Carbocyclic nucleoside-containing siRNA conjugate as well as pharmaceutical composition and application thereof

Abstract

The invention provides a carbocyclic nucleoside-containing siRNA conjugate as well as a pharmaceutical composition and application thereof, and belongs to the technical field of medicines. A positive-sense strand and / or an antisense strand of the siRNA conjugate at least comprise one carbocyclic nucleoside with a structure as shown in a formula (I), and the positive-sense strand comprises at least 16 continuous nucleotides of a 5 '-GUCAUCACAAUGAGAGUACA-3' nucleotide sequence; the antisense strand comprises at least 18 consecutive nucleotides of a 5 '-UGUACUUCAUUGUGAUGACGA-3' nucleotide sequence. The carbocyclic nucleoside-containing siRNA conjugate provided by the invention can be used for treating, preventing and / or alleviating diseases related to AGT, such as hypertension. # imgabs0 # (I).

Description

Technical Field

[0001] The present invention belongs to the field of medical technology, and in particular relates to a siRNA conjugate containing a carbocyclic nucleoside, a pharmaceutical composition and use thereof. Background Art

[0002] Factors that contribute to poor control of hypertension include poor compliance, the need for multiple antihypertensive drugs, difficulty in adopting a healthy lifestyle, and health disparities. In addition, more than 10% of hypertensive patients are resistant to current antihypertensive drugs. The reasons for poor drug response may be related to the failure of drugs to target the appropriate pathways for regulating blood pressure, incomplete pathway inhibition, and the formation of compensatory systems when a single system is completely downregulated. These reasons are mainly related to the renin-angiotensin system (RAS), which plays a key role in long-term control of blood pressure and is the main target of antihypertensive therapy. RAS upregulation increases blood pressure by changing vascular tone, blood volume, electrolyte balance, and aldosterone synthesis, leading to tissue degeneration and end-organ damage. Therefore, inhibition of angiotensinogen is an effective way to weaken RAS to lower blood pressure.

[0003] Angiotensinogen is encoded by the gene AGT, a glycoprotein secreted by liver cells. 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 angiotensinogen and angiotensin II to hypertension: the more copies of the angiotensinogen gene, the higher the blood pressure. Traditional small molecule drugs cannot effectively reduce AGT levels, and oligonucleotide compounds targeting the AGT gene have shown significant and lasting antihypertensive effects in animal models.

[0004] Two strategies that have been recently used for therapeutic gene silencing targeting AGT are antisense oligonucleotides (ASOs) and small interfering RNA (siRNA). Of these, the siRNA strategy is the most effective, with a single dose lowering blood pressure for up to several months. Its mechanism of action is that it enters cells via endocytosis, escapes from endosomes, and then binds 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 a 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, resulting in the cleavage of the mRNA, silencing its target gene, and reducing the translation of its protein. siRNA produces a long-term, effective antihypertensive effect by silencing AGT production in the liver, inhibiting angiotensinogen, and weakening the RAS system.

[0005] Reasonable modification can give siRNA unique characteristics such as in vivo distribution, degradation, endocytosis and release, and maintain the high activity of siRNA while increasing its targeting, safety and durability, which is the key to the success of siRNA drugs. Backbone modification is the most basic chemical modification. Thiophosphorothioate (PS) modification is the most commonly used backbone modification, also known as the first generation of chemical modification. Replacing one of the non-bridging phosphate oxygen atoms with a sulfur atom can enhance the hydrophobicity of oligonucleotides, resist degradation by nucleases and increase protein affinity. However, some studies have shown that excessive PS modification can enhance the toxicity of siRNA or oligonucleic acids and reduce their ability to silence genes. Ribose modification is a 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 ability to bind to complementary nucleotide chains. Chinese patent CN114981431 A discloses methods and compositions for treating angiotensinogen (AGT)-related diseases. The invention relates to a method for inhibiting the expression of the AGT gene in a subject, and a method for treating a subject with an AGT-related disorder (e.g., hypertension) using an RNAi agent (e.g., a double-stranded RNAi agent) targeting the AGT gene. The invention also relates to a method for using such an RNAi agent to inhibit the expression of the AGT gene to reduce the blood pressure level of the subject. The double-stranded RNAi agent composition described in the invention adopts the three most commonly used modifications (2′-OMe, 2′-F, PS modifications). Summary of the invention

[0006] In order to solve the above problems, the present invention provides a siRNA conjugate containing a carbocyclic nucleoside.

[0007] In one aspect, the present invention provides a siRNA conjugate containing a carbocyclic nucleoside.

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

[0009] (I) Wherein, 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 is completely or partially complementary to the antisense strand.

[0013] More specifically, the sense strand comprises 2'-O-methyl modified (2'-OMe) nucleosides and / or 2'-fluorine modified (2'-F) nucleosides.

[0014] More specifically, the antisense strand comprises 2'-O-methyl modified (2'-OMe) nucleosides and / or 2'-fluorine modified (2'-F) nucleosides.

[0015] More specifically, the 3' end and / or the 5' end of the sense strand independently contain 1-5 phosphorothioate linkages.

[0016] Preferably, the 3' end and / or the 5' end of the sense strand independently comprises two phosphorothioate linkages.

[0017] More specifically, the 3' end and / or the 5' end of the antisense strand independently contain 1-5 phosphorothioate linkages.

[0018] Preferably, the 3' end and / or the 5' end of the antisense strand independently contain two phosphorothioate linkages.

[0019] Specifically, the siRNA can be optionally conjugated to one or more ligands, moieties or conjugates that enhance its activity, cellular distribution or cellular uptake, wherein the ligand can be linked to the 3' end and / or 5' end of the sense strand; and / or, the ligand can be linked to the 3' end and / or 5' end of the antisense strand.

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

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

[0022] Formula (II)

[0023] Formula (III)

[0024] Formula (IV)

[0025] Formula (V)

[0026] Formula (VI)

[0027] Formula (VII) In another aspect, the present invention provides a pharmaceutical composition comprising a siRNA conjugate of a carbocyclic nucleoside.

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

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

[0030] Preferably, the carrier includes, but is not limited to, liposomes, sterile aqueous solution, physiological saline, buffer, surfactant, osmotic pressure regulator or preservative.

[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 carbocyclic nucleoside or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition in the preparation of a drug for reducing the level of AGT protein.

[0033] In another aspect, the present invention also provides the use of the above-mentioned siRNA conjugate containing carbocyclic nucleoside or its pharmaceutically acceptable salt, or its pharmaceutical composition in the preparation of drugs for treating and / or preventing and / or alleviating diseases related to AGT.

[0034] The AGT-related diseases include, but are not limited to, hypertension, kidney disease, atherosclerosis, heart failure, stroke or fatty degeneration of the liver.

[0035] Compared with the prior art, the present invention has the following advantages: The invention introduces conformationally locked carbocyclic nucleosides into siRNA to improve its stability to nucleases, increase base pairing affinity, improve specificity, reduce off-target effects, improve pharmacokinetic properties, and have better biological activity. Compared with publicly disclosed compounds, it has more therapeutic value and can be used to treat and / or prevent and / or alleviate diseases related to AGT.

[0036] The siRNA conjugate of the present invention is modified with carbocyclic nucleosides on the sense strand and / or the antisense strand. The small nucleic acid modified with carbocyclic nucleosides is easy to form a stable double strand with the target nucleic acid due to the locked conformation of the carbocyclic nucleosides, has a high binding affinity and good nuclease resistance, and has a long residence time in the body. Therefore, the introduction of conformationally locked carbocyclic nucleosides into siRNA can improve its stability to nucleases, increase the affinity of base pairing, improve specificity, reduce off-target effects, improve pharmacokinetic properties, and have better biological activity. BRIEF DESCRIPTION OF THE DRAWINGS

[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.

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

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

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

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

[0042] Figure 6 The inhibition rate of hAGT in the serum of B6-Rosa26-hAGT model mice in Example 6 is changed. DETAILED DESCRIPTION

[0043] It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the embodiments, as claimed.

[0044] Unless otherwise indicated, the following terms have the following meanings. Any undefined term has its art-recognized meaning.

[0045] As used herein, AGT mRNA refers to mRNA having a sequence shown in Genbank Accession No. NM000029.3. Preferably, if not otherwise specified, the term "target gene" used in the present invention refers to a gene expressing AGT mRNA, and the term "target mRNA" refers to the above-mentioned AGT mRNA.

[0046] As used herein, "siRNA" refers to an RNA or RNA-like (e.g., chemically modified RNA) oligonucleotide molecule that can reduce or inhibit the translation of messenger RNA (mRNA) in a sequence-specific manner. siRNA can act through an RNA interference mechanism (e.g., by inducing mRNA degradation through interaction with the mRNA interference pathway mechanism (RNA-induced silencing complex RISC) of mammalian cells), or other arbitrary mechanisms or pathways. Although it is believed that the term siRNA drug used herein mainly acts through an RNA interference mechanism, the siRNA drug is not limited to or confined to any specific pathway or mechanism of action. The siRNA drug described herein is composed of an oligonucleotide chain that is at least partially complementary to the mRNA as a target. In some embodiments, the siRNA drug described herein is double-stranded and consists of an antisense strand and a sense strand that is at least partially complementary to the antisense strand.

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

[0048] As used herein, the "2'-OMe, 2'-O-methylated modified nucleotide" refers to a nucleotide in which the 2-hydroxyl group of the ribose group is replaced by a methoxy group.

[0049] As used herein, the term "cLNA" refers to a carbon ring-containing nucleoside comprising the structure of formula (I) herein.

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

[0051] As used herein, "phosphorothioate linkage" means 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, "sense strand" refers to the strand on the RNA molecule that carries the nucleotide sequence encoding protein amino acid information, which is called the sense strand, also known as the coding strand, sense strand or positive strand, and the other nucleotide sequence that is complementary to it is the antisense strand.

[0053] As used herein, the "antisense strand" is substantially reverse complementary or essentially reverse complementary to a nucleotide sequence in the mRNA expressed by the target gene that has the same length as the antisense strand.

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

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

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

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

[0058] As used herein, "nucleoside" is a compound formed by a ribose (or deoxyribose) and a nucleobase (purine or pyrimidine) connected by a glycosidic bond, and is the basic building block of nucleic acid. Nucleosides include natural nucleosides and also include modified nucleosides with modified base moieties and / or modified sugar moieties.

[0059] As used herein, "base" refers to the conventional DNA and RNA bases (uracil, thymine, adenine, guanine, and cytosine), and also includes modified bases.

[0060] As used herein, "pharmaceutically acceptable salts" refers to physiologically and pharmaceutically acceptable salts of compounds that retain the desired biological activity of the parent compound and do not impart undesired toxicological effects thereto.

[0061] As used herein, "pharmaceutically acceptable carrier" refers to a medium or diluent that does not interfere with the structure or function of an oligonucleotide, an additive other than an oligonucleotide that is administered to an animal or a human, some of which enable the pharmaceutical composition to be formulated into oral preparations such as tablets, capsules, liquids, suspensions, and some of which enable the pharmaceutical composition to be formulated into injectable preparations for injection or infusion, for example, a pharmaceutically acceptable carrier may be a sterile aqueous solution or physiological saline.

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

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

[0064] The siRNA conjugates provided by the present invention exhibit significantly improved stability in plasma and low off-target effects, while also exhibiting higher 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 following table.

[0065] Table 1 siRNA sequences disclosed in the present invention

[0066] Among them, "C, G, U, A, T" represent the base composition of nucleotides; "k" represents cLNA modification; "m" represents 2ʹ-OMe modification; "f" represents 2ʹ-F modification; "s" represents PS connection, and PO connection is used unless otherwise specified; GalNAc ” indicates ligand conjugation.

[0067] The carbocyclic nucleoside-modified siRNA conjugates or pharmaceutical compositions thereof described in the present invention can 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 a 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 activities of the carbocyclic nucleoside-modified siRNA conjugates for regulating AGT protein expression of the present invention, but the following examples are only used to illustrate the compounds described herein and are not intended to limit the present invention.

[0069] Example 1: Synthesis of Carbocyclic Nucleoside Phosphoramidite Monomer G

[0070] (1) Synthesis of Compound 19

[0071] Under nitrogen atmosphere, compound 18 (24 g, 0.07 mol), cesium fluoride (31.9 g, 0.21 mol), O-6-benzylguanine (33.8 g, 0.14 mol) were suspended in 240 mL of dry DMF, heated to 90 ° C for 3 h, and the reaction was completed after LCMS monitoring. The temperature was lowered, 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, and the eluent was petroleum ether: ethyl acetate = 1:2~1:3, to obtain 23.3 g of oil, 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 nitrogen atmosphere, compound 19 (23.2 g, 0.04 mol) was dissolved in 230 mL of dry pyridine, cooled to 0 °C, and trimethylsilyl chloride (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 mixture was 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 2 h. 30 mL of concentrated ammonia was added dropwise, and the mixture was stirred at room temperature for 1 h. After the reaction was completed by LCMS monitoring, 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, and the eluent was petroleum ether: ethyl acetate = 1:1~1:2, to obtain 26.3 g of an oily liquid with a yield of 97%. [M+1] + =652.3.

[0073] (3) Synthesis of Compound 21

[0074] Under nitrogen atmosphere, compound 20 (25.5 g, 0.039 mol) was dissolved in 200 mL of dry pyridine, 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. When the reaction was monitored by LCMS until the product content was maximum, water was added to quench the reaction, and ethyl acetate was extracted twice. 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 with an eluent of petroleum ether: ethyl acetate = 1:1 to obtain 19.1 g of oil with a yield of 56.8%. 4.5 g of raw material was recovered. [M+1] + =730.3.

[0075] (4) Synthesis of Compound 22

[0076] Under nitrogen atmosphere, 60% sodium hydride (3.5 g, 87.9 mmol) was carefully added to 210 mL of dry tetrahydrofuran and stirred for 10 min. The temperature was lowered 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. After the reaction was completed by LCMS monitoring, the reaction was quenched with acetic acid, saturated sodium bicarbonate solution was added, and ethyl acetate was extracted twice. The organic phases were combined, washed with saturated brine in turn, dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was purified by column chromatography with the eluent being petroleum ether: ethyl acetate = 1:1 to obtain 15.8 g of an oily substance with a yield of 85%. [M+1] + =634.3.

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

[0078] Under nitrogen atmosphere, compound 22 (5 g, 7.9 mmol) was dissolved in 50 mL of dichloromethane, cooled to -78 °C, and 71 mL of 1 M dichloromethane solution of boron tribromide was slowly added dropwise. After the addition was complete, the temperature was slowly raised to -40 °C and stirred for 2 h. After the reaction was completed by LCMS monitoring, methanol was added dropwise to quench, the temperature was raised to room temperature, and the mixture was concentrated. The crude product was purified by column chromatography with the eluent being dichloromethane:methanol = 10:1, to obtain 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 nitrogen atmosphere, compound I-1-3 (3.1 g, 9.1 mmol) and 4,4'-bismethoxytrityl chloride (6.8 g, 20.2 mmol) were dissolved in 30 mL of dry dichloromethane, cooled to 0°C, and N,N-diisopropylethylamine (5.89 g, 45.5 mmol) was slowly added dropwise. After the addition was complete, the mixture was heated to room temperature and stirred for 2 h. The reaction was completed after LCMS monitoring, and the mixture was concentrated. The crude product was purified by column chromatography, and the eluent was dichloromethane: methanol: triethylamine = 30:1:0.1%, to obtain 4.6 g of light 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 nitrogen atmosphere, compound 24 (1.0 g, 1.5 mmol) and 4,5-dicyanoimidazole (443 mg, 3.75 mmol) were dissolved in 10 mL of dry dichloromethane, and 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 completed by LCMS monitoring, dichloromethane and 0.1 mL of triethylamine were added, and the organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was separated by high-pressure preparative liquid phase, the chromatographic column was C18, and the mobile phase was acetonitrile (containing 0.0035% diisopropylamine): water (containing 0.0035% diisopropylamine) = 7:3, and 660 mg of white solid was obtained, with a yield of 51%. [M+1] + =866.4; 31 PNMR:147.27,147.17.

[0083] Example 2: Synthesis and purification of siRNA conjugates 1-9 containing carbocyclic nucleosides By using an automatic oligonucleotide synthesizer, a solid phase carrier and a phosphoramidite nucleoside monomer were used to perform deprotection, coupling, oxidation / thiolation, capping, aminolysis and other multi-step solid phase synthesis of oligonucleic acid analogs containing carbocyclic nucleoside phosphoramidite monomers at the order of 0.2 μmol, which are compounds 1-9, respectively, and the sequences and modifications are shown in Table 2. Taking the synthesis and purification of compound 1 as an example, the synthesis and purification of siRNA conjugates containing carbocyclic nucleoside modifications are further described.

[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 nucleotides; "k" represents cLNA modification; "m" represents 2ʹ-OMe modification; "f" represents 2ʹ-F modification; "s" represents PS connection, and PO connection is used unless otherwise specified; GalNAc ” indicates ligand conjugation.

[0086] Synthesis and purification of compound 1: (1) Synthesis of single-stranded oligoribonucleotides: Oligoribonucleotides were synthesized according to the phosphoramidite solid phase synthesis technique. The solid phase synthesis carrier was selected as the universal controlled porous glass CPG (500Å). The carrier with pre-loaded ligand conjugates such as GalNAc was selected as needed. In addition to the homemade carbocyclic nucleoside phosphoramidite, other 2'-modified RNA phosphoramidite monomers (phosphoramidite), ligand conjugates such as GalNAc phosphoramidite monomers and auxiliary reagents were commercially available reagents. All phosphoramidite monomers were dissolved in anhydrous acetonitrile and molecular sieves (3Å) were added for standby use. Deprotection was performed using 3% dichloroacetic acid and monitored by ultraviolet absorption. During the coupling process, 0.6M 5-ethylthio-1H-tetrazole (ETT) was used as an activator, the monomer concentration was 0.2M, and the coupling time was 12 minutes. Oxidation / sulfurization process: Use 0.05M iodine solution (dissolved in pyridine / water = 9:1) to construct phosphate bonds; use 0.2M hydrogenated xanthan gum in anhydrous acetonitrile / pyridine (v / v = 1 / 1) solution to generate thiophosphate bonds, and the reaction time is 5 minutes. Acetic anhydride is used as a capping agent in the capping step. The target sequence compound is synthesized in a cycle, and the sequence is completed after the DMTr group is removed at the end.

[0087] (2) Cleavage and deprotection of oligoribonucleotides bound to CPG: After the solid phase synthesis is terminated, the protecting groups are removed by treating with acetonitrile solution containing 20% ​​diethylamine for 10 minutes. The obtained CPG carrier is then heated with concentrated ammonia water at 55°C for 16-20 hours to remove the protecting groups on the carrier and the base, and the solution containing the product is obtained after filtration.

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

[0089] Reverse phase ion pair purification: Column purification was performed using a C18 reverse phase column. Buffer A was 0.1 M hexafluoroisopropanol + 0.007 M triethylamine; buffer B was 70% A + 30% acetonitrile, the elution gradient was 0-60% B, and the target product was separated in 20CV.

[0090] (4) The sense chain and antisense chain obtained by chemical synthesis were subjected to base complementary pairing in a molar ratio of 1:1. The reaction conditions were 70°C 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°C, 4h), freezing (-15°C, 1h), primary drying (-5°C, 1h), secondary drying (40°C, 99h), to obtain the final product compound 1, the product purity was 95.3% as characterized by HPLC.

[0092] Example 3: Inhibitory effect of siRNA conjugates containing carbocyclic nucleosides on human angiotensinogen (AGT) in Huh7 cells siRNA conjugates 2 to 9 targeting human AGT containing carbocyclic nucleosides were designed, and the sequence modifications were as shown in Table 2 of Example 2. Their inhibitory effects on AGT mRNA in vitro were tested in Huh7 cells. The starting concentration of the siRNA conjugate containing carbocyclic nucleosides to be tested was 0.500 nM, 4-fold dilution, 7 concentration points were set, and 2 replicate wells were measured in parallel. Each siRNA conjugate was diluted to a concentration 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 conjugate was mixed with the Huh7 cell suspension, and the cell concentration was 2×10 4 cells / well, at 37°C, 5% CO 2 After culturing for 48 h under the same conditions, the cells were collected and RNA was isolated and extracted from the cells. 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 carbocyclic nucleosides

[0094] Example 4: Inhibitory effect of siRNA conjugates containing carbocyclic nucleosides 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, and subcutaneously injected with compounds 1 to 7 and 9 in Table 2 of Example 3, with a single dose of 1 mpk. Before administration (D-1), D7, D14, and D21 after administration, all mice were blooded and serum was separated, and the hAGT level was detected by ELISA. Another mouse was injected with PBS as a negative control group, and the siRNA conjugate injection group containing carbocyclic nucleosides was compared with it. The hAGT protein inhibition rate was calculated by normalization method. The calculation formula of hAGT protein inhibition rate is as follows:

[0095] The protein level data and inhibition rate data of hAGT in mouse serum are shown in Table 4 and Table 5, respectively. The changes of hAGT protein level and inhibition rate in mouse serum are shown in Figure 1 , Figure 2 . Compared with the negative control group, the test samples in each group could significantly inhibit the hAGT protein level 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, which was close to 80% 21 days after administration. Compound 7 could maintain an inhibition rate of more than 60% for 21 days, and compound 2 could maintain an inhibition rate of more than 50% 21 days after administration.

[0096] Table 4 hAGT protein level data in mouse serum

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

[0098] Example 5: Inhibitory effect of siRNA conjugates containing carbocyclic nucleosides on hAGT protein in B6-Rosa26-hAGT model mice B6-Rosa26-hAGT model mice containing humanized AGT were subcutaneously injected with compound 2 in Table 2 of Example 3, and the dosage was single. Compound 2 was divided into low, medium and high dose groups, with doses of 1 mpk, 3 mpk and 9 mpk, respectively, and 6 mice in each dose group. Before administration (D -1), D7, D14 and D21 after administration, all mice were blooded and serum was separated, and ELISA was used to detect hAGT levels. Another mouse was injected with PBS as a negative control group, and the siRNA conjugate injection group containing carbocyclic nucleosides was compared with it. The hAGT protein inhibition rate was calculated by normalization method. The calculation formula of hAGT protein inhibition rate is as follows:

[0099] The protein level data and inhibition rate data of hAGT in mouse serum are shown in Table 6 and Table 7, respectively. The changes of hAGT protein level and inhibition rate in mouse serum are shown in Figure 3 , Figure 4 . Compared with the negative control group, the test samples in each group can significantly inhibit the protein level of hAGT in the serum of B6-Rosa26-hAGT mice on D7, D14, and D21 after administration. Among them, the inhibition rate of the low-dose group of compound 2 can be maintained at more than 49% for 21 days, the inhibition rate of the medium-dose group of compound 2 can be maintained at more than 77% for 21 days, and the inhibition rate of the high-dose group of compound 2 can be maintained at more than 90% for 21 days. The inhibition rates of Day 7, Day 14, and Day 21 after administration of the test samples in each dosage group are relatively close, indicating that the inhibitory effect of each test sample on hAGT in mouse serum is not only significant, but also maintains a good effect.

[0100] Table 6 hAGT protein level data in mouse serum

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

[0102] Example 6: Inhibitory effect of siRNA conjugates containing carbocyclic nucleosides on hAGT protein in B6-Rosa26-hAGT model mice B6-Rosa26-hAGT model mice containing humanized AGT were subcutaneously injected with compounds 8-9 in Table 2 of Example 3, and the dosage was 3 mpk for each compound 8-9, with 3 mice in each group. Before administration (D-1), D7, D14, and D21 after administration, blood was collected from all mice to separate serum, and hAGT levels were detected by ELISA. Another mouse was injected with PBS as a negative control group, and the siRNA conjugate injection group containing carbocyclic nucleosides was compared with it. The hAGT protein inhibition rate was calculated by normalization method, and the hAGT protein inhibition rate calculation formula is as follows:

[0103] The protein level data and inhibition rate data of hAGT in mouse serum are shown in Table 8 and Table 9, respectively. The changes of hAGT protein level and inhibition rate in mouse serum are shown in Figure 5 , Figure 6 . Compared with the negative control group, the test samples in each group can significantly inhibit the hAGT protein level in the serum of B6-Rosa26-hAGT mice on D7, D14, and D21 after administration. The inhibition rate of compound 8 can be maintained at more than 86% for 21 days; the inhibition rate of compound 9 can be maintained at more than 90% for 21 days. The inhibition rates of the test samples in each group on Day 7, Day 14, and Day 21 after administration are relatively close, indicating that the inhibitory effect of each test sample on hAGT in mouse serum is not only significant, but also maintains a good effect.

[0104] Table 8 hAGT protein level data in mouse serum

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

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

[0107] The sequence modification of siRNA conjugate-compound 10 without carbocyclic nucleoside is shown in Table 10, and its inhibitory effect on AGT mRNA in vitro was tested in Huh7 cells. The starting concentration of the test compound 10 was 0.500 nM, and it was diluted 4 times. Seven concentration points were set, and two replicate wells were measured in parallel. The dilution concentrations were 0.00012 nM, 0.0005 nM, 0.002 nM, 0.008 nM, 0.031 nM, 0.125 nM, and 0.500 nM. The transfected siRNA conjugate was mixed with the Huh7 cell suspension at an equal volume of 2×10 4 cells / well, at 37°C, 5% CO 2 After culturing for 48 h under the above conditions, the cells were collected and RNA was isolated and extracted from the cells. 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 carbocyclic nucleoside modification on hAGT protein in B6-Rosa26-hAGT model mice B6-Rosa26-hAGT model mice containing humanized AGT were subcutaneously injected with compound 10 in Table 10 of Comparative Example 1, with a single dose of 1 mpk. Before administration (D-1), D7, D14, and D21 after administration, blood was collected from all mice to separate serum, and hAGT levels were detected by ELISA. Another mouse was injected with PBS as a negative control group. There were 3 mice in each group. The hAGT protein inhibition rate was calculated by normalization method, and the hAGT protein inhibition rate calculation formula is as follows:

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

[0111] Table 12 hAGT protein level data in mouse serum

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

[0113] Obviously, the above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the invention.

Claims

1. A siRNA conjugate containing a carbocyclic nucleoside, characterized in that The siRNA conjugate comprises a sense strand and an antisense strand, and comprises at least one carbocyclic nucleoside having a structure of the following formula (I): （I） Wherein, 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.

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. The siRNA conjugate according to claim 1, characterized in that The sense strand is at least 19 consecutive nucleotides, the antisense strand is at least 21 consecutive nucleotides, and the sense strand is completely or partially complementary to the antisense strand.

4. The siRNA conjugate according to claim 1, characterized in that The sense strand comprises 2'-O-methyl modified nucleosides and / or 2'-fluoro modified nucleosides.

5. The siRNA conjugate according to claim 1, characterized in that The antisense strand comprises 2'-O-methyl modified nucleosides and / or 2'-fluoro modified nucleosides.

6. The siRNA conjugate according to claim 1, characterized in that The 3' end and / or the 5' end of the sense strand independently contain 1 to 5 phosphorothioate linkages.

7. The siRNA conjugate according to claim 1, characterized in that The 3' end and / or 5' end of the antisense strand independently contain 1 to 5 phosphorothioate linkages.

8. The siRNA conjugate according to claim 1, characterized in that Also comprises at least one ligand.

9. The siRNA conjugate according to claim 8, characterized in that The ligand includes GalNAc and / or its derivatives.

10. The siRNA conjugate according to any one of claims 8 to 9, characterized in that The ligand is conjugated to the 3' end and / or 5' end of the siRNA sense strand.

11. The siRNA conjugate according to any one of claims 8 to 9, characterized in that The ligand is conjugated to the 3' end and / or 5' end of the antisense strand of the siRNA.

12. A pharmaceutical composition containing a siRNA conjugate of a carbocyclic nucleoside, characterized in that: The siRNA conjugate pharmaceutical composition comprises the carbocyclic nucleoside-containing siRNA conjugate according to any one of claims 1 to 11 or a pharmaceutically acceptable salt thereof.

13. The pharmaceutical composition according to claim 12, characterized in that Pharmaceutically acceptable carriers and / or diluents are also included.

14. Use of the siRNA conjugate containing carbocyclic nucleoside according to any one of claims 1 to 11 or a pharmaceutically acceptable salt thereof and / or the siRNA conjugate pharmaceutical composition according to any one of claims 12 to 13 in the preparation of a drug for reducing AGT protein levels.

15. Use of the siRNA conjugate containing carbocyclic nucleoside according to any one of claims 1 to 11 or a pharmaceutically acceptable salt thereof and / or the siRNA conjugate pharmaceutical composition according to any one of claims 12 to 13 in the preparation of a drug for treating, preventing and / or alleviating AGT-related diseases.

16. The use according to claim 15, characterized in that The AGT-related diseases include: hypertension, kidney disease, atherosclerosis, heart failure, stroke or fatty degeneration of the liver.

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