Double-stranded oligonucleotide comprising lipid-modified monomer and use thereof

Abstract

The present disclosure provides a double-stranded oligonucleotide comprising a lipid-modified monomer and use thereof and pertains to the technical field of oligonucleotide drugs. In the present disclosure, a lipid group is introduced into a nucleotide monomer to prepare a modified nucleotide monomer, and the lipid-modified nucleotide monomer is utilized to prepare a double-stranded oligonucleotide, a double-stranded oligonucleotide conjugate, or a pharmaceutical composition. The modified double-stranded oligonucleotide, the double-stranded oligonucleotide conjugate, or the pharmaceutical composition can effectively treat and / or prevent pathological conditions or diseases caused by abnormal expression of a specific gene in target tissue-related cells.

Description

Double-stranded oligonucleotides containing lipid-modified monomers and their applications

[0001] Cross-references to related applications

[0002] This disclosure claims priority to Chinese Patent Application No. 202410481984.3, filed on April 22, 2024, entitled "Double-stranded oligonucleotides containing lipid-modified monomers and their uses", the entire contents of which are incorporated herein by reference. Technical Field

[0003] This disclosure belongs to the field of oligonucleotide drug technology, specifically relating to lipid-modified nucleoside monomers and double-stranded oligonucleotides containing such monomers, as well as their uses. Background Technology

[0004] RNA interference (RNAi) refers to a highly conserved evolutionary phenomenon characterized by the efficient and specific degradation of homologous mRNA induced by double-stranded small interfering RNA (siRNA). Currently, RNAi agents containing oligonucleotides can be delivered into cells in vivo to treat diseases caused by the abnormal expression of specific genes in target tissue cells. For the treatment of liver-related disorders, oligonucleotides in RNAi agents can be targeted and delivered to liver cells to exert therapeutic effects, showing promising application prospects; however, for delivery to extrahepatic tissues, oligonucleotides typically do not achieve satisfactory delivery results.

[0005] Since conjugated lipid groups can modulate the hydrophobicity of siRNA and enhance its tissue accumulation and cellular uptake, conjugation with molecules such as fatty acids, cholesterol, and tocopherol can be used to improve the extrahepatic delivery of siRNA. Although siRNAs conjugated with various lipid classes already exist, there is still a need to develop highly efficient extrahepatic siRNA delivery systems to achieve better therapeutic effects in various organs and / or tissues outside the liver, such as the nervous system, heart, fat, and muscle. Summary of the Invention

[0006] This disclosure provides lipid-modified nucleoside monomers, double-stranded oligonucleotides containing said modified nucleoside monomers, conjugates, pharmaceutical compositions, and uses thereof. This disclosure enhances activity by introducing lipid-modified nucleoside monomers into double-stranded oligonucleotides.

[0007] In a first aspect, this disclosure provides a modified nucleoside monomer having the structure shown in formula (I), or a stereoisomer thereof, a tautomer thereof, or a pharmaceutically acceptable salt thereof:

[0008] Wherein, X, p, q, Z1, Z2, R1, R2 and L are as defined in equation (i) of the second aspect of this disclosure;

[0009] Base is selected from modified or unmodified bases; if an amino group is present in the Base, the amino group is protected with an amino protecting group;

[0010] R6 is selected from H or a hydroxyl protecting group;

[0011] R7 is selected from H or phosphorus-containing leaving groups.

[0012] In a second aspect, this disclosure provides a double-stranded oligonucleotide comprising a sense strand and an antisense strand, the antisense strand at least partially base-pairing with the sense strand to form a complementary double-stranded region, the sense strand and / or antisense strand containing at least one modified nucleotide having a structure of formula (i), or a stereoisomer thereof, or a tautomer thereof, or a pharmaceutically acceptable salt thereof.

[0013] Where Q is selected from hydroxyl or mercapto groups;

[0014] X is selected from O or S;

[0015] p and q are each independently selected from integers from 1 to 4;

[0016] Base is selected from H, or modified or unmodified bases;

[0017] R1 is selected from H or substituted or unsubstituted C1-C6 alkyl groups, wherein the substituents in the substituted C1-C6 alkyl groups are selected from halogens, hydroxyl groups or C1-C6 alkoxy groups;

[0018] R2 exists or does not exist; when R2 exists, R2 is selected from... R3 and R4 are independently selected from H or substituted or unsubstituted C1-C6 alkyl groups, wherein the substituents in the substituted C1-C6 alkyl groups are selected from halogens, hydroxyl groups or C1-C6 alkoxy groups; m is selected from 1;

[0019] One of Z1 and Z2 may exist, or both Z1 and Z2 may exist simultaneously.

[0020] Z1 and Z2 are selected independently. Among them, R z1 R z2 R z3 R z4 R z5 R z6 R z7 R z8 R z9 Rz10 Each is independently selected from H or C1-C3 alkyl groups;

[0021] L is selected from optionally substituted C10-C23 alkyl groups.

[0022] In a third aspect of this disclosure, a composition is provided comprising the double-stranded oligonucleotide described in the second aspect of this disclosure.

[0023] In a fourth aspect of this disclosure, this disclosure provides the use of the double-stranded oligonucleotide described in the second aspect of this disclosure and the composition described in the third aspect in the preparation of a medicament for treating and / or preventing pathological conditions or diseases caused by the abnormal expression of a specific gene in cells related to a target tissue.

[0024] In some alternative implementations, the target tissue is selected from the central nervous system, cardiac muscle, skeletal muscle, and fat.

[0025] In a fifth aspect of this disclosure, a pharmaceutical composition is provided comprising the double-stranded oligonucleotides described in the second aspect of this disclosure, and / or the compositions described in the third aspect, and optionally one or more pharmaceutically acceptable carriers or excipients.

[0026] In a sixth aspect of this disclosure, this disclosure provides a method for inhibiting the expression of a specific gene in target tissue-related cells, the method comprising contacting the target tissue-related cells with the double-stranded oligonucleotide described in the second aspect of this disclosure, and / or the composition described in the third aspect, and / or the pharmaceutical composition described in the fifth aspect;

[0027] Optionally, the target tissue is selected from the central nervous system, cardiac muscle, skeletal muscle, and fat.

[0028] In a seventh aspect of this disclosure, this disclosure provides a method for preventing and / or treating a pathological condition or disease caused by the abnormal expression of a specific gene in cells associated with a target tissue, the method comprising administering to a subject a pharmaceutically acceptable dose of the double-stranded oligonucleotide described in the second aspect of this disclosure, and / or the composition described in the third aspect, and / or the pharmaceutical composition described in the fifth aspect;

[0029] In some alternative embodiments, the target tissue is selected from the central nervous system, myocardium, skeletal muscle, and fat.

[0030] In an eighth aspect of this disclosure, this disclosure provides a method for inhibiting the expression of a target gene in central nervous system cells, skeletal muscle cells, cardiomyocytes, or adipocytes, the method comprising: contacting the cells with the double-stranded oligonucleotides described in the second aspect of this disclosure, and / or the composition described in the third aspect, and / or the pharmaceutical composition described in the fifth aspect.

[0031] In a ninth aspect of this disclosure, this disclosure provides a method for preventing and / or treating diseases or conditions related to the central nervous system, myocardium, skeletal muscle, and adipose tissue, the method comprising: administering to the subject a therapeutically effective amount of the double-stranded oligonucleotide of the second aspect of this disclosure, and / or the composition of the third aspect, and / or the pharmaceutical composition of the fifth aspect, thereby treating the subject.

[0032] In some alternative implementations, the subject is a human being.

[0033] This disclosure discloses the preparation of modified nucleotide monomers by introducing lipid groups into nucleotide monomers, and the preparation of double-stranded oligonucleotides, double-stranded oligonucleotide conjugates, or pharmaceutical compositions by introducing lipid-modified nucleoside monomers into double-stranded oligonucleotides. The modified double-stranded oligonucleotides, double-stranded oligonucleotide conjugates, or pharmaceutical compositions can effectively treat and / or prevent pathological conditions or diseases caused by the abnormal expression of specific genes in target tissue-related cells. Attached Figure Description

[0034] Figure 1 shows the relative residual expression level of the target gene SOD1 mRNA in the mouse brain after administration of siRNA conjugates RZ899093, RZ899115, and RZ899116 in Example 1.

[0035] Figure 2 shows the relative residual expression level of the target gene SOD1 mRNA in the mouse brain after administration of siRNA conjugates RZ899111, RZ899112, RZ899113, and RZ899114 in Example 2.

[0036] Figure 3 shows the relative residual expression levels of the target gene SOD1 mRNA in mouse heart, fat, and skeletal muscle after administration of siRNA conjugates RZ899111, RZ899112, RZ899113, and RZ899114 in Example 3.

[0037] Figure 4 shows the relative residual expression level of the target gene SOD1 mRNA in the rat lumbar spine after administration of siRNA conjugates RZ899093 and RZ899116 in Example 4.

[0038] Figure 5 shows the relative residual expression level of the target gene SOD1 mRNA in the rat thoracic vertebrae after administration of siRNA conjugates RZ899093 and RZ899116 in Example 4.

[0039] Figure 6 shows the relative residual expression level of the target gene SOD1 mRNA in the rat cervical spine after administration of siRNA conjugates RZ899093 and RZ899116 in Example 4.

[0040] Figure 7 shows the relative residual expression level of the target gene SOD1 mRNA in the rat cerebellum after administration of siRNA conjugates RZ899093 and RZ899116 in Example 4.

[0041] Figure 8 shows the relative residual expression level of the target gene SOD1 mRNA in the rat hippocampus after administration of siRNA conjugates RZ899093 and RZ899116 in Example 4.

[0042] Figure 9 shows the relative residual expression level of the target gene SOD1 mRNA in the rat cerebral cortex after administration of siRNA conjugates RZ899093 and RZ899116 in Example 4. Detailed Implementation

[0043] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Those skilled in the art can refer to the content of this document and appropriately improve the process parameters to achieve the desired results. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this application. The methods and applications of this application have been described through preferred embodiments. Those skilled in the art can obviously modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of this application to implement and apply the technology of this application.

[0044] Terminology Explanation

[0045] In this disclosure, the terms “comprising” or “including” are open-ended expressions, meaning that they include the contents specified in this disclosure but do not exclude other contents.

[0046] In this disclosure, the terms “optionally,” “optionally,” or “optionally” generally refer to an event or condition that may, but may not, occur, and the description includes both cases in which the event or condition occurs and cases in which the event or condition does not occur.

[0047] In this disclosure, the term "double-stranded oligonucleotide" refers to a double-stranded structure formed by two oligonucleotides through partial or complete base pairing. The two oligonucleotides include a sense strand and an antisense strand, which may or may not be of the same length. As long as at least some base-pairing regions exist to form a double-stranded region, the oligonucleotide having a double-stranded structure is considered a double-stranded oligonucleotide as described in this disclosure. The nucleotides constituting the double-stranded oligonucleotide in this disclosure can be modified or unmodified nucleotides. When referring to modified nucleotides, unless otherwise specified, the modification does not specifically refer to the modified site. In addition to the modification of the nucleotides, the linking bonds between the nucleotides in the double-stranded oligonucleotide in this disclosure may also be modified. Double-stranded oligonucleotides containing modified linking bonds between nucleotides are also considered double-stranded oligonucleotides as described in this invention. Besides the nucleotide portion, the double-stranded oligonucleotide in this disclosure may also contain compounds or modifiers acceptable in the art to improve the properties of the double-stranded oligonucleotide, such as linking ligands to form conjugates.

[0048] In this disclosure, the terms "ligand" or "conjugation group" refer to an atom or group of atoms that binds to an oligonucleotide or other oligomer. Generally, a conjugation group modifies one or more properties of the compound to which it is linked, including but not limited to pharmacodynamic, pharmacokinetic, binding, absorption, cellular distribution, cellular uptake, charge, and / or clearance properties. When referring to a link between two molecules, the term "link" as used herein means that the two molecules are directly or indirectly connected by a covalent bond, or that the two molecules are associated by a non-covalent bond (e.g., a hydrogen bond or an ionic bond).

[0049] In this disclosure, the term "pharmaceutical composition" or "composition" can refer to something used for the treatment of a disease or for use in in vitro cell culture experiments. When used for the treatment of a disease, the term "pharmaceutical composition" generally refers to a unit dose form and can be prepared by any method well known in the pharmaceutical industry. All methods involve the step of combining the active ingredient with excipients constituting one or more adjunct components. Typically, compositions are prepared by uniformly and adequately combining active siRNA with liquid excipients, finely pulverized solid excipients, or both.

[0050] In this disclosure, the term "pharmaceutical acceptable" means that a substance or composition must be chemically and / or toxicologically compatible with other components of the formulation and / or the mammals to which it is treated. Preferably, "pharmaceutical acceptable" as used in this disclosure means approved by a federal regulatory agency or national government, or listed in the United States Pharmacopeia or other generally recognized pharmacopoeia for use in animals, particularly in humans.

[0051] In this disclosure, the term "pharmaceutically acceptable carrier or excipient" may include any solvent, solid excipient, diluent, or other liquid excipient, etc., suitable for a specific target dosage form. The use of any conventional excipients that are incompatible with the siRNA of this disclosure, such as those that produce any adverse biological effects or interactions with any other component of the pharmaceutically acceptable composition in a harmful manner, is also within the scope of this disclosure.

[0052] In this disclosure, the term "small interfering RNA (siRNA)" refers to a double-stranded RNA of 17 to 25 nucleotides in length, comprising a sense strand and an antisense strand. siRNA mediates targeted cleavage of RNA transcripts via the RISC pathway by forming an RNA-induced silencing complex (RISC). Specifically, siRNA directs the specific degradation of mRNA sequences through a known RNA interference (RNAi) process, inhibiting the translation of mRNA into amino acids and its conversion into proteins.

[0053] In this disclosure, the term "antisense strand (or guide strand)" includes a region substantially complementary to a target sequence. The term "sense strand (or follower strand)" refers to an iRNA strand containing an iRNA strand substantially complementary to the antisense strand. The term "substantially complementary" means completely complementary or at least partially complementary, for example, the antisense strand being completely complementary or at least partially complementary to the target sequence. In the case of partial complementarity, mismatches may be present in the internal or terminal regions of the molecule, wherein the most tolerant mismatches are present in the terminal regions, for example, within 5, 4, 3, or 2 nucleotides at the 5'- and / or 3' ends of the iRNA. It should be noted that "at least partially substantially complementary" of the antisense strand to the mRNA means that the antisense strand has a polynucleotide substantially complementary to a continuous portion of the mRNA of interest.

[0054] In this disclosure, the term "complementary" refers to the ability of an oligonucleotide of the first sequence to hybridize with an oligonucleotide of the second sequence under certain conditions and form a double-stranded structure.

[0055] In this disclosure, the terms "nucleotide difference," "nucleotide base difference," and "nucleotide sequence difference" are used interchangeably. A nucleotide difference refers to a change in the type of bases of nucleotides at the same or corresponding positions compared to the original nucleotide sequence. For example, if a nucleotide base in the original nucleotide sequence is A, and the nucleotide base at the same or corresponding position is changed to U, C, G, or dT, dC, dG, etc., then a nucleotide sequence difference is considered to exist at that position. It should be noted that if, compared to the original nucleotide sequence, the nucleotides at the same or corresponding positions differ only in the presence or type of modification, then a nucleotide sequence difference is not considered to exist at that position.

[0056] In this disclosure, the terms “treatment,” “relief,” or “improvement” are used interchangeably. These terms refer to methods of achieving beneficial or desired outcomes, including, but not limited to, treatment benefits. A “treatment benefit” means the eradication or improvement of the underlying disorder being treated. Here, a treatment benefit is achieved by eradicating or improving one or more physical symptoms associated with the underlying disorder, thereby observing improvement in the subject, although the subject may still be suffering from the underlying disorder.

[0057] In this disclosure, the terms “prevention” and “avoidance” are used interchangeably to refer to methods for obtaining beneficial or desired results, including but not limited to preventive benefits. To obtain a “preventive benefit,” the conjugate, RNAi reagent, or composition may be given to a subject at risk of developing a specific disease, or to a subject who reports one or more physiological symptoms of a disease, even if a diagnosis of the disease may not have been made.

[0058] In this disclosure, the term "administration" generally refers to the introduction of a pharmaceutical preparation of this disclosure into the body of a subject by any route of introduction or delivery. Any method known to those skilled in the art for contacting cells, organs, or tissues with the drug may be employed. Administration may include, but is not limited to, intravenous, intra-arterial, intranasal, intraperitoneal, intramuscular, subcutaneous, or oral administration. A daily dose may be divided into one, two, or more doses in suitable forms to be administered at one, two, or more times during a period of time.

[0059] As in this disclosure, the term "regulation of gene expression" means that the expression of a gene, or the level of an RNA molecule or equivalent RNA molecule encoding one or more proteins or protein subunits, is upregulated or downregulated such that the expression, level, or activity is greater or less than that observed in the absence of a regulator. For example, the term "regulation" may mean "inhibition," but the use of the word "regulation" is not limited to this definition.

[0060] In addition to any conventional excipients, the use of any range of siRNAs incompatible with the present disclosure, such as any adverse biological effects produced or interactions with any other component of a pharmaceutically acceptable composition in a harmful manner, is also within the scope of this disclosure.

[0061] Modified nucleotide monomers

[0062] In a first aspect, this disclosure provides a modified nucleoside monomer, characterized in that the modified nucleoside monomer has the structure shown in formula (I), or a stereoisomer thereof, or a tautomer thereof, or a pharmaceutically acceptable salt thereof:

[0063] Wherein, X, p, q, Z1, Z2, R1, R2 and L are defined as in formula (i) as described in the second aspect of this disclosure;

[0064] Base is selected from H, or modified or unmodified bases; if an amino group is present in Base, the amino group is protected with an amino protecting group;

[0065] R6 is selected from H or a hydroxyl protecting group;

[0066] R7 is selected from H or phosphorus-containing leaving groups.

[0067] In some alternative embodiments, the amino protecting group is selected from acyl amino protecting groups.

[0068] In some alternative embodiments, the acyl amino protecting group is selected from phthaloyl (Pht), p-toluenesulfonyl (Tos), trifluoroacetyl (Tfa), o-(or p-)nitrobenzenesulfonyl (Ns), pivaloyl (Piv), acetyl (Ac) or benzoyl (Bz).

[0069] In some alternative embodiments, the amino protecting group is selected from benzoyl.

[0070] In some alternative implementations, Base is selected from the bases A, U, T, C, and G.

[0071] In some alternative embodiments, the structural formula of the hydroxyl protecting group is as follows: Among them, R 1a R 1b and R 1c Each is independently selected from H, halogen, C1-C3 alkyl or C1-C3 alkoxy.

[0072] In some alternative implementations, the R 1a The R 1b and the R 1cEach is independently selected from H or C1-C3 alkoxy groups.

[0073] In some alternative implementations, the R 1a The R 1b and the R 1c Each is independently selected from H or methoxy.

[0074] In some alternative embodiments, the hydroxyl protecting group is selected from triphenylmethyl (Tr), 4-methoxytriphenylmethyl (MMTr), 4,4'-dimethoxytriphenylmethyl (DMTr), or 4,4',4”-trimethoxytriphenylmethyl (TMTr).

[0075] In some alternative embodiments, the hydroxyl protecting group is selected from 4,4'-dimethoxytriphenylmethyl (DMTr).

[0076] In some alternative embodiments, the structural formula of the phosphorus-containing leaving group is as follows: Among them, R 2a Selected from secondary amino groups, R 2b Selected from secondary amino or cyano-substituted C1-C3 alkoxy groups.

[0077] In some alternative implementations, the R 2a Selected from

[0078] In some alternative implementations, the R 2b Selected from

[0079] In some alternative embodiments, the phosphorus-containing leaving group is selected from...

[0080] In some alternative embodiments, the modified nucleoside monomer has the structure shown in formula (Ia) or (Ib), or a stereoisomer thereof, or a tautomer thereof, or a pharmaceutically acceptable salt thereof:

[0081] The definitions of X, R2, R6, R7, Z1 and Z2 are the same as above, and Z1 and Z2 exist simultaneously in the (Ib) structural formula;

[0082] p and q are each independently selected from 0 or 1.

[0083] In some alternative implementations, p is selected from 1 and q is selected from 0.

[0084] In some alternative embodiments, R1 is selected from H or substituted or unsubstituted C1-C3 alkyl groups; the substituents in the substituted C1-C3 alkyl groups are selected from halogens, hydroxyl groups or C1-C3 alkoxy groups.

[0085] In some alternative embodiments, R1 is selected from H or methyl.

[0086] In some alternative implementations, Base is selected from H or bases A, U, T, C, and G.

[0087] In some alternative embodiments, L is selected from optionally substituted C15-C23 alkyl groups.

[0088] In some alternative embodiments, the modified nucleoside monomer has the structure shown in formula (II-a1), or a stereoisomer thereof, or a tautomer thereof, or a pharmaceutically acceptable salt thereof:

[0089] X is selected from O or S;

[0090] Y is selected from O or S;

[0091] The definitions of R6 and R7 are the same as above;

[0092] p and q are each independently selected from 0 or 1.

[0093] In some alternative implementations, p is selected from 1 and q is selected from 0;

[0094] L is selected from optionally substituted C15-C23 alkyl groups;

[0095] Base is selected from H or bases A, U, T, C or G;

[0096] R5 is selected from H or substituted or unsubstituted C1-C6 alkyl groups, wherein the substituents in the substituted C1-C6 alkyl groups are selected from halogens, hydroxyl groups or C1-C6 alkoxy groups.

[0097] In some alternative embodiments, R5 is selected from H or substituted or unsubstituted C1-C3 alkyl groups; the substituents in the substituted C1-C3 alkyl groups are selected from halogens, hydroxyl groups or C1-C3 alkoxy groups.

[0098] In some alternative embodiments, the modified nucleoside monomer has the structure shown in formula (II-a1-1), or a stereoisomer thereof, or a tautomer thereof, or a pharmaceutically acceptable salt thereof:

[0099] In some alternative embodiments, the modified nucleoside monomer has the structure shown in formula (III-a1), or a stereoisomer thereof, or a tautomer thereof, or a pharmaceutically acceptable salt thereof:

[0100] In some alternative embodiments, the modified nucleoside monomer has the structure shown in formula (III-a1-1), or a stereoisomer thereof, or a tautomer thereof, or a pharmaceutically acceptable salt thereof:

[0101] In some alternative embodiments, the modified nucleoside monomer has the structure shown in formula (IIII-a1), or a stereoisomer thereof, or a tautomer thereof, or a pharmaceutically acceptable salt thereof:

[0102] In some alternative embodiments, the modified nucleoside monomer has the structure shown in formula (IIII-a1-1), or a stereoisomer thereof, or a tautomer thereof, or a pharmaceutically acceptable salt thereof:

[0103] In some alternative embodiments, the modified nucleoside monomer is selected from any of the following compounds:

[0104] In some alternative embodiments, the modified nucleoside monomer has the structure shown in formula (II-a2), or a stereoisomer thereof, or a tautomer thereof, or a pharmaceutically acceptable salt thereof:

[0105] X is selected from O or S;

[0106] Y is selected from O or S;

[0107] The definitions of R6 and R7 are the same as above;

[0108] p and q are each independently selected from 0 or 1.

[0109] In some alternative implementations, p is selected from 1 and q is selected from 0;

[0110] L is selected from optionally substituted C15-C23 alkyl groups;

[0111] Base is selected from H or bases A, U, T, C or G;

[0112] R5 is selected from H or substituted or unsubstituted C1-C6 alkyl groups, wherein the substituents in the substituted C1-C6 alkyl groups are selected from halogens, hydroxyl groups or C1-C6 alkoxy groups.

[0113] In some alternative embodiments, R5 is selected from H or substituted or unsubstituted C1-C3 alkyl groups; the substituents in the substituted C1-C3 alkyl groups are selected from halogens, hydroxyl groups or C1-C3 alkoxy groups.

[0114] In some alternative embodiments, the modified nucleoside monomer has the structure shown in formula (II-a2-1), or a stereoisomer thereof, or a tautomer thereof, or a pharmaceutically acceptable salt thereof:

[0115] In some alternative embodiments, the modified nucleoside monomer has the structure shown in formula (III-a2), or a stereoisomer thereof, or a tautomer thereof, or a pharmaceutically acceptable salt thereof:

[0116] In some alternative embodiments, the modified nucleoside monomer has the structure shown in formula (III-a2-1), or a stereoisomer thereof, or a tautomer thereof, or a pharmaceutically acceptable salt thereof:

[0117] In some alternative embodiments, the modified nucleoside monomer has the structure shown in formula (IIII-a1), or a stereoisomer thereof, or a tautomer thereof, or a pharmaceutically acceptable salt thereof:

[0118] In some alternative embodiments, the modified nucleoside monomer has the structure shown in formula (IIII-a2-1), or a stereoisomer thereof, or a tautomer thereof, or a pharmaceutically acceptable salt thereof:

[0119] In some alternative embodiments, the modified nucleoside monomer is selected from any of the following compounds:

[0120] In some alternative embodiments, the modified nucleoside monomer has the structure shown in formula (II-b), or a stereoisomer thereof, or a tautomer thereof, or a pharmaceutically acceptable salt thereof:

[0121] X is selected from O or S;

[0122] Y is selected from O or S;

[0123] The definitions of R6 and R7 are the same as above;

[0124] p and q are each independently selected from 0 or 1.

[0125] In some alternative implementations, p is selected from 1 and q is selected from 0;

[0126] L is selected from optionally substituted C15-C23 alkyl groups;

[0127] Base is selected from H or bases A, U, T, C or G;

[0128] R5 is selected from H or substituted or unsubstituted C1-C6 alkyl groups, wherein the substituents in the substituted C1-C6 alkyl groups are selected from halogens, hydroxyl groups or C1-C6 alkoxy groups.

[0129] In some alternative embodiments, R5 is selected from H or substituted or unsubstituted C1-C3 alkyl groups; the substituents in the substituted C1-C3 alkyl groups are selected from halogens, hydroxyl groups or C1-C3 alkoxy groups;

[0130] R2 is selected from R3 and R4 are independently selected from H or substituted or unsubstituted C1-C3 alkyl groups, wherein the substituents in the substituted C1-C3 alkyl groups are selected from halogens, hydroxyl groups or C1-C3 alkoxy groups; m is selected from 1.

[0131] In some alternative embodiments, the modified nucleoside monomer has the structure shown in formula (II-b-1), or a stereoisomer thereof, or a tautomer thereof, or a pharmaceutically acceptable salt thereof:

[0132] In some alternative embodiments, the modified nucleoside monomer has the structure shown in formula (III-b), or a stereoisomer thereof, or a tautomer thereof, or a pharmaceutically acceptable salt thereof:

[0133] In some alternative embodiments, the modified nucleoside monomer has the structure shown in formula (III-b-1), or a stereoisomer thereof, or a tautomer thereof, or a pharmaceutically acceptable salt thereof:

[0134] In some alternative embodiments, the modified nucleoside monomer has the structure shown in formula (IIII-b), or a stereoisomer thereof, or a tautomer thereof, or a pharmaceutically acceptable salt thereof:

[0135] In some alternative embodiments, the modified nucleoside monomer has the structure shown in formula (IIII-b-1), or a stereoisomer thereof, or a tautomer thereof, or a pharmaceutically acceptable salt thereof:

[0136] In some alternative embodiments, the modified nucleoside monomer is selected from any of the following compounds:

[0137] Double-stranded oligonucleotides

[0138] In a second aspect, this disclosure provides a double-stranded oligonucleotide, characterized in that the double-stranded oligonucleotide comprises a sense strand and an antisense strand, the antisense strand at least partially base-pairing with the sense strand to form a complementary double-stranded region, the sense strand and / or antisense strand containing at least one modified nucleotide having the structure shown in formula (i), or a stereoisomer thereof, or a tautomer thereof, or a pharmaceutically acceptable salt thereof:

[0139] Where Q is selected from hydroxyl or mercapto groups;

[0140] X is selected from O or S;

[0141] p and q are each independently selected from integers from 1 to 4;

[0142] Base is selected from H, or modified or unmodified bases;

[0143] R1 is selected from H or substituted or unsubstituted C1-C6 alkyl groups, wherein the substituents in the substituted C1-C6 alkyl groups are selected from halogens, hydroxyl groups or C1-C6 alkoxy groups;

[0144] R2 exists or does not exist; when R2 exists, R2 is selected from... R3 and R4 are independently selected from H or substituted or unsubstituted C1-C6 alkyl groups, wherein the substituents in the substituted C1-C6 alkyl groups are selected from halogens, hydroxyl groups or C1-C6 alkoxy groups; m is selected from 1;

[0145] One of Z1 and Z2 may exist, or both Z1 and Z2 may exist simultaneously.

[0146] Z1 and Z2 are selected independently. Among them, R z1 R z2 R z3 R z4 R z5 R z6 R z7 R z8 R z9 R z10 Each is independently selected from H or C1-C3 alkyl groups;

[0147] L is selected from optionally substituted C10-C23 alkyl groups or C10-C23 olefins containing at least one unsaturated bond.

[0148] In some alternative embodiments, the modified nucleotide has the structure shown in formula (ia) or (ib), or a stereoisomer thereof, a tautomer thereof, or a pharmaceutically acceptable salt thereof.

[0149] The substitutions of Q, X, R2, Z1 and Z2 are the same as in claim 1, and Z1 and Z2 are present simultaneously in the (ib) structural formula;

[0150] p and q are each independently selected from 0 or 1.

[0151] In some alternative implementations, p is selected from 1 and q is selected from 0.

[0152] In some alternative embodiments, R1 is selected from H or substituted or unsubstituted C1-C3 alkyl groups; the substituents in the substituted C1-C3 alkyl groups are selected from halogens, hydroxyl groups or C1-C3 alkoxy groups.

[0153] In some alternative embodiments, R1 is selected from H or methyl;

[0154] Base is selected from H or bases A, U, T, C or G;

[0155] L is selected from optionally substituted C15-C23 alkyl groups.

[0156] In some alternative embodiments, the antisense strand does not contain the modified nucleotide at positions 1-19, and the sense strand contains at least one of the modified nucleotides.

[0157] In some alternative embodiments, the modified nucleotide is located at positions 1-17 of the positive strand starting from the 5' end and positions 1-2 of the positive strand starting from the 3' end.

[0158] In some alternative embodiments, the modified nucleotide is located at positions 1-8 of the positive strand starting from the 5' end and position 1 of the positive strand starting from the 3' end.

[0159] In some alternative embodiments, the modified nucleotide is located at position 1, 4, 5, or 6 of the positive strand starting from the 5' end, or at position 1 of the positive strand starting from the 3' end.

[0160] Optionally, the modified nucleotide is located at the first or second position of the antisense strand, starting from the 3' end.

[0161] In some alternative implementations, the length of the sense strand and the antisense strand is each independently 15-25 nucleotides.

[0162] In some alternative embodiments, the double-stranded oligonucleotide is selected from siRNA.

[0163] In some alternative embodiments, the modified nucleotide has the structure shown in formula (ii-a1), or a stereoisomer thereof, or a tautomer thereof, or a pharmaceutically acceptable salt thereof:

[0164] Q is selected from hydroxyl or thiol groups;

[0165] X is selected from O or S;

[0166] Y is selected from O or S;

[0167] p and q are each independently selected from 0 or 1.

[0168] In some alternative implementations, p is selected from 1 and q is selected from 0;

[0169] L is selected from optionally substituted C15-C23 alkyl groups;

[0170] Base is selected from H or bases A, U, T, C or G;

[0171] R5 is selected from H or substituted or unsubstituted C1-C6 alkyl groups, wherein the substituents in the substituted C1-C6 alkyl groups are selected from halogens, hydroxyl groups or C1-C6 alkoxy groups.

[0172] In some alternative embodiments, R5 is selected from H or substituted or unsubstituted C1-C3 alkyl groups; the substituents in the substituted C1-C3 alkyl groups are selected from halogens, hydroxyl groups or C1-C3 alkoxy groups.

[0173] In some alternative embodiments, the modified nucleotide has the structure shown in formula (ii-a1-1), or a stereoisomer thereof, or a tautomer thereof, or a pharmaceutically acceptable salt thereof:

[0174] In some alternative embodiments, the modified nucleotide has the structure shown in formula (iii-a1), or a stereoisomer thereof, or a tautomer thereof, or a pharmaceutically acceptable salt thereof:

[0175] In some alternative embodiments, the modified nucleotide has the structure shown in formula (iii-a1-1), or a stereoisomer thereof, or a tautomer thereof, or a pharmaceutically acceptable salt thereof:

[0176] In some alternative embodiments, the modified nucleotide has the structure shown in formula (iiii-a1), or a stereoisomer thereof, or a tautomer thereof, or a pharmaceutically acceptable salt thereof:

[0177] In some alternative embodiments, the modified nucleotide has the structure shown in formula (iiii-a1-1), or a stereoisomer thereof, or a tautomer thereof, or a pharmaceutically acceptable salt thereof:

[0178] In some alternative embodiments of this disclosure, the modified nucleotide is selected from any of the following compounds:

[0179] In some alternative embodiments, the modified nucleotide has the structure shown in formula (ii-a2), or a stereoisomer thereof, or a tautomer thereof, or a pharmaceutically acceptable salt thereof:

[0180] Q is selected from hydroxyl or thiol groups;

[0181] X is selected from O or S;

[0182] Y is selected from O or S;

[0183] p and q are each independently selected from 0 or 1.

[0184] In some alternative implementations, p is selected from 1 and q is selected from 0;

[0185] L is selected from optionally substituted C15-C23 alkyl groups;

[0186] Base is selected from H or bases A, U, T, C or G;

[0187] R5 is selected from H or substituted or unsubstituted C1-C6 alkyl groups, wherein the substituents in the substituted C1-C6 alkyl groups are selected from halogens, hydroxyl groups or C1-C6 alkoxy groups.

[0188] In some alternative embodiments, R5 is selected from H or substituted or unsubstituted C1-C3 alkyl groups; the substituents in the substituted C1-C3 alkyl groups are selected from halogens, hydroxyl groups or C1-C3 alkoxy groups.

[0189] In some alternative embodiments, the modified nucleotide has the structure shown in formula (ii-a2-1), or a stereoisomer thereof, or a tautomer thereof, or a pharmaceutically acceptable salt thereof:

[0190] In some alternative embodiments, the modified nucleotide has the structure shown in formula (iii-a2), or a stereoisomer thereof, or a tautomer thereof, or a pharmaceutically acceptable salt thereof:

[0191] In some alternative embodiments, the modified nucleotide has the structure shown in formula (iii-a2-1), or a stereoisomer thereof, or a tautomer thereof, or a pharmaceutically acceptable salt thereof:

[0192] In some alternative embodiments, the modified nucleotide has the structure shown in formula (iiii-a1), or a stereoisomer thereof, or a tautomer thereof, or a pharmaceutically acceptable salt thereof:

[0193] In some alternative embodiments, the modified nucleotide has the structure shown in formula (iiii-a2-1), or a stereoisomer thereof, or a tautomer thereof, or a pharmaceutically acceptable salt thereof:

[0194] In some alternative embodiments, the modified nucleotide is selected from any of the following compounds:

[0195] In some alternative embodiments, the modified nucleotide has the structure shown in formula (ii-b), or a stereoisomer thereof, or a tautomer thereof, or a pharmaceutically acceptable salt thereof:

[0196] Q is selected from hydroxyl or thiol groups;

[0197] X is selected from O or S;

[0198] Y is selected from O or S;

[0199] p and q are each independently selected from 0 or 1.

[0200] In some alternative implementations, p is selected from 1 and q is selected from 0;

[0201] L is selected from optionally substituted C15-C23 alkyl groups;

[0202] Base is selected from H or bases A, U, T, C or G;

[0203] R5 is selected from H or substituted or unsubstituted C1-C6 alkyl groups, wherein the substituents in the substituted C1-C6 alkyl groups are selected from halogens, hydroxyl groups or C1-C6 alkoxy groups.

[0204] In some alternative embodiments, R5 is selected from H or substituted or unsubstituted C1-C3 alkyl groups; the substituents in the substituted C1-C3 alkyl groups are selected from halogens, hydroxyl groups or C1-C3 alkoxy groups;

[0205] R2 is selected from R3 and R4 are independently selected from H or substituted or unsubstituted C1-C3 alkyl groups, wherein the substituents in the substituted C1-C3 alkyl groups are selected from halogens, hydroxyl groups or C1-C3 alkoxy groups; m is selected from 1.

[0206] In some alternative embodiments, the modified nucleotide has the structure shown in formula (ii-b-1), or a stereoisomer thereof, or a tautomer thereof, or a pharmaceutically acceptable salt thereof:

[0207] In some alternative embodiments, the modified nucleotide has the structure shown in formula (iii-b), or a stereoisomer thereof, or a tautomer thereof, or a pharmaceutically acceptable salt thereof:

[0208] In some alternative embodiments, the modified nucleotide has the structure shown in formula (iii-b-1), or a stereoisomer thereof, or a tautomer thereof, or a pharmaceutically acceptable salt thereof:

[0209] In some alternative embodiments, the modified nucleotide has the structure shown in formula (iiii-b), or a stereoisomer thereof, or a tautomer thereof, or a pharmaceutically acceptable salt thereof:

[0210] In some alternative embodiments, the modified nucleotide has the structure shown in formula (iiii-b-1), or a stereoisomer thereof, or a tautomer thereof, or a pharmaceutically acceptable salt thereof:

[0211] In some alternative embodiments, the modified nucleotide is selected from any of the following compounds:

[0212] Composition

[0213] In a third aspect of this disclosure, a composition is provided comprising the double-stranded oligonucleotide described in the second aspect of this disclosure.

[0214] use

[0215] In a fourth aspect of this disclosure, this disclosure provides the use of the double-stranded oligonucleotide described in the second aspect of this disclosure, and the composition described in the third aspect, in the preparation of a medicament for treating and / or preventing pathological conditions or diseases caused by the abnormal expression of specific genes in target tissue-related cells.

[0216] In some alternative implementations, the target tissue is selected from the central nervous system, cardiac muscle, skeletal muscle, and fat.

[0217] Pharmaceutical Composition

[0218] In a fifth aspect of this disclosure, a pharmaceutical composition is provided comprising the double-stranded oligonucleotides described in the second aspect of this disclosure, and / or the compositions described in the third aspect, and optionally one or more pharmaceutically acceptable carriers or excipients.

[0219] Methods to suppress the expression of specific genes in cells

[0220] In a sixth aspect of this disclosure, a method for inhibiting the expression of a specific gene in target tissue-related cells is provided, the method comprising contacting the target tissue-related cells with the double-stranded oligonucleotide described in the second aspect of this disclosure, and / or the composition described in the third aspect, and / or the pharmaceutical composition described in the fifth aspect.

[0221] In some alternative implementations, the target tissue is selected from the central nervous system, cardiac muscle, skeletal muscle, and fat.

[0222] Treatment methods for diseases

[0223] In a seventh aspect of this disclosure, this disclosure provides a method for preventing and / or treating a pathological condition or disease caused by the abnormal expression of a specific gene in cells associated with a target tissue, the method comprising administering to a subject a pharmaceutically acceptable dose of the double-stranded oligonucleotide described in the second aspect of this disclosure, and / or the composition described in the third aspect, and / or the pharmaceutical composition described in the fifth aspect.

[0224] In some alternative embodiments, the target tissue is selected from the central nervous system, myocardium, skeletal muscle, and fat.

[0225] Methods to inhibit the expression of target genes in central nervous system cells, skeletal muscle cells, cardiomyocytes, or adipocytes

[0226] In an eighth aspect of this disclosure, a method for inhibiting the expression of a target gene in central nervous system cells, skeletal muscle cells, cardiomyocytes, or adipocytes is provided, the method comprising: contacting the cells with the double-stranded oligonucleotides described in the second aspect of this disclosure, and / or the composition described in the third aspect, and / or the pharmaceutical composition described in the fifth aspect.

[0227] Methods for the prevention and / or treatment of diseases or conditions related to the central nervous system, myocardium, skeletal muscle, and adipose tissue.

[0228] In a ninth aspect of this disclosure, this disclosure provides a method for preventing and / or treating diseases or conditions related to the central nervous system, myocardium, skeletal muscle, and adipose tissue, the method comprising: administering to the subject a therapeutically effective amount of the double-stranded oligonucleotide of the second aspect of this disclosure, and / or the composition of the third aspect, and / or the pharmaceutical composition of the fifth aspect, thereby treating the subject.

[0229] In some alternative implementations, the subject is a human being.

[0230] In some alternative implementations, the skeletal muscle-related disease or condition is muscular dystrophy.

[0231] In some alternative embodiments, the muscular atrophy is selected from the group consisting of: Duchenne muscular dystrophy, myotonic muscular atrophy, Becker muscular dystrophy, limb-girdle muscular atrophy, facioscapulohumeral muscular atrophy, congenital muscular atrophy, oculopharyngeal muscular atrophy, distal muscular atrophy, Emery-Dreifuss muscular dystrophy, myostatin-associated hypertrophy, congenital myasthenia gravis, and facioscapulohumeral muscular dystrophy (FSHD).

[0232] In some alternative embodiments, the myocardial-related diseases or conditions comprise the group consisting of: obstructive hypertrophic cardiomyopathy (HOCM); familial hypertrophic cardiomyopathy (FHC); heart failure with preserved ejection fraction (HFPEF); atrial fibrillation (AFIB); ventricular fibrillation (VFIB); angina pectoris; myocardial infarction (MI); heart failure with reduced ejection fraction (HFREF); supraventricular tachycardia (SVT); hypertrophic cardiomyopathy (HCM), dilated cardiomyopathy (DCM), arrhythmias, and congestive heart failure (CHF).

[0233] In some alternative embodiments, the adipose tissue-related disease or condition is a metabolic disease; the metabolic disease is selected from the group consisting of: lipid metabolism disorders, hypertension, cardiovascular disease, or overweight-related conditions.

[0234] In some alternative embodiments, the administration is selected from: intrathecal administration, subcutaneous administration, intramuscular administration, intravenous administration, intraperitoneal administration, or intravitreal administration.

[0235] In some alternative embodiments, the application results in the expression of the target gene being no more than 40%, no more than 50%, no more than 60%, or no more than 70% of the level at which no application was performed.

[0236] To make the objectives, technical solutions, and advantages of this application clearer, the implementation schemes of this application will be further described in detail below with reference to the embodiments.

[0237] Preparation of nucleoside monomers

[0238] Preparation Example 1: Synthesis of Compound NM075

[0239] In this preparation example, the synthetic route of compound NM075 is shown below:

[0240] (1-1) Synthesis of compound NM075-2

[0241] Compound NM075-1 (890 mg, 1.63 mmol, 1 eq, CAS No. 174221-86-4) was dissolved in dichloromethane (10 mL, abbreviated as DCM), and octadecanoic acid (585.1 mg, 1.96 mmol, 1.2 eq, CAS No. 57-11-4), 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (1.24 g, 3.26 mmol, 2 eq, abbreviated as HATU, CAS No. 148893-10-1), and triethylamine (493.9 mg, 4.89 mmol, 3 eq, abbreviated as TEA) were added. The mixture was stirred at 25 °C for 0.5 hours. After the reaction was complete, saturated ammonium chloride aqueous solution (30 mL) was added to the reaction solution, followed by extraction with dichloromethane (3 × 20 mL). The organic phases were combined, washed with saturated sodium chloride aqueous solution (2 × 20 mL), dried over anhydrous sodium sulfate, filtered, concentrated, and purified by reversed-phase column chromatography (eluent: methanol / water = 1 / 0, v / v) to give a yellow solid compound NM077-2 (1.26 g, yield 95.4%). MS ESI (m / z) = 812.5 [M+H] + .

[0242] (1-2) Synthesis of compound NM075

[0243] Compound NM075-2 (1.26 g, 1.55 mmol, 1 eq) was dried three times with acetonitrile (10 mL each time) and then dissolved in dichloromethane (15 mL). A solution of bis(diisopropylamino)(2-cyanoethoxy)phosphine (701.5 mg, 2.33 mmol, 1.5 eq) dried three times with acetonitrile (10 mL each time) in dichloromethane (5 mL) was added. 1H-imidazolium-4,5-dianitrile (146.3 mg, 1.24 mmol, 0.8 eq, CAS No. 1122-28-7) was added. The mixture was purged with nitrogen three times, and the reaction system was stirred at 25 °C for 1 hour under a nitrogen atmosphere. After the reaction was complete, the reaction solution was diluted with 20 mL of saturated sodium bicarbonate aqueous solution, extracted with dichloromethane (3 × 10 mL), and the organic phases were combined. The organic phases were washed with 2 × 10 mL of saturated sodium chloride aqueous solution, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by reversed-phase column chromatography (elution: acetonitrile / water = 1 / 0, v / v) to give a white solid, compound NM075 (1.02 g, yield 65.1%). MS ESI (m / z) = 1012.6 [M+H]+ .

[0244] Preparation Example 2: Synthesis of Compound NM076

[0245] In this preparation example, the synthetic route of compound NM076 is shown below:

[0246] (2-1) Synthesis of compound NM076-2

[0247] Compound 1 (890 mg, 1.63 mmol, 1 eq, CAS No. 174221-86-4) was dissolved in dichloromethane (10 mL), and hexadecanoic acid (501.7 mg, 1.96 mmol, 1.2 eq, CAS No. 57-10-3), HATU (1.24 g, 3.26 mmol, 2 eq), and triethylamine (493.9 mg, 4.89 mmol, 3 eq) were added. The mixture was stirred at 25 °C for 0.5 hours. After the reaction was complete, saturated ammonium chloride aqueous solution (30 mL) was added to the reaction solution, and the mixture was extracted with dichloromethane (3 × 20 mL). The organic phases were combined, washed with saturated sodium chloride aqueous solution (2 × 20 mL), dried over anhydrous sodium sulfate, filtered, concentrated, and purified by reversed-phase column chromatography (eluent: methanol / water = 1 / 0, v / v) to give compound NM076-2 (1.14 g, yield 89.4%) as a yellow solid. MS ESI (m / z) = 785.5 [M+H] + .

[0248] (2-2) Synthesis of compound NM076

[0249] Compound NM076-2 (1.14 g, 1.45 mmol, 1 eq) was dried three times with acetonitrile (10 mL each time) and dissolved in dichloromethane (15 mL). A solution of bis(diisopropylamino)(2-cyanoethoxy)phosphine (658.3 mg, 2.18 mmol, 1.5 eq) in dichloromethane (5 mL) was added, dried three times with acetonitrile (10 mL each time). 1H-imidazolium-4,5-dianitrile (137.2 mg, 1.16 mmol, 0.8 eq) was added. The mixture was purged with nitrogen three times, and the reaction system was stirred at 25 °C for 1 hour under a nitrogen atmosphere. After the reaction was complete, the reaction solution was diluted with 20 mL of saturated sodium bicarbonate aqueous solution, extracted with dichloromethane (3 × 10 mL), and the organic phases were combined. The organic phases were washed with 2 × 10 mL of saturated sodium chloride aqueous solution, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography (elution: petroleum ether / ethyl acetate = 2 / 3, v / v) to give a white solid, compound NM076 (700 mg, yield 49.0%). MS ESI (m / z) = 984.4 [M+H] + .

[0250] 1 H NMR (400MHz, DMSO-d6) δ11.40(dd,J=4.9,2.2Hz,1H),7.99(t,J=9.4Hz,1H),7.65(d,J=8.1Hz,1H),7.43–7.29(m,4H),7.26( dt,J=8.9,3.3Hz,5H),6.94–6.82(m,4H),5.98–5.88(m,1H),5.41(dt,J=8.1,2.4Hz,1H),4.83(dq,J=27.3,7.8Hz,1H),4.51– 4.10(m,2H),3.74(s,7H),3.53(dq,J=37.4,6.4Hz,2H),3.27(d,J=3.5Hz,1H),2.78–2.63(m,2H),2.20–2.00(m,2H),1.47(s, 2H), 1.22 (d, J = 8.8Hz, 28H), 1.11 (dd, J = 6.8, 2.4Hz, 5H), 1.06 (d, J = 6.7Hz, 3H), 0.97 (d, J = 6.7Hz, 2H), 0.85 (t, J = 6.5Hz, 3H).

[0251] Preparation Example 3: Synthesis of Compound NM077

[0252] In this preparation example, the synthetic route of compound NM077 is shown below:

[0253] (3-1) Synthesis of compound NM077-2

[0254] Compound 1 (940 mg, 1.72 mmol, 1 eq, CAS No. 174221-86-4) was dissolved in dichloromethane (10 mL), and then nonadecanoic acid (617.6 mg, 2.06 mmol, 1.2 eq, CAS No. 646-30-0), 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (1.31 g, 3.44 mmol, 2 eq, abbreviated as HATU, CAS No. 148893-10-1) and triethylamine (522.4 mg, 5.16 mmol, 3 eq, CAS No. 121-44-8) were added. The mixture was stirred at 25 °C for 0.5 hours. After the reaction was complete, saturated ammonium chloride aqueous solution (30 mL) was added to the reaction solution, followed by extraction with dichloromethane (3 × 20 mL). The organic phases were combined, washed with saturated brine (2 × 20 mL), dried over anhydrous sodium sulfate, filtered, concentrated, and purified by reversed-phase column chromatography (elution gradient, methanol:water = 1:0) to give compound NM077-2 (1.32 g, yield 93.3%) as a yellow solid. MS ESI (m / z) = 826.5 [M+H] + .

[0255] (3-2) Synthesis of compound NM-077

[0256] Compound NM077-2 (1.22 g, 1.48 mmol, 1 eq) was repeatedly dried with acetonitrile (3 × 10 mL) and dissolved in dichloromethane (15 mL). A solution of bis(diisopropylamino)(2-cyanoethoxy)phosphine (669.5 mg, 2.22 mmol, 1.5 eq, CAS No. 102691-36-1) in dichloromethane (5 mL) was added, followed by 1H-imidazolium-4,5-dianitrile (139.5 mg, 1.18 mmol, 0.8 eq, CAS No. 1122-28-7). The mixture was purged with nitrogen three times, and the reaction solution was stirred at 25 °C for 1 hour under a nitrogen atmosphere. After the reaction was complete, the reaction solution was diluted with 20 mL of saturated sodium bicarbonate aqueous solution, extracted with dichloromethane (3 × 10 mL), the organic phases were combined, washed with 2 × 10 mL of saturated sodium chloride aqueous solution, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by reversed-phase column chromatography (elution gradient, acetonitrile:water = 1:0) to give compound NM077 (1.15 g, yield 75.9%) as a white solid. MS ESI (m / z) = 1026.6 [M+H] + .

[0257] 1H NMR (400MHz, DMSO-d6) δ11.40(t,J=3.1Hz,1H),7.98(t,J=9.5Hz,1H),7.65(d,J=8.1Hz,1H),7.36(ddt,J=40.5,7.8,3.9Hz,4H),7.26 (dt,J=8.8,3.2Hz,5H),6.88(dd,J=8.9,2.4Hz,4H),6.02–5.89(m,1H),5.41(dt,J=8.3,2.3Hz,1H),4.99–4.75(m,1H),4.47–4.10(m, 2H),3.73(s,8H),3.63–3.41(m,2H),3.28(d,J=3.4Hz,1H),2.75(t,J=6.0Hz,1H),2.65(t,J=6.1Hz,1H),2.21–1.98(m,2H),1.48(d,J =15.5Hz,2H),1.22(d,J=8.5Hz,32H),1.11(dd,J=6.8,2.4Hz,6H),1.06(d,J=6.7Hz,3H),0.97(d,J=6.7Hz,2H),0.85(t,J=6.7Hz,3H).

[0258] Preparation Example 4: Preparation of Compound NM078

[0259] In this preparation example, the synthetic route of compound NM078 is shown below:

[0260] (4-1) Synthesis of compound NM078-2

[0261] Compound 1 (940 mg, 1.72 mmol, 1 eq, CAS No. 174221-86-4) was dissolved in 10 mL of dichloromethane. Eicosanoic acid (646.6 mg, 2.06 mmol, 1.2 eq, CAS No. 506-30-9), HATU (1.31 g, 3.44 mmol, 2 eq, CAS No. 148893-10-1), and triethylamine (522.4 mg, 5.16 mmol, 3 eq) were added, and the mixture was stirred at 25 °C for 0.5 hours. After the reaction was complete, 30 mL of saturated ammonium chloride aqueous solution was added to the reaction mixture, followed by extraction with 3 × 20 mL of dichloromethane. The organic phases were combined, washed with 2 × 20 mL of saturated sodium chloride aqueous solution, dried over anhydrous sodium sulfate, filtered, concentrated, and slurried with acetonitrile to obtain a yellow solid compound NM078-2 (1.22 g, yield 84.3%). MS ESI (m / z) = 840.5 [M+H] + .

[0262] (4-2) Synthesis of compound NM078

[0263] Compound NM078-2 (1.22 g, 1.45 mmol, 1 eq) was repeatedly dried with acetonitrile (3 × 10 mL), dissolved in dichloromethane (15 mL), and a solution of bis(diisopropylamino)(2-cyanoethoxy)phosphine (658.3 mg, 2.18 mmol, 1.5 eq, CAS No. 102691-36-1) in dichloromethane (5 mL) was added. 1H-imidazolium-4,5-dianitrile (137.2 mg, 1.16 mmol, 0.8 eq, CAS No. 1122-28-7) was added. The mixture was purged with nitrogen three times, and the reaction solution was stirred at 25 °C for 1 hour under a nitrogen atmosphere. After the reaction was complete, the reaction solution was diluted with 20 mL of saturated sodium bicarbonate aqueous solution, extracted with dichloromethane (3 × 10 mL), the organic phases were combined, washed with 2 × 10 mL of saturated sodium chloride aqueous solution, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography (eluent: petroleum ether / ethyl acetate = 2 / 3, v / v) to obtain NM078 as a white solid (723 mg, yield 47.8%). MS ESI (m / z) = 1040.4 [M+H] + .

[0264] 1 H NMR (400MHz, DMSO-d6) δ11.40(dd,J=4.9,2.2Hz,1H),7.99(t,J=9.4Hz,1H),7.65(d,J=8.1Hz,1H),7.43–7.29(m,4H),7.26( dt,J=8.9,3.3Hz,5H),6.94–6.82(m,4H),5.98–5.88(m,1H),5.41(dt,J=8.1,2.4Hz,1H),4.83(dq,J=27.3,7.8Hz,1H),4.51– 4.10(m,2H),3.74(s,7H),3.53(dq,J=37.4,6.4Hz,2H),3.27(d,J=3.5Hz,1H),2.78–2.63(m,2H),2.20–2.00(m,2H),1.47(s, 2H), 1.22 (d, J = 8.8Hz, 36H), 1.11 (dd, J = 6.8, 2.4Hz, 5H), 1.06 (d, J = 6.7Hz, 3H), 0.97 (d, J = 6.7Hz, 2H), 0.85 (t, J = 6.5Hz, 3H).

[0265] Preparation Example 5: Synthesis of Compound NM088

[0266] In this preparation example, the synthetic route of compound NM088 is shown below:

[0267] (5-1) Synthesis of compound NM088-2

[0268] Compound 1 (5.0 g, 9.17 mmol, 1 eq, CAS No. 174221-86-4) was dissolved in N,N-dimethylformamide (100 mL, abbreviated as DMF), and N-fluorenylmethoxycarbonyl-L-alanine (3.42 g, 11.0 mmol, 1.2 eq, CAS No. 35661-39-3), HATU (6.97 g, 18.34 mmol, 2 eq, CAS No. 148893-10-1) and triethylamine (2.78 g, 27.51 mmol, 3 eq) were added. The mixture was stirred at 25 °C for 0.5 hours. After the reaction was complete, 100 mL of saturated ammonium chloride aqueous solution was added to the reaction solution, followed by extraction with dichloromethane (3 × 120 mL). The organic phases were combined, washed with 2 × 120 mL of saturated sodium chloride aqueous solution, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by reversed-phase column chromatography (eluent: methanol / water = 1 / 0) to give a yellow solid compound NM088-2 (5.2 g, yield 67.6%). MS ESI (m / z) = 839.5 [M+H] + .

[0269] (5-2) Synthesis of compound NM088-3

[0270] Compound NM088-2 (5.2 g, 6.2 mmol, 1 eq) was dissolved in DMF (50 mL), and piperidine (1.06 g, 12.4 mmol, 2 eq, CAS No. 110-89-4) was added dropwise. The mixture was purged with nitrogen three times, and the reaction system was stirred at 25 °C for 1 hour under a nitrogen atmosphere. After the reaction was complete, the reaction solution was diluted with saturated sodium bicarbonate aqueous solution (20 mL), extracted with dichloromethane (3 × 100 mL), the organic phases were combined, washed with saturated sodium chloride aqueous solution (2 × 100 mL), dried over anhydrous sodium sulfate, filtered, concentrated, and purified by reversed-phase column chromatography (elution: acetonitrile / water = 1 / 0) to give compound NM088-3 (3.2 g, yield 83.8%) as a white solid. MS ESI (m / z) = 617.6 [M+H] + .

[0271] (5-3) Synthesis of compound NM088-4

[0272] Compound NM088-3 (3.2 g, 5.19 mmol, 1 eq) was dissolved in DMF (32 mL), and tetradecanoic acid (1.86 g, 6.23 mmol, 1.2 eq, CAS No. 646-30-0), HATU (3.94 g, 10.38 mmol, 2 eq), and triethylamine (1.57 g, 15.57 mmol, 3 eq) were added. The mixture was stirred at 25 °C for 0.5 hours. After the reaction was complete, saturated ammonium chloride aqueous solution (100 mL) was added to the reaction solution, and the mixture was extracted with dichloromethane (3 × 120 mL). The organic phases were combined, washed with saturated sodium chloride aqueous solution (2 × 120 mL), dried over anhydrous sodium sulfate, filtered, concentrated, and purified by reversed-phase column chromatography (eluent: acetonitrile / water = 1 / 0, v / v) to give compound NM088-4 (4.2 g, yield 90.3%) as a yellow solid. MS ESI (m / z) = 897.5 [M+H] + .

[0273] (5-4) Synthesis of compound NM088

[0274] Compound NM088-4 (4.2 g, 4.69 mmol, 1 eq) was repeatedly dried with acetonitrile (3 × 40 mL) and dissolved in dichloromethane (80 mL). A solution of bis(diisopropylamino)(2-cyanoethoxy)phosphine (2.12 g, 7.04 mmol, 1.5 eq) in dichloromethane (20 mL) was added, along with 4,5-dicyanimidazole (442.7 mg, 3.75 mmol, 0.8 eq, CAS No. 1122-28-7). The mixture was purged with nitrogen three times, and the reaction system was stirred at 25 °C for 1 hour under a nitrogen atmosphere. After the reaction was complete, the reaction solution was diluted with 50 mL of saturated sodium bicarbonate aqueous solution, extracted with dichloromethane (3 × 100 mL), the organic phases were combined, washed with 2 × 50 mL of saturated sodium chloride aqueous solution, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by reversed-phase column chromatography (elution: acetonitrile / water = 1 / 0) to give a white solid compound NM088 (4.15 g, yield 80.7%). MS ESI (m / z) = 1097.6 [M+H] + .

[0275] Preparation Example 6: Synthesis of Compound NM089

[0276] In this preparation example, the synthetic route of compound NM089 is shown below:

[0277] (6-1) Synthesis of compound NM089-2

[0278] Compound 1 (3 g, 15.3 mmol, 1.0 eq) was dissolved in pyridine (150 mL), and triisopropylchlorosilane (6 g, 3.0 eq, TIDPSCl, CAS No. 13154-24-0) was added under ice bath conditions. The mixture was stirred at 25 °C for 4 hours, and then quenched with methanol (100 mL). After the reaction was complete, the reaction solution was extracted twice with ethyl acetate (100 mL × 2), the organic phases were combined, washed with saturated sodium chloride aqueous solution (2 × 50 mL), dried over anhydrous sodium sulfate, filtered, concentrated, and purified by normal phase chromatography (EA / PE = 0–40%, v / v) to give a white solid compound NM089-2 (3 g, yield 75%). MS ESI (m / z) = 487 [M+H] + .

[0279] (6-2) Synthesis of compound NM089-3

[0280] Compound NM089-2 (3 g, 11.19 mmol, 1.0 eq) was dissolved in acetonitrile (30 mL), and 2-iodobenzoic acid (3.7 g, 11.19 mmol, 1.0 eq, IBX, CAS No. 64297-64-9) was added. The mixture was stirred at 80 °C for 5 hours. After the reaction was complete, the reaction solution was filtered, and the filter cake was washed twice with ethyl acetate. The filtrate was concentrated to obtain a pale yellow solid, compound NM089-3 (1.9 g), which was used directly in the next reaction without purification. MS ESI (m / z) = 485 [M+H] + .

[0281] (6-3) Synthesis of compound NM089-4

[0282] Methyltriphenylphosphine bromide (200 mg, 0.33 mmol, 1 eq, CAS No. 1779-49-3) was dissolved in anhydrous tetrahydrofuran (THF, 10 mL), purged three times with nitrogen, and potassium tert-butoxide (67 mg, 0.66 mmol, 2 eq) was added under nitrogen protection in an ice bath with stirring for 0.5 hours. A THF solution of compound NM089-3 (1.9 g) was added dropwise, and the reaction was carried out at 25 °C for 2 hours. After the reaction was complete, the reaction solution was purified by normal phase (EA / PE = 0–40%, v / v) to give a white powder of compound NM089-4 (160 mg). MS ESI (m / z) = 505 [M + Na] + .

[0283] (6-4) Synthesis of compound NM089-5

[0284] The compound NM089-4 (500 mg, 1.03 mmol, 1 eq) and 1,1,2,2-tetramethyl-1,2-diaminoethane-N,N'-bis(3,5-di-tert-salicyl)cobalt(II) (20 mg, 0.035 mmol, 0.035 eq, CAS No. 157472-96-3) were dissolved in p-toluenesulfonyl azide (CAS No. 941-55-9). The mixture was stirred at 25 °C for 5 minutes, and a 3 ml ethanol solution of phenylsilane (121 mg, 1.11 mmol, CAS No. 694-53-1) was added dropwise. The reaction was carried out at 25 °C for 0.5 hours and quenched with saturated sodium chloride solution. After the reaction was complete, the reaction solution was extracted three times with ethyl acetate. The combined organic phases were washed with saturated sodium chloride aqueous solution (2 × 50 mL), dried over anhydrous sodium sulfate, filtered, concentrated, and purified by normal-phase chromatography to give compound NM089-5 (197 mg). MS ESI (m / z) = 526 [M + H] + .

[0285] (6-5) Synthesis of compound NM089-6

[0286] Compound NM089-5 (190 mg, 0.33 mmol, 1 eq) was dissolved in anhydrous THF (2 ml), and tetrabutylammonium fluoride (32 mg, 1.5 eq, TBAF, CAS No. 429-41-4) was added. The reaction was carried out at room temperature for 2 hours. After the reaction was completed, the reaction solution was purified by reverse phase to give compound NM089-6 (100 mg). MS ESI (m / z) = 284 [M+H] + .

[0287] (6-6) Synthesis of compound NM089-7

[0288] Compound NM089-6 (100 mg, 0.35 mmol, 1 eq) was dissolved in pyridine (5 mL), and 4,4'-dimethoxytriphenylchloromethane (155 mg, 1.3 eq, DMTrCl, CAS No. 40615-36-9) was added at low temperature. The reaction was carried out at 25 °C for 3 hours, and quenched with methanol. After the reaction was completed, the reaction solution was extracted three times with ethyl acetate (20 mL each time). The organic phases were combined, washed with saturated sodium chloride aqueous solution (2 × 50 mL), dried over anhydrous sodium sulfate, filtered, concentrated, and purified in normal phase to give compound NM089-7 (200 mg) as a yellow solid. MS ESI (m / z) = 586 [M+H] + .

[0289] (6-7) Synthesis of compound NM089-8

[0290] Compound NM089-8 (200 mg, 0.34 mmol, 1 eq) was dissolved in methanol (10 mL), and wet palladium on carbon (20 mg, 10 wt%) was added. The mixture was purged with hydrogen three times, and the reaction was carried out at 25 °C for 5 hours under a hydrogen atmosphere. After the reaction was complete, the reaction solution was filtered, and the filtrate was concentrated to give compound NM089-8 (100 mg) as a white solid. MS ESI (m / z) = 560 [M+H] + .

[0291] (6-8) Synthesis of compound NM089-9

[0292] Compound NM089-8 (100 mg, 0.18 mmol, 1 eq) was dissolved in DMF (5 ml), and HATU (100 mg, 1.5 eq), N,N'-diisopropylethylamine (70 mg, 3 eq, DIEA, CAS No. 7087-68-5), and stearic acid (53 mg, 1.1 eq, CAS No. 57-11-4) were added separately. The reaction was carried out at 25 °C for 2 hours. After the reaction was completed, the reaction solution was purified by reverse phase to obtain a white solid compound NM089-9 (150 mg). MS ESI (m / z) = 812 [M+H] + .

[0293] (6-9) Synthesis of compound NM089

[0294] Compound NM089-9 (100 mg, 0.12 mmol, 1 eq) was dissolved in ultra-dry DCM (2 ml), and 4,5-dicyanimidazole (12 mg, 0.8 eq, DCI, CAS No. 1122-28-7) and bis(diisopropylamino)(2-cyanoethoxy)phosphine (40 mg, 1.1 eq) were added separately. The reaction mixture was purged with nitrogen three times, and the reaction was carried out at 25 °C for 2 hours under a nitrogen atmosphere. After the reaction was completed, the mixture was extracted with DCM and saturated sodium bicarbonate aqueous solution. The organic phase was dried over anhydrous sodium sulfate, filtered, concentrated, and purified by reverse phase to obtain compound NM089 (80 mg) as a white solid. MS ESI (m / z) = 1012 [M+H] + .

[0295] siRNA synthesis

[0296] Unless otherwise stated, all reagents, reagent consumables, and instruments used in this disclosure are commercially available. The main reagents and consumables are shown in Table 1, and the main instruments and equipment are shown in Table 2.

[0297] Table 1 Main Reagents and Consumables

[0298] Table 2 Main Instruments and Equipment

[0299] Preparation Example 7: Synthesis of siRNA

[0300] (7-1) Synthesis of the Chain of Justice (SS)

[0301] The phosphoramidite solid-phase synthesis method involves sequentially linking nucleoside monomers along the 3'-5' direction according to the nucleotide sequence. Each linking of a nucleoside monomer involves four steps: deprotection, coupling, capping, and oxidation or sulfidation. The synthetic conditions are given below:

[0302] The nucleoside monomer was prepared into an acetonitrile solution with a concentration of 0.1 M.

[0303] The deprotection reaction conditions were the same for each step. The deprotection reaction conditions were: temperature 25℃, reaction time 70 seconds, deprotection reagent was a dichloromethane solution of dichloroacetic acid (3% by volume), and the molar ratio of dichloroacetic acid to the 4,4'-dimethoxytriphenylmethyl protecting group on the solid support was 5:1.

[0304] The conditions for each coupling reaction were identical. The coupling reaction conditions were as follows: temperature 25℃, molar ratio of nucleic acid sequence to nucleoside monomer on the solid-phase support 1:10, molar ratio of nucleic acid sequence to coupling reagent on the solid-phase support 1:65, reaction time 600 seconds, coupling reagent 0.5M acetonitrile solution of 5-ethylthio-1H-tetrazole, and thioreagent 0.2mol / L acetonitrile / pyridine mixed solution of hydrogenated xanthanin (acetonitrile and pyridine volume ratio 1:1).

[0305] The conditions for each capping reaction were identical. The conditions for the capping reaction were: temperature 25℃; reaction time 2 minutes; the capping reagent solution was a 1:1 molar ratio of Cap1 and Cap2, where Cap1 was a 20% (v / v) N-methylimidazole pyridine / acetonitrile mixture with a pyridine to acetonitrile volume ratio of 3:5, and Cap2 was a 20% (v / v) acetic anhydride acetonitrile solution; the molar ratio of N-methylimidazole in Cap1 and acetic anhydride in Cap2 to the nucleic acid sequence linked on the solid-phase support was 1:1:1.

[0306] The conditions for each oxidation reaction were identical. The oxidation reaction conditions were: temperature 25°C; reaction time 3 seconds; oxidizing agent concentration of 0.05M iodine solution, with a molar ratio of iodine to the nucleic acid sequence linked on the solid support in the coupling reaction of 30:1; the oxidation reaction was carried out in a water / pyridine mixed solvent (water to pyridine volume ratio 1:9). The sulfidation reaction conditions were: temperature 25°C; reaction time 360 ​​seconds; thioreagent concentration of 0.2M hydroflavin in pyridine solution, with a molar ratio of thioreagent to the nucleic acid sequence linked on the solid support in the coupling reaction of 4:1; the thioreagent reaction was carried out in a water / pyridine mixed solvent (water to pyridine volume ratio 1:9).

[0307] After the last nucleoside monomer was ligated, the nucleic acid sequence ligated on the solid-phase support was sequentially cut, deprotected, purified, and desalted, and then freeze-dried to obtain the positive strand, wherein:

[0308] The cleavage and deprotection conditions were as follows: The synthesized nucleotide sequence linked to a solid-phase support was added to 25% (w / w) ammonia solution at a concentration of 0.5 mL / μmol. The reaction was carried out at 55 °C for 16 hours. The solvent was removed, and the solution was concentrated to dryness under vacuum. After ammonia treatment, the product was dissolved in 0.4 mL / μmol N-methylpyrrolidone relative to the amount of single-stranded nucleic acid. Subsequently, 0.3 mL / μmol triethylamine and 0.6 mL / μmol triethylamine trifluoride were added to deprotect the 2'-O-TBDMS protection on the ribose.

[0309] Purification and desalting conditions: Nucleic acid purification was performed using a preparative ion chromatography column (Source 15Q) with a NaCl gradient elution. Specifically: eluent 1 was 20 mM sodium phosphate (pH = 8.1), and the solvent was a water / acetonitrile mixture (water to acetonitrile volume ratio of 9:1); eluent 2 was 1.5 M sodium chloride, 20 mM sodium phosphate (pH = 8.1), and the solvent was a water / acetonitrile mixture (water to acetonitrile volume ratio of 9:1); the elution gradient was eluent 1: eluent 2 = (100:0) - (50:50). The product eluates were collected and combined, and desalting was performed using a reverse chromatographic purification column. Desalting conditions included using a dextran gel column (g25 dextran gel) and elution with deionized water.

[0310] Detection: Purity was determined using ion exchange chromatography (IEX-HPLC); molecular weight was determined using liquid chromatography-mass spectrometry (LC-MS). The measured molecular weight was compared with the theoretical value. If the measured value and the theoretical value were consistent, it indicated that the compound was conjugated to the 3' end of the positive strand of the siRNA.

[0311] (7-2) Synthesis of antisense strand (AS)

[0312] The phosphoramidite solid-phase synthesis method for nucleic acids utilizes the compounds (CR01004Z, CR01005Z, CR01007Z) linked to the solid-phase support as the starting cycle, and sequentially links nucleoside monomers along the 3'-5' direction according to the nucleotide sequence. Each linkage of a nucleoside monomer involves four steps: deprotection, coupling, capping, and oxidation or sulfidation. The deprotection, coupling, capping, oxidation or sulfidation reaction conditions, cleavage and deprotection conditions, purification and desalting conditions in the solid-phase synthesis of the antisense strand are the same as those in step (7-1) for the synthesis of the sense strand.

[0313] Detection: Purity was determined using ion exchange chromatography (IEX-HPLC); molecular weight was determined using liquid chromatography-mass spectrometry (LC-MS). The measured molecular weight was compared with the theoretical value. If the measured value and the theoretical value were consistent, it indicated that the siRNA antisense strand had been obtained.

[0314] (7-3) Synthesis of siRNA

[0315] The sense strand synthesized in step (7-1) and the antisense strand synthesized in step (7-2) are mixed in an equimolar ratio, dissolved in water for injection, and heated to 95°C. The mixture is then slowly cooled to room temperature and kept at room temperature for 10 minutes to allow the sense and antisense strands to form a double-stranded structure through hydrogen bonds, thereby obtaining siRNA with the sense and antisense strands shown in Table 3.

[0316] Table 3a Information on the unmodified nucleotide sequence of siRNA formation

[0317] Table 3 Sequence information of siRNA

[0318] Unless otherwise specified, the base composition and modifications described in this disclosure have the following meanings: Uppercase letters A, U, G, C, and T represent the base composition of a nucleotide; lowercase letter m indicates that the nucleotide adjacent to the left of m is 2'-O-methyl modified; lowercase letter f indicates that the nucleotide adjacent to the left of f is 2'-fluoro modified; (moe) indicates that the nucleotide adjacent to the left of (moe) is 2'-O-methoxyethyl modified; lowercase letter s indicates that the two nucleotides adjacent to s are linked by a phosphate thioester bond. VP indicates that the 5' end of the antisense strand in siRNA is modified with 5'-(E)-vinylphosphonate (5'-(E)-VP).

[0319] The structural formula of the nucleotide modified with 2'-O-methyl is

[0320] The structural formula of the 2'-fluorinated nucleotide is

[0321] The structural formula of the nucleotide modified with 2'-O-methoxyethyl is:

[0322] The structural formula of VPUm is:

[0323] (Uhd) represents a nucleotide with the following structural formula:

[0324] The structural formula of its nucleoside monomer Uhd is as follows:

[0325] The nucleotide structure represented by (NM075) is:

[0326] The nucleotide structure represented by (NM077) is as follows:

[0327] The nucleotide structure represented by (NM076) is:

[0328] The nucleotide structure represented by (NM078) is:

[0329] The nucleotide structure represented by (NM088) is:

[0330] The nucleotide structure represented by (NM089) is:

[0331] Where Base represents the bases A, U, G, C, and T. Q represents a hydroxyl or thiol group.

[0332] Table 4. Detection results of siRNA

[0333] As can be seen from the data in Table 4, the siRNA obtained in Table 3 of this disclosure can maintain a high purity.

[0334] Biological testing experiments

[0335] Unless otherwise stated, all siRNA sequences used in this disclosure were synthesized by Suzhou Beixin Biotechnology Co., Ltd.; all PCR primers used in this disclosure were synthesized by Beijing Qingke Biotechnology Co., Ltd.; and all experimental animals used in this disclosure, namely C57BL / 6J mice and SD rats, were purchased from Spiford (Beijing) Biotechnology Co., Ltd.

[0336] A method for evaluating the inhibitory activity of siRNA conjugates on target genes in the left and right hemispheres of mice based on lateral ventricle administration:

[0337] Six- to eight-week-old C57BL / 6j mice were randomly divided into several test groups and one blank control group (PBS control group) according to body weight. Drug administration was performed via the lateral ventricle. Each mouse in the test group was given PBS buffer containing the siRNA conjugate; each mouse in the PBS control group was given PBS buffer without the siRNA conjugate. Using the Bregma point in the mouse brain as the zero point, the coordinates of the right ventricle were located using a stereotaxic instrument, and the position was marked on the skull surface (lateral ventricle location: AP (anteroposterior): -0.58mm, ML (mediolateral): -1.2mm, DV (dorsoventral): -2.2mm). A hole was drilled in the skull above the target location using a dental drill. The drug was then injected into the lateral ventricle using a micro-infusion pump, with a total injection volume of 5μL over 10 minutes. The needle was left in place for 5 minutes after injection, then slowly withdrawn. When the DV reached -1.0mm, the needle was left in place for 1 minute, and then slowly withdrawn until completely removed. The needle hole was then sealed with bio-adhesive, and the head skin was secured. After the animal awoke, it was returned to the animal house for continued care. The day of drug administration was designated as day 0 (D0). Mice were euthanized at the observation point after drug administration. The left and right hemispheres were harvested and preserved in RNAlaters, respectively.

[0338] A method for evaluating the inhibitory activity of siRNA conjugates on target genes in mice based on subcutaneous abdominal administration:

[0339] Six- to eight-week-old C57BL / 6J mice (all female) were randomly assigned to groups based on body weight, including several test groups and one control group (PBS control group). Drug administration was performed subcutaneously via abdominal injection. Mice in the test groups received PBS buffer containing the siRNA conjugate, while mice in the PBS control group received PBS buffer without the siRNA conjugate. The day of administration was designated as day 0 (D0). At a predetermined time after administration, five mice from each group were sacrificed. Gross dissection was performed on the sacrificed mice, and target tissue was collected from each mouse. The target tissue was cut into pieces approximately 2 mm in size. 3 Small pieces, stored using RNA Later.

[0340] A method for evaluating the inhibitory activity of siRNA conjugates on target genes in mice based on intrathecal administration:

[0341] Six- to eight-week-old SD rats were randomly divided into several test groups and one blank control group (PBS control group) according to body weight. Intrathecal administration was used. Each rat in the test groups was given PBS buffer containing siRNA conjugates, while each rat in the PBS control group was given PBS buffer without siRNA conjugates. The day of administration was designated D0. At a predetermined time after administration, five mice from each group were sacrificed. Different spinal cord segments (lumbar, thoracic, and cervical vertebrae), as well as cerebral cortex, hippocampus, and cerebellum tissues were collected and preserved in RNA Later.

[0342] RNA extraction and detection

[0343] RNA extraction: The tissue samples were removed from the RNAlater and homogenized for 60 seconds in a Tissuelyser II fully automated tissue homogenizer. Total RNA was extracted from each tissue sample using a fully automated nucleic acid extractor and nucleic acid extraction kit from Zhejiang Hanwei Technology Co., Ltd., following the method described in the instruction manual.

[0344] Reverse transcription reaction: 1 μg of total RNA was extracted from each tissue sample and transduced using the Promega Reverse Transcription System (A3500) with Oligo(dT) as the target. 15 Prepare a 20 μL reverse transcription system according to the kit instructions and complete the reverse transcription reaction. After the reaction, add 80 μL of RNase-free water to the reverse transcription system to obtain the cDNA solution for Real-time PCR detection.

[0345] Real-time PCR testing: using ABI SYBR Green. TM SelectMaster Mix (Catalog number: 4472908) reagents. Prepare 20 μL Real-time PCR reaction mixtures per PCR well according to the kit instructions. Each reaction mixture contains 5 μL of cDNA template obtained from the reverse transcription reaction and 10 μL of SYBR. TMSelect Master Mix, 0.5 μL 10 μM upstream primer, 0.5 μL 10 μM downstream primer, and 4 μL RNase-Free H2O. Place the prepared reaction mixture on an ABI StepOnePlus PCR instrument and perform real-time PCR amplification using a three-step method. The amplification program is: 95℃ pre-denaturation for 10 min, followed by 95℃ denaturation for 30 s, 60℃ annealing for 30 s, and 72℃ extension for 30 s. Repeat the denaturation, annealing, and extension process for 40 cycles. After the program is complete, gene expression differences are calculated using the ΔΔCt method.

[0346] In Real-time PCR, the ΔΔCt method was used to relatively quantify the expression level and inhibition rate of the target gene mRNA in each test group. The calculation method is as follows:

[0347] ΔCt(test group) = Ct(target gene in test group) - Ct(internal reference gene in test group)

[0348] ΔCt) (blank control group) = Ct (target gene in blank control group) - Ct (internal reference gene in blank control group)

[0349] ΔCt(test group) = ΔCt(test group) - ΔCt(mean of blank control group)

[0350] ΔCt(blank control group) = ΔCt(blank control group) - ΔCt(blank control group average)

[0351] Here, ΔCt (mean of the blank control group) is the arithmetic mean of the ΔCt values ​​of the five mice sacrificed at the same time point in the blank control group. Therefore, each sample in both the test group and the blank control group corresponds to a ΔCt value.

[0352] The relative expression level of the target gene mRNA in the test group = 2 - ΔΔCt(test group) × 100%

[0353] Using the blank control group as a baseline, the relative expression level of the target gene mRNA in the test group was normalized, and the relative expression level of the target gene mRNA in the blank control group was defined as 100%.

[0354] The inhibition rate of target gene mRNA expression in the test group = 100% - the relative expression level of target gene mRNA in the test group

[0355] Unless otherwise stated, all data on inhibition activity are expressed as X±STDEV and were plotted and analyzed using GraphPad Prism 8.0 software.

[0356] Example 1: Evaluation of the inhibitory activity of siRNA conjugates on the target gene superoxide dismutase 1 (SOD1) in the left and right hemispheres of mice based on lateral ventricle administration:

[0357] This embodiment uses a method based on lateral ventricle administration to evaluate the inhibitory activity of siRNA conjugates on target genes in the left and right hemispheres of mice. The method evaluated the inhibitory activity of the following siRNA conjugates in the left and right hemispheres of mice: RZ899115 (NM077), RZ899116 (NM078), and RZ899093 (Uhd) on the target gene SOD1.

[0358] Six- to eight-week-old C57BL / 6j mice were randomly divided into three groups (n=5 per group) based on body weight: three test groups and one PBS control group. In the test groups, each mouse received 150 μg of the drug at a volume of 5 μL. In the PBS control group, each mouse received 5 μL of the drug. Day 0 (D0) was the day of drug administration. Mice were sacrificed on day 5 (D5). The left and right hemispheres were preserved in RNAlaters, respectively. RNA was extracted from each tissue, reverse transcribed, and quantitatively analyzed using real-time fluorescence. Gene expression differences were calculated using the ΔΔCt method described above.

[0359] Table 5. Sequence listing of primers used in Example 1:

[0360] The experimental results of Example 1 (Figure 1, Table 6) show that: in the left brain, the inhibitory effect of siRNA conjugate RZ899116 on the target gene is superior to that of siRNA conjugate RZ899093; in the right brain, the inhibitory effects of siRNA conjugate RZ899116 and siRNA conjugate RZ899093 on the target gene are comparable. The inhibitory effect of siRNA conjugate RZ899115 on the target gene in both the left and right brains is basically comparable to that of siRNA conjugate RZ899093, and the inhibitory activity can reach about 80%.

[0361] Table 6. Inhibitory activity of siRNA conjugates RZ899093, RZ899115, and RZ899116 on the target gene SOD1 in the left and right hemispheres of mice:

[0362] Example 2: Evaluation of the inhibitory activity of siRNA conjugates on the target gene SOD1 in the left and right hemispheres of mice based on lateral ventricle administration:

[0363] This embodiment uses a method based on lateral ventricle administration to evaluate the inhibitory activity of siRNA conjugates on target genes in the left and right hemispheres of mice. The method evaluated the inhibitory activity of the following siRNA conjugates in the left and right hemispheres of mice: RZ899111 (NM076), RZ899112 (NM075), RZ899113 (NM076), and RZ899114 (NM075) on the target gene SOD1.

[0364] Six- to eight-week-old C57BL / 6j mice were randomly divided into four test groups and one PBS control group, with five mice in each group. In the test groups, each mouse received 150 μg of the drug at a volume of 5 μL. In the PBS control group, each mouse received 5 μL of the drug. The day of administration was designated D0, and mice were sacrificed on D5. The left and right hemispheres were preserved in RNAlaters, respectively. RNA was extracted from each tissue, reverse transcribed, and quantitatively analyzed using real-time fluorescence PCR. Gene expression differences were calculated using the ΔΔCt method described above. Primers for quantitative PCR are shown in Table 5 of Example 1.

[0365] The results of Example 2 (Figure 2, Table 7) show that the siRNA conjugates RZ899111, RZ899112, RZ899113, and RZ899114, which are terminal conjugates of the sense strand, can all significantly inhibit the target gene SOD1 in mouse brain tissue.

[0366] Table 7. Inhibitory activity of siRNA conjugates RZ899111, RZ899112, RZ899113, and RZ899114 on the target gene SOD1 in the left and right hemispheres of mice.

[0367] Example 3: Evaluation of the inhibitory activity of siRNA conjugates on the target gene SOD1 in mouse heart, adipose tissue, and muscle tissue based on subcutaneous abdominal administration:

[0368] This embodiment evaluated the inhibitory activity of siRNA conjugates against the target gene SOD1 in mouse heart, adipose tissue, and muscle tissue using the method of "evaluating the inhibitory activity of siRNA conjugates against the target gene in mice based on intravenous administration". The results showed that the siRNA conjugates RZ899111 (NM076) starting at the 5' end of the positive strand, RZ899112 (NM075) starting at the 5' end of the positive strand, RZ899113 (NM076) starting at the 3' end of the positive strand, and RZ899114 (NM075) starting at the 3' end of the positive strand were evaluated.

[0369] Six- to eight-week-old C57BL / 6j mice were randomly divided into five groups based on body weight: four test groups and one PBS control group, with five mice in each group. In the test groups, each mouse received 5 mg (as siRNA) / kg (based on mouse body weight) of the drug, administered in a volume of 5 ml / kg (based on mouse body weight). In the PBS control group, each mouse received 5 ml / kg (based on mouse body weight). The day of administration was designated D0. Five mice were sacrificed on D14, and gross dissection was performed. Heart, fat, and skeletal muscle tissue were collected and cut into several 2 mm pieces. 3 The small fragments were preserved using RNA Later. RNA extraction, reverse transcription, and Real-time PCR detection methods were as described above, and the relative quantification of target gene mRNA in each test group was calculated according to the aforementioned ΔΔCt method. Primers were as shown in Example 1.

[0370] The results of Example 3 (Figure 3, Table 8) showed that siRNA conjugates RZ899111, RZ899112, RZ899113, and RZ899114 all exhibited high inhibitory activity against the target gene SOD1 in mouse heart, adipose tissue, and skeletal muscle tissue. Among these, in adipose tissue, the siRNA conjugate RZ899111, with the first position (NM076) at the 5' end of the positive strand, showed the highest inhibitory effect on the target gene SOD1, reaching 85.15%. In heart and skeletal muscle tissue, the siRNA conjugates RZ899112 and RZ899114, conjugated with (NM075), showed superior inhibitory activity against the target gene SOD1 compared to the siRNA conjugates RZ899111 and RZ899113, conjugated with (NM076).

[0371] Table 8. Inhibitory activity of siRNA conjugates RZ899111, RZ899112, RZ899113, and RZ899114 against the target gene SOD1 in mouse heart, adipose tissue, and skeletal muscle tissue:

[0372] Example 4: Evaluation of the inhibitory activity of siRNA conjugates on the target gene SOD1 in different rat tissues based on intrathecal administration:

[0373] This embodiment uses the "Method for Evaluating the Inhibitory Activity of siRNA Conjugates on Target Genes in Mice Based on Intrathecal Administration" to evaluate the inhibitory activity of the siRNA conjugate RZ899116 (NM078) conjugated at position 4 (Uhd) from the 5' end of the positive strand and the siRNA conjugate RZ899093 conjugated at position 4 (Uhd) from the 5' end of the positive strand on the 5' end of the positive strand in the spinal cord and brain of rats on the target gene SOD1.

[0374] Six- to eight-week-old SD rats were randomly divided into two groups based on body weight: two test groups and one PBS control group, with 25 rats in each group. In the test groups, each rat received 900 μg of the drug at a volume of 40 μL. In the PBS control group, each rat received 40 μL of the drug. The day of administration was designated D0. Rats were sacrificed on D14, D28, D56, D84, and D147. Tissue samples from different spinal cord segments (lumbar, thoracic, and cervical), cerebellum, hippocampus, and cerebral cortex were collected and preserved in RNAlater. RNA was extracted from each tissue, reverse transcribed, and quantitatively analyzed in real-time. Gene expression differences were calculated using the ΔΔCt method.

[0375] Table 9. Primer sequence listing:

[0376] The results of Example 4 showed that at each observation time point, the NM078-conjugated siRNA sequence RZ899116 and the Uhd-conjugated control sequence RZ899093 had comparable inhibitory activity, and maintained about 50% inhibitory activity in the thoracic spine, cervical spine, and cerebellum on day D147 (Figures 4-9, Tables 10-15).

[0377] Table 10. Inhibitory activity of siRNA conjugates RZ899093 and RZ899116 against the target gene SOD1 in the rat lumbar spine:

[0378] Table 11. Inhibitory activity of siRNA conjugates RZ899093 and RZ899116 against the target gene SOD1 in the rat thoracic vertebrae:

[0379] Table 12. Inhibitory activity of siRNA conjugates RZ899093 and RZ899116 against the target gene SOD1 in the rat cervical spine:

[0380] Table 13. Inhibitory activity of siRNA conjugates RZ899093 and RZ899116 on the target gene SOD1 in the rat cerebellum:

[0381] Table 14. Inhibitory activity of siRNA conjugates RZ899093 and RZ899116 against the target gene SOD1 in the rat hippocampus:

[0382] Table 15. Inhibitory activity of siRNA conjugates RZ899093 and RZ899116 on the target gene SOD1 in the rat cerebral cortex:

[0383] The above specific embodiments are merely illustrative of the present invention and do not represent a limitation thereof. For those skilled in the art, various modifications and improvements can be made without departing from the spirit and substance of this disclosure, and these modifications and improvements are also considered to be within the scope of protection of this disclosure.

Claims

1. A double-stranded oligonucleotide, characterized in that, The double-stranded oligonucleotide comprises a sense strand and an antisense strand, wherein the antisense strand is at least partially base-paired with the sense strand to form a complementary double-stranded region, and the sense strand and / or antisense strand contains at least one modified nucleotide having the structure shown in formula (i), or a stereoisomer thereof, a tautomer thereof, or a pharmaceutically acceptable salt thereof. Where Q is selected from hydroxyl or mercapto groups; X is selected from O or S; p and q are each independently selected from integers from 1 to 4; Base is selected from H, or modified or unmodified bases; R1 is selected from H or substituted or unsubstituted C1-C6 alkyl groups, wherein the substituents in the substituted C1-C6 alkyl groups are selected from halogens, hydroxyl groups or C1-C6 alkoxy groups; R2 exists or does not exist; when R2 exists, R2 is selected from... R3 and R4 are independently selected from H or substituted or unsubstituted C1-C6 alkyl groups, wherein the substituents in the substituted C1-C6 alkyl groups are selected from halogens, hydroxyl groups or C1-C6 alkoxy groups; m is selected from 1; One of Z1 and Z2 may exist, or both Z1 and Z2 may exist simultaneously. Z1 and Z2 are selected independently. Among them, R z1 R z2 R z3 R z4 R z5 R z6 R z7 R z8 R z9 R z10 Each is independently selected from H or C1-C3 alkyl groups; L is selected from optionally substituted C10-C23 alkyl groups or C10-C23 olefins containing at least one unsaturated bond.

2. The double-stranded oligonucleotide as described in claim 1, characterized in that, The modified nucleotide has the structure shown in formula (ia) or (ib), or a stereoisomer thereof, a tautomer thereof, or a pharmaceutically acceptable salt thereof: Q, X, R2, Z1 and Z2 as defined in claim 1, wherein Z1 and Z2 exist simultaneously in equation (ib); p and q are each independently selected from 0 or 1; Optionally, p is selected from 1, and q is selected from 0; Optionally, R1 is selected from H or substituted or unsubstituted C1-C3 alkyl groups; the substituents in the substituted C1-C3 alkyl groups are selected from halogens, hydroxyl groups or C1-C3 alkoxy groups; Base is selected from H or bases A, U, T, C or G; L is selected from optionally substituted C15-C23 alkyl groups.

3. The double-stranded oligonucleotide as described in claim 1, characterized in that, The antisense strand does not contain the modified nucleotide at positions 1-19, and the sense strand contains at least one of the modified nucleotides; Optionally, the modified nucleotide is located at positions 1-17 of the positive strand starting from the 5' end and positions 1-2 of the positive strand starting from the 3' end; Optionally, the modified nucleotide is located at positions 1-8 of the positive strand starting from the 5' end and position 1 of the positive strand starting from the 3' end; Optionally, the modified nucleotide is located at position 1, 4, 5, or 6 of the positive strand starting from the 5' end, or at position 1 of the positive strand starting from the 3' end; Optionally, the modified nucleotide is located at the first or second position of the antisense strand, starting from the 3' end; Optionally, the length of the sense strand and the antisense strand is each independently 15-25 nucleotides; Optionally, the double-stranded oligonucleotide is selected from siRNA.

4. The double-stranded oligonucleotide according to any one of claims 1-3, characterized in that, The modified nucleotide has the structure shown in formula (ii-a1), or a stereoisomer thereof, or a tautomer thereof, or a pharmaceutically acceptable salt thereof: p, q, Q, X, L, and Base are as defined in claim 2; Y is selected from O or S; R5 is selected from H or substituted or unsubstituted C1-C6 alkyl groups, wherein the substituents in the substituted C1-C6 alkyl groups are selected from halogens, hydroxyl groups or C1-C6 alkoxy groups; Optionally, R5 is selected from H or substituted or unsubstituted C1-C3 alkyl groups; the substituents in the substituted C1-C3 alkyl groups are selected from halogens, hydroxyl groups or C1-C3 alkoxy groups; Optionally, the modified nucleotide has the structure shown in formula (ii-a1-1), or a stereoisomer thereof, or a tautomer thereof, or a pharmaceutically acceptable salt thereof: Optionally, the modified nucleotide has the structure shown in formula (iii-a1-1), or a stereoisomer thereof, or a tautomer thereof, or a pharmaceutically acceptable salt thereof:

5. The double-stranded oligonucleotide as described in claim 4, characterized in that, The modified nucleotide is selected from any of the following compounds:

6. The double-stranded oligonucleotide according to any one of claims 1-3, characterized in that, The modified nucleotide has the structure shown in formula (ii-a2), or a stereoisomer thereof, or a tautomer thereof, or a pharmaceutically acceptable salt thereof: The definitions of p, q, Q, X, L, and Base are the same as in claim 2; Y is selected from O or S; R5 is selected from H or substituted or unsubstituted C1-C6 alkyl groups, wherein the substituents in the substituted C1-C6 alkyl groups are selected from halogens, hydroxyl groups or C1-C6 alkoxy groups; Optionally, R5 is selected from H or substituted or unsubstituted C1-C3 alkyl groups; the substituents in the substituted C1-C3 alkyl groups are selected from halogens, hydroxyl groups or C1-C3 alkoxy groups; Optionally, the modified nucleotide has the structure shown in formula (iii-a2-1), or a stereoisomer thereof, or a tautomer thereof, or a pharmaceutically acceptable salt thereof: Optionally, the modified nucleotide has the structure shown in formula (iiii-a2-1), or a stereoisomer thereof, or a tautomer thereof, or a pharmaceutically acceptable salt thereof:

7. The double-stranded oligonucleotide as described in claim 6, characterized in that, The modified nucleotide is selected from any of the following compounds:

8. The double-stranded oligonucleotide according to any one of claims 1-3, characterized in that, The modified nucleotide has the structure shown in formula (ii-b), or a stereoisomer thereof, or a tautomer thereof, or a pharmaceutically acceptable salt thereof: The definitions of p, q, Q, X, L, and Base are the same as in claim 2; Y is selected from O or S; R5 is selected from H or substituted or unsubstituted C1-C6 alkyl groups, wherein the substituents in the substituted C1-C6 alkyl groups are selected from halogens, hydroxyl groups or C1-C6 alkoxy groups; Optionally, R5 is selected from H or substituted or unsubstituted C1-C3 alkyl groups; the substituents in the substituted C1-C3 alkyl groups are selected from halogens, hydroxyl groups or C1-C3 alkoxy groups; R2 is selected from R3 and R4 are independently selected from H or substituted or unsubstituted C1-C3 alkyl groups, wherein the substituents in the substituted C1-C3 alkyl groups are selected from halogens, hydroxyl groups or C1-C3 alkoxy groups; m is selected from 1; Optionally, the modified nucleotide has the structure shown in formula (iii-b), or a stereoisomer thereof, or a tautomer thereof, or a pharmaceutically acceptable salt thereof: Optionally, the modified nucleotide has the structure shown in formula (iiii-b-1), or a stereoisomer thereof, or a tautomer thereof, or a pharmaceutically acceptable salt thereof:

9. The double-stranded oligonucleotide as described in claim 8, characterized in that, The modified nucleotide is selected from any of the following compounds:

10. A composition, characterized in that, The composition comprises the double-stranded oligonucleotide according to any one of claims 1-9.

11. The use of the double-stranded oligonucleotide as described in any one of claims 1-9, and / or the composition as described in claim 10, in the preparation of a medicament for treating and / or preventing pathological conditions or diseases caused by abnormal expression of a specific gene in cells associated with a target tissue; Optionally, the target tissue is selected from the central nervous system, cardiac muscle, skeletal muscle, and fat.

12. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises the double-stranded oligonucleotide of any one of claims 1-9, and / or the composition of claim 10, and optionally one or more pharmaceutically acceptable carriers or excipients.

13. A method for inhibiting the expression of a specific gene in cells related to a target tissue, characterized in that, The method comprises contacting the target tissue-associated cells with the double-stranded oligonucleotide of any one of claims 1-9, and / or the composition of claim 10, and / or the pharmaceutical composition of claim 12; Optionally, the target tissue is selected from the central nervous system, cardiac muscle, skeletal muscle, and fat.

14. A method for preventing and / or treating pathological conditions or diseases caused by the abnormal expression of a specific gene in cells related to a target tissue, characterized in that, The method includes administering to a subject a pharmaceutically acceptable dose of the double-stranded oligonucleotide of any one of claims 1-9, and / or the composition of claim 10, and / or the pharmaceutical composition of claim 12; Optionally, the target tissue is selected from the central nervous system, cardiac muscle, skeletal muscle, and fat.

15. A method for inhibiting the expression of target genes in central nervous system cells, skeletal muscle cells, cardiomyocytes, or adipocytes, characterized in that, It includes: The cells are brought into contact with the double-stranded oligonucleotide of any one of claims 1-9, and / or the composition of claim 10, and / or the pharmaceutical composition of claim 12.

16. A method for preventing and / or treating diseases or conditions related to the central nervous system, myocardium, skeletal muscle, and adipose tissue, characterized in that, It includes: administering to the subject a therapeutically effective amount of the double-stranded oligonucleotide of any one of claims 1-9, and / or the composition of claim 10, and / or the pharmaceutical composition of claim 12, thereby treating the subject; Optionally, the subject is a human being.

17. The method of claim 16, characterized in that, The skeletal muscle-related disease or condition is muscular atrophy. Optionally, the muscular atrophy is selected from the group consisting of: Duchenne muscular dystrophy, myotonic muscular atrophy, Becker muscular dystrophy, limb-girdle muscular atrophy, facioscapulohumeral muscular atrophy, congenital muscular atrophy, oculopharyngeal muscular atrophy, distal muscular atrophy, Emery-Dreifuss muscular dystrophy, myostatin-associated hypertrophy, congenital myasthenia gravis, and facioscapulohumeral muscular dystrophy (FSHD).

18. The method of claim 16, characterized in that, The myocardial-related diseases or conditions comprise the following group: obstructive hypertrophic cardiomyopathy (HOCM); familial hypertrophic cardiomyopathy (FHC); heart failure with preserved ejection fraction (HFPEF); atrial fibrillation (AFIB); ventricular fibrillation (VFIB); angina pectoris; myocardial infarction (MI); heart failure with heart failure or reduced ejection fraction (HFREF); supraventricular tachycardia (SVT); hypertrophic cardiomyopathy (HCM), dilated cardiomyopathy (DCM), arrhythmias, and congestive heart failure (CHF).

19. The method of claim 16, characterized in that, The diseases or conditions related to adipose tissue are metabolic diseases; Optionally, the metabolic disease is selected from the group consisting of: lipid metabolism disorders, hypertension, cardiovascular disease, or overweight-related disorders.

20. The method according to any one of claims 16-19, characterized in that, The double-stranded oligonucleotide agent is administered to the subject, the administration being selected from: intrathecal administration, subcutaneous administration, intramuscular administration, intravenous administration, intraperitoneal administration, or intravitreal administration.

21. The method as described in claim 20, characterized in that, The application of the double-stranded oligonucleotide agent results in the expression of the target gene being no more than 40%, 50%, 60%, or 70% of that without application.

22. A modified nucleoside monomer, characterized in that, The modified nucleoside monomer has the structure shown in formula (I), or a stereoisomer thereof, or a tautomer thereof, or a pharmaceutically acceptable salt thereof: Wherein, X, p, q, Z1, Z2, R1, R2 and L are as defined in claim 1; Base is selected from H, or modified or unmodified bases; if an amino group is present in Base, the amino group is protected with an amino protecting group; Base is selected from the bases A, U, T, C, and G; R6 is selected from H or a hydroxyl protecting group; R7 is selected from H or phosphorus-containing leaving groups; The structural formula of the phosphorus-containing leaving group is as follows: Among them, R 2a Selected from secondary amino groups, R 2b Selected from secondary amino or cyano-substituted C1-C3 alkoxy groups.

23. The modified nucleoside monomer according to claim 22, characterized in that, The modified nucleoside monomer has the structure shown in formula (Ia) or (Ib), or a stereoisomer thereof, or a tautomer thereof, or a pharmaceutically acceptable salt thereof. The substitutions of X, R2, R6, R7, Z1 and Z2 are the same as in claim 22, (Ib) where Z1 and Z2 coexist in the structural formula; p and q are each independently selected from 0 or 1; R1 is selected from H or substituted or unsubstituted C1-C3 alkyl groups; the substituents in the substituted C1-C3 alkyl groups are selected from halogens, hydroxyl groups or C1-C3 alkoxy groups; Base is selected from H or bases A, U, T, C or G; L is selected from optionally substituted C15-C23 alkyl groups.

24. The modified nucleoside monomer according to any one of claims 22-23, characterized in that, The modified nucleoside monomer has the structure shown in formula (II-a1), or a stereoisomer thereof, or a tautomer thereof, or a pharmaceutically acceptable salt thereof: The definitions of p, q, R6, R7, X, L, and Base are the same as in claim 23; Y is selected from O or S; R5 is selected from H or substituted or unsubstituted C1-C6 alkyl groups, wherein the substituents in the substituted C1-C6 alkyl groups are selected from halogens, hydroxyl groups or C1-C6 alkoxy groups; Optionally, R5 is selected from H or substituted or unsubstituted C1-C3 alkyl groups; the substituents in the substituted C1-C3 alkyl groups are selected from halogens, hydroxyl groups or C1-C3 alkoxy groups; Optionally, the modified nucleoside monomer has the structure shown in formula (IIII-a1-1), or a stereoisomer thereof, or a tautomer thereof, or a pharmaceutically acceptable salt thereof:

25. The modified nucleoside monomer according to claim 24, characterized in that, The modified nucleoside monomer is selected from any of the following compounds:

26. The modified nucleoside monomer according to any one of claims 22-23, characterized in that, The modified nucleoside monomer has the structure shown in formula (II-a2), or a stereoisomer thereof, or a tautomer thereof, or a pharmaceutically acceptable salt thereof: The definitions of p, q, R6, R7, X, L, and Base are the same as in claim 23; Y is selected from O or S; R5 is selected from H or substituted or unsubstituted C1-C3 alkyl groups; the substituents in the substituted C1-C3 alkyl groups are selected from halogens, hydroxyl groups or C1-C3 alkoxy groups; Optionally, the modified nucleoside monomer has the structure shown in formula (IIII-a2-1), or a stereoisomer thereof, or a tautomer thereof, or a pharmaceutically acceptable salt thereof:

27. The modified nucleoside monomer according to claim 26, characterized in that, The modified nucleoside monomer is selected from any of the following compounds:

28. The modified nucleoside monomer according to any one of claims 22-23, characterized in that, The modified nucleoside monomer has the structure shown in formula (II-b), or a stereoisomer thereof, or a tautomer thereof, or a pharmaceutically acceptable salt thereof: The definitions of p, q, R6, R7, X, L, and Base are the same as in claim 23; Y is selected from O or S; R5 is selected from H or substituted or unsubstituted C1-C3 alkyl groups; the substituents in the substituted C1-C3 alkyl groups are selected from halogens, hydroxyl groups or C1-C3 alkoxy groups; R2 is selected from R3 and R4 are independently selected from H or substituted or unsubstituted C1-C3 alkyl groups, wherein the substituents in the substituted C1-C3 alkyl groups are selected from halogens, hydroxyl groups or C1-C3 alkoxy groups; m is selected from 1; Optionally, the modified nucleoside monomer has the structure shown in formula (IIII-b-1), or a stereoisomer thereof, or a tautomer thereof, or a pharmaceutically acceptable salt thereof:

29. The modified nucleoside monomer according to claim 28, characterized in that, The modified nucleoside monomer is selected from any of the following compounds:

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