Sirna nucleic acid conjugate, preparation method therefor, and use thereof

Abstract

The present invention relates to an siRNA nucleic acid conjugate, a preparation method therefor, and use thereof. The siRNA nucleic acid conjugate comprises: 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 by means of the linker, the linker is connected to the first nucleic acid molecule and the second nucleic acid molecule by means of phosphate (p) or phosphorothioate (s), and the linker is selected from the structure represented by formula (I):

Description

siRNA nucleic acid conjugates, their preparation methods and applications Technical Field

[0001] This invention relates to the field of medicine, and more specifically, to an siRNA nucleic acid conjugate, its preparation method, and its application. 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 an siRNA nucleic acid conjugate, its preparation method and application.

[0005] In a first aspect, the present invention provides an siRNA 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 The R 3 R 4 Together, they cyclize into substitution. The replacement 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... c, d, and e are the connection positions with the first or second nucleic acid molecule;

[0009] Wherein, x, m, n, p are independently selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, and q is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10.

[0010] As a preferred technical solution of the present invention, the C 3-8 The heterocyclic alkyl group is selected from cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl. The heterocyclic alkyl group refers to the cycloalkyl group in which the carbon atom is replaced by a heteroatom, which is selected from nitrogen, oxygen, or sulfur.

[0011] As a preferred embodiment of the present invention, the connector is selected from:

[0012] The connector includes a first connection site and a second connection site. The first connection site can connect to either a first nucleic acid molecule or a second nucleic acid molecule. The second connection site can connect to either a first nucleic acid molecule or a second nucleic acid molecule. For example, when the first connection site connects to the first nucleic acid molecule, the second connection site connects to the second nucleic acid molecule, or when the first connection site connects to the second nucleic acid molecule, the second connection site connects to the first nucleic acid molecule.

[0013] In a preferred embodiment of the present invention, the carboxylic acid of the linker is linked to the ligand of the desialyl glycoprotein receptor ASGPR via an amide bond.

[0014] As a preferred embodiment of the present invention, the compound in which the carboxylic acid of the linker is linked to the ligand of the desialyl glycoprotein receptor ASGPR via an amide bond is selected from the structure shown in formula (II):

[0015] Z 1 Z 2 Selected independently from N or CH;

[0016] Y is selected from alkyl, C 3-8 Cycloalkyl groups

[0017] L 1 L 2 L 3 Selected by oneself

[0018] W is selected from the target portion that can bind to cell surface receptors or the target portion containing a protective group;

[0019] Wherein, u1, u2, v, t1, and t3 are independently selected from 0, 1, 2, 3, 4, 5, and 6; t2 is independently selected from 1, 2, 3, 4, 5, and 6;

[0020] Among them, T and R1 R 2 x is as defined above.

[0021] As a preferred technical solution of the present invention, the L 1 L 2 L 3 Selected by oneself

[0022] As a preferred embodiment of the present invention, the ligand is selected from... The Selected from:

[0023] As a preferred embodiment of the present invention, the ligand Further selections from: The ligand Further selections from:

[0024] As a preferred embodiment of the present invention, the compound in which the carboxylic acid of the linker is linked to the ligand of the desialyl glycoprotein receptor ASGPR via an amide bond is selected from the structures shown in Tables 1 and 2.

[0025] As a preferred embodiment of the present invention, the compound in which the carboxylic acid of the linker is linked to the ligand of the desialyl glycoprotein receptor ASGPR via an amide bond is selected from compounds A8, A9, A10, D8, D9, and D10.

[0026] As a preferred embodiment of the present invention, the first nucleic acid molecule is used to inhibit the expression of the first target gene, 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 may be the same or different.

[0027] Among them, the first target gene and the second target gene can be independently selected from PCSK9, ANGPTL3, LPA, apo3, AGT, factor B, factor C3, factor MASP2, factor PNP, HSD17B13, NR1H3, SOD1, HCV, HBV, TTR, FVII, etc.

[0028] In a second aspect, the present invention provides an siRNA conjugate for inhibiting gene expression, comprising:

[0029] Connector

[0030] The first nucleic acid molecule, which is used to inhibit the expression of the first target gene, and

[0031] A second nucleic acid molecule, which is used to inhibit the expression of a second target gene, is connected to the first nucleic acid molecule and the second nucleic acid molecule through the adapter, which is connected by a phosphate ester (p) or a thiophosphate ester (s), as described above;

[0032] The first target gene and the 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, and FVII.

[0033] As a preferred embodiment of the present invention, the connector is selected from the structures shown in Tables 1 and 2, and preferably the structures of compounds A8, A9, A10, D8, D9, and D10.

[0034] Thirdly, the present invention provides an siRNA conjugate for inhibiting the expression of ANGPTL3 and PCSK9, comprising:

[0035] The first nucleic acid molecule, used to inhibit PCSK9 expression; and

[0036] The second nucleic acid molecule is used to inhibit the expression of ANGPTL3;

[0037] The first nucleic acid molecule and the second nucleic acid molecule are connected by the linker, which is a phosphate ester (p) or a thiophosphate ester (s) linking the first nucleic acid molecule and the second nucleic acid molecule, and the linker is selected from the structure shown in formula (I):

[0038] The T is selected from -NH-C(O)- or -C(O)-NH-;

[0039] The R 1 Selected from The R 3 R 4 Together, they cyclize into substitution. The replacement is selected from a and b are the connection positions with the first or second nucleic acid molecule;

[0040] 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... c, d, and e are the connection positions with the first or second nucleic acid molecule;

[0041] Wherein, x, m, n, p are independently selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, and q is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10.

[0042] As a preferred technical solution of the present invention, the C 3-8 The heterocyclic alkyl group is selected from cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl. The heterocyclic alkyl group refers to the cycloalkyl group in which the carbon atom is replaced by a heteroatom, which is selected from nitrogen, oxygen, or sulfur.

[0043] As a preferred embodiment of the present invention, the connector is selected from: B1′, B2′, B3′, B4′.

[0044] In a preferred embodiment of the present invention, the carboxylic acid of the linker is linked to the ligand of the desialyl glycoprotein receptor ASGPR via an amide bond.

[0045] As a preferred embodiment of the present invention, the compound in which the carboxylic acid of the linker is linked to the ligand of the desialyl glycoprotein receptor ASGPR via an amide bond is selected from the structure shown in formula (II).

[0046] Z 1 Z 2 Selected independently from N or CH;

[0047] Y is selected from alkyl, C 3-8 Cycloalkyl groups

[0048] L 1 L 2 L 3 Selected by oneself

[0049] W is selected from the target portion that can bind to cell surface receptors or the target portion containing a protective group;

[0050] Wherein, u1, u2, v, t1, and t3 are independently selected from 0, 1, 2, 3, 4, 5, and 6; t2 is independently selected from 1, 2, 3, 4, 5, and 6;

[0051] Among them, T and R 1 R 2 x is as defined above.

[0052] As a preferred embodiment of the present invention, the ligand is selected from... The Selected from:

[0053] As a preferred embodiment of the present invention, the compound in which the carboxylic acid of the linker is linked to the ligand of the desialyl glycoprotein receptor ASGPR via an amide bond is selected from the structures shown in Tables 1 and 2, with the structures of compounds A8, A9, A10, D8, D9, and D10 being preferred.

[0054] The present invention further provides compounds of siRNA conjugates selected from the structures shown in Tables 1 and 2, preferably compounds 1-10 and the structures shown in C1, C2, C3, C4, C5, C6, C7, C8, C9, and C10, and more preferably the structures shown in compounds 8, 9, 10, C8, C9, and C10.

[0055] In a preferred embodiment of the present invention, the first nucleic acid molecule comprises a sense strand and an antisense strand, and the second nucleic acid molecule comprises a sense strand and an antisense strand. The sense strand of the first nucleic acid molecule is connected to the sense strand of the second nucleic acid molecule via the nucleic acid adapter or covalent bond. For example, the nucleic acid adapter or covalent bond is connected to a nucleotide at the 5′ or 3′ end of the sense strand.

[0056] In a preferred embodiment of the present invention, the first nucleic acid molecule comprises a sense strand and an antisense strand, and the second nucleic acid molecule comprises a sense strand and an antisense strand. The antisense strand of the first nucleic acid molecule is connected to the antisense strand of the second nucleic acid molecule via the nucleic acid adapter or covalent bond. For example, the nucleic acid adapter or covalent bond is connected to a nucleotide at the 5′ or 3′ end of the antisense strand.

[0057] As a preferred embodiment of the present invention, the first nucleic acid molecule and the second nucleic acid molecule are selected from the sequences shown in Table 5.

[0058] In a preferred embodiment of the present invention, the positive strand of the first nucleic acid molecule and / or the second nucleic acid molecule is connected to a targeting ligand. For example, the nucleotide at the 5′ or 3′ end of the positive strand of the first nucleic acid molecule is connected to the targeting ligand, or the nucleotide at the 5′ or 3′ end of the positive strand of the second nucleic acid molecule is connected to the targeting ligand.

[0059] As a preferred embodiment of the present invention, at least one nucleotide in the double-stranded RNA conjugate is modified, wherein the modification is selected from: alkyl nucleotides, methoxy nucleotides, ethoxy nucleotides, methoxyethyl nucleotides, amino nucleotides, fluoronucleotides, deoxynucleotides, 5′-methylphosphonucleotides, 5′-C-methylphosphonucleotides, 2′-deoxy-2′-fluoronucleotides, vinylphosphonate nucleotides (VP), 5′-phosphonucleotides (P), thiophosphonucleotides, dithiophosphonucleotides, locked nucleic acids (LNA), morpholino oligonucleotides (PMO), ethylene glycol nucleic acid modification (GNA), hypoxanthine modification (I), reverse debasing deoxyribose residues (invAb), and 5′-methyldeoxycytosine nucleotides (m5dC).

[0060] As a preferred embodiment of the present invention, the siRNA conjugate is selected from the nucleotide sequences described in Tables 6-8.

[0061] Fourthly, the present invention also provides a compound selected from the structures shown in compounds 1-10, B1, B2, B3, B4, and C1-C10.

[0062] Fifthly, the present invention also provides a pharmaceutical composition for inhibiting the expression of ANGPTL3 and PCSK9 genes, said pharmaceutical composition comprising the siRNA conjugate as described above.

[0063] As a preferred embodiment of the present invention, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient.

[0064] In a sixth aspect, the present invention also provides the use of the above-described double-stranded RNA conjugate in the preparation of a treatment for diseases, conditions or symptoms mediated at least in part by the expression of ANGPTL3 and / or PCSK9 genes, TTR and / or ANGPTL3, TTR and / or FVII.

[0065] The double-stranded RNA conjugates provided by this invention, which inhibit the expression of ANGPTL3 and PCSK9, TTR and ANGPTL3, and TTR and FVII, respectively, exhibit good inhibitory activity against ANGPTL3 and PCSK9, TTR and ANGPTL3, and TTR and FVII, and can be used to prevent and / or treat diseases mediated by the expression of ANGPTL3 and / or PCSK9 genes, TTR and / or ANGPTL3 genes, and TTR and / or FVII genes. Detailed Implementation

[0066] The present invention will be further described in detail below with reference to embodiments, but the implementation of the invention is not limited thereto.

[0067] 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.

[0068] 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.

[0069] 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.

[0070] 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.

[0071] 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.

[0072] The term "ANGPTL3" refers to angiopoietin-like protein 3 (NM_014495,4), a secreted protein 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 a significant factor contributing to lipid metabolism dysregulation. It has multiple potential points of action. Loss-of-function mutations in ANGPTL3 can lead to decreased LDL-C, VLDL-C, HDL-C, and triglyceride (TG) levels, thereby reducing the risk of cardiovascular disease based on GWAS, without any known adverse phenotypes of genetic defects. Therefore, inhibiting ANGPTL3 activity can effectively prevent or treat dyslipidemia.

[0073] The nucleic acid molecule used to inhibit ANGPTL3 gene expression in this invention can be selected from the nucleic acid sequences disclosed in the patent applications or publication numbers: WO2021188795A1, CN201480068063.9, CN201280041047.1, CN201680034444.4, CN201880048600.1, CN202180007277.5, CN201880073901.X, WO2020099482A2, WO2022068923A1, WO2022187435A1, WO2022268054A1, CN202210705962.1, WO2023045994A1, and WO2023134705A1.

[0074] In this invention, the phrase "inhibit ANGPTL3 expression" includes inhibiting the expression of any ANGPTL3 gene (e.g., mouse ANGPTL3 gene, rat ANGPTL3 gene, monkey ANGPTL3 gene, or human ANGPTL3 gene) along with variants or mutants of the ANGPTL3 gene encoding the ANGPTL3 protein.

[0075] The phrase "inhibit PCSK9 expression" includes inhibiting the expression of any PCSK9 gene (such as, for example, the mouse PCSK9 gene, rat PCSK9 gene, monkey PCSK9 gene, or human PCSK9 gene) and variants (e.g., naturally occurring variants) or mutants of the PCSK9 gene. Therefore, the PCSK9 gene can be a wild-type PCSK9 gene, a mutant PCSK9 gene, or, in the case of genetically manipulated cells, cell groups, or organisms, a transgenic PCSK9 gene.

[0076] "Inhibition of ANGPTL3 and PCSK9 expression" includes inhibition of the ANGPTL3 and PCSK9 genes at any level, such as at least partially stopping the expression of the ANGPTL3 and PCSK9 genes, for example, 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 this invention, nucleic acid can refer to double-stranded siRNA or single-stranded ASO. In a double-stranded nucleic acid molecule, the bases on one strand pair with the bases on 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 consists of one purine and one pyrimidine. When adenine on one strand always pairs with uracil on the other strand, and guanine always pairs with cytosine, the two strands are considered complementary, and the sequence of the complementary strand can be inferred from its sequence.

[0077] In this invention, the modified nucleotides include, but are not limited to: alkyl nucleotides, methoxy nucleotides, ethoxy nucleotides, methoxyethyl nucleotides, amino nucleotides, fluoronucleotides, deoxynucleotides, 5′-methylphosphonucleotides, 5′-C-methylphosphonucleotides, 2′-deoxy-2′-fluoronucleotides, vinylphosphonate nucleotides (VP), thiophosphonucleotides, dithiophosphonucleotides, locked nucleic acids (LNA), morpholino oligonucleotides (PMO), and inverse debased deoxyribose residues (invAb).

[0078] Among them, alkyl-modified nucleotides, such as 2′-methyl nucleotides, 2′-Ethylnucleotide, The nucleotide modified with 2′-methoxy group has the following structure: 2′-Methoxyethyl nucleotide, with the following structure: 2′-Methoxyethoxynucleotide, with the following structure: 2′-Fluoronucleotide, with the following structure: 5′-C-methylphosphonucleotide, with the following structure:

[0079] (E)-vinylphosphonate deoxyribonucleotide (VP), with the following structure: Phosphonucleotide (p), structure: Phosphophosphate nucleotide (S), with the following structure: 2′-Deoxyribonucleotide, with the following structure: The reverse debasing deoxyribose residue (invAb) has the following structure: Glycol nucleic acids (GNAs), including (S)-glycol nucleic acids ((S)-GNAs), have the following structure: And (R)-ethylene glycol nucleic acid ((R)-GNA), the structure is: Inosine monophosphate (I) has the following structure: 5-Methyldeoxycytosine nucleotide (m5dC), where m5dC is 2'-deoxy-5-methylcytidine-3'-phosphate.

[0080] Where Base represents a base, R represents an alkyl or alkoxy group, Me represents a methyl group, and Et represents an ethyl group.

[0081] The term "locked nucleic acid" is a nucleotide with a modified ribose moiety, wherein the ribose moiety includes an additional bridge connecting the 2′ and 4′ carbons. This structure effectively "locks" the ribose in a 3′-endonucleotide conformation. Adding locked nucleic acids to siRNA has been shown to increase the stability of siRNA in serum and reduce off-target effects (Elmen, J. et al., (2005) Nucleic Acids Research 33(1): 439-447; Mook, OR. et al., (2007) MolCancTher 6(3): 833-843; Grunweller, A. et al., (2003) Nucleic Acids Research 31(12): 3185-3193).

[0082] Representative U.S. patents for 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.

[0083] The structure of the locked nucleic acid is as follows:

[0084] In some embodiments, the sugar substitute comprises a ring having more than 5 atoms and more than 1 heteroatom. For example, nucleosides comprising morpholine sugar moieties and their use in oligomeric compounds have been reported (see, for example: 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).

[0085] The term "morpholino" refers to a sugar substitute having the following formula:

[0086] In some embodiments, the morpholino group can be modified, for example, by adding or changing various substituents according to the morpholino structure described above. Such sugar substitutes are referred to herein as "modified morpholino groups".

[0087] In this invention, unless otherwise specified, uppercase letters C, G, U, and A represent the base composition of nucleotides. Lowercase letter m indicates that the nucleotide adjacent to the left of letter m is methoxylated; lowercase letter f indicates that the nucleotide adjacent to the left of letter f is fluorinated; LNA indicates that the nucleotide adjacent to its right is locked nucleic acid (LNA); lowercase letter s indicates that the two nucleotides to the left and right of this letter are linked by a phosphate thioester group; VP indicates that the nucleotide to the right of letter VP is (E)-vinyl phosphate modified. invAb represents an inverted debased deoxyribonucleotide; dN represents any deoxyribonucleotide; dA represents deoxyadenine nucleotide; dT represents deoxythymidine nucleotide; dU represents deoxyurine nucleotide; dC represents deoxycytosine nucleotide; and dG represents deoxyguanine nucleotide.

[0088] It should be emphasized that the “modification” of nucleotides described in this disclosure includes, but is not limited to, the examples above. Nucleotides may also be replaced with other nucleotides, such as (S)-glycerol nucleic acids.

[0089] The term "targeting ligand" can include naturally occurring substances such as proteins (e.g., human serum albumin (HAS), low-density lipoprotein (LDL), or globulins); carbohydrates (e.g., dextran, styrax, chitosan, chitosan, inulin, cyclodextrin, N-acetylglucosamine, N-acetylglucosamine, or hyaluronic acid); or lipids. Ligands can also be recombinant or synthetic molecules, such as synthetic polymers, for example, synthetic polyamino acids. Examples of polyamino acids include the following: polylysine (PLL), poly-L-aspartic acid, poly-L-glutamic acid, styrene-maleic anhydride copolymer, poly(L-lactide-co-glycolic acid) 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, peptide-mimicking polyamine, dendritic polyamine, arginine, amidine, protamine, cationic lipids, cationic porphyrins, quaternary salts of polyamines, or α-helical peptides.

[0090] Targeting ligands can also be cell or tissue-targeting agents that bind to specific cell types, such as kidney cells, for example, lectins, glycoproteins, lipids, or proteins, such as antibodies. Targeting groups can be thyroid-stimulating hormone, melanocyte-stimulating hormone, lectins, glycoproteins, surfactant protein A, mucin carbohydrates, polylactose, polygalactose, N-acetylgalactosamine, N-acetylglucosamine, polymannose, polyfucose, glycosylated polyamino acids, polygalactose, transferrin, bisphosphonates, polyglutamic acid, polyaspartic acid, lipids, cholesterol, steroids, bile acids, folic acid, vitamin B12, vitamin A, biotin, or RGD peptides or RGD peptide mimics.

[0091] Targeting ligands can also be proteins, such as glycoproteins, or peptides, such as molecules with specific affinity for co-ligands, or antibodies, such as antibodies that bind to a specific cell type, such as hepatocytes. Ligands can also include hormones and hormone receptors. They can also include non-peptide species, such as lipids, lectins, carbohydrates, vitamins, cofactors, polylactose, polygalactose, N-acetylgalactosamine, N-acetylglucosamine, polymannose, or polyfucose. Ligands can be, for example, lipopolysaccharides, activators of p38MAP kinase, or activators of NF-κB.

[0092] Targeting ligands can be substances that can increase the uptake of iRNA into cells, for example, by disrupting the cell's cytoskeleton (e.g., by disrupting cellular microtubules, microfilaments, and / or intermediate filaments), such as drugs. Drugs can be, for example, taxon, vincristine, vinblastine, pinocembrin, nocodazole, iaplakinolide, red sea sponge A, phalloidin, swinholide A, indanocine, or myoservin.

[0093] The term "pharmaceuticalally acceptable excipient" refers to a substance other than the active pharmaceutical ingredient (API, therapeutic product, such as ANGPTL3) that is intentionally included in a drug delivery system. Excipients are not intended, nor are they designed, to exert a therapeutic effect at the intended dose. Excipients may serve to: a) facilitate handling of the drug delivery system during preparation; b) protect, support, or enhance the stability, bioavailability, or patient acceptability of the API; c) facilitate product identification; and / or d) enhance any other properties of the API, including overall safety, efficacy, or delivery, during storage or use.

[0094] The excipients include (but are not limited to): absorption enhancers, anti-sticking agents, defoamers, antioxidants, adhesives, buffers, carriers, coating agents, colorants, delivery enhancers, delivery polymers, detergents, dextran, dextran, diluents, disintegrants, emulsifiers, swelling agents, fillers, flavoring agents, flow aids, wetting agents, oils, polymers, preservatives, brine, salts, solvents, sugars, surfactants, suspending agents, sustained-release matrices, sweeteners, thickeners, tensioning agents, mediators, waterproofing agents, wetting agents, lubricants such as sodium lauryl sulfate and magnesium stearate, flavoring agents, and fragrances.

[0095] The pharmaceutical compositions disclosed herein include those suitable for oral, nasal, topical, sublingual, sublingual, rectal, and / or parenteral administration. The formulations can be conveniently present in unit dosage form and can be prepared by any method known in the pharmaceutical field. The amount of active ingredient that can be combined with a carrier substance to prepare a single-dose form is generally the amount of the compound that produces the therapeutic effect. Generally, in percent units, this amount is from about 1% to about 99% of the active ingredient, preferably from about 5% to about 70%, and most preferably from about 10% to about 30%.

[0096] The term "blood lipids" refers to any major lipids present in the blood. Blood lipids can exist in the blood in their free form or as part of protein complexes, such as lipoprotein complexes. Non-limiting examples of blood lipids may 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).

[0097] The term "dysregulation of lipid metabolism" refers to any disorder that is associated with or caused by disturbances in lipid metabolism. For example, the term includes any disorder, disease, or condition that can lead to hyperlipidemia, or a condition characterized by abnormally elevated levels of any or all lipids and / or lipoproteins in the blood. The term refers to hereditary disorders, such as familial hypertriglyceridemia, or acquired disorders, such as those acquired due to diet or the use of certain medications. Exemplary lipid metabolism disorders include, but are not limited to, arteriosclerosis, dyslipidemia, hypertriglyceridemia (including drug-induced hypertriglyceridemia, diuretic-induced hypertriglyceridemia, alcohol-induced hypertriglyceridemia, β-adrenergic blocker-induced hypertriglyceridemia, estrogen-induced hypertriglyceridemia, glucocorticoid-induced hypertriglyceridemia, retinoid-induced hypertriglyceridemia, metformin-induced hypertriglyceridemia, and familial hypertriglyceridemia), acute pancreatitis associated with hypertriglyceridemia, chylomicron syndrome, familial chylomicronemia, Apo-E deficiency or resistance, LPL deficiency or decreased activity, hyperlipidemia (including familial complex hyperlipidemia), hypercholesterolemia, gout associated with hypercholesterolemia, and xanthoma (subcutaneous cholesterol deposition).

[0098] The term "treatment" is used to refer to achieving a desired pharmacological and / or physiological effect. This effect may be preventative in terms of complete or partial prevention of disease or its symptoms, and / or therapeutic in terms of partial or complete cure of disease and / or adverse effects caused by disease. As used herein, "treatment" covers diseases in mammals, particularly humans, including: (a) prevention of disease or the onset of disease in individuals susceptible to disease but not yet diagnosed with it; (b) inhibition of disease, such as blocking disease progression; or (c) relief of disease, such as reducing disease-related symptoms. As used herein, "treatment" encompasses any administration of a drug or compound to an individual to treat, cure, relieve, improve, reduce, or inhibit the individual's disease, including but not limited to administration of a drug containing a compound described herein to an individual in need.

[0099] The term “inhibition” is used interchangeably with “reduction,” “silence,” “downregulation,” “stop,” and other similar terms and includes any level of inhibition.

[0100] ANGPTL3 gene expression can be assessed based on the level of any variable associated with ANGPTL3 gene expression, such as ANGPTL3 mRNA level or ANGPTL3 protein level. ANGPTL3 expression can also be indirectly assessed based on the levels 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 baseline levels before administration or levels determined from similar subjects, cells, or samples that have never been treated or have been treated with a control (e.g., a buffer-only control or a non-active agent control).

[0101] "PCSK9-related diseases" are intended to include any disease associated with the PCSK9 gene or protein. Such diseases can be caused, for example, by an overproduction of the PCSK9 protein, mutations in the PCSK9 gene, abnormal cleavage of the PCSK9 protein, or abnormal interactions between PCSK9 and other proteins or other endogenous or exogenous substances. Exemplary PCSK9-related diseases include lipemias, such as hyperlipidemia, and other forms of lipid imbalances, such as hypercholesterolemia, hypertriglyceridemia, and pathological conditions associated with these imbalances, such as heart and circulatory system diseases.

[0102] Detailed Implementation

[0103] Preparation of intermediate 1 (B2)

[0104] The specific synthesis route is as follows:

[0105] Step A: Synthesis of 1,5-dioxane-2,8-dione

[0106] Under nitrogen protection at 0°C, N,N-dicyclohexylcarboimide (8.40 g, 40.71 mmol, 8.23 ​​mL) was added to a solution of 3-(2-carboxyethoxy)propionic acid (6 g, 37.01 mmol) in ethyl acetate (60 mL). The reaction mixture was stirred at room temperature for 2 hours until the reaction was substantially complete as monitored by TLC.

[0107] The reaction solution was filtered, and the filtrate was concentrated under vacuum to obtain 5.33 g of a light yellow oily product, 1,5-dioxane-2,8-dione.

[0108] Step B: Synthesis of 3-(3-methoxy-3-oxypropoxy)propionic acid

[0109] At room temperature, 5.33 g (36.98 mmol) of 1,5-dioxane-2,8-dione and sodium methoxide solution (5.4 mol / L, 136.97 μL) were dissolved in 50 mL of methanol. The reaction mixture was purged with nitrogen three times, the temperature was raised to 70 °C, and the mixture was stirred for 1 hour under a nitrogen atmosphere until the reaction was substantially complete as monitored by TLC.

[0110] The reaction was cooled to room temperature, and the reaction solution was concentrated under vacuum. The crude product was purified by column chromatography (eluent: petroleum ether / ethyl acetate = 1 / 0-1 / 1) to give 3.10 g of the yellow oily product 3-(3-methoxy-3-oxypropoxy)propionic acid.

[0111] Step C: Synthesis of methyl 3-[3-[(2S)-2-(hydroxymethyl)pyrrolidone-1-yl]-3-oxopropoxy]propionate

[0112] Under nitrogen protection at room temperature, 3-(3-methoxy-3-oxypropoxy)propionic 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), and 1-hydroxybenzotriazole (4.76 g, 35.19 mmol) were dissolved in dichloromethane (5 mL), cooled to 0°C, and stirred for 0.5 h. Then, a dichloromethane solution (5 mL) of L-prolyl (1.78 g, 17.6 mmol) was slowly added to the reaction system, and the mixture was brought back to room temperature and stirred for 2 h until the reaction was complete as monitored by TLC.

[0113] The reaction mixture was concentrated under reduced pressure to remove the solvent. It was diluted with 60 mL of water and then extracted with dichloromethane (50 mL × 3 times). The combined organic phases were washed with saturated brine (50 mL × 2 times), 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 / 0-0 / 1) to give 2.73 g of a yellow oil, methyl 3-[3-[(2S)-2-(hydroxymethyl)pyrrolidine-1-yl]-3-oxopropoxy]propionate.

[0114] Step D: Synthesis of methyl 3-[3-[(2S)-2-[[bis(4-methoxyphenyl)-phenylmethoxy]methyl]pyronin-1-yl]-3-oxypropoxy]propionate

[0115] Under zero temperature and nitrogen protection, 4-dimethylaminopyridine (105.07 mg, 860.01 μmol) and pyridine (3.40 g, 43.00 mmol, 3.47 mL) and 1-[chloro-(4-methoxyphenyl)-phenylmethyl]-4-methoxybenzene (3.50 g, 10.32 mmol) were added to a solution of methyl 3-[3-[(2S)-2-(hydroxymethyl)pyrrolidine-1-acyl]-3-oxypropoxy]propionate (2.23 g, 8.60 mmol) in dichloromethane (20 mL). The mixture was stirred at room temperature for 2 hours until the reaction was complete as monitored by LC-MS and TLC.

[0116] The reaction solution was diluted with water (50 mL) and then extracted with dichloromethane (80 mL). The combined organic phases were washed with saturated sodium bicarbonate solution (50 mL × 2 times) and saturated brine (50 mL × 2 times), dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum to obtain the crude product. The crude product was purified by column chromatography (eluent: petroleum ether / ethyl acetate = 1 / 0-1 / 1) to give 6.2 g of a yellow oily product, methyl 3-[3-[(2S)-2-[[bis(4-methoxyphenyl)-phenylmethoxy]methyl]pyronan-1-yl]-3-oxypropoxy]propionate. LC-MS: RT = 0.750 min, [M+H] + =562.3.

[0117] Step E: Synthesis of 3-[3-[(2S)-2-[[bis(4-methoxyphenyl)-phenylmethoxy]methyl]pyrrolidine-1-yl]-3-oxypropoxy]propionic acid

[0118] At room temperature, methyl 3-[3-[(2S)-2-[[bis(4-methoxyphenyl)-phenylmethoxy]methyl]pyronin-1-yl]-3-oxypropoxy]propionate (5.70 g, 10.15 mmol) was dissolved in tetrahydrofuran (45 mL) and water (15 mL), cooled to 0°C, and then lithium hydroxide monohydrate (511 mg, 12.18 mmol) was added. The mixture was then brought back to room temperature and stirred for 2 hours until the reaction was complete as monitored by LC-MS.

[0119] The reaction solution was concentrated to give 4.88 g of the product 3-[3-[(2S)-2-[[bis(4-methoxyphenyl)-phenylmethoxy]methyl]pyrrolid-1-yl]-3-oxypropoxy]propionic acid. LC-MS: RT = 0.456 min, [MH] + =546.5.

[0120] Step F: Synthesis of methyl(2S)-2-(9H-fluorene-9-ylmethoxycarbonylamino)-6-[(4-hydroxycyclohexanecarbonyl)amino]hexanoate

[0121] Under nitrogen protection, 26.25 g (182 mmol) of 4-hydroxycyclohexylcarboxylic acid, 70.6 g (546 mmol, 95.14 mL) of diisopropylethylamine, 45.38 g (236.7 mmol) of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, and 31.98 g (236 mmol) of 1-hydroxybenzotriazole were dissolved in 840 mL of dichloromethane and cooled to 0°C. Then, 83.9 g (200.29 mmol) of methyl(2S)-6-amino-2-(9H-fluorene-9-ylmethoxycarbonylamino)hexanoate was added to the reaction mixture, and the mixture was brought back to room temperature and stirred for 2 hours until the reaction was complete as monitored by LC-MS.

[0122] The reaction mixture was concentrated under reduced pressure to remove the solvent. It was diluted with 400 mL of water and then extracted with dichloromethane (500 mL × 2 times). The combined organic phases were washed with saturated brine (300 mL × 2 times), 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 / 0-0 / 1) to give 11.5 g (98.8% purity) methyl(2S)-2-(9H-fluorene-9-ylmethoxycarbonylamino)-6-[(4-hydroxycyclohexanecarbonyl)amino]hexanoate. LC-MS: RT = 0.600 min, [M+H] + =509.3. 32.5 g (92.6% purity) of a yellow solid product, methyl (2S)-2-(9H-fluorene-9-ylmethoxycarbonylamino)-6-[(4-hydroxycyclohexanecarbonyl)amino]hexanoate. LC-MS: RT = 0.604 min, [M+H] + =509.3.

[0123] Step G: Synthesis of methyl(2S)-2-amino-6-[(4-hydroxycyclohexanecarbonyl)amino]hexanoate

[0124] Under nitrogen protection, ethylenediamine (32.36 g, 442.39 mmol, 45.57 mL) was added to a tetrahydrofuran (500 mL) solution of methyl (2S)-2-(9H-fluorene-9-ylmethoxycarbonylamino)-6-[(4-hydroxycyclohexanecarbonyl)amino]hexanoate (45 g, 88.48 mmol). The mixture was stirred at room temperature for 2 hours until the reaction was complete as monitored by LC-MS.

[0125] The reaction mixture was filtered, and the filter cake was concentrated under reduced pressure to remove the solvent. 6.7 g (91% purity) of a pale yellow oil, methyl(2S)-2-amino-6-[(4-hydroxycyclohexanecarbonyl)amino]hexanoate, was given. LC-MS: RT = 0.296 min, [M+H] +=287.1. 17.0 g (89.7% purity) yellow oily product methyl (2S)-2-amino-6-[(4-hydroxycyclohexanecarbonyl)amino]hexanoate.

[0126] Step H: Synthesis of methyl(2S)-2-[3-[3-[3-[(2S)-2-[[bis(4-methoxyphenyl)-phenylmethoxy]methyl]pyronin-1-yl]-3-oxypropoxy]propionamide]-6-[(4-hydroxycyclohexyl)amino]hexanoate

[0127] Under nitrogen protection, 3-[3-[(2S)-2-[[bis(4-methoxyphenyl)-phenylmethoxy]methyl]pyrrolidine-1-yl]-3-oxypropoxy]propionic acid (4.68 g, 8.55 mmol), diisopropylethylamine (3.31 g, 25.64 mmol, 4.47 mL), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (3.28 g, 17.09 mmol), and 1-hydroxybenzotriazole (2.31 g, 17.09 mmol) were dissolved in dichloromethane (50 mL), cooled to 0°C, and stirred for 0.5 h. Subsequently, methyl(2S)-2-amino-6-[(4-hydroxycyclohexanecarbonyl)amino]hexanoate (2.94 g, 10.25 mmol) was added to the reaction system, and the mixture was then brought to room temperature and stirred for 2 hours until the reaction was complete as monitored by LC-MS.

[0128] The reaction mixture was concentrated under reduced pressure to remove the solvent. It was diluted with 100 mL of water and then extracted with ethyl acetate (100 mL × 3 times). The combined organic phases were washed with saturated sodium bicarbonate solution (100 mL × 2 times) and saturated brine (75 mL × 2 times), dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum to obtain the crude product. The crude product was purified by reversed-phase chromatography (column: 330 g Flash Column Welch Ultimate XB_C1820-40 μm; 120 A, 0.1% ammonia system) to give 3.6 g of methyl(2S)-2-[3-[3-[3-[(2S)-2-[[bis(4-methoxyphenyl)-phenylmethoxy]methyl]pyronin-1-yl]-3-oxypropoxy]propionamide]-6-[(4-hydroxycyclohexyl)amino]hexanoate. LC-MS: RT = 0.661 min, [MH] + =814.5.

[0129] Step I: Synthesis of (2S)-2-[3-[3-[(2S)-2-[[bis(4-methoxyphenyl)-phenylmethoxy]methyl]pyrrolidone-1-yl]-3-oxypropoxy]propionamide]-6-[(4-hydroxycyclohexyl)amino]hexanoic acid (intermediate 1)

[0130] At room temperature, methyl(2S)-2-[3-[3-[3-[(2S)-2-[[bis(4-methoxyphenyl)-phenylmethoxy]methyl]pyronin-1-yl]-3-oxypropoxy]propionamide]-6-[(4-hydroxycyclohexyl)amino]hexanoate (3.60 g, 4.41 mmol) was dissolved in tetrahydrofuran (30 mL) and water (10 mL), cooled to 0°C, and then lithium hydroxide monohydrate (222 mg, 5.29 mmol) was added. The mixture was then brought back to room temperature and stirred for 2 hours until the reaction was complete as monitored by LC-MS.

[0131] The reaction solution was diluted with 20 mL of water and then extracted with ethyl acetate (50 mL × 2 times). The aqueous phase was lyophilized to remove water, yielding 3.26 g of the (lithium salt) product (2S)-2-[3-[3-[(2R)-2-[[bis(4-methoxyphenyl)-phenylmethoxy]methyl]pyrrolidone-1-yl]-3-oxypropoxy]propionamide]-6-[(4-hydroxycyclohexyl)amino]hexanoic acid. LC-MS: RT = 0.450 min, [MH] + =800.3, SFC:RT=1.480min. 1 HNMR(400MHz,DMSO-d6)δ7.68(br t,J=5.60Hz,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).

[0132] Preparation of intermediate 2 (B4)

[0133] The specific synthesis route is as follows:

[0134] Step A: Synthesis of benzyl(S)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)pyrrolidine-1-carboxylate

[0135] At room temperature, 4-dimethylaminopyridine (0.52 g, 4.25 mmol) and pyridine (16.81 mg, 212.5 mmol) were added sequentially to 100 mL of dichloromethane containing benzyl(S)-2-(hydroxymethyl)pyrrolidine-1-carboxylate (10.0 g, 42.50 mmol). At 0°C, 4,4'-bismethoxytriphenylmethyl chloride (17.28 g, 51.0 mmol) was added, and the reaction was allowed to proceed overnight at room temperature.

[0136] After the reaction was complete, the solvent was evaporated, the solution was diluted with dichloromethane, washed twice with saturated sodium bicarbonate aqueous solution, and once with saturated brine. The solution was then dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: n-hexane / ethyl acetate = 3 / 1, containing 0.5% triethylamine) to give 20.0 g of benzyl(S)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)pyrrolidine-1-carboxylate. LCMS: RT = 2.44 min, [M + Na] + =560.0.

[0137] Step B: Synthesis of (S)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)pyrrolidine

[0138] At room temperature, benzyl(S)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)pyrrolidine-1-carboxylate (20.0 g, 37.2 mmol) was dissolved in tetrahydrofuran (500 mL), wet palladium on carbon (2.0 g) was added, hydrogen was purged twice, and the reaction was carried out at 45°C for 3 hours under a hydrogen atmosphere.

[0139] After the reaction was complete, hydrogen was replaced with nitrogen, palladium on carbon was removed by filtration, and the mixture was concentrated to give 15.0 g of (S)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)pyrrolidine. LC-MS: RT = 1.77 min, [M + Na] + =426.3.

[0140] Step C: Synthesis of 3-[3-[(2S)-2-[[bis(4-methoxyphenyl)-phenylmethoxy]methyl]pyrrolidine-1-yl]-3-oxypropoxy]propionic acid

[0141] Under nitrogen protection, DIEA (47.8 g, 370 mmol) was added to 240 mL of dichloromethane containing 12 g (74 mmol), followed by the addition of EDCI (18.44 g, 96.21 mmol) and HOBt (14.73 g, 96.21 mmol) in portions, and the mixture was stirred for 0.5 h. Subsequently, a solution of (S)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)pyrrolidine (29.86 g, 74 mmol) in 60 mL of dichloromethane was added dropwise, and the mixture was stirred overnight.

[0142] The reaction mixture was concentrated under reduced pressure to remove the solvent. EA was added, and the mixture was washed twice with saturated NaCl:H₂O at a ratio of 1:1. The combined aqueous phases were extracted once with EA. The combined organic phases were then added to an aqueous solution of K₃PO₄ and stirred for 30 minutes (the amount of water should be twice the volume of water capable of dissolving potassium phosphate). After settling, three layers slowly formed, with the product mainly in the middle layer; sometimes only two layers were separated, requiring readjustment of the water volume to form three layers). The organic phase was discarded after separation. The pH of the aqueous phase was adjusted to approximately 6 with solid citric acid. The aqueous phase was extracted with EA, and the organic phase was dried. After rotary evaporation, 16.2 g of a colorless colloidal substance, 3-[3-[(2S)-2-[[bis(4-methoxyphenyl)-phenylmethoxy]methyl]pyrrolidine-1-yl]-3-oxypropoxy]propionic acid, was obtained. LC-MS: RT = 2.10 min, [MH] - =545.79.

[0143] Step D: Synthesis of methyl(1R,3R)-3-(2-(benzyloxy)ethoxy)cyclobutane-1-carboxylic acid ester

[0144] At room temperature, methyl trans-3-hydroxycyclobutane carboxylate (5.0 g, 38.42 mmol) was dissolved in tetrahydrofuran (50.0 mL), and sodium hydrogen (1.84 g, 76.84 mmol) was slowly added under ice bath conditions while stirring for 30 minutes. Finally, benzyl 2-bromoethyl ether (9.92 g, 46.10 mmol) was added, and the mixture was reacted overnight at room temperature.

[0145] After the reaction was complete, the reaction system was quenched dropwise with 1M hydrochloric acid aqueous solution at 0°C. Extraction was performed with ethyl acetate (50 mL × 2 times). The organic phases were combined, washed twice with saturated sodium bicarbonate and twice with saturated sodium chloride, dried, and purified by rotary evaporation (hexane / ethyl acetate = 20 / 1) to give 4.5 g of an oily product, methyl(1R,3R)-3-(2-(benzyloxy)ethoxy)cyclobutane-1-carboxylic acid ester. LCMS: RT = 2.21 min, [M + H] + =265.17.

[0146] Step E: Synthesis of (1R,3R)-3-(2-(benzyloxy)ethoxy)cyclobutane-1-carboxylic acid

[0147] 4.5 g (16.98 mmol) of methyl (1R,3R)-3-(2-(benzyloxy)ethoxy)cyclobutane-1-carboxylic acid ester was dissolved in a mixed solvent of tetrahydrofuran and water (4 / 1) (27.0 mL), and then lithium hydroxide monohydrate (1.4 g, 33.96 mmol) was added. The mixture was reacted at room temperature for 2 hours.

[0148] After the reaction was complete, it was quenched with 2N hydrochloric acid aqueous solution, and then extracted with ethyl acetate (30 mL × 2 times). The organic phases were combined, washed twice with saturated sodium chloride, dried, and then directly evaporated to dryness to give 3.0 g of (1R,3R)-3-(2-(benzyloxy)ethoxy)cyclobutane-1-carboxylic acid. LCMS: RT = 1.98 min, [M + H] + =251.15.

[0149] Step F: Synthesis of methyl N 2 -(((9H-fluorene-9-yl)methoxy)carbonyl)-N 6 -((1R,3S)-3-(2-(benzyloxy)ethoxy)cyclobutane-1-carbonyl)-L-lysine ester

[0150] At room temperature, (S)-2-((((9H-fluorene-9-yl)methoxy)carbonyl)amino)-6-aminohexanoate methyl hydrochloride (1.37 g, 3.27 mmol) and N,N-diisopropylethylamine (1.35 mL, 8.18 mmol) were added to dichloromethane (20.0 mL) containing (1R,3R)-3-(2-(benzyloxy)ethoxy)cyclobutane-1-carboxylic acid (0.90 g, 3.60 mmol) in 3-( ... in 3-(2-(1R,3R)-3-(2-(benzyloxy)ethoxy) in 3-(2-(1R,3R)-3-(2-(benzyloxy)ethoxy) in 3-(2-(1R,3R)-3-(2-(benzyloxy)ethoxy) in 3-(2-(1R,3R)-3-(2-(benzyloxy)ethoxy) in 3-(2-(1R,3R)-3-(2-(benzyloxy)ethoxy) in 3-(2-(1R,3R)-3-(2-(benzyloxy)ethoxy) in 3-(2-

[0151] After the reaction was complete, water was added to quench the reaction, and the mixture was extracted twice with dichloromethane. The organic phases were combined, washed twice with saturated sodium chloride, dried, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: n-hexane / ethyl acetate = 3 / 5) to give 900.0 mg of methyl N. 2 -(((9H-fluorene-9-yl)methoxy)carbonyl)-N 6 -((1R,3S)-3-(2-(benzyloxy)ethoxy)cyclobutane-1-carbonyl)-L-lysine ester. LCMS: RT = 2.049 min, [M+H] + =615.4.

[0152] Step G: Synthesis of methyl N 6-((1R,3S)-3-(2-hydroxyethoxy)cyclobutane-1-carbonyl)-L-lysine ester

[0153] At room temperature, to a substance containing methyl N 2 -(((9H-fluorene-9-yl)methoxy)carbonyl)-N 6 500.0 mg (0.81 mmol) of 1-((1R,3S)-3-(2-(benzyloxy)ethoxy)cyclobutane-1-carbonyl)-L-lysine ester (500.0 mL) was added to tetrahydrofuran (50.0 mL), and 50.0 mg of palladium hydroxide on carbon and wet palladium on carbon (50.0 mg) were added. The reaction was carried out at 65°C for 24 hours under hydrogen atmosphere.

[0154] After the reaction was complete, the mixture was filtered through a diatomaceous earth filter onto palladium on carbon. The residue was concentrated under reduced pressure and purified by silica gel column chromatography (eluent: dichloromethane / methanol = 10 / 1) to give 220.89 mg of a colorless oily substance, methyl N. 6 -((1R,3S)-3-(2-hydroxyethoxy)cyclobutane-1-carbonyl)-L-lysine ester. LCMS: RT = 0.51 min, [M+H] + =303.00.

[0155] Step H: Synthesis of methyl N 6 -((1R,3S)-3-(2-hydroxyethoxy)cyclobutane-1-carbonyl)-N 2 -(3-(3-((S)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)pyrrolid-1-yl)-3-oxopropoxy)propionyl)-L-lysine ester

[0156] At room temperature, N,N-diisopropylethylamine (0.40 mL, 2.4 mmol), 1-hydroxybenzotriazole (0.22 g, 1.6 mmol), and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (0.31 g, 1.6 mmol) were added sequentially to 20.0 mL of dichloromethane containing (S)-3-(3-(2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)pyrrolidine-1-yl)-3-oxopropoxy)propionic acid (445.0 mg, 0.80 mmol). After stirring for 30 minutes, methyl N 6 -((1R,3S)-3-(2-hydroxyethoxy)cyclobutane-1-carbonyl)-L-lysine ester (242.0 mg, 0.80 mmol), reacted overnight at room temperature.

[0157] After the reaction was complete, water was added to quench the reaction. The mixture was extracted twice with dichloromethane, the organic phases were combined, washed twice with saturated sodium chloride, dried, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: dichloromethane / methanol = 20 / 1) to give 400.0 mg of methyl N.6 -((1R,3S)-3-(2-hydroxyethoxy)cyclobutane-1-carbonyl)-N 2 -(3-(3-((S)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)pyrrolidine-1-yl)-3-oxopropoxy)propionyl)-L-lysine ester. LCMS:RT=2.081min, [M+H] + =832.0.

[0158] Step I: Synthesize N 6 -((1R,3S)-3-(2-hydroxyethoxy)cyclobutane-1-carbonyl)-N 2 -(3-(3-((S)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)pyrrolid-1-yl)-3-oxopropoxy)propionyl)-L-lysine

[0159] At room temperature, to a substance containing methyl N 6 -((1R,3S)-3-(2-hydroxyethoxy)cyclobutane-1-carbonyl)-N 2 In a solution of 400.0 mg (0.48 mmol) of 3-(3-((S)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)pyrrolidine-1-yl)-3-oxopropoxy)propionyl)-L-lysine ester (4.0 mL) of tetrahydrofuran and 1.0 mL of water, lithium hydroxide monohydrate (24.2 mg, 0.58 mmol) was added, and the mixture was reacted at room temperature for 30 minutes.

[0160] After the reaction was completed, the residue was concentrated under reduced pressure and then lyophilized to give 349.0 mg N. 6 -((1R,3S)-3-(2-hydroxyethoxy)cyclobutane-1-carbonyl)-N 2 -(3-(3-((S)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)pyrrolidine-1-yl)-3-oxopropoxy)propionyl)-L-lysine. LCMS:RT=3.371min, [MH] - =816.4.

[0161] Example 1: Synthesis of Compound 1

[0162] The specific synthesis route is as follows:

[0163] Step A: Synthesize intermediate C1

[0164] The synthesis route of SM1 was prepared with reference to the international patent publication number WO2009073809.

[0165] SM1 (1.5 g, 0.84 mmol) and B1 (0.68 g, 0.84 mmol) were dissolved in dry, anhydrous dichloromethane (20 mL), then DIEA (0.22 g, 1.68 mmol) was added, and finally HATU (0.64 g, 1.68 mmol) was added. The mixture was reacted at 45°C for 24 hours.

[0166] After the reaction was complete, the mixture was quenched with water, then extracted with dichloromethane (20 mL × 2 times), dried, and directly concentrated to obtain an oily substance. This oil was purified by silica gel column chromatography (eluent: dichloromethane / methanol = 15 / 1) to give 600 mg of a white solid (C1 yield: 28%). LCMS: RT = 1.95 min, [(M-DMTr+2H) / 2] + =1143.

[0167] Step B: Synthesize compound 1

[0168] At room temperature, C1 (613 mg, 0.24 mmol), diisopropyltetrazolium ammonium salt (210 mg, 1.20 mmol) was dissolved in dichloromethane (10 mL), an appropriate amount of activated molecular sieve was added, nitrogen was purged, and bis(diisopropylamino)(2-cyanoethoxy)phosphine (140 mg, 0.48 mmol) was added with stirring. The mixture was stirred at room temperature for 2 hours.

[0169] After the reaction was complete, the reaction solution was added dropwise to a pre-cooled saturated sodium bicarbonate / saturated brine solution (6 ml / 6 ml) in an ice-water bath with stirring. The organic phase was then washed with saturated sodium bicarbonate / saturated brine solution (10 ml / 10 ml), dried over sodium sulfate, filtered, and concentrated to obtain an oily substance. Purification was achieved by silica gel column chromatography (basified with triethylamine) (eluent: dichloromethane / methanol = 20:1) to give 269 mg of compound 1 (yield: 41%). LCMS: RT = 2.03 min, (MW-Propiononitrile-HH)²⁻ / ² = 1366.1 H⁻¹ NMR(400MHz, DMSO-d6)δ8.06-7.95(m,4H),7.91(t,J=5.7Hz,2H),7.42-7.31(m,2H),7.30-7.21(m, 4H),6.93(ddd,J=8.8,6.4,2.0Hz,3H),5.26(d,J=3.4Hz,2H),5.02(dd,J=11.2,3.4Hz,2H),4.57(d, J=8.5Hz,3H),4.06(q,J=4.4Hz,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=14.1,6.5Hz,23H),1.20-1.15(m,12H).31P NMR (400MHz, DMSO-d6): ppmδ144.93.

[0170] Example 2: Synthesis of Compound 2

[0171] The specific synthesis route is as follows:

[0172] Step A: Synthesize intermediate C2

[0173] At room temperature, N,N-diisopropylethylamine (478.0 μL, 2.75 mmol), benzotriazole-N,N,N'2,N'-tetramethylurea hexafluorophosphate (312.7 mg, 0.825 mmol), and N2-(3-(3-((S)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)pyrrolidine-1-yl)-3-oxopropoxy)propionyl)-N6-((1S,4R)-4-hydroxycyclohexane-1-carbonyl)-L-lysine (intermediate 1, also known as B2) (447.0 mg, 0.55 mmol) were added to 15.0 mL of dichloromethane containing intermediate SM1 (1.3 g, 0.72 mmol), and the reaction was carried out overnight at 45°C.

[0174] 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 give 710.0 mg of white foamy solid intermediate C2 (yield: 38.3%). LCMS: RT = 1.916 min, [M-DMTr+H+H]2+ / 2 = 1138.6.

[0175] Step B: Synthesize compound 2

[0176] At 0°C, dichloromethane (10.0 mL) containing intermediate C2 (710.0 mg, 0.275 mmol) was added to a three-necked flask containing 4A molecular sieve and N,N-diisopropylammonium tetrazolium salt (236.0 mg, 1.38 mmol), followed by bis(diisopropylamino)(2-cyanoethoxy)phosphine (874.0 μL, 2.75 mmol), and the reaction was carried out at room temperature for 1 hour.

[0177] After the reaction was complete, the mixture was quenched at -20°C with an ice-cold aqueous solution of sodium bicarbonate and sodium chloride (1:1), extracted with DCM, washed three times with the mixed aqueous solution, dried, concentrated to 1 mL, and added dropwise to ice-cold MTBE. A solid precipitated, was centrifuged, and the supernatant was discarded. This process was repeated 5 times. The solid was dissolved in dichloromethane and evaporated to dryness to give 330.0 mg of compound 2 (yield: 43.2%). LCMS:RT: 2.200 min, [M-DMTr-Diisopropylamine+H+H]2+ / 2=1188.2. 31P NMR (162 MHz, DMSO-d6) δ 145.01.

[0178] Example 3: Synthesis of Compound 3

[0179] The specific synthesis route is as follows:

[0180] Step A: Synthesize intermediate C3

[0181] The preparation route of SM2 can be referenced from the synthesis route of the patent with patent publication number CN202280016262.X.

[0182] SM2 (2.67 g, 1.78 mmol) was dissolved in dry, anhydrous dichloromethane (30 mL), and DIEA (1.13 mL, 6.85 mmol), HBTU (0.78 g, 2.06 mmol), and B3 (1.1 g, 1.37 mmol) were added sequentially. The mixture was reacted at 45°C for 24 hours.

[0183] After the reaction was completed, the solution was quenched with saturated sodium bicarbonate aqueous solution, then extracted with dichloromethane (20 mL × 2 times), dried, and directly concentrated to obtain an oily substance. This oil was purified by silica gel column chromatography (eluent: dichloromethane / methanol = 15 / 1), and then further purified by high-performance liquid chromatography to obtain 0.7 g of white foamed solid C3 (yield: 22%). LCMS: RT = 4.065 min, [MH] / 2- = 1139.4.

[0184] Step B: Synthesize compound 3

[0185] At room temperature, C3 (700 mg, 0.31 mmol), diisopropyltetrazolium ammonium salt (160 mg, 0.93 mmol) was dissolved in dichloromethane (15 mL), an appropriate amount of activated molecular sieve was added, nitrogen was purged, and bis(diisopropylamino)(2-cyanoethoxy)phosphine (280 mg, 0.93 mmol) was added dropwise at room temperature, and the mixture was stirred at room temperature for 2 hours.

[0186] After the reaction was complete, the reaction solution was cooled to -20°C, and saturated sodium bicarbonate / saturated saline solution (6 ml / 6 ml) was added dropwise with stirring. After stirring for 2 minutes, the mixture was stirred at room temperature for 5 minutes. The liquid was separated, and the organic phase was dried over sodium sulfate, filtered, and concentrated to obtain a white gel. 3 ml of dichloromethane was added to the white gel, and then it was added dropwise to 30 ml of methyl ether. The mixture was centrifuged and concentrated, and the supernatant was discarded. The gel was then dissolved in 3 ml of dichloromethane. The above operation was repeated 3 times. Finally, the obtained gel was concentrated to obtain 596 mg of compound 3 (yield: 78%). LCMS: RT = 4.839 min, [M-cyanoethyl-H] / 2- = 1213.031 P NMR (400 MHz, DMSO-d6): ppm δ 144.95.

[0187] Example 4: Synthesis of Compound 4

[0188] The specific synthesis route is as follows:

[0189] Step A: Synthesize intermediate C4

[0190] The preparation route of SM3 can be referred to the literature with patent numbers CN201780042047.6 or CN202380010110.3.

[0191] SM3 (0.52 g, 0.59 mmol) was dissolved in dry, anhydrous dichloromethane (20 mL), and DIEA (0.49 mL, 2.95 mmol), HBTU (0.34 g, 0.89 mmol), and B2 (0.90 g, 0.59 mmol) were added sequentially. The mixture was reacted at 45°C for 24 hours.

[0192] After the reaction was completed, the mixture was quenched with saturated sodium bicarbonate aqueous solution, then extracted with dichloromethane (20 mL × 2 times), dried and concentrated directly to obtain an oily substance, which was purified by silica gel column chromatography (eluent: dichloromethane / methanol = 15 / 1), and then purified by high performance liquid chromatography to obtain 0.5 g of compound C4 (yield: 37%).

[0193] Step B: Synthesize compound 4

[0194] At room temperature, C4 (500 mg, 0.22 mmol), diisopropyltetrazolium ammonium salt (110 mg, 0.66 mmol) was dissolved in dichloromethane (15 mL), an appropriate amount of activated molecular sieve was added, nitrogen was purged, and bis(diisopropylamino)(2-cyanoethoxy)phosphine (270 mg, 0.88 mmol) was added dropwise at room temperature, and the mixture was stirred at room temperature for 2 hours.

[0195] After the reaction was complete, the reaction solution was cooled to -20°C, and saturated sodium bicarbonate / saturated saline solution (6 ml / 6 ml) was added dropwise with stirring. After stirring for 2 minutes, the mixture was stirred at room temperature for 5 minutes. The liquid was separated, and the organic phase was dried over sodium sulfate, filtered, and concentrated to obtain a white gel. 3 ml of dichloromethane was added to the white gel, and then it was added dropwise to 30 ml of methyl ether. The mixture was centrifuged and concentrated, and the supernatant was discarded. The gel was then dissolved in 3 ml of dichloromethane. The above operation was repeated 3 times. Finally, the obtained gel was concentrated to obtain 305 mg of compound 4 (yield: 56%). LCMS: RT = 2.155 min, [M-cyanoethyl-H] / 2- = 1227.2. 31 PNMR (400MHz, DMSO-d6): ppmδ144.98.

[0196] Example 5: Synthesis of Compound 5

[0197] The specific synthesis route is as follows:

[0198] Step A: Synthesis of 3-(3-(4-hydroxypiperidin-1-yl)-3-oxopropoxy)propionic acid

[0199] At 0°C, di(2-carboxyethyl) ether (5 g, 30.84 mmol) and 4-hydroxypiperidine (3.43 g, 33.92 mmol) were dissolved in dry, anhydrous dichloromethane (110 mL), followed by the addition of DIEA (7.65 mL, 46.26 mmol) and HBTU (14.03 g, 37.01 mmol), and the reaction was carried out at room temperature for 24 hours.

[0200] After the reaction was completed, the solution was quenched with saturated sodium bicarbonate aqueous solution, then extracted with dichloromethane (20 mL × 2 times), dried and concentrated directly to obtain an oily substance, which was purified by silica gel column chromatography (eluent: dichloromethane / methanol = 15 / 1), and then purified by high performance liquid chromatography to obtain 7.55 g of white foam solid 3-(3-(4-hydroxypiperidin-1-yl)-3-oxopropoxy)propionic acid (yield: 99%).

[0201] Step B: Synthesize 005-2

[0202] 29 g (118.24 mmol) of 3-(3-(4-hydroxypiperidin-1-yl)-3-oxopropoxy)propionic acid was dissolved in 145 mL of dry, anhydrous pyridine, and 25 g (73.78 mmol) of 4,4'-bismethoxytriphenylmethyl chloride was added. The reaction was carried out at room temperature for 24 hours.

[0203] After the reaction was completed, the product was dried and concentrated to obtain an oily substance, which was then purified by silica gel column chromatography (eluent: dichloromethane / methanol = 15 / 1), and then purified by high performance liquid chromatography to obtain 60 g of white foam solid 005-2 (yield: 93%).

[0204] Step C: Synthesize 005-4

[0205] At 0°C, 005-2 (12 g, 21.91 mmol) was dissolved in dry, anhydrous dichloromethane (120 mL), followed by the addition of DIEA (5.43 mL, 32.87 mmol), HOPO (0.49 g, 4.38 mmol), EDCI (5.46 g, 28.48 mmol), and 005-3 (7.45 g, 26.02 mmol). The mixture was reacted at room temperature for 24 hours.

[0206] After the reaction was completed, the mixture was quenched with saturated sodium bicarbonate aqueous solution, then extracted with dichloromethane (20 mL × 2 times), dried and directly concentrated to obtain an oily substance, which was purified by silica gel column chromatography (eluent: dichloromethane / methanol = 15 / 1), and then purified by high performance liquid chromatography to obtain 8.37 g of white foam solid 005-4 (yield: 47%).

[0207] Step D: Synthesize B3

[0208] At room temperature, 005-4 (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. The system was heated to 35°C and stirred for 3 hours.

[0209] After the reaction was completed, the system temperature was maintained below 40 degrees Celsius, and the tetrahydrofuran was concentrated until no fraction flowed out. The remaining system was directly freeze-dried to obtain 8.2 g of B3 (yield: 99%).

[0210] Step E: Synthesize intermediate C5

[0211] At 0°C, SM1 (0.95 g, 0.48 mmol) was dissolved in dry, anhydrous dichloromethane (16 mL), followed by the addition of DIEA (0.12 mL, 0.72 mmol), HBTU (0.27 g, 0.75 mmol), and B3 (0.42 g, 0.53 mmol) in sequence, and the reaction was carried out at room temperature for 24 hours.

[0212] After the reaction was completed, the solution was quenched with saturated sodium bicarbonate aqueous solution, then extracted with dichloromethane (20 mL × 2 times), dried and concentrated directly to obtain an oily substance, which was purified by silica gel column chromatography (eluent: dichloromethane / methanol = 15 / 1), and then purified by high performance liquid chromatography to obtain 481 mg of intermediate C5 (yield: 39%).

[0213] Step F: Synthesize compound 5

[0214] At room temperature, C5 (481 mg, 0.19 mmol), diisopropyltetrazolium ammonium salt (39 mg, 0.23 mmol) was dissolved in dichloromethane (10 mL), an appropriate amount of activated molecular sieve was added, nitrogen was purged, and bis(diisopropylamino)(2-cyanoethoxy)phosphine (110 mg, 0.38 mmol) was added dropwise at room temperature, and the mixture was stirred at room temperature for 2 hours.

[0215] After the reaction was complete, the reaction solution was cooled to -20°C, and saturated sodium bicarbonate / saturated saline solution (6 ml / 6 ml) was added dropwise with stirring. After stirring for 2 minutes, the mixture was stirred at room temperature for 5 minutes. The liquid was separated, and the organic phase was dried over sodium sulfate, filtered, and concentrated to obtain a white gel. 3 ml of dichloromethane was added to the white gel, and then it was added dropwise to 30 ml of methyl ether. The mixture was centrifuged and concentrated, and the supernatant was discarded. The gel was then dissolved in 3 ml of dichloromethane. The above operation was repeated 3 times. Finally, the obtained gel was concentrated to obtain 514 mg of compound 5 (yield: 99%). LCMS: RT = 2.268 min, [M-cyanoethyl-H] / 2- = 1361.0.31 P NMR (400 MHz, DMSO-d6): ppm δ 144.97.

[0216] Example 6: Synthesis of Compound 6

[0217] The specific synthesis route is as follows:

[0218] Step A: Synthesize intermediate C6

[0219] At 0°C, SM2 (0.72 g, 0.48 mmol) was dissolved in dry, anhydrous dichloromethane (15 mL), and DIEA (0.12 mL, 0.72 mmol), HBTU (0.27 g, 0.72 mmol), and B3 (0.42 g, 0.53 mmol) were added sequentially. The mixture was reacted at room temperature for 24 hours.

[0220] After the reaction was completed, the solution was quenched with saturated sodium bicarbonate aqueous solution, then extracted with dichloromethane (20 mL × 2 times), dried and concentrated directly to obtain an oily substance, which was purified by silica gel column chromatography (eluent: dichloromethane / methanol = 15 / 1), and then purified by high performance liquid chromatography to obtain 371 mg of C6 (yield: 34%).

[0221] Step B: Synthesize compound 6

[0222] At room temperature, C6 (371 mg, 0.16 mmol) and diisopropyltetrazolium ammonium salt (33 mg, 0.19 mmol) were dissolved in dichloromethane (10 mL), an appropriate amount of activated molecular sieve was added, nitrogen was purged, and bis(diisopropylamino)(2-cyanoethoxy)phosphine (96 mg, 0.32 mmol) was added dropwise at room temperature, and the mixture was stirred at room temperature for 2 hours.

[0223] After the reaction was complete, the reaction solution was cooled to -20°C, and saturated sodium bicarbonate / saturated saline solution (6 ml / 6 ml) was added dropwise with stirring. After stirring for 2 minutes, the mixture was stirred at room temperature for 5 minutes. The liquid was separated, and the organic phase was dried over sodium sulfate, filtered, and concentrated to obtain a white gel. 3 ml of dichloromethane was added to the white gel, and then it was added dropwise to 30 ml of methyl ether. The mixture was centrifuged and concentrated, and the supernatant was discarded. The gel was then dissolved in 3 ml of dichloromethane. The above operation was repeated 3 times. Finally, the obtained gel was concentrated to obtain 399 mg of compound 6 (yield: 98%). LCMS: RT = 2.064 min, [M-cyanoethyl-H] / 2- = 1213.2.31 P NMR (400 MHz, DMSO-d6): ppm δ 144.97.

[0224] Example 7: Synthesis of Compound 7

[0225] Synthesis of [(2R,3R,4R,5R,6R)-3,4-bis(acetoxy)-6-(2-(2-(2-[(2S)-2-(3-(3-[(2S)-2-[(bis(4-methoxyphenyl)(phenyl)methoxy)methyl]pyrrolidine-1-yl]-3-oxopropoxy)propionamide)-N-(3-(bis({[(2-(2-{[(2R,)3R,4R,5R,6R) )-4,5-bis(acetoxy)-6-[(acetoxy)methyl]-3-acetaminooxyimida-2-yl]ethoxy)amino)amino)propyl)-6-{[(1s,3s)-3-(2-{[(bis(propyl-2-yl)amino)(2-cyanoethoxy)phosphophenyl]oxy}ethoxy)cyclobutyl]formamido}hexylamino]acetamino)ethoxy)-5-acetaminooxypyrimidin-2-yl]methyl acetate

[0226] The specific synthesis route is as follows:

[0227] Step A: Synthesis of (1R,3R)-3-(2-hydroxyethoxy)cyclobutane-1-carboxylic acid

[0228] (1R,3R)-3-(2-(benzyloxy)ethoxy)cyclobutane-1-carboxylic acid (574 mg, 2.3 mmol) was dissolved in tetrahydrofuran (12 mL) under ice bath conditions, and two drops of 6N hydrochloric acid were added. Then, Pd / C (100 mg, 10% wt) and Pd(OH)₂ / C (100 mg, 10% wt) were added, followed by hydrogen displacement. After the addition was complete, the mixture was heated to 65 °C and reacted for 12 hours. After the reaction was complete, the mixture was cooled to room temperature and filtered. The filter cake was washed with methanol, and the concentrated organic phase yielded 352 mg of crude (1R,3R)-3-(2-hydroxyethoxy)cyclobutane-1-carboxylic acid (yield: 95.6%) without further treatment.

[0229] Step B: Synthesis of methyl N2-((benzyloxy)carbonyl)-N6-((1R,3S)-3-(2-hydroxyethoxy)cyclobutane-1-carbonyl)-L-lysine ester

[0230] (1R,3R)-3-(2-hydroxyethoxy)cyclobutane-1-carboxylic acid (352 mg, 2.20 mmol) was dissolved in dichloromethane (25 mL) under ice bath conditions. 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-lysine ester hydrochloride (800 mg, 2.42 mmol) was added, and the mixture was then heated to room temperature and stirred for 3 hours. 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) to give 600 mg of white solid methyl N2-((benzyloxy)carbonyl)-N6-((1R,3S)-3-(2-hydroxyethoxy)cyclobutane-1-carbonyl)-L-lysine ester (yield: 62.7%).

[0231] Step C: Synthesis of N2-((benzyloxy)carbonyl)-N6-((1R,3S)-3-(2-hydroxyethoxy)cyclobutane-1-carbonyl)-L-lysine

[0232] 600 mg (1.38 mmol) of methyl N2-((benzyloxy)carbonyl)-N6-((1R,3S)-3-(2-hydroxyethoxy)cyclobutane-1-carbonyl)-L-lysine ester was dissolved in a mixture of tetrahydrofuran (12 mL) and water (3 mL) at room temperature. Lithium hydroxide monohydrate (174 mg, 4.13 mmol) was added, and the reaction was carried out for 5 hours. After the reaction was completed, 0.5 mL of 6N hydrochloric acid was added, and the mixture was concentrated under reduced pressure and evaporated to dryness to give 577 mg of white solid N2-((benzyloxy)carbonyl)-N6-((1R,3S)-3-(2-hydroxyethoxy)cyclobutane-1-carbonyl)-L-lysine (yield: 97.8%).

[0233] Step D: Synthesis of methyl ethoxy[(2R,3R,4R,5R,6R)-3,4-bis(acetoxy)-6-(2-(2-(2-)[(2S)-2-{[(benzyloxy)carbonyl]amino}-N-(3-(bis({[(2-{[(2R,3R,4R,5R,6R)-4,5-bis(acetoxy)-6-[(acetoxy)methyl]-3-acetaminooxyethoxy)ethyl)carbamoyl]methyl})amino)propyl)-6-{[(1S,3S)-3-(2-hydroxyethoxy)cyclobutyl]formamido}hexylamino]acetamino)ethoxy)ethoxy)-5-acetaminooxaoxane-2-yl]ethoxyacetate

[0234] At room temperature, N2-((benzyloxy)carbonyl)-N6-((1R,3S)-3-(2-hydroxyethoxy)cyclobutane-1-carbonyl)-L-lysine (577 mg, 1.35 mmol) was dissolved in N,N-dimethylformamide (7.5 mL), followed by the addition of dichloromethane (60 mL). N,N-diisopropylethylamine (760 mg, 5.89 mmol) and HATU (726 mg, 1.91 mmol) were then added and stirred for 3 minutes. Then, methyl [(3R,4R,6R)-3,4-bis(acetoxy)-6-(2-(2-({[(2-{[(2R,4R,5R)-4,5-bis(acetyl)-6-[(acetoxy)methyl]-3-acetaminooxy-2-yl]oxy}ethoxy)ethyl)carbamoyl]methyl}(3-({[(2-(2-{[(2R,4R,5R)-4,5-bis(acetoxy)-6-[(acetoxy)methyl]-3-acetaminooxy-2-yl]oxy}ethoxy)ethyl)carbamoyl]methyl}amino)propyl)amino)acetamamido)ethoxyethoxy]-5-acetaminooxycarbonyl-2-yl]acetate (2.65 g, 1.77 mmol) was added and reacted for 12 hours. After the reaction was completed, saturated sodium bicarbonate aqueous solution (50 mL) and dichloromethane (50 mL) were added for extraction. The organic phase was washed again with saturated sodium bicarbonate aqueous solution (50 mL × 3), then washed once with saturated sodium chloride aqueous solution (50 mL), and then dried with anhydrous sodium sulfate. The crude product obtained by concentrated organic phase under reduced pressure was subjected to column chromatography (eluent: methanol / dichloromethane = 1 / 20) to give 1.30 g of white solid [(2R,3R,4R,5R,6R)-3,4-bis(acetoxy)-6-(2-(2-(2-)[(2S)-2-{[(benzyloxy)carbonyl]amino}-N-(3-(bis({[(2-{[(2R,3R,4R,5R,6R)-4,5-bis(acetoxy)-6-[(acetoxy)methyl]-3-acetaminooxyethoxy)ethyl)carbamoyl]methyl})amino)propyl)-6-{[(1S,3S)-3-(2-hydroxyethoxy)cyclobutyl]formamido}hexylamino]acetamino)ethoxy)ethoxy)-5-acetaminooxaoxane-2-yl]ethoxyacetyl acetate (yield: 46.3%).

[0235] Step E: Synthesis of methyl [(2R,3R,4R,5R,6R)-3,4-bis(acetoxy)-6-(2-(2-(2-[(2S)-2-amino-N-(3-(bis({[(2-(2-{[(2R,3R,4R,5R,6R)-4,5-bis(acetoxy)-6-[(acetoxy)methyl]-3-acetaminooxo-2-yl]ethoxy)ethyl)carbamoyl]methyl})amino)propyl)-6-{[(1S,3S)-3-(2-hydroxyethoxy)cyclobutyl]formamido}hexylamino]acetamino)ethoxy)ethoxy)-5-acetaminooxo-2-yl]ethoxyethoxy)-5-acetaminooxo-2-yl]ethoxyacet ...

[0236] At room temperature, [(2R,3R,4R,5R,6R)-3,4-bis(acetoxy)-6-(2-(2-(2-)[(2S)-2-{[(benzyloxy)carbonyl]amino}-N-(3-(bis({[(2-{[(2R,3R,4R,5R,6R)-4,5-bis(acetoxy)-6-[(acetoxy)methyl]-3-acetaminooxyethoxy)ethyl) Methyl ethoxyacetate (1.30 g, 0.68 mmol) was dissolved in tetrahydrofuran (40 mL), Pd / C (400 mg, 10% wt) was added, and hydrogen was substituted. The mixture was then heated to 40 °C and reacted for 12 hours. After the reaction was completed, the mixture was filtered, the filter cake was washed with methanol, and the organic phase was concentrated under reduced pressure to give 1.20 g of crude product as a white solid [(2R,3R,4R,5R,6R)-3,4-bis(acetoxy)-6-(2-(2-(2-[(2S)-2-amino-N-(3-(bis(bis({[(2-(2-{[(2R,3R,4R,5R,6R)-4,5-bis(acetoxy)-6-[(acetoxy) Methyl acetaminophen(methyl)-3-acetaminophen(2-yl)ethoxyphen(ethyl)carbamoyl]methyl})amino)propyl)-6-{[(1S,3S)-3-(2-hydroxyethoxy)cyclobutyl]formamido}hexylaminophen(ethoxy)ethoxyphen(5-acetaminophen(2-yl)ethoxyphen(5-acetaminophen(2-yl)ethoxyacetaminophen(2-yl)ethoxyacetyl]ethoxyacetyl ...

[0237] Step F: Synthesis of [(2R,3R,4R,5R,6R)-3,4-bis(acetoxy)-6-(2-(2-(2-[(2S)-2-(3-(3-[(2S)-2-[(bis(4-methoxyphenyl)(phenyl)methoxy)methyl]pyrrolidine-1-yl]-3-oxopropoxy)propionamide)-N-(3-(bis({[(2-(2-{[(2R,)3R,4R,5R, 6R)-4,5-bis(acetoxy)-6-[(acetoxy)methyl]-3-acetaminooxyimidazol-2-yl]ethoxy)amino)amino)propyl)-6-{[(1s,3s)-3-(2-{[(bis(propyl-2-yl)amino)(2-cyanoethoxy)phosphophenyl]oxy}ethoxy)cyclobutyl]formamido}hexylamino]acetamino)ethoxy)-5-acetaminooxypyrimidin-2-yl]methyl acetate

[0238] At room temperature, [(2R,3R,4R,5R,6R)-3,4-bis(acetoxy)-6-(2-(2-(2-[(2S)-2-amino-N-(3-(bis(bis({[(2-(2-{[(2R,3R,4R,5R,6R)-4,5-bis(acetoxy)-6-[(acetoxy)methyl]-3-acetaminooxy-2-yl]ethoxy)ethyl)carbamoyl]methyl})amino)propyl)-6-{[(1S Methyl ethoxyacetyl(3S)-3-(2-hydroxyethoxy)cyclobutyl]formamido}hexylamino]acetamido)ethoxy)ethoxy)-5-acetamioxa-2-yl]ethoxyethoxy)-5-acetamioxa-2-yl]ethoxyacetate (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 stirred 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 obtained organic phase was washed again with saturated sodium bicarbonate aqueous solution (25 mL × 2), then washed once with saturated sodium chloride solution (25 mL), and finally dried with 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 white solid [(2R,3R,4R,5R,6R)-3,4-bis(acetoxy)-6-(2-(2-(2-[(2S)-2-(3-(3-[(2S)-2-[(bis(4-methoxyphenyl)(phenyl)methoxy)methyl]pyrrolidine-1-yl]-3-oxopropoxy)propionamide)-N-(3-(bis({[(2-(2- {[(2R,)3R,4R,5R,6R)-4,5-bis(acetoxy)-6-[(acetoxy)methyl]-3-acetaminooxyimidazol-2-yl]ethoxy)amino)amino)propyl)-6-{[(1s,3s)-3-(2-{[(bis(propyl-2-yl)amino)(2-cyanoethoxy)phosphophenyl]oxy}ethoxy)cyclobutyl]formamido}hexylamino]acetamino)ethoxy)-5-acetaminooxypyrimidin-2-yl]methyl acetate (yield: 31.8%).

[0239] Step G: Synthesis of [(2R,3R,4R,5R,6R)-3,4-bis(acetoxy)-6-(2-(2-(2-[(2S)-2-(3-(3-[(2S)-2-[(bis(4-methoxyphenyl)(phenyl)methoxy)methyl]pyrrolidine-1-yl]-3-oxopropoxy)propionamide)-N-(3-(bis({[(2-(2-{[(2R,)3R,4R,5R, 6R)-4,5-bis(acetoxy)-6-[(acetoxy)methyl]-3-acetaminooxyimidazol-2-yl]ethoxy)amino)amino)propyl)-6-{[(1s,3s)-3-(2-{[(bis(propyl-2-yl)amino)(2-cyanoethoxy)phosphophenyl]oxy}ethoxy)cyclobutyl]formamido}hexylamino]acetamino)ethoxy)-5-acetaminooxypyrimidin-2-yl]methyl acetate

[0240] At room temperature, [(2R,3R,4R,)5R,6R)-3,4-bis(acetoxy)-6-(2-(2-(2-[(2S)-2-(3-(3-[(2S)-2-[(bis(4-methoxyphenyl)(phenyl)methoxy)methyl]pyrrolidine-1-yl]-3-oxopropoxy)propionamide)-N-(3-(bis({[(2-{[(2R,3R,4R,5R,6R)-4,5-bis(acetoxy)-6-[(ethyl [Acyloxy)methyl]-3-acetaminooxa-2-yl]ethoxy)ethyl)amino]methyl)amino)propyl)-6-{[(1S,3S)-3-(2-hydroxyethoxy)cyclobutyl]formamido}hexyl]ethoxy)ethoxy)-5-acetaminooxa-2-yl]ethoxy)ethoxy)-5-acetaminooxa-2-yl]ethoxyethoxy)-5-acetaminooxa-2-yl]ethoxyethoxy)-5-acetaminooxa-2-yl]ethoxyethoxy) (469 mg, 0.2 mmol) and In a 20 mL solution of molecular sieve dichloromethane, bis(diisopropylamino)(2-cyanoethoxy)phosphine (181 mg, 0.6 mmol) and diisopropylammonium tetrazolium (103 mg, 0.6 mmol) were added, and the mixture was reacted 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, and once with saturated brine. It was then dried with anhydrous sodium sulfate, concentrated under reduced pressure, and the residue was slurried in an ice bath (MTBE / DCM = 8:1). The mixture was centrifuged (3500 rpm, 30 seconds), 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 [(2R,3R,4R,5R,6R)-3,4-bis(acetoxy)-6-(2-(2-(2-[(2S)-2-(3-(3-[(2S)-2-[(bis(4-methoxyphenyl)(phenyl)methoxy)methyl]pyrrolidine-1-yl]-3-oxopropoxy)propionamide)-N-(3-(bis({[( 2-(2-{[(2R,)3R,4R,5R,6R)-4,5-bis(acetoxy)-6-[(acetoxy)methyl]-3-acetaminooxyimidazol-2-yl]ethoxy)amino)amino)propyl)-6-{[(1s,3s)-3-(2-{[(bis(propyl-2-yl)amino)(2-cyanoethoxy)phosphophenyl]oxy}ethoxy)cyclobutyl]formamido}hexylamino]acetamino)ethoxy)-5-acetaminooxypyrimidin-2-yl]methyl acetate (yield: 42.5%). LCMS: RT = 4.725 min, [(M-54) / 2]2- = 1220.8.31 PNMR (400 MHz, DMSO-d6): ppm δ 147.39.

[0241] Example 8 Compound 8

[0242] The specific synthesis route is as follows:

[0243] Step A: Synthesize intermediate C8

[0244] At room temperature, N,N-diisopropylethylamine (0.75 mL, 4.47 mmol) and HBTU (672 mg, 1.79 mmol) were added sequentially to dichloromethane (50 mL) containing SM3 (2.27 g, 1.49 mmol) and B4 (intermediate 2) (1.21 g, 1.49 mmol). The reaction was stirred at 40°C for 3 hours, then diluted with dichloromethane, washed twice with saturated sodium bicarbonate solution, washed once with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was purified by silica gel column chromatography (eluent: dichloromethane / methanol = 9 / 1) to give 1.46 g of C8 (yield: 42%).

[0245] Step B: Synthesize compound 8

[0246] At room temperature, the mixture containing C8 (1.45 g, 0.62 mmol) and Diisopropylammonium tetrazolium (427 mg, 2.48 mmol) and bis(diisopropylamino)(2-cyanoethoxy)phosphine (376 μL, 1.86 mmol) were added to dichloromethane (20 mL) containing molecular sieves, and the reaction was carried out at room temperature for 4 hours. After the reaction was completed, the reaction was quenched with a saturated sodium bicarbonate solution at low temperature, extracted once with dichloromethane, dried over anhydrous sodium sulfate, filtered, and the organic phase was concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (eluent: dichloromethane / methanol = 30 / 1), and concentrated to give 700 mg of compound 8 (yield: 44%). LCMS: RT = 4.965 min, [M-DMTr-Diisopropylamine+H+H]2+ / 2 = 1061.4.

[0247] Example 9: Synthesis of Compound 9

[0248] The specific synthesis route is as follows:

[0249] Step A: Synthesize intermediate C9

[0250] The preparation method of SM4 can be found in the patent document with patent publication number WO2023088227.

[0251] At room temperature, N,N-diisopropylethylamine (0.22 mL, 1.32 mmol), benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate (380.0 mg, 0.99 mmol), and finally N2-(3-(3-((S)-2-(bis(4-methoxyphenyl)(phenyl)methoxy)methyl)pyrrolidine-1-yl)-3-oxopropoxy)propionyl)-N6-((1S,4R)-4-hydroxycyclohexane-1-carbonyl)-L-lysine (533.0 mg, 0.66 mmol) were added to 20.0 mL of dichloromethane containing intermediate SM4 (1.29 g, 0.79 mmol) in sequence, and the reaction was carried out at 45°C for 48 hours.

[0252] After the reaction was completed, water was added to quench the reaction, and the mixture was extracted twice with dichloromethane. The organic phases were combined, washed twice with saturated sodium chloride, dried, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: dichloromethane / methanol = 10 / 1) to give 700.0 mg C9 (yield: 43.6%). LCMS:RT: 4.020 min, [M+H]+ / 2 = 1205.6.

[0253] Step B: Synthesize compound 9

[0254] At 0°C, dichloromethane (10 mL) containing intermediate C9 (700.0 mg, 0.29 mmol) was added to a three-necked flask containing 4A molecular sieve and N,N-diisopropylammonium tetrazolium salt (250.0 mg, 1.45 mmol), followed by bis(diisopropylamino)(2-cyanoethoxy)phosphine (460.0 μL, 1.45 mmol), and the reaction was carried out at room temperature for 3 hours.

[0255] After the reaction was complete, the mixture was quenched at 0°C with an ice-cold aqueous solution of sodium bicarbonate and sodium chloride (1:1), extracted with DCM, washed three times with the aqueous solution, dried, concentrated to 10 mL, and added dropwise to ice-cold MTBE. A solid precipitated, was centrifuged, and the supernatant was discarded. This process was repeated twice. The solid was dissolved in dichloromethane and evaporated to dryness to give 400.0 mg of compound 9 (yield: 52.8%). LCMS: 5.089 min, [M-cyanoethyl-HH]²⁺ / ² = 1279.4. 31P NMR (162 MHz, DMSO-d⁶) δ 145.00.

[0256] Example 10: Synthesis of Compound 10

[0257] The specific synthesis route is as follows:

[0258] Step A: Synthesize intermediate 10-1

[0259] At room temperature, N,N-diisopropylethylamine (1.83 mL, 11.0 mmol) was added sequentially to 50.0 mL of dichloromethane containing (2R,3R,5R,6R)-5-acetamido-2-(acetyoxymethyl)-6-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)tetrahydro-2H-pyran-3,4-dimethyldiacetate (5.06 g, 10.6 mmol). 1-Hydroxybenzotriazole (1.5 g, 11.0 mmol) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (2.1 g, 11.0 mmol) were added dropwise, followed by N,N-dimethylformamide (10.0 mL) of ((S)-4-((benzyloxy)carbonyl)amino)-4-carboxybutyryl)-L-glutamic acid (1.14 g, 2.8 mmol). The mixture was reacted overnight at room temperature.

[0260] After the reaction was completed, a saturated sodium bicarbonate aqueous solution was added to quench the reaction. The mixture was extracted twice with dichloromethane, and the organic phases were combined, washed twice with saturated sodium chloride, dried, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: dichloromethane / methanol = 10 / 1) to give 3.08 g of intermediate 10⁻¹ (yield: 60.7%). LCMS: RT: 3.644 min; [M+H] + =1792.2.

[0261] Step B: Synthesize intermediate SM5

[0262] Palladium on carbon (600.0 mg) was added to 120.0 mL of ethanol containing intermediate 10-1 (3.08 g, 1.7 mmol) at room temperature, and the reaction was carried out at room temperature for 3 hours under hydrogen atmosphere.

[0263] After the reaction was completed, palladium on carbon was filtered through diatomaceous earth and concentrated under reduced pressure to obtain 2.85 g of intermediate SM5 (yield: 87.7%). LCMS:RT: 3.246 min; [MH]- = 1655.8.

[0264] Step C: Synthesize intermediate C10

[0265] At room temperature, N,N-diisopropylethylamine (0.25 mL, 1.51 mmol), benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate (428.9 mg, 1.1 mmol), were added sequentially to dichloromethane (20.0 mL) and N,N-dimethylformamide (5.0 mL) containing intermediate SM5 (1.5 g, 0.91 mmol), and finally N... 6 -((1R,3S)-3-(2-hydroxyethoxy)cyclobutane-1-carbonyl)-N 2 -(3-(3-((S)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)pyrrolidine-1-yl)-3-oxopropoxy)propionyl)-L-lysine (616.8 mg, 0.75 mmol), reacted overnight at 45°C.

[0266] After the reaction was completed, a saturated sodium bicarbonate aqueous solution was added to quench the reaction. The mixture was extracted twice with dichloromethane, and the organic phases were combined, washed twice with saturated sodium chloride, dried, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: dichloromethane / methanol = 10 / 1) to give 1.5 g of intermediate C10 (yield: 81.1%). LCMS:RT: 4.213 min; [M-3O3+2H]+ / 2 = 1078.0.

[0267] Step D: Synthesize compound 10

[0268] At 0°C, dichloromethane (50 mL) containing intermediate C10 (1.5 mg, 0.61 mmol) was added to a three-necked flask containing 4A molecular sieve and N,N-diisopropylammonium tetrazolium salt (522.0 mg, 3.05 mmol), followed by bis(diisopropylamino)(2-cyanoethoxy)phosphine (967.4 μL, 3.05 mmol), and the reaction was carried out at room temperature for 1 hour.

[0269] After the reaction was complete, the mixture was quenched at 0°C with an ice-cold aqueous solution of sodium bicarbonate and sodium chloride (1:1), extracted with DCM, washed three times with the mixed aqueous solution, dried, concentrated to 10 mL, and added dropwise to ice-cold MTBE. A solid precipitated, which was centrifuged, and the supernatant was discarded. This process was repeated twice. The solid was dissolved in dichloromethane and evaporated to dryness to give 800.0 mg of compound 10 (yield: 49.4%). LCMS:RT: 4.975 min; [M-cyanoethyl]- / 2 = 1301.2. 31 PNMR (162MHz, DMSO-d6) δ 144.96.

[0270] The structures of compounds 1-10 conjugated with siRNA sequences are shown in Table 1:

[0271] Table 1 shows the structure of A1-A10.

[0272] The structures of A1-A10 linked to nucleotides via phosphate esters or thiophosphate esters are shown in Table 2.

[0273] Table 2 shows the structures of D1-D10 linked with phosphate esters.

[0274] Example 11 Synthesis of modified sequences for nucleic acid molecules

[0275] Modified nucleic acid molecules were prepared using a Dr.48 DNA / RNA automated synthesizer. The specific synthetic route can be found in Example 12. The sequences of the modified nucleic acid molecules are shown in Table 3.

[0276] Table 3 shows the sequence listing of modified nucleic acid molecules.

[0277] 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′-thiophosphate; Ams = 2′-O-methyladenosine-3′-thiophosphate; Ums = 2′-O-methyluridine-3′-thiophosphate; Cms = 2′-O-methylcytidine-3′-thiophosphate; Gms = 2′-O-methylguanosine-3′-thiophosphate; Af = 2′-fluoroadenosine-3′-phosphate; Uf = 2′-fluorouridine-3′-phosphate; Cf = 2′-fluorocytidine-3′-phosphate; Gf = 2′-fluoroguanosine-3′-phosphate; Afs = 2′-fluoroadenosine-3′-thiophosphate; Ufs = 2′-fluorouridine-3′-thiophosphate; Cfs = 2′-fluorocytidine-3′-thiophosphate; Gfs = 2′-fluoroguanosine-3′-thiophosphate; dT = 2′-deoxythymidine-3′-phosphate; m = 2′-O-methyl; f = 2′-fluorine; s = thiophosphate bond.

[0278] Example 12 Preparation of double-stranded RNA conjugates

[0279] This preparation example synthesized a double-stranded RNA conjugate. The RNA conjugated in this double-stranded RNA conjugate has the sense and antisense strand sequences corresponding to those in Tables 4, 5, and 6.

[0280] The positive strand of synthesized double-stranded RNA conjugates

[0281] Nucleoside monomers were sequentially linked from 3' to 5' along the positive strand nucleotide arrangement as shown in Tables 4, 5, and 6 using a solid-phase phosphoramide method. Each linkage of a nucleoside monomer involved four steps: deprotection, coupling, capping, and oxidation or sulfidation. Specifically, when two nucleotides were linked using a phosphate ester, the linkage of the subsequent nucleoside monomer involved deprotection, coupling, capping, and oxidation. When two nucleotides were linked using a thiophosphate ester, the linkage of the subsequent nucleoside monomer involved deprotection, coupling, capping, and sulfidation. The synthetic conditions are given below:

[0282] Commercial nucleoside monomers and compounds (1-10) prepared by the above steps were dissolved in ultra-dry anhydrous acetonitrile to prepare 0.05-0.1M solutions, and 4A molecular sieves were added. The deprotection reaction conditions were the same for each step, namely, 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′-dimethoxytriphenylmethyl protecting group on the solid support was 150:1.

[0283] The coupling reaction conditions were identical for each step, including a temperature of 25°C, a molar ratio of nucleic acid sequence to nucleoside monomer linked on the solid-phase support of 1:20, a molar ratio of nucleic acid sequence to coupling reagent linked on the solid-phase support of 1:120, a reaction time of 300 seconds, and a 0.3 M acetonitrile solution of 5-benzylthio-1H-tetrazole as the coupling reagent.

[0284] Each capping step was performed under identical conditions, including a temperature of 25°C and a reaction time of 30 seconds. The capping reagent solution was a 1:1 molar mixture of CapA and CapB, and the molar ratio of the capping reagent to the nucleic acid sequence linked on the solid-phase support was acetic anhydride:N-methylimidazole:nucleic acid sequence linked on the solid-phase support = 100:100:1.

[0285] Each oxidation step was performed under identical conditions, including a temperature of 25°C, a reaction time of 30 seconds, and 0.05M iodine solution as the oxidizing agent. The molar ratio of iodine to the nucleic acid sequence linked on the solid-phase support in the coupling step was 30:1. The reaction was carried out in a mixed solvent of tetrahydrofuran:water:pyridine = 7:2:1.

[0286] The conditions for each sulfidation reaction were identical, including a temperature of 25°C, a reaction time of 300 seconds, and a pyridine solution of 0.2 M hydroflavin as the sulfidation reagent. The molar ratio of the sulfidation reagent to the nucleic acid sequence linked on the solid-phase support in the coupling step was 120:1.

[0287] After the last nucleoside monomer was ligated, 250 μL of 20% diethylamine / acetonitrile solution was added to each column and allowed to pass through for 5 minutes, followed by complete flushing. The washing was repeated twice more, for a total of 7500 μL and 15 minutes. The oligonucleotide / solid support was then washed with 3 x 300 μL of acetonitrile and dried under vacuum. The nucleic acid sample was purified using a preparative C18 column via reversed-phase ion-pairing chromatography. 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. The purity of the eluent was determined, and nucleic acid samples meeting the purity requirements were combined. A precipitation method was then used to change the salt, concentrating the sample to dryness, adding sodium acetate aqueous solution and ethanol solution, collecting the precipitate, and washing with ethanol.

[0288] Antisense strand of synthetic double-stranded RNA conjugate

[0289] The antisense strand was synthesized via the solid-phase phosphoramide method, using a universal solid-phase support to initiate the cycle, following the nucleotide arrangement sequence in Table 4-6. The deprotection, coupling, capping, oxidation or sulfidation reaction conditions, cleavage and deprotection, purification and desalting conditions in the solid-phase synthesis method were the same as those for the synthesis of the sense strand.

[0290] 3. Deprotection

[0291] The synthesized nucleotide sequence linked with the vector was added to ammonia water with a concentration of 25 wt% (1 ml / μmol of ammonia water). The reaction was carried out at 35 °C for 16 hours. The remaining vector was removed by filtration, and the supernatant was concentrated to dryness under vacuum.

[0292] 4. Purification and desalting

[0293] Nucleic acid purification was achieved using a preparative ion chromatography column (Source 15Q) and gradient elution with NaBr.

[0294] 5. Annealing and freeze-drying

[0295] Take equimolar amounts of the sense and antisense chains, heat at 95°C for 2 minutes, and slowly cool to room temperature. Aliquot according to pharmacological department requirements, lyophilize into a solid powder, and store at -20°C for later use.

[0296] The sense and antisense strand sequences of the double-stranded RNA conjugates are shown in Tables 4, 5, and 6.

[0297] Table 4 shows the dual-target sequences of PCSK9 and ANGPTL3.

[0298] Table 5 shows the dual-target sequences of TTR and ANGPTL3.

[0299] Table 6 shows the dual-target sequences of TTR and FVII.

[0300] 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′-thiophosphate; Ams = 2′-O-methyladenosine-3′-thiophosphate; Ums = 2′-O-methyluridine-3′-thiophosphate; Cms = 2′-O-methylcytidine-3′-thiophosphate; Gms = 2′-O-methyl Guanosine-3′-thiophosphate; Af = 2′-fluoroadenosine-3′-phosphate; Uf = 2′-fluorouridine-3′-phosphate; Cf = 2′-fluorocytidine-3′-phosphate; Gf = 2′-fluoroguanosine-3′-phosphate; Afs = 2′-fluoroadenosine-3′-thiophosphate; Ufs = 2′-fluorouridine-3′-thiophosphate; Cfs = 2′-fluorocytidine-3′-thiophosphate; Gfs = 2′-fluoroguanosine-3′-thiophosphate; dT = 2′-deoxythymidine-3′-phosphate; m = 2′-O-methyl; f = 2′-fluorine; s = thiophosphate; invAb = reverse debasing deoxyribose residue; VP structure is: The example structure is as follows:

[0301] The D1 structure is The s-D1 structure is as follows: The D1-s structure is: The s-D1-s structure is: The structures of D1-D10 are shown in Table 2, and they are linked to RNA via phosphate esters or thiophosphate esters.

[0302] Example 13: In vivo efficacy of double-stranded RNA conjugates

[0303] To evaluate the in vivo efficacy of the double-stranded RNA conjugates in Table 6, 6-8 week old PCSK9 humanized mice (purchased from Jicui Pharmaceutical) were fed a high-fat diet at least 2 weeks in advance and were grouped according to their baseline low-density lipoprotein cholesterol levels, with 4 mice in each group. The grouping design is as follows:

[0304] A. Solvent group (2mg / kg);

[0305] B, ANGPTL3 single-target siRNA compound (1 mg / kg) test group;

[0306] C, PCSK9 single-target (Inclisiran) siRNA compound (1 mg / kg) test group;

[0307] D. The double-stranded RNA conjugate (2 mg / kg) test group in Table 4.

[0308] On day -1 (the day before administration), after a 4-hour fast, mouse plasma samples were obtained to measure baseline target protein and lipid levels. The siRNA compounds for each group were diluted with physiological saline and then administered subcutaneously on day 1 according to the experimental design, at doses of 1 mg / kg or 2 mg / kg, concentrations of 0.1 mg / mL or 0.2 mg / mL, and a volume of 10 mL / kg. Plasma samples were obtained on days 7, 14, 21, 28, 35, and 42 after a 4-hour fast. The levels of ANGPTL3 and PCSK9 proteins in the plasma were detected by ELISA according to the protocol provided by the supplier (R&D Systems), and the inhibition rate was calculated using the following formula. The results are shown in Table 7.

[0309] Target protein inhibition rate (%) relative to the solvent group = (1 - target protein expression level of a specific mouse / mean target protein expression level of mice in the solvent group) × 100

[0310] Inhibition rate (%) of a specific mouse target protein relative to baseline = (1 - expression level of the specific mouse target protein / baseline expression level of the corresponding target protein in the specific mouse) × 100

[0311] Table 7 shows the in vivo test results of the RNA conjugate at a dosage of 2 mg / kg.

[0312] As shown in Table 7, the double-stranded RNA conjugate of the present invention has excellent inhibitory activity against both ANGPTL3 and PCSK9.

[0313] The levels of four lipid parameters in mouse plasma, namely total triglycerides (TG), total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C), were detected using a biochemical analyzer (Mindray) or a kit provided by the supplier (Nanjing Jiancheng). The results showed that the double-stranded RNA conjugate in this invention has good inhibitory activity against the four lipid parameters in mice.

[0314] Example 14: Drug Efficacy in Mice

[0315] To evaluate the in vivo efficacy of the RNA conjugates in Table 5, 6-8 week old male C57BL / 6 mice (purchased from Guangdong Vital River) were used for drug administration and indicator detection. Plasma samples were obtained from mice on day -1, the day before drug administration, and baseline expression levels of murine TTR (mTTR) and murine Angptl3 (mAngptl3) proteins were detected using kits provided by the supplier (ALPCO and R&D). Mice were randomly assigned to groups of 4-5 mice each according to their baseline mAngptl3 protein levels. The siRNA compounds in each group were diluted with physiological saline and administered subcutaneously on day 0 according to the experimental design, with a dose of 2 mg / kg. The solvent group received an equal volume of physiological saline directly. Plasma was collected from mice on days 7, 14, 21, 28, and 35, and mTTR and mAngptl3 protein expression levels were detected. The inhibition rate at specific time points was then calculated using the following formula:

[0316] mTTR inhibition rate (%) of a specific mouse relative to baseline = (1 - mTTR expression level of the specific mouse / baseline mTTR expression level of that mouse) × 100

[0317] The inhibition rate of mAngptl3 in a specific mouse relative to the solvent group (%) = (1 - mAngptl3 expression level of the specific mouse / mean of the baseline expression level of mAngptl3 in the solvent group) × 100. The inhibition level of each compound on the target protein was taken as the average of the inhibition rates of the target protein in each group. The results are shown in Table 8.

[0318] Table 8 shows the in vivo test results.

[0319] Where A≥40%, mTTR is the inhibition rate relative to baseline, and ANGPTL3 is the inhibition rate relative to the solvent group.

[0320] As shown in Table 8, the double-stranded RNA conjugate of the present invention has excellent inhibitory activity against both mTTR and ANGPTL3.

[0321] Example 15 In vivo efficacy

[0322] To evaluate the in vivo efficacy of the RNA conjugates in Table 6, 6-8 week old male C57BL / 6 mice (purchased from Vital River Pharmaceuticals, Guangdong) were used for drug administration and indicator detection. Mice were randomly divided into 12 groups of 4 mice each. On the day of administration (day 0), the compounds were pre-dissolved in sterile saline and then administered subcutaneously to mice at doses of 3 mg / kg or 6 mg / kg, either the RNA conjugate solution or the solvent (sterile saline). Two weeks after administration (day 14), the expression of human F7 (FVII) and Ttr genes in the mouse liver was detected by RT-PCR. Gene expression levels were assessed by normalizing the relative expression levels of F7 or Ttr mRNA corresponding to Gapdh mRNA in the corresponding mice, and the inhibition rate of F7 or Ttr gene expression in a specific mouse (relative to the solvent group) was calculated using the following formula:

[0323] Inhibition rate (%) of F7 gene expression in a specific mouse = (1 - relative expression level of F7 mRNA in that mouse / mean relative expression level of F7 mRNA in the solvent group) × 100

[0324] Inhibition rate (%) of Ttr gene expression in a specific mouse = (1 - relative expression level of Ttr mRNA in that mouse / mean relative expression level of Ttr mRNA in the solvent group) × 100

[0325] Then the average inhibition rate of F7 and Ttr genes in each group of mice was calculated, and the results are shown in Table 9.

[0326] Table 9

[0327] Among them, B ≥ 60%.

[0328] As shown in Table 9, the double-stranded RNA conjugate of the present invention has excellent inhibitory activity against both mTTR and FVII.

[0329] Example 16 In vivo efficacy

[0330] To evaluate the in vivo efficacy of the RNA conjugates in Tables 5 and 6, 6-8 week old male C57BL / 6 mice (purchased from Guangdong Vital River) were used for drug administration and indicator detection. Mice were randomly divided into 12 groups of 4 mice each. Plasma was collected from mice before administration for baseline target protein level detection. On the day of administration (day 0), the compounds were pre-dissolved in sterile saline and then administered subcutaneously to mice at doses of 2 mg / kg, 4 mg / kg, or 10 mg / kg, either the RNA conjugate solution or the solvent (sterile saline). On days 7, 14, 22, and 28 post-administration, the concentrations of FactorVII, TTR, and Angptl3 proteins in mouse plasma were detected using ELISA according to the methods provided by the suppliers (Mouse Factor VII assay kit purchased from Solarbio, Mouse TTR assay kit purchased from ABCAM, Mouse Angptl3 assay kit purchased from R&D Systems). The inhibition rates of Factor VII (F7), TTR, and Angptl3 proteins were calculated using the following formulas:

[0331] Inhibition rate (%) of Factor VII expression in mice = (1 - plasma concentration of Factor VII at a specific time point in mice / baseline plasma concentration of Factor VII in mice) × 100

[0332] Inhibition rate of mouse TTR expression (%) = (1 - mouse plasma TTR concentration at a specific time point / mouse baseline plasma TTR concentration) × 100

[0333] Inhibition rate of Angptl3 expression in mice (%) = (1 - Plasma concentration of Angptl3 at a specific time point in mice / Baseline plasma concentration of Angptl3 in mice) × 100

[0334] The results are shown in Tables 10 and 11.

[0335] Table 10

[0336] Table 11

[0337] Among them, A ≥ 40%.

[0338] As shown in Tables 10 and 11, the double-stranded RNA conjugates of the present invention exhibit excellent inhibitory activity against mTTR and FVII, and mTTR and ANGPTL3.

[0339] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A siRNA conjugate, characterized in that, include: 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): The T is selected from -NH-C(O)- or -C(O)-NH-; The R 1 selected from said R 3 , R 4 together cyclize to a substituted said substituents are selected from the group consisting of a and b are the connection positions with the first or 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 c, d, and e are the connection positions with the first or second nucleic acid molecule; Wherein, x, m, n, p are independently selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, and q is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10.

2. The siRNA conjugate according to claim 1, characterized in that, 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 siRNA conjugate according to claim 1, wherein the linker is selected from the group consisting of:

4. The siRNA conjugate according to any one of claims 1 to 3, characterized in that, The carboxylic acid of the linker is linked to the ligand of the desialyl glycoprotein receptor ASGPR via an amide bond.

5. The siRNA conjugate according to claim 4, characterized in that, Compounds in which the carboxylic acid of the linker is linked via an amide bond to the ligand of the asialoglycoprotein receptor ASGPR are selected from the structures shown in formula (II), Z 1 , Z 2 is individually selected from N or CH; Y is selected from the group consisting of alkyl, C 3-8 cycloalkyl, L 1 , L 2 , L 3 is independently selected from W is selected from the target portion that can bind to cell surface receptors or the target portion containing a protective group; Wherein, u1, u2, v, t1, and t3 are independently selected from 0, 1, 2, 3, 4, 5, and 6; t2 is independently selected from 1, 2, 3, 4, 5, and 6; wherein T, R 1 , R 2 , x are as defined in claim 1.

6. The siRNA conjugate according to claim 5, characterized in that, The L 1 , L 2 , L 3 is individually selected from 7. The siRNA conjugate according to claim 5, wherein The ligand is selected from The is selected from:

8. The siRNA conjugate according to claim 4, wherein The compounds in which the carboxylic acid of the linker is linked to the ligand of the desialyl glycoprotein receptor ASGPR via an amide bond are selected from the structures shown in Tables 1 and 2.

9. The siRNA conjugate according to any one of claims 1-8, wherein, 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. The first target gene and the second target gene may be the same or different.

10. The siRNA conjugate according to any one of claims 1 to 8, characterized in that, The first nucleic acid molecule includes a sense strand and an antisense strand, and the second nucleic acid molecule includes a sense strand and an antisense strand. The sense strand of the first nucleic acid molecule is connected to the sense strand of the second nucleic acid molecule through the connector, or the antisense strand of the first nucleic acid molecule is connected to the antisense strand of the second nucleic acid molecule through the connector.

11. The siRNA conjugate according to any one of claims 1 to 8, characterized in that, At least one nucleotide in the first nucleic acid molecule and the second nucleic acid molecule is modified, and the modification is selected from: alkyl nucleotides, methoxy nucleotides, ethoxy nucleotides, methoxyethyl nucleotides, amino nucleotides, fluoronucleotides, deoxynucleotides, 5′-methylphosphonucleotides, 5′-C-methylphosphonucleotides, 2′-deoxy-2′-fluoronucleotides, vinylphosphonate nucleotides (VP), 5′-phosphonucleotides (P), thiophosphonucleotides, dithiophosphonucleotides, locked nucleic acids (LNA), morpholino oligonucleotides (PMO), ethylene glycol nucleic acid modification (GNA), hypoxanthine modification (I), reverse debasing deoxyribose residues (invAb), and 5′-methyldeoxycytosine nucleotides (m5dC).

12. An siRNA conjugate for inhibiting gene expression, characterized by, include: Connector The first nucleic acid molecule, which is used to inhibit the expression of the first target gene, and A second nucleic acid molecule, the second nucleic acid molecule being used to inhibit the expression of a second target gene, the first nucleic acid molecule and the second nucleic acid molecule being connected by the adapter, the adapter being connected by a phosphate ester (p) or a thiophosphate ester (s), the adapter being as described in any one of claims 1-11; The first target gene and the 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, and FVII.

13. A compound, characterized in that, selected from the structures shown in compounds 1-10, B1, B2, B3, B4, C1-C10.

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