Oligomeric nucleic acid and compound for reducing off-target activity

Abstract

The invention relates to an oligomeric nucleic acid and a compound for reducing off-target activity. The invention provides a single-stranded or double-stranded oligomeric nucleic acid, each strand has 15 to 35 nucleotides, and at least one nucleotide position of the oligomeric nucleic acid, which is not the head end, comprises a chemical modification shown in a formula (I) or a tautomer modification thereof. According to the modification method disclosed by the invention, the off-target effect of the siRNA is reduced, and meanwhile, the in-target activity of the siRNA is not influenced to the greatest extent. # imgabs0 #

Description

Oligonucleic acid and compound for reducing off-target activity Technical Field

[0001] The present invention relates to the fields of molecular biology and biochemistry, and in particular to an oligonucleic acid and a compound for reducing off-target activity. Background Art

[0002] siRNA has a very important prospect for drug development. However, in vivo applications, siRNA often has varying degrees of off-target effects, and there may be cases where siRNA targets non-target genes. Off-target effects similar to the microRNA pathway are one of the main causes of siRNA off-target effects, namely the inhibitory activity on mRNA generated by the complete or incomplete pairing of the siRNA antisense strand (also known as the AS strand) seed region (positions 2-8 at the 5' end) with the target mRNA. The off-target effect of a single siRNA molecule may affect multiple mRNAs. Therefore, unpredictable toxic side effects may occur, which is the main reason for the toxic side effects of siRNA drugs.

[0003] To reduce off-target toxicity and improve the safety of siRNA drugs, the antisense strand seed region of the siRNA drug can be chemically modified at specific sites, thereby reducing the off-target toxicity of small nucleic acid drugs. By introducing chemical modifications, the strength of the interaction between the seed region and the target gene can be adjusted, and efforts are made to reduce off-target effects without affecting the silencing effect of the target mRNA (Mark K. Schlegel, Chirality Dependent Potency Enhancement and Structural Impact of Glycol Nucleic Acid Modification on siRNA. J. Am. Chem. Soc. 2017, 139, 8537-8546).

[0004] In the prior art, chemical modifications, such as GNA or UNA, introduced into the 5'-end seed region of the antisense strand of siRNA can reduce off-target toxicity, but also significantly reduce on-target activity. Therefore, there is a need for a chemical modification method that can minimize the on-target activity of siRNA while reducing off-target effects.

[0005] Summary of the Invention

[0006] In view of this, the purpose of the present invention is to provide an oligonucleic acid modified to reduce off-target activity and a compound for reducing off-target activity. The solution of the present invention can reduce the off-target effect of the oligonucleic acid while minimizing the impact on the on-target activity of the oligonucleic acid. At the same time, the present invention can also improve the stability of single-stranded oligonucleic acids or double-stranded oligonucleic acids.

[0007] Based on the above purpose, the first aspect of the present invention provides a single-stranded or double-stranded oligonucleic acid, each chain having 15 to 35 nucleotides, and at least one non-terminal nucleotide position of the oligonucleic acid comprises a chemical modification represented by formula (I) or a tautomer modification thereof,

[0008] wherein B is a natural nucleobase, a modified nucleobase, a universal base or an H atom; preferably, B is a natural nucleobase or a modified nucleobase;

[0009] Z is selected from O, NH and S; preferably, Z is O;

[0010] R1, R2, R3, R4, R5, R6 and R7 are independently selected from H, OH, halogen, NH2, C1-C6 alkyl, C1-C6 alkoxy, C2-C6 alkenyl, C2-C6 alkynyl, S-CH3, NCH3(CH3), OCH2CH2OCH3 and -O-alkylamino; preferably, R1, R2, R3, R4, R5, R6 and R7 are independently selected from H, C1-C6 alkyl, C1-C6 alkoxy, C2-C6 alkenyl, C2-C6 alkynyl;

[0011] X is O or S;

[0012] n is 1, 2 or 3; preferably, n is 1 or 2.

[0013] In a preferred embodiment of the present invention, the oligonucleic acid is siRNA, which comprises a sense strand and an antisense strand.

[0014] In a preferred embodiment of the present invention, the antisense strand comprises a chemical modification represented by formula (I-1) or a tautomer modification thereof at at least one nucleotide position from position 2 to position 8 in its 5' region,

[0015] Wherein: B1 and B2 are each independently a natural nucleobase, a modified nucleobase, a universal base or an H atom; preferably, B1 and B2 are each independently a natural nucleobase or a modified nucleobase;

[0016] Z is selected from O, NH and S; preferably, Z is O;

[0017] R1, R2, R3, R4, R5, R6 and R7 are each independently selected from H, OH, halogen, NH2, C1-C6 alkyl, C1-C6 alkoxy, C2-C6 alkenyl, C2-C6 alkynyl, S-CH3, NCH3(CH3), OCH2CH2OCH3 and -O-alkylamino; preferably, R1, R2, R3, R4, R5, R6 and R7 are each independently H;

[0018] X is O or S;

[0019] M1 and M2 are independently selected from H, OH, F, OCH3, OCH2CH2OCH3 and -O-alkyl; preferably, M1 and M2 are independently selected from H, OH, F and OCH3;

[0020] n is 1, 2 or 3, preferably, n is 1 or 2.

[0021] In a preferred embodiment of the present invention, the antisense strand comprises a chemical modification represented by formula (I-2) or a tautomer modification thereof at at least one nucleotide position from position 2 to position 8 in its 5' region,

[0022] wherein B1 and B2 are each independently a natural nucleobase, a modified nucleobase, a universal base or an H atom; preferably, B1 and B2 are each independently a natural nucleobase or a modified nucleobase;

[0023] R3 and R4 are each independently selected from H, OH, halogen, NH2, C1-C6 alkyl, C1-C6 alkoxy, C2-C6 alkenyl, C2-C6 alkynyl, S-CH3, NCH3(CH3), OCH2CH2OCH3 and -O-alkylamino; preferably, R3 and R4 are each independently H;

[0024] X is O or S; preferably, X is O;

[0025] M is selected from H, OH, F, OCH3, OCH2CH2OCH3 and -O-alkyl; preferably, M is selected from H, OH, F and OCH3.

[0026] In a preferred embodiment of the present invention, the antisense strand comprises a chemical modification represented by formula (I-3), (I-4) or (I-5) or a tautomer modification thereof at at least one nucleotide position from position 2 to position 8 in the 5' region of the antisense strand.

[0027] wherein B1 and B2 are each independently a natural nucleobase, a modified nucleobase, a universal base or an H atom; preferably, B1 and B2 are each independently a natural nucleobase or a modified nucleobase;

[0028] R3 and R4 are each independently selected from H, OH, halogen, NH2, C1-C6 alkyl, C1-C6 alkoxy, C2-C6 alkenyl, C2-C6 alkynyl, S-CH3, NCH3(CH3), OCH2CH2OCH3 and -O-alkylamino; preferably, R3 and R4 are each independently H;

[0029] X is O or S; preferably, X is O;

[0030] M is selected from OH, F, OCH3, OCH2CH2OCH3 and -O-alkyl; preferably, M is selected from OH, F and OCH3.

[0031] In a preferred embodiment of the present invention, the chemical modification is selected from any one of the following structures:

[0032] In a preferred embodiment of the present invention, the antisense strand comprises any one of the chemical modifications defined above or a tautomer modification thereof at at least one nucleotide position from position 5 to position 8 in its 5' region;

[0033] Preferably, the antisense strand comprises any one of the chemical modifications defined above or a tautomer modification thereof at at least one of the nucleotide positions 6 and 7 in the 5' region.

[0034] In a preferred embodiment of the present invention, in addition to the nucleotides comprising any one of the chemical modifications or tautomer modifications as defined above, at least one of the sense strand and / or antisense strand further comprises at least one other modified nucleotide;

[0035] Preferably, in addition to the nucleotides comprising any one of the chemical modifications or tautomer modifications defined above, the remaining nucleotides in the sense strand and / or antisense strand are other modified nucleotides.

[0036] In a preferred embodiment of the present invention, the other modified nucleotides are independently selected from the group consisting of 2'-fluoro-modified nucleotides, 2'-alkoxy-modified nucleotides, 2'-substituted alkoxy-modified nucleotides, 2'-alkyl-modified nucleotides, 2'-substituted alkyl-modified nucleotides, 2'-deoxynucleotides, 2'-amino-modified nucleotides and 2'-substituted amino-modified nucleotides;

[0037] Preferably, the other modified nucleotides are independently selected from the group consisting of 2'-F modified nucleotides, 2'-O-CH3 modified nucleotides, 2'-O-CH2-CH2-O-CH3 modified nucleotides, 2'-O-CH2-CH=CH2 modified nucleotides, 2'-CH2-CH2-CH=CH2 modified nucleotides and 2'-deoxynucleotides;

[0038] More preferably, the other modified nucleotides are independently selected from: 2'-F modified nucleotides and 2'-O-CH3 modified nucleotides.

[0039] In a preferred embodiment of the present invention, the single-stranded or double-stranded oligonucleic acid / siRNA has at least one of the following characteristics:

[0040] (i) the antisense strand comprises 2, 3, 4, 5 or 6 2'-fluoro modifications;

[0041] (ii) the antisense strand comprises 1, 2, 3, or 4 phosphorothioate internucleotide linkages;

[0042] (iv) the sense strand contains 2, 3, 4, or 5 2'-fluoro modifications;

[0043] (v) the sense strand contains 1, 2, 3, or 4 phosphorothioate internucleotide linkages;

[0044] (vi) the dsRNA comprises at least four 2'-fluoro modifications;

[0045] (vii) The siRNA comprises a duplex region of 12-40 nucleotide pairs in length.

[0046] The second aspect of the present invention provides an siRNA conjugate, which comprises the above-mentioned single-stranded or double-stranded oligonucleic acid / siRNA and a conjugated group conjugated to the siRNA.

[0047] Preferably, the conjugate group comprises a pharmaceutically acceptable targeting group and a linker, and the single-stranded or double-stranded oligonucleic acid / siRNA, the linker and the targeting group are sequentially covalently or non-covalently linked.

[0048] The third aspect of the present invention provides a pharmaceutical composition comprising the above-mentioned single-stranded or double-stranded oligonucleic acid / siRNA, or the above-mentioned siRNA conjugate, and a pharmaceutically acceptable carrier.

[0049] The fourth aspect of the present invention provides a kit comprising the above-mentioned single-stranded or double-stranded oligonucleic acid / siRNA, or the above-mentioned siRNA conjugate, or the above-mentioned pharmaceutical composition.

[0050] The fifth aspect of the present invention provides a compound represented by the following formula (III) or its tautomer,

[0051] wherein B1 and B2 are each independently a natural nucleobase, a modified nucleobase, a universal base or an H atom;

[0052] R3 and R4 are each independently selected from H, OH, halogen, NH2, C1-C6 alkyl, C1-C6 alkoxy, C2-C6 alkenyl, C2-C6 alkynyl, S-CH3, NCH3(CH3), OCH2CH2OCH3 and -O-alkylamino;

[0053] X is O or S;

[0054] M is selected from OH, F, OCH3, OCH2CH2OCH3 and -O-alkyl;

[0055] E is a leaving group; preferably, E is MMTr or DMTr;

[0056] Q is a phosphorus-containing reactive group; preferably, Q is

[0057] In a preferred embodiment of the present invention, the compound represented by the above formula (III) or (IV) or its tautomer is selected from any of the following structures:

[0058] A sixth aspect of the present invention provides a method for preparing the above-mentioned single-stranded or double-stranded oligonucleic acid / siRNA or the above-mentioned siRNA conjugate, comprising the following steps:

[0059] 1) synthesizing the compound represented by the above formula (III) or its tautomer;

[0060] 2) synthesizing the siRNA or siRNA conjugate using the compound of step 1) or its tautomer.

[0061] The seventh aspect of the present invention provides a single-stranded or double-stranded oligonucleic acid, each chain having 15 to 60 nucleotides, wherein at least one non-terminal nucleotide position of the oligonucleic acid comprises a chemical modification represented by formula (V) or a tautomer modification thereof,

[0062] wherein B1 and B2 are independently selected from natural nucleobases, modified nucleobases, universal bases or H atoms;

[0063] Z is selected from O, NH and S;

[0064] G is selected from -(CR8R9)m- and -CR10=CR11-;

[0065] R1, R2, R5, R6, R7, R8, R9, R8, R9, R10 and R11 are each independently selected from H, OH, halogen, NH2, C1-C6 alkyl, C1-C6 alkoxy, C2-C6 alkenyl, C2-C6 alkynyl, S-CH3, NCH3(CH3), OCH2CH2OCH3 and -O-alkylamino;

[0066] M1 and M2 are independently selected from H, OH, F and OCH3;

[0067] n is 1, 2, or 3;

[0068] m is 1, 2, 3, 4 or 5;

[0069] y is 0, 1, 2, 3;

[0070] X is O or S.

[0071] The eighth aspect of the present invention provides an siRNA comprising a sense strand and an antisense strand, each strand having 15 to 60 nucleotides, wherein at least one nucleotide position of the antisense strand comprises a chemical modification represented by formula (V-2), (V-3), (V-4) or (V-5) or a tautomer modification thereof,

[0072] wherein: B1 and B2 are each independently a natural nucleobase, a modified nucleobase, a universal base or an H atom; preferably, B1 and B2 are each independently a natural nucleobase, a modified or unmodified nucleobase;

[0073] M1 is selected from H, OH, F and OCH3; preferably, M1 is selected from F and OCH3;

[0074] X is O or S.

[0075] In a preferred embodiment of the present invention, the chemical modification is selected from any one of the following:

[0076] In a preferred embodiment of the present invention, the antisense strand comprises a chemical modification represented by formula (V), (V-2), (V-3) or (V-4) as defined above, or a tautomer modification thereof, at at least one nucleotide position from position 5 to position 8 in its 5' region;

[0077] Preferably, the antisense strand comprises a chemical modification represented by formula (V), (V-2), (V-3) or (V-4) as defined above or a tautomer modification thereof at at least one of nucleotide positions 6 and 7 in its 5' region.

[0078] In a preferred embodiment of the present invention, in addition to the chemically modified nucleotides represented by formula (V), (V-2), (V-3) or (V-4) as defined above, or modified tautomers thereof, at least one of the sense strand and / or antisense strand further comprises at least one other modified nucleotide;

[0079] Preferably, in addition to the chemically modified nucleotides represented by formula (V), (V-2), (V-3) or (V-4) defined above or their tautomer-modified nucleotides, the remaining nucleotides in the sense strand and / or antisense strand are other modified nucleotides.

[0080] In a preferred embodiment of the present invention, the other modified nucleotides are independently selected from the group consisting of 2'-fluoro-modified nucleotides, 2'-alkoxy-modified nucleotides, 2'-substituted alkoxy-modified nucleotides, 2'-alkyl-modified nucleotides, 2'-substituted alkyl-modified nucleotides, 2'-deoxynucleotides, 2'-amino-modified nucleotides and 2'-substituted amino-modified nucleotides;

[0081] Preferably, the other modified nucleotides are independently selected from the group consisting of 2'-F modified nucleotides, 2'-O-CH3 modified nucleotides, 2'-O-CH2-CH2-O-CH3 modified nucleotides, 2'-O-CH2-CH=CH2 modified nucleotides, 2'-CH2-CH2-CH=CH2 modified nucleotides and 2'-deoxynucleotides;

[0082] More preferably, the other modified nucleotides are independently selected from: 2'-F modified nucleotides and 2'-O-CH3 modified nucleotides.

[0083] In a preferred embodiment of the present invention, at least one of the following features is present:

[0084] (i) the antisense strand comprises 2, 3, 4, 5 or 6 2'-fluoro modifications;

[0085] (ii) the antisense strand comprises 1, 2, 3, or 4 phosphorothioate internucleotide linkages;

[0086] (iii) the sense strand contains 2, 3, 4, or 5 2'-fluoro modifications;

[0087] (iv) the sense strand contains 1, 2, 3, or 4 phosphorothioate internucleotide linkages;

[0088] (v) the dsRNA comprises at least four 2'-fluoro modifications;

[0089] (vi) The siRNA comprises a duplex region of 12-40 nucleotide pairs in length.

[0090] The ninth aspect of the present invention provides an siRNA conjugate, which comprises the above-mentioned single-stranded or double-stranded oligonucleic acid / siRNA and a conjugated group conjugated to the siRNA.

[0091] Preferably, the conjugate group comprises a pharmaceutically acceptable targeting group and a linker, and the single-stranded or double-stranded oligonucleic acid / siRNA, the linker and the targeting group are sequentially covalently or non-covalently linked.

[0092] The tenth aspect of the present invention provides a pharmaceutical composition comprising the above-mentioned single-stranded or double-stranded oligonucleic acid, or the above-mentioned siRNA conjugate, and a pharmaceutically acceptable carrier.

[0093] The eleventh aspect of the present invention provides a kit comprising the above-mentioned single-stranded or double-stranded oligonucleic acid, or the above-mentioned siRNA conjugate, or the above-mentioned pharmaceutical composition.

[0094] The twelfth aspect of the present invention provides a compound represented by the following formula (VI) or its tautomer:

[0095] wherein B1 and B2 are each independently a natural nucleobase, a modified nucleobase, a universal base or an H atom;

[0096] G is selected from -(CR8R9)n- and -CR10=CR11-;

[0097] X is O or S;

[0098] M is selected from H, OH, F, OCH3, OCH2CH2OCH3 and -O-alkyl;

[0099] E is a leaving group; preferably, E is MMTr or DMTr;

[0100] Q is a phosphorus-containing reactive group; preferably, Q is

[0101] In a preferred embodiment of the present invention, the compound represented by formula (VI) or its tautomer is selected from any one of the following structures:

[0102] The thirteenth aspect of the present invention provides a method for preparing the above-mentioned single-stranded or double-stranded oligonucleic acid or the above-mentioned siRNA conjugate, which comprises the following steps:

[0103] 1) synthesizing the compound represented by the above formula (VI) or its tautomer;

[0104] 2) synthesizing the siRNA or siRNA conjugate using the compound of step 1) or its tautomer.

[0105] The fourteenth aspect of the present invention provides a method for inhibiting a target gene in a cell, which comprises the step of introducing the above-mentioned single-stranded or double-stranded oligonucleic acid, or the above-mentioned siRNA conjugate into the cell.

[0106] The fifteenth aspect of the present invention provides a method for modifying positions 2-8 of the antisense chain of siRNA to reduce off-target activity, wherein the above-mentioned chemical modification or its tautomer modification, or the above-mentioned chemical modification or its tautomer modification is introduced into positions 2-8 of the antisense chain of the siRNA molecule.

[0107] The beneficial effects of the present invention are:

[0108] The present invention introduces a special compound into the 5'-end seed region of the antisense strand of siRNA to reduce the off-target effect of siRNA while minimizing the impact on the on-target activity of siRNA, thereby improving the safety of siRNA as a drug. BRIEF DESCRIPTION OF THE DRAWINGS

[0109] FIG1 is a graph showing the HBsAg levels before and after administration in Example 5. DETAILED DESCRIPTION

[0110] It should be noted that, unless otherwise defined, the technical terms or scientific terms used in this application should be understood by those skilled in the art in the ordinary sense. The experimental methods in the following examples are all conventional methods unless otherwise specified. The medicinal raw materials, reagent materials, etc. used in the following examples are all commercially available products unless otherwise specified. When used herein and in the appended claims, the singular forms "one", "another", and "the" include plural referents unless the context clearly indicates otherwise.

[0111] definition

[0112] In the context of the present invention, capital letters A, U, C, and G represent the base composition of a nucleotide; lowercase letter m indicates that the nucleotide adjacent to the left of letter m is a methoxy-modified nucleotide; lowercase letter f indicates that the nucleotide adjacent to the left of letter f is a fluorinated modified nucleotide; lowercase letter s indicates that the two nucleotides adjacent to the left and right of letter s are connected by a phosphorothioate group; the letter combination VP indicates that the nucleotide adjacent to the right of letter combination VP is a vinyl phosphate-modified nucleotide, as shown below. The structure of the phosphorothioate group is shown in formula (1):

[0113] As used herein, the term "natural nucleobase" refers to a nucleobase that has not been modified from its naturally occurring form in RNA or DNA. Examples of "natural nucleobases" include the purine nucleobases adenine (A) and guanine (G) and the pyrimidine nucleobases thymine (T), cytosine (C), and uracil (U). In addition to "natural nucleobases," many modified nucleobases or nucleobase mimetics known to those skilled in the art are suitable for use in the compounds described herein.

[0114] As used herein, the term "modified nucleobase" refers to a nucleobase that is structurally very similar to a parent nucleobase, such as 7-deazapurine, 5-methylcytosine, or the G-clamp.

[0115] As used herein, a universal nucleic acid base is a base that can complementarily pair with at least two commonly used bases, such as hypoxanthine (whose nucleoside is inosine), which can pair with any of A, T, G, or C, with a binding affinity of I:C>I:A>I:G>I:T; or 5-Bromouridine (BrU), which can pair with A or G. Alternatively, other universal bases that can complementarily pair with at least two commonly used bases can also be used in the present invention, such as 3-nitropyrrole, 5-nitroindole, 7-azaindole, and the like.

[0116] As used herein, the term "seed region" is the region of the antisense strand of an RNAi agent that is responsible for recognizing the target mRNA and responds to, for example, nucleotides 2-8 from the 5' end of the antisense strand.

[0117] As used herein, the term "nucleotide position" refers to the position of a nucleotide in an oligonucleotide as counted from the nucleotide at the 5' end. For example, nucleotide position 1 refers to the 5' terminal nucleotide of the oligonucleotide.

[0118] As used herein, "oligonucleotide" or "oligonucleic acid" refers to a polymeric form of nucleotides in the range of 2 to 2500 nucleotides. Oligonucleotides may be single-stranded or double-stranded. In certain embodiments, the oligonucleotide has 500 to 1500 nucleotides, typically, for example, wherein the oligonucleotide is used for gene therapy. In certain embodiments, the oligonucleotide is single-stranded or double-stranded and has 7 to 100 nucleotides. In certain embodiments, the oligonucleotide is single-stranded or double-stranded and has 15 to 100 nucleotides. In another embodiment, the oligonucleotide is single-stranded or double-stranded with 15 to 50 nucleotides, typically, for example, wherein the oligonucleotide is a nucleic acid inhibitor molecule. In another embodiment, the oligonucleotide is single-stranded or double-stranded with 25 to 40 nucleotides, typically, for example, wherein the oligonucleotide is a nucleic acid inhibitor molecule. In another embodiment, the oligonucleotide is single-stranded or double-stranded and has 19 to 40 or 19 to 25 nucleotides, typically, for example, wherein the oligonucleotide is a double-stranded nucleic acid inhibitor molecule and forms a double helix with at least 18 to 25 base pairs. In other embodiments, the oligonucleotide is single-stranded and has 15 to 25 nucleotides, typically, for example, wherein the oligonucleotide nucleotide is a single-stranded RNAi inhibitor molecule. Typically, as described herein, the oligonucleotide contains one or more phosphorus-containing internucleotide linking groups. In other embodiments, as described herein, the internucleotide linking group is a phosphite amide group.

[0119] As used herein, a "fluorinated nucleotide" refers to a nucleotide in which the hydroxyl group at the 2' position of the ribose group of the nucleotide is substituted with fluorine, and has a structure as shown in the following formula (7). In some embodiments, the 2'-alkyl modified nucleotide is a methoxy modified nucleotide (2'-OMe), as shown in formula (8).

[0120] Here, base represents a base, such as A, U, G, C or T.

[0121] As used herein, "non-fluorinated modified nucleotide" refers to a nucleotide in which the hydroxyl group at the 2'-position of the ribose group of the nucleotide is replaced by a non-fluorinated group.

[0122] As used herein, "conjugation" refers to the covalent attachment of two or more chemical moieties, each with a specific function, to one another; accordingly, "conjugate" refers to a compound formed by covalent attachment of these chemical moieties. Furthermore, "siRNA conjugate" refers to a compound formed by covalent attachment of one or more chemical moieties with a specific function to an siRNA.

[0123] In the context of the present invention, particularly when describing the siRNA of the application, containing the compositions of siRNA or the preparation method of siRNA conjugate, unless otherwise stated, described nucleoside monomer (nucleoside monomer) refers to, according to the kind and order of Nucleotide in the siRNA for preparation or the siRNA conjugate, the modification used in the phosphoramidite solid phase synthesis or unmodified nucleoside phosphoramidite monomer (unmodified or modified RNA phosphoramidites, RNA phosphoramidites is also referred to as Nucleoside phosphoramidites sometimes).Phosphoramidite solid phase synthesis is the method used in RNA synthesis known to those skilled in the art.The application's used nucleoside monomer all can be commercially obtained.

[0124] 1. Single-stranded or double-stranded oligonucleic acid / siRNA of the present invention

[0125] The present invention provides a single-stranded or double-stranded oligonucleic acid, each strand having 15 to 35 nucleotides, wherein at least one non-terminal nucleotide position of the oligonucleic acid comprises a chemical modification represented by formula (I) or (V) or a tautomer modification thereof,

[0126] In the present invention, "non-first end of an oligonucleotide" refers to any position other than the first and last ends of an oligonucleotide, in other words, any position other than the first and last nucleotides (5'-3' direction) of an oligonucleotide.

[0127] The siRNA of the present application contains a nucleotide group as a basic structural unit. It is well known to those skilled in the art that the nucleotide group contains a phosphate group, a ribose group and a base.

[0128] The present invention introduces one of these compounds into the 5' end seed region of the antisense strand of siRNA for chemical modification, which reduces the off-target effect of siRNA while minimizing the impact on the on-target activity of siRNA, thereby improving the safety of siRNA as a drug.

[0129] In the prior art, GNA or UNA modifications are usually introduced into the 5'-end seed region of the antisense strand of siRNA, which can reduce off-target toxicity but also result in a significant loss of on-target activity.

[0130] Wherein, UNA is shown in formula (3), and GNA is shown in formula (4).

[0131] In the above formulae (3) and (4), R is selected from H, OH or alkoxy (O-alkyl).

[0132] In the present application, different siRNA sequences for multiple targets were designed, and the on-target activity and off-target activity of siRNA were compared when the chemical modification of the present application and the GNA modification were introduced at the same position in the 5' end seed region of the antisense strand of siRNA composed of the same base sequence. The results showed that the introduction of the chemical modification and GNA modification of the present application can reduce the off-target effect of siRNA, improve the safety of siRNA as a drug, and the introduction of the chemical modification of the present application is superior to the GNA modification in reducing off-target activity; and the siRNA introduced with the chemical modification of the present application has better on-target activity than the GNA modification or can still ensure effective on-target activity, which shows that the introduction of the chemical modification of the present application can not affect the on-target activity of siRNA to the greatest extent. The effect is the same in multiple siRNAs, which also shows that the effect achieved by the siRNA of the present invention is unrelated to the siRNA sequence itself.

[0133] The present application provides many chemical modifications, such as Agt, Agt01, Uut, Agt02, Aut, Aut01, Agz, Agy, Agx, etc., and the introduction of one of these chemical modifications at at least one nucleotide position from the 2nd to the 8th position in the 5' region of the antisense chain can retain the on-target activity basically the same as the original sequence and significantly reduce the off-target activity.

[0134] In the examples of the present application (including in vitro and in vivo experiments), it was verified that the inclusion of the above-mentioned chemical modification at the 6th or 7th nucleotide position in the 5'-end seed region of the siRNA antisense strand can reduce the off-target effect of the siRNA while minimizing the impact on the on-target activity of the siRNA.

[0135] 2. siRNA conjugates of the present invention

[0136] The present invention provides an siRNA conjugate comprising the above-mentioned single-stranded or double-stranded oligonucleic acid / siRNA and a conjugated group conjugated to the siRNA. Preferably, the conjugated group comprises a pharmaceutically acceptable targeting group and a linker, and the single-stranded or double-stranded oligonucleic acid / siRNA, the linker, and the targeting group are sequentially covalently or non-covalently linked.

[0137] In a preferred embodiment of the present invention, the conjugated group conjugated to the siRNA is TIN, L96 or SA51, etc., wherein the structural formula of TIN is shown in the figure below, and the structural formulas of L96 and SA51 are shown in Example 5.

[0138] TIN.

[0139] 3. Pharmaceutical compositions and kits of the present invention

[0140] The present application provides a pharmaceutical composition, which contains the single-stranded or double-stranded oligonucleic acid / siRNA or siRNA conjugate as described above as an active ingredient and a pharmaceutically acceptable carrier.

[0141] The pharmaceutically acceptable carrier may be a carrier conventionally used in the field of siRNA administration, such as, but not limited to, magnetic nanoparticles (e.g., nanoparticles based on Fe3O4 or Fe2O3), carbon nanotubes, mesoporous silicon, calcium phosphate nanoparticles, polyethylenimine (PEI), polyamidoamine (PAMAM) dendrimer, poly(L-lysine, PLL), chitosan, 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), poly (D&L-lactic / glycolic acid) copolymer (PLGA), poly(2-aminoethyl ethylene phosphate), poly(L-lysine, PLL ... phosphate), PPEEA) and poly (methacrylate-N, N-dimethylaminoethyl ester) (poly (2-dimethylaminoethyl methacrylate), PDMAEMA) and one or more of their derivatives.

[0142] In the pharmaceutical composition, there is no particular requirement for the content of the single-stranded or double-stranded oligonucleic acid / siRNA or siRNA conjugate and the pharmaceutically acceptable carrier, and the conventional content of each component can be used.

[0143] The present disclosure provides a kit comprising the above-mentioned single-stranded or double-stranded oligonucleic acid / siRNA or the above-mentioned siRNA conjugate or the above-mentioned pharmaceutical composition.

[0144] In some embodiments, the kits described herein may provide single-stranded or double-stranded oligonucleic acid / siRNA or siRNA conjugates in one container. In some embodiments, the kits described herein may include a container for providing a pharmaceutically acceptable excipient. In some embodiments, the kits may also include other ingredients, such as stabilizers or preservatives. In some embodiments, the kits described herein may include at least one other therapeutic agent in other containers other than the container providing single-stranded or double-stranded oligonucleic acid / siRNA or siRNA conjugates described herein. In some embodiments, the kits may include instructions for mixing single-stranded or double-stranded oligonucleic acid / siRNA or siRNA conjugates with pharmaceutically acceptable carriers and / or excipients or other ingredients (if any).

[0145] In the kit of the present application, the single-stranded or double-stranded oligonucleic acid / siRNA, siRNA conjugate and pharmaceutically acceptable carrier and / or adjuvant and / or pharmaceutical composition, and / or pharmaceutically acceptable carrier and / or adjuvant can be provided in any form, such as liquid form, dried form or lyophilized form. In some embodiments, the single-stranded or double-stranded oligonucleic acid / siRNA, siRNA conjugate and pharmaceutically acceptable carrier and / or adjuvant and the pharmaceutical composition and pharmaceutically acceptable carrier and / or adjuvant are substantially pure and / or sterile. In some embodiments, sterile water can be provided in the kit of the present application.

[0146] 4. Intermediates

[0147] The present invention provides a compound represented by the following formula (III) or formula (VI) or a tautomer thereof,

[0148] R3 and R4 are each independently selected from H, C1-C6 alkyl, C1-C6 alkoxy, C2-C6 alkenyl and C2-C6 alkynyl;

[0149] X is O or S;

[0150] M is selected from H, OH, F, OCH3, OCH2CH2OCH3 and -O-alkyl;

[0151] E is a leaving group; preferably, E is MMTr or DMTr;

[0152] Q is a phosphorus-containing reactive group; preferably, Q is

[0153] G is selected from -(CR8R9)n- and -CR10=CR11-.

[0154] The above intermediates can be used to synthesize the single-stranded or double-stranded oligonucleic acid / siRNA or siRNA conjugate of the present invention.

[0155] The technical solutions of the present invention are further described below with reference to specific examples, but the present invention is not limited to these examples. Unless otherwise specified, the preparations and devices used herein are all commercially available products.

[0156] Example 1 Preparation of Compound SA000003

[0157] In this embodiment, the synthesis route of compound SA000003 is shown in the figure below:

[0158] 1.1.1 Preparation of Intermediate 9-2

[0159] Compound 9-1 (commercially available, purchased from Shanghai Zhaowei Technology Development Co., Ltd.) (8.0 g, 11.9 mmol, 1.0 equiv) was dissolved in 56 mL of acetonitrile and the temperature was lowered to 0°C. Tert-butyldimethylsilyl chloride (3.6 g, 23.8 mmol, 2.93 mL, 2.0 equiv) and imidazole (3.25 g, 47.7 mmol, 4.0 equiv) were added at this temperature. After the addition was complete, the mixture was allowed to warm to room temperature and stirred for 16 hours. TLC (developing system: petroleum ether:ethyl acetate = 1:1) confirmed the complete reaction and the disappearance of compound 9-1. The reaction mixture was cooled to 0°C, and isopropanol (2.87 g, 47.7 mmol, 3.66 mL, 4.0 equiv) was added. The mixture was stirred for 1 hour and used directly in the next step without purification.

[0160] 1.1.2 Preparation of Intermediate 9

[0161] Dodecanethiol (7.25 g, 35.8 mmol, 8.58 mL, 3.0 equiv) and trifluoroacetic acid (13.6 g, 119.0 mmol, 8.84 mL, 10.0 equiv) were added to the reaction mixture from the previous step. The reaction mixture was stirred at 0°C for 2 hours. TLC (petroleum ether:ethyl acetate = 1:1) indicated complete reaction of the starting material 9-2. N-methylimidazole (11.76 g, 143.27 mmol, 11.4 mL, 12.0 equiv) was added at 0°C. The reaction mixture was filtered and the filter cake residue was washed with acetonitrile. The reaction mixture and the washing solution were combined, and 10 mL of saturated sodium bicarbonate solution was added. The resulting solution was washed three times with a mixed solvent of n-heptane / methyl tert-butyl ether (4 / 1), and the aqueous phase was extracted twice with methyl tert-butyl ether. The organic phases were combined and washed twice with water and saturated brine, respectively. The organic phase was dried and the solvent was evaporated to obtain yellow solid compound 9 (5.0 g, 10.38 mmol, 87% yield), which was used directly in the next step without purification.

[0162] LC-MS:C 21 H 35 N5O6Si, molecular weight 481.2, 482.2 (M+H).

[0163] 1 H NMR (CDCl3, 400MHz): δppm 7.86(s,1H),5.80(d,J=6.8Hz,1H),4.49-4.47(m,1H),4.29-4.26(m,1H),4.14(d,J=2.0Hz,1H),3.98- 3.95(m,1H),3.74-3.70(m,1H),2.75-2.69(m,1H),1.27-1.18(m,6H),0.93(s,8H),0.13-0.09(m,6H).

[0164] 1.2.1 Preparation of Compound 2

[0165] Compound 1 (commercially available, purchased from Adamas) (6.0 g, 81.0 mmol, 5.36 mL, 1.0 equiv) was dissolved in pyridine. 4,4'-bismethoxytrityl chloride (32.9 g, 97.1 mmol, 1.2 equiv) was added at 0°C and stirred for 1 hour. TLC (petroleum ether / ethyl acetate = 10 / 1) confirmed the complete disappearance of compound 1. The reaction was quenched by the addition of 200 mL of saturated sodium bicarbonate solution at 0°C. 100 mL of ethyl acetate was added, and the mixture was allowed to stand for 24 hours. The organic phase was separated, and the remaining aqueous phase was extracted twice with ethyl acetate. The organic phases were combined. The organic phases were washed twice with saturated brine, dried over anhydrous sodium sulfate, and filtered. The filtrate was evaporated under reduced pressure to remove the solvent. The crude product was purified by silica gel column chromatography (developing system: petroleum ether / ethyl acetate = 20 / 1 to 10 / 1) to afford compound 2 (20.0 g, 53.1 mmol, 65.6% yield) as a colorless oil.

[0166] LC-MS:C 24 H 24 O4 molecular weight 376.1,377.2 (M+H).

[0167] 1 H NMR (CDCl3, 400MHz): δppm 7.49(d,J=7.2Hz,2H),7.39-7.20(m,8H),6.86(d,J=8.8Hz,4H),3.82(s,6H) ,3.37-3.34(m,1H),3.19-3.12(m,2H),2.82-2.80(m,1H),2.67-2.65(m,1H).

[0168] 1.2.2 Preparation of Intermediate 3

[0169] Adenine (7.11 g, 52.6 mmol, 1.1 equiv) was dissolved in N-methylformamide, and potassium carbonate (6.61 g, 47.8 mmol, 1.0 equiv) was added at 0°C. The reaction mixture was warmed to room temperature and stirred for 2 hours. A solution of compound 2 (18.0 g, 47.8 mmol, 1.0 equiv) in N,N-dimethylformamide was added, and the reaction mixture was warmed to 90°C and stirred for 16 hours. TLC (petroleum ether / ethyl acetate = 3 / 1) showed the disappearance of compound 2. The reaction mixture was cooled to 0°C and quenched by the addition of 500 mL of saturated ammonium chloride solution. 500 mL of ethyl acetate was added, and the layers were allowed to stand. The aqueous phase was extracted twice with ethyl acetate. The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the solvent in the filtrate was evaporated under reduced pressure. The crude product was used directly in the next step.

[0170] LC-MS:C29 H 29 N5O4, molecular weight 511.2, 512.2 (M+H).

[0171] 1 H NMR (CDCl3, 400MHz): δppm 7.96(d,J=11.2Hz,3H),7.43-7.38(m,3H),7.33-7.16(m,12H),6.89-6.84(m,6H),5.36(d,J=5.2Hz, 1H),4.32-4.26(m,1H),4.14-4.07(m,2H),3.73(s,8H),2.99-2.96(m,1H),2.88(s,8H),2.72(s,6H).

[0172] 1.2.3 Preparation of intermediate 4

[0173] Under nitrogen, compound 3 (23.0 g, 44.9 mmol, 1.0 equiv) and pyridine (100 mL x 2) were added to a dry round-bottom flask. The pyridine was evaporated at elevated temperature to remove water from compound 3. 160 mL of pyridine was then added to the reaction flask. The reaction mixture was cooled to 0-5°C, and trimethylsilyl chloride (19.5 g, 179 mmol, 22.8 mL, 4.00 equiv) was added. The mixture was heated to 25-30°C and stirred for 2 hours. LCMS confirmed the reaction was complete. 100 mL of water and 200 mL of concentrated aqueous ammonia were added dropwise, and the mixture was stirred for half an hour. The aqueous phase was extracted with ethyl acetate, and the organic phase was washed with 100 mL of saturated brine. The organic phase was dried, filtered, and the filtrate was concentrated. The resulting residue was purified by column chromatography (dichloromethane:methanol = 50 / 1 to 10 / 1) to afford compound 4 (9.0 g, 14.6 mmol, 33% yield) as a light yellow oil.

[0174] LC-MS:C 36 H 33 N5O5, molecular weight 615.2, 616.3 (M+H).

[0175] 1HNMR(DMSO-d6,400MHz): δppm 11.13(s,1H),8.72(s,1H),8.36(s,1H),8.06(d,J=7.2Hz,2H),7.90-7.88(m,2H),7.65-7.20(m,17H),6.90-6.87(m, 4H),5.44(d,J=5.6Hz,1H),4.49-4.45(m,1H),4.30-4.25(m,1H),4.15-4.13(m,1H),3.73(s,6H),3.08-2.87(m,2H).

[0176] 1.2.4 Preparation of Intermediate 5

[0177] Compound 4 (7.0 g, 11.3 mmol, 1.0 equiv) was dissolved in N,N-dimethylformamide, and sodium hydride (1.36 g, 34.1 mmol, 3.0 equiv) was added at 0°C. After 30 minutes, a solution of dimethyl p-toluenesulfonyloxymethylphosphonate (10.0 g, 34.1 mmol, 3.0 equiv) in N,N-dimethylformamide was added dropwise to the solution. The mixture was stirred at 25°C for 12 hours. LC-MS analysis showed the disappearance of the starting compound 4. The reaction was quenched with saturated ammonium chloride solution at 0°C, diluted with ethyl acetate, and the mixture allowed to stand for separation. The organic phase was separated, and the aqueous phase was extracted twice with ethyl acetate, dried, and the organic phase concentrated. The resulting crude yellow oil, compound 5, was used directly in the next step without purification.

[0178] LC-MS:C 39 H 40 N5O8P, molecular weight 737.2, 738.3 (M+H).

[0179] 1.2.5 Preparation of Intermediate 6

[0180] Compound 5 (8.39 g, 11.3 mmol, 1.0 equiv) was dissolved in a mixture of 48 mL of pyridine and 32 mL of water, heated to 60°C, and stirred for 16 hours. The reaction was quenched by the addition of 150 mL of saturated sodium bicarbonate solution at 0°C and diluted with 150 mL of ethyl acetate. The mixture was allowed to separate into layers, and the aqueous phase was extracted twice with 150 mL of ethyl acetate each. The organic phases were combined and washed twice with saturated brine, dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated by vacuum distillation to remove the organic solvent. The crude product was purified by silica gel column chromatography (elution system: dichloromethane / methanol = 10 / 1) to obtain Compound 6 (3.8 g, 4.61 mmol, 42% yield) as a brown solid.

[0181] LC-MS:C 38 H 38 N5O8P, molecular weight 723.2,724.3 (M+H).

[0182] 1 H NMR (CDCl3, 400MHz): δppm 8.68(s,1H),8.42(s,1H),7.97(d,J=7.6Hz,2H),7.55-7.43(m,3H),7. 36-7.29(m,2H),7.23-7.10(m,10H),6.73(d,J=8.8Hz,4H),4.58-4.53( m,1H),4.37-4.32(m,1H),3.83-3.78(m,2H),3.70-3.65(m,6H),3.52-3 .39(m,4H),3.17-3.08(m,2H),2.94-2.89(m,10H),1.29-1.17(m,14H). 31 P NMR (CDCl3, 162MHz): δppm 15.65.

[0183] 1.2.6 Preparation of Intermediate 7

[0184] The triethylamine salt of compound 6 (2.8 g, 3.39 mmol, 1.0 equiv) was dissolved in 30 mL of pyridine. The temperature was cooled to 0°C, and 2,4,6-triisopropylbenzenesulfonyl chloride (3.08 g, 10.1 mmol, 3.0 equiv) was added. The reaction solution was stirred at 0°C for 15 minutes. Methylimidazole (1.39 g, 16.9 mmol, 1.35 mL, 5.0 equiv) was added, followed by a solution of compound 9 (1.55 g, 3.22 mmol, 0.95 equiv) in pyridine (18 mL) dropwise. After the addition was complete, the reaction solution was warmed to room temperature and stirred for 16 hours. LCMS analysis showed the disappearance of compound 6. The solvent was evaporated, and the resulting residue was purified by C18 reverse-phase column chromatography to afford compound 7 (1.5 g, 1.26 mmol, 37% yield) as a yellow solid.

[0185] LC-MS:C 59 H 71 N 10 O 13 PSi, molecular weight 1186.4, 1187.5 (M+H).

[0186] 1H NMR(DMSO-d6,400MHz): δppm 12.10(s,1H),11.58(s,1H),11.12(s,1H),8.67(s,1H),8.33-8.28(m,2H),8.05-8.02(m,2H), 7.65-7.51(m,3H),7.39-7.19(m,10H),6.86-6.82(m,4H),5.89-5.86(m,1H),4.49-4.32(m,4H) ,4.25-3.90(m,6H),3.74-3.71(m,6H),3.58-3.47(m,3H),3.34(s,1H),3.26-3.24(m,3H),3.1 9-3.16(m,1H),3.03-2.99(m,1H),1.10(d,J=6.8Hz,6H),0.86-0.85(m,9H),0.07-0.06(m,9H). 31 P NMR (DMSO-d6, 162MHz): δppm 23.17, 22.79.

[0187] 1.2.7 Preparation of Intermediate 8

[0188] Compound 7 (1.5 g, 1.26 mmol, 1.0 equiv) was dissolved in tetrahydrofuran (9.0 mL), cooled to 0°C, and triethylamine trihydrofluoride (1.22 g, 7.58 mmol, 1.24 mL, 6.0 equiv) and 3 mL of tetrahydrofuran were added. The reaction mixture was warmed to room temperature and stirred for 24 hours. HPLC indicated that approximately 3% of compound 7 remained. 50 mL of ethyl acetate was added to the reaction mixture, and the resulting organic phase was washed with saturated sodium bicarbonate solution and saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate concentrated. The resulting residue was purified by preparative liquid chromatography to yield compound 8 (1.0 g, 931.9 μmol, 74% yield) as a white solid.

[0189] LC-MS:C 53 H 57 N 10 O 13 P, molecular weight 1072.3,1073.4 (M+H).

[0190] 1H NMR(DMSO-d6,400MHz): δppm 12.16-12.01(m,1H),11.70-11.57(m,1H),11.17-11.01(m,1H),8.66(s,1H ),8.33(s,1H),8.24-8.21(m,1H),8.04(d,J=7.2Hz,2H),7.65-7.48(m,3H), 7.42-7.14(m,9H),6.90-6.79(m,4H),5.93-5.85(m,1H),5.49-5.45(m,1H),4.55-4.47(m,2H),4.35-3.87(m,8H), 3.56-3.39(m,3H),3.32-3.24(m,7H),3.20-3.14(m,1H),3.04-2.96(m,1H),2.80-2.66(m,1H),1.17-1.09(m,6H). 31 P NMR (DMSO-d6, 162MHz): δppm 23.08, 22.66.

[0191] 1.2.8 Preparation of compound SA000003

[0192] Compound 8 (1.0 g, 0.93 mmol, 1.0 equiv) was dissolved in 10 mL of dichloromethane, and bis(diisopropylamino)(2-cyanoethoxy)phosphine (561.0 mg, 1.86 mmol, 2.0 equiv) and 4,5-dicyanoimidazole (165.0 mg, 1.4 mmol, 1.5 equiv) were added. The reaction mixture was allowed to react at 25°C for 2 hours. LCMS showed the disappearance of compound 8. 40 mL of saturated sodium bicarbonate solution was added in two portions. The layers were allowed to stand, and the organic phase was separated. The organic phase was washed with saturated brine and dried over anhydrous sodium sulfate. The dried organic phase was filtered and the filtrate was concentrated. The crude product was then slurried and washed three times in a mixed solvent system of n-hexane / dichloromethane (100 mL / 10 mL). The solid was collected by suction to obtain product SA000003 as a pale yellow solid.

[0193] LC-MS:C 62 H 74 N 12 O 14 P2, molecular weight 1272.4, 1273.5 (M+H).

[0194] Example 2 Preparation of Compound 035

[0195] In this example, the synthesis route of compound 035 (Ag147) is shown in the figure below:

[0196] 1.1 Preparation of intermediate 035S2M1

[0197] Compound 035S2S1 (commercially available, purchased from Shanghai Titan Technology Co., Ltd.) (2.10 g, 3.14 mmol, 1.0 equiv) and imidazole (0.64 g, 9.41 mmol, 3.0 equiv) were placed in a clean, dry reaction flask, 20 mL of acetonitrile was added, and tert-butyldimethylsilyl chloride (0.95 g, 6.27 mmol, 2.0 equiv) was slowly added at room temperature. The mixture was then stirred at room temperature for 16 hours. After the reaction, 50 mL of ethyl acetate was added to the reaction solution, which was then washed with 50 mL of saturated sodium bicarbonate solution and 50 mL of saturated brine. The organic phase was dried, filtered, and concentrated. The crude product was purified by silica gel column chromatography (gradient elution: methanol / dichloromethane = 0 / 100-5 / 100) to obtain compound 035S2M1 (2.4 g, 3.06 mmol, 97.6% yield) as a white solid. Compound 035S2M1 Molecular formula: C 42 H 53 N5O8Si, molecular weight: 783.3, LC-MS found 784.4 (M+H).

[0198] 1.2 Preparation of intermediate 035S2

[0199] Compound 035S2M1 (2.4 g, 3.06 mmol, 1.0 equiv) was placed in a clean, dry reaction flask, and DCA (3.5% V / V, 36 mL, 15.3 mmol, 5.0 equiv) and Cysteine ​​(0.45 g, 3.67 mmol, 1.2 equiv) were added. The mixture was stirred at room temperature for 16 hours. The reaction solution was washed with 30 mL of saturated sodium bicarbonate solution, and the organic phase was dried, filtered, and concentrated. The crude product was purified by silica gel column chromatography (gradient elution: methanol / dichloromethane = 0 / 100 to 5 / 100) to obtain compound 035S2 (1.21 g, 2.51 mmol, 82.3% yield) as a light yellow foam. Compound 035S2 molecular formula: C 21 H 35 N5O6S, molecular weight: 481.2, LC-MS found 482.4 (M+H).

[0200] 1.3 Preparation of intermediate 035M1

[0201] 5-Hydroxymethyl-2,2-dimethyl-1,3-dioxane (commercially available, purchased from Shanghai Titan Technology Co., Ltd.) (9.1 g, 62.2 mmol, 1.0 equiv) was placed in a clean, dry reaction flask, 100 mL of dichloromethane and 17.3 mL of triethylamine were added, and the mixture was cooled to 0°C in an ice-salt bath. p-Toluenesulfonyl chloride (12.46 g, 65.4 mmol, 1.05 equiv) was added dropwise, and stirred at room temperature for 5 hours. After the reaction, 80 mL of an aqueous solution of sodium carbonate (7.4 g) was added to the reaction solution. The aqueous phase was extracted with 80 mL of dichloromethane. The organic phases were combined and washed with 50 mL of saturated brine. The organic phases were dried, filtered, and concentrated. The resulting crude product, a light yellow solid compound 035M1 (18.7 g, 62.26 mmol, 100% yield), was directly used in the next step. Compound 035M1 Molecular formula: C 14 H 20 O5S, molecular weight: 300.1, LC-MS found 301.3 (M+H).

[0202] 1.4 Preparation of intermediate 035M2

[0203] N6-Benzoyladenine (16.38 g, 68.48 mmol, 1.1 equiv) was placed in a clean, dry reaction flask, and 200 mL of DMF was added. The reaction was cooled to 0°C in an ice-salt bath, and NaH (3.49 g, 60%, 87.16 mmol, 1.4 equiv) was slowly added. The mixture was stirred at 0°C for 0.5 hours. A solution of 035M1 (18.7 g, 62.26 mmol, 1.0 equiv) in 50 mL of DMF was added, and the temperature was raised to 60°C and stirred overnight. After cooling, 500 mL of ethyl acetate was added to the reaction mixture, and the mixture was washed three times with saturated brine (200 mL each). The organic phase was dried, filtered, and concentrated. The crude product was purified by silica gel column chromatography (gradient elution: methanol / dichloromethane = 0 / 100 to 6 / 100) to afford compound 035M2 (11.7 g, 31.85 mmol, 51.1% yield) as a white solid. Compound 035M2 Molecular formula: C 19 H 21 N5O3, molecular weight: 367.1, LC-MS found 366.3 (MH).

[0204] 1.5 Preparation of intermediate 035M3

[0205] Compound 035M2 (11.7 g, 31.85 mmol, 1.0 equiv) was placed in a clean, dry reaction flask, 125 mL of 80% acetic acid was added, and the temperature was raised to 60°C for 0.5 hours. The solvent was removed by concentration, and the crude product was purified by reverse-phase column chromatography (gradient elution: acetonitrile / water = 1 / 20-1 / 4) to obtain compound 035M3 (7.1 g, 21.69 mmol, 68.1% yield) as a white solid. Compound 035M3 molecular formula: C 16 H 17 N5O3, molecular weight: 327.1, LC-MS found 326.1 (MH).

[0206] 1.6 Preparation of intermediate 035M4

[0207] Compound 035M3 (7.1 g, 21.69 mmol, 1.0 equiv) was placed in a clean, dry reaction flask, and 100 mL of pyridine was added. 4,4'-bismethoxytrityl chloride (10.29 g, 30.4 mmol) and DMAP (0.13 g, 1.08 mmol, 0.05 equiv) were then added at room temperature. Stirring was continued at room temperature for 16 hours. After the reaction, 150 mL of ethyl acetate was added to the reaction solution, which was then washed with 150 mL of saturated sodium bicarbonate solution and 150 mL of saturated brine. The organic phase was dried, filtered, and concentrated. The resulting crude product was purified by silica gel column chromatography (gradient elution: methanol / dichloromethane = 0 / 100-2 / 100) to obtain a light yellow foam compound 035M4 (4.8 g, 7.62 mmol, 35.1% yield). Compound 035M4 molecular formula: C 37 H 35 N5O5, molecular weight: 629.2, LC-MS found 628.4 (MH).

[0208] 1.7 Preparation of intermediate 035M5

[0209] Compound 035M4 (4.8 g, 7.62 mmol, 1.0 equiv) was placed in a clean, dry reaction flask and dissolved in 50 mL of dichloromethane. Dess-Martin periodinane (6.47 g, 15.24 mmol, 2.0 equiv) was added at room temperature and stirred for 2 hours. After the reaction, the mixture was filtered and the filter cake was washed with 20 mL of dichloromethane. The combined filtrates were concentrated and the crude product was purified by silica gel column chromatography (gradient elution: methanol / dichloromethane = 0 / 100 to 5 / 100) to obtain Compound 035M5 (3.6 g, 5.74 mmol, 75.3% yield) as a foamy solid. Compound 035M5 molecular formula: C 37 H 33N5O5, molecular weight: 627.2, LC-MS found 628.3 (M+H).

[0210] 1.8 Preparation of intermediate 035M6

[0211] Under argon, 50 mL of toluene and sodium hydride (0.50 g, 12.62 mmol, 2.2 equiv) were added to a clean, dry reaction flask. After stirring for a short while, tetramethylmethylene diphosphate (3.33 g, 14.34 mmol, 2.5 equiv) was slowly added and stirred at room temperature for 0.5 hour. A 50 mL toluene solution of 035M5 (3.6 g, 5.74 mmol, 1.0 equiv) was added and stirred at room temperature for 2 hours. After the reaction, 100 mL of ethyl acetate was added to the reaction solution, which was then washed sequentially with 100 mL of saturated sodium bicarbonate solution and 100 mL of saturated brine. The organic phase was dried, filtered, and concentrated. The crude product was purified by silica gel column chromatography (gradient elution: methanol / dichloromethane = 0 / 100 to 4 / 100) to obtain Compound 035M6 (2.4 g, 3.27 mmol, 57.0% yield) as a foamy solid. Compound 035M6 Molecular Formula: C 40 H 40 N5O7P, molecular weight: 733.2, LC-MS found 734.3 (M+H).

[0212] 1.9 Preparation of intermediate 035M7

[0213] Compound 035M6 (1.1 g, 1.50 mmol, 1.0 equiv) was placed in a clean, dry reaction flask, 6 mL of pyridine and 4 mL of water were added, and the temperature was raised to 60°C for 16 hours. The solvent was removed by concentration, and the crude product was purified on a C18 column (gradient elution: acetonitrile / water = 1 / 20 - 100 / 0) to obtain compound 035M7 (0.74 g, 1.03 mmol, 68.5% yield) as a white solid. Compound 035M7 molecular formula: C 39 H 38 N5O7P, molecular weight: 719.2, LC-MS found 720.3 (M+H).

[0214] 1.10 Preparation of intermediate 035M8

[0215] Under argon, a clean, dry reaction flask was charged with 5 mL of acetonitrile, 035M7 (540 mg, 0.75 mmol, 1.0 equiv), 035S2 (343.3 mg, 0.71 mmol, 0.95 equiv), N,N,N',N'-tetramethylchloroformamidine hexafluorophosphate (505 mg, 1.8 mmol, 2.4 equiv), and N-methylimidazole (431 mg, 5.25 mmol, 7.0 equiv). The mixture was stirred at room temperature for 16 hours. After the reaction, 20 mL of ethyl acetate was added to the reaction mixture, which was then washed sequentially with 20 mL of saturated sodium bicarbonate solution and 20 mL of saturated brine. The organic phase was dried, filtered, and concentrated. The crude product was purified by silica gel column chromatography (gradient elution: methanol / dichloromethane = 0 / 100 to 5 / 100) to afford compound 035M8 (843 mg, 0.71 mmol, 94.9% yield) as a foamy solid. Compound 035M8 Molecular formula: C 60 H 71 N 10 O 12 PSi, molecular weight: 1182.4, LC-MS found 1183.5 (M+H).

[0216] 1.11 Preparation of intermediate 035M9

[0217] Compound 035M8 (843 mg, 0.71 mmol, 1.0 equiv) was placed in a clean, dry reaction flask, and 8 mL of tetrahydrofuran was added. Tetrabutylammonium fluoride (1 M, 1.4 mL, 1.4 mmol, 2.0 equiv) was then added at room temperature, followed by stirring at room temperature for 2 hours. After the reaction, 20 mL of ethyl acetate was added to the reaction solution, which was then washed sequentially with 20 mL of saturated sodium bicarbonate solution and 20 mL of saturated brine. The organic phase was dried, filtered, and concentrated. The resulting crude product was purified by silica gel column chromatography (gradient elution: methanol / dichloromethane = 0 / 100-10 / 100) and further purified using a C18 column (gradient elution: acetonitrile / water = 1 / 20-100 / 0) to obtain compound 035M9 (482 mg, 0.45 mmol, 63.2% yield) as a white foamy solid. Compound 035M9 molecular formula: C 54 H 57 N 10 O 12 P, molecular weight: 1068.3, LC-MS found 1069.4 (M+H). 1H NMR (400MHz, DMSO-d6): δ12.08(s,1H),11.63(s,1H),11.14(s,1H),8.70(d,J=2 .3Hz,1H),8.37–8.35(m,1H),8.22–8.18(m,1H),8.03(d,J=8.0Hz,2H),7.64(t, J=6.8Hz,1H),7.55(dd,J=10.5,4.6Hz,2H),7.33–7.25(m,4H),7.17(dt,J=8.7, 4.3Hz, 5H), 6.84 (d, J = 8.9Hz, 4H), 6.80–6.67 (m, 1H), 5.93–5.91 (m, 1H), 5.88–5. 79(m,1H),5.76(s,1H),5.50–5.47(m,1H),4.47–4.41(m,2H),4.33–4.29(m,1H) ,4.25–4.19(m,1H),4.08–3.94(m,2H),3.72(s,6H),3.50(dd,J=11.1,9.5Hz,2H) ,3.40(t,J=10.7Hz,1H),3.32–3.29(m,2H),3.26–3.22(m,1H),3.16–3.05(m,2H ),2.75(dt,J=13.5,6.9Hz,1H),1.25–1.23(m,1H),1.11(dd,J=10.7,4.0Hz,6H). 31 P NMR (162MHz, DMSO-d6): δ19.16 (s), 18.98 (d, J = 8.7Hz).

[0218] 1.12 Preparation of Compound 035

[0219] Compound 035M9 (471 mg, 0.44 mmol, 1.0 equiv) was placed in a clean, dry reaction flask, and 5 mL of anhydrous dichloromethane was added. 2-Cyanoethyl N,N,N',N'-tetraisopropylphosphorodiamidite (265.6 mg, 0.88 mmol, 2.0 equiv) and 4,5-dicyanimidazole (78.1 mg, 0.66 mmol, 1.5 equiv) were added under argon protection at room temperature and stirred at room temperature for 1 hour. After the reaction, 10 mL of dichloromethane was added to the reaction solution, and the solution was washed with 20 mL of saturated sodium bicarbonate solution. The organic phase was dried, filtered, and concentrated. The crude product was purified by chromatography on a C18 reverse-phase column (specification: 30 μm; Commercially available, purchased from Shanghai Boyun Biotechnology Co., Ltd.) (acetonitrile / water = 5 / 95-85 / 15) to prepare white foamy solid compound 035 (400 mg, 0.32 mmol, 71.5% yield). Compound 035 molecular formula: C 63 H 74 N 12 O 13 P2, molecular weight: 1268.5, LC-MS found 1269.5 (M+H). 1 H NMR (400MHz, DMSO-d6): δ12.07(s,1H),11.62(s,1H),11.16(s,1H),8.70(t,J=3.7Hz,1H),8.38–8.33(m,1H),8.28–8.21(m,1H),8.04( d,J=7.7Hz,2H),7.64(t,J=7.3Hz,1H),7.55(t,J=7.6Hz,2H),7.33–7.25(m,4H),7.18–7.14(m,5H),6.84–6.82(m,4H),6.79–6.69(m,1 H),5.94–5.73(m,2H),4.56–4.38(m,4H),4.31–3.98(m,3H),3.78(dd,J=12.8,6.7Hz,2H),3.71(s,6H),3.69–3.57(m,3H),3.54–3.48( m,2H),3.41(dd,J=12.6,11.3Hz,1H),3.34–3.31(m,1H),3.30–3.26(m,2H),3.16–3.06(m,2H),2.81–2.71(m,3H),1.17–1.07(m,18H). 31 P NMR (162MHz, DMSO-d6): δ150.36(d,J=9.8Hz), 149.93(d,J=9.3Hz), 19.32(d,J=15.0Hz), 19.16(d,J=8.0Hz).

[0220] Example 3 Preparation of siRNA and siRNA conjugates

[0221] Using the solid-phase phosphoramidite method, the specially modified compounds prepared in the above steps and commercially available conventionally modified monomers (purchased from Shanghai Zhaowei Technology Development Co., Ltd.) were used to link nucleoside monomers one by one in the 3'-5' direction according to the nucleotide arrangement order. The specially modified anti-off-target compound was placed in the seed region of the antisense strand (any position 4-8 from the 5' end). Each linking of a nucleoside monomer involved a four-step reaction: deprotection, coupling, capping, and oxidation or sulfurization. Synthesis conditions for the sense and antisense strands were used.

[0222] Instrument model: MerMade 12 Oligonucleotide syntheizer solid phase synthesizer, Beijing Haijing 1ml synthesis column, Si Tuofan SourceTM 15Q 4.6 / 100PE purification column.

[0223] The reagents used to synthesize siRNA conjugates were purchased from Suzhou Kelema.

[0224] The synthesis is briefly described as follows:

[0225] The single-strand synthesis reaction process is extended from 3' to 5' and is completed on a solid phase synthesizer. It includes four main reaction steps:

[0226] a. De-DMT reaction: Use dichloroacetic acid to remove the DMT protecting group on the nucleotide to obtain the 5'-hydroxyl end;

[0227] b. Coupling reaction: The protected nucleotide phosphoramidite monomer is mixed with the activator ethylthiotetrazolium. The phosphoramidite group is activated, and the 5'-hydroxyl group, still protected by DMT, undergoes a condensation reaction with the 5'-hydroxyl group attached to the solid support to form a phosphite triester.

[0228] c. Oxidation reaction: Under the action of iodine, the phosphite triester obtained in the previous condensation reaction is converted into a more stable phosphate ester (i.e., trivalent phosphorus is oxidized to pentavalent phosphorus);

[0229] d. Thio-reaction: Under the action of the thio-reagent PADS, the phosphite triester obtained in the previous condensation reaction is converted into a thiophosphate (oxidation or thio-reaction is selected according to the sequence design).

[0230] e. Capping reaction: There may be a very small number of 5'-hydroxyl groups (less than 2%) that do not participate in the condensation reaction. They are reacted with acetic anhydride and 1-methylimidazole to form acetate end-capping that cannot participate in subsequent reactions, preventing further reactions. This short fragment can be separated during purification.

[0231] Repeat the above four steps until the desired sequence is synthesized. The main chemical reaction equation is as follows

[0232] After the last nucleoside monomer is connected, the nucleic acid sequence connected to the solid phase support is cut, deprotected, purified, desalted, and then freeze-dried to obtain the sense chain and antisense chain, wherein:

[0233] The cleavage and deprotection conditions are as follows: First, prepare the aminolysis solution (a mixture of ammonia and ethanol = 3:1 to a volume of 2 mL), add the solid phase support to the above reaction flask, and shake thoroughly to evenly distribute. Aminolysis is carried out in a constant temperature water bath at 50°C for 16 hours. After 16 hours of aminolysis, the water bath is cooled to a temperature of 25°C ± 2°C, filtered through a fritted funnel, and the filtrate is collected in a round-bottom flask. The residue is rinsed with 50% ethanol aqueous solution, the filtrate is collected, concentrated on a rotary evaporator, and transferred to a glass bottle. A small sample of the crude product is sent to the analytical department for crude LC-MS analysis. The detection method is as follows: A Waters Acquity UPLC-LTQ LCMS (column: ACQUITY UPLC BEH C18) is used to detect the purity of the above sense and antisense chains and analyze the molecular weight. The measured values ​​are consistent with the theoretical values.

[0234] Purification and desalting conditions are as follows: purification using an ion exchange chromatography column and desalting using a Stovepane HiPrep™ 26 / 10 desalting gel column, followed by single-strand lyophilization. After lyophilization, the single-stranded product needs to be sampled for LC-MS analysis.

[0235] Finally, the obtained sense and antisense strands need to be annealed into double strands.

[0236] Annealing was performed as follows: the purified sense and antisense strands were dissolved in water for injection to prepare a 0.1 mg / mL to 40 mg / mL solution. The mixture was then calibrated using a thermometer Nanodrop Eight to determine the molar ratio. The mixture was heated at 90°C for 5 minutes, then cooled naturally to allow hydrogen bonding to form a double-stranded structure. Samples were collected and sent for SEC purity testing. The double-stranded sample was lyophilized.

[0237] Example 4 In vitro evaluation of on-target and off-target activities of Safe02 and Safe03

[0238] In this example, the effects of the chemical modification represented by formula (I) or (V) at the 7th nucleotide position in the 5' region of different antisense strands on the on-target activity and anti-off-target activity of siRNA were verified.

[0239] In formula (I), B may be A, G, C or U. In formula (V), B1 and B2 may be A, G, C or U, respectively.

[0240] When B, B1, and B2 are different, the structures formed are different. For the convenience of describing this embodiment, the general term Safe02 is used, which includes Agt in Table 1 or Table 2 (when B is base A, the chemically modified base A is called Agt), and its specific structure has been given above. For the convenience of describing this embodiment, the general term Safe03 is used, which includes Ag147 in Table 1 or Table 2 (when B1 is base G and B2 is base A, the chemically modified base A is called Ag147), and its specific structure has been given above.

[0241] In this example, GNA was chemically modified into base A to form Agn.

[0242] use Dual luciferase assays were used to detect on-target and off-target activity in Safe02 or Safe03 sequences. On-target activity was assessed by co-transfecting 10 ng of an on-target reporter plasmid (containing a sequence GSCM that is fully complementary to the antisense strand inserted into the 3' untranslated region of the Renilla luciferase reporter plasmid) with siRNA at three-fold dilutions ranging from 10 nM to 0.000169 nM using Lipofectamine 2000 (ThermoFisher, 11668019). Off-target activity was assessed by co-transfecting 10 ng of an off-target reporter plasmid (containing a sequence GSSM that is fully complementary to the antisense strand inserted into the 3' untranslated region of the Renilla luciferase reporter plasmid) with siRNA at three-fold dilutions ranging from 90 nM to 0.00152 nM into HEK293 cells using Lipofectamine 2000. HEK293 cells were cultured in DMEM high-glucose medium supplemented with 10% fetal bovine serum at 37°C, 5% CO2. The modified siRNA sequences are shown in Table 1. The sense strand of the naked siRNA sequence is 5'-UGACAAGAAUCCUCACAAU-3' (SEQ ID NO: 1), and the antisense strand is 5'-AUUGUGAGGAUUCUU GUCAAC-3' (SEQ ID NO: 2). Detection was performed 24 hours after transfection using a dual-luciferase assay kit (Promega, E1980). The reporter plasmids all contain firefly luciferase as an internal control. A group transfected with only the plasmid served as a control.

[0243] The Renilla luciferase signal readings in each well were normalized to the firefly luciferase (control) signal, and then compared with cells transfected with the same plasmid but not treated with siRNA to calculate the relative levels at different concentrations, and the IC 50The test results are shown in Table 2. The results show that siRNA containing Safe02 and Safe03 retains the on-target activity that is basically the same as or even better than the original sequence in vitro, and significantly reduces the off-target activity; and the on-target activity of siRNA containing Safe02 and Safe03 is significantly better than that of GNA.

[0244] Table 1

[0245] Table 2

[0246] Example 5 Evaluation of the activity of Safe02 modified conjugates using C57BL / 6-HBV (hepatitis B virus) transgenic mice

[0247] C57BL / 6-HBV transgenic mice were selected from Beijing Weitongda Biotechnology Co., Ltd.: 1.28 times the length of HBV (type A, GeneBank: AF305422.1) linearized fragment was injected into the pronucleus of C57BL / 6NCrl mouse embryos to obtain transgenic positive mice. Peripheral blood HBV copy number analysis showed that the copy number remained at 10 7 ~10 8 eq / ml founder mice were used to establish an HBV transgenic mouse strain by mating hemizygous wild-type C57BL / 6NCrl mice. The test sequences are shown in Table 3 below. The corresponding naked sequence has a sense strand of 5'-GUGUGCACUUCGCUUCACA-3' (SEQ ID NO: 3) and an antisense strand of 5'-UGUGAAGCGAAGUGCACACUU-3' (SEQ ID NO: 4).

[0248] C57BL / 6-HBV transgenic mice were divided into three groups, with five mice in each group. Blood was collected from the animals submandibularly on day -3 and centrifuged at 5000 rpm at 4°C for 10 minutes. Serum was diluted with PBS, with the dilution factor determined based on the patient's specific needs. The diluted samples were then sent to Beijing Dian Medical Testing Laboratory Co., Ltd. for HBsAg testing, and the results were back-calculated based on the dilution factor. HBsAg quantification was performed using direct electrochemiluminescence. Animals were grouped according to HBsAg levels and received a single subcutaneous injection of 3 mg / kg body weight on day 0. Blood was collected on days 7, 14, 21, and 28 to measure HBsAg levels. The anti-HBV activity of siRNA containing Safe02 was compared with that of the positive control, VIR-2218.

[0249] The HBsAg levels before and after administration are shown in Figure 1. The results showed that the compound SD004191 containing Safe02 significantly reduced HBsAg levels, which was better than the positive compound VIR-2218.

[0250] Table 3

[0251] In this embodiment, the conjugated group L96 conjugated to the siRNA is:

[0252] In this embodiment, SA51SA51SA51 represents three compounds SA51 conjugated to the 3' end of the sense chain, and the conjugate structure is For the synthesis method of SA51, please refer to invention patent application number 2024100522838.

[0253] Some embodiments of the present application are described in detail above. However, the present application is not limited to the specific details of the above embodiments. Within the technical concept of the present application, various simple modifications can be made to the technical solution of the present application, and these simple modifications all fall within the scope of protection of the present application.

[0254] It should also be noted that the various specific technical features described in some of the above embodiments can be combined in any appropriate manner unless there is any contradiction. In order to avoid unnecessary repetition, this application will not further describe various possible combinations.

[0255] In addition, the various implementation methods of the present application may be arbitrarily combined, and as long as they do not violate the concept of the present application, they should also be regarded as the contents disclosed in the present application.

Claims

1. A single-stranded or double-stranded oligonucleic acid, each strand having 15 to 35 nucleotides, wherein at least one nucleotide position other than the first and last nucleotides of the oligonucleic acid comprises a chemical modification represented by formula (I) or a tautomer modification thereof, in, B is a natural nucleobase, a modified nucleobase, a universal base or an H atom; Z is selected from O, NH and S; R1, R2, R3, R4, R5, R6 and R7 are independently selected from H, OH, halogen, NH2, C1-C6 alkyl, C1-C6 alkoxy, C2-C6 alkenyl, C2-C6 alkynyl, S-CH3, NCH3(CH3), OCH2CH2OCH3 and -O-alkylamino; X is O or S; n is 1, 2 or 3.

2. The single-stranded or double-stranded oligonucleic acid according to claim 1, wherein The oligonucleic acid is siRNA, which comprises a sense strand and an antisense strand; wherein: B is a natural nucleobase or a modified nucleobase; Z is O; R1, R2, R3, R4, R5, R6 and R7 are independently selected from H, C1-C6 alkyl, C1-C6 alkoxy, C2-C6 alkenyl, C2-C6 alkynyl; X is O or S; n is 1 or 2.

3. The single-stranded or double-stranded oligonucleic acid according to claim 1, wherein The oligonucleic acid is siRNA, which comprises a sense strand and an antisense strand; the antisense strand comprises a chemical modification represented by formula (I-1) or a tautomer modification thereof at at least one nucleotide position from the 2nd to the 8th nucleotide position in the 5' region of the antisense strand, Wherein: B1 and B2 are independently natural nucleobases, modified nucleobases, universal bases or H atoms; Z is selected from O, NH and S; R1, R2, R3, R4, R5, R6 and R7 are independently selected from H, OH, halogen, NH2, C1-C6 alkyl, C1-C6 alkoxy, C2-C6 alkenyl, C2-C6 alkynyl, S-CH3, NCH3(CH3), OCH2CH2OCH3 and -O-alkylamino; X is O or S; M1 and M2 are independently selected from H, OH, F, OCH3, OCH2CH2OCH3 and -O-alkyl; n is 1, 2 or 3.

4. The single-stranded or double-stranded oligonucleic acid according to claim 3, wherein B1 and B2 are each independently a natural nucleobase or a modified nucleobase; Z is O; R1, R2, R3, R4, R5, R6 and R7 are each independently H; X is O or S; M1 and M2 are independently selected from H, OH, F and OCH3; n is 1 or 2.

5. The single-stranded or double-stranded oligonucleic acid according to claim 1, wherein The oligonucleic acid is siRNA, which comprises a sense strand and an antisense strand; the antisense strand comprises a chemical modification represented by formula (I-2) or a tautomer modification thereof at at least one nucleotide position from the 2nd to the 8th nucleotide position in the 5' region of the antisense strand, wherein B1 and B2 are independently a natural nucleobase, a modified nucleobase, a universal base or an H atom; R3 and R4 are independently selected from H, OH, halogen, NH2, C1-C6 alkyl, C1-C6 alkoxy, C2-C6 alkenyl, C2-C6 alkynyl, S-CH3, NCH3(CH3), OCH2CH2OCH3 and -O-alkylamino; X is O or S; M is selected from H, OH, F, OCH3, OCH2CH2OCH3 and -O-alkyl.

6. The single-stranded or double-stranded oligonucleic acid according to claim 5, wherein B1 and B2 are each independently a natural nucleobase or a modified nucleobase; R3 and R4 are each independently H; X is O; M is selected from H, OH, F and OCH3.

7. The single-stranded or double-stranded oligonucleic acid according to claim 1, wherein The oligonucleic acid is siRNA, comprising a sense strand and an antisense strand; the antisense strand comprises a chemical modification represented by formula (I-3), (I-4) or (I-5) or a tautomer modification thereof at at least one nucleotide position from the 2nd to the 8th nucleotide position in the 5' region of the antisense strand, wherein B1 and B2 are independently a natural nucleobase, a modified nucleobase, a universal base or an H atom; R3 and R4 are independently selected from H, OH, halogen, NH2, C1-C6 alkyl, C1-C6 alkoxy, C2-C6 alkenyl, C2-C6 alkynyl, S-CH3, NCH3(CH3), OCH2CH2OCH3 and -O-alkylamino; X is O or S; M is selected from OH, F, OCH3, OCH2CH2OCH3 and -O-alkyl; M is selected from OH, F and OCH3.

8. The single-stranded or double-stranded oligonucleic acid according to claim 7, wherein B1 and B2 are each independently a natural nucleobase or a modified nucleobase; X is O; R3 and R4 are each independently H; M is selected from F and OCH3.

9. The single-stranded or double-stranded oligonucleic acid according to claim 1, wherein the chemical modification is selected from any of the following structures:

10. The single-stranded or double-stranded oligonucleic acid according to any one of claims 1 to 9, wherein The oligonucleic acid is siRNA, which comprises a sense strand and an antisense strand; the chemical modification or its tautomer modifies at least one nucleotide position from position 5 to position 8 in the 5' region of the antisense strand.

11. The single-stranded or double-stranded oligonucleic acid according to claim 10, wherein The chemical modification or tautomer thereof modifies at least one nucleotide position in positions 6 and 7 of the 5' region of the antisense strand.

12. The single-stranded or double-stranded oligonucleic acid according to claim 10, wherein In addition to the chemically modified or tautomer-modified nucleotides, at least one of the sense strand and / or antisense strand further comprises at least one other modified nucleotide; Preferably, in addition to the nucleotides comprising the chemical modification or tautomer modification thereof, the remaining nucleotides in the sense strand and / or antisense strand are other modified nucleotides.

13. The single-stranded or double-stranded oligonucleic acid according to claim 12, wherein The other modified nucleotides are independently selected from the group consisting of 2'-fluoro modified nucleotides, 2'-alkoxy modified nucleotides, 2'-substituted alkoxy modified nucleotides, 2'-alkyl modified nucleotides, 2'-substituted alkyl modified nucleotides, 2'-deoxy nucleotides, 2'-amino modified nucleotides and 2'-substituted amino modified nucleotides; Preferably, the other modified nucleotides are independently selected from the group consisting of 2'-F modified nucleotides, 2'-O-CH3 modified nucleotides, 2'-O-CH2-CH2-O-CH3 modified nucleotides, 2'-O-CH2-CH=CH2 modified nucleotides, 2'-CH2-CH2-CH=CH2 modified nucleotides and 2'-deoxynucleotides; More preferably, the other modified nucleotides are independently selected from: 2'-F modified nucleotides and 2'-O-CH3 modified nucleotides.

14. The single-stranded or double-stranded oligonucleic acid according to any one of claims 10 to 13, wherein Has at least one of the following characteristics: (i) the antisense strand comprises 2, 3, 4, 5 or 6 2'-fluoro modifications; (ii) the antisense strand comprises 1, 2, 3 or 4 phosphorothioate internucleotide linkages; (iii) the sense strand contains 2, 3, 4 or 5 2'-fluoro modifications; (iv) the sense strand contains 1, 2, 3 or 4 phosphorothioate internucleotide linkages; (v) the dsRNA comprises at least four 2'-fluoro modifications; (vi) The siRNA comprises a duplex region of 12-40 nucleotide pairs in length.

15. A siRNA conjugate comprising siRNA and a conjugated group conjugated to the siRNA, wherein: The siRNA is a single-stranded or double-stranded oligonucleotide according to any one of claims 1 to 14. 16 . A pharmaceutical composition comprising the single-stranded or double-stranded oligonucleic acid according to any one of claims 1 to 14 or the siRNA conjugate according to claim 15 , and a pharmaceutically acceptable carrier.

17. A kit comprising the single-stranded or double-stranded oligonucleic acid according to any one of 1 to 14, or the siRNA conjugate according to claim 15, or the pharmaceutical composition according to claim 16.

18. A compound represented by the following formula (III) or its tautomer, in, B1 and B2 are each independently a natural nucleobase, a modified nucleobase, a universal base or an H atom; R3 and R4 are independently selected from H, C1-C6 alkyl, C1-C6 alkoxy, C2-C6 alkenyl and C2-C6 alkynyl; X is O or S; M is selected from H, OH, F, OCH3, OCH2CH2OCH3 and -O-alkyl; E is a leaving group; Q is a phosphorus-containing reactive group.

19. The compound represented by formula (III) according to claim 18 or its tautomer, which is selected from any of the following structures:

20. A method for preparing the single-stranded or double-stranded oligonucleotide according to any one of claims 1 to 14 or the siRNA conjugate according to claim 15, comprising the following steps: 1) synthesizing the compound represented by formula (III) or its tautomer according to any one of claims 18 to 19; 2) synthesizing the oligonucleic acid or siRNA conjugate using the compound of step 1) or its tautomer.

21. A single-stranded or double-stranded oligonucleic acid, each strand having 15 to 60 nucleotides, wherein at least one nucleotide position other than the first and last nucleotides of the oligonucleic acid comprises a chemical modification represented by formula (V) or a tautomer modification thereof, in, B1 and B2 are independently selected from natural nucleobases, modified nucleobases, universal bases or H atoms; Z is selected from O, NH and S; G is selected from -(CR8R9)m- and -CR10=CR11-; R1, R2, R5, R6, R7, R8, R9, R8, R9, R10 and R11 are independently selected from H, OH, halogen, NH2, C1-C6 alkyl, C1-C6 alkoxy, C2-C6 alkenyl, C2-C6 alkynyl, S-CH3, NCH3(CH3), OCH2CH2OCH3 and -O-alkylamino; M1 and M2 are independently selected from H, OH, F and OCH3; n is 1, 2 or 3; m is 1, 2, 3, 4 or 5; y is 0, 1, 2 or 3; X is O or S.

22. The single-stranded or double-stranded oligonucleic acid according to claim 21, wherein The oligonucleic acid is siRNA, which comprises a sense strand and an antisense strand, wherein at least one nucleotide position of the antisense strand comprises a chemical modification represented by formula (V-2), (V-3), (V-4) or (V-5) or a tautomer modification thereof, Wherein: B1 and B2 are independently natural nucleobases, modified nucleobases, universal bases or H atoms; M1 is selected from H, OH, F and OCH 3； X is O or S.

23. The single-stranded or double-stranded oligonucleic acid according to claim 22, wherein B1 and B2 are each independently a natural nucleobase, a modified or an unmodified nucleobase; M1 is selected from F and OCH3; X is O or S.

24. The single-stranded or double-stranded oligonucleic acid according to claim 22, wherein the chemical modification is selected from any of the following structures:

25. The single-stranded or double-stranded oligonucleotide according to any one of claims 21 to 24, wherein The antisense strand comprises the chemical modification or a tautomer modification thereof at at least one nucleotide position among positions 5 to 8 in the 5' region thereof.

26. The single-stranded or double-stranded oligonucleic acid according to claim 25, wherein The antisense strand comprises the chemical modification or a tautomer modification thereof at at least one nucleotide position in positions 6 and 7 of the 5' region thereof.

27. The single-stranded or double-stranded oligonucleic acid according to claim 25 or 26, wherein In addition to the chemically modified or tautomer-modified nucleotides, at least one of the sense strand and / or antisense strand further comprises at least one other modified nucleotide; Preferably, in addition to the nucleotides comprising the chemical modification or tautomer modification thereof, the remaining nucleotides in the sense strand and / or antisense strand are other modified nucleotides.

28. The single-stranded or double-stranded oligonucleic acid according to claim 27, wherein The other modified nucleotides are independently selected from the group consisting of 2'-fluoro modified nucleotides, 2'-alkoxy modified nucleotides, 2'-substituted alkoxy modified nucleotides, 2'-alkyl modified nucleotides, 2'-substituted alkyl modified nucleotides, 2'-deoxy nucleotides, 2'-amino modified nucleotides and 2'-substituted amino modified nucleotides; Preferably, the other modified nucleotides are independently selected from the group consisting of 2'-F modified nucleotides, 2'-O-CH3 modified nucleotides, 2'-O-CH2-CH2-O-CH3 modified nucleotides, 2'-O-CH2-CH=CH2 modified nucleotides, 2'-CH2-CH2-CH=CH2 modified nucleotides and 2'-deoxynucleotides; More preferably, the other modified nucleotides are independently selected from: 2'-F modified nucleotides and 2'-O-CH3 modified nucleotides.

29. The single-stranded or double-stranded oligonucleic acid according to claim 27 or 28, wherein Has at least one of the following characteristics: (i) the antisense strand comprises 2, 3, 4, 5 or 6 2'-fluoro modifications; (ii) the antisense strand comprises 1, 2, 3 or 4 phosphorothioate internucleotide linkages; (iii) the sense strand contains 2, 3, 4 or 5 2'-fluoro modifications; (iv) the sense strand contains 1, 2, 3 or 4 phosphorothioate internucleotide linkages; (v) the dsRNA comprises at least four 2'-fluoro modifications; (vi) The siRNA comprises a duplex region of 12-40 nucleotide pairs in length.

30. A siRNA conjugate comprising siRNA and a conjugated group conjugated to the siRNA, wherein: The siRNA is a single-stranded or double-stranded oligonucleotide according to any one of claims 21 to 29.

31. A pharmaceutical composition comprising the single-stranded or double-stranded oligonucleic acid according to any one of claims 21 to 29 or the siRNA conjugate according to claim 30, and a pharmaceutically acceptable carrier.

32. A kit comprising the single-stranded or double-stranded oligonucleic acid according to any one of 21-29, or the siRNA conjugate according to claim 30, or the pharmaceutical composition according to claim 31.

33. A compound represented by the following formula (VI) or its tautomer, in, B1 and B2 are each independently a natural nucleobase, a modified nucleobase, a universal base or an H atom; G is selected from -(CR8R9)n- and -CR10=CR11-; X is O or S; M is selected from H, OH, F, OCH3, OCH2CH2OCH3 and -O-alkyl; E is a leaving group; Q is a phosphorus-containing reactive group.

34. A compound or tautomer thereof of formula (VI) according to claim 33, selected from any of the following structures:

35. A method for preparing the single-stranded or double-stranded oligonucleotide according to any one of claims 21 to 29 or the siRNA conjugate according to claim 30, comprising the following steps: 1) synthesizing the compound represented by formula (VI) or its tautomer according to any one of claims 33-34; 2) synthesizing the oligonucleic acid or siRNA conjugate using the compound of step 1) or its tautomer.

36. A method for inhibiting a target gene in a cell, characterized in that: The method comprises the step of introducing the single-stranded or double-stranded oligonucleotide according to any one of claims 1 to 14 or claims 21 to 29, or the siRNA conjugate according to claim 15 or claim 30 into the cell.

37. A method for modifying positions 2-8 of the antisense strand of siRNA to reduce off-target activity, characterized in that: The chemical modification or tautomer modification thereof according to any one of claims 1 to 9, or the chemical modification or tautomer modification thereof according to any one of claims 21 to 24 is introduced into positions 2-8 of the antisense strand of the siRNA molecule.

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