Double-stranded SIRNA, conjugate thereof and application of double-stranded SIRNA

Abstract

The invention discloses a series of double-stranded siRNA (small interfering Ribonucleic Acid) for inhibiting LPA (Lipoprotein A) expression as well as conjugates and application thereof.

Description

Double-stranded siRNA, its conjugate and its application

[0001] Citation of Related Applications

[0002] This application claims the Chinese invention patent application No. 202310598735.8 filed with the State Intellectual Property Office of China on May 24, 2023, the Chinese invention patent application No. 202311140396.5 filed with the State Intellectual Property Office of China on September 5, 2023, the Chinese invention patent application No. 202311601195.0 filed with the State Intellectual Property Office of China on November 27, 2023, and the Chinese invention patent application No. 202311601195.0 filed with the State Intellectual Property Office of China on January 25, 2024. The priority and benefits of Chinese invention patent application No. 202410112975.7 filed with the State Intellectual Property Office of China, Chinese invention patent application No. 202410505495.7 filed with the State Intellectual Property Office of China on April 25, 2024, and Chinese invention patent application No. 202410621293.9 filed with the State Intellectual Property Office of China on May 17, 2024, the entire contents of which are incorporated herein by reference in their entirety. Technical Field

[0003] The present invention relates to the field of medicine, and in particular to a series of double-stranded siRNAs for inhibiting LPA expression, conjugates thereof and applications thereof. Background Art

[0004] Lp(a) is a lipoprotein particle synthesized in the liver and consists of low-density lipoprotein (LDL)-like particles and apolipoprotein a [Apo(a), also known as LPA], which are covalently bound by disulfide bonds. Apo(a) is encoded by the highly polymorphic LPA gene, in which a variable number of KIV type 2 repeats results in apo(a) isoforms of various sizes. The LPA gene evolved from the plasminogen gene (PLG), and the two genes have highly homologous sequences (see Konrad Schmidt et al., J. Lipid Res 2016(57), 1339-1359). Plasma Lp(a) levels vary nearly 1000-fold between individuals, with approximately 20-30% of the population having highly elevated Lp(a) levels (≥50 mg / dL). For related literature, see Hopewell et al., J. Intern. Med., (2013) 273(1):260-8; Wilson et al., Clinical Lipidology (2019) 13(3):374–92. High Lp(a) levels and low copy number KIV type 2 apo(a) subtypes are associated with an increased risk of cardiovascular disease. High Lp(a) is an independent high-risk factor for atherosclerotic cardiovascular disease (ASCVD), including coronary heart disease, myocardial infarction, stroke, peripheral artery disease, calcified aortic valve disease, and atherosclerosis.

[0005] In RNA interference (RNAi) technology, small interfering RNA (siRNA) binds to the RNA-induced silencing complex (RISC), displacing the sense strand and allowing the antisense strand to bind to the complementary RNA (target RNA) within the RISC. Once bound, the target RNA is cleaved by the RNA endonuclease Argonaute (AGO) within the RISC and then further degraded by RNA exonucleases. RNAi has been used to develop a new class of therapeutic agents for diseases caused by genetic abnormalities or aberrant gene expression. Elevated Lp(a) levels are an independent risk factor for atherosclerotic cardiovascular disease (ASCVD), and Lp(a) inhibitors are urgently needed to address the high prevalence of some cardiovascular diseases. RNA-targeted therapies, with their high selectivity and affinity for target mRNAs, have been successfully applied in various disease areas and represent a promising intervention for lowering Lp(a). siRNA targeting liver LPA can effectively reduce Lp(a) levels by silencing the expression of the Apo(a) gene and blocking Apo(a) protein synthesis.

[0006] Summary of the Invention

[0007] The present invention provides a double-stranded siRNA or a pharmaceutically acceptable salt thereof for inhibiting LPA expression, comprising a sense strand and an antisense strand capable of forming a double-stranded region, wherein the sense strand comprises any one of the sequences shown in SEQ ID NOs: 1-18, and the antisense strand comprises any one of the sequences shown in SEQ ID NOs: 19-26, and each nucleotide on the sense strand and the antisense strand is optionally a modified nucleotide.

[0008] In some embodiments of the present invention, at least one nucleotide on the sense strand and the antisense strand is a modified nucleotide.

[0009] In some embodiments of the present invention, there are 0, 1, 2, 3 or 4 unmodified nucleotides on the sense strand and the antisense strand, and the rest are modified nucleotides.

[0010] The present invention provides a double-stranded siRNA or a pharmaceutically acceptable salt thereof, which comprises a sense strand and an antisense strand capable of forming a double-stranded region, wherein the antisense strand comprises any one of the antisense strand sequences shown in Table 1, and each nucleotide in the antisense strand sequence is optionally a modified nucleotide.

[0011] In some embodiments of the present invention, in the double-stranded siRNA or a pharmaceutically acceptable salt thereof, the sense strand comprises any sense strand sequence shown in Table 1, and each nucleotide in the sense strand sequence is optionally a modified nucleotide.

[0012] In some embodiments of the present invention, the double-stranded siRNA comprises any of the double strands shown in Table 1, wherein each nucleotide on the double strand is independently optionally a modified nucleotide.

[0013] In some embodiments of the present invention, the modified nucleotides are selected from 2'-OMe modified nucleotides, 2'-F modified nucleotides and 2'-deoxynucleotides.

[0014] In some embodiments of the present invention, the 5' end of the antisense strand optionally comprises a nucleotide selected from mDVPU, B29, B30, B33, B34 and B36, wherein

[0015] The mDVPU is

[0016] The B29 is

[0017] The B30 is

[0018] The B33 is

[0019] The B34 is

[0020] The B36 is

[0021] In some embodiments of the present invention, the 5' end of the sense strand optionally comprises a nucleotide selected from invAb, wherein the invAb is

[0022] In some embodiments of the present invention, each internucleoside linking group of the double-stranded region of the double-stranded siRNA is independently a phosphodiester internucleoside linking group or a phosphorothioate internucleoside linking group.

[0023] The present invention provides a double-stranded siRNA or a pharmaceutically acceptable salt thereof, comprising a sense strand and an antisense strand capable of forming a double-stranded region, wherein each nucleotide in the double-stranded siRNA is independently a modified or unmodified nucleotide, and the antisense strand comprises any one of the antisense strand sequences shown in Table 1.

[0024] In some embodiments of the present invention, in the double-stranded siRNA or a pharmaceutically acceptable salt thereof, the sense strand comprises any one of the sense strand sequences shown in Table 1.

[0025] In some embodiments of the present invention, the double-stranded siRNA comprises any of the double strands shown in Table 1, wherein each nucleotide on the double strand is independently optionally a modified nucleotide.

[0026] In some embodiments of the present invention, the double-stranded siRNA is as shown in Table 1, for example, U-S1 is a double-stranded structure consisting of SEQ ID NO: 1 and SEQ ID NO: 19.

[0027] Table 1 Double-stranded siRNA

[0028] The Z of the present invention is

[0029] In some embodiments of the present invention, the intermediate structure for synthesizing the above Z is shown in formula (ZM):

[0030] The R of the present invention is

[0031] The dR of the present invention is

[0032] In some embodiments of the present invention, the intermediate structure for synthesizing the above dR is shown in formula (dR-M):

[0033] In some embodiments of the invention, the sense strand of the double-stranded siRNA comprises 0, 1, 2, 3, 4, 5 or 6 unmodified nucleotides.

[0034] In some embodiments of the invention, the antisense strand of the double-stranded siRNA comprises 0, 1, 2, 3, 4, 5 or 6 unmodified nucleotides.

[0035] In some embodiments of the present invention, the sense strand of the double-stranded siRNA comprises 0, 1 or 2 nucleosides substituted with Z nucleotides.

[0036] In some embodiments of the present invention, the antisense strand of the double-stranded siRNA comprises 0, 1 or 2 nucleosides substituted with Z nucleotides.

[0037] In some embodiments of the present invention, the sense strand of the double-stranded siRNA comprises 0, 1, 2 or 3 nucleosides substituted with R nucleotides.

[0038] In some embodiments of the present invention, the antisense strand of the double-stranded siRNA comprises 0, 1, 2 or 3 nucleosides substituted with R nucleotides.

[0039] In some embodiments of the present invention, the double-stranded siRNA comprises a double-stranded structure shown in Table 2, for example: S1 represents a double-stranded structure consisting of SEQ ID NO: 27 and SEQ ID NO: 56.

[0040] Table 2 Modified double-stranded siRNA

[0041] In some embodiments of the present invention, the double-stranded siRNA is S26, S41, S42, S49, S51, S55, S57, S58, S59, S61, S62, S63, or S64.

[0042] The mDVPU of the present invention is B29 is B30 is B33 is B34 B36 invAb is

[0043] In some embodiments of the present invention, the intermediate structures for synthesizing the above-mentioned mDVPU, B29, B30, B33, B34 and B36 are shown in the formula mDVPU-M, B29-M, B30-M, B33-M, B34-M and B36-M, respectively:

[0044] The present invention also provides a double-stranded siRNA conjugate or a pharmaceutically acceptable salt thereof for inhibiting LPA expression. The double-stranded siRNA conjugate is formed by the double-stranded siRNA conjugated ligand defined in any of the above technical solutions.

[0045] In some embodiments of the present invention, the double-stranded siRNA conjugate or a pharmaceutically acceptable salt thereof, wherein the double-stranded siRNA comprises a sense strand and an antisense strand capable of forming a double-stranded region, wherein the antisense strand comprises any one of the antisense strand sequences shown in Table 1, and each nucleotide in the antisense strand sequence is optionally a modified nucleotide.

[0046] In some embodiments of the present invention, the double-stranded siRNA conjugate or a pharmaceutically acceptable salt thereof, wherein the sense strand comprises any sense strand sequence shown in Table 1, and each nucleotide in the antisense strand sequence is optionally a modified nucleotide.

[0047] In some embodiments of the present invention, in the double-stranded siRNA conjugate or a pharmaceutically acceptable salt thereof, each nucleotide on the double strand is independently optionally a modified nucleotide.

[0048] In some embodiments of the present invention, in the double-stranded siRNA conjugate or a pharmaceutically acceptable salt thereof, the modified nucleotide is selected from 2'-OMe modified nucleotides, 2'-F modified nucleotides, and 2'-deoxynucleotides.

[0049] In some embodiments of the present invention, in the double-stranded siRNA conjugate or a pharmaceutically acceptable salt thereof, the 5' end of the antisense strand optionally comprises a nucleotide selected from mDVPU, B29, B30, B33, B34 or B36.

[0050] In some embodiments of the present invention, in the double-stranded siRNA conjugate or a pharmaceutically acceptable salt thereof, the 5' end of the sense strand optionally comprises a nucleotide selected from invAb.

[0051] In some embodiments of the present invention, the double-stranded siRNA conjugate or a pharmaceutically acceptable salt thereof, wherein the double-stranded siRNA comprises any one of the double strands shown in Table 2.

[0052] In some embodiments of the present invention, in the double-stranded siRNA conjugate or a pharmaceutically acceptable salt thereof, the ligand is conjugated to any position of the double-stranded siRNA.

[0053] In some embodiments of the present invention, in the double-stranded siRNA conjugate or a pharmaceutically acceptable salt thereof, the ligand is conjugated to the 3' end of the sense strand of the double-stranded siRNA.

[0054] In some embodiments of the present invention, in the double-stranded siRNA conjugate or a pharmaceutically acceptable salt thereof, the ligand is conjugated to the double-stranded siRNA via a phosphodiester bond or a phosphorothioate bond.

[0055] In some embodiments of the present invention, the double-stranded siRNA conjugate or a pharmaceutically acceptable salt thereof, wherein the ligand is one or more GalNAc derivatives attached using a divalent, trivalent or tetravalent branched linker.

[0056] In some embodiments of the present invention, the double-stranded siRNA conjugate or a pharmaceutically acceptable salt thereof, wherein the ligand is selected from D01, D08, D10, D11, D18, D21 or L96,

[0057] In some embodiments of the present invention, the intermediate structure for synthesizing the above-mentioned D08 is shown in formula (D08-M):

[0058] In some embodiments of the present invention, the intermediate structure for synthesizing the above-mentioned D10 is shown in formula (D10-M):

[0059] In some embodiments of the present invention, the intermediate structure for synthesizing the above-mentioned D11 is shown in formula (D11-M):

[0060] In some embodiments of the present invention, the intermediate structure of the synthesis of the above-mentioned D18 is shown in formula (D18-M):

[0061] In some embodiments of the present invention, the intermediate structure for synthesizing the above-mentioned D21 is shown in formula (D21-M):

[0062] Some other solutions of the present invention are obtained by arbitrarily combining the above solutions.

[0063] In the present invention, the connection method of the double-stranded siRNA conjugate is as follows:

[0064] Among them, SS represents the sense strand and AS represents the antisense strand;

[0065] X is selected from O and S.

[0066] The double-stranded siRNA conjugates provided by the present invention are shown in Table 3.

[0067] Table 3 Double-stranded siRNA conjugates

[0068] In some embodiments of the present invention, the double-stranded siRNA conjugate is selected from Z27, Z42, Z43, Z46, Z50, Z52, Z56, Z58, Z59, Z60, Z62, Z63, Z64 and Z65.

[0069] In some embodiments of the present invention, the double-stranded siRNA conjugate is as shown in Formula I:

[0070] SS is the sense strand, AS is the antisense strand; wherein X is O; the sense strand and the antisense strand are selected from the combination shown in any one of (1) to (13):

[0071] (1) the sense strand is as shown in SEQ ID NO: 49, and the antisense strand is as shown in SEQ ID NO: 80;

[0072] (2) the sense strand is as shown in SEQ ID NO: 49, and the antisense strand is as shown in SEQ ID NO: 81;

[0073] (3) the sense strand is as shown in SEQ ID NO:43, and the antisense strand is as shown in SEQ ID NO:66;

[0074] (4) the sense strand is as shown in SEQ ID NO: 51, and the antisense strand is as shown in SEQ ID NO: 86;

[0075] (5) the sense strand is as shown in SEQ ID NO: 53, and the antisense strand is as shown in SEQ ID NO: 88;

[0076] (6) the sense strand is as shown in SEQ ID NO:43, and the antisense strand is as shown in SEQ ID NO:92;

[0077] (7) the sense strand is as shown in SEQ ID NO:43, and the antisense strand is as shown in SEQ ID NO:94;

[0078] (8) the sense strand is as shown in SEQ ID NO:43, and the antisense strand is as shown in SEQ ID NO:95;

[0079] (9) the sense strand is as shown in SEQ ID NO:43, and the antisense strand is as shown in SEQ ID NO:96;

[0080] (10) the sense strand is as shown in SEQ ID NO: 49, and the antisense strand is as shown in SEQ ID NO: 94;

[0081] (11) the sense strand is as shown in SEQ ID NO: 49, and the antisense strand is as shown in SEQ ID NO: 95;

[0082] (12) the sense strand is as shown in SEQ ID NO: 49, and the antisense strand is as shown in SEQ ID NO: 96;

[0083] (13) The positive strand is shown in SEQ ID NO: 55, and the antisense strand is shown in SEQ ID NO: 95.

[0084] The present invention also provides the use of the double-stranded siRNA, its conjugate, its salt or the salt of its conjugate in the preparation of a drug for reducing Lp(a) levels.

[0085] In some embodiments of the present invention, the above application is characterized in that the drug for lowering Lp(a) levels is a drug for treating cardiovascular diseases.

[0086] In some embodiments of the present invention, the above-mentioned cardiovascular diseases include but are not limited to: Berger's disease, peripheral artery disease, coronary artery disease, metabolic syndrome, aortic valve stenosis, aortic valve regurgitation, aortic dissection, retinal artery occlusion, mesenteric ischemia, superior mesenteric artery occlusion, renal artery stenosis, stable / unstable angina, acute coronary syndrome, heterozygous or homozygous familial hypercholesterolemia, hyperlipoprotein betalipoproteinemia, cerebral vascular atherosclerosis and venous thrombosis.

[0087] Technical Effects

[0088] The double-stranded siRNA, conjugates, salts thereof, or salts of their conjugates of the present invention have excellent activity in reducing Lp(a) levels. They demonstrate excellent efficacy in an in vitro mouse HDI model and a cynomolgus monkey PD model, significantly reducing LPA mRNA. Furthermore, the double-stranded siRNA conjugates are stable and exhibit long-lasting efficacy in in vivo experiments. They can be stored in vivo for long periods of time and slowly released, enabling long-term intermittent dosing. The double-stranded siRNA conjugates have low off-target risk, no cytotoxicity risk, low immunogenicity risk, and excellent safety. Furthermore, they are easy to synthesize, facilitating industrial production.

[0089] Definition and Description

[0090] Unless otherwise indicated, the following terms and phrases used herein are intended to have the following meanings. The absence of a specific definition for a particular term or phrase should not be construed as undefined or ambiguous, but rather should be understood as meaning understood by one of ordinary skill in the art. When a trade name appears herein, it is intended to refer to the corresponding commercial product or its active ingredient.

[0091] Unless otherwise stated, the terms "comprises," "includes," and "contains" or equivalents herein are open-ended expressions, meaning that in addition to the listed elements, components, or steps, other unspecified elements, components, or steps may also be included.

[0092] The terms "optional" or "optionally" mean that the subsequently described event or circumstance may but need not occur, and that the description includes instances where said event or circumstance occurs and instances where it does not.

[0093] Unless otherwise indicated, the term "nucleic acid" as used herein refers to a polymer containing at least two nucleotides (i.e., deoxyribonucleotides or ribonucleotides) in single-stranded or double-stranded form, and includes DNA and RNA. A "nucleotide" contains the sugar deoxyribose (DNA) or ribose (RNA), a base, and a phosphate group. The nucleotides are linked together by the phosphate group. A "base" includes purines and pyrimidines, which also include the natural compounds adenine, thymine, guanine, cytosine, uracil, inosine, and natural analogs.

[0094] Unless otherwise indicated, the term "oligonucleotide" or "oligonucleotide" herein refers to a polymer or oligomer of nucleotide or nucleoside monomers composed of naturally occurring bases, sugars, and intersugar (backbone) linkages, and also includes polymers or oligomers containing non-naturally occurring monomers or portions thereof, which function similarly.

[0095] Unless otherwise specified, the "oligonucleotide" or "oligonucleotide" of the present invention is a nucleotide sequence containing 14 to 30 nucleotides or nucleotide base pairs. In some embodiments of the present invention, the oligonucleotide has a nucleobase sequence that is at least partially complementary to a coding sequence in a target nucleic acid or target gene expressed in a cell. The nucleotides may be optionally modified. In some embodiments of the present invention, after the oligonucleotide is delivered to a cell expressing a gene, the oligonucleotide is able to inhibit or block the expression of the gene in vitro or in vivo. "Oligonucleotide" includes, but is not limited to, single-stranded oligonucleotides, single-stranded antisense oligonucleotides, short interfering RNA (siRNA), double-stranded RNA (dsRNA), microRNA (miRNA), short hairpin RNA (shRNA), ribozymes, interfering RNA molecules, and Dicer enzyme substrates.

[0096] Unless otherwise specified, "short interfering RNA (siRNA)" as used herein refers to a class of RNA molecules, 14-30 base pairs in length, similar to miRNA, that operate within the RNA interference (RNAi) pathway, interfering with the translation of mRNA of a specific gene with a complementary nucleotide sequence, leading to mRNA degradation. Short interfering RNA (siRNA) as used herein includes double-stranded siRNA (including both sense and antisense strands) and single-stranded siRNA (e.g., including only the antisense strand).

[0097] Unless otherwise specified, "inhibit" as used herein, when referring to the expression of a given gene, means that gene expression is reduced when the cell, cell population or tissue is treated with the oligonucleotides of the invention, compared to cells, cell populations or tissues that have not been so treated.

[0098] Unless otherwise specified, a "sequence" or "nucleotide sequence" as used herein refers to an order or sequence of nucleobases or nucleotides described by a sequence of letters using standard nucleotide nomenclature.

[0099] Unless otherwise specified, the "antisense strand" (AS), template strand, or "guide strand" of the present invention refers to the strand in an oligonucleotide compound that is substantially complementary to the corresponding region of the target sequence (eg, LPA mRNA).

[0100] Unless otherwise specified, the "sense strand," "sense strand," or "coding strand" (SS) herein refers to a strand that forms a substantially complementary region with the antisense strand. "Substantially complementary" means that corresponding positions of the two sequences can be completely complementary or contain one or more mismatches. When mismatches exist, they typically contain no more than 3, 2, or 1 base pairs. In a double-stranded nucleic acid molecule, bases on one strand pair with 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 (G) always pairs with the pyrimidine base cytosine (C).

[0101] Unless otherwise indicated, the "pharmaceutically acceptable conjugating groups" of the present invention facilitate the in vivo delivery of nucleic acids and compositions suitable for in vivo therapeutic use. In some embodiments, the "pharmaceutically acceptable conjugating groups" of the present invention facilitate enhancing the affinity of nucleic acids and compositions suitable for in vivo therapeutic use for targets (target tissues / cells).

[0102] In some embodiments of the present invention, the "modification" of the present invention refers to the modification of the base, sugar ring, nucleotide and / or the linkage between the groups of the nucleotide or the replacement nucleotide. Exemplarily, the "modification" of the present invention includes but is not limited to MOE modification, methoxy modification, fluoro modification, (E)-vinyl phosphate modification, thiophosphate modification or replacement of the nucleotide with GNA (glycerol nucleic acid) and the like. Exemplarily, the "modification" of the present invention may include one or more locked nucleic acids (LNA). Locked nucleic acids are nucleotides with a modified ribose moiety, wherein the ribose moiety includes an additional bridge connecting the 2' carbon and the 4' carbon. This structure effectively "locks" the ribose in a 3'-endo conformation.

[0103] Unless otherwise specified, the "nucleotides are optionally modified" described in the present invention means that each nucleotide can independently be an unmodified nucleotide or a modified nucleotide, and the modification on each modified nucleotide is also independent. The "modification" includes but is not limited to modifications to nucleobases, modifications to ribose, and modifications to phosphates. The "unmodified nucleotides" refer to nucleotides composed of naturally occurring nucleobases, natural sugar rings, and natural phosphates. The "modified nucleotides" refer to nucleotides containing at least one of modified nucleobases, modified sugar rings, and modified phosphates. In some embodiments of the present invention, "modified nucleotides" refer to nucleotides composed of modified nucleobases, and / or modified sugar rings, and / or modified phosphates. In some embodiments of the present invention, the "modified nucleotides" consist of modified nucleobases, natural sugar rings and natural phosphates; in some embodiments of the present invention, the "modified nucleotides" consist of natural nucleobases, modified sugar rings and natural phosphates; in some embodiments of the present invention, the "modified nucleotides" consist of natural nucleobases, natural sugar rings and modified phosphates; in some embodiments of the present invention, the "modified nucleotides" consist of natural nucleobases, modified sugar rings and modified phosphates; in some embodiments of the present invention, the "modified nucleotides" consist of modified nucleobases, natural sugar rings and modified phosphates; in some embodiments of the present invention, the "modified nucleotides" consist of modified nucleobases, modified sugar rings and natural phosphates; in some embodiments of the present invention, the "modified nucleotides" consist of modified nucleobases, modified sugar rings and modified phosphates.

[0104] Unless otherwise specified, the "natural sugar ring" of the present invention is selected from the five-membered sugar ring of 2'-OH.

[0105] Unless otherwise specified, the "natural bases" of the present invention are selected from the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U).

[0106] Unless otherwise specified, the "modified nucleobase" of the present invention refers to a 5-12 membered saturated, partially unsaturated or aromatic heterocycle other than a natural base, including a monocyclic or condensed ring, specific examples of which include but are not limited to thiophene, thianthrene, furan, pyran, isobenzofuran, benzothiazine, pyrrole, imidazole, substituted or unsubstituted triazole, pyrazole, isothiazole, isoxazole, pyridazine, indolizine, indole, isoindole, isoquinoline, quinoline, naphthopyridine, quinazoline, carbazole, phenanthridine, piperidine, phenazine, phenazine, phenothiazine, furane, phenoxazine, pyrrolidine, pyrroline, imidazolidine, imidazoline, pyrazolidine, 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 2-aminoadenine, 2-aminoguanidine Purine, 2-propyl adenine and guanine and other alkyl derivatives, 2-thiouracil, 2-thiothymine, 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azouracil, 6-azocytosine, 6-azothymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-sulfhydryl, 8-sulfanyl, 8-hydroxy and other 8-substituted adenine and guanine, 5-halo, especially 5-bromo, 5-trifluoromethyl and other 5-substituted uracil and cytosine, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine, and 3-deazaguanine and 3-deazaadenine, etc.

[0107] Unless otherwise specified, the "modified sugar ring" of the present invention may include, but is not limited to, one of the following modifications at the 2' position: H; F; O-, S- or N-alkyl; O-, S- or N-alkenyl; O-, S- or N-alkynyl; or O-alkyl-O-alkyl, wherein alkyl, alkenyl and alkynyl groups may be substituted or unsubstituted C1 to C 10 Alkyl or C2 to C 10 Exemplary suitable modifications include O[(CH2) n O] m CH3, O(CH2) n OCH3, O(CH2) n NH2, O(CH2) n CH3, O(CH2) n ONH2, and O(CH2) n ON[(CH2) n CH3)]2, wherein n and m are from 1 to 10. In other embodiments, the 2' position includes but is not limited to one of the following modifications: substituted or unsubstituted C1 to C 10Lower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3, SO2CH3, ONO2, NO2, N3, NH2, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, RNA cleavage group, reporter group, intercalator, group for improving the pharmacokinetic properties of iRNA, or group for improving the pharmacodynamic characteristics of iRNA, and other substituents with similar properties.

[0108] Unless otherwise specified, the "modified phosphates" of the present invention include but are not limited to: 1. thiophosphate modification, wherein the "thiophosphate" includes (R)- and (S)-isomers and / or mixtures thereof; 2. (E)-vinyl phosphate modification (E-VP or VP), unless otherwise specified, VP in the present invention refers to E-type vinyl phosphate modification, for example: the structure of 5'-terminal mVPU is

[0109] In some embodiments of the present invention, the modified nucleotides may comprise one or more locked nucleic acids (LNAs). LNAs are nucleotides with a modified ribose moiety, wherein the ribose moiety includes an additional bridge connecting the 2' carbon and the 4' carbon. This structure effectively "locks" the ribose in a 3'-endo conformation.

[0110] In some embodiments of the present invention, the modified nucleotides include one or more monomers that are UNA (unlocked nucleic acid) nucleotides. UNA is an unlocked acyclic nucleic acid in which any sugar bonds have been removed, thereby forming an unlocked "sugar" residue. In one example of the present invention, UNA also encompasses monomers in which the bond between C1'-C4' has been removed (i.e., a covalent carbon-oxygen-carbon bond between the C1' and C4' carbons). In another example of the present invention, the C2'-C3' bond of the sugar (i.e., a covalent carbon-carbon bond between the C2' and C3' carbons) has been removed.

[0111] In some embodiments of the present invention, the modified nucleotide comprises one or more monomers of GNA (glycerol nucleic acid) nucleotides. GNA includes GNA-A, GNA-T, GNA-C, GNA-G and GNA-U. The structure of GNA-A is The structure of GNA-T is The structure of GNA-C is The structure of GNA-G is The structure of GNA-U is

[0112] In some embodiments of the present invention, the nucleotide of modification also can comprise one or more bicyclic sugar moieties." bicyclic sugar " is furanyl (furanosyl) ring modified by the bridge connection of two atoms." bicyclic nucleoside " (" BNA ") is the nucleoside with sugar moiety, and described sugar moiety comprises the bridge of two carbon atoms connecting sugar ring, forms dicyclic system thus.In specific embodiments, bridge connects 4 '-carbon and 2 '-carbon of sugar ring.

[0113] Unless otherwise indicated, the term "double-stranded siRNA" as used herein refers to a complex of ribonucleic acid molecules having a double-stranded structure comprising two antiparallel and substantially complementary nucleotide chains that have "sense" and "antisense" orientations relative to the target RNA. In the present invention, "complementary" has the meaning known to those skilled in the art, i.e., in a double-stranded nucleic acid molecule, the bases of one chain are paired with the bases on the other chain in a complementary manner. The purine base adenine (A) always pairs with the pyrimidine base uracil (U); the purine base guanine (G) always pairs with the pyrimidine base cytosine (C). Each base pair includes a purine and a pyrimidine. When adenine on one chain always pairs with uracil on the other chain, and guanine always pairs with cytosine, the two chains are considered to be complementary to each other, and the sequence of the chain can be inferred from the sequence of its complementary chain.

[0114] Unless otherwise indicated, the term "substantially complementary" in the present invention means that the corresponding positions of the two sequences can be completely complementary or there can be one or more mismatches. When mismatches exist, there are usually no more than 5, 4, 3, 2 or 1 mismatched base pairs.

[0115] Unless otherwise indicated, the term "conjugation" in the present invention refers to the covalent linkage of two or more chemical moieties, each with a specific function, to each other; accordingly, "conjugate" refers to a compound formed by covalent linkage of the chemical moieties.

[0116] Unless otherwise specified, the term "double-stranded siRNA conjugate" of the present invention refers to a compound formed by linking double-stranded siRNA and a pharmaceutically acceptable conjugating group, and the double-stranded siRNA and the pharmaceutically acceptable conjugating group are covalently linked.

[0117] Unless otherwise indicated, the "covalent linkage" described in the present invention includes but is not limited to "phosphate bond", "phosphodiester bond", "phosphorothioate bond" and the like.

[0118] In some embodiments of the present invention, the "modification" of the nucleotides described herein includes modification of the nucleobase, modification of the sugar ring, modification of the internucleotide linkage, etc. Exemplarily, the "modification" of the nucleotides described herein includes, but is not limited to, methoxy modification, fluorination modification, (E)-vinyl phosphate modification, phosphorothioate modification, or replacement of the nucleotide with (S)-glycerol nucleic acid, etc.

[0119] In some embodiments of the present invention, "A", "U", "C" and "G" in the sequence represent adenosine, uridine, cytidine and guanosine, respectively. Before the "A", "U", "C" and "G", "m" represents that the nucleotide or the group replacing the nucleotide is modified with 2'-O-methyl (belonging to methoxy modification), such as mA representing 2'-O-methyladenosine; "f" represents that the nucleotide or the group replacing the nucleotide is modified with 2'-fluoro nucleotide (belonging to fluorinated modification), such as fG representing 2'-fluoroguanosine; "d" represents that the nucleotide or the group replacing the nucleotide is deoxy-modified at the 2' position, such as dA representing 2'-deoxyadenosine, and dT representing 2'-deoxythymidine.

[0120] In some embodiments of the present invention, the "s" between "Z," "R," "A," "U," "C," and "G" indicates that the nucleotides are linked via a phosphorothioate group. For example, "fGs,mA" indicates that fG and mA are linked via a phosphorothioate bond, i.e., 2'-fluoroguanosine and 2'-O-methyladenosine are linked via a phosphorothioate bond. For example, the chemical structure of 5'-fCs,mG,fU,mA,Z-3' is as follows:

[0121] Unless otherwise specified, the term "multiple" and "multivalent" in the present invention refers to an integer greater than or equal to 2, including but not limited to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20, up to the theoretical upper limit of the number of GalNAc derivatives attached to the siRNA or branched linker.

[0122] The sense strand or antisense strand of the double-stranded siRNA of the present invention may also include "overhangs," e.g., unpaired protruding nucleotides that are not directly involved in the RNA double helix structure, wherein the RNA double helix structure is generally formed by a "sense strand" and an "antisense strand" pair as defined herein. Exemplarily, such overhangs may include one or more modified or unmodified C, G, U, A, Z, R, and T.

[0123] The compounds of the present invention may exist in specific geometric or stereoisomeric forms. The present invention contemplates all such compounds, including (R)- and (S)-enantiomers, diastereomers, and racemic mixtures and other mixtures thereof, such as enantiomerically or diastereomerically enriched mixtures, all of which are within the scope of the present invention. Additional asymmetric carbon atoms may be present in substituents such as alkyl groups. All such isomers and mixtures thereof are encompassed within the scope of the present invention.

[0124] Unless otherwise indicated, the term "enantiomer" or "optical isomer" refers to stereoisomers that are mirror images of one another.

[0125] Unless otherwise indicated, the term "diastereomer" refers to stereoisomers that have two or more chiral centers and that are not mirror images of each other.

[0126] Unless otherwise specified, use a solid wedge key. and dotted wedge key To indicate the absolute configuration of a stereocenter, use a straight solid bond and straight dashed key Indicate the relative configuration of stereocenters with a wavy line Indicates a wedge-shaped solid key or dotted wedge key Or use a wavy line Indicates a straight solid bond and / or straight dashed key

[0127] Unless otherwise indicated, the terms "enriched in one isomer," "isomerically enriched," "enriched in one enantiomer," or "enantiomerically enriched" mean that the content of one isomer or enantiomer is less than 100%, and the content of that isomer or enantiomer is greater than or equal to 60%, or greater than or equal to 70%, or greater than or equal to 80%, or greater than or equal to 90%, or greater than or equal to 95%, or greater than or equal to 96%, or greater than or equal to 97%, or greater than or equal to 98%, or greater than or equal to 99%, or greater than or equal to 99.5%, or greater than or equal to 99.6%, or greater than or equal to 99.7%, or greater than or equal to 99.8%, or greater than or equal to 99.9%.

[0128] Unless otherwise indicated, the term "isomer excess" or "enantiomeric excess" refers to the difference between the relative percentages of two isomers or two enantiomers. For example, if the content of one isomer or enantiomer is 90% and the content of the other isomer or enantiomer is 10%, the isomer or enantiomeric excess (ee value) is 80%.

[0129] Optically active (R)- and (S)-isomers as well as D and L isomers can be prepared by chiral synthesis or chiral reagents or other conventional techniques. If one enantiomer of a compound of the present invention is desired, it can be prepared by asymmetric synthesis or derivatization with a chiral auxiliary, wherein the resulting diastereomeric mixture is separated and the auxiliary group is cleaved to provide the pure desired enantiomer. Alternatively, when the molecule contains a basic functional group (such as an amino group) or an acidic functional group (such as a carboxyl group), a diastereomeric salt is formed with an appropriate optically active acid or base, and then the diastereoisomers are split by conventional methods known in the art and then recovered to obtain the pure enantiomer. In addition, the separation of enantiomers and diastereoisomers is typically accomplished by using chromatography, which employs a chiral stationary phase and is optionally combined with a chemical derivatization method (e.g., carbamate generated from an amine). The compounds of the present invention may contain non-natural ratios of atomic isotopes on one or more atoms constituting the compound. For example, the compound may be labeled with a radioactive isotope, such as tritium ( 3 H), iodine-125( 125 I) or C-14( 14 C). For example, deuterated drugs can be formed by replacing hydrogen with heavy hydrogen. The bond between deuterium and carbon is stronger than the bond between ordinary hydrogen and carbon. Compared with non-deuterated drugs, deuterated drugs have advantages such as reduced toxic side effects, increased drug stability, enhanced efficacy, and prolonged drug biological half-life. All isotopic variations of the compounds of the present invention, whether radioactive or not, are included within the scope of this invention.

[0130] The term "salt" refers to salts of the compounds of the present invention, prepared by reacting the compounds of the present invention with relatively nontoxic acids or bases. When the compounds of the present invention contain relatively acidic functional groups, base addition salts can be obtained by contacting the compounds with a sufficient amount of base in a neat solution or a suitable inert solvent. Pharmaceutically acceptable base addition salts include sodium, potassium, calcium, ammonium, organic amine, or magnesium salts, or similar salts. When the compounds of the present invention contain relatively basic functional groups, acid addition salts can be obtained by contacting the compounds with a sufficient amount of acid in a neat solution or a suitable inert solvent. Examples of pharmaceutically acceptable acid addition salts include inorganic acid salts such as hydrochloric acid, hydrobromic acid, nitric acid, carbonic acid, bicarbonate, phosphoric acid, monohydrogen phosphate, dihydrogen phosphate, sulfuric acid, bisulfate, hydroiodic acid, phosphorous acid, and the like; and organic acid salts such as acetic acid, propionic acid, isobutyric acid, maleic acid, malonic acid, benzoic acid, succinic acid, suberic acid, fumaric acid, lactic acid, mandelic acid, phthalic acid, benzenesulfonic acid, p-toluenesulfonic acid, citric acid, tartaric acid, and methanesulfonic acid; and salts of amino acids (such as arginine) and organic acids such as glucuronic acid. Certain specific compounds of the present invention contain both basic and acidic functional groups and can be converted into either base or acid addition salts.

[0131] The salts of the present invention can be synthesized by conventional chemical methods from parent compounds containing acid radicals or bases. Generally, such salts are prepared by reacting these compounds in free acid or base form with a stoichiometric amount of an appropriate base or acid in water or an organic solvent or a mixture of the two.

[0132] The compounds of the present invention can be prepared by a variety of synthetic methods well known to those skilled in the art, including the specific embodiments listed below, embodiments formed by combining them with other chemical synthesis methods, and equivalent substitutions well known to those skilled in the art. Preferred embodiments include but are not limited to the examples of the present invention.

[0133] The structures of the compounds of the present invention can be confirmed by conventional methods well known to those skilled in the art. If the present invention relates to the absolute configuration of the compounds, the absolute configuration can be confirmed by conventional techniques in the art. For example, single crystal X-ray diffraction (SXRD) is used to collect diffraction intensity data on the cultured single crystal using a Bruker D8 venture diffractometer, using CuKα radiation as the light source and scanning mode: / ω scanning, and after collecting relevant data, the crystal structure was further analyzed using the direct method (Shelxs97) to confirm the absolute configuration.

[0134] The solvent used in the present invention is commercially available.

[0135] Unless otherwise specified, the solvent ratios used in the column chromatography and preparative thin-layer silica gel chromatography of the present invention are all volume ratios.

[0136] The present invention uses the following abbreviations: DMSO represents dimethyl sulfoxide; Cbz represents benzyloxycarbonyl; Boc represents tert-butyloxycarbonyl; Ac represents acetyl; DMTr represents dimethoxytrityl; DCM represents dichloromethane; i-PrOH represents isopropyl alcohol; Bn represents benzyl; Bz represents benzoyl; Me represents methyl; i-Pr represents isopropyl; OSu represents M is the concentration unit, indicating mol / L; TsOH stands for p-toluenesulfonic acid; TBS stands for tert-butyldimethylsilyl; i-Pr stands for isopropyl; and LPS stands for lipopolysaccharide.

[0137] Compounds are named according to the conventional nomenclature in the art or using Software naming, commercially available compounds use supplier catalog names. DETAILED DESCRIPTION

[0138] The present invention is described in detail below by examples, but it is not intended to limit the present invention in any way. The compounds of the present invention can be prepared by a variety of synthetic methods well known to those skilled in the art, including the specific embodiments listed below, the embodiments formed by combining them with other chemical synthesis methods, and equivalent replacement modes well known to those skilled in the art. Preferred embodiments include but are not limited to the embodiments of the present invention. It will be apparent to those skilled in the art that various changes and modifications will be made to the specific embodiments of the present invention without departing from the spirit and scope of the present invention.

[0139] Example 1: Synthesis of double-stranded siRNA or its conjugate

[0140] Synthesis of single-stranded oligoribonucleotides containing conjugated groups: oligoribonucleotides were synthesized using phosphoramidite solid phase synthesis technology. ) and the conjugated intermediate compound are covalently bonded to form a solid support. All RNA phosphoramidite monomers in the sequence are dissolved in anhydrous acetonitrile in the order of sequence 3' to 5' and molecular sieves are added. 5-Ethylthio-1H-tetrazole (ETT) was used for activation for 5 minutes. A 50 mM I2-water / pyridine (1 / 9 by volume) solution was reacted for 3 minutes to generate phosphate bonds. A 50 mM 3-((dimethylamino-methylene)amino)-3H-1,2,4-dithiazole-3-thione (DDTT) solution in anhydrous acetonitrile / pyridine (1 / 1 by volume) was reacted for 3 minutes to generate phosphorothioate bonds. The resulting sequence was synthesized after the final removal of the DMT group.

[0141] Synthesis of single-stranded oligoribonucleotides without conjugated groups: Oligoribonucleotides were synthesized using the phosphoramidite solid phase synthesis technique. Synthesis was performed on a solid support made of controlled pore glass (CPG). All RNA phosphoramidite monomers in the sequence were dissolved in anhydrous acetonitrile in the order of 3' to 5' and molecular sieves were added. The coupling time was 5 minutes using 5-ethylthio-1H-tetrazole (ETT) as an activator. A 50 mM I2-water / pyridine (1 / 9 by volume) solution was reacted for 3 minutes to generate the phosphate bond. A 50 mM 3-((dimethylamino-methylene)amino)-3H-1,2,4-dithiazole-3-thione (DDTT) solution in anhydrous acetonitrile / pyridine (1 / 1 by volume) was reacted for 3 minutes to generate the phosphorothioate bond. The resulting sequence was synthesized after the final removal of the DMT group.

[0142] Cleavage and Deprotection of CPG-Bound Oligomers: After solid-phase synthesis is terminated, the protecting groups are removed by treatment with 10% diethylamine in acetonitrile for 30 minutes, without cleaving the nucleotides from the CPG. Subsequently, the dried CPG is treated with concentrated aqueous ammonia at 40°C for 18 hours. After centrifugation, the supernatant is transferred to a new tube and the CPG is washed with water. The combined solutions are concentrated to yield a solid mixture. If natural nucleotides are present, the solid mixture is dissolved in DMSO (10V), followed by the addition of triethylammonium hydrofluoride (2V) and stirring at 40°C for 16 hours. Purification is performed after the reaction is complete.

[0143] Purification of single-stranded oligoribonucleotides: Oligomers were purified by HPLC using a NanoQ anion exchange column. Buffer A consisted of 10 mM sodium perchlorate, 20 mM Tris, 1 mM EDTA, pH 7.4, and 20% acetonitrile, and buffer B consisted of 500 mM sodium perchlorate, 20 mM Tris, 1 mM EDTA, pH 7.4, and 20% acetonitrile. The desired product was isolated and desalted using a reversed-phase C18 column.

[0144] Annealing of single-stranded oligoribonucleotides to produce siRNA: Prepare the single-stranded oligoribonucleotides to be annealed at a concentration of 200 μM using sterile RNase-free H2O (RNA hydrolase-free). Set up the annealing reaction as follows: Place 10 nmol of the 100 μL mixture in a 95°C waterbath for 10 minutes (amounts ≥ 100 nmol require 20 minutes at high temperature). Immediately place the mixture in a 60°C waterbath and allow to cool naturally. Do not store the annealed solution at high temperatures. Complementary strands are formed by combining equimolar amounts of the single-stranded oligoribonucleotide solutions.

[0145] Example 2: Synthesis of ZM

[0146] Step A: Dissolve 1-1 (10 g, 19.82 mmol) in acetonitrile (120 mL) and 1,2-dichloroethane (80 mL). Add 1-2 (6.02 g, 42.62 mmol) and trimethylsilyl trifluoromethanesulfonate (11.01 g, 49.56 mmol) at 0°C. The mixture is stirred at 35°C for 12 hours. The reaction mixture is quenched by slowly adding saturated aqueous sodium bicarbonate (100 mL) at 0°C and extracted with dichloromethane (100 mL x 2). The resulting organic phase is washed with saturated aqueous sodium chloride (200 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain the crude product. The crude product is purified on a silica gel column (eluent: petroleum ether / ethyl acetate = 20:1) to obtain 1-3.

[0147] Step B: Dissolve 1-3 (6.7 g, 13.05 mmol) in 7 M ammonia methanol solution (50 mL), and stir the mixture at 40°C for 12 hours. The reaction mixture is concentrated under reduced pressure to obtain a crude product, which is then purified on a silica gel column (eluent: ethyl acetate / methanol = 50 / 1 to 10 / 1) to afford 1-4.

[0148] Step C: Dissolve 1-4 (2.3 g, 11.43 mmol) in anhydrous pyridine (25 mL) and add 1,3-dichloro-1,1,3,3-tetraisopropyldisiloxane (3.64 g, 11.55 mmol) at 0°C. The mixture is stirred at 20°C for 12 hours. The reaction mixture is quenched by adding water (30 mL) and extracted with ethyl acetate (30 mL × 2). The resulting organic phase is washed sequentially with hydrochloric acid (1 M, 30 mL × 3) and saturated brine (30 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain a crude product. The crude product is purified on a silica gel column (eluent, petroleum ether / ethyl acetate = 7 / 1 to 5 / 1) to obtain 1-5.

[0149] Step D: 1-5 (4 g, 9.02 mmol) was dissolved in acetonitrile (40 mL), and silver oxide (8.36 g, 36.06 mmol) was added in sequence. Molecular sieves (3 g), anhydrous pyridine (1.78 g, 22.54 mmol) and iodomethane (6.4 g, 45.08 mmol) were added and the mixture was stirred at 25°C for 12 hours. Ethyl acetate (50 mL) was added to the reaction solution and stirred at 20°C for 1 hour. The filtrate was filtered and collected, and the filtrate was extracted with water (100 mL) and ethyl acetate (100 mL × 2). The resulting organic phase was washed with saturated brine (200 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain a crude product. The crude product was purified on a silica gel column (eluent: petroleum ether / ethyl acetate = 15 / 1 to 10 / 1 to 8 / 1) to obtain 1-6.

[0150] Step E: Dissolve 1-6 (2 g, 4.37 mmol) in anhydrous tetrahydrofuran (20 mL), add triethylamine trihydrogen fluoride (1.55 g, 9.61 mmol, 1.57 mL), and stir the mixture at 20°C for 12 hours. The reaction mixture is concentrated under reduced pressure to obtain a crude product, which is then purified on a silica gel column (eluent: ethyl acetate / methanol = 100 / 1 to 50 / 1) to obtain 1-7.

[0151] Step F: Dissolve 1-7 (1 g, 4.65 mmol) in anhydrous pyridine (10 mL), add 4,4-bismethoxytrityl chloride (1.57 g, 4.65 mmol), and stir the mixture at 20°C for 12 hours. The reaction mixture is quenched by adding water (20 mL) and extracted with ethyl acetate (20 mL x 2). The resulting organic phase is washed with saturated brine (40 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain the crude product. The crude product is purified on a silica gel column (eluent: petroleum ether / ethyl acetate = 3 / 1 to 1 / 1) to obtain 1-8. 1 H NMR (400MHz, DMSO-d6): δ = 8.78 (s, 1H), 8.14-8.03 (m, 1H), 7.41-7.31 (m, 2H), 7.29-7.16 (m, 7H), 6.92-6.76 (m, 4H), 6.08 ( d,J=3.2Hz,1H),5.25(d,J=6.5Hz,1H),4.48-4.34(m,1H),4.13-4.01(m,2H),3.73(s,6H),3.38(s,3H),3.18-3.03(m,2H).

[0152] Step G: Compounds 1-8 (3 g, 5.80 mmol) and 1-9 (1.92 g, 6.38 mmol) were dissolved in anhydrous dichloromethane (30 mL). 4,5-dicyanoimidazole (342.26 mg, 2.90 mmol) was added under a nitrogen atmosphere. The mixture was stirred at 25°C for 2 hours. 15 mL of saturated aqueous sodium bicarbonate solution and 15 mL of water were added to the reaction solution, and the mixture was extracted with dichloromethane (30 mL x 2). The combined organic phases were washed with saturated brine (30 mL x 2), dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the crude product. ZM was obtained by purification via silica gel column chromatography (eluent: petroleum ether / ethyl acetate (0.1% triethylamine) = 1 / 0 to 2 / 1). 1 H NMR (400MHz, DMSO-d6): δ = 8.83 (s, 1H), 8.11 (d, J = 6.2Hz, 1H), 7.38 (br t,J=6.9Hz,2H),7.32-7.15(m,7H),6.92-6.76(m,4H),6.26-6.05(m,1H),4.78-4.56(m,1H),4.31(br d,J=3.8Hz,1H),4.25-4.12(m,1H),3.85-3.77(m,1H),3.73(br s,6H),3.66-3.46(m,3H),3.39(br d,J=15.7Hz,3H),3.32-3.19(m,1H),3.10(br dd,J=5.4,10.0Hz,1H),2.78(br t,J=5.7Hz,1H),2.60(br t,J=5.5Hz,1H),1.22-1.04(m,9H),0.98(br d,J=6.5Hz,3H). 31 P NMR (162MHz, DMSO-d6): δ = 149.33 (br d, J = 11.7Hz, 1P). LCMS(ESI)m / z:718[M+H] + .

[0153] Example 3: Synthesis of D08-M

[0154] Step A: Under nitrogen, sodium hydroxide (2.75 g, 68.68 mmol, 60% purity) was added gradually to a solution of diethyl malonate (10 g, 62.43 mmol) in tetrahydrofuran (100 mL) at 0°C. After the addition, the mixture was stirred for 0.5 hours. Then, a solution of compound 2-11-1 (17.86 g, 62.43 mmol) in tetrahydrofuran (50 mL) was added dropwise at 0°C. The mixture was stirred at 25°C for 15.5 hours. The reaction was quenched by the addition of saturated ammonium chloride (20 mL) at 10-25°C, diluted with water (20 mL), extracted with ethyl acetate (50 mL x 2), washed with saturated brine (30 mL x 2), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain the crude product. The crude product was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 1 / 0 to 9 / 1) to obtain compound 2-11-2.

[0155] Step B: Under nitrogen protection, sodium borohydride (15.2 g, 401.77 mmol) was added portionwise to a solution of compound 2-11-2 (15 g, 41.06 mmol) in methanol (150 mL) at 60°C over 0.5 hours. The reaction was stirred for 0.5 hours, and the mixture was stirred at 65°C for 1 hour. Water was added to quench the reaction at 0-25°C, stirred for 0.5 hours, and diluted with water (50 mL). The reaction solution was concentrated under reduced pressure at 30-35°C to remove methanol, extracted with ethyl acetate (100 mL*2), washed with saturated brine (50 mL*2), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain compound 2-11-3.

[0156] Step C: Compound 2-11-3 (8.5 g, 30.22 mmol) and potassium phthalimide (14.00 g, 75.56 mmol) were mixed in N,N-dimethylformamide (90 mL). The atmosphere was purged with nitrogen three times and the reaction mixture was incubated at 100°C under nitrogen for 16 hours. The reaction mixture was diluted with water (300 mL). Solids precipitated and the filter cake was collected by filtration. The filter cake was purified by silica gel column (petroleum ether / ethyl acetate = 1 / 0 to 1 / 1) to obtain compound 2-11-4.

[0157] Step D: Compound 2-11-4 (6.1 g, 17.56 mmol), 4,4-dimethoxytrityl chloride (7.14 g, 21.07 mmol), and triethylenediamine (2.95 g, 26.33 mmol) were mixed in dichloromethane (60 mL). The atmosphere was replaced with nitrogen three times. Under nitrogen protection, the reaction mixture was reacted at 25°C for 16 hours. The reaction mixture was diluted with water (30 mL) and extracted with dichloromethane (50 mL). The organic phase was washed with saturated brine (30 mL x 2), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain the crude product. The crude product was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 1 / 0 to 4 / 1, supplemented with 0.1% triethylamine) to obtain compound 2-11-5.

[0158] Step E: Compound 2-11-5 (7.1 g, 10.93 mmol) and hydrazine monohydrate (1.52 g, 29.76 mmol, 98% purity) were mixed in ethanol (80 mL) and the atmosphere was replaced with nitrogen three times. Under nitrogen protection, the reaction mixture was incubated at 70°C for 16 hours. The ethanol was removed by concentration under reduced pressure at 35-40°C to obtain intermediate 2-11.

[0159] Step F: To a toluene solution (150 mL) of compound 2-2 (13.24 g, 50.93 mmol) was added triethylamine (10.10 g, 99.86 mmol) and a toluene solution (100 mL) of compound 2-1 (12 g, 49.93 mmol) at 25°C. The reaction was stirred at 100°C for 16 hours. Saturated aqueous sodium carbonate (200 mL) was added to the reaction solution, and the layers were separated. The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the crude product, which was separated by preparative HPLC (column: Agela Innoval ODS-2 250 mm*100 mm*10 μm; mobile phase: [water (formic acid)-acetonitrile]; acetonitrile percentage: 15%-35%, 25 minutes) to obtain compound 2-3.

[0160] Step G: A solution of compound 2-3 (12.6 g, 31.04 mmol), palladium on carbon (5 g, 10% palladium content), and di-tert-butyl carbonic anhydride (14.90 g, 68.28 mmol) in methanol (120 mL) was purged with argon three times and then with hydrogen three times. The reaction mixture was stirred at 25°C under a hydrogen atmosphere at atmospheric pressure for 16 hours. The reaction mixture was filtered and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography to yield compound 2-4. 1 H NMR (400MHz, CDCl3) δppm 3.60-3.95 (m, 7H) 3.36-3.50 (m, 2H) 3.19-3.34 (m, 2H) 2.96-3.12 (m, 1H) 1.46 (s, 18H).

[0161] Step H: Compound 2-4 (7.5 g, 18.66 mmol) was dissolved in hydrochloric acid ethyl acetate solution (4 mol / L, 18.66 mL), replaced with nitrogen three times, and stirred at 25°C for 1 hour under nitrogen protection. The mixture was concentrated under reduced pressure to obtain compound 2-5.

[0162] Step I: A solution of compound 2-6 (2.2 g, 5.69 mmol), compound 2-5 (2.24 g, 9.68 mmol), triethylamine (2.88 g, 28.47 mmol), and 1-propylphosphonic anhydride (5.06 g, 7.95 mmol, 50% ethyl acetate) in N,N-dimethylformamide (25 mL) was purged with nitrogen three times and stirred at 25°C under nitrogen for 1 hour. Water (50 mL) was added to the reaction solution, which was then extracted with ethyl acetate (80 mL x 2). The combined organic phases were washed with saturated brine (100 mL), dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was purified by preparative HPLC (column: Phenomenex luna C18 150 x 40 mm x 15 μm; mobile phase: [water (0.1% formic acid)-acetonitrile]; acetonitrile percentage: 14%-44%; 15 minutes) to afford compound 2-7. 1 H-NMR (400MHz, CDCl3) δppm 7.30-7.46(m,10H)5.59-5.92(m,2H)4.97-5.27(m,4H)3.79-3.92(m,1H) 3.67-3.78 (m, 3H) 3.36-3.65 (m, 4H) 3.09-3.32 (m, 4H) 2.79-3.04 (m, 4H).

[0163] Step J: A solution of compound 2-7 (2.2 g, 4.06 mmol), compound 2-8 (1.50 g, 6.09 mmol), O-benzotriazole-N,N,N',N'-tetramethyluronium tetrafluoroborate (2.22 g, 6.90 mmol), and triethylamine (1.23 g, 12.18 mmol) in N,N-dimethylformamide (20 mL) was purged with nitrogen three times and stirred at 25°C under nitrogen for 1 hour. Water (80 mL) was added to the reaction solution, which was then extracted with ethyl acetate (80 mL x 2). The combined organic phases were washed with saturated brine (100 mL), dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a crude product. The crude product was separated by silica gel column chromatography (eluent: petroleum ether / ethyl acetate, gradient elution) to obtain compound 2-9. 1H NMR (400MHz, CDCl3) δppm 7.29-7.56 (m, 15H) 5.72 (br s, 2H) 4.93-5.32 (m, 6H) 4.00-4.07 (m, 1H) 3.82-3.99 (m, 2H) 3.76 (s, 3H) 3.56-3.70 (m, 2H) 3.06-3.54 (m, 8H) 2.98 (s, 1H).

[0164] Step K: To a mixed solution of compound 2-9 (2.5 g, 3.43 mmol) in methanol (22 mL) and water (7 mL) was added lithium hydroxide monohydrate (504.37 mg, 12.02 mmol). The reaction solution was stirred at 25°C for 12 hours. Water (50 mL) was added to the reaction solution. The aqueous phase was adjusted to pH 4-5 with 2M hydrochloric acid and extracted with ethyl acetate (100 mL*2). The combined organic phases were washed with saturated brine (50 mL). After drying over anhydrous sodium sulfate, the filtrate was filtered and concentrated to obtain compound 2-10.

[0165] Step L: A solution of compound 2-10 (500 mg, 710.49 μmol), compound 2-11 (406.18 mg, 781.54 μmol), benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate (485.01 mg, 1.28 mmol), and triethylamine (215.68 mg, 2.13 mmol) in N,N-dimethylformamide (5 mL) was purged with nitrogen three times and stirred at 25°C under a nitrogen atmosphere for 1 hour. Water (80 mL) was added to the reaction solution, which was then extracted with dichloromethane (80 mL x 2). The combined organic phase was washed with saturated brine (100 mL * 1), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated. Preparative HPLC (column: Waters Xbridge C18 150*50 mm*10 μm; mobile phase: [water (0.1% ammonium bicarbonate)-acetonitrile]; acetonitrile%: 57%-87%, 10 minutes) was used to obtain compound 2-12. 1 H NMR (400MHz, DMSO-d6) δppm 7.21-7.38 (m, 24H) 6.79-6.95 (m, 4H) 4.74-5.19 (m, 6H) 4.35 (t, J=4.83Hz, 1H) 3.59-4. 07 (m, 13H) 3.39-3.55 (m, 6H) 2.87-3.23 (m, 10H) 2.58-2.78 (m, 6H) 1.04-1.28 (m, 14H).

[0166] Step M: To a solution of compound 2-12 (320 mg, 203.93 μmol) in methanol (10 mL) was added palladium on carbon (100 mg, 10% content). The atmosphere was then replaced with argon three times and then with hydrogen three times. The reaction mixture was stirred at 25°C under a hydrogen atmosphere at atmospheric pressure for 16 hours. The reaction mixture was filtered and the filtrate was concentrated under reduced pressure to obtain compound 2-13. 1 H NMR (400MHz, DMSO-d6) δppm 8.02-8.28(m,1H)7.34-7.43(m,2H)7.27-7.33(m,2H)7.18-7.27(m,5H)6.64-7.00(m,4H)3 .86-4.21(m,3H)2.82-3.56(m,24H)1.55-1.69(m,2H)1.37-1.45(m,2H)1.05-1.29(m,14H).

[0167] Step N: A solution of compound 2-13 (178 mg, 221.66 μmol), compound 2-14 (413.14 mg, 775.80 μmol), benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate (336.25 mg, 886.63 μmol), and triethylamine (179.43 mg, 1.77 mmol) in N,N-dimethylformamide (4 mL) was purged with nitrogen three times. The reaction mixture was stirred at 25°C under a nitrogen atmosphere for 1 hour. Water (100 mL) was added to the reaction mixture and extracted twice with 50 mL of DCM / i-PrOH (v / v = 3 / 1). The combined organic phases were washed with saturated brine (150 mL*1), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure and purified by preparative HPLC (column: Waters Xbridge C18 150*50mm*10μm; mobile phase: [water (0.1% ammonium bicarbonate)-acetonitrile]; acetonitrile%: 36%-66%, 10 minutes) to give compound 2-15. 1H-NMR (400MHz, DMSO-d6) δppm 8.10-8.23 (m, 1H) 7.92-8.01 (m, 1H) 7.66-7.91 (m, 8H) 7.35-7.40 (m, 2H) 7.30 (t, J=7 .64Hz, 2H) 7.21-7.27 (m, 5H) 6.88 (d, J = 8.80Hz, 4H) 5.22 (d, J = 3.30Hz, 3H) 4.97 (dd, J=11.25, 3.30Hz, 3H) 4.49 (d, J=8.56Hz, 3H) 4.36 (t, J=4.89Hz, 1H) 3.94-4.10 (m, 12 H)3.81-3.92(m,5H)3.64-3.80(m,12H)3.37-3.49(m,6H)2.84-3.23(m,17H)2.53(br d, J=1.96Hz, 5H) 2.08-2.16 (m, 11H) 2.00 (s, 12H) 1.89 (s, 9H) 1.78 (s, 9H) 1.54-1.67 (m, 7H) 1.38-1.53 ​​(m, 14H) 1.01-1.32 (m, 16H).

[0168] Step O: A dichloromethane solution (5 mL) of compound 2-15 (240 mg, 97.49 μmol), compound 2-16 (34.15 mg, 341.21 μmol), 4-dimethylaminopyridine (11.91 mg, 97.49 μmol), and triethylamine (9.86 mg, 97.49 μmol) was purged with nitrogen three times and stirred at 25°C under nitrogen for 16 hours. The reaction solution was concentrated and purified by preparative HPLC (column: Waters Xbridge C18 150*50 mm*10 μm; mobile phase: [water (0.1% ammonium bicarbonate)-acetonitrile]; acetonitrile percentage: 19%-49%, 10 minutes) to obtain compound D08-M. 1H NMR (400MHz, DMSO-d6) δppm 7.65-8.20 (m, 11H) 7.28-7.39 (m, 4H) 7.17-7.28 (m, 5H) 6.78-7.03 (m, 4H) 5.13-5.33 (m, 3H) 4.89-5.10 (m, 3H) 4.39-4.60 (m, 3H) 3.94-4.15 (m, 15H) 3.82-3.92 (m, 5H) 3.64-3.81 (m, 12H) 2.83-3.19 (m, 16H) 2.08-2.19 (m, 14 H) 1.96-2.07 (m, 19H) 1.86-1.92 (m, 9H) 1.74-1.81 (m, 10H) 1.54-1.65 (m, 7H) 1.34-1.54 (m, 16H) 1.03-1.33 (m, 16H); LCMS (ESI): 1221.8.

[0169] Example 4: Synthesis of D10-M

[0170] Step A: Dissolve compound 3-1 (2 g, 10.68 mmol) in a mixture of acetonitrile (20 mL) and water (20 mL). Add sodium bicarbonate (1.79 g, 21.36 mmol, 831.06 μL) and N-(9-fluorenylmethyloxycarbonyloxy)succinimide (4.32 g, 12.82 mmol) in this order. The reaction mixture is allowed to react at 25°C for 12 hours. Dilute the reaction mixture with 1 M aqueous hydrochloric acid (200 mL), extract with ethyl acetate (100 mL x 2), dry over anhydrous sodium sulfate, filter, and concentrate under reduced pressure to obtain a crude product. Add the crude product to a mixture of petroleum ether (10 mL) and ethyl acetate (10 mL) and slurry for 12 hours to obtain compound 3-2.

[0171] Step B: Dissolve compound 3-2 (3.2 g, 7.81 mmol) in dichloromethane (50 mL). Add N,N-diisopropylethylamine (23.44 mmol, 4.08 mL), 1-hydroxy-7-azabenzotriazole (1.60 g, 11.72 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (2.25 g, 11.72 mmol), and compound 3-3 (3.11 g, 7.42 mmol) in sequence. The reaction mixture was allowed to react at 25°C for 12 hours. The reaction mixture was diluted with water (200 mL), extracted with dichloromethane (100 mL x 2), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain the crude product. The crude product was purified by silica gel column to obtain compound 3-4.

[0172] Step C: Dissolve compound 3-4 (3.8 g, 4.69 mmol) and diethylamine (13.71 g, 187.42 mmol) in dichloromethane (30 mL), and react at 25°C for 12 hours. Concentrate under reduced pressure to obtain compound 3-5.

[0173] Step D: Dissolve compound 3-6 (37.7 g, 68.72 mmol, oxalate salt) and compound 3-7 (22.45 g, 82.47 mmol) and triethylamine (240.53 mmol, 33.48 mL) in acetonitrile (400 mL) and replace the atmosphere with nitrogen three times. Stir the mixture at 25°C for 3.5 hours. Dilute the mixture with methyl tert-butyl ether (400 mL) and wash with saturated sodium bicarbonate solution (400 mL*2), brine (400 mL), saturated ammonium chloride (400 mL*2), and brine (400 mL), respectively. Dry the organic phase over anhydrous sodium sulfate, filter, and concentrate under reduced pressure to obtain compound 3-8.

[0174] Step E: Dissolve compound 3-8 (42.31 g, 68.72 mmol) in methanol (200 mL) and water (50 mL), then add sodium hydroxide (6.87 g, 171.79 mmol) at 5°C. Stir the mixture at 25°C for 2 hours. Concentrate the mixture to remove most of the methanol, dilute with water (100 mL), adjust the pH to 4-5 with 1M hydrochloric acid, and extract with dichloromethane (300 mL x 2) at 5°C. Dry the organic phase over anhydrous sodium sulfate, filter, and concentrate under reduced pressure to obtain compound 3-9.

[0175] Step F: Compound 3-9 (5 g, 8.31 mmol) was dissolved in dichloromethane (50 mL), and 1,8-diazabicyclo[5.4.0]undec-7-ene (9.97 mmol, 1.50 mL) and benzyl bromide (9.97 mmol, 1.18 mL) were added sequentially. The reaction mixture was allowed to react at 25°C for 12 hours. The reaction mixture was concentrated under reduced pressure to remove dichloromethane, and aqueous solution (100 mL) was added. The mixture was extracted with ethyl acetate (100 mL x 2), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain the crude product. The crude product was purified by preparative HPLC (column: Kromasil Eternity XT 250 x 80 mm x 10 μm; mobile phase: water (0.1% ammonium bicarbonate)-acetonitrile; gradient: 43% to 73% acetonitrile over 20 minutes) to obtain compound 3-10.

[0176] Step G: Compound 3-10 (3.7 g, 5.35 mmol) was added to a 2M hydrochloric acid and ethyl acetate solution (92.50 mL). The reaction mixture was reacted at 25°C for 12 hours. The reaction mixture was concentrated under reduced pressure to obtain compound 3-11.

[0177] Step H: Compound 2-14 (9.47 g, 17.79 mmol) was dissolved in dichloromethane (60 mL). N,N-diisopropylethylamine (48.52 mmol, 8.45 mL), O-(7-azabenzotriazol-1-yl)-N,N,N,N-tetramethyluronium hexafluorophosphonate (8.20 g, 21.56 mmol), and compound 3-11 (2.7 g, 5.39 mmol) were added sequentially. The reaction mixture was incubated at 25°C for 12 hours. The mixture was concentrated under reduced pressure to obtain a crude product, which was then purified by preparative HPLC (column: Kromasil Eternity XT 250*80 mm*10 μm; mobile phase: [water (0.1% ammonium bicarbonate)-acetonitrile]; gradient: 20% to 50% acetonitrile over 20 minutes) to afford compound 3-12.

[0178] Step I: Dissolve compound 3-12 (5.2 g, 2.69 mmol) in methanol (50 mL). Under a nitrogen atmosphere, add Pd / C (1 g, 939.67 μmol, 10% palladium content). Replace the reaction mixture with hydrogen three times. React under hydrogen (15 psi) at 25°C for 12 hours. Remove the palladium on carbon by filtration, and concentrate the filtrate under reduced pressure to obtain compound 3-13.

[0179] Step J: Compound 3-13 (1 g, 542.03 μmol) was dissolved in dichloromethane (10 mL), and N,N-diisopropylethylamine (1.63 mmol, 283.24 μL), 1-hydroxy-7-azabenzotriazole (110.67 mg, 813.05 μmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (155.86 mg, 813.05 μmol), and compound 3-5 (462.52 mg, 542.03 μmol) were added in sequence. The reaction mixture was reacted at 25°C for 12 hours. The crude product was concentrated under reduced pressure and purified by preparative HPLC (column: Waters Xbridge C18 150*50mm*10μm; mobile phase: [water (0.1% ammonium bicarbonate)-acetonitrile]; gradient: 31%-61% acetonitrile; 10 minutes) to give compound 3-14.

[0180] Step K: Compound 3-14 (0.47 g, 194.56 μmol) was dissolved in dichloromethane (5 mL). Triethylamine (1.07 mmol, 148.94 μL), 4-dimethylaminopyridine (23.77 mg, 194.56 μmol), and succinic anhydride (97.35 mg, 972.82 μmol) were added sequentially. The reaction mixture was incubated at 25°C for 12 hours, and the reaction was detected to be complete. The mixture was concentrated under reduced pressure to obtain the crude product, which was then purified by preparative HPLC (column: Waters Xbridge C18 150*50 mm*10 μm; mobile phase: water (0.1% ammonium bicarbonate)-acetonitrile; gradient: 22% to 52% acetonitrile over 10 minutes) to yield compound D10-M. 1 H NMR (400MHz, DMSO-d6) δ = 8.46-7.60 (m, 9H), 7.38-7.09 (m, 9H), 6.93-6.80 (m, 4H), 5.21 (d, J = 3.3Hz, 3 H),4.97(dd,J=3.4,11.1Hz,3H),4.48(d,J=8.4Hz,3H),4.26-4.15(m,1H),4.08-3.93(m,10H),3.92- 3.78(m,5H),3.78-3.63(m,11H),3.57-3.39(m,6H),3.16-2.86(m,18H),2.40-2.19(m,6H),2.17-2.08(m,13H),2.07- 2.01(m,8H),1.99(s,10H),1.89(s,9H),1.77(s,9H),1.65-1.33(m,22H),1.33-1.08(m,11H); LCMS(ESI) m / z: 1256.8.

[0181] Example 5: Synthesis of D11-M

[0182] Step A: Dissolve compound 3-2 (3 g, 7.33 mmol) in tetrahydrofuran (30 mL). Add N,N-diisopropylethylamine (1.14 g, 8.79 mmol, 1.53 mL) and 2-succinimidyl-1,1,3,3-tetramethyluronium tetrafluoroborate (2.43 g, 8.06 mmol) sequentially. Incubate the reaction at 25°C for 12 hours. Dilute the reaction solution with water (100 mL) and filter to obtain the crude product. The crude product is purified on a silica gel column (eluent: petroleum ether:ethyl acetate = 2:1) to obtain compound 4-1.

[0183] Step B: Dissolve compound 4-1 (2.86 g, 5.65 mmol) in dichloromethane (30 mL) and add compound 4-2 (1.44 g, 9.03 mmol). The reaction mixture is allowed to react at 25°C for 12 hours. The reaction mixture is diluted with water (200 mL), extracted with dichloromethane (100 mL x 2), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain the crude product. The crude product is purified by preparative HPLC (column: Waters Xbridge C18 150 x 50 mm x 10 μm; mobile phase: [water (0.1% ammonium bicarbonate)-acetonitrile]; gradient: 40% to 70% acetonitrile over 10 minutes) to obtain compound 4-3.

[0184] Step C: Compound 4-3 (2 g, 3.63 mmol) was dissolved in dichloromethane (20 mL). 4A molecular sieves (2 g), triethylenediamine (488.84 mg, 4.36 mmol, 479.25 μL), and 4,4-dimethoxytrityl chloride (1.35 g, 3.99 mmol) were added sequentially. The reaction mixture was allowed to react at 25°C for 16 hours. The reaction mixture was diluted with water (200 mL), extracted with dichloromethane (100 mL x 2), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain the crude product. The crude product was purified by preparative HPLC (column: Kromasil Eternity XT 250 x 80 mm x 10 μm; mobile phase: [water (ammonia)-acetonitrile]; gradient: 60% to 90% acetonitrile over 25 minutes) to obtain compound 4-4.

[0185] Step D: Dissolve compound 4-4 (1.78 g, 2.09 mmol) and diethylamine (7.90 g, 108.00 mmol) in dichloromethane (15 mL). Incubate the reaction at 25°C for 12 hours. Check the reaction is complete. Concentrate under reduced pressure to obtain compound 4-5. LCMS (M+1, m / z: 632)

[0186] Step E: Dissolve compound 3-13 (1.5 g, 813.05 μmol) in dichloromethane (15 mL). Add N,N-diisopropylethylamine (315.24 mg, 2.44 mmol), 1-hydroxy-7-azabenzotriazole (166.00 mg, 1.22 mmol), and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (233.79 mg, 1.22 mmol) sequentially. Stir thoroughly before adding compound 4-5 (854.86 mg, 813.05 μmol). Incubate the reaction at 25°C for 12 hours, and check for completion. The crude product was concentrated under reduced pressure and purified by preparative HPLC (column: Waters Xbridge C18 150*50mm*10μm; mobile phase: [water (0.1% ammonium bicarbonate)-acetonitrile]; gradient: 34%-64% acetonitrile, 10 minutes) to give compound 4-6.

[0187] Step F: Compound 4-6 (450 mg, 183.09 μmol) was dissolved in dichloromethane (3 mL), and triethylamine (166.74 mg, 1.65 mmol), 4-dimethylaminopyridine (22.37 mg, 183.09 μmol), and succinic anhydride (146.58 mg, 1.46 mmol) were added sequentially. Under nitrogen, the reaction mixture was incubated at 35°C for 24 hours. The mixture was concentrated under reduced pressure to obtain the crude product, which was then purified by preparative HPLC (column: Waters Xbridge C18 150*50 mm*10 μm; mobile phase: [water (0.1% ammonium bicarbonate)-acetonitrile]; gradient: 23% to 53% acetonitrile over 10 minutes) to afford compound D11-M. 1 H NMR (400MHz, DMSO-d6) δ=8.47-7.70(m,10H),7.41-7.15(m,9H),6.87(br d,J=7.1Hz,4H),5.21(d,J=3.2Hz,3H),4.97(br dd,J=2.6,11.2Hz,3H),4.49(br d,J=8.3Hz,3H),4.18-3.91(m,12H),3.91-3.79(m,5H),3.76-3.61(m,14H),3. 22-2.82(m,27H),2.10(s,11H),1.99(s,18H),1.88(s,9H),1.77(s,8H),1.66- 1.30 (m, 22H), 1.23 (br d, J = 6.2Hz, 10H), 1.11-1.02 (m, 3H), 0.97 (br d, J = 2.8Hz, 3H); LCMS (ESI) m / z: 1278.2.

[0188] Example 6: Synthesis of D18-M

[0189] Step A: To a solution of compound 5-1 (10 g, 25.68 mmol) in 1,2-dichloroethane (100 mL), scandium trifluoromethanesulfonate (348.84 mg, 1.8 mmol) and compound 5-2 (2.83 g, 28.25 mmol) were added. The reaction was stirred at 60°C for 6 hours. The reaction was detected to be complete. The reaction solution was quenched by adding water (200 mL) at 25°C, extracted, and the organic phase was washed with 5% sodium bicarbonate (200 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain a crude product. The crude product was recrystallized from a mixed solution of ethyl acetate / n-heptane (volume ratio: 1 / 3, 80 mL) to obtain compound 5-3.

[0190] Step B: Sodium periodate (19.92 g, 93.14 mmol) was added to a solution of compound 5-3 (8 g, 18.63 mmol) and ruthenium trichloride trihydrate (82.8 mg, 316.68 μmol) in dichloromethane (24 mL), acetonitrile (24 mL), and water (40 mL). The reaction was stirred at 25°C for 2 hours. The reaction was detected to be complete. The reaction solution was filtered, and the filtrate was adjusted to pH 8 with saturated sodium bicarbonate. The filtrate was extracted with dichloromethane (50 mL x 2). The aqueous phase was retained and the pH was adjusted to 3 with citric acid. The filtrate was extracted with a dichloromethane / isopropanol mixture (volume ratio: 3 / 1, 50 mL x 3). The organic phases were combined, washed with saturated brine and 10% sodium bisulfite, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain the crude product. The crude product was recrystallized from a mixture of ethyl acetate (32 mL) and n-heptane (24 mL) to obtain compound 5-4.

[0191] Step C: To a solution of compound 5-5 (150 g, 976.52 mmol) in acetonitrile (1500 mL), triethylamine (242.09 g, 2.39 mol) and trifluoroacetic anhydride (332.98 g, 2.34 mol) were added. The reaction was stirred at 25°C for 12 hours. The reaction was detected to be complete. The reaction solution was filtered, the filter cake was rinsed with ethyl acetate (200 mL*2), and the mother liquor was concentrated under reduced pressure to obtain an oil. The oil was slurried with ethyl acetate (1500 mL) at 20°C for 1 hour, filtered, the filter cake was rinsed with ethyl acetate (200 mL*2), and the mother liquor was concentrated under reduced pressure to obtain compound 5-6.

[0192] Step D: To a solution of compound 5-6 (6 g, 28.15 mmol) in dichloromethane (40 mL) was added pyridine (22.27 g, 281.49 mmol) and 4,4-dimethoxytrityl chloride (9.54 g, 28.15 mmol) at 0°C. The reaction was stirred at 0-25°C for 12 hours. The reaction was detected to be complete. The reaction solution was diluted with water (100 mL), extracted with dichloromethane (100 mL), and the organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the crude product. The crude product was purified by silica gel column (eluted with ethyl acetate / petroleum ether = 15 / 1 to 5 / 1, with 1% triethylamine added to the eluent) to obtain compound 5-7.

[0193] Step E: To a methanol solution of compound 5-7 (2 g, 3.88 mmol), potassium hydroxide (435.33 mg, 7.76 mmol) and water (5 mL) were added. The reaction was stirred at 25°C for 1 hour. The reaction was detected to be complete. The reaction solution was concentrated under reduced pressure to remove methanol, and then extracted with water (80 mL) and dichloromethane (50 mL x 2). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain compound 3-3.

[0194] Step F: Under nitrogen, set up a water trap at room temperature and mix compound 5-8 (100.85 g, 740.75 mmol), trimethyl orthoacetate (89.00 g, 740.75 mmol), and p-toluenesulfonic acid monohydrate (7.05 g, 37.04 mmol) in toluene (500 mL). Heat to 110°C and stir for 20 hours. The reaction was completed. The toluene was concentrated under reduced pressure, and triethylamine (10 mL) was added. The mixture was concentrated under reduced pressure to dryness to obtain compound 5-9.

[0195] Step G: Under nitrogen, add compound 5-9 (25 g, 156.09 mmol), 4-dimethylaminopyridine (1.91 g, 15.61 mmol), and p-toluenesulfonyl chloride (32.73 g, 171.70 mmol) to dichloromethane (250 mL). Then, add triethylamine (23.69 g, 234.13 mmol) while controlling the temperature at 10-25°C. Stir and react at 25°C for 16 hours. The reaction is complete upon detection. Dilute with water (500 mL) and extract with dichloromethane (100 mL x 2). Combine the organic phases, wash with saturated brine (200 mL x 2), dry over anhydrous sodium sulfate, filter, and concentrate under reduced pressure to obtain the crude product. The product was then purified by a reverse phase column (neutral conditions, column model: Kromasil Eternity XT 250*80mm*10μm; mobile phase: [water (0.1% ammonium bicarbonate)-acetonitrile]; gradient: 25%-55%, 20 minutes) to obtain compound 5-10.

[0196] Step H: Under nitrogen protection, compound 5-10 (50g, 159.06mmol), compound 5-11 (32.25g, 174.96mmol) and potassium hydroxide (35.70g, 636.23mmol) were added to tetrahydrofuran (500mL), and the temperature was raised to 50°C for 16 hours. The reaction was detected to be complete. Diluted with water (500mL) and extracted with methyl tert-butyl ether (500mL*2). The organic phases were combined, washed with saturated brine (200mL*2), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain a crude product. Purified by silica gel column (eluted with ethyl acetate / petroleum ether = 1 / 0 to 10 / 1, and 0.1% triethylamine was added to the eluent) to obtain compound 5-12.

[0197] Step I: Under nitrogen, add compound 5-12 (28 g, 85.77 mmol) and 37% concentrated hydrochloric acid (6 mL) to methanol (300 mL) and water (60 mL). Stir at 25°C for 2 hours. Check for completion. Concentrate under reduced pressure, then evaporate methanol and then tetrahydrofuran to remove water and methanol to obtain compound 5-13.

[0198] Step J: Under nitrogen, compound 5-13 (25.94 g, 85.77 mmol), 2,2-dimethoxyacetone (10.72 g, 102.82 mmol), and p-toluenesulfonyl chloride monohydrate (1.63 g, 8.58 mmol) were added to N,N-dimethylformamide (250 mL) and stirred at 25°C for 16 hours. The reaction was detected to be complete. The reaction solution was quenched with saturated sodium bicarbonate (10 mL), diluted with water (10 mL), and extracted with ethyl acetate (50 mL x 2). The organic phases were combined, washed with saturated brine (30 mL x 2), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain the crude product. The product was purified on a silica gel column (eluted with ethyl acetate / petroleum ether = 1 / 0 to 1 / 1, with 0.1% triethylamine added to the eluent) to obtain compound 5-14.

[0199] Step K: Under nitrogen protection, compound 5-14 (9.8 g, 28.61 mmol) and Dess-Martin periodinane (14.56 g, 34.33 mmol) were added to dichloromethane (98 mL) and stirred at 25°C for 16 hours. The reaction was detected to be complete. Saturated sodium bicarbonate (20 mL) was added to the reaction solution for dilution and extracted with dichloromethane (50 mL*2). The organic phases were combined, washed with saturated brine (30 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain the crude product. Compound 5-15 was purified by column chromatography (eluting with ethyl acetate / petroleum ether = 1 / 0 to 1 / 5) to obtain compound 5-15.

[0200] Step L: Under nitrogen, compound 5-15 (6.8 g, 19.97 mmol), disodium hydrogen phosphate (4.19 g, 34.95 mmol), 2-methyl-2-butene (9.8 g, 139.80 mmol), and sodium chlorite (8.31 g, 91.87 mmol) were added to tert-butyl alcohol (40 mL) and water (10 mL). The reaction was stirred at 25°C for 16 hours. The reaction was detected to be complete. Water (100 mL) was added to the reaction solution for dilution, and the mixture was extracted with dichloromethane (100 mL x 2). The mixture was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain compound 5-16.

[0201] Step M: Under nitrogen, compound 5-16 (7.12 g, 19.97 mmol) and 37% concentrated hydrochloric acid (0.7 mL) were added to methanol (35 mL) and water (7 mL). The mixture was stirred at 25°C for 16 hours. The reaction was completed. The methanol was removed by concentration under reduced pressure. The methanol was then evaporated from tetrahydrofuran to remove water and methanol, yielding compound 5-17.

[0202] Step N: Under nitrogen, compound 5-17 (6.32 g, 19.97 mmol), compound 5-18 (3.19 g, 21.97 mmol), triethylamine (3.03 g, 29.96 mmol), and O-(7-azabenzotriazol-1-yl)-N,N,N,N-tetramethyluronium hexafluorophosphonate (9.11 g, 23.97 mmol) were added to dichloromethane (60 mL) and stirred at 25°C for 16 hours. The reaction was detected to be complete. Water (20 mL) was added to the reaction solution for dilution and extraction was performed with dichloromethane (20 mL x 2). The organic phases were combined, washed with saturated brine (20 mL x 2), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain the crude product. The product was purified on a silica gel column (ethyl acetate / petroleum ether = 1 / 0 to 1 / 1) to obtain compound 5-19.

[0203] Step O: Under nitrogen protection, compound 5-19 (3 ​​g, 6.76 mmol), tert-butyl acrylate (2.60 g, 20.29 mmol) and potassium hydroxide (37.95 mg, 676.26 μmol) were added to dimethyl sulfoxide (6 mL) and water (0.6 mL) and stirred at 25 ° C for 16 hours. The reaction was detected to be complete. Water (20 mL) was added to the reaction solution for dilution and extracted with ethyl acetate (50 mL). The organic phases were combined, washed with saturated brine (30 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain a crude product. Purification by silica gel column (ethyl acetate / petroleum ether = 1 / 0 to 4 / 1) gave compound 5-20.

[0204] Step P: Under nitrogen protection, compound 5-20 (1.9 g, 2.71 mmol) was added to dioxane hydrochloride (2 M, 20 mL) and stirred at 25°C for 16 hours. The reaction was detected to be complete and concentrated under reduced pressure to dryness to obtain compound 5-21.

[0205] Step Q: Under nitrogen, compound 5-21 (1 g, 1.88 mmol), compound 5-22 (1.23 g, 7.05 mmol), triethylamine (1.14 g, 11.29 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (1.35 g, 7.05 mmol) and 1-hydroxy-7-azabenzotriazole (960.09 mg, 7.05 mmol) were added to 2-methyltetrahydrofuran (10 mL) and stirred at 25° C. for 16 hours. The reaction was detected to be complete. Water (50 mL) was added to the reaction solution for dilution, and the mixture was extracted with 2-methyltetrahydrofuran (50 mL*2). The organic phases were combined and washed with 10% citric acid (30 mL*2), saturated brine (30 mL), 10% sodium bicarbonate (30 mL), and saturated brine (50 mL*2), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain compound 5-23.

[0206] Step R: Under nitrogen, compound 5-23 (1.88 g, 1.88 mmol) and p-toluenesulfonic acid monohydrate (2.14 g, 11.28 mmol) were added to 1,2-dichloroethane (9 mL) and stirred at 50°C for 1.5 hours. The reaction was completed. Compound 5-24 was obtained and used directly in the next reaction.

[0207] Step S: Under nitrogen, compound 5-24 (reaction solution from the previous step), triethylamine (2.86 g, 28.29 mmol), compound 5-4 (2.78 g, 6.22 mmol), 1-hydroxy-7-azabenzotriazole (898.38 mg, 6.6 mmol), and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (1.27 g, 6.60 mmol) were added to 1,2-dichloroethane (9 mL) and stirred at 25°C for 2 hours. The reaction was checked for completion. Dichloromethane (50 mL) was added to the reaction solution, and the mixture was treated with 10% citric acid (50 mL*2), 10% sodium bicarbonate (50 mL*2), and saturated brine (50 mL). The mixture was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain compound 5-25.

[0208] Step T: Sodium periodate (1.78 g, 8.30 mmol) was slowly added to a solution of dioxane (10 mL), water (10 mL), compound 5-25 (3 g, 1.51 mmol), and ruthenium trichloride trihydrate (19.73 mg, 75.44 μmol) at 25°C. The mixture was stirred at 25°C for 2 hours. The reaction was detected to be complete. The reaction solution was filtered, rinsed with dichloromethane (50 mL), diluted with water (30 mL), adjusted to pH 8-9 with saturated sodium bicarbonate, and extracted with dichloromethane (50 mL*2). The aqueous phase was retained and adjusted to pH 3-4 with 10% citric acid. The mixture was extracted with dichloromethane / isopropanol = 3 / 1 (50 mL*3). The organic phases were combined, washed with 10% sodium bisulfite (20 mL) and saturated brine (30 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain compound 5-26.

[0209] Step U: Compound 5-26 (1.6 g, 797.53 μmol), compound 3-3 (351.30 mg, 837.41 μmol), N,N-diisopropylethylamine (309.23 mg, 2.39 mmol), 1-hydroxy-7-azabenzotriazole (54.28 mg, 398.77 μmol), and O-(7-azabenzotriazol-1-yl)-N,N,N,N-tetramethyluronium hexafluorophosphonate (363.90 mg, 957.04 μmol) were added to dichloromethane (15 mL) and stirred at 25°C under nitrogen for 16 hours. The reaction was detected as complete. The reaction solution was diluted with dichloromethane (50 mL), washed with 10% saturated sodium hydroxide (50 mL x 2) and saturated brine (50 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain compound 5-27.

[0210] Step V: Under nitrogen, compound 5-27 (1 g, 415.34 μmol), triethylamine (210.14 mg, 2.08 μmol), 4-lutidine (5.07 mg, 41.53 μmol), and succinic anhydride (166.26 mg, 1.66 mmol) were added to dichloromethane (5 mL) and stirred at 25°C for 2 hours. The reaction was detected as complete. The crude product was concentrated under reduced pressure and purified via a reverse phase column (neutral conditions; column model: Waters Xbridge C18 150*50 mm*10 μm; mobile phase: water (0.1% ammonium bicarbonate)-acetonitrile; gradient: 22% to 52% over 10 minutes) to obtain D18-M. 1H NMR (400MHz, DMSO-d6) δ=12.45-12.01(m,1H),7.88-7.79(m,6H),7.74(br t,J=5.4Hz,3H),7.51(br t,J=5.0Hz,1H),7.37-7.26(m,4H),7.26-7.16(m,5H),6.95-6.78(m,4H),5.22(d,J=3.4Hz,3H),4.97(dd,J=3.4,11.2H z,3H),4.49(d,J=8.6Hz,3H),4.08-3.98(m,9H),3.88(td,J=9.0,10.9Hz,3H),3.74(s,6H),3.72-3.67(m,3H),3.53(br t,J=6.2Hz,4H),3.47-3.44(m,4H),3.43-3.39(m,4H),3.27-3.19(m,3H),3.03(br d,J=5.5Hz,13H),2.49-2.44(m,3H),2.32-2.18(m,9H),2.10(s,9H),2.08(s,7H),2.04(br t, J=7.1Hz, 6H), 2.00 (s, 9H), 1.89 (s, 9H), 1.78 (s, 9H), 1.56-1.38 (m, 23H), 1.23 (br s, 13H); LCMS (ESI) m / z: 2507.76.

[0211] Example 7: Synthesis of mDVPU-M

[0212] Step A: Under nitrogen, add n-butyllithium (2.5 M, THF, 94.57 mL) to a solution of compound 6-2 (78 g, 236.43 mmol) in tetrahydrofuran (1 L) at -78°C over 10 minutes. After addition, stir at the same temperature for 20 minutes. Add a solution of compound 6-1 (98.94 g, 236.43 mmol) in tetrahydrofuran (500 mL) at -78°C. Stir and react at -78°C for 1.5 hours. Quench the reaction by adding saturated ammonium chloride (500 mL) at 0°C. Extract with ethyl acetate (1 L), wash with saturated brine (500 mL x 2), dry over anhydrous sodium sulfate, filter, and concentrate under reduced pressure to obtain the crude product. The crude product is then purified on a silica gel column (petroleum ether / ethyl acetate = 1 / 0 to 3 / 1) to obtain compound 6-3.

[0213] Step B: Under nitrogen, boron trifluoride etherate (217.33 mL) and triethylsilyl hydrochloride (282.24 mL) were added to a solution of compound 6-3 (110 g, 176.71 mmol) in dichloromethane (1.1 L) at -78°C. The mixture was stirred at -78°C for 2 hours. The reaction was quenched by adding 5% sodium carbonate (500 mL) at 0°C. The mixture was extracted with dichloromethane (300 mL x 2). The organic phases were combined, washed with saturated sodium chloride (200 mL x 2), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain the crude product. The crude product was purified on a silica gel column (petroleum ether / ethyl acetate = 1 / 0 to 5 / 1) to obtain compound 6-4.

[0214] Step C: Under nitrogen, compound 6-4 (73 g, 120.36 mmol), compound 6-5 (21.08 g, 132.40 mmol), cuprous iodide (2.29 g, 12.04 mmol), N,N'-diisopropylethylamine (601.82 mmol, 104.83 mL), and 1,1-bis(diphenylphosphino)ferrocenepalladium chloride (8.81 g, 12.04 mmol) were added to acetonitrile (400 mL). The mixture was stirred at 25°C for 2 hours. The reaction solution was diluted with water (300 mL) and extracted with ethyl acetate (500 mL x 2). The organic phases were combined, washed with saturated sodium chloride (300 mL x 2), dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the crude product. The crude product was purified on a silica gel column (petroleum ether / ethyl acetate = 1 / 0 to 3 / 1) to obtain compound 6-6.

[0215] Step D: Under nitrogen, compound 6-6 (70 g, 109.76 mmol) and cesium carbonate (71.52 g, 219.52 mmol) were added to acetonitrile (700 mL) and stirred at 80°C for 16 hours. The reaction solution was concentrated under reduced pressure to remove the acetonitrile, diluted with water (100 mL), and extracted with ethyl acetate (100 mL*2). The organic phases were combined, washed with saturated sodium chloride (50 mL*2), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain the crude product. The crude product was purified on a silica gel column (petroleum ether / ethyl acetate = 1 / 0 to 3 / 1) to obtain compound 6-7.

[0216] Step E: Under nitrogen protection, boron trichloride (1M, diethyl ether solution, 627.19 mL) was added to a solution of compound 6-7 (50 g, 78.40 mmol) in dichloromethane (500 mL) at -78 ° C. The reaction was stirred at -78 ° C for 2 hours. The reaction was detected to be complete. At -78 ° C, methanol (100 mL) was slowly added dropwise to quench the reaction solution, and then concentrated under reduced pressure to obtain a crude product. The crude product was purified by silica gel column (petroleum ether / ethyl acetate = 1 / 0 to 1 / 1, then ethyl acetate / ethanol = 10 / 1 to 5 / 1) to obtain compound 6-8.

[0217] Step F: Under nitrogen, compound 6-8 (28.8 g, 78.39 mmol), 1,3-dichloro-1,1,3,3-tetraisopropyldimethylsilyl ether (29.67 g, 94.07 mmol), and imidazole (21.35 g, 313.56 mmol) were added to dichloromethane (280 mL) and stirred at 25°C for 16 hours. The reaction solution was diluted with water (200 mL) and extracted with dichloromethane (200 mL x 2). The organic phases were combined, washed with saturated sodium chloride (200 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain the crude product. The crude product was purified on a silica gel column (petroleum ether / ethyl acetate = 1 / 0 to 4 / 1) to obtain compound 6-9.

[0218] Step G: Under nitrogen, sodium hydroxide (2.20 g, 55.09 mmol, 60% purity) was slowly added to a solution of compound 6-9 (28 g, 45.91 mmol) in N,N-dimethylformamide (140 mL) at 0°C. After addition, the reaction was stirred at 0°C for 0.5 hours. Iodomethane (137.73 mmol, 8.57 mL) was added and stirred at 25°C for 1.5 hours. The reaction mixture was quenched with saturated ammonium chloride (100 mL) at 10-25°C, diluted with water (200 mL), and extracted with ethyl acetate (200 mL x 2). The organic phases were combined, washed with saturated sodium chloride (30 mL x 2), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain the crude product. The crude product was purified on a silica gel column (petroleum ether / ethyl acetate = 1 / 0 to 4 / 1) to obtain compound 6-10.

[0219] Step H: Under nitrogen, add compound 6-10 (22.3 g, 35.74 mmol) and triethylamine hydrofluoride (9.53 g, 78.63 mmol) to a solution of tetrahydrofuran (160 mL). Stir and react at 25°C for 16 hours. The reaction solution is concentrated under reduced pressure to obtain a crude product. The crude product is purified on a silica gel column (petroleum ether / ethyl acetate = 1 / 0 to 0 / 1) to obtain compound 6-11.

[0220] Step I: Under nitrogen, compound 6-11 (10.6 g, 27.79 mmol), 4,4-dimethoxytrityl chloride (11.30 g, 33.35 mmol), and triethylenediamine (4.68 g, 41.69 mmol) were mixed in dichloromethane (60 mL) and stirred at 25°C for 16 hours. The reaction solution was diluted with water (30 mL) and extracted with dichloromethane (50 mL x 2). The combined organic phases were washed with saturated sodium chloride (20 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain the crude product. The crude product was purified on a silica gel column (petroleum ether / ethyl acetate = 1 / 0 to 1 / 1, 0.1% triethylamine) to obtain compound 6-12.

[0221] Step J: Under nitrogen, compound 6-12 (17 g, 24.86 mmol), imidazole (3.39 g, 49.72 mmol), and tert-butyldiphenylsilyl chloride (8.20 g, 29.83 mmol) were added to dichloromethane (170 mL) and stirred at 25°C for 16 hours. The reaction solution was diluted with water (20 mL) and extracted with dichloromethane (30 mL x 2). The organic phases were combined, washed with saturated sodium chloride (30 mL), dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the crude product. The crude product was purified on a silica gel column (petroleum ether / ethyl acetate = 1 / 0 to 2 / 1) to obtain compound 6-13.

[0222] Step K: Under nitrogen, compound 6-13 (21 g, 22.77 mmol) and p-toluenesulfonyl chloride monohydrate (2.17 g, 11.39 mmol) were added to dichloromethane (200 mL) and methanol (60 mL). The mixture was stirred at 5°C for 1 hour. The reaction mixture was quenched with saturated sodium bicarbonate (20 mL) and extracted with dichloromethane (50 mL x 2). The organic phases were combined, washed with saturated sodium chloride (30 mL x 2), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain the crude product. The crude product was purified on a silica gel column (petroleum ether / ethyl acetate = 1 / 0 to 2 / 1) to obtain compound 6-14.

[0223] Step L: Under nitrogen, compound 6-14 (13.7 g, 22.10 mmol) and Dess-Martin periodinane (11.25 g, 26.52 mmol) were added to dichloromethane (100 mL) and stirred at 25°C for 1 hour. The reaction mixture was quenched with saturated sodium bicarbonate (20 mL) and extracted with dichloromethane (50 mL x 2). The organic phases were combined, washed with saturated sodium chloride (30 mL x 2), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain the crude product. The crude product was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 1 / 0 to 2 / 1) to obtain compound 6-15.

[0224] Step M: Under nitrogen, compound 6-15 (2 g, 3.24 mmol), tetraethylmethylene diphosphate (1.12 g, 3.88 mmol), lithium chloride (274.48 mg, 6.47 mmol), and 1.8-diazabicyclo[5.4.0]undec-7-ene (1.48 g, 9.71 mmol) were added to dichloromethane (20 mL) and stirred at 25°C for 3 hours. The reaction mixture was quenched with water (10 mL) at 25°C and extracted with dichloromethane (30 mL x 2). The combined organic phases were washed with saturated sodium chloride (30 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain the crude product. The crude product was purified on a silica gel column (petroleum ether / ethyl acetate = 1 / 0 to 1 / 1) to obtain compound 6-16.

[0225] Step N: Under nitrogen, compound 6-16 (2.1 g, 2.79 mmol) and triethylamine hydrofluoride (676.97 mg, 5.59 mmol) were added to tetrahydrofuran (10 mL). The mixture was stirred at 40°C for 16 hours. The reaction solution was concentrated under reduced pressure to obtain a crude product. The crude product was purified on a silica gel column (petroleum ether / ethyl acetate = 1 / 0 to 0 / 1, then ethyl acetate / ethanol = 1 / 0 to 5 / 1) to obtain compound 6-17.

[0226] Step O: Under nitrogen, compound 6-17 (1.3 g, 2.53 mmol), bis(diisopropylamino)(2-cyanoethoxy)phosphine (1.07 g, 3.54 mmol), and 4,5-dicyanoimidazole (149.48 mg, 1.27 mmol) were added to dichloromethane (15 mL) and reacted at 25°C for 1 hour. The reaction solution was diluted with water (10 mL) and extracted with dichloromethane (50 mL x 2). The organic phases were combined, washed with saturated sodium chloride (30 mL x 2), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain the crude product. The crude product was purified by reverse-phase column chromatography (neutral conditions; column: Waters Xbridge C18 150*50 mm x 10 μm; mobile phase: water (0.1% ammonium bicarbonate)-acetonitrile]; gradient: acetonitrile % = 57% - 87%) to obtain compound mDVPU-M. MS=774.5(M+HCO3 - ), 1 H NMR (400MHz, DMSO-d6)δ=8.00-7.82(m,2H),7.58-7.44(m,3H),7.43-7.23(m,4H),7.04(s,1H),6.90-6.65(m,1H),6.10(br dd,J=18.0,19.4Hz,1H),4.89(dd,J=5.9,15.8Hz,1H),4.69-4.53(m,1H),4.33-4.19(m,1H),4.15(s,2H), 4.04-3.92(m,4H),3.86-3.53(m,5H),3.34-3.29(m,3H),2.80(td,J=5.4,11.0Hz,2H),1.30-1.06(m,18H).

[0227] Example 8: Synthesis of D21-M

[0228] Step A: Sodium carbonate (14.91 g, 140.65 mmol) was added to a solution of compound 7-1 (27.7 g, 93.76 mmol) and compound 7-2 (21.58 g, 98.45 mmol) in tetrahydrofuran (300 mL). The mixture was stirred at 25°C for 12 hours. After completion of the reaction, the mixture was diluted with ethyl acetate (800 mL) and washed with saturated brine (500 mL*3). The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain the crude product. The crude product was purified by silica gel column (eluted with ethyl acetate / petroleum ether = 0:1 to 1:10) to obtain compound 7-3.

[0229] Step B: Under nitrogen protection, palladium / carbon (14 g, 10% palladium content) and palladium hydroxide / carbon (14 g, 20% purity) were added to a solution of compound 7-3 (27.9 g, 65.57 mmol) and p-toluenesulfonic acid monohydrate (27.44 g, 144.25 mmol) in tetrahydrofuran (1 L). The reaction system was replaced with hydrogen three times and reacted under a hydrogen atmosphere of 50°C and 50 psi for 12 hours. After the reaction was completed, the reaction solution was filtered through celite, the filter cake was washed with a mixed solution of dichloromethane / methanol (400 mL / 400 mL), and the filtrate was concentrated under reduced pressure to obtain a crude product. The crude product was slurried with ethyl acetate (600 mL) at 25°C for 12 hours, filtered, and the filter cake was concentrated under reduced pressure to obtain compound 7-4.

[0230] Step C: To a solution of compound 7-5 (9 g, 39.08 mmol) in dichloromethane (150 mL) were added N,N-diisopropylethylamine (7.58 g, 58.62 mmol), 1-hydroxy-7-azabenzotriazole (7.98 g, 58.62 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (11.24 g, 58.62 mmol) and compound 3-3 (18.03 g, 42.99 mmol) in sequence, and stirred at 25°C for 12 hours. After completion of the reaction, the mixture was diluted with water (250 mL) and extracted with dichloromethane (200 mL). The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain a crude product. The residue was then purified by a reverse phase column (neutral conditions, column model: Phenomenex luna c18 250mm*100mm*10μm; mobile phase: [water (10mM ammonium bicarbonate)-acetonitrile]; gradient: acetonitrile% = 60%-80%, 30 minutes) to obtain compound 7-6.

[0231] Step D: To a mixed solution of compound 7-6 (20.7 g, 32.76 mmol) in methanol (65 mL), tetrahydrofuran (65 mL) and water (65 mL) was added lithium hydroxide (941.56 mg, 39.32 mmol), and the mixture was stirred at 25°C for 12 hours, followed by additional lithium hydroxide (392.32 mg, 16.38 mmol). The mixture was stirred at 25°C for 8 hours. After the reaction was completed, the mixture was concentrated under reduced pressure to obtain a crude product, which was purified by a reverse phase column (alkaline conditions, column model: Kromasil Eternity XT 250*80mm*10μm; mobile phase: [water (0.05% ammonia water)-acetonitrile]; gradient: acetonitrile% = 15%-45%, 20 minutes) to obtain compound 7-7.

[0232] Step E: To a solution of compound 7-8 (7 g, 26.29 mmol) and triethylamine (13.3 g, 131.46 mmol) in N,N-dimethylformamide (70 mL) was added O-(7-azabenzotriazole-1-yl)-N,N,N,N-tetramethyluronium hexafluorophosphonate (23.99 g, 63.1 mmol), and the mixture was stirred at 25°C for 0.5 hour. Then, compound 5-22 (10.99 g, 63.1 mmol) was added and stirred at 25°C for 12 hours. After the reaction was completed, the mixture was diluted with ethyl acetate (400 mL), washed successively with hydrochloric acid (0.5 M, 300 mL*2), saturated brine (300 mL*2), sodium bicarbonate solution (300 mL*2) and saturated brine (300 mL*2), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain a crude product. The crude product was purified by silica gel column (eluted with ethyl acetate / petroleum ether = 0 / 1 to 1 / 0) to obtain compound 7-9.

[0233] Step F: To a mixed solution of compound 7-9 (18 g, 31.1 mmol) in methanol (60 mL) and water (30 mL) was added sodium hydroxide (1.74 g, 43.55 mmol), stirred at 30°C for 3 hours, and then concentrated under reduced pressure to remove methanol. The mixture was then diluted with water (40 mL) and extracted with dichloromethane (60 mL*3). The organic phase was discarded, and the aqueous phase was adjusted to pH 2-3 with hydrochloric acid (1 M) and extracted with a mixed solution of dichloromethane / isopropanol (volume ratio: 4 / 1, 50 mL*4). The combined organic phases were dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain a crude compound 7-10.

[0234] Step G: Benzotriazole-N,N,N,N-tetramethyluronium hexafluorophosphate (6.91 g, 18.23 mmol) was added to a solution of compound 7-10 (7.42 g) and triethylamine (9.22 g, 91.13 mmol) in N,N-dimethylformamide (100 mL), and the mixture was stirred at 25°C for 0.5 hours. Compound 7-4 (17.91 g) was then added and stirred at 25°C for 12 hours. After the reaction was completed, ethyl acetate (300 mL) and 2-methyltetrahydrofuran (300 mL) were added for dilution and the pH was adjusted to 10. The mixture was then washed with saturated brine (300 mL*3), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give a crude product. The crude product was purified by silica gel column (eluted with methanol / dichloromethane = 0 / 1 to 1 / 10) to give compound 7-11.

[0235] Step H: To a solution of compound 7-11 (3.62 g, 13.91 mmol) and triethylamine (3.52 g, 34.78 mmol) in N,N-dimethylformamide (80 mL) was added O-(7-azabenzotriazole-1-yl)-N,N,N,N-tetramethyluronium hexafluorophosphonate (6.17 g, 16.23 mmol), and the mixture was stirred at 25°C for 0.5 hours, and then compound 7-12 (8.3 g, 11.59 mmol) was added. The mixture was stirred at 5°C for 12 hours. After the reaction was completed, 2-methyltetrahydrofuran (400 mL) was added for dilution, followed by washing with aqueous citric acid solution (10%, 300 mL*2), saturated brine (300 mL*2), aqueous sodium bicarbonate solution (300 mL*2) and saturated brine (300 mL*2), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain a crude product, which was purified by silica gel column (eluted with methanol / dichloromethane = 0 / 1 to 12 / 100) to obtain compound 7-13.

[0236] Step I: To a mixed solution of compound 7-13 (8.52 g, 8.89 mmol) in tetrahydrofuran (50 mL) and ethanol (50 mL) was added hydrazine hydrate (7.66 g, 149.96 mmol, 98% purity), and the mixture was stirred at 45°C for 6 hours. After the reaction was completed, it was concentrated under reduced pressure to obtain a crude product. The crude product was slurried with a mixed solution of acetonitrile (50 mL) and dichloromethane (50 mL) at 25°C for 0.5 hours, filtered, and the filtrate was concentrated under reduced pressure to obtain compound 7-14.

[0237] Step J: To a solution of compound 7-14 (7.9 g, 9.54 mmol) and N,N-diisopropylethylamine (2.47 g, 19.08 mmol) in tetrahydrofuran (70 mL) was added benzyloxycarbonyl succinimide (2.85 g, 11.45 mmol), and the mixture was stirred at 25°C for 12 hours. After the reaction was completed, 2-methyltetrahydrofuran (400 mL) was added for dilution, and the solution was washed with citric acid aqueous solution (10%, 200 mL*2), saturated brine (200 mL*2), sodium bicarbonate aqueous solution (200 mL*2) and saturated brine (200 mL*2) in sequence. The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain a crude product. The crude product was purified by silica gel column (eluted with methanol / dichloromethane = 0 / 1 to 12 / 100) to obtain compound 7-15.

[0238] Step K: A solution of compound 7-15 (8 g, 8.31 mmol) in formic acid (80 mL, 98% purity) was stirred at 45° C. for 2 hours. After the reaction was completed, the mixture was concentrated under reduced pressure to obtain the crude formate salt of compound 7-16.

[0239] Step L: To a mixed solution of the formate salt of compound 7-16 (6.65 g) and compound 7-17 (20.82 g, 38.24 mmol), tetrahydrofuran (170 mL) and water (170 mL) was added N, N-diisopropylethylamine (16.12 g, 124.71 mmol), and stirred at 30 ° C for 12 hours. After the reaction was completed, it was extracted with a mixed solution of dichloromethane / isopropanol (volume ratio: 5 / 1, 100 mL * 3), and the combined organic phases were dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain a crude product. The crude product was then dissolved in acetic anhydride (27.18 g, 266.19 mmol, 25 mL) and pyridine (100 mL), stirred at 25 ° C for 12 hours, and concentrated under reduced pressure to obtain a crude product, which was then purified by reverse phase column (acidic conditions, column type: Phenomenex luna c18 250mm*80mm*10μm; mobile phase: [water (0.225% formic acid)-acetonitrile]; gradient: acetonitrile% = 25%-45%, 20 minutes) to obtain compound 7-18.

[0240] Step M: Under nitrogen, palladium on carbon (1 g, 10% palladium content) was added to a solution of compound 7-18 (2.5 g, 1.28 mmol) and p-toluenesulfonic acid monohydrate (243.86 mg, 1.28 mmol) in tetrahydrofuran (50 mL). The reaction system was replaced with hydrogen three times and reacted under a hydrogen atmosphere of 20 psi at 25°C for 12 hours. After completion of the reaction, the reaction solution was filtered through celite, and the filtrate was concentrated under reduced pressure to obtain the crude p-toluenesulfonic acid salt of compound 7-19.

[0241] Step N: A solution of compound 7-7 (950.84 mg, 1.54 mmol), 1-hydroxy-7-azabenzotriazole (226.95 mg, 1.67 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (319.64 mg, 1.67 mmol) and N,N-diisopropylethylamine (994.62 mg, 7.7 mmol) in N,N-dimethylformamide (25 mL) was replaced three times with nitrogen, stirred at 25°C for 0.5 hours, and then p-toluenesulfonate of compound 7-19 (2.55 g) was added and stirred at 25°C for 12 hours. After the reaction was completed, the reaction solution was directly purified by a reverse phase column (neutral conditions, column model: Waters Xbridge C18 150*50mm*10μm; mobile phase: [water (10mM ammonium bicarbonate)-acetonitrile]; gradient: acetonitrile% = 28%-58%, 10 minutes) to obtain compound 7-20.

[0242] Step O: A solution of compound 7-20 (1.86 g, 769.97 μmol), succinic anhydride (385.27 mg, 3.85 mmol), and triethylamine (623.3 mg, 6.16 mmol) in dichloromethane (5 mL) was purged with nitrogen three times and stirred at 30°C under nitrogen for 2 hours. After completion of the reaction, the mixture was concentrated under reduced pressure to obtain the crude product, which was then purified via a reverse phase column (neutral conditions; column model: Waters Xbridge C18 150*50 mm*10 μm; mobile phase: [water (10 mM ammonium bicarbonate)-acetonitrile]; gradient: acetonitrile % = 20% - 50% over 10 minutes) to obtain D21-M.

[0243] 1H NMR (400MHz, DMSO-d6) δ = 8.04-7.63 (m, 9H), 7.34-7.13 (m, 9H), 6.93-6.80 (m, 4H), 5.42-5.31 (m, 1H), 5.21 (d, J = 3. 2Hz,3H),4.97(dd,J=3.2,11.2Hz,3H),4.49(d,J=8.4Hz,3H),4.25–4.15(m,1H),4.07-3.95(m,9H),3.93-3.82(m, 3H),3.79-3.64(m,12H),3.62-3.50(m,4H),3.48-3.17(m,14H),3.11-2.91(m,14H),2.48-2.28(m,8H),2.26-2.15 (m,4H),2.10(s,9H),2.07-2.01(m,8H),1.99(s,10H),1.89(s,9H),1.77(s,9H),1.46(s,19H),1.31-1.09(m,10H).

[0244] Example 9: Synthesis of B29-M

[0245] Step A: Under nitrogen, compound 8-1 (1.83 g, 6.44 mmol), imidazole (2.63 g, 38.63 mmol), tert-butyldimethylsilyl chloride (3.88 g, 25.75 mmol), and 4-dimethylaminopyridine (235.94 mg, 1.93 mmol) were mixed in N,N-dimethylformamide (30 mL) and the reaction mixture was heated to 60°C for 16 hours. The reaction mixture was diluted with water (100 mL) and extracted with ethyl acetate (150 mL × 2). The organic phases were combined, washed with 5% aqueous citric acid solution (50 mL × 2) and sodium chloride solution (100 mL × 2), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain compound 8-2.

[0246] Step B: Under nitrogen, 8-2 (3.3 g, 6.44 mmol) and trifluoroacetic acid (23.03 g, 201.93 mmol) were mixed in a solution of tetrahydrofuran (60 mL) and water (15 mL). The mixture was stirred at 25°C for 2 hours. The reaction solution was diluted with water (30 mL) and extracted with ethyl acetate (50 mL × 2). The organic phases were combined, washed with saturated sodium bicarbonate (50 mL × 2) and saturated sodium chloride (50 mL × 2), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain the crude product. The crude product was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 1 / 0 to 1 / 1) to obtain compound 8-3.

[0247] Step C: Under nitrogen protection, compound 8-3 (1.29 g, 3.24 mmol) and 2-iodobenzoic acid (1.81 g, 6.47 mmol) were mixed in acetonitrile (20 mL), and the reaction solution was stirred at 50°C for 12 hours. The reaction solution was filtered and the filtrate was concentrated under reduced pressure to dryness to obtain compound 8-4.

[0248] Step D: Under nitrogen, tetrakis[[pivaloyloxymethyl]methylenediphosphonate (4.79 g, 7.57 mmol) was added to a solution of sodium hydrogen phosphate (363.13 mg, 9.08 mmol, 60% purity) in tetrahydrofuran (5 mL) at -78°C. The mixture was reacted at -78°C for 0.5 hour. A solution of compound 8-4 (1.2 g, 3.03 mmol) in tetrahydrofuran (5 mL) was then added at -78°C. The reaction mixture was heated to 25°C and stirred for 2.5 hours. The reaction mixture was quenched by the addition of saturated ammonium chloride (30 mL) at 10-25°C, diluted with water (10 mL), and extracted with ethyl acetate (100 mL × 2). The organic phases were combined, washed with saturated sodium chloride (50 mL × 2), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain the crude product. The crude product was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 1 / 0 to 1 / 1) to obtain compound 8-5.

[0249] Step E: Under nitrogen protection, compound 8-5 (1.1 g, 1.57 mmol) and formic acid (10 mL) were mixed in water (10 mL), and the reaction solution was reacted at 40°C for 12 hours. The reaction solution was diluted with water (50 mL) and extracted with ethyl acetate (100 mL × 2). The organic phases were combined, washed with saturated sodium bicarbonate (50 mL × 2) and saturated sodium chloride (50 mL × 2), respectively, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain a crude product. The crude product was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 1 / 0 to 0 / 1) to obtain compound 8-6.

[0250] Step F: Under nitrogen, compound 8-6 (430 mg, 730.62 μmol), compound 1-9 (330.32 mg, 1.1 mmol), and 4,5-dicyanoimidazole (430 mg, 730.62 μmol) were mixed in dichloromethane (5 mL). The mixture was reacted at 25°C for 2 hours. The mixture was concentrated under reduced pressure to obtain a crude product, which was then purified by reverse-phase column chromatography (column: Waters Xbridge C18 150 × 50 mm × 10 μm; mobile phase: [10 mmol / L aqueous ammonium bicarbonate-acetonitrile]; gradient: acetonitrile % = 56% - 76% over 10 minutes) to obtain compound B29-M. LCMS (ESI) m / z: 789.5 [M+H] + . 1H NMR (400MHz, DMSO-d6) δ = 11.48 (s, 1H), 7.70-7.42 (m, 1H), 7.11-6.76 (m, 1H), 6.30- 6.07(m,1H),5.70-5.54(m,5H),4.53-4.16(m,2H),3.99-3.74(m,3H),3.72-3.50(m ,3H),2.92-2.73(m,2H),2.12-1.99(m,1H),1.96-1.58(m,3H),1.25-1.06(m,30H).

[0251] Example 10: Synthesis of B30-M

[0252] Step A: Compound 9-1 (5 g, 10.02 mmol) was dissolved in tetrahydrofuran (40 mL), trifluoroacetic acid (10 mL) and water (10 mL), and the reaction solution was stirred at 25 ° C for 2 hours. The reaction solution was diluted with 40 mL of water, and ethyl acetate (40 mL × 2) was added and extracted twice. The organic phases were combined and washed with a mixture of saturated brine / saturated sodium bicarbonate aqueous solution (50 mL / 10 mL × 4), dried over anhydrous sodium sulfate, filtered and concentrated to obtain a crude product. The crude product was purified by silica gel column chromatography (ethyl acetate / petroleum ether = 0 / 1-3 / 5) to obtain compound 9-2. LCMS (ESI) m / z: 385.1 [M+H] + .

[0253] Step B: Dissolve compound 9-2 (3.52 g, 9.15 mmol) in acetonitrile (40 mL) and add 2-iodoacylbenzoic acid (5.40 g, 18.31 mmol, 95% purity). Stir the reaction mixture at 50°C for 12 hours. Cool the reaction mixture to room temperature, filter, and concentrate the filtrate in vacuo to obtain compound 9-3. LCMS (ESI) m / z: 383.2 [M+H] + .

[0254] Step C: Sodium hydroxide (1.10 g, 27.45 mmol, 60% purity) was added to tetrahydrofuran (30 mL). The reaction mixture was cooled to -70°C and protected with a nitrogen atmosphere. Compound tetrakis[[pivaloyloxy)methyl]methylene diphosphonate (14.47 g, 22.88 mmol) was added to the reaction mixture, and the reaction mixture was stirred at -70°C for 0.5 hours. Compound 9-3 (3.5 g, 9.15 mmol) was dissolved in tetrahydrofuran (30 mL) at -70°C and added dropwise to the above reaction mixture. The reaction mixture was stirred at 30°C for 12 hours. The reaction mixture was slowly poured into saturated aqueous ammonium chloride solution (200 mL) and extracted with ethyl acetate (300 mL). The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo to obtain the crude product. The crude product was purified by silica gel column chromatography (ethyl acetate / petroleum ether = 0 / 1 to 1 / 2) to obtain compound 9-4. LCMS (ESI) m / z: 689.2 [M+H] + .

[0255] Step D: Compound 9-4 (3 g, 4.36 mmol) was dissolved in formic acid (15 mL) and water (15 mL), and the reaction solution was stirred at 45 ° C for 12 hours. The reaction solution was cooled to room temperature, 40 mL of water was added, and extracted with ethyl acetate (40 mL × 2). The organic phases were combined and washed with a mixture of saturated brine / saturated sodium bicarbonate aqueous solution (50 mL / 10 mL × 5), dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo to obtain a crude product. The crude product was purified by silica gel column chromatography (ethyl acetate / petroleum ether = 0 / 1 to 17 / 20) to obtain compound 9-5. LCMS (ESI) m / z: 575.2 [M+H] + .

[0256] Step 5: Dissolve compound 9-5 (1.2 g, 2.09 mmol) in dichloromethane (10 mL), add compound 1-9 (944.33 mg, 3.13 mmol) and 4,5-dicyanoimidazole (123.34 mg, 1.04 mmol). Stir the reaction mixture at 25°C for 3 hours. Concentrate the mixture under vacuum to obtain a crude product. The crude product is purified by reverse-phase column chromatography (column: Waters Xbrid ge C18 150 × 50 mm × 10 μm; mobile phase: [aqueous ammonium bicarbonate solution (10 mmol / L)-acetonitrile]; gradient: 48% to 68% acetonitrile over 10 minutes) to obtain B30-M.

[0257] 1H NMR (400MHz, DMSO-d6) δ = 11.48 (s, 1H), 7.78-7.55 (m, 1H), 7.01-6.64 (m, 1H), 6.22-6.04 (m, 1H), 5.91 ( s,1H),5.71-5.53(m,5H),4.58-4.15(m,4H),3.95-3.70(m,2H),2.97-2.52(m,4H),1.26-1.10(m,32H).

[0258] 31 P NMR (162MHz, DMSO-d6) δ = 153.98-144.66 (m, 1P), 17.24-16.44 (m, 1P).

[0259] Example 11: Synthesis of B33-M

[0260] Step A: Compound 10-1 (1.9 g, 6.68 mmol) was dissolved in N,N-dimethylformamide (30 mL), and imidazole (2.73 g, 40.10 mmol), 4-dimethylaminopyridine (244.97 mg, 2.01 mmol), and tert-butyldimethylsilyl chloride (4.03 g, 26.74 mmol) were added. The mixture was stirred at 65°C for 16 hours. The reaction solution was diluted with ethyl acetate (100 mL) and washed with saturated brine (100 mL × 2). The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain a crude product. The crude product was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 1 / 1 to 1 / 2) to obtain compound 10-2. LCMS (ESI) m / z: 513 [M+H] + .

[0261] Step B: Compound 10-2 (2.5 g, 4.88 mmol) was dissolved in tetrahydrofuran (24 mL), and trifluoroacetic acid aqueous solution (24 mL, V / V = 1 / 1) was added. The mixture was stirred at 25°C for 2 hours. The reaction solution was diluted with ethyl acetate (120 mL), washed with saturated sodium bicarbonate aqueous solution (120 mL) and water (120 mL). The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain a crude product. The crude product was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 2 / 1 to 1 / 1) to obtain compound 10-3. LCMS (ESI) m / z: 399 [M+H] + .

[0262] Step C: Dissolve compound 10-3 (1.8 g, 4.52 mmol) in acetonitrile (30 mL), add 2-iodobenzoic acid (2.53 g, 9.03 mmol), and stir the mixture at 50°C for 16 hours. The reaction mixture was filtered, and the filtrate was concentrated to obtain crude compound 10-4. LCMS (ESI) m / z: 397 [M+H] + .

[0263] Step D: Under nitrogen, sodium hydroxide (411.55 mg, 10.29 mmol, 60% purity) and tetrakis[[pivaloyloxy]methyl]methylene diphosphonate (5.42 g, 8.57 mmol) were added to tetrahydrofuran (15 mL) at -78°C. The mixture was stirred at -78°C for 0.5 hour. A solution of compound 10-4 (1.7 g, 4.29 mmol) in tetrahydrofuran (15 mL) was then added, and the mixture was stirred at 25°C for 1 hour. The reaction mixture was quenched by adding saturated aqueous ammonium chloride (30 mL) and extracted with ethyl acetate (30 mL). The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain the crude product. The crude product was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 2 / 1 to 1 / 1) to obtain compound 10-5.

[0264] Step E: Compound 10-5 (1.3 g, 1.85 mmol) was added to formic acid (13 mL) and water (13 mL). The mixture was stirred at 65°C for 12 hours. The reaction solution was diluted with ethyl acetate (120 mL) and washed with saturated aqueous sodium bicarbonate (120 mL) and water (120 mL). The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain the crude product. The crude product was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 2 / 1 to 1 / 1) to obtain compound 10-6.

[0265] Step F: Under nitrogen, compound 10-6 (520 mg, 883.54 μmol) was dissolved in dichloromethane (10 mL). 4,5-Dicyanoimidazole (520 mg, 883.54 μmol) and compound 1-9 (399.46 mg, 1.33 mmol) were added. The mixture was stirred at 25°C for 16 hours. The reaction mixture was concentrated under reduced pressure to obtain the crude product. The crude product was purified by HPLC (column: Waters Xbridge C18 150 × 50 mm × 10 μm; mobile phase: [10 mmol / L aqueous ammonium bicarbonate-acetonitrile]; acetonitrile gradient: 54% to 84% over 10 minutes) to obtain compound B33-M.

[0266] 1 H NMR (400 MHz, DMSO-d 6)δ=11.45(s,1H),7.62-7.58(m,1H),6.99-6.75(m,1H),6.22-6.10(m,1H),6.04(s,1H),5.66-5.59(m,5H),4.58-4.51(m,1H),4.17-4.01 (m,1H),3.84-3.68(m,3H),3.65-3.51(m,3H),2.84–2.78(m,2H),2.39-2.16(m,1H),1.94–1.80(m,2H),1.77-1.64(m,1H),1.17(s,30H).

[0267] Example 12: Synthesis of B34-M

[0268] Step A: Dissolve compound 11-1 (2.5 g, 9.83 mmol) in N,N-dimethylformamide (70 mL), add imidazole (4.02 g, 59.00 mmol), 4-dimethylaminopyridine (360.39 mg, 2.95 mmol), and tert-butyldimethylsilyl chloride (5.93 g, 39.33 mmol). Stir the mixture at 65°C for 16 hours. Dilute the reaction solution with ethyl acetate (200 mL) and wash with saturated brine (200 mL x 2). Dry the organic phase over anhydrous sodium sulfate, filter, and concentrate under reduced pressure to obtain crude compound 11-2.

[0269] Step B: Dissolve compound 11-2 (4.7 g, 9.74 mmol) in tetrahydrofuran (50 mL), add trifluoroacetic acid aqueous solution (50 mL, V / V = 1 / 1), and stir the mixture at 25°C for 2 hours. The reaction solution is diluted with ethyl acetate (200 mL) and washed with saturated sodium bicarbonate aqueous solution (200 mL) and water (200 mL). The organic phase is dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain the crude product. The crude product is purified by silica gel column chromatography (petroleum ether / ethyl acetate = 2 / 1 to 1 / 1) to obtain compound 11-3.

[0270] Step C: Dissolve compound 11-3 (1.8 g, 4.88 mmol) in acetonitrile (40 mL), add 2-iodoacylbenzoic acid (3.42 g, 12.21 mmol), and stir the mixture at 55° C. for 16 hours. The reaction mixture is filtered and the filtrate is concentrated to obtain crude compound 11-4.

[0271] Step D: Under nitrogen, sodium hydroxide (458.41 mg, 11.46 mmol) and tetrakis[[pivaloyloxy]methyl]methylene diphosphonate (6.04 g, 9.55 mmol) were added to tetrahydrofuran (20 mL) at -78°C. After stirring at -78°C for 0.5 hour, a solution of compound 11-4 (1.75 g, 4.78 mmol) in tetrahydrofuran (20 mL) was added, and the mixture was stirred at 25°C for 4 hours. The reaction solution was quenched by adding saturated aqueous ammonium chloride (100 mL) and extracted with ethyl acetate (100 mL). The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain the crude product. The crude product was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 2 / 1 to 1 / 1) to obtain compound 11-5.

[0272] Step E: Compound 11-5 (2.2 g, 3.27 mmol) was added to formic acid (20 mL) and water (20 mL). The mixture was stirred at 65°C for 12 hours. The reaction solution was diluted with ethyl acetate (120 mL) and washed with saturated aqueous sodium bicarbonate (120 mL) and water (120 mL). The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain the crude product. The crude product was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 2 / 1 to 1 / 1) to obtain compound 11-6.

[0273] Step F: Under nitrogen, compound 11-6 (600 mg, 1.07 mmol) was dissolved in dichloromethane (12 mL). 4,5-Dicyanoimidazole (63.44 mg, 537.14 μmol) and compound 1-9 (485.70 mg, 1.61 mmol) were added. The mixture was stirred at 25°C for 16 hours. The reaction mixture was concentrated under reduced pressure to obtain a crude product. The crude product was purified by HPLC (neutral conditions; column: Waters Xbridge C18 150 x 50 mm x 10 μm; mobile phase: [water (ammonium bicarbonate - 10 mmol / L) - acetonitrile]; acetonitrile gradient: 53% to 83% over 10 minutes) to obtain compound B34-M.

[0274] 1 H NMR (400MHz, DMSO-d6) δ = 11.34 (s, 1H), 7.67-7.62 (m, 1H), 7.02-6.77 (m, 1H), 6.18-6.07 (m, 1H), 5.92 (d, J = 9.2Hz, 1H), 5.64-5.59 (m,5H),4.53-4.38(m,2H),3.79-3.53(m,4H),2.83(t,J=8.4Hz,1H),2.78(t,J=8.4Hz,1H),1.31–1.12(m,31H),0.92–0.56(m,3H).

[0275] Example 13: Synthesis of B36-M

[0276] Step A: Under nitrogen, potassium tert-butoxide (61.90 mL, 1 M) was added to compound 12-1 (22.11 g, 61.90 mmol) in tetrahydrofuran (60 mL) at 25°C. After addition, the mixture was stirred at 25°C for 0.5 h. A solution of methyltriphenylphosphonium bromide (15 g, 30.95 mmol) in tetrahydrofuran (40 mL) was then added at 25°C. The mixture was stirred at 35°C for 15.5 h. 5% citric acid (100 mL) was added to the reaction solution to quench the reaction and extract with ethyl acetate (200 mL x 2). The organic phase was washed with saturated sodium chloride (100 mL x 2), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain the crude product. The crude product was purified on a silica gel column (petroleum ether / ethyl acetate = 1 / 0 to 1 / 1) to obtain compound 12-2.

[0277] Step B: Under nitrogen, compound 12-2 (12 g, 24.86 mmol) and triethylamine trihydrofluoride (12.02 g, 74.58 mmol) were mixed in tetrahydrofuran (100 mL). The mixture was reacted at 35°C for 3 hours. The reaction solution was concentrated under reduced pressure. The crude product was purified on a silica gel column (petroleum ether / ethyl acetate = 1 / 0 to 0 / 1, ethanol / ethyl acetate = 0 / 1 to 1 / 4) to obtain compound 12-3.

[0278] Step C: Under nitrogen, compound 12-3 (3.9 g, 16.24 mmol), 4-dimethylaminopyridine (595.05 mg, 4.87 mmol), imidazole (6.63 g, 97.41 mmol), and tert-butyldimethylsilyl chloride (9.79 g, 64.94 mmol) were mixed in N,N-dimethylformamide (40 mL). The mixture was reacted at 60°C for 5 hours. The reaction solution was diluted with water (50 mL) and extracted with ethyl acetate (100 mL x 2). The organic phases were combined, washed with saturated sodium bicarbonate (100 mL x 2) and saturated sodium chloride (100 mL x 2), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain compound 12-4.

[0279] Step D: Under nitrogen, compound 12-4 (6.61 g, 14.10 mmol) and trifluoroacetic acid (10.15 g, 88.99 mmol) were mixed in a solution of tetrahydrofuran (66 mL) and water (13 mL). The mixture was reacted at 15°C for 6 hours. The reaction solution was diluted with water (50 mL) and extracted with ethyl acetate (100 mL x 2). The organic phases were combined, washed with saturated sodium bicarbonate (100 mL x 2), then with saturated sodium chloride (100 mL x 2), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain the crude product. The crude product was purified on a silica gel column (petroleum ether / ethyl acetate = 1 / 0 to 1 / 1) to obtain compound 12-5.

[0280] Step E: Under nitrogen protection, compound 12-5 (3.3 g, 9.31 mmol) and 2-iodoacylbenzoic acid (5.21 g, 18.62 mmol) were mixed in acetonitrile (40 mL), and the mixture was reacted at 50° C. for 16 hours. The reaction solution was filtered, and the filtrate was concentrated under reduced pressure to obtain compound 12-6.

[0281] Step F: Under nitrogen, tetrakis[(pivaloyloxy)methyl]methylenediphosphonate (7.36 g, 11.63 mmol) was added to a solution of sodium hydrogen phosphate (558.31 mg, 13.96 mmol, 60% purity) in tetrahydrofuran (30 mL) at -78°C. After addition, the mixture was reacted at -78°C for 0.5 hour. Then, a solution of compound 12-6 (1.64 g, 4.65 mmol) in tetrahydrofuran (10 mL) was added at -78°C. The mixture was reacted at 25°C for 2.5 hours. The reaction mixture was quenched by the addition of saturated ammonium chloride (50 mL) at 10-25°C, diluted with water (50 mL), and extracted with ethyl acetate (100 mL x 2). The organic phases were combined, washed with saturated sodium chloride (50 mL x 2), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain the crude product. The crude product was purified by silica gel column (petroleum ether / ethyl acetate = 1 / 0 to 1 / 1) to obtain compound 12-7.

[0282] Step G: Under nitrogen, compound 12-7 (2 g, 3.04 mmol) was mixed with formic acid (20 mL) and water (20 mL). The mixture was stirred at 40°C for 4 hours. The reaction solution was diluted with water (100 mL) and extracted with ethyl acetate (100 mL x 2). The organic phases were combined, washed with saturated sodium bicarbonate (50 mL x 2) and brine (50 mL x 2), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain the crude product. The crude product was purified on a silica gel column (petroleum ether / ethyl acetate = 1 / 0 to 0 / 1) to obtain compound 12-8.

[0283] Step H: Under nitrogen, compound 12-8 (1 g, 1.84 mmol), bis(diisopropylamino)(2-cyanoethoxy)phosphine (830.34 mg, 2.75 mmol), and 4,5-dicyanoimidazole (108.45 mg, 918.29 mmol) were mixed in dichloromethane (20 mL). The mixture was reacted at 25°C for 2 hours. The reaction solution was concentrated under reduced pressure to obtain a crude product. The crude product was purified by reverse phase preparative chromatography (neutral conditions, column: Waters xbridge 150*25 mm*10 μm; mobile phase: [water(ammonium bicarbonate)-acetonitrile]; gradient: acetonitrile % = 62% - 82% over 8 minutes) to obtain compound B36-M.

[0284] 1 H NMR(400MHz,DMSO-d6)δ=11.42(br s,1H),7.56(d,J=8.1Hz,1H),7.04-6.74(m,1H),6.48(dd,J=1.5,8.9Hz,1H),6.23-6.05(m,1H),5.73-5.53(m,6H), 5.46-5.41(m,1H),4.98-4.81(m,1H),4.47-4.30(m,1H),3.92-3.54(m,4H),2.88-2.76(m,2H),1.19-1.14(m,30H).

[0285] Example 14: Synthesis of dR-M

[0286] Step A: Compound 13-1 (250.00 g, 785.48 mmol), compound 13-2 (119.8 g, 942.58 mmol), and trifluoromethanesulfonic acid (1.18 g, 7.85 mmol) were mixed in a bottle and stirred at 125°C under nitrogen for 3 hours. After the reaction, ethyl acetate (2.5 L) was added, followed by water (2.5 L), 5% aqueous sodium bicarbonate solution (2.5 L x 2), and saturated brine (2.5 L). The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain a crude product. The crude product was recrystallized from ethanol (1 L) and n-heptane (1.75 L) at 70°C, filtered, and the filter cake was collected and dried under vacuum to obtain compound 13-3.

[0287] Step B: To a solution of compound 13-3 (100.00 g, 259.52 mmol) in methanol (700 mL) was added triethylamine (26.26 g, 259.52 mmol). The mixture was stirred at 65°C under nitrogen for 10 hours. After the reaction was complete, a 2 M solution of hydrogen chloride in methanol (130 mL) was added to the reaction mixture, and the mixture was concentrated under reduced pressure to obtain compound 13-4.

[0288] Step C: To a solution of compound 13-4 (67.27 g, 259.51 mmol) in dichloromethane (670 mL) at 0°C, imidazole (88.33 g, 1.3 mol) and 1,3-dichloro-1,1,3,3-tetraisopropyldisiloxane (90.04 g, 285.47 mmol) were added dropwise. After the addition, the reaction mixture was naturally warmed to 25°C and stirred for 16 hours. After the reaction, the organic phase was washed with water (1 L), 10% aqueous citric acid solution (1 L), and saturated brine (1 L), dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain the crude product. The crude compound was recrystallized from n-heptane (350 mL) and tert-methyl ether (350 mL) at 80°C, filtered, and the filter cake was collected and dried under vacuum to obtain compound 13-5.

[0289] Step D: Dissolve 13-5 (2 g, 3.99 mmol) and thiocarbonyldiimidazole (1.07 g, 5.98 mmol) in dichloromethane (20 mL). Add 4-dimethylaminopyridine (97.4 mg, 0.797 mmol). Stir the mixture at 25°C for 16 hours. After completion of the reaction, concentrate the reaction mixture under reduced pressure to obtain crude 13-6.

[0290] Step E: Dissolve the crude product of 13-6 (2.44 g), azobisisobutyronitrile (163.71 mg, 996.97 μmol), and tri-n-butyltin hydride (7.55 g, 25.94 mmol) in anhydrous toluene (30 mL) and replace the atmosphere with nitrogen three times. The reaction mixture was stirred at 100°C for 6 hours. After completion, the reaction mixture was cooled to 25°C and quenched with water (100 mL). The mixture was extracted with ethyl acetate (100 mL x 2). The organic phase was washed with saturated sodium chloride solution (100 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain the crude product. The crude product was purified on a silica gel column (eluent: petroleum ether / ethyl acetate = 100 / 1 to 1 / 1) to obtain 13-7.

[0291] Step F: Dissolve 13-7 (1.4 g, 2.88 mmol) in anhydrous tetrahydrofuran (20 mL), add triethylamine trihydrofluoride (1.02 g, 6.34 mmol, 1.03 mL), and stir at 25°C for 16 hours. The reaction mixture is concentrated under reduced pressure to obtain a crude product, which is then purified on a silica gel column (eluent: dichloromethane / methanol = 100 / 1 to 10 / 1) to obtain 13-8.

[0292] Step G: Dissolve 13-8 (0.53 g, 2.18 mmol), 4,4-dimethoxytrityl chloride (886.02 mg, 2.61 mmol), and triethylenediamine (366.66 mg, 3.27 mmol) in dichloromethane (10 mL). The atmosphere was replaced with nitrogen three times, and the mixed solution was stirred at 25°C under nitrogen for 4 hours. Methanol (2 mL) was added to quench the mixture, and the mixture was concentrated under reduced pressure to obtain a crude product. The crude product was purified on a silica gel column (eluent: petroleum ether / ethyl acetate = 100 / 1 to 1 / 1) to provide 13-9.

[0293] Step H: Dissolve 13-9 (0.6 g, 1.1 mmol), 1-9 (497.2 mg, 1.65 mmol), and 4,5-dicyanoimidazole (64.94 mg, 549.87 μmol) in dichloromethane (2 mL). The mixture was purged with nitrogen three times, and stirred at 25°C under nitrogen for 3 hours. The reaction mixture was quenched by adding 2 mL of saturated sodium bicarbonate solution, diluted with 20 mL of water, and extracted with dichloromethane (30 mL × 2). The combined organic phases were washed with saturated sodium chloride (30 mL × 2), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain the crude product. The crude product was purified on a silica gel column (eluent: petroleum ether / ethyl acetate = 100 / 1 to 1 / 1) to obtain compound dR-M.

[0294] LCMS (ESI) m / z: 746.5 [M+H] + .

[0295] 1 H NMR (400MHz, DMSO-d6): δ = 8.92-8.91 (d, J = 4.0Hz, 1H), 7.31-7.27 (m, 2H), 7.26-7.13 (m, 7H), 6.89-6 .73(m,4H),6.48-6.35(m,1H),4.80-4.55(m,1H),4.18-4.08(m,1H),3.82(s,3H),3.76-3.68(m,7H), 3.64-3.59(m,1H),3.56-3.42(m,2H),3.21-3.07(m,2H),2.81–2.72(m,2H),2.66 –2.63(m,1H),2.61-2.52(m,1H),1.14-1.09(m,9H),1.03-0.95(d,J=6.8Hz,3H).

[0296] Biological testing section

[0297] Experimental Example 1: Evaluation of siRNA Inhibitory Activity against LPA mRNA Using the psiCHECK-2 Dual Fluorescence Reporter Gene Vector

[0298] 1. Experimental Materials:

[0299] 1.1. Test compounds

[0300] Dissolve in PBS to prepare stock solution and store at -20℃.

[0301] 1.2. psiCHECK-2 reporter construct

[0302] The human LPA gene (Genbank number: NM_005577.4) was cloned into psiCHECK TM -2 vector.

[0303] Cell culture

[0304] Huh7 cells were cultured in DMEM (Gibco-11995-065, 500 mL) supplemented with FBS (ExCellBio-FSP500, 57 mL), penicillin-streptomycin solution (Hyclone-SV30010, 5.7 mL), and GlutaMAX TM (Gibco-35050-061, 5.7 mL) and non-essential amino acid solution (Gibco-11140050, 5.7 mL).

[0305] 1.4. Main Reagents

[0306] Lipofectamine RNAiMAX (Invitrogen-13778-150); Dulbecco's Modified Eagle Medium (Gibco-11995-065); Fetal Bovine Serum (ExCellBio-FSP500); FuGENE-HD (Promega-E2311); Opti-MEM Medium (Gibco-31985070); MEM NEAA (100X) (Gibco-11140050); Penicillin-Streptomycin Solution (PS) (Hyclone-SV30010); Luciferase Assay System (Promega-E2980).

[0307] 1.5. Main equipment

[0308] Multi-mode microplate reader (Perkin 2105)

[0309] 2. Experimental Procedure

[0310] 2.1 Transfection of LPA_psiCHECK(TM)-2 plasmid

[0311] On day one, the LPA_PSICHECK(TM)-2 plasmid transfection method is briefly described as follows: Huh7 cells were washed with DPBS, digested with 0.05% trypsin, and gently pipetted in DMEM supplemented with 10% FBS until single cells were counted. Plasmid DNA was then transferred into Huh7 cells using Fugene HD. Transfected cells were seeded at a density of 10,000 cells per well in a 96-well plate with 100 μL of culture medium per well. Cells were cultured overnight in a 5% CO2, 37°C incubator.

[0312] 2.2 Compound transfection and drug addition

[0313] On the next day, the test compound was serially diluted into 3 concentrations using PBS.

[0314] Prepare RNAiMAX transfection reagent by dispensing the desired volume in a 15 mL centrifuge tube at a ratio of 3:47 RNAiMAX:Opti-MEM. Mix thoroughly. Add the compound to the RNAiMAX / Opti-MEM mixture (1:1) and incubate for 15 minutes. Dilute the compound-containing RNAiMAX / Opti-MEM mixture 10-fold with fresh culture medium and mix thoroughly.

[0315] The culture medium in each well was aspirated, and then 100 μL of new culture medium containing the compound was added. After addition, the wells were placed in a 5% CO 2 , 37° C. incubator for culture.

[0316] 2.3 Dual luciferase assay system

[0317] Experimental steps reference The instructions for the dual-luciferase assay system are as follows: remove the cells from the incubator, discard the supernatant, add 75 μL of fresh culture medium and 75 μL of detection reagent, shake in the dark for 10 minutes, and after the cells are fully lysed, transfer 100 μL of the sample to an opaque white plate to detect the luminescent signal of fireflies; add 50 μL of Stop& Add the detection reagent and shake in the dark for 10 minutes before measuring the Renilla luciferase signal. Calculate the ratio of the reporter gene signal to the internal reference reporter gene signal in each well.

[0318] 2.4 Analysis

[0319] Measurement ratio = (Renilla luminescence - background Renilla luminescence) / (Firefly luminescence - background Firefly luminescence)

[0320] Inhibition % = (1-sample ratio / RNAiMAX control ratio) × 100%

[0321] 3. Experimental results are shown in Table 4

[0322] Table 4 Inhibitory activity of psiCHECK-2 dual-fluorescein reporter gene vector against LPA mRNA

[0323] 4. Experimental Conclusion

[0324] The double-stranded siRNA conjugate of the present invention can effectively inhibit human LPA mRNA.

[0325] Experimental Example 2: Evaluation of the in vivo efficacy of compounds using the LPA-HDI mouse model

[0326] 1. Experimental Principle

[0327] The knockdown effect of the target gene LPA by the double-stranded siRNA conjugate in mice was evaluated by rapid injection of physiological saline containing the target gene recombinant plasmid pcDNA-LPA through high-pressure tail vein.

[0328] 2. Experimental Materials

[0329] pcDNA-LPA plasmid, BALB / c mice, PBS (phosphate buffered saline), and the conjugate of the present invention.

[0330] 3. Experimental Methods

[0331] Six-week-old BALB / c female mice were ordered and acclimatized to quarantine for three days after arrival at the animal house.

[0332] On day 0, mice were randomly divided into groups according to body weight data. After grouping, all mice were administered subcutaneous injection with a single dose of 10 mL / kg. Mice in group 1 were given PBS; mice in other groups were given the conjugate (dissolved in PBS).

[0333] On the 13th day after administration, all mice were injected with physiological saline containing pcDNA-LPA plasmid via the tail vein within 5 seconds. The injection volume (mL) = mouse body weight (g) × 8%. The mass of the injected plasmid for each mouse was 10 μg.

[0334] On day 14 after administration, mice in all groups were euthanized by CO2 inhalation, and two liver samples were collected from each mouse. The liver samples were treated with RNAlater overnight at 4°C, then the RNAlater was removed and stored at -80°C for detection of LPA gene expression levels.

[0335] 4. Experimental results are shown in Table 5

[0336] Table 5 In vivo efficacy test results of the conjugates of the present invention (4 mice per group)

[0337] Note: "LPA mRNA down-regulation percentage" refers to the down-regulation percentage of LPA-mRNA in the liver of mice in the drug-treated group relative to the PBS blank group.

[0338] 5. Experimental Conclusion

[0339] The double-stranded siRNA conjugates of the present invention can effectively inhibit LPA mRNA in the LPA-HDI mouse model.

[0340] Experimental Example 3: Stability of double-stranded siRNA conjugates

[0341] 1. Experimental Methods

[0342] The Tm value of DNA / RNA was determined by recording the change in UV absorption at 260 nm in phosphate buffer solution as the temperature increased.

[0343] 2. Experimental Parameters

[0344] The instrument parameters used in the experiment are shown in Table 6.

[0345] Table 6 Melting temperature (Tm) test instrument parameters

[0346] 3. Experimental Results

[0347] The melting temperature (Tm) was calculated from the first derivative of the smoothed heating curves, and the reported values ​​are the average of at least two independent measurements. The results are shown in Table 7.

[0348] Table 7 Melting temperature (Tm) of the conjugates of the present invention

[0349] 4. Experimental Conclusion

[0350] The double-stranded siRNA conjugate of the present invention has a high melting temperature and good stability.

[0351] Experimental Example 4: Off-target evaluation of double-stranded siRNA conjugates

[0352] 1 Experimental Materials

[0353] 1.1 Double-stranded siRNA conjugates

[0354] 1.2 PHH cells were purchased from Red Liver Disease Research (Shanghai) Co., Ltd.

[0355] 1.3 Main reagents: InvitroGRO CP Medium (Cat. No.: BIOIVT-S03316), Fetal Bovine Serum (Cat. No.: ExCellBio-FSP500), Penicilin-Streptomycin (Cat. No.: Hyclone-SV30010), TRLzol Reagent (Cat. No.: Ambion-15596018).

[0356] 2 Experimental methods

[0357] 2.1 Cell recovery and compound treatment

[0358] Remove the frozen PHH from liquid nitrogen and thaw in a 37°C water bath. Transfer the completely thawed PHH to 10% FBS InvitrogRO CP medium and mix well. Count the cells using a cell counter and adjust the cell density to 6×10 5 cells / mL.

[0359] Dilute siRNA to 10 times the final concentration with RNase-free PBS (for example, if the test concentration is 100nM, dilute to 1000nM), then add the diluted siRNA to a collagen-coated 12-well plate at a volume of 100μL / well, 1 concentration point, 3 replicates, and blank PBS as a negative control group. 5 PHH cells were added to a collagen-coated 12-well plate at a density of 10 cells / mL at a volume of 900 μL / well. Final concentrations of test compounds were 20 nM, 100 nM, and 500 nM. The cell plates were incubated in a 5% CO2, 37°C incubator for 48 hours. After 48 hours, the medium was removed and 600 μL of Trizol was added to each well to lyse the cells. The cells were then sent to Novogene (Beijing) for RNA sequencing.

[0360] 2.2 Library construction and quality control

[0361] NEB general library construction: Using fragmented mRNA as a template and random oligonucleotides as primers, the first cDNA chain is synthesized in the M-MuLV reverse transcriptase system, followed by RNaseH degradation of the RNA chain, and the second cDNA chain is synthesized using dNTPs as raw materials in the DNA polymerase I system. The purified double-stranded cDNA is end-repaired, A-tailed, and ligated to sequencing adapters. AMPure XP beads are used to screen cDNA of about 250-300bp, PCR amplification is performed, and the PCR product is purified again using AMPure XP beads to finally obtain the library. The kit used for library construction is Ultra TMRNA Library Prep Kit for Illumina

[0362] After library construction, a Qubit2.0 Fluorometer was used for preliminary quantification, and the library was diluted to 1.5 ng / μL. The insert size of the library was then detected using an Agilent 2100 bioanalyzer. Once the insert size was in line with expectations, qRT-PCR was used to accurately quantify the effective concentration of the library (the effective concentration of the library was greater than 2 nM) to ensure library quality.

[0363] 2.3 Sequencing

[0364] After passing the library inspection, different libraries are pooled according to effective concentration and target data volume requirements before Illumina sequencing. The basic principle of sequencing is sequencing by synthesis. Four fluorescently labeled dNTPs, DNA polymerase, and adapter primers are added to the sequencing flow cell for amplification. As each sequencing cluster extends the complementary chain, each added fluorescently labeled dNTP releases corresponding fluorescence. The sequencer captures the fluorescent signal and converts it into sequencing peaks through computer software, thereby obtaining the sequence information of the fragment to be tested.

[0365] 3 Sequencing data analysis

[0366] Obtain experimental results through software analysis

[0367] 4 Experimental results:

[0368] The threshold for differential gene analysis was DESeq2 padj≤0.05|log2FoldChange|≥1.0, and the statistical results are shown in Table 8.

[0369] Table 8. Statistical results of differentially expressed genes

[0370] 5 Experimental Conclusions

[0371] The double-stranded siRNA conjugate of the present invention has low off-target risk.

[0372] Experimental Example 5: Cytotoxicity assay of double-stranded siRNA conjugates

[0373] 1 Experimental Materials

[0374] 1.1 Double-stranded siRNA conjugates

[0375] 1.2 PHH cells were purchased from Reed Liver Disease Research (Shanghai) Co., Ltd. HEK293 cells were purchased from ATCC (Cat. No. CRL-1573)

[0376] 1.3 Main reagents: InvitroGRO CP Medium (Cat. No.: BIOIVT-S03316), EMEM medium (Cat. No.: ATCC-30-2003), Fetal Bovine Serum (Cat. No.: ExCellBio-FSP500), Penicilin-Streptomycin (Cat. No.: Hyclone-SV30010), TRLzol Reagent (Cat. No.: Ambion-15596018), Luminescent Cell Viability Assay (Product No.: Promega-G7573)

[0377] 2 Experimental methods

[0378] 2.1 Compound treatment

[0379] PHH: Dilute the test siRNA to 10 times the final concentration with RNase-free PBS (if the test final concentration is 100 nM, dilute to 1000 nM). Add the diluted siRNA to a collagen-coated 96-well cell plate at a volume of 10 μL / well, for a total of 9 concentration points, in duplicate wells. RNase-free PBS is also added as a control.

[0380] HEK293 cells: Transfect siRNA using the reverse transfection method. First, add the transfection reagent and siRNA mixture to the culture plate and incubate them together before adding the cells to the culture medium. A 20 μM stock solution was serially diluted five-fold with RNase-free PBS to nine concentrations, resulting in final concentrations of 500 nM, 100 nM, 20 nM, 4 nM, 0.8 nM, 0.16 nM, 0.032 nM, 0.064 nM, and 0.00128 nM after addition to the cell culture system. A PBS control well was also set up. Prepare a mixture of RNAiMAX transfection reagent and Opti-MEM® (1.5:48.5) as needed and incubate at room temperature for 15 minutes. Add 10 μL of the diluted compound to 10 μL of RNAiMAX:Opti-MEM® medium, mix thoroughly, and incubate for 15 minutes. Add 10 μL of the Opti-MEM:RNAiMAX and compound or PBS mixture to the cell culture plate.

[0381] 2.2 Cell treatment

[0382] PHH cells: Take out the frozen cells and thaw them in a 37℃ water bath. Then transfer them to 10% FBS InvitroGRO CP medium and mix them evenly. Count them using a cell counter and adjust the cell density to 6×10 5 cells / mL. Add the cells after adjusting the density to the corresponding 96-well cell plates at a volume of 90 μL / well, with a final volume of 100 μL per well. Incubate the cell plates in a 5% CO2, 37°C incubator for 48 hours.

[0383] HEK293 cells: Trypsinize HEK293 cells to 80% confluency, centrifuge, resuspend, and count. Resuspend HEK293 cells in EMEM complete medium to a concentration of 75,000 cells / mL. Add 90 μL of the suspension to each well of a 96-well plate for a final volume of 100 μL.

[0384] 2.3 Luminescence signal detection

[0385] Remove the 96-well cell culture plate and allow it to equilibrate to room temperature for 5 minutes. Add 50 μL of CellTiter Glo reagent to each well of the 96-well plate and shake at 500 rpm for 5 minutes. Allow to equilibrate to room temperature for 5 minutes. Read the plate using the *USLum-US LUM 96 (cps) program on a microplate reader.

[0386] 2.4 Data Processing

[0387] 1) Cell viability: Viability% = (sample value - blank well mean) / (control well mean - blank well mean) × 100%

[0388] 2) Substitute the Viability% and the corresponding siRNA concentration into the GraphPad Prism 8.3 statistical software to fit the curve and obtain CC 50 The calculation formula used is: Y = Bottom + (Top-Bottom) / (1 + 10^((LogIC 50 -X)*HillSlope)), where IC 50 In this experiment, it is equivalent to CC 50 , absolutely CC 50 It refers to the siRNA concentration corresponding to 50% cell viability.

[0389] 3 Experimental results:

[0390] Table 9. Cytotoxicity of the compounds of the present invention on PHH cells and HEK293 cells

[0391] 4 Experimental conclusions:

[0392] The double-stranded siRNA conjugate of the present invention has a low risk of cytotoxicity.

[0393] Experimental Example 6: hPBMC immunogenicity test of the compound of the present invention

[0394] 1 Experimental Materials

[0395] 1.1 Double-stranded siRNA conjugates

[0396] 1.2 hPBMCs, purchased from Shanghai Saili Biotechnology Co., Ltd.

[0397] 1.3 Main reagents: Penicillin-Streptomycin (Cat. No.: 15140122), RNase-free water (Cat. No.: AM9938), IL-6 ELISA kit (Cat. No.: 555220), TNF-α ELISA kit (Cat. No.: 555212), LPS (Cat. No.: L2630), etc.

[0398] 2 Experimental methods

[0399] 2.1 Compound Preparation: Prepare the compound to the stock concentration in enzyme-free water (compound weight = powder weight * purity). The concentration after preparation is 900 μg / mL. Then, dilute the solution three-fold with enzyme-free water. Dilute each concentration 10-fold with 1640 medium. The final content of enzyme-free water is 5.0%.

[0400] 2.2 Experimental steps:

[0401] 2.2.1 Resuscitate PBMC cells one day in advance and incubate them with 1640 complete medium overnight.

[0402] 2.2.2 On the day of the experiment, PBMC cells were centrifuged at 300 × g for 8 minutes and the cell density was adjusted to 1.6 × 10 6 mL, added to a 96-well plate, 125 μL per well, and incubated in a 37°C, 5% CO2 incubator for 15 min.

[0403] 2.2.3 Add the diluted test compound to a 96-well plate according to the plate map, in duplicate, with 125 μL added to each well; add equal amounts of 1640 complete culture medium with the same enzyme-free water content to the positive control group and blank group.

[0404] 2.2.4 Gently mix the plate and incubate at 37°C for 18-20 hours. Collect the supernatant by centrifugation and assay the secretion of TNF-α and IL-6 by ELISA.

[0405] 2.2.5 Analyze data using GraphPad Prism 8 software.

[0406] 3 Experimental results

[0407] Table 10. IL-6 and TNF-α levels in the supernatant of donor 1 hPBMC after treatment with different doses of test compounds

[0408] Table 11. IL-6 and TNF-α levels in the supernatant of 2hPBMC from donors after treatment with different doses of test compounds

[0409] 4 Experimental Conclusions

[0410] The compounds of the present invention have a low risk of immunogenicity.

[0411] Experimental Example 7: Evaluation of the in vivo activity of the compounds of the present invention using cynomolgus monkeys

[0412] 1 Experimental Materials

[0413] 1.1 Double-stranded siRNA conjugates of the present invention

[0414] 1.2 Cynomolgus macaque

[0415] 1.3 Experimental Reagents

[0416] PBS as solvent, Lp(a) ELISA Kit, Catalog Number: Mercodia-10-1106-01

[0417] 2 Experimental methods

[0418] 2.1 Cynomolgus monkeys (weighing 3.5-5 kg) were recruited, with two animals per group. Each animal was subcutaneously injected with 2 mg / kg of the test article (double-stranded siRNA conjugate dissolved in PBS). After fasting overnight, blood was collected on days -15 (pre-dose), -7 (pre-dose), 0 (pre-dose), and 7, 14, 21, and 28 after dosing. The collected blood samples were allowed to clot at room temperature for at least 30 minutes and then centrifuged at 3500 rpm for 10 minutes at 4°C. The collected serum (approximately 0.5 mL) was used for ELISA assay.

[0419] 3 Experimental results

[0420] Table 12. Effects of the compounds of the present invention on reducing Lp(a) in cynomolgus monkeys

[0421] 4 Experimental Conclusions

[0422] The compound of the present invention has a significant effect of reducing the Lp(a) level in cynomolgus monkeys and has a long-lasting effect.

Claims

1. A double-stranded siRNA or a pharmaceutically acceptable salt thereof for inhibiting LPA expression, comprising a sense strand and an antisense strand capable of forming a double-stranded region, wherein: The sense strand comprises any one of the sequences shown in SEQ ID NOs: 1-18, and the antisense strand comprises any one of the sequences shown in SEQ ID NOs: 19-26, and each nucleotide on the sense strand and the antisense strand is optionally a modified nucleotide. 2 . The double-stranded siRNA or a pharmaceutically acceptable salt thereof according to claim 1 , wherein at least one nucleotide on the sense strand and the antisense strand is a modified nucleotide.

3. The double-stranded siRNA or a pharmaceutically acceptable salt thereof according to claim 1, wherein 0, 1, 2, 3 or 4 nucleotides on the sense strand and the antisense strand are unmodified nucleotides, and the rest are modified nucleotides. The double-stranded siRNA or a pharmaceutically acceptable salt thereof according to claim 1 , wherein the modified nucleotide is selected from the group consisting of 2′-OMe modified nucleotides, 2′-F modified nucleotides and 2′-deoxy nucleotides.

5. The double-stranded siRNA or a pharmaceutically acceptable salt thereof according to claim 1, wherein the 5' end of the antisense strand optionally comprises a nucleotide selected from mDVPU, B29, B30, B33, B34 and B36, wherein: The mDVPU is The B29 is The B30 is The B33 is The B34 is The B36 is 6. The double-stranded siRNA or a pharmaceutically acceptable salt thereof according to claim 1, wherein the 5' end of the sense strand optionally comprises a nucleotide selected from invAb, wherein invAb is 7 . The double-stranded siRNA or a pharmaceutically acceptable salt thereof according to claim 1 , comprising any double-stranded chain shown by S1-S37, S39-S44 or S46-S64.

8. A double-stranded siRNA conjugate or a pharmaceutically acceptable salt thereof for inhibiting LPA expression, wherein the double-stranded siRNA conjugate is formed by conjugating a ligand to the double-stranded siRNA or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 7.

9. The double-stranded siRNA conjugate according to claim 8 or a pharmaceutically acceptable salt thereof, wherein the ligand is one or more GalNAc derivatives attached using a divalent, trivalent or tetravalent branched linker.

10. The double-stranded siRNA conjugate according to claim 9 or a pharmaceutically acceptable salt thereof, wherein the ligand is selected from D01, D08, D10, D11, D18, D21 and L96 with the structural formula shown below: The double-stranded siRNA conjugate or a pharmaceutically acceptable salt thereof according to claim 8 , wherein the ligand is conjugated to the 3′ end of the sense strand. 12 . The double-stranded siRNA conjugate or a pharmaceutically acceptable salt thereof according to claim 8 , wherein the ligand is conjugated to the double-stranded siRNA via a phosphodiester bond or a phosphorothioate bond.

13. The double-stranded siRNA conjugate or a pharmaceutically acceptable salt thereof according to claim 8, wherein the double-stranded siRNA conjugate is selected from any one of the conjugates shown in Z1-Z65.

14. The double-stranded siRNA conjugate or a pharmaceutically acceptable salt thereof according to claim 8, wherein the double-stranded siRNA conjugate is as shown in Formula I: in, X is O; SS is the sense strand, and AS is the antisense strand; the sense strand and the antisense strand are selected from the combination shown in any one of (1) to (13): (1) the sense strand is as shown in SEQ ID NO:49, and the antisense strand is as shown in SEQ ID NO:80; (2) the sense strand is as shown in SEQ ID NO:49, and the antisense strand is as shown in SEQ ID NO:81; (3) the sense strand is as shown in SEQ ID NO:43, and the antisense strand is as shown in SEQ ID NO:66; (4) the sense strand is as shown in SEQ ID NO:51, and the antisense strand is as shown in SEQ ID NO:86; (5) the sense strand is as shown in SEQ ID NO:53, and the antisense strand is as shown in SEQ ID NO:88; (6) the sense strand is as shown in SEQ ID NO:43, and the antisense strand is as shown in SEQ ID NO:92; (7) the sense strand is as shown in SEQ ID NO:43, and the antisense strand is as shown in SEQ ID NO:94; (8) the sense strand is as shown in SEQ ID NO:43, and the antisense strand is as shown in SEQ ID NO:95; (9) the sense strand is as shown in SEQ ID NO:43, and the antisense strand is as shown in SEQ ID NO:96; (10) the sense strand is as shown in SEQ ID NO:49, and the antisense strand is as shown in SEQ ID NO:94; (11) the sense strand is as shown in SEQ ID NO:49, and the antisense strand is as shown in SEQ ID NO:95; (12) the sense strand is as shown in SEQ ID NO:49, and the antisense strand is as shown in SEQ ID NO:96; (13) The sense strand is shown in SEQ ID NO:55, and the antisense strand is shown in SEQ ID NO:

95.

15. Use of the double-stranded siRNA or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 7, or the double-stranded siRNA conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 8 to 14 in the preparation of a drug for treating cardiovascular diseases.

16. The use according to claim 15, wherein: The cardiovascular disease is selected from Buerger's disease, peripheral artery disease, coronary artery disease, metabolic syndrome, aortic stenosis, aortic regurgitation, aortic dissection, retinal artery occlusion, mesenteric ischemia, superior mesenteric artery occlusion, renal artery stenosis, stable / unstable angina, acute coronary syndrome, heterozygous or homozygous familial hypercholesterolemia, hyperlipoprotein betalipoproteinemia, cerebrovascular atherosclerosis and venous thrombosis.