the system was stirred at room temperature for 1 hour.

[0137] After the reaction was completed, the reaction system was washed twice with saturated aqueous sodium bicarbonate solution (50 mL) and the organic phase was washed once with saturated aqueous sodium chloride solution (50 mL). The organic phase was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated and dried, and then purified by silica gel column chromatography (eluent: dichloromethane / methanol = 10 / 1) to obtain 457 mg of yellow foamy solid intermediate C3 (yield: 12%).

[0138] Step F: Synthesis of compound 3

[0139] 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) at 0°C under nitrogen atmosphere. Bis(diisopropylamino)(2-cyanoethoxy)phosphine (90 mg, 0.3 mmol) was added and the system was stirred at room temperature for 1 hour.

[0140] After the reaction was completed, the reaction system was washed twice with saturated aqueous sodium bicarbonate solution (12 mL) and the organic phase was washed once with saturated aqueous sodium chloride solution (12 mL). The organic phase was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated and dried, and then purified by silica gel column chromatography (eluent: dichloromethane / methanol = 10 / 1) to obtain 160 mg of compound 3 (yield: 39%). 1H NMR (400 MHz, DMSO-d6) δ 7.91 - 7.82 (m, 5H), 7.77 (t, J = 5.7 Hz, 2H), 7.33 (dd, J = 11.6, 7.1 Hz, 2H), 7.23 (t, J = 7.9 Hz, 4H), 6.90 (t, J = 6.6 Hz, 2H), 5.23 (d, J = 3.3 Hz, 2H), 4.98 (dd, J = 11.1, 3.3 Hz, 1H), 4.50 (d, J = 8.5 Hz, 2H), 4.04 (s, 11H), 3.73 (d, J = 18.5 Hz, 11H), 3.59 - 3.50 (m, 15H), 3.05 (p, J = 6.4, 6.0 Hz, 13H), 2.47 (t, J = 7.1 Hz, 14H), 2.29 (t, J = 6.4 Hz, 5H), 2.12 (s, 9H), 2.06 (t, J = 7.1 Hz, 5H), 2.01 (s, 11H), 1.91 (s, 9H), 1.79 (s, 9H), 1.50 (dt, J = 16.6, 8.1 Hz, 21H), 1.25 (s, 6H), 1.19 (t, J = 7.1 Hz, 3H), 1.14 (d, J = 6.8 Hz, 9H), 0.96 (t, J = 7.2 Hz, 25H). 31 PNMR (162 MHz, DMSO) δ 144.96. LCMS: RT = 2.08 min, [M-DMTr + H + H] 2+ = 1166.6.

[0141] Example 4 Synthesis of compound 4

[0142] Compound 4 is different from compound 2A in that the intermediate SM2 is different, and the synthesis route of SM2 can be prepared by referring to the literature with patent application number CN202280016262.X, wherein the characterization data of compound 4 are as follows: LCMS: RT = 1.98 min, (M.W.-Propiononitrile-H-H)2- / 2 = 1198.1H NMR (400 MHz, Chloroform-d) δ 7.88 (s, 1H), 7.42 (d, J = 7.6 Hz, 2H), 7.36-7.29 (m, 7H), 6.85 (t, J = 8.9 Hz, 5H), 5.38 (s, 3H), 5.34 (d, J = 3.5 Hz, 3H), 5.26 (d, J = 10.6 Hz, 2H), 4.78 (d, J = 9.0 Hz, 2H), 4.34 (s, 3H), 4.13 (dt, J = 30.4, 10.6 Hz, 13H), 3.98 (d, J = 11.8 Hz, 6H), 3.88-3.71 (m, 12H), 3.55 (t, J = 38.6 Hz, 25H), 3.26 (d, J = 13.9 Hz, 6H), 2.67 (s, 5H), 2.18 (d, J = 3.7 Hz, 9H), 2.11-2.06 (m, 12H), 2.06-1.96 (m, 24H), 1.89 (d, J = 12.2 Hz, 7H), 1.62 (d, J = 49.4 Hz, 8H), 1.21 (d, J = 5.8 Hz, 12H).31P NMR (400 MHz, CDCl3): ppm δ 145.84.

[0143] Example 5 Synthesis of compound 5

[0144] Compound 5 can be prepared by referring to the preparation methods of examples 1-3, and the characterization data of compound 5 are as follows: LCMS: RT = 2.036 min, [M-DMTr-Diisopropylamine+H+H] 2+ / 2 = 1187.0. 31 P NMR (162 MHz, DMSO-d6) δ 145.01 (d, J = 8.0 Hz).

[0145] Example 6 Synthesis of compound 6

[0146] The specific synthesis route is as follows:

[0147] Compound 6 can be prepared by referring to the preparation methods of examples 1-3, and the characterization data of compound 6 are as follows: LCMS: RT = 2.155 min, [M-cyanoethyl] - = 1347.4. 31PNMR (162 MHz, DMSO-d6) δ 141.54.

[0148] Example 7 Synthesis of compound 7

[0149] wherein the synthesis route of compound 7 was prepared by referring to Example 2A, compound 7 was characterized: 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).

[0150] Example 8 Synthesis of compound 8

[0151] The specific synthesis route is as follows:

[0152] wherein the specific synthesis route of compound 8 was prepared by referring to Examples 1-3, compound 8 was characterized: LCMS: RT = 1.981 min, [M-DMTr-Diisopropylamine + H + H]2+ / 2 = 1160.2. 2+ 31 P NMR (162 MHz, DMSO-d6) δ 145.06.

[0153] Example 9A Synthesis of compound 9A

[0154] wherein compound 9A can be prepared by referring to the preparation method of Example 2A, LCMS: RT = 4.987 min, [M-DMTr-Diisopropylamine + H + H]2+ / 2 = 1060.0. 31 P NMR (162 MHz, Chloroform-d) δ 149.74.

[0155] Example 9B Synthesis of compound 9B

[0156] The specific synthesis route is as follows:

[0157] wherein compound 9B can be prepared by referring to the preparation method of Example 2A, compound 9B: LCMS: RT = 4.748 min, [M-cyanoethyl-H] - = 1527.8. 31 P NMR (400 MHz, DMSO-d6): ppm δ 146.65 (dd, J = 140 Hz).

[0158] Example 10 Synthesis of compound 10 ​

[0159] The specific synthesis route is as follows:

[0160] The preparation method of compound 10 can be prepared according to the synthesis route of Example 1, and the characterization of compound 10 is as follows:

[0161] LCMS: RT = 1.98 min, (M. W. -Propiononitrile-H-H) 2- / 2 = 1198. 1 H NMR (400 MHz, DMSO-d6) δ 8.07 (d, J = 5.5 Hz, 2H), 7.85 (d, J = 9.2 Hz, 2H), 7.37 - 7.31 (m, 3H), 7.23 (td, J = 9.3, 2.7 Hz, 5H), 6.99 - 6.79 (m, 3H), 5.24 (d, J = 3.4 Hz, 3H), 5.01 (dt, J = 11.3, 2.9 Hz, 2H), 4.57 (d, J = 8.5 Hz, 3H), 4.04 (d, J = 5.5 Hz, 10H), 3.90 (dt, J = 11.0, 8.7 Hz, 3H), 3.75 (s, 11H), 3.64 - 3.47 (m, 11H), 3.39 (d, J = 22.7 Hz, 26H), 3.26 (d, J = 5.6 Hz, 7H), 3.13 - 2.89 (m, 7H), 2.12 (d, J = 1.1 Hz, 11H), 2.01 (d, J = 1.3 Hz, 9H), 1.91 (s, 10H), 1.80 (s, 10H), 1.47 (s, 11H), 1.15 (dd, J = 6.8, 2.8 Hz, 13H). 31 P NMR (400 MHz, DMSO-d6): ppm δ 145.00.

[0162] Example 11 Synthesis of compound 11

[0163] The specific synthesis route is as follows:

[0164] The preparation route of compound 11 can be prepared according to Examples 1-3, and the characterization of compound 11 is as follows: LCMS: RT = 2.132 min, [M-cyanoethyl] - = 1367.4. 31 PNMR (162 MHz, DMSO-d6) δ 144.94.

[0165] Example 12 Synthesis of compound 12

[0166] The specific synthesis route is as follows:

[0167] The preparation route of compound 12 can be prepared by referring to Examples 1-3, compound 12: LCMS: RT = 2.132 min, [M - cyanoethyl] - = 1353.2. 31 P NMR (162 MHz, DMSO-d6) δ 144.80.

[0168] Example 13 Synthesis of compound 13

[0169] wherein the preparation route of compound 13 can be prepared by referring to Example 1, compound 13: LCMS: RT = 2.03 min, (M.W. - Propiononitrile-H-H) 2- / 2 = 1366. 1 H NMR (400 MHz, DMSO-d6) δ 8.06 - 7.95 (m, 4H), 7.91 (t, J = 5.7 Hz, 2H), 7.42 - 7.31 (m, 2H), 7.30 - 7.21 (m, 4H), 6.93 (ddd, J = 8.8, 6.4, 2.0 Hz, 3H), 5.26 (d, J = 3.4 Hz, 2H), 5.02 (dd, J = 11.2, 3.4 Hz, 2H), 4.57 (d, J = 8.5 Hz, 3H), 4.06 (q, J = 4.4 Hz, 23H), 3.90 - 3.65 (m, 42H), 3.52 - 3.21 (m, 38H), 2.33 (s, 6H), 2.14 (s, 9H), 2.04 (s, 9H), 1.93 (s, 9H), 1.82 (s, 9H), 1.54 (dt, J = 14.1, 6.5 Hz, 23H), 1.20 - 1.15 (m, 12H). 31 P NMR (400 MHz, DMSO-d6): ppm δ 144.93.

[0170] Example 14 Synthesis of compound 14

[0171] The specific synthesis route is as follows:

[0172] Step A: Synthesis of 1,5-dioxane-2,8-dione

[0173] N,N-dicyclohexylcarbodiimide (8.40 g, 40.71 mmol, 8.23 mL) was added to a solution of 3-(2-carboxyethoxy)propanoic acid (6 g, 37.01 mmol) in ethyl acetate (60 mL) at zero degrees under nitrogen protection. The reaction solution was stirred at room temperature for 2 hours until the reaction was substantially complete by TLC monitoring.

[0174] The reaction solution was filtered and the filtrate was concentrated in vacuo to give 5.33 g of the product 1,5-dioxane-2,8-dione as a light yellow oil.

[0175] Step B: Synthesis of 3-(3-methoxy-3-oxopropoxy)propanoic acid

[0176] At room temperature, 1,5-dioxane-2,8-dione (5.33 g, 36.98 mmol) and sodium methoxide solution (5.4 mol / L, 136.97 μL) were dissolved in methanol (50 mL). The reaction mixture was replaced with nitrogen for 3 times, the temperature was raised to 70 °C, and stirred for 1 hour under nitrogen atmosphere until the reaction was substantially complete as monitored by TLC.

[0177] The reaction was allowed to cool to room temperature, and the reaction solution was concentrated in vacuo. The crude product was purified by column chromatography (eluent: petroleum ether / ethyl acetate = 1 / 0-1 / 1) to give 3.10 g of the product 3-(3-methoxy-3-oxopropoxy)propanoic acid as a yellow oil.

[0178] Step C: Synthesis of methyl 3-[3-[(2S)-2-(hydroxymethyl)pyrrolidin-1-yl]-3- oxopropoxy]propanoate

[0179] At room temperature and under nitrogen protection, 3-(3-methoxy-3-oxopropoxy)propanoic acid (3.10 g, 17.6 mmol), diisopropylethylamine (5.69 g, 43.99 mmol, 7.66 mL), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (6.75 g, 35.19 mmol), 1-hydroxybenzotriazole (4.76 g, 35.19 mmol) were dissolved in dichloromethane (5 mL), cooled to 0 °C, and stirred for 0.5 hour. Then a solution of L-prolinol (1.78 g, 17.6 mmol) in dichloromethane (5 mL) was slowly added to the reaction system, and then the temperature was raised to room temperature, and stirred for 2 hours until the reaction was complete as monitored by TLC.

[0180] The reaction mixture was concentrated under reduced pressure to remove the solvent. It was diluted with water 60 mL, and then extracted with dichloromethane (50 mL*3 times). The combined organic phase was washed with saturated brine (50 mL*2 times), dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo to give the crude product. The crude product was purified by column chromatography (eluent: petroleum ether / ethyl acetate = 1 / 0-0 / 1) to give 2.73 g of the product methyl 3-[3-[(2S)-2-(hydroxymethyl)pyrrolidin-1-yl]-3-oxopropoxy]propanoate as a yellow oil.

[0181] Step D: Synthesis of methyl 3-[3-[(2S)-2-[[bis(4-methoxyphenyl)-phenylmethoxy]methyl]pyrrolidin-1-yl]-3-oxopropoxy]propanoate

[0182] To a solution of 3-[3-[(2S)-2-[[bis(4-methoxyphenyl)-phenylmethoxy]methyl]pyrrolidin-1- yl]-3-oxopropoxy]propanoic acid (4.88 g, 8.99 mmol) in tetrahydrofuran (40 mL) and water (20 mL) was added 4-fluorobenzenesulfonyl chloride (1.70 g, 9.99 mmol) at room temperature. The reaction mixture was stirred at room temperature for 2 hours to LC-MS monitoring reaction complete.

[0183] The reaction mixture was concentrated to give 3.50 g of product 3-[3-[(2S)-2-[[bis(4- methoxyphenyl)-phenylmethoxy]methyl]pyrrolidin-1-yl]-3-oxopropoxy]-4- fluoropropanoic acid. LC-MS: RT = 0.657 min, [M+H]+ = 576.3.

[0184] Step E: Synthesis of 3-[3-[(2S)-2-[[bis(4-methoxyphenyl)-phenylmethoxy]methyl]pyrrolidin-1- yl]-3-oxopropoxy]propanoic acid

[0185] The reaction mixture was concentrated to give 3.50 g of product 3-[3-[(2S)-2-[[bis(4- methoxyphenyl)-phenylmethoxy]methyl]pyrrolidin-1-yl]-3-oxopropoxy]-4- fluoropropanoic acid. LC-MS: RT = 0.657 min, [M+H]+ = 576.3.

[0186] The reaction mixture was concentrated to give 3.50 g of product 3-[3-[(2S)-2-[[bis(4- methoxyphenyl)-phenylmethoxy]methyl]pyrrolidin-1-yl]-3-oxopropoxy]-4- fluoropropanoic acid. LC-MS: RT = 0.657 min, [M+H]+ = 576.3.

[0187] Step F: Synthesis of methyl (2S)-2-(9H-fluoren-9-ylmethoxycarbonylamino)-6-[(4- hydroxycyclohexanecarbonyl)amino]hexanoate

[0188] A solution of 4-hydroxycyclohexanecarboxylic acid (26.25 g, 182 mmol), diisopropylethylamine (70.6 g, 546 mmol, 95.14 mL), l-(3-dimethylaminopropyl)-3- ethylcarbodiimide hydrochloride (45.38 g, 236.7 mmol), and l-hydroxybenzotriazole (31.98 g, 236 mmol) in dichloromethane (840 mL) was cooled to 0 °C under nitrogen. Methyl (2S)-6-amino-2-(9H-fluoren-9-ylmethoxycarbonylamino)hexanoate (83.9 g, 200.29 mmol) was then added to the reaction mixture, which was then allowed to warm to room temperature and stirred for 2 h until the reaction was complete by LC-MS.

[0189] The reaction mixture was concentrated under reduced pressure to remove the solvent. It was diluted with water (400 mL) and extracted with dichloromethane (500 mL x 2). The combined organic phases were washed with saturated brine (300 mL x 2), dried over anhydrous sodium sulfate, filtered and concentrated under vacuum to give the crude product. The crude product was purified by column chromatography (eluent: petroleum ether / ethyl acetate = 1 / 0 to 0 / 1) to give 11.5 g (98.8% purity) of methyl (2S)-2-(9H-fluoren-9-ylmethoxycarbonylamino)-6-[(4- hydroxycyclohexanecarbonyl)amino]hexanoate. LC-MS: RT = 0.600 min, [M+H]+= 509.3. 32.5 g (92.6% purity) of the yellow solid product methyl (2S)-2-(9H-fluoren-9- ylmethoxycarbonylamino)-6-[(4-hydroxycyclohexanecarbonyl)amino]hexanoate. LC-MS: RT = 0.604 min, [M+H]+= 509.3.

[0190] Step G: Synthesis of methyl (2S)-2-amino-6-[(4-hydroxycyclohexanecarbonyl)amino]hexanoate

[0191] A solution of methyl (2S)-2-(9H-fluoren-9-ylmethoxycarbonylamino)-6-[(4- hydroxycyclohexanecarbonyl)amino]hexanoate (45 g, 88.48 mmol) in tetrahydrofuran (500 mL) was added ethylenediamine (32.36 g, 442.39 mmol, 45.57 mL) at 0 °C under nitrogen. It was stirred at room temperature for 2 h until the reaction was complete by LC-MS.

[0192] The reaction mixture was filtered and the filter cake was concentrated under reduced pressure to remove the solvent. This gave 6.7 g (91% purity) of the light yellow oily product methyl (2S)-2-amino-6-[(4-hydroxycyclohexanecarbonyl)amino]hexanoate. LC-MS: RT = 0.296 min, [M+H]+= 287.1. 17.0 g (89.7% purity) of the yellow oily product methyl (2S)-2-amino-6-[(4- hydroxycyclohexanecarbonyl)amino]hexanoate.

[0193] Step H: Synthesis of methyl (2S)-2-[3-[3-[3-[(2S)-2-[[bis(4- methoxyphenyl)-phenylmethoxy]methyl]pyrrolidin-1-yl]-3-oxopropoxy]propanamide]-6- [(4-hydroxycyclohexyl)amino]hexanoate

[0194] To a solution of methyl (2S)-2-[3-[3-[3-[(2S)-2-[[bis(4- methoxyphenyl)-phenylmethoxy]methyl]pyrrolidin-1-yl]-3-oxopropoxy]propanamide]-6- [(4-hydroxycyclohexyl)amino]hexanoate (3.6 g, 4.41 mmol) in dichloromethane (50 mL) was added trifluoroacetic acid (5 mL) at room temperature under nitrogen. The reaction mixture was stirred at room temperature for 2 hours. The reaction mixture was concentrated under reduced pressure to remove the solvent. The residue was diluted with water (100 mL) and extracted with ethyl acetate (100 mL*3 times). The organic phase was washed with saturated sodium bicarbonate solution (100 mL*2 times), saturated brine (75 mL*2 times), dried over anhydrous sodium sulfate, filtered and concentrated under vacuum to give the crude product. The crude product was purified by reverse phase chromatography (Chromatography column: 330 g Flash Column Welch Ultimate XB CI 8 20-40 pm; 120 A, 0.1% ammonia system) to give 2.9 g of methyl (2S)-2-[3-[3-[3-[(2S)-2-[[bis(4- methoxyphenyl)-phenylmethoxy]methyl]pyrrolidin-1-yl]-3-oxopropoxy]propanamide]-6- [(4-hydroxycyclohexyl)amino]hexanoate. LC-MS: RT = 0.661 min, [M-H]+ = 814.5.

[0195] The reaction mixture was concentrated under reduced pressure to remove the solvent. It was diluted with water (100 mL) and extracted with ethyl acetate (100 mL*3 times). The organic phase was washed with saturated sodium bicarbonate solution (100 mL*2 times), saturated brine (75 mL*2 times), dried over anhydrous sodium sulfate, filtered and concentrated under vacuum to give the crude product. The crude product was purified by reverse phase chromatography (Chromatography column: 330 g Flash Column Welch Ultimate XB CI 8 20-40 pm; 120 A, 0.1% ammonia system) to give 2.9 g of methyl (2S)-2-[3-[3-[3-[(2S)-2-[[bis(4- methoxyphenyl)-phenylmethoxy]methyl]pyrrolidin-1-yl]-3-oxopropoxy]propanamide]-6- [(4-hydroxycyclohexyl)amino]hexanoate. LC-MS: RT = 0.661 min, [M-H]+ = 814.5.

[0196] Step I: Synthesis of (2S)-2-[3-[3-[(2S)-2-[[bis(4-methoxyphenyl)- phenylmethoxy]methyl]pyrrolidin-1-yl]-3-oxopropoxy]propanamide]-6-[(4- hydroxycyclohexyl)amino]hexanoic acid (B10)

[0197] Methyl (2S)-2-[3-[3-[3-[(2S)-2-[[bis(4-methoxyphenyl)-phenylmethoxy]methyl]pyrrolidin-1-yl]-3-oxopropoxy]propanamido]-6-[(4-hydroxycyclohexyl)amino]hexanoate (3.60 g, 4.41 mmol) was dissolved in tetrahydrofuran (30 mL) and water (10 mL) at room temperature, cooled to 0 °C, then lithium hydroxide monohydrate (222 mg, 5.29 mmol) was added, allowed to warm to room temperature and stirred for 2 h until the reaction was complete by LC-MS monitoring.

[0198] The reaction was diluted with 20 mL water and extracted with ethyl acetate (50 mL x 2). The aqueous phase was lyophilized to remove water to give 3.26 g (lithium salt) of the product (2S)-2-[3-[3-[(2R)-2-[[bis(4-methoxyphenyl)-phenylmethoxy]methyl]pyrrolidin-1-yl]-3-oxopropoxy]propanamido]-6-[(4-hydroxycyclohexyl)amino]hexanoic acid. LC-MS: RT = 0.450 min, [M-H]+ = 800.3, SFC: RT = 1.480 min.1H NMR (400 MHz, DMSO-d6) δ 7.68 (br t, J = 5.60 Hz, 1H) 7.51 - 7.41 (m, 1H) 7.37 - 7.25 (m, 4H) 7.25 - 7.16 (m, 5H) 6.93 - 6.84 (m, 4H) 4.40 (br s, 1H) 4.10 (br s, 1H) 3.93 - 3.85 (m, 1H) 3.72 (s, 7H) 3.60 - 3.52 (m, 3H) 3.21 - 3.09 (m, 1H) 3.09 - 3.00 (m, 1H) 2.99 - 2.87 (m, 3H) 2.47 - 2.37 (m, 1H) 2.36 - 2.20 (m, 2H) 2.08 - 2.03 (m, 1H) 1.91 - 1.72 (m, 6H) 1.70 - 1.56 (m, 5H) 1.53 - 1.10 (m, 10H).

[0199] Step J: Synthesis of Intermediate C14

[0200] To 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'N'-tetramethyluronium hexafluorophosphate (312.7 mg, 0.825 mmol) and B10 (447.0 mg, 0.55 mmol) at room temperature and allowed to react overnight at 45 °C. 2

[0201] ​After the reaction was completed, 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 710.0 mg of intermediate C14 (yield: 38.3%). LCMS: RT = 1.916 min, [M-DMTr+H+H] 2+ / 2 = 1138.6.

[0202] Step K: Synthesis of compound 14

[0203] To a three-necked flask containing 4A molecular sieves and N,N-diisopropylammonium tetrazolide (236.0 mg, 1.38 mmol) at 0°C, intermediate C14 (710.0 mg, 0.275 mmol) in dichloromethane (10.0 mL) was added, followed by bis(diisopropylamino)(2-cyanoethoxy)phosphine (874.0 μL, 2.75 mmol) and the reaction was allowed to proceed at room temperature for 1 hour.

[0204] After the reaction was completed, an ice mixture of sodium bicarbonate and sodium chloride aqueous solution (1:1) was added at -20°C to quench the reaction, extracted with DCM, washed with an aqueous mixture three times, dried, concentrated to 1 mL, and added dropwise to ice MTBE, and the solid was separated by centrifugation, the supernatant was discarded, and the operation was repeated 5 times, dissolved in dichloromethane, and evaporated to dryness to obtain 330.0 mg of compound 14 (yield: 43.2%). LCMS: RT: 2.200 min, [M-DMTr-Diisopropylamine+H+H] 2+ / 2 = 1188.2. 31 PNMR (162 MHz, DMSO-d6) δ 145.01.

[0205] Example 15 Synthesis of compound 15

[0206] wherein the preparation route of compound 15 can be prepared according to Example 1, compound 15: LCMS: RT = 2.007 min, [M-DMTr-Diisopropylamine+H+H]+ / 2 = 1187.4. 31 P NMR (400 MHz, DMSO-d6): ppm δ 144.99.

[0207] Example 16 Synthesis of compound 16

[0208] wherein the preparation route of compound 16 can be prepared according to Example 2A, compound 16: LCMS: RT = 1.98 min, (M.W.-Propiononitrile-H-H) 2- / 2 = 1212. 1H NMR (400 MHz, DMSO-d6) δ 7.97 (d, J = 7.0 Hz, 4H), 7.87 (dd, J = 20.8, 7.7 Hz, 5H), 7.42 - 7.27 (m, 4H), 7.23 (td, J = 9.0, 7.9, 3.1 Hz, 5H), 6.95 - 6.85 (m, 4H), 5.23 (d, J = 3.4 Hz, 3H), 5.00 (dd, J = 11.2, 3.4 Hz, 3H), 4.63 - 4.55 (m, 3H), 4.19 (d, J = 6.9 Hz, 4H), 4.04 (d, J = 4.3 Hz, 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 = 1.3 Hz, 9H), 1.91 (s, 15H), 1.79 (s, 9H), 1.72 (s, 5H), 1.47 (d, J = 31.4 Hz, 3H), 1.15 (d, J = 6.8 Hz, 12H). 31 P NMR (400 MHz, DMSO-d6): ppm δ 144.99, 144.95.

[0209] Example 17 Synthesis of compound 17

[0210] The specific synthesis route is as follows:

[0211] wherein, the preparation route of compound 17 can be prepared by referring to example 1, compound 17: LCMS: RT = 1.98 min, (M.W. - Propiononitrile-H-H) 2- / 2 = 1198. 1H NMR (400 MHz, DMSO-d6) δ 8.06 (d, J = 5.3 Hz, 2H), 7.84 (d, J = 9.3 Hz, 3H), 7.45 (d, J = 7.7 Hz, 1H), 7.33 (d, J = 8.0 Hz, 5H), 6.90 (d, J = 8.5 Hz, 3H), 5.24 (d, J = 3.4 Hz, 3H), 5.00 (dt, J = 11.2, 2.9 Hz, 3H), 4.57 (dd, J = 8.5, 3.9 Hz, 3H), 4.05 (s, 10H), 3.90 (q, J = 9.5 Hz, 3H), 3.75 (s, 8H), 3.71 (dd, J = 10.6, 7.1 Hz, 3H), 3.63 - 3.47 (m, 11H), 3.41 (d, J = 7.9 Hz, 7H), 3.26 (s, 7H), 3.09 (s, 12H), 2.12 (d, J = 2.5 Hz, 10H), 2.01 (d, J = 3.1 Hz, 9H), 1.91 (s, 9H), 1.80 (d, J = 1.7 Hz, 10H), 1.15 (dd, J = 6.8, 2.8 Hz, 12H). 31 P NMR (400 MHz, DMSO-d6): ppm δ 144.97.

[0212] Example 18 Synthesis of compound 18

[0213] The specific synthesis route is as follows:

[0214] Step A: Synthesis of intermediate C18

[0215] SM2 (2.67 g, 1.78 mmol) was dissolved in dry and anhydrous dichloromethane (30 mL), DIEA (1.13 mL, 6.85 mmol), HBTU (0.78 g, 2.06 mmol), B10 (1.1 g, 1.37 mmol) were added successively and reacted at 45 degrees for 24 hours.

[0216] After the reaction was completed, it was quenched with saturated aqueous sodium bicarbonate solution, extracted with dichloromethane (20 mL x 2 times), dried and directly concentrated to an oil, which was purified by silica gel column chromatography (eluent: dichloromethane / methanol = 15 / 1), and then purified by high performance liquid chromatography column to obtain 0.7 g of white foam solid C18 (yield: 22%). LCMS: RT = 4.065 min, [M-H] / 2 = 1139.4. -

[0217] Step B: Synthesis of compound 18

[0218] ​C18 (700 mg, 0.31 mmol), diisopropyl tetrazolyl ammonium salt (160 mg, 0.93 mmol) were dissolved in dichloromethane (15 ml) at room temperature, activated molecular sieves were added, nitrogen was bubbled through, and bis(diisopropylamino)(2-cyanoethoxy) phosphine (280 mg, 0.93 mmol) was added dropwise at room temperature. The reaction was stirred at room temperature for 2 hours.

[0219] After the reaction was completed, the reaction solution was cooled to -20 °C, and saturated sodium bicarbonate / saturated brine (6 ml / 6 ml) was added dropwise with stirring. After stirring for 2 minutes, the reaction solution was stirred at room temperature for 5 minutes, and then separated. The organic phase was dried over sodium sulfate, filtered, and concentrated to give a white gum. The white gum was dissolved in 3 ml of dichloromethane, and then added dropwise to 30 ml of methyl tert-butyl ether. The mixture was centrifuged and concentrated, and the supernatant was discarded. The gum was dissolved in 3 ml of dichloromethane, and the above operation was repeated three times. Finally, the gum was concentrated to give 596 mg of compound 18 (yield: 78%). LCMS: RT = 4.839 min, [M-cyanoethyl-H] / 2 = 1213.03 1H NMR (400 MHz, DMSO-d6): ppm δ 144.95.

[0220] Example 19 Synthesis of compound 19

[0221] The specific synthesis route is as follows:

[0222] Step A: Synthesis of intermediate C19

[0223] SM3B (0.52 g, 0.59 mmol) was dissolved in dry and anhydrous dichloromethane (20 ml), and DIEA (0.49 ml, 2.95 mmol), HBTU (0.34 g, 0.89 mmol), and B10 (0.90 g, 0.59 mmol) were added in sequence. The reaction was carried out at 45 °C for 24 hours.

[0224] After the reaction was completed, the reaction solution was cooled to -20 °C, and saturated sodium bicarbonate / saturated brine (6 ml / 6 ml) was added dropwise with stirring. After stirring for 2 minutes, the reaction solution was stirred at room temperature for 5 minutes, and then separated. The organic phase was dried over sodium sulfate, filtered, and concentrated to give a white gum. The white gum was dissolved in 3 ml of dichloromethane, and then added dropwise to 30 ml of methyl tert-butyl ether. The mixture was centrifuged and concentrated, and the supernatant was discarded. The gum was dissolved in 3 ml of dichloromethane, and the above operation was repeated three times. Finally, the gum was concentrated to give 596 mg of compound 18 (yield: 78%). LCMS: RT = 4.839 min, [M-cyanoethyl-H] / 2 = 1213.03 1H NMR (400 MHz, DMSO-d6): ppm δ 144.95.

[0225] Step B: Synthesis of compound 19

[0226] C19 (500 mg, 0.22 mmol), diisopropyl tetrazolyl ammonium salt (110 mg, 0.66 mmol) were dissolved in dichloromethane (15 ml) at room temperature, activated molecular sieves were added, nitrogen was bubbled through, and bis(diisopropylamino)(2-cyanoethoxy) phosphine (270 mg, 0.88 mmol) was added dropwise at room temperature. The reaction was stirred at room temperature for 2 hours.

[0227] After the reaction was completed, the reaction solution was cooled to -20 °C, and saturated sodium bicarbonate / saturated brine (6 ml / 6 ml) was added dropwise with stirring. After stirring for 2 minutes, stirring was performed at room temperature for 5 minutes, and the solution was separated. The organic phase was dried over sodium sulfate, filtered, and concentrated to obtain a white gum. To the white gum was added 3 ml of dichloromethane, which was then added dropwise to 30 ml of tert-butyl methyl ether. The mixture was centrifuged and concentrated, and the supernatant was discarded. The gum was dissolved again in 3 ml of dichloromethane, and the above operation was repeated three times. Finally, the obtained gum was concentrated to obtain 305 mg of compound 19 (yield: 56%). LCMS: RT = 2.155 min, [M-cyanoethyl-H] / 2- = 1227.2.31P NMR (400 MHz, DMSO-d6): ppm δ 144.98.

[0228] Example 20 Synthesis of compound 20

[0229] The specific synthesis route is as follows:

[0230] Step A: Synthesis of 3-(3-(4-hydroxypiperidin-1-yl)-3-oxopropoxy)propanoic acid

[0231] At 0 °C, bis(2-carboxyethyl) ether (5 g, 30.84 mmol) and 4-hydroxypiperidine (3.43 g, 33.92 mmol) were dissolved in dry and anhydrous dichloromethane (110 ml), and DIEA (7.65 ml, 46.26 mmol), HBTU (14.03 g, 37.01 mmol) were added in sequence. The reaction was performed at room temperature for 24 hours.

[0232] After the reaction was completed, the reaction solution was cooled to -20 °C, and saturated sodium bicarbonate / saturated brine (6 ml / 6 ml) was added dropwise with stirring. After stirring for 2 minutes, stirring was performed at room temperature for 5 minutes, and the solution was separated. The organic phase was dried over sodium sulfate, filtered, and concentrated to obtain a white gum. To the white gum was added 3 ml of dichloromethane, which was then added dropwise to 30 ml of tert-butyl methyl ether. The mixture was centrifuged and concentrated, and the supernatant was discarded. The gum was dissolved again in 3 ml of dichloromethane, and the above operation was repeated three times. Finally, the obtained gum was concentrated to obtain 305 mg of compound 19 (yield: 56%). LCMS: RT = 2.155 min, [M-cyanoethyl-H] / 2- = 1227.2.31P NMR (400 MHz, DMSO-d6): ppm δ 144.98.

[0233] Step B: Synthesis of compound 0020-2

[0234] 3-(3-(4-hydroxypiperidin-1-yl)-3-oxopropoxy)propanoic acid (29 g, 118.24 mmol) was dissolved in dry and anhydrous pyridine (145 ml), and 4,4'-dimethoxytrityl chloride (25 g, 73.78 mmol) was added. The reaction was performed at room temperature for 24 hours.

[0235] After the reaction was completed, the mixture was directly concentrated to get an oil, which was purified by silica gel column chromatography (eluent: dichloromethane / methanol = 15 / 1) and then by high performance liquid chromatography column to obtain 60 g of white foam solid 0020-2 (yield: 93%).

[0236] Step C: Synthesis of 0020-4

[0237] At 0 degrees, 0020-2 (12 g, 21.91 mmol) was dissolved in dry and anhydrous dichloromethane (120 ml), and then DIEA (5.43 ml, 32.87 mmol), HOPO (0.49 g, 4.38 mmol), EDCI (5.46 g, 28.48 mmol), and 0020-3 (7.45 g, 26.02 mmol) were added in sequence, and the reaction was carried out at room temperature for 24 hours.

[0238] After the reaction was completed, the mixture was directly concentrated to get an oil, which was purified by silica gel column chromatography (eluent: dichloromethane / methanol = 15 / 1) and then by high performance liquid chromatography column to obtain 60 g of white foam solid 0020-2 (yield: 93%).

[0239] Step D: Synthesis of B12

[0240] At room temperature, 0020-4 (8.37 g, 10.26 mmol) was dissolved in a mixed solution of tetrahydrofuran (8.25 ml) and water (2.75 ml), and lithium hydroxide monohydrate (820 mg, 19.6 mmol) was added, and the system was heated to 35 degrees Celsius and stirred for 3 hours.

[0241] After the reaction was completed, the system was kept below 40 degrees Celsius, and the tetrahydrofuran was concentrated to no distillate, and the remaining system was directly freeze-dried to obtain 8.2 g of B12 (yield: 99%).

[0242] Step E: Synthesis of intermediate C20

[0243] At 0 degrees, SM1 (0.95 g, 0.48 mmol) was dissolved in dry and anhydrous dichloromethane (16 ml), and then DIEA (0.12 ml, 0.72 mmol), HBTU (0.27 g, 0.75 mmol), and B12 (0.42 g, 0.53 mmol) were added in sequence, and the reaction was carried out at room temperature for 24 hours.

[0244] After the reaction was completed, the mixture was directly concentrated to get an oil, which was purified by silica gel column chromatography (eluent: dichloromethane / methanol = 15 / 1) and then by high performance liquid chromatography column to obtain 60 g of white foam solid 0020-2 (yield: 93%).

[0245] Step F: Synthesis of compound 20

[0246] C20 (481 mg, 0.19 mmol), diisopropyl tetrazolyl ammonium salt (39 mg, 0.23 mmol) were dissolved in dichloromethane (10 ml) at room temperature, activated molecular sieve was added, nitrogen was replaced, bis(diisopropylamino)(2-cyanoethoxy) phosphine (110 mg, 0.38 mmol) was added dropwise at room temperature, stirred at room temperature for 2 hours.

[0247] After the reaction was completed, the reaction solution was cooled to minus 20 degrees Celsius, saturated sodium bicarbonate / saturated brine (6 ml / 6 ml) was added dropwise with stirring, stirred for 2 minutes at room temperature, stirred for 5 minutes at room temperature, separated, the organic phase was dried with sodium sulfate, filtered, concentrated to white gum. 3 ml of dichloromethane was added to the white gum, then added dropwise into 30 ml of methyl tert-butyl ether, centrifuged and concentrated, the supernatant was discarded, the gum was dissolved in 3 ml of dichloromethane, and the above operation was repeated for 3 times, finally the obtained gum was concentrated to 514 mg of compound 20 (yield: 99%). LCMS: RT = 2.268 min, [M-cyanoethyl-H] / 2- = 1361.0.31P NMR (400 MHz, DMSO-d6): ppm δ 144.97.

[0248] Example 21 Synthesis of compound 21

[0249] The specific synthesis route is as follows:

[0250] Wherein, the preparation route of compound 21 can be prepared by referring to example 20, compound 21: LCMS: RT = 2.064 min, [M-cyanoethyl-H] / 2- = 1213.2.31P NMR (400 MHz, DMSO-d6): ppm δ 144.97.

[0251] Example 22 Synthesis of compound 22

[0252] The specific synthesis route is as follows:

[0253] Step A: Synthesis of (1R, 3R)-3-(2-hydroxyethoxy)cyclobutane-1-carboxylic acid

[0254] (1R,3R)-3-(2-(benzyloxy)ethoxy)cyclobutane-1-carboxylic acid (574 mg, 2.3 mmol) was dissolved in tetrahydrofuran (12 mL) under ice bath, and two drops of 6N hydrochloric acid were added. After addition of Pd / C (100 mg, 10% wt) and Pd(OH)2 / C (100 mg, 10% wt), hydrogen gas was replaced. After the addition was completed, the temperature was raised to 65°C and the reaction was allowed to proceed for 12 hours. After the reaction was completed, the temperature was lowered to room temperature and then filtered. The filter cake was washed with methanol, and the organic phase was concentrated without further treatment to give the crude product 352 mg of (1R,3R)-3-(2-hydroxyethoxy)cyclobutane-1-carboxylic acid (yield: 95.6%)

[0255] Step B: Synthesis of methyl N2-((benzyloxy)carbonyl)-N6-((1R,3S)-3-(2- hydroxyethoxy)cyclobutane-1-carboxyI)-L-lysinate

[0256] (1R,3R)-3-(2-hydroxyethoxy)cyclobutane-1-carboxylic acid (352 mg, 2.20 mmol) was dissolved in dichloromethane (25 mL) under ice bath, and N,N- diisopropylethylamine (852 mg, 6.60 mmol) and HATU (1.00 g, 2.64 mmol) were added. After stirring for 3 minutes, methyl ((benzyloxy)carbonyl)-L- lysinate hydrochloride (800 mg, 2.42 mmol) was added, and then the temperature was raised to room temperature and stirred for 3 hours. After the reaction was completed, dichloromethane (25 mL) and saturated aqueous sodium bicarbonate solution (30 mL) were added and extracted. The resulting organic phase was washed with saturated aqueous sodium bicarbonate solution (25 mL x 2), and then with saturated sodium chloride solution (25 mL) once, and finally dried over anhydrous sodium sulfate. The organic phase was concentrated under reduced pressure, and the resulting crude product was subjected to column chromatography (eluent: methanol / dichloromethane = 1 / 20) to give 600 mg of methyl N2-((benzyloxy)carbonyl)-N6-((1R,3S)-3-(2-hydroxyethoxy)cyclobutane-1-carboxyI)-L-lysinate (yield: 62.7%).

[0257] Step C: Synthesis of N2-((benzyloxy)carbonyl)-N6-((1R,3S)-3-(2- hydroxyethoxy)cyclobutane-1-carboxyI)-L-lysine

[0258] N2-((benzyloxy)carbonyl)-N6-((lR,3S)-3-(2-hydroxyethoxy)cyclobutane-l- carbonyl)-L-lysine (577 mg, 1.35 mmol) was dissolved in N,N-dimethylformamide (7.5 mL) at room temperature, and dichloromethane (60 mL) was added. N,N- diisopropylethylamine (760 mg, 5.89 mmol) and HATU (726 mg, 1.91 mmol) were added and stirred for 3 minutes. Then, SM2 (2.65 g, 1.77 mmol) was added and reacted for 12 hours. After the reaction was completed, saturated aqueous sodium bicarbonate solution (50 mL) and dichloromethane (50 mL) were added and extracted. The organic phase was washed with saturated aqueous sodium bicarbonate solution (50 mL x 3) and then with saturated aqueous sodium chloride solution (50 mL) once, and then dried with anhydrous sodium sulfate. The organic phase was concentrated under reduced pressure, and the obtained crude product was subjected to column chromatography (eluent: methanol / dichloromethane = 1 / 20) to obtain 1.30 g of compound 022-6 (yield: 46.3%).

[0259] Step D: Synthesis of compound 022-6

[0260] N2-((benzyloxy)carbonyl)-N6-((lR,3S)-3-(2-hydroxyethoxy)cyclobutane-l- carbonyl)-L-lysine (577 mg, 1.35 mmol) was dissolved in N,N-dimethylformamide (7.5 mL) at room temperature, and dichloromethane (60 mL) was added. N,N- diisopropylethylamine (760 mg, 5.89 mmol) and HATU (726 mg, 1.91 mmol) were added and stirred for 3 minutes. Then, SM2 (2.65 g, 1.77 mmol) was added and reacted for 12 hours. After the reaction was completed, saturated aqueous sodium bicarbonate solution (50 mL) and dichloromethane (50 mL) were added and extracted. The organic phase was washed with saturated aqueous sodium bicarbonate solution (50 mL x 3) and then with saturated aqueous sodium chloride solution (50 mL) once, and then dried with anhydrous sodium sulfate. The organic phase was concentrated under reduced pressure, and the obtained crude product was subjected to column chromatography (eluent: methanol / dichloromethane = 1 / 20) to obtain 1.30 g of compound 022-6 (yield: 46.3%).

[0261] Step E: Synthesis of compound 022-7

[0262] Compound 022-6 (1.30 g, 0.68 mmol) was dissolved in tetrahydrofuran (40 mL) at room temperature, Pd / C (400 mg, 10% wt) was added, and hydrogen was replaced. Then, it was heated to 40°C and reacted for 12 hours. After the reaction was completed, it was filtered, the filter cake was rinsed with methanol, and the organic phase was concentrated under reduced pressure to obtain 1.20 g of compound 022-7 (yield: 99.9%).

[0263] Step F: Synthesis of intermediate C22

[0264] At room temperature, C22 (1.20 g, 0.67 mmol) was dissolved in dichloromethane (40 mL), and N,N-diisopropylethylamine (351 mg, 2.72 mmol) and HATU (336 mg, 0.88 mmol) were added and stirred for 3 minutes. Then (S)-3-(3-(2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)pyrrolidine-1-yl)-3-oxopropoxy)propionic acid (447 mg, 0.82 mmol) was added, and stirring continued for 4 hours at room temperature. After the reaction was complete, dichloromethane (25 mL) and saturated sodium bicarbonate aqueous solution (30 mL) were added for extraction. The resulting organic phase was washed with saturated sodium bicarbonate aqueous solution (25 mL × 2), then washed once with saturated sodium chloride solution (25 mL), and finally dried over anhydrous sodium sulfate. The crude product obtained by vacuum concentration of the organic phase was subjected to column chromatography (eluent: methanol / dichloromethane = 1 / 20-1 / 10) to give 469 mg of intermediate C22 (yield: 31.8%).

[0265] Step G: Synthesize compound 22

[0266] At room temperature, the mixture containing intermediate C22 (469 mg, 0.2 mmol) and In a 20 mL solution of dichloromethane containing molecular sieves, bis(diisopropylamino)(2-cyanoethoxy)phosphine (181 mg, 0.6 mmol) and diisopropylammonium tetrazolium (103 mg, 0.6 mmol) were added, and the reaction was carried out at room temperature for 4 hours. After the reaction was complete, dichloromethane was added, and the mixture was washed twice with ice-cold saturated sodium bicarbonate solution, once with saturated brine, and then dried over anhydrous sodium sulfate. The residue was concentrated under reduced pressure and slurried in an ice bath (MTBE / DCM = 8:1). The mixture was centrifuged (3500 rpm, 30 s), the supernatant was discarded, and the residue was redissolved in dichloromethane and added dropwise to MTBE. This process was repeated five times to obtain 212 mg of compound 22. LCMS:RT = 4.725 min, [(M-54) / 2] 2- =1220.8. 31 PNMR (400MHz, DMSO-d6): ppmδ147.39.

[0267] The structures of compounds 1-22 conjugated with siRNA sequences are shown in Table 1.

[0268] Table 1 shows the structure of A1-A22.

[0269] The intermediate structures used in the synthesis of compounds 1-22 are B1, B2A, B2B, B3, B4, B5, B6, B7, B8, B9, B10, B11, B12, and B13, respectively. Compounds 1, 9A, 9B, and 10 use the same intermediate B1, compounds 7, 4, and 2 use the same intermediate structure B2, compounds 6 and 16 use intermediate B5, compounds 11 and 17 use intermediate B7, compound 12 uses intermediate B8, compound 13 uses intermediate B9, compounds 14, 18, and 19 use intermediate B10, compound 15 uses intermediate B11, compounds 20 and 21 use intermediate B12, and compound 22 uses intermediate B13. The intermediate structures C1, C2, C3, C4, C5, C6, C7, C8, C9A, C9B, C10, C11, C12, C13, C14, C15, C16, C17, C18, C19, C20, C21, and C22 are shown in Table 2:

[0270] Table 2 shows the intermediate structures

[0271] When A1-A22 are connected to nucleotides via phosphates or thiophosphates, the structures are shown in Table 3:

[0272] Table 3 shows the structures of D1-D22 connected with phosphates

[0273] Synthesis of nucleic acid molecules

[0274] The nucleic acid molecules, including the first nucleic acid molecule and the second nucleic acid molecule, were prepared using a Dr. 48 model DNA / RNA automatic synthesizer. The specific synthesis route can be referred to Example 25, and the sequences of the nucleic acid molecules are shown in Table 4.

[0275] Table 4 shows the sequence table of the nucleic acid molecules

[0276] Synthesis of modified sequences of nucleic acid molecules

[0277] The nucleic acid molecules containing modifications were prepared using a Dr. 48 model DNA / RNA automatic synthesizer. The specific synthesis route can be referred to Example 25, and the sequences of the modified nucleic acid molecules are shown in Table 5:

[0278] Table 5 shows the sequence table of the modified nucleic acid molecules

[0279] 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, VP = vinyl phosphonate ribonucleotide; invAb is an inverted abasic deoxyribose residue.

[0280] Example 25 Preparation of double-stranded RNA conjugate

[0281] The double-stranded RNA conjugate was synthesized in this preparation example. The RNA conjugated in the double-stranded RNA conjugate has the sense strand and antisense strand sequences corresponding to Table 6, Table 7, and Table 8.

[0282] Synthesis of sense strand of double-stranded RNA conjugate

[0283] The nucleoside monomers were sequentially connected from 3'-5' direction according to the corresponding sense strand nucleotide arrangement sequence in Table 6, Table 7, and Table 8 by solid-phase phosphoramidite method. Each connection of a nucleoside monomer includes four steps of deprotection, coupling, capping, oxidation or sulfurization. When the phosphate linkage is used between two nucleotides, the connection of the next nucleoside monomer includes four steps of deprotection, coupling, capping, and oxidation. When the phosphorothioate linkage is used between two nucleotides, the connection of the next nucleoside monomer includes four steps of deprotection, coupling, capping, and sulfurization. The synthesis conditions are given as follows:

[0284] Commercial nucleoside monomers and compound (1-22) prepared by the above steps were dissolved in super-dry anhydrous acetonitrile to prepare a 0.05-0.1 M solution, and 4A molecular sieves were added. The conditions of the deprotection reaction of each step were the same, i.e. the temperature was 25°C, the reaction time was 70 seconds, the deprotection reagent was a dichloromethane solution of trichloroacetic acid (3% v / v), and the molar ratio of trichloroacetic acid to the 4,4'-dimethoxytrityl protecting group on the solid support was 150:1.

[0285] The conditions of each coupling reaction were the same, 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, and a reaction time of 300 seconds. The coupling reagent was a 0.3 M acetonitrile solution of 5-benzylthio-1H-tetrazole.

[0286] The conditions of each capping step were the same, including a temperature of 25°C and a reaction time of 30 seconds. The capping reagent solution was a mixture of CapA and CapB at a molar ratio of 1:1, and the molar ratio of the capping reagent to the nucleic acid sequence attached to the solid support was acetic anhydride:N-methylimidazole:nucleic acid sequence attached to the solid support = 100:100:1.

[0287] The conditions of each oxidation reaction were the same, including a temperature of 25°C, a reaction time of 30 seconds, and an oxidation reagent of 0.05 M aqueous iodine. The molar ratio of iodine to the nucleic acid sequence attached to the solid support in the coupling step was 30:1. The reaction was carried out in a mixed solvent of tetrahydrofuran:water:pyridine = 7:2:1.

[0288] The conditions of each sulfurization reaction were the same, including a temperature of 25°C, a reaction time of 300 seconds, and a sulfurization reagent of a 0.2 M pyridine solution of hydrogenated xanthate. The molar ratio of the sulfurization reagent to the nucleic acid sequence attached to the solid support in the coupling step was 120:1.

[0289] After the last nucleoside monomer was attached, 250 uL of a 20% diethylamine / acetonitrile solution was added to each column and allowed to drain for 5 minutes, and then completely drained. The washing was repeated twice more, for a total of 7500 uL and 15 minutes. The oligonucleotide / solid support was then washed with 3 x 300 uL of acetonitrile and dried by vacuum suction. The nucleic acid sample was purified using a preparative C18 chromatographic column using reverse-phase ion pair method. Specifically: eluent A: 100 mM TEAA aqueous solution, eluent B: 100 mM TEAA acetonitrile solution, elution gradient: eluent A: eluent B = 90:10-80:20 gradient elution. After the product eluate was collected, the purity was determined, and the nucleic acid samples with a purity meeting the requirements were combined. Salt exchange was then performed using a precipitation method. After the sample was concentrated to dryness, a sodium acetate aqueous solution and an ethanol solution were added, the precipitate was collected, and ethanol was used for washing.

[0290] Antisense strand of the double-stranded RNA conjugate

[0291] The antisense strand was synthesized by solid phase phosphoramidite method, using universal solid phase support to initiate the cycle, according to the order of nucleotide arrangement of the antisense strand in Table 6-8. The reaction conditions of deprotection, coupling, capping, oxidation or sulfuration in the solid phase synthesis method, cleavage and deprotection, purification and desalination conditions are the same as those of the synthesis of the sense strand.

[0292] 3. Deprotection

[0293] The synthesized nucleotide sequence with support was added to 25wt% ammonia solution, the amount of ammonia was 1ml / μmol, and the reaction was carried out at 35°C for 16 hours. The remaining support was removed by filtration, and the supernatant was vacuum concentrated to dryness.

[0294] 4. Purification and desalination

[0295] The nucleic acid was purified by gradient elution of NaBr using a preparative ion chromatography purification column (Source 15Q).

[0296] 5. Annealing and aliquot freezing

[0297] Equal molar sense and antisense strands were heated at 95°C for 2 minutes and slowly cooled to room temperature. Quantitative aliquot was prepared according to the requirements of the pharmacological department, and freeze-dried to solid powder, which was stored at -20°C for standby use.

[0298] The sequences of the sense and antisense strands of the double-stranded RNA conjugate are shown in Table 6, Table 7 and Table 8.

[0299] Table 6 is a double-stranded RNA conjugate

[0300] Table 7 is a double target sequence of TTR and ANGPTL3

[0301] Table 8 is a double target sequence of TTR and FVII (F7)

[0302] 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,

[0303] wherein example structures are as follows:

[0304] D1 structure is s-D1 structure is: D1-s structure is: s-D1-s structure is: wherein the structures of D1, D2A, D2B, D3, D4, D7, D8, D9A, D9B, D10, D11, D12, D13, D14, D15, D16, D17, D18, D19, D20, D21, D22 are shown in Table 3, which are linked to RNA via phosphate or phosphorothioate.

[0305] Example 26 In vivo efficacy of double-stranded RNA conjugates

[0306] To evaluate the in vivo efficacy of double-stranded RNA conjugates in Table 6, 6-8 week PCSK9 humanized mice (purchased from Janvier Labs) were fed with high-fat diet at least 2 weeks in advance, and were grouped according to the baseline value of low-density lipoprotein cholesterol level of the mice, 4 mice per group, and the grouping design is as follows:

[0307] A, vehicle group (3 mg / kg);

[0308] B, ANGPTL3 single-target siRNA compound (3 mg / kg) test group;

[0309] C. PCSK9 single target (Inclisiran) siRNA compound (3 mg / kg) test group;

[0310] D. Double-stranded RNA conjugate (6 mg / kg) test group in Table 6.

[0311] On day -1 (the day before administration), the pre-dose mouse plasma sample was obtained after 4 hours of fasting, to measure the baseline target protein and blood lipid levels. The siRNA compound of each group was diluted with normal saline, and then subcutaneously injected on day 1 according to the experimental grouping design, with a dose of 2 mg / kg, 3 mg / kg or 6 mg / kg, a concentration of 0.3 mg / mL or 0.6 mg / mL, and a volume of 10 mL / kg. The plasma samples were obtained after 4 hours of fasting on days 7, 14, 21, 28, 35, and 42. The ANGPTL3 and PCSK9 protein levels in the plasma were detected by ELISA according to the experimental protocol provided by the supplier (R&D Systems), and the inhibition rate was calculated according to the following formula. The results are shown in Tables 9 and 10.

[0312] Target protein inhibition rate (%) relative to the vehicle group = (1 - target protein expression level of a particular mouse / average of target protein expression levels of vehicle group mice) * 100

[0313] Inhibition rate (%) of a particular mouse target protein relative to baseline = (1 - expression level of a particular mouse target protein / baseline expression level of the corresponding target protein of the particular mouse) * 100

[0314] Table 9 is the in vivo test result of the RNA conjugate administered at 6 mg / kg in group D

[0315] Table 10 is the in vivo test result of the RNA conjugate administered at 2 mg / kg in group D

[0316] As shown in Tables 9 and 10, the double-stranded RNA conjugate of the present application has good inhibition activity on ANGPTL3 and PCSK9.

[0317] The four blood lipids in mouse plasma, namely total triglycerides (TG), total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C) levels were detected by a biochemical analyzer (Mindray) or a kit provided by the supplier (Nanjing Jiancheng). The detection results show that the double-stranded RNA conjugate of the present application has good inhibition activity on the four blood lipids in mice.

[0318] Example 27 In vivo efficacy in mice

[0319] To evaluate the in vivo efficacy of the RNA conjugates in Table 7, 6-8 week old male C57BL / 6 mice (purchased from Guangdong Vantons Lihua) were used for dosing and index detection. On the day before dosing, i.e. day -1, pre-dose mouse plasma samples were obtained, and the baseline expression levels of murine TTR (mTTR) and murine Angptl3 (mAngptl3) proteins were detected using kits provided by the suppliers (ALPCO and R&D). The mice were randomly grouped according to the baseline levels of mAngptl3 protein, with 4-5 mice per group. The siRNA compounds of each group were diluted with normal saline, and then subcutaneously injected on day 0 according to the experimental grouping design, with dosages of 1 mg / kg and 2 mg / kg, respectively, and the vehicle 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, and then the inhibition rates at specific time points were calculated according to the following formula:

[0320] mTTR inhibition rate (%) of a specific mouse relative to the baseline = (1 - mTTR expression level of the specific mouse / mTTR baseline expression level of the mouse) * 100

[0321] mAngptl3 inhibition rate (%) of a specific mouse relative to the vehicle group = (1 - mAngptl3 expression level of the specific mouse / mAngptl3 baseline expression level of the vehicle group) * 100, and the inhibition level of each compound on the target protein was taken as the average of the target protein inhibition rates of each group, and the results are shown in Table 11.

[0322] Table 11 is the in vivo test results at a dosage of 2 mg / kg

[0323] wherein A ≥ 50%, mTTR is the inhibition rate relative to the baseline, and ANGPTL3 is the inhibition rate relative to the vehicle group.

[0324] As can be seen from Table 11, the double-stranded RNA conjugates of the present application have good inhibition activity on mTTR and ANGPTL3.

[0325] Example 28 in vivo efficacy

[0326] To evaluate the in vivo efficacy of the RNA conjugates in Table 8, 6-8 week old male C57BL / 6 mice (purchased from Guangdong Vital River Laboratory Animal Technology Co., Ltd.) were used for the dosing and the index detection. The mice were randomly divided into 12 groups, 4 mice in each group. On the day of dosing (day 0), the compounds were dissolved in sterile saline in advance, and then the RNA conjugate solution or vehicle (sterile saline) was administered to the mice at a dose of 3 mg / kg or 6 mg / kg by subcutaneous injection. Two weeks after dosing (day 14), the human F7 (coagulation factor FVII) and Ttr gene expression in the liver of the mice was detected by RT-PCR. The expression level of the gene was evaluated by normalizing the relative expression level of F7 or Ttr mRNA of the corresponding mouse Gapdh mRNA, and the inhibition rate (relative to the vehicle group) of F7 or Ttr gene expression of a particular mouse was calculated by the following formula:

[0327] Inhibition rate (%) of F7 gene expression of a particular mouse = (1 - F7 mRNA relative expression level of the mouse / average of F7 mRNA relative level of the vehicle group) x 100

[0328] Inhibition rate (%) of Ttr gene expression of a particular mouse = (1 - Ttr mRNA relative expression level of the mouse / average of Ttr mRNA relative level of the vehicle group) x 100, and then the average inhibition rate of F7 and Ttr genes of the mice in each group was calculated, and the results are shown in Table 12.

[0329] Table 12 is the in vivo efficacy of the RNA conjugates administered at 6 mg / kg

[0330] wherein B≥60%.

[0331] As can be seen from Table 12, the double-stranded RNA conjugates of the present application have good inhibitory activity on mTtr and F7.

[0332] Example 29 In vivo efficacy in cynomolgus monkeys

[0333] To evaluate the in vivo efficacy of the RNA conjugates of the present application, male cynomolgus monkeys were used to test the in vivo activity of the RNA conjugates, and the specific operation was as follows: The serum samples of the cynomolgus monkeys were obtained one day before the administration of the RNA conjugates (day -1), and the expression levels of serum lipids (TC, TG, LDL-C and HDL-C), ANGPTL3 and PCSK9 were detected, and the measurement data on day -1 were taken as the baseline of each detection index.

[0334] Subsequently, the RNA conjugate was diluted with normal saline, and then subcutaneously injected at a dose of 2 mg / kg, 4 mg / kg, 6 mg / kg, 10 mg / kg or 20 mg / kg on day 0. Serum samples were obtained from the cynomolgus monkeys at 7, 14, 21, 28, 35, 42, 49, 56, 63 days after administration, and the expression levels of blood lipid four items, ANGPTL3 and PCSK9 were measured. The relative inhibition level of the RNA conjugate on the specific index of the cynomolgus monkey was calculated by the following formula:

[0335] Inhibition rate (%) relative to baseline = (1 - serum expression level of the specific cynomolgus monkey at a specific time point for a specific detection index / the baseline level of the index of the cynomolgus monkey) * 100%.

[0336] The inhibition rate of each group of cynomolgus monkeys was represented by the average value of the relative baseline inhibition rate of the animals in the group, and the results are shown in Tables 13 and 14.

[0337] Table 13 is the in vivo data of the RNA conjugate administered at 6 mg / kg on PCSK9

[0338] Table 14 is the in vivo data of the RNA conjugate administered at 6 mg / kg on ANGPTL3

[0339] Wherein, the inhibition rate C is greater than 40%.

[0340] As can be seen from Tables 13 and 14, the preferred RNA conjugate of the present application has good inhibition activity on PCSK9 and ANGPTL3.

[0341] The above examples are preferred embodiments of the present application, but the embodiments of the present application are not limited by the above examples, and any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present application should be equivalent replacement methods, and are all included in the protection scope of the present application.

Claims

1. A double stranded RNA conjugate, characterized in that, comprising: a first nucleic acid molecule, a second nucleic acid molecule, and a linker, the first nucleic acid molecule connecting the second nucleic acid molecule through the linker, the linker connecting the first nucleic acid molecule and the second nucleic acid molecule through a phosphoester (p) or a phosphorothioate (s), the linker being selected from the structures shown in Formula (I): said T is selected from -NH-C(O)-, or -C(O)-NH-; The R 1 selected from the group consisting of wherein y1, y2, y3 are R 1 the R 3 , R 4 together cyclize to a substituted said substituents are selected from the group consisting of a and b are the attachment positions to the first nucleic acid molecule or the second nucleic acid molecule; The R 2 selected from wherein said R 5 selected from substituted C 3-8 heterocycloalkyl, said substitution being selected from y4 is R 2 Connection positions, c, d, e are connection positions to the first nucleic acid molecule or the second nucleic acid molecule; wherein, said x, m, n, p, q, r are independently selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10.

2. The double stranded RNA conjugate of claim 1, wherein, The C 3-8 Cycloalkyl is selected from cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, and heterocycloalkyl means that a carbon atom in the cycloalkyl is replaced by a heteroatom selected from nitrogen, oxygen or sulfur.

3. The double stranded RNA conjugate of claim 1, wherein, the linker is selected from the group consisting of:

4. The double stranded RNA conjugate according to any one of claims 1 to 3, characterized in that, the carboxylic acid of said linker is connected to the ligand of asialoglycoprotein receptor ASGPR through an amide bond.

5. The double stranded RNA conjugate of claim 4, wherein, The ligand is selected from the group consisting of:

6. The double stranded RNA conjugate of claim 4, wherein, the compound wherein the carboxylic acid of said linker is connected to the ligand of asialoglycoprotein receptor ASGPR through an amide bond is selected from the structures shown in Table 1 and Table 3.

7. The double stranded RNA conjugate according to any one of claims 1 to 6, characterized in that, said first nucleic acid molecule is for inhibiting the expression of a first target gene, said second nucleic acid molecule is for inhibiting the expression of a second target gene, said first target gene and said second target gene are the same or different, and said first target gene and said second target gene are independently selected from PCSK9, ANGPTL3, LPA, apo3, AGT, Factor B, Factor C3, Factor MASP2, Factor PNP, HSD17B13, NR1H3, SOD1, HCV, HBV, TTR, FVII.

8. The double stranded RNA conjugate according to any one of claims 1 to 6, characterized in that, at least one nucleotide in said first nucleic acid molecule, second nucleic acid molecule is modified, said modification is selected from alkyl nucleotide, methoxy nucleotide, ethoxy nucleotide, methoxy ethyl nucleotide, amino nucleotide, fluorinated nucleotide, deoxy nucleotide, 5'-methylphosphonate nucleotide, 5'-C-methylphosphonate nucleotide, 2'-deoxy-2'-fluoro nucleotide, vinyl phosphonate nucleotide (VP), 5'-phosphate nucleotide (P), phosphorothioate nucleotide, phosphorodithioate nucleotide, locked nucleic acid (LNA), morpholino oligonucleotide (PMO), glycol nucleic acid modification (GNA), hypoxanthine modification (I), inverted abasic deoxyribose residue (invAb), 5'-methyldeoxycytosine nucleotide (m5dC).

9. A double-stranded RNA conjugate for inhibiting gene expression, characterized by, comprising: a linker, a first nucleic acid molecule for inhibiting the expression of a first target gene; and a second nucleic acid molecule for inhibiting the expression of a second target gene; wherein, said first nucleic acid molecule and said second nucleic acid molecule are connected through said linker, said linker connects said first nucleic acid molecule and said second nucleic acid molecule through a phosphonate (p) or a thiophosphonate (s), said linker is as described in any one of claims 1-9; wherein, said first target gene and second target gene are independently selected from PCSK9, ANGPTL3, LPA, apo3, AGT, Factor B, Factor C3, Factor MASP2, Factor PNP, HSD17B13, NR1H3, SOD1, HCV, HBV, TTR, FVII.

10. A compound, characterized in that, selected from the structures shown in Table 2 and compounds 1-22.

Images