Nucleotide dimer as well as preparation method and application thereof

Abstract

The invention provides a nucleotide dimer as well as a preparation method and application thereof, and belongs to the technical field of nucleic acid medicines. The nucleotide dimer can be used as a whole for synthesis of the nucleotide chain, and compared with two-step one-by-one nucleotide coupling, the method for synthesizing the nucleotide chain by using the nucleotide dimer provided by the invention has the advantages of few synthesis steps, improved synthesis efficiency, less generated impurities, higher purity of the obtained product and good application prospect.

Description

Technical Field

[0001] The present disclosure relates to the technical field of nucleic acid drugs, and in particular to a nucleotide dimer and a preparation method and use thereof. Background Art

[0002] In recent years, breakthroughs in nucleic acid modification and delivery vectors have brought about a wave of innovative transformative therapies. Among them, nucleic acid drugs, which are considered to be the third generation of innovative drugs after small molecule drugs and antibody drugs, have ushered in rapid growth. Their advantages include a wide range of druggable targets, strong specificity, high safety, long-lasting effects, high development success rate and low manufacturing costs.

[0003] Oligonucleic acid drugs are single-stranded or double-stranded structures composed of 20-60 nucleotide units. They regulate the transcription and translation of disease genes and inhibit gene expression by acting on mRNA. At present, most oligonucleic acid drugs are synthesized by solid-phase phosphoramidite chemistry. Chemical synthesis is carried out in the 3'-5' direction. Each cycle of solid-phase synthesis mainly includes four steps: deprotection, coupling, oxidation and capping. Impurities are inevitably generated during synthesis, such as truncated sequences, long sequences, diastereomers produced by phosphorothioate bonds (PS), and PO impurities produced by oxidation, base depurination impurities, 2'-5' connection isomer impurities, sequence isomers, etc. Moreover, as the synthesis steps proceed further, more impurities are generated, especially the connection step and oxidation step of the last two nucleotides and the additional sulfurization step have much lower reaction efficiency and more impurity generation than the connection of the previous nucleotides. These impurities not only reduce the synthesis efficiency, but also affect the effectiveness of nucleic acid drugs. Therefore, improving the synthesis efficiency and effectively reducing impurities are urgent problems to be solved in the preparation stage of nucleic acid drugs.

[0004] In view of this, the present disclosure is proposed. Summary of the invention

[0005] In order to solve the above technical problems, the present disclosure provides a nucleotide dimer, which is connected to the chain end as a whole during the solid phase synthesis of an oligonucleotide chain. Compared with coupling nucleotides one by one, the synthesis process provided by the present disclosure has fewer steps, high reaction efficiency, less impurities generated, and higher purity of the obtained product.

[0006] The present disclosure includes the following technical solutions:

[0007] In the first aspect of the present disclosure, the present disclosure provides a nucleotide dimer, characterized in that the nucleotide dimer is selected from the structure shown in Formula I, or a pharmaceutically acceptable salt thereof:

[0008]

[0009] Wherein, Base1 and Base2 are independently selected from nucleotide bases A, U, G, C, T and their derivatives, preferably A, U, G or C.

[0010] The derivatives are selected from nucleotide bases substituted by one or more of the following substituents: halogen, C1-C3 alkyl, C1-C3 alkyloxy, or substituted iminoacyl.

[0011] If there is an amino group in the Base1 and the Base2, the amino group is protected by an amino protecting group; the amino protecting group is selected from an alkoxycarbonyl amino protecting group, an acyl amino protecting group or an alkyl amino protecting group.

[0012] P1 is selected from H or a hydroxyl protecting group.

[0013] Optionally, the hydroxy protecting group is selected from a trityl group (Tr group), a 4-methoxytrityl group (MMTr group), a 4,4'-dimethoxytrityl group (DMTr group) or a 4,4',4"-trimethoxytrityl group (TMTr group).

[0014] In a specific embodiment of the present disclosure, the hydroxyl protecting group is selected from 4,4'-dimethoxytrityl (DMTr group).

[0015] P2 is selected from H or a phosphorus-containing leaving group.

[0016] Optionally, the phosphorus-containing leaving group has the structural formula Among them, R 2a Selected from secondary amino groups, R 2b Selected from secondary amino or cyano-substituted C1-C3 alkoxy.

[0017] Optionally, the R 2a Selected from

[0018] Optionally, the R 2b Selected from

[0019] Optionally, the phosphorus-containing leaving group is selected from

[0020] In a specific embodiment of the present disclosure, the phosphorus-containing leaving group is selected from

[0021] The 2' position of one nucleotide ribose group in the nucleotide dimer is substituted by R1 and R2, and the 2' position of the other nucleotide ribose group is substituted by R3 and R4, and the 2' substituents of the two nucleotide ribose groups are the same or different; R1, R2 and R3, R4 are each independently selected from halogen, C1-C3 alkyl or C1-C3 alkoxy; such as F, -CH3, -CH2CH3, -CH2CH2CH3, -OCH3, -OCH2CH3.

[0022] Optionally, the nucleotide dimer is selected from the structure shown in Formula II, or a pharmaceutically acceptable salt thereof:

[0023]

[0024] In some embodiments of the present disclosure, R1 and R2 are the same or different, and R1 and R2 are independently selected from F, -CH3, and -CH2CH3.

[0025] In some embodiments of the present disclosure, R1 and R2 are different, and R1 and R2 are selected from F and -CH3, respectively.

[0026] In some embodiments of the present disclosure, R3 and R4 are the same or different, and R3 and R4 are independently selected from F, -CH3, and -CH2CH3.

[0027] In some embodiments of the present disclosure, R3 and R4 are different, and R3 and R4 are selected from F and -CH3, respectively.

[0028] Optionally, the nucleotide dimer is selected from the structure shown in Formula III, or a pharmaceutically acceptable salt thereof:

[0029]

[0030] wherein Base1 and Base2 are independently selected from A, U, G or C.

[0031] In some embodiments of the present disclosure, Base1 and Base2 form a combination selected from AA, UU, GG, CC, AU, AG, AC, UG, UC or GC.

[0032] Optionally, the nucleotide dimer is selected from the structure shown below, or a pharmaceutically acceptable salt thereof:

[0033]

[0034] Wherein, Ra is selected from an amino protecting group.

[0035] In a specific embodiment of the present disclosure, the amino protecting group is selected from one of the following:

[0036]

[0037] In a second aspect of the present disclosure, the present disclosure provides a method for preparing a nucleotide dimer, characterized in that the method comprises the following steps:

[0038] The synthetic route is as follows:

[0039]

[0040] Wherein, the definitions of Base1, Base2, R1, R2, R3, and R4 are as described in the first aspect of the present disclosure.

[0041] S1: Compound 1 and Compound 2 were dissolved in dichloromethane, 5-benzylthiotetrazolyl (5-BTT) was added, and the mixture was stirred at room temperature for 2-3 hours. The organic phase of the reaction solution was dried and concentrated, and purified to obtain Compound 3;

[0042] S2: dissolving compound 4 in tetrahydrofuran, adding tetrabutylammonium fluoride solution to remove the protecting group, drying and concentrating the organic phase of the reaction solution, and purifying to obtain compound 4;

[0043] S3: Compound 4 was dissolved in dichloromethane, bis(diisopropylamino)(2-cyanoethoxy)phosphine and 4,5-dicyanoimidazole were added, and the mixture was stirred at room temperature for 2-3 hours. The organic phase of the reaction solution was dried and concentrated, and purified to obtain the target compound.

[0044] In a third aspect of the present disclosure, the present disclosure provides a use of a nucleotide dimer in preparing a nucleic acid drug. The nucleic acid drug is selected from antisense oligonucleotides (ASOs), small interfering RNA (siRNA) and micro RNA (miRNA).

[0045] Optionally, the present disclosure provides use of the nucleotide dimer in preparing small interfering RNA (siRNA), the antisense strand of the small interfering RNA comprising at least one of the nucleotide dimers.

[0046] Optionally, the 3' end of the antisense strand of the small interfering RNA comprises at least one of the nucleotide dimers.

[0047] In a fourth aspect of the present disclosure, the present disclosure provides a siRNA, characterized in that the siRNA is prepared using at least one of the nucleotide dimers.

[0048] Optionally, at least one of the nucleotide dimers is located at the 3' end of the antisense strand of the siRNA.

[0049] In a specific embodiment of the present disclosure, one of the nucleotide dimers is located at the 3' end of the antisense strand of the siRNA.

[0050] Optionally, the antisense strand of the siRNA is 21 nt, and starting from the 5' end, the nucleotide dimer is located at position 20-21 of the antisense strand.

[0051] Optionally, the siRNA is coupled to a ligand to form a siRNA conjugate.

[0052] Optionally, the ligand comprises one or more GalNAc.

[0053] In the fifth aspect of the present disclosure, the present disclosure provides a pharmaceutical composition, characterized in that the pharmaceutical composition comprises the siRNA described in the fourth aspect of the present disclosure, and a pharmaceutically acceptable excipient.

[0054] Compared with the prior art, the present invention has the following beneficial effects:

[0055] The present disclosure provides a nucleotide dimer, which can be used as a whole to synthesize a nucleotide chain. Compared with the two-step coupling of nucleotides one by one, the present disclosure provides a nucleotide dimer for synthesizing a nucleotide chain with fewer synthesis steps, which can significantly improve the synthesis efficiency by about 5%, generate less impurities, and obtain a higher purity product, which has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0056] Figure 1 This is the inhibitory activity of the target gene in mouse liver primary cells after administration of the siRNA conjugate described in Example 1.

[0057] Figure 2 This is the inhibitory activity of the target gene in mice after administration of the siRNA conjugate described in Example 2.

[0058] Figure 3 This is the inhibitory activity of the target gene in mice after administration of the siRNA conjugate described in Example 3.

[0059] Figure 4 Relative expression level of SEAP in the serum of mice after a single administration of siRNA conjugates.

[0060] Figure 5 Relative expression level of SEAP in the serum of mice after a single administration of siRNA conjugates.

[0061] Figure 6 Relative expression level of SEAP in the serum of mice after a single administration of siRNA conjugates.

[0062] Figure 7 Relative expression level of SEAP in the serum of mice after a single administration of siRNA conjugates.

[0063] Figure 8This is the inhibitory activity of the target gene in mice after administration of the siRNA conjugate described in Example 8. DETAILED DESCRIPTION

[0064] The technical solution of the present disclosure will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are part of the embodiments of the present disclosure, rather than all of the embodiments. Based on the embodiments in the present disclosure, all other embodiments obtained by ordinary technicians in the field without creative work are within the scope of protection of the present disclosure.

[0065] Explanation of terms

[0066] In the present disclosure, the terms “comprise” or “include” are open expressions, that is, including the contents specified in the present disclosure but not excluding other contents.

[0067] In this disclosure, the terms "optionally", "optional" or "optionally" generally mean that the subsequently described event or circumstance can but need not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not occur.

[0068] In the present disclosure, the term "small interfering RNA (siRNA)" is a double-stranded RNA of 17 to 25 nucleotides in length, comprising a sense strand and an antisense strand. siRNA mediates targeted cleavage of RNA transcripts of the RISC pathway by forming a silencing complex (RISC). Specifically, siRNA guides the specific degradation of mRNA sequences through the known RNA interference (RNAi) process, inhibiting the translation of mRNA into amino acids and into proteins. For example, siRNA can regulate (e.g., inhibit) the expression of LPA in cells.

[0069] In the present disclosure, a double-stranded oligonucleotide consists of two strands, wherein the strand that binds to the target sequence is called the antisense strand or guide strand, and the other strand is called the sense strand or passenger strand.

[0070] In the present disclosure, the term "antisense strand" refers to a strand of a double-stranded oligonucleotide that includes a region that is completely or substantially complementary to a target sequence.

[0071] In the present disclosure, the term "sense strand" refers to that strand of a double-stranded oligonucleotide that includes a region that is substantially complementary to a region as that term is defined herein as the antisense strand.

[0072] In the present disclosure, the term "pharmaceutically acceptable salt" includes pharmaceutically acceptable acid addition salts and pharmaceutically acceptable base addition salts.

[0073] In the present disclosure, the term "pharmaceutically acceptable acid addition salt" refers to a salt formed with an inorganic acid or organic acid that retains the biological effectiveness of the free base without other side effects. Inorganic acid salts include, but are not limited to, hydrochlorides, hydrobromates, sulfates, nitrates, phosphates, and the like; organic acid salts include, but are not limited to, formates, acetates, 2,2-dichloroacetates, trifluoroacetates, propionates, caproates, caprylates, analytes, undecylenates, glycolates, gluconates, lactates, sebacates, adipates, glutarates, malonates, oxalates, maleates, succinates, fumarates, tartrates, citrates, palmitates, stearates, oleates, cinnamates, laurates, malates, glutamates, pyroglutamates, aspartates, benzoates, methanesulfonates, benzenesulfonates, p-toluenesulfonates, alginate, ascorbates, salicylates, 4-aminosalicylates, and the like. These salts can be prepared by methods known in the art.

[0074] In the present disclosure, the term "pharmaceutically acceptable base addition salt" refers to a salt formed with an inorganic base or an organic base that can maintain the biological effectiveness of the free acid without other side effects. Salts derived from inorganic bases include, but are not limited to, sodium salts, potassium salts, sylvite salts, ammonium salts, calcium salts, magnesium salts, iron salts, zinc salts, copper salts, manganese salts, aluminum salts, etc. Preferred inorganic salts are ammonium salts, sodium salts, potassium salts, calcium salts and magnesium salts, preferably sodium salts. Salts derived from organic bases include, but are not limited to, salts of primary, secondary and tertiary amines, substituted amines, including naturally substituted amines, cyclic amines and basic ion exchange resins, such as ammonia, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, diethanolamine, triethanolamine, dimethylethanolamine, 2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, choline, betaine, ethylenediamine, glucosamine, methylglucosamine, theobromine, purine, piperazine, piperidine, N-ethylpiperidine, polyamine resins, etc. These salts can be prepared by methods known in the art.

[0075] In the present disclosure, the term "alkyl" refers to a saturated aliphatic hydrocarbon group, non-limiting examples of which include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, and the like, and various branched chain isomers thereof. In some embodiments selected from the group consisting of alkyl groups containing 1 to 6 carbon atoms, non-limiting examples of which include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2-methylpropyl, 1,1,2-trimethylpropyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 2,2-dimethylbutyl, 1,3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2.3-dimethylbutyl, and the like. By way of example only, "C1-4 alkyl" means having one to four carbon atoms in the alkyl chain, i.e., the C1-4 alkyl is selected from methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, sec-butyl or tert-butyl. The alkyl group may be substituted or unsubstituted.

[0076] In the present disclosure, the term "alkoxy" refers to -O-alkyl, wherein alkyl is as defined above. Non-limiting examples of the alkoxy group include: methoxy, ethoxy, propoxy, butoxy, cyclopropyloxy, cyclobutyloxy, cyclopentyloxy, cyclohexyloxy. By way of example only, "C 1-10 "Alkylene" means an alkyl group having one to ten carbon atoms in the alkyl chain. 1-10 The alkylene group is selected from methylene (-CH2-), ethylene (=CH2CH3), 1,2-ethylene (-CH2CH2-), n-propylene (-CH2CH2CH2-), isopropylene (-CH2CH(CH3)-), and the like.

[0077] In this disclosure, the term "hydroxyl" refers to an -OH group.

[0078] In the present disclosure, the term "halogen" or "halo" refers to any of the radio-stable atoms of Group VII of the Periodic Table of Elements, such as fluorine, chlorine, bromine or iodine, among which fluorine and chlorine are preferred, and fluorine is more preferred.

[0079] In the present disclosure, the term "connection", when referring to the connection between two molecules, refers to the connection of two molecules by covalent bonds or the association of two molecules via non-covalent bonds (e.g., hydrogen bonds or ionic bonds), including direct connection and indirect connection. The term "direct connection" refers to the connection of a first compound or group to a second compound or group without any intervening atoms or atomic groups. The term "indirect connection" refers to the connection of a first compound or group to a second compound or group through an intermediate group, compound or molecule (e.g., a linking group).

[0080] In this disclosure, the term "optionally substituted" is used to define a variable, which may be unsubstituted or substituted.

[0081] In this disclosure, the term "unsubstituted" means that the designated group bears no substituents.

[0082] In this disclosure, the terms "substituted", "substituted" and "substituted" are used interchangeably to indicate that any one or more hydrogen atoms in a given structure are replaced by a specified substituent (e.g., C 1-3 Alkyl, C 1-3 alkoxy or halogen), provided that the normal valence of the designated atom is not exceeded and the substitution results in a stable compound. Unless otherwise indicated, a substituted group may have a substituent at each substitutable position of the group. When more than one position in a given structural formula can be substituted by one or more substituents selected from a specific group, then the substituents may be substituted the same or differently at each substitutable position.

[0083] In the present disclosure, in the structural formulas of "compound", "ligand" and "carrier", the bond Indicates that the configuration is not specified. If chiral isomerism exists in the chemical structure, the bond Can be or include both Although all of the above structural formulae are drawn in certain isomeric forms for simplicity, the present disclosure may include all isomers, for example, tautomers, rotational isomers, geometric isomers, diastereomers, racemates, and enantiomers.

[0084] In the present disclosure, "conjugation" refers to the connection between two or more chemical moieties by covalent bonding; "conjugate" refers to a compound formed by covalent bonding between chemical moieties; "conjugated molecule" is understood to be a specific compound that can be conjugated to an oligonucleotide through a reaction to ultimately form the oligonucleotide conjugate of the present disclosure.

[0085] In the present disclosure, "pharmaceutical composition" may refer to a composition used for the treatment of a disease, and may also be used for in vitro culture experiments of cells. When used for the treatment of a disease, the term "pharmaceutical composition" generally refers to a unit dosage form, and may be prepared by any of the methods well known in the pharmaceutical field. All methods include the step of combining the active ingredient with an excipient that constitutes one or more accessory ingredients. Typically, the composition is prepared by uniformly and fully combining the active siRNA with a liquid excipient, a finely divided solid excipient, or both.

[0086] In the present disclosure, the term "pharmaceutically acceptable" means that the substance or composition must be chemically and / or toxicologically compatible with the other ingredients of the formulation and / or the mammals treated therewith. Preferably, the "pharmaceutically acceptable" described in the present disclosure refers to those approved by federal regulatory agencies or national governments or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeias for use in animals, especially humans.

[0087] In the present disclosure, the term "pharmaceutically acceptable excipient" may include any solvent, solid excipient, diluent or other liquid excipient, etc., suitable for a specific target dosage form. Except for any conventional excipients that are incompatible with the siRNA of the present disclosure, such as any adverse biological effects produced or interactions with any other components of the pharmaceutically acceptable composition in a harmful manner, their use is also within the scope of the present disclosure.

[0088] In the present disclosure, the terms "patient," "subject," or "individual" are used interchangeably and include humans or non-human animals, such as mammals, such as humans or monkeys.

[0089] In this disclosure, the terms "treat," "alleviate," or "amortize" are used interchangeably herein. These terms refer to an approach for obtaining beneficial or desired results, including but not limited to a therapeutic benefit. "Therapeutic benefit" means eradication or amelioration of the underlying disorder being treated. Here, a therapeutic benefit is obtained by eradication or amelioration of one or more physiological symptoms associated with the underlying disorder, such that an improvement is observed in the subject, although the subject may still be afflicted with the underlying disorder.

[0090] In this disclosure, the terms "prevention" and "prevention" are used interchangeably to refer to an approach to obtaining a beneficial or desired result, including but not limited to a prophylactic benefit. To obtain a "prophylactic benefit," a conjugate, RNAi agent, or composition can be administered to a subject at risk for a particular disease, or to a subject reporting one or more physiological symptoms of a disease, even though a diagnosis of the disease may not have been made.

[0091] In addition to any conventional excipients, to the extent that they are incompatible with the siRNA of the present disclosure, such as producing any adverse biological effects or interacting in a deleterious manner with any other component of the pharmaceutically acceptable composition, their use is also contemplated by the present disclosure.

[0092] The present disclosure is further described below by means of specific examples. However, it should be understood that these examples are only used for more detailed description and should not be construed as limiting the present disclosure in any form.

[0093] Preparation of compounds

[0094] Unless otherwise specified, the siRNA sequences used in this disclosure were commissioned to Suzhou Xuanjing Biotechnology Co., Ltd. and Suzhou Beixin Biotechnology Co., Ltd. for synthesis; the PCR primers used in this disclosure were commissioned to Beijing Qingke Biotechnology Co., Ltd. for synthesis; the experimental animals C57BL / 6J mice used in this disclosure were purchased from Sibeifu (Beijing) Biotechnology Co., Ltd. Among them, the information of the main reagents, consumables and instruments and equipment is shown in Tables 1 and 2.

[0095] Table 1 Main reagents and consumables

[0096] name factory 1× PBS Zhongke Maichen (Beijing) Technology Co., Ltd. Hanwei RNA Extraction Kit Zhejiang Hanwei Technology Co., Ltd. Reverse Transcription System Promega Corporation SYBR Select Master Mix ABI TaqMan Fast Advanced Master Mix ABI RNALater Thermo Fisher Scientific

[0097] Table 2 Main instruments and equipment

[0098] name Manufacturer Fully automatic nucleic acid extraction instrument Zhejiang Hanwei Technology Co., Ltd. High-speed refrigerated centrifuge Eppendorf NANODROP OneC Thermo Fisher Scientific Gradient PCR Amplifier Eppendorf Fluorescence quantitative PCR instrument ABI StepOne Plus Fluorescence quantitative PCR instrument Bio-Rad CFX Opus 384 Gel Imager Shanghai Tianneng Life Sciences Co., Ltd. Electrophoresis Apparatus Beijing Liuyi Instrument Factory Tissuelyser II fully automatic tissue homogenizer Shanghai Jingxin Industrial Development Co., Ltd. R540IE Small Animal Anesthesia Machine Shenzhen Ruiwode Life Science Co., Ltd.

[0099] Preparation Example 1, Synthesis of Compound NM054 and Compound NM142:

[0100] In this preparation example, the synthetic routes of compound NM054 and compound NM142 are as follows:

[0101]

[0102] (1-1) Synthesis of compound NM054-2:

[0103] Compound NM054-1 (10 g, 1.0 eq, (2'R)-2'-deoxy-2'-fluoro-2'-methyluridine, CAS No. 863329-66-2) was dissolved in pyridine (100 ml), and 4,4'-dimethoxytrityl chloride (16.9 g, 1.3 eq, DMTrCl for short, CAS No. 40615-36-9) was added in an ice bath; the mixture was stirred at room temperature for 3 hours in a nitrogen atmosphere until the reaction was complete. The reaction solution was concentrated and purified by reverse phase chromatography (eluent: acetonitrile / water = 70 / 30, v / v) to obtain compound NM054-2 (20.5 g, yield 94.9%). .MSESI (m / z) = 563.3 [M+H] + .

[0104] (1-2) Synthesis of compound NM054:

[0105] Compound NM054-2 (10.25 g, 1 eq) was dissolved in anhydrous dichloromethane (100 ml), and bis(diisopropylamino)(2-cyanoethoxy)phosphine (8.25 g, 1.5 eq, CAS No. 102691-36-1) and 4,5-dicyanoimidazole (1.72 g, 0.8 eq, referred to as DCI, CAS No. 1122-28-7) were added. The mixture was stirred at room temperature for 2 hours, and the reaction was completed. Saturated sodium bicarbonate aqueous solution was added to the reaction solution, and the organic phase was dried and concentrated, and purified by reverse phase chromatography (eluent: acetonitrile / water = 95 / 5, v / v) to obtain compound NM054 (10.5 g, yield 75.5%) as a white solid. MS ESI (m / z) = 763.2 [M+H] + .

[0106] (1-3) Synthesis of compound NM142-1:

[0107] Compound NM054-2 (10.25 g, 1.0 eq) was dissolved in DMF (100 ml), and imidazole (3.1 g, 2.5 eq, CAS No. 288-32-4) and tert-butyldimethylsilyl chloride (4.13 g, 1.5 eq, abbreviated as TBDMSCl, CAS No. 18162-48-6) were added in sequence. The mixture was reacted at room temperature for 8 hours until the reaction was completed. Ethyl acetate (400 ml) and water (150 ml) were added to the reaction solution, and the mixture was extracted and separated. The organic phase was washed with saturated sodium chloride aqueous solution 5 times (100 ml each time), and the organic phase was concentrated and purified by normal phase chromatography (eluent: petroleum ether / ethyl acetate = 4 / 1, v / v) to obtain compound NM142-1 (11.05 g, yield 89.5%) as a white solid. MS ESI (m / z) = 676.3 [M+H] + .

[0108] (1-3) Synthesis of compound NM142-2:

[0109] Compound NM142-1 (11.05 g, 1.0 eq) was dissolved in dichloromethane (100 ml), p-toluenesulfonic acid (5.6 g, 2.0 eq, CAS No. 104-15-4) was added, and the mixture was stirred at room temperature for 3 hours in a nitrogen atmosphere to complete the reaction. The reaction solution was washed with a saturated sodium bicarbonate aqueous solution 3 times (50 ml each time), the organic phase was concentrated, and purified by normal phase chromatography (eluent: petroleum ether / ethyl acetate = 1 / 3, v / v) to obtain compound NM142-2 (4.5 g, yield 73.8%) as a white solid. MS ESI (m / z) = 375.2 [M+H] + .

[0110] (1-5) Synthesis of compound NM142-3:

[0111] Compound NM054 (5 g, 1 eq) and compound NM142-2 (3.68 g, 1.5 eq) were dissolved in anhydrous dichloromethane (100 ml), and 5-benzylthiotetrazolyl (1.89 g, 1.5 eq, CAS No. 21871-47-6) was added. The mixture was stirred at room temperature for 2 hours to complete the reaction. The reaction solution was washed with a saturated sodium bicarbonate aqueous solution, the organic phase was dried and concentrated, and purified by reverse phase chromatography (eluent: acetonitrile / water = 95 / 5, v / v) to obtain compound NM142-3 (4.1 g, yield 60.3%) as a white solid. MS ESI (m / z) = 1036.2 [M+H] + .

[0112] (1-6) Synthesis of compound NM142-4:

[0113] Compound NM142-3 (4 g, 1 eq) was dissolved in tetrahydrofuran (40 ml), and tetrabutylammonium fluoride solution (1 Min THF, 3.7 ml, 1.0 eq) was added. The mixture was stirred at room temperature for 2 hours and the reaction was completed. Ethyl acetate (200 ml) was added to the reaction solution, and the mixture was washed with purified water 5 times (50 ml each time). The organic phase was dried and concentrated, and purified by reverse phase chromatography (eluent: acetonitrile / water = 90 / 10, v / v) to obtain compound NM142-4 (2 g, yield 56.2%) as a white solid. MS ESI (m / z) = 922.2 [M+H] + .

[0114] (1-7) Synthesis of compound NM142:

[0115] Compound NM142-4 (2 g, 1 eq) was dissolved in anhydrous dichloromethane (20 ml), and bis(diisopropylamino)(2-cyanoethoxy)phosphine (0.98 g, 1.5 eq) and 4,5-dicyanoimidazole (0.2 g, 0.8 eq) were added. The mixture was stirred at room temperature for 2 hours and the reaction was completed. Saturated sodium bicarbonate aqueous solution was added to the reaction solution, and the extract was separated. The organic phase was dried and concentrated, and purified by reverse phase chromatography (eluent: acetonitrile / water = 95 / 5, v / v) to obtain compound NM142 (1 g, yield 41.1%) as a white solid. MS ESI (m / z) = 1121.2 [M+H] + .

[0116] Preparation Example 2, Preparation of Compound NM118:

[0117] In this preparation example, the synthesis route of compound NM118 is as follows:

[0118]

[0119] (2-1) Synthesis of Compound NM118-2

[0120] Compound NM118-1 (20 g, 1.0 eq, 2'-C-methyluridine, CAS No. 31448-54-1) was dissolved in pyridine (100 ml), 1,3-dichloro-1,1,3,3-tetraisopropyldisiloxane (28.6 g, 1.2 eq, TiPDSCl2, CAS No. 69304-37-6) was added, and the mixture was stirred at room temperature for 12 hours to complete the reaction. The reaction solution was concentrated, water (100 ml) was added, and the mixture was extracted with ethyl acetate twice (100 ml each time). The organic phase was dried and concentrated, and purified by normal phase (eluent: ethyl acetate / petroleum ether = 34 / 66, volume ratio V / V) to obtain compound NM118-2 (39 g) as a white solid. MS ESI (m / z) = 501 [M+H] + .

[0121] (2-2) Synthesis of Compound NM118-3

[0122] Compound NM118-2 (5.0 g, 1 eq) was dissolved in anhydrous tetrahydrofuran (50 ml), and a 60 wt% solution of NaH (1.6 g, 4 eq) in tetrahydrofuran was added under ice bath, and nitrogen was replaced 3 times. The mixture was stirred under ice bath for 1 hour. Methyl iodide (3 g, 2 eq) was added, and the mixture was reacted at room temperature for 4 hours. The reaction was completed. Saturated aqueous ammonium chloride solution (20 ml) was added to the reaction solution, and the mixture was extracted 3 times with ethyl acetate (20 ml each time). The organic phase was dried and concentrated, and purified by reverse phase (eluent: acetonitrile / water = 25 / 75, V / V) to obtain compound NM118-3 (3 g) as a white powder. MS ESI (m / z) = 515 [M+H] + .

[0123] (2-3) Synthesis of Compound NM118-4

[0124] Compound NM118-3 (3.0 g, 1 eq) was dissolved in tetrahydrofuran (30 ml), and a 1 M tetrabutylammonium fluoride solution in tetrahydrofuran (3 ml, 0.5 eq) was added. The mixture was stirred at room temperature for 1 hour to complete the reaction. Water (20 ml) was added to the reaction solution, and the mixture was extracted with ethyl acetate three times (20 ml each time). The organic phase was dried and concentrated, and purified by reverse phase (eluent: acetonitrile / water = 36 / 64, V / V) to obtain compound NM118-4 (1.1 g) as a white powder. MS ESI (m / z) = 295 [M+Na] + .

[0125] (2-4) Synthesis of Compound NM118-5

[0126] Compound NM118-4 (1.1 g, 1.0 eq) was dissolved in pyridine (20 ml), 4,4'-bismethoxytrityl chloride (1.7 g, 1.3 eq) was added under ice bath, nitrogen was replaced 3 times, stirred at room temperature for 3 hours, methanol (20 ml) was added to quench, and the reaction was completed. The reaction solution was concentrated, water (20 ml) was added, and extracted with ethyl acetate 3 times (20 ml each time), the organic phase was dried and concentrated, and normal phase purification (eluent: ethyl acetate / petroleum ether = 50 / 50, V / V) was obtained to obtain compound NM118-5 (1.8 g) as a light yellow solid. MS ESI (m / z) = 575 [M+H] + .

[0127] (2-5) Synthesis of Compound NM118

[0128] Compound NM118-5 (1.6 g, 1.0 eq) was dissolved in anhydrous dichloromethane (20 ml), and 4,5-dicyanoimidazole (264.4 mg, 0.8 eq) and bis(diisopropylamino)(2-cyanoethoxy)phosphine (0.92 g, 1.1 eq) were added respectively. The nitrogen was replaced 3 times, and the mixture was stirred at room temperature for 2 hours. The reaction was completed. A saturated aqueous solution of sodium bicarbonate (20 ml) was added to the reaction solution, and the mixture was extracted with dichloromethane 3 times (20 ml each time). The organic phase was dried and concentrated, and purified by reverse phase (eluent: acetonitrile / water = 75 / 25, V / V) to obtain compound NM118 (1.7 g, yield 78.7%) as a white powder. MS ESI (m / z) = 775 [M+H] + .

[0129] 1 H NMR(400MHz, DMSO-d6)δ11.47(s,1H),7.99(dd,J=8.1,1.6Hz,1H),7.44–7.18(m,9H),6.92(ddd, J=10.7,7.4,3.5Hz,4H),5.96(d,J=9.5Hz,1H),5.77(s,1H),4.97(t,J=8.7Hz,1H),4.38–4.07(m, 2H),3.76(d,J=3.1Hz,7H),3.50(ddt,J=13.9,11.6,7.1Hz,4H),3.40(d,J=6.7Hz,3H),2.81(t,J =5.8Hz,1H),2.64–2.54(m,1H),1.25(d,J=3.3Hz,3H),1.19–1.03(m,10H),0.92(d,J=6.7Hz,2H).

[0130] Preparation Example 3, Preparation of Compound CR01008 and Compound CR01008Z:

[0131] In this preparation example, the synthetic routes of compound CR01008 and compound CR01008Z are as follows:

[0132]

[0133] (3-1) Synthesis of Compound CR01008-2

[0134] Compound CR01008-1 (10.0 g, 1.0 eq, trans-4-(Boc-amino)cyclohexylcarboxaldehyde, CAS No. 181308-57-6) and 37 wt% formaldehyde aqueous solution (8.9 g, 2.4 eq) were dissolved in methanol (33 ml), and 45.3 wt% KOH aqueous solution (13 ml) was added dropwise. After the addition was completed, the mixture was stirred at 25°C for 30 minutes, heated to 60°C and refluxed at 60°C for 2 hours to terminate the reaction. After the reaction solution was cooled to room temperature, the reaction solution was evaporated under reduced pressure to obtain a crude product as a white solid. A small amount of water was added to the crude product for pulping, and filtered to obtain compound CR01008-2 (9 g, yield 78.9%) as a white solid. MS-ESI (m / z) = 260 [M+H] + .

[0135] (3-2) Synthesis of Compound CR01008-3

[0136] Compound CR01008-2 (9 g, 1 eq) was dissolved in 1,4-dioxane (70 ml), and a 4M solution of hydrogen chloride in 1,4-dioxane (45 ml) was added, and the mixture was stirred at 25°C for 1 hour to complete the reaction. The reaction solution was evaporated under reduced pressure to obtain compound CR01008-3 (6.8 g, yield 100%) as a white solid.

[0137] (3-3) Synthesis of Compound CR01008-5

[0138] Compound CR01008-3 (1.8 g, 2.0 eq), compound CR01008-4 (2.1 g, 1.0 eq, 5-[[(2R, 3R, 4R, 5R, 6R)-3-acetylamino-4,5-diacetoxy-6-(acetoxymethyl)-2-tetrahydropyranyl]oxy]pentanoic acid, CAS No. 1159408-54-4) and N,N-diisopropylethylamine (3.5 g, 6.0 eq, abbreviated as DIEA, CAS No. 7087-68-5) were dissolved in DMF (15 ml), and benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate (1.9 g, 1.1 eq, abbreviated as HBTU, CAS No. 94790-37-1) was added. The reaction system was stirred at 25°C in a nitrogen atmosphere for 3 hours, and the reaction was completed. The reaction solution was evaporated to dryness under reduced pressure and purified by reverse phase (eluent: acetonitrile / water = 22 / 78, v / v) to obtain compound CR01008-5 (1.78 g, yield 64.4%) as a white solid. MS-ESI (m / z) = 589 [M+H] + .

[0139] (3-4) Synthesis of Compound CR01008-6

[0140] Compound CR01008-5 (1.54 g, 1.0 eq) was dissolved in pyridine (15 ml), the reaction system was cooled to 0°C with an ice-water bath, DMTrCl (1.32 g, 1.5 eq) was added at 0°C, the reaction was allowed to react for 3 hours at 25°C, methanol (15 ml) was added to quench the reaction, and the reaction was completed. The reaction solution was evaporated to dryness under reduced pressure, and reverse phase purification (eluent: acetonitrile / water = 60 / 40, v / v) was performed to obtain compound CR01008-6 (1 g, yield 42.7%) as a yellow solid. MS-ESI (m / z) = 891 [M+H] + .

[0141] (3-5) Synthesis of Compound CR01008

[0142] Compound CR01008-6 (1.08 g, 1.0 eq) was dissolved in anhydrous dichloromethane (20 ml), and 4,5-dicyanoimidazole (115 mg, 0.8 eq, referred to as DCI, CAS No. 1122-28-7) and bis(diisopropylamino)(2-cyanoethoxy)phosphine (732 mg, 2.1 eq, CAS No. 102691-36-1) were added respectively, and nitrogen was replaced 3 times. The reaction system was stirred at 25° C. in a nitrogen atmosphere for 2 hours, and the reaction was completed. Saturated sodium bicarbonate aqueous solution (20 ml) was added to the reaction solution, and dichloromethane was extracted 3 times (20 ml each time), and the organic phases were combined, dried and concentrated, and reverse phase purified (eluent: acetonitrile / water=72 / 28, v / v) to obtain compound CR01008 (1 g, yield 76.0%) as a white powder. MS-ESI (m / z) = 1091 [M + Na] + .

[0143] 1 H NMR(400MHz, DMSO-d6)δ1.05(d,J=6.7Hz,6H).1.14(d,J=6.7Hz,6H),1.37–1.17(m,5H),1.60–1.40(m,6H),1.68–1.62(m,1H),1.80(s,3H), 1.80(s,3H),1.92(s,3H),2.02(s,5H),2.13(s,3H),2.71(t,J=5.9Hz,2H),2.79(d,J=8.4Hz,1H),2.87(d,J=8.4Hz,1H),3.36(s,1H),3.58– 3.39(m,3H),3.69–3.60(m,2H),3.75(s,7H),3.90(dt,J=11.2,8.8Hz,1H),4.05(s,3H),4.51(d,J=8.4Hz,1H),4.99(dd,J=11.3,3.4Hz,1H) ,5.24(d,J=3.4Hz,1H),5.78(s,1H),6.93–6.87(m,4H),7.35–7.21(m,7H),7.44–7.37(m,2H),7.66(d,J=7.8Hz,1H),7.84(d,J=9.2Hz,1H).

[0144] (3-6) Synthesis of Compound CR01008-7

[0145] Compound CR01008-6 (500 mg) was dissolved in dichloromethane (10 ml), and succinic anhydride (112 mg), 4-dimethylaminopyridine (6.8 mg, DMAP, CAS No. 1122-58-3) and triethylamine (226.2 mg) were added. The nitrogen atmosphere was replaced three times, and the reaction system was stirred at 25°C in a nitrogen atmosphere for 16 hours. The reaction was completed. The reaction solution was purified by FLASH to obtain compound CR01008-7 (300 mg, yield 53.6%). MS-ESI (m / z) = 1013 [M+Na] + .

[0146] (3-7) Synthesis of Compound CR01008Z

[0147] Compound CR01008-7 (50 mg, amino CPG (1.25 g, 0.1 mmol), HBTU (27 mg), and DIEA (12 mg) were added to a 20 ml sample bottle and shaken for 16 hours to complete the reaction. The reaction solution was filtered, and the filter cake was washed once with acetonitrile (10 ml) and then vacuum dried. The dried filter cake, DMAP (3 mg), Cap1 (10 ml) and Cap2 (1 ml) were added to a 20 ml sample bottle and shaken for 6 hours to complete the reaction. The reaction solution was filtered, and the filter cake was washed once with 10 ml of acetonitrile and then vacuum dried to obtain compound CR01008Z (1.03 g, loading 20-30 μmol / g).

[0148] Among them, the structural formula of amino CPG (Aminoalkyl-CPG) is: Model C3006-1000, 100-200 mesh, loading capacity 80 μmol / g.

[0149] Cap1 and Cap2 are capping reagents, Cap1 is a 20 volume % N-methylimidazole mixed solution in pyridine / acetonitrile, with the volume ratio of pyridine to acetonitrile being 3:5; Cap2 is a 20 volume % acetic anhydride solution in acetonitrile.

[0150] Preparation Example 4: Preparation of siRNA conjugates:

[0151] Compound L96-PS was purchased from Ailiying Pharmaceutical Group (Tianjin) Co., Ltd., with a loading of 120±12 μmol / g (detection method: UV / HPLC). The structural formula of compound L96-PS is as follows:

[0152]

[0153] Wherein, PS represents polystyrene resin solid phase carrier.

[0154] (1-1) Synthesis of sense strand (SS) and antisense strand (AS):

[0155] By the phosphoramidite nucleic acid solid phase synthesis method, the above compounds connected to the solid phase carrier (such as CPG carrier, PS carrier, compound L96-PS, compound CR01008Z) are used to start the cycle, and the nucleoside monomers are connected one by one in the direction from the 3' end to the 5' end of the nucleotide sequence. During the synthesis process, compounds CR01008, NM054, NM142 and NM118 are respectively regarded as a nucleoside monomer.

[0156] Each connection of a nucleoside monomer includes four steps of deprotection, coupling, capping, oxidation or sulfurization. The synthesis conditions are given as follows:

[0157] The nucleoside monomer was prepared into an acetonitrile solution with a concentration of 0.1 M.

[0158] The conditions of the deprotection reaction in each step are the same. The conditions of the deprotection reaction are: temperature of 25° C., reaction time of 70 seconds, deprotection reagent of dichloroacetic acid in dichloromethane solution (3% by volume), and the molar ratio of dichloroacetic acid to 4,4'-dimethoxytrityl protecting group on the solid phase carrier is 5:1.

[0159] The conditions of each coupling reaction are the same. The coupling reaction conditions are: temperature is 25°C, the molar ratio of the nucleic acid sequence connected to the solid phase support to the nucleoside monomer is 1:10, the molar ratio of the nucleic acid sequence connected to the solid phase support to the coupling reagent is 1:65, the reaction time is 600 seconds, the coupling reagent is an acetonitrile solution of 5-ethylthio-1H-tetrazole with a concentration of 0.5M, and the thiolation reagent is an acetonitrile / pyridine mixed solution of hydrogenated xanthanol with a concentration of 0.2mol / L (the volume ratio of acetonitrile and pyridine is 1:1).

[0160] The conditions of each capping reaction are the same. The conditions of the capping reaction are: temperature is 25°C; reaction time is 2 minutes; the capping reagent solution is a mixed solution of Cap1 and Cap2 with a molar ratio of 1:1, Cap1 is a pyridine / acetonitrile mixed solution of N-methylimidazole with a concentration of 20 volume%, the volume ratio of pyridine to acetonitrile is 3:5, Cap2 is an acetonitrile solution of acetic anhydride with a concentration of 20 volume%; the molar ratio of N-methylimidazole in Cap1 capping reagent, acetic anhydride in Cap2 capping reagent to the nucleic acid sequence connected to the solid phase carrier is 1:1:1.

[0161] The conditions for each step of the oxidation reaction are the same. The conditions for the oxidation reaction are: temperature of 25°C; reaction time of 3 seconds; oxidation reagent concentration of 0.05M iodine water, the molar ratio of iodine to the nucleic acid sequence connected to the solid phase carrier in the coupling reaction is 30:1; oxidation reaction is carried out in a water / pyridine mixed solvent (the volume ratio of water to pyridine is 1:9). The conditions for the sulfidation reaction are: temperature of 25°C; reaction time of 360 seconds; thiolation reagent concentration of 0.2M hydrogenated xanthan gum pyridine solution, the molar ratio of thiolation reagent to the nucleic acid sequence connected to the solid phase carrier in the coupling reaction is 4:1; thiolation reaction is carried out in a water / pyridine mixed solvent (the volume ratio of water to pyridine is 1:9).

[0162] After the last nucleoside monomer is connected, the nucleic acid sequence connected to the solid phase carrier is cut, deprotected, purified, desalted, and then freeze-dried to obtain the positive chain, wherein:

[0163] The cleavage and deprotection conditions are as follows: the synthesized nucleotide sequence connected to the solid phase carrier is added to 25% by mass ammonia water, the amount of ammonia water is 0.5 ml / μmol, the reaction is carried out at 55°C for 16 hours, the solvent is removed, and the product is concentrated to dryness in vacuo. After the ammonia water treatment, the product is dissolved with 0.4 ml / μmol N-methylpyrrolidone relative to the amount of single-stranded nucleic acid, and then 0.3 ml / μmol triethylamine and 0.6 ml / μmol triethylamine trihydrofluoride are added to remove the 2'-O-TBDMS protection on the ribose.

[0164] Purification and desalting conditions: The nucleic acid was purified by gradient elution of NaCl using a preparative ion chromatography purification column (Source 15Q). Specifically, eluent 1 was 20 mM sodium phosphate (pH = 8.1), and the solvent was a water / acetonitrile mixed solution (the volume ratio of water to acetonitrile was 9:1); eluent 2 was 1.5 M sodium chloride, 20 mM sodium phosphate (pH = 8.1), and the solvent was a water / acetonitrile mixed solution (the volume ratio of water to acetonitrile was 9:1); the elution gradient was eluent 1: eluent 2 = (100:0)-(50:50). The product eluates were collected and combined, and desalted using a reverse chromatography purification column. The desalting conditions included desalting using a dextran gel column, the filler was dextran gel G25, and eluted with deionized water.

[0165] Detection: Use ion exchange chromatography (IEX-HPLC) for purity detection; use liquid chromatography-mass spectrometry (LC-MS) for molecular weight detection, and compare the measured value of the molecular weight with the theoretical value. If the measured value is consistent with the theoretical value, it indicates that the sense chain and the target antisense chain are obtained.

[0166] (4-2) Synthesis of siRNA conjugates

[0167] The sense chain and antisense chain synthesized in step (4-1) are mixed in an equimolar ratio, dissolved in water for injection and heated to 95°C, slowly cooled to room temperature and kept at room temperature for 10 minutes, so that the sense chain and antisense chain form a double-stranded structure through hydrogen bonds, thereby obtaining a siRNA conjugate.

[0168] Wherein, when the delivery vector is L96, the structural formula of the siRNA conjugate is:

[0169]

[0170] When the delivery vector is three clusters of CR01008 (denoted as: (CR01008)(CR01008)(CR01008Z) or (CR01008×3)), the structural formula of the siRNA conjugate is:

[0171] in, Indicates siRNA.

[0172] The siRNA conjugates prepared in the present disclosure are shown in Table 4, wherein the unmodified nucleotide sequence information of the siRNA conjugates is shown in Table 3.

[0173] Table 3 Unmodified sequence information of siRNA conjugates

[0174]

[0175] Table 4 Sequence information of siRNA conjugates

[0176]

[0177]

[0178] Unless otherwise specified, the base composition and modification meanings used in the present disclosure are as follows: capital letters A, U, G, C, and T represent the base composition of nucleotides; lowercase letter d represents that the nucleotide represented by the capital letter adjacent to its left is a 2'-deoxy-modified nucleotide; lowercase letter m represents that the nucleotide represented by the capital letter adjacent to its left is a 2'-O-Me-modified nucleotide; lowercase letter f represents that the nucleotide represented by the capital letter adjacent to its left is a 2'-F-modified nucleotide; (moe) represents that the nucleotide represented by the capital letter adjacent to its left is a 2'-O-methoxyethyl-modified nucleotide; lowercase letter s represents that the internucleoside bond between the two adjacent nucleotides on its left and right sides is a phosphorothioate bond.

[0179] (NM054) represents a nucleotide, which is obtained by synthesizing the siRNA conjugate with the compound NM054. The structural formula of (NM054) is

[0180] (NM054)s(NM054) represents two nucleotides, which are synthesized by compound NM142 participating in the siRNA conjugate. The structural formula of (NM054)s(NM054) is

[0181] (NM118) represents a nucleotide, which is obtained by synthesizing the siRNA conjugate with the compound NM118. The structural formula of (NM118) is

[0182] Biological detection experiments

[0183] Unless otherwise stated, the reagents, consumables and instruments used in the biological detection experiments disclosed herein are all commercially available products.

[0184] Method for evaluating the inhibitory activity of siRNA conjugates on target genes in primary mouse liver

[0185] Isolation of primary mouse hepatocytes:

[0186] Mouse primary hepatocytes were extracted from fresh liver tissue of C56BL / 6j mice. The specific operation steps were as follows: mice were anesthetized by intraperitoneal injection of 10% chloral hydrate solution, fixed and their abdomen and chest were disinfected with 75% ethanol. The surgical instruments were sterilized, and the abdominal cavity was opened to expose the portal vein and inferior vena cava. The indwelling needle was equipped with a heparin cap, connected to a scalp needle connected to an infusion pump bottle (0.5mM EDTA HBSS perfusion solution), inserted into the inferior vena cava, and perfused at a rate of 120 drops / min. The portal vein was cut open, and the perfusion solution was allowed to flow out of the cut portal vein. The perfusion was performed for 4 minutes, and then replaced with 0.8mg / mL type IV collagenase HBSS solution (Sigma, C5138) (containing 0.08% DNAⅠ enzyme (sigma, DN25)) and continued to perfuse for 8 minutes. The perfused liver was removed from the animal, washed with HBSS (containing Ca2+, Mg2+, MACGENE, CC016), placed in a sterile culture dish, added with DMEM complete medium (DMEM medium + 10% serum) to shred the liver, filtered the cell suspension with a cell sieve to remove undigested tissue and connective tissue, centrifuged at 800 rpm for 3 min, discarded the supernatant, added with DMEM complete medium again, suspended and centrifuged to obtain primary mouse hepatocytes.

[0187] Cell culture and transfection:

[0188] Add DMEM complete medium to adjust the cell density to 2 × 10 5 cells / mL to obtain a mouse primary hepatocyte suspension. The cells were then inoculated into a 12-well culture plate pre-coated with rat tail collagen type I (the coating method was described in the solarbio (C8062) manual at 2 μg / cm 2The volume of cell suspension added was 1000 μL / well, i.e., the cell amount was 2×10 5 cells / well.

[0189] Dilute each group of conjugates with PBS to a working solution of 1000 times the final test concentration (in terms of siRNA). Add 1 μL / well of siRNA conjugate working solution to the above 12-well culture plate, and set 2-3 culture wells for each siRNA conjugate. Add 1 μL / well of PBS to another 2-3 culture wells as blank control wells. Shake the culture plate to mix evenly. Place the culture plate in a cell culture incubator at 37°C and 5% CO2 and continue to culture for 24 hours.

[0190] RNA extraction: The total RNA of each group of primary hepatocyte samples was extracted using the fully automatic nucleic acid extractor and nucleic acid extraction kit of Zhejiang Hanwei Technology Co., Ltd. according to the method described in the instructions.

[0191] Method for evaluating the inhibitory activity of siRNA conjugates on target genes in mice

[0192] 6-8 week old C57BL / 6J mice were randomly divided into groups according to body weight (all female). The mice in each group were given a single dose of drug according to body weight and administered by subcutaneous injection in the abdomen. Each siRNA conjugate was configured with a PBS solution to a corresponding concentration (calculated as siRNA) for administration, and the administration volume was 5ml (in terms of siRNA) / kg (in terms of mice). The PBS control group was given 5ml / kg (in terms of mice) of PBS solution (without drug conjugate). The day of administration was recorded as day 0 (recorded as D0), and 5 mice were killed in each group at the preset time after administration. The mice killed were grossly dissected and the liver tissue of each killed mouse was collected, and the liver tissue was cut into about 2mm 3 Small pieces were stored with RNA Later.

[0193] Liver tissue samples at different time points in different experimental groups were taken from the above RNA later, and the liver tissue samples were broken in a Tissuelyser II fully automatic tissue homogenizer for 60 seconds, and then total RNA was extracted using a fully automatic nucleic acid extractor (purchased from Zhejiang Hanwei Technology Co., Ltd.) and a nucleic acid extraction kit (purchased from Zhejiang Hanwei Technology Co., Ltd.) according to the standard operating procedures for total RNA extraction.

[0194] mRNA expression level detection:

[0195] Take the above 1 μg total RNA, use the reverse transcription kit (Promega, Reverse Transcription System, A3500) and select Oligo (dT) 15 reverse transcription primer, prepare 20 μL reverse transcription system according to the method described in the reverse transcription kit manual and complete the reverse transcription reaction. After the reaction is completed, add 80 μL RNase-Free water to the reverse transcription system to obtain cDNA solution. Then use the real-time fluorescence quantitative PCR kit (ABI, SYBR TM Select MasterMix, Catalog number: 4472908) to detect the expression of target gene mRNA. In this real-time fluorescence quantitative PCR method, primers for the target gene and primers for the internal reference gene are used to detect the target gene and the internal reference gene respectively. According to the method described in the instruction manual of the real-time fluorescence quantitative PCR kit, 20μL real-time PCR reaction system is configured for each PCR detection well, and each reaction system contains 5μL of the cDNA solution obtained by the above reverse transcription reaction, 10μL SYBR TM SelectMaster Mix, 0.5 μL 10 μM upstream primer, 0.5 μL 10 μM downstream primer, 4 μL RNase-Free H2O. The prepared reaction system was placed in a real-time fluorescence quantitative PCR instrument (ABI, StepOnePlus TM ) was used for Real-time PCR amplification using a three-step method. The amplification program was pre-denaturation at 95°C for 10 min, followed by denaturation at 95°C for 30 s, annealing at 60°C for 30 s, and extension at 72°C for 30 s. The denaturation, annealing, and extension process was repeated for 40 cycles.

[0196] Alternatively, a reverse transcription kit (Thermo Fisher Scientific, RevertAid FirstStrand cDNA Synthesis Kit, K1622) was used and Oligo (dT) 18 reverse transcription primer was selected, and a 20 μL reverse transcription system was configured according to the method described in the reverse transcription kit manual and the reverse transcription reaction was completed. Then, a real-time fluorescence quantitative PCR kit (Thermo Fisher Scientific, TaqMan Fast Advanced Master Mix, 4444557) was used to detect the expression of the target gene mRNA on a fluorescence quantitative PCR instrument (Bio-Rad, CFX Opus 384). In the real-time fluorescence quantitative PCR method, the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene was used as an internal reference gene, and primers for the target gene and the GAPDH internal reference gene were used to detect the target gene and the GAPDH internal reference gene, respectively.

[0197] In the real-time fluorescence quantitative PCR method, the ΔΔCt method was used to perform relative quantitative calculation of the expression level and inhibition rate of the target gene mRNA in each test group. The calculation method is as follows:

[0198] ΔCt(test group) = Ct(test group target gene) – Ct(test group reference gene)

[0199] ΔCt(control group) = Ct(control group target gene) – Ct(control group internal reference gene)

[0200] ΔΔCt(test group) = ΔCt(test group) – ΔCt(average of control group)

[0201] ΔΔCt(control group) = ΔCt(control group) – ΔCt(control group average)

[0202] In cell experiments, ΔCt (control group average) is the arithmetic mean of ΔCt (control group) of several replicate wells in the control group. In animal experiments, ΔCt (control group average) is the arithmetic mean of ΔCt (control group) of 5 mice sacrificed at the same time point in the control group. Therefore, each sample in the test group and the control group corresponds to a ΔΔCt value.

[0203] Relative expression level of target gene mRNA in the test group = 2 -ΔΔCt( Test group) × 100%

[0204] The target gene mRNA expression level of the test group was normalized with the control group as the benchmark, and the target gene mRNA expression level of the control group was defined as 100%.

[0205] Test group target gene mRNA expression inhibition rate (%) = 100% - test group target gene mRNA relative expression level

[0206] Unless otherwise stated, the in vivo activity data are based on The experimental data were plotted and analyzed using GraphPadprism 8.0 software.

[0207] Example 1 Effect of NM054 modified sequence at the 3' end of the antisense strand on superoxide dismutation of the target gene in mouse primary liver cells Evaluation of the inhibitory activity of superoxide dismutase 1 (SOD1)

[0208] Example 1 The above mouse liver primary culture target gene inhibitory activity evaluation experimental method was used to evaluate the inhibitory activity of the antisense chain 20-position NM054 modified sequence RZ599034 and the reference sequence RZ599001 on the target gene SOD1 in mouse primary culture liver cells.

[0209] Mouse primary hepatocytes were cultured at a cell count of 2×10 5 Cells / well were inoculated in 12-well culture plates, and each group of double-stranded siRNA conjugates were gradiently diluted with PBS to 10μM and 1μM working solutions (in terms of siRNA). 1μL / well of siRNA conjugate working solution of each concentration was added to the above 12-well culture plates, which was equivalent to a final transfection concentration of 10nM and 1nM (in terms of siRNA) for siRNA conjugates. 2-3 culture wells were set for each concentration of siRNA. In addition, 1μL / well of PBS was added to 2-3 culture wells as blank control wells (BLANK).

[0210] Table 5 Primer sequence information in Example 1

[0211]

[0212] Table 6 Inhibitory activity of target genes in mouse liver primary cells after administration of the siRNA conjugates described in Example 1

[0213]

[0214] The results of Example 1 show that the sequence RZ599034 modified with NM054 at position 20 of the antisense strand has a stronger target gene inhibition effect than the reference sequence RZ599001. Figure 1 , Table 6)

[0215] Example 2. Evaluation of the inhibitory activity of the NM054 modified sequence at the 3' end of the antisense strand on the target gene SOD1 in mice

[0216] In this example, the target gene inhibitory activity evaluation method in vivo in mice was used to evaluate the inhibitory activity of the conjugates RZ599060 containing the NM054 group at position 20 of the SOD1 target siRNA antisense chain, the conjugate RZ599061 containing NM054 at positions 20 and 21, and the conjugate RZ599001 not containing this group on the target gene SOD1 in mice.

[0217] 6-8 week old C57BL / 6j mice were randomly divided into 4 groups according to body weight, 15 mice in each group, and the above siRNA conjugate was administered subcutaneously in the abdomen. The dose of each mouse in the PBS control group was 5 mL / kg, and the dose of each mouse in the siRNA conjugate experimental group was 3 mg / kg (in terms of siRNA) and the administration volume was 5 mL / kg. The day of administration was recorded as day 0 (D0). After administration, 5 mice in each group were killed on the 7th day (D7), the 28th day (D28), and the 49th day (D49). The animals were grossly dissected, and the liver tissue was collected and cut into several 2 mm 3 The small pieces were preserved with RNAlater. RNA extraction, reverse transcription reaction, and fluorescence quantitative PCR were performed as described above, and the gene expression difference was calculated by the ΔΔCt method. The primers are shown in Table 5 of Example 1.

[0218] Table 7 Inhibitory activity of target genes in mice after administration of the siRNA conjugates described in Example 2

[0219]

[0220]

[0221] The results of Example 2 show that compared with the control conjugate RZ599001 not containing the NM054 group, the conjugates RZ599060 and RZ599061 containing the NM054 group have higher inhibitory activity at D7, D28 and D49. At D49, the inhibitory activity of RZ599060 and RZ599061 is about 15% higher than that of the control conjugate RZ599001. Figure 2 , Table 7).

[0222] Example 3. Effect of NM054 modified sequence at the 3' end of the antisense strand on the target gene angiopoietin-like protein 3 in mice Evaluation of the inhibitory activity of angiopoietin-like 3 (ANGPTL3)

[0223] In this example, the target gene inhibitory activity evaluation method in vivo in mice was used to evaluate the inhibitory activity of the conjugates RZ597115 containing the NM054 group at position 20 of the ANGPTL3 target siRNA antisense chain, the conjugate RZ597116 containing NM054 at positions 20 and 21, and the conjugate RZ597114 not containing this group on the target gene ANGPTL3 in mice.

[0224] 6-8 week old C57BL / 6j mice were randomly divided into 4 groups according to body weight, 15 mice in each group, and the above siRNA conjugate was administered subcutaneously in the abdomen. The dose of each mouse in the PBS control group was 5 mL / kg, and the dose of each mouse in the siRNA conjugate experimental group was 3 mg / kg (in terms of siRNA) and the administration volume was 5 mL / kg. The day of administration was recorded as D0. After administration, 5 mice in each group were killed on D7, D28, and D56, and the animals were grossly dissected, liver tissues were collected, and cut into several 2 mm 3 The small pieces were preserved with RNAlater. RNA extraction, reverse transcription reaction, and real-time PCR detection were as described above, and the gene expression difference was calculated by the ΔΔCt method.

[0225] Table 8 Primer sequence information in Example 3

[0226]

[0227] Table 9 Inhibitory activity of target genes in mice after administration of the siRNA conjugates described in Example 3

[0228]

[0229] The results of Example 3 show that the conjugates RZ597115 and RZ597116 containing the NM054 group have higher inhibitory activity at D7, D28 and D56 than the control conjugate RZ597114, which does not contain the NM054 group. At D56, the inhibitory activity of the conjugate RZ597115 containing the NM054 group at position 20 of the antisense chain is about 25% higher than that of the control conjugate RZ597114; the inhibitory activity of the conjugate RZ597116 containing the NM054 group at positions 20 and 21 of the antisense chain is about 35% higher than that of the control conjugate RZ597114. Figure 3 , Table 9)

[0230] Example 4. Effect of the sequence containing NM054 at the 3' end of the antisense strand on the SEAP protein in serum in the BALB / c-SEAP model Evaluation of the inhibitory activity of

[0231] In this example, the relative expression of SEAP protein in serum of the sequence RZ003075 containing NM054 at positions 20 and 21 of the antisense strand and the reference sequence RZ003089 were evaluated in BALB / c-SEAP model mice.

[0232] The BALB / c-SEAP model was constructed by inserting the AGT transcript sequence into the transposon plasmid (Shanghai Langjing Biotechnology Co., Ltd.), and injecting 1.6 mL of the plasmid mixed injection solution (25 μg of the transposon plasmid fused with the AGT transcript and 25 μg of the transposase Super PiggyBac Transposase plasmid) into the mouse through the tail vein hydrodynamics within 3 to 5 seconds. After 2 weeks, a mouse model that stably expressed the SEAP reporter gene was obtained.

[0233] Animal grouping, drug administration and serum sample collection:

[0234] Female BALB / c-SEAP stable mice were grouped according to SEAP levels, with 5 mice in each group. Each test group was given a predetermined dose of drug and a saline control group was added. RZ003089 and RZ003075 sequences were administered once by subcutaneous injection in the abdomen. The drug dose for all mice was calculated based on body weight, with a dosing volume of 10 mL / kg (mouse body weight), and the dose was 0.3 mg / kg (calculated as siRNA / mouse body weight). The saline control group was given 10 mL / kg (mouse body weight) of saline solution without siRNA conjugates. The serum of mice in the saline group and the RZ003089 and RZ003075 groups was collected on D5 after administration. Phospha-Light TM The SEAP reporter gene detection system (Thermo, T1017) was used to detect the SEAP levels in the serum of mice in all groups.

[0235] Table 10 Relative expression levels of SEAP in mouse serum after single administration of siRNA conjugates

[0236]

[0237] The results of Example 4 show that compared with the reference sequence RZ003089, the NM054-modified RZ003075 has a higher inhibitory effect on SEAP in mouse serum. Figure 4 , Table 10)

[0238] Example 5. Inhibition of SEAP protein in serum by BALB / c-SEAP containing the sequence NM054 at the 3' end of the antisense strand Activity evaluation

[0239] The mouse model of SEAP reporter gene used in this example was used to evaluate the effect of the sequence RZ003079 containing NM054 at positions 20 and 21 of the antisense strand and the reference sequence RZ003087 on the relative expression of SEAP in serum.

[0240] The construction of the BALB / c-SEAP model is shown in Example 6. Female BALB / c-SEAP stable mice were grouped according to SEAP levels, with 5 mice in each group. Each test group was given a predetermined dose of drug and a saline control group was added. RZ003087 and RZ003079 were administered in a single sequence by subcutaneous injection in the abdomen. The drug dose of all mice was calculated according to body weight, the administration volume was 10 mL / kg (mouse body weight), and the dosage was 3 mg / kg (calculated as siRNA / mouse body weight). The saline control group was given 10 mL / kg (mouse body weight) of saline solution without siRNA conjugate. The serum of mice in the saline group and the RZ003087 and RZ003079 administration groups was collected before administration (pre-dose) and on D7, D14, D21, D28, and D35 after administration. Phospha-Light TM The SEAP reporter gene detection system (Thermo, T1017) was used to detect the SEAP levels in the serum of mice in all groups.

[0241] Table 11 Relative expression levels of SEAP in mouse serum after single administration of siRNA conjugates

[0242]

[0243] The results of Example 5 showed that compared with the reference sequence RZ003087, the NM054-modified RZ003079 had a higher inhibitory effect on SEAP protein expression in mouse serum at various time points and showed a longer protein inhibition level. Figure 5 , Table 11)

[0244] Example 6. Inhibition of SEAP protein in serum by the sequence containing NM054 at the 3' end of the antisense strand in BALB / c-SEAP Activity evaluation

[0245] The mouse model of SEAP reporter gene used in this example was used to evaluate the effect of the conjugate RZ003080 containing NM054 at positions 20 and 21 of the antisense strand and the reference sequence RZ003088 on the relative expression of SEAP in serum.

[0246] The construction of the BALB / c-SEAP model is shown in Example 4. Female BALB / c-SEAP stable mice were grouped according to SEAP levels, with 5 mice in each group. Each test group was given a predetermined dose of drug and a saline control group was added. RZ003088 and RZ003080 sequences were administered once by subcutaneous injection in the abdomen. The drug dose for all mice was calculated based on body weight. The administration volume was 10 mL / kg (mouse body weight), and the administration dose was 3 mg / kg (calculated as siRNA / mouse body weight). The saline control group was given 10 mL / kg (mouse body weight) of saline solution without siRNA conjugate. The serum of mice in the saline group and the RZ003080 and RZ003088 administration groups was collected before administration (pre-dose) and at D7, D14, D21, D28, and D35 after administration. Phospha-Light TM The SEAP reporter gene detection system (Thermo, T1017) was used to detect the SEAP levels in the serum of mice in all groups.

[0247] Table 12 Relative expression levels of SEAP in mouse serum after single administration of siRNA conjugates

[0248]

[0249] The results of Example 6 showed that compared with the reference sequence RZ003088, the NM054-modified RZ003080 had a comparable or higher inhibitory effect on SEAP protein expression in mouse serum at each time point, and exhibited a longer-term protein inhibition level. Figure 6 , Table 12)

[0250] Example 7. Inhibition of SEAP protein in serum by BALB / c-SEAP containing the sequence NM054 at the 3' end of the antisense strand Activity evaluation

[0251] The mouse model of SEAP reporter gene used in this example was used to evaluate the effect of the conjugate RZ003076 containing NM054 at positions 20 and 21 of the antisense strand and the reference sequence RZ003090 on the relative expression of SEAP in serum.

[0252] The construction of the BALB / c-SEAP model is shown in Example 4. Female BALB / c-SEAP stable mice were grouped according to SEAP levels, with 5 mice in each group. Each test group was given a predetermined dose of drug and a saline control group was added. RZ003076 and RZ003090 sequences were administered once by subcutaneous injection in the abdomen. The drug dose for all mice was calculated based on body weight, the administration volume was 10 mL / kg (mouse body weight), and the administration dose was 3 mg / kg (calculated as siRNA / mouse body weight). The saline control group was given 10 mL / kg (mouse body weight) of saline solution without siRNA conjugate. The serum of mice in the saline group and the RZ003076 and RZ003090 administration groups was collected before administration (pre-dose) and at D7, D14, D21, D28, and D35 after administration. Phospha-Light TM The SEAP reporter gene detection system (Thermo, T1017) was used to detect the SEAP levels in the serum of mice in all groups.

[0253] Table 13 Relative expression levels of SEAP in mouse serum after single administration of siRNA conjugates

[0254]

[0255] The results of Example 7 showed that compared with the reference sequence RZ003090, the NM054-modified RZ003076 had a comparable or higher inhibitory effect on SEAP protein expression in mouse serum at each time point, and exhibited a longer-term protein inhibition level. Figure 7 , Table 13)

[0256] Example 8. Evaluation of the inhibitory activity of the NM118 modified sequence at the 3' end of the antisense strand on ANGPTL3 in mice

[0257] In this example, the in vivo target gene inhibitory activity evaluation method for mice was used to evaluate the inhibitory activity of the conjugate RZ597174 containing the NM118 group at positions 20 and 21 of the ANGPTL3 target siRNA antisense strand and the conjugate RZ597114 not containing the NM118 group on the target gene ANGPTL3 in mice.

[0258] 6-8 week old C57BL / 6j mice were randomly divided into 3 groups according to body weight, 5 mice in each group, and the above siRNA conjugate was administered subcutaneously in the abdomen. The dose of each mouse in the PBS control group was 5 mL / kg, and the dose of each mouse in the siRNA conjugate experimental group was 1 mg / kg (in terms of siRNA) and the administration volume was 5 mL / kg. The day of administration was recorded as D0, and the mice were killed on D7 after administration. The animals were dissected, and the liver tissue was collected and cut into several 2 mm 3The small pieces were preserved with RNAlater. RNA extraction, reverse transcription reaction, and Real-time PCR detection were as described above, and the gene expression difference was calculated by the ΔΔCt method. The primers were as shown in Example 3.

[0259] Table 14 Inhibitory activity of target genes in mice after administration of the siRNA conjugates described in Example 8

[0260] Group Average % inhibition rate ±STDEV value PBS 0.00 16.94 RZ597114 69.60 8.99 RZ597174 64.73 5.37

[0261] The results of Example 8 showed that the sequence RZ597174 containing the NM118 group at the 3' end of the antisense strand had slightly lower inhibitory activity than the reference sequence RZ597114 ( Figure 8 , Table 14).

[0262] The above specific implementations are only illustrative of the content of the present disclosure and do not represent limitations of the content of the present disclosure. For those skilled in the art, various modifications and improvements can be made without departing from the spirit and essence of the present disclosure, and these modifications and improvements are also considered to be within the scope of protection of the present disclosure.

Claims

1. A nucleotide dimer, characterized in that The nucleotide dimer is selected from the structure shown in Formula I, or a pharmaceutically acceptable salt thereof: wherein Base1 and Base2 are independently selected from the nucleotide bases A, U, G, C, T and their derivatives; P1 is selected from H or a hydroxyl protecting group, wherein the hydroxyl protecting group is selected from trityl, 4-methoxytrityl, 4,4'-dimethoxytrityl or 4,4',4"-trimethoxytrityl; P2 is selected from H or a phosphorus-containing leaving group, the phosphorus-containing leaving group having the structural formula Among them, R 2a Selected from secondary amino groups, R 2b Selected from secondary amino or cyano-substituted C1-C3 alkoxy; The 2' position of one nucleotide ribose group in the nucleotide dimer is substituted by R1 and R2, and the 2' position of the other nucleotide ribose group is substituted by R3 and R4, and the 2' substituents of the two nucleotide ribose groups are the same or different; R1, R2 and R3, R4 are independently selected from halogen, C1-C3 alkyl or C1-C3 alkoxy.

2. The nucleotide dimer according to claim 1, characterized in that The hydroxyl protecting group is selected from 4,4'-dimethoxytrityl; The phosphorus-containing leaving group is selected from 3. The nucleotide dimer according to claim 2, characterized in that: The nucleotide dimer is selected from the structure shown in Formula II, or a pharmaceutically acceptable salt thereof: R1 and R2 are the same or different, and R1 and R2 are independently selected from F, -CH3, -CH2CH3; R3 and R4 are the same or different, and R3 and R4 are independently selected from F, -CH3, and -CH2CH3.

4. The nucleotide dimer according to claim 3, characterized in that R1 and R2 are different, and R1 and R2 are selected from F and -CH3 respectively; R3 and R4 are different, and R3 and R4 are selected from F and -CH3 respectively.

5. The nucleotide dimer according to claim 4, characterized in that The nucleotide dimer is selected from the structure shown in Formula III, or a pharmaceutically acceptable salt thereof: wherein Base1 and Base2 are independently selected from A, U, G or C; Further, Base1 and Base2 form a combination, and the combination is selected from AA, UU, GG, CC, AU, AG, AC, UG, UC or GC.

6. The nucleotide dimer according to claim 5, characterized in that The nucleotide dimer is selected from the following structures, or a pharmaceutically acceptable salt thereof: Ra is selected from amino protecting groups.

7. The method for preparing a nucleotide dimer according to any one of claims 1 to 6, characterized in that: The method comprises the following steps: The synthetic route is as follows: Wherein, Base1, Base2, R1, R2, R3, and R4 are defined as in claim 1; S1: Compound 1 and Compound 2 were dissolved in dichloromethane, 5-benzylthiotetrazolyl (5-BTT) was added, and the mixture was stirred at room temperature for 2-3 hours. The organic phase of the reaction solution was dried and concentrated, and purified to obtain Compound 3; S2: dissolving compound 4 in tetrahydrofuran, adding tetrabutylammonium fluoride solution to remove the protecting group, drying and concentrating the organic phase of the reaction solution, and purifying to obtain compound 4; S3: Compound 4 was dissolved in dichloromethane, bis(diisopropylamino)(2-cyanoethoxy)phosphine and 4,5-dicyanoimidazole were added, and the mixture was stirred at room temperature for 2-3 hours. The organic phase of the reaction solution was dried and concentrated, and purified to obtain the target compound.

8. Use of the nucleotide dimer according to any one of claims 1 to 6 in the preparation of nucleic acid drugs.

9. siRNA, characterized in that The siRNA is prepared using at least one nucleotide dimer according to any one of claims 1-6.

10. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the siRNA according to claim 9 and pharmaceutically acceptable excipients.

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