Double-stranded rnas containing nucleotide analogs that reduce off-target toxicity

Abstract

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

Description

[0001] This invention claims priority to Chinese applications CN202210744263.8 filed on June 27, 2022, CN202210948347.3 filed on August 8, 2022, and CN202211483699.2 filed on November 24, 2022, all of which are incorporated herein by reference in their entirety. Invention Field

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

[0003] RNA interference is a phenomenon in which target mRNA is efficiently and specifically degraded by double-stranded RNA (dsRNA). Incorporating thermally unstable nucleotides (e.g., glycerol nucleotides (GNA)) into the antisense seed region of double-stranded RNA can help improve interference efficiency and reduce off-target toxicity; see, for example, PCT Publication No. WO2018098328A1.

[0004] Therefore, there is a need in this field to develop a new nucleotide analog that, when incorporated into double-stranded RNA, helps reduce off-target toxicity. 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 provides a nucleotide dimer of formula (A):

[0007]

[0008] The groups are defined below.

[0009] In another aspect, the present invention provides 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, and said antisense strand comprises one or more nucleotide monomers of formula (III) or (IV):

[0010]

[0011] in,

[0012] The nucleotide monomers shown are arranged in the order of 5' => 3' from... to connect;

[0013] Each group is defined below.

[0014] In another aspect, the present invention provides nucleic acid molecules whose nucleotide sequences contain one or more nucleotide dimers and / or nucleotide monomers as described herein.

[0015] In another aspect, the present invention provides a vector comprising a nucleotide sequence encoding the aforementioned double-stranded RNA.

[0016] In another aspect, the present invention provides a cell containing the aforementioned double-stranded RNA or the aforementioned vector.

[0017] 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.

[0018] In another aspect, the present invention relates to a kit comprising a double-stranded RNA molecule as described herein.

[0019] 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.

[0020] In another aspect, the present invention relates to a method for inhibiting the expression of a target gene in a cell, comprising expressing a double-stranded RNA molecule as described herein in the cell.

[0021] In another aspect, the present invention relates to a method for reducing off-target toxicity in cells, comprising the step of introducing the double-stranded RNA molecule described herein into the cell.

[0022] In another aspect, the present invention relates to a method for reducing off-target toxicity in cells, comprising expressing the double-stranded RNA molecule described herein in the cells.

[0023] When the nucleotides of the present invention are incorporated into the antisense strand of dsRNA, the resulting double-stranded RNA exhibits one or more of the following: enhanced stability, reduced off-target toxicity, and enhanced efficacy. Invention Details

[0025] definition

[0026] Chemical definition

[0027] The definitions of specific functional groups and chemical terms are described in more detail below.

[0028] When listing a range of values, it is assumed that each value and the subranges within that range are included. For example, "C 1-6 Alkyl groups include C1, C2, C3, C4, C5, C6, and C6. 1-6 C 1-5 C 1-4 C1-3 C 1-2 C 2-6 C 2-5 C 2-4 C 2-3 C 3-6 C 3-5 C 3-4 C 4-6 C 4-5 and C 5-6 alkyl.

[0029] “C 1-6 "Alkyl" refers to a straight-chain or branched saturated hydrocarbon group having 1 to 6 carbon atoms. In some embodiments, C 1-4 Alkyl and C 1-2 Alkyl groups are preferred. C 1-6 Examples of alkyl groups include: methyl (C1), ethyl (C2), n-propyl (C3), isopropyl (C3), n-butyl (C4), tert-butyl (C4), sec-butyl (C4), isobutyl (C4), n-pentyl (C5), 3-pentyl (C5), pentyl (C5), neopentyl (C5), 3-methyl-2-butyl (C5), tert-pentyl (C5), and n-hexyl (C6). The term "C" is used in conjunction with the preceding text. 1-6 "Alkyl" also includes heteroalkyl, wherein one or more (e.g., 1, 2, 3, or 4) carbon atoms are replaced by heteroatoms (e.g., oxygen, sulfur, nitrogen, boron, silicon, phosphorus). The alkyl group may be optionally substituted by one or more substituents, for example, by 1 to 5 substituents, 1 to 3 substituents, or 1 substituent. Common alkyl abbreviations include: Me(-CH3), Et(-CH2CH3), iPr(-CH(CH3)2), nPr(-CH2CH2CH3), n-Bu(-CH2CH2CH2CH3) or i-Bu(-CH2CH(CH3)2).

[0030] “C 2-6 "Alkenyl" refers to a straight-chain or branched hydrocarbon group having 2 to 6 carbon atoms and at least one carbon-carbon double bond. In some embodiments, C 2-4 Alkenyl groups are preferred. C 2-6 Examples of alkenyl groups include: vinyl (C2), 1-propenyl (C3), 2-propenyl (C3), 1-butenyl (C4), 2-butenyl (C4), butadienyl (C4), pentenyl (C5), pentadienyl (C5), hexenyl (C6), and so on. The term "C" is used in conjunction with these groups. 2-6 "Alkenyl" also includes heteroalkenyl groups, wherein one or more (e.g., 1, 2, 3, or 4) carbon atoms are replaced by heteroatoms (e.g., oxygen, sulfur, nitrogen, boron, silicon, phosphorus). The alkenyl group may be optionally substituted by one or more substituents, for example, by 1 to 5 substituents, 1 to 3 substituents, or 1 substituent.

[0031] “C 2-6 "Alkyne" refers to a straight-chain or branched hydrocarbon group having 2 to 6 carbon atoms, at least one carbon-carbon triple bond, and optionally one or more carbon-carbon double bonds. In some embodiments, C 2-4 Alkyne groups are preferred. C 2-6 Examples of alkynyl groups include, but are not limited to: ethynyl (C2), 1-propynyl (C3), 2-propynyl (C3), 1-butynyl (C4), 2-butynyl (C4), pentyynyl (C5), hexynyl (C6), etc. The term "C" is used in conjunction with other alkynyl groups. 2-6 "Alkyne" also includes heteroyne, wherein one or more (e.g., 1, 2, 3 or 4) carbon atoms are replaced by heteroatoms (e.g., oxygen, sulfur, nitrogen, boron, silicon, phosphorus). The alkynyl group may be optionally substituted by one or more substituents, for example, by 1 to 5 substituents, 1 to 3 substituents or 1 substituent.

[0032] "Halogen" or "halogen" refers to fluorine (F), chlorine (Cl), bromine (Br), and iodine (I).

[0033] Therefore, "C" 1-6 "Halogenated alkyl" refers to the above "C 1-6 "alkyl" is substituted with one or more halogen groups. In some embodiments, C 1-4 Haloalkyl groups are particularly preferred, and C4 groups are more preferred. 1-2 Halogenated alkyl groups. Exemplary alkyl halogenated groups include, but are not limited to: -CF3, -CH2F, -CHF2, -CHFCH2F, -CH2CHF2, -CF2CF3, -CCl3, -CH2Cl, -CHCl2, 2,2,2-trifluoro-1,1-dimethyl-ethyl, etc. The alkyl halogenated group can be substituted at any available connection point, for example, 1 to 5 substituents, 1 to 3 substituents, or 1 substituent.

[0034] “C 1-6 "Alkoxy" refers to the -OR group, where R is as described above as "C". 1-6 "alkyl" and "C" 1-6 The definition of "halogenated alkyl".

[0035] “C 3-10 "Cycloalkyl" refers to a non-aromatic cyclic hydrocarbon group having 3 to 10 ring carbon atoms and zero heteroatoms. In some embodiments, C 4-7 cycloalkyl and C 3-6 Cycloalkyl groups are particularly preferred, and C10 is more preferred. 5-6Cycloalkyl groups. Cycloalkyl groups also include ring systems in which the aforementioned cycloalkyl ring is fused with one or more aryl or heteroaryl groups, wherein the connecting point is on the cycloalkyl ring, and in such cases, the number of carbons continues to represent the number of carbons in the cycloalkyl system. Exemplary cycloalkyl groups include, but are not limited to: cyclopropyl (C3), cyclopropenyl (C3), cyclobutyl (C4), cyclobutenyl (C4), cyclopentyl (C5), cyclopentenyl (C5), cyclohexyl (C6), cyclohexenyl (C6), cyclohexadienyl (C6), cycloheptyl (C7), cycloheptenyl (C7), cycloheptadienyl (C7), cyclohepttrienyl (C7), etc. The cycloalkyl group may optionally be substituted with one or more substituents, for example, substituted with 1 to 5 substituents, 1 to 3 substituents, or 1 substituent.

[0036] "3-10 membered heterocyclic groups" refer to groups with a 3- to 10-membered non-aromatic ring system having a cyclic carbon atom and 1 to 5 cyclic heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen, sulfur, boron, phosphorus, and silicon. In heterocyclic groups containing one or more nitrogen atoms, the linkage can be a carbon or nitrogen atom, provided that the valence allows. In some embodiments, a 4-10 membered heterocyclic group is preferred, which is a 4-10 membered non-aromatic ring system having a cyclic carbon atom and 1 to 5 cyclic heteroatoms; in some embodiments, a 3-8 membered heterocyclic group is preferred, which is a 3-8 membered non-aromatic ring system having a cyclic carbon atom and 1 to 4 cyclic heteroatoms; a 3-6 membered heterocyclic group is preferred, which is a 3-6 membered non-aromatic ring system having a cyclic carbon atom and 1 to 3 cyclic heteroatoms; a 4-7 membered heterocyclic group is preferred, which is a 4-7 membered non-aromatic ring system having a cyclic carbon atom and 1 to 3 cyclic heteroatoms; and a 5-6 membered heterocyclic group is preferred, which is a 5-6 membered non-aromatic ring system having a cyclic carbon atom and 1 to 3 cyclic heteroatoms. Heterocyclic groups also include ring systems in which the aforementioned heterocyclic ring is fused with one or more cycloalkyl groups, wherein the linking point is on the cycloalkyl ring, or ring systems in which the aforementioned heterocyclic ring is fused with one or more aryl or heteroaryl groups, wherein the linking point is on the heterocyclic ring; and in such cases, the number of ring members continues to represent the number of ring members in the heterocyclic ring system. Exemplary 3-membered heterocyclic groups containing one heteroatom include, but are not limited to: azirropropyl, oxetane, and thiorenyl. Exemplary 4-membered heterocyclic groups containing one heteroatom include, but are not limited to: azirrobutyl, oxetane, and thiorenyl. Exemplary 5-membered heterocyclic groups containing one heteroatom include, but are not limited to: tetrahydrofuranyl, dihydrofuranyl, tetrahydrothiophenyl, dihydrothiophenyl, pyrrolidinyl, dihydropyrrolidinyl, and pyrrolidin-2,5-dione. Exemplary 5-membered heterocyclic groups containing two heteroatoms include, but are not limited to: dioxasulfuranyl, oxasulfuranyl, disulfuranyl, and oxazolidin-2-one. Exemplary 5-membered heterocyclic groups containing three heteroatoms include, but are not limited to: triazolinyl, oxadiazolinyl, and thiadiazolinyl. Exemplary 6-membered heterocyclic groups containing one heteroatom include, but are not limited to: piperidinyl, tetrahydropyranyl, dihydropyridinyl, and thianyl. Exemplary 6-membered heterocyclic groups containing two heteroatoms include, but are not limited to: piperazinyl, morpholinyl, disulfuranyl, and dioxalyl. Exemplary 6-membered heterocyclic groups containing three heteroatoms include, but are not limited to: triazinanyl. Exemplary 7-membered heterocyclic groups containing one heteroatom include, but are not limited to: azirheptanyl, oxasulfuranyl, and thioheptanyl. Exemplary 5-membered heterocyclic groups fused with a C6 aryl ring (also referred to herein as 5,6-bicyclic heterocyclic groups) include, but are not limited to: dihydroindolyl, isodihydroindolyl, dihydrobenzofuranyl, dihydrobenzothiophenyl, benzoxazolinone, etc.Exemplary 6-membered heterocyclic groups fused with a C6 aryl ring (also referring to 6,6-bicyclic heterocyclic groups herein) include, but are not limited to, tetrahydroquinolinyl, tetrahydroisoquinolinyl, etc. The heterocyclic group may be optionally substituted by one or more substituents, for example, by 1 to 5 substituents, 1 to 3 substituents, or 1 substituent.

[0037] “C 6-10 "Aryl" refers to a monocyclic or polycyclic (e.g., bicyclic) 4n+2 aromatic ring system (e.g., having 6 or 10 shared π electrons arranged in a ring) having 6-10 ring carbon atoms and zero heteroatoms. In some embodiments, the aryl group has six ring carbon atoms ("C6 aryl"; e.g., phenyl). In some embodiments, the aryl group has ten ring carbon atoms ("C6 aryl"). 10 "Aryl"; for example, naphthyl, such as 1-naphthyl and 2-naphthyl). Aryl also includes a ring system in which the above-mentioned aryl ring is fused with one or more cycloalkyl or heterocyclic groups, and the connection point is on the aryl ring, in which case the number of carbon atoms continues to represent the number of carbon atoms in the aryl ring system. The aryl group may be optionally substituted by one or more substituents, for example, by 1 to 5 substituents, 1 to 3 substituents, or 1 substituent.

[0038] "5-14 membered heteroaryl" refers to a 4n+2 aromatic ring system of a 5-14 membered monocyclic or bicyclic ring (e.g., having 6, 10, or 14 shared π electrons arranged in a ring) having a ring carbon atom and 1-4 ring heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur. In heteroaryl containing one or more nitrogen atoms, the bonding point can be a carbon or nitrogen atom, provided the valence allows. A heteroaryl bicyclic system may include one or more heteroatoms in one or both rings. Heteroaryl also includes ring systems in which the aforementioned heteroaryl ring is fused with one or more cycloalkyl or heterocyclic groups, and the bonding point is on the heteroaryl ring, in which case the number of carbon atoms continues to represent the number of carbon atoms in the heteroaryl ring system. In some embodiments, 5-10 membered heteroaryl is preferred, which is a 4n+2 aromatic ring system of a 5-10 membered monocyclic or bicyclic ring having a ring carbon atom and 1-4 ring heteroatoms. In other embodiments, 5-6 membered heteroaryl groups are particularly preferred, which are 4n+2 aromatic ring systems of 5-6 membered monocyclic or bicyclic rings having a cyclic carbon atom and 1-4 cyclic heteroatoms. Exemplary 5-membered heteroaryl groups containing one heteroatom include, but are not limited to: pyrrole, furanyl, and thiophene. Exemplary 5-membered heteroaryl groups containing two heteroatoms include, but are not limited to: imidazolyl, pyrazolyl, oxazolyl, isoxazolyl, thiazolyl, and isothiazolyl. Exemplary 5-membered heteroaryl groups containing three heteroatoms include, but are not limited to: triazolyl, oxadiazolyl (e.g., 1,2,4-oxadiazolyl), and thiadiazolyl. Exemplary 5-membered heteroaryl groups containing four heteroatoms include, but are not limited to: tetrazolyl. Exemplary 6-membered heteroaryl groups containing one heteroatom include, but are not limited to: pyridinyl. Exemplary 6-membered heteroaryl groups containing two heteroatoms include, but are not limited to: pyridazinyl, pyrimidinyl, and pyrazinyl. Exemplary 6-membered heteroaryl groups containing three or four heteroatoms include, but are not limited to, triazinyl and tetraazinyl. Exemplary 7-membered heteroaryl groups containing one heteroatom include, but are not limited to, azirmonoheptatrienyl, oxadiazinyl, and thioheptatrienyl. Exemplary 5,6-bicyclic heteroaryl groups include, but are not limited to, indolyl, isoindolyl, indazole, benzotriazolyl, benzothiophene, isobenzothiophene, benzofuranyl, benzoisofuranyl, benzoimidazolyl, benzoxazolyl, benzoisoxazolyl, benzoxadiazolyl, benzothiazolyl, benzoisothiazolyl, benzothiadiazolyl, indazinyl, and purinyl. Exemplary 6,6-bicyclic heteroaryl groups include, but are not limited to, naphthidyl, pteridinyl, quinolinyl, isoquinolinyl, zolinyl, quinoxolinyl, phthalazinyl, and quinazolinyl. The heteroaryl group may be optionally substituted by one or more substituents, for example, by 1 to 5 substituents, 1 to 3 substituents or 1 substituent.

[0039] The alkyl, alkenyl, ynyl, cycloalkyl, heterocyclic, aryl, and heteroaryl groups defined in this article are optional substituted groups.

[0040] Exemplary substituents on carbon atoms include, but are not limited to: halogen, -CN, -NO2, -N3, -SO2H, -SO3H, -OH, -OR aa -ON(R) bb )2、-N(R bb )2、-N(R bb )3 + X - -N(OR) cc )R bb -SH, -SR aa -SSR cc -C(=O)R aa -CO2H, -CHO, -C(OR) cc )2、-CO2R aa -OC(=O)R aa -OCO2R aa -C(=O)N(R) bb )2、-OC(=O)N(R bb )2、-NR bb C(=O)R aa -NR bb CO2R aa -NR bb C(=O)N(R bb )2、-C(=NR bb )R aa -C(=NR) bb OR aa -OC(=NR) bb )R aa -OC(=NR) bb OR aa -C(=NR) bb )N(R bb )2、-OC(=NR bb )N(R bb )2、-NR bb C(=NR bb )N(R bb )2、-C(=O)NR bb SO2R aa -NR bb SO2R aa -SO2N(R) bb )2、-SO2R aa -SO2OR aa -OSO2R aa -S(=O)R aa -OS(=O)R aa 、-Si(R 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, ynyl, cycloalkyl, heterocyclic, aryl, and heteroaryl, wherein each alkyl, alkenyl, ynyl, cycloalkyl, heterocyclic, aryl, and heteroaryl is independently bounded by 0, 1, 2, 3, 4, or 5 R groups. dd Group substitution;

[0041] Or the two hydrogen-bearing groups on the carbon atom: =O, =S, =NN(R) bb )2、=NNR bb C(=O)R aa =NNR bb C(=O)OR aa =NNR bb S(=O)2R aa =NR bb or = NOR cc replace;

[0042] R aa Each of them is independently selected from alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, heterocyclic, aryl, and heteroaryl, or two R aa Groups are combined to form heterocyclic or heteroaryl rings, wherein each alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclic, aryl, and heteroaryl group is independently bounded by 0, 1, 2, 3, 4, or 5 R groups. dd Group substitution;

[0043] R bb Each is independently selected from: hydrogen, -OH, -OR aa -N(R) cc )2、-CN、-C(=O)R aa -C(=O)N(R) cc )2、-CO2R aa -SO2R aa -C(=NR) cc OR aa -C(=NR) cc )N(R cc )2、-SO2N(R cc )2、-SO2R cc -SO2OR cc -SOR aa -C(=S)N(R) cc )2、-C(=O)SR cc -C(=S)SR cc -P(=O)2R aa -P(=O)(R aa )2、-P(=O)2N(R cc )2、-P(=O)(NR cc 2. Alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, heterocyclic, aryl and heteroaryl, or two R bb Groups are combined to form heterocyclic or heteroaryl rings, wherein each alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclic, aryl, and heteroaryl group is independently bounded by 0, 1, 2, 3, 4, or 5 R groups. dd Group substitution;

[0044] R cc Each is independently selected from hydrogen, alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, heterocyclic, aryl, and heteroaryl, or two R cc Groups are combined to form heterocyclic or heteroaryl rings, wherein each alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclic, aryl, and heteroaryl group is independently bounded by 0, 1, 2, 3, 4, or 5 R groups. dd Group substitution;

[0045] R ddEach is independently selected from: halogen, -CN, -NO2, -N3, -SO2H, -SO3H, -OH, -OR ee -ON(R) ff )2、-N(R ff )2,、-N(R ff )3 + X - -N(OR) ee )R ff -SH, -SR ee -SSR ee -C(=O)R ee -CO2H, -CO2R ee -OC(=O)R ee -OCO2R ee -C(=O)N(R) ff )2、-OC(=O)N(R ff )2、-NR ff C(=O)R ee -NR ff CO2R ee -NR ff C(=O)N(R ff )2、-C(=NR ff OR ee -OC(=NR) ff )R ee -OC(=NR) ff OR ee -C(=NR) ff )N(R ff )2、-OC(=NR ff )N(R ff )2、-NR ff C(=NR ff )N(R ff )2、-NR ff SO2R ee -SO2N(R) ff )2、-SO2R ee -SO2OR ee -OSO2R ee -S(=O)R ee 、-Si(R ee )3、-OSi(R ee 3. -C(=S)N(R) ff )2、-C(=O)SR ee -C(=S)SR ee -SC(=S)SR ee -P(=O)2Ree -P(=O)(R ee )2、-OP(=O)(R ee )2、-OP(=O)(OR ee 2. Alkyl, haloalkyl, alkenyl, ynyl, cycloalkyl, heterocyclic, aryl, heteroaryl, wherein each alkyl, alkenyl, ynyl, cycloalkyl, heterocyclic, aryl, and heteroaryl group is independently marked by 0, 1, 2, 3, 4, or 5 R groups. gg Group substitution, or two geminal radicals dd Substituents can combine to form =O or =S;

[0046] R ee Each is independently selected from alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, aryl, heterocyclic, and heteroaryl, wherein each alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclic, aryl, and heteroaryl is independently surrounded by 0, 1, 2, 3, 4, or 5 R groups. gg Group substitution;

[0047] R ff Each is independently selected from hydrogen, alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, heterocyclic, aryl, and heteroaryl, or two R ff The groups combine to form a heterocyclic or heteroaryl ring, wherein each alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclic, aryl, and heteroaryl group is independently bounded by 0, 1, 2, 3, 4, or 5 R groups. gg Group substitution;

[0048] R gg Each of these is independently: halogen, -CN, -NO2, -N3, -SO2H, -SO3H, -OH, -OC 1-6 Alkyl, -ON(C) 1-6 Alkyl)2, -N(C 1-6 Alkyl)2, -N(C 1-6 Alkyl)3 + X - -NH(C 1-6 Alkyl)2 + X - -NH2(C 1-6 alkyl) + X - -NH3 + X - -N(OC) 1-6 Alkyl)(C 1-6 Alkyl), -N(OH)(C 1-6 Alkyl groups, -NH(OH), -SH, -SC 1-6 Alkyl, -SS(C 1-6 Alkyl), -C(=O)(C 1-6Alkyl group, -CO2H, -CO2(C 1-6 Alkyl), -OC (=O)(C 1-6 Alkyl), -OCO2(C 1-6 Alkyl groups, -C(=O)NH2, -C(=O)N(C 1-6 Alkyl)2、-OC(=O)NH(C 1-6 Alkyl), -NHC(=O)(C 1-6 alkyl), -N(C) 1-6 Alkyl)C(=O)(C 1-6 alkyl), -NHCO2(C 1-6 Alkyl), -NHC(=O)N(C 1-6 Alkyl)2、-NHC(=O)NH(C 1-6 Alkyl groups, -NHC(=O)NH2, -C(=NH)O(C 1-6 Alkyl), -OC (=NH)(C 1-6 Alkyl group), -OC (=NH)OC 1-6 Alkyl group, -C(=NH)N(C 1-6 Alkyl)2、-C(=NH)NH(C 1-6 Alkyl groups, -C(=NH)NH2, -OC(=NH)N(C 1-6 Alkyl)2、-OC(NH)NH(C 1-6 Alkyl groups, -OC(NH)NH2, -NHC(NH)N(C 1-6 Alkyl)2, -NHC(=NH)NH2, -NHSO2(C 1-6 alkyl), -SO2N(C 1-6 alkyl)2、-SO2NH(C 1-6 Alkyl groups, -SO2NH2, -SO2C 1-6 Alkyl, -SO2OC 1-6 Alkyl, -OSO2C 1-6 Alkyl, -SOC 1-6 Alkyl, -Si(C) 1-6 Alkyl)3、-OSi(C 1-6 Alkyl)3, -C(=S)N(C 1-6 Alkyl)2、C(=S)NH(C 1-6 Alkyl), C(=S)NH2, -C(=O)S(C 1-6 Alkyl), -C(=S)SC 1-6 Alkyl, -SC (=S)SC 1-6 Alkyl group, -P(=O)2(C 1-6 Alkyl), -P(=O)(C 1-6 Alkyl)2、-OP(=O)(C 1-6Alkyl)2、-OP(=O)(OC 1-6 Alkyl)2, C 1-6 Alkyl, C 1-6 Haloalkyl, C2-C6 alkenyl, C2-C6 ynyl, C3-C7 cycloalkyl, C6-C 10 Aryl, C3-C7 heterocyclic, C5-C 10 heteroaryl; or two ethryl groups gg Substituents can combine to form =O or =S; where X - It is a counterion.

[0049] Exemplary substituents on the nitrogen atom include, but are not limited to: hydrogen, -OH, -OR aa -N(R) cc )2、-CN、-C(=O)R aa -C(=O)N(R) cc )2、-CO2R aa -SO2R aa -C(=NR) bb )R aa -C(=NR) cc OR aa -C(=NR) cc )N(R cc )2、-SO2N(R cc )2、-SO2R cc -SO2OR cc -SOR aa -C(=S)N(R) cc )2、-C(=O)SR cc -C(=S)SR cc -P(=O)2R aa -P(=O)(R aa )2、-P(=O)2N(R cc )2、-P(=O)(NR cc 2. Alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, heterocyclic, aryl, and heteroaryl, or two R atoms attached to a nitrogen atom. cc The groups combine to form a heterocyclic or heteroaryl ring, wherein each alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclic, aryl, and heteroaryl group is independently bounded by 0, 1, 2, 3, 4, or 5 R groups. dd Group substitution, wherein R aa R bb R cc and R dd As stated above.

[0050] Other definitions

[0051] The term "siRNA" in this article refers to a class of double-stranded RNA molecules that can mediate the silencing of a complementary target RNA (e.g., mRNA, such as transcripts of genes encoding proteins). siRNA is typically double-stranded, consisting of an antisense strand complementary to the target RNA and a sense strand complementary to that antisense strand. For convenience, such mRNA is also referred to herein as the mRNA to be silenced. Such genes are also referred to as target genes. Typically, the RNA to be silenced is an endogenous gene or a pathogen gene. Additionally, RNAs other than mRNA (e.g., tRNA) and viral RNA can also be targeted.

[0052] The term "antisense strand" refers to a strand of siRNA that contains regions that are completely, sufficiently, or substantially complementary to the target sequence. The term "sense strand" refers to a strand of siRNA that includes regions that are completely, sufficiently, or substantially complementary to the regions defined herein as antisense strands.

[0053] The term "complementary region" refers to a region on the antisense strand that is completely, fully, or substantially complementary to the target mRNA sequence. In cases where the complementary region is not perfectly complementary to the target sequence, mismatches can occur within the molecule or at the ends. Typically, the most tolerant mismatches are located in the end regions, for example, within 5, 4, 3, 2, or 1 nucleotides at the 5' and / or 3' ends. The portion of the antisense strand most sensitive to mismatches is called the "seed region." For example, in a siRNA containing a 19-nt strand, the 19th position (from 5' to 3') can tolerate some mismatches.

[0054] The term "complementary" refers to the ability of a first polynucleotide to hybridize with a second polynucleotide under certain conditions, such as stringent conditions. For example, stringent conditions may include 400 mM NaCl, 40 mM PIPES at pH 6.4, and 1 mM EDTA at 50 or 70°C for 12–16 hours. In terms of meeting the above requirements regarding their hybridization ability, a "complementary" sequence may also include base pairs formed entirely from non-Watson-Crick base pairs and / or from non-natural and modified nucleotides. Such non-Watson-Crick base pairs include, but are not limited to, G:U swing base pairings or Hoogstein base pairs.

[0055] A polynucleotide that is “at least partially complementary,” “fully complementary,” or “substantially complementary” to messenger RNA (mRNA) refers to a polynucleotide that is substantially complementary to a continuous portion of the mRNA of interest. For example, a polynucleotide is at least partially complementary to PCSK9 mRNA if the sequence is substantially complementary to an uninterrupted portion of the mRNA encoding PCSK9. The terms “complementary,” “fully complementary,” “fully complementary,” and “substantially complementary” can be used relative to base pairing between the sense and antisense strands of the siRNA, or between the antisense strand of the siRNA reagent and the target sequence.

[0056] "Perfect complementarity" means that in order to maintain the overall double-stranded character of the molecule, the sense strand only needs to be complementary to the antisense strand to a certain extent. In other words, although perfect complementarity is usually required, in some cases, especially in the antisense strand, there may be one or more mismatches (relative to the target mRNA), such as 6, 5, 4, 3, 2 or 1, but the sense and antisense strands can still maintain the overall double-stranded character of the molecule.

[0057] "shRNA" refers to short hairpin RNA. shRNA consists of two short inverted repeat sequences. The shRNA cloned into the shRNA expression vector comprises two short inverted repeat sequences separated by a stem-loop sequence, forming a hairpin structure controlled by the polIII promoter. Subsequently, 5-6 T molecules are added as a transcription terminator for RNA polymerase III.

[0058] "Nucleoside" is a compound composed of two substances: a purine or pyrimidine base and ribose or deoxyribose. "Nucleotide" is a compound composed of three substances: a purine or pyrimidine base, ribose or deoxyribose, and phosphate. "Oligonucleotide" refers to nucleic acid molecules (RNA or DNA) with a length of less than 100, 200, 300, or 400 nucleotides.

[0059] A "base" is the basic building block for the synthesis of nucleosides, nucleotides, and nucleic acids. Its constituent elements include nitrogen, hence it is also called a "nitrogenous base." In this article, unless otherwise specified, the capital letters A, U, T, G, and C represent the base composition of nucleotides, namely adenine, uracil, thymine, guanine, and cytosine, respectively.

[0060] The term "modification" of nucleotides as used herein includes, but is not limited to, methoxy modification, fluorination modification, thiophosphate linkage, or conventional protecting group protection. For example, a fluorinated nucleotide refers to a nucleotide in which the hydroxyl group at the 2' position of the ribosome is replaced by fluorine, and a methoxy modified nucleotide refers to a nucleotide in which the 2'-hydroxyl group of the ribosome is replaced by a methoxy group.

[0061] In this document, "modified nucleotides" include, but are not limited to, nucleotides modified with 2'-O-methyl, nucleotides modified with 2'-fluorine, nucleotides modified with 2'-deoxy-, inosine ribonucleotides, debased nucleotides, reverse abased deoxyribonucleotides, nucleotides containing a thiophosphate group, nucleotides modified with vinyl phosphate, locked nucleotides, nucleotides modified with 2'-amino, nucleotides modified with 2'-alkyl, morpholinonucleotides, aminophosphates, non-natural bases containing nucleotides, and terminal nucleotides linked to cholesterol derivatives or dodecanoic acid diecamide groups, deoxyribonucleotides, or those protected by conventional protecting groups. For example, a nucleotide modified with 2'-fluorine refers to a nucleotide in which the hydroxyl group at the 2' position of the ribosyl group is replaced by fluorine. A nucleotide modified with 2'-deoxy- refers to a nucleotide in which the 2'-hydroxyl group of the ribosyl group is replaced by a methoxy group.

[0062] "Reactive phosphorus group" refers to a phosphorus-containing group contained in a nucleotide unit or nucleotide analog unit that can react with a hydroxyl or amino group contained in another molecule, particularly in another nucleotide unit or another nucleotide analog, via a nucleophilic attack reaction. Typically, such a reaction produces an ester-type nucleoside bond linking the first nucleotide unit or the first nucleotide analog unit to the second nucleotide unit or the second nucleotide analog unit. The reactive phosphorus group can be selected from phosphorous amides, H-phosphonates, alkyl-phosphonates, phosphate esters, or phosphate ester analogs, including but not limited to: native phosphate esters, thiophosphate esters, dithiophosphate esters, boron phosphate esters, boron thiophosphate esters, phosphonates, halogen-substituted phosphonates and phosphate esters, aminophosphate esters, phosphate diesters, phosphate triesters, thiophosphate diesters, thiophosphate triesters, diphosphate esters, and triphosphate esters, preferably -P(OCH2CH2CN)(N(iPr)2).

[0063] A "protecting group," also known as a "protecting element," is any atom or group of atoms added to a molecule to prevent existing groups in the molecule from undergoing undesirable chemical reactions. Protecting groups can be unstable chemical moieties known in the art, used to protect reactive groups, such as hydroxyl, amino, and thiol groups, to prevent unwanted or untimely reactions during chemical synthesis. Protecting groups are typically used selectively and / or orthogonally to protect sites during reactions at other reactive sites, and can then be removed to leave unprotected groups intact or usable for further reactions.

[0064] A non-limiting list of protecting groups includes benzyl; substituted benzyl; alkyl carbonyl and alkoxy carbonyl (e.g., tert-butoxy carbonyl (BOC), acetyl, or isobutyryl); arylalkyl carbonyl and arylalkoxy carbonyl (e.g., benzyloxy carbonyl); substituted methyl ether (e.g., methoxymethyl ether); substituted diethyl ether; substituted benzyl ether; tetrahydropyranyl ether; silyl (e.g., trimethylsilyl, triethylsilyl, triisopropylsilyl, tert-butyldimethylsilyl, tri-isopropylsilyloxymethyl, [2-(trimethylsilyl)ethoxy)). Methyl or tert-butyldiphenylsilyl); esters (e.g., benzoates); carbonates (e.g., methoxymethyl carbonate); sulfonates (e.g., toluenesulfonates or methanesulfonates); noncyclic ketals (e.g., dimethyl acetal); cyclic ketals (e.g., 1,3-dioxane, 1,3-dioxolane, and those described herein); noncyclic acetals; cycloacetals (e.g., those described herein); noncyclic hemiacetals; cyclic hemiacetals; cyclic dithioketals (e.g., 1,3-dithiane or 1,3-dithiopentane); orthoesters (e.g., those described herein); and triarylmethyl groups. (For example, triphenylmethyl; monomethoxytriphenylmethyl (MMTr); 4,4′-dimethoxytriphenylmethyl (DMTr); 4,4′,4″-trimethoxytriphenylmethyl (TMTr); and those described herein). Preferred protecting groups are selected from acetyl (Ac), benzoyl (Bzl), benzyl (Bn), isobutyryl (iBu), phenylacetyl, benzyloxymethylacetal (BOM), β-methoxyethoxymethyl ether (MEM), methoxymethyl ether (MOM), p-methoxybenzyl ether (PMB), methyl thiomethyl ether, new Vanoyl (Piv), Tetrahydropyranyl (THP), Triphenylmethyl (Trt), Methoxytriphenylmethyl[(4-methoxyphenyl)diphenylmethyl] (MMT), Dimethoxytriphenylmethyl, [bis-(4-methoxyphenyl)phenylmethyl (DMT), Trimethylsilyl ether (TMS), Tert-butyldimethylsilyl ether (TBDMS), Tri-isopropylsilyloxymethyl ether (TOM), Tri-isopropylsilyl ether (TIPS), Methyl ether, Ethoxyethyl ether (EE), N,N-dimethylformamidinium and 2-cyanoethyl (CE).

[0065] A "hydroxyl protecting group" is a group that prevents the hydroxyl group from undergoing chemical reactions and can be removed under specific conditions to restore the hydroxyl group. These mainly include silane-type protecting groups, acyl-type protecting groups, or ether-type protecting groups, with the following being preferred:

[0066] 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 (Troc), Benzyloxycarbonyl (Cbz), Tert-Butyloxycarbonyl (Boc) Benzyl (Bn), p-methoxybenzyl (PMB), allyl, triphenylmethyl (Tr), bis-p-methoxytriphenylmethyl (DMTr), methoxymethyl (MOM), phenoxymethyl (BOM), 2,2,2-trichloroethoxymethyl, 2-methoxyethoxymethyl (MEM), methylthiomethyl (MTM), p-methoxybenzylmethyl (PMBM), -C(O)CH2CH2C(O)OH or 4,4'-dimethoxytriphenylmethyl, preferably -C(O)CH2CH2C(O)OH or 4,4'-dimethoxytriphenylmethyl, more preferably -C(O)CH2CH2C(O)OH.

[0067] As used herein, the term "pharmaceutically acceptable salt" refers to carboxylates and amino acid addition salts of the compounds of the present invention that are suitable for contact with patient tissues within the limits of reliable medical judgment, without producing undue toxicity, irritation, allergic reactions, etc., and are effective for their intended use in proportion to a reasonable benefit / risk ratio, including (where possible) zwitterionic forms of the compounds of the present invention.

[0068] This invention includes tautomers, which are functional group isomers resulting from the rapid movement of an atom in a molecule to two positions. A compound exists in different tautomer forms, and a compound is not limited to any particular tautomer, but is intended to encompass all tautomer forms.

[0069] The compounds of this invention may include one or more asymmetric centers and therefore may exist in a variety of stereoisomeric forms, such as enantiomers and / or diastereomers. For example, the compounds of this invention may be individual enantiomers, diastereomers, or geometric isomers (e.g., cis and trans isomers), or may be in the form of mixtures of stereoisomers, including racemic mixtures and mixtures rich in one or more stereoisomers. The isomers can be separated from the mixture by methods known to those skilled in the art, including chiral high-performance liquid chromatography (HPLC) and the formation and crystallization of chiral salts; or preferred isomers may be prepared by asymmetric synthesis.

[0070] The present invention also includes isotopically labeled compounds (isotopic variants) that are equivalent to those described in formula (I), but in which one or more atoms are replaced by atoms with atomic masses or mass numbers different from those commonly found in nature. Examples of isotopes that can be introduced into the compounds of the present invention include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, and chlorine, respectively, for example... 2 H, 3 H, 13 C 11 C 14 C 15 N、 18 O、 17 O、 31 P, 32 P, 35 S, 18 F and 36 Cl. Other isotopes of the present invention containing the aforementioned isotopes and / or other atoms, their prodrugs, and pharmaceutically acceptable salts of said compounds or said prodrugs are all within the scope of this invention. Certain isotope-labeled compounds of the present invention, for example, those incorporating radioactive isotopes (e.g.,...) 3 H and 14 Those in category C) can be used for drug and / or substrate tissue distribution determination. Tritium, i.e. 3 H and carbon-14, i.e. 14 Carbon isotopes are particularly preferred because they are easy to prepare and detect. Subsequently, they are replaced by heavier isotopes, such as deuterium, i.e., 2 H, because higher metabolic stability can provide therapeutic benefits, such as prolonged in vivo half-life or reduced dosage requirements, may be preferred in some cases. Isotope-labeled compounds of formula (I) of the present invention and their prodrugs can generally be prepared by using readily available isotope-labeled reagents instead of non-isotope-labeled reagents when performing the processes described below and / or the techniques disclosed in the examples and preparation examples.

[0071] Compounds of the present invention

[0072] This invention specifically relates to nucleotide dimers represented by formula (A):

[0073]

[0074] in,

[0075] One of Q1 and Q2 is R4, and the other is O-L2;

[0076] L1 is H or P1, or a chemical bond that connects to the 2' or 3' end of the ribose of another nucleotide or oligonucleotide, preferably P1;

[0077] L2 is H or P2, or a chemical bond, preferably P2, attached to the 5' end of the ribose of another nucleotide or oligonucleotide.

[0078] X is selected from -(CR1R2) m -or -CR1 = CR2-;

[0079] Y1 is O, S, or NR;

[0080] Y2 is an O, S, or chemical bond;

[0081] R1 and R2 are independently selected from H, D, halogens, OH, CN, and C. 1-6 Alkyl, C 1-6 Haloalkyl, C 2-6 alkenyl, C 2-6 alkynyl group, C 3-10 Cycloalkyl, 3-10 membered heterocyclic groups, C 6-10 Aryl or 5-14 heteroaryl, which may optionally be substituted by 1, 2, 3, 4, 5, 6, 7, 8 or more R';

[0082] R3 is selected from H and C. 1-6 Alkyl, C 1-6 Haloalkyl, C 2-6 alkenyl, C 2-6 alkynyl group, C 3-10 Cycloalkyl, 3-10 membered heterocyclic groups, C 6-10 Aryl or 5-14 heteroaryl, which may optionally be substituted by 1, 2, 3, 4, 5, 6, 7, 8 or more R';

[0083] R4 and R5 are independently selected from H, D, OH, halogens, and C. 1-6 Alkyl, C 1-6 Halogenated alkyl or C 1-6 Alkyl groups, preferably H, F, or OMe.

[0084] P1 is a hydroxyl protecting group, such as 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 (Troc), benzyloxy Carbonyl (Cbz), tert-butoxycarbonyl (Boc), benzyl (Bn), p-methoxybenzyl (PMB), allyl, triphenylmethyl (Tr), bis-p-methoxytriphenylmethyl (DMTr), methoxymethyl (MOM), phenoxymethyl (BOM), 2,2,2-trichloroethoxymethyl, 2-methoxyethoxymethyl (MEM), methylthiomethyl (MTM), p-methoxybenzylmethyl (PMBM), 4,4'-dimethoxytriphenylmethyl, -P(OCH2CH2CN)(N(iPr)2) or -C(O)CH2CH2C(O)OH, preferably DMTr;

[0085] P2 is a reactive phosphorus group, such as phosphoramide, H-phosphonate, alkyl-phosphonate, phosphate ester or phosphate ester analog, such as natural phosphate ester, thiophosphate ester, dithiophosphate ester, borane phosphate ester, borane thiophosphate ester, phosphonate ester, halogen-substituted phosphonate ester and phosphate ester, aminophosphate ester, phosphate diester, phosphate triester, thiophosphate diester, thiophosphate triester, diphosphate ester or triphosphate ester, preferably -P(OCH2CH2CN)(N(iPr)2);

[0086] Base and Base' are independently selected from H, modified or unmodified bases or leaving groups, preferably modified or unmodified A, U, T, G and C;

[0087] R is selected from H and C. 1-6 Alkyl or C 1-6 Halogenated alkyl groups;

[0088] R' is selected from D, halogen, CN, C. 1-6 Alkyl, C 1-6 Haloalkyl, C 2-6 alkenyl, C 2-6 alkynyl group, C 3-10 Cycloalkyl, 3-10 membered heterocyclic groups, C 6-10 Aryl, 5-14 heteroaryl, -OR a -OC(O)R a -OC(O)OR b -OC(O)NR a Rb -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 -OS(O) n 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 ;

[0089] R a and R b Independently selected from H and C 1-6 Alkyl, C 1-6 Haloalkyl, C 2-6 alkenyl, C 2-6 alkynyl group, C 3-10 Cycloalkyl, 3-10 membered heterocyclic groups, C 6-10 aryl or 5-14 heteroaryl; or R a and R b And the nitrogen atoms they connect to form 3-10 membered heterocyclic groups;

[0090] m is selected from 1, 2, 3, 4, or 5;

[0091] n is independently selected from 1 or 2.

[0092] The present invention also 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, and said antisense strand comprises one or more nucleotide monomers of formula (III) or (IV):

[0093]

[0094] in,

[0095] The nucleotide monomers shown are arranged in the order of 5' => 3' from... to connect;

[0096] Each group is as defined in the context;

[0097] Preferably, the nucleotide monomer is selected from:

[0098]

[0099]

[0100] Where Base is selected from

[0101] Q1 and Q2

[0102] In one implementation, Q1 is R4 and Q2 is O-L2; in another implementation, Q1 is O-L2 and Q2 is R4.

[0103] X

[0104] In one implementation, X is -(CR1R2). m -; In another implementation, X is -CR1 = CR2-.

[0105] In one more specific embodiment, X is -CH2-; in another more specific embodiment, X is -CH(OH)-; in another more specific embodiment, X is -CH2-CH2-; in another more specific embodiment, X is -CH=CH-.

[0106] Y1 and Y2

[0107] In one implementation, Y1 is O; in another implementation, Y1 is S; in yet another implementation, Y1 is NR.

[0108] In one embodiment, Y2 is O; in another embodiment, Y2 is S; in yet another embodiment, Y2 is a chemical bond.

[0109] L1 and L2

[0110] In one embodiment, L1 is H; in another embodiment, L1 is P1; in yet another embodiment, L1 is a chemical bond to a phosphate P atom at the 2' or 3' end of the ribose of another nucleotide or oligonucleotide.

[0111] In one embodiment, L2 is H; in another embodiment, L2 is P2; in yet another embodiment, L2 is a chemical bond to the 5' end of the phosphate P atom of the ribose of another nucleotide or oligonucleotide.

[0112] R1 and R2

[0113] In one embodiment, R1 is H; in another embodiment, R1 is D; in another embodiment, R1 is a halogen; in another embodiment, R1 is OH; in another embodiment, R1 is CN; in another embodiment, R1 is C. 1-6 Alkyl; in another embodiment, R1 is C 1-4 Alkyl; in another embodiment, R1 is C 1-6 Halogenated alkyl; in another embodiment, R1 is C 1-4 Halogenated alkyl; in another embodiment, R1 is C 2-6 Alkenyl; in another embodiment, R1 is C 2-6 Alkyne group; in another embodiment, R1 is C 3-10 Cycloalkyl; in another embodiment, R1 is a 3-10 membered heterocyclic group; in another embodiment, R1 is a C 6-10 Aryl; in another embodiment, R1 is a 5-14 member heteroaryl.

[0114] In one embodiment, R1 is not replaced; in another embodiment, R1 is replaced by 1 R'; in another embodiment, R1 is replaced by 2 R'; in another embodiment, R1 is replaced by 3 R'; in another embodiment, R1 is replaced by 4 R'; in another embodiment, R1 is replaced by 5 R'; in another embodiment, R1 is replaced by 6 R'; in another embodiment, R1 is replaced by 7 R'; in another embodiment, R1 is replaced by 8 R'; in another embodiment, R1 is replaced by multiple R'.

[0115] In one embodiment, R2 is H; in another embodiment, R2 is D; in another embodiment, R2 is a halogen; in another embodiment, R2 is OH; in another embodiment, R2 is CN; in another embodiment, R2 is C. 1-6 Alkyl; in another embodiment, R2 is C 1-4 Alkyl; in another embodiment, R2 is C 1-6 Halogenated alkyl; in another embodiment, R2 is C 1-4 Halogenated alkyl; in another embodiment, R2 is C 2-6 Alkenyl; in another embodiment, R2 is C 2-6 Alkyne group; in another embodiment, R2 is C 3-10 Cycloalkyl; in another embodiment, R2 is a 3-10 membered heterocyclic group; in another embodiment, R2 is a C 6-10Aryl; in another embodiment, R2 is a 5-14 member heteroaryl.

[0116] In one embodiment, R2 is not replaced; in another embodiment, R2 is replaced by 1 R'; in another embodiment, R2 is replaced by 2 R'; in another embodiment, R2 is replaced by 3 R'; in another embodiment, R2 is replaced by 4 R'; in another embodiment, R2 is replaced by 5 R'; in another embodiment, R2 is replaced by 6 R'; in another embodiment, R2 is replaced by 7 R'; in another embodiment, R2 is replaced by 8 R'; in another embodiment, R2 is replaced by multiple R'.

[0117] R3

[0118] In one implementation, R3 is H; in another implementation, R3 is C. 1-6 Alkyl; in another embodiment, R3 is C 1-4 Alkyl, such as Me; in another embodiment, R3 is C 1-6 Halogenated alkyl; in another embodiment, R3 is C 1-4 Halogenated alkyl; in another embodiment, R3 is C 2-6 Alkenyl; in another embodiment, R3 is C 2-6 Alkyne group; in another embodiment, R3 is C 3-10 Cycloalkyl; in another embodiment, R3 is a 3-10 membered heterocyclic group; in another embodiment, R3 is a C 6-10 Aryl; in another embodiment, R3 is a 5-14 member heteroaryl.

[0119] In one embodiment, R3 is not replaced; in another embodiment, R3 is replaced by 1 R'; in another embodiment, R3 is replaced by 2 R'; in another embodiment, R3 is replaced by 3 R'; in another embodiment, R3 is replaced by 4 R'; in another embodiment, R3 is replaced by 5 R'; in another embodiment, R3 is replaced by 6 R'; in another embodiment, R3 is replaced by 7 R'; in another embodiment, R3 is replaced by 8 R'; in another embodiment, R3 is replaced by multiple R'.

[0120] R4 and R5

[0121] In one embodiment, R4 is H; in another embodiment, R4 is D; in another embodiment, R4 is OH; in another embodiment, R4 is a halogen; in another embodiment, R4 is C. 1-6 Alkyl; in another embodiment, R4 is C1-4 Alkyl; in another embodiment, R4 is C 1-6 Halogenated alkyl; in another embodiment, R4 is C 1-4 Halogenated alkyl; in another embodiment, R4 is C 1-6 alkoxy group; in another embodiment, R4 is C 1-4 Alkyl group, such as OMe; in another embodiment, R4 is C 1-6 Haloalkoxy; in another embodiment, R4 is C 1-4 Halogenated alkoxy groups.

[0122] In one embodiment, R5 is H; in another embodiment, R5 is D; in another embodiment, R5 is OH; in another embodiment, R5 is a halogen, such as F; in another embodiment, R5 is C. 1-6 Alkyl; in another embodiment, R5 is C 1-4 Alkyl; in another embodiment, R5 is C 1-6 Halogenated alkyl; in another embodiment, R5 is C 1-4 Halogenated alkyl; in another embodiment, R5 is C 1-6 alkoxy group; in another embodiment, R5 is C 1-4 Alkoxy, such as OMe; in another embodiment, R5 is C 1-6 Haloalkoxy; in another embodiment, R5 is C 1-4 Halogenated alkoxy groups.

[0123] P1 and P2

[0124] In one embodiment, P1 is a protecting group; in another embodiment, P1 is a hydroxyl protecting group, such as 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-trichloro Ethoxycarbonyl (Troc), benzyloxycarbonyl (Cbz), tert-butoxycarbonyl (Boc), benzyl (Bn), p-methoxybenzyl (PMB), allyl, triphenylmethyl (Tr), bis-p-methoxytriphenylmethyl (DMTr), methoxymethyl (MOM), phenoxymethyl (BOM), 2,2,2-trichloroethoxymethyl, 2-methoxyethoxymethyl (MEM), methylthiomethyl (MTM), p-methoxybenzylmethyl (PMBM), 4,4'-dimethoxytriphenylmethyl, -P(OCH2CH2CN)(N(iPr)2) or -C(O)CH2CH2C(O)OH, preferably DMTr.

[0125] In one embodiment, P2 is a reactive phosphorus group, such as phosphoramide, H-phosphonate, alkyl-phosphonate, phosphate ester or phosphate ester analogue, such as natural phosphate ester, thiophosphate ester, dithiophosphate ester, borane phosphate ester, borane thiophosphate ester, phosphonate ester, halogen-substituted phosphonate ester and phosphate ester, aminophosphate ester, phosphate diester, phosphate triester, thiophosphate diester, thiophosphate triester, diphosphate ester or triphosphate ester, preferably -P(OCH2CH2CN)(N(iPr)2).

[0126] Base and Base'

[0127] In one embodiment, Base is H; in another embodiment, Base is a modified or unmodified base or leaving group, such as preferably modified or unmodified A, U, T, G and C.

[0128] In one embodiment, Base' is H; in another embodiment, Base' is a modified or unmodified base or leaving group, such as preferably modified or unmodified A, U, T, G and C.

[0129] In a more specific implementation, Base is... In another, more specific implementation, Base is In another, more specific implementation, Base is In another, more specific implementation, Base is In another, more specific implementation, Base is In another, more specific implementation, Base is In another, more specific implementation, Base is In another, more specific implementation, Base is In another, more specific implementation, Base is In another, more specific implementation, Base is

[0130] In a more specific implementation, Base' is... In another, more specific implementation, Base' is In another, more specific implementation, Base' is In another, more specific implementation, Base' is In another, more specific implementation, Base' is In another, more specific implementation, Base' is In another, more specific implementation, Base' is In another, more specific implementation, Base' is In another, more specific implementation, Base' is In another, more specific implementation, Base' is

[0131] R

[0132] In one implementation, R is H; in another implementation, R is C. 1-6 Alkyl; in another embodiment, R is C 1-6 Halogenated alkyl groups.

[0133] R'

[0134] In one embodiment, R' is D; in another embodiment, R' is a halogen; in yet another embodiment, R' is CN; in still another embodiment, R' is C. 1-6 Alkyl; in another embodiment, R' is C 1-6 Halogenated alkyl; in another embodiment, R' is C 2-6 Alkenyl; in another embodiment, R' is C 2-6 Alkyne group; in another embodiment, R' is C 3-10 Cycloalkyl; in another embodiment, R' is a 3-10 membered heterocyclic group; in another embodiment, R' is a C 6-10aryl; in another embodiment, R' is a 5-14 membered heteroaryl; in another embodiment, R' is -OR a For example, OH; in another embodiment, R' is -OC(O)R a In another implementation, R' is -OC(O)OR b In another implementation, R' is -OC(O)NR a R b In another implementation, R' is -C(O)R a In another implementation, R' is -C(O)OR a In another implementation, R' is -C(O)NR a R b In another implementation, R' is -S(O). n R a In another implementation, R' is -S(O). n OR a In another implementation, R' is -S(O). n NR a R b In another implementation, R' is -OS(O). n R b In another implementation, R' is -NR a R b In another implementation, R' is -NR a C(O)R b In another implementation, R' is -NR a -C(O)OR b In another implementation, R' is -NR a -S(O) n R b In another implementation, R' is -NR a C(O)NR a R b .

[0135] Among them, each R a Selected independently from: H, C 1-6 Alkyl, C 1-6 Haloalkyl, C 2-6 alkenyl, C 2-6 alkynyl group, C 3-10 Cycloalkyl, 3-10 membered heterocyclic groups, C 6-10 aryl or 5-14 heteroaryl; or R a and R b And the nitrogen atoms they connect to form 3-10 membered heterocyclic groups;

[0136] Each R b Selected independently from: H, C 1-6 Alkyl, C 1-6 Haloalkyl, C 2-6 alkenyl, C 2-6 alkynyl group, C 3-10 Cycloalkyl, 3-10 membered heterocyclic groups, C 6-10 aryl or 5-14 heteroaryl; or R a and R b And the nitrogen atoms they connect to form 3-10 membered heterocyclic groups.

[0137] m

[0138] In one implementation, m is 0; in another implementation, m is 1; in another implementation, m is 2; in another implementation, m is 3; in another implementation, m is 4; in another implementation, m is 5.

[0139] n

[0140] In one implementation, n is 0; in another implementation, n is 1; in another implementation, n is 2; in another implementation, n is 3; in another implementation, n is 4; in another implementation, n is 5.

[0141] GalNAc

[0142] In one embodiment, GalNAc is a conjugated group as shown in formula (X):

[0143]

[0144] in,

[0145] Indicates the location where it connects to a biomolecule;

[0146] Q is independent of H,

[0147] Where L G1 For chemical bonds, -CH2-, -CH2CH2-, -C(O)-, -CH2O-, -CH2O-CH2CH2O-, or -NHC(O)-(CH2NHC(O)) a -;

[0148] L G2 It is a chemical bond or -CH2CH2C(O)-;

[0149] L G3It is a chemical bond, -(NHCH2CH2) b -、-(NHCH2CH2CH2) b -or-C(O)CH2-;

[0150] L G4 -(OCH2CH2) c -、-(OCH2CH2CH2) c -、-(OCH2CH2CH2CH2) c -、-(OCH2CH2CH2CH2CH2) c -or -NHC(O)-(CH2) d -;

[0151] Where a = 0, 1, 2 or 3;

[0152] b = 1, 2, 3, 4 or 5;

[0153] c = 1, 2, 3, 4 or 5;

[0154] d = 1, 2, 3, 4, 5, 6, 7 or 8;

[0155] A is a chemical bond, -CH2O- or -NHC(O)-;

[0156] A' is a chemical bond, -C(O)NH-, -NHC(O)-, or -O(CH2CH2O). e -;

[0157] Where e is 1, 2, 3, 4 or 5;

[0158] B is a chemical bond, -CH2-, -C(O)-, -M-, -CH2-M-, or -C(O)-M-;

[0159] Where M is

[0160] R G1 and R G2 Together they form -CH2CH2O- or -CH2CH(R) G )-O-, and R G3 For H;

[0161] Or R G1 and R G3 Together form -C 1-2 alkylene-, and R G2 For H;

[0162] Where R G For -OR G '、-CH2OR G 'or -CH2CH2ORG ', where R G The 'protecting group' is H, a hydroxyl protecting group, or a solid support, wherein the hydroxyl protecting group is preferably -C(O)CH2CH2C(O)OH or 4,4'-dimethoxytriphenylmethyl;

[0163] m1 = 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

[0164] n1 = 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0165] In another implementation, GalNAc is the combination group shown in formula (I'):

[0166]

[0167] in,

[0168] Indicates the location where it connects to a biomolecule;

[0169] Q is independent of H,

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

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

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

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

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

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

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

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

[0178] A is either -CH2O- or -NHC(O)-;

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

[0180] R G1 and R G2 Together they form -CH2CH2O- or -CH2CH(R) G )-O-, and R G3 For H;

[0181] Or R G1 and R G3 Together form -C 1-2 alkylene-, and R G2 For H;

[0182] Where R G For -OR G '、-CH2OR G 'or -CH2CH2OR G ', where R G The 'protecting group' is H, a hydroxyl protecting group, or a solid support, wherein the hydroxyl protecting group is preferably -C(O)CH2CH2C(O)OH or 4,4'-dimethoxytriphenylmethyl;

[0183] m1 = 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

[0184] n1 = 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0185] In another embodiment, GalNAc is a conjugated group as shown in formula (X), wherein,

[0186] Q is independent of H,

[0187] Where L G1 For chemical bonds, -CH2-, -CH2CH2-, -C(O)-, -CH2O-, -CH2O-CH2CH2O-, or -NHC(O)-(CH2NHC(O)) a -;

[0188] L G2 It is a chemical bond or -CH2CH2C(O)-;

[0189] L G3It is a chemical bond, -(NHCH2CH2) b -、-(NHCH2CH2CH2) b -or-C(O)CH2-;

[0190] L G4 -(OCH2CH2) c -、-(OCH2CH2CH2) c -、-(OCH2CH2CH2CH2) c -、-(OCH2CH2CH2CH2CH2) c -or -NHC(O)-(CH2) d -;

[0191] Where a = 0, 1, 2 or 3;

[0192] b = 1, 2, 3, 4 or 5;

[0193] c = 1, 2, 3, 4 or 5;

[0194] d = 1, 2, 3, 4, 5, 6, 7 or 8;

[0195] A is a chemical bond, -CH2O- or -NHC(O)-;

[0196] A' is a chemical bond, -C(O)NH-, -NHC(O)-, or -O(CH2CH2O). e -;

[0197] Where e is 1, 2, 3, 4 or 5;

[0198] B is a chemical bond, -CH2-, -M-, -CH2-M-, or -C(O)-M-;

[0199] Where M is

[0200] R G1 and R G2 Together they form -CH2CH2O- or -CH2CH(R) G )-O-, and R G3 For H;

[0201] Or R G1 and R G3 Together form -C 1-2 alkylene-, and R G2 For H;

[0202] Where R G For -OR G '、-CH2OR G 'or -CH2CH2OR G', where R G The 'protecting group' is H, a hydroxyl protecting group, or a solid support, wherein the hydroxyl protecting group is preferably -C(O)CH2CH2C(O)OH or 4,4'-dimethoxytriphenylmethyl;

[0203] m1 = 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

[0204] n1 = 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0205] In another embodiment, GalNAc is a conjugated group of formula (X), wherein:

[0206] Q is independent of H,

[0207]

[0208] Where L G1 For chemical bonds, -CH2-, -CH2CH2-, -C(O)-, -CH2O-, -CH2O-CH2CH2O-, or -NHC(O)-(CH2NHC(O)) a -;

[0209] L G2 It is a chemical bond or -CH2CH2C(O)-;

[0210] L G3 It is a chemical bond, -(NHCH2CH2) b -、-(NHCH2CH2CH2) b -or-C(O)CH2-;

[0211] L G4 -(OCH2CH2) c -、-(OCH2CH2CH2) c -、-(OCH2CH2CH2CH2) c -、-(OCH2CH2CH2CH2CH2) c -or -NHC(O)-(CH2) d -;

[0212] Where a = 0, 1, 2 or 3;

[0213] b = 1, 2, 3, 4 or 5;

[0214] c = 1, 2, 3, 4 or 5;

[0215] d = 1, 2, 3, 4, 5, 6, 7 or 8;

[0216] A is a chemical bond, -CH2O- or -NHC(O)-;

[0217] A' is -O(CH2CH2O) e -;

[0218] Where e is 1, 2, 3, 4 or 5;

[0219] B is a chemical bond, -CH2-, -C(O)-, -M-, -CH2-M-, or -C(O)-M-;

[0220] Where M is

[0221] R G1 and R G2 Together they form -CH2CH2O- or -CH2CH(R) G )-O-, and R G3 For H;

[0222] Or R G1 and R G3 Together form -C 1-2 alkylene-, and R G2 For H;

[0223] Where R G For -OR G '、-CH2OR G 'or -CH2CH2OR G ', where R G The 'protecting group' is H, a hydroxyl protecting group, or a solid support, wherein the hydroxyl protecting group is preferably -C(O)CH2CH2C(O)OH or 4,4'-dimethoxytriphenylmethyl;

[0224] m1 = 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

[0225] n1 = 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0226] 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 X can be combined with any technical solution or any combination thereof of Q1, Q2, Y1, Y2, L1, L2, R1, R2, R3, R4, R5, Base, and Base', etc. This invention aims to include combinations of all these technical solutions; due to space limitations, they will not be listed one by one.

[0227] The present invention also provides a vector comprising a nucleotide sequence encoding the siRNA described herein. The vector of the present invention is capable of amplifying or expressing the nucleotides encoding the siRNA linked thereto.

[0228] For example, siRNA targeting the PCSK9 gene can be expressed from transcriptional units inserted into DNA or RNA vectors. Expression can be transient (lasting from hours to weeks) or persistent (lasting from weeks to months or longer), depending on the specific construct used and the target tissue or cell type. The coding nucleotides of the siRNA can be introduced into linear constructs, circular plasmids, or viral vectors. The siRNA nucleotides can be stably expressed by integration into the cellular genome or by stable extrachromosomal inheritance. Generally, siRNA expression vectors are typically DNA plasmids or viral vectors.

[0229] Viral vector systems containing coding sequences of siRNA include, but are not limited to: (a) adenovirus vectors; (b) retrovirus vectors; (c) adeno-associated virus vectors; (d) herpes simplex virus vectors; (e) SV40 vectors; (f) polyomavirus vectors; (g) papillomavirus vectors; (h) microRNA virus vectors; (i) poxvirus vectors; and (j) helper virus-dependent adenoviruses or enteroviruses.

[0230] The present invention also provides a cell containing the siRNA or vector described herein, wherein the siRNA or vector described herein is capable of transcription in the cell.

[0231] This invention specifically relates to the following technical solutions:

[0232] 1. The nucleotide dimer represented by formula (A):

[0233]

[0234] in,

[0235] One of Q1 and Q2 is R4, and the other is O-L2;

[0236] L1 is H or P1, or a chemical bond that connects to the 2' or 3' end of the ribose of another nucleotide or oligonucleotide, preferably P1;

[0237] L2 is H or P2, or a chemical bond, preferably P2, attached to the 5' end of the ribose of another nucleotide or oligonucleotide.

[0238] X is selected from -(CR1R2) m -or -CR1 = CR2-;

[0239] Y1 is O, S, or NR;

[0240] Y2 is an O, S, or chemical bond;

[0241] R1 and R2 are independently selected from H, D, halogens, OH, CN, and C. 1-6 Alkyl, C 1-6 Haloalkyl, C 2-6 alkenyl, C 2-6 alkynyl group, C 3-10 Cycloalkyl, 3-10 membered heterocyclic groups, C 6-10 Aryl or 5-14 heteroaryl, which may optionally be substituted by 1, 2, 3, 4, 5, 6, 7, 8 or more R';

[0242] R3 is selected from H and C. 1-6 Alkyl, C 1-6 Haloalkyl, C 2-6 alkenyl, C 2-6 alkynyl group, C 3-10 Cycloalkyl, 3-10 membered heterocyclic groups, C 6-10 Aryl or 5-14 heteroaryl, which may optionally be substituted by 1, 2, 3, 4, 5, 6, 7, 8 or more R';

[0243] R4 and R5 are independently selected from H, D, OH, halogens, and C. 1-6 Alkyl, C 1-6 Halogenated alkyl or C 1-6 Alkyl groups, preferably H, F, or OMe.

[0244] P1 is a hydroxyl protecting group, preferably DMTr;

[0245] P2 is a reactive phosphorus group, preferably -P(OCH2CH2CN)(N(iPr)2);

[0246] Base and Base' are independently selected from H, modified or unmodified bases or leaving groups, preferably modified or unmodified A, U, T, G and C;

[0247] R is selected from H and C. 1-6 Alkyl or C 1-6 Halogenated alkyl groups;

[0248] R' is selected from D, halogen, CN, C. 1-6 Alkyl, C 1-6 Haloalkyl, C 2-6 alkenyl, C 2-6 alkynyl group, C 3-10 Cycloalkyl, 3-10 membered heterocyclic groups, C 6-10 Aryl, 5-14 heteroaryl, -OR a -OC(O)R a -OC(O)OR b -OC(O)NRa R b -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 -OS(O) n 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 ;

[0249] R a and R b Independently selected from H and C 1-6 Alkyl, C 1-6 Haloalkyl, C 2-6 alkenyl, C 2-6 alkynyl group, C 3-10 Cycloalkyl, 3-10 membered heterocyclic groups, C 6-10 aryl or 5-14 heteroaryl; or R a and R b And the nitrogen atoms they connect to form 3-10 membered heterocyclic groups;

[0250] m is selected from 1, 2, 3, 4, or 5;

[0251] n is independently selected from 1 or 2.

[0252] 2. The nucleotide dimer of technical solution 1 has the structure of formula (I) or (II):

[0253]

[0254] Wherein, each group is as defined in technical solution 1.

[0255] 3. The nucleotide dimer of technical solution 2, wherein,

[0256] X is selected from -(CR1R2) m-or -CR1 = CR2-;

[0257] R1 and R2 are independently selected from H, D, halogens, OH, CN, and C. 1-6 Alkyl, C 1-6 Haloalkyl, C 2-6 alkenyl or C 2-6 The alkynyl group, which is optionally substituted with 1, 2, 3, 4, 5 or more R';

[0258] R' is selected from D, halogen, CN, C. 1-6 Alkyl, C 1-6 Haloalkyl, C 2-6 alkenyl, C 2-6 Alkyne group, -OR a -OC(O)R a -OC(O)OR b -OC(O)NR a R b -C(O)R a -C(O)OR a -C(O)NR a R b -NR a R b -NR a C(O)R b -NR a -C(O)OR b or -NR a C(O)NR a R b ;

[0259] R a and R b Independently selected from H and C 1-6 Alkyl, C 1-6 Haloalkyl, C 2-6 alkenyl or C 2-6 alkynyl group;

[0260] m is selected from 1, 2, 3, 4, or 5;

[0261] Preferably,

[0262] X is selected from -(CR1R2) m -or -CR1 = CR2-;

[0263] R1 and R2 are independently selected from H, D, halogens, OH, CN, and C. 1-6 Alkyl or C 1-6 Haloalkyl, which is optionally substituted with 1, 2, 3 or more R';

[0264] R' is selected from D, halogen, CN, C.1-6 Alkyl, C 1-6 Haloalkyl, C 2-6 alkenyl, C 2-6 Alkyne group, -OR a or -NR a R b ;

[0265] R a and R b Independently selected from H and C 1-6 Alkyl or C 1-6 Halogenated alkyl groups;

[0266] m is selected from 1, 2, or 3;

[0267] More preferably,

[0268] X is selected from -CH2-, -CH(OH)-, -CH2-CH2-, or -CH=CH-.

[0269] 4. The nucleotide dimer of technical solution 2 or 3, wherein,

[0270] Y1 is either O or NR;

[0271] Y2 is an O, S, or chemical bond;

[0272] R is selected from H and C. 1-6 Alkyl or C 1-6 Halogenated alkyl groups;

[0273] Preferably,

[0274] Y1 is either O or NR;

[0275] Y2 is an O, S, or chemical bond;

[0276] R is selected from H or C. 1-6 alkyl;

[0277] More preferably,

[0278] Y1 is 0;

[0279] Y2 is 0.

[0280] 5. A nucleotide dimer of any one of technical solutions 2-4, wherein,

[0281] R3 is selected from H and C. 1-6 Alkyl, C 1-6 Haloalkyl, C 2-6 alkenyl or C 2-6 The alkynyl group, which is optionally substituted with 1, 2, 3, 4, 5 or more R';

[0282] Preferably,

[0283] R3 is selected from H and C. 1-6 Alkyl or C 1-6 Haloalkyl, which is optionally substituted with 1, 2, 3 or more R';

[0284] More preferably,

[0285] R3 is C 1-4 Alkyl group, preferably Me.

[0286] 6. A nucleotide dimer of any one of technical solutions 2-5, wherein,

[0287] R4 and R5 are independently selected from H, D, OH, halogens, and C. 1-6 Alkyl, C 1-6 Halogenated alkyl or C 1-6 Alkoxy;

[0288] Preferably,

[0289] R4 and R5 are independently selected from H, OH, halogens, and C. 1-6 Alkyl, C 1-6 Halogenated alkyl or C 1-6 Alkyl groups, preferably H, F, OH or OMe;

[0290] More preferably,

[0291] R4 is selected from H, OH, or C. 1-4 Alkyl groups, preferably H or OMe;

[0292] R5 is selected from halogen, OH, or C. 1-4 Alkyl groups, preferably F or OMe.

[0293] 7. A nucleotide dimer of any one of technical solutions 2-6, wherein,

[0294] Base and Base' are selected independently.

[0295] 8. A nucleotide dimer of any one of technical solutions 2-7, wherein,

[0296] L1 is H or P1, or a chemical bond that connects to the 2' or 3' end of the ribose of another nucleotide or oligonucleotide, preferably P1;

[0297] L2 is H or P2, or a chemical bond, preferably P2, attached to the 5' end of the ribose of another nucleotide or oligonucleotide.

[0298] X is selected from -(CR1R2) m -or -CR1 = CR2-;

[0299] Y1 is O, S, or NR;

[0300] Y2 is an O, S, or chemical bond;

[0301] R1 and R2 are independently selected from H, D, halogens, OH, CN, and C. 1-6 Alkyl, C 1-6 Haloalkyl, C 2-6 alkenyl or C 2-6 The alkynyl group, which is optionally substituted with 1, 2, 3, 4, 5 or more R';

[0302] R3 is selected from H and C. 1-6 Alkyl, C 1-6 Haloalkyl, C 2-6 alkenyl or C 2-6 The alkynyl group, which is optionally substituted with 1, 2, 3, 4, 5 or more R';

[0303] R4 and R5 are independently selected from H, D, OH, halogens, and C. 1-6 Alkyl, C 1-6 Halogenated alkyl or C 1-6 Alkyl groups, preferably H, F, or OMe;

[0304] P1 is a hydroxyl protecting group, preferably DMTr;

[0305] P2 is a reactive phosphorus group, preferably -P(OCH2CH2CN)(N(iPr)2);

[0306] Base and Base' are independently selected from H, modified or unmodified bases or leaving groups, preferably modified or unmodified A, U, T, G and C;

[0307] R is selected from H and C. 1-6 Alkyl or C 1-6 Halogenated alkyl groups;

[0308] R' is selected from D, halogen, CN, C. 1-6 Alkyl, C 1-6 Haloalkyl, C 2-6 alkenyl, C 2-6 Alkyne group, -OR a -OC(O)R a -OC(O)OR b -OC(O)NR a R b -C(O)R a -C(O)OR a -C(O)NR a R b -NR a R b -NR aC(O)R b -NR a -C(O)OR b or -NR a C(O)NR a R b ;

[0309] R a and R b Independently selected from H and C 1-6 Alkyl, C 1-6 Haloalkyl, C 2-6 alkenyl or C 2-6 alkynyl group;

[0310] m is selected from 1, 2, 3, 4 or 5.

[0311] 9. A nucleotide dimer of any one of technical solutions 2-8, wherein,

[0312] L1 is H or P1, or a chemical bond that connects to the 2' or 3' end of the ribose of another nucleotide or oligonucleotide, preferably P1;

[0313] L2 is H or P2, or a chemical bond, preferably P2, attached to the 5' end of the ribose of another nucleotide or oligonucleotide.

[0314] X is selected from -(CR1R2) m -or -CR1 = CR2-;

[0315] Y1 is either O or NR;

[0316] Y2 is an O, S, or chemical bond;

[0317] R1 and R2 are independently selected from H, D, halogens, OH, CN, and C. 1-6 Alkyl or C 1-6 Haloalkyl, which is optionally substituted with 1, 2, 3 or more R';

[0318] R3 is selected from H and C. 1-6 Alkyl or C 1-6 Haloalkyl, which is optionally substituted with 1, 2, 3 or more R';

[0319] R4 and R5 are independently selected from H, D, halogens, and C. 1-6 Alkyl, C 1-6 Halogenated alkyl or C 1-6 Alkyl groups, preferably H, F, or OMe;

[0320] P1 is a hydroxyl protecting group, preferably DMTr;

[0321] P2 is a reactive phosphorus group, preferably -P(OCH2CH2CN)(N(iPr)2);

[0322] Base and Base' are independently selected from H, modified or unmodified bases or leaving groups, preferably modified or unmodified A, U, T, G and C;

[0323] R is selected from H and C. 1-6 Alkyl or C 1-6 Halogenated alkyl groups;

[0324] R' is selected from D, halogen, CN, C. 1-6 Alkyl, C 1-6 Haloalkyl, C 2-6 alkenyl, C 2-6 Alkyne group, -OR a or -NR a R b ;

[0325] R a and R b Independently selected from H and C 1-6 Alkyl or C 1-6 Halogenated alkyl groups;

[0326] m is selected from 1, 2, or 3.

[0327] 10. A nucleotide dimer of any one of technical solutions 2-9, wherein,

[0328] L1 is H or P1, or a chemical bond that connects to the 2' or 3' end of the ribose of another nucleotide or oligonucleotide, preferably P1;

[0329] L2 is H or P2, or a chemical bond, preferably P2, attached to the 5' end of the ribose of another nucleotide or oligonucleotide.

[0330] X is selected from -(CR1R2) m -or -CR1=CR2-, preferably -CH2-, -CH(OH)-, -CH2-CH2- or -CH=CH-;

[0331] Y1 is 0;

[0332] Y2 is 0;

[0333] R1 and R2 are independently selected from H, D, halogens, OH, CN, and C. 1-4 Alkyl or C 1-4 Halogenated alkyl groups;

[0334] R3 is C 1-4 Alkyl group, preferably Me;

[0335] R4 is selected from H, OH, or C.1-4 Alkyl groups, preferably H or OMe;

[0336] R5 is selected from halogen, OH, or C. 1-4 Alkyl groups, preferably F or OMe;

[0337] P1 is a hydroxyl protecting group, preferably DMTr;

[0338] P2 is a reactive phosphorus group, preferably -P(OCH2CH2CN)(N(iPr)2);

[0339] Base and Base' are selected independently.

[0340] m is selected from 1, 2, or 3.

[0341] 11. The nucleotide dimer of any one of technical solutions 1-10 is selected from:

[0342]

[0343] Wherein, Base and Base' are as defined in any one of technical solutions 1-10, preferably...

[0344] R5 is defined as in any of the technical solutions 1-10, and is preferably F or OMe.

[0345] 12. A double-stranded RNA molecule or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof, comprising a sense strand and an antisense strand, each strand having 14 to 30 nucleotides, and said antisense strand comprising one or more nucleotide monomers of formula (III) or (IV):

[0346]

[0347] in,

[0348] The nucleotide monomers shown are arranged in the order of 5' => 3' from... to connect;

[0349] Each group is as defined in any one of technical solutions 1-11;

[0350] Preferably, the nucleotide monomer is selected from:

[0351]

[0352] Where Base is selected from

[0353] 13. The double-stranded RNA molecule of technical solution 12, wherein the sense strand and the antisense strand each have 20 to 25 nucleotides.

[0354] 14. The double-stranded RNA molecule of technical solution 12 or 13, wherein the nucleotide monomer is located at position 2-8, preferably position 6 or 7, more preferably position 7, at the 5' end of the antisense strand.

[0355] 15. A double-stranded RNA molecule of any one of claims 12-14, wherein the double-stranded RNA exhibits enhanced stability compared to a double-stranded RNA having the same sequence but not containing the nucleotide monomers described in claim 12.

[0356] 16. A double-stranded RNA molecule of any one of claims 12-15, wherein the double-stranded RNA has a melting temperature from about 40°C to about 80°C, preferably from about 55°C to 67°C.

[0357] 17. A double-stranded RNA molecule of any one of claims 12-16, wherein the double-stranded RNA exhibits reduced off-target toxicity compared to a double-stranded RNA having the same sequence but not containing the nucleotide monomers described in claim 12.

[0358] 18. A double-stranded RNA molecule of any one of claims 12-17, wherein the double-stranded RNA exhibits enhanced effectiveness compared to a double-stranded RNA having the same sequence but not containing the nucleotide monomers described in claim 12.

[0359] 19. A double-stranded RNA molecule of any one of technical solutions 12-18, 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 the degradation of the target mRNA.

[0360] 20. The double-stranded RNA molecule of technical solutions 12-19, wherein the target mRNA is encoded by an endogenous gene or by a pathogen gene.

[0361] 21. A double-stranded RNA molecule of any one of technical solutions 12-20, wherein the sense strand and / or antisense strand comprises a 3' and / or 5' overhang.

[0362] 22. A double-stranded RNA molecule of any one of technical solutions 12-21, wherein the double-stranded RNA is further coupled to a ligand, preferably, the ligand comprising one or more GalNAc.

[0363] 23. A nucleic acid molecule, wherein the nucleotide sequence of the nucleic acid molecule contains one or more nucleotide monomers as described in technical solution 12.

[0364] 24. The nucleic acid molecule of technical solution 23, wherein the nucleic acid is selected from DNA, RNA and DNA / RNA hybrids.

[0365] 25. The nucleic acid molecule of technical solution 24, wherein the nucleic acid molecule is single-stranded or double-stranded.

[0366] 26. The nucleic acid molecule of any one of technical solutions 23-25, wherein the nucleic acid molecule is selected from small interfering RNA (siRNA) and short hairpin RNA (shRNA).

[0367] 27. A vector comprising a nucleotide sequence encoding a double-stranded RNA as described in any one of the preceding technical solutions 12-22.

[0368] 28. A cell containing double-stranded RNA as described in any one of technical solutions 12-22 or a vector as described in technical solution 27.

[0369] 29. A pharmaceutical composition comprising a double-stranded RNA molecule as described in any one of technical solutions 12-22, and a pharmaceutically acceptable carrier or excipient.

[0370] 30. A kit comprising a double-stranded RNA molecule as described in any one of technical solutions 12-22.

[0371] 31. A method for inhibiting the expression of a target gene in a cell, comprising the step of introducing a double-stranded RNA molecule as described in any one of technical solutions 12-22 into the cell.

[0372] 32. A method for inhibiting the expression of a target gene in a cell, comprising expressing a double-stranded RNA molecule as described in any one of techniques 12-22 in the cell.

[0373] 33. A method for reducing off-target toxicity in cells, comprising the step of introducing a double-stranded RNA molecule, as described in any one of technical solutions 12-22, into the cell.

[0374] 34. A method for reducing off-target toxicity in cells, comprising expressing a double-stranded RNA molecule as described in any one of techniques 12-22 in the cells. Example

[0375] The following examples are for illustrative purposes only and are not intended to limit the scope of the invention.

[0376]

[0377] Example 1: Preparation of compound E1

[0378]

[0379] 1. Preparation of Compound 2

[0380]

[0381] Compound 1 (50.0 g, 263 mmol) was dissolved in DCM (800 mL) at 25 °C, followed by the sequential addition of imidazole (26.9 g, 394 mmol) and TBDPSCl (75.2 mL, 289 mmol). The reaction mixture was stirred at 25 °C for 18 hours. Thin-layer chromatography (DCM / MeOH = 10 / 1, PE / EA = 3 / 1) showed complete consumption of the reactants and the formation of new spots. The reaction mixture was evaporated to dryness to obtain the crude product, which was purified by MPLC (PE / EA = 1 / 0-5 / 1) to give compound 2 (95.0 g, yield 84.31%), a colorless oily liquid.

[0382] 1 H NMR (400MHz, CDCl3) δ7.70(dd,J=7.6,1.6Hz,4H),7.35-7.46(m,6H),5.86(d,J=3.6Hz,1H),4.61(dd,J=4.8,4.0H z,1H),4.15(td,J=8.8,5.2Hz,1H),3.93-4.01(m,1H),3.81-3.92(m,2H),1.60(s,3H),1.39(s,3H),1.06(s,9H).

[0383] 2. Preparation of Compound 3

[0384]

[0385] Compound 2 (95.0 g, 222 mmol) was dissolved in toluene (1.50 L) at 25 °C. Imidazole (30.2 g, 443 mmol), triphenylphosphine (116 g, 443 mmol), and iodine (84.4 g, 322 mmol) were added sequentially, and the reaction mixture was stirred at 100 °C for 18 hours. Thin-layer chromatography (PE / EA = 5 / 1) showed complete consumption of the reactants and the formation of new spots. 20.0 mL of saturated NaHSO3 solution was added to the reaction mixture, followed by 500 mL of water. The reaction mixture separated into layers, and the organic phase was washed with saturated NaCl solution (50.0 mL × 3). The solution was dried over anhydrous Na2SO4 and evaporated to dryness to obtain the crude product. The crude product was purified by MPLC (PE / EA = 1 / 0-10 / 1) to obtain a colorless oily liquid compound 3 (115 g, yield 96.35%).

[0386] 1H NMR (400MHz, CD3OD) δ7.64-7.72(m,4H),7.37-7.49(m,6H),5.95(d,J=3.6Hz,1H),5.06(d,J=3.6Hz,1H),4.42(d,J=3.2Hz,1H),3. 87(dd,J=10.4,5.6Hz,1H),3.66(dd,J=10.4,6.4Hz,1H),3.53(td,J=6.0,3.2Hz,1H),1.44(s,3H),1.29(s,3H),1.02-1.07(m,9H).

[0387] 3. Preparation of Compound 4

[0388]

[0389] Compound 3 (12.5 g, 23.2 mmol) was dissolved in a mixed solvent of MeOH (225 mL) and EtOAc (25 mL) at 25 °C. TEA (6.45 mL, 46.4 mmol) and 10% Pd / C (2.00 g, 18.8 mmol) were added. The reaction mixture was stirred at 25 °C under a hydrogen atmosphere (14.696 psi) for 14 hours. Thin-layer chromatography (PE / EA = 10 / 1) showed complete consumption of the reactants and the formation of new spots. The reaction mixture was filtered, and the filtrate was evaporated to dryness to obtain the crude product. The crude product was purified by MPLC (PE / EA = 1 / 0-10 / 1) to obtain a colorless oily liquid compound 4 (8.75 g, yield 83.06%).

[0390] 1 H NMR (400MHz, CD3OD) δ7.64-7.72(m,4H),7.36-7.47(m,6H),5.80(d,J=3.6Hz,1H),4.77(t,J=4.4Hz,1H),4.25-4.32 (m,1H),3.68-3.82(m,2H),2.00(dd,J=13.6,4.8Hz,1H),1.78-1.87(m,1H),1.45(s,3H),1.30(s,3H),1.04(s,9H).

[0391] 4. Preparation of Compound 5

[0392]

[0393] Compound 4 (35.0 g, 84.8 mmol) was dissolved in THF (500 mL) at 25 °C, and tetraethylammonium fluoride (63.3 g, 424 mmol) was added. The reaction mixture was stirred at 25 °C for 18 hours. Thin-layer chromatography (PE / EA = 10 / 1, DCM / MeOH = 10 / 1) showed that the reactants were completely consumed and new spots were formed. The reaction mixture was concentrated under vacuum to obtain a crude product, which was purified by MPLC (DCM / MeOH = 1 / 0-10 / 1) to give a white solid compound 5 (12 g, yield 81.21%).

[0394] 1 H NMR (400MHz, CD3OD) δ5.79(d,J=3.6Hz,1H),4.77(t,J=4.0Hz,1H),4.21-4.28(m,1H),3.70(dd,J=12.0,3.6Hz,1H ),3.54(dd,J=12.0,4.8Hz,1H),1.95-2.02(m,1H),1.74(ddd,J=13.2,10.8,4.8Hz,1H),1.46(s,3H),1.30(s,3H).

[0395] 5. Preparation of Compound 6

[0396]

[0397] Compound 5 (12.0 g, 68.9 mmol) was dissolved in toluene (500 mL) at 25 °C. Imidazole (9.38 g, 138 mmol), triphenylphosphine (36.1 g, 138 mmol), and iodine (26.2 g, 103 mmol) were added sequentially, and the reaction mixture was stirred at 100 °C for 3 hours. Thin-layer chromatography (PE / EA = 5 / 1) showed complete consumption of the reactants and the formation of new spots. 100 mL of saturated NaHSO3 solution was added to the reaction mixture, followed by 100 mL of water. The reaction mixture separated into layers, and the organic phase was washed with saturated NaCl solution (100 mL × 3), dried over anhydrous Na2SO4, and evaporated to dryness to obtain the crude product. The crude product was purified by MPLC (PE / EA = 1 / 0-10 / 1) to give compound 6 (16.6 g, yield 84.82%) as a white solid.

[0398] 1 H NMR(400MHz, CDCl3)δ5.88(d,J=3.6Hz,1H),4.77(t,J=4.2Hz,1H),4.13-4.21(m,1H),3.2 5-3.38(m,2H),2.31(dd,J=13.6,4.4Hz,1H),1.61-1.67(m,1H),1.52(s,3H),1.33(s,3H).

[0399] 6. Preparation of Compound 7

[0400]

[0401] Compound 6 (40.0 g, 141 mmol) was dissolved in trimethyl phosphite (500 mL) at 25 °C, and the reaction mixture was stirred at 120 °C for 11 hours. Thin-layer chromatography (ethyl acetate / acetone = 3 / 1, PE / EA = 10 / 1) showed residual starting material and the formation of new spots. The reaction mixture was evaporated to dryness to obtain a crude product, which was purified by MPLC (ethyl acetate / acetone = 1 / 0-20 / 1) to give compound 7 (8.90 g, yield 23.74%), a pale yellow oily liquid, and the recovered starting material, a white solid compound 7 (30.0 g).

[0402] 1 H NMR (400MHz, CDCl3) δ5.81(d,J=4.0Hz,1H),4.74(t,J=4.4Hz,1H),4.40-4.52(m,1H),3.76(dd,J=10.8,0.8H z,6H),2.23-2.35(m,2H),1.95-2.07(m,1H),1.63(ddd,J=13.6,10.8,4.8Hz,1H),1.52(s,3H),1.32(s,3H).

[0403] 7. Preparation of Compound 8

[0404]

[0405] Compound 7 (13 g, 48.830 mmol), Ac₂O (23.036 mL, 244.150 mmol), and H₂SO₄ (2.615 mL, 48.830 mmol) were added sequentially to AcOH (260 mL). The reaction was carried out at 25 °C for 6 hours. A new spot was detected by TLC (ethyl acetate: acetone = 3:1). The reaction solution was quenched with ice water, extracted with DCM (500 mL x 3), dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure to obtain the crude product. The crude product was purified by column chromatography TLC (ethyl acetate: acetone = 50:1-3:1) to give a yellow oily compound 8 (7.9 g, 25.464 mmol, 52.15%).

[0406] 1H NMR (400MHz, CDCl3) δ6.10(d,J=1.2Hz,1H),5.18(d,J=5.2Hz,1H),4.58-4.72(m,1H),3.75-3 .79(m,3H),3.71-3.74(m,3H)2.16-2.38(m,3H),2.07(s,3H),2.05(s,3H),1.96-2.04(m,1H).

[0407] 8. Preparation of Compound 9

[0408]

[0409] Compound 8 (7.9 g, 25.464 mmol) and compound 8A (6.09 g, 25.464 mmol) were added to CH3CN (316 mL), and SnCl4 (8.781 mL, 76.392 mmol) was slowly added dropwise at 0 °C. The reaction was carried out at 25 °C for 2 hours. TLC (ethyl acetate:acetone = 10:1, PMA) showed that the starting compound 8 disappeared and a new spot was formed. LCMS (RW0006-267-P1A) showed that 31.3% of the product was formed. The reaction solution was cooled to 0℃, and the pH was adjusted to 8 with saturated NaHCO3 aqueous solution. The aqueous phase was then extracted with DCM (200 mL x 3), and the organic phase was dried with anhydrous Na2SO4. The mixture was filtered and evaporated to dryness to obtain the crude product. The crude product was purified by column chromatography (ethyl acetate:acetone = 20:1-3:1) to give a yellow oily compound 9 (6.1 g, 12.464 mmol, 48.95%).

[0410] 1 H NMR(400MHz, CDCl3)δ8.75-8.84(m,1H),8.17(s,1H),8.01-8.10(m,2H), 7.59-7.70(m,1H),7.50-7.57(m,2H),6.07(s,1H),5.66-5.73(m,1H),4.9 8-5.12(m,1H),4.70-4.81(m,1H),3.71-3.82(m,12H),2.70-2.81(m,1H), 2.34-2.52(m,2H),2.21-2.27(m,1H),2.16(s,3H),2.06(d,J=4.4Hz,4H).

[0411] 9. Preparation of Compound 10

[0412]

[0413] Compound 9 (6.1 g, 12.259 mmol) was dissolved in pyridine (30 mL) and water (20 mL) and reacted at 60 °C for 12 h. TLC (DCM:MeOH = 10:1) showed that the starting material disappeared. The reaction solution was directly evaporated to dryness to give the crude product, which was purified by Prep-HPLC (MeCN / H2O = 30 / 1-80 / 1; flow rate: 30 mL / min) to give a yellow oily compound 10 (3.85 g, 8.098 mmol, 66.06%).

[0414] 10. Preparation of Compound 11

[0415]

[0416] Compound 10 (3.7 g, 7.783 mmol) and compound SM2 (10.24 g, 15.566 mmol) were dissolved in pyridine (37 mL). 2,4,6-triisopropylbenzenesulfonyl chloride (14.14 g, 46.698 mmol) was added at 0 °C, and the reaction was carried out at 0 °C for 60 min. N-methylimidazolium (5.11 g, 62.263 mmol) was then slowly added dropwise to the reaction solution. The reaction was continued at 25 °C for 13 h, and the product was detected by LCMS (RW0006-284-P1C). New spots were observed by TLC (DCM:MeOH = 10:1, UV). The reaction solution was cooled to 0 °C, quenched with 50 mL of saturated sodium carbonate solution, and the layers separated. The organic phase was diluted with 300 mL of DCM, washed with water (50 mL x 3) and saturated NaCl aqueous solution (50 mL x 1), dried over anhydrous Na2SO4, filtered, and evaporated to dryness to obtain the crude product. The crude product was purified by column chromatography (DCM: MeOH = 2%-4%, TEA) to give brown oily compound 11 (2100 mg, 1.883 mmol, 24.20%).

[0417] 11. Preparation of Compound 12

[0418]

[0419] Compound 11 (2.4 g, 2.152 mmol) was dissolved in MeOH (36 mL), and NH3 / MeOH (12.299 mL, 7 M) was added. The reaction was carried out at 0 °C for 3 hours. LCMS (RW0006-290-P1A) showed that the product was formed. The reaction solution was diluted with 150 mL of DCM, washed with 30 mL of water, and the organic phase was dried over anhydrous Na2SO4. The crude product was obtained by filtration and rotary evaporation. The crude product was purified by Prep-HPLC (column: 01-Waters Xbridge BEH C18 19*150 mm; mobile phase: TEAA-ACN; gradient: 33%-58.5% / 15 min; flow rate: 15 mL / min) to obtain a yellow solid compound 12 (380 mg, 0.354 mmol, 16.45%).

[0420] 12. Preparation of E1

[0421]

[0422] Compound 12 (380 mg, 0.354 mmol) was bubbled three times with acetonitrile, dissolved in DCM (6.0 mL), and then added sequentially to 5A molecular sieve, DCI (41.82 mg, 0.354 mmol), and compound 13 (426.95 mg, 1.417 mmol). The reaction mixture was purged with nitrogen three times and reacted at 25 °C for 1 hour. LCMS (RW00006-291-P1A) showed 5.4% of the starting material remaining. The reaction mixture was quenched dropwise by adding 20 mL of ice-cold saturated NaHCO3 aqueous solution, diluted with 30 mL of DCM, separated, and the organic phase was washed with 20 mL of saturated NaHCO3 aqueous solution and 20 mL of saturated NaCl aqueous solution. The organic phase was dried with anhydrous Na2SO4, filtered, and evaporated to dryness to obtain the crude product. The crude product was purified by reverse phase (acetonitrile / water: 20-80%, 30 min, 20 mL / min) to obtain a yellow solid E1 (210 mg, 0.165 mmol, 46.57%).

[0423] 1H NMR (400MHz, CDCl3) δ11.96-12.03(m,1H),10.07-10.20(m,1H),8.99-9.12(m,1H),8.62-8.80(m,1H),8.07-8.16(m,1H),8.01-8.05(m,2 H),7.78(d,J=1.2Hz,1H),7.60-7.66(m,1H),7.51-7.58(m,2H),7.21-7.26(m,2H),7.09-7.20(m,7H),6.66-6.75(m,4H),6.37-6.56(m,1 H),5.75-6.11(m,3H),5.07-5.19(m,1H),4.60-4.74(m,1H),4.16-4.29(m,1H),3.81-3.89(m,1H),3.76-3.81(m,3H),3.70(d,J=1.6Hz,7 H),3.54-3.69(m,3H),3.05-3.15(m,1H),2.54-2.65(m,2H),2.03-2.53(m,5H),1.43-1.50(m,1H),1.14-1.21(m,11H),1.01-1.13(m,6H).

[0424] Example 2: Preparation of siRNA

[0425] The siRNA of the present invention was prepared using the solid-phase phosphoramide method well known in the art. Specific methods can be found, for example, in PCT publications WO2016081444 and WO2019105419, and are briefly described below.

[0426] 1. Synthesis of the Justice Chain (SS Chain)

[0427] The oligonucleotide synthesis method, using a solid-phase phosphoramide, employs a blank CPG solid support or an L96-linked solid support as the starting cycle. Nucleoside monomers are sequentially linked from the 3'-5' direction according to the nucleotide arrangement of the positive strand. Each linkage of a nucleoside monomer involves four steps: deprotection, coupling, capping, and oxidation or thiolation. The synthesis conditions for oligonucleotides at a scale of 5 μmol are as follows:

[0428] The nucleoside monomer was provided in a 0.05 mol / L acetonitrile solution. The reaction conditions were identical for each step: 25°C. Deprotection was performed three times using a 3% trichloroacetic acid-dichloromethane solution. The coupling reaction was activated twice using a 0.25 mol / L ETT-acetonitrile solution. Capping was performed twice using a 10% acetic anhydride-acetonitrile and pyridine / N-methylimidazole / acetonitrile mixture (10:14:76, v / v / v). Oxidation was performed twice using a 0.05 mol / L iodine / tetrahydrofuran / pyridine / water mixture (70 / 20 / 10, v / v / v). Thiolation was performed twice using a 0.2 mol / L PADS mixture of acetonitrile / 3-methylpyridine (1 / 1, v / v).

[0429] 2. Synthesis of the antisense chain (AS chain)

[0430] The solid-phase phosphoramide synthesis method utilizes a blank CPG solid-phase support as the starting cycle, and sequentially links nucleoside monomers or nucleotide dimers of the present invention from the 3'-5' direction according to the antisense strand nucleotide arrangement sequence. Each linking of a nucleoside monomer or nucleotide dimer of the present invention involves four steps: deprotection, coupling, capping, oxidation, or thiolation. The synthesis conditions for 5 μmol oligonucleotides of the antisense strand are the same as those for the sense strand.

[0431] 3. Purification and Annealing of Oligonucleotides

[0432] 3.1 Ammonolysis

[0433] The synthesized solid support (sense or antisense chain) was added to a 5 mL centrifuge tube, and 3% diethylamine / ammonia (v / v) was added. The mixture was reacted in a constant temperature water bath at 35℃ (or 55℃) for 16 hours (or 8 hours). After filtration, the solid support was washed three times with ethanol / water, 1 mL each time. The filtrate was concentrated by centrifugation and the crude product was purified.

[0434] 3.2 Purification

[0435] Purification and desalting methods are well known to those skilled in the art. For example, a column packed with strong anion exchange material can be used for elution purification with a sodium chloride-sodium hydroxide system, and the product can be collected and piped. Desalting can be performed using a gel-packed purification column with pure water as the elution system.

[0436] 3.3 Annealing

[0437] According to Table 6, the sense chain (SS chain) and the antisense chain (AS chain) are mixed in a molar ratio (SS chain / AS chain = 1 / 1.05), heated in a water bath to 70-95℃, held for 3-5 minutes, and then naturally cooled to room temperature. The system is then freeze-dried to obtain the product.

[0438] The siRNA sequence used in this invention is as follows:

[0439]

[0440] The meanings of the abbreviations in this article are as follows:

[0441] The distributions A, U, G, and C represent naturally occurring adenine ribonucleotides, uracil ribonucleotides, guanine ribonucleotides, and cytosine ribonucleotides.

[0442] The 'm' indicates that the nucleotide adjacent to it on the left is a nucleotide modified with 2'-OCH3. For example, Am, Um, Gm, and Cm represent A, U, G, and C modified with 2'-OCH3.

[0443] The 'f' indicates that the nucleotide adjacent to it on the left is a 2'-F modified nucleotide. For example, Af, Uf, Gf, and Cf represent 2'-F modified A, U, G, and C, respectively.

[0444] “s” or “s-” indicates that the two adjacent nucleotides and / or delivery carriers are linked by a phosphate thioester.

[0445] L96 represents a GalNAc delivery vector with the following structure well known in the art, wherein... The location indicated by the phosphate ester group or thiophosphate ester group linked to siRNA can be found, for example, in PCT publications WO2009073809 and WO2009082607.

[0446]

[0447] ROR14 represents a nucleotide substitution of the structure described above, where Base can be any base, for example, ROR14-A indicates that Base is adenine.

[0448] Specifically, the ROR14-A structure is as follows: Base is adenine.

[0449] Example 3: Detection of the target and off-target activities of the siRNA compound psi-CHECK2

[0450] 1. Plasmid preparation:

[0451] In the target plasmid: The corresponding antisense target plasmid was designed according to the compound sequence. The psiCHECK2GSCM recombinant plasmid was prepared by Sangon Biotech (Shanghai) Co., Ltd. and diluted to 1000 ng / μL for later use.

[0452] Off-target plasmids: Based on the compound sequence, the corresponding antisense off-target plasmids were designed. The psiCHECK2GSSM-5Hits recombinant plasmid was prepared by Sangon Biotech (Shanghai) Co., Ltd. and diluted to 1000 ng / μL for later use.

[0453] 2. Cell transfection:

[0454] HEK293A cells (Nanjing Kebai, catalog number CBP60436) were seeded in 96-well plates with 100 μL of cell resuspended solution; cell quantity: 8 × 10⁶ cells / well. 3 cells / pores.

[0455] On the second day, the complete culture medium in the wells was first aspirated and replaced with 80 μL of Opti-MEM culture medium per well, and then starved for about 1.5 hours.

[0456] Plasmid mixture: Single-well preparation amount: 0.01 μL plasmid / well, 8.99 μL Opti-MEM / well.

[0457] Lipo mixing: Dilute Lipo 2000 (Lipofectamine) with Opti-MEM. TM 2000 transfection reagent (Thermo, 11668019), let stand at room temperature for 5 minutes, specific preparation volume of Lipo mixture: Lipo 0.2 μL / well, Opti-MEM 9.8 μL / well.

[0458] 22 μL of the prepared Lipo mixture, 2.2 μL of the compound, and 19.8 μL of the plasmid mixture were aliquoted into corresponding wells and named Well A. After mixing thoroughly by pipetting, the mixture was incubated at room temperature for 20 minutes before co-transfection. Finally, 20 μL of Well A mixture was added to each well, along with the existing 80 μL of Opti-MEM, for a final volume of 100 μL per well. After incubation at 37°C and 5% CO2 for 4 hours, 100 μL of DMEM medium containing 20% ​​fetal bovine serum was added to each well. After incubation at 37°C and 5% CO2 for 24 hours, the cells were analyzed.

[0459] 3. Result Detection:

[0460] Before the experiment, mix the ingredients. Luciferase The Luciferase Assay System (Promega, E2940) was reconstituted and, after equilibration to room temperature, DMEM was added to each tube at a 1:1 ratio to prepare substrate I, to be used immediately. Stop & Remelt the buffer and allow it to equilibrate to room temperature before mixing with... Stop & Prepare Substrate II at a ratio of 100:1 and use immediately after preparation.

[0461] The vacuum pump removes the original culture medium from the 96-well culture plate;

[0462] Add 150 μL of substrate I to each well and incubate on a shaker at room temperature for 10 min.

[0463] Take 120 μL of substrate I, transfer it to a 96-well microplate, and read the Firefly chemiluminescence value on a microplate reader (Tecan, Infinite 200);

[0464] Add 60 μL of substrate II to each well, incubate on a shaker at room temperature for 10 min, and read the Renilla chemiluminescence value on a microplate reader.

[0465] 4. Data Analysis and Processing

[0466] Fluorescence activity was measured using a microplate reader. The collected Renilla signals were normalized using the Firefly signal standard. The inhibitory effect of siRNA was determined by comparing the results without treatment (residual inhibitory activity). The calculation process is as follows:

[0467] Homogenized Ren / Fir ratio: Ratio = Renilla (renilla luciferase) / Firefly (firefly luciferase).

[0468] Residual inhibition rate: 2 replicates (Ratio) siRNA / Ratio control The mean is calculated as () * 100% of the original value, where Ratio control To calculate the average ratio of the two duplicate wells (excluding siNRA), calculate the ratio of each duplicate well separately. siRNA / Ratio control Then, the average value is taken as the residual inhibition rate;

[0469] Plotting: Using Graphpad Prism

[0470] Half-maximal inhibitory concentration (IC50): In this experiment, the top and bottom were plotted. The IC50 value was obtained by formula Y = Bottom + (Top - Bottom) / (1 + 10^((LogIC50 - X) * HillSlope)), where Y = 50 and X = log(concentration).

[0471] In the target activity assay, HEK293A (Nanjing Kebai, catalog number CBP60436) cell line was transfected with psiCHECK2-GSCM recombinant plasmid. The initial concentration of the compound was 10 nM, and 11 concentration points (10 nM, 3.33 nM, 1.11 nM, 0.37 nM, 0.123 nM, 0.041 nM, 0.0136 nM, 0.0045 nM, 0.00152 nM, 0.000508 nM, 0.000169 nM) were selected for siRNA compound activity screening. The results are shown in Table 1.

[0472] Table 1. Results of psi-CHECK2 target activity assay

[0473]

[0474] Off-target activity assays were performed using HEK293A (Nanjing Kebai, catalog number CBP60436) cell line transfected with the psiCHECK2-GSSM-5Hits recombinant plasmid. The initial concentration of the compound was 10 nM, and 11 concentrations were obtained by 3-fold dilution (10 nM, 3.33 nM, 1.11 nM, 0.37 nM, 0.123 nM, 0.041 nM, 0.0136 nM, 0.0045 nM, 0.00152 nM, 0.000508 nM, 0.000169 nM) for siRNA compound activity screening. The results are shown in Table 2.

[0475] Table 2. Results of psi-CHECK2 off-target activity assay

[0476]

[0477]

[0478] The results showed that siRNA carrying ROR14 effectively reduced off-target activity while maintaining target activity.

[0479] Example 4: Validation of the long-lasting efficacy of the compound in the C57BL / 6 mouse model

[0480] C57BL / 6 mice (male, 18–21 g, 6–8 weeks old) were randomly divided into groups of 6 according to Table 3. The dosage for each animal was calculated based on its body weight. The medication was administered subcutaneously as a single dose. The siRNA compound was first prepared as a 1 mg / mL solution (using 0.9% sodium chloride aqueous solution as the solvent). Before the experiment, the siRNA compound was dissolved in 0.9% sodium chloride aqueous solution and the volume was adjusted to the required concentration and volume. The administration volume of physiological saline and siRNA compound was 5 mL / kg.

[0481] Table 3 Grouping of Animal Experiments

[0482]

[0483] Blood samples were collected from the orbital venous plexus of mice before administration (day 0) and on days 14, 28, 42 and 56 after administration. Serum mTTR protein was detected at each time point using an ELISA kit (Abcam, ab282297). At the last experimental time point, 10 mg of liver was collected and placed in RNAlater solution to detect liver mTTR mRNA.

[0484] Table 4. Results of the long-term efficacy validation of the compounds in the C57BL / 6 mouse model.

[0485]

[0486] The results showed that siRNA carrying ROR14 could reduce the expression of target genes in vivo for a long period of time.

Claims

1. Nucleotide dimers, which are: ， in, Base and Base' are , , , or ; R5 is either F or OMe.

2. A double-stranded RNA molecule or a pharmaceutically acceptable salt thereof, comprising a sense strand and an antisense strand, wherein each strand has 14 to 30 nucleotides, and said antisense strand comprises one or more nucleotide monomers with the following structure: ROR14 in, Base selected , , , or .

3. The double-stranded RNA molecule of claim 2, wherein the sense strand and the antisense strand each have 20 to 25 nucleotides.

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

5. The double-stranded RNA molecule of claim 2, wherein the nucleotide monomer is located at the 6th or 7th position at the 5' end of the antisense strand.

6. The double-stranded RNA molecule of claim 2, wherein the nucleotide monomer is located at the 7th position at the 5' end of the antisense strand.

7. The double-stranded RNA molecule of claim 2, wherein the double-stranded RNA has a melting temperature from 40°C to 80°C.

8. The double-stranded RNA molecule of claim 2, wherein the double-stranded RNA has a melting temperature from 55°C to 67°C.

9. The double-stranded RNA molecule of claim 2, wherein the antisense strand has a sequence that is fully complementary to the sense strand and the target mRNA, and has the ability to induce the degradation of the target mRNA.

10. The double-stranded RNA molecule of claim 2, wherein the target mRNA is encoded by an endogenous gene or by a pathogen gene.

11. The double-stranded RNA molecule of claim 2, wherein the sense strand and / or antisense strand comprises 3' and / or 5' overhangs.

12. The double-stranded RNA molecule of claim 2, wherein the double-stranded RNA is further coupled to a ligand.

13. The double-stranded RNA molecule of claim 12, wherein the ligand comprises one or more GalNAc.

14. A nucleic acid molecule, wherein the nucleotide sequence of the nucleic acid molecule comprises one or more nucleotide monomers as described in claim 2.

15. The nucleic acid molecule of claim 14, wherein the nucleic acid is selected from DNA, RNA and DNA / RNA hybrids.

16. The nucleic acid molecule of claim 14, wherein the nucleic acid molecule is single-stranded or double-stranded.

17. The nucleic acid molecule of claim 14, wherein the nucleic acid molecule is selected from small interfering RNA and short hairpin RNA.

18. A pharmaceutical composition comprising a double-stranded RNA molecule as described in any one of claims 2-13, and a pharmaceutically acceptable carrier or excipient.

19. A kit comprising a double-stranded RNA molecule as described in any one of claims 2-13.

20. Use of the double-stranded RNA molecule of any one of claims 2-13 in the preparation of a medicament for inhibiting the expression of target genes in cells.

21. Use of the double-stranded RNA molecule according to any one of claims 2-13 in the preparation of a medicament for reducing off-target toxicity in cells.

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