Nucleic acids, compositions and complexes containing the nucleic acids, and preparation methods and uses

Abstract

To provide a siRNA for suppressing hepatitis B virus gene expression, and a pharmaceutical composition and conjugate containing the siRNA.SOLUTION: Each nucleotide in a siRNA for suppressing hepatitis B virus gene expression is an independent modified nucleotide. The siRNA contains a sense strand and an antisense strand. The sense strand includes a specific nucleotide sequence. The antisense strand includes a nucleotide sequence having the specific sequence that is equal in a length and differs by no more than three nucleotides from the nucleotide sequence.SELECTED DRAWING: Figure 26

Description

[Technical field]

[0001] The present disclosure relates to nucleic acids, compositions and complexes comprising the nucleic acids, and methods of preparation and use. . [Background technology]

[0002] Viral hepatitis B (also known as hepatitis B) poses a serious threat to the whole world, especially China. Currently, the only drugs approved worldwide for preventing hepatitis B are Interleukin 1 (IL-1), There are two types of drugs, feron and nucleoside analogues, but these two types of drugs have been shown to have drug resistance after use. There are various disadvantages, such as the tendency for interferograms to occur and limitations to their use. Nucleoside drugs are prone to drug resistance and relapse after discontinuation. Therefore, it is necessary to silence the gene expression of the virus at the genetic level. By blocking the production and replication of HBV, it is possible to fundamentally inhibit viral metabolism and infection of liver cells. If we could lower this, it would undoubtedly be the most ideal treatment for hepatitis B. In addition, small interfering RNA (siRNA) is based on a mechanism called RNA interference (RNAi), Expression of any gene of interest (e.g., a gene that induces a disease such as cancer) can be sequence-specifically inhibited or blocked to achieve the goal of treating disease. Summary of the Invention [Problem to be solved by the invention]

[0003] In the development of small RNA drugs, stabilization of siRNA and its delivery system are two key factors. It's technology. [Means for solving the problem]

[0004] In some embodiments, the present disclosure provides an siRNA having the structure shown in formula (1). A complex is provided.

[0005] [Chemical formula] Wherein n1 is an integer selected from 1 to 3, and n3 is an integer selected from 0 to 4 , m1, m2 and m3 are each independently an integer selected from 2 to 10, R 10 , R 11 , R 12 , R 13 , R 14 and R 15 are each independently H , or a C1-C 10 alkyl group, a C1-C 10 halogenated alkyl group and a C1-C 10 alk oxy group, selected from the group consisting of R3 is a group having the structure shown in formula A59.

[0006] [Chemical formula] Wherein E1 is OH, SH or BH2, and Nu is siRNA.

[0007] Each nucleotide in the siRNA is independently a modified or unmodified nucleotide. The siRNA includes a sense strand and an antisense strand. The sense strand includes nucleotide sequence 1, and the antisense strand includes nucleotide sequence 2. Nucleotide sequence 1 and nucleotide sequence 2 are at least partially reverse complementary to form a double-stranded region. ​​​forms a domain, and the nucleotide sequence 1 and the nucleotide sequence shown in SEQ ID NO: 155 have the same length and three or fewer nucleotide differences, and the nucleotide sequence 2 and the nucleotide sequence shown in SEQ ID NO: 156 have the same length and three or fewer nucleotide differences, 5’-CCUUGAGGCAUACUUCAAZ-3’ (SEQ ID NO: 155), 5’-Z’UUGAAGUAUGCCUCAAGG-3’ (SEQ ID NO: 156) wherein Z is A and Z’ is U, the nucleotide sequence 1 contains the nucleotide Z corresponding to the position Z, A and the nucleotide sequence 2 contains the nucleotide Z’ corresponding to the position Z’, B and the Z’ B is the first nucleotide at the 5’ end of the antisense strand.

[0008] R2 is a straight-chain alkylene group having 1 to 20 carbon atoms, and one or more carbon atoms are optionally substituted with one or more selected from the group consisting of C(O), NH, O, S, CH=N, S(O)2, C2-C 10 alkenylene group, C2 -C 10 alkynylene group, C6-C 10 arylene group, C3-C 18 heterocyclylene group and C5-C 10 heteroarylene group, and R2 is C1-C alkyl group, C6-C 10 aryl group, C5-C 10 hetero 10 aryl group, C1-C halogenated alkyl group, -OC1-C 10 alkyl group, -OC 10 alkyl group, -OC 1-C 10 alkylphenyl group, -C1-C 10Alkyl-OH, -OC1-C 10 Halo Alkyl halide group, -SC1-C 10 Alkyl group, -SC1-C 10 Alkylphenyl group , -C1-C 10 Alkyl-SH, -SC1-C 10 Halogenated alkyl group, halogen substitution group, -OH, -SH, -NH2, -C1-C 10 Alkyl-NH2, -N(C1-C1 0 alkyl group)(C1-C 10 alkyl group), -NH(C1-C 10 alkyl group), cyano group, nitro group, -CO2H, -C(O)O(C1-C 10 alkyl group), -CON(C 1-C 10 alkyl group)(C1-C 10 alkyl group), -CONH(C1-C 10 alkyl group), -CONH2, -NHC(O)(C1-C 10 alkyl group), -NHC(O)( phenyl group), -N(C1-C 10 alkyl)C(O)(C1-C 10 alkyl group), - N(C1-C 10 alkyl)C(O)(phenyl group), -C(O)C1-C 10 alkyl group, -C(O)C1-C 10 alkylphenyl group, -C(O)C1-C 10 haloalkyl group, -OC(O)C1-C 10 alkyl group, -SO2(C1-C 10 alkyl group), -S O2(phenyl group), -SO2(C1-C 10 halogenated alkyl group), -SO2NH2 , -SO2NH(C1-C 10 alkyl group), -SO2NH(phenyl group), -NHSO 2(C1-C 10(alkyl group), -NHSO2(phenyl group) and -NHSO2(C1- C 10 optionally having any one or more substituents selected from the group consisting of (haloalkyl group) and may be.

[0009] Each L1 is a straight-chain alkylene group of 1 to 70 carbon atoms, and one or more carbon atoms are C(O), NH, O, S, CH=N, S(O)2, C2-C 10 alkenylene group, C 2-C 10 alkynylene group, C6-C 10 arylene group, C3-C 18 heterocyclylene group and C5-C 10 heteroarylene group, and are optionally substituted with one or more selected from the group consisting of L1 is a C1-C 10 alkyl group, C6-C 10 aryl group, C5-C 10 hetero aryl group, C1-C 10 haloalkyl group, -OC1-C 10 alkyl group, -O C1-C 10 alkylphenyl group, -C1-C 10 alkyl-OH, -OC1-C 10 halo alkyl group, -SC1-C 10 alkyl group, -SC1-C 10 alkylphenyl group, -C1-C 10 alkyl-SH, -SC1-C 10 haloalkyl group, halogen substituent, -OH, -SH, -NH2, -C1-C 10 alkyl-NH2, -N(C1-C 10 alkyl group)(C1-C 10 alkyl group), -NH(C1-C 10 alkyl group), cyano group, nitro group, -CO2H, -C(O)O(C1-C alkyl group)10 (alkyl group), -CON( C1-C 10 (alkyl group)(C1-C 10 (alkyl group), -CONH(C1-C 10 al kyl group), -CONH2, -NHC(O)(C1-C 10 (alkyl group), -NHC(O) (phenyl group), -N(C1-C 10 alkyl)C(O)(C1-C 10 (alkyl group), -N(C1-C 10 alkyl)C(O)(phenyl group), -C(O)C1-C 10 al kyl group, -C(O)C1-C 10 alkylphenyl group, -C(O)C1-C 10 haloal kyl group, -OC(O)C1-C 10 (alkyl group), -SO2(C1-C 10 (alkyl group), - SO2(phenyl group), -SO2(C1-C 10 (halogenated alkyl group), -SO2NH 2, -SO2NH(C1-C 10 (alkyl group), -SO2NH(phenyl group), -NHS O2(C1-C 10 (alkyl group), -NHSO2(phenyl group) and -NHSO2(C1 -C 10 (halogenated alkyl group) and may optionally have any one or more substituents selected from the group consisting of thereof. TIFF0007702159000003.tif4147 represents the site where the group is attached to the rest of the molecule, and M1 represents the target group.

[0010] In some embodiments, the present disclosure provides, under the conditions of phosphoramidite solid-phase synthesis, respectively, depending on the nucleotide types and sequences of the sense strand and the antisense strand of siRNA, 3' or Towards the 5'-end, nucleoside monomers are sequentially linked, and the linkage of each nucleoside monomer includes four reactions of deprotection, coupling, capping, oxidation or sulfidation, and a method for preparing a complex including isolating the sense strand and the antisense strand of siRNA and performing annealing. Each nucleotide in the siRNA is independently a modified or unmodified nucleotide. The siRNA includes a sense strand and an antisense strand. The sense strand has the same length as the nucleotide sequence shown in SEQ ID NO: 155 and contains a nucleotide sequence 1 with three or fewer nucleotide differences. The antisense strand has the same length as the nucleotide sequence shown in SEQ ID NO: 156 and contains a nucleotide sequence 2 with three or fewer nucleotide differences. The nucleotide sequence 1 and the nucleotide sequence 2 form a double-stranded region that is at least partially reverse complementary, 5'-CCUUGAGGCAUACUUCAAZ-3' (SEQ ID NO: 155), 5'-Z'UUGAAGUAUGCCUCAAGG-3' (SEQ ID NO: 156) where Z is A and Z' is U. However, Z is A and Z' is U.

[0011] The nucleotide sequence 1 contains a nucleotide Z at a position corresponding to Z, A and the nucleotide sequence 2 contains a nucleotide Z' at a position corresponding to Z', where Z' B is B the first nucleotide at the 5'-end of the antisense strand.

[0012] Also, the method involves binding a compound represented by formula (3 21) to a nucleoside monomer or a nucleotide ​Contact with the d-array and subject the compound represented by formula (321) to a coupling reaction to obtain a nucleotide which further includes binding to the d-array. Hereinafter, the compound represented by formula (321) is also referred to as a complex molecule.

[0013]

Chemical formula

[0014] In some embodiments, the present disclosure provides an siRNA capable of suppressing HBV gene expression, and the siRNA includes a sense strand and an antisense strand, and the sense strand and the antisense ​​​​​​​​The sense strands each contain both fluorine-modified nucleotides and non-fluorine-modified nucleotides. , the sense strand contains nucleotide sequence I, and the antisense strand contains nucleotide sequence II, at least a part of the nucleotide sequence I and the nucleotide sequence II form a double-stranded region that is reverse complementary, the nucleotide sequence I contains nucleotide sequence A , the nucleotide sequence A has the same length as the nucleotide sequence shown in SEQ ID NO: 155 and has 3 or fewer nucleotide differences, the nucleotide sequence II contains nucleotide sequence B, the nucleotide sequence B has the same length as the nucleotide sequence shown in SEQ ID NO: 156 and has 3 or fewer nucleotide differences, 5'-CCUUGAGGCAUACUUCAAZ-3' (SEQ ID NO: 155), 5'-Z'UUGAAGUAUGCCUCAAGG-3' (SEQ ID NO: 156) where Z is A and Z' is U. The nucleotide Z corresponding to the position Z is included in the nucleotide sequence A, and the nucleotide Z' corresponding to the position Z' is included in the nucleotide sequence B, and the Z' A is the first nucleotide at the 5'-end of the antisense strand. B B

[0015] The fluorine-modified nucleotides are located in the nucleotide sequence A and the nucleotide sequence B, and from the 5'-end to the 3'-end, the 7th, 8th, and 9th nucleotides of the nucleotide sequence 1 are fluorine-modified nucleotides, and from the 5'-end to the 3'-end, the 2nd, 6th, 14th, and 16th nucleotides of the nucleotide sequence 2 are fluorine-modified nucleotides. nucleotides.

[0016] ​​​​​​​​ In some embodiments, the present disclosure provides a drug composition comprising the siRNA of the present disclosure and a pharmaceutically acceptable carrier.

[0017] In some embodiments, the present disclosure provides an siRNA complex comprising the siRNA provided by the present disclosure and a complexing group that binds to and complexes with the siRNA. The siRNA comprises a sense strand and an antisense strand, and both the sense strand and the antisense strand comprise both fluorine-modified nucleotides and non-fluorine-modified nucleotides. The sense strand comprises a nucleotide sequence I, the antisense strand comprises a nucleotide sequence II, and the nucleotide sequence I and the nucleotide sequence II form at least a partially reverse complementary double-stranded region. The nucleotide sequence I comprises a nucleotide sequence A, the nucleotide sequence A has the same length as the nucleotide sequence shown in SEQ ID NO: 155 and has 3 or fewer nucleotide differences, the nucleotide sequence II comprises a nucleotide sequence B, the nucleotide sequence B has the same length as the nucleotide sequence shown in SEQ ID NO: 156 and has 3 or fewer nucleotide differences, 5'-CCUUGAGGCAUACUUCAAZ-3' (SEQ ID NO: 155), 5'-Z'UUGAAGUAUGCCUCAAGG-3' (SEQ ID NO: 156) wherein Z is A and Z' is U. The nucleotide sequence A contains a nucleotide Z at the position corresponding to Z, the nucleotide sequence B contains a nucleotide Z' at the position corresponding to Z', and the Z' is 5'-CCUUGAGGCAUACUUCAAZ-3' (SEQ ID NO: 155), 5'-Z'UUGAAGUAUGCCUCAAGG-3' (SEQ ID NO: 156) wherein Z is A and Z' is U.

[0018] The nucleotide sequence A contains a nucleotide Z at the position corresponding to Z, and the nucleotide sequence B A contains a nucleotide Z' at the position corresponding to Z'. The nucleotide sequence B contains a nucleotide Z' at the position corresponding to Z', and the Z' B is B such that It is the first nucleotide at the 5'-end of the antisense strand.

[0019] The fluorine-modified nucleotide is located in nucleotide sequence A and nucleotide sequence B and, from the 5'-end to the 3'-end, the 7th, 8th, and 9th nucleotides of nucleotide sequence 1 are fluorine-modified nucleotides, and from the 5'-end to the 3'-end, the 2nd, 6th, 14th, and 16th nucleotides of nucleotide sequence 2 are fluorine-modified nucleotides. are.

[0020] In some embodiments, the present disclosure provides the use of the siRNA of the present disclosure, and / or the pharmaceutical composition and / or the siRNA complex, in the preparation of a medicament for the treatment and / or prevention of a pathological condition or disease caused by hepatitis B virus infection.

[0021] In some embodiments, the present disclosure provides a method for treating and / or preventing a pathological condition or disease caused by hepatitis B virus infection, comprising administering to a patient in need thereof an effective amount of the siRNA of the present disclosure, and / or the pharmaceutical composition and / or the siRNA complex.

[0022] In some embodiments, the present disclosure provides a method for suppressing the expression of HBV gene, comprising contacting an effective amount of the modified siRNA, pharmaceutical composition and / or siRNA complex of the present disclosure with hepatitis cells infected with the hepatitis B virus.

[0023] In some embodiments, the present disclosure provides a kit comprising the siRNA of the present disclosure, and / or the pharmaceutical composition and / or the siRNA complex.

[0024] [Incorporation by reference] All publications, patents, and patent applications mentioned in this specification are hereby incorporated by reference into this specification to the same extent as if each individual publication, patent, and patent application were specifically and individually indicated to be incorporated by reference. is incorporated by reference into this specification to the same extent as if each individual publication, patent, and patent application were specifically and individually indicated to be incorporated by reference. is incorporated by reference into this specification by citation.

Brief Description of the Drawings

[0025]

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Figure 28A - 28D

Mode for Carrying Out the Invention

[0026] The following describes in detail the mode for carrying out the invention of the present disclosure. Also, the mode for carrying out the invention described herein is merely for explaining or interpreting the present disclosure and should be understood not to be for limiting the present disclosure.

[0027] (Definition) In the present disclosure, the HBV gene refers to the gene whose DNA sequence is shown in Genbank accession number NC_00 3977.1.

[0028] In the context, unless otherwise specified, the capital letters C, G, U, A, and T represent the nucleotide base sequence, the small letter d represents that one nucleotide adjacent to the right side of the letter d is deoxy ribonucleotide, the small letter m represents that one nucleotide adjacent to the left side of the letter m is methoxy-modified nucleotide, the small letter f represents that one nucleotide adjacent to the left side of the letter f is fluoro-modified nucleotide, the small letter s represents that the two nucleotides adjacent to the left and right sides of the letter s are linked by a represents being combined, and P1 is one nucleotide adjacent to the right side of said P1 being 5 '-phosphate nucleotide or 5'-phosphate analog-modified nucleotide, in particular, vinyl phosphate ester-modified nucleotide (represented by VP in the following examples), 5'-phosphate nu cleotide (represented by P in the following examples) or 5'-thiophosphate ester-modified nu cleotide (represented by Ps in the following examples).

[0029] In the context, "fluoro-modified nucleotide" refers to a nucleotide in which the hydroxy group at the 2' position of the ribose group of the nucleotide is substituted with fluorine, and "non-fluoro-modified nu cleotide" refers to a nucleotide or nucleotide analog in which the hydroxy group at the 2' position of the ribose group of the nucleotide is substituted with a non-fluorinated group. "Nucleotide analog" refers to a group that can replace a nucleotide in nucleic acid but has a structure different from that of adenosine ribonucleotide, guanosine ribonucleotide, cytidine ribonucleotide, uracil ribonucleotide or thymine deoxyribonucleotide. For example, it includes isonucleotide, bridged nucleic acid (abbreviated as BNA) or acyclic nucleotide. The "methoxy-modified nucleotide" refers to a nucleotide in which the 2 '-hydroxy group of the ribose group is substituted with methoxy. In the context of this specification, the terms "complementary" or "reverse complementary" may be used interchangeably and have the meaning well-known to those skilled in the art, that is, in a double-stranded nucleic acid molecule, the bases on one strand pair complementarily with the bases on the other strand. In DNA, purine bases are bridged nucleic acid (abbreviated as BNA) or acyclic nucleotide. The "methoxy-modified nucleotide" refers to a nucleotide in which the 2 '-hydroxy group of the ribose group is substituted with methoxy.

[0030] In the context of this specification, the terms "complementary" or "reverse complementary" may be used interchangeably and have the meaning well-known to those skilled in the art, that is, in a double-stranded nucleic acid molecule, the bases on one strand pair complementarily with the bases on the other strand. In DNA, purine bases are complementary to pyrimidine bases, and in RNA, adenine and uracil are complementary to each other, and guanine and cytosine are complementary to each other. The term "complementary" also includes Watson-Crick base pairing and wobble base pairing. The term "reverse complementary" means that the base sequence of one strand is the reverse complement of the base sequence of the other strand. For example, if one strand has a base sequence of 5'-ATGC-3', its reverse complement strand has a base sequence of 3'-TACG-5'. The adenine (A) always pairs with the pyrimidine base thymine (T) (or uracil (U) in RNA), and the purine base guanine (C) always pairs with the pyrimidine base cytosine (G). Each base pair contains one purine and one pyrimidine. When adenine on one strand always pairs with thymine (or uracil) on the other strand and guanine always pairs with cytosine, the two strands are considered complementary, and it is thought that the sequence of one strand can be inferred from the sequence of its complementary strand. Accordingly, in this field, a "mismatch" means that in double-stranded nucleic acids, the bases at corresponding positions do not pair complementarily. In the context, unless otherwise specified, "substantially reverse complementary" means that there are three or fewer base mismatches between two related nucleotide sequences, "substantially reverse complementary" means that there is one or fewer base mismatches between two nucleotide sequences, and "fully complementary" means that there are no base mismatches between two nucleotide sequences. In the context, when there is a "nucleotide difference" between one nucleotide sequence and another nucleotide sequence, it means that the base type of the nucleotide at the same position has changed in the former compared to the latter. For example, when one nucleotide base is A in the latter, and at this time, if the corresponding nucleotide base at the same position in the former is U, C, G, or T, a nucleotide difference is considered to exist between the two nucleotide sequences at this position. In some embodiments, instead of the nucleotide at the original position, a abasic nucleotide or its The adenine on one strand always pairs with thymine (or uracil) on the other strand, and guanine always pairs with cytosine. In this case, the two strands are complementary, and it is considered that the sequence of this strand can be inferred from the sequence of its complementary strand. Accordingly, in this field, a "mismatch" means that in double-stranded nucleic acids, the bases at corresponding positions do not pair complementarily. When adenine on one strand always pairs with thymine (or uracil) on the other strand and guanine always pairs with cytosine, the two strands are complementary, and it is considered that the sequence of this strand can be inferred from the sequence of its complementary strand. Accordingly, in this field, a "mismatch" means that in double-stranded nucleic acids, the bases at corresponding positions do not pair complementarily. In the context, unless otherwise specified, "substantially reverse complementary" means that there are three or fewer base mismatches between two related nucleotide sequences, "substantially reverse complementary" means that there is one or fewer base mismatches between two nucleotide sequences, and "fully complementary" means that there are no base mismatches between two nucleotide sequences. In the context, when there is a "nucleotide difference" between one nucleotide sequence and another nucleotide sequence, it means that the base type of the nucleotide at the same position has changed in the former compared to the latter. For example, when one nucleotide base is A in the latter, and at this time, if the corresponding nucleotide base at the same position in the former is U, C, G, or T, a nucleotide difference is considered to exist between the two nucleotide sequences at this position.

[0031] In the context, unless otherwise specified, "substantially reverse complementary" means that there are three or fewer base mismatches between two related nucleotide sequences, "substantially reverse complementary" means that there is one or fewer base mismatches between two nucleotide sequences, and "fully complementary" means that there are no base mismatches between two nucleotide sequences. In the context, unless otherwise specified, "substantially reverse complementary" means that there are three or fewer base mismatches between two related nucleotide sequences, "substantially reverse complementary" means that there is one or fewer base mismatches between two nucleotide sequences, and "fully complementary" means that there are no base mismatches between two nucleotide sequences. In the context, unless otherwise specified, "substantially reverse complementary" means that there are three or fewer base mismatches between two related nucleotide sequences, "substantially reverse complementary" means that there is one or fewer base mismatches between two nucleotide sequences, and "fully complementary" means that there are no base mismatches between two nucleotide sequences. In the context, unless otherwise specified, "substantially reverse complementary" means that there are three or fewer base mismatches between two related nucleotide sequences, "substantially reverse complementary" means that there is one or fewer base mismatches between two nucleotide sequences, and "fully complementary" means that there are no base mismatches between two nucleotide sequences. In the context, unless otherwise specified, "substantially reverse complementary" means that there are three or fewer base mismatches between two related nucleotide sequences, "substantially reverse complementary" means that there is one or fewer base mismatches between two nucleotide sequences, and "fully complementary" means that there are no base mismatches between two nucleotide sequences.

[0032] In the context, when there is a "nucleotide difference" between one nucleotide sequence and another nucleotide sequence, it means that the base type of the nucleotide at the same position has changed in the former compared to the latter. For example, when one nucleotide base is A in the latter, and at this time, if the corresponding nucleotide base at the same position in the former is U, C, G, or T, a nucleotide difference is considered to exist between the two nucleotide sequences at this position. In the context, when there is a "nucleotide difference" between one nucleotide sequence and another nucleotide sequence, it means that the base type of the nucleotide at the same position has changed in the former compared to the latter. For example, when one nucleotide base is A in the latter, and at this time, if the corresponding nucleotide base at the same position in the former is U, C, G, or T, a nucleotide difference is considered to exist between the two nucleotide sequences at this position. In the context, when there is a "nucleotide difference" between one nucleotide sequence and another nucleotide sequence, it means that the base type of the nucleotide at the same position has changed in the former compared to the latter. For example, when one nucleotide base is A in the latter, and at this time, if the corresponding nucleotide base at the same position in the former is U, C, G, or T, a nucleotide difference is considered to exist between the two nucleotide sequences at this position. In the context, when there is a "nucleotide difference" between one nucleotide sequence and another nucleotide sequence, it means that the base type of the nucleotide at the same position has changed in the former compared to the latter. For example, when one nucleotide base is A in the latter, and at this time, if the corresponding nucleotide base at the same position in the former is U, C, G, or T, a nucleotide difference is considered to exist between the two nucleotide sequences at this position. In the context, when there is a "nucleotide difference" between one nucleotide sequence and another nucleotide sequence, it means that the base type of the nucleotide at the same position has changed in the former compared to the latter. For example, when one nucleotide base is A in the latter, and at this time, if the corresponding nucleotide base at the same position in the former is U, C, G, or T, a nucleotide difference is considered to exist between the two nucleotide sequences at this position. In some embodiments, instead of the nucleotide at the original position, a abasic nucleotide or its When using equivalents, it is also considered that nucleotide differences occurred at that position.

[0033] In the context, particularly when describing the method for preparing the composite molecule or the siRNA complex of the present disclosure, unless otherwise specified, the nucleoside monomer refers to an unmodified or modified RNA phosphoramidite monomer (RNA phosphoramidites are sometimes referred to as Nucleoside phosphoramidites) used in phosphoramidite solid-phase synthesis according to the type and order of nucleotides in the siRNA or siRNA complex to be prepared. Phosphoramidite solid-phase synthesis is a method used for RNA synthesis known to those skilled in the art. All nucleoside monomers used in the present disclosure can be purchased as commercial products. In the context of the present disclosure, unless otherwise specified, "composite" refers to the covalent bonding of two or more chemical moieties each having a specific function to each other, and accordingly, " complex" refers to a compound formed by the covalent bonding of these chemical moieties to each other. Further, "siRNA complex" represents a compound formed by the covalent bonding of one or more chemical moieties having a specific function to siRNA. Hereinafter, the siRNA complex of the present disclosure may also be simply referred to as "complex". The siRNA complex can be understood as a general term for siRNA complexes, the first type of siRNA complex, or the second type of siRNA complex depending on the context. In the context of the present disclosure, "composite molecule" is a molecule that, when reacted, forms siRNA. Phosphoramidite solid-phase synthesis is a method used for RNA synthesis known to those skilled in the art. All nucleoside monomers used in the present disclosure can be purchased as commercial products. In the context of the present disclosure, unless otherwise specified, "composite" refers to the covalent bonding of two or more chemical moieties each having a specific function to each other, and accordingly, "

[0034] complex" refers to a compound formed by the covalent bonding of these chemical moieties to each other. Further, "siRNA complex" represents a compound formed by the covalent bonding of one or more chemical moieties having a specific function to siRNA. Hereinafter, the siRNA complex of the present disclosure may also be simply referred to as "complex". The siRNA complex can be understood as a general term for siRNA complexes, the first type of siRNA complex, or the second type of siRNA complex depending on the context. In the context of the present disclosure, "composite molecule" is a molecule that, when reacted, forms siRNA. In the context of the present disclosure, unless otherwise specified, "composite" refers to the covalent bonding of two or more chemical moieties each having a specific function to each other, and accordingly, " complex" refers to a compound formed by the covalent bonding of these chemical moieties to each other. Further, "siRNA complex" represents a compound formed by the covalent bonding of one or more chemical moieties having a specific function to siRNA. Hereinafter, the siRNA complex of the present disclosure may also be simply referred to as "complex". The siRNA complex can be understood as a general term for siRNA complexes, the first type of siRNA complex, or the second type of siRNA complex depending on the context. In the context of the present disclosure, "composite molecule" is a molecule that, when reacted, forms siRNA. In the context of the present disclosure, "composite molecule" is a molecule that, when reacted, forms Specific compounds that can be combined and ultimately form the siRNA complexes of the present disclosure should be understood to be.

[0035] As used herein, a dash ( "-") that is not between two alphabetic characters or symbols is used to indicate the position of the attachment point of a substituent. For example, -C1 -C -C 10 alkyl-NH2 is attached by a C1-C 10 alkyl group.

[0036] As used herein, "optional" or "optionally" means that the subsequently described event or circumstance may or may not occur, and the foregoing description includes both the case where the event or circumstance occurs and the case where it does not occur. For example, "optionally substituted alkyl" includes both "alkyl" and "substituted alkyl" as defined in the following text. With respect to any group containing one or more substituents, it is not intended to introduce any substitution or substitution pattern that is sterically unrealistic, synthetically infeasible and / or essentially unstable, as will be understood by those skilled in the art. or essentially unstable, and it is understood by those skilled in the art that no substitution or substitution pattern is intended that would introduce any sterically unrealistic, synthetically infeasible and / or essentially unstable situations.

[0037] As used herein, "alkyl" refers to straight-chain and branched-chain groups having a specific number of carbon atoms, and the specific number is usually 1 to 20 carbon atoms, for example, 1 to 8 or 1 to 6 carbon atoms. For example, C1-C6 alkyl includes straight-chain and branched-chain alkyls having 1 to 6 carbon atoms. When naming an alkyl residue having a specific number of carbons, it is intended to include all branched-chain and straight-chain forms having that number of carbons. Thus, for example, "butyl" is intended to include n-butyl, sec-butyl, isobutyl, and tert-butyl. For example, C1-C6 alkyl includes straight-chain and branched-chain alkyls having 1 to 6 carbon atoms. When naming an alkyl residue having a specific number of carbons, it is intended to include all branched-chain and straight-chain forms having that number of carbons. Thus, for example, "butyl" is intended to include all branched-chain and straight-chain forms having that number of carbons. Thus, for example, "butyl" is intended to include n-butyl, sec-butyl, isobutyl, and tert-butyl. ​"Alkyl" means, and "propyl" includes n-propyl and isopropyl. Alkylene is a subset of alkyl and refers to a residue that is the same as alkyl but has two attachment points. When used herein, "alkenyl" refers to an unsaturated branched or straight-chain alkyl having at least one carbon-carbon double bond, where the carbon-carbon double bond is obtained by removing one hydrogen molecule from adjacent carbon atoms of the parent alkyl. The group may be in the cis or trans configuration of the double bond. Typical alkenyls include vinyl,

[0038] prop-1-en-1-yl, prop-1-en-2-yl, prop-2-en-1-yl (allyl), prop-2-en-2-yl and other propenyls, and but-1-en-1-yl, but-1-en-2-yl, 2-methylprop-1-en-1-yl, but-2-en-1-yl, but-2-en-2-yl, but-1,3-dien-1-yl, but-1,3-dien-2-yl and other butenyls, including but not limited to these. In certain embodiments, alkenyl has 2 to 20 carbon atoms, while in other embodiments, it has 2 to 10, 2 to 8 or 2 to 6 carbon atoms. Alkenylene is a subset of alkenyl and refers to a residue that is the same as alkenyl but has two attachment points. When used herein, "alkynyl" refers to an unsaturated branched or straight-chain alkyl group having at least one carbon-carbon triple bond, where the carbon-carbon triple bond is obtained by removing two hydrogen molecules from adjacent carbon atoms of the parent alkyl. Typical alkynyls include ethynyl, prop-1-yn-1-yl, prop-2-yn-1-yl, In certain embodiments, alkynyl has 2 to 20 carbon atoms, while in other embodiments, it has 2 to 10, 2 to 8 or 2 to 6 carbon atoms. Alkynylene is a subset of alkynyl and refers to a residue that is the same as alkynyl but has two attachment points.

[0039] Propynyl such as yl, and butynyl such as but-1-yn-1-yl, but-1-yn-3-yl, but- 3-yn-1-yl, etc., including but not limited to these. In certain embodiments wherein the alkynyl has 2 to 20 carbon atoms, but in other embodiments, 2 to 10, 2 to 8 or 2 to 6 carbon atoms. Alkynylene is a subset of alkynyl and refers to a residue that is the same as alkynyl but has two attachment points.

[0040] As used herein, "alkoxy" refers to an alkyl of a specific number of carbon atoms bonded by an oxygen bridge, for example, methoxy, ethoxy, propoxy, isopropoxy, n-butoxy, sec-butoxy, tert-butoxy, pentyloxy, 2-pentyloxy, isopentyloxy, neopentyloxy, hexyloxy, 2-hexyloxy, 3-hexyloxy, 3-methylpentyloxy, etc. Alkoxy usually has 1 to 10, 1 to 8, 1 to 6 or 1 to 4 carbon atoms bonded by an oxygen bridge.

[0041] As used herein, "aryl" refers to a radical formed by removing a hydrogen atom from a ring carbon atom, derived from an aromatic monocyclic or polycyclic hydrocarbon ring system. The said aromatic monocyclic or polycyclic hydrocarbon ring system contains only hydrogen and carbon of 6 to 18 carbon atoms, and at least one of the rings in this ring system is completely unsaturated, that is, it contains a cyclic, delocalized (4n + 2)π-electron system in accordance with Hückel's theory. Examples of aryl include, but are not limited to, groups such as phenyl, fluorenyl and naphthyl. Arylene is a subset of aryl and refers to a residue that is the same as aryl but has two attachment points.

[0042] As used herein, "cycloalkyl" refers to a group that typically contains 3 to 7 cyclic carbon atoms. The ring may be saturated or may contain one or more carbon-carbon dimers. Examples of cycloalkyl include cyclopropyl and cyclobutyl. , cyclopentyl, cyclopentenyl, cyclohexyl and cyclohexenyl, and no These include bridged and cage cyclic groups such as norbornane.

[0043] As used herein, a "halogen substituent" or "halo" refers to any group including fluoro, chloro, , bromo and iodo, and the term "halogen" includes fluorine, chlorine, bromine and iodine. .

[0044] As used herein, "halogenated alkyl" refers to an alkyl group having a specified number of carbon atoms. is an alkyl group as defined above, substituted with up to the maximum permitted number of halogen atoms. Examples of halogenated alkyl groups include trifluoromethyl, difluoromethyl, and , 2-fluoroethyl, and pentafluoroethyl.

[0045] The term "heterocyclic group" refers to a group having 2 to 12 carbon atoms and 1 to 2 carbon atoms selected from nitrogen, oxygen, and sulfur. It refers to a stable 3- to 18-membered non-aromatic cyclic group containing 6 heteroatoms. Unless otherwise specified, heterocyclic groups may be monocyclic, bicyclic, tricyclic or tetracyclic ring systems, fused or bridged. Heteroatoms in heterocyclic radicals may be optionally oxidized. One or more nitrogen atoms, if present, are optionally quaternized. The heterocyclic group is partially saturated. Heterocyclic groups can be attached to the rest of the molecule through any atom of the ring. It can be. Examples of such heterocyclic groups include dioxanyl, thiophenyl [1 ,3]disulfonyl, decahydroisoquinolinyl, imidazolinyl, imidazolidinyl, isothiazolidinyl, isoxazolidinyl, morpholinyl, octahydroindole, octahydroisoindole, 2-oxapiperazinyl, 2-oxapiperidyl, 2-o oxapyrimidinyl, oxazolidinyl, piperidyl, piperazinyl, 4-piperidonyl, pyrrolidinyl, pyrazolidyl, quinuclidinyl, thiazolidinyl, tetrahydrofuryl, trisulfonyl, tetrahydropyranyl, thiomorpholinyl, thiamorpholinyl, 1-o xothiomorpholinyl and 1,1-dioxothiomorpholinyl are included, but are not limited to these.

[0046] "Heteroaryl" refers to a group derived from a 3- to 18-membered aromatic ring radical containing 2 to 17 carbon atoms and 1 to 6 heteroatoms selected from nitrogen, oxygen and sulfur. As used herein, heteroaryl may be a monocyclic, bicyclic, tricyclic or tetracyclic system, and at least one of the rings in this ring system is completely unsaturated, i.e., it contains a cyclic delocalized (4n + 2)π-electron system according to Hückel's theory. Heteroaryl includes fused rings or bridged ring systems. The heteroatoms in heteroaryl are optionally oxidized. One or more nitrogen atoms (if present) are optionally quaternized. Heteroaryl is bonded to the rest of the molecule through any atom in the ring. Examples of heteroaryl include azepinyl , acridinyl, benzimidazolyl, benzoindole, 1,3-benzodioxazolyl , benzofuryl, benzoxazolyl, benzo[d]thiazolyl, benzothiadiazolyl , etc. yl, etc. lu, benzothieno[b][1,4]dioxazolyl, benzothieno[b][1,4]oxazolyl, 1 ,4-benzodioxazolyl, benzonaphthofuranyl, benzodiazolyl, benzodiox aphenyl, benzopyranyl, benzopyranonyl (benzopyranonyl), benzofuryl, benzofuranonyl, benzothiophene nyl, benzothieno[3,2-d]pyrimidinyl, benzotriazolyl, benzo[4,6 imidazo[1,2-a]pyridyl, carbazolyl, cinnolyl, cyclopenta[d]py rimidinyl, 6,7-dihydro-5H-cyclopenta[4,5]thieno[2,3-d]py rimidinyl, 5,6-dihydrobenz[h]quinazolinyl, 5,6-dihydrobenz[h cinnolinyl, 6,7-dihydro-5H-benzo[6,7]cyclohepta[1,2-c pyridazinyl, dibenzofuryl, dibenzothiophenyl, furyl, furanonyl, furo 3,2-c]pyridyl, 5,6,7,8,9,10-hexahydrocyclohepta[d]py rimidinyl, 5,6,7,8,9,10-hexahydrocycloocta[d]pyridazinyl 、5,6,7,8,9,10-hexahydrocycloocta[d]pyridyl, isothiazole ryl, indazolyl, imidazolyl, indole, isoindole, indolinyl, isoindolinyl 、isoquinolyl, indolizinyl, isoxazolyl, 5,8-methano-5, 6,7,8-tetrahydroquinazolinyl, naphthyridinonyl, 1,6-naphthyridinonyl 、oxadiazolyl, 2-oxoazepinyl, oxazolyl, oxiranyl, 5,6,6 a,7,8,9,10,10a-octahydrobenz[H]quinazolinyl, 1-phenyl -1H-pyrrolyl, phenazinyl, phenothiazinyl, phenoxazinyl, phthaloyl, pteridinyl, purinyl, pyrrolyl, pyrazolyl, pyrazolo[3,4-d]pyrimidinyl , pyridyl, pyrido[3,2-d]pyrimidinyl, pyrido[3,4-d]pyrimidinyl, pyrazinyl, pyrimidinyl, pyridazinyl, pyrrolyl, quinazolinyl, quinoxalinyl, quinolyl, isoquinolyl, tetrahydroquinolyl, 5,6,7,8-tetrahydroquinazo linyl, 5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimi nil, 6,7,8,9-tetrahydro-5H-cyclohepta[4,5]thieno[2,3-d pyrimidinyl, 5,6,7,8-tetrahydropyrido[4,5-c]pyridazinyl, th iazolyl, thiadiazolyl, triazolyl, tetrazolyl, triazinyl, thieno[2, 3-d]pyrimidinyl, thieno[3,2-d]pyrimidinyl, thieno[2,3-c]pur dinyl and thiophenyl, including but not limited to these.

[0047] In the present disclosure, various hydroxy protecting groups can be used. Generally, a protecting group can render chemical functionalities insensitive to specific reaction conditions and can be added to and then removed from such functionalities in a molecule without substantially damaging the remainder of the molecule. Representative hydroxy protecting groups are disclosed in Beaucage et al., Tetrahedron 1992, 48, 2223-2311, and Greene and Wuts, Protective Groups in Organic Synthesis, Chapter 2, 2d ed, John Wiley & Sons, New York, 1991, which are hereby incorporated by reference in their entirety. In some embodiments, the protecting group is stable under basic conditions but can be removed under acidic conditions. In some embodiments, non-exclusive examples of hydroxy protecting groups that can be used herein include dimethoxytrityl (DMT), monomethoxytrityl, 9-phenylxanthin-9-yl (Pixyl), and 9-(p-methoxyphenyl)xanthin-9-yl (Mox). In some embodiments, non-exclusive examples of hydroxy protecting groups that can be used herein include Tr (trityl), MMTr (4-methoxytrityl), DMTr (4,4'-dimethoxytrityl), and TMTr (4,4',4''-trimethoxytrityl).

[0048] As used herein, the term "subject" refers to any animal, e.g., a mammal or a marsupial. Subjects of the present disclosure include, but are not limited to, humans, non-human primates (e.g., rhesus monkeys or other species of macaque monkeys), mice, pigs, horses, donkeys, cows, sheep, rats, and any kind of poultry.

[0049] As used herein, the terms "treatment method", "treatment", "alleviation", or "improvement" may be used interchangeably herein. These terms refer to a method of obtaining a beneficial or desired result and , including, but not limited to, therapeutic effects. "Therapeutic effect" means eradicating or improving the potential disorder to be treated. Also, a therapeutic effect can be obtained by observing an improvement in the patient by eradicating or improving one or more physiological symptoms related to the potential disorder, even though the patient may still be susceptible to the pain of the potential disorder. and eradicating or improving the potential disorder. Also, a therapeutic effect can be obtained by observing an improvement in the patient by eradicating or improving one or more physiological symptoms related to the potential disorder, even though the patient may still be susceptible to the pain of the potential disorder. and eradicating or improving the potential disorder. Also, a therapeutic effect can be obtained by observing an improvement in the patient by eradicating or improving one or more physiological symptoms related to the potential disorder, even though the patient may still be susceptible to the pain of the potential disorder. and eradicating or improving the potential disorder. Also, a therapeutic effect can be obtained by observing an improvement in the patient by eradicating or improving one or more physiological symptoms related to the potential disorder, even though the patient may still be susceptible to the pain of the potential disorder.

[0050] As used herein, "prevention" and "prevention" may be used interchangeably. These terms refer to a method of obtaining a beneficial or desired result, including, but not limited to, a preventive effect. To obtain a "preventive effect", a diagnosis of the disease may not have been made, but the complex or composition can be administered to a patient at risk of developing a particular disease, or to a patient in whom one or more pathological symptoms of the disease have been reported. As used herein, "prevention" and "prevention" may be used interchangeably. These terms refer to a method of obtaining a beneficial or desired result, including, but not limited to, a preventive effect. To obtain a "preventive effect", a diagnosis of the disease may not have been made, but the complex or composition can be administered to a patient at risk of developing a particular disease, or to a patient in whom one or more pathological symptoms of the disease have been reported. As used herein, "prevention" and "prevention" may be used interchangeably. These terms refer to a method of obtaining a beneficial or desired result, including, but not limited to, a preventive effect. To obtain a "preventive effect", a diagnosis of the disease may not have been made, but the complex or composition can be administered to a patient at risk of developing a particular disease, or to a patient in whom one or more pathological symptoms of the disease have been reported. As used herein, "prevention" and "prevention" may be used interchangeably. These terms refer to a method of obtaining a beneficial or desired result, including, but not limited to, a preventive effect. To obtain a "preventive effect", a diagnosis of the disease may not have been made, but the complex or composition can be administered to a patient at risk of developing a particular disease, or to a patient in whom one or more pathological symptoms of the disease have been reported. As used herein, "prevention" and "prevention" may be used interchangeably. These terms refer to a method of obtaining a beneficial or desired result, including, but not limited to, a preventive effect. To obtain a "preventive effect", a diagnosis of the disease may not have been made, but the complex or composition can be administered to a patient at risk of developing a particular disease, or to a patient in whom one or more pathological symptoms of the disease have been reported.

[0051] <Modified siRNA> The siRNA of the present disclosure contains nucleotide groups as basic structural units, and it is known to those skilled in the art that the nucleotide groups contain phosphate groups, ribose groups and bases, so the description is omitted here. The siRNA of the present disclosure contains nucleotide groups as basic structural units, and it is known to those skilled in the art that the nucleotide groups contain phosphate groups, ribose groups and bases, so the description is omitted here. The siRNA of the present disclosure contains nucleotide groups as basic structural units, and it is known to those skilled in the art that the nucleotide groups contain phosphate groups, ribose groups and bases, so the description is omitted here.

[0052] CN102140458B discloses siRNAs that specifically suppress HBV genes, and studies are being conducted on multiple chemical modification strategies for the siRNAs. According to the study, it was found that different modification strategies have completely different effects on indicators such as the stability, biological activity and cytotoxicity of siRNAs. In the study, seven effective modification methods were demonstrated, and the siRNA obtained by one of the modification methods improved blood stability compared to the unmodified siRNA and maintained substantially the same inhibitory activity as the unmodified siRNA. CN102140458B discloses siRNAs that specifically suppress HBV genes, and studies are being conducted on multiple chemical modification strategies for the siRNAs. According to the study, it was found that different modification strategies have completely different effects on indicators such as the stability, biological activity and cytotoxicity of siRNAs. In the study, seven effective modification methods were demonstrated, and the siRNA obtained by one of the modification methods improved blood stability compared to the unmodified siRNA and maintained substantially the same inhibitory activity as the unmodified siRNA. CN102140458B discloses siRNAs that specifically suppress HBV genes, and studies are being conducted on multiple chemical modification strategies for the siRNAs. According to the study, it was found that different modification strategies have completely different effects on indicators such as the stability, biological activity and cytotoxicity of siRNAs. In the study, seven effective modification methods were demonstrated, and the siRNA obtained by one of the modification methods improved blood stability compared to the unmodified siRNA and maintained substantially the same inhibitory activity as the unmodified siRNA. CN102140458B discloses siRNAs that specifically suppress HBV genes, and studies are being conducted on multiple chemical modification strategies for the siRNAs. According to the study, it was found that different modification strategies have completely different effects on indicators such as the stability, biological activity and cytotoxicity of siRNAs. In the study, seven effective modification methods were demonstrated, and the siRNA obtained by one of the modification methods improved blood stability compared to the unmodified siRNA and maintained substantially the same inhibitory activity as the unmodified siRNA. CN102140458B discloses siRNAs that specifically suppress HBV genes, and studies are being conducted on multiple chemical modification strategies for the siRNAs. According to the study, it was found that different modification strategies have completely different effects on indicators such as the stability, biological activity and cytotoxicity of siRNAs. In the study, seven effective modification methods were demonstrated, and the siRNA obtained by one of the modification methods improved blood stability compared to the unmodified siRNA and maintained substantially the same inhibitory activity as the unmodified siRNA. CN102140458B discloses siRNAs that specifically suppress HBV genes, and studies are being conducted on multiple chemical modification strategies for the siRNAs. According to the study, it was found that different modification strategies have completely different effects on indicators such as the stability, biological activity and cytotoxicity of siRNAs. In the study, seven effective modification methods were demonstrated, and the siRNA obtained by one of the modification methods improved blood stability compared to the unmodified siRNA and maintained substantially the same inhibitory activity as the unmodified siRNA.

[0053] The present disclosure provides a modified siRNA capable of suppressing hepatitis B virus gene expression. The siRNA includes a sense strand and an antisense strand, and each nucleotide in the siRNA is a modified nucleotide. Both the sense strand and the antisense strand contain both fluoromodified nucleotides and non-fluoromodified nucleotides. The sense strand contains nucleotide sequence I, the antisense strand contains nucleotide sequence II, and the nucleotide sequence I and the nucleotide sequence II form a double-stranded region that is at least partially reverse complementary, the nucleotide sequence I contains nucleotide sequence A, the nucleotide sequence A has the same length as the nucleotide sequence shown in SEQ ID NO: 155 and has three or fewer nucleotide differences. The nucleotide sequence II contains nucleotide sequence B, and the nucleotide sequence B has the same length as the nucleotide sequence shown in SEQ ID NO: 156 and has three or fewer nucleotide differences, 5’-CCUUGAGGCAUACUUCAAZ-3’ (SEQ ID NO: 155), 5’-Z’UUGAAGUAUGCCUCAAGG-3’ (SEQ ID NO: 156) where Z is A and Z’ is U.

[0054] The nucleotide sequence A contains a nucleotide Z at a position corresponding to Z, A and the nucleotide sequence B contains a nucleotide Z’ at a position corresponding to Z’, B and Z’ B is the first nucleotide at the 5’ end of the antisense strand.

[0055] The fluoromodified nucleotides are located in the nucleotide sequence A and the nucleotide sequence B, , from the 5'-end to the 3'-end, the nucleotides at positions 7, 8, and 9 of the nucleotide sequence A are fluorinated nucleotides, and from the 5'-end to the 3'-end, the nucleotides at positions 2, 6, 14, and 16 of the nucleotide sequence B are fluorinated nucleotides. In some embodiments, the number of fluorinated nucleotides in the nucleotide sequence A is 5 or less, and the number of fluorinated nucleotides in the nucleotide sequence B is 7 or less.

[0056] In the context, "corresponding positions" means being at the same position in the nucleotide sequence starting from the same end of the nucleotide sequence. For example, the first nucleotide at the 3'-end of the nucleotide sequence A corresponds to the first nucleotide at the 3'-end of the nucleotide sequence with SEQ ID NO: 155.

[0057] In some embodiments, the sense strand contains only the nucleotide sequence I, and the antisense strand contains only the nucleotide sequence II.

[0058] In some embodiments, there is 1 or less nucleotide difference between the nucleotide sequence A and the nucleotide sequence shown in SEQ ID NO: 155, and / or there is 1 or less nucleotide difference between the nucleotide sequence B and the nucleotide sequence shown in SEQ ID NO: 156.

[0059] In some embodiments, the nucleotide difference between the nucleotide sequence B and the nucleotide sequence shown in SEQ ID NO: 156 includes the difference at the position of Z', B where Z' B is A. , is selected from C or G. In some embodiments, the nucleotide difference is Z ’ B at the position of, and Z’ B is selected from A, C or G. In some embodiments wherein, Z A is a nucleotide complementary to Z’ B . These nucleotide differences do not significantly reduce the ability of siRNA to suppress the target gene, and siRNAs containing these nu cleotide differences are also within the protection scope of the present disclosure.

[0060] In some embodiments, the nucleotide sequence A and the nucleotide sequence B are substantially reverse complementary, substantially reverse complementary or completely reverse complementary, and the substantially reverse complementary means that there are three or fewer base mismatches between the two nucleotide sequences , and the substantially reverse complementary means that there is one or fewer base mismatches between the two nucleotide sequences, and the completely reverse complementary means that there is no mismatch between the two nucleotide sequences.

[0061] In some embodiments, the nucleotide sequence A is the nucleotide sequence shown in SEQ ID NO: 1, and the nucleotide sequence B is the nucleotide sequence shown in SEQ ID NO: 2 , , 5'-CCUUGAGGCAUACUUCAAZ A -3'(SEQ ID NO: 1), 5'-Z' B UUGAAGUAUGCCUCAAGG-3'(SEQ ID NO: 2) wherein, the Z' B is the first nucleotide at the 5' end of the antisense strand, and Z A is selected from A, U, G or C, and Z' B is ZA and is a nucleotide complementary to From the 5'-end to the 3'-end, the 7th, 8th, and 9th nucleotides of the nucleotide sequence A are fluorine-modified nucleotides, and from the 5'-end to the 3'-end, the 2nd, 6th, 14th, and 16th nucleotides of the nucleotide sequence B are fluorine-modified nucleotides.

[0062] In some embodiments, the siRNA comprises a sense strand and an antisense strand, the sense strand comprises a nucleotide sequence I, the antisense strand comprises a nucleotide sequence II, the nucleotide sequence I and the nucleotide sequence II form a double-stranded region in an inverse complementary manner, the nucleotide sequence I comprises the nucleotide sequence shown in SEQ ID NO: 1, the nucleotide sequence II comprises the nucleotide sequence shown in SEQ ID NO: 2, 5'-CCUUGAGGCAUACUUCAAZ A -3' (SEQ ID NO: 1), 5'-Z' B UUGAAGUAUGCCUCAAGG-3' (SEQ ID NO: 2) Here, the Z' B is the first nucleotide at the 5'-end of the antisense strand, and Z A is selected from A, U, G, or C, and Z' B is a nucleotide complementary to Z A In some embodiments, Z A is A, and Z' B is U.

[0063] From the 5'-end to the 3'-end, the 7th, 8th, and 9th nucleotides of SEQ ID NO: 1 in the sense strand of the siRNA are fluorine-modified nucleotides, and the sense strand of the siRNA ​​​wherein the nucleotides at other positions are non-fluorinated modified nucleotides, and from the 5' end to the 3 ' end, in the antisense strand of the siRNA, the 2nd, 6th, 1st 4th, and 16th nucleotides of SEQ ID NO: 2 are fluorinated modified nucleotides, and the nucleotides at other positions in the antisense strand of the siRNA are non-fluorinated modified nucleotides.

[0064] The sense strand and the antisense strand may have the same or different lengths, the length of the sense strand is 19 to 23 nucleotides, and the length of the antisense strand is 20 to 26 nucleotides. Thus, the length ratio of the sense strand to the antisense strand of the siRNA provided by the present disclosure is 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 / 2 5, 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 strand to the antisense strand of the siRN A is 19 / 21, 21 / 23 or 23 / 25. .

[0065] According to one embodiment of the present disclosure, the sense strand and the antisense strand have the same length and the nucleotide sequence I further includes the nucleotide sequence III, the nucleotide sequence II further includes the nucleotide sequence IV, and the nucleotide sequences III and nucle otide sequences IV each independently have a length of 1 to 4 nucleotides, and the nucleo The nucleotide sequence III is linked to the 5'-end of the nucleotide sequence A, and the nucleotide sequence I V is linked to the 3'-end of the nucleotide sequence B, and the nucleotide sequence III and the nu cleotide sequence IV have equal lengths.

[0066] The nucleotide sequence III and the nucleotide sequence IV may or may not be complementary. In some embodiments, to improve the stability of the siRNA, the nucleotide sequence III and the nucleotide sequence IV are at least partially complementary, and in some embodiments, 8 0% or more or 90% or more of the bases of the nucleotide sequence III and the nucleotide sequence IV are complementary. In some embodiments, the nu cleotide sequence III and the nucleotide sequence IV are substantially reverse-complementary or completely reverse-complementary. By substantially reverse-complementary is meant that there is one or fewer base mismatches between the two nucleotide sequences, and by completely reverse-complementary is meant that there are no mismatches between the two nucleotide sequences. In some embodiments, the nucleotide sequence III and the nu cleotide sequence IV are completely reverse-complementary. Thereby, the sense strand and the antisense strand of the siRNA have equal lengths, and the length ratio is 20 / 20, 21 / 21, 22 / 22 or 23 / 23. In some embodiments, the length ratio of the sense strand and the antisense strand of the siRNA is 21 / 21 or 23 / 23. In some embodiments, the nucleotide sequence III and the nucleotide sequence IV are both 1 nucleotide in length, the base of the nucleotide sequence III is A, and the nu In some embodiments, the length ratio of the sense strand and the antisense strand of the siRNA is 21 / 21 or 23 / 23. sense strand and the antisense strand of the siRNA have equal lengths, and the length ratio is 20 / 20, 21 / 21, 22 / 22 or

[0067] In some embodiments, the nucleotide sequence III and the nucleotide sequence IV are both 1 nucleotide in length, the base of the nucleotide sequence III is A, and the nu The base of nucleotide sequence IV is U, and at this time, the length ratio of the sense strand to the antisense strand is 20 / 20, or nucleotide sequences III and IV are both 2 nucleotides in length, and from the 5' end to the 3' end, the base sequence of nucleotide sequence III is GA, the base sequence of nucleotide sequence IV is UC, and at this time, the length ratio of the sense strand to the anti- sense strand is 21 / 21, or nucleotide sequences III and IV are both 3 nucleotides in length, and from the 5' end to the 3' end, the base sequence of nucleotide sequence III is CGA, the base sequence of nucleotide sequence IV is UCG, and at this time, the length ratio of the sense strand to the anti- sense strand is 22 / 22, or nucleotide sequences III and IV are both 4 nucleotides in length, and from the 5' end to the 3' end, the base sequence of nucleotide sequence III is CCGA, the base sequence of nucleotide sequence IV is UCGG, and at this time, the length ratio of the sense strand to the anti- sense strand is 23 / 23. In some embodiments, the nucleotide sequence III and the nucleotide sequence IV are both 2 nucleotides in length, and from the 5' end to the 3' end, the base sequence of nucleotide sequence III is GA, the base sequence of nucleotide sequence IV is UC, and at this time, the length ratio of the sense strand to the anti- sense strand is 21 / 21. In some embodiments, the nucleotide sequence III and the nucleotide sequence IV are of the same length and are completely reverse complementary, so that when the base of nucleotide sequence III is given, the base of nucleotide sequence IV is also determined.

[0068] In some embodiments, nucleotide sequences III and IV are of the same length and are completely reverse complementary, so that when the base of nucleotide sequence III is given, the base of nucleotide sequence IV is also determined.

[0069] In some embodiments, the sense and antisense strands are different lengths, The nucleotide sequence II further comprises a nucleotide sequence V, the nucleotide sequence V having a length The length of the antisense strand is 1 to 3 nucleotides, and the antisense strand is bound to the 3' end of the antisense strand. This constitutes the 3' overhang end of the siRNA provided by the present disclosure. The length ratio of the sense strand to the antisense strand was 19 / 20, 19 / 21, 19 / 22, and 20 / 21, 20 / 22, 20 / 23, 21 / 22, 21 / 23, 21 / 24, 22 / 23, 2 It may be 2 / 24, 22 / 25, 23 / 24, 23 / 25 or 23 / 26. In some embodiments, the nucleotide sequence V is 2 nucleotides in length, Thus, the length ratio of the sense strand to the antisense strand of the siRNA provided by the present disclosure is 1 It may be 9 / 21, 21 / 23 or 23 / 25.

[0070] Each nucleotide in the nucleotide sequence V may be any nucleotide. In some embodiments, the nucleotide sequence V is a sequence of two consecutive thymine determinants. Oxyribonucleotide (TT) or two consecutive uracil ribonucleotides (UU) and in some embodiments, the nucleotide sequence V is is complementary to the nucleotide at position

[0071] In some embodiments, the sense strand of the siRNA is the nucleotide sequence set forth in SEQ ID NO:1. The antisense strand of the siRNA comprises the nucleotide sequence shown in SEQ ID NO:3. comprising a leutidine sequence, 5'-CCUUGAGGCAUACUUCAAZ A -3' (SEQ ID NO: 1), 5'-Z'B UUGAAGUAUGCCUCAAGGUU-3’ (SEQ ID NO: 3) Alternatively, the sense strand of the siRNA contains the nucleotide sequence shown in SEQ ID NO: 1 and the antisense strand of the siRNA contains the nucleotide sequence shown in SEQ ID NO: 4, 5’-CCUUGAGGCAUACUUCAAZ A -3’ (SEQ ID NO: 1), 5’-Z’ B UUGAAGUAUGCCUCAAGGUC-3’ (SEQ ID NO: 4) wherein said Z’ B is the first nucleotide at the 5’ end of the antisense strand, and Z A is selected from A, U, G or C, and Z’ B is a nucleotide complementary to Z A and is a nucleotide complementary to Z

[0072] According to some specific embodiments of the present disclosure, the siRNA described in the present disclosure is siH Ba1 or siHBa2. siHBa1 Sense strand: 5’-CCUUGAGGCAUACUUCAAA-3’ (SEQ ID NO: 5) Antisense strand: 5’-UUUGAAGUAUGCCUCAAGGUU-3’ (SEQ ID NO: 6) siHBa2 Sense strand: 5’-GACCUUGAGGCAUACUUCAAA-3’ (SEQ ID NO: 7) Antisense strand: 5’-UUUGAAGUAUGCCUCAAGGUCGG-3’ (SEQ NO: 8)

[0073] As described above, all of the nucleotides in the siRNA of the present disclosure are modified nucleotides, and these modifications on the nucleotide groups do not significantly weaken or lose the function of the siRNA complex of the present disclosure to inhibit hepatitis B virus gene expression. For example Well, J.K. Watts, G.F. Deleavey, and M.J. Damh a, Chemically modified siRNA: tools and ap plications. Drug Discov Today, 2008, 13(19 -20): 842-55, the modified nucleotides disclosed therein may be selected.

[0074] In some embodiments, from the 5'-end towards the 3'-end, in the sense strand, the nucleotides at positions 7, 8, 9 or 5, 7, 8, 9 of the nucleotide sequence A are fluoro-modified nucleotides, the nucleotides at other positions in the sense strand are non-fluoro-modified nucleotides, and in the antisense strand, the nucleotides at positions 2, 6, 14, 16 or positions 2, 6, 8, 9, 14, 16 of the nucleotide sequence B are fluoro-modified nucleotides, and the nucleotides at other positions in the antisense strand are non-fluoro-modified nucleotides.

[0075] In the context of the present disclosure, a fluoro-modified nucleotide refers to a nucleotide having the structure shown in the following formula (107), in which the hydroxy group at the 2'-position of the ribose group of the nucleotide is replaced by fluorine. A non-fluoro-modified nucleotide refers to a nucleotide or nucleotide analog in which the hydroxy group at the 2'-position of the ribose group of the nucleotide is replaced by a non-fluorinated group. In some embodiments, each non-fluoro-modified nucleotide is independently selected from any one of nucleotides or nucleotide analogs in which the hydroxy group at the 2'-position of the ribose group of the nucleotide is replaced by a non-fluorinated group.

[0076] Nucleotides in which the 2'-hydroxy group of these ribose groups is substituted with a non-fluorinated group are known to those skilled in the art, and these nucleotides may be one selected from 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 nu cleotides, 2'-deoxynucleotides.

[0077] In some embodiments, the 2'-alkoxy-modified nucleotide is a methoxy-modified nucleotide (2'-OMe) represented by formula (108). In some embodiments the 2'-substituted alkoxy-modified nucleotide may be, for example, a 2'- O-methoxyethyl-modified nucleotide (2'-MOE) represented by formula (109). In some embodiments the 2'-amino-modified nucleotide (2'-NH2) is represented by formula (110). In some embodiments the 2'-deoxynucleotide (DNA) is represented by formula (11 1).

[0078]

Chemical formula

[0079] A nucleotide analog is a group that can replace a nucleotide in a nucleic acid but has a structure different from that of an adenine ribonucleotide, a guanine ribonucleotide, a cytosine ribonucleotide, a uracil ribonucleotide or a thymine deoxyribonucleotide. In some embodiments, the nucleotide analog is an isonucleotide, a bridged nucleic acid (abbreviated as BNA), or an acyclic . In some embodiments nucleic acid.​ It may be a cyclic nucleotide.

[0080] BNA refers to nucleotides that are constrained or not close to each other. BNA may include a cross-linked structure having a "fixed" C3'-endo sugar packing of a five-membered ring, a six-membered ring, or a seven-membered ring. Usually, the bridge is introduced at the 2'- and 4'-positions of the ribose to provide a 2',4'-BNA nucleotide. In some embodiments, BNA may be LNA represented by formula (112), ENA represented by formula (113), cET BNA represented by formula (114) and the like. etc.

[0081]

Chemical Formula

[0082] An acyclic nucleotide is a nucleotide in which the sugar ring of the nucleotide is opened. In some embodiments, the acyclic nucleotide may be unlocked nucleic acid (UNA) represented by formula (115) or glycerol nucleic acid (GNA) represented by formula (116)

[0083]

Chemical Formula

[0084] In the above formulas (115) and (116), R is selected from H, OH or alkoxy (O-alkyl ).

[0085] An isonucleotide refers to a compound in which the position of the base in the ribose ring of the nucleotide is changed . In some embodiments, the isonucleotide may be of formula (117) or The compound shown in (118) may be one in which the base migrates from the 1'-position to the 2'-position or 3'-position of the ribose ring. It may also be a compound.

[0086]

Chemical formula

[0087] In the compounds of the above formulas (117) to (118), Base represents a base such as A, U, G, C or T, and R is selected from H, OH, F or the non-fluorinated groups described above.

[0088] In some embodiments, the nucleotide analog is any one selected from isonucleotide, LNA, ENA, cET, UNA and GNA. In some embodiments Each non-fluoro-modified nucleotide is a methoxy-modified nucleotide. In the context, the methoxy-modified nucleotide refers to a nucleotide in which the 2'-hydroxy group of the ribose group is substituted with methoxy. In the context, "fluoro-modified nucleotide", "2'-fluoro-modified nucleotide"

[0089] "nucleotide in which the 2'-hydroxy group of the ribose group is substituted with fluorine" and "2'- fluororibose group" have the same meaning, and all refer to a compound having the structure shown in formula (107) in which the 2'-hydroxy group of the nucleotide is substituted with fluorine. "Methoxy modified nucleotide", "2'-methoxy modified nucleotide", "nucleotide in which the 2'-hydroxy group of the ribose group is substituted with methoxy" and "2'-methoxyribose group" have the same meaning, and all refer to a compound having the structure shown in formula (108) in which the 2'-hydroxy group of the ribose group of the nucleotide is substituted with methoxy. ​

[0090] In some embodiments, the fluorine-modified nucleotide is located in nucleotide sequence A and nucleotide sequence B, the number of fluorine-modified nucleotides in the nucleotide sequence A is 5 or less, and from the 5'-end to the 3'-end, the 7th, 8th, and 9th nucleotides of the nucleotide sequence A are fluorine-modified nucleotides, the number of fluorine-modified nucleotides in the nucleotide sequence B is 7 or less, and the 2nd, 6th, 14th, and 16th nucleotides of the nucleotide sequence B are fluorine-modified nucleotides.

[0091] In some embodiments, from the 5'-end to the 3'-end, in the sense strand, the 7th, 8th, 9th or 5th, 7th, 8th, 9th nucleotides of the nucleotide sequence A are fluorine-modified nucleotides, and the nucleotides at other positions in the sense strand are non-fluorine-modified nucleotides. From the 5'-end to the 3'-end, in the antisense strand, the 2nd, 6th, 14th, 16th or 2nd, 6th, 8th, 9th, 14th, 16th nucleotides of the nucleotide sequence B are fluorine-modified nucleotides, and the nucleotides at other positions in the antisense strand are non-fluorine-modified nucleotides.

[0092] In some embodiments, the siRNA of the present disclosure is an siRNA having the following modifications. That is, from the 5'-end to the 3'-end, in the sense strand, the 7th, 8th, 9th or 5th, 7th, 8th, 9th nucleotides of the nucleotide sequence A are fluorine-modified nucleotides, and the nucleotides at other positions in the sense strand are methoxy-modified nucleotides. ​​​​​​​​​a nucleotide, and in the antisense strand, the 2nd, 6th, 1 4th, 16th positions or the 2nd, 6th, 8th, 9th, 14th, 16th positions of the nucleotide sequence B are fluorine-modified nucleotides, and the nucleotides at other positions in the antisense strand are methoxy-modified nucleotides is. In some embodiments, the siRNA of the present disclosure is an siRNA having the following modifications

[0093] That is, from the 5' end to the 3' end, the 5th, 7th, 8th, and 9th nucleotides of the nucleotide sequence A in the sense strand of the siRNA are fluorine-modified nucleotides and the nucleotides at other positions in the sense strand of the siRNA are methoxy-modified nucleotides In addition, from the 5' end to the 3' end, the 2nd, 6th, 8th, 9th, 14th, and 16th nucleotides of the nucleotide sequence B in the antisense strand of the siRNA are fluorine modified nucleotides, and the nucleotides at other positions in the antisense strand of the siRNA are methoxy-modified nucleotides Or, from the 5' end to the 3' end, the 5th, 7th, 8th, and 9th nucleotides of the nucleotide sequence A in the sense strand of the siRNA are fluorine-modified nucleotides and the nucleotides at other positions in the sense strand of the siRNA are methoxy-modified nucleotides In addition, from the 5' end to the 3' end, the 2nd, 6th, 14th, and 16th nucleotides of the nucleotide sequence B in the antisense strand of the siRNA are fluorine modified nucleotides, and the nucleotides at other positions in the antisense strand of the siRNA are methoxy-modified nucleotides Or, from the 5' end to the 3' end, the 5th, 7th, 8th, and 9th nucleotides of the nucleotide sequence A in the sense strand of the siRNA are fluorine-modified nucleotides and the nucleotides at other positions in the sense strand of the siRNA are methoxy-modified nucleotides In addition, from the 5' end to the 3' end, the 2nd, 6th, 14th, and 16th nucleotides of the nucleotide sequence B in the antisense strand of the siRNA are fluorine modified nucleotides, and the nucleotides at other positions in the antisense strand of the siRNA are methoxy-modified nucleotides Or, from the 5' end to the 3' end, the 5th, 7th, 8th, and 9th nucleotides of the nucleotide sequence A in the sense strand of the siRNA are fluorine-modified nucleotides and the nucleotides at other positions in the sense strand of the siRNA are methoxy-modified nucleotides is. Or, from the 5' end to the 3' end, the nucleotides in the sense strand of the siRNA The nucleotides at positions 7, 8, and 9 of the oligonucleotide sequence A are fluorinated nucleotides, and s the nucleotides at other positions of the sense strand of the siRNA are methoxylated nucleotides, and also , from the 5'-end to the 3'-end, the nucleo the nucleotides at positions 2, 6, 14, and 16 of the nucleotide sequence B of the antisense strand of the siRNA are fluorinated nucleotides and the nucleotides at other positions of the antisense strand of the siRNA are methoxylated nucleotides .

[0094] In other words, the ribose groups in the phosphate-sugar backbone of the siRNA each have the following mod ifying groups. From the 5'-end to the 3'-end, the nucleo the glycosyl groups at positions 5, 7, 8, and 9 of the nucleotide sequence A of the sense strand of the siRNA are 2'-fluororibose groups and the glycosyl groups of the nucleotides at other positions of the sense strand of the siRNA are 2'-methoxy ribose groups. Also, from the 5'-end to the 3'-end, the nucleo the glycosyl groups at positions 2, 6, 8, 9, 14, and 16 of the nucleotide sequence B of the antisense strand of the siRNA are 2'-fluororibose groups and the glycosyl groups of the nucleotides at other positions of the antisense strand of the siRNA are 2'-methoxyribose groups, or, from the 5'-end to the 3'-end, the nucleo the glycosyl groups at positions 5, 7, 8, and 9 of the nucleotide sequence A of the sense strand of the siRNA are 2'-fluororibose groups and the glycosyl groups of the nucleotides at other positions of the sense strand of the siRNA are 2'-methoxyribo se groups. Also, from the 5'-end to the 3'-end, the antisense the glycosyl groups at positions 2, 6, 14, and 16 of the nucleotide sequence B of the antisense strand of the siRNA are 2'-fluoro is an orolibose group, and the glycosyl groups of nucleotides at other positions of the antisense strand of siRNA is a 2'-methoxyribose group, alternatively, from the 5'-end to the 3'-end, the nucleotides in the sense strand of the siRNA the glycosyl groups at the 7th, 8th, and 9th positions of nucleotide sequence A are 2'-fluororibose groups, and s the glycosyl groups of nucleotides at other positions of the sense strand of iRNA are 2'-methoxyribose groups, and also from the 5'-end to the 3'-end, in the antisense strand of the siRNA the glycosyl groups at the 2nd, 6th, 14th, and 16th positions of nucleotide sequence B are 2'-fluoro ribose groups, and the glycosyl groups of nucleotides at other positions of the antisense strand of siRNA are 2'-methoxyribose groups.

[0095] In some embodiments, the siRNA provided by the present disclosure is siHBa1M 1, siHBa1M2, siHBa2M1 or siHBa2M2. siHBa1M1 Sense strand: 5'-CmCmUmUmGmAmGfGfCfAmUmAmCmUmUmC mAmAmAm-3'(SEQ ID NO: 9) Antisense strand: 5'-UmUfUmGmAmAfGmUmAmUmGmCmCmUf CmAfAmGmGmUmUm-3'(SEQ ID NO: 10) siHBa1M2 Sense strand: 5'-CmCmUmUmGfAmGfGfCfAmUmAmCmUmUmC mAmAmAm-3'(SEQ ID NO: 11) Antisense strand: 5'-UmUfUmGmAmAfGmUfAfUmGmCmCmUf CmAfAmGmGmUmUm-3'(SEQ ID NO: 12) siHBa2M1 Sense strand: 5’-GmAmCmCmUmUmGmAmGfGfCfAmUmAmCmU mUmCmAmAmAm-3’ (SEQ ID NO: 13) Antisense strand: 5’-UmUfUmGmAmAfGmUmAmUmGmCmCmUf CmAfAmGmGmUmCmGmGm-3’ (SEQ ID NO: 14) siHBa2M2 Sense strand: 5’-GmAmCmCmUmUmGfAmGfGfCfAmUmAmCmU mUmCmAmAmAm-3’ (SEQ ID NO: 15) Antisense strand: 5’-UmUfUmGmAmAfGmUfAfUmGmCmCmUf CmAfAmGmGmUmCmGmGm-3’ (SEQ ID NO: 16)

[0096] However, the capital letters C, G, U, and A represent the nucleotide base sequences, and the lowercase letter m represents that the single nucleotide adjacent to the left of the letter m is a 2’-methoxy-modified nucleotide and the lowercase letter f represents that the single nucleotide adjacent to the left of the letter f is a 2’-fluoro modified nucleotide. The siRNA having the above modifications not only has a low cost but also can make it difficult for ribonucleases in the blood to cleave the nucleic acid, thereby improving the stability of the nucleic acid and endowing the nucleic acid with strong nuclease hydrolysis resistance .

[0097] In some embodiments, in the sense strand and antisense strand of the siRNA provided by the present disclosure, at least some of the phosphate ester groups in at least one single-stranded phosphate-sugar backbone are phosphate ester groups having a modifying group. In some embodiments , the phosphate ester group having a modifying group is a phosphodiester bond in the phosphate ester group It is a thiophosphate ester group in which at least one oxygen atom is replaced by a sulfur atom. In several embodiments, the phosphate ester group having the modifying group is a thiophosphate ester group having a structure represented by formula (101).

[0098]

Chemical formula

[0099] By such modification, the double-stranded structure of siRNA can be stabilized, and high specificity of base pairing and high affinity can be maintained.

[0100] In some embodiments, in the siRNA provided by the present disclosure, the thiophosphate ester group is present bonded to at least one selected from the group consisting of between the first nucleotide and the second nucleotide at any one end of the sense strand or the antisense strand, between the second nucleotide and the third nucleotide at any one end of the sense strand or the antisense strand, or any combination thereof. In some embodiments, the thiophosphate ester group is present bonded to all of the above positions except the 5' end of the sense strand. In some embodiments, the thiophosphate ester group is present bonded to all of the above positions except the 3' end of the sense strand. In some embodiments, the thiophosphate ester group is present bonded to at least one of the following positions. Between the first nucleotide and the second nucleotide from the 5' end of the sense strand, Between the second nucleotide and the third nucleotide from the 5' end of the sense strand,

[0101] In some embodiments, the thiophosphate ester group is present bonded to at least one of the following positions. Between the first nucleotide and the second nucleotide from the 5' end of the sense strand, Between the second nucleotide and the third nucleotide from the 5' end of the sense strand, ​​​​Between the first nucleotide and the second nucleotide from the 3'-end of the sense strand, Between the second nucleotide and the third nucleotide from the 3'-end of the sense strand, Between the first nucleotide and the second nucleotide from the 5'-end of the antisense strand and Between the second nucleotide and the third nucleotide from the 5'-end of the antisense strand and Between the first nucleotide and the second nucleotide from the 3'-end of the antisense strand and Between the second nucleotide and the third nucleotide from the 3'-end of the antisense strand and

[0102] In some embodiments, the siRNA provided by the present disclosure is siHBa1M 1S, siHBa1M2S, siHBa2M1S or siHBa2M2S. siHBa1M1S Sense strand: 5'-CmsCmsUmUmGmAmGfGfCfAmUmAmCmUmU mCmAmAmAm-3'(SEQ ID NO: 17) Antisense strand: 5'-UmsUfsUmGmAmAfGmUmAmUmGmCmCm UfCmAfAmGmGmsUmsUm-3'(SEQ ID NO: 18) siHBa1M2S Sense strand: 5'-CmsCmsUmUmGfAmGfGfCfAmUmAmCmUmU mCmAmAmAm-3'(SEQ ID NO: 19) Antisense strand: 5'-UmsUfsUmGmAmAfGmUfAfUmGmCmCm UfCmAfAmGmGmsUmsUm-3'(SEQ ID NO: 20) siHBa2M1S Sense strand: 5'-GmsAmsCmCmUmUmGmAmGfGfCfAmUmAmC mUmUmCmAmAmAm-3'(SEQ ID NO: 21) Antisense strand: 5’-UmsUfsUmGmAmAfGmUmAmUmGmCmCm UfCmAfAmGmGmUmCmsGmsGm-3’ (SEQ ID NO: 22) siHBa2M2S Sense strand: 5’-GmsAmsCmCmUmUmGfAmGfGfCfAmUmAmC mUmUmCmAmAmAm-3’ (SEQ ID NO: 23) Antisense strand: 5’-UmsUfsUmGmAmAfGmUfAfUmGmCmCm UfCmAfAmGmGmUmCmsGmsGm-3’ (SEQ ID NO: 24)

[0103] However, capital letters C, G, U, and A represent the nucleotide base sequences, and lowercase letter m indicates that one nucleotide adjacent to the left of the letter m is a methoxy-modified nucleotide, lowercase letter f indicates that one nucleotide adjacent to the left of the letter f is a fluoro-modified nucleotide, lowercase letter s indicates that the two nucleotides on the left and right of the letter are linked by a phosphorothioate ester group.

[0104] In some embodiments, the nucleotide at the 5’ end of the antisense strand of the siRNA is a 5’-phosphate nucleotide or a 5’-phosphate analog-modified nucleotide.

[0105] The conventional 5’-phosphate nucleotide or 5’-phosphate analog-modified nucleotide is known to those skilled in the art. For example, the 5’-phosphate nucleotide may have the following structure

[0106]

Chemical formula

[0107] ​​​Also, for example, Anastasia Khvorova and Jonathan K. Watts, The chemical evolution of oligo nucleotide therapies of clinical utility . Nature Biotechnology, 2017, 35(3): 238~48 discloses the following four types of 5'-phosphate analog-modified nucleotides.

[0108]

Chemical formula

[0109] In some embodiments, the 5'-phosphate nucleotide is a nucleotide containing a 5'-phosphate modification shown in formula (102), and the 5'-phosphate analog-modified nucleotide is a nucleotide containing a vinyl phosphate ester (5'-(E)-vinylphosphonate, E-VP) modification shown in formula (103), or a thiophosphate ester-modified nucleotide shown in formula (105). is a nucleotide containing a vinyl phosphate ester (5'-(E)-vinylphosphonate, E-VP) modification shown in formula (103), or a thiophosphate ester-modified nucleotide shown in formula (105). sphonate, E-VP) modification shown in formula (103), or a thiophosphate ester-modified nucleotide shown in formula (105). is a thiophosphate ester-modified nucleotide shown in formula (105).

[0110] In some embodiments, the siRNA provided by the present disclosure is any one of siHBa1M 1P1, siHBa1M2P1, siHBa2M1P1, siHBa2M2P1, siH Ba1M1SP1, siHBa1M2SP1, siHBa2M1SP1, siHBa2M 2SP1. siHBa1M1P1 Sense strand: 5'-CmCmUmUmGmAmGfGfCfAmUmAmCmUmUmC mAmAmAm-3’(SEQ ID NO: 25) Antisense strand: 5’-P1-UmUfUmGmAmAfGmUmAmUmGmCmC mUfCmAfAmGmGmUmUm-3’(SEQ ID NO: 26) siHBa1M2P1 Sense strand: 5’-CmCmUmUmGfAmGfGfCfAmUmAmCmUmUmC mAmAmAm-3’(SEQ ID NO: 27) Antisense strand: 5’-P1-UmUfUmGmAmAfGmUfAfUmGmCmC mUfCmAfAmGmGmUmUm-3’(SEQ ID NO: 28) siHBa2M1P1 Sense strand: 5’-GmAmCmCmUmUmGmAmGfGfCfAmUmAmCmU mUmCmAmAmAm-3’(SEQ ID NO: 29) Antisense strand: 5’-P1-UmUfUmGmAmAfGmUmAmUmGmCmC mUfCmAfAmGmGmUmCmGmGm-3’(SEQ ID NO: 30) siHBa2M2P1 Sense strand: 5’-GmAmCmCmUmUmGfAmGfGfCfAmUmAmCmU mUmCmAmAmAm-3’(SEQ ID NO: 31) Antisense strand: 5’-P1-UmUfUmGmAmAfGmUfAfUmGmCmC mUfCmAfAmGmGmUmCmGmGm-3’(SEQ ID NO: 32) siHBa1M1SP1 Sense strand: 5’-CmsCmsUmUmGmAmGfGfCfAmUmAmCmUmU mCmAmAmAm-3’(SEQ ID NO: 33) Antisense strand: 5’-P1-UmsUfsUmGmAmAfGmUmAmUmGmC mCmUfCmAfAmGmGmsUmsUm-3’(SEQ ID NO: 34) siHBa1M2SP1 Sense strand: 5’-CmsCmsUmUmGfAmGfGfCfAmUmAmCmUmU mCmAmAmAm-3’ (SEQ ID NO: 35) Antisense strand: 5’-P1-UmsUfsUmGmAmAfGmUfAfUmGmC mCmUfCmAfAmGmGmsUmsUm-3’ (SEQ ID NO: 36) siHBa2M1SP1 Sense strand: 5’-GmsAmsCmCmUmUmGmAmGfGfCfAmUmAmC mUmUmCmAmAmAm-3’ (SEQ ID NO: 37) Antisense strand: 5’-P1-UmsUfsUmGmAmAfGmUmAmUmGmC mCmUfCmAfAmGmGmUmCmsGmsGm-3’ (SEQ ID NO: 38) siHBa2M2SP1 Sense strand: 5’-GmsAmsCmCmUmUmGfAmGfGfCfAmUmAmC mUmUmCmAmAmAm-3’ (SEQ ID NO: 39) Antisense strand: 5’-P1-UmsUfsUmGmAmAfGmUfAfUmGmC mCmUfCmAfAmGmGmUmCmsGmsGm-3’ (SEQ ID NO: 40)

[0111] However, capital letters C, G, U, and A represent the nucleotide base sequences, and lowercase letter m represents that the one nucleotide adjacent to the left of the letter m is a 2’-methoxy-modified nucleotide. Lowercase letter f represents that the one nucleotide adjacent to the left of the letter f is a 2’-fluoro modified nucleotide. Lowercase letter s represents that the two nucleotides adjacent to the left and right of the letter s are linked by a phosphorothioate group. P1 represents that the one nucleotide adjacent to the right of the letter P1 is a 5’-phosphate nucleotide or a 5’-phosphate analog-modified nucleotide.

[0112] The inventors of the present disclosure have found that the siRNA provided by the present disclosure has significantly improved plasma and lysosomal stability, not only reduces off-target effects, but also unexpectedly maintains very high gene silencing activity.

[0113] The siRNA provided by the present disclosure can be obtained by ordinary siRNA preparation methods in the art (e.g., solid-phase synthesis method and liquid-phase synthesis method). Here, solid-phase synthesis already has a commercial customization service. By using nucleoside monomers with corresponding modifications, modified nucleotide groups can be introduced into the siRNA described in the present disclosure, and the methods for preparing nucleoside monomers with corresponding modifications and the methods for introducing modified nucleotide groups into siRNA are also well known to those skilled in the art.

[0114] <Drug Composition> The present disclosure provides a drug composition comprising the above-described siRNA as an active ingredient and a pharmaceutically acceptable carrier.

[0115] The pharmaceutically acceptable carrier may be a carrier commonly used in the field of siRNA administration, such as, for example, magnetic nanoparticles (e.g., nanoparticles based on Fe3O4 or Fe2O3), carbon nanotubes (carbon nanotubes), mesoporous silicon (mesoporous silico n), calcium phosphate nanoparticles (calcium phosphate nanopa rticles), polyethyleneimine (PEI) polyamidoamine dendrimer (PAMAM) d endrimer), poly(L-lysine) (PLL), chitosan (chitosan), 1,2-dioleoyl-3-trimethylammonium-propane 1,2-dioleoyl-3-trimethylammonium-propane 、DOTAP), poly(D- or L-lactic / glycolic acid) copolymer (PLGA), poly(2-aminoethyl ethylene phosphate) (PPEEA), and poly(N,N-dimethylaminoethyl methacrylate) (PDMAEMA), and one or more of their derivatives, but not limited to these. actic / glycolic acid)copolymer, PLGA), poly(2-aminoethyl ethylene phosphate) (PPEEA) and poly(N,N-dimethylaminoethyl methacrylate) (PDMAEMA) and one or more of these derivatives, but not limited to these. lene phosphate), PPEEA) and poly(N,N-dimethylaminoethyl meth acrylate), PDMAEMA) and one or more of these derivatives, but not limited to these. These are not limited to these.

[0116] In some embodiments, there are no special requirements for the content of siRNA and pharmaceutically acceptable carriers in the drug composition, but in some embodiments, the weight ratio of siRNA to pharmaceutically acceptable carriers may be 1:(1 - 500). In some embodiments, the above weight ratio is 1:(1 - 50). In some embodiments, there are no special requirements for the content of siRNA and pharmaceutically acceptable carriers in the drug composition, but in some embodiments, the weight ratio of siRNA to pharmaceutically acceptable carriers may be 1:(1 - 500). In some embodiments, the above weight ratio is 1:(1 - 50). NA and pharmaceutically acceptable carriers may be 1:(1 - 500). In some embodiments, the above weight ratio is 1:(1 - 50). In some embodiments, the above weight ratio is 1:(1 - 50).

[0117] In some embodiments, the drug composition may contain other pharmaceutically acceptable additives, which may be one or more of various formulations or compounds commonly used in this field. For example, the other pharmaceutically acceptable additives may include at least one of pH buffer, In some embodiments, the drug composition may contain other pharmaceutically acceptable additives, which may be one or more of various formulations or compounds commonly used in this field. For example, the other pharmaceutically acceptable additives may include at least one of pH buffer, In some embodiments, the drug composition may contain other pharmaceutically acceptable additives, which may be one or more of various formulations or compounds commonly used in this field. For example, the other pharmaceutically acceptable additives may include at least one of pH buffer, protective agent, and osmotic pressure regulator.

[0118] The pH buffer may be tris (hydroxymethyl) aminomethane hydrochloride with a pH of 7.5 to 8.5 buffer (tris (hydroxymethyl) aminomethane hyd rochloride buffer) and / or a phosphate buffer with a pH of 5.5 to 8.5 and may be, for example, a phosphate buffer with a pH of 5.5 to 8.5.

[0119] The protectant may be at least one of inositol, sorbitol, sucrose, trehalose, mannose , maltose, lactose and glucose. Based on the total weight of the drug composition , the content of the protectant may be 0.01 to 30% by weight.

[0120] The osmotic pressure regulator may be sodium chloride and / or potassium chloride. The content of the osmotic pressure regulator is determined such that the osmotic pressure of the drug composition is 200 to 700 milliosmoles / kilogram ram. Depending on the desired osmotic pressure, those skilled in the art can easily determine the content of the osmotic pressure regulator .

[0121] In some embodiments, the drug composition may be a liquid preparation such as an injection solution, or may be a freeze-dried powder injection, which is mixed with a liquid additive during administration to form a liquid preparation . The above liquid preparation can be used for subcutaneous, intramuscular or intravenous injection administration, but is not limited thereto . It can also be administered to the lungs by spraying, or through the lungs to other organ tissues (e.g., the liver ) by spraying, but is not limited thereto. In some embodiments, the above drug composition is used for intravenous injection administration .

[0122] In some embodiments, the drug composition may be in the form of a liposome preparation​​ Yes. In some embodiments, the pharmaceutically acceptable carrier used in the liposomal formulation comprises an amine-containing transfection compound (hereinafter also referred to as an organic amine), a co-lipid and / or a polyethylene glycol (PEG)-ylated lipid. Here, the organic amine , co-lipid and PEG-ylated lipid may each be one or more selected from the amine-containing transfection compounds or pharmaceutically acceptable salts or derivatives thereof, co-lipids and PEG-ylated lipids described in CN103380113A (incorporated herein by reference in its entirety).

[0123] In some embodiments, the organic amine may be a compound represented by formula (201) described in CN103380113A or a pharmaceutically acceptable salt thereof.

[0124]

Chemical formula

[0125]

Chemical formula

[0126] In some embodiments, R 103 is a polyamine. In other embodiments, R 103 is a ketal. In some embodiments, R in formula (201) 10 1 and R 102 each are independently an optionally substituted or unsubstituted, branched or linear alkyl or alkenyl, wherein the alkyl or alkenyl has 3 to about 20 carbon atoms, for example, 8 to about 18 carbon atoms, and 0 to 4 double bonds, for example, 0 to 2 double bonds are present.

[0127] In some embodiments, when each of n and m is independently a value of 1 or 3, R 103 may be any one of the following formulas (204) to (213).

[0128] [Chemical formula] In Formulas (204) to (213), g, e, and f are each independently an integer from 1 to 6 , each "HCC" represents a hydrocarbon chain, and each * is R 103 and the bondable point with the nitrogen atom in Formula (201) is shown, and each H at any * position may be substituted to achieve the bond with the nitrogen atom in Formula (201).

[0129] The compound shown in Formula (201) may be prepared according to the description in CN103380113A as well.

[0130] In some embodiments, the organic amine is the organic amine shown in Formula (214) and / or the organic amine shown in Formula (215).

[0131] [Chemical formula] The co-lipid is cholesterol, an analog of cholesterol, and / or a derivative of cholesterol , and the PEGylated lipid is 1,2-dipalmitamide-sn-glycero-3-phosphatidyl ethanolamine-N-[methoxy(polyethylene glycol)]-2000.

[0132] In some embodiments, in the pharmaceutical composition, the molar ratio of the organic amine, the co-lipid and the PEGylated lipid is (19.7 to 80):(19.7 to 80):(0.3 to 50), for example, (50 to 70):(20 to 40):(3 to 20) may also be acceptable.

[0133] In some embodiments, the drug composition particles formed by the siRNA of the present disclosure and the amine-containing transfection reagent have an average diameter of about 30 nm to about 200 nm and are generally about 40 nm to about 135 nm, and more generally, the average diameter of the liposome particles is about 50 nm to about 120 nm, about 50 nm to about 100 nm, about 60 nm to about 90 nm or about 70 nm to about 90 nm. For example, the average diameter of the liposome particles is about 30, 4 0, 50, 60, 70, 75, 80, 85, 90, 100, 110, 120, 130, 1 40, 150 or 160 nm. In some embodiments, in the drug composition formed by the siRNA of the present disclosure and the amine-containing transfection reagent, the weight ratio (weight / weight ratio) of siRNA to total lipid (e.g., organic amine

[0134] , co-lipid and / or PEGylated lipid) is about 1:1 to about 1:5 0, about 1:1 to about 1:30, about 1:3 to about 1:20, about 1:4 to about 1:18, about 1:5 to about 1:17, about 1:5 to about 1:15, about 1:5 to about 1:12, about 1:6 to about 1:12 or about 1:6 to about 1:10, and for example, the weight ratio of the siRNA of the present disclosure to total lipid is about 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13 , 1:14, 1:15, 1:16, 1:17 or 1:18. In some embodiments, the drug composition may exist with each component independently when marketed, or may exist as a liquid formulation when in use. In some embodiments, the drug composition formed by the siRNA provided by the present disclosure and the pharmaceutically acceptable carrier

[0135] In some embodiments, the drug composition may exist with each component independently when marketed, or may exist as a liquid formulation when in use. In some embodiments, the drug composition formed by the siRNA provided by the present disclosure and the pharmaceutically acceptable carrier formed by the siRNA provided by the present disclosure and the pharmaceutically acceptable carrier It may be prepared according to various known methods, and the siRNA provided by the present disclosure may be used instead of the conventional siRNA. In some embodiments, it may be prepared according to the following methods. Mix the organic amine, co-lipid, and PEGylated lipid in the above molar ratio and suspend them in alcohol to obtain a homogeneous mixture to obtain a lipid solution. The dosage of alcohol is determined such that the total mass concentration of the obtained lipid solution is 2 to 25 mg / mL, for example, 8 to 18 mg / mL. The alcohol is one or more selected from pharmaceutically acceptable alcohols such as ethanol, propylene glycol, benzyl alcohol, glycerin, polyethylene glycol 200, polyethylene glycol 300, polyethylene glycol 400, etc., which are liquids near room temperature, and may be, for example, ethanol. It may be prepared according to the following methods.

[0136] The organic amine, co-lipid, and PEGylated lipid are suspended in alcohol in the above molar ratio and uniformly mixed to obtain a lipid solution. The dosage of alcohol is determined such that the total mass concentration of the obtained lipid solution is 2 to 25 mg / mL, for example, 8 to 18 mg / mL. The alcohol is selected from one or more pharmaceutically acceptable alcohols such as ethanol, propylene glycol, benzyl alcohol, glycerin, polyethylene glycol 200, polyethylene glycol 300, polyethylene glycol 400, etc., which are liquids near room temperature, and may be, for example, ethanol. The alcohol is selected from one or more pharmaceutically acceptable alcohols such as ethanol, propylene glycol, benzyl alcohol, glycerin, polyethylene glycol 200, polyethylene glycol 300, polyethylene glycol 400, etc., which are liquids near room temperature, and may be, for example, ethanol. The alcohol is selected from one or more pharmaceutically acceptable alcohols such as ethanol, propylene glycol, benzyl alcohol, glycerin, polyethylene glycol 200, polyethylene glycol 300, polyethylene glycol 400, etc., which are liquids near room temperature, and may be, for example, ethanol. The alcohol is selected from one or more pharmaceutically acceptable alcohols such as ethanol, propylene glycol, benzyl alcohol, glycerin, polyethylene glycol 200, polyethylene glycol 300, polyethylene glycol 400, etc., which are liquids near room temperature, and may be, for example, ethanol. The alcohol is selected from one or more pharmaceutically acceptable alcohols such as ethanol, propylene glycol, benzyl alcohol, glycerin, polyethylene glycol 200, polyethylene glycol 300, polyethylene glycol 400, etc., which are liquids near room temperature, and may be, for example, ethanol.

[0137] Dissolve the siRNA provided by the present disclosure in a buffer salt solution to obtain an aqueous siRNA solution. The concentration of the buffer salt solution is 0.05 to 0.5 M, and may be, for example, 0.1 to 0.2 M. Adjust the pH of the buffer salt solution to 4.0 to 5.5, and may be, for example, 5.0 to 5.2. The dosage of the buffer salt solution is determined such that the concentration of siRNA is 0.6 mg / mL or less, for example, 0.2 to 0.4 mg / mL. The buffer salt is one or more selected from soluble acetates and soluble citrates, and may be, for example, sodium acetate and / or potassium acetate. The buffer salt is one or more selected from soluble acetates and soluble citrates, and may be, for example, sodium acetate and / or potassium acetate. The buffer salt is one or more selected from soluble acetates and soluble citrates, and may be, for example, sodium acetate and / or potassium acetate.

[0138] After mixing the lipid solution and the aqueous siRNA solution, the obtained product is cultured at 40 to 60 °C for at least 2 minutes, for example, 5 to 30 minutes, to obtain a cultured liposome formulation. After mixing the lipid solution and the aqueous siRNA solution, the obtained product is cultured at 40 to 60 °C for at least 2 minutes, for example, 5 to 30 minutes, to obtain a cultured liposome formulation. The volume ratio of the iRNA aqueous solution may be 1:(2 - 5), for example, 1:4.

[0139] The cultured liposome preparation is concentrated or diluted, impurities are removed, sterilized, and the drug composition provided by the present disclosure is obtained. Its physicochemical parameters include a pH of 6.5 - 8, an encapsulation efficiency of 80% or more, a particle size of 40 - 200 nm, a polydispersity index of 0.30 or less, and an osmotic pressure of 250 - 400 mOsm / kg. For example, the physicochemical parameters may include a pH of 7.2 - 7.6, an encapsulation efficiency of 90% or more, a particle size of 60 - 100 nm, a polydispersity index of 0.20 or less, and an osmotic pressure of 300 - 400 mOsm / kg. Here, concentration or dilution may be performed before removing impurities, after removing impurities, or simultaneously. As a method for removing impurities, various conventional methods may be adopted. For example, using a tangential flow system and a hollow fiber column, ultrafiltration is performed under the condition of 100KDa, and the ultrafiltration exchange solution may be a phosphate buffer solution (PBS) with a pH of 7.4. As a sterilization method, various conventional methods may be adopted. For example, it may be sterilized by filtration with a 0.22μm filter. The encapsulation efficiency is 80% or more, the particle size is 40 - 200 nm, the polydispersity index is 0.30 or less, and the osmotic pressure is 250 - 400 mOsm / kg. For example, the physicochemical parameters may include a pH of 7.2 - 7.6, an encapsulation efficiency of 90% or more, a particle size of 60 - 100 nm, a polydispersity index of 0.20 or less, and an osmotic pressure of 300 - 400 mOsm / kg. Here, concentration or dilution may be performed before removing impurities, after removing impurities, or simultaneously. As a method for removing impurities, various conventional methods may be adopted. For example, using a tangential flow system and a hollow fiber column, ultrafiltration is performed under the condition of 100KDa, and the ultrafiltration exchange solution may be a phosphate buffer solution (PBS) with a pH of 7.4. As a sterilization method, various conventional methods may be adopted. For example, it may be sterilized by filtration with a 0.22μm filter. The physicochemical parameters include a pH of 6.5 - 8, an encapsulation efficiency of 80% or more, a particle size of 40 - 200 nm, a polydispersity index of 0.30 or less, and an osmotic pressure of 250 - 400 mOsm / kg. For example, the physicochemical parameters may include a pH of 7.2 - 7.6, an encapsulation efficiency of 90% or more, a particle size of 60 - 100 nm, a polydispersity index of 0.20 or less, and an osmotic pressure of 300 - 400 mOsm / kg. The encapsulation efficiency is 90% or more, the particle size is 60 - 100 nm, the polydispersity index is 0.20 or less, and the osmotic pressure is 300 - 400 mOsm / kg. It may also be like this.

[0140] Here, concentration or dilution may be performed before removing impurities, after removing impurities, or simultaneously. As a method for removing impurities, various conventional methods may be adopted. For example, using a tangential flow system and a hollow fiber column, ultrafiltration is performed under the condition of 100KDa, and the ultrafiltration exchange solution may be a phosphate buffer solution (PBS) with a pH of 7.4. As a sterilization method, various conventional methods may be adopted. For example, it may be sterilized by filtration with a 0.22μm filter. Here, concentration or dilution may be performed before removing impurities, after removing impurities, or simultaneously. As a method for removing impurities, various conventional methods may be adopted. For example, using a tangential flow system and a hollow fiber column, ultrafiltration is performed under the condition of 100KDa, and the ultrafiltration exchange solution may be a phosphate buffer solution (PBS) with a pH of 7.4. As a sterilization method, various conventional methods may be adopted. For example, it may be sterilized by filtration with a 0.22μm filter. For example, using a tangential flow system and a hollow fiber column, ultrafiltration is performed under the condition of 100KDa, and the ultrafiltration exchange solution may be a phosphate buffer solution (PBS) with a pH of 7.4. The ultrafiltration exchange solution may be a phosphate buffer solution (PBS) with a pH of 7.4. As a sterilization method, various conventional methods may be adopted. For example, it may be sterilized by filtration with a 0.22μm filter. As a sterilization method, various conventional methods may be adopted. For example, it may be sterilized by filtration with a 0.22μm filter. It may be sterilized by filtration.

[0141] <The first type of siRNA complex> In one embodiment, the present disclosure provides a first type of siRNA complex comprising the above siRNA and a complexing group bound to the siRNA. In general, the complexing group includes at least one pharmaceutically acceptable targeting group and an optional linker, and the siRNA, the linker, and the targeting group are bound in sequence.

[0142] In general, the complexing group includes at least one pharmaceutically acceptable targeting group and an optional linker, and the siRNA, the linker, and the targeting group are bound in sequence. The siRNA, the linker, and the targeting group are bound in sequence. They are combined. In some embodiments, the number of said target groups is from 1 to 6. In some embodiments, the number of said target groups is from 2 to 4. The siRNA molecule may be non-covalently or covalently combined with the composite group. For example, it may be covalently combined with the composite group. The composite site between the siRNA and the composite group may be at the 3'-end or 5'-end of the sense strand of the siRNA, may be at the 5'-end of the antisense strand, or may be in the internal sequence of the siRNA. In some embodiments, the composite site between the siRNA and the composite group is at the 3'-end of the sense strand of the siRNA.

[0143] In some embodiments, the composite group may be bound to the phosphate group of the nucleotide, the 2'-hydroxy group or the base. In some embodiments, the composite group may be bound to the 3'-hydroxy group. In this case, the nucleotides are linked by 2'-5' phosphodiester bonds. When the composite group is bound to the end of the siRNA strand, it is usually bound to the phosphate group of the nucleotide. When it is bound to the internal sequence of the siRNA, it is usually bound to the ribose sugar ring or the base. For various binding methods, reference can be made to Muthiah Manoharan et.al. siRNA conjugates carr ying sequentially assembled trivalent N- acetylgalactosamine linked through nucle osides elicit robust gene silencing in v ivo in hepatocytes. ACS Chemical biology ,2015,10 (5):1181~7.

[0144] In some embodiments, the siRNA and the complexing group may be linked by an acid-labile or reducible chemical bond, and in the acidic environment of the cell endosome, these chemical bonds can be cleaved, allowing the siRNA to be released. For complexing methods that cannot be cleaved, the complexing group is bound to the sense strand of the siRNA, and the influence on siRNA activity due to complexing can be reduced as much as possible.

[0145] In some embodiments, the pharmaceutically acceptable targeting group is a ligand commonly used in the field of double-stranded oligonucleotide administration, for example, various ligands described in WO2009082607A2, the disclosure of which is hereby incorporated by reference in its entirety into this specification and made a part hereof.

[0146] In some embodiments, the pharmaceutically acceptable targeting group is a lipophilic molecule such as cholesterol, bile acid, vitamins (e.g., tocopherol), lipid molecules with different chain lengths, etc., a polymer such as polyethylene glycol, a polypeptide such as a membrane-permeable peptide, an aptamer, an antibody, a quantum dot, sugars such as lactose, polylactose, mannose, galactose, N-acetylgalactosamine (GalNAc), etc., folate, asialoorosomucoid, asialo sugar residues, lipoproteins (e.g., high-density lipoprotein, low-density lipoprotein, etc.), glucagon, neurotransmitters (e.g., adrenaline), growth factors, transferrin, etc., and one or more selected from ligands formed by target molecules such as receptor ligands expressed on hepatocytes or derivatives thereof may also be used.

[0147] In some embodiments, each of the ligands is independently selected from ligands capable of binding to receptors on the cell surface. In some embodiments, at least one ligand is a ligand capable of binding to a receptor on the surface of hepatocytes. In some embodiments, at least one ligand is a ligand capable of binding to a receptor on the surface of mammalian hepatocytes. In some embodiments, at least one ligand is a ligand capable of binding to a receptor on the surface of human hepatocytes. In some embodiments, at least one ligand is a ligand capable of binding to the asialoglycoprotein receptor (ASGPR) on the liver surface. The types of these ligands are known to those skilled in the art, and as their function, they generally bind to specific receptors on the target cell surface and mediate the delivery of the double-stranded oligonucleotide bound to the ligand to the target cell.

[0148] In some embodiments, the pharmaceutically acceptable target group may be any one ligand that binds to the asialoglycoprotein receptor (ASGPR) on the surface of mammalian hepatocytes. In some embodiments, each ligand is independently an asialoglycoprotein, for example, asialoorosomucoid (ASOR) or asialofetuin (ASF). In some embodiments, the ligand is a sugar or a derivative of a sugar. In some embodiments, at least one ligand is a sugar. In some embodiments, each ligand is a sugar. In some embodiments, at least

[0149] one ligand is a sugar. In some embodiments, each ligand is a sugar. In some embodiments, at least one ligand is a sugar. In some embodiments, each ligand is a sugar. In some embodiments, at least ​​​​One ligand is a monosaccharide, polysaccharide, modified monosaccharide, modified polysaccharide or sugar derivative. Some In embodiments, at least one of the ligands may be a monosaccharide, disaccharide or trisaccharide. In some embodiments, at least one ligand is a modified sugar. In some embodiments, each ligand is a modified sugar. In some embodiments, each ligand is independently selected from polysaccharides, modified polysaccharides, monosaccharides, modified monosaccharides, polysaccharide derivatives or monosaccharide derivatives. In some embodiments, each or at least one of the ligands is selected from the group consisting of glucose and its derivatives, mannan and its derivatives, galactose and its derivatives, xylose and its derivatives, ribose and its derivatives, fucose and its derivatives, lactose and its derivatives, maltose and its derivatives, arabinose and its derivatives, fructose and its derivatives, and sialic acid.

[0150] In some embodiments, each of the ligands is D-mannopyranose, L-manno pyranose, D-arabinose, D-xylofuranose, L-xylofuranose, D-glu cose, L-glucose, D-galactose, L-galactose, α-D-mannofura nose, β-D-mannofuranose, α-D-mannopyranose, β-D-mannopyran ose, α-D-glucopyranose, β-D-glucopyranose, α-D-glucofura nose, β-D-glucofuranose, α-D-fructofuranose, α-D-fructopyran ose, α-D-galactopyranose, β-D-galactopyranose, α-D-galactof ranose, β-D-galactofuranose, glucosamine, sialic acid, galactosamine, N -acetylgalactosamine, N-trifluoroacetylgalactosamine, N-propionyl galactosamine, N-n-butyrylgalactosamine, N-isobutyrylgalactosamine , 2-amino-3-O-[(R)-1-carboxyethyl]-2-deoxy-β-D-glu copyranose, 2-deoxy-2-methylamino-L-glucopyranose, 4,6-di deoxy-4-formamido-2,3-di-O-methyl-D-mannopyranose, 2-de oxy-2-sulfoamino-D-glucopyranose, N-glycolyl-α-neuraminic acid , 5-thio-β-D-glucopyranose, methyl 2,3,4-tris-O-acetyl-1 -thio-6-O-trityl-α-D-glucopyranoside (methyl 2,3,4-t ris-O-acetyl-1-thio-6-O-trityl-α-D-gluco pyranoside), 4-thio-β-D-galactopyranose, ethyl 3,4,6, 7-tetra-O-acetyl-2-deoxy-1,5-dithio-α-D-glucoheptopyr anoside, 2,5-anhydro-D-allonitrile, ribose, D-ribose, D-4-thio ribose, L-ribose or L-4-thioribose may be independently selected. The other options for the above-mentioned ligands may refer to, for example, the description in CN105378082A , the disclosure of which is incorporated herein by reference in its entirety.

[0151] In some embodiments, the pharmaceutically acceptable target group in the first siRNA complex may be galactose or N-acetylgalactosamine, and the galact ose or N-acetylgalactosamine molecule may be monovalent, divalent, trivalent or tetravalent. Here, monovalent, divalent, trivalent, and tetravalent refer to the molar ratio of the double-stranded oligonucleotide molecule to the galactose or N-acetylgalactosamine molecule as the target group in the oligonucleotide complex formed from the complex group being 1:1, 1:2, 1:3, or 1:4, respectively. It should be understood as such. In some embodiments, the pharmaceutically acceptable target group is N-acetylgalactosamine. In some embodiments, when the double-stranded oligonucleotide described in the present disclosure complexes with a complex group containing N-acetylgalactosamine, the N-acetylgalactosamine molecule is trivalent or tetravalent. In some embodiments, when the double-stranded oligonucleotide described in the present disclosure complexes with a complex group containing N-acetylgalactosamine, the N-acetylgalactosamine molecule is trivalent. The target group may be bound to the siRNA molecule via a suitable linker, and those skilled in the art can select a suitable linker according to the specific type of the target group. For these linkers, the type of the target group, and the binding method to the siRNA, reference may be made to the disclosure of WO2015006740A2, the content of which is incorporated herein by reference in its entirety. In some embodiments, when the target group is N-acetylgalactosamine, the suitable linker may have the structure shown in formula (301). Here, the monovalent, divalent, trivalent, and tetravalent refer to the molar ratio of the double-stranded oligonucleotide molecule to the galactose or N-acetylgalactosamine molecule as the target group in the oligonucleotide complex formed from the complex group being 1:1, 1:2, 1:3, or 1:4, respectively. It should be understood as such. In some embodiments, the pharmaceutically acceptable target group is N-acetylgalactosamine. In some embodiments, when the double-stranded oligonucleotide described in the present disclosure complexes with a complex group containing N-acetylgalactosamine, the N-acetylgalactosamine molecule is trivalent or tetravalent. In some embodiments, when the double-stranded oligonucleotide described in the present disclosure complexes with a complex group containing N-acetylgalactosamine, the N-acetylgalactosamine molecule is trivalent.

[0152] The target group may be bound to the siRNA molecule via a suitable linker, and those skilled in the art can select a suitable linker according to the specific type of the target group. For these linkers, the type of the target group, and the binding method to the siRNA, reference may be made to the disclosure of WO2015006740A2, the content of which is incorporated herein by reference in its entirety.

[0153] In some embodiments, when the target group is N-acetylgalactosamine, the suitable linker may have the structure shown in formula (301).

[0154]

Chemical formula

[0155]

Chemical formula

[0156]

Chemical formula

[0157] In some embodiments, when n = 3 and L C is a tetravalent linker group based on trihydroxymethylaminomethane -(L A )3 trihydroxy methylaminomethane-L B - binds the N-acetylgalactosamine molecule and the siRNA molecule The first type of siRNA complex formed by combination has a structure shown in the following formula (304). as follows.

[0158] [Chemical formula] In the formula, the double helix structure represents siRNA.

[0159] Similarly, the complex site of siRNA and the complex group may be at the 3'-end or 5'-end of the sense strand of siRNA, may be at the 5'-end of the antisense strand, or may be in the internal sequence of siRNA. It may be at the 3'-end or 5'-end of the sense strand of siRNA, may be at the 5'-end of the antisense strand, or may be in the internal sequence of siRNA. as follows.

[0160] In some embodiments, the 3'-end of the sense strand of the siRNA described in the present disclosure is covalently complexed with three N- A acetylgalactosamine (GalNAc) molecules by a linker-(L B )3-tris(hydroxymethyl)aminomethane-L to obtain a first type of siRNA complex (hereinafter also referred to as (GalNAc)3-siRNA) having a structure shown in the following formula (305), in which the molar ratio of the siRNA molecule to the GalNAc molecule is 1:3. In some embodiments, the first type of siRNA complex (hereinafter also referred to as (GalNAc)3-siRNA) having a structure shown in the following formula (305), in which the molar ratio of the siRNA molecule to the GalNAc molecule is 1:3. is obtained.

[0161] [Chemical formula] In the formula, the double helix structure represents the siRNA, and the linker is bound to the 3'-end of the sense strand of the siRNA. as follows.

[0162] In some embodiments, when the target group is N-acetylgalactosamine, a suitable linker may have a structure shown in formula (306).

[0163] [Chemical formula] In the formula, l is an integer from 0 to 3, * represents a site in the linker that is bonded to the target group by an ether bond, # represents a site in the linker that is bonded to the siRNA by a phosphate ester bond .

[0164] In some embodiments, when l = 2, the siRNA complex has a structure represented by formula (30 7).

[0165]

Chemical formula

[0166] The above complex may be synthesized by a method already described in detail in the prior art. For example, WO2015006740A2 describes in detail a method for preparing a plurality of types of complexes. The first type of siRNA complex of the present disclosure can be obtained by a method well known to those skilled in the art. For example, WO2014025805A1 describes a method for preparing a structure represented by formula (305), and Rajeev et al. describe a method for preparing a structure represented by formula (307) in ChemBioChem 20 15, 16, 903 - 908.

[0167] <The second type of siRNA complex> In some embodiments, the present disclosure provides a second type of s iRNA complex having a structure represented by formula (1).

[0168]

Chemical formula

[0169]

Chemical formula

[0170] Each nucleotide in the siRNA represented by Nu is each independently a modified or un modified nucleotide, and the siRNA represented by Nu contains a sense strand and an antisense strand . The sense strand contains nucleotide sequence 1, and the antisense strand contains nucleotide sequence 2. The nucleotide sequence 1 and the nucleotide sequence 2 form a double-stranded region that is at least partially reverse complementary. The nucleotide sequence 1 and the nucleotide sequence shown in SEQ ID NO: 155 have the same length and three or fewer nucleotide differences. The nucleotide sequence 2 and the nucleotide sequence shown in SEQ ID NO: 156 have the same length and three or fewer nucleotide differences, 5’-CCUUGAGGCAUACUUCAAZ-3’ (SEQ ID NO: 155), 5’-Z’UUGAAGUAUGCCUCAAGG-3’ (SEQ ID NO: 156) However, Z is A and Z’ is U, the nucleotide sequence 1 contains a nucleotide Z at the position corresponding to Z, A and the nucleotide sequence 2 contains a nucleotide Z’ at the position corresponding to Z’, B and the Z’ B is the first nucleotide at the 5’ end of the antisense strand.

[0171] R2 is a linear alkylene group having 1 to 20 carbon atoms, and one or more carbon atoms are , C(O), NH, O, S, CH=N, S(O)2, C2-C 10 alkenylene group, C2 -C 10 alkynylene group, C6-C 10 arylene group, C3-C 18 heterocyclylene group and C5-C 10 heteroarylene group, and is optionally substituted with one or more selected from the group consisting of and R2 is C1-C 10 alkyl group, C6-C 10 aryl group, C5-C 10 hetero aryl group, C1-C 10 halogenated alkyl group, -OC1-C 10 alkyl group, -OC 1-C 10 alkylphenyl group, -C1-C 10 alkyl-OH, -OC1-C 10 halo genated alkyl group, -SC1-C 10 alkyl group, -SC1-C 10 alkylphenyl group , -C1-C 10 alkyl-SH, -SC1-C 10 halogenated alkyl group, halogenated , -OH, -SH, -NH2, -C1-C 10Alkyl-NH2, -N(C1-C 10 A lkyl group)(C1-C 10 lkyl group), -NH(C1-C 10 lkyl group), cyano group , nitro group, -CO2H, -C(O)O(C1-C 10 lkyl group), -CON(C1- C 10 lkyl group)(C1-C 10 lkyl group), -CONH(C1-C 10 lkyl group ), -CONH2, -NHC(O)(C1-C 10 lkyl group), -NHC(O)(phe nyl group), -N(C1-C 10 lkyl)C(O)(C1-C 10 lkyl group), -N( C1-C 10 lkyl)C(O)(phenyl group), -C(O)C1-C 10 lkyl group, , -C(O)C1-C 10 lkylphenyl group, -C(O)C1-C 10 haloalkyl group, , -OC(O)C1-C 10 lkyl group, -SO2(C1-C 10 lkyl group), -SO2 (phenyl group), -SO2(C1-C 10 halogenated alkyl group), -SO2NH2, - SO2NH(C1-C 10 lkyl group), -SO2NH(phenyl group), -NHSO2( C1-C 10 lkyl group), -NHSO2(phenyl group) and -NHSO2(C1-C1 0 halogenated alkyl group) and may optionally have any one or more substituents selected from the group consisting of Yes.

[0172] Each L1 is a straight-chain alkylene group of 1 to 70 carbon atoms in length, and one or more carbon atoms are C(O), NH, O, S, CH=N, S(O)2, C2-C10 An alkenylene group, C 2-C 10 An alkynylene group, C6-C 10 An arylene group, C3-C 18 A heterocyclylene group and C5-C 10 selected from the group consisting of a heteroarylene group, and optionally substituted with one or more, L1 is a C1-C alkyl group, C6-C 10 alkyl group, C6-C 10 aryl group, C5-C 10 Hete roaryl group, C1-C 10 halogenated alkyl group, -OC1-C 10 alkyl group, -O C1-C 10 alkylphenyl group, -C1-C 10 alkyl-OH, -OC1-C 10 Ha logenated alkyl group, -SC1-C 10 alkyl group, -SC1-C 10 alkylphenyl group, -C1-C 10 alkyl-SH, -SC1-C 10 halogenated alkyl group, halogen ated, -OH, -SH, -NH2, -C1-C 10 alkyl-NH2, -N(C1-C 10 alkyl group)(C1-C 10 alkyl group), -NH(C1-C 10 alkyl group), cyano group, nitro group, -CO2H, -C(O)O(C1-C 10 alkyl group), -CON(C1 -C 10 alkyl group)(C1-C 10 alkyl group), -CONH(C1-C 10 alkyl group), -CONH2, -NHC(O)(C1-C 10 alkyl group), -NHC(O)(ph enyl group), -N(C1-C 10 alkyl)C(O)(C1-C 10(alkyl group), -N (C1-C 10 (alkyl)C(O)(phenyl group), -C(O)C1-C 10 alkyl group , -C(O)C1-C 10 alkylphenyl group, -C(O)C1-C 10 haloalkyl group , -OC(O)C1-C 10 alkyl group, -SO2(C1-C 10 alkyl group), -SO 2(phenyl group), -SO2(C1-C 10 halogenated alkyl group), -SO2NH2, -SO2NH(C1-C 10 alkyl group), -SO2NH(phenyl group), -NHSO2 (C1-C 10 alkyl group), -NHSO2(phenyl group) and -NHSO2(C1-C 10 halogenated alkyl group) and may optionally have any one or more substituents selected from the group consisting of .

[0173] In some embodiments, L1 may be selected from the group consisting of the groups of A1 to A26 or any combination thereof, and the structures and definitions of A1 to A26 are as follows. where j1 is an integer from 1 to 20, j2 is an integer from 1 to 20,

[0174] [Chemical formula] provided that R’ is a C1-C10 alkyl group, and Ra is selected from the group consisting of the groups of formula A27 to A45 or any combination thereof.

[0175] [Chemical formula] Rb is a C1-C 10 alkyl group, TIFF0007702159000027.tif4170 represents the site where the base is attached to the rest of the molecule.

[0176] For convenience, L1 is defined as a linear alkylene group, but it is understood by those skilled in the art that, for example, amino or alkenyl groups resulting from the above-mentioned substitution and / or replacement may not be linear groups or may have different names. For the purposes of the present disclosure, the length of L1 is the number of atoms in the chain connecting the two bonding points. For this purpose, a ring obtained by substituting the carbon atoms of the linear alkylene (for example, heterocyclylene or heteroarylene) is regarded as one atom.

[0177] M1 represents a target group, and its definition and selectable range are the same as those of the above-mentioned target group. In some embodiments, each M1 is independently selected from one of the ligands having an affinity for the asialoglycoprotein receptor on the surface of mammalian liver cells.

[0178] When M1 is a ligand having an affinity for the asialoglycoprotein receptor on the surface of mammalian liver cells, in some embodiments, n1 may be an integer from 1 to 3, n3 may be an integer from 0 to 4, and the number of M1 ligands in the complex is ensured to be at least 2. In some embodiments, n1 + n3 ≧ 2, so that the number of M1 ligands is at least 3, the M1 ligand binds more easily to the asialoglycoprotein receptor on the liver surface, and further the complex can be promoted to be taken up by cells by the action of endocytosis. As can be seen from experiments, when the number of M1 ligands is 3 or more, the improvement in the ease of binding between the M1 ligand and the asialoglycoprotein receptor on the liver surface is not obvious, so the synthesis capacity Considering various aspects such as ease of use, structure / processing cost, and delivery efficiency, several In an embodiment, n1 is an integer of 1 to 2, n3 is an integer of 0 to 1, and n1 +n3=2~3.

[0179] In some embodiments, m1, m2, and m3 are independently selected from integers from 2 to 10. When the M1 ligand is selected, the spatial positions of multiple M1 ligands are determined by the M1 ligand and the asialoglycoprotein on the liver surface. The conjugates provided by the present disclosure can be adapted to bind to protein receptors. In order to make the process simpler, more synthetically accessible, and / or less costly, some implementations have been In the embodiment, m1, m2, and m3 are each independently an integer of 2 to 5, In this embodiment, m1=m2=m3.

[0180] R 10 , R 11 , R 12 , R 13 , R 14 and R 15 But, H, C1-C 10 Alkyl group , C1-C 10 Halogenated alkyl groups and C1-C 10 each independently from alkoxy Any one of these, if selected, may alter the properties of the complexes disclosed herein. It will be understood by those skilled in the art that the objectives of the present disclosure can be achieved without the need for some In the embodiment, R 10 , R 11 , R 12 , R 13 , R 14 and R 15 are respectively In some embodiments, R1 is independently selected from H, a methyl group, and an ethyl group. 0, R 11 , R 12 , R13 , R 14 and R 15 are both H.

[0181] R3 is a group having the structure shown in Formula A59, wherein E1 is OH, SH or BH2 and, considering the availability of the preparation raw materials, in some embodiments, E1 is OH or is SH.

[0182] In some embodiments, R2 is selected to realize the bonding of N on the nitrogen-containing skeleton and A59. In the context of the present disclosure, the "nitrogen-containing skeleton" refers to a chain 10 R 11 , R 12 R 13 R 14 and R 15 in which the carbon atom to which R is bonded and N are bonded to each other. Therefore, R2 may be any linker group that can bond the A59 group to N on the nitrogen-containing skeleton in a suitable manner. In some embodiments, when preparing the siRNA complex of the present disclosure by a solid-phase synthesis process, the R2 group needs to include both a binding site for binding to N on the nitrogen-containing skeleton and a binding site for binding to P in R3. In some embodiments, the site for binding to N in the nitrogen-containing skeleton in R2 forms an amide bond with N, and the site for binding to P on R3 forms a phosphoester bond with P. In some embodiments, R2 may be B5, B6, B5' or B6'. In some embodiments, the site for binding to N in the nitrogen-containing skeleton in R2 forms an amide bond with N, and the site for binding to P on R3 forms a phosphoester bond with P. In some embodiments, R2 may be B5, B6, B5' or B6'.

[0183]

Chemical formula

[0184] The value range of q2 may be an integer from 1 to 10, and in some embodiments, q 2 is an integer from 1 to 5.

[0185] L1 binds to N on the nitrogen-containing backbone with the M1 ligand and plays a role in providing liver targeting function to the second type of siRNA complex of the present disclosure. In some embodiments, L1 is one or a combination of a plurality of bonds selected from the groups of formulas A1 to A26. In some embodiments it is one or a combination of a plurality of bonds selected from A1, A4, A5, A6, A8, A10, A11 and A13 from. In some embodiments, L1 is one or a combination of a plurality of bonds selected from A 1, A4, A8, A10 and A11 is at least a combination of two bonds . In some embodiments, L1 is at least a combination of two bonds selected from A1, A8, A10.

[0186] In some embodiments, the length of L1 is 3 to 25 atoms, 3 to 20 atoms, 4 to 15 atoms or 5 to 12 atoms may also be possible. In some embodiments, the length of L1 is 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60 atoms.

[0187] In some embodiments, j1 is an integer from 2 to 10, and in some embodiments Here, j1 is an integer from 3 to 5. j2 is an integer from 2 to 10, and in some embodiments here, j2 is an integer from 3 to 5. R’ is a C1-C4 alkyl group, and in some embodiments, R’ is one of a methyl group, an ethyl group, and an isopropyl group. Ra is one of A27, A28, A29, A30, and A31, and in some embodiments here, Ra is A27 or A28. Rb is a C1-C5 alkyl group, and in some embodiments, Rb is one of a methyl group, an ethyl group, an isopropyl group, and a butyl group. In some embodiments, by selecting j1, j2 , R’, Ra, and Rb in formulas A1 to A26 respectively, the binding between the M1 ligand and N on the nitrogen-containing skeleton is realized, and the spatial positions between the M1 ligands are further adapted to the binding between the M1 ligand and the asialoglycoprotein receptor on the liver surface.

[0188] In some embodiments, the second type of siRNA complex of the present disclosure has a structure shown in formula (3), (4 ), (5), (6), (7), (8), (9), (10), (11), (12), (13 ), (14), (15), (16), (17), (18), (19), (20), (21 ), or (22).

[0189]

Chemical formula

[0190] In some embodiments, P in formula A59 may be attached to any possible position in the siRNA (shown as Nu in the above formulas) sequence. For example, formula A5 ​ In 9, P may be bound to any one nucleotide of the sense strand or the antisense strand of the siRNA. In some embodiments, P in formula A59 is bound to any one nucleotide of the sense strand of siRNA. In some embodiments it is bound to any one nucleotide of the sense strand of siRNA. In some embodiments P in formula A59 is bound to an end of the sense strand or the antisense strand of the siRNA. In some embodiments P in formula A59 is bound to the end of the sense strand or the antisense strand of the siRNA. In some embodiments P in formula A59 is bound to the end of the sense strand of the siRNA. The end refers to the first four nucleotides from one end in the sense strand or the antisense strand. In some embodiments P in formula A59 is bound to the end of the sense strand or the antisense strand of the siRNA. In some embodiments P in formula A59 is bound to the 3'-end of the sense strand of the siRNA. When bound to the above position of the sense strand of the siRNA, the siRNA complex provided by the present disclosure after entering the cell, when unwound, releases the antisense strand of a single siRNA and blocks the process of HBV mRNA translating into protein, and can suppress hepatitis B virus (HBV) gene expression. P in formula A59 may be bound to any possible position on the nucleotide in the siRNA represented by Nu, for example, the 5'-position of the nucleotide, the 2'-position of the nucleotide, the 3'-position of the nucleotide or the base of the nucleotide. In some embodiments, P in formula A59 is bound to the 2'-position, 3'-position or 5'-position of the nucleotide in the siRNA by forming a phosphodiester bond. In some embodiments it is bound to the 2'-position, 3'-position or 5'-position of the nucleotide in the siRNA by forming a phosphodiester bond. In some embodiments P in formula A59 may be bound to the 2'-position, 3'-position or 5'-position of the nucleotide in the siRNA by forming a phosphodiester bond. In some embodiments it is bound to the 2'-position, 3'-position or 5'-position of the nucleotide in the siRNA by forming a phosphodiester bond. In some embodiments

[0191] P in formula A59 may be bound to any possible position on the nucleotide in the siRNA represented by Nu, for example, the 5'-position of the nucleotide, the 2'-position of the nucleotide, the 3'-position of the nucleotide or the base of the nucleotide. In some embodiments, P in formula A59 is bound to the 2'-position, 3'-position or 5'-position of the nucleotide in the siRNA by forming a phosphodiester bond. In some embodiments it is bound to the 2'-position, 3'-position or 5'-position of the nucleotide in the siRNA by forming a phosphodiester bond. In some embodiments P in formula A59 is bound to the 2'-position, 3'-position or 5'-position of the nucleotide in the siRNA by forming a phosphodiester bond. In some embodiments it is bound to the 2'-position, 3'-position or 5'-position of the nucleotide in the siRNA by forming a phosphodiester bond. In some embodiments , in Formula A59, P is bonded to an oxygen atom obtained by dehydrogenating the 3'-hydroxy group of the nucleotide at the 3'-end of the sense strand of the siRNA, or in Formula A59, P is bonded to a nucleotide by substituting the hydrogen in the 2'-hydroxy group of one nucleotide in the sense strand of the siRNA, or in Formula A59, P is bonded to a nucleotide by substituting the hydrogen in the 5'-hydroxy group of the nucleotide at the 5'-end of the sense strand of the siRNA. In some embodiments, the nucleotide sequence 1 and the nucleotide sequence shown in SEQ ID NO: 1 have at most one nucleotide difference, and / or the nucleotide sequence 2 and the nucleotide sequence shown in SEQ ID NO: 2 have at most one nucleotide difference. In some embodiments, the nucleotide difference between the nucleotide sequence 2 and the nucleotide sequence shown in SEQ ID NO: 2 includes a difference at the position of Z', where Z' is selected from A, C

[0192] or G. In some embodiments, the nucleotide difference is a difference at the position of Z', where Z' is selected from A, C or G. In some embodiments, Z is a nucleotide complementary to Z'. These nucleotide differences

[0193] do not significantly reduce the target gene suppression ability by the second type of siRNA complex, and the second type of siRNA complex containing these specific nucleotide differences is also within the protection scope of the present disclosure. B In some embodiments, the nucleotide difference between the nucleotide sequence 2 and the nucleotide sequence shown in SEQ ID NO: 2 B includes a difference at the position of Z', where Z' is selected from A, C B or G. In some embodiments, the nucleotide difference is a difference at the position of Z', where Z' B is selected from A, C or G. In some embodiments, Z is a nucleotide complementary to Z'. These nucleotide differences A do not significantly reduce the target gene suppression ability by the second type of siRNA complex, and the second type of siRNA complex containing these specific nucleotide differences is also within the protection scope B of the present disclosure. do not significantly reduce the target gene suppression ability by the second type of siRNA complex, and the second type of siRNA complex containing these specific nucleotide differences is also within the protection scope of the present disclosure. These nucleotide differences do not significantly reduce the target gene suppression ability by the second type of siRNA complex, and the second type of siRNA complex containing these specific nucleotide differences is also within the protection scope

[0194] In some embodiments, the nucleotide sequence 1 and the nucleotide sequence 2 are basically reverse complementary, substantially reverse complementary or completely reverse complementary. The basically reverse complementary means that there are three or fewer base mismatches between the two nucleotide sequences, the substantially reverse complementary means that there is one or fewer base mismatches between the two nucleotide sequences, and the completely reverse complementary means that there is no mismatch between the two nucleotide sequences.

[0195] In some embodiments, the sense strand further comprises a nucleotide sequence 3, and the anti-sense strand further comprises a nucleotide sequence 4. The nucleotide sequence 3 and the nucleotide sequence 4 are each independently 1 to 4 nucleotides in length, and the nucleotide sequence 3 and the nucleotide sequence 4 are in corresponding positions. In some embodiments, the nucleotides at the corresponding positions of the nucleotide sequence 4 and the target mRNA are at least partially complementary, and in some embodiments, the nucleotides at the corresponding positions of the nucleotide sequence 4 and the target mRNA are completely complementary.

[0196] In some embodiments, the nucleotide sequence 3 is linked to the 5' end of the nucleotide sequence 1, and the nucleotide sequence 4 is linked to the 3' end of the nucleotide sequence 2. In some embodiments, the nucleotide sequence 3 and the nucleotide sequence 4 are of equal length and reverse complementary. Therefore, the lengths of the sense strand and the anti-sense strand may be 19 to 23 nucleotides.

[0197] ​In some embodiments, the nucleotide sequence 3 and the nucleotide sequence 4 both have a length of 1 nucleotide, the base of the nucleotide sequence 3 is A, and when this is the case, the length of the double-stranded region may be 20 nucleotides, that is, the ratio of the length of the sense strand to the length of the antisense strand may be 20 / 20, or the nucleotide sequence 3 and the nucleotide sequence 4 both have a length of 2 nucleotides and, from the 5'-end to the 3'-end, the bases of the nucleotide sequence 3 are G and A in sequence and, when this is the case, the length of the double-stranded region may be 21 nucleotides, that is, the ratio of the length of the sense strand to the length of the antisense strand may be 21 / 21, or the nucleotide sequence 3 and the nucleotide sequence 4 both have a length of 3 nucleotides and, from the 5'-end to the 3'-end, the bases of the nucleotide sequence 3 are C, G and A in sequence, and, when this is the case, the length of the double-stranded region may be 22 nucleotides, that is, the ratio of the length of the sense strand to the length of the antisense strand may be 22 / 22, or the nucleotide sequence 3 and the nucleotide sequence 4 both have a length of 4 nucleotides and, from the 5'-end to the 3'-end, the bases of the nucleotide sequence 3 are C, C G and A in sequence, and, when this is the case, the length of the double-stranded region may be 23 nucleotides and, that is, the ratio of the length of the sense strand to the length of the antisense strand may be 23 / 23.

[0198] In some embodiments, the nucleotide sequence 3 has a length of 2 nucleotides and, from the 5'-end to the 3'-end, the bases of the nucleotide sequence 3 are G and G in sequence and there is. ​

[0199] The nucleotide sequence 3 and the nucleotide sequence 4 have the same length and are complementary, so it should be understood that when the bases of the nucleotide sequence 3 are given, the bases of the nucleotide sequence 4 are also determined.

[0200] In some embodiments, the siRNA represented by Nu in formula (1) has a length of 1 to 3 nucleotides, is bound to the 3'-end of the antisense strand, and further includes a nucleotide sequence 5 that constitutes the 3'-overhang end of the antisense strand. In some embodiments, the nucleotide sequence 5 has a length of 1 or 2 nucleotides. Thus, the ratio of the length of the sense strand to the antisense strand of the siRNA represented by Nu may be 19 / 20, 19 / 21, 20 / 21, 20 / 22, 21 / 22, 21 / 23, 22 / 23, 22 / 24, 23 / 24 or 23 / 25.

[0201] In some embodiments, the nucleotide sequence 5 has a length of 2 nucleotides, and from the 5'-end to the 3'-end, the nucleotide sequence 5 is two consecutive thymidine deoxyribonucleotides, two consecutive uracil ribonucleotides, or two nucleotides complementary to the target mRNA. Thus, in some embodiments, the ratio of the length of the sense strand to the antisense strand of the siRNA represented by Nu is 19 / 21 or 21 / 23, and at this time, the complex containing the siRNA has better APOC3 mRNA silencing activity. / 23, and at this time, the complex containing the siRNA has better APOC3 mRNA silencing activity.

[0202] In some embodiments, the sense strand has the nucleotide sequence shown in SEQ ID NO: 1. ​​​​​​​​​comprising columns, wherein the antisense strand has the nucleotide sequence shown in SEQ ID NO: 3 or SEQ ID NO: 4 comprising columns, 5’-CCUUGAGGCAUACUUCAAZ A -3’ (SEQ ID NO: 1), 5’-Z’ B UUGAAGUAUGCCUCAAGGUU-3’ (SEQ ID NO: 3), 5’-Z’ B UUGAAGUAUGCCUCAAGGUC-3’ (SEQ ID NO: 4) However, said Z’ B is the first nucleotide at the 5’ end of the antisense strand, and Z A is selected from A, U, G or C, and Z’ B is a nucleotide complementary to Z A and is the complementary nucleotide of Z.

[0203] In some embodiments, the siRNA represented by Nu is siHBa1 or siH Ba2. siHBa1 Sense strand: 5’-CCUUGAGGCAUACUUCAAA-3’ (SEQ ID NO: 5) Antisense strand: 5’-UUUGAAGUAUGCCUCAAGGUU-3’ (SEQ ID NO: 6) siHBa2 Sense strand: 5’-GACCUUGAGGCAUACUUCAAA-3’ (SEQ ID NO: 7) Antisense strand: 5’-UUUGAAGUAUGCCUCAAGGUCGG-3’ (SEQ NO: 8)

[0204] As described above, the nucleotides in the siRNA represented by Nu in formula (1) are each independently modified or unmodified nucleotides. In some embodiments the nucleotides in the siRNA represented by Nu are unmodified nucleotides, and some In some embodiments, some or all of the nucleotides in the siRNA shown as Nu are modified nucleotides, and these modifications on the nucleotide groups do not significantly weaken or lose the function of the second type of siRNA complex of the present disclosure to inhibit HBV gene expression. In some embodiments, the siRNA in the complex contains at least one modified nucleotide. In the context of the present disclosure, the term "modified nucleotide" refers to a nucleotide in which the hydroxy group at the 2'-position of the ribose group of the nucleotide is replaced by another group or a nucleotide analog, or a nucleotide in which the base on the nucleotide is a modified base. The modified nucleotide does not significantly weaken or lose the function of the siRNA complex to inhibit gene expression. For example, the modified nucleotides disclosed in J.K. Watts, G.F. Deleavey, and M.J. Damha, Chemically modified siRNA: tools and applications. Drug Discov Today, 2008, 13(19-20):842-55 may be selected. In some embodiments, at least one of the sense strand or the antisense strand has at least one nucleotide that is a modified nucleotide and / or at least one phosphate ester group that is a phosphate ester group having a modifying group. In other words, at least some of the phosphate ester groups and / or ribose groups in at least one single-stranded phosphate-sugar backbone in the sense strand and the antisense strand have modifying groups.

[0205]

[0206] It is a ribose group having a decorating group.

[0207] In some embodiments, all the nucleotides in the sense strand and / or the antisense strand are modified nucleotides. In some embodiments, all the nucleotides in the sense strand and the antisense strand are modified nucleotides. In some embodiments, each nucleotide in the sense strand and the antisense strand is independently a fluorine-modified nucleotide or a non-fluorine-modified nucleotide. In some embodiments, each nucleotide in the sense strand and the antisense strand is independently a fluorine-modified nucleotide or a non-fluorine-modified nucleotide. In some embodiments, all the nucleotides in the sense strand and the antisense strand are modified nucleotides. In some embodiments, each nucleotide in the sense strand and the antisense strand is independently a fluorine-modified nucleotide or a non-fluorine-modified nucleotide.

[0208] The inventors of the present disclosure have surprisingly found that the second type of siRNA complex of the present disclosure achieves a high balance between plasma stability and gene silencing efficiency in animal experiments. In some embodiments, the fluorine-modified nucleotides are located at nucleotide sequence 1 and nucleotide sequence 2. From the 5'-end to the 3'-end, the 7th, 8th, and 9th nucleotides of nucleotide sequence 1 are fluorine-modified nucleotides, and from the 5'-end to the 3'-end, the 2nd, 6th, 14th, and 16th nucleotides of nucleotide sequence 2 are fluorine-modified nucleotides. In some embodiments, the fluorine-modified nucleotides are located at nucleotide sequence 1 and nucleotide sequence 2. From the 5'-end to the 3'-end, the 7th, 8th, and 9th nucleotides of nucleotide sequence 1 are fluorine-modified nucleotides, and from the 5'-end to the 3'-end, the 2nd, 6th, 14th, and 16th nucleotides of nucleotide sequence 2 are fluorine-modified nucleotides.

[0209] In some embodiments, the fluorine-modified nucleotides are located at nucleotide sequence 1 and nucleotide sequence 2. From the 5'-end to the 3'-end, the 7th, 8th, and 9th nucleotides of nucleotide sequence 1 are fluorine-modified nucleotides, and from the 5'-end to the 3'-end, the 2nd, 6th, 14th, and 16th nucleotides of nucleotide sequence 2 are fluorine-modified nucleotides. In some embodiments, the fluorine-modified nucleotides are located at nucleotide sequence 1 and nucleotide sequence 2. From the 5'-end to the 3'-end, the 7th, 8th, and 9th nucleotides of nucleotide sequence 1 are fluorine-modified nucleotides, and from the 5'-end to the 3'-end, the 2nd, 6th, 14th, and 16th nucleotides of nucleotide sequence 2 are fluorine-modified nucleotides. In some embodiments, the fluorine-modified nucleotides are located at nucleotide sequence 1 and nucleotide sequence 2. From the 5'-end to the 3'-end, the 7th, 8th, and 9th nucleotides of nucleotide sequence 1 are fluorine-modified nucleotides, and from the 5'-end to the 3'-end, the 2nd, 6th, 14th, and 16th nucleotides of nucleotide sequence 2 are fluorine-modified nucleotides. In some embodiments, the fluorine-modified nucleotides are located at nucleotide sequence 1 and nucleotide sequence 2. From the 5'-end to the 3'-end, the 7th, 8th, and 9th nucleotides of nucleotide sequence 1 are fluorine-modified nucleotides, and from the 5'-end to the 3'-end, the 2nd, 6th, 14th, and 16th nucleotides of nucleotide sequence 2 are fluorine-modified nucleotides. In some embodiments, the fluorine-modified nucleotides are located at nucleotide sequence 1 and nucleotide sequence 2. From the 5'-end to the 3'-end, the 7th, 8th, and 9th nucleotides of nucleotide sequence 1 are fluorine-modified nucleotides, and from the 5'-end to the 3'-end, the 2nd, 6th, 14th, and 16th nucleotides of nucleotide sequence 2 are fluorine-modified nucleotides.

[0210] In some embodiments, the number of fluorine-modified nucleotides in nucleotide sequence 1 is 5 or less. In some embodiments, the number of fluorine-modified nucleotides in nucleotide sequence 2 is 7 or less. In some embodiments, the number of fluorine-modified nucleotides in nucleotide sequence 1 is 5 or less. In some embodiments, the number of fluorine-modified nucleotides in nucleotide sequence 2 is 7 or less. In some embodiments, the number of fluorine-modified nucleotides in nucleotide sequence 1 is 5 or less. In some embodiments, the number of fluorine-modified nucleotides in nucleotide sequence 2 is 7 or less.

[0211] In some embodiments, from the 5'-end to the 3'-end, in the sense strand, the 7th, 8th, and 9th nucleotides or the 5th, 7th, 8th, and 9th nucleotides of nucleotide sequence 1 are fluorine-modified nucleotides, and the nucleotides at other positions in the sense strand are non-fluorine-modified nucleotides. In some embodiments, from the 5'-end to the 3'-end, in the sense strand, the 7th, 8th, and 9th nucleotides or the 5th, 7th, 8th, and 9th nucleotides of nucleotide sequence 1 are fluorine-modified nucleotides, and the nucleotides at other positions in the sense strand are non-fluorine-modified nucleotides. In some embodiments, from the 5'-end to the 3'-end, in the sense strand, the 7th, 8th, and 9th nucleotides or the 5th, 7th, 8th, and 9th nucleotides of nucleotide sequence 1 are fluorine-modified nucleotides, and the nucleotides at other positions in the sense strand are non-fluorine-modified nucleotides. It is a fluoro-modified nucleotide, and from the 5'-end to the 3'-end, in the antisense strand wherein the nucleotides at the 2nd, 6th, 14th, 16th positions or the 2nd, 6th, 8th, 9th, 14th , 16th positions of the nucleotide sequence 2 are fluoro-modified nucleotides, and the nucleotides at other positions in the antisense strand are non-fluoro-modified nucleotides.

[0212] The definitions and selectable ranges of the fluoro-modified nucleotides and non-fluoro-modified nucleotides are as described above.

[0213] In some embodiments, from the 5'-end to the 3'-end, the nucleotides at the 5th, 7th, 8th and 9th positions of the nucleotide sequence 1 in the sense strand of the siRNA shown as Nu are fluoro-modified nucleotides, the nucleotides at other positions in the sense strand of the siRNA are methoxy-modified nucleotides, and also, from the 5'-end to the 3'-end, the nucleotides at the 2nd, 6th, 8th, 9th, 14th and 16th positions of the nucleotide sequence 2 in the antisense strand of the siRNA shown as Nu are fluoro-modified nucleotides, the nucleotides at other positions in the antisense strand of the siRNA are methoxy-modified nucleotides, or, from the 5'-end to the 3'-end, the nucleotides at the 7th, 8th and 9th positions of the nucleotide sequence 1 in the sense strand of the siRNA shown as Nu are fluoro-modified nucleotides, the nucleotides at other positions in the sense strand of the siRNA are methoxy-modified nucleotides, and also, from the 5'-end to the 3'-end, the nucleotides at the 2nd, 6th, 14th and 16th positions of the nucleotide sequence 2 in the antisense strand of the siRNA shown as Nu are fluoro-modified nucleotides, the nucleotides at other positions in the antisense strand of the siRNA are methoxy-modified nucleotides, or, from the 5'-end to the 3'-end, the nucleotides at the 7th, 8th and 9th positions of the nucleotide sequence 1 in the sense strand of the siRNA shown as Nu are fluoro-modified nucleotides, the nucleotides at other positions in the sense strand of the siRNA are methoxy-modified nucleotides, and also, from the 5'-end to the 3'-end, the nucleotides at the 2nd, 6th, 14th and 16th positions of the nucleotide sequence 2 in the antisense strand of the siRNA shown as Nu are fluoro-modified nucleotides, It is a modified nucleotide, and the nucleotides at other positions of the antisense strand of siRNA are meth oxy-modified nucleotides, or, from the 5'-end to the 3'-end, the nucleotides at positions 5, 7, 8, and 9 of nucleotide sequence 1 in the sense strand of the siRNA shown by Nu are fluoro-modified nucleotides, and the nucleotides at other positions of the sense strand of the siRNA are methoxy-modified nucleotides. Also, from the 5'-end to the 3'-end, the nucleotides at positions 2, 6, 14, and 16 of nucleotide sequence 2 in the antisense strand of the siRNA shown by Nu are fluoro-modified nucleotides, and the nucleotides at other positions of the antisense strand of the siRNA are methoxy-modified nucleotides. methoxy-modified nucleotides.

[0214] In some embodiments, the nucleotide has a phosphate group modification. In some embodiments, the phosphate group modification is a phosphorothioate modification represented by the following formula (101), that is, by substituting a non-bridging oxygen atom in the phosphodiester bond with one sulfur atom, the phosphodiester bond is replaced with a phosphorothioate diester bond. By this modification, the structure of the siRNA can be stabilized, and high specificity and high affinity of base pairing can be maintained.

[0215]

Chemical formula

[0216] In some embodiments, in the siRNA shown by Nu, the phosphorothioate group is the first nucleotide and the second nucleotide at any one end of the sense strand or the antisense strand. Between nucleotides, and with the second nucleotide at either end of the sense strand or the antisense strand Between the third nucleotide, or at least one selected from the group consisting of any combination thereof Is present bonded to at least one. In some embodiments, the thiophosphate ester group Is present bonded to all of the above positions except the 5' end of the sense strand. In some embodiments In, the thiophosphate ester group is bonded to all of the above positions except the 3' end of the sense strand Is present. In some embodiments, the thiophosphate ester group is present at least at one of the following positions Is present bonded to at least one location. Between the first nucleotide and the second nucleotide from the 5' end of the sense strand, Between the second nucleotide and the third nucleotide from the 5' end of the sense strand, Between the first nucleotide and the second nucleotide from the 3' end of the sense strand, Between the second nucleotide and the third nucleotide from the 3' end of the sense strand, Between the first nucleotide and the second nucleotide from the 5' end of the antisense strand Between, Between the second nucleotide and the third nucleotide from the 5' end of the antisense strand Between, Between the first nucleotide and the second nucleotide from the 3' end of the antisense strand Between, and Between the second nucleotide and the third nucleotide from the 3' end of the antisense strand Between.

[0217] In some embodiments, the nucleotide at the 5' end of the antisense strand sequence of the siRNA molecule represented by Nu Is a 5'-phosphate nucleotide or a 5'-phosphate analog-modified nu Cleotide.

[0218] In some embodiments, the 5'-phosphate nucleotide or 5'-phosphate analog-modified nucleotide is a nucleotide containing a 5'-phosphate modification as shown in formula (102), a nucleotide containing vinyl phosphate ester (E-vinylphosphonate , E-VP) as shown in formula (103), or a nucleotide containing a 5'-thiophosphate modification as shown in formula (105).

[0219] The inventors of the present disclosure have surprisingly found that the second type of siRNA complex of the present disclosure has significantly improved plasma stability, a low off-target effect, and further shows an HBV mRNA silencing activity that is not significantly reduced, and has a higher lipid suppression effect. Therefore, in some embodiments, the siRNA shown as Nu in the second type of siRNA complex of the present disclosure may be the siRNA shown in Table 1. In the siRNA or siRNA complex described in the present disclosure, each adjacent nucleotide

[0220]

Table 1

[0221] In the siRNA or siRNA complex described in the present disclosure, each adjacent nucleotide is linked by a phosphodiester bond or a thiophosphate diester bond, and the non-bridging oxygen atom or sulfur atom in the phosphodiester bond or thiophosphate diester bond is negatively charged and may exist as a hydroxy group or a sulfhydryl group, and the hydrogen ion in the hydroxy group or sulfhydryl group may be partially or completely substituted with a cation. The cation is any cation, for example, a metal cation, an ammonium ion NH4 + ​ It may also be one of the organic ammonium cations. Considering the improvement of solubility, in some embodiments, the cation is selected from one or more of alkali metal ions, ammonium cations formed by tertiary amines and quaternary ammonium cations. The alkali metal ion may be K and / or Na + and the cation formed by the tertiary amine may be an ammonium ion formed by triethylamine and / or + an ammonium ion formed by N,N - diisopropylethylamine. Therefore, the siRNA or siRNA complex described in the present disclosure may exist at least in part as a salt. In one aspect, the non - bridging oxygen atom or sulfur atom in the phosphodiester bond or thiophosphate diester bond is at least in part bound to a sodium ion, and the siRNA or siRNA complex described in the present disclosure exists as a sodium salt or a partial sodium salt. As will be clearly known to those skilled in the art, a modified nucleotide group can be introduced into the siRNA described in the present disclosure by using a corresponding modified nucleoside monomer. Methods for preparing the corresponding modified nucleoside monomers and methods for introducing the modified nucleotide group into siRNA are also well known to those skilled in the art. All modified nucleoside monomers may be purchased as commercial products or prepared by known methods. / or an ammonium ion formed by N,N - diisopropylethylamine. Therefore, the siRNA or siRNA complex described in the present disclosure may exist at least in part as a salt. In one aspect, the non - bridging oxygen atom or sulfur atom in the phosphodiester bond or thiophosphate diester bond is at least in part bound to a sodium ion, and the siRNA or siRNA complex described in the present disclosure exists as a sodium salt or a partial sodium salt. As will be clearly known to those skilled in the art, a modified nucleotide group can be introduced into the siRNA described in the present disclosure by using a corresponding modified nucleoside monomer. Methods for preparing the corresponding modified nucleoside monomers and methods for introducing the modified nucleotide group into siRNA are also well known to those skilled in the art. All modified nucleoside monomers may be purchased as commercial products or prepared by known methods. As will be clearly known to those skilled in the art, a modified nucleotide group can be introduced into the siRNA described in the present disclosure by using a corresponding modified nucleoside monomer. Methods for preparing the corresponding modified nucleoside monomers and methods for introducing the modified nucleotide group into siRNA are also well known to those skilled in the art. All modified nucleoside monomers may be purchased as commercial products or prepared by known methods. As will be clearly known to those skilled in the art, a modified nucleotide group can be introduced into the siRNA described in the present disclosure by using a corresponding modified nucleoside monomer. Methods for preparing the corresponding modified nucleoside monomers and methods for introducing the modified nucleotide group into siRNA are also well known to those skilled in the art. All modified nucleoside monomers may be purchased as commercial products or prepared by known methods. As will be clearly known to those skilled in the art, a modified nucleotide group can be introduced into the siRNA described in the present disclosure by using a corresponding modified nucleoside monomer. Methods for preparing the corresponding modified nucleoside monomers and methods for introducing the modified nucleotide group into siRNA are also well known to those skilled in the art. All modified nucleoside monomers may be purchased as commercial products or prepared by known methods. As will be clearly known to those skilled in the art, a modified nucleotide group can be introduced into the siRNA described in the present disclosure by using a corresponding modified nucleoside monomer. Methods for preparing the corresponding modified nucleoside monomers and methods for introducing the modified nucleotide group into siRNA are also well known to those skilled in the art. All modified nucleoside monomers may be purchased as commercial products or prepared by known methods.

[0222] As will be clearly known to those skilled in the art, a modified nucleotide group can be introduced into the siRNA described in the present disclosure by using a corresponding modified nucleoside monomer. Methods for preparing the corresponding modified nucleoside monomers and methods for introducing the modified nucleotide group into siRNA are also well known to those skilled in the art. All modified nucleoside monomers may be purchased as commercial products or prepared by known methods. As will be clearly known to those skilled in the art, a modified nucleotide group can be introduced into the siRNA described in the present disclosure by using a corresponding modified nucleoside monomer. Methods for preparing the corresponding modified nucleoside monomers and methods for introducing the modified nucleotide group into siRNA are also well known to those skilled in the art. All modified nucleoside monomers may be purchased as commercial products or prepared by known methods. As will be clearly known to those skilled in the art, a modified nucleotide group can be introduced into the siRNA described in the present disclosure by using a corresponding modified nucleoside monomer. Methods for preparing the corresponding modified nucleoside monomers and methods for introducing the modified nucleotide group into siRNA are also well known to those skilled in the art. All modified nucleoside monomers may be purchased as commercial products or prepared by known methods. As will be clearly known to those skilled in the art, a modified nucleotide group can be introduced into the siRNA described in the present disclosure by using a corresponding modified nucleoside monomer. Methods for preparing the corresponding modified nucleoside monomers and methods for introducing the modified nucleotide group into siRNA are also well known to those skilled in the art. All modified nucleoside monomers may be purchased as commercial products or prepared by known methods. As will be clearly known to those skilled in the art, a modified nucleotide group can be introduced into the siRNA described in the present disclosure by using a corresponding modified nucleoside monomer. Methods for preparing the corresponding modified nucleoside monomers and methods for introducing the modified nucleotide group into siRNA are also well known to those skilled in the art. All modified nucleoside monomers may be purchased as commercial products or prepared by known methods.

[0223] <Preparation of the second type of siRNA complex> The second type of siRNA complex may be prepared by any reasonable synthetic route.

[0224] In some embodiments, the second type of siRNA complex can be prepared by the following method. The method involves binding nucleoside monomers in sequence from 3' to 5' according to the nucleotide types and sequences of the sense strand and antisense strand of siRNA respectively under the conditions of phosphoramidite solid-phase synthesis. The binding of each nucleoside monomer involves four reactions: deprotection, cleavage, capping, oxidation or sulfurization. Isolate the sense strand and antisense strand of siRNA and perform annealing. Each nucleotide in the above is independently a modified or unmodified nucleotide. The siRNA represented by Nu includes a sense strand and an antisense strand. The sense strand includes nucleotide sequence 1, and the antisense strand includes nucleotide sequence 2. The nucleotide sequence 1 and the nucleotide sequence 2 form a double-stranded region that is at least partially reverse complementary. The nucleotide sequence 1 and the nucleotide sequence shown in SEQ ID NO: 155 have the same length and have three or fewer nucleotide differences. The nucleotide sequence 2 and the nucleotide sequence shown in SEQ ID NO: 156 have the same length and have three or fewer nucleotide differences. 5'-CCUUGAGGCAUACUUCAAZ-3' (SEQ ID NO: 155), 5'-Z'UUGAAGUAUGCCUCAAGG-3' (SEQ ID NO: 156) where Z is A and Z' is U. The nucleotide sequence 1 contains a nucleotide Z at the position corresponding to Z, and the nucleotide sequence 2 contains a nucleotide Z' at the position corresponding to Z'. The Z' is The nucleotide sequence 1 and the nucleotide sequence 2 form a double-stranded region that is at least partially reverse complementary. The nucleotide sequence 1 and the nucleotide sequence shown in SEQ ID NO: 155 have the same length and have three or fewer nucleotide differences. The nucleotide sequence 2 and the nucleotide sequence shown in SEQ ID NO: 156 have the same length and have three or fewer nucleotide differences. The nucleotide sequence 1 and the nucleotide sequence shown in SEQ ID NO: 155 have the same length and have three or fewer nucleotide differences. The nucleotide sequence 2 and the nucleotide sequence shown in SEQ ID NO: 156 have the same length and have three or fewer nucleotide differences. 5'-CCUUGAGGCAUACUUCAAZ-3' (SEQ ID NO: 155), 5'-Z'UUGAAGUAUGCCUCAAGG-3' (SEQ ID NO: 156) 5'-CCUUGAGGCAUACUUCAAZ-3' (SEQ ID NO: 155), 5'-Z'UUGAAGUAUGCCUCAAGG-3' (SEQ ID NO: 156) where Z is A and Z' is U.

[0225] The nucleotide sequence 1 contains a nucleotide Z at the position corresponding to Z, and the nucleotide sequence 2 contains a nucleotide Z' at the position corresponding to Z'. A The nucleotide Z corresponding to the position of Z is included in the nucleotide sequence 1. The nucleotide sequence 2 contains a nucleotide Z' at the position corresponding to Z'. B The nucleotide Z' corresponding to the position of Z' is included in the nucleotide sequence 2. B The Z' It is the first nucleotide at the 5' end of the antisense strand.

[0226] The method also includes reacting a compound of formula (3) under coupling reaction conditions and in the presence of a coupling reagent. 21) is added to a nucleoside monomer or a nucleoside bound to a solid support. and contacting the compound represented by formula (321) with a nucleotide sequence, to form a nucleotide sequence. The compound shown in formula (321) below is a conjugate molecule. Also called a child.

[0227] [ka] During the ceremony, R4 is a moiety capable of binding to the siRNA shown in Nu. where R4 is a moiety that can bind to the siRNA represented by Nu via a covalent bond. In some embodiments, R4 is linked to s by a phosphodiester bond via a reaction. A moiety that can be conjugated to any functional group of an iRNA, Each S1 was independently prepared by substituting all active hydroxy groups in M1 with YCOO- groups. each Y is a methyl group, a trifluoromethyl group, a difluoromethyl group, a fluoromethyl group, ethyl group, trichloromethyl group, dichloromethyl group, chloromethyl group, ethyl group, n-propyl group Independent of propyl, isopropyl, phenyl, halophenyl and alkylphenyl groups It is the one that is selected.

[0228] n1, n3, m1, m2, m3, R 10 , R 11 , R 12 , R 13 , R 14 , R 15 , The definitions and selectable ranges of L1 and M1 are as described above.

[0229] R4 is selected to achieve a bond with N on the nitrogen-containing backbone and provide an appropriate reaction site for the synthesis of the siRNA complex of formula (1). In some embodiments, R4 contains an R2 linker group or a protected R2 linker group, and a functional group capable of forming a structure shown in A59 by reacting with siRNA. In some embodiments, R4 contains a first functional group capable of forming a phosphite ester with a group on the siRNA or nucleoside monomer, and a second functional group capable of reacting with a hydroxy group or an amino group to form a covalent bond, or includes a solid-phase support bound by said covalent bond. In some embodiments, the first functional group is a phosphoramidite, a hydroxy group or a protected hydroxy group. In some embodiments, the second functional group is a phosphoramidite, a carboxylic acid or a carboxylate salt. In some embodiments, the second functional group is a solid-phase support bound to another part of the molecule via a covalent bond, and said covalent bond is formed by a hydroxy group or an amino group. In some embodiments, the solid-phase support is bound via a phosphoester bond, a carboxylic acid ester bond or an amide bond. In some embodiments, the solid-phase support is a resin. In some embodiments, the first functional group contains a hydroxy group, -OR or a group shown in formula ( C3), and the second functional group is of formula (C1), (C2), (C3), (

[0230] In some embodiments, R4 is a group on the siRNA or nucleoside monomer that can form a phosphite ester with a group on the siRNA or nucleoside monomer, and a second functional group that can react with a hydroxy group or an amino group to form a covalent bond, or includes a solid-phase support bound by said covalent bond. In some embodiments, the first functional group is a phosphoramidite, a hydroxy group or a protected hydroxy group. In some embodiments, the second functional group is a phosphoramidite, a carboxylic acid or a carboxylate salt. In some embodiments, the second functional group is a solid-phase support bound to another part of the molecule via a covalent bond, and said covalent bond is formed by a hydroxy group or an amino group. In some embodiments, the solid-phase support is bound via a phosphoester bond, a carboxylic acid ester bond or an amide bond. In some embodiments, the solid-phase support is a resin. In some embodiments, the first functional group contains a hydroxy group, -OR or a group shown in formula ( C3), and the second functional group is of formula (C1), (C2), (C3), ( In some embodiments, R4 contains a first functional group capable of forming a phosphite ester with a group on the siRNA or nucleoside monomer, and a second functional group capable of reacting with a hydroxy group or an amino group to form a covalent bond, or includes a solid-phase support bound by said covalent bond. In some embodiments, the first functional group is a phosphoramidite, a hydroxy group or a protected hydroxy group. In some embodiments, the second functional group is a phosphoramidite, a carboxylic acid or a carboxylate salt. In some embodiments, the second functional group is a solid-phase support bound to another part of the molecule via a covalent bond, and said covalent bond is formed by a hydroxy group or an amino group. In some embodiments, the solid-phase support is bound via a phosphoester bond, a carboxylic acid ester bond or an amide bond. In some embodiments, the solid-phase support is a resin. In some embodiments, the first functional group is a phosphoramidite, a hydroxy group or a protected hydroxy group. In some embodiments, the second functional group is a phosphoramidite, a carboxylic acid or a carboxylate salt. In some embodiments, the second functional group is a solid-phase support bound to another part of the molecule via a covalent bond, and said covalent bond is formed by a hydroxy group or an amino group. In some embodiments, the solid-phase support is bound via a phosphoester bond, a carboxylic acid ester bond or an amide bond. In some embodiments, the solid-phase support is a resin. In some embodiments, the second functional group is a phosphoramidite, a carboxylic acid or a carboxylate salt. In some embodiments, the second functional group is a solid-phase support bound to another part of the molecule via a covalent bond, and said covalent bond is formed by a hydroxy group or an amino group. In some embodiments, the solid-phase support is bound via a phosphoester bond, a carboxylic acid ester bond or an amide bond. In some embodiments, the solid-phase support is a resin. In some embodiments, the second functional group is a solid-phase support bound to another part of the molecule via a covalent bond, and said covalent bond is formed by a hydroxy group or an amino group. In some embodiments, the solid-phase support is bound via a phosphoester bond, a carboxylic acid ester bond or an amide bond. In some embodiments, the solid-phase support is a resin. In some embodiments, the second functional group is a solid-phase support bound to another part of the molecule via a covalent bond, and said covalent bond is formed by a hydroxy group or an amino group. In some embodiments, the solid-phase support is bound via a phosphoester bond, a carboxylic acid ester bond or an amide bond. In some embodiments, the solid-phase support is a resin. In some embodiments, the solid-phase support is bound via a phosphoester bond, a carboxylic acid ester bond or an amide bond. In some embodiments, the solid-phase support is a resin. In some embodiments, the solid-phase support is bound via a phosphoester bond, a carboxylic acid ester bond or an amide bond. In some embodiments, the solid-phase support is a resin. In some embodiments, the solid-phase support is a resin.

[0231] In some embodiments, the first functional group contains a hydroxy group, -OR k or a group shown in formula ( C3), and the second functional group is of formula (C1), (C2), (C3), ( It contains the structure shown in (C1’) or (C3’).

[0232]

Chemical formula

[0233] In some embodiments, the first functional group contains a phosphoramidite functional group as shown in formula (C3), and the phosphoramidite group can undergo a coupling reaction with a hydroxy group at any position on the nucleotide, for example, the 2'-hydroxy group or the 3'-hydroxy group, to form a phosphite ester, which is then oxidized or sulfided to form a phosphodiester bond or a thiophosphate ester bond as shown in formula A59, and the composite molecule can be complexed with siRNA. In this case, even if the second functional group is absent, the compound of formula (321) can be complexed with the nucleotide and has no effect on the acquisition of the siRNA complex shown in formula (1). In this case, after obtaining the sense strand or antisense strand of siRNA by methods such as phosphoramidite solid-phase synthesis, the compound of formula (321) is reacted with the hydroxy group on the terminal nucleotide in the nucleotide sequence, and a phosphodiester bond or a thiophosphate ester bond is formed during the subsequent oxidation or sulfidation process, and the compound of formula (321) is complexed with siRNA. contains a phosphoramidite functional group, and the phosphoramidite group can undergo a coupling reaction with a hydroxy group at any position on the nucleotide, for example, the 2'-hydroxy group or the 3'-hydroxy group, to form a phosphite ester, which is then oxidized or sulfided to form a phosphodiester bond or a thiophosphate ester bond as shown in formula A59, and the composite molecule can be complexed with siRNA. In this case, even if the second functional group is absent, the compound of formula (321) can be complexed with the nucleotide and has no effect on the acquisition of the siRNA complex shown in formula (1). In this case, after obtaining the sense strand or antisense strand of siRNA by methods such as phosphoramidite solid-phase synthesis, the compound of formula (321) is reacted with the hydroxy group on the terminal nucleotide in the nucleotide sequence, and a phosphodiester bond or a thiophosphate ester bond is formed during the subsequent oxidation or sulfidation process, and the compound of formula (321) is complexed with siRNA. position, such as the 2'-hydroxy group or the 3'-hydroxy group, to form a phosphite ester, which is then oxidized or sulfided to form a phosphodiester bond or a thiophosphate ester bond as shown in formula A59, and the composite molecule can be complexed with siRNA. In this case, even if the second functional group is absent, the compound of formula (321) can be complexed with the nucleotide and has no effect on the acquisition of the siRNA complex shown in formula (1). In this case, after obtaining the sense strand or antisense strand of siRNA by methods such as phosphoramidite solid-phase synthesis, the compound of formula (321) is reacted with the hydroxy group on the terminal nucleotide in the nucleotide sequence, and a phosphodiester bond or a thiophosphate ester bond is formed during the subsequent oxidation or sulfidation process, and the compound of formula (321) is complexed with siRNA. to form a phosphite ester, which is then oxidized or sulfided to form a phosphodiester bond or a thiophosphate ester bond as shown in formula A59, and the composite molecule can be complexed with siRNA. In this case, even if the second functional group is absent, the compound of formula (321) can be complexed with the nucleotide and has no effect on the acquisition of the siRNA complex shown in formula (1). In this case, after obtaining the sense strand or antisense strand of siRNA by methods such as phosphoramidite solid-phase synthesis, the compound of formula (321) is reacted with the hydroxy group on the terminal nucleotide in the nucleotide sequence, and a phosphodiester bond or a thiophosphate ester bond is formed during the subsequent oxidation or sulfidation process, and the compound of formula (321) is complexed with siRNA. to form a phosphodiester bond or a thiophosphate ester bond, and the composite molecule can be complexed with siRNA. In this case, even if the second functional group is absent, the compound of formula (321) can be complexed with the nucleotide and has no effect on the acquisition of the siRNA complex shown in formula (1). The compound can be complexed with the nucleotide, and it does not affect the acquisition of the siRNA complex shown in formula (1). In this case, after obtaining the sense strand or antisense strand of siRNA by methods such as phosphoramidite solid-phase synthesis, the compound of formula (321) is reacted with the hydroxy group on the terminal nucleotide in the nucleotide sequence, and a phosphodiester bond or a thiophosphate ester bond is formed during the subsequent oxidation or sulfidation process, and the compound of formula (321) is complexed with siRNA. to form a phosphodiester bond or a thiophosphate ester bond, and the compound of formula (321) is complexed with siRNA.

[0234] In some embodiments, the first functional group contains a protected hydroxy group. In some embodiments, the second functional group includes a group capable of reacting with a solid support, and provides a composite molecule including the solid support through the reaction described above. In some embodiments, the second functional group includes a carboxy group, a carboxylate, or a phosphoramidite, as represented by formula (C1), (C2), or (C3). When the second functional group includes a carboxy group or a carboxylate, an esterification reaction or an amidation reaction is carried out with a hydroxy group or an amino group in a solid support, such as a resin, in the compound of formula (321), to form a composite molecule including the solid support bonded by a carboxylic acid ester bond or an amide bond. When the second functional group includes a phosphoramidite functional group, a coupling reaction is carried out with a hydroxy group in a general solid support, such as a resin, in the compound of formula (321), and then oxidized to form a composite molecule including the solid support bonded by a phosphoric acid diester bond. Then, starting from the product to which the above solid support is bonded, nucleoside monomers are sequentially bonded according to the phosphoramidite solid-phase synthesis method to obtain a sense strand or an antisense strand of siRNA to which a composite group is bonded. In the phosphoramidite solid-phase synthesis process, the first functional group is deprotected and then coupled with a phosphoramidite group in a nucleoside monomer under coupling reaction conditions. In some embodiments, the first functional group includes a hydroxy group or a protected hydroxy group, and the second functional group is a solid support bonded by a carboxylic acid ester bond or a solid support bonded by an amide bond, as represented by formula (C1') or (C3'). When the second functional group includes a carboxy group or a carboxylate, an esterification reaction or an amidation reaction is carried out with a hydroxy group or an amino group in a solid support, such as a resin, in the compound of formula (321), to form a composite molecule including the solid support bonded by a carboxylic acid ester bond or an amide bond. When the second functional group includes a phosphoramidite functional group, a coupling reaction is carried out with a hydroxy group in a general solid support, such as a resin, in the compound of formula (321), and then oxidized to form a composite molecule including the solid support bonded by a phosphoric acid diester bond. Then, starting from the product to which the above solid support is bonded, nucleoside monomers are sequentially bonded according to the phosphoramidite solid-phase synthesis method to obtain a sense strand or an antisense strand of siRNA to which a composite group is bonded. In the phosphoramidite solid-phase synthesis process, the first functional group is deprotected and then coupled with a phosphoramidite group in a nucleoside monomer under coupling reaction conditions. When the second functional group includes a carboxy group or a carboxylate, an esterification reaction or an amidation reaction is carried out with a hydroxy group or an amino group in a solid support, such as a resin, in the compound of formula (321), to form a composite molecule including the solid support bonded by a carboxylic acid ester bond or an amide bond. When the second functional group includes a phosphoramidite functional group, a coupling reaction is carried out with a hydroxy group in a general solid support, such as a resin, in the compound of formula (321), and then oxidized to form a composite molecule including the solid support bonded by a phosphoric acid diester bond. Then, starting from the product to which the above solid support is bonded, nucleoside monomers are sequentially bonded according to the phosphoramidite solid-phase synthesis method to obtain a sense strand or an antisense strand of siRNA to which a composite group is bonded. In the phosphoramidite solid-phase synthesis process, the first functional group is deprotected and then coupled with a phosphoramidite group in a nucleoside monomer under coupling reaction conditions. When the second functional group includes a carboxy group or a carboxylate, an esterification reaction or an amidation reaction is carried out with a hydroxy group or an amino group in a solid support, such as a resin, in the compound of formula (321), to form a composite molecule including the solid support bonded by a carboxylic acid ester bond or an amide bond. When the second functional group includes a phosphoramidite functional group, a coupling reaction is carried out with a hydroxy group in a general solid support, such as a resin, in the compound of formula (321), and then oxidized to form a composite molecule including the solid support bonded by a phosphoric acid diester bond. Then, starting from the product to which the above solid support is bonded, nucleoside monomers are sequentially bonded according to the phosphoramidite solid-phase synthesis method to obtain a sense strand or an antisense strand of siRNA to which a composite group is bonded. In the phosphoramidite solid-phase synthesis process, the first functional group is deprotected and then coupled with a phosphoramidite group in a nucleoside monomer under coupling reaction conditions. When the second functional group includes a carboxy group or a carboxylate, an esterification reaction or an amidation reaction is carried out with a hydroxy group or an amino group in a solid support, such as a resin, in the compound of formula (321), to form a composite molecule including the solid support bonded by a carboxylic acid ester bond or an amide bond. When the second functional group includes a phosphoramidite functional group, a coupling reaction is carried out with a hydroxy group in a general solid support, such as a resin, in the compound of formula (321), and then oxidized to form a composite molecule including the solid support bonded by a phosphoric acid diester bond. Then, starting from the product to which the above solid support is bonded, nucleoside monomers are sequentially bonded according to the phosphoramidite solid-phase synthesis method to obtain a sense strand or an antisense strand of siRNA to which a composite group is bonded. In the phosphoramidite solid-phase synthesis process, the first functional group is deprotected and then coupled with a phosphoramidite group in a nucleoside monomer under coupling reaction conditions. When the second functional group includes a carboxy group or a carboxylate, an esterification reaction or an amidation reaction is carried out with a hydroxy group or an amino group in a solid support, such as a resin, in the compound of formula (321), to form a composite molecule including the solid support bonded by a carboxylic acid ester bond or an amide bond. When the second functional group includes a phosphoramidite functional group, a coupling reaction is carried out with a hydroxy group in a general solid support, such as a resin, in the compound of formula (321), and then oxidized to form a composite molecule including the solid support bonded by a phosphoric acid diester bond. Then, starting from the product to which the above solid support is bonded, nucleoside monomers are sequentially bonded according to the phosphoramidite solid-phase synthesis method to obtain a sense strand or an antisense strand of siRNA to which a composite group is bonded. In the phosphoramidite solid-phase synthesis process, the first functional group is deprotected and then coupled with a phosphoramidite group in a nucleoside monomer under coupling reaction conditions. When the second functional group includes a carboxy group or a carboxylate, an esterification reaction or an amidation reaction is carried out with a hydroxy group or an amino group in a solid support, such as a resin, in the compound of formula (321), to form a composite molecule including the solid support bonded by a carboxylic acid ester bond or an amide bond. When the second functional group includes a phosphoramidite functional group, a coupling reaction is carried out with a hydroxy group in a general solid support, such as a resin, in the compound of formula (321), and then oxidized to form a composite molecule including the solid support bonded by a phosphoric acid diester bond. Then, starting from the product to which the above solid support is bonded, nucleoside monomers are sequentially bonded according to the phosphoramidite solid-phase synthesis method to obtain a sense strand or an antisense strand of siRNA to which a composite group is bonded. In the phosphoramidite solid-phase synthesis process, the first functional group is deprotected and then coupled with a phosphoramidite group in a nucleoside monomer under coupling reaction conditions. When the second functional group includes a carboxy group or a carboxylate, an esterification reaction or an amidation reaction is carried out with a hydroxy group or an amino group in a solid support, such as a resin, in the compound of formula (321), to form a composite molecule including the solid support bonded by a carboxylic acid ester bond or an amide bond. When the second functional group includes a phosphoramidite functional group, a coupling reaction is carried out with a hydroxy group in a general solid support, such as a resin, in the compound of formula (321), and then oxidized to form a composite molecule including the solid support bonded by a phosphoric acid diester bond. Then, starting from the product to which the above solid support is bonded, nucleoside monomers are sequentially bonded according to the phosphoramidite solid-phase synthesis method to obtain a sense strand or an antisense strand of siRNA to which a composite group is bonded. In the phosphoramidite solid-phase synthesis process, the first functional group is deprotected and then coupled with a phosphoramidite group in a nucleoside monomer under coupling reaction conditions. Then, starting from the product to which the above solid support is bonded, nucleoside monomers are sequentially bonded according to the phosphoramidite solid-phase synthesis method to obtain a sense strand or an antisense strand of siRNA to which a composite group is bonded. In the phosphoramidite solid-phase synthesis process, the first functional group is deprotected and then coupled with a phosphoramidite group in a nucleoside monomer under coupling reaction conditions. Then, starting from the product to which the above solid support is bonded, nucleoside monomers are sequentially bonded according to the phosphoramidite solid-phase synthesis method to obtain a sense strand or an antisense strand of siRNA to which a composite group is bonded. In the phosphoramidite solid-phase synthesis process, the first functional group is deprotected and then coupled with a phosphoramidite group in a nucleoside monomer under coupling reaction conditions. Then, starting from the product to which the above solid support is bonded, nucleoside monomers are sequentially bonded according to the phosphoramidite solid-phase synthesis method to obtain a sense strand or an antisense strand of siRNA to which a composite group is bonded. In the phosphoramidite solid-phase synthesis process, the first functional group is deprotected and then coupled with a phosphoramidite group in a nucleoside monomer under coupling reaction conditions. Then, starting from the product to which the above solid support is bonded, nucleoside monomers are sequentially bonded according to the phosphoramidite solid-phase synthesis method to obtain a sense strand or an antisense strand of siRNA to which a composite group is bonded. In the phosphoramidite solid-phase synthesis process, the first functional group is deprotected and then coupled with a phosphoramidite group in a nucleoside monomer under coupling reaction conditions. Then, starting from the product to which the above solid support is bonded, nucleoside monomers are sequentially bonded according to the phosphoramidite solid-phase synthesis method to obtain a sense strand or an antisense strand of siRNA to which a composite group is bonded. In the phosphoramidite solid-phase synthesis process, the first functional group is deprotected and then coupled with a phosphoramidite group in a nucleoside monomer under coupling reaction conditions.

[0235] In some embodiments, the first functional group includes a hydroxy group or a protected hydroxy group, and the second functional group is a solid support bonded by a carboxylic acid ester bond or a solid support bonded by an amide bond, as represented by formula (C1') or (C3'). In some embodiments, the first functional group includes a hydroxy group or a protected hydroxy group, and the second functional group is a solid support bonded by a carboxylic acid ester bond or a solid support bonded by an amide bond, as represented by formula (C1') or (C3'). In some embodiments, the first functional group includes a hydroxy group or a protected hydroxy group, and the second functional group is a solid support bonded by a carboxylic acid ester bond or a solid support bonded by an amide bond, as represented by formula (C1') or (C3'). It contains a carrier or a solid-phase carrier bonded by a phosphoric acid ester bond. In this case, starting with using the compound of formula (321) instead of the solid-phase carrier, and following the phosphoramidite solid-phase synthesis method successively bond the corresponding nucleoside monomers to obtain the sense strand or the antisense strand of siRNA to which the composite group is bonded. In some embodiments, the carboxylate salt is -COO - M + where M + is a cation, for example, one selected from metal cations, ammonium cation NH4 + and organic ammonium cations. In some embodiments, the metal ion is one selected from alkali metal ions, for example, K + or Na + . Considering improving solubility and performing the reaction smoothly , in some embodiments, the organic ammonium ion is an ammonium cation formed by a tertiary amine or a quaternary ammonium cation, for example, an ammonium ion formed by triethylamine or an ammonium ion formed by N,N-diisopropylethylamine. In some embodiments, the carboxylate salt is tri ethylamine carboxylate or N,N-diisopropylethylamine carboxylate . .

[0236] In some embodiments, R4 contains a structure represented by formula (B9), (B10), (B9’), (B1 0’), (B11), (B12), (B11’) or (B12’). .

[0237]

Chemical Formula

[0238] In some embodiments, R k is one or more of Tr (trityl group), MMTr (4-methoxy trityl group), DMTr (4,4'-bis-methoxytrityl group), TMTr (4,4', 4'-trimethoxyphenylmethyl group). In some embodiments R k may be DMTr, i.e., 4,4'-bis-methoxytrityl (4,4'-dimet hoxytrityl).

[0239] The definition of L1 is as described above.

[0240] In some embodiments, L1 binds the M1 ligand to the N atom on the nitrogen-containing backbone and is used to provide a liver targeting function to the oligonucleotide complex. In some embodiments L1 contains any one or a combination thereof of A1 to A26.

[0241] As can be easily understood by those skilled in the art from the above description, phosphoramide known in the art Compared with the amidite solid-phase synthesis method, the first functional group and any second functional group enable the formation of a complex molecule bound to any possible position of the nucleotide sequence, such as the end or terminus of the nucleotide sequence, to obtain an siRNA complex. Accordingly, unless otherwise specified, in the following descriptions regarding the preparation of the complex, when referring to reactions such as "deprotection", "cleavage", "capping", "oxidation", "sulfidation", etc., it should be understood that the reaction conditions and reagents according to the known amidite nucleic acid solid-phase synthesis method in this field are also applied to these reactions. Exemplary reaction conditions and reagents are described in detail below. In some embodiments, each S1 is independently M1. In some embodiments, each S1 is a group in which at least one active hydroxy group in M1 is protected by a hydroxy protecting group. In some embodiments, each S1 is a group in which all active hydroxy groups present in M1 are protected by hydroxy protecting groups. In some embodiments, any hydroxy protecting group known to those skilled in the art can be used to protect the active hydroxy groups in M1. In some embodiments, the protected hydroxy group may be represented by the formula YCOO-, and each Y is independently selected from the group consisting of C1-C alkyl groups and C6-C aryl groups, and the C1-C alkyl group and C6-C aryl group are optionally substituted with one or more substituents, and the substituents are selected from the group consisting of halogen and C1-C6 alkyl groups. In some embodiments,

[0242] In some embodiments, each S1 is independently M1. In some embodiments, each S1 is a group in which at least one active hydroxy group in M1 is protected by a hydroxy protecting group. In some embodiments, each S1 is a group in which all active hydroxy groups present in M1 are protected by hydroxy protecting groups. In some embodiments, any hydroxy protecting group known to those skilled in the art can be used to protect the active hydroxy groups in M1. In some embodiments, the protected hydroxy group may be represented by the formula YCOO-, and each Y is independently selected from the group consisting of C1-C alkyl groups and C6-C aryl groups, and the C1-C alkyl group and C6-C aryl group are optionally substituted with one or more substituents, and the substituents are selected from the group consisting of halogen and C1-C6 alkyl groups. In some embodiments, the protected hydroxy group may be represented by the formula YCOO-, and each Y is independently selected from the group consisting of C1-C 10 alkyl groups and C6-C 10 aryl groups, and the C1-C 10 alkyl group and C6-C 10 aryl group are optionally substituted with one or more substituents, and the substituents are selected from the group consisting of halogen and C1-C6 alkyl groups. In some embodiments, wherein each Y is independently selected from the group consisting of a methyl group, a trifluoromethyl group, a difluoromethyl group, a monofluoromethyl group, a trichloromethyl group, a dichloromethyl group, a chloromethyl group, an ethyl group, an n-propyl group, an isopropyl group, a phenyl group, a halophenyl group, and a C1-C6 alkylphenyl group.

[0243] In some embodiments, each S1 is independently selected from the group consisting of Formulas A46 to A54.

[0244]

Chemical formula

[0245] In some embodiments, S1 is Formula A49 or A50.

[0246] In some embodiments, each Y is one independently selected from a methyl group, a trifluoromethyl group, a difluoromethyl group, a fluoromethyl group, a trichloromethyl group, a dichloromethyl group, a chloromethyl group, an ethyl group, an n-propyl group, an isopropyl group, a phenyl group, a halophenyl group, and an alkylphenyl group. For the purpose of simplifying the composite molecule of the present disclosure, in some embodiments, Y is a methyl group.

[0247] As described above, the method for preparing the second type of siRNA complex further includes synthesizing the other strand of siRNA (for example, when the sense strand of siRNA bound to the composite molecule is synthesized in the above step, further including synthesizing the antisense strand of siRNA according to the solid-phase synthesis method, and vice versa), isolating the sense strand and the antisense strand, and annealing. ​​​​​​​​​​​It also includes. Specifically, in the isolation step, it binds to a nucleotide sequence and / or a complex molecule The solid support to which it is bound is cleaved and the necessary protecting groups are removed (in this case, each S1 group in the compound of formula (321 ) is converted to the corresponding M1 ligand), and the complex molecule binds The sense strand (or antisense strand) of the siRNA to which the complex molecule is bound and the corresponding antisense strand (or sense strand) are obtained, and the sense strand and the antisense strand are annealed to form a double-stranded RNA structure to obtain the siRNA complex shown in formula (1).

[0248] In some embodiments, the method for preparing the second type of siRNA complex is coupling Under coupling reaction conditions and in the presence of a coupling reagent, the compound of formula (321) is brought into contact with the first nucleoside monomer at the 3'-end of the sense strand or antisense strand, and the compound of formula (321) is bound to the first nucleotide in the sequence, and phosphoramidite Under the conditions of solid-phase synthesis, according to the type and order of the nucleotides of the desired sense strand or antisense strand The nucleoside monomers are sequentially bound from 3' to 5' to synthesize the sense strand or antisense strand of siRNA. The compound of (321) has a first functional group containing a protected hydroxy group at R4 and a second functional group having the structure shown in formula (C1') or (C3'). The compound of formula (321) is deprotected before binding to the first nucleo side monomer, and the binding of each nucleoside monomer includes four reactions: deprotection, coupling, capping, oxidation or sulfurization. To obtain the sense strand or antisense strand of the nucleic acid to which the complex molecule is bound; phosphoramidite Before binding to the first nucleoside monomer, the compound of formula (321) is deprotected, and the binding of each nucleoside mono mer includes four reactions: deprotection, coupling, capping, oxidation or sulfurization. To obtain the sense strand or antisense strand of the nucleic acid to which the complex molecule is bound; phosphoramidite Under the conditions of solid-phase synthesis, depending on the types and sequences of nucleotides of the antisense strand or sense strand, nucleoside monomers are sequentially linked from 3' to 5' to synthesize the antisense strand or sense strand of nucleic acid. The binding of each nucleoside monomer includes four reactions: deprotection, coupling, capping, oxidation or sulfidation. The protecting group is removed and cleaved from the solid-phase support, and then isolated and purified to obtain the sense strand and antisense strand of nucleic acid, including the step of annealing.

[0249] In some embodiments, the method for preparing the siRNA complex is based on the types and sequences of nucleotides of the sense strand or antisense strand in the double-stranded siR NA. Nucleoside monomers are sequentially linked from 3' to 5' to synthesize the sense strand and antisense strand. Each nucleoside monomer binding includes four reactions: deprotection, coupling, capping, oxidation or sulfur ization. A step of obtaining the sense strand bound to the solid-phase support and the antisense strand bound to the solid-phase support, and under coupling reaction conditions and in the presence of a coupling reagent, a compound represented by formula (321) is contacted with the sense strand bound to the solid-phase support or the antisense strand bound to the solid-phase support, and a compound of formula (321) containing a first functional group which is a phosphoramidite group at R4 is bound to the sense strand or antisense strand. A step of removing the protecting group and cleaving from the solid-phase support, isolating and purifying them respectively to obtain the sense strand or antisense strand of siRNA to which the composite molecule is bound, and including the step of annealing. (321) is contacted with the sense strand bound to the solid-phase support or the antisense strand bound to the solid-phase support, and a compound of formula (321) containing a first functional group which is a phosphoramidite group at R4 is bound to the sense strand or antisense strand. A step of removing the protecting group and cleaving from the solid-phase support, isolating and purifying them respectively to obtain the sense strand or antisense strand of siRNA to which the composite molecule is bound, and including the step of annealing. (321) is contacted with the sense strand bound to the solid-phase support or the antisense strand bound to the solid-phase support, and a compound of formula (321) containing a first functional group which is a phosphoramidite group at R4 is bound to the sense strand or antisense strand. A step of removing the protecting group and cleaving from the solid-phase support, isolating and purifying them respectively to obtain the sense strand or antisense strand of siRNA to which the composite molecule is bound, and including the step of annealing. The step of contacting the compound of formula (321) containing a first functional group which is a phosphoramidite group at R4 with the sense strand or antisense strand, removing the protecting group and cleaving from the solid-phase support, isolating and purifying them respectively to obtain the sense strand or antisense strand of siRNA to which the composite molecule is bound, and including the step of annealing. The step of contacting the compound of formula (321) containing a first functional group which is a phosphoramidite group at R4 with the sense strand or antisense strand, removing the protecting group and cleaving from the solid-phase support, isolating and purifying them respectively to obtain the sense strand or antisense strand of siRNA to which the composite molecule is bound, and including the step of annealing. The step of removing the protecting group and cleaving from the solid-phase support, isolating and purifying them respectively to obtain the sense strand or antisense strand of siRNA to which the composite molecule is bound, and including the step of annealing. The step of removing the protecting group and cleaving from the solid-phase support, isolating and purifying them respectively to obtain the sense strand or antisense strand of siRNA to which the composite molecule is bound, and including the step of annealing.

[0250] In some embodiments, P in formula A59 is bound to the 3' end of the sense strand in the siRNA. The method for preparing the siRNA complex of the present disclosure is as follows. (1) In the compound of formula (321) (the compound of formula (321) contains a first functional group including a protected hydroxy group OR and a second functional group having a structure represented by formula (C1’) or (C3’)), removing the hydroxy protecting group R k and contacting the deprotected product with a nucleoside monomer under a coupling reaction condition and in the presence of a coupling reagent to obtain a nucleoside monomer bound to a solid support by a composite molecule, (2) Starting from the nucleoside monomer bound to the solid support by the composite molecule, synthesizing the sense strand of siRNA by a phosphoramidite solid-phase synthesis method in the 3’-5’ direction, k (3) Synthesizing the antisense strand of siRNA by a phosphoramidite solid-phase synthesis method, (4) Isolating and annealing the sense strand and the antisense strand of siRNA to obtain the siRNA complex of the present disclosure. (5) In step (1), the method for removing the protecting group R in the compound of formula (321) includes contacting the compound of formula (321) with a deprotecting reagent under deprotection conditions. The deprotection conditions are such that the temperature is 0 to 50 °C, and in some embodiments, 15 to 35 °C, the reaction time is 30 to 300 seconds, and in some embodiments, 50 to 150 seconds, and the deprotecting reagent may be one or more selected from trifluoroacetic acid, trichloroacetic acid, dichloroacetic acid, and chloroacetic acid, and in some embodiments, dichloroacetic acid. The molar ratio of the deprotecting reagent to the compound of formula (321) is 10:1 to 1000:1. (6) In step (1), the method for removing the protecting group R in the compound of formula (321) includes contacting the compound of formula (321) with a deprotecting reagent under deprotection conditions. The deprotection conditions are such that the temperature is 0 to 50 °C, and in some embodiments, 15 to 35 °C, the reaction time is 30 to 300 seconds, and in some embodiments, 50 to 150 seconds, and the deprotecting reagent may be one or more selected from trifluoroacetic acid, trichloroacetic acid, dichloroacetic acid, and chloroacetic acid, and in some embodiments, dichloroacetic acid. The molar ratio of the deprotecting reagent to the compound of formula (321) is 10:1 to 1000:1. (2) Starting from the nucleoside monomer bound to the solid support by the composite molecule, synthesizing the sense strand of siRNA by a phosphoramidite solid-phase synthesis method in the 3’-5’ direction, (3) Synthesizing the antisense strand of siRNA by a phosphoramidite solid-phase synthesis method, (4) Isolating and annealing the sense strand and the antisense strand of siRNA to obtain the siRNA complex of the present disclosure. (3) In step (1), the method for removing the protecting group R in the compound of formula (321) includes contacting the compound of formula (321) with a deprotecting reagent under deprotection conditions. The deprotection conditions are such that the temperature is 0 to 50 °C, and in some embodiments, 15 to 35 °C, the reaction time is 30 to 300 seconds, and in some embodiments, 50 to 150 seconds, and the deprotecting reagent may be one or more selected from trifluoroacetic acid, trichloroacetic acid, dichloroacetic acid, and chloroacetic acid, and in some embodiments, dichloroacetic acid. The molar ratio of the deprotecting reagent to the compound of formula (321) is 10:1 to 1000:1. (4) In step (1), the method for removing the protecting group R in the compound of formula (321) includes contacting the compound of formula (321) with a deprotecting reagent under deprotection conditions. The deprotection conditions are such that the temperature is 0 to 50 °C, and in some embodiments, 15 to 35 °C, the reaction time is 30 to 300 seconds, and in some embodiments, 50 to 150 seconds, and the deprotecting reagent may be one or more selected from trifluoroacetic acid, trichloroacetic acid, dichloroacetic acid, and chloroacetic acid, and in some embodiments, dichloroacetic acid. The molar ratio of the deprotecting reagent to the compound of formula (321) is 10:1 to 1000:1. (4) Isolating and annealing the sense strand and the antisense strand of siRNA to obtain the siRNA complex of the present disclosure. (5) In step (1), the method for removing the protecting group R in the compound of formula (321) includes contacting the compound of formula (321) with a deprotecting reagent under deprotection conditions. The deprotection conditions are such that the temperature is 0 to 50 °C, and in some embodiments, 15 to 35 °C, the reaction time is 30 to 300 seconds, and in some embodiments, 50 to 150 seconds, and the deprotecting reagent may be one or more selected from trifluoroacetic acid, trichloroacetic acid, dichloroacetic acid, and chloroacetic acid, and in some embodiments, dichloroacetic acid. The molar ratio of the deprotecting reagent to the compound of formula (321) is 10:1 to 1000:1.

[0251] In step (1), the method for removing the protecting group R in the compound of formula (321) includes contacting the compound of formula (321) with a deprotecting reagent under deprotection conditions. The deprotection conditions are such that the temperature is 0 to 50 °C, and in some embodiments, 15 to 35 °C, the reaction time is 30 to 300 seconds, and in some embodiments, 50 to 150 seconds, and the deprotecting reagent may be one or more selected from trifluoroacetic acid, trichloroacetic acid, dichloroacetic acid, and chloroacetic acid, and in some embodiments, dichloroacetic acid. The molar ratio of the deprotecting reagent to the compound of formula (321) is 10:1 to 1000:1. k The method for removing the protecting group R in the compound of formula (321) includes contacting the compound of formula (321) with a deprotecting reagent under deprotection conditions. The deprotection conditions are such that the temperature is 0 to 50 °C, and in some embodiments, 15 to 35 °C, the reaction time is 30 to 300 seconds, and in some embodiments, 50 to 150 seconds, and the deprotecting reagent may be one or more selected from trifluoroacetic acid, trichloroacetic acid, dichloroacetic acid, and chloroacetic acid, and in some embodiments, dichloroacetic acid. The molar ratio of the deprotecting reagent to the compound of formula (321) is 10:1 to 1000:1. The deprotection conditions include contacting the compound of formula (321) with a deprotecting reagent under deprotection conditions. The deprotection conditions are such that the temperature is 0 to 50 °C, and in some embodiments, 15 to 35 °C, the reaction time is 30 to 300 seconds, and in some embodiments, 50 to 150 seconds, and the deprotecting reagent may be one or more selected from trifluoroacetic acid, trichloroacetic acid, dichloroacetic acid, and chloroacetic acid, and in some embodiments, dichloroacetic acid. The molar ratio of the deprotecting reagent to the compound of formula (321) is 10:1 to 1000:1. The deprotection conditions are such that the temperature is 0 to 50 °C, and in some embodiments, 15 to 35 °C, the reaction time is 30 to 300 seconds, and in some embodiments, 50 to 150 seconds, and the deprotecting reagent may be one or more selected from trifluoroacetic acid, trichloroacetic acid, dichloroacetic acid, and chloroacetic acid, and in some embodiments, dichloroacetic acid. The molar ratio of the deprotecting reagent to the compound of formula (321) is 10:1 to 1000:1. The reaction time is 30 to 300 seconds, and in some embodiments, 50 to 150 seconds, and the deprotecting reagent may be one or more selected from trifluoroacetic acid, trichloroacetic acid, dichloroacetic acid, and chloroacetic acid, and in some embodiments, dichloroacetic acid. The molar ratio of the deprotecting reagent to the compound of formula (321) is 10:1 to 1000:1. The deprotecting reagent may be one or more selected from trifluoroacetic acid, trichloroacetic acid, dichloroacetic acid, and chloroacetic acid, and in some embodiments, dichloroacetic acid. The molar ratio of the deprotecting reagent to the compound of formula (321) is 10:1 to 1000:1. The molar ratio of the deprotecting reagent to the compound of formula (321) is 10:1 to 1000:1. In some embodiments, dichloroacetic acid. The molar ratio of the deprotecting reagent to the compound of formula (321) is 10:1 to 1000:1. In some embodiments, it is from 50:1 to 500:1.

[0252] As the coupling reaction conditions and the coupling reagent, any conditions and reagents suitable for the above coupling reaction may be used. In some embodiments, the same conditions and reagents as those in the coupling reaction in the solid-phase synthesis method employed may be used. In some embodiments, as the coupling reaction conditions, the reaction temperature is 0 to 50 °C, and in some embodiments, it is 15 to 35 °C. The molar ratio of the compound of formula (321) to the nucleoside monomer is 1:1 to 1:50, and in some embodiments, it is 1:2 to 1:5. The molar ratio of the compound of formula (321) to the coupling reagent may be 1:1 to 1:50, and in some embodiments, it is 1:3 to 1:10. The reaction time is 200 to 3000 seconds, and in some embodiments, it is 500 to 1500 seconds. The coupling reagent is one or more selected from 1H-tetrazole, 5-ethylthio-1H-tetrazole, and 5-benzylthio-1H-tetrazole, and in some embodiments, it is 5-ethylthio-1H-tetrazole. The coupling reaction may be carried out in an organic solvent. The organic solvent is one or more selected from anhydrous acetonitrile, anhydrous DMF, and anhydrous dichloromethane, and in some embodiments, it is anhydrous acetonitrile. The dosage of the organic solvent is 3 to 50 L / mol with respect to the compound of formula (321), and in some embodiments, it is 5 to 20 L / mol. In some embodiments, the same conditions and reagents as those in the coupling reaction in the solid-phase synthesis method employed may be used.

[0253] In some embodiments, as the coupling reaction conditions, the reaction temperature is 0 to 50 °C, and in some embodiments, it is 15 to 35 °C. The molar ratio of the compound of formula (321) to the nucleoside monomer is 1:1 to 1:50, and in some embodiments, it is 1:2 to 1:5. The molar ratio of the compound of formula (321) to the coupling reagent may be 1:1 to 1:50, and in some embodiments, it is 1:3 to 1:10. The reaction time is 200 to 3000 seconds, and in some embodiments, it is 500 to 1500 seconds. The coupling reagent is one or more selected from 1H-tetrazole, 5-ethylthio-1H-tetrazole, and 5-benzylthio-1H-tetrazole, and in some embodiments, it is 5-ethylthio-1H-tetrazole. The coupling reaction may be carried out in an organic solvent. The organic solvent is one or more selected from anhydrous acetonitrile, anhydrous DMF, and anhydrous dichloromethane, and in some embodiments, it is anhydrous acetonitrile. The dosage of the organic solvent is 3 to 50 L / mol with respect to the compound of formula (321), and in some embodiments, it is 5 to 20 L / mol. In some embodiments, it is 5-ethylthio-1H-tetrazole. The coupling reaction may be carried out in an organic solvent. The organic solvent is one or more selected from anhydrous acetonitrile, anhydrous DMF, and anhydrous dichloromethane, and in some embodiments, it is anhydrous acetonitrile. The dosage of the organic solvent is 3 to 50 L / mol with respect to the compound of formula (321), and in some embodiments, it is 5 to 20 L / mol. In some embodiments, it is 5 to 20 L / mol.

[0254] In step (2), starting from the nucleoside monomer bound to the solid support by the phosphoramidite nucleic acid solid-phase synthesis method, the sense strand S of the siRNA complex is synthesized in the 3'-5' direction. In this case, the complex molecule is bound to the 3'-end of the obtained sense strand.

[0255] As other conditions for the solid-phase synthesis in steps (2) and (3), the deprotection conditions of the nucleoside monomer, the type and dosage of the deprotection reagent, the coupling reaction conditions, the type and dosage of the coupling reagent, the capping reaction conditions, the type and dosage of the capping reagent, the oxidation reaction conditions, the type and dosage of the oxidation reagent, the sulfurization reaction conditions, the sulfurization reagent and dosage are included, and various reagents, dosages and conditions commonly used in this field are adopted.

[0256] For example, in some embodiments, in steps (2) and (3), the following conditions may be used for the solid-phase synthesis.

[0257] As the deprotection conditions of the nucleoside monomer, the temperature is 0 to 50 ° C, and in some embodiments, it is 15 to 35 ° C, the reaction time is 30 to 300 seconds, and in some embodiments, it is 50 to 150 seconds. The deprotection reagent may be one or more selected from trifluoroacetic acid, trichloroacetic acid, dichloroacetic acid, and chloroacetic acid, and in some embodiments, it is dichloroacetic acid. The molar ratio of the deprotection reagent to the 4,4'-dimethoxytrityl protecting group in the solid support is 2:1 to 100:1, and in some embodiments, it is 3:1 to 50:1.

[0258] ​​​​​​​​​​​​​​The coupling reaction conditions are such that the temperature is 0 to 50 °C, and in some embodiments, it is 15 to 35 °C. The molar ratio of the nucleic acid sequence bound to the solid support to the nucleoside monomer is 1:1 to 1:50, and in some embodiments, it is 1:5 to 1:15. The molar ratio of the nucleic acid sequence bound to the solid support to the coupling reagent is 1:1 to 1:100, and in some embodiments, it is 1:50 to 1:80. The selection of the reaction time and the coupling reagent is the same as described above. The molar ratio of the nucleic acid sequence bound to the solid support to the nucleoside monomer is 1:1 to 1:50, and in some embodiments, it is 1:5 to 1:15. The molar ratio of the nucleic acid sequence bound to the solid support to the nucleoside monomer is 1:1 to 1:50, and in some embodiments, it is 1:5 to 1:15. The molar ratio of the nucleic acid sequence bound to the solid support to the coupling reagent is 1:1 to 1:100, and in some embodiments, it is 1:50 to 1:80. The molar ratio of the nucleic acid sequence bound to the solid support to the coupling reagent is 1:1 to 1:100, and in some embodiments, it is 1:50 to 1:80. The selection of the reaction time and the coupling reagent is the same as described above. The selection of the reaction time and the coupling reagent is the same as described above.

[0259] The capping reaction conditions are such that the temperature is 0 to 50 °C, and in some embodiments, it is 15 to 35 °C. The reaction time is 5 to 500 seconds, and in some embodiments, it is 10 to 100 seconds. The selection of the capping reagent is the same as described above. The molar ratio of the total amount of the capping reagent to the nucleic acid sequence bound to the solid support is 1:100 to 100:1, and in some embodiments, it is 1:10 to 10:1. When using equimolar amounts of acetic anhydride and N-methylimidazole as the capping reagent, the molar ratio of acetic anhydride, N-methylimidazole, and the nucleic acid sequence bound to the solid support is 1:1:10 to 10:10:1, and in some embodiments, it is 1:1:2 to 2:2:1. The capping reaction conditions are such that the temperature is 0 to 50 °C, and in some embodiments, it is 15 to 35 °C. The reaction time is 5 to 500 seconds, and in some embodiments, it is 10 to 100 seconds. The selection of the capping reagent is the same as described above. The molar ratio of the total amount of the capping reagent to the nucleic acid sequence bound to the solid support is 1:100 to 100:1, and in some embodiments, it is 1:10 to 10:1. When using equimolar amounts of acetic anhydride and N-methylimidazole as the capping reagent, the molar ratio of acetic anhydride, N-methylimidazole, and the nucleic acid sequence bound to the solid support is 1:1:10 to 10:10:1, and in some embodiments, it is 1:1:2 to 2:2:1. The molar ratio of the total amount of the capping reagent to the nucleic acid sequence bound to the solid support is 1:100 to 100:1, and in some embodiments, it is 1:10 to 10:1. When using equimolar amounts of acetic anhydride and N-methylimidazole as the capping reagent, the molar ratio of acetic anhydride, N-methylimidazole, and the nucleic acid sequence bound to the solid support is 1:1:10 to 10:10:1, and in some embodiments, it is 1:1:2 to 2:2:1. The molar ratio of the total amount of the capping reagent to the nucleic acid sequence bound to the solid support is 1:100 to 100:1, and in some embodiments, it is 1:10 to 10:1. When using equimolar amounts of acetic anhydride and N-methylimidazole as the capping reagent, the molar ratio of acetic anhydride, N-methylimidazole, and the nucleic acid sequence bound to the solid support is 1:1:10 to 10:10:1, and in some embodiments, it is 1:1:2 to 2:2:1. When using equimolar amounts of acetic anhydride and N-methylimidazole as the capping reagent, the molar ratio of acetic anhydride, N-methylimidazole, and the nucleic acid sequence bound to the solid support is 1:1:10 to 10:10:1, and in some embodiments, it is 1:1:2 to 2:2:1. When using equimolar amounts of acetic anhydride and N-methylimidazole as the capping reagent, the molar ratio of acetic anhydride, N-methylimidazole, and the nucleic acid sequence bound to the solid support is 1:1:10 to 10:10:1, and in some embodiments, it is 1:1:2 to 2:2:1. When using equimolar amounts of acetic anhydride and N-methylimidazole as the capping reagent, the molar ratio of acetic anhydride, N-methylimidazole, and the nucleic acid sequence bound to the solid support is 1:1:10 to 10:10:1, and in some embodiments, it is 1:1:2 to 2:2:1.

[0260] The oxidation reaction conditions are such that the temperature is 0 to 50 °C, and in some embodiments, it is 15 to 35 °C. The reaction time is 1 to 100 seconds, and in some embodiments, it is 5 to 50 seconds. The oxidation reagent is iodine in some embodiments (provided as iodine water in some embodiments). The oxidation reagent and the solid phase in the coupling step The oxidation reaction conditions are such that the temperature is 0 to 50 °C, and in some embodiments, it is 15 to 35 °C. The reaction time is 1 to 100 seconds, and in some embodiments, it is 5 to 50 seconds. The oxidation reagent is iodine in some embodiments (provided as iodine water in some embodiments). The oxidation reagent and the solid phase in the coupling step The oxidation reagent is iodine in some embodiments (provided as iodine water in some embodiments). The oxidation reagent and the solid phase in the coupling step The molar ratio with the nucleic acid sequence bound to the carrier may be 1:1 to 100:1, and in some embodiments, it is 5:1 to 50:1. In some embodiments, the oxidation reaction is carried out in a mixed solvent of tetrahydrofuran: water: pyridine = 3:1:1 to 1:1:3 . As the sulfurization reaction conditions, the temperature is 0 to 50 °C, and in some embodiments, it is 15 to 35 °C, the reaction time is 50 to 2000 seconds, and in some embodiments , it is 100 to 1000 seconds. The sulfurization reagent is, in some embodiments, xanthane hydride. The molar ratio of the sulfurization reagent to the nucleic acid sequence bound to the solid-phase carrier in the coupling step is 10:1 to 1000:1, and in some embodiments, it is 10:1 to 5 00:1. In some embodiments, the sulfurization reaction is carried out in a mixed solvent of acetonitrile: pyr idine = 1:3 to 3:1. After binding all the nucleoside monomers and before annealing, the method further includes isolating the sense strand and the antisense strand of the siRNA. The isolation method is known to those skilled in the art

[0261] and generally includes cleaving the synthesized nucleotide sequence from the solid-phase carrier, removing the protecting groups on the bases, phosphate groups and ligands, and purifying and desalting.

[0262] To cleave the synthesized nucleotide sequence from the solid-phase carrier and remove the protecting groups on the bases, phosphate groups and ligands, it may be carried out by the usual cleavage and deprotection methods in siRNA synthesis . For example, the nucleotide sequence bound to the obtained solid-phase carrier is contacted with concentrated ammonia water , and in the deprotection process, the protecting group YCOO- of the A46 - A54 group is converted to a hydroxy group . Convert it to S1 group and convert the S1 group to the corresponding M1 group to generate the complex shown in formula (1). . Here, the concentrated aqueous ammonia may be 25-30 wt% aqueous ammonia, and the concentration The dosage of the concentrated aqueous ammonia may be 0.2 ml / μmol to 0. 8 ml / μmol with respect to the target siRNA sequence.

[0263] When there is at least some 2'-TBDMS protection in the synthesized nucleotide sequence , the method further includes removing the 2'-TBDMS protection by contacting the nucleotide sequence from which the solid support has been removed with triethylamine hydrofluoride acid salt. In this case, the obtained target siRNA sequence has a nucleoside corresponding to the free 2'-hydroxy group . The dosage of pure triethylamine hydrofluoride acid salt is 0.4 ml / μmol to 1.0 ml / μmol with respect to the target siRNA sequence. In this way, the siRNA complex of formula (1) can be obtained.

[0264] Methods for purification and desalting are well known to those skilled in the art. For example, using a preparative ion chromatography purification column, nucleic acid purification is completed by gradient elution of NaBr or NaCl, and the products are recovered and combined, and then desalted by a reverse phase chromatography purification column .

[0265] In the siRNA complex thus obtained, the non-bridging oxygen atom or sulfur atom in the phosphodiester bond or phosphorothioate diester bond between nucleotides is basically bound to a sodium lithium ion, and the siRNA complex basically exists as a sodium salt . By well-known ion exchange methods, hydrogen ions and / or other cations are used to replace the sodium ion.​​ Mu ions can be replaced to obtain other forms of siRNA complexes. The cation is as described above.

[0266] During the synthesis process, the purity and molecular weight of the nucleic acid sequence are always detected to better control the synthesis quality which can be achieved. Such detection methods are known to those skilled in the art. For example, nucleic acid purity can be detected by ion exchange chromatography and the molecular weight can be measured by liquid chromatography tandem mass spectrometry.

[0267] The annealing method is also well known to those skilled in the art. For example, the simply synthesized sense strand (S strand) and the antisense strand (AS strand) are mixed in an equimolar ratio in water for injection and heated to 70 - 95 °C and then cooled to room temperature to form a double-stranded structure by hydrogen bonding. In this way a second type of siRNA complex can be obtained.

[0268] After obtaining the complex, in some embodiments, for example, methods such as liquid chromatography tandem mass spectrometry are used to clarify the characteristics of the second type of siRNA complex synthesized by molecular weight detection, etc., and it can be confirmed that the synthesized siRNA complex is the second type of siR NA complex of the target design, and the sequence of the synthesized siRNA is consistent with the sequence of the siRN A to be synthesized and is, for example, one of the sequences shown in Table 1.

[0269] The compound shown in formula (321) is, in an organic solvent, under esterification reaction conditions and in the presence of a base and an esterification catalyst, the compound shown in formula (313) is contacted with a cyclic acid anhydride for ion exchange and isolation to obtain the compound shown in formula (321) in a preparation method It can be obtained by

[0270]

Chemical formula

[0271]

Chemical formula

[0272] The esterification reaction conditions are such that the reaction temperature is 0 to 100 °C and the reaction time is 8 to 4 8 hours. In some embodiments, the esterification reaction conditions are such that the reaction temperature is 10 to 40 °C and the reaction time is 20 to 30 hours.

[0273] In some embodiments, the organic solvent is an epoxy solvent, an ether solvent, a halogen An alkyl halide solvent, dimethyl sulfoxide, N,N-dimethylformamide, and N ,N-diisopropylethylamine, or one or more of these. In some embodiments , the epoxy solvent is dioxane and / or tetrahydrofuran, and the ether solvent is ethyl ether and / or methyl tert-butyl ether, and the alkyl halide solvent is dichloromethane, trichloromethane, and 1,2-dichloro ethane, or one or more of these. In some embodiments, the organic solvent is dichloro methane. For the compound represented by the formula (313), the dosage of the organic solvent is 3 to 50 L / mol, and in some embodiments, it is 5 to 20 L / mol.

[0274] In some embodiments, the cyclic acid anhydride is one of succinic anhydride, glutaric anhydride, adipic anhydride, or pimelic anhydride, and in some embodiments , it is succinic anhydride. The molar ratio of the cyclic acid anhydride to the compound represented by the formula (313) is 1:1 to 10:1, and in some embodiments, it is 2:1 to 5:1.

[0275] The esterification catalyst may be any catalyst that catalyzes the esterification reaction. For example , the catalyst may be 4-dimethylaminopyridine. The molar ratio of the catalyst to the compound represented by the formula (313 ) is 1:1 to 10:1, and in some embodiments , it is 2:1 to 5:1.

[0276] In some embodiments, the base may be any inorganic base, organic base, or a combination thereof. Considering solubility and the stability of the product, the base is, for example, a group. It may be a tertiary amine organic base. In some embodiments, the tertiary amine organic base is triethylamine or N,N-diisopropylethylamine. The molar ratio of the tertiary amine organic base to the compound represented by formula (313) is 1:1 to 20:1 and, in some embodiments, 3:1 to 10:1.

[0277] The ion exchange action is to convert the compound of formula (321) into the desired carboxylic acid or carboxylate form. The ion exchange method is known to those skilled in the art. By using an appropriate ion exchange solution and exchange conditions, a composite molecule in which the aforementioned cation is M + can be obtained, and detailed description thereof is omitted here. In some embodiments, the ion exchange reaction is carried out using a triethylamine phosphate solution, and the concentration of the triethylamine phosphate solution is 0 .2 to 0.8 M, and in some embodiments, 0.4 to 0.6 M. For the compound of formula (3 13), the dosage of the triethylamine phosphate solution is 3 to 6 L / mol and, in some embodiments, 4 to 5 L / mol.

[0278] The compound of formula (321) can be isolated from the reaction mixture by any suitable isolation method. In some embodiments, after evaporating and removing the solvent, the compound of formula (321) can be isolated by chromatography method. For example, (1) normal phase purification silica gel: silica gel packing agent of 200 - 300 mesh is gradient eluted with dichloromethane:methanol = 100:18 to 100:20 containing 1 wt‰ triethylamine, or (2) reverse phase purification: C , C8 reverse phase packing agent, with methanol:acetonitrile = 0.1:1 to 1:0. 18 ​ It can be isolated under chromatographic conditions of gradient elution with 1. In some examples the crude product of the compound of formula (321) can be obtained by directly removing the solvent, and the crude product can be used directly in subsequent reactions.

[0279] In some embodiments, the method for preparing the compound of formula (321) further comprises contacting, under condensation reaction conditions, in an organic solvent, in the presence of a condensing agent and a tertiary amine organic base, the product obtained by the above ion exchange reaction with a solid phase support containing an amino group or a hydroxy group. In this case, R4 contains a first functional group and a second functional group, the first functional group contains a hydroxy protecting group, and the second functional group contains a structure represented by formula (C1’), and a compound of formula (321) is obtained.

[0280] The solid phase support is one of the supports used in the solid phase synthesis of siRNA, and some of them are known to those skilled in the art. For example, the solid phase support may be selected from solid phase supports containing an active hydroxy group or an amino functional group. In some embodiments, the solid phase support is an amino resin or a hydroxy resin. In order to facilitate subsequent nucleic acid solid phase synthesis, in some embodiments, the amino resin or hydroxy resin has parameters of a particle size of 100 to 400 mesh and an amino group or hydroxy group loading amount on the surface of 0.2 to 0.5 mmol / g. The dosage ratio of the compound represented by formula (321) to the solid phase support is 10 to 400 μmol compound / 1 gram of solid phase support (μmol / g). In some embodiments, the dosage ratio of the compound represented by formula (321) to the solid phase support is 50 to 200 μmol / g. ​

[0281] The organic solvent may be any suitable solvent or mixed solvent known to those skilled in the art. In some embodiments, the organic solvent is one or more of acetonitrile, epoxy solvents, ether solvents, alkyl halide solvents, dimethyl sulfoxide, N,N-dimethylformamide and N,N-diisopropylethylamine. In some embodiments, the epoxy solvent is dioxane and / or tetrahydrofuran, the ether solvent is ethyl ether and / or methyl tert-butyl ether, and the alkyl halide solvent is one or more of dichloromethane, trichloromethane and 1,2- dichloroethane. In some embodiments, the organic solvent is acetonitrile. For the compound of formula (321), the dosage of the organic solvent is 20~200 L / mol, and in some embodiments, it is 50~100 L / mol.

[0282] The condensing agent may be (benzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate, 3-diethoxyphosphoryl-1,2,3-benzoxazol-4(3H)-one and / or O-benzotriazole-tetramethyluronium hexafluorophosphate. In some embodiments, it is O-benzotriazole-tetramethyluronium hexafluorophosphate. The molar ratio of the condensing agent to the compound shown in formula (321) is 1:1~20:1, and in some embodiments, it is 1:1~5:1.

[0283] ​​​​In some embodiments, the tertiary amine organic base is triethylamine and / or N,N - diisopropylethylamine. In some embodiments, it is N, N - diisopropylethylamine. The molar ratio of the tertiary amine organic base to the compound represented by formula (321) is 1:1 to 20:1. In some embodiments, it is 1 :1 to 5:1. :1 to 5:1.

[0284] In some embodiments, the preparation method of the compound of formula (321) may further include contacting the obtained condensation product with a capping reagent and an acylation catalyst in an organic solvent under capping reaction conditions and isolating to obtain the compound represented by formula (321). The function of the capping reaction is to remove any active reaction functional groups that have not yet fully reacted in order to avoid the formation of unnecessary by - products in subsequent reactions. The conditions of the above - mentioned capping reaction are that the reaction temperature is 0 to 50 °C. In some embodiments it is 15 to 35 °C, the reaction time is 1 to 10 h, and in some embodiments it is 3 to 6 h. As the capping reagent, a capping reagent known to those skilled in the art and used in the solid - phase synthesis of siRNA may be used. The above - mentioned capping reaction conditions are that the reaction temperature is 0 to 50 °C. In some embodiments it is 15 to 35 °C, the reaction time is 1 to 10 h, and in some embodiments it is 3 to 6 h. As the capping reagent, a capping reagent known to those skilled in the art and used in the solid - phase synthesis of siRNA may be used. it is 3 to 6 h. As the capping reagent, a capping reagent known to those skilled in the art and used in the solid - phase synthesis of siRNA may be used. it is 3 to 6 h. As the capping reagent, a capping reagent known to those skilled in the art and used in the solid - phase synthesis of siRNA may be used.

[0285] In some embodiments, the capping reagent consists of capping reagent A (capA) and capping reagent B (capB). Capping reagent A is N-methylimidazole. In some embodiments, it is provided as a pyridine / acetonitrile mixed solution of N-methylimidazole, and the volume ratio of pyridine to acetonitrile is 1:10 to 1:1. In some embodiments, it is 1:3 to 1:1. The ratio of the total volume of pyridine and acetonitrile to the volume of N-methylimidazole is 1:1 to 10:1. In some embodiments, it is 3:1 to 7:1. The capping reagent B is acetic anhydride. In some embodiments, it is provided as an acetonitrile solution of acetic anhydride, and the volume ratio of acetic anhydride to acetonitrile is 1:1 to 1:10. In some embodiments, it is 1:2 to 1:6.

[0286] In some embodiments, the ratio of the volume of the pyridine / acetonitrile mixed solution of N-methylimidazole to the mass of the compound of formula (321) is 5 ml / g to 50 ml / g. In some embodiments, it is 15 ml / g to 30 ml / g. The ratio of the volume of the acetonitrile solution of acetic anhydride to the mass of the compound of formula (321) is 0.5 ml / g to 10 ml / g. In some embodiments, it is 1 ml / g to 5 ml / g. In some embodiments, equimolar amounts of acetic anhydride and N-methylimidazole are used as the capping reagent. The organic solvent is one or more of acetonitrile, epoxy solvents, ether solvents, alkyl halide solvents, dimethyl sulfoxide, N,N-dimethylformamide, and N,N-diisopropylethylamine. In some embodiments, the organic solvent is acetonitrile.

[0287] In some embodiments, equimolar amounts of acetic anhydride and N-methylimidazole are used as the capping reagent. The organic solvent is one or more of acetonitrile, epoxy solvents, ether solvents, alkyl halide solvents, dimethyl sulfoxide, N,N-dimethylformamide, and N,N-diisopropylethylamine. In some embodiments, the organic solvent is acetonitrile. For the compound of formula (321), In some embodiments, equimolar amounts of acetic anhydride and N-methylimidazole are used as the capping reagent. The organic solvent is one or more of acetonitrile, epoxy solvents, ether solvents, alkyl halide solvents, dimethyl sulfoxide, N,N-dimethylformamide, and N,N-diisopropylethylamine. In some embodiments, the organic solvent is acetonitrile. In contrast, the dosage of the organic solvent is 10 to 50 L / mol, and in some embodiments, it is 5 to 30 L / mol.

[0288] The acylation catalyst may be any catalyst that can be used in esterification condensation or amidation condensation, for example, it may be selected from alkali heterocyclic compounds. In some embodiments, the acyl ation catalyst is 4-dimethylaminopyridine. The mass ratio of the catalyst to the compound represented by formula (321) is 0.001:1 to 1:1, and in some embodiments, it is 0.0 1:1 to 0.1:1.

[0289] The compound of formula (321) can be isolated from the reaction mixture by any suitable isolation method. In some embodiments, by thoroughly washing with an organic solvent, filtering, and removing unreacted reactants, excess capping reagent and other impurities, the compound of formula (321) can be obtained. The organic solvent is selected from acetonitrile, dichloromethane, methanol, and in some embodiments, it is acetonitrile.

[0290] In some embodiments, the method for preparing the composite molecule represented by formula (321) is to contact the compound represented by formula (313) with phosphorodiamidite in an organic solvent under coupling reaction conditions and in the presence of a coupling reagent, and isolate to obtain the compound represented by formula (321). In this case, R4 contains a first functional group and a second functional group, the first functional group contains a hydroxy protecting group, and the second functional group contains a structure represented by formula (C3), and a compound of formula ( 321) can be obtained.

[0291] In some embodiments, the coupling reaction conditions are such that the temperature is 0 to 50 °C, for example, 15 to 35 °C, and the molar ratio of the compound of formula (313) to the phosphoramidite may be 1:1 to 1:50, for example, 1:5 to 1:15, and the molar ratio of the compound of formula (313) to the coupling reagent may be 1:1 to 1:100, for example, 1:50 to 1:80, and the reaction time may be 200 to 3000 seconds, for example, 500 to 1500 seconds. The phosphoramidite may be, for example, bis(diisopropylamino) (2-cyanoethoxy)phosphine, or a commercially available product may be purchased, or it may be synthesized by a method known in the art. (2-cyanoethoxy)phosphine may be used, or a commercially available product may be purchased, or it may be synthesized by a method known in the art. The coupling reagent is one or more selected from 1H-tetrazole, 5-ethylthio-1H-tetrazole, 5-benzylthio-1H-tetrazole, for example, 5-ethylthio-1H-tetrazole. The coupling reaction may be carried out in an organic solvent, and the organic solvent is one or more selected from anhydrous acetonitrile, anhydrous DMF, anhydrous dichloromethane, for example, anhydrous acetonitrile. In some embodiments, the dosage of the organic solvent with respect to the compound of formula (313) is 3 to 50 L / mol, for example, 5 to 20 L / mol may also be acceptable. By carrying out the coupling reaction, the hydroxy group in the compound of formula (313) reacts with the phosphoramidite to form a phosphoramidite group. In some embodiments, the solvent can be directly removed to obtain a crude product of the compound of formula (321), and the crude product can be directly used in the subsequent reaction. In some embodiments, the method for preparing the compound of formula (321) is a coupling reaction where the dosage of the organic solvent is 3 to 50 L / mol with respect to the compound of formula (313), for example, 5 to 20 L / mol may also be acceptable. By carrying out the coupling reaction, the hydroxy group in the compound of formula (313) reacts with the phosphoramidite to form a phosphoramidite group. In some embodiments, the hydroxy group in the compound of formula (313) reacts with the phosphoramidite to form a phosphoramidite group. By carrying out the coupling reaction, the hydroxy group in the compound of formula (313) reacts with the phosphoramidite to form a phosphoramidite group. In some embodiments, the solvent can be directly removed to obtain a crude product of the compound of formula (321), and the crude product can be directly used in the subsequent reaction. can be directly used in the subsequent reaction.

[0292] In some embodiments, the method for preparing the compound of formula (321) is a coupling reaction The product obtained by isolating the product obtained by reacting the product with the product obtained by reacting ... and then contacting the solid phase carrier with a hydroxy group-containing solid phase carrier. The compound of formula (321) is obtained by carrying out a cycloaddition reaction, an oxidation reaction, and isolating the compound. and a second functional group, the first functional group comprising a hydroxy protecting group and the second functional group comprising a A compound of formula (321) in which the functional group has the structure shown in formula (C3') is obtained.

[0293] In some embodiments, the solid support is a suitable substrate for solid phase synthesis of nucleic acids as known in the art. For example, a commercially available general-purpose solid phase support (NittoPhase) that has been deprotected is ase(R)HL UnyLinker TM 300 Oligonucleotides ide Synthesis Support, Kinovate Life Science Nces Corporation, the structure of which is shown in formula B80.

[0294] [ka]

[0295] Deprotection reactions are known to those skilled in the art. In some embodiments, deprotection conditions include The temperature is 0 to 50°C, for example 15 to 35°C, and the reaction time is 30 to 300 seconds. For example, 50 to 150 seconds. The deprotection reagent is trifluoroacetic acid, trichloroacetic acid, dichloroacetic acid, In some embodiments, the acetic acid may be one or more selected from the group consisting of acetic acid, acetic acid, and chloroacetic acid. In this embodiment, the deprotection reagent is dichloroacetic acid. The molar ratio of the r(4,4'-dimethoxytrityl) protecting group is 2:1 to 100:1, e.g. For example, it is 3:1 to 50:1. By performing the deprotection, a free hydroxy group having reactive activity is obtained on the surface of the solid support, facilitating the subsequent coupling reaction. By performing the deprotection, a free hydroxy group having reactive activity is obtained on the surface of the solid support, facilitating the subsequent coupling reaction.

[0296] The coupling reaction conditions and the selection of the coupling reagent are as described above. By performing the coupling reaction, a free hydroxy group formed in the deprotection reaction is reacted with a phosphoramidite group to form a phosphite ester bond. By performing the coupling reaction, a free hydroxy group formed in the deprotection reaction is reacted with a phosphoramidite group to form a phosphite ester bond. By performing the coupling reaction, a free hydroxy group formed in the deprotection reaction is reacted with a phosphoramidite group to form a phosphite ester bond.

[0297] In some embodiments, the capping reaction conditions are such that the temperature is 0 to 50 °C, for example 15 to 35 °C, the reaction time is 5 to 500 seconds, for example 10 to 100 seconds, and the capping reaction is carried out in the presence of a capping reagent. The selection and dosage of the capping reagent are as described above. In some embodiments, the capping reaction conditions are such that the temperature is 0 to 50 °C, for example 15 to 35 °C, the reaction time is 5 to 500 seconds, for example 10 to 100 seconds, and the capping reaction is carried out in the presence of a capping reagent. The selection and dosage of the capping reagent are as described above. In some embodiments, the capping reaction conditions are such that the temperature is 0 to 50 °C, for example 15 to 35 °C, the reaction time is 5 to 500 seconds, for example 10 to 100 seconds, and the capping reaction is carried out in the presence of a capping reagent. The selection and dosage of the capping reagent are as described above. In some embodiments, the capping reaction conditions are such that the temperature is 0 to 50 °C, for example 15 to 35 °C, the reaction time is 5 to 500 seconds, for example 10 to 100 seconds, and the capping reaction is carried out in the presence of a capping reagent. The selection and dosage of the capping reagent are as described above.

[0298] The oxidation reaction conditions are such that the temperature is 0 to 50 °C, for example, it may be 15 to 35 °C, the reaction time is 1 to 100 seconds, for example, it may be 5 to 50 seconds, and the oxidation reagent may be, for example, iodine (in some embodiments, provided as iodine water). In some embodiments, the molar ratio of the oxidation reagent to the phosphite ester group is 1:1 to 100:1, for example, it may be 5:1 to 50:1. In some embodiments, the oxidation reaction is carried out in a mixed solvent of tetrahydrofuran:water:pyridine = 3:1:1 to 1:1:3. The oxidation reaction conditions are such that the temperature is 0 to 50 °C, for example, it may be 15 to 35 °C, the reaction time is 1 to 100 seconds, for example, it may be 5 to 50 seconds, and the oxidation reagent may be, for example, iodine (in some embodiments, provided as iodine water). In some embodiments, the molar ratio of the oxidation reagent to the phosphite ester group is 1:1 to 100:1, for example, it may be 5:1 to 50:1. In some embodiments, the oxidation reaction is carried out in a mixed solvent of tetrahydrofuran:water:pyridine = 3:1:1 to 1:1:3. The oxidation reaction conditions are such that the temperature is 0 to 50 °C, for example, it may be 15 to 35 °C, the reaction time is 1 to 100 seconds, for example, it may be 5 to 50 seconds, and the oxidation reagent may be, for example, iodine (in some embodiments, provided as iodine water). In some embodiments, the molar ratio of the oxidation reagent to the phosphite ester group is 1:1 to 100:1, for example, it may be 5:1 to 50:1. In some embodiments, the oxidation reaction is carried out in a mixed solvent of tetrahydrofuran:water:pyridine = 3:1:1 to 1:1:3. The oxidation reaction conditions are such that the temperature is 0 to 50 °C, for example, it may be 15 to 35 °C, the reaction time is 1 to 100 seconds, for example, it may be 5 to 50 seconds, and the oxidation reagent may be, for example, iodine (in some embodiments, provided as iodine water). In some embodiments, the molar ratio of the oxidation reagent to the phosphite ester group is 1:1 to 100:1, for example, it may be 5:1 to 50:1. In some embodiments, the oxidation reaction is carried out in a mixed solvent of tetrahydrofuran:water:pyridine = 3:1:1 to 1:1:3. The oxidation reaction conditions are such that the temperature is 0 to 50 °C, for example, it may be 15 to 35 °C, the reaction time is 1 to 100 seconds, for example, it may be 5 to 50 seconds, and the oxidation reagent may be, for example, iodine (in some embodiments, provided as iodine water). In some embodiments, the molar ratio of the oxidation reagent to the phosphite ester group is 1:1 to 100:1, for example, it may be 5:1 to 50:1. In some embodiments, the oxidation reaction is carried out in a mixed solvent of tetrahydrofuran:water:pyridine = 3:1:1 to 1:1:3. The oxidation reaction conditions are such that the temperature is 0 to 50 °C, for example, it may be 15 to 35 °C, the reaction time is 1 to 100 seconds, for example, it may be 5 to 50 seconds, and the oxidation reagent may be, for example, iodine (in some embodiments, provided as iodine water). In some embodiments, the molar ratio of the oxidation reagent to the phosphite ester group is 1:1 to 100:1, for example, it may be 5:1 to 50:1. In some embodiments, the oxidation reaction is carried out in a mixed solvent of tetrahydrofuran:water:pyridine = 3:1:1 to 1:1:3. The oxidation reaction conditions are such that the temperature is 0 to 50 °C, for example, it may be 15 to 35 °C, the reaction time is 1 to 100 seconds, for example, it may be 5 to 50 seconds, and the oxidation reagent may be, for example, iodine (in some embodiments, provided as iodine water). In some embodiments, the molar ratio of the oxidation reagent to the phosphite ester group is 1:1 to 100:1, for example, it may be 5:1 to 50:1. In some embodiments, the oxidation reaction is carried out in a mixed solvent of tetrahydrofuran:water:pyridine = 3:1:1 to 1:1:3.

[0299] In some embodiments, R6 is one of the groups of formula B7 or B8.

[0300]

Chemical formula

[0301] In this case, the compound represented by formula (313) is, in an organic solvent, under amidation reaction conditions and in the presence of an amidation reaction condensing agent and a tertiary amine organic base, contacted with the compound represented by formula (314) and the compound represented by formula (A-1) or the compound of formula (A-2), and then isolated, and can be obtained by the preparation method described above.

[0302]

Chemical formula

[0303] The amidation reaction conditions are such that the reaction temperature is 0 to 100 °C and the reaction time may be 1 to 48 hours. In some embodiments, the amidation reaction conditions are such that the reaction temperature is 10 to 40 °C and the reaction time is 2 to 16 hours.

[0304] In some embodiments, the organic solvent is one or more of an alcohol solvent, an epoxy solvent, an ether solvent, a halogenated alkyl solvent, dimethyl sulfoxide, N,N-dimethyl formamide, and N,N-diisopropylethylamine. The alcohol solvent is, in some embodiments, one or more of methanol, ethanol, and propanol and, in some embodiments, ethanol. The epoxy In some embodiments, the xylyl solvents are dioxane and / or tetrahydrofuran The ether solvents are, in some embodiments, ethyl ether and / or methyl tert-butyl ether. The alkyl halide solvents are, in some embodiments, one or more of dichloromethane, trichloromethane, and 1,2-dichloroethane In some embodiments, the organic solvent is dichloromethane For the compound of formula (314), the dosage of the organic solvent is 3 to 50 L / mol, and in some embodiments, it is 3 to 20 L / mol

[0305] In some embodiments, the amidation reaction condensing agent is (benzotriazol-1 -yloxy)tripyrrolidinophosphonium hexafluorophosphate, 3-diethoxy phosphoryl-1,2,3-benzoxazol-4(3H)-one, 4-(4,6-dimethoxy triazin-2-yl)-4-methylmorpholine hydrochloride (4-(4,6-dimethoxy triazin-2-yl)-4-methylmorpholine hydrochloride), 2-ethoxy-1-ethoxycarbonyl-1,2-dihydro quinoline (EEDQ), or O-benzotriazol-tetramethyluronium hexafluorophosphate, and in a further embodiment, it is 3-diethoxyphosphoryl-1,2 ,3-benzoxazol-4(3H)-one. The molar ratio of the amidation reaction condensing agent to the compound represented by formula (31 4) may be 1:1 to 10:1, and in some embodiments, it is 2.5:1 to 5:1 ,3-benzoxazol-4(3H)-one. The molar ratio of the amidation reaction condensing agent to the compound represented by formula (31 4) may be 1:1 to 10:1, and in some embodiments, it is 2.5:1 to 5:1 In some embodiments, it is 2.5:1 to 5:1

[0306] In some embodiments, the tertiary amine organic base is triethylamine or N,N-diisopropylethylamine, and in some embodiments, N,N-di isopropylethylamine. The molar ratio of the tertiary amine organic base to the compound represented by formula (314) is 3:1 to 20:1, and in some embodiments, 5:1 to 10:1.

[0307] The compounds of formula (A-1) and formula (A-2) may be prepared by any suitable method . For example, when R k is a DMTr group, calcium glycerate and DMTrCl can be reacted to prepare the compound of formula (A-1). Similarly, after contacting 3-amino-1,2-prop anediol with a cyclic acid anhydride and then reacting with DMTrCl, the compound of formula (A-2) can be prepared, and the cyclic acid anhydride may be a cyclic acid anhydride having 4 to 13 carbon atoms, and in some embodiments, 4 to 8. As can be easily understood by those skilled in the art , the selection of the cyclic acid anhydride corresponds to different values of q2 in the compound of (A~2). For example, when the cyclic acid anhydride is succinic anhydride, q2 = 1, and when the cyclic acid anhydride is glutaric anhydride, q2 = 2, and so on.

[0308] In some variations, the compound represented by formula (314) is reacted with the cyclic acid anhydride, 3- amino-1,2-propanediol and DMTrCl in sequence to prepare the compound of formula ( 313). As can be easily understood by those skilled in the art, these variations do not affect the structure and function of the compound of formula (313), and It is easily realized by the above method by the operator.

[0309] Similarly to the above, the compound of formula (313) can be isolated from the reaction mixture by any suitable isolation method. In some embodiments, after evaporating and removing the solvent, the compound of formula (313) can be isolated by a chromatography method. For example, (1) normal-phase purification: silica gel filler of 200-300 mesh is eluted with a gradient of petroleum ether: ethyl acetate: dichloromethane: N,N-dimethylformamide = 1:1:1:0.5-1:1:1:0.6, (2) reverse-phase purification: C18 or C8 reverse-phase filler is eluted with a gradient of methanol: acetonitrile = 0.1:1-1:0.1 under two chromatography conditions. In some embodiments, the compound of formula (313) can be isolated by a chromatography method. For example, (1) normal-phase purification: silica gel filler of 200-300 mesh is eluted with a gradient of petroleum ether: ethyl acetate: dichloromethane: N,N-dimethylformamide = 1:1:1:0.5-1:1:1:0.6, (2) reverse-phase purification: C18 or C8 reverse-phase filler is eluted with a gradient of methanol: acetonitrile = 0.1:1-1:0.1 under two chromatography conditions. : dichloromethane: N,N-dimethylformamide = 1:1:1:0.5~1:1:1:0.6 gradient elution, (2) reverse-phase purification: C18, C8 reverse-phase filler, methanol: acetonitrile = 0.1:1~1:0.1 gradient elution, and the compound of formula (313) can be isolated under these two chromatography conditions. : dichloromethane: N,N-dimethylformamide = 1:1:1:0.5~1:1:1:0.6 gradient elution, (2) reverse-phase purification: C18, C8 reverse-phase filler, methanol: acetonitrile = 0.1:1~1:0.1 gradient elution, and the compound of formula (313) can be isolated under these two chromatography conditions. : 0.6 gradient elution, (2) reverse-phase purification: C18, C8 reverse-phase filler, methanol: acetonitrile = 0.1:1~1:0.1 gradient elution, and the compound of formula (313) can be isolated under these two chromatography conditions. 18 : C18, C8 reverse-phase filler, methanol: acetonitrile = 0.1:1~1:0.1 gradient elution, and the compound of formula (313) can be isolated under these two chromatography conditions. : acetonitrile = 0.1:1~1:0.1 gradient elution, and the compound of formula (313) can be isolated under these two chromatography conditions. In some embodiments, the solvent can be directly removed to obtain a crude product of the compound of formula (313), and the crude product can be directly used in the subsequent reaction. In some embodiments, the solvent can be directly removed to obtain a crude product of the compound of formula (313), and the crude product can be directly used in the subsequent reaction. In some embodiments, the solvent can be directly removed to obtain a crude product of the compound of formula (313), and the crude product can be directly used in the subsequent reaction.

[0310] In some embodiments, the compound represented by formula (314) can be obtained by a preparation method including contacting the compound represented by formula (315) with haloacetic acid under deprotection reaction conditions in an organic solvent and then isolating it. In some embodiments, the compound represented by formula (314) can be obtained by a preparation method including contacting the compound represented by formula (315) with haloacetic acid under deprotection reaction conditions in an organic solvent and then isolating it. In some embodiments, the compound represented by formula (314) can be obtained by a preparation method including contacting the compound represented by formula (315) with haloacetic acid under deprotection reaction conditions in an organic solvent and then isolating it.

[0311]

Chemical formula

[0312]

Chemical formula

[0313] The haloacetic acid may be selected from the group consisting of dichloroacetic acid, trichloroacetic acid, chloroacetic acid and trifluoroacetic acid. In some embodiments, the dichloroacetate may be one or more selected from the group consisting of dichloroacetate, dichloromethane ... It is an acid.

[0314] The deprotection reaction conditions are a reaction temperature of 0 to 100° C. and a reaction time of 0.1 to 2 4 hours, and in some embodiments, the reaction temperature is 10 to 40° C., and the reaction time is is 0.5 to 16 hours.

[0315] In some embodiments, the organic solvent is an epoxy solvent, an ether solvent, a haloperidol solvent, or a cyclohexane solvent. Alkyl halogenated solvents, dimethyl sulfoxide, N,N-dimethylformamide and N , N-diisopropylethylamine. In some embodiments, the ether is dioxane and / or tetrahydrofuran. The tert-butyl ether solvent may, in some embodiments, be ethyl ether and / or methyl tert-butyl ether. In some embodiments, the halogenated alkyl solvent is butyl ether. One or more of dichloromethane, trichloromethane and 1,2-dichloroethane In some embodiments, the organic solvent is dichloromethane. For the compound of 5), the amount of organic solvent is 3 to 50 L / mol, and in some embodiments In some embodiments, the concentration is 5 to 20 L / mol.

[0316] The molar ratio of the haloacetic acid to the compound represented by the formula (315) is 5:1 to 100:1, and in some embodiments, it is 10:1 to 50:1.

[0317] Similar to the above, the compound of formula (314) can be isolated from the reaction mixture by any suitable isolation method. In some embodiments, after the solvent is removed by evaporation, the compound of formula (314) can be isolated by a chromatographic method. For example, (1) normal phase purification: silica gel filler of 200-300 mesh is eluted with a gradient of dichloromethane:methanol = 100:30 to 100:40, (2) reverse phase purification: The C, C8 reverse phase filler is eluted with a gradient of methanol:acetonitrile = 0.1:1 to 1:0.1, and the compound of formula (314) can be isolated under these two chromatographic conditions. In some embodiments, 18 the solvent can be directly removed to obtain a crude product of the compound of formula (314), and the crude product can be used directly in the subsequent reaction. The compound represented by the formula (315) can be obtained by a preparation method including contacting the compound represented by the formula (317) with the compound represented by the formula (3 16) in an organic solvent in the presence of an amidation reaction condensing agent and a tertiary amine organic base and under condensation reaction conditions, and then isolating.

[0318] In the formula, n1, n3, m1, m2, m3, R7, R , R , R

[0319]

Chemical formula

[0320] As the compound of formula (316), for example, the compounds disclosed in J. Am. Chem. Soc. 20 14, 136, 16958 - 16961 may be used, or the compound of formula (316) can be prepared by those skilled in the art by various methods. For example, referring to the method disclosed in Example 1 of US Patent US8106022B2, some compounds of formula (316 ) can be prepared, and by reference, all the contents of the above - mentioned literature are incorporated herein by reference in their entirety.

[0321] In some embodiments, the condensation reaction conditions are such that the reaction temperature is 0 - 100 °C and the reaction time is 0.1 - 24 hours. In some embodiments, the reaction temperature is 1 0 - 40 °C and the reaction time is 0.5 - 16 hours.

[0322] The molar ratio of the compound represented by formula (316) to the compound represented by formula (317) may be 2:1 - 10:1, and in some embodiments, it is 2.5:1 - 5:1.

[0323] In some embodiments, the organic solvent is one or more of acetonitrile, epoxy solvents, ether solvents, alkyl halide solvents, dimethyl sulfoxide, N,N - dimethylformamide, and N,N - diisopropylethylamine. The epoxy solvents are, in some embodiments, dioxane and / or tetrahydrofuran. The ether solvents are, in some embodiments, ethyl ether and / or tetrahydrofuran, and the alkyl halide solvents are, in some embodiments, dichloromethane and / or chloroform. is methyl tert-butyl ether, and the alkyl halide solvent is several In some embodiments, one or more of dichloromethane, trichloromethane, and 1,2-dichloroethane In some embodiments, the organic solvent is acetonitrile . For the compound of formula (317), the dosage of the organic solvent is 3 to 50 L / mol , and in some embodiments, it is 5 to 20 L / mol.

[0324] The amidation reaction condensing agent is (benzotriazol-1-yloxy)tripyrrolidino phosphonium hexafluorophosphate, 3-diethoxyphosphoryl-1,2,3-benz oxazolone 4(3H)-one (DEPBT), O-benzotriazole-tetram thyluronium hexafluorophosphate or 4-(4,6-dimethoxytriazin- 2-yl)-4-methylmorpholine hydrochloride, and in some embodiments, it is 4-( 4,6-dimethoxytriazin-2-yl)-4-methylmorpholine hydrochloride. The molar ratio of the amidation reaction condensing agent to the compound represented by formula (317) is 2:1 to 10:1 , and in some embodiments, it is 2.5:1 to 5:1.

[0325] The tertiary amine organic base is N-methylmorpholine, triethylamine, or N,N -diisopropylethylamine, and in some embodiments, it is N-methylmorpho line. The molar ratio of the tertiary amine organic base to the compound represented by formula (317) is 3:1 to 20:1, and in some embodiments, it is 5:1 to 10:1.

[0326] Similar to the above, the compound of formula (315) is isolated from the reaction mixture by any suitable isolation method It can be separated. In some embodiments, after the solvent is removed by evaporation, chromatography The compound of formula (315) can be isolated by the flash method. For example, (1) normal-phase purification Silica gel: A silica gel packing agent of 200-300 mesh is gradient eluted with dichloromethane: methanol = 100:5 to 100:7, (2) reverse-phase purification: C 18 , C8 reverse-phase packing Agent is gradient eluted with methanol: acetonitrile = 0.1:1 to 1:0.1, and can be isolated under two chromatography conditions. In some embodiments, the solvent can be directly removed to obtain a crude product of the compound of formula (315), and the crude product can be used as it is in subsequent reactions.

[0327] In some embodiments, the compound of formula (317) and a sufficient amount of the compound of formula (316) are reacted once to produce the desired compound of formula (315). In this case, each S1- L1 part is the same. In some embodiments, if necessary, the compound of formula (317) is batch-reacted with a compound of a different formula (316), that is, a compound of formula (31 6) in which L1 and / or S1 are different, so that the resulting compound of formula (315) can contain two or more types of S1 and / or L1. For example, for 1 eq of the compound of formula (317), it is contacted with 2 eq of the first compound of formula (316), and after the first S1-L1 part is bonded to the two terminal primary amine groups in the compound of formula (317), it is subsequently (n3 + n1 - 1) eq of the second compound of formula (316) is contacted (the definitions and value ranges of n3 and n1 are as described above), and in the compound of formula (317), (n3 + n1 The second S1-L1 moiety can be attached to the (-1) secondary amine group.

[0328] In some embodiments, the compound represented by formula (317) is contacted with an aqueous solution of methylamine with the compound represented by formula (318) in the presence of an organic solvent and under deprotection reaction conditions, and then can be obtained by a preparation method including isolation.

[0329] [Chemical formula] wherein n1, n3, m1, m2, m3, R7, R 10 , R 11 , R 12 , R 13 , R1 4, R 15 The respective definitions and selectable ranges are as described above.

[0330] The deprotection reaction conditions are such that the reaction temperature is 0 to 150 °C and the reaction time is 5 to 72 hours. In some embodiments, the reaction temperature is 20 to 80 °C and the reaction time is 1 0 to 30 hours.

[0331] The organic solvent is selected from alcohols and, in some embodiments, is one of methanol, ethanol, and isopropanol. In some embodiments, it is methanol, and the dosage of the organic solvent is 1 to 20 L / mol relative to the compound of formula (318). In some embodiments, it is 1.5 to 10 L / mol.

[0332] The concentration of the aqueous methylamine solution may be 30 to 40% by mass, and the molar ratio of methylamine to the compound represented by formula (318) may be 10:1 to 500:1. In some embodiments, it is 50:1 to 200:1.

[0333] Similarly to the above, the compound of formula (317) can be isolated from the reaction mixture by any suitable isolation method. In some embodiments, after removing the solvent by evaporation, the compound of formula (317) can be isolated by chromatography, for example, normal phase purification silica gel: (1) a silica gel packing agent of 200-300 mesh is gradient eluted with dichloromethane: methanol: aqueous ammonia (25 wt%) = 1:1:0.05-1:1:0.25, (2) reverse phase purification: C, C8 reverse phase packing agent is isolated under two chromatography conditions of gradient elution with methanol: acetonitrile = 0.1 :1-1:0.1. In some embodiments, the solvent can be directly removed to obtain a crude product of the compound of formula (317), and the crude product can be directly used in subsequent reactions. 18

[0334] In some embodiments, the compound represented by formula (318) is obtained by a preparation method including contacting the compound represented by formula (319) with triphenylchloromethane (TrCl), diphenylethylphenylchloromethane, phenyldiethylphenylchloromethane or triethylphenylchloromethane, and in some embodiments triphenylchloromethane (TrCl) in the presence of an organic solvent and under substitution reaction conditions, and then isolating.

[0335]

Chemical formula

[0336] As the substitution reaction conditions, the reaction temperature is 0 to 100 °C, and the reaction time is 5 to 72 hours and it may be so. In some embodiments, as the reaction conditions, the reaction temperature is 10 to 4 0 °C, and the reaction time is 10 to 30 hours.

[0337] Triphenylchloromethane (TrCl), diphenylethylphenylchloromethane, phenyl diethylphenylchloromethane or triethylphenylchloromethane can be purchased commercially, and the molar ratio of triphenylchloromethane (TrCl), diphenylethylphenyl chloromethane, phenyldiethylphenylchloromethane or triethylphenylchloromethane to the compound represented by formula (319) may be 1:1 to 10:1, and in some embodiments, it is 1:1 to 3:1.

[0338] The organic solvent may be one or more of epoxy solvents, ether solvents, alkyl halide solvents, dimethyl sulfoxide, N,N-dimethylformamide and N,N-diisopropylethylamine. In some embodiments, the epoxy solvent may be dioxane and / or tetrahydrofuran, and in some embodiments, the ether solvent is ethyl ether and / or methyl tert-butyl ether, and in some embodiments, the alkyl halide solvent is one or more of dichloromethane, trichloromethane and 1,2-dichloroethane, and in some embodiments, the organic solvent is dichloromethane. The compound of formula (319)​​​​ For this, the dosage of the organic solvent may be 3 to 50 L / mol, and in some embodiments it is 5 to 20 L / mol.

[0339] Similarly to the above, the compound of formula (318) can be isolated from the reaction mixture by any suitable isolation method. In some embodiments, after evaporating and removing the solvent, the compound of formula (318) can be isolated by a chromatography method. For example, (1) normal phase purification: silica gel filler of 200 - 300 mesh is eluted with a gradient of methanol:dichloromethane = 0.01:1 to 0.5:1, or methanol:dichloromethane:ethyl acetate:petroleum ether = 0.1:1:1:1 to 1:1:1:1; (2) reverse phase purification: C , C8 reverse phase filler is eluted with a gradient of methanol:acetonitrile = 0.1:1 to 1:0.1 under two chromatography conditions. In some embodiments, the solvent can be directly removed to obtain a crude product of the compound of formula (318), and this crude product can be directly used in subsequent reactions. For example, (1) normal phase purification: silica gel filler of 200 - 300 mesh is eluted with a gradient of methanol:dichloromethane = 0.01:1 to 0.5:1, or methanol:dichloromethane:ethyl acetate:petroleum ether = 0.1:1:1:1 to 1:1:1:1; (2) reverse phase purification: C , C8 reverse phase filler is eluted with a gradient of methanol:acetonitrile = 0.1:1 to 1:0.1 under two chromatography conditions. In some embodiments, the solvent can be directly removed to obtain a crude product of the compound of formula (318), and this crude product can be directly used in subsequent reactions. , dichloromethane = 0.01:1 to 0.5:1, or methanol:dichloromethane:ethyl acetate:petroleum ether = 0.1:1:1:1 to 1:1:1:1; (2) reverse phase purification: C , C8 reverse phase filler is eluted with a gradient of methanol:acetonitrile = 0.1:1 to 1:0.1 under two chromatography conditions. In some embodiments, the solvent can be directly removed to obtain a crude product of the compound of formula (318), and this crude product can be directly used in subsequent reactions. 18 , C8 reverse phase filler is eluted with a gradient of methanol:acetonitrile = 0.1:1 to 1:0.1. The compound of formula (318) can be isolated under these two chromatography conditions. In some embodiments, the solvent can be directly removed to obtain a crude product of the compound of formula (318), and this crude product can be directly used in subsequent reactions. In some embodiments, the solvent can be directly removed to obtain a crude product of the compound of formula (318), and this crude product can be directly used in subsequent reactions.

[0340] In some embodiments, the compound shown in formula (319) can be obtained by a preparation method including contacting the compound shown in formula (320) with ethyl trifluoroacetate under substitution reaction conditions in an organic solvent and then isolating it. For example, (1) normal phase purification: silica gel filler of 200 - 300 mesh is eluted with a gradient of methanol:dichloromethane = 0.01:1 to 0.5:1, or methanol:dichloromethane:ethyl acetate:petroleum ether = 0.1:1:1:1 to 1:1:1:1; (2) reverse phase purification: C , C8 reverse phase filler is eluted with a gradient of methanol:acetonitrile = 0.1:1 to 1:0.1 under two chromatography conditions. In some embodiments, the solvent can be directly removed to obtain a crude product of the compound of formula (318), and this crude product can be directly used in subsequent reactions.

[0341]

Chemical formula

[0342] In some embodiments, the organic solvent is acetonitrile, an epoxy solvent, an ethylene oxide solvent, Ether solvents, halogenated alkyl solvents, dimethyl sulfoxide, N,N-dimethyl ether The preferred amines are one or more of: dimethylformamide and N,N-diisopropylethylamine. In the embodiment, the epoxy solvent is dioxane and / or tetrahydrofuran. In some embodiments, the ether solvent is ethyl ether and / or is methyl tert-butyl ether, and in some embodiments, the halogen The alkyl chlorinated solvents are dichloromethane, trichloromethane and 1,2-dichloroethane. In some embodiments, the organic solvent is acetonitrile. The amount of the organic solvent is 1 to 50 L / mol relative to the compound of formula (320). In some embodiments, the concentration is 1 to 20 L / mol.

[0343] The conditions for the substitution reaction are a reaction temperature of 0 to 100° C. and a reaction time of 5 to 72 hours. In some embodiments, the substitution reaction conditions include a reaction temperature of The temperature is 10 to 40°C and the reaction time is 10 to 30 hours.

[0344] Compounds of formula (320) can be obtained commercially or by methods known to those skilled in the art. For example, m1 = m2 = m3 = 3, n1 = 1, n3 = 2, and R 10 , R 11 , R 12 , R 13 , R 14 , R 15When both are H, the compound of formula (320) The compound can be purchased commercially from Alfa Aesar.

[0345] The molar ratio of the ethyl trifluoroacetate to the compound represented by formula (320) is 2:1 to 1 0:1, and in some embodiments, 3:1 to 5:1.

[0346] Similarly as above, the compound of formula (319) can be isolated from the reaction mixture by any suitable isolation method. In some embodiments, after the solvent is evaporated, the chromatograph may be The compound of formula (319) can be isolated by a chromatography method, for example: (1) normal phase purification Silica gel: 200-300 mesh silica gel packing material is dissolved in methanol:dichloromethane. or methanol:dichloromethane:ethyl acetate:silane = 0.01:1 to 0.5:1 (2) Reverse phase purification: C 18 , C8 reversed-phase packing, methanol:acetonitrile = 0.1:1 to 1:0.1 gradient The compound can be isolated under two chromatographic conditions: In the above embodiment, the solvent can be directly removed to obtain the crude compound of formula (319); The crude product can be used as is in the subsequent reaction.

[0347] The first or second siRNA complex of the present disclosure may be administered in combination with other pharma- ceutically acceptable excipients. The additive may be one or more of various agents or compounds commonly used in the art. It may be of multiple types, and for details, please refer to the description of the drug composition of the present disclosure above. .

[0348] <Modified siRNA, pharmaceutical composition, first siRNA complex and second siRNA complex of the present disclosure> <Use of RNA complex> In some embodiments, the present invention provides for the use of the siRNA, pharmaceutical composition, first siRNA complex and / or second siRNA complex provided by the present invention in the preparation of a drug for the treatment and / or prevention of a pathological condition or disease caused by infection with the hepatitis B virus.

[0349] According to some embodiments of the present invention, the present invention provides a method for treating a pathological condition or disease caused by infection with the hepatitis B virus, comprising administering to a patient the siRNA, pharmaceutical composition, first siRNA complex and / or second siRNA complex provided by the present invention.

[0350] According to some other embodiments of the present invention, the present invention provides a method for suppressing the expression of hepatitis B virus genes in hepatocytes infected with chronic hepatitis B virus, comprising contacting the siRNA, pharmaceutical composition, first siRNA complex and / or second siRNA complex provided by the present invention with hepatocytes infected with chronic hepatitis B virus.

[0351] The pathological condition or disease caused by infection with the hepatitis B virus is selected from chronic liver disease, inflammation, fibrotic disease and hyperplastic disease.

[0352] By administering the siRNA and / or pharmaceutical composition, first siRNA complex and / or second siRNA complex of the present invention to a patient in need thereof, the purpose of treating hepatitis B by the RNA interference mechanism can be achieved. Therefore, the siRN of the present invention ​​​​​​​​A, and / or a pharmaceutical composition and / or an siRNA complex is used for the prevention and / or treatment of hepatitis B, or can be used for the preparation of a drug for the prevention and / or treatment of hepatitis B. It can be used for the prevention and / or treatment of hepatitis B, or can be used for the preparation of a drug for the prevention and / or treatment of hepatitis B.

[0353] As used herein, the term "drug administration / dosing" refers to introducing the modified siRNA, pharmaceutical composition, first siRNA complex and / or second siRNA complex of the present disclosure into the body of a subject by at least a part of the method or route that localizes at a desired site to produce a desired effect. The administration routes suitable for the methods of the present disclosure include local administration and systemic administration. Generally, local administration delivers more of the modified siRNA, pharmaceutical composition, first siRNA complex and / or second siRNA complex of the present disclosure to a specific site than the whole body of the subject, while systemic administration delivers the modified siRNA, pharmaceutical composition, first siRNA complex and / or second siRNA complex of the present disclosure to almost the whole body of the subject. Considering that the present disclosure is intended to provide means for the prevention and / or treatment of dyslipidemia, in some embodiments, the administration method can deliver the drug to the liver. It can be administered to a subject by any suitable route known in the art, including, but not limited to, oral administration or parenteral (non-oral) routes, such as intravenous administration, intramuscular administration, subcutaneous administration, transdermal administration, intratracheal administration (aerosol), pulmonary administration, nasal administration, rectal administration, and local administration (including intraoral administration and sublingual administration). The dosing frequency is once a day.

[0354] It can be administered to a subject by any suitable route known in the art, including, but not limited to, oral administration or parenteral (non-oral) routes, such as intravenous administration, intramuscular administration, subcutaneous administration, transdermal administration, intratracheal administration (aerosol), pulmonary administration, nasal administration, rectal administration, and local administration (including intraoral administration and sublingual administration). ​​​​​​​​​​​​​​It may be once or multiple times per week, every two weeks, every three weeks, once a month, or once a year.

[0355] The dosages of the siRNA, pharmaceutical composition, first siRNA complex, and / or second siRNA complex described in the present disclosure may be normal dosages in the art, and the dosages may be determined by various parameters, particularly the age, weight, and gender of the subject. Toxicity and therapeutic effects may be measured by standard pharmaceutical procedures in cell culture or experimental animals. For example, LD50 (the dosage that kills 50% of the population) and ED50 (in a quantitative response, it refers to the dosage that can cause 50% of the maximum response intensity, and in a qualitative response, it refers to the dosage when a positive response occurs in 50% of the experimental subjects) may be measured. A range of human dosages can be obtained based on the data obtained from cell culture

[0356] When administering the pharmaceutical composition or siRNA complex described in the present invention, for example, for male or female C57BL / 6J or C3H / HeNCrl Vr mice at 6 - 12 weeks of age and weighing 18 - 25 g, as the amount of siRNA in the pharmaceutical composition or siRNA complex, (i) for the first siRNA complex and / or the second siRNA complex, the siRNA dosage may be 0.001 - 100 mg / kg body weight. In a further embodiment, it is 0.01 - 50 mg / kg body weight. In an even further embodiment, it is 0.0 5 - 20 mg / kg body weight. In yet another further embodiment, it is 0.1 - 10 mg / kg body weight. (ii) For the pharmaceutical composition formed by siRNA and a pharmaceutically acceptable carrier, the siRNA dosage may be 0.001 - 50 mg / kg body weight. Further In a certain embodiment, it is 0.01 to 10 mg / kg body weight, and in a further embodiment, it is 0.05 to 5 mg / kg body weight, and in yet a further embodiment, it is 0.1 to 3 m g / kg body weight.

[0357] In addition, by introducing the siRNA, and / or drug composition and / or siRNA complex of the present invention into hepatocytes infected with chronic HBV, and further by the RNA interference mechanism, the object of suppressing the expression of the HBV gene in hepatocytes infected with chronic HBV can also be achieved. In some preferred embodiments, the cells are HepG2.2.15 cells . The expression of the HBV gene in cells is suppressed by the method provided by the present invention. Regardless of whether any of the provided siRNA, drug composition, first type of siRNA complex and / or second type of siRNA complex is used, the siRNA dosage generally can reduce the expression of the target gene and has an extracellular concentration of 1 pM to 1 μM, 0.01 nM to 100 nM, 0.05 nM

[0358] to 50 nM, or 0.05 nM to about 5 nM on the surface of the target cells. The amount required to achieve the local concentration varies depending on various factors including the delivery method, delivery site, number of cell layers between the delivery site and the target cells or tissues, whether the delivery is local or systemic, etc. The concentration at the delivery site may be significantly higher than the concentration on the surface of the target cells or tissues.

[0359]

[0360] <Kit> The present disclosure provides a kit containing an effective amount of at least one of the modified siRNA, drug composition, first type of siRNA complex and second type of siRNA complex of the present disclosure.

[0360] In some embodiments, the kit described herein comprises a modified siRNA in a container and can provide it. In some embodi...

Claims

1. An siRNA that contains a sense strand and an antisense strand, both of which contain fluorinated nucleotides and non-fluorinated nucleotides, and can suppress the expression of hepatitis B virus (HBV) genes, wherein the sense strand contains nucleotide sequence I, the antisense strand contains nucleotide sequence II, nucleotide sequence I and nucleotide sequence II form a double-stranded region that is at least partially reverse complementary, nucleotide sequence I consists only of nucleotide sequence A, nucleotide sequence A has the same length as the nucleotide sequence shown in SEQ ID NO: 155, nucleotide sequence II consists only of nucleotide sequence B, and nucleotide sequence B has the same length as the nucleotide sequence shown in SEQ ID NO: 156, 5'-CCUUGAGGCAUACUUCAAZ-3' (SEQ ID NO: 155), 5'-Z'UUUGAAGUAGCCUCAAGG-3' (SEQ ID NO: 156) provided that Z is A and Z' is U, The nucleotide sequence A contains a nucleotide Z whose position corresponds to Z, Ａ and the nucleotide sequence B contains a nucleotide Z' whose position corresponds to Z', Ｂ wherein the Z' Ｂ is the first nucleotide at the 5'-end of the antisense strand, the fluorinated nucleotides are located in nucleotide sequence A and nucleotide sequence B, and the 7th, 8th, and 9th nucleotides of nucleotide sequence A from the 5'-end to the 3'-end are fluorinated nucleotides, and the 2nd, 6th, 14th, and 16th nucleotides of nucleotide sequence B from the 5'-end to the 3'-end are fluorinated nucleotides, the sense strand contains the nucleotide sequence shown in SEQ ID NO: 1, and the antisense strand contains the nucleotide sequence shown in SEQ ID NO: 3 or SEQ ID NO: 4, 5'-CCUUGAGGCAUACUUCAAZ A-3' (SEQ ID NO: 1), 5'-Z' B UUUGAAGUAGCCUCAAGGUU-3' (SEQ ID NO: 3), 5'-Z' B UUUGAAGUAGCCUCAAGGUC-3' (SEQ ID NO: 4) provided that Z' B is the first nucleotide at the 5'-end of the antisense strand, Z A is selected from A, U, G, or C, and Z' B is a nucleotide complementary to Z A, all of the non-fluorinated nucleotides are methoxy-modified nucleotides, and the methoxy-modified nucleotides refer to nucleotides in which the 2'-hydroxy group of the ribose group is substituted with methoxy, siRNA.

2. The nucleotide sequence I further includes the nucleotide sequence III, the nucleotide sequence II further includes the nucleotide sequence IV, the lengths of the nucleotide sequence III and the nucleotide sequence IV are each independently 1 to 4 nucleotides, the nucleotide sequence III is bound to the 5' end of the nucleotide sequence A, the nucleotide sequence IV is bound to the 3' end of the nucleotide sequence B, and the nucleotide sequence III and the nucleotide sequence IV have the same length and are reverse complementary. The siRNA according to claim 1.

3. Both the nucleotide sequence III and the nucleotide sequence IV have a length of 1 nucleotide, and the base of the nucleotide sequence III is A, or Both the nucleotide sequence III and the nucleotide sequence IV have a length of 2 nucleotides, and the bases of the nucleotide sequence III are sequentially G and A from the 5' end to the 3' end, or Both the nucleotide sequence III and the nucleotide sequence IV have a length of 3 nucleotides, and the bases of the nucleotide sequence III are sequentially C, G, and A from the 5' end to the 3' end, or Both the nucleotide sequence III and the nucleotide sequence IV have a length of 4 nucleotides, and the bases of the nucleotide sequence III are sequentially C, C, G, and A from the 5' end to the 3' end. The siRNA according to claim 2.

4. The nucleotide sequence II further includes the nucleotide sequence V, the nucleotide sequence V has a length of 1 to 3 nucleotides, is bound to the 3' end of the antisense strand, and constitutes the 3' overhang end of the antisense strand. The siRNA according to claim 1.

5. The length of the nucleotide sequence V is 2 nucleotides, the nucleotide sequence V is complementary to the target mRNA, or the nucleotide sequence V is two consecutive thymidine deoxyribonucleotides or two consecutive uracil ribonucleotides from the 5' end to the 3' end. The siRNA according to claim 4.

6. The siRNA is siHBa1 or siHBa2. The siRNA according to claim 1: siHBa1 Sense strand: 5'-CCUUGAGGCAUACUUCAA A-3' (SEQ ID NO: 5) Antisense strand: 5'-UUUGAAGU AUGCCUCAAGGUU-3' (SEQ ID NO: 6) siHBa2 Sense strand: 5'-GACCUUGAGGCAUACUUCAAA-3' (SEQ ID NO: 7) Antisense strand: 5'-UUUGAAGU AUGCCUCAAGGUCGG-3' (SEQ ID NO: 8).

7. From the 5'-end towards the 3'-end, in the sense strand, the nucleotides at positions 7, 8, 9 or 5, 7, 8, 9 of the nucleotide sequence A are fluorine-modified nucleotides, the nucleotides at other positions in the sense strand are non-fluorine-modified nucleotides, in the antisense strand, the nucleotides at positions 2, 6, 14, 16 or positions 2, 6, 8, 9, 14, 16 of the nucleotide sequence B are fluorine-modified nucleotides, and the nucleotides at other positions in the antisense strand are non-fluorine-modified nucleotides, the siRNA according to claim 1.

8. The siRNA is siHBa1M1, siHBa1M2, siHBa2M1 or siHBa2M2, the siRNA according to claim 1: siHBa1M1 Sense strand: 5'-CmCmUmUmGmAmGfGfCfAmUmAmCmUmUmCmAmAmAm-3' (SEQ ID NO: 9) Antisense strand: 5'-UmUfUmGmAmAfGmUmAmUmGmCmCmUfCmAfAmGmGmUmUm-3' (SEQ ID NO: 10) siHBa1M2 Sense strand: 5'-CmCmUmUmGfAmGfGfCfAmUmAmCmUmUmCmAmAmAm-3' (SEQ ID NO: 11) Antisense strand: 5'-UmUfUmGmAmAfGmUfAfUmGmCmCmUfCmAfAmGmGmUmUm-3' (SEQ ID NO: 12) siHBa2M1 Sense strand: 5'-GmAmCmCmUmUmGmAmGfGfCfAmUmAmCmUmUmCmAmAmAm-3' (SEQ ID NO: 13) Antisense strand: 5'-UmUfUmGmAmAfGmUmAmUmGmCmCmUfCmAfAmGmGmUmCmGmGm-3' (SEQ ID NO: 14) siHBa2M2 Sense strand: 5'-GmAmCmCmUmUmGfAmGfGfCfAmUmAmCmUmUmCmAmAmAm-3' (SEQ ID NO: 15) or, Antisense strand: 5'-UmUfUmGmAmAfGmUfAfUmGmCmCmUfCmAfAmGmGmUmCmGmGm-3' (SEQ ID NO: 16) However, the capital letters C, G, U, and A represent the nucleotide base sequences, the small letter m represents that one nucleotide adjacent to the left side of the letter m is a 2'-methoxy modified nucleotide, and the small letter f represents that one nucleotide adjacent to the left side of the letter f is a 2'-fluoro modified nucleotide.

9. In the siRNA, at least one phosphate group is a phosphorothioate group, and the phosphorothioate group is between the first nucleotide and the second nucleotide from the 5'-end of the sense strand, between the second nucleotide and the third nucleotide from the 5'-end of the sense strand, between the first nucleotide and the second nucleotide from the 3'-end of the sense strand, between the second nucleotide and the third nucleotide from the 3'-end of the sense strand, between the first nucleotide and the second nucleotide from the 5'-end of the antisense strand, between the second nucleotide and the third nucleotide from the 5'-end of the antisense strand, between the first nucleotide and the second nucleotide from the 3'-end of the antisense strand, and the siRNA according to claim 1, which is bound to at least one selected from the group consisting of between the second nucleotide and the third nucleotide from the 3'-end of the antisense strand.

10. The siRNA according to claim 1, wherein the siRNA is siHBa1M1S, siHBa1M2S, siHBa2M1S or siHBa2M2S: siHBa1M1S Sense strand: 5'-CmsCmsUmUmGmAmGfGfCfAmUmAmCmUmUmCmAmAmAm-3' (SEQ ID NO: 17) Antisense strand: 5'-UmsUfsUmGmAmAfGmUmAmUmGmCmCmUfCmAfAmGmGmsUmsUm-3' (SEQ ID NO: 18) siHBa1M2S Sense strand: 5'-CmsCmsUmUmGfAmGfGfCfAmUmAmCmUmUmCmAmAmAm-3' (SEQ ID NO: 19) Antisense strand: 5'-UmsUfsUmGmAmAfGmUfAfUmGmCmCmUfCmAfAmGmGmsUmsUm-3' (SEQ ID NO: 20) siHBa2M1S Sense strand: 5'-GmsAmsCmCmUmUmGmAmGfGfCfAmUmAmCmUmUmCmAmAmAm-3' (SEQ ID NO: 21) Antisense strand: 5'-UmsUfsUmGmAmAfGmUfAfUmGmCmCmUfCmAfAmGmGmUmCm sGm sGm-3' (SEQ ID NO: 22) siHBa2M2S Sense strand: 5'-GmsAmsCmCmUmUmGfAmGfGfCfAmUmAmCmUmUmCmAmAmAm-3' (SEQ ID NO: 23) Antisense strand: 5'-UmsUfsUmGmAmAfGmUfAfUmGmCmCmUfCmAfAmGmGmUmCm sGm sGm-3' (SEQ ID NO: 24) However, capital letters C, G, U, and A represent the nucleotide base sequences, lowercase letter m indicates that one nucleotide adjacent to the left of the letter m is a methoxy-modified nucleotide, lowercase letter f indicates that one nucleotide adjacent to the left of the letter f is a fluoro-modified nucleotide, and lowercase letter s indicates that the two nucleotides on the left and right of the letter are linked by a phosphorothioate group.

11. The siRNA according to claim 1, wherein the nucleotide at the 5'-end of the antisense strand is a 5'-phosphate nucleotide or a 5'-phosphate analog-modified nucleotide.

12. The 5'-phosphate nucleotide is a nucleotide having a structure represented by formula (102), and the 5'-phosphate analog-modified nucleotide is selected from nucleotides having a structure represented by any one of formulas (103) to (106), 【Chemical 1】 However, R is selected from H, OH, a methoxy group, or fluorine, and Base represents a base selected from A, U, C, G, or T. The siRNA according to claim 11.

13. The siRNA according to claim 1, wherein the siRNA is any one of siHBa1M1P1, siHBa1M2P1, siHBa2M1P1, siHBa2M2P1, siHBa1M1SP1, siHBa1M2SP1, siHBa2M1SP1, siHBa2M2SP1: siHBa1M1P1 Sense strand: 5'-CmCmUmUmGmAmGfGfCfAmUmAmCmUmUmCmAmAmAm-3' (SEQ ID NO: 25) Antisense strand: 5'-P1-UmUfUmGmAmAfGmUmAmUmGmCmCmUfCmAfAmGmGmUmUm-3' (SEQ ID NO: 26) siHBa1M2P1 Sense strand: 5'-CmCmUmUmGfAmGfGfCfAmUmAmCmUmUmCmAmAmAm-3' (SEQ ID NO: 27) Antisense strand: 5'-P1-UmUfUmGmAmAfGmUmAmUmGmCmCmUfCmAfAmGmGmUmUm-3' (SEQ ID NO: 28) siHBa2M1P1 Sense strand: 5'-GmAmCmCmUmUmGmAmGfGfCfAmUmAmCmUmUmCmAmAmAm-3' (SEQ ID NO: 29) Antisense strand: 5'-P1-UmUfUmGmAmAfGmUmAmUmGmCmCmUfCmAfAmGmGmUmCmGmGm-3' (SEQ ID NO: 30) siHBa2M2P1 Sense strand: 5'-GmAmCmCmUmUmGfAmGfGfCfAmUmAmCmUmUmCmAmAmAm-3' (SEQ ID NO: 31) Antisense strand: 5'-P1-UmUfUmGmAmAfGmUfAfUmGmCmCmUfCmAfAmGmGmUmCmGmGm-3' (SEQ ID NO: 32) siHBa1M1SP1 Sense strand: 5'-CmsCmsUmUmGmAmGfGfCfAmUmAmCmUmUmCmAmAmAm-3' (SEQ ID NO: 33) Antisense strand: 5'-P1-UmsUfsUmGmAmAfGmUmAmUmGmCmCmUfCmAfAmGmGmsUmsUm-3' (SEQ ID NO: 34) siHBa1M2SP1 Sense strand: 5'-CmsCmsUmUmGfAmGfGfCfAmUmAmCmUmUmCmAmAmAm-3' (SEQ ID NO: 35) Antisense strand: 5'-P1-UmsUfsUmGmAmAfGmUfAfUmGmCmCmUfCmAfAmGmGmsUmsUm-3' (SEQ ID NO: 36) siHBa2M1SP1 Sense strand: 5'-GmsAmsCmCmUmUmGmAmGfGfCfAmUmAmCmUmUmCmAmAmAm-3' (SEQ ID NO: 37) Antisense strand: 5'-P1-UmsUfsUmGmAmAfGmUmAmUmGmCmCmUfCmAfAmGmGmUmCmsGmsGm-3' (SEQ ID NO: 38) siHBa2M2SP1 Sense strand: 5'-GmsAmsCmCmUmUmGfAmGfGfCfAmUmAmCmUmUmCmAmAmAm-3' (SEQ ID NO: 39) Antisense strand: 5'-P1-UmsUfsUmGmAmAfGmUfAfUmGmCmCmUfCmAfAmGmGmUmCmsGmsGm-3' (SEQ ID NO: 40) However, the capital letters C, G, U, and A represent the nucleotide base sequences, the lowercase letter m indicates that one nucleotide adjacent to the left side of the letter m is a 2'-methoxy modified nucleotide, the lowercase letter f indicates that one nucleotide adjacent to the left side of the letter f is a 2'-fluoro modified nucleotide, the lowercase letter s indicates that the two nucleotides adjacent to the left and right sides of the letter are linked by a phosphorothioate group, and P1 indicates that one nucleotide adjacent to the right side of the letter P1 is a 5'-phosphate nucleotide or a 5'-phosphate analog modified nucleotide.

14. A pharmaceutical composition comprising the siRNA according to Claim 1 and a pharmaceutically acceptable carrier, wherein the weight ratio of the siRNA to the pharmaceutically acceptable carrier is 1:(1 - 500).

15. An siRNA complex comprising the siRNA according to Claim 1 and a complex group that binds to the siRNA in a complex manner.

16. For the treatment and / or prevention of pathological conditions or diseases caused by hepatitis B virus infection, A composition comprising the siRNA according to Claim 1, and / or a pharmaceutical composition comprising the siRNA according to Claim 1, and / or an siRNA complex comprising the siRNA according to Claim 1.

17. The composition according to Claim 16, wherein the pathological condition or disease caused by hepatitis B virus infection is selected from chronic liver diseases, hepatitis, liver fibrotic diseases, or liver hyperplastic diseases.

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