Uses of double-stranded sirna and conjugate thereof

Abstract

A double-stranded siRNA and a use thereof. Specifically disclosed are uses of a double-stranded siRNA containing non-natural nucleotides and a conjugate of the double-stranded siRNA, targeting an inhibin βE gene, and being capable of being applied to treatment of metabolic disorder-related diseases.

Description

Applications of double-stranded siRNA and its conjugates

[0001] This application claims priority to Chinese Patent Application No. 2024101154015 filed on January 26, 2024, Chinese Patent Application No. 2024104636471 filed on April 16, 2024, Chinese Patent Application No. 202410518440X filed on April 26, 2024, Chinese Patent Application No. 2024112876053 filed on September 13, 2024, and Chinese Patent Application No. 2025100845612 filed on January 17, 2025. The entire contents of the aforementioned Chinese patent applications are incorporated herein by reference. Technical Field

[0002] The present invention relates to double-stranded siRNA and applications thereof, and in particular to applications of a class of double-stranded siRNA containing non-natural nucleotides and conjugates thereof. Background Art

[0003] Abdominal obesity, also known as abdominal obesity, is a condition characterized by the accumulation of fat tissue around abdominal organs, the mesentery, and the aorta. It primarily manifests as an increase in waist circumference. According to the Chinese Industrial Health Standards, abdominal obesity is diagnosed when a waist circumference exceeds 90 cm for men or 85 cm for women.

[0004] Abdominal obesity is a common condition in China. A survey conducted between 2012 and 2015 showed that the prevalence of abdominal obesity in China was 29.1%, including 28.6% for men and 29.6% for women. Based on this proportion, the number of adults with abdominal obesity nationwide is approximately 277.8 million.

[0005] Abdominal obesity is a manifestation of metabolic syndrome and is closely associated with type 2 diabetes and coronary heart disease. A health survey of over 310,000 Koreans showed that general obesity alone does not increase the risk of major adverse cardiovascular events. However, those with only abdominal obesity or a combination of both obesity and abdominal obesity showed a significantly increased risk of major adverse cardiovascular events.

[0006] However, no drugs have been approved for abdominal obesity, indicating that there is an unmet clinical need in this area.

[0007] The inhibin E (INHBE) gene encodes the inhibin βE subunit, an activin / inhibin component of the TGF-β superfamily. Studies of people with loss-of-function mutations in this gene have shown a close correlation between INHBE expression and abdominal fat and fat distribution. Individuals with INHBE loss-of-function mutations have a lower waist-to-hip ratio adjusted for body mass index (WHRadjBMI), a metric commonly used to measure abdominal obesity. Furthermore, individuals with INHBE loss-of-function mutations have a lower incidence of coronary heart disease and type 2 diabetes. Importantly, individuals whose INHBE mutations result in an approximately 50% decrease in INHBE expression have lower triglyceride levels, higher high-density lipoprotein cholesterol levels, and an overall healthier metabolic phenotype.

[0008] Since its discovery in 1998, the RNA interference (RNAi, or siRNA) mechanism has garnered significant attention. This mechanism works by downregulating target mRNA levels to control the function of the corresponding target, thereby achieving the desired pharmacological effect. In recent years, several RNAi drugs have been approved for marketing, demonstrating their feasibility for human drug application. Drugs that achieve pharmacological effects through this mechanism are known as RNAi drugs. Summary of the Invention

[0009] The present invention provides a double-stranded siRNA 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, the sense strand comprising at least 16 consecutive nucleotides from the sequence shown in any one of SEQ ID NOs: 1-80 and SEQ ID NOs: 161-188; the antisense strand comprising at least 16 consecutive nucleotides from the sequence shown in any one of SEQ ID NOs: 81-160 and SEQ ID NOs: 189-230;

[0010] The nucleotides included in the sense strand and the nucleotides included in the antisense strand are modified or unmodified nucleotides.

[0011] In some embodiments of the present invention, the above-mentioned double-stranded siRNA or a pharmaceutically acceptable salt thereof, wherein the sense strand comprises nucleotides 1-17, 1-18, 1-19, 1-20, 1-21, 2-17, 2-18, 2-19, 2-20, 2-21, 3-18, 3-19, 3-20, 3-21, 4-19, 4-20, 4-21 or 5-21 of the 5' end of the sequence shown in any one of SEQ ID NOs: 1-80, SEQ ID NOs: 161-188; preferably, the sense strand comprises nucleotides 1-17, 1-18, 1-19, 1-20, 1-21, 2-17, 2-18, 2-19, 2-20, 2-21, 3-18, 3-19, 3-20, 3-21, 4-19, 4-20, 4-21 or 5-21 of the 5' end of the sequence shown in any one of SEQ ID NOs: 1-80, SEQ ID NOs: 161-188; Nucleotides at positions 1-17, 1-18, 1-19, 2-17, 2-18, 2-19, 2-20, 3-18, 3-19, 3-20, 4-19, 4-20, 4-21 or 5-21 of the 5' end of any one of the sequences shown in NOs: 161-188.

[0012] In some embodiments of the present invention, the above-mentioned double-stranded siRNA or a pharmaceutically acceptable salt thereof, wherein the antisense strand comprises nucleotides 1-17, 1-18, 1-19, 1-20, 1-21, 1-22, 1-23, 2-18, 2-19, 2-20, 2-21, 2-22, 2-23, 3-19, 3-20, 3-21, 3-22, 3-23, 4-20, 4-21, 4-22, 4-23, 5-21, 5-22, 5-23, 6-22, 6-23 or 7-23 of the 5' end of any one of the sequences shown in SEQ ID NOs: 81-160, SEQ ID NOs: 189-230; preferably, the antisense strand comprises nucleotides 1-17, 1-18, 1-19, 1-20, 1-21, 1-22, 1-23, 2-18, 2-19, 2-20, 2-21, 2-22, 2-23, 3-19, 3-20, 3-21, 3-22, 3-23, 4-20, 4-21, 4-22, 4-23, 5-21, 5-22, 5-23, 6-22, 6-23 or 7-23 of the 5' end of any one of the sequences shown in SEQ ID NOs: Nucleotides at positions 1-19, 1-20, 1-21, 1-22, 1-23, 2-18, 2-19, 2-20, 2-21, 3-20 or 3-21 of the 5' end of any one of the sequences shown in NO: 189-230.

[0013] In some embodiments of the present invention, the double-stranded siRNA or a pharmaceutically acceptable salt thereof, wherein the sense strand, the antisense strand, or at least one nucleotide residue on the sense strand and the antisense strand is independently optionally replaced by dR, R or Z, wherein dR is The R is The Z is

[0014] In some embodiments of the present invention, in the above-mentioned double-stranded siRNA or a pharmaceutically acceptable salt thereof, 1-4 nucleotide residues at positions 2, 5-7, 12, and 14-18 of the 5' end of the antisense strand are independently optionally replaced by dR, R, or Z.

[0015] In some embodiments of the present invention, in the double-stranded siRNA or a pharmaceutically acceptable salt thereof, 1-4 nucleotides at positions 2, 5-7, 12, and 14-18 of the 5' end of the antisense strand are independently selected as 2' deoxynucleotides.

[0016] In some embodiments of the present invention, in the double-stranded siRNA or a pharmaceutically acceptable salt thereof, the first nucleoside at the 5' end of the antisense strand can be replaced by a nucleoside analog of vinyl phosphate.

[0017] In some embodiments of the present invention, in the double-stranded siRNA or a pharmaceutically acceptable salt thereof, the vinyl phosphate nucleoside analog is selected from the following structural fragments:

[0018] 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 the sense strand comprises nucleotides at positions 1-17, 1-18, 1-19, 1-20, 1-21, 2-17, 2-18, 2-19, 2-20, 2-21, 3-18, 3-19, 3-20, 3-21, 4-19, 4-20, 4-21 or 5-21 of the 5' end of any one of the sequences shown in SEQ ID NOs: 1-80, and 1-4 nucleotide residues on the sense strand are independently and optionally replaced by dR, R or Z, wherein dR is The R is The Z is The nucleotides on the sense strand are optionally modified.

[0019] 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 the antisense strand comprises nucleotides at positions 1-17, 1-18, 1-19, 1-20, 1-21, 1-22, 1-23, 2-18, 2-19, 2-20, 2-21, 2-22, 2-23, 3-19, 3-20, 3-21, 3-22, 3-23, 4-20, 4-21, 4-22, 4-23, 5-21, 5-22, 5-23, 6-22, 6-23 or 7-23 of the 5' end of any one of the sequences shown in SEQ ID NOs: 81-160, and 1-4 nucleotide residues on the antisense strand are independently optionally replaced by dR, R or Z, wherein dR is The R is The Z is The nucleotides on the antisense strand are optionally modified.

[0020] 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 the sense strand comprises nucleotides at positions 1-17, 1-18, 1-19, 1-20, 1-21, 2-17, 2-18, 2-19, 2-20, 2-21, 3-18, 3-19, 3-20, 3-21, 4-19, 4-20, 4-21 or 5-21 of the 5' end of any one of the sequences shown in SEQ ID NOs: 161-188, and 1-4 nucleotide residues on the sense strand are independently and optionally replaced by dR, R or Z, wherein dR is The R is The Z is The nucleotides on the sense strand are optionally modified.

[0021] 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 the antisense strand comprises nucleotides at positions 1-17, 1-18, 1-19, 1-20, 1-21, 1-22, 1-23, 2-18, 2-19, 2-20, 2-21, 2-22, 2-23, 3-19, 3-20, 3-21, 3-22, 3-23, 4-20, 4-21, 4-22, 4-23, 5-21, 5-22, 5-23, 6-22, 6-23 or 7-23 of the 5' end of any one of the sequences shown in SEQ ID NOs: 189-230, and 1-4 nucleotide residues on the antisense strand are independently optionally replaced by dR, R or Z, wherein dR is The R is The Z is The nucleotides on the antisense strand are optionally modified.

[0022] 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 the sense strand comprises nucleotides at positions 1-17, 1-18, 1-19, 1-20, 1-21, 2-17, 2-18, 2-19, 2-20, 2-21, 3-18, 3-19, 3-20, 3-21, 4-19, 4-20, 4-21 or 5-21 of the 5' end of any one of the sequences shown in SEQ ID NOs: 161-188, and 1-4 nucleotide residues on the sense strand are independently and optionally replaced by dR, R or Z, wherein dR is The R is The Z is

[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 the antisense strand comprises nucleotides at positions 1-17, 1-18, 1-19, 1-20, 1-21, 1-22, 1-23, 2-18, 2-19, 2-20, 2-21, 2-22, 2-23, 3-19, 3-20, 3-21, 3-22, 3-23, 4-20, 4-21, 4-22, 4-23, 5-21, 5-22, 5-23, 6-22, 6-23 or 7-23 of the 5' end of any one of the sequences shown in SEQ ID NOs: 189-230, and 1-4 nucleotide residues on the antisense strand are independently optionally replaced by dR, R or Z, wherein dR is The R is The Z is

[0024] In some embodiments of the present invention, the sense strand comprises nucleotides 1-17, 1-18, 1-19, 2-17, 2-18, 2-19, 2-20, 3-18, 3-19, 3-20, 4-19, 4-20, 4-21 or 5-21 from the 5' end of any one of SEQ ID NOs: 1-80, and 1-4 nucleotide residues on the sense strand are independently optionally replaced by dR, R or Z, wherein dR is The R is The Z is The nucleotides on the sense strand contain at least one modified nucleotide.

[0025] In some embodiments of the present invention, the antisense strand comprises nucleotides 1-19, 1-20, 1-21, 1-22, 1-23, 2-18, 2-19, 2-20, 2-21, 3-20 or 3-21 from the 5' end of any one of the sequences shown in SEQ ID NOs: 81-160, and 1-4 nucleotide residues on the antisense strand are independently optionally replaced by dR, R or Z, wherein dR is The R is The Z is The nucleotides on the antisense strand comprise at least one modified nucleotide.

[0026] In some embodiments of the present invention, the above-mentioned positive chain comprises nucleotides at positions 1-17, 1-18, 1-19, 2-17, 2-18, 2-19, 2-20, 3-18, 3-19, 3-20, 4-19, 4-20, 4-21 or 5-21 from the 5' end of any one of the sequences shown in SEQ ID NOs: 161-188, and 1-4 nucleotide residues on the positive chain are independently optionally replaced by dR, R or Z.

[0027] In some embodiments of the present invention, the antisense strand comprises nucleotides at positions 1-19, 1-20, 1-21, 1-22, 1-23, 2-18, 2-19, 2-20, 2-21, 3-20 or 3-21 of the 5' end of any one of the sequences shown in SEQ ID NOs: 189-230, and 1-4 nucleotide residues on the antisense strand are independently optionally replaced by dR, R or Z.

[0028] In some embodiments of the present invention, the positive strand comprises nucleotides 1-17, 1-18, 1-19, 2-17, 2-18, 2-19, 2-20, 3-18, 3-19, 3-20, 4-19, 4-20, 4-21 or 5-21 of the 5' end of any one of SEQ ID NOs: 1-80.

[0029] In some embodiments of the present invention, the positive strand comprises nucleotides 1-17, 1-18, 1-19, 2-17, 2-18, 2-19, 2-20, 3-18, 3-19, 3-20, 4-19, 4-20, 4-21 or 5-21 of the 5' end of any one of SEQ ID NOs: 161-188.

[0030] In some embodiments of the present invention, the antisense strand comprises nucleotides 1-19, 1-20, 1-21, 1-22, 1-23, 2-18, 2-19, 2-20, 2-21, 3-20 or 3-21 of the 5' end of any one of SEQ ID NOs: 81-160.

[0031] In some embodiments of the present invention, the antisense strand comprises nucleotides 1-19, 1-20, 1-21, 1-22, 1-23, 2-18, 2-19, 2-20, 2-21, 3-20 or 3-21 of the 5' end of any one of SEQ ID NOs: 189-230.

[0032] In some embodiments of the present invention, 1-4 nucleotide residues at positions 2, 5-7, 12, and 14-18 of the 5' end of the antisense strand are independently replaced by dR, R, or Z, respectively.

[0033] In some embodiments of the present invention, 1-4 nucleotide residues at positions 2, 5-7, and 12 at the 5' end of the antisense strand are independently and optionally replaced by dR, R, or Z, respectively.

[0034] In some embodiments of the present invention, 1 to 4 nucleotides at positions 2, 5 to 7, 12, and 14 to 18 at the 5' end of the antisense strand are independently selected to be 2' deoxynucleotides.

[0035] In some embodiments of the present invention, 1-4 nucleotides at positions 2, 5-7, 12, and 14-18 of the 5' end of the antisense strand are independently selected to be adenine deoxyribonucleotide, guanine deoxyribonucleotide, cytosine deoxyribonucleotide, or thymine deoxyribonucleotide.

[0036] In some embodiments of the present invention, 1-4 nucleotides at positions 2, 5-7 and 12 of the 5' end of the antisense strand are independently selected to be adenine deoxyribonucleotide, guanine deoxyribonucleotide, cytosine deoxyribonucleotide or thymine deoxyribonucleotide.

[0037] In some aspects of the present invention, the 5' end or 3' end of the sense strand or antisense strand may optionally include an overhang, respectively and independently.

[0038] In some embodiments of the present invention, the overhang comprises 1-5 nucleotides.

[0039] In some embodiments of the present invention, the overhang comprises 2 nucleotides.

[0040] In some embodiments of the present invention, the 3' end of the antisense strand optionally comprises an overhang, and the overhang comprises 1-5 nucleotides.

[0041] In some embodiments of the present invention, the 3' end of the antisense strand optionally comprises an overhang, and the overhang comprises 2 nucleotides.

[0042] In some embodiments of the present invention, the first nucleoside at the 5' end of the antisense strand can be replaced by a nucleoside analog of vinyl phosphate.

[0043] In some embodiments of the present invention, the nucleoside analog of the vinyl phosphate is selected from the following structural fragments:

[0044] In some embodiments of the present invention, the nucleoside analog of vinyl phosphate is selected from B19 and B33, wherein B19 is The B33 is

[0045] In some embodiments of the present invention, the nucleoside analog of vinyl phosphate is selected from B33, wherein B33 is

[0046] In some embodiments of the present invention, the sense strand is selected from any one of the sequences shown in SEQ ID NOs: 161-188, and the antisense strand is selected from any one of the sequences shown in SEQ ID NOs: 189-230.

[0047] In some embodiments of the present invention, the double-stranded siRNA is selected from S1-S81.

[0048] The present invention provides a double-stranded siRNA or a pharmaceutically acceptable salt thereof, which comprises a sense chain and an antisense chain capable of forming a double-stranded region, wherein the nucleotide sequence of the sense chain and the nucleotide sequence of the antisense chain are selected from any one of the nucleotide sequence combinations shown in S83-S129.

[0049] In some embodiments of the present invention, the double-stranded siRNA is selected from Table 1 and Table 2:

[0050] Table 1 Unmodified double-stranded siRNA

[0051] Table 2 Modified double-stranded siRNA

[0052] The present invention provides a conjugate or a pharmaceutically acceptable salt thereof, comprising any of the above-mentioned double-stranded siRNAs and a pharmaceutically acceptable conjugation group.

[0053] In some embodiments of the present invention, the conjugate or a pharmaceutically acceptable salt thereof may contain 1, 2, 3, 4 or 5 pharmaceutically acceptable conjugating groups.

[0054] In some embodiments of the present invention, the conjugate or a pharmaceutically acceptable salt thereof comprises one pharmaceutically acceptable conjugating group.

[0055] In some embodiments of the present invention, the pharmaceutically acceptable conjugate group is selected from antibodies, polypeptides and ligands.

[0056] In some embodiments of the present invention, the pharmaceutically acceptable conjugated groups can be independently linked to any nucleotide of the sense strand and / or antisense strand of the double-stranded siRNA.

[0057] In some embodiments of the present invention, the pharmaceutically acceptable conjugate group can be linked to the 3' end and / or 5' end of the sense strand and / or antisense strand of the double-stranded siRNA.

[0058] In some embodiments of the present invention, the pharmaceutically acceptable conjugated groups independently contain 1, 2, 3, 4 or 5 GalNAc groups, and the GalNAc groups can bind to ASGPR.

[0059] In some embodiments of the present invention, the conjugated group is selected from the following structures:

[0060] In the present invention, the connection mode of the double-stranded siRNA and the conjugated group in the conjugate is as follows:

[0061] Wherein, SS represents the sense strand, AS represents the antisense strand; and X is selected from O and S.

[0062] In some embodiments of the present invention, the conjugate is selected from C1-C47.

[0063] In some embodiments of the present invention, the conjugate is selected from Table 3:

[0064] Table 3 Conjugates

[0065] In some embodiments of the present invention, the conjugate is represented by formula (I):

[0066] Wherein, X is O or S; 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 (9):

[0067] (1) the sense strand is as shown in SEQ ID NO: 161, and the antisense strand is as shown in SEQ ID NO: 189;

[0068] (2) the sense strand is shown in SEQ ID NO: 161, and the antisense strand is shown in SEQ ID NO: 210;

[0069] (3) the sense strand is as shown in SEQ ID NO: 186, and the antisense strand is as shown in SEQ ID NO: 226;

[0070] (4) the sense strand is as shown in SEQ ID NO: 188, and the antisense strand is as shown in SEQ ID NO: 225;

[0071] (5) the sense strand is as shown in SEQ ID NO: 164, and the antisense strand is as shown in SEQ ID NO: 192;

[0072] (6) the sense strand is as shown in SEQ ID NO: 171, and the antisense strand is as shown in SEQ ID NO: 199;

[0073] (7) the sense strand is shown in SEQ ID NO: 161, and the antisense strand is shown in SEQ ID NO: 202;

[0074] (8) the sense strand is shown in SEQ ID NO: 185, and the antisense strand is shown in SEQ ID NO: 220;

[0075] (9) The positive strand is shown in SEQ ID NO: 188, and the antisense strand is shown in SEQ ID NO: 228.

[0076] In some embodiments of the present invention, the conjugate is represented by formula (I):

[0077] Wherein, 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 (4):

[0078] (1) the sense strand is as shown in SEQ ID NO: 161, and the antisense strand is as shown in SEQ ID NO: 189;

[0079] (2) the sense strand is shown in SEQ ID NO: 161, and the antisense strand is shown in SEQ ID NO: 210;

[0080] (3) the sense strand is as shown in SEQ ID NO: 186, and the antisense strand is as shown in SEQ ID NO: 226;

[0081] (4) The positive strand is shown in SEQ ID NO: 188, and the antisense strand is shown in SEQ ID NO: 225.

[0082] 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 drugs for treating diseases related to INHBE gene expression.

[0083] In some embodiments of the present invention, the above application is characterized in that the disease related to INHBE gene expression is a metabolic disease caused by abnormal body fat distribution.

[0084] In some embodiments of the present invention, the above-mentioned applications, wherein the metabolic diseases caused by abnormal body fat distribution include but are not limited to type 2 diabetes, obesity, abdominal obesity, coronary heart disease, non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, hyperlipidemia, dyslipidemia, atherosclerosis, etc.

[0085] The present invention also provides the following test method

[0086] Test method 1: PHH cell free uptake experiment

[0087] 1. Experimental principle:

[0088] The test samples were incubated with primary human hepatocytes to evaluate the degree of downregulation of INHBE mRNA by the test samples.

[0089] 2. Experimental Materials:

[0090] Primary human hepatocytes (PHH), 96Kit, FastKing RT Kit (With gDNase), TaqMan Gene Expression Assay.

[0091] 3. Experimental methods:

[0092] The conjugate of the present invention was diluted to 10 times the concentration to be tested with PBS solution. 10 μL of siRNA was transferred to a 96-well plate. PHH cells were thawed and transplanted into a 96-well plate to a final cell density of 5.4×10 5 The conjugate of the present invention was tested at 10 concentration points, with the highest concentration being 500 nM, and 4-fold dilution.

[0093] The cells were incubated at 37°C in 5% CO2 for 48 h, and the cell status was examined under a microscope.

[0094] After incubation, the cells were lysed and the lysate was obtained. All RNA was extracted using 96Kit and reverse transcribed using FastKing RT Kit (with gDNase) to obtain cDNA. INHBE cDNA was detected by qPCR.

[0095] 4. Experimental conclusion: The conjugate of the present invention can significantly downregulate the INHBE mRNA level in PHH cells.

[0096] Test Method 2: In vivo PD test in non-human primates, cynomolgus monkeys

[0097] 1. Experimental principle:

[0098] Using the non-human primate cynomolgus macaque model, which is highly homologous to the human INHBE gene, the degree of downregulation of cynomolgus macaque plasma INHBE protein by the test sample was observed.

[0099] 2. Experimental materials: cynomolgus monkeys, 1x PBS (phosphate buffered saline), and the conjugate of the present invention.

[0100] 3. Experimental methods:

[0101] Order 10 cynomolgus macaques weighing 2-3 kg and acclimate them to quarantine for one week upon arrival at the animal house.

[0102] On the 6th day before administration, the cynomolgus macaques were randomly divided into groups according to body weight data, with 2 monkeys in each group. After grouping, the plasma INHBE protein concentration of all cynomolgus macaques was tested as the experimental data baseline for each group.

[0103] Dosing was performed on day 1, and blood was collected on days 1, 2, 4, 8, 15, 22, 29, 32, 35, 43, 57, and 71 after dosing to measure plasma INHBE protein concentration in each cynomolgus monkey. The plasma INHBE protein concentration after dosing was compared with the INHBE protein concentration on day 6 before dosing to determine the relative downregulation of INHBE protein in each group of animals.

[0104] 4. Experimental conclusion: The conjugate of the present invention can significantly downregulate the INHBE protein concentration in cynomolgus monkey plasma.

[0105] Test Method 3: In vitro RNA sequencing studies

[0106] 1. Experimental Introduction:

[0107] The transcriptome refers to the sum of all RNA transcribed by a specific tissue or cell at a specific time or state, primarily including mRNA and non-coding RNA. Transcriptome sequencing, based on the Illumina sequencing platform, studies all mRNA transcribed by a specific tissue or cell at a specific time. It is fundamental to the study of gene function and structure and plays a crucial role in understanding biological development and disease. With the advancement of gene sequencing technology and the reduction in sequencing costs, RNA-seq has become a leading method for transcriptome research due to its high throughput, high sensitivity, and wide range of applications. The RNA-seq technical process primarily consists of two parts: library construction and sequencing, and bioinformatics analysis.

[0108] 2. RNA extraction and detection:

[0109] RNA is extracted from tissues or cells using standard extraction methods, and then the RNA samples are strictly quality controlled, mainly through the Agilent 2100 bioanalyzer: accurately detecting RNA integrity.

[0110] 3. Library construction and quality control:

[0111] There are two main methods for obtaining mRNA: 1. Taking advantage of the structural characteristic of most eukaryotic mRNAs with polyA tails, mRNA with polyA tails can be enriched using Oligo(dT) magnetic beads. 2. mRNA can be obtained by removing ribosomal RNA from total RNA. The resulting mRNA is then randomly fragmented with divalent cations in NEB Fragmentation Buffer and constructed using either the NEB standard library construction method or the strand-specific library construction method.

[0112] 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 TM RNA Library Prep Kit for

[0113] Chain-specific library construction: The method for reverse transcription synthesis of the first chain of cDNA is the same as the NEB general library construction method. The difference is that when synthesizing the second chain, dTTP in dNTPs is replaced by dUTP. After that, the cDNA end repair, A tail addition, sequencing adapter connection and length screening are also performed. Then, the second chain of cDNA containing U is degraded with USER enzyme and PCR amplification is performed to obtain the library. Chain-specific libraries have many advantages, such as obtaining more effective information with the same amount of data; obtaining more accurate gene quantification, positioning and annotation information; and providing the expression level of antisense transcripts and a single exon in each isoform. The kit used for library construction is Ultra TM Directional RNA Library Prep Kit for

[0114] Note: Sequencing adapters consist of three parts: P5 / P7, index, and Rd1 / Rd2SP. P5 / P7 are the PCR amplification primers and the primer binding sites on the flow cell. The index provides information to distinguish different libraries. Rd1 / Rd2SP, or read1 / read2 sequence primers, is the sequencing primer binding region. Theoretically, sequencing proceeds backward from Rd1 / Rd2SP.

[0115] 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.

[0116] 4. Sequencing:

[0117] 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.

[0118] 5. Experimental conclusion: The conjugate of the present invention significantly down-regulated the INHBE gene in RNA sequencing studies.

[0119] Test method 4: Huh7 cell transfection experiment

[0120] 1. Experimental purpose: The purpose of this study is to use Huh7 cells to evaluate the effect of the conjugate described in this patent on inhibiting target genes, using the IC 50 The value was used as an indicator to evaluate the inhibitory activity of the compound on the target gene INHBE.

[0121] 2. Experimental Materials:

[0122] 2.1 Cell line: Huh7 cells, conjugate of the present invention

[0123] Huh7 cells were cultured in DMEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin.

[0124] 2.2 Reagents:

[0125] The main reagents used in this experiment include Lipofectamine TM iRNAiMAX transfection reagent, FastStart Universal Probe Mast, RNA extraction kit, GAPDH gene expression kit, INHBE gene expression kit, FastKing cDNA first-strand synthesis kit, 96-well plate.

[0126] 2.3 Consumables and instruments:

[0127] The main instruments used in this experiment include a fluorescence qPCR instrument (Applied Biosystems, model QuantStudio 6 Flex) and a cell counter (Vi-cellTM XR).

[0128] 3. Experimental steps and methods:

[0129] 3.1 Transfection

[0130] 1) On the first day, prepare a mixture of RNAiMAX transfection reagent and Opti-MEM as needed and incubate at room temperature for 15 minutes.

[0131] 2) For each well of cells, add a certain amount of diluted compound to an equal amount of RNAiMAX Optim-MEM medium, mix well, and incubate for 15 minutes;

[0132] 3) Take Huh7 cells, wash them with DPBS, add trypsin for digestion, and adjust the cells to an appropriate density;

[0133] 4) 20 μL of the mixture of Opti-MEM RNAiMax and the compound was added to the cell culture plate at the same time, and the cells were seeded into a 96-well plate at a density of 20,000 cells per well. The final culture medium per well was 120 μL.

[0134] 5) The cells were cultured in a 5% CO2, 37°C incubator for 24 h.

[0135] 3.2 RNA extraction and reverse transcription

[0136] The cells were collected, RNA was extracted according to the instructions of the extraction kit, and RNA was reverse transcribed into cDNA according to the instructions of the FastKing cDNA First Strand Synthesis Kit.

[0137] 3.3 QPCR detection

[0138] Target gene cDNA was detected by qPCR, using GAPDH as an internal reference gene. qPCR was performed in a 384-well plate. The qPCR reaction program was as follows: heating at 95°C for 10 minutes, followed by cycling at 95°C for 15 seconds, followed by 60°C for 1 minute, for a total of 40 cycles.

[0139] 3.4 Data Analysis: ΔΔCt Method (Ct Difference Comparison Method)

[0140] This method requires the introduction of the internal reference gene GAPDH. This gene is expressed in all cells, its product is essential for cell survival, and its expression level or genomic copy number is constant, making it less susceptible to environmental influences. After qRT-PCR, the CT value of the internal reference is recorded, referred to as Ct(GAPDH), and the CT value of the sample is referred to as Ct(sample).

[0141] ΔCt(sample)=Ct(sample)-Ct(GAPDH)

[0142] ΔCt(control)=Ct(control)-Ct(GAPDH)

[0143] ΔΔCt = ΔCt(sample) - ΔCt(control)

[0144] Relative gene expression = 2^-ΔΔCt

[0145] 4. Experimental conclusion: The conjugate of the present invention has a good knockout effect on INHBE mRNA in Huh7 cells.

[0146] Technical Effects

[0147] The double-stranded siRNA and conjugate of the present invention can significantly inhibit the expression of INHBE mRNA in vitro and in vivo, have low off-target risk and good stability, and can be used to treat diseases related to the INHBE gene and protein.

[0148] Definition and Description

[0149] 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.

[0150] Unless otherwise stated, the terms "comprises, includes and contains" or equivalents are open-ended expressions and mean that in addition to the listed elements, components or steps, other unspecified elements, components or steps may also be included.

[0151] Unless otherwise indicated, 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.

[0152] Double-stranded siRNA refers to a complex of ribonucleic acid molecules having a double-stranded structure comprising two antiparallel and substantially complementary nucleic acid strands having a "sense" and "antisense" orientation 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 strand are paired with bases on the other strand in a complementary manner. The purine base adenine (A) is always paired with the pyrimidine bases uracil (U) or thymine (T); the purine base guanine (C) is always paired with the pyrimidine base cytosine (G). Each base pair includes a purine and a pyrimidine. When adenine on one strand is always paired with uracil on the other strand, and guanine is always paired with cytosine, the two strands are considered to be complementary to each other, and the sequence of the strand can be inferred from the sequence of its complementary strand.

[0153] The term "single-stranded oligonucleotide" as used herein refers to a single-stranded oligonucleotide having a sequence that is at least partially complementary to a target mRNA, which is capable of hybridizing to the target mRNA through hydrogen bonding under mammalian physiological conditions (or a comparable in vitro environment). In some embodiments of the present invention, the single-stranded oligonucleotide is a single-stranded antisense oligonucleotide.

[0154] The term "double-stranded oligonucleotide" as used herein refers to a duplex structure comprising two antiparallel and substantially complementary nucleotide chains, one of which is a sense strand and the other is an antisense strand, wherein the antisense strand is a strand that is substantially complementary to the corresponding region of the target sequence (e.g., INHBE mRNA) and is capable of hybridizing to the target mRNA through hydrogen bonds under mammalian physiological conditions (or equivalent in vitro environments). The term "substantially complementary" 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 typically no more than 3, 2, or 1 mismatched base pairs. In a double-stranded nucleic acid molecule, the bases of one chain pair with the bases on the other chain in a complementary manner. The purine base adenine (A) always pairs with the pyrimidine base uracil (U); the purine base guanine (C) always pairs with the pyrimidine base cytosine (G). In some embodiments of the present invention, the double-stranded oligonucleotide is a double-stranded siRNA.

[0155] The "short interfering RNA (siRNA)" described herein is a type of RNA molecule with a double-stranded region of 17 to 25 base pairs in length, similar to miRNA, and operates 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. The short interfering RNA (siRNA) described herein includes double-stranded siRNA (including both sense and antisense strands) and single-stranded siRNA (antisense strand only).

[0156] In the present invention, unless otherwise specified, capital letters T, C, G, U, and A represent the base composition of nucleotides.

[0157] "Modifications" of the nucleotides described herein include, but are not limited to, methoxy modifications, fluoro modifications, (E)-vinyl phosphate modifications, phosphorothioate linkages, or replacement of the nucleotides with monomers of GNA (glycerol nucleic acid) nucleotides, wherein the GNAs include GNA-A, GNA-T, GNA-C, GNA-G, and GNA-U. The sequences described herein may include those listed in the "further modified sequences" in Table 2 below.

[0158] The fluorinated modified nucleotides of the present invention refer to nucleotides in which the hydroxyl group at the 2' position of the ribose group is replaced by fluorine, and the methoxy-modified nucleotides refer to nucleotides in which the 2'-hydroxyl group of the ribose group is replaced by a methoxy group.

[0159] The monomers of the GNA (glycerol nucleic acid) nucleotides of the present invention (GNA-A, GNA-T, GNA-C, GNA-G and GNA-U):

[0160] 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

[0161] Unless otherwise specified, the term "optionally modified nucleotides" as used herein means that the nucleotides may be unmodified or modified. The "unmodified nucleotides" are nucleotides composed of natural nucleobases, sugar rings, and phosphates. The "modified nucleotides" are nucleotides composed of modified nucleobases, modified sugar rings, and / or 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 phosphates and natural sugar rings; 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 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 modified nucleobases, modified sugar rings and modified phosphates.

[0162] Unless otherwise specified, the "natural sugar ring" of the present invention is selected from the five-membered sugar ring of 2'-OH.

[0163] 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).

[0164] 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-aminoguanine, 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 adenines and guanines, 5-halo, especially 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine, and 3-deazaguanine and 3-deazaadenine,

[0165] 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, heterocycloalkylaryl, 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. In some embodiments, the modification includes but is not limited to 2'-methoxyethoxy (2'-O-CH2CH2OCH3, also known as 2'-O-(2-methoxyethyl) or 2'-MOE).

[0166] Unless otherwise specified, the "modified phosphate" of the present invention includes but is not limited to: phosphorothioate modification, and the "phosphorothioate" includes (R)- and (S)-isomers and / or mixtures thereof.

[0167] Unless otherwise indicated, the term "phosphate" is used in its ordinary sense as understood by those skilled in the art and includes its protonated forms (e.g., ).

[0168] Unless otherwise indicated, the terms "phosphothioate" and "phosphothioate" refer to phosphothioates of the formula Its protonated form (e.g. ) and its tautomers (e.g. ) compounds.

[0169] In some embodiments of the present invention, the modified nucleotides include one or more dX (deoxynucleotide) nucleotide monomers. dX includes dA, dT, dC and dG. The structure of dA is The dT structure is The structure of dC is The dG structure is

[0170] Unless otherwise specified, the "overhang" of the present invention refers to at least one unpaired nucleotide protruding from the double-stranded region structure of a double-stranded compound. For example, the 3'-end of one chain extends beyond the 5'-end of the other chain, or the 5'-end of one chain extends beyond the 3'-end of the other chain. The overhang may comprise at least one nucleotide; or the overhang may comprise at least two nucleotides, at least three nucleotides, at least four nucleotides, at least five or more nucleotides. The nucleotides at the nucleotide overhang are optionally modified nucleotides. The overhang may be located on the sense strand, the antisense strand, or any combination thereof. In addition, the overhang may be present at the 5'-end, the 3'-end, or both ends of the antisense or sense strand of the double-stranded compound. In some embodiments of the present invention, the antisense strand has an overhang of 1 to 10 nucleotides (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides) at the 3'-end and / or the 5'-end. In some embodiments of the present invention, the sense strand has an overhang of 1 to 10 nucleotides (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides) at the 3'-end and / or the 5'-end. In some embodiments of the present invention, the antisense strand is at the 3'-end and the sense strand has an overhang of 1 to 10 nucleotides (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides) at the 3'-end. In some embodiments of the present invention, the antisense strand is at the 5'-end and the sense strand has an overhang of 1 to 10 nucleotides (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides) at the 5'-end.

[0171] In the present invention, the double-stranded siRNA conjugate (also referred to as "conjugate") is 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.

[0172] In the present invention, pharmaceutically acceptable conjugated groups include but are not limited to antibodies, polypeptides, ligands, and the like.

[0173] In the present invention, the number of pharmaceutically acceptable conjugated groups is 1, 2, 3, 4 or 5, and the pharmaceutically acceptable conjugated groups can be independently linked to any nucleotide of the sense strand and / or antisense strand of the double-stranded siRNA.

[0174] In the present invention, the pharmaceutically acceptable conjugated group can be linked to the 3' end and / or 5' end of the sense strand and / or antisense strand of the double-stranded siRNA.

[0175] In the present invention, the number of pharmaceutically acceptable conjugated groups is 1, 2, 3, 4 or 5, and the pharmaceutically acceptable conjugated groups can be independently linked to the 3' end and / or 5' end of the sense strand and / or antisense strand of the double-stranded siRNA.

[0176] In the context of the present invention, unless otherwise specified, "conjugation" 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.

[0177] As used herein, unless otherwise specified, "linker" refers to an organic moiety that connects two parts of a compound, e.g., covalently attaches two parts of a compound. The linker typically comprises a direct bond or an atom (e.g., oxygen or sulfur), an atom group (e.g., NRR, C(O), C(O)NH, SO, SO2, SO2NH), a substituted or unsubstituted alkyl, a substituted or unsubstituted alkenyl, a substituted or unsubstituted alkynyl, a substituted or unsubstituted aryl, a substituted or unsubstituted heteroaryl, a substituted or unsubstituted cycloalkyl, or a substituted or unsubstituted heterocycloalkyl, wherein one or more carbon atoms in the substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, or substituted or unsubstituted alkynyl group can be replaced by a substituted or unsubstituted aryl, a substituted or unsubstituted heteroaryl, a substituted or unsubstituted cycloalkyl, or a substituted or unsubstituted heterocycloalkyl group.

[0178] The cleavable linker is sufficiently stable outside the cell but is cleaved upon entry into the target cell to release the two moieties to which the linker co-immobilizes.

[0179] 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.

[0180] Unless otherwise indicated, the term "enantiomer" or "optical isomer" refers to stereoisomers that are mirror images of one another.

[0181] 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.

[0182] 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 or straight dashed key

[0183] 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%.

[0184] 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%.

[0185] 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 resolved by conventional methods known in the art, and then the pure enantiomer is recovered. In addition, the separation of enantiomers and diastereomers is typically accomplished by using chromatography, which employs a chiral stationary phase and is optionally combined with a chemical derivatization method (e.g., carbamate formation from an amine).

[0186] Unless otherwise specified, when a group has one or more linkable sites, any one or more sites of the group can be linked to other groups via chemical bonds. The chemical bonds linking the sites to other groups can be represented by wavy lines. For example, The wavy lines in the figure indicate that the double-stranded siRNA is connected to other nucleotides through the oxygen atoms at positions 3 and 5 in dR.

[0187] The compounds of the present invention may contain unnatural proportions of atomic isotopes at one or more of the atoms that constitute the compound. For example, the compounds may be labeled with radioactive isotopes, 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.

[0188] As used herein, the term "pharmaceutically acceptable salt" refers to those carboxylate salts, amino acid addition salts of the compounds of the present invention that are suitable for use in contact with patient tissues within the scope of sound medical judgment, do not produce undue toxicity, irritation, allergic response, etc., are commensurate with a reasonable benefit / risk ratio, and are effective for their intended use, including (where possible) zwitterionic forms of the compounds of the present invention.

[0189] 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.

[0190] 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.

[0191] 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.

[0192] The compounds of the present invention may contain unnatural proportions of atomic isotopes at one or more of the atoms that constitute the compound. For example, the compounds may be labeled with radioactive isotopes, 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.

[0193] When the linking group is listed without specifying its linking direction, its linking direction is arbitrary, for example, The connecting group L is -MW-, in which case -MW- can connect ring A and ring B in the same direction as the reading order from left to right to form You can also connect ring A and ring B in the opposite direction of reading from left to right to form Combinations of linkers, substituents, and / or variations thereof are permissible only if such combinations result in stable compounds.

[0194] 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.

[0195] The term "substituted" means that any one or more hydrogen atoms on a particular atom are replaced by a substituent, which may include deuterium and hydrogen variants, as long as the valence state of the particular atom is normal and the substituted compound is stable. When the substituent is oxygen (i.e., =O), it means that two hydrogen atoms are replaced. Oxygen substitution does not occur on aromatic groups. The term "optionally substituted" means that it may be substituted or unsubstituted, and unless otherwise specified, the type and number of substituents can be any on the basis of chemical achievable.

[0196] When any variable (e.g., R) occurs more than once in a compound's composition or structure, its definition at each occurrence is independent. Thus, for example, if a group is substituted with 0-2 Rs, the group may be optionally substituted with up to two Rs, with each occurrence of R being an independent choice. Furthermore, combinations of substituents and / or their variants are permissible only if such combinations result in stable compounds.

[0197] When the number of a linking group is 0, such as -(CRR)0-, it means that the linking group is a single bond.

[0198] When a substituent is vacant, it means that the substituent does not exist. For example, when X in AX is vacant, it means that the structure is actually A. When the substituent is listed without specifying which atom it is connected to the substituted group, the substituent can be bonded through any atom of the substituent. For example, a pyridyl substituent can be connected to the substituted group through any carbon atom on the pyridine ring.

[0199] 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.

[0200] 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: After scanning and collecting relevant data, the crystal structure is further analyzed using the direct method (Shelxs97) to confirm the absolute configuration.

[0201] 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.

[0202] The solvent used in the present invention is commercially available.

[0203] The present invention uses the following abbreviations: aq represents water; HATU represents O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate; EDC represents N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride; m-CPBA represents 3-chloroperoxybenzoic acid; eq represents equivalent; CDI represents carbonyldiimidazole; DCM represents dichloromethane; PE represents petroleum ether; DIAD represents diisopropyl azodicarboxylate; DMF represents N,N-dimethylformamide; DMSO represents dimethyl sulfoxide; EtOAc represents ethyl acetate; EtOH represents ethanol; MeOH represents methanol; CBz represents benzyloxycarbonyl, which is an amine protecting group; Boc represents methylbenzyloxycarbonyl, which is an amine protecting group. represents tert-butyloxycarbonyl, an amino protecting group; HOAc represents acetic acid; NaCNBH3 represents sodium cyanoborohydride; rt represents room temperature; O / N represents overnight; THF represents tetrahydrofuran; Boc2O represents di-tert-butyl dicarbonate; TFA represents trifluoroacetic acid; DIPEA represents diisopropylethylamine; SOCl2 represents thionyl chloride; CS2 represents carbon disulfide; TsOH represents p-toluenesulfonic acid; NFSI represents N-fluoro-N-(phenylsulfonyl)benzenesulfonamide; NCS represents 1-chloropyrrolidine-2,5-dione; n-Bu4NF represents tetrabutylammonium fluoride; iPrOH represents 2-propanol; mp represents melting point; LDA represents lithium diisopropylamide; the GalNAc group represents PBS stands for phosphate-buffered saline; ASGPR stands for anti-asialoglycoprotein receptor, a liver-specific transmembrane glycoprotein.

[0204] In some embodiments of the present invention, the "A", "U", "C" and "G" are replaced by lowercase letters "a", "u", "c" and "g" to indicate that the nucleotide is modified with 2'-O-methyl (methoxy modification); the "A", "U", "C" and "G" are followed by "f" to indicate that the nucleotide is modified with 2'-fluoro nucleotide (fluorinated modification); the lowercase letter "s" indicates that the nucleotides are connected by phosphorothioate groups; the "A", "U", "C" and "G" are preceded by "d" to indicate that the nucleotide adjacent to the right of the letter d is a deoxyribonucleotide. For example, the chemical structure of 5'-us, Ufs, U, g, dA-3' is as follows:

[0205] Compounds are named according to the conventional nomenclature in the art or using Software naming, commercially available compounds use supplier catalog names. DETAILED DESCRIPTION

[0206] 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.

[0207] Example 1: Synthesis of Z1 and Z2

[0208] 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, 8.95 mL) at 0°C. Stir at 35°C for 12 h. Quench the mixture by slowly adding saturated aqueous sodium bicarbonate (100 mL) at 0°C and extract with dichloromethane (100 mL x 2). The 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 by silica gel column chromatography (eluent: petroleum ether / ethyl acetate = 20:1) to obtain 1-3.

[0209] Step B: Dissolve 1-3 (6.7 g, 13.05 mmol) in methanolic ammonia (7 mol / L, 50 mL). Stir at 40°C for 12 h. Concentrate the reaction mixture under reduced pressure to obtain a crude product. Purify the crude product by silica gel column chromatography (eluent: ethyl acetate / methanol = 50 / 1 to 10 / 1) to obtain 1-4.

[0210] Step C: Dissolve 1-4 (2.3 g, 11.43 mmol) in anhydrous pyridine (25 mL). Add 1,3-dichloro-1,1,3,3-tetraisopropyldisiloxane (3.64 g, 11.55 mmol, 3.69 mL) at 0°C. Stir the mixture at 20°C for 12 h. Quench the mixture with water (30 mL) and extract with ethyl acetate (30 mL x 2). The organic phase is washed sequentially with hydrochloric acid (1 mol / L, 30 mL x 3) and saturated brine (30 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to afford the crude product. The crude product is purified by silica gel column chromatography (eluent: petroleum ether / ethyl acetate = 7 / 1 to 5 / 1) to afford 1-5.

[0211] 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, 1.82 mL), and iodomethane (6.4 g, 45.08 mmol, 2.81 mL) were stirred at 25°C for 12 h. Ethyl acetate (50 mL) was added and stirred at 20°C for 1 h. The mixture was filtered through a Buchner funnel with a pad of celite and the filtrate was collected. The filtrate was extracted with water (100 mL) and ethyl acetate (100 mL x 2). The organic phase was washed with saturated brine (200 mL), 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 (eluent: petroleum ether / ethyl acetate = 15 / 1 to 10 / 1 to 8 / 1) to obtain 1-6.

[0212] Step E: Dissolve 1-6 (2 g, 4.37 mmol) in anhydrous tetrahydrofuran (20 mL) and add triethylamine trihydrofluoride (1.55 g, 9.61 mmol, 1.57 mL). Stir at 20°C for 12 h. The reaction mixture is concentrated under reduced pressure to give a crude product. The crude product is purified by silica gel column chromatography (eluent: ethyl acetate / methanol = 100 / 1 to 50 / 1) to give 1-7.

[0213] 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 at 20°C for 12 h. Quench the mixture with water (20 mL) and extract with ethyl acetate (20 mL x 2). The 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 by silica gel column chromatography (eluent: petroleum ether / ethyl acetate = 3 / 1 to 1 / 1) to obtain Z1. 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).

[0214] Step G: In a 10 L dry reactor at room temperature, nitrogen was used as a protective layer. Dichloromethane (5.5 L), raw material Z1 (1.1 g, 2.13 mmol), bis(diisopropylamino)(2-cyanoethoxy)phosphine (0.7 g, 2.34 mmol), and 4,5-dicyanoimidazole (0.13 g, 1.06 mmol) were added separately and the temperature was controlled at 20-25°C for 3-4 hours. After completion of the reaction, 1 mL of saturated sodium bicarbonate was added to quench the reaction at 20-25°C, and the reaction was diluted with 10 mL of water. The mixture was allowed to stand, separated, and washed with 10 mL of water. The organic phase was dried over 1 g of anhydrous sodium sulfate, filtered, and the mother liquor was concentrated under reduced pressure to obtain the crude product. The concentrate was transferred to a round-bottom flask, 15 mL of toluene and 10 mL of n-heptane were added, stirred and dissolved, washed three times with 5 mL*3 N, N-dimethylformamide / water = 7 / 3, then washed three times with 5 mL*3 water, and then washed with 5 mL of saturated brine. The organic phase was dried over 11 g of anhydrous sodium sulfate, filtered, and the mother liquor was concentrated and distilled with acetonitrile to obtain Z2.1H NMR (400MHz, DMSO-d6) δ = 8.82 (s, 1H), 8.11 (d, J = 6.6Hz, 1H), 7.42-7.32 (m, 2H ),7.30-7.16(m,7H),6.90-6.76(m,4H),6.20-6.08(m,1H),4.76-4.51(m,1H) ,4.29(q,J=4.2Hz,1H),4.23-4.09(m,1H),3.83-3.75(m,1H),3.72(d,J=2.4H z,6H),3.61-3.45(m,3H),3.38(d,J=15.2Hz,3H),3.30-3.16(m,1H),3.07(br dd,J=5.6,10.4Hz,1H),2.77(t,J=5.9Hz,1H),2.63-2.55(m,1H),1.16-1.07(m,9H),0.96(d,J=6.6Hz,3H).

[0215] Z2 is an intermediate for preparing a sense chain, an antisense chain or a conjugate containing Z.

[0216] Example 2: B19-M

[0217] Step 1: Under nitrogen, n-butyllithium (2.5 mol / L, 94.57 mL) was added to a solution of compound 2-2 (78 g, 236.43 mmol) in tetrahydrofuran (1000 mL) at -78°C for 10 minutes. After the addition, the mixture was stirred at this temperature for 20 minutes. A solution of compound 2-1 (98.94 g, 236.43 mmol) in tetrahydrofuran (500 mL) was added at -78°C. The reaction was stirred at -78°C for 1.5 hours. Saturated ammonium chloride (500 mL) was added to quench the reaction at 0°C. The mixture was extracted with ethyl acetate (1000 mL), washed with saturated brine (500 mL × 2), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain the crude product. The crude product was then purified by silica gel column chromatography (petroleum ether / ethyl acetate = 1 / 0 to 3 / 1) to obtain compound 2-3.

[0218] Step 2: Under nitrogen protection, boron trifluoride etherate (250.80 g, 1.77 mol / L, 217.33 mL) and triethylsilyl hydrochloride (205.47 g, 1.77 mol / L) were added to a solution of compound 2-3 (110 g, 176.71 mmol) in dichloromethane (1100 mL) 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, and the mixture was extracted with dichloromethane (300 mL × 2). The organic phases were combined, washed with saturated sodium chloride (200 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 chromatography (petroleum ether / ethyl acetate = 1 / 0 to 5 / 1) to obtain compound 2-4.

[0219] Step 3: Under nitrogen, compound 2-4 (73 g, 120.36 mmol), compound 2-5 (21.08 g, 132.40 mmol), cuprous iodide (2.29 g, 12.04 mmol), N,N-diisopropylethylamine (104.83 mL), and 1,1-bis(diphenylphosphino)ferrocenepalladium chloride (8.81 g, 12.04 mmol) were added to acetonitrile (400 mL) and stirred at 25°C for 2 hours. The reaction solution was diluted with water (300 mL) and extracted with ethyl acetate (500 mL × 2). The organic phases were combined, washed with saturated sodium chloride (300 mL × 2), dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the crude product. The crude product was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 1 / 0 to 3 / 1) to obtain compound 2-6.

[0220] Step 4: Under nitrogen protection, compound 2-6 (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 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 a crude product. The crude product was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 1 / 0 to 3 / 1) to obtain compound 2-7.

[0221] Step 5: Under nitrogen protection, boron trichloride (1 mol / L, 627.19 mL) was added to a solution of compound 2-7 (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 chromatography (petroleum ether / ethyl acetate = 1 / 0 to 1 / 1, then ethyl acetate / ethanol = 10 / 1 to 5 / 1) to obtain compound 2-8.

[0222] Step 6: Under nitrogen protection, compound 2-8 (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 × 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 a crude product. The crude product was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 1 / 0 to 4 / 1) to obtain compound 2-9.

[0223] Step 7: Under nitrogen protection, sodium hydroxide (2.20 g, 55.09 mmol, 60% purity) was slowly added to a solution of compound 2-9 (28 g, 45.91 mmol) in N,N-dimethylformamide (140 mL) at 0°C. After the addition, the reaction was stirred at 0°C for 0.5 hours. Iodomethane (8.57 mL) was added and the reaction was stirred at 25°C for 1.5 hours. The reaction solution 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 by silica gel column chromatography (petroleum ether / ethyl acetate = 1 / 0 to 4 / 1) to obtain compound 2-10.

[0224] Step 8: Under nitrogen, add compound 2-10 (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 by silica gel column chromatography (petroleum ether / ethyl acetate = 1 / 0 to 0 / 1) to obtain compound 2-11.

[0225] Step 9: Under nitrogen protection, compound 2-11 (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 × 2). The organic phases were combined, 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 by silica gel column chromatography (petroleum ether / ethyl acetate = 1 / 0 to 1 / 1, 0.1% triethylamine) to obtain compound 2-12.

[0226] Step 10: Under nitrogen protection, compound 2-12 (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 × 2). The organic phases were combined, washed with saturated sodium chloride (30 mL), dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a crude product. The crude product was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 1 / 0 to 2 / 1) to obtain compound 2-13.

[0227] Step 11: Under nitrogen, add compound 2-13 (21 g, 22.77 mmol) and p-toluenesulfonyl chloride monohydrate (2.17 g, 11.39 mmol) to dichloromethane (200 mL) and methanol (60 mL). Stir and react at 5°C for 1 hour. The reaction solution is quenched with saturated sodium bicarbonate (20 mL) and extracted with dichloromethane (50 mL x 2). The organic phases are 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 is purified by silica gel column chromatography (petroleum ether / ethyl acetate = 1 / 0 to 2 / 1) to obtain compound 2-14.

[0228] Step 12: Under nitrogen, compound 2-14 (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 × 2). The organic phases were combined, 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 by silica gel column chromatography (petroleum ether / ethyl acetate = 1 / 0 to 2 / 1) to obtain compound 2-15.

[0229] Step 13: Under nitrogen, compound 2-15 (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 solution was quenched with water (10 mL) at 25°C 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 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 2-16.

[0230] Step 14: Under nitrogen, compound 2-16 (2.1 g, 2.79 mmol) and triethylamine trihydrofluoride (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 the crude product. The crude product was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 1 / 0 to 0 / 1, then ethyl acetate / ethanol = 1 / 0 to 5 / 1) to obtain compound 2-17.

[0231] Step 15: Under nitrogen, compound 2-17 (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 (column: Waters Xbridge C18 150 × 50 mm × 10 μm; mobile phase: aqueous ammonium bicarbonate (10 mmol / L)-acetonitrile; acetonitrile gradient: 57%-87%) to obtain B19-M. 1H 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).

[0232] B19-M is an intermediate used to prepare sense chains, antisense chains, or conjugates containing B19.

[0233] Example 3: B33-M

[0234] Step 1: Dissolve compound 3-1 (1.9 g, 6.68 mmol) in N,N-dimethylformamide (30 mL), add 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). 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 3-2. LCMS (ESI) m / z: 513 [M+H] + .

[0235] Step 2: Compound 3-2 (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 3-3. LCMS (ESI) m / z: 399 [M+H] + .

[0236] Step 3: Dissolve compound 3-3 (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 3-4. LCMS (ESI) m / z: 397 [M+H] + .

[0237] Step 4: Under nitrogen protection, sodium hydroxide (411.55 mg, 10.29 mmol, 60% purity) and compound 3-4a (5.42 g, 8.57 mmol) were added to tetrahydrofuran (15 mL) at -78°C and stirred for 0.5 hours at -78°C. A solution of compound 3-4 (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 solution 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 3-5.

[0238] Step 5: Compound 3-5 (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 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 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 3-6.

[0239] Step 6: Under nitrogen, compound 3-6 (520 mg, 883.54 μmol) was dissolved in dichloromethane (10 mL). 4,5-Dicyanoimidazole (520 mg, 883.54 μmol) and compound 3-7 (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%-84%) to obtain compound B33-M. 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).

[0240] B33-M is an intermediate used to prepare sense chains, antisense chains, or conjugates containing B33.

[0241] Example 3: Preparation of conjugate

[0242] Preparation method: oligoribonucleotides were synthesized by phosphoramidite solid phase synthesis technique. ) were synthesized on a solid support. All 2'-modified RNA phosphoramidites and auxiliary reagents were commercially available reagents. All amides were dissolved in anhydrous acetonitrile and molecular sieves were added. Benzyl-1H-tetrazole (BTT) was used as an activating agent, with a coupling time of 5 minutes. ((Dimethylamino-methylene)amino)-3H-1,2,4-dithiazole-3-thione (DDTT) (1 g / 100 mL) in anhydrous acetonitrile / pyridine (v / v = 1 / 2) was used to generate phosphate or phosphorothioate bonds, with a reaction time of 3 minutes. All sequences were synthesized after the final removal of the DMT group.

[0243] Cleavage and Deprotection of CPG-Bound Oligomers: After completion of solid-phase synthesis, the protecting groups were removed by treatment with 10% diethylamine in acetonitrile for 30 minutes, without cleaving the nucleotides from the CPG. Subsequently, the dried CPG was treated with concentrated aqueous ammonia at 40°C for 18 hours. After centrifugation, the supernatant was transferred to a new tube and the CPG was washed with water. The combined solutions were concentrated to yield a solid mixture.

[0244] Purification: The oligomers were purified using NanoQ anion exchange HPLC. 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.

[0245] Annealing: Prepare the RNA oligomers to be annealed at a concentration of 200 μM in sterile RNase-free water (without RNA hydrolases). Complementary strands are formed by combining equimolar RNA solutions. The annealing reaction is set up as follows: Place 10 nmol of the 100 μL mixture in a 95°C water bath for 10 minutes, then quickly place it in a 60°C water bath and allow to cool naturally. Do not store the annealed solution at high temperatures.

[0246] For example, Examples 4 and 5 can be prepared by following the above method by adjusting the order of the raw materials according to the sequence of nucleotides, dR, R and Z. The conjugates shown in Table 3 were all prepared using this method.

[0247] Example 4: Preparation of Conjugate C1

[0248] The sense strand is 5'-cs,cs,a,g,u,c,g,u,Cf,Cf,Cf,a,g,a,a,u,a,a,c,u,a,D01-3' (SEQ ID NO:161), the antisense strand is 5'-us,dAs,g,u,dT,a,dT,u,c,u,g,dG,g,Af,c,g,a,c,u,g,gs,us,c-5' (SEQ ID NO:189),

[0249] Wherein, D01 and a are connected via a phosphodiester bond.

[0250] Example 5: Preparation of Conjugate C25

[0251] The sense strand is 5'-cs,cs,a,g,u,c,g,u,Cf,Cf,Cf,a,g,a,a,u,a,a,c,u,a,D01-3' (SEQ ID NO: 161),

[0252] The antisense strand is 5'-us,dAs,g,u,dT,a,dR,u,c,u,g,dG,g,Af,c,g,a,c,R,g,gs,us,c-3' (SEQ ID NO: 210),

[0253] Wherein, D01 and a are connected via a phosphodiester bond.

[0254] Biological testing

[0255] Test Example 1: INHBE mRNA Inhibitory Activity in Huh7 Cells

[0256] 1. Experimental purpose: The purpose of this study is to evaluate the activity of siRNA in inhibiting the target gene INHBE mRNA in vitro using Huh7 cells, and to use the E maxThe value was used as an indicator to evaluate the inhibitory activity of the compound on INHBE mRNA.

[0257] 2. Experimental Materials:

[0258] 2.1 Cell line: Huh7 cells

[0259] Huh7 cells were provided by Shanghai WuXi AppTec Pharmaceutical Development Co., Ltd. Huh7 cells were cultured in DMEM medium (Invitrogen-11965118) supplemented with 10% fetal bovine serum (ExCellBio, product number FSP500) and 1% penicillin-streptomycin (HyClone product number SV30010).

[0260] 2.2 Reagents:

[0261] The main reagents used in this experiment include Lipofectamine TM iRNAiMAX transfection reagent, FastStart Universal Probe Mast, RNA extraction kit, GAPDH gene expression kit, INHBE gene expression kit, FastKing cDNA first-strand synthesis kit, 96-well plate.

[0262] 2.3 Consumables and instruments:

[0263] The main instruments used in this experiment include a fluorescence qPCR instrument (Applied Biosystems, model QuantStudio 6 Flex) and a cell counter (Vi-cellTM XR).

[0264] 3. Experimental steps and methods:

[0265] 3.1 Transfection

[0266] 1) On the first day, prepare a mixture of RNAiMAX transfection reagent and Opti-MEM (1.5:48.5) as needed and incubate at room temperature for 15 minutes.

[0267] 2) For each well of cells, add a certain amount of diluted compound to an equal amount of RNAiMAX Optim-MEM medium, mix well, and incubate for 15 minutes;

[0268] 3) Take Huh7 cells, wash them with DPBS, add trypsin for digestion, and adjust the cells to an appropriate density;

[0269] 4) 20 μL of the mixture of Opti-MEM RNAiMax and the compound was added to the cell culture plate at the same time, and the cells were seeded into a 96-well plate at a density of 20,000 cells per well. The final culture medium per well was 120 μL.

[0270] 5) The cells were cultured in a 5% CO2, 37°C incubator for 24 h.

[0271] 3.2 RNA extraction and reverse transcription

[0272] The cells were collected, and RNA was extracted according to the instructions of the Qiagen-74182 RNA extraction kit. The RNA was reverse transcribed into cDNA according to the instructions of the FastKing cDNA first-strand synthesis kit.

[0273] 3.3 QPCR detection

[0274] Target gene cDNA was detected using qPCR. The qPCR reaction system was prepared as shown in Table 4. GAPDH was used as an internal reference gene, and qPCR was performed in a 384-well plate. The qPCR reaction program was as follows: heating at 95°C for 10 minutes, followed by cycling at 95°C for 15 seconds, followed by 60°C for 1 minute, for a total of 40 cycles.

[0275] Table 4. RT-PCR reaction system

[0276] 3.4 Data Analysis: ΔΔCt Method (Ct Difference Comparison Method)

[0277] This method requires the introduction of the housekeeping gene GAPDH. This gene is expressed in all cells, its product is essential for cell survival, and its expression level, or genomic copy number, is constant in cells, making it less susceptible to environmental influences. After qRT-PCR, the CT value of the internal reference is recorded, referred to as Ct(GAPDH), and the CT value of the sample is referred to as Ct(sample).

[0278] ΔCt(sample)=Ct(sample)-Ct(GAPDH)

[0279] ΔCt(control)=Ct(control)-Ct(GAPDH)

[0280] ΔΔCt = ΔCt(sample) - ΔCt(control)

[0281] Relative gene expression = 2^-ΔΔCt

[0282] Inhibition rate % = (1-relative expression level of sample / average relative expression level of control group) × 100

[0283] 4. The experimental results are shown in Table 5.

[0284] Table 5. Inhibitory activity of the conjugates of the present invention in reducing INHBE mRNA in Huh7 cells

[0285] 5. Conclusion: The conjugate of the present invention can significantly reduce the level of INHBE mRNA in Huh7 cells.

[0286] Test Example 2: PHH cell free uptake experiment

[0287] 1. Experimental principle:

[0288] The test samples were incubated with primary human hepatocytes to evaluate the degree of downregulation of INHBE mRNA by the test samples.

[0289] 2. Experimental Materials:

[0290] Primary human hepatocytes (PHH), 96Kit(12)(QIAGEN-74182), FastKing RT Kit(With gDNase)(Tiangen-KR116-02).

[0291] 3. Experimental methods:

[0292] The conjugate of the present invention was diluted to 10 times the concentration to be tested with PBS solution. 10 μL of siRNA was transferred to a 96-well plate. PHH cells were thawed and transplanted into a 96-well plate to a final cell density of 5.4×10 4 The conjugate of the present invention was tested at 10 concentration points, with the highest concentration being 500 nM, and 5-fold dilution.

[0293] The cells were incubated at 37°C in 5% CO2 for 48 hours, and the cell status was examined under a microscope.

[0294] After incubation, the cells were lysed and the lysate was obtained. Total RNA was extracted using the QIAGEN 96 Kit (QIAGEN-74182) and reverse transcribed using the FastKing RT Kit (with gDNase) (Tiangen-KR116-02) to obtain cDNA. INHBE cDNA was detected by qPCR.

[0295] 4. The experimental results are shown in Table 6.

[0296] Table 6 Results of the PHH cell free uptake experiment of the conjugates of the present invention

[0297] 5. Experimental conclusion: The conjugate of the present invention can significantly downregulate the level of INHBE mRNA in PHH cells.

[0298] Test Example 3: Mouse hINHBE HDI Model

[0299] 1. Experimental principle:

[0300] The mouse model of high-pressure tail vein injection of pcDNA-INHBE plasmid was used to evaluate the in vivo targeting and inhibitory effect of the test sample on the target gene.

[0301] 2. Experimental Materials:

[0302] pcDNA-INHBE plasmid, BALB / c female mice, DPBS (Dulbecco's phosphate buffered saline), and the conjugate of the present invention.

[0303] 3. Experimental methods:

[0304] Order 6-8 week old BALB / c female mice and acclimate them to quarantine for one week after arrival at the animal house.

[0305] On day 0, mice were randomly divided into groups according to body weight data, with 4-6 mice in each group. After grouping, all mice were administered with subcutaneous injection, a single dose, and a dosing volume of 10 mL / kg. Mice in group 1 were administered with DPBS; mice in other groups were administered with the conjugate of the present invention.

[0306] On the 7th day after administration, all mice were injected with pcDNA-INHBE plasmid at a volume of 8% of their body weight via the tail vein within 5 seconds (injection volume (mL) = mouse body weight (g) × 8%), and the mass of the injected plasmid per mouse was 10 μg.

[0307] On day 8 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 analysis of INHBE gene expression levels.

[0308] 4. The experimental results are shown in Tables 7-9.

[0309] Table 7. Inhibition of INHBE mRNA expression in mouse liver by conjugates C1 and C23 of the present invention

[0310] “ / ” indicates no data, and p<0.05 indicates a statistically significant difference

[0311] Table 8. Inhibition of INHBE mRNA expression in mouse liver by the conjugate C25 of the present invention

[0312] “ / ” indicates no data, and p<0.05 indicates a statistically significant difference

[0313] Table 9. Inhibition of INHBE mRNA expression in mouse liver by the conjugates C42-C45 and C47 of the present invention

[0314] “ / ” indicates no data, and p<0.05 indicates a statistically significant difference

[0315] 5. Experimental conclusion: The conjugate of the present invention can significantly downregulate the INHBE mRNA level in mouse liver.

[0316] Test Example 4: Mouse hINHBE HDI Model

[0317] 1. Experimental principle:

[0318] The mouse model of high-pressure tail vein injection of pcDNA-INHBE plasmid was used to evaluate the in vivo targeting and inhibitory effect of the test sample on the target gene.

[0319] 2. Experimental Materials:

[0320] pcDNA-INHBE plasmid, BALB / c female mice, DPBS (Dulbecco's phosphate buffered saline), and the conjugate of the present invention.

[0321] 3. Experimental methods:

[0322] Order 6-8 week old BALB / c female mice and acclimate them to quarantine for one week after arrival at the animal house.

[0323] On day 0, mice were randomly divided into groups according to body weight data, with 6-7 mice in each group. After grouping, all mice were given subcutaneous injections, a single dose, and a dosing volume of 10 mL / kg. Group 1 mice were given DPBS; Group 2 mice were given conjugate C43; and Group 3 mice were given conjugate C47.

[0324] On the 14th day after administration, all mice were injected with pcDNA-INHBE plasmid at a volume of 8% of their body weight (injection volume (mL) = mouse body weight (g) × 8%) via the tail vein within 5 seconds. The mass of the injected plasmid for each mouse was 10 μg.

[0325] On day 15 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 analysis of INHBE gene expression levels.

[0326] 4. The experimental results are shown in Table 10.

[0327] Table 10. Relative expression of INHBE mRNA in the liver of mice in the drug-treated group compared with that in the phosphate buffer group

[0328] “ / ” indicates no data, and p<0.05 indicates a statistically significant difference

[0329] 5. Experimental conclusion: The conjugate of the present invention can significantly downregulate the INHBE mRNA level in mouse liver and has a long-term effect.

[0330] Test Method 5: INHBE mRNA Knockdown Experiment in Non-Human Primate Cynomolgus Monkeys

[0331] 1. Experimental principle:

[0332] Using a non-human primate cynomolgus macaque model that is highly homologous to the human INHBE gene, the degree of downregulation of INHBE mRNA in the liver of the cynomolgus macaque by the test sample was observed.

[0333] 2. Experimental materials: cynomolgus monkeys, 1x PBS (phosphate buffered saline), and the conjugate of the present invention.

[0334] 3. Experimental methods:

[0335] Order 4 cynomolgus macaques weighing 2-4 kg and acclimate them to quarantine for one week after arrival at the animal house.

[0336] Three days before administration, cynomolgus monkeys were randomly divided into groups according to body weight data, with 2 monkeys in each group.

[0337] On day 1, the conjugate of the present invention was administered subcutaneously at a dose of 3 mpk. Blood and liver tissue were collected on days 15 and 29 after administration, and liver INHBE mRNA levels in cynomolgus monkeys were measured using the aforementioned qPCR method. Comparison of pre- and post-dose liver INHBE mRNA levels revealed the percentage of INHBE mRNA downregulation achieved by the conjugate in each group of animals.

[0338] 4. The experimental results are shown in Table 11.

[0339] Table 11 Inhibition of INHBE mRNA expression in cynomolgus monkey liver by the conjugates of the present invention

[0340] 5. Experimental conclusion: The conjugate of the present invention can significantly reduce the expression of INHBE mRNA in the liver of cynomolgus monkeys and has good in vivo inhibitory activity.

[0341] Test Method 6: Liver Homogenate Stability Test

[0342] 1. Experimental principle:

[0343] The test compound was co-incubated with mouse, rat, cynomolgus monkey and human liver homogenates to observe the stability of the test compound in the liver homogenate.

[0344] 2. Experimental materials: liver homogenates of mice, rats, cynomolgus monkeys and humans, and the conjugate of the present invention.

[0345] 3. Experimental methods:

[0346] 3.1 Prepare 250 μL of working solution of the test compound at a concentration of 20 μM.

[0347] 3.2 Thaw the frozen liver homogenate in a 37°C water bath.

[0348] 3.3 Add 190 μL of liver homogenate to each well of a 96-well plate.

[0349] 3.4 Add 10 μL of the working solution of the test compound to 190 μL of liver homogenate.

[0350] 3.5 Place the 96-well plate containing the liver homogenate and the test substance and incubate at 37°C for a maximum of 48 hours.

[0351] 3.6 Terminate the incubation at different time points. Extract the test compound, and detect the concentration of the sense and antisense strands of the test compound by LC-MS / MS, and calculate the remaining percentage.

[0352] 4. The experimental results are shown in Table 12. The data in the table are all percentages.

[0353] Table 12 Remaining amounts of sense and antisense strands of the test substance in liver homogenates of different species after incubation

[0354] 5. Experimental conclusion: The antisense chain of the conjugate of the present invention has good stability in mouse, rat, cynomolgus monkey and human liver homogenate, indicating that it can exist in the body for a long time and exert its pharmaceutical effect.

[0355] Test Example 7: In vitro RNA sequencing study

[0356] 1. Experimental principle:

[0357] The conjugate of the present invention was incubated with primary human hepatocytes for 48 hours, and the cells were lysed to obtain cell fluid. RNA was extracted using a standard extraction method. After processing through a standard process, RNA was sequenced to detect the gene regulation of the conjugate of the present invention in primary human hepatocytes and evaluate its off-target properties.

[0358] 2. Experimental materials: conjugate of the present invention, primary human hepatocytes

[0359] 3. Experimental Methods

[0360] 3.1 Test conjugates were treated in human primary hepatocytes by free uptake (48 h).

[0361] 1) Plating medium configuration: 10% FBS InvitroGRO CP Medium contains: 10% FBS, 1% double antibody.

[0362] 2) Remove the frozen primary human hepatocytes from the liquid nitrogen tank and immediately thaw them in a 37°C water bath until only a small piece of ice remains in the tube.

[0363] 3) Add 2 mL of thawed human primary hepatocytes to 18 mL of plating medium and gently invert to mix.

[0364] 4) 100 μL of compound dilution was added to a 12-well collagen-coated cell culture plate, and 900 μL of cell suspension was added to each well of the 12-well plate.

[0365] 5) After plating, incubate at 37°C, 5% CO2 for 48 hours.

[0366] 3.2 Lysate Collection

[0367] 1) After 48 hours of cell culture, remove the cell plate and observe the cell status under a microscope.

[0368] 2) Remove the culture medium, add 500 μL PBS to each well to wash once, and add 400 μL TRIzol (RNA extraction reagent) to each well of the 12-well plate.

[0369] 3) Observe under a microscope to ensure complete cell lysis. Combine the duplicate wells of each block, transfer to a 1.5 mL centrifuge tube, and store in a -80°C refrigerator.

[0370] 3.3. Sequencing: Total RNA was extracted from TRIzol lysate and library was constructed, and sequencing was performed using a high-throughput sequencer.

[0371] 4. The experimental results are shown in Table 13.

[0372] Table 13. Regulation of genes in human primary hepatocytes by the conjugates of the present invention

[0373] 5. Experimental conclusion: The conjugate of the present invention showed low off-target risk and good targeting in in vitro RNA sequencing studies.

Claims

1. A double-stranded siRNA 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. The sense strand comprises at least 16 consecutive nucleotides in any one of the sequences shown in SEQ ID NO: 1-80 and SEQ ID NO: 161-188; the antisense strand comprises at least 16 consecutive nucleotides in any one of the sequences shown in SEQ ID NO: 81-160 and SEQ ID NO: 189-230. The nucleotides comprised in the sense strand and the nucleotides comprised in the antisense strand are modified or unmodified nucleotides.

2. The double-stranded siRNA or a pharmaceutically acceptable salt thereof according to claim 1, wherein, The sense strand comprises the nucleotides at positions 1-17, 1-18, 1-19, 1-20, 1-21, 2-17, 2-18, 2-19, 2-20, 2-21, 3-18, 3-19, 3-20, 3-21, 4-19, 4-20, 4-21 or 5-21 starting from the 5'-end of any one of the sequences shown in SEQ ID NO: 1-80 and SEQ ID NO: 161-188; preferably, the sense strand comprises the nucleotides at positions 1-17, 1-18, 1-19, 2-17, 2-18, 2-19, 2-20, 3-18, 3-19, 3-20, 4-19, 4-20, 4-21 or 5-21 starting from the 5'-end of any one of the sequences shown in SEQ ID NO: 1-80 and SEQ ID NO: 161-188.

3. The double-stranded siRNA or a pharmaceutically acceptable salt thereof according to claim 1, wherein, The antisense strand comprises the nucleotides at positions 1-17, 1-18, 1-19, 1-20, 1-21, 1-22, 1-23, 2-18, 2-19, 2-20, 2-21, 2-22, 2-23, 3-19, 3-20, 3-21, 3-22, 3-23, 4-20, 4-2l, 4-22, 4-23, 5-21, 5-22, 5-23, 6-22, 6-23 or 7-23 starting from the 5'-end of any one of the sequences shown in SEQ ID NO: 81-160 and SEQ ID NO: 189-230; preferably, the antisense strand comprises the nucleotides at positions 1-19, 1-20, 1-21, 1-22, 1-23, 2-18, 2-19, 2-20, 2-21, 3-20 or 3-21 starting from the 5'-end of any one of the sequences shown in SEQ ID NO: 81-160 and SEQ ID NO: 189-230.

4. The double-stranded siRNA or a pharmaceutically acceptable salt thereof according to claims 1 to 3, wherein, At least one nucleotide residue on the sense strand, the antisense strand, or both the sense strand and the antisense strand is independently optionally replaced by dR, R, or Z, where dR is R is Z is 5. The double-stranded siRNA or a pharmaceutically acceptable salt thereof according to claim 4, wherein, One to four nucleotide residues at positions 2, 5-7, 12 and 14-18 at the 5'-end of the antisense strand are each independently optionally replaced by dR, R or Z.

6. The double-stranded siRNA or a pharmaceutically acceptable salt thereof according to claim 4, wherein, One to four nucleotides at positions 2, 5-7, 12 and 14-18 at the 5'-end of the antisense strand are each independently optionally 2'-deoxynucleotides.

7. The double-stranded siRNA or a pharmaceutically acceptable salt thereof according to claim 1, wherein, The first nucleoside at the 5'-end of the antisense strand can be replaced by a nucleoside analogue of vinyl phosphate.

8. The double-stranded siRNA or a pharmaceutically acceptable salt thereof according to claim 7, wherein, The nucleoside analogs of vinyl phosphate are selected from the following structural fragments:

9. 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, and the combination of the nucleotide sequence of the sense strand and the nucleotide sequence of the antisense strand is selected from any one of the nucleotide sequence combinations shown in S83-S129.

10. A conjugate or a pharmaceutically acceptable salt thereof, wherein, The conjugate or a pharmaceutically acceptable salt thereof comprises the double-stranded siRNA according to any one of claims 1 to 9 and a pharmaceutically acceptable conjugating group.

11. The conjugate or a pharmaceutically acceptable salt thereof according to claim 10, wherein, The conjugating group is selected from one of the following structures:

12. The conjugate or a pharmaceutically acceptable salt thereof according to claim 11, wherein, The conjugate is selected from C1-C47.

13. The conjugate or a pharmaceutically acceptable salt thereof according to claim 10, wherein the conjugate is represented by formula (I): Among them, X is O or S; SS is the sense strand, and AS is the antisense strand; the sense strand and the antisense strand are selected from the combinations shown in any one of (1) to (9): (1) The sense strand is as shown in SEQ ID NO: 161, and the antisense strand is as shown in SEQ ID NO: 189; (2) The sense strand is as shown in SEQ ID NO: 161, and the antisense strand is as shown in SEQ ID NO: 210; (3) The sense strand is as shown in SEQ ID NO: 186, and the antisense strand is as shown in SEQ ID NO: 226; (4) The sense strand is as shown in SEQ ID NO: 188, and the antisense strand is as shown in SEQ ID NO: 225; (5) The sense strand is as shown in SEQ ID NO: 164, and the antisense strand is as shown in SEQ ID NO: 192; (6) The sense strand is as shown in SEQ ID NO: 171, and the antisense strand is as shown in SEQ ID NO: 199; (7) The sense strand is as shown in SEQ ID NO: 161, and the antisense strand is as shown in SEQ ID NO: 202; (8) The sense strand is as shown in SEQ ID NO: 185, and the antisense strand is as shown in SEQ ID NO: 220; (9) The sense strand is as shown in SEQ ID NO: 188, and the antisense strand is as shown in SEQ ID NO:

228.

14. Use of the double-stranded siRNA according to claims 1-9 or a pharmaceutically acceptable salt thereof, and the conjugate according to claims 10-13 or a pharmaceutically acceptable salt thereof in the preparation of a drug for treating a disease related to INHBE gene expression.

15. The application according to claim 14, wherein, The disease related to INHBE gene expression is a metabolic disease caused by abnormal body fat distribution.

16. The application according to claim 15, wherein, The metabolic diseases caused by abnormal body fat distribution are selected from type 2 diabetes, obesity, abdominal obesity, coronary heart disease, non-alcoholic fatty liver, non-alcoholic steatohepatitis, hyperlipidemia, dyslipidemia, and atherosclerosis.