Modified oligonucleotide and use thereof

Abstract

An oligonucleotide containing the modification group as represented by formula (I). The present invention further relates to an RNAi agent containing the modification group, a composition containing the oligonucleotide or RNAi agent, a kit and the related use.

Description

Modified oligonucleotides and their uses Technical Field

[0001] This invention relates to peptides that target tumor-associated fibroblast activation protein (FAP).

[0002] Invention Field

[0003] This invention belongs to the pharmaceutical field, specifically relating to oligonucleotides containing off-target modification groups, as well as RNAi drugs containing them and related applications. Background Technology

[0004] Oligonucleotides are polymeric sequences of nucleotides (RNA, DNA, and their analogues). Nucleic acid inhibitor molecules are oligonucleotides that regulate intracellular RNA levels and have shown early promise in the treatment of cancer, viral infections, and genetic disorders. Nucleic acid inhibitor molecules can regulate RNA expression through a different set of mechanisms, including RNA interference (RNAi).

[0005] RNAi is a conserved pathway found in most eukaryotes, in which a double-stranded RNA molecule (dsRNA) inhibits the expression of a target gene having a sequence complementary to that dsRNA. In a typical RNAi pathway, the longer dsRNA is cleaved by the cleavage enzyme Dicer into a shorter RNA duplex called small interfering RNA (“siRNA”). siRNA has been shown to associate with cleavage enzyme, trans-activating response RNA-binding protein (TRBP), and Argonaute 2 (“Ago2”) to form a complex, sometimes referred to as the RNA-induced silencing complex (“RISC”). Ago2 is a nuclease that uses the antisense strand (also known as the guide strand) of the siRNA to guide the sequence-specific cleavage of the target mRNA.

[0006] However, siRNAs often exhibit varying degrees of off-target effects. One type of off-target effect is miRNA-like, where the seed region (positions 2-8 at the 5' end) of the siRNA antisense strand (AS strand) forms a complete or incomplete pair with the target mRNA, resulting in inhibitory activity against the mRNA. The off-target effect of one siRNA molecule may affect multiple mRNAs. Therefore, unpredictable toxic side effects may occur, which is a major cause of toxic side effects from siRNA drugs (Janas, MM, Schlegel, MK, Harbison, CE et al. Selection of GalNAc-conjugated siRNAs with limited off-target-driven rat hepatotoxicity. Nat Commun 9, 723 (2018)).

[0007] Therefore, reducing off-target risks while maintaining target activity is crucial for improving the safety and efficacy of small nucleic acid drugs. Summary of the Invention

[0008] This invention provides oligonucleotides and RNAi agents containing off-target modifications, which can be used to reduce off-target toxicity, for example, to improve drug safety and efficacy to achieve therapeutic purposes. The modification patterns described herein can be universally applied to various oligonucleotides or RNAi agents with different sequences and targets.

[0009] Specifically, in one aspect, the present invention provides an oligonucleotide or a pharmaceutically acceptable salt thereof, wherein the oligonucleotide internally comprises one, two, three, four, five or more modifying groups of the structure shown in formula (I), or a pharmaceutically acceptable salt, isotopic variant, tautomer or stereoisomer thereof:

[0010] The variables are as defined in this paper.

[0011] In another aspect, the present invention provides a compound of formula (I'), or a pharmaceutically acceptable salt, isotopic variant, tautomer, or stereoisomer thereof, or a mixture thereof:

[0012] The variables are as defined in this paper.

[0013] In another aspect, the present invention provides an RNAi agent, wherein the RNAi agent comprises one, two, three, four, five or more modifying groups of formula (I) described herein;

[0014] Preferably, the nucleotide chain of the RNAi agent includes one, two, three, four, five, six, seven, or eight modifying groups of formula (I) described herein, or pharmaceutically acceptable salts, isotopic variants, tautomers, or stereoisomers thereof.

[0015] In another aspect, the present invention provides a composition comprising one or more oligonucleotides described herein, or RNAi agents described herein.

[0016] In another aspect, the present invention provides a kit comprising one or more oligonucleotides described herein, or RNAi agents described herein.

[0017] In another aspect, the present invention provides a method for inhibiting the expression of a target nucleic acid, the method comprising administering a subject a therapeutic amount of the oligonucleotide, RNAi agent, or composition described herein.

[0018] In another aspect, the present invention provides a method for reducing off-target toxicity in cells, comprising the step of introducing an oligonucleotide or RNAi agent as described herein into the cell.

[0019] In another aspect, the present invention provides a method for reducing off-target toxicity in cells, comprising expressing an oligonucleotide or RNAi agent described herein in the cells.

[0020] In another aspect, the present invention provides the use of compounds of formula (I') described herein, or pharmaceutically acceptable salts, isotopic variants, tautomers or stereoisomers thereof, or mixtures thereof, in reducing off-target effects of oligonucleotides.

[0021] In another aspect, the present invention provides the use of compounds of formula (I') described herein, or pharmaceutically acceptable salts, isotopic variants, tautomers or stereoisomers thereof, or mixtures thereof, in the preparation of oligonucleotide drugs (e.g., RNAi agents).

[0022] In another aspect, the present invention provides a method for treating a disease, the method comprising administering a therapeutic amount of the oligonucleotide, RNAi agent, or composition described herein to a subject who requires treatment for the disease. Attached Figure Description

[0023] Figure 1 shows the expression level of siRNA conjugate TTR mRNA in the liver of C57BL / 6 mice as detected by real-time quantitative PCR. Detailed Implementation

[0024] The above-described features and advantages of the present invention, as well as their additional features and advantages, will become more clearly understood below by taking into account the accompanying drawings and the detailed description of the following embodiments. The embodiments described herein with reference to the accompanying drawings are illustrative, exemplary, and intended for a general understanding of the invention. The embodiments should not be construed as limiting the scope of the invention. Identical or similar elements and elements having the same or similar functions are represented by the same reference numerals throughout the specification.

[0025] In this invention, unless otherwise stated, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the terms and laboratory procedures related to nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, and immunology used herein are all widely used terms and routine procedures in their respective fields. To better understand this invention, definitions and explanations of relevant terms are provided below.

[0026] Invention Details

[0027] definition

[0028] Chemical definition

[0029] The definitions of specific functional groups and chemical terms are described in more detail below.

[0030] 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 C 5-6 alkyl.

[0031] “C 1-6 "Alkyl" refers to a straight-chain or branched saturated hydrocarbon group having 1 to 6 carbon atoms. 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).

[0032] “C 1-6 "Heteroalkyl" refers to a C-type compound containing at least one heteroatom. 1-6 Alkyl groups, with suitable heteroatoms including, but not limited to: O, N, Si, P, Se, B, and S. C 1-6 Examples of heteroalkyl groups can be selected from: -OC 1-6 Alkyl, -C 0-3 Alkyl-OC 0-3 Alkyl, -C 0-2 Alkylene-OC 0-2 Alkylene-OC 0-2 Alkyl groups, for example, -OCH2CH3, -OCH2-OCH2CH3, -CH2OCH2CH3, -CH2CH2OCH2CH3, -CH2CH2O-CH2CH2O-CH2CH3 or -OCH2CHCH2OCH2CH2CH3, etc.

[0033] “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. 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.

[0034] 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. 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.

[0035] “C 6-30 "Hydrocarbon group" refers to the group with C removed. 6-30 The divalent group formed by the other hydrogen atom of the hydrocarbon group can be substituted or unsubstituted.

[0036] “C 1-6 "Alkylene" refers to the removal of C 1-6 The alkyl group is a divalent group formed by another hydrogen atom, and can be substituted or unsubstituted. An exemplary C 1-6 Alkylenes can be selected from C 1-4 Alkylene, C 2-4 Alkylene and C 1-3 Alkylenes. Unsubstituted alkylenes include, but are not limited to: methylene (-CH2-), ethylene (-CH2CH2-), propylene (-CH2CH2CH2-), butylene (-CH2CH2CH2CH2-), pentylene (-CH2CH2CH2CH2CH2-), hexylene (-CH2CH2CH2CH2CH2CH2-), etc. Exemplary substituted alkylenes, for example, alkylenes substituted with one or more alkyl (methyl) groups, include, but are not limited to: substituted methylene (-CH(CH3)-, -C(CH3)2-), substituted ethylene (-CH(CH3)CH2-, -CH2CH(CH3)-, -C(CH3)2CH2-, -CH2C(CH3) 2- ), substituted propylidenes (-CH(CH3)CH2CH2-, -CH2CH(CH3)CH2-, -CH2CH2CH(CH3)-, -C(CH3)2CH2CH2-, -CH2C(CH3)2CH2-, -CH2CH2C(CH3)2-), etc.

[0037] “C 1-6 "Heteroalkyl" refers to the alkyl group after the removal of C24. 1-6 It is a divalent group formed by another hydrogen atom of a heteroalkyl group, and can be substituted or unsubstituted.

[0038] “C 2-6 "Alkenyl" refers to the group that has been de-carbonied. 2-6The alkenyl group is a divalent group formed by the other hydrogen atom, and it can be substituted or unsubstituted.

[0039] “C 2-6 "Iso-ynyl" refers to the group that has the C group removed. 2-6 The alkynyl group is a divalent group formed by the other hydrogen atom, and it can be substituted or unsubstituted.

[0040] "Halogen" or "halogen" refers to fluorine (F), chlorine (Cl), bromine (Br), and iodine (I).

[0041] Therefore, "C" 1-6 "Halogenated alkyl" refers to the above "C 1-6 "alkyl" is substituted with one or more halogen groups. An example C 1- 6-Hydroalkyl groups can be selected from C 1-4 Halogenated alkyl groups and C 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.

[0042] “C 1-6 "Alkoxy" refers to the -OR group, where R is as described above. 1-6 As defined by "alkyl".

[0043] “C 1-6 "Haloalkoxy" refers to the -OR group, where R is as described above as "C". 1-6 The definition of "halogenated alkyl".

[0044] “C 3-10 "Cycloalkyl" refers to a monocyclic or polycyclic non-aromatic cyclic hydrocarbon group having 3 to 10 ring carbon atoms and zero heteroatoms. 3-10 When the cycloalkyl group is monocyclic, it represents a saturated carbocyclic ring with 3 to 10 cyclic carbon atoms and zero heteroatoms. An exemplary C 3-10 Cycloalkyl groups can be selected from C 3-7 cycloalkyl, C 4-7 cycloalkyl, C 3-6 cycloalkyl and C 5-6Cycloalkyl groups. Cycloalkyl groups also include ring systems in which the aforementioned cycloalkyl ring is fused with one or more aryl or heteroaryl groups, wherein the linkage 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.

[0045] "3-10 membered heterocyclic groups" refer to monocyclic or polycyclic groups with a 3- to 10-membered non-aromatic ring system having a ring carbon atom and 1 to 5 ring 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 may be selected, 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 may be selected, 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 may be selected, 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 may be selected, which is a 4-7 membered non-aromatic ring system having a cyclic carbon atom and 1 to 3 cyclic heteroatoms; a 5-6 membered heterocyclic group may be selected, 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 to a C6 aryl ring (also referred to herein as 5,6-bicyclic heterocyclic groups) include, but are not limited to: dihydroindolyl, isodihydroindolyl, dihydrobenzofuranyl, dihydrobenzothiophenyl, benzoxazolinoneyl, etc. Exemplary 6-membered heterocyclic groups fused to a C6 aryl ring (also referred to herein as 6,6-bicyclic heterocyclic groups) include, but are not limited to: tetrahydroquinolinyl, tetrahydroisoquinolinyl, etc. The heterocyclic group may be optionally substituted with one or more substituents, for example, substituted with 1 to 5 substituents, 1 to 3 substituents, or 1 substituent.

[0046] “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.

[0047] "5-10-membered heteroaryl" refers to a 4n+2 aromatic ring system of a 5-10-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. Exemplary 5-10-membered heteroaryl includes 5-6-membered heteroaryl, which is a 4n+2 aromatic ring system of a 5-6-membered monocyclic or bicyclic ring having a ring carbon atom and 1-4 ring 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 thiazolyl. 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, respectively. Exemplary 7-membered heteroaryl groups containing one heteroatom include, but are not limited to: azirheptatrienyl, oxaheptatrienyl, and thioheptatrienyl. Exemplary 5,6-bicyclic heteroaryl groups include, but are not limited to: indolyl, isoindolyl, indazole, benzotriazolyl, benzothiophenyl, isobenzothiophenyl, benzofuranyl, benzoisofuranyl, benzoimidazolyl, benzoxazolyl, benzoisoxazolyl, benzoxadiazolyl, benzothiazolyl, benzoisothiazolyl, benzothiadiazolyl, indazinyl, and purinel. Exemplary 6,6-bicyclic heteroaryl groups include, but are not limited to: naphridinyl, pteridinyl, quinolinyl, isoquinolinyl, zenolinyl, quinoxalinyl, phthalazinyl, and quinazolinyl. The heteroaryl group may be optionally substituted with one or more substituents, for example, substituted with 1 to 5 substituents, 1 to 3 substituents, or 1 substituent.

[0048] The terms "hydrocarbon group", "alkyl group", "alkenyl group", "alkynyl group", "cycloalkyl group", "heterocyclic group", "aryl group" and "heteroaryl group" as defined in this article are optional substituted groups.

[0049] Exemplary substituents on carbon atoms include, but are not limited to: halogen, -CN, -NO2, -N3, -SO2H, -SO3H, -OH, -OR aa、-ON(R bb )2、-N(R bb )2、-N(R bb )3 + X - 、-N(OR cc )R bb 、-SH、-SR aa 、-SSR cc 、-C(=O)R aa 、-CO2H、-CHO、-C(OR cc )2、-CO2R aa 、-OC(=O)R aa 、-OCO2R aa 、-C(=O)N(R bb )2、-OC(=O)N(R bb )2、-NR bb C(=O)R aa 、-NR bb CO2R aa 、-NR bb C(=O)N(R bb )2、-C(=NR bb )R aa 、-C(=NR bb )OR aa 、-OC(=NR bb )R aa 、-OC(=NR bb )OR aa 、-C(=NR bb )N(R bb )2、-OC(=NR bb )N(R bb )2、-NR bb C(=NR bb )N(R bb )2、-C(=O)NR bb SO2R aa 、-NR bb SO2R aa 、-SO2N(R bb )2、-SO2R aa 、-SO2OR aa 、-OSO2R aa 、-S(=O)R aa 、-OS(=O)R aa 、-Si(R aa )3、-OSi(R aa )3、-C(=S)N(R bb )2、-C(=O)SR aa-C(=S)SR aa -SC(=S)SR aa -SC(=O)SR aa -OC(=O)SR aa -SC(=O)OR aa -SC(=O)R aa -P(=O)2R aa -OP(=O)2R aa -P(=O)(R aa )2、-OP(=O)(R aa )2、-OP(=O)(OR cc )2、-P(=O)2N(R bb )2、-OP(=O)2N(R bb )2、-P(=O)(NR bb )2、-OP(=O)(NR bb )2、-NR bb P(=O)(OR cc )2、-NR bb P(=O)(NR bb )2、-P(R cc )2、-P(R cc )3、-OP(R cc )2、-OP(R cc )3、-B(R aa 2. -B(OR) cc )2、-BR aa (OR cc ), alkyl, haloalkyl, alkenyl, ynyl, cycloalkyl, heterocyclic, aryl, and heteroaryl, wherein each alkyl, alkenyl, ynyl, cycloalkyl, heterocyclic, aryl, and heteroaryl is independently bounded by 0, 1, 2, 3, 4, or 5 R groups. dd Group substitution;

[0050] 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;

[0051] R aa Each of them is independently selected from alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, heterocyclic, aryl, and heteroaryl, or two R aaGroups 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;

[0052] 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;

[0053] R cc Each is independently selected from hydrogen, alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, heterocyclic, aryl, and heteroaryl, or two R cc Groups are combined to form heterocyclic or heteroaryl rings, wherein each alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclic, aryl, and heteroaryl group is independently bounded by 0, 1, 2, 3, 4, or 5 R groups. dd Group substitution;

[0054] R dd Each 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)2R ee 、-P(=O)(R ee )2、-OP(=O)(R ee )2、-OP(=O)(OR ee2. 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;

[0055] 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;

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

[0057] 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 1-6 Alkyl group, -CO2H, -CO2(C 1-6 Alkyl), -OC (=O)(C 1-6 Alkyl), -OCO2(C 1-6Alkyl 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 1-6 Alkyl)2、-OP(=O)(OC 1-6 Alkyl)2, C 1-6 Alkyl, C 1-6Haloalkyl, 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.

[0058] 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.

[0059] As used herein, unless otherwise stated, the divalent structure can be attached to the remainder of the compound in either a left-to-right or right-to-left direction. In one embodiment, the divalent structure can be attached to the remainder of the compound in a left-to-right direction. For example, when the linking group L in ALB is -MW-, -MW- can be attached to variables A and B in the same direction in a left-to-right reading order, forming A-MW-B, or it can be attached to rings A and B in a right-to-left reading order, forming A-WM-B.

[0060] When one of the variables is a single bond (i.e., a chemical bond), it indicates that the two groups connected by that single bond are directly linked. For example, when L in ALB represents a single bond, the structure of ALB is actually AB. In the groups of formulas (II-1), (II-2), or (II-3) described herein, when R2 is a chemical bond, it indicates that the compound exists in ionic form.

[0061] The term “substitution” refers to the replacement of one or more (e.g., 1, 2, 3, or 4) hydrogen atoms on a specified atom by a choice of indicated groups, provided that the substitution does not exceed the normal valence of the specified atom in the present case and the substitution forms a stable compound.

[0062] The term "optionally substituted" means that a group is not substituted or is substituted by a specific group or atomic group.

[0063] If a substituent is described as being “independently selected” from a group, then each substituent is selected independently of the others. Therefore, each substituent may be the same as or different from another (other) substituent.

[0064] As used in (II-1), (II-2) or (II-3) and their sub-formulas, the tilde connecting key Or dashed connection key This indicates the linking site of the group.

[0065] When the bond of a substituent is such that it passes through the ring and connects two atoms, then such a substituent can be bonded to any cyclic atom in the substituted ring.

[0066] This article uses solid lines (—) and solid wedges. Or virtual wedge The covalent chemical bonds of the compounds of the present invention are depicted. When solid lines are used to depict bonds to chiral atoms, it indicates that all possible stereoisomers at that chiral atom are included (e.g., specific enantiomers, racemic mixtures, etc.). When solid or dashed wedges are used to depict bonds to chiral atoms, it indicates the presence of the shown stereoisomers. Unless otherwise specified, the stereoisomers of the compounds of the present invention may encompass specific enantiomers, diastereomers, racemic mixtures, or mixtures thereof in any proportion.

[0067] The terms “including,” “comprising,” “having,” “containing,” or “involving,” and their other variations herein, are inclusive or open-ended and do not exclude other unlisted elements or method steps.

[0068] It should also be understood that certain compounds of the present invention may be present in a free form for therapeutic purposes, or, where appropriate, in the form of their pharmaceutically acceptable derivatives. According to the present invention, pharmaceutically acceptable derivatives include, but are not limited to, pharmaceutically acceptable salts, solvates, metabolites, or prodrugs, which, upon administration to a patient in need, can directly or indirectly provide the compounds of the present invention or their metabolites or residues.

[0069] The term "prevention" refers to methods implemented to prevent or delay the occurrence of a disease, condition, or symptom (e.g., a tumor) in a subject's body.

[0070] The terms “disease,” “disorder,” and “symptom” are used interchangeably herein. The term “treatment” aims to alleviate or eliminate the targeted disease state or symptom. A subject is successfully “treated” if, in accordance with the methods described herein, a subject receives a therapeutic amount of a compound, its optical isomer, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof, and the subject exhibits an observable and / or detectable reduction or improvement in one or more indications and symptoms. It should also be understood that treatment of the disease state or symptom includes not only complete treatment but also the achievement of some biological or medically relevant outcome without achieving complete treatment.

[0071] The term "subject" in the administration includes, but is not limited to: humans (i.e., men or women of any age group, e.g., pediatric subjects (e.g., infants, children, adolescents) or adult subjects (e.g., young adults, middle-aged adults, or older adults)) and / or non-human animals, such as mammals, e.g., primates (e.g., cynomolgus monkeys, rhesus monkeys), cattle, pigs, horses, sheep, goats, rodents, cats, and / or dogs. In some embodiments, the subject is a human. In some embodiments, the subject is a non-human animal. The terms "human," "patient," and "subject" are used interchangeably herein.

[0072] Generally, the "effective amount" of a compound refers to the amount sufficient to elicit a target biological response. As will be understood by those skilled in the art, the effective amount of the compounds of the present invention can vary depending on factors such as the biological target, the pharmacokinetics of the compound, the disease being treated, the administration method, and the age, health status, and symptoms of the subject. Effective amounts include therapeutic effective amounts and prophylactic effective amounts.

[0073] Other definitions

[0074] The term “oligonucleotide” refers to a nucleic acid molecule (RNA or DNA) having a length of less than 100, 200, 300 or 400 nucleotides, for example. The oligonucleotide can be a single-stranded oligonucleotide (e.g., antisense oligonucleotide) or a double-stranded oligonucleotide (e.g., small interfering RNA or short hairpin RNA).

[0075] The term "antisense oligonucleotide" (ASO) refers to a single-stranded DNA or RNA sequence consisting of 15-25 nucleotides that are paired with a target gene. It achieves gene regulation by specifically blocking the transcription or translation of the target gene.

[0076] 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.

[0077] The term "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. This is followed by the addition of 5-6 T molecules as a transcription terminator for RNA polymerase III.

[0078] In some embodiments, the oligonucleotides described herein are RNAi agents. An “RNAi agent” is an agent containing an RNA or RNA-like (e.g., chemically modified RNA) oligonucleotide molecule capable of reducing or inhibiting the translation of mRNA transcripts of target messenger RNA (mRNA) in a sequence-specific manner. As used herein, RNAi agents can function through RNA interference mechanisms (e.g., by inducing RNA interference through interaction with RNA interference pathway mechanisms in mammalian cells (RNA-induced silencing complexes or RISC)) or any other mechanism or pathway. While the term RNAi agent as used herein is considered to function primarily through RNA interference mechanisms, the RNAi agent is not limited to or restricted to any particular pathway or mechanism of action. RNAi agents include, but are not limited to, single-stranded oligonucleotides, single-stranded antisense oligonucleotides, short interfering RNA (siRNA), double-stranded RNA (dsRNA), microRNA (miRNA), short hairpin RNA (shRNA), and dicer enzyme substrates. The RNAi agents described herein consist of oligonucleotides having a strand at least partially complementary to the target mRNA. In some embodiments, the RNAi agent described herein is double-stranded and consists of an antisense strand and a sense strand at least partially complementary to the antisense strand. The RNAi agent may be composed of modified nucleotides and / or one or more non-phosphodiester linkages. In some embodiments, the RNAi agent is single-stranded.

[0079] The terms “silence,” “reduction,” “inhibition,” “downregulation,” or “knockdown” when referring to the expression of a given gene mean that, when a cell, cell cluster, tissue, organ, or object is treated with an oligomer compound such as an RNAi agent described herein, the expression of the gene is reduced compared to a second cell, cell cluster, tissue, organ, or object that has not been so treated, and the expression of the gene is determined by the level of RNA transcribed from the gene or the level of polypeptides, proteins, or protein subunits translated from mRNA in a cell, cell cluster, tissue, organ, or object in which the transcription of the gene has occurred.

[0080] 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.

[0081] 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.

[0082] 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.

[0083] A polynucleotide that is “at least partially complementary,” “fully complementary,” or “substantially complementary” to messenger RNA (mRNA) refers to a polynucleotide that is substantially complementary to a continuous portion of the mRNA of interest. For example, a polynucleotide is at least partially complementary to PCSK9 mRNA if its sequence is substantially complementary to an uninterrupted portion of the mRNA encoding PCSK9. The terms “complementary,” “fully complementary,” “fully complementary,” and “substantially complementary” can be used relative to base pairing between the sense and antisense strands of the siRNA, or between the antisense strand of the siRNA reagent and the target sequence.

[0084] "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.

[0085] "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.

[0086] Nucleosides are compounds composed of purine or pyrimidine bases and ribose or deoxyribose, while nucleotides are compounds composed of purine or pyrimidine bases, ribose or deoxyribose, and phosphate.

[0087] 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.

[0088] 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.

[0089] 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.

[0090] A "reactive phosphorus group" refers to a phosphorus-containing group contained in a nucleotide unit or a nucleotide analog unit that can react with a hydroxyl or amino group contained in another molecule, particularly in another nucleotide unit or another nucleotide analog, via a nucleophilic attack reaction. Typically, such a reaction produces an ester-type nucleoside bond linking the first nucleotide unit or the first nucleotide analog unit to the second nucleotide unit or the second nucleotide analog unit. The reactive phosphorus group can be selected from phosphorous amides, H-phosphonates, alkyl-phosphonates, phosphate esters, or phosphate ester analogs, including but not limited to: native phosphate esters, thiophosphate esters, dithiophosphate esters, boron phosphate esters, boron thiophosphate esters, phosphonates, halogen-substituted phosphonates and phosphate esters, aminophosphate esters, phosphate diesters, phosphate triesters, thiophosphate diesters, thiophosphate triesters, diphosphate esters, and triphosphate esters, such as -P(OCH2CH2CN)(N(iPr)2).

[0091] 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.

[0092] 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). Exemplary 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, and 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).

[0093] 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. Examples of hydroxyl protecting groups are as follows:

[0094] Trimethylsilyl (TMS), Triethylsilyl (TES), Dimethylisopropylsilyl (DMIPS), Diethylisopropylsilyl (DEIPS), Tert-butyldimethylsilyl (TBDMS), Tert-butyldiphenylsilyl (TBDPS), Triisopropylsilyl (TIPS), Acetyl (Ac), Chloroacetyl, Dichloroacetyl, Trichloroacetyl, Trifluoroacetyl (TFA), Benzoyl, p-Methoxybenzoyl, 9-fluorenylmethoxycarbonyl (Fmoc), Allyloxycarbonyl (Alloc), 2,2,2-Trichloroethoxycarbonyl The following are listed: Troc, benzyloxycarbonyl (Cbz), tert-butyloxycarbonyl (Boc), benzyl (Bn), p-methoxybenzyl (PMB), allyl, triphenylmethyl (Tr), bis-p-methoxytriphenylmethyl (DMTr), methoxymethyl (MOM), phenoxymethyl (BOM), 2,2,2-trichloroethoxymethyl, 2-methoxyethoxymethyl (MEM), methylthiomethyl (MTM), p-methoxybenzyloxymethyl (PMBM), -C(O)CH2CH2C(O)OH, or 4,4'-dimethoxytriphenylmethyl.

[0095] 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.

[0096] 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.

[0097] 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.

[0098] The present invention also includes isotopically labeled compounds (isotopic variants) that are equivalent to those described in formula (I) or formula (II'), 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.

[0099] Oligonucleotides containing the modified group of formula (I)

[0100] In this article, “formula (I) modifying group” and “formula (I) group” refer to formula (I) group (including sub-formulas, such as formula (I-1), (I-2), (II), (II-1), (II-2), (III), (III-1) or (III-2)), or pharmaceutically acceptable salts, isotopic variants, tautomers or stereoisomers, and mixtures thereof.

[0101] In one embodiment, the present invention relates to an oligonucleotide or a pharmaceutically acceptable salt thereof, wherein the oligonucleotide internally comprises one, two, three, four, five or more (e.g., one, two, three, four, five, six, seven, eight, nine or ten) modifying groups of the structure shown in Formula (I), or a pharmaceutically acceptable salt, isotopic variant, tautomer or stereoisomer thereof:

[0102] in,

[0103] Ring A is selected from C 0-6 Alkylene-C 3-10 Cycloalkyl-C 0-6 Alkylene, C 0-6 alkylene-3-10-membered heterocyclic-C 0-6 Alkylene, C 0-6 Alkylene-C 6-10 Aspartic-C 0-6 Alkylene or C 0-6 alkylene-5-10-membered heteroaryl-C 0-6 Alkylene, wherein the ring A is optionally surrounded by 1, 2, 3, 4, 5 or 6 R atoms. a replace;

[0104] L1 is selected from C 1-6 Alkylene, C 1-6 Heteroalkyl, C 2-6 imidene group, C 2-6 Ethyne group, C 3-10 Cycloalkylene, 3-10 membered heterocyclic alkylene, C 6-10 The L1 is optionally surrounded by 1, 2, 3, 4, 5, or 6 R atoms. b replace;

[0105] L2 is selected from C 1-6 Alkylene, NH-C 0-6 Alkylene, C(O)-C 0-6 Alkylene, NHC(O)-C 0-6 Alkylene, C 2-6 imidene group, C 2-6 Ethyne group, C 3-10 Cycloalkylene, 3-10 membered heterocyclic alkylene, C 6-10 The L2 is optionally surrounded by 1, 2, 3, 4, 5, or 6 R groups. c replace;

[0106] X is selected from O or S;

[0107] Y is OR d ;

[0108] Base is selected from modified or unmodified bases;

[0109] Each R a Independently selected from H, halogen, OR, CN, NRR', C 1-6 Alkyl, C 1-6 Haloalkyl, C 1-6 Alkoxy or C 1-6 Halogenated alkoxy groups;

[0110] Each R b Independently selected from H, halogen, OR, CN, NRR', C 1-6 Alkyl, C 1-6 Haloalkyl, C 1-6 Alkoxy, C 1-6 Haloalkoxy groups, or two R atoms on the same or different carbon atoms. b They are connected, and together with the carbon atoms they are connected to, they form C. 3-7 Cycloalkylene;

[0111] Each R c Independently selected from H, halogen, OR, CN, NRR', C 1-6 Alkyl, C 1-6 Haloalkyl, C 1-6 Alkoxy, C 1-6 Haloalkoxy groups, or two R atoms on the same or different carbon atoms. c They are connected, and together with the carbon atoms they are connected to, they form C. 3-7 Cycloalkylene;

[0112] R d Selected from H, halogens, C 1-6 Alkyl or C 1-6 Halogenated alkyl groups;

[0113] R and R' are independently selected from H, halogens, and C. 1-6 Alkyl, C 2-6 alkenyl, C 2-6 alkynyl or C 1-6 Haloalkyl groups, or R and R', are attached to the nitrogen atom to which they are attached, together forming 3-7 membered heterocyclic groups;

[0114] The compound of formula (I) may optionally be further substituted by one, two, three, four or five substituents selected from the following:

[0115] H, halogens, OH, CN, NH2, C 1-6 Alkyl, C 2-6 alkenyl, C 2-6 alkynyl group, C 1-6 Haloalkyl, C 1-6 Alkoxy or C1-6 Halogenated alkoxy groups.

[0116] In one embodiment, the present invention relates to the above-described oligonucleotide or a pharmaceutically acceptable salt thereof, wherein the modifying group of the structure shown in formula (I) has the following structure:

[0117] The variables are as defined in this paper.

[0118] X

[0119] In one implementation, X is O; in another implementation, X is S.

[0120] Y

[0121] In one implementation, Y is OR d For example, OH.

[0122] L1

[0123] In one implementation, L1 is selected from C. 1-6 Alkylene, C 1-6 Heteroalkyl, C 2-6 imidene group, C 2-6 Ethyne group, C 3-10 The L1 is optionally surrounded by 1, 2, 3, 4, or 5 R groups, or a cycloalkyl or 3-10-membered heterocyclic group. b replace.

[0124] In one implementation, L1 is selected from C. 1-6 Alkylene, C 1-6 Heteroalkyl, C 2-6 imidene group, C 2-6 Ethyne group, C 3-10 The L1 is optionally surrounded by 1, 2, 3, 4, or 5 R groups, or a cycloalkyl or 3-10-membered heterocyclic group. b replace.

[0125] In one implementation, L1 is selected from C. 1-6 Alkylene, C 2-6 imide or C 2-6 The ynylene group, wherein the L1 is optionally surrounded by one, two, or three R groups. b replace.

[0126] In one specific implementation, L1 is C 1-6 Alkylene, such as -CH2-; in another embodiment, L1 is C 1- 6 heteroalkylene, such as -OCH2-; in another embodiment, L1 is C 2-6 Ideonyl; in another embodiment, L1 is C2-6 Idemynyl; in another embodiment, L1 is C 3-10 Cycloalkylene, such as C 3-7 Cycloalkylene, preferably C 3-5 Cycloalkylene; in another embodiment, L1 is a 3-10 membered heterocyclic group, for example a 3-7 membered heterocyclic group, preferably a 3-5 membered heterocyclic group.

[0127] In one specific implementation, L1 is not replaced; in another specific implementation, L1 is replaced by one, two, three, four, five, or six independently selected R... b replace.

[0128] L2

[0129] In one implementation, L2 is selected from C 1-6 Alkylene, NH-C 0-6 Alkylene, C(O)-C 0-6 Alkylene, NHC(O)-C 0- 6-alkylene, C 2-6 imidene group, C 2-6 Ethyne group, C 3-10 The L2 is optionally surrounded by 1, 2, 3, 4, or 5 R groups. c replace.

[0130] In one implementation, L2 is selected from C 1-6 Alkylene, NH-C 0-6 Alkylene, C(O)-C 0-6 Alkylene, NHC(O)-C 0- 6-alkylene, C 2-6 imidene group, C 2-6 Ethyne group, C 3-10 The L2 is optionally surrounded by 1, 2, 3, 4, or 5 R groups. c replace.

[0131] In one implementation, L2 is selected from C 1-6 Alkylene, C 2-6 imidene group, C 2-6 Ethyne group or NHC(O)-C 1-6 Alkylene, wherein the L2 is optionally surrounded by one, two or three R atoms. c replace.

[0132] In one specific implementation, L2 is C 1-6 Alkylene, such as -CH2-; in another embodiment, L2 is -NH-C 0-6Alkylene, such as -NH-; in another embodiment, L2 is -C(O)-C 0-6 Alkylene-, for example -C(O)-; in another embodiment, L2 is -NHC(O)-C 0-6 Alkylene, such as -NHC(O)-; in another embodiment, L2 is C 2-6 Ideonyl; in another embodiment, L2 is C 2-6 Alkyne group; in another embodiment, L2 is C 3-10 Cycloalkylene, such as C 3-7 Cycloalkylene, preferably C 3-5 Cycloalkylene; in another embodiment, L2 is a 3-10 membered heterocyclic group, for example a 3-7 membered heterocyclic group, preferably a 3-5 membered heterocyclic group.

[0133] In one specific implementation, L2 is not replaced; in another specific implementation, L2 is replaced by one, two, three, four, five, or six independently selected R... c replace.

[0134] Ring A

[0135] In one implementation, ring A is selected from C. 0-6 Alkylene-C 3-10 Cycloalkyl-C 0-6 Alkylene, C 0-6 alkylene-3-10-membered heterocyclic-C 0-6 Alkylene, C 0-6 Alkylene-C 6-10 Aspartic-C 0-6 Alkylene or C 0-6 alkylene-5-10-membered heteroaryl-C 0- 6 alkylene groups, wherein the ring A is optionally surrounded by 1, 2, 3, 4, 5, or 6 R groups. a replace.

[0136] In one implementation, ring A is C. 3-10 Cycloalkylene, 3-10 membered heterocyclic alkylene, C 6-10 arylene or 5-10 heteroarylene, wherein the ring A is optionally surrounded by one, two or three R atoms. a replace.

[0137] In one implementation, ring A is selected from C. 3-7 Cycloalkylene or 3-7 membered heterocyclic group, wherein ring A is optionally surrounded by 1, 2 or 3 R groups. a replace.

[0138] In one implementation, ring A is C. 3-5Cycloalkylene, preferably cyclopropylene, optionally surrounded by one or two atoms selected from H and C. 1-4 Alkyl substituents.

[0139] In one specific implementation, ring A is C. 0-6 Alkylene-C 3-10 Cycloalkyl-C 0-6 Alkylene; in another specific embodiment, ring A is C 3-10 Cycloalkylene, such as C 3-7 Cycloalkylene, preferably C 3-5 Cycloalkylene; in another specific embodiment, ring A is C 0-6 alkylene-3-10-membered heterocyclic-C 0-6 Alkylene; in another specific embodiment, ring A is a 3-10 membered heterocyclic group, for example a 3-7 membered heterocyclic group, preferably a 3-5 membered heterocyclic group; in another specific embodiment, ring A is C 0-6 Alkylene-C 6-10 Aspartic-C 0-6 Alkylene, such as C 6-10 Aromatic; in another specific embodiment, ring A is C 0-6 alkylene-5-10-membered heteroaryl-C 0-6 Alkylenes, such as 5-10 alkylenes.

[0140] In one specific implementation, ring A is not replaced; in another specific implementation, ring A is replaced by one, two, three, four, five, or six independently selected R... a replace.

[0141] Base

[0142] In one implementation, Base is selected from modified or unmodified bases.

[0143] In one implementation, Base is selected from...

[0144] R a

[0145] In one implementation scheme, each R a Independently selected from H, halogens, OR (e.g., OH), CN, NRR' (e.g., NH2), C 1-6 Alkyl, C 1-6 Haloalkyl, C 1-6 Alkoxy or C 1-6 Halogenated alkoxy groups.

[0146] In one implementation scheme, each R a Independently selected from H, halogens, C1-4 Alkyl or C 1-4 Halogenated alkyl groups.

[0147] In one specific implementation plan, R a For H; in another specific implementation, R a For halogen; in another specific implementation, R a For example, OH; in another specific implementation, R is OR. a For CN; in another specific implementation, R a For NRR', such as NH2; in another specific embodiment, R a C 1-6 Alkyl, such as C 1-4 Alkyl; in another specific embodiment, R a C 1-6 Haloalkyl, such as C 1-4 Halogenated alkyl; in another specific embodiment, R a C 1-6 Alkyl group; in another specific embodiment, R a C 1-6 Halogenated alkoxy groups.

[0148] R b and R b '

[0149] In one implementation scheme, each R b Independently selected from H, halogens, OR (e.g., OH), CN, NRR' (e.g., NH2), C 1-6 Alkyl, C 1-6 Haloalkyl, C 1-6 Alkoxy, C 1-6 Haloalkoxy groups, or two R atoms on the same or different carbon atoms. b They are connected, and together with the carbon atoms they are connected to, they form C. 3-7 Cycloalkylene.

[0150] In one implementation scheme, each R b and R b 'Independently selected from H, halogens, OH, CN, NH2, C 1-6 Alkyl, C 1- 6-Hydroalkyl, C 1-6 Alkoxy, C 1-6 Haloalkoxy groups, or two R atoms on the same or different carbon atoms. b (or R) b and R b ') are connected together, and together with the carbon atoms they are connected to, they form C. 3-7 Cycloalkylene.

[0151] In one specific implementation plan, R b For H; in another specific implementation, R b For halogen; in another specific implementation, R b For example, OH; in another specific implementation, R is OR. b For CN; in another specific implementation, R b For NRR', such as NH2; in another specific embodiment, R b C 1-6 Alkyl, such as C 1-4 Alkyl; in another specific embodiment, R b C 1-6 Haloalkyl, such as C 1-4 Halogenated alkyl; in another specific embodiment, R b C 1-6 Alkyl group; in another specific embodiment, R b C 1-6 Halogenated alkoxy groups; in another specific embodiment, two R groups on the same or different carbon atoms b They are connected, and together with the carbon atoms they are connected to, they form C. 3-7 Cycloalkylene, such as C 3-5 Cycloalkylene.

[0152] In one specific implementation plan, R b 'For H; in another specific implementation, R b 'For halogen; in another specific implementation, R b 'For OR, such as OH; in another specific implementation, R b 'For CN; in another specific implementation, R b 'For NRR', for example, NH2; in another specific implementation, R b 'For C 1-6 Alkyl, such as C 1-4 Alkyl; in another specific embodiment, R b 'For C 1-6 Haloalkyl, such as C 1-4 Halogenated alkyl; in another specific embodiment, R b 'For C 1-6 Alkyl group; in another specific embodiment, R b 'For C 1-6 Halogenated alkoxy groups; in another specific embodiment, R on the same or different carbon atoms b and R b'Connected, together with the carbon atoms they are connected to, form C' 3-7 Cycloalkylene, such as C 3-5 Cycloalkylene.

[0153] R c and R c '

[0154] In one implementation scheme, each R c Independently selected from H, halogens, OR (e.g., OH), CN, NRR' (e.g., NH2), C 1-6 Alkyl, C 1-6 Haloalkyl, C 1-6 Alkoxy, C 1-6 Haloalkoxy groups, or two R atoms on the same or different carbon atoms. c They are connected, and together with the carbon atoms they are connected to, they form C. 3-7 Cycloalkylene.

[0155] In one implementation scheme, each R c and R c 'Independently selected from H, halogens, OH, CN, NH2, C 1-6 Alkyl, C 1- 6-Hydroalkyl, C 1-6 Alkoxy, C 1-6 Haloalkoxy groups, or two R atoms on the same or different carbon atoms. c (or R) c and R c ') are connected together, and together with the carbon atoms they are connected to, they form C. 3-7 Cycloalkylene.

[0156] In one specific implementation plan, R c For H; in another specific implementation, R c For halogen; in another specific implementation, R c For example, OH; in another specific implementation, R is OR. c For CN; in another specific implementation, R c For NRR', such as NH2; in another specific embodiment, R c C 1-6 Alkyl, such as C 1-4 Alkyl; in another specific embodiment, R c C 1-6 Haloalkyl, such as C 1-4 Halogenated alkyl; in another specific embodiment, R c C 1-6 Alkyl group; in another specific embodiment, R c C1-6 Halogenated alkoxy groups; in another specific embodiment, two R groups on the same or different carbon atoms c They are connected, and together with the carbon atoms they are connected to, they form C. 3-7 Cycloalkylene, such as C 3-5 Cycloalkylene.

[0157] In one specific implementation plan, R c 'For H; in another specific implementation, R c 'For halogen; in another specific implementation, R c 'For OR, such as OH; in another specific implementation, R c 'For CN; in another specific implementation, R c 'For NRR', for example, NH2; in another specific implementation, R c 'For C 1-6 Alkyl, such as C 1-4 Alkyl; in another specific embodiment, R c 'For C 1-6 Haloalkyl, such as C 1-4 Halogenated alkyl; in another specific embodiment, R c 'For C 1-6 Alkyl group; in another specific embodiment, R c 'For C 1-6 Halogenated alkoxy groups; in another specific embodiment, R on the same or different carbon atoms c and R c 'Connected, together with the carbon atoms they are connected to, form C' 3-7 Cycloalkylene, such as C 3-5 Cycloalkylene.

[0158] R d

[0159] In one implementation, R d Selected from H, halogens, C 1-6 Alkyl or C 1-6 Halogenated alkyl; in another embodiment, R d Selected from H, halogens, C 1-4 Alkyl or C 1-4 Halogenated alkyl groups.

[0160] In one specific implementation plan, R d For H; in another implementation, R d For halogen; in another embodiment, R d C 1-6 Alkyl, such as C 1-4Alkyl; in another embodiment, R d C 1-6 Haloalkyl, such as C 1-4 Halogenated alkyl groups.

[0161] R and R'

[0162] In one implementation, R and R' are independently selected from H, halogen, C. 1-6 Alkyl, C 2-6 alkenyl, C 2-6 alkynyl or C 1-6 Haloalkyl groups, or R and R', are attached to the nitrogen atom to which they are attached, together forming 3-7 membered heterocyclic groups.

[0163] In one specific implementation, R is H; in another implementation, R is a halogen; in yet another implementation, R is C. 1-6 Alkyl, such as C 1-4 Alkyl; in another embodiment, R is C 2-6 Alkenyl; in another embodiment, R is C 2-6 Alkyne group; in another embodiment, R is C 1-6 Haloalkyl, such as C 1-4 Halogenated alkyl groups.

[0164] In one specific implementation, R' is H; in another implementation, R' is a halogen; in yet another implementation, R' is C. 1-6 Alkyl, such as C 1-4 Alkyl; in another embodiment, R' is C 2-6 Alkenyl; in another embodiment, R' is C 2-6 Alkyne group; in another embodiment, R' is C 1-6 Haloalkyl, such as C 1-4 Halogenated alkyl groups.

[0165] In another specific embodiment, R and R' are attached to the nitrogen atom to which they are attached, together forming a 3-7 membered heterocyclic group, such as a 3-5 membered heterocyclic group.

[0166] m

[0167] In one implementation, m is selected from 0, 1, 2, or 3.

[0168] Any technical solution or any combination thereof in any of the above specific embodiments can be combined with any technical solution or any combination thereof in other specific embodiments. For example, any technical solution or any combination thereof of X can be combined with Y, L1, L2, ring A, Base, R. a R b R b'、R c R c '、R d This invention relates to any technical solution, or any combination thereof, including R, R', and m. The invention aims to include combinations of all these technical solutions; however, due to space limitations, they are not listed individually.

[0169] In one specific embodiment, the present invention relates to the above-mentioned oligonucleotide or a pharmaceutically acceptable salt thereof, wherein...

[0170] Ring A is C 3-10 Cycloalkylene, 3-10 membered heterocyclic alkylene, C 6-10 arylene or 5-10 heteroarylene, wherein the ring A is optionally surrounded by one, two or three R atoms. a replace;

[0171] Each R a Independently selected from H, halogens, OH, CN, NH2, C 1-6 Alkyl, C 1-6 Haloalkyl, C 1-6 Alkoxy, C 1-6 Halogenated alkoxy groups;

[0172] Preferably,

[0173] Ring A is selected from C 3-7 Cycloalkylene or 3-7 membered heterocyclic group, wherein ring A is optionally surrounded by 1, 2 or 3 R groups. a replace;

[0174] Each R a Independently selected from H, halogens, C 1-4 Alkyl or C 1-4 Halogenated alkyl groups;

[0175] More preferably,

[0176] Ring A is C 3-5 Cycloalkylene, preferably cyclopropylene, optionally surrounded by one or two atoms selected from H and C. 1-4 Alkyl substituents.

[0177] In one specific embodiment, the present invention relates to the above-mentioned oligonucleotide or a pharmaceutically acceptable salt thereof, wherein...

[0178] Ring A is C 3-10 Cycloalkylene, 3-10 membered heterocyclic alkylene, C 6-10 arylene or 5-10 heteroarylene, wherein the ring A is optionally surrounded by one, two or three R atoms. a replace;

[0179] L1 is selected from C 1-6 Alkylene, C2-6 imide or C 2-6 The ynylene group, wherein the L1 is optionally surrounded by one, two, or three R groups. b replace;

[0180] L2 is selected from C 1-6 Alkylene, C 2-6 imidene group, C 2-6 Ethyne group or NHC(O)-C 1-6 Alkylene, wherein the L2 is optionally surrounded by one, two or three R atoms. c replace;

[0181] X is selected from O or S;

[0182] Y is OR d ;

[0183] Base is selected from modified or unmodified bases;

[0184] Each R a Independently selected from H, halogens, OH, CN, NH2, C 1-6 Alkyl, C 1-6 Haloalkyl, C 1-6 Alkoxy, C 1-6 Halogenated alkoxy groups;

[0185] Each R b and R b 'Independently selected from H, halogens, OH, CN, NH2, C 1-6 Alkyl, C 1-6 Haloalkyl, C 1-6 Alkoxy, C 1-6 Haloalkoxy groups, or two R atoms on the same or different carbon atoms. b (or R) b and R b ') are connected together, and together with the carbon atoms they are connected to, they form C. 3-7 Cycloalkylene;

[0186] Each R c and R c 'Independently selected from H, halogens, OH, CN, NH2, C 1-6 Alkyl, C 1-6 Haloalkyl, C 1-6 Alkoxy, C 1-6 Haloalkoxy groups, or two R atoms on the same or different carbon atoms. c (or R) c and R c ') are connected together, and together with the carbon atoms they are connected to, they form C. 3-7 Cycloalkylene;

[0187] Rd Selected from H, halogens, C 1-6 Alkyl or C 1-6 Halogenated alkyl groups.

[0188] In one specific embodiment, the present invention relates to the above-mentioned oligonucleotide or a pharmaceutically acceptable salt thereof, wherein...

[0189] Ring A is selected from C 3-7 Cycloalkylene or 3-7 membered heterocyclic group, wherein ring A is optionally surrounded by 1, 2 or 3 R groups. a replace;

[0190] L1 is C 1-6 Alkylene, wherein the L1 is optionally surrounded by one, two or three R atoms. b replace;

[0191] L2 is C 1-6 Alkylene, wherein the L2 is optionally surrounded by one, two or three R atoms. c replace;

[0192] X is selected from O or S, preferably O;

[0193] Y is OR d ;

[0194] Base is selected from modified or unmodified bases;

[0195] Each R a Independently selected from H, halogens, C 1-4 Alkyl or C 1-4 Halogenated alkyl groups;

[0196] Each R b and R b 'Independently selected from H, halogen, C 1-4 Alkyl or C 1-4 Halogenated alkyl groups;

[0197] Each R c and R c 'Independently selected from H, halogen, C 1-4 Alkyl or C 1-4 Halogenated alkyl groups;

[0198] R d Selected from H, halogens, C 1-4 Alkyl or C 1-4 Halogenated alkyl groups;

[0199] Preferably,

[0200] Ring A is C 3-5 Cycloalkylene, preferably cyclopropylene;

[0201] L1 is C 1-4Alkylene, preferably -CH2-;

[0202] L2 is C 1-4 Alkylene, preferably -CH2-;

[0203] X is selected from O or S, preferably O;

[0204] Y is selected from OH or C. 1-4 Alkyl group, preferably OH;

[0205] Base selected

[0206] In one specific embodiment, the present invention relates to the above-mentioned oligonucleotide or a pharmaceutically acceptable salt thereof, wherein the modifying group of formula (I) is selected from the structure of formula (III), formula (III-1) or formula (III-2), a pharmaceutically acceptable salt thereof, an isotopic variant, a tautomer or a stereoisomer:

[0207] in,

[0208] Ring A is selected from C 3-7 Cycloalkylene or 3-7 membered heterocyclic group, wherein ring A is optionally surrounded by 1, 2 or 3 R groups. a replace;

[0209] X is selected from O or S, preferably O;

[0210] Base is selected from modified or unmodified bases;

[0211] Each R a Independently selected from H, halogens, C 1-4 Alkyl or C 1-4 Halogenated alkyl groups;

[0212] R b and R b 'Independently selected from H, halogen, C 1-4 Alkyl or C 1-4 Halogenated alkyl groups;

[0213] R c and R c 'Independently selected from H, halogen, C 1-4 Alkyl or C 1-4 Halogenated alkyl groups;

[0214] R d Selected from H, halogens, C 1-4 Alkyl or C 1-4 Halogenated alkyl groups;

[0215] Preferably,

[0216] Ring A is C 3-5Cycloalkylene, preferably cyclopropylene, wherein ring A is optionally surrounded by one R a replace;

[0217] R a Selected from H, halogen or C 1-4 alkyl;

[0218] R b and R b 'Independently selected from H, halogen or C 1-4 alkyl;

[0219] R c and R c 'Independently selected from H, halogen or C 1-4 alkyl;

[0220] X is selected from O or S, preferably O;

[0221] Y is selected from OH or C. 1-4 Alkyl group, preferably OH;

[0222] Base selected

[0223] In one specific embodiment, the present invention relates to the above-mentioned oligonucleotide or a pharmaceutically acceptable salt thereof, wherein the modifying group is selected from the following structures, pharmaceutically acceptable salts thereof, isotopic variants, tautomers, or stereoisomers:

[0224] Where X is selected from O or S;

[0225] Base selected

[0226] Preferably, the modifying group is selected from pharmaceutically acceptable salts, isotopic variants, tautomers, or stereoisomers of the following structures:

[0227] In one specific embodiment, the present invention relates to the above-mentioned oligonucleotide or a pharmaceutically acceptable salt thereof, wherein the oligonucleotide is a single strand having 14 to 30 nucleotides.

[0228] In one specific embodiment, the present invention relates to the above-mentioned oligonucleotide or a pharmaceutically acceptable salt thereof, wherein the oligonucleotide comprises 1, 2, 3, 4, 5, 6, 7 or 8 modifying groups of formula (I) described herein or pharmaceutically acceptable salts, isotopic variants, tautomers or stereoisomers thereof;

[0229] Preferably, the oligonucleotide includes at least one of the following nucleotide positions from the 2nd to the 20th (preferably the 2nd to the 10th, more preferably the 3rd to the 8th) of its 5' end, a modifying group of formula (I) as described herein, or a pharmaceutically acceptable salt, isotopic variant, tautomer or stereoisomer thereof;

[0230] Preferably, the oligonucleotide includes a modifying group of formula (I) as described herein or a pharmaceutically acceptable salt, isotope variant, tautomer or stereoisomer thereof at the 4th, 5th, 6th or 7th (preferably 5th) nucleotide position from its 5' end.

[0231] In one specific embodiment, the present invention relates to the above-mentioned oligonucleotide or a pharmaceutically acceptable salt thereof, wherein the oligonucleotide is a double strand comprising a sense strand and an antisense strand, wherein each strand has 14 to 30 nucleotides;

[0232] Preferably, the oligonucleotide is selected from short interfering RNA (siRNA), double-stranded RNA (dsRNA), microRNA (miRNA) or short hairpin RNA (shRNA), and is preferably siRNA.

[0233] In one specific embodiment, the present invention relates to the above-mentioned oligonucleotide or a pharmaceutically acceptable salt thereof, wherein the oligonucleotide comprises, within the sense strand and / or antisense strand, one, two, three, four, five, six, seven or eight modifying groups of formula (I) described herein or pharmaceutically acceptable salts, isotopic variants, tautomers or stereoisomers thereof;

[0234] Preferably, the oligonucleotide includes one, two, three, four, five, six, seven, or eight modifying groups of formula (I) described herein or pharmaceutically acceptable salts, isotopic variants, tautomers, or stereoisomers within the antisense strand;

[0235] More preferably, the oligonucleotide includes one, two, three, four, five, six, seven or eight modifying groups of formula (I) described herein or their pharmaceutically acceptable salts, isotopic variants, tautomers or stereoisomers at positions 2-20 (preferably positions 2-10, preferably positions 3-8) from the 5' end of the antisense strand.

[0236] More preferably, the oligonucleotide includes a modifying group of formula (I) as described herein or a pharmaceutically acceptable salt, isotope variant, tautomer or stereoisomer thereof at the 4th, 5th, 6th or 7th (preferably 5th) nucleotide position starting from the 5' end of the antisense strand.

[0237] In one specific embodiment, the present invention relates to the above-mentioned oligonucleotide or a pharmaceutically acceptable salt thereof, wherein the oligonucleotide comprises one or more targeting ligands or delivery carriers; preferably, the delivery carrier is selected from peptide delivery carriers or antibody delivery carriers; preferably, the targeting ligand is an N-acetylgalactosamine (GalNAc) targeting ligand, such as compound 6, compound 7, and L96:

[0238] Compound 6:

[0239] Compound 7:

[0240] L96:

[0241] In one specific embodiment, the present invention relates to the above-mentioned oligonucleotide or a pharmaceutically acceptable salt thereof, wherein the oligonucleotide comprises 5 or more, 7 or more, or 10 or more modified nucleotide monomers, for example, all nucleotide monomers of the oligonucleotide are modified, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 nucleotide monomers of the oligonucleotide are modified;

[0242] Preferably, the modification is selected from one or more of 2'-O-methyl modification, 2'-F modification, and 5'-spirocyclic phosphonate modification;

[0243] In one specific embodiment, the present invention relates to the above-mentioned oligonucleotide or a pharmaceutically acceptable salt thereof, wherein the oligonucleotide contains phosphate thioester bonds, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20.

[0244] RNAi agents containing formula (I) group

[0245] In one embodiment, the present invention relates to an RNAi agent, wherein the RNAi agent comprises one, two, three, four, five or more modifying groups of formula (I) described herein;

[0246] Preferably, the nucleotide chain of the RNAi agent includes one, two, three, four, five, six, seven, or eight modifying groups of formula (I) described herein, or pharmaceutically acceptable salts, isotopic variants, tautomers, or stereoisomers thereof.

[0247] In one specific embodiment, the present invention relates to the above-mentioned RNAi agent, wherein the RNAi agent is double-stranded, comprising a sense strand and an antisense strand, wherein each strand has 14 to 30 nucleotides;

[0248] Preferably, the RNAi agent is selected from short interfering RNA (siRNA), double-stranded RNA (dsRNA), microRNA (miRNA) or short hairpin RNA (shRNA), and is preferably siRNA.

[0249] In one specific embodiment, the present invention relates to the above-mentioned RNAi agent, wherein the RNAi agent comprises 1, 2, 3, 4, 5, 6, 7 or 8 modifying groups of formula (I) described herein or their pharmaceutically acceptable salts, isotopic variants, tautomers or stereoisomers within the sense strand and / or antisense strand;

[0250] Preferably, the RNAi agent includes one, two, three, four, five, six, seven, or eight modifying groups of formula (I) described herein or their pharmaceutically acceptable salts, isotopic variants, tautomers, or stereoisomers within the antisense strand;

[0251] More preferably, the RNAi agent includes 1, 2, 3, 4, 5, 6, 7 or 8 modifying groups of formula (I) described herein or their pharmaceutically acceptable salts, isotopic variants, tautomers or stereoisomers at positions 2-20 (preferably positions 2-10, preferably positions 3-8) from the 5' end of the antisense strand.

[0252] More preferably, the RNAi agent includes a modifying group of formula (I) as described herein or a pharmaceutically acceptable salt, isotope variant, tautomer or stereoisomer thereof at the 4th, 5th, 6th or 7th (preferably the 5th) nucleotide position starting from the 5' end of the antisense strand.

[0253] In one specific embodiment, the present invention relates to the above-mentioned RNAi agent, wherein the RNAi agent comprises one or more targeting ligands or delivery carriers; preferably, the delivery carrier is selected from peptide delivery carriers or antibody delivery carriers; preferably, the targeting ligand is an N-acetylgalactosamine (GalNAc) targeting ligand; preferably, the GalNAc targeting ligand can be selected from GalNAc targeting ligands known in the art, or GalNAc targeting ligands described herein, such as compounds 6, 7, and L96:

[0254] Compound 6:

[0255] Compound 7:

[0256] L96:

[0257] In one specific embodiment, the present invention relates to the above-mentioned RNAi agent, wherein the RNAi agent comprises 5 or more, 7 or more, or 10 or more modified nucleotide monomers, for example, all nucleotide monomers of the oligonucleotide are modified, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 nucleotide monomers of the oligonucleotide are modified;

[0258] Preferably, the modification is selected from one or more of 2'-O-methyl modification, 2'-F modification and 5'-spirocyclic phosphonate modification.

[0259] In one specific embodiment, the present invention relates to the above-mentioned RNAi agent, wherein the RNAi agent contains phosphate thioester bonds, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20.

[0260] In one specific embodiment, the present invention relates to the above-mentioned RNAi agent, wherein the antisense strand of the RNAi agent is selected from or includes the following sequences:

[0261] 1) Having a sequence or a fragment thereof as shown in SEQ ID NO.4, or a modified sequence thereof; or

[0262] 2) Having a sequence or a fragment thereof as shown in SEQ ID NO.5, or a modified sequence thereof;

[0263] Preferably, the sense and antisense strands of the RNAi agent are selected from or contain the following sequences:

[0264] (1) The positive chain has a sequence as shown in SEQ ID NO.1 or a fragment thereof, or a modified sequence of said sequence or a fragment thereof; and the negative chain has a sequence as shown in SEQ ID NO.4 or a fragment thereof, or a modified sequence of said sequence or a fragment thereof; or

[0265] (2) The sense chain has a sequence or a fragment thereof as shown in SEQ ID NO.1, or a modified sequence thereof; and the antisense chain has a sequence or a fragment thereof as shown in SEQ ID NO.5, or a modified sequence thereof;

[0266] More preferably, the sense and antisense strands of the RNAi agent are selected from or contain the following sequences:

[0267] (1) The sense chain has the sequence shown in SEQ ID NO.1; and the antisense chain has the sequence shown in SEQ ID NO.4; or

[0268] (2) The sense chain has the sequence shown in SEQ ID NO.1; and the antisense chain has the sequence shown in SEQ ID NO.5.

[0269] 5'-spirocyclic phosphonate modified nucleotide monomers

[0270] The 5'-spirocyclic phosphonate-modified nucleotide monomers described herein have the structures shown in formulas (XI), (XI-1), (X-II), (X-II-1), (X-II-2), (X-III), (X-III-1), or (X-III-2):

[0271] in,

[0272] Indicates a single bond or a double bond;

[0273] Ring A1 is selected from C 3-10 Cycloalkylene, 3-10 membered heterocyclic alkylene, C 6-10 Arenes or 5-10 quinone heteroaryl compounds;

[0274] Ring B1 is selected from C 3-10 Cycloalkyl, 3-10 membered heterocyclic groups, C 6-10 Aryl or 5-10 heteroaryl groups;

[0275] Ring A1 and ring B1 can each be replaced by 1, 2, 3, 4 or 5 R*s;

[0276] R* is selected from H, halogens, OH, CN, NH2, C 1-6 Alkyl, C 2-6 alkenyl, C 2-6 alkynyl group, C 1-6 Haloalkyl, C 1-6 Alkoxy or C 1-6 Halogenated alkoxy groups;

[0277] R1 is selected from O, S, or NR. ax ;

[0278] R2 and R3 were independently selected from OR bx or SR cx ;

[0279] R ax Selected from H, C 1-6 Alkyl or C 1-6 Halogenated alkyl groups;

[0280] R bx and R cx Independently selected from H and C 1-6 Alkyl, C 1-6 Haloalkyl, C 3-7 cycloalkyl or 3-7 membered heterocyclic groups;

[0281] X0 is selected from -O-, -S-, -NR dx -、-CR e R f -、-CR e R f -CR e R f -or -CH = CH-;

[0282] R dx R e and R f Independently selected from H, halogens, C 1-6 Alkyl or C 1-6 Halogenated alkyl groups;

[0283] Y1, Y2, Y3, and Y4 are independently selected from H, halogens, OH, CN, NH2, and C. 1-6 Alkyl, C 2-6 alkenyl, C 2-6 alkynyl group, C 1-6 Haloalkyl, C 1-6 Alkoxy, C 1-6 Halogenated alkoxy groups, C 3-7 cycloalkyl or 3-7 membered heterocyclic groups;

[0284] Z is selected from H, halogen, OR j NR k R l C 1-18 Alkyl, C 2-18 alkenyl, C 2-18 alkynyl or C 1-18 Halogenated alkyl groups, preferably selected from H, halogens, and OR. j NR k R l C 1-10 Alkyl, C 2-10 alkenyl, C 2-10 alkynyl or C 1-10 The alkyl halogroup, wherein the Z is optionally further substituted by one, two or three R#;

[0285] R j R k and R l Independently selected from H, halogens, C 1-6 Alkyl or C 1-6 Halogenated alkyl groups;

[0286] R# is selected from H, halogens, OH, NH2, CN, C 1-6 Alkoxy or C 1-6 Halogenated alkoxy groups;

[0287] Q is selected from -O-, -S-, -NR g -or-CR h R i -;

[0288] R g R h and R i Independently selected from H, halogens, C 1-6 Alkyl or C 1-6 Halogenated alkyl groups;

[0289] Base is selected from H, modified or unmodified bases;

[0290] m is selected from 0, 1, or 2;

[0291] The compound of formula (I) may optionally be further substituted by one, two, three, four or five substituents selected from the following:

[0292] H, halogens, OH, CN, NH2, C 1-6 Alkyl, C 2-6 alkenyl, C 2-6 alkynyl group, C 1-6 Haloalkyl, C 1-6 Alkoxy or C 1-6 Halogenated alkoxy groups;

[0293] Preferably,

[0294] Indicates a single bond or a double bond;

[0295] Ring A1 is selected from C 3-10 Cycloalkylene, 3-10 membered heterocyclic alkylene, C 6-10 Arenes or 5-10 quinone heteroaryl compounds;

[0296] Ring B1 is selected from C 3-10 Cycloalkyl, 3-10 membered heterocyclic groups, C 6-10 Aryl or 5-10 heteroaryl groups;

[0297] Ring A1 and ring B1 are each optionally replaced by one, two or three R*;

[0298] R* is selected from H, halogens, C 1-6 Alkyl, C 1-6 Haloalkyl, C 1-6 Alkoxy or C 1-6 Halogenated alkoxy groups;

[0299] R1 is selected from O, S, or NR. ax ;

[0300] R2 and R3 were independently selected from OR bx or SR cx ;

[0301] R ax Selected from H, C 1-6 Alkyl or C 1-6 Halogenated alkyl groups;

[0302] Each R bx and R cx Independently selected from H and C 1-6 Alkyl or C 1-6 Halogenated alkyl groups;

[0303] X0 is selected from -O-, -S-, -NR dx -or-CR e R f -;

[0304] R dx R e and R f Independently selected from H, halogens, C 1-6 Alkyl or C 1-6 Halogenated alkyl groups;

[0305] Y1, Y2, Y3, and Y4 are independently selected from H, halogens, and C. 1-6 Alkyl, C 1-6 Haloalkyl, C 1-6 Alkoxy or C 1-6 Halogenated alkoxy groups;

[0306] Z is selected from H, halogen, OR j C 1-6 Alkyl or C 1-6 Haloalkyl, wherein Z is optionally substituted with 1, 2 or 3 R#;

[0307] R j Selected from H, C 1-6 Alkyl or C 1-6 Halogenated alkyl groups;

[0308] R# is selected from H, halogen, OH, C. 1-6 Alkoxy or C 1-6 Halogenated alkoxy groups;

[0309] Q is selected from -O-, -S-, -NR g -or-CR h R i -;

[0310] R g R hand R i Independently selected from H and C 1-6 Alkyl or C 1-6 Halogenated alkyl groups;

[0311] Base is selected from H, modified or unmodified bases.

[0312] Preferably,

[0313] Indicates a single bond or a double bond, preferably a single bond;

[0314] Ring A1 is selected from C 3-7 Cycloalkylene or 3-7 membered heterocyclic alkylene;

[0315] Ring B1 is selected from C 3-7 cycloalkyl or 3-7 membered heterocyclic groups;

[0316] Ring A1 and ring B1 are each optionally replaced by one, two or three R*;

[0317] R* is selected from H, halogens, C 1-6 Alkyl or C 1-6 Halogenated alkyl groups;

[0318] R1 is selected from O or S, preferably O;

[0319] R2 and R3 are independently selected from OH and C. 1-6 Alkoxy or C 1-6 Halogenated alkoxy groups;

[0320] X0 is selected from -O-, -S-, -NH- or -CH2-;

[0321] Y1, Y2, Y3, and Y4 are independently selected from H, halogens, and C. 1-6 Alkyl or C 1-6 Halogenated alkyl groups;

[0322] Z is selected from C 1-6 Alkoxy or C 1-6 Haloalkoxy group, wherein the Z is optionally substituted with one or two R#;

[0323] R# is selected from H, halogen, OH, C. 1-6 Alkoxy or C 1-6 Halogenated alkoxy groups;

[0324] Q is selected from -O-, -S-, -NH-, or -CH2-;

[0325] Base is selected from H, modified or unmodified bases.

[0326] More preferably,

[0327] Indicates a single bond or a double bond, preferably a single bond;

[0328] Ring A1 is selected from C 3-5 Cycloalkylene or 3-5 membered heterocyclic alkylene, preferably C 3-5 Cycloalkylene compounds, such as cyclopropylene, cyclobutylene, or cyclopentylene;

[0329] Ring B1 is selected from C 3-5 Cycloalkyl or 3-5 membered heterocyclic groups, preferably C 3-5 Cycloalkyl groups, such as cyclopropyl, cyclobutyl, or cyclopentyl;

[0330] Preferably, ring A1 and ring B1 together form the following structure: For example

[0331] R1 is selected from O or S, preferably O;

[0332] R2 and R3 are independently selected from OH or C. 1-4 Alkyl groups, such as OCH3 or OCH2CH3;

[0333] X0 is selected from -O-, -S- or -CH2-, preferably O;

[0334] Y1, Y2, Y3, and Y4 are independently selected from H or C. 1-4 Alkyl group, preferably H;

[0335] Z is C 1-4 Alkyl group, wherein the Z is optionally substituted with one or two R#;

[0336] R# is selected from H, halogen, or C. 1-4 Alkoxy;

[0337] Z is preferably -OCH3 or -OCH2CH2OCH3;

[0338] Q is selected from -O-, -S-, or -CH2-;

[0339] Base is selected from H or a base, preferably H or a base.

[0340] The 5'-spirocyclic phosphonate-modified nucleotide monomers described in this article are selected from the following structures:

[0341] Where Base is selected from

[0342] Preferably, the 5'-spirocyclic phosphonate-modified nucleotide monomers described herein are selected from the following structures:

[0343] The 5'-spirocyclic phosphonate-modified nucleotide monomers described in this article are selected from the following structures:

[0344] Where Base is selected from

[0345] Preferably, the 5'-spirocyclic phosphonate-modified nucleotide monomers described herein are selected from the following structures:

[0346] Compound of formula (I')

[0347] In this document, “compound of formula (I')” means a compound of formula (I') (including sub-formulas such as (I'-1), (I'-2), (II'), (II'-1), (II'-2), (III'), (III'-1) or (III'-2)), or a pharmaceutically acceptable salt, isotopic variant, tautomer or stereoisomer thereof, and mixtures thereof.

[0348] In one embodiment, the present invention relates to a compound of formula (I'), or a pharmaceutically acceptable salt, isotopic variant, tautomer, or stereoisomer thereof, or a mixture thereof:

[0349] in,

[0350] Ring A is selected from C 0-6 Alkylene-C 3-10 Cycloalkyl-C 0-6 Alkylene, C 0-6 alkylene-3-10-membered heterocyclic-C 0-6 Alkylene, C 0-6 Alkylene-C 6-10 Aspartic-C 0-6 Alkylene or C 0-6 alkylene-5-10-membered heteroaryl-C 0-6 Alkylene, wherein the ring A is optionally surrounded by 1, 2, 3, 4, 5 or 6 R atoms. a replace;

[0351] L1 is selected from C 1-6 Alkylene, C 1-6 Heteroalkyl, C 2-6 imidene group, C 2-6 Ethyne group, C 3-10 Cycloalkylene, 3-10 membered heterocyclic alkylene, C 6-10 The L1 is optionally surrounded by 1, 2, 3, 4, 5, or 6 R atoms. b replace;

[0352] L2 is selected from C 1-6 Alkylene, NH-C 0-6 Alkylene, C(O)-C 0-6 Alkylene, NHC(O)-C 0-6 Alkylene, C 2-6 imidene group, C 2-6 Ethyne group, C 3-10 Cycloalkylene, 3-10 membered heterocyclic alkylene, C 6-10 The L2 is optionally surrounded by 1, 2, 3, 4, 5, or 6 R groups. c replace;

[0353] P1 is selected from H or a reactive phosphorus group, preferably -P(OCH2CH2CN)(N(iPr)2);

[0354] P2 is selected from H or a protecting group, preferably a hydroxyl protecting group;

[0355] Base' is selected from H, modified or unmodified bases;

[0356] Each R a Independently selected from H, halogen, OR, CN, NRR', C 1-6 Alkyl, C 1-6 Haloalkyl, C 1-6 Alkoxy or C 1-6 Halogenated alkoxy groups;

[0357] Each R b Independently selected from H, halogen, OR, CN, NRR', C 1-6 Alkyl, C 1-6 Haloalkyl, C 1-6 Alkoxy, C 1-6 Haloalkoxy groups, or two R atoms on the same or different carbon atoms. b They are connected, and together with the carbon atoms they are connected to, they form C. 3-7 Cycloalkylene;

[0358] Each R c Independently selected from H, halogen, OR, CN, NRR', C 1-6 Alkyl, C 1-6 Haloalkyl, C 1-6 Alkoxy, C 1-6 Haloalkoxy groups, or two R atoms on the same or different carbon atoms. c They are connected, and together with the carbon atoms they are connected to, they form C. 3-7 Cycloalkylene;

[0359] R and R' are independently selected from H, halogens, and C. 1-6 Alkyl, C2-6 alkenyl, C 2-6 alkynyl or C 1-6 Haloalkyl groups, or R and R', are attached to the nitrogen atom to which they are attached, together forming 3-7 membered heterocyclic groups;

[0360] The compound of formula (I') may optionally be further substituted by one, two, three, four or five substituents selected from the following:

[0361] H, halogens, OH, CN, NH2, C 1-6 Alkyl, C 2-6 alkenyl, C 2-6 alkynyl group, C 1-6 Haloalkyl, C 1-6 Alkoxy or C 1-6 Halogenated alkoxy groups.

[0362] L1, L2, Ring A, R a R b R b '、R c R c '、R d The definitions of R, R' and m are as described above.

[0363] Base'

[0364] In one implementation, Base' is selected from H, modified or unmodified bases.

[0365] In one implementation, Base' is selected from H, unmodified base, or C. 1-4 Alkyl or protecting group modified bases.

[0366] In one implementation, Base' is selected from...

[0367] P1 and P2

[0368] In one embodiment, P1 is H; in another embodiment, P1 is a reactive phosphorus group, such as -P(OCH2CH2CN)(N(iPr)2); in yet another embodiment, P1 is a hydroxyl protecting group.

[0369] In one embodiment, P2 is H; in another embodiment, P2 is a hydroxyl protecting group, such as DMTr; in yet another embodiment, P2 is a reactive phosphorus group.

[0370] 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 in L1 can be combined with L2, ring A, Base', R a R b R b '、R c R c '、R d This invention relates to any combination of technical solutions such as R, R', m, P1, P2, etc. The present invention aims to include combinations of all these technical solutions; however, due to space limitations, they are not listed individually.

[0371] In one embodiment, the present invention relates to a compound of formula (I') above, or a pharmaceutically acceptable salt, isotopic variant, tautomer, or stereoisomer thereof, or a mixture thereof, wherein the compound has the following structure:

[0372] in,

[0373] Ring A is selected from C 3-10 Cycloalkylene, 3-10 membered heterocyclic alkylene, C 6-10 The ring is arylene or 5-10 heteroarylene, wherein the ring A is optionally surrounded by 1, 2, 3, 4 or 5 R groups. a replace;

[0374] L1 is selected from C 1-6 Alkylene, C 1-6 Heteroalkyl, C 2-6 imidene group, C 2-6 Ethyne group, C 3-10 The L1 is optionally surrounded by 1, 2, 3, 4, or 5 R groups, or a cycloalkyl or 3-10-membered heterocyclic group. b replace;

[0375] L2 is selected from C 1-6 Alkylene, NH-C 0-6 Alkylene, C(O)-C 0-6 Alkylene, NHC(O)-C 0-6 Alkylene, C 2-6 imidene group, C 2-6 Ethyne group, C 3-10 The L2 is optionally surrounded by 1, 2, 3, 4, or 5 R groups. c replace;

[0376] P1 is selected from H or a reactive phosphorus group, preferably -P(OCH2CH2CN)(N(iPr)2);

[0377] P2 is selected from H or a protecting group, preferably a hydroxyl protecting group;

[0378] Base' is selected from H, modified or unmodified bases;

[0379] Each R a Independently selected from H, halogens, OH, CN, NH2, C 1-6 Alkyl, C 1-6 Haloalkyl, C 1-6 Alkoxy or C 1-6 Halogenated alkoxy groups;

[0380] Each R b and R b 'Independently selected from H, halogens, OH, CN, NH2, C 1-6 Alkyl, C 1-6 Haloalkyl, C 1-6 Alkoxy, C 1-6 Haloalkoxy groups, or two R atoms on the same or different carbon atoms. b (or R) b and R b ') are connected together, and together with the carbon atoms they are connected to, they form C. 3-7 Cycloalkylene (preferably C16) 3-5 (cycloalkylene);

[0381] Each R c and R c 'Independently selected from H, halogens, OH, CN, NH2, C 1-6 Alkyl, C 1-6 Haloalkyl, C 1-6 Alkoxy, C 1-6 Haloalkoxy groups, or two R atoms on the same or different carbon atoms. c (or R) c and R c ') are connected together, and together with the carbon atoms they are connected to, they form C. 3-7 Cycloalkylene (preferably C16) 3-5 (cycloalkylene);

[0382] m is selected from 0, 1, 2 or 3.

[0383] In one specific embodiment, the present invention relates to a compound of formula (I') above, or a pharmaceutically acceptable salt, isotopic variant, tautomer, or stereoisomer thereof, or a mixture thereof, wherein,

[0384] Ring A is C 3-10 Cycloalkylene, 3-10 membered heterocyclic alkylene, C 6-10 arylene or 5-10 heteroarylene, wherein the ring A is optionally surrounded by one, two or three R atoms. a replace;

[0385] L1 is selected from C 1-6 Alkylene, C 2-6 imide or C 2-6 The ynylene group, wherein the L1 is optionally surrounded by one, two, or three R groups. b replace;

[0386] L2 is selected from C 1-6 Alkylene, C 2-6 imidene group, C 2-6 Ethyne group or NHC(O)-C 1-6 Alkylene, wherein the L2 is optionally surrounded by one, two or three R atoms. c replace;

[0387] P1 is selected from H or a reactive phosphorus group, preferably -P(OCH2CH2CN)(N(iPr)2);

[0388] P2 is selected from H or a protecting group, preferably a hydroxyl protecting group;

[0389] Base' is selected from H, modified or unmodified bases;

[0390] Each R a Independently selected from H, halogens, OH, CN, NH2, C 1-6 Alkyl, C 1-6 Haloalkyl, C 1-6 Alkoxy, C 1-6 Halogenated alkoxy groups;

[0391] Each R b and R b 'Independently selected from H, halogens, OH, CN, NH2, C 1-6 Alkyl, C 1-6 Haloalkyl, C 1-6 Alkoxy, C 1-6 Haloalkoxy groups, or two R atoms on the same or different carbon atoms. b They are connected, and together with the carbon atoms they are connected to, they form C. 3-7 Cycloalkylene;

[0392] Each R c and R c 'Independently selected from H, halogens, OH, CN, NH2, C 1-6 Alkyl, C 1-6 Haloalkyl, C 1-6 Alkoxy, C 1-6 Haloalkoxy groups, or two R atoms on the same or different carbon atoms. c They are connected, and together with the carbon atoms they are connected to, they form C. 3-7 Cycloalkylene;

[0393] Preferably,

[0394] Ring A is selected from C 3-7 Cycloalkylene or 3-7 membered heterocyclic alkylene, wherein ring A is optionally surrounded by 1, 2 or 3 R groups. a replace;

[0395] L1 is C 1-6 Alkylene, wherein the L1 is optionally surrounded by one, two or three R atoms. b replace;

[0396] L2 is C 1-6 Alkylene, wherein the L2 is optionally surrounded by one, two or three R atoms. c replace;

[0397] P1 is selected from H or a reactive phosphorus group, preferably -P(OCH2CH2CN)(N(iPr)2);

[0398] P2 is selected from H or a protecting group, preferably a hydroxyl protecting group, and more preferably DMTr;

[0399] Base' is selected from H, modified or unmodified bases;

[0400] Each R a Independently selected from H, halogens, C 1-4 Alkyl or C 1-4 Halogenated alkyl groups;

[0401] Each R b and R b 'Independently selected from H, halogen, C 1-4 Alkyl or C 1-4 Halogenated alkyl groups;

[0402] Each R c and R c 'Independently selected from H, halogen, C 1-4 Alkyl or C 1-4 Halogenated alkyl groups;

[0403] R d Selected from H, halogens, C 1-4 Alkyl or C 1-4 Halogenated alkyl groups;

[0404] More preferably,

[0405] Ring A is C 3-5 Cycloalkylene, preferably cyclopropylene;

[0406] L1 is C 1-4 Alkylene, preferably -CH2-;

[0407] L2 is C 1-4Alkylene, preferably -CH2-;

[0408] P1 is selected from H or a reactive phosphorus group, preferably -P(OCH2CH2CN)(N(iPr)2);

[0409] P2 is selected from H or a protecting group, preferably a hydroxyl protecting group;

[0410] Base' is selected from

[0411] In one specific embodiment, the present invention relates to compounds of formula (I') above, or pharmaceutically acceptable salts, isotopic variants, tautomers or stereoisomers thereof, or mixtures thereof, wherein the compounds have a structure of formula (III'), (III'-1) or (III'-2):

[0412] in,

[0413] Ring A is selected from C 3-7 Cycloalkylene or 3-7 membered heterocyclic group, wherein ring A is optionally surrounded by 1, 2 or 3 R groups. a replace;

[0414] P1 is selected from H or a reactive phosphorus group, preferably -P(OCH2CH2CN)(N(iPr)2);

[0415] P2 is selected from H or a protecting group, preferably a hydroxyl protecting group, and more preferably DMTr;

[0416] Base' is selected from H, unmodified base, or C. 1-4 Alkyl or protecting group modified bases;

[0417] Each R a Independently selected from H, halogens, C 1-4 Alkyl or C 1-4 Halogenated alkyl groups;

[0418] R b and R b 'Independently selected from H, halogen, C 1-4 Alkyl or C 1-4 Halogenated alkyl groups;

[0419] R c and R c 'Independently selected from H, halogen, C 1-4 Alkyl or C 1-4 Halogenated alkyl groups;

[0420] Preferably,

[0421] Ring A is C 3-5Cycloalkylene, preferably cyclopropylene, wherein ring A is optionally surrounded by one R a replace;

[0422] R a Selected from H, halogen or C 1-4 alkyl;

[0423] R b and R b 'Independently selected from H, halogen or C 1-4 alkyl;

[0424] R c and R c 'Independently selected from H, halogen or C 1-4 alkyl;

[0425] P1 is selected from H or a reactive phosphorus group, preferably -P(OCH2CH2CN)(N(iPr)2);

[0426] P2 is selected from H or a protecting group, preferably a hydroxyl protecting group, and more preferably DMTr;

[0427] Base' is selected from H, unmodified base, or C. 1-4 Alkyl or protecting bases, for example

[0428] In one specific embodiment, the present invention relates to compounds of formula (I') above, or pharmaceutically acceptable salts, isotopic variants, tautomers, or stereoisomers thereof, or mixtures thereof, wherein the compounds are selected from the following structures:

[0429] in,

[0430] P1 is selected from H or a reactive phosphorus group, preferably -P(OCH2CH2CN)(N(iPr)2);

[0431] P2 is selected from H or a hydroxyl protecting group, preferably DMTr;

[0432] Base' is selected from

[0433] Preferably, the compound is selected from the following structures:

[0434] Pharmaceutical Composition

[0435] The present invention provides pharmaceutical compositions comprising one or more oligonucleotides described herein, or RNAi agents described herein, and pharmaceutically acceptable carriers or excipients.

[0436] The oligonucleotide or RNAi agents of the present invention (also referred to herein as “active compounds”) may be incorporated into pharmaceutical compositions suitable for administration. Such compositions typically comprise one or more of the oligonucleotides or RNAi agents described herein, along with a pharmaceutically acceptable carrier. As used herein, the term “pharmaceutically acceptable carrier” is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents, and absorption delay agents compatible with drug administration. Preferred examples of such carriers or excipients include, but are not limited to, water, saline, Ringer's solution, dextran solution, and 5% human serum albumin. Liposomes and non-aqueous media such as non-volatile oils may also be used. The use of such media and agents for pharmaceutically active substances is well known in the art. Unless any conventional media or agent is incompatible with the active compound, its use in the composition is contemplated. Additional active compounds may also be incorporated into the composition.

[0437] The pharmaceutical compositions of the present invention can be formulated to be compatible with their intended route of administration. Examples of routes of administration include parenteral administration, such as intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (i.e., topical), mucosal, and rectal administration. Solutions or suspensions for parenteral, intradermal, or subcutaneous application may include components such as sterile diluents, antibacterial agents, antioxidants, chelating agents, buffers, and agents for adjusting tension. The pH value can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. Parenteral formulations can be packaged in ampoules, disposable syringes, or multi-dose vials made of glass or plastic.

[0438] Pharmaceutical compositions suitable for injectable applications include sterile aqueous solutions (if water-soluble) or dispersions and sterile powders for the ad hoc preparation of sterile injectable solutions or dispersions. In all cases, the composition must be sterile and should be a fluid present in a manner easily injectable. It must be stable under preparation and storage conditions and must be protected against contamination by microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium. Microbial action can be prevented by various antimicrobial and antifungal agents.

[0439] Oral compositions typically include inert diluents or edible carriers. They may be encapsulated in gelatin capsules or compressed into tablets. For oral therapeutic administration, the active compound may be mixed with excipients and administered in tablet, lozenge, or capsule form. Pharmaceutically compatible binders and / or adjuvant materials may be included as part of the composition. Tablets, pills, capsules, lozenges, etc., may contain any of the following components or compounds with similar properties: binders, disintegrants, lubricants, glidants, sweeteners, etc.

[0440] For inhalation administration, the compound is delivered as an aerosol spray from a pressure vessel or dispenser or sprayer containing a suitable propellant (e.g., a gas such as carbon dioxide).

[0441] Systemic application can also be performed via transmucosal or transdermal routes. Transmucosal application can be accomplished using nasal sprays or suppositories. For transdermal application, the active compound is formulated as an ointment, cream, gel, or lotion, as is known in the art.

[0442] Active compounds can also be formulated as suppositories (e.g., using conventional suppository bases such as cocoa butter and other glycerides) or as retention enemas for rectal delivery.

[0443] This invention provides therapeutic compositions comprising the oligonucleotide or RNAi agents of this invention. The therapeutic compositions according to the invention will be administered together with suitable carriers, excipients, and other agents incorporated into the formulation. Many suitable formulations are available in all formulations known to medicinal chemists: Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, PA. These formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, vesicle-containing lipids (cationic or anionic) (such as LIPOFECTINTM), DNA conjugates, anhydrous absorbent pastes, oil-in-water and water-in-oil emulsions, polyethylene glycol emulsions (polyethylene glycol of various molecular weights), semi-solid gels, and semi-solid mixtures containing polyethylene glycol. See also Powell et al., “Compendium of excipients for parenteral formulations”, PDA (1998), J Pharm Sci Technol 52:238-311.

[0444] Treatment methods and uses

[0445] The present invention provides a method for reducing the expression of a target gene in a cell, the method comprising contacting the cell with an oligonucleotide or a pharmaceutically acceptable salt thereof described herein, an RNAi agent described herein, or a pharmaceutical composition described herein.

[0446] The present invention also provides the use of the oligonucleotides described herein or pharmaceutically acceptable salts thereof, the RNAi agents described herein, or the pharmaceutical compositions described herein in the preparation of medicaments for reducing the expression of target genes in cells.

[0447] The present invention also provides oligonucleotides or pharmaceutically acceptable salts thereof described herein, RNAi agents described herein, or pharmaceutical compositions described herein for reducing the expression of target genes in cells.

[0448] The present invention also provides a method for reducing the expression of target genes in a subject, the method comprising administering to the subject the oligonucleotide described herein or a pharmaceutically acceptable salt thereof, the RNAi agent described herein, or the pharmaceutical composition described herein.

[0449] The present invention also provides the use of the oligonucleotides described herein or pharmaceutically acceptable salts thereof, the RNAi agents described herein, or the pharmaceutical compositions described herein in the preparation of medicaments for reducing the expression of target genes in subjects.

[0450] The present invention also provides oligonucleotides or pharmaceutically acceptable salts thereof described herein, RNAi agents described herein, or pharmaceutical compositions described herein for reducing the expression of target genes in subjects.

[0451] The present invention also provides a method for treating a disease, the method comprising administering to a subject in need the oligonucleotide described herein or a pharmaceutically acceptable salt thereof, the RNAi agent described herein, or a pharmaceutical composition described herein.

[0452] The present invention also provides the use of the oligonucleotides described herein or pharmaceutically acceptable salts thereof, the RNAi agents described herein, or the pharmaceutical compositions described herein in the preparation of medicaments for treating diseases.

[0453] The present invention also provides the use of compounds of formula (I') described herein, or pharmaceutically acceptable salts, isotopic variants, tautomers or stereoisomers thereof, or mixtures thereof, in reducing off-target effects of oligonucleotides.

[0454] The present invention also provides the use of compounds of formula (I') described herein, or pharmaceutically acceptable salts, isotopic variants, tautomers or stereoisomers thereof, or mixtures thereof, in the preparation of oligonucleotide drugs (e.g., RNAi drugs).

[0455] The present invention also provides oligonucleotides or pharmaceutically acceptable salts thereof described herein, RNAi agents described herein, or pharmaceutical compositions described herein for the treatment of diseases.

[0456] In some implementations, the disease is cancer, an autoimmune disease, an inflammatory disease, a metabolic disease, a genetic disease, or a rare disease.

[0457] In some implementations, the disease is cancer, which may be selected from neuroendocrine tumors, gastric cancer, colon cancer, rectal cancer, small bowel cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, hidradenoma, sebaceous gland carcinoma, papillary carcinoma, papillary gland carcinoma, cystic adenocarcinoma, medullary carcinoma, bronchial cancer, esophageal cancer, renal cell carcinoma, liver cancer, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, embryonal carcinosarcoma, cervical cancer, uterine cancer, testicular cancer, lung cancer, small cell lung cancer, non-small cell lung cancer, bladder cancer, or epithelial cancer.

[0458] In some implementations, the disease is an autoimmune disease or an inflammatory disease, selectable from sepsis, septic shock, Crohn's disease, rheumatoid arthritis, asthma, allergy, atopic disease, multiple sclerosis, pertussis, gonorrhea, inflammatory bowel disease, scleroderma, lupus, polymyositis, dermatomyositis, fibromyalgia, psoriatic arthritis, ankylosing spondylitis, Reiter's syndrome, or juvenile rheumatoid arthritis.

[0459] In some implementations, the disease is a metabolic disease, selectable from type 1 diabetes, type 2 diabetes, impaired glucose tolerance, abnormal fasting blood glucose, hyperglycemia, postprandial hyperglycemia, overweight, obesity, hypertension, insulin resistance, and / or metabolic syndrome.

[0460] In some implementations, the disease is a hereditary disorder, selectable from cystic fibrosis (CF), Duchenne muscular dystrophy (DMD), ataxia-angiotensinosis, Hurler syndrome, hemophilia A, hemophilia B, Usher syndrome, Tay-Sachs disease, Becker muscular dystrophy (BMD), congenital muscular dystrophy (CMD), familial atrial fibrillation, Haley-Haley disease, McCardy's disease, mucopolysaccharidosis, nephrotic cystinemia, polycystic kidney disease, Rett syndrome, spinal muscular atrophy (SMA), X-linked nephrogenic diabetes insipidus (XNDI), or X-linked retinitis pigmentosa.

[0461] In some implementations, the disease is a rare disease, which may be selected from albinism, acromegaly, idiopathic pulmonary hypertension, phenylketonuria, or mitochondrial disease.

[0462] In some implementations, a therapeutically effective amount of one (or more) different types of RNAi agents described herein is administered to a subject to inhibit the expression of a target gene in the subject (e.g., to the extent that the expression of a target gene in the subject is effectively inhibited).

[0463] In some implementations, the dose administered to the subject may vary depending on the implementation method, the drug used, the method of administration, and the site of treatment and the subject. However, the dose should be sufficient to provide a therapeutic response. Clinicians can determine the effective amount to administer to humans or other subjects to treat a medical condition. The precise amount required for effective treatment may depend on many factors, such as the activity of the oligonucleotide or RNAi agent and the route of administration.

[0464] The oligonucleotides, RNAi agents, or compositions described herein can be administered to mammals once or in a series of sub-dose over appropriate time periods, such as daily, bi-weekly, weekly, bi-weekly, bi-weekly, bi-monthly, semi-annually, or annually as needed. Dosage units containing an effective amount of the oligonucleotide, RNAi agent, or composition can be administered as a single daily dose, or the total daily dose can be administered as needed in two, three, four, or more sub-dose administrations per day.

[0465] The appropriate route of administration can be chosen by the physician. Administration routes may include parenteral administration, such as by injection, nasal administration, pulmonary administration, or percutaneous administration. Systemic or local administration may be performed via intravenous injection, intramuscular injection, intraperitoneal injection, or subcutaneous injection. In some embodiments, oligonucleotides, RNAi agents, or compositions are selected for parenteral delivery, inhalation, or delivery via the digestive tract, such as oral administration. The dosage and method of administration can vary depending on the subject's weight, age, condition, etc., and can be appropriately selected.

[0466] Reagent kit / drug delivery device

[0467] The present invention provides a kit or delivery device comprising the oligonucleotides, RNAi agents or pharmaceutical compositions disclosed herein.

[0468] In some embodiments, the kit or delivery device includes one or more containers containing one or more components of the pharmaceutical composition described herein, such as oligonucleotides or RNAi agents disclosed herein.

[0469] In a specific embodiment, the kit includes a first container containing an oligonucleotide or RNAi agent disclosed herein. In another specific embodiment, the kit includes a first container which is a vial containing an oligonucleotide or RNAi agent as a lyophilized sterile powder under vacuum, and the kit also includes a second container containing a pharmaceutically acceptable fluid.

[0470] In a specific embodiment, this document provides an injection device containing an oligonucleotide or RNAi agent. In a specific embodiment, the injection device contains an oligonucleotide or RNAi agent in a sterile solution. In a specific embodiment, the injection device is a syringe.

[0471] In one embodiment, the kit includes instructional material disclosing how to use the oligonucleotide or RNAi reagents of the present invention. The instructional material may be in written, electronic (e.g., computer floppy disk or optical disc) or visual (e.g., video file) form. The kit may also include additional components to facilitate the application for which the kit is designed. Thus, for example, the kit may additionally contain tools for detecting labels (e.g., enzyme substrates for enzymatic labeling, filter sets for detecting fluorescent labels, suitable secondary labels such as secondary antibodies, etc.). The kit may also include buffers and other reagents conventionally used to perform a particular method. Such kits and suitable contents are well known to those skilled in the art.

[0472] II. Compound Examples

[0473] The structure of the compound was determined by nuclear magnetic resonance (NMR) or mass spectrometry (MS). Chemical shift δ is expressed in terms of 10⁻⁶. -6 The measurements are given in ppm. NMR measurements were performed using a Bruker NMR spectrometer. The solvents used were deuterated dimethyl sulfoxide (DMSO-d6), deuterated chloroform (CDCl3), and deuterated methanol (CD3OD). The internal standard was tetramethylsilane (TMS).

[0474] LCMS measurements were performed using an Agilent 1260 Infinity II (ESI) mass spectrometer, a Waters UPLC H Class plus (ESI) or a Shimadzu LCMS-2020 (ESI).

[0475] High-performance liquid chromatography (HPLC) analysis was performed using an Agilent 1260 or a Shimadzu LC-20AD.

[0476] Preparative high-performance liquid chromatography (pre-HPLC) uses a GILSON GX-281 or an Agilent 1260 Infinity II preparative liquid chromatograph.

[0477] Chiral preparations were performed using supercritical fluid chromatography (SFC) with a Shimadzu LC-30Adsf or Shimadzu LC-20AD instrument.

[0478] The silica gel plates used for thin-layer chromatography are GF254 acrylic adhesive silica gel plates from Anhui Liangchen Silicon Source Materials Co., Ltd. The silica gel plates used in thin-layer chromatography (TLC) are 0.2 mm in diameter, while the silica gel plates used for thin-layer chromatography separation and purification are 0.5 mm in diameter.

[0479] Column chromatography typically uses 200-300 mesh silica gel from Anhui Liangchen Silicon Source Materials Co., Ltd. as a carrier.

[0480] Mean inhibition rate of kinases and IC 50 The values ​​were determined using a SpectraMax i3X microplate reader (MD, Inc., USA).

[0481] The known starting materials disclosed herein can be synthesized using or in accordance with methods known in the art, or can be purchased from companies such as Bid Pharmaceuticals, Leyan, Shaoyuan Chemical Technology, and Anaiji Chemicals.

[0482] Unless otherwise specified, all reactions in the following examples were carried out under an argon or nitrogen atmosphere.

[0483] Argon or nitrogen atmosphere refers to a reaction flask connected to an argon or nitrogen gas balloon with a volume of approximately 1L.

[0484] A hydrogen atmosphere refers to a reaction flask connected to a hydrogen balloon with a volume of approximately 1L.

[0485] The hydrogenation reaction is usually carried out under vacuum, filled with hydrogen gas, and repeated 3 times.

[0486] An oxygen atmosphere refers to a reaction flask connected to an oxygen balloon with a volume of approximately 1L.

[0487] Unless otherwise specified in the following examples, the solution refers to an aqueous solution, and the reaction temperature is room temperature, which is 20℃-30℃.

[0488] The reaction process in the examples was monitored using thin-layer chromatography (TLC). The developing solvent used in the reaction, the eluent system used for column chromatography to purify the compounds, and the developing solvent system for TLC included: A: dichloromethane / methanol system, B: petroleum ether / ethyl acetate system. The volume ratio of the solvent was adjusted according to the polarity of the compounds, and small amounts of basic or acidic reagents such as triethylamine and acetic acid could also be added for adjustment.

[0489] Example 1-1: Preparation of compounds 1A and 1B

[0490] first step

[0491] Dissolve 1-1 (32.00 g, 170.01 mmol) in dry tetrahydrofuran (400 mL), add tetraisopropyl titanate (67.65 g, 238.02 mmol), cool to 0 °C under nitrogen protection, and then slowly add a tetrahydrofuran solution of ethyl magnesium bromide (476.04 mL, 1 mol / L). After the addition is complete, heat the resulting mixture to 25 °C and stir for 3 hours. Slowly pour the reaction solution into ice water (600 mL), extract with ethyl acetate (400 mL * 3), combine the organic phases, wash with saturated brine (500 mL), dry to anhydrous sodium sulfate, filter, concentrate the filtrate by vacuum distillation, and purify the residue by normal-phase silica gel column chromatography (mobile phase: A: petroleum ether, B: ethyl acetate, gradient elution: B%: 10%-70%) to give title product 1-2 (16.41 g, yield: 53%) as a white solid.

[0492] Step 2

[0493] Dissolve 1-2 (16.40 g, 95.22 mmol) in dry dichloromethane (200 ml), add 4-dimethylaminopyridine (2.31 g, 19.04 mmol) and triethylamine (38.54 g, 380.91 mmol), and then slowly add benzoyl chloride (26.77 g, 190.45 mmol). After the addition is complete, heat the resulting mixture to 35 °C and stir for 16 hours. Wash the reaction solution with water (200 ml * 2), separate the organic phase, dry it with anhydrous sodium sulfate, filter, concentrate the filtrate by vacuum distillation, and purify the residue by normal-phase silica gel column chromatography (mobile phase: A: petroleum ether, B: ethyl acetate, gradient elution: B%: 5%-40%) to give title product 1-3 (24.73 g, yield: 84%) as a colorless oil.

[0494] MS m / z(ESI):277.3[M+1].

[0495] Step 3

[0496] Products 1-3 (24.70 g, 89.38 mmol) were dissolved in a mixed solution of acetic acid (150 mL) and water (30 mL). The resulting mixture was heated to 60 °C and stirred for 1 hour. The reaction solution was directly concentrated under reduced pressure, and the residue was purified by normal-phase silica gel column chromatography (mobile phase: A: petroleum ether, B: 2-methyltetrahydrofuran, gradient elution: B%: 20%-90%) to give title products 1-4 (20.25 g, yield: 91%) as a colorless oil.

[0497] MS m / z(ESI):237.3[M+1].

[0498] Step 4

[0499] Dissolve 1-4 (20.20 g, 85.49 mmol) in dry dichloromethane (2 L), add imidazole (11.64 g, 170.99 mmol), and then slowly add tert-butyldimethylchlorosilane (11.60 g, 76.94 mmol) in portions. Stir the mixture at 25 °C for 10 min. Wash the reaction solution with water (400 ml * 2), separate the organic phase, dry it with anhydrous sodium sulfate, filter, concentrate the filtrate by vacuum distillation, and purify the residue by normal-phase silica gel column chromatography (mobile phase: A: petroleum ether, B: ethyl acetate, gradient elution: B%: 0%-20%) to give title products 1-5 (17.22 g, yield: 57%) as a colorless oil.

[0500] MS m / z(ESI): 351.2 [M+1].

[0501] Step 5

[0502] Dissolve 1-5 (1.95 g, 5.56 mmol) in dry tetrahydrofuran (50 mL). Under a nitrogen atmosphere, add 1-6 (1.68 g, 7.79 mmol) and triphenylphosphine (2.92 g, 11.13 mmol). After the addition is complete, cool the reaction solution to 0 °C in an ice bath, and then slowly add diisopropyl azodicarbonate (2.36 g, 11.68 mmol). The resulting mixture is stirred at 25 °C for 3 hours. The reaction solution is directly concentrated under reduced pressure, and the residue is purified by normal-phase silica gel column chromatography (mobile phase: A: petroleum ether, B: ethyl acetate, gradient elution: B%: 5%-40%) to give the title product 1-7 (2.55 g, yield: 83%) as a colorless oil.

[0503] MS m / z(ESI): 549.4 [M+1].

[0504] Step 6

[0505] Dissolve 1-7 (2.55 g, 4.65 mmol) in methanol (30 mL), then add sodium methoxide methanol solution (5.57 mL, 27.88 mmol, 5 mol / L). The resulting mixture is stirred at 25 °C for 1 hour. The reaction solution is directly concentrated under reduced pressure, and the residue is purified by C18 reversed-phase chromatography (mobile phase: A-water (0.1% trifluoroacetic acid), B-acetonitrile, gradient elution: B%: 10%-70%), to give title product 1-8 (1.02 g, yield: 64%) as a yellow oil.

[0506] MS m / z(ESI):341.2[M+1].

[0507] Step 7

[0508] Dissolve 1-8 (900 mg, 2.65 mmol) in dichloromethane (10 mL), then add trifluoroacetic acid (5 mL). After the addition is complete, stir the mixture at 25 °C for 0.5 h. The reaction solution is directly concentrated under reduced pressure, and the residue is purified by C18 reversed-phase chromatography (mobile phase: A-water (0.1% trifluoroacetic acid), B-acetonitrile, gradient elution: B%: 0%-30%), to give title product 1-9 (310 mg, yield: 51%) as a white solid.

[0509] MS m / z(ESI):227.2[M+1].

[0510] Step 8

[0511] Dissolve 1-9 (310 mg, 1.37 mmol) in a mixed solvent of dry pyridine (2 mL) and dry dichloromethane (6 mL). Add 4,4'-dimethoxytriphenylmethyl chloride (557 mg, 1.65 mmol) under a nitrogen atmosphere. Stir the mixture at 25 °C for 10 min. Quench the reaction mixture with methanol (3 mL) and then concentrate under reduced pressure. Purify the residue by normal-phase silica gel column chromatography (mobile phase: A: petroleum ether (0.1% triethylamine), B: ethyl acetate (0.1% triethylamine), gradient elution: B%: 40%-90%) to give title product 1-10 (510 mg, yield: 68%) as a white solid.

[0512] 1-10 (510 mg, 0.95 mmol) was chirally separated by SFC to obtain monoconfiguration compounds 1-10a and 1-10b.

[0513] 1-10a White solid, 232 mg, yield: 45%.

[0514] MS m / z(ESI): 529.3 [M+1].

[0515] 1-10b, white solid, 227 mg, yield: 44%.

[0516] MS m / z(ESI): 529.3 [M+1].

[0517] Step 9

[0518] 1-10a (220 mg, 0.42 mmol) was dissolved in dry acetonitrile (5 mL), and 4,5-dicyanimidazole (49 mg, 0.42 mmol) and 1-11 (377 mg, 1.25 mmol) were added under a nitrogen atmosphere. After the addition was complete, the mixture was stirred at 40 °C for 1 hour. The reaction solution was directly purified by C18 reversed-phase chromatography (mobile phase: A-water (10 mmol / L ammonium bicarbonate), B-acetonitrile, gradient elution: B%: 30%-95%), to give title product 1A (281 mg, yield: 89%) as a white solid.

[0519] MS m / z(ESI): 729.4 [M+1].

[0520] 1 H NMR(400MHz, DMSO-d6)δ11.21(dd,J=7.6,2.2Hz,1H),7.41(dd,J=9.1,7.8Hz,1H),7.38-7 .32(m,2H),7.31-7.25(m,2H),7.25-7.15(m,5H),6.93-6.74(m,4H),5.59-5.45(m,1H),4. 23-4.05(m,1H),3.72(d,J=1.5Hz,6H),3.62-3.31(m,6H),3.11-2.95(m,1H),2.70(t,J=5. 8Hz,1H),2.59(t,J=5.8Hz,1H),1.83-1.63(m,1H),1.12-0.85(m,14H),0.62-0.40(m,2H). 31 PNMR(400MHz,DMSO-d6)δ142.99,141.44.

[0521] Step 10

[0522] 1-10b (220 mg, 0.42 mmol) was dissolved in dry acetonitrile (5 mL), and 4,5-dicyanimidazole (49 mg, 0.42 mmol) and 1-11 (377 mg, 1.25 mmol) were added under a nitrogen atmosphere. After the addition was complete, the mixture was stirred at 40 °C for 1 hour. The reaction solution was directly purified by C18 reversed-phase chromatography (mobile phase: A-water (10 mmol / L ammonium bicarbonate), B-acetonitrile, gradient elution: B%: 30%-95%), to give the title product 1B (280 mg, yield: 89%) as a white solid.

[0523] MS m / z(ESI): 729.4 [M+1].

[0524] 1H NMR(400MHz, DMSO-d6)δ11.20(dd,J=7.5,2.2Hz,1H),7.41(dd,J=9.1,7.9Hz,1H),7.38-7 .32(m,2H),7.31-7.25(m,2H),7.25-7.16(m,5H),6.91-6.79(m,4H),5.57-5.46(m,1H),4. 24-4.01(m,1H),3.72(d,J=1.5Hz,6H),3.64-3.30(m,6H),3.12-2.97(m,1H),2.70(t,J=5. 8Hz,1H),2.59(t,J=5.9Hz,1H),1.79-1.65(m,1H),1.14-0.84(m,14H),0.63-0.40(m,2H). 31 PNMR(400MHz,DMSO-d6)δ142.99,141.44.

[0525] Examples 1-2: Preparation of compounds 2A and 2B

[0526] first step

[0527] Dissolve 1-5 (1.95 g, 5.56 mmol) in dry tetrahydrofuran (50 mL). Under a nitrogen atmosphere, add 2-1 (1.13 g, 6.68 mmol) and triphenylphosphine (2.19 g, 8.34 mmol). After the addition is complete, cool the reaction solution to 0 °C in an ice bath, and then slowly add diisopropyl azodicarbonate (1.69 g, 8.34 mmol). The resulting mixture is stirred at 25 °C for 16 hours. The reaction solution is directly concentrated under reduced pressure, and the residue is purified by normal-phase silica gel column chromatography (mobile phase: A: petroleum ether, B: ethyl acetate, gradient elution: B%: 10%-70%) to give the title product 2-2 (2.52 g, yield: 90%) as a white solid.

[0528] MS m / z(ESI): 502.3 [M+1].

[0529] Step 2

[0530] 2-2 (2.52 g, 5.02 mmol) was dissolved in a mixed solvent of tetrahydrofuran (5 mL), water (5 mL), and trifluoroacetic acid (15 mL). The resulting mixture was heated to 70 °C and stirred for 2 hours. The reaction solution was directly concentrated under reduced pressure, and the residue was purified by C18 reversed-phase chromatography (mobile phase: A-water (0.1% trifluoroacetic acid), B-acetonitrile, gradient elution: B%: 5%-50%), to give the title product 2-3 (1.81 g, yield: 97%) as a white solid.

[0531] MS m / z(ESI): 370.2 [M+1].

[0532] Step 3

[0533] Dissolve 2-3 (1.81 g, 4.90 mmol) in dry pyridine (30 mL), and slowly add isobutyryl chloride (1.57 g, 14.71 mmol) dropwise under a nitrogen atmosphere. After the addition is complete, stir the mixture at 25 °C for 1 hour. Dilute the reaction solution with dichloromethane (100 mL), then wash with water (30 mL * 2), and concentrate the organic phase under reduced pressure. The title product 2-4 (2.85 g, crude product) is given as a white solid.

[0534] MS m / z(ESI): 510.4 [M+1].

[0535] Step 4

[0536] Dissolve 2-4 (2.85 g, crude product) in pyridine (40 mL), cool in an ice bath to below 5 °C, and then slowly add sodium hydroxide solution (1 mol / L water / methanol = 1 / 4, 24.50 mL). After the addition is complete, stir the reaction mixture at below 5 °C for 20 minutes. Add saturated ammonium chloride solution (10 mL) to the reaction mixture, and then concentrate under reduced pressure. The resulting residue is purified by C18 reversed-phase chromatography (mobile phase: A-water (0.1% trifluoroacetic acid), B-acetonitrile, gradient elution: B%: 5%-50%), giving the title product 2-5 (1.31 g, overall yield of two steps: 75%) as a white solid.

[0537] MS m / z(ESI): 336.2 [M+1].

[0538] Step 5

[0539] Product 2-5 (1.31 g, 3.91 mmol) was dissolved in a mixed solvent of dry pyridine (20 mL) and dry dichloromethane (20 mL). 4,4'-bismethoxytriphenylmethyl chloride (1.46 g, 4.30 mmol) was added under a nitrogen atmosphere, and the mixture was stirred at 25 °C for 0.5 h. The reaction was quenched with methanol (5 mL), and then concentrated under reduced pressure. The reaction solution was directly purified by C18 reversed-phase chromatography (mobile phase: A-water (10 mmol / L ammonium bicarbonate), B-acetonitrile, gradient elution: B%: 20%-90%) to give the title product 2-6 (1.52 g, yield: 60%) as a white solid.

[0540] 2-6 (1.52 g, 2.38 mmol) was chirally separated by SFC to obtain mono-configuration compounds 2-6a and 2-6b.

[0541] Compound 1 with a single configuration of 2-6 (2-6a, short retention time in SFC analysis), white solid, 721 mg, yield: 47%.

[0542] MS m / z(ESI): 638.2 [M+1].

[0543] Compound 2-6 (2-6b, with a longer retention time in SFC analysis), white solid, 670 mg, yield: 44%.

[0544] MS m / z(ESI): 638.2 [M+1].

[0545] Step 6

[0546] The monoconfiguration of compound 2-6 (2-6a, with a short retention time in SFC analysis) (206 mg, 0.32 mmol) was dissolved in dry acetonitrile (4 mL). Under a nitrogen atmosphere, 4,5-dicyanimidazole (38 mg, 0.32 mmol) and 1-11 (292 mg, 0.97 mmol) were added. After the addition was complete, the mixture was stirred at 40 °C for 5 hours. The reaction solution was directly purified by C18 reversed-phase chromatography (mobile phase: A-water (10 mmol / L ammonium bicarbonate), B-acetonitrile, gradient elution: B%: 30%-95%), yielding the title product 2A (218 mg, yield: 78%) as a white solid.

[0547] MS m / z(ESI): 838.6 [M+1].

[0548] 1 H NMR (400MHz, DMSO-d6) δ12.06 (s, 1H), 11.38 (s, 1H), 7.78 (d, J = 9.3Hz, 1H), 7.28-7.21 (m, 4H), 7 .21-7.15(m,1H),7.15-7.06(m,4H),6.86-6.75(m,4H),4.31-4.13(m,2H),3.71(s,6H),3.69-3 .30(m,5H),3.12-2.99(m,1H),2.88-2.78(m,1H),2.72(t,J=5.8Hz,1H),2.61(t,J=5.8Hz,1H), 1.98-1.89(m,1H),1.15-0.94(m,19H),0.94-0.80(m,1H),0.68-0.51(m,1H),0.46-0.34(m,1H). 31 PNMR(400MHz,DMSO-d6)δ142.90,141.81.

[0549] Step 7

[0550] Compound 2-6b (220 mg, 0.34 mmol) of the single configuration of 2-6 was dissolved in dry acetonitrile (4 mL). 4,5-Dicyanimidazole (41 mg, 0.34 mmol) and 1-11 (312 mg, 1.03 mmol) were added under a nitrogen atmosphere. After the addition was complete, the mixture was stirred at 40 °C for 5 hours. The reaction solution was directly purified by C18 reversed-phase chromatography (mobile phase: A-water (10 mmol / L ammonium bicarbonate), B-acetonitrile, gradient elution: B%: 30%-95%), yielding the title product 2B (239 mg, yield: 81%) as a white solid.

[0551] MS m / z(ESI): 838.6 [M+1].

[0552] 1 H NMR (400MHz, DMSO-d6) δ12.06 (s, 1H), 11.38 (s, 1H), 7.78 (d, J = 9.3Hz, 1H), 7.28-7.21 (m, 4H), 7.2 1-7.16(m,1H),7.15-7.06(m,4H),6.86-6.75(m,4H),4.31-4.13(m,2H),3.71(t,J=1.9Hz,6H),3. 69-3.30(m,5H),3.12-2.99(m,1H),2.88-2.77(m,1H),2.72(t,J=5.8Hz,1H),2.61(t,J=5.8Hz,1H ),1.98-1.89(m,1H),1.15-0.94(m,19H),0.93-0.81(m,1H),0.68-0.50(m,1H),0.46-0.35(m,1H). 31 PNMR(400MHz,DMSO-d6)δ142.90,141.82.

[0553] Examples 1-3: Preparation of compounds 3A and 3B

[0554] first step

[0555] Dissolve 1-5 (2.50 g, 7.13 mmol) in dry tetrahydrofuran (80 mL). Under a nitrogen atmosphere, add 3-1 (2.87 g, 8.56 mmol) and triphenylphosphine (2.81 g, 10.70 mmol). After the addition is complete, cool the reaction solution to 0 °C in an ice bath, and then slowly add diisopropyl azodicarbonate (2.16 g, 10.70 mmol). Stir the mixture at 25 °C for 1 hour. Concentrate the reaction solution directly under reduced pressure. The residue is purified by normal-phase silica gel column chromatography (mobile phase: A: petroleum ether, B: ethyl acetate, gradient elution: B%: 5%-40%) to give the title product 3-2 (4.21 g, yield: 86%) as a colorless oil.

[0556] MS m / z(ESI): 668.4 [M+1].

[0557] Step 2

[0558] 3-2 (4.21 g, 6.30 mmol) was dissolved in dichloromethane (25 mL), and trifluoroacetic acid (15 mL) was added. The resulting mixture was stirred at 25 °C for 2 hours. The reaction solution was directly concentrated under reduced pressure, and the residue was purified by C18 reversed-phase chromatography (mobile phase: A-water (0.1% trifluoroacetic acid), B-acetonitrile, gradient elution: B%: 0%-40%), to give the title product 3-3 (2.12 g, yield: 94%) as a white solid.

[0559] MS m / z(ESI): 354.2 [M+1].

[0560] Step 3

[0561] 3-3 (2.12 g, 6.00 mmol) was dissolved in dry pyridine (40 mL), and benzoyl chloride (3.20 g, 30.00 mmol) was slowly added dropwise under a nitrogen atmosphere. After the addition was complete, the mixture was stirred at 25 °C for 1 hour. The reaction solution was diluted with dichloromethane (100 mL), then washed with water (30 mL * 2), and the organic phase was separated and concentrated under reduced pressure. The title product 3-4 (3.92 g, crude product) was given as a white solid.

[0562] MS m / z(ESI): 562.3 [M+1].

[0563] Step 4

[0564] Dissolve 3-4 (3.92 g, crude product) in a mixed solvent of pyridine (40 mL) and methanol (20 mL), cool in an ice bath to below 5 °C, and then slowly add sodium hydroxide solution (1 mol / L - water / methanol = 1 / 4, 36.00 mL). After the addition is complete, stir the reaction mixture at below 5 °C for 20 minutes. Add saturated ammonium chloride solution (20 mL) to the reaction mixture, and then concentrate under reduced pressure. The resulting residue is purified by C18 reversed-phase chromatography (mobile phase: A - water (0.1% trifluoroacetic acid), B - acetonitrile, gradient elution: B%: 5%-50%), giving the title product 3-5 (2.03 g, total yield of two steps: 89%) as a white solid.

[0565] MS m / z(ESI): 354.2 [M+1].

[0566] Step 5

[0567] Dissolve 3-5 (2.03 g, 6.05 mmol) in a mixed solvent of dry pyridine (30 mL) and dry dichloromethane (30 mL), and add 4,4'-bismethoxytriphenylmethyl chloride (2.09 g, 6.17 mmol) under a nitrogen atmosphere. Stir the mixture at 25 °C for 0.5 h. Quench the reaction mixture with methanol (5 mL), then concentrate under reduced pressure. Purify the reaction mixture directly by C18 reversed-phase chromatography (mobile phase: A-water (10 mmol / L ammonium bicarbonate), B-acetonitrile, gradient elution: B%: 20%-90%) to give title product 3-6 (2.91 g, yield: 73%) as a white solid.

[0568] 3-6 (2.91 g, 4.43 mmol) was chirally separated by SFC to obtain mono-configuration compounds 3-6a and 3-6b.

[0569] 3-6a, white solid, 1.33 g, yield: 45%.

[0570] MS m / z(ESI): 656.2 [M+1].

[0571] 3-6b, yellow solid, 1.37 g, yield: 47%.

[0572] MS m / z(ESI): 656.2 [M+1].

[0573] Step 6

[0574] 3-6a (320 mg, 0.49 mmol) was dissolved in dry acetonitrile (5 mL), and 4,5-dicyanimidazolium (57 mg, 0.49 mmol) and 1-11 (441 mg, 1.46 mmol) were added under a nitrogen atmosphere. After the addition was complete, the mixture was stirred at 40 °C for 1 hour. The reaction solution was directly purified by C18 reversed-phase chromatography (mobile phase: A-water (10 mmol / L ammonium bicarbonate), B-acetonitrile, gradient elution: B%: 30%-95%), to give the title product 3A (411 mg, yield: 96%) as a white solid.

[0575] MS m / z(ESI): 856.6 [M+1].

[0576] 1 H NMR (400MHz, DMSO-d6) δ11.14(s,1H),8.71(d,J=6.9Hz,1H),8.33(d,J=4.7Hz,1H),8.14-7.99(m,2H),7.70-7 .60(m,1H),7.60-7.51(m,2H),7.28-7.14(m,5H),7.13-7.03(m,4H),6.86-6.76(m,4H),4.67-4.47(m,1H),4. 47-4.31(m,1H),3.71(d,J=3.0Hz,6H),3.69-3.36(m,5H),3.18-3.00(m,1H),2.73(t,J=5.8Hz,1H),2.64(t,J =5.9Hz,1H),2.09-2.00(m,1H),1.14-0.96(m,13H),0.96-0.79(m,1H),0.63-0.44(m,1H),0.40-0.23(m,1H). 31 PNMR(400MHz,DMSO-d6)δ142.96,141.80.

[0577] Step 7

[0578] 3-6b (305 mg, 0.47 mmol) was dissolved in dry acetonitrile (4 mL), and 4,5-dicyanimidazolium (55 mg, 0.47 mmol) and 1-11 (420 mg, 1.40 mmol) were added under a nitrogen atmosphere. After the addition was complete, the mixture was stirred at 40 °C for 1 hour. The reaction solution was directly purified by C18 reversed-phase chromatography (mobile phase: A-water (10 mmol / L ammonium bicarbonate), B-acetonitrile, gradient elution: B%: 30%-95%), to give the title product 3B (356 mg, yield: 88%) as a pale yellow solid.

[0579] MS m / z(ESI): 856.6 [M+1].

[0580] 1 H NMR (400MHz, DMSO-d6) δ11.13(s,1H),8.71(d,J=6.9Hz,1H),8.33(d,J=4.7Hz,1H),8.05(d,J=8.0Hz,2H),7.6 9-7.60(m,1H),7.60-7.51(m,2H),7.29-7.13(m,5H),7.15-7.02(m,4H),6.87-6.76(m,4H),4.67-4.47(m,1H), 4.45-4.31(m,1H),3.71(d,J=3.0Hz,6H),3.69-3.35(m,6H),3.17-2.99(m,1H),2.73(t,J=5.9Hz,1H),2.64(t, J=5.9Hz,1H),2.09-1.99(m,1H),1.14-0.96(m,13H),0.96-0.78(m,1H),0.63-0.45(m,1H),0.39-0.24(m,1H). 31 PNMR(400MHz,DMSO-d6)δ142.96,141.81.

[0581] Examples 1-4: Preparation of compounds 4A and 4B

[0582] first step

[0583] Dissolve 1-10 (2.20 g, 4.04 mmol) in dry dichloromethane (30 mL). Under a nitrogen atmosphere, add imidazole (1.92 g, 28.29 mmol), 4-dimethylaminopyridine (493 mg, 4.04 mmol), and triethylchlorosilane (3.05 g, 20.22 mmol) sequentially. Stir the mixture at 25 °C for 1 hour. Dilute the reaction solution with dichloromethane (100 mL) and wash with water (30 mL x 2). Separate the organic phase and concentrate under reduced pressure. Purify the residue by normal-phase silica gel column chromatography (mobile phase: A: petroleum ether (0.1% triethylamine), B: 2-methyltetrahydrofuran (0.1% triethylamine), gradient elution: B%: 20%-70%) to give the title product 4-1 (2.41 g, yield: 90%) as a white solid.

[0584] MS m / z(ESI): 643.4 [M+1].

[0585] Step 2

[0586] 4-1 (2.40 g, 3.69 mmol) was dissolved in dry acetonitrile (30 mL). Under a nitrogen atmosphere, triethylamine (1.12 g, 11.07 mmol), 4-dimethylaminopyridine (901 mg, 7.38 mmol), and 2,4,6-triisopropylbenzenesulfonyl chloride (2.24 g, 7.38 mmol) were added sequentially. The resulting mixture was stirred at 25 °C for 1 hour. Then, concentrated ammonia (30 mL) was added to the reaction solution, and the reaction was continued at 25 °C for 5 hours. The reaction solution was directly concentrated under reduced pressure, and the residue was purified by C18 reversed-phase chromatography (mobile phase: A-water (10 mmol / L ammonium bicarbonate), B-acetonitrile, gradient elution: B%: 20%-100%) to give the title product 4-2 (1.85 g, yield: 70%) as a white solid.

[0587] MS m / z(ESI): 642.5 [M+1].

[0588] Step 3

[0589] 4-2 (1.85 g, 2.89 mmol) was dissolved in dry pyridine (15 mL). Under a nitrogen atmosphere, 4-dimethylaminopyridine (35 mg, 0.29 mmol) and benzoyl chloride (1.31 g, 5.77 mmol) were added, and the mixture was stirred at 25 °C for 1 hour. The reaction was quenched by adding methanol (5 mL), and then concentrated under reduced pressure. The residue was purified by C18 reversed-phase chromatography (mobile phase: A-water (10 mmol / L ammonium bicarbonate), B-acetonitrile, gradient elution: B%: 30%-100%), giving the title product 4-3 (1.78 g, yield: 81%) as a white solid.

[0590] MS m / z(ESI): 746.5 [M+1].

[0591] Step 4

[0592] 4-3 (1.78 g, 2.39 mmol) was dissolved in dry tetrahydrofuran (15 mL). Under a nitrogen atmosphere, a tetrahydrofuran solution of tetrabutylammonium fluoride (3.58 mL, 1 mol / L) was added. The resulting mixture was stirred at 25 °C for 0.5 h. The reaction solution was concentrated under reduced pressure, and the residue was purified by C18 reversed-phase chromatography (mobile phase: A-water (10 mmol / L ammonium bicarbonate), B-acetonitrile, gradient elution: B%: 30%-100%), to give the title product 4-4 (1.40 g, yield: 91%) as a white solid.

[0593] 4-4 (1.40 g, 2.21 mmol) was chirally separated by SFC to obtain mono-configuration compounds 4-4a and 4-4b.

[0594] 4-4a White solid, 635 mg, yield: 45%.

[0595] MS m / z(ESI): 632.3 [M+1].

[0596] 4-4b, yellow solid, 628 mg, yield: 45%.

[0597] MS m / z(ESI): 632.3 [M+1].

[0598] Step 5

[0599] 4-4a (300 mg, 0.47 mmol) was dissolved in dry acetonitrile (5 mL), and 4,5-dicyanimidazolium (55 mg, 0.47 mmol) and 1-11 (426 mg, 1.41 mmol) were added under a nitrogen atmosphere. After the addition was complete, the mixture was stirred at 40 °C for 1 hour. The reaction solution was directly purified by C18 reversed-phase chromatography (mobile phase: A-water (10 mmol / L ammonium bicarbonate), B-acetonitrile, gradient elution: B%: 30%-95%), to give the title product 4A (356 mg, yield: 91%) as a white solid.

[0600] MS m / z(ESI): 832.5 [M+1].

[0601] 1 H NMR(400MHz,DMSO-d6)δ11.20(s,1H),8.07-7.97(m,2H),7.90-7.82(m,1H),7.66-7.58(m,1H),7 .56-7.48(m,2H),7.36-7.22(m,5H),7.22-7.12(m,5H),6.90-6.78(m,4H),4.27-4.05(m,1H),3.9 5-3.83(m,1H),3.72-3.67(m,3H),3.67-3.64(m,3H),3.55-3.35(m,5H),3.19-2.98(m,1H),2.71( t,J=5.9Hz,1H),2.60(t,J=5.8Hz,1H),2.01-1.85(m,1H),1.31-0.76(m,14H),0.66-0.41(m,2H). 31 PNMR(400MHz,DMSO-d6)δ143.15,141.51.

[0602] Step 6

[0603] 4-4b (300 mg, 0.47 mmol) was dissolved in dry acetonitrile (5 mL), and 4,5-dicyanimidazolium (55 mg, 0.47 mmol) and 1-11 (426 mg, 1.41 mmol) were added under a nitrogen atmosphere. After the addition was complete, the mixture was stirred at 40 °C for 1 hour. The reaction solution was directly purified by C18 reversed-phase chromatography (mobile phase: A-water (10 mmol / L ammonium bicarbonate), B-acetonitrile, gradient elution: B%: 30%-95%), to give the title product 4B (348 mg, yield: 88%) as a white solid.

[0604] MS m / z(ESI): 832.5 [M+1].

[0605] 1 H NMR(400MHz,DMSO-d6)δ11.20(s,1H),8.07-7.97(m,2H),7.90-7.82(m,1H),7.66-7.58(m,1H),7 .56-7.48(m,2H),7.36-7.22(m,5H),7.22-7.12(m,5H),6.90-6.78(m,4H),4.27-4.05(m,1H),3.9 5-3.83(m,1H),3.72-3.67(m,3H),3.67-3.64(m,3H),3.55-3.35(m,5H),3.19-2.98(m,1H),2.71( t,J=5.9Hz,1H),2.60(t,J=5.8Hz,1H),2.01-1.85(m,1H),1.31-0.76(m,14H),0.66-0.41(m,2H). 31 PNMR(400MHz,DMSO-d6)δ143.15,141.51.

[0606] Examples 1-5: Preparation of compounds 5A and 5B

[0607] first step

[0608] 5-2 (3.25 g, 10.35 mmol) was dissolved in dry dimethyl sulfoxide (20 mL), and sodium hydride (410 mg, 10.35 mmol, 60%) was added under a nitrogen atmosphere. After the addition was complete, the mixture was stirred at 25 °C for 0.5 hours. Then, 5-1 (1.7 g, 3.45 mmol, prepared by the method disclosed in steps A to F of Example 2 on page 84 of patent application "WO 2017 / 214112 A1") was dissolved in dry dimethyl sulfoxide (10 mL) and added dropwise to the reaction solution. The resulting mixture was stirred at 25 °C under a nitrogen atmosphere for 1 hour. The reaction was quenched by slowly adding saturated ammonium chloride aqueous solution (30 mL) to the reaction solution, followed by the addition of water (160 mL), and then extraction with ethyl acetate (200 mL × 2). The combined organic phases were washed with saturated brine (200 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (mobile phase: A-petroleum ether, B-ethyl acetate; gradient elution: B%: 20%-80%) to give the title product 5-3 (701 mg, yield: 33%) as a yellow solid.

[0609] MS m / z(ESI): 561.4 [M+1].

[0610] Step 2

[0611] Dissolve 5-3 (701 mg, 1.57 mmol) in a mixture of tetrahydrofuran (15 mL) and water (15 mL), cool in an ice bath to below 5 °C, then add potassium peroxide monosulfonate (1641 mg, 2.67 mmol). After the addition is complete, allow the mixture to rise naturally to 25 °C and stir for 2 hours. Pour the reaction mixture into water (50 mL), then extract with ethyl acetate (60 mL × 2). Combine the organic phases, wash with saturated brine (60 mL), dry the organic phase with anhydrous sodium sulfate, filter, and concentrate the filtrate under reduced pressure. The resulting residue is purified by C18 reversed-phase chromatography (mobile phase: A-water (10 mmol / L ammonium bicarbonate), B-acetonitrile; gradient elution: B%: 20%-90%), yielding the title product 5-4 (405 mg, yield: 46%) as a white solid.

[0612] MS m / z(ESI): 545.4 [M+1].

[0613] Step 3

[0614] 5-4 (405 mg, 0.74 mmol) was dissolved in a mixed solution of formic acid (3 mL), water (2 mL), and acetonitrile (2 mL), and the mixture was stirred at 25 °C for 15 hours. The reaction solution was directly purified by Prep-HPLC (column: InfinityLab Poroshell 120SB-C18 21.2 × 150 mm, 4 μm; mobile phase: A-water (0.1% TFA), B-acetonitrile, gradient elution, B%: 5%-40%), yielding a pair of isomers of the title product as white solids.

[0615] 5-5a (115mg, yield: 35%).

[0616] HPLC analysis: Retention time 13.626 min. (Column: Agilent ZORBAX BONUS RP, 4.6*150mm, 3.5μm; Mobile phase: A-water (0.05% trifluoroacetic acid), B-acetonitrile; Gradient elution: B%: 5%-35%, 20 min; Flow rate: 1 mL / min; Instrument: Agilent 1260).

[0617] MS m / z(ESI):431.3[M+1].

[0618] 5-5b (173 mg, yield: 53%).

[0619] HPLC analysis: Retention time 14.179 min. (Column: Agilent ZORBAX BONUS RP, 4.6*150mm, 3.5μm; Mobile phase: A-water (0.05% trifluoroacetic acid), B-acetonitrile; Gradient elution: B%: 5%-35%, 20 min; Flow rate: 1 mL / min; Instrument: Agilent 1260).

[0620] MS m / z(ESI):431.3[M+1].

[0621] Step 4

[0622] 5-5a (115 mg, 0.27 mmol) was dissolved in dry acetonitrile (5 mL). 4,5-Dicyanimidazole (31.56 mg, 0.27 mmol) and 1-11 (241.61 mg, 0.80 mmol) were added under a nitrogen atmosphere. After addition, the mixture was stirred at 25 °C for 1 hour. The reaction solution was diluted with dichloromethane (20 mL), washed with sodium bicarbonate aqueous solution (0.5 mmol / mL, 10 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by Prep-HPLC (column: InfinityLab Poroshell 120SB-C18 21.2 × 150 mm, 4 μm; mobile phase: A-water (10 mmol / L ammonium bicarbonate), B-acetonitrile, gradient elution, B%: 20%-80%), yielding product 5A (105 mg, 60%) as a white solid.

[0623] MS m / z(ESI): 631.3 [M+1].

[0624] Step 5

[0625] 5-5b (173 mg, 0.40 mmol) was dissolved in dry acetonitrile (7 mL). Under a nitrogen atmosphere, 4,5-dicyanimidazolium (47.47 mg, 0.40 mmol) and 1-11 (363.47 mg, 1.21 mmol) were added. After addition, the mixture was stirred at 25 °C for 1 hour. The reaction solution was diluted with dichloromethane (30 mL), washed with sodium bicarbonate aqueous solution (0.5 mmol / mL, 15 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by Prep-HPLC (column: InfinityLab Poroshell 120SB-C18 21.2 × 150 mm, 4 μm; mobile phase: A-water (10 mmol / L ammonium bicarbonate), B-acetonitrile, gradient elution, B%: 20%-80%), yielding product 5B (166 mg, 63%) as a white solid.

[0626] MS m / z(ESI): 631.3 [M+1].

[0627] 1H NMR (400MHz, DMSO-d6) δ7.40 (dd, J=8.2, 7.0Hz, 1H), 5.97 (dd, J=13.3, 5.0Hz, 1H) ,5.76(dd,J=8.1,1.4Hz,1H),4.34-4.23(m,1H),4.19-4.04(m,4H),3.99-3.85(m ,2H),3.83-3.61(m,3H),3.49(d,J=4.2Hz,3H),2.72-2.62(m,2H),2.09-2.00(m, 1H),1.43-1.28(m,8H),1.25-1.16(m,12H),1.14-0.98(m,3H),0.94-0.82(m,1H). 31 PNMR(400MHz,DMSO-d6)δ150.16,150.09,27.74,27.35.

[0628] Examples 1-6: Preparation of Compound 6

[0629] first step

[0630] 6-1 (5.00 g, 34.9 mmol) was dissolved in 20 mL of ethanol, and ethyl 2-bromoacetate (5.83 g, 34.9 mmol) was added. The mixture was stirred at 80 °C for 15 hours. The reaction solution was concentrated by vacuum distillation, and 25 mL of isopropanol was added to the residue. The mixture was purified by recrystallization to give the title product 6-2 (7.34 g, yield: 91%) as a yellow solid.

[0631] MS m / z(ESI):230.1[M+1].

[0632] Step 2

[0633] 6-2 (6.84 g, 29.7 mmol) was dissolved in 50 mL of acetonitrile under nitrogen protection. 2-Cyclopentenone (11.7 g, 142 mmol) and triethylamine (3.31 g, 32.6 mmol) were added, and the mixture was stirred at 25 °C for 24 hours under nitrogen protection. 50 mL of water was added to the reaction mixture, and the mixture was extracted with ethyl acetate (80 mL × 3). The organic phase was washed with saturated sodium chloride solution (60 mL × 2), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated by vacuum distillation. The residue was purified by silica gel column chromatography to give the title product 6-3 (5.81 g, yield: 61%) as a yellow solid.

[0634] MS m / z(ESI):312.1[M+1].

[0635] Step 3

[0636] 6-3 (5.31 g, 16.5 mmol) was dissolved in 60 mL of toluene under nitrogen protection. Tributyltin hydride (11.9 g, 40.9 mmol) and azobisisobutyronitrile (543 mg, 3.31 mmol) were added, and the reaction was carried out at 120 °C for 6 hours under nitrogen protection. The reaction solution was concentrated by vacuum distillation. 80 mL of ethyl acetate and 40 mL of hydrochloric acid (1 N) were added to the residue. The resulting liquid was left to stand at 25 °C for 14 hours. The organic phase was removed, and the remaining aqueous phase was washed with ethyl acetate (100 mL × 3). The resulting aqueous phase was an unpurified yellow liquid to give crude product 6-4 (3.27 g). The product was used directly in the next reaction without purification.

[0637] Step 4

[0638] Add sodium bicarbonate solution (25 mL), acetonitrile (25 mL), and 9-fluorenylmethyl-N-succinimide carbonate (6.71 g, 19.9 mmol) to 6-4 (3.27 g, 16.6 mmol). React at 25 °C for 2 h under nitrogen protection. Extract the reaction mixture with ethyl acetate (80 mL × 3). Wash the organic phase with saturated sodium chloride solution (100 mL × 2). Dry the organic phase with anhydrous sodium sulfate, filter, concentrate the filtrate by vacuum distillation, and purify the residue by silica gel column chromatography to give the title product 6-5 (2.01 g, yield: 27%) as a yellow oil.

[0639] 6-5 (2.01 g, 4.80 mmol) was separated by SFC (separation conditions: column: DAICEL CHIRALPAK AD (250 mm × 30 mm, 10 μm); mobile phase: A-carbon dioxide: B-methanol, isocratic elution: B: 35%) to give mono-configuration compounds 6-5a and 6-5b.

[0640] 6-5a: Yellow gelatinous substance, 775 mg, yield: 39%. SFC analysis: retention time 1.830 min. (Column: Chiralpak AD-3 50×4.6 mm ID, 3 μm; mobile phase: A-carbon dioxide, B-methanol (0.05% diethylamine); gradient elution: B: 5%-40%; flow rate: 3 mL / min; instrument: Shimadzu LC-30ADsf).

[0641] MS m / z(ESI):420.1[M+1].

[0642] 6-5b: Yellow gelatinous substance, 750 mg, yield: 37%. SFC analysis: retention time 2.193 min. (Column: Chiralpak AD-3 50×4.6 mm ID, 3 μm; mobile phase: A-carbon dioxide, B-methanol (0.05% diethylamine); gradient elution: B: 5%-40%; flow rate: 3 mL / min; instrument: Shimadzu LC-30ADsf).

[0643] MS m / z(ESI):420.1[M+1].

[0644] Step 5

[0645] (R)-5,5-biphenyl-2-methyl-3,4-propanol-1,3,2-oxazoloborane (79.3 mg, 286 μmol) was dissolved in 2 mL of toluene under nitrogen protection and cooled to 0 °C. Borane dimethyl sulfide complex (10 M, 171 μL) was added dropwise, and the reaction was carried out at 0 °C for 15 min. Then, a toluene solution of 6-5b (600 mg, 1.43 mmol) (8 mL) was added, and the reaction was carried out at 0 °C for 30 min. At 0 °C, 5 mL of methanol and 8 mL of water were added to the reaction solution. The reaction solution was extracted with dichloromethane (10 mL × 3). The organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated by vacuum distillation. The residue was purified by silica gel column chromatography to give the title product 6-6 (500 mg, yield: 81%) as a yellow oil.

[0646] MS m / z(ESI):422.0[M+1].

[0647] Step 6

[0648] Dissolve 6-6 (500 mg, 1.15 mmol) in 10 mL of tetrahydrofuran under nitrogen protection, cool to 0 °C, and add dropwise a 2 M, 864 μL solution of lithium borohydride in tetrahydrofuran. Stir at 25 °C for 1 hour. Add 1 mL of ammonium chloride solution and 5 mL of water at 0 °C. Extract the reaction mixture with dichloromethane (8 mL × 3). Dry the organic phase with anhydrous sodium sulfate, filter, concentrate the filtrate by vacuum distillation, and purify the residue by silica gel column chromatography to give the title product 6-7 (189 mg, yield: 63%) as a white solid.

[0649] MS m / z(ESI):380.0[M+1].

[0650] Step 7

[0651] Dissolve 6-7 (25 mg, 64.57 μmol) in 3 mL of dry pyridine. Under nitrogen protection, cool to below 5 °C. Then, dissolve 4,4'-bismethoxytriphenylmethyl chloride (21.88 mg, 64.57 μmol) in 5 mL of dichloromethane and slowly add it dropwise to the reaction solution. Allow the mixture to rise naturally to 25 °C and stir for 12 hours. Quench the reaction solution with 1 mL of methanol. Concentrate the reaction solution by vacuum distillation. The residue is purified by preparative high-performance liquid chromatography (HPLC) (separation conditions: column: InfinityLab Poroshell 120SB-C18 21.2 × 250 mm, 4 μm; mobile phase: A-water (0.05% ammonium bicarbonate): B-acetonitrile, gradient elution: B%: 20%-100%), yielding the title product 6-8 (27 mg, yield: 58%) as a white solid.

[0652] Step 8

[0653] 6-8 (26 mg, 38 μmol) and hexahydropyridine (6 mg, 73 μmol) were dissolved in 3 mL of N,N-dimethylformamide and reacted at 25 °C for 12 h. The reaction solution was directly purified by C18 reversed-phase column (mobile phase: A-water (0.05% ammonium bicarbonate), B-acetonitrile; gradient elution: B%: 10%-80%) to give the title product 6-9 (16 mg, yield: 91%) as a white solid.

[0654] Step 9

[0655] Dissolve 6-9 (15 mg, 31.00 μmol) and monomethyl dodecanoate (7.58 mg, 31.02 μmol) in 2 mL of dichloromethane, add 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (14.15 mg, 37.21 μmol) and N,N-diisopropylethylamine (20.03 mg, 155.0 μmol), and react at 25 °C for 2 hours. The reaction solution was concentrated by vacuum distillation, and the residue was purified by preparative high-performance liquid chromatography (HPLC) (separation conditions: column: InfinityLab Poroshell 120SB-C18 21.2×250mm, 4μm; mobile phase: A-water (0.05% ammonium bicarbonate): B-acetonitrile, gradient elution: B%: 30%-100%), to give the title product 6-10 (14 mg, yield: 62%) as a white solid.

[0656] Step 10

[0657] Dissolve 6-10 (12 mg, 16.62 μmol) in 2 mL of tetrahydrofuran and 2 mL of water, add lithium hydroxide (1.99 mg, 83.09 μmol), and react at 25 °C for 12 hours. Concentrate the reaction solution under reduced pressure by distillation, and purify the residue by preparative high performance liquid chromatography (HPLC) (separation conditions: column: InfinityLab Poroshell 120SB-C18 21.2 × 250 mm, 4 μm; mobile phase: A-water (0.05% ammonium bicarbonate): B-acetonitrile, gradient elution: B%: 10%-70%), to give the title product 6-11 (10 mg, yield: 85%) as a white solid.

[0658] Step 11

[0659] 6-12b (280 mg, 1.40 mmol, prepared by the method disclosed in Example 2 on page 24 of patent application "WO2016155545 A1") and 6-12a (200 mg, 425 μmol, prepared by the method disclosed in Example 4 on page 512 of patent application "WO2014179620 A1") were dissolved in 10 mL of dichloromethane, and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (532 mg, 1.40 mmol) and N,N-diisopropylethylamine (197 mg, 1.53 mmol) were added. The mixture was stirred at 20 °C for 2 hours. The reaction solution was concentrated by vacuum distillation, and the residue was purified by C18 reversed-phase column (separation conditions: mobile phase: A-water (0.05% ammonium bicarbonate): B-acetonitrile, gradient elution: B%: 20%-80%), to give the title product 6-12c (400 mg, yield: 93%) as a white solid.

[0660] Step Twelve

[0661] 6-12c (400 mg, 393 μmol) was dissolved in 1 mL of methanol, and a dioxane solution of hydrochloric acid (4 N, 20 mL) was added. The mixture was stirred at 20 °C for 6 hours. The reaction solution was concentrated by vacuum distillation, and the residue was adjusted to neutral with sodium carbonate solution. The resulting mixture was purified by C18 reversed-phase column chromatography (separation conditions: mobile phase: A-water (0.05% ammonium bicarbonate): B-acetonitrile, gradient elution: B%: 5%-50%), yielding the title product 6-12d (270 mg, yield: 96%) as a white solid.

[0662] MS m / z(ESI):359.83[1 / 2(M+2)].

[0663] Step Thirteen

[0664] 6-12e (144 mg, 322 μmol, prepared by the method disclosed in Example 1 on page 161 of patent application "WO2009073809 A2") and 6-12d (70 mg, 97 μmol) were dissolved in 5 mL of dichloromethane. 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (122 mg, 322 μmol) and N,N-diisopropylethylamine (88 mg, 682 μmol) were added, and the mixture was stirred at 20 °C for 2 hours. The reaction solution was concentrated by vacuum distillation, and the residue was purified by a C18 reverse-phase column (separation conditions: mobile phase: A-water (0.05% ammonium bicarbonate): B-acetonitrile, gradient elution: B%: 20%-70%), yielding the title product 6-12f (120 mg, yield: 61%) as a white solid.

[0665] MS m / z(ESI):1004.37[1 / 2(M+2)].

[0666] Step Fourteen

[0667] 6-12f (120 mg, 60 μmol) and acetic acid (17 mg, 283 μmol) were dissolved in 10 mL of methanol and 10 mL of ethyl acetate. Wet palladium on carbon (60 mg, 10%) was added, and the mixture was purged three times with hydrogen. The reaction mixture was stirred at 25 °C for 6 hours under hydrogen (15 Psi). The reaction mixture was filtered through diatomaceous earth, and the filtrate was concentrated by vacuum distillation. The residue was purified by reversed-phase liquid chromatography (separation conditions: mobile phase: A-water (0.1% formic acid): B-acetonitrile, gradient elution: B%: 10%-50%), yielding the title product 6-12 (90 mg, yield: 76%) as a white solid.

[0668] MS m / z(ESI):937.12[1 / 2(M+2)].

[0669] Step 15

[0670] Dissolve 6-12 (5 mg, 7.07 μmol) and 6-11 (14.71 mg, 7.07 μmol) in 2 mL of dichloromethane, add 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (4.03 mg, 10.6 μmol) and N,N-diisopropylethylamine (4.57 mg, 35.36 μmol), and react at 25 °C for 2 hours. Concentrate the reaction solution under reduced pressure by distillation, and purify the residue by preparative high-performance liquid chromatography (HPLC) (separation conditions: column: InfinityLab Poroshell 120SB-C18 21.2 × 250 mm, 4 μm; mobile phase: A-water (0.05% ammonium bicarbonate): B-acetonitrile, gradient elution: B%: 20%-70%), to give the title product 6-13 (11 mg, yield: 55%) as a white solid.

[0671] Step Sixteen

[0672] Dissolve 6-13 (10 mg, 3.94 μmol) in 3 mL of dichloromethane under nitrogen protection. Add 4-dimethylaminopyridine (0.1 mg, 0.79 μmol), succinic anhydride (1.97 mg, 19.7 μmol), and N,N-diisopropylethylamine (5.09 mg, 39.4 μmol). Stir at 25 °C for 15 hours. Concentrate the reaction solution under reduced pressure. The residue is purified by preparative high-performance liquid chromatography (HPLC) (separation conditions: column: InfinityLab Poroshell 120SB-C18 21.2 × 250 mm, 4 μm; mobile phase: A-water (0.05% ammonium bicarbonate): B-acetonitrile, gradient elution: B%: 10%-90%) to give title product 6 (4 mg, yield: 36%) as a creamy white solid.

[0673] MS m / z(ESI):1162.9[1 / 2(M-301)].

[0674] 1H NMR (400MHz, CD3OD) δ7.39-7.36(m,2H),7.30-7.28(m,1H),7.27-7.24(m,5H),7.21-7.18(m,1H),6.87-6.82(m,4H),5.33-5.31(m, 3H),5.08-5.03(m,4H),4.57-4.54(m,3H),4.17-4.04(m,12H),4.02-3.97(m,4H),3.89-3.84(m,4H),3.78-3.77(m,6H),3.70-3.65 (m,12H),3.56-3.50(m,4H),3.48-3.46(m,1H),3.33-3.31(m,2H),3.15-3.11(m,12H),2.46-2.42(m,8H),2.25-2.21(m,6H),2.13- 2.11(m,9H),2.02-2.00(m,9H),1.94-1.91(m,18H),1.71-1.49(m,22H),1.48-1.42(m,2H),1.31-1.25(m,12H),0.50-0.46(m,12H).

[0675] Examples 1-7: Preparation of Compound 7

[0676] first step

[0677] (S)-5,5-biphenyl-2-methyl-3,4-propanol-1,3,2-oxazoloborane (85.9 mg, 310 μmol) was dissolved in 10 mL of toluene under nitrogen protection and cooled to 0 °C. Borane dimethyl sulfide complex (10 M, 186 μL) was added dropwise and the reaction was carried out at 0 °C for 15 min. Then 6-5a (650 mg, 1.55 mmol) was added and the reaction was carried out at 0 °C for 30 min. At 0 °C, 5 mL of methanol and 8 mL of water were added to the reaction solution. The reaction solution was extracted with dichloromethane (10 mL × 3). The organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated by vacuum distillation. The residue was purified by silica gel column chromatography to give the title product 7-1 (503 mg, yield: 74%) as a yellow oil.

[0678] MS m / z(ESI):422.1[M+1].

[0679] Step 2

[0680] 7-1 (503 mg, 1.15 mmol) was dissolved in 8 mL of tetrahydrofuran under nitrogen protection and cooled to 0 °C. A 2 M solution of lithium borohydride in tetrahydrofuran (862 μL) was added dropwise, and the mixture was stirred at 25 °C for 1 hour. 1 mL of ammonium chloride solution and 5 mL of water were added at 0 °C. The reaction mixture was extracted with dichloromethane (8 mL × 3). The organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated by vacuum distillation. The residue was purified by silica gel column chromatography to give the title product 7-2 (51.83 mg, yield: 38%) as a white solid.

[0681] MS m / z(ESI): 380.1 [M+1].

[0682] Step 3

[0683] Dissolve 7-2 (20 mg, 52.71 μmol) in 1 mL of dry pyridine. Under nitrogen protection, cool to below 5 °C. Then, dissolve 4,4'-dimethoxytriphenylmethyl chloride (27 mg, 79.07 μmol) in 1 mL of dichloromethane and slowly add it dropwise to the reaction solution. Allow the mixture to rise naturally to room temperature and stir for 15 hours. Quench the reaction solution with 1 mL of methanol, concentrate the reaction solution by vacuum distillation, and purify the residue by preparative high-performance liquid chromatography (HPLC) (separation conditions: column: InfinityLab Poroshell 120SB-C182 1.2 × 250 mm, 4 μm; mobile phase: A-water (0.05% ammonium bicarbonate): B-acetonitrile, gradient elution: B%: 20%-100%) to obtain the title product 7-3 (21 mg, yield: 58%) as a pale yellow oil.

[0684] Step 4

[0685] 7-3 (21 mg, 30.8 μmol) was dissolved in 1 mL of N,N-dimethylformamide, and then piperidine (13 mg, 153.96 μmol) was added. The mixture was stirred at room temperature for 1 hour. The reaction solution was concentrated by vacuum distillation, and the residue was purified by preparative high-performance liquid chromatography (HPLC) (separation conditions: column: InfinityLab Poroshell 120SB-C18 21.2 × 250 mm, 4 μm; mobile phase: A-water (0.05% ammonium bicarbonate): B-acetonitrile, gradient elution: B%: 20%-100%), yielding the title product 7-4 (12 mg, yield: 85%) as a pale yellow oil.

[0686] Step 5

[0687] 7-4 (12 mg, 26.11 μmol) and monomethyl dodecanoate (7.02 mg, 28.73 μmol) were dissolved in 1 mL of dichloromethane, and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (11.91 mg, 31.31 μmol) and N,N-diisopropylethylamine (13.50 mg, 104.46 μmol) were added. The mixture was reacted at 25 °C for 2 hours. The reaction solution was concentrated by vacuum distillation, and the residue was purified by preparative high-performance liquid chromatography (HPLC) (separation conditions: column: InfinityLab Poroshell 120SB-C182 1.2×250mm, 4μm; mobile phase: A-water (0.05% ammonium bicarbonate): B-acetonitrile, gradient elution: B%: 20%-100%), yielding the title product 7-5 (15mg, yield: 84%) as a pale yellow oil.

[0688] Step 6

[0689] Dissolve 7-5 (15 mg, 21.87 μmol) in 1 mL of methanol and 0.3 mL of water, add lithium hydroxide (2.62 mg, 109.39 μmol), and react at 25 °C for 2 hours. Concentrate the reaction solution under reduced pressure by distillation, and purify the residue by preparative high-performance liquid chromatography (HPLC) (separation conditions: column: InfinityLab Poroshell 120SB-C18 21.2 × 250 mm, 4 μm; mobile phase: A-water (0.05% ammonium bicarbonate): B-acetonitrile, gradient elution: B%: 5%-80%), to give the title product 7-6 (9 mg, yield: 61%) as a white solid.

[0690] Step 7

[0691] Dissolve 7-6 (8 mg, 11.9 μmol) and 6-12 (22.48 mg, 12.0 μmol) in 1 mL of dichloromethane, add 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (5.43 mg, 14.28 μmol) and N,N-diisopropylethylamine (6.16 mg, 47.66 μmol), and react at 25 °C for 2 hours. Concentrate the reaction solution under reduced pressure by distillation, and purify the residue by preparative high-performance liquid chromatography (HPLC) (separation conditions: column: InfinityLab Poroshell 120SB-C18 21.2 × 250 mm, 4 μm; mobile phase: A-water (0.05% ammonium bicarbonate): B-acetonitrile, gradient elution: B%: 5%-90%), to give the title product 7-7 (17 mg, yield: 56%) as a white solid.

[0692] MS m / z(ESI):2224.8[M-301].

[0693] Step 8

[0694] 7-7 (17 mg, 6.73 μmol) was dissolved in 1 mL of dichloromethane under nitrogen protection. 4-Dimethylaminopyridine (0.16 mg, 1.35 μmol), succinic anhydride (3.37 mg, 33.68 μmol), and N,N-diisopropylethylamine (8.7 mg, 67.31 μmol) were added, and the mixture was stirred at 25 °C for 15 hours. The reaction solution was concentrated by vacuum distillation, and the residue was purified by preparative high-performance liquid chromatography (HPLC) (separation conditions: column: InfinityLab Poroshell 120SB-C18 21.2 × 250 mm, 4 μm; mobile phase: A-water (0.05% ammonium bicarbonate): B-acetonitrile, gradient elution: B%: 10%-90%), yielding the title product 7 (16 mg, yield: 90%) as a white solid.

[0695] MS m / z(ESI):2324.8[M-301].

[0696] 1H NMR (400MHz, CDCl3) δ7.51-7.49(m,1H),7.32-7.30(m,1H),7.30-7.28(m,3H),7.20-7 .16(m,4H),6.86-6.84(m,2H),6.83-6.82(m,2H),5.39-5.34(m,3H),5.21-5.17(m,3H) ,5.09-4.97(m,1H),4.66-4.58(m,3H),4.55-4.44(m,1H),4.21-4.05(m,10H),3.99-3. 90(m,6H),3.82-3.80(m,6H),3.72-3.67(m,12H),3.55-3.49(m,4H),3.19-3.05(m,12H ),2.63-2.55(m,4H),2.50-2.45(m,6H),2.38-2.21(m,10H),2.20-2.17(m,2H),2.17-2 .15(m,9H),2.13-2.08(m,2H),2.07-2.05(m,9H),2.01-1.99(m,9H),1.98-1.95(m,9H) ,1.85-1.77(m,4H),1.76-1.67(m,6H),1.66-1.61(m,6H),1.57-1.53(m,2H),1.39-1.3 3(m,2H),1.31-1.29(m,2H),1.28-1.24(m,8H),0.92-0.82(m,2H),0.54-0.47(m,12H).

[0697] Example 2: Preparation of a Customized GalNAc Mounting Loader

[0698] Weigh N,N-diisopropylethylamine (3 eq) and dissolve it in anhydrous N,N-dimethylformamide to prepare an N,N-diisopropylethylamine solution with a concentration of 8.1 mg / mL. Weigh 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (2 eq) and dissolve it in anhydrous acetonitrile to prepare a 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate solution with a concentration of 14.6 mg / mL. Dissolve the GalNAc compounds (compounds 6 and 7 above) in the above N,N-diisopropylethylamine N,N-dimethylformamide solution (3 eq), and then add the 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate acetonitrile solution (2 eq). Take CPG (blank loading of 180 μmol / g, target loading of 40 μmol / g) and add it dropwise to the above mixed solution containing GalNAc. Shake gently to mix. Place the reaction solution on a shaker (temperature: 25℃, speed: 200 rpm) and shake overnight. Add dichloromethane to the reaction solution to flush the CPG adhering to the flask wall to the bottom. Wash the solution three times with dichloromethane and twice with anhydrous acetonitrile. After each wash, wait for the CPG to precipitate to the bottom of the flask, carefully aspirate the supernatant, and dry the solid obtained from the last wash under vacuum for 1 hour. Weigh 1 mg of 4-dimethylaminopyridine and add 1000 μL of anhydrous acetonitrile to prepare a 1 mg / mL acetonitrile solution of 4-dimethylaminopyridine; take 20 μL of N-methylimidazole and add 80 μL of anhydrous acetonitrile to prepare a CapA solution; take 40 μL of acetic anhydride and add 60 μL of anhydrous acetonitrile to prepare a CapB1 solution; take 60 μL of anhydrous pyridine and add 40 μL of anhydrous acetonitrile to prepare a CapB2 solution; add 65.4 μL of the above acetonitrile solution of 4-dimethylaminopyridine, 12.95 μL of CapA, 56.95 μL of CapB1, and 6.33 μL of CapB2 sequentially to a CPG support linked with different GalNAc compounds, and place the reaction solution on a shaker (temperature: 25℃, speed: 200 rpm) and shake for 2 hours. Anhydrous acetonitrile was added to the reaction solution to flush the CPG adhering to the bottle wall to the bottom of the bottle. The mixture was then washed three times with anhydrous acetonitrile. The solid was collected, dried under vacuum overnight, and CPG containing the GalNAc compound was obtained (labeled as CPG-L6 and CPG-L7, respectively). The loading was then determined.

[0699] The structures of CPG-L6 and CPG-L7 are as follows:

[0700] Example 3: Preparation of siRNA

[0701] The siRNA of this invention was prepared using the solid-phase phosphoramide method well-known in the art. The specific method is briefly described below.

[0702] 1. Synthesis of the Justice Chain (SS Chain)

[0703] The oligonucleotide synthesis method, using a solid-phase phosphoramide, employs a blank CPG solid support or a solid support linked with GalNAc as the starting cycle. Nucleotide 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 0.5 μmol oligonucleotides are as follows:

[0704] The nucleoside monomer was provided in a 0.05 mol / L acetonitrile solution. The reaction conditions were the same for each step, i.e., the temperature was 25 °C. Deprotection was performed four times using a 3% trichloroacetic acid-dichloromethane solution. The coupling reaction was activated with a 0.30 mol / L 5-BTT-acetonitrile solution and was performed three times. Capping was performed twice using 20% ​​acetic anhydride-acetonitrile and 15% N-methylimidazolium-acetonitrile. Oxidation was performed twice using a 0.05 mol / L iodine-tetrahydrofuran / pyridine / water solution (70 / 20 / 10, v / v / v). Thiolysis was performed three times using a 0.05 mol / L LDDTT solution of acetonitrile / pyridine (l / 1, v / v).

[0705] 2. Synthesis of the antisense chain (AS chain)

[0706] 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 0.5 μmol oligonucleotide of the antisense strand are the same as those for the sense strand.

[0707] Hydrolysis of 3,5'-phosphonate modified nucleotides

[0708] The synthesized solid support with 5'-phosphonate modified nucleotides was added to a 3 mL centrifuge tube, and a mixture of trimethylsilyl / pyridine / acetonitrile (1 mL, 1:13:40, v / v / v) was added. The mixture was reacted in a water bath at 25 °C for 1 hour. After filtration, the solid support was washed three times with acetonitrile / water (1 / 1, v / v), 2 mL each time, and then the solid support was collected.

[0709] 4. Ammonolysis

[0710] The synthesized solid-phase support (sense or antisense chain) was added to a 3 mL centrifuge tube, and ammonia / ethanol (0.5 mL, 3 / 1, v / v) was added. The mixture was reacted in a constant temperature water bath at 45 °C for 16 hours, filtered, and the crude product was purified by centrifugation and concentration of the filtrate.

[0711] 5. Purification

[0712] 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.

[0713] 6. Annealing

[0714] According to the table below, the sense chain (SS chain) and the antisense chain (AS chain) are mixed in a molar ratio (SS chain / AS chain = 1 / 1), 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.

[0715] Table 1: The siRNA sequences used in this invention are as follows:

[0716] The meanings of the abbreviations in this article are as follows:

[0717] The distributions A, U, G, and C represent naturally occurring adenine ribonucleotides, uracil ribonucleotides, guanine ribonucleotides, and cytosine ribonucleotides.

[0718] 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.

[0719] 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.

[0720] “s” or “s-” indicates that the two adjacent nucleotides and / or delivery carriers are linked by a phosphate thioester.

[0721] (S)-GNA-A is a well-known non-natural nucleic acid analog in the art, and can be prepared with reference to patent WO2011133876 A2. SPP1b-Um is prepared from compound 5B in the compound examples using the above-described siRNA preparation method. (S)-hcpNA-A and (R)-hcpNA-A are prepared from compounds 3A and 3B in the compound examples using the above-described siRNA preparation method, respectively. The structures of (S)-GNA-A, SPP1b-Um, (S)-hcpNA-A, and (R)-hcpNA-A are as follows:

[0722] III. Test Case

[0723] Biological evaluation

[0724] Test Example 1: Inhibitory activity test of the disclosed siRNA against INHBE mRNA in Hep3b cells.

[0725] The effect of siRNA targeting INHBE on the mRNA expression level of INHBE was tested in vitro. The inhibitory effect of different siRNA modifications on INHBE mRNA was determined; for each siRNA, the concentrations measured were 10.0, 2.0, 0.4, 0.08, 0.016, 0.032 and 0.00064 nM, for a total of 7 concentrations, 5-fold dilutions and 2 biological replicates.

[0726] 1) Cell culture and plating: Hep3b cells were cultured in MEM medium containing 10% fetal bovine serum at 37°C and 5% CO2. 24 hours before transfection, Hep3b cells were collected and seeded into 96-well plates at a density of 20,000 cells per well with 100 μL of medium per well.

[0727] 2) Transfection: Refer to the product instruction manual and use lipofectamine RNAiMAX (ThermoFisher, 13778150) to transfect siRNA. The final concentration of siRNA transfection is 10 nM, and three replicates are used.

[0728] 3) RNA extraction: Continue culturing cells for 24 hours, and use Thermo Fisher's automated nucleic acid extractor in conjunction with Tiangen's magnetic bead-based tissue / cell / blood total RNA extraction kit (DP761) to extract RNA. Refer to the product instruction manual for specific steps.

[0729] 3) RNA reverse transcription to cDNA: using Novizan III. Reverse transcription was performed using the RT SuperMix for qPCR (+gDNAwiper) kit. A 10 μL reverse transcription reaction mixture was prepared according to the kit instructions to reverse transcribe the total RNA from the cells. The transcribed cDNA was then diluted 3-fold with 20 μL of DEPC water. The sample was used for subsequent experiments or stored at -20°C.

[0730] 4) qPCR: For each reverse transcription reaction system, take 4 μL of the diluted cDNA solution as a template and prepare 10 μL of qPCR reaction system using the reagents provided by the Novizan AceQ Universal U+Probe Master Mix V2 kit. The PCR primer sequences for amplifying the target gene INHBE and the internal reference gene GAPDH are shown in Table 2, and the TaqMan probe sequences for amplifying the target gene INHBE and the internal reference gene GAPDH are shown in Table 3. The final concentration of each primer and probe is 10 μM. The Ct values ​​of the target gene INHBE and the internal reference gene GAPDH are obtained using a real-time quantitative PCR system based on the TaqMan method.

[0731] Table 2. PCR primer sequences

[0732] Table 3. Taqman probe sequences used in real-time quantitative PCR

[0733] 6-FAM and VIC represent fluorescent groups, and BHQ1 represents a quenching group.

[0734] The expression levels of the target gene INHBE in each test group and control group were relatively quantitatively calculated using the Ct (ΔΔCt) method. The calculation method is as follows:

[0735] ΔCt(test group) = Ct(target gene in test group) - ΔCt(internal reference gene in test group)

[0736] ΔCt(control group) = Ct(target gene in control group) - ΔCt(internal reference gene in control group)

[0737] ΔCt(test group) = ΔCt(test group) - ΔCt(control group average)

[0738] ΔCt(control group) = ΔCt(control group) - ΔCt(control group average)

[0739] Wherein, ΔCt (control group average) is the arithmetic mean of ΔCt (control group) for each of the three culture wells in the control group. Therefore, each culture well in both the test group and the control group corresponds to a ΔCt value. The relative expression level of INHBE mRNA in the test group = 2 - ΔCt (test group) × 100%.

[0740] Table 4. Inhibitory activity of different siRNAs against INHBE mRNA in Hep3b cells

[0741] Conclusion: The siRNA conjugates tested above exhibited significant inhibitory activity against INHBE mRNA expression in Hep3b cells in vitro. Furthermore, the on-target activities of dsRNA-3 and dsRNA-4, modified at position 5' of the AS strand (as shown in Table 1) using compounds (S)-hcpNA-A and (R)-hcpNA-A, were no weaker than, and even superior to, the parent sequence dsRNA-1 and the reference sequence dsRNA-2 modified with (S)-GNA-A.

[0742] Test Example 2: Off-target activity test of the disclosed siRNA against psi-CHECK2

[0743] 1) Plasmid preparation: Design the corresponding antisense off-target plasmid based on the siRNA sequence, and dilute the recombinant plasmid psiCHECK-2-I1008 containing the complementary sequence of the guide strand seed region to 1000 μg / μL for later use.

[0744] 2) Cell transfection: HEK293T cells were seeded in 96-well plates with 100 μL of cell resuspended solution. Cell quantity: 2 × 10⁶ cells / well. 4 Cells / well. On the second day, 0.5 μL of Lipofectamine 2000 (ThermoFisher, 11668019) was diluted in 25 μL of serum-free Opti-MEM, gently mixed, and allowed to stand for 5 min. DNA-siRNA mixed dilution buffer was prepared by adding 30 μL of plasmid to 5 mL of Opti-MEM. A 96-well U-shaped plate was used as the dilution plate. 69 μL of the plasmid-Opti-MEM mixed dilution buffer was added to well A, and 60 μL of the same buffer was added to wells B and G. The siRNA was started at 2 pmol / well and diluted 1:4 (5-fold) to set 7 concentrations. Specifically, 6 μL of siRNA was added to well A, mixed thoroughly, and then 15 μL was transferred to well B for further dilution. This process was repeated until well G was reached, and then 15 μL of each diluted siRNA buffer was discarded. Mix 60 μL of lipofectamine 2000 dilution buffer with 60 μL of DNA-siRNA dilution buffer and let stand for 10 min. Add 50 μL of the transfection complex to a 96-well plate and incubate at 37°C with 5% CO2 for 48 h.

[0745] 3) Result Detection: Melt Duo-Lite Luciferase Buffer and Duo-Lite Stop&Lite (Vazyme, DD1205-01) at 2–8℃ or room temperature. Add the melted Duo-Lite Luciferase Buffer to Duo-Lite Luciferase Substrate, gently inverting and mixing 3–5 times to ensure complete substrate dissolution. Dilute Duo-Lite Stop&Lite Substrate 1:100 to the corresponding volume of Duo-Lite Stop&Lite. Discard all supernatant from the 96-well plate and add 40 μL of Duo-Lite Luciferase assay reagent. Incubate at room temperature for 5 min to detect firefly luciferase luminescence. Add 40 μL of Duo-Lite Stop&Lite assay reagent (freshly prepared), and detect Renilla luciferase luminescence after 5 min.

[0746] 4) Data Analysis: To ensure accuracy, the luminescence values ​​of both firefly and Renilla luciferase were subtracted from the corresponding background values ​​during calculation. Cells were transfected with the psiCHECK2 plasmid and the negative siRNA sequence to standardize the results (i.e., control group Firefly and control group Renilla). The collected Renilla signal was normalized using the Firefly signal standard, and the inhibitory effect of the siRNA was determined by comparing the results without treatment (residual inhibitory activity).

[0747] Residual inhibition rate = ((experimental group Renilla - background Renilla) / (experimental group Firefly - background Firefly)) / ((control group Renilla - background Renilla) / (control group Firefly - background Firefly)).

[0748] Table 5. Off-target activities of different siRNAs

[0749] Conclusion: Modifying the 5th position of the 5' end of the AS strand with compounds (S)-hcpNA-A and (R)-hcpNA-A (sequences shown in Table 1) to form dsRNA-3 and dsRNA-4 significantly reduced the off-target activity of siRNA compared to the parent sequence. Furthermore, at 10 nM, the off-target activity of compound dsRNA-4 was significantly lower than that of compound dsRNA-2, indicating that compound dsRNA-4 has better safety.

[0750] Test Example 3: On-target and off-target activity test of the disclosed siRNA against different target sequences

[0751] This test case used siRNAs targeting different gene (HBV, HAO1, TTR) mRNAs (sequence information is shown in Table 1). These sequences were modified at position 6 or 7 of the 5' end of the AS strand using compounds (S)-hcpNA-A and (R)-hcpNA-A, and then compared with the parent sequence for on-target and off-target activities. The experimental methods were the same as in Test Case 1 and Test Case 2.

[0752] Table 6. Target activity results of siRNAs targeting different target sequences

[0753] Table 7. Off-target activity results of siRNAs targeting different target sequences

[0754] The results of the target activity experiments are shown in Table 6. The results show that siRNAs modified with compounds (S)-hcpNA-A and (R)-hcpNA-A to different target sequences maintained or even improved their target activity compared to their unmodified parent sequences, demonstrating no significant sequence dependence and strong general applicability. The results of the off-target activity experiments are shown in Table 7. It can be seen that, compared to the parent sequence, siRNAs modified with compounds (S)-hcpNA-A and (R)-hcpNA-A significantly reduced off-target activity.

[0755] Test Example 4: Efficacy evaluation of the disclosed siRNA conjugate in the C57BL / 6 mouse model

[0756] C57BL / 6 mice were randomly divided into groups of four. The dosage for each animal was calculated based on its body weight. The drug was administered subcutaneously as a single dose. Blood samples were collected from the orbital venous plexus of mice before administration (day 0) and on days 7 and 14 after administration. At each time point, the expression level of TTR protein in the serum of mice was detected by an ELISA kit (Abcam, ab282297). After the experiment, total RNA was extracted from liver tissue using Trizol (catalog number: 15596026CN, Thermo Fisher). After reverse transcription, the level of TTR mRNA in the liver of mice was detected by real-time quantitative PCR (qPCR) (primer information is shown in Table 8). The experimental results are shown in Table 10 and Figure 1.

[0757] Table 8: Primer Information

[0758] Data statistics and analysis

[0759] Calculate the 2-ΔΔα value and convert it to a percentage to obtain the residual inhibition rate;

[0760] ΔΔCt=[(Ct experimental group target gene - Ct experimental group internal reference) - (Ct control group target gene - Ct control group internal reference)].

[0761] The target gene is mTTR, and the internal reference is mGAPDH.

[0762] Table 9: Compound Information

[0763] Table 10: Pharmacodynamic results of the compounds in the C57BL / 6 mouse model

[0764] The above test results indicate that all the tested siRNA conjugates can effectively knock down the level of TTR mRNA in mouse liver. At the same dose, compound dsRNA-14 can maintain in vivo knockdown activity comparable to the parent sequence dsRNA-11, and the in vivo knockdown effect is better than that of the reference compound dsRNA-12.

[0765] While preferred embodiments of the invention have been shown and described herein, these embodiments will be apparent to those skilled in the art only as examples. Many variations, changes, and substitutions will occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments described herein may be employed. The following claims are intended to define the scope of the invention and cover the methods and structures within the scope of these claims and their equivalents.

Claims

An oligonucleotide or a pharmaceutically acceptable salt thereof, wherein, The oligonucleotide includes one, two, three, four, five or more modifying groups of the structure shown in formula (I), or pharmaceutically acceptable salts, isotopic variants, tautomers or stereoisomers thereof: in, Ring A is selected from C 0-6 Alkylene-C 3-10 Cycloalkyl-C 0-6 Alkylene, C 0-6 alkylene-3-10-membered heterocyclic-C 0-6 Alkylene, C 0-6 Alkylene-C 6-10 Aspartic-C 0-6 Alkylene or C 0-6 alkylene-5-10-membered heteroaryl-C 0-6 Alkylene, wherein the ring A is optionally surrounded by 1, 2, 3, 4, 5 or 6 R atoms. a replace; L1 is selected from C 1-6 Alkylene, C 1-6 Heteroalkyl, C 2-6 imidene group, C 2-6 Ethyne group, C 3-10 Cycloalkylene, 3-10 membered heterocyclic alkylene, C 6-10 The L1 is optionally surrounded by 1, 2, 3, 4, 5, or 6 R atoms. b replace; L2 is selected from C 1-6 Alkylene, NH-C 0-6 Alkylene, C(O)-C 0-6 Alkylene, NHC(O)-C 0-6 Alkylene, C 2-6 imidene group, C 2-6 Ethyne group, C 3-10 Cycloalkylene, 3-10 membered heterocyclic alkylene, C 6-10 The L2 is optionally surrounded by 1, 2, 3, 4, 5, or 6 R groups. c replace; X is selected from O or S; Y is OR d ; Base is selected from modified or unmodified bases; Each R a Independently selected from H, halogen, OR, CN, NRR', C 1-6 Alkyl, C 1-6 Haloalkyl, C 1-6 Alkoxy or C 1-6 Halogenated alkoxy groups; Each R b Independently selected from H, halogen, OR, CN, NRR', C 1-6 Alkyl, C 1-6 Haloalkyl, C 1-6 Alkoxy, C 1-6 Haloalkoxy groups, or two R atoms on the same or different carbon atoms. b They are connected, and together with the carbon atoms they are connected to, they form C. 3-7 Cycloalkylene; Each R c Independently selected from H, halogen, OR, CN, NRR', C 1-6 Alkyl, C 1-6 Haloalkyl, C 1-6 Alkoxy, C 1-6 Haloalkoxy groups, or two R atoms on the same or different carbon atoms. c They are connected, and together with the carbon atoms they are connected to, they form C. 3-7 Cycloalkylene; R d Selected from H, halogens, C 1-6 Alkyl or C 1-6 Halogenated alkyl groups; R and R' are independently selected from H, halogens, and C. 1-6 Alkyl, C 2-6 alkenyl, C 2-6 alkynyl or C 1-6 Haloalkyl groups, or R and R', are attached to the nitrogen atom to which they are attached, together forming 3-7 membered heterocyclic groups. The oligonucleotide of claim 1 or a pharmaceutically acceptable salt thereof, wherein, The modifying group of formula (I) has the following structure: in, Ring A is selected from C 3-10 Cycloalkylene, 3-10 membered heterocyclic alkylene, C 6-10 The ring is arylene or 5-10 heteroarylene, wherein the ring A is optionally surrounded by 1, 2, 3, 4 or 5 R groups. a replace; L1 is selected from C 1-6 Alkylene, C 1-6 Heteroalkyl, C 2-6 imidene group, C 2-6 Ethyne group, C 3-10 The L1 is optionally surrounded by 1, 2, 3, 4, or 5 R groups, or a cycloalkyl or 3-10-membered heterocyclic group. b replace; L2 is selected from C 1-6 Alkylene, NH-C 0-6 Alkylene, C(O)-C 0-6 Alkylene, NHC(O)-C 0-6 Alkylene, C 2-6 imidene group, C 2-6 Ethyne group, C 3-10 The L2 is optionally surrounded by 1, 2, 3, 4, or 5 R groups. c replace; X is selected from O or S; Y is OR d ; Base is selected from modified or unmodified bases; Each R a Independently selected from H, halogens, OH, CN, NH2, C 1-6 Alkyl, C 1-6 Haloalkyl, C 1-6 Alkoxy or C 1-6 Halogenated alkoxy groups; Each R b and R b 'Independently selected from H, halogens, OH, CN, NH2, C 1-6 Alkyl, C 1-6 Haloalkyl, C 1-6 Alkoxy, C 1-6 Haloalkoxy groups, or two R atoms on the same or different carbon atoms. b (or R) b and R b ') are connected together, and together with the carbon atoms they are connected to, they form C. 3-7 Cycloalkylene (preferably C16) 3-5 (cycloalkylene); Each R c and R c 'Independently selected from H, halogens, OH, CN, NH2, C 1-6 Alkyl, C 1-6 Haloalkyl, C 1-6 Alkoxy, C 1-6 Haloalkoxy groups, or two R atoms on the same or different carbon atoms. c (or R) c and R c ') are connected together, and together with the carbon atoms they are connected to, they form C. 3-7 Cycloalkylene (preferably C16) 3-5 (cycloalkylene); R d Selected from H, halogens, C 1-6 Alkyl or C 1-6 Halogenated alkyl groups; m is selected from 0, 1, 2 or 3. The oligonucleotide of claim 1 or 2 or a pharmaceutically acceptable salt thereof, wherein, Ring A is C 3-10 Cycloalkylene, 3-10 membered heterocyclic alkylene, C 6-10 arylene or 5-10 heteroarylene, wherein the ring A is optionally surrounded by one, two or three R atoms. a replace; Each R a Independently selected from H, halogens, OH, CN, NH2, C 1-6 Alkyl, C 1-6 Haloalkyl, C 1-6 Alkoxy, C 1-6 Halogenated alkoxy groups; Preferably, Ring A is selected from C 3-7 Cycloalkylene or 3-7 membered heterocyclic group, wherein ring A is optionally surrounded by 1, 2 or 3 R groups. a replace; Each R a Independently selected from H, halogens, C 1-4 Alkyl or C 1-4 Halogenated alkyl groups; More preferably, Ring A is C 3-5 Cycloalkylene, preferably cyclopropylene, optionally surrounded by one or two atoms selected from H and C. 1-4 Alkyl substituents. The oligonucleotide or a pharmaceutically acceptable salt thereof as described in any one of claims 1-3, wherein, Ring A is C 3-10 Cycloalkylene, 3-10 membered heterocyclic alkylene, C 6-10 arylene or 5-10 heteroarylene, wherein the ring A is optionally surrounded by one, two or three R atoms. a replace; L1 is selected from C 1-6 Alkylene, C 2-6 imide or C 2-6 The ynylene group, wherein the L1 is optionally surrounded by one, two, or three R groups. b replace; L2 is selected from C 1-6 Alkylene, C 2-6 imidene group, C 2-6 Ethyne group or NHC(O)-C 1-6 Alkylene, wherein the L2 is optionally surrounded by one, two or three R atoms. c replace; X is selected from O or S; Y is OR d ; Base is selected from modified or unmodified bases; Each R a Independently selected from H, halogens, OH, CN, NH2, C 1-6 Alkyl, C 1-6 Haloalkyl, C 1-6 Alkoxy, C 1-6 Halogenated alkoxy groups; Each R b and R b 'Independently selected from H, halogens, OH, CN, NH2, C 1-6 Alkyl, C 1-6 Haloalkyl, C 1-6 Alkoxy, C 1-6 Haloalkoxy groups, or two R atoms on the same or different carbon atoms. b (or R) b and R b ') are connected together, and together with the carbon atoms they are connected to, they form C. 3-7 Cycloalkylene; Each R c and R c 'Independently selected from H, halogens, OH, CN, NH2, C 1-6 Alkyl, C 1-6 Haloalkyl, C 1-6 Alkoxy, C 1-6 Haloalkoxy groups, or two R atoms on the same or different carbon atoms. c (or R) c and R c ') are connected together, and together with the carbon atoms they are connected to, they form C. 3-7 Cycloalkylene; R d Selected from H, halogens, C 1-6 Alkyl or C 1-6 Halogenated alkyl groups. The oligonucleotide or a pharmaceutically acceptable salt thereof as described in any one of claims 1-4, wherein, Ring A is selected from C 3-7 Cycloalkylene or 3-7 membered heterocyclic group, wherein ring A is optionally surrounded by 1, 2 or 3 R groups. a replace; L1 is C 1-6 Alkylene, wherein the L1 is optionally surrounded by one, two or three R atoms. b replace; L2 is C 1-6 Alkylene, wherein the L2 is optionally surrounded by one, two or three R atoms. c replace; X is selected from O or S, preferably O; Y is OR d ; Base is selected from modified or unmodified bases; Each R a Independently selected from H, halogens, C 1-4 Alkyl or C 1-4 Halogenated alkyl groups; Each R b and R b 'Independently selected from H, halogen, C 1-4 Alkyl or C 1-4 Halogenated alkyl groups; Each R c and R c 'Independently selected from H, halogen, C 1-4 Alkyl or C 1-4 Halogenated alkyl groups; R d Selected from H, halogens, C 1-4 Alkyl or C 1-4 Halogenated alkyl groups; Preferably, Ring A is C 3-5 Cycloalkylene, preferably cyclopropylene; L1 is C 1-4 Alkylene, preferably -CH2-; L2 is C 1-4 Alkylene, preferably -CH2-; X is selected from O or S, preferably O; Y is selected from OH or C. 1-4 Alkyl group, preferably OH; Base selected The oligonucleotide of claim 1 or 2 or a pharmaceutically acceptable salt thereof, wherein, The modifying group of formula (I) is selected from the structure of formula (III), formula (III-1) or formula (III-2), its pharmaceutically acceptable salt, isotopic variant, tautomer or stereoisomer: in, Ring A is selected from C 3-7 Cycloalkylene or 3-7 membered heterocyclic group, wherein ring A is optionally surrounded by 1, 2 or 3 R groups. a replace; X is selected from O or S, preferably O; Base is selected from modified or unmodified bases; Each R a Independently selected from H, halogens, C 1-4 Alkyl or C 1-4 Halogenated alkyl groups; R b and R b 'Independently selected from H, halogen, C 1-4 Alkyl or C 1-4 Halogenated alkyl groups; R c and R c 'Independently selected from H, halogen, C 1-4 Alkyl or C 1-4 Halogenated alkyl groups; R d Selected from H, halogens, C 1-4 Alkyl or C 1-4 Halogenated alkyl groups; Preferably, Ring A is C 3-5 Cycloalkylene, preferably cyclopropylene, wherein ring A is optionally surrounded by one R a replace; R a Selected from H, halogen or C 1-4 alkyl; R b and R b 'Independently selected from H, halogen or C 1-4 alkyl; R c and R c 'Independently selected from H, halogen or C 1-4 alkyl; X is selected from O or S, preferably O; Y is selected from OH or C. 1-4 Alkyl group, preferably OH; Base selected The oligonucleotide or a pharmaceutically acceptable salt thereof as described in any one of claims 1-6, wherein, The modifying group is selected from the following structures, pharmaceutically acceptable salts, isotopic variants, tautomers, or stereoisomers: Where X is selected from O or S; Base selected Preferably, the modifying group is selected from pharmaceutically acceptable salts, isotopic variants, tautomers, or stereoisomers of the following structures: The oligonucleotide of any one of claims 1-7, wherein, The oligonucleotide is a single strand having 14 to 30 nucleotides. The oligonucleotide or a pharmaceutically acceptable salt thereof as described in any one of claims 1-8, wherein, The oligonucleotide contains one, two, three, four, five, six, seven, or eight modifying groups as described in any one of claims 1-7, or their pharmaceutically acceptable salts, isotopic variants, tautomers, or stereoisomers. Preferably, the oligonucleotide comprises at least one of the following nucleotide positions from the 2nd to the 20th (preferably the 2nd to the 10th, preferably the 3rd to the 8th) from its 5' end, a modifying group as defined in any one of claims 1-7 or a pharmaceutically acceptable salt, isotopic variant, tautomer or stereoisomer thereof. Preferably, the oligonucleotide includes a modifying group as defined in any one of claims 1-7 or a pharmaceutically acceptable salt, isotope variant, tautomer or stereoisomer thereof at the nucleotide position 4, 5, 6 or 7 (preferably 5) from its 5' end. The oligonucleotide of any one of claims 1-7, wherein, The oligonucleotide is double-stranded, comprising a sense strand and an antisense strand, each strand having 14 to 30 nucleotides; Preferably, the oligonucleotide is selected from short interfering RNA (siRNA), double-stranded RNA (dsRNA), microRNA (miRNA) or short hairpin RNA (shRNA), and is preferably siRNA. The oligonucleotide of claim 10, wherein, The oligonucleotide includes, within the sense strand and / or antisense strand, one, two, three, four, five, six, seven, or eight modifying groups as defined in any one of claims 1-7, or their pharmaceutically acceptable salts, isotopic variants, tautomers, or stereoisomers. Preferably, the oligonucleotide includes, within the antisense strand, one, two, three, four, five, six, seven, or eight modifying groups as defined in any one of claims 1-7, or their pharmaceutically acceptable salts, isotopic variants, tautomers, or stereoisomers. More preferably, the oligonucleotide comprises 1, 2, 3, 4, 5, 6, 7 or 8 modifying groups as defined in any one of claims 1-7 or pharmaceutically acceptable salts, isotopic variants, tautomers or stereoisomers thereof at positions 2-20 (preferably positions 2-10, preferably positions 3-8) from the 5' end of the antisense strand. More preferably, the oligonucleotide includes, at the 4th, 5th, 6th or 7th (preferably 5th) nucleotide position starting from the 5' end of the antisense strand, a modifying group as defined in any one of claims 1-7 or a pharmaceutically acceptable salt, isotopic variant, tautomer or stereoisomer thereof. The oligonucleotide of any one of claims 1-11, wherein the oligonucleotide comprises one or more targeting ligands or delivery vectors; preferably, the delivery vector is selected from peptide delivery vectors or antibody delivery vectors; preferably, the targeting ligand is an N-acetylgalactosamine (GalNAc) targeting ligand, such as compound 6, compound 7, and L96. Compound 6: Compound 7: L96： The oligonucleotide of any one of claims 1-12, wherein the oligonucleotide comprises a modified nucleotide monomer, the modification being selected from one or more of 2'-O-methyl modification, 2'-F modification and 5'-spirocyclic phosphonate modification. The oligonucleotide of any one of claims 1-13, wherein the oligonucleotide contains a phosphate thioester bond. Compound of formula (I'), or a pharmaceutically acceptable salt, isotopic variant, tautomer or stereoisomer thereof, or a mixture thereof: in, Ring A is selected from C 0-6 Alkylene-C 3-10 Cycloalkyl-C 0-6 Alkylene, C 0-6 alkylene-3-10-membered heterocyclic-C 0-6 Alkylene, C 0-6 Alkylene-C 6-10 Aspartic-C 0-6 Alkylene or C 0-6 alkylene-5-10-membered heteroaryl-C 0-6 Alkylene, wherein the ring A is optionally surrounded by 1, 2, 3, 4, 5 or 6 R atoms. a replace; L1 is selected from C 1-6 Alkylene, C 1-6 Heteroalkyl, C 2-6 imidene group, C 2-6 Ethyne group, C 3-10 Cycloalkylene, 3-10 membered heterocyclic alkylene, C 6-10 The L1 is optionally surrounded by 1, 2, 3, 4, 5, or 6 R atoms. b replace; L2 is selected from C 1-6 Alkylene, NH-C 0-6 Alkylene, C(O)-C 0-6 Alkylene, NHC(O)-C 0-6 Alkylene, C 2-6 imidene group, C 2-6 Ethyne group, C 3-10 Cycloalkylene, 3-10 membered heterocyclic alkylene, C 6-10 The L2 is optionally surrounded by 1, 2, 3, 4, 5, or 6 R groups. c replace; P1 is selected from H or a reactive phosphorus group, preferably -P(OCH2CH2CN)(N(iPr)2); P2 is selected from H or a protecting group, preferably a hydroxyl protecting group; Base' is selected from H, modified or unmodified bases; Each R a Independently selected from H, halogen, OR, CN, NRR', C 1-6 Alkyl, C 1-6 Haloalkyl, C 1-6 Alkoxy or C 1-6 Halogenated alkoxy groups; Each R b Independently selected from H, halogen, OR, CN, NRR', C 1-6 Alkyl, C 1-6 Haloalkyl, C 1-6 Alkoxy, C 1-6 Haloalkoxy groups, or two R atoms on the same or different carbon atoms. b They are connected, and together with the carbon atoms they are connected to, they form C. 3-7 Cycloalkylene; Each R c Independently selected from H, halogen, OR, CN, NRR', C 1-6 Alkyl, C 1-6 Haloalkyl, C 1-6 Alkoxy, C 1-6 Haloalkoxy groups, or two R atoms on the same or different carbon atoms. c They are connected, and together with the carbon atoms they are connected to, they form C. 3-7 Cycloalkylene; R and R' are independently selected from H, halogens, and C. 1-6 Alkyl, C 2-6 alkenyl, C 2-6 alkynyl or C 1-6 Haloalkyl groups, or R and R', are attached to the nitrogen atom to which they are attached, together forming 3-7 membered heterocyclic groups. The compound of claim 15, or a pharmaceutically acceptable salt, isotopic variant, tautomer, or stereoisomer thereof, or a mixture thereof, wherein, The compound has the following structure: in, Ring A is selected from C 3-10 Cycloalkylene, 3-10 membered heterocyclic alkylene, C 6-10 The ring is arylene or 5-10 heteroarylene, wherein the ring A is optionally surrounded by 1, 2, 3, 4 or 5 R groups. a replace; L1 is selected from C 1-6 Alkylene, C 1-6 Heteroalkyl, C 2-6 imidene group, C 2-6 Ethyne group, C 3-10 The L1 is optionally surrounded by 1, 2, 3, 4, or 5 R groups, or a cycloalkyl or 3-10-membered heterocyclic group. b replace; L2 is selected from C 1-6 Alkylene, NH-C 0-6 Alkylene, C(O)-C 0-6 Alkylene, NHC(O)-C 0-6 Alkylene, C 2-6 imidene group, C 2-6 Ethyne group, C 3-10 The L2 is optionally surrounded by 1, 2, 3, 4, or 5 R groups. c replace; P1 is selected from H or a reactive phosphorus group, preferably -P(OCH2CH2CN)(N(iPr)2); P2 is selected from H or a protecting group, preferably a hydroxyl protecting group; Base' is selected from H, modified or unmodified bases; Each R a Independently selected from H, halogens, OH, CN, NH2, C 1-6 Alkyl, C 1-6 Haloalkyl, C 1-6 Alkoxy or C 1-6 Halogenated alkoxy groups; Each R b and R b 'Independently selected from H, halogens, OH, CN, NH2, C 1-6 Alkyl, C 1-6 Haloalkyl, C 1-6 Alkoxy, C 1-6 Haloalkoxy groups, or two R atoms on the same or different carbon atoms. b (or R) b and R b ') are connected together, and together with the carbon atoms they are connected to, they form C. 3-7 Cycloalkylene (preferably C16) 3-5 (cycloalkylene); Each R c and R c 'Independently selected from H, halogens, OH, CN, NH2, C 1-6 Alkyl, C 1-6 Haloalkyl, C 1-6 Alkoxy, C 1-6 Haloalkoxy groups, or two R atoms on the same or different carbon atoms. c (or R) c and R c ') are connected together, and together with the carbon atoms they are connected to, they form C. 3-7 Cycloalkylene (preferably C16) 3-5 (cycloalkylene); m is selected from 0, 1, 2 or 3. The compound of claim 15 or 16, or a pharmaceutically acceptable salt, isotopic variant, tautomer, or stereoisomer thereof, or a mixture thereof, wherein, Ring A is C 3-10 Cycloalkylene, 3-10 membered heterocyclic alkylene, C 6-10 arylene or 5-10 heteroarylene, wherein the ring A is optionally surrounded by one, two or three R atoms. a replace; L1 is selected from C 1-6 Alkylene, C 2-6 imide or C 2-6 The ynylene group, wherein the L1 is optionally surrounded by one, two, or three R groups. b replace; L2 is selected from C 1-6 Alkylene, C 2-6 imidene group, C 2-6 Ethyne group or NHC(O)-C 1-6 Alkylene, wherein the L2 is optionally surrounded by one, two or three R atoms. c replace; P1 is selected from H or a reactive phosphorus group, preferably -P(OCH2CH2CN)(N(iPr)2); P2 is selected from H or a protecting group, preferably a hydroxyl protecting group; Base' is selected from H, modified or unmodified bases; Each R a Independently selected from H, halogens, OH, CN, NH2, C 1-6 Alkyl, C 1-6 Haloalkyl, C 1-6 Alkoxy, C 1-6 Halogenated alkoxy groups; Each R b and R b 'Independently selected from H, halogens, OH, CN, NH2, C 1-6 Alkyl, C 1-6 Haloalkyl, C 1-6 Alkoxy, C 1-6 Haloalkoxy groups, or two R atoms on the same or different carbon atoms. b (or R) b and R b ') are connected together, and together with the carbon atoms they are connected to, they form C. 3-7 Cycloalkylene; Each R c and R c 'Independently selected from H, halogens, OH, CN, NH2, C 1-6 Alkyl, C 1-6 Haloalkyl, C 1-6 Alkoxy, C 1-6 Haloalkoxy groups, or two R atoms on the same or different carbon atoms. c (or R) c and R c ') are connected together, and together with the carbon atoms they are connected to, they form C. 3-7 Cycloalkylene; Preferably, Ring A is selected from C 3-7 Cycloalkylene or 3-7 membered heterocyclic group, wherein ring A is optionally surrounded by 1, 2 or 3 R groups. a replace; L1 is C 1-6 Alkylene, wherein the L1 is optionally surrounded by one, two or three R atoms. b replace; L2 is C 1-6 Alkylene, wherein the L2 is optionally surrounded by one, two or three R atoms. c replace; P1 is selected from H or a reactive phosphorus group, preferably -P(OCH2CH2CN)(N(iPr)2); P2 is selected from H or a protecting group, preferably a hydroxyl protecting group, and more preferably DMTr; Base' is selected from H, modified or unmodified bases; Each R a Independently selected from H, halogens, C 1-4 Alkyl or C 1-4 Halogenated alkyl groups; Each R b and R b 'Independently selected from H, halogen, C 1-4 Alkyl or C 1-4 Halogenated alkyl groups; Each R c and R c 'Independently selected from H, halogen, C 1-4 Alkyl or C 1-4 Halogenated alkyl groups; R d Selected from H, halogens, C 1-4 Alkyl or C 1-4 Halogenated alkyl groups; More preferably, Ring A is C 3-5 Cycloalkylene, preferably cyclopropylene; L1 is C 1-4 Alkylene, preferably -CH2-; L2 is C 1-4 Alkylene, preferably -CH2-; P1 is selected from H or a reactive phosphorus group, preferably -P(OCH2CH2CN)(N(iPr)2); P2 is selected from H or a protecting group, preferably a hydroxyl protecting group, and more preferably DMTr; Base is selected from The compound of any one of claims 15-17, or a pharmaceutically acceptable salt, isotopic variant, tautomer, or stereoisomer thereof, or a mixture thereof, wherein, The compound has a structure of formula (III'), (III'-1), or (III'-2): in, Ring A is selected from C 3-7 Cycloalkylene or 3-7 membered heterocyclic group, wherein ring A is optionally surrounded by 1, 2 or 3 R groups. a replace; P1 is selected from H or a reactive phosphorus group, preferably -P(OCH2CH2CN)(N(iPr)2); P2 is selected from H or a protecting group, preferably a hydroxyl protecting group, and more preferably DMTr; Base' is selected from H, unmodified base, or C. 1-4 Alkyl or protecting group modified bases; Each R a Independently selected from H, halogens, C 1-4 Alkyl or C 1-4 Halogenated alkyl groups; R b and R b 'Independently selected from H, halogen, C 1-4 Alkyl or C 1-4 Halogenated alkyl groups; R c and R c 'Independently selected from H, halogen, C 1-4 Alkyl or C 1-4 Halogenated alkyl groups; Preferably, Ring A is C 3-5 Cycloalkylene, preferably cyclopropylene, wherein ring A is optionally surrounded by one R a replace; R a Selected from H, halogen or C 1-4 alkyl; R b and R b 'Independently selected from H, halogen or C 1-4 alkyl; R c and R c 'Independently selected from H, halogen or C 1-4 alkyl; P1 is selected from H or a reactive phosphorus group, preferably -P(OCH2CH2CN)(N(iPr)2); P2 is selected from H or a protecting group, preferably a hydroxyl protecting group, and more preferably DMTr; Base' is selected from H, unmodified base, or C. 1-4 Alkyl or protecting bases, for example The compound of any one of claims 15-18, or a pharmaceutically acceptable salt, isotopic variant, tautomer, or stereoisomer thereof, or a mixture thereof, wherein, The compound is selected from the following structures: in, P1 is selected from H or a reactive phosphorus group, preferably -P(OCH2CH2CN)(N(iPr)2); P2 is selected from H or a hydroxyl protecting group, preferably DMTr; Base is selected from Preferably, the compound is selected from the following structures: An RNAi drug, wherein, The RNAi agent comprises one, two, three, four, five or more modifying groups according to any one of claims 1-7; Preferably, the nucleotide chain of the RNAi agent includes one, two, three, four, five, six, seven, or eight modifying groups as defined in any one of claims 1-7, or pharmaceutically acceptable salts, isotopic variants, tautomers, or stereoisomers thereof. The RNAi agent of claim 20, wherein, The RNAi agent is double-stranded, comprising a sense strand and an antisense strand, each strand having 14 to 30 nucleotides; Preferably, the RNAi agent is selected from short interfering RNA (siRNA), double-stranded RNA (dsRNA), microRNA (miRNA) or short hairpin RNA (shRNA), and is preferably siRNA. The RNAi agent according to claim 20 or 21, wherein, The RNAi agent includes, within the sense strand and / or antisense strand, one, two, three, four, five, six, seven, or eight modifying groups as defined in any one of claims 1-7, or their pharmaceutically acceptable salts, isotopic variants, tautomers, or stereoisomers. Preferably, the RNAi agent includes one, two, three, four, five, six, seven, or eight modifying groups as defined in any one of claims 1-7, or their pharmaceutically acceptable salts, isotopic variants, tautomers, or stereoisomers within the antisense strand; More preferably, the RNAi agent comprises 1, 2, 3, 4, 5, 6, 7 or 8 modifying groups as defined in any one of claims 1-7 or their pharmaceutically acceptable salts, isotopic variants, tautomers or stereoisomers at positions 2-20 (preferably positions 2-10, preferably positions 3-8) starting from the 5' end of the antisense strand. More preferably, the RNAi agent includes a modifying group as defined in any one of claims 1-7 or a pharmaceutically acceptable salt, isotope variant, tautomer or stereoisomer thereof at the 4th, 5th, 6th or 7th (preferably the 5th) nucleotide position starting from the 5' end of the antisense strand. The RNAi agent according to any one of claims 20-22, wherein, The RNAi agent comprises one or more targeting ligands or delivery vectors; preferably, the delivery vector is selected from peptide delivery vectors or antibody delivery vectors; preferably, the targeting ligand is an N-acetylgalactosamine (GalNAc) targeting ligand, such as compound 6, compound 7 and L96. The RNAi agent according to claims 20-23, wherein, The RNAi agent comprises a modified nucleotide monomer, the modification being selected from one or more of 2'-O-methyl modification, 2'-F modification, and 5'-spirocyclic phosphonate modification. The RNAi agent according to any one of claims 20-24, wherein, The RNAi agent contains thiophosphate bonds. The RNAi agent according to any one of claims 20-25, wherein, The antisense strand of the RNAi agent is selected from or contains the following sequences: 1) Having a sequence or a fragment thereof as shown in SEQ ID NO.4, or a modified sequence thereof; or 2) Having a sequence or a fragment thereof as shown in SEQ ID NO.5, or a modified sequence thereof; Preferably, the sense and antisense strands of the RNAi agent are selected from or contain the following sequences: (1) The positive chain has a sequence as shown in SEQ ID NO.1 or a fragment thereof, or a modified sequence of said sequence or a fragment thereof; and the negative chain has a sequence as shown in SEQ ID NO.4 or a fragment thereof, or a modified sequence of said sequence or a fragment thereof; or (2) The sense chain has a sequence or a fragment thereof as shown in SEQ ID NO.1, or a modified sequence thereof; and the antisense chain has a sequence or a fragment thereof as shown in SEQ ID NO.5, or a modified sequence thereof; More preferably, the sense and antisense strands of the RNAi agent are selected from or contain the following sequences: (1) The sense chain has the sequence shown in SEQ ID NO.1; and the antisense chain has the sequence shown in SEQ ID NO.4; or (2) The sense chain has the sequence shown in SEQ ID NO.1; and the antisense chain has the sequence shown in SEQ ID NO.

5. A composition comprising one or more oligonucleotides according to any one of claims 1-14, or an RNAi agent according to any one of claims 20-26. A kit comprising one or more oligonucleotides according to any one of claims 1-14, or an RNAi agent according to any one of claims 20-26. A method for inhibiting the expression of a target nucleic acid, the method comprising administering to a subject a therapeutic amount of any oligonucleotide of claims 1-14, any RNAi agent of claims 20-26, or the composition of claim 27. A method for reducing off-target toxicity in cells, comprising the step of introducing an oligonucleotide of any one of claims 1-14 or an RNAi agent of any one of claims 20-26 into the cell. A method for reducing off-target toxicity in cells, comprising expressing in the cells an oligonucleotide of any one of claims 1-14 or an RNAi agent of any one of claims 20-26. Use of the compound of formula (I') according to any one of claims 15-19, or a pharmaceutically acceptable salt, isotopic variant, tautomer or stereoisomer, or mixture thereof, in reducing the off-target effects of oligonucleotides. Use of the compound of formula (I') according to any one of claims 15-19, or a pharmaceutically acceptable salt, isotopic variant, tautomer or stereoisomer, or mixture thereof, in the preparation of oligonucleotide drugs (e.g., RNAi drugs). A method of treating a disease, the method comprising administering to a subject requiring treatment of the disease a therapeutic amount of any one of claims 1-14, any one of claims 20-26, or the composition of claim 27. The method of claim 34, wherein, The diseases mentioned are selected from cancer, autoimmune diseases, inflammatory diseases, metabolic diseases, genetic diseases, or rare diseases.

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