Nucleoside and nucleotide analogs, double-stranded oligonucleotides containing nucleotide analogs, conjugates and uses thereof

Abstract

The present disclosure provides a nucleotide analogue, a double-stranded oligonucleotide containing the nucleotide analogue and a conjugate and application, belonging to the technical field of small nucleic acid drugs. The nucleotide analogue has a structure shown in formula (200), or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof. The double-stranded oligonucleotide contains a sense strand and an antisense strand, each strand having 17-35 nucleotides; at least one nucleotide in positions 2-8 of the antisense strand is replaced by the nucleotide analogue in the 5' end to 3' end direction. The double-stranded oligonucleotide containing the nucleotide analogue and the conjugate provided by the present disclosure can effectively treat and / or prevent the disease symptoms related to the mRNA level expressed by the target gene, while also having a low off-target effect, reducing the toxic reactions caused by the off-target effect.

Description

Nucleoside and nucleotide analogs, double-stranded oligonucleotides and conjugates containing nucleotide analogs and their applications

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to the Chinese patent application with application number 202311421634.X filed with the State Intellectual Property Office of China on October 30, 2023, and entitled “Nucleoside and nucleotide analogs, double-stranded oligonucleotides and conjugates containing nucleotide analogs and their applications”, the entire contents of which are incorporated by reference into this application. Technical Field

[0003] The present disclosure belongs to the technical field of small nucleic acid drugs, and particularly relates to nucleoside and nucleotide analogs, double-stranded oligonucleotides containing the nucleotide analogs, double-stranded oligonucleotide conjugates, and uses thereof. Background Art

[0004] Double-stranded oligonucleotides are a class of drugs synthesized artificially. They act on mRNA through complementary base pairing, interfering with gene unwinding, replication, transcription, mRNA splicing and processing, and even output and translation. This causes abnormally coded genes to lose their function, thereby preventing the expression of "wrong" proteins, thereby exerting a unique mechanism for regulating the transcription and translation process of disease genes at the genetic level.

[0005] Due to the unique chemical structure of oligonucleotide drugs, they exhibit poor drugability: large molecular weight, strong hydrophilicity, high negative charge, do not follow Lipinski's principle, and have poor pharmacokinetic characteristics, cannot pass through biological membranes, and may have off-target effects.

[0006] The field has been developing double-stranded oligonucleotides with both good activity and low off-target effects. In order to develop double-stranded oligonucleotides with both good pharmaceutical activity and low off-target effects, the inventors found that double-stranded oligonucleotides containing nucleotide analogs can have lower off-target effects.

[0007] Summary of the Invention

[0008] The present disclosure provides nucleoside analogs, nucleotide analogs, double-stranded oligonucleotides containing the nucleotide analogs, and uses thereof. The present disclosure improves the off-target protection of double-stranded oligonucleotides by introducing nucleotide analogs into double-stranded oligonucleotides, thereby significantly improving toxicity without substantially reducing the activity of the double-stranded oligonucleotides.

[0009] In the first aspect of the present disclosure, the present disclosure provides a nucleotide analogue selected from the structure represented by formula (II-i), or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof:

[0010] Among them, B 200 selected from bases or modified bases;

[0011] Z is selected from hydroxyl or thiol (SH);

[0012] n is selected from 1, 2 or 3;

[0013] X is selected from Each R' is independently selected from optionally substituted C 1-3 Alkyl, the optionally substituted C 1-3 The alkyl group contains substituents independently selected from halogen, C 1-3 Alkoxy, hydroxy or amino groups;

[0014] m is selected from 1, 2, 3 or 4;

[0015] r is selected from 1, 2, 3 or 4;

[0016] Each R A and each R B are each independently selected from H or optionally substituted C 1-3 alkyl; and R A and R B There is at least one optionally substituted C 1-3 Alkyl; if the optionally substituted C 1-3 The alkyl group contains substituents independently selected from halogen, C 1-3 alkoxy, hydroxy or amino.

[0017] Each * independently represents a covalent bonding site.

[0018] In some optional embodiments of the present disclosure, each R A and each R B are each independently selected from H or optionally substituted C 1-3 alkyl; and R A and R B There is at least one optionally substituted C 1-3 Alkyl; if the optionally substituted C 1-3 The alkyl group contains substituents independently selected from halogen, C 1-3 Alkoxy, hydroxy or amino. If one R A Selected from optionally substituted C 1-3 Alkyl, and the rest of R A and all R B Selected from H; or one R B Selected from optionally substituted C 1-3 Alkyl, and the rest of R B and all R A Selected from H.

[0019] In some optional embodiments of the present disclosure, each R A and each R B Each independently selected from H or C 1-3 alkyl; and each R A and each R B Any one of C 1-3 Alkyl. Such as an R A Selected from C 1-3 Alkyl, and the rest of R A and all R B Selected from H; or one R B Selected from C 1-3 Alkyl, and the rest of R B and all R A Selected from H.

[0020] In some optional embodiments of the present disclosure, the C 1-3 The alkyl group is selected from methyl, ethyl, n-propyl and isopropyl.

[0021] In some specific embodiments of the present disclosure, the C 1-3 The alkyl group is selected from methyl.

[0022] In some optional embodiments of the present disclosure, each R A and each R B are each independently selected from H or methyl; and each R A and each R B Any one of them is selected from methyl. A is selected from methyl, and the remaining R A and all R B Selected from H; or one R B is selected from methyl, and the remaining R B and all R A Selected from H.

[0023] In some specific embodiments of the present disclosure, m is selected from 2, and r is selected from 2.

[0024] In some optional embodiments of the present disclosure, the nucleotide analog is selected from the structure represented by formula (II-ii), or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof:

[0025] Among them, B 200 selected from bases or modified bases;

[0026] Z is selected from hydroxyl or thiol (SH);

[0027] n is selected from 1, 2 or 3;

[0028] X is selected from Each R' is independently selected from optionally substituted C 1-3 Alkyl, the optionally substituted C 1-3 The alkyl group contains substituents independently selected from halogen, C 1-3 Alkoxy, hydroxy or amino groups;

[0029] Each * independently represents a covalent bonding site.

[0030] R2, R3, R5, R6 are each independently selected from H or optionally substituted C 1-3 and at least one of R2, R3, R5 and R6 is selected from optionally substituted C 1-3 Alkyl; if the optionally substituted C 1-3 The alkyl group contains substituents independently selected from halogen, C 1-3 alkoxy, hydroxy or amino.

[0031] In some optional embodiments of the present disclosure, R2, R3, R5, and R6 are each independently selected from H or optionally substituted C 1-3 and any one of R2, R3, R5 and R6 is selected from optionally substituted C 1-3 Alkyl, such as R2 is selected from optionally substituted C 1-3 and R3, R5 and R6 are selected from H, or R3 is selected from optionally substituted C 1-3 and R2, R5 and R6 are selected from H, or R5 is selected from optionally substituted C 1-3 and R2, R3 and R6 are selected from H, or R6 is selected from optionally substituted C 1-3 Alkyl and R2, R3 and R5 are selected from H.

[0032] In some optional embodiments of the present disclosure, R2, R3, R5, and R6 are each independently selected from H or C 1-3 and any one of R2, R3, R5 and R6 is selected from C 1-3 Alkyl, such as R2 is selected from C 1-3 Alkyl and R3, R5 and R6 are selected from H, or R3 is selected from C 1-3 Alkyl and R2, R5 and R6 are selected from H, or R5 is selected from C 1-3 Alkyl and R2, R3 and R6 are selected from H, or R6 is selected from C 1-3 Alkyl and R2, R3 and R5 are selected from H.

[0033] In some optional embodiments of the present disclosure, the C 1-3 The alkyl group is selected from methyl, ethyl, n-propyl and isopropyl.

[0034] In some specific embodiments of the present disclosure, the C 1-3 The alkyl group is selected from methyl.

[0035] In some specific embodiments of the present disclosure, R2, R3, R5, and R6 are each independently selected from H or methyl; and any one of R2, R3, R5, and R6 is selected from methyl, such as R2 is selected from methyl and R3, R5, and R6 are selected from H, or R3 is selected from methyl and R2, R5, and R6 are selected from H, or R5 is selected from methyl and R2, R3, and R6 are selected from H, or R6 is selected from methyl and R2, R3, and R5 are selected from H.

[0036] In another aspect of the present disclosure, the present disclosure provides a nucleotide analog having a structure represented by formula (200), or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof:

[0037] Among them, B 200 selected from bases or modified bases;

[0038] Z is selected from hydroxyl or thiol (SH);

[0039] n is selected from 1, 2 or 3;

[0040] X is selected from Each R' is independently selected from optionally substituted C 1-3 Alkyl, the optionally substituted C 1-3 The alkyl group contains substituents independently selected from halogen, C 1-3 Alkoxy, hydroxy or amino groups;

[0041] m is selected from 1, 2, 3 or 4;

[0042] R2 and R3 are independently selected from H or optionally substituted C 1-3 and at least one of R2 and R3 is selected from optionally substituted C 1-3 Alkyl; if the optionally substituted C 1-3 The alkyl group contains substituents independently selected from halogen, C 1-3 Alkoxy, hydroxy or amino groups;

[0043] Each * independently represents a covalent bonding site.

[0044] The present disclosure also provides a nucleotide analog having a structure represented by formula (500), or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof:

[0045] wherein, is selected from a base or a modified base;

[0046] Z is selected from hydroxyl or thiol (SH);

[0047] n is selected from 1, 2 or 3;

[0048] X is selected from Each R' is independently selected from optionally substituted C 1-3 Alkyl, the optionally substituted C 1-3 The alkyl group contains substituents independently selected from halogen, C 1-3 Alkoxy, hydroxy or amino groups;

[0049] r is selected from 1, 2, 3 or 4;

[0050] R5 and R6 are independently selected from H or optionally substituted C 1-3 and at least one of R5 and R6 is selected from optionally substituted C 1-3 Alkyl; if the optionally substituted C 1-3 The alkyl group contains substituents independently selected from halogen, C 1-3 Alkoxy, hydroxy or amino groups;

[0051] Each * independently represents a covalent bond site. In one aspect of the present disclosure, the present disclosure provides a double-stranded oligonucleotide comprising a sense strand and an antisense strand, each strand having 17-35 nucleotides; in a 5' to 3' direction, at least one nucleotide at positions 2-8 of the antisense strand is replaced by a nucleotide analogue as described above.

[0052] In one aspect of the present disclosure, the present disclosure further provides a double-stranded oligonucleotide conjugate, wherein the oligonucleotide conjugate contains the double-stranded oligonucleotide provided by the present disclosure and one or more ligands capable of binding to a cell surface receptor.

[0053] In one aspect of the present disclosure, the present disclosure provides a composition comprising the double-stranded oligonucleotide and / or the double-stranded oligonucleotide conjugate described in the present disclosure.

[0054] In one aspect of the present disclosure, the present disclosure further provides a pharmaceutical composition comprising the double-stranded oligonucleotide, and / or the double-stranded oligonucleotide conjugate, and / or the composition described in the present disclosure.

[0055] In one aspect of the present disclosure, the present disclosure provides a nucleoside analogue, characterized in that it has a structure represented by formula (100), or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof:

[0056] Among them, B 100 Selected from bases or modified bases, if B 100 Contains an amino group, and the amino group is protected by an amino protecting group;

[0057] n is selected from 1, 2 or 3;

[0058] X is selected from Each R' is independently selected from optionally substituted C 1-3 Alkyl, the optionally substituted C 1-3 The alkyl group contains substituents independently selected from halogen, C 1-3 Alkoxy, hydroxy or amino groups;

[0059] m is selected from 1, 2, 3 or 4;

[0060] R1 is selected from H or a hydroxyl protecting group;

[0061] R2 and R3 are independently selected from H or optionally substituted C 1-3 and at least one of R2 and R3 is selected from optionally substituted C 1-3 Alkyl; if the optionally substituted C 1-3 The alkyl group contains substituents independently selected from halogen, C 1-3 Alkoxy, hydroxy or amino groups;

[0062] R4 is selected from H or Each R 4a Independently selected from or C containing a cyano substituent 1-6 Alkoxy, and at least one R 4a Selected from Each R 4a 'Independently selected from optionally substituted C 1-6 alkyl.

[0063] In one aspect of the present disclosure, the present disclosure also provides the use of nucleotide analogs, and / or double-stranded oligonucleotides, and / or double-stranded oligonucleotide conjugates, and / or compositions, and / or pharmaceutical compositions, and / or nucleoside analogs in the preparation of drugs for treating and / or preventing diseases or symptoms associated with the level of mRNA expressed by a target gene.

[0064] In one aspect of the present disclosure, the present disclosure also provides a method for treating and / or preventing a disease or symptom associated with the level of mRNA expressed by a target gene, the method comprising administering a double-stranded oligonucleotide, and / or double-stranded oligonucleotide conjugate, and / or composition, and / or pharmaceutical composition of the present disclosure to a subject in need thereof.

[0065] In one aspect of the present disclosure, the present disclosure also provides a method for regulating the expression level of a target gene in a cell, the method comprising contacting an effective amount of a double-stranded oligonucleotide, and / or double-stranded oligonucleotide conjugate, and / or composition, and / or pharmaceutical composition of the present disclosure with the cell.

[0066] In one aspect of the present disclosure, the present disclosure further provides a kit comprising the double-stranded oligonucleotide, and / or double-stranded oligonucleotide conjugate, and / or composition, and / or pharmaceutical composition of the present disclosure.

[0067] The double-stranded oligonucleotides, double-stranded oligonucleotide conjugates, compositions and / or pharmaceutical compositions provided by the present disclosure can effectively treat and / or prevent disease symptoms related to the level of mRNA expressed by the target gene, while also having low off-target effects and reducing toxic reactions caused by off-target effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0068] FIG1 shows the relative expression levels of target genes in mice after administration of siRNA conjugates in Example 1.

[0069] FIG2 shows the relative expression levels of target genes in mice after administration of siRNA conjugates in Example 2.

[0070] Figure 3 shows serum ALT levels in ICR mice after administration of siRNA conjugates in Example 3.

[0071] FIG4 shows serum AST in ICR mice after administration of siRNA conjugates in Example 3.

[0072] FIG5 shows the pathological scores of mouse liver tissues after administration of siRNA conjugates in Example 3.

[0073] Figure 6 shows pathological sections of mouse liver tissue after administration of siRNA conjugates in Example 3.

[0074] FIG7 shows the IC50 curve of the target gene ANGPTL3 in HEK 293T cells after administration of RZ597002 in Example 4.

[0075] FIG8 shows the IC50 curve of the target gene ANGPTL3 in HEK 293T cells after administration of RZ597113 in Example 4.

[0076] FIG9 shows the IC50 curve of the target gene ANGPTL3 in HEK 293T cells after administration of RZ597007 in Example 4.

[0077] FIG10 shows the IC50 curve of the target gene ANGPTL3 in HEK 293T cells after administration of RZ597112 in Example 4.

[0078] FIG11 shows the IC50 curve of the target gene ANGPTL3 in HEK 293T cells after administration of RZ597102 in Example 4.

[0079] FIG12 shows the IC50 curve of the target gene ANGPTL3 in HEK 293T cells after administration of RZ597103 in Example 4.

[0080] FIG13 shows the IC75 curve of the target gene ANGPTL3 in HEK 293T cells after administration of RZ597002 in Example 4.

[0081] FIG14 shows the IC75 curve of the target gene ANGPTL3 in HEK 293T cells after administration of RZ597113 in Example 4.

[0082] FIG15 shows the IC75 curve of the target gene ANGPTL3 in HEK 293T cells after administration of RZ597007 in Example 4.

[0083] FIG16 shows the IC75 curve of the target gene ANGPTL3 in HEK 293T cells after administration of RZ597112 in Example 4.

[0084] FIG17 shows the IC75 curve of the target gene ANGPTL3 in HEK 293T cells after administration of RZ597102 in Example 4.

[0085] FIG18 shows the IC75 curve of the target gene ANGPTL3 in HEK 293T cells after administration of RZ597103 in Example 4.

[0086] FIG19 shows the IC50 curve of the target gene ANGPTL3 in HEK 293T cells after administration of RZ597136 in Example 5.

[0087] FIG20 shows the IC50 curve of the target gene ANGPTL3 in HEK 293T cells after administration of RZ597157 in Example 5.

[0088] FIG21 shows the IC50 curve of the target gene ANGPTL3 in HEK 293T cells after administration of RZ597161 in Example 5.

[0089] FIG22 shows the IC60 curve of the target gene ANGPTL3 in HEK 293T cells after administration of RZ597136 in Example 5.

[0090] FIG23 shows the IC60 curve of the target gene ANGPTL3 in HEK 293T cells after administration of RZ597157 in Example 5.

[0091] FIG24 shows the IC60 curve of the target gene ANGPTL3 in HEK 293T cells after administration of RZ597161 in Example 5. DETAILED DESCRIPTION

[0092] The following describes the specific embodiments of the present disclosure in detail. It should be understood that the specific embodiments described herein are only used to illustrate and explain the present disclosure and are not intended to limit the present disclosure.

[0093] Explanation of terms

[0094] In this document, the terms “include” or “comprising” are open expressions, that is, including the contents specified in the present disclosure, but not excluding other contents.

[0095] As used herein, the terms "optionally," "optional," or "optionally" generally mean that the subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

[0096] As used herein, the term "optionally substituted" is used to define a variable that can be unsubstituted or substituted.

[0097] As used herein, the term "unsubstituted" means that the designated group bears no substituents.

[0098] The terms "substituted," "substituted," and "substituted" are used interchangeably herein to indicate that any one or more hydrogen atoms in a given structure are replaced by a specified substituent (e.g., C 1-3 Alkyl, C 1-3 alkoxy or halogen), provided that the normal valence of the designated atom is not exceeded and the substitution results in a stable compound. Unless otherwise indicated, a substituted group may have a substituent at each substitutable position of the group. When more than one position in a given structural formula can be substituted with one or more substituents selected from a specified group, the substituents may be the same or different at each substitutable position.

[0099] In this document, the terms "each... is independently selected from" and "... are each independently selected from" and "... are independently selected from" are interchangeable and should be understood in a broad sense. They can mean that in different groups, the specific options expressed by the same symbols do not affect each other, or that in the same group, the specific options expressed by the same symbols do not affect each other.

[0100] As used herein, the term "stereoisomers" refers to compounds that have identical chemical constitution but differ in the way the atoms or groups are arranged in space. Stereoisomers include enantiomers, diastereomers, conformers (rotamers), geometric isomers (cis / trans) isomers, atropisomers, and the like.

[0101] As used herein, the term "chiral" refers to a molecule that is non-superimposable on its mirror image, while "achiral" refers to a molecule that is superimposable on its mirror image.

[0102] As used herein, the term "enantiomers" refers to two non-superimposable isomers of a compound that are mirror images of each other.

[0103] As used herein, the term "diastereoisomer" refers to stereoisomers that have two or more chiral centers and whose molecules are not mirror images of each other. Diastereoisomers have different physical properties, such as melting points, boiling points, spectral properties, and reactivity. Diastereomeric mixtures can be separated by high-resolution analytical procedures such as electrophoresis and chromatography, for example, HPLC.

[0104] As used herein, the term "conjugation" refers to the covalent attachment of two or more chemical moieties, each with a specific function, to one another; accordingly, a "conjugate" refers to a compound formed by covalent attachment of two or more chemical moieties. Furthermore, a "drug conjugate" refers to a compound formed by covalent attachment of one or more chemical moieties with specific functions to an active drug. Sometimes, and especially in the examples, the "drug conjugate" of the present disclosure is also referred to as a "conjugate," "double-stranded oligonucleotide conjugate," or "siRNA conjugate." Drug conjugate should be understood as a general term for drug conjugates or a specific drug conjugate represented by a specific structural formula, depending on the context.

[0105] As used herein, the term "small interfering RNA (siRNA)" refers to a double-stranded RNA (dsRNA) of 17 to 25 nucleotides in length, comprising both a sense and an antisense strand. siRNAs mediate the targeted cleavage of RNA transcripts via the RNA-induced silencing complex (RISC) pathway by forming a silencing complex. Specifically, siRNAs direct the specific degradation of mRNA sequences through a process known as RNA interference (RNAi), inhibiting the translation of mRNA into amino acids and protein.

[0106] In this article, the term "antisense strand (or guide strand)" includes a region that is substantially complementary to a target sequence. The term "sense strand (or trailing strand)" refers to an iRNA chain that is substantially complementary to the antisense strand. The term "substantially complementary" refers to complete complementarity or at least partial complementarity, for example, the antisense strand is completely complementary to the target sequence or at least partially complementary. In the case of partial complementarity, mismatches can exist in the interior or terminal regions of the molecule, wherein the most tolerant mismatches exist in the terminal regions, for example, within 5, 4, 3 or 2 nucleotides of the 5'- and / or 3' end of the iRNA. It should be noted that "at least partially substantially complementary" between the antisense strand and the mRNA means that the antisense strand has a polynucleotide that is substantially complementary to a continuous portion of the mRNA of interest.

[0107] In this article, "substantially reverse complementary" means that there are no more than 3 base mismatches between the two nucleotide sequences involved; "substantially reverse complementary" means that there is no more than 1 base mismatch between the two nucleotide sequences involved; and "completely reverse complementary" means that there is no base mismatch between the two nucleotide sequences involved.

[0108] As used herein, the terms "treat," "treat," or "alleviate" are used interchangeably herein. These terms refer to an approach for obtaining beneficial or desired results, including but not limited to a therapeutic benefit. "Therapeutic benefit" means eradication or amelioration of the underlying disorder being treated. As used herein, a therapeutic benefit is achieved by eradication or amelioration of one or more physiological symptoms associated with the underlying disorder, such that an improvement is observed in the subject, although the subject may still be afflicted with the underlying disorder.

[0109] As used herein, the terms "prevent" and "prevent" are used interchangeably to refer to an approach for obtaining beneficial or desired results, including but not limited to prophylactic benefit. To obtain a "prophylactic benefit," a conjugate, RNAi agent, or composition can be administered to a subject at risk for a particular disease, or to a subject reporting one or more physiological symptoms of a disease, even though a diagnosis of the disease may not have yet been made.

[0110] As used herein, the term "ligand" generally refers to any compound or molecule that can covalently or otherwise chemically bind to a biologically active substance (such as an oligonucleotide). In certain embodiments, a ligand can interact directly or indirectly with another compound, such as a receptor. The receptor that interacts with the ligand can be present on the cell surface, or alternatively can be an intracellular and / or intercellular receptor. The interaction of the ligand with the receptor can result in a biochemical reaction, or can be simply a physical interaction or binding.

[0111] As used herein, the term "administer" generally refers to introducing the disclosed pharmaceutical formulation into a subject's body by any introduction or delivery route. 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, intraarterial, intranasal, intraperitoneal, intramuscular, subcutaneous, or oral administration. The daily dose may be divided into one, two, or more suitable dosage forms for administration at one, two, or more times during a certain time period.

[0112] As used herein, "pharmaceutical composition" may refer to a composition for use in treating a disease or in vitro cell culture experiments. When used in treating a disease, the term "pharmaceutical composition" generally refers to a unit dosage form and can be prepared by any of the methods well known in the pharmaceutical art. All methods include the step of combining the active ingredient with an excipient that constitutes one or more adjunct ingredients. Typically, the composition is prepared by uniformly and thoroughly combining the active siRNA with a liquid excipient, a finely divided solid excipient, or both.

[0113] As used herein, the term "pharmaceutically acceptable" means that the substance or composition must be chemically and / or toxicologically compatible with the other ingredients of the formulation and / or the mammal to be treated therewith. Preferably, the "pharmaceutically acceptable" herein refers to a substance approved by a federal regulatory agency or a state government or listed in the U.S. Pharmacopoeia or other generally recognized pharmacopeia for use in animals, particularly humans.

[0114] As used herein, the term "pharmaceutically acceptable excipient" may include any solvent, solid excipient, diluent, or other liquid excipient, etc., suitable for the particular intended dosage form. Except to the extent that any conventional excipient is incompatible with the siRNA of the present disclosure, such as by producing any adverse biological effect or interacting in a deleterious manner with any other component of the pharmaceutically acceptable composition, their use is also contemplated by the present disclosure.

[0115] In addition to any conventional excipients, to the extent that they are incompatible with the siRNA of the present disclosure, such as by producing any adverse biological effects or interacting in a deleterious manner with any other component of the pharmaceutically acceptable composition, their use is also contemplated by the present disclosure.

[0116] Nucleotide analogs

[0117] In a first aspect of the present disclosure, the present disclosure provides a nucleotide analogue,

[0118] It is selected from the structure represented by formula (II-i), or its stereoisomers, or its pharmaceutically acceptable salts:

[0119] Among them, B200 selected from bases or modified bases;

[0120] Z is selected from hydroxyl or thiol (SH);

[0121] n is selected from 1, 2 or 3;

[0122] X is selected from Each R' is independently selected from optionally substituted C 1-3 Alkyl, the optionally substituted C 1-3 The alkyl group contains substituents independently selected from halogen, C 1-3 Alkoxy, hydroxy or amino groups;

[0123] m is selected from 1, 2, 3 or 4;

[0124] r is selected from 1, 2, 3 or 4;

[0125] Each R A and each R B are each independently selected from H or optionally substituted C 1-3 alkyl; and R A and R B There is at least one optionally substituted C 1-3 Alkyl; if the optionally substituted C 1-3 The alkyl group contains substituents independently selected from halogen, C 1-3 alkoxy, hydroxy or amino.

[0126] Each * independently represents a covalent bonding site.

[0127] In some optional embodiments of the present disclosure, each R A and each R B are each independently selected from H or optionally substituted C 1-3 alkyl; and each R A and each R B Any one of which is selected from optionally substituted C 1-3 Alkyl; if the optionally substituted C 1-3 The alkyl group contains substituents independently selected from halogen, C 1-3 Alkoxy, hydroxy or amino. If one R A Selected from optionally substituted C 1-3 Alkyl, and the rest of R A and all R B Selected from H; or one R B Selected from optionally substituted C 1-3 Alkyl, and the rest of R B and all R A Selected from H.

[0128] In some optional embodiments of the present disclosure, each RA and each R B Each independently selected from H or C 1-3 alkyl; and each R A and each R B Any one of C 1-3 Alkyl. Such as an R A Selected from C 1-3 Alkyl, and the rest of R A and all R B Selected from H; or one R B Selected from C 1-3 Alkyl, and the rest of R B and all R A Selected from H.

[0129] In some optional embodiments of the present disclosure, the C 1-3 The alkyl group is selected from methyl, ethyl, n-propyl and isopropyl.

[0130] In some specific embodiments of the present disclosure, the C 1-3 The alkyl group is selected from methyl.

[0131] In some optional embodiments of the present disclosure, each R A and each R B are each independently selected from H or methyl; and each R A and each R B Any one of them is selected from methyl. A is selected from methyl, and the remaining R A and all R B Selected from H; or one R B is selected from methyl, and the remaining R B and all R A Selected from H.

[0132] In some specific embodiments of the present disclosure, m is selected from 2, and r is selected from 2.

[0133] In some optional embodiments of the present disclosure, the nucleotide analog is selected from the structure represented by formula (II-ii), or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof:

[0134] Among them, B 200 selected from bases or modified bases;

[0135] Z is selected from hydroxyl or thiol (SH);

[0136] n is selected from 1, 2 or 3;

[0137] X is selected from Each R' is independently selected from optionally substituted C 1-3Alkyl, the optionally substituted C 1-3 The alkyl group contains substituents independently selected from halogen, C 1-3 Alkoxy, hydroxy or amino groups;

[0138] Each * independently represents a covalent bonding site.

[0139] R2, R3, R5, R6 are each independently selected from H or optionally substituted C 1-3 and at least one of R2, R3, R5 and R6 is selected from optionally substituted C 1-3 Alkyl; if the optionally substituted C 1-3 The alkyl group contains substituents independently selected from halogen, C 1-3 alkoxy, hydroxy or amino.

[0140] In some optional embodiments of the present disclosure, R2, R3, R5, and R6 are each independently selected from H or optionally substituted C 1-3 and any one of R2, R3, R5 and R6 is selected from optionally substituted C 1-3 Alkyl, such as R2 is selected from optionally substituted C 1-3 and R3, R5 and R6 are selected from H, or R3 is selected from optionally substituted C 1-3 and R2, R5 and R6 are selected from H, or R5 is selected from optionally substituted C 1-3 and R2, R3 and R6 are selected from H, or R6 is selected from optionally substituted C 1-3 Alkyl and R2, R3 and R5 are selected from H.

[0141] In some optional embodiments of the present disclosure, R2, R3, R5, and R6 are each independently selected from H or C 1-3 Alkyl; and any one of R2, R3, R5 and R6 is selected from C 1-3 Alkyl, such as R2 is selected from C 1-3 Alkyl and R3, R5 and R6 are selected from H, or R3 is selected from C 1-3 Alkyl and R2, R5 and R6 are selected from H, or R5 is selected from C 1-3 Alkyl and R2, R3 and R6 are selected from H, or R6 is selected from C 1-3 Alkyl and R2, R3 and R5 are selected from H.

[0142] In some optional embodiments of the present disclosure, the C 1-3 The alkyl group is selected from methyl, ethyl, n-propyl and isopropyl.

[0143] In some specific embodiments of the present disclosure, the C 1-3 The alkyl group is selected from methyl.

[0144] In some specific embodiments of the present disclosure, R2, R3, R5, and R6 are each independently selected from H or methyl; and any one of R2, R3, R5, and R6 is selected from methyl, such as R2 is selected from methyl and R3, R5, and R6 are selected from H, or R3 is selected from methyl and R2, R5, and R6 are selected from H, or R5 is selected from methyl and R2, R3, and R6 are selected from H, or R6 is selected from methyl and R2, R3, and R5 are selected from H.

[0145] Furthermore, the nucleotide analog has a structure represented by formula (200), or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof:

[0146] Among them, B 200 selected from bases or modified bases;

[0147] Z is selected from hydroxyl or thiol (SH);

[0148] n is selected from 1, 2 or 3;

[0149] X is selected from Each R' is independently selected from optionally substituted C 1-3 Alkyl, the optionally substituted C 1-3 The alkyl group contains substituents independently selected from halogen, C 1-3 Alkoxy, hydroxy or amino groups;

[0150] m is selected from 1, 2, 3 or 4;

[0151] R2 and R3 are independently selected from H or optionally substituted C 1-3 and at least one of R2 and R3 is selected from optionally substituted C 1-3 Alkyl; if the optionally substituted C 1-3 The alkyl group contains substituents independently selected from halogen, C 1-3 Alkoxy, hydroxy or amino groups;

[0152] Each * independently represents a covalent bonding site.

[0153] In some embodiments of the present disclosure, the base is selected from uracil U, thymine T, cytosine C, adenine A or guanine G.

[0154] In some embodiments of the present disclosure, the modified base is selected from

[0155] In some embodiments of the present disclosure, B 200 Select from any of the following structures:

[0156] In some embodiments of the present disclosure, B200 Selected from

[0157] In some embodiments of the present disclosure, B 200 Selected from

[0158] In some embodiments of the present disclosure, B 200 Selected from

[0159] In some embodiments of the present disclosure, B 200 Selected from

[0160] In some embodiments of the present disclosure, B 200 Selected from

[0161] In some embodiments of the present disclosure, in formula (200), n is selected from 1 or 2.

[0162] In some embodiments of the present disclosure, n is selected from 1.

[0163] In some embodiments of the present disclosure, in formula (200), m is selected from 1, 2 or 3.

[0164] In some embodiments of the present disclosure, m is selected from 2.

[0165] In some embodiments of the present disclosure, X in the above formula is selected from

[0166] In some embodiments of the present disclosure, in formula (200), R2 and R3 are independently selected from H or optionally substituted C 1-3 and R2 and R3 are not simultaneously selected from H and optionally substituted C 1-3 Alkyl (ie: R2 is selected from H, and R3 is selected from optionally substituted C 1-3 Alkyl; or R2 is selected from optionally substituted C 1-3 alkyl, and R3 is selected from H).

[0167] In some embodiments of the present disclosure, R2 and R3 are independently selected from H or C 1-3 Alkyl; and R2 and R3 are not simultaneously selected from H and C 1-3 alkyl.

[0168] In some embodiments of the present disclosure, R2 and R3 are independently selected from H or methyl; and R2 and R3 are not simultaneously selected from H and methyl.

[0169] In some embodiments of the present disclosure, the nucleotide analog has a structure represented by formula (201), or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof:

[0170] In some embodiments of the present disclosure, the nucleotide analog has any of the following structures, or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof:

[0171] wherein R2 is selected from optionally substituted C 1-3 alkyl;

[0172] wherein R3 is selected from optionally substituted C 1-3 alkyl.

[0173] In some embodiments of the present disclosure, the nucleotide analog has a structure represented by formula (202), or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof:

[0174] wherein R2 is selected from optionally substituted C 1-3 alkyl.

[0175] In some embodiments of the present disclosure, the nucleotide analog has a structure represented by formula (203), or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof:

[0176] wherein R3 is selected from optionally substituted C 1-3 alkyl.

[0177] In some embodiments of the present disclosure, the nucleotide analog has any of the following structures, or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof:

[0178] wherein R2 and R3 are independently selected from optionally substituted C 1-3 alkyl.

[0179] In some embodiments of the present disclosure, the nucleotide analog has a structure represented by Formula (202A), or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof:

[0180] wherein R2 is selected from optionally substituted C 1-3 alkyl.

[0181] In some embodiments of the present disclosure, the nucleotide analog has a structure represented by formula (202B), or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof:

[0182] wherein R2 is selected from optionally substituted C 1-3 alkyl.

[0183] In some embodiments of the present disclosure, the nucleotide analog has a structure represented by formula (203A), or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof:

[0184] wherein R3 is selected from optionally substituted C 1-3 alkyl.

[0185] In some embodiments of the present disclosure, the nucleotide analog has a structure represented by formula (203B), or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof:

[0186] wherein R3 is selected from optionally substituted C 1-3 alkyl.

[0187] In some embodiments of the present disclosure, the nucleotide analog is selected from any one of the following structures, or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof:

[0188] Furthermore, the nucleotide analog has a structure represented by formula (500), or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof:

[0189] Among them, B 200 , Z, n, X and * are as defined above;

[0190] r is selected from 1, 2, 3 or 4;

[0191] R5 and R6 are independently selected from H or optionally substituted C 1-3 and at least one of R5 and R6 is selected from optionally substituted C 1-3 Alkyl; if the optionally substituted C 1-3 The alkyl group contains substituents independently selected from halogen, C 1-3 alkoxy, hydroxy or amino.

[0192] In some embodiments of the present disclosure, r is selected from 1, 2, or 3.

[0193] In some embodiments of the present disclosure, r is selected from 2.

[0194] In some embodiments of the present disclosure, R5 and R6 are independently selected from H or optionally substituted C 1-3 and R5 and R6 are not H or optionally substituted C 1-3 alkyl.

[0195] In some embodiments of the present disclosure, R5 and R6 are independently selected from H or C 1-3Alkyl; and R5 and R6 are not H or C 1-3 alkyl.

[0196] In some embodiments of the present disclosure, R5 and R6 are independently selected from H or methyl; and R5 and R6 are not H or methyl at the same time.

[0197] In some embodiments of the present disclosure, the nucleotide analog has a structure represented by formula (501), or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof:

[0198] In some embodiments of the present disclosure, the nucleotide analog has any of the following structures, or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof:

[0199] wherein R5 is selected from optionally substituted C 1-3 alkyl;

[0200] wherein R6 is selected from optionally substituted C 1-3 alkyl.

[0201] In some embodiments of the present disclosure, the nucleotide analog has any of the following structures, or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof:

[0202] wherein R5 and R6 are independently selected from optionally substituted C 1-3 alkyl.

[0203] In some embodiments of the present disclosure, the nucleotide analog is selected from any one of the following structures, or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof:

[0204] Double-stranded oligonucleotides

[0205] In the second aspect of the present disclosure, the present disclosure provides a double-stranded oligonucleotide comprising a sense strand and an antisense strand, each strand having 17-35 nucleotides; and in the 5' to 3' direction, at least one nucleotide in positions 2-8 of the antisense strand is replaced by the nucleotide analogue described above.

[0206] In some embodiments of the present disclosure, the present disclosure provides a double-stranded oligonucleotide, wherein at least one nucleotide in positions 3 to 8 of the antisense strand is replaced by the nucleotide analog in the 5' to 3' direction.

[0207] In some embodiments, in the 5' to 3' direction, any one of the nucleotides at positions 3, 4, 5, 6, 7, and 8 of the antisense strand is replaced by the nucleotide analog.

[0208] In some embodiments, the 5th, 6th, 7th or 8th nucleotide of the antisense strand is replaced by the nucleotide analogue in the 5' to 3' direction.

[0209] In some embodiments, the third nucleotide of the antisense strand is replaced by the nucleotide analog in the 5' to 3' direction.

[0210] In some embodiments, the 4th nucleotide of the antisense strand is replaced by the nucleotide analog in the 5' to 3' direction.

[0211] In some embodiments, the 5th nucleotide of the antisense strand is replaced by the nucleotide analog in the 5' to 3' direction.

[0212] In some embodiments, the 6th nucleotide of the antisense strand is replaced by the nucleotide analog in the 5' to 3' direction.

[0213] In some embodiments, the 7th nucleotide of the antisense strand is replaced by the nucleotide analog in the 5' to 3' direction.

[0214] In some embodiments, the 8th nucleotide of the antisense strand is replaced by the nucleotide analog in the 5' to 3' direction.

[0215] In some embodiments, in the double-stranded oligonucleotide provided by the present disclosure, the Base in each of the nucleotide analogs is the same as the base in the nucleotide it replaces.

[0216] In some embodiments, in the double-stranded oligonucleotide provided by the present disclosure, in the direction from the 5' end to the 3' end, the 5th nucleotide of the nucleotide sequence in the sense strand is selected from a 2'-O-methyl modified nucleotide or a 2'-O-methoxyethyl modified nucleotide, at least three nucleotides from positions 7 to 10 are 2'-fluoro modified nucleotides, and the nucleotides at the remaining positions are independently selected from 2'-O-methyl modified nucleotides; and / or

[0217] In some embodiments, in the double-stranded oligonucleotide provided by the present disclosure, in the direction from the 5' end to the 3' end, at least one nucleotide at positions 3 to 8 of the nucleotide sequence in the antisense strand is independently selected from the nucleotide analog, at least four nucleotides at positions 2, 6, 9, 12, 14, and 16 are selected from 2'-fluoro-modified nucleotides, the nucleotide at position 15 is selected from a 2'-O-methoxyethyl-modified nucleotide or a 2'-O-methyl-modified nucleotide, and the nucleotides at the remaining positions are independently selected from 2'-O-methyl-modified nucleotides; wherein, when the nucleotide at position 6 is selected from the nucleotide analog, the nucleotides at positions 2, 9, 12, 14, and 16 are selected from 2'-fluoro-modified nucleotides; when the nucleotide at position 6 is selected from a 2'-fluoro-modified nucleotide, at least one nucleotide at positions 3 to 5 and 7 to 8 is independently selected from the nucleotide analog;

[0218] In some embodiments, in the double-stranded oligonucleotide provided by the present disclosure, at least three nucleotides at positions 7 to 10 of the nucleotide sequence in the sense strand from the 5' end to the 3' end are 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions are independently selected from 2'-O-methyl-modified nucleotides.

[0219] In some embodiments, in the double-stranded oligonucleotide provided by the present disclosure, in the direction from the 5' end to the 3' end, at least one nucleotide at positions 3 to 8 of the nucleotide sequence in the antisense strand is independently selected from the nucleotide analogues, at least four nucleotides at positions 2, 6, 9, 12, 14 and 16 are selected from 2'-fluoro-modified nucleotides, the nucleotide at position 15 is selected from 2'-O-methoxyethyl-modified nucleotides or 2'-O-methyl-modified nucleotides, and the nucleotides at the remaining positions are independently selected from 2'-O-methyl-modified nucleotides; wherein, when the nucleotide at position 6 is selected from the nucleotide analogues, the nucleotides at positions 2, 9, 12, 14 and 16 are selected from 2'-fluoro-modified nucleotides; when the nucleotide at position 6 is selected from 2'-fluoro-modified nucleotides, at least one nucleotide at positions 3 to 5 and 7 to 8 is independently selected from the nucleotide analogues.

[0220] In some embodiments, in the double-stranded oligonucleotide provided by the present disclosure, in the direction from the 5' end to the 3' end, at least three nucleotides in the 7th to 10th positions of the nucleotide sequence in the positive strand are 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions are independently selected from 2'-O-methyl-modified nucleotides.

[0221] In some embodiments, in the double-stranded oligonucleotide provided by the present disclosure, in the direction from the 5' end to the 3' end, at least one nucleotide at positions 3 to 8 of the nucleotide sequence in the antisense strand is independently selected from the nucleotide analog, at least three nucleotides at positions 2, 6, 14 and 16 are selected from 2'-fluoro-modified nucleotides, and the nucleotide at position 9 or 12 is selected from a 2'-fluoro-modified nucleotide, the nucleotide at position 15 is selected from a 2'-O-methoxyethyl-modified nucleotide or a 2'-O-methyl-modified nucleotide, and the nucleotides at the remaining positions are independently selected from 2'-O-methyl-modified nucleotides; wherein, when the nucleotide at position 6 is selected from the nucleotide analog, the nucleotides at positions 2, 9, 12, 14 and 16 are selected from 2'-fluoro-modified nucleotides; when the nucleotide at position 6 is selected from a 2'-fluoro-modified nucleotide, at least one nucleotide at positions 3 to 5 and 7 to 8 is independently selected from the nucleotide analog.

[0222] In some embodiments, in the double-stranded oligonucleotide provided by the present disclosure, the modifications of the sense strand and the antisense strand in the double-stranded oligonucleotide are selected from one of the following (1) to (24):

[0223] (1) In the direction from the 5' end to the 3' end, the nucleotides at positions 7 to 10 in the nucleotide sequence of the sense strand are independently selected from 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions are independently selected from 2'-O-methyl-modified nucleotides;

[0224] In the direction from the 5' end to the 3' end, the 3rd nucleotide of the nucleotide sequence in the antisense strand is selected from the nucleotide analog, the 2nd, 6th, 9th, 14th and 16th nucleotides are independently selected from 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions are independently selected from 2'-O-methyl-modified nucleotides;

[0225] (2) in the direction from the 5' end to the 3' end, the nucleotides at positions 7 to 10 in the nucleotide sequence of the sense strand are independently selected from 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions are independently selected from 2'-O-methyl-modified nucleotides;

[0226] In the direction from the 5' end to the 3' end, the 4th nucleotide of the nucleotide sequence in the antisense strand is selected from the nucleotide analog, the 2nd, 6th, 9th, 14th and 16th nucleotides are independently selected from 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions are independently selected from 2'-O-methyl-modified nucleotides;

[0227] (3) In the direction from the 5' end to the 3' end, the nucleotides at positions 7 to 10 in the nucleotide sequence of the sense strand are independently selected from 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions are independently selected from 2'-O-methyl-modified nucleotides;

[0228] In the direction from the 5' end to the 3' end, the 5th nucleotide of the nucleotide sequence in the antisense strand is selected from the nucleotide analog, the 2nd, 6th, 9th, 14th and 16th nucleotides are independently selected from 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions are independently selected from 2'-O-methyl-modified nucleotides;

[0229] (4) In the direction from the 5' end to the 3' end, the nucleotides at positions 7 to 10 in the nucleotide sequence of the sense strand are independently selected from 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions are independently selected from 2'-O-methyl-modified nucleotides;

[0230] In the direction from the 5' end to the 3' end, the 6th nucleotide of the nucleotide sequence in the antisense strand is selected from the nucleotide analog, the 2nd, 9th, 14th and 16th nucleotides are independently selected from 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions are independently selected from 2'-O-methyl-modified nucleotides;

[0231] (5) In the direction from the 5' end to the 3' end, the nucleotides at positions 7 to 10 in the nucleotide sequence of the sense strand are independently selected from 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions are independently selected from 2'-O-methyl-modified nucleotides;

[0232] In the direction from the 5' end to the 3' end, the 7th nucleotide of the nucleotide sequence in the antisense strand is selected from the nucleotide analog, the 2nd, 6th, 9th, 14th and 16th nucleotides are independently selected from 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions are independently selected from 2'-O-methyl-modified nucleotides;

[0233] (6) In the direction from the 5' end to the 3' end, the nucleotides at positions 7 to 10 in the nucleotide sequence of the sense strand are independently selected from 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions are independently selected from 2'-O-methyl-modified nucleotides;

[0234] In the direction from the 5' end to the 3' end, the 8th nucleotide of the nucleotide sequence in the antisense strand is selected from the nucleotide analog, the 2nd, 6th, 9th, 14th and 16th nucleotides are independently selected from 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions are independently selected from 2'-O-methyl-modified nucleotides;

[0235] (7) In the direction from the 5' end to the 3' end, the nucleotides at positions 7 to 10 in the nucleotide sequence of the sense strand are independently selected from 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions are independently selected from 2'-O-methyl-modified nucleotides;

[0236] In the direction from the 5' end to the 3' end, the nucleotide at position 3 of the nucleotide sequence in the antisense strand is selected from the nucleotide analog, the nucleotides at positions 2, 6, 9, 14, and 16 are independently selected from 2'-fluoro-modified nucleotides, the nucleotide at position 15 is selected from 2'-O-methoxyethyl-modified nucleotides, and the nucleotides at the remaining positions are independently selected from 2'-O-methyl-modified nucleotides;

[0237] (8) In the direction from the 5' end to the 3' end, the nucleotides at positions 7 to 10 in the nucleotide sequence of the sense strand are independently selected from 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions are independently selected from 2'-O-methyl-modified nucleotides;

[0238] In the direction from the 5' end to the 3' end, the 4th nucleotide of the nucleotide sequence in the antisense strand is selected from the nucleotide analog, the 2nd, 6th, 9th, 14th and 16th nucleotides are independently selected from 2'-fluoro-modified nucleotides, the 15th nucleotide is selected from 2'-O-methoxyethyl-modified nucleotides, and the nucleotides at the remaining positions are independently selected from 2'-O-methyl-modified nucleotides;

[0239] (9) In the direction from the 5' end to the 3' end, the nucleotides at positions 7 to 10 in the nucleotide sequence of the sense strand are independently selected from 2'-fluoro-modified nucleotides, the nucleotide at position 15 is selected from 2'-O-methoxyethyl-modified nucleotides, and the nucleotides at the remaining positions are independently selected from 2'-O-methyl-modified nucleotides;

[0240] In the direction from the 5' end to the 3' end, the 5th nucleotide of the nucleotide sequence in the antisense strand is selected from the nucleotide analog, the 2nd, 6th, 9th, 14th and 16th nucleotides are independently selected from 2'-fluoro-modified nucleotides, the 15th nucleotide is selected from 2'-O-methoxyethyl-modified nucleotides, and the nucleotides at the remaining positions are independently selected from 2'-O-methyl-modified nucleotides;

[0241] (10) In the direction from the 5' end to the 3' end, the nucleotides at positions 7 to 10 in the nucleotide sequence of the sense strand are independently selected from 2'-fluoro-modified nucleotides, the nucleotide at position 15 is selected from 2'-O-methoxyethyl-modified nucleotides, and the nucleotides at the remaining positions are independently selected from 2'-O-methyl-modified nucleotides;

[0242] In the direction from the 5' end to the 3' end, the 6th nucleotide of the nucleotide sequence in the antisense strand is selected from the nucleotide analog, the 2nd, 9th, 14th and 16th nucleotides are independently selected from 2'-fluoro-modified nucleotides, the 15th nucleotide is selected from 2'-O-methoxyethyl-modified nucleotides, and the nucleotides at the remaining positions are independently selected from 2'-O-methyl-modified nucleotides;

[0243] (11) In the direction from the 5' end to the 3' end, the nucleotides at positions 7 to 10 in the nucleotide sequence of the sense strand are independently selected from 2'-fluoro-modified nucleotides, the nucleotide at position 15 is selected from 2'-O-methoxyethyl-modified nucleotides, and the nucleotides at the remaining positions are independently selected from 2'-O-methyl-modified nucleotides;

[0244] In the direction from the 5' end to the 3' end, the 7th nucleotide of the nucleotide sequence in the antisense strand is selected from the nucleotide analog, the 2nd, 6th, 9th, 14th and 16th nucleotides are independently selected from 2'-fluoro-modified nucleotides, the 15th nucleotide is selected from 2'-O-methoxyethyl-modified nucleotides, and the nucleotides at the remaining positions are independently selected from 2'-O-methyl-modified nucleotides;

[0245] (12) In the direction from the 5' end to the 3' end, the nucleotides at positions 7 to 10 in the nucleotide sequence of the sense strand are independently selected from 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions are independently selected from 2'-O-methyl-modified nucleotides;

[0246] In the direction from the 5' end to the 3' end, the 8th nucleotide of the nucleotide sequence in the antisense strand is selected from the nucleotide analog, the 2nd, 6th, 9th, 14th and 16th nucleotides are independently selected from 2'-fluoro-modified nucleotides, the 15th nucleotide is selected from 2'-O-methoxyethyl-modified nucleotides, and the nucleotides at the remaining positions are independently selected from 2'-O-methyl-modified nucleotides;

[0247] (13) In the direction from the 5' end to the 3' end, the nucleotides at positions 7 to 10 in the nucleotide sequence of the sense strand are independently selected from 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions are independently selected from 2'-O-methyl-modified nucleotides;

[0248] In the direction from the 5' end to the 3' end, the 3rd nucleotide of the nucleotide sequence in the antisense strand is selected from the nucleotide analog, the 2nd, 6th, 12th, 14th and 16th nucleotides are independently selected from 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions are independently selected from 2'-O-methyl-modified nucleotides;

[0249] (14) In the direction from the 5' end to the 3' end, the nucleotides at positions 7 to 10 in the nucleotide sequence of the sense strand are independently selected from 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions are independently selected from 2'-O-methyl-modified nucleotides;

[0250] In the direction from the 5' end to the 3' end, the 4th nucleotide of the nucleotide sequence in the antisense strand is selected from the nucleotide analog, the 2nd, 6th, 12th, 14th and 16th nucleotides are independently selected from 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions are independently selected from 2'-O-methyl-modified nucleotides;

[0251] (15) In the direction from the 5' end to the 3' end, the nucleotides at positions 7 to 10 in the nucleotide sequence of the sense strand are independently selected from 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions are independently selected from 2'-O-methyl-modified nucleotides;

[0252] In the direction from the 5' end to the 3' end, the 5th nucleotide of the nucleotide sequence in the antisense strand is selected from the nucleotide analog, the 2nd, 6th, 12th, 14th and 16th nucleotides are independently selected from 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions are independently selected from 2'-O-methyl-modified nucleotides;

[0253] (16) In the direction from the 5' end to the 3' end, the nucleotides at positions 7 to 10 in the nucleotide sequence of the sense strand are independently selected from 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions are independently selected from 2'-O-methyl-modified nucleotides;

[0254] In the direction from the 5' end to the 3' end, the 6th nucleotide of the nucleotide sequence in the antisense strand is selected from the nucleotide analog, the 2nd, 12th, 14th, and 16th nucleotides are independently selected from 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions are independently selected from 2'-O-methyl-modified nucleotides;

[0255] (17) In the direction from the 5' end to the 3' end, the nucleotides at positions 7 to 10 in the nucleotide sequence of the sense strand are independently selected from 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions are independently selected from 2'-O-methyl-modified nucleotides;

[0256] In the direction from the 5' end to the 3' end, the 7th nucleotide of the nucleotide sequence in the antisense strand is selected from the nucleotide analog, the 2nd, 6th, 12th, 14th, and 16th nucleotides are independently selected from 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions are independently selected from 2'-O-methyl-modified nucleotides;

[0257] (18) In the direction from the 5′ end to the 3′ end, the nucleotides at positions 7 to 10 in the nucleotide sequence of the sense strand are independently selected from 2′-fluoro-modified nucleotides, and the nucleotides at the remaining positions are independently selected from 2′-O-methyl-modified nucleotides;

[0258] In the direction from the 5' end to the 3' end, the 8th nucleotide of the nucleotide sequence in the antisense strand is selected from the nucleotide analog, the 2nd, 6th, 12th, 14th and 16th nucleotides are independently selected from 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions are independently selected from 2'-O-methyl-modified nucleotides;

[0259] (19) In the direction from the 5' end to the 3' end, the nucleotides at positions 7 to 10 in the nucleotide sequence of the sense strand are independently selected from 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions are independently selected from 2'-O-methyl-modified nucleotides;

[0260] In the direction from the 5' end to the 3' end, the nucleotide at position 3 of the nucleotide sequence in the antisense strand is selected from the nucleotide analog, the nucleotides at positions 2, 6, 12, 14, and 16 are independently selected from 2'-fluoro-modified nucleotides, the nucleotide at position 15 is selected from 2'-O-methoxyethyl-modified nucleotides, and the nucleotides at the remaining positions are independently selected from 2'-O-methyl-modified nucleotides;

[0261] (20) In the direction from the 5′ end to the 3′ end, the nucleotides at positions 7 to 10 in the nucleotide sequence of the sense strand are independently selected from 2′-fluoro-modified nucleotides, and the nucleotides at the remaining positions are independently selected from 2′-O-methyl-modified nucleotides;

[0262] In the direction from the 5' end to the 3' end, the 4th nucleotide of the nucleotide sequence in the antisense strand is selected from the nucleotide analog, the 2nd, 6th, 12th, 14th and 16th nucleotides are independently selected from 2'-fluoro-modified nucleotides, the 15th nucleotide is selected from 2'-O-methoxyethyl-modified nucleotides, and the nucleotides at the remaining positions are independently selected from 2'-O-methyl-modified nucleotides;

[0263] (21) In the direction from the 5' end to the 3' end, the nucleotides at positions 7 to 10 in the nucleotide sequence of the sense strand are independently selected from 2'-fluoro-modified nucleotides, the nucleotide at position 15 is selected from 2'-O-methoxyethyl-modified nucleotides, and the nucleotides at the remaining positions are independently selected from 2'-O-methyl-modified nucleotides;

[0264] In the direction from the 5' end to the 3' end, the 5th nucleotide of the nucleotide sequence in the antisense strand is selected from the nucleotide analog, the 2nd, 6th, 12th, 14th and 16th nucleotides are independently selected from 2'-fluoro-modified nucleotides, the 15th nucleotide is selected from 2'-O-methoxyethyl-modified nucleotides, and the nucleotides at the remaining positions are independently selected from 2'-O-methyl-modified nucleotides;

[0265] (22) In the direction from the 5′ end to the 3′ end, the nucleotides at positions 7 to 10 in the nucleotide sequence of the sense strand are independently selected from 2′-fluoro-modified nucleotides, the nucleotide at position 15 is selected from 2′-O-methoxyethyl-modified nucleotides, and the nucleotides at the remaining positions are independently selected from 2′-O-methyl-modified nucleotides;

[0266] In the direction from the 5' end to the 3' end, the 6th nucleotide of the nucleotide sequence in the antisense strand is selected from the nucleotide analog, the 2nd, 12th, 14th, and 16th nucleotides are independently selected from 2'-fluoro-modified nucleotides, the 15th nucleotide is selected from 2'-O-methoxyethyl-modified nucleotides, and the nucleotides at the remaining positions are independently selected from 2'-O-methyl-modified nucleotides;

[0267] (23) In the direction from the 5′ end to the 3′ end, the nucleotides at positions 7 to 10 in the nucleotide sequence of the sense strand are independently selected from 2′-fluoro-modified nucleotides, the nucleotide at position 15 is selected from 2′-O-methoxyethyl-modified nucleotides, and the nucleotides at the remaining positions are independently selected from 2′-O-methyl-modified nucleotides;

[0268] In the direction from the 5' end to the 3' end, the 7th nucleotide of the nucleotide sequence in the antisense strand is selected from the nucleotide analog, the 2nd, 6th, 12th, 14th, and 16th nucleotides are independently selected from 2'-fluoro-modified nucleotides, the 15th nucleotide is selected from 2'-O-methoxyethyl-modified nucleotides, and the nucleotides at the remaining positions are independently selected from 2'-O-methyl-modified nucleotides;

[0269] (24) In the direction from the 5′ end to the 3′ end, the nucleotides at positions 7 to 10 in the nucleotide sequence of the sense strand are independently selected from 2′-fluoro-modified nucleotides, and the nucleotides at the remaining positions are independently selected from 2′-O-methyl-modified nucleotides;

[0270] In the direction from the 5' end to the 3' end, the 8th nucleotide of the nucleotide sequence in the antisense strand is selected from the nucleotide analog, the 2nd, 6th, 12th, 14th and 16th nucleotides are independently selected from 2'-fluoro-modified nucleotides, the 15th nucleotide is selected from 2'-O-methoxyethyl-modified nucleotides, and the nucleotides at the remaining positions are independently selected from 2'-O-methyl-modified nucleotides.

[0271] In some embodiments of the present disclosure, in the double-stranded oligonucleotide provided by the present disclosure, the double-stranded oligonucleotide is selected from siRNA.

[0272] In some embodiments of the present disclosure, at least one of the phosphate groups in the phosphate-sugar backbone of at least one single strand in the sense strand and the antisense strand of the double-stranded oligonucleotide provided by the present disclosure is a phosphate group having a modified group. In some embodiments, the phosphate group having a modified group is a phosphorothioate group formed by replacing at least one oxygen atom in the phosphodiester bond in the phosphate group with a sulfur atom. In some embodiments, in the double-stranded oligonucleotide, the phosphate group having a modified group is present in at least one of the group consisting of the following positions:

[0273] between the first and second nucleotides at the 5' end of the sense strand;

[0274] between the second and third nucleotides at the 5' end of the sense strand;

[0275] between the first and second nucleotides at the 3' end of the sense strand;

[0276] between the second and third nucleotides at the 3' end of the sense strand;

[0277] between the first and second nucleotides at the 5' end of the antisense strand;

[0278] between the second and third nucleotides at the 5' end of the antisense strand;

[0279] between the first nucleotide and the second nucleotide at the 3' end of the antisense strand; and between the second nucleotide and the third nucleotide at the 3' end of the antisense strand.

[0280] Double-stranded oligonucleotide conjugates (siRNA conjugates)

[0281] In a third aspect of the present disclosure, the present disclosure provides a double-stranded oligonucleotide conjugate, wherein the conjugate comprises the double-stranded oligonucleotide described above in the present disclosure and one or more ligands capable of binding to cell surface receptors.

[0282] In some embodiments of the present disclosure, the ligand is selected from asialoglycoprotein receptor ligands (ASGPR ligands).

[0283] In some embodiments of the present disclosure, the ASGPR ligand comprises galactose, a galactose derivative, or a galactose cluster.

[0284] In some embodiments of the present disclosure, the galactose derivative comprises a galactose derivative having an affinity for asialoglycoprotein receptor equal to or greater than that of galactose.

[0285] In some embodiments of the present disclosure, the galactose cluster comprises molecules having 2-4 terminal galactose and / or galactose derivatives.

[0286] In some embodiments of the present disclosure, the galactose derivative is selected from galactosamine, N-formylgalactosamine, N-acetylgalactosamine, N-propionylgalactosamine, N-n-butyrylgalactosamine and N-isobutyrylgalactosamine.

[0287] In some embodiments of the present disclosure, the number of the ligands is selected from one, and one ligand is conjugated to the 3' end of the sense strand of the double-stranded oligonucleotide.

[0288] In some embodiments of the present disclosure, in the conjugates described herein, each of the ligands is selected from the structure represented by formula (300), or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof:

[0289] Wherein, * represents the covalent attachment site of the ligand on the sense strand or the antisense strand;

[0290] Said j is selected from 1, 2, 3 or 4;

[0291] Each of said Z' is independently selected from hydroxyl or thiol;

[0292] Each of said p is independently selected from 1, 2 or 3;

[0293] Each of said q is independently selected from 1, 2 or 3;

[0294] Each R is independently selected from H, optionally substituted C 1-6 Alkyl or optionally substituted C 1-6 alkoxy;

[0295] Each of said L is independently selected from optionally substituted C 2-20 Alkylene or R La and R Lb Independently selected from optionally substituted C 1-10 Alkylene, k is selected from 1, 2, 3, 4 or 5;

[0296] Each Y is independently selected from O, S or NH.

[0297] In some embodiments of the present disclosure, said m is selected from 1, 2 or 3;

[0298] In some embodiments of the present disclosure, m is selected from 3.

[0299] In some embodiments of the present disclosure, the Z' is selected from hydroxyl.

[0300] In some embodiments of the present disclosure, the p is selected from 1.

[0301] In some embodiments of the present disclosure, q is selected from 1.

[0302] In some embodiments of the present disclosure, the R is selected from H.

[0303] In some embodiments of the present disclosure, each of said L is independently selected from optionally substituted C 2-10 Alkylene or Among them, R La and R Lb Independently selected from optionally substituted C 1-10 Alkylene, k is selected from 1, 2 or 3.

[0304] In some embodiments of the present disclosure, k is selected from 1.

[0305] In some embodiments of the present disclosure, each L is independently selected from

[0306] In some embodiments of the present disclosure, each L is independently selected from

[0307] In some embodiments of the present disclosure, each L is independently selected from

[0308] In some embodiments of the present disclosure, each L is independently selected from

[0309] In some embodiments of the present disclosure, Y is selected from O.

[0310] In some embodiments of the present disclosure, in the conjugate of the present disclosure, each ligand is independently selected from the structure represented by formula (302), or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof:

[0311] In some embodiments of the present disclosure, in the conjugates described herein, each ligand is independently selected from any of the following structures, or stereoisomers thereof, or pharmaceutically acceptable salts thereof:

[0312] In some embodiments of the present disclosure, in the conjugate described herein, each ligand is independently selected from the structure represented by formula (CR01008×3), or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof:

[0313] Composition

[0314] In a fourth aspect of the present disclosure, the present disclosure provides a composition comprising the double-stranded oligonucleotide and / or the double-stranded oligonucleotide conjugate described in the present disclosure.

[0315] Pharmaceutical composition

[0316] In a fifth aspect of the present disclosure, the present disclosure also provides a pharmaceutical composition comprising the double-stranded oligonucleotide, and / or the double-stranded oligonucleotide conjugate, and / or the composition described in the present disclosure.

[0317] In some embodiments of the present disclosure, the pharmaceutical composition further includes pharmaceutically acceptable excipients or adjuvants.

[0318] The excipients or auxiliary agents may be one or more of various preparations or compounds conventionally used in the art. For example, the pharmaceutically acceptable excipients or auxiliary agents include at least one of a pH buffer, a protective agent, and an osmotic pressure regulator.

[0319] The pH buffer may be a tris hydrochloride buffer with a pH value of 7.5-8.5 and / or a phosphate buffer with a pH value of 5.5-8.5, for example, a phosphate buffer with a pH value of 5.5-8.5.

[0320] The protective agent may be at least one of inositol, sorbitol, sucrose, trehalose, mannose, maltose, lactose and glucose. The content of the protective agent may be 0.01-30% by weight based on the total weight of the pharmaceutical composition.

[0321] The osmotic pressure regulator can be sodium chloride and / or potassium chloride. The content of the osmotic pressure regulator is such that the osmotic pressure of the pharmaceutical composition is 200-700 milliosmoles / kilogram (mOsm / kg). According to the desired osmotic pressure, those skilled in the art can easily determine the content of the osmotic pressure regulator. In some embodiments, the dosage of the preparation made from the pharmaceutical composition during administration may be adjusted due to different administration methods.

[0322] In some embodiments of the present disclosure, the pharmaceutical composition may be a liquid preparation, such as an injection; or a lyophilized powder injection, which is mixed with a liquid excipient during administration to form a liquid preparation. The liquid preparation may be administered subcutaneously, intramuscularly, or intravenously, and may be delivered by spray administration to the lungs, spray administration to other organs (such as the liver) through the lungs, or by oropharyngeal inhalation, or nasal administration.

[0323] Nucleoside analogs

[0324] In a sixth aspect of the present disclosure, the present disclosure provides a nucleoside analogue selected from the structure represented by formula (Ii), or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof:

[0325] Among them, B 100 Selected from bases or modified bases, if B 100 Contains an amino group, and the amino group is protected by an amino protecting group;

[0326] n is selected from 1, 2 or 3;

[0327] X is selected from Each R5 is independently selected from optionally substituted C 1-3 Alkyl, the optionally substituted C 1-3 The alkyl group contains substituents independently selected from halogen, C 1-3 Alkoxy, hydroxy or amino groups;

[0328] R1 is selected from H or a hydroxyl protecting group;

[0329] R4 is selected from H or Each R 4a Independently selected from or C containing a cyano substituent 1-6 Alkoxy, and at least one R 4a Selected from Each R 4a 'Independently selected from optionally substituted C 1-6 alkyl;

[0330] m is selected from 1, 2, 3 or 4;

[0331] r is selected from 1, 2, 3 or 4;

[0332] Each R A and each R B are each independently selected from H or optionally substituted C 1-3 alkyl; and each R A and each R B At least one of the following is selected from optionally substituted C 1-3 Alkyl; if the optionally substituted C 1-3 The alkyl group contains substituents independently selected from halogen, C 1-3 alkoxy, hydroxy or amino.

[0333] In some optional embodiments of the present disclosure, each R A and each R B are each independently selected from H or optionally substituted C 1-3 alkyl; and each R A and each R BAny one of which is selected from optionally substituted C 1-3 Alkyl; if the optionally substituted C 1-3 The alkyl group contains substituents independently selected from halogen, C 1-3 Alkoxy, hydroxy or amino. If one R A Selected from optionally substituted C 1-3 Alkyl, the rest R A and all R B Select H, or one R B Selected from optionally substituted C 1-3 Alkyl, the rest R B and all R A Selected from H.

[0334] In some specific embodiments of the present disclosure, m is selected from 2 and r is selected from 2.

[0335] In some specific embodiments of the present disclosure, the nucleoside analog is selected from the structure represented by formula (I-ii), or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof:

[0336] wherein R2, R3, R5, and R6 are each independently selected from H or an optionally substituted C 1-3 and at least one of R2, R3, R5 and R6 is selected from optionally substituted C 1-3 Alkyl; if the optionally substituted C 1-3 The alkyl group contains substituents independently selected from halogen, C 1-3 alkoxy, hydroxy or amino.

[0337] In some optional embodiments of the present disclosure, R2, R3, R5, and R6 are each independently selected from H or optionally substituted C 1-3 and any one of R2, R3, R5 and R6 is selected from optionally substituted C 1-3 Alkyl, such as R2 is selected from optionally substituted C 1-3 and R3, R5 and R6 are selected from H, or R3 is selected from optionally substituted C 1-3 and R2, R5 and R6 are selected from H, or R5 is selected from optionally substituted C 1-3 and R2, R3 and R6 are selected from H, or R6 is selected from optionally substituted C 1-3 Alkyl and R2, R3 and R5 are selected from H.

[0338] In some optional embodiments of the present disclosure, R2, R3, R5, and R6 are each independently selected from H or C 1-3 Alkyl; and any one of R2, R3, R5 and R6 is selected from C 1-3 Alkyl, such as R2 is selected from C 1-3Alkyl and R3, R5 and R6 are selected from H, or R3 is selected from C 1-3 Alkyl and R2, R5 and R6 are selected from H, or R5 is selected from C 1-3 Alkyl and R2, R3 and R6 are selected from H, or R6 is selected from C 1-3 Alkyl and R2, R3 and R5 are selected from H.

[0339] In some optional embodiments of the present disclosure, the C 1-3 The alkyl group is selected from methyl, ethyl, n-propyl or isopropyl.

[0340] In some specific embodiments of the present disclosure, the C 1-3 The alkyl group is selected from methyl.

[0341] In some specific embodiments of the present disclosure, R2, R3, R5, and R6 are each independently selected from H or methyl; and any one of R2, R3, R5, and R6 is selected from methyl, such as R2 is selected from methyl and R3, R5, and R6 are selected from H, or R3 is selected from methyl and R2, R5, and R6 are selected from H, or R5 is selected from methyl and R2, R3, and R6 are selected from H, or R6 is selected from methyl and R2, R3, and R5 are selected from H.

[0342] Furthermore, the nucleoside analog has a structure represented by formula (100), or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof:

[0343] Among them, B 100 Selected from bases or modified bases, if B 100 Contains an amino group, and the amino group is protected by an amino protecting group;

[0344] n is selected from 1, 2 or 3;

[0345] X is selected from Each R5 is independently selected from optionally substituted C 1-3 Alkyl, the optionally substituted C 1-3 The alkyl group contains substituents independently selected from halogen, C 1-3 Alkoxy, hydroxy or amino groups;

[0346] m is selected from 1, 2, 3 or 4;

[0347] R1 is selected from H or a hydroxyl protecting group;

[0348] R2 and R3 are independently selected from H or optionally substituted C 1-3 and at least one of R2 and R3 is selected from optionally substituted C 1-3 Alkyl; if the optionally substituted C 1-3 The alkyl group contains substituents independently selected from halogen, C 1-3Alkoxy, hydroxy or amino groups;

[0349] R4 is selected from H or Each R 4a Independently selected from or C containing a cyano substituent 1-6 Alkoxy, and at least one R 4a Selected from Each R 4a 'Independently selected from optionally substituted C 1-6 alkyl.

[0350] In some optional embodiments of the present disclosure, the hydroxy protecting group is selected from trityl, 4-methoxytrityl, 4,4'-dimethoxytrityl or 4,4',4"-trimethoxytrityl.

[0351] In some optional embodiments of the present disclosure, the hydroxyl protecting group is selected from 4,4'-dimethoxytrityl.

[0352] In some optional embodiments of the present disclosure, R 4a ' is selected from isopropyl.

[0353] In some optional embodiments of the present disclosure, the C containing a cyano substituent 1-6 Alkoxy is selected from

[0354] In some optional embodiments of the present disclosure, Selected from

[0355] In some optional embodiments of the present disclosure, Selected from

[0356] In some optional embodiments of the present disclosure, Selected from

[0357] In some optional embodiments of the present disclosure, the base is selected from uracil U, thymine T, cytosine C, adenine A or guanine G.

[0358] In some optional embodiments of the present disclosure, the modified base is selected from

[0359] In some optional embodiments of the present disclosure, B 100 is selected from the bases uracil U, thymine T, cytosine C, adenine A or guanine G, if B 100 The amino group is protected by an amino protecting group.

[0360] In some optional embodiments of the present disclosure, the amino protecting group is selected from an alkoxycarbonyl amino protecting group, an acyl amino protecting group or an alkyl amino protecting group.

[0361] In some optional embodiments of the present disclosure, the amino protecting group is selected from acyl amino protecting groups.

[0362] In some optional embodiments of the present disclosure, the acyl amino protecting group is selected from phthaloyl, p-toluenesulfonyl, trifluoroacetyl, o-nitrobenzenesulfonyl, p-nitrobenzenesulfonyl, pivaloyl, isobutyryl, acetyl (Ac) or benzoyl (Bz).

[0363] In some optional embodiments of the present disclosure, the amino protecting group is selected from isobutyryl, acetyl (Ac) or benzoyl (Bz).

[0364] In some optional embodiments of the present disclosure, B 100 Select from any of the following structures:

[0365] In some optional embodiments of the present disclosure, B 100 Selected from

[0366] In some optional embodiments of the present disclosure, B 100 Selected from

[0367] In some optional embodiments of the present disclosure, B 100 Selected from

[0368] In some optional embodiments of the present disclosure, B 100 Selected from

[0369] In some optional embodiments of the present disclosure, B 100 Selected from

[0370] In some optional embodiments of the present disclosure, B 100 Selected from

[0371] In some optional embodiments of the present disclosure, B 100 Selected from

[0372] In some optional embodiments of the present disclosure, B 100 Selected from

[0373] In some optional embodiments of the present disclosure, B 100 Selected from

[0374] In some optional embodiments of the present disclosure, B 100 Selected from

[0375] In some optional embodiments of the present disclosure, B 100 Selected from

[0376] In some optional embodiments of the present disclosure, in formula (100), n is selected from 1 or 2.

[0377] In some optional embodiments of the present disclosure, n is selected from 1.

[0378] In some optional embodiments of the present disclosure, in formula (100), m is selected from 1, 2 or 3.

[0379] In some optional embodiments of the present disclosure, m is selected from 2.

[0380] In some optional embodiments of the present disclosure, in formula (100), X is selected from

[0381] In some optional embodiments of the present disclosure, in formula (100), R2 and R3 are independently selected from H or optionally substituted C 1-3 and R2 and R3 are not simultaneously selected from H and optionally substituted C 1-3 Alkyl (ie: R2 is selected from H, and R3 is selected from optionally substituted C 1-3 Alkyl; or R2 is selected from optionally substituted C 1-3 alkyl, and R3 is selected from H).

[0382] In some optional embodiments of the present disclosure, R2 and R3 are independently selected from H or C 1-3 Alkyl; and R2 and R3 are not simultaneously selected from H and C 1-3 alkyl.

[0383] In some optional embodiments of the present disclosure, R2 and R3 are independently selected from H or methyl; and R2 and R3 are not simultaneously selected from H and methyl.

[0384] In some optional embodiments of the present disclosure, the nucleoside analog has a structure represented by formula (101), or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof:

[0385] In some embodiments of the present disclosure, the nucleoside analog has any of the following structures, or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof:

[0386] wherein R2 is selected from optionally substituted C 1-3 alkyl;

[0387] wherein R3 is selected from optionally substituted C 1-3 alkyl.

[0388] In some optional embodiments of the present disclosure, the nucleoside analog has the structure shown in (102), or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof:

[0389] wherein R2 is selected from optionally substituted C 1-3 alkyl.

[0390] In some optional embodiments of the present disclosure, the nucleoside analog has the structure shown in (103), or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof:

[0391] wherein R3 is selected from optionally substituted C 1-3 alkyl.

[0392] In some embodiments of the present disclosure, the nucleoside analog has any of the following structures, or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof:

[0393] wherein R2 and R3 are independently selected from optionally substituted C 1-3 alkyl.

[0394] In some optional embodiments of the present disclosure, the nucleoside analog has a structure represented by formula (102A), or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof:

[0395] wherein R2 is selected from optionally substituted C 1-3 alkyl.

[0396] In some optional embodiments of the present disclosure, the nucleoside analog has a structure represented by formula (102B), or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof:

[0397] wherein R2 is selected from optionally substituted C 1-3 alkyl.

[0398] In some optional embodiments of the present disclosure, the nucleoside analog has a structure represented by formula (103A), or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof:

[0399] wherein R3 is selected from optionally substituted C 1-3 alkyl.

[0400] In some optional embodiments of the present disclosure, the nucleoside analog has a structure represented by formula (103B), or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof:

[0401] wherein R3 is selected from optionally substituted C 1-3 alkyl.

[0402] In some embodiments of the present disclosure, the nucleoside analog is selected from any one of the following structures, or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof:

[0403] Furthermore, the nucleoside analog has a structure represented by formula (400), or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof:

[0404] Among them, R1, R4, B 100 , n, X and * are as defined above;

[0405] r is selected from 1, 2, 3 or 4;

[0406] R5 and R6 are independently selected from H or optionally substituted C 1-3 and at least one of R5 and R6 is selected from optionally substituted C 1-3 Alkyl; if the optionally substituted C 1-3 The alkyl group contains substituents independently selected from halogen, C 1-3 alkoxy, hydroxy or amino.

[0407] In some optional embodiments of the present disclosure, r is selected from 1, 2 or 3.

[0408] In some specific embodiments of the present disclosure, r is selected from 2.

[0409] In some optional embodiments of the present disclosure, R5 and R6 are independently selected from H or optionally substituted C 1-3 and R5 and R6 are not H or optionally substituted C 1-3 alkyl.

[0410] In some optional embodiments of the present disclosure, R5 and R6 are independently selected from H or C 1-3 Alkyl; and R5 and R6 are not H or C 1-3 alkyl;

[0411] In some optional embodiments of the present disclosure, R5 and R6 are independently selected from H or methyl; and R5 and R6 are not H or methyl at the same time, such as R5 is selected from H and R6 is selected from methyl, or R5 is selected from methyl and R6 is selected from H.

[0412] In some optional embodiments of the present disclosure, the nucleoside analog has a structure represented by formula (401), or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof:

[0413] In some optional embodiments of the present disclosure, the nucleoside analog has any of the following structures, or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof:

[0414] wherein R5 is selected from optionally substituted C 1-3 alkyl;

[0415] wherein R6 is selected from optionally substituted C 1-3 alkyl.

[0416] In some optional embodiments of the present disclosure, the nucleoside analog has any of the following structures, or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof:

[0417] wherein R5 and R6 are independently selected from optionally substituted C 1-3 alkyl.

[0418] In some optional embodiments of the present disclosure, the nucleoside analog is selected from any one of the following structures, or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof:

[0419] application

[0420] In a seventh aspect of the present disclosure, the present disclosure further 提供 Use of the nucleoside analog, and / or the nucleotide analog, and / or the double-stranded oligonucleotide, and / or the double-stranded oligonucleotide conjugate, and / or the composition, and / or the pharmaceutical composition in the preparation of a medicament for treating and / or preventing a disease or condition associated with dysregulation of mRNA levels expressed by a specific gene.

[0421] In some embodiments of the present disclosure, the specific gene is a gene abnormally expressed in hepatocytes. In some embodiments, the specific gene is an endogenous gene expressed in the liver. In some embodiments, the specific gene is a gene of a pathogen that reproduces in the liver. In some embodiments, the specific gene is a gene expressed in lung epithelial cells. In some embodiments, the specific gene is a gene expressed in the central nervous system. In some embodiments, the specific gene is a gene expressed in tumor cells.

[0422] Exemplarily, the mRNA expressed by the specific gene includes but is not limited to one selected from the group consisting of mRNAs transcribed from the following genes: ACE2, ANGPTL3, ApoA, ApoB, ApoC, AR, ASK1, C5, Col1A1, CTGF, Ebola, FOXO1, FTO, FVII, FXI, FXII, GCGR, HBV, HCV, HSD17B13, p53, PCSK9, PNP, PLG, PKK, KNG, SARS-CoV-2, SCD1, SCNN1A, SOD1, STAT3, TIMP-1, TMPRSS6, XO, INSR, SREBF1, HDV, RPTOR, TLK2, LPA, C3, and AGT.

[0423] Furthermore, the mRNA expressed by the specific gene is selected from the mRNA expressed by the hepatitis B virus gene (HBV), the mRNA expressed by the angiopoietin-like protein 3 (ANGPTL3) gene, or the mRNA expressed by the apolipoprotein C3 (ApoC3) gene.

[0424] In some embodiments of the present disclosure, the disease or condition associated with the level of mRNA expressed by a specific gene is chronic liver disease, hepatitis, liver fibrosis, liver proliferative disease, and / or dyslipidemia. In some embodiments, the disease or condition associated with the level of mRNA expressed by a specific gene is hepatitis B or dyslipidemia. In some embodiments, the dyslipidemia is hypercholesterolemia, hypertriglyceridemia, or atherosclerosis.

[0425] Methods for regulating the expression of specific genes in target cells

[0426] In the eighth aspect of the present disclosure, the present disclosure also provides a method for regulating the expression of a specific gene in a target cell, the method comprising: contacting the double-stranded oligonucleotide, and / or the double-stranded oligonucleotide conjugate, and / or the composition, and / or the pharmaceutical composition provided by the present disclosure with the target cell.

[0427] In some embodiments, the modulation refers to the inhibition of expression of a specific gene in a cell.

[0428] By introducing the double-stranded oligonucleotides disclosed herein, and / or the double-stranded oligonucleotide conjugates, and / or the compositions, and / or the pharmaceutical compositions into cells in which a specific gene is abnormally expressed, the purpose of inhibiting the expression of a specific gene in the cell can also be achieved through the mechanism of gene expression regulation. In some embodiments, the cells are hepatocytes. In some embodiments, the hepatocytes can be cells selected from hepatoma cell lines such as Hep3B, HepG2, Huh7, or isolated primary hepatocytes. In some embodiments, the hepatocytes are primary hepatocytes.

[0429] The method provided by the present disclosure is used to inhibit the expression of a specific gene in a cell. The dosage of the double-stranded oligonucleotide provided, and / or the double-stranded oligonucleotide conjugate, and / or the composition, and / or the pharmaceutical composition is easily determined by those skilled in the art according to the effect desired to be obtained. For example, in some embodiments, the double-stranded oligonucleotide conjugate dosage is such an amount that it is enough to reduce the expression of the target gene and result in an extracellular concentration of 1pM to 1 μM, or 0.01nM to 100nM, or 0.05nM to 50nM, or to about 5nM at the target cell surface. The amount required to reach this local concentration will vary with various factors, including delivery method, delivery site, the number of cell layers between the delivery site and the target cell or tissue, whether delivery is local or systemic, etc. The concentration at the delivery site can be significantly higher than the concentration at the surface of the target cell or tissue.

[0430] Disease prevention and / or treatment methods

[0431] In the ninth aspect of the present disclosure, the present disclosure also provides a method for preventing and / or treating a disease or condition associated with dysregulation of mRNA levels expressed by a specific gene in target cells in a subject, characterized in that the method comprises: administering to the subject a pharmaceutically acceptable dose of the double-stranded oligonucleotide, and / or the double-stranded oligonucleotide conjugate, and / or the composition, and / or the pharmaceutical composition described in the present disclosure.

[0432] By administering the double-stranded oligonucleotides, and / or the double-stranded oligonucleotide conjugates, and / or the compositions, and / or the pharmaceutical compositions provided by the present disclosure to subjects in need, the purpose of preventing and / or treating pathological conditions or diseases caused by the expression of specific genes in cells can be achieved through the mechanism of regulating gene expression.

[0433] The administration refers to a method or approach that places the double-stranded oligonucleotide, and / or the double-stranded oligonucleotide conjugate, and / or the composition, and / or the pharmaceutical composition into the subject's body by causing the double-stranded oligonucleotide, and / or the double-stranded oligonucleotide conjugate, and / or the composition, and / or the pharmaceutical composition to be at least partially positioned at a desired site to produce a desired effect. Routes of administration suitable for the disclosed methods include topical administration and systemic administration. In general, topical administration results in more double-stranded oligonucleotide, and / or the double-stranded oligonucleotide conjugate, and / or the composition, and / or the pharmaceutical composition being delivered to a specific site compared to the entire body of the subject; whereas systemic administration results in the double-stranded oligonucleotide, and / or the double-stranded oligonucleotide conjugate, and / or the composition, and / or the pharmaceutical composition being delivered to substantially the entire body of the subject.

[0434] The drug may be administered to a subject by any suitable route known in the art, including but not limited to oral or parenteral routes, such as intravenous administration, intramuscular administration, subcutaneous administration, transdermal administration, airway administration (aerosol), pulmonary administration, nasal administration, rectal administration, and topical administration (including buccal administration and sublingual administration). The frequency of administration may be once or more daily, weekly, biweekly, three-weekly, monthly, or annually.

[0435] The dosage of the double-stranded oligonucleotides, and / or the double-stranded oligonucleotide conjugates, and / or the compositions, and / or the pharmaceutical compositions disclosed herein may be conventional dosages in the art, and the dosages may be determined based on various parameters, particularly the age, weight, and sex of the subject. Toxicity and efficacy may be determined by standard pharmaceutical procedures in cell culture or experimental animals, such as determining LD50 (the dose that causes 50% of the population to be lethal) and ED50 (the dose that causes 50% of the maximum reaction intensity in a quantitative reaction, and the dose that causes a positive reaction in 50% of the experimental subjects in a qualitative reaction). The range of human dosages may be derived based on data obtained from cell culture analysis and animal studies.

[0436] When administering the double-stranded oligonucleotide, and / or the double-stranded oligonucleotide conjugate, and / or the composition, and / or the pharmaceutical composition of the present disclosure, for example, to male or female C57BL / 6J or C3H / HeNCrlVr mice aged 6-12 weeks and weighing 18-25 g, based on the amount of the double-stranded oligonucleotide, and / or the double-stranded oligonucleotide conjugate, and / or the composition, and / or the pharmaceutical composition: for the oligonucleotide conjugate formed by the double-stranded oligonucleotide and the pharmaceutically acceptable conjugating molecule, the amount of the double-stranded oligonucleotide can be 0.001-100 mg / kg body weight, in some embodiments, 0.01-50 mg / kg body weight, in further embodiments, 0.05-20 mg / kg body weight, in still further embodiments, 0.1-15 mg / kg body weight, and in yet further embodiments, 0.1-10 mg / kg body weight. When administering the double-stranded oligonucleotide, and / or the double-stranded oligonucleotide conjugate, and / or the composition, and / or the pharmaceutical composition of the present disclosure, the above dosage may be preferred.

[0437] Reagent test kit

[0438] In the tenth aspect of the present disclosure, the present disclosure provides a kit comprising the double-stranded oligonucleotide, and / or the double-stranded oligonucleotide conjugate, and / or the composition, and / or the pharmaceutical composition provided by the present disclosure.

[0439] In some embodiments, the kit described in the present disclosure may provide the double-stranded oligonucleotide, and / or the double-stranded oligonucleotide conjugate, and / or the composition, and / or the pharmaceutical composition in one container. In some embodiments, the kit described in the present disclosure may include a container for providing a pharmaceutically acceptable excipient. In some embodiments, the kit may also include other ingredients, such as stabilizers or preservatives. In some embodiments, the kit described in the present disclosure may include at least one other therapeutic agent in a container other than the container for providing the double-stranded oligonucleotide, and / or the double-stranded oligonucleotide conjugate, and / or the composition, and / or the pharmaceutical composition described herein. In some embodiments, the kit may include instructions for mixing the double-stranded oligonucleotide, and / or the double-stranded oligonucleotide conjugate, and / or the composition, and / or the pharmaceutical composition with pharmaceutically acceptable excipients or adjuvants or other ingredients (if any).

[0440] In the kit disclosed herein, the double-stranded oligonucleotide, and / or the double-stranded oligonucleotide conjugate, and / or the composition, and / or the pharmaceutical composition and pharmaceutically acceptable adjuvant or auxiliary agent can be provided in any form, such as liquid form, dried form or lyophilized form. In some embodiments, the double-stranded oligonucleotide, and / or the double-stranded oligonucleotide conjugate, and / or the composition, and / or the pharmaceutical composition and optional pharmaceutically acceptable adjuvant or auxiliary agent are substantially pure and / or sterile. In some embodiments, sterile water can be provided in the kit disclosed herein.

[0441] Example

[0442] Unless otherwise stated, the ratios of reagents used in the present disclosure are calculated on a volume basis (v / v).

[0443] Unless otherwise stated, all reagents used in the synthesis of nucleoside analogs and ligands / carriers were purchased from Beijing Coupling Technology Co., Ltd. The main reagents are shown in Table 1.

[0444] Table 1 Main reagents

[0445] Among them, CPG stands for controlled pore glass (Controlled Pore Glass) carrier.

[0446] Unless otherwise stated, the reagents, consumables, and instruments used in the biological detection experiments of this disclosure are all commercially available products. Among them, the main reagents and consumables are shown in Table 2, and the main instruments and equipment are shown in Table 3.

[0447] Table 2 Main reagents and consumables

[0448] Table 3 Main instruments and equipment

[0449] Synthesis of nucleoside analogs

[0450] Preparation Example 1: Synthesis of Compound NM022

[0451] In this preparation example, the synthetic route of compound NM022 is as follows:

[0452] (1-1) Synthesis of Compound NM-U

[0453] KOH (36.04 g, 642.35 mmol, 4.0 eq) was dissolved in 180 ml of water. Uracil (18 g, 160.59 mmol, 1.0 eq) was slowly added to the aqueous KOH solution. The temperature was raised to 80°C and the reaction was stirred at 80°C for 30 min. Bromoacetic acid (24.54 g, 176.65 mmol, 1.1 eq) was added and the reaction was stirred at 80°C for 6 hours. After the reaction, 4 M aqueous hydrogen chloride solution was added to adjust the pH to 2. The solid was filtered to obtain a solid. The solid was washed with water (2 × 50 ml) and then with saturated aqueous ethyl acetate (2 × 50 ml) to obtain compound NM-U (12 g, 70.58 mmol, 44.4% yield, CAS number 4113-97-7) as a white solid. MS-ESI (m / z) = 171 [M+H] + .

[0454] (1-2) Synthesis of Compound NM022-2

[0455] 2-Bromoethanol (20 g, 160.045 mmol, 1.0 eq.), triethylamine (40.49 g, 400.112 mmol, 2.5 eq.), and DMTrCl (58.93 g, 192.054 mmol, 1.2 eq.) were dissolved in tetrahydrofuran (200 mL) and stirred at 25°C under an argon atmosphere for 6 hours. After completion of the reaction, the reaction solution was concentrated to dryness in vacuo, and the residue was purified by silica gel column chromatography (eluent: ethyl acetate / petroleum ether = 40 / 60, v / v) to obtain compound NM022-2 (50 g, yield 73.11%) as a white solid. MS ESI (m / z) = 427.2 [M+H] + .

[0456] 1 H NMR(300MHz,Chloroform-d)δ:7.48-7.43(m,2H),7.37-7.15(m,7H),6.85-6.80(m,4H),3.78(s,6H),3.43-3.37(m,4H).

[0457] (1-3) Synthesis of Compound NM022-3

[0458] Ethyl-(2R)-2-aminopropionic acid hydrochloride (5.03 g, 32.76 mmol, 1.0 eq.) and K2CO3 (6.79 g, 49.14 mmol, 1.5 eq.) were dissolved in acetonitrile (70 mL) and stirred at 25 ° C for 30 min. Then, compound NM022-2 (14 g, 32.761 mmol, 1.0 eq.) was added and stirred at 80 ° C for 16 h. After that, the reaction solution was cooled to 0 ° C and quenched with saturated NaHCO3 aqueous solution (300 ml). After completion of the reaction, the reaction solution was extracted with ethyl acetate (500 ml x 3). The organic phases were combined, washed with saturated aqueous sodium chloride solution (500 ml), dried over anhydrous sodium sulfate, filtered, and concentrated to dryness in vacuo. The residue was purified by silica gel column chromatography (eluent: ethyl acetate / petroleum ether = 20 / 80, v / v) to obtain compound NM022-3 (11 g, 72.43% yield) as a yellow semisolid. MS ESI (m / z) = 464.2 [M+H] + .

[0459] 1 H NMR(300MHz,Chloroform-d)δ:7.45-7.41(m,2H),7.34-7.16(m,7H),6.84-6.79(m,4H),4.24-4.14(m,2H), 3.78(s,6H),3.39-3.32(m,1H),3.21-3.17(m,2H),2.89-2.81(m,1H),2.70-2.63(m,1H),1.31-1.25(m,6H).

[0460] (1-4) Synthesis of Compound NM022-4

[0461] (2,4-Dioxy-3H-pyrimidin-1-yl)acetic acid (4.04 g, 23.747 mmol, 1.0 eq.) and HATU (18.04 g, 47.458 mmol, 2.0 eq.) were dissolved in N,N-dimethylformamide (80 ml), stirred at 25 ° C for 20 min, and then compound NM022-3 (11 g, 23.729 mmol, 1.0 eq.) and DIEA (10.73 g, 83.052 mmol, 3.5 eq.) were added. The mixture was stirred at 25 ° C for 2 hours, and then saturated NaHCO3 aqueous solution (400 ml) was added at 0 ° C to quench the mixture. After the reaction, the mixture was extracted with ethyl acetate (500 ml × 3). The organic phases were combined, washed with saturated aqueous sodium chloride (400 ml × 3), dried over anhydrous sodium sulfate, filtered, and concentrated to dryness in vacuo. The residue was purified by silica gel column chromatography (eluent: ethyl acetate / petroleum ether = 65 / 35, v / v) to obtain compound NM022-4 (10.2 g, 69.82% yield) as a yellow semisolid. MS ESI (m / z) = 614.20 [MH] - .

[0462] 1 H NMR(400MHz,Chloroform-d)δ:7.40-7.35(m,2H),7.32-7.18(m,7H),6.90-6.79(m,5H),5.63(d,J=7.6Hz,1H),4.67-4.46(m,2H),4 .33-4.28(m,1H),4.13-4.04(m,2H),3.78-3.75(m,7H),3.58-3.54(m,1H),3.47-3.40(m,2H),1.34-1.30(m,3H),1.20-1.15(m,3H).

[0463] (1-5) Synthesis of Compound NM022-5

[0464] Under argon atmosphere, LiBH4 (546.7 mg, 24.851 mmol, 1.5 equiv) was added to tetrahydrofuran (120 mL) at 0°C, followed by the addition of compound NM022-4 (10.2 g, 16.567 mmol, 1.0 equiv) with stirring. The mixture was stirred at 0°C for 1 hour and quenched by the addition of water (500 mL) at 0°C. After completion of the reaction, the mixture was extracted with ethyl acetate (500 mL x 3). The combined organic phases were washed with saturated aqueous sodium chloride (500 mL), dried over anhydrous sodium sulfate, filtered, and concentrated to dryness in vacuo. The residue was purified by reverse phase column chromatography (AQ-C18 column; eluent: acetonitrile / water = 5 / 95, v / v; detector: UV-254) to afford compound NM022-5 (6.6 g, 69.45% yield) as an off-white semisolid. MS ESI (m / z) = 572.15 [MH] - .

[0465] 1 H NMR(400MHz, Acetonitrile-d3)δ:7.48-7.43(m,2H),7.37-7.05(m,8H),6.92-6.87(m, 4H),5.57-5.54(m,1H),4.69-4.49(m,2H),3.96-3.85(m,1H),3.79(s,6H),3.55-3.11(m,6H),1.09-1.03(m,3H).

[0466] (1-6) Synthesis of Compound NM022

[0467] Compound NM022-5 (1.5 g, 2.615 mmol, 1.0 eq.) was dissolved in dry dichloromethane (15 ml), bis(diisopropylamino)(2-cyanoethoxy)phosphine (1.18 g, 3.923 mmol, 1.5 eq.) was added, and 4,5-dicyanoimidazole (0.25 g, 2.092 mmol, 0.8 eq.) was added to the reaction system in an argon atmosphere. The reaction was carried out at 25 ° C for 3 hours and quenched by adding cold saturated NaHCO3 aqueous solution (100 mL). After completion of the reaction, the reaction solution was extracted with dichloromethane (100 ml x 3), and the organic phases were combined; the organic phases were first washed with a saturated aqueous sodium chloride solution (100 ml x 2), then dried over anhydrous sodium sulfate, filtered, and concentrated to dryness under reduced pressure. The residue was purified by reverse phase column chromatography (C18 spherical column; eluent: acetonitrile / water = 10 / 90, v / v; detector: UV-254) to obtain compound NM022 (1.2912 g, yield 61.38%) as a colorless oil. MS ESI (m / z) = 772.30 [MH] - .

[0468] 1 H NMR((300MHz, Acetonitrile-d3)δ:9.01(br s,1H),7.43-7.37(m,2H),7.31-7.21(m,7H),7.15-6.95(m,1H),6.87-6.81(m,4H),5.54-5.47(m,1H ),4.61-4.38(m,2H),4.16-3.84(m,1H),3.77-3.13(m,16H),2.61-2.52(m,2H),1.20-0.90(m,15H).

[0469] Preparation Example 2: Synthesis of Compound NM023

[0470] In this preparation example, the synthetic route of compound NM023 is as follows:

[0471] (2-1) Synthesis of Compound NM023-1

[0472] Ethyl-(2S)-2-aminopropionic acid hydrochloride (6.47 g, 42.14 mmol, 1.0 eq.) and K2CO3 (8.73 g, 63.19 mmol, 1.5 eq.) were stirred in acetonitrile (90 ml) for 30 min, and then compound NM022-2 (18 g, 42.121 mmol, 1.0 eq.) was added and stirred at 80 ° C for 16 h. Saturated NaHCO3 aqueous solution (300 ml) was added at 0 ° C to quench. After the reaction, the reaction solution was extracted with ethyl acetate (500 mL × 3), and the organic phases were combined; the organic phase was first washed with a saturated aqueous sodium chloride solution (500 ml), then dried over anhydrous sodium sulfate, filtered, and concentrated to dryness in vacuo. The residue was purified by silica gel column chromatography (eluent: ethyl acetate / petroleum ether = 40 / 60, v / v) to obtain compound NM023-1 (12.4 g, yield 63.50%) as a yellow semisolid.

[0473] 1 H NMR(400MHz,Chloroform-d)δ:7.45-7.42(m,2H),7.34-7.17(m,7H),6.83-6.79(m,4H),4.23-4.13(m,2H), 3.78(s,6H),3.37-3.32(m,1H),3.20-3.17(m,2H),2.86-2.84(m,1H),2.69-2.66(m,1H),1.33-1.29(m,6H).

[0474] (2-2) Synthesis of Compound NM023-2

[0475] To N,N-dimethylformamide (80 ml) were added compound NM-U (4.55 g, 26.749 mmol, 1.0 eq.) and HATU (20.34 g, 53.498 mmol, 2.0 eq.), and the mixture was stirred at 25°C for 20 min. Then, compound NM023-1 (12.4 g, 26.749 mmol, 1.0 eq.) and DIEA (12.10 g, 93.621 mmol, 3.5 eq.) were added, and the mixture was stirred at 25°C for 2 hours. A saturated aqueous NaHCO3 solution (400 ml) at 0°C was added to quench the mixture. After completion of the reaction, the reaction solution was extracted with ethyl acetate (500 mL × 3), and the organic phases were combined; the organic phases were first washed with saturated aqueous sodium chloride (400 mL × 3), then dried over anhydrous Na2SO4, filtered, and concentrated to dryness in vacuo. The residue was purified by silica gel column chromatography (eluent: ethyl acetate / petroleum ether = 60 / 40, v / v) to obtain compound NM023-2 (12 g, yield 72.87%) as a yellow semisolid. MS ESI (m / z) = 614.15 [MH] - .

[0476] (2-3) Synthesis of Compound NM023-3

[0477] Under a nitrogen atmosphere at 0°C, LiBH4 (583.69 mg, 26.799 mmol, 1.5 eq.) was added to tetrahydrofuran (120 mL). Compound NM023-2 (11 g, 17.866 mmol, 1.0 eq.) was then added to the solution with stirring. The reaction was stirred at 0°C for 1 hour and quenched by the addition of water (500 mL) at 0°C. After completion of the reaction, the reaction solution was extracted with ethyl acetate (500 mL × 3), and the organic phases were combined; the organic phases were washed with saturated aqueous sodium chloride (500 mL), dried over anhydrous Na2SO4, and filtered. The filtrate was concentrated to dryness in vacuo, and the residue was purified by RP-Combi-flash (chromatographic column: AQ-C18; eluent: acetonitrile / water = 5 / 95, v / v; detector: UV-254) to obtain compound NM023-3 (6.2 g, yield 60.49%). MS ESI (m / z) = 572.20 [MH] - .

[0478] 1H NMR(400MHz,DMSO-d6)δ:11.25(br s,1H),7.42-7.13(m,10H),6.87-6.83(m,4H),5.50-5.48(m,1H),4.92-4.62(m,2H),3.96 -3.74(m,1H),3.69-3.68(m,6H),3.43-3.29(m,3H),3.26-2.98(m,2H),1.04-0.85(m,3H).

[0479] (2-4) Synthesis of Compound NM023

[0480] Compound NM023-3 (2.7 g, 4.707 mmol, 1.0 eq.) was dissolved in dry dichloromethane (15 ml), bis(diisopropylamino)(2-cyanoethoxy)phosphine (2.13 g, 7.06 mmol, 1.5 eq.) was added, and 4,5-dicyanoimidazole (389.10 mg, 3.295 mmol, 0.7 eq.) was added in an argon atmosphere. The mixture was reacted at 25 ° C for 3 hours and quenched by adding saturated NaHCO3 aqueous solution (100 mL) at 0 ° C. After the reaction, the reaction solution was extracted with dichloromethane (100 mL × 3), and the organic phases were combined; the organic phases were first washed with saturated sodium chloride aqueous solution (100 mL × 2), then dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated to dryness under reduced pressure, and the residue was purified by RP-Flash (C18 spherical column; eluent: acetonitrile / water = 10 / 90, v / v; detector: UV-254) to obtain NM023 (1.88 g, yield 51.88%) as a colorless oil. MS ESI (m / z) = 772.30 [MH] - .

[0481] 1 H NMR(400MHz, Acetonitrile-d3)δ:8.97(br s,1H),7.38-7.32(m,2H),7.26-7.13(m,7H),7.05-6.92(m,1H),6.83-6.75(m,4H),5.51-5.41(m,1H),4.60-4.26(m,2H),4.15-3.82(m ,1H),3.68(s,6H),3.64-3.56(m,2H),3.52-3.36(m,4H),3.33-3.20(m,3H),3.17-3.04(m,1H),2.54-2.48(m,2H),1.13-0.97(m,15H).

[0482] Preparation Example 3: Synthesis of Compound NM036

[0483] In this preparation example, the synthetic route of compound NM036 is as follows:

[0484] (3-1) Synthesis of Compound NM036-2

[0485] Compound NM036-1 (3.0 g, 39.96 mmol, 1.0 eq, (R)-1-aminopropan-2-ol, CAS No. 2799-17-8) was dissolved in 30 ml of dichloromethane. Imidazole (4.08 g, 59.94 mmol, 1.5 eq) was added, and the mixture was cooled to 0°C in an ice bath. Tert-butyldimethylsilyl chloride (7.84 g, 51.95 mmol, 1.3 eq, TBSCl) was added, and the mixture was stirred at 25°C for 16 hours. After completion of the reaction, the reaction mixture was diluted with dichloromethane (30 ml), washed with saturated aqueous ammonium chloride (2 x 50 ml) and saturated aqueous sodium chloride (2 x 50 ml), dried over anhydrous sodium sulfate, filtered, and concentrated to afford compound NM036-2 (7.5 g) as a yellow oil, which was used directly in the next step without purification. MS-ESI (m / z) = 190 [M+H] + .

[0486] (3-2) Synthesis of Compound NM036-3

[0487] Compound NM036-2 (3.88 g, 22.65 mmol, 1.0 eq) was dissolved in 100 ml of acetonitrile, and KCO (4.68 g, 33.98 mmol, 1.5 eq) and compound NM022-2 (9.65 g, 22.65 mmol, 1.0 eq) were added. The mixture was stirred at 80°C for 16 hours. After completion of the reaction, saturated aqueous sodium bicarbonate (100 ml) was added to the reaction solution, and the mixture was extracted with ethyl acetate (2 x 100 mL). The organic phases were combined and washed with saturated aqueous sodium chloride (2 x 50 ml), dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography (eluent: petroleum ether / ethyl acetate = 5 / 1, v / v) to afford compound NM036-3 (3.48 g, 6.50 mmol, yield 28.70%) as a white solid. MS-ESI (m / z) = 536 [M+H] + .

[0488] (3-3) Synthesis of Compound NM036-4

[0489] Compound NM036-3 (1.91 g, 11.2 mmol, 2.0 eq) was dissolved in N,N-dimethylformamide (30 ml), the ice bath was cooled to 0°C, and EDCI (1.61 g, 8.4 mmol, 1.5 eq) and triethylamine (1.98 g, 19.6 mmol, 3.5 eq) were added at 0°C, and stirred at 0°C for 10 minutes. 1-Hydroxybenzotriazole (1.14 g, 8.4 mmol, 1.5 eq) was added at 0°C, and the temperature was slowly raised to 25°C and compound NM-U (3.0 g, 5.6 mmol, 1.0 eq) was added at 25°C. The reaction was stirred at 25°C for 16 hours. After the reaction, saturated aqueous sodium bicarbonate (200 ml) was added to the reaction solution, and the mixture was extracted with ethyl acetate (2 × 100 ml). The organic phases were combined; the organic phases were washed with saturated aqueous sodium chloride (2 × 50 ml), dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography (eluent: petroleum ether / ethyl acetate = 2 / 3, v / v) to obtain compound NM036-4 (3.83 g, 5.57 mmol, yield 99.5%) as a white solid. MS-ESI (m / z) = 688 [M+H] + .

[0490] (3-4) Synthesis of Compound NM036-5

[0491] Compound NM036-4 (3.34 g, 4.86 mmol, 1.0 eq) was dissolved in 30 ml of tetrahydrofuran, and a 1 M solution of tetrabutylammonium fluoride in tetrahydrofuran (14.6 ml, 14.6 mmol, 3.0 eq) was added. The mixture was stirred at 25°C for 16 hours. After completion of the reaction, saturated aqueous sodium bicarbonate (100 ml) was added to the reaction solution, and the mixture was extracted with ethyl acetate (2 x 100 ml). The organic phases were combined and washed with saturated brine (2 x 50 ml), dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography (eluent: dichloromethane / methanol = 20 / 1, v / v) to obtain compound NM036-5 (2.56 g, 4.46 mmol, yield 91.77%) as a white solid. MS-ESI (m / z) = 574 [M+H] + .

[0492] (3-5) Synthesis of Compound NM036

[0493] Compound NM036-5 (2.33 g, 4.06 mmol, 1.0 eq) was dissolved in 30 ml of anhydrous dichloromethane. DCI (384 mg, 3.25 mmol, 0.8 eq) and bis(diisopropylamino)(2-cyanoethoxy)phosphine (1.48 g, 4.87 mmol, 1.2 eq) were added, respectively. The atmosphere was purged with nitrogen three times and the mixture was stirred at 25°C for 1 hour. After completion of the reaction, the reaction solution was washed with saturated aqueous sodium bicarbonate (2 x 20 ml) and then with saturated brine (20 ml). The organic phase was separated and dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography (eluent: water / acetonitrile = 5 / 95, v / v) to afford compound NM-036 (2.7 g, 3.49 mmol, yield 85.96%) as a white solid. MS-ESI (m / z) = 774 [M+H] + .

[0494] 1 H NMR (400MHz, DMSO-d6) δ: 11.30 (d, J = 11.3Hz, 1H), 7.34 (dt, J = 16.1, 8.0Hz, 4H), 7.22 (ddt, J = 12.1, 9.1, 4.4Hz, 5H), 6.89 (d, J = 8.7Hz, 4H), 4.59 (q, J=10.9,8.8Hz,2H),3.73(s,6H),3.71–3.36(m,7H),3.36–3.20(m,3H),3. 15–3.03(m,1H),2.72(ddt,J=24.1,11.9,5.8Hz,2H),1.32–0.99(m,15H).

[0495] Preparation Example 4: Synthesis of Compound NM037

[0496] In this preparation example, the synthetic route of compound NM037 is as follows:

[0497] (4-1) Synthesis of Compound NM037-2

[0498] Compound NM037-1 (3 g, 39.96 mmol, 1.0 eq, (S)-1-aminopropan-2-ol, CAS No. 2799-17-9) was dissolved in 30 ml of dichloromethane. Imidazole (4.08 g, 59.94 mmol, 1.5 eq) was added, and the mixture was cooled to 0°C in an ice bath. Tert-butyldimethylsilyl chloride (7.84 g, 51.95 mmol, 1.3 eq) was added at 0°C, and the mixture was stirred at 25°C for 16 hours. After completion of the reaction, the reaction mixture was diluted with dichloromethane (30 ml), washed with saturated aqueous ammonium chloride (2 x 50 ml) and saturated aqueous sodium chloride (2 x 50 ml), dried over anhydrous sodium sulfate, filtered, and concentrated to afford compound NM037-2 (5 g) as a yellow oil. MS-ESI (m / z) = 190 [M+H] + .

[0499] (4-2) Synthesis of Compound NM037-3

[0500] Compound NM037-2 (3 g, 15.84 mmol, 1.0 eq) was dissolved in 100 ml of acetonitrile, and KCO (3.28 g, 23.76 mmol, 1.5 eq) and compound NM022-2 (6.77 g, 15.84 mmol, 1.0 eq) were added. The mixture was stirred at 80°C for 30 hours. After completion of the reaction, saturated aqueous sodium bicarbonate (100 ml) was added to the reaction solution, and the mixture was extracted with ethyl acetate (2 x 100 ml). The organic phases were combined, washed with saturated aqueous sodium chloride (2 x 50 ml), dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography (eluent: petroleum ether / ethyl acetate = 5 / 1, v / v) to obtain compound NM037-3 (3.5 g, 6.50 mmol, yield 41.2%) as a white solid. MS-ESI (m / z) = 536 [M+H] + .

[0501] (4-3) Synthesis of Compound NM037-4

[0502] Compound NM037-3 (0.95 g, 6.16 mmol, 1.1 eq) was dissolved in 30 ml of N,N-dimethylformamide, cooled to 0°C in an ice bath and stirred at 0°C for 10 minutes. HATU (3.19 g, 8.4 mmol, 1.5 eq) and DIEA (2.05 g, 16.8 mmol, 3 eq) were then added at 0°C. The temperature was slowly raised to 25°C and compound NM-U (3.0 g, 5.6 mmol, 1.0 eq) was added at 25°C. The reaction was stirred at 25°C for 16 hours. After the reaction, saturated aqueous sodium bicarbonate solution (200 ml) was added to the reaction solution, and the mixture was extracted with ethyl acetate (2 × 100 ml). The organic phases were combined, washed with saturated aqueous sodium chloride solution (2 × 50 ml), dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography (eluent: petroleum ether / ethyl acetate = 2 / 3, v / v) to obtain compound NM037-4 (1.5 g, 2.18 mmol, yield 56.5%) as a white solid. MS-ESI (m / z) = 688 [M+H] + .

[0503] (4-4) Synthesis of Compound NM037-5

[0504] Compound NM037-4 (1.4 g, 2.03 mmol, 1.0 eq) was dissolved in 30 ml of tetrahydrofuran, and a solution of tetrabutylammonium fluoride in tetrahydrofuran (1 M, 6.09 ml, 6.09 mmol, 3.0 eq) was added. The mixture was stirred at 25°C for 16 hours. After completion of the reaction, saturated aqueous sodium bicarbonate (100 ml) was added to the reaction solution, and the mixture was extracted with ethyl acetate (2 x 100 ml). The organic phases were combined, washed with saturated brine (2 x 50 ml), dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography (eluent: dichloromethane / methanol = 20 / 1, v / v) to obtain compound NM037-5 (1 g, 1.74 mmol, yield 71.4%) as a white solid. MS-ESI (m / z) = 574 [M+H] + .

[0505] (4-5) Synthesis of Compound NM037

[0506] Compound NM037-5 (1 g, 1.74 mmol, 1.0 eq) was dissolved in 30 ml of anhydrous dichloromethane, and DCI (161 mg, 1.39 mmol, 0.8 eq) and bis(diisopropylamino)(2-cyanoethoxy)phosphine (629 mg, 2.08 mmol, 1.2 eq) were added, respectively. The atmosphere was purged with nitrogen three times, and the mixture was stirred at 25°C for 1 hour. After completion of the reaction, the reaction solution was washed with saturated aqueous sodium bicarbonate (2 x 20 ml) and saturated aqueous sodium chloride (20 mL), dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography (eluent: water / acetonitrile = 5 / 95, v / v) to obtain compound NM-037 (670 mg, 3.49 mmol, 50% yield) as a white solid. MS-ESI (m / z) = 774 [M+H] + .

[0507] 1 H NMR(400MHz, DMSO-d6)δ:11.30(d,J=11.3Hz,1H),7.45(dd,J=7.7,4.5Hz,2H),7.39–7.15(m,7H),6.98–6.85(m,5H),5.69–5.47(m,1H),4.89–4 .44(m,2H),3.63-3.5(ddt,J=19.9,13.6,6.5Hz,10H),3.47–3.15(m,4H ),2.64(ddt,J=17.7,12.6,5.9Hz,2H),2.17(s,2H),1.39–1.04(m,15H).

[0508] Preparation Example 5: Synthesis of Compound NM084

[0509] In this preparation example, the synthetic route of compound NM084 is as follows:

[0510] (5-1) Synthesis of Compound NM084-1

[0511] Compound NM022-2 (64 g, 0.15 mol) and L-alanine ethyl ester hydrochloride (23 g, 0.15 mol) were added to a 2000 ml reactor. Potassium carbonate (62 g, 0.45 mol) and acetonitrile (640 ml) were also added. The temperature was raised to 80°C and stirred at 80°C for 16 hours. After completion of the reaction, the reaction solution was filtered, the filtrate was concentrated, and purified by column chromatography to obtain compound NM084-1 (31 g, 44.5% yield) as a yellow oil. MS ESI (m / z) = 464.0 [M+H] + .

[0512] (5-2) Synthesis of Compound NM084-2

[0513] Compound NM084-1 (7 g, 0.015 mol) and tetrahydrofuran (42 ml) were added to a 100 ml reactor, the temperature was lowered to 0°C, and a 2M solution of lithium aluminum hydride in tetrahydrofuran (15 ml, 0.0486 mol) was added dropwise at 0°C. The atmosphere was replaced with nitrogen three times and stirred at 25°C for 1 hour. 20 ml of purified water was slowly added, and the mixture was extracted with ethyl acetate. The organic phase was separated, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain compound NM084-2 (5 g, yield 78.6%) as a white solid. MS ESI (m / z) = 422 [M+H] + .

[0514] (5-3) Synthesis of Compound NM084-3

[0515] To a 250 ml reactor was added compound NM084-2 (3 g, 6.5 mmol), compound NM-A (2.12 g, 7.1 mmol), HATU (3.69 g, 9.7 mmol), DIEA (1.67 g, 13 mmol), and N,N-dimethylformamide (30 ml). The atmosphere was purged with nitrogen three times and stirred at 25°C for 3 hours. After completion of the reaction, the reaction mixture was extracted with purified water (30 ml) and ethyl acetate (30 ml). The organic phase was separated, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography to obtain compound NM084-3 (2 g, 40.2% yield) as a white solid. MS ESI (m / z) = 701 [M+H] + .

[0516] (5-4) Synthesis of Compound NM084

[0517] Compound NM084-3 (1.6 g, 2.3 mmol) was added to a 100 ml reaction vessel, cooled to 0°C, and bis(diisopropylamino)(2-cyanoethoxy)phosphine (0.83 g, 2.77 mmol), 4,5-dicyanoimidazole (0.215 g, 1.8 mmol), and dichloromethane (16 ml) were added portionwise at 0°C. The atmosphere was purged with nitrogen three times and stirred at 25°C for 3 hours. After completion of the reaction, 20 ml of saturated aqueous sodium bicarbonate solution was added to the reaction solution. The organic phase was separated, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by reverse phase column chromatography (eluent: acetonitrile / water = 80 / 20, v / v) to afford compound NM084 (1.6 g, 77.7% yield) as a white solid. MS ESI (m / z) = 901 [M+H] + .

[0518] 1H NMR(400MHz,DMSO-d6)δ11.13(s,1H),8.72–8.56(m,1H),8.28–8.15(m,1H),8.05(d,J=7.6Hz ,2H),7.70–7.61(m,1H),7.61–7.49(m,2H),7.49–7.12(m,9H),6.93–6.79(m,4H),5.18(d,J= 17.8Hz,2H),3.71(d,J=4.3Hz,7H),3.57(dt,J=19.4,9.1Hz,5H),3.34(s,2H),3. 14(s,1H),2.70(q,J=8.0,6.1Hz,2H),2.07(d,J=1.4Hz,2H),1.39–0.84(m,15H).

[0519] Preparation Example 6: Synthesis of Compound NM085

[0520] In this preparation example, the synthetic route of compound NM085 is as follows:

[0521] (6-1) Synthesis of Compound NM-G

[0522] In this preparation example, the synthetic route of compound NM-G is as follows:

[0523] (6-1-1) Synthesis of Compound NM-g

[0524] A 250 ml reaction vessel was charged with N2-isobutyrylguanine (15 g, 0.0679 mol), tert-butyl bromoacetate (14.55 g, 0.0746 mol), potassium carbonate (18.73 g, 0.136 mol), and N,N-dimethylformamide (150 ml). The atmosphere was purged with nitrogen three times and stirred at 25°C for 6 hours. After completion of the reaction, the reaction solution was filtered, and the filtrate was extracted with purified water (300 ml) and ethyl acetate (300 ml). The organic phase was separated, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography to obtain compound NM-g (7.7 g, 34% yield). MS ESI (m / z) = 336 [M+H] + .

[0525] (6-1-2) Synthesis of Compound NM-G

[0526] Compound NM-g (7.7 g, 0.023 mol), trifluoroacetic acid (77 ml), and water (0.77 ml) were added to a 250 ml reactor and stirred at 25°C for 1 hour. After the reaction, the reaction solution was concentrated, slurried with ethyl acetate, and filtered to obtain compound NM-G (4 g, yield 62.5%). MS ESI (m / z) = 280 [M+H] + .

[0527] (6-2) Synthesis of Compound NM085-1

[0528] To a 250 ml reactor were added compound NM084-2 (5 g, 0.012 mol), compound NM-G (2.77 g, 0.01 mol), HATU (5.64 g, 0.015 mol), DIEA (2.55 g, 0.02 mol), and N,N-dimethylformamide (28 ml). The atmosphere was purged with nitrogen three times and stirred at 25°C for 3 hours. After completion of the reaction, the reaction mixture was extracted with purified water (80 ml) and ethyl acetate (80 ml). The organic phase was separated, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography to obtain compound NM085-1 (3.1 g, 45.8% yield). MS ESI (m / z) = 683 [M+H] + .

[0529] (6-3) Synthesis of Compound NM085

[0530] Compound NM085-1 (3.1 g, 0.0045 mol) was added to a 100 ml reaction vessel, cooled to 0°C, and bis(diisopropylamino)(2-cyanoethoxy)phosphine (1.78 g, 0.0055 mol), 4,5-dicyanoimidazole (0.428 g, 0.0036 mol), and dichloromethane (31 ml) were added portionwise at 0°C. The atmosphere was purged with nitrogen three times, and the mixture was stirred at 25°C for 3 hours. After completion of the reaction, saturated aqueous sodium bicarbonate (30 ml) was added to the reaction solution, and the organic phase was separated, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by reverse phase column chromatography (eluent: acetonitrile / water = 85 / 15, v / v) to obtain compound NM085 (2.2 g, 55% yield). MS ESI (m / z) = 884 [M+H] + .

[0531] 1H NMR(400MHz,Chloroform-d)δ11.31–11.07(s,1H),δ7.44–7.15(m,9H),6.88(dd,J=18.4,8.1Hz,5H),5.14–4 .85(m,2H),3.71(s,8H),3.60–3.33(m,5H),3.10(s,1H),2.83–2.64(m,2H),2.50(s,2H),1.25–0.99(m,21H).

[0532] Preparation Example 7: Synthesis of Compound NM086

[0533] In this preparation example, the synthetic route of compound NM086 is as follows:

[0534] (7-1) Synthesis of Compound NM-C

[0535] In this preparation example, the synthetic route of compound NM-C is as follows:

[0536] (7-1-1) Synthesis of Compound NM-c

[0537] To a 250 ml reaction vessel were added N4-benzoylcytosine (9.5 g, 0.0442 mol), tert-butyl bromoacetate (9.04 g, 0.0464 mol), potassium carbonate (12.2 g, 0.0884 mol), and N,N-dimethylformamide (95 ml). The atmosphere was purged with nitrogen three times and stirred at 25°C for 3 hours. After completion of the reaction, the reaction solution was filtered and extracted with purified water (100 ml) and ethyl acetate (100 ml). The organic phase was separated, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography to obtain compound NM-c (8.5 g, 58.6% yield). MS ESI (m / z) = 330 [M+H] + .

[0538] (7-1-2) Synthesis of Compound NM-C

[0539] Compound NM-c (8.5 g, 0.0258 mol), trifluoroacetic acid (85 ml), and water (0.85 ml) were added to a 250 ml reactor and stirred at 25°C for 1 hour. After the reaction, the reaction solution was concentrated to obtain compound NM-C (5.4 g, yield 77.1%). MS ESI (m / z) = 274 [M+H] + .

[0540] (7-2) Synthesis of Compound NM086-1

[0541] To a 250 ml reactor were added compound NM084-2 (4.68 g, 0.0111 mol), compound NM-C (2.33 g, 0.0085 mol), HATU (4.86 g, 0.0128 mol), DIEA (2.2 g, 0.017 mol), and N,N-dimethylformamide (47 ml). The atmosphere was purged with nitrogen three times and stirred at 25°C for 3 hours. After completion of the reaction, the reaction mixture was extracted with purified water (80 ml) and ethyl acetate (80 ml). The organic phase was separated, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by reverse phase column chromatography (eluent: acetonitrile / water = 70 / 30, v / v) to afford compound NM086-1 (2.5 g, 43.3% yield). MS ESI (m / z) = 677 [M+H] + .

[0542] (7-3) Synthesis of Compound NM086

[0543] Compound NM086-1 (2.5 g, 0.0037 mol) was added to a 100 ml reaction vessel, cooled to 0°C, and bis(diisopropylamino)(2-cyanoethoxy)phosphine (1.4 g, 0.0046 mol), 4,5-dicyanoimidazole (0.334 g, 0.0028 mol), and dichloromethane (25 ml) were added portionwise at 0°C. The atmosphere was purged with nitrogen three times, and the mixture was stirred at 25°C for 3 hours. After completion of the reaction, saturated aqueous sodium bicarbonate solution (30 ml) was added to the reaction solution. The organic phase was separated, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by reverse phase column chromatography (eluent: acetonitrile / water = 85 / 15) to obtain compound NM086 (1.4 g, 38.9% yield). MS ESI (m / z) = 977 [M+H] + .

[0544] 1H NMR (400MHz, DMSO-d6) δ11.31–11.07(s,1H),8.07–8.00(d,J=7.7Hz,2H),7.94–7.81(d,J=7.2Hz,1H),7. 71–7.61(t,J=7.4Hz,1H),7.56–7.49(d,J=7.6Hz,2H),7.48–7.20(m,10H),6.97–6.85(t,J=8.1Hz,4H),4 .77–4.60(m,1H),4.22–4.03(tt,J=13.5,7.2Hz,1H),3.82–3.69(s,7H),3.68–3.44(m,7H),3.44–3.36(q ,J=11.2,7.4Hz,2H),3.19–3.10(d,J=8.3Hz,1H),2.84–2.67(dt,J=23.5,5.6Hz,2H),1.21–1.02(m,15H).

[0545] Preparation Example 8: Synthesis of Compound NM102

[0546] In this preparation example, the synthetic route of compound NM102 is as follows:

[0547] (8-1) Synthesis of Compound NM102-2

[0548] Compound NM102-1 (10 g, 0.13 mol, 1 eq) was dissolved in 100 mL of a mixture of tetrahydrofuran and water (the volume ratio of tetrahydrofuran to water was 1:4). Sodium bicarbonate (21.8 g, 0.26 mol, 2 eq) and benzyl chloroformate (23.3 g, 0.14 mol, 1.05 eq) were added sequentially at 0°C. The mixture was heated to 25°C and stirred for 2 hours. After completion of the reaction, the reaction solution was extracted with ethyl acetate (3 × 100 mL). The organic phases were combined, washed with saturated aqueous sodium chloride (2 × 50 mL), dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography to obtain compound NM102-2 (20 g, 69.4% yield) as a white solid. MS-ESI (m / z) = 210.2 [M+H] + .

[0549] (8-2) Synthesis of Compound NM102-3

[0550] Compound NM102-2 (20 g, 95.7 mmol, 1.0 eq) was dissolved in 200 mL of pyridine and cooled to 0°C in an ice bath. DMTrCl (4,4'-dimethoxytriphenylmethane chloride, 48.7 g, 143.6 mmol, 1.5 eq) was added portionwise and stirred at 0°C for 1 hour. The mixture was quenched by adding methanol. After completion of the reaction, the reaction solution was concentrated to remove pyridine, and saturated aqueous ammonium chloride (150 mL) was added. The mixture was extracted with ethyl acetate (2 x 250 mL). The organic phases were combined, washed with saturated brine (150 mL), dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography (eluent: petroleum ether / ethyl acetate = 1 / 1, v / v) to afford compound NM102-3 (35 g, 71.7% yield) as a yellow solid. MS-ESI (m / z) = 512.2 [M+H] + .

[0551] (8-3) Synthesis of Compound NM102-4

[0552] Compound NM102-3 (35 g, 68.5 mmol, 1.0 eq) was dissolved in 350 mL of methanol, and 3.5 g of wet palladium on carbon (10%, 0.1 g / g) was added. The atmosphere was purged with hydrogen three times. After completion of the purging, the mixture was stirred at 25°C for 6 hours. After completion of the reaction, the reaction mixture was filtered and the filtrate was concentrated to obtain compound NM102-4 (20 g, yield 77.5%) as a yellow solid. MS-ESI (m / z) = 378.3 [M+H] + .

[0553] (8-4) Synthesis of Compound NM102-5

[0554] Compound NM102-4 (20 g, 52.9 mmol, 1 eq) was dissolved in 200 mL of acetonitrile, and potassium carbonate (14.6 g, 105.8 mmol, 2 eq) and (2-bromoethoxy)-tert-butyldimethylsilane (19.0 g, 79.4 mmol, 1.5 eq) were added sequentially. The mixture was stirred at 80°C for 12 hours. After completion of the reaction, the temperature was lowered to 25°C, 100 mL of water was added, and the mixture was extracted with ethyl acetate (3 × 150 mL). The organic phases were combined, washed with saturated brine (2 × 50 mL), dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography to obtain compound NM102-5 (25 g, 88.4% yield) as a white solid. MS-ESI (m / z) = 536.2 [M+H] + .

[0555] (8-5) Synthesis of Compound NM102-6

[0556] Compound NM102-5 (10 g, 18.7 mmol, 1 eq) was dissolved in 100 mL of DMF, and HATU (10.6 g, 28.1 mmol, 1.5 eq), compound NM-U (4.8 g, 28.1 mmol, 1.5 eq), and DIEA (6.03 g, 46.75 mmol, 2.5 eq) were added sequentially. The mixture was stirred at 25°C for 2 hours. After completion of the reaction, 100 mL of water was added to the reaction solution, and the mixture was extracted with ethyl acetate (3 × 100 mL). The organic phases were combined, washed with saturated brine (5 × 50 mL), dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography to obtain compound NM102-6 (10 g, yield 78.1%) as a white solid. MS-ESI (m / z) = 688.2 [M+H] + .

[0557] (8-6) Synthesis of Compound NM102-7

[0558] Compound NM102-6 (10 g, 14.5 mmol, 1 eq) was dissolved in 100 mL of tetrahydrofuran, and a 1 M solution of tetrabutylammonium fluoride in tetrahydrofuran (21.8 mL, 21.8 mmol, 1.5 eq) was added. The mixture was stirred at 25°C for 2 hours. After completion of the reaction, 100 mL of water was added to the reaction solution, and the mixture was extracted with ethyl acetate (3 × 100 mL). The organic phases were combined, washed with water (4 × 50 mL), dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography to obtain compound NM102-7 (6.5 g, yield 78.3%) as a white solid. MS-ESI (m / z) = 574.2 [M+H] + .

[0559] (8-7) Synthesis of Compound NM102

[0560] Compound NM102-7 (3.0 g, 5.23 mmol, 1.0 eq) was dissolved in 30 ml of anhydrous dichloromethane, and DCI (493 mg, 4.18 mmol, 0.8 eq) and bis(diisopropylamino)(2-cyanoethoxy)phosphine (1.65 g, 5.49 mmol, 1.05 eq) were added, respectively. The atmosphere was purged with nitrogen three times, and the mixture was stirred at 25°C for 1 hour. After completion of the reaction, the reaction mixture was washed with saturated aqueous sodium bicarbonate (2 × 30 mL). The organic phase was washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography (eluent: water / acetonitrile = 10 / 90, v / v) to afford compound NM102 (2.5 g, 62.5% yield) as a white solid. MS-ESI (m / z) = 774 [M+H] + .

[0561] Preparation Example 9: Synthesis of Compound NM103

[0562] In this preparation example, the synthetic route of compound NM103 is as follows:

[0563] (9-1) Synthesis of Compound NM103-2

[0564] Compound NM103-1 (20 g, 95.7 mmol, 1.0 eq) was dissolved in 200 mL of pyridine and cooled to 0°C in an ice bath. DMTrCl (4,4'-dimethoxytriphenylmethane chloride, 48.7 g, 143.6 mmol, 1.5 eq) was added portionwise and stirred at 0°C for 1 hour. The mixture was quenched by adding methanol. After completion of the reaction, the reaction solution was concentrated to remove the pyridine, and saturated aqueous ammonium chloride (150 mL) was added. The mixture was then extracted with ethyl acetate (2 x 250 mL). The organic phases were combined, washed with saturated brine (150 mL), dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography (eluent: petroleum ether / ethyl acetate = 1 / 1, v / v) to afford compound NM103-2 (30 g, 61.3% yield) as a yellow solid. MS-ESI (m / z) = 512.2 [M+H] + .

[0565] (9-2) Synthesis of Compound NM103-3

[0566] Compound NM103-2 (30 g, 58.6 mmol, 1.0 eq) was dissolved in 300 mL of methanol, and 3.0 g of wet palladium on carbon (10%, 0.1 g / g) was added. The mixture was purged with hydrogen three times. After completion of the purging, the mixture was stirred at 25°C for 6 hours. After completion of the reaction, the reaction mixture was filtered and the filtrate was concentrated to obtain compound NM103-3 (20 g, 90.9% yield) as a yellow solid. MS-ESI (m / z) = 378.3 [M+H] + .

[0567] (9-3) Synthesis of Compound NM103-4

[0568] Compound NM103-3 (20 g, 52.9 mmol, 1 eq) was dissolved in 200 mL of acetonitrile, and potassium carbonate (14.6 g, 105.8 mmol, 2 eq) and (2-bromoethoxy)-tert-butyldimethylsilane (19.0 g, 79.4 mmol, 1.5 eq) were added sequentially. The mixture was stirred at 80°C for 12 hours. After completion of the reaction, the temperature was lowered to 25°C, 100 mL of water was added, and the mixture was extracted with ethyl acetate (3 × 150 mL). The organic phases were combined, washed with saturated brine (2 × 50 mL), dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography to obtain compound NM103-4 (25 g, 88.4% yield) as a white solid. MS-ESI (m / z) = 536.2 [M+H] + .

[0569] (9-4) Synthesis of Compound NM103-5

[0570] Compound NM103-4 (10 g, 18.7 mmol, 1 eq) was dissolved in 100 mL of DMF, and HATU (10.6 g, 28.1 mmol, 1.5 eq), compound NM-U (4.8 g, 28.1 mmol, 1.5 eq), and DIEA (6.03 g, 46.75 mmol, 2.5 eq) were added sequentially. The mixture was stirred at 25°C for 2 hours. After completion of the reaction, 100 mL of water was added to the reaction solution, and the mixture was extracted with ethyl acetate (3 × 100 mL). The organic phases were combined, washed with saturated brine (5 × 50 mL), dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography to obtain compound NM103-5 (10 g, yield 78.1%) as a white solid. MS-ESI (m / z) = 688.2 [M+H] + .

[0571] (9-5) Synthesis of Compound NM103-6

[0572] Compound NM103-5 (10 g, 14.5 mmol, 1 eq) was dissolved in 100 mL of tetrahydrofuran, and a 1 M solution of tetrabutylammonium fluoride in tetrahydrofuran (21.8 mL, 21.8 mmol, 1.5 eq) was added. The mixture was stirred at 25°C for 2 hours. After completion of the reaction, 100 mL of water was added to the reaction solution, and the mixture was extracted with ethyl acetate (3 × 100 mL). The organic phases were combined, washed with water (4 × 50 mL), dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography to obtain compound NM103-6 (6.5 g, 78.3% yield) as a white solid. MS-ESI (m / z) = 574.2 [M+H] + .

[0573] (9-6) Synthesis of Compound NM103

[0574] Compound NM103-6 (3.0 g, 5.23 mmol, 1.0 eq) was dissolved in 30 ml of anhydrous dichloromethane. DCI (493 mg, 4.18 mmol, 0.8 eq) and bis(diisopropylamino)(2-cyanoethoxy)phosphine (1.65 g, 5.49 mmol, 1.05 eq) were added, respectively. The atmosphere was purged with nitrogen three times and the mixture was stirred at 25°C for 1 hour. After completion of the reaction, the reaction solution was washed with saturated aqueous sodium bicarbonate (2 × 30 mL). The organic phase was washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography (elution gradient, water:acetonitrile = 10:85) to obtain compound NM103 (2.7 g, yield 67.5%) as a white solid. MS-ESI (m / z) = 774 [M+H] + .

[0575] Preparation Example 10: Synthesis of Compound NM104

[0576] In this preparation example, the synthetic route of compound NM104 is as follows:

[0577] (10-1) Synthesis of Compound NM104-2

[0578] Compound NM104-1 (10 g, 0.13 mol, 1 eq) was dissolved in 100 mL of a mixture of tetrahydrofuran and water (the volume ratio of tetrahydrofuran to water was 1:4). Sodium bicarbonate (21.8 g, 0.26 mol, 2 eq) and benzyl chloroformate (23.3 g, 0.14 mol, 1.05 eq) were added sequentially at 0°C. The mixture was heated to 25°C and stirred for 2 hours. After completion of the reaction, the reaction solution was extracted with ethyl acetate (3 × 100 mL). The combined organic phases were washed with saturated brine (2 × 50 mL), dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography to obtain compound NM104-2 (20 g, 69.4% yield) as a white solid. MS-ESI (m / z) = 210.2 [M+H] + .

[0579] (10-2) Synthesis of Compound NM104-3

[0580] Compound NM104-2 (20 g, 95.7 mmol, 1.0 eq) was dissolved in 200 mL of pyridine and cooled to 0°C in an ice bath. DMTrCl (4,4'-dimethoxytriphenylmethane chloride, 48.7 g, 143.6 mmol, 1.5 eq) was added portionwise and stirred at 0°C for 1 hour. The mixture was quenched by adding methanol. After completion of the reaction, the reaction solution was concentrated to remove pyridine, and saturated aqueous ammonium chloride (150 mL) was added. The mixture was extracted with ethyl acetate (2 x 250 mL). The organic phases were combined, washed with saturated brine (150 mL), dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography (eluent: petroleum ether / ethyl acetate = 1 / 1, v / v) to obtain compound NM104-3 (37 g, 75.7% yield) as a yellow solid. MS-ESI (m / z) = 512.2 [M+H] + .

[0581] (10-3) Synthesis of Compound NM104-4

[0582] Compound NM104-3 (35 g, 68.5 mmol, 1.0 eq) was dissolved in 350 mL of methanol, and 3.5 g of wet palladium on carbon (10%, 0.1 g / g) was added. The atmosphere was purged with hydrogen three times. After completion of the purging, the mixture was stirred at 25°C for 6 hours. After completion of the reaction, the reaction mixture was filtered and the filtrate was concentrated to obtain compound NM104-4 (20 g, yield 77.5%) as a yellow solid. MS-ESI (m / z) = 378.3 [M+H] + .

[0583] (10-4) Synthesis of Compound NM104-5

[0584] Compound NM104-4 (20 g, 52.9 mmol, 1 eq) was dissolved in 200 mL of acetonitrile, and potassium carbonate (14.6 g, 105.8 mmol, 2 eq) and (2-bromoethoxy)-tert-butyldimethylsilane (19.0 g, 79.4 mmol, 1.5 eq) were added sequentially. The mixture was stirred at 80°C for 12 hours. After completion of the reaction, the temperature was lowered to 25°C, 100 mL of water was added, and the mixture was extracted with ethyl acetate (3 × 150 mL). 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 column chromatography to obtain compound NM104-5 (24 g, 84.8% yield) as a white solid. MS-ESI (m / z) = 536.2 [M+H] + .

[0585] (10-5) Synthesis of Compound NM104-6

[0586] Compound NM104-5 (10 g, 18.7 mmol, 1 eq) was dissolved in 100 mL of DMF, and HATU (10.6 g, 28.1 mmol, 1.5 eq), compound NM-U (4.8 g, 28.1 mmol, 1.5 eq), and DIEA (6.03 g, 46.75 mmol, 2.5 eq) were added sequentially. The mixture was stirred at 25°C for 2 hours. After completion of the reaction, 100 mL of water was added to the reaction solution, and the mixture was extracted with ethyl acetate (3 × 100 mL). The organic phases were combined, washed with saturated brine (5 × 50 mL), dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography to obtain compound NM104-6 (11 g, yield 85.9%) as a white solid. MS-ESI (m / z) = 688.2 [M+H] + .

[0587] (10-6) Synthesis of Compound NM104-7

[0588] Compound NM104-6 (10 g, 14.5 mmol, 1 eq) was dissolved in 100 mL of tetrahydrofuran, and a 1 M tetrabutylammonium fluoride solution in tetrahydrofuran (21.8 mL, 21.8 mmol, 1.5 eq) was added. The mixture was stirred at 25°C for 2 hours. After completion of the reaction, 100 mL of water was added to the reaction solution, and the mixture was extracted with ethyl acetate (3 × 100 mL). The organic phases were combined, washed with water (4 × 50 mL), dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography to obtain compound NM104-7 (7.0 g, yield 83.9%) as a white solid. MS-ESI (m / z) = 574.2 [M+H] + .

[0589] (10-7) Synthesis of Compound NM104

[0590] Compound NM104-7 (3.0 g, 5.23 mmol, 1.0 eq) was dissolved in 30 ml of anhydrous dichloromethane. DCI (493 mg, 4.18 mmol, 0.8 eq) and bis(diisopropylamino)(2-cyanoethoxy)phosphine (1.65 g, 5.49 mmol, 1.05 eq) were added, respectively. The atmosphere was purged with nitrogen three times and the mixture was stirred at 25°C for 1 hour. After completion of the reaction, the reaction mixture was washed with saturated aqueous sodium bicarbonate (2 x 30 mL). The organic phase was washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography (eluent: water / acetonitrile = 10 / 90, v / v) to afford compound NM104 (2.45 g, 60.6% yield) as a white solid. MS-ESI (m / z) = 774 [M+H] + .

[0591] Preparation Example 11: Synthesis of Compound NM105

[0592] In this preparation example, the synthetic route of compound NM105 is as follows:

[0593] (11-1) Synthesis of Compound NM105-2

[0594] Compound NM105-1 (20 g, 95.7 mmol, 1.0 eq) was dissolved in 200 mL of pyridine and cooled to 0°C in an ice bath. DMTrCl (4,4'-dimethoxytriphenylmethane chloride, 48.7 g, 143.6 mmol, 1.5 eq) was added portionwise and stirred at 0°C for 1 hour. The mixture was quenched by adding methanol. After completion of the reaction, the reaction solution was concentrated to remove pyridine, and saturated aqueous ammonium chloride (150 mL) was added. The mixture was then extracted with ethyl acetate (2 x 250 mL). The organic phases were combined, washed with saturated brine (150 mL), dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography (eluent: petroleum ether / ethyl acetate = 1 / 1, v / v) to afford compound NM105-2 (32.5 g, 66.3% yield) as a yellow solid. MS-ESI (m / z) = 512.2 [M+H] + .

[0595] (11-2) Synthesis of Compound NM105-3

[0596] Compound NM105-2 (30 g, 58.6 mmol, 1.0 eq) was dissolved in 300 mL of methanol, and 3.0 g of palladium on carbon (10%, 0.1 g / g) was added. The mixture was purged with hydrogen three times. After completion of the purging, the mixture was stirred at 25°C for 6 hours. After completion of the reaction, the mixture was filtered and the filtrate was concentrated to obtain compound NM105-3 (20 g, 90.9% yield) as a yellow solid. MS-ESI (m / z) = 378.3 [M+H] + .

[0597] (11-3) Synthesis of Compound NM105-4

[0598] Compound NM105-3 (20 g, 52.9 mmol, 1 eq) was dissolved in 200 mL of acetonitrile, and potassium carbonate (14.6 g, 105.8 mmol, 2 eq) and (2-bromoethoxy)-tert-butyldimethylsilane (19.0 g, 79.4 mmol, 1.5 eq) were added sequentially. The mixture was stirred at 80°C for 12 hours. After completion of the reaction, the temperature was lowered to 25°C, and 100 mL of water was added to the reaction solution. The mixture was extracted with ethyl acetate (3 × 150 mL). 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 column chromatography to obtain compound NM105-4 (19 g, 67.1% yield) as a white solid. MS-ESI (m / z) = 536.2 [M+H] + .

[0599] (11-4) Synthesis of Compound NM105-5

[0600] Compound NM105-4 (10 g, 18.7 mmol, 1 eq) was dissolved in 100 mL of DMF. HATU (10.6 g, 28.1 mmol, 1.5 eq), compound NM-U (4.8 g, 28.1 mmol, 1.5 eq), and DIEA (6.03 g, 46.75 mmol, 2.5 eq) were added sequentially. The mixture was stirred at 25°C for 2 hours. After completion of the reaction, 100 mL of water was added to the reaction solution, and the mixture was extracted with ethyl acetate (3 × 100 mL). The organic phases were combined, washed with saturated brine (5 × 50 mL), dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography to obtain compound NM105-5 (9.4 g, 73.4% yield) as a white solid. MS-ESI (m / z) = 688.2 [M+H] + .

[0601] (11-5) Synthesis of Compound NM105-6

[0602] Compound NM105-5 (10 g, 14.5 mmol, 1 eq) was dissolved in 100 mL of tetrahydrofuran, and a 1 M solution of tetrabutylammonium fluoride in tetrahydrofuran (21.8 mL, 21.8 mmol, 1.5 eq) was added. The mixture was stirred at 25°C for 2 hours. After completion of the reaction, 100 mL of water was added to the reaction solution, and the mixture was extracted with ethyl acetate (3 × 100 mL). The organic phases were combined, washed with water (4 × 50 mL), dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography to obtain compound NM105-6 (6.1 g, 73.1% yield) as a white solid. MS-ESI (m / z) = 574.2 [M+H] + .

[0603] (11-6) Synthesis of Compound NM105

[0604] Compound NM105-6 (3.0 g, 5.23 mmol, 1.0 eq) was dissolved in 30 ml of anhydrous dichloromethane. DCI (493 mg, 4.18 mmol, 0.8 eq) and bis(diisopropylamino)(2-cyanoethoxy)phosphine (1.65 g, 5.49 mmol, 1.05 eq) were added, respectively. The atmosphere was purged with nitrogen three times and the mixture was stirred at 25°C for 1 hour. After completion of the reaction, the reaction solution was washed with saturated aqueous sodium bicarbonate (2 x 30 mL) and saturated brine (20 mL), dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography (eluent: water / acetonitrile = 10 / 90, v / v) to afford compound NM105 (2.23 g, 55.8% yield) as a white solid. MS-ESI (m / z) = 774 [M+H] + .

[0605] Synthesis of ligands

[0606] Preparation Example 12: Synthesis of Compound CR01008 and Compound CR01008Z

[0607] (12-1) Synthesis of Compound CR01008

[0608] In this preparation example, the synthetic route of compound CR01008 is as follows:

[0609] (12-1-1) Synthesis of Compound 2

[0610] Compound 1 (trans-4-(Boc-amino)cyclohexylcarboxaldehyde, 10.0 g, 1.0 eq) and formaldehyde solution (8.9 g, 37 mass %, 2.4 eq) were dissolved in 33 ml of methanol, and 13 ml of a 45.3 mass % KOH aqueous solution was added dropwise. After the addition was complete, the mixture was stirred at 25°C for 30 minutes, then heated to 60°C and refluxed at 60°C for 2 hours. After the reaction was completed, the reaction solution was cooled to room temperature and evaporated to dryness under reduced pressure to obtain a crude white solid. A small amount of water was added to the crude product to slurry it, and the mixture was filtered to obtain compound 2 (9 g, yield 78.9%) as a white solid. MS-ESI (m / z) = 260 [M+H] + .

[0611] (12-1-2) Synthesis of Compound 3

[0612] Compound 2 (9 g, 1 eq) was dissolved in 70 ml of 1,4-dioxane, and a 45 ml, 4 M solution of hydrogen chloride in 1,4-dioxane was added. The mixture was stirred at 25°C for 1 hour. After completion of the reaction, the reaction solution was evaporated to dryness under reduced pressure to obtain compound 3 (6.8 g, 100% yield) as a white solid.

[0613] (12-1-3) Synthesis of Compound 5

[0614] Compound 3 (1.8 g, 2.0 eq), compound 4 (5-[[(2R,3R,4R,5R,6R)-3-acetylamino-4,5-diacetoxy-6-(acetoxymethyl)-2-tetrahydropyranyl]oxy]pentanoic acid, 2.1 g, 1.0 eq), and DIEA (N,N-diisopropylethylamine, 3.5 g, 6.0 eq) were dissolved in 15 ml of DMF. HBTU (1.9 g, 1.1 eq) was added, and the mixture was stirred at 25° C. under a N atmosphere for 3 hours. After completion of the reaction, the reaction solution was evaporated to dryness under reduced pressure and reverse-phase purified (22% acetonitrile in water) to obtain compound 5 (1.78 g, 64.4% yield) as a white solid. MS-ESI (m / z) = 589 [M+H] + .

[0615] (12-1-4) Synthesis of Compound 6

[0616] Compound 5 (1.54 g, 1.0 eq) was dissolved in 15 ml of pyridine. The reaction system was cooled to 0°C using an ice-water bath and DMTrCl (4,4'-dimethoxytriphenylmethane chloride, 1.32 g, 1.5 eq) was added at 0°C. The reaction was allowed to react at 25°C for 3 hours. 15 ml of methanol was added to the reaction solution to quench the reaction. After completion of the reaction, the reaction solution was evaporated to dryness under reduced pressure and reverse-phase purified (60% acetonitrile in water) to obtain compound 6 (1 g, 42.7% yield) as a yellow solid. MS-ESI (m / z) = 891 [M+H] + .

[0617] (12-1-5) Synthesis of Compound CR01008

[0618] Compound 6 (1.08 g, 1.0 eq) was dissolved in 20 ml of anhydrous dichloromethane, and DCI (115 mg, 0.8 eq) and compound 7 (bis(diisopropylamino)(2-cyanoethoxy)phosphine, 732 mg, 2.1 eq) were added, respectively. The atmosphere was purged with nitrogen three times, and the mixture was stirred at 25°C for 2 hours. After completion of the reaction, 20 ml of saturated aqueous sodium bicarbonate solution was added to the reaction solution, and the mixture was extracted three times with 20 ml of dichloromethane (3×20 ml). The organic phases were combined and evaporated to dryness under reduced pressure. After reverse purification (72 vol% acetonitrile in water), the mixture was dried under vacuum for 12 hours to obtain compound CR01008 (1 g, 76.0% yield) as a white powder. MS-ESI (m / z) = 1091 [M+Na] + .

[0619] 1H NMR(400MHz, DMSO-d6)δ1.05(d,J=6.7Hz,6H).1.14(d,J=6.7Hz,6H),1.37–1.17(m,5H),1.60–1.40(m,6H),1.68–1.62(m,1H),1.80(s,3H), 1.80(s,3H),1.92(s,3H),2.02(s,5H),2.13(s,3H),2.71(t,J=5.9Hz,2H),2.79(d,J=8.4Hz,1H),2.87(d,J=8.4Hz,1H),3.36(s,1H),3.58– 3.39(m,3H),3.69–3.60(m,2H),3.75(s,7H),3.90(dt,J=11.2,8.8Hz,1H),4.05(s,3H),4.51(d,J=8.4Hz,1H),4.99(dd,J=11.3,3.4Hz,1H) ,5.24(d,J=3.4Hz,1H),5.78(s,1H),6.93–6.87(m,4H),7.35–7.21(m,7H),7.44–7.37(m,2H),7.66(d,J=7.8Hz,1H),7.84(d,J=9.2Hz,1H).

[0620] (12-2) Synthesis of Compound CR01008Z

[0621] In this preparation example, the synthetic route of compound CR01008Z is as follows:

[0622] (12-2-1) Synthesis of Compound 9

[0623] Compound 6 (500 mg) was dissolved in 10 ml of dichloromethane, and compound 8 (succinic anhydride, 112 mg), DMAP (6.8 mg), and TEA (226.2 mg) were added. The atmosphere was purged with nitrogen three times, and the mixture was stirred at 25°C for 16 hours. Flash purification was performed to obtain compound 9 (300 mg, yield 53.6%). MS-ESI (m / z) = 1013 [M+Na] + .

[0624] (12-2-2) Synthesis of Compound CR01008Z

[0625] Compound 9 (50 mg), aminoCPG (1.25 g, 80 μmol / g, 0.1 mmol), HBTU (27 mg), and DIEA (12 mg) were added to a 20 ml vial and shaken for 16 hours. After the reaction, the reaction mixture was filtered to obtain a filter cake, which was washed once with 10 ml of acetonitrile (1 × 10 ml) and then dried under vacuum. The dried filter cake, DMAP (3 mg), Cap 1 (10 ml, 200 V), and Cap 2 (1 ml, 20 V) were added to a 20 ml vial and shaken for 6 hours. After the reaction, the reaction mixture was filtered to obtain a filter cake, which was washed once with 10 ml of acetonitrile (1 × 10 ml) and then dried under vacuum to obtain compound CR01008Z (1.03 g, loading 20-30 μmol / g).

[0626] Cap1 and Cap2 are capping reagents, Cap1 is a 20% by volume N-methylimidazole mixed solution in pyridine / acetonitrile, with a volume ratio of pyridine to acetonitrile of 3:5; Cap2 is a 20% by volume acetic anhydride solution in acetonitrile.

[0627] Preparation Example 13: Synthesis of Compound CR01013 and Compound CR01013Z

[0628] (13-1) Preparation of Compound CR01013

[0629] In this preparation example, the synthetic route of compound CR01013 is as follows:

[0630] (13-1-1) Synthesis of Compound 11

[0631] Compound 3 (3.6 g) was dissolved in 36 ml of DMF, followed by the addition of TEA (5.62 g), compound 10 (N-benzyloxycarbonyl-4-aminobutyric acid, 5.28 g), and HBTU (8.43 g). The mixture was stirred at 25°C for 16 hours. After completion of the reaction, the reaction solution was added to 200 ml of saturated aqueous sodium bicarbonate solution and extracted three times with 100 ml of ethyl acetate (3 × 100 ml). The organic phases were combined, washed once with 50 ml of saturated aqueous sodium chloride solution (1 × 50 ml), and then dried over anhydrous sodium sulfate. The organic phase was evaporated to dryness under reduced pressure and purified by normal phase column chromatography (eluent: dichloromethane / methanol = 10 / 1, v / v) to obtain compound 11 (2.3 g, 33.0% yield) as a white solid. ESIMS (m / z) = 379.5 [M+H] + .

[0632] (13-1-2) Synthesis of Compound 12

[0633] Compound 11 (2.3 g) was dissolved in 23 ml of methanol, and wet palladium on carbon (230 mg, 10% loading) was added. The atmosphere was replaced with hydrogen three times, and the reaction system was stirred at 25°C under a hydrogen atmosphere (15 psi) for 16 hours. After completion of the reaction, the reaction solution was filtered to obtain a filtrate, which was evaporated to dryness under reduced pressure to obtain compound 12 (1.48 g, 99.8% yield) as a yellow oil.

[0634] (13-1-3) Synthesis of Compound 13

[0635] Compound 12 (1.48 g) was dissolved in 15 ml of DMF, and triethylamine (1.22 g), compound 4 (1.35 g), and HBTU (3.45 g) were added. The mixture was stirred at 25°C for 16 hours. After completion of the reaction, the reaction solution was added to 150 ml of saturated aqueous sodium bicarbonate solution and extracted three times with 50 ml of ethyl acetate (3 x 50 ml). The organic phases were combined, washed once with 30 ml of saturated aqueous sodium chloride solution (1 x 30 ml), and then dried over anhydrous sodium sulfate. The organic phase was evaporated to dryness under reduced pressure and purified by reverse phase column chromatography (C18 column, eluent: water / acetonitrile = 5 / 1, v / v) to obtain compound 13 (1.3 g, yield 31.8%) as a white solid. ESI-MS (m / z) = 674.3 [M+H] + .

[0636] (13-1-4) Synthesis of Compound 14

[0637] Compound 13 (1.1 g) was dissolved in 11 ml of pyridine. The reaction system was cooled to 0°C using an ice-water bath and DMTrCl (813 mg) was added portionwise at 0°C. The reaction system was stirred at 0°C for 1 hour. After the reaction was completed, methanol was added to the reaction solution to quench the reaction. The solvent was evaporated and the solution was purified by reverse phase column chromatography (eluent: water / acetonitrile = 1 / 4, v / v) to obtain compound 14 (800 mg, yield 50.3%) as a white solid. ESI-MS (m / z) = 976.5 [M+H] + .

[0638] (13-1-5) Synthesis of Compound CR01013

[0639] At 25°C, compound 14 (550 mg) was dissolved in 5 ml of dichloromethane (DCM), and 4,5-dicyanoimidazole (53.2 mg) and compound 7 (2-cyanoethyl N,N,N',N'-tetraisopropylphosphorodiamidite, 255.4 mg) were added. The atmosphere was purged with nitrogen three times, and the reaction system was stirred at 25°C for 1 hour. After completion of the reaction, the reaction solution was washed twice with 5 ml of saturated sodium bicarbonate solution (2 × 5 ml) and once with 30 ml of saturated sodium chloride solution (1 × 30 ml). The organic phase was separated and dried over anhydrous sodium sulfate. The organic phase solvent was evaporated to dryness under reduced pressure and purified by normal phase column chromatography (eluent: dichloromethane / methanol = 20 / 1, v / v) to obtain compound CR01013 (532 mg, 80.4% yield) as a white solid. ESI-MS (m / z) = 1176.7 [M+H] + .

[0640] 1H NMR(400MHz, DMSO-d6)δ0.95–1.05(d,J=6.7Hz,5H),1.06–1.15(q,J=7.6Hz,8H),1.15–1.21(t,J=7.2Hz,14H),1.72–1 .80(s,3H),1.84–1.92(s,3H),1.94–2.07(d,J=16.0Hz,7H),2.07–2.14(s,3H),2.64–2.72(q,J=5.8Hz,2H),2.74–2.89 (d,J=8.5Hz,2H),3.35–3.56(m,4H),3.57–3.70(m,4H),3.71–3.77(s,6H),3.81–3.93(m,1H),3.96–4.09(d,J=6.4Hz, 3H), 6.82–6.97 (d, J = 8.7Hz, 4H), 7.17–7.27 (t, J = 8.7Hz, 5H), 7.27–7.34 (t, J = 7.6Hz, 2H), 7.34–7.43 (d, J = 7.5Hz, 2H).

[0641] (13-2) Preparation of Compound CR01013Z

[0642] In this preparation example, the synthetic route of compound CR01013Z is as follows:

[0643] (13-2-1) Synthesis of Compound 15

[0644] Compound 14 (100 mg, 0.10 mmol) was dissolved in 2 ml of dichloromethane at 25°C, and triethylamine (25.9 mg, 0.25 mmol), DMAP (1.25 mg, 0.01 mmol), and compound 8 (succinic anhydride, 15.4 mg, 0.15 mmol) were added. The reaction system was stirred at 25°C for 16 hours. After completion of the reaction, the solvent was evaporated from the reaction solution and the product was purified by reverse phase column chromatography (C18 column, eluent: water / acetonitrile = 2 / 1, v / v) to obtain compound 15 (110 mg, 0.10 mmol, yield 100%) as a yellow oil. ESI-MS (m / z) = 1099.3 [M+Na] + .

[0645] (13-2-2) Synthesis of Compound CR01013Z

[0646] Compound 15 (50 mg, 0.04 mmol) was dissolved in 10 ml of acetonitrile, and HBTU (24.2 mg, 0.06 mmol), DIEA (11.0 mg, 0.08 mmol), and aminoCPG (1.06 g, loading 80 μmol / g) were added. The reaction system was stirred at 25°C for 16 hours. After completion of the reaction, the reaction solution was filtered to obtain a filter cake, which was washed twice with 50 ml of dichloromethane (2 × 50 ml), three times with 50 ml of acetonitrile (3 × 50 ml), and once with 50 ml of ethyl acetate (1 × 50 ml), and then vacuum dried. Cap 1 (4.8 ml), Cap 2 (0.54 ml), and DMAP (2.59 mg) were added to the dried filter cake, and the reaction system was stirred at 25°C for 5 hours. After the reaction was completed, the reaction solution was filtered to obtain a filter cake, which was washed three times with 50 ml of acetonitrile (3×50 ml) and dried in vacuo to obtain compound CR01013Z (900 mg, loading amount of 20-30 μmol / g).

[0647] Cap1 and Cap2 are capping reagents, Cap1 is a 20% by volume N-methylimidazole mixed solution in pyridine / acetonitrile, with a volume ratio of pyridine to acetonitrile of 3:5; Cap2 is a 20% by volume acetic anhydride solution in acetonitrile.

[0648] Compound L96-PS

[0649] Compound L96-PS was purchased from Asymchem Pharmaceuticals (Tianjin) Co., Ltd. with a loading of 120±12 μmol / g (detection method: UV / HPLC). The structural formula of compound L96-PS is shown below:

[0650] Wherein, PS represents polystyrene resin solid phase carrier.

[0651] Synthesis of siRNA conjugates

[0652] Unless otherwise specified, the siRNA sequences used in this disclosure were commissioned to Suzhou Beixin Biotechnology Co., Ltd. for synthesis; the PCR primers used in this disclosure were commissioned to Beijing Qingke Biotechnology Co., Ltd. for synthesis.

[0653] Preparation Example 14: Synthesis of siRNA Conjugates

[0654] (14-1) Synthesis of the Sense Chain (SS)

[0655] The method of phosphoramidite nucleic acid solid phase synthesis is used to initiate the cycle using the above-mentioned compounds (such as CR01008Z, CR01013Z, and L96-PS) connected to the solid phase support. Nucleoside monomers are connected one by one in the 3'-5' direction according to the nucleotide sequence (in the synthesis process, compound CR01008 and compound CR01013 are each considered as a nucleoside monomer). Each connection of a nucleoside monomer includes four steps: deprotection, coupling, capping, oxidation or sulfurization. The synthesis conditions are given as follows:

[0656] The nucleoside monomer was prepared into an acetonitrile solution with a concentration of 0.1 M.

[0657] The deprotection reaction conditions for each step were identical: 25°C, 70 seconds, a 3% vol. dichloroacetic acid solution in dichloromethane as the deprotection reagent, and a 5:1 molar ratio of dichloroacetic acid to the 4,4'-dimethoxytriphenylmethyl protecting group on the solid support.

[0658] The conditions for each coupling reaction were the same. The coupling reaction conditions were: a temperature of 25°C, a molar ratio of the nucleic acid sequence attached to the solid support to the nucleoside monomer of 1:10, a molar ratio of the nucleic acid sequence attached to the solid support to the coupling reagent of 1:65, a reaction time of 600 seconds, a 0.5 M solution of 5-ethylthio-1H-tetrazole in acetonitrile as the coupling reagent, and a 0.2 mol / L solution of hydrogenated xanthan gum in acetonitrile / pyridine (1:1 volume ratio of acetonitrile to pyridine) as the thiolation reagent.

[0659] The capping reaction conditions were identical for each step. The capping reaction conditions were: 25°C; 2 minutes; a 1:1 molar ratio of Cap1 and Cap2; Cap1: a 20% by volume N-methylimidazole solution in pyridine / acetonitrile (with a 3:5 volume ratio of pyridine to acetonitrile); and Cap2: a 20% by volume solution of acetic anhydride in acetonitrile. The molar ratio of the N-methylimidazole in Cap1 to the acetic anhydride in Cap2 to the nucleic acid sequence attached to the solid support was 1:1:1.

[0660] The oxidation reaction conditions were identical for each step. The oxidation reaction conditions were: temperature, 25°C; reaction time, 3 seconds; oxidizing agent concentration, 0.05 M iodine solution; a molar ratio of iodine to the nucleic acid sequence attached to the solid support during the coupling reaction, 30:1; and the oxidation reaction was performed in a water / pyridine mixture (1:9 by volume). The sulfidation reaction conditions were: temperature, 25°C; reaction time, 360 seconds; thiolation agent concentration, 0.2 M hydrogenated xanthan gum in pyridine solution; a molar ratio of thiolation agent to the nucleic acid sequence attached to the solid support during the coupling reaction, 4:1; and the sulfidation reaction was performed in a water / pyridine mixture (1:9 by volume).

[0661] After the last nucleoside monomer is connected, the nucleic acid sequence connected to the solid phase support is cut, deprotected, purified, desalted, and then freeze-dried to obtain the positive chain, wherein:

[0662] Cleavage and deprotection conditions were as follows: the synthesized nucleotide sequence attached to a solid support was added to 0.5 ml / μmol of 25% ammonia water at 55°C for 16 hours, the solvent was removed, and the product was concentrated to dryness in vacuo. After the ammonia treatment, the product was dissolved in 0.4 ml / μmol of N-methylpyrrolidone relative to the amount of single-stranded nucleic acid, followed by the addition of 0.3 ml / μmol of triethylamine and 0.6 ml / μmol of triethylamine trihydrofluoride to remove the 2'-O-TBDMS protection from the ribose.

[0663] Purification and desalting conditions: Nucleic acid purification was achieved using a preparative ion chromatography column (Source 15Q) with a NaCl gradient elution. Specifically, eluent 1 consisted of 20 mM sodium phosphate (pH 8.1) in a water / acetonitrile mixture (9:1 volume ratio of water to acetonitrile); eluent 2 consisted of 1.5 M sodium chloride, 20 mM sodium phosphate (pH 8.1) in a water / acetonitrile mixture (9:1 volume ratio of water to acetonitrile); the elution gradient was eluent 1:eluent 2 = (100:0) to (50:50). The product eluates were collected and combined, and desalted using a reversed-phase chromatography column. Desalting conditions included using a Sephadex column with Sephadex G25 as the filler and eluting with deionized water.

[0664] 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 and theoretical values ​​were consistent, the compound was conjugated to the 3' end of the siRNA sense strand.

[0665] During the synthesis of the positive chain, three clusters of CR01008 (denoted as (CR01008×3) or (CR01008)×3) and three clusters of CR01013 (denoted as (CR01013×3) or (CR01013)×3) were obtained.

[0666] Among them, the structural formula of the three clusters of CR01008 is as follows:

[0667] The structural formula of the three-cluster CR01013 is as follows:

[0668] (14-2) Synthesis of antisense strand (AS)

[0669] The antisense chain was synthesized using a universal solid phase support. The deprotection, coupling, capping, oxidation or sulfurization reaction conditions, cleavage and deprotection conditions, purification and desalting conditions in the solid phase synthesis method of the antisense chain are the same as those in step (14-1) for synthesizing the sense chain.

[0670] Detection: Purity was determined by ion exchange chromatography (IEX-HPLC); molecular weight was determined by liquid chromatography-mass spectrometry (LC-MS). The measured molecular weight was compared with the theoretical value. If the measured value was consistent with the theoretical value, the siRNA antisense strand was obtained.

[0671] (14-3) Synthesis of siRNA Conjugates

[0672] The sense chain synthesized in step (14-1) and the antisense chain synthesized in step (14-2) were mixed in an equimolar ratio, dissolved in water for injection and heated to 95° C., slowly cooled to room temperature and maintained at room temperature for 10 minutes to allow the sense chain and antisense chain to form a double-stranded structure through hydrogen bonds, thereby obtaining an siRNA conjugate having the sense chain and antisense chain shown in Table 5.

[0673] The structural formula of the siRNA conjugate with L96 conjugated to the 3' end of the sense strand is shown below:

[0674] in, Represents siRNA, in which L96 is conjugated to the 3' end of the siRNA sense strand via a phosphodiester bond.

[0675] The structural formula of the siRNA conjugate with the 3' end of the sense strand (CR01008×3) is shown below:

[0676] in, Represents siRNA. (CR01008×3) is conjugated to the 3' end of the sense strand of the siRNA via a phosphodiester bond.

[0677] The structural formula of the siRNA conjugate with the 3' end of the sense strand (CR01008×3) is shown below:

[0678] in, Represents siRNA. (CR01013×3) is conjugated to the 3' end of the sense strand of the siRNA via a phosphodiester bond.

[0679] In the siRNA conjugates prepared in the specific embodiments of the present disclosure, the unmodified nucleotide sequences forming the siRNA conjugates are shown in the following table:

[0680] Table 4 Unmodified nucleotide sequence information for forming siRNA conjugates

[0681] In the siRNA conjugates prepared in the specific embodiments of the present disclosure, the modified nucleotide sequence information of the siRNA conjugates is shown in the following table:

[0682] Table 5 Sequence information of siRNA conjugates

[0683] Exemplarily, "CmsAmsGmAmCmAmGfAdCfAmAmGmAmCmCmAmUmCmUm_L96" represents L96 linked to the 3' end of the modified nucleotide sequence of SEQ ID NO. 1 via a phosphodiester bond. From the 5' end to the 3' end, the modified nucleotide sequence of SEQ ID NO. 1 is CmsAmsGmAmCmAmGfAdCfAmAmGmAmCmCmAmUmCmUm.

[0684] Unless otherwise indicated, the base composition and modification meanings in the synthetic sequences disclosed herein are as follows: capital letters A, U, G, C, and T represent the base composition of the nucleotides, respectively; a lowercase letter m indicates that the nucleotide represented by the capital letter adjacent to the left of the letter m is a 2'-O-methyl-modified (also known as: 2'-methoxy-modified) nucleotide; a lowercase letter f indicates that the nucleotide represented by the capital letter adjacent to the left of the letter f is a 2'-fluoro-modified nucleotide; a lowercase letter indicates that the nucleotide represented by the capital letter adjacent to the left of the letter d is a 2'-deoxy-modified nucleotide; a combination symbol (moe) indicates that the nucleotide represented by the capital letter adjacent to the left of the combination symbol (moe) is a 2'-O-methoxyethyl-modified nucleotide; a lowercase letter s indicates that the two nucleotides adjacent to the left and right of the letter s are connected by a phosphorothioate bond.

[0685] (NM022), (NM023), (NM036), (NM037), (NM084), (NM085), (NM086), (NM102), (NM103), (NM104), (NM105), and (SNB) each represent a nucleotide.

[0686] Among them, after the compound NM022 is synthesized by the above-mentioned "siRNA conjugate", the structural formula in the above sequence is as shown in (NM022):

[0687] After compound NM023 is synthesized through the above-mentioned "siRNA conjugate", the structural formula in the above sequence is shown as (NM023):

[0688] After compound NM036 is synthesized through the above-mentioned "siRNA conjugate", the structural formula in the above sequence is shown as (NM036):

[0689] After compound NM037 is synthesized through the above-mentioned "siRNA conjugate", the structural formula in the above sequence is shown as (NM037):

[0690] After compound NM084 was synthesized by the above-mentioned "siRNA conjugate", the structural formula in the above sequence is shown as (NM084):

[0691] After compound NM085 is synthesized through the above-mentioned "siRNA conjugate", the structural formula in the above sequence is shown as (NM085):

[0692] After compound NM086 was synthesized through the above-mentioned "siRNA conjugate", the structural formula in the above sequence is shown as (NM086):

[0693] After compound NM102 is synthesized through the above-mentioned "siRNA conjugate", the structural formula in the above sequence is shown as (NM102):

[0694] After compound NM103 is synthesized through the above-mentioned "siRNA conjugate", the structural formula in the above sequence is shown as (NM103):

[0695] After compound NM104 is synthesized through the above-mentioned "siRNA conjugate", the structural formula in the above sequence is shown as (NM104):

[0696] After compound NM105 is synthesized through the above-mentioned "siRNA conjugate", the structural formula in the above sequence is shown as (NM105):

[0697] The structural formula of (SNB) is: (SNB) is derived from the nucleoside analog SNB, the structural formula of SNB is

[0698] Table 6 Detection results of siRNA conjugates

[0699] It can be seen from the data shown in Table 6 that the present disclosure obtains the siRNA conjugates shown in Table 5 while maintaining a relatively high purity.

[0700] Biological detection experiments

[0701] Unless otherwise specified, the experimental animals C57BL / 6J mice used in this disclosure were purchased from Spef (Beijing) Biotechnology Co., Ltd.; the serum biochemical tests in this disclosure were commissioned to Beijing Sinoin Biotechnology Co., Ltd.; the pathological sections, staining and film reading in this disclosure were commissioned to Wuhan Saiweier Biotechnology Co., Ltd.

[0702] Method for evaluating the target gene inhibition activity of siRNA conjugates in mice

[0703] 6-8 week old C57BL / 6J mice were randomly divided into groups (all female) by body weight. The mice in each group were dosed according to body weight, and the mice were given a single dose of subcutaneous injection in the abdomen. Each siRNA conjugate was configured into a corresponding concentration (calculated as siRNA) solution with a PBS solution for administration, and the administration volume was 5 ml (in terms of siRNA) / kg (in terms of mice). The PBS control group was given a PBS solution (not containing drug conjugate) of 5 ml / kg (in terms of mice). The administration day was recorded as day 0 (recorded as D0), and at the preset time after administration, 5 mice were killed in each group. The mice that were killed were respectively subjected to gross dissection and the liver tissue of each mouse that was killed was collected, and the liver tissue was cut into about 2 mm 3 Small pieces were preserved with RNALater.

[0704] Liver tissue samples were collected from the RNAlater at different time points in different experimental groups and disrupted in a Tissuelyser II fully automatic tissue homogenizer for 60 seconds. Total RNA was then extracted using a fully automatic nucleic acid extractor (purchased from Zhejiang Hanwei Technology Co., Ltd.) and a nucleic acid extraction kit (purchased from Zhejiang Hanwei Technology Co., Ltd.) according to the standard operating procedures for total RNA extraction.

[0705] Take 1 μg of total RNA and use a reverse transcription kit (Promega, Reverse Transcription System, A3500) and select Oligo (dT) 15 reverse transcription primer. Prepare 20 μL reverse transcription system according to the instructions of the reverse transcription kit and complete the reverse transcription reaction. After the reaction is completed, add 80 μL RNase-Free water to the reverse transcription system to obtain cDNA solution. Then use a real-time fluorescence quantitative PCR kit (ABI, SYBR TM Select Master Mix, Catalog number: 4472908) to detect the expression level of target gene mRNA in liver tissue. In this real-time fluorescence quantitative PCR method, primers for the target gene and primers for the internal reference gene are used to detect the target gene and the internal reference gene respectively. According to the method described in the instructions of the real-time fluorescence quantitative PCR kit, 20μL Real-time PCR reaction system is configured for each PCR detection well. Each reaction system contains 5μL of the cDNA solution obtained by the above reverse transcription reaction, 10μL SYBR TM SelectMaster Mix, 0.5 μL 10 μM upstream primer, 0.5 μL 10 μM downstream primer, 4 μL RNase-Free H2O. The prepared reaction system was placed in a real-time fluorescence quantitative PCR instrument (ABI, StepOnePlus TM ) were used for real-time PCR amplification using a three-step method: pre-denaturation at 95°C for 10 minutes, followed by denaturation at 95°C for 30 seconds, annealing at 60°C for 30 seconds, and extension at 72°C for 30 seconds. The denaturation, annealing, and extension process was repeated for 40 cycles. In this real-time fluorescence quantitative PCR method, the expression level and inhibition rate of the target gene mRNA in each test group were relatively quantitatively calculated using the ΔΔCt method. The calculation method is as follows:

[0706] ΔCt(test group) = Ct(test group target gene) – Ct(test group reference gene)

[0707] ΔCt(control group) = Ct(control group target gene) – Ct(control group internal reference gene)

[0708] ΔΔCt(test group) = ΔCt(test group) – ΔCt(average of control group)

[0709] ΔΔCt(control group) = ΔCt(control group) – ΔCt(average of control group)

[0710] Here, ΔCt (control group average) is the arithmetic mean of the ΔCt (control group) values ​​of five mice sacrificed at the same time point in the control group. Therefore, each mouse in the test group and the control group corresponds to a ΔΔCt value.

[0711] The target gene mRNA expression level in the test group was normalized with the control group as the benchmark, and the remaining expression level of the target gene mRNA in the control group was defined as 100%.

[0712] Relative residual expression level of target gene mRNA in the test group = 2-ΔΔCt (test group) × 100%

[0713] Test group target gene mRNA expression inhibition rate = (1-test group target gene mRNA relative remaining expression level) × 100%

[0714] Unless otherwise stated, the in vivo activity data are based on The experimental data were plotted and analyzed using GraphPadprism 8.0 software.

[0715] In this disclosure, unless otherwise stated, the in vivo activity experimental data used in this disclosure are based on The experimental data were plotted and analyzed using GraphPad prism 8.0 software.

[0716] Example 1 Evaluation of the inhibitory activity of siRNA conjugates against the target gene complement component 3 (CC3) in mice

[0717] This example used an in vivo target gene inhibition activity assessment method to evaluate the inhibitory activity of siRNA conjugates RZ502025, whose antisense strand contains the NM023 group, and RZ502051, which contains the NM022 group, in the CC3 target site. The NM022 group and the NM023 group are isomers of each other.

[0718] 6-8 week old C57BL / 6j mice were randomly divided into four groups of 10 mice each based on body weight. Each group was administered the above-mentioned siRNA conjugates subcutaneously in the abdomen. The PBS control group received a dose of 5 ml / kg per mouse, while the siRNA conjugate experimental group received a dose of 3 mg / kg (as siRNA) per mouse in a 5 ml / kg volume. The day of administration was designated as day 0 (D0). Five mice from each group were sacrificed on day 7 (D7) and day 21 (D21) after administration. The animals were dissected, and liver tissues were collected for RNA extraction, reverse transcription, and real-time PCR analysis. Relative quantification of target gene mRNA in each test group was performed using the aforementioned ΔΔCt method.

[0719] Table 7 Primer sequence information in Example 1

[0720] Table 8 Inhibitory activity of target genes in mice after administration of the siRNA conjugates described in Example 1

[0721] The results of Example 1 are shown in FIG1 and Table 8. The conjugates RZ502025 containing NM023 groups and RZ502051 containing NM022 groups in the antisense chain were more active at D7 and D21 than the control conjugate RZ502017 containing no replacement groups.

[0722] Example 2 Evaluation of the inhibitory activity of siRNA conjugates against the target gene angiopoietin-like protein 3 (ANGPTL3) in mice

[0723] In this example, the in vivo target gene inhibitory activity evaluation method for mice was used to evaluate the inhibitory activity of siRNA conjugates RZ597007 containing an NM023 group, siRNA conjugate RZ597112 containing an NM022 group, siRNA conjugate RZ597102 containing an NM036 group, siRNA conjugate RZ597103 containing an NM037 group, reference conjugate RZ597113 containing an SNB group, and reference conjugate RZ597002 containing no substituted group against the target gene ANGPTL3 in mice. The difference between the NM036 group and the NM023 group lies in the position of the methyl group in the open-ring structure, and the difference between the NM037 group and the NM022 group lies in the position of the methyl group in the open-ring structure. The NM036 group and the NM037 group are isomers of each other.

[0724] 6-8 week old C57BL / 6j mice were randomly divided into 7 groups according to body weight, with 10 mice in each group. Each group of mice was given the above siRNA conjugate by subcutaneous administration in the abdomen, wherein each mouse in the PBS control group was given a dose of 5 ml / kg, and each mouse in the siRNA conjugate experimental group was given a dose of 3 mg / kg (in terms of siRNA) in a volume of 5 ml / kg. The day of administration was recorded as D0, and 5 mice in each group were sacrificed on D7 and D28 after administration. The animals were subjected to gross anatomy, and liver tissue was collected and cut into several 2 mm 3 The small pieces were preserved in RNAlater. RNA extraction, reverse transcription reaction, 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.

[0725] Table 9 Primer sequence information in Example 2

[0726] Table 10 Inhibitory activity of target genes in mice after administration of the siRNA conjugates described in Example 2

[0727] The results of Example 2 are shown in Figure 2 and Table 10. The activity of the conjugate RZ597007 containing the NM023 group in the antisense chain was basically equivalent to that of the control conjugate RZ597113 containing the SNB group and the control conjugate RZ597002 containing no replacement group at D7 and D28; at D7 and D28, the inhibitory activity of the conjugate RZ597112 containing the NM022 group was 8%-10% different from that of the control conjugate; at D7, the inhibitory activity of the conjugate RZ597102 containing the NM036 group was basically equivalent to that of the control conjugates RZ597113 and RZ597002, and the inhibitory activity was better than that of the control conjugate at D28; the inhibitory activity of the conjugate RZ597103 containing the NM037 group in the antisense chain was weaker than that of the control conjugate at D7 and D28. Overall, the results showed that compared with the control sequence, the siRNA conjugate containing the NM036 group maintained better inhibitory activity, the conjugate containing the NM023 group maintained comparable inhibitory activity, the NM023 configuration was superior to the NM022 configuration, and the conjugate containing the NM037 group was second.

[0728] Example 3 Toxicological evaluation of siRNA in mice

[0729] In this example, subcutaneous injection was used to evaluate the nature and extent of toxic reactions in ICR mice of the conjugates RZ597007, whose antisense chains contain the NM023 group, the conjugate RZ597112, whose antisense chains contain the NM022 group, the conjugate RZ597102, whose antisense chains contain the NM036 group, the conjugate RZ597103, whose antisense chains contain the NM037 group, the reference conjugate RZ597113, which contains the SNB group, and the reference conjugate RZ597002, which does not contain the alternative group.

[0730] 6-8 week old ICR mice were randomly divided into groups according to body weight, with 3 mice per group. Each test group was given the above-mentioned dose of drug conjugate and a PBS control group was added. The drug dose was calculated for all mice according to body weight and a single dose was administered by subcutaneous injection in the abdomen. Each drug conjugate was administered in the form of a 60 mg / ml (calculated as siRNA) PBS solution, and the administration volume was 10 ml / kg mouse body weight. That is, the dosage of each drug conjugate was 600 mg / kg mouse body weight (calculated as siRNA). The PBS control group was given the same volume of PBS solution (without drug conjugate) and observed for 7 days. The day of administration was recorded as day 0 (denoted as D0). Clinical observation was performed at least once a day after the first administration during the trial; blood was collected from all animals on the 7th day of the trial (denoted as D7) (mice were fasted for at least 12 hours before sampling and testing, and water was not forbidden) for blood biochemical tests. Blood samples (without anticoagulation) were collected and placed at room temperature for approximately 30 minutes. After the blood coagulated, it was centrifuged at 2000 g for 10 minutes at 4°C. Blood biochemistry analysis was performed using a fully automatic biochemical analyzer (Mindray BS-430). The change rate of each serum biochemical index was calculated as follows: Change rate = (experimental group - blank control group (PBS)) / blank control group.

[0731] Figures 3 and 4 are scatter plots of ALT and AST concentrations in mouse serum following administration of 600 mg / kg of RZ597007, RZ597112, RZ597102, RZ597103, and the reference conjugates RZ597002 and RZ597113, respectively, along with a blank control group (PBS). As shown in Figures 3 and 4, compared to the blank control group, administration of the conjugate RZ597002, which does not contain a substituted group, increased ALT levels in mouse serum from 18.10 U / L in the blank control group to 450.10 U / L, a 23.9-fold increase. AST levels also increased significantly, from 62.60 U / L to 478.90 U / L, a 6.7-fold increase, indicating significant hepatotoxicity.

[0732] After administration of the siRNA conjugate of this example, the serum ALT and AST concentrations decreased to varying degrees, indicating that the siRNA conjugate of this example has a certain effect of reducing hepatotoxicity. Specifically, after administration of the conjugate containing the NM023 group (RZ597007), the serum ALT concentration of mice decreased to 34.30 U / L, causing the ALT change rate of the conjugate to decrease from 23.9 times (RZ597002) to 0.9 times, and the AST concentration decreased to 74.40 U / L, causing the AST change rate of the conjugate to decrease from 6.7 times (RZ597002) to 0.2 times, which was basically the same as that of the PBS group; after administration of the conjugate containing NM036 (RZ597102), the serum ALT change rate of mice was 1.9 times, and the AST change rate was 0.1 times; after administration of the conjugate containing NM037 (RZ597103), the serum ALT change rate of mice was 2.2 times, and the AST change rate was 0.5 times; these two groups also did not show a significant increase in ALT and AST levels; the NM023 group, NM036 group, and NM037 group have a significant effect in reducing hepatotoxicity.

[0733] After administration of the conjugate containing the NM022 group (RZ597112), the change rate of ALT in the mouse serum was 5.5 times, and the change rate of AST was 0.7 times; compared with the control conjugate (RZ597002) of 23.9 times (ALT) and 6.7 times (AST), it still showed a certain effect of reducing liver toxicity.

[0734] The reference conjugate RZ597113 containing the SNB group showed an 11.1-fold and 5.2-fold change in mouse serum ALT and AST, respectively. Although it showed a certain reduction in hepatotoxicity compared with the control conjugate (RZ597002), it still showed a greater degree of hepatotoxicity compared with the other siRNA conjugates in this example.

[0735] Table 11: Blood biochemical test results of mice after administration of the siRNA conjugate described in Example 3

[0736] All animals were dissected on the seventh day of the experiment (designated D7). Mice were fasted for at least 12 hours prior to dissection and anesthetized using a Reward R540IE small animal anesthesia machine. After blood collection, animals were euthanized by exsanguination of the abdominal aorta. Gross anatomical observations were performed. The livers of all animals were preserved in 4% cell fixative and histopathologically examined (stained with hematoxylin and eosin). The severity of hepatocyte degeneration in pathological sections was evaluated and graded using a four-level grading system (reference document: [US] Peter. Mann et al. International Standards for Terminology and Diagnostic Criteria for Pathological Changes in Rats and Mice (INHAND) [M]. Translated by Yang Lifeng, Zhou Xiangmei, and Zhao Deming. Beijing: China Agriculture Press, 2019). Comparisons were made.

[0737] As shown in the pathological section results of Figures 5 and 6, compared with the blank control, among the three mice administered with the reference conjugate RZ597002, which does not contain any substituent groups, one mouse exhibited moderate to severe hepatocyte degeneration, specifically, a large number of hepatocytes with watery degeneration, severe cell swelling, loose and lightly stained cytoplasm (▲), irregular arrangement of hepatocytes, frequent hepatocyte necrosis, nuclear dissolution (arrows), connective tissue hyperplasia (*) around a large number of veins and hepatic sinusoids, disordered hepatic lobule structure, accompanied by scattered lymphocyte infiltration (#); one mouse exhibited mild to moderate hepatocyte degeneration, a large number of hepatocytes with swelling, loose and lightly stained cytoplasm, punctate necrosis of hepatocytes and a small amount of granulocyte and lymphocyte infiltration; one mouse exhibited mild hepatocyte degeneration, with some hepatocytes undergoing watery degeneration and a small amount of hepatocyte necrosis. In summary, the hepatocyte degeneration was more severe than that of the blank control group. Among the three mice treated with the reference conjugate RZ597113 containing the SNB group, two showed mild to mild hepatocellular degeneration, specifically manifested by a small amount of watery hepatocellular degeneration, some punctate necrosis of hepatocellular cells and punctate lymphocytic infiltration, and one had mild hepatocellular degeneration, manifested by some mild swelling of hepatocellular cells and loose and lightly stained cytoplasm.

[0738] After administration of conjugates containing the substituted groups NM023 (RZ597007), NM022 (RZ597112), NM036 (RZ597102), and NM037 (RZ597103), various indicators of hepatocyte degeneration in pathological sections were attenuated, especially the level of hepatocyte necrosis, where each siRNA conjugate showed a lower rating than the reference conjugates RZ597113 and RZ597002 ( Figures 5 and 6 ). Specifically, the two groups of mice given the conjugate containing the NM022 group (RZ597112) and the conjugate containing the NM036 group (RZ597102) showed mild hepatocellular degeneration. In both groups, only one mouse showed a small amount of punctate hepatocellular necrosis, slight lymphocyte infiltration, and a small amount of hepatocellular hydropic degeneration. The other two mice showed only mild hepatocellular hydropic degeneration and no hepatocellular necrosis. Among the three mice given the conjugate containing the NM023 group (RZ597007), two showed mild to mild hepatocellular degeneration. The 3 mice in the group treated with the conjugate containing the NM037 group (RZ597103) showed mild to mild hepatocyte degeneration, specifically partial hepatocyte swelling, one of which was accompanied by a small amount of punctate hepatocyte necrosis; and one mouse in the blank control group also showed very little punctate hepatocyte necrosis, partial inflammatory cell infiltration and hydropic degeneration. It was assessed that the cause of the mild hepatocyte degeneration was the animal's own cell metabolism.

[0739] In summary, compared to the conjugate RZ597002, which does not contain a substituted group, and the reference conjugate RZ597113, the siRNA conjugates disclosed herein can, to a certain extent, reduce hepatotoxic reactions caused by off-target effects and exhibit higher safety. Specifically, the conjugate RZ597102, which contains the NM036 group, exhibits improved safety while maintaining superior inhibitory activity.

[0740] Method for evaluating the inhibitory activity of siRNA conjugates on target genes in cells in vitro

[0741] Plasmid construction: siRNA seed region on-target / off-target sequences were designed and inserted into the multiple cloning sites downstream of the hRluc gene in the psi-CHECK2 plasmid to construct the antisense chain on-target plasmid (ASC) and off-target plasmid (ASM).

[0742] Cell culture and transfection: DMEM complete medium was used to culture cells normally at 37°C and 5% CO2. 2When the cell culture flask density reaches 80-90%, discard the culture medium and rinse with 0.25% trypsin. After trypsin digestion, stop the digestion with culture medium, resuspend the cells, centrifuge at 800 rpm for 5 minutes, resuspend with fresh culture medium and count, dilute the cell density to 8 × 10 3 cells / well, inoculated into 96-well cell plates, 100 μL / well, cultured for 24 h before transfection.

[0743] Preparation of siRNA serial dilutions: Prepare each conjugate to a 20 μM stock solution (based on siRNA) in PBS. Then, serially dilute the stock solution with PBS to working concentrations of 4000 nM, 1000 nM, 250 nM, 62.5 nM, 15.625 nM, 3.906 nM, 0.9766 nM, 0.2441 nM, and 0.06104 nM, respectively. Preparation of the transfection mixture: Add 0.2 μL of Lipo2000 to 9.8 μL of Opti-MEM per well to make a 10 μL Lipo2000 mixture. Incubate at room temperature for 5 minutes. Add 10 ng of ASC / ASM plasmid to each well. Add 0.05 μL of a 200 ng / μL stock solution to 8.95 μL of Opti-MEM to create a 9 μL plasmid mixture. Mix 1 μL of siRNA working solution, 9 μL of the plasmid mixture, and 10 μL of Lipo2000 to create a transfection mixture. Simultaneously, set up a 0 nM transfection control well by mixing 1 μL of PBS, 9 μL of the plasmid mixture, and 10 μL of Lipo2000 to create a 20 μL control transfection mixture (control 1). Mix thoroughly by pipetting and incubate at room temperature for 20 minutes before performing subsequent transfections.

[0744] Aspirate the complete medium from the culture wells and replace each well with 80 μL of Opti-MEM medium. Add 20 μL of the transfection mixture. Set up three replicates for each conjugate concentration. Incubate in a 37°C, 5% CO₂ incubator for 4 hours. Add 100 μL of DMEM medium supplemented with 10% fetal bovine serum to each well. Incubate the plate in a 37°C, 5% CO₂ incubator for an additional 24 hours.

[0745] Detection and analysis: Aspirate the culture medium in the 96-well plate, add 150 μL Firfly reaction solution to each well (prepared at a 1:1 ratio per well, i.e., 75 μL Firfly substrate and 75 μL DMEM / (well)), shake in a shaker in the dark for 10 minutes, pipette 120 μL of reaction solution to the detection plate, read the plate on a microplate reader, and obtain the Firfly value; then add 60 μL stop solution (prepared at a 1:100 ratio per well, i.e., 1 μL Renilla substrate and 100 μL Buffer / (well) (prepared immediately for use)) to the detection plate, shake in a shaker in the dark for 10 minutes, read the plate on a microplate reader, and obtain the Renilla value; and calculate the normalized ratio: Ratio = Renilla / Firfly.

[0746] Calculation of residual inhibition rate: [1-Mean(Ratio siRNA缀合物 / Ratio control )]*100%=inhibited activity (%); where Ratio control is the average value of Ratio of three replicate wells in the control well (without siRNA conjugate); Ratio siRNA缀合物 Represents the mean value of Ratio of three replicate wells in the test well (containing siRNA conjugate); inhibited activity represents the residual inhibition rate; Mean (Ratio siRNA / Ratio control ) refers to 3 replicate wells (Ratio siRNA缀合物 / Ratio control )*100% mean.

[0747] IC50 curve fitting was performed using the four-parameter log(inhibitor) vs. response-variable slope (four parameters) model in GraphPad Prism 8.0. The IC50 concentration was calculated using the formula X = 10^(LogIC50 - Log((Top-Bottom) / (50-Bottom) - 1) / HillSlope). The parameters in this formula can be found in the GraphPad Prism 8.0 analysis results table.

[0748] IC60 curve fitting was performed using the four-parameter log(inhibitor) vs. response-variable slope (four parameters) model in GraphPad Prism 8.0. The IC60 concentration was calculated using the formula X = 10^(LogIC50 - Log((Top-Bottom) / (60-Bottom) - 1) / HillSlope). The parameters in this formula can be found in the GraphPad Prism 8.0 analysis results table.

[0749] IC⁷⁵ curve fitting was performed using the four-parameter log(inhibitor) vs. response-variable slope (four parameters) model in GraphPad Prism 8.0. The IC⁷⁵ concentration was calculated using the formula X = 10^(LogIC⁷⁵ - Log((Top-Bottom) / (IC⁷⁵ - Bottom) - 1) / HillSlope). The parameters in this formula can be found in the GraphPad Prism 8.0 analysis results table.

[0750] Example 4 Evaluation of the inhibitory activity of siRNA conjugates against the target gene angiopoietin-like protein 3 (ANGPTL3) in HEK293T cells

[0751] This example uses an in vitro cell-based method to evaluate the inhibitory activity of siRNA conjugates RZ597007, RZ597112, RZ597102, RZ597103, RZ597103, RZ597113, and RZ597002, containing no substituted groups, against the target gene ANGPTL3 in HEK293T cells. The NM022 and NM023 groups are isomers of each other; the NM036 and NM037 groups are isomers of each other; and the difference between the NM036 and NM023 groups lies in the position of the methyl groups in the open ring structure.

[0752] According to the above-mentioned in vitro cell target gene inhibitory activity evaluation method, on-target and off-target tests of siRNA conjugates were performed respectively, and the IC50 value of the on-target test and the IC75 value of the off-target test were calculated.

[0753] The results of Example 4 show that in the target (ASC) evaluation, the IC50 values ​​of the siRNA conjugates RZ597007, RZ597112, RZ597102, and RZ597103 were 0.0832 nM, 0.0695 nM, 0.0655 nM, and 0.0611 nM, respectively, which were superior to the reference conjugate RZ597002 (0.1345 nM) that did not contain the substituted group; and were basically equivalent to the reference conjugate RZ597113 (0.0535 nM) that contained the SNB group. ; However, the IC75 value calculation in the off-target (ASM) activity assessment found that RZ597007 and RZ597102 had basically no off-target activity; compared with RZ597002 (16.3539nM) and RZ597113 (9.0746nM), the IC75 value of RZ597112 was 16.1609nM, which was superior to RZ597113 in reducing off-target activity; the IC75 value of RZ597103 was 37.6409nM, which showed a certain anti-off-target trend (Figure 7-18, Table 12).

[0754] Table 12 Evaluation of IC50 and IC75 values ​​of siRNA conjugates in target gene ANGPTL3 in HEK293T cells

[0755] Example 5 Evaluation of the inhibitory activity of siRNA conjugates against the target gene ANGPTL3 in HEK293T cells

[0756] In this example, the inhibitory activity of the target gene in vitro in cells was evaluated using a method for evaluating the inhibitory activity of the target gene ANGPTL3 in HEK293T cells using conjugates RZ597157 containing an NM102 group at position 5 of the antisense strand of the ANGPTL3 target site, conjugate RZ597161 containing an NM103 group at position 7 of the antisense strand, and a reference conjugate RZ597136 that does not contain any alternative groups.

[0757] The on-target and off-target assays of the siRNA conjugates were performed according to the above-mentioned in vitro cell target gene inhibitory activity evaluation method, and the IC50 and IC60 values ​​of the on-target and off-target assays were calculated.

[0758] The results of Example 5 show that the IC50 values ​​of the on-target (ASC) activity assessments for the siRNA conjugates RZ597157 and RZ597161 were 0.0559 nM and 0.0455 nM, respectively, which are essentially comparable to the reference conjugate RZ597136 (0.0313 nM). No IC60 values ​​were fitted for the off-target (ASM) activity assessments for RZ597157 and RZ597161, while the reference conjugate RZ597136 had an IC60 value of 9.5713 nM, indicating that RZ597157 and RZ597161 have a certain tendency to prevent off-target effects (Figures 19-24, Table 13).

[0759] Table 13 Evaluation of IC50 and IC60 values ​​of siRNA conjugates in target gene ANGPTL3 in HEK293T cells

[0760] It is understood that the above embodiments are merely exemplary embodiments for illustrating the principles of the present disclosure, and the present disclosure is not limited thereto. Those skilled in the art may make various modifications and improvements without departing from the spirit and substance of the present disclosure, and such modifications and improvements are also considered to be within the scope of protection of the present disclosure.

Claims

1. A nucleotide analogue, characterized in that It is selected from the structure represented by the following formula (II-i), or its stereoisomers, or its pharmaceutically acceptable salts: Among them, B 200 selected from bases or modified bases; Z is selected from hydroxyl or thiol; n is selected from 1, 2 or 3; X is selected from Each R' is independently selected from optionally substituted C 1-3 Alkyl, the optionally substituted C 1-3 The alkyl group contains substituents independently selected from halogen, C 1-3 Alkoxy, hydroxy or amino; m is selected from 1, 2, 3 or 4; r is selected from 1, 2, 3 or 4; Each R A and each R B are each independently selected from H or optionally substituted C 1-3 Alkyl, and R A and R B There is at least one optionally substituted C 1-3 Alkyl; if the optionally substituted C 1-3 The alkyl group contains substituents independently selected from halogen, C 1-3 Alkoxy, hydroxy or amino; Each * independently represents a covalent bonding site; Optionally, the nucleotide analog represented by formula (II-i) has a structure represented by the following formula (200), or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof: wherein R2 and R3 are independently selected from H or optionally substituted C 1-3 and at least one of R2 and R3 is selected from optionally substituted C 1-3 Alkyl; if the optionally substituted C 1-3 The alkyl group contains substituents independently selected from halogen, C 1-3 Alkoxy, hydroxy or amino; the remaining substituents are as defined above.

2. The nucleotide analogue according to claim 1, characterized in that The base is selected from uracil U, thymine T, cytosine C, adenine A or guanine G; Optionally, the modified base is selected from Optionally, B 200 Select from any of the following structures:

3. The nucleotide analogue according to any one of claims 1 to 2, characterized in that n is selected from 1 or 2; Optionally, n is selected from 1.

4. The nucleotide analogue according to any one of claims 1 to 3, characterized in that m is selected from 1, 2 or 3; Optionally, m is selected from 2.

5. The nucleotide analogue according to any one of claims 1 to 4, characterized in that X is selected from 6. The nucleotide analogue according to any one of claims 1 to 5, characterized in that R2 and R3 are independently selected from H or optionally substituted C 1-3 and R2 and R3 are not H or optionally substituted C 1-3 alkyl; Optionally, R2 and R3 are independently selected from H or C 1-3 Alkyl; and R2 and R3 are not H or C at the same time 1-3 alkyl; Optionally, R2 and R3 are independently selected from H or methyl; and R2 and R3 are not H or methyl at the same time.

7. The nucleotide analogue according to any one of claims 1 to 6, characterized in that The nucleotide analog has a structure represented by formula (201), or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof:

8. The nucleotide analogue according to any one of claims 1 to 7, characterized in that The nucleotide analog has any of the following structures, or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof: wherein R2 is selected from optionally substituted C 1-3 alkyl; wherein R3 is selected from optionally substituted C 1-3 alkyl.

9. The nucleotide analogue according to any one of claims 1 to 8, characterized in that The nucleotide analog has any of the following structures, or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof: wherein R2 and R3 are independently selected from optionally substituted C 1-3 alkyl.

10. The nucleotide analogue according to any one of claims 1 to 9, characterized in that The nucleotide analog is selected from any of the following structures, or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof:

11. The nucleotide analog according to claim 1, characterized in that The nucleotide analog represented by formula (II-i) has a structure represented by the following formula (500), or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof: Among them, B 200 , Z, n, X and * are as defined in any one of claims 1 to 10; r is selected from 1, 2, 3 or 4; R5 and R6 are independently selected from H or optionally substituted C 1-3 and at least one of R5 and R6 is selected from optionally substituted C 1-3 Alkyl; if the optionally substituted C 1-3 The alkyl group contains substituents independently selected from halogen, C 1-3 Alkoxy, hydroxy or amino.

12. The nucleotide analog according to claim 11, characterized in that r is selected from 1, 2 or 3; Optionally, r is selected from 2; Optionally, R5 and R6 are independently selected from H or optionally substituted C 1-3 and R5 and R6 are not H or optionally substituted C 1-3 alkyl; Optionally, R5 and R6 are independently selected from H or C 1-3 Alkyl; and R5 and R6 are not H or C 1-3 alkyl; Optionally, R5 and R6 are independently selected from H or methyl; and R5 and R6 are not H or methyl at the same time; Optionally, the nucleotide analog has a structure represented by formula (501), or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof: Optionally, the nucleotide analog has any of the following structures, or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof: Wherein R5 is selected from optionally substituted C 1-3 alkyl; wherein R6 is selected from optionally substituted C 1-3 alkyl; Optionally, the nucleotide analog has any of the following structures, or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof: wherein R5 and R6 are independently selected from optionally substituted C 1-3 alkyl; Optionally, the nucleotide analog is selected from any of the following structures, or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof:

13. A double-stranded oligonucleotide, characterized in that The double-stranded oligonucleotide comprises a sense strand and an antisense strand, each strand having 17-35 nucleotides; from the 5' end to the 3' end, at least one nucleotide in positions 2-8 of the antisense strand is replaced by a nucleotide analogue according to any one of claims 1-12.

14. The double-stranded oligonucleotide according to claim 13, characterized in that At least one nucleotide in positions 3 to 8 of the antisense strand is replaced by the nucleotide analogue in the direction from the 5' end to the 3' end; Optionally, in the direction from 5' to 3', any one of the nucleotides at position 3, 4, 5, 6, 7 or 8 of the antisense strand is replaced by the nucleotide analog; Optionally, in the 5' to 3' direction, any one of the 5th, 6th, 7th or 8th nucleotides of the antisense strand is replaced by the nucleotide analog; Optionally, in the direction from 5' to 3', the 3rd nucleotide of the antisense strand is replaced by the nucleotide analog; Optionally, in the direction from 5' to 3', the 4th nucleotide of the antisense strand is replaced by the nucleotide analog; Optionally, in the direction from 5' to 3', the 5th nucleotide of the antisense strand is replaced by the nucleotide analog; Optionally, in the direction from 5' to 3', the 6th nucleotide of the antisense strand is replaced by the nucleotide analog; Optionally, in the direction from 5' to 3', the 7th nucleotide of the antisense strand is replaced by the nucleotide analog; Optionally, the 8th nucleotide of the antisense strand is replaced by the nucleotide analogue in the 5' to 3' direction.

15. The double-stranded oligonucleotide according to any one of claims 13 to 14, characterized in that The Base in each of the nucleotide analogs is the same as the base in the nucleotide it replaces.

16. The double-stranded oligonucleotide according to any one of claims 13 to 15, characterized in that In the direction from the 5' end to the 3' end, the 5th nucleotide of the nucleotide sequence in the sense strand is selected from a 2'-O-methyl modified nucleotide or a 2'-O-methoxyethyl modified nucleotide, at least three nucleotides from the 7th to the 10th positions are 2'-fluoro modified nucleotides, and the nucleotides at the remaining positions are independently selected from 2'-O-methyl modified nucleotides; And / or, in the direction from the 5' end to the 3' end, at least one nucleotide in positions 3 to 8 of the nucleotide sequence in the antisense strand is independently selected from the nucleotide analogs, at least four nucleotides in positions 2, 6, 9, 12, 14 and 16 are selected from 2'-fluoro-modified nucleotides, the nucleotide in position 15 is selected from 2'-O-methoxyethyl-modified nucleotides or 2'-O-methyl-modified nucleotides, and the nucleotides in the remaining positions are independently selected from 2'-O-methyl-modified nucleotides; wherein, when the nucleotide in position 6 is selected from the nucleotide analogs, the nucleotides in positions 2, 9, 12, 14 and 16 are selected from 2'-fluoro-modified nucleotides; when the nucleotide in position 6 is selected from 2'-fluoro-modified nucleotides, at least one nucleotide in positions 3 to 5 and 7 to 8 is independently selected from the nucleotide analogs; Optionally, in the direction from the 5' end to the 3' end, at least three nucleotides in positions 7 to 10 of the nucleotide sequence in the sense strand are 2'-fluoro-modified nucleotides, and the nucleotides in the remaining positions are independently selected from 2'-O-methyl-modified nucleotides; and / or, in the direction from the 5' end to the 3' end, at least one nucleotide in positions 3 to 8 of the nucleotide sequence in the antisense strand is independently selected from the nucleotide analogs, at least four nucleotides in positions 2, 6, 9, 12, 14 and 16 are selected from 2'-fluoro-modified nucleotides, the nucleotide in position 15 is selected from 2'-O-methoxyethyl-modified nucleotides or 2'-O-methyl-modified nucleotides, and the nucleotides in the remaining positions are independently selected from 2'-O-methyl-modified nucleotides; wherein, when the nucleotide in position 6 is selected from the nucleotide analogs, the nucleotides in positions 2, 9, 12, 14 and 16 are selected from 2'-fluoro-modified nucleotides; when the nucleotide in position 6 is selected from 2'-fluoro-modified nucleotides, at least one nucleotide in positions 3 to 5 and 7 to 8 is independently selected from the nucleotide analogs.

17. The double-stranded oligonucleotide according to any one of claims 13 to 16, characterized in that In the direction from the 5' end to the 3' end, at least three nucleotides in the 7th to 10th positions of the nucleotide sequence in the sense strand are 2'-fluoro-modified nucleotides, and the nucleotides in the remaining positions are independently selected from 2'-O-methyl-modified nucleotides; In the direction from the 5' end to the 3' end, at least one nucleotide in positions 3 to 8 of the nucleotide sequence in the antisense strand is independently selected from the nucleotide analogs, at least three nucleotides in positions 2, 6, 14 and 16 are selected from 2'-fluoro-modified nucleotides, and the nucleotide in position 9 or 12 is selected from 2'-fluoro-modified nucleotides, the nucleotide in position 15 is selected from 2'-O-methoxyethyl-modified nucleotides or 2'-O-methyl-modified nucleotides, and the nucleotides in the remaining positions are independently selected from 2'-O-methyl-modified nucleotides; wherein, when the nucleotide in position 6 is selected from the nucleotide analogs, the nucleotides in positions 2, 9, 12, 14 and 16 are selected from 2'-fluoro-modified nucleotides; when the nucleotide in position 6 is selected from 2'-fluoro-modified nucleotides, at least one nucleotide in positions 3 to 5 and 7 to 8 is independently selected from the nucleotide analogs.

18. The double-stranded oligonucleotide according to any one of claims 13 to 17, characterized in that The modifications of the sense strand and the antisense strand in the double-stranded oligonucleotide are selected from one of the following (1) to (24): (1) In the direction from the 5' end to the 3' end, the 7th to 10th nucleotides of the nucleotide sequence in the sense strand are independently selected from 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions are independently selected from 2'-O-methyl-modified nucleotides; In the direction from the 5' end to the 3' end, the nucleotide at position 3 of the nucleotide sequence in the antisense strand is selected from the nucleotide analog, the nucleotides at positions 2, 6, 9, 14, and 16 are independently selected from 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions are independently selected from 2'-O-methyl-modified nucleotides; (2) In the direction from the 5' end to the 3' end, the nucleotides at positions 7 to 10 in the nucleotide sequence of the sense strand are independently selected from 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions are independently selected from 2'-O-methyl-modified nucleotides; In the direction from the 5' end to the 3' end, the 4th nucleotide of the nucleotide sequence in the antisense strand is selected from the nucleotide analog, the 2nd, 6th, 9th, 14th and 16th nucleotides are independently selected from 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions are independently selected from 2'-O-methyl-modified nucleotides; (3) In the direction from the 5' end to the 3' end, the nucleotides at positions 7 to 10 in the nucleotide sequence of the sense strand are independently selected from 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions are independently selected from 2'-O-methyl-modified nucleotides; In the direction from the 5' end to the 3' end, the 5th nucleotide of the nucleotide sequence in the antisense strand is selected from the nucleotide analog, the 2nd, 6th, 9th, 14th and 16th nucleotides are independently selected from 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions are independently selected from 2'-O-methyl-modified nucleotides; (4) In the direction from the 5' end to the 3' end, the nucleotides at positions 7 to 10 in the nucleotide sequence of the sense strand are independently selected from 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions are independently selected from 2'-O-methyl-modified nucleotides; In the direction from the 5' end to the 3' end, the 6th nucleotide of the nucleotide sequence in the antisense strand is selected from the nucleotide analog, the 2nd, 9th, 14th and 16th nucleotides are independently selected from 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions are independently selected from 2'-O-methyl-modified nucleotides; (5) In the direction from the 5' end to the 3' end, the nucleotides at positions 7 to 10 in the nucleotide sequence of the sense strand are independently selected from 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions are independently selected from 2'-O-methyl-modified nucleotides; In the direction from the 5' end to the 3' end, the 7th nucleotide of the nucleotide sequence in the antisense strand is selected from the nucleotide analog, the 2nd, 6th, 9th, 14th and 16th nucleotides are independently selected from 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions are independently selected from 2'-O-methyl-modified nucleotides; (6) In the direction from the 5' end to the 3' end, the nucleotides at positions 7 to 10 in the nucleotide sequence of the sense strand are independently selected from 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions are independently selected from 2'-O-methyl-modified nucleotides; In the direction from the 5' end to the 3' end, the 8th nucleotide of the nucleotide sequence in the antisense strand is selected from the nucleotide analog, the 2nd, 6th, 9th, 14th and 16th nucleotides are independently selected from 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions are independently selected from 2'-O-methyl-modified nucleotides; (7) In the direction from the 5' end to the 3' end, the nucleotides at positions 7 to 10 in the nucleotide sequence of the sense strand are independently selected from 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions are independently selected from 2'-O-methyl-modified nucleotides; In the direction from the 5' end to the 3' end, the nucleotide at position 3 of the nucleotide sequence in the antisense strand is selected from the nucleotide analog, the nucleotides at positions 2, 6, 9, 14, and 16 are independently selected from 2'-fluoro-modified nucleotides, the nucleotide at position 15 is selected from 2'-O-methoxyethyl-modified nucleotides, and the nucleotides at the remaining positions are independently selected from 2'-O-methyl-modified nucleotides; (8) In the direction from the 5' end to the 3' end, the nucleotides at positions 7 to 10 in the nucleotide sequence of the sense strand are independently selected from 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions are independently selected from 2'-O-methyl-modified nucleotides; In the direction from the 5' end to the 3' end, the 4th nucleotide of the nucleotide sequence in the antisense strand is selected from the nucleotide analog, the 2nd, 6th, 9th, 14th and 16th nucleotides are independently selected from 2'-fluoro modified nucleotides, and the 15th nucleotide is selected from 2'-O-methoxyethyl modified nucleotides. The nucleotides at the remaining positions are independently selected from 2'-O-methyl modified nucleotides; (9) In the direction from the 5' end to the 3' end, the nucleotides at positions 7 to 10 of the nucleotide sequence in the sense strand are independently selected from 2'-fluoro modified nucleotides, the nucleotide at position 15 is selected from 2'-O-methoxyethyl modified nucleotides, and the nucleotides at the remaining positions are independently selected from 2'-O-methyl modified nucleotides; In the direction from the 5' end to the 3' end, the 5th nucleotide of the nucleotide sequence in the antisense strand is selected from the nucleotide analog, the 2nd, 6th, 9th, 14th and 16th nucleotides are independently selected from 2'-fluoro-modified nucleotides, the 15th nucleotide is selected from 2'-O-methoxyethyl-modified nucleotides, and the nucleotides at the remaining positions are independently selected from 2'-O-methyl-modified nucleotides; (10) In the direction from the 5' end to the 3' end, the nucleotides at positions 7 to 10 of the nucleotide sequence in the sense strand are independently selected from 2'-fluoro modified nucleotides, the nucleotide at position 15 is selected from 2'-O-methoxyethyl modified nucleotides, and the nucleotides at the remaining positions are independently selected from 2'-O-methyl modified nucleotides; In the direction from the 5' end to the 3' end, the 6th nucleotide of the nucleotide sequence in the antisense strand is selected from the nucleotide analog, the 2nd, 9th, 14th and 16th nucleotides are independently selected from 2'-fluoro-modified nucleotides, the 15th nucleotide is selected from 2'-O-methoxyethyl-modified nucleotides, and the nucleotides at the remaining positions are independently selected from 2'-O-methyl-modified nucleotides; (11) In the direction from the 5' end to the 3' end, the nucleotides at positions 7 to 10 in the nucleotide sequence of the sense strand are independently selected from 2'-fluoro modified nucleotides, the nucleotide at position 15 is selected from 2'-O-methoxyethyl modified nucleotides, and the nucleotides at the remaining positions are independently selected from 2'-O-methyl modified nucleotides; In the direction from the 5' end to the 3' end, the 7th nucleotide of the nucleotide sequence in the antisense strand is selected from the nucleotide analog, the 2nd, 6th, 9th, 14th and 16th nucleotides are independently selected from 2'-fluoro-modified nucleotides, the 15th nucleotide is selected from 2'-O-methoxyethyl-modified nucleotides, and the nucleotides at the remaining positions are independently selected from 2'-O-methyl-modified nucleotides; (12) In the direction from the 5' end to the 3' end, the nucleotides at positions 7 to 10 in the nucleotide sequence of the sense strand are independently selected from 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions are independently selected from 2'-O-methyl-modified nucleotides; In the direction from the 5' end to the 3' end, the 8th nucleotide of the nucleotide sequence in the antisense strand is selected from the nucleotide analog, the 2nd, 6th, 9th, 14th and 16th nucleotides are independently selected from 2'-fluoro-modified nucleotides, the 15th nucleotide is selected from 2'-O-methoxyethyl-modified nucleotides, and the nucleotides at the remaining positions are independently selected from 2'-O-methyl-modified nucleotides; (13) In the direction from the 5' end to the 3' end, the nucleotides at positions 7 to 10 in the nucleotide sequence of the sense strand are independently selected from 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions are independently selected from 2'-O-methyl-modified nucleotides; In the direction from the 5' end to the 3' end, the nucleotide at position 3 of the nucleotide sequence in the antisense strand is selected from the nucleotide analog, the nucleotides at positions 2, 6, 12, 14, and 16 are independently selected from 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions are independently selected from 2'-O-methyl-modified nucleotides; (14) In the direction from the 5' end to the 3' end, the nucleotides at positions 7 to 10 in the nucleotide sequence of the sense strand are independently selected from 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions are independently selected from 2'-O-methyl-modified nucleotides; In the direction from the 5' end to the 3' end, the 4th nucleotide of the nucleotide sequence in the antisense strand is selected from the nucleotide analog, the 2nd, 6th, 12th, 14th and 16th nucleotides are independently selected from 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions are independently selected from 2'-O-methyl-modified nucleotides; (15) In the direction from the 5' end to the 3' end, the nucleotides at positions 7 to 10 in the nucleotide sequence of the sense strand are independently selected from 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions are independently selected from 2'-O-methyl-modified nucleotides; In the direction from the 5' end to the 3' end, the 5th nucleotide of the nucleotide sequence in the antisense strand is selected from the nucleotide analog, the The nucleotides at positions 2, 6, 12, 14, and 16 are independently selected from 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions are independently selected from 2'-O-methyl-modified nucleotides; (16) In the direction from the 5' end to the 3' end, the nucleotides at positions 7 to 10 in the nucleotide sequence of the sense strand are independently selected from 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions are independently selected from 2'-O-methyl-modified nucleotides; In the direction from the 5' end to the 3' end, the 6th nucleotide of the nucleotide sequence in the antisense strand is selected from the nucleotide analog, the 2nd, 12th, 14th and 16th nucleotides are independently selected from 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions are independently selected from 2'-O-methyl-modified nucleotides; (17) In the direction from the 5' end to the 3' end, the nucleotides at positions 7 to 10 in the nucleotide sequence of the sense strand are independently selected from 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions are independently selected from 2'-O-methyl-modified nucleotides; In the direction from the 5' end to the 3' end, the 7th nucleotide of the nucleotide sequence in the antisense strand is selected from the nucleotide analog, the 2nd, 6th, 12th, 14th and 16th nucleotides are independently selected from 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions are independently selected from 2'-O-methyl-modified nucleotides; (18) In the direction from the 5' end to the 3' end, the nucleotides at positions 7 to 10 in the nucleotide sequence of the sense strand are independently selected from 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions are independently selected from 2'-O-methyl-modified nucleotides; In the direction from the 5' end to the 3' end, the 8th nucleotide of the nucleotide sequence in the antisense strand is selected from the nucleotide analog, the 2nd, 6th, 12th, 14th and 16th nucleotides are independently selected from 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions are independently selected from 2'-O-methyl-modified nucleotides; (19) In the direction from the 5' end to the 3' end, the nucleotides at positions 7 to 10 in the nucleotide sequence of the sense strand are independently selected from 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions are independently selected from 2'-O-methyl-modified nucleotides; In the direction from the 5' end to the 3' end, the nucleotide at position 3 of the nucleotide sequence in the antisense strand is selected from the nucleotide analog, the nucleotides at positions 2, 6, 12, 14, and 16 are independently selected from 2'-fluoro-modified nucleotides, the nucleotide at position 15 is selected from 2'-O-methoxyethyl-modified nucleotides, and the nucleotides at the remaining positions are independently selected from 2'-O-methyl-modified nucleotides; (20) In the direction from the 5' end to the 3' end, the nucleotides at positions 7 to 10 in the nucleotide sequence of the sense strand are independently selected from 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions are independently selected from 2'-O-methyl-modified nucleotides; In the direction from the 5' end to the 3' end, the 4th nucleotide of the nucleotide sequence in the antisense strand is selected from the nucleotide analog, the 2nd, 6th, 12th, 14th and 16th nucleotides are independently selected from 2'-fluoro-modified nucleotides, the 15th nucleotide is selected from 2'-O-methoxyethyl-modified nucleotides, and the nucleotides at the remaining positions are independently selected from 2'-O-methyl-modified nucleotides; (21) In the direction from the 5' end to the 3' end, the nucleotides at positions 7 to 10 of the nucleotide sequence in the sense strand are independently selected from 2'-fluoro modified nucleotides, the nucleotide at position 15 is selected from 2'-O-methoxyethyl modified nucleotides, and the nucleotides at the remaining positions are independently selected from 2'-O-methyl modified nucleotides; In the direction from the 5' end to the 3' end, the 5th nucleotide of the nucleotide sequence in the antisense strand is selected from the nucleotide analog, the 2nd, 6th, 12th, 14th and 16th nucleotides are independently selected from 2'-fluoro-modified nucleotides, the 15th nucleotide is selected from 2'-O-methoxyethyl-modified nucleotides, and the nucleotides at the remaining positions are independently selected from 2'-O-methyl-modified nucleotides; (22) In the direction from the 5' end to the 3' end, the nucleotides at positions 7 to 10 of the nucleotide sequence in the sense strand are independently selected from 2'-fluoro modified nucleotides, the nucleotide at position 15 is selected from 2'-O-methoxyethyl modified nucleotides, and the nucleotides at the remaining positions are independently selected from 2'-O-methyl modified nucleotides; In the direction from the 5' end to the 3' end, the 6th nucleotide of the nucleotide sequence in the antisense strand is selected from the nucleotide analog, the The nucleotides at positions 2, 12, 14, and 16 are independently selected from 2'-fluoro modified nucleotides, the nucleotide at position 15 is selected from 2'-O-methoxyethyl modified nucleotides, and the nucleotides at the remaining positions are independently selected from 2'-O-methyl modified nucleotides; (23) In the direction from the 5' end to the 3' end, the 7th to 10th nucleotides of the nucleotide sequence in the sense strand are independently selected from 2'-fluoro modified nucleotides, the 15th nucleotide is selected from 2'-O-methoxyethyl modified nucleotides, and the remaining nucleotides are independently selected from 2'-O-methyl modified nucleotides; In the direction from the 5' end to the 3' end, the 7th nucleotide of the nucleotide sequence in the antisense strand is selected from the nucleotide analog, the 2nd, 6th, 12th, 14th and 16th nucleotides are independently selected from 2'-fluoro-modified nucleotides, the 15th nucleotide is selected from 2'-O-methoxyethyl-modified nucleotides, and the nucleotides at the remaining positions are independently selected from 2'-O-methyl-modified nucleotides; (24) In the direction from the 5' end to the 3' end, the nucleotides at positions 7 to 10 in the nucleotide sequence of the sense strand are independently selected from 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions are independently selected from 2'-O-methyl-modified nucleotides; In the direction from the 5' end to the 3' end, the 8th nucleotide of the nucleotide sequence in the antisense strand is selected from the nucleotide analog, the 2nd, 6th, 12th, 14th and 16th nucleotides are independently selected from 2'-fluoro-modified nucleotides, the 15th nucleotide is selected from 2'-O-methoxyethyl-modified nucleotides, and the nucleotides at the remaining positions are independently selected from 2'-O-methyl-modified nucleotides.

19. The double-stranded oligonucleotide according to any one of claims 13 to 18, characterized in that The double-stranded oligonucleotide is selected from siRNA.

20. A double-stranded oligonucleotide conjugate, characterized in that The conjugate comprises the double-stranded oligonucleotide of any one of claims 13 to 19 and one or more ligands capable of binding to a cell surface receptor; Optionally, the ligand is selected from asialoglycoprotein receptor ligands (ASGPR ligands); Optionally, the ASGPR ligand comprises galactose, a galactose derivative, or a galactose cluster; The galactose derivative comprises a galactose derivative having an affinity for asialoglycoprotein receptor equal to or greater than that of galactose; The galactose cluster comprises molecules having 2-4 terminal galactose and / or galactose derivatives; Optionally, the galactose derivative is selected from galactosamine, N-formylgalactosamine, N-acetylgalactosamine, N-propionylgalactosamine, N-n-butyrylgalactosamine and N-isobutyrylgalactosamine.

21. The conjugate according to claim 20, characterized in that The number of the ligands is selected from one, and the ligand is conjugated to the 3' end of the sense strand of the double-stranded oligonucleotide.

22. The conjugate according to any one of claims 20 to 21, characterized in that Each of the ligands is selected from the structure represented by formula (300), or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof: Wherein, * represents the covalent attachment site of the ligand on the sense strand or the antisense strand; The j is selected from 1, 2, 3 or 4; Each of said Z' is independently selected from hydroxyl or thiol; Each of said p is independently selected from 1, 2 or 3; Each of said q is independently selected from 1, 2 or 3; Each R is independently selected from H, optionally substituted C 1-6 Alkyl or optionally substituted C 1-6 Alkoxy; Each of said L is independently selected from optionally substituted C 2-20 Alkylene or R La and R Lb are independently selected from optionally substituted C 1-10 Alkylene, k is selected from 1, 2, 3, 4 or 5; Each Y is independently selected from O, S or NH; Optionally, m is selected from 1, 2 or 3; Optionally, said m is selected from 3; Optionally, said Z' is selected from hydroxyl; Optionally, said p is selected from 1; Optionally, q is selected from 1; Optionally, R is selected from H; Optionally, each of said L is independently selected from optionally substituted C 2-10 Alkylene or Among them, R La and R Lb are independently selected from optionally substituted C 1-10 Alkylene, k is selected from 1, 2 or 3; Optionally, k is selected from 1; Optionally, each L is independently selected from Optionally, each L is independently selected from Optionally, each L is independently selected from Optionally, each L is independently selected from Optionally, Y is selected from O.

23. The conjugate according to claims 20-22, characterized in that Each of the ligands is independently selected from the structure represented by formula (301), or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof:

24. The conjugate according to any one of claims 20 to 23, characterized in that Each ligand is independently selected from any of the following structures, or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof:

25. The conjugate according to any one of claims 20 to 24, characterized in that Each ligand is independently selected from the structure represented by formula (CR01008×3), or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof:

26. A composition, characterized in that The composition comprises the double-stranded oligonucleotide according to any one of claims 13 to 19 and / or the double-stranded oligonucleotide conjugate according to any one of claims 20 to 25.

27. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the double-stranded oligonucleotide according to any one of claims 13 to 19, and / or the double-stranded oligonucleotide conjugate according to any one of claims 20 to 25, and / or the composition according to claim 26.

28. A nucleoside analogue, characterized in that It is selected from the structure represented by formula (Ii), or its stereoisomers, or its pharmaceutically acceptable salts: Among them, B 100 Same as B in claim 1 200 The definition of B 100 contains an amino group, and the amino group is protected by an amino protecting group; n is selected from 1, 2 or 3; X is selected from Each R5 is independently selected from optionally substituted C 1-3 Alkyl, the optionally substituted C 1-3 The alkyl group contains substituents independently selected from halogen, C 1-3 Alkoxy, hydroxy or amino; R1 is selected from H or a hydroxyl protecting group; Optionally, the hydroxy protecting group is selected from trityl, 4-methoxytrityl, 4,4'-dimethoxytrityl or 4,4',4"-trimethoxytrityl; R4 is selected from H or Each R 4a Independently selected from or C containing a cyano substituent 1-6 alkoxy, and at least one R 4a Selected from Each R 4a ' is independently selected from optionally substituted C 1-6 alkyl; m is selected from 1, 2, 3 or 4; r is selected from 1, 2, 3 or 4; Each R A and each R B are each independently selected from H or optionally substituted C 1-3 Alkyl; and R A and R B There is at least one optionally substituted C 1-3 Alkyl; if the optionally substituted C 1-3 The alkyl group contains substituents independently selected from halogen, C 1-3 Alkoxy, hydroxy or amino; Optionally, the nucleoside analog represented by formula (Ii) has a structure represented by formula (100), or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof: wherein R2 and R3 are independently selected from H or optionally substituted C 1-3 and at least one of R2 and R3 is selected from optionally substituted C 1-3 Alkyl; if the optionally substituted C 1-3 The alkyl group contains substituents independently selected from halogen, C 1-3 Alkoxy, hydroxy or amino; the remaining substituents are as defined above.

29. The nucleoside analog according to claim 28, characterized in that The hydroxy protecting group is selected from 4,4'-dimethoxytrityl.

30. The nucleoside analogue according to any one of claims 28 to 29, characterized in that R 4a 'Selected from isopropyl, C containing a cyano substituent 1-6 Alkoxy is selected from 31. The nucleoside analogue according to any one of claims 28 to 30, characterized in that Selected from Optionally, Selected from 32. The nucleoside analogue according to any one of claims 28 to 31, characterized in that Selected from 33. The nucleoside analogue according to any one of claims 28 to 32, characterized in that The nucleoside analog is selected from any of the following structures, or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof:

34. The nucleoside analog according to claim 28, characterized in that The nucleoside analog represented by formula (Ii) has a structure represented by the following formula (400), or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof: Among them, R1, R4, B 100 , n, X and * are as defined in any one of claims 11-12; r is selected from 1, 2, 3 or 4; R5 and R6 are independently selected from H or optionally substituted C 1-3 and at least one of R5 and R6 is selected from optionally substituted C 1-3 Alkyl; if the optionally substituted C 1-3 The alkyl group contains substituents independently selected from halogen, C 1-3 Alkoxy, hydroxy or amino.

35. The nucleoside analogue according to claim 34, characterized in that r is selected from 1, 2 or 3; Optionally, r is selected from 2; Optionally, R5 and R6 are independently selected from H or optionally substituted C 1-3 and R5 and R6 are not H or optionally substituted C 1-3 alkyl; Optionally, R5 and R6 are independently selected from H or C 1-3 Alkyl; and R5 and R6 are not H or C 1-3 alkyl; Optionally, R5 and R6 are independently selected from H or methyl; and R5 and R6 are not H or methyl at the same time; Optionally, the nucleoside analog has a structure represented by formula (401), or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof: Optionally, the nucleoside analog has any of the following structures, or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof: Wherein R5 is selected from optionally substituted C 1-3 alkyl; wherein R6 is selected from optionally substituted C 1-3 alkyl; Optionally, the nucleoside analog has any of the following structures, or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof: wherein R5 and R6 are independently selected from optionally substituted C 1-3 alkyl; Optionally, the nucleoside analog is selected from any of the following structures, or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof:

36. Use of the nucleotide analogue of any one of claims 1-12, and / or the double-stranded oligonucleotide of any one of claims 13-19, and / or the double-stranded oligonucleotide conjugate of any one of claims 20-25, and / or the composition of claim 26, and / or the pharmaceutical composition of claim 27, and / or the nucleoside analogue of any one of claims 28-35 in the preparation of a medicament for treating and / or preventing a disease or condition associated with dysregulated mRNA levels of specific gene expression.

37. A method for regulating the expression of a specific gene in a target cell, characterized in that: The method includes: The double-stranded oligonucleotide according to any one of claims 13 to 19, and / or the double-stranded oligonucleotide conjugate according to any one of claims 20 to 25, and / or the composition according to claim 26, and / or the pharmaceutical composition according to claim 27 are contacted with the target cell.

38. A method for preventing and / or treating a disease or condition associated with dysregulated mRNA levels expressed by a specific gene in a target cell in a subject, characterized in that: The method includes: Administering a pharmaceutically acceptable dose of the double-stranded oligonucleotide according to any one of claims 13 to 19, and / or the double-stranded oligonucleotide conjugate according to any one of claims 20 to 25, and / or the composition according to claim 26, and / or the pharmaceutical composition according to claim 27 to a subject.

39. A kit comprising the double-stranded oligonucleotide according to any one of claims 13 to 19, and / or the double-stranded oligonucleotide conjugate according to any one of claims 20 to 25, and / or the composition according to claim 26, and / or the pharmaceutical composition according to claim 27.

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