Double-stranded RNA with nucleotide analogs

Abstract

The present invention provides a double-stranded RNA having a nucleotide analogue. The double stranded RNAs of the present invention exhibit 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 with application date of January 4, 2023 and application number 202310009472.2. Field of the Invention

[0002] The present invention belongs to the field of medicine, and in particular relates to double-stranded RNA with nucleotide analogs. Background Art

[0003] RNA interference is a phenomenon of efficient and specific degradation of target mRNA induced by double-stranded RNA (dsRNA). The incorporation of thermally unstable nucleotides (e.g., glycerol nucleic acid (GNA)) into the antisense strand seed region of double-stranded RNA helps to 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), the incorporation of GNA-G or GNA-C into the center of double-stranded RNA results in a decrease in the stability of double-stranded RNA relative to GNA-A or GNA-T.

[0004] Therefore, there is a need in the art to develop nucleotide analogs with 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 a nucleotide dimer represented by formula (A),

[0007]

[0008] in,

[0009] L2 is H or P2;

[0010] L3 is 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 O, S or a chemical bond;

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

[0016] R3 is selected from H, C 1-6 Cyanoalkyl, C 1-6 Alkyl, C 1-6 Haloalkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 3-7 Cycloalkyl, 3-7 membered heterocyclic group, C 6-10 aryl or 5-10 membered heteroaryl, which is optionally substituted with 1, 2, 3, 4 or 5 R';

[0017] R8 is selected from H, D, OH, halogen, C 1-6 Alkyl, C 1-6 Haloalkyl or C 1-6 Alkoxy;

[0018] Base and Base' are independently selected from H, a modified or unmodified base, or a leaving group;

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

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

[0021] R is selected from H, C 1-6 Alkyl or C 1-6 Haloalkyl;

[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, C 3-10 Cycloalkyl, 3-10 membered heterocyclic group, C 6-10 Aryl or 5-14 membered heteroaryl;

[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, wherein each strand has 14 to 30 nucleotides, the antisense strand has a sequence that is sufficiently complementary to the sense strand and the target mRNA, and has the ability to induce degradation of the target mRNA, and the antisense strand comprises one or more nucleotide monomers represented by formula (IV):

[0025]

[0026] in,

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

[0028] Each group is as defined above and below

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

[0034] After the nucleotides of the present invention are incorporated into the antisense strand of dsRNA, the resulting double-stranded RNA exhibits one or more of enhanced stability, reduced off-target toxicity, and enhanced effectiveness. DETAILED DESCRIPTION OF THE INVENTION

[0036] definition

[0037] Chemical Definition

[0038] Definitions of specific functional groups and chemical terms are described in more detail below.

[0039] When a numerical range is listed, it is intended to include every value and sub-range within the stated range. For example, "C 1-6 "Alkyl" includes C1, C2, C3, C4, C5, C6, C 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.

[0040] “C 1-6 "Alkyl" refers to a straight or branched chain saturated hydrocarbon group having 1 to 6 carbon atoms. In some embodiments, C 1-4 Alkyl and C 1-2 Alkyl groups are preferred. 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 1-6 "Alkyl" also includes heteroalkyl groups in which 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. Conventional alkyl abbreviations include: Me(-CH3), Et(-CH2CH3), iPr(-CH(CH3)2), nPr(-CH2CH2CH3), n-Bu(-CH2CH2CH2CH3) or i-Bu(-CH2CH(CH3)2).

[0041] “C 2-6 "Alkenyl" refers to a straight 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 is preferred. 2-6 Examples of alkenyl groups include ethenyl (C2), 1-propenyl (C3), 2-propenyl (C3), 1-butenyl (C4), 2-butenyl (C4), butadienyl (C4), pentenyl (C5), pentadienyl (C5), hexenyl (C6), and the like. The term “C 2-6 "Alkenyl" also includes heteroalkenyl groups in which 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 with one or more substituents, for example, with 1 to 5 substituents, 1 to 3 substituents, or 1 substituent.

[0042] “C 2-6 "Alkynyl" refers to a straight or branched chain 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 2-4Alkynyl is preferred. 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), pentynyl (C5), hexynyl (C6), and the like. The term “C 2-6 "Alkynyl" also includes heteroalkynyl groups, in which one or more (e.g., 1, 2, 3 or 4) carbon atoms are replaced by heteroatoms (e.g., oxygen, sulfur, nitrogen, boron, silicon, phosphorus). Alkynyl groups may be optionally substituted with one or more substituents, for example, with 1 to 5 substituents, 1 to 3 substituents, or 1 substituent.

[0043] “C 1-6 "Alkylene" refers to the removal of C 1-6 The other hydrogen of C alkyl forms a divalent group, and may be substituted or unsubstituted. 1-4 Alkylene, C 2-4 Alkylene and C 1-3 Alkylene is preferred. Unsubstituted alkylene includes, but is not limited to, methylene (-CH2-), ethylene (-CH2CH2-), propylene (-CH2CH2CH2-), butylene (-CH2CH2CH2CH2-), pentylene (-CH2CH2CH2CH2CH2-), hexylene (-CH2CH2CH2CH2CH2CH2-), and the like. Exemplary substituted alkylenes, for example, alkylenes 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 propylene (-CH(CH3)CH2CH2-, -CH2CH(CH3)CH2-, -CH2CH2CH(CH3)-, -C(CH3)2CH2CH2-, -CH2C(CH3)2CH2-, -CH2CH2C(CH3)2-), and the like.

[0044] "Halo" or "halogen" refers to fluorine (F), chlorine (Cl), bromine (Br) and iodine (I).

[0045] Therefore, “C 1-6 "Haloalkyl" refers to the above-mentioned "C 1-6 "alkyl", which is substituted by one or more halogen groups. In some embodiments, C 1-4 Haloalkyl is particularly preferred, more preferably C 1-2Haloalkyl. Exemplary haloalkyls include, but are not limited to, -CF3, -CH2F, -CHF2, -CHFCH2F, -CH2CHF2, -CF2CF3, -CCl3, -CH2Cl, -CHCl2, 2,2,2-trifluoro-1,1-dimethyl-ethyl, and the like. The haloalkyl group may be substituted at any available point of attachment, for example, 1 to 5 substituents, 1 to 3 substituents, or 1 substituent.

[0046] “C 1-6 "Alkoxy" refers to an -OR group, where R is as described above in "C 1-6 Alkyl" and "C 1-6 "Haloalkyl" is defined as

[0047] “C 1-6 "Cyanoalkyl" refers to -C 1-6 Alkylene -CN, where "C 1-6 "Alkylene" is as defined above. In some embodiments, C 1-4 Cyanoalkyl is particularly preferred, more preferably C 1-2 Cyanoalkyl groups, such as cyanoethyl groups (-CH2CH2CN).

[0048] “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 is particularly preferred, more preferably C 5-6 Cycloalkyl. Cycloalkyl also includes a ring system in which the above-mentioned cycloalkyl ring is fused with one or more aryl or heteroaryl groups, wherein the connection point is on the cycloalkyl ring, and in such a case, the number of carbons continues to represent the number of carbons in the cycloalkyl system. Exemplary cycloalkyls 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), cycloheptatrienyl (C7), etc. The cycloalkyl group may be optionally substituted by one or more substituents, for example, substituted by 1 to 5 substituents, 1 to 3 substituents or 1 substituent.

[0049] "3-10 membered heterocyclyl" refers to a group of a 3- to 10-membered non-aromatic ring system having ring carbon atoms and 1 to 5 ring heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen, sulfur, boron, phosphorus and silicon. In heterocyclyl groups containing one or more nitrogen atoms, the point of attachment may be a carbon or nitrogen atom as long as the valence permits. In some embodiments, a 4-10 membered heterocyclyl is preferred, which is a 4 to 10 membered non-aromatic ring system having ring carbon atoms and 1 to 5 ring heteroatoms; in some embodiments, a 3-8 membered heterocyclyl is preferred, which is a 3 to 8 membered non-aromatic ring system having ring carbon atoms and 1 to 4 ring heteroatoms; a 3-6 membered heterocyclyl is preferred, which is a 3 to 6 membered non-aromatic ring system having ring carbon atoms and 1 to 3 ring heteroatoms; a 4-7 membered heterocyclyl is preferred, which is a 4 to 7 membered non-aromatic ring system having ring carbon atoms and 1 to 3 ring heteroatoms; and a 5-6 membered heterocyclyl is more preferred, which is a 5 to 6 membered non-aromatic ring system having ring carbon atoms and 1 to 3 ring heteroatoms. Heterocyclyl also includes a ring system in which the above-mentioned heterocyclyl ring is fused to one or more cycloalkyl groups, wherein the point of attachment is on the cycloalkyl ring, or a ring system in which the above-mentioned heterocyclyl ring is fused to one or more aryl or heteroaryl groups, wherein the point of attachment is on the heterocyclyl ring; and in such a case, the number of ring members continues to represent the number of ring members in the heterocyclyl ring system. Exemplary 3-membered heterocyclyls containing one heteroatom include, but are not limited to, aziridine, oxirane, and thiorenyl. Exemplary 4-membered heterocyclyls containing one heteroatom include, but are not limited to, azetidine, oxirane, and thiorenyl. Exemplary 5-membered heterocyclyls containing one heteroatom include, but are not limited to, tetrahydrofuranyl, dihydrofuranyl, tetrahydrothienyl, dihydrothienyl, pyrrolidinyl, dihydropyrrolyl, and pyrrolyl-2,5-dione. Exemplary 5-membered heterocyclic groups containing two heteroatoms include, but are not limited to, dioxolanyl, 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, dithianyl, and dioxanyl. Exemplary 6-membered heterocyclic groups containing three heteroatoms include, but are not limited to, hexahydrotriazinyl. Exemplary 7-membered heterocyclic groups containing one heteroatom include, but are not limited to, azepanyl, oxepanyl, and thianyl. Exemplary 5-membered heterocyclyl groups fused to a C6 aryl ring (also referred to herein as 5,6-bicyclic heterocyclyl) include, but are not limited to, indolinyl, isoindolinyl, dihydrobenzofuranyl, dihydrobenzothiophenyl, benzoxazolinonyl, and the like.Exemplary 6-membered heterocyclyl groups fused to a C6 aryl ring (also referred to herein as 6,6-bicyclic heterocyclyl groups) include, but are not limited to, tetrahydroquinolinyl, tetrahydroisoquinolinyl, etc. The heterocyclyl group may be optionally substituted with one or more substituents, for example, 1 to 5 substituents, 1 to 3 substituents, or 1 substituent.

[0050] “C 6-10 "Aryl" refers to a monocyclic or polycyclic (e.g., bicyclic) 4n+2 aromatic ring system (e.g., having 6 or 10 π electrons shared in a cyclic arrangement) having 6-10 ring carbon atoms and zero heteroatoms. In some embodiments, aryl has six ring carbon atoms ("C6 aryl"; e.g., phenyl). In some embodiments, aryl has ten ring carbon atoms ("C 10 Aryl also includes ring systems in which the above aryl ring is fused to one or more cycloalkyl or heterocyclyl groups, and the point of attachment is on the aryl ring, in which case the number of carbon atoms continues to refer to the number of carbon atoms in the aryl ring system. Aryl groups can be optionally substituted with one or more substituents, for example, 1 to 5 substituents, 1 to 3 substituents, or 1 substituent.

[0051] "5-14 membered heteroaryl" refers to a group of a 5-14 membered monocyclic or bicyclic 4n+2 aromatic ring system (e.g., having 6, 10, or 14 π electrons shared in a cyclic arrangement) having ring carbon atoms and 1-4 ring heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur. In heteroaryl groups containing one or more nitrogen atoms, the point of attachment may be a carbon or nitrogen atom as long as the valence permits. Heteroaryl bicyclic ring systems may include one or more heteroatoms in one or both rings. Heteroaryl also includes a ring system in which the above-mentioned heteroaryl ring is fused to one or more cycloalkyl or heterocyclic groups, and the point of attachment 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, a 5-10 membered heteroaryl is preferred, which is a 5-10 membered monocyclic or bicyclic 4n+2 aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms. In other embodiments, 5-6 membered heteroaryls are particularly preferred, which are 5-6 membered monocyclic or bicyclic 4n+2 aromatic ring systems having ring carbon atoms and 1-4 ring heteroatoms. Exemplary 5 membered heteroaryls containing one heteroatom include, but are not limited to, pyrrolyl, furanyl, and thienyl. Exemplary 5 membered heteroaryls containing two heteroatoms include, but are not limited to, imidazolyl, pyrazolyl, oxazolyl, isoxazolyl, thiazolyl, and isothiazolyl. Exemplary 5 membered heteroaryls containing three heteroatoms include, but are not limited to, triazolyl, oxadiazolyl (e.g., 1,2,4-oxadiazolyl), and thiadiazolyl. Exemplary 5 membered heteroaryls containing four heteroatoms include, but are not limited to, tetrazolyl. Exemplary 6 membered heteroaryls containing one heteroatom include, but are not limited to, pyridinyl. Exemplary 6 membered heteroaryls 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 tetrazinyl, respectively. Exemplary 7-membered heteroaryl groups containing one heteroatom include, but are not limited to, azacycloheptatrienyl, oxacycloheptatrienyl, and thiacycloheptatrienyl. Exemplary 5,6-bicyclic heteroaryl groups include, but are not limited to, indolyl, isoindolyl, indazolyl, benzotriazolyl, benzothienyl, isobenzothienyl, benzofuranyl, benzisofuranyl, benzimidazolyl, benzoxazolyl, benzisoxazolyl, benzoxadiazolyl, benzothiazolyl, benzisothiazolyl, benzothiadiazolyl, indazinyl, and purinyl. Exemplary 6,6-bicyclic heteroaryl groups include, but are not limited to, naphthyridinyl, pteridinyl, quinolyl, isoquinolyl, cinnolinyl, quinoxalinyl, phthalazinyl, and quinazolinyl. A heteroaryl group can be optionally substituted with one or more substituents, for example, with 1 to 5 substituents, 1 to 3 substituents, or 1 substituent.

[0052] Alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl and heteroaryl groups and the like as defined herein are optionally substituted groups.

[0053] 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 S02R aa 、-NR bb S02R aa 、-SO2N(R bb 2. -SO2R aa 、-SO2OR aa 、-OSO2R aa 、-S(=O)R aa 、-OS(=O)R aa 、-Si(R aa)3.-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, alkynyl, cycloalkyl, heterocyclyl, aryl and heteroaryl, wherein each alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl and heteroaryl is independently substituted by 0, 1, 2, 3, 4 or 5 R dd Group substitution;

[0054] Or the two geminal hydrogen atoms on the carbon atom are replaced by groups =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;

[0055] R aa Each of R is independently selected from alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl and heteroaryl, or two R aa The groups are combined to form a heterocyclyl or heteroaryl ring, wherein each alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl and heteroaryl group is independently substituted by 0, 1, 2, 3, 4 or 5 R dd Group substitution;

[0056] R bb Each of 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, heterocyclyl, aryl and heteroaryl, or two R bb The groups are combined to form a heterocyclyl or heteroaryl ring, wherein each alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl and heteroaryl group is independently substituted by 0, 1, 2, 3, 4 or 5 R dd Group substitution;

[0057] R cc Each of R is independently selected from hydrogen, alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl and heteroaryl, or two R cc The groups are combined to form a heterocyclyl or heteroaryl ring, wherein each alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl and heteroaryl group is independently substituted by 0, 1, 2, 3, 4 or 5 R dd Group substitution;

[0058] R ddEach of 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 S02R 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, alkynyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, wherein each alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl and heteroaryl is independently substituted by 0, 1, 2, 3, 4 or 5 R gg group substitution, or two geminal R dd Substituents may combine to form =O or =S;

[0059] R ee Each of R is independently selected from alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, aryl, heterocyclyl and heteroaryl, wherein each alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl and heteroaryl is independently substituted by 0, 1, 2, 3, 4 or 5 R gg Group substitution;

[0060] R ff Each of R is independently selected from hydrogen, alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl and heteroaryl, or two R ff The groups are combined to form a heterocyclic or heteroaryl ring, wherein each alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclic, aryl and heteroaryl group is independently substituted by 0, 1, 2, 3, 4 or 5 R gg Group substitution;

[0061] R gg Each of 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), -NH(OH), -SH, -SC 1-6 Alkyl, -SS(C 1-6 alkyl), -C(=O)(C 1-6alkyl), -CO2H, -CO2(C 1-6 alkyl), -OC(=O)(C 1-6 Alkyl), -OCO2(C 1-6 alkyl), -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), -NHC(=O)NH2, -C(=NH)O(C 1-6 alkyl), -OC(=NH)(C 1-6 alkyl), -OC(=NH)OC 1-6 Alkyl, -C(=NH)N(C 1-6 Alkyl)2, -C(=NH)NH(C 1-6 alkyl), -C(=NH)NH2, -OC(=NH)N(C 1-6 Alkyl)2, -OC(NH)NH(C 1-6 alkyl), -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), -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, -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 Halogenated alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C7 cycloalkyl, C6-C 10 Aryl, C3-C7 heterocyclic, C5-C 10 heteroaryl; or two geminal R gg Substituents may combine to form =O or =S; wherein X - For the counter ion.

[0062] Exemplary substituents on nitrogen atoms 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, heterocyclyl, aryl and heteroaryl, or two R attached to the nitrogen atom 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 substituted by 0, 1, 2, 3, 4 or 5 R dd substituted with a group, and wherein R aa , R bb , R cc and R dd As mentioned above.

[0063] Other definitions

[0064] The term "siRNA" herein refers to a class of double-stranded RNA molecules that can mediate the silencing of a target RNA (e.g., mRNA, e.g., a transcript of a gene encoding a protein) that is complementary thereto. siRNA is typically double-stranded, comprising an antisense strand complementary to the target RNA, and a sense strand complementary to the antisense strand. For convenience, such mRNA is also referred to herein as mRNA to be silenced. Such a gene is also referred to as a target gene. Typically, the RNA to be silenced is an endogenous gene or a pathogen gene. In addition, RNA (e.g., tRNA) and viral RNA other than mRNA can also be targeted.

[0065] The term "antisense strand" refers to a strand of an siRNA that includes a region that is completely, fully or substantially complementary to a target sequence. The term "sense strand" refers to a strand of an siRNA that includes a region that is completely, fully or substantially complementary to a region that is the antisense strand as defined herein.

[0066] 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 the case where the complementary region is not completely complementary to the target sequence, mispairing can be located in the interior or terminal region of the molecule. Typically, the most tolerated mispairing is located in the terminal region, for example, within 5, 4, 3, 2 or 1 nucleotides at 5' and / or 3' ends. The antisense strand portion that is most sensitive to mispairing is referred to as a "seed region". For example, in a siRNA comprising a 19nt chain, the 19th position (from 5' to 3') can tolerate some mispairing.

[0067] 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 400mM NaCl, 40mM PIPES pH 6.4, 1mM EDTA at 50°C or 70°C for 12-16 hours. In terms of meeting the above requirements relative to their ability to hybridize, "complementary" sequences may also include or be completely formed from non-Watson-Crick base pairs and / or base pairs formed from non-natural and modified nucleotides. Such non-Watson-Crick base pairs include, but are not limited to, G:U wobble base pairing or Hoogstein base pairing.

[0068] A polynucleotide that is "at least partially complementary," "fully complementary," or "substantially complementary" to a messenger RNA (mRNA) refers to a polynucleotide that is substantially complementary to a contiguous portion of an mRNA of interest. For example, a polynucleotide is complementary to at least a portion of a PCSK9 mRNA if the sequence is substantially complementary to a non-interrupted portion of an mRNA encoding PCSK9. The terms "complementary," "fully complementary," "fully complementary," and "substantially complementary" herein can be used with respect to base pairing between the sense strand and the antisense strand of an siRNA, or between the antisense strand of an siRNA agent and a target sequence.

[0069] "Fully complementary" refers to the extent to which the sense strand only needs to be complementary to the antisense strand in order to maintain the overall double-stranded characteristics of the molecule. In other words, although perfect complementarity is generally required, in some cases, particularly in the antisense strand, one or more, such as 6, 5, 4, 3, 2 or 1 mismatches (relative to the target mRNA) may be included, but the sense strand and the antisense strand can still maintain the overall double-stranded characteristics of the molecule.

[0070] "shRNA" refers to short hairpin RNA. shRNA includes two short inverted repeat sequences. The shRNA cloned into the shRNA expression vector includes two short inverted repeat sequences separated by a stem-loop sequence in the middle, forming a hairpin structure controlled by the polⅢ promoter. Then 5-6 Ts are connected as the transcription terminator of RNA polymerase Ⅲ.

[0071] "Nucleoside" is a compound composed of two substances, a purine base or a pyrimidine base, and ribose or deoxyribose, "nucleotide" is a compound composed of three substances, a purine base or a pyrimidine base, ribose or deoxyribose, and phosphate, and "oligonucleotide" refers to a nucleic acid molecule (RNA or DNA) with a length of, for example, less than 100, 200, 300 or 400 nucleotides.

[0072] "Base" is the basic unit of synthesis of nucleosides, nucleotides and nucleic acids. It contains nitrogen and is also called "nitrogenous base". In this article, unless otherwise specified, capital letters A, U, T, G and C represent the base composition of nucleotides, which are adenine, uracil, thymine, guanine and cytosine respectively.

[0073] The "modification" of the nucleotides described herein includes, but is not limited to, methoxy modification, fluorine modification, phosphorothioate linkage, or conventional protecting group protection, etc. For example, the fluorine-modified nucleotide refers to a nucleotide in which the 2'-hydroxyl group of the ribose group of the nucleotide is replaced by fluorine, and the methoxy-modified nucleotide refers to a nucleotide in which the 2'-hydroxyl group of the ribose group is replaced by a methoxy group.

[0074] "Modified nucleotides" herein include but are not limited to 2'-O-methyl modified nucleotides, 2'-fluoro modified nucleotides, 2'-deoxy-modified nucleotides, inosine ribonucleotides, abasic nucleotides, reverse abasic deoxyribonucleotides, nucleotides comprising thiophosphate groups, vinyl phosphate modified nucleotides, locked nucleotides, 2'-amino-modified nucleotides, 2'-alkyl-modified nucleotides, morpholino nucleotides, phosphoramidates, non-natural bases comprising nucleotides, and terminal nucleotides, deoxyribonucleotides or conventional protecting groups that are connected to cholesterol derivatives or dodecanoic acid didecylamide groups. For example, the 2'-fluoro modified nucleotides refer to nucleotides in which the hydroxyl group at the 2' position of the ribose group of the nucleotide is replaced by fluorine. The 2'-deoxy-modified nucleotides refer to nucleotides in which the 2'-hydroxyl group of the ribose group is replaced by methoxy.

[0075] "Leaving group" is also called leaving group. It is an atom or functional group that breaks away from a larger molecule in a chemical reaction. It is a term used in nucleophilic substitution reactions and elimination reactions.

[0076] "Reactive phosphorus group" refers to a phosphorus-containing group contained in a nucleotide unit or in a nucleotide analog unit, which can react with a hydroxyl or an amine group contained in another molecule, especially in another nucleotide unit or in another nucleotide analog by a nucleophilic attack reaction. Typically, such a reaction produces an ester-type internucleoside bond connecting the first nucleotide unit or the first nucleotide analog unit to the second nucleotide unit or the second nucleotide analog unit. Reactive phosphorus group can be selected from phosphoramidites, H-phosphonates, alkyl-phosphonates, phosphates or phosphate mimetics, including but not limited to: natural phosphates, thiophosphates, dithiophosphates, borane phosphates, borane thiophosphates, phosphonates, halogen-substituted phosphonates and phosphates, phosphoramidates, phosphodiester, phosphotriester, thiophosphorodiester, thiophosphorothioates, diphosphates and triphosphates, preferably-P(OCH2CH2CN)(N(iPr)2).

[0077] "Protecting group" is also called "protecting group", which refers to any atom or group of atoms added to a molecule to prevent the existing groups in the molecule from undergoing undesirable chemical reactions. "Protecting group" can be an unstable chemical moiety known in the art, which is used to protect reactive groups, such as hydroxyl, amino and thiol groups, to prevent undesirable or inappropriate reactions during chemical synthesis. Protecting groups are often used selectively and / or orthogonally to protect sites during reactions at other reactive sites, and can then be removed to leave the unprotected group intact or available for further reactions.

[0078] A non-limiting list of protecting groups includes benzyl; substituted benzyl; alkylcarbonyl and alkoxycarbonyl (e.g., tert-butyloxycarbonyl (BOC), acetyl or isobutyryl); arylalkylcarbonyl and arylalkoxycarbonyl (e.g., benzyloxycarbonyl); substituted methyl ethers (e.g., methoxymethyl ether); substituted ethyl ethers; substituted benzyl ethers; tetrahydropyranyl ethers; silyl (e.g., trimethylsilyl, triethylsilyl, triisopropylsilyl, tert-butyldimethylsilyl, tri-isopropylsilyloxymethyl, [2-(trimethylsilyl)ethoxy] methyl or tert-butyldiphenylsilyl); esters (e.g., benzoate); carbonates (e.g., methoxymethyl carbonate); sulfonates (e.g., tosylate or mesylate); acyclic ketals (e.g., dimethyl acetal); cyclic ketals (e.g., 1,3-dioxane, 1,3-dioxolane, and those described herein); acyclic acetals; cyclic acetals (e.g., those described herein); acyclic hemiacetals; cyclic hemiacetals; cyclic dithioketals (e.g., 1,3-dithiane or 1,3-dithiolane); orthoesters (e.g., those described herein) and triarylmethyl groups (e.g., trityl; monomethoxytrityl (MMTr); 4,4′-dimethoxytrityl (DMTr); 4,4′,4″-trimethoxytrityl (TMTr); and those described herein). Preferred protecting groups are selected from acetyl (Ac), benzoyl (Bzl), benzyl (Bn), isobutyryl (iBu), phenylacetyl, benzyloxymethyl acetal (BOM), β-methoxyethoxymethyl ether (MEM), methoxymethyl ether (MOM), p-methoxybenzyl ether (PMB), methylthiomethyl ether, neopentyl (V), benzyloxymethyl acetal (BOM), benzyloxymethyl ether (BOM ... Valeryl (Piv), tetrahydropyranyl (THP), triphenylmethyl (Trt), methoxytrityl [(4-methoxyphenyl) diphenylmethyl] (MMT), dimethoxytrityl, [bis-(4-methoxyphenyl) phenylmethyl (DMT), trimethylsilyl ether (TMS), tert-butyldimethylsilyl ether (TBDMS), tri-iso-propylsilyloxymethyl ether (TOM), tri-isopropylsilyl ether (TIPS), methyl ether, ethoxyethyl ether (EE) N,N-dimethylformamidine and 2-cyanoethyl (CE).

[0079] "Hydroxy protecting group" refers to a group that can protect the hydroxyl group from chemical reactions and can be removed under specific conditions to restore the hydroxyl group. It mainly includes silane type protecting groups, acyl type protecting groups or ether type protecting groups, preferably the following:

[0080] 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 yl (Troc), benzyloxycarbonyl (Cbz), tert-butyloxycarbonyl (Boc), benzyl (Bn), p-methoxybenzyl (PMB), allyl, triphenylmethyl (Tr), di-p-methoxytrityl (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'-dimethoxytrityl.

[0081] The term "pharmaceutically acceptable salt" as used herein refers to those carboxylates, amino acid addition salts of the compounds of the present invention which are suitable for use in contact with patient tissues within the scope of sound medical judgment, do not produce undue toxicity, irritation, allergic response, etc., are commensurate with a reasonable benefit / risk ratio, and are effective for their intended use, including (where possible) zwitterionic forms of the compounds of the present invention.

[0082] The present invention includes tautomers, which are functional isomers produced by rapid movement of an atom in two positions in a molecule. Compounds that exist in different tautomeric forms are not limited to any specific tautomer, but are intended to cover all tautomeric forms.

[0083] The compounds of the present invention may include one or more asymmetric centers and may therefore exist in a variety of stereoisomeric forms, for example, enantiomers and / or diastereoisomeric forms. For example, the compounds of the present invention may be individual enantiomers, diastereomers or geometric isomers (e.g., cis and trans isomers), or may be in the form of a mixture of stereoisomers, including racemic mixtures and mixtures enriched in one or more stereoisomers. Isomers may be separated from the mixture by methods known to those skilled in the art, including chiral high pressure liquid chromatography (HPLC) and the formation and crystallization of chiral salts; or preferred isomers may be prepared by asymmetric synthesis.

[0084] The present invention also includes isotopically labeled compounds (isotopic variants) which are identical to those described in formula (I) but in which one or more atoms are replaced by atoms having an atomic mass or mass number different from the atomic mass or mass number 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, 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. Compounds of the present invention containing the above isotopes and / or other isotopes of other atoms, their prodrugs and pharmaceutically acceptable salts of the compounds or prodrugs are within the scope of the present invention. Certain isotopically labeled compounds of the present invention, such as those in which a radioactive isotope (e.g. 3 H and 14 C) can be used for drug and / or substrate tissue distribution assays. 3 H and carbon-14, i.e. 14 C isotopes are particularly preferred because they are easy to prepare and detect. 2 H, because higher metabolic stability can provide therapeutic benefits, such as prolonged half-life in vivo or reduced dosage requirements, and thus may be preferred in some cases. Isotope-labeled compounds of formula (I) of the present invention and their prodrugs can generally be prepared by replacing non-isotope-labeled reagents with readily available isotope-labeled reagents when carrying out the processes disclosed in the following schemes and / or Examples and Preparation Examples.

[0085] Compounds of the present invention

[0086] The present invention specifically relates to a nucleotide dimer represented by formula (A):

[0087]

[0088] wherein each group is as defined above and below.

[0089] Q

[0090] In one embodiment, Q is -X-; in another embodiment, Q is -XO-.

[0091] X

[0092] In one embodiment, X is a chemical bond; in another embodiment, X is -(CR1R2) m -; In another embodiment, X is -CR1=CR2-.

[0093] Y1 and Y2

[0094] In one embodiment, Y1 is O; in another embodiment, Y1 is S; in another embodiment, Y1 is NR.

[0095] In one embodiment, Y2 is O; in another embodiment, Y2 is S; in another embodiment, Y2 is a chemical bond.

[0096] L2 and L3

[0097] In one embodiment, L2 is H; in another embodiment, L2 is P2.

[0098] In one embodiment, L3 is H; in another embodiment, L3 is P3.

[0099] R1, R2, R4, R5, R6, and R7

[0100] In one embodiment, R1 is H; in another embodiment, R1 is D; in another embodiment, R1 is halogen; in another embodiment, R1 is OH; in another embodiment, R1 is CN; in another embodiment, R1 is C 1-6 Alkyl, such as C 1-4 Alkyl; in another embodiment, R1 is C 1-6 Haloalkyl, such as C 1-4 haloalkyl; in another embodiment, R1 is C 2-6 In another embodiment, R1 is C 2-6 Alkynyl.

[0101] In one embodiment, R2 is H; in another embodiment, R2 is D; in another embodiment, R2 is halogen; in another embodiment, R2 is OH; in another embodiment, R2 is CN; in another embodiment, R2 is C 1-6 Alkyl, such as C 1-4 Alkyl; in another embodiment, R2 is C 1-6 Haloalkyl, such as C 1-4 haloalkyl; in another embodiment, R2 is C 2-6 In another embodiment, R2 is C 2-6 Alkynyl.

[0102] In one embodiment, R4 is H; in another embodiment, R4 is D; in another embodiment, R4 is halogen; in another embodiment, R4 is OH; in another embodiment, R4 is CN; in another embodiment, R4 is C 1-6 Alkyl, such as C 1-4 Alkyl; in another embodiment, R4 is C 1-6 Haloalkyl, such as C 1-4 haloalkyl; in another embodiment, R4 is C 2-6 In another embodiment, R4 is C 2-6 Alkynyl.

[0103] In one embodiment, R5 is H; in another embodiment, R5 is D; in another embodiment, R5 is halogen; in another embodiment, R5 is OH; in another embodiment, R5 is CN; in another embodiment, R5 is C 1-6 Alkyl, such as C 1-4 Alkyl; in another embodiment, R5 is C 1-6 Haloalkyl, such as C 1-4 haloalkyl; in another embodiment, R2 is C 2-6 In another embodiment, R5 is C 2-6 Alkynyl.

[0104] In one embodiment, R6 is H; in another embodiment, R6 is D; in another embodiment, R6 is halogen; in another embodiment, R6 is OH; in another embodiment, R6 is CN; in another embodiment, R6 is C 1-6 Alkyl, such as C 1-4 Alkyl; in another embodiment, R6 is C 1-6 Haloalkyl, such as C 1-4 haloalkyl; in another embodiment, R6 is C 2-6 In another embodiment, R6 is C 2-6 Alkynyl.

[0105] In one embodiment, R7 is H; in another embodiment, R7 is D; in another embodiment, R7 is halogen; in another embodiment, R7 is OH; in another embodiment, R7 is CN; in another embodiment, R7 is C 1-6 Alkyl, such as C 1-4 In another embodiment, R7 is C 1-6 Haloalkyl, such as C 1-4 haloalkyl; in another embodiment, R7 is C 2-6In another embodiment, R7 is C 2-6 Alkynyl.

[0106] In one embodiment, R1, R2, R4, R5, R6 and R7 are each independently optionally substituted; in another embodiment, R1, R2, R4, R5, R6 and R7 are each independently optionally substituted with 1, 2, 3, 4, 5, 6, 7, 8 or more R'.

[0107] R3

[0108] In one embodiment, R3 is H; in another embodiment, R3 is C 1-6 Alkyl, preferably C 1-4 Alkyl; in another embodiment, R3 is C 1-6 In another embodiment, R3 is C 1-4 In another embodiment, R3 is C 1-6 Halogenated alkyl, preferably C 1-4 haloalkyl; in another embodiment, R3 is C 2-6 In another embodiment, R3 is C 2-6 In another embodiment, R3 is C 3-10 cycloalkyl; in another embodiment, R3 is 3-10 membered heterocyclyl; in another embodiment, R3 is C 6-10 Aryl; in another embodiment, R3 is 5-14 membered heteroaryl.

[0109] In one embodiment, R3 is unsubstituted; in another embodiment, R3 is optionally substituted with 1, 2, 3, 4, 5, 6, 7, 8 or more R'.

[0110] R8

[0111] In one embodiment, R8 is H; in another embodiment, R8 is D; in another embodiment, R8 is OH; in another embodiment, R8 is halogen, such as fluorine; in another embodiment, R8 is C 1-6 Alkyl, preferably C 1-4 Alkyl; in another embodiment, R8 is C 1-6 Halogenated alkyl, preferably C 1-4 haloalkyl; in another embodiment, R8 is C 1-6 Alkoxy, preferably C 1-4 Alkoxy groups, such as methoxy groups.

[0112] Base and Base'

[0113] In one embodiment, Base is H; in another embodiment, Base is a modified or unmodified base or a leaving group; in another embodiment, Base is selected from modified or unmodified A, U, T, G and C, for example

[0114] In one embodiment, Base' is H; in another embodiment, Base' is a modified or unmodified base or a leaving group; in another embodiment, Base' is selected from modified or unmodified A, U, T, G and C, for example

[0115] P2 and P3

[0116] In one embodiment, P2 is a hydroxy protecting group; in another embodiment, P2 is a reactive phosphorus group, such as -P(OCH2CH2CN)(N(iPr)2).

[0117] In one embodiment, P3 is a hydroxy protecting group, preferably a silane protecting group, an acyl protecting group or an ether protecting group, such as DMTr.

[0118] R and R'

[0119] In one embodiment, R is H; in another embodiment, R is C 1-6 Alkyl groups, such as C 1-4 Alkyl; in another embodiment, R is C 1-6 Halogenated alkyl.

[0120] In one embodiment, R' is D; in another embodiment, R' is halogen; in another embodiment, R' is CN; in another embodiment, R' is C 1-6 Alkyl; in another embodiment, R' is C 1-6 haloalkyl; in another embodiment, R' is C 2-6 In another embodiment, R' is C 2-6 Alkynyl; in another embodiment, R' is C 3-10 cycloalkyl; in another embodiment, R' is 3-10 membered heterocyclyl; in another embodiment, R' is 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 .

[0121] R a and R b Independently selected from H, C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 3-10 Cycloalkyl, 3-10 membered heterocyclic group, C 6-10 Aryl or 5-14 membered heteroaryl; or R a and R b and the nitrogen atom to which they are attached form a 3-10 membered heterocyclic group.

[0122] n is independently selected from 1 or 2.

[0123] m

[0124] In one embodiment, m is 1; in another embodiment, m is 2; in another embodiment, m is 3; in another embodiment, m is 4; in another embodiment, m is 5.

[0125] GalNAc

[0126] In one embodiment, GalNAc is a conjugated group represented by formula (X):

[0127]

[0128] in,

[0129] Indicates the location of attachment to a biomolecule;

[0130] Q G Independently for H,

[0131] Where L G1 is a chemical bond, -CH2-, -CH2CH2-, -C(O)-, -CH2O-, -CH2O-CH2CH2O-, or -NHC(O)-(CH2NHC(O)) a -;

[0132] L G2 is a chemical bond or -CH2CH2C(O)-;

[0133] L G3 For chemical bonds, -(NHCH2CH2) b -、-(NHCH2CH2CH2) b - or -C(O)CH2-;

[0134] L G4 -(OCH2CH2) c -、-(OCH2CH2CH2) c -、-(OCH2CH2CH2CH2) c -、-(OCH2CH2CH2CH2CH2CH2) c -or-NHC(O)-(CH2) d -;

[0135] Where a = 0, 1, 2 or 3;

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

[0137] c = 1, 2, 3, 4 or 5;

[0138] d = 1, 2, 3, 4, 5, 6, 7 or 8;

[0139] A is a chemical bond, -CH2O- or -NHC(O)-;

[0140] A' is a chemical bond, -C(O)NH-, -NHC(O)- or -O(CH2CH2O) e -;

[0141] Where e is 1, 2, 3, 4 or 5;

[0142] B is a chemical bond, -CH2-, -C(O)-, -M-, -CH2-M- or -C(O)-M-;

[0143] Where M is

[0144] R G1 and R G2 Together they form -CH2CH2O- or -CH2CH(R G )-O-, and RG3 is H;

[0145] or R G1 and R G3 Together they form -C 1-2 Alkylene-, and R G2 is H;

[0146] Where R G For-OR G ', -CH2OR G 'or-CH2CH2OR G ', where R G ' is H, a hydroxyl protecting group or a solid phase carrier, wherein the hydroxyl protecting group is preferably -C(O)CH2CH2C(O)OH or 4,4'-dimethoxytrityl;

[0147] m1=0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

[0148] n1=0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0149] In another embodiment, GalNAc is a conjugate group represented by formula (I'):

[0150]

[0151] in,

[0152] Indicates the location of attachment to a biomolecule;

[0153] Q G Independently for H,

[0154] Where L G1 is a chemical bond, -CH2-, -CH2CH2-, -C(O)-, -CH2O-, -CH2O-CH2CH2O-, or -NHC(O)-(CH2NHC(O)) a -;

[0155] L G2 is a chemical bond or -CH2CH2C(O)-;

[0156] L G3 For chemical bonds, -(NHCH2CH2) b -、-(NHCH2CH2CH2) b - or -C(O)CH2-;

[0157] L G4 -(OCH2CH2) c-、-(OCH2CH2CH2) c -、-(OCH2CH2CH2CH2) c -、-(OCH2CH2CH2CH2CH2CH2) c -or-NHC(O)-(CH2) d -;

[0158] Where a = 0, 1, 2 or 3;

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

[0160] c = 1, 2, 3, 4 or 5;

[0161] d = 1, 2, 3, 4, 5, 6, 7 or 8;

[0162] A is -CH2O- or -NHC(O)-;

[0163] A' is a chemical bond, -C(O)NH- or -NHC(O)-;

[0164] R G1 and R G2 Together they form -CH2CH2O- or -CH2CH(R G )-O-, and R G3 is H;

[0165] or R G1 and R G3 Together they form -C 1-2 Alkylene-, and R G2 is H;

[0166] Where R G For-OR G ', -CH2OR G 'or-CH2CH2OR G ', where R G ' is H, a hydroxyl protecting group or a solid phase carrier, wherein the hydroxyl protecting group is preferably -C(O)CH2CH2C(O)OH or 4,4'-dimethoxytrityl;

[0167] m1=0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

[0168] n1=0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0169] In another embodiment, GalNAc is a conjugated group represented by formula (X), wherein,

[0170] Q G Independently for H,

[0171] Where L G1 is a chemical bond, -CH2-, -CH2CH2-, -C(O)-, -CH2O-, -CH2O-CH2CH2O-, or -NHC(O)-(CH2NHC(O)) a -;

[0172] L G2 is a chemical bond or -CH2CH2C(O)-;

[0173] L G3 For chemical bonds, -(NHCH2CH2) b -、-(NHCH2CH2CH2) b - or -C(O)CH2-;

[0174] L G4 -(OCH2CH2) c -、-(OCH2CH2CH2) c -、-(OCH2CH2CH2CH2) c -、-(OCH2CH2CH2CH2CH2CH2) c -or-NHC(O)-(CH2) d -;

[0175] Where a = 0, 1, 2 or 3;

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

[0177] c = 1, 2, 3, 4 or 5;

[0178] d = 1, 2, 3, 4, 5, 6, 7 or 8;

[0179] A is a chemical bond, -CH2O- or -NHC(O)-;

[0180] A' is a chemical bond, -C(O)NH-, -NHC(O)- or -O(CH2CH2O) e -;

[0181] Where e is 1, 2, 3, 4 or 5;

[0182] B is a chemical bond, -CH2-, -M-, -CH2-M- or -C(O)-M-;

[0183] Where M is

[0184] R G1 and R G2 Together they form -CH2CH2O- or -CH2CH(R G)-O-, and R G3 is H;

[0185] or R G1 and R G3 Together they form -C 1-2 Alkylene-, and R G2 is H;

[0186] Where R G For-OR G ', -CH2OR G 'or-CH2CH2OR G ', where R G ' is H, a hydroxyl protecting group or a solid phase carrier, wherein the hydroxyl protecting group is preferably -C(O)CH2CH2C(O)OH or 4,4'-dimethoxytrityl;

[0187] m1=0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

[0188] n1=0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0189] In another embodiment, GalNAc is a conjugated group represented by formula (X), wherein:

[0190] Q G Independently for H,

[0191] Where L G1 is a chemical bond, -CH2-, -CH2CH2-, -C(O)-, -CH2O-, -CH2O-CH2CH2O-, or -NHC(O)-(CH2NHC(O)) a -;

[0192] L G2 is a chemical bond or -CH2CH2C(O)-;

[0193] L G3 For chemical bonds, -(NHCH2CH2) b -、-(NHCH2CH2CH2) b - or -C(O)CH2-;

[0194] L G4 -(OCH2CH2) c -、-(OCH2CH2CH2) c -、-(OCH2CH2CH2CH2) c -、-(OCH2CH2CH2CH2CH2CH2) c -or-NHC(O)-(CH2)d -;

[0195] Where a = 0, 1, 2 or 3;

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

[0197] c = 1, 2, 3, 4 or 5;

[0198] d = 1, 2, 3, 4, 5, 6, 7 or 8;

[0199] A is a chemical bond, -CH2O- or -NHC(O)-;

[0200] A' is -O(CH2CH2O) e -;

[0201] Where e is 1, 2, 3, 4 or 5;

[0202] B is a chemical bond, -CH2-, -C(O)-, -M-, -CH2-M- or -C(O)-M-;

[0203] Where M is

[0204] R G1 and R G2 Together they form -CH2CH2O- or -CH2CH(R G )-O-, and R G3 is H;

[0205] or R G1 and R G3 Together they form -C 1-2 Alkylene-, and R G2 is H;

[0206] Where R G For-OR G ', -CH2OR G 'or-CH2CH2OR G ', where R G ' is H, a hydroxyl protecting group or a solid phase carrier, wherein the hydroxyl protecting group is preferably -C(O)CH2CH2C(O)OH or 4,4'-dimethoxytrityl;

[0207] m1=0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

[0208] n1=0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0209] 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., or any combination thereof. The present invention is intended to include all combinations of these technical solutions, which are not listed one by one due to space limitations.

[0210] In one aspect, the present invention specifically relates to the following technical solutions:

[0211] A1. Nucleotide monomers shown in formula I,

[0212]

[0213] in,

[0214] L1 is OH or OP1, or a chemical bond to the hydroxyl group at the 2' or 3' end of the ribose of another nucleotide, nucleoside or oligonucleotide;

[0215] L2 is H or P2, or a chemical bond to the phosphate P atom at the 5' end of the ribose of another nucleotide, nucleoside or oligonucleotide;

[0216] X is a chemical bond or CR1R2;

[0217] Y1 is O, S or NR;

[0218] Y2 is O, S or a chemical bond;

[0219] 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, C 3-10 Cycloalkyl, 3-10 membered heterocyclic group, C 6-10 aryl or 5-14 membered heteroaryl, which is optionally substituted with 1, 2, 3, 4, 5, 6, 7, 8 or more R';

[0220] R3 is selected from H, C 1-6 Alkyl, C 1-6 Haloalkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 3-10 Cycloalkyl, 3-10 membered heterocyclic group, C 6-10 aryl or 5-14 membered heteroaryl, which is optionally substituted with 1, 2, 3, 4, 5, 6, 7, 8 or more R';

[0221] Base is H, a modified or unmodified base or a leaving group;

[0222] Wherein P1 and P2 are independently selected from hydroxyl protecting groups, preferably silane protecting groups, acyl protecting groups or ether protecting groups, more preferably

[0223] trimethylsilyl (TMS), triethylsilyl (TES), dimethylisopropylsilyl (DMIPS), diethylisopropylsilyl (DEIPS), tert-butyldimethylsilyl (TBDMS), tert-butyldiphenylsilyl (TBDPS), triisopropylsilyl (TIPS),

[0224] 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),

[0225] Benzyl (Bn), p-methoxybenzyl (PMB), allyl, triphenylmethyl (Tr), di-p-methoxytrityl (DMTr), methoxymethyl (MOM), phenoxymethyl (BOM), 2,2,2-trichloroethoxymethyl, 2-methoxyethoxymethyl (MEM), methylthiomethyl (MTM), p-methoxybenzyloxymethyl (PMBM);

[0226] R is selected from H, C 1-6 Alkyl or C 1-6 Haloalkyl;

[0227] R' is selected from D, halogen, CN, C 1-6 Alkyl, C 1-6 Haloalkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 3-10 Cycloalkyl, 3-10 membered heterocyclic group, C 6-10 Aryl, 5-14 membered 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 ;

[0228] n is independently 1 or 2;

[0229] R a and R b Independently selected from H, C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 3-10 Cycloalkyl, 3-10 membered heterocyclic group, C 6-10 Aryl or 5-14 membered heteroaryl; or R a and R b and the nitrogen atom to which they are attached form a 3-10 membered heterocyclic group.

[0230] A2. The nucleotide monomer of technical solution A1, wherein L1 is a chemical bond connected to the hydroxyl group at the 2' or 3' end of the ribose of another nucleotide, nucleoside or oligonucleotide, preferably L1 is a chemical bond connected to the hydroxyl group at the 3' end of the ribose of another nucleotide, nucleoside or oligonucleotide.

[0231] A3. The nucleotide monomer of technical solution A1 or A2, wherein L2 is H, or a chemical bond to a phosphate P atom at the 5' end of the ribose of another nucleotide, nucleoside or oligonucleotide; preferably L2 is H; preferably L2 is a chemical bond to a phosphate P atom at the 5' end of the ribose of another nucleotide, nucleoside or oligonucleotide.

[0232] A4. The nucleotide monomer of any one of technical solutions A1-A3, wherein X is a chemical bond.

[0233] A5. The nucleotide monomer of any one of technical solutions A1-A3, wherein X is CR1R2; preferably CH2.

[0234] A6. The nucleotide monomer of any one of technical solutions A1-A5, wherein Y1 is O.

[0235] A7. The nucleotide monomer of any one of technical solutions A1-A6, wherein Y2 is O.

[0236] A8. The 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, C1-6 Alkyl, C 1-6 Haloalkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 4-8 Cycloalkyl, 4-7 membered heterocyclic group, C 6-10 Aryl and 5-6 membered heteroaryl, preferably H, D, halogen, CN, C 1-6 Alkyl, C 1-6 Haloalkyl, C 2-6 Alkenyl or C 2-6 Alkynyl, preferably H, D, halogen or C 1-6 Alkyl, more preferably H or D.

[0237] A9. A nucleotide monomer according to 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, C 3-10 Cycloalkyl, 3-10 membered heterocyclic group, C 6-10 Aryl or 5-14 membered heteroaryl, preferably H, C 1-6 Alkyl or C 1-6 Halogenated alkyl.

[0238] A10. The 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.

[0239] A11. A nucleotide monomer according to any one of technical solutions A1-A9, wherein Base is a leaving group; preferably selected from halogen, hydroxyl, -OCOR", -OTs or -ONO2, wherein R" is C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 3-10 Cycloalkyl, 3-10 membered heterocyclic group, C 6-10 Aryl or 5-14 membered heteroaryl; preferably halogen or -OTs; more preferably -OTs.

[0240] A12. The nucleotide monomer of technical solution A1 has one of the following structures:

[0241]

[0242] in,

[0243] L1 is OH or OP1, or a chemical bond to the hydroxyl group at the 3' end of the ribose of another nucleotide, nucleoside or oligonucleotide;

[0244] L2 is H or P2, or a chemical bond to the phosphate P atom at the 5' end of the ribose of another nucleotide, nucleoside or oligonucleotide;

[0245] R3 is selected from H, C 1-6 Alkyl or C 1-6 Haloalkyl;

[0246] 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.

[0247] A13. A nucleotide dimer represented by formula III,

[0248]

[0249] in,

[0250] L3 is H or P2, or a chemical bond to the phosphate P atom at the 2' or 3' end of the ribose of another nucleotide, nucleoside or oligonucleotide;

[0251] R8 is selected from H, OH, halogen, C 1-6 Alkyl, C 1-6 Haloalkyl or C 1-6 Alkoxy, preferably fluoro or methoxy, preferably H;

[0252] Base' is H, a modified or unmodified base or a leaving group, preferably modified or unmodified A, U, T, G and C;

[0253] The other groups are as defined in any one of technical solutions A1-A12.

[0254] A14. The nucleotide dimer of technical solution A13 has the following structure:

[0255]

[0256] A15. A nucleic acid molecule, the nucleotide sequence of which contains 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.

[0257] A16. The nucleic acid molecule of technical solution A15, wherein the nucleic acid is selected from DNA, RNA and DNA / RNA hybrid.

[0258] A17. The nucleic acid molecule of technical solution A15 or A16, wherein the nucleic acid molecule is single-stranded or double-stranded.

[0259] A18. The 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).

[0260] A19. A double-stranded RNA molecule comprising a sense strand and an antisense strand, wherein each strand has 14 to 30 nucleotides, and the 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.

[0261] A20. The double-stranded RNA molecule of technical solution A19, wherein the sense strand and the antisense strand each have 20 to 25 nucleotides; preferably, the sense strand is 20 nucleotides in length; preferably, the sense strand is 21 nucleotides in length; preferably, the sense strand is 22 nucleotides in length; preferably, the sense strand is 23 nucleotides in length; preferably, the sense strand is 24 nucleotides in length; preferably, the sense strand is 25 nucleotides in length; preferably, the antisense strand is 20 nucleotides in length; preferably, the antisense strand is 21 nucleotides in length; preferably, the antisense strand is 22 nucleotides in length; preferably, the antisense strand is 23 nucleotides in length; preferably, the antisense strand is 24 nucleotides in length; preferably, the antisense strand is 25 nucleotides in length.

[0262] A21. The 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 among positions 1-30 of the 5' end of the sense strand, preferably any one or more positions among 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.

[0263] A22. A double-stranded RNA molecule according to any one of technical solutions A19-A21, wherein the nucleotide duplex is located at any one or more positions on the sense strand or the antisense strand, including but not limited to positions 1-30 at the 5' end of the antisense strand and / or any one or more positions among positions 1-30 at the 5' end of the sense strand, preferably positions 2-9 at the 5' end of the antisense strand, preferably positions 6 and 7 at the 5' end of the antisense strand, preferably positions 7 and 8 at the 5' end of the antisense strand.

[0264] A23. A double-stranded RNA molecule according to any one of technical solutions A19-A22, wherein the double-stranded RNA shows enhanced stability compared to a double-stranded RNA having the same sequence but not containing the nucleotide monomers according to any one of technical solutions 1-12 or the nucleotide duplexes according to technical solutions 13 or 14.

[0265] A24. The 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 about 55°C to 67°C.

[0266] A25. A double-stranded RNA molecule according to any one of technical solutions A19-A24, wherein the double-stranded RNA shows reduced off-target toxicity compared to a double-stranded RNA having the same sequence but not containing the nucleotide monomers according to any one of technical solutions A1-A12 or the nucleotide duplexes according to technical solutions A13 or A14.

[0267] A26. A double-stranded RNA molecule according to any one of technical solutions A19-A25, wherein the double-stranded RNA shows enhanced effectiveness compared to a double-stranded RNA having the same sequence but not containing the nucleotide monomers according to any one of technical solutions A1-A12 or the nucleotide duplexes according to technical solutions A13 or A14.

[0268] A27. A double-stranded RNA molecule according to 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 degradation of the target mRNA.

[0269] A28. The double-stranded RNA molecule of technical solution A27, wherein the target mRNA is encoded by an endogenous gene or a pathogen gene.

[0270] A29. The 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.

[0271] A30. The 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 comprises one or more GalNAc.

[0272] 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.

[0273] A32. A kit comprising a double-stranded RNA molecule as described in any one of technical solutions A19-A30.

[0274] A33. A method for inhibiting the expression of a target gene in a cell, comprising the step of introducing a double-stranded RNA molecule described in any one of technical solutions A19-A30 into the cell.

[0275] A34. A method for inhibiting the expression of a target gene in a cell, comprising expressing a double-stranded RNA molecule described in any one of technical schemes A19-A30 in the cell.

[0276] In another aspect, the present invention relates to the following technical solutions:

[0277] B1. Nucleotide dimer shown in formula III:

[0278]

[0279] in,

[0280] L2 is H or P2, or a chemical bond to the phosphate P atom at the 5' end of the ribose of another nucleotide or oligonucleotide;

[0281] L3 is H or P3, or a chemical bond to a phosphate P atom at the 2' or 3' end of the ribose of another nucleotide or oligonucleotide;

[0282] X is a chemical bond or CR1R2;

[0283] Y1 is O, S or NR;

[0284] Y2 is O, S or a chemical bond;

[0285] 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, C 3-10 Cycloalkyl, 3-10 membered heterocyclic group, C 6-10 aryl or 5-14 membered heteroaryl, which is optionally substituted with 1, 2, 3, 4, 5, 6, 7, 8 or more R';

[0286] R3 is selected from H, C 1-6 Alkyl, C 1-6 Haloalkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 3-10 Cycloalkyl, 3-10 membered heterocyclic group, C 6-10 aryl or 5-14 membered heteroaryl, which is optionally substituted with 1, 2, 3, 4, 5, 6, 7, 8 or more R';

[0287] R8 is selected from H, OH, halogen, C 1-6 Alkyl, C1-6 Haloalkyl or C 1-6 Alkoxy, preferably fluoro or methoxy, preferably H;

[0288] Base is H, a modified or unmodified base or a leaving group;

[0289] Base' is H, a modified or unmodified base or a leaving group, preferably modified or unmodified A, U, T, G and C;

[0290] Wherein P2 and P3 are independently selected from hydroxyl protecting groups, preferably silane protecting groups, acyl protecting groups or ether protecting groups, more preferably

[0291] trimethylsilyl (TMS), triethylsilyl (TES), dimethylisopropylsilyl (DMIPS), diethylisopropylsilyl (DEIPS), tert-butyldimethylsilyl (TBDMS), tert-butyldiphenylsilyl (TBDPS), triisopropylsilyl (TIPS),

[0292] 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),

[0293] Benzyl (Bn), p-methoxybenzyl (PMB), allyl, triphenylmethyl (Tr), di-p-methoxytrityl (DMTr), methoxymethyl (MOM), phenoxymethyl (BOM), 2,2,2-trichloroethoxymethyl, 2-methoxyethoxymethyl (MEM), methylthiomethyl (MTM), p-methoxybenzyloxymethyl (PMBM);

[0294] R is selected from H, C 1-6 Alkyl or C 1-6 Haloalkyl;

[0295] R' is selected from D, halogen, CN, C 1-6 Alkyl, C 1-6 Haloalkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 3-10 Cycloalkyl, 3-10 membered heterocyclic group, C 6-10 Aryl, 5-14 membered 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 ;

[0296] n is independently 1 or 2;

[0297] R a and R b Independently selected from H, C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 3-10 Cycloalkyl, 3-10 membered heterocyclic group, C 6-10 Aryl or 5-14 membered heteroaryl; or R a and R b and the nitrogen atom to which they are attached form a 3-10 membered heterocyclic group.

[0298] B2. The nucleotide dimer of technical solution B1, wherein L3 is a chemical bond connected to the hydroxyl group at the 2' or 3' end of the ribose of another nucleotide or oligonucleotide, preferably L3 is a chemical bond connected to the hydroxyl group at the 3' end of the ribose of another nucleotide or oligonucleotide.

[0299] B3. The nucleotide dimer of technical solution B1 or B2, wherein L2 is H, or a chemical bond to a phosphate P atom at the 5' end of the ribose of another nucleotide or an oligonucleotide; preferably L2 is H; preferably L2 is a chemical bond to a phosphate P atom at the 5' end of the ribose of another nucleotide or an oligonucleotide.

[0300] B4. The nucleotide dimer of any one of technical solutions B1-B3, wherein X is a chemical bond.

[0301] B5. The nucleotide duplex of any one of technical solutions B1-B4, wherein X is CR1R2; preferably CH2.

[0302] B6. The nucleotide dimer of any one of technical solutions B1-B5, wherein Y1 is O.

[0303] B7. The nucleotide dimer of any one of technical solutions B1-B6, wherein Y2 is O.

[0304] B8. The 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, C 1-6 Alkyl, C 1-6 Haloalkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 4-8 Cycloalkyl, 4-7 membered heterocyclic group, C 6-10 Aryl and 5-6 membered heteroaryl, preferably H, D, halogen, CN, C 1-6 Alkyl, C 1-6 Haloalkyl, C 2-6 Alkenyl or C 2-6 Alkynyl, preferably H, D, halogen or C 1-6 Alkyl, more preferably H or D.

[0305] B9. The 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, C 3-10 Cycloalkyl, 3-10 membered heterocyclic group, C 6-10 Aryl or 5-14 membered heteroaryl, preferably H, C 1-6 Alkyl or C 1-6 Halogenated alkyl.

[0306] B10. The nucleotide duplex 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.

[0307] B11. The nucleotide dimer of any one of technical solutions B1-B9, wherein Base is a leaving group; preferably selected from halogen, hydroxyl, -OCOR", -OTs or -ONO2, wherein R" is C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 3-10 Cycloalkyl, 3-10 membered heterocyclic group, C 6-10 Aryl or 5-14 membered heteroaryl; preferably halogen or -OTs; more preferably -OTs.

[0308] B12. The nucleotide dimer of any one of technical solutions B1-B11, which has one of the following structures:

[0309]

[0310] in,

[0311] L2 is H or P2, or a chemical bond to the phosphate P atom at the 5' end of the ribose of another nucleotide or oligonucleotide;

[0312] L3 is H or P3, or a chemical bond to a phosphate P atom at the 2' or 3' end of the ribose of another nucleotide or oligonucleotide;

[0313] R3 is selected from H, C 1-6 Alkyl or C 1-6 Haloalkyl;

[0314] R8 is selected from H, OH, halogen, C 1-6 Alkyl, C 1-6 Haloalkyl or C 1-6 Alkoxy, preferably fluoro or methoxy, preferably H;

[0315] 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;

[0316] Base' is H, a modified or unmodified base or a leaving group, preferably modified or unmodified A, U, T, G and C.

[0317] In another aspect, the present invention relates to the following technical solutions:

[0318] C1. A nucleotide dimer represented by formula (A),

[0319]

[0320] in,

[0321] L2 is H or P2;

[0322] L3 is H or P3;

[0323] Q is -X- or -XO-;

[0324] X is a chemical bond, -(CR1R2) m -or-CR1=CR2-;

[0325] Y1 is O, S or NR;

[0326] Y2 is O, S or a chemical bond;

[0327] 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 or C 2-6alkynyl, which is optionally substituted with 1, 2, 3, 4 or 5 R';

[0328] R3 is selected from H, C 1-6 Cyanoalkyl, C 1-6 Alkyl, C 1-6 Haloalkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 3-7 Cycloalkyl, 3-7 membered heterocyclic group, C 6-10 aryl or 5-10 membered heteroaryl, which is optionally substituted with 1, 2, 3, 4 or 5 R';

[0329] R8 is selected from H, D, OH, halogen, C 1-6 Alkyl, C 1-6 Haloalkyl or C 1-6 Alkoxy;

[0330] Base and Base' are independently selected from H, a modified or unmodified base, or a leaving group;

[0331] P2 is selected from reactive phosphorus groups, preferably -P(OCH2CH2CN)(N(iPr)2);

[0332] P3 is selected from a hydroxyl protecting group, preferably DMTr;

[0333] R is selected from H, C 1-6 Alkyl or C 1-6 Haloalkyl;

[0334] 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, C 3-10 Cycloalkyl, 3-10 membered heterocyclic group, C 6-10 Aryl or 5-14 membered heteroaryl;

[0335] m is selected from 1, 2, 3, 4 or 5.

[0336] C2. The nucleotide dimer of technical solution C1, wherein

[0337] L2 is P2;

[0338] L3 is P3;

[0339] Q is -X- or -XO-;

[0340] X is a chemical bond, -(CR1R2) m -or-CR1=CR2-;

[0341] Y1 is O or S;

[0342] Y2 is O or a chemical bond;

[0343] R1, R2, R4, R5, R6 and R7 are independently selected from H, D, halogen, CN, C 1-6 Alkyl or C 1-6 Haloalkyl, preferably H, optionally substituted with 1, 2 or 3 R';

[0344] R3 is selected from H, C 1-6 Alkyl, C 1-6 Cyanoalkyl, C 1-6 Haloalkyl, C 2-6 Alkenyl or C 2-6 Alkynyl, optionally substituted with 1, 2 or 3 R';

[0345] R8 is selected from H, OH, halogen or C 1-6 Alkoxy, preferably fluorine or methoxy, preferably fluorine;

[0346] Base and Base' are independently selected from H, modified or unmodified bases or leaving groups. Preferably, Base and Base' are independently selected from

[0347] P2 is selected from reactive phosphorus groups, preferably -P(OCH2CH2CN)(N(iPr)2);

[0348] P3 is selected from a hydroxyl protecting group, preferably DMTr;

[0349] R' is selected from D, halogen, OH, CN, NH2, C 1-6 Alkyl or C 1-6 Haloalkyl;

[0350] m is selected from 1, 2 or 3.

[0351] C3. The nucleotide dimer of technical solution C1 or C2, wherein,

[0352] L2 is P2;

[0353] L3 is P3;

[0354] Q is -X- or -XO-;

[0355] X is -CH2-, -CH2-CH2- or -CH=CH-;

[0356] Y1 is O;

[0357] Y2 is O;

[0358] R1, R2, R4, R5, R6 and R7 are independently selected from H, halogen or C1-4 Alkyl, preferably H;

[0359] R3 is selected from H, C 1-4 Alkyl or C 1-4 Cyanoalkyl, preferably methyl or cyanoethyl;

[0360] R8 is selected from halogen or C 1-4 Alkoxy, preferably fluorine or methoxy, preferably fluorine;

[0361] Base and Base' are independently selected from

[0362] P2 is selected from reactive phosphorus groups, preferably -P(OCH2CH2CN)(N(iPr)2);

[0363] P3 is selected from hydroxyl protecting groups, preferably DMTr.

[0364] C4. The nucleotide dimer of any one of technical solutions C1-C3, wherein Q is -CH2-O-.

[0365] C5. The nucleotide dimer of any one of technical solutions C1-C3, which has the following structure:

[0366]

[0367] in,

[0368] R3 is methyl or cyanoethyl;

[0369] The other groups are as defined in any one of Technical Schemes C1-C3.

[0370] C6. The nucleotide dimer of any one of technical solutions C1-C5, which has the following structure:

[0371]

[0372] C7. A double-stranded RNA molecule or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, comprising a sense strand and an antisense strand, wherein each strand has 14 to 30 nucleotides, wherein the antisense strand has a sequence that is sufficiently complementary to the sense strand and the target mRNA and has the ability to induce degradation of the target mRNA, and the antisense strand comprises one or more nucleotide monomers represented by formula (IV):

[0373]

[0374] in,

[0375] The nucleotide monomers are shown in the order 5'=>3' from to connect;

[0376] Each group is as defined in any one of technical solutions C1-C6;

[0377] Preferably, the nucleotide monomer is selected from:

[0378]

[0379] Among them, Base is selected from

[0380] C8. The double-stranded RNA molecule of technical solution C7, wherein the double-stranded RNA molecule is prepared using the nucleotide dimer of any one of technical solutions C1-C6.

[0381] C9. The double-stranded RNA molecule of technical solution C7 or C8, wherein the sense strand and the antisense strand each have 20 to 30 nucleotides.

[0382] C10. The 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, and more preferably position 7, of the 5' end of the antisense strand.

[0383] C11. The double-stranded RNA molecule of any one of technical solutions C7-C10, wherein the double-stranded RNA shows enhanced stability compared to a double-stranded RNA having the same sequence but not containing the nucleotide monomer described in technical solution C7.

[0384] C12. The 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 about 55°C to 67°C.

[0385] C13. The double-stranded RNA molecule of any one of technical solutions C7-C12, wherein the double-stranded RNA shows reduced off-target toxicity compared to a double-stranded RNA having the same sequence but not containing the nucleotide monomer described in technical solution C7.

[0386] C14. The double-stranded RNA molecule of any one of technical solutions C7-C13, wherein the double-stranded RNA shows enhanced effectiveness compared to a double-stranded RNA having the same sequence but not containing the nucleotide monomer described in technical solution C7.

[0387] C15. The 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.

[0388] C16. The 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 comprises one or more GalNAc.

[0389] 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 using the nucleotide dimer according to any one of technical solutions C1-C6.

[0390] C18. A nucleic acid molecule, the nucleotide sequence of which contains one or more nucleotide monomers as described in technical solution C7.

[0391] C19. The nucleic acid molecule of technical solution C18, wherein the nucleic acid is selected from DNA, RNA and DNA / RNA hybrid.

[0392] C20. The nucleic acid molecule of technical solution C19, wherein the nucleic acid molecule is single-stranded or double-stranded.

[0393] C21. The 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).

[0394] 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.

[0395] C22. A kit comprising a double-stranded RNA molecule as described in any one of technical solutions C7-C16.

[0396] C23. A method for inhibiting the expression of a target gene in a cell, comprising the step of introducing a double-stranded RNA molecule described in any one of technical solutions C7-C16 into the cell.

[0397] C24. A method for inhibiting the expression of a target gene in a cell, comprising expressing in the cell a double-stranded RNA molecule described in any one of technical schemes C7-C16. Example

[0398] The following examples are intended to illustrate the present invention but are not intended to limit the scope of the present invention.

[0399] Example 1: Synthesis of Nucleotide Dimer of Compound 14

[0400] Synthesis route:

[0401]

[0402] Experimental steps:

[0403] 1. Synthesis of Compound 2

[0404] Compound 1 (25 g, 189.165 mmol) was dissolved in 250 mL of dichloromethane, followed by the 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), and the reaction mixture was stirred overnight at room temperature. TLC monitored the reaction of the raw materials to be completely reacted. The reaction system was then diluted with dichloromethane (300 mL), washed with saturated NaHCO3 aqueous solution (3 x 300 mL), citric acid (1 x 200 mL) and saturated brine (300 mL) in sequence, the organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain compound 2 (42.0 g, 286.34 mmol, 77.54%) as a yellow oil.

[0405] m / z=286.2[M+H] +

[0406] 2. Synthesis of Compound 3

[0407] Under argon protection, adenine (23.78 g, 176.015 mmol) was dissolved in 600 mL N, N-dimethylformamide, and sodium hydrogen (5.28 g, 220.018 mmol) was added in batches under an ice-water bath. The reaction mixture was heated to 100 degrees Celsius and stirred for 2 hours. Compound 2 (42 g, 146.679 mmol) was then dissolved in 200 mL N, N-dimethylformamide and added dropwise to the above reaction system, and continued to stir at 100 ° C overnight. TLC monitored the reaction of the raw materials, and then methanol (20 mL) was used to quench the reaction. The reaction solvent was decompressed and dried, and the crude product was purified by 100-200 mesh silica gel column chromatography (methanol: dichloromethane = 0-10%, 30 min) to obtain compound 3 (1 g, 249.27 mmol, 49.23%) as a white solid.

[0408] m / z=250.2[M+H] +

[0409] 3. Synthesis of Compound 4

[0410] Compound 3 (18 g, 72.211 mmol) was dissolved in 200 mL of anhydrous pyridine, and benzoyl chloride (20.30 g, 144.422 mmol) was added at room temperature and stirred overnight. TLC monitored that the reaction raw materials reacted completely, and then ammonia methanol solution (7 M, 200 mL) was added. After stirring for 10 minutes, the reaction system was decompressed and dried, and the crude product was purified by 100-200 mesh 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.

[0411] m / z=354.2[M+H] +

[0412] 4. Synthesis of Compound 5

[0413] 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 system was stirred at room temperature for 3 hours. TLC monitored the reaction of the raw materials to be complete. The reaction system was then dried under reduced pressure, and the crude product was purified by 100-200 mesh 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.

[0414] m / z=314.0[M+H] +

[0415] 5. Synthesis of Compound 6

[0416] Compound 5 (3.9 g, 12.447 mmol) was washed with anhydrous pyridine three times and then dissolved in anhydrous pyridine (40 mL). 4,4-dimethoxytriphenylmethane (5.06 g, 14.937 mmol) was added at room temperature under argon protection. The reaction system was stirred overnight at room temperature. The conversion of the raw materials was detected by LC-MS. The reaction system was diluted with ethyl acetate (200 mL), washed with water twice, 50 mL each time, the organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The crude product was purified by 100-200 mesh silica gel column chromatography (ethyl acetate: petroleum ether = 0-50%, 40 minutes) to obtain compound 6 (6.2 g, 10.077 mmol, 80.96%) as a yellow solid.

[0417] m / z=616.2[M+H] +

[0418] 6. Synthesis of Compound 7

[0419] Dissolve dimethyl hydroxymethylphosphite (9.3g, 66.395mmol) in dichloromethane (100mL), cool the reaction solution to 0 degrees, slowly add triethylamine (11.997mL, 86.314mmol) and p-toluenesulfonyl chloride (13.92g, 73.035mmol) under argon protection, and stir the reaction mixture at room temperature overnight. LC-MS monitoring shows that the reaction of the raw material is complete, the reaction solution is diluted with dichloromethane, washed with water, dried over anhydrous sodium sulfate, filtered, and the filtrate is concentrated under reduced pressure. The crude product is purified by 100-200 mesh silica gel column chromatography (methanol: dichloromethane = 0-20%, 40 minutes) to obtain compound 7 (11g, 37.382mmol, 56.30%) as a light yellow solid.

[0420] m / z=294.8[M+H] +

[0421] 7. Synthesis of Compound 8

[0422] Compound 6 (400 mg, 0.650 mmol) was dissolved in anhydrous N, N-dimethylformamide (20 mL) under nitrogen protection, sodium hydrogen (46.77 mg, 1.950 mmol) was added under ice-water bath, and the above reaction mixture was stirred under ice-water bath for 30 minutes. 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. After the reaction mixture was stirred at room temperature for 72 hours, LC-MS monitored that the raw material was completely converted. 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 obtain crude compound 8 (510 mg) as a yellow solid.

[0423] m / z=294.8[M+H] +

[0424] 8. Synthesis of Compound 9

[0425] 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 then concentrated under reduced pressure. The crude product was purified by 100-200 mesh silica gel column chromatography (methanol: dichloromethane = 0-10%, 40 minutes) to obtain compound 9 (145 mg, 49.14%) as a white solid.

[0426] 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).

[0427] 9. Synthesis of Compound 10

[0428] Compound 9 (650 mg, 1.49 mmol) was dissolved in anhydrous N,N-dimethylformamide (5 mL) under nitrogen protection, and imidazole (304.8 mg, 4.48 mmol) and tert-butyldimethylsilyl chloride (336.2 mg, 2.24 mmol) were added at room temperature. The reaction mixture was stirred at room temperature overnight, and the conversion of the raw materials was completed after LC-MS monitoring. The reaction mixture was quenched with ice water, extracted with ethyl acetate (3x50 mL), and 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 100-200 mesh silica gel column chromatography (methanol: dichloromethane = 0-3%, 40 minutes) to obtain compound 10 (710 mg, 1.29 mmol, 86%) as an off-white solid.

[0429] m / z=550.4[M+H] +

[0430] 10. Synthesis of Compound 11

[0431] Compound 10 (710 mg, 1.29 mmol) was dissolved in pyridine aqueous solution (3:2, 20 mL), and the reaction mixture was stirred at 50 degrees for 6 hours. LC-MS monitored the conversion of the raw materials, and the reaction solution was concentrated under reduced pressure and the crude product was directly used for the next reaction without purification.

[0432] m / z=536.2[M+H] +

[0433] 11. Synthesis of Compound 12

[0434] Under argon protection, the crude compound 11 (450 mg) was dissolved in anhydrous pyridine (5 mL), triisopropylsilyl chloride (763.94 mg, 2.522 mmol) was added under ice bath, and the reaction system was stirred at room temperature for 15 minutes. Then 5'-O-(4,4'-dimethoxytrityl)-N2-isobutyryl-2'-fluorodeoxyguanosine (580.26 mg, 0.883 mmol) and N-methylimidazole (345.15 mg, 4.204 mmol) were dissolved in anhydrous pyridine (5 mL) and slowly added dropwise to the above reaction system. The reaction mixture was stirred at room temperature overnight, and LC-MS monitored the conversion of the raw materials. 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 reverse phase chromatography (5 M ammonium bicarbonate aqueous solution / acetonitrile) to obtain compound 12 (480 mg, 63%) as a yellow solid.

[0435] m / z=1175.6[M+H] +

[0436] 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).

[0437] 19 F NMR(DMSO-d6):δ-204.82,-204.53

[0438] 31 P NMR (DMSO-d6): δ 22.94, 22.72

[0439] 12. Synthesis of Compound 13

[0440] Compound 12 (200 mg, 0.170 mmol) was dissolved in anhydrous tetrahydrofuran (10 mL) under nitrogen protection. A solution of triethylamine hydrofluoride (137.26 mg, 0.851 mmol) in tetrahydrofuran (2 mL) was slowly added dropwise under an ice-water bath. The reaction system was heated to room temperature for 1 hour. The conversion of the reaction raw materials was completed after monitoring by LC-MS. The reaction system was diluted with ethyl acetate and quenched with water. The organic phase was separated and dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The crude product was purified by reverse phase preparation (preparation conditions: chromatographic 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 in 5.5min; wavelength: 254 / 210nm; RT1(min): 4.5,5.1) to give compound 13 (69mg, 0.065mmol, 38.21%) as a white solid.

[0441] m / z=1062.3[M+H] +

[0442] 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).

[0443] 19 F NMR (400MHz, DMSO-d6): δ-204.54

[0444] 31 P NMR (400 MHz, DMSO-d6): δ 23.17

[0445] 13. Synthesis of Compound 14

[0446] First, bis(diisopropylamino)(2-cyanoethoxy)phosphine (23.45 mg, 0.078 mmol) was washed three times with ultra-dry acetonitrile, and then compound 13 (55 mg, 0.052 mmol) was washed 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-dicyanoimidazole (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 LC-MS monitored the conversion of the raw materials. The reaction system was added dropwise to an icy sodium bicarbonate aqueous solution, extracted with dichloromethane, the organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The crude product was purified by reverse phase (pure water and acetonitrile, about 70% product), the collected liquid 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.

[0447] m / z=1261.7[M+H] +

[0448] 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).

[0449] 31 P NMR (400MHz, CD3CN) δ148.69,148.57,23.54,23.50.

[0450] 19 F NMR(400MHz,CD3CN)δ-203.63,-203.69.

[0451] Example 2 Preparation of siRNA

[0452] The siRNA of the present invention is prepared using the solid phase phosphoramidite method well known in the art. The specific method can be found in, for example, PCT Publication Nos. WO2016081444 and WO2019105419, and is briefly described as follows.

[0453] 1Synthesis of the positive sense chain (SS chain)

[0454] Through solid phase phosphoramidite synthesis, using a blank CPG solid phase carrier or a solid phase carrier connected with L96 as the starting cycle, the nucleoside monomers are connected one by one from the 3'-5' direction according to the arrangement order of the positive chain nucleotides. Each connection of a nucleoside monomer includes four steps of deprotection, coupling, capping, oxidation or thiolation. The synthesis conditions of oligonucleotides with a synthesis scale of 5umol are as follows:

[0455] The nucleoside monomer was provided with a 0.05 mol / L acetonitrile solution. The reaction conditions for each step were the same, i.e., the temperature was 25°C. A 3% trichloroacetic acid-dichloromethane solution was used for deprotection, and the deprotection was repeated 3 times. The activator used in the coupling reaction was a 0.25 mol / L ETT-acetonitrile solution, and the coupling was repeated 2 times. The capping reaction was performed with 10% acetic anhydride-acetonitrile and pyridine / N-methylimidazole / acetonitrile (10:14:76, v / v / v) and the capping was repeated 2 times. The oxidation reaction was performed with 0.05 mol / L iodine / tetrahydrofuran / pyridine / water (70 / 20 / 10, v / v / v) and the oxidation was repeated 2 times. The thiolation reaction was performed with 0.2 mol / L PADS in acetonitrile / 3-methylpyridine (1 / 1, v / v) and the thiolation was repeated 2 times.

[0456] 2 Synthesis of antisense strand (AS strand)

[0457] By solid phase phosphoramidite synthesis, using a blank CPG solid phase carrier as the starting cycle, nucleoside monomers or nucleotide duplexes of the present invention are connected one by one from the 3'-5' direction according to the arrangement order of the antisense chain nucleotides. Each connection of a nucleoside monomer or a nucleotide duplex of the present invention includes four steps of deprotection, coupling, capping, oxidation or thiolation, and the synthesis conditions of 5umol oligonucleotides of the antisense chain are the same as those of the sense chain.

[0458] 3 Oligonucleotide purification and annealing

[0459] 3.1 Aminolysis

[0460] Add the synthesized solid phase carrier (sense chain or antisense chain) to a 5 mL centrifuge tube, add 3% diethylamine / ammonia water (v / v), react in a constant temperature water bath at 35°C (or 55°C) for 16 hours (or 8 hours), filter, wash the solid phase carrier three times with ethanol / water, 1 mL each time, and purify the crude product after centrifugation and concentration of the filtrate.

[0461] 3.2 Purification

[0462] The methods of purification and desalination are well known to those skilled in the art. For example, a column filled with strong anion fillers can be used, and a sodium chloride-sodium hydroxide system can be used for elution and purification, and the product can be collected and piped. A gel filler purification column can be used for desalination, and the elution system is pure water.

[0463] 3.3 Annealing

[0464] According to the table below, the sense strand (SS strand) and the antisense strand (AS strand) were mixed at a molar ratio (SS strand / AS strand = 1 / 1.05), heated in a water bath to 70-95°C, maintained for 3-5 min, cooled naturally to room temperature, and the system was lyophilized to obtain the product.

[0465] The siRNA sequences used in the present invention are as follows:

[0466]

[0467] In this article, the meanings of the abbreviations are as follows:

[0468] The A, U, G and C distribution represents the natural adenine, uracil, guanine and cytosine ribonucleotides.

[0469] m indicates that the adjacent nucleotide on the left is a 2'-OCH3-modified nucleotide. For example, Am, Um, Gm, and Cm represent 2'-OCH3-modified A, U, G, and C.

[0470] f indicates that the adjacent nucleotide on its left is a 2'-F modified nucleotide. For example, Af, Uf, Gf, and Cf represent 2'-F modified A, U, G, and C, respectively.

[0471] "s" or "s-" indicates that the two adjacent nucleotides and / or delivery vectors are linked by phosphorothioate.

[0472] L96 represents a GalNAc delivery vector of the following structure well known in the art, wherein Refer to the position of the siRNA linked via a phosphate group or a phosphorothioate group, for example, PCT Publication Nos. WO2009073809 and WO2009082607.

[0473]

[0474] ROR1 represents a nucleotide substitute of the structure described above, wherein Base can be any base, for example, ROR1-A represents that Base is adenine.

[0475] Example 3 Detection of on-target and off-target activities of siRNA compound psi-CHECK2

[0476] 1. Plasmid preparation:

[0477] On-target plasmid: The corresponding antisense strand on-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.

[0478] Off-target plasmid: The corresponding antisense chain off-target plasmid was designed according to the compound sequence. The psiCHECK2 GSSM-5Hits recombinant plasmid was prepared by Sangon Biotech (Shanghai) Co., Ltd. and the recombinant plasmid was diluted to 1000 ng / μL for use.

[0479] 2. Cell transfection:

[0480] HEK293A cells (Nanjing Kebai, Cat. No. CBP60436) were used to plate 96-well plates with 100 μL of cell resuspension. Cell number: 8×10 3 cells / well.

[0481] On the second day, the complete medium in the wells was firstly discarded and replaced with Opti-MEM medium 80 μL / well, and the cells were starved for about 1.5 h.

[0482] Plasmid mixture: single well preparation volume: plasmid 0.01μL / well, Opti-MEM 8.99μL / well.

[0483] Lipo Mix: Dilute Lipo 2000 (Lipofectamine TM 2000 transfection reagent, Thermo, 11668019), let stand at room temperature for 5 minutes, the specific preparation amount of Lipo mixture: Lipo 0.2μL / well, Opti-MEM 9.8μL / well.

[0484] 22μL of the prepared Lipo mixture, 2.2μL of the compound, and 19.8μL of the plasmid mixture were dispensed into the corresponding wells: named Well A. After mixing, incubate at room temperature for 20 minutes before co-transfection. Finally, add wellAmixture to each well of cells, 20μL / well, plus the original 80μL Opti-MEM, and the final volume of each well is 100μL. After culturing in a 37℃5% CO2 incubator for 4h, 100μL of DMEM medium containing 20% ​​fetal bovine serum was added to each well. Detection was performed after culturing in a 37℃5% CO2 incubator for 24h.

[0485] 3. Result detection:

[0486] Before the experiment, mix Luciferase( Luciferase Assay System, Promega, E2940) was reconstituted and after equilibration to room temperature, DMEM was added to each tube at a ratio of 1:1 to prepare substrate I for immediate use. Stop& Buffer was reconstituted and then equilibrated to room temperature. Stop& Substrate 100:1 is prepared into Substrate II and is ready for use.

[0487] The vacuum pump sucks away the original culture medium in the 96-well culture plate;

[0488] Add 150 μL of substrate I to each well and incubate on a shaker at room temperature for 10 min;

[0489] Take 120 μL of substrate I, transfer to a 96-well ELISA plate, and read the Firefly chemiluminescence value on an ELISA reader (Tecan, Infinite 200);

[0490] Then, 60 μL of substrate II was added to each well, incubated on a shaker at room temperature for 10 min, and the Renilla chemiluminescence value was read on a microplate reader.

[0491] 4. Data analysis and processing

[0492] The fluorescence activity was measured by an ELISA instrument, and the collected Renilla signal was normalized by the Firefly signal standard. The inhibitory effect of siRNA was obtained by comparing the untreated results (residual inhibitory activity). The calculation process is as follows:

[0493] Normalized Ren / Fir ratio: Ratio = Renilla (Renilla luciferase) / Firefly (Firefly luciferase).

[0494] Residual inhibition rate: 2 replicate wells (Ratio siRNA / Ratio control )*100% mean: where Ratio control is the average value of the Ratio of the two replicate wells of the control well (without siNRA), and the Ratio of the two replicate wells is calculated respectively. siRNA / Ratio control , and then take the mean value as the residual inhibition rate;

[0495] Plotting: Plotting with Graphpad Prism

[0496] Half maximal inhibitory concentration (IC50): This experiment was plotted using Top and Bottom plots, and the IC50 value was calculated using the formula Y = Bottom + (Top-Bottom) / (1 + 10^((LogIC50-X)*HillSlope)), where Y = 50 and X = log(concentration).

[0497] In the target activity detection experiment, HEK293A (Nanjing Kebai, catalog number CBP60436) cell line was selected for psiCHECK2-GSCM recombinant plasmid transfection, and the starting concentration of the compound was selected as 10nM, and 11 concentration points (10nM, 3.33nM, 1.11nM, 0.37nM, 0.123nM, 0.041nM, 0.0136nM, 0.0045nM, 0.00152nM, 0.000508nM, 0.000169nM) were diluted 3 times to perform siRNA compound activity screening. The results are shown in Table 1.

[0498] Table 1 Results of psi-CHECK2 on-target activity detection experiments

[0499]

[0500] For the off-target activity detection experiment, HEK293A (Nanjing Kebai, catalog number CBP60436) cell line was selected for psiCHECK2-GSSM-5Hits recombinant plasmid transfection, and the starting concentration of the compound was selected as 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 diluted 3 times to perform siRNA compound activity screening. The results are shown in Table 2.

[0501] Table 2 Psi-CHECK2 off-target activity detection experimental results

[0502]

[0503] The results show that the siRNA carrying the nucleotide analog monomers of the present invention effectively reduces off-target activity while maintaining target activity.

[0504] Example 4 Verification of the long-term efficacy of the compound in the C57BL / 6 mouse model

[0505] C57BL / 6 mice (male, 18-21 g, 6-8 weeks) were randomly divided into groups according to Table 3, with 6 animals in each group. The dosage of each animal was calculated according to the body weight, and a single administration was performed by subcutaneous injection. The siRNA compound was first prepared as a 1 mg / mL solution (0.9% sodium chloride aqueous solution as solvent). Before the experiment, the siRNA compound was dissolved in 0.9% sodium chloride aqueous solution and fixed to the required solution concentration and volume. The administration volume of physiological saline and siRNA compound was 5 mL / kg.

[0506] Table 3 Animal experiment groups

[0507] Serial number Compound-Group 1 Normal saline 2 DR002220 3 DR005760

[0508] Blood was collected from the orbital venous plexus of mice before administration (recorded as day 0) and on days 14, 28, 42, and 56 after administration, and serum mTTR protein was detected using an ELISA kit (Abcam, ab282297) at each time point. At the last experimental time point, 10 mg of liver was collected and placed in RNAlater solution to detect liver mTTR mRNA.

[0509] Table 4 Results of long-term efficacy verification of compounds in C57BL / 6 mouse model

[0510]

[0511] The results show that siRNA carrying the nucleotide analog monomers of the present invention can reduce the expression of the target gene for a long time in vivo.

Claims

1. A nucleotide dimer represented by formula (A), in, L2 is H or P2; L3 is H or P3; Q is -X- or -XO-; X is a chemical bond, -(CR1R2) m -or-CR1=CR2-; Y1 is O, S or NR; Y2 is O, S or a chemical bond; 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 or C 2-6 alkynyl, which is optionally substituted with 1, 2, 3, 4 or 5 R'; R3 is selected from H, C 1-6 Cyanoalkyl, C 1-6 Alkyl, C 1-6 Haloalkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 3-7 Cycloalkyl, 3-7 membered heterocyclic group, C 6-10 aryl or 5-10 membered heteroaryl, which is optionally substituted with 1, 2, 3, 4 or 5 R'; R8 is selected from H, D, OH, halogen, C 1-6 Alkyl, C 1-6 Haloalkyl or C 1-6 Alkoxy; Base and Base' are independently selected from H, a modified or unmodified base, or a leaving group; P2 is selected from reactive phosphorus groups, preferably -P(OCH2CH2CN)(N(iPr)2); P3 is selected from a hydroxyl protecting group, preferably DMTr; R is selected from H, C 1-6 Alkyl or C 1-6 Haloalkyl; 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, C 3-10 Cycloalkyl, 3-10 membered heterocyclic group, C 6-10 Aryl or 5-14 membered heteroaryl; m is selected from 1, 2, 3, 4 or 5.

2. The nucleotide dimer according to claim 1, wherein L2 is P2; L3 is P3; Q is -X- or -XO-; X is a chemical bond, -(CR1R2) m -or-CR1=CR2-; Y1 is O or S; Y2 is O or a chemical bond; R1, R2, R4, R5, R6 and R7 are independently selected from H, D, halogen, CN, C 1-6 Alkyl or C 1-6 Haloalkyl, preferably H, optionally substituted with 1, 2 or 3 R'; R3 is selected from H, C 1-6 Alkyl, C 1-6 Cyanoalkyl, C 1-6 Haloalkyl, C 2-6 Alkenyl or C 2-6 Alkynyl, optionally substituted with 1, 2 or 3 R'; R8 is selected from H, OH, halogen or C 1-6 Alkoxy, preferably fluorine or methoxy, preferably fluorine; Base and Base' are independently selected from H, modified or unmodified bases or leaving groups. Preferably, Base and Base' are independently selected from P2 is selected from reactive phosphorus groups, preferably -P(OCH2CH2CN)(N(iPr)2); P3 is selected from a hydroxyl protecting group, preferably DMTr; R' is selected from D, halogen, OH, CN, NH2, C 1-6 Alkyl or C 1-6 Haloalkyl; m is selected from 1, 2 or 3.

3. The nucleotide dimer according to claim 1 or 2, wherein L2 is P2; L3 is P3; Q is -X- or -XO-; X is -CH2-, -CH2-CH2- or -CH=CH-; Y1 is O; Y2 is O; R1, R2, R4, R5, R6 and R7 are independently selected from H, halogen or C 1-4 Alkyl, preferably H; R3 is selected from H, C 1-4 Alkyl or C 1-4 Cyanoalkyl, preferably methyl or cyanoethyl; R8 is selected from halogen or C 1-4 Alkoxy, preferably fluorine or methoxy, preferably fluorine; Base and Base' are independently selected from P2 is selected from reactive phosphorus groups, preferably -P(OCH2CH2CN)(N(iPr)2); P3 is selected from hydroxyl protecting groups, preferably DMTr.

4. The nucleotide dimer according to any one of claims 1 to 3, wherein Q is -CH2-O-.

5. The nucleotide dimer according to any one of claims 1 to 3, having the following structure: in, R3 is methyl or cyanoethyl; The other groups are as defined in any one of claims 1 to 3.

6. The nucleotide duplex according to any one of claims 1 to 5, having the following structure:

7. A double-stranded RNA molecule or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, comprising a sense strand and an antisense strand, wherein each strand has 14 to 30 nucleotides, the antisense strand has a sequence that is sufficiently complementary to the sense strand and the target mRNA, and has the ability to induce degradation of the target mRNA, and the antisense strand comprises one or more nucleotide monomers represented by formula (IV): in, The nucleotide monomers shown are in the order of 5'=>3' from ----- to connect; Each group is defined as in any one of claims 1 to 6; Preferably, the nucleotide monomer is selected from: Among them, Base is selected from 8. The double-stranded RNA molecule of claim 7, wherein The double-stranded RNA molecule is prepared using the nucleotide duplex according to any one of claims 1-6.

9. The double-stranded RNA molecule according to claim 7 or 8, wherein the nucleotide monomer is located at position 2-8, preferably position 6 or 7, more preferably position 7, of the 5' end of the antisense strand.

10. The double-stranded RNA molecule according to any one of claims 7 to 9, wherein the double-stranded RNA is further coupled to a ligand, preferably, the ligand comprises one or more GalNAc.

11. The method for preparing a double-stranded RNA molecule according to any one of claims 7 to 10, wherein: The double-stranded RNA molecule is prepared using the nucleotide duplex according to any one of claims 1-6.

12. A pharmaceutical composition comprising the double-stranded RNA molecule according to any one of claims 7 to 10, and a pharmaceutically acceptable carrier or excipient.

13. A method for inhibiting the expression of a target gene in a cell, comprising the step of introducing the double-stranded RNA molecule according to any one of claims 7 to 10 into the cell.

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