Nucleotide dimers, their preparation methods and uses

Abstract

This disclosure provides a nucleotide dimer, its preparation method, and its uses, belonging to the field of nucleic acid drug technology. The nucleotide dimer can be synthesized as a whole into a nucleotide chain. Compared to synthesizing nucleotides one by one through two steps, the synthesis method using the nucleotide dimer provided in this disclosure involves fewer steps, higher synthesis efficiency, less impurities, and higher product purity, showing promising application prospects.

Description

Technical Field

[0001] This disclosure relates to the field of nucleic acid drug technology, specifically to a nucleotide dimer and its preparation method and uses. Background Technology

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

[0003] Oligonucleotide drugs are single- 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. Currently, most oligonucleotide drugs are synthesized using a solid-phase phosphoramide chemical method. Chemical synthesis proceeds along 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 generated by phosphate thioester (PS) bonds, PO impurities from oxidation, base depurination impurities, 2'-5' linker isomers, and sequence isomers. Moreover, the more advanced the synthesis steps, the more impurities are generated, especially in the last two nucleotide linking steps, the oxidation step, and the additional sulfidation step, which have significantly lower reaction efficiency and generate more impurities than the earlier nucleotide linking steps. These impurities not only reduce synthesis efficiency but also affect the efficacy of nucleic acid drugs. Therefore, improving synthesis efficiency and effectively reducing impurities are urgent problems to be solved in the preparation of nucleic acid drugs.

[0004] In view of this, this disclosure is hereby made. Summary of the Invention

[0005] To address the aforementioned technical problems, this disclosure provides a nucleotide dimer in which the nucleotide dimer is linked as a whole to the end of the chain during the solid-phase synthesis of oligonucleotide chains. Compared with coupling each nucleotide individually, the synthesis process provided by this disclosure has fewer steps, higher reaction efficiency, less impurities generated, and higher product purity.

[0006] This disclosure includes the following technical solutions:

[0007] In a first aspect, this 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] 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 derivative is selected from nucleotide bases substituted with one or more of the following substituents: halogen, C1-C3 alkyl, C1-C3 alkyloxy, or substituted iminoyl.

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

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

[0013] Optionally, the hydroxyl protecting group is selected from triphenylmethyl (Tr), 4-methoxytriphenylmethyl (MMTr), 4,4'-dimethoxytriphenylmethyl (DMTr), or 4,4',4”-trimethoxytriphenylmethyl (TMTr).

[0014] In specific embodiments of this disclosure, the hydroxyl protecting group is selected from 4,4'-dimethoxytriphenylmethyl (DMTr).

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

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

[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 specific embodiments of this disclosure, the phosphorus-containing leaving group is selected from...

[0021] In the nucleotide dimer, the 2' position of one nucleotide ribosome is replaced by R1 and R2, and the 2' position of the other nucleotide ribosome is replaced by R3 and R4. The substituents at the 2' positions of the two nucleotide ribosomes may be the same or different. R1, R2 and R3, R4 are each independently selected from halogens, C1-C3 alkyl groups, or C1-C3 alkoxy groups, such as F, -CH3, -CH2CH3, -CH2CH2CH3, -OCH3, and -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 this disclosure, R1 and R2 may be the same or different, and R1 and R2 are independently selected from F, -CH3, and -CH2CH3.

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

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

[0027] In some embodiments of this 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] Base1 and Base2 are independently selected from A, U, G or C.

[0031] In some embodiments of this 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 structures shown below, or pharmaceutically acceptable salts thereof:

[0033]

[0034] Ra is selected from amino protecting groups.

[0035] In specific embodiments of this disclosure, the amino protecting group is selected from one of the following:

[0036]

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

[0038] The synthesis route is as follows:

[0039]

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

[0041] S1: Compound 1 and Compound 2 were dissolved in dichloromethane, 5-benzylthiotetrazole (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: Compound 4 was dissolved in tetrahydrofuran, tetrabutylammonium fluoride solution was added to remove the protecting group, the organic phase of the reaction solution was dried and concentrated, and purified to obtain compound 4;

[0043] S3: Dissolve compound 4 in dichloromethane, add bis(diisopropylamino)(2-cyanoethoxy)phosphine and 4,5-dicyanimidazole, stir at room temperature for 2-3 hours, dry and concentrate the organic phase of the reaction solution, purify, and obtain the target compound.

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

[0045] Optionally, this disclosure provides the use of the nucleotide dimer in the preparation of small interfering RNA (siRNA), the antisense strand of which comprises at least one of the nucleotide dimers.

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

[0047] In a fourth aspect, this disclosure provides an 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 this 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 the nucleotide dimer is located at position 20-21 of the antisense strand, starting from the 5' end.

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

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

[0053] In a fifth aspect of this disclosure, a pharmaceutical composition is provided, characterized in that the pharmaceutical composition comprises the siRNA described in the fourth aspect of this disclosure, and pharmaceutically acceptable excipients.

[0054] Compared with the prior art, this disclosure has the following beneficial effects:

[0055] This disclosure provides a nucleotide dimer that can be used as a whole to synthesize nucleotide chains. Compared with the two-step individual coupling of nucleotides, the synthesis of nucleotide chains using nucleotide dimers provided in this disclosure has fewer steps, significantly improves synthesis efficiency by about 5%, generates fewer impurities, and yields products with higher purity, showing good application prospects. Attached Figure Description

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

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

[0058] Figure 3 To assess 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 dose of siRNA conjugate.

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

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

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

[0063] Figure 8To assess the inhibitory activity of the target gene in mice after administration of the siRNA conjugate described in Example 8. Detailed Implementation

[0064] The technical solutions of this disclosure will be clearly and completely described below with reference to the embodiments. Obviously, the described embodiments are only some embodiments of this disclosure, not all embodiments. Based on the embodiments of this disclosure, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this disclosure.

[0065] Terminology Explanation

[0066] In this disclosure, the terms “comprising” or “including” are open-ended expressions, meaning that they include the contents specified in this disclosure but do not exclude other contents.

[0067] In this disclosure, the terms “optionally,” “optionally,” or “optionally” generally refer to an event or condition that may, but may not, occur, and the description includes both cases in which the event or condition occurs and cases in which the event or condition does not occur.

[0068] In this 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 via the RISC pathway by forming an RNA-induced silencing complex (RISC). Specifically, siRNA directs the specific degradation of mRNA sequences through known RNA interference (RNAi) processes, inhibiting the translation of mRNA into amino acids and its conversion into proteins. For example, siRNA can regulate (e.g., inhibit) the expression of LPA in cells.

[0069] In this disclosure, the double-stranded oligonucleotide consists of two strands, one of which binds to the target sequence and is called the antisense strand or guide strand, and the other strand is called the sense strand or guest strand.

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

[0071] In this disclosure, the term "sense chain" refers to a chain of double-stranded oligonucleotides that includes a region substantially complementary to the region referred to herein as the antisense chain.

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

[0073] In this disclosure, the term "pharmaceutically acceptable acid addition salt" refers to a salt formed with an inorganic or organic acid that retains the bioavailability of the free base without other side effects. Inorganic acid salts include, but are not limited to, hydrochlorides, hydrobromic acid sulfates, nitrates, and phosphates; organic acid salts include, but are not limited to, formates, acetates, 2,2-dichloroacetate, trifluoroacetate, propionate, hexanoate, octanoate, undecenoate, glycolate, gluconate, lactate, sebate, adipate, glutarate, malonate, oxalate, maleate, succinate, fumarate, tartrate, citrate, palmitate, stearate, oleate, cinnamate, laurate, malate, glutamate, pyroglutamate, aspartate, benzoate, methanesulfonate, benzenesulfonate, p-toluenesulfonate, alginate, ascorbate, salicylate, and 4-aminosalicylic acid. These salts can be prepared by methods known in the art.

[0074] In this disclosure, the term "pharmaceutically acceptable base addition salt" refers to a salt formed with an inorganic or organic base that retains the bioavailability of the free acid without other side effects. Salts derived from inorganic bases include, but are not limited to, sodium, potassium, thiocyanate, calcium, magnesium, iron, zinc, copper, manganese, and aluminum salts. Preferred inorganic salts are ammonium, sodium, potassium, calcium, and magnesium salts, with sodium salts being the most preferred. Salts derived from organic bases include, but are not limited to, the following: primary amines, secondary amines, and tertiary amines; substituted amines, including naturally occurring 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 this disclosure, the term "alkyl" refers to a saturated aliphatic hydrocarbon group, and non-limiting examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, and their various branched isomers. In some embodiments, the alkyl group selected is an alkyl group containing 1 to 6 carbon atoms. Non-limiting examples 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, etc. By way of example only, "C1-4 alkyl" means having one to four carbon atoms in an 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 can be substituted or unsubstituted.

[0076] In this disclosure, the term "alkoxy" refers to -O-alkyl, wherein the definition of alkyl is as shown above. Non-limiting examples of said alkoxy groups include: methoxy, ethoxy, propoxy, butoxy, cyclopropoxy, cyclobutoxy, cyclopentoxy, and cyclohexyloxy. By way of example only, "C..." 1-10 "alkylene" indicates that the alkyl chain has one to ten carbon atoms, i.e., the C atoms. 1-10 The alkylene group is selected from methylene (-CH2-), ethylene (=CH2CH3), 1,2-ethylene (-CH2CH2-), n-propylene (-CH2CH2CH2-), isopropylene (-CH2CH(CH3)-), etc.

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

[0078] In this disclosure, the term "halogen" or "halogenated" refers to any radioactive-stable atom in Group VII of the periodic table, such as fluorine, chlorine, bromine, or iodine, wherein fluorine and chlorine are preferred, and fluorine is more preferred.

[0079] In this disclosure, the term "link," when referring to a connection between two molecules, means that the two molecules are linked by a covalent bond or by a non-covalent bond (e.g., a hydrogen bond or an ionic bond), including direct and indirect links. The term "direct link" refers to the connection between a first compound or group and a second compound or group without any intercalating atoms or groups. The term "indirect link" refers to the connection between a first compound or group and a second compound or group via 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 that may be unsubstituted or substituted.

[0081] In this disclosure, the term "unsubstituted" means that the specified group does not contain substituents.

[0082] In this disclosure, the terms “substituted,” “replaced,” and “substituted” are used interchangeably to indicate that any one or more hydrogen atoms in the given structure are specifically substituented (e.g., C). 1-3 Alkyl, C 1-3 The substituted group may be replaced by an alkoxy or halogen group, provided that the normal valence of the specified atom does not exceed the valence of the substituted atom and the substitution produces a stable compound. Unless otherwise indicated, a substituted group may have one substituent at each substituted position of the group. When more than one position in the given structural formula can be substituted by one or more substituents selected from a particular group, then the substituents may be substituted at each substituted position in the same or different manner.

[0083] In this disclosure, the structural formulas of "compound", "ligand" and "support" contain bonds. This indicates that the configuration is not specified. If chiral isomers exist in the chemical structure, the bond... It can be Or simultaneously include Two configurations. Although all the above structural formulas are shown in some isomer forms for simplicity, this disclosure can include all isomers, such as tautomers, rotational isomers, geometric isomers, diastereomers, racemates, and enantiomers.

[0084] In this disclosure, "conjugation" refers to the connection between two or more chemical parts through covalent linkage; "conjugated compound" refers to a compound formed by the covalent linkage between the chemical parts; and "conjugated molecule" is understood as a specific compound that can be reactively conjugated to an oligonucleotide to ultimately form the oligonucleotide conjugated compound of this disclosure.

[0085] In this disclosure, "pharmaceutical composition" can refer to a drug for the treatment of a disease or for use in in vitro cell culture experiments. When used for the treatment of a disease, the term "pharmaceutical composition" generally refers to a unit dose form and can be prepared by any method well known in the pharmaceutical industry. All methods involve the step of combining the active ingredient with excipients constituting one or more adjunct components. Typically, the composition is prepared by uniformly and adequately combining active siRNA with liquid excipients, finely pulverized solid excipients, or both.

[0086] In this disclosure, the term "pharmaceutical acceptable" means that a substance or composition must be chemically and / or toxicologically compatible with other components of the formulation and / or the mammals to which it is treated. Preferably, "pharmaceutical acceptable" as used in this disclosure means approved by a federal regulatory agency or national government, or listed in the United States Pharmacopeia or other generally recognized pharmacopoeia for use in animals, particularly in humans.

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

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

[0089] In this disclosure, the terms “treatment,” “relief,” or “improvement” are used interchangeably. These terms refer to methods of achieving beneficial or desired outcomes, including, but not limited to, treatment benefits. A “treatment benefit” means the eradication or improvement of the underlying disorder being treated. Here, a treatment benefit is achieved by eradicating or improving one or more physical symptoms associated with the underlying disorder, thereby observing improvement in the subject, although the subject may still be suffering from the underlying disorder.

[0090] In this disclosure, the terms “prevention” and “avoidance” are used interchangeably to refer to methods for obtaining beneficial or desired results, including but not limited to preventive benefits. To obtain a “preventive benefit,” the conjugate, RNAi reagent, or composition may be given to a subject at risk of developing a specific disease, or to a subject who reports one or more physiological symptoms of a disease, even if a diagnosis of the disease may not have been made.

[0091] In addition to any conventional excipients, the use of any siRNA incompatible with the present disclosure, such as any adverse biological effects produced or interactions with any other component of a pharmaceutically acceptable composition in a harmful manner, is also within the scope of this disclosure.

[0092] The present disclosure is further illustrated below with specific embodiments. However, it should be understood that these embodiments are merely for the purpose of more detailed illustration and should not be construed as limiting the present disclosure in any way.

[0093] Preparation of compounds

[0094] Unless otherwise stated, the siRNA sequences used in this disclosure were synthesized by Suzhou Xuanjing Biotechnology Co., Ltd. and Suzhou Beixin Biotechnology Co., Ltd.; the PCR primers used in this disclosure were synthesized by Beijing Qingke Biotechnology Co., Ltd.; and the experimental animals, C57BL / 6J mice, used in this disclosure were purchased from Spiford (Beijing) Biotechnology Co., Ltd. Information on the main reagents, consumables, and instruments 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 automated nucleic acid extractor Zhejiang Hanwei Technology Co., Ltd. High-speed refrigerated centrifuge Eppendorf NANODROP OneC Thermo Fisher Scientific Gradient PCR Amplification Instrument Eppendorf Real-time PCR instrument ABI StepOne Plus Real-time PCR instrument Bio-Rad CFX Opus 384 Gel Imaging Shanghai Tianneng Life Science Co., Ltd. Electrophoresis apparatus Beijing Liuyi Instrument Factory Tissuelyser II Fully Automated Tissue Homogenizer Shanghai Jingxin Industrial Development Co., Ltd. R540IE Small Animal Anesthesia Machine Shenzhen Ruiwode Life Technology Co., Ltd.

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

[0100] In this preparation example, the synthetic routes for compounds NM054 and NM142 are shown below:

[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'-methylurea, CAS No. 863329-66-2) was dissolved in pyridine (100 ml). 4,4'-bismethoxytriphenylmethylchloro (16.9 g, 1.3 eq, abbreviated as DMTrCl, CAS No. 40615-36-9) was added in an ice bath. The mixture was stirred at room temperature under a nitrogen atmosphere for 3 hours until the reaction was complete. The reaction solution was concentrated and purified by reversed-phase chromatography (elution: acetonitrile / water = 70 / 30, v / v) to give 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-dicyanimidazole (1.72 g, 0.8 eq, abbreviated as DCI, CAS No. 1122-28-7) were added. The mixture was stirred at room temperature for 2 hours until the reaction was complete. A saturated sodium bicarbonate aqueous solution was added to the reaction solution, and the mixture was extracted and separated. The organic phase was dried and concentrated, and purified by reversed-phase chromatography (elution: acetonitrile / water = 95 / 5, v / v) to give 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-butyldimethylchlorosilane (4.13 g, 1.5 eq, abbreviated as TBDMSCl, CAS No. 18162-48-6) were added sequentially. The reaction was carried out at room temperature for 8 hours until completion. 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 five times with saturated sodium chloride aqueous solution (100 ml each time), concentrated, and purified by normal phase chromatography (eluent: petroleum ether / ethyl acetate = 4 / 1, v / v) to give 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), and p-toluenesulfonic acid (5.6 g, 2.0 eq, CAS No. 104-15-4) was added. The mixture was stirred at room temperature under a nitrogen atmosphere for 3 hours until the reaction was complete. The reaction solution was washed three times with saturated sodium bicarbonate aqueous solution (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 give 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] Compounds NM054 (5 g, 1 eq) and NM142-2 (3.68 g, 1.5 eq) were dissolved in anhydrous dichloromethane (100 mL), and 5-benzylthiotetrazole (1.89 g, 1.5 eq, CAS No. 21871-47-6) was added. The mixture was stirred at room temperature for 2 hours until the reaction was complete. The reaction solution was washed with saturated sodium bicarbonate aqueous solution, the organic phase was dried and concentrated, and purified by reversed-phase chromatography (elution: acetonitrile / water = 95 / 5, v / v) to give 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 until the reaction was complete. Ethyl acetate (200 mL) was added to the reaction solution, and the mixture was washed five times with purified water (50 mL each time). The organic phase was dried and concentrated, and purified by reversed-phase chromatography (eluent: acetonitrile / water = 90 / 10, v / v) to give 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-dicyanimidazole (0.2 g, 0.8 eq) were added. The mixture was stirred at room temperature for 2 hours until the reaction was complete. A saturated sodium bicarbonate aqueous solution was added to the reaction mixture, and the mixture was extracted and separated. The organic phase was dried and concentrated, and purified by reversed-phase chromatography (elution: acetonitrile / water = 95 / 5, v / v) to give 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 synthetic 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), and 1,3-dichloro-1,1,3,3-tetraisopropyldisiloxane (28.6 g, 1.2 eq, TiPDSCl2, CAS No. 69304-37-6) was added. The mixture was stirred at room temperature for 12 hours until the reaction was complete. The reaction solution was concentrated, and water (100 ml) was added. The mixture was extracted twice with ethyl acetate (100 ml each time). The organic phase was dried and concentrated, and purified by normal phase (elution: ethyl acetate / petroleum ether = 34 / 66, v / v) to give 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). A 60 wt% NaH solution (1.6 g, 4 eq) in tetrahydrofuran was added under ice bath conditions. The mixture was purged with nitrogen three times and stirred under ice bath conditions for 1 hour. Iodomethane (3 g, 2 eq) was then added, and the reaction was carried out at room temperature for 4 hours until completion. A saturated ammonium chloride aqueous solution (20 ml) was added to the reaction solution, and the mixture was extracted three times with ethyl acetate (20 ml each time). The organic phase was dried and concentrated, and then purified by reverse-phase chromatography (elution: acetonitrile / water = 25 / 75, v / v) to obtain a white powder, compound NM118-3 (3 g). 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 until the reaction was complete. Water (20 mL) was added to the reaction solution, and the mixture was extracted three times with ethyl acetate (20 mL each time). The organic phase was dried and concentrated, and then purified by reverse-phase extraction (elution: acetonitrile / water = 36 / 64, v / v) to give 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'-bismethoxytriphenylmethyl chloride (1.7 g, 1.3 eq) was added under ice bath conditions. The mixture was purged with nitrogen three times, stirred at room temperature for 3 hours, and then quenched with methanol (20 mL). The reaction was complete. The reaction solution was concentrated, and water (20 mL) was added. The mixture was extracted three times with ethyl acetate (20 mL each time). The organic phase was dried and concentrated, and purified by normal phase purification (elution: ethyl acetate / petroleum ether = 50 / 50, V / V) to give compound NM118-5 (1.8 g) as a pale 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-dicyanimidazole (264.4 mg, 0.8 eq) and bis(diisopropylamino)(2-cyanoethoxy)phosphine (0.92 g, 1.1 eq) were added separately. The mixture was purged with nitrogen three times, and stirred at room temperature for 2 hours until the reaction was complete. A saturated aqueous solution of sodium bicarbonate (20 mL) was added to the reaction solution, and the mixture was extracted three times with dichloromethane (20 mL each time). The organic phase was dried and concentrated, and purified by reverse-phase chromatography (elution: acetonitrile / water = 75 / 25, v / v) to give a white powder, compound NM118 (1.7 g, 78.7% yield). 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 compounds CR01008 and CR01008Z:

[0131] In this preparation example, the synthetic routes for compounds CR01008 and 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)cyclohexylformaldehyde, CAS No. 181308-57-6) and a 37 wt% formaldehyde aqueous solution (8.9 g, 2.4 eq) were dissolved in methanol (33 ml). A 45.3 wt% KOH aqueous solution (13 ml) was added dropwise. After the addition was complete, the mixture was stirred at 25 °C for 30 minutes, then heated to 60 °C and refluxed at 60 °C for 2 hours. The reaction was then complete. After the reaction solution cooled to room temperature, it was evaporated under reduced pressure to obtain a white solid crude product. A small amount of water was added to the crude product to form a slurry, which was then filtered to obtain a white solid compound CR01008-2 (9 g, yield 78.9%). 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 4 M solution of 1,4-dioxane hydrogen chloride (45 ml) was added. The mixture was stirred at 25 °C for 1 hour until the reaction was complete. The reaction solution was evaporated under reduced pressure to obtain a white solid compound CR01008-3 (6.8 g, 100% yield).

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

[0138] Compounds CR01008-3 (1.8 g, 2.0 eq), CR01008-4 (2.1 g, 1.0 eq, 5-[[(2R,3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)-2-tetrahydropyranyl]oxy]valeric 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). Benzotriazole-N,N,N',N'-tetramethylurea 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 for 3 hours under a nitrogen atmosphere until the reaction was completed. The reaction mixture was evaporated to dryness under reduced pressure, and purified by reverse-phase extraction (elution buffer: acetonitrile / water = 22 / 78, v / v) to give a white solid, CR01008-5 (1.78 g, yield 64.4%). 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 using an ice-water bath, and DMTrCl (1.32 g, 1.5 eq) was added at 0 °C. The reaction was carried out at 25 °C for 3 hours, and then quenched with methanol (15 mL). The reaction was complete. The reaction solution was evaporated to dryness under reduced pressure, and purified by reverse-phase chromatography (elution: acetonitrile / water = 60 / 40, v / v) to give 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). 4,5-Dicyanoimidazole (115 mg, 0.8 eq, abbreviated 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 separately. The mixture was purged with nitrogen three times, and the reaction system was stirred at 25 °C for 2 hours under a nitrogen atmosphere until the reaction was complete. A saturated sodium bicarbonate aqueous solution (20 ml) was added to the reaction solution, and the mixture was extracted three times with dichloromethane (20 ml each time). The organic phases were combined, dried, and purified by reverse-phase extraction (elution: acetonitrile / water = 72 / 28, v / v) to obtain a white powder, compound CR01008 (1 g, yield 76.0%). 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, abbreviated as DMAP, CAS No. 1122-58-3), and triethylamine (226.2 mg) were added. The mixture was purged with nitrogen three times, and the reaction system was stirred at 25 °C under a nitrogen atmosphere for 16 hours until the reaction was complete. The reaction solution was purified by FLASH to give 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), aminoCPG (1.25 g, 0.1 mmol), HBTU (27 mg), and DIEA (12 mg) were added to a 20 ml sample vial. The mixture was reacted on a shaker for 16 hours until the reaction was complete. The reaction solution was filtered, and the filter cake was washed once with acetonitrile (10 ml) and then dried under vacuum. The dried filter cake, DMAP (3 mg), Cap1 (10 ml), and Cap2 (1 ml) were added to a 20 ml sample vial. The mixture was reacted on a shaker for 6 hours until the reaction was complete. The reaction solution was filtered, and the filter cake was washed once with 10 ml of acetonitrile and then dried under vacuum to obtain compound CR01008Z (1.03 g, loading 20-30 μmol / g).

[0148] The structural formula of amino-CPG (Aminoalkyl-CPG) is as follows: Model C3006-1000, 100-200 mesh, loading 80μmol / g.

[0149] Cap1 and Cap2 are capping reagents. Cap1 is a 20 vol% N-methylimidazolium pyridine / acetonitrile mixed solution with a pyridine to acetonitrile volume ratio of 3:5. Cap2 is a 20 vol% acetic anhydride acetonitrile solution.

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

[0151] Compound L96-PS was purchased from Asymchem Laboratories (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] PS represents polystyrene resin solid carrier.

[0154] (1-1) Composition of the Justice Chain (SS) and Antisense Chain (AS):

[0155] The phosphoramide solid-phase synthesis method for nucleic acids involves starting a cycle using compounds linked to a solid support (e.g., CPG support, PS support, compound L96-PS, compound CR01008Z), and sequentially linking nucleoside monomers one by one along the nucleotide sequence from the 3' end to the 5' end. During the synthesis, compounds CR01008, NM054, NM142, and NM118 are each considered as a nucleoside monomer.

[0156] Each connection of a nucleoside monomer involves four steps: deprotection, coupling, capping, and oxidation or sulfidation. The synthetic conditions are given below:

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

[0158] The deprotection reaction conditions were the same for each step. The deprotection reaction conditions were: temperature 25℃, reaction time 70 seconds, deprotection reagent was a dichloromethane solution of dichloroacetic acid (3% by volume), and the molar ratio of dichloroacetic acid to the 4,4'-dimethoxytriphenylmethyl protecting group on the solid support was 5:1.

[0159] The conditions for each coupling reaction were identical. The coupling reaction conditions were as follows: temperature 25℃, molar ratio of nucleic acid sequence to nucleoside monomer on the solid-phase support 1:10, molar ratio of nucleic acid sequence to coupling reagent on the solid-phase support 1:65, reaction time 600 seconds, coupling reagent 0.5M acetonitrile solution of 5-ethylthio-1H-tetrazole, and thioreagent 0.2mol / L acetonitrile / pyridine mixed solution of hydrogenated xanthanin (acetonitrile and pyridine volume ratio 1:1).

[0160] The conditions for each capping reaction were identical. The conditions for the capping reaction were: temperature 25℃; reaction time 2 minutes; the capping reagent solution was a 1:1 molar ratio of Cap1 and Cap2, Cap1 being a 20% (v / v) N-methylimidazole pyridine / acetonitrile mixture with a pyridine to acetonitrile volume ratio of 3:5, and Cap2 being a 20% (v / v) acetic anhydride acetonitrile solution; the molar ratio of N-methylimidazole in Cap1 and acetic anhydride in Cap2 to the nucleic acid sequence linked on the solid-phase support was 1:1:1.

[0161] The conditions for each oxidation reaction were identical. The oxidation reaction conditions were: temperature 25°C; reaction time 3 seconds; oxidizing agent concentration of 0.05M iodine solution, with a molar ratio of iodine to the nucleic acid sequence linked on the solid support in the coupling reaction of 30:1; the oxidation reaction was carried out in a water / pyridine mixed solvent (water to pyridine volume ratio 1:9). The sulfidation reaction conditions were: temperature 25°C; reaction time 360 ​​seconds; thioreagent concentration of 0.2M hydroflavin in pyridine solution, with a molar ratio of thioreagent to the nucleic acid sequence linked on the solid support in the coupling reaction of 4:1; the thioreagent reaction was carried out in a water / pyridine mixed solvent (water to pyridine volume ratio 1:9).

[0162] After the last nucleoside monomer was ligated, the nucleic acid sequence ligated on the solid-phase support was sequentially cut, deprotected, purified, and desalted, and then freeze-dried to obtain the positive strand, wherein:

[0163] The cleavage and deprotection conditions were as follows: The synthesized nucleotide sequence linked to a solid-phase support was added to 25% (w / w) ammonia solution at a concentration of 0.5 mL / μmol. The reaction was carried out at 55 °C for 16 hours. The solvent was removed, and the solution was concentrated to dryness under vacuum. After ammonia treatment, the product was dissolved in 0.4 mL / μmol N-methylpyrrolidone relative to the amount of single-stranded nucleic acid. Subsequently, 0.3 mL / μmol triethylamine and 0.6 mL / μmol triethylamine trifluoride were added to deprotect the 2'-O-TBDMS protection on the ribose.

[0164] Purification and desalting conditions: Nucleic acid purification was performed using a preparative ion chromatography column (Source 15Q) with a NaCl gradient elution. Specifically: eluent 1 was 20 mM sodium phosphate (pH = 8.1), and the solvent was a water / acetonitrile mixture (water to acetonitrile volume ratio of 9:1); eluent 2 was 1.5 M sodium chloride, 20 mM sodium phosphate (pH = 8.1), and the solvent was a water / acetonitrile mixture (water to acetonitrile volume ratio of 9:1); the elution gradient was eluent 1: eluent 2 = (100:0) - (50:50). The product eluates were collected and combined, and desalting was performed using a reverse chromatographic purification column. Desalting conditions included using a dextran gel column (g25 dextran gel) and elution with deionized water.

[0165] Detection: Purity was determined using ion exchange chromatography (IEX-HPLC); molecular weight was determined using liquid chromatography-mass spectrometry (LC-MS). The measured molecular weight was compared with the theoretical value. If the measured value and the theoretical value were consistent, it indicated that the sense strand and the target antisense strand had been obtained.

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

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

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

[0169]

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

[0171] in, This indicates siRNA.

[0172] The siRNA conjugates prepared in this disclosure are shown in Table 4, and 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 stated, the base composition and modifications used in this disclosure have the following meanings: uppercase letters A, U, G, C, and T represent the base composition of a nucleotide; lowercase letter d indicates that the nucleotide represented by the uppercase letter to its left is a nucleotide modified with 2'-deoxy; lowercase letter m indicates that the nucleotide represented by the uppercase letter to its left is a nucleotide modified with 2'-O-Me; lowercase letter f indicates that the nucleotide represented by the uppercase letter to its left is a nucleotide modified with 2'-F; (moe) indicates that the nucleotide represented by the uppercase letter to its left is a nucleotide modified with 2'-O-methoxyethyl; lowercase letter s indicates that the nucleoside bond between two adjacent nucleotides on its left and right sides is a phosphate thioester bond.

[0179] (NM054) represents a nucleotide obtained by the synthesis of siRNA conjugates using the compound NM054. The structural formula of (NM054) is as follows:

[0180] (NM054)s(NM054) represents two nucleotides, obtained by the synthesis of an siRNA conjugate using compound NM142. The structural formula of (NM054)s(NM054) is as follows:

[0181] (NM118) represents a nucleotide obtained by synthesizing an siRNA conjugate containing the compound NM118. The structural formula of (NM118) is as follows:

[0182] Biological testing experiments

[0183] Unless otherwise stated, all reagents, consumables and instruments used in biological testing experiments in this disclosure are commercially available products.

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

[0185] Isolation of primary mouse hepatocytes:

[0186] Primary hepatocytes were extracted from fresh liver tissue of C56BL / 6j mice. The specific procedure involved anesthetizing the mice with an intraperitoneal injection of 10% chloral hydrate solution, fixing the mice, and disinfecting their abdomen and chest with 75% ethanol. Surgical instruments were sterilized, and the abdominal cavity was opened to expose the portal vein and inferior vena cava. A heparin cap was attached to an indwelling needle, which was then connected to a scalp vein needle attached to an infusion pump vial (0.5 mM EDTA HBSS perfusion solution). The needle was inserted through the inferior vena cava, and perfusion was performed at a rate of 120 drops / min. The portal vein was then cut open to allow the perfusion solution to flow out. Perfusion continued for 4 minutes, followed by replacement with 0.8 mg / mL type IV collagenase HBSS solution (Sigma, C5138) (containing 0.08% DN1 enzyme (Sigma, DN25)) for another 8 minutes. The perfused liver was removed from the animal and washed with HBSS (containing Ca2+, Mg2+, MACGENE, CC016). The liver was placed in a sterile culture dish, and DMEM complete medium (DMEM medium + 10% serum) was added to shred the liver. The cell suspension was filtered through a cell sieve to remove undigested tissue and connective tissue. The liver was centrifuged at 800 rpm for 3 min and the supernatant was discarded. The liver was then resuspended in DMEM complete medium and centrifuged again to obtain primary mouse hepatocytes.

[0187] Cell culture and transfection:

[0188] Add DMEM complete medium to adjust cell density to 2×10⁶ 5 Primary mouse hepatocyte suspension was obtained by measuring cells / mL. The cells were then seeded into 12-well culture plates pre-coated with type I rat tail collagen (coating method according to SolarBio (C8062) instructions at 2 μg / mL). 2(Concentration coating), the volume of cell suspension added was 1000 μL / well, i.e., the cell quantity was 2 × 10⁻⁶. 5 Cells / pores.

[0189] Dilute each conjugate to a final experimental concentration 1000 times with PBS (based on siRNA). Add 1 μL / well of the siRNA conjugate working solution to each of the above 12-well culture plates, with 2-3 wells for each siRNA conjugate. Add 1 μL / well of PBS to the remaining 2-3 wells as blank control wells. Shake the culture plate to mix thoroughly. Incubate the culture plate in a cell culture incubator at 37°C and 5% CO2 for 24 hours.

[0190] RNA extraction: Total RNA was extracted from each group of primary hepatocyte samples using a fully automated nucleic acid extractor and nucleic acid extraction kit from Zhejiang Hanwei Technology Co., Ltd., following the method described in the instruction manual.

[0191] Methods for assessing the inhibitory activity of siRNA conjugates on target genes in mice

[0192] Six- to eight-week-old C57BL / 6J mice (all female) were randomly divided into groups according to body weight. The drug dosage for each group was calculated based on body weight and administered via a single subcutaneous injection. Each siRNA conjugate was prepared with PBS solution to the corresponding concentration (based on siRNA) for administration, at a volume of 5 ml (based on siRNA) / kg (based on mice). The PBS control group received 5 ml / kg (based on mice) of PBS solution (without drug conjugates). The day of administration was designated as day 0 (D0). At a predetermined time after administration, five mice from each group were sacrificed. The sacrificed mice were grossly dissected, and liver tissue was collected from each mouse. The liver tissue was cut into pieces approximately 2 mm in size. 3 Small pieces, stored using RNA Later.

[0193] Liver tissue samples were taken from different experimental groups at different time points from the RNA later sample. The liver tissue samples were homogenized in a Tissuelyser II fully automated tissue homogenizer for 60 seconds, and then total RNA was extracted using a fully automated 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 procedure for total RNA extraction.

[0194] mRNA expression level detection:

[0195] Take 1 μg of total RNA, and use a reverse transcription kit (Promega, Reverse Transcription System, A3500) with Oligo(dT)15 reverse transcription primers. Prepare a 20 μL reverse transcription system according to the instructions of the reverse transcription kit and complete the reverse transcription reaction. After the reaction, add 80 μL of RNase-free water to the reverse transcription system to obtain a cDNA solution. Then, use a real-time quantitative PCR kit (ABI, SYBR). TM Select MasterMix (Catalog number: 4472908) was used to detect the expression level of target gene mRNA. In this real-time quantitative PCR method, primers targeting the target gene and primers targeting the internal reference gene were used to detect the target gene and the internal reference gene, respectively. A 20 μL Real-time PCR reaction system was prepared for each PCR detection well according to the instructions of the real-time quantitative PCR kit. Each reaction system contained 5 μL of cDNA solution obtained from the above reverse transcription reaction and 10 μL of SYBR Green. TM SelectMaster Mix, 0.5 μL 10 μM upstream primer, 0.5 μL 10 μM downstream primer, and 4 μL RNase-Free H2O. Place the prepared reaction mixture in a real-time quantitative PCR instrument (ABI, StepOnePlus). TM Real-time PCR amplification was performed using a three-step method. The amplification program was 95℃ pre-denaturation for 10 min, followed by 95℃ denaturation for 30 s, 60℃ annealing for 30 s, and 72℃ extension 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 with Oligo(dT)18 reverse transcription primers. A 20 μL reverse transcription system was prepared according to the kit's instructions, and the reverse transcription reaction was completed. Then, a real-time quantitative PCR kit (Thermo Fisher Scientific, TaqMan Fast Advanced Master Mix, 4444557) was used on a quantitative PCR instrument (Bio-Rad, CFX Opus 384) to detect the expression level of the target gene mRNA. In this real-time quantitative PCR method, the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene was used as an internal control gene. Primers targeting the target gene and primers targeting the GAPDH internal control gene were used to detect the target gene and the GAPDH internal control gene, respectively.

[0197] In this real-time quantitative PCR method, the ΔΔCt method was used to calculate the relative quantitative levels and inhibition rates of target gene mRNA in each test group. The calculation method is as follows:

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

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

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

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

[0202] In cell experiments, ΔCt (control group mean) is the arithmetic mean of ΔCt (control group) values ​​from several replicates in the control group. In animal experiments, ΔCt (control group mean) is the arithmetic mean of ΔCt (control group) values ​​from each of the five mice sacrificed at the same time point in the control group. Therefore, each sample in both the test and control groups corresponds to a ΔCt value.

[0203] The relative expression level of the target gene mRNA in the test group was 2. -ΔΔCt( (Test group) × 100%

[0204] Using the control group as a baseline, the expression level of the target gene mRNA in the test group was normalized, and the expression level of the target gene mRNA in the control group was defined as 100%.

[0205] The inhibition rate (%) of target gene mRNA expression in the test group = 100% – the relative expression level of target gene mRNA in the test group

[0206] Unless otherwise stated, in vivo activity data are presented in [year]. It is noted that all experimental data were plotted and analyzed using GraphPadprism 8.0 software.

[0207] Example 1: Superoxide dismutation of target gene in mouse primary liver cells by the NM054 modification sequence at the 3' end of the antisense strand. Evaluation of the inhibitory activity of enzyme 1 (Superoxide dismutase 1, SOD1)

[0208] Example 1 uses the above-described experimental method for evaluating the inhibitory activity of primary mouse liver cells against the target gene SOD1 to evaluate the inhibitory activity of the antisense strand NM054 modified sequence RZ599034 and the reference sequence RZ599001 in primary mouse hepatocytes.

[0209] Primary mouse hepatocytes with a cell mass of 2 × 10⁻⁶ 5 Cells were seeded in 12-well plates. The double-stranded siRNA conjugates were serially diluted with PBS to 10 μM and 1 μM working solutions (based on siRNA). 1 μL of each concentration of siRNA conjugate working solution was added to each well of the 12-well plate, equivalent to a final transfection concentration of 10 nM and 1 nM (based on siRNA). Two to three wells were used for each siRNA concentration. An additional 2 to 3 wells contained 1 μL of PBS as blank control wells.

[0210] Table 5 Primer sequence information in Example 1

[0211]

[0212] Table 6 shows the inhibitory activity of the target gene in mouse primary liver cells after administration of the siRNA conjugate described in Example 1.

[0213]

[0214] The results of Example 1 indicate that the sequence RZ599034, modified by NM054 at position 20 of the antisense strand, exhibits stronger target gene repression than the reference sequence RZ599001. Figure 1 (Table 6)

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

[0216] In this embodiment, the inhibitory activity of the conjugate RZ599060 containing the NM054 group at position 20 of the antisense strand of the SOD1 target siRNA, the conjugate RZ599061 containing the NM054 group at positions 20 and 21, and the conjugate RZ599001 without the NM054 group were evaluated in mice using a mouse target gene inhibitory activity assessment method.

[0217] Six- to eight-week-old C57BL / 6j mice were randomly divided into four groups of 15 mice each, based on body weight. Each group received the aforementioned siRNA conjugate via subcutaneous abdominal administration. In the PBS control group, each mouse received 5 mL / kg of siRNA, while in the siRNA conjugate experimental group, each mouse received 3 mg / kg (based on siRNA) at a dose of 5 mL / kg. The day of administration was designated as day 0 (D0). Five mice from each group were sacrificed on days 7 (D7), 28 (D28), and 49 (D49). Gross dissection was performed, and liver tissue was collected and cut into several 2 mm segments. 3 The small fragments were preserved using RNAlater. RNA extraction, reverse transcription, and quantitative real-time PCR were performed as described above, and gene expression differences were calculated using the ΔΔCt method. Primers are shown in Table 5 of Example 1.

[0218] Table 7 shows the inhibitory activity of the target gene in mice after administration of the siRNA conjugate described in Example 2.

[0219]

[0220]

[0221] The results of Example 2 show that, compared with the control conjugate RZ599001 which does not contain the NM054 group, the conjugates RZ599060 and RZ599061 containing the NM054 group both exhibited higher inhibitory activity at D7, D28, and D49. At D49, the inhibitory activity of RZ599060 and RZ599061 was approximately 15% higher than that of the control conjugate RZ599001. Figure 2 (Table 7).

[0222] Example 3. The NM054 modification sequence at the 3' end of the antisense strand inhibits the target gene angiopoietin-like protein 3 in mice. Evaluation of the inhibitory activity of (angiopoietin-like3, ANGPTL3)

[0223] In this embodiment, the inhibitory activity of conjugates RZ597115 (containing the NM054 group at position 20 of the antisense strand of the ANGPTL3 target siRNA), RZ597116 (containing the NM054 group at positions 20 and 21), and RZ597114 (not containing the NM054 group) on the target gene ANGPTL3 in mice was evaluated using a mouse in vivo target gene inhibitory activity assessment method.

[0224] Six- to eight-week-old C57BL / 6j mice were randomly divided into four groups of 15 mice each, based on body weight. Each group received the aforementioned siRNA conjugate via subcutaneous abdominal administration. In the PBS control group, each mouse received 5 mL / kg of siRNA, while in the siRNA conjugate experimental group, each mouse received 3 mg / kg (based on siRNA) at a dose of 5 mL / kg. The day of administration was designated D0. Five mice from each group were sacrificed on D7, D28, and D56. The animals were grossly dissected, and liver tissue was collected and cut into several 2 mm sections. 3 The small fragments were preserved using RNAlater. RNA extraction, reverse transcription, and real-time PCR detection were performed as described above, and gene expression differences were calculated using the ΔΔCt method.

[0225] Table 8 Primer sequence information in Example 3

[0226]

[0227] Table 9 shows the inhibitory activity of the target gene in mice after administration of the siRNA conjugate described in Example 3.

[0228]

[0229] The results of Example 3 showed that, compared with the control conjugate RZ597114 which did not contain the NM054 group, the conjugates RZ597115 and RZ597116 containing the NM054 group exhibited higher inhibitory activity at D7, D28, and D56. At D56, the inhibitory activity of the conjugate RZ597115 containing the NM054 group at position 20 of the antisense chain was approximately 25% higher than that of the control conjugate RZ597114; the inhibitory activity of the conjugate RZ597116 containing NM054 at positions 20 and 21 of the antisense chain was approximately 35% higher than that of the control conjugate RZ597114. Figure 3 (Table 9)

[0230] Example 4. A sequence containing NM054 at the 3' end of the antisense strand was used to suppress SEAP protein in serum in a BALB / c-SEAP model. Evaluation of white's inhibitory activity

[0231] In this embodiment, 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 was evaluated in BALB / c-SEAP model mice.

[0232] The BALB / c-SEAP model was constructed by inserting the AGT transcript sequence into a transposon plasmid (Shanghai Langjing Biotechnology Co., Ltd.). 1.6 mL of plasmid mixture (25 μg of transposon plasmid fused with the AGT transcript and 25 μg of Super PiggyBac Transposase plasmid) was injected into mice via tail vein hydrodynamics within 3–5 seconds. A mouse model stably expressing the SEAP reporter gene was obtained 2 weeks later.

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

[0234] Female BALB / c-SEAP stable mice were divided into groups of five mice each according to their SEAP levels. Each test group received a predetermined dose of the drug, plus a saline control group. RZ003089 and RZ003075 sequences were administered via subcutaneous abdominal injection. The drug dosage was calculated based on body weight, with an administration volume of 10 mL / kg (mice body weight) and a dosage of 0.3 mg / kg (calculated as siRNA / mice body weight). The saline control group received 10 mL / kg (mice body weight) of saline solution without the siRNA conjugate. Serum was collected from mice in the saline group and the RZ003089 and RZ003075 administration groups on day 5 after drug administration. Phospha-Light was used to analyze the results. TM The SEAP reporter gene assay system (Thermo, T1017) was used to detect SEAP levels in the serum of mice in all groups.

[0235] Table 10. Relative expression levels of SEAP in the serum of mice after a single dose of siRNA conjugate.

[0236]

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

[0238] Example 5. The NM054 sequence at the 3' end of the antisense strand inhibits the effect of BALB / c-SEAP on serum SEAP protein. Activity evaluation

[0239] In this embodiment, a mouse model of the SEAP reporter gene 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 BALB / c-SEAP model was constructed as shown in Example 6. Female BALB / c-SEAP stable mice were divided into groups of five mice each according to their SEAP levels. Each test group received a predetermined dose of the drug, with a saline control group added. RZ003087 and RZ003079 sequences were administered via subcutaneous abdominal injection. The drug dosage for all mice was calculated based on body weight, with an administration volume of 10 mL / kg (mice body weight) and a dosage of 3 mg / kg (calculated as siRNA / mice body weight). The saline control group received 10 mL / kg (mice body weight) of saline solution without the siRNA conjugate. Serum samples were collected from mice in the saline group and the RZ003087 and RZ003079 administration groups before administration and at post-administration levels (D7, D14, D21, D28, and D35). Phospha-Light was used to analyze the serum. TM The SEAP reporter gene assay system (Thermo, T1017) was used to detect SEAP levels in the serum of mice in all groups.

[0241] Table 11. Relative expression levels of SEAP in the serum of mice after a single dose of siRNA conjugate.

[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 all time points and exhibited a longer duration of protein inhibition. Figure 5 (Table 11)

[0244] Example 6. The NM054 sequence at the 3' end of the antisense strand inhibits the effect of BALB / c-SEAP on serum SEAP protein. Activity evaluation

[0245] In this embodiment, a mouse model of the SEAP reporter gene 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 BALB / c-SEAP model was constructed as shown in Example 4. Female BALB / c-SEAP stable mice were divided into groups of five mice each according to their SEAP levels. Each test group received a predetermined dose of the drug, with a saline control group added. RZ003088 and RZ003080 sequences were administered via subcutaneous abdominal injection, respectively. The drug dosage for all mice was calculated based on body weight, with an administration volume of 10 mL / kg (mice body weight) and a dosage of 3 mg / kg (calculated as siRNA / mice body weight). The saline control group received 10 mL / kg (mice body weight) of saline solution without the siRNA conjugate. Serum samples were collected from mice in the saline group and the RZ003080 and RZ003088 administration groups before administration and at D7, D14, D21, D28, and D35 after administration. Phospha-Light was used to analyze the serum. TM The SEAP reporter gene assay system (Thermo, T1017) was used to detect SEAP levels in the serum of mice in all groups.

[0247] Table 12. Relative SEAP expression levels in the serum of mice after a single dose of siRNA conjugate.

[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 all time points, and exhibited a longer duration of protein inhibition. Figure 6 (Table 12)

[0250] Example 7. The NM054 sequence at the 3' end of the antisense strand inhibits the effect of BALB / c-SEAP on serum SEAP protein. Activity evaluation

[0251] In this embodiment, a mouse model of the SEAP reporter gene 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 BALB / c-SEAP model was constructed as shown in Example 4. Female BALB / c-SEAP stable mice were divided into groups of five mice each according to their SEAP levels. Each test group received a predetermined dose of the drug, with a saline control group added. RZ003076 and RZ003090 sequences were administered via subcutaneous abdominal injection. The drug dosage for all mice was calculated based on body weight, with an administration volume of 10 mL / kg (mice body weight) and a dosage of 3 mg / kg (calculated as siRNA / mice body weight). The saline control group received 10 mL / kg (mice body weight) of saline solution without the siRNA conjugate. Serum samples were collected from mice in the saline group and the RZ003076 and RZ003090 administration groups before administration and at D7, D14, D21, D28, and D35 after administration. Phospha-Light was used to analyze the serum. TM The SEAP reporter gene assay system (Thermo, T1017) was used to detect SEAP levels in the serum of mice in all groups.

[0253] Table 13. Relative SEAP expression levels in the serum of mice after a single dose of siRNA conjugate.

[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 all time points, and exhibited a longer duration of protein inhibition. Figure 7 (Table 13)

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

[0257] In this embodiment, the inhibitory activity of the conjugate RZ597174 containing the NM118 group at positions 20 and 21 of the antisense strand of the ANGPTL3 target siRNA and the conjugate RZ597114 without the NM118 group in mice was evaluated using a mouse in vivo target gene inhibitory activity assessment method.

[0258] Six- to eight-week-old C57BL / 6j mice were randomly divided into three groups of five mice each, based on body weight. Each group received the aforementioned siRNA conjugate via subcutaneous abdominal administration. The PBS control group received 5 mL / kg of siRNA per mouse, while the siRNA conjugate experimental group received 1 mg / kg (based on siRNA) per mouse, administered at a volume of 5 mL / kg. The day of administration was designated D0. Mice were sacrificed on D7, and gross dissection was performed. Liver tissue was collected and cut into several 2 mm sections. 3The small fragments were preserved using RNAlater. RNA extraction, reverse transcription, and real-time PCR detection were performed as described above. Gene expression differences were calculated using the ΔΔCt method, with primers as shown in Example 3.

[0259] Table 14 shows the inhibitory activity of the target gene in mice after administration of the siRNA conjugate described in Example 8.

[0260] Group % Inhibition rate average ±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, which contains an NM118 group at the 3' end of the antisense strand, had slightly lower inhibitory activity compared to the reference sequence RZ597114. Figure 8 (Table 14).

[0262] The above specific embodiments are merely illustrative of the content of this disclosure and do not represent a limitation thereof. For those skilled in the art, various modifications and improvements can be made without departing from the spirit and substance of this disclosure, and these modifications and improvements are also considered to be within the scope of protection of this disclosure.

Claims

1. A nucleotide dimer, characterized in that, The nucleotide dimer is selected from the structure shown in Formula III, or a pharmaceutically acceptable salt thereof: （Ⅲ） Base1 and Base2 are independently selected from A, U, G or C.

2. The nucleotide dimer according to claim 1, characterized in that, Base1 and Base2 form a combination, wherein the combination is selected from AA, UU, GG, CC, AU, AG, AC, UG, UC or GC.

3. A nucleotide dimer, characterized in that, The nucleotide dimer is selected from the structures shown below, or pharmaceutically acceptable salts thereof: 、 、 、 ， Ra is selected from amino protecting groups.

4. The method for preparing the nucleotide dimer according to any one of claims 1-2, characterized in that, The method includes the following steps: The synthesis route is as follows: Wherein, Base1 and Base2 are defined as described in claim 1 or 2; R1 and R2 are different, and R1 and R2 are respectively selected from F and -CH3; R3 and R4 are different, and R3 and R4 are respectively selected from F and -CH3. S1: Compound 1 and Compound 2 were dissolved in dichloromethane, 5-benzylthiotetrazole (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: Compound 3 was dissolved in tetrahydrofuran, tetrabutylammonium fluoride solution was added to remove the protecting group, the organic phase of the reaction solution was dried and concentrated, and purified to obtain compound 4; S3: Dissolve compound 4 in dichloromethane, add bis(diisopropylamino)(2-cyanoethoxy)phosphine and 4,5-dicyanimidazole, stir at room temperature for 2-3 hours, dry and concentrate the organic phase of the reaction solution, purify, and obtain the target compound.

5. Use of the nucleotide dimer according to any one of claims 1-3 in the preparation of nucleic acid drugs.

6. siRNA, characterized in that, The siRNA is prepared using at least one nucleotide dimer as described in any one of claims 1-3.

7. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises the siRNA of claim 6, and pharmaceutically acceptable excipients.

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