Double-stranded rnas with nucleotide analogs

Abstract

The present invention provides a double-stranded RNA having nucleotide analogs. The double-stranded RNA of the present invention exhibits one or more of enhanced stability, reduced off-target toxicity, and enhanced effectiveness.

Description

[0001] This application is a divisional application of the invention patent application filed on January 4, 2023, with application number 202310009472.2. Invention Field

[0002] This invention belongs to the field of medicine, specifically relating to double-stranded RNA having nucleotide analogs. Background Technology

[0003] RNA interference (RNAi) is a phenomenon characterized by the highly efficient and specific degradation of target mRNA induced by double-stranded RNA (dsRNA). Incorporation of thermally unstable nucleotides (e.g., glycerol nucleotides (GNA)) into the antisense seed region of dsRNA can improve interference efficiency and reduce off-target toxicity (see, for example, PCT Publication No. WO2018098328A1). However, as described in Schlegel et al. Nucleic Acids Research (2021), incorporation of GNA-G or GNA-C into the center of dsRNA, relative to GNA-A or GNA-T, leads to a decrease in dsRNA stability.

[0004] Therefore, there is a need in the art to develop a nucleotide analog that exhibits enhanced stability when incorporated into double-stranded RNA. Summary of the Invention

[0005] The present invention solves the above problems by providing a novel nucleotide analog.

[0006] In one aspect, the present invention relates to nucleotide dimers represented by formula (A),

[0007]

[0008] in,

[0009] L2 is either H or P2;

[0010] L3 is either H or P3;

[0011] Q is -X- or -XO-;

[0012] X is a chemical bond, -(CR1R2) m -or -CR1 = CR2-;

[0013] Y1 is O, S, or NR;

[0014] Y2 is an O, S, or chemical bond;

[0015] R1, R2, R4, R5, R6, and R7 are independently selected from H, D, halogens, CN, and C. 1-6 Alkyl, C 1-6Haloalkyl, C 2-6 alkenyl or C 2-6 The alkynyl group, which is optionally substituted with 1, 2, 3, 4 or 5 R' groups;

[0016] R3 is selected from H and C. 1-6 Cyanoalkyl, C 1-6 Alkyl, C 1-6 Haloalkyl, C 2-6 alkenyl, C 2-6 alkynyl group, C 3-7 Cycloalkyl, 3-7 membered heterocyclic groups, C 6-10 Aryl or 5-10 heteroaryl groups, which are optionally substituted by 1, 2, 3, 4 or 5 R's;

[0017] R8 is selected from H, D, OH, halogens, and C. 1-6 Alkyl, C 1-6 Halogenated alkyl or C 1-6 Alkoxy;

[0018] Base and Base' are independently selected from H, modified or unmodified bases, or leaving groups;

[0019] P2 is selected from reactive phosphorus groups, preferably -P(OCH2CH2CN)(N(iPr)2);

[0020] P3 is selected from hydroxyl protecting groups, preferably DMTr;

[0021] R is selected from H and C. 1-6 Alkyl or C 1-6 Halogenated alkyl groups;

[0022] R' is selected from D, halogen, OH, CN, NH2, C 1-6 Alkyl, C 1-6 Haloalkyl, C 2-6 alkenyl, C 2-6 alkynyl group, C 3-10 Cycloalkyl, 3-10 membered heterocyclic groups, C 6-10 Aryl or 5-14 heteroaryl groups;

[0023] m is selected from 1, 2, 3, 4 or 5.

[0024] In another aspect, the present invention relates to a double-stranded RNA molecule or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof, comprising a sense strand and an antisense strand, each strand having 14 to 30 nucleotides, the antisense strand having a sequence sufficiently complementary to the sense strand and the target mRNA and having the ability to induce the degradation of the target mRNA, and the antisense strand comprising one or more nucleotide monomers of formula (IV):

[0025]

[0026] in,

[0027] The nucleotide monomers shown are arranged in the order of 5' => 3' from... to connect;

[0028] Each group is as defined in the context.

[0029] In another aspect, the present invention relates to a nucleic acid molecule whose nucleotide sequence comprises one or more nucleotide monomers and / or nucleotide dimers as described herein.

[0030] In another aspect, the present invention relates to a pharmaceutical composition comprising a double-stranded RNA molecule as described herein, and a pharmaceutically acceptable carrier or excipient.

[0031] In another aspect, the present invention relates to a kit comprising a double-stranded RNA molecule as described herein.

[0032] In another aspect, the present invention relates to a method for inhibiting the expression of a target gene in a cell, comprising the step of introducing a double-stranded RNA molecule as described herein into the cell.

[0033] In another aspect, the present invention relates to a method for inhibiting the expression of a target gene in a cell, comprising expressing a double-stranded RNA molecule as described herein in the cell.

[0034] When the nucleotides of the present invention are incorporated into the antisense strand of dsRNA, the resulting double-stranded RNA exhibits one or more of the following: enhanced stability, reduced off-target toxicity, and enhanced efficacy. Invention Details

[0035] definition

[0036] Chemical definition

[0037] The definitions of specific functional groups and chemical terms are described in more detail below.

[0038] When listing a range of values, it is assumed that each value and the subranges within that range are included. For example, "C 1-6 Alkyl groups include C1, C2, C3, C4, C5, C6, and C6. 1-6 C 1-5 C 1-4 C 1-3 C 1-2 C 2-6 C 2-5 C 2-4 C 2-3 C 3-6 C3-5 C 3-4 C 4-6 C 4-5 and C 5-6 alkyl.

[0039] “C 1-6 "Alkyl" refers to a straight-chain or branched saturated hydrocarbon group having 1 to 6 carbon atoms. In some embodiments, C 1-4 Alkyl and C 1-2 Alkyl groups are preferred. C 1-6 Examples of alkyl groups include: methyl (C1), ethyl (C2), n-propyl (C3), isopropyl (C3), n-butyl (C4), tert-butyl (C4), sec-butyl (C4), isobutyl (C4), n-pentyl (C5), 3-pentyl (C5), pentyl (C5), neopentyl (C5), 3-methyl-2-butyl (C5), tert-pentyl (C5), and n-hexyl (C6). The term "C" is used in conjunction with the preceding text. 1-6 "Alkyl" also includes heteroalkyl, wherein one or more (e.g., 1, 2, 3, or 4) carbon atoms are replaced by heteroatoms (e.g., oxygen, sulfur, nitrogen, boron, silicon, phosphorus). The alkyl group may be optionally substituted by one or more substituents, for example, by 1 to 5 substituents, 1 to 3 substituents, or 1 substituent. Common alkyl abbreviations include: Me(-CH3), Et(-CH2CH3), iPr(-CH(CH3)2), nPr(-CH2CH2CH3), n-Bu(-CH2CH2CH2CH3) or i-Bu(-CH2CH(CH3)2).

[0040] “C 2-6 "Alkenyl" refers to a straight-chain or branched hydrocarbon group having 2 to 6 carbon atoms and at least one carbon-carbon double bond. In some embodiments, C 2-4 Alkenyl groups are preferred. C 2-6 Examples of alkenyl groups include: vinyl (C2), 1-propenyl (C3), 2-propenyl (C3), 1-butenyl (C4), 2-butenyl (C4), butadienyl (C4), pentenyl (C5), pentadienyl (C5), hexenyl (C6), and so on. The term "C" is used in conjunction with these groups. 2-6 "Alkenyl" also includes heteroalkenyl groups, wherein one or more (e.g., 1, 2, 3, or 4) carbon atoms are replaced by heteroatoms (e.g., oxygen, sulfur, nitrogen, boron, silicon, phosphorus). The alkenyl group may be optionally substituted by one or more substituents, for example, by 1 to 5 substituents, 1 to 3 substituents, or 1 substituent.

[0041] “C 2-6 "Alkyne" refers to a straight-chain or branched hydrocarbon group having 2 to 6 carbon atoms, at least one carbon-carbon triple bond, and optionally one or more carbon-carbon double bonds. In some embodiments, C 2-4The alkynyl group is preferred. C 2-6 Examples of alkynyl groups include, but are not limited to: ethynyl (C2), 1-propynyl (C3), 2-propynyl (C3), 1-butynyl (C4), 2-butynyl (C4), pentyynyl (C5), hexynyl (C6), etc. The term "C" is used in conjunction with other alkynyl groups. 2-6 "Alkyne" also includes heteroyne, wherein one or more (e.g., 1, 2, 3 or 4) carbon atoms are replaced by heteroatoms (e.g., oxygen, sulfur, nitrogen, boron, silicon, phosphorus). The alkynyl group may be optionally substituted by one or more substituents, for example, by 1 to 5 substituents, 1 to 3 substituents or 1 substituent.

[0042] “C 1-6 "Alkylene" refers to the removal of C 1-6 The alkyl group is a divalent group formed by another hydrogen atom, and can be substituted or unsubstituted. In some embodiments, C 1-4 Alkylene, C 2-4 Alkylene and C 1-3 Alkylenes are preferred. Unsubstituted alkylenes include, but are not limited to: methylene (-CH2-), ethylene (-CH2CH2-), propylene (-CH2CH2CH2-), butylene (-CH2CH2CH2CH2-), pentylene (-CH2CH2CH2CH2CH2-), hexylene (-CH2CH2CH2CH2CH2CH2-), and so on. Exemplary substituted alkylenes, for example, those substituted with one or more alkyl (methyl) groups, include, but are not limited to: substituted methylene (-CH(CH3)-, -C(CH3)2-), substituted ethylene (-CH(CH3)CH2-, -CH2CH(CH3)-, -C(CH3)2CH2-, -CH2C(CH3) 2- ), substituted propylidenes (-CH(CH3)CH2CH2-, -CH2CH(CH3)CH2-, -CH2CH2CH(CH3)-, -C(CH3)2CH2CH2-, -CH2C(CH3)2CH2-, -CH2CH2C(CH3)2-), etc.

[0043] "Halogen" or "halogen" refers to fluorine (F), chlorine (Cl), bromine (Br), and iodine (I).

[0044] Therefore, "C" 1-6 "Halogenated alkyl" refers to the above "C 1-6 "alkyl" is substituted with one or more halogen groups. In some embodiments, C 1-4 Halogenated alkyl groups are particularly preferred, and C4 groups are more preferred. 1-2Halogenated alkyl groups. Exemplary alkyl halogenated groups include, but are not limited to: -CF3, -CH2F, -CHF2, -CHFCH2F, -CH2CHF2, -CF2CF3, -CCl3, -CH2Cl, -CHCl2, 2,2,2-trifluoro-1,1-dimethyl-ethyl, etc. The alkyl halogenated group can be substituted at any available connection point, for example, 1 to 5 substituents, 1 to 3 substituents, or 1 substituent.

[0045] “C 1-6 "Alkoxy" refers to the -OR group, where R is as described above as "C". 1-6 "alkyl" and "C" 1-6 The definition of "halogenated alkyl".

[0046] “C 1-6 "Cyanalkyl" refers to -C 1-6 Alkylene-CN, where "C" 1-6 "alkylene" is as defined above. In some embodiments, C 1-4 Cyanoalkyl groups are particularly preferred, and C14 is more preferred. 1-2 Cyanoalkyl, such as cyanoethyl (-CH2CH2CN).

[0047] “C 3-10 "Cycloalkyl" refers to a non-aromatic cyclic hydrocarbon group having 3 to 10 ring carbon atoms and zero heteroatoms. In some embodiments, C 4-7 cycloalkyl and C 3-6 Cycloalkyl groups are particularly preferred, and C10 is more preferred. 5-6 Cycloalkyl groups. Cycloalkyl groups also include ring systems in which the aforementioned cycloalkyl ring is fused with one or more aryl or heteroaryl groups, wherein the connecting point is on the cycloalkyl ring, and in such cases, the number of carbons continues to represent the number of carbons in the cycloalkyl system. Exemplary cycloalkyl groups include, but are not limited to: cyclopropyl (C3), cyclopropenyl (C3), cyclobutyl (C4), cyclobutenyl (C4), cyclopentyl (C5), cyclopentenyl (C5), cyclohexyl (C6), cyclohexenyl (C6), cyclohexadienyl (C6), cycloheptyl (C7), cycloheptenyl (C7), cycloheptadienyl (C7), cyclohepttrienyl (C7), etc. The cycloalkyl group may optionally be substituted with one or more substituents, for example, substituted with 1 to 5 substituents, 1 to 3 substituents, or 1 substituent.

[0048] "3-10 membered heterocyclic groups" refer to groups with a 3- to 10-membered non-aromatic ring system having a cyclic carbon atom and 1 to 5 cyclic heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen, sulfur, boron, phosphorus, and silicon. In heterocyclic groups containing one or more nitrogen atoms, the linkage can be a carbon or nitrogen atom, provided that the valence allows. In some embodiments, a 4-10 membered heterocyclic group is preferred, which is a 4-10 membered non-aromatic ring system having a cyclic carbon atom and 1 to 5 cyclic heteroatoms; in some embodiments, a 3-8 membered heterocyclic group is preferred, which is a 3-8 membered non-aromatic ring system having a cyclic carbon atom and 1 to 4 cyclic heteroatoms; a 3-6 membered heterocyclic group is preferred, which is a 3-6 membered non-aromatic ring system having a cyclic carbon atom and 1 to 3 cyclic heteroatoms; a 4-7 membered heterocyclic group is preferred, which is a 4-7 membered non-aromatic ring system having a cyclic carbon atom and 1 to 3 cyclic heteroatoms; and a 5-6 membered heterocyclic group is preferred, which is a 5-6 membered non-aromatic ring system having a cyclic carbon atom and 1 to 3 cyclic heteroatoms. Heterocyclic groups also include ring systems in which the aforementioned heterocyclic ring is fused with one or more cycloalkyl groups, wherein the linking point is on the cycloalkyl ring, or ring systems in which the aforementioned heterocyclic ring is fused with one or more aryl or heteroaryl groups, wherein the linking point is on the heterocyclic ring; and in such cases, the number of ring members continues to represent the number of ring members in the heterocyclic ring system. Exemplary 3-membered heterocyclic groups containing one heteroatom include, but are not limited to: azirropropyl, oxetane, and thiorenyl. Exemplary 4-membered heterocyclic groups containing one heteroatom include, but are not limited to: azirrobutyl, oxetane, and thiorenyl. Exemplary 5-membered heterocyclic groups containing one heteroatom include, but are not limited to: tetrahydrofuranyl, dihydrofuranyl, tetrahydrothiophenyl, dihydrothiophenyl, pyrrolidinyl, dihydropyrrolidinyl, and pyrrolidin-2,5-dione. Exemplary 5-membered heterocyclic groups containing two heteroatoms include, but are not limited to: dioxasulfuranyl, oxasulfuranyl, disulfuranyl, and oxazolidin-2-one. Exemplary 5-membered heterocyclic groups containing three heteroatoms include, but are not limited to: triazolinyl, oxadiazolinyl, and thiadiazolinyl. Exemplary 6-membered heterocyclic groups containing one heteroatom include, but are not limited to: piperidinyl, tetrahydropyranyl, dihydropyridinyl, and thianyl. Exemplary 6-membered heterocyclic groups containing two heteroatoms include, but are not limited to: piperazinyl, morpholinyl, disulfuranyl, and dioxalyl. Exemplary 6-membered heterocyclic groups containing three heteroatoms include, but are not limited to: triazinanyl. Exemplary 7-membered heterocyclic groups containing one heteroatom include, but are not limited to: azirheptanyl, oxasulfuranyl, and thioheptanyl. Exemplary 5-membered heterocyclic groups fused with a C6 aryl ring (also referred to herein as 5,6-bicyclic heterocyclic groups) include, but are not limited to: dihydroindolyl, isodihydroindolyl, dihydrobenzofuranyl, dihydrobenzothiophenyl, benzoxazolinone, etc.Exemplary 6-membered heterocyclic groups fused with a C6 aryl ring (also referring to 6,6-bicyclic heterocyclic groups herein) include, but are not limited to, tetrahydroquinolinyl, tetrahydroisoquinolinyl, etc. The heterocyclic group may be optionally substituted by one or more substituents, for example, by 1 to 5 substituents, 1 to 3 substituents, or 1 substituent.

[0049] “C 6-10 "Aryl" refers to a monocyclic or polycyclic (e.g., bicyclic) 4n+2 aromatic ring system (e.g., having 6 or 10 shared π electrons arranged in a ring) having 6-10 ring carbon atoms and zero heteroatoms. In some embodiments, the aryl group has six ring carbon atoms ("C6 aryl"; e.g., phenyl). In some embodiments, the aryl group has ten ring carbon atoms ("C6 aryl"). 10 "Aryl"; for example, naphthyl, such as 1-naphthyl and 2-naphthyl). Aryl also includes a ring system in which the above-mentioned aryl ring is fused with one or more cycloalkyl or heterocyclic groups, and the connection point is on the aryl ring, in which case the number of carbon atoms continues to represent the number of carbon atoms in the aryl ring system. The aryl group may be optionally substituted by one or more substituents, for example, by 1 to 5 substituents, 1 to 3 substituents, or 1 substituent.

[0050] "5-14 membered heteroaryl" refers to a 4n+2 aromatic ring system of a 5-14 membered monocyclic or bicyclic ring (e.g., having 6, 10, or 14 shared π electrons arranged in a ring) having a ring carbon atom and 1-4 ring heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur. In heteroaryl containing one or more nitrogen atoms, the bonding point can be a carbon or nitrogen atom, provided the valence allows. A heteroaryl bicyclic system may include one or more heteroatoms in one or both rings. Heteroaryl also includes ring systems in which the aforementioned heteroaryl ring is fused with one or more cycloalkyl or heterocyclic groups, and the bonding point is on the heteroaryl ring, in which case the number of carbon atoms continues to represent the number of carbon atoms in the heteroaryl ring system. In some embodiments, 5-10 membered heteroaryl is preferred, which is a 4n+2 aromatic ring system of a 5-10 membered monocyclic or bicyclic ring having a ring carbon atom and 1-4 ring heteroatoms. In other embodiments, 5-6 membered heteroaryl groups are particularly preferred, which are 4n+2 aromatic ring systems of 5-6 membered monocyclic or bicyclic rings having a cyclic carbon atom and 1-4 cyclic heteroatoms. Exemplary 5-membered heteroaryl groups containing one heteroatom include, but are not limited to: pyrrole, furanyl, and thiophene. Exemplary 5-membered heteroaryl groups containing two heteroatoms include, but are not limited to: imidazolyl, pyrazolyl, oxazolyl, isoxazolyl, thiazolyl, and isothiazolyl. Exemplary 5-membered heteroaryl groups containing three heteroatoms include, but are not limited to: triazolyl, oxadiazolyl (e.g., 1,2,4-oxadiazolyl), and thiadiazolyl. Exemplary 5-membered heteroaryl groups containing four heteroatoms include, but are not limited to: tetrazolyl. Exemplary 6-membered heteroaryl groups containing one heteroatom include, but are not limited to: pyridinyl. Exemplary 6-membered heteroaryl groups containing two heteroatoms include, but are not limited to: pyridazinyl, pyrimidinyl, and pyrazinyl. Exemplary 6-membered heteroaryl groups containing three or four heteroatoms include, but are not limited to, triazinyl and tetraazinyl. Exemplary 7-membered heteroaryl groups containing one heteroatom include, but are not limited to, azirmonoheptatrienyl, oxadiazinyl, and thioheptatrienyl. Exemplary 5,6-bicyclic heteroaryl groups include, but are not limited to, indolyl, isoindolyl, indazole, benzotriazolyl, benzothiophene, isobenzothiophene, benzofuranyl, benzoisofuranyl, benzoimidazolyl, benzoxazolyl, benzoisoxazolyl, benzoxadiazolyl, benzothiazolyl, benzoisothiazolyl, benzothiadiazolyl, indazinyl, and purinyl. Exemplary 6,6-bicyclic heteroaryl groups include, but are not limited to, naphthidyl, pteridinyl, quinolinyl, isoquinolinyl, zolinyl, quinoxolinyl, phthalazinyl, and quinazolinyl. The heteroaryl group may be optionally substituted by one or more substituents, for example, by 1 to 5 substituents, 1 to 3 substituents or 1 substituent.

[0051] The alkyl, alkenyl, ynyl, cycloalkyl, heterocyclic, aryl, and heteroaryl groups defined in this article are optional substituted groups.

[0052] Exemplary substituents on carbon atoms include, but are not limited to: halogen, -CN, -NO2, -N3, -SO2H, -SO3H, -OH, -OR aa -ON(R) bb )2、-N(R bb )2、-N(R bb )3 + X - -N(OR) cc )R bb -SH, -SR aa -SSR cc -C(=O)R aa -CO2H, -CHO, -C(OR) cc )2、-CO2R aa -OC(=O)R aa -OCO2R aa -C(=O)N(R) bb )2、-OC(=O)N(R bb )2、-NR bb C(=O)R aa -NR bb CO2R aa -NR bb C(=O)N(R bb )2、-C(=NR bb )R aa -C(=NR) bb OR aa -OC(=NR) bb )R aa -OC(=NR) bb OR aa -C(=NR) bb )N(R bb )2、-OC(=NR bb )N(R bb )2、-NR bb C(=NR bb )N(R bb )2、-C(=O)NR bb SO2R aa -NR bb SO2R aa -SO2N(R) bb )2、-SO2R aa -SO2OR aa -OSO2R aa -S(=O)R aa -OS(=O)R aa 、-Si(R aa3. -OSi(R) aa 3. -C(=S)N(R) bb )2、-C(=O)SR aa -C(=S)SR aa -SC(=S)SR aa -SC(=O)SR aa -OC(=O)SR aa -SC(=O)OR aa -SC(=O)R aa -P(=O)2R aa -OP(=O)2R aa -P(=O)(R aa )2、-OP(=O)(R aa )2、-OP(=O)(OR cc )2、-P(=O)2N(R bb )2、-OP(=O)2N(R bb )2、-P(=O)(NR bb )2、-OP(=O)(NR bb )2、-NR bb P(=O)(OR cc )2、-NR bb P(=O)(NR bb )2、-P(R cc )2、-P(R cc )3、-OP(R cc )2、-OP(R cc )3、-B(R aa 2. -B(OR) cc )2、-BR aa (OR cc ), alkyl, haloalkyl, alkenyl, ynyl, cycloalkyl, heterocyclic, aryl, and heteroaryl, wherein each alkyl, alkenyl, ynyl, cycloalkyl, heterocyclic, aryl, and heteroaryl is independently bounded by 0, 1, 2, 3, 4, or 5 R groups. dd Group substitution;

[0053] Or the two hydrogen-bearing groups on the carbon atom: =O, =S, =NN(R) bb )2、=NNR bb C(=O)R aa =NNR bb C(=O)OR aa =NNR bb S(=O)2R aa =NR bb or = NOR cc replace;

[0054] R aa Each of them is independently selected from alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, heterocyclic, aryl, and heteroaryl, or two R aa The groups are combined to form a heterocyclic or heteroaryl ring, wherein each alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclic, aryl, and heteroaryl group is independently bounded by 0, 1, 2, 3, 4, or 5 R groups. dd Group substitution;

[0055] R bb Each is independently selected from: hydrogen, -OH, -OR aa -N(R) cc )2、-CN、-C(=O)R aa -C(=O)N(R) cc )2、-CO2R aa -SO2R aa -C(=NR) cc OR aa -C(=NR) cc )N(R cc )2、-SO2N(R cc )2、-SO2R cc -SO2OR cc -SOR aa -C(=S)N(R) cc )2、-C(=O)SR cc -C(=S)SR cc -P(=O)2R aa -P(=O)(R aa )2、-P(=O)2N(R cc )2、-P(=O)(NR cc 2. Alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, heterocyclic, aryl and heteroaryl, or two R bb The groups are combined to form a heterocyclic or heteroaryl ring, wherein each alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclic, aryl, and heteroaryl group is independently bounded by 0, 1, 2, 3, 4, or 5 R groups. dd Group substitution;

[0056] R cc Each is independently selected from hydrogen, alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, heterocyclic, aryl, and heteroaryl, or two R cc The groups are combined to form a heterocyclic or heteroaryl ring, wherein each alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclic, aryl, and heteroaryl group is independently bounded by 0, 1, 2, 3, 4, or 5 R groups. dd Group substitution;

[0057] R ddEach is independently selected from: halogen, -CN, -NO2, -N3, -SO2H, -SO3H, -OH, -OR ee -ON(R) ff )2、-N(R ff )2,、-N(R ff )3 + X - -N(OR) ee )R ff -SH, -SR ee -SSR ee -C(=O)R ee -CO2H, -CO2R ee -OC(=O)R ee -OCO2R ee -C(=O)N(R) ff )2、-OC(=O)N(R ff )2、-NR ff C(=O)R ee -NR ff CO2R ee -NR ff C(=O)N(R ff )2、-C(=NR ff OR ee -OC(=NR) ff )R ee -OC(=NR) ff OR ee -C(=NR) ff )N(R ff )2、-OC(=NR ff )N(R ff )2、-NR ff C(=NR ff )N(R ff )2、-NR ff SO2R ee -SO2N(R) ff )2、-SO2R ee -SO2OR ee -OSO2R ee -S(=O)R ee 、-Si(R ee 3. -OSi(R) ee 3. -C(=S)N(R) ff )2、-C(=O)SR ee -C(=S)SR ee -SC(=S)SR ee -P(=O)2Ree -P(=O)(R ee )2、-OP(=O)(R ee )2、-OP(=O)(OR ee 2. Alkyl, haloalkyl, alkenyl, ynyl, cycloalkyl, heterocyclic, aryl, heteroaryl, wherein each alkyl, alkenyl, ynyl, cycloalkyl, heterocyclic, aryl, and heteroaryl group is independently marked by 0, 1, 2, 3, 4, or 5 R groups. gg Group substitution, or two geminal radicals dd Substituents can combine to form =O or =S;

[0058] R ee Each is independently selected from alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, aryl, heterocyclic, and heteroaryl, wherein each alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclic, aryl, and heteroaryl is independently surrounded by 0, 1, 2, 3, 4, or 5 R groups. gg Group substitution;

[0059] R ff Each is independently selected from hydrogen, alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, heterocyclic, aryl, and heteroaryl, or two R ff The groups combine to form a heterocyclic or heteroaryl ring, wherein each alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclic, aryl, and heteroaryl group is independently bounded by 0, 1, 2, 3, 4, or 5 R groups. gg Group substitution;

[0060] R gg Each of these is independently: halogen, -CN, -NO2, -N3, -SO2H, -SO3H, -OH, -OC 1-6 Alkyl, -ON(C) 1-6 Alkyl)2, -N(C 1-6 Alkyl)2, -N(C 1-6 Alkyl)3 + X - -NH(C 1-6 Alkyl)2 + X - -NH2(C 1-6 alkyl) + X - -NH3 + X - -N(OC) 1-6 Alkyl)(C 1-6 Alkyl), -N(OH)(C 1-6 Alkyl groups, -NH(OH), -SH, -SC 1-6 Alkyl, -SS(C 1-6 Alkyl), -C(=O)(C 1-6Alkyl group, -CO2H, -CO2(C 1-6 Alkyl), -OC (=O)(C 1-6 Alkyl), -OCO2(C 1-6 Alkyl groups, -C(=O)NH2, -C(=O)N(C 1-6 Alkyl)2、-OC(=O)NH(C 1-6 Alkyl), -NHC(=O)(C 1-6 alkyl), -N(C) 1-6 Alkyl)C(=O)(C 1-6 alkyl), -NHCO2(C 1-6 Alkyl), -NHC(=O)N(C 1-6 Alkyl)2、-NHC(=O)NH(C 1-6 Alkyl groups, -NHC(=O)NH2, -C(=NH)O(C 1-6 Alkyl), -OC (=NH)(C 1-6 Alkyl group), -OC (=NH)OC 1-6 Alkyl group, -C(=NH)N(C 1-6 Alkyl)2、-C(=NH)NH(C 1-6 Alkyl groups, -C(=NH)NH2, -OC(=NH)N(C 1-6 Alkyl)2、-OC(NH)NH(C 1-6 Alkyl groups, -OC(NH)NH2, -NHC(NH)N(C 1-6 Alkyl)2, -NHC(=NH)NH2, -NHSO2(C 1-6 alkyl), -SO2N(C 1-6 alkyl)2、-SO2NH(C 1-6 Alkyl groups, -SO2NH2, -SO2C 1-6 Alkyl, -SO2OC 1-6 Alkyl, -OSO2C 1-6 Alkyl, -SOC 1-6 Alkyl, -Si(C) 1-6 Alkyl)3、-OSi(C 1-6 Alkyl)3, -C(=S)N(C 1-6 Alkyl)2、C(=S)NH(C 1-6 Alkyl), C(=S)NH2, -C(=O)S(C 1-6 Alkyl), -C(=S)SC 1-6 Alkyl, -SC (=S)SC 1-6 Alkyl group, -P(=O)2(C 1-6 Alkyl), -P(=O)(C 1-6 Alkyl)2、-OP(=O)(C 1-6Alkyl)2、-OP(=O)(OC 1-6 Alkyl)2, C 1-6 Alkyl, C 1-6 Haloalkyl, C2-C6 alkenyl, C2-C6 ynyl, C3-C7 cycloalkyl, C6-C 10 Aryl, C3-C7 heterocyclic, C5-C 10 heteroaryl; or two aryl groups gg Substituents can combine to form =O or =S; where X - It is a counterion.

[0061] Exemplary substituents on the nitrogen atom include, but are not limited to: hydrogen, -OH, -OR aa -N(R) cc )2、-CN、-C(=O)R aa -C(=O)N(R) cc )2、-CO2R aa -SO2R aa -C(=NR) bb )R aa -C(=NR) cc OR aa -C(=NR) cc )N(R cc )2、-SO2N(R cc )2、-SO2R cc -SO2OR cc -SOR aa -C(=S)N(R) cc )2、-C(=O)SR cc -C(=S)SR cc -P(=O)2R aa -P(=O)(R aa )2、-P(=O)2N(R cc )2、-P(=O)(NR cc 2. Alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, heterocyclic, aryl, and heteroaryl, or two R atoms attached to a nitrogen atom. cc The groups combine to form a heterocyclic or heteroaryl ring, wherein each alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclic, aryl, and heteroaryl group is independently bounded by 0, 1, 2, 3, 4, or 5 R groups. dd Group substitution, wherein R aa R bb R cc and R dd As stated above.

[0062] Other definitions

[0063] The term "siRNA" in this article refers to a class of double-stranded RNA molecules that can mediate the silencing of a complementary target RNA (e.g., mRNA, such as transcripts of genes encoding proteins). siRNA is typically double-stranded, consisting of an antisense strand complementary to the target RNA and a sense strand complementary to that antisense strand. For convenience, such mRNA is also referred to herein as the mRNA to be silenced. Such genes are also referred to as target genes. Typically, the RNA to be silenced is an endogenous gene or a pathogen gene. Additionally, RNAs other than mRNA (e.g., tRNA) and viral RNA can also be targeted.

[0064] The term "antisense strand" refers to a strand of siRNA that contains regions that are completely, sufficiently, or substantially complementary to the target sequence. The term "sense strand" refers to a strand of siRNA that includes regions that are completely, sufficiently, or substantially complementary to the regions defined herein as antisense strands.

[0065] The term "complementary region" refers to a region on the antisense strand that is completely, fully, or substantially complementary to the target mRNA sequence. In cases where the complementary region is not perfectly complementary to the target sequence, mismatches can occur within the molecule or at the ends. Typically, the most tolerant mismatches are located in the end regions, for example, within 5, 4, 3, 2, or 1 nucleotides at the 5' and / or 3' ends. The portion of the antisense strand most sensitive to mismatches is called the "seed region." For example, in a siRNA containing a 19-nt strand, the 19th position (from 5' to 3') can tolerate some mismatches.

[0066] The term "complementary" refers to the ability of a first polynucleotide to hybridize with a second polynucleotide under certain conditions, such as stringent conditions. For example, stringent conditions may include 400 mM NaCl, 40 mM PIPES at pH 6.4, and 1 mM EDTA at 50 or 70°C for 12–16 hours. In terms of meeting the above requirements regarding their hybridization ability, a "complementary" sequence may also include base pairs formed entirely from non-Watson-Crick base pairs and / or from non-natural and modified nucleotides. Such non-Watson-Crick base pairs include, but are not limited to, G:U swing base pairings or Hoogstein base pairs.

[0067] A polynucleotide that is “at least partially complementary,” “fully complementary,” or “substantially complementary” to messenger RNA (mRNA) refers to a polynucleotide that is substantially complementary to a continuous portion of the mRNA of interest. For example, a polynucleotide is at least partially complementary to PCSK9 mRNA if its sequence is substantially complementary to an uninterrupted portion of the mRNA encoding PCSK9. The terms “complementary,” “fully complementary,” “fully complementary,” and “substantially complementary” can be used relative to base pairing between the sense and antisense strands of the siRNA, or between the antisense strand of the siRNA reagent and the target sequence.

[0068] "Perfect complementarity" means that in order to maintain the overall double-stranded character of the molecule, the sense strand only needs to be complementary to the antisense strand to a certain extent. In other words, although perfect complementarity is usually required, in some cases, especially in the antisense strand, there may be one or more mismatches (relative to the target mRNA), such as 6, 5, 4, 3, 2 or 1, but the sense and antisense strands can still maintain the overall double-stranded character of the molecule.

[0069] "shRNA" refers to short hairpin RNA. shRNA consists of two short inverted repeat sequences. The shRNA cloned into the shRNA expression vector comprises two short inverted repeat sequences separated by a stem-loop sequence, forming a hairpin structure controlled by the polIII promoter. Subsequently, 5-6 T molecules are added as a transcription terminator for RNA polymerase III.

[0070] "Nucleoside" is a compound composed of two substances: a purine or pyrimidine base and ribose or deoxyribose. "Nucleotide" is a compound composed of three substances: a purine or pyrimidine base, ribose or deoxyribose, and phosphate. "Oligonucleotide" refers to nucleic acid molecules (RNA or DNA) with a length of less than 100, 200, 300, or 400 nucleotides.

[0071] A "base" is the basic building block for the synthesis of nucleosides, nucleotides, and nucleic acids. Its constituent elements include nitrogen, hence it is also called a "nitrogenous base." In this article, unless otherwise specified, the capital letters A, U, T, G, and C represent the base composition of nucleotides, namely adenine, uracil, thymine, guanine, and cytosine, respectively.

[0072] The term "modification" of nucleotides as used herein includes, but is not limited to, methoxy modification, fluorination modification, thiophosphate linkage, or conventional protecting group protection. For example, a fluorinated nucleotide refers to a nucleotide in which the hydroxyl group at the 2' position of the ribosome is replaced by fluorine, and a methoxy modified nucleotide refers to a nucleotide in which the 2'-hydroxyl group of the ribosome is replaced by a methoxy group.

[0073] In this document, "modified nucleotides" include, but are not limited to, nucleotides modified with 2'-O-methyl, nucleotides modified with 2'-fluorine, nucleotides modified with 2'-deoxy-, inosine ribonucleotides, debased nucleotides, reverse abased deoxyribonucleotides, nucleotides containing a thiophosphate group, nucleotides modified with vinyl phosphate, locked nucleotides, nucleotides modified with 2'-amino, nucleotides modified with 2'-alkyl, morpholinonucleotides, aminophosphates, non-natural bases containing nucleotides, and terminal nucleotides linked to cholesterol derivatives or dodecanoic acid diecamide groups, deoxyribonucleotides, or those protected by conventional protecting groups. For example, a nucleotide modified with 2'-fluorine refers to a nucleotide in which the hydroxyl group at the 2' position of the ribosyl group is replaced by fluorine. A nucleotide modified with 2'-deoxy- refers to a nucleotide in which the 2'-hydroxyl group of the ribosyl group is replaced by a methoxy group.

[0074] A "leaving group" is an atom or functional group that leaves a larger molecule during a chemical reaction. It is a term used in nucleophilic substitution and elimination reactions.

[0075] "Reactive phosphorus group" refers to a phosphorus-containing group contained in a nucleotide unit or nucleotide analog unit that can react with a hydroxyl or amino group contained in another molecule, particularly in another nucleotide unit or another nucleotide analog, via a nucleophilic attack reaction. Typically, such a reaction produces an ester-type nucleoside bond linking the first nucleotide unit or the first nucleotide analog unit to the second nucleotide unit or the second nucleotide analog unit. The reactive phosphorus group can be selected from phosphorous amides, H-phosphonates, alkyl-phosphonates, phosphate esters, or phosphate ester analogs, including but not limited to: native phosphate esters, thiophosphate esters, dithiophosphate esters, boron phosphate esters, boron thiophosphate esters, phosphonates, halogen-substituted phosphonates and phosphate esters, aminophosphate esters, phosphate diesters, phosphate triesters, thiophosphate diesters, thiophosphate triesters, diphosphate esters, and triphosphate esters, preferably -P(OCH2CH2CN)(N(iPr)2).

[0076] A "protecting group," also known as a "protecting element," is any atom or group of atoms added to a molecule to prevent existing groups in the molecule from undergoing undesirable chemical reactions. Protecting groups can be unstable chemical moieties known in the art, used to protect reactive groups, such as hydroxyl, amino, and thiol groups, to prevent unwanted or untimely reactions during chemical synthesis. Protecting groups are typically used selectively and / or orthogonally to protect sites during reactions at other reactive sites, and can then be removed to leave unprotected groups intact or usable for further reactions.

[0077] A non-limiting list of protecting groups includes benzyl; substituted benzyl; alkyl carbonyl and alkoxy carbonyl (e.g., tert-butoxy carbonyl (BOC), acetyl, or isobutyryl); arylalkyl carbonyl and arylalkoxy carbonyl (e.g., benzyloxy carbonyl); substituted methyl ether (e.g., methoxymethyl ether); substituted diethyl ether; substituted benzyl ether; tetrahydropyranyl ether; silyl (e.g., trimethylsilyl, triethylsilyl, triisopropylsilyl, tert-butyldimethylsilyl, tri-isopropylsilyloxymethyl, [2-(trimethylsilyl)ethoxy)). Methyl or tert-butyldiphenylsilyl); esters (e.g., benzoates); carbonates (e.g., methoxymethyl carbonate); sulfonates (e.g., toluenesulfonates or methanesulfonates); noncyclic ketals (e.g., dimethyl acetal); cyclic ketals (e.g., 1,3-dioxane, 1,3-dioxolane, and those described herein); noncyclic acetals; cycloacetals (e.g., those described herein); noncyclic hemiacetals; cyclic hemiacetals; cyclic dithioketals (e.g., 1,3-dithiane or 1,3-dithiopentane); orthoesters (e.g., those described herein); and triarylmethyl groups. (For example, triphenylmethyl; monomethoxytriphenylmethyl (MMTr); 4,4′-dimethoxytriphenylmethyl (DMTr); 4,4′,4″-trimethoxytriphenylmethyl (TMTr); and those described herein). Preferred protecting groups are selected from acetyl (Ac), benzoyl (Bzl), benzyl (Bn), isobutyryl (iBu), phenylacetyl, benzyloxymethylacetal (BOM), β-methoxyethoxymethyl ether (MEM), methoxymethyl ether (MOM), p-methoxybenzyl ether (PMB), methyl thiomethyl ether, new Vanoyl (Piv), Tetrahydropyranyl (THP), Triphenylmethyl (Trt), Methoxytriphenylmethyl[(4-methoxyphenyl)diphenylmethyl] (MMT), Dimethoxytriphenylmethyl, [bis-(4-methoxyphenyl)phenylmethyl (DMT), Trimethylsilyl ether (TMS), Tert-butyldimethylsilyl ether (TBDMS), Tri-isopropylsilyloxymethyl ether (TOM), Tri-isopropylsilyl ether (TIPS), Methyl ether, Ethoxyethyl ether (EE), N,N-dimethylformamidinium and 2-cyanoethyl (CE).

[0078] A "hydroxyl protecting group" is a group that prevents the hydroxyl group from undergoing chemical reactions and can be removed under specific conditions to restore the hydroxyl group. These mainly include silane-type protecting groups, acyl-type protecting groups, or ether-type protecting groups, with the following being preferred:

[0079] Trimethylsilyl (TMS), Triethylsilyl (TES), Dimethylisopropylsilyl (DMIPS), Diethylisopropylsilyl (DEIPS), Tert-butyldimethylsilyl (TBDMS), Tert-butyldiphenylsilyl (TBDPS), Triisopropylsilyl (TIPS), Acetyl (Ac), Chloroacetyl, Dichloroacetyl, Trichloroacetyl, Trifluoroacetyl (TFA), Benzoyl, p-Methoxybenzoyl, 9-fluorenylmethoxycarbonyl (Fmoc), Allyloxycarbonyl (Alloc), 2,2,2-Trichloroethoxycarbonyl The following are listed: Troc, benzyloxycarbonyl (Cbz), tert-butyloxycarbonyl (Boc), benzyl (Bn), p-methoxybenzyl (PMB), allyl, triphenylmethyl (Tr), bis-p-methoxytriphenylmethyl (DMTr), methoxymethyl (MOM), phenoxymethyl (BOM), 2,2,2-trichloroethoxymethyl, 2-methoxyethoxymethyl (MEM), methylthiomethyl (MTM), p-methoxybenzyloxymethyl (PMBM), -C(O)CH2CH2C(O)OH, or 4,4'-dimethoxytriphenylmethyl.

[0080] As used herein, the term "pharmaceutically acceptable salt" refers to carboxylates and amino acid addition salts of the compounds of the present invention that are suitable for contact with patient tissues within the limits of reliable medical judgment, without producing undue toxicity, irritation, allergic reactions, etc., and are effective for their intended use in proportion to a reasonable benefit / risk ratio, including (where possible) zwitterionic forms of the compounds of the present invention.

[0081] This invention includes tautomers, which are functional group isomers resulting from the rapid movement of an atom in a molecule to two positions. A compound exists in different tautomer forms, and a compound is not limited to any particular tautomer, but is intended to encompass all tautomer forms.

[0082] The compounds of this invention may include one or more asymmetric centers and therefore may exist in a variety of stereoisomeric forms, such as enantiomers and / or diastereomers. For example, the compounds of this invention may be individual enantiomers, diastereomers, or geometric isomers (e.g., cis and trans isomers), or may be in the form of mixtures of stereoisomers, including racemic mixtures and mixtures rich in one or more stereoisomers. The isomers can be separated from the mixture by methods known to those skilled in the art, including chiral high-performance liquid chromatography (HPLC) and the formation and crystallization of chiral salts; or preferred isomers may be prepared by asymmetric synthesis.

[0083] The present invention also includes isotopically labeled compounds (isotopic variants) that are equivalent to those described in formula (I), but in which one or more atoms are replaced by atoms with atomic masses or mass numbers different from those commonly found in nature. Examples of isotopes that can be introduced into the compounds of the present invention include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, and chlorine, respectively, for example... 2 H, 3 H, 13 C 11 C 14 C 15 N、 18 O、 17 O、 31 P, 32 P, 35 S, 18 F and 36 Cl. Other isotopes of the present invention containing the aforementioned isotopes and / or other atoms, their prodrugs, and pharmaceutically acceptable salts of said compounds or said prodrugs are all within the scope of this invention. Certain isotope-labeled compounds of the present invention, for example, those incorporating radioactive isotopes (e.g.,...) 3 H and 14 Those in category C) can be used for drug and / or substrate tissue distribution determination. Tritium, i.e. 3 H and carbon-14, i.e. 14 Carbon isotopes are particularly preferred because they are easy to prepare and detect. Subsequently, they are replaced by heavier isotopes, such as deuterium, i.e., 2 H, because higher metabolic stability can provide therapeutic benefits, such as prolonged in vivo half-life or reduced dosage requirements, may be preferred in some cases. Isotope-labeled compounds of formula (I) of the present invention and their prodrugs can generally be prepared by using readily available isotope-labeled reagents instead of non-isotope-labeled reagents when performing the processes described below and / or the techniques disclosed in the examples and preparation examples.

[0084] Compounds of the present invention

[0085] This invention specifically relates to nucleotide dimers represented by formula (A):

[0086]

[0087] Each group is defined as in the context.

[0088] Q

[0089] In one implementation, Q is -X-; in another implementation, Q is -XO-.

[0090] X

[0091] In one embodiment, X is a chemical bond; in another embodiment, X is -(CR1R2). m -; In another implementation, X is -CR1 = CR2-.

[0092] Y1 and Y2

[0093] In one implementation, Y1 is O; in another implementation, Y1 is S; in yet another implementation, Y1 is NR.

[0094] In one embodiment, Y2 is O; in another embodiment, Y2 is S; in yet another embodiment, Y2 is a chemical bond.

[0095] L2 and L3

[0096] In one implementation, L2 is H; in another implementation, L2 is P2.

[0097] In one implementation, L3 is H; in another implementation, L3 is P3.

[0098] R1, R2, R4, R5, R6 and R7

[0099] In one embodiment, R1 is H; in another embodiment, R1 is D; in another embodiment, R1 is a halogen; in another embodiment, R1 is OH; in another embodiment, R1 is CN; in another embodiment, R1 is C. 1-6 Alkyl groups, such as C 1-4 Alkyl; in another embodiment, R1 is C 1-6 Halogenated alkyl groups, such as C 1-4 Halogenated alkyl; in another embodiment, R1 is C 2-6 Alkenyl; in another embodiment, R1 is C 2-6 Alkyne group.

[0100] In one embodiment, R2 is H; in another embodiment, R2 is D; in another embodiment, R2 is a halogen; in another embodiment, R2 is OH; in another embodiment, R2 is CN; in another embodiment, R2 is C. 1-6 Alkyl groups, such as C 1-4 Alkyl; in another embodiment, R2 is C 1-6 Halogenated alkyl groups, such as C 1-4 Halogenated alkyl; in another embodiment, R2 is C 2-6 Alkenyl; in another embodiment, R2 is C 2-6 Alkyne group.

[0101] In one embodiment, R4 is H; in another embodiment, R4 is D; in another embodiment, R4 is a halogen; in another embodiment, R4 is OH; in another embodiment, R4 is CN; in another embodiment, R4 is C. 1-6 Alkyl groups, such as C 1-4 Alkyl; in another embodiment, R4 is C 1-6 Halogenated alkyl groups, such as C 1-4 Halogenated alkyl; in another embodiment, R4 is C 2-6 Alkenyl; in another embodiment, R4 is C 2-6 Alkyne group.

[0102] In one embodiment, R5 is H; in another embodiment, R5 is D; in another embodiment, R5 is a halogen; in another embodiment, R5 is OH; in another embodiment, R5 is CN; in another embodiment, R5 is C. 1-6 Alkyl groups, such as C 1-4 Alkyl; in another embodiment, R5 is C 1-6 Halogenated alkyl groups, such as C 1-4 Halogenated alkyl; in another embodiment, R2 is C 2-6 Alkenyl; in another embodiment, R5 is C 2-6 Alkyne group.

[0103] In one embodiment, R6 is H; in another embodiment, R6 is D; in another embodiment, R6 is a halogen; in another embodiment, R6 is OH; in another embodiment, R6 is CN; in another embodiment, R6 is C. 1-6 Alkyl groups, such as C 1-4 Alkyl; in another embodiment, R6 is C 1-6 Halogenated alkyl groups, such as C 1-4 Halogenated alkyl; in another embodiment, R6 is C 2-6 Alkenyl; in another embodiment, R6 is C 2-6 Alkyne group.

[0104] In one embodiment, R7 is H; in another embodiment, R7 is D; in another embodiment, R7 is a halogen; in another embodiment, R7 is OH; in another embodiment, R7 is CN; in another embodiment, R7 is C. 1-6 Alkyl groups, such as C 1-4 Alkyl; in another embodiment, R7 is C 1-6 Halogenated alkyl groups, such as C 1-4 Halogenated alkyl; in another embodiment, R7 is C 2-6Alkenyl; in another embodiment, R7 is C 2-6 Alkyne group.

[0105] In one embodiment, R1, R2, R4, R5, R6 and R7 are each independently and optionally not substituted; in another embodiment, R1, R2, R4, R5, R6 and R7 are each independently and optionally substituted by 1, 2, 3, 4, 5, 6, 7, 8 or more R'.

[0106] R3

[0107] In one implementation, R3 is H; in another implementation, R3 is C. 1-6 Alkyl group, preferably C 1-4 Alkyl; in another embodiment, R3 is C 1-6 Cyanoalkyl; in another embodiment, R3 is C 1-4 Cyanoalkyl, such as cyanoethyl; in another embodiment, R3 is C 1-6 Halogenated alkyl groups, preferably C 1-4 Halogenated alkyl; in another embodiment, R3 is C 2-6 Alkenyl; in another embodiment, R3 is C 2-6 Alkyne group; in another embodiment, R3 is C 3-10 Cycloalkyl; in another embodiment, R3 is a 3-10 membered heterocyclic group; in another embodiment, R3 is a C 6-10 Aryl; in another embodiment, R3 is a 5-14 member heteroaryl.

[0108] In one implementation, R3 is not replaced; in another implementation, R3 is optionally replaced by 1, 2, 3, 4, 5, 6, 7, 8 or more R'.

[0109] R8

[0110] In one embodiment, R8 is H; in another embodiment, R8 is D; in another embodiment, R8 is OH; in another embodiment, R8 is a halogen, such as fluorine; in another embodiment, R8 is C. 1-6 Alkyl group, preferably C 1-4 Alkyl; in another embodiment, R8 is C 1-6 Halogenated alkyl groups, preferably C 1-4 Halogenated alkyl; in another embodiment, R8 is C 1-6 Alkoxy, preferably C 1-4 Alkyl groups, such as methoxy groups.

[0111] Base and Base'

[0112] In one embodiment, Base is H; in another embodiment, Base is a modified or unmodified base or leaving group; in yet another embodiment, Base is selected from modified or unmodified A, U, T, G, and C, for example...

[0113] In one embodiment, Base' is H; in another embodiment, Base' is a modified or unmodified base or leaving group; in yet another embodiment, Base' is selected from modified or unmodified A, U, T, G, and C, for example...

[0114] P2 and P3

[0115] In one embodiment, P2 is a hydroxyl protecting group; in another embodiment, P2 is a reactive phosphorus group, such as -P(OCH2CH2CN)(N(iPr)2).

[0116] In one embodiment, P3 is a hydroxyl protecting group, preferably a silane protecting group, an acyl protecting group, or an ether protecting group, such as DMTr.

[0117] R and R'

[0118] In one implementation, R is H; in another implementation, R is C. 1-6 Alkyl, such as C 1-4 Alkyl; in another embodiment, R is C 1-6 Halogenated alkyl groups.

[0119] In one embodiment, R' is D; in another embodiment, R' is a halogen; in yet another embodiment, R' is CN; in still another embodiment, R' is C. 1-6 Alkyl; in another embodiment, R' is C 1-6 Halogenated alkyl; in another embodiment, R' is C 2-6 Alkenyl; in another embodiment, R' is C 2-6 Alkyne group; in another embodiment, R' is C 3-10 Cycloalkyl; in another embodiment, R' is a 3-10 membered heterocyclic group; in another embodiment, R' is a C 6-10 aryl; in another embodiment, R' is a 5-14 membered heteroaryl; in another embodiment, R' is selected from -OR a -OC(O)R a -C(O)R a -C(O)OR a -C(O)NR a Rb -S(O) n R a -S(O) n OR a -S(O) n NR a R b -NR a R b -NR a C(O)R b -NR a -C(O)OR b -NR a -S(O) n R b or -NR a C(O)NR a R b .

[0120] R a and R b Independently selected from H and C 1-6 Alkyl, C 2-6 alkenyl, C 2-6 alkynyl group, C 3-10 Cycloalkyl, 3-10 membered heterocyclic groups, C 6-10 aryl or 5-14 heteroaryl; or R a and R b And the nitrogen atoms they connect to form 3-10 membered heterocyclic groups.

[0121] n is independently selected from 1 or 2.

[0122] m

[0123] In one implementation, m is 1; in another implementation, m is 2; in another implementation, m is 3; in another implementation, m is 4; in another implementation, m is 5.

[0124] GalNAc

[0125] In one embodiment, GalNAc is a conjugated group as shown in formula (X):

[0126]

[0127] in,

[0128] Indicates the location where it connects to a biomolecule;

[0129] Q G Independently for H,

[0130] Where L G1 For chemical bonds, -CH2-, -CH2CH2-, -C(O)-, -CH2O-, -CH2O-CH2CH2O-, or -NHC(O)-(CH2NHC(O)) a -;

[0131] L G2 It is a chemical bond or -CH2CH2C(O)-;

[0132] L G3 It is a chemical bond, -(NHCH2CH2) b -、-(NHCH2CH2CH2) b -or-C(O)CH2-;

[0133] L G4 -(OCH2CH2) c -、-(OCH2CH2CH2) c -、-(OCH2CH2CH2CH2) c -、-(OCH2CH2CH2CH2CH2) c -or -NHC(O)-(CH2) d -;

[0134] Where a = 0, 1, 2 or 3;

[0135] b = 1, 2, 3, 4 or 5;

[0136] c = 1, 2, 3, 4 or 5;

[0137] d = 1, 2, 3, 4, 5, 6, 7 or 8;

[0138] A is a chemical bond, -CH2O- or -NHC(O)-;

[0139] A' is a chemical bond, -C(O)NH-, -NHC(O)-, or -O(CH2CH2O). e -;

[0140] Where e is 1, 2, 3, 4 or 5;

[0141] B is a chemical bond, -CH2-, -C(O)-, -M-, -CH2-M-, or -C(O)-M-;

[0142] Where M is

[0143] R G1 and R G2 Together they form -CH2CH2O- or -CH2CH(R) G )-O-, and RG3 For H;

[0144] Or R G1 and R G3 Together form -C 1-2 alkylene-, and R G2 For H;

[0145] Where R G For -OR G '、-CH2OR G 'or -CH2CH2OR G ', where R G The 'protecting group' is H, a hydroxyl protecting group, or a solid support, wherein the hydroxyl protecting group is preferably -C(O)CH2CH2C(O)OH or 4,4'-dimethoxytriphenylmethyl;

[0146] m1 = 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

[0147] n1 = 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0148] In another implementation, GalNAc is the combination group shown in formula (I'):

[0149]

[0150] in,

[0151] Indicates the location where it connects to a biomolecule;

[0152] Q G Independently for H,

[0153] Where L G1 For chemical bonds, -CH2-, -CH2CH2-, -C(O)-, -CH2O-, -CH2O-CH2CH2O-, or -NHC(O)-(CH2NHC(O)) a -;

[0154] L G2 It is a chemical bond or -CH2CH2C(O)-;

[0155] L G3 It is a chemical bond, -(NHCH2CH2) b -、-(NHCH2CH2CH2) b -or-C(O)CH2-;

[0156] L G4 -(OCH2CH2) c-、-(OCH2CH2CH2) c -、-(OCH2CH2CH2CH2) c -、-(OCH2CH2CH2CH2CH2) c -or -NHC(O)-(CH2) d -;

[0157] Where a = 0, 1, 2 or 3;

[0158] b = 1, 2, 3, 4 or 5;

[0159] c = 1, 2, 3, 4 or 5;

[0160] d = 1, 2, 3, 4, 5, 6, 7 or 8;

[0161] A is -CH2O- or -NHC(O)-;

[0162] A' is a chemical bond, -C(O)NH- or -NHC(O)-;

[0163] R G1 and R G2 Together they form -CH2CH2O- or -CH2CH(R) G )-O-, and R G3 For H;

[0164] Or R G1 and R G3 Together form -C 1-2 alkylene-, and R G2 For H;

[0165] Where R G For -OR G '、-CH2OR G 'or -CH2CH2OR G ', where R G The 'protecting group' is H, a hydroxyl protecting group, or a solid support, wherein the hydroxyl protecting group is preferably -C(O)CH2CH2C(O)OH or 4,4'-dimethoxytriphenylmethyl;

[0166] m1 = 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

[0167] n1 = 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0168] In another embodiment, GalNAc is a conjugated group of formula (X), wherein,

[0169] Q G Independently for H,

[0170] Where L G1 For chemical bonds, -CH2-, -CH2CH2-, -C(O)-, -CH2O-, -CH2O-CH2CH2O-, or -NHC(O)-(CH2NHC(O)) a -;

[0171] L G2 It is a chemical bond or -CH2CH2C(O)-;

[0172] L G3 It is a chemical bond, -(NHCH2CH2) b -、-(NHCH2CH2CH2) b -or-C(O)CH2-;

[0173] L G4 -(OCH2CH2) c -、-(OCH2CH2CH2) c -、-(OCH2CH2CH2CH2) c -、-(OCH2CH2CH2CH2CH2) c -or -NHC(O)-(CH2) d -;

[0174] Where a = 0, 1, 2 or 3;

[0175] b = 1, 2, 3, 4 or 5;

[0176] c = 1, 2, 3, 4 or 5;

[0177] d = 1, 2, 3, 4, 5, 6, 7 or 8;

[0178] A is a chemical bond, -CH2O- or -NHC(O)-;

[0179] A' is a chemical bond, -C(O)NH-, -NHC(O)-, or -O(CH2CH2O). e -;

[0180] Where e is 1, 2, 3, 4 or 5;

[0181] B is a chemical bond, -CH2-, -M-, -CH2-M-, or -C(O)-M-;

[0182] Where M is

[0183] R G1 and R G2 Together they form -CH2CH2O- or -CH2CH(R) G)-O-, and R G3 For H;

[0184] Or R G1 and R G3 Together form -C 1-2 alkylene-, and R G2 For H;

[0185] Where R G For -OR G '、-CH2OR G 'or -CH2CH2OR G ', where R G The 'protecting group' is H, a hydroxyl protecting group, or a solid support, wherein the hydroxyl protecting group is preferably -C(O)CH2CH2C(O)OH or 4,4'-dimethoxytriphenylmethyl;

[0186] m1 = 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

[0187] n1 = 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0188] In another embodiment, GalNAc is a conjugated group of formula (X), wherein:

[0189] Q G Independently for H,

[0190] Where L G1 For chemical bonds, -CH2-, -CH2CH2-, -C(O)-, -CH2O-, -CH2O-CH2CH2O-, or -NHC(O)-(CH2NHC(O)) a -;

[0191] L G2 It is a chemical bond or -CH2CH2C(O)-;

[0192] L G3 It is a chemical bond, -(NHCH2CH2) b -、-(NHCH2CH2CH2) b -or-C(O)CH2-;

[0193] L G4 -(OCH2CH2) c -、-(OCH2CH2CH2) c -、-(OCH2CH2CH2CH2) c -、-(OCH2CH2CH2CH2CH2) c -or -NHC(O)-(CH2)d -;

[0194] Where a = 0, 1, 2 or 3;

[0195] b = 1, 2, 3, 4 or 5;

[0196] c = 1, 2, 3, 4 or 5;

[0197] d = 1, 2, 3, 4, 5, 6, 7 or 8;

[0198] A is a chemical bond, -CH2O- or -NHC(O)-;

[0199] A' is -O(CH2CH2O) e -;

[0200] Where e is 1, 2, 3, 4 or 5;

[0201] B is a chemical bond, -CH2-, -C(O)-, -M-, -CH2-M-, or -C(O)-M-;

[0202] Where M is

[0203] R G1 and R G2 Together they form -CH2CH2O- or -CH2CH(R) G )-O-, and R G3 For H;

[0204] Or R G1 and R G3 Together form -C 1-2 alkylene-, and R G2 For H;

[0205] Where R G For -OR G '、-CH2OR G 'or -CH2CH2OR G ', where R G The 'protecting group' is H, a hydroxyl protecting group, or a solid support, wherein the hydroxyl protecting group is preferably -C(O)CH2CH2C(O)OH or 4,4'-dimethoxytriphenylmethyl;

[0206] m1 = 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

[0207] n1 = 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0208] Any technical solution or any combination thereof in any of the above specific embodiments can be combined with any technical solution or any combination thereof in other specific embodiments. For example, any technical solution or any combination thereof of Q can be combined with any technical solution or any combination thereof of X, Y1, Y2, L2, L3, R1, R2, R3, R4, R5, R6, R7, R8, Base, and Base', etc. This invention aims to include combinations of all these technical solutions; due to space limitations, they will not be listed one by one.

[0209] In one aspect, the present invention specifically relates to the following technical solutions:

[0210] A1. The nucleotide monomer shown in Formula I,

[0211]

[0212] in,

[0213] L1 is OH or OP1, or a chemical bond that connects to the 2' or 3' end of the ribose of another nucleotide, nucleoside, or oligonucleotide;

[0214] L2 is H or P2, or a chemical bond that connects to the 5' end of the phosphate P atom of the ribose of another nucleotide, nucleoside, or oligonucleotide.

[0215] X is a chemical bond or CR1R2;

[0216] Y1 is O, S, or NR;

[0217] Y2 is an O, S, or chemical bond;

[0218] R1, R2, R4, R5, R6, and R7 are independently selected from H, D, halogens, CN, and C. 1-6 Alkyl, C 1-6 Haloalkyl, C 2-6 alkenyl, C 2-6 alkynyl group, C 3-10 Cycloalkyl, 3-10 membered heterocyclic groups, C 6-10 Aryl or 5-14 heteroaryl, which may optionally be substituted by 1, 2, 3, 4, 5, 6, 7, 8 or more R';

[0219] R3 is selected from H and C. 1-6 Alkyl, C 1-6 Haloalkyl, C 2-6 alkenyl, C 2-6 alkynyl group, C 3-10 Cycloalkyl, 3-10 membered heterocyclic groups, C 6-10 Aryl or 5-14 heteroaryl, which may optionally be substituted by 1, 2, 3, 4, 5, 6, 7, 8 or more R';

[0220] Base is H, a modified or unmodified base, or a leaving group;

[0221] Wherein P1 and P2 are independently selected from hydroxyl protecting groups, preferably silane protecting groups, acyl protecting groups, or ether protecting groups, more preferably...

[0222] Trimethylsilyl (TMS), Triethylsilyl (TES), Dimethylisopropylsilyl (DMIPS), Diethylisopropylsilyl (DEIPS), Tert-butyldimethylsilyl (TBDMS), Tert-butyldiphenylsilyl (TBDPS), Triisopropylsilyl (TIPS)

[0223] Acetyl (Ac), chloroacetyl, dichloroacetyl, trichloroacetyl, trifluoroacetyl (TFA), benzoyl, p-methoxybenzoyl, 9-fluorenylmethoxycarbonyl (Fmoc), allyloxycarbonyl (Alloc), 2,2,2-trichloroethoxycarbonyl (Troc), benzyloxycarbonyl (Cbz), tert-butyloxycarbonyl (Boc)

[0224] Benzyl (Bn), p-methoxybenzyl (PMB), allyl, triphenylmethyl (Tr), bis-p-methoxytriphenylmethyl (DMTr), methoxymethyl (MOM), phenoxymethyl (BOM), 2,2,2-trichloroethoxymethyl, 2-methoxyethoxymethyl (MEM), methylthiomethyl (MTM), p-methoxybenzylmethyl (PMBM);

[0225] R is selected from H and C. 1-6 Alkyl or C 1-6 Halogenated alkyl groups;

[0226] R' is selected from D, halogen, CN, C. 1-6 Alkyl, C 1-6 Haloalkyl, C 2-6 alkenyl, C 2-6 alkynyl group, C 3-10 Cycloalkyl, 3-10 membered heterocyclic groups, C 6-10 Aryl, 5-14 heteroaryl, -OR a -OC(O)R a -C(O)R a -C(O)OR a -C(O)NR a R b -S(O) n R a -S(O) n OR a -S(O) n NR a R b -NR a R b-NR a C(O)R b -NR a -C(O)OR b -NR a -S(O) n R b or -NR a C(O)NR a R b ;

[0227] n is either 1 or 2 independently;

[0228] R a and R b Independently selected from H and C 1-6 Alkyl, C 2-6 alkenyl, C 2-6 alkynyl group, C 3-10 Cycloalkyl, 3-10 membered heterocyclic groups, C 6-10 aryl or 5-14 heteroaryl; or R a and R b And the nitrogen atoms they connect to form 3-10 membered heterocyclic groups.

[0229] A2. The nucleotide monomer of technical solution A1, wherein L1 is a chemical bond attached to the 2' or 3' end hydroxyl group of the ribose of another nucleotide, nucleoside or oligonucleotide, preferably L1 is a chemical bond attached to the 3' end hydroxyl group of the ribose of another nucleotide, nucleoside or oligonucleotide.

[0230] A3. A nucleotide monomer of technical solution A1 or A2, wherein L2 is H, or a chemical bond attached to the 5' end of the ribose of another nucleotide, nucleoside or oligonucleotide; preferably L2 is H; preferably L2 is a chemical bond attached to the 5' end of the ribose of another nucleotide, nucleoside or oligonucleotide.

[0231] A4. A nucleotide monomer of any one of the technical solutions A1-A3, wherein X is a chemical bond.

[0232] A5. A nucleotide monomer of any one of technical solutions A1-A3, wherein X is CR1R2; preferably CH2.

[0233] A6. A nucleotide monomer of any one of the technical solutions A1-A5, wherein Y1 is O.

[0234] A7. A nucleotide monomer of any one of technical solutions A1-A6, wherein Y2 is O.

[0235] A8. A nucleotide monomer of any one of technical solutions A1-A7, wherein R1, R2, R4, R5, R6 and R7 are independently selected from H, D, halogen, CN, C.1-6 Alkyl, C 1-6 Haloalkyl, C 2-6 alkenyl, C 2-6 alkynyl group, C 4-8 Cycloalkyl, 4-7 membered heterocyclic groups, C 6-10 Aryl and 5-6 quinone heteroaryl groups, preferably H, D, halogen, CN, or C. 1-6 Alkyl, C 1-6 Haloalkyl, C 2-6 alkenyl or C 2-6 Alkyne group, preferably H, D, halogen or C 1-6 Alkyl, more preferably H or D.

[0236] A9. A nucleotide monomer of any one of technical solutions A1-A8, wherein R3 is selected from H, C 1-6 Alkyl, C 1-6 Haloalkyl, C 2-6 alkenyl, C 2-6 alkynyl group, C 3-10 Cycloalkyl, 3-10 membered heterocyclic groups, C 6-10 Aryl or 5-14 quinone heteroaryl, preferably H or C 1-6 Alkyl or C 1-6 Halogenated alkyl groups.

[0237] A10. A nucleotide monomer of any one of technical solutions A1-A9, wherein Base is a modified or unmodified base, preferably modified or unmodified A, U, T, G and C, more preferably modified or unmodified G or C.

[0238] A11. A nucleotide monomer of any one of technical solutions A1-A9, wherein Base is a leaving group; preferably selected from halogens, hydroxyl groups, -OCOR", -OTs or -ONO2, wherein R" is C 1-6 Alkyl, C 2-6 alkenyl, C 2-6 alkynyl group, C 3-10 Cycloalkyl, 3-10 membered heterocyclic groups, C 6-10 Aryl or 5-14 aryl; preferably halogen or -OTs; more preferably -OTs.

[0239] A12. The nucleotide monomer of technical solution A1 has one of the following structures:

[0240]

[0241] in,

[0242] L1 is OH or OP1, or a chemical bond that connects to the 3' end of the ribose of another nucleotide, nucleoside, or oligonucleotide;

[0243] L2 is H or P2, or a chemical bond that connects to the 5' end of the phosphate P atom of the ribose of another nucleotide, nucleoside, or oligonucleotide.

[0244] R3 is selected from H and C. 1-6 Alkyl or C 1-6 Halogenated alkyl groups;

[0245] Base is a modified or unmodified base, preferably modified or unmodified A, U, T, G and C, more preferably modified or unmodified G or C.

[0246] A13. The nucleotide dimer shown in Formula III,

[0247]

[0248] in,

[0249] L3 is H or P2, or a chemical bond that connects to the 2' or 3' end of the ribose of another nucleotide, nucleoside, or oligonucleotide;

[0250] R8 is selected from H, OH, halogens, and C. 1-6 Alkyl, C 1-6 Halogenated alkyl or C 1-6 Alkyl group, preferably fluorine or methoxy group, preferably H;

[0251] Base' is H, a modified or unmodified base or a leaving group, preferably modified or unmodified A, U, T, G and C;

[0252] Other groups are as defined in any of technical solutions A1-A12.

[0253] A14. The nucleotide dimer of technical solution A13 has the following structure:

[0254]

[0255] A15. A nucleic acid molecule, wherein the nucleotide sequence of the nucleic acid molecule comprises one or more nucleotide monomers as described in any one of technical solutions A1-A12 and / or nucleotide dimers as described in technical solutions A13 or A14.

[0256] A16. The nucleic acid molecule of technical solution A15, wherein the nucleic acid is selected from DNA, RNA and DNA / RNA hybrids.

[0257] A17. The nucleic acid molecule of technical solution A15 or A16, wherein the nucleic acid molecule is single-stranded or double-stranded.

[0258] A18. A nucleic acid molecule of any one of technical solutions A15-A17, wherein the nucleic acid molecule is selected from small interfering RNA (siRNA) and short hairpin RNA (shRNA).

[0259] A19. A double-stranded RNA molecule comprising a sense strand and an antisense strand, wherein each strand has 14 to 30 nucleotides, and said antisense strand comprises one or more nucleotide monomers as described in any one of technical solutions A1-A12 and / or one or more nucleotide dimers as described in technical solutions A13 or A14.

[0260] A20. A double-stranded RNA molecule of technical solution A19, wherein the sense strand and antisense strand each have 20 to 25 nucleotides; preferably, the length of the sense strand is 20 nucleotides; preferably, the length of the sense strand is 21 nucleotides; preferably, the length of the sense strand is 22 nucleotides; preferably, the length of the sense strand is 23 nucleotides; preferably, the length of the sense strand is 24 nucleotides; preferably, the length of the sense strand is 25 nucleotides; preferably, the length of the antisense strand is 20 nucleotides; preferably, the length of the antisense strand is 21 nucleotides; preferably, the length of the antisense strand is 22 nucleotides; preferably, the length of the antisense strand is 23 nucleotides; preferably, the length of the antisense strand is 24 nucleotides; preferably, the length of the antisense strand is 25 nucleotides.

[0261] A21. A double-stranded RNA molecule of technical solution A19 or A20, wherein the nucleotide monomer is located at any one or more positions on the sense strand or the antisense strand, including but not limited to positions 1-30 of the 5' end of the antisense strand and / or any one or more positions 1-30 of the 5' end of the sense strand, preferably any one or more positions 2-8 of the 5' end of the antisense strand, preferably position 6 or 7 of the 5' end of the antisense strand, more preferably position 7 of the 5' end of the antisense strand.

[0262] A22. A double-stranded RNA molecule of any one of technical solutions A19-A21, wherein the nucleotide dimer is located at any one or more positions on the sense strand or the antisense strand, including but not limited to positions 1-30 of the 5' end of the antisense strand and / or any one or more positions 1-30 of the 5' end of the sense strand, preferably positions 2-9 of the 5' end of the antisense strand, preferably positions 6 and 7 of the 5' end of the antisense strand, and preferably positions 7 and 8 of the 5' end of the antisense strand.

[0263] A23. A double-stranded RNA molecule of any one of technical solutions A19-A22, wherein the double-stranded RNA exhibits enhanced stability compared to a double-stranded RNA having the same sequence but not containing a nucleotide monomer of any one of technical solutions 1-12 or a nucleotide dimer of technical solutions 13 or 14.

[0264] A24. A double-stranded RNA molecule of technical solution A23, wherein the double-stranded RNA has a melting temperature from about 40°C to about 80°C, preferably from about 55°C to 67°C.

[0265] A25. A double-stranded RNA molecule of any one of technical solutions A19-A24, wherein the double-stranded RNA exhibits reduced off-target toxicity compared to a double-stranded RNA having the same sequence but not containing a nucleotide monomer of any one of technical solutions A1-A12 or a nucleotide dimer of technical solutions A13 or A14.

[0266] A26. A double-stranded RNA molecule of any one of technical solutions A19-A25, wherein the double-stranded RNA exhibits enhanced effectiveness compared to a double-stranded RNA having the same sequence but not containing a nucleotide monomer or a nucleotide dimer as described in any one of technical solutions A1-A12 or technical solutions A13 or A14.

[0267] A27. A double-stranded RNA molecule of any one of technical solutions 19-26, wherein the antisense strand has a sequence that is fully complementary to the sense strand and the target mRNA, and has the ability to induce the degradation of the target mRNA.

[0268] A28. A double-stranded RNA molecule of technical solution A27, wherein the target mRNA is encoded by an endogenous gene or by a pathogen gene.

[0269] A29. A double-stranded RNA molecule of any one of technical solutions A19-A28, wherein the sense strand and / or antisense strand comprises a 3' and / or 5' overhang.

[0270] A30. A double-stranded RNA molecule of any one of technical solutions A19-A29, wherein the double-stranded RNA is further coupled to a ligand, preferably, the ligand comprising one or more GalNAc.

[0271] A31. A pharmaceutical composition comprising a double-stranded RNA molecule as described in any one of technical solutions A19-A30, and a pharmaceutically acceptable carrier or excipient.

[0272] A32. A kit comprising a double-stranded RNA molecule as described in any one of technical solutions A19-A30.

[0273] A33. A method for inhibiting the expression of a target gene in a cell, comprising the step of introducing a double-stranded RNA molecule, as described in any one of technical solutions A19-A30, into the cell.

[0274] A34. A method for inhibiting the expression of a target gene in a cell, comprising expressing a double-stranded RNA molecule as described in any one of technical solutions A19-A30 in the cell.

[0275] In another aspect, the present invention relates to the following technical solutions:

[0276] B1. Nucleotide dimers represented by Formula III:

[0277]

[0278] in,

[0279] L2 is H or P2, or a chemical bond that connects to the 5' end of the phosphate P atom of the ribose of another nucleotide or oligonucleotide;

[0280] L3 is H or P3, or a chemical bond that connects to the 2' or 3' end of the ribose of another nucleotide or oligonucleotide;

[0281] X is a chemical bond or CR1R2;

[0282] Y1 is O, S, or NR;

[0283] Y2 is an O, S, or chemical bond;

[0284] R1, R2, R4, R5, R6, and R7 are independently selected from H, D, halogens, CN, and C. 1-6 Alkyl, C 1-6 Haloalkyl, C 2-6 alkenyl, C 2-6 alkynyl group, C 3-10 Cycloalkyl, 3-10 membered heterocyclic groups, C 6-10 Aryl or 5-14 heteroaryl, which may optionally be substituted by 1, 2, 3, 4, 5, 6, 7, 8 or more R';

[0285] R3 is selected from H and C. 1-6 Alkyl, C 1-6 Haloalkyl, C 2-6 alkenyl, C 2-6 alkynyl group, C 3-10 Cycloalkyl, 3-10 membered heterocyclic groups, C 6-10 Aryl or 5-14 heteroaryl, which may optionally be substituted by 1, 2, 3, 4, 5, 6, 7, 8 or more R';

[0286] R8 is selected from H, OH, halogens, and C. 1-6 Alkyl, C1-6 Halogenated alkyl or C 1-6 Alkyl group, preferably fluorine or methoxy group, preferably H;

[0287] Base is H, a modified or unmodified base, or a leaving group;

[0288] Base' is H, a modified or unmodified base or a leaving group, preferably modified or unmodified A, U, T, G and C;

[0289] Wherein P2 and P3 are independently selected from hydroxyl protecting groups, preferably silane protecting groups, acyl protecting groups, or ether protecting groups, more preferably...

[0290] Trimethylsilyl (TMS), Triethylsilyl (TES), Dimethylisopropylsilyl (DMIPS), Diethylisopropylsilyl (DEIPS), Tert-butyldimethylsilyl (TBDMS), Tert-butyldiphenylsilyl (TBDPS), Triisopropylsilyl (TIPS)

[0291] Acetyl (Ac), chloroacetyl, dichloroacetyl, trichloroacetyl, trifluoroacetyl (TFA), benzoyl, p-methoxybenzoyl, 9-fluorenylmethoxycarbonyl (Fmoc), allyloxycarbonyl (Alloc), 2,2,2-trichloroethoxycarbonyl (Troc), benzyloxycarbonyl (Cbz), tert-butyloxycarbonyl (Boc)

[0292] Benzyl (Bn), p-methoxybenzyl (PMB), allyl, triphenylmethyl (Tr), bis-p-methoxytriphenylmethyl (DMTr), methoxymethyl (MOM), phenoxymethyl (BOM), 2,2,2-trichloroethoxymethyl, 2-methoxyethoxymethyl (MEM), methylthiomethyl (MTM), p-methoxybenzylmethyl (PMBM);

[0293] R is selected from H and C. 1-6 Alkyl or C 1-6 Halogenated alkyl groups;

[0294] R' is selected from D, halogen, CN, C. 1-6 Alkyl, C 1-6 Haloalkyl, C 2-6 alkenyl, C 2-6 alkynyl group, C 3-10 Cycloalkyl, 3-10 membered heterocyclic groups, C 6-10 Aryl, 5-14 heteroaryl, -OR a -OC(O)R a -C(O)R a -C(O)OR a -C(O)NR a R b -S(O)n R a -S(O) n OR a -S(O) n NR a R b -NR a R b -NR a C(O)R b -NR a -C(O)OR b -NR a -S(O) n R b or -NR a C(O)NR a R b ;

[0295] n is either 1 or 2 independently;

[0296] R a and R b Independently selected from H and C 1-6 Alkyl, C 2-6 alkenyl, C 2-6 alkynyl group, C 3-10 Cycloalkyl, 3-10 membered heterocyclic groups, C 6-10 aryl or 5-14 heteroaryl; or R a and R b And the nitrogen atoms they connect to form 3-10 membered heterocyclic groups.

[0297] B2. The nucleotide dimer of technical solution B1, wherein L3 is a chemical bond attached to the 2' or 3' end hydroxyl group of the ribose of another nucleotide or oligonucleotide, preferably L3 is a chemical bond attached to the 3' end hydroxyl group of the ribose of another nucleotide or oligonucleotide.

[0298] B3. A nucleotide dimer of technical solution B1 or B2, wherein L2 is H, or a chemical bond attached to a phosphate P atom at the 5' end of the ribose of another nucleotide or oligonucleotide; preferably L2 is H; preferably L2 is a chemical bond attached to a phosphate P atom at the 5' end of the ribose of another nucleotide or oligonucleotide.

[0299] B4. A nucleotide dimer of any one of technical solutions B1-B3, wherein X is a chemical bond.

[0300] B5. A nucleotide dimer of any one of technical solutions B1-B4, wherein X is CR1R2; preferably CH2.

[0301] B6. A nucleotide dimer of any one of technical solutions B1-B5, wherein Y1 is O.

[0302] B7. A nucleotide dimer of any one of technical solutions B1-B6, wherein Y2 is O.

[0303] B8. A nucleotide dimer of any one of technical solutions B1-B7, wherein R1, R2, R4, R5, R6 and R7 are independently selected from H, D, halogen, CN, and C. 1-6 Alkyl, C 1-6 Haloalkyl, C 2-6 alkenyl, C 2-6 alkynyl group, C 4-8 Cycloalkyl, 4-7 membered heterocyclic groups, C 6-10 Aryl and 5-6 quinone heteroaryl groups, preferably H, D, halogen, CN, or C. 1-6 Alkyl, C 1-6 Haloalkyl, C 2-6 alkenyl or C 2-6 Alkyne group, preferably H, D, halogen or C 1-6 Alkyl, more preferably H or D.

[0304] B9. A nucleotide dimer of any one of technical solutions B1-B8, wherein R3 is selected from H, C 1-6 Alkyl, C 1-6 Haloalkyl, C 2-6 alkenyl, C 2-6 alkynyl group, C 3-10 Cycloalkyl, 3-10 membered heterocyclic groups, C 6-10 Aryl or 5-14 quinone heteroaryl, preferably H or C 1-6 Alkyl or C 1-6 Halogenated alkyl groups.

[0305] B10. A nucleotide dimer of any one of technical solutions B1-B9, wherein Base is a modified or unmodified base, preferably modified or unmodified A, U, T, G and C, more preferably modified or unmodified G or C.

[0306] B11. A nucleotide dimer of any one of technical solutions B1-B9, wherein Base is a leaving group; preferably selected from halogens, hydroxyl groups, -OCOR", -OTs or -ONO2, wherein R" is C 1-6 Alkyl, C 2-6 alkenyl, C 2-6 alkynyl group, C 3-10 Cycloalkyl, 3-10 membered heterocyclic groups, C 6-10 Aryl or 5-14 aryl; preferably halogen or -OTs; more preferably -OTs.

[0307] B12. A nucleotide dimer of any one of technical solutions B1-B11, having one of the following structures:

[0308]

[0309] in,

[0310] L2 is H or P2, or a chemical bond that connects to the 5' end of the phosphate P atom of the ribose of another nucleotide or oligonucleotide;

[0311] L3 is H or P3, or a chemical bond that connects to the 2' or 3' end of the ribose of another nucleotide or oligonucleotide;

[0312] R3 is selected from H and C. 1-6 Alkyl or C 1-6 Halogenated alkyl groups;

[0313] R8 is selected from H, OH, halogens, and C. 1-6 Alkyl, C 1-6 Halogenated alkyl or C 1-6 Alkyl group, preferably fluorine or methoxy group, preferably H;

[0314] Base is a modified or unmodified base, preferably modified or unmodified A, U, T, G and C, more preferably modified or unmodified G or C;

[0315] Base' is H, a modified or unmodified base, or a leaving group, preferably modified or unmodified A, U, T, G, and C.

[0316] In another aspect, the present invention relates to the following technical solutions:

[0317] C1. The nucleotide dimer represented by formula (A),

[0318]

[0319] in,

[0320] L2 is either H or P2;

[0321] L3 is either H or P3;

[0322] Q is -X- or -XO-;

[0323] X is a chemical bond, -(CR1R2) m -or -CR1 = CR2-;

[0324] Y1 is O, S, or NR;

[0325] Y2 is an O, S, or chemical bond;

[0326] R1, R2, R4, R5, R6, and R7 are independently selected from H, D, halogens, CN, and C. 1-6 Alkyl, C 1-6 Haloalkyl, C 2-6 alkenyl or C 2-6The alkynyl group, which is optionally substituted with 1, 2, 3, 4 or 5 R' groups;

[0327] R3 is selected from H and C. 1-6 Cyanoalkyl, C 1-6 Alkyl, C 1-6 Haloalkyl, C 2-6 alkenyl, C 2-6 alkynyl group, C 3-7 Cycloalkyl, 3-7 membered heterocyclic groups, C 6-10 Aryl or 5-10 heteroaryl groups, which are optionally substituted by 1, 2, 3, 4 or 5 R's;

[0328] R8 is selected from H, D, OH, halogens, and C. 1-6 Alkyl, C 1-6 Halogenated alkyl or C 1-6 Alkoxy;

[0329] Base and Base' are independently selected from H, modified or unmodified bases, or leaving groups;

[0330] P2 is selected from reactive phosphorus groups, preferably -P(OCH2CH2CN)(N(iPr)2);

[0331] P3 is selected from hydroxyl protecting groups, preferably DMTr;

[0332] R is selected from H and C. 1-6 Alkyl or C 1-6 Halogenated alkyl groups;

[0333] R' is selected from D, halogen, OH, CN, NH2, C 1-6 Alkyl, C 1-6 Haloalkyl, C 2-6 alkenyl, C 2-6 alkynyl group, C 3-10 Cycloalkyl, 3-10 membered heterocyclic groups, C 6-10 Aryl or 5-14 heteroaryl groups;

[0334] m is selected from 1, 2, 3, 4 or 5.

[0335] C2. The nucleotide dimer of technical solution C1, wherein,

[0336] L2 is P2;

[0337] L3 is P3;

[0338] Q is -X- or -XO-;

[0339] X is a chemical bond, -(CR1R2) m -or -CR1 = CR2-;

[0340] Y1 is O or S;

[0341] Y2 is O or a chemical bond;

[0342] R1, R2, R4, R5, R6, and R7 are independently selected from H, D, halogens, CN, and C. 1-6 Alkyl or C 1-6 Haloalkyl, preferably H, which is optionally substituted with 1, 2 or 3 R';

[0343] R3 is selected from H and C. 1-6 Alkyl, C 1-6 Cyanoalkyl, C 1-6 Haloalkyl, C 2-6 alkenyl or C 2-6 The alkynyl group, which is optionally substituted by 1, 2 or 3 R';

[0344] R8 is selected from H, OH, halogen, or C. 1-6 Alkyl group, preferably fluorine or methoxy group, preferably fluorine;

[0345] Base and Base' are independently selected from H, modified or unmodified bases, or leaving groups. Preferably, Base and Base' are independently selected from...

[0346] P2 is selected from reactive phosphorus groups, preferably -P(OCH2CH2CN)(N(iPr)2);

[0347] P3 is selected from hydroxyl protecting groups, preferably DMTr;

[0348] R' is selected from D, halogen, OH, CN, NH2, C 1-6 Alkyl or C 1-6 Halogenated alkyl groups;

[0349] m is selected from 1, 2, or 3.

[0350] C3. A nucleotide dimer of technical solution C1 or C2, wherein,

[0351] L2 is P2;

[0352] L3 is P3;

[0353] Q is -X- or -XO-;

[0354] X is -CH2-, -CH2-CH2-, or -CH=CH-;

[0355] Y1 is O;

[0356] Y2 is O;

[0357] R1, R2, R4, R5, R6, and R7 are independently selected from H, halogens, or C.1-4 Alkyl group, preferably H;

[0358] R3 is selected from H and C. 1-4 Alkyl or C 1-4 Cyanoalkyl, preferably methyl or cyanoethyl;

[0359] R8 is selected from halogen or C. 1-4 Alkyl group, preferably fluorine or methoxy group, preferably fluorine;

[0360] Base and Base' are selected independently.

[0361] P2 is selected from reactive phosphorus groups, preferably -P(OCH2CH2CN)(N(iPr)2);

[0362] P3 is selected from hydroxyl protecting groups, preferably DMTr.

[0363] C4. A nucleotide dimer of any one of the technical solutions C1-C3, wherein Q is -CH2-O-.

[0364] C5. A nucleotide dimer of any one of technical solutions C1-C3, having the following structure:

[0365]

[0366] in,

[0367] R3 is methyl or cyanoethyl;

[0368] Other groups are as defined in any of technical solutions C1-C3.

[0369] C6. A nucleotide dimer of any one of technical solutions C1-C5, having the following structure:

[0370]

[0371] C7. A double-stranded RNA molecule or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof, comprising a sense strand and an antisense strand, each strand having 14 to 30 nucleotides, wherein the antisense strand has a sequence sufficiently complementary to the sense strand and the target mRNA and has the ability to induce the degradation of the target mRNA, and wherein the antisense strand comprises one or more nucleotide monomers of formula (IV):

[0372]

[0373] in,

[0374] The nucleotide monomers shown are arranged in the order of 5' => 3' from... to connect;

[0375] Each group is as defined in any one of technical solutions C1-C6;

[0376] Preferably, the nucleotide monomer is selected from:

[0377]

[0378] Where Base is selected from

[0379] C8. The double-stranded RNA molecule of technical solution C7, wherein the double-stranded RNA molecule is prepared by using a nucleotide dimer of any one of technical solutions C1-C6.

[0380] C9. A double-stranded RNA molecule of C7 or C8, wherein the sense strand and the antisense strand each have 20 to 30 nucleotides.

[0381] C10. A double-stranded RNA molecule of any one of technical solutions C7-C9, wherein the nucleotide monomer is located at position 2-8, preferably position 6 or 7, more preferably position 7, at the 5' end of the antisense strand.

[0382] C11. A double-stranded RNA molecule of any one of technical solutions C7-C10, wherein the double-stranded RNA exhibits enhanced stability compared to a double-stranded RNA having the same sequence but not containing the nucleotide monomers described in technical solution C7.

[0383] C12. A double-stranded RNA molecule of technical solution C11, wherein the double-stranded RNA has a melting temperature from about 40°C to about 80°C, preferably from about 55°C to 67°C.

[0384] C13. A double-stranded RNA molecule of any one of technical solutions C7-C12, wherein the double-stranded RNA exhibits reduced off-target toxicity compared to a double-stranded RNA having the same sequence but not containing the nucleotide monomers described in technical solution C7.

[0385] C14. A double-stranded RNA molecule of any one of technical solutions C7-C13, wherein the double-stranded RNA exhibits enhanced effectiveness compared to a double-stranded RNA having the same sequence but not containing the nucleotide monomers described in technical solution C7.

[0386] C15. A double-stranded RNA molecule of any one of technical solutions C7-C14, wherein the sense strand and / or antisense strand comprises a 3' and / or 5' overhang.

[0387] C16. A double-stranded RNA molecule of any one of technical solutions C7-C15, wherein the double-stranded RNA is further coupled to a ligand, preferably, the ligand comprising one or more GalNAc.

[0388] C17. A method for preparing a double-stranded RNA molecule according to any one of technical solutions C7-C16, wherein the double-stranded RNA molecule is prepared by using a nucleotide dimer according to any one of technical solutions C1-C6.

[0389] C18. A nucleic acid molecule, wherein the nucleotide sequence of the nucleic acid molecule contains one or more nucleotide monomers as described in technical solution C7.

[0390] C19. The nucleic acid molecule of technical solution C18, wherein the nucleic acid is selected from DNA, RNA and DNA / RNA hybrids.

[0391] C20. The nucleic acid molecule of technical solution C19, wherein the nucleic acid molecule is single-stranded or double-stranded.

[0392] C21. A nucleic acid molecule of any one of technical solutions C18-C20, wherein the nucleic acid molecule is selected from small interfering RNA (siRNA) and short hairpin RNA (shRNA).

[0393] C21. A pharmaceutical composition comprising a double-stranded RNA molecule as described in any one of technical solutions C7-C16, and a pharmaceutically acceptable carrier or excipient.

[0394] C22. A kit comprising a double-stranded RNA molecule as described in any one of technical solutions C7-C16.

[0395] C23. A method for inhibiting the expression of a target gene in a cell, comprising the step of introducing a double-stranded RNA molecule, as described in any one of technical solutions C7-C16, into the cell.

[0396] C24. A method for inhibiting the expression of a target gene in a cell, comprising expressing a double-stranded RNA molecule as described in any one of the technical solutions C7-C16 in the cell. Example

[0397] The following examples are for illustrative purposes only and are not intended to limit the scope of the invention.

[0398] Example 1: Synthesis of nucleotide dimers of compound 14

[0399] Synthesis route:

[0400]

[0401] Experimental steps:

[0402] 1. Synthesis of Compound 2

[0403] Compound 1 (25 g, 189.165 mmol) was dissolved in 250 mL of dichloromethane, followed by the sequential addition of p-toluenesulfonyl chloride (54.09 g, 283.747 mmol), triethylamine (47.85 g, 472.912 mmol), and 4-dimethylaminopyridine (2.31 g, 18.916 mmol). The reaction mixture was stirred overnight at room temperature. The reaction mixture was monitored by TLC until complete. The reaction mixture was then diluted with 300 mL of dichloromethane and washed successively with saturated NaHCO3 aqueous solution (3 x 300 mL), citric acid (1 x 200 mL), and saturated brine (300 mL). The organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give compound 2 (42.0 g, 286.34 mmol, 77.54%) as a yellow oil.

[0404] m / z = 286.2[M+H] +

[0405] 2. Synthesis of Compound 3

[0406] Under argon protection, adenine (23.78 g, 176.015 mmol) was dissolved in 600 mL of N,N-dimethylformamide. Sodium hydroxide (5.28 g, 220.018 mmol) was added dropwise in an ice-water bath. The reaction mixture was heated to 100 °C and stirred for 2 hours. Then, compound 2 (42 g, 146.679 mmol) was dissolved in 200 mL of N,N-dimethylformamide and added dropwise to the above reaction system. The mixture was stirred overnight at 100 °C. The reaction mixture was monitored by TLC until complete. The reaction was then quenched with methanol (20 mL). The reaction solvent was evaporated to dryness under reduced pressure. The crude product was purified by silica gel column chromatography (100-200 mesh, methanol:dichloromethane = 0-10%, 30 min) to obtain compound 3 (1 g, 249.27 mmol, 49.23%) as a white solid.

[0407] m / z = 250.2[M+H] +

[0408] 3. Synthesis of Compound 4

[0409] Compound 3 (18 g, 72.211 mmol) was dissolved in 200 mL of anhydrous pyridine. Benzoyl chloride (20.30 g, 144.422 mmol) was added at room temperature and the mixture was stirred overnight. The reaction mixture was monitored by TLC until the reactants were completely reacted. Then, ammonia-methanol solution (7 M, 200 mL) was added, and the mixture was stirred for 10 minutes. The reaction mixture was then evaporated to dryness under reduced pressure. The crude product was purified by silica gel column chromatography (methanol:dichloromethane = 0-10%, 30 min) to obtain compound 4 (16 g, 31.513 mmol, 43.64%) as a yellow solid.

[0410] m / z = 354.2[M+H] +

[0411] 4. Synthesis of Compound 5

[0412] Compound 4 (25 g, 70.740 mmol) was dissolved in a mixed solution of trifluoroacetic acid (180 mL) and water (60 mL), and the reaction mixture was stirred at room temperature for 3 hours. The reaction mixture was monitored by TLC until complete. The reaction mixture was then evaporated to dryness under reduced pressure, and the crude product was purified by silica gel column chromatography (methanol:dichloromethane = 0-15%, 30 min) to obtain compound 5 (16 g, 51.066 mmol, 72.19%) as a white solid.

[0413] m / z = 314.0 [M+H] +

[0414] 5. Synthesis of Compound 6

[0415] Compound 5 (3.9 g, 12.447 mmol) was dissolved in anhydrous pyridine (40 mL) after being rinsed three times with water. 4,4-Dimethoxytriphenylchloromethane (5.06 g, 14.937 mmol) was added at room temperature under argon protection. The reaction mixture was stirred overnight at room temperature, and the conversion of the starting material was confirmed by LC-MS. The reaction mixture was diluted with ethyl acetate (200 mL), washed twice with 50 mL of water each time, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to dryness under reduced pressure. The crude product was purified by silica gel column chromatography (ethyl acetate: petroleum ether = 0-50%, 40 min) to give compound 6 (6.2 g, 10.077 mmol, 80.96%) as a yellow solid.

[0416] m / z = 616.2[M+H] +

[0417] 6. Synthesis of Compound 7

[0418] Dimethyl hydroxymethyl phosphite (9.3 g, 66.395 mmol) was dissolved in dichloromethane (100 mL). The reaction solution was cooled to 0°C, and triethylamine (11.997 mL, 86.314 mmol) and p-toluenesulfonyl chloride (13.92 g, 73.035 mmol) were slowly added under argon protection. The reaction mixture was stirred overnight at room temperature. The reaction was monitored by LC-MS until the reactants were fully reacted. The reaction solution was diluted with dichloromethane, washed with water, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (methanol:dichloromethane = 0-20%, 40 min) to give compound 7 (11 g, 37.382 mmol, 56.30%) as a pale yellow solid.

[0419] m / z = 294.8 [M+H] +

[0420] 7. Synthesis of Compound 8

[0421] Compound 6 (400 mg, 0.650 mmol) was dissolved in anhydrous N,N-dimethylformamide (20 mL) under nitrogen protection. Sodium hydroxide (46.77 mg, 1.950 mmol) was added under an ice-water bath, and the reaction mixture was stirred for 30 minutes under an ice-water bath. Compound 7 (382.35 mg, 1.300 mmol) was dissolved in anhydrous N,N-dimethylformamide (2 mL) and slowly added dropwise to the above reaction system. The reaction mixture was stirred at room temperature for 72 hours, and the conversion of the starting material was monitored by LC-MS to ensure complete conversion. The reaction was quenched with ice water, diluted with dichloromethane (100 mL), the organic phase was washed with ice water, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give crude compound 8 (510 mg) as a yellow solid.

[0422] m / z = 294.8 [M+H] +

[0423] 8. Synthesis of Compound 9

[0424] Compound 8 (500 mg, 0.678 mmol) was dissolved in methanol / dichloromethane (1:3, 8 mL), and p-toluenesulfonic acid (64.46 mg, 0.339 mmol) was added at room temperature. The reaction mixture was stirred at room temperature for 2 hours and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (methanol:dichloromethane = 0-10%, 40 min) to give compound 9 (145 mg, 49.14%) as a white solid.

[0425] 1H NMR (400MHz, DMSO-d6): δ11.14(s,1H),8.74(s,1H),8.39(s,1H),8.03-8.06(m,2H),7.52-7.68(m,3 H),4.99-5.03(m,1H),4.31-4.53(m,2H),3.83-4.12(m,3H),3.57(s,3H),3.53(s,3H),3.46(s,2H).

[0426] 9. Synthesis of Compound 10

[0427] Compound 9 (650 mg, 1.49 mmol) was dissolved in anhydrous N,N-dimethylformamide (5 mL) under nitrogen protection. Imidazole (304.8 mg, 4.48 mmol) and tert-butyldimethylchlorosilane (336.2 mg, 2.24 mmol) were added at room temperature. The reaction mixture was stirred overnight at room temperature. The reaction mixture was monitored by LC-MS until the starting material was completely converted. The reaction mixture was quenched with ice water and extracted with ethyl acetate (3 x 50 mL). The organic phase was washed with brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (methanol:dichloromethane = 0.3%, 40 min) to give compound 10 (710 mg, 1.29 mmol, 86%) as an off-white solid.

[0428] m / z = 550.4 [M+H] +

[0429] 10. Synthesis of Compound 11

[0430] Compound 10 (710 mg, 1.29 mmol) was dissolved in an aqueous pyridine solution (3:2, 20 mL), and the reaction mixture was stirred at 50°C for 6 hours. LC-MS was used to monitor the completion of the starting material conversion. The crude product was concentrated under reduced pressure and then directly proceeded to the next step of the reaction without purification.

[0431] m / z = 536.2 [M+H] +

[0432] 11. Synthesis of Compound 12

[0433] Under argon protection, crude compound 11 (450 mg) was dissolved in anhydrous pyridine (5 mL), and triisopropylchlorosilane (763.94 mg, 2.522 mmol) was added in an ice bath. The reaction mixture was stirred at room temperature for 15 minutes. Then, 5'-O-(4,4'-dimethoxytriphenylmethyl)-N2-isobutyryl-2'-fluorodeoxyguanosine (580.26 mg, 0.883 mmol) and N-methylimidazolium (345.15 mg, 4.204 mmol) dissolved in anhydrous pyridine (5 mL) were slowly added dropwise to the above reaction mixture. The reaction mixture was stirred at room temperature overnight. LC-MS was used to monitor the conversion of the starting material. The reaction was quenched with sodium bicarbonate aqueous solution (1.0 M), and the reaction mixture was concentrated under reduced pressure. The crude product was purified by reversed-phase chromatography (5 M ammonium bicarbonate aqueous solution / acetonitrile) to give compound 12 (480 mg, 63%) as a yellow solid.

[0434] m / z = 1175.6 [M+H] +

[0435] 1 H NMR (400MHz, DMSO-d6): δ8.65(s,1H),8.26(s,1H),8.08-8.20(m,1H),7.82-7.96(m,2H ),7.55-7.62(m,1H),7.45-7.50(m,2H),7.06-7.28(m,9H),7.70-7.82(m,4H),6.16-6.2 2(m,1H),5.68-5.85(m,1H),5.30-5.45(m,1H),3.86-4.42(m,5H),3.52-3.76(m,7H),3 .22-3.46(m,9H),2.03(s,2H),1.05-1.11(m,6H),0.81-0.83(m,9H),0.00-0.04(m,6H).

[0436] 19 F NMR(DMSO-d6):δ-204.82,-204.53

[0437] 31 P NMR (DMSO-d6): δ 22.94, 22.72

[0438] 12. Synthesis of Compound 13

[0439] Compound 12 (200 mg, 0.170 mmol) was dissolved in anhydrous tetrahydrofuran (10 mL) under nitrogen protection. A solution of triethylamine hydrofluoric acid (137.26 mg, 0.851 mmol) in tetrahydrofuran (2 mL) was slowly added dropwise under an ice-water bath. The reaction system was brought to room temperature and reacted for 1 hour. The conversion of the reactants was monitored by LC-MS. The reaction system was diluted with ethyl acetate, quenched with water, and the organic phase was separated. The mixture was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The crude product was purified by reverse-phase chromatography (preparation conditions: column: XBridge Shield RP18 OBD column, 19*150mm, 5μm; mobile phase A: water (10mmol / L NH4HCO3), mobile phase B: ACN; flow rate: 20mL / min; gradient: 50% B to 60% B, 60% B over 5.5 min; wavelength: 254 / 210nm; RT1 (min): 4.5, 5.1) to obtain compound 13 (69mg, 0.065mmol, 38.21%) as a white solid.

[0440] m / z = 1062.3 [M+H] +

[0441] 1 H NMR (400MHz, DMSO-d6): δ8.70(s,1H),8.31(s,1H),8.15(s,1H),7.98-8.00(m,2H),7.46-7.62(m,3H),7.16-7.40(m,9H),6.70-6.65(m,4H),6.2 5-6.20(m,1H),5.68-5.90(m,1H),5.38-5.46(m,1H),4.96-5.02(m,1H) ,3.82-4.48(m,12H),3.70(s,2H),3.25-3.46(m,9H),1.11-1.13(m,6H).

[0442] 19 F NMR (400MHz, DMSO-d6): δ-204.54

[0443] 31 P NMR (400MHz, DMSO-d6): δ 23.17

[0444] 13. Synthesis of Compound 14

[0445] First, bis(diisopropylamino)(2-cyanoethoxy)phosphine (23.45 mg, 0.078 mmol) was rinsed three times with ultra-dry acetonitrile, and then compound 13 (55 mg, 0.052 mmol) was rinsed three times with ultra-dry acetonitrile. Under argon protection, bis(diisopropylamino)(2-cyanoethoxy)phosphine (23.45 mg, 0.078 mmol) was dissolved in anhydrous dichloromethane (1.5 mL), and 4,5-dicyanimidazole (4.90 mg, 0.041 mmol) was added at room temperature. Then, a solution of compound 13 (55 mg, 0.052 mmol) in anhydrous dichloromethane (1 mL) was added to the above reaction system. The reaction mixture was stirred at room temperature for 2 hours, and the conversion of the starting materials was monitored by LC-MS. The reaction system was added dropwise to an ice-cold aqueous solution of sodium bicarbonate, extracted with dichloromethane, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The crude product was purified by reverse-phase extraction (using pure water and acetonitrile, yielding approximately 70% product). The collected solution was extracted with dichloromethane, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain compound 14 (30 mg, 0.024 mmol, 45.89%) as a white solid.

[0446] m / z = 1261.7 [M+H] +

[0447] 1 H NMR (400MHz, CD3CN): δ8.55(s,1H),8.09(s,1H),7.72-7.89(m,3H),7.41-7.53(m,3H),7.03-7.08(m,5 H),6.91-7.01(m,4H),6.50-6.73(m,4H),6.16-6.22(m,1H),5.95-6.00(m,1H),5.62-5.72(m,1H),4.44 -4.48(m,1H),4.22-4.26(m,1H),4.12-4.18(m,1H),3.91-4.08(m,2H),3.59-3.88(m,10H),3.50-3.54( m,2H),3.27-3.35(m,4H),2.98-3.02(m,1H),2.53-2.64(m,3H),2.00-2.10(m,1H),1.05-1.10(m,18H).

[0448] 31 P NMR (400MHz, CD3CN) δ148.69,148.57,23.54,23.50.

[0449] 19 F NMR(400MHz,CD3CN)δ-203.63,-203.69.

[0450] Example 2: Preparation of siRNA

[0451] The siRNA of the present invention was prepared using the solid-phase phosphoramide method well known in the art. Specific methods can be found, for example, in PCT publications WO2016081444 and WO2019105419, and are briefly described below.

[0452] 1. Synthesis of the Justice Chain (SS Chain)

[0453] The oligonucleotide synthesis method, using a solid-phase phosphoramide, employs a blank CPG solid support or an L96-linked solid support as the starting cycle. Nucleoside monomers are sequentially linked from the 3'-5' direction according to the nucleotide arrangement of the positive strand. Each linkage of a nucleoside monomer involves four steps: deprotection, coupling, capping, and oxidation or thiolation. The synthesis conditions for oligonucleotides at a scale of 5 μmol are as follows:

[0454] The nucleoside monomer was provided in a 0.05 mol / L acetonitrile solution. The reaction conditions were identical for each step: 25°C. Deprotection was performed three times using a 3% trichloroacetic acid-dichloromethane solution. The coupling reaction was activated twice using a 0.25 mol / L ETT-acetonitrile solution. Capping was performed twice using a 10% acetic anhydride-acetonitrile and pyridine / N-methylimidazole / acetonitrile mixture (10:14:76, v / v / v). Oxidation was performed twice using a 0.05 mol / L iodine / tetrahydrofuran / pyridine / water mixture (70 / 20 / 10, v / v / v). Thiolation was performed twice using a 0.2 mol / L PADS mixture of acetonitrile / 3-methylpyridine (1 / 1, v / v).

[0455] 2. Synthesis of the antisense chain (AS chain)

[0456] The solid-phase phosphoramide synthesis method utilizes a blank CPG solid-phase support as the starting cycle, and sequentially links nucleoside monomers or nucleotide dimers of the present invention from the 3'-5' direction according to the antisense strand nucleotide arrangement sequence. Each linking of a nucleoside monomer or nucleotide dimer of the present invention involves four steps: deprotection, coupling, capping, oxidation, or thiolation. The synthesis conditions for 5 μmol oligonucleotides of the antisense strand are the same as those for the sense strand.

[0457] 3. Purification and Annealing of Oligonucleotides

[0458] 3.1 Ammonolysis

[0459] The synthesized solid support (sense or antisense chain) was added to a 5 mL centrifuge tube, and 3% diethylamine / ammonia (v / v) was added. The mixture was reacted in a constant temperature water bath at 35℃ (or 55℃) for 16 hours (or 8 hours). After filtration, the solid support was washed three times with ethanol / water, 1 mL each time. The filtrate was concentrated by centrifugation and the crude product was purified.

[0460] 3.2 Purification

[0461] Purification and desalting methods are well known to those skilled in the art. For example, a column packed with strong anion exchange material can be used for elution purification with a sodium chloride-sodium hydroxide system, and the product can be collected and piped. Desalting can be performed using a gel-packed purification column with pure water as the elution system.

[0462] 3.3 Annealing

[0463] According to the table below, the sense chain (SS chain) and the antisense chain (AS chain) are mixed at a molar ratio (SS chain / AS chain = 1 / 1.05), heated in a water bath to 70-95℃, held for 3-5 minutes, and then naturally cooled to room temperature. The system is then freeze-dried to obtain the product.

[0464] The siRNA sequence used in this invention is as follows:

[0465]

[0466] The meanings of the abbreviations in this article are as follows:

[0467] The distributions A, U, G, and C represent naturally occurring adenine ribonucleotides, uracil ribonucleotides, guanine ribonucleotides, and cytosine ribonucleotides.

[0468] The 'm' indicates that the nucleotide adjacent to it on the left is a nucleotide modified with 2'-OCH3. For example, Am, Um, Gm, and Cm represent A, U, G, and C modified with 2'-OCH3.

[0469] The 'f' indicates that the nucleotide adjacent to it on the left is a 2'-F modified nucleotide. For example, Af, Uf, Gf, and Cf represent 2'-F modified A, U, G, and C, respectively.

[0470] “s” or “s-” indicates that the two adjacent nucleotides and / or delivery carriers are linked by a phosphate thioester.

[0471] L96 represents a GalNAc delivery vector with the following structure well known in the art, wherein... The location indicated by the phosphate ester group or thiophosphate ester group linked to siRNA can be found, for example, in PCT publications WO2009073809 and WO2009082607.

[0472]

[0473] ROR1 represents a nucleotide substitution of the structure described above, where Base can be any base, for example, ROR1-A indicates that Base is adenine.

[0474] Example 3: Detection of the target and off-target activities of the siRNA compound psi-CHECK2

[0475] 1. Plasmid preparation:

[0476] In the target plasmid: The corresponding antisense target plasmid was designed according to the compound sequence. The psiCHECK2 GSCM recombinant plasmid was prepared by Sangon Biotech (Shanghai) Co., Ltd. and the recombinant plasmid was diluted to 1000 ng / μL for later use.

[0477] Off-target plasmids: Based on the compound sequence, the corresponding antisense off-target plasmids were designed. The psiCHECK2 GSSM-5Hits recombinant plasmid was prepared by Sangon Biotech (Shanghai) Co., Ltd. and diluted to 1000 ng / μL for later use.

[0478] 2. Cell transfection:

[0479] HEK293A cells (Nanjing Kebai, catalog number CBP60436) were seeded in 96-well plates with 100 μL of cell resuspended solution; cell quantity: 8 × 10⁶ cells / well. 3 cells / pores.

[0480] On the second day, the complete culture medium in the wells was first aspirated and replaced with 80 μL of Opti-MEM culture medium per well, and then starved for about 1.5 hours.

[0481] Plasmid mixture: Single-well preparation amount: 0.01 μL plasmid / well, 8.99 μL Opti-MEM / well.

[0482] Lipo mixing: Dilute Lipo 2000 (Lipofectamine) with Opti-MEM. TM Transfection reagent 2000 (Thermo, 11668019), let stand at room temperature for 5 minutes, specific preparation volume of Lipo mixture: Lipo 0.2 μL / well, Opti-MEM 9.8 μL / well.

[0483] 22 μL of the prepared Lipo mixture, 2.2 μL of the compound, and 19.8 μL of the plasmid mixture were aliquoted into corresponding wells and named Well A. After mixing thoroughly by pipetting, the mixture was incubated at room temperature for 20 minutes before co-transfection. Finally, 20 μL of well A mixture was added to each well, along with the original 80 μL of Opti-MEM, for a final volume of 100 μL per well. After incubation at 37°C and 5% CO2 for 4 hours, 100 μL of DMEM medium containing 20% ​​fetal bovine serum was added to each well. After incubation at 37°C and 5% CO2 for 24 hours, the cells were analyzed.

[0484] 3. Result Detection:

[0485] Before the experiment, mix the ingredients. Luciferase The Luciferase Assay System (Promega, E2940) was reconstituted and, after equilibration to room temperature, DMEM was added to each tube at a 1:1 ratio to prepare substrate I, to be used immediately. Stop & Remelt the buffer and allow it to equilibrate to room temperature before mixing with... Stop & Prepare Substrate II using a 100:1 ratio and use immediately after preparation.

[0486] The vacuum pump removes the original culture medium from the 96-well culture plate;

[0487] Add 150 μL of substrate I to each well and incubate on a shaker at room temperature for 10 min.

[0488] Take 120 μL of substrate I, transfer it to a 96-well microplate, and read the Firefly chemiluminescence value on a microplate reader (Tecan, Infinite 200);

[0489] Add 60 μL of substrate II to each well, incubate on a shaker at room temperature for 10 min, and read the Renilla chemiluminescence value on a microplate reader.

[0490] 4. Data Analysis and Processing

[0491] Fluorescence activity was measured using a microplate reader. The collected Renilla signals were normalized using the Firefly signal standard. The inhibitory effect of siRNA was determined by comparing the results without treatment (residual inhibitory activity). The calculation process is as follows:

[0492] Homogenized Ren / Fir ratio: Ratio = Renilla (renilla luciferase) / Firefly (firefly luciferase).

[0493] Residual inhibition rate: 2 replicates (Ratio) siRNA / Ratio control The mean is calculated as () * 100%: where Ratio control To calculate the average ratio of the two duplicate wells (excluding siNRA), calculate the ratio of each duplicate well separately. siRNA / Ratio control Then, the average value is taken as the residual inhibition rate;

[0494] Plotting: Using Graphpad Prism

[0495] Half-maximal inhibitory concentration (IC50): In this experiment, the top and bottom were plotted. The IC50 value was obtained by formula Y = Bottom + (Top - Bottom) / (1 + 10^((LogIC50 - X) * HillSlope)), where Y = 50 and X = log(concentration).

[0496] In the target activity assay, HEK293A (Nanjing Kebai, catalog number CBP60436) cell line was transfected with psiCHECK2-GSCM recombinant plasmid. The initial concentration of the compound was 10 nM, and 11 concentration points (10 nM, 3.33 nM, 1.11 nM, 0.37 nM, 0.123 nM, 0.041 nM, 0.0136 nM, 0.0045 nM, 0.00152 nM, 0.000508 nM, 0.000169 nM) were selected for siRNA compound activity screening. The results are shown in Table 1.

[0497] Table 1. Results of psi-CHECK2 target activity assay.

[0498]

[0499] Off-target activity assays were performed using HEK293A (Nanjing Kebai, catalog number CBP60436) cell line transfected with the psiCHECK2-GSSM-5Hits recombinant plasmid. The initial concentration of the compound was 10 nM, and 11 concentrations were obtained by 3-fold dilution (10 nM, 3.33 nM, 1.11 nM, 0.37 nM, 0.123 nM, 0.041 nM, 0.0136 nM, 0.0045 nM, 0.00152 nM, 0.000508 nM, 0.000169 nM) for siRNA compound activity screening. The results are shown in Table 2.

[0500] Table 2. Results of psi-CHECK2 off-target activity assay.

[0501]

[0502] The results showed that the siRNA carrying the nucleotide analog monomer of the present invention effectively reduced off-target activity while maintaining target activity.

[0503] Example 4: Validation of the long-term efficacy of the compound in the C57BL / 6 mouse model

[0504] C57BL / 6 mice (male, 18–21 g, 6–8 weeks old) were randomly divided into groups of 6 according to Table 3. The dosage for each animal was calculated based on its body weight. The medication was administered subcutaneously as a single dose. The siRNA compound was first prepared as a 1 mg / mL solution (using 0.9% sodium chloride aqueous solution as the solvent). Before the experiment, the siRNA compound was dissolved in 0.9% sodium chloride aqueous solution and the volume was adjusted to the required concentration and volume. The administration volume of physiological saline and siRNA compound was 5 mL / kg.

[0505] Table 3 Grouping of Animal Experiments

[0506] 1 physiological saline 2 DR002220 3 DR005760

[0507] Blood samples were collected from the orbital venous plexus of mice before administration (day 0) and on days 14, 28, 42 and 56 after administration. Serum mTTR protein was detected at each time point using an ELISA kit (Abcam, ab282297). At the last experimental time point, 10 mg of liver was collected and placed in RNAlater solution to detect liver mTTR mRNA.

[0508] Table 4 shows the results of efficacy and long-term efficacy verification of the compounds in the C57BL / 6 mouse model.

[0509]

[0510] The results showed that siRNA carrying the nucleotide analog monomer of the present invention can reduce the expression of target genes in vivo for a long time.

Claims

1. The nucleotide dimer represented by formula (A), (A) in, L2 is P2; L3 is P3; Q is -CH2-O-; Y1 is O; Y2 is O; R3 is H or C 1-4 alkyl; R4, R5, R6, and R7 are independently selected from H, halogens, or C. 1-4 alkyl; R8 is selected from halogens; Base and Base' are selected independently. , , , or ; P2 is -P(OCH2CH2CN)(N(iPr)2); P3 is DMTr.

2. The nucleotide dimer of claim 1, wherein, R4, R5, R6, and R7 are H.

3. The nucleotide dimer of claim 1, wherein, R8 stands for fluorine.

4. The nucleotide dimer of claim 1, having the following structure: (A-1) in, R3 is a methyl group; R8 is fluorine; The other groups are as defined in claim 1.

5. A double-stranded RNA molecule or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof, comprising a sense strand and an antisense strand, each strand having 14 to 30 nucleotides, said antisense strand having a sequence sufficiently complementary to said sense strand and target mRNA and having the ability to induce degradation of target mRNA, and said antisense strand comprising one or more nucleotide monomers of formula (IV): (IV) in, The nucleotide monomers shown are arranged in the order of 5' => 3' from... to connect; Each group is defined as described in any one of claims 1-4. The double-stranded RNA molecule is prepared using the nucleotide dimer of any one of claims 1-4.

6. The double-stranded RNA molecule of claim 5, wherein the nucleotide monomer is located at positions 2-8 of the 5' end of the antisense strand.

7. The double-stranded RNA molecule of claim 5, wherein the nucleotide monomer is located at the 6th or 7th position at the 5' end of the antisense strand.

8. The double-stranded RNA molecule of claim 5, wherein the nucleotide monomer is located at the 7th position at the 5' end of the antisense strand.

9. The double-stranded RNA molecule of claim 5, wherein the double-stranded RNA is further coupled to a ligand comprising one or more GalNAc.

10. A method for preparing a double-stranded RNA molecule according to any one of claims 5-9, wherein, The double-stranded RNA molecule is prepared using the nucleotide dimer of any one of claims 1-4.

11. A pharmaceutical composition comprising a double-stranded RNA molecule as described in any one of claims 5-9, and a pharmaceutically acceptable excipient.

12. Use of the double-stranded RNA molecule of any one of claims 5-9 in the preparation of a medicament for inhibiting the expression of a target gene in a cell.

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