Prodrugs for delivery of siRNA into cells

Abstract

The present invention provides an oligonucleotide comprising a compound of Formula (Ia), or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof. The compound of Formula (I) is comprised within an oligonucleotide, or at the 5' end and / or 3' end, for improving the ability of double stranded RNA to cross the cell membrane. The present invention also relates to double stranded RNA, cells, pharmaceutical compositions, and kits comprising the oligonucleotide.

Description

[0001] This invention claims priority to Chinese application 202211374509.3 filed on October 31, 2022; Chinese application 202310101722.5 filed on February 9, 2023; and Chinese application 202310552663.3 filed on May 16, 2023, all of which are incorporated herein by reference in their entirety. Invention Field

[0002] This invention pertains to the pharmaceutical field and specifically relates to a masking group having the ability to enhance the ability of double-stranded RNA to cross cell membranes, such as the TXL- group having the structure of formula (I), and a compound of formula (I) having the masking group attached to a nucleotide, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof. Background Technology

[0003] RNA interference is a phenomenon in which target mRNA is efficiently and specifically degraded by double-stranded RNA (dsRNA).

[0004] Research has found that by preparing small interfering RNA (siRNA) into a prodrug form, the negative charge of oligonucleotide phosphate groups can be masked with cell-cleavable masking groups, thereby enhancing cell penetration. For example, WO2022147214A2 discloses a prodrug based on a disulfide ring.

[0005] However, prodrug approaches remain substantially challenging, partly due to the difficulty in selecting the optimal masking group. For example, cell lysis of the masking group often yields products considered undesirable or even toxic. Furthermore, a balance must be struck between allowing absorption in the intestine and allowing lysis in the blood or target cells.

[0006] Therefore, there is a need in this field to develop more prodrugs for efficient in vivo delivery of siRNA. Summary of the Invention

[0007] In one aspect, the present invention provides an oligonucleotide, comprising a compound of formula (II), or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof:

[0008]

[0009] The variables are defined below.

[0010] In another aspect, the present invention provides an oligonucleotide comprising one, two, or more compounds of formula (Ia), or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof:

[0011]

[0012] The variables are defined below.

[0013] In another aspect, the present invention provides a double-stranded RNA having a sense strand and an antisense strand, each strand having 14 to 30 nucleotides, wherein the antisense strand contains a sequence sufficiently complementary to the sense strand and the target mRNA, wherein the sense strand and / or antisense strand contains one or more of the aforementioned compound of formula (II) or the aforementioned compound of formula (Ia), or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof.

[0014] In another aspect, the present invention provides a cell containing the aforementioned double-stranded RNA.

[0015] In another aspect, the present invention provides a pharmaceutical composition comprising the aforementioned double-stranded RNA, the aforementioned cells, and optionally a pharmaceutically acceptable carrier or excipient.

[0016] In another aspect, the present invention provides a kit comprising the aforementioned double-stranded RNA, the aforementioned cells, or the aforementioned pharmaceutical composition.

[0017] In another aspect, the present invention provides compounds of formula (IIb), or pharmaceutically acceptable salts, tautomers, or stereoisomers thereof:

[0018]

[0019] The variables are defined below. Invention Details

[0021] definition

[0022] Chemical definition

[0023] The definitions of specific functional groups and chemical terms are described in more detail below.

[0024] When listing a range of values, it is assumed that each value and the subranges within that range are included. For example, "C 1-6 Alkyl groups include C1, C2, C3, C4, C5, C6, and C6. 1-6 C 1-5 C 1-4 C 1-3 C 1-2 C 2-6 C 2-5 C 2-4 C 2-3 C 3-6 C 3-5 C 3-4 C 4-6 C 4-5 and C5-6 alkyl.

[0025] “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).

[0026] “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.

[0027] “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 2-6Examples 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.

[0028] "Halogen" or "halogen" refers to fluorine (F), chlorine (Cl), bromine (Br), and iodine (I).

[0029] 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.

[0030] “C 3-10 "Cycloalkyl" refers to a non-aromatic cyclic hydrocarbon group having 3 to 10 ring carbon atoms and zero heteroatoms. In some embodiments, C 4-7 cycloalkyl and C 3-6 Cycloalkyl groups are particularly preferred, and C10 is more preferred. 5-6 Cycloalkyl groups. Cycloalkyl groups also include ring systems in which the aforementioned cycloalkyl ring is fused with one or more aryl or heteroaryl groups, wherein the connecting point is on the cycloalkyl ring, and in such cases, the number of carbons continues to represent the number of carbons in the cycloalkyl system. Exemplary cycloalkyl groups include, but are not limited to: cyclopropyl (C3), cyclopropenyl (C3), cyclobutyl (C4), cyclobutenyl (C4), cyclopentyl (C5), cyclopentenyl (C5), cyclohexyl (C6), cyclohexenyl (C6), cyclohexadienyl (C6), cycloheptyl (C7), cycloheptenyl (C7), cycloheptadienyl (C7), cyclohepttrienyl (C7), etc. The cycloalkyl group may optionally be substituted with one or more substituents, for example, substituted with 1 to 5 substituents, 1 to 3 substituents, or 1 substituent.

[0031] "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.

[0032] “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.

[0033] "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.

[0034] The alkyl, alkenyl, ynyl, cycloalkyl, heterocyclic, aryl, and heteroaryl groups defined in this article are optional substituted groups.

[0035] Exemplary substituents on carbon atoms include, but are not limited to: halogen, -CN, -NO2, -N3, -SO2H, -SO3H, -OH, -OR aa -ON(R) bb )2、-N(R bb )2、-N(R bb )3 + X - -N(OR) cc )R bb -SH, -SR aa -SSR cc -C(=O)R aa -CO2H, -CHO, -C(OR) cc )2、-CO2R aa -OC(=O)R aa -OCO2R aa -C(=O)N(R) bb )2、-OC(=O)N(R bb )2、-NR bb C(=O)R aa -NR bb CO2R aa -NR bb C(=O)N(R bb )2、-C(=NR bb )R aa -C(=NR) bb OR aa -OC(=NR) bb )R aa -OC(=NR) bb OR aa -C(=NR) bb )N(R bb )2、-OC(=NR bb )N(R bb )2、-NR bb C(=NR bb )N(R bb )2、-C(=O)NR bb SO2R aa -NR bb SO2R aa -SO2N(R) bb )2、-SO2R aa -SO2OR aa -OSO2R aa -S(=O)R aa -OS(=O)R aa 、-Si(R aa3. -OSi(R) aa 3. -C(=S)N(R) bb )2、-C(=O)SR aa -C(=S)SR aa -SC(=S)SR aa -SC(=O)SR aa -OC(=O)SR aa -SC(=O)OR aa -SC(=O)R aa -P(=O)2R aa -OP(=O)2R aa -P(=O)(R aa )2、-OP(=O)(R aa )2、-OP(=O)(OR cc )2、-P(=O)2N(R bb )2、-OP(=O)2N(R bb )2、-P(=O)(NR bb )2、-OP(=O)(NR bb )2、-NR bb P(=O)(OR cc )2、-NR bb P(=O)(NR bb )2、-P(R cc )2、-P(R cc )3、-OP(R cc )2、-OP(R cc )3、-B(R aa 2. -B(OR) cc )2、-BR aa (OR cc ), alkyl, haloalkyl, alkenyl, ynyl, cycloalkyl, heterocyclic, aryl, and heteroaryl, wherein each alkyl, alkenyl, ynyl, cycloalkyl, heterocyclic, aryl, and heteroaryl is independently bounded by 0, 1, 2, 3, 4, or 5 R groups. dd Group substitution;

[0036] 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;

[0037] 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;

[0038] 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;

[0039] R cc Each is independently selected from hydrogen, alkyl, haloalkyl, alkenyl, ynyl, 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;

[0040] 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;

[0041] 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;

[0042] R ff Each is independently selected from hydrogen, alkyl, haloalkyl, alkenyl, ynyl, 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;

[0043] 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.

[0044] 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.

[0045] Other definitions

[0046] 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.

[0047] 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.

[0048] 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.

[0049] 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.

[0050] 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.

[0051] "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.

[0052] "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.

[0053] "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.

[0054] Ribose is a pentose aldose composed of five carbon atoms. The carbon atom adjacent to the oxygen atom is numbered 1' (or simply 1' end), and the carbon atoms are numbered sequentially in a clockwise direction. A base is attached to 1', and a phosphate group is attached to 5'. The structural formula and carbon atom numbering are as follows:

[0055]

[0056] 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.

[0057] 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.

[0058] 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.

[0059] 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.

[0060] 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).

[0061] 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:

[0062] 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.

[0063] 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.

[0064] 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.

[0065] 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.

[0066] 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.

[0067] Compounds of the present invention

[0068] This invention specifically relates to compounds of formula (I), or pharmaceutically acceptable salts, tautomers, or stereoisomers thereof:

[0069]

[0070] in,

[0071] X1 is selected from OR a OP1, SP1 or NR b R c ;

[0072] R a Selected from H, C 1-6 Alkyl, C1-6 Haloalkyl, C 2-6 alkenyl or C 2-6 The alkynyl group, which is optionally deuterated, up to and including complete deuteration;

[0073] R b and R c Independently selected from H and C 1-6 Alkyl or C 1-6 Halogenated alkyl, wherein R b and R c Can be optionally D, C 6-10 Aryl or 5-10 heteroaryl substitutions, up to complete deuteration;

[0074] X2 is OH, OP1, or SP1, or X2 is a nucleoside moiety that is attached to P via a hydroxyl or thiol group at the 2', 3', or 5' end of the ribose;

[0075] X3 is independently selected from O or S;

[0076] T is selected from

[0077] Each R T1 Independently selected from H, D, halogens, CN, C 1-6 Alkyl, C 1-6 Haloalkyl, C 1-6 Haloalkyl, C 2-6 alkenyl, C 2-6 Alkyne group or chain containing GalNAc, which is optionally deuterated up to fully deuterated;

[0078] Each R T2 Independently selected from H, D, halogens, CN, C 1-6 Alkyl, C 1-6 Haloalkyl, C 2-6 alkenyl or C 2-6 The alkynyl group, which is optionally deuterated, up to and including complete deuteration;

[0079] Each R T3 Independently selected from H, D, halogens, CN, C 1-6 Alkyl, C 1-6 Haloalkyl, C 2-6 alkenyl or C 2-6 The alkynyl group, which is optionally deuterated, up to and including complete deuteration;

[0080] Each R T4 Independently selected from H, D, halogens, CN, C 1-6 Alkyl, C 1-6 Haloalkyl, C 2-6 alkenyl or C 2-6 The alkynyl group, which is optionally deuterated, up to and including complete deuteration;

[0081] m can be 0, 1, 2, 3, 4, or 5;

[0082] n is 0, 1, 2, 3, 4 or 5;

[0083] p is 0, 1, 2, 3, 4 or 5;

[0084] X is selected from chemical bonds, -O-, -S-, -C(O)-, -C(O)O-, -OC(O)-, -OC(O)NR X1 -、-NR X1 C(O)O-、-NR X1 C(O)- or -C(O)NR X1 -;

[0085] R X1 Selected from H, halogens, C 1-6 Alkyl or C 1-6 Halogenated alkyl groups, optionally deuterated, up to and including complete deuteration;

[0086] L stands for -Ar-(CH2) 1-6 -O-, where each CH2 group may optionally be replaced by R#, which is selected from H, D, halogens, CN, and C. 1-6 Alkyl, C 1-6 Haloalkyl, C 2-6 alkenyl or C 2-6 The alkynyl group, which is optionally deuterated, up to and including complete deuteration;

[0087] In L, Ar is bonded to X, and oxygen atom is bonded to phosphorus atom;

[0088] Ar is selected from C 3-10 Cycloalkyl, 3-10 membered heterocyclic groups, C 6-10 Aryl or 5-14 heteroaryl, wherein C 3-10 Cycloalkyl, 3-10 membered heterocyclic groups, C 6-10 The aryl or 5-10 heteroaryl group can be optionally replaced by 1, 2, 3, 4 or 5 R*.

[0089] R* is selected from H, D, halogens, CN, and C. 1-6 Alkyl, C 1-6 Haloalkyl, C 2-6 alkenyl or C 2-6 The alkynyl group, which is optionally deuterated, up to and including complete deuteration;

[0090] Wherein P1 is selected from protecting groups, preferably hydroxyl protecting groups, 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), 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), 4,4'-dimethoxytriphenylmethyl, -P(OCH2CH2CN)(N(iPr)2) or -C(O)CH2CH2C(O)OH, preferably -P(OCH2CH2CN)(N(iPr)2) or -C(O)CH2CH2C(O)OH.

[0091] This invention also relates to oligonucleotides comprising one, two, or more compounds of formula (Ia), or pharmaceutically acceptable salts, tautomers, or stereoisomers thereof; and to double-stranded RNA having a sense strand and an antisense strand, each strand having 14 to 30 nucleotides, wherein the antisense strand comprises a sequence sufficiently complementary to the sense strand and the target mRNA, wherein the sense strand and / or antisense strand comprises one or more compounds of formula (Ia), or pharmaceutically acceptable salts, tautomers, or stereoisomers thereof.

[0092]

[0093] in,

[0094] Selected from OR a or NR b R c , or represents a chemical bond that connects to the 5' end hydroxyl or thiol group of the ribose of an adjacent nucleotide;

[0095] X1 is selected from OR a or NR b R c Or a chemical bond linked to a hydroxyl or thiol group at the 2' or 3' end of the ribose of another adjacent nucleotide;

[0096] and Unlike X1, OR is not used at the same time. a or NR b R c ;

[0097] R a Selected from H, C 1-6 Alkyl, C 1-6 Haloalkyl, C 2-6 alkenyl or C 2-6 The alkynyl group, which is optionally deuterated, up to and including complete deuteration;

[0098] R b and R c Independently selected from H and C 1-6 Alkyl or C 1-6 Halogenated alkyl, wherein R b and R c Can be optionally D, C 6-10 Aryl or 5-10 heteroaryl substitutions, up to complete deuteration;

[0099] X3 is independently selected from O or S;

[0100] T is selected from

[0101] Each R T1 Independently selected from H, D, halogens, CN, C 1-6 Alkyl, C 1-6 Haloalkyl, C 1-6 Haloalkyl, C 2-6 alkenyl, C 2-6 Alkyne group or chain containing GalNAc, which is optionally deuterated up to fully deuterated;

[0102] Each R T2 Independently selected from H, D, halogens, CN, C 1-6 Alkyl, C 1-6 Haloalkyl, C 2-6 alkenyl or C 2-6 The alkynyl group, which is optionally deuterated, up to and including complete deuteration;

[0103] Each R T3 Independently selected from H, D, halogens, CN, C 1-6 Alkyl, C 1-6 Haloalkyl, C 2-6 alkenyl or C 2-6 The alkynyl group, which is optionally deuterated, up to and including complete deuteration;

[0104] Each R T4 Independently selected from H, D, halogens, CN, C 1-6 Alkyl, C 1-6 Haloalkyl, C 2-6 alkenyl or C2-6 The alkynyl group, which is optionally deuterated, up to and including complete deuteration;

[0105] m can be 0, 1, 2, 3, 4, or 5;

[0106] n is 0, 1, 2, 3, 4 or 5;

[0107] p is 0, 1, 2, 3, 4 or 5;

[0108] X is selected from chemical bonds, -O-, -S-, -C(O)-, -C(O)O-, -OC(O)-, -OC(O)NR X1 -、-NR X1 C(O)O-、-NR X1 C(O)- or -C(O)NR X1 -;

[0109] R X1 Selected from H, halogens, C 1-6 Alkyl or C 1-6 Halogenated alkyl groups, optionally deuterated, up to and including complete deuteration;

[0110] L stands for -Ar-(CH2) 1-6 -O-, where each CH2 group may optionally be replaced by R#, which is selected from H, D, halogens, CN, and C. 1-6 Alkyl, C 1-6 Haloalkyl, C 2-6 alkenyl or C 2-6 The alkynyl group, which is optionally deuterated, up to and including complete deuteration;

[0111] In L, Ar is bonded to X, and oxygen atom is bonded to phosphorus atom;

[0112] Ar is selected from C 3-10 Cycloalkyl, 3-10 membered heterocyclic groups, C 6-10 Aryl or 5-14 heteroaryl, wherein C 3-10 Cycloalkyl, 3-10 membered heterocyclic groups, C 6-10 The aryl or 5-10 heteroaryl group can be optionally replaced by 1, 2, 3, 4 or 5 R*.

[0113] R* is selected from H, D, halogens, CN, and C. 1-6 Alkyl, C 1-6 Haloalkyl, C 2-6 alkenyl or C 2-6 The alkynyl group is optionally deuterated, up to and including complete deuteration.

[0114] This invention also relates to oligonucleotides, compounds comprising formula (II), or pharmaceutically acceptable salts, tautomers, or stereoisomers thereof:

[0115]

[0116] in,

[0117] X1 is selected from OR a or NR b R c ;

[0118] R a Selected from H, C 1-6 Alkyl or C 1-6 Halogenated alkyl groups, optionally deuterated, up to and including complete deuteration;

[0119] R b and R c Selected from H, C 1-6 Alkyl or C 1-6 Halogenated alkyl, wherein R b and R c Can be optionally D, C 6-10 Aryl or 5-10 heteroaryl substitutions, up to complete deuteration;

[0120] X2 is the remaining part of the oligonucleotide, which is linked to P by a hydroxyl or thiol group on the 2', 3', or 5' carbon atom of the ribose of the first nucleotide at the 5' end;

[0121] X3 is independently selected from O or S;

[0122] Each R T1 Independently selected from H, D, and C 1-6 Alkyl, C 1-6 Haloalkyl, C 2-6 alkenyl, C 2-6 Alkyne group or chain containing GalNAc, which is optionally deuterated up to fully deuterated;

[0123] Each R T2 Independently selected from H, D, and C 1-6 Alkyl, C 1-6 Haloalkyl, C 2-6 alkenyl or C 2-6 The alkynyl group, which is optionally deuterated, up to and including complete deuteration;

[0124] m can be 0, 1, 2, 3, 4, or 5;

[0125] X is selected from chemical bonds, -O-, -S-, -C(O)-, -C(O)O-, -OC(O)-, -OC(O)NR X1 -、-NR X1 C(O)O-、-NR X1 C(O)- or -C(O)NR X1 -;

[0126] RX1 Selected from H, C 1-6 Alkyl or C 1-6 Halogenated alkyl groups, optionally deuterated, up to and including complete deuteration;

[0127] L stands for -Ar-(CH2) 1-6 -O-, where each CH2 may optionally be replaced by one or two R#, where R# is selected from H, D, C. 1-6 Alkyl or C 1-6 Halogenated alkyl groups, optionally deuterated, up to and including complete deuteration;

[0128] In L, Ar is bonded to X, and oxygen atom is bonded to phosphorus atom;

[0129] Ar is selected from C 6-10 Aryl or 5-10 heteroaryl, wherein C 6-10 The aryl or 5-10 heteroaryl group can be optionally replaced by 1, 2, 3, 4 or 5 R*.

[0130] R* is selected from C 1-6 Alkyl, C 1-6 Haloalkyl, C 2-6 alkenyl or C 2-6 The alkynyl group is optionally deuterated, up to and including complete deuteration.

[0131] This invention also relates to oligonucleotides comprising one, two, or more compounds of formula (Ia), or pharmaceutically acceptable salts, tautomers, or stereoisomers thereof:

[0132]

[0133] in,

[0134] Selected from OR a or NR b R c , or represents a chemical bond that connects to a hydroxyl or thiol group on the 5' carbon atom of the ribose of an adjacent nucleotide;

[0135] X1 is a chemical bond that connects to a hydroxyl or thiol group on the 2' or 3' carbon atom of the ribose of another adjacent nucleotide;

[0136] R a Selected from H, C 1-6 Alkyl, C 1-6 Haloalkyl, C 2-6 alkenyl or C 2-6 The alkynyl group, which is optionally deuterated, up to and including complete deuteration;

[0137] R b and R c Independently selected from H and C1-6 Alkyl or C 1-6 Halogenated alkyl, wherein R b and R c Can be optionally D, C 6-10 Aryl or 5-10 heteroaryl substitutions, up to complete deuteration;

[0138] X3 is independently selected from O or S;

[0139] T is

[0140] Each R T1 Independently selected from H, D, halogens, CN, C 1-6 Alkyl, C 1-6 Haloalkyl, C 1-6 Haloalkyl, C 2-6 alkenyl, C 2-6 Alkyne group or chain containing GalNAc, which is optionally deuterated up to fully deuterated;

[0141] Each R T2 Independently selected from H, D, halogens, CN, C 1-6 Alkyl, C 1-6 Haloalkyl, C 2-6 alkenyl or C 2-6 The alkynyl group, which is optionally deuterated, up to and including complete deuteration;

[0142] m can be 0, 1, 2, 3, 4, or 5;

[0143] X is selected from chemical bonds, -O-, -S-, -C(O)-, -C(O)O-, -OC(O)-, -OC(O)NR X1 -、-NR X1 C(O)O-、-NR X1 C(O)- or -C(O)NR X1 -;

[0144] R X1 Selected from H, halogens, C 1-6 Alkyl or C 1-6 Halogenated alkyl groups, optionally deuterated, up to and including complete deuteration;

[0145] L stands for -Ar-(CH2) 1-6 -O-, where each CH2 group may optionally be replaced by R#, which is selected from H, D, halogens, CN, and C. 1-6 Alkyl, C 1-6 Haloalkyl, C 2-6 alkenyl or C 2-6 The alkynyl group, which is optionally deuterated, up to and including complete deuteration;

[0146] In L, Ar is bonded to X, and oxygen atom is bonded to phosphorus atom;

[0147] Ar is selected from C 3-10 Cycloalkyl, 3-10 membered heterocyclic groups, C 6-10 Aryl or 5-14 heteroaryl, wherein C 3-10 Cycloalkyl, 3-10 membered heterocyclic groups, C 6-10 The aryl or 5-10 heteroaryl group can be optionally replaced by 1, 2, 3, 4 or 5 R*.

[0148] R* is selected from H, D, halogens, CN, and C. 1-6 Alkyl, C 1-6 Haloalkyl, C 2-6 alkenyl or C 2-6 The alkynyl group is optionally deuterated, up to and including complete deuteration.

[0149] X1, X2, X3 and

[0150] In one implementation, X1 is OR a In another embodiment, X1 is OP1; in another embodiment, X1 is SP1; in another embodiment, X1 is NR. b R c In another embodiment, X1 is a chemical bond connected to a hydroxyl or thiol group at the 5' end of the ribose of another adjacent nucleotide; in another embodiment, X1 is a chemical bond connected to a hydroxyl or thiol group at the 2' end of the ribose of another adjacent nucleotide; in another embodiment, X1 is a chemical bond connected to a hydroxyl or thiol group at the 3' end of the ribose of another adjacent nucleotide.

[0151] In one embodiment, X1 is a chemical bond that connects to a hydroxyl or thiol group on the 2' or 3' carbon atom of the ribose of another adjacent nucleotide.

[0152] In a more specific embodiment, X1 is OH; in another more specific embodiment, X1 is

[0153] In one embodiment, X2 is OH; in another embodiment, X2 is OP1; in another embodiment, X2 is SP1; in another embodiment, X2 is a nucleoside moiety connected to P via a hydroxyl or thiol group at the 2' end of the ribose; in another embodiment, X2 is a nucleoside moiety connected to P via a hydroxyl or thiol group at the 3' end of the ribose; in another embodiment, X2 is a nucleoside moiety connected to P via a hydroxyl or thiol group at the 5' end of the ribose.

[0154] In one embodiment, X2 is the remaining portion of the oligonucleotide that is linked to P via a hydroxyl or thiol group on the 2', 3', or 5' carbon atom of the ribose of the first nucleotide at the 5' end, for example, via a hydroxyl or thiol group on the 5' carbon atom of the ribose of the first nucleotide at the 5' end.

[0155] In one implementation, X3 is 0; in another implementation, X3 is S.

[0156] In one implementation scheme OR a In another implementation scheme, For NR b R c In another implementation scheme, A chemical bond that links the ribose of another adjacent nucleotide to a hydroxyl or thiol group at the 5' end; in another embodiment, Unlike X1, OR is not used at the same time. a or NR b R c .

[0157] In one implementation scheme This indicates a chemical bond that connects to a hydroxyl or thiol group on the 5' carbon atom of the ribose of an adjacent nucleotide.

[0158] T

[0159] In one implementation, T is... In another implementation, T is In another implementation, T is In another implementation, T is In another implementation, T is

[0160] In one implementation, T is... In another implementation, T is In another implementation, T is

[0161] In a more specific implementation, T is... In another, more specific implementation, T is... In another, more specific implementation, T is... In another, more specific implementation, T is... In another, more specific implementation, T is... In another, more specific implementation, T is...

[0162] R T1 R T2R T3 R T4 R T m, n and p

[0163] In one implementation, R T1 For H; in another implementation, R T1 For D; in another implementation, R T1 For halogen; in another embodiment, R T1 For CN; in another implementation, R T1 C 1-6 Alkyl, such as C 1-4 Alkyl; in another embodiment, R T1 C 1-6 Halogenated alkyl groups, such as C 1-4 Halogenated alkyl; in another embodiment, R T1 C 1-6 Halogenated alkyl; in another embodiment, R T1 C 2-6 alkenyl; in another embodiment, R T1 C 2-6 alkynyl group; in another embodiment, R T1 For a chain containing GalNAc; in another embodiment, the R T1 It can be selectively deuterated, until it is completely deuterated.

[0164] In one implementation, R T2 For H; in another implementation, R T2 For D; in another implementation, R T2 For halogen; in another embodiment, R T2 For CN; in another implementation, R T2 C 1-6 Alkyl, such as C 1-4 Alkyl; in another embodiment, R T2 C 1-6 Halogenated alkyl groups, such as C 1-4 Halogenated alkyl; in another embodiment, R T2 C 2-6 alkenyl; in another embodiment, R T2 C 2-6 Alkyne group; in another embodiment, the R T2 It can be selectively deuterated, until it is completely deuterated.

[0165] In one implementation, R T3 For H; in another implementation, R T3 For D; in another implementation, RT3 For halogen; in another embodiment, R T3 For CN; in another implementation, R T3 C 1-6 Alkyl, such as C 1-4 Alkyl; in another embodiment, R T3 C 1-6 Halogenated alkyl groups, such as C 1-4 Halogenated alkyl; in another embodiment, R T3 C 2-6 alkenyl; in another embodiment, R T3 C 2-6 Alkyne group; in another embodiment, the R T3 It can be selectively deuterated, until it is completely deuterated.

[0166] In one implementation, R T4 For H; in another implementation, R T4 For D; in another implementation, R T4 For halogen; in another embodiment, R T4 For CN; in another implementation, R T4 C 1-6 Alkyl, such as C 1-4 Alkyl; in another embodiment, R T4 C 1-6 Halogenated alkyl groups, such as C 1-4 Halogenated alkyl; in another embodiment, R T4 C 2-6 alkenyl; in another embodiment, R T4 C 2-6 Alkyne group; in another embodiment, the R T4 It can be selectively deuterated, until it is completely deuterated.

[0167] In one implementation, R T For H; in another implementation, R T For D; in another implementation, R T CH3; in another embodiment, R T For a chain containing GalNAc; in another embodiment, the R T It can be selectively deuterated, until it is completely deuterated.

[0168] 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.

[0169] 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.

[0170] In one implementation, p is 0; in another implementation, p is 1; in another implementation, p is 2; in another implementation, p is 3; in another implementation, p is 4; in another implementation, p is 5.

[0171] X

[0172] In one embodiment, X is a chemical bond; in another embodiment, X is -O-; in another embodiment, X is -S-; in another embodiment, X is -C(O)-; in another embodiment, X is -C(O)O-; in another embodiment, X is -OC(O)-; in another embodiment, X is -OC(O)NR X1 -; In another implementation, X is -NR X1 C(O)O-; in another embodiment, X is -NR X1 C(O)-; in another embodiment, X is -C(O)NR X1 -

[0173] In a more specific embodiment, X is a chemical bond; in another more specific embodiment, X is -O-; in another more specific embodiment, X is -NHC(O)O-; in another more specific embodiment, X is -OC(O)NH-; in another more specific embodiment, X is -N(CH3)C(O)O-; in another more specific embodiment, X is -C(O)O-.

[0174] L

[0175] In one implementation, L stands for -Ar-(CH2). 1-6 -O-, wherein each CH2 atom may optionally be replaced by R#, and the Ar atom in L is bonded to X, and the oxygen atom is bonded to the phosphorus atom; in another embodiment, L is -Ar-(CH2). 1-4 -O-, where each CH2 can be optionally replaced by one or two R#, Ar in L is connected to X, and oxygen atom is connected to phosphorus atom.

[0176] In one implementation, Ar is C 3-10 Cycloalkyl; in another embodiment, Ar is a 3-10 membered heterocyclic group; in another embodiment, Ar is a C 6-10Aryl; in another embodiment, Ar is a 5-14-membered heteroaryl, such as a 5-10-membered heteroaryl, or phenyl; in another embodiment, the Ar may optionally be substituted with 1, 2, 3, 4 or 5 R*.

[0177] In a more specific implementation scheme, L is... In another, more specific implementation, L is... In another, more specific implementation, L is... In another, more specific implementation, L is... In another, more specific implementation, L is... In another, more specific implementation, L is... In another, more specific implementation, L is... In another, more specific implementation, L is...

[0178] R a R b and R c

[0179] In one implementation, R a For H; in another implementation, R a C 1-6 Alkyl, such as C 1-4 Alkyl; in another embodiment, R a C 1-6 Halogenated alkyl groups, such as C 1-4 Halogenated alkyl; in another embodiment, R a C 2-6 alkenyl; in another embodiment, R a C 2-6 Alkyne group; in another embodiment, the R a It can be selectively deuterated, until it is completely deuterated.

[0180] In one implementation, R b For H; in another implementation, R b C 1-6 Alkyl, such as C 1-4 Alkyl; in another embodiment, R b C 1-6 Halogenated alkyl groups, such as C 1-4 Halogenated alkyl; in another embodiment, the R b Can be optionally D, C 6-10 Aryl or 5-10 heteroaryl substitutions are made up to complete deuteration.

[0181] In one implementation, R cFor H; in another implementation, R c C 1-6 Alkyl, such as C 1-4 Alkyl; in another embodiment, R c C 1-6 Halogenated alkyl groups, such as C 1-4 Halogenated alkyl; in another embodiment, the R c Can be optionally D, C 6-10 Aryl or 5-10 heteroaryl substitutions are made up to complete deuteration.

[0182] R X1

[0183] In one implementation, R X1 For H; in another implementation, R X1 C 1-6 Alkyl, such as C 1-4 Alkyl; in another embodiment, R X1 C 1-6 Halogenated alkyl groups, such as C 1-4 Halogenated alkyl; in another embodiment, the R X1 It can be selectively deuterated, until it is completely deuterated.

[0184] R#

[0185] In one embodiment, R# is H; in another embodiment, R# is D; in yet another embodiment, R# is halogen; in another embodiment, R# is CN; in yet another embodiment, R# is C. 1-6 Alkyl, such as C 1-4 Alkyl; in another embodiment, R# is C 1-6 Halogenated alkyl groups, such as C 1-4 Halogenated alkyl; in another embodiment, R# is C 2-6 Alkenyl; in another embodiment, R# is C 2-6 Alkyne group; in another embodiment, the R# is optionally deuterated, up to and including complete deuteration.

[0186] R*

[0187] In one embodiment, R* is H; in another embodiment, R* is D; in yet another embodiment, R* is a halogen; in another embodiment, R* is CN; in yet another embodiment, R* is C. 1-6 Alkyl, such as C 1-4 Alkyl; in another embodiment, R* is C 1-6 Halogenated alkyl groups, such as C 1-4 Halogenated alkyl; in another embodiment, R* is C2-6 Alkenyl; in another embodiment, R* is C 2-6 Alkyne group; in another embodiment, the R* is optionally deuterated, up to and including complete deuteration.

[0188] PG and P1

[0189] In one embodiment, PG is a protecting group; in another embodiment, PG 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), benzyloxycarbonyl (Cb) z), 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 -P(OCH2CH2CN)(N(iPr)2) or -C(O)CH2CH2C(O)OH.

[0190] 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-trichloroethoxycarbonyl (Troc), benzyloxycarbonyl (Cb) z), 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 -P(OCH2CH2CN)(N(iPr)2) or -C(O)CH2CH2C(O)OH.

[0191] Chains containing GalNAc

[0192] In one embodiment, the chain containing GalNAc comprises a conjugated group as shown in formula (X'):

[0193]

[0194] in,

[0195] Indicates the location where it connects to a biomolecule;

[0196] Q is independent of H,

[0197] Where L1 is a chemical bond, -CH2-, -CH2CH2-, -C(O)-, -CH2O-, -CH2O-CH2CH2O-, or -NHC(O)-(CH2NHC(O)). a -;

[0198] L2 is a chemical bond or -CH2CH2C(O)-;

[0199] L3 is a chemical bond, -(NHCH2CH2) b -、-(NHCH2CH2CH2) b-or-C(O)CH2-;

[0200] L4 is -(OCH2CH2) c -、-(OCH2CH2CH2) c -、-(OCH2CH2CH2CH2) c -、-(OCH2CH2CH2CH2CH2) c -or -NHC(O)-(CH2) d -;

[0201] Where a = 0, 1, 2 or 3;

[0202] b = 1, 2, 3, 4 or 5;

[0203] c = 1, 2, 3, 4 or 5;

[0204] d = 1, 2, 3, 4, 5, 6, 7 or 8;

[0205] A is a chemical bond, -CH2O- or -NHC(O)-;

[0206] A' is a chemical bond, -C(O)NH-, -NHC(O)-, or -O(CH2CH2O). e -;

[0207] Where e is 1, 2, 3, 4 or 5;

[0208] B is a chemical bond, -CH2-, -C(O)-, -M-, -CH2-M-, or -C(O)-M-;

[0209] Where M is

[0210] R1 and R2 together form -CH2CH2O- or -CH2CH(R)-O-, and R3 is H;

[0211] Or R1 and R3 together form -C 1-2 Alkylene-, and R2 is H;

[0212] Wherein R is -OR', -CH2OR' or -CH2CH2OR', where R' is H, a hydroxyl protecting group or a solid support, and the hydroxyl protecting group is preferably -C(O)CH2CH2C(O)OH or 4,4'-dimethoxytriphenylmethyl;

[0213] m1 = 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

[0214] n1 = 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0215] In another aspect, the present invention relates to a ligand comprising N-acetylgalactosamine, wherein the conjugating group is as shown in formula (I'):

[0216]

[0217] in,

[0218] Indicates the location where it connects to a biomolecule;

[0219] Q is independent of H,

[0220] Where L1 is a chemical bond, -CH2-, -CH2CH2-, -C(O)-, -CH2O-, -CH2O-CH2CH2O-, or -NHC(O)-(CH2NHC(O)). a -;

[0221] L2 is a chemical bond or -CH2CH2C(O)-;

[0222] L3 is a chemical bond, -(NHCH2CH2) b -、-(NHCH2CH2CH2) b -or-C(O)CH2-;

[0223] L4 is -(OCH2CH2) c -、-(OCH2CH2CH2) c -、-(OCH2CH2CH2CH2) c -、-(OCH2CH2CH2CH2CH2) c -or -NHC(O)-(CH2) d -;

[0224] Where a = 0, 1, 2 or 3;

[0225] b = 1, 2, 3, 4 or 5;

[0226] c = 1, 2, 3, 4 or 5;

[0227] d = 1, 2, 3, 4, 5, 6, 7 or 8;

[0228] A is either -CH2O- or -NHC(O)-;

[0229] A' is a chemical bond, -C(O)NH- or -NHC(O)-;

[0230] R1 and R2 together form -CH2CH2O- or -CH2CH(R)-O-, and R3 is H;

[0231] Or R1 and R3 together form -C 1-2 Alkylene-, and R2 is H;

[0232] Wherein R is -OR', -CH2OR' or -CH2CH2OR', where R' is H, a hydroxyl protecting group or a solid support, and the hydroxyl protecting group is preferably -C(O)CH2CH2C(O)OH or 4,4'-dimethoxytriphenylmethyl;

[0233] m1 = 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

[0234] n1 = 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0235] In another embodiment, the chain containing GalNAc is a conjugated group containing the formula (X'), wherein,

[0236] Q is independent of H,

[0237] Where L1 is a chemical bond, -CH2-, -CH2CH2-, -C(O)-, -CH2O-, -CH2O-CH2CH2O-, or -NHC(O)-(CH2NHC(O)). a -;

[0238] L2 is a chemical bond or -CH2CH2C(O)-;

[0239] L3 is a chemical bond, -(NHCH2CH2) b -、-(NHCH2CH2CH2) b -or-C(O)CH2-;

[0240] L4 is -(OCH2CH2) c -、-(OCH2CH2CH2) c -、-(OCH2CH2CH2CH2) c -、-(OCH2CH2CH2CH2CH2) c -or -NHC(O)-(CH2) d -;

[0241] Where a = 0, 1, 2 or 3;

[0242] b = 1, 2, 3, 4 or 5;

[0243] c = 1, 2, 3, 4 or 5;

[0244] d = 1, 2, 3, 4, 5, 6, 7 or 8;

[0245] A is a chemical bond, -CH2O- or -NHC(O)-;

[0246] A' is a chemical bond, -C(O)NH-, -NHC(O)-, or -O(CH2CH2O). e -;

[0247] Where e is 1, 2, 3, 4 or 5;

[0248] B is a chemical bond, -CH2-, -M-, -CH2-M-, or -C(O)-M-;

[0249] Where M is

[0250] R1 and R2 together form -CH2CH2O- or -CH2CH(R)-O-, and R3 is H;

[0251] Or R1 and R3 together form -C 1-2 Alkylene-, and R2 is H;

[0252] Wherein R is -OR', -CH2OR' or -CH2CH2OR', where R' is H, a hydroxyl protecting group or a solid support, and the hydroxyl protecting group is preferably -C(O)CH2CH2C(O)OH or 4,4'-dimethoxytriphenylmethyl;

[0253] m1 = 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

[0254] n1 = 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0255] In another embodiment, the chain containing GalNAc is a conjugated group containing the formula (X'), wherein:

[0256] Q is independent of H,

[0257] Where L1 is a chemical bond, -CH2-, -CH2CH2-, -C(O)-, -CH2O-, -CH2O-CH2CH2O-, or -NHC(O)-(CH2NHC(O)). a -;

[0258] L2 is a chemical bond or -CH2CH2C(O)-;

[0259] L3 is a chemical bond, -(NHCH2CH2) b -、-(NHCH2CH2CH2) b -or-C(O)CH2-;

[0260] L4 is -(OCH2CH2) c -、-(OCH2CH2CH2) c -、-(OCH2CH2CH2CH2) c -、-(OCH2CH2CH2CH2CH2) c -or -NHC(O)-(CH2) d -;

[0261] Where a = 0, 1, 2 or 3;

[0262] b = 1, 2, 3, 4 or 5;

[0263] c = 1, 2, 3, 4 or 5;

[0264] d = 1, 2, 3, 4, 5, 6, 7 or 8;

[0265] A is a chemical bond, -CH2O- or -NHC(O)-;

[0266] A' is -O(CH2CH2O) e -;

[0267] Where e is 1, 2, 3, 4 or 5;

[0268] B is a chemical bond, -CH2-, -C(O)-, -M-, -CH2-M-, or -C(O)-M-;

[0269] Where M is

[0270] R1 and R2 together form -CH2CH2O- or -CH2CH(R)-O-, and R3 is H;

[0271] Or R1 and R3 together form -C 1-2 Alkylene-, and R2 is H;

[0272] Wherein R is -OR', -CH2OR' or -CH2CH2OR', where R' is H, a hydroxyl protecting group or a solid support, and the hydroxyl protecting group is preferably -C(O)CH2CH2C(O)OH or 4,4'-dimethoxytriphenylmethyl;

[0273] m1 = 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

[0274] n1 = 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0275] 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 X1 can be combined with any technical solution or any combination thereof of X2, X3, T, X, and L, etc. This invention aims to include combinations of all these technical solutions; due to space limitations, they will not be listed one by one.

[0276] 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.

[0277] 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.

[0278] 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.

[0279] 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.

[0280] This invention specifically relates to the following technical solutions:

[0281] A1. A compound of formula (I), or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof:

[0282]

[0283] in,

[0284] X1 is selected from OR a OP1, SP1 or NR b R c ;

[0285] R aSelected from H, C 1-6 Alkyl, C 1-6 Haloalkyl, C 2-6 alkenyl or C 2-6 The alkynyl group, which is optionally deuterated, up to and including complete deuteration;

[0286] R b and R c Independently selected from H and C 1-6 Alkyl or C 1-6 Halogenated alkyl, wherein R b and R c Can be optionally D, C 6-10 Aryl or 5-10 heteroaryl substitutions, up to complete deuteration;

[0287] X2 is OH, OP1, or SP1, or X2 is a nucleoside moiety that is attached to P via a hydroxyl or thiol group at the 2', 3', or 5' end of the ribose;

[0288] X3 is independently selected from O or S;

[0289] T is selected from

[0290] Each R T1 Independently selected from H, D, halogens, CN, C 1-6 Alkyl, C 1-6 Haloalkyl, C 1-6 Haloalkyl, C 2-6 alkenyl, C 2-6 Alkyne group or chain containing GalNAc, which is optionally deuterated up to fully deuterated;

[0291] Each R T2 Independently selected from H, D, halogens, CN, C 1-6 Alkyl, C 1-6 Haloalkyl, C 2-6 alkenyl or C 2-6 The alkynyl group, which is optionally deuterated, up to and including complete deuteration;

[0292] Each R T3 Independently selected from H, D, halogens, CN, C 1-6 Alkyl, C 1-6 Haloalkyl, C 2-6 alkenyl or C 2-6 The alkynyl group, which is optionally deuterated, up to and including complete deuteration;

[0293] Each R T4 Independently selected from H, D, halogens, CN, C 1-6 Alkyl, C 1-6 Haloalkyl, C 2-6 alkenyl or C 2-6The alkynyl group, which is optionally deuterated, up to and including complete deuteration;

[0294] m can be 0, 1, 2, 3, 4, or 5;

[0295] n is 0, 1, 2, 3, 4 or 5;

[0296] p is 0, 1, 2, 3, 4 or 5;

[0297] X is selected from chemical bonds, -O-, -S-, -C(O)-, -C(O)O-, -OC(O)-, -OC(O)NR X1 -、-NR X1 C(O)O-、-NR X1 C(O)- or -C(O)NR X1 -;

[0298] R X1 Selected from H, C 1-6 Alkyl or C 1-6 Halogenated alkyl groups, optionally deuterated, up to and including complete deuteration;

[0299] L stands for -Ar-(CH2) 1-6 -O-, where each CH2 group may optionally be replaced by R#, which is selected from H, D, halogens, CN, and C. 1-6 Alkyl, C 1-6 Haloalkyl, C 2-6 alkenyl or C 2-6 The alkynyl group, which is optionally deuterated, up to and including complete deuteration;

[0300] In L, Ar is bonded to X, and oxygen atom is bonded to phosphorus atom;

[0301] Ar is selected from C 3-10 Cycloalkyl, 3-10 membered heterocyclic groups, C 6-10 Aryl or 5-14 heteroaryl, wherein C 3-10 Cycloalkyl, 3-10 membered heterocyclic groups, C 6-10 The aryl or 5-10 heteroaryl group can be optionally replaced by 1, 2, 3, 4 or 5 R*.

[0302] R* is selected from H, D, halogens, CN, and C. 1-6 Alkyl, C 1-6 Haloalkyl, C 2-6 alkenyl or C 2-6 The alkynyl group, which is optionally deuterated, up to and including complete deuteration;

[0303] P1 is selected from the protecting base.

[0304] A2. The compound of formula (I) of technical solution A1, or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof, wherein,

[0305] X1 is selected from OR a OP1, SP1 or NR b R c ;

[0306] R a Selected from H, C 1-4 Alkyl or C 1-4 Halogenated alkyl groups, optionally deuterated, up to and including complete deuteration;

[0307] R b and R c Independently selected from H and C 1-4 Alkyl or C 1-4 Halogenated alkyl groups, and the R b and R c Can be optionally D, C 6-10 Aryl substitution, which is optionally deuterated, up to and including complete deuteration;

[0308] X2 is OH, OP1, or SP1, or X2 is a nucleoside moiety that is attached to P via a hydroxyl or thiol group at the 2', 3', or 5' end of the ribose;

[0309] X3 is independently selected from O or S;

[0310] T is selected from

[0311] Each R T1 Independently selected from H, D, and C 1-4 Alkyl, C 1-4 Halogenated alkyl groups or chains containing GalNAc, which are optionally deuterated up to fully deuterated;

[0312] Each R T2 Independently selected from H, D, and C 1-4 Alkyl or C 1-4 Halogenated alkyl groups, optionally deuterated, up to and including complete deuteration;

[0313] Each R T3 Independently selected from H, D, and C 1-4 Alkyl or C 1-4 Halogenated alkyl groups, optionally deuterated, up to and including complete deuteration;

[0314] Each R T4 Independently selected from H, D, and C 1-4 Alkyl or C 1-4 Halogenated alkyl groups, optionally deuterated, up to and including complete deuteration;

[0315] m can be 0, 1, 2, or 3;

[0316] n is 0, 1, 2, or 3;

[0317] p is 0, 1, 2 or 3;

[0318] X is selected from chemical bonds, -O-, -S-, and -OC(O)NR. X1 -、-NR X1 C(O)O-, -C(O)O-, -OC(O)-, -NR X1 C(O)- or -C(O)NR X1 -;

[0319] R X1 Selected from H, C 1-4 Alkyl or C 1-4 Halogenated alkyl groups, optionally deuterated, up to and including complete deuteration;

[0320] L stands for -Ar-(CH2) 1-4 -O-, where each CH2 may optionally be replaced by one or two R#, where R# is selected from H, D, C. 1-4 Alkyl or C 1-4 Halogenated alkyl groups, optionally deuterated, up to and including complete deuteration;

[0321] In L, Ar is bonded to X, and oxygen atom is bonded to phosphorus atom;

[0322] Ar is selected from C 6-10 Aryl or 5-10 heteroaryl, wherein C 6-10 The aryl or 5-10 heteroaryl group can be optionally replaced by one, two or three R*.

[0323] R* is selected from C 1-4 Alkyl or C 1-4 Halogenated alkyl groups, optionally deuterated, up to and including complete deuteration;

[0324] P1 is selected from the protecting base.

[0325] A3. A compound of formula (I) of technical solution A1 or A2, or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof, wherein,

[0326] X1 is selected from OR a OP1, SP1 or NR b R c ;

[0327] R a Selected from H, C 1-4 Alkyl or C 1-4 Halogenated alkyl groups, optionally deuterated, up to and including complete deuteration;

[0328] R b and R c Independently selected from H and C 1-4Alkyl, C 1-4 Halogenated alkyl, wherein R b and R c Optionally substituted with D or phenyl, up to complete deuteration;

[0329] X2 is OH, OP1, or SP1, or X2 is a nucleoside moiety that is attached to P via a hydroxyl or thiol group at the 2', 3', or 5' end of the ribose;

[0330] X3 is independently selected from O or S;

[0331] T is selected from Where R T Chains selected from H, D, CH3 or containing GalNAc, which are optionally deuterated up to and including complete deuteration;

[0332] X is selected from chemical bonds, -O-, -S-, -C(O)-, -C(O)O-, -OC(O)-, -OC(O)NR X1 -、-NR X1 C(O)O-、-NR X1 C(O)- or -C(O)NR X1 -;

[0333] R X1 Selected from H, C 1-4 Alkyl or C 1-4 Halogenated alkyl groups, optionally deuterated, up to and including complete deuteration;

[0334] L is selected from

[0335] P1 is selected from the protecting base.

[0336] A4. A compound of formula (I) from any one of technical solutions A1-A3, or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof, wherein,

[0337] X1 is selected from OH, OP1, SP1, or

[0338] X2 is OH, OP1, or SP1, or X2 is a nucleoside moiety that is attached to P via a hydroxyl or thiol group at the 2', 3', or 5' end of the ribose;

[0339] X3 is independently selected from O or S;

[0340] T is selected from

[0341] X is selected from chemical bonds, -O-, -NHC(O)O-, -OC(O)NH-, -N(CH3)C(O)O-, or -C(O)O-;

[0342] L is selected from

[0343] P1 is selected from the protecting base.

[0344] A5. A compound of formula (I) according to any one of technical solutions A1-A4, or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof, wherein the compound of formula (I) has the following structure:

[0345]

[0346] The groups are as defined in technical solutions A1-A4.

[0347] A6. The compound of formula (I) of technical solution A5, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, wherein the compound of formula (I) has the structure of formula (II):

[0348]

[0349] in,

[0350] X1 is selected from OR a or NR b R c ;

[0351] R a Selected from H, C 1-6 Alkyl or C 1-6 Halogenated alkyl groups, optionally deuterated, up to and including complete deuteration;

[0352] R b and R c Selected from H, C 1-6 Alkyl or C 1-6 Halogenated alkyl, wherein R b and R c Can be optionally D, C 6-10 Aryl or 5-10 heteroaryl substitutions, up to complete deuteration;

[0353] X2 is OH, OP1, or SP1, or X2 is a nucleoside moiety that is attached to P via a hydroxyl or thiol group at the 2', 3', or 5' end of the ribose;

[0354] X3 is independently selected from O or S;

[0355] Each R T1 Independently selected from H, D, and C 1-6 Alkyl, C 1-6 Haloalkyl, C 2-6 alkenyl, C 2-6Alkyne group or chain containing GalNAc, which is optionally deuterated up to fully deuterated;

[0356] Each R T2 Independently selected from H, D, and C 1-6 Alkyl, C 1-6 Haloalkyl, C 2-6 alkenyl or C 2-6 The alkynyl group, which is optionally deuterated, up to and including complete deuteration;

[0357] m can be 0, 1, 2, 3, 4, or 5;

[0358] X is selected from chemical bonds, -O-, -S-, -C(O)-, -C(O)O-, -OC(O)-, -OC(O)NR X1 -、-NR X1 C(O)O-、-NR X1 C(O)- or -C(O)NR X1 -;

[0359] R X1 Selected from H, C 1-6 Alkyl or C 1-6 Halogenated alkyl groups, optionally deuterated, up to and including complete deuteration;

[0360] L stands for -Ar-(CH2) 1-6 -O-, where each CH2 may optionally be replaced by one or two R#, where R# is selected from H, D, C. 1-6 Alkyl or C 1-6 Halogenated alkyl groups, optionally deuterated, up to and including complete deuteration;

[0361] In L, Ar is bonded to X, and oxygen atom is bonded to phosphorus atom;

[0362] Ar is selected from C 6-10 Aryl or 5-10 heteroaryl, wherein C 6-10 The aryl or 5-10 heteroaryl group can be optionally replaced by 1, 2, 3, 4 or 5 R*.

[0363] R* is selected from C 1-6 Alkyl, C 1-6 Haloalkyl, C 2-6 alkenyl or C 2-6 The alkynyl group, which is optionally deuterated, up to and including complete deuteration;

[0364] P1 is selected from the protecting base.

[0365] A7. A compound of formula (I) of technical solution A6, or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof, wherein,

[0366] X1 is selected from ORa or NR b R c ;

[0367] R a Selected from H, C 1-4 Alkyl or C 1-4 Halogenated alkyl groups, optionally deuterated, up to and including complete deuteration;

[0368] R b and R c Independently selected from H and C 1-4 Alkyl, C 1-4 Halogenated alkyl, wherein R b and R c Can be optionally D, C 6-10 Aryl substitution, up to complete deuteration;

[0369] X2 is OH, OP1, or SP1, or X2 is a nucleoside moiety that is attached to P via a hydroxyl or thiol group at the 2', 3', or 5' end of the ribose;

[0370] X3 is independently selected from O or S;

[0371] Each R T1 Independently selected from H, D, and C 1-4 Alkyl, C 1-4 Halogenated alkyl groups or chains containing GalNAc, which are optionally deuterated up to fully deuterated;

[0372] Each R T2 Independently selected from H, D, and C 1-4 Alkyl or C 1-4 Halogenated alkyl groups, optionally deuterated, up to and including complete deuteration;

[0373] m can be 0, 1, 2, or 3;

[0374] X is selected from chemical bonds, -O-, -S-, -C(O)-, -C(O)O-, -OC(O)-, -OC(O)NR X1 -、-NR X1 C(O)O-、-NR X1 C(O)- or -C(O)NR X1 -;

[0375] R X1 Selected from H, C 1-4 Alkyl or C 1-4 Halogenated alkyl groups, optionally deuterated, up to and including complete deuteration;

[0376] L stands for -Ar-(CH2) 1-4 -O-, where each CH2 may optionally be replaced by one or two R#, where R# is selected from H, D, C.1-4 Alkyl or C 1-4 Halogenated alkyl groups, optionally deuterated, up to and including complete deuteration;

[0377] In L, Ar is bonded to X, and oxygen atom is bonded to phosphorus atom;

[0378] Ar is selected from C 6-10 Aryl or 5-10 heteroaryl, wherein C 6-10 The aryl or 5-10 heteroaryl group can be optionally replaced by one, two or three R*.

[0379] R* is selected from C 1-4 Alkyl or C 1-4 Halogenated alkyl groups, optionally deuterated, up to and including complete deuteration;

[0380] P1 is selected from the protecting base.

[0381] A8. A compound of formula (I) of technical solution A6 or A7, or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof, wherein,

[0382] X1 is selected from OR a or NR b R c ;

[0383] R a Selected from H, C 1-4 Alkyl or C 1-4 Halogenated alkyl groups, optionally deuterated, up to and including complete deuteration;

[0384] R b and R c Independently selected from H and C 1-4 Alkyl, C 1-4 Halogenated alkyl, wherein R b and R c Optionally substituted with D or phenyl, up to complete deuteration;

[0385] X2 is OH, OP1, or SP1, or X2 is a nucleoside moiety that is attached to P via a hydroxyl or thiol group at the 2', 3', or 5' end of the ribose;

[0386] X3 is independently selected from O or S;

[0387] Selected from

[0388] Where R T Chains selected from H, D, CH3 or containing GalNAc, which are optionally deuterated up to and including complete deuteration;

[0389] X is selected from chemical bonds, -O-, -S-, -C(O)-, -C(O)O-, -OC(O)-, -OC(O)NR X1 -、-NR X1 C(O)O-、-NR X1 C(O)- or -C(O)NR X1 -;

[0390] R X1 Selected from H, C 1-4 Alkyl or C 1-4 Halogenated alkyl groups, optionally deuterated, up to and including complete deuteration;

[0391] L is selected from

[0392] P1 is selected from the protecting base.

[0393] A9. A compound of formula (I) from any one of technical solutions A6-A8, or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof, wherein,

[0394] X1 is selected from OH or

[0395] X2 is OH, OP1, or SP1, or X2 is a nucleoside moiety that is attached to P via a hydroxyl or thiol group at the 2', 3', or 5' end of the ribose;

[0396] X3 is independently selected from O or S;

[0397] Selected from

[0398] X is selected from chemical bonds, -O-, -NHC(O)O-, -OC(O)NH-, -N(CH3)C(O)O-, or -C(O)O-;

[0399] L is selected from

[0400] P1 is selected from the protecting base.

[0401] A10. The compound of formula (I) of technical solution A5, or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof, wherein the compound of formula (I) has a structure of formula (III) or (IV):

[0402]

[0403] in,

[0404] X1 is selected from OR aOP1, SP1 or NR b R c ;

[0405] R a Selected from H, C 1-6 Alkyl or C 1-6 Halogenated alkyl groups, optionally deuterated, up to and including complete deuteration;

[0406] R b and R c Selected from H, C 1-6 Alkyl or C 1-6 Halogenated alkyl, wherein R b and R c Can be optionally D, C 6-10 Aryl or 5-10 heteroaryl substitutions, up to complete deuteration;

[0407] X2 is OH, OP1, or SP1, or X2 is a nucleoside moiety that is attached to P via a hydroxyl or thiol group at the 2', 3', or 5' end of the ribose;

[0408] X3 is independently selected from O or S;

[0409] Each R T3 Independently selected from H, D, and C 1-6 Alkyl, C 1-6 Haloalkyl, C 2-6 alkenyl or C 2-6 The alkynyl group, which is optionally deuterated, up to and including complete deuteration;

[0410] Each R T4 Independently selected from H, D, and C 1-6 Alkyl, C 1-6 Haloalkyl, C 2-6 alkenyl or C 2-6 The alkynyl group, which is optionally deuterated, up to and including complete deuteration;

[0411] n is 0, 1, 2, 3, 4 or 5;

[0412] p is 0, 1, 2, 3, 4 or 5;

[0413] X is selected from chemical bonds, -O-, -S-, -C(O)-, -C(O)O-, -OC(O)-, -OC(O)NR X1 -、-NR X1 C(O)O-、-NR X1 C(O)- or -C(O)NR X1 -;

[0414] R X1 Selected from H, C 1-6 Alkyl or C 1-6Halogenated alkyl groups, optionally deuterated, up to and including complete deuteration;

[0415] L stands for -Ar-(CH2) 1-6 -O-, where each CH2 may optionally be replaced by one or two R#, where R# is selected from H, D, C. 1-6 Alkyl or C 1-6 Halogenated alkyl groups, optionally deuterated, up to and including complete deuteration;

[0416] In L, Ar is bonded to X, and oxygen atom is bonded to phosphorus atom;

[0417] Ar is selected from C 6-10 Aryl or 5-10 heteroaryl, wherein C 6-10 The aryl or 5-10 heteroaryl group can be optionally replaced by one, two or three R*.

[0418] R* is selected from C 1-6 Alkyl, C 1-6 Haloalkyl, C 2-6 alkenyl or C 2-6 The alkynyl group, which is optionally deuterated, up to and including complete deuteration;

[0419] P1 is selected from the protecting base.

[0420] A11. The compound of formula (I) of technical solution A10, or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof, wherein,

[0421] X1 is selected from OR a OP1, SP1 or NR b R c ;

[0422] R a Selected from H, C 1-4 Alkyl or C 1-4 Halogenated alkyl groups, optionally deuterated, up to and including complete deuteration;

[0423] R b and R c Independently selected from H and C 1-4 Alkyl, C 1-4 Halogenated alkyl, wherein R b and R c Can be optionally D, C 6-10 Aryl substitution, up to complete deuteration;

[0424] X2 is OH, OP1, or SP1, or X2 is a nucleoside moiety that is attached to P via a hydroxyl or thiol group at the 2', 3', or 5' end of the ribose;

[0425] X3 is independently selected from O or S;

[0426] Each R T3 Independently selected from H, D, and C 1-6 Alkyl or C 1-6 Halogenated alkyl groups, optionally deuterated, up to and including complete deuteration;

[0427] Each R T4 Independently selected from H, D, and C 1-6 Alkyl or C 1-6 Halogenated alkyl groups, optionally deuterated, up to and including complete deuteration;

[0428] n is 0, 1, 2, or 3;

[0429] p is 0, 1, 2 or 3;

[0430] X is selected from chemical bonds, -O-, -S-, -C(O)-, -C(O)O-, -OC(O)-, -OC(O)NR X1 -、-NR X1 C(O)O-、-NR X1 C(O)- or -C(O)NR X1 -;

[0431] R X1 Selected from H, C 1-4 Alkyl or C 1-4 Halogenated alkyl groups, optionally deuterated, up to and including complete deuteration;

[0432] L stands for -Ar-(CH2) 1-4 -O-, where each CH2 may optionally be replaced by one or two R#, where R# is selected from H, D, C. 1-4 Alkyl or C 1-4 Halogenated alkyl groups, optionally deuterated, up to and including complete deuteration;

[0433] In L, Ar is bonded to X, and oxygen atom is bonded to phosphorus atom;

[0434] Ar is selected from C 6-10 Aryl or 5-10 heteroaryl, wherein C 6-10 The aryl or 5-10 heteroaryl group can be optionally replaced by one, two or three R*.

[0435] R* is selected from C 1-4 Alkyl or C 1-4 Halogenated alkyl groups, optionally deuterated, up to and including complete deuteration;

[0436] P1 is selected from the protecting base.

[0437] A12. A compound of formula (I) of technical solution A10 or A11, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, wherein the compound of formula (I) has the structure of formula (III-1) or (IV-1).

[0438]

[0439] in,

[0440] X1 is selected from OR a OP1, SP1 or NR b R c ;

[0441] R a Selected from H, C 1-4 Alkyl or C 1-4 Halogenated alkyl groups, optionally deuterated, up to and including complete deuteration;

[0442] R b and R c Independently selected from H and C 1-4 Alkyl, C 1-4 Halogenated alkyl, wherein R b and R c Optionally substituted with D or phenyl, up to complete deuteration;

[0443] X2 is OH, OP1, or SP1, or X2 is a nucleoside moiety that is attached to P via a hydroxyl or thiol group at the 2', 3', or 5' end of the ribose;

[0444] X3 is independently selected from O or S;

[0445] Each R T3 Independently selected from H, D, and C 1-4 Alkyl or C 1-4 Halogenated alkyl groups, optionally deuterated, up to and including complete deuteration;

[0446] Each R T4 Independently selected from H, D, and C 1-4 Alkyl or C 1-4 Halogenated alkyl groups, optionally deuterated, up to and including complete deuteration;

[0447] n is 0, 1, 2, or 3;

[0448] p is 0, 1, 2 or 3;

[0449] X is selected from chemical bonds, -O-, -S-, -C(O)-, -C(O)O-, -OC(O)-, -OC(O)NR X1 -、-NR X1 C(O)O-、-NR X1C(O)- or -C(O)NR X1 -;

[0450] R X1 Selected from H, C 1-4 Alkyl or C 1-4 Halogenated alkyl groups, optionally deuterated, up to and including complete deuteration;

[0451] L is selected from

[0452] P1 is selected from the protecting base.

[0453] A13. The compound of formula (I) of technical solution A12, or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof, wherein,

[0454] X1 is selected from OH, OP1, SP1, or

[0455] X2 is OH, OP1, or SP1, or X2 is a nucleoside moiety that is attached to P via a hydroxyl or thiol group at the 2', 3', or 5' end of the ribose;

[0456] X3 is independently selected from O or S;

[0457] R T3 Selected from H, D or CH3, which are optionally deuterated, up to and including complete deuteration;

[0458] R T4 For H;

[0459] n is 3;

[0460] p is 0;

[0461] X is selected from chemical bonds, -O-, -NHC(O)O-, -OC(O)NH-, -N(CH3)C(O)O-, or -C(O)O-;

[0462] L is selected from

[0463] P1 is selected from the protecting base.

[0464] A14. A compound of formula (I) from any one of technical solutions A1-A13, or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof, wherein the compound of formula (I) is selected from:

[0465]

[0466]

[0467]

[0468]

[0469] in,

[0470] X1 is selected from OH, OP1, or SP1 or Preferably OH or

[0471] X2 is an OH, OP1, or SP1 chemical bond, or a hydroxyl or thiol group attached to the 2' or 3' end of the ribose of another nucleotide, nucleoside, or oligonucleotide.

[0472] X3 is independently selected from O or S;

[0473] P1 is selected from a protecting group;

[0474] Preferably, the compound of formula (I) is selected from:

[0475]

[0476] X2 is the nucleoside moiety, which is linked to P via a hydroxyl group at the 2', 3', or 5' end of the ribose.

[0477] A15. A compound of formula (I) of any one of technical solutions A1-A14, or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof, wherein P1 is selected from 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), benzyloxycarbonyl (Cbz), tert-butyloxycarbonyl (Boc), benzyl (Bn), p-methoxybenzyl (PMB), allyl, triphenylmethyl (Tr), bis-p-methoxytriphenylmethyl (DMTr), methoxymethyl (MOM), phenoxymethyl (BOM), 2,2,2-trichloroethoxymethyl, 2-methoxyethoxymethyl (MEM), methylthiomethyl (MTM), p-methoxybenzyloxymethyl (PMBM), 4,4'-dimethoxytriphenylmethyl, -P(OCH2CH2CN)(N(iPr)2) or -C(O)CH2CH2C(O)OH, preferably -P(OCH2CH2CN)(N(iPr)2) or -C(O)CH2CH2C(O)OH.

[0478] A16. Oligonucleotides comprising one, two or more compounds of formula (Ia), or pharmaceutically acceptable salts, tautomers or stereoisomers thereof:

[0479]

[0480] in,

[0481] OR a or NR b R c Or a chemical bond that connects to the 5' end hydroxyl or thiol group of the ribose of an adjacent nucleotide;

[0482] X1 is selected from OR a or NR b R c Or a chemical bond linked to a hydroxyl or thiol group at the 2' or 3' end of the ribose of another adjacent nucleotide;

[0483] and Unlike X1, OR is not used at the same time. a or NR b R c ;

[0484] R a Selected from H, C 1-6 Alkyl, C 1-6 Haloalkyl, C 2-6 alkenyl or C 2-6 The alkynyl group, which is optionally deuterated, up to and including complete deuteration;

[0485] R b and R c Independently selected from H and C 1-6 Alkyl or C 1-6 Halogenated alkyl, wherein R b and R c Can be optionally D, C 6-10 Aryl or 5-10 heteroaryl substitutions, up to complete deuteration;

[0486] X3 is independently selected from O or S;

[0487] T is selected from

[0488] Each R T1 Independently selected from H, D, halogens, CN, C 1-6 Alkyl, C 1-6 Haloalkyl, C 1-6 Haloalkyl, C 2-6 alkenyl, C 2-6 Alkyne group or chain containing GalNAc, which is optionally deuterated up to fully deuterated;

[0489] Each R T2 Independently selected from H, D, halogens, CN, C 1-6 Alkyl, C 1-6 Haloalkyl, C 2-6 alkenyl or C 2-6 The alkynyl group, which is optionally deuterated, up to and including complete deuteration;

[0490] Each R T3 Independently selected from H, D, halogens, CN, C 1-6 Alkyl, C 1-6 Haloalkyl, C 2-6 alkenyl or C 2-6 The alkynyl group, which is optionally deuterated, up to and including complete deuteration;

[0491] Each R T4 Independently selected from H, D, halogens, CN, C 1-6 Alkyl, C 1-6 Haloalkyl, C 2-6 alkenyl or C 2-6 The alkynyl group, which is optionally deuterated, up to and including complete deuteration;

[0492] m can be 0, 1, 2, 3, 4, or 5;

[0493] n is 0, 1, 2, 3, 4 or 5;

[0494] p is 0, 1, 2, 3, 4 or 5;

[0495] X is selected from chemical bonds, -O-, -S-, -C(O)-, -C(O)O-, -OC(O)-, -OC(O)NR X1 -、-NR X1 C(O)O-、-NR X1 C(O)- or -C(O)NR X1 -;

[0496] R X1 Selected from H, halogens, C 1-6 Alkyl or C 1-6 Halogenated alkyl groups, optionally deuterated, up to and including complete deuteration;

[0497] L stands for -Ar-(CH2) 1-6 -O-, where each CH2 group may optionally be replaced by R#, which is selected from H, D, halogens, CN, and C. 1-6 Alkyl, C 1-6 Haloalkyl, C 2-6 alkenyl or C 2-6 The alkynyl group, which is optionally deuterated, up to and including complete deuteration;

[0498] In L, Ar is bonded to X, and oxygen atom is bonded to phosphorus atom;

[0499] Ar is selected from C 3-10 Cycloalkyl, 3-10 membered heterocyclic groups, C 6-10 Aryl or 5-14 heteroaryl, wherein C 3-10 Cycloalkyl, 3-10 membered heterocyclic groups, C 6-10 The aryl or 5-10 heteroaryl group can be optionally replaced by 1, 2, 3, 4 or 5 R*.

[0500] R* is selected from H, D, halogens, CN, and C. 1-6 Alkyl, C 1-6 Haloalkyl, C 2-6 alkenyl or C 2-6 The alkynyl group is optionally deuterated, up to and including complete deuteration.

[0501] A17. The oligonucleotide in technical solution A16, wherein,

[0502] A chemical bond that connects to the 5' end hydroxyl or thiol group of the ribose of an adjacent nucleotide;

[0503] X1 is selected from OR a or NR b R c Or a chemical bond linked to a hydroxyl or thiol group at the 2' or 3' end of the ribose of another adjacent nucleotide;

[0504] R a Selected from H, C 1-4 Alkyl or C 1-4 Halogenated alkyl groups, optionally deuterated, up to and including complete deuteration;

[0505] R b and R c Independently selected from H and C 1-4 Alkyl or C 1-4 Halogenated alkyl groups, and the R b and R c Can be optionally D, C 6-10 Aryl substitution, which is optionally deuterated, up to and including complete deuteration;

[0506] X2 and X3 are independently selected from O or S;

[0507] T is selected from

[0508] Each R T1 Independently selected from H, D, and C 1-4 Alkyl, C 1-4 Halogenated alkyl groups or chains containing GalNAc, which are optionally deuterated up to fully deuterated;

[0509] Each R T2 Independently selected from H, D, and C 1-4 Alkyl or C 1-4 Halogenated alkyl groups, optionally deuterated, up to and including complete deuteration;

[0510] Each R T3 Independently selected from H, D, and C 1-4 Alkyl or C 1-4 Halogenated alkyl groups, optionally deuterated, up to and including complete deuteration;

[0511] Each R T4 Independently selected from H, D, and C 1-4 Alkyl or C 1-4 Halogenated alkyl groups, optionally deuterated, up to and including complete deuteration;

[0512] m can be 0, 1, 2, or 3;

[0513] n is 0, 1, 2, or 3;

[0514] p is 0, 1, 2 or 3;

[0515] X is selected from chemical bonds, -O-, -S-, and -OC(O)NR. X1 -、-NR X1 C(O)O-, -C(O)O-, -OC(O)-, -NR X1 C(O)- or -C(O)NR X1 -;

[0516] R X1 Selected from H, C 1-4 Alkyl or C 1-4 Halogenated alkyl groups, optionally deuterated, up to and including complete deuteration;

[0517] L stands for -Ar-(CH2) 1-4 -O-, where each CH2 may optionally be replaced by one or two R#, where R# is selected from H, D, C. 1-4 Alkyl or C 1-4 Halogenated alkyl groups, optionally deuterated, up to and including complete deuteration;

[0518] In L, Ar is bonded to X, and oxygen atom is bonded to phosphorus atom;

[0519] Ar is selected from C 6-10 Aryl or 5-10 heteroaryl, wherein C 6-10 The aryl or 5-10 heteroaryl group can be optionally replaced by one, two or three R*.

[0520] R* is selected from C 1-4 Alkyl or C 1-4Halogenated alkyl groups, optionally deuterated, up to and including complete deuteration.

[0521] A18. The oligonucleotide of technical solution A16 or A17, wherein,

[0522] A chemical bond that connects to the 5' end hydroxyl or thiol group of the ribose of an adjacent nucleotide;

[0523] X1 is selected from OR a or NR b R c Or a chemical bond linked to a hydroxyl or thiol group at the 2' or 3' end of the ribose of another adjacent nucleotide;

[0524] R a Selected from H, C 1-4 Alkyl or C 1-4 Halogenated alkyl groups, optionally deuterated, up to and including complete deuteration;

[0525] R b and R c Independently selected from H and C 1-4 Alkyl or C 1-4 Halogenated alkyl, wherein R b and R c Optionally substituted with D or phenyl, up to complete deuteration;

[0526] X3 is independently selected from O or S;

[0527] T is selected from Where R T Chains selected from H, D, CH3 or containing GalNAc, which are optionally deuterated up to and including complete deuteration;

[0528] X is selected from chemical bonds, -O-, -S-, -C(O)-, -C(O)O-, -OC(O)-, -OC(O)NR X1 -、-NR X1 C(O)O-、-NR X1 C(O)- or -C(O)NR X1 -;

[0529] R X1 Selected from H, C 1-4 Alkyl or C 1-4 Halogenated alkyl groups, optionally deuterated, up to and including complete deuteration;

[0530] L is selected from

[0531] A19. An oligonucleotide from any one of technical solutions A16-A18, wherein,

[0532] A chemical bond that connects to the 5' end hydroxyl or thiol group of the ribose of an adjacent nucleotide;

[0533] X1 is selected from OH or Or a chemical bond linked to a hydroxyl or thiol group at the 2' or 3' end of the ribose of another adjacent nucleotide;

[0534] X3 is selected from O or S;

[0535] T is selected from

[0536] X is selected from chemical bonds, -O-, -NHC(O)O-, -OC(O)NH-, -N(CH3)C(O)O-, or -C(O)O-;

[0537] L is selected from

[0538] A20. An oligonucleotide of any one of technical solutions A16-A19, wherein the compound of formula (Ia) is selected from the following structures:

[0539]

[0540] The groups are as defined in technical solutions 16-19.

[0541] A21. The oligonucleotide of technical solution A20, wherein the compound of formula (Ia) has the structure of formula (IIa):

[0542]

[0543] in,

[0544] A chemical bond that connects to the 5' end hydroxyl or thiol group of the ribose of an adjacent nucleotide;

[0545] X1 is selected from OR a or NR b R c ;

[0546] R a Selected from H, C 1-6 Alkyl or C 1-6 Halogenated alkyl groups, optionally deuterated, up to and including complete deuteration;

[0547] R b and R c Selected from H, C 1-6 Alkyl or C 1-6 Halogenated alkyl, wherein R b and R cCan be optionally D, C 6-10 Aryl or 5-10 heteroaryl substitutions, up to complete deuteration;

[0548] X3 is independently selected from O or S;

[0549] Each R T1 Independently selected from H, D, and C 1-6 Alkyl, C 1-6 Haloalkyl, C 2-6 alkenyl, C 2-6 Alkyne group or chain containing GalNAc, which is optionally deuterated up to fully deuterated;

[0550] Each R T2 Independently selected from H, D, and C 1-6 Alkyl, C 1-6 Haloalkyl, C 2-6 alkenyl or C 2-6 The alkynyl group, which is optionally deuterated, up to and including complete deuteration;

[0551] m can be 0, 1, 2, or 3;

[0552] X is selected from chemical bonds, -O-, -S-, -C(O)-, -C(O)O-, -OC(O)-, -OC(O)NR X1 -、-NR X1 C(O)O-、-NR X1 C(O)- or -C(O)NR X1 -;

[0553] R X1 Selected from H, C 1-6 Alkyl or C 1-6 Halogenated alkyl groups, optionally deuterated, up to and including complete deuteration;

[0554] L stands for -Ar-(CH2) 1-6 -O-, where each CH2 may optionally be replaced by one or two R#, where R# is selected from H, D, C. 1-6 Alkyl or C 1-6 Halogenated alkyl groups, optionally deuterated, up to and including complete deuteration;

[0555] In L, Ar is bonded to X, and oxygen atom is bonded to phosphorus atom;

[0556] Ar is selected from C 6-10 Aryl or 5-10 heteroaryl, wherein C 6-10 The aryl or 5-10 heteroaryl group can be optionally replaced by one, two or three R*.

[0557] R* is selected from C 1-6 Alkyl, C 1-6 Haloalkyl, C2-6 alkenyl or C 2-6 The alkynyl group is optionally deuterated, up to and including complete deuteration.

[0558] A22. The oligonucleotide in technical solution A21, wherein,

[0559] A chemical bond that connects to the 5' end hydroxyl or thiol group of the ribose of an adjacent nucleotide;

[0560] X1 is selected from OR a or NR b R c ;

[0561] R a Selected from H, C 1-4 Alkyl or C 1-4 Halogenated alkyl groups, optionally deuterated, up to and including complete deuteration;

[0562] R b and R c Independently selected from H and C 1-4 Alkyl, C 1-4 Halogenated alkyl, wherein R b and R c Can be optionally D, C 6-10 Aryl substitution, up to complete deuteration;

[0563] X3 is independently selected from O or S;

[0564] Each R T1 Independently selected from H, D, and C 1-4 Alkyl, C 1-4 Halogenated alkyl groups or chains containing GalNAc, which are optionally deuterated up to fully deuterated;

[0565] Each R T2 Independently selected from H, D, and C 1-4 Alkyl or C 1-4 Halogenated alkyl groups, optionally deuterated, up to and including complete deuteration;

[0566] m is 0, 1, or 2;

[0567] X is selected from chemical bonds, -O-, -S-, -C(O)-, -C(O)O-, -OC(O)-, -OC(O)NR X1 -、-NR X1 C(O)O-、-NR X1 C(O)- or -C(O)NR X1 -;

[0568] R X1 Selected from H, C 1-4 Alkyl or C1-4 Halogenated alkyl groups, optionally deuterated, up to and including complete deuteration;

[0569] L stands for -Ar-(CH2) 1-4 -O-, where each CH2 may optionally be replaced by one or two R#, where R# is selected from H, D, C. 1-4 Alkyl or C 1-4 Halogenated alkyl groups, optionally deuterated, up to and including complete deuteration;

[0570] In L, Ar is bonded to X, and oxygen atom is bonded to phosphorus atom;

[0571] Ar is selected from C 6-10 Aryl or 5-10 heteroaryl, wherein C 6-10 The aryl or 5-10 heteroaryl group can be optionally replaced by one, two or three R*.

[0572] R* is selected from C 1-4 Alkyl or C 1-4 Halogenated alkyl groups, optionally deuterated, up to and including complete deuteration.

[0573] A23. The oligonucleotide of technical solution A21 or A22, wherein,

[0574] A chemical bond that connects to the 5' end hydroxyl or thiol group of the ribose of an adjacent nucleotide;

[0575] X1 is selected from OR a or NR b R c ;

[0576] R a Selected from H, C 1-4 Alkyl or C 1-4 Halogenated alkyl groups, optionally deuterated, up to and including complete deuteration;

[0577] R b and R c Independently selected from H and C 1-4 Alkyl, C 1-4 Halogenated alkyl, wherein R b and R c Optionally substituted with D or phenyl, which may be deuterated until fully deuterated;

[0578] X3 is independently selected from O or S;

[0579] Selected from

[0580] Where R TChains selected from H, D, CH3 or containing GalNAc, which are optionally deuterated up to and including complete deuteration;

[0581] X is selected from chemical bonds, -O-, -S-, -C(O)-, -C(O)O-, -OC(O)-, -OC(O)NR X1 -、-NR X1 C(O)O-、-NR X1 C(O)- or -C(O)NR X1 -;

[0582] R X1 Selected from H, C 1-4 Alkyl or C 1-4 Halogenated alkyl groups, optionally deuterated, up to and including complete deuteration;

[0583] L is selected from

[0584] A24. An oligonucleotide from any one of technical solutions A21-A23, wherein,

[0585] A chemical bond that connects to the 5' end hydroxyl or thiol group of the ribose of an adjacent nucleotide;

[0586] X1 is selected from OH or

[0587] X3 is selected from O or S;

[0588] Selected from

[0589] X is selected from chemical bonds, -O-, -NHC(O)O-, -OC(O)NH-, -N(CH3)C(O)O-, or -C(O)O-;

[0590] L is selected from

[0591] A25. An oligonucleotide of any one of technical solutions A16-A24, wherein the compound of formula (Ia) is selected from the following structures:

[0592]

[0593]

[0594] in,

[0595] This represents a chemical bond connecting the ribose of an adjacent nucleotide to a hydroxyl or thiol group at the 5' end; X1 is selected from OH or...

[0596] X3 is selected from O or S;

[0597] Preferably, the compound of formula (Ia) is selected from:

[0598]

[0599]

[0600] This represents a chemical bond that connects to the 5' end hydroxyl group of the ribose of an adjacent nucleotide.

[0601] A26. The oligonucleotide of technical solution A20, wherein the compound of formula (Ia) has the structure of formula (IIIa) or (IVa):

[0602]

[0603] in,

[0604] Selected from OR a or NR b R c , or it represents a chemical bond that connects to the 5' end hydroxyl or thiol group of the ribose of an adjacent nucleotide;

[0605] X1 is selected from OR a or NR b R c Or a chemical bond linked to a hydroxyl or thiol group at the 2' or 3' end of the ribose of another adjacent nucleotide;

[0606] and Unlike X1, OR is not used at the same time. a or NR b R c ;

[0607] R a Selected from H, C 1-6 Alkyl or C 1-6 Halogenated alkyl groups, optionally deuterated, up to and including complete deuteration;

[0608] R b and R c Selected from H, C 1-6 Alkyl or C 1-6 Halogenated alkyl, wherein R b and R c Can be optionally D, C 6-10 Aryl or 5-10 heteroaryl substitutions, up to complete deuteration;

[0609] X3 is independently selected from O or S;

[0610] Each R T3 Independently selected from H, D, and C 1-6 Alkyl, C 1-6 Haloalkyl, C 2-6 alkenyl or C 2-6 The alkynyl group, which is optionally deuterated, up to and including complete deuteration;

[0611] Each R T4 Independently selected from H, D, and C 1-6 Alkyl, C 1-6 Haloalkyl, C 2-6 alkenyl or C 2-6 The alkynyl group, which is optionally deuterated, up to and including complete deuteration;

[0612] n is 0, 1, 2, 3, 4 or 5;

[0613] p is 0, 1, 2, 3, 4 or 5;

[0614] X is selected from chemical bonds, -O-, -S-, -C(O)-, -C(O)O-, -OC(O)-, -OC(O)NR X1 -、-NR X1 C(O)O-、-NR X1 C(O)- or -C(O)NR X1 -;

[0615] R X1 Selected from H, C 1-6 Alkyl or C 1-6 Halogenated alkyl groups, optionally deuterated, up to and including complete deuteration;

[0616] L stands for -Ar-(CH2) 1-6 -O-, where each CH2 may optionally be replaced by one or two R#, where R# is selected from H, D, C. 1-6 Alkyl or C 1-6 Halogenated alkyl groups, optionally deuterated, up to and including complete deuteration;

[0617] In L, Ar is bonded to X, and oxygen atom is bonded to phosphorus atom;

[0618] Ar is selected from C 6-10 Aryl or 5-10 heteroaryl, wherein C 6-10 The aryl or 5-10 heteroaryl group can be optionally replaced by one, two or three R*.

[0619] R* is selected from C 1-6 Alkyl, C 1-6 Haloalkyl, C 2-6 alkenyl or C 2-6 The alkynyl group is optionally deuterated, up to and including complete deuteration.

[0620] A27. The oligonucleotide in technical solution A26, wherein,

[0621] Selected from OR a or NR b R c , or it represents a chemical bond that connects to the 5' end hydroxyl or thiol group of the ribose of an adjacent nucleotide;

[0622] X1 is selected from OR a or NR b R c Or a chemical bond linked to a hydroxyl or thiol group at the 2' or 3' end of the ribose of another adjacent nucleotide;

[0623] and Unlike X1, OR is not used at the same time. a or NR b R c ;

[0624] R a Selected from H, C 1-4 Alkyl or C 1-4 Halogenated alkyl groups;

[0625] R b and R c Independently selected from H and C 1-4 Alkyl, C 1-4 Halogenated alkyl, wherein R b and R c Can be optionally C 6-10 Aryl substitution;

[0626] X2 and X3 are independently selected from O or S;

[0627] Each R T3 Independently selected from H and C 1-6 Alkyl or C 1-6 Halogenated alkyl groups;

[0628] Each R T4 Independently selected from H and C 1-6 Alkyl or C 1-6 Halogenated alkyl groups;

[0629] n is 0, 1, 2, or 3;

[0630] p is 0, 1, 2 or 3;

[0631] X is selected from chemical bonds, -O-, -S-, -C(O)-, -C(O)O-, -OC(O)-, -OC(O)NR X1 -、-NR X1 C(O)O-、-NR X1C(O)- or -C(O)NR X1 -;

[0632] R X1 Selected from H, C 1-4 Alkyl or C 1-4 Halogenated alkyl groups;

[0633] L stands for -Ar-(CH2) 1-4 -O-, where each CH2 may optionally be replaced by one or two R#, where R# is selected from H, C 1-4 Alkyl or C 1-4 Halogenated alkyl groups;

[0634] In L, Ar is bonded to X, and oxygen atom is bonded to phosphorus atom;

[0635] Ar is selected from C 6-10 Aryl or 5-10 heteroaryl, wherein C 6-10 The aryl or 5-10 heteroaryl group can be optionally replaced by one, two or three R*.

[0636] R* is selected from C 1-4 Alkyl or C 1-4 Halogenated alkyl groups.

[0637] A28. The oligonucleotide of technical solution A26 or A27, wherein the compound of formula (Ia) has a (III-1a) or (IV-1a) structure:

[0638]

[0639] in,

[0640] Selected from OR a or NR b R c , or it represents a chemical bond that connects to the 5' end hydroxyl or thiol group of the ribose of an adjacent nucleotide;

[0641] X1 is selected from OR a or NR b R c Or a chemical bond linked to a hydroxyl or thiol group at the 2' or 3' end of the ribose of another adjacent nucleotide;

[0642] and Unlike X1, OR is not used at the same time. a or NR b R c ;

[0643] R a Selected from H, C 1-4 Alkyl or C 1-4Halogenated alkyl groups, optionally deuterated, up to and including complete deuteration;

[0644] R b and R c Independently selected from H and C 1-4 Alkyl, C 1-4 Halogenated alkyl, wherein R b and R c Optionally substituted with D or phenyl, up to complete deuteration;

[0645] X3 is independently selected from O or S;

[0646] Each R T3 Independently selected from H, D, and C 1-4 Alkyl or C 1-4 Halogenated alkyl groups, optionally deuterated, up to and including complete deuteration;

[0647] Each R T4 Independently selected from H, D, and C 1-4 Alkyl or C 1-4 Halogenated alkyl groups, optionally deuterated, up to and including complete deuteration;

[0648] n is 0, 1, 2, or 3;

[0649] p is 0, 1, 2 or 3;

[0650] X is selected from chemical bonds, -O-, -S-, -C(O)-, -C(O)O-, -OC(O)-, -OC(O)NR X1 -、-NR X1 C(O)O-、-NR X1 C(O)- or -C(O)NR X1 -;

[0651] R X1 Selected from H, C 1-4 Alkyl or C 1-4 Halogenated alkyl groups, optionally deuterated, up to and including complete deuteration;

[0652] L is selected from

[0653] A29. The oligonucleotides in technical solution A28, wherein,

[0654] Selected from OH or Or it can represent a chemical bond that connects to the 5' end hydroxyl or thiol group of the ribose of an adjacent nucleotide;

[0655] X1 is selected from OH or Or a chemical bond linked to a hydroxyl or thiol group at the 2' or 3' end of the ribose of another adjacent nucleotide;

[0656] and Unlike X1, it is not OH or

[0657] X3 is selected from O or S;

[0658] R T3 Selected from H, D or CH3, which are optionally deuterated, up to and including complete deuteration;

[0659] R T4 For H;

[0660] n is 3;

[0661] p is 0;

[0662] X is selected from chemical bonds, -O-, -NHC(O)O-, -OC(O)NH-, -N(CH3)C(O)O-, or -C(O)O-;

[0663] L is selected from

[0664] A30. An oligonucleotide of any one of technical solutions A16-A20 and A26-A29, wherein the compound of formula (Ia) is selected from the following structures:

[0665]

[0666]

[0667] in,

[0668] Selected from OH or Or it can represent a chemical bond that connects to the 5' end hydroxyl or thiol group of the ribose of an adjacent nucleotide;

[0669] X1 is selected from OH or Or a chemical bond linked to a hydroxyl or thiol group at the 2' or 3' end of the ribose of another adjacent nucleotide;

[0670] and Unlike X1, it is not OH or

[0671] X3 is selected from O or S.

[0672] A31. An oligonucleotide of any one of technical solutions A16-A30, having 14 to 30 nucleotides.

[0673] A32. An oligonucleotide of any one of technical solutions A16-A30, wherein the 5' end contains a compound of formula (Ia), or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof.

[0674] A33. An oligonucleotide of any one of technical solutions A16-A30, wherein the 3' end contains a compound of formula (Ia), or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof.

[0675] A34. An oligonucleotide of any one of technical solutions A16-A30, comprising a compound of formula (Ia) at its 5' end and 3' end, respectively, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof.

[0676] A35. An oligonucleotide of any one of technical solutions A16-A20 and A26-A30, wherein the oligonucleotide contains one or more compounds of formula (I'), or pharmaceutically acceptable salts, tautomers or stereoisomers thereof.

[0677] A36. A double-stranded RNA having a sense strand and an antisense strand, each strand having 14 to 30 nucleotides, wherein the antisense strand contains a sequence fully complementary to the sense strand and the target mRNA, wherein the sense strand and / or antisense strand comprises one or more compounds of formula (Ia) as described in any one of technical solutions A16-A35, or pharmaceutically acceptable salts, tautomers or stereoisomers thereof.

[0678] A37. The double-stranded RNA of technical solution A36, wherein the sense strand comprises at its 5' end a compound of formula (Ia) as described in any one of technical solutions A16-A35, or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof.

[0679] A38. A double-stranded RNA of technical solution A36 or A37, wherein the sense strand comprises a compound of formula (Ia) as described in any one of technical solutions A16-A35, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, at its 3' end.

[0680] A39. A double-stranded RNA of any one of technical solutions A36-A38, wherein the sense strand comprises a compound of formula (Ia) of any one of technical solutions A16-A35, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, at the 5' end and 3' end, respectively.

[0681] A40. A double-stranded RNA of any one of technical solutions A36-A39, wherein the sense strand comprises, within the oligonucleotide, one or more compounds of formula (I') as described in any one of technical solutions A16-A20, A26-A30, or pharmaceutically acceptable salts, tautomers or stereoisomers thereof.

[0682] A41. A double-stranded RNA of any one of technical solutions A36-A40, wherein the antisense strand comprises at its 5' end a compound of formula (Ia) as described in any one of technical solutions A16-A35, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof.

[0683] A42. A double-stranded RNA of any one of technical solutions A36-A41, wherein the antisense strand comprises a compound of formula (Ia) of any one of technical solutions A16-A35, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, at its 3' end.

[0684] A43. A double-stranded RNA of any one of technical solutions A36-A42, wherein the antisense strand comprises a compound of formula (Ia) of any one of technical solutions A16-A35, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, at the 5' end and 3' end, respectively.

[0685] A44. A double-stranded RNA of any one of technical solutions A36-A43, wherein the antisense strand comprises, within the oligonucleotide, one or more compounds of formula (I') as described in any one of technical solutions A16-A20, A26-A30, or pharmaceutically acceptable salts, tautomers or stereoisomers thereof.

[0686] A45. A double-stranded RNA selected from any one of technical solutions A36-A44, wherein the RNA is selected from small interfering RNA (siRNA) and short hairpin RNA (shRNA).

[0687] A46. A vector comprising a nucleotide sequence encoding a double-stranded RNA as described in any one of the preceding technical solutions A36-A45.

[0688] A47. A cell containing double-stranded RNA as described in any one of technical solutions A36-A45 or a vector as described in technical solution 54.

[0689] A48. A pharmaceutical composition comprising, as described in any one of technical solutions A36-A45, a vector as described in technical solution 46, or a cell as described in technical solution 47, and optionally a pharmaceutically acceptable carrier or excipient.

[0690] A49. A kit comprising double-stranded RNA as described in any one of claims A36-A45, a vector as described in claim 46, or cells as described in claim 47.

[0691] A50. A method for improving the effectiveness of siRNA in cells, comprising the step of introducing a double-stranded RNA or a vector as described in any one of technical solutions A36-A45 into the cell.

[0692] A51. A compound of formula (IIIb) or (IVb), or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof:

[0693]

[0694] in,

[0695] Each R T3 Independently selected from H, D, and C 1-6 Alkyl, C 1-6 Haloalkyl, C 2-6 alkenyl or C 2-6 The alkynyl group, which is optionally deuterated, up to and including complete deuteration;

[0696] Each R T4 Independently selected from H, D, and C 1-6 Alkyl, C 1-6 Haloalkyl, C 2-6 alkenyl or C 2-6 The alkynyl group, which is optionally deuterated, up to and including complete deuteration;

[0697] n is 0, 1, 2, 3, 4 or 5;

[0698] p is 0, 1, 2, 3, 4 or 5;

[0699] X is selected from chemical bonds, -O-, -S-, -C(O)-, -C(O)O-, -OC(O)-, -OC(O)NR X1 -、-NR X1 C(O)O-、-NR X1 C(O)- or -C(O)NR X1 -;

[0700] R X1 Selected from H, C 1-6 Alkyl or C 1-6 Halogenated alkyl groups, optionally deuterated, up to and including complete deuteration;

[0701] L stands for -Ar-(CH2) 1-6 -O-, where each CH2 may optionally be replaced by one or two R#, where R# is selected from H, D, C. 1-6 Alkyl or C 1-6 Halogenated alkyl groups, optionally deuterated, up to and including complete deuteration;

[0702] Ar is selected from C 6-10 Aryl or 5-10 heteroaryl, wherein C 6-10 The aryl or 5-10 heteroaryl group can be optionally replaced by one, two or three R*.

[0703] R* is selected from C1-6 Alkyl, C 1-6 Haloalkyl, C 2-6 alkenyl or C 2-6 The alkynyl group, which is optionally deuterated, up to and including complete deuteration;

[0704] PG is selected from the protective base.

[0705] A52. The compound of technical solution A51, or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof, wherein...

[0706] Each R T3 Independently selected from H, D, and C 1-6 Alkyl or C 1-6 Halogenated alkyl groups, optionally deuterated, up to and including complete deuteration;

[0707] Each R T4 Independently selected from H, D, and C 1-6 Alkyl or C 1-6 Halogenated alkyl groups, optionally deuterated, up to and including complete deuteration;

[0708] n is 0, 1, 2, or 3;

[0709] p is 0, 1, 2 or 3;

[0710] X is selected from chemical bonds, -O-, -S-, -C(O)-, -C(O)O-, -OC(O)-, -OC(O)NR X1 -、-NR X1 C(O)O-、-NR X1 C(O)- or -C(O)NR X1 -;

[0711] R X1 Selected from H, C 1-4 Alkyl or C 1-4 Halogenated alkyl groups, optionally deuterated, up to and including complete deuteration;

[0712] L stands for -Ar-(CH2) 1-4 -O-, where each CH2 may optionally be replaced by one or two R#, where R# is selected from H, D, C. 1-4 Alkyl or C 1-4 Halogenated alkyl groups, optionally deuterated, up to and including complete deuteration;

[0713] Ar is selected from C 6-10 Aryl or 5-10 heteroaryl, wherein C 6-10 The aryl or 5-10 heteroaryl group can be optionally replaced by one, two or three R*.

[0714] R* is selected from C 1-4 Alkyl or C1-4 Halogenated alkyl groups, optionally deuterated, up to and including complete deuteration;

[0715] PG is selected from the protective base.

[0716] A53. The compound of technical solution A51 or A52, or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof, wherein PG is selected from 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 (Tro c) 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 -P(OCH2CH2CN)(N(iPr)2) or -C(O)CH2CH2C(O)OH.

[0717] A54. A compound of any one of technical solutions A51-A53, or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof, wherein the compound of formula (IIIb) or (IVb) has the structure of formula (III-1b) or (IV-1b).

[0718]

[0719] in,

[0720] Each R T3 Independently selected from H, D, and C 1-4 Alkyl or C 1-4 Halogenated alkyl groups, optionally deuterated, up to and including complete deuteration;

[0721] Each R T4 Independently selected from H, D, and C 1-4 Alkyl or C 1-4 Halogenated alkyl groups, optionally deuterated, up to and including complete deuteration;

[0722] n is 0, 1, 2, or 3;

[0723] p is 0, 1, 2 or 3;

[0724] X is selected from chemical bonds, -O-, -S-, -C(O)-, -C(O)O-, -OC(O)-, -OC(O)NR X1 -、-NR X1 C(O)O-、-NR X1 C(O)- or -C(O)NR X1 -;

[0725] R X1 Selected from H, C 1-4 Alkyl or C 1-4 Halogenated alkyl groups, optionally deuterated, up to and including complete deuteration;

[0726] L is selected from

[0727] PG is a protecting group, selected from DMTr, -P(OCH2CH2CN)(N(iPr)2) or -C(O)CH2CH2C(O)OH.

[0728] A55. The compound of technical solution A54, or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof, wherein...

[0729] R T3 Selected from H, D or CH3, which are optionally deuterated, up to and including complete deuteration;

[0730] R T4 Selected from H or D;

[0731] n is 3;

[0732] p is 0;

[0733] X is selected from chemical bonds, -O-, -NHC(O)O-, -OC(O)NH-, -N(CH3)C(O)O-, or -C(O)O-;

[0734] L is selected from

[0735] PG is selected from DMTr, -P(OCH2CH2CN)(N(iPr)2) or -C(O)CH2CH2C(O)OH.

[0736] A56. A compound of any one of technical solutions A51-A55, or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof, wherein the compound of formula (IIIb) or (IVb) is selected from:

[0737]

[0738]

[0739] in,

[0740] PG is selected from protecting groups, preferably hydroxyl protecting groups, 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), 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), 4,4'-dimethoxytriphenylmethyl, -P(OCH2CH2CN)(N(iPr)2) or -C(O)CH2CH2C(O)OH, preferably -P(OCH2CH2CN)(N(iPr)2) or -C(O)CH2CH2C(O)OH.

[0741] A57. A compound of formula (I) from any one of technical solutions A1-A3 and A6-A8, or an oligonucleotide from any one of technical solutions A16-18 and A21-23, wherein the chain containing GalNAc is a conjugating group represented by formula (X'):

[0742]

[0743] in,

[0744] Indicates the location where it connects to a biomolecule;

[0745] Q is independent of H,

[0746] Where L1 is a chemical bond, -CH2-, -CH2CH2-, -C(O)-, -CH2O-, -CH2O-CH2CH2O-, or -NHC(O)-(CH2NHC(O)). a -;

[0747] L2 is a chemical bond or -CH2CH2C(O)-;

[0748] L3 is a chemical bond, -(NHCH2CH2) b -、-(NHCH2CH2CH2) b -or-C(O)CH2-;

[0749] L4 is -(OCH2CH2) c -、-(OCH2CH2CH2) c -、-(OCH2CH2CH2CH2) c -、-(OCH2CH2CH2CH2CH2) c -or -NHC(O)-(CH2) d -;

[0750] Where a = 0, 1, 2 or 3;

[0751] b = 1, 2, 3, 4 or 5;

[0752] c = 1, 2, 3, 4 or 5;

[0753] d = 1, 2, 3, 4, 5, 6, 7 or 8;

[0754] A is a chemical bond, -CH2O- or -NHC(O)-;

[0755] A' is a chemical bond, -C(O)NH-, -NHC(O)-, or -O(CH2CH2O). e -;

[0756] Where e is 1, 2, 3, 4 or 5;

[0757] B is a chemical bond, -CH2-, -C(O)-, -M-, -CH2-M-, or -C(O)-M-;

[0758] Where M is

[0759] R1 and R2 together form -CH2CH2O- or -CH2CH(R)-O-, and R3 is H;

[0760] Or R1 and R3 together form -C 1-2 Alkylene-, and R2 is H;

[0761] Wherein R is -OR', -CH2OR' or -CH2CH2OR', where R' is H, a hydroxyl protecting group or a solid support, and the hydroxyl protecting group is preferably -C(O)CH2CH2C(O)OH or 4,4'-dimethoxytriphenylmethyl;

[0762] m1 = 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

[0763] n1 = 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0764] This invention also specifically relates to the following technical solutions:

[0765] B1. An oligonucleotide, comprising a compound of formula (II), or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof:

[0766]

[0767] in,

[0768] X1 is selected from OR a or NR b R c ;

[0769] R a Selected from H, C 1-6 Alkyl or C 1-6 Halogenated alkyl groups, optionally deuterated, up to and including complete deuteration;

[0770] R b and R c Selected from H, C 1-6 Alkyl or C 1-6 Halogenated alkyl, wherein R b and R c Can be optionally D, C 6-10 Aryl or 5-10 heteroaryl substitutions, up to complete deuteration;

[0771] X2 is the remaining part of the oligonucleotide, which is linked to P by a hydroxyl or thiol group on the 2', 3', or 5' carbon atom of the ribose of the first nucleotide at the 5' end;

[0772] X3 is independently selected from O or S;

[0773] Each R T1 Independently selected from H, D, and C 1-6 Alkyl, C 1-6 Haloalkyl, C 2-6 alkenyl, C 2-6 Alkyne group or chain containing GalNAc, which is optionally deuterated up to fully deuterated;

[0774] Each R T2 Independently selected from H, D, and C 1-6 Alkyl, C 1-6 Haloalkyl, C 2-6 alkenyl or C 2-6The alkynyl group, which is optionally deuterated, up to and including complete deuteration;

[0775] m can be 0, 1, 2, 3, 4, or 5;

[0776] X is selected from chemical bonds, -O-, -S-, -C(O)-, -C(O)O-, -OC(O)-, -OC(O)NR X1 -、-NR X1 C(O)O-、-NR X1 C(O)- or -C(O)NR X1 -;

[0777] R X1 Selected from H, C 1-6 Alkyl or C 1-6 Halogenated alkyl groups, optionally deuterated, up to and including complete deuteration;

[0778] L stands for -Ar-(CH2) 1-6 -O-, where each CH2 may optionally be replaced by one or two R#, where R# is selected from H, D, C. 1-6 Alkyl or C 1-6 Halogenated alkyl groups, optionally deuterated, up to and including complete deuteration;

[0779] In L, Ar is bonded to X, and oxygen atom is bonded to phosphorus atom;

[0780] Ar is selected from C 6-10 Aryl or 5-10 heteroaryl, wherein C 6-10 The aryl or 5-10 heteroaryl group can be optionally replaced by 1, 2, 3, 4 or 5 R*.

[0781] R* is selected from C 1-6 Alkyl, C 1-6 Haloalkyl, C 2-6 alkenyl or C 2-6 The alkynyl group is optionally deuterated, up to and including complete deuteration.

[0782] B2. The oligonucleotide of technical solution B1, or its pharmaceutically acceptable salt, tautomer, or stereoisomer, wherein...

[0783] X1 is selected from OR a or NR b R c ;

[0784] R a Selected from H, C 1-4 Alkyl or C 1-4 Halogenated alkyl groups, optionally deuterated, up to and including complete deuteration;

[0785] R b and R cIndependently selected from H and C 1-4 Alkyl, C 1-4 Halogenated alkyl, wherein R b and R c Can be optionally D, C 6-10 Aryl substitution, up to complete deuteration;

[0786] X2 is the remaining part of the oligonucleotide, which is linked to P by a hydroxyl or thiol group on the 5' carbon atom of the ribose of the first nucleotide at the 5' end;

[0787] X3 is independently selected from O or S;

[0788] Each R T1 Independently selected from H, D, and C 1-4 Alkyl, C 1-4 Halogenated alkyl groups or chains containing GalNAc, which are optionally deuterated up to fully deuterated;

[0789] Each R T2 Independently selected from H, D, and C 1-4 Alkyl or C 1-4 Halogenated alkyl groups, optionally deuterated, up to and including complete deuteration;

[0790] m can be 0, 1, 2, or 3;

[0791] X is selected from chemical bonds, -O-, -S-, -C(O)-, -C(O)O-, -OC(O)-, -OC(O)NR X1 -、-NR X1 C(O)O-、-NR X1 C(O)- or -C(O)NR X1 -;

[0792] R X1 Selected from H, C 1-4 Alkyl or C 1-4 Halogenated alkyl groups, optionally deuterated, up to and including complete deuteration;

[0793] L is -Ar-(CH2)-O-, where CH2 can be optionally replaced by one or two R#, and R# is selected from H, D, and C. 1-4 Alkyl or C 1-4 Halogenated alkyl groups, optionally deuterated, up to and including complete deuteration;

[0794] In L, Ar is bonded to X, and oxygen atom is bonded to phosphorus atom;

[0795] Ar is selected from C 6-10 Aryl or 5-10 heteroaryl, wherein C 6-10 The aryl or 5-10 heteroaryl group can be optionally replaced by one, two or three R*.

[0796] R* is selected from C 1-4 Alkyl or C 1-4 Halogenated alkyl groups, optionally deuterated, up to and including complete deuteration.

[0797] B3. The oligonucleotide of technical solution B1 or B2, or its pharmaceutically acceptable salt, tautomer, or stereoisomer, wherein...

[0798] X1 is selected from OR a or NR b R c ;

[0799] R a Selected from H, C 1-4 Alkyl or C 1-4 Halogenated alkyl groups, optionally deuterated, up to and including complete deuteration;

[0800] R b and R c Independently selected from H and C 1-4 Alkyl, C 1-4 Halogenated alkyl, wherein R b and R c Optionally substituted with D or phenyl, up to complete deuteration;

[0801] X2 is the remaining part of the oligonucleotide, which is linked to P by a hydroxyl or thiol group on the 5' carbon atom of the ribose of the first nucleotide at the 5' end;

[0802] X3 is independently selected from O or S;

[0803] Selected from

[0804] Where R T Chains selected from H, D, CH3 or containing GalNAc, which are optionally deuterated up to and including complete deuteration;

[0805] X is selected from chemical bonds, -O-, -S-, -C(O)-, -C(O)O-, -OC(O)-, -OC(O)NR X1 -、-NR X1 C(O)O-、-NR X1 C(O)- or -C(O)NR X1 -;

[0806] R X1 Selected from H, C 1-4 Alkyl or C 1-4 Halogenated alkyl groups, optionally deuterated, up to and including complete deuteration;

[0807] L is selected from

[0808] B4. An oligonucleotide of any one of technical solutions B1-B3, or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof, wherein...

[0809] X1 is selected from OH or

[0810] X2 is the remaining part of the oligonucleotide, which is linked to P by a hydroxyl or thiol group on the 5' carbon atom of the ribose of the first nucleotide at the 5' end;

[0811] X3 is independently selected from O or S;

[0812] Selected from

[0813] X is selected from chemical bonds, -O-, -NHC(O)O-, -OC(O)NH-, -N(CH3)C(O)O-, or -C(O)O-;

[0814] L is selected from

[0815] B5. An oligonucleotide of any one of technical solutions B1-B4, or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof, wherein the compound of formula (II) is selected from:

[0816]

[0817]

[0818] in,

[0819] X1 is selected from OH or

[0820] X2 is the remaining portion of the oligonucleotide, which is linked to P via a hydroxyl or thiol group on the 5' carbon atom of the ribose of the first nucleotide at the 5' end;

[0821] X3 is independently selected from O or S;

[0822] Preferably, the compound of formula (I) is selected from:

[0823]

[0824] X2 is the remaining part of the oligonucleotide, which is linked to P by a hydroxyl group on the 5' carbon atom of the ribose of the first nucleotide at the 5' end.

[0825] B6. Oligonucleotides comprising one, two or more compounds of formula (Ia), or pharmaceutically acceptable salts, tautomers or stereoisomers thereof:

[0826]

[0827] in,

[0828] Selected from OR a or NR b R c , or represents a chemical bond that connects to a hydroxyl or thiol group on the 5' carbon atom of the ribose of an adjacent nucleotide;

[0829] X1 is a chemical bond that connects to a hydroxyl or thiol group on the 2' or 3' carbon atom of the ribose of another adjacent nucleotide;

[0830] R a Selected from H, C 1-6 Alkyl, C 1-6 Haloalkyl, C 2-6 alkenyl or C 2-6 The alkynyl group, which is optionally deuterated, up to and including complete deuteration;

[0831] R b and R c Independently selected from H and C 1-6 Alkyl or C 1-6 Halogenated alkyl, wherein R b and R c Can be optionally D, C 6-10 Aryl or 5-10 heteroaryl substitutions, up to complete deuteration;

[0832] X3 is independently selected from O or S;

[0833] T is

[0834] Each R T1 Independently selected from H, D, halogens, CN, C 1-6 Alkyl, C 1-6 Haloalkyl, C 1-6 Haloalkyl, C 2-6 alkenyl, C 2-6 Alkyne group or chain containing GalNAc, which is optionally deuterated up to fully deuterated;

[0835] Each R T2 Independently selected from H, D, halogens, CN, C 1-6 Alkyl, C 1-6 Haloalkyl, C 2-6 alkenyl or C 2-6 The alkynyl group, which is optionally deuterated, up to and including complete deuteration;

[0836] m can be 0, 1, 2, 3, 4, or 5;

[0837] X is selected from chemical bonds, -O-, -S-, -C(O)-, -C(O)O-, -OC(O)-, -OC(O)NR X1 -、-NR X1 C(O)O-、-NR X1 C(O)- or -C(O)NR X1 -;

[0838] R X1 Selected from H, halogens, C 1-6 Alkyl or C 1-6 Halogenated alkyl groups, optionally deuterated, up to and including complete deuteration;

[0839] L stands for -Ar-(CH2) 1-6 -O-, where each CH2 group may optionally be replaced by R#, which is selected from H, D, halogens, CN, and C. 1-6 Alkyl, C 1-6 Haloalkyl, C 2-6 alkenyl or C 2-6 The alkynyl group, which is optionally deuterated, up to and including complete deuteration;

[0840] In L, Ar is bonded to X, and oxygen atom is bonded to phosphorus atom;

[0841] Ar is selected from C 3-10 Cycloalkyl, 3-10 membered heterocyclic groups, C 6-10 Aryl or 5-14 heteroaryl, wherein C 3-10 Cycloalkyl, 3-10 membered heterocyclic groups, C 6-10 The aryl or 5-10 heteroaryl group can be optionally replaced by 1, 2, 3, 4 or 5 R*.

[0842] R* is selected from H, D, halogens, CN, and C. 1-6 Alkyl, C 1-6 Haloalkyl, C 2-6 alkenyl or C 2-6 The alkynyl group is optionally deuterated, up to and including complete deuteration.

[0843] B7. The oligonucleotide in technical solution B6, wherein,

[0844] Selected from OR a or NR b R c , or represents a chemical bond that connects to a hydroxyl or thiol group on the 5' carbon atom of the ribose of an adjacent nucleotide;

[0845] X1 is a chemical bond that connects to a hydroxyl or thiol group on the 2' or 3' carbon atom of the ribose of another adjacent nucleotide;

[0846] Ra Selected from H, C 1-4 Alkyl or C 1-4 Halogenated alkyl groups, optionally deuterated, up to and including complete deuteration;

[0847] R b and R c Independently selected from H and C 1-4 Alkyl or C 1-4 Halogenated alkyl groups, and the R b and R c Can be optionally D, C 6-10 Aryl substitution, which is optionally deuterated, up to and including complete deuteration;

[0848] X2 and X3 are independently selected from O or S;

[0849] T is selected from

[0850] Each R T1 Independently selected from H, D, and C 1-4 Alkyl, C 1-4 Halogenated alkyl groups or chains containing GalNAc, which are optionally deuterated up to fully deuterated;

[0851] Each R T2 Independently selected from H, D, and C 1-4 Alkyl or C 1-4 Halogenated alkyl groups, optionally deuterated, up to and including complete deuteration;

[0852] m can be 0, 1, 2, or 3;

[0853] X is selected from chemical bonds, -O-, -S-, and -OC(O)NR. X1 -、-NR X1 C(O)O-, -C(O)O-, -OC(O)-, -NR X1 C(O)- or -C(O)NR X1 -;

[0854] R X1 Selected from H, C 1-4 Alkyl or C 1-4 Halogenated alkyl groups, optionally deuterated, up to and including complete deuteration;

[0855] L is -Ar-(CH2)-O-, where CH2 can be optionally replaced by one or two R#, and R# is selected from H, D, and C. 1-4 Alkyl or C 1-4 Halogenated alkyl groups, optionally deuterated, up to and including complete deuteration;

[0856] In L, Ar is bonded to X, and oxygen atom is bonded to phosphorus atom;

[0857] Ar is selected from C 6-10 Aryl or 5-10 heteroaryl, wherein C 6-10 The aryl or 5-10 heteroaryl group can be optionally replaced by one, two or three R*.

[0858] R* is selected from C 1-4 Alkyl or C 1-4 Halogenated alkyl groups, optionally deuterated, up to and including complete deuteration.

[0859] B8. Oligonucleotides of technical solution B6 or B7, wherein,

[0860] Selected from OR a or NR b R c , or represents a chemical bond that connects to a hydroxyl or thiol group on the 5' carbon atom of the ribose of an adjacent nucleotide;

[0861] X1 is a chemical bond that connects to a hydroxyl or thiol group on the 2' or 3' carbon atom of the ribose of another adjacent nucleotide;

[0862] R a Selected from H, C 1-4 Alkyl or C 1-4 Halogenated alkyl groups, optionally deuterated, up to and including complete deuteration;

[0863] R b and R c Independently selected from H and C 1-4 Alkyl or C 1-4 Halogenated alkyl, wherein R b and R c Optionally substituted with D or phenyl, up to complete deuteration;

[0864] X3 is independently selected from O or S;

[0865] T is selected from Where R T Chains selected from H, D, CH3 or containing GalNAc, which are optionally deuterated up to and including complete deuteration;

[0866] X is selected from chemical bonds, -O-, -S-, -C(O)-, -C(O)O-, -OC(O)-, -OC(O)NR X1 -、-NR X1 C(O)O-、-NR X1 C(O)- or -C(O)NR X1 -;

[0867] R X1 Selected from H, C 1-4 Alkyl or C 1-4Halogenated alkyl groups, optionally deuterated, up to and including complete deuteration;

[0868] L is selected from

[0869] B9. An oligonucleotide from any one of technical solutions B6-B8, wherein,

[0870] This refers to the chemical bond that connects to a hydroxyl or thiol group on the 5' carbon atom of the ribose of an adjacent nucleotide;

[0871] X1 is a chemical bond that connects to a hydroxyl or thiol group on the 2' or 3' carbon atom of the ribose of another adjacent nucleotide;

[0872] X3 is selected from O or S;

[0873] T is selected from

[0874] X is selected from chemical bonds, -O-, -NHC(O)O-, -OC(O)NH-, -N(CH3)C(O)O-, or -C(O)O-;

[0875] L is selected from B10. An oligonucleotide of any one of technical solutions B6-B9, wherein the compound of formula (Ia) is selected from the following structures:

[0876]

[0877]

[0878] in,

[0879] This refers to the chemical bond that connects to a hydroxyl or thiol group on the 5' carbon atom of the ribose of an adjacent nucleotide;

[0880] X1 is a chemical bond that connects to a hydroxyl or thiol group on the 2' or 3' carbon atom of the ribose of another adjacent nucleotide;

[0881] X3 is selected from O or S;

[0882] Preferably, the compound of formula (Ia) is selected from:

[0883]

[0884] This represents the chemical bond that connects the ribose of the adjacent nucleotide to the hydroxyl group on the 5' carbon atom.

[0885] X1 is a chemical bond that connects to a hydroxyl or thiol group on the 2' or 3' carbon atom of the ribose of another adjacent nucleotide;

[0886] X3 is selected from O or S.

[0887] B11. An oligonucleotide of any one of technical solutions B6-B10, having 14 to 30 nucleotides.

[0888] B12. An oligonucleotide of any one of technical solutions B6-B11, wherein the oligonucleotide contains one or more compounds of formula (Ia), or pharmaceutically acceptable salts, tautomers or stereoisomers thereof.

[0889] B13. A double-stranded RNA having a sense strand and an antisense strand, each strand having 14 to 30 nucleotides, wherein the antisense strand contains a sequence fully complementary to the sense strand and the target mRNA, wherein the sense strand and / or antisense strand contains one or more compounds of formula (II) as described in any one of technical solutions B1-B5 or compounds of formula (Ia) as described in any one of technical solutions B6-B10, or pharmaceutically acceptable salts, tautomers or stereoisomers thereof.

[0890] B14. The double-stranded RNA of technical solution B13, wherein the sense strand contains one or more compounds of formula (Ia) as described in any one of technical solutions B6-B10, or pharmaceutically acceptable salts, tautomers or stereoisomers thereof, within an oligonucleotide.

[0891] B15. The double-stranded RNA of technical solution B13, wherein the antisense strand comprises at its 5' end a compound of formula (II) as described in any one of technical solutions B1-B5, or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof.

[0892] B16. The double-stranded RNA of technical solution B13, wherein the antisense strand contains one or more compounds of formula (Ia) as described in any one of technical solutions B6-B10, or pharmaceutically acceptable salts, tautomers or stereoisomers thereof, within the oligonucleotide.

[0893] B17. A double-stranded RNA selected from any one of technical solutions B13-B16, wherein the RNA is selected from small interfering RNA (siRNA) and short hairpin RNA (shRNA).

[0894] B18. A cell containing double-stranded RNA as described in any one of technical solutions B14-B17.

[0895] B19. A pharmaceutical composition comprising double-stranded RNA as described in any one of technical solutions B14-B17, or cells as described in technical solution B18, and optionally a pharmaceutically acceptable carrier or excipient.

[0896] B20. A kit comprising double-stranded RNA as described in any one of technical solutions B14-B17, cells as described in technical solution B18, or a pharmaceutical composition as described in technical solution B19.

[0897] B21. A compound of formula (IIb), or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof:

[0898]

[0899] in,

[0900] Each R T1 Independently selected from H, D, and C 1-6 Alkyl, C 1-6 Haloalkyl, C 2-6 alkenyl, C 2-6 Alkyne group or chain containing GalNAc, which is optionally deuterated up to fully deuterated;

[0901] Each R T2 Independently selected from H, D, and C 1-6 Alkyl, C 1-6 Haloalkyl, C 2-6 alkenyl or C 2-6 The alkynyl group, which is optionally deuterated, up to and including complete deuteration;

[0902] m can be 0, 1, 2, 3, 4, or 5;

[0903] X is selected from chemical bonds, -O-, -S-, -C(O)-, -C(O)O-, -OC(O)-, -OC(O)NR X1 -、-NR X1 C(O)O-、-NR X1 C(O)- or -C(O)NR X1 -;

[0904] R X1 Selected from H, C 1-6 Alkyl or C 1-6 Halogenated alkyl groups, optionally deuterated, up to and including complete deuteration;

[0905] L is -Ar-(CH2)-O-, where CH2 can be optionally replaced by one or two R#, and R# is selected from H, D, and C. 1-6 Alkyl or C 1-6 Halogenated alkyl groups, optionally deuterated, up to and including complete deuteration;

[0906] Ar is selected from C 6-10 Aryl or 5-10 heteroaryl, wherein C 6-10 The aryl or 5-10 heteroaryl group can be optionally replaced by one, two or three R*.

[0907] R* is selected from C 1-6 Alkyl, C 1-6 Haloalkyl, C 2-6 alkenyl or C 2-6 The alkynyl group, which is optionally deuterated, up to and including complete deuteration;

[0908] PG is selected from the protective base.

[0909] B22. The compound of technical solution B21, or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof, wherein...

[0910] Each R T1 Independently selected from H, D, and C 1-6 Alkyl, C 1-6 Halogenated alkyl groups or chains containing GalNAc, which are optionally deuterated up to fully deuterated;

[0911] Each R T2 Independently selected from H, D, and C 1-6 Alkyl, C 1-6 Halogenated alkyl groups, optionally deuterated, up to and including complete deuteration;

[0912] m can be 0, 1, 2, or 3;

[0913] X is selected from chemical bonds, -O-, -S-, -C(O)-, -C(O)O-, -OC(O)-, -OC(O)NR X1 -、-NR X1 C(O)O-、-NR X1 C(O)- or -C(O)NR X1 -;

[0914] R X1 Selected from H, C 1-4 Alkyl or C 1-4 Halogenated alkyl groups, optionally deuterated, up to and including complete deuteration;

[0915] L is -Ar-(CH2)-O-, where CH2 can be optionally replaced by one or two R#, and R# is selected from H, D, and C. 1-4 Alkyl or C 1-4 Halogenated alkyl groups, optionally deuterated, up to and including complete deuteration;

[0916] Ar is selected from C 6-10 Aryl or 5-10 heteroaryl, wherein C 6-10 The aryl or 5-10 heteroaryl group can be optionally replaced by one, two or three R*.

[0917] R* is selected from C 1-4 Alkyl or C 1-4Halogenated alkyl groups, optionally deuterated, up to and including complete deuteration;

[0918] PG is selected from the protective base.

[0919] B23. The compound of technical solution B21 or B22, or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof, wherein PG is selected from 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 (Tro c) 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 -P(OCH2CH2CN)(N(iPr)2) or -C(O)CH2CH2C(O)OH.

[0920] B24. A compound of any one of technical solutions B21-B23, or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof, wherein the compound of formula (IIb) is selected from:

[0921]

[0922]

[0923] in,

[0924] PG is -P(OCH2CH2CN)(N(iPr)2) or H.

[0925] B25. An oligonucleotide of any one of technical solutions B1-B3 and B6-B8, or a compound of formula (IIb) of technical solutions B21 or B22, wherein the chain containing GalNAc is a conjugating group represented by formula (X'):

[0926]

[0927] in,

[0928] Indicates the location where it connects to a biomolecule;

[0929] Q is independent of H,

[0930] Where L1 is a chemical bond, -CH2-, -CH2CH2-, -C(O)-, -CH2O-, -CH2O-CH2CH2O-, or -NHC(O)-(CH2NHC(O)). a -;

[0931] L2 is a chemical bond or -CH2CH2C(O)-;

[0932] L3 is a chemical bond, -(NHCH2CH2) b -、-(NHCH2CH2CH2) b -or-C(O)CH2-;

[0933] L4 is -(OCH2CH2) c -、-(OCH2CH2CH2) c -、-(OCH2CH2CH2CH2) c -、-(OCH2CH2CH2CH2CH2) c -or -NHC(O)-(CH2) d -;

[0934] Where a = 0, 1, 2 or 3;

[0935] b = 1, 2, 3, 4 or 5;

[0936] c = 1, 2, 3, 4 or 5;

[0937] d = 1, 2, 3, 4, 5, 6, 7 or 8;

[0938] A is a chemical bond, -CH2O- or -NHC(O)-;

[0939] A' is a chemical bond, -C(O)NH-, -NHC(O)-, or -O(CH2CH2O). e -;

[0940] Where e is 1, 2, 3, 4 or 5;

[0941] B is a chemical bond, -CH2-, -C(O)-, -M-, -CH2-M-, or -C(O)-M-;

[0942] Where M is

[0943] R1 and R2 together form -CH2CH2O- or -CH2CH(R)-O-, and R3 is H;

[0944] Or R1 and R3 together form -C 1-2 Alkylene-, and R2 is H;

[0945] Wherein R is -OR', -CH2OR' or -CH2CH2OR', where R' is H, a hydroxyl protecting group or a solid support, and the hydroxyl protecting group is preferably -C(O)CH2CH2C(O)OH or 4,4'-dimethoxytriphenylmethyl;

[0946] m1 = 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

[0947] n1 = 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. Example

[0948] The following examples are for illustrative purposes only and are not intended to limit the scope of the invention.

[0949] abbreviation

[0950]

[0951] Example 1: Preparation of DN0110

[0952]

[0953] 1. Preparation of compound 1c

[0954]

[0955] Compound 1a (40 g, 425 mmol, 37.3 mL) was dissolved in CH3SO3H (240 mL), and compound 1b (72.7 g, 637 mmol, 77.8 mL) was added. The mixture was stirred at 70 °C for 96 hours. The reaction mixture was cooled to 0 °C and quenched with water (500 mL), then extracted with ethyl acetate (500 mL x 2). The organic phase was washed with brine (100 mL x 2), dried over anhydrous Na2SO4, filtered, concentrated under reduced pressure, and purified by silica gel column chromatography (petroleum ether: ethyl acetate = 10:1) to give compound 1c (43.1 g, 57%).

[0956] 1H NMR (400MHz CDCl3) δ7.63(dd,J=1.6Hz,3.6Hz,1H),7.24-7.29(m,1H),7.14-7.18(m,1H),7.63(dd,J=1.2Hz,8.0Hz,1H),2.64(s,2H),1.37(s,6H).

[0957] m / z: ES+[M+H]+177.0.

[0958] 2. Preparation of compound 1d

[0959]

[0960] Lithium aluminum hydride (8.61 g, 227 mmol) was suspended in tetrahydrofuran (350 mL) and purged three times with nitrogen. Then, a solution of compound 1c (20.0 g, 113 mmol) dissolved in tetrahydrofuran (140 mL) was added dropwise at 0 °C. The mixture was then stirred at 0 °C for 1.5 hours. A saturated ammonium chloride solution (150 mL) was slowly added to the reaction mixture. The reaction mixture was filtered through diatomaceous earth. The mixture was diluted with ethyl acetate (200 x 2), washed twice with brine (300 mL x 2), dried over anhydrous sodium sulfate, filtered, and distilled under reduced pressure to give compound 4 (20.4 g, 97%).

[0961] 1 H NMR(400MHz CDCl3)δ7.22(dd,J=0.8Hz,4.0Hz,1H),7.04-7.10(m,1H),6.82-6.09(m,1H),6.64(dd,J=0.8 Hz,4.0Hz,1H),6.17-6.31(m,1H),3.53(t,J=4.0Hz,2H),2.23(t,J=4.0Hz,2H),1.42(6H,s).

[0962] 3. Preparation of compound 1f

[0963]

[0964] A mixture of compound 1d (15.0 g, 83.2 mmol) and cesium carbonate (81.3 g, 249 mmol) was purged with nitrogen three times in DMF (150 mL). Then, compound 1e (20.0 g, 124 mmol, 17.4 mL) was added, and the reaction mixture was stirred at 25 °C for 1 hr. The reaction mixture was quenched with water (100 mL) and then extracted with ethyl acetate (100 mL x 2). The combined organic layers were washed with brine (200 mL x 2), dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and purified by silica gel column chromatography (petroleum ether: ethyl acetate = 3:1) to give compound 1f (10.8 g, 53.9%).

[0965] 1 H NMR (400MHz CDCl3) δ7.17-7.28(m,2H),6.88-6.98(m,2H),5.2(s,2H),3.45(t,J=4.0Hz,2H),2.31(s,3H),2.20(t,J=7.2Hz,2H),1.3(s,6H).

[0966] 4. Preparation of 1g of compound

[0967]

[0968] Compound 1f (10.8 g, 44.9 mmol) was dissolved in pyridine (100 mL), and acetic anhydride (13.7 g, 134 mmol, 12.6 mL) and DMAP (548 mg, 4.49 mmol) were added sequentially. The reaction mixture was stirred at 25 °C for 2 hours. Ethanol (50 mL) was added to the solution, and the mixture was concentrated under reduced pressure. The residue was dissolved in ethyl acetate (100 mL), and the solution was washed with saturated sodium bicarbonate solution (120 mL) and brine (120 mL). The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give compound 1 g (12.0 g, 94.5%).

[0969] 1 H NMR(400MHz CDCl3)δ7.16-7.26(m,2H),6.88-6.97(m,2H),5.19(s,2H),3.88(t,J=4. 0Hz, 2H), 2.31 (s, 3H), 2.25 (t, J = 4.0Hz, 1.6H), 1.89 (s, 3H), 1.43 (s, 6H).

[0970] 5. Preparation of compound 1h

[0971]

[0972] Under a nitrogen atmosphere, SO₂Cl₂ (2.22 g, 16.4 mmol, 1.65 mL) was slowly added to a 1 g (5.00 g, 17.7 mmol) solution of the compound in dichloromethane (50 mL). The mixture was stirred at 25 °C for 2 hours. The reaction mixture was evaporated to dryness under vacuum. The mixture was dissolved in dichloromethane (20 mL) and DMF (12 mL). p-TsSK (6.01 g, 26.5 mmol) was added to the solution, and the mixture was stirred at 25 °C for 1 hour. Then tert-butyl mercaptan (3.19 g, 35.4 mmol, 3.99 mL) was added, and the reaction mixture was stirred at 25 °C under a nitrogen atmosphere for 18 hours. The reaction solution was diluted with ethyl acetate (120 mL), washed with water (100 mL x 3), dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure and purified by silica gel column chromatography (petroleum ether: ethyl acetate = 10:1) to give compound 1h (4.42 g, 69.7%).

[0973] 1 H NMR(400MHz CDCl3)δ7.14-7.26(m,2H),6.87-6.98(m,2H),5.34(m,2H),3.85(t,J=4.0Hz,2H),2.25(t,J=4.0Hz,2H),1.89(s,3H),1.42(s,6H),1.37(s,9H).

[0974] 6. Preparation of compound 1i

[0975]

[0976] Compound 1h (4.4 g, 12.3 mmol) and potassium carbonate (3.41 g, 24.6 mmol) were dissolved in methanol (40 mL) and stirred at 25 °C for 2 hours. The reaction mixture was diluted with ethyl acetate (50 mL), washed twice with brine (100 mL × 2), dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and purified by silica gel column chromatography (petroleum ether: ethyl acetate = 5:1) to give compound 1i (1.19 g, 30.8%).

[0977] 1 H NMR (400MHz CDCl3) δ7.18-7.27(m,2H),6.89-6.99(m,2H),5.35(s,2H),3.43(t,J=2.0Hz,2H),2.20(t,J=4.0Hz,2H),1.42(s,6H),1.38(s,9H).

[0978] 7. Preparation of compound DN0110

[0979]

[0980] Compound 1i (1.00 g, 3.18 mmol) and DCI (187 mg, 1.59 mmol) were dissolved in dichloromethane (10 mL), and compound 1j (1.92 g, 6.36 mmol, 2.02 mL) was added. The reaction mixture was stirred at 25 °C for 1 hour. Triethylamine (0.5 mL) and triethylamine-based silica gel were added to the reaction mixture, and the mixture was concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (petroleum ether (1% triethylamine) / ethyl acetate = 98 / 2) to give compound DN0110 (1.03 g, 62.9%).

[0981] 1 H NMR(400MHz CDCl3)δ7.16-7.27(m,2H),6.89-6.99(m,2H),5.33(m,2H),3.67-3.80(m,2H),3.47-3.59(m,2H),3.30-3.45(m,2H ),2.58(t,J=4.0Hz,2H),2.17-2.25(m,2H),1.41(s,6H),1.37(s,9H),1.15(t,J=4.0Hz,6H),1.09(t,J=4.0Hz,6H)

[0982] 31 P NMR (400MHz CDCl3) δ 146.50.

[0983] Example 2 Preparation of DN0111

[0984]

[0985] The synthesis steps of Example 2 are the same as those of Example 1, except that the compound isopropylthiol is used instead of the compound tert-butylthiol.

[0986] 1 H NMR (400MHz, CDCl3) δ7.09-7.17(m,2H),6.82-6.91(m,2H),5.21(s,2H),3.61-3.70(m,2H),3.440-3.57(m,4H),3.18-3.27(m,1H),2. 99-3.05(m,1H),2.51(t,J=4Hz,1H),2.111-2.21(m,2H),1.33-1.35(m,6H),1.24-1.26(m,6H),1.18-1.22(m,6H),1.05-1.08(m,6H).

[0987] 31 P NMR (400MHz CDCl3) δ 146.52.

[0988] Example 3 Preparation of compound AE1

[0989]

[0990] 1. Preparation of Compound 3

[0991]

[0992] Compound 1 (13.0 g, 104 mmol) and cesium carbonate (102 g, 314 mmol) were dissolved in DMF (130 mL). After purging with nitrogen three times, compound 2 (15.2 g, 157 mmol, 13.2 mL) was added to the previous solution and reacted at 25 °C for 2 hours. TLC (petroleum ether: ethyl acetate = 3:1) showed that the starting material disappeared and new spots formed. The reaction solution was diluted with ethyl acetate and extracted (50 mL x 3). The organic phase was washed successively with saturated sodium bicarbonate (50 mL) and saturated sodium chloride (50 mL), dried over anhydrous sodium sulfate, and evaporated to dryness. The resulting yellow oily compound 3 (10.0 g, 54.3 mmol, 51.82%) was obtained by column chromatography (petroleum ether: ethyl acetate = 5:1-3:1).

[0993] 1 H NMR (400MHz, CDCl3) δ7.32-7.27(m,2H),6.96-6.94(m,2H),5.20(s,2H),4.62(s,2H),2.25(s,3H).

[0994] 2. Preparation of Compound 4

[0995]

[0996] Compound 3 (10 g, 54.27 mmol) was dissolved in pyridine (100 mL), and acetic anhydride (6.65 g, 65.13 mmol, 6.12 mL) and 4-dimethylaminopyridine were added. The reaction was carried out at 25 °C for 3 hours. TLC (petroleum ether: ethyl acetate = 3:1) showed that the starting material disappeared and new spots formed. The reaction solution was quenched with ethanol (10 mL), and the solvent was evaporated to dryness. The solution was then extracted with ethyl acetate (100 mL x 3). The organic phase was washed successively with saturated sodium bicarbonate (50 mL) and saturated sodium chloride (50 mL), dried over anhydrous sodium sulfate, and evaporated to dryness. The solution was then passed through a silica gel column (petroleum ether: ethyl acetate = 5:1-3:1) to give a colorless oily compound 4 (4 g, 17.32 mmol, 31.92%).

[0997] 1H NMR (400MHz, DMSO) δ7.31 (d, J = 8.78Hz, 2H), 6.99 (d, J = 8.53Hz, 2H), 5.27 (s, 2H), 5.00 (s, 2H), 2.16 (s, 3H), 2.03 (s, 3H).

[0998] 3. Preparation of Compound 5

[0999]

[1000] Compound 4 (5.0 g, 22.1 mmol) was dissolved in 50 mL of CH₂Cl₂ under a nitrogen atmosphere. SO₂Cl₂ (2.98 g, 22.1 mmol, 2.21 mL) dissolved in 5 mL of CH₂Cl₂ was slowly added. The mixture was stirred at 25 °C for 2 hours. The reaction mixture was evaporated to dryness under vacuum. The mixture was then dissolved in 20 mL of CH₂Cl₂ and 12 mL of DMF. P-TsSK (7.50 g, 33.1 mmol) was added to the solution, and the mixture was stirred at 25 °C for 1 hour. Then tert-butyl mercaptan (3.99 g, 44.2 mmol, 4.98 mL) was added, and the reaction mixture was stirred at 25 °C under a nitrogen atmosphere for 18 hours. TLC (petroleum ether: ethyl acetate = 10:1) showed the formation of new spots. The reaction solution was diluted with ethyl acetate (60 mL), washed with water (50 mL x 3), the organic layer was dried with Na2SO4, filtered, the organic phase was concentrated under reduced pressure and purified by normal phase column chromatography (petroleum ether: ethyl acetate = 10:1) to give a yellow oily compound 5 (4.90 g, 16.3 mmol, 73.81%). 1 H NMR (400MHz, CD3OD) δ7.32-7.27(m,2H),6.96-6.94(m,2H),5.20(s,2H),4.62(s,2H),2.25(s,3H).

[1001] 4. Preparation of Compound 6

[1002]

[1003] Compound 5 (4.90 g, 16.3 mmol) and K₂CO₃ (4.51 g, 32.6 mmol) were dissolved in MeOH (50 mL) and stirred at 25 °C for 2 hours. TLC (petroleum ether: ethyl acetate = 3:1) showed the formation of new spots. The mixture was concentrated under reduced pressure, and the reaction mixture was diluted with ethyl acetate (20 mL), washed twice with brine (50 mL x 2), and the organic layer was dried over Na₂SO₄. The organic phase was concentrated under reduced pressure and purified by normal-phase column chromatography (petroleum ether: ethyl acetate = 3:1) to give a yellow oily compound 6 (2.1 g, 7.09 mmol, 43.50%).

[1004] 1 H NMR: (400MHz, CD3OD) δ7.31-7.29(m,2H),6.95–6.91(m,2H),5.33(s,2H),4.50(s,2H),1.32(s,9H).

[1005] 5. Preparation of compound AE1

[1006]

[1007] Compound 6 (800 mg, 3.10 mmol) and DCI (183 mg, 1.55 mmol) were dissolved in CH2Cl2 (8 mL) solution, and compound 7 (1.87 g, 6.19 mmol) was added. The reaction mixture was stirred at 25 °C for 1 hour. TLC (petroleum ether (1% triethylamine) / ethyl acetate = 10 / 1) showed that the starting material had reacted completely. Triethylamine (0.5 mL) and triethylamine-based silica gel were added to the reaction mixture, and the mixture was concentrated under reduced pressure. The crude product was separated by normal-phase column chromatography (petroleum ether (1% triethylamine) / ethyl acetate = 98 / 2) to give a colorless oil AE1 (560 mg, 1.22 mmol, 39.44%).

[1008] 1 H NMR (400MHz, CDCl3) δ7.29-7.31(m,2H),6.91-6.93(m,2H),5.29(s,2H),4.59-4.74(m,2H),3 .81-3.88(m,2H),3.61-3.70(m,2H),2.63(t,J=6.8Hz,2H),1.35(s,9H),1.18-1.22(m,12H).

[1009] Example 4 Preparation of compound AE2

[1010]

[1011] 1. Preparation of Compound 2

[1012]

[1013] Compound 1 (5.00 g, 40.9 mmol) was dissolved in THF (40 mL), and isopropyl magnesium chloride (i-PrMgCl) (2 M, 81.9 mL) was added at 0 °C. The reaction was then carried out at 0 °C for 3 h. New spots were observed by TLC (petroleum ether: ethyl acetate = 3:1). The reaction mixture was cooled to 0 °C and quenched with ammonium chloride (80 mL), then extracted with ethyl acetate (100 mL x 2). The organic phase was washed with brine (100 mL x 2), dried over anhydrous Na₂SO₄, and filtered to give a brown solid, compound 2 (6.70 g, crude).

[1014] 1 H NMR (400MHz, CD3OD) δ7.11(d,J=8.4Hz,2H),6.73(d,J=8.4Hz,2H),4.15(d,J =7.2Hz,1H),1.83-1.89(m,1H),0.98(d,J=7.6Hz,3H),0.72(d,J=7.6Hz,3H).

[1015] 2. Preparation of Compound 4

[1016]

[1017] Compound 2 (2 g, 12.0 mmol) and cesium carbonate (11.8 g, 36.1 mmol) were dissolved in DMF (20 mL) and purged three times with nitrogen. Then, compound 3 (1.74 g, 18.1 mmol, 1.51 mL) was added. The mixture was then stirred at 25 °C for 1 hour. TLC (petroleum ether: ethyl acetate = 3:1) showed the formation of new spots. The mixture was diluted with ethyl acetate (40 mL), washed twice with brine (30 mL), dried over Na₂SO₄, filtered, and evaporated to dryness to give compound 4 (2.8 g, crude) as a yellow oil.

[1018] 1 H NMR (400MHz, CD3OD) δ7.23(d,J=8.8Hz,2H),6.93(d,J=8.8Hz,2H),5.18(s,2H),4.22(d,J =7.2Hz,1H),2.21(s,3H),1.83-1.93(m,1H),0.99(d,J=6.4Hz,3H),0.74(d,J=6.8Hz,3H).

[1019] 3. Preparation of Compound 5

[1020]

[1021] Compound 4 (2.70 g, 11.9 mmol) was dissolved in pyridine (25 mL), and acetic anhydride (3.65 g, 35.8 mmol, 3.36 mL) and DMAP (146 mg, 1.19 mmol) were added sequentially. The reaction mixture was stirred at 25 °C for 2 hours. TLC (petroleum ether: ethyl acetate = 3:1) showed the formation of new spots. The mixture was evaporated under vacuum. The residue was dissolved in ethyl acetate (30 mL), and the solution was washed with NaHCO3 solution (120 mL) and brine (120 mL). The organic layer was dried over Na2SO4, and concentrated by vacuum filtration to give a yellow oily compound 5 (2.87 g, 10.69 mmol, 89.65%).

[1022] 1 H NMR(400MHz,CD3OD)δ7.22(d,J=8.8Hz,2H),6.94(d,J=8.8Hz,2H),5.18(s,2H),2.20 (s,3H),2.07-2.13(m,1H),2.02(s,3H),1.97(d,J=8.0Hz,3H),0.78(d,J=6.8Hz,3H).

[1023] 4. Preparation of Compound 6

[1024]

[1025] Compound 5 (2.8 g, 10.4 mmol) was dissolved in 30 mL of CH₂Cl₂ under a nitrogen atmosphere. SO₂Cl₂ (1.31 g, 9.70 mmol, 970 μL) dissolved in 5 mL of CH₂Cl₂ was slowly added. The mixture was stirred at 25 °C for 2 hours. The reaction mixture was evaporated to dryness under vacuum. The mixture was then dissolved in 10 mL of CH₂Cl₂ and 6 mL of DMF. P-TsSK (3.54 g, 15.7 mmol) was added to the solution, and the mixture was stirred at 25 °C for 1 hour. Then tert-butyl mercaptan (941 mg, 10.4 mmol, 1.17 mL) was added, and the reaction mixture was stirred at 25 °C under a nitrogen atmosphere for 18 hours. TLC (petroleum ether: ethyl acetate = 10:1) showed the formation of new spots. The reaction solution was diluted with ethyl acetate (60 mL), washed with water (50 mL x 3), the organic layer was dried with Na2SO4, filtered, the organic phase was concentrated under reduced pressure and purified by normal phase column chromatography (petroleum ether: ethyl acetate = 10:1) to give a yellow oily compound 6 (2.10 g, 6.13 mmol, 58.7%).

[1026] 1H NMR (400MHz, CD3OD) δ7.23-7.26(m,2H),6.91-6.94(m,2H),5.37(d,J=8.0Hz,1H),5.32(s,2H ),2.06-2.12(m,1H),2.04(s,3H),1.32(s,9H),0.97(d,J=6.4Hz,3H),0.78(d,J=6.4Hz,3H).

[1027] 5. Preparation of Compound 7

[1028]

[1029] Compound 6 (2.10 g, 6.13 mmol) and K₂CO₃ (1.69 g, 12.3 mmol) were dissolved in MeOH (20 mL) and stirred at 25 °C for 2 hours. TLC (petroleum ether: ethyl acetate = 10:1) showed the formation of new spots. The reaction mixture was diluted with ethyl acetate (20 mL), washed twice with brine (100 mL × 2), and the organic layer was dried over Na₂SO₄. The organic phase was concentrated under reduced pressure and purified by normal-phase column chromatography (petroleum ether: ethyl acetate = 10:1) to give a yellow oily compound 7 (1.2 g, 3.88 mmol, 63.31%).

[1030] 1 H NMR (400MHz, CD3OD) δ7.23-7.26(m,2H),6.90-6.93(m,2H),5.33(s,2H),4.22(d,J=7. 2Hz,1H),1.85-1.93(m,1H),1.32(s,9H),0.98(d,J=6.8Hz,3H),0.74(d,J=6.8Hz,3H).

[1031] 6. Preparation of compound AE2

[1032]

[1033] Compound 7 (500 mg, 1.66 mmol) and DCI (98.3 mg, 832 μmol) were dissolved in CH₂Cl₂ (10 mL), and compound 8 (1.00 g, 3.33 mmol, 1.06 mL) was added. The reaction mixture was stirred at 25 °C for 1 hour. TLC (petroleum ether (1% triethylamine) / ethyl acetate = 10 / 1) showed that the starting material had reacted completely. Triethylamine (0.5 mL) and triethylamine-based silica gel were added to the reaction mixture, and the mixture was concentrated under reduced pressure. The crude product was separated by normal-phase column chromatography (petroleum ether (1% triethylamine) / ethyl acetate = 98 / 2) to give a colorless oily compound AE₂ (310 mg, 619 μmol).

[1034] 1 H NMR (400MHz, CDCl3) δ7.22(d,J=8.4Hz,2H),6.86-6.91(m,2H),5.28(d,J=5.6Hz,2H),4.40-4.46(m,1H),3.62-3.89(m,2H),3.48-3.56(m, 2H),2.64(t,J=6.4Hz,1H),2.33(t,J=6.4Hz,1H),194-1.99(m,1H),1.35(s,9H),1.12-1.23(m,9H),0.91-0.97(m,6H),0.78-0.80(m,3H).

[1035] Example 5 Preparation of compound AE3

[1036]

[1037] 1. Preparation of compound 124-1

[1038]

[1039] 30% hydrogen peroxide (20 mL) was added dropwise to a 50 mL ethanol solution of benzo[b]thiophene-2-boric acid (10.0 g, 56.2 mmol) over a period of 30 minutes. The reaction mixture was then stirred for 16 hours. TLC showed that the reaction was complete. The reaction mixture was then carefully washed three times with water and extracted twice with dichloromethane. The organic phase was dried and concentrated to obtain a crude product, which was then purified by silica gel column chromatography (PE / EA = 5:1) to obtain compound 124-1 (7.8 g, 92.1%) as a pale yellow solid.

[1040] 1 H NMR (400MHz, CDCl3) δ7.50–7.08 (m, 4H), 3.99 (s, 2H).

[1041] 2. Preparation of compound 124-2

[1042]

[1043] A solution of LiAlH4 (3.9 g, 102.7 mmol) in THF (80 mL) was cooled to 0 °C, and then compound 124-1 was dissolved in THF (30 mL) over a 1-hour period and added to the reaction mixture. The reaction mixture was stirred for 16 hours. TLC showed that the reaction was complete, and the reaction mixture was then carefully quenched with saturated Na2SO4 solution, followed by the addition of 1 M HCl solution until the reaction mixture became clear. The organic phase was dried and concentrated to give a crude product, which was then purified by silica gel column chromatography (PE / EA = 2:1) to obtain a colorless oily compound 124-2 (6.0 g, 75.2%).

[1044] 1 H NMR (400MHz, CDCl3) δ7.23 (dd, J=7.3, 1.8Hz, 1H), 7.12 (dd, J=7.2, 2.0Hz, 1H), 7.0 4(pd,J=7.3,1.8Hz,2H),3.81(t,J=6.6Hz,2H),3.34(s,1H),2.90(t,J=6.6Hz,2H).

[1045] 3. Preparation of compound 124-3

[1046]

[1047] A methanol solution (20 mL) of compound 124-2 (6.0 g, 38.9 mmol) was added to a methanol solution (100 mL) of 2,2'-dithiopyridine (PySSPy, 21.4 g, 97.1 mmol) and acetic acid (1.2 mL). The reaction mixture was stirred for 16 hours. TLC showed that the reaction was complete. The reaction mixture was then directly concentrated to obtain a crude product, which was purified by silica gel column chromatography to obtain a colorless oily compound 124-3 (5.7 g, 56.0%).

[1048] 1 H NMR (400MHz, CDCl3) δ8.50 (dt, J=4.9, 1.4Hz, 1H), 7.64 (ddt, J=8.2, 7.2, 3.2Hz, 3H) ,7.27–7.17(m,3H),7.15–7.11(m,1H),3.97(t,J=6.6Hz,2H),3.18(t,J=6.6Hz,2H).

[1049] 4. Preparation of compound 124-4

[1050]

[1051] Methyl trifluoromethanesulfonate (MeOTf, 7.1 g, 43.4 mmol) was added dropwise to a solution of compound 124-3 (5.7 g, 21.7 mmol) in dichloromethane (60 mL), and the reaction mixture was stirred for 15 min. Tert-butyl mercaptan (2.0 g, 21.7 mmol) and DIPEA (7.6 mL, 43.4 mmol) were then added to the reaction mixture. The mixture was stirred for 30 min. TLC showed that the reaction was complete. The reaction mixture was directly concentrated to obtain a crude product, which was purified by silica gel column chromatography (PE / EA = 6:1) to give compound 124-4 (2.9 g, 55.6%) as a colorless oil.

[1052] 1 H NMR (400MHz, CDCl3) δ7.77(dd,J=7.9,1.1Hz,1H),7.19–7.05(m,3H),3.84(t,J=6.8Hz,2H),3.05(t,J=6.7Hz,2H),1.23(s,9H).

[1053] 5. Preparation of compound 124-5

[1054]

[1055] Phosphorous acid (9.8 g, 120.0 mmol) was co-evaporated three times with anhydrous pyridine and then redissolved in pyridine (60 mL). Compound 124-4 (2.9 g, 12.0 mmol) was added to the reaction mixture, which was then cooled to 0 °C and stirred for 10 min. Trimethylacetyl chloride (7.2 g, 60.0 mmol) was added to the reaction mixture and stirred for 3 h. The reaction was quenched with triethylammonium bicarbonate buffer (30 mL, 1 M) and diluted twice with ethyl acetate. The organic phases were then combined and concentrated to give a crude product, which was purified by silica gel column chromatography (MeOH:DCM = 10:1, 1% TEA) to give a colorless oily compound 124-5 (860 mg, 59.7%).

[1056] 6. Preparation of compound 124-7

[1057]

[1058] Solutions of compounds 124-5 (860 mg, 2.1 mmol) and 124-6 (465.0 mg, 1.8 mmol) were co-evaporated three times with anhydrous pyridine and redissolved in pyridine (15 mL). The reaction mixture was cooled to -15 °C and trimethylacetyl chloride (0.5 mL, 3.6 mmol) was added dropwise while stirring at -15 °C for 2 hours. The reaction mixture was diluted with dichloromethane and the reaction was quenched with saturated NH4Cl solution. The organic phases were combined and concentrated to give a crude product, which was purified by silica gel column chromatography (MeOH:DCM = 5:1, 1% acetic acid) to give compound 124-7 (650 mg, 56.2%) as a white solid.

[1059] 7. Preparation of compound 124-8

[1060]

[1061] Benzylamine (0.6 mL, 5.5 mmol) was added to a solution of compound 124-7 (650 mg, 1.2 mmol) in dichloromethane and carbon tetrachloride (v / v = 1:1, 16 mL), and the reaction mixture was stirred for 16 hours. TLC showed the reaction was complete. The reaction mixture was then directly concentrated to obtain a crude product, which was purified by silica gel column chromatography (MeOH:DCM = 8:1) to obtain compound 124-8 (580 mg, 75.0%) as a white solid.

[1062] 8. Preparation of compound AE3

[1063]

[1064] Compound 124-9 (352 mg, 1.2 mmol) and DCI (159.6 mg, 1.4 mmol) were added to a dichloromethane (8 mL) solution of compound 124-8 (580 mg, 0.9 mmol), and the reaction mixture was stirred for 2 hours. TLC showed the reaction was complete. The mixture was then washed with water and extracted with dichloromethane. The organic phases were combined and concentrated to obtain a crude product, which was purified by silica gel column chromatography (PE:EA = 6:1, 1% pyridine) to obtain compound AE3 (640 mg, 85.2%) as a white solid.

[1065] 1H NMR(400MHz, CDCl3)δ7.91(s,1H),7.69(dd,J=8.2,5.0Hz,1H),7.58(dd,J=8.2,3.1Hz,1H),7.35–7.27(m,3H),7.25–7.15(m,2H ),6.97–6.91(m,1H),6.87(d,J=5.0Hz,1H),6.00–5.92(m,1H),5.64(dd,J=8.2,6.6Hz,1H),5.56(dd,J=8.2,6.7Hz,1H),5.31–5. 29(m,2H),4.00(dtd,J=12.9,6.8,6.4,2.8Hz,3H),3.88–3.74(m,5H),3.69(tt,J=12.5,6.3Hz,2H),3.62(tt,J=12.5,6.3Hz,3H) ,3.49(d,J=1.0Hz,1H),3.45(d,J=6.0Hz,2H),2.29(t,J=7.2Hz,3H),1.34(d,J=1.2Hz,9H),1.19(ddd,J=15.2,6.9,3.6Hz,12H). 31 P NMR (162MHz, CDCl3) δ151.07,150.78,150.28,150.23,8.92,8.73,8.42,8.31.

[1066] Example 6 Preparation of compound AE4

[1067]

[1068] 1. Preparation of compound 125-4

[1069]

[1070] Trimethylacetyl chloride (4.07 mL, 33.73 mmol) was added to an anhydrous pyridine (Py, 50 mL) solution of phosphonic acid (5.53 g, 67.45 mmol) and compound 125-3 (6.70 g, 13.49 mmol) and stirred. The reaction mixture was then subjected to TLC. After 3 hours, the reaction was quenched by careful addition of 2 M triethylamine bicarbonate buffer (TEAB, 20 mL). After carbon dioxide evaporation stopped, the reaction mixture was evaporated to dryness and the residue was separated between dichloromethane and 0.5 M TEAB. The organic layer was evaporated and the residue was purified on a silica gel column to obtain compound 125-4 (8.40 g, 94%).

[1071] 2. Preparation of compound 125-5

[1072]

[1073] Trimethylacetyl chloride (1.4 g, 11.3 mmol) was added to a stirred solution of compound 125-4 (2.5 g, 3.8 mmol) and compound 1i (1.2 g, 3.8 mmol) in anhydrous pyridine (10 mL). After TLC analysis showed that the reaction was complete, 2 M TEAB (1 mL) was added. The reaction mixture was concentrated and the residue was separated between dichloromethane and 0.5 M TEAB. The organic layer was collected, evaporated using Na2SO4, and then purified on a silica gel column to give a mixture of non-corresponding isomers of compound 125-5 (crude, 1.7 g, 53%).

[1074] 3. Preparation of compound 125-6

[1075]

[1076] Benzylamine (0.3 g, 3 mmol) was added dropwise to a solution of compound 125-5 (1.7 g, 2.0 mmol) in dichloromethane and carbon tetrachloride (v / v = 1:1, 4 mL), and the resulting mixture was stirred for 3 hours. The volatiles were removed under vacuum to obtain a residue, which was then purified by flash silica gel column chromatography to obtain compound 125-6 (1.5 g, 79%) as a white solid.

[1077] 4. Preparation of compound 125-7

[1078]

[1079] Compound 125-6 was dissolved in 0.5 mL of 1 M TBAF in THF and stirred overnight. The reaction mixture was then separated between water and diethyl ether. The organic layer was collected, dried over Na2SO4, and evaporated. Purification on a silica gel column yielded a mixture of non-corresponding isomers of compound 125-7 (crude, 1.1 g, 97%).

[1080] 5. Preparation of compound AE4

[1081]

[1082] Compound 125-8 (0.37 g, 1.24 mmol) was added to a solution of compound 125-7 (0.60 g, 0.83 mmol) and 5-(ethylthio)-1H-tetrazole (ETT, 0.08 g, 0.66 mmol) in anhydrous dichloromethane (5 mL). The reaction mixture was stirred at room temperature for 2 hours. The reaction mixture was filtered, concentrated, and loaded onto a silica gel column to obtain a white, foamy compound AE4 (0.50 g, 65%).

[1083] 1H NMR (400MHz, CDCl3) δ7.91(s,1H),7.69(dd,J=8.2,5.0Hz,1H),7.58(dd,J=8.2,3.1Hz,1H),7.35–7.27(m,3H),7.25–7.15(m,4H),6.97–6.91(m ,1H),6.87(d,J=5.0Hz,1H),6.00–5.92(m,1H),5.64(dd,J=8.2,6.6Hz,1H),5.56(dd,J=8.2,6.7Hz,1H),5.31–5.29(m,2H),4.41–4.25(m,3H), 4.24–4.13(m,3H),4.00(dtd,J=12.9,6.8,6.4,2.8Hz,3H),3.88–3.74( m,5H),3.62(tt,J=12.5,6.3Hz,3H),3.49(d,J=1.0Hz,1H),3.45(d,J=6 .0Hz,2H),2.98–2.72(m,3H),2.66–2.54(m,3H),2.29(t,J=7.2Hz,3H), 1.40(s,6H),1.34(d,J=1.2Hz,9H),1.19(ddd,J=15.2,6.9,3.6Hz,12H). 31 P NMR (162MHz, CDCl3) δ151.07,150.78,150.28,150.23,8.92,8.73,8.42,8.31.

[1084] Example 7 Preparation of compound AE5

[1085]

[1086] 1. Preparation of compound 3b

[1087]

[1088] LiAlH4 (57.08 mg, 142.69 mmol) was added to THF (150 mL), and the reaction solution was cooled to 0 °C. Then, compound 3a (10.00 g, 64.86 mmol) was added. The reaction was carried out at 20 °C for 14 h, and then at 25 °C for 24 h. The reaction solution was cooled to 0 °C and quenched with 2N hydrochloric acid solution. The aqueous phase was extracted twice with 100 mL of ethyl acetate. The organic phase was filtered and evaporated to dryness to give compound 3b (8.90 g, 63.48 mmol, 97.9%).

[1089] 1H NMR (400MHz, CD3OD) δ7.18-7.28(m,4H),4.54(s,2H).

[1090] 2. Preparation of compound 3c

[1091]

[1092] Compound 3b (5.00 g, 35.7 mmol) was dissolved in EtOH (100 mL), and tert-butyl mercaptan (36.95 mL, 340.1 mmol) was added. Iodine (6.79 g, 26.7 mmol) in EtOH (100 mL) was added dropwise at 0 °C, followed by reaction at 20 °C for 2 hours. After the reaction was complete, the pH was adjusted to 7 with saturated sodium bicarbonate, and some ethanol was removed by evaporation. The aqueous phase was extracted with ethyl acetate (300 mL x 3). The organic phase was filtered and evaporated to dryness to obtain the crude product. The crude product was purified by column chromatography (petroleum ether / ethyl acetate) to obtain compound 3c (3.20 g, 14.0 mmol, 39.3%).

[1093] 1 H NMR (400MHz, CD3OD) δ7.50-7.59(m,2H),7.27-7.35(m,2H),4.57(s,2H),1.28(s,9H).

[1094] 3. Preparation of compound AE5

[1095]

[1096] Compound 3c (1.00 g, 4.38 mmol) was dissolved in DCM (10.0 mL). The reaction solution was cooled to 0 °C, and DCI (0.57 g, 4.8 mmol) and compound 3d (2.64 g, 8.76 mmol) were added. The reaction was carried out at 0 °C for 1 hour. TLC (petroleum ether: ethyl acetate = 3:1) showed that the starting material disappeared. 0.3 mL of triethylamine and basic silica gel were added to the reaction solution, and the solution was evaporated to dryness. The crude product was purified by column chromatography (petroleum ether: ethyl acetate) to give compound AE5 (1.10 g, 2.57 mmol, 58.61%).

[1097] 1 H NMR (400MHz, CDCl3) δ7.51-7.56(m,2H),7.25-7.35(m,2H),4.55-4.80(m,2H),3.76- 3.93(m,2H),3.58-3.71(m,2H),2.60-2.66(m,2H),1.30(s,9H),1.16-1.23(m,12H).

[1098] Example 8 Preparation of siRNA

[1099] 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.

[1100] 1. Synthesis of the Justice Chain (SS Chain)

[1101] 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:

[1102] 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).

[1103] 2. Synthesis of the antisense chain (AS chain)

[1104] 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 the 5 μmol oligonucleotide of the antisense strand are the same as those for the sense strand.

[1105] 3. Purification and Annealing of Oligonucleotides

[1106] 3.1 Ammonolysis

[1107] 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.

[1108] 3.2 Purification

[1109] 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.

[1110] 3.3 Annealing

[1111] According to the table below, the sense chain (SS chain) and the antisense chain (AS chain) are mixed at a molar ratio (SS chain / AS chain = 1 / 1.05), heated in a water bath to 70-95℃, held for 3-5 minutes, and then naturally cooled to room temperature. The system is then freeze-dried to obtain the product.

[1112] The siRNA sequence used in this invention is as follows:

[1113]

[1114]

[1115] The meanings of the abbreviations in this article are as follows:

[1116] The distributions A, U, G, and C represent naturally occurring adenine ribonucleotides, uracil ribonucleotides, guanine ribonucleotides, and cytosine ribonucleotides.

[1117] 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.

[1118] 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.

[1119] “s” or “s-” indicates that the two adjacent nucleotides and / or delivery carriers are linked by a phosphate thioester.

[1120] 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.

[1121]

[1122] RPD1-RPD5 represents the following structure, where The chemical bond connecting the ribose of the nucleotide on the right to the hydroxyl group at the 5' carbon atom:

[1123]

[1124] Example 9: Screening Experiment of C57BL / 6 Wild-type Mouse Primary Hepatocytes (PMH)

[1125] 1. Free intake or transfection

[1126] C57BL / 6 wild-type mouse primary liver cells were isolated, counted, seeded in 24-well plates, 900 μL / well, 8 × 10⁸ cells / well. 4 Cells / well; 96-well plate deposition, 100 μL / well, 1×10⁻⁶ 4 Cells / wells. Then, choose between free uptake or transfection.

[1127] Free uptake: Add 10 μL of the diluted compound to 90 μL of Opti-MEM, mix well, and then add to the corresponding well. Incubate at 37°C in a 5% CO2 incubator for 24 hours. The control group did not receive siRNA.

[1128] Transfection: Add 10 μL of the diluted compound to 40 μL of Opti-MEM and mix well. Add 3 μL of RNAiMAX to 47 μL of Opti-MEM and mix well. Incubate for 5 min, then mix with the diluted compound. Let stand at room temperature for 10 min, then add to the corresponding wells and incubate at 37°C with 5% CO2 for 24 hours. No siRNA was added to the control group.

[1129] 2. Quantitative Real-Time PCR

[1130] Total RNA was extracted using a high-throughput nucleic acid extractor-magnetic bead method (Fanzhi Medical, FG0412; Hangzhou Aosheng, Auto-pure96), followed by reverse transcription (PrimeScript). TM After using the 1st Strand cDNA Synthesis Kit (Takara, 6210B), quantitative real-time PCR was performed (TaqMan). TM Fast Advanced Master Mix (ABI, 4444965)).

[1131] Table 1 Primer Information

[1132]

[1133] 3. Data Statistics

[1134] Calculate 2 -△△Ct The values ​​are then converted to percentages to obtain the residual inhibition rate;

[1135] △△Ct=[(Ct experimental group target gene - Ct experimental group internal reference) - (Ct control group target gene - Ct control group internal reference)].

[1136] The target gene is mAPOB or mFXII, and the control gene is mGAPDH.

[1137] 4. mAPOB gene experimental results

[1138] Using transfection, primary hepatocytes of wild-type mice (C57BL / 6) were selected, seeded in 24-well plates, and the initial concentration of the compound was 40 nM. Five concentrations (40 nM, 4 nM, 0.4 nM, 0.04 nM, and 0.004 nM) were diluted 10-fold to screen the IC50 activity of C57BL / 6 wild-type mouse primary hepatocytes at five points. The experimental results are shown in Table 2.

[1139] Table 2. Results of IC50 activity screening experiment of PMH, a siRNA compound targeting the mAPOB gene carrying the compounds of the present invention.

[1140]

[1141] 5. mFXII gene experimental results

[1142] Using the free uptake method, primary hepatocytes of C57BL / 6 wild-type mice were selected, plated in 96-well plates, and the initial concentration of the compound was 100 nM. Five concentration points (100 nM, 10 nM, 1 nM, 0.1 nM, 0.01 nM) were selected for IC50 activity screening of C57BL / 6 wild-type mouse primary hepatocytes under free uptake. The experimental results are shown in Table 3.

[1143] Table 3. Results of IC50 activity screening experiment of PMH, a siRNA compound targeting the mFXII gene carrying the compounds of this invention.

[1144]

[1145] Example 10: Validation of compound efficacy in C57BL / 6 mouse model

[1146] C57BL / 6 mice (male, 18–21 g, 6–8 weeks old, from Spifort (Suzhou) Biotechnology Co., Ltd.) were randomly divided into groups of 9 animals each (mice were euthanized on day 14 and day 28 (N=3 / group) for liver harvesting). The dosage for each animal was calculated based on body weight and administered via a single subcutaneous injection. The siRNA conjugate was provided to the CRO company in a 10 mg / mL solution (0.9% sodium chloride aqueous solution as solvent). Specifically, before the experiment, the siRNA conjugate was dissolved in 0.9% sodium chloride aqueous solution and brought to the required solution concentration and volume. The administration volume of physiological saline and siRNA conjugate was 5 mL / kg.

[1147] Blood samples were collected before drug administration (referred to as day -2) and grouped according to LDL levels on day 2. Remaining samples were retained for the detection of the target protein. Blood samples were also collected from the orbital venous plexus of mice on days 7, 14, 21, 28, and 42 after drug administration (after a 5-hour starvation period before each collection). Serum LDL concentration (using a Meikang brand, low-density lipoprotein cholesterol detection reagent) was measured at each time point using a direct method (Neusoft fully automated biochemical analyzer, NT-1000) at each time point. Remaining samples were retained for the ELISA method to detect the target protein (Mouse ApoB ELISA Kit, Abcam, ab230932). On days 14, 28, and 42, 10 mg of liver tissue (n = 3 / group / time point) was collected and placed in RNAlater solution, frozen at -80℃, and sent on dry ice for extraction and detection of liver mRNA expression (detection primers are shown in Table 4, requiring updates at subsequent time points). Experimental results are shown in Table 5.

[1148] Table 4 Primer Information

[1149]

[1150] Table 5. Pharmacological effects of siRNA compounds in the C57BL / 6 mouse model.

[1151]

[1152] Example 11: Screening Experiment of C57BL / 6 Wild-type Mouse Primary Hepatocytes (PMH)

[1153] More compounds of the present invention were tested according to the method of Example 9.

[1154] Table 6. Results of IC50 activity screening experiment of PMH, a siRNA compound targeting the mAPOB gene carrying the compounds of the present invention.

[1155]

[1156]

[1157] Table 7. Results of IC50 activity screening experiment of PMH, a siRNA compound targeting the mFXII gene carrying the compound of the present invention.

[1158]

Claims

1. Double-stranded RNA, selected from: i) The justice chain is SEQ ID NO: 1, and the antisense chain is SEQ ID NO: 3; or ii) The justice chain is SEQ ID NO: 7, and the antisense chain is SEQ ID NO:

9. in, SEQ ID NO: 1 is CfsCmsUfGmGfAmCfAmUfUfCfAmGfAmAfCmAfAmGfAmAf-L96; SEQ ID NO: 3 is RPD1-UmsUfsCmUfUmGfUmUfCmUfGmAmAmUfGmUfCmCfAmGf GmsGfsUm; SEQ ID NO: 7 is CmsAmsCmAmGmAmAfAmCfUfCfAmAmUmAmAmAmGmUmGmAm-L96; SEQ ID NO: 9 is RPD1-UmsCfsAmCmUmUfUmAfUfUmGmAmGmUfUmUfCmUmGm UmGmsCmsCm, RPD1 is .

2. A cell containing the double-stranded RNA as described in claim 1.

3. A pharmaceutical composition comprising the double-stranded RNA as described in claim 1, or the cells as described in claim 2, and optionally a pharmaceutically acceptable excipient.

4. A kit comprising the double-stranded RNA as claimed in claim 1, the cells as claimed in claim 2, or the pharmaceutical composition as claimed in claim 3.

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