Modified nucleotide monomers and uses thereof

Abstract

The present application relates to a kind of nucleotide monomer compounds shown in formula (I) and its application in nucleic acid drug molecules.Introducing the nucleotide monomer of the present application in siRNA antisense strand seed region, the potential off-target effect of antisense strand seed region can be significantly reduced, and the activity of siRNA can be maintained or improved, so that the target gene mRNA is more selectively silenced, the safety window of siRNA drug is expanded, the drug property of siRNA can be improved, so that the related diseases caused by target gene mRNA can be more effectively treated.

Description

Modified nucleotide monomers and their uses Technical Field

[0001] The present invention relates to the field of biochemistry, and in particular to a modified nucleotide monomer and application thereof in siRNA molecules. Background Art

[0002] In recent years, considerable progress has been made in the development of siRNA drugs. siRNA is well known as an active pharmaceutical ingredient, and its corresponding siRNA mechanism of action is characterized by an endonuclease complex-mediated RNA interference. At the genetic level, siRNA can treat nearly all diseases, including tumors, infectious diseases, and genetic disorders, making it a hot area of ​​biomedical research. In theory, siRNA therapy can target target genes in any pathogenic genome, with potent and long-lasting therapeutic effects. It can significantly reduce dosing frequency and improve patient compliance, thereby achieving better therapeutic outcomes.

[0003] Studies have shown that a major factor in RNAi off-target effects is that some fragments of the siRNA sequence that binds to the target mRNA may function like microRNA (miRNA). They can bind to the 3'-untranscribed region (3'-UTR) of other mRNAs and inhibit the translation and stability of mRNA. MiRNA mainly recognizes target genes through base pairing between the seed region (position 2-8 at the 5'-end) and the target mRNA. Studies have also shown that the off-target effects caused by siRNA mainly come from the base complementarity between the antisense chain seed region of siRNA and one or more mRNAs. Therefore, the sequence of the antisense chain seed region may affect the expression of multiple genes and become a major factor in the side effects of RNAi drugs.

[0004] To effectively address off-target effects in the antisense strand seed region, Alnylam modified the nucleotide monomers located in the seed region and discovered a new class of nucleoside monomers (glycerol nucleic acid, or GNA). Introduced into the siRNA antisense strand seed region, this new class of nucleoside monomers can reduce RNAi off-target effects and improve drug safety. Therefore, off-target effects in the antisense strand seed region can be effectively addressed by modifying the nucleoside monomers within this region.

[0005] The present invention introduces nucleotide monomers into specific locations in the seed region of the siRNA antisense strand. This modification of the siRNA sequence significantly reduces off-target effects while preserving the silencing effect of the siRNA target gene, thereby expanding the safety window of the siRNA drug molecule. Therefore, this invention is of great value to the development and clinical application of siRNA drugs. Summary of the Invention

[0006] The present invention first provides a nucleotide monomer compound represented by Formula I, or a stereoisomer thereof, or a deuterated compound thereof, or a fluorinated compound thereof, or a usable salt thereof:

[0007] in:

[0008] Bx is selected from

[0009] R is selected from hydrogen or a hydroxy protecting group;

[0010] m is 1, 2 or 3; preferably, m is 1;

[0011] n is 1, 2 or 3; preferably, n is 1;

[0012] Z is selected from a hydroxyl group, a phosphate group, a phosphorothioate group or a phosphoramidite group.

[0013] Furthermore, the compound represented by formula I is represented by formula II:

[0014] in,

[0015] Bx is selected from

[0016] R is selected from hydrogen or a hydroxy protecting group;

[0017] m is 1, 2 or 3; preferably, m is 1;

[0018] n is 1, 2 or 3; preferably, n is 1;

[0019] Z is selected from a hydroxyl group, a phosphate group, a phosphorothioate group or a phosphoramidite group.

[0020] Preferably,

[0021] R is selected from hydrogen, 4,4'-bismethoxytrityl or 4-methoxytrityl;

[0022] Z is selected from hydroxyl or

[0023] In some specific embodiments of the present invention, the compound is specifically:

[0024] The present invention also provides use of any of the above-mentioned nucleotide monomer compounds as an intermediate in the preparation of oligonucleotides; preferably, the oligonucleotide is siRNA.

[0025] Preferably, the present invention provides the use of any of the above-mentioned nucleotide monomer compounds as an intermediate in the preparation of an siRNA antisense strand seed region. More specifically, the siRNA antisense strand seed region is the 2nd to 8th nucleotides from the 5' end of the siRNA antisense strand.

[0026] The present invention also provides an siRNA comprising a sense strand and an antisense strand; the sense strand and the antisense strand each comprise 15 to 45 modified or unmodified nucleotides, and the sense strand and the antisense strand are partially complementary to form a double-stranded region; wherein the seed region of the antisense strand contains at least one nucleotide having a structure shown in Formula V, and Site covalently linked to the rest of the siRNA:

[0027] in,

[0028] Bx is selected from

[0029] m is 1, 2 or 3; preferably, m is 1;

[0030] n is 1, 2 or 3; preferably, n is 1;

[0031] X 1 、X 2 are independently selected from O or S.

[0032] Furthermore, the nucleotides of the structure represented by Formula V in the siRNA are represented by Formula VI:

[0033] in,

[0034] Bx is selected from

[0035] m is 1, 2 or 3; preferably, m is 1;

[0036] n is 1, 2 or 3; preferably, n is 1;

[0037] X 1 、X 2 are independently selected from O or S.

[0038] In some specific embodiments of the present invention, the nucleotide structure of the structure represented by Formula V or Formula VI in the siRNA is specifically:

[0039] In some embodiments, at least one nucleotide in the sense strand and / or the antisense strand is a nucleotide having a structure shown in Formula V or Formula VI.

[0040] In some embodiments, in the direction from the 5' end to the 3' end, the 5th, 6th, 7th, and 8th positions of the seed region of the antisense strand contain at least one nucleotide having the structure shown in Formula V or Formula VI.

[0041] In some embodiments, from the 5' end to the 3' end, the 5th, 6th, and 7th positions of the seed region of the antisense strand contain at least one nucleotide having the structure shown in Formula V or Formula VI.

[0042] In some embodiments, in the direction from the 5' end to the 3' end, the 5th position of the seed region of the antisense strand is a nucleotide having a structure shown in Formula V or Formula VI.

[0043] In some embodiments, in the direction from the 5' end to the 3' end, the 6th position of the seed region of the antisense strand is a nucleotide having a structure shown in Formula V or Formula VI.

[0044] In some embodiments, in the direction from the 5' end to the 3' end, the 7th position of the seed region of the antisense strand is a nucleotide having a structure shown in Formula V or Formula VI.

[0045] In some embodiments, in the direction from the 5' end to the 3' end, the 8th position of the seed region of the antisense strand is a nucleotide having a structure shown in Formula V or Formula VI.

[0046] In some embodiments, in the direction from the 5' end to the 3' end, the 5th position of the seed region of the antisense strand is a nucleotide having a structure represented by Formula V or Formula VI;

[0047] The nucleotides at positions 2, 6, 14 and 16 of the antisense strand are each independently a 2'-fluoro-modified nucleotide.

[0048] In some embodiments, in the direction from the 5' end to the 3' end, the 6th position of the seed region of the antisense strand is a nucleotide having a structure represented by Formula V or Formula VI;

[0049] The nucleotides at positions 2, 14 and 16 of the antisense strand are each independently a 2'-fluoro-modified nucleotide.

[0050] In some embodiments, in the direction from the 5' end to the 3' end, the 7th position of the seed region of the antisense strand is a nucleotide having a structure represented by Formula V or Formula VI;

[0051] The nucleotides at positions 2, 6, 14 and 16 of the antisense strand are each independently a 2'-fluoro-modified nucleotide.

[0052] In some embodiments, in the direction from the 5' end to the 3' end, the 8th position of the seed region of the antisense strand is a nucleotide having a structure represented by Formula V or Formula VI;

[0053] The nucleotides at positions 2, 6, 14 and 16 of the antisense strand are each independently a 2'-fluoro-modified nucleotide.

[0054] In some embodiments, the remaining nucleotides of the antisense strand are 2-methoxy-modified nucleotides.

[0055] In some embodiments, at least one phosphodiester group in the sense strand and / or antisense strand is a phosphodiester group with a modifying group. The modifying group allows the siRNA to have increased stability in a biological sample or environment. In some embodiments, the phosphodiester group with a modifying group is a thiophosphate diester group.

[0056] In some embodiments, the phosphodiester group having a modifying group is present in at least one position selected from the group consisting of:

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

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

[0059] between the first and second nucleotides at the 5' end of the antisense strand;

[0060] between the second and third nucleotides at the 5' end of the antisense strand;

[0061] The 5' end of the antisense strand is between the 21st nucleotide and the 22nd nucleotide.

[0062] In some embodiments, the sense strand and / or the antisense strand includes a plurality of phosphodiester groups having modifying groups.

[0063] In some embodiments, the sense strand and / or antisense strand comprises a plurality of phosphodiester groups with modifying groups, wherein the phosphorothioate diester groups are present in:

[0064] between the first and second nucleotides at the 5' end of the sense strand;

[0065] between the second and third nucleotides at the 5' end of the sense strand;

[0066] between the first and second nucleotides at the 5' end of the antisense strand;

[0067] between the second and third nucleotides at the 5' end of the antisense strand;

[0068] The 5' end of the antisense strand is between the 21st nucleotide and the 22nd nucleotide.

[0069] In some embodiments, the sense strand is selected from a nucleotide sequence (5'-3') comprising the following formula:

[0070] mN*mN*mNmNmNmN / fN / mN / fN / fN / fN / mNmNmNmNmNmNmNmNmNmNmN,

[0071] In some embodiments, the antisense strand is selected from a nucleotide sequence (5'-3') comprising the following formula:

[0072] mN* / fN / *mNmN / V / fN / mNmNmNmNmNmNmN / fN / mN / fN / mNmNmNmNmN*mN*mN;

[0073] or

[0074] mN* / fN / *mNmNmN / V / mNmNmNmNmNmNmN / fN / mN / fN / mNmNmNmNmN*mN*mN;

[0075] Or mN* / fN / *mNmNmN / fN / V / mNmNmNmNmNmN / fN / mN / fN / mNmNmNmNmN*mN*mN.

[0076] In the above-mentioned sense strand and antisense strand, mN represents any 2'-methoxy-modified nucleoside, such as 2'-methoxy-modified C, G, U, A, and T; fN represents any 2'-fluoro-modified nucleoside, such as 2'-fluoro-modified C, G, U, A, and T;

[0077] V represents a nucleotide having a structure represented by formula V of the present invention.

[0078] “*” indicates that the connection between the two adjacent nucleotides is a phosphorothioate connection;

[0079] The present invention also provides a pharmaceutical composition comprising any of the above siRNAs and a pharmaceutically acceptable carrier.

[0080] The present invention also provides a siRNA conjugate, which is formed by conjugating any of the above siRNAs and a conjugated molecule.

[0081] The present invention also provides use of any of the above-mentioned siRNAs, and / or the above-mentioned pharmaceutical compositions, and / or the above-mentioned siRNA conjugates in the preparation of drugs.

[0082] In another aspect, the present invention further provides a method for inhibiting the expression level of a target gene in a cell, comprising contacting an effective amount of the siRNA, pharmaceutical composition and / or siRNA conjugate of the present invention with the cell.

[0083] The modified nucleotides of the present invention can improve the selectivity of RNAi drug molecules for target gene silencing and reduce off-target effects.

[0084] After the nucleotide monomers of the present invention are introduced into the antisense chain seed region of siRNA, the potential off-target effects of the antisense chain seed region can be significantly reduced, and the activity of siRNA can be maintained or enhanced, thereby achieving more selective silencing of the target gene mRNA, expanding the safety window of siRNA drugs, and improving the drugability of siRNA, thereby more effectively treating related diseases caused by target gene mRNA.

[0085] The modified nucleotides described herein can improve the drugability of siRNA drug molecules. In some embodiments, the modified nucleotides described herein can enhance the effectiveness of siRNA in degrading target genes. Therefore, the modified nucleotides described herein can enhance the pharmacological effects of siRNA silencing.

[0086] The siRNA conjugate provided by the present invention is formed by conjugating siRNA and a conjugation molecule, and comprises the siRNA of the present invention and a conjugation group conjugated to the siRNA, wherein the conjugation group comprises a linker and a pharmaceutically acceptable targeting group and / or a delivery auxiliary group, and the siRNA, the linker and the targeting group or the delivery auxiliary group are sequentially covalently or non-covalently linked, each of the targeting groups is selected from a ligand capable of binding to a cell surface receptor, and each of the delivery auxiliary groups is selected from a group capable of increasing the biocompatibility of the siRNA conjugate in the target organ or tissue for delivery.

[0087] In some embodiments, the siRNA drug comprising the modified nucleotides of the present invention is double-stranded. With respect to double-stranded siRNA drugs, the lengths of the sense and antisense strands of the siRNA drug are independently 15-45 nucleotides. In some embodiments, the double-stranded siRNA drug comprises a sense strand and an antisense strand that are at least partially complementary to each other (at least 70% complementary). The antisense strand comprises a region having a sequence that is perfectly complementary (100% complementary) or at least partially complementary (at least 85% complementary) to the sequence in the target mRNA. The sense and antisense strands can be the same length or different lengths.

[0088] In some embodiments, the sense strand is about 19 nucleotides in length, while the antisense strand is about 21 nucleotides in length. In some embodiments, the sense strand is about 21 nucleotides in length, while the antisense strand is about 23 nucleotides in length. The perfect or partially complementary region between the sense strand and the antisense strand is typically 15-25 nucleotides in length and is located at or near the 5'-end of the antisense strand (e.g., the region is 1, 2, 3, or 4 nucleotides away from the 5'-end of the antisense strand that is imperfect or partially complementary).

[0089] For double-stranded siRNA drugs, if there are other sense strand nucleotides, they can be identical or different from the corresponding sequence in the target mRNA. If there are other antisense strand nucleotides, they can be complementary or non-complementary to the other nucleotides (if any) of the corresponding sense strand.

[0090] In some embodiments, the sense strand and antisense strand of the double-stranded siRNA drug comprising the modified nucleotides of the present invention contain the same number of nucleotides. In some embodiments, the sense strand and antisense strand of the siRNA drug of the present invention contain different numbers of nucleotides.

[0091] In some embodiments, both the sense and antisense strands contain 1 to 4 phosphorothioate linkages.

[0092] In some embodiments, in the siRNA of the present invention, each nucleotide is a modified or unmodified nucleotide. In the context of the present disclosure, the term "modified nucleotide" used refers to a nucleotide or nucleotide analog formed by replacing the 2' hydroxyl group of the ribose group of the nucleotide with other groups, or a nucleotide in which the base on the nucleotide is a modified base. The modified nucleotide will not cause the function of the siRNA to regulate gene expression to be significantly weakened or lost. For example, the modified nucleotide disclosed in JK Watts, GF Deleavey, and MJ Damha, Chemically modified siRNA: tools and applications. Drug Discov. Today, 2008, 13 (19-20): 842-55 can be selected.

[0093] In some embodiments, the siRNA drug comprising the modified nucleotide inhibits the expression of the target mRNA in a cell, tissue or body. In some embodiments, a therapeutically effective amount of the siRNA drug comprising the modified nucleotide of the present invention is administered into the body, thereby inhibiting the expression of the target mRNA in the body.

[0094] In some embodiments, the siRNA drug is used to treat, prevent or control clinical manifestations related to the expression of the target mRNA. In some embodiments, a therapeutically or prophylactically effective amount of one or more siRNA drugs is administered to a subject in need of such treatment, prevention or control.

[0095] The siRNA drug comprising the modified nucleotides of the present invention and the composition comprising the siRNA drug of the present invention can be delivered to cells, tumors, tissues or objects using oligonucleotide delivery technologies known in the art. In general, any method suitable for delivering nucleic acid molecules (in vitro or in vivo) in the art can be applied to the siRNA drug comprising one or more modified nucleotides of the present invention.

[0096] In some embodiments where the siRNA drug is double-stranded, a targeting ligand or targeting group, a linker or a delivery vehicle is covalently linked to the sense strand to form a conjugate. In some embodiments, the targeting ligand, linker and / or delivery vehicle is directly or indirectly linked to the 3' or 5' end of the sense strand via a linker.

[0097] In some embodiments, the targeting ligand or targeting group contains two to four terminal N-acetylgalactosamine (GalNAc) groups, which are linked to a siRNA drug comprising one or more modified nucleotides. It is known that triantennary (GalNAc) structures bind to ASGPR with greater affinity than diantennary or monoantennary structures (Connolly et al., J. Biol. Chem., 1982, 257, 939-945).

[0098] Definitions of terms used in the present invention: Unless otherwise stated, the initial definitions provided for groups or terms in this document apply to the groups or terms throughout the specification; for terms that are not specifically defined herein, they should be given the meaning that a person skilled in the art would give them based on the disclosure and context.

[0099] In the present invention, unless otherwise specified, capital letters C, G, U, A, and T represent the base composition of nucleotides, including modified and unmodified nucleotides; m indicates that the nucleotide adjacent to the right of the identifier m is a 2'-methoxy nucleotide; f indicates that the nucleotide adjacent to the right of the identifier f is a 2'-fluoro nucleotide; d indicates that the nucleotide adjacent to the right of the identifier d is a 2'-deoxy nucleotide; gn indicates that the nucleotide adjacent to the right of the identifier gn is a glycerol nucleotide (GNA); the identifier * indicates that the two nucleotides adjacent to the left and right of the identifier * (or between the nucleotide and the linker-targeting ligand portion) are connected by a thiophosphate group; eVP indicates that the nucleotide adjacent to the right is a (E)-vinyl phosphate-modified nucleotide; and iab indicates an inverted abasic residue.

[0100] In the present invention, "oligonucleotide" refers to a polymer of linked nucleosides, each of which can be independently modified or unmodified, and comprises an oligonucleotide sequence of about 10-50 single-stranded nucleotides or double-stranded nucleotide base pairs. In some embodiments, the oligonucleotide has a core base sequence that is at least partially complementary to the core sequence of a target gene expressed in a cell. In some embodiments, the oligonucleotide can regulate the expression of the corresponding target gene after delivery to a cell expressing the gene. Target gene expression can be regulated in vitro or in vivo. "Oligonucleotide" includes, but is not limited to, single-stranded antisense oligonucleotides, short interfering RNA (siRNA), double-stranded RNA (dsRNA), microRNA (miRNA), short hairpin RNA (shRNA), ribozymes, interfering RNA molecules, and dicer enzyme substrates.

[0101] In the present invention, "siRNA" refers to an oligonucleotide molecule containing RNA or RNA-like (eg, chemically modified RNA) that can reduce or inhibit the translation of messenger RNA (mRNA) in a sequence-specific manner.

[0102] In the present invention, the "seed region" refers to a specific region from the second base to the eighth base from the 5'-end in the oligonucleotide sequence.

[0103] siRNA can act through an RNA interference mechanism (e.g., by inducing mRNA degradation through interaction with the mRNA interference pathway mechanism (RNA-induced silencing complex RISC) of mammalian cells), or other arbitrary mechanisms or pathways. Although it is believed that the term siRNA drug used in the present invention primarily acts through an RNA interference mechanism, the siRNA drug is not limited to or confined to any particular pathway or mechanism of action. siRNA drugs include, but are not limited to, single-stranded antisense oligonucleotides, short interfering RNA (siRNA), double-stranded RNA (dsRNA), microRNA (miRNA), short hairpin RNA (shRNA), and dicer enzyme substrates. The siRNA drug of the present invention is composed of an oligonucleotide chain that is at least partially complementary to the target mRNA. In some embodiments, the siRNA drug of the present invention is double-stranded and consists of an antisense chain and a sense chain that is at least partially complementary to the antisense chain.

[0104] The terms "silencing," "reducing," "inhibiting," "downregulating," or "knockdown" refer to a decrease or reduction in the expression of a given gene when the siRNA drug molecules of the present invention are used to treat cells, tissues, organs, or animals for direct administration, compared to administration to cells, tissues, organs, or animals that have not been treated in this manner.

[0105] The term "sequence" or "nucleotide sequence" refers to the order or sequence of nucleobases or nucleotides, expressed in alphabetical order using standard nucleotide nomenclature.

[0106] The term "complementary" is used to describe the relationship between a first nucleotide sequence (e.g., a siRNA drug sense strand or a target mRNA) and a second nucleotide sequence (e.g., a single-stranded antisense oligonucleotide or a double-stranded siRNA drug antisense strand), and refers to the ability of an oligonucleotide or polynucleotide comprising the first nucleotide sequence to hybridize with an oligonucleotide or polynucleotide comprising the second nucleotide sequence under certain conditions (under mammalian physiological conditions or similar in vitro conditions) to form base pairing and form a duplex or double helical structure. Complementary sequences contain Watson-Crick base pairing or non-Watson-Crick base pairing, and contain natural or modified nucleotides or nucleotide analogs to at least the extent that they meet the above-mentioned hybridization requirements. For example, for the purpose of determining identity or complementarity, monomers a and Af are complementary to U (or T) and are equivalent to A.

[0107] The term "sense strand" refers to the strand on the RNA molecule that carries the nucleotide sequence encoding protein amino acid information, which is called the sense strand, also known as the coding strand, sense strand or positive strand, and the other nucleotide sequence that is complementary to it is the antisense strand.

[0108] The term "antisense strand" refers to a nucleotide sequence in the mRNA expressed by the target gene that is substantially reverse complementary or essentially reverse complementary to a nucleotide sequence having the same length as the antisense strand.

[0109] The term "optionally" or "optionally" means that the subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not. For example, "a heterocyclic group optionally substituted with an alkyl group" means that an alkyl group may but need not be present, and that the description includes instances where the heterocyclic group is substituted with an alkyl group and instances where the heterocyclic group is not substituted with an alkyl group.

[0110] The term "substituted" refers to the replacement of one or more hydrogen atoms, preferably up to 5, more preferably 1 to 3, in a group with a corresponding number of substituents, independently of one another. It goes without saying that the substituents are only in their possible chemical positions, and those skilled in the art will be able to determine (by experiment or theory) which substitutions are possible or impossible using existing technical means and experimental conditions. For example, an amino or hydroxyl group with free hydrogen may be unstable when combined with a carbon atom with an unsaturated (e.g., olefinic) bond.

[0111] The minimum and maximum carbon atom content in a hydrocarbon group is indicated by a prefix, for example, the prefix C a～ C b Alkyl refers to any alkyl group containing from "a" to "b" carbon atoms. Thus, for example, C 1～6The alkyl group refers to a straight-chain or branched alkyl group containing 1 to 6 carbon atoms.

[0112] Alkyl refers to a straight or branched hydrocarbon group in an alkane molecule, such as methyl -CH3, ethyl -CH2CH3, and methylene -CH2-. Alkyl groups can also be part of other groups, such as C1-C6 alkoxy and C1-C6 alkylamino. Non-limiting examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, and n-hexyl. Alkyl groups can be substituted or unsubstituted. When substituted, the substituents can be substituted at any available point of attachment. The substituents are preferably one or more of the following groups independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxy, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkyloxy, heterocycloalkyloxy, cycloalkylthio, heterocycloalkylthio, oxo, carboxyl, or carboxylate groups.

[0113] In the present invention, "protecting group" refers to an unstable chemical moiety known in the art that is used to prevent reactive groups (such as hydroxyl, amino, carboxyl and sulfhydryl) from reacting undesirably during synthesis. Protecting groups are typically used selectively and / or orthogonally to protect sites at other reactive sites in a reaction and are subsequently removed to release the unprotected group, making it available for further reaction. In some embodiments, a "substituted" group or substituent comprises a protecting group.

[0114] Representative hydroxy protecting groups in the present invention 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, each of which is incorporated herein by reference in its entirety. In some embodiments, the protecting group is stable under alkaline conditions but can be removed under acidic conditions. In some embodiments, non-exclusive examples of hydroxy protecting groups that can be used in the present invention include dimethoxytrityl (DMT), monomethoxytrityl, 9-phenylxanthen-9-yl (Pixyl) and 9-(p-methoxyphenyl)xanthen-9-yl (Mox). In some embodiments, non-exclusive examples of hydroxy protecting groups that may be used in the present invention include Tr (trityl), MMTr (4-methoxytrityl), DMTr (4,4'-dimethoxytrityl), and TMTr (4,4',4"-trimethoxytrityl).

[0115] The compounds and compositions described herein may have certain atoms (e.g., N, O, or S atoms) in a protonated or deprotonated state, depending on the environment in which the compound or composition is located. Thus, as used herein, the structures described herein take into account that certain functional groups, such as OH, SH, or NH, may be protonated or deprotonated. The present disclosure is intended to encompass the above-described compounds and compositions, regardless of their protonation state based on the ambient pH, as will be readily understood by those of ordinary skill in the art.

[0116] The terms "salts" and "usable salts" refer to acidic and / or basic salts of the above-mentioned compounds or their stereoisomers, formed with inorganic and / or organic acids and bases, including zwitterionic salts (inner salts), and also quaternary ammonium salts, such as alkylammonium salts. These salts can be obtained directly during the final separation and purification of the compounds. They can also be obtained by mixing the above-mentioned compounds, or their stereoisomers, with a suitable amount of acid or base (e.g., an equivalent amount). These salts may be precipitated in solution and collected by filtration, or recovered after evaporation of the solvent, or obtained by freeze-drying after reaction in an aqueous medium.

[0117] The following further describes the above content of the present invention in detail through specific embodiments in the form of examples. However, this should not be construed as limiting the scope of the above subject matter of the present invention to the following examples. All technologies implemented based on the above content of the present invention fall within the scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0118] Figure 1 shows the structure of compound HG_NA02 1 H NMR spectrum;

[0119] Figures 2 and 3 show the structures of compounds HG_NA03a and HG_NA03b. 1 H NMR spectrum;

[0120] FIG4 is an LCMS diagram of compound HG_NA02;

[0121] Figures 5 and 6 are LCMS images of compounds HG_NA03a and HG_NA03b;

[0122] Figure 7 is a diagram of the detection of siRNA on-target activity in Experimental Example 1;

[0123] FIG8 is a diagram of the detection of siRNA off-target activity in Experimental Example 1. DETAILED DESCRIPTION

[0124] The structures of the compounds were confirmed by nuclear magnetic resonance (NMR) and mass spectrometry (MS). NMR shifts (δ) are given in units of 10⁻⁶ (ppm). NMR measurements were performed using a Bruker Avance III 400 and Bruker Avance 300 NMR spectrometer. The solvents used were deuterated dimethyl sulfoxide (DMSO-d₆), deuterated chloroform (CDCl₃), and deuterated methanol (CD₃OD), with tetramethylsilane (TMS) as the internal standard.

[0125] LC-MS analysis was performed using a Shimadzu LC-MS2020 (ESI) liquid chromatography-mass spectrometer. HPLC analysis was performed using a Shimadzu LC-20A high-pressure liquid chromatograph. MPLC (medium-pressure preparative chromatography) was performed using a Gilson GX-281 reverse-phase preparative chromatograph. Thin-layer chromatography silica gel plates were Yantai Huanghai HSGF254 or Qingdao GF254. The specifications used for thin-layer chromatography separation and purification products were 0.4 mm to 0.5 mm. Column chromatography typically used Yantai Huanghai 200-300 mesh silica gel as the carrier.

[0126] The known starting materials of the present invention can be synthesized by methods known in the art, or can be purchased from companies such as Anaiji Chemical, Chengdu Kelon Chemical, Shaoyuan Chemical Technology, and Bailingwei Technology. Unless otherwise specified, the materials and reagents used in the examples of the present invention are commercially available products.

[0127] Unless otherwise specified, reactions were carried out under a nitrogen atmosphere. Unless otherwise specified, solutions are aqueous solutions. Unless otherwise specified, reactions were performed at room temperature. Unless otherwise specified, M is moles per liter.

[0128] The following further describes the above content of the present invention in detail through specific embodiments in the form of examples. However, this should not be construed as limiting the scope of the above subject matter of the present invention to the following examples. All technologies implemented based on the above content of the present invention fall within the scope of the present invention.

[0129] Example 1. Synthesis of (R)-1-(bis(4-methoxyphenyl)(phenyl)methoxy)-3-(3-carbonyl-1H-1,2,4-triazol-1-yl)propan-2-yl(2-cyanoethyl)bisisopropylphosphinamide

[0130] Step 1. Synthesis of compound HG_NA01-2

[0131] To a 250 mL single-necked flask, add 1H-1,2,4-triazole-3-carboxamide (5.00 g, 44.60 mmol) and dissolve in DMF (100 mL). Then add (S)-(-)-4-chloromethyl-2,2-dimethyl-1,3-dioxolane (8.60 g, 57.10 mmol) and cesium carbonate (29.08 g, 89.21 mmol). After the addition is complete, heat to 150°C and stir overnight, monitoring by LC-MS. After completion of the reaction, the reaction solution is directly analyzed by MPLC to obtain HG_NA01-2 (1.00 g, 10.40% yield). LCMS (E+) m / z: 227.1 [M+H] + .

[0132] Step 2. Synthesis of compound HG_NA01-3

[0133] To a 100 mL single-necked flask, add HG_NA01-2 (1.00 g, 4.42 mmol) and dissolve in THF (30 mL). Slowly add concentrated hydrochloric acid (15 mL, 12 M) with stirring. After addition, heat the reaction overnight and monitor with LC-MS. After completion of the reaction, concentrate the reaction solution, add dry pyridine (50 mL) to the residue, and concentrate under reduced pressure to obtain HG_NA01-3 (0.89 g, 108.14% yield). LCMS (E+) m / z: 187.1 [M+H] + .

[0134] Step 3. Synthesis of compound HG_NA01-4

[0135] To a 100 mL three-necked flask, add HG_NA01-3 (0.89 g, 4.78 mmol), dissolve it in pyridine (30 mL), and then add DMTrCl (1.62 g, 4.78 mmol). After the addition, stir at room temperature for 3 hours and monitor with LC-MS. After the reaction is complete, the reaction solution is concentrated, and the residue is purified by MPLC to yield HG_NA01-4 (2.20 g, 94.30% yield). LCMS (E+) m / z: 489.2 [M+H] + .

[0136] Step 4. Synthesis of compound HG_NA01

[0137] To a 100 mL three-necked flask, add HG_NA01-4 (1.20 g, 2.46 mmol) and dissolve in dry DCM (20 mL). Then add CEPC1 (0.70 g, 2.94 mmol) and DIPEA (0.63 g, 4.92 mmol). After the addition, stir at room temperature for 2 hours and monitor with LC-MS. After the reaction is complete, directly obtain HG_NA01 (0.24 g, 14.19% yield) by MPLC. LCMS (E+) m / z: 689.2 [M+H] + . 1 H NMR(400MHz,Chloroform-d)δ8.27–8.00(m,1H),7.50–7.41(m,2H),7.36–7.18(m,8H),6.97(s,1H),6.83(t,J=7.8Hz,4H),4.69–4.25( m,3H),3.79(d,J=2.8Hz,6H),3.56(dtd,J=47.8,15.1,7.1Hz,4H),3.37–3.05(m,2H),2.46(dt,J=58.1,6.8Hz,2H),1.17–0.98(m,12H).

[0138] Example 2. Synthesis of (R)-1-(bis(4-methoxyphenyl)(phenyl)methoxy)-3-(4-carbonyl-1H-imidazol-1-yl)propan-2-yl(2-cyanoethyl)diisopropylphosphinamide

[0139] Step 1. Synthesis of compound HG_NA02-2

[0140] Imidazole-4-carboxamide (4.95 g, 44.60 mmol) was added to a 250 mL single-necked flask and dissolved in DMF (100 mL). (S)-(-)-4-chloromethyl-2,2-dimethyl-1,3-dioxolane (8.60 g, 57.10 mmol) and cesium carbonate (29.08 g, 89.21 mmol) were then added. After the addition was complete, the temperature was raised to 150°C and stirred for overnight reaction, monitored by LC-MS. After the reaction was complete, the reaction solution was directly analyzed by MPLC to obtain HG_NA02-2 (1.40 g, 14.00% yield). LCMS (E+) m / z: 226.1 [M+H] + .

[0141] Step 2. Synthesis of compound HG_NA02-3

[0142] To a 100 mL single-necked flask, add HG_NA02-2 (1.00 g, 4.42 mmol) and dissolve in THF (30 mL). Slowly add concentrated hydrochloric acid (15 mL, 12 M) with stirring. After addition, the temperature was raised and the reaction was allowed to react overnight, monitored by LC-MS. After completion of the reaction, the reaction solution was concentrated, and the residue was added with dry pyridine (50 mL). The solution was concentrated under reduced pressure to obtain HG_NA02-3 (0.87 g, 98.35% yield). LCMS (E+) m / z: 186.1 [M+H] + .

[0143] Step 3. Synthesis of compound HG_NA02-4

[0144] To a 100 mL three-necked flask, add HG_NA02-3 (0.87 g, 4.69 mmol), dissolve it in pyridine (20 mL), and then add DMTrCl (1.58 g, 4.69 mmol). After the addition, stir at room temperature for 3 hours and monitor with LC-MS. After the reaction is complete, the reaction solution is concentrated, and the residue is purified by MPLC to obtain HG_NA02-4 (0.90 g, 39.38% yield). LCMS (E+) m / z: 488.2 [M+H] + .

[0145] Step 4. Synthesis of compound HG_NA02

[0146] HG_NA02-4 (0.55 g, 1.13 mmol) was added to a 100 mL three-necked flask and dissolved in dry DCM (10 mL). Bis(diisopropylamino)(2-cyanoethoxy)phosphine (0.34 g, 1.13 mmol) and diisopropylammonium tetrazolium salt (0.10 g, 0.56 mmol) were then added. After the addition, the reaction was stirred at room temperature for 1 hour and monitored by LC-MS. After the reaction was completed, the reaction solution was extracted with saturated sodium bicarbonate and DCM, and the organic phase was concentrated and then prepared by HPLC to obtain HG_NA02 (0.38 g, 48.00% yield). LCMS (E+) m / z: 688.2 [M+H] + . 1H NMR(400MHz,Chloroform-d)δ7.55(dd,J=7.5,1.2Hz,1H),7.51–7.12(m,11H),6.97–6.76(m,5H),4.35–4.04(m,3H),3.80(d,J=2.7Hz,6H),3.73 –3.43(m,4H),3.27(ddd,J=32.1,9.8,4.5Hz,1H),2.95(ddd,J=28.6,9. 6,7.3Hz,1H),2.69–2.32(m,2H),1.52–1.44(m,1H),1.19–0.97(m,12H).

[0147] Example 3. Synthesis of 1-(bis(4-methoxyphenyl)(phenyl)methoxy)-3-(1-methyl-2,4-dioxo-1,2,3,4-tetrahydropyridin-5-yl)propan-2-yl(2-cyanoethyl)diisopropylphosphinamide

[0148] Step 1. Synthesis of compound HG_NA03-2

[0149] To a 250 mL reaction flask, add 5-bromo-1-methylpyrimidine-2,4(1H,3H)-dione (6.00 g, 29.27 mmol) and dissolve in DMF (100 mL). Then add allyltributyltin (14.54 g, 43.90 mmol), Pd(PPh3)2Cl2 (2.05 g, 2.93 mmol), and lithium fluoride (0.76 mg, 2.93 mmol). After the addition, replace the atmosphere with nitrogen three times, heat to 110°C, stir, and react overnight. Monitor by LC-MS. After the reaction, the reaction solution is directly analyzed by MPLC to obtain HG_NA03-2 (1.00 g, 20.56% yield). LCMS (E+) m / z: 167.1 [M+H] + .

[0150] Step 2. Synthesis of compound HG_NA03-3

[0151] HG_NA03-2 (1.00 g, 6.50 mmol) was added to a 100 mL single-necked flask and dissolved in 1,4-dioxane (10 mL). NMO (0.24 g, 2.07 mmol) and potassium osmate dihydrate (0.60 g, 0.20 mmol) were then added. After the addition was complete, the reaction was stirred at room temperature for half an hour and monitored by LC-MS. After the reaction was completed, the reaction solution was directly purified by MPLC to obtain HG_NA03-3 (613.00 mg, 50.61% yield). LCMS (E+) m / z: 201.1 [M+H] + .

[0152] Step 3. Synthesis of compound HG_NA03-4

[0153] To a 100 mL single-necked flask, add HG_NA03-3 (613.00 mg, 3.06 mmol) and dissolve in dry pyridine (20 mL). DMTrCl (1.04 g, 3.06 mmol) was then added. After the addition, the reaction was stirred at room temperature for 2 hours and monitored by LC-MS. After completion of the reaction, the reaction solution was concentrated and purified by column chromatography to obtain HG_NA03-4 (1.24 g, 80.58% yield). LCMS (E+) m / z: 503.2 [M+H] + .

[0154] Step 4. Synthesis of compounds HG_NA03a and HG_NA03b

[0155] To a 100 mL single-necked flask, add HG_NA03-4 (489.00 mg, 0.97 mmol) and dissolve in DCM (20 mL). DIPEA (251.51 mg, 1.95 mmol) and CEPC1 (253.32 mg, 1.07 mmol) were then added. After the addition, the reaction was stirred at room temperature for 2 hours and monitored by LC-MS. After completion of the reaction, column chromatography was used to purify HG_NA03a and HG_NA03b (150.00 mg each, 46.65% yield). LCMS (E+) m / z: 703.2 [M+H] + . HG_NA03a:1H NMR(400MHz,Chloroform-d)δ7.51–7.40(m,2H),7.39–7.23(m,6H),7.22–7.1 5(m,1H),7.08(dd,J=34.1,7.8Hz,1H),6.90–6.72(m,4H),5.67(dd,J=7.8,6.0 Hz,1H),4.56–4.29(m,2H),3.97(ddd,J=29.7,12.3,3.9Hz,1H),3.86–3.42(m ,10H),3.41–3.00(m,5H),2.67–2.29(m,2H),1.33–0.96(m,12H).HG_NA03b:1H NMR(400MHz,DMSO-d6)δ7.64(t,J=7.5Hz,1H),7.45–7.35(m,2H),7.35–7.16(m,7H) ,6.88(dd,J=8.9,3.1Hz,4H),5.64(dd,J=12.1,7.8Hz,1H),4.40–4.07(m,2H),3.86 –3.70(m,7H),3.69–3.36(m,4H),3.24(d,J=13.1Hz,3H),3.13(ddd,J=25.7,9.7,4. 8Hz, 1H), 2.94 (ddd, J=40.0, 9.7, 5.1Hz, 1H), 2.74–2.53 (m, 2H), 1.31–0.75 (m, 12H).

[0156] Example 4. siRNA Synthesis

[0157] For the sense and antisense strands of the siRNA duplexes and the sense and antisense strands of the modified duplexes disclosed herein, deoxynucleoside CPG was used as a solid support to synthesize the sense strand and universal CPG to synthesize the antisense strand.

[0158] Sequence synthesis was performed on a 48-channel synthesizer at a 0.2 μmol scale. The phosphoramidite monomer was used at a concentration of 0.05 M, and the activator was 0.3 M BTT.

[0159] Sequences were cleaved and deprotected in 1.5 ml tubes using AMA in the first step and triethylamine trihydrofluoride to remove the 2-position protecting group in the second step. For sequences containing all 2 modifications, ammonia solution was required. Sequences after cleavage and deprotection were precipitated using an acetone:ethanol (80:20) mixture and dissolved in RNase-free water. Each sequence was analyzed by LC-MS to determine sequence accuracy, spectrophotometrically quantified, and HPLC to determine purity.

[0160] After HPLC purification, lyophilization and quality inspection, the salt was replaced by sodium acetate alcohol precipitation and desalted using a 3KD ultrafiltration tube. After desalting, the sense chain and antisense chain were quantitatively determined by spectrophotometer and mixed in a 1:1 ratio and annealed to form siRNA duplexes.

[0161] The siRNA sequences used in the present invention are as follows:

[0162] SS represents the sense strand, AS represents the antisense strand, the lowercase letter m represents that the nucleotide adjacent to the right of the letter m is a 2'-methoxy-modified nucleotide, the lowercase letter f represents that the nucleotide adjacent to the right of the letter f is a 2-fluoro-modified nucleotide, and "*" represents that the connection between the two adjacent nucleotides on the left and right is a phosphorothioate connection; GNA represents (S)-ethylene glycol nucleotide; HG_NA02 is the nucleoside phosphoramidite monomer HG_NA02 obtained by solid phase synthesis in Example 2; HG_NA03a and HG_NA03b are the nucleoside phosphoramidite monomers HG_NA03a and HG_NA03b obtained by solid phase synthesis in Example 3.

[0163] Experimental Example 1: Detection of on-target and off-target siRNA activity at the cellular level based on psiCHECK

[0164] On-target and off-target psiCHECK plasmid construction:

[0165] For each siRNA compound, a pair of psiCHECK2-based plasmids were constructed: psiCHECK-cm, which contains a perfect match to the antisense strand of the target siRNA compound, and psiCHECK-sm, which contains a seed region match (nucleotides 2-8 in the 5'-3' direction) of the siRNA antisense strand. The psiCHECK-cm and psiCHECK-sm plasmids are used to detect on-target and off-target activity of the siRNA compound, respectively.

[0166] Cell culture, transfection, and dual-luciferase reporter assay:

[0167] HEK293T cell culture: HEK293T cells were cultured in DMEM complete medium (Gibco, supplemented with 10% FBS) at 37°C in a 5% CO2 environment until near confluence, and then the cells were trypsinized and plated in 96-well plates.

[0168] HEK293T cells were plated and reverse transfected with siRNA and plasmid at the same time. The siRNA compound to be tested was set up with multiple concentration gradients and 3-4 replicates. The wells where only cells and Opti-MEM were added were used as the background group of the experiment, and the wells where only plasmid was transfected were used as the control group. After diluting the siRNA and plasmid to be tested, the siRNA and plasmid were diluted with Lipofectamine TM 2000 transfected HEK293T cell suspensions were incubated in a 37°C, 5% CO2 incubator for 24 h. The recommended method of Luciferase Assay System (Vazyme) is used to detect the corresponding Renilla and Firefly luciferase activities.

[0169] The test data were processed as follows, and the results indicated the remaining percentage of target sequence mRNA expression:

[0170] Relative Renilla / Luciferase expression%

[0171] =[(experimental group Renilla-background group Renilla) / (experimental group Firefly-background group Firefly)] /

[0172] [(Renilla in the control group - Renilla in the background group) / (Firefly in the control group - Firefly in the background group)]

[0173] In the target activity detection experiment, HEK293T cell line was selected for transfection of psiCHECK-cm recombinant plasmid and siRNA compound. The starting concentration of the compound was 50 nM, and 8 concentration points (50 nM, 7.14286 nM, 1.02041 nM, 0.14577 nM, 0.02082 nM, 0.00297 nM, 0.00042 nM, 0.00006 nM) were selected for siRNA compound activity screening experiment. The results are shown in Table 1.

[0174] Table 1 Results of psi-CHECK2 on-target activity detection experiments

[0175] In the active sequence of TTR-targeting siRNA, after replacing the A bases at positions 5 and 7 in the AS seed region with HG_NA02, the target activity was maintained well (sequence numbers: TTR-2 and TTR-6), and even better than the modified sequences replaced with GNA(A) (sequence numbers: TTR-1 and TTR-5); after replacing the G base at position 6 in the AS seed region with HG_NA02, the target activity was still maintained (sequence number: TTR-4). This result shows that monomer HG_NA02 can replace both A and G bases and maintain target activity. In the active siRNA sequences targeting AGT, replacing the T base at position 6 in the AS seed region with HG_NA03a and HG_NA03b (sequence numbers: AGT-2 and AGT-3) maintained good on-target activity. Replacing the C base at position 7 in the AS seed region with HG_NA03a and HG_NA03b (sequence numbers: AGT-5 and AGT-6) also maintained good on-target activity, with AGT-6 showing superior activity to AGT-4 (GNA(C)). These results demonstrate that HG_NA03 can simultaneously replace both T and C bases while maintaining on-target activity.

[0176] For the off-target activity detection experiment, HEK293T cell lines were transfected with psiCHECK-sm recombinant plasmid and siRNA compounds. The starting concentration of the compound was 50 nM, and 8 concentration points (50 nM, 7.14286 nM, 1.02041 nM, 0.14577 nM, 0.02082 nM, 0.00297 nM, 0.00042 nM, and 0.00006 nM) were diluted 7-fold to perform siRNA compound activity screening. The results are shown in Table 2.

[0177] Table 2 Results of psi-CHECK2 off-target activity detection experiments

[0178] In the active sequence of siRNA targeting TTR, replacing the A bases at positions 5 and 7 of the AS seed region with HG_NA02 significantly improved the siRNA off-target activity; replacing the G base at position 6 of the AS seed region with HG_NA02 significantly improved the siRNA off-target activity. In the active sequence of siRNA targeting AGT, replacing the T base at position 6 of the AS seed region with HG_NA03a and HG_NA03b significantly improved the siRNA off-target activity; replacing the C base at position 7 of the AS seed region with HG_NA03a and HG_NA03b significantly improved the siRNA off-target activity. The results show that siRNA carrying the nucleotide analog monomer of the present invention effectively reduces off-target activity.

Claims

1. A nucleotide monomer compound represented by Formula I, or a stereoisomer thereof, or a deuterated compound thereof, or a fluorinated compound thereof, or a pharmaceutically acceptable salt thereof: Wherein: Bx is selected from R is selected from hydrogen or a hydroxyl protecting group; m is 1, 2 or 3; preferably, m is 1; n is 1, 2 or 3; preferably, n is 1; Z is selected from a hydroxyl group, a phosphate group, a thiophosphate group or a phosphoramidite group.

2. The compound according to claim 1, wherein: The compound shown in Formula I is as shown in Formula II: Wherein, Bx is selected from R is selected from hydrogen or a hydroxyl protecting group; m is 1, 2 or 3; preferably, m is 1; n is 1, 2 or 3; preferably, n is 1; Z is selected from a hydroxyl group, a phosphate group, a thiophosphate group or a phosphoramidite group.

3. The compound according to claim 1 or 2, wherein: R is selected from hydrogen, 4,4'-dimethoxytriphenylmethyl or 4-methoxytriphenylmethyl; Z is selected from a hydroxyl group or 4. The compound according to any one of claims 1 to 3, characterized in that: The specific compound is as follows:

5. Use of the nucleotide monomer compound according to any one of claims 1 to 4 as an intermediate in the preparation of an oligonucleotide; preferably, the oligonucleotide is siRNA.

6. The use according to claim 5, wherein: Use of the nucleotide monomer compound according to any one of claims 1 to 4 as an intermediate in the preparation of the seed region of the antisense strand of siRNA.

7. An siRNA, which comprises a sense strand and an antisense strand; each of the sense strand and the antisense strand comprises 15 to 45 modified or unmodified nucleotides, and the sense strand and the antisense strand are partially complementary to form a double-stranded region; wherein, The seed region of the antisense strand contains at least one nucleotide having the structure shown in Formula V and is covalently linked to the rest of the siRNA at the site: Wherein, Bx is selected from m is 1, 2 or 3; preferably, m is 1; n is 1, 2 or 3; preferably, n is 1; X 1 and X 2 are each independently selected from O or S.

8. The siRNA according to claim 7, wherein: The nucleotides of the structure shown by formula V in the siRNA are shown by formula VI: Wherein, Bx is selected from m is 1, 2 or 3; preferably, m is 1; n is 1, 2 or 3; preferably, n is 1; X 1 、X 2 are each independently selected from O or S.

9. The siRNA according to claim 7 or 8, wherein: The nucleotide structure of the siRNA with the structure shown in Formula V or Formula VI is specifically as follows:

10. The siRNA according to claim 7 or 8, characterized in that: In the direction from the 5'-end to the 3'-end, at least one nucleotide having the structure shown in Formula V or Formula VI is contained in the 5th, 6th and 7th positions of the seed region of the antisense strand.

11. The siRNA according to claim 7 or 8, characterized in that: In the direction from the 5'-end to the 3'-end, the nucleotide at the 5th position of the seed region of the antisense strand has the structure shown in Formula V or Formula VI.

12. The siRNA according to claim 7 or 8, wherein: In the direction from the 5'-end to the 3'-end, the nucleotide at the 6th position of the seed region of the antisense strand has the structure shown in Formula V or Formula VI.

13. The siRNA according to claim 7 or 8, characterized in that: In the direction from the 5'-end to the 3'-end, the nucleotide at the 7th position of the seed region of the antisense strand has the structure shown in Formula V or Formula VI.

14. The siRNA according to claim 7 or 8, wherein: In the direction from the 5'-end to the 3'-end, the nucleotide at the 5th position of the seed region of the antisense strand has the structure shown in Formula V or Formula VI; The nucleotides at the 2nd, 6th, 14th and 16th positions of the antisense strand are each independently a 2'-fluoro-modified nucleotide.

15. The siRNA according to claim 7 or 8, characterized in that: In the direction from the 5'-end to the 3'-end, the nucleotide at the 6th position of the seed region of the antisense strand has the structure shown in Formula V or Formula VI; The nucleotides at the 2nd, 14th and 16th positions of the antisense strand are each independently a 2'-fluoro-modified nucleotide.

16. The siRNA according to claim 7 or 8, wherein: In the direction from the 5'-end to the 3'-end, the nucleotide at the 7th position of the seed region of the antisense strand has the structure shown in Formula V or Formula VI; The nucleotides at the 2nd, 6th, 14th and 16th positions of the antisense strand are each independently a 2'-fluoro-modified nucleotide.

17. A pharmaceutical composition comprising the siRNA according to any one of claims 7 to 16 and a pharmaceutically acceptable carrier.

18. An siRNA conjugate formed by conjugating the siRNA according to any one of claims 7 to 16 and a conjugating molecule.

19. Use of the siRNA according to any one of claims 7 to 16, and / or the pharmaceutical composition according to claim 17, and / or the siRNA conjugate according to claim 18 in the preparation of a drug.

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