A siRNA conjugate containing a carbon ring nucleoside, and pharmaceutical compositions and uses thereof

Abstract

The application provides a kind of carbon ring nucleoside-containing siRNA conjugate and its pharmaceutical composition and use, belong to medical technology field.The sense strand and / or antisense strand of the siRNA conjugate at least include one carbon ring nucleoside of the following formula (I) structure, the sense strand includes, 5'-GUCAUCCACAAUGAGAGUACA-3' nucleotide sequence at least 16 consecutive nucleotides;Antisense strand includes 5'-UGUACUCUCAUUGUGGAUGACGA-3' nucleotide sequence at least 18 consecutive nucleotides.The carbon ring nucleoside-containing siRNA conjugate provided by the application can be used for treating, preventing and / or relieving AGT-related diseases, such as hypertension and the like.(I).

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to a carbon-cyclic nucleoside-containing siRNA conjugate, its pharmaceutical composition, and its uses. Background Technology

[0002] Factors contributing to poor hypertension control include poor adherence, the need for multiple antihypertensive medications, difficulty adopting a healthy lifestyle, and health disparities. Furthermore, over 10% of hypertensive patients develop resistance to their current antihypertensive drugs. Poor drug response may be related to the drug's failure to target the appropriate pathway for blood pressure regulation, incomplete pathway inhibition, and the formation of compensatory systems when a single system is completely downregulated. These factors are primarily related to the renin-angiotensin system (RAS), which plays a crucial role in long-term blood pressure control and is a major target for antihypertensive therapy. RAS upregulation increases blood pressure by altering vascular tone, blood volume, electrolyte balance, and aldosterone synthesis, leading to tissue degeneration and end-organ damage. Therefore, inhibiting angiotensinogen is an effective way to weaken the RAS and lower blood pressure.

[0003] Angiotensinogen, encoded by the gene AGT, is a glycoprotein secreted by hepatocytes. It is cleaved by renin to produce angiotensin I, which is further cleaved to produce angiotensin II. Angiotensinogen is the rate-limiting factor in the production of angiotensin II, and there is strong evidence linking both angiotensinogen and angiotensin II to hypertension: the higher the copy number of the angiotensinogen gene, the higher the blood pressure. Traditional small-molecule drugs are ineffective in lowering AGT levels, while oligonucleotide compounds targeting the AGT gene have shown significant and durable antihypertensive effects in animal models.

[0004] Two recent strategies for therapeutic gene silencing targeting AGT have been antisense oligonucleotides (ASOs) and small interfering RNA (siRNA). The siRNA strategy is the most effective, with a single dose lowering blood pressure for months. Its mechanism of action involves endocytosis into the cell, escaping from the endosome, and then binding to the RNA-induced silencing complex (RISC), which contains a functional core endonuclease. The antisense strand is recognized as the guide strand and retained to form the mature RISC, while the sense, non-guide strand is released. The RISC complex then aligns with the complementary target mRNA sequence of the guide strand, leading to mRNA cleavage, silencing its target gene, and reducing protein translation. siRNA produces a long-term, effective blood pressure-lowering effect by silencing AGT production in the liver, inhibiting angiotensinogen, and weakening the RAS system.

[0005] Appropriate modifications can endow siRNA with unique characteristics in vivo distribution, degradation, endocytosis, and release, maintaining high siRNA activity while increasing its targeting, safety, and persistence, which is key to the success of siRNA drugs. Backbone modification is the most basic chemical modification; phosphate thiocyanate (PS) modification is the most widely used backbone modification, also known as first-generation chemical modification. Replacing one non-bridging phosphate oxygen atom with a sulfur atom enhances the hydrophobicity of oligonucleotides, resists exonuclease degradation, and increases protein affinity. However, some studies have shown that excessive PS modification can enhance the toxicity of siRNA or oligonucleotides and reduce their gene silencing ability. Ribose modification refers to the modification of the ribose structure. The most common 2′-OMe and 2′-F modifications in siRNA can increase resistance to nuclease degradation and increase the binding ability to complementary nucleotide chains. Chinese patent CN114981431 A discloses a method and composition for treating angiotensinogen (AGT)-related diseases. This invention relates to a method for inhibiting AGT gene expression in a subject, and a method for treating a subject with an AGT-related condition (e.g., hypertension) using an RNAi agent targeting the AGT gene (e.g., a double-stranded RNAi agent). The invention also relates to a method for using such an RNAi agent to inhibit AGT gene expression to lower the blood pressure level of a subject. The double-stranded RNAi agent composition of this invention employs three of the most commonly used modifications (2′-OMe, 2′-F, PS modification). Summary of the Invention

[0006] To address the above problems, the present invention provides a siRNA conjugate containing a carbon-cyclic nucleoside.

[0007] On the one hand, the present invention provides a siRNA conjugate containing a carbon-cyclic nucleoside.

[0008] Specifically, the siRNA conjugate comprises a sense strand and an antisense strand, and contains at least one carbocyclic nucleoside of formula (I):

[0009] (I) Where Base is a heterocyclic base; The sense strand comprises at least 16 consecutive nucleotides of the nucleotide sequence shown in SEQ ID NO.1; the antisense strand comprises at least 18 consecutive nucleotides of the nucleotide sequence shown in SEQ ID NO.2.

[0010] SEQ ID NO.1: 5'-GUCAUCCACAAUGAGAGUACA-3'; SEQ ID NO. 2: 5'-UGUACUCUCAUUGUGGAUGACGA-3'.

[0011] More specifically, the heterocyclic base is selected from any one of thymine, cytosine, adenine, guanine, or uracil.

[0012] Preferably, the sense strand comprises at least 19 consecutive nucleotides of the 5'-GUCAUCCACAAUGAGAGUACA-3' nucleotide sequence; the antisense strand comprises at least 21 consecutive nucleotides of the 3'-AGCAGUAGGUGUUACUCUCAUGU-5' nucleotide sequence, and the sense strand and antisense strand are completely or partially complementary.

[0013] More specifically, the positive chain comprises a 2'-O-methylated (2'-OMe) nucleoside and / or a 2'-fluorinated (2'-F) nucleoside.

[0014] More specifically, the antisense chain comprises a 2'-O-methylated (2'-OMe) nucleoside and / or a 2'-fluorinated (2'-F) nucleoside.

[0015] More specifically, the 3' end and / or 5' end of the positive chain independently contain 1-5 thiophosphate groups linked together.

[0016] Preferably, the 3' end and / or 5' end of the positive chain independently contain two thiophosphate groups.

[0017] More specifically, the 3' end and / or 5' end of the antisense chain each independently contain 1-5 thiophosphate groups.

[0018] Preferably, the 3' end and / or 5' end of the antisense chain independently contain two thiophosphate groups linked together.

[0019] Specifically, the siRNA may optionally be conjugated to one or more ligands, portions or conjugates that enhance its activity, cellular distribution or cellular uptake, said ligands being attached to the 3' end and / or 5' end of the sense strand; and / or said ligands being attached to the 3' end and / or 5' end of the antisense strand.

[0020] Preferably, the ligand is a GalNAc derivative, conjugated to the 3' and / or 5' end of the sense or antisense strand of the siRNA. For example, the ligand may conjugate to the sense strand. In some embodiments, the ligand conjugates to the 3' end of the sense strand.

[0021] In some embodiments, the ligands used in this invention are selected from the following:

[0022] Formula (II)

[0023] Formula (III)

[0024] Equation (Ⅳ)

[0025] Formula (V)

[0026] Formula (VI)

[0027] Formula (VII) On the other hand, the present invention provides a pharmaceutical composition of a siRNA conjugate containing a carboxycyclic nucleoside.

[0028] Specifically, the pharmaceutical composition comprises the above-mentioned siRNA conjugate containing a carbocyclic nucleoside or a pharmaceutically acceptable salt thereof.

[0029] More specifically, the composition further includes a pharmaceutically acceptable carrier and / or diluent.

[0030] Preferably, the carrier includes, but is not limited to: liposomes, sterile aqueous solutions, physiological saline, buffer solutions, surfactants, osmotic pressure regulators, or preservatives.

[0031] Preferably, the diluent includes, but is not limited to, starch, dextrin, or cellulose.

[0032] In another aspect, the present invention also provides the use of the above-mentioned siRNA conjugate containing a carbon-cyclic nucleoside or its pharmaceutically acceptable salt, or pharmaceutical composition, in the preparation of a drug for reducing AGT protein levels.

[0033] In another aspect, the present invention also provides the use of the above-mentioned siRNA conjugates containing carbon-cyclic nucleosides or pharmaceutically acceptable salts thereof, or pharmaceutical compositions thereof, in the preparation of medicaments for treating and / or preventing and / or alleviating diseases related to AGT.

[0034] The diseases associated with AGT include, but are not limited to: hypertension, kidney disease, atherosclerosis, heart failure, stroke, or hepatic steatosis.

[0035] Compared with the prior art, the present invention has the following advantages: This invention introduces conformationally locked carbocyclic nucleosides into siRNA, which improves its stability to nucleases, increases base pairing affinity, enhances specificity, reduces off-target effects, improves pharmacokinetic properties, and exhibits better biological activity. Compared to publicly disclosed compounds, it has greater therapeutic value and can be used to treat and / or prevent and / or alleviate diseases associated with AGT.

[0036] The siRNA conjugates described in this invention involve C-cyclic nucleoside modification of the sense and / or antisense strands. Small nucleic acids modified with C-cyclic nucleosides, due to the conformational locking of the C-cyclic nucleoside, readily form stable double strands with target nucleic acids, exhibiting high binding affinity and good nuclease resistance, and long residence time in vivo. Therefore, introducing conformationally locked C-cyclic nucleosides into siRNA can improve its stability against nucleases, increase base pairing affinity, enhance specificity, reduce off-target effects, improve pharmacokinetic properties, and result in better biological activity. Attached Figure Description

[0037] Figure 1 The changes in hAGT protein levels in the serum of B6-Rosa26-hAGT model mice in Example 4 and Comparative Example 2 are shown.

[0038] Figure 2 The changes in the inhibition rate of hAGT in the serum of B6-Rosa26-hAGT model mice in Example 4 and Comparative Example 2 are shown.

[0039] Figure 3 The change in hAGT protein level in the serum of B6-Rosa26-hAGT model mice in Example 5.

[0040] Figure 4 The change in the inhibition rate of hAGT in the serum of B6-Rosa26-hAGT model mice in Example 5.

[0041] Figure 5 The change in hAGT protein level in the serum of B6-Rosa26-hAGT model mice in Example 6.

[0042] Figure 6 The change in the inhibition rate of hAGT in the serum of B6-Rosa26-hAGT model mice in Example 6. Detailed Implementation

[0043] It should be understood that the foregoing overview and the following detailed description are merely exemplary and illustrative, and do not limit the embodiments as claimed.

[0044] Unless otherwise specified, the following terms have the following meanings. Any undefined term has the meaning generally accepted in its field.

[0045] As used herein, AGT mRNA refers to mRNA having the sequence shown in GenBank accession number NM000029.3. Further, unless otherwise specified, the term "target gene" as used in this invention refers to the gene that records AGT mRNA, and the term "target mRNA" refers to the aforementioned AGT mRNA.

[0046] As used herein, “siRNA” refers to an RNA or RNA-like (e.g., chemically modified RNA) oligonucleotide molecule that contains the ability to reduce or inhibit the translation of messenger RNA (mRNA) in a sequence-specific manner. siRNAs may function through RNA interference mechanisms (e.g., by interacting with the mRNA interference pathway mechanism in mammalian cells (RNA-induced silencing complex RISC)) or any other mechanism or pathway. While the term siRNA drug as used herein is considered to function primarily through RNA interference mechanisms, the siRNA drug is not limited to or restricted to any particular pathway or mechanism of action. The siRNA drugs described herein consist of an oligonucleotide chain having at least a partial complementarity to the mRNA that serves as the target. In some embodiments, the siRNA drugs described herein are double-stranded and consist of an antisense strand and a sense strand that is at least partially complementary to the antisense strand.

[0047] As used herein, "2'-F, fluorinated nucleotide" refers to a nucleotide in which the hydroxyl group at the 2' position of the ribosome is replaced by fluorine, and "non-fluorinated nucleotide" refers to a nucleotide or nucleotide analog in which the hydroxyl group at the 2' position of the ribosome is replaced by a non-fluorinated group. "Nucleotide analog" refers to a group that can replace a nucleotide in nucleic acids but whose structure differs from adenine ribonucleotide, guanine ribonucleotide, cytosine ribonucleotide, uracil ribonucleotide, or thymine deoxyribonucleotide.

[0048] As used herein, “2’-OMe,2’-O-methylated nucleotide” refers to a nucleotide formed by replacing the 2-hydroxyl group of the ribosome with a methoxy group.

[0049] As used herein, “cLNA” refers to a nucleoside containing a carbon ring with the structure of formula (I) in this article.

[0050] As used herein, “PS link” refers to a thiophosphate link, meaning a modified phosphate link in which one of the non-bridging oxygen atoms is replaced by a sulfur atom.

[0051] As used in this article, "thiophosphate linkage" refers to a modified phosphate linkage in which one of the non-bridging oxygen atoms is replaced by a sulfur atom.

[0052] As used in this article, the "sense strand" refers to the nucleotide sequence on an RNA molecule that carries the information of the amino acids that encode the protein. It is also called the coding strand, sense strand, or positive strand, while the other nucleotide sequence that is complementary to it is called the antisense strand.

[0053] As used herein, the “antisense strand” is substantially or substantially anticomplementary to a nucleotide sequence of the same length in the mRNA of the target gene expression.

[0054] As used herein, the terms "complementary" and "reverse complementary" are used interchangeably and have the meaning known to those skilled in the art: in a double-stranded nucleic acid molecule, the bases of one strand are paired complementaryly with the bases of the other strand. In DNA, the purine base adenine (A) always pairs with the pyrimidine base thymine (T) (or uracil (U) in RNA); the purine base guanine (C) always pairs with the pyrimidine base cytosine (G). Each base pair consists of one purine and one pyrimidine. When adenine on one strand always pairs with thymine (or uracil) on the other strand, and guanine always pairs with cytosine, the two strands are considered complementary, and the sequence of the complementary strand can be inferred from its sequence. Correspondingly, "mismatch" in the art means, in a double-stranded nucleic acid, that the bases at corresponding positions are not paired complementaryly.

[0055] As used herein, "sequence" or "nucleotide sequence" refers to the order or sequence of nucleobases or nucleotides, expressed alphabetically using standard nucleotide nomenclature.

[0056] As used herein, unless otherwise specified, uppercase letters C, G, U, A, and T indicate the base composition of nucleotides, including modified and unmodified nucleotides.

[0057] As used herein, an "oligonucleotide" is composed of 15-30 linked nucleosides. In some embodiments of the invention, the oligonucleotide has a nucleobase sequence that is at least partially complementary to the coding sequence of a target gene expressed in the cell. The nucleotide may optionally be modified. In some embodiments of the invention, after delivery of the oligonucleotide to a cell expressing a gene, the oligonucleotide is able to inhibit or block gene expression in vitro or in vivo.

[0058] As used herein, a nucleoside is a compound consisting of a ribose (or deoxyribose) and a nucleobase (purine or pyrimidine) linked by a glycosidic bond, and is a basic building block of nucleic acids. Nucleosides include natural nucleosides as well as modified nucleosides with modified base moieties and / or modified sugar moieties.

[0059] As used in this article, "base" refers to the standard DNA and RNA bases (uracil, thymine, adenine, guanine, and cytosine), as well as modified bases.

[0060] As used in this article, "pharmaceutically acceptable salt" means a physiologically and pharmaceutically acceptable salt of a compound that retains the desired biological activity of the parent compound and does not impart undesirable toxicological effects.

[0061] As used herein, "pharmaceuticalally acceptable carrier" refers to a medium or diluent that does not interfere with the structure or function of oligonucleotides, an adjunct other than oligonucleotides administered to animals or humans, some of which enable the formulation of pharmaceutical compositions into oral preparations such as tablets, capsules, liquids, suspensions, etc., and some of which enable the formulation of pharmaceutical compositions into injectable or infusion preparations. For example, pharmaceutically acceptable carriers may be sterile aqueous solutions or physiological saline.

[0062] As used herein, "pharmaceutical composition" means a mixture of substances suitable for individual administration. For example, a pharmaceutical composition may comprise one or more compounds or salts thereof and a sterile aqueous solution.

[0063] The siRNA conjugates provided by this invention did not exhibit significant off-target effects. Off-target effects can be, for example, the inhibition of normal expression of non-target genes. It is believed that an off-target effect is insignificant if the binding / inhibition of off-target gene expression is less than 50%, 40%, 30%, 20%, or 10% compared to the effect on the target gene.

[0064] The siRNA conjugates provided by this invention exhibit significantly improved plasma stability and low off-target effects, while also demonstrating high AGT protein inhibitory activity. Therefore, in some embodiments, the siRNA conjugates disclosed herein may be one or more of the siRNA conjugates shown in the table below.

[0065] Table 1. siRNA sequences disclosed in this invention

[0066] Wherein, "C, G, U, A, T" represent the base composition of the nucleotide; "k" indicates cLNA modification; "m" indicates 2ʹ-OMe modification; "f" indicates 2ʹ-F modification; "s" indicates PS linkage, and unless otherwise specified, all are PO linkages; GalNAc "" indicates ligand fusion.

[0067] The carbon-cyclic nucleoside-modified siRNA conjugates or pharmaceutical compositions thereof described in this invention reduce AGT mRNA expression by at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%, or the range defined by any two of these values.

[0068] The following examples illustrate in more detail the synthesis and in vitro and in vivo biological activity of the carbon-cyclic nucleoside-modified siRNA conjugates for regulating AGT protein expression according to the present invention. However, the following examples are only for illustrating the compounds described herein and are not intended to limit the present invention.

[0069] Example 1: Synthesis of C-cyclic nucleoside phosphoramidide monomer G

[0070] (1) Synthesis of compound 19

[0071] Under a nitrogen atmosphere, compound 18 (24 g, 0.07 mol), cesium fluoride (31.9 g, 0.21 mol), and O-6-benzylguanine (33.8 g, 0.14 mol) were suspended in 240 mL of dry DMF. The mixture was heated to 90 °C and reacted for 3 h. The reaction was monitored by LCMS until completion. The mixture was then cooled, water was added, and the mixture was extracted twice with ethyl acetate. The combined organic phases were washed twice with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was purified by column chromatography using petroleum ether:ethyl acetate as the eluent in a ratio of 1:2 to 1:3, yielding 23.3 g of an oily substance, with a yield of 56.8%. [M+1] + =582.2; 1H NMR (400 MHz, Chloroform-d) δ 7.64 (s, 1H), 7.57– 7.47 (m, 2H), 7.46 – 7.29 (m, 13H), 5.58 (s, 2H), 4.94 (d, J = 11.4 Hz, 1H), 4.88 (s, 2H), 4.70 (dd, J = 10.8, 3.0 Hz, 2H), 4.57 (dd, J = 8.4, 5.6Hz, 1H), 4.52 (s, 2H), 4.15 (d, J = 5.6 Hz, 1H), 3.85 (d, J = 11.3 Hz, 1H),3.66 (d, J = 11.3 Hz, 1H), 3.41 (s, 2H), 2.38 (dd, J = 13.3, 8.7 Hz, 1H), 2.09 – 1.96 (m, 2H).

[0072] (2) Synthesis of compound 20 G10 G11 Under a nitrogen atmosphere, compound 19 (23.2 g, 0.04 mol) was dissolved in 230 mL of dry pyridine. The solution was cooled to 0 °C, and trimethylchlorosilane (26.1 g, 0.24 mol) was slowly added dropwise. After the addition was complete, the mixture was stirred at room temperature for 2 h. The solution was then cooled to 0 °C, and isobutyryl chloride (4.7 g, 0.044 mol) was slowly added dropwise. After the addition was complete, the mixture was stirred for another 2 h. 30 mL of concentrated ammonia was added dropwise, and the mixture was stirred at room temperature for 1 h. The reaction was monitored by LCMS until completion. Water was added, and the mixture was extracted twice with ethyl acetate. The organic phases were combined, washed twice with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was purified by column chromatography using petroleum ether:ethyl acetate as the eluent, yielding 26.3 g of an oily liquid, with a yield of 97%. [M+1] + =652.3.

[0073] (3) Synthesis of compound 21

[0074] Under a nitrogen atmosphere, compound 20 (25.5 g, 0.039 mol) was dissolved in 200 mL of dry pyridine. The solution was cooled to 0 °C, and a tetrahydrofuran solution of methanesulfonic anhydride (8.7 g, 0.051 mol) was slowly added dropwise. After the addition was complete, stirring was continued for 2 h. The reaction was monitored by LCMS until the product concentration reached its maximum. Water was added to quench the reaction, and the product was extracted twice with ethyl acetate. The combined organic phases were washed twice with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was purified by column chromatography using petroleum ether:ethyl acetate (1:1) as the eluent, yielding 19.1 g of oil (56.8%). 4.5 g of the starting material was recovered. [M+1] + =730.3.

[0075] (4) Synthesis of compound 22

[0076] Under a nitrogen atmosphere, 3.5 g (87.9 mmol) of 60% sodium hydride was carefully added to 210 mL of dry tetrahydrofuran, and the mixture was stirred for 10 min. The mixture was cooled to 0 °C, and a tetrahydrofuran solution of compound 21 (21.4 g, 29.3 mmol) was slowly added dropwise. After the addition was complete, stirring was continued for 2 h. The reaction was monitored by LCMS until complete. The reaction was quenched with acetic acid, and the mixture was extracted twice with ethyl acetate after adding saturated sodium bicarbonate solution. The combined organic phases were washed successively with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was purified by column chromatography using petroleum ether:ethyl acetate as the eluent, yielding 15.8 g of an oily substance in 85% yield. [M+1] + =634.3.

[0077] (5) Synthesis of compound I-1-3

[0078] Under a nitrogen atmosphere, compound 22 (5 g, 7.9 mmol) was dissolved in 50 mL of dichloromethane. The solution was cooled to -78 °C, and 71 mL of a 1 M boron tribromide solution in dichloromethane was slowly added dropwise. After the addition was complete, the solution was slowly heated to -40 °C and stirred for 2 h. The reaction was monitored by LCMS until complete. The reaction was quenched dropwise with methanol, and the solution was heated to room temperature and concentrated. The crude product was purified by column chromatography using dichloromethane:methanol (10:1) as the eluent to give 2.5 g of a white solid, with a yield of 87.2%. [M+1] + = 364.1; 1H NMR (400 MHz, DMSO-d6) δ 8.14(s, 1H), 7.36 – 7.21 (m, 1H), 5.17 (d, J = 4.2 Hz, 1H), 4.59 (t, J = 5.2 Hz,1H), 4.44 (dd, J = 9.7, 5.4 Hz, 1H), 4.03 (d, J = 4.4 Hz, 1H), 3.96 (s, 1H), 3.78 – 3.68 (m, 2H), 2.79 (p, J = 6.8 Hz, 1H), 2.23 (ddd, J = 26.1, 14.8,10.2 Hz, 2H), 1.99 (dd, J = 10.2, 6.6 Hz, 1H), 1.12 (d, J = 6.8 Hz, 5H).

[0079] (6) Synthesis of compound 24

[0080] Under a nitrogen atmosphere, compound I-1-3 (3.1 g, 9.1 mmol) and 4,4'-dimethoxytriphenylmethyl chloride (6.8 g, 20.2 mmol) were dissolved in 30 mL of dry dichloromethane. The solution was cooled to 0 °C, and N,N-diisopropylethylamine (5.89 g, 45.5 mmol) was slowly added dropwise. After the addition was complete, the solution was heated to room temperature and stirred for 2 h. The reaction was monitored by LCMS until completion. The solution was concentrated, and the crude product was purified by column chromatography using dichloromethane:methanol:triethylamine = 30:1:0.1% as the eluent, yielding 4.6 g of a pale yellow solid, with a yield of 76%. [M+1] +=666.3; 1H NMR (400 MHz, Chloroform-d) δ 12.10 (s, 1H), 9.40 (s, 1H), 7.75 (s,1H), 7.46 – 7.40 (m, 2H), 7.34 – 7.25 (m, 5H), 7.23 – 7.17 (m, 2H), 6.83(ddd, J = 9.0, 4.2, 2.0 Hz, 4H), 4.66 (s, 1H), 4.48 (t, J = 7.6 Hz, 1H), 4.27(s, 1H), 3.95 (d, J = 7.1 Hz, 1H), 3.82 (s, 1H), 3.78 (s, 6H), 3.68 (d, J =7.0 Hz, 1H), 3.46 (d, J = 9.6 Hz, 1H), 3.27 (d, J = 9.6 Hz, 1H), 2.53 (p, J =6.9 Hz, 1H), 2.32 (d, J = 7.6 Hz, 2H), 1.19 (dd, J = 9.7, 6.8 Hz, 6H).

[0081] (7) Synthesis of compound I-1-7

[0082] Under a nitrogen atmosphere, compound 24 (1.0 g, 1.5 mmol) and 4,5-dicyanimidazole (443 mg, 3.75 mmol) were dissolved in 10 mL of dry dichloromethane. Bis(diisopropylamino)(2-cyanoethoxy)phosphine (1.1 g, 3.75 mmol) was added dropwise at room temperature, and stirring was continued for 2 h. After the reaction was monitored by LCMS, dichloromethane and 0.1 mL of triethylamine were added. The organic phase was washed sequentially with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was separated using preparative high-performance liquid chromatography (HPLC) on a C18 column with a mobile phase of acetonitrile (containing 0.0035% diisopropylamine):water (containing 0.0035% diisopropylamine) = 7:3, yielding 660 mg of a white solid, in 51% yield. [M+1] + =866.4; 31 PNMR: 147.27, 147.17.

[0083] Example 2: Synthesis and purification of siRNA conjugates 1-9 containing carbon-cyclic nucleosides Oligonucleotide analogs containing carbocyclic nucleoside phosphoramide monomers were synthesized in a multi-step solid-phase process using an automated oligonucleotide synthesizer on a solid-phase support and at a rate of 0.2 μmol / L. These processes included deprotection, coupling, oxidation / thiolation, capping, and ammonolysis. The resulting compounds were 1-9, and their sequences and modifications are shown in Table 2. The synthesis and purification of compound 1 are used as an example to further illustrate the synthesis and purification of siRNA conjugates modified with carbocyclic nucleosides.

[0084] Table 2. Several siRNA conjugates targeting human AGT with 2'-F, 2'-OMe, and cLNA modifications.

[0085] Note: "C, G, U, A, T" represent the base composition of the nucleotide; "k" indicates cLNA modification; "m" indicates 2ʹ-OMe modification; "f" indicates 2ʹ-F modification; "s" indicates PS linkage, unless otherwise specified, all are PO linkage; GalNAc "" indicates ligand fusion.

[0086] Synthesis and purification of compound 1: (1) Synthesis of single-stranded oligonucleotides: Oligonucleotides were synthesized using phosphoramidite solid-phase synthesis technology. A general-purpose controllable porous glass CPG (500 Å) was selected as the solid-phase synthesis carrier. The carrier was chosen based on whether or not it was pre-loaded with ligand conjugates such as GalNAc. Except for the self-made carbocyclic nucleoside phosphoramidite, other 2'-modified RNA phosphoramidite monomers, GalNAc ligand conjugate phosphoramidite monomers, and auxiliary reagents were all commercially available. All phosphoramidite monomers were dissolved in anhydrous acetonitrile and added to a molecular sieve (3 Å) for later use. Deprotection was performed using 3% dichloroacetic acid, monitored by UV absorption. During coupling, 0.6 M 5-ethylthio-1H-tetrazole (ETT) was used as the activator, with a monomer concentration of 0.2 M and a coupling time of 12 minutes. Oxidation / sulfidation process: Phosphate ester bonds were constructed using a 0.05M iodine solution (dissolved in pyridine / water = 9:1); thiophosphate ester bonds were generated using a 0.2M hydroflavin solution in anhydrous acetonitrile / pyridine (v / v = 1 / 1) for 5 minutes. Acetic anhydride was used as the capping reagent in the capping step. This process was repeated sequentially to synthesize the target sequence compound. The synthesis was complete after the final removal of the DMTr group.

[0087] (2) Cleavage and deprotection of oligonucleotides bound to CPG: After the solid-phase synthesis was terminated, the protecting groups were removed by treating with an acetonitrile solution containing 20% ​​diethylamine for 10 minutes. The obtained CPG carrier was then subjected to ammonolysis with concentrated ammonia at 55°C for 16-20 hours to remove the protecting groups on the carrier and bases. After filtration, a solution containing the product was obtained.

[0088] (3) Ion purification of single-stranded oligonucleotides: The nucleotides were purified by HPLC using NanoQ anion exchange. Buffer A consisted of 20 mM phosphate + 10% acetonitrile aqueous solution; and buffer B consisted of 20 mM phosphate + 2 M sodium chloride + 10% acetonitrile aqueous solution. The elution gradient was 0-60% B, 20 CV, to separate the target product. The obtained target product was then desalted by gel electrophoresis.

[0089] Reverse-phase ion-pair purification: Column purification was performed using a C18 reverse-phase column. Buffer A consisted of 0.1 M hexafluoroisopropanol + 0.007 M triethylamine; Buffer B consisted of 70% A + 30% acetonitrile, with an elution gradient of 0-60% B. The target product was obtained by separation at 20 CV.

[0090] (4) The sense and antisense chains obtained by chemical synthesis are paired with each other in a molar ratio of 1:1. The reaction conditions are 70℃ for 10 min and then slowly restored to room temperature to finally obtain the target product.

[0091] (5) Product freeze drying: Temperature and time parameters, pre-freezing (-50℃, 4h), freezing (-15℃, 1h), first drying (-5℃, 1h), second drying (40℃, 99h) to obtain the final product compound 1. The purity of the product was characterized by HPLC as 95.3%.

[0092] Example 3: Inhibitory effect of C-cyclic nucleoside-containing siRNA conjugates on human angiotensinogen (AGT) in Huh7 cells Designed siRNA conjugates 2-9 containing carbon-cyclic nucleosides targeting human AGT, with sequence modifications as shown in Table 2 of Example 2, to test their inhibitory effect on AGT mRNA in vitro in Huh7 cells. The initial concentration of the siRNA conjugates to be tested was 0.500 nM, diluted 4-fold, with 7 concentration points set up, and measured in duplicate. Each siRNA conjugate was diluted to concentrations of 0.00012 nM, 0.0005 nM, 0.002 nM, 0.008 nM, 0.031 nM, 0.125 nM, and 0.500 nM, and mixed with an equal volume of RNAiMAX. The transfected siRNA conjugates were then mixed with Huh7 cell suspension to a cell concentration of 2 × 10⁻⁶ cells / well. 4 Cells / wells were cultured at 37°C and 5% CO2 for 48 hours. Cells were then collected and RNA was extracted from them. The expression level of AGT mRNA was measured by quantitative real-time PCR. The experimental results are shown in Table 3.

[0093] Table 3. Inhibition of AGT mRNA by siRNA conjugates containing carbon-cyclic nucleosides

[0094] Example 4: Inhibitory effect of C-cyclic nucleoside-containing siRNA conjugates on hAGT protein in B6-Rosa26-hAGT model mice B6-Rosa26-hAGT model mice containing humanized AGT were used, with 3 mice in each group. Compounds 1-7 and 9 from Table 2 of Example 3 were subcutaneously injected once at a dose of 1 mpk. Blood samples were collected from all mice before administration (D-1), and on D7, D14, and D21 after administration to separate serum, and hAGT levels were detected by ELISA. Mice injected with PBS served as a negative control group, and were compared with the group injected with the C-cyclic nucleoside siRNA conjugate. The hAGT protein inhibition rate was calculated using the normalization method, and the formula for calculating the hAGT protein inhibition rate is shown below:

[0095] The protein level and inhibition rate of hAGT in mouse serum are shown in Tables 4 and 5, respectively. Changes in hAGT protein level and inhibition rate in mouse serum are shown in... Figure 1 , Figure 2 Compared with the negative control group, all test compounds significantly inhibited the protein level of hAGT in the serum of B6-Rosa26-hAGT mice on Day 7, Day 14, and Day 21 after administration. Among them, compound 9 had the highest inhibition rate, approaching 80% after 21 days of administration; compound 7 maintained an inhibition rate greater than 60% after 21 days; and compound 2 maintained an inhibition rate greater than 50% after 21 days.

[0096] Table 4. Data on hAGT protein levels in mouse serum

[0097] Table 5. Inhibition rate data of hAGT in mouse serum

[0098] Example 5: Inhibitory effect of C-cyclic nucleoside-containing siRNA conjugates on hAGT protein in B6-Rosa26-hAGT model mice. B6-Rosa26-hAGT model mice containing humanized AGT were subcutaneously injected with compound 2 from Table 2 of Example 3, as a single dose. Compound 2 was divided into low, medium, and high dose groups, with doses of 1 mpk, 3 mpk, and 9 mpk, respectively, with 6 mice in each dose group. Blood samples were collected from all mice before administration (D-1), and on D7, D14, and D21 after administration to separate serum, and hAGT levels were detected by ELISA. Mice injected with PBS served as a negative control group, and the group injected with the C-cyclic nucleoside siRNA conjugate was compared with this group. The hAGT protein inhibition rate was calculated using the normalization method, and the formula for calculating the hAGT protein inhibition rate is shown below:

[0099] The protein level and inhibition rate of hAGT in mouse serum are shown in Tables 6 and 7, respectively. Changes in hAGT protein level and inhibition rate in mouse serum are shown in... Figure 3 , Figure 4 Compared with the negative control group, all test products significantly inhibited the protein level of hAGT in the serum of B6-Rosa26-hAGT mice on days 7, 14, and 21 after administration. Specifically, the inhibition rate of the low-dose group of compound 2 remained greater than 49% for 21 days, the medium-dose group maintained an inhibition rate greater than 77% for 21 days, and the high-dose group maintained an inhibition rate greater than 90% for 21 days. The inhibition rates of each dose group on days 7, 14, and 21 after administration were relatively similar, indicating that the inhibitory effect of each test product on hAGT in mouse serum was not only significant but also well-maintained.

[0100] Table 6. Protein level data of hAGT in mouse serum

[0101] Table 7. Inhibition rate data of hAGT in mouse serum

[0102] Example 6: Inhibitory effect of C-cyclic nucleoside-containing siRNA conjugates on hAGT protein in B6-Rosa26-hAGT model mice B6-Rosa26-hAGT model mice containing humanized AGT were subcutaneously injected with compounds 8-9 from Table 2 of Example 3, as a single dose. The dosage of compounds 8-9 was 3 mpk, with 3 mice in each group. Blood samples were collected from all mice before administration (D-1), and on D7, D14, and D21 after administration to separate serum, and hAGT levels were detected by ELISA. Mice injected with PBS served as a negative control group, and were compared with the group injected with the C-cyclic nucleoside siRNA conjugate. The hAGT protein inhibition rate was calculated using the normalization method, and the formula for calculating the hAGT protein inhibition rate is shown below:

[0103] The protein level and inhibition rate of hAGT in mouse serum are shown in Tables 8 and 9, respectively. Changes in hAGT protein level and inhibition rate in mouse serum are shown in... Figure 5 , Figure 6Compared with the negative control group, all test products significantly inhibited the protein level of hAGT in the serum of B6-Rosa26-hAGT mice on days 7, 14, and 21 after administration. Compound 8 maintained an inhibition rate greater than 86% for 21 days, while compound 9 maintained an inhibition rate greater than 90% for 21 days. The inhibition rates on days 7, 14, and 21 after administration were relatively similar across all test products, indicating that the inhibitory effects of each test product on hAGT in mouse serum were not only significant but also well-maintained.

[0104] Table 8. Data on hAGT protein levels in mouse serum

[0105] Table 9. Inhibition rate data of hAGT in mouse serum

[0106] Comparative Example 1: Inhibitory effect of carbocyclic nucleoside-free siRNA conjugates on human angiotensinogen (AGT) in Huh7 cells Table 10. siRNA conjugates targeting human AGT without cLNA modification

[0107] The sequence modification of compound 10, a siRNA conjugate without carbocyclic nucleosides, is shown in Table 10. Its inhibitory effect on AGT mRNA in vitro was tested in Huh7 cells. The initial concentration of compound 10 was 0.500 nM, diluted 4-fold, with 7 concentration points set up. The assay was performed in duplicate with 2 replicates at concentrations of 0.00012 nM, 0.0005 nM, 0.002 nM, 0.008 nM, 0.031 nM, 0.125 nM, and 0.500 nM. An equal volume of RNAi MAX was mixed with the siRNA conjugate, and the transfected siRNA conjugate was then mixed with Huh7 cell suspension to a cell concentration of 2 × 10⁻⁶ cells / well. 4 Cells / wells were cultured at 37°C and 5% CO2 for 48 hours. Cells were then collected and RNA was extracted from them. The expression level of AGT mRNA was measured by quantitative real-time PCR. The experimental results are shown in Table 11.

[0108] Table 11 Inhibition of AGT mRNA by siRNA conjugates without carbocyclic nucleosides

[0109] Comparative Example 2: Inhibitory effect of siRNA conjugates without C-cyclic nucleoside modification on hAGT protein in B6-Rosa26-hAGT model mice B6-Rosa26-hAGT model mice containing humanized AGT were used. Compound 10 from Table 10 of Comparative Example 1 was administered subcutaneously as a single dose of 1 MPk. Blood samples were collected from all mice before administration (D⁻¹), and on D7, D14, and D21 after administration. Serum was separated, and hAGT levels were detected by ELISA. Mice injected with PBS served as a negative control group. Three mice were used in each group. The hAGT protein inhibition rate was calculated using a normalization method, as shown in the formula below:

[0110] The protein level and inhibition rate of hAGT in mouse serum are shown in Tables 12 and 13, respectively. The changes in hAGT protein level and inhibition rate in mouse serum are shown in... Figure 1 and Figure 2 .

[0111] Table 12. Data on hAGT protein levels in mouse serum.

[0112] Table 13 Inhibition rate data of hAGT in mouse serum

[0113] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A siRNA conjugate containing a carbon-cyclic nucleoside, characterized in that, The siRNA conjugate is selected from compound 7 or compound 9. The positive chain of compound 7 is 5'-G. m sU m sC m A m U m C m C f A m C f A f A f U m G m A m G m A m G m U m A m C m A k - GalNAc -3', the antisense strand of compound 7 is 3'-A k sG m sC m A m G m U m A m G f G m U f G m U m U m A m C m U m C m U f C m A m U m sG f sU m -5'; The positive chain of compound 9 is 5'-G. k sU m sC m A m U m C m C f A m C f A f A f U m G m A m G m A m G m U m A m C m A m - GalNAc -3', the antisense strand of compound 9 is 3'-A m sG k sC m A m G m U m A m G f G m U f G m U m U m A m C m U m C m U f C m A m U m sG f sU m -5'; Where C, G, U, and A are the base composition of the nucleotide; k is cLNA modification, where cLNA is a nucleoside containing a carbon ring in the structure of formula (I); m is 2 -OMe modifier; f is 2 -F modifier; s indicates PS connection; GalNAc For ligand fusion; （I） Base is a heterocyclic base.

2. The siRNA conjugate according to claim 1, characterized in that, The heterocyclic base is selected from any one of thymine, cytosine, adenine, guanine, or uracil.

3. A pharmaceutical composition containing a carboxycyclic nucleoside siRNA conjugate, characterized in that, The siRNA conjugate pharmaceutical composition comprises the siRNA conjugate containing a carbon-cyclic nucleoside as described in any one of claims 1-2, or a pharmaceutically acceptable salt thereof.

4. The pharmaceutical composition according to claim 3, characterized in that, It also includes pharmaceutically acceptable carriers and / or diluents.

Images