TW202229549A - Sirnas and compositions for inhibiting the expression of coagulation factor xi and medical use thereof - Google Patents

Abstract

The present disclosure relates to siRNAs and compositions for inhibiting the expression of coagulation factor XI and medical use thereof. Specifically, the present disclosure relates to a siRNA comprising a sense strand and an antisense strand, wherein the sense strand contains a nucleotide sequence A, the antisense strand contains a nucleotide sequence B, and the nucleotide sequence A and the nucleotide sequence B are at least partially reverse complementary to form a double-stranded region; wherein the nucleotide sequence A differs from the sense strand nucleotide sequence provided in Table 1 in no more than 3 nucleotides; and the nucleotide sequence B differs from the antisense strand nucleotide sequence provided in Table 1 in no more than 3 nucleotides. The present disclosure also relates to pharmaceutical compositions, cells or kits containing the siRNA, and the use of the siRNA for the treatment and/or prevention of a subject suffering from coagulation factor XI-related diseases.

Description

siRNA for inhibiting the expression of coagulation factor XI, composition and medical use thereof

This application requires the Chinese patent application CN202010772542.6 with the filing date of August 04, 2020, the Chinese patent application CN202110244977.8 with the filing date of March 05, 2021, and the Chinese patent application with the filing date of April 2, 2021. CN202110361502.7 and Chinese patent application CN202110559411.4 with an application date of May 21, 2021 have priority rights, and this application cites the original text of the above Chinese patent application.

The present disclosure belongs to the field of biomedicine, and in particular relates to siRNA for inhibiting the expression of coagulation factor XI, compositions and medical uses thereof.

This section merely provides background information related to the present disclosure and does not necessarily constitute prior art.

The blood circulatory system not only has a coagulation mechanism to prevent blood loss, but also has an anticoagulation mechanism to counteract improper intravascular embolism. The dynamic balance between the two is the key to the normal body to maintain the state of blood flow in the body and prevent blood loss. The coagulation process is a process in which a series of coagulation factors are activated by successive enzymatic hydrolysis, and finally thrombin is generated to form a fibrin clot. There are two activation pathways in the human body, exogenous and endogenous. Endogenous pathways include: when the blood vessel wall is damaged, the subendothelial tissue is exposed, the negatively charged subendothelial collagen fibers are in contact with coagulation factors, factor XII is bound to it, and it is activated to factor XIIa with the participation of HK and PK; Factor XIIa activates factor XI to factor XIa in the presence of calcium ions independent of calcium ions; in the presence of calcium ions, activated factor XIa activates factor IX; IXa alone is quite ineffective in activating factor X, and it is much more potent than VIIIa. Combine to form a 1:1 complex, also known as the factor X enzyme complex; this reaction must also involve Ca ²⁺ and PL. Exogenous pathways include: tissue factor (TF) released after tissue injury combines with calcium ions, factor VII (or factor VIIa) in plasma to form a TF-Ca ²⁺ -FVII/FVIIa complex, which binds factor X Activated as factor Xa. The two pathways converge upon activation of factor X. Activated factor Xa and factor V form a prothrombin activator, which hydrolyzes prothrombin into thrombin, which converts fibrinogen into fibrin, and forms a fibrin clot under the action of platelets. Studies have found that factor XI in the endogenous pathway is associated with the formation of venous thrombosis (JOOST CMMEIJERS et al., The New England Journal of Medicine, 2000, Vol. 342, No. 10, 696-701). There is a natural loss-of-function mutation in the population with reduced risk of thrombosis and no significant risk of bleeding.

Thromboembolism can lead to conditions such as deep vein thrombosis, pulmonary embolism, myocardial infarction, and stroke. Anticoagulants can reduce the risk of thromboembolism. Current anticoagulants such as warfarin, heparin and low molecular weight heparin (LMWH), coagulation factor X inhibitors, etc. have significant shortcomings, such as lack of predictability and specificity, Careful monitoring of patients is therefore required to prevent adverse side effects such as bleeding complications. At present, there are no anticoagulant drugs that only target the endogenous pathway or the exogenous pathway.

RNA interference is an effective way to silence gene expression. According to statistics, among the disease-related proteins in the human body, about more than 80% of the proteins cannot be detected by the current conventional small molecules. The target of drugs and biological macromolecular preparations is a non-drugable protein. Antisense oligonucleotides (ASOs) and small interfering RNAs (siRNAs) are known strategies for silencing gene expression. CN102245186A describes ASO targeting coagulation factor XI. There are no reports on siRNA against coagulation factor XI. Although some algorithms for siRNA design are known, such as mFOLD, these algorithms only consider the primary structure and roughly predicted secondary structure of mRNA, and do not consider the tertiary structure of mRNA and the interaction of mRNA with RNA-binding proteins on siRNA activity and selectivity, so existing algorithms alone are not enough to obtain siRNAs with sufficient activity and selectivity.

Based on this, the present disclosure provides mRNA and/or protein level compounds, compositions and methods capable of modulating factor XI.

The present disclosure provides an siRNA comprising a sense strand and an antisense strand, wherein the sense strand contains a segment of nucleotide sequence A, the antisense strand contains a segment of nucleotide sequence B, nucleotide sequence A and nucleotide sequence B at least partially reverse complementary to form a double-stranded region;

Wherein, nucleotide sequence A is equal to any one of the nucleotide sequences of the sense strand provided in Table 1, and there are no more than 4, no more than 3, no more than 2 or no more than each other. less than 1 nucleotide difference; and nucleotide sequence B is the same length as any of the antisense strand nucleotide sequences provided in Table 1, and there are no more than 4, no more than 3, No more than 2 or no more than 1 nucleotide difference.

The present disclosure also provides an siRNA capable of inhibiting the expression of coagulation factor XI, comprising a sense strand and an antisense strand, wherein the sense strand contains a nucleotide sequence A, and the antisense strand contains a nucleotide sequence A. There is a segment of nucleotide sequence B, and nucleotide sequence A and nucleotide sequence B are at least partially reverse complementary to form a double-stranded region;

Wherein, nucleotide sequence A is equal to any one of the nucleotide sequences of the sense strand provided in Table 1, and there are no more than 4, no more than 3, no more than 2 or no more than each other. less than 1 nucleotide difference; and nucleotide sequence B is the same length as any of the antisense strand nucleotide sequences provided in Table 1, and there are no more than 4, no more than 3, No more than 2 or no more than 1 nucleotide difference.

In some embodiments, there is no more than 1 nucleotide difference between nucleotide sequence A and any of the sense strand nucleotide sequences provided in Table 1; and/or the nucleotide sequence B differs from Table 1. There is no more than 1 nucleotide difference between any of the antisense strand nucleotide sequences provided in 1.

In some embodiments, there is no more than 3 base mismatches between nucleotide sequence A and the nucleotide sequence B. In some embodiments, there is no more than 2 base mismatches between nucleotide sequence A and the nucleotide sequence B. In some embodiments, there is no more than 1 base mismatch between nucleotide sequence A and the nucleotide sequence B.

In some embodiments, there is no mismatch between nucleotide sequence A and the nucleotide sequence B.

In some embodiments, the sense strand and the antisense strand are each less than 30, less than 29, less than 28, less than 27, less than 26, less than 25, less than 24, less than 23, less than 22 in length each , less than 21, or less than 20 nucleotides.

In some embodiments, nucleotide sequence A is the nucleotide sequence set forth in any one of SEQ ID NOs: 1-23.

In some embodiments, nucleotide sequence B is the nucleotide sequence set forth in any one of SEQ ID NOs: 24-46.

The present disclosure also provides an siRNA, which is the modified siRNA described above, wherein at least one nucleotide in the sense strand and/or the antisense strand is a modified nucleotide. In some embodiments, all nucleotides are modified nucleotides.

In some embodiments, the modified nucleotides are independently selected from the group consisting of deoxy-nucleotides, 3'-terminal deoxy-thymidine nucleotides, 2'-O-methyl modified nucleotides, 2'- Fluorine modified nucleotides, 2'-deoxy-modified nucleotides, locked nucleotides, unlocked nucleotides, conformationally restricted nucleotides, restricted ethyl nucleotides, abasic nucleotides , 2'-amino-modified nucleotides, 2'-O-allyl-modified nucleotides, 2'-C-alkyl-modified nucleotides, 2'-hydroxy-modified nucleotides nucleotides, 2'-methoxyethyl modified nucleotides, 2'-O-alkyl-modified nucleotides, morpholino nucleotides, phosphoramidates, cores containing unnatural bases nucleotides, tetrahydropyran-modified nucleotides, 1,5-anhydrohexitol-modified nucleotides, cyclohexenyl-modified nucleotides, phosphorothioate-containing nucleotides, methyl-containing phosphonate-containing nucleotides, 5'-phosphate-containing nucleotides, and 5'-phosphate mimetic-containing nucleotides.

In some embodiments, the modified nucleotides are independently selected from: 2'-alkoxy modified nucleotides, 2'-substituted alkoxy modified nucleotides, 2'-alkyl modified nucleotides nucleotides, 2'-substituted alkyl-modified nucleotides, 2'-amino-modified nucleotides, 2'-substituted amine-modified nucleotides, 2'-fluoro-modified nucleotides Nucleotides, 2'-deoxynucleotides, 2'-deoxy-2'-fluoromodified nucleotides, 3'-deoxy-thymidine nucleotides, isonucleotides, LNA, ENA, cET, UNA and GNA.

In some embodiments, the modified nucleotides are independently selected from the group consisting of: 2'-methoxy-modified nucleotides, 2'-fluoro-modified nucleotides, and 2'-deoxy-modified nucleotides.

In the context of the present disclosure, a fluorine-modified nucleotide refers to a nucleotide formed by replacing the hydroxyl group at the 2' position of the ribosyl group of the nucleotide with a fluorine. In some embodiments, the 2'-alkoxy-modified nucleotide is a methoxy-modified nucleotide (2'-OMe). In some embodiments, a 2'-substituted alkoxy-modified nucleotide, eg, can be a 2'-O-methoxyethyl-modified nucleotide (2'-MOE). In some embodiments, 2'-amino modified nucleotides (2'- _NH2 ).

In some embodiments, at least one phosphate group in the sense and/or antisense strand is a phosphate group with a modifying group that provides increased stability of the siRNA in a biological sample or environment.

In some embodiments, the phosphate group with the modifying group is a phosphorothioate group. In some embodiments, a phosphorothioate group refers to a phosphodiester group in which a non-bridging oxygen atom is replaced by a sulfur atom.

In some embodiments, the nucleotides at positions 2, 6, 9, 12, and 14 of the nucleotide sequence B are each independently 2'-deoxynucleotides in the 5'-end to 3'-end direction or 2'-fluoro-modified nucleotides.

In some embodiments, the nucleotides at positions 2, 4, 6, 9, 12, 14 and 18 of the nucleotide sequence B are each independently 2'- Deoxynucleotides or 2'-fluoro-modified nucleotides.

In some embodiments, the nucleotides at positions 2, 4, 6, 9, 12, 14, 16 and 18 of the nucleotide sequence B are each independently 2 in the 5'-end to 3'-end direction '-deoxynucleotides or 2'-fluoro-modified nucleotides.

In some embodiments, the sense and antisense strands are the same or different in length, the sense strand is 19-23 nucleotides in length and the antisense strand is 20-26 nucleotides in length. In this way, the length ratio of the sense and antisense strands of the siRNA provided by the present disclosure can be 19/20, 19/21, 19/22, 19/23, 19/24, 19/25, 19/26, 20/20 , 20/21, 20/22, 20/23, 20/24, 20/25, 20/26, 21/20, 21/21, 21/22, 21/23, 21/24, 21/25, 21 /26, 22/20, 22/21, 22/22, 22/23, 22/24, 22/25, 22/26, 23/20, 23/21, 23/22, 23/23, 23/24, 23/25 or 23/26. In some embodiments, the length ratio of the sense and antisense strands of the siRNA is 19/21, 21/23, or 23/25.

In some embodiments, the phosphorothioate group is present in at least one position selected from the group consisting of:

Between the first nucleotide and the second nucleotide in the 5'-3' direction of the sense strand;

Between the second nucleotide and the third nucleotide in the 5'-3' direction of the sense strand;

The first nucleotide end in the 3'-5' direction of the sense strand;

Between the first nucleotide and the second nucleotide in the 3'-5' direction of the sense strand;

Between the second nucleotide and the third nucleotide in the 3'-5' direction of the sense strand;

Between the first nucleotide and the second nucleotide in the 5'-3' direction of the antisense strand;

Between the second nucleotide and the third nucleotide in the 5'-3' direction of the antisense strand;

The first nucleotide end of the 3'-5' direction of the antisense strand;

Between the first nucleotide and the second nucleotide in the 3'-5' direction of the antisense strand; or

Between the 2nd nucleotide and the 3rd nucleotide in the 3'-5' direction of the antisense strand.

In some embodiments, the sense strand binds to at least one targeting ligand.

The present disclosure also provides an siRNA comprising a sense strand and an antisense strand, wherein the sense strand contains a segment of nucleotide sequence I, the antisense strand contains a segment of nucleotide sequence II, the nucleotide sequence I and nucleoside sequences The acid sequence II is at least partially reverse complementary to form a double-stranded region, and the nucleotide sequence II is represented by the formula:

5'-N _a 'N _b 'N _a 'X'N _a 'N _b 'N _a 'N _a 'N _b 'N _a 'N _a 'N _b 'N _a 'N _b 'N _a 'Y'N _a 'X'N _a 'N _a 'N _a '-3' (nucleotide sequence II)

wherein each _Na ' and _Nb ' independently represents a modified nucleotide or an unmodified nucleotide, wherein the modifications on Na' and _Nb ' are different; each _X ' is independently _Na ' or _Nb ';Y' is _Na , or _Nb '.

The present disclosure also provides an siRNA comprising a sense strand and an antisense strand, wherein the sense strand contains a segment of nucleotide sequence I, the antisense strand contains a segment of nucleotide sequence II, the nucleotide sequence I and nucleoside sequences The acid sequence II is at least partially reverse complementary to form a double-stranded region, and the nucleotide sequence II is represented by the formula:

5'-N _a 'N _b 'N _a 'X'N _a 'N _b 'N _a 'N _a 'N _b 'N _a 'N _a 'N _b 'N _a 'N _b 'N _a 'Y'N _a 'X'N _a 'N _a 'N _a '-3' (nucleotide sequence II)

Wherein, each N _a ' independently represents a modified nucleotide or an unmodified nucleotide; each N _b ' independently represents a 2'-fluoro-modified nucleotide or a 2'-deoxy-modified nucleoside acid; each X' is independently _Na ' or _Nb ';Y' is _Na ' or _Nb '.

In some embodiments, the modified nucleotides are independently selected from the group consisting of: 2'-alkoxy modified nucleotides, 2'-substituted alkoxy modified nucleotides, 2'-alkyl modified nucleotides nucleotides, 2'-substituted alkyl-modified nucleotides, 2'-amino-modified nucleotides, 2'-substituted amine-modified nucleotides, 2'-fluoro-modified nucleotides Nucleotides, 2'-deoxynucleotides, 2'-deoxy-2'-fluoromodified nucleotides, 3'-deoxy-thymidine nucleotides, isonucleotides, LNA, ENA, cET, UNA and GNA.

In some embodiments, the modified nucleotides are independently of each other 2'-methoxy modified nucleotides.

In the context of the present disclosure, "unmodified ()nucleotides" refer to nucleotides consisting of naturally occurring nucleoside bases, sugar groups, and intranucleoside linkages. In some embodiments, Unmodified nucleotides are RNA nucleotides (ie β-D-ribonucleosides) or DNA nucleotides (ie β-D-deoxyribonucleosides).

In some embodiments, X' is _Nb '.

In some embodiments, Y' is _Nb '.

In some embodiments, the modification on Na' is different from the modification on _Nb '; in some embodiments, _Na ' is _a 2'-methoxy-modified nucleotide.

In some embodiments, the nucleotide sequence I is represented by the formula:

5'-N _a N _a N _a N _a N _b N _a N _b N _b N _b N _a N _a N _a N _a N _a N _a N _a N _a N _a N _a -3' (nucleotide sequence I )

Wherein, each _Na and _Nb independently represent modified nucleotides or unmodified nucleotides, and the modifications on _Na and _Nb are different;

In some embodiments, the modified nucleotides are independently selected from the group consisting of: 2'-alkoxy-modified nucleotides, 2'-substituted alkoxy-modified nucleotides, 2'-alkyl Modified Nucleotides, 2'-Substituted Alkyl Modified Nucleotides, 2'-Amino Modified Nucleotides, 2'-Substituted Amino Modified Nucleotides, 2'-Fluoro Modified nucleotides, 2'-deoxynucleotides, 2'-deoxy-2'-fluoromodified nucleotides, 3'-deoxy-thymine nucleotides, isonucleotides, LNA, ENA, cET, UNA and GNA;

In some embodiments, the modified nucleotides are independently selected from the group consisting of: 2'-methoxy-modified nucleotides, 2'-fluoro-modified nucleotides, and 2'-deoxy-modified nucleotides ;

In some embodiments, Na is _a 2'-methoxy-modified nucleotide and _Nb is a 2'-fluoro-modified nucleotide or a 2'-deoxy-modified nucleotide.

In some embodiments, nucleotide sequence II is represented by the formula:

pMD-AS19: 5'-NmNfNmNfNmNfNmNmNfNmNmNfNmNfNmNfNmNfNmNmNm-3'.

In some embodiments, nucleotide sequence II is represented by the formula:

pMD-AS18: 5'-NmNfNmN _DNA NmNfNmNmN _DNA NmNmN _DNA NmNfNmNfNmNmNmNmNm-3', wherein N _DNA indicates that the nucleotide at this position is a 2'-deoxy-modified nucleotide.

In some embodiments, the nucleotide sequence I is represented by the formula:

pMD-SS3: 5’-NmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNmNm-3’.

In some embodiments, there is no more than 3 base mismatches between nucleotide sequence I and the nucleotide sequence II; in some embodiments, the nucleotide sequence I and the nucleotide sequence II There is no more than 2 base mismatches between; in some embodiments, there is no more than 1 base mismatch between nucleotide sequence I and the nucleotide sequence II; in some embodiments , there is no mismatch between nucleotide sequence I and this nucleotide sequence II.

In some embodiments, at least one phosphate group in the sense strand and/or antisense strand is a phosphate group with a modifying group.

In some embodiments, the phosphate group with the modifying group is a phosphorothioate group.

In some embodiments, the phosphorothioate group is present in at least one position selected from the group consisting of:

Between the first nucleotide and the second nucleotide in the 5'-3' direction of the sense strand;

Between the second nucleotide and the third nucleotide in the 5'-3' direction of the sense strand;

The first nucleotide end in the 3'-5' direction of the sense strand;

Between the first nucleotide and the second nucleotide in the 3'-5' direction of the sense strand;

Between the second nucleotide and the third nucleotide in the 3'-5' direction of the sense strand;

Between the first nucleotide and the second nucleotide in the 5'-3' direction of the antisense strand;

Between the second nucleotide and the third nucleotide in the 5'-3' direction of the antisense strand;

The first nucleotide end of the 3'-5' direction of the antisense strand;

Between the first nucleotide and the second nucleotide in the 3'-5' direction of the antisense strand; or

Between the 2nd nucleotide and the 3rd nucleotide in the 3'-5' direction of the antisense strand.

In some embodiments, nucleotide sequence II is represented by the formula:

MD-AS19: 5'-NmsNfsNmNfNmNfNmNmNfNmNmNfNmNfNmNfNmNfNmsNmsNm-3'.

In some embodiments, nucleotide sequence II is represented by the formula:

MD-AS18: 5'-NmsNfsNmN _DNA NmNfNmNmN _DNA NmNmN _DNA NmNfNmNfNmNmNmsNmsNm-3', where N _DNA indicates that the nucleotide at this position is a 2'-deoxy-modified nucleotide.

In some embodiments, the nucleotide sequence I is represented by the formula:

MD-SS3: 5'-NmsNmsNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNmNm-3'.

In some embodiments, the siRNA comprises one or two blunt ends.

The terms "blunt end" or "blunt end" are used interchangeably and refer to the absence of unpaired nucleotides or nucleotide analogs at a given end of an siRNA, ie, no nucleotide overhang. In most cases, an siRNA that is blunt-ended at both ends will be double-stranded over its entire length.

In some embodiments, the siRNA comprises an overhang having 1 to 4 unpaired nucleotides, eg, an overhang of 2 or 3 unpaired nucleotides.

In some embodiments, the siRNA comprises an overhang located at the 3'-end of the antisense strand of the siRNA.

In some embodiments, the sense strand binds to at least one targeting ligand.

In some embodiments, the present disclosure provides an siRNA comprising a sense strand and an antisense strand, wherein the sequence of the sense strand is shown in the formula:

MD-SS3: 5'-NmsNmsNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNmNm-3';

And the sequence of the antisense strand is shown in the following formula:

MD-AS19: 5'-NmsNfsNmNfNmNfNmNmNfNmNmNfNmNfNmNfNmNfNmsNmsNm-3'.

The present disclosure also provides an siRNA capable of inhibiting the expression of a target gene, comprising a sense strand and an antisense strand, wherein the sense strand contains a segment of nucleotide sequence I, the antisense strand contains a segment of nucleotide sequence II, and the nucleoside sequence Acid sequence I and nucleotide sequence II are at least partially reverse complementary to form a double-stranded region, and the nucleotide sequence II is represented by the following formula:

5'-N _a 'N _b 'N _a 'X'N _a 'N _b 'N _a 'N _a 'N _b 'N _a 'N _a 'N _b 'N _a 'N _b 'N _a 'Y'N _a 'X'N _a 'N _a 'N _a '-3' (nucleotide sequence II)

Wherein, each N _a ' independently represents a modified nucleotide or an unmodified nucleotide; each N _b ' independently represents a 2'-fluoro-modified nucleotide or a 2'-deoxy-modified nucleoside acid; each X' is independently _Na ' or _Nb ';Y' is _Na ' or _Nb '.

In some embodiments, the modified nucleotides are independently selected from the group consisting of: 2'-alkoxy-modified nucleotides, 2'-substituted alkoxy-modified nucleotides, 2'-alkyl Modified Nucleotides, 2'-Substituted Alkyl Modified Nucleotides, 2'-Amino Modified Nucleotides, 2'-Substituted Amino Modified Nucleotides, 2'-Fluoro Modified nucleotides, 2'-deoxynucleotides, 2'-deoxy-2'-fluoromodified nucleotides, 3'-deoxy-thymine nucleotides, isonucleotides, LNA, ENA, cET, UNA and GNA.

In some embodiments, the modified nucleotides are independently of each other a 2'-methoxy modified nucleotide.

The present disclosure also provides an siRNA comprising a sense strand and an antisense strand, wherein the sense strand contains a nucleotide sequence III, the antisense strand contains a nucleotide sequence IV, a nucleotide sequence III and a nucleotide sequence IV is at least partially reverse complementary to form a double-stranded region, and the nucleotide sequence IV is selected from the following sequences:

pMD-AS1: 5’-NmNfNmNfNmNfNmNfNmNmNmNfNmNfNmNfNmNfNmNfNm-3’;

pMD-AS2: 5'-NmNfNmNfNmNfNmNfNmNfNmNfNmNfNmNfNmNfNmNmNm-3';

pMD-AS4: 5'-NmNfNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmNm-3';

pMD-AS5: 5'-NmNfNmNmNmNfNmNmNmNmNmNmNmNfNmNmNmNmNmNmNm-3';

pMD-AS6: 5’-NmNfNmNmNmNmNmNmNmNmNmNmNfNmNmNmNmNmNmNm-3’;

pMD-AS8: 5'-NmNfNmNmNmNfNmNmNmN _DNA NmN _DNA NmNfNmNfNmNmNmNmNmNm-3';

pMD-AS9: 5'-NmNfNmNmNmNfNmN _DNA N _DNA NmNmNmNmNfNmNfNmNmNmNmNm-3';

pMD-AS10: 5'-NmNfNmNfNmNfNmNmN _DNA NmNmN _DNA NmNfNmNfNmN _DNA NmNmNm-3';

pMD-AS11: 5’-NmNfNmNfNmNfNmNfNmNfNmNfNmNfNmNfNmNmNmNmNm-3’;

pMD-AS12: 5'-NmNfNmNfNmNfNmNfNmNfNmNfNmNfNmNmNmNmNmNmNm-3';

pMD-AS17: 5'-NmNfNmNmNfNmNmNfNmNmNfNmNmNfNmNmNfNmNmNfNm-3';

pMD-AS18: 5'-NmNfNmN _DNA NmNfNmNmN _DNA NmNmN _DNA NmNfNmNfNmNmNmNmNm-3'; and

pMD-AS19: 5'-NmNfNmNfNmNfNmNmNfNmNmNfNmNfNmNfNmNfNmNmNm-3'.

In some embodiments, nucleotide sequence III is selected from the following sequences:

pMD-1: 5'-NfNmNfNmNfNmNfNfNfNmNfNmNfNmNfNmNfNmNf-3';

pMD-2: 5'-NmNmNmNmNmNmNfNfNfNmNmNmNmNmNmNmNmNmNm-3';

pMD-3: 5'-NmNfNmNmNmNfNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmNm-3';

pMD-4: 5'-NmNmNfNmNfNmNfNfNfNmNmNmNmNmNmNmNmNmNm-3';

pMD-5: 5'-NmNmNmNmNmNfNfNfNfNmNmNmNmNmNmNmNmNmNm-3';

pMD-6: 5'-NmNmNmNmNfNmNfNfN _DNA NmNmNmNmNmNmNmNmNmNm-3';

pMD-7: 5'-NmNmNmNmNfNmN _DNA NfNfNmNmNmNmNmNmNmNmNmNm-3';

pMD-8: 5'-NmNmNmNmNfNmNfN _DNA NfNmNmNmNmNmNmNmNmNmNm-3';

pMD-9: 5'-NmNmNmNmNfNmN _DNA NfN _DNA NmNmNmNmNmNmNmNmNmNm-3';

pMD-10: 5'-NmNmNmNmN _DNA NmNfNfNfNmNmNmNmNmNmNmNmNmNm-3';

pMD-11: 5'-NmNmNmNmN _DNA NmNfNfN _DNA NmNmNmNmNmNmNmNmNmNm-3';

pMD-12: 5'-NmNmNmNmNmNmN _DNA NfNfNmNmNmNmNmNmNmNmNmNm-3';

and pMD-13: 5'-NmNmNmNmNfNmNmNfNfNmNmNmNmNmNmNmNmNm-3'.

In some embodiments, the iRNA comprises a sense strand and an antisense strand selected from the group consisting of:

(1) Sense strand: 5'-NfNmNfNmNfNmNfNfNfNmNfNmNfNmNfNmNfNmNf-3';

Antisense strand: 5'-NmNfNmNmNmNfNmNmNmNmNmNmNmNfNmNfNmNmNmNmNm-3';

(2) Positive strand: 5'-NmNmNmNmNmNmNfNfNfNmNmNmNmNmNmNmNmNmNm-3';

Antisense strand: 5'-NmNfNmNmNmNfNmNmNmNmNmNmNmNfNmNfNmNmNmNmNm-3';

(3) Positive strand: 5'-NmNmNfNmNfNmNfNfNfNmNmNmNmNmNmNmNmNmNm-3';

Antisense strand: 5'-NmNfNmNmNmNfNmNmNmNmNmNmNmNfNmNfNmNmNmNmNm-3';

(4) Positive strand: 5'-NmNmNmNmNmNfNfNfNfNmNmNmNmNmNmNmNmNmNm-3';

Antisense strand: 5'-NmNfNmNmNmNfNmNmNmNmNmNmNmNfNmNfNmNmNmNmNm-3';

(5) Sense strand: 5'-NmNmNmNmNfNmNfNfN _DNA NmNmNmNmNmNmNmNmNmNm-3';

Antisense strand: 5'-NmNfNmNmNmNfNmNmNmNmNmNmNmNfNmNfNmNmNmNmNm-3';

(6) Sense strand: 5'-NmNmNmNmNfNmN _DNA NfNfNmNmNmNmNmNmNmNmNmNm-3';

Antisense strand: 5'-NmNfNmNmNmNfNmNmNmNmNmNmNmNfNmNfNmNmNmNmNm-3';

(7) Sense strand: 5'-NmNmNmNmNfNmNfN _DNA NfNmNmNmNmNmNmNmNmNmNm-3';

Antisense strand: 5'-NmNfNmNmNmNfNmNmNmNmNmNmNmNfNmNfNmNmNmNmNm-3';

(8) Sense strand: 5'-NmNmNmNmNfNmN _DNA NfN _DNA NmNmNmNmNmNmNmNmNmNm-3';

Antisense strand: 5'-NmNfNmNmNmNfNmNmNmNmNmNmNmNfNmNfNmNmNmNmNm-3';

(9) Sense strand: 5'-NmNmNmNmN _DNA NmNfNfNfNmNmNmNmNmNmNmNmNmNm-3';

Antisense strand: 5'-NmNfNmNmNmNfNmNmNmNmNmNmNmNfNmNfNmNmNmNmNm-3';

(10) Sense strand: 5'-NmNmNmNmN _DNA NmNfNfN _DNA NmNmNmNmNmNmNmNmNmNm-3';

Antisense strand: 5'-NmNfNmNmNmNfNmNmNmNmNmNmNmNfNmNfNmNmNmNmNm-3';

(11) Sense strand: 5'-NmNmNmNmNmNmN _DNA NfNfNmNmNmNmNmNmNmNmNmNm-3';

Antisense strand: 5'-NmNfNmNmNmNfNmNmNmNmNmNmNmNfNmNfNmNmNmNmNm-3';

(12) Positive strand: 5'-NmNmNmNmNfNmNmNfNfNmNmNmNmNmNmNmNmNmNm-3';

Antisense strand: 5'-NmNfNmNmNmNfNmNmNmNmNmNmNfNmNfNmNmNmNmNm-3'

(13) Positive strand: 5'-NmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNmNm-3';

Antisense strand: 5'-NmNfNmNfNmNfNmNfNmNmNmNfNmNfNmNfNmNfNmNfNm-3';

(14) Positive strand: 5'-NmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNmNm-3';

Antisense strand: 5'-NmNfNmNfNmNfNmNfNmNfNmNfNmNfNmNfNmNfNmNmNm-3';

(15) Positive strand: 5'-NmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNmNm-3';

Antisense strand: 5'-NmNfNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNm-3';

(16) Positive strand: 5'-NmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNmNm-3';

Antisense strand: 5'-NmNfNmNmNmNfNmNmNmNmNmNmNmNfNmNmNmNmNmNmNm-3';

(17) Positive strand: 5'-NmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNmNm-3';

Antisense strand: 5'-NmNfNmNmNmNmNmNmNmNmNmNmNfNmNmNmNmNmNmNm-3';

(18) Positive strand: 5'-NmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNmNm-3';

Antisense strand: 5'-NmNfNmNmNmNfNmNmNmN _DNA NmN _DNA NmNfNmNfNmNmNmNmNm-3';

(19) Positive strand: 5'-NmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNmNm-3';

Antisense strand: 5'-NmNfNmNmNmNfNmN _DNA N _DNA NmNmNmNmNfNmNfNmNmNmNmNm-3';

(20) Positive strand: 5'-NmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNmNm-3';

Antisense strand: 5'-NmNfNmNfNmNfNmNmN _DNA NmNmN _DNA NmNfNmNfNmN _DNA NmNmNm-3';

(21) Positive strand: 5'-NmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNmNm-3';

Antisense strand: 5'-NmNfNmNfNmNfNmNfNmNfNmNfNmNfNmNfNmNmNmNmNmNm-3';

(22) Positive strand: 5'-NmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNmNm-3';

Antisense strand: 5'-NmNfNmNfNmNfNmNfNmNfNmNfNmNfNmNmNmNmNmNmNm-3';

(23) Positive strand: 5'-NmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNmNm-3';

Antisense strand: 5'-NmNfNmNmNfNmNmNfNmNmNfNmNmNfNmNmNfNmNmNfNm-3';

(24) Positive strand: 5'-NmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNmNm-3';

Antisense strand: 5'-NmNfNmN _DNA NmNfNmNmN _DNA NmNmN _DNA NmNfNmNfNmNmNmNmNm-3'; and

(25) Positive strand: 5'-NmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNmNm-3';

Antisense strand: 5'-NmNfNmNfNmNfNmNmNfNmNmNfNmNfNmNfNmNfNmNmNm-3'.

In some embodiments, at least one phosphate group in the sense strand and/or antisense strand is a phosphate group with a modifying group;

In some embodiments, the phosphate group with the modifying group is a phosphorothioate group.

In some embodiments, the phosphorothioate group is present in at least one position selected from the group consisting of:

Between the first nucleotide and the second nucleotide in the 5'-3' direction of the sense strand;

Between the second nucleotide and the third nucleotide in the 5'-3' direction of the sense strand;

The first nucleotide end in the 3'-5' direction of the sense strand;

Between the first nucleotide and the second nucleotide in the 3'-5' direction of the sense strand;

Between the second nucleotide and the third nucleotide in the 3'-5' direction of the sense strand;

Between the first nucleotide and the second nucleotide in the 5'-3' direction of the antisense strand;

Between the second nucleotide and the third nucleotide in the 5'-3' direction of the antisense strand;

The first nucleotide end of the 3'-5' direction of the antisense strand;

Between the first nucleotide and the second nucleotide in the 3'-5' direction of the antisense strand; or

Between the 2nd nucleotide and the 3rd nucleotide in the 3'-5' direction of the antisense strand.

In some embodiments, the nucleotide sequence IV is selected from the following sequences:

MD-AS1: 5'-NmsNfsNmNfNmNfNmNfNmNmNmNfNmNfNmNfNmNfNmsNfsNm-3';

MD-AS2: 5'-NmsNfsNmNfNmNfNmNfNmNfNmNfNmNfNmNfNmNfNmsNmsNm-3';

MD-AS3: 5’-NmsNfsNmNmNmNfNmNmNmNmNmNmNfNmNfNmNmNmsNmsNm-3’;

MD-AS4: 5'-NmsNfsNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmsNmsNm-3';

MD-AS5: 5’-NmsNfsNmNmNmNfNmNmNmNmNmNmNfNmNmNmNmNmsNmsNm-3’;

MD-AS6: 5’-NmsNfsNmNmNmNmNmNmNmNmNmNmNfNmNmNmNmNmsNmsNm-3’;

MD-AS8: 5'-NmsNfsNmNmNmNfNmNmNmN _DNA NmN _DNA NmNfNmNfNmNmNmsNmsNm-3';

MD-AS9: 5'-NmsNfsNmNmNmNfNmN _DNA N _DNA NmNmNmNmNfNmNfNmNmNmsNmsNm-3';

MD-AS10: 5'-NmsNfsNmNfNmNfNmNmN _DNA NmNmN _DNA NmNfNmNfNmN _DNA NmsNmsNm-3';

MD-AS11: 5'-NmsNfsNmNfNmNfNmNfNmNfNmNfNmNfNmNfNmNmNmsNmsNm-3';

MD-AS12: 5'-NmsNfsNmNfNmNfNmNfNmNfNmNfNmNfNmNmNmNmNmsNmsNm-3';

MD-AS17: 5'-NmsNfsNmNmNfNmNmNfNmNmNfNmNmNfNmNmNfNmNmsNfsNm-3';

MD-AS18: 5'-NmsNfsNmN _DNA NmNfNmNmN _DNA NmNmN _DNA NmNfNmNfNmNmNmsNmsNm-3';

and MD-AS19: 5'-NmsNfsNmNfNmNfNmNmNfNmNmNfNmNfNmNfNmNfNmsNmsNm-3'.

In some embodiments, nucleotide sequence III is selected from the following sequences:

MD-SS1: 5'-NfsNmsNfNmNfNmNfNfNfNmNfNmNfNmNfNmNfsNmsNf-3';

MD-SS2: 5’-NmsNmsNmNmNmNmNfNfNfNmNmNmNmNmNmNmsNmsNm-3’;

MD-SS3: 5'-NmsNmsNmNmNfNmNfNfNfNmNmNmNmNmNmNmsNmsNm-3';

MD-SS4: 5'-NmsNmsNfNmNfNmNfNfNfNmNmNmNmNmNmNmsNmsNm-3';

MD-SS5: 5'-NmsNmsNmNmNmNfNfNfNfNmNmNmNmNmNmNmnsNmsNm-3';

MD-SS6: 5'-NmsNmsNmNmNfNmNfNfN _DNA NmNmNmNmNmNmNmnsNmsNm-3';

MD-SS7: 5'-NmsNmsNmNmNfNmN _DNA NfNfNmNmNmNmNmNmNmnsNmsNm-3';

MD-SS8: 5'-NmsNmsNmNmNfNmNfN _DNA NfNmNmNmNmNmNmNmNmsNmsNm-3';

MD-SS9: 5'-NmsNmsNmNmNfNmN _DNA NfN _DNA NmNmNmNmNmNmNmnsNmsNm-3';

MD-SS10: 5'-NmsNmsNmNmN _DNA NmNfNfNfNmNmNmNmNmNmNmnsNmsNm-3';

MD-SS11: 5'-NmsNmsNmNmN _DNA NmNfNfN _DNA NmNmNmNmNmNmNmNmsNmsNm-3';

MD-SS12: 5'-NmsNmsNmNmNmNmN _DNA NfNfNmNmNmNmNmNmNmNmsNmsNm-3';

MD-SS13: 5’-NmsNmsNmNmNfNmNmNfNfNmNmNmNmNmNmNmNmsNmsNm-3’.

In some embodiments, the siRNA comprises a sense strand and an antisense strand selected from the group consisting of:

(1) Sense strand: 5'-NfsNmsNfNmNfNmNfNfNfNmNfNmNfNmNfNmNfsNmsNf-3';

Antisense strand: 5'-NmsNfsNmNmNmNfNmNmNmNmNmNmNmNfNmNfNmNmNmsNmsNm-3';

(2) Positive strand: 5'-NmsNmsNmNmNmNmNfNfNfNmNmNmNmNmNmNmsNmsNm-3';

Antisense strand: 5'-NmsNfsNmNmNmNfNmNmNmNmNmNmNmNfNmNfNmNmNmsNmsNm-3';

(3) Positive strand: 5’-NmsNmsNfNmNfNmNfNfNfNmNmNmNmNmNmNmNmsNmsNm-3’;

Antisense strand: 5'-NmsNfsNmNmNmNfNmNmNmNmNmNmNmNfNmNfNmNmNmsNmsNm-3';

(4) Positive strand: 5'-NmsNmsNmNmNmNfNfNfNfNmNmNmNmNmNmNmnsNmsNm-3';

Antisense strand: 5'-NmsNfsNmNmNmNfNmNmNmNmNmNmNmNfNmNfNmNmNmsNmsNm-3';

(5) Sense strand: 5'-NmsNmsNmNmNfNmNfNfN _DNA NmNmNmNmNmNmNmnsNmsNm-3';

Antisense strand: 5'-NmsNfsNmNmNmNfNmNmNmNmNmNmNmNfNmNfNmNmNmsNmsNm-3';

(6) Sense strand: 5'-NmsNmsNmNmNfNmN _DNA NfNfNmNmNmNmNmNmNmsNmsNm-3';

Antisense strand: 5'-NmsNfsNmNmNmNfNmNmNmNmNmNmNmNfNmNfNmNmNmsNmsNm-3';

(7) Sense strand: 5'-NmsNmsNmNmNfNmNfN _DNA NfNmNmNmNmNmNmNmnsNmsNm-3';

Antisense strand: 5'-NmsNfsNmNmNmNfNmNmNmNmNmNmNmNfNmNfNmNmNmsNmsNm-3';

(8) Sense strand: 5'-NmsNmsNmNmNfNmN _DNA NfN _DNA NmNmNmNmNmNmNmnsNmsNm-3';

Antisense strand: 5'-NmsNfsNmNmNmNfNmNmNmNmNmNmNmNfNmNfNmNmNmsNmsNm-3';

(9) Sense strand: 5'-NmsNmsNmNmN _DNA NmNfNfNfNmNmNmNmNmNmNmNmsNmsNm-3';

Antisense strand: 5'-NmsNfsNmNmNmNfNmNmNmNmNmNmNmNfNmNfNmNmNmsNmsNm-3';

(10) Sense strand: 5'-NmsNmsNmNmN _DNA NmNfNfN _DNA NmNmNmNmNmNmNmNmsNmsNm-3';

Antisense strand: 5'-NmsNfsNmNmNmNfNmNmNmNmNmNmNmNfNmNfNmNmNmsNmsNm-3';

(11) Sense strand: 5'-NmsNmsNmNmNmNmN _DNA NfNfNmNmNmNmNmNmNmNmsNmsNm-3';

Antisense strand: 5'-NmsNfsNmNmNmNfNmNmNmNmNmNmNmNfNmNfNmNmNmsNmsNm-3';

(12) Positive strand: 5'-NmsNmsNmNmNfNmNmNfNfNmNmNmNmNmNmNmNmsNmsNm-3';

Antisense strand: 5'-NmsNfsNmNmNmNfNmNmNmNmNmNmNmNfNmNfNmNmNmsNmsNm-3';

(13) Positive strand: 5’-NmsNmsNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmsNmsNm-3’;

Antisense strand: 5'-NmsNfsNmNfNmNfNmNfNmNmNmNfNmNfNmNfNmNfNmsNfsNm-3';

(14) Positive strand: 5'-NmsNmsNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmsNmsNm-3';

Antisense strand: 5'-NmsNfsNmNfNmNfNmNfNmNfNmNfNmNfNmmNfNmNfNmsNmsNm-3';

(15) Positive strand: 5'-NmsNmsNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmsNmsNm-3';

Antisense strand: 5'-NmsNfsNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmsNmsNm-3';

(16) Positive strand: 5’-NmsNmsNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmsNmsNm-3’;

Antisense strand: 5'-NmsNfsNmNmNmNfNmNmNmNmNmNmNmNfNmNmNmNmNmsNmsNm-3';

(17) Positive strand: 5'-NmsNmsNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmsNmsNm-3';

Antisense strand: 5'-NmsNfsNmNmNmNmNmNmNmNmNmNmNfNmNmNmNmNmsNmsNm-3';

(18) Positive strand: 5'-NmsNmsNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmsNmsNm-3';

Antisense strand: 5'-NmsNfsNmNmNmNfNmNmNmN _DNA NmN _DNA NmNfNmNfNmNmNmsNmsNm-3';

(19) Positive strand: 5'-NmsNmsNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmsNmsNm-3';

Antisense strand: 5'-NmsNfsNmNmNmNfNmN _DNA N _DNA NmNmNmNmNfNmNfNmNmNmsNmsNm-3';

(20) Positive strand: 5’-NmsNmsNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmsNmsNm-3’;

Antisense strand: 5'-NmsNfsNmNfNmNfNmNmN _DNA NmNmN _DNA NmNfNmNfNmN _DNA NmsNmsNm-3';

(21) Positive strand: 5'-NmsNmsNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmsNmsNm-3';

Antisense strand: 5'-NmsNfsNmNfNmNfNmNfNmNfNmNfNmNfNmNfNmNmNmsNmsNm-3';

(22) Positive strand: 5'-NmsNmsNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmsNmsNm-3';

Antisense strand: 5'-NmsNfsNmNfNmNfNmNfNmNfNmNfNmNfNmNmNmNmNmsNmsNm-3';

(23) Positive strand: 5'-NmsNmsNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmsNmsNm-3';

Antisense strand: 5'-NmsNfsNmNmNfNmNmNfNmNmNfNmNmNfNmNmNfNmNmsNfsNm-3';

(24) Positive strand: 5'-NmsNmsNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmsNmsNm-3';

Antisense strand: 5'-NmsNfsNmN _DNA NmNfNmNmN _DNA NmNmN _DNA NmNfNmNfNmNmNmsNmsNm-3';

and

(25) Positive strand: 5'-NmsNmsNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmsNmsNm-3';

Antisense strand: 5'-NmsNfsNmNfNmNfNmNmNfNmNmNfNmNfNmNfNmNfNmsNmsNm-3'.

The present disclosure also provides an siRNA conjugate comprising the above-mentioned siRNA and a conjugation group attached to the siRNA.

In some embodiments, the conjugation group comprises a pharmaceutically acceptable targeting ligand and a linker, and the siRNA, the linker, and the targeting ligand are sequentially covalently or non-covalently linked.

In some embodiments, the linker is attached to the 3' end of the sense strand of the siRNA.

In some embodiments, in order to promote the entry of siRNA into cells, a lipophilic group such as cholesterol can be introduced at the end of the sense strand of siRNA. Protein, vitamin E, etc., to facilitate the interaction with intracellular mRNA through the cell membrane composed of lipid bilayers. At the same time, siRNA can also be modified by non-covalent bonds, such as binding phospholipid molecules, polypeptides, cationic polymers, etc. through hydrophobic bonds or ionic bonds to increase stability and biological activity.

The present disclosure also provides a method for preparing the aforementioned siRNA or siRNA conjugate, comprising: synthesizing the aforementioned siRNA or siRNA conjugate; and purifying the siRNA or the siRNA conjugate.

In some embodiments, the method comprises the steps of: (1) oligoribonucleotide synthesis; (2) deprotection; (3) purification isolation; (4) annealing.

In some embodiments, step (1) comprises: synthesizing sense and antisense oligoribose, respectively, on an RNA synthesizer (eg, Dr. Oligo48 synthesizer (Biolytic)) using solid support-mediated phosphamidite chemistry Nucleotides can be synthesized on a scale of 200 nanomolar (nmol); the coupling time of all phosphamidites (50 mM in acetonitrile) is 6 minutes (min) using 5-ethylthio-1H-tetrazole ( ETT) as activator (0.6M acetonitrile solution), using 0.22M PADS dissolved in 1:1 volume ratio of acetonitrile and collidine solution as sulfurization reagent, sulfurization reaction time is 3 minutes (min), using iodopyridine/ The aqueous solution is used as an oxidant, and the oxidation reaction time is 2 minutes (min); According to whether the target product has 5'-phosphorothioate modification, select the above-mentioned vulcanization reaction conditions or oxidation reaction conditions;

Step (2) includes: after the solid-phase synthesis is completed, the oligoribonucleotide is cleaved from the solid support, and soaked in 28% ammonia water and ethanol solution with a volume ratio of 3:1 at 50° C. for 16 hours. Then high-speed centrifugation, the supernatant was transferred to another centrifuge tube, concentrated and evaporated to dryness to obtain the crude oligonucleotide;

Step (3) includes: purifying the obtained crude oligonucleotide using C18 reverse chromatography, the mobile phase is 0.1M TEAA and acetonitrile, and using 3% trifluoroacetic acid solution to remove DMTr, collecting the target product and lyophilizing;

Step (4) includes: the sense strand and antisense strand synthesized respectively according to the above steps, annealing according to equimolar ratio, so that they form a double-stranded structure by hydrogen bonding, and finally dissolving the obtained double-stranded siRNA in 1× PBS and adjusted to the desired concentration for the experiment.

In another aspect, the present disclosure provides a siRNA comprising a sense strand and an antisense strand forming a double-stranded region; the sense strand comprises at least 15 nucleotide sequences that differ from any one of the sense strands in Table 1 by no more than 3 nucleotide sequences Contiguous nucleotides; the antisense strand contains at least 15 contiguous nucleotides that differ by no more than 3 nucleotide sequences from any of the antisense strands in Table 1; the siRNA contains one or more modified nucleotides.

In some embodiments, the antisense strand is at least partially reverse complementary to the target sequence to mediate RNA interference; in some embodiments, there are no more than 5, no more than 4, No more than 3, no more than 2, no more than 1 mismatch; in some embodiments, the antisense strand is fully reverse complementary to the target sequence.

In some embodiments, the sense and antisense strands are at least partially reverse complementary to form a double-stranded region; More than 4, no more than 3, no more than 2, no more than 1 mismatch; in some embodiments, the sense and antisense strands are fully reverse complementary.

In some embodiments, the sense strand of the siRNA contains three consecutive nucleotides that are 2'-fluoro modified nucleotides.

In some embodiments, the nucleotides at positions 2, 6, and 14 of the siRNA antisense strand of the present disclosure are each independently 2'-deoxynucleotides or 2'-deoxynucleotides in the 5'-end to 3'-end direction. '-Fluoro-modified nucleotides.

In some embodiments, the nucleotides at positions 2, 6, 14, and 16 of the antisense strand are each independently 2'-deoxynucleotides or 2'-fluoronucleotides in the 5'-end to 3'-end direction Modified Nucleotides.

In some embodiments, the nucleotides at positions 2, 6, 9, 12, and 14 of the antisense strand are each independently 2'-deoxynucleotides or 2', in the 5'-end to 3'-end orientation - Fluorine modified nucleotides.

In some embodiments, the nucleotides at positions 2, 6, 10, 12, and 14 of the antisense strand are each independently 2'-deoxynucleotides or 2', in the 5'-end to 3'-end orientation - Fluorine modified nucleotides.

In some embodiments, the nucleotides at positions 2, 4, 6, 9, 12, 14, and 18 of the antisense strand are each independently a 2'-deoxynucleoside in the 5'-end to 3'-end orientation Acid or 2'-fluoro modified nucleotides.

In some embodiments, the nucleotides at positions 2, 4, 6, 10, 12, 14, and 18 of the antisense strand are each independently a 2'-deoxynucleoside in the 5' to 3' direction Acid or 2'-fluoro modified nucleotides.

In some embodiments, the nucleotides at positions 2, 4, 6, 9, 12, 14, 16, and 18 of the antisense strand are each independently 2'-deoxy, in the 5' to 3' direction Nucleotides or 2'-fluoro-modified nucleotides.

In some embodiments, the nucleotides at positions 2, 4, 6, 10, 12, 14, 16 and 18 of the antisense strand are each independently 2'-deoxy, in the 5' to 3' direction Nucleotides or 2'-fluoro-modified nucleotides.

In some embodiments, the nucleotides at positions 2, 4, 6, 9, 10, 12, 14, 16 and 18 of the antisense strand are each independently 2' in the 5' to 3' direction - Deoxynucleotides or 2'-fluoro-modified nucleotides.

In some embodiments, nucleotides 2, 6 and 14 of the antisense strand are each independently 2'-fluoro modified nucleotides in the 5'-end to 3'-end direction;

In some embodiments, the nucleotides at positions 2, 6, 14 and 16 of the antisense strand are each independently 2'-fluoro modified nucleotides in the 5'-end to 3'-end direction;

In some embodiments, the nucleotides at positions 2, 6, 12 and 14 of the antisense strand are each independently 2'-fluoro modified nucleotides in the 5'-end to 3'-end direction;

In some embodiments, the nucleotides at positions 2, 4, 6, 12, 14, 16 and 18 of the antisense strand are each independently 2'-fluoro modified in the 5'-end to 3'-end orientation Nucleotides;

In some embodiments, the nucleotides at positions 2, 4, 6, 9, 12, 14, 16, and 18 of the antisense strand are each independently 2'-fluoro in the 5'-end to 3'-end orientation modified nucleotides;

In some embodiments, the nucleotides at positions 2, 4, 6, 10, 12, 14, 16 and 18 of the antisense strand are each independently 2'-fluoro, in the 5' to 3' direction Modified Nucleotides.

In some embodiments, the sense and antisense strands each independently have 16 to 35, 16 to 34, 17 to 34, 17 to 33, 18 to 33, 18 to 32, 18 to 31, 18 to 30, 18 to 29, 18 to 28, 18 to 27, 18 to 26 18 to 25, 18 to 24, 18 to 23, 19 to 25, 19 to 24, or 19 to 23 nucleotides.

In some embodiments, the sense and antisense strands are the same or different in length, the sense strand is 19-23 nucleotides in length and the antisense strand is 19-26 nucleotides in length. In this way, the length ratio of the sense and antisense strands of the siRNA provided by the present disclosure can be 19/20, 19/21, 19/22, 19/23, 19/24, 19/25, 19/26, 20/20 , 20/21, 20/22, 20/23, 20/24, 20/25, 20/26, 21/20, 21/21, 21/22, 21/23, 21/24, 21/25, 21 /26, 22/20, 22/21, 22/22, 22/23, 22/24, 22/25, 22/26, 23/20, 23/21, 23/22, 23/23, 23/24 , 23/25 or 23/26. In some embodiments, the length ratio of the sense and antisense strands of the siRNA is 19/21, 21/23, or 23/25. In some embodiments, the length ratio of the sense and antisense strands of the siRNA is 19/21.

In some embodiments, the siRNA comprises one or two blunt ends.

In some specific embodiments, the siRNA comprises an overhang having 1 to 4 unpaired nucleotides, eg, 1, 2, 3, 4.

In some embodiments, the siRNA of the present disclosure comprises an overhang located at the 3' end of the antisense strand of the siRNA.

In some embodiments, at least one additional nucleotide in the sense and/or antisense strand is a modified nucleotide.

In some embodiments, the modified nucleotides are independently selected from the group consisting of deoxy-nucleotides, 3'-terminal deoxy-thymidine nucleotides, 2'-O-methyl modified nucleotides, 2'- Fluorine modified nucleotides, 2'-deoxy-modified nucleotides, locked nucleotides, unlocked nucleotides, conformationally restricted nucleotides, restricted ethyl nucleotides, abasic nucleotides , 2'-amino-modified nucleotides, 2'-O-allyl-modified nucleotides, 2'-C-alkyl-modified nucleotides, 2'-hydroxy-modified nucleotides nucleotides, 2'-methoxyethyl modified nucleotides, 2'-O-alkyl-modified nucleotides, morpholino nucleotides, Phosphoramidates, nucleotides containing unnatural bases, tetrahydropyran-modified nucleotides, 1,5-anhydrohexitol-modified nucleotides, cyclohexenyl-modified nucleotides, containing Phosphorothioate-containing nucleotides, methylphosphonate-containing nucleotides, 5'-phosphate-containing nucleotides, and 5'-phosphate mimetic-containing nucleotides, comprising the formula of the present disclosure The chemically modified nucleotide shown in (I) or its tautomer modified.

In some embodiments, the modified nucleotides are independently selected from the group consisting of: 2'-alkoxy modified nucleotides, 2'-substituted alkoxy modified nucleotides, 2'-alkyl modified nucleotides nucleotides, 2'-substituted alkyl-modified nucleotides, 2'-amino-modified nucleotides, 2'-substituted amine-modified nucleotides, 2'-fluoro-modified nucleotides Nucleotides, 2'-deoxynucleotides, 2'-deoxy-2'-fluoromodified nucleotides, 3'-deoxy-thymidine nucleotides, isonucleotides, LNA, ENA, cET, UNA , GNA, or a nucleotide comprising a chemical modification of formula (I) of the present disclosure or a tautomer modification thereof.

In the context of the present disclosure, a 2'-fluoro modified nucleotide refers to a nucleotide formed by substituting the hydroxyl group at the 2' position of the ribosyl group of the nucleotide with fluorine. In some embodiments, the 2'-alkoxy-modified nucleotide is a 2'-methoxy-modified nucleotide (2'-OMe). In some embodiments, a 2'-substituted alkoxy-modified nucleotide, for example, can be a 2'-O-methoxyethyl-modified nucleotide (2'-MOE) or a 2'-amine base-modified nucleotides (2'- _NH2 ).

In some embodiments, the modified nucleotides are independently selected from: 2'-methoxy-modified nucleotides, 2'-fluoro-modified nucleotides, 2'-deoxy-modified nucleotides, or Nucleotides containing chemical modifications represented by formula (I) of the present disclosure or modified tautomers thereof.

； In the present disclosure, formula (I) is selected from

;

wherein Y is selected from O, NH and S;

Each X is independently selected from CR ₄ (R ₄ ′), S, NR ₅ and NH-CO, wherein R ₄ , R ₄ ′, R ₅ are each independently H or C ₁ -C ₆ alkyl;

J ₂ is H or C ₁ -C ₆ alkyl;

n=0, 1 or 2; m=0, 1 or 2; s=0 or 1;

R ₃ is selected from H, OH, halogen, NH ₂ , C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, C ₂ -C ₆ alkenyl, C ₂ -C ₆ alkynyl, S-CH ₃ , NCH ₃ (CH ₃ ), OCH ₂ CH ₂ OCH ₃ , -O-alkylamino and (CH ₂ ) _p R ₆ ; wherein R ₆ is selected from OH, halogen, methoxy, ethoxy, N ₃ , C ₂ -C ₆ alkenyl and C ₂ -C ₆ alkynyl, p=1, 2 or 3;

，Q₂為R₂；或者Q₁為R₂，Q₂為

； _Q1 is

, Q ₂ is R ₂ ; or Q ₁ is R ₂ , and Q ₂ is

;

in,

R ₁ is selected from H, C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, C ₂ -C ₆ alkenyl, C ₂ -C ₆ alkynyl and (CH ₂ ) _q R ₇ ; wherein R ₇ selected from OH, halogen, methoxy, ethoxy, N ₃ , C ₂ -C ₆ alkenyl and C ₂ -C ₆ alkynyl, q=1, 2 or 3;

J ₁ is H or C ₁ -C ₆ alkyl;

R ₂ is selected from H, OH, halogen, NH ₂ , C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, C ₂ -C ₆ alkenyl, C ₂ -C ₆ alkynyl, S-CH ₃ , NCH ₃ (CH ₃ ), OCH ₂ CH ₂ OCH ₃ , -O-alkylamino and (CH ₂ ) _r R ₈ ; wherein R ₈ is selected from OH, halogen, methoxy, ethoxy, N ₃ , C ₂ -C ₆ alkenyl and C ₂ -C ₆ alkynyl, r=1, 2 or 3;

Optionally, R ₁ and R ₂ are directly connected to form a ring;

B is a base or a base analog;

。 Wherein, the chemical modification shown in the formula (I) is not

.

In some embodiments, when X is NH _- CO, R1 is not H.

In some embodiments, B is a base or a base analog; eg, those selected from the group consisting of purine bases, pyrimidine bases, indole, 5-nitroindole, and 3-nitropyrrole;

In some embodiments, B is selected from the group consisting of adenine, guanine, isoguanine, hypoxanthine, xanthine, C2 modified purine, N8 modified purine, 2,6-diaminopurine, 6-dimethylamine purine, 2-aminopurine, N6-alkyladenine, O6-alkylguanine, 7-deazapurine, cytosine, 5-methylcytosine, isocytosine, pseudocytosine, uracil, Pseudouracil, 2-thiouridine, 4-thiouridine, C5-modified pyrimidine, thymine, indole, 5-nitroindole, and 3-nitropyrrole;

In some embodiments, B is selected from adenine, guanine, 2,6-diaminopurine, 6-dimethylaminopurine, 2-aminopurine, cytosine, uracil, thymine, indole, 5-nitroindole and 3-nitropyrrole;

In some embodiments, B is selected from the base at the position corresponding to the modified nucleotide of the antisense strand.

In some embodiments, Formula (I) is selected from Formula (I-1):

wherein Y is selected from O, NH and S;

Each X is independently selected from CR ₄ (R ₄ ′), S, NR ₅ and NH-CO, wherein R ₄ , R ₄ ′, R ₅ are each independently H or C ₁ -C ₆ alkyl;

Each J ₁ , J ₂ is independently H or C ₁ -C ₆ alkyl;

n=0, 1 or 2; m=0, 1 or 2; s=0 or 1;

R ₃ is selected from H, OH, halogen, NH ₂ , C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, C ₂ -C ₆ alkenyl, C ₂ -C ₆ alkynyl, S-CH ₃ , NCH ₃ (CH ₃ ), OCH ₂ CH ₂ OCH ₃ , -O-alkylamino and (CH ₂ ) _p R ₆ ; wherein R ₆ is selected from OH, halogen, methoxy, ethoxy, N ₃ , C ₂ -C ₆ alkenyl and C ₂ -C ₆ alkynyl, p=1, 2 or 3;

R ₁ is selected from H, C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, C ₂ -C ₆ alkenyl, C ₂ -C ₆ alkynyl and (CH ₂ ) _q R ₇ ; wherein R ₇ selected from OH, halogen, methoxy, ethoxy, N ₃ , C ₂ -C ₆ alkenyl and C ₂ -C ₆ alkynyl, q=1, 2 or 3;

R ₂ is selected from H, OH, halogen, NH ₂ , C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, C ₂ -C ₆ alkenyl, C ₂ -C ₆ alkynyl, S-CH ₃ , NCH ₃ (CH ₃ ), OCH ₂ CH ₂ OCH ₃ , -O-alkylamino and (CH ₂ ) _r R ₈ ; wherein R ₈ is selected from OH, halogen, methoxy, ethoxy, N ₃ , C ₂ -C ₆ alkenyl and C ₂ -C ₆ alkynyl, r=1, 2 or 3;

Optionally, R ₁ and R ₂ are directly connected to form a ring;

B is as defined in formula (I).

In some embodiments, B is a base or a base analog; eg, those selected from the group consisting of purine bases, pyrimidine bases, indole, 5-nitroindole, and 3-nitropyrrole;

In some embodiments, B is selected from the group consisting of adenine, guanine, isoguanine, hypoxanthine, xanthine, C2 modified purine, N8 modified purine, 2,6-diaminopurine, 6-dimethylamine Alkylpurine, 2-aminopurine, N6-alkyladenine, O6-alkylguanine, 7-deazapurine, cytosine, 5- Methylcytosine, isocytosine, pseudocytosine, uracil, pseudouracil, 2-thiouridine, 4-thiouridine, C5-modified pyrimidine, thymine, indole, 5-nitroindole Indoles and 3-nitropyrroles;

In some embodiments, B is selected from adenine, guanine, 2,6-diaminopurine, 6-dimethylaminopurine, 2-aminopurine, cytosine, uracil, thymine, indole, 5-nitroindole and 3-nitropyrrole;

In some embodiments, B is selected from the base at the position corresponding to the modified nucleotide of the antisense strand.

In some embodiments, Formula (I) is selected from Formula (I-2):

wherein Y is selected from O, NH and S;

Each X is independently selected from CR ₄ (R ₄ '), S, NR ₅ and NH-CO, wherein R ₄ , R ₄ ', R ₃ are each independently H or C ₁ -C ₆ alkyl;

n=0, 1 or 2; m=0, 1 or 2; s=0 or 1;

Each J ₁ , J ₂ is independently H or C ₁ -C ₆ alkyl;

R ₃ is selected from H, OH, halogen, NH ₂ , C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, C ₂ -C ₆ alkenyl, C ₂ -C ₆ alkynyl, S-CH ₃ , NCH ₃ (CH ₃ ), OCH ₂ CH ₂ OCH ₃ , -O-alkylamino and (CH ₂ ) _p R ₆ ; wherein R ₆ is selected from OH, halogen, methoxy, ethoxy, N ₃ , C ₂ -C ₆ alkenyl and C ₂ -C ₆ alkynyl, p=1, 2 or 3;

R ₁ is selected from H, C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, C ₂ -C ₆ alkenyl, C ₂ -C ₆ alkynyl and (CH ₂ ) _q R ₇ ; wherein R ₇ selected from OH, halogen, methoxy, ethoxy, N ₃ , C ₂ -C ₆ alkenyl and C ₂ -C ₆ alkynyl, q=1, 2 or 3;

R ₂ is selected from H, C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, S-CH ₃ , NCH ₃ (CH ₃ ), OCH ₂ CH ₂ OCH ₃ , -O-alkylamino and (CH ₂ ) _r R ₈ ; wherein R ₈ is selected from OH, halogen, methoxy, ethoxy, N ₃ , C ₂ -C ₆ alkenyl and C ₂ -C ₆ alkynyl; r=1, 2 or 3;

Optionally, R ₁ and R ₂ are directly connected to form a ring;

B is as defined in formula (I).

In some embodiments, B is a base or a base analog; eg, those selected from the group consisting of purine bases, pyrimidine bases, indole, 5-nitroindole, and 3-nitropyrrole;

In some embodiments, B is selected from the group consisting of adenine, guanine, isoguanine, hypoxanthine, xanthine, C2 modified purine, N8 modified purine, 2,6-diaminopurine, 6-dimethylamine purine, 2-aminopurine, N6-alkyladenine, O6-alkylguanine, 7-deazapurine, cytosine, 5-methylcytosine, isocytosine, pseudocytosine, uracil, Pseudouracil, 2-thiouridine, 4-thiouridine, C5-modified pyrimidine, thymine, indole, 5-nitroindole, and 3-nitropyrrole;

In some embodiments, B is selected from adenine, guanine, 2,6-diaminopurine, 6-dimethylaminopurine, 2-aminopurine, cytosine, uracil, thymine, indole, 5-nitroindole and 3-nitropyrrole;

In some embodiments, B is selected from the base at the position corresponding to the modified nucleotide of the antisense strand.

。 In some embodiments, the chemical modifications described above are not

.

In some embodiments, each X is independently selected from CR ₄ (R ₄ ′), S, NR ₅ and NH-CO, wherein R ₄ , R ₄ ′, R ₅ are each independently H or C ₁ -C ₃ alkyl;

n=0, 1 or 2; m=0, 1 or 2; s=0 or 1;

Each J ₁ , J ₂ is independently H or C ₁ -C ₃ alkyl;

R ₃ is selected from H, OH, halogen, NH ₂ , C ₁ -C ₃ alkyl, C ₁ -C ₃ alkoxy, C ₂ -C ₄ alkenyl, C ₂ -C ₄ alkynyl, S-CH ₃ , NCH ₃ (CH ₃ ), OCH ₂ CH ₂ OCH ₃ , -O-alkylamino and (CH ₂ ) _p R ₆ ; wherein R ₆ is selected from OH, halogen, methoxy, ethoxy, N ₃ , C ₂ -C ₆ alkenyl and C ₂ -C ₆ alkynyl, p=1, 2 or 3;

R ₁ is selected from H, C ₁ -C ₃ alkyl, C ₁ -C ₃ alkoxy, C ₂ -C ₄ alkenyl, C ₂ -C ₄ alkynyl and (CH ₂ ) _q R ₇ ; wherein R ₇ selected from OH, halogen, methoxy, ethoxy, N ₃ , C ₂ -C ₄ alkenyl and C ₂ -C ₄ alkynyl, q=1, 2 or 3;

R ₂ is selected from H, OH, halogen, NH ₂ , C ₁ -C ₃ alkyl, C ₁ -C ₃ alkoxy, C ₂ -C ₄ alkenyl, C ₂ -C ₄ alkynyl, S-CH ₃ , NCH ₃ (CH ₃ ), OCH ₂ CH ₂ OCH ₃ , -O-alkylamino and (CH ₂ ) _r R ₈ ; wherein R ₈ is selected from OH, halogen, methoxy, ethoxy, N ₃ , C ₂ -C ₄ alkenyl and C ₂ -C ₄ alkynyl, r=1, 2 or 3;

Optionally, R1 and _R2 are directly connected to form _a ring.

B is as defined in formula (I).

In some embodiments, B is a base or a base analog; eg, those selected from the group consisting of purine bases, pyrimidine bases, indole, 5-nitroindole, and 3-nitropyrrole;

In some embodiments, B is selected from the group consisting of adenine, guanine, isoguanine, hypoxanthine, xanthine, C2 modified purine, N8 modified purine, 2,6-diaminopurine, 6-dimethylamine purine, 2-aminopurine, N6-alkyladenine, O6-alkylguanine, 7-deazapurine, cytosine, 5-methylcytosine, isocytosine, pseudocytosine, uracil, Pseudouracil, 2-thiouridine, 4-thiouridine, C5-modified pyrimidine, thymine, indole, 5-nitroindole, and 3-nitropyrrole;

In some embodiments, B is selected from adenine, guanine, 2,6-diaminopurine, 6-dimethylaminopurine, 2-aminopurine, cytosine, uracil, thymine, indole, 5-nitroindole and 3-nitropyrrole;

In some embodiments, B is selected from the base at the position corresponding to the modified nucleotide of the antisense strand.

In some embodiments, each X is independently selected from CR ₄ (R ₄ ′), S, NR ₅ and NH-CO, wherein R ₄ , R ₄ ′, R ₅ are each independently H, methyl, B radical, n-propyl or isopropyl;

n=0, 1 or 2; m=0, 1 or 2; s=0 or 1;

Each J ₁ , J ₂ is independently H or methyl;

R ₃ is selected from H, OH, F, Cl, NH ₂ , methyl, ethyl, n-propyl, isopropyl, methoxy, ethoxy, n-propoxy, isopropoxy, vinyl, Allyl, ethynyl, propargyl, S- _CH3 , _NCH3 ( _CH3 ), _OCH2CH2OCH3 , -O _- methylamino, -O-ethylamino and ( _{CH2 ) p} R ₆ ; wherein R ₆ is selected from OH, F, Cl, methoxy, ethoxy, N ₃ , vinyl, allyl, ethynyl and propargyl, p=1 or 2;

R ₁ is selected from H, methyl, ethyl, n-propyl, isopropyl, methoxy, ethoxy, n-propoxy, isopropoxy, vinyl, allyl, ethynyl, propargyl and (CH ₂ ) _q R ₇ ; wherein R ₇ is selected from OH, F, Cl, methoxy, ethoxy, N ₃ , vinyl, allyl, ethynyl and propargyl, q=1 or 2;

R ₂ is selected from H, OH, F, Cl, NH ₂ , methyl, ethyl, n-propyl, isopropyl, methoxy, ethoxy, n-propoxy, isopropoxy, vinyl, Allyl, ethynyl, propargyl, S- _CH3 , _NCH3 ( _CH3 ), _OCH2CH2OCH3 , -O _- methylamino, -O-ethylamino and ( _{CH2 ) r} R ₈ ; wherein R ₈ is selected from OH, F, Cl, methoxy, ethoxy, N ₃ , vinyl, allyl, ethynyl and propargyl, r=1 or 2;

Optionally, R1 and _R2 are directly connected to form _a ring.

B is as defined in formula (I).

In some embodiments, B is a base or a base analog; eg, those selected from the group consisting of purine bases, pyrimidine bases, indole, 5-nitroindole, and 3-nitropyrrole;

In some embodiments, B is selected from the group consisting of adenine, guanine, isoguanine, hypoxanthine, xanthine, C2 modified purine, N8 modified purine, 2,6-diaminopurine, 6-dimethylamine purine, 2-aminopurine, N6-alkyladenine, O6-alkylguanine, 7-deazapurine, cytosine, 5-methylcytosine, isocytosine, pseudocytosine, uracil, Pseudouracil, 2-thiouridine, 4-thiouridine, C5-modified pyrimidine, thymine, indole, 5-nitroindole, and 3-nitropyrrole;

In some embodiments, B is selected from adenine, guanine, 2,6-diaminopurine, 6-dimethylaminopurine, 2-aminopurine, cytosine, uracil, thymine, indole, 5-nitroindole and 3-nitropyrrole;

In some embodiments, B is selected from the base at the position corresponding to the modified nucleotide of the antisense strand.

In some embodiments, each X is independently selected from CR ₄ (R ₄ ′), S, NR ₅ and NH-CO, wherein R ₄ , R ₄ ′, R ₅ are each independently H, methyl, B radical, n-propyl or isopropyl;

n=0, 1 or 2; m=0, 1 or 2; s=0 or 1;

Each J ₁ , J ₂ is independently H or methyl;

R ₃ is selected from H, OH, F, Cl, NH ₂ , methyl, ethyl, n-propyl, isopropyl, methoxy, ethoxy, n-propoxy, isopropoxy, vinyl, Allyl, ethynyl, propargyl, S- _CH3 , _NCH3 ( _CH3 ), _OCH2CH2OCH3 , -O _- methylamino, -O-ethylamino and ( _{CH2 ) p} R ₆ ; wherein R ₆ is selected from OH, F, Cl, methoxy, ethoxy, N ₃ , vinyl, allyl, ethynyl and propargyl, p=1 or 2;

R ₁ is selected from H, methyl, ethyl, n-propyl, isopropyl, methoxy, ethoxy, n-propoxy, isopropoxy, vinyl, allyl, ethynyl, propargyl and (CH ₂ ) _q R ₇ ; wherein R ₇ is selected from OH, F, Cl, methoxy, ethoxy, N ₃ , vinyl, allyl, ethynyl and propargyl, q=1 or 2;

R ₂ is selected from H, OH, F, Cl, NH ₂ , methyl, ethyl, n-propyl, isopropyl, methoxy, ethoxy, n-propoxy, isopropoxy, vinyl, Allyl, ethynyl, propargyl, S- _CH3 , _NCH3 ( _CH3 ), _OCH2CH2OCH3 , -O _- methylamino, -O-ethylamino and ( _{CH2 ) r} R ₈ ; wherein R ₈ is selected from OH, F, Cl, methoxy, ethoxy, N ₃ , vinyl, allyl, ethynyl and propargyl, r=1 or 2;

Optionally, R1 and _R2 are directly connected to form _a ring.

B is as defined in formula (I).

In some embodiments, B is a base or a base analog; eg, those selected from the group consisting of purine bases, pyrimidine bases, indole, 5-nitroindole, and 3-nitropyrrole;

In some embodiments, B is selected from the group consisting of adenine, guanine, isoguanine, hypoxanthine, xanthine, C2 modified purine, N8 modified purine, 2,6-diaminopurine, 6-dimethylamine purine, 2-aminopurine, N6-alkyladenine, O6-alkylguanine, 7-deazapurine, cytosine, 5-methylcytosine, isocytosine, pseudocytosine, uracil, Pseudouracil, 2-thiouridine, 4-thiouridine, C5-modified pyrimidine, thymine, indole, 5-nitroindole, and 3-nitropyrrole;

In some embodiments, B is selected from adenine, guanine, 2,6-diaminopurine, 6-dimethylaminopurine, 2-aminopurine, cytosine, uracil, thymine, indole, 5-nitroindole and 3-nitropyrrole;

In some embodiments, B is selected from the base at the position corresponding to the modified nucleotide of the antisense strand.

In some embodiments, Y is O or NH; each X is independently selected from NH-CO, _CH and NH;

n=0 or 1; m=0 or 1; s=0 or 1;

Each J ₁ , J ₂ is independently H;

R ₁ is selected from H, methyl and CH ₂ OH;

R ₂ is selected from H, OH, NH ₂ , methyl and CH ₂ OH;

_{R3 is} selected from H, OH, _NH2 , methyl and CH2OH _;

Optionally, R ₁ and R ₂ are directly connected to form a ring;

B is as defined in formula (I).

In some embodiments, B is a base or a base analog; eg, those selected from the group consisting of purine bases, pyrimidine bases, indole, 5-nitroindole, and 3-nitropyrrole;

In some embodiments, B is selected from the group consisting of adenine, guanine, isoguanine, hypoxanthine, xanthine, C2 modified purine, N8 modified purine, 2,6-diaminopurine, 6-dimethylamine purine, 2-aminopurine, N6-alkyladenine, O6-alkylguanine, 7-deazapurine, cytosine, 5-methylcytosine, isocytosine, pseudocytosine, uracil, Pseudouracil, 2-thiouridine, 4-thiouridine, C5-modified pyrimidine, thymine, indole, 5-nitroindole, and 3-nitropyrrole;

In some embodiments, B is selected from adenine, guanine, 2,6-diaminopurine, 6-dimethylaminopurine, 2-aminopurine, cytosine, uracil, thymine, indole, 5-nitroindole and 3-nitropyrrole;

In some embodiments, B is selected from the base at the position corresponding to the modified nucleotide of the antisense strand.

In some embodiments, Y is O or NH; each X is independently selected from NH-CO, _CH and NH;

n=0 or 1; m=0 or 1; s=0 or 1;

Each J ₁ , J ₂ is independently H;

R ₁ is selected from H, methyl and CH ₂ OH;

R ₂ is selected from H, methyl and CH ₂ OH;

_{R3 is} selected from H, OH, _NH2 , methyl and CH2OH _;

Optionally, R ₁ and R ₂ are directly connected to form a ring;

In some embodiments, B is a base or a base analog; eg, those selected from the group consisting of purine bases, pyrimidine bases, indole, 5-nitroindole, and 3-nitropyrrole;

In some embodiments, B is selected from the group consisting of adenine, guanine, isoguanine, hypoxanthine, xanthine, C2 modified purine, N8 modified purine, 2,6-diaminopurine, 6-dimethylamine purine, 2-aminopurine, N6-alkyladenine, O6-alkylguanine, 7-deazapurine, cytosine, 5-methylcytosine, isocytosine, pseudocytosine, uracil, Pseudouracil, 2-thiouridine, 4-thiouridine, C5-modified pyrimidine, thymine, indole, 5-nitroindole, and 3-nitropyrrole;

In some embodiments, B is selected from adenine, guanine, 2,6-diaminopurine, 6-dimethylaminopurine, 2-aminopurine, cytosine, uracil, thymine, indole, 5-nitroindole and 3-nitropyrrole;

In some embodiments, B is selected from the base at the position corresponding to the modified nucleotide of the antisense strand.

In some embodiments, the chemical modification of formula (I) is selected from:

wherein B is a base or a base analog; for example, those selected from purine bases, pyrimidine bases, indole, 5-nitroindole and 3-nitropyrrole;

In some embodiments, B is selected from the group consisting of adenine, guanine, isoguanine, hypoxanthine, xanthine, C2 modified purine, N8 modified purine, 2,6-diaminopurine, 6-dimethylamine purine, 2-aminopurine, N6-alkyladenine, O6-alkylguanine, 7-deazapurine, cytosine, 5-methylcytosine, isocytosine, pseudocytosine, uracil, Pseudouracil, 2-thiouridine, 4-thiouridine, C5-modified pyrimidine, thymine, indole, 5-nitroindole, and 3-nitropyrrole;

In some embodiments, B is selected from adenine, guanine, 2,6-diaminopurine, 6-dimethylaminopurine, 2-aminopurine, cytosine, uracil, thymine, indole, 5-nitroindole and 3-nitropyrrole;

In some embodiments, B is selected from the base at the position corresponding to the modified nucleotide of the antisense strand.

In some embodiments, the chemical modification of formula (I) is selected from:

wherein B is a base or a base analog; for example, those selected from purine bases, pyrimidine bases, indole, 5-nitroindole and 3-nitropyrrole;

In some embodiments, B is selected from the group consisting of adenine, guanine, isoguanine, hypoxanthine, xanthine, C2 modified purine, N8 modified purine, 2,6-diaminopurine, 6-dimethylamine purine, 2-aminopurine, N6-alkyladenine, O6-alkylguanine, 7-deazapurine, cytosine, 5-methylcytosine, isocytosine, pseudocytosine, uracil, Pseudouracil, 2-thiouridine, 4-thiouridine, C5-modified pyrimidine, thymine, indole, 5-nitroindole, and 3-nitropyrrole;

In some embodiments, B is selected from adenine, guanine, 2,6-diaminopurine, 6-dimethylaminopurine, 2-aminopurine, cytosine, uracil, thymine, indole, 5-nitroindole and 3-nitropyrrole;

In some embodiments, B is selected from the base at the position corresponding to the modified nucleotide of the antisense strand.

In some embodiments, the nucleotides comprising the chemical modifications represented by formula (I) or tautomer modifications thereof are selected from nucleotides comprising chemical modifications represented by formula (I') or tautomer modifications thereof ,

wherein Y is selected from O, NH and S;

Each X is independently selected from CR ₄ (R ₄ ′), S, NR ₅ and NH-CO, wherein R ₄ , R ₄ ′, R ₅ are each independently H or C ₁ -C ₆ alkyl;

J ₂ is H or C ₁ -C ₆ alkyl;

n=0, 1 or 2; m=0, 1 or 2; s=0 or 1;

R ₃ is selected from H, OH, halogen, NH ₂ , C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, C ₂ -C ₆ alkenyl, C ₂ -C ₆ alkynyl, S-CH ₃ , NCH ₃ (CH ₃ ), OCH ₂ CH ₂ OCH ₃ , -O-alkylamino and (CH ₂ ) _p R ₆ ; wherein R ₆ is selected from OH, halogen, methoxy, ethoxy, N ₃ , C ₂ -C ₆ alkenyl and C ₂ -C ₆ alkynyl, p=1, 2 or 3;

，Q_2’為R₂；或者Q_1’為R₂，Q_2’為

； Q1 _' is

, Q _2' is R ₂ ; or Q _1' is R ₂ , Q _2' is

;

in,

R ₁ is selected from H, C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, C ₂ -C ₆ alkenyl, C ₂ -C ₆ alkynyl and (CH ₂ ) _q R ₇ ; wherein R ₇ selected from OH, halogen, methoxy, ethoxy, N ₃ , C ₂ -C ₆ alkenyl and C ₂ -C ₆ alkynyl, q=1, 2 or 3;

J ₁ is H or C ₁ -C ₆ alkyl;

R ₂ is selected from H, OH, halogen, NH ₂ , C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, C ₂ -C ₆ alkenyl, C ₂ -C ₆ alkynyl, S-CH ₃ , NCH ₃ (CH ₃ ), OCH ₂ CH ₂ OCH ₃ , -O-alkylamino and (CH ₂ ) _r R ₈ ; wherein R ₈ is selected from OH, halogen, methoxy, ethoxy, N ₃ , C ₂ -C ₆ alkenyl and C ₂ -C ₆ alkynyl, r=1, 2 or 3;

Optionally, R ₁ and R ₂ are directly connected to form a ring;

B is a base or a base analog;

M is O or S;

。 Wherein, the chemical modification represented by the formula (I') is not

.

In some embodiments, when X is NH _- CO, R1 is not H.

In some embodiments, B is a base or a base analog; eg, those selected from the group consisting of purine bases, pyrimidine bases, indole, 5-nitroindole, and 3-nitropyrrole;

In some embodiments, B is selected from the group consisting of adenine, guanine, isoguanine, hypoxanthine, xanthine, C2 modified purine, N8 modified purine, 2,6-diaminopurine, 6-dimethylamine purine, 2-aminopurine, N6-alkyladenine, O6-alkylguanine, 7-deazapurine, cytosine, 5-methylcytosine, isocytosine, pseudocytosine, uracil, Pseudouracil, 2-thiouridine, 4-thiouridine, C5-modified pyrimidine, thymine, indole, 5-nitroindole, and 3-nitropyrrole;

In some embodiments, B is selected from adenine, guanine, 2,6-diaminopurine, 6-dimethylaminopurine, 2-aminopurine, cytosine, uracil, thymine, indole, 5-nitroindole and 3-nitropyrrole;

In some embodiments, B is selected from the base at the position corresponding to the modified nucleotide of the antisense strand.

In some embodiments, formula (I') is selected from formula (I'-1):

wherein Y is selected from O, NH and S;

Each X is independently selected from CR ₄ (R ₄ ′), S, NR ₅ and NH-CO, wherein R ₄ , R ₄ ′, R ₅ are each independently H or C ₁ -C ₆ alkyl;

Each J ₁ , J ₂ is independently H or C ₁ -C ₆ alkyl;

n=0, 1 or 2; m=0, 1 or 2; s=0 or 1;

R ₃ is selected from H, OH, halogen, NH ₂ , C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, C ₂ -C ₆ alkenyl, C ₂ -C ₆ alkynyl, S-CH ₃ , NCH ₃ (CH ₃ ), OCH ₂ CH ₂ OCH ₃ , -O-alkylamino and (CH ₂ ) _p R ₆ ; wherein R ₆ is selected from OH, halogen, methoxy, ethoxy, N ₃ , C ₂ -C ₆ alkenyl and C ₂ -C ₆ alkynyl, p=1, 2 or 3;

R ₁ is selected from H, C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, C ₂ -C ₆ alkenyl, C ₂ -C ₆ alkynyl and (CH ₂ ) _q R ₇ ; wherein R ₇ selected from OH, halogen, methoxy, ethoxy, N ₃ , C ₂ -C ₆ alkenyl and C ₂ -C ₆ alkynyl, q=1, 2 or 3;

R ₂ is selected from H, OH, halogen, NH ₂ , C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, C ₂ -C ₆ alkenyl, C ₂ -C ₆ alkynyl, S-CH ₃ , NCH ₃ (CH ₃ ), OCH ₂ CH ₂ OCH ₃ , -O-alkylamino and (CH ₂ ) _r R ₈ ; wherein R ₈ is selected from OH, halogen, methoxy, ethoxy, N ₃ , C ₂ -C ₆ alkenyl and C ₂ -C ₆ alkynyl, r=1, 2 or 3;

M is O or S;

Optionally, R ₁ and R ₂ are directly connected to form a ring;

B is as defined in formula (I').

In some embodiments, B is a base or a base analog; eg, those selected from the group consisting of purine bases, pyrimidine bases, indole, 5-nitroindole, and 3-nitropyrrole;

In some embodiments, B is selected from the group consisting of adenine, guanine, isoguanine, hypoxanthine, xanthine, C2 modified purine, N8 modified purine, 2,6-diaminopurine, 6-dimethylamine Alkylpurine, 2-aminopurine, N6-alkyladenine, O6-alkylguanine, 7-deazapurine, cytosine, 5- Methylcytosine, isocytosine, pseudocytosine, uracil, pseudouracil, 2-thiouridine, 4-thiouridine, C5-modified pyrimidine, thymine, indole, 5-nitroindole Indoles and 3-nitropyrroles;

In some embodiments, B is selected from adenine, guanine, 2,6-diaminopurine, 6-dimethylaminopurine, 2-aminopurine, cytosine, uracil, thymine, indole, 5-nitroindole and 3-nitropyrrole;

In some embodiments, B is selected from the base at the position corresponding to the modified nucleotide of the antisense strand.

In some embodiments, formula (I') is selected from formula (I'-2):

wherein Y is selected from O, NH and S;

Each X is independently selected from CR ₄ (R ₄ ′), S, NR ₅ and NH-CO, wherein R ₄ , R ₄ ′, R ₅ are each independently H or C ₁ -C ₆ alkyl;

n=0, 1 or 2; m=0, 1 or 2; s=0 or 1;

Each J ₁ , J ₂ is independently H or C ₁ -C ₆ alkyl;

R ₃ is selected from H, OH, halogen, NH ₂ , C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, C ₂ -C ₆ alkenyl, C ₂ -C ₆ alkynyl, S-CH ₃ , NCH ₃ (CH ₃ ), OCH ₂ CH ₂ OCH ₃ , -O-alkylamino and (CH ₂ ) _p R ₆ ; wherein R ₆ is selected from OH, halogen, methoxy, ethoxy, N ₃ , C ₂ -C ₆ alkenyl and C ₂ -C ₆ alkynyl, p=1, 2 or 3;

R ₁ is selected from H, C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, C ₂ -C ₆ alkenyl, C ₂ -C ₆ alkynyl and (CH ₂ ) _q R ₇ ; wherein R ₇ selected from OH, halogen, methoxy, ethoxy, N ₃ , C ₂ -C ₆ alkenyl and C ₂ -C ₆ alkynyl, q=1, 2 or 3;

R ₂ is selected from H, C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, S-CH ₃ , NCH ₃ (CH ₃ ), OCH ₂ CH ₂ OCH ₃ , -O-alkylamino and (CH ₂ ) _r R ₈ ; wherein R ₈ is selected from OH, halogen, methoxy, ethoxy, N ₃ , C ₂ -C ₆ alkenyl and C ₂ -C ₆ alkynyl; r=1, 2 or 3;

Optionally, R ₁ and R ₂ are directly connected to form a ring;

M is O or S;

B is as defined in formula (I').

In some embodiments, B is a base or a base analog; eg, those selected from the group consisting of purine bases, pyrimidine bases, indole, 5-nitroindole, and 3-nitropyrrole;

In some embodiments, B is selected from the group consisting of adenine, guanine, isoguanine, hypoxanthine, xanthine, C2 modified purine, N8 modified purine, 2,6-diaminopurine, 6-dimethylamine purine, 2-aminopurine, N6-alkyladenine, O6-alkylguanine, 7-deazapurine, cytosine, 5-methylcytosine, isocytosine, pseudocytosine, uracil, Pseudouracil, 2-thiouridine, 4-thiouridine, C5-modified pyrimidine, thymine, indole, 5-nitroindole, and 3-nitropyrrole;

In some embodiments, B is selected from adenine, guanine, 2,6-diaminopurine, 6-dimethylaminopurine, 2-aminopurine, cytosine, uracil, thymine, indole, 5-nitroindole and 3-nitropyrrole;

In some embodiments, B is selected from the base at the position corresponding to the modified nucleotide of the antisense strand.

。 In some embodiments, the chemical modifications described above are not

.

In some embodiments, when X is NH _- CO, R1 is not H.

In some embodiments, each X is independently selected from CR ₄ (R ₄ ′), S, NR ₅ and NH-CO, wherein R ₄ , R ₄ ′, R ₅ are each independently H or C ₁ -C ₃ alkyl;

n=0, 1 or 2; m=0, 1 or 2; s=0 or 1;

Each J ₁ , J ₂ is independently H or C ₁ -C ₃ alkyl;

R ₃ is selected from H, OH, halogen, NH ₂ , C ₁ -C ₃ alkyl, C ₁ -C ₃ alkoxy, C ₂ -C ₄ alkenyl, C ₂ -C ₄ alkynyl, S-CH ₃ , NCH ₃ (CH ₃ ), OCH ₂ CH ₂ OCH ₃ , -O-alkylamino and (CH ₂ ) _p R ₆ ; wherein R ₆ is selected from OH, halogen, methoxy, ethoxy, N ₃ , C ₂ -C ₆ alkenyl and C ₂ -C ₆ alkynyl, p=1, 2 or 3;

R ₁ is selected from H, C ₁ -C ₃ alkyl, C ₁ -C ₃ alkoxy, C ₂ -C ₄ alkenyl, C ₂ -C ₄ alkynyl and (CH ₂ ) _q R ₇ ; wherein R ₇ selected from OH, halogen, methoxy, ethoxy, N ₃ , C ₂ -C ₄ alkenyl and C ₂ -C ₄ alkynyl, q=1, 2 or 3;

R ₂ is selected from H, OH, halogen, NH ₂ , C ₁ -C ₃ alkyl, C ₁ -C ₃ alkoxy, C ₂ -C ₄ alkenyl, C ₂ -C ₄ alkynyl, S-CH ₃ , NCH ₃ (CH ₃ ), OCH ₂ CH ₂ OCH ₃ , -O-alkylamino and (CH ₂ ) _r R ₈ ; wherein R ₈ is selected from OH, halogen, methoxy, ethoxy, N ₃ , C ₂ -C ₄ alkenyl and C ₂ -C ₄ alkynyl, r=1, 2 or 3;

Optionally, R1 and _R2 are directly connected to form _a ring.

In some embodiments, each X is independently selected from CR ₄ (R ₄ ′), S, NR ₅ and NH-CO, wherein R ₄ , R ₄ ′, R ₅ are each independently H, methyl, B radical, n-propyl or isopropyl;

n=0, 1 or 2; m=0, 1 or 2; s=0 or 1;

Each J ₁ , J ₂ is independently H or methyl;

R ₃ is selected from H, OH, F, Cl, NH ₂ , methyl, ethyl, n-propyl, isopropyl, methoxy, ethoxy, n-propoxy, isopropoxy, vinyl, Allyl, ethynyl, propargyl, S- _CH3 , _NCH3 ( _CH3 ), _OCH2CH2OCH3 , -O _- methylamino, -O-ethylamino and ( _{CH2 ) p} R ₆ ; wherein R ₆ is selected from OH, F, Cl, methoxy, ethoxy, N ₃ , vinyl, allyl, ethynyl and propargyl, p=1 or 2;

R ₁ is selected from H, methyl, ethyl, n-propyl, isopropyl, methoxy, ethoxy, n-propoxy, isopropoxy, vinyl, allyl, ethynyl, propargyl and (CH ₂ ) _q R ₇ ; wherein R ₇ is selected from OH, F, Cl, methoxy, ethoxy, N ₃ , vinyl, allyl, ethynyl and propargyl, q=1 or 2;

R ₂ is selected from H, OH, F, Cl, NH ₂ , methyl, ethyl, n-propyl, isopropyl, methoxy, ethoxy, n-propoxy, isopropoxy, vinyl, Allyl, ethynyl, propargyl, S- _CH3 , _NCH3 ( _CH3 ), _OCH2CH2OCH3 , -O _- methylamino, -O-ethylamino and ( _{CH2 ) r} R ₈ ; wherein R ₈ is selected from OH, F, Cl, methoxy, ethoxy, N ₃ , vinyl, allyl, ethynyl and propargyl, r=1 or 2;

Optionally, R1 and _R2 are directly connected to form _a ring.

In some embodiments, B is a base or a base analog; eg, those selected from the group consisting of purine bases, pyrimidine bases, indole, 5-nitroindole, and 3-nitropyrrole;

In some embodiments, B is selected from the group consisting of adenine, guanine, isoguanine, hypoxanthine, xanthine, C2 modified purine, N8 modified purine, 2,6-diaminopurine, 6-dimethylamine purine, 2-aminopurine, N6-alkyladenine, O6-alkylguanine, 7-deazapurine, cytosine, 5-methylcytosine, isocytosine, pseudocytosine, uracil, Pseudouracil, 2-thiouridine, 4-thiouridine, C5-modified pyrimidine, thymine, indole, 5-nitroindole, and 3-nitropyrrole;

In some embodiments, B is selected from adenine, guanine, 2,6-diaminopurine, 6-dimethylaminopurine, 2-aminopurine, cytosine, uracil, thymine, indole, 5-nitroindole and 3-nitropyrrole;

In some embodiments, B is selected from the base at the position corresponding to the modified nucleotide of the antisense strand.

In some embodiments, each X is independently selected from CR ₄ (R ₄ ′), S, NR ₅ and NH-CO, wherein R ₄ , R ₄ ′, R ₅ are each independently H, methyl, B radical, n-propyl or isopropyl;

n=0, 1 or 2; m=0, 1 or 2; s=0 or 1;

Each J ₁ , J ₂ is independently H or methyl;

R ₃ is selected from H, OH, F, Cl, NH ₂ , methyl, ethyl, n-propyl, isopropyl, methoxy, ethoxy, n-propoxy, isopropoxy, vinyl, Allyl, ethynyl, propargyl, S- _CH3 , _NCH3 ( _CH3 ), _OCH2CH2OCH3 , -O _- methylamino, -O-ethylamino and ( _{CH2 ) p} R ₆ ; wherein R ₆ is selected from OH, F, Cl, methoxy, ethoxy, N ₃ , vinyl, allyl, ethynyl and propargyl, p=1 or 2;

R ₁ is selected from H, methyl, ethyl, n-propyl, isopropyl, methoxy, ethoxy, n-propoxy, isopropoxy, vinyl, allyl, ethynyl, propargyl and (CH ₂ ) _q R ₇ ; wherein R ₇ is selected from OH, F, Cl, methoxy, ethoxy, N ₃ , vinyl, allyl, ethynyl and propargyl, q=1 or 2;

R ₂ is selected from H, OH, F, Cl, NH ₂ , methyl, ethyl, n-propyl, isopropyl, methoxy, ethoxy, n-propoxy, isopropoxy, vinyl, Allyl, ethynyl, propargyl, S- _CH3 , _NCH3 ( _CH3 ), _OCH2CH2OCH3 , -O _- methylamino, -O-ethylamino and ( _{CH2 ) r} R ₈ ; wherein R ₈ is selected from OH, F, Cl, methoxy, ethoxy, N ₃ , vinyl, allyl, ethynyl and propargyl, r=1 or 2;

Optionally, R1 and _R2 are directly connected to form _a ring.

In some embodiments, B is a base or a base analog; eg, those selected from the group consisting of purine bases, pyrimidine bases, indole, 5-nitroindole, and 3-nitropyrrole;

In some embodiments, B is selected from the group consisting of adenine, guanine, isoguanine, hypoxanthine, xanthine, C2 modified purine, N8 modified purine, 2,6-diaminopurine, 6-dimethylamine purine, 2-aminopurine, N6-alkyladenine, O6-alkylguanine, 7-deazapurine, cytosine, 5-methylcytosine, isocytosine, pseudocytosine, uracil, pseudouracil , 2-thiouridine, 4-thiouridine, C5-modified pyrimidine, thymine, indole, 5-nitroindole and 3-nitropyrrole;

In some embodiments, B is selected from adenine, guanine, 2,6-diaminopurine, 6-dimethylaminopurine, 2-aminopurine, cytosine, uracil, thymine, indole, 5-nitroindole and 3-nitropyrrole;

In some embodiments, B is selected from the base at the position corresponding to the modified nucleotide of the antisense strand.

In some embodiments, Y is O or NH; each X is independently selected from NH-CO, _CH and NH;

n=0 or 1; m=0 or 1; s=0 or 1;

Each J ₁ , J ₂ is independently H;

R ₁ is selected from H, methyl and CH ₂ OH;

R ₂ is selected from H, OH, NH ₂ , methyl and CH ₂ OH;

_{R3 is} selected from H, OH, _NH2 , methyl and CH2OH _;

Optionally, R1 and _R2 are directly connected to form _a ring.

In some embodiments, B is a base or a base analog; eg, those selected from the group consisting of purine bases, pyrimidine bases, indole, 5-nitroindole, and 3-nitropyrrole;

In some embodiments, B is selected from the group consisting of adenine, guanine, isoguanine, hypoxanthine, xanthine, C2 modified purine, N8 modified purine, 2,6-diaminopurine, 6-dimethylamine purine, 2-aminopurine, N6-alkyladenine, O6-alkylguanine, 7-deazapurine, cytosine, 5-methylcytosine, isocytosine, pseudocytosine, uracil, Pseudouracil, 2-thiouridine, 4-thiouridine, C5-modified pyrimidine, thymine, indole, 5-nitroindole, and 3-nitropyrrole;

In some embodiments, B is selected from adenine, guanine, 2,6-diaminopurine, 6-dimethylaminopurine, 2-aminopurine, cytosine, uracil, thymine, indole, 5-nitroindole and 3-nitropyrrole;

In some embodiments, B is selected from the base at the position corresponding to the modified nucleotide of the antisense strand.

In some embodiments, Y is O or NH; each X is independently selected from NH-CO, _CH and NH;

n=0 or 1; m=0 or 1; s=0 or 1;

Each J ₁ , J ₂ is independently H;

R ₁ is selected from H, methyl and CH ₂ OH;

R ₂ is selected from H, methyl and CH ₂ OH;

_{R3 is} selected from H, OH, _NH2 , methyl and CH2OH _;

Optionally, R1 and _R2 are directly connected to form _a ring.

In some embodiments, B is a base or a base analog; eg, those selected from the group consisting of purine bases, pyrimidine bases, indole, 5-nitroindole, and 3-nitropyrrole;

In some embodiments, B is selected from the group consisting of adenine, guanine, isoguanine, hypoxanthine, xanthine, C2 modified purine, N8 modified purine, 2,6-diaminopurine, 6-dimethylamine purine, 2-aminopurine, N6-alkyladenine, O6-alkylguanine, 7-deazapurine, cytosine, 5-methylcytosine, isocytosine, pseudocytosine, uracil, Pseudouracil, 2-thiouridine, 4-thiouridine, C5-modified pyrimidine, thymine, indole, 5-nitroindole, and 3-nitropyrrole;

In some embodiments, B is selected from adenine, guanine, 2,6-diaminopurine, 6-dimethylaminopurine, 2-aminopurine, cytosine, uracil, thymine, indole, 5-nitroindole and 3-nitropyrrole;

In some embodiments, B is selected from the base at the position corresponding to the modified nucleotide of the antisense strand.

In some embodiments, the chemical modification of formula (I') is selected from:

Among them, M is O or S;

B is as defined in formula (I').

In some embodiments, B is a base or a base analog; eg, those selected from the group consisting of purine bases, pyrimidine bases, indole, 5-nitroindole, and 3-nitropyrrole;

In some embodiments, B is selected from the group consisting of adenine, guanine, isoguanine, hypoxanthine, xanthine, C2 modified purine, N8 modified purine, 2,6-diaminopurine, 6-dimethylamine purine, 2-aminopurine, N6-alkyladenine, O6-alkylguanine, 7-deazapurine, cytosine, 5-methylcytosine, isocytosine, pseudocytosine, uracil, Pseudouracil, 2-thiouridine, 4-thiouridine, C5-modified pyrimidine, thymine, indole, 5-nitroindole, and 3-nitropyrrole;

In some embodiments, B is selected from adenine, guanine, 2,6-diaminopurine, 6-dimethylaminopurine, 2-aminopurine, cytosine, uracil, thymine, indole, 5-nitroindole and 3-nitropyrrole;

In some embodiments, B is selected from the base at the position corresponding to the modified nucleotide of the antisense strand.

In some embodiments, the chemical modification of formula (I') is selected from:

Among them, M is O or S;

B is as defined in formula (I').

In some embodiments, B is a base or a base analog; eg, those selected from the group consisting of purine bases, pyrimidine bases, indole, 5-nitroindole, and 3-nitropyrrole;

In some embodiments, B is selected from the group consisting of adenine, guanine, isoguanine, hypoxanthine, xanthine, C2 modified purine, N8 modified purine, 2,6-diaminopurine, 6-dimethylamine purine, 2-aminopurine, N6-alkyladenine, O6-alkylguanine, 7-deazapurine, cytosine, 5-methylcytosine, isocytosine, pseudocytosine, uracil, Pseudouracil, 2-thiouridine, 4-thiouridine, C5-modified pyrimidine, thymine, indole, 5-nitroindole, and 3-nitropyrrole;

In some embodiments, B is selected from adenine, guanine, 2,6-diaminopurine, 6-dimethylaminopurine, 2-aminopurine, cytosine, uracil, thymine, indole, 5-nitroindole and 3-nitropyrrole;

In some embodiments, B is selected from the base at the position corresponding to the modified nucleotide of the antisense strand.

In some embodiments, the chemical modification of formula (I') is selected from:

Among them, M is O or S;

B is as defined in formula (I').

In some embodiments, B is a base or a base analog; eg, those selected from the group consisting of purine bases, pyrimidine bases, indole, 5-nitroindole, and 3-nitropyrrole;

In some embodiments, B is selected from the group consisting of adenine, guanine, isoguanine, hypoxanthine, xanthine, C2 modified purine, N8 modified purine, 2,6-diaminopurine, 6-dimethylamine purine, 2-aminopurine, N6-alkyladenine, O6-alkylguanine, 7-deazapurine, cytosine, 5-methylcytosine, isocytosine, pseudocytosine, uracil, Pseudouracil, 2-thiouridine, 4-thiouridine, C5-modified pyrimidine, thymine, indole, 5-nitroindole, and 3-nitropyrrole;

In some embodiments, B is selected from adenine, guanine, 2,6-diaminopurine, 6-dimethylaminopurine, 2-aminopurine, cytosine, uracil, thymine, indole, 5-nitroindole and 3-nitropyrrole;

In some embodiments, B is selected from the base at the position corresponding to the modified nucleotide of the antisense strand.

In some embodiments, the chemical modification represented by the formula (I') includes, but is not limited to:

and those in which the adenine in their structure is replaced by a guanine, cytosine, uracil or thymine.

In some embodiments, the modified nucleotides are located at positions 2 to 8 of the antisense strand in its 5' region.

In some embodiments, the modified nucleotide is located at position 5, 6 or 7 in the 5' region of the antisense strand.

In some embodiments, when the chemical modification represented by formula (I) or its tautomer modification is at the 5th position in its 5' region, B is selected from adenine, guanine, 2,6-diaminopurine , 6-dimethylaminopurine, 2-aminopurine, cytosine, uracil, thymine, indole, 5-nitroindole and 3-nitropyrrole; preferably, B is the antisense of the The base of the chain at the corresponding position in position 5 of its 5' region.

When the chemical modification represented by formula (I) or its tautomer is modified at the 6th position in its 5' region, B is selected from adenine, guanine, 2,6-diaminopurine, 6-dimethyl aminopurine, 2-aminopurine, cytosine, uracil, thymine, indole, 5-nitroindole and 3-nitropyrrole; preferably, B is the antisense strand in its 5' region The base at the corresponding position in the 6th position;

When the chemical modification represented by formula (I) or its tautomer modification is at the 7th position in its 5' region, B is selected from adenine, guanine, 2,6-diaminopurine, 6-dimethyl aminopurine, 2-aminopurine, cytosine, uracil, thymine, indole, 5-nitroindole and 3-nitropyrrole; preferably, B is the antisense strand in its 5' region The base at the corresponding position in position 7.

In some embodiments, all nucleotides are modified nucleotides.

In some embodiments, the sense strand contains a nucleotide sequence (5'-3') of the formula:

N _a N _a N _a N _a XN _a N _b N _b N _b N _a N _a N _a N _a N _a N _a N _a N _a N _a N _a

wherein each _X is independently Na or _Nb ;

Wherein, each X' is independently Na ' or N _b ', and Y is _Na ' or N _b ';W' represents _a 2'-methoxy-modified nucleotide or a nucleotide represented by formula (I) The chemical modification of or its tautomer modified nucleotide; Na is a 2'-methoxy modified nucleotide, and N _b is _a 2'-fluoro modified nucleotide.

In some embodiments, the antisense strand contains a nucleotide sequence (5'-3') of the formula:

N _a 'N _b 'N _a 'X'N _a 'N _b 'W'N _a 'X'Y'N _a 'X'N _a 'N _b 'N _a 'X'N _a 'X'N _a ' N _a 'N _a ';

Wherein, each X' is independently _Na ' or _Nb ', Y' is Na' or _Nb ';W' represents _a 2'-methoxy-modified nucleotide or a nucleotide represented by formula (I) The chemical modification of or its tautomer modified nucleotide; Na is a 2'-methoxy modified nucleotide, and N _b is _a 2'-fluoro modified nucleotide.

In some embodiments, the sense strand contains a nucleotide sequence of the formula:

5'-N _a N _a N _a N _a N _a N _a N _b N _b N _b N _a N _a N _a N _a N _a N _a N _a N _a N _a N _a -3'; or,

5'-N _a N _a N _a N _a NbN _a N _b N _b N _b N _a N _a N _a N _a N _a N _a N _a N _a N _a N _a -3';

Wherein, Na is a 2'-methoxy-modified nucleotide, and N _b is _a 2'-fluoro-modified nucleotide.

In some embodiments, the antisense strand contains a nucleotide sequence of the formula:

5'-N _a 'N _b 'N _a 'N _b 'N _a 'N _b 'W'N _a 'X'Y'N _a 'N _b 'N _a 'N _b 'N _a 'N _b 'N _a 'N _b 'N _a 'N _a 'N _a '-3';

Wherein, each X' is independently _Na ' or _Nb ', Y' is _Na ' or _Nb ';Na' is _a 2'-methoxy-modified nucleotide, and _Nb ' is 2' -Fluorine-modified nucleotide; W' represents a 2'-methoxy-modified nucleotide or a nucleotide modified by a chemical modification represented by formula (I) or a tautomer thereof.

In some embodiments, the antisense strand contains a nucleotide sequence of the formula:

5'-N _a 'N _b 'N _a 'N _b 'N _a 'N _b 'W'N _a 'N _a 'N _b 'N _a 'N _b 'N _a 'N _b 'N _a 'N _b ' N _a 'N _b 'N _a 'N _a 'N _a '-3'; or,

5'-N _a 'N _b 'N _a 'N _b 'N _a 'N _b 'W'N _a 'N _b 'N _a 'N _a 'N _b 'N _a 'N _b 'N _a 'N _b ' N _a 'N _b 'N _a 'N _a 'N _a '-3';

Wherein, N _a ' is a nucleotide modified by 2'-methoxyl group, and N _b ' is a nucleotide modified by 2'-fluoro;W' represents a nucleotide modified by 2'-methoxyl group or a nucleotide containing formula ( The chemically modified nucleotides shown in I) or their tautomers.

In some specific embodiments, W' represents a 2'-methoxy-modified nucleotide or a nucleotide comprising a chemical modification represented by formula (I) or a tautomer modification thereof;

In some specific embodiments, formula (I) is selected from:

、

或

；其中，B選自鳥嘌呤、腺嘌呤、胞嘧啶或尿嘧啶；在一些具體的實施方案中，B選自反義鏈在其5’區域的第7位中對應位置的鹼基。

,

or

; wherein, B is selected from guanine, adenine, cytosine or uracil; in some specific embodiments, B is selected from the base at the corresponding position in the 7th position of the 5' region of the antisense strand.

In some specific embodiments, formula (I) is selected from:

、

，和

；其中，M為O或S；其中，B選自鳥嘌呤、腺嘌呤、胞嘧啶或尿嘧啶；在一些具體的實施方案中，B選自反義鏈在其5’區域的第7位中對應位置的鹼基。

,

,and

wherein, M is O or S; wherein, B is selected from guanine, adenine, cytosine or uracil; In some specific embodiments, B is selected from the antisense strand in the 7th position in its 5' region base at the corresponding position.

In some specific embodiments, M is S. In some specific embodiments, M is O.

In some embodiments, at least one phosphate group in the sense strand and/or antisense strand is a phosphate group with a modifying group that imparts increased stability to the siRNA in a biological sample or environment; in In some embodiments, the phosphate group with the modifying group is a phosphorothioate group. Specifically, a phosphorothioate group refers to a phosphodiester group in which a non-bridging oxygen atom is replaced by a sulfur atom.

In some embodiments, the phosphorothioate group is present in at least one position selected from the group consisting of:

Between the first nucleotide and the second nucleotide in the 5'-3' direction of the sense strand;

Between the second nucleotide and the third nucleotide in the 5'-3' direction of the sense strand;

The first nucleotide end in the 3'-5' direction of the sense strand;

Between the first nucleotide and the second nucleotide in the 3'-5' direction of the sense strand;

Between the second nucleotide and the third nucleotide in the 3'-5' direction of the sense strand;

Between the first nucleotide and the second nucleotide in the 5'-3' direction of the antisense strand;

Between the second nucleotide and the third nucleotide in the 5'-3' direction of the antisense strand;

The first nucleotide end of the 3'-5' direction of the antisense strand;

Between the first nucleotide and the second nucleotide in the 3'-5' direction of the antisense strand; or

Between the 2nd nucleotide and the 3rd nucleotide in the 3'-5' direction of the antisense strand.

In some embodiments, a plurality of phosphorothioate groups are included in the sense and/or antisense strands, the phosphorothioate groups being present in:

between the 1st nucleotide and the 2nd nucleotide in the 5'-3' direction of the sense strand; and

between the 2nd nucleotide and the 3rd nucleotide in the 5'-3' direction of the sense strand; and

between the 1st nucleotide and the 2nd nucleotide in the 5'-3' direction of the antisense strand; and

between the 2nd nucleotide and the 3rd nucleotide in the 5'-3' direction of the antisense strand; and

between the 1st nucleotide and the 2nd nucleotide in the 3'-5' direction of the antisense strand; and

between the 2nd nucleotide and the 3rd nucleotide in the 3'-5' direction of the antisense strand; and

and optionally, the first nucleotide end in the 3'-5' direction of the sense strand, and/or,

Optionally, the sense strand is between the first nucleotide and the second nucleotide in the 3'-5' direction.

In some embodiments, the sense strand has a phosphorothioate group at:

i) between the first nucleotide and the second nucleotide in the 5'-3' direction of the sense strand; and

Between the second nucleotide and the third nucleotide in the 5'-3' direction of the sense strand;

ii) between the first nucleotide and the second nucleotide in the 5'-3' direction of the sense strand;

between the 2nd nucleotide and the 3rd nucleotide in the 5'-3' direction of the sense strand; and

The first nucleotide end in the 3'-5' direction of the sense strand;

iii) between the first nucleotide and the second nucleotide in the 5'-3' direction of the sense strand;

between the 2nd nucleotide and the 3rd nucleotide in the 5'-3' direction of the sense strand; and

Between the first nucleotide and the second nucleotide in the 3'-5' direction of the sense strand; or

iv) between the first nucleotide and the second nucleotide in the 5'-3' direction of the sense strand;

Between the second nucleotide and the third nucleotide in the 5'-3' direction of the sense strand;

between the first nucleotide and the second nucleotide in the 3'-5' direction of the sense strand; and

The first nucleotide end of the sense strand in the 3'-5' direction.

In some embodiments, the antisense strand has a phosphorothioate group at:

Between the first nucleotide and the second nucleotide in the 5'-3' direction of the antisense strand;

Between the second nucleotide and the third nucleotide in the 5'-3' direction of the antisense strand;

between the 1st nucleotide and the 2nd nucleotide in the 3'-5' direction of the antisense strand; and

Between the 2nd nucleotide and the 3rd nucleotide in the 3'-5' direction of the antisense strand.

In some embodiments, the sense strand is selected from a nucleotide sequence of the formula:

5’-NmsNmsNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNmNm-3’, or,

5’-NmsNmsNmNmNmNmNfNfNfNmNmNmNmNmNmNmNmNmNm-3’, or,

5’-NmsNmsNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNmns-3’, or,

5’-NmsNmsNmNmNmNmNfNfNfNmNmNmNmNmNmNmNmNmns-3’, or,

5’-NmsNmsNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNmsNm-3’, or,

5’-NmsNmsNmNmNmNmNfNfNfNmNmNmNmNmNmNmNmNmsNm-3’, or,

5’-NmsNmsNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmnsNms-3’, or,

5’-NmsNmsNmNmNmNmNfNfNfNmNmNmNmNmNmNmNmsNms-3’,

Wherein, Nm represents any nucleotide modified by 2'-methoxy group, such as C, G, U, A, T modified by 2'-methoxy group; Nf represents any nucleotide modified by 2'-fluoro, such as 2'-Fluorine modified C, G, U, A, T;

When the lowercase letter s is in the middle of the uppercase letter, it means that there is a phosphorothioate connection between the two nucleotides adjacent to the letter s; The adjacent one nucleotide end is a phosphorothioate group.

In some embodiments, the antisense strand has a nucleotide sequence of the formula:

5’-Nms’Nfs’Nm’Nf’Nm’Nf’W’Nm’Nm’Nf’Nm’Nf’Nm’Nf’Nm’Nf’Nm’Nf’Nms’Nms’Nm’-3’or,

5’-Nms’Nfs’Nm’Nf’Nm’Nf’W’Nm’Nf’Nm’Nm’Nf’Nm’Nf’Nm’Nf’Nm’Nf’Nms’Nms’Nm’-3’

Wherein, Nm' represents any nucleotide modified by 2'-methoxy group, such as C, G, U, A, T modified by 2'-methoxy group; Nf' represents any nucleotide modified by 2'-fluoro , such as 2'-fluoro modified C, G, U, A, T;

When the lowercase letter s is in the middle of the uppercase letter, it means that there is a phosphorothioate connection between the two nucleotides adjacent to the letter s. One of the adjacent nucleotide ends is a phosphorothioate group;

W' represents a 2'-methoxy-modified nucleotide or a nucleotide modified by a chemical modification represented by formula (I) or a tautomer thereof;

In some embodiments, formula (I) is selected from:

、

或

；其中，B選自鳥嘌呤、腺嘌呤、胞嘧啶或尿嘧啶，在一些實施方案中選自反義鏈在其5’區域的第7位中對應位置的鹼基。

,

or

; wherein, B is selected from guanine, adenine, cytosine or uracil, and in some embodiments is selected from the base at the corresponding position in the 7th position of the antisense strand in its 5' region.

In some embodiments, formula (I) is selected from:

、

，和

；其中，M為O或S；其中，B選自鳥嘌呤、腺嘌呤、胞嘧啶或尿嘧啶；在一些具體的實施方案中，B選自反義鏈在其5’區域的第7位中對應位置的鹼基。

,

,and

wherein, M is O or S; wherein, B is selected from guanine, adenine, cytosine or uracil; In some specific embodiments, B is selected from the antisense strand in the 7th position in its 5' region base at the corresponding position.

In some specific embodiments, M is S. In some specific embodiments, M is O.

In some embodiments, the sense strand comprises at least 15 contiguous nucleotides that differ from any of SEQ ID NO: 1 to SEQ ID NO: 23 by no more than 3 nucleotide sequences;

In some embodiments, the antisense strand comprises at least 15 contiguous nucleotides that differ from any of SEQ ID NO: 24 to SEQ ID NO: 46 by no more than 3 nucleotide sequences;

In some embodiments, the sense strand comprises at least 19 contiguous nucleotides that differ from any of SEQ ID NO: 1 to SEQ ID NO: 23 by no more than 3 nucleotide sequences; in some embodiments, the nucleoside The acid sequences differ by no more than 1 nucleotide.

In some embodiments, the antisense strand comprises at least 21 contiguous nucleotides that differ from any of SEQ ID NO: 24 to SEQ ID NO: 46 by no more than 3 nucleotide sequences; in some embodiments, the core The nucleotide sequences differ by no more than 1 nucleotide.

In some embodiments, the sense strand nucleotide sequence is selected from the group consisting of: the nucleotide sequence of any one of EQ ID NO: 1 to SEQ ID NO: 23;

In some embodiments, the antisense strand nucleotide sequence is selected from the group consisting of: the nucleotide sequence of any one of SEQ ID NO: 47 to SEQ ID NO: 69, wherein W' represents a 2'-methoxy-modified core nucleotides, or nucleotides comprising chemical modifications represented by formula (I) or modified tautomers thereof;

In some embodiments, formula (I) is selected from:

、

或

；其中，B選自鳥嘌呤、腺嘌呤、胞嘧啶或尿嘧啶，在一些實施方案中選自反義鏈在其5’區域的第7位中對應位置的鹼基。

,

or

; wherein, B is selected from guanine, adenine, cytosine or uracil, and in some embodiments is selected from the base at the corresponding position in the 7th position of the antisense strand in its 5' region.

In some embodiments, formula (I) is selected from:

、

，和

；其中，M為O或S；其中，B選自鳥嘌呤、腺嘌呤、胞嘧啶或尿嘧啶；在一些具體的實施方案中，B選自反義鏈在其5’區域的第7位中對應位置的鹼基。

,

,and

wherein, M is O or S; wherein, B is selected from guanine, adenine, cytosine or uracil; In some specific embodiments, B is selected from the antisense strand in the 7th position in its 5' region base at the corresponding position.

In some specific embodiments, M is S. In some specific embodiments, M is O.

In some specific embodiments, the sense strand is selected from any one of SEQ ID NOs: 70-77, 86-93, 102-161, 282-293, 306-425;

In some specific embodiments, the antisense strand is selected from any one of SEQ ID NOs: 78-85, 94-101, 162-261, 294-305;

In some specific embodiments, the siRNA of the present disclosure is selected from,

the sense strand is selected from SEQ ID NO: 142, and the antisense strand is selected from SEQ ID NO: 202, SEQ ID NO: 222, or SEQ ID NO: 242;

the sense strand is selected from SEQ ID NO: 143, and the antisense strand is selected from SEQ ID NO: 203, SEQ ID NO: 223, or SEQ ID NO: 243;

the sense strand is selected from SEQ ID NO: 144, and the antisense strand is selected from SEQ ID NO: 204, SEQ ID NO: 224, or SEQ ID NO: 244;

the sense strand is selected from SEQ ID NO: 145, and the antisense strand is selected from SEQ ID NO: 205, SEQ ID NO: 225, or SEQ ID NO: 245;

the sense strand is selected from SEQ ID NO: 146, and the antisense strand is selected from SEQ ID NO: 206, SEQ ID NO: 226, or SEQ ID NO: 246;

the sense strand is selected from SEQ ID NO: 147, and the antisense strand is selected from SEQ ID NO: 207, SEQ ID NO: 227, or SEQ ID NO: 247;

the sense strand is selected from SEQ ID NO: 148, and the antisense strand is selected from SEQ ID NO: 208, SEQ ID NO: 228, or SEQ ID NO: 248;

the sense strand is selected from SEQ ID NO: 149, and the antisense strand is selected from SEQ ID NO: 209, SEQ ID NO: 229, or SEQ ID NO: 249;

the sense strand is selected from SEQ ID NO: 150, and the antisense strand is selected from SEQ ID NO: 210, SEQ ID NO: 230, or SEQ ID NO: 250;

the sense strand is selected from SEQ ID NO: 151, and the antisense strand is selected from SEQ ID NO: 211, SEQ ID NO: 231, or SEQ ID NO: 251;

the sense strand is selected from SEQ ID NO: 152, and the antisense strand is selected from SEQ ID NO: 212, SEQ ID NO: 232, or SEQ ID NO: 252;

the sense strand is selected from SEQ ID NO: 153, and the antisense strand is selected from SEQ ID NO: 213, SEQ ID NO: 233, or SEQ ID NO: 253;

the sense strand is selected from SEQ ID NO: 154, and the antisense strand is selected from SEQ ID NO: 214, SEQ ID NO: 234, or SEQ ID NO: 254;

the sense strand is selected from SEQ ID NO: 155, and the antisense strand is selected from SEQ ID NO: 215, SEQ ID NO: 235, or SEQ ID NO: 255;

the sense strand is selected from SEQ ID NO: 156, and the antisense strand is selected from SEQ ID NO: 216, SEQ ID NO: 236, or SEQ ID NO: 256;

the sense strand is selected from SEQ ID NO: 157, and the antisense strand is selected from SEQ ID NO: 217, SEQ ID NO: 237, or SEQ ID NO: 257;

the sense strand is selected from SEQ ID NO: 158, and the antisense strand is selected from SEQ ID NO: 218, SEQ ID NO: 238, or SEQ ID NO: 258;

the sense strand is selected from SEQ ID NO: 159, and the antisense strand is selected from SEQ ID NO: 219, SEQ ID NO: 239, or SEQ ID NO: 259;

the sense strand is selected from SEQ ID NO: 160, and the antisense strand is selected from SEQ ID NO: 220, SEQ ID NO: 240, or SEQ ID NO: 260;

the sense strand is selected from SEQ ID NO: 161, and the antisense strand is selected from SEQ ID NO: 221, SEQ ID NO: 241, or SEQ ID NO: 261;

In some specific embodiments, the siRNA of the present disclosure is selected from,

the sense strand is selected from SEQ ID NO:346, and the antisense strand is selected from SEQ ID NO:202, SEQ ID NO:222, or SEQ ID NO:242;

the sense strand is selected from SEQ ID NO:347, and the antisense strand is selected from SEQ ID NO:203, SEQ ID NO:223, or SEQ ID NO:243;

the sense strand is selected from SEQ ID NO:348, and the antisense strand is selected from SEQ ID NO:204, SEQ ID NO:224, or SEQ ID NO:244;

the sense strand is selected from SEQ ID NO:349, and the antisense strand is selected from SEQ ID NO:205, SEQ ID NO:225, or SEQ ID NO:245;

the sense strand is selected from SEQ ID NO:350, and the antisense strand is selected from SEQ ID NO:206, SEQ ID NO:226, or SEQ ID NO:246;

the sense strand is selected from SEQ ID NO:351, and the antisense strand is selected from SEQ ID NO:207, SEQ ID NO:227, or SEQ ID NO:247;

the sense strand is selected from SEQ ID NO:352, and the antisense strand is selected from SEQ ID NO:208, SEQ ID NO:228, or SEQ ID NO:248;

the sense strand is selected from SEQ ID NO:353, and the antisense strand is selected from SEQ ID NO:209, SEQ ID NO:229, or SEQ ID NO:249;

the sense strand is selected from SEQ ID NO:354, and the antisense strand is selected from SEQ ID NO:210, SEQ ID NO:230, or SEQ ID NO:250;

the sense strand is selected from SEQ ID NO:355, and the antisense strand is selected from SEQ ID NO:211, SEQ ID NO:231, or SEQ ID NO:251;

the sense strand is selected from SEQ ID NO:356, and the antisense strand is selected from SEQ ID NO:212, SEQ ID NO:232, or SEQ ID NO:252;

the sense strand is selected from SEQ ID NO:357, and the antisense strand is selected from SEQ ID NO:213, SEQ ID NO:233, or SEQ ID NO:253;

the sense strand is selected from SEQ ID NO:358, and the antisense strand is selected from SEQ ID NO:214, SEQ ID NO:234, or SEQ ID NO:254;

the sense strand is selected from SEQ ID NO:359, and the antisense strand is selected from SEQ ID NO:215, SEQ ID NO:235, or SEQ ID NO:255;

the sense strand is selected from SEQ ID NO:360, and the antisense strand is selected from SEQ ID NO:216, SEQ ID NO:236, or SEQ ID NO:256;

the sense strand is selected from SEQ ID NO:361, and the antisense strand is selected from SEQ ID NO:217, SEQ ID NO:237, or SEQ ID NO:257;

the sense strand is selected from SEQ ID NO:362, and the antisense strand is selected from SEQ ID NO:218, SEQ ID NO:238, or SEQ ID NO:258;

the sense strand is selected from SEQ ID NO:363, and the antisense strand is selected from SEQ ID NO:219, SEQ ID NO:239, or SEQ ID NO:259;

the sense strand is selected from SEQ ID NO:364, and the antisense strand is selected from SEQ ID NO:220, SEQ ID NO:240, or SEQ ID NO:260;

the sense strand is selected from SEQ ID NO:365, and the antisense strand is selected from SEQ ID NO:221, SEQ ID NO:241, or SEQ ID NO:261;

In some specific embodiments, the siRNA of the present disclosure is selected from,

the sense strand is selected from SEQ ID NO:406, and the antisense strand is selected from SEQ ID NO:202, SEQ ID NO:222, or SEQ ID NO:242;

the sense strand is selected from SEQ ID NO:407, and the antisense strand is selected from SEQ ID NO:203, SEQ ID NO:223, or SEQ ID NO:243;

the sense strand is selected from SEQ ID NO:408, and the antisense strand is selected from SEQ ID NO:204, SEQ ID NO:224, or SEQ ID NO:244;

the sense strand is selected from SEQ ID NO:409, and the antisense strand is selected from SEQ ID NO:205, SEQ ID NO:225, or SEQ ID NO:245;

the sense strand is selected from SEQ ID NO:410, and the antisense strand is selected from SEQ ID NO:206, SEQ ID NO:226, or SEQ ID NO:246;

the sense strand is selected from SEQ ID NO:411, and the antisense strand is selected from SEQ ID NO:207, SEQ ID NO:227, or SEQ ID NO:247;

the sense strand is selected from SEQ ID NO:412, and the antisense strand is selected from SEQ ID NO:208, SEQ ID NO:228, or SEQ ID NO:248;

the sense strand is selected from SEQ ID NO:413, and the antisense strand is selected from SEQ ID NO:209, SEQ ID NO:229, or SEQ ID NO:249;

the sense strand is selected from SEQ ID NO:414, and the antisense strand is selected from SEQ ID NO:210, SEQ ID NO:230, or SEQ ID NO:250;

the sense strand is selected from SEQ ID NO:415, and the antisense strand is selected from SEQ ID NO:211, SEQ ID NO:231, or SEQ ID NO:251;

the sense strand is selected from SEQ ID NO:416, and the antisense strand is selected from SEQ ID NO:212, SEQ ID NO:232, or SEQ ID NO:252;

the sense strand is selected from SEQ ID NO:417, and the antisense strand is selected from SEQ ID NO:213, SEQ ID NO:233, or SEQ ID NO:253;

the sense strand is selected from SEQ ID NO:418, and the antisense strand is selected from SEQ ID NO:214, SEQ ID NO:234, or SEQ ID NO:254;

the sense strand is selected from SEQ ID NO:419, and the antisense strand is selected from SEQ ID NO:215, SEQ ID NO:235, or SEQ ID NO:255;

the sense strand is selected from SEQ ID NO:420, and the antisense strand is selected from SEQ ID NO:216, SEQ ID NO:236, or SEQ ID NO:256;

the sense strand is selected from SEQ ID NO:421, and the antisense strand is selected from SEQ ID NO:217, SEQ ID NO:237, or SEQ ID NO:257;

the sense strand is selected from SEQ ID NO:422, and the antisense strand is selected from SEQ ID NO:218, SEQ ID NO:238, or SEQ ID NO:258;

the sense strand is selected from SEQ ID NO:423, and the antisense strand is selected from SEQ ID NO:219, SEQ ID NO:239, or SEQ ID NO:259;

the sense strand is selected from SEQ ID NO:424, and the antisense strand is selected from SEQ ID NO:220, SEQ ID NO:240, or SEQ ID NO:260;

The sense strand is selected from SEQ ID NO:425, and the antisense strand is selected from SEQ ID NO:221, SEQ ID NO:241, or SEQ ID NO:261.

In some embodiments, the siRNA described above, when contacted with cells expressing the target gene, is screened for activity by, for example, psiCHECK and luciferase reporter gene assays, other methods such as PCR or branched DNA (bDNA) based methods, or protein based methods The above-mentioned siRNA inhibits the expression of the target gene by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% , at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%.

In some embodiments, the siRNA described above, when contacted with cells expressing the target gene, is screened for activity by, for example, psiCHECK and luciferase reporter gene assays, other methods such as PCR or branched DNA (bDNA) based methods, or protein based methods method, such as immunofluorescence analysis, such as Western Blot or flow cytometry, the remaining expression percentage of target gene mRNA caused by the above siRNA is not higher than 99%, not higher than 95%, not higher than 90%, Not higher than 85%, Not higher than 80%, Not higher than 75%, Not higher than 70%, Not higher than 65%, Not higher than 60%, Not higher than 55%, Not higher than 50%, Not high less than 45%, no more than 40%, no more than 35%, no more than 30%, no more than 25%, no more than 20%, no more than 15%, or no more than 10%.

In some embodiments, siRNAs comprising chemical modifications of the present disclosure, when contacted with cells expressing the target gene, are screened for, for example, psiCHECK activity and luciferase reporter gene assays, others such as PCR or branched DNA (bDNA) based assays. Methods, or protein-based methods, such as assayed by immunofluorescence assays, such as Western Blot, or flow cytometry, comprising chemical modifications of the present disclosure, such as siRNAs comprising chemical modifications of formula (I), remain at target activity. while reducing off-target activity by at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70% % or at least 75%.

In some embodiments, siRNAs comprising chemical modifications of the present disclosure, when contacted with cells expressing the target gene, are screened for, for example, psiCHECK activity and luciferase reporter gene assays, others such as PCR or branched DNA (bDNA) based assays. Methods, or protein-based methods, such as assayed by immunofluorescence assays, such as Western Blot, or flow cytometry, comprising chemical modifications of the present disclosure, such as siRNAs comprising chemical modifications of formula (I), reduce on-target activity At least 20%, at least 25%, at least 30%, at least 35%, at least 40% while reducing off-target activity by up to 20%, up to 19%, up to 15%, up to 10%, up to 5%, or more than 1% , at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, or at least 75%.

In some embodiments, siRNAs comprising chemical modifications of the present disclosure, when contacted with cells expressing the target gene, are screened for, for example, psiCHECK activity and luciferase reporter gene assays, others such as PCR or branched DNA (bDNA) based assays. Methods, or protein-based methods, such as assayed by immunofluorescence assays, such as Western Blot, or flow cytometry, comprising chemical modifications of the present disclosure, such as siRNAs comprising chemical modifications of formula (I), increase on-target activity at least 1%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, or at least 80% while reducing off-target activity by at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, At least 50%, at least 55%, at least 60%, at least 65%, at least 70% or at least 75%.

The present disclosure also provides an siRNA conjugate comprising any one of the siRNAs described above and a targeting ligand linked to the siRNA.

In some embodiments, the siRNA and the targeting ligand are covalently or non-covalently linked.

In some embodiments, the targeting ligand targets the liver; in some embodiments, the targeting ligand binds the asialoglycoprotein receptor (ASGPR); in some embodiments, the targeting ligand comprises a galactose cluster or a cluster of galactose derivatives selected from the group consisting of N-acetyl-galactosamine, N-trifluoroacetylgalactosamine, N-propionylgalactosamine, N-n-butylacylgalactosamine amine or N-isobutyrylgalactosamine.

In some embodiments, the targeting ligand is attached to the 3' end of the sense strand of the siRNA.

In some embodiments, in order to promote the entry of siRNA into cells, a lipophilic group such as cholesterol can be introduced at the end of the sense strand of siRNA. Protein, vitamin E, etc., to facilitate the interaction with intracellular mRNA through the cell membrane composed of lipid bilayers. At the same time, siRNA can also be modified by non-covalent bonds, such as binding phospholipid molecules, polypeptides, cationic polymers, etc. through hydrophobic bonds or ionic bonds to increase stability and biological activity.

In some embodiments, the targeting ligand is attached to the end of the siRNA via a phosphate group, phosphorothioate group, or phosphonate group. In some embodiments, the targeting ligand is indirectly attached to the end of the siRNA via a phosphate group, phosphorothioate group, or phosphonate group. In some embodiments, the targeting ligand is bound to the siRNA via a phosphate group, phosphorothioate group, or phosphonate group The ends are directly connected. In some embodiments, the targeting ligand is directly attached to the end of the siRNA via a phosphate group or phosphorothioate group. In some embodiments, the targeting ligand is directly attached to the 3' end of the siRNA sense strand via a phosphate or phosphorothioate group.

In some embodiments, the targeting ligand structure is shown in the following formula (II),

wherein T is the targeting moiety, E is the branching group, L1 is the linker moiety, L2 is the tethering moiety between the targeting moiety and the branching group, wherein i is selected from an integer from ₁ to 10, such as 1, ₂ , 3, 4, 5, 6, 7, 8, 9, 10.

In some embodiments, i is selected from an integer from 2 to 8.

In some embodiments, i is selected from an integer from 3 to 5.

In some embodiments, L ₁ is

R ⁹ and R ¹⁰ are each independently selected from -S-, -NH-, -O-, -C(O)-, -OC(O)-, -C(O)O-, -NHC(O)- , -C(O)NH-, -CH ₂ -, -CH ₂ NH-, -CH ₂ O-, -NH-C(O)-CH ₂ -, -C(O)-CH ₂ -NH-, -NH(CO)NH-, a 3-12-membered heterocyclic group, the -CH ₂ - is optionally substituted by a substituent selected from halogen, alkyl, alkoxy, and alkylamino, and the alkyl group is optionally further substituted by Substituents selected from hydroxyl, amino, and halogen are substituted;

R ¹¹ is selected from deuterium, halogen, alkyl, amino, cyano, nitro, alkenyl, alkynyl, carboxyl, hydroxyl, mercapto, alkylmercapto, alkoxy, alkylamino, -C(O)-alkane radical, -C(O)-O-alkyl, -CONH ₂ , -CONH-alkyl, -OC(O)-alkyl, -NH-C(O)-alkyl, -S(O)O- Alkyl, -S(O)ONH ₂ , -S(O)ONH-alkyl, the alkyl, alkenyl, alkynyl, alkylmercapto, alkyloxy, -C(O)-alkyl, - C(O)-O-Alkyl, -CONH-Alkyl, -OC(O)-Alkyl, -NH-C(O)-Alkyl, -S(O)O-Alkyl, -S(O ) ONH-alkyl, optionally further substituted by a substituent selected from halogen, hydroxyl, amino, mercapto;

the k is selected from 0, 1, 2, 3, 4;

The j is selected from an integer from 1 to 20 (eg 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 , 20).

In some embodiments, L ₁ is

wherein R ¹¹ is selected from deuterium, halogen, alkyl, amino, cyano, nitro, alkenyl, alkynyl, carboxyl, hydroxyl, mercapto, alkylmercapto, alkoxy, alkylamino, -C(O)- Alkyl, -C(O)-O-Alkyl, -CONH ₂ , -CONH-Alkyl, -OC(O)-Alkyl, -NH-C(O)-Alkyl, -S(O)O -Alkyl, -S(O)ONH2, -S(O)ONH _- alkyl, the alkyl, alkenyl, alkynyl, carboxyl, alkylmercapto, alkyloxy, -C(O)-alkane Base, -C(O)-O-Alkyl, -CONH-Alkyl, -OC(O)-Alkyl, -NH-C(O)-Alkyl, -S(O)O-Alkyl, - S(O)ONH-alkyl, optionally further substituted with a substituent selected from halogen, hydroxyl, amino, mercapto;

the k is selected from 0, 1, 2, 3, 4;

In some embodiments, L ₁ is

In some embodiments, L ₁ is

In some embodiments, L ₁ is

In some embodiments, E in the targeting ligand is

The R ¹² , R ¹³ , R ¹⁴ and R ¹⁵ are each independently selected from -C(O)NH-, -C(O)-, the carbonyl group is optionally further substituted with an alkyl group, and the alkyl group is optionally further selected Substituted from alkyl, hydroxyl, -C(O)O-, -C(O)O-alkyl-, -C(O)NH-;

The X ² , X ³ , X ⁴ and X ⁵ are each independently an integer selected from 0 to 10 (eg, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10).

In some embodiments, E in the targeting ligand is

The R ¹² , R ¹³ , R ¹⁴ and R ¹⁵ are each independently selected from -C(O)NH-, -C(O)-, the -C(O)NH-, -C(O)- are optionally further substituted with an alkyl group optionally further substituted by a group selected from the group consisting of alkyl, hydroxy, -C(O)O-, -C(O)O-alkyl-, -C(O)NH-;

The X ² , X ³ , X ⁴ and X ⁵ are each independently an integer selected from 0 to 10 (eg, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10).

In some embodiments, E in the targeting ligand is

、

、

、

、

、

、

、

的取代基取代，該X²、X³、X⁴及X⁵各自獨立的選自0到10的整數(例如0，1、2、3、4、5、6、7、8、9、10)。 The R ¹² , R ¹³ , R ¹⁴ and R ¹⁵ are each independently selected from -C(O)NH-, -C(O)-,

,

,

,

,

,

,

,

substituted by a substituent, the X ² , X ³ , X ⁴ and X ⁵ are each independently an integer selected from 0 to 10 (for example, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 ).

In some embodiments, E in the targeting ligand is

In some embodiments, E in the targeting ligand is selected from

或

。 In some embodiments, E in the targeting ligand is selected from

or

.

。 In some embodiments, E in the targeting ligand is selected from

.

，L₁選自如下結構： In some embodiments, E in the targeting ligand is

, L ₁ is selected from the following structures:

或者

，

or

,

R ⁹ and R ¹⁰ are each independently selected from -S-, -NH-, -O-, -S-, -C(O)-, -OC(O)-, -C(O)O-, -NHC (O)-, -C(O)NH-, _-CH2- , -CH2NH-, _-CH2O- , -NH-C(O) _-CH2- , _-C (O) _-CH2 -NH-, -NH(CO)NH-, 3-12-membered heterocyclic group, the -CH ₂ - is optionally substituted by a substituent selected from halogen, alkyl, alkoxy, alkylamino, the alkyl optionally further substituted with a substituent selected from hydroxy, amine, halogen;

R ¹¹ is selected from deuterium, halogen, alkyl, amino, cyano, nitro, alkenyl, alkynyl, carboxyl, hydroxyl, mercapto, alkylmercapto, alkoxy, alkylamino, -C(O)-alkane radical, -C(O)-O-alkyl, -CONH ₂ , -CONH-alkyl, -OC(O)-alkyl, -NH-C(O)-alkyl, -S(O)O- Alkyl, -S(O)ONH ₂ , -S(O)ONH-alkyl, the alkyl, alkenyl, alkynyl, alkylmercapto, alkyloxy, -C(O)-alkyl, - C(O)-O-Alkyl, -CONH-Alkyl, -OC(O)-Alkyl, -NH-C(O)-Alkyl, -S(O)O-Alkyl, -S(O ) ONH-alkyl, optionally further substituted by a substituent selected from halogen, hydroxyl, amino, mercapto;

the k is selected from 0, 1, 2, 3, 4;

The j is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20.

In some embodiments, E in the targeting ligand is selected from:

、

或

，L₁選自：

，或

。

,

or

, L ₁ is selected from:

,or

.

In some embodiments, E in the targeting ligand is selected from:

，L₁選自：

，即E-L₁為

。

, L1 is selected from _:

, that is, EL ₁ is

.

In some embodiments, E in the targeting ligand is selected from:

，L₁選自：

或

，即E-L₁為

或

。

, L ₁ is selected from:

or

, that is, EL ₁ is

or

.

或

，L₁選自

，即E-L₁為

或

。 In some embodiments, E in the targeting ligand is selected from

or

, L ₁ is selected from

, that is, EL ₁ is

or

.

或

，L₁選自

或

，即E-L₁為

或

。 In some embodiments, E in the targeting ligand is selected from

or

, L ₁ is selected from

or

, that is, EL ₁ is

or

.

In the present disclosure, L ₂ is the tethering moiety between the targeting moiety and the branching group, and L ₂ plays the role of linking and spacing between the branching group and each targeting moiety.

_In some embodiments, one end of L2 is directly attached to the targeting ligand and the other end is directly attached to the branching group E.

In some embodiments, one end of L ₂ is directly attached to the targeting ligand and the other end is indirectly attached to the branching group E.

_In some embodiments, one end of L2 is indirectly linked to the targeting ligand and the other end is indirectly linked to the branching group E.

In some embodiments, the targeting ligands disclosed herein include 2 L2 and ₂ targeting moieties.

In some embodiments, the targeting ligands disclosed herein include 3 L2 and ₃ targeting moieties.

In some embodiments, the targeting ligands disclosed herein include 4 L2 and ₄ targeting moieties.

In some embodiments, the targeting ligands disclosed herein include multiple L _2s and multiple targeting moieties.

In some embodiments, L2 in the present disclosure is selected from 1 or a combination of 2-20 covalently linked groups (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20):

取代的或未取代的環烷基(例如環己基、環丙基、環丁基、環戊基、環庚基、環辛基等)、取代的或未取代的環烯基(例如環己烯基、環丁烯基、環戊烯基、 環庚烯基、環辛烯基、環己二烯基、環戊二烯基、環庚二烯基、環辛二烯基等)、取代的或未取代的芳基(例如苯基、萘基、聯萘基、蒽基等)、取代的或未取代的雜芳基(例如吡啶基、嘧啶基、吡咯、咪唑、呋喃、苯并呋喃、吲哚等)和取代的或未取代的雜環基(例如四氫呋喃、四氫吡喃、哌啶、吡咯烷等)的基團的共價組合。

Substituted or unsubstituted cycloalkyl (eg cyclohexyl, cyclopropyl, cyclobutyl, cyclopentyl, cycloheptyl, cyclooctyl, etc.), substituted or unsubstituted cycloalkenyl (eg cyclohexene cyclobutenyl, cyclobutenyl, cyclopentenyl, cycloheptenyl, cyclooctenyl, cyclohexadienyl, cyclopentadienyl, cycloheptadienyl, cyclooctadienyl, etc.), substituted or unsubstituted aryl (such as phenyl, naphthyl, binaphthyl, anthracenyl, etc.), substituted or unsubstituted heteroaryl (such as pyridyl, pyrimidinyl, pyrrole, imidazole, furan, benzofuran, indole, etc.) and substituted or unsubstituted heterocyclyl (eg, tetrahydrofuran, tetrahydropyran, piperidine, pyrrolidine, etc.) groups.

In some embodiments, there are from 1 to 20 L2 in the present disclosure (eg, 1, ₂ , 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 , 17, 18, 19 or 20) optional from

的基團的共價組合。

A covalent combination of groups.

，其中x⁶是從1至20的整數(例如，1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19或20)。 _In some embodiments, the targeting ligand comprises L2 having the structure shown below,

, where ^x6 is an integer from 1 to 20 (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20).

。 _In some embodiments, the targeting ligand comprises L2 having the structure shown below,

.

。 _In some embodiments, the targeting ligand comprises L2 having the structure shown below,

.

。 _In some embodiments, the targeting ligand comprises L2 having the structure shown below,

.

。 _In some embodiments, the targeting ligand comprises L2 having the structure shown below,

.

。 _In some embodiments, the targeting ligand comprises L2 having the structure shown below,

.

，其中，x⁷是從1至20的整數(例如，1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19或20)，並且Z是

。 _In some embodiments, the targeting ligand comprises L2 having the structure shown below,

, where x ⁷ is an integer from 1 to 20 (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 , 19 or 20), and Z is

.

。 _In some embodiments, the targeting ligand has L2 of the structure shown below,

.

， _In some embodiments, the targeting ligand has L2 of the structure shown below,

,

。 where x8 is an integer from 1 to 20 (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, ¹⁶ , 17, 18, 19 or 20), and Z is

.

，其中x⁹和X¹⁰各自獨立選自從1至20的整數(例如，1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19或20)，並且Z是

。 _In some embodiments, the targeting ligand has L2 of the structure shown below,

, where x ⁹ and X ¹⁰ are each independently selected from integers from 1 to 20 (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 , 17, 18, 19, or 20), and Z is

.

。 _In some embodiments, the targeting ligand has L2 of the structure shown below,

.

_In some embodiments, the targeting ligand has L2 of the structure shown below,

。 wherein ^x7 and ^X8 are each independently selected from integers from 1 to 20 (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 , 17, 18, 19, or 20), and Z is

.

In some specific embodiments, the targeting ligand has the following structure:

In some specific embodiments, the targeting ligand has the following structure:

In some specific embodiments, the targeting ligand has the following structure:

In some specific embodiments, the targeting ligand has the following structure:

In some embodiments, the targeting moiety of the targeting ligand is comprised of one or more targeting groups or targeting moieties that assist in directing delivery of the therapeutic agent attached thereto to the desired target site. In some cases, the targeting moiety can bind to a cell or cellular receptor and initiate endocytosis to facilitate entry of the therapeutic agent into the cell. Targeting moieties can include compounds that have affinity for cell receptors or cell surface molecules or antibodies. Various targeting ligands containing targeting moieties can be linked to therapeutic agents and other compounds to target the agents to cells and specific cellular receptors.

In some embodiments, types of targeting moieties include carbohydrates, cholesterol, and cholesteryl groups or steroids. Targeting moieties that can bind to cellular receptors include carbohydrates such as galactose, derivatives of galactose (eg N-acetyl-galactosamine, N-trifluoroacetylgalactosamine, N-propionylgalactose amine, N-n-butyl galactosamine, N-isobutyl galactosamine), mannose and mannose derivatives).

Targeting moieties known to bind the asialoglycoprotein receptor (ASGPR) are particularly useful for directing delivery of oligomeric compounds to the liver. The asialoglycoprotein receptor is abundantly expressed on liver cells (hepatocytes). Cell receptor targeting moieties targeting ASCPR include galactose and galactose derivatives. Specifically, clusters of galactose derivatives, including clusters consisting of 2, 3, 4, or more than 4 N-acetyl-galactosamine (GalNAc or NAG), can promote the uptake of certain compounds in hepatocytes. GalNAc clusters coupled to oligomeric compounds were used to direct the composition to the liver, where N-acetyl-galactosamine sugars were able to bind to asialoglycoprotein receptors on the surface of liver cells. Binding of the asialoglycoprotein receptor is thought to initiate receptor-mediated endocytosis, thereby facilitating the entry of compounds into the cell interior.

In some embodiments, a targeting ligand may include 2, 3, 4, or more than 4 targeting moieties. In some embodiments, the targeting ligands disclosed herein can include 1, ₂ , 3, 4, or more than 4 targeting moieties linked to a branching group through L2.

In some embodiments, the targeting ligand is in the form of a galactose cluster.

In some embodiments, each targeting moiety includes a galactosamine derivative, which is N-acetyl-galactosamine. Other saccharides that can be used as targeting moieties and have affinity for the asialoglycoprotein receptor can be selected from galactose, galactosamine, N-formyl-galactosamine, N-acetyl-galactosamine , N-propionyl-galactosamine, N-n-butyryl-galactosamine and N-isobutyryl-galactosamine, etc.

In some embodiments, the targeting ligands of the present disclosure include N-acetylgalactosamine as a targeting moiety,

。

.

In some embodiments, the targeting ligand includes three terminal galactosamine or galactosamine derivatives (such as N-acetyl-galactosamine), each of which has an affinity for the sialoglycoprotein receptor. In some embodiments, the targeting ligand includes three terminal N-acetyl-galactosamine (GalNAc or NAG) as targeting moieties.

In some embodiments, the targeting ligand includes four terminal galactosamine or galactosamine derivatives (such as N-acetyl-galactosamine), each of which has an affinity for the asialoglycoprotein receptor . In some embodiments, the targeting ligand includes four terminal N-acetyl-galactosamine (GalNAc or NAG) as targeting moieties.

Terms commonly used in the art when referring to the three-terminal N-acetyl-galactosamine include tri-antennary, tri-valent, and trimer.

Terms commonly used in the art when referring to the four terminal N-acetyl-galactosamines include tetra-antennary, tetra-valent, and tetramer.

In some specific embodiments, the targeting ligands provided by the present disclosure have the following structures,

或

。

or

.

In some specific embodiments, the targeting ligands provided by the present disclosure have the following structures,

或

。

or

.

In some specific embodiments, the targeting ligands provided by the present disclosure have the following structures,

。

.

In some specific embodiments, the targeting ligands provided by the present disclosure have the following structures,

或

。

or

.

In some embodiments, the siRNA of the present disclosure is linked to a targeting ligand of the present disclosure to form a siRNA conjugate having the following,

，

,

wherein T is the targeting moiety, E is the branching group, L1 is the linker moiety, L2 is the tethering moiety between the targeting moiety and the branching group, wherein x is an integer from ₁ to 10, and D is the targeting coagulation factor XI siRNA.

In some embodiments, D is an siRNA targeting factor XI.

In some embodiments, D is any siRNA of the present disclosure. In some embodiments, the L1 is linked to the 3' end of the sense strand of the siRNA.

In some embodiments, the targeting ligand is attached to the end of the siRNA via a phosphate group, phosphorothioate group, or phosphonate group.

In some embodiments, the targeting ligand is indirectly attached to the end of the siRNA via a phosphate group, phosphorothioate group, or phosphonate group.

In some embodiments, the targeting ligand is directly attached to the siRNA terminus via a phosphate group, phosphorothioate group, or phosphonate group.

In some embodiments, the targeting ligand is directly attached to the end of the siRNA via a phosphate group, a phosphorothioate group.

In some embodiments, the targeting ligand is directly attached to the 3' end of the siRNA sense strand via a phosphate group, a phosphorothioate group.

In some specific embodiments, the present disclosure provides an siRNA conjugate,

或

，

or

,

Wherein, D is siRNA targeting coagulation factor XI.

In some embodiments, D is an siRNA targeting factor XI; in some embodiments, D is any siRNA of the disclosure. In some specific embodiments, the targeting ligand is directly attached to the 3' end of the siRNA sense strand via a phosphate group or a phosphorothioate group.

In some specific embodiments, the present disclosure provides an siRNA conjugate,

或

，其中，D為靶向凝血因子XI的siRNA；在一些實施方式中，在一些實施方式中，D為本公開任一siRNA。

or

, wherein D is an siRNA targeting factor XI; in some embodiments, in some embodiments, D is any siRNA of the present disclosure.

In some specific embodiments, the targeting ligand is directly attached to the 3' end of the siRNA sense strand via a phosphate group or a phosphorothioate group.

In some specific embodiments, the present disclosure provides an siRNA conjugate,

或

，其中，D為靶向凝血因子XI的siRNA；在一些實施方式中，D為本公開任一siRNA。在一些具體的實施方案中，靶向配體藉由磷酸酯基團或硫代磷酸酯基團與siRNA正義鏈3’末端直接連接。

or

, wherein D is an siRNA targeting coagulation factor XI; in some embodiments, D is any siRNA of the present disclosure. In some specific embodiments, the targeting ligand is directly attached to the 3' end of the siRNA sense strand via a phosphate group or a phosphorothioate group.

In some specific embodiments, the present disclosure provides an siRNA conjugate,

，其中，D為靶向凝血因子XI的siRNA；在一些實施方式中，D為本公開任一siRNA。在一些具體的實施方案中，靶向配體藉由磷酸酯基團或硫代磷酸酯基團與siRNA正義鏈3’末端直接連接。

, wherein D is an siRNA targeting coagulation factor XI; in some embodiments, D is any siRNA of the present disclosure. In some specific embodiments, the targeting ligand is directly attached to the 3' end of the siRNA sense strand via a phosphate group or a phosphorothioate group.

In some specific embodiments, the L1 and D are linked via a phosphate group, a phosphorothioate group, or a phosphonic acid group.

In some specific embodiments, the L ₁ and the 3' end of the D sense strand are linked by a phosphate group, a phosphorothioate group, or a phosphonic acid group.

In some specific embodiments, the L ₁ and D sense strands are directly linked at the 3' end via a phosphate group or a phosphorothioate group.

In some specific embodiments, the L1 is indirectly linked to the 3' terminus of the D sense strand via a phosphate group or a phosphorothioate group.

In some specific embodiments, the siRNA conjugate sense strand is selected from the group consisting of: any one of SEQ ID NO:262 to SEQ ID NO:281.

In some specific embodiments, the siRNA conjugate antisense strand is selected from the group consisting of: any one of SEQ ID NO:202 to SEQ ID NO:261.

In some specific embodiments, the siRNA conjugate is selected from:

the sense strand is selected from SEQ ID NO:262, and the antisense strand is selected from SEQ ID NO:202, SEQ ID NO:222, or SEQ ID NO:242;

the sense strand is selected from SEQ ID NO:263, and the antisense strand is selected from SEQ ID NO:203, SEQ ID NO:223, or SEQ ID NO:243;

the sense strand is selected from SEQ ID NO:264, and the antisense strand is selected from SEQ ID NO:204, SEQ ID NO:224, or SEQ ID NO:244;

the sense strand is selected from SEQ ID NO:265, and the antisense strand is selected from SEQ ID NO:205, SEQ ID NO:225, or SEQ ID NO:245;

the sense strand is selected from SEQ ID NO:266, and the antisense strand is selected from SEQ ID NO:206, SEQ ID NO:226, or SEQ ID NO:246;

the sense strand is selected from SEQ ID NO:267, and the antisense strand is selected from SEQ ID NO:207, SEQ ID NO:227, or SEQ ID NO:247;

the sense strand is selected from SEQ ID NO:268, and the antisense strand is selected from SEQ ID NO:208, SEQ ID NO:228, or SEQ ID NO:248;

the sense strand is selected from SEQ ID NO:269, and the antisense strand is selected from SEQ ID NO:209, SEQ ID NO:229, or SEQ ID NO:249;

the sense strand is selected from SEQ ID NO:270, and the antisense strand is selected from SEQ ID NO:210, SEQ ID NO:230, or SEQ ID NO:250;

the sense strand is selected from SEQ ID NO:271, and the antisense strand is selected from SEQ ID NO:211, SEQ ID NO:231, or SEQ ID NO:251;

the sense strand is selected from SEQ ID NO:272, and the antisense strand is selected from SEQ ID NO:212, SEQ ID NO:232, or SEQ ID NO:252;

the sense strand is selected from SEQ ID NO:273, and the antisense strand is selected from SEQ ID NO:213, SEQ ID NO:233, or SEQ ID NO:253;

the sense strand is selected from SEQ ID NO:274, and the antisense strand is selected from SEQ ID NO:214, SEQ ID NO:234, or SEQ ID NO:254;

the sense strand is selected from SEQ ID NO:275, and the antisense strand is selected from SEQ ID NO:215, SEQ ID NO:235, or SEQ ID NO:255;

the sense strand is selected from SEQ ID NO:276, and the antisense strand is selected from SEQ ID NO:216, SEQ ID NO:236, or SEQ ID NO:256;

the sense strand is selected from SEQ ID NO:277, and the antisense strand is selected from SEQ ID NO:217, SEQ ID NO:237, or SEQ ID NO:257;

the sense strand is selected from SEQ ID NO:278, and the antisense strand is selected from SEQ ID NO:218, SEQ ID NO:238, or SEQ ID NO:258;

the sense strand is selected from SEQ ID NO:279, and the antisense strand is selected from SEQ ID NO:219, SEQ ID NO:239, or SEQ ID NO:259;

the sense strand is selected from SEQ ID NO:280, and the antisense strand is selected from SEQ ID NO:220, SEQ ID NO:240, or SEQ ID NO:260;

The sense strand is selected from SEQ ID NO:281, and the antisense strand is selected from SEQ ID NO:221, SEQ ID NO:241, or SEQ ID NO:261.

Another aspect of the present disclosure provides a composition comprising the above-described conjugate, and one or more pharmaceutically acceptable excipients, such as a carrier, vehicle, diluent, and/or delivery polymer.

Various delivery systems are known and can be used for the siRNA or siRNA conjugates of the present disclosure, such as encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the compound, receptor-mediated endocytosis , construct nucleic acid as part of retrovirus or other vector.

Another aspect of the present disclosure provides a use of the above-mentioned conjugate or a composition containing the conjugate in the manufacture of a medicament for treating a disease in a subject, which in some embodiments is selected from liver-derived diseases.

Another aspect of the present disclosure provides a method of treating a disease in a subject, comprising administering to the subject the above-described conjugate, or composition.

Another aspect of the present disclosure provides a method of inhibiting mRNA expression in a subject, the method comprising administering to the subject the above-described conjugate, or composition.

Another aspect of the present disclosure provides a method of delivering an expression-inhibiting oligomeric compound to the liver in vivo by administering the above-described conjugate, or composition, to a subject.

The conjugates, compositions, and methods disclosed herein can reduce the level of a target mRNA in a cell, cell population, cell population, tissue, or subject, comprising: administering to the subject a therapeutically effective amount of an expression-inhibiting oligomer described herein The expression-inhibiting oligomer is linked to a targeting ligand, thereby inhibiting the expression of the target mRNA in the subject.

In some embodiments, the subject has been previously identified as having pathogenic upregulation of the target gene in the targeted cell or tissue.

A subject as described in the present disclosure refers to a subject suffering from a disease or disorder that would benefit from reduction or inhibition of target mRNA expression.

Delivery can be by local administration (eg, direct injection, implantation, or topical administration), systemic administration, or subcutaneous, intravenous, intraperitoneal, or parenteral routes, including intracranial (eg, intraventricular, parenchymal) intramuscular, transdermal, airway (aerosol), nasal, oral, rectal, or topical (including buccal and sublingual) administration.

In alternative embodiments, the pharmaceutical compositions provided by the present disclosure can be administered by injection, eg, intravenous, intramuscular, intradermal, subcutaneous, intraduodenal, or intraperitoneal injection.

In alternative embodiments, after the targeting ligand is linked to the expression-inhibiting polymeric compound to form a conjugate, the conjugate can be packaged in a kit.

The present disclosure also provides a pharmaceutical composition comprising the siRNA or siRNA conjugate of the present disclosure.

In some embodiments, the pharmaceutical composition may further include pharmaceutically acceptable adjuvants and/or adjuvants, and the adjuvants may be one or more various formulations or compounds conventionally used in the art. For example, the pharmaceutically acceptable adjuvant may include at least one of pH buffering agents, protecting agents and osmotic pressure adjusting agents.

In some embodiments, the siRNA conjugates or pharmaceutical compositions described above, when contacted with cells expressing the target gene, are screened for, for example, psiCHECK activity and luciferase reporter gene assays, others such as PCR or branched DNA (bDNA) based assays. ), or protein-based methods, such as immunofluorescence assays, such as Western Blot or flow cytometry, the siRNA conjugates or pharmaceutical compositions described above inhibit the expression of the target gene by at least 5%, at least 10% , at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75% %, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% .

In some embodiments, the siRNA conjugates or pharmaceutical compositions described above, when contacted with cells expressing the target gene, are screened for, for example, psiCHECK activity and luciferase reporter gene assays, others such as PCR or branched DNA (bDNA) based assays. ) method, or a protein-based method, such as an immunofluorescence assay, such as Western Blot or flow cytometry, the residual expression percentage of target gene mRNA caused by the above-mentioned siRNA conjugate or pharmaceutical composition is not higher than 99% %, not higher than 95%, not higher than 90%, not higher than 85%, not higher than 80%, not higher than 75%, not higher than 70%, not higher than 65%, not higher than 60%, Not higher than 55%, Not higher than 50%, Not higher than 45%, Not higher than 40%, Not higher than 35%, Not higher than 30%, Not higher than 25%, Not higher than 20%, Not high at 15%, or not higher than 10%.

In some embodiments, the siRNA conjugate or pharmaceutical composition, when contacted with cells expressing the target gene, is screened for, for example, psiCHECK activity and luciferase reporter gene assays, others such as PCR or branched DNA (bDNA) based assays. methods, or protein-based methods, such as immunofluorescence assays, such as Western Blot, or flow cytometry, the siRNA conjugate reduces off-target activity by at least 20%, at least 20%, while maintaining on-target activity. 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, or at least 75%.

In some embodiments, the siRNA conjugate or pharmaceutical composition, when contacted with cells expressing the target gene, is screened for, for example, psiCHECK activity and luciferase reporter gene assays, others such as PCR or branched DNA (bDNA) based assays. methods, or protein-based methods, siRNA conjugates reduce on-target activity by up to 20%, up to 19%, up to 15%, up to 10%, up to 5%, or more than 1%, as determined by immunofluorescence assays, such as Western Blot, or flow cytometry % while reducing off-target activity by at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70% or at least 75%.

In some embodiments, the siRNA conjugate or pharmaceutical composition, when contacted with cells expressing the target gene, is screened for, for example, psiCHECK activity and luciferase reporter gene assays, others such as PCR or branched DNA (bDNA) based assays. methods, or protein-based methods, such as immunofluorescence assays, such as Western Blot, or flow cytometry, the siRNA conjugate increases on-target activity by at least 1%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75% or at least 80% while reducing off-target activity by at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65% %, at least 70%, or at least 75%.

The present disclosure also provides a cell comprising the siRNA or siRNA conjugate of the present disclosure.

The present disclosure also provides a kit comprising the siRNA or siRNA conjugate of the present disclosure.

The present disclosure also provides a method for silencing a target gene or mRNA of a target gene in a cell, the method comprising the step of introducing into the cell an siRNA, siRNA conjugate and/or pharmaceutical composition according to the present disclosure.

The present disclosure also provides a method for silencing a target gene or mRNA of a target gene in a cell in vivo or in vitro, the method comprising introducing into the cell an siRNA, siRNA conjugate and/or pharmaceutical composition according to the present disclosure A step of.

The present disclosure also provides a method for inhibiting mRNA expression of a target gene or target gene, the method comprising administering to a subject in need thereof an effective amount or an effective dose of an siRNA, siRNA conjugate and/or according to the present disclosure or pharmaceutical compositions.

In some embodiments, administration is by means of administration including intramuscular, intrabronchial, intrapleural, intraperitoneal, intraarterial, lymphatic, intravenous, subcutaneous, cerebrospinal, or a combination thereof.

In some embodiments, the effective amount or effective dose of the siRNA, siRNA conjugate and/or pharmaceutical composition is about 0.001 mg/kg body weight to about 200 mg/kg body weight, about 0.01 mg/kg body weight to about 100 mg/kg body weight Or about 0.5 mg/kg body weight to about 50 mg/kg body weight.

In some embodiments, the target gene is the factor XI gene.

The present disclosure also provides a pharmaceutical composition comprising the siRNA or siRNA conjugate of the present disclosure.

In some embodiments, the pharmaceutical composition may further include pharmaceutically acceptable adjuvants and/or adjuvants, and the adjuvants may be one or more various formulations or compounds conventionally used in the art. For example, the pharmaceutically acceptable adjuvant may include at least one of pH buffering agents, protecting agents and osmotic pressure adjusting agents.

In some embodiments, the siRNA conjugates or pharmaceutical compositions described above, when contacted with cells expressing the target gene, are analyzed by, for example, PCR or branched DNA (bDNA)-based methods, or protein-based methods, such as immunofluorescence analysis The aforementioned siRNA conjugate or pharmaceutical composition inhibits the expression of the target gene by at least 5%, as determined by Western Blot or flow cytometry, for example, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70% %, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%.

In some embodiments, the siRNA conjugates or pharmaceutical compositions described above, when contacted with cells expressing the target gene, are analyzed by, for example, PCR or branched DNA (bDNA)-based methods, or protein-based methods, such as immunofluorescence analysis For example, as determined by Western Blot or flow cytometry, the residual expression percentage of target gene mRNA caused by the above-mentioned siRNA conjugate or pharmaceutical composition is not higher than 99%, not higher than 95%, not higher than 90%, not higher than 90%. higher than 85%, not higher than 80%, not higher than 75%, not higher than 70%, not higher than 65%, not higher than 60%, not higher than 55%, not higher than 50%, not higher than 45%, no more than 40%, no more than 35%, no more than 30%, no more than 25%, no more than 20%, no more than 15%, or no more than 10%.

The present disclosure also provides a cell comprising the siRNA or siRNA conjugate of the present disclosure.

The present disclosure also provides a kit comprising the siRNA or siRNA conjugate of the present disclosure.

The present disclosure also provides an siRNA, a siRNA conjugate, or a pharmaceutical composition comprising the same, for treating and/or preventing a subject suffering from a coagulation factor XI-related disease.

The present disclosure also provides use of the siRNA, siRNA conjugate or pharmaceutical composition according to the present disclosure in the manufacture of a medicament for the treatment and/or prevention of a subject suffering from a factor XI-related disease.

The present disclosure also provides a method of treating/preventing a coagulation factor XI-related disease, comprising administering to a subject in need thereof a therapeutically and/or prophylactically effective amount of the siRNA, siRNA conjugate, or medicament according to the present disclosure composition.

In some embodiments, administration is by means of administration including intramuscular, intrabronchial, intrapleural, intraperitoneal, intraarterial, lymphatic, intravenous, subcutaneous, cerebrospinal, or a combination thereof.

In some embodiments, the factor XI-related disorder is a thromboembolic complication, particularly deep vein thrombosis, pulmonary embolism, myocardial infarction, or stroke. In some embodiments, the individual is at risk for blood clotting disorders, including, but not limited to, infarction, thrombosis, embolism, and thromboembolism, such as, for example, deep vein thrombosis, pulmonary embolism, myocardial infarction, and stroke. This includes individuals with acquired problems, diseases or disorders that pose a risk of thrombosis, such as surgery, cancer, immobility, sepsis, atherosclerosis, atrial fibrillation, etc., as well as factors such as antiphospholipid syndrome and autosomal dominant inheritance of Factor V Leiden Genetic predisposition such as mutation. In some embodiments, the individual is identified as in need of anticoagulation therapy. Examples of such individuals include, but are not limited to, those undergoing major orthopaedic surgery (eg, hip/knee replacement or hip fracture surgery) and patients requiring long-term treatment such as those experiencing atrial fibrillation to prevent stroke.

The present disclosure also provides a method of inhibiting FXI gene expression in a cell. The method includes contacting a cell with an siRNA, siRNA conjugate, or a pharmaceutical composition comprising the same of the present disclosure, and maintaining for a time sufficient to achieve degradation of the mRNA transcript of the FXI gene, thereby inhibiting the expression of the FXI gene in the cell.

The present disclosure also provides a method for silencing a target gene or mRNA of a target gene in a cell, the method comprising the step of introducing an siRNA, a siRNA conjugate, or a pharmaceutical composition according to the present disclosure into the cell.

The present disclosure also provides a method for silencing a target gene or mRNA of a target gene in a cell in vivo or in vitro, the method comprising introducing an siRNA, an siRNA conjugate, or a pharmaceutical composition according to the present disclosure into the steps in cells.

The present disclosure also provides a method for inhibiting a target gene or mRNA expression of a target gene, the method comprising administering to a subject in need thereof an siRNA, an siRNA conjugate, or a pharmaceutical composition comprising the same according to the present disclosure.

In some embodiments, the target gene is the human factor XI gene, TTR gene.

The present disclosure also provides an siRNA or siRNA conjugate, characterized in that a base T is used to replace one or more bases U of any siRNA or siRNA conjugate of the present disclosure, such as 1, 2, 3 bases , 3, 5, 6, 7, 8, 9, 10.

The pharmaceutically acceptable salts of the compounds described in the present disclosure are selected from inorganic salts or organic salts, and the compounds described in the present disclosure can react with acidic or basic substances to form corresponding salts.

On the other hand, where no configuration is indicated, the compounds of the present disclosure may exist in particular geometric or stereoisomeric forms. This disclosure contemplates all such compounds, including cis and trans isomers, (-)- and (+)-enantiomers, (R)- and (S)-enantiomers, diastereomers isomers, (D)-isomers, (L)-isomers, and racemic and other mixtures thereof, such as enantiomerically or diastereomerically enriched mixtures, all of which are within the scope of this disclosure. Additional asymmetric carbon atoms may be present in substituents such as alkyl. All such isomers, as well as mixtures thereof, are included within the scope of this disclosure.

In addition, without specifying configuration, the compounds and intermediates of the present disclosure may also exist in different tautomeric forms, and all such forms are included within the scope of the present disclosure. The term "tautomer" or "tautomeric form" refers to a form that is interconvertible via a low energy barrier Structural isomers of different energies. For example, proton tautomers (also known as proton tautomers) include interconversions via migration of protons, such as keto-enol and imine-enamine, lactamine-lactamine isomerizations. An example of a lactam-lactamine equilibrium is between A and B as shown below.

All compounds in this disclosure can be drawn as either A or B type. All tautomeric forms are within the scope of this disclosure. The naming of compounds does not exclude any tautomers.

The compounds of the present disclosure may be asymmetric, eg, have one or more stereoisomers. Unless otherwise specified, all stereoisomers include, such as enantiomers and diastereomers. Compounds of the present disclosure containing asymmetric carbon atoms can be isolated in optically pure or racemic forms. Optically pure forms can be resolved from racemic mixtures or synthesized by using chiral starting materials or chiral reagents.

Optically active (R)- and (S)-isomers, as well as D and L isomers, can be prepared by chiral synthesis or chiral reagents or other conventional techniques. If an enantiomer of a compound of the present disclosure is desired, it can be prepared by asymmetric synthesis or derivatization with a chiral auxiliary, wherein the resulting mixture of diastereomers is separated and the auxiliary group is cleaved to provide pure desired enantiomer. Alternatively, when the molecule contains a basic functional group (such as an amine group) or an acidic functional group (such as a carboxyl group), a diastereomeric salt is formed with an appropriate optically active acid or base, and then the diastereoisomeric salt is formed by conventional methods known in the art. The method performs the resolution of diastereomers, followed by recovery of the pure enantiomers. Furthermore, the separation of enantiomers and diastereomers is usually accomplished by the use of chromatography, The chromatography employs a chiral stationary phase and is optionally combined with chemical derivatization (eg, carbamate from amines).

The present disclosure also includes certain isotopically-labeled compounds of the present disclosure which are identical to those described herein, but wherein one or more atoms are replaced by an atom having an atomic weight or mass number different from that normally found in nature. Examples of isotopes that can be incorporated into the compounds of the present disclosure include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, iodine, and chlorine, such as 2H, ^3H , ^11C , ^13C , ^14C , ¹³ , ^respectively N, ¹⁵ N, ¹⁵ O, ¹⁷ O, ¹⁸ O, ³¹ P, ³² P, ³⁵ S, ¹⁸ F, ¹²³ I, ¹²⁵ I and ³⁶ Cl and the like.

Unless otherwise stated, when a position is specifically designated as deuterium (D), the position is understood to have an abundance of deuterium (ie, at least 10 times greater than the natural abundance of deuterium, which is 0.015%), at least 1000 times greater % deuterium incorporation). Exemplary compounds having natural abundance greater than deuterium may be at least 1000 times more abundant deuterium, at least 2000 times more abundant deuterium, at least 3000 times more abundant deuterium, at least 4000 times more abundant deuterium, at least 4000 times more abundant 5000 times more abundant deuterium, at least 6000 times more abundant deuterium or more abundant deuterium. The present disclosure also includes compounds of Formula I in various deuterated forms. Each available hydrogen atom attached to a carbon atom can be independently replaced by a deuterium atom. One of ordinary skill in the art can refer to the relevant literature to synthesize the compound of formula I in deuterated form. Commercially available deuterated starting materials can be used in preparing deuterated forms of the compounds of formula I, or they can be synthesized using conventional techniques using deuterated reagents including, but not limited to, deuterated borane, trideuterated borane Tetrahydrofuran solution, deuterated lithium aluminum hydride, deuterated iodoethane and deuterated iodomethane, etc.

”或“

”，或者同時包含“

”和“

”兩種構型。雖然為簡便起見將全 部上述結構式畫成某些異構體形式，但是本公開可以包括所有的異構體，如互變異構體、旋轉異構體、幾何異構體、非對映異構體、外消旋體和對映異構體。本公開所述化合物的化學結構中，鍵“

”並未指定構型，即鍵“

”的構型可以為E型或Z型，或者同時包含E和Z兩種構型。 Without specifying the configuration, in the chemical structure of the compound described in the present disclosure, the bond "/" represents the unspecified configuration, that is, if there is a chiral isomer in the chemical structure, the bond "/" can be "/"

"or"

", or both "

"and"

" two configurations. Although all of the above structural formulae are drawn as certain isomeric forms for simplicity, the present disclosure may include all isomers, such as tautomers, rotamers, geometric isomers isomers, diastereomers, racemates and enantiomers. In the chemical structures of the compounds described in this disclosure, the bond "

"No configuration specified, i.e. bond"

The configuration of " can be E or Z, or both E and Z configurations.

Terminology Explanation

For an easier understanding of the present disclosure, some technical and scientific terms are specifically defined below. Unless explicitly defined otherwise herein, all other technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

In the context of this disclosure, the terms "factor XI", "factor 11", "factor XI", "factor 11", "FXI" and "F11" are used interchangeably. "Factor XI nucleic acid" refers to any nucleic acid encoding Factor XI. For example, in some embodiments, a factor XI nucleic acid includes a DNA sequence encoding factor XI such as a "factor XI gene," an RNA sequence transcribed from DNA encoding factor XI (including intron- and exon-containing genomic DNA), and mRNA sequence encoding factor XI. "Factor XI gene" is any of the following sequences: GENBANK Accession No. NM_000128:3, GENBANK Accession No. NT_022792.17, 19598000 to 19624000 deletion, GENBANK Accession No. NM_028066.1, exons 1-15, GENBANK Accession No. NW_001118167 .1. "Factor XI mRNA" refers to mRNA encoding Factor XI protein.

As used herein, in the context of RNA-mediated gene silencing, the sense strand (also known as SS, SS or sense strand) of an siRNA refers to the strand comprising the same or substantially the same sequence as the target mRNA sequence; The antisense strand (also called AS or AS strand) of an siRNA refers to the strand having a sequence complementary to the target mRNA sequence.

Unless otherwise specified, in the context of the present disclosure, capital letters C, G, U, A, T represent the base composition of nucleotides; lowercase letter d represents a nucleoside adjacent to the right side of the letter d Acid is deoxyribonucleotide; lowercase letter m indicates that the adjacent nucleotide to the left of the letter m is a methoxy-modified nucleotide; lowercase letter f indicates that the adjacent nucleotide to the left of the letter f is fluorine Modified nucleotides; the lowercase letter s indicates a phosphorothioate linkage between the two nucleotides adjacent to the letter s.

As used herein, the term "fluorine-modified nucleotide" refers to a nucleotide formed by substituting the hydroxyl group at the 2' position of the ribosyl of a nucleotide with fluorine, and "non-fluorine-modified nucleotide" refers to a nucleotide of Nucleotides or nucleotide analogs in which the hydroxyl group at the 2' position of the ribosyl group is replaced by a non-fluorine group. "Nucleotide analogs" refer to the ability to replace nucleotides in nucleic acids, but the structure is different from adenine ribose nucleus A group of nucleotides, guanine ribonucleotides, cytosine ribonucleotides, uracil ribonucleotides, or thymidine ribonucleotides. Such as isonucleotides, bridged nucleotides (bridged nucleic acid, BNA for short) or acyclic nucleotides. The methoxy-modified nucleotide refers to a nucleotide in which the 2'-hydroxyl group of the ribosyl group is substituted with a methoxy group. Isonucleotides refer to compounds formed by changing the position of the base in the nucleotide on the ribose ring. In some embodiments, the isonucleotide may be a compound formed by moving the base from the 1'-position to the 2'-position or the 3'-position of the ribose ring. BNA refers to constrained or inaccessible nucleotides. The BNA may contain a five-, six-, or seven-membered ring bridged structure with "fixed" C3'-endoglycan constriction. The bridge is typically incorporated at the 2'-,4'-position of the ribose sugar to provide a 2',4'-BNA nucleotide. In some embodiments, the BNA can be LNA, ENA, cETBNA, or the like. Acyclic nucleotides are a class of nucleotides formed by opening the sugar ring of a nucleotide. In some embodiments, an acyclic nucleotide can be an unlocked nucleic acid (UNA) or a glycerol nucleic acid (GNA).

In the context of the present disclosure, a "nucleotide difference" between a nucleotide sequence and another nucleotide sequence means that the base type of the nucleotide at the same position has changed in the former compared with the latter, such as , when one nucleotide base is A in the latter, and the corresponding nucleotide base at the same position of the former is U, C, G or T, it is considered that the distance between the two nucleotide sequences is There is a nucleotide difference at this position. In some embodiments, abasic nucleotides are A nucleotide difference at that position is also considered to have occurred when the nucleotide at the original position is replaced by an equivalent or an equivalent thereof.

In the context of describing the sense strands of siRNAs described herein, the term "' at least 15 contiguous nucleotides that differ by no more than 3 nucleotide sequences from any of the sense strands in Table 1" is intended to mean the siRNAs described herein The sense strand comprises at least 15 contiguous nucleotides of any of the sense strands in Table 1, or differs by no more than 3 nucleotide sequences from at least 15 contiguous nucleotides of any of the sense strands in Table 1, optionally , differ by no more than 2 nucleotide sequences, and optionally, differ by 1 nucleotide sequence. Optionally, the siRNA sense strands described herein comprise at least 16 contiguous nucleotides of any one of the sense strands in Table 1, or differ by no more than 3 from at least 16 contiguous nucleotides of any one of the sense strands in Table 1 nucleotide sequences, optionally, differ by no more than 2 nucleotide sequences, optionally, differ by 1 nucleotide sequence;

Optionally, the siRNA sense strands described herein comprise at least 17 contiguous nucleotides of any one of the sense strands in Table 1, or differ by no more than 3 from at least 17 contiguous nucleotides of any one of the sense strands in Table 1 nucleotide sequences, optionally, differ by no more than 2 nucleotide sequences, optionally, differ by 1 nucleotide sequence;

Optionally, the siRNA sense strands described herein comprise at least 18 contiguous nucleotides of any one of the sense strands in Table 1, or differ by no more than 3 from at least 18 contiguous nucleotides of any one of the sense strands in Table 1 nucleotide sequences, optionally, differ by no more than 2 nucleotide sequences, optionally, differ by 1 nucleotide sequence;

Optionally, the siRNA sense strands described herein comprise all 19 contiguous nucleotides of any one of the sense strands in Table 1, or differ by no more than 3 from all 19 contiguous nucleotides of any one of the sense strands in Table 1 The nucleotide sequences, optionally, differ by no more than 2 nucleotide sequences, optionally, by 1 nucleotide sequence.

In the context of describing the siRNA antisense strands described herein, the term "at least 15 contiguous nucleotides that differ by no more than 3 nucleotide sequences from any of the antisense strands in Table 1" is intended to mean the The siRNA antisense strand comprises at least 15 contiguous nucleotides of any antisense strand in Table 1, or differs by no more than 3 nucleotide sequences from at least 15 contiguous nucleotides of any antisense strand in Table 1 , optionally, differ by no more than 2 nucleotide sequences, optionally, differ by 1 nucleotide sequence.

Optionally, the siRNA antisense strands described herein comprise or differ from at least 16 contiguous nucleotides of any antisense strand in Table 1, or differ from at least 16 contiguous nucleotides of any antisense strand in Table 1 no more than 3 nucleotide sequences, optionally, differing by no more than 2 nucleotide sequences, optionally, differing by 1 nucleotide sequence;

Optionally, the siRNA antisense strands described herein comprise or differ from at least 17 contiguous nucleotides of any antisense strand in Table 1, or differ from at least 17 contiguous nucleotides of any antisense strand in Table 1 no more than 3 nucleotide sequences, optionally, differing by no more than 2 nucleotide sequences, optionally, differing by 1 nucleotide sequence;

Optionally, the siRNA antisense strands described herein comprise or differ from at least 18 contiguous nucleotides of any antisense strand in Table 1, or differ from at least 18 contiguous nucleotides of any antisense strand in Table 1 no more than 3 nucleotide sequences, optionally, differing by no more than 2 nucleotide sequences, optionally, differing by 1 nucleotide sequence;

Optionally, the siRNA antisense strands described herein comprise or differ from at least 19 contiguous nucleotides of any antisense strand in Table 1, or differ from at least 19 contiguous nucleotides of any antisense strand in Table 1 no more than 3 nucleotide sequences, optionally, differing by no more than 2 nucleotide sequences, optionally, differing by 1 nucleotide sequence;

Optionally, the siRNA antisense strands described herein comprise at least 20 contiguous nucleotides of any antisense strand in Table 1, or at least 20 contiguous nucleotides of any antisense strand in Table 1 The acids differ by no more than 3 nucleotide sequences, optionally, by no more than 2 nucleotide sequences, optionally by 1 nucleotide sequence;

Optionally, the siRNA antisense strands described herein comprise, or differ from, all 21 contiguous nucleotides of any antisense strand in Table 1 No more than 3 nucleotide sequences, optionally, no more than 2 nucleotide sequences, optionally 1 nucleotide sequence.

As used herein, the terms "complementary" or "reverse complementary" are used interchangeably and have the meaning known to those of ordinary skill in the art, ie, in a double-stranded nucleic acid molecule, the bases of one strand are Pairs complementary to bases on the other strand. 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) pairs. Each base pair consists of a purine and a pyrimidine. When adenine on one chain is always paired with thymine (or uracil) on the other chain, and guanine is always paired with cytosine, the two chains are considered complementary to each other, and from their complementary chains The sequence of this chain can be deduced from the sequence. Correspondingly, "mismatch" in the art means that in a double-stranded nucleic acid, the bases at corresponding positions are not paired in complementary form.

As used herein, the term "inhibit", is used interchangeably with "reduce," "silence," "down-regulate," "repression," and other similar terms, and includes any level of inhibition.

As used herein, the term "inhibiting factor XI expression" includes inhibiting the expression of the factor XI gene and variants (eg, naturally occurring variants) or mutants of the factor XI gene, inhibiting the expression of factor XI mRNA, And/or inhibit the expression of coagulation factor XI protein. The factor XI gene can be a wild-type human factor XI gene, a mutant human factor XI gene, or a transgenic human factor XI gene in the context of genetically manipulated cells, cell populations, or organisms. Inhibit factor XI gene expression including any level of factor XI Inhibition of a gene, such as at least partial inhibition of the expression of the factor XI gene, such as inhibition of at least about 5%, at least about 10%, at least about 15%, 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%. Factor XI gene expression can be assessed based on the level of any variable associated with factor XI gene expression, such as factor XI mRNA levels or factor XI protein levels. Inhibition can be assessed by a reduction in absolute or relative levels of one or more of these variables compared to control levels. The control level can be any type of control level used in the art, such as a pre-dose baseline level or from a similar untreated or controlled (eg buffer only control or inert control) treated subject, cell , or the level determined by the sample. For example, the residual expression of mRNA can be used to characterize the degree of inhibition of target gene expression by siRNA, for example, the residual expression of mRNA is not higher than 99%, not higher than 95%, not higher than 90%, not higher than 85%, not higher than More than 80%, less than 75%, less than 70%, less than 65%, less than 60%, less than 55%, less than 50%, less than 45%, less than 40%, no more than 35%, no more than 30%, no more than 25%, no more than 20%, no more than 15%, or no more than 10%. The inhibition rate of target gene expression can be detected by Dual-Glo® Luciferase Assay System, read firefly (Firfly) chemiluminescence value and Renilla (Renilla) chemiluminescence value respectively, calculate the relative value Ratio=Ren/Fir, inhibition rate (% )=1-(Ratio+siRNA/Ratioreporter only)*100%; in the present disclosure, the remaining mRNA expression ratio=100%-inhibition rate (%).

Unless otherwise specified, the "compounds", "ligands", "nucleic acid ligand conjugates", and "nucleic acids" of the present disclosure can be independently expressed as salts, mixed salts or non-salts (eg, free acids or free bases). form exists. When present in the form of a salt or mixed salts, it can be a pharmaceutically acceptable salt.

The term "pharmaceutically acceptable salt" includes pharmaceutically acceptable acid addition salts and pharmaceutically acceptable base addition salts.

"Pharmaceutically acceptable acid addition salts" refers to salts with inorganic or organic acids that retain the biological effectiveness of the free base without other side effects. Inorganic acid salts include but are not limited to hydrochloride, hydrobromide, sulfate, nitrate, phosphate, etc.; organic acid salts include but are not limited to formate, acetate, 2,2-dichloroacetate, trichloroacetate Fluoroacetate, propionate, caproate, caprylate, caprate, undecylenate, glycolate, gluconate, lactate, sebacate, adipate, glutarate acid salt, malonate, oxalate, maleate, succinate, fumarate, tartrate, citrate, palmitate, stearate, oleate, cinnamate, laurate, malate, glutamate, pyroglutamate, aspartate, benzoate, mesylate, benzenesulfonate, p-toluenesulfonate, alginate, Ascorbate, salicylate, 4-aminosalicylate, naphthalene disulfonate, etc. These salts can be prepared by methods known in the art.

"Pharmaceutically acceptable base addition salts" refers to salts with inorganic or organic bases that retain the biological availability of the free acid without other adverse effects. Salts derived from inorganic bases include, but are not limited to, sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum, and the like. Preferred inorganic salts are ammonium, sodium, potassium, calcium and magnesium salts, preferably sodium salts. Salts derived from organic bases include, but are not limited to, the following: primary, secondary, and tertiary amines, substituted amines, including natural substituted amines, cyclic amines, and basic ions Exchange resins such as amine, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, diethanolamine, triethanolamine, dimethylethanolamine, 2-dimethylaminoethanol, 2-diethylamine Ethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, choline, betaine, ethylenediamine, glucosamine, methylglucamine, theobromine, purine, Piperazine, Piperidine, N-Ethyl Piperidine, polyamine resin, etc. Preferred organic bases include isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline and caffeine. These salts can be prepared by methods known in the art.

An "effective amount" or "effective dose" refers to the amount of a drug, compound, or pharmaceutical composition necessary to obtain any one or more beneficial or desired therapeutic results. For prophylactic use, beneficial or desired results include elimination or reduction of risk, reduction in severity, or delay in onset of disorders, including biochemical, tissue academic and/or behavioral symptoms. For therapeutic applications, beneficial or desired outcomes include clinical outcomes, such as reducing the incidence or amelioration of one or more symptoms of the various target gene, target mRNA or target protein-related disorders of the present disclosure, reducing the need to treat the disorder The dosage of the other agent, enhances the therapeutic effect of the other agent, and/or delays the progression of the target gene, target mRNA or target protein-related disorder of the present disclosure in the patient.

In some embodiments, the effective amount or effective dose of siRNA is about 0.001 mg/kg body weight to about 200 mg/kg body weight, about 0.01 mg/kg body weight to about 100 mg/kg body weight, or about 0.5 mg/kg body weight to about 50 mg/kg body weight. kg body weight.

A "pharmaceutical composition" comprises the siRNA or siRNA conjugate of the present disclosure and a pharmaceutically acceptable adjuvant and/or adjuvant, and the adjuvant may be one or more various formulations or compounds conventionally used in the art. For example, the pharmaceutically acceptable adjuvant may include at least one of pH buffering agents, protecting agents and osmotic pressure adjusting agents.

As used herein, "subject", "patient", "subject" or "individual" are used interchangeably and include humans or non-human animals, eg, mammals, eg, humans or monkeys.

The siRNA provided by the present disclosure can be obtained by conventional preparation methods in the art (eg, solid-phase synthesis and liquid-phase synthesis methods). Among them, solid-phase synthesis has already had commercial customized services. Modified nucleotide groups can be introduced into the present disclosure by using nucleoside monomers with corresponding modifications Among the siRNAs, methods for preparing nucleoside monomers with corresponding modifications and methods for introducing modified nucleotide groups into siRNAs are also well known to those of ordinary skill in the art.

The term "chemical modification" or "modification" includes all alterations of nucleotides by chemical means, such as the addition or removal of chemical moieties, or the substitution of one chemical moiety for another.

The term "base" includes any known DNA and RNA bases, base analogs such as purines or pyrimidines, which also includes the natural compounds adenine, thymine, guanine, cytosine, uracil, inosine and natural analogues.

In the present disclosure, base analogs are generally purine or pyrimidine bases, excluding common bases: guanine (G), cytosine (C), adenine (A), thymine (T), and uracil ( U). Non-limiting examples of bases include hypoxanthine (I), xanthine (X), 3β-D-ribofuranosyl-(2,6-diaminopyrimidine) (K), 3-β-D-furan Ribosyl-(1-methyl-pyrazolo[4,3-d]pyrimidine-5,7(4H,6H)-dione) (P), isocytosine (iso-C), isoguanine ( iso-G), 1-β-D-ribofuranosyl-(5-nitroindole), 1-β-D-ribofuranosyl-(3-nitropyrrole), 5-bromouracil, 2- Aminopurine, 4-thio-dT, 7-(2-thienyl)-imidazo[4,5-b]pyridine (Ds) and pyrrole-2-carbaldehyde (Pa), 2-amino-6- (2-thienyl)purine (S), 2-oxypyridine (Y), difluorotolyl, 4-fluoro-6-methylbenzimidazole, 4-methylbenzimidazole, 3-methylhydroxy Isoquinolyl, 5-methylhydroxyisoquinolyl and 3-methyl-7-propynylhydroxyisoquinolyl, 7-azaindolyl, 6-methyl-7-azaindole base, imidazopyridyl, 9-methyl-imidazopyridyl, pyrrolopyrazinyl, hydroxyisoquinolinyl, 7-propynylhydroxyisoquinolinyl, propynyl-7-azaindole base, 2,4,5-trimethylphenyl, 4-methylindolyl, 4,6-dimethylindolyl, phenyl, naphthyl, anthracenyl, phenanthryl, pyrenyl, stilbene (stilbenzyl), tetraphenyl, pentacyl and their structural derivatives. A base analog can also be a universal base.

The term "universal base" refers to a heterocyclic moiety located at the 1' position of a nucleotide sugar moiety in a modified nucleotide or an equivalent position in a nucleotide sugar moiety substitution, which heterocyclic moiety, when present on a nucleic acid duplex In vivo, it can be positioned relative to more than one base without changing the double helix junction structure (eg, the structure of the phosphate backbone). Furthermore, the universal base does not disrupt the ability of the single-stranded nucleic acid in which it resides to form a duplex with the target nucleic acid. The ability of a single-stranded nucleic acid containing a universal base to form a duplex with a target nucleic acid can be determined by methods obvious to those of ordinary skill in the art (eg, UV absorbance, circular dichroism, gel shift, single-stranded nucleic acid enzyme sensitivity, etc.). Additionally, the conditions under which duplex formation is observed can be varied to determine duplex stability or formation, eg, temperature, such as melting temperature (Tm), correlates with nucleic acid duplex stability. Compared to a reference single-stranded nucleic acid that is precisely complementary to the target nucleic acid, the Tm of the duplex formed by the single-stranded nucleic acid containing the universal base with the target nucleic acid is lower than that of the duplex formed with the complementary nucleic acid. However, compared to the reference single-stranded nucleic acid in which the universal base is replaced by a base to generate a single mismatch, the Tm of the duplex formed by the single-stranded nucleic acid containing the universal base and the target nucleic acid is higher than that of the single-stranded nucleic acid with the mismatched base. nucleic acid duplexes.

Some universal bases can be combined with all bases guanine (G), cytosine (C), adenine (A), thymine (T) and uracil (U) under base pairing conditions Hydrogen bonds are formed to achieve base pairing. Universal bases are not bases that only form base pairs with a single complementary base. In a duplex, a universal base may not hydrogen bond, form one hydrogen bond, or form more than one hydrogen bond with each of the G, C, A, T, and U opposing it on the opposite strand of the duplex. In some embodiments, the universal base does not interact with the opposite base on the opposite strand of the duplex. In duplexes, base pairing with universal bases does not alter the double helix structure of the phosphate backbone. Universal bases can also interact with bases in adjacent nucleotides on the same nucleic acid strand via stacking interactions. This stacking interaction can stabilize the duplex, especially if the universal base does not form any hydrogen bonds with bases positioned opposite it on the opposite strand of the duplex. Non-limiting examples of universal binding nucleotides include inosine, 1-beta-D-ribofuranosyl-5-nitroindole, and/or 1-beta-D-ribofuranosyl-3-nitropyrrole.

The terms "blunt end" or "blunt end" are used interchangeably and refer to the absence of unpaired nucleotides or nucleotide analogs at a given end of an siRNA, ie, no nucleotide overhangs. In most cases, an siRNA that is blunt-ended at both ends will be double-stranded over its entire length.

The terms "about", "approximately" mean that the index value is within an acceptable error range of the particular value determined by one of ordinary skill in the art, which value depends in part on how it is measured or determined (ie, the limits of the measurement system). For example, "about" can mean within 1 or more than 1 standard deviation. Alternatively, "about" or "substantially comprising" may mean a range of up to 20%, such as between 1% and 15%, between 1% and 10%, between 1% and 5%, between 0.5% Varies between 0.5% and 1%, and in this disclosure, each instance of a number or numerical range preceded by the term "about" also includes embodiments of the given number. Furthermore, particularly with respect to biological systems or processes, the term can mean at most one order of magnitude or at most five times the value. Unless otherwise stated, when a specific value appears in this application and claims, the meaning of "about" or "substantially comprising" should be assumed to be within an acceptable error range for the specific value.

The terms "optionally" or "optionally" are meant to mean that the subsequently described event or circumstance can, but need not, occur, and that the specification includes instances where the event or circumstance does or does not occur. For example, "C _1-6 alkyl optionally substituted by halogen or cyano" means that halogen or cyano may but need not be present, and the description includes the case where the alkyl is substituted by halogen or cyano and the case where the alkyl is not substituted by halogen and cyano substitution.

The term "alkyl" refers to a saturated aliphatic hydrocarbon group that is a straight or branched chain group containing 1 to 20 carbon atoms, in some embodiments selected from alkyl groups containing 1 to 12 carbon atoms. Non-limiting examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1,1-dimethylpropyl , 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl- 2-Methylpropane base, 1,1,2-trimethylpropyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 2,2-dimethylbutyl, 1,3-dimethylbutyl butyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2,3-dimethylbutyl, n-heptyl, 2-methyl Hexyl, 3-methylhexyl, 4-methylhexyl, 5-methylhexyl, 2,3-dimethylpentyl, 2,4-dimethylpentyl, 2,2-dimethylpentyl, 3,3-dimethylpentyl, 2-ethylpentyl, 3-ethylpentyl, n-octyl, 2,3-dimethylhexyl, 2,4-dimethylhexyl, 2,5- Dimethylhexyl, 2,2-dimethylhexyl, 3,3-dimethylhexyl, 4,4-dimethylhexyl, 2-ethylhexyl, 3-ethylhexyl, 4-ethylhexyl, 2-methyl-2-ethylpentyl, 2-methyl-3-ethylpentyl, n-nonyl, 2-methyl-2-ethylhexyl, 2-methyl-3-ethylhexyl, 2,2-diethylpentyl, n-decyl, 3,3-diethylhexyl, 2,2-diethylhexyl, and various branched isomers thereof, etc. In some embodiments selected are alkyl groups containing 1 to 6 carbon atoms, non-limiting examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tertiary Butyl, dibutyl, n-pentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2 -Methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2-methylpropyl, 1,1,2-trimethylpropyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 2,2-dimethylbutyl, 1,3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl , 4-methylpentyl, 2,3-dimethylbutyl, etc. Alkyl groups may be substituted or unsubstituted, and when substituted, the substituents may be substituted at any available point of attachment, which in some embodiments is selected from one or more of the following groups, independently is selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxy, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, hetero Aryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, pendant oxy, carboxyl or carboxylate.

The term "alkoxy" refers to -O-(alkyl) and -O-(unsubstituted cycloalkyl), wherein alkyl is as defined above. Non-limiting examples of alkoxy groups include: methoxy, ethoxy, propoxy, butoxy, cyclopropoxy, cyclobutoxy, cyclopentyloxy, cyclohexyloxy. Alkoxy can be optionally substituted or unsubstituted, and when substituted, the substituents are in some embodiments selected from one or more of the following groups independently selected from halogen, deuterium, hydroxy, pendant oxy , nitro, cyano, C _1-6 alkyl, C _1-6 alkoxy, C _2-6 alkenyloxy, C _2-6 alkynyloxy, C _3-6 cycloalkoxy, 3 to 6 Member heterocycloalkoxy, C _3-8 cycloalkenyloxy, 5- to 6-membered aryl or heteroaryl, the C _1-6 alkyl, C _1-6 alkoxy, C _2-6 alkenyloxy , C _2-6 alkynyloxy, C _3-6 cycloalkoxy, 3- to 6-membered heterocycloalkoxy, C _3-8 cycloalkenyloxy, 5- to 6-membered aryl or heteroaryl are optionally One or more substituted by halogen, deuterium, hydroxyl, pendant oxy, nitro, cyano. Similarly, the definitions of "alkynyloxy", "alkenyloxy", "cycloalkoxy", "heterocycloalkoxy" and "cycloalkenyloxy" are as defined above for "alkoxy".

The term "alkenyl" refers to a straight or branched chain non-aromatic hydrocarbon group containing at least one carbon-carbon double bond and having from 2 to 10 carbon atoms. Up to 5 carbon-carbon double bonds may be present in such groups. For example, "C2 _{- C6} " alkenyl is defined as an alkenyl group having 2-6 carbon atoms. Examples of alkenyl groups include, but are not limited to, vinyl, propenyl, butenyl, and cyclohexenyl. The straight, branched or cyclic portion of the alkenyl group can contain double bonds and is optionally mono-, di-, tri-, tetra- or penta-substituted at any position allowed by normal valences.

The term "cycloalkenyl" refers to a monocyclic hydrocarbon group having the specified number of carbon atoms and at least one carbon-carbon double bond.

The term "alkynyl" refers to a straight or branched chain hydrocarbon group containing from 2 to 10 carbon atoms and containing at least one carbon-carbon triple bond. Up to 5 carbon-carbon triple bonds can be present. Thus, "C2 _{- C6alkynyl} " means an alkenyl group having 2-6 carbon atoms. Examples of alkynyl groups include, but are not limited to, ethynyl, 2-propynyl, and 2-butynyl. The straight-chain, branched portion of the alkynyl group can contain triple bonds as normal valences allow, and is optionally mono-, di-, tri-, tetra- or penta-substituted at any position permitted by normal valences.

The term "ketone" refers to any alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, heterocyclyl, heteroaryl, or aryl group described herein attached through a carbonyl bridge. Examples of keto groups include, but are not limited to: alkanoyl (eg, acetyl, propionyl, butyryl, pentamyl, hexyl), alkenyl (eg, acryl) alkynyl (eg, ethynyl, propynyl, butynyl, pentynyl, hexynyl), aryl (eg, benzyl), heteroaryl (eg, pyrrolidyl, imidazolinyl) group, quinolinyl group, pyridyl group).

The term "alkoxycarbonyl" refers to any alkoxy group as defined above attached through a carbonyl bridge (ie, -C(O)O-alkyl). Examples of alkoxycarbonyl include, but are not limited to: methoxycarbonyl, ethoxycarbonyl, isopropoxycarbonyl, n-propoxycarbonyl, tert-butoxycarbonyl, benzyloxycarbonyl, or n-pentoxycarbonyl .

The term "aryloxycarbonyl" refers to any aryl group as defined above attached through an oxycarbonyl bridge (ie, -C(O)O-aryl). Examples of aryloxycarbonyl groups include, but are not limited to: phenoxycarbonyl and naphthoxycarbonyl.

The term "heteroaryloxycarbonyl" refers to any heteroaryl group as defined above attached through an oxycarbonyl bridge (ie, -C(O)O-heteroaryl). Examples of heteroaryloxycarbonyl include, but are not limited to: 2-pyridyloxycarbonyl, 2-oxazolyloxycarbonyl, 4-thiazolyloxycarbonyl, or pyrimidinyloxycarbonyl.

The term "cycloalkyl" or "carbocycle" refers to a saturated or partially unsaturated monocyclic or polycyclic cyclic hydrocarbon substituent, the cycloalkyl ring containing from 3 to 20 carbon atoms, and in some embodiments selected from the group consisting of 3 to 20 carbon atoms. 7 carbon atoms. Non-limiting examples of monocyclic cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cyclohexadienyl, and the like; polycyclic cycloalkyl groups include spiro Ring, fused and bridged cycloalkyl groups. Cycloalkyl groups may be substituted or unsubstituted, and when substituted, the substituents may be substituted at any available point of attachment, and in some embodiments are selected from one or more of the following groups, independently selected from Halogen, deuterium, hydroxyl, pendant oxy, nitro, cyano, C _1-6 alkyl, C _1-6 alkoxy, C _2-6 alkenyloxy, C _2-6 alkynyloxy, C _{3- 6} -cycloalkoxy, 3- to 6-membered heterocycloalkoxy, C _3-8 cycloalkenyloxy, 5- to 6-membered aryl or heteroaryl, the C _1-6 alkyl, C _1-6 alkoxy base, C _2-6 alkenyloxy, C _2-6 alkynyloxy, C _3-6 cycloalkoxy, 3-6 membered heterocycloalkoxy, C _3-8 cycloalkenyloxy, 5-6 membered Aryl or heteroaryl groups are optionally substituted with one or more groups selected from halogen, deuterium, hydroxy, pendant oxy, nitro, cyano.

The cycloalkyl ring can be fused to an aryl or heteroaryl ring, wherein the ring attached to the parent structure is a cycloalkyl, non-limiting examples include indanyl, tetrahydronaphthyl, benzocycloheptyl Alkyl etc. Cycloalkyl can be optionally substituted or unsubstituted, and when substituted, the substituents are in some embodiments selected from one or more of the following groups independently selected from halogen, deuterium, hydroxy, pendant oxy , nitro, cyano, C _1-6 alkyl, C _1-6 alkoxy, C _2-6 alkenyloxy, C _2-6 alkynyloxy, C _3-6 cycloalkoxy, 3 to 6 Member heterocycloalkoxy, C _3-8 cycloalkenyloxy, 5- to 6-membered aryl or heteroaryl, the C _1-6 alkyl, C _1-6 alkoxy, C _2-6 alkenyloxy , C _2-6 alkynyloxy, C _3-6 cycloalkoxy, 3- to 6-membered heterocycloalkoxy, C _3-8 cycloalkenyloxy, 5- to 6-membered aryl or heteroaryl are optionally One or more substituted by halogen, deuterium, hydroxyl, pendant oxy, nitro, cyano.

The term "heterocycloalkyl" or "heterocycle" or "heterocyclyl" refers to a saturated or partially unsaturated monocyclic or polycyclic cyclic hydrocarbon substituent containing from 3 to 20 ring atoms, of which one or more rings Atoms are heteroatoms selected from nitrogen, oxygen or S(O) _m (where m is an integer from 0 to 2), but excluding ring moieties of -OO-, -OS- or -SS-, the remaining ring atoms are carbon. In some embodiments selected from containing 3 to 12 ring atoms, of which 1-4 are heteroatoms; in some embodiments selected from containing 3 to 7 ring atoms. Non-limiting examples of monocyclic heterocycloalkyl include pyrrolidinyl, imidazolidinyl, tetrahydrofuranyl, tetrahydrothienyl, dihydroimidazolyl, dihydrofuranyl, dihydropyrazolyl, dihydropyrrolyl, piper pyridyl, piperazinyl, morpholinyl, thiomorpholinyl, homopiperazinyl, etc. Polycyclic heterocycloalkyl groups include spiro, fused and bridged ring heterocycloalkyl groups. Non-limiting examples of "heterocycloalkyl" include:

，等等。

,and many more.

The heterocycloalkyl ring can be fused to an aryl or heteroaryl ring, wherein the ring attached to the parent structure is a heterocycloalkyl, non-limiting examples of which include:

和

等。

and

Wait.

Heterocycloalkyl can be optionally substituted or unsubstituted, and when substituted, the substituents are in some embodiments selected from one or more of the following groups independently selected from halogen, deuterium, hydroxyl, pendant oxygen group, nitro, cyano, C _1-6 alkyl, C _1-6 alkoxy, C _2-6 alkenyloxy, C _2-6 alkynyloxy, C _3-6 cycloalkoxy, 3 to 6-membered heterocycloalkoxy, C _3-8 cycloalkenyloxy, 5- to 6-membered aryl or heteroaryl, the C _1-6 alkyl, C _1-6 alkoxy, C _2-6 alkenyloxy group, C _2-6 alkynyloxy, C _3-6 cycloalkoxy, 3- to 6-membered heterocycloalkoxy, C _3-8 cycloalkenyloxy, 5- to 6-membered aryl or heteroaryl optional Substituted with one or more selected from halogen, deuterium, hydroxy, pendant oxy, nitro, cyano.

The term "aryl" refers to a 6- to 14-membered all-carbon monocyclic or fused polycyclic (ie, rings sharing adjacent pairs of carbon atoms) groups having a conjugated pi-electron system, in some embodiments selected from 6 to 14 12 members, such as phenyl and naphthyl. The aryl ring can be fused to a heteroaryl, heterocycloalkyl or cycloalkyl ring, wherein the ring linked to the parent structure is an aryl ring, non-limiting examples of which include:

Aryl groups may be substituted or unsubstituted, and when substituted, the substituents are in some embodiments selected from one or more of the following groups independently selected from halogen, deuterium, hydroxy, pendant oxy, nitro , cyano, C _1-6 alkyl, C _1-6 alkoxy, C _2-6 alkenyloxy, C _2-6 alkynyloxy, C _3-6 cycloalkoxy, 3-6 membered heterocycle Alkoxy, C _3-8 cycloalkenyloxy, 5- to 6-membered aryl or heteroaryl, the C _1-6 alkyl, C _1-6 alkoxy, C _2-6 alkenyloxy, C _{2 -6} alkynyloxy, C _3-6 cycloalkoxy, 3- to 6-membered heterocycloalkoxy, C _3-8 cycloalkenyloxy, 5- to 6-membered aryl or heteroaryl optionally replaced by one or more One is substituted by halogen, deuterium, hydroxyl, pendant oxy, nitro, cyano.

，

、

等。 The term "heteroaryl" refers to a heteroaromatic system comprising 1 to 4 heteroatoms, 5 to 14 ring atoms, wherein the heteroatoms are selected from oxygen, sulfur and nitrogen. Heteroaryl is selected from 6 to 12 members in some embodiments, more in some embodiments from 5 or 6 members. E.g. Non-limiting examples thereof include: imidazolyl, furyl, thienyl, thiazolyl, pyrazolyl, oxazolyl, isoxazolyl, pyrrolyl, tetrazolyl, pyridyl, pyrimidinyl , thiadiazole, pyrazinyl, triazolyl, indazolyl, benzimidazolyl,

,

,

Wait.

The heteroaryl ring can be fused to an aryl, heterocycloalkyl or cycloalkyl ring, wherein the ring linked to the parent structure is a heteroaryl ring, non-limiting examples of which include:

Heteroaryl groups can be optionally substituted or unsubstituted, and when substituted, the substituents are in some embodiments selected from one or more of the following groups independently selected from halogen, deuterium, hydroxy, pendant oxy , nitro, cyano, C _1-6 alkyl, C _1-6 alkoxy, C _2-6 alkenyloxy, C _2-6 alkynyloxy, C _3-6 cycloalkoxy, 3 to 6 Member heterocycloalkoxy, C _3-8 cycloalkenyloxy, 5- to 6-membered aryl or heteroaryl, the C _1-6 alkyl, C _1-6 alkoxy, C _2-6 alkenyloxy , C _2-6 alkynyloxy, C _3-6 cycloalkoxy, 3- to 6-membered heterocycloalkoxy, C _3-8 cycloalkenyloxy, 5- to 6-membered aryl or heteroaryl are optionally One or more substituted by halogen, deuterium, hydroxyl, pendant oxy, nitro, cyano.

The term "hydroxy" refers to the -OH group.

The term "halogen" refers to fluorine, chlorine, bromine or iodine.

The term "haloalkyl" refers to an alkyl group substituted with halogen, wherein alkyl is as defined above.

The term "cyano" refers to -CN.

The term "nitro" refers to _-NO2 .

The term "pendant oxy" refers to a =O group, eg, a carbon atom is attached to an oxygen atom by a double bond, wherein a ketone or aldehyde group is formed.

The term "amino" refers to _-NH2 .

The term "carboxy" refers to -C(O)OH.

The term "aldehyde group" refers to -CHO.

”或

其可以根據本文所述發明範圍連接一個或多個任何基團；星號“*”表示手性中心。 In the chemical structural formula of the present disclosure, "

"or

It can be attached to one or more of any group in accordance with the scope of the invention described herein; an asterisk "*" indicates a chiral center.

中的

部分可以替換為能夠與相鄰核苷酸實現連接的任意基團。 In the context of this disclosure, the group

middle

A moiety can be replaced with any group that enables linkage to adjacent nucleotides.

The term "connected," when referring to a connection between two molecules, refers to the connection of two molecules by covalent bonds or the association of two molecules by non-covalent bonds (eg, hydrogen bonds or ionic bonds), including direct connection, indirect connection.

The term "directly attached" refers to the attachment of a first compound or group to a second compound or group without any intervening atoms or groups of atoms.

The term "indirectly attached" refers to the attachment of a first compound or group to a second compound or group through an intermediate group, compound or molecule (eg, a linking group).

The term "substituted" means that any one or more hydrogen atoms on the designated atom (usually carbon, oxygen, and nitrogen atoms) are replaced by any group as defined herein, provided that the designated atom's normal valence is not exceeded and the substitution Generate stable compounds. Non-limiting examples of substituents include C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, cyano, hydroxy, pendant oxy, carboxyl, cycloalkyl, cycloalkenyl, heterocyclyl, heterocyclyl Aryl, aryl, ketone, alkoxycarbonyl, aryloxycarbonyl, heteroaryloxycarbonyl, or halogen (eg, F, Cl, Br, I). When the substituent is a ketone or pendant oxygen (ie, =O), then two (2) hydrogens are replaced on the atom.

"Substituted with one or more" means that it may be substituted with single or multiple substituents. When substituted by a plurality of substituents, it may be a plurality of the same substituents, or a combination of one or a plurality of different substituents.

Some abbreviations in this disclosure are defined as follows:

DCE: dichloroethane;

Sc(OTf) ₃ : scandium trifluoromethanesulfonate;

TFH: tetrahydrofuran;

Pd/C: palladium-carbon;

TFA: trifluoroacetic acid;

DMF: dimethylformamide;

DIPEA: N-ethyldiisopropylamine;

HoBt: 1-hydroxybenzotriazole;

EDCI: 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride;

DMTrCl: 4,4'-bismethoxytrityl chloride;

DIEA: N,N-diisopropylethylamine;

HATU: 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate;

LiOH: lithium hydroxide;

DMAP: 4-dimethylaminopyridine;

HBTU: benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate;

DMTrCl: 1-[Chloro(4-methoxyphenyl)benzyl]-4-methoxybenzene

CF ₃ SO ₃ H: trifluoromethanesulfonic acid;

BnBr: benzyl bromide;

DEPBT: 3-(diethoxyphosphoryloxy)-1,2,3-benzotriazin-4-one;

Bz: benzyl protecting group;

MMTr: methoxyphenyl benzhydryl;

DMTr: dimethoxytrityl protecting group.

Fig. 1 is the verification result of on-target activity and off-target level of siRNA according to the present disclosure;

Figure 2 is the inhibitory activity of FXI expression according to modified siRNA;

Figure 3 shows the inhibitory activity of GalNAc-conjugated siRNA on mTTR gene in murine primary hepatocytes;

Figure 4 shows the in vivo inhibitory activity of GalNAc-conjugated siRNA for mouse mTTR gene;

Figure 5 shows the in vivo long-term inhibitory activity of aminogalactose cluster-conjugated siRNA on mouse mTTR gene.

The present disclosure is further described below in conjunction with the examples, but these examples do not limit the scope of the present disclosure. In the case where specific experimental conditions and/or methods are not indicated in the examples of the present disclosure, it is usually carried out in accordance with conventional conditions or in accordance with conditions and/or methods suggested by raw material or commodity manufacturers. Reagents not specifically sourced are available from suppliers of molecular biology reagents in quality/purity for molecular biology applications.

Example 1 Design and synthesis of factor XI siRNA

1) siRNA design , the human coagulation factor XI gene (NM_000128.4) was used as the target gene to design siRNA. The unmodified sense and antisense strand sequences are shown in Table 1.

2) siRNA synthesis , the siRNA sequences of Tables 5 and 6 were synthesized using solid support mediated phosphamide chemistry on a Dr. Oligo48 synthesizer (Biolytic) at 200 nanomolar (nmol) scale. The solid support is a general solid support (Shenzhen Comma Bio). Nucleoside monomer raw materials 2'-F-RNA, 2'-O-methyl RNA and other nucleoside phosphoramidite monomers were purchased from Shanghai Zhaowei or Suzhou Zima. Coupling time for all phosphamidites (50 mM in acetonitrile) was 6 minutes (min) using 5-ethylthio-1H-tetrazole (ETT) as the activator (0.6 M in acetonitrile) in 0.22 M in PADS. In 1:1 volume ratio of acetonitrile and collema (Suzhou Kelema) solution as sulfurization reagent, sulfurization reaction time is 3 minutes (min), using iodopyridine/water solution (Suzhou Kelema) as oxidant, oxidation reaction Time 2 minutes (min). The above sulfidation reaction conditions or oxidation reaction conditions are selected according to whether the target product has 5'-phosphorothioate modification.

After the solid-phase synthesis was completed, the oligoribonucleotides were cleaved from the solid support, and immersed in a 3:1 volume ratio of 28% ammonia water and ethanol solution at 50°C for 16 hours. Then high-speed centrifugation, the supernatant was transferred to another centrifuge tube, concentrated and evaporated to dryness, purified using C18 reverse-phase chromatography, the mobile phase was 0.1M TEAA and acetonitrile, and DMTr was removed using 3% trifluoroacetic acid solution. The target oligonucleotides were collected and lyophilized, identified as the target product by LC-MS, and quantified by UV (260 nm).

The sense and antisense strands synthesized according to the above steps were annealed according to the equimolar ratio to form a double-stranded structure by hydrogen bonding. Finally, the obtained double-stranded siRNA was dissolved in 1×PBS and adjusted to The concentration required for the experiment.

Table 1. Unmodified sense and antisense strands of factor XI siRNA, and antisense strands containing chemical modifications

、

或

所示的化學修飾或其互變異構體修飾的核苷酸； In Table 1, W' in SEQ ID NOs: 47-69 is selected from: 2'-methoxy-modified nucleotides or comprising

,

or

The indicated chemically modified nucleotides or their tautomer modified nucleotides;

Wherein, M is O or S; wherein, B is selected from the bases at the corresponding positions in the 7th position of the 5' region of SEQ ID NO: 47-69 and SEQ ID NO: 24-46 in Table 2, such as SEQ ID NO : 47 corresponds to SEQ ID NO:24, SEQ ID NO:69 corresponds to SEQ ID NO:46.

The following Tables 2-3 represent the modified factor XI siRNA sense and antisense strands, wherein

The lowercase letter m indicates that a nucleotide adjacent to the left of the letter m is a 2'-methoxy modified nucleotide; the lowercase letter f indicates that the adjacent nucleotide to the left of the letter f is a 2'-fluorine modified nucleotide Nucleotides;

When the lowercase letter s is in the middle of the uppercase letter, it means that the connection between the two nucleotides adjacent to the letter s is phosphorothioate; A nucleotide end adjacent to the left of the parent s is a phosphorothioate group. m, s and f used elsewhere in this manual have the same meaning.

Table 2. Modified sense and antisense strands of factor XI siRNA

Table 3. Modified sense and antisense strands of factor XI siRNA

Example 2 Inhibition of human coagulation factor XI by siRNA in Huh7 cells-screening of single-concentration point inhibition activity

The effect of siRNA targeting factor XI on factor XI mRNA expression levels was tested in vitro. Huh7 cells were cultured in DMEM high-glucose medium containing 10% fetal bovine serum at 37°C under 5% CO ₂ conditions. 24h before transfection, Huh7 cells were seeded in 24-well plates at a seeding density of 100,000 cells per well and 500 μL of medium per well.

Referring to the product instruction manual, Lipofeetamine RNAiMAX (ThermoFisher, 13778150) was used to transfect siRNA, and the final concentration of siRNA transfection was 10 nM, with two replicate wells. After 24 hours of treatment, RNA was isolated from cells using a cellular RNA extraction kit (Fanzhi Medical Technology (Jiangsu) Co., Ltd., FG0410-L), and a reverse transcription kit (Takara PrimeScript ^™ II 1st Strand cDNA Synthesis Kit, 6210A) was used. ) was reverse transcribed, and quantitative real-time PCR detection was performed with a qPCR reaction kit (ThermoFisher TaqMan Fast Advanced Master Mix, 4444964) to determine the mRNA level of coagulation factor XI, and the mRNA level of coagulation factor XI was corrected according to the GAPDH internal reference gene level. The results are expressed as the remaining percentage of factor XI mRNA expression in the treated cells relative to the blank control group, and the results are shown in Table 4.

Table 4. Single-concentration point screening results for the inhibitory effect of siRNA on human coagulation factor XI in Huh7 cells

Example 3 Inhibition of human coagulation factor XI by siRNA in Huh7 cells-double concentration point activity

siRNA was screened in Huh7 cells at 2 concentrations (10 nM, 1 nM).

Huh7 cells were cultured in DMEM high-glucose medium containing 10% fetal bovine serum at 37°C under 5% CO ₂ conditions. 24h before transfection, Huh7 cells were seeded in 96-well plates at a seeding density of 10,000 cells per well and 100 μL of medium per well.

Referring to the product instruction manual, Lipofectamine RNAiMAX (ThermoFisher, 13778150) was used to transfect siRNA, and the final concentrations of siRNA transfection were 10 nM and 1 nM. After 24 hours of treatment, quantitative real-time PCR was used to detect the mRNA level of coagulation factor XI using TaqMan ^TM Fast Advanced Cells-to-CT ^TM Kit (ThermoFisher, A35378). Correction.

The results were expressed as the remaining percentage of factor XI mRNA expression relative to the blank control group-treated cells, and the results are shown in Table 5.

Table 5. Double-concentration point screening results for the inhibitory effect of siRNA on human coagulation factor XI in Huh7 cells

Example 4 Inhibition of human coagulation factor XI in Huh7 cells by siRNA - five-concentration point inhibitory activity

siRNA was screened in Huh7 cells using 5 concentration gradients. Each siRNA sample was always at a concentration of 10 nM from transfection in a 10-fold serial dilution. For the experimental method, refer to Example 3, wherein the final concentrations of siRNA transfection were 10 nM, 1 nM, 0.1 nM, 0.01 nM, and 0.001 nM.

Results are expressed as the remaining percentage of factor XI mRNA expression relative to blank control-treated cells. The IC50 results of the inhibition rate are shown in Table 6.

Table 6 Multi-dose inhibitory activity of siRNA on human coagulation factor XI in Huh7 cells

Example 5 Inhibition of human coagulation factor XI in Huh7 cells by siRNA-dose-dependent experiment

siRNA was screened in Huh7 cells using 11 concentration gradients. Each siRNA sample was always at a concentration of 50 nM from transfection in a 10-fold serial dilution. The experimental method refers to Example 2.

Results are expressed as the remaining percentage of factor XI mRNA expression relative to blank control-treated cells. The IC50 results of the inhibition rate are shown in Table 7.

Example 6 Validation of siRNA sequence activity and off-target level

In HEK293A cells, 11 concentration gradients were used to screen siRNA at the on-target and off-target levels in vitro. The results show that the siRNA of the present disclosure has high activity and low off-target activity.

The on-target and off-target sequences corresponding to the siRNA sequences were constructed and inserted into the psiCHECK-2 plasmid. The plasmid contains Renilla luciferase gene and firefly luciferase gene. As a dual reporter gene system, the target sequence of the siRNA is inserted into the 3'UTR region of the Renilla luciferase gene, and the activity of the siRNA against the target sequence can be expressed by the Renilla luciferase calibrated by firefly luciferase The detection of the condition was reflected using the Dual-Luciferase Reporter Assay System (Promega, E2940).

The target plasmid construction rules corresponding to the siRNA sequence are as follows:

For the antisense strand of siRNA, construct the on-target plasmid GSCM that is completely complementary to the AS strand; construct the off-target plasmid GSSM that is completely complementary to the 5' end of the antisense strand at positions 1-8, but the bases at other positions are completely mismatched. The corresponding rules for base mismatch are A and C interfit, G and T interfit.

For the sense strand of siRNA, construct off-target plasmid PSCM that is completely complementary to the SS strand; construct off-target plasmid PSSM that is completely complementary to the 5' end of the sense strand at positions 1-8, but the bases at other positions are completely mismatched. The corresponding rules are that A and C are paired with each other, and G and T are paired with each other.

HEK293A cells were cultured in DMEM high-glucose medium containing 10% fetal bovine serum at 37°C under 5% CO ₂ conditions. 24h before transfection, HEK293A cells were seeded in 96-well plates at a seeding density of 10,000 cells per well and 100 μL of medium per well.

According to the instructions, cells were co-transfected with siRNA and corresponding plasmids using Lipofectamine2000 (ThermoFisher, 11668019), and 0.2 μL of Lipofectamine2000 was used in each well. The amount of plasmid transfection was 10 ng per well. For on-target plasmids, a total of 11 concentration points were set for siRNA, the final concentration of the highest concentration point was 1nM, 2-fold gradient dilution; for off-target sequence plasmids, siRNA was set to 11 concentration points, and the final concentration of the highest concentration point was 40nM, 4-fold Gradient dilution. 24h after transfection, the off-target level was detected by Dual-Luciferase Reporter Assay System (Promega, E2940). The results are shown in Figure 1.

Example 7 siRNA of different modification schemes inhibits FXI expression

Four siRNA sequences targeting human F11 factor mRNA were selected as follows:

siRNA1

Sense strand: 5'-AGUGGUACAUUUCAUUUUA-3' (SEQ ID NO: 1)

Antisense strand: 5'-UAAAAUGAAAUGUACCACUUG-3' (SEQ ID NO: 24)

siRNA2

Sense strand: 5'-GAUGAUUUUCUUAUAUCAA-3' (SEQ ID NO: 2)

Antisense strand: 5'-UUGAUAUAAGAAAUCAUCCU-3' (SEQ ID NO: 25)

siRNA3

Sense strand: 5'-GGAUAUUGUUGCUGCAAAA-3' (SEQ ID NO: 3)

Antisense Strand: 5'-UUUUGCAGCAACAAUAUCCAG-3' (SEQ ID NO: 26)

siRNA4

Sense strand: 5'-CAGGAUGAUUUUCUUAUAU-3' (SEQ ID NO: 5)

Antisense strand: 5'-AUAUAAGAAAAUCAUCCUGAA-3' (SEQ ID NO: 28)

According to the method of Example 1, each nucleotide position of the sense strand and antisense strand was synthesized according to the modification scheme in Table 8, and the combination of the sense strand and antisense strand in the siRNA modification scheme shown in Table 9 was obtained by annealing to obtain 12 sets of siRNA.

Activity assessment was performed in Huh7 cell line (96-well plate, starting concentration of 10 nM, 5 concentration points, 10-fold serial dilution).

The primer sequences used in the Taqman probe Q-PCR detection experiment are as follows:

Target gene-Probe-F (hFXI-PF): 5'-TTTGCTGGGAGAGGGTGTTG-3' (SEQ ID NO: 426)

Target gene-Probe-R (hFXI-PR): 5'-TACAAACACCAAGCCCCTTCA-3' (SEQ ID NO: 427)

Target gene-Probe (hFXI-P): 5'-CCAGCATGCTTCCTCCACAGTAACACG-3' (SEQ ID NO: 428)

(6-FAM modification at the 5' end and BHQ1 modification at the 3' end)

Internal reference gene-Probe-F (h-GAPDH-PF): 5'-CCAGGTGGTCTCCTCTGACTTC-3' (SEQ ID NO: 429)

Internal reference gene-Probe-R (h-GAPDH-PR): 5'-GTGGTCGTTGAGGGCAATG-3' (SEQ ID NO: 430)

Internal reference gene-Probe (h-GAPDH-P): 5'-ACAGCGACACCCACTCCTCCACCTT-3' (SEQ ID NO: 431)

(TET modification at the 5' end and BHQ2 modification at the 3' end)

The experimental results are shown in Table 10 and Figure 2 (Graph Prism 5 software uses the Mean & SD value as a bar graph), the 4 groups of siRNA targeting FXI using the MD31 modification scheme all have lower _IC50 , indicating that the MD31 modification scheme has better activity.

Example 8 Preparation of chemical modification

8.1 Synthesis of Compound 1-1a and Compound 1-1b

Compound 1 (500 mg, 3.42 mmol) and triethylamine (Et ₃ N, 692 mg, 6.84 mmol, 0.95 mL) were dissolved in dichloromethane (DCM, 10 mL), and 4-toluenesulfonyl chloride ( TsCl, 717 mg, 3.76 mmol) in dichloromethane (10 mL), the reaction was stirred overnight at room temperature after the dropwise addition, quenched with water after the reaction was completed, the aqueous phase was extracted three times with dichloromethane (15 mL), and the combined The organic phase was washed with saturated aqueous sodium bicarbonate solution (10 mL), then with saturated brine (20 mL), and then the solvent was evaporated to dryness under reduced pressure to obtain crude product 2 (820 mg, 80%), which was directly used in the next reaction. MS m/z: _C14H21O5S , [M+H] ⁺ theory: _301.10 found: _301.2 .

Compound 3 (239 mg, 1.22 mmol) was dissolved in dimethylformamide (DMF, 10 mL), NaH (60% dissolved in mineral oil, 93 mg, 2.33 mmol) solution was added under ice bath, and the reaction was stirred for 30 minutes, and then dropwise added compound 2 (350 mg, 1.16 mmol), after the completion of the dropwise addition, the reaction was stirred at 60 ° C for 5 hours, after the completion of the reaction, water was added to quench, the aqueous phase was extracted three times with ethyl acetate (15 mL), and the combined organic The phase was washed three times with water (10 mL), then with saturated brine (10 mL), and then the solvent was evaporated to dryness under reduced pressure. Preparative HPLC (C ¹⁸ , conditions: 5-50% (A: H ₂ O, B: CH ₃ CN), flow rate: 70 mL/min), 220 mg of compound 4 was obtained after lyophilization. MS m/z: _C19H21N5O3Na , [ _M +Na] ⁺ theory: _390.16 , found: _390.3 .

Compound 4 (1.50 g, 4.08 mmol) was dissolved in 20 mL of a mixed solution of acetic acid and water (4:1) at room temperature, and stirred at 60° C. for 30 minutes. After the reaction was completed, the solvent was evaporated under reduced pressure. Preparative HPLC (C ¹⁸ , condition: 5-25% (A: H ₂ O, B: CH ₃ CN), flow rate: 70 mL/min) gave 1.10 g of compound 5 after lyophilization. MS m/z: _C16H18N5O3 , [ _M +H] ⁺ theory: _328.13 , found: _328.4 .

Compound 5 (1.00 g, 3.05 mmol) was dissolved in pyridine (Py, 10 mL), and 4,4'-bismethoxytrityl chloride (DMTrCl, 1.50 g, 4.58 mmol) in pyridine ( 5mL) solution, after the dropwise addition, the reaction was stirred at room temperature overnight, after the completion of the reaction, quenched with water, evaporated to dryness under reduced pressure, and prepared by reverse-phase HPLC (C ¹⁸ , conditions: 5-80% (A:H) ₂ O, B: CH ₃ CN), flow rate: 70 mL/min), 1.00 g of compound 6 was obtained after lyophilization. MS m/z: _C37H36N5O5 , [ _MH ] ⁺ theory: _630.26 , found: _630.5 . The racemic compound 6 was resolved on a chiral column (Daicel CHIRALPAK® IE 250*4.6 mm, 5 μm, A: n-hexane, B: ethanol) to give 410 mg of 6A(-) and 435 mg of 6B(+).

Compound 6A(-) (200 mg, 0.32 mmol), tetrazole (11 mg, 0.16 mmol), N-methylimidazole (5 mg, 0.06 mmol), 3A molecular sieve (500 mg) were dissolved in 10 mL of acetonitrile, and added at room temperature Compound 7 (144 mg, 0.48 mmol) was stirred at room temperature overnight. After the reaction was completed, the molecular sieves were filtered off, dichloromethane (30 mL) was added, saturated aqueous sodium bicarbonate solution (10 mL) was washed three times, and then saturated brine ( ²⁰ mL) was washed. , conditions: 5-100% (A: water, B: CH ₃ CN), flow rate: 70 mL/min), 200 mg of compound 1-1a was obtained after lyophilization. MS m/z: C ₄₀ H ₃₉ N ₆ O ₇ P, [M-diisopropyl+OH] ⁺ theory: 747.26, found: 747.6. 1H NMR (400 MHz, Acetonitrile- d ₃ ) δ 7.56, 7.54 (2s ,1H),7.36-7.27(m,2H),7.24-7.21(m,7H),6.83-6.80(m,4H),4.12-4.10(m,2H),3.75-3.68(m,10H),3.20 -2.80(m, 2H), 2.68-2.54(m, 4H), 1.22-1.04(m, 18H).

Compound 6B(+) (200 mg, 0.32 mmol), tetrazole (11 mg, 0.16 mmol), N-methylimidazole (5 mg, 0.06 mmol), 3A molecular sieve (500 mg) were dissolved in 10 mL of acetonitrile, and added at room temperature Compound 7 (144 mg, 0.48 mmol) was stirred at room temperature overnight. After the reaction was completed, the molecular sieves were filtered off, dichloromethane (30 mL) was added, saturated aqueous sodium bicarbonate solution (10 mL) was washed three times, and then saturated brine ( ²⁰ mL) was washed. , conditions: 5-100% (A: water, B: CH ₃ CN), flow rate: 70 mL/min), 200 mg of compound 1-1b was obtained after lyophilization. MS m/z: _C40H39N6O7P , [M _- diisopropyl+OH] ⁺ theory: _747.26 , found: 747.5.

8.2 Synthesis of Compounds 1-2

Compound 1 (2 g, 8.36 mmol) was dissolved in DMF (20 mL), and NaH (0.37 g, 9.2 mmol, 60% dissolved in mineral oil) was slowly added under argon at room temperature. After stirring at room temperature for 2 hours, compound 2 (3.3 g, 16.72 mmol) was added to the reaction solution. After stirring at room temperature for 12 h, the reaction solution was concentrated, and the residue was recrystallized with ethanol (EtOH, 50 mL) to obtain the target product 3A (1.3 g, yield 44.0%) (dichloromethane:ethyl acetate=2:1, Rf = 0.2) and target product 3B (0.6 g, mixture of compound 1) (dichloromethane:ethyl acetate=2:1, Rf=0.18).

Compound 3A (1.3 g, 3.68 mmol) was dissolved in a mixture of trifluoroacetic acid (TFA, 4 mL) and DCM (20 mL), stirred at room temperature for 12 h, the reaction solution was concentrated, and the obtained residue was purified by reverse column ( C ¹⁸ , H ₂ O + acetonitrile) to give the target product 4 (1 g, 91.44% yield). MS m/z: _C39H38N6O6 , _[ M+ _H ] ⁺ : _687.5 .

The compound (D-Threoninol 5 , 1.2 g, 11.4 mmol) was dissolved in pyridine (10 mL) and a solution of DMTrCl (4.64 g, 13.70 mmol) in pyridine (15 mL) was added slowly. After stirring at room temperature for 16 h, the reaction was quenched with _H2O (10 mL) and concentrated. The reaction solution was concentrated, and the obtained residue was purified by reverse column (C ¹⁸ , H ₂ O + acetonitrile) to obtain the target product 6 (4.0 g, yield 86.0%). MS m/z: _C25H29NO4 , [M ₊ Na] ⁺ : _430.4 .

Compound 6 (600 mg, 2.02 mmol), compound 4 (822.5 mg, 2.02 mmol) and dihydroquinoline (EEDQ, 998.2 mg, 4.04 mmol) were dissolved in DCM (10 mL) and methanol (MeOH, 5 mL). After the mixture was stirred at room temperature for 16 h, the solid was filtered off and the filtrate was diluted with DCM (100 mL). The organic phase was washed three times with _H2O (30 mL), dried _over anhydrous _Na2SO4 , filtered and concentrated. The resulting residue was purified by reverse column (C ¹⁸ , H ₂ O + acetonitrile) to give the desired product 7 (780 mg, 56.3% yield). MS m/z: _C39H38N6O6 , _[ M+ _H ] ⁺ : _687.5 .

Compound 7 (780 mg, 1.13 mmol), tetrazole (39.8 mg, 0.57 mmol), N-methylimidazole (18.7 mg, 0.23 mmol) were dissolved in _CH3CN (10 mL), and 3A molecular sieves (700 mg) were added. After stirring at room temperature for 5 min under argon, compound 8 (513.5 mg, 1.70 mmol) was added. After stirring at room temperature for 1 h, the molecular sieves were filtered off and the solid was rinsed three times with DCM (30 mL). The filtrate was washed with saturated aqueous NaHCO ₃ (30 mL×4) and H ₂ O (30 mL×4), respectively; the organic phase was concentrated at 30°C. The obtained residue was purified by reverse column (C ¹⁸ , H ₂ O + acetonitrile, acetonitrile 90%), and the target compound 1-2 (700 mg, yield 69.5%) was obtained after lyophilization. MS m/z: _C48H55N8O7P , [M _{- cyanoethyl} -diisopropyl+OH] ^- : _749.3 .

8.3 Synthesis of Compounds 1-3

Compound 1 (2 g, 8.36 mmol) was dissolved in DMF (20 mL), and NaH (0.37 g, 9.2 mmol, 60% dissolved in mineral oil) was slowly added under argon at room temperature. After stirring at room temperature for 2 hours, compound 2 (3.3 g, 16.72 mmol) was added to the reaction solution. After stirring at room temperature for 12 h, the reaction was also concentrated, and the residue was recrystallized with EtOH (50 mL) to obtain the target product 3A (1.3 g, yield 44.0%) (dichloromethane:ethyl acetate=2:1, Rf=0.2 ) and the target product 3B (0.6 g, a mixture of compound 1) (dichloromethane:ethyl acetate=2:1, Rf=0.18).

Compound 3A (1.3 g, 3.68 mmol) was dissolved in a mixture of TFA (4 mL) and DCM (20 mL), stirred at room temperature for 12 h, the reaction solution was concentrated, and the obtained residue was purified by reverse column (C ¹⁸ , H ₂ O + acetonitrile) to give the target product 4 (1 g, yield 91.44%). MS m/z: _C39H38N6O6 , _[ M+ _H ] ⁺ : _687.5 .

The compound L-Threoninol (L-Threoninol 5 , 1.2 g, 11.4 mmol) was dissolved in pyridine (10 mL), then a solution of DMTrCl (4.64 g, 13.70 mmol) in pyridine (15 mL) was slowly added. After stirring at room temperature for 16 h, the reaction was quenched with _H2O (10 mL) and concentrated. The reaction solution was concentrated, and the obtained residue was purified by reverse column (C ¹⁸ , H ₂ O + acetonitrile) to obtain the target product 6 (4.0 g, yield 86.0%). MS m/z: _C25H29NO4 , [M ₊ Na] ⁺ : _430.4 .

Compound 6 (600 mg, 2.02 mmol), compound 4 (822.5 mg, 2.02 mmol), tetramethylurea hexafluorophosphate (HATU, 1.15 g, 3.03 mmol) and diisopropylethylamine (DIEA, 1 mL, 6.05 mmol) was dissolved in DMF (10 mL). After stirring at room temperature for 16 h, the reaction solution was filtered, and the filtrate was diluted with DCM (100 mL). The organic phase was washed three times with _H2O (30 mL), dried _over anhydrous _Na2SO4 , filtered and concentrated. The obtained residue was purified by reverse column (C ¹⁸ , H ₂ O + acetonitrile, acetonitrile 60%) to obtain the target compound 7 (1.0 g, yield 72.1%) after lyophilization. MS m/z: _C39H38N6O6 , _[ M+ _H ] ⁺ : _687.5 .

Compound 7 (1.2 g, 1.75 mmol), tetrazole (61.2 mg, 0.87 mmol), N-methylimidazole (28.7 mg, 0.35 mmol) were dissolved in _CH3CN (10 mL), and 3A molecular sieves (700 mg) were added. After stirring at room temperature for 5 min under argon, compound 8 (0.79 g, 2.62 mmol) was added. After stirring at room temperature for 1 h, the molecular sieves were filtered off and the solid was rinsed three times with DCM (30 mL). The filtrate was washed with saturated aqueous NaHCO ₃ (30 mL×4) and H ₂ O (30 mL×4), respectively. The organic phase was concentrated at 30°C. The obtained residue was purified by reverse column (C ¹⁸ , H ₂ O + acetonitrile, 90% acetonitrile), and the target compound 1-3 (1.2 g, yield 77.4%) was obtained after lyophilization. MS m/z: _C48H55N8O7P , [M _{- cyanoethyl} -diisopropyl+OH] ^- : _749.3 .

8.4 Synthesis of compound 1-4a and compound 1-4b

Compound 1A (6.73 g, 28.14 mmol) was dissolved in dry DMF (80 mL), and NaH (60%, 1.24 g, 30.95 mmol) was added slowly under argon. After the mixed solution was stirred at room temperature for 30 min, the reaction solution was added to a solution containing tetrakis(triphenylphosphine) palladium (Pd(PPh ₃ ) ₄ , 1.95 g, 1.69 mmol), triphenyl phosphine (PPh ₃ , 0.74 g) , 2.81 mmol) and compound 1 (4.0 g, 28.14 mmol) in tetrahydrofuran (THF, 60 mL). After the reaction was stirred at 55°C for 16 h, the solid was filtered off and washed three times with DCM (60 mL). The filtrate was concentrated, and the resulting residue was purified with a forward column (the column was flushed with ethyl acetate, then ethyl acetate:methanol (12:1) to give the desired product 2 (7 g, crude).

Compound 2 (8 g, crude) and DMTrCl (12.65 g, 37.34 mmol) were dissolved in pyridine (10 mL). After the mixture was stirred at room temperature for 16 h, it was quenched with water (80 mL) and concentrated. The obtained residue was purified by reverse column (C ¹⁸ , water+acetonitrile) and lyophilized to obtain the target compound 3 (13 g, yield 83.7%).

Compound 3 (5 g, 8.02 mmol) was dissolved in methanol (MeOH, 20 mL) and ammonia water (6 mL), the mixture was stirred at room temperature for 16 h, and the reaction solution was concentrated. The obtained residue was purified with a forward column (DCM:MeOH=20:1) to obtain the target compound 4 (4 g, yield 96.0%).

Under argon, a solution of borane (BH ₃ ) in tetrahydrofuran (1.0 M in THF, 38.54 mL, 38.54 mmol) was added dropwise to a solution of compound 4 (4.00 g, 7.71 mmol) in THF ( 12mL) solution. After the compound was stirred at 0°C for 6 h under argon, _H2O (27 mL) was added dropwise. Then, a 3M NaOH aqueous solution (52 mL, 156 mmol) was added dropwise to the reaction solution at 0°C, and then a 30% H ₂ O ₂ (106 mL) aqueous solution was added dropwise to the reaction solution, and EtOH (10 mL) was added dropwise. ). After the reaction solution was stirred at room temperature for 48 h, saturated Na ₂ S ₂ O ₃ was added slowly at 0° C. until no bubbles were formed. _H2O (300 mL) was added to the reaction and extracted with DCM (4 x 200 mL). The organic phase was dried _over anhydrous _Na2SO4 , filtered and concentrated. The obtained residue was purified by reverse column column (C ¹⁸ , acetonitrile+H2O, 50%), and after lyophilization, the target product 5a (730 mg, yield 17.6%) and target product 5b (1.1 g, 26.6%) were obtained.

Compound 5a (730 mg, 1.36 mmol) was dissolved in pyridine (8 mL) and TMSCl (0.67 g, 6.14 mmol) was added under argon protection at room temperature. After stirring at room temperature for 1 h, BzCl (0.29 mL, 2.46 mmol) was added to the reaction solution. After stirring at room temperature for 16 h, the reaction was quenched with _H2O (10 mL) and concentrated. The resulting residue was dissolved in THF (30 mL) and tetrabutylammonium fluoride (TBAF, 1 mL) was added. After stirring at room temperature for 1 h, ammonia water (0.5 mL) was added, and the mixture was stirred at room temperature for 5 h. The reaction solution was diluted with ethanol (EA, 100 mL) and washed five times with saturated saline (30 mL). The organic phase was concentrated, and the residue was purified by reverse phase column (C18, H ₂ O + acetonitrile, acetonitrile 60%) and lyophilized to obtain the target product 6a (480 mg, yield 74.8%). MS m/ _z : _C38H35N5O5 , [M+ _H ] ⁺ : _642.6 .

Compound 5b (1.1 g, 2.05 mmol) was dissolved in pyridine (20 mL) and TMSCl (1.34 g, 1.28 mmol) was added under argon protection at room temperature. After stirring at room temperature for 1 h, benzyl chloride (BzCl, 0.59 mL, 5.92 mmol) was added to the reaction solution. After stirring at room temperature for 16 h, the reaction was quenched with _H2O (10 mL) and concentrated. The resulting residue was dissolved in THF (30 mL) and TBAF (2 mL) was added. After stirring at room temperature for 1 h, ammonia water (0.5 mL) was added, and the mixture was stirred at room temperature for 5 h. The reaction solution was diluted with EA (100 mL) and washed five times with saturated saline (30 mL). The organic phase was concentrated, and the residue was purified by reverse phase column (C18, H ₂ O + acetonitrile, acetonitrile 60%) and lyophilized to obtain the target product 6b (1.4 g, yield 82.1%). MS m/ _z : _C38H35N5O5 , [M+ _H ] ⁺ : _642.5 .

Compound 6a (700 mg, 1.04 mmol), tetrazole (26.2 mg, 0.37 mmol), and N-methylimidazole were dissolved in CH ₃ CN (10 mL), and 3A molecular sieves (500 mg) were added. Under argon protection, after stirring at room temperature for 5 min, compound 7 (470.4 mg, 1.56 mmol) was added. After stirring at room temperature for 1 h, the molecular sieves were filtered off and the solid was rinsed three times with DCM (50 mL). The filtrate was washed with saturated aqueous NaHCO ₃ (50 mL×4) and H ₂ O (50 mL×4), respectively. The organic phase was concentrated at 30°C. The obtained residue was purified by reverse column (C ¹⁸ , H ₂ O + acetonitrile, 90% acetonitrile), and the target compound 1-4A (600 mg, yield 66.1%) was obtained after lyophilization. MS m/z: C ₄₇ H ₅₂ N ₇ O ₆ P, [M-cyanoethyl-diisopropyl+OH] ⁻ : 704.3.

Compound 6b (1.3 g, 2.03 mmol), tetrazole (71.0 mg, 1.01 mmol), and N-methylimidazole (33.3 mg, 0.41 mmol) were dissolved in CH ₃ CN (20 mL), and 3A molecular sieves (700 mg) were added. Under argon protection, after stirring at room temperature for 5 min, compound 7 (0.92 g, 3.04 mmol) was added. After stirring at room temperature for 1 h, the molecular sieves were filtered off and the solid was rinsed three times with DCM (50 mL). The filtrate was washed with saturated aqueous NaHCO ₃ (50 mL×4) and H ₂ O (50 mL×4), respectively. The organic phase was concentrated at 30°C. The obtained residue was purified by reverse column column (C ¹⁸ , H ₂ O + acetonitrile, acetonitrile 90%) to obtain the target compound 1-4b (1.4 g, yield 82.1%) after lyophilization. MS m/z: _C47H52N7O6P , _[ M _- cyanoethyl-diisopropyl] ^- : _704.3 .

8.5 Synthesis of Compounds 1-5

Compound 1A (6.73 g, 28.14 mmol) was dissolved in dry DMF (80 mL), and NaH (60%, 1.24 g, 30.95 mmol) was added slowly under argon. After the mixture was stirred at room temperature for 30 min, the reaction solution was added to a solution containing Pd(PPh ₃ ) ₄ (1.95 g, 1.69 mmol), PPh ₃ (0.74 g, 2.81 mmol) and compound 1 (4.0 g, 28.14 mmol) in THF (60 mL). After the reaction was stirred at 55°C for 16 h, the solid was filtered off and washed three times with DCM (60 mL). The filtrate was concentrated and the resulting residue was purified with a forward column (the column was flushed with ethyl acetate, then ethyl acetate:methanol (12:1) to give the desired solid 2 (7 g, crude).

Compound 2 (8 g, crude) and DMTrCl (12.65 g, 37.34 mmol) were dissolved in pyridine (10 mL). After the mixture was stirred at room temperature for 16 h, it was quenched with water (80 mL) and concentrated. The obtained residue was purified by reverse column (C ¹⁸ , water+acetonitrile) and lyophilized to obtain the target compound 3 (13 g, yield 83.7%).

Compound 3 (1 g, 1.60 mmol), KHCO ₃ (0.48 g, 4.81 mmol) and ethylene glycol (0.40 g, 6.41 mmol) were dissolved in acetone (50 mL), and KMnO ₄ (40% dissolved) was slowly added at -30 °C in water, 0.67 g, 1.68 mmol). After stirring for 1 h at -30°C, the reaction was quenched with saturated aqueous sodium thiosulfate solution (30 mL). The mixture was extracted four times with DCM (30 mL). The organic phase was dried _over anhydrous _Na2SO4 , filtered and concentrated. The residue was purified by reverse column (C18, H ₂ O + acetonitrile, acetonitrile 60%) and lyophilized to give the desired product 4 (600 mg, yield 56.9%). MS m/ _z : _C38H35N5O6 , _[ M+H] ⁺ : _658.5 .

Add reactant 4 (5.0g, 7.601mmol), _NaIO4 and 1,4-dioxane/water (50mL/5mL) to a 250mL round-bottomed flask, react at room temperature for 2 hours, remove the solvent under reduced pressure, and obtain a white The solid (6.0 g) was then dissolved in methanol (50 mL), sodium borohydride (1.62 g, 38 mmol) was added, and after stirring at room temperature for 2 hours, 10% ammonium chloride solution (10 mL) was added, and the solvent was removed under reduced pressure, C18 Column chromatography (water/acetonitrile: 5%-95%) gave product P1 as colorless oil 5 (2.0 g, 3.0315 mmol, 39%), LCMS, MS+, [M+H]+: 660.

Compound 5 (1.7 g, 2.58 mmol) and DBU (0.77 mL, 5.15 mmol) were dissolved in DCM (20 mL) and BzCl (0.5 M in DCM, 0.8 mL) was added dropwise to the reaction under argon at -70°C middle. The reaction was placed at -70°C for 1 h, and the reaction was quenched with ethanol (5 mL). The quenched reaction was diluted with DCM (100 mL) and washed three times with water (30 mL), the organic phase was dried over anhydrous Na ₂ SO ₄ , filtered and concentrated. The resulting residue was purified with a forward column (DCM:EA=1:1) to give 6 (80 mg, 4.14% yield) as a white solid. MS m/ _z : _C45H41N5O7 , [M+ _H ] ⁺ : _764.5 .

Compound 4 (380 mg, 0.50 mmol), tetrazole (17.43 mg, 0.25 mmol), N-methylimidazole (8.17 mg, 0.10 mmol) were dissolved in _CH3CN (10 mL), and 3A molecular sieves (500 mg) were added. Under argon protection, after stirring at room temperature for 5 min, compound 7 (224.95 mg, 0.75 mmol) was added. After stirring at room temperature for 1 h, the molecular sieves were filtered off and the solid was rinsed three times with DCM (50 mL). The filtrate was washed with saturated aqueous NaHCO ₃ (50 mL×4) and H ₂ O (50 mL×4), respectively. The organic phase was concentrated at 30°C. The obtained residue was purified by reverse column (C ¹⁸ , H ₂ O + acetonitrile, 90% acetonitrile), and the target product 1-5 was obtained after lyophilization (330 mg, yield 68.8%). MS m/z: _C54H58N7O8P , [M _{- cyanoethyl} -diisopropyl] ^- : _826.3 .

8.6 Synthesis of compounds 1-6a

Compound 1 (10 g, 68.404 mmol), compound 2 (15 g, 62.186 mmol) and triphenylphosphine (32.62 g, 124.371 mmol) were dissolved in anhydrous THF (30 mL), DIAD (24.656 mL) was slowly added dropwise at 0 °C, 124.371 mmol). The reaction solution is at 25 degrees The reaction took 12h. LCMS showed the reaction was complete. The reaction solution was extracted with ethyl acetate (200 mL) and water (200 mL), the organic phase was dried, the filtrate was concentrated, and the obtained residue was purified with a forward column (DCM/MeOH=10/1) to obtain the target product 3 (20 g) ).

Compound 3 (20 g, 28.585 mmol) was dissolved in acetic acid (24 mL, 426.016 mmol) and H2O (12 mL) and stirred at 60°C for 1 hour. Then, the reaction solution was spin-dried, THF (12 mL) and H 2 O (12 mL) were added, and the mixture was stirred at 80° C. for 7 hours. LCMS showed the reaction was complete. The reaction solution was added with ethyl acetate (200 mL) and water (100 mL) for extraction, and solid sodium carbonate was added to the aqueous phase until a large amount of solid was precipitated in the aqueous phase. The solid was filtered, washed with water, and the filter cake was pulled dry with an oil pump to obtain the target compound 5 (9 g).

Under nitrogen protection, compound 5 (6.8 g, 18.581 mmol) was dissolved in pyridine (80 mL), TMSCl (14.250 mL, 111.489 mmol) was slowly added at 0 degrees, and stirred for 2 h. Then, Isobutyryl chloride (2.044 mL, 19.511 mmol) was added at 0 degrees, and the mixture was stirred at 25 degrees for 1 h. LCMS showed the reaction was complete. Extracted with dichloromethane (200 mL) and water (200 mL), the organic phase was dried and spin-dried, and the sample was mixed, and purified with a forward column (DCM:MeOH=10:1), passed through the column, and the peak was at 4.8%), Compound 6 (12 g) was obtained as a yellow oil.

Under nitrogen protection, compound 6 (5.5 g, 12.392 mmol) was dissolved in pyridine (30 mL), MOLECULAR SIEVE 4A 1/16 (7 g, 12.392 mmol) was added, then DMTrCl (5.04 g, 14.870 mmol) was added in portions at 0 °C mmol) solid, reacted at 25°C for 2 h. TLC (PE:EtOAc=1:1, Rf=0.69) showed that the reaction was complete. The reaction solution and TJN200879-040-P1 were combined and processed together. The reaction solution was extracted with ethyl acetate (200 mL) and water (200 mL), the organic phase was dried and spin-dried, and the mixed sample was purified with a forward column (PE: EtOAc was passed through the column, peak at 84%) to obtain a yellow oil as compound 7 (12 g).

Compound 7 (12 g, 15.389 mmol) was dissolved in EtOAc (140 mL), wet palladium carbon Pd/C (7 g, 15.389 mmol) was added, and the reaction solution was reacted at 25 degrees under hydrogen (15 Psi) for 2 hours. TLC (PE:EtOAc=0:1, Rf=0.09) showed that the reaction was complete. The reaction solution was filtered, the filter cake was washed three times with ethyl acetate (30 mL), and the filtrate was collected. After the filtrate was spin-dried, 50 mL of dichloromethane and 2 mL of triethylamine were added, and the sample was mixed and purified with a forward column (DCM:MeOH=10:1 passed through the column, peak at 0.5%) to obtain 9g (yellow foamy solid) . racemize the resulting The compound was resolved by SFC to obtain the target compound 7A(-)(3.9g) and the target compound 7B(+)(3.8g).

Compound 7A(-) (3.30 g, 5.40 mmol), tetrazole (190 mg, 2.70 mmol), 1-methylimidazole (90 mg, 1.10 mmol), 3A molecular sieve (500 mg) were dissolved in 30 mL of acetonitrile, at room temperature Compound 8 (2.50 g, 8.10 mmol) was added and stirred at room temperature for 2 h. After the reaction was completed, the molecular sieves were filtered off, DCM (150 mL) was added, washed with saturated aqueous sodium bicarbonate solution (30 mL*3), and then with saturated brine (30 mL), the filtrate was spin-dried and subjected to reverse-phase preparative HPLC (C18, condition : 5-100% (A: water, B: CH3CN), flow rate: 70 mL/min), 1-6a (2.9 g, 66%) was obtained after lyophilization. MS m/z: C43H55N7O7P[M+H]+, theory: 812.38, found: 812.5. 1H NMR (400MHz, Acetonitrile-d3) δ 7.56, 7.54 (2s, 1H), 7.36-7.27 (m, 2H), 7.24 -7.21(m,7H),6.83-6.80(m,4H),4.12-4.10(m,2H),3.75-3.68(m,10H),3.20-2.80(m,2H),2.68-2.54(m, 4H), 1.22-1.04 (m, 18H).

8.7 Synthesis of compounds 1-7a

Under nitrogen protection, compound 1 (5 g, 23.1272 mmol), compound 2 (6.76 g, 46.254 mmol) and triphenylphosphine (7.28 g, 27.753 mmol) were dissolved in 30 mL of dioxane, and slowly added dropwise at 0 °C DEAD (5.502 mL, 27.753 mmol). After the dropwise addition was completed, the reaction was slowly heated to 25 °C and continued for 1 h. 100 mL of H2O and 100 mL of EtOAc were added to the reaction solution for extraction, the organic phases were combined, dried, filtered and concentrated, and the samples were mixed and passed through a column, and purified with a forward column (PE: EtOAc). = 1:1 through the column to obtain the target product (4g).

Compound 3 (3.3 g) was dissolved in HOAc (16 mL) and H 2 O (4 mL) and heated in an oil bath at 60° C. for 0.5 h. The residue obtained by rotating the reaction solution was purified with a forward column (PE:EtOAc=0:1 through the column) to obtain the target product 4 (3 g).

Compound 4 (3 g, 8.873 mmol) was dissolved in 5 mL of pyridine, and a solution of DMTrCl (3.91 g, 11.535 mmol) in 10 mL of pyridine was slowly added dropwise at 0°C under nitrogen protection. After the dropwise addition, the reaction temperature was raised to 25°C and the reaction was continued for 1 h. 50 mL of water and 100 mL of ethyl acetate were added to the reaction solution for extraction. The aqueous phase was extracted three times with 100 mL of ethyl acetate, and the organic phase Combine, dry, filter, concentrate and purify with forward column (with PE:EtOAc=2:1). The target product 5 (4 g) was obtained.

Compound 5 (4 g, 5.769 mmol) was dissolved in methanol (10 mL), saturated NH3 methanol solution (40 mL) was added, and the reaction was carried out at 0 °C for 6 h. 1) 2.4 g of the racemic compound was obtained by SFC resolution to obtain the target product 6A (750 mg, 100% purity) and the target product 6B (400 mg, 99.16% purity).

Compound 6A(-) (700 mg, 1.40 mmol), tetrazole (50 mg, 0.70 mmol), 1-methylimidazole (23 mg, 0.28 mmol), 3A molecular sieve (500 mg) were dissolved in 10 mL of acetonitrile, and added at room temperature Compound 7 (630 mg, 2.10 mmol) was stirred at room temperature for 2 h. After the reaction, the molecular sieves were filtered off, DCM (50 mL) was added, saturated aqueous sodium bicarbonate solution (10 mL*3) was washed, and then saturated brine (20 mL) was washed, the filtrate was spin-dried and subjected to reverse-phase preparative HPLC (C18, condition : 5-100% (A: water, B: CH3CN), flow rate: 70 mL/min), 1-7a (700 mg, 72%) was obtained after lyophilization. MS m/z: C38H47N4O7PNa[M+Na]+, theoretical: 725.32, found: 725.5.

8.8 Synthesis of compounds 1-8a

Compound 1 (8.5 g, 76.508 mmol) and compound 2 (30.64 g, 91.809 mmol) were dissolved in DMF (150 mL), CS2CO3 (29.91 g, 91.809 mmol) was added, and the reaction was carried out at 90° C. for 12 h under nitrogen protection. The reaction was complete by LCMS. The reaction solution was filtered, the oil pump was spin-dried, and the column was separated and purified (80 g, DCM/MeOH=10/1~5/1) to obtain the target product 3 (13.5 g, 80% purity).

Compound 3 (10.5 g, 35.105 mmol) was dissolved in pyridine (65 mL) and CH3CN (65 mL), BzCl (4.894 mL, 42.126 mmol) was added dropwise to the solution, and the reaction was carried out at 25° C. for 2 h. The reaction of most of the raw materials was detected by LCMS, quenched by adding H2O (100 mL), extracted with EtOAc (100 mL × 3), dried and spin-dried, and purified by column separation (combined TJN200872-101) (80 g, PE/EtOAc=10/1~0 /1, DCM/MeOH=10/1) to obtain the target product 4 (14 g, 90% purity).

Compound 4 (14 g, 36.694 mmol) was dissolved in HOAc (56 mL, 314.796 mmol) and H 2 O (14 mL) and reacted at 60° C. for 2 h. LCMS showed that the reaction was complete. Oil pump concentration, forward column separation (40g, DCM/MeOH=1/0~5/1) to obtain the target product 5 (8.4g, 90% purity & 2.4g, 80% purity).

Compound 5 (7.4 g, 21.957 mmol), DMAP (0.54 g, 4.391 mmol), MOLECULAR SIEVE 4A (11.1 g, 2.967 mmol) were dissolved in pyridine (60 mL), stirred under ice bath for 10 min, and then DMTrCl (8.93 g, 26.348 mmol), the reaction was stirred for 1.8 h. LCMS detected about 19% of the raw material remaining, about 60% of the target MS. Combined (TJN200872-105 & 106) and purified together. H2O (50 mL) was added to the reaction solution, extracted with DCM (50 mL×3), dried, spin-dried, and separated by column (120 g, PE/(EA:DCM:TEA=1:1:0.05) =1/0~0/1 to DCM/MeOH=10/1) to obtain yellow solid compound 6 (11 g, 89% purity, TJN200872-105&106&107), and the raw material (3.0 g, 70% purity) was recovered.

Compound 6 (15g, 22.041mmol) was isolated by SFC (DAICEL CHIRALPAK AD (250mm*50mm, 10um); 0.1% NH3H2O EtOH, B: 45%-45%; 200ml/min) to obtain the target product 6A (5.33g, 94.29%) purity), the target product 6B (6.14 g, 97.91% purity), 1.0 g of compound 6 was recovered.

Compound 6B(-) (5.4 g, 8.92 mmol), tetrazole (312 mg, 4.46 mmol), 1-methylimidazole (146 mg, 1.78 mmol), 3A molecular sieve (500 mg) were dissolved in 40 mL of acetonitrile, at room temperature Compound 7 (4 g, 13.4 mmol) was added and stirred at room temperature for 2 h. After the reaction was completed, the molecular sieves were filtered off, DCM (200 mL) was added, saturated aqueous sodium bicarbonate solution (30 mL*3) was washed, and then saturated brine (50 mL) was washed, the filtrate was spin-dried and subjected to reverse phase preparative HPLC (C18, condition : 5-100% (A: water, B: CH3CN), flow rate: 70 mL/min), 1-8a (5.8 g, 80%) was obtained after lyophilization. MS m/z: C45H51N5O7P, [M+H]+, theory: 804.36, found: 804.4.

Example 9 psiCHECK activity screening experiment

The siRNA sample synthesis was described above, and the plasmid was obtained from Sangon Bioengineering (Shanghai) Co., Ltd. Using psiCHECK activity screening, Dual-Glo® Luciferase Assay System detection.

psiCHECK activity screening experimental steps: cell plating, preparation of transfection complex (Lipofectamine 2000: 0.2 μL/well; Plasmid: 0.05 μL/well), cell transfection, and diluted to different concentrations as working solution according to different experimental requirements in Table 11 Spare, ready to use.

Table 11 Multi-concentration point siRNA dilution scheme

24h after transfection, the assay was performed according to the experimental protocol of the Dual-Glo® Luciferase Assay System detection kit. Dual-Glo® Luciferase Assay System detection, using Dual luciferase reporter gene assay kit (Promega, cat.E2940), read Firfly chemiluminescence value and Renilla chemiluminescence value;

Calculate the relative value Ratio=Ren/Fir; calculate the inhibition rate: 1-(Ratio+siRNA/Ratioreporter only)*100%=inhibition rate (%); in the present disclosure, the remaining mRNA expression ratio=100%-inhibition rate (% ).

Example 10 On-target and off-target activity experiments of siRNAs containing different chemical modifications

Using the compounds in Table 12, and verifying the on-target activity and off-target activity of each siRNA by the method of Example 9, each siRNA uses the same sense strand, and the 7th position at the 5' end of the antisense strand is the following modified nucleosides. acid/chemical modification,

Among them, we define hmpNA from nucleotides synthesized from 2-hydroxymethyl-1,3-propanediol as the starting material;

TJ-NA019(A) is obtained by solid-phase synthesis of nucleoside phosphoramidite monomers 1-2 in Example 8.1;

TJ-NA020(A) is obtained by solid-phase synthesis of nucleoside phosphoramidite monomers 1-3 in Example 8.1;

TJ-NA026(A) is obtained by solid-phase synthesis of nucleoside phosphoramidite monomer 1-4a in Example 8.1;

TJ-NA027(A) is obtained by solid-phase synthesis of nucleoside phosphoramidite monomer 1-4b in Example 8.1;

TJ-NA038(A) is obtained by solid-phase synthesis of nucleoside phosphoramidite monomers 1-5 in Example 8.1;

(+)hmpNA(A) is obtained by solid-phase synthesis of nucleoside phosphoramidite monomer 1-1b in Example 8.1, and its absolute configuration is ( S )-hmpNA(A);

(-)hmpNA(A) is obtained by solid-phase synthesis of nucleoside phosphoramidite monomer 1-1a in Example 8.1, and its absolute configuration is ( R )-hmpNA(A);

Similarly, substituting the base species of hmpNA, the following structure was obtained by solid phase synthesis and the absolute configuration was confirmed:

(+)hmpNA(G), the absolute configuration is ( S )-hmpNA(G);

(-)hmpNA(G), the absolute configuration is ( R )-hmpNA(G);

(+)hmpNA(C), the absolute configuration is ( S )-hmpNA(C);

(-)hmpNA(C), the absolute configuration is ( R )-hmpNA(C);

(+)hmpNA(U), the absolute configuration is ( R )-hmpNA(U);

(-)hmpNA(U), the absolute configuration is ( S )-hmpNA(U).

( S )-hmpNA(G), ( R )-hmpNA(G), ( S )-hmpNA(C), ( R )-hmpNA(C), ( S )-hmpNA(U) and ( R )-hmpNA The absolute configuration of (U) was confirmed by X-Ray diffraction of its intermediates or derivatives.

The structure of the intermediate or derivative is:

TJ-NA067: The detected crystal is a colorless block (0.30×0.10×0.04mm3), belonging to the monoclinic system P21 space group. Unit cell parameters a=16.0496(5)Å, b=4.86260(10)Å, c=16.4686(5)Å, α =90°, β =118.015(4)°, γ =90°,V=1134.65(7 )Å3,Z=4. The calculated density Dc=1.389g/cm3, the number of electrons in the unit cell F (000)=504.0, the linear absorption coefficient of the unit cell μ (Cu Kα)=0.840mm-1, and the diffraction experiment temperature T=150.00(11)K.

6A(+): The detected crystal is a colorless block (0.30×0.20×0.10mm3), belonging to the monoclinic system P21 space group. Unit cell parameters a=22.6688(7)Å, b=8.5595(2)Å, c=23.3578(5)Å, α =90°, β =113.876(3)°, γ =90°,V=4144.3(2 )Å3,Z=2. The calculated density Dc=0.999g/cm3, the number of electrons in the unit cell F (000)=1318.0, the linear absorption coefficient of the unit cell μ (Cu Kα)=0.570mm-1, and the diffraction experiment temperature T=100.01(18)K.

TJ-NA048: The detected crystal is colorless needle-like (0.30×0.04×0.04mm3), belonging to the monoclinic system P1 space group. Unit cell parameters a=7.6165(4)Å, b=11.3423(5)Å, c=17.3991(8)Å, α =85.007(4)°, β =88.052(4)°, γ =70.532(4)° , V=1411.75(12)Å3, Z=2. The calculated density Dc=1.366g/cm3, the number of electrons in the unit cell F (000)=620.0, the linear absorption coefficient of the unit cell μ (Cu Kα)=0.856mm-1, and the diffraction experiment temperature T=150.00(13)K.

TJ-NA092: The detection crystal is colorless prism (0.30×0.10×0.10mm3), belonging to the P1 space group of the triclinic system. Unit cell parameters a=5.17960(10)Å, b=8.0667(2)Å, c=12.4077(2)Å, α =93.146(2)°, β =101.266(2)°, γ =96.134(2)° , V=503.993(18)Å3, Z=2. The calculated density Dc=1.412g/cm3, the number of electrons in the unit cell F (000)=228.0, the linear absorption coefficient of the unit cell μ (Cu Kα)=0.945mm-1, and the diffraction experiment temperature T=100.00(10)K.

Table 12 Sequence and modification information of siRNA targeting HBV-S

See Table 13 for the results of the on-target activity test and Table 14 for the results of the off-target activity test. The compounds in the current experiment all show activities equivalent to or slightly better than the parent sequence in the test sequence, indicating that the modification has no effect on the on-target activity, with the most active compounds Preferred are siRNAs comprising GNA/Abasic/Id, TJ-NA019 (A) , TJ-NA020 (A) , TJ-NA026 (A) , (+)hmpNA(A) and (-)hmpNA(A) , respectively. In addition, the parent sequence has obvious off-target activity, and all modifications showed a significant effect of inhibiting off-target activity, especially for TJ-NA027 (A) , (+)hmpNA(A) and (-)hmpNA(A), respectively siRNA, no off-target activity was observed.

Example 11 Sequence-dependent experiments of siRNAs containing different chemical modifications

Abasic modifications are known to be siRNA sequence dependent, so the inventors tested experimental compounds of the present disclosure on a number of different sequences. Using siRNA targeting four different genes (ANGPTL3, HBV-S, HBV-X, TTR) mRNA (sequences are shown in Table 15), using compounds TJ-NA020 (A), TJ-NA027 ( A) , (+)hmpNA(A) , (-)hmpNA(A) and GNA (A) as a control, Id compound modifies the 7th position of the 5' end of the AS chain (the sequence is shown in Table 16), and then mixes with the parent Sequence comparison of on-target and off-target activity.

The results of the on-target activity experiments are shown in Table 17. GNA (A) showed obvious sequence dependence, and the on-target activities of different sequences were significantly different. The experimental compounds of the present disclosure do not show obvious sequence dependence, and are more universally applicable.

The off-target activity experimental results of siRNA2 and siRNA3 are shown in Table 18. It can be seen that, compared with the parent sequence, the experimental compounds of the present disclosure significantly reduce the off-target activity of siRNA.

Example 12 Galactosamine compound 1-t linked to solid support

The synthetic route is as follows:

1) Synthetic route of compound 1-g

2) Synthetic route of compound 1-h

3) Synthetic route of compound 1-I

4) Synthesis of compound 1-q

5) Synthesis of aminogalactose compound 1-t linked to solid support

step one

Raw material 1-a (297g, 763mmol,) and raw material 1-b (160g, 636mmol) were dissolved in 960ml DCE, at 15°C, Sc(OTf) ₃ (15.6g, 31.8mmol, ) was added, and the reaction was elevated The temperature reached 85°C, and the reaction was stirred for 2 h. After the reaction was completed, 1.5 L of saturated NaHCO ₃ was added to stop the reaction. The organic phase was separated and washed with 1.5 L of saturated brine. The organic phase was dried over anhydrous Na ₂ SO ₄ , and the filtered solution was reduced After distillation under pressure, it was purified by silica gel column chromatography (petroleum ether:ethyl acetate 5:1-0:1) to obtain a pale yellow oily product 1-c (328 g, 544 mmol, yield 85.5%, purity 96.4%) ).

¹ HNMR: (400MHz, CDCl ₃ )δ 7.44-7.29(m, 5H), 5.83(d, J=8.8Hz, 1H), 5.40-5.23(m, 2H), 5.18-5.06(m, 2H), 4.86 (s,1H),4.66(d,J=8.4Hz,1H),4.21-4.07(m,2H),4.04-3.77(m,3H),3.51-3.45(m,1H),3.31-3.11(m ,2H),2.18(d,J=2.0Hz,1H),2.14(s,3H),2.06(s,3H),2.03-1.99(m,3H),1.95(s,3H),1.64-1.46( m, 4H), 1.43-1.29 (m, 4H).

^{MS ,} _C28H40N2O11 , found M _{+ 581.3} .

Step 2

The compound obtained in step 1 was divided into two parts in parallel: each reaction contained compound 1-c (72.0 g, 124 mmol) was added to 432 mL of THF, and Pd/C (20.0 g, 10% purity) was added under argon protection, and then added TFA (14.1 g, 124 mmol, 9.18 mL), passed hydrogen into the reaction solution, kept the gas pressure at 30 Psi, heated to 30 °C and stirred the reaction for 16 h. After the reaction was complete, the two parallel reactions were combined, filtered, and the filtrate was concentrated under reduced pressure. The residue was diluted with dichloromethane and concentrated under reduced pressure repeatedly three times. After drying under reduced pressure, the target compound 1-d (139 g) was obtained.

¹ HNMR(400MHz, DMSO- d ₆ )δ 7.85(d, J =9.2Hz,1H),7.74(s,3H),5.21(d, J =3.6Hz,1H),4.97(dd, J =2.8, 10.8Hz, 1H), 4.48(d, J =8.8Hz, 1H), 4.06-3.98(m, 3H), 3.93-3.82(m, 1H), 3.73-3.68(m, 1H), 3.63-3.56(m ,1H),3.43-3.38(m,1H),2.82-2.71(m,2H),2.13-2.09(m,3H),2.01-1.97(m,3H),1.91-1.87(m,3H),1.77 (s, 3H), 1.76-1.73 (m, 1H), 1.52-1.44 (m, 4H), 1.28 (s, 4H).

Step 3

Compound 1-d (139 g, 247 mmol) and compound 1-e (75.3 g, 223 mmol) were added to DMF solution (834 mL), and then DIPEA (41.6 g, 322 mmol, 56.1 mL), HOBt (36.8 g, 272mmol) and EDCI (52.2g, 272mmol), keep 15 ℃ of stirring reaction for 16h, after the reaction is completed, the reaction solution is diluted with dichloromethane ((400mL), then with saturated ammonium chloride solution (1L), saturated NaHCO ₃ ( 1.00L), washed with saturated brine in turn, separated the organic phase and dried with anhydrous sodium sulfate, filtered and distilled under reduced pressure to remove the solvent, and the residue was purified by silica gel column chromatography (petroleum ether: ethyl acetate = 5: 1-0: 1) to obtain the target compound 1-f (108 g, yield 56.8%).

¹ HNMR(40(400MHz, _DMSO - d6 )δ 7.89-7.78(m,2H),7.41-7.27(m,6H),5.21(d, J =3.2Hz,1H),5.08-4.92(m,3H) ), 4.48(d, J =8.4Hz, 1H), 4.07-3.99(m, 3H), 3.97-3.81(m, 2H), 3.75-3.64(m, 1H), 3.42-3.37(m, 1H), 3.13-2.93(m, 2H), 2.20(t, J =8.0Hz, 2H), 2.10(s, 3H), 1.99(s, 3H), 1.89(s, 3H), 1.87-1.79(m, 1H) ,1.76(s,3H),1.74-1.64(m,1H),1.48-1.41(m,2H),1.38(s,12H),1.29-1.20(m,4H),1.19-1.14(m,1H) .

_MS , _C37H55N3O14 , found _M ⁺ _766.4 .

Step 4

The above obtained compound 1-f was divided into two parts in parallel: each reaction contained compound 6 (47.0 g, 61.3 mmol) was added to 280 mL of THF, Pd/C (15.0 g, 10% purity) was added under argon protection, and then TFA (7.00 g, 61.3 mmol, 4.54 mL) was added, and hydrogen was passed into the reaction solution, keeping the gas pressure at 30 Psi, heating to 30° C. and stirring the reaction for 16 h. After the reaction was complete, the two parallel reactions were combined, filtered, and the filtrate was concentrated under reduced pressure. The residue was diluted with dichloromethane and concentrated under reduced pressure repeatedly three times. After drying under reduced pressure, the target compound 1-g (94.0 g, crude) was obtained.

¹ HNMR(400MHz,DMSO-d6)δ 8.38(s,1H),8.10(s,3H),7.83(d,J=9.2Hz,1H),5.21(d,J=3.2Hz,1H),4.96( dd,J=3.6,11.2Hz,1H),4.47(d,J=8.4Hz,1H),4.06-3.98(m,3H),3.92-3.82(m,1H),3.75-3.67(m,2H) ,3.60(s,1H),3.43-3.37(m,1H),3.18-3.04(m,2H),2.30-2.24(m,2H),2.10(s,3H),2.00(s,3H),1.95 -1.90(m, 2H), 1.89(s, 3H), 1.78-1.75(m, 3H), 1.49-1.41(m, 3H), 1.40(s, 9H), 1.26(s, 4H).

Step 5

The compound 1-f obtained above was divided into two parts in parallel: each reaction contained compound 1-f (46.0 g, 60 mmol) added to HCl-EtOAc (2.00 M, 276 mL), and the reaction was stirred at 15 °C for 16 h. After completion of the reaction, the two reaction solutions were combined, concentrated by distillation under reduced pressure, the residue was diluted with dichloromethane and concentrated under reduced pressure was repeated three times. After drying under reduced pressure, light red compound 1-h (91.0 g, crude product) was obtained.

¹ HNMR (400MHz, DMSO- d ₆ )δ 7.91-7.80(m, 2H), 7.42-7.26(m, 6H), 5.21(d, J =3.2Hz, 1H), 5.07-4.92(m, 4H), 4.48(d, J =8.4Hz, 1H), 4.06-3.98(m, 3H), 3.98-3.82(m, 3H), 3.73-3.65(m, 1H), 3.44-3.35(m, 1H), 3.12- 2.94(m,2H),2.22(t,J=8.0Hz,2H),2.10(s,3H),2.01-1.97(m,4H),1.94-1.90(m,1H),1.89(s,3H) , 1.87-1.79(m, 2H), 1.76(s, 3H), 1.74-1.67(m, 1H), 1.49-1.40(m, 2H), 1.40-1.32(m, 2H), 1.24(d, J = 4.0Hz, 4H), 1.19-1.13 (m, 1H).

_MS , _C33H47N3O14 , found _M ⁺ _710.3 .

Step 6

Two reactions were performed in parallel: each reaction containing compound 1-g (45.0 g, 60.3 mmol) and compound 1-h (38.5 g, 54.3 mmol) was added to 270 mL of DMF followed by DIPEA (10.1 g, 78.4 mmol) at 0°C mmol, 13.6 mL) followed by HOBt (8.97 g, 66.3 mmol) and EDCI (12.7 g, 66.3 mmol). The reaction was stirred at 15°C for 16h. After the completion of the reaction, the two reaction solutions were combined, diluted with 300 mL of DCM, washed with saturated ammonium chloride (800 mL), saturated NaHCO ₃ (800 mL) and saturated brine (800 mL) in sequence, and the organic phase was dried over anhydrous Na ₂ SO ₄ . After filtration, concentrated by evaporation under pressure, the residue was purified by silica gel column chromatography (petroleum ether:ethyl acetate=5:1-0:1) to obtain white compound 1-i (66.0 g, 47.4 mmol, the yield was 39.3% with a purity of 95.1%).

¹ HNMR (400MHz, DMSO- d ₆ )δ 7.96-7.78(m, 5H), 7.41-7.25(m, 6H), 5.21(d, J =3.6Hz, 2H), 5.05-4.92(m, 4H), 4.48(d, J =8.8Hz, 2H), 4.22-4.12(m, 1H), 4.02(s, 6H), 3.94-3.80(m, 3H), 3.74-3.64(m, 2H), 3.45-3.35( m,2H),3.11-2.92(m,4H),2.20-2.12(m,4H),2.10(s,6H),1.99(s,6H),1.89(s,6H),1.82-1.79(m, 2H), 1.76(s, 6H), 1.74-1.63(m, 2H), 1.44(d, J =6.0Hz, 4H), 1.37(s, 12H), 1.24(s, 9H).

_MS : _C62H94N6O25 , found m/ _{z 1323.8} .

Step seven

It was divided into 11 reactions: Compound 1-i (5.00 g, 3.78 mmol) and toluene (300 mL) were added to each reaction, and silica gel (45.0 g) was added. The reaction was stirred at 100 °C for 40 h, and the 11 reaction mixtures were combined after completion of the reaction. After the solvent was distilled off under reduced pressure, isopropanol and dichloromethane were added to the residue, followed by stirring for 20 min. The insolubles were removed by filtration, and the filter cake was washed with isopropanol until no product was eluted. The obtained solution was desolventized and dried to obtain a pale yellow compound 1-j (43.2 g, 34.0 mmol, 82.0% yield).

¹ HNMR: (400MHz, DMSO-d6)δ 8.01(d, J=7.6Hz, 1H), 7.93-7.79(m, 2H), 7.39-7.27(m, 3H), 5.21(d, J=3.2Hz, 1H), 5.06-4.91(m, 2H), 4.48(d, J=8.0Hz, 1H), 4.07-3.97(m, 3H), 3.94-3.82(m, 2H), 3.73-3.65(m, 1H) ,3.45-3.36(m,2H),3.10-2.94(m,2H),2.15(d,J=7.6Hz,2H),2.10(s,3H),1.99(s,3H),1.89(s,3H ),1.86-1.79(m,1H),1.77(s,3H),1.74-1.65(m,1H),1.44(s,2H),1.37(d,J=5.2Hz,2H),1.24(s, 4H).

_MS : _C58H86N6O25 , found m/ _z = _1267.8 .

Step 8

This step was divided into two reactions in parallel: each reaction contained compound 1-d (11.8 g, 21.0 mmol) and compound 1-j (21.3 g, 16.8 mmol) were added to 70 mL of DMF, followed by DIPEA (3.54 g at 0°C) g, 27.3 mmol, 4.77 mL) followed by HOBt (3.13 g, 23.1 mmol) and EDCI (4.44 g, 23.1 mmol). The reaction was stirred at 15°C for 16h. After the completion of the reaction, the two reaction solutions were combined, diluted with 500 mL of DCM, washed with saturated ammonium chloride (1.5 L), saturated NaHCO ₃ (1.5 mL) and saturated brine (1.5 mL) in sequence, and the organic phase was washed with anhydrous Na ₂ SO ₄ Dry. After filtration, concentrated by evaporation under pressure, the residue was purified by silica gel column chromatography (dichloromethane:methanol=50:1-10:1) to obtain pale yellow compound 1-k (54.0 g, 31.8 mmol, the yield was 75.6%).

¹ HNMR(400MHz, DMSO- d ₆ )δ 7.91(d,J=7.6Hz,1H),7.87-7.78(m,5H),7.73(t,J=5.2Hz,1H),7.42-7.24(m, 6H), 5.21(d, J=3.6Hz, 3H), 5.06-4.92(m, 5H), 4.48(d, J=8.4Hz, 3H), 4.19-4.09(m, 2H), 4.07-3.97(m ,10H),3.94-3.80(m,4H),3.76-3.64(m,3H),3.42-3.37(m,4H),3.08-2.94(m,6H),2.20-2.12(m,2H),2.10 (s,9H),2.08-2.01(m,2H),1.99(s,9H),1.89(s,9H),1.87-1.79(m,2H),1.77(s,9H),1.74-1.63(m , 2H), 1.44(d, J=5.6Hz, 6H), 1.40-1.31(m, 6H), 1.24(s, 13H).

_MS : _C78H118N8O33 , found m/ _z = _1696.1 .

Step nine

This step was divided into 3 reactions in parallel: Compound 1-k (17.0 g, 10.0 mmol) and THF (100 mL) were added to each reaction, Pd/C (5.0 g, 10% purity) was added under argon protection, Then TFA (1.14g, 10.0mmol, 742uL) was added, hydrogen was passed into the reaction solution, the gas pressure was kept at 15Psi, heated to 30°C and stirred for 4h. After the reaction was complete, the 3 parallel reactions were combined, filtered, and the filtrate was concentrated under reduced pressure. The residue was diluted with dichloromethane and concentrated under reduced pressure repeatedly three times. The residue was purified using preparative liquid chromatography (C18, mobile phase A 0.1% TFA-water, mobile phase B: 10-40% CAN, 20 min) to give white compound 1-1 (17.3 g, 10.2 mmol, 34.0% yield) ).

¹ HNMR: (400MHz, DMSO- d ₆ )δ 8.45(t, J =5.2Hz, 1H), 8.14(d, J =5.2Hz, 3H), 7.97(t, J =5.2Hz, 1H), 7.90- 7.77(m, 4H), 5.21(d, J =2.8Hz, 3H), 4.96(dd, J =3.2, 11.6Hz, 3H), 4.47(d, J =8.4Hz, 3H), 4.20-4.10(m ,1H),4.02(s,8H),3.87(q, J =9.6Hz,3H),3.75-3.61(m,4H),3.46-3.34(m,3H),3.21-2.93(m,6H), 2.21(s, 2H), 2.14-2.02(m, 11H), 1.99(s, 9H), 1.96-1.82(m, 12H), 1.80-1.65(m, 10H), 1.44(d, J =5.6Hz, 8H), 1.36(d, J =6.4Hz, 4H), 1.30-1.17(m, 12H)

MS: C ₇₀ H ₁₁₂ N ₈ O ₃₁ , found m/2z=781.8

Step ten

Compound 1-m (2 g, 12.64 mmol) was dissolved in pyridine (10 mL), a solution of DMTrCl (4.71 g, 13.90 mmol) in pyridine (10 mL) was added dropwise at room temperature, and the reaction was stirred at room temperature for 5 hours. After completion, quenched with methanol, concentrated under reduced pressure to obtain the crude product, purified with silica gel (petroleum ether:ethyl acetate=10:1 elution), collected the product eluent, evaporated the solvent under reduced pressure to obtain 4g of compound 1-n .

MS m/ _z : _C29H32O5 , [M+H] ⁺ found: _461.3 .

step eleven

Compound 1-n (2 g, 4.34 mmol), N,N-diisopropylethylamine (DIEA, 1.43 mL, 8.68 mmol) and HATU (2.47 g, 6.51 mmol) were dissolved in DMF (10 mL) at room temperature A solution of compound 1-o in DMF (5 mL) was added and the reaction was stirred at room temperature for 8 hours. After the reaction was completed, water was added to quench, the aqueous phase was extracted with ethyl acetate, the combined organic phases were washed first with water, then with saturated brine (20 mL), and then the solvent was evaporated to dryness under reduced pressure. Green ODS 150*30mm*5um, condition: 25-80% (A: water 0.075% NH ₃ .H ₂ O, B: CH ₃ CN), flow rate: 55mL/min), after lyophilization, 2.4g of compound 1- p .

MS m/ _z : _C33H39NO7 , [M+H] ⁺ found: _562.4 .

Step 12

Compound 1-p (2.4 g, 4.27 mmol) was dissolved in 15 mL of a mixed solution of methanol and water (2:1), LiOH (0.36 g, 8.54 mmol) was added at room temperature and stirred overnight. After the reaction was completed, the solvent was evaporated to dryness under reduced pressure, preparative HPLC (column: Boston Green ODS 150*30mm*5um, conditions: 25-75% (A: water 0.075% NH ₃ .H ₂ O, B: CH _{3 )} CN), flow rate: 55 mL/min), 2 g of compound 1-q was obtained after lyophilization.

MS m/ _z : _C32H37NO7 , [M+H] ⁺ found: _548.6 .

Step Thirteen

Compound 1-q (0.37 g, 0.69 mmol), DIEA (0.19 mL, 1.15 mmol) and HATU (0.32 g, 0.86 mmol) were dissolved in 2 mL of DMF, and compound 1-1 (0.9 g, 0.69 mmol) was added at room temperature mmol) in DMF (2 mL) and stirred at room temperature overnight. After the reaction was completed, the reaction solution was diluted with dichloromethane (10 mL), washed with saturated NaHCO ₃ (20 mL) and saturated brine (20 mL) successively, the organic phase was dried over anhydrous Na ₂ SO ₄ , filtered and concentrated under reduced pressure. Purified by reverse phase preparative HPLC (column: Boston Green ODS 150*30mm*5um, conditions: 25-65% ( _A : water 0.075% NH3.H2O, B: _{CH3CN )} , flow rate: 45 mL/min) , 0.5 g of compound 1-r was obtained after lyophilization.

MS m/z: _{C102H147N9O37} , [ _MH ] ⁺ found: _{2088.5 .}

Step Fourteen

Compound 1-r (300 mg, 0.14 mmol) and succinic anhydride (28.70 mg, 0.28 mmol) were dissolved in tetrahydrofuran, DMAP (3.50 mg, 0.028 mmol) was added to the reaction solution and stirred at 40°C overnight. After the reaction was completed, methanol (18.8 mg) was added, and the reaction was stirred for 10 min, then the reaction solution was diluted with dichloromethane (3 mL) and washed twice with saturated NaHCO 3 (5 mL). The organic phase was concentrated to dryness under reduced pressure, preparative HPLC by reverse phase (column: Boston Green ODS 150*30mm*5um, conditions: 25-65% ( _A : water 0.075% NH3.H2O, B: _{CH3CN )} ), flow rate: 35 mL/min), purified, and obtained 140 mg of compound 1-s after lyophilization.

MS m/ _z : _{C106H151N9O40} , [ _MH ] ⁺ found: _2189.4 .

Step fifteen

The compound 1-r (140mg, 64ummol) obtained in the previous step was added to acetonitrile (5mL), then HBTU (48.7mg, 128umol) was added, the surface amine group-modified solid phase support (CPG-NH2, 2.3g) was added, DIEA (41.5mg, 320umol, 55uL) was kept at 30°C for 16h shaking reaction. After the reaction was completed, it was filtered and washed sequentially with methanol (8 mL x 4), dichloromethane (8 mL x 4). The solid was continuously added to pyridine:acetic anhydride (v:v=4:1, 10.0 mL), and the reaction was continued to be kept at 30°C for 16h with shaking. After the reaction was completed, it was filtered and washed sequentially with methanol (8 mL x 4), dichloromethane (8 mL x 4). 2.1 g of compound 1-t attached to a solid support was obtained.

Example 13 Galactosamine compound 2-e linked to solid support

The synthetic route is as follows:

1) Synthesis of compound 2-b

2) Synthesis of compound 2-e

step one

Compound 2-a (1.00 g, 2.37 mmol) was added to THF (7.5 mL) and H ₂ O (7.5 mL), then LiOH.H ₂ O (109 mg, 2.60 mmol) was added, and the reaction was stirred at 16° C. for 16 h. After the reaction was completed, the solvent was evaporated under reduced pressure, and the residue was further lyophilized to obtain a white compound 2-b (960 mg, 2.32 mmol, 97.8% yield).

¹ HNMR: (400MHz, DMSO-d6)δ 7.44(d,J=8.4Hz,2H),7.34-7.23(m,6H),7.22-7.15(m,1H),6.86(d,J=8.0Hz, 4H),3.73(s,6H),3.66(d,J=6.4Hz,1H),3.32(d,J=12.0Hz,1H),3.11(dd,J=2.0,9.2Hz,1H),2.85( t,J=8.8Hz,1H).

MS m/ _z : _C24H24O6 , found m/z: _407.2 .

Step 2

Compound 1-1 (500 mg, 0.30 mmol) was added to dichloromethane (3 mL), and at 15 °C, compound 2-b (0.14 g, 0.34 mmol) was added to the reaction, and HBTU (142 mg) was added at 0 °C. , 375umol) and DIEA ((115mg, 895umol), kept at 15°C for 16h. After the reaction, the reaction solution was diluted with dichloromethane (10mL), and washed with saturated NaHCO ₃ (20mL) and saturated brine (20mL) successively , the organic phase was dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure, and the residue was purified by preparative liquid phase (column: Welch Xtimate C18 250*70mm#10um; mobile phase: [water-ACN]; B%: 40%-66%, 18 min) to obtain compound 2-c .

MS m/ _z : _C94H134N8O36 , [ _MH ]+ found: _1952.1 .

Step 3

Compound 2-c (230 mg, 0.12 mmol) and succinic anhydride (23.5 mg, 0.26 mmol) were dissolved in a dichloromethane solution (2 mL), and DMAP (43.1 mg, 0.35 mmol) was added to the reaction solution, and kept at 15 Stir at ℃ for 16h. After the reaction was completed, methanol (18.8 mg) was added, and the reaction was stirred for 10 min, then the reaction solution was diluted with dichloromethane (3 mL), and washed twice with saturated NaHCO ₃ . The reaction solution was concentrated under reduced pressure to dry to obtain compound 2-d (240 mg, crude product).

MS m/z: C106H151N9O40, [M-H]+ measured: m/2z: 2070.2

Step 4

The compound 2-d (240mg, 116ummol) obtained in the previous step was added to acetonitrile (8mL), then HBTU (88.7mg, 233umol) was added, the surface amine group-modified solid support (CPG-NH2, 4g) was added, and DIEA was added. (75.5mg, 584umol, 101uL), kept at 30°C for 16h shaking reaction. After the reaction was completed, it was filtered and washed sequentially with methanol (8 mL x 4), dichloromethane (8 mL x 4). The solid was continuously added to pyridine:acetic anhydride (v:v=4:1, 10.0 mL), and the reaction was continued to be kept at 30°C for 16h with shaking. After the reaction was completed, it was filtered and washed sequentially with methanol (8 mL x 4), dichloromethane (8 mL x 4). 3.7 g of compound 2 -e in which the target product was linked to a solid support was obtained.

Example 14 Galactosamine compound 3-n linked to solid support

The synthetic route is as follows:

1) Synthesis of compound 3-d

2) Synthesis of compound 3-g

3) Synthesis of compound 3-n

step one

Raw material 3-a (78.8 g, 202 mmol) and raw material 3-b (40 g, 168 mmol) were dissolved in DCE (250 mL), CF ₃ SO ₃ H (4.15 g, 8.43 mmol, ) was added at 15° C., and then Raise the reaction temperature to 75°C, stir the reaction for 2 h, add 1 L saturated NaHCO ₃ to stop the reaction after the reaction, separate the organic phase, wash with 1 L saturated brine, dry the organic phase over anhydrous Na ₂ SO ₄ , and filter the solution After distillation under reduced pressure, it was purified by silica gel column chromatography (petroleum ether:ethyl acetate 5:1-0:1) to obtain the target product 3-c (63.2 g, 107 mmol, 63.5% yield).

¹ H NMR: (400 MHz, CDCl ₃ ) δ 7.35-7.26 (m, 5H), 5.88 (s, 1H), 5.34-5.25 (m, 2H), 4.65 (d, J =8.4Hz, 1H), 4.16-4.13 (m,2H),3.92-3.87(m,3H),3.18-3.17(m,1H),3.15-3.14(m,2H),2.16-1.91(m,15H),1.58-1.50(m,5H) , 1.49-1.36 (m, 2H).

MS m/ _{z : C24H40N2O11} , found m/z: _567.4 .

Step 2

Compound 3-c (60.0 g, 106 mmol) obtained above was added to 360 mL of THF, Pd/C (15.0 g, 10% purity) was added under argon protection, TFA (12.1 g, 106 mmol, 7.84 mL) was added, and Pass hydrogen into the reaction solution, keep the gas pressure at 30 Psi, heat to 30° C. and stir the reaction for 16 h. After the reaction was completed, it was filtered, and the filtrate was concentrated under reduced pressure. The residue was diluted with dichloromethane and concentrated under reduced pressure for three repetitions (500 mL x 3). After drying under reduced pressure, a pale yellow compound 3-d (44 g, 102 mmol, 96.1% yield) was obtained.

Step 3

Compound 3-e (60.0 g, 447 mmol) was dissolved in DMF (300 mL), K ₂ CO ₃ (92.7 g, 671 mmol) was added, and BnBr (115 g, 671 mmol, 79.7 mL) was added dropwise at 0°C. The reaction was stirred at 25 °C for 6 h. The reaction solution was poured into crushed ice, then extracted with ethyl acetate (100 mL*6), and the organic phase was washed with water (100 mL*2) and saturated brine (100 mL*3) successively. The organic phase was dried over anhydrous sodium sulfate, and then the solvent was distilled off under reduced pressure. The residue was purified by silica gel column chromatography (petroleum ether:ethyl acetate=2:1-0:1) to obtain a white solid compound 3-f (60.3 g, 269 mmol, yield 60.1%).

¹ H NMR: (400 MHz, CDCl ₃ ) δ 7.37-7.26 (m, 5H), 5.18 (d, J=4.4 Hz, 2H), 3.95-3.90 (m, 2H), 3.75-3.71 (m, 2H), 1.08 (s, 1H).

MS m/z: _C12H16O4 , found m/z: _{223.5 .}

Step 4

Compound 3-f (50.0 g, 223 mmol) was dissolved in dichloromethane (300 mL), pyridine (73.5 g, 929 mmol, 75 mL) and p-nitrophenyl chloroformate dissolved in dichloromethane (50 mL) were added (180 g, 892 mmol), the reaction was stirred at 25 °C under nitrogen protection for 24 h. After the reaction was completed, dichloromethane (250 mL) was added to dilute, and then NaHSO ₄ solution (30 mL*3) and saturated brine (30 mL*2) were used to wash in turn, the organic phase was dried with MgSO ₄ , filtered, and the solvent was evaporated under reduced pressure. . The obtained crude product was purified by silica gel column chromatography (petroleum ether:ethyl acetate=3:1) to obtain the target compound 3-g (37.0 g, 66.7 mmol, 29.9% yield).

MS m/ _{z : C26H22N2O12} , found m/z: _553.4 .

Step 5

Compound 3-g (22.0 g, 39.7 mmol) was added to acetonitrile (120 mL), and under nitrogen protection, triethylamine (24.1 g, 238 mmol, 33, 1 mL) was added, the reaction solution was cooled to 0 °C, dropwise Compound 3-d (42.1 g, 40 mmol) dissolved in acetonitrile (120 mL) was added, the temperature was raised to 25°C, and the reaction was stirred for 1 h. After the reaction was completed, the solvent was concentrated under reduced pressure to remove the solvent, and then purified by silica gel column chromatography (petroleum ether:ethyl acetate=2:1) to obtain the target compound 3-h (37.0 g, 12.0 mmol, 30.2% yield).

MS m/ _z : _C52H76N4O24 , found m/ _z : _1141.8 .

Step 6

Compound 3-h (11.0 g, 9.64 mmol) was dissolved in ethyl acetate (60 mL), Pd/C (2.00 g, 10% purity) was added, hydrogen was passed through the reaction solution, and the gas pressure was maintained at 40 Psi and the temperature was 25 The reaction was stirred for 8h. After the reaction was completed, it was filtered, and evaporated to dryness under reduced pressure to obtain the target compound 3-i (10.0 g, 9.42 mmol, 97.7% yield).

¹ HNMR: (400MHz, DMSO- d ₆ )δ 7.79(d, J=9.2Hz, 2H), 7.10(s, 2H), 5.74(t, J=1.6Hz, 2H), 5.21(d, J=3.6 Hz,2H),4.98-4.95(m,2H),4.48(d,J=8.4Hz,2H),4.02(d,J=4.8Hz,11H),3.87-3.84(m,2H),3.69-3.67 (m,2H),3.41-3.39(m,2H),2.94-2.90(m,4H),2.10(s,5H),1.99(s,7H),1.89(s,6H),1.77(s,6H) ), 1.47-1.35(m, 8H), 1.26-1.24(m, 4H), 1.23-1.08(m, 3H).

MS m/z: C ₄₅ H ₇₀ N ₄ O ₂₄ , found m/z: 1051.4.

Step seven

Compound 3-i (5.00 g, 4.76 mmol) was added to a mixed solvent of dichloromethane (30 mL) and DMF (30 mL), compound 33 (312 mg, 2.38 mmol) was added, and HBTU (1.80 g, 4.76 mmol) was added and DIEA (615 mg, 4.76 mmol), and the reaction was stirred at 25 °C for 12 h. After the completion of the reaction, the reaction solution was poured into ethyl acetate (100 mL), then washed with saturated brine, dried over anhydrous Na ₂ SO ₄ , filtered and evaporated under reduced pressure to remove the solvent, and the residue was purified by preparative HPLC to obtain the target compound 3-k (2.1 g, 956 umol, 20.1% yield).

¹ HNMR: (400MHz, DMSO- d ₆ )δ 7.84-7.81 (m, 5H), 7.12-7.07 (m, 3H), 5.21 (d, J=3.6Hz, 4H), 4.99-4.96 (m, 4H) ,4.49(d,J=8.4Hz,4H),4.06-4.00(m,24H),3.88-3.86(m,4H),3.55-3.52(m,4H),3.49-3.43(m,4H),3.25 -3.05(m, 4H), 2.94-2.93(m, 8H), 2.11(s, 12H), 2.00(s, 16H), 1.90(s, 12H), 1.78(s, 12H), 1.46-1.44(m , 8H), 1.38-1.35 (m, 8H), 1.26-1.24 (m, 8H), 1.18-1.16 (m, 6H), 1.09-0.99 (m, 2H).

MS m/ _z : _{C96H153N11O46} , found m/ _z : _2197.5 .

Step 8

Compound 3-k (100 mg, 45.5 umol) was added to DMF (1 mL), then compound 2-b (21.1 mg, 54 umol) was added to the reaction, followed by HBTU (21.8 mg, 57.3 umol) and DIEA ((17.7 mg) , 136 umol), kept at 15 ° C for 16 h. After the reaction was completed, the reaction solution was diluted with dichloromethane (10 mL), and washed with saturated NaHCO ₃ and saturated brine in turn, the organic phase was dried over anhydrous Na ₂ SO ₄ , filtered and reduced pressure Concentrated, the residue was purified by preparative liquid phase (column: Phenomenex Gemini-NX 150*30mm*5um; mobile phase: [water-ACN]; B%: 35%-75%, 12min) to obtain compound 3-1 . MS m/z: C ₁₂₀ H ₁₇₅ N ₁₁ O ₅₁ , found: 2586.9.

Step nine

Compound 3-1 (14 mg, 5.4 umol) and succinic anhydride (1.08 mg, 10.8 umol) were dissolved in dichloromethane solution (1 mL), and DMAP (2.0 mg, 16 umol) and TEA (1.1 mg) were added to the reaction solution. , 10.8umol, 1.5uL), kept stirring at 15°C for 16h. After the reaction was completed, methanol (0.9 mg) was added, and the reaction was stirred for 10 min, then the reaction solution was diluted with dichloromethane, and washed twice with saturated NaHCO ₃ . The reaction solution was concentrated under reduced pressure to dryness to obtain compound 3-m ( 18 mg).

MS m/z: C ₁₂₄ H ₁₇₉ N ₁₁ O ⁵⁴ , found: 2687.2.

Step ten

The compound 3-m (18mg, 6.7ummol) obtained in the previous step was added to acetonitrile (3mL), then HBTU (5.1mg, 13.4umol) was added, and the surface amine group-modified solid support (CPG-NH2, 200mg) was added, DIEA (4.3 mg, 33.5 umol, 5.8 uL) was added, and the reaction was kept at 30° C. for 16 h with shaking. After the reaction was completed, it was filtered and washed sequentially with methanol (2 mL x 4), dichloromethane (2 mL x 4). The solid was added to pyridine:acetic anhydride (v:v=4:1, 2mL), and the reaction was kept at 30°C for 16h with shaking. After the reaction was completed, it was filtered and washed with methanol and dichloromethane successively. 200 mg of compound 3-n in which the target product was linked to a solid support was obtained.

Example 15 Galactosamine compound 4-c linked to solid support

The synthetic route is as follows:

1) Synthesis of compound 4-c

step one

Compound 3-k (149.5 mg, 68 ummol), DIEA (141.0 mg, 1.09 mmol), 3A molecular sieves (500 mg) and DEPBT (163.4 mg, 0.55 mmol) were dissolved in 5 mL of DCM, and compound 1-q was added at room temperature (400 mg, 0.18 mmol) and stirred at room temperature overnight. After the reaction was completed, the molecular sieves were filtered off, the filtrate was spin-dried and subjected to reverse-phase preparative HPLC (column: Boston Green ODS 150*30mm*5um, condition: 5-50% (A: water, B: CH3CN), flow rate: 45mL/ min), lyophilized to obtain compound 4-a (118 mg, 32 ummol, 62.6% yield).

MS m/z: C ₁₂₈ H ₁₈₈ N ₁₂ O ₅₂ , found [M+HCOO ⁻ ]=2770.6.

Step 2

Compound 4-a (110mg, 4.0umol), DMAP (7.4mg, 40umol), 3A molecular sieves (100mg) and succinic anhydride (11.9mg, 120umol) were dissolved in 5mL of THF, under argon protection, stirring at 40 °C 4h. After the reaction, the molecular sieves were filtered off, the filtrate was spin-dried and purified by reverse-phase preparative HPLC (column: Boston Green ODS 150*30mm*5um, condition: 5-50% (A: water, B: CH ₃ CN), flow rate : 45 mL/min), and lyophilized to obtain compound 4-b ( 80 mg, 28.3 ummol, 70.8% yield).

MS m/ _z : _{C132H192N12O55} , [ _MH ] ⁺ found: _2824.6 .

Step 3

The compound 38 (71mg, 25ummol) obtained in the previous step was added to acetonitrile (5mL), then HBTU (19.0mg, 50umol) was added, the surface amine group-modified solid phase support (CPG-NH2, 0.86g) was added, and DIEA ( 16.2mg, 125umol, 21.6uL), kept at 30°C for 16h shaking reaction. After the reaction was completed, it was filtered and washed sequentially with methanol (5 mL x 4), dichloromethane (5 mL x 4). The solid was further added to pyridine:acetic anhydride (v:v=4:1, 6.0 mL), and the reaction was continued at 30°C for 16h with shaking. After the reaction was completed, it was filtered and washed with methanol and dichloromethane successively. 0.74 g of compound 4-c linked to a solid support was obtained.

The preparation of embodiment 16 control compound L96

Control compounds were prepared according to the method described in patent WO2014025805A1. According to the same method as the above-mentioned connection to the solid phase carrier, it was connected to the CPG solid phase carrier to obtain compound L96.

Example 17 Synthesis of aminogalactose cluster-conjugated siRNA

The siRNA used for the test, the siRNA targeting mouse TTR gene mRNA (Molecular Therapy Vol. 26 No 3 March 2018) was as follows, and the 3'-end of the SS chain was linked to the aminogalactose molecular cluster M by a covalent bond.

SS chain (5'-3'): CmsAmsGm UmGfUm UfCfUfUmGmCm UmCmUm AmUmAm Am-M

AS chain (5'-3'): UmsUfsAm UmAmGf AmGmCm AmAmGm AmAfCm AfCmUm GmsUmsUm

The synthesis of siRNA is the same as the general phosphamide solid-phase synthesis method, the difference is that when synthesizing the SS chain of siRNA, the above-synthesized aminogalactose cluster is used. The CPG vector replaces the usual Universal-CPG vector. A brief description is as follows: On a Dr. Oligo48 synthesizer (Biolytic), starting from the above-synthesized aminogalactose-linked CPG vector, the nucleoside phosphoramidite monomers were linked one by one according to the synthetic procedure. Nucleoside monomer raw materials 2'-F RNA, 2'-O-methyl RNA and other nucleoside phosphoramidite monomers were purchased from Shanghai Zhaowei or Suzhou Zima. 5-Ethylthio-1H-tetrazole (ETT) was used as the activator (0.6M acetonitrile solution), and 0.22M PADS was used to dissolve in a 1:1 volume ratio of acetonitrile and melidine (Suzhou Kelema) solution As the sulfiding reagent, iodopyridine/water solution (Colema) was used as the oxidizing agent.

After the solid-phase synthesis was completed, the oligoribonucleotides were cleaved from the solid support, and soaked in a 3:1 solution of 28% ammonia water and ethanol at 50°C for 16 hours. After centrifugation, the supernatant was transferred to another centrifuge tube, concentrated and evaporated to dryness, and purified using C18 reverse-phase chromatography with 0.1 MTEAA and acetonitrile as mobile phases, and DMTr was removed using 3% trifluoroacetic acid solution. The target oligonucleotides were collected and lyophilized, identified as the target product by LC-MS, and then quantified by UV (260 nm).

The obtained single-stranded oligonucleotides were annealed to AS strands according to the equimolar ratio and complementary pairing, and finally the obtained double-stranded siRNA was dissolved in 1X PBS and adjusted to the concentration required for the experiment.

A galactosamine cluster-conjugated siRNA was synthesized, and the siRNA used in the experiment targeted mouse TTR mRNA.

M=

Table 19 Targeting ligand activity evaluation siRNA number and sequence

Example 18 Inhibition of mRNA expression by aminogalactose cluster-conjugated siRNA in primary liver cells

According to the method reported by Severgini et al. (Cytotechnology. 2012; 64(2): 187-195.), fresh mouse primary hepatocytes were isolated and obtained.

After primary hepatocytes were isolated, 100,000 per well were seeded into 24-well plates, and the siRNA to be tested were added at final concentrations of 50 nM, 10 nM, 2 nM, 0.4 nM, 0.08 nM, 0.016 nM, 0.0032 nM, and 0.00064 nM, respectively. Subsequently, the primary hepatocytes were cultured at 37°C in a 5% CO ₂ environment for 24 h. After 24 hours, the mRNA expression level of mTTR was detected by qPCR.

As shown in Figure 3, S-1, S-2, S-3, and S-4 all showed excellent mTTR gene expression inhibition efficiency. The IC50 values of S-1 and S-4 were lower than those of the other two groups. Compared with the IC50 value of S-L96 of the control group, which was 0.280 nM, the IC50 value of S-1 was 0.131 nM, and the IC50 value of S-4 was 0.135 nM. Shows that siRNA conjugated to S-1 and S-4 compounds is freely taken up by primary hepatocytes in vitro The extraction efficiency was better than that of the control group, and the S-1 and S-4 compounds could more efficiently mediate the entry of siRNA into primary hepatocytes.

Example 19 Inhibition of mRNA expression in vivo by aminogalactose cluster-conjugated siRNA

8-week-old C57BL/6 mice (Shao derivative, SPF grade, female) were used to deliver the siRNAs described above by subcutaneous injection. On day 1, a subcutaneous injection of 100 μl of a solution containing 1 mg/kg (mpk) in PBS or PBS at 0.2 mpk dose of the corresponding siRNA (S-L96, S -3, S-2, S-4 or S-1). Six mice were injected in each group. After 3 days of administration, the mice were sacrificed by short neck, and the mRNA expression level of mTTR in mouse liver tissue was detected by qPCR.

As shown in Figure 4, S-1, S-2, S-3, and S-4 all showed excellent mTTR gene expression inhibition efficiency. Among them, the activities of S-2, S-3 and S-4 were similar to those of the control group S-L96 at 1 mpk and 0.2 mpk. The activity levels of S-1 at 1mpk and 0.2mpk were better than those of the control group S-L96.

Eight-week-old C57BL/6 mice (Shao derivative, SPF grade, female) were used to deliver subcutaneous injection of siRNA conjugated with the above-described galactosamine cluster. On day 0, on the loose skin of the shoulder and neck of the mice, a subcutaneous injection of 100 μl of the solution containing PBS (called the Mock group, ie the blank control group) or the corresponding dose of 1 mg/kg (mpk) in PBS was administered of galactosamine cluster-conjugated siRNAs (S-1-2 and S-L96-2). Nine mice were injected in each group.

After 7 days, 14 days, and 28 days of administration, three mice were sacrificed by cervical dislocation, and two liver tissue samples were taken from each mouse, and the mRNA expression level of mTTR in the liver tissue of the mice was detected by qPCR.

As shown in Figure 5, the mRNA ratio (%) compared with the PBS group, the expression level of mRNA in the liver tissue of mice after administration of Compound S-1-2 was: 7 days (0.13), 14 days (0.12) and 28 days (0.21);

The expression levels of mRNA in the liver tissue of mice after administration of compound S-L96-2 were: 7 days (0.17), 14 days (0.13) and 28 days (0.29); compared with the control group S-L96-2, S-1-2 had a higher inhibition rate.

Example 20 Design and synthesis of human factor XI siRNA

1) See Table 1 for the unmodified sense and antisense sequences of factor XI;

See Table 2-3 and Table 20 for the sequences of the sense and antisense strands of siRNA modified with 2'-fluoro, 2'-methoxy, etc.; see Table 21-1 for changes in the optical rotation of the 7-position modification of the antisense strand. The modification changes are detailed in Table 21-2;

The modified sense and antisense strand sequences of factor XI siRNA conjugates are shown in Table 22.

、

或

所示的化學修飾或其互變異構體修飾的核苷酸； When synthesizing the nucleotide modified at the 7th position of the 5' of the AS chain, the phosphamide monomer synthesized in Example 8 or the replacement parent sequence original nucleotide of the 2'-methoxy-modified phosphamide monomer was used. The sequence of the antisense strand modified at position 7 of the 5' of the AS chain is shown in Table 1, wherein W' is selected from: 2'-methoxy-modified nucleotides or containing

,

or

The indicated chemically modified nucleotides or their tautomer modified nucleotides;

Wherein, M is O or S; wherein, B is selected from the bases at the corresponding positions in the 7th position of the 5' region of SEQ ID NO: 47-69 and SEQ ID NO: 24-46 in Table 2, such as SEQ ID NO : 47 corresponds to SEQ ID NO:24, SEQ ID NO:69 corresponds to SEQ ID NO:46.

2) siRNA synthesis , the siRNA sequences of Tables 5 and 6 were synthesized on a Dr. Oligo48 synthesizer (Biolytic) at a scale of 200 nanomoles using solid support mediated phosphamidite chemistry. The solid support is a general solid support (Shenzhen Comma Bio). Nucleoside monomer raw materials 2'-F-RNA, 2'-O-methyl RNA and other nucleoside phosphoramidite monomers were purchased from Shanghai Zhaowei or Suzhou Zima. Coupling time for all phosphamidites (50 mM in acetonitrile) was 6 minutes (min) using 5-ethylthio-1H-tetrazole (ETT) as the activator (0.6 M in acetonitrile) in 0.22 M in PADS. In 1:1 volume ratio of acetonitrile and collema (Suzhou Kelema) solution as sulfurization reagent, sulfurization reaction time is 3 minutes (min), using iodopyridine/water solution (Suzhou Kelema) as oxidant, oxidation reaction Time 2 minutes (min). The above sulfidation reaction conditions or oxidation reaction conditions are selected according to whether the target product has 5'-phosphorothioate modification.

After the solid-phase synthesis was completed, the oligoribonucleotides were cleaved from the solid support, and immersed in a 3:1 volume ratio of 28% ammonia water and ethanol solution at 50°C for 16 hours. Then high-speed centrifugation, the supernatant was transferred to another centrifuge tube, concentrated and evaporated to dryness, purified using C18 reverse-phase chromatography, the mobile phase was 0.1M TEAA and acetonitrile, and DMTr was removed using 3% trifluoroacetic acid solution. The target oligonucleotides were collected and lyophilized, identified as the target product by LC-MS, and then quantified by UV (260 nm).

The sense and antisense strands synthesized according to the above steps were annealed according to the equimolar ratio to form a double-stranded structure by hydrogen bonding. Finally, the obtained double-stranded siRNA was dissolved in 1×PBS and adjusted to The concentration required for the experiment.

、

或

所示的化學修飾或其互變異構體修飾的核苷酸； In Table 1, W' is selected from: 2'-methoxy-modified nucleotides or comprising

,

or

The indicated chemically modified nucleotides or their tautomer modified nucleotides;

Wherein, M is O or S; wherein, B is selected from the bases at the corresponding positions in the 7th position of the 5' region of SEQ ID NO: 47-69 and SEQ ID NO: 24-46 in Table 2, such as SEQ ID NO : 47 corresponds to SEQ ID NO:24, SEQ ID NO:69 corresponds to SEQ ID NO:46.

In Table 20-22, the nucleotides synthesized from 2-hydroxymethyl-1,3-propanediol are defined as hmpNA; hmpNA is a racemic structure;

(-)hmpNA(A) is the nucleoside phosphoramidite monomer 1-1a obtained by solid-phase synthesis in Section 8.1; (+)hmpNA(A) is the optical isomer;

(-)hmpNA(G) is the nucleoside phosphoramidite monomer 1-6a obtained by solid-phase synthesis in Example 8.6; (+)hmpNA(G) is the optical isomer;

(-)hmpNA(C) is the nucleoside phosphoramidite monomer 1-8a obtained by solid-phase synthesis in Section 8.8; (+)hmpNA(C) is the optical isomer;

(-)hmpNA(U) is the nucleoside phosphoramidite monomer 1-7a obtained by solid-phase synthesis in Example 8.7; (+)hmpNA(U) is the optical isomer.

The lowercase letter m indicates that a nucleotide adjacent to the left of the letter m is a 2'-methoxy modified nucleotide; the lowercase letter f indicates that the adjacent nucleotide to the left of the letter f is a 2'-fluorine modified nucleotide Nucleotides;

When the lowercase letter s is in the middle of the uppercase letter, it indicates that the connection between the two nucleotides adjacent to the letter s is a phosphorothioate connection;

The lowercase letter s is the first at the 3' end, indicating that a nucleotide end adjacent to the left side of the letter s is a phosphorothioate group.

In Table 22, the structure of NAG1 is:

Example 21 Screening of the inhibitory activity of siRNA by psiCHECK at the target activity-11 concentration point

The siRNA sample synthesis was described above, and the plasmid was obtained from Sangon Bioengineering (Shanghai) Co., Ltd. To prepare HEK 293A cells, the specific preparation amount of Plasmid Mix is shown in Table 23; components of the transfection complex:

Table 23 Amount of transfection complex required for each well of 96-well plate

See Table 24 for the siRNA dilution scheme. After 24 hours of transfection, the detection was carried out according to the experimental operation scheme of the Dual-Glo® Luciferase Assay System Detection Kit. In the present disclosure, remaining activity % (also referred to as mRNA remaining expression % or mRNA remaining expression ratio)=100%-inhibition rate (%).

Table 24 Multi-concentration siRNA dilution scheme

The results are shown in Table 25. The results show that the mRNA levels of FXI treated with siRNA were decreased in a dose-dependent manner, and all of the siRNAs had high levels of on-target inhibitory activity in vitro.

Table 25. Screening results of psiCHECK inhibitory activity of siRNA at target activity-11 concentration point

Example 22. In vivo FXI silencing effect of siRNA in hFXI expressing mice

The stable transfection plasmid carrying human FXI and capable of expressing SEAP protein was introduced into C57BL/6 mice by high pressure tail vein injection to construct a mouse model expressing human FXI (hFXI). The specific operation is to select 6-8 week old female C57BL/6 mice, inject 50 μg of the plasmid carrying the cDNA sequence of human FXI and the full-length sequence of SEAP protein 3'UTR through the high pressure tail vein, and test the target knockdown of siRNA two weeks later. reduce ability.

All mice were administered subcutaneously on the 15th day, and the dose was 0.3, 1 or 3 mg/kg, and the dose was 200 μL/20 g per mouse. One day before administration, the expression of SEAP protein in mouse serum was detected by Phospha-Light SEAP reporter gene detection kit, and the mice were randomly divided into seven groups according to the results of the average expression level of SEAP protein. The day of administration was the 0th day, and the submandibular blood was collected from all mice on the 8th, 15th, 22nd and 29th days after administration to collect plasma, which was used to detect the expression of SEAP protein in serum.

Table 26. In vivo SEAP protein expression and FXI silencing effect of siRNA in hFXI mice

Example 23. Evaluation of different modifications at positions 9 and 10 of AS chains

This experiment investigated the inhibition efficiency of the 2'-fluorine modified siRNA conjugates at different sites of the present disclosure on the mRNA expression of target genes in vivo. Male 6-8 week old C57BL/6 mice were randomly divided into 6 mice in each group and 3 mice at each time point. Conjugate (TRD002218) and PBS. The dose of all animals was calculated according to the total body, and the dose was subcutaneously injected in a single dose. The dose of siRNA conjugate (calculated as the amount of siRNA) was 1 mg/kg, and the dose was 5 mL/kg. After 7 days of administration, the mice were sacrificed, and the livers were collected and stored with RNA later (Sigma Aldrich Company); subsequently, the liver tissue was homogenized with a tissue homogenizer, and then the tissue RNA extraction kit (Fanzhi Medical Technology, FG0412) was used according to the operation. The operation steps indicated in the instructions were used to extract total RNA from liver tissue. The total RNA was reverse transcribed into cDNA and the expression of TTR mRNA in liver tissue was detected by real-time fluorescence quantitative PCR. In this fluorescent quantitative PCR method, the glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene was used as an internal reference gene, and Taqman probe primers for TTR and GAPDH were used to detect the mRNA expression levels of TTR and GAPDH, respectively. See Table 27 for compound information, Table 28 for grouping information of experimental compounds in mice, and Table 29 for primers.

Table 27. AS Chain 9 and 10 Modified Compounds

Table 28. In vivo experimental compound grouping information in mice

Table 29. Detection primer sequences

Table 30. 7-day and 28-day test results

TTR mRNA expression was calculated according to the following equation:

TTR mRNA expression = [(TTR mRNA expression in test group / GAPDH mRNA expression in test group) / (TTR mRNA expression in control group / GAPDH mRNA expression in control group)] x 100%

After 28 days of administration, the in vivo inhibition efficiency of the fluorine-modified siRNA conjugates at different sites of the present disclosure on the mRNA expression of target genes is shown in Table 30. Referring to the positive control compound TRD002218, the siRNA compounds modified with fluorine at different sites inhibited the expression of TTR mRNA at 28 days after administration than the reference positive compound. Both modification methods showed high inhibition efficiency and no significant difference. It shows that the two modification methods can mediate more efficient siRNA inhibition efficiency.

Claims (25)

A siRNA for inhibiting the expression of coagulation factor XI, comprising a sense strand and an antisense strand forming a double-stranded region; wherein The sense strand comprises at least 15 contiguous nucleotides that differ by no more than 3 nucleotide sequences from any of the sense strands in Table 1; the antisense strand comprises no more than 3 cores from any of the antisense strands in Table 1 at least 15 consecutive nucleotides of the nucleotide sequence; and The sense strand contains three consecutive nucleotides that are 2'-fluoro-modified nucleotides; and The nucleotides at positions 2, 6 and 14 of the antisense strand are each independently 2'-fluoro-modified nucleotides in the direction from the 5' end to the 3' end; Preferably, the nucleotides at positions 2, 6, 14 and 16 of the antisense strand are independently 2'-fluoro-modified nucleotides in the direction from the 5' end to the 3' end; Preferably, the nucleotides at positions 2, 6, 12 and 14 of the antisense strand are independently 2'-fluoro-modified nucleotides in the direction from the 5' end to the 3' end; More preferably, the nucleotides at positions 2, 4, 6, 12, 14, 16 and 18 of the antisense strand are each independently a 2'-fluoro modified core in the direction from the 5' end to the 3' end. Glycosides; Still more preferably, the nucleotides at positions 2, 4, 6, 9, 12, 14, 16 and 18 of the antisense strand are each independently 2'-fluoro in the direction from the 5' end to the 3' end modified nucleotides; Still more preferably, the nucleotides at positions 2, 4, 6, 10, 12, 14, 16 and 18 of the antisense strand are each independently 2'-fluoro in the direction from the 5' end to the 3' end Modified Nucleotides. The siRNA for inhibiting the expression of coagulation factor XI according to claim 1, wherein at least one other nucleotide in the sense strand and/or antisense strand is a modified nucleotide, and the modified nucleotide is selected from: 2 '-methoxy-modified nucleotides, 2'-substituted alkoxy-modified nucleotides, 2'-alkyl-modified nucleotides, 2'-substituted alkyl-modified nucleotides , 2'-amino-modified nucleotides, 2'-substituted amino-modified nucleotides, 2'-fluoro-modified nucleotides, 2'-deoxynucleotides, 2'-deoxynucleotides Oxy-2'-fluoro-modified nucleotides, 3'-deoxy-thymine nucleotides, isonucleotides, LNA, ENA, cET, UNA, GNA or chemical modifications comprising formula (I) or tautomer-modified nucleotides; Wherein, the chemical modification shown in formula (I) is selected from:

Wherein, B is a base; preferably, B is the base at the corresponding position of the modified nucleotide of the sense strand and/or antisense strand; Preferably, the modified nucleotides are selected from: 2'-methoxy-modified nucleotides or nucleotides modified by chemical modifications represented by formula (I) or tautomers thereof. The siRNA for inhibiting the expression of coagulation factor XI as claimed in claim 2, wherein the chemical modification represented by the formula (I) is selected from:

Among them, B is a base; Preferably, B is the base at the position corresponding to the modified nucleotide of the sense strand and/or antisense strand. The siRNA for inhibiting the expression of coagulation factor XI according to claim 2 or 3, wherein the modified nucleotides are located in the 2nd to 8th positions of the antisense strand in its 5' region; preferably, the modified nucleotides are located in Position 5, 6 or 7 of the 5' region of the antisense strand. The siRNA for inhibiting the expression of coagulation factor XI according to any one of claims 1 to 4, wherein the sense strand contains a nucleotide sequence (5'-3') represented by the following formula: N _a N _a N _a N _a XN _a N _b N _b N _b N _a N _a N _a N _a N _a N _a N _a N _a N _a N _a wherein each _X is independently Na or _Nb ; and/or, The antisense strand contains the nucleotide sequence (5'-3') of the following formula: N _a 'N _b 'N _a 'X'N _a 'N _b 'W'N _a 'X'Y'N _a 'X'N _a 'N _b 'N _a 'X'N _a 'X'N _a ' N _a 'N _a '; wherein, each X' is independently _Na ' or _Nb ', and Y' is Na' or _Nb ';W' represents _a 2'-methoxy-modified nucleotide or contains as claimed in items 2 to 4 The nucleotides defined in any one of the definitions include the chemical modification represented by formula (I) or its tautomer modification; _Na and _Na ' are 2'-methoxy-modified nucleotides, and N _b , _Nb ' is a 2'-fluoro modified nucleotide. The siRNA for inhibiting the expression of coagulation factor XI according to any one of claims 1 to 5, wherein the sense strand contains a nucleotide sequence represented by the following formula: 5'-N _a N _a N _a N _a N _a N _a N _b N _b N _b N _a N _a N _a N _a N _a N _a N _a N _a N _a N _a -3'; or, 5'-N _a N _a N _a N _a N _b N _a N _b N _b N _b N _a N _a N _a N _a N _a N _a N _a N _a N _a N _a -3'; Wherein, Na is a 2'-methoxy-modified nucleotide, and N _b is _a 2'-fluoro-modified nucleotide. The siRNA for inhibiting the expression of coagulation factor XI according to any one of claims 1 to 6, wherein the antisense strand contains a nucleotide sequence represented by the following formula: 5'-N _a 'N _b 'N _a 'N _b 'N _a 'N _b 'W'N _a 'X'Y'N _a 'N _b 'N _a 'N _b 'N _a 'N _b 'N _a 'N _b 'N _a 'N _a 'N _a '-3'; Wherein, each X' is independently _Na ' or _Nb ', Y' is _Na ' or _Nb ';Na' is _a 2'-methoxy-modified nucleotide, and _Nb ' is 2' -Fluorine-modified nucleotide; W' represents a 2'-methoxy-modified nucleotide or a chemical modification comprising formula (I) as defined in any one of claims 2-4 or a tautomer thereof body-modified nucleotides; Preferably, the antisense strand contains the nucleotide sequence shown in the following formula: 5'-N _a 'N _b 'N _a 'N _b 'N _a 'N _b 'W'N _a 'N _a 'N _b 'N _a 'N _b 'N _a 'N _b 'N _a 'N _b ' N _a 'N _b 'N _a 'N _a 'N _a '-3'; or, 5'-N _a 'N _b 'N _a 'N _b 'N _a 'N _b 'W'N _a 'N _b 'N _a 'N _a 'N _b 'N _a 'N _b 'N _a 'N _b ' N _a 'N _b 'N _a 'N _a 'N _a '-3'. The siRNA for inhibiting the expression of coagulation factor XI according to claim 7, wherein W' represents a 2'-methoxy-modified nucleotide or comprises

,

or

Modified or tautomer-modified nucleotides; wherein, B is a base; Preferably, B is selected from the base at the corresponding position in the 7th position of the 5' region of the antisense strand. The siRNA for inhibiting the expression of coagulation factor XI according to any one of claims 1 to 8, wherein at least one phosphate group in the sense strand and/or antisense strand is a phosphate group with a modified group, preferably Phosphorothioate group. The siRNA for inhibiting the expression of coagulation factor XI as claimed in claim 9, wherein the phosphorothioate group is present in at least one position selected from the group consisting of: Between the first nucleotide and the second nucleotide in the 5'-3' direction of the sense strand; Between the second nucleotide and the third nucleotide in the 5'-3' direction of the sense strand; The first nucleotide end in the 3'-5' direction of the sense strand; Between the first nucleotide and the second nucleotide in the 3'-5' direction of the sense strand; Between the second nucleotide and the third nucleotide in the 3'-5' direction of the sense strand; Between the first nucleotide and the second nucleotide in the 5'-3' direction of the antisense strand; Between the second nucleotide and the third nucleotide in the 5'-3' direction of the antisense strand; The first nucleotide end of the 3'-5' direction of the antisense strand; Between the first nucleotide and the second nucleotide in the 3'-5' direction of the antisense strand; or Between the 2nd nucleotide and the 3rd nucleotide in the 3'-5' direction of the antisense strand. The siRNA for inhibiting the expression of coagulation factor XI according to any one of claims 1 to 10, wherein the sense strand comprises a nucleotide sequence that differs from any one of SEQ ID NO: 1 to SEQ ID NO: 23 by no more than 3 nucleotides at least 15 consecutive nucleotides; and/or, the antisense strand comprises at least 15 contiguous nucleotides that differ from any of SEQ ID NO: 24 to SEQ ID NO: 46 by no more than 3 nucleotide sequences; Preferably, the sense strand comprises at least 19 consecutive nucleotides that differ from any one of SEQ ID NO: 1 to SEQ ID NO: 23 by no more than 3 nucleotide sequences; preferably, the nucleotide sequence differs by no more than 1 nucleotides; and/or, The antisense strand comprises at least 21 contiguous nucleotides that differ from any of SEQ ID NO: 24 to SEQ ID NO: 46 by no more than 3 nucleotide sequences; preferably the nucleotide sequence differs by no more than 1 nucleotide . The siRNA for inhibiting the expression of coagulation factor XI according to any one of claims 1 to 11, wherein the nucleotide sequence of the sense strand contains: the nucleotide sequence of any one of SEQ ID NO: 1 to SEQ ID NO: 23; and / or, The nucleotide sequence of the antisense strand comprises: the nucleotide sequence of any one of SEQ ID NO: 47 to SEQ ID NO: 69, wherein W' represents a 2'-methoxy-modified nucleotide or comprises as claimed in the claim The nucleotides defined in any one of 2 to 4 comprising the chemical modification represented by formula (I) or its tautomer modification; Preferably, the sense strand contains any one of SEQ ID NOs: 70-77, 86-93, 102-161, 282-293, 306-425; and/or the antisense strand contains SEQ ID NOs: 78-85, 94 - Any of 101, 162-261, 294-305. The siRNA for inhibiting the expression of coagulation factor XI according to any one of claims 1 to 12, selected from: The sense strand contains SEQ ID NO: 142, and the antisense strand contains SEQ ID NO: 202, SEQ ID NO: 222, or SEQ ID NO: 242; The sense strand contains SEQ ID NO: 143, and the antisense strand contains SEQ ID NO: 203, SEQ ID NO: 223, or SEQ ID NO: 243; The sense strand contains SEQ ID NO: 144, and the antisense strand contains SEQ ID NO: 204, SEQ ID NO: 224, or SEQ ID NO: 244; The sense strand contains SEQ ID NO: 145, and the antisense strand contains SEQ ID NO: 205, SEQ ID NO: 225, or SEQ ID NO: 245; The sense strand contains SEQ ID NO: 146, and the antisense strand contains SEQ ID NO: 206, SEQ ID NO: 226, or SEQ ID NO: 246; The sense strand contains SEQ ID NO: 147, and the antisense strand contains SEQ ID NO: 207, SEQ ID NO: 227, or SEQ ID NO: 247; The sense strand contains SEQ ID NO: 148, and the antisense strand contains SEQ ID NO: 208, SEQ ID NO: 228, or SEQ ID NO: 248; The sense strand contains SEQ ID NO: 149, and the antisense strand contains SEQ ID NO: 209, SEQ ID NO: 229, or SEQ ID NO: 249; The sense strand contains SEQ ID NO: 150, and the antisense strand contains SEQ ID NO: 210, SEQ ID NO: 230, or SEQ ID NO: 250; The sense strand contains SEQ ID NO: 151, and the antisense strand contains SEQ ID NO: 211, SEQ ID NO: 231, or SEQ ID NO: 251; The sense strand contains SEQ ID NO: 152, and the antisense strand contains SEQ ID NO: 212, SEQ ID NO: 232, or SEQ ID NO: 252; The sense strand contains SEQ ID NO: 153, and the antisense strand contains SEQ ID NO: 213, SEQ ID NO: 233, or SEQ ID NO: 253; The sense strand contains SEQ ID NO: 154, and the antisense strand contains SEQ ID NO: 214, SEQ ID NO: 234, or SEQ ID NO: 254; The sense strand contains SEQ ID NO: 155, and the antisense strand contains SEQ ID NO: 215, SEQ ID NO: 235, or SEQ ID NO: 255; The sense strand contains SEQ ID NO: 156, and the antisense strand contains SEQ ID NO: 216, SEQ ID NO: 236, or SEQ ID NO: 256; The sense strand contains SEQ ID NO: 157, and the antisense strand contains SEQ ID NO: 217, SEQ ID NO: 237, or SEQ ID NO: 257; The sense strand contains SEQ ID NO: 158, and the antisense strand contains SEQ ID NO: 218, SEQ ID NO: 238, or SEQ ID NO: 258; The sense strand contains SEQ ID NO: 159, and the antisense strand contains SEQ ID NO: 219, SEQ ID NO: 239, or SEQ ID NO: 259; The sense strand contains SEQ ID NO: 160, and the antisense strand contains SEQ ID NO: 220, SEQ ID NO: 240, or SEQ ID NO: 260; The sense strand contains SEQ ID NO: 161, and the antisense strand contains SEQ ID NO: 221, SEQ ID NO: 241, or SEQ ID NO: 261; Preferably, at least one phosphate group in the sense chain is a phosphorothioate group; still more preferably, the phosphorothioate group is present in at least one of the following positions: Between the first nucleotide and the second nucleotide in the 5'-3' direction of the sense strand; Between the second nucleotide and the third nucleotide in the 5'-3' direction of the sense strand; The first nucleotide end in the 3'-5' direction of the sense strand; Between the first nucleotide and the second nucleotide in the 3'-5' direction of the sense strand; Between the 2nd nucleotide and the 3rd nucleotide in the 3'-5' direction of the sense strand. A siRNA for inhibiting the expression of coagulation factor XI, comprising a sense strand and an antisense strand, wherein the sense strand contains a segment of nucleotide sequence A, the antisense strand contains a segment of nucleotide sequence B, the nucleotide sequence A and The nucleotide sequence B is at least partially reverse complementary to form a double-stranded region, wherein the nucleotide sequence A is the nucleotide sequence shown in any one of SEQ ID NOs: 1-23; and/or the nucleotide sequence Sequence B is the nucleotide sequence shown in any one of SEQ ID NOs: 24-46. A siRNA conjugate comprising the siRNA for inhibiting the expression of coagulation factor XI as described in any one of claims 1 to 14 and a targeting ligand connected to the end of the siRNA; preferably, the targeting ligand target To the liver; preferably, the targeting ligand binds asialoglycoprotein receptor (ASGPR); still more preferably, the targeting ligand comprises a galactose cluster or a galactose derivative cluster, the galactose derivative is selected from N-acetyl-galactosamine, N-trifluoroacetone galactosamine, N-propyl galactosamine, N-n-butyl galactosamine or N-isobutyl galactosamine. The siRNA conjugate of claim 15, wherein the targeting ligand is attached to the end of the siRNA via a phosphorothioate or phosphate group. A pharmaceutical composition comprising the siRNA for inhibiting the expression of coagulation factor XI as described in any one of claim 1 to 14 or the siRNA conjugate as described in claim 15 or 16, and a pharmaceutically acceptable carrier . A cell comprising the siRNA for inhibiting the expression of coagulation factor XI as claimed in any one of claims 1 to 14 or comprising the siRNA conjugate as claimed in claim 15 or 16. A kit comprising the siRNA for inhibiting the expression of coagulation factor XI as described in any one of claim 1 to 14 or the siRNA conjugate as claimed in claim 15 or 16 or the medicine as claimed in claim 17 composition. A siRNA for inhibiting the expression of coagulation factor XI as described in any one of claim 1 to 14 or a siRNA conjugate comprising the siRNA conjugate as described in claim 15 or 16 or the pharmaceutical composition as claimed in claim 17 in preparation Use in drugs for inhibiting the expression of coagulation factor XI. A siRNA for inhibiting the expression of coagulation factor XI as described in any one of claim 1 to 14 or a siRNA conjugate comprising the siRNA conjugate as described in claim 15 or 16 or the pharmaceutical composition as claimed in claim 17 in preparation Use in a medicament for the treatment and/or prophylaxis of a subject suffering from a disease; preferably, the disease is a thromboembolic complication, more preferably deep vein thrombosis, pulmonary embolism, myocardial infarction or stroke. A method for treating/preventing the expression of coagulation factor XI, comprising administering a therapeutically and/or prophylactically effective amount of the inhibitor as described in any one of claims 1 to 14 to an object in need thereof. The siRNA for inhibiting the expression of coagulation factor XI or the siRNA conjugate as claimed in claim 15 or 16 or the pharmaceutical composition as claimed in claim 17. A method for treating/preventing disease, comprising administering the siRNA for inhibiting the expression of coagulation factor XI as described in any one of claim 1 to 14 or comprising as claimed The siRNA conjugate of 15 or 16 or the pharmaceutical composition of claim 17; preferably, the disease is a thromboembolic complication, more preferably deep vein thrombosis, pulmonary embolism, myocardial infarction or stroke. A method for silencing a target gene in a cell, the method comprising adding the siRNA for inhibiting the expression of coagulation factor XI as described in any one of claims 1 to 14 or a siRNA conjugate comprising the siRNA as described in claim 15 or 16 Or the step of introducing the pharmaceutical composition of claim 17 into the cell. A method for preparing siRNA or siRNA conjugate, comprising: synthesizing the siRNA for inhibiting the expression of coagulation factor XI as described in any one of claim 1 to 14 or comprising the siRNA conjugate as described in claim 15 or 16 and purifying the siRNA or the siRNA conjugate.

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