Double-stranded oligonucleotides containing terminal nucleotide modifications and their application in inhibiting AGT gene expression

Abstract

The present disclosure provides a double-stranded oligonucleotide containing terminal nucleotide modification and its application in inhibiting AGT gene expression, belonging to the field of nucleic acid drug technology. The double-stranded oligonucleotide provided by the present disclosure can induce silencing complex (RISC)-mediated cleavage of RNA transcripts of angiotensinogen (AGT) gene, inhibit the expression of AGT gene, thereby helping to treat or prevent diseases or conditions mediated by angiotensinogen (AGT) gene, and has good application prospects.

Description

Technical Field

[0001] The present disclosure relates to the technical field of nucleic acid drugs, and in particular to a double-stranded oligonucleotide containing terminal nucleotide modifications and its application in inhibiting AGT gene expression. Background Art

[0002] Angiotensinogen (AGT), also known as SERPINA8 or ANHU, is a member of the serpin family and is a component of the renin-angiotensin-aldosterone system (RAAS), which plays a key role in blood pressure regulation. The juxtaglomerular cells of the kidney secrete renin into the circulation. It is primarily produced in the liver and released into the circulation where renin converts it to angiotensin I. Angiotensin I is subsequently converted to angiotensin II by angiotensin converting enzyme (ACE). Angiotensin II is a peptide hormone that causes vasoconstriction, which in turn can increase blood pressure. Angiotensin II also stimulates the secretion of the hormone aldosterone by the adrenal cortex. Aldosterone causes the kidneys to increase the reabsorption of sodium and water, leading to an increase in the volume of fluid in the body, which in turn can increase blood pressure. Overstimulation or activity of the RAAS pathway can lead to high blood pressure. Chronically high blood pressure is called hypertension. The high blood pressure in people with hypertension requires the heart to work harder to circulate blood through the blood vessels.

[0003] The World Health Organization (WHO) has identified hypertension as a major cause of cardiovascular morbidity. Hypertension is a major risk factor for a variety of diseases, disorders, and conditions, such as shortened life expectancy, chronic kidney disease, stroke, myocardial infarction, heart failure, vascular aneurysms (e.g., aortic aneurysms), peripheral arterial disease, heart damage (e.g., cardiac enlargement or hypertrophy), and other cardiovascular-related diseases, disorders, and / or conditions.

[0004] Despite the large number of antihypertensive drugs available for the treatment of hypertension, more than two-thirds of subjects cannot be controlled with one antihypertensive drug and require two or more antihypertensive drugs selected from different drug classes. Since compliance and side effects increase with increased medication, this further reduces the number of subjects with controlled blood pressure. Therefore, alternative and combination therapies for subjects with angiotensinogen-related diseases remain to be studied.

[0005] In view of this, the present disclosure is proposed. Summary of the invention

[0006] The present disclosure provides a double-stranded oligonucleotide, which can induce silencing complex (RISC)-mediated cleavage of RNA transcripts of angiotensinogen (AGT) gene, inhibit the expression of AGT gene, and can be used to treat or prevent diseases or conditions mediated by angiotensinogen (AGT) gene. The present disclosure also provides conjugates, pharmaceutical compositions, kits including the double-stranded oligonucleotide, and methods and uses thereof for inhibiting or reducing AGT gene expression or treating AGT gene-mediated diseases or symptoms.

[0007] The present disclosure includes the following technical solutions:

[0008] In a first aspect of the present disclosure, the present disclosure provides a double-stranded oligonucleotide for inhibiting the expression of an AGT gene, the double-stranded oligonucleotide comprising a sense strand and an antisense strand, the antisense strand comprising or being selected from a nucleotide sequence of at least 15, at least 16, at least 17, at least 18, at least 19, at least 20 or at least 21 consecutive nucleotides in any sequence as shown in formula (A), or comprising a nucleotide sequence having 1, 2 or 3 nucleotide differences with the consecutive nucleotides; the sense strand comprises at least 17 nucleotides, and the sense strand is complementary or substantially complementary to the antisense strand to form a duplex region, wherein the substantially complementary means that the mismatch between the sense strand and the antisense strand in the duplex region does not exceed 3 nucleotides;

[0009] Formula (A) 5'-X1GUUUCUUCAUCCAGUUGA(X2) m (X3) n -3';

[0010] Wherein, X1 is selected from A or U;

[0011] (X2) m (X3) n represents the protruding base at the 3' end, X2 and X3 are each independently selected from A, U, G, C or T, m and n are independently selected from 0 or 1, and m+n≥1.

[0012] Furthermore, at least one of the nucleotides X2 and X3 is a [2'-R1-2'-R2] disubstituted modified nucleotide, wherein the disubstituted modified nucleotide refers to a nucleotide in which the hydroxyl group and hydrogen at the 2' position of the ribose of the nucleotide are substituted, wherein R1 and R2 are each independently selected from halogen (preferably F), optionally substituted C1-C6 alkyl (preferably methyl or ethyl) or optionally substituted C1-C6 alkoxy (preferably methoxy).

[0013] In some specific embodiments of the present disclosure, X1 is selected from A. In other specific embodiments of the present disclosure, X1 is selected from U.

[0014] In some optional embodiments of the present disclosure, (X2) m (X3) n is selected from GG, UU, TT, CC, AA, G, U, T, C or A. In other optional embodiments of the present disclosure, (X2) m (X3) n is selected from UU or U. In some embodiments of the present disclosure, (X2) m (X3) n is selected from UU. In some other embodiments of the present disclosure, (X2) m (X3) n Selected from U.

[0015] According to an embodiment of the present disclosure, the antisense strand comprises or is selected from any one of the sequences shown in A1)-A3), or a nucleotide sequence having 1 or 2 nucleotide differences from any of the above sequences:

[0016] A1) 5'-AGUUUCUUCCAGUUGAUU-3';

[0017] A2) 5'-AGUUUCUUCAUCCAGUUGAU-3';

[0018] A3) 5'-UGUUUCUUCCAGUUGAUU-3'.

[0019] According to an embodiment of the present disclosure, the sense strand comprises or is selected from a nucleotide sequence of at least 15, at least 16, at least 17, at least 18 or at least 19 consecutive nucleotides in any one of the sequences shown in B1) to B2), or comprises a nucleotide sequence having 1, 2 or 3 nucleotide differences with the consecutive nucleotides;

[0020] B1) 5'-UCAACUGGAUGAAGAAACU-3';

[0021] B2) 5'-UCAACUGGAUGAAGAAACA-3'.

[0022] In a specific implementation of the present disclosure, the antisense strand comprises or is selected from any one of the sequences shown in A1)-A3), and the sense strand comprises or is selected from any one of the sequences shown in B1)-B2).

[0023] In some specific embodiments of the present disclosure, the double-stranded oligonucleotide is selected from one or more of the following groups:

[0024] 1) The antisense strand has a nucleotide sequence as shown in A1 (SEQ ID NO.3), and the sense strand has a nucleotide sequence as shown in B1 (SEQ ID NO.1);

[0025] 2) the antisense strand has a nucleotide sequence as shown in A2 (SEQ ID NO. 4), and the sense strand has a nucleotide sequence as shown in B1 (SEQ ID NO. 1);

[0026] 3) The antisense strand has a nucleotide sequence as shown in A3 (SEQ ID NO. 5), and the sense strand has a nucleotide sequence as shown in B2 (SEQ ID NO. 2).

[0027] Table 1 Naked sequence nucleotide information of double-stranded oligonucleotides

[0028]

[0029] In some embodiments of the present disclosure, each nucleotide in the double-stranded oligonucleotide is independently selected from modified or unmodified nucleotides.

[0030] In some embodiments of the present disclosure, at least one nucleotide in the sense strand or the antisense strand of the double-stranded oligonucleotide is a modified nucleotide, for example, the modified nucleotide is a nucleotide group in which the ribose group and optionally the phosphate group are modified, but is not limited thereto.

[0031] According to an embodiment of the present disclosure, all nucleotides in the double-stranded oligonucleotide are substantially selected from modified nucleotides. Wherein, "all nucleotides in the double-stranded oligonucleotide are substantially selected from modified nucleotides" means that most but not all of the nucleotides in the double-stranded oligonucleotide are modified nucleotides, and may contain no more than 5, 4, 3, 2 or 1 unmodified nucleotides.

[0032] According to an embodiment of the present disclosure, all nucleotides in the double-stranded oligonucleotide are selected from modified nucleotides, and the modification includes one or both of a single substitution or a double substitution modification at the 2' position of the ribose group of the nucleotide.

[0033] Among them, the structural formula of the unmodified nucleotide is: , Base represents a nucleoside base, and the nucleoside base on each nucleotide is independently selected from uracil U, thymine T, cytosine C, adenine A or guanine G.

[0034] In an optional embodiment of the present disclosure, each nucleotide in the double-stranded oligonucleotide is independently selected from the following modified nucleotides:

[0035] 2'-Fluoro-modified nucleotides, 2'-deoxy-modified nucleotides, 2'-O-methyl-modified nucleotides, 2'-O-(CH2) x-O-R3 modified nucleotides, 2'-O-Si(R4)3 modified nucleotides, 2'-amino modified nucleotides, abasic nucleotides, quasi-nucleotides or [2'-F-2'-methyl] disubstituted modified nucleotides.

[0036] The quasi-nucleotides disclosed herein are selected from one or more of peptide nucleic acid (PNA), morpholino nucleic acid (MNA), bridged nucleic acid (BNA), locked nucleic acid (LNA), glycol nucleic acid / glycerol nucleic acid (GNA), threose nucleic acid (TNA) or unlocked nucleic acid (UNA).

[0037] wherein x is selected from 1 or 2, R3 is selected from optionally substituted C 1-6 Alkyl or optionally substituted C 1-6 Alkoxy, if R3 contains a substituent, the substituent is selected from halogen, C 1-6 Alkoxy, hydroxy or amino.

[0038] R4 is independently selected from optionally substituted C 1-6 Alkyl, if R4 contains a substituent, the substituent is selected from halogen, C1-C3 alkyl or C1-C3 alkoxy.

[0039] In the present disclosure, 2'-O-(CH2) x -R3 modified nucleotides refer to nucleotides whose 2' hydroxyl group of the ribose group is replaced by -O-(CH2) x -R3 substitution. Wherein, when x is selected from 1, the 2'-O-(CH2) x -R3 modified nucleotides are selected from 2'-O-ethoxymethyl modified nucleotides or 2'-O-2,2,2-trifluoroethoxymethyl modified nucleotides. When x is selected from 2, the 2'-O-(CH2) x -R3 modified nucleotides are selected from 2'-O-methoxyethyl modified nucleotides (also known as 2'-O-moe modified nucleotides).

[0040] In some specific embodiments of the present disclosure, the 2'-O-(CH2) x The -O-R3 modified nucleotide is selected from a 2'-O-methoxyethyl modified nucleotide or a 2'-O-ethoxymethyl modified nucleotide.

[0041] In the present disclosure, "2'-O-Si(R4)3 modified nucleotide" refers to a nucleotide in which the 2' hydroxyl group of the ribose group of the nucleotide is replaced by -O-Si(R4)3. Exemplarily, 2'-O-TBDMS modified nucleotide, 2'-O-TIPS modified nucleotide or 2'-O-TOM modified nucleotide; wherein the structural formula of TBDMS is The structural formula of TIPS is , the structural formula of TOM is .

[0042] In some specific embodiments of the present disclosure, the 2'-O-Si(R4)3 modified nucleotide is selected from 2'-O-TBDMS modified nucleotide, 2'-O-TIPS modified nucleotide or 2'-O-TOM modified nucleotide.

[0043] In some specific embodiments of the present disclosure, the structural formula of the [2'-F-2'-methyl] disubstituted modified nucleotide is ; Base represents the nucleobase A, U, G, C or T.

[0044] According to an embodiment of the present disclosure, all nucleotides in the double-stranded oligonucleotide are selected from modified nucleotides, wherein the modification includes a single substitution modification or a double substitution modification at the 2' position of the ribose group of the nucleotide, and the nucleotides in the double-stranded oligonucleotide are each independently selected from the following modified nucleotides: at least three of 2'-fluorine-modified nucleotides, 2'-O-methyl-modified nucleotides, 2'-O-methoxyethyl-modified nucleotides, and [2'-F-2'-methyl] double-substituted modified nucleotides.

[0045] In some specific embodiments of the present disclosure, the antisense strand of the double-stranded oligonucleotide contains at least one 2'-O-methoxyethyl modified nucleotide.

[0046] In some specific embodiments of the present disclosure, in the direction from the 5' end to the 3' end, the 20th and / or 21st nucleotides of the antisense strand are [2'-F-2'-methyl] disubstituted modified nucleotides.

[0047] In some specific embodiments of the present disclosure, in the direction from the 5' end to the 3' end, the 19th nucleotide of the sense strand is a [2'-F-2'-methyl] disubstituted modified nucleotide.

[0048] In some embodiments of the present disclosure, in the direction from the 5' end to the 3' end, at least 3 nucleotides in positions 7-10 of the nucleotide sequence in the sense strand are selected from 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions are independently selected from 2'-O-methyl-modified nucleotides.

[0049] In some embodiments of the present disclosure, in the direction from the 5' end to the 3' end, at least 3 nucleotides in positions 7-10 of the nucleotide sequence in the positive strand are selected from 2'-fluoro-modified nucleotides, the nucleotide at position 19 is selected from [2'-F-2'-methyl] disubstituted modified nucleotides, and the nucleotides at the remaining positions are independently selected from 2'-O-methyl modified nucleotides.

[0050] According to an embodiment of the present disclosure, in the direction from the 5' end to the 3' end, at least four nucleotides among the nucleotides at positions 2, 6, 9-12, 14 and 16 of the nucleotide sequence in the antisense strand are selected from 2'-fluoro-modified nucleotides, and the nucleotides at positions 2, 6, 14 and 16 are selected from 2'-fluoro-modified nucleotides, the nucleotides at positions 20 and / or 21 are selected from [2'-F-2'-methyl] disubstituted modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl modified nucleotides.

[0051] According to an embodiment of the present disclosure, at least four nucleotides among the nucleotides at positions 2, 6, 9-12, 14 and 16 of the nucleotide sequence in the antisense strand are selected from 2'-fluoro-modified nucleotides, and the nucleotides at positions 2, 6, 14 and 16 are selected from 2'-fluoro-modified nucleotides, the nucleotides at positions 8 and / or 15 are selected from 2'-O-methoxyethyl-modified nucleotides, the nucleotides at positions 20 and / or 21 are selected from [2'-F-2'-methyl] disubstituted modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified nucleotides.

[0052] According to an embodiment of the present disclosure, each nucleotide of the double-stranded oligonucleotide is independently selected from a modified nucleotide, and the modification is selected from any one of the following (1)-(2):

[0053] 1) In the direction from the 5' end to the 3' end, the nucleotides at positions 7 to 10 of the nucleotide sequence in the sense strand are selected from 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified nucleotides; and the nucleotides at positions 2, 6, 9, 14 and 16 of the nucleotide sequence in the antisense strand are selected from 2'-fluoro-modified nucleotides, the nucleotides at positions 20 and / or 21 are selected from [2'-F-2'-methyl] disubstituted modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified nucleotides.

[0054] 2) In the direction from the 5' end to the 3' end, the nucleotides at positions 7 to 10 of the nucleotide sequence in the sense strand are selected from 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified nucleotides; and the nucleotides at positions 2, 6, 9, 14 and 16 of the nucleotide sequence in the antisense strand are selected from 2'-fluoro-modified nucleotides, the nucleotide at position 15 is selected from 2'-O-methoxyethyl-modified nucleotides, the nucleotides at positions 20 and / or 21 are selected from [2'-F-2'-methyl] disubstituted modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified nucleotides.

[0055] According to an embodiment of the present disclosure, the sense strand and / or the antisense strand independently comprises one or more phosphorothioate bonds.

[0056] According to an embodiment of the present disclosure, at least one or at least two of the following connections between nucleotides in the sense strand are phosphorothioate bonds:

[0057] The connection between the first nucleotide and the second nucleotide at the 5' end of the sense strand;

[0058] The connection between the second nucleotide and the third nucleotide at the 5' end of the sense strand;

[0059] The connection between the first nucleotide and the second nucleotide at the 3' end of the sense strand;

[0060] The connection between the second nucleotide and the third nucleotide at the 3' end of the sense strand;

[0061] as well as,

[0062] The following nucleotides in the antisense strand are linked by phosphorothioate bonds:

[0063] the junction between the first and second nucleotides, and the junction between the second and third nucleotides at the 5' end of the antisense strand;

[0064] The connection between the first nucleotide and the second nucleotide at the 3' end of the antisense strand, and the connection between the second nucleotide and the third nucleotide.

[0065] In some embodiments of the present disclosure, in the direction from the 5' end to the 3' end, the internucleoside bond between the 10th nucleotide and the 11th nucleotide of the antisense strand is selected from a phosphorothioate bond.

[0066] According to an embodiment of the present disclosure, from the 5' end to the 3' end, the sense strand of the double-stranded oligonucleotide comprises or is selected from any one of the modified nucleotide sequences shown in MB1)-MB4):

[0067] MB1) UmsCmsAmAmCmUmGfGfAfUfGmAmAmGmAmAmAmCmsUm;

[0068] MB2) UmsCmAmAmCmUmGfGfAfUfGmAmAmGmAmAmAmsCmsUm;

[0069] MB3) UmsCmAmAmCmUmGfGfAfUfGmAmAmGmAmAmAmsCms(NM);

[0070] MB4) UmsCmsAmAmCmUmGfGfAfUfGmAmAmGmAmAmAmCmsAm.

[0071] According to an embodiment of the present disclosure, in the direction from the 5' end to the 3' end, the antisense strand of the double-stranded oligonucleotide comprises or is selected from any one of the modified nucleotide sequences shown in MA1)-MA5):

[0072] MA1) AmsGfsUmUmUmCfUmUmCfAmsUmCmCmAfGmUfUmGmAms(NM)s(NM);

[0073] MA2) AmsGfsUmUmUmCfUmUmCfAmsUmCmCmAfG(moe)UfUmGmAms(NM)s(NM);

[0074] MA3) AmsGfsUmUmUmCfUmUmCfAmUmCmCmAfG(moe)UfUmGmsAms(NM);

[0075] MA4) AmsGfsUmUmUmCfUmUmCfAmUmCmCmAfG(moe)UfUmGmAms(NM)s(NM);

[0076] MA5)UmsGfsUmUmUmCfUmUmCfAmUmCmCmAfG(moe)UfUmGmAms(NM)s(NM).

[0077] In some specific embodiments of the present disclosure, the double-stranded oligonucleotide includes one or more groups from Group 1) to Group 5) shown in Table 2.

[0078] Table 2 Nucleotide information of double-stranded oligonucleotide modified sequences

[0079]

[0080] Wherein, C, G, U, A, T represent cytidine-3'-phosphate, guanosine-3'-phosphate, uridine-3'-phosphate, adenosine-3'-phosphate, thymidine-3'-phosphate, respectively; m represents that the nucleotide represented by the capital letter adjacent to the left of the letter m is a 2'-O-methyl modified nucleotide; f represents that the nucleotide represented by the capital letter adjacent to the left of the letter f is a 2'-fluorine modified nucleotide; (moe) represents that the nucleotide represented by the capital letter adjacent to the left of the combination mark (moe) is a 2'-O-methoxyethyl modified nucleotide; s represents that the internucleoside bond between the nucleotide adjacent to the left and the nucleotide adjacent to the right is a phosphorothioate bond;

[0081] (NM) indicates a nucleotide in which the 2'-hydroxyl group and hydrogen of the ribose are replaced by [2'-F-2'-methyl], and the structural formula is .

[0082] In an optional embodiment, the double-oligonucleotide is selected from siRNA.

[0083] In a second aspect of the present disclosure, the present disclosure provides a conjugate, which comprises the double-stranded oligonucleotide according to the first aspect of the present disclosure, and one or more ligands capable of binding to a cell receptor conjugated to the double-stranded oligonucleotide.

[0084] The ligand can be covalently or chemically conjugated to the double-stranded oligonucleotide, and the binding of the ligand to the cell receptor can promote the specific targeting of the conjugate to the target cell and internalization of the conjugate into the target cell, so as to inhibit the translation of AGT mRNA into amino acids and proteins in the target cell, and can effectively alleviate, prevent and / or treat diseases or conditions mediated by the angiotensinogen (AGT) gene.

[0085] In some optional embodiments of the present disclosure, the ligand is selected from asialoglycoprotein receptor ligands (ASGPR ligands).

[0086] In some optional embodiments of the present disclosure, the ligand comprises at least one N-acetyl galactosamine (GalNAc).

[0087] In some optional embodiments of the present disclosure, the ligand is selected from the structure shown in formula (I), or its stereoisomers, or its tautomers, or its pharmaceutically acceptable salts:

[0088]

[0089] Ⅰ

[0090] In formula (I), * represents the conjugation site of the ligand and the double-stranded oligonucleotide, and j is selected from 1, 2, 3 or 4;

[0091] In some optional embodiments of the present disclosure, each Z is independently selected from hydroxyl or thiol. In some specific embodiments of the present disclosure, each Z is hydroxyl.

[0092] In some embodiments of the present disclosure, each p is independently 1 or 2.

[0093] In some specific embodiments of the present disclosure, each p is 1.

[0094] In some embodiments of the present disclosure, each q is independently 1 or 2.

[0095] In some specific embodiments of the present disclosure, each q is 1.

[0096] In some specific embodiments of the present disclosure, each p is 1, and each q is 1.

[0097] Each L is independently selected from C1-C 30 Alkylene or ; where each R L2a Each independently selected from C1-C 10 Alkylene, each R L2b Each is independently selected from O, S, NH or -NH-C(O)-, and k is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0098] Further, in some specific embodiments of the present disclosure, the ligand has a structure as shown in formula (II), or a stereoisomer thereof, or a tautomer thereof, or a pharmaceutically acceptable salt thereof:

[0099]

[0100] Ⅱ

[0101] In formula (II), j is selected from 1, 2, 3 or 4; L is independently selected from -CH2-, -CH2-CH2-, -CH2-CH2-CH2-, -(CH2)4-, -(CH2)5-, -(CH2)6-, -(CH2)7-, -(CH2)8-, -(CH2)9-, -(CH2) 10 -, -CH2-NH-CO-(CH2)5-, -(CH2)2-NH-CO-(CH2)4-, -(CH2)3-NH-CO-(CH2)3-, -(CH2)4-NH-CO-(CH2)2-, or -(CH2)5-NH-CO-CH2-.

[0102] In some alternative embodiments of the present disclosure, each L is independently selected from -CH2-CH2- or -(CH2)2-NH-CO-(CH2)4-.

[0103] In some specific embodiments of the present disclosure, L is selected from -CH2-CH2-.

[0104] In some specific embodiments of the present disclosure, L is selected from -(CH2)2-NH-CO-(CH2)4-.

[0105] In some specific embodiments of the present disclosure, the ligand has a structure represented by formula (III), formula (IV), formula (V) or formula (VI), or a stereoisomer thereof, or a tautomer thereof, or a pharmaceutically acceptable salt thereof:

[0106] , , , .

[0107] According to an embodiment of the present disclosure, the number of the ligand is selected from one, and one of the ligands is conjugated to the 3' end of the sense strand of the double-stranded oligonucleotide.

[0108] According to an embodiment of the present disclosure, the number of the ligands is selected from two, and the two ligands are conjugated to the 5' end of the sense strand and the 3' end of the antisense strand of the double-stranded oligonucleotide, respectively.

[0109] In some specific embodiments of the present disclosure, the conjugate includes one or more of R303079, R303082, R303086, R303087, R303088, and R303089 shown in Table 3.

[0110] Table 3 Nucleotide information of conjugates

[0111]

[0112] Among them, (CR01008×2), (CR01008×3) and (CR01008×4) all represent ligands; if the ligand is located at the 5' end of the sense chain, it means that the ligand is conjugated to the 5' end of the sense chain; if the ligand is located at the 3' end of the sense chain, it means that the ligand is conjugated to the 3' end of the sense chain; if the ligand is located at the 3' end of the antisense chain, it means that the ligand is conjugated to the 3' end of the antisense chain.

[0113] The structural formula of (CR01008×2) is: ;

[0114] The structural formula of (CR01008×3) is: ;

[0115] The structural formula of (CR01008×4) is: .

[0116] In a third aspect of the present disclosure, the present disclosure provides a pharmaceutical composition, comprising any of the following items and a pharmaceutically acceptable excipient:

[0117] (I) the double-stranded oligonucleotide described in the first aspect of the present disclosure; and / or

[0118] (II) The conjugate according to the second embodiment of the present disclosure.

[0119] Both the double-stranded oligonucleotide and the conjugate can degrade AGT mRNA and inhibit the expression of AGT. Therefore, the pharmaceutical composition disclosed herein can effectively prevent and / or treat diseases or conditions mediated by the angiotensinogen (AGT) gene.

[0120] The pharmaceutical compositions disclosed herein include preparations suitable for parenteral administration. The preparations can be conveniently present in unit dosage form and can be prepared by any method known in the pharmaceutical field. The amount of active ingredient that can be combined with excipients to prepare a single dose form is generally the amount of siRNA that produces a therapeutic effect.

[0121] In a fourth aspect of the present disclosure, the present disclosure provides the use of any of the following items in the preparation of a medicament for alleviating, preventing and / or treating a disease or condition mediated by the AGT gene:

[0122] (I) the double-stranded oligonucleotide described in the first aspect of the present disclosure; and / or

[0123] (II) the conjugate according to the second aspect of the present disclosure; and / or

[0124] (III) The pharmaceutical composition according to the third aspect of the present disclosure.

[0125] According to an embodiment of the present disclosure, the diseases or conditions include hypertension, critical hypertension, essential hypertension, secondary hypertension, hypertensive crisis, hypertensive emergency state, isolated systolic and diastolic hypertension, pregnancy-related hypertension, diabetic hypertension, resistant hypertension, refractory hypertension, paroxysmal hypertension, renovascular hypertension, Goldblatt's hypertension, ocular hypertension, glaucoma, pulmonary hypertension, portal hypertension, systemic venous hypertension, systolic hypertension, unstable hypertension; hypertensive heart disease, hypertensive nephropathy, atherosclerosis, arteriosclerosis, vascular disease, diabetic nephropathy, diabetic retinopathy, chronic heart failure, cardiomyopathy, diabetic cardiomyopathy, glomerulosclerosis, aortic coarctation, aortic aneurysm and ventricular fibrosis.

[0126] The effective amount of the double-stranded oligonucleotide, conjugate or pharmaceutical composition disclosed herein may vary with the mode of administration and the severity of the disease to be treated. The selection of the preferred effective amount can be determined by a person of ordinary skill in the art based on various factors (e.g., through clinical trials). The factors include, but are not limited to: pharmacokinetic parameters of the active ingredient such as bioavailability, metabolism, half-life, etc.; the severity of the disease to be treated by the patient, the patient's weight, the patient's immune status, the route of administration, etc. For example, depending on the urgency of the treatment situation, several divided doses may be administered daily, for example, four times a day, three times a day, twice a day, once a day, or once every other day, or several doses administered daily may be proportionally reduced.

[0127] The drug can be administered to a subject by any suitable route known in the art, including but not limited to oral or parenteral routes, including intravenous administration, intramuscular administration, subcutaneous administration, transdermal administration, airway administration (aerosol), pulmonary administration, nasal administration, rectal administration and topical administration (including buccal administration and sublingual administration), preferably intravenous injection.

[0128] In a fifth aspect of the present disclosure, the present disclosure provides a kit, the kit comprising any of the following items:

[0129] (I) the double-stranded oligonucleotide described in the first aspect of the present disclosure; and / or

[0130] (II) the conjugate according to the second aspect of the present disclosure; and / or

[0131] (III) The pharmaceutical composition according to the third aspect of the present disclosure.

[0132] The double-stranded oligonucleotides, conjugates and pharmaceutical compositions provided by the present disclosure can induce silencing complex (RISC)-mediated cleavage of RNA transcripts of the angiotensinogen (AGT) gene, inhibit the expression of the AGT gene, and help treat or prevent diseases or disorders mediated by the angiotensinogen (AGT) gene. BRIEF DESCRIPTION OF THE DRAWINGS

[0133] Figure 1 The following is the change in the level of AGT protein in the serum of cynomolgus monkeys after administration of the siRNA conjugate in Example 1. DETAILED DESCRIPTION

[0134] The technical solution of the present disclosure will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are part of the embodiments of the present disclosure, rather than all of the embodiments. Based on the embodiments in the present disclosure, all other embodiments obtained by ordinary technicians in the field without creative work are within the scope of protection of the present disclosure.

[0135] Explanation of terms

[0136] In the present disclosure, the terms “comprise” or “include” are open expressions, that is, including the contents specified in the present disclosure but not excluding other contents.

[0137] In this disclosure, the terms "optionally", "optional" or "optionally" generally mean that the subsequently described event or circumstance can but need not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not occur.

[0138] In the present disclosure, "2'-position monosubstituted modification" means that the 2'-position hydroxyl group of ribose in a nucleotide is substituted.

[0139] In the present disclosure, "2'-position double substitution modification" means that the 2'-hydroxyl group and the 2'-hydrogen group of the ribose in the nucleotide are simultaneously substituted. For example, a [2'-F-2'-methyl] substituted modified nucleotide has the structural formula .

[0140] In the present disclosure, the term "small interfering RNA (siRNA)" is a double-stranded RNA of 17 to 25 nucleotides in length, comprising a sense strand and an antisense strand. siRNA mediates targeted cleavage of RNA transcripts of the RISC pathway by forming a silencing complex (RISC). Specifically, siRNA guides the specific degradation of mRNA sequences through the known RNA interference (RNAi) process, inhibiting the translation of mRNA into amino acids and proteins. For example, siRNA can regulate (e.g., inhibit) the expression of AGT in cells.

[0141] In the present disclosure, the term "complementary" refers to the ability of an oligonucleotide of a first sequence to hybridize with an oligonucleotide of a second sequence under certain conditions and form a double-stranded structure.

[0142] In the present disclosure, the term "nucleotide difference" and the term "nucleotide base difference" and the term "nucleotide sequence difference" can be used interchangeably. It means that compared with the original nucleotide sequence, the base type of the nucleotide at the same or corresponding position has changed. For example, when one nucleotide base in the original nucleotide sequence is A, when the nucleotide base at the same or corresponding position is changed to U, C, G or dT, dC, dG, etc., it is considered that there is a difference in the nucleotide sequence at this position. It should be noted here that, when compared with the original nucleotide sequence, the nucleotide at the same or corresponding position only differs in the presence or absence of modification or the type of modification, it is not considered that there is a difference in the nucleotide sequence at this position.

[0143] In the present disclosure, the term "overhang" refers to at least one unpaired nucleotide protruding from the double-stranded oligonucleotide double helix structure, and is also a nucleotide sequence in the siRNA structure other than the double-stranded region. For example: when the 3' end of one strand in the sense strand and / or the antisense strand exceeds the 5' end of the other strand, or when the 5' end of one strand in the sense strand and / or the antisense strand exceeds the 3' end of the other strand, there is a nucleotide overhang. The overhang may contain at least one nucleotide, at least two nucleotides, at least three nucleotides, at least four nucleotides, at least five nucleotides or more nucleotides. The nucleotide overhang may contain or be composed of nucleotide / nucleoside analogs, including deoxynucleotides / nucleosides. The overhang may be located in the sense strand, the antisense strand, or any combination thereof. In addition, the nucleotides of the overhang may appear at the 5' end, the 3' end, or both ends of the antisense or sense strand.

[0144] In the present disclosure, the term "inhibiting the expression of the AGT gene" includes any level of inhibition of the AGT gene, for example, at least partial inhibition of the expression of the AGT gene, such as inhibition of at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99%. Wherein, the expression of the AGT gene can be evaluated based on the level of any variable related to the expression of the AGT gene, for example, the mRNA level or protein level of AGT. Inhibition can be evaluated by a reduction in the absolute or relative level of one or more of these variables compared to the control level. The control level can be any type of control level utilized in the art, e.g., a baseline level prior to administration, or a level measured from a similar subject, cell, or sample that has not been treated or that has been treated with a control (e.g., a buffer-only control or a no active agent control).

[0145] In the present disclosure, "conjugation" refers to the connection between two or more chemical moieties by covalent bonding; "conjugate" refers to a compound formed by covalent bonding between chemical moieties; "conjugated molecule" is understood to be a specific compound that can be conjugated to an oligonucleotide through a reaction to ultimately form the oligonucleotide conjugate of the present disclosure.

[0146] In the present disclosure, "pharmaceutical composition" may refer to a composition used for the treatment of a disease, and may also be used for in vitro culture experiments of cells. When used for the treatment of a disease, the term "pharmaceutical composition" generally refers to a unit dosage form, and may be prepared by any of the methods well known in the pharmaceutical field. All methods include the step of combining the active ingredient with an excipient that constitutes one or more accessory ingredients. Typically, the composition is prepared by uniformly and fully combining the active siRNA with a liquid excipient, a finely divided solid excipient, or both.

[0147] In the present disclosure, the term "pharmaceutically acceptable" means that the substance or composition must be chemically and / or toxicologically compatible with the other ingredients of the formulation and / or the mammals treated therewith. Preferably, the "pharmaceutically acceptable" described in the present disclosure refers to those approved by federal regulatory agencies or national governments or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeias for use in animals, especially humans.

[0148] In the present disclosure, the term "pharmaceutically acceptable excipient" may include any solvent, solid excipient, diluent or other liquid excipient, etc., suitable for a specific target dosage form. Except for any conventional excipients that are incompatible with the siRNA of the present disclosure, such as any adverse biological effects produced or interactions with any other components of the pharmaceutically acceptable composition in a harmful manner, their use is also within the scope of the present disclosure.

[0149] In addition to any conventional excipients, to the extent that they are incompatible with the siRNA of the present disclosure, such as producing any adverse biological effects or interacting in a deleterious manner with any other component of the pharmaceutically acceptable composition, their use is also contemplated by the present disclosure.

[0150] The present disclosure is further described below by means of specific examples. However, it should be understood that these examples are only used for more detailed description and should not be construed as limiting the present disclosure in any form.

[0151] Unless otherwise specified, the siRNA sequences used in the present disclosure were commissioned to Kunshan Aotai Biotechnology Co., Ltd. for synthesis; the PCR primers used in the present disclosure were commissioned to Sangon Biotechnology (Shanghai) Co., Ltd. for synthesis.

[0152] The detection data of AGT protein at different time points in the in vivo activity experiment involved in the present disclosure and the calculation method are summarized as follows:

[0153] ;

[0154] In the context of this disclosure, unless otherwise stated, in vivo activity experimental data are presented as The experimental data were plotted and analyzed using GraphPad prism 8.0 software.

[0155] In the context of the present disclosure, unless otherwise stated, the reagent ratios provided below are calculated on a volume basis (v / v).

[0156] Preparation of compounds

[0157] Unless otherwise specified, the reagents used in the preparation of the compounds disclosed herein were purchased from Beijing Coupling Technology Co., Ltd. The information of the main reagents is shown in Table 4.

[0158] Table 4

[0159]

[0160] Among them, CPG stands for controlled aperture glass (Controlled Pore Glass) carrier.

[0161] Preparation Example 1 Preparation of compound CR01008:

[0162] (1-1) The synthetic structure of compound CR01008 is shown below:

[0163]

[0164] The synthetic route of compound CR01008 is as follows:

[0165] .

[0166] (1-1-1) Synthesis of compound 2:

[0167] Compound 1 (trans-4-(Boc-amino)cyclohexylcarboxaldehyde, 10.0 g, 1.0 eq) and formaldehyde aqueous solution (8.9 g, 37 mass%, 2.4 eq) were dissolved in 33 ml of methanol, and 13 ml of KOH aqueous solution with a concentration of 45.3 mass% was added dropwise. After the addition was completed, the reaction was stirred at 25°C for 30 minutes, the temperature was raised to 60°C and refluxed at 60°C for 2 hours. After the reaction was completed, the reaction solution was cooled to room temperature and evaporated to dryness under reduced pressure to obtain a crude product as a white solid. A small amount of water was added to the crude product for pulping and filtration to obtain compound 2 as a white solid (9 g, yield 78. 9%). MS-ESI (m / z) = 260 [M + H] + .

[0168] (1-1-2) Synthesis of compound 3:

[0169] Compound 2 (9 g, 1 eq) was dissolved in 70 ml of 1,4-dioxane, and a solution of hydrogen chloride in 1,4-dioxane (45 ml, 4 M) was added, and the mixture was stirred at 25°C for 1 hour. After the reaction, the reaction solution was evaporated under reduced pressure to obtain compound 3 (6.8 g, yield 100%) as a white solid.

[0170] (1-1-3) Synthesis of compound 5:

[0171] Compound 3 (1.8 g, 2.0 eq), compound 4 (5-[[(2R,3R,4R,5R,6R)-3-acetylamino-4,5-diacetoxy-6-(acetoxymethyl)-2-tetrahydropyranyl]oxy]pentanoic acid, 2.1 g, 1.0 eq) and DIEA ( N,N -Diisopropylethylamine, 3.5 g, 6.0 eq) was dissolved in 15 ml of DMF, HBTU (1.9 g, 1.1 eq) was added, and the mixture was stirred at 25 °C for 3 hours in a N2 atmosphere. After the reaction, the reaction solution was evaporated under reduced pressure and purified by reverse phase column chromatography (22% acetonitrile aqueous solution) to obtain compound 5 (1.78 g, yield 64.4%) as a white solid. MS-ESI (m / z) = 589[M + H] + .

[0172] (1-1-4) Synthesis of compound 6:

[0173] Compound 5 (1.54 g, 1.0 eq) was dissolved in 15 ml of pyridine, the reaction system was cooled to 0°C with an ice-water bath, and DMTrCl (4,4'-dimethoxytriphenylmethane chloride, 1.32 g, 1.5 eq) was added at 0°C, and the reaction was carried out at 25°C for 3 hours. 15 ml of methanol was added to the reaction solution to quench the reaction. After the reaction was completed, the reaction solution was evaporated under reduced pressure and purified by reverse phase column chromatography (60% by volume of acetonitrile in water) to obtain compound 6 (1 g, yield 42.7%) as a yellow solid. MS-ESI ( m / z ) = 891 [M + H] + .

[0174] (1-1-5) Synthesis of compound CR01008:

[0175] Compound 6 (1.08 g, 1.0 eq) was dissolved in 20 ml of anhydrous dichloromethane, and DCI (115 mg, 0.8 eq) and compound 7 (bis(diisopropylamino)(2-cyanoethoxy)phosphine, 732 mg, 2.1 eq) were added respectively. The atmosphere was replaced with nitrogen three times and stirred at 25°C for 2 hours. After the reaction, 20 ml of saturated sodium bicarbonate aqueous solution was added to the reaction solution, and the mixture was extracted three times with 20 ml of dichloromethane (3×20 ml). The organic phases were combined and evaporated to dryness under reduced pressure. The organic phases were purified by reverse phase chromatography (72% by volume acetonitrile aqueous solution) and then vacuum dried for 12 hours to obtain compound CR01008 (1 g, yield 76.0%) as a white powder. MS-ESI (m / z) =1091 [M + Na] + .

[0176] H NMR (400 MHz, DMSO- d 6) δ 1.05 (d, J = 6.7 Hz, 6H).1.14 (d, J = 6.7 Hz,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 = 5.9 Hz,2H), 2.79 (d, J = 8.4 Hz, 1H), 2.87 (d, J = 8.4 Hz, 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, 8.8 Hz, 1H),4.05 (s, 3H),4.51 (d, J = 8.4 Hz, 1H),4.99 (dd, J = 11.3, 3.4 Hz, 1H), 5.24 (d, J =3.4 Hz, 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= 7.8 Hz, 1H), 7.84 (d, J = 9.2 Hz, 1H).

[0177] (1-2) Synthesis of compound CR01008Z:

[0178]

[0179] The synthetic route of compound CR01008Z is as follows:

[0180]

[0181] (1-2-1) Synthesis of compound 9:

[0182] Compound 6 (500 mg) was dissolved in 10 ml of dichloromethane, and compound 8 (succinic anhydride, 112 mg), DMAP (6.8 mg) and TEA (226.2 mg) were added. The nitrogen atmosphere was replaced three times, and the mixture was stirred at 25°C for 16 hours. The mixture was flash purified to obtain compound 9 (300 mg, yield 53.6%). MS-ESI (m / z) = 1013 [M + Na] + .

[0183] (1-2-2) Synthesis of compound CR01008Z:

[0184] Compound 9 (50 mg, aminoCPG (1.25 g, 80 μmol / g, 0.1 mmol), HBTU (27 mg), and DIEA (12 mg) were added to a 20 ml sample bottle and shaken for 16 hours. After the reaction, the reaction solution was filtered to obtain a filter cake, which was first washed once with 10 ml acetonitrile (1×10 ml) and then vacuum dried. The dried filter cake, DMAP (3 mg), Cap1 (10 ml, 200 V) and Cap2 (1 ml, 20 V) were added to a 20 ml sample bottle and shaken for 6 hours. After the reaction, the reaction solution was filtered to obtain a filter cake, which was first washed once with 10 ml acetonitrile (1×10 ml) and then vacuum dried to obtain compound CR01008Z (1.03 g, loading 20-30 μmol / g).

[0185] Among them, Cap1 and Cap2 are capping reagents, Cap1 is a 20 volume % N- A pyridine / acetonitrile mixed solution of methylimidazole, with a volume ratio of pyridine to acetonitrile of 3:5; Cap2 is an acetonitrile solution of acetic anhydride with a concentration of 20 volume %.

[0186] Preparation Example 2 Synthesis of Compound NM054:

[0187] In this preparation example, the synthesis route of compound NM054 is as follows:

[0188]

[0189] (2-1) Synthesis of compound NM054-2:

[0190] Compound NM054-1 (3 g, 11.54 mmol, 1.0 eq, (2'R)-2'-deoxy-2'-fluoro-2'-methyluridine, CAS No. 863329-66-2) and pyridine (30 ml) were added to a 500 ml reactor, the temperature was lowered to 0°C, 4,4'-dimethoxytrityl chloride (4.29 g, 12.7 mmol, 1.1 eq) was added in batches, nitrogen was replaced 3 times, the reaction system was stirred at 25°C for 3 hours in a nitrogen atmosphere, and HPLC showed no raw material. After the reaction was completed, the reaction solution was concentrated, purified water (50 ml) and ethyl acetate (50 ml) were added for extraction, the organic phase was separated, the organic phase was dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography to obtain compound NM054-2 (2.7 g, yield 41.7%). MS ESI (m / z) =563.0 [M + H] + .

[0191] (2-2) Synthesis of compound NM054:

[0192] Compound NM054-2 (2.7 g, 4.8 mmol, 1.0 eq) was added to a 100 ml reaction kettle, and bis(diisopropylamino)(2-cyanoethoxy)phosphine (1.74 g, 5.76 mmol, 1.2 eq) was added in batches, and then 4,5-dicyanoimidazole (0.45 g, 3.8 mmol, 0.8 eq, referred to as DCI, CAS No. 1122-28-7) and dichloromethane (27 ml) were added. The nitrogen atmosphere was replaced three times, and the reaction system was stirred at 25 ° C for 3 hours in a nitrogen atmosphere. After the reaction was completed, sodium bicarbonate aqueous solution (20 ml) was added to the reaction solution, and the organic phase was separated. The organic phase was dried over anhydrous sodium sulfate, filtered, concentrated, and purified by reverse phase column chromatography (eluent: acetonitrile / water = 90 / 10, v / v) to obtain compound NM054 (3.0 g). MS ESI (m / z) = 763 [M + H] + .

[0193] Preparation Example 3 Preparation of siRNA:

[0194] (3-1) Synthesis of the positive chain (SS):

[0195] By the phosphoramidite nucleic acid solid phase synthesis method, the compound on the solid phase carrier is used as the starting cycle to connect nucleoside monomers one by one in the 3'-5' direction according to the nucleotide sequence. In the synthesis process, the compound NM054 is regarded as a nucleoside monomer.

[0196] Each connection of a nucleoside monomer includes four steps of deprotection, coupling, capping, oxidation or sulfurization. The synthesis conditions are given as follows:

[0197] The nucleoside monomer was prepared into an acetonitrile solution with a concentration of 0.1 M.

[0198] The conditions of the deprotection reaction in each step are the same. The conditions of the deprotection reaction are: temperature of 25°C, reaction time of 70 seconds, deprotection reagent of dichloroacetic acid in dichloromethane solution (3 volume %), and the molar ratio of dichloroacetic acid to 4,4'-dimethoxytrityl protecting group on the solid phase carrier is 5:1.

[0199] The conditions of each coupling reaction are the same. The coupling reaction conditions are as follows: temperature is 25°C, the molar ratio of the nucleic acid sequence connected to the solid phase support to the nucleoside monomer is 1:10, the molar ratio of the nucleic acid sequence connected to the solid phase support to the coupling reagent is 1:65, the reaction time is 600 seconds, the coupling reagent is a 0.5M 5-ethylthio-1H-tetrazole acetonitrile solution, and the thio reagent is a 0.2M acetonitrile / pyridine mixed solution of hydrogenated xanthanone (the volume ratio of acetonitrile and pyridine is 1:1).

[0200] The conditions of each capping reaction are the same. The conditions of the capping reaction are: temperature is 25°C; reaction time is 2 minutes; the capping reagent solution is a mixed solution of Cap1 and Cap2 with a molar ratio of 1:1, Cap1 is a pyridine / acetonitrile mixed solution of N-methylimidazole with a concentration of 20 volume%, the volume ratio of pyridine to acetonitrile is 3:5, Cap2 is an acetonitrile solution of acetic anhydride with a volume ratio of 20 volume%; the molar ratio of N-methylimidazole in Cap1 capping reagent, acetic anhydride in Cap2 capping reagent to the nucleic acid sequence connected to the solid phase carrier is 1:1:1.

[0201] The conditions for each step of the oxidation reaction are the same. The conditions for the oxidation reaction are: temperature 25°C; reaction time 3 seconds; oxidation reagent concentration is 0.05M iodine water, and the molar ratio of iodine to the nucleic acid sequence connected to the solid phase carrier in the coupling reaction is 30:1; the oxidation reaction is carried out in a water / pyridine mixed solvent (the volume ratio of water to pyridine is 1:9). The conditions for the sulfidation reaction are: temperature 25°C; reaction time 360 ​​seconds; thiolation reagent concentration is 0.2M hydrogenated xanthin pyridine solution, and the molar ratio of thiolation reagent to the nucleic acid sequence connected to the solid phase carrier in the coupling reaction is 4:1; the sulfidation reaction is carried out in a water / pyridine mixed solvent (the volume ratio of water to pyridine is 1:9).

[0202] After the last nucleoside monomer is connected, the nucleic acid sequence connected to the solid phase carrier is cut, deprotected, purified, desalted, and then freeze-dried to obtain the positive chain, wherein:

[0203] The cleavage and deprotection conditions are as follows: the synthesized nucleotide sequence connected to the solid phase carrier is added to 25% by mass ammonia water, the amount of ammonia water is 0.5 ml / μmol, the reaction is carried out at 55°C for 16 hours, the solvent is removed, and the product is concentrated to dryness in vacuo. After the ammonia water treatment, the product is dissolved with 0.4 ml / μmol N-methylpyrrolidone relative to the amount of single-stranded nucleic acid, and then 0.3 ml / μmol triethylamine and 0.6 ml / μmol triethylamine trihydrofluoride are added to remove the 2'-O-TBDMS protection on the ribose.

[0204] Purification and desalting conditions: The nucleic acid was purified by gradient elution of NaCl using a preparative ion chromatography purification column (Source 15Q). Specifically, eluent 1 was 20 mM sodium phosphate (pH = 8.1), and the solvent was a water / acetonitrile mixed solution (the volume ratio of water to acetonitrile was 9:1); eluent 2 was 1.5 M sodium chloride, 20 mM sodium phosphate (pH = 8.1), and the solvent was a water / acetonitrile mixed solution (the volume ratio of water to acetonitrile was 9:1); the elution gradient was eluent 1: eluent 2 = (100:0)-(50:50). The product eluates were collected and combined, and desalted using a reverse phase chromatography purification column. The desalting conditions included desalting using a dextran gel column, the filler was dextran gel G25, and eluted with deionized water.

[0205] Detection: Use ion exchange chromatography (IEX-HPLC) for purity detection; use liquid chromatography-mass spectrometry (LC-MS) for molecular weight detection, and compare the measured value of the molecular weight with the theoretical value. If the measured value is consistent with the theoretical value, it indicates that the positive chain of siRNA is obtained.

[0206] (3-2) Synthesis of antisense strand (AS):

[0207] The antisense chain was synthesized using a universal solid phase carrier. The deprotection, coupling, capping, oxidation or sulfurization reaction conditions, cleavage and deprotection conditions, purification and desalting conditions in the solid phase synthesis method of the antisense chain are the same as those in step (3-1) for synthesizing the sense chain.

[0208] Detection: Use ion exchange chromatography (IEX-HPLC) for purity detection; use liquid chromatography-mass spectrometry (LC-MS) for molecular weight detection, and compare the measured value of the molecular weight with the theoretical value. If the measured value is consistent with the theoretical value, it indicates that the antisense chain of siRNA is obtained.

[0209] (3-3) Synthesis of siRNA:

[0210] The sense chain synthesized in step (3-1) and the antisense chain synthesized in step (3-2) are mixed in an equimolar ratio, dissolved in water for injection and heated to 95° C., slowly cooled to room temperature and kept at room temperature for 10 minutes, so that the sense chain and the antisense chain form a double-stranded structure through hydrogen bonds, thereby obtaining siRNA having the sense chain and antisense chain shown in Table 2.

[0211] Preparation Example 4 Synthesis of siRNA conjugates

[0212] (4-1) Synthesis of positive chain

[0213] By using the phosphoramidite nucleic acid solid phase synthesis method, starting with a carrier compound (such as Universal CPG carrier, Universal PS carrier, compound CR01008Z), nucleoside monomers are connected one by one in the direction from the 3' end to the 5' end of the nucleotide sequence. During the synthesis process, compound CR01008 and compound NM054 are each regarded as a nucleoside monomer.

[0214] Each connection of a nucleoside monomer includes four steps of reaction: deprotection, coupling, capping, oxidation or sulfurization. The deprotection, coupling, capping, oxidation or sulfurization reaction conditions, cleavage and deprotection conditions, purification and desalting conditions in the synthesis of the sense chain of this preparation example are the same as those of the synthesis of the sense chain in step (3-1).

[0215] During the synthesis of the positive chain, two clusters of CR01008 vectors (denoted as (CR01008×2) or (CR01008)×2), three clusters of CR01008 vectors (denoted as (CR01008×3) or (CR01008)×3), and four clusters of CR01008 vectors (denoted as (CR01008×4) or (CR01008)×4) were obtained.

[0216] The structural formula of the two-cluster CR01008 is: .

[0217] The structural formula of the three-cluster CR01008 is: .

[0218] The structural formula of four clusters of CR01008 is: .

[0219] (4-2) Synthesis of antisense strand

[0220] The antisense strand of this preparation example was synthesized according to the antisense strand synthesis method shown in step (3-2) of preparation example 3.

[0221] (4-3) Synthesize siRNA conjugate according to the method shown in step (3-3) of Preparation Example 3.

[0222] The structural formula of the siRNA conjugate in which the (CR01008)×3 vector is conjugated to the 3' end of the sense strand is as follows:

[0223] ;in, represents siRNA, SS represents the sense strand of siRNA, and AS represents the antisense strand of siRNA. (CR01008×3) is conjugated to the 3' end of the sense strand of siRNA through a phosphodiester bond.

[0224] The structural formula of the siRNA conjugate with (CR01008)×4 vector conjugated to the 3' end of the sense strand is as follows:

[0225] ;in, represents siRNA, SS represents the sense strand of siRNA, and AS represents the antisense strand of siRNA. (CR01008×4) is conjugated to the 3' end of the sense strand of siRNA through a phosphodiester bond.

[0226] The structural formula of the siRNA conjugate with two (CR01008)×2 vectors conjugated to the 5' end of the sense strand and the 3' end of the antisense strand is shown below:

[0227] ;in, represents siRNA, SS represents the sense strand of siRNA, and AS represents the antisense strand of siRNA. Two (CR01008×2) are conjugated to the 5' end of the sense strand and the 3' end of the antisense strand of siRNA respectively through phosphodiester bonds.

[0228] Biological detection experiments:

[0229] Unless otherwise stated, the reagents, consumables and instruments used in the biological detection experiments disclosed herein are all derived from commercially available products.

[0230] Example 1 Comparison of the efficacy of siRNA conjugates in normal cynomolgus monkeys:

[0231] In this example, the ELISA method was used to determine the expression of AGT protein in the serum of cynomolgus monkeys at different time points after a single administration of RZ003089, R303079, R303082, R303086, R303087, R303088, and R303089.

[0232] The nucleotide information of conjugate RZ003089 is as follows:

[0233] Sense strand (5'-3'): UmsCmsAmAmCmUmGfGfAfUfGmAmAmGmAmAmAmCmUm_(CR01008×3);

[0234] Antisense strand (5'-3'):AmsGfsUmUmUmCfUmUmCfAmUmCmCmAfG(moe)UfUmGmAmsGmsGm.

[0235] Animal grouping, drug administration and tissue sample collection:

[0236] Healthy cynomolgus monkeys weighing 3-5 kg ​​were divided into groups according to serum AGT protein levels, with 3 males in each group. Each test group was given a predetermined dose of drug conjugate and a vehicle control group was added. The dosage of all animals was calculated according to body weight, and a single dose was given by subcutaneous injection in the interscapular region of the back. Each drug conjugate was administered in the form of 6 mg (calculated as siRNA) / mL of 0.9% sodium chloride injection, and the administration volume was 1 mL / kg (cynomolgus monkey body weight), that is, the dosage of each drug conjugate was 6 mg (calculated as siRNA) / kg (cynomolgus monkey body weight). The vehicle control group was given 1 mL / kg (cynomolgus monkey body weight) of 0.9% sodium chloride injection without siRNA conjugate. The serum of cynomolgus monkeys was collected on the day of administration (recorded as D0, before administration), the 7th day (recorded as D7), the 14th day (recorded as D14), the 21st day (recorded as D21), the 28th day (recorded as D28), the 35th day (recorded as D35), and the 42nd day (recorded as D42) after administration, and the AGT protein expression was determined using the human angiotensinogen (AGT) kit (IBL, 27412). The experiment is in progress.

[0237] Table 5 Changes in AGT protein levels in cynomolgus monkey serum after administration of the siRNA conjugates

[0238]

[0239] The results of Example 1 are shown in Table 5 and Figure 1 As shown, the results showed that a single dose of 6 mg / kg of RZ003089, R303079, R303082, R303086, R303087, R303088, and R303089 could significantly reduce the AGT protein level in the serum of crab-eating monkeys; as the observation time prolonged, the effects of R303082 and R303089 on serum AGT protein were slightly better than those of RZ003089.

[0240] The above specific implementations are only illustrative of the content of the present invention and do not represent limitations of the content of the present invention. For those skilled in the art, various modifications and improvements can be made without departing from the spirit and essence of the present disclosure, and these modifications and improvements are also considered to be within the scope of protection of the present disclosure.

Claims

1. A conjugate, characterized in that The conjugate is selected from R303082 or R303089, From 5' to 3', the positive strand of R303082 is: UmsCmsAmAmCmUmGfGfAfUfGmAmAmGmAmAmAmCmsUm_(CR01008×4); The antisense strand of R303082 is: AmsGfsUmUmUmCfUmUmCfAmsUmCmCmAfGmUfUmGmAms(NM)s(NM); From 5' to 3', the positive strand of R303089 is: UmsCmsAmAmCmUmGfGfAfUfGmAmAmGmAmAmAmCmsAm_(CR01008×4); The antisense strand of R303089 is: UmsGfsUmUmUmCfUmUmCfAmUmCmCmAfG(moe)UfUmGmAms(NM)s(NM); Wherein, m indicates that the nucleotide represented by the capital letter adjacent to the left of the letter m is a 2'-O-methyl modified nucleotide; f indicates that the nucleotide represented by the capital letter adjacent to the left of the letter f is a 2'-fluorine modified nucleotide; (moe) indicates that the nucleotide represented by the capital letter adjacent to the left of the combination mark (moe) is a 2'-O-methoxyethyl modified nucleotide; s indicates that the internucleoside bond between the nucleotide adjacent to the left of the letter s and the nucleotide adjacent to the right of the letter s is selected from a phosphorothioate bond; (NM) indicates a nucleotide in which the 2'-hydroxyl group and hydrogen of the ribose are replaced by [2'-F-2'-methyl], and the structural formula is ; The structural formula of (CR01008×4) is: .

2. A pharmaceutical composition comprising the conjugate according to claim 1 and a pharmaceutically acceptable excipient.

3. Use of any of the following in the preparation of a medicament for alleviating, preventing and / or treating a disease or condition mediated by the AGT gene: (I) the conjugate according to claim 1; and / or (II) the pharmaceutical composition according to claim 2; The disease or condition mediated by the AGT gene is hypertension.

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