Modified nucleotide monomers and uses thereof

Abstract

The invention relates to a modified nucleotide monomer compound shown as a formula Ia or a formula Ib, siRNA containing modified nucleotide, a pharmaceutical composition, siRNA conjugate and application of a modified nucleotide monomer in siRNA pharmaceutical molecules. After siRNA is introduced into the modified nucleotide monomer, the silence efficiency of siRNA on target gene mRNA can be remarkably improved. The druggability of siRNA can be improved, and related diseases caused by target gene mRNA can be more effectively treated. # imgabs0 #

Description

Technical Field

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

[0002] In recent years, considerable progress has been made in the development of siRNA drugs. siRNA as an active ingredient in drugs is already known to the public. The corresponding siRNA mechanism of action is an RNA interference method mediated by a characteristic endonuclease complex. From a genetic level, siRNA can treat almost all diseases, including tumors, infectious diseases, genetic diseases, etc., so siRNA has become the hottest field of biomedical research. In theory, siRNA therapy can target target genes in any pathogenic genome, has a strong and lasting therapeutic effect, can significantly reduce the frequency of dosing, and improve patient compliance with medication, thereby achieving better therapeutic effects.

[0003] However, due to the unique physical and chemical properties of oligonucleic acid substances, siRNA therapy has not been rapidly developed in clinical practice. Its in vivo stability and very limited bioavailability have become the main obstacles to the development of new oligonucleic acid drugs.

[0004] In order to comprehensively improve the activity, in vivo stability and pharmacokinetic properties of siRNA and enhance its bioavailability, technicians in this field often choose to chemically modify specific nucleotides in siRNA to improve the physicochemical properties of siRNA and enhance its in vivo bioavailability, thereby enhancing the silencing efficiency of siRNA on target gene mRNA.

[0005] The present invention synthesizes modified nucleotide monomers by means of medicinal chemistry and introduces them into a special position of siRNA - the first position of the 5'-end of the sense strand, thereby improving the efficacy of siRNA. Therefore, the present invention has a very important value for the development of siRNA drugs. Summary of the invention

[0006] To solve the above problems, the present invention aims to solve the above problems or provide an alternative drug development and treatment method.

[0007] In one aspect, the present invention provides a nucleotide monomer compound represented by Formula Ia or Formula Ib, or a deuterated compound thereof, or a fluorinated compound thereof, or a usable salt thereof:

[0008]

[0009] in,

[0010] X is O or S;

[0011] R1 It has the structure shown in formula II:

[0012]

[0013] in,

[0014] R 2 , R 3 are independently selected from hydrogen, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C2-C6 alkenyl or substituted or unsubstituted C2-C6 alkynyl;

[0015] R 4 Selected from C1-C6 alkyl, cyanoethyl, cyanopropyl or cyanobutyl;

[0016] Z 1 , Z 2 , Z 3 , Z 4 , J are independently selected from hydrogen, halogen, C1-C6 alkyl, C1-C6 alkoxy, -O-(C1-C6 alkylene)-O-(C1-C6 alkyl), -O-(C1-C6 alkylene)-C(O)NR Z1 R Z2 ;

[0017] Or, J and Z 1 or Z 2 connected to each other, and together with the ring in which they are located, form a bridged heterocycle;

[0018] R Z1 , R Z2 Each is independently selected from hydrogen, C1-C6 alkyl, and cycloalkyl;

[0019] Bx is selected from a heterocyclic base;

[0020] Y 3 Selected from substituted or unsubstituted C1-C6 alkylene, substituted or unsubstituted C2-C6 alkenylene, substituted or unsubstituted C2-C6 alkynylene, substituted or unsubstituted 3-8 membered carbocyclic ring, substituted or unsubstituted 4-8 membered heterocyclic ring; wherein one or more methylene groups in the alkylene, alkenylene and alkynylene are optionally substituted by heteroatoms; preferably, the heteroatoms are O, S or NH;

[0021] Y is selected from -OR Y1 、-SR Y1 or -NR Y1 R Y2 ;

[0022] R Y1 , R Y2are independently selected from hydrogen, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, -(C0-C6 alkylene)-(3-10 membered carbocyclic ring), -(C0-C6 alkylene)-(4-10 membered heterocyclic ring), -(C0-C6 alkylene)-(5-10 membered bridged ring), -(C0-C6 alkylene)-(5-10 membered bridged heterocyclic ring), -(C0-C6 alkylene)-(5-10 membered spirocyclic ring), -(C0-C6 alkylene)-(5-10 membered spiroheterocyclic ring), -(C0-C6 alkylene)-(6-10 membered aromatic ring), -(C0-C6 alkylene)-(5-10 membered aromatic heterocyclic ring); wherein the alkylene, alkyl, alkenyl, alkynyl, carbocyclic ring, heterocyclic ring, bridged ring, bridged heterocyclic ring, spirocyclic ring, spiroheterocyclic ring, aromatic ring, aromatic heterocyclic ring are optionally substituted by one or more P;

[0023] Or, R Y1 , R Y2 The nitrogen atom connected thereto together forms a 4- to 10-membered heterocycle, a 5- to 10-membered bridged heterocycle, a 5- to 10-membered spiro heterocycle, or a 5- to 10-membered aromatic heterocycle; wherein the heterocycle, bridged heterocycle, spiro heterocycle, or aromatic heterocycle is optionally substituted by one or more P;

[0024] Each P is independently selected from hydrogen, C1-C3 alkyl, and 3-10 membered carbon ring.

[0025] Further,

[0026] The heterocyclic base is selected from conventional bases, or unconventional nucleoside bases and their derivatives;

[0027] The conventional nucleoside base is selected from adenine, guanine, cytosine, uracil or thymine;

[0028] The unconventional base is selected from hypoxanthine, xanthine, and 2-thiouracil;

[0029] The derivatives include halides, thiols, methylations and their metabolites.

[0030] Furthermore, the heterocyclic base is selected from:

[0031]

[0032] Wherein, PG is selected from hydrogen or a hydroxyl / amine protecting group; preferably, the hydroxyl / amine protecting group is selected from an acyl group, a benzyl group or an alkylsilyl group.

[0033] In some embodiments of the present invention, Y is selected from the following structures:

[0034]

[0035] in,

[0036] The methylene group in the above structure is optionally replaced by a heteroatom; preferably, the heteroatom is O, S or NH;

[0037] P 1 , P 2 , P 3 , P 4 are independently selected from hydrogen, C1-C3 alkyl, cycloalkyl; preferably, P 1 , P 2 , P 3 , P 4 All are hydrogen.

[0038] In some embodiments of the present invention, further, Y is selected from the following structures:

[0039]

[0040] In some embodiments of the present invention, Y 3 Selected from methylene.

[0041] In some embodiments of the present invention, X is O.

[0042] In some embodiments of the present invention, Z 1 is selected from methoxy or -OCH2CH2OCH3; Z 2 , Z 3 , Z 4 , J is hydrogen.

[0043] In some embodiments of the present invention, R 2 , R 3 are independently selected from isopropyl or isobutyl; R 4 Selected from cyanoethyl or cyanopropyl.

[0044] In some embodiments of the present invention, specifically, R 1 for

[0045] In some specific embodiments of the present invention, the compound of formula Ia or formula Ib is specifically:

[0046]

[0047]

[0048] Wherein, PG is selected from hydrogen or a hydroxyl / amine protecting group; preferably, the hydroxyl / amine protecting group is selected from an alkyl acyl group, an alkoxy acyl group, an aryl acyl group, and an aryloxy acyl group.

[0049] The present invention also provides the use of any of the above monomeric compounds as an intermediate in the preparation of an oligonucleotide; preferably, the oligonucleotide is siRNA.

[0050] Furthermore, the present invention provides the use of any of the above monomers as an intermediate in the preparation of the first nucleotide at the 5'-terminus of the sense strand of siRNA.

[0051] In another aspect, the present invention provides an siRNA comprising a sense strand and an antisense strand; the sense strand and the antisense strand each comprise 15 to 45 modified or unmodified nucleotides, and the sense strand and the antisense strand are partially complementary to form a double-stranded region; wherein the first nucleotide at the 5' end of the sense strand has a structure shown in Formula Va or Formula Vb, and Site covalently linked to the rest of the siRNA:

[0052]

[0053] in,

[0054] X is O or S;

[0055] Z 1 , Z 2 , Z 3 , Z 4 , J are independently selected from hydrogen, halogen, C1-C6 alkyl, C1-C6 alkoxy, -O-(C1-C6 alkylene)-O-(C1-C6 alkyl), -O-(C1-C6 alkylene)-C(O)NR Z1 R Z2 ;

[0056] Or, J and Z 1 or Z 2 connected to each other, and together with the ring in which they are located, form a bridged heterocycle;

[0057] R Z1 , R Z2 Each is independently selected from hydrogen, C1-C6 alkyl, and cycloalkyl;

[0058] Bx is selected from a heterocyclic base;

[0059] Y 3 Selected from substituted or unsubstituted C1-C6 alkylene, substituted or unsubstituted C2-C6 alkenylene, substituted or unsubstituted C2-C6 alkynylene, substituted or unsubstituted 3-8 membered carbocyclic ring, substituted or unsubstituted 4-8 membered heterocyclic ring; wherein one or more methylene groups in the alkylene, alkenylene and alkynylene are optionally substituted by heteroatoms; preferably, the heteroatoms are O, S or NH;

[0060] Y is selected from -OR Y1 、-SR Y1 or -NR Y1 R Y2 ;

[0061] RY1 , R Y2 are independently selected from hydrogen, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, -(C0-C6 alkylene)-(3-10 membered carbocyclic ring), -(C0-C6 alkylene)-(4-10 membered heterocyclic ring), -(C0-C6 alkylene)-(5-10 membered bridged ring), -(C0-C6 alkylene)-(5-10 membered bridged heterocyclic ring), -(C0-C6 alkylene)-(5-10 membered spirocyclic ring), -(C0-C6 alkylene)-(5-10 membered spiroheterocyclic ring), -(C0-C6 alkylene)-(6-10 membered aromatic ring), -(C0-C6 alkylene)-(5-10 membered aromatic heterocyclic ring); wherein the alkylene, alkyl, alkenyl, alkynyl, carbocyclic ring, heterocyclic ring, bridged ring, bridged heterocyclic ring, spirocyclic ring, spiroheterocyclic ring, aromatic ring, aromatic heterocyclic ring are optionally substituted by one or more P;

[0062] Or, R Y1 , R Y2 The nitrogen atom connected thereto together forms a 4- to 10-membered heterocycle, a 5- to 10-membered bridged heterocycle, a 5- to 10-membered spiro heterocycle, or a 5- to 10-membered aromatic heterocycle; wherein the heterocycle, bridged heterocycle, spiro heterocycle, or aromatic heterocycle is optionally substituted by one or more P;

[0063] Each P is independently selected from hydrogen, C1-C3 alkyl, 3-10 membered carbocyclic ring;

[0064] Y 4 Selected from O or S;

[0065] Y 5 Selected from -OH, -O(CH3), -SH, -S(CH3) or -N(CH3)2.

[0066] Further,

[0067] The heterocyclic base is selected from conventional bases, or unconventional nucleoside bases and their derivatives;

[0068] The conventional nucleoside base is selected from adenine, guanine, cytosine, uracil or thymine;

[0069] The unconventional base is selected from hypoxanthine, xanthine, and 2-thiouracil;

[0070] The derivatives include halides, thiols, methylations and their metabolites.

[0071] Furthermore, the heterocyclic base is selected from:

[0072]

[0073] Wherein, PG is selected from hydrogen or a hydroxyl / amine protecting group; preferably, the hydroxyl / amine protecting group is selected from an acyl group, a benzyl group or an alkylsilyl group.

[0074] In some embodiments of the present invention, Y is selected from the following structures:

[0075]

[0076] in,

[0077] The methylene group in the above structure is optionally replaced by a heteroatom; preferably, the heteroatom is O, S or NH;

[0078] P 1 , P 2 , P 3 , P 4 are independently selected from hydrogen, C1-C3 alkyl, cycloalkyl; preferably, P 1 , P 2 , P 3 , P 4 All are hydrogen.

[0079] In some embodiments of the present invention, further, Y is selected from the following structures:

[0080]

[0081] In some embodiments of the present invention, Y 3 Selected from methylene.

[0082] In some embodiments of the present invention, X is O; Y 4 S; Y 5 It is -OH.

[0083] In some embodiments of the present invention, Z 1 is selected from methoxy or -OCH2CH2OCH3; Z 2 , Z 3 , Z 4 , J is hydrogen. In some specific embodiments of the present invention, the structure represented by formula Va or formula Vb in the siRNA is specifically:

[0084]

[0085]

[0086] Wherein, PG is selected from hydrogen or a hydroxyl / amine protecting group; preferably, the hydroxyl / amine protecting group is selected from an alkyl acyl group, an alkoxy acyl group, an aryl acyl group, and an aryloxy acyl group.

[0087] The present invention also provides a pharmaceutical composition comprising any of the above siRNAs and a pharmaceutically acceptable carrier.

[0088] The present invention also provides a siRNA conjugate, which is formed by conjugating any of the above siRNAs and a conjugated molecule.

[0089] The present invention also provides a kit comprising any of the above siRNAs, and / or the above pharmaceutical compositions and / or the above siRNA conjugates.

[0090] The present invention also provides the use of any of the above siRNAs, and / or the above pharmaceutical compositions, and / or the above siRNA conjugates, and / or the above kits in the preparation of drugs.

[0091] In another aspect, the present invention also provides a method for inhibiting the expression level of a target gene in a cell, the method comprising contacting an effective amount of the siRNA, pharmaceutical composition and / or siRNA conjugate of the present invention with the cell.

[0092] The modified nucleotides provided by the present invention can improve the efficiency of silencing the target gene for siRNA drug molecules. The inventor unexpectedly found that the 4'\5' position of the ribose and the ribose base of the nucleoside monomer are modified, and the siRNA containing the modified nucleotides prepared from the modified nucleoside monomer has high bioavailability, better inhibitory activity on the mRNA expressed by the target gene, and lower toxicity.

[0093] The modified nucleotides of the present invention can improve the drugability of siRNA drug molecules. In some embodiments, the modified nucleotides of the present invention can enhance the effect of siRNA in degrading target genes. Therefore, the modified nucleotides of the present invention can enhance the pharmacodynamic effect of siRNA silencing.

[0094] The siRNA conjugate provided by the present invention is formed by conjugating siRNA and a conjugation molecule, and comprises the siRNA of the present invention and a conjugation group conjugated to the siRNA, wherein the conjugation group comprises a linker and a pharmaceutically acceptable targeting group and / or a delivery auxiliary group, and the siRNA, the linker and the targeting group or the delivery auxiliary group are sequentially covalently or non-covalently connected, each of the targeting groups is selected from a ligand capable of binding to a cell surface receptor, and each of the delivery auxiliary groups is selected from a group capable of increasing the biocompatibility of the siRNA conjugate in the target organ or tissue for delivery.

[0095] In some embodiments, the siRNA drug comprising the modified nucleotides of the present invention is double-stranded. With respect to double-stranded siRNA drugs, the length of the sense and antisense strands of the siRNA drug is independently 15-45 nucleotides. In some embodiments, the double-stranded siRNA drug comprises a sense strand and an antisense strand that are at least partially complementary to each other (at least 70% complementary). The antisense strand comprises a region having a sequence that is perfectly complementary (100% complementary) or at least partially complementary (at least 85% complementary) to a sequence in the target mRNA. The sense and antisense strands can be the same length or different lengths.

[0096] In some embodiments, the sense strand is about 19 nucleotides in length, while the antisense strand is about 21 nucleotides in length. In some embodiments, the sense strand is about 21 nucleotides in length, while the antisense strand is about 23 nucleotides in length. The perfect or partially complementary region between the sense strand and the antisense strand is typically 15-25 nucleotides in length and is located at or near the 5'-end of the antisense strand (e.g., the region is 1, 2, 3, or 4 nucleotides away from the 5'-end of the antisense strand, which is imperfect or partially complementary).

[0097] For double-stranded siRNA drugs, if there are other sense strand nucleotides, they can be identical or different from the corresponding sequence in the target mRNA. If there are other antisense strand nucleotides, they can be complementary or non-complementary to other nucleotides (if present) of the corresponding sense strand.

[0098] In some embodiments, the sense strand and antisense strand of the double-stranded siRNA drug comprising the modified nucleotides of the present invention contain the same number of nucleotides. In some embodiments, the sense strand and antisense strand of the siRNA drug of the present invention contain different numbers of nucleotides.

[0099] In some embodiments, both the sense and antisense strands contain 1 to 4 phosphorothioate linkages.

[0100] In some embodiments, in the siRNA of the present invention, each nucleotide is a modified or unmodified nucleotide. In the context of the present disclosure, the term "modified nucleotide" used refers to a nucleotide or nucleotide analog formed by replacing the 2' hydroxyl group of the ribose group of the nucleotide with other groups, or a nucleotide in which the base on the nucleotide is a modified base. The modified nucleotide will not cause the function of siRNA to regulate gene expression to be significantly weakened or lost. For example, the modified nucleotide disclosed in JK Watts, GF Deleavey, and MJ Damha, Chemically modified siRNA: tools and applications. Drug Discov. Today, 2008, 13 (19-20): 842-55 can be selected.

[0101] In some embodiments, the siRNA drug comprising the modified nucleotide inhibits the expression of the target mRNA in cells, tissues or in vivo. In some embodiments, a therapeutically effective amount of the siRNA drug comprising the modified nucleotide of the present invention is administered into the body, thereby inhibiting the expression of the target mRNA in the body.

[0102] In some embodiments, the siRNA drug is used to treat, prevent or control clinical manifestations related to the expression of the target mRNA. In some embodiments, a therapeutically or prophylactically effective amount of one or more siRNA drugs is administered to a subject in need of such treatment, prevention or control.

[0103] The siRNA medicine comprising the modified nucleotides of the present invention and the composition comprising the siRNA medicine of the present invention can be delivered to cells, tumors, tissues or objects using oligonucleotide delivery techniques known in the art. In general, any method suitable for delivering nucleic acid molecules (in vitro or in vivo) in the art can be applied to the siRNA medicine comprising one or more modified nucleotides of the present invention.

[0104] In some embodiments where the siRNA drug is double-stranded, a targeting ligand or targeting group, a linking group or a delivery vehicle is covalently linked to the sense strand to form a conjugate. In some embodiments, the targeting ligand, linking group and / or delivery vehicle is directly or indirectly linked to the 3' or 5' end of the sense strand via a linker.

[0105] In some embodiments, the targeting ligand or targeting group contains two to four terminal N-acetylgalactosamines (GalNAc), which are linked to a siRNA drug comprising one or more of the modified nucleotides. It is known that the triantennary (GalNAc) binds to ASGPR with greater affinity than the diantennary or monoantennary structures (Connolly et al., J. Biol. Chem., 1982, 257, 939-945).

[0106] Definitions of terms used in the present invention: Unless otherwise stated, the initial definitions provided for groups or terms in this document apply to the groups or terms throughout the specification; for terms that are not specifically defined in this document, the meaning that a person skilled in the art can give them should be given based on the disclosure and context.

[0107] In the present invention, unless otherwise specified, capital letters C, G, U, A, and T represent the base composition of nucleotides, including modified and unmodified nucleotides; m represents that a nucleotide adjacent to the right side of the mark m is a 2'-methoxy nucleotide; f represents that a nucleotide adjacent to the right side of the mark f is a 2'-fluoro nucleotide; d represents that a nucleotide adjacent to the right side of the mark d is a 2'-deoxy nucleotide; gn represents that a nucleotide adjacent to the right side of the mark gn is a glycerol nucleotide (GNA); the mark * represents that the two nucleotides adjacent to the left and right of the mark * (or between the nucleotide and the linker-targeting ligand part) are connected by thiophosphate groups; eVP represents that a nucleotide adjacent to the right side is a (E)-vinyl phosphate-modified nucleotide; and iab represents a reverse abasic residue.

[0108] In the present invention, "oligonucleotide" refers to a polymer of linked nucleosides, each of which may be independently modified or unmodified, and comprises an oligonucleotide sequence of about 10-50 single-stranded nucleotides or double-stranded nucleotide base pairs. In some embodiments, the oligonucleotide has a core base sequence that is at least partially complementary to a target gene core sequence expressed in a cell. In some embodiments, the oligonucleotide can regulate the expression of the corresponding target gene after being delivered to a cell expressing the gene. Target gene expression can be regulated in vitro or in vivo. "Oligonucleotide" includes, but is not limited to: single-stranded antisense oligonucleotides, short interfering RNA (siRNA), double-stranded RNA (dsRNA), micro RNA (miRNA), short hairpin RNA (shRNA), ribozymes, interfering RNA molecules, and dicer enzyme substrates.

[0109] In the present invention, "siRNA" refers to an oligonucleotide molecule containing RNA or RNA-like (eg, chemically modified RNA) that can reduce or inhibit the translation of messenger RNA (mRNA) in a sequence-specific manner.

[0110] siRNA can act through RNA interference mechanism (for example, by inducing mRNA degradation through interaction with the mRNA interference pathway mechanism of mammalian cells (RNA induced silencing complex RISC)), or other arbitrary mechanisms or pathways. Although it is believed that the term siRNA drug used in the present invention mainly acts through the RNA interference mechanism, the siRNA drug is not limited to or confined to any specific pathway or mechanism of action. siRNA drugs include, but are not limited to: single-stranded antisense oligonucleotides, short interfering RNA (siRNA), double-stranded RNA (dsRNA), microRNA (miRNA), short hairpin RNA (shRNA) and dicer enzyme substrates. The siRNA drug of the present invention is composed of an oligonucleotide chain that is at least partially complementary to the mRNA as a target. In some embodiments, the siRNA drug of the present invention is double-stranded and consists of an antisense chain and a sense chain that is at least partially complementary to the antisense chain.

[0111] The terms "silencing", "reducing", "inhibiting", "down-regulating", or "knockdown" refer to the expression of a given gene, when the siRNA drug molecules described in the present invention are used to treat cells, tissues, organs or animals for direct administration, compared with cells, tissues, organs or animals that have not been treated in this way, The level of expression of the gene is reduced or decreased.

[0112] The term "sequence" or "nucleotide sequence" refers to the order or sequence of nucleobases or nucleotides, expressed in alphabetical order using standard nucleotide nomenclature.

[0113] The term "heterocyclic base" is a nucleobase or modified nucleobase as defined herein. In some embodiments, the heterocyclic base moiety is a pyrimidine, a substituted pyrimidine, a purine or a substituted purine and a nitrogen-containing heterocycle. In some embodiments, the heterocyclic base moiety is an unconventional purine or a substituted purine. In some embodiments, the heterocyclic base moiety is an unconventional pyrimidine or a substituted pyrimidine. In some embodiments, the heterocyclic base moiety contains a non-natural five-membered nitrogen-containing heterocycle. In some embodiments, the heterocyclic base moiety may include one or more protecting groups.

[0114] In the present invention, "nucleotide base" or "nucleobase" is a heterocyclic pyrimidine or purine compound, wherein the standard components of conventional nucleic acids include the bases of conventional nucleotides of adenine (A), guanine (G), cytosine (C), thymine (T) and uracil (U). Nucleobases can be modified or replaced by novel unconventional nucleobases. Nucleobases include naturally occurring nucleotide bases and non-naturally occurring nucleotide bases. It should be obvious to those skilled in the art that various nucleotide bases that were previously considered to be "non-naturally occurring" have been found in nature later. Thus, "nucleotide bases" include not only known purine and pyrimidine heterocycles, but also heterocyclic analogs and tautomers thereof. Exemplary nucleotide bases include adenine, guanine, thymine, cytosine, uracil, purine, xanthine, diaminopurine, 8-oxo-N6-methyladenine, 7-diazaxanthine, 7-diazaguanine, N4, N4-ethanocytosine, N6, N6-ethano-2,6-diaminopurine, 5-methylcytosine, 5-(C3-C6)-alkynylcytosine, 5-fluorouracil, 5-bromouracil, pseudoisocytosine, 2-hydroxy-5-methyl-4-triazolopyridine, isocytosine, isoguanine, inosine, and the "non-naturally occurring" nucleotide bases described in U.S. Pat. No. 5,432,272 to Benner et al. The term "nucleotide base" includes each and all of these examples and their analogs and tautomers. Particularly important nucleotide bases are adenine, guanine, thymine, cytosine and uracil, which are considered to be the naturally occurring nucleotide bases relevant to human therapeutic and diagnostic applications.

[0115] The term "complementary" is used to describe the relationship between a first nucleotide sequence (e.g., a siRNA drug sense strand or a target mRNA) and a second nucleotide sequence (e.g., a single-stranded antisense oligonucleotide or a double-stranded siRNA drug antisense strand), and refers to the ability of an oligonucleotide or polynucleotides comprising a first nucleotide sequence to hybridize with an oligonucleotide or polynucleotides comprising a second nucleotide sequence under certain conditions (under mammalian physiological conditions or similar in vitro conditions) to form base pairing and form a duplex or double helix structure. Complementary sequences contain Watson-Crick base pairing or non-Watson-Crick base pairing, and contain natural or modified nucleotides or nucleotide analogs to at least the extent that they meet the above hybridization requirements. For example, for the purpose of determining identity or complementarity, monomers a and Af are complementary to U (or T) and are equivalent to A.

[0116] The term "sense strand" refers to the strand on the RNA molecule that carries the nucleotide sequence encoding protein amino acid information, which is called the sense strand, also known as the coding strand, sense strand or positive strand, and the other nucleotide sequence that is complementary to it is the antisense strand.

[0117] The term "antisense strand" refers to a nucleotide sequence in mRNA expressed by a target gene that is substantially reverse complementary or essentially reverse complementary to the antisense strand and has the same length as the antisense strand.

[0118] The term "optional" or "optionally" means that the subsequently described event or circumstance may but need not occur, and the description includes instances where the event or circumstance occurs or does not occur. For example, "a heterocyclic group optionally substituted with an alkyl group" means that the alkyl group may but need not be present, and the description includes instances where the heterocyclic group is substituted with an alkyl group and instances where the heterocyclic group is not substituted with an alkyl group.

[0119] The term "substituted" means that one or more hydrogen atoms, preferably up to 5, more preferably 1 to 3 hydrogen atoms in a group are replaced independently of each other by a corresponding number of substituents. It goes without saying that the substituents are only in their possible chemical positions, and those skilled in the art can determine (by experiment or theory) possible or impossible substitutions by means of the prior art and experimental conditions. For example, an amino or hydroxyl group with free hydrogen may be unstable when combined with a carbon atom with an unsaturated (e.g. olefinic) bond.

[0120] The minimum and maximum carbon atom content in a hydrocarbon group is indicated by a prefix, for example, the prefix C a～ C b Alkyl refers to any alkyl group containing from "a" to "b" carbon atoms. Thus, for example, C 1～6 The alkyl group refers to a straight chain or branched chain alkyl group containing 1 to 6 carbon atoms.

[0121] Alkyl refers to a straight or branched hydrocarbon group in an alkane molecule, such as methyl-CH3, ethyl-CH2CH3, methylene-CH2-; the alkyl group may also be part of other groups, such as C1-C6 alkoxy, C1-C6 alkylamino. Non-limiting examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, n-hexyl, etc. The alkyl group may be substituted or unsubstituted, and when substituted, the substituent may be substituted at any available connection point, and the substituent is preferably one or more of the following groups, which are independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkyloxy, heterocycloalkyloxy, cycloalkylthio, heterocycloalkylthio, oxy, carboxyl or carboxylate.

[0122] "Alkenyl" refers to a straight or branched chain hydrocarbon group having at least 2 carbon atoms and at least one site of vinyl unsaturation (>C=C<). a-b Alkenyl refers to an alkenyl group having from a to b carbon atoms and is intended to include, for example, ethenyl, propenyl, isopropenyl, 1,3-butadienyl, and the like.

[0123] "Alkynyl" refers to a straight chain monovalent hydrocarbon radical or a branched monovalent hydrocarbon radical containing at least one triple bond. The term "alkynyl" is also intended to include those hydrocarbon radicals having one triple bond and one double bond. For example, C 2-6 Alkynyl is meant to include ethynyl, propynyl, and the like.

[0124] "Alkoxy" refers to-O-(alkyl) or-O-(cycloalkyl), wherein the definition of alkyl is as described above. Non-limiting examples of alkoxy include: methoxy, ethoxy, propoxy, butoxy, cyclopropyloxy, cyclobutyloxy, cyclopentyloxy, cyclohexyloxy. Alkoxy can be optionally substituted or unsubstituted, and when substituted, substituents are preferably one or more of the following groups, which are independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, sulfhydryl, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkyloxy, heterocycloalkyloxy, cycloalkylthio, heterocycloalkylthio, carboxyl or carboxylate.

[0125] "Halogen" is fluorine, chlorine, bromine or iodine.

[0126] "Halo-substituted alkyl" refers to an alkyl group substituted with one or more halogens, wherein alkyl is as defined above.

[0127] "Ester" refers to a -C(O)O(alkyl) or -C(O)O(cycloalkyl) group wherein alkyl and cycloalkyl are as defined above.

[0128] "Acyl" refers to a compound containing a -C(O)R group, where R is alkyl, cycloalkyl, heterocyclyl, aryl or heteroaryl.

[0129] "Heteroatoms" Heteroatoms refer to atoms other than carbon and hydrogen in organic compounds. Generally, they refer to atoms that replace carbon in the molecular skeleton (especially the ring system). Nitrogen, oxygen, sulfur, phosphorus, boron, chlorine, bromine, iodine, etc. are all common heteroatoms. If there is a ring containing heteroatoms in an organic compound, it is called a heterocyclic compound.

[0130] In the present invention, "protecting group" refers to an unstable chemical moiety known in the art for preventing reactive groups (e.g., hydroxyl, amino, carboxyl, and sulfhydryl) from reacting undesirably during synthesis. Protecting groups are typically used selectively and / or orthogonally to protect sites at other reactive sites in the reaction, and are subsequently removed to release the unprotected group, enabling it to be used in further reactions. In some embodiments, a "substituted" group or substituent comprises a protecting group.

[0131] Representative hydroxyl protecting groups in the present invention are disclosed in Beaucage et al., Tetrahedron 1992, 48, 2223-2311, and Greene and Wuts, Protective Groups in Organic Synthesis, Chapter 2, 2d ed, John Wiley & Sons, New York, 1991, each of which is incorporated herein by reference in its entirety. In some embodiments, the protecting group is stable under alkaline conditions, but can be removed under acidic conditions. In some embodiments, non-exclusive examples of hydroxyl protecting groups that can be used in the present invention include dimethoxytrityl (DMT), monomethoxytrityl, 9-phenyloxanthene-9-yl (Pixyl) and 9-(p-methoxyphenyl)oxanthene-9-yl (Mox). In some embodiments, non-exclusive examples of hydroxy protecting groups that may be used in the present invention include Tr (trityl), MMTr (4-methoxytrityl), DMTr (4,4'-dimethoxytrityl), and TMTr (4,4',4"-trimethoxytrityl).

[0132] The compounds and compositions described herein may have certain atoms (e.g., N, O, or S atoms) in a protonated or deprotonated state, depending on the environment in which the compound or composition is located. Thus, as used herein, the structures described herein take into account that certain functional groups, such as OH, SH, or NH, may be protonated or deprotonated. The disclosure of the present invention is intended to encompass the above-described compounds and compositions, regardless of their protonation state based on the ambient pH, as will be readily appreciated by those of ordinary skill in the art.

[0133] The terms "salts" and "usable salts" refer to acidic and / or basic salts of the above-mentioned compounds or their stereoisomers, formed with inorganic and / or organic acids and bases, and also include zwitterionic salts (inner salts), and also include quaternary ammonium salts, such as alkylammonium salts. These salts can be obtained directly in the final separation and purification of the compounds. They can also be obtained by mixing the above-mentioned compounds, or their stereoisomers, with a certain amount of acid or base appropriately (e.g., equivalent amounts). These salts may be precipitated in the solution and collected by filtration, or recovered after solvent evaporation, or obtained by freeze drying after reaction in an aqueous medium.

[0134] The above contents of the present invention are further described in detail below through specific implementation methods in the form of embodiments. However, this should not be understood as the scope of the above subject matter of the present invention being limited to the following examples. All technologies realized based on the above contents of the present invention belong to the scope of the present invention. Specific implementation plan

[0135] Obviously, according to the above contents of the present invention, in accordance with common technical knowledge and customary means in the art, without departing from the above basic technical ideas of the present invention, other various forms of modification, replacement or change may be made.

[0136] The above contents of the present invention are further described in detail below through specific implementation methods in the form of embodiments. However, this should not be understood as the scope of the above subject matter of the present invention being limited to the following examples. All technologies realized based on the above contents of the present invention belong to the scope of the present invention.

[0137] The raw materials and equipment used in the present invention are all known products, which are obtained by purchasing commercially available products.

[0138] The compounds disclosed in the present invention can be prepared by the following synthetic methods. BRIEF DESCRIPTION OF THE DRAWINGS

[0139] Figure 1 This is the MS spectrum of monomer XY-013-3;

[0140] Figure 2 This is the 3H-NMR spectrum of monomer XY-013;

[0141] Figure 3 This is the monomer XY-013-3P-NMR spectrum;

[0142] Figure 4 This is the MS spectrum of monomer XY-013-5;

[0143] Figure 5 This is the 5H-NMR spectrum of monomer XY-013;

[0144] Figure 6 This is the monomer XY-013-5P-NMR spectrum;

[0145] Figure 7 This is the MS spectrum of monomer XY-014-1;

[0146] Figure 8 This is the 1H-NMR spectrum of monomer XY-014;

[0147] Fig. 9 This is the P-NMR spectrum of monomer XY-014-1;

[0148] Fig.10 A schematic diagram of the solid phase synthesis route for synthesizing siRNA sequences using a solid phase carrier;

[0149] Fig.11 The structural diagram of the solid phase support (L96-PS) modified with GalNAc (L96);

[0150] Fig.12The figure is a flow chart of the synthesis process of siRNA sequences using solid phase carriers.

[0151] Example

[0152] Hereinafter, in order to help understand the present disclosure, exemplary embodiments will be provided. However, the following embodiments are provided only to make it easier to understand the present disclosure and are not intended to limit the present disclosure.

[0153] Unless otherwise specified, the materials and reagents used in the examples are commercially available. The nucleic acid electrophoresis, real-time PCR and other operations used are all performed according to conventional protocols. For example, they can be performed according to the procedures described in Molecular Cloning (Cold Spring Harbor Laboratory Press (1989)).

[0154] Example 1. Synthesis of (2R,3R,4R,5R)-2-((adamantane-1-methyl)(methyl)amino)methyl)-5-(4-isobutylamino-2-oxopyrimidin-1(2H)-yl)-4-methoxytetrahydrofuran-3-yl(2-cyanoethyl)diisopropylphosphoramide

[0155]

[0156] Step 1. Synthesis of compound XY-013-3-6

[0157] Into a dry 250mL single-necked bottle, add the crude substrate XY-013-1-5 (4.0g, 9.10mmol), add anhydrous methanol (40mL) to dissolve, then add 1-adamantanemethane (1.96g, 11.83mmol) and acetic acid (545.98mg, 9.10mmol) at 0°C under nitrogen protection. After the addition, keep the reaction system stirring at 0°C for 0.5h, then add sodium cyanoborohydride (1.14g, 18.20mmol). After the addition, keep the reaction system stirring at room temperature for 16h. Then saturated sodium bicarbonate (100 mL) was added at 0°C, extracted with dichloromethane (100 mL*3), and washed with saturated brine (100 mL). The organic phase was dried over anhydrous Na2SO4, filtered, and concentrated. The residue was purified by column chromatography (petroleum ether: ethyl acetate = 3:1) and concentrated to give the product XY-013-3-6 (1.94 g, 36.21%), LCMS (ESI) m / z: 589.4 [M+H] + .

[0158] Step 2. Synthesis of compound XY-013-3-7

[0159] In a dry 100mL single-necked bottle, add the crude substrate XY-013-3-6 (1.94g, 3.29mmol), add anhydrous methanol (15mL) to dissolve, then add formaldehyde aqueous solution (37%, 401.2mg) and acetic acid (197.67mg, 3.29mmol) at 0°C under nitrogen protection. After the addition, keep the reaction system stirring at 0°C for 0.5h, then add sodium cyanoborohydride (414.06mg, 6.59mmol), after the addition, keep the reaction system stirring at room temperature for 16h. Then saturated sodium bicarbonate (50 mL) was added at 0°C, extracted with dichloromethane (50 mL*3), and washed with saturated brine (100 mL). The organic phase was dried over anhydrous Na2SO4, filtered, and concentrated. The residue was purified by column chromatography (dichloromethane) and concentrated to give the product XY-013-3-7 (1.98 g, 99.69%), LCMS (ESI) m / z: 603.7 [M+H] + .

[0160] Step 3. Synthesis of compound XY-013-3-8

[0161] Substrate 7 (1.98 g, 3.28 mmol) was added to a dry 50 mL single-mouth bottle, tetrahydrofuran (36 mL) was added to dissolve, and then triethylamine (479.56 mg, 4.93 mmol) and triethylamine hydrofluoride (3.70 g, 22.99 mmol) were added at room temperature. After the addition was completed, the reaction system was stirred at room temperature for 16 h. After concentration, the residue was purified by C18 (33% ACN in 0.05% TFA / H2O) and concentrated to obtain the product XY-013-3-8 (1.3 g, 81.01%), 1 H NMR (400MHz, Methanol-d4) δ8.06(d,J=7.5Hz,1H),7.50(d,J=7.3Hz,1H),5.88(s,1H),4.34(s,1H),4.11(s,1H),4.01(s,1H),3.61(s, 3H),3.35(s,1H),3.16-2.90(m,5H),2.76-2.63(m,1H),2.04(s,3H),1.85-1.63(m,13H),1.22-1.11(m,6H).LCMS(ESI)m / z:489.6[M+H] + .

[0162] Step 4. Synthesis of compound XY-013-3-9

[0163] At room temperature, compound XY-013-3-8 (579 mg, 1.18 mmol) was dissolved in toluene and concentrated (20 mL*3), then dissolved in dry anhydrous acetonitrile (8 mL), and then N, N-diisopropylethylamine (612.58 mg, 4.74 mmol) and 2-cyanoethyl N, N-diisopropylchlorophosphoramidite (560.92 mg, 2.37 mmol) were added under nitrogen protection. After the addition, the reaction system was stirred at room temperature for 1 h under nitrogen protection. Concentration was performed to obtain a crude product, which was purified by C18 (100% ACN in 0.05% NH4HCO3 / H2O), concentrated, and the residue was purified again by column chromatography (petroleum ether: ethyl acetate = 2:1-1:2), and concentrated to obtain the product XY-013-3-9 (335 mg, 41.04%).

[0164] LCMS (ESI) m / z: 689.3 [M+H] +. Figure 1 .

[0165] 1 H NMR (400MHz, DMSO-d6) δ10.83(s,1H),8.00(dd,J=7.5,5.2Hz,1H),7.22(dd,J=7.5,0.8Hz,1H) ,5.80(s,1H),4.07-3.97(m,2H),3.88-3.83(m,1H),3.75-3.47(m,4H),3.36(d,J=11.1Hz,3H) ,2.80-2.58(m,5H),2.23(d,J=6.8Hz,3H),2.09-1.94(m,2H),1.83(d,J=4.1Hz,3H),1.59(d,J =12.2Hz, 3H), 1.50 (d, J = 12.1Hz, 3H), 1.40 (s, 6H), 1.10-1.04 (m, 12H), 0.99 (d, J = 6.8Hz, 6H). See Figure 2 .

[0166] 31 P NMR (162 MHz, DMSO-d6) δ 149.00 (d, J = 127.4 Hz). Figure 3 .

[0167] Example 2. Synthesis of Compound XY-013-5

[0168]

[0169] Synthesis of compound XY-013-5-3 :

[0170] Add cyclohexylmethylmagnesium bromide (0.5M, 150ml) to a dry 250mL single-mouth bottle, add lithium tetrachlorocuprate (0.1M, 5mL) to dissolve, then add substrate XY-013-5-2 (5.0g, 10.4mmol) under nitrogen protection, and keep the reaction system stirring at room temperature for 2.0h. LCMS detected that the raw material completely disappeared and the reaction was over. Then add a few drops of methanol at 0℃, extract with ethyl acetate (200mL*3), and wash with saturated brine (200Ml). The organic phase is dried over anhydrous Na2SO4, filtered, and concentrated to obtain the product XY-014-1-3 (4.0g, 85.3% yield)

[0171] ESI-LCMS: m / z 453.2[M+H] + .. 1 H NMR(400MHz,DMSO-d6)δ11.31(s,1H),7.53-7.48(m,1H),5.67-5.64(m,1H),5.60-5.55(m,1H),3.40-3.94(m,1 H),3.79-3.76(m,1H),3.63-3.57(m,1H),1.62-1.46(m,9H),1.20-1.04(m,6H),0.79(s,9H),0.02-0.00(m,6H).

[0172] Synthesis of compound XY-013-5-4:

[0173] Add crude substrate XY-013-5-3 (4.0 g, 8.85 mmol) to a dry 250 mL single-mouth bottle, add anhydrous acetonitrile (40 mL) to dissolve, then add TEA (1.79 g, 17.7 mmol), DMAP (2.16 g, 17.7 mmol) and TPSCl (5.36 g, 17.7 mmol) at room temperature, after addition, the reaction system is kept stirring at room temperature for 2 hours. LCMS and TLC detection of the raw material completely disappeared, ammonia water (20 mL) was added to the reaction system, after addition, the reaction system was kept stirring at room temperature for 1 hour. LCMS and TLC detection of the raw material completely disappeared. Extract with ethyl acetate (200 mL*3), and wash with saturated brine (200 mL), the organic phase is dried over anhydrous Na2SO4, filtered, and concentrated to obtain the crude product XY-014-1-3 (4.5 g). ESI-LCMS: m / z 452.2 [M+H] + .

[0174] Synthesis of compound XY-013-5-5 :

[0175] Substrate XY-013-5-4 (4.5 g, 9.95 mmol) was added to a dry 100 mL single-mouth bottle and dissolved in anhydrous DCM (40 mL). Then, pyridine (7.86 g, 99.5 mmol) and BzCl (1.41 g, 9.95 mmol) were added at 0 ° C under nitrogen protection. After the addition, the reaction system was kept stirring at room temperature overnight. LCMS detected that the raw material disappeared completely and the reaction was completed. Then 50 mL of dichloromethane was added to the reaction solution, washed with water (100 mL*3), and washed with saturated brine (100 mL). The organic phase was dried over anhydrous Na2SO4, filtered, and concentrated to obtain a crude product. Purification was performed with reverse phase C18 (CH3CN / H2O (0.05% NH4HCO3) = 1 / 0) and concentrated to obtain a colorless and transparent oily product XY-013-5-5 (2.2 g, 3.96 mmol, 57.8% yield). ESI-LCMS: m / z 556.2 [M+H] + .

[0176] Synthesis of compound XY-013-5-6 :

[0177] In a dry 100mL single-mouth bottle, add the substrate XY-013-5-5 (2.2g, 3.96mmol) dissolved in THF (30mL), then add TBAF (4.36ml, 4.36mmol), after addition, keep the reaction system at room temperature and stir for 30min. LCMS detected that the raw material completely disappeared, and the reaction was completed. Then add 50mL of ethyl acetate to the reaction solution, wash with water (100mL*3), and wash with saturated brine (100mL), the organic phase is dried over anhydrous Na2SO4, filtered, and concentrated to obtain a crude product, which is purified by reverse phase C18 (CH3CN / H2O (0.05% NH4HCO3) = 1 / 0), and concentrated to obtain a white solid product XY-013-5-6 (1.0g, 2.27mmol, 57.3% yield).

[0178] ESI-LCMS: m / z 442.2[M+H] - . 1 H NMR(400MHz,DMSO-d6)δ11.29(s,1H),8.06-7.99(m,3H),7.68-7.60(m,1H),7.55-7.49(m,2H),7.44-7.39(m,1H),5.86-5.85(m,1 H),5.11-5.09(m,1H),3.82-3.74(m,3H),3.45(s,3H),1.76-1.60(m,7H),1.42-1.32(m,1H),1.30-1.12(m,5H),0.94-0.83(m,2H).

[0179] Synthesis of compound XY-013-5 :

[0180] Substrate XY-013-5-6 (1.0 g, 2.27 mmol) was added to a dry 100 mL single-mouth bottle and dissolved in anhydrous DCM (10 mL), and then DCI (228 mg, 1.93 mmol) and CEP[N(iPr)2]2 (821 mg, 2.72 mmol) were added at 0°C under nitrogen protection. After the addition, the reaction system was stirred at room temperature for 1.0 h. The reaction was completed when the raw material disappeared completely by LCMS. Then add 50mL of dichloromethane to the reaction solution, wash with water (100mL*3), and wash with saturated brine (100mL), dry the organic phase over anhydrous Na2SO4, filter and concentrate to obtain a crude product which is purified by reverse phase C18 (CH3CN / H2O (0.05% NH4HCO3) = 1 / 0) and concentrated to obtain a white solid product XY-013-5-5 (1.0 g, 1.56mmol, 68.7% yield).

[0181] ESI-LCMS: m / z 640.7[MH] + .See Figure 4 .

[0182] 1 H NMR(400MHz,DMSO-d6)δ11.30(s,1H),8.10-7.99(m,3H),7.66-7.60(m,1H),7.55-7.49(m,2H),7.44-7.39(m,1H),5.92-5.88(m,1H),4.14-3.89 (m,3H),3.83-3.71(m,2H),3.68-3.60(m,2H),3.48-3.40(m,3H),3.81- 2.79(m,2H),1.83-1.59(m,7H),1.40-1.10(m,18H),0.94-0.81(m,2H).See Figure 5 . 31 P NMR (400MHz, DMSO-d6) δ 149.23, 148.91. Figure 6 .

[0183] Example 3. Synthesis of (2R,3R,4R,5R)-2-((2-oxa-7-azaspiro[3.5]nonan-7-yl)methyl)-5-(4-isobutylamino-2-oxopyrimidin-1(2H)-yl)-4-methoxytetrahydrofuran-3-yl(2-cyanoethyl)diisopropylphosphoramide

[0184]

[0185] Step 1. Synthesis of compound 2

[0186] Substrate 1 (15.30 g, 59.48 mmol) was added to a dry 500 mL single-mouth bottle, and anhydrous N, N-dimethylformamide (150 mL) was added to dissolve, followed by the addition of tert-butyldimethylsilyl chloride (24.59 g, 297.38 mmol) and imidazole (28.34 g, 416.34 mmol) under nitrogen protection. After the addition, the reaction system was stirred at room temperature overnight. Then 150 mL of dichloromethane was added to the reaction solution, washed with water (300 mL*3), and washed with saturated brine (300 mL), the organic phase was dried over anhydrous Na2SO4, filtered, and concentrated to obtain a crude product, which was purified by C18 (98% ACN in 0.05% NH4HCO3 / H2O) and concentrated to obtain product 2 (26.0 g, 89.99%), LCMS (ESI) m / z: 486.5 [M+H] + .

[0187] Step 2. Synthesis of compound 3

[0188] Substrate 2 (26.0 g, 53.52 mmol) was added to a dry 500 mL single-mouth bottle, and anhydrous pyridine (260 mL) was added to dissolve. Then, isobutyryl chloride (14.26 g, 133.81 mmol) was added at 0 ° C under nitrogen protection. After the addition, the reaction system was kept at room temperature and stirred for 4 h. Concentration was performed to obtain a crude product, ice water was added, and then 25 mL of dichloromethane was added to the solution, and it was washed with water (250 mL*3) and saturated brine (200 mL). The organic phase was dried over anhydrous Na2SO4, filtered, and concentrated. The residue was purified by column chromatography (petroleum ether: ethyl acetate = 5:3) and concentrated to obtain product 3 (29 g, 97.47%), LCMS (ESI) m / z: 556.6 [M+H] + .

[0189] Step 3. Synthesis of compound 4

[0190] Substrate 3 (29.0 g, 52.17 mmol) was added to a dry 1L single-mouth bottle, and then tetrahydrofuran (300 mL) was added to dissolve, followed by ice-cold trifluoroacetic acid (75 mL) and water (75 mL) mixed solution at 0°C. After the addition was completed, the reaction system was kept at 0°C and stirred for 3 h. Saturated sodium bicarbonate was then added at 0°C to pH = 7-8, extracted with dichloromethane (300 mL*3), and washed with saturated brine (300 mL). The organic phase was dried over anhydrous Na2SO4, filtered, and concentrated. The residue was purified by column chromatography (petroleum ether: ethyl acetate = 5:3-1:1) and concentrated to obtain product 4 (13.10 g, 56.86%).1 H NMR (400MHz, DMSO-d6) δ10.89(s,1H),8.46(d,J=7.5Hz,1H),7.24(d,J=7.5Hz,1H),5.83(d,J=2.4Hz,1H),4.23(dd,J=7.0,4.8Hz,1H),3.89(m,1H),3 .82-3.71(m,2H),3.56(dd,J=12.3,2.8Hz,1H),3.44(s,3H),2.72(m,1H),1 .06(d,J=6.8Hz,6H),0.86(s,9H),0.06(s,6H).LCMS(ESI)m / z:442.1[M+H] + .

[0191] Step 4. Synthesis of compound 5

[0192] Substrate 4 (13.1 g, 29.70 mmol) was added to a dry 1L single-mouth bottle, and dimethyl sulfoxide (220 mL) was added to dissolve it. Then, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (22.86 g, 118.80 mmol), pyridine (4.71 g, 59.40 mmol) and trifluoroacetic acid (3.40 g, 29.7 mmol) were added at 0°C under nitrogen protection. After the addition, the reaction system was stirred at room temperature for 4 h. Saturated sodium bicarbonate (400 mL) and dichloromethane (300 mL) were slowly added to the mixture, and the mixture was washed with saturated sodium bicarbonate (250 mL*3). The organic layer was washed with saturated brine (200 mL), dried over anhydrous Na2SO4, filtered, and concentrated to obtain crude compound 5 (15 g), LCMS (ESI) m / z: 440.5 [M+H] + .

[0193] Step 5. Synthesis of compound 6

[0194] The crude substrate 5 (3.10 g, 7.05 mmol) was added to a dry 250 mL single-necked bottle, and dichloroethane (60 mL) was added to dissolve it. Then, 2-oxazole-7-azaspiro[3.5]nonane (896.93 mg, 7.05 mmol) was added under nitrogen protection. After the addition was complete, the reaction system was stirred at room temperature for 1.0 h. Then, sodium triacetoxyborohydride (2.99 g, 14.10 mmol) was added. After the addition was complete, the reaction system was stirred at room temperature for 16 h. Then saturated sodium bicarbonate (100 mL) was added at 0°C, extracted with dichloromethane (100 mL*3), and washed with saturated brine (100 mL). The organic phase was dried over anhydrous Na2SO4, filtered, and concentrated. The residue was purified by column chromatography (petroleum ether: ethyl acetate = 1:1) and concentrated to give product 6 (1.73 g, 44.54%), LCMS (ESI) m / z: 551.6 [M+H] + .

[0195] Step 6. Synthesis of compound 7

[0196] Substrate 6 (1.47 g, 2.67 mmol) was added to a dry 100 mL single-mouth bottle, tetrahydrofuran (34 mL) was added to dissolve, and then triethylamine (404.91 mg, 4.00 mmol) and triethylamine hydrofluoride (3.23 g, 20.04 mmol) were added at room temperature. After the addition was completed, the reaction system was kept stirring at room temperature for 16 h. After concentration, the obtained residue was purified by C18 (8% ACN in 0.05% TFA / H2O) and concentrated to obtain product 7 (1.0 g, 85.83%). 1 H NMR(400MHz, Methanol-d4)δ8.06(s,1H),7.57-7.39(m,1H),5.87(d,J=3.1Hz,1H),4.48(d,J=52.6Hz,4H),4.36(s,1H),4.09(s,1H),4.00(d,J=3.0Hz, 1H),3.57(d,J=11.9Hz,7H),3.08(d,J=12.2Hz,2H),2.69(p,J=6.8Hz,1H), 2.34(s,2H),2.02(s,2H),1.18(d,J=6.8Hz,6H).LCMS(ESI)m / z:437.4[M+H] + .

[0197] Step 7. Synthesis of Compound 8

[0198] At room temperature, compound 7 (1 g, 1.06 mmol) was dissolved in toluene and concentrated (20 mL*3), then dissolved in dry anhydrous acetonitrile (7 mL), and then N, N-diisopropylethylamine (545.98 mg, 4.22 mmol) and 2-cyanoethyl N, N-diisopropylchlorophosphoramidite (499.92 mg, 2.11 mmol) were added under nitrogen protection. After addition, the reaction system was stirred at room temperature for 1 h under nitrogen protection. Concentration was performed to obtain a crude product, which was purified by C18 (54% ACN in 0.05% NH4HCO3 / H2O), concentrated, and the residue was purified again by column chromatography (petroleum ether: ethyl acetate = 1:2-ethyl acetate), and concentrated to obtain product 8 (190 mg, 13.0%).

[0199] LCMS (ESI) m / z: 637.3 [M+H] + .See Figure 7 .

[0200] 1 H NMR (400MHz, DMSO-d6) δ10.90 (s, 1H), 8.25 (dd, J=24.6, 7.5Hz, 1H), 7.28 (dd, J= 7.5,2.4Hz,1H),5.86(s,1H),4.27(d,J=3.4Hz,4H),4.20-4.02(m,2H),3.90(td, J=9.1,4.7,2.8Hz,1H),3.81-3.52(m,4H),3.44(d,J=11.7Hz,3H),2.81-2.53(m, 5H), 2.38 (s, 4H), 1.82-1.68 (m, 4H), 1.18-1.10 (m, 12H), 1.06 (d, J = 6.8Hz, 6H). See Figure 8 .

[0201] 31 P NMR (162MHz, DMSO-d6) δ149.07 (d, J=66.1Hz). Fig. 9 .

[0202] Example 2: siRNA Synthesis

[0203] For the sense and antisense strands of the siRNA sequence of the present invention and the sense and antisense strands of the modified duplex, a solid phase support was used to start the chain synthesis; a solid phase support modified with GalNAc (L96) was used as the starting cycle of the sense chain synthesis, and a universal solid phase support (Primer support 5G unylinker 350) was used as the starting cycle of the antisense chain synthesis. Fig.10 ).

[0204] The structure of the GalNAc(L96) modified solid support (L96-PS) is as follows Fig.11 : Primer support 5 Gunylinker 350 vector structure is as follows:

[0205]

[0206] ●PS stands for polystyrene solid phase carrier

[0207] For the sense and antisense strands of the siRNA sequence of the present invention and the sense and antisense strands of the modified duplex, PS was used as a solid phase carrier; a GalNAc-modified PS solid phase was used as the starting cycle for the synthesis of the sense strand, and a universal PS was used as the starting cycle for the antisense strand ( Fig.10 ).

[0208] Using a YB-192S synthesizer and phosphoramidite triester solid phase synthesis method, starting with a solid phase carrier, nucleoside monomers were sequentially connected in the 3'-5' direction to perform sequence synthesis with a synthesis scale of 0.2umol.

[0209] See the process flow Fig.12 .

[0210] Process Description:

[0211] Oligo synthesis starts with Universal-PS, and uses 3'-0-(2-cyanoethyl)phosphoramidite / 4,4'dimethoxytrityl (dimethoxytrityl, DMT) group protection to assemble oligonucleotide chains on the solid phase support PS. Each synthesis cycle includes 5'-hydroxyl deprotection, coupling, capping and oxidation (thiolation). Each coupling reaction is carried out by activating the appropriate phosphoramidite monomer and reacting with the free 5'-hydroxyl of the protected nucleotide or oligonucleotide fixed on the carrier. According to the sequence information, the corresponding crude oligonucleotide single-strand PS-Oligo is synthesized cyclically, and the crude oligonucleotide single-strand is cleaved from the solid phase support and the relevant protecting groups are removed. Then, the final product is obtained through preparative chromatography purification, ultrafiltration desalting, annealing, and freeze-drying steps.

[0212] (1) Synthesis procedure

[0213] The following units are included:

[0214] 1) De-blocking: The 5'-OH end of the ribonucleotide is protected by a DMT group (dimethoxytrityl). In the first step of the synthesis, trichloroacetic acid (TCA) is used to remove the DMT protecting group from the solid phase support and the ribonucleotide, so that the 5'-OH of the naked ribonucleotide can be coupled with a new base.

[0215] 2) Coupling: Nucleotide monomers are mixed with activation reagents and react with PS-Oligo in the synthesis column. The activation reagent provides a proton to the N atom of diisopropylamide on the 3'-phosphoric acid to form a phosphoramidite tetrazole active intermediate. When phosphoramidite tetrazole comes into contact with PS-Oligo, it undergoes a nucleophilic reaction with its 5'-hydroxyl group, coupling occurs and tetrazole is removed, extending a nucleotide.

[0216] 3) Capping: Since the coupling efficiency cannot reach 100%, in order to prevent the uncoupled PS-Oligo from continuing to the next step of coupling, the 5'-hydroxyl group of PS-Oligo is capped with a Cap reagent.

[0217] 4) Oxidation or thiolation: After the coupling reaction, the nucleotide is connected to the oligonucleotide on the PS through a phosphite bond (trivalent phosphorus). This phosphite bond is unstable and easily hydrolyzed by acid and alkali. The trivalent phosphorus here is oxidized to pentavalent phosphorus by an oxidizing agent. Thiolation refers to the reaction of the trivalent phosphorus of the phosphite bond to form a phosphorus-sulfur bond under weak alkaline conditions by a thiolation agent.

[0218] 5) VP protecting group removal and aminolysis

[0219] Transfer the synthesized PS-oligo from the synthesis column to a centrifuge tube, prepare a removal reagent according to the volume ratio of 3:2:100 = TMS-I: pyridine: DCM, and add the removal reagent to the centrifuge tube for reaction for 30 minutes. Prepare TEA / acetonitrile = 1:1 solution, add 2-mercaptoethanol (final concentration 2M), mix well, and add to the reaction to terminate the reaction. Remove the supernatant and wash the PS-oligo for 5min×2 times. After washing, add aminolysis solution (2-mercaptoethanol (2M) / 28% ammonia water) for aminolysis for 10h. After the aminolysis is completed, vacuum centrifuge to remove the ammonia water and purify.

[0220] LC-MS detection after conjugate synthesis

[0221]

[0222] (3) Purification

[0223] The sample diluted by ammonolysis was purified by anion column exchange chromatography, and the liquid phase system adopted high performance liquid chromatography. The chromatographic conditions were as follows.

[0224] Chromatographic column: PS-15Q10*250mm

[0225] Flow rate: 4ml / min

[0226] Detection wavelength: 260nm

[0227] Ion column preparation purification gradient program:

[0228] Mobile phase: Phase A: 10mM NaOH solution (PH=10) Phase B: 10mM NaOH solution (PH=10) + 2M NaCl

[0229]

[0230] After preparation, the samples were confirmed by mass spectrometry and purity.

[0231] (4) Ultrafiltration

[0232] After purification, add 4 ml PBS to dissolve the sample, transfer it to a 1K ultrafiltration tube, centrifuge it at 5000 r / min for 45 min, and then use Nanodrop to detect whether the liquid in the centrifuge tube contains the sample. If the sample is detected at a wavelength of 260 nm, it means that the ultrafiltration membrane is damaged and the ultrafiltration tube needs to be replaced and ultrafiltration is repeated. If the sample is not detected at a wavelength of 260 nm, add 4 ml RNase water and ultrafiltration again. Ultrafiltration is performed 3 times, each time for 45 min.

[0233] (5) Annealing

[0234] After ultrafiltration of the sense and antisense chains, dilute the samples at a concentration of 3 mg / ml, and mix the sense and antisense chains at a molar ratio of 1:1. Heat the water bath to 90°C, place the sense and antisense chains mixed in proportion in the water bath, and anneal for 30 minutes. Turn off the water bath and let it cool to room temperature for 16 hours. Take 10 μL of the sample for HPLC detection.

[0235] The present invention uses a PC sequence known to have an ANGPTL3 gene inhibitory effect as a tool sequence to compare the effects before and after modification.

[0236] The PC-1 sequence before modification is:

[0237] Justice chain: 5'- / mC* / mA* / mU / mA / mU / fU / mU / fG / fA / fU / mC / mA / mG / mU / mC / mU /

[0238] mU / mU / mU / mU / L96-3'(SEQ ID NO:1)

[0239] Antisense strand: 5'- / mA* / fA* / mA / mA / mA / fG / mA / mC / mU / mG / mA / mU / mC / fA / mA / fA /

[0240] mU / mA / mU / mG* / mU* / mU / -3'(SEQ ID NO:2)

[0241] The sequence after PC-1 modification is:

[0242] Sense strand: 5'-mG-B1* / mA* / mU / mA / mU / fU / mU / fG / fA / fU / mC / mA / mG / mU / mC / mU /

[0243] mU / mU / mU / mU / L96-3’(SEQ ID NO:3)

[0244] Antisense strand: 5'- / mA* / fA* / mA / mA / mA / fG / mA / mC / mU / mG / mA / mU / mC / fA / mA / fA /

[0245] mU / mA / mU / mG* / mU* / mU / -3’(SEQ ID NO:2)

[0246] The sequence of PC-2 before modification is:

[0247] Sense strand: 5'- / mC* / mA* / mU / mA / mU / fU / mU / fG / fA / fU / mC / mA / mG / mU / mC / mU /

[0248] mU / mU / mU / mA / L96-3’(SEQ ID NO:4)

[0249] Antisense strand: 5'- / mU* / fA* / mA / mA / mA / fG / mA / mC / mU / mG / mA / mU / mC / fA / mA / fA /

[0250] mU / mA / mU / mG* / mU* / mU / -3’(SEQ ID NO:5)

[0251] The sequence after PC-2 modification is:

[0252] Sense strand: 5'-mG-B1* / mA* / mU / mA / mU / fU / mU / fG / fA / fU / mC / mA / mG / mU / mC / mU /

[0253] mU / mU / mU / mA / L96-3’(SEQ ID NO:6)

[0254] Antisense strand: 5'- / mU* / fA* / mA / mA / mA / fG / mA / mC / mU / mG / mA / mU / mC / fA /

[0255] mA / fA / mU / mA / mU / mG* / mU* / mU / -3’(SEQ ID NO:5)

[0256]

[0257] Among them, the expression of nucleoside monomer modification is as follows:

[0258] mA represents 2'-O-methyladenosine, mU represents 2'-O-methyluridine, mG represents 2'-O-methylguanosine, mC represents 2'-O-methylcytosine, fA represents 2'-fluoroadenosine, fU represents 2'-fluorouridine, fG represents 2'-fluoroguanosine, fC represents 2'-fluorocytosine, mG-B1 represents the 5'-block nucleotides of the present invention (such as 013-4, 013-5, 014-1), * represents connection with thiophosphate bonds, and there is no symbol between directly adjacent nucleotides.

[0259] Example 2: In vitro activity detection

[0260] Cell culture and transfection

[0261] Cell culture: Hep3B cells (ATCC) were cultured in MEM complete medium (Gibco, supplemented with 10% FBS) at 37°C and 5% CO2 until nearly confluent, and then the cells were digested with trypsin and plated. 2.0×10 5 Hep3B cells and 0.09 mL MEM complete medium (Gibco, supplemented with 10% FBS) were cultured at 37° C. and 5% CO 2 for 16-24 h before transfection.

[0262] Cell transfection: 0.15 μL lipofectamine RNAiMax (Invitrogen) was added to 4.85 μL opti-MEM per well, followed by 5.0 μL siRNA, mixed, added to a PCR tube and incubated at room temperature for 5 minutes, and finally the siRNA mixture was added to the above cells and cultured for 24 hours before RNA extraction. The experiment was performed at 1.0 nM and 0.1 nM siRNA concentrations.

[0263] RNA extraction

[0264] Use the total RNA isolation kit (Omega, cat: R6834-02): collect cells, wash with 1% PBS, then add 400 μL of lysis buffer (containing 2% β-mercaptoethanol) to lyse the cells, and follow the instructions of the RNA isolation kit for subsequent steps. Finally, add 30 μL of RNase-free water, let stand for 2 minutes, and then centrifuge at 14000g for 2 minutes to collect RNA.

[0265] cDNA synthesis

[0266] cDNA synthesis was performed using the gDNA removal cDNA synthesis kit of Quanshijin (Beijing Quanshijin Biotechnology Co., Ltd., Beijing, China Cat#AE311-03). 1 μg of total RNA was added to each sample, and cDNA synthesis was performed using a gradient thermal cycler (LongGene, A600) according to the instructions.

[0267] Real-time fluorescence quantitative PCR

[0268] The synthesized cDNA and mixed master solution (including primers, qPCR premix and ultrapure water) were added to a 384-well plate (Biocon Media, Cat# PC-0040-9U) so that the final real-time fluorescence quantitative PCR system contained 0.25 μM of upstream and downstream primers of the target gene (ANGPTL3) or internal reference gene (GADPH) and 1×SYBR Green premix (Applied Biosystem).

[0269] Using the ΔΔCt assay in ABIQuantStudio TM Real-time fluorescence PCR was performed in a 6-well real-time fluorescence PCR system. Each duplex was tested for 3-4 independent transfections, and each transfection was tested in triplicate.

[0270] Conjugate Hep3B single dose test results 1

[0271]

[0272] Conjugate Hep3B single dose test results 2

[0273]

[0274] Example 3 Activity detection in wild-type mice

[0275] 6-8 week old male C57BL / 6J mice (Chengdu Weitonglihua Experimental Animal Technology Co., Ltd.). Blood was collected and serum was separated before grouping and administration, and used for ELISA to detect the level of mouse ANGPTL3 protein. Mice were randomly divided into 4 groups according to the level of ANGPTL3, defined as day0. On Day1, PBS, PC, conjugate 2, conjugate 3, and conjugate 4 were subcutaneously injected, respectively, with a drug dose of 1 mpk. Serum was separated from blood on day 8, day 15, day 22 after administration or on day 8, day 12, day 21, day 28 after administration, and used for ELISA to detect ANGPTL3. The serum protein concentration of each mouse at each time point was compared with the protein concentration of the mouse before administration (day 0), and the detection results of different conjugates are shown in the table.

[0276] Changes in ANGPTL3 protein expression before and after administration of different siRNA modified duplexes

[0277]

[0278] Changes in ANGPTL3 protein expression before and after administration of different siRNA modified duplexes

[0279]

[0280] The above experimental results show that after the nucleotide monomer of the present invention is introduced into the first position of the 5'-end of the siRNA sense chain, the activity of siRNA can be significantly improved, thereby silencing the target gene mRNA with higher selectivity and reducing the dosage of siRNA, thereby more effectively treating related diseases caused by the target gene mRNA.

Claims

1. A nucleotide monomer compound represented by Formula Ia or Formula Ib, or a deuterated compound thereof, or a fluorinated compound thereof, or a usable salt thereof: in, X is O or S; R 1 It has the structure shown in formula II: in, R 2 , R 3 are independently selected from hydrogen, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C2-C6 alkenyl or substituted or unsubstituted C2-C6 alkynyl; R 4 Selected from C1-C6 alkyl, cyanoethyl, cyanopropyl or cyanobutyl; Z 1 , Z 2 , Z 3 , Z 4 , J are independently selected from hydrogen, halogen, C1-C6 alkyl, C1-C6 alkoxy, -O-(C1-C6 alkylene)-O-(C1-C6 alkyl), -O-(C1-C6 alkylene)-C(O)NR Z1 R Z2 ; Or, J and Z 1 or Z 2 connected to each other, and together with the ring in which they are located, form a bridged heterocycle; R Z1 , R Z2 Each is independently selected from hydrogen, C1-C6 alkyl, and cycloalkyl; Bx is selected from a heterocyclic base; Y 3 Selected from substituted or unsubstituted C1-C6 alkylene, substituted or unsubstituted C2-C6 alkenylene, substituted or unsubstituted C2-C6 alkynylene, substituted or unsubstituted 3-8 membered carbocyclic ring, substituted or unsubstituted 4-8 membered heterocyclic ring; wherein one or more methylene groups in the alkylene, alkenylene and alkynylene are optionally substituted by heteroatoms; preferably, the heteroatoms are O, S or NH; Y is selected from -OR Y1 、-SR Y1 or -NR Y1 R Y2 ; R Y1 , R Y2 are independently selected from hydrogen, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, -(C0-C6 alkylene)-(3-10 membered carbocyclic ring), -(C0-C6 alkylene)-(4-10 membered heterocyclic ring), -(C0-C6 alkylene)-(5-10 membered bridged ring), -(C0-C6 alkylene)-(5-10 membered bridged heterocyclic ring), -(C0-C6 alkylene)-(5-10 membered spirocyclic ring), -(C0-C6 alkylene)-(5-10 membered spiroheterocyclic ring), -(C0-C6 alkylene)-(6-10 membered aromatic ring), -(C0-C6 alkylene)-(5-10 membered aromatic heterocyclic ring); wherein the alkylene, alkyl, alkenyl, alkynyl, carbocyclic ring, heterocyclic ring, bridged ring, bridged heterocyclic ring, spirocyclic ring, spiroheterocyclic ring, aromatic ring, aromatic heterocyclic ring are optionally substituted by one or more P; Or, R Y1 , R Y2 The nitrogen atom connected thereto together forms a 4- to 10-membered heterocycle, a 5- to 10-membered bridged heterocycle, a 5- to 10-membered spiro heterocycle, or a 5- to 10-membered aromatic heterocycle; wherein the heterocycle, bridged heterocycle, spiro heterocycle, or aromatic heterocycle is optionally substituted by one or more P; Each P is independently selected from hydrogen, C1-C3 alkyl, and 3-10 membered carbon ring.

2. The monomer compound according to claim 1, characterized in that: The heterocyclic base is selected from conventional bases, or unconventional nucleoside bases and their derivatives; The conventional nucleoside base is selected from adenine, guanine, cytosine, uracil or thymine; The unconventional base is selected from hypoxanthine, xanthine, and 2-thiouracil; The derivatives include halides, thiols, methylations and their metabolites.

3. The monomer compound according to claim 2, characterized in that , the heterocyclic base is selected from: Wherein, PG is selected from hydrogen or a hydroxyl / amine protecting group; preferably, the hydroxyl / amine protecting group is selected from an acyl group, a benzyl group or an alkylsilyl group.

4. The monomer compound according to claim 1, characterized in that: Y can be selected from the following structures: in, The methylene group in the above structure is optionally replaced by a heteroatom; preferably, the heteroatom is O, S or NH; P 1 , P 2 , P 3 , P 4 are independently selected from hydrogen, C1-C3 alkyl, cycloalkyl; preferably, P 1 , P 2 , P 3 , P 4 All are hydrogen.

5. The monomer compound according to claim 1, characterized in that: Y is selected from the following structures:

6. The monomer compound according to any one of claims 1 to 5, characterized in that: Y 3 Selected from methylene.

7. The monomer compound according to any one of claims 1 to 5, characterized in that: X is O.

8. The monomer compound according to any one of claims 1 to 5, characterized in that: Z 1 is selected from methoxy or -OCH2CH2OCH3; Z 2 , Z 3 , Z 4 , J is hydrogen.

9. The monomer compound according to any one of claims 1 to 5, characterized in that: R 2 , R 3 are independently selected from isopropyl or isobutyl; R 4 Selected from cyanoethyl or cyanopropyl.

10. The monomer compound according to claim 9, characterized in that: R 1 for 11. The monomer compound according to any one of claims 1 to 10, characterized in that: The compound of formula Ia or formula Ib is specifically: Wherein, PG is selected from hydrogen or a hydroxyl / amine protecting group; preferably, the hydroxyl / amine protecting group is selected from an alkyl acyl group, an alkoxy acyl group, an aryl acyl group, and an aryloxy acyl group.

12. Use of the monomer compound according to any one of claims 1 to 11 as an intermediate in the preparation of an oligonucleotide; preferably, the oligonucleotide is siRNA.

13. The use according to claim 12, characterized in that: Use of the monomer according to any one of claims 1 to 11 as an intermediate in the preparation of the first nucleotide at the 5'-terminus of the sense strand of siRNA.

14. An siRNA comprising a sense strand and an antisense strand; the sense strand and the antisense strand each comprise 15 to 45 modified or unmodified nucleotides, and the sense strand and the antisense strand are partially complementary to form a double-stranded region; wherein, The first nucleotide at the 5' end of the sense strand has a structure shown in Formula Va or Formula Vb, and Site covalently linked to the rest of the siRNA: in, X is O or S; Z 1 , Z 2 , Z 3 , Z 4 , J are independently selected from hydrogen, halogen, C1-C6 alkyl, C1-C6 alkoxy, -O-(C1-C6 alkylene)-O-(C1-C6 alkyl), -O-(C1-C6 alkylene)-C(O)NR Z1 R Z2 ; Or, J and Z 1 or Z 2 connected to form a bridged heterocycle together with the ring where it is located; R Z1 , R Z2 Each is independently selected from hydrogen, C1-C6 alkyl, and cycloalkyl; Bx is selected from a heterocyclic base; Y 3 Selected from substituted or unsubstituted C1-C6 alkylene, substituted or unsubstituted C2-C6 alkenylene, substituted or unsubstituted C2-C6 alkynylene, substituted or unsubstituted 3-8 membered carbocyclic ring, substituted or unsubstituted 4-8 membered heterocyclic ring; wherein one or more methylene groups in the alkylene, alkenylene and alkynylene are optionally substituted by heteroatoms; preferably, the heteroatoms are O, S or NH; Y is selected from -OR Y1 、-SR Y1 or -NR Y1 R Y2 ; R Y1 , R Y2 are independently selected from hydrogen, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, -(C0-C6 alkylene)-(3-10 membered carbocyclic ring), -(C0-C6 alkylene)-(4-10 membered heterocyclic ring), -(C0-C6 alkylene)-(5-10 membered bridged ring), -(C0-C6 alkylene)-(5-10 membered bridged heterocyclic ring), -(C0-C6 alkylene)-(5-10 membered spirocyclic ring), -(C0-C6 alkylene)-(5-10 membered spiroheterocyclic ring), -(C0-C6 alkylene)-(6-10 membered aromatic ring), -(C0-C6 alkylene)-(5-10 membered aromatic heterocyclic ring); wherein the alkylene, alkyl, alkenyl, alkynyl, carbocyclic ring, heterocyclic ring, bridged ring, bridged heterocyclic ring, spirocyclic ring, spiroheterocyclic ring, aromatic ring, aromatic heterocyclic ring are optionally substituted by one or more P; Or, R Y1 , R Y2 The nitrogen atom connected thereto together forms a 4- to 10-membered heterocycle, a 5- to 10-membered bridged heterocycle, a 5- to 10-membered spiro heterocycle, or a 5- to 10-membered aromatic heterocycle; wherein the heterocycle, bridged heterocycle, spiro heterocycle, or aromatic heterocycle is optionally substituted by one or more P; Each P is independently selected from hydrogen, C1-C3 alkyl, 3-10 membered carbocyclic ring; Y 4 Selected from O or S; Y 5 Selected from -OH, -O(CH3), -SH, -S(CH3) or -N(CH3)2.

15. The siRNA according to claim 14, characterized in that: The heterocyclic base is selected from conventional bases, or unconventional nucleoside bases and their derivatives; The conventional nucleoside base is selected from adenine, guanine, cytosine, uracil or thymine; The unconventional base is selected from hypoxanthine, xanthine, and 2-thiouracil; The derivatives include halides, thiols, methylations and their metabolites.

16. The siRNA according to claim 15, characterized in that: The heterocyclic base is selected from: Wherein, PG is selected from hydrogen or a hydroxyl / amine protecting group; preferably, the hydroxyl / amine protecting group is selected from an acyl group, a benzyl group or an alkylsilyl group.

17. The siRNA according to claim 14, characterized in that: Y is selected from the following structures: in, The methylene group in the above structure is optionally replaced by a heteroatom; preferably, the heteroatom is O, S or NH; P 1 , P 2 , P 3 , P 4 are independently selected from hydrogen, C1-C3 alkyl, cycloalkyl; preferably, P 1 , P 2 , P 3 , P 4 All are hydrogen.

18. The siRNA according to claim 17, characterized in that: Y is selected from the following structures:

19. The siRNA according to any one of claims 14 to 18, characterized in that: Y 3 Selected from methylene.

20. The siRNA according to any one of claims 14 to 18, characterized in that: X is O; Y 4 S; Y 5 It is -OH.

21. The siRNA according to any one of claims 14 to 18, characterized in that: Z 1 is selected from methoxy or -OCH2CH2OCH3; Z 2 , Z 3 , Z 4 , J is hydrogen.

22. The siRNA according to any one of claims 14 to 21, characterized in that: The structure shown in Formula Va or Formula Vb in the siRNA is specifically: Wherein, PG is selected from hydrogen or a hydroxyl / amine protecting group; preferably, the hydroxyl / amine protecting group is selected from an alkyl acyl group, an alkoxy acyl group, an aryl acyl group, and an aryloxy acyl group.

23. A pharmaceutical composition comprising the siRNA according to any one of claims 14 to 22 and a pharmaceutically acceptable carrier.

24. An siRNA conjugate, formed by conjugating the siRNA according to any one of claims 14 to 22 and a conjugated molecule.

25. A kit comprising the siRNA according to any one of claims 14 to 22, and / or the pharmaceutical composition according to claim 23, and / or the siRNA conjugate according to claim 24.

26. Use of the siRNA according to any one of claims 14 to 22, and / or the pharmaceutical composition according to claim 23, and / or the siRNA conjugate according to claim 24, and / or the kit according to claim 25 in the preparation of a drug.

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