Double-stranded RNA containing nucleotide analogs

Abstract

The present invention provides a double-stranded RNA having a nucleotide analogue. The double stranded RNAs of the present invention exhibit reduced off-target toxicity.

Description

Double-stranded RNA containing nucleotide analogs

[0001] The present invention claims priority to Chinese patent application CN202311011321.7 filed on August 10, 2023, which is incorporated herein by reference in its entirety as part of the disclosure of the present invention. Field of the Invention

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

[0003] RNA interference (RNAi) is a phenomenon in which double-stranded RNA (dsRNA) induces the efficient and specific degradation of target mRNA. Incorporating heat-labile nucleotides (e.g., glycerol nucleic acid (GNA)) into the antisense strand of dsRNA can help improve interference efficiency and reduce off-target toxicity (see, for example, PCT Publication No. WO2018098328A1).

[0004] There is a need in the art to modify siRNA molecules to improve their stability and effectiveness in vivo. Based on long-term experience, the vast majority of current siRNA therapeutics utilize a fully modified model, where all nucleotides in the siRNA duplex are modified. Commonly used modifications are 2'-F and 2'-OCH3. The development of more modified nucleotides is desirable in this area.

[0005] Summary of the Invention

[0006] The present invention solves the above problems by providing a novel nucleotide analog.

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

[0008] The variables are defined as follows.

[0009] In another aspect, the present invention provides a double-stranded RNA having a sense strand and an antisense strand, each strand having 14 to 30 nucleotides, the antisense strand comprising a sequence sufficiently complementary to the sense strand and the target mRNA, wherein the sense strand and / or the antisense strand comprises one or more compounds of formula (I) as described herein, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof:

[0010] in,

[0011] The variables are defined below.

[0012] In another aspect, the present invention provides a vector comprising a nucleotide sequence encoding a double-stranded RNA as described herein.

[0013] In another aspect, the present invention provides a cell containing the double-stranded RNA as described herein or the vector as described herein.

[0014] In another aspect, the present invention relates to a pharmaceutical composition comprising a double-stranded RNA molecule as described herein, and a pharmaceutically acceptable carrier or excipient.

[0015] In another aspect, the present invention relates to a kit comprising a double-stranded RNA molecule as described herein.

[0016] In another aspect, the present invention relates to a method for inhibiting the expression of a target gene in a cell, comprising the step of introducing a double-stranded RNA molecule as described herein into the cell.

[0017] In another aspect, the present invention relates to a method for inhibiting the expression of a target gene in a cell, comprising expressing a double-stranded RNA molecule as described herein in said cell.

[0018] In another aspect, the present invention relates to a method for reducing off-target toxicity in a cell, comprising the step of introducing the double-stranded RNA molecule described herein into the cell.

[0019] In another aspect, the present invention relates to a method for reducing off-target toxicity in a cell, comprising expressing a double-stranded RNA molecule described herein in said cell.

[0020] The nucleotide analogs of the present invention can be used to replace conventional 2'-F and 2'-OCH3 modifications. In addition, after being incorporated into the antisense strand of dsRNA, the resulting double-stranded RNA can also exhibit reduced off-target toxicity.

[0021] Detailed Description of the Invention

[0022] definition

[0023] Chemical definition

[0024] Definitions of specific functional groups and chemical terms are described in more detail below.

[0025] When a numerical range is listed, it is intended to include every value and sub-range within the stated range. For example, "C 1-6 "Alkyl" includes C1, C2, C3, C4, C5, C6, C 1-6 、C 1-5 、C 1-4 、C 1-3 、C 1-2 、C 2-6 、C2-5 、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.

[0026] “C 1-10 "Alkyl" refers to a straight or branched chain saturated hydrocarbon group having 1 to 10 carbon atoms. 1-6 "Alkyl" refers to a straight or branched chain saturated hydrocarbon group having 1 to 6 carbon atoms. In some embodiments, C 1-6 Alkyl, C 1-4 Alkyl and C 1-2 Alkyl groups are preferred. 1-6 Examples of alkyl groups include: methyl (C1), ethyl (C2), n-propyl (C3), isopropyl (C3), n-butyl (C4), tert-butyl (C4), sec-butyl (C4), isobutyl (C4), n-pentyl (C5), 3-pentyl (C5), pentyl (C5), neopentyl (C5), 3-methyl-2-butyl (C5), tert-pentyl (C5), and n-hexyl (C6). The term “C 1-6 "Alkyl" also includes heteroalkyl groups in which one or more (e.g., 1, 2, 3, or 4) carbon atoms are replaced by heteroatoms (e.g., oxygen, sulfur, nitrogen, boron, silicon, phosphorus). The alkyl group may be optionally substituted with one or more substituents, for example, 1 to 5 substituents, 1 to 3 substituents, or 1 substituent. Conventional alkyl abbreviations include: Me(-CH3), Et(-CH2CH3), iPr(-CH(CH3)2), nPr(-CH2CH2CH3), n-Bu(-CH2CH2CH2CH3), or i-Bu(-CH2CH(CH3)2).

[0027] “C 2-10 "Alkenyl" refers to a straight or branched chain hydrocarbon group having 2 to 10 carbon atoms and at least one carbon-carbon double bond. 2-6 "Alkenyl" refers to a straight or branched chain hydrocarbon group having 2 to 6 carbon atoms and at least one carbon-carbon double bond. In some embodiments, C 2-6 Alkenyl and C 2-4 Alkenyl is preferred. 2-6 Examples of alkenyl groups include ethenyl (C2), 1-propenyl (C3), 2-propenyl (C3), 1-butenyl (C4), 2-butenyl (C4), butadienyl (C4), pentenyl (C5), pentadienyl (C5), hexenyl (C6), and the like. The term "C 2-6"Alkenyl" also includes heteroalkenyl groups in which one or more (e.g., 1, 2, 3, or 4) carbon atoms are replaced by heteroatoms (e.g., oxygen, sulfur, nitrogen, boron, silicon, phosphorus). An alkenyl group may be optionally substituted with one or more substituents, for example, with 1 to 5 substituents, 1 to 3 substituents, or 1 substituent.

[0028] “C 2-10 "Alkynyl" refers to a straight or branched chain hydrocarbon group having 2 to 10 carbon atoms, at least one carbon-carbon triple bond, and optionally one or more carbon-carbon double bonds. 2-6 "Alkynyl" refers to a straight or branched chain hydrocarbon group having 2 to 6 carbon atoms, at least one carbon-carbon triple bond, and optionally one or more carbon-carbon double bonds. In some embodiments, C 2-6 Alkynyl and C 2-4 Alkynyl is preferred. 2-6 Examples of alkynyl groups include, but are not limited to, ethynyl (C2), 1-propynyl (C3), 2-propynyl (C3), 1-butynyl (C4), 2-butynyl (C4), pentynyl (C5), hexynyl (C6), and the like. The term "C 2-6 "Alkynyl" also includes heteroalkynyl groups in which one or more (e.g., 1, 2, 3, or 4) carbon atoms are replaced by heteroatoms (e.g., oxygen, sulfur, nitrogen, boron, silicon, phosphorus). Alkynyl groups can be optionally substituted with one or more substituents, for example, 1 to 5 substituents, 1 to 3 substituents, or 1 substituent.

[0029] "Halo" or "halogen" refers to fluorine (F), chlorine (Cl), bromine (Br) and iodine (I).

[0030] Therefore, “C 1-6 "Haloalkyl" refers to the above-mentioned "C 1-6 Alkyl", which is substituted by one or more halogen groups. In some embodiments, C 1-4 Halogenated alkyl is particularly preferred, more preferably C 1-2 Haloalkyl. Exemplary haloalkyl groups include, but are not limited to, -CF3, -CH2F, -CHF2, -CHFCH2F, -CH2CHF2, -CF2CF3, -CCl3, -CH2Cl, -CHCl2, 2,2,2-trifluoro-1,1-dimethyl-ethyl, and the like. The haloalkyl group can be substituted at any available point of attachment, for example, with 1 to 5 substituents, 1 to 3 substituents, or 1 substituent.

[0031] “C 1-6 "Alkoxy" refers to a -OR group, wherein R is as described above in "C 1-6 Alkyl" and "C 1-6 "Haloalkyl" is defined.

[0032] “C1-10 "Alkylene" refers to the group with the C 1-10 In some embodiments, C 2-8 Alkylene, C 3-7 Alkylene, C 1-6 Alkylene, C 4-6 Alkylene, C 1-4 Alkylene, C 2-4 Alkylene and C 1-3 Alkylene is preferred. Unsubstituted alkylene includes but is not limited to: methylene (-CH2-), ethylene (-CH2CH2-), propylene (-CH2CH2CH2-), butylene (-CH2CH2CH2CH2-), pentylene (-CH2CH2CH2CH2CH2-), hexylene (-CH2CH2CH2CH2CH2CH2-), etc. Exemplary substituted alkylene groups, for example, alkylene groups substituted with one or more alkyl (methyl) groups, include, but are not limited to, substituted methylene groups (-CH(CH3)-, -C(CH3)2-), substituted ethylene groups (-CH(CH3)CH2-, -CH2CH(CH3)-, -C(CH3)2CH2-, -CH2C(CH3)2-), substituted propylene groups (-CH(CH3)CH2CH2-, -CH2CH(CH3)CH2-, -CH2CH2CH(CH3)-, -C(CH3)2CH2CH2-, -CH2C(CH3)2CH2-), and the like.

[0033] “C 2-10 "Alkenylene" refers to the group with the C 2-10 In some embodiments, C 2-8 Alkenylene, C 3-7 Alkenylene, C 2-6 Alkenylene, C 4-6 Alkenylene, C 1-4 Alkenylene, C 2-4 Alkenylene is preferred.

[0034] “C 2-10 "Alkynylidene" refers to the group with the C 2-10 In some embodiments, C 2-8 Alkynylidene, C 3-7 Alkynylidene, C 2-6 Alkynylidene, C 4-6 Alkynylidene, C 1-4 Alkynylidene, C 2-4Alkynylene is preferred.

[0035] Alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, and the like are defined herein as optionally substituted groups.

[0036] 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 、-SO2ORaa 、-OSO2R aa 、-S(=O)R aa 、-OS(=O)R aa 、-Si(R aa )3、-OSi(R aa )3、-C(=S)N(R bb )2, -C(=O)SR aa 、-C(=S)SR aa 、-SC(=S)SR aa 、-SC(=O)SR aa 、-OC(=O)SR aa 、-SC(=O)OR aa 、-SC(=O)R aa 、-P(=O)2R aa 、-OP(=O)2R aa 、-P(=O)(R aa )2、-OP(=O)(R aa )2、-OP(=O)(OR cc )2、-P(=O)2N(R bb )2、-OP(=O)2N(R bb )2、-P(=O)(NR bb )2、-OP(=O)(NR bb )2、-NR bb P(=O)(OR cc )2、-NR bb P(=O)(NR bb )2、-P(R cc )2、-P(R cc )3、-OP(R cc )2、-OP(R cc )3、-B(R aa )2、-B(OR cc )2, -BR aa (OR cc ), alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl and heteroaryl, wherein each alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl and heteroaryl is independently replaced by 0, 1, 2, 3, 4 or 5 R dd group substitution;

[0037] Or the two geminal hydrogen atoms on the carbon atom are replaced by groups =O, =S, =NN(R bb )2, =NNR bb C(=O)R aa 、=NNR bb C(=O)OR aa 、=NNRbb S(=O)2R aa 、=NR bb or = NOR cc replace;

[0038] R aa Each of R is independently selected from alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl and heteroaryl, or two R aa The groups are combined to form a heterocyclyl or heteroaryl ring, wherein each alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl and heteroaryl group is independently replaced by 0, 1, 2, 3, 4 or 5 R dd group substitution;

[0039] R bb Each of the following is independently selected from: hydrogen, -OH, -OR aa 、-N(R cc )2, -CN, -C(=O)R aa 、-C(=O)N(R cc )2, -CO2R aa 、-SO2R aa 、-C(=NR cc )OR aa 、-C(=NR cc )N(R cc )2、-SO2N(R cc )2, -SO2R cc 、-SO2OR cc 、-SOR aa 、-C(=S)N(R cc )2, -C(=O)SR cc 、-C(=S)SR cc 、-P(=O)2R aa 、-P(=O)(R aa )2、-P(=O)2N(R cc )2、-P(=O)(NR cc )2, alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl and heteroaryl, or two R bb The groups are combined to form a heterocyclyl or heteroaryl ring, wherein each alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl and heteroaryl group is independently replaced by 0, 1, 2, 3, 4 or 5 R dd group substitution;

[0040] R cc Each of R is independently selected from hydrogen, alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl and heteroaryl, or two R ccThe groups are combined to form a heterocyclyl or heteroaryl ring, wherein each alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl and heteroaryl group is independently replaced by 0, 1, 2, 3, 4 or 5 R dd group substitution;

[0041] R dd Each of the 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(Ree )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 ee )2, alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, wherein each alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl and heteroaryl is independently substituted by 0, 1, 2, 3, 4 or 5 R gg Group substitution, or two geminal R dd Substituents may combine to form =O or =S;

[0042] R ee Each of R is independently selected from alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, aryl, heterocyclyl and heteroaryl, wherein each alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl and heteroaryl is independently replaced by 0, 1, 2, 3, 4 or 5 R gg group substitution;

[0043] R ff Each of R is independently selected from hydrogen, alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl and heteroaryl, or two R ff The groups are combined to form a heterocyclic or heteroaryl ring, wherein each alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclic, aryl and heteroaryl group is independently replaced by 0, 1, 2, 3, 4 or 5 R gg group substitution;

[0044] R gg Each of the 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)(C1-6 Alkyl), -N(OH)(C 1-6 Alkyl), -NH(OH), -SH, -SC 1-6 Alkyl, -SS(C 1-6 alkyl), -C(=O)(C 1-6 alkyl), -CO2H, -CO2(C 1-6 alkyl), -OC(=O)(C 1-6 Alkyl), -OCO2(C 1-6 alkyl), -C(=O)NH2, -C(=O)N(C 1-6 alkyl)2, -OC(=O)NH(C 1-6 alkyl), -NHC(=O)(C 1-6 Alkyl), -N(C 1-6 alkyl)C(=O)(C 1-6 Alkyl), -NHCO2(C 1-6 alkyl), -NHC(=O)N(C 1-6 Alkyl)2, -NHC(=O)NH(C 1-6 alkyl), -NHC(=O)NH2, -C(=NH)O(C 1-6 alkyl), -OC(=NH)(C 1-6 alkyl), -OC(=NH)OC 1-6 Alkyl, -C(=NH)N(C 1-6 Alkyl)2, -C(=NH)NH(C 1-6 alkyl), -C(=NH)NH2, -OC(=NH)N(C 1-6 Alkyl)2, -OC(NH)NH(C 1-6 alkyl), -OC(NH)NH2, -NHC(NH)N(C 1-6 Alkyl)2, -NHC(=NH)NH2, -NHSO2(C 1-6 Alkyl), -SO2N(C 1-6 Alkyl)2, -SO2NH(C 1-6 alkyl), -SO2NH2, -SO2C 1-6 Alkyl, -SO2OC 1-6 Alkyl, -OSO2C 1-6 Alkyl, -SOC 1-6 Alkyl, -Si(C 1-6 alkyl)3, -OSi(C 1-6 alkyl)3, -C(=S)N(C 1-6 alkyl)2、C(=S)NH(C 1-6 alkyl), C(=S)NH2, -C(=O)S(C 1-6 alkyl), -C(=S)SC1-6 Alkyl, -SC(=S)SC 1-6 Alkyl, -P(=O)2(C 1-6 alkyl), -P(=O)(C 1-6 alkyl)2, -OP(=O)(C 1-6 alkyl)2, -OP(=O)(OC 1-6 Alkyl)2, C 1-6 Alkyl, C 1-6 Halogenated alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C7 cycloalkyl, C6-C 10 Aryl, C3-C7 heterocyclic, C5-C 10 heteroaryl; or two geminal R gg Substituents may combine to form =O or =S; wherein X - For the counter ion.

[0045] Exemplary substituents on nitrogen atoms include, but are not limited to, hydrogen, -OH, -OR aa 、-N(R cc )2, -CN, -C(=O)R aa -C(=O)N(R cc )2, -CO2R aa 、-SO2R aa 、-C(=NR bb )R aa 、-C(=NR cc )OR aa 、-C(=NR cc )N(R cc )2、-SO2N(R cc )2, -SO2R cc 、-SO2OR cc 、-SOR aa 、-C(=S)N(R cc )2, -C(=O)SR cc 、-C(=S)SR cc 、-P(=O)2R aa 、-P(=O)(R aa )2、-P(=O)2N(R cc )2、-P(=O)(NR cc )2, alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl and heteroaryl, or two R attached to the nitrogen atom cc The groups are combined to form a heterocyclic or heteroaryl ring, wherein each alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclic, aryl and heteroaryl group is independently replaced by 0, 1, 2, 3, 4 or 5 R dd Group substituted, and wherein R aa 、Rbb 、R cc and R dd As mentioned above.

[0046] Other definitions

[0047] The term "siRNA" herein refers to a class of double-stranded RNA molecules that can mediate the silencing of a target RNA (e.g., mRNA, e.g., a transcript of a gene encoding a protein) that is complementary thereto. siRNA is typically double-stranded, comprising an antisense strand complementary to the target RNA and a sense strand complementary to the antisense strand. For convenience, such mRNA is also referred to herein as mRNA to be silenced. Such a gene is also referred to as a target gene. Typically, the RNA to be silenced is an endogenous gene or a pathogen gene. In addition, RNA (e.g., tRNA) and viral RNA other than mRNA can also be targeted.

[0048] The term "antisense strand" refers to a strand of an siRNA that includes a region that is completely, fully, or substantially complementary to a target sequence. The term "sense strand" refers to a strand of an siRNA that includes a region that is completely, fully, or substantially complementary to a region that is, as the term is defined herein, an antisense strand.

[0049] The term "complementary region" refers to a region on the antisense strand that is completely, fully or substantially complementary to the target mRNA sequence. In the case where the complementary region is not completely complementary to the target sequence, mispairing can be located in the interior or terminal regions of the molecule. Typically, the most tolerated mispairing is located in the terminal regions, for example, in 5' and / or 3' ends within 5, 4, 3, 2 or 1 nucleotide. The antisense strand portion that is most sensitive to mispairing is referred to as a "seed region." For example, in a siRNA comprising a 19nt chain, the 19th position (from 5' to 3') can tolerate some mispairing.

[0050] The term "complementary" refers to the ability of a first polynucleotide to hybridize to a second polynucleotide under certain conditions, such as stringent conditions. For example, stringent conditions may include 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA at 50° C. or 70° C. for 12-16 hours. In terms of meeting the above requirements relative to their ability to hybridize, "complementary" sequences may also include or be completely formed from non-Watson-Crick base pairs and / or base pairs formed from non-natural and modified nucleotides. Such non-Watson-Crick base pairs include, but are not limited to, G:U wobble base pairing or Hoogstein base pairing.

[0051] A polynucleotide that is "at least partially complementary," "substantially complementary," or "at least partially complementary" to a messenger RNA (mRNA) refers to a polynucleotide that is substantially complementary to a contiguous portion of the mRNA of interest. For example, a polynucleotide is complementary to at least a portion of a PCSK9 mRNA if the sequence is substantially complementary to a non-interrupted portion of the mRNA encoding PCSK9. The terms "complementary," "fully complementary," "substantially complementary," and "substantially complementary" as used herein can be used with respect to base pairing between the sense and antisense strands of an siRNA, or between the antisense strand of an siRNA agent and a target sequence.

[0052] "Fully complementary" refers to the extent to which the sense strand only needs to be complementary to the antisense strand in order to maintain the overall double-stranded nature of the molecule. In other words, while perfect complementarity is generally desired, in some cases, particularly in the antisense strand, one or more, for example, 6, 5, 4, 3, 2, or 1 mismatches (relative to the target mRNA) may be included, but the sense and antisense strands can still maintain the overall double-stranded nature of the molecule.

[0053] "shRNA" stands for short hairpin RNA. shRNA consists of two short inverted repeats. When cloned into an shRNA expression vector, the two short inverted repeats are separated by a stem-loop sequence, forming a hairpin structure controlled by a Pol III promoter. Five to six Ts are then attached to serve as a transcriptional terminator for RNA polymerase III.

[0054] A "nucleoside" is a compound composed of a purine or pyrimidine base and ribose or deoxyribose; a "nucleotide" is a compound composed of a purine or pyrimidine base, ribose or deoxyribose, and phosphate; an "oligonucleotide" is a nucleic acid molecule (RNA or DNA) having, for example, fewer than 100, 200, 300, or 400 nucleotides in length.

[0055] "Bases" are the fundamental building blocks of nucleosides, nucleotides, and nucleic acids. They contain nitrogen and are also called "nitrogenous bases." Unless otherwise specified, the capital letters A, U, T, G, and C represent the bases of nucleotides, representing adenine, uracil, thymine, guanine, and cytosine, respectively.

[0056] The "modification" of nucleotides herein includes, but is not limited to, methoxy modification, fluorination, phosphorothioate linkage, or conventional protecting group protection. For example, the fluorination-modified nucleotide refers to a nucleotide in which the hydroxyl group at the 2' position of the ribose group of the nucleotide is replaced by fluorine, and the methoxy-modified nucleotide refers to a nucleotide in which the 2'-hydroxyl group of the ribose group is replaced by a methoxy group.

[0057] " modified nucleotide " herein includes but is not limited to 2 '-O-methyl modified nucleotides, 2 '-fluoro modified nucleotides, 2 '-deoxy-modified nucleotides, inosine ribonucleotides, abasic nucleotides, reverse abasic deoxyribonucleotides, nucleotides comprising thiophosphate groups, vinyl phosphate modified nucleotides, locked nucleotides, 2 '-amino-modified nucleotides, 2 '-alkyl-modified nucleotides, morpholino nucleotides, phosphoramidates, the non-natural bases comprising nucleotides and the terminal nucleotides, deoxyribonucleotides or conventional protecting group protections on a cholesterol derivative or a dodecanoic acid didecylamide group. For example, the 2 '-fluoro modified nucleotides refer to nucleotides in which the hydroxyl group at the ribose group 2 ' position is replaced by fluorine. The 2 '-deoxy-modified nucleotides refer to nucleotides in which the 2 '-hydroxyl group at the ribose group is replaced by methoxy and formed.

[0058] " reactive phosphorus group " refers to the phosphorus-containing group contained in the nucleotide unit or in the nucleotide analog unit, and it can react with the hydroxyl or the amido reaction contained in another molecule, especially in another nucleotide unit or in another nucleotide analog by nucleophilic attack reaction.Usually, such reaction produces the ester type internucleoside bond that the first nucleotide unit or the first nucleotide analog unit are connected with the second nucleotide unit or the second nucleotide analog unit.Reactive phosphorus group can be selected from phosphoramidites, H-phosphonates, alkyl-phosphonates, phosphate or phosphate analogs, include but not limited to: natural phosphate, thiophosphate, phosphorodithioate, borane phosphate, borane thiophosphate, phosphonate, halogen-substituted phosphonate and phosphate, phosphoramidate, phosphodiester, phosphotriester, thiophosphate diester, thiophosphate triester, diphosphate and triphosphate, preferably-P(OCH2CH2CN)(N(iPr)2).

[0059] A "protecting group," also known as a "blocking group," refers to any atom or group of atoms added to a molecule to prevent existing groups in the molecule from undergoing undesirable chemical reactions. A "protecting group" may be an unstable chemical moiety known in the art that is used to protect reactive groups, such as hydroxyl, amino, and thiol groups, from undesirable or inappropriate 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 the unprotected group intact or available for further reactions.

[0060] A non-limiting list of protecting groups includes benzyl; substituted benzyl; alkylcarbonyl and alkoxycarbonyl (e.g., tert-butyloxycarbonyl (BOC), acetyl, or isobutyryl); arylalkylcarbonyl and arylalkoxycarbonyl (e.g., benzyloxycarbonyl); substituted methyl ethers (e.g., methoxymethyl ether); substituted ethyl ethers; substituted benzyl ethers; tetrahydropyranyl ethers; silyl (e.g., trimethylsilyl, triethylsilyl, triisopropylsilyl, tert-butyldimethylsilyl, tri-isopropylsilyloxymethyl, [2-(trimethylsilyl)ethoxy] methyl or tert-butyldiphenylsilyl); esters (e.g., benzoate); carbonates (e.g., methoxymethyl carbonate); sulfonates (e.g., tosylate or mesylate); acyclic ketals (e.g., dimethyl acetal); cyclic ketals (e.g., 1,3-dioxane, 1,3-dioxolane, and those described herein); acyclic acetals; cyclic acetals (e.g., those described herein); acyclic hemiacetals; cyclic hemiacetals; cyclic dithioketals (e.g., 1,3-dithiane or 1,3-dithiolane); orthoesters (e.g., those described herein), and triarylmethyl groups (e.g., trityl; monomethoxytrityl (MMTr); 4,4′-dimethoxytrityl (DMTr); 4,4′,4″-trimethoxytrityl (TMTr); and those described herein). Preferred protecting groups are selected from acetyl (Ac), benzoyl (Bzl), benzyl (Bn), isobutyryl (iBu), phenylacetyl, benzyloxymethyl acetal (BOM), β-methoxyethoxymethyl ether (MEM), methoxymethyl ether (MOM), p-methoxybenzyl ether (PMB), methylthiomethyl ether, neopentyl (Vl), methylthiomethyl ether, ... Valeryl (Piv), tetrahydropyranyl (THP), triphenylmethyl (Trt), methoxytrityl [(4-methoxyphenyl)diphenylmethyl] (MMT), dimethoxytrityl, [bis-(4-methoxyphenyl)phenylmethyl] (DMT), trimethylsilyl ether (TMS), tert-butyldimethylsilyl ether (TBDMS), tri-iso-propylsilyloxymethyl ether (TOM), tri-isopropylsilyl ether (TIPS), methyl ether, ethoxyethyl ether (EE) N,N-dimethylformamidine and 2-cyanoethyl (CE).

[0061] A "hydroxyl protecting group" is a group that protects the hydroxyl group from chemical reactions and can be removed under specific conditions to restore the hydroxyl group. These groups primarily include silane-type protecting groups, acyl-type protecting groups, or ether-type protecting groups, with the following being preferred:

[0062] Trimethylsilyl (TMS), triethylsilyl (TES), dimethylisopropylsilyl (DMIPS), diethylisopropylsilyl (DEIPS), tert-butyldimethylsilyl (TBDMS), tert-butyldiphenylsilyl (TBDPS), triisopropylsilyl (TIPS), acetyl (Ac), chloroacetyl, dichloroacetyl, trichloroacetyl, trifluoroacetyl (TFA), benzoyl, p-methoxybenzoyl, 9-fluorenylmethoxycarbonyl (Fmoc), allyloxycarbonyl (Alloc), 2,2,2-trichloroethoxycarbonyl (Troc), benzyloxycarbonyl (Cbz), tert-butyloxycarbonyl (Boc) , benzyl (Bn), p-methoxybenzyl (PMB), allyl, triphenylmethyl (Tr), bis-p-methoxytrityl (DMTr), methoxymethyl (MOM), phenoxymethyl (BOM), 2,2,2-trichloroethoxymethyl, 2-methoxyethoxymethyl (MEM), methylthiomethyl (MTM), p-methoxybenzyloxymethyl (PMBM), -C(O)CH2CH2C(O)OH or 4,4'-dimethoxytrityl, preferably -C(O)CH2CH2C(O)OH or 4,4'-dimethoxytrityl, more preferably -C(O)CH2CH2C(O)OH.

[0063] As used herein, the term "pharmaceutically acceptable salt" refers to those carboxylate salts, amino acid addition salts of the compounds of the present invention that are suitable for use in contact with patient tissues within the scope of sound medical judgment, do not produce undue toxicity, irritation, allergic response, etc., are commensurate with a reasonable benefit / risk ratio, and are effective for their intended use, including (where possible) zwitterionic forms of the compounds of the present invention.

[0064] The present invention includes tautomers, which are functional isomers produced by the rapid movement of an atom in two positions in a molecule. Compounds that exist in different tautomeric forms are not limited to any specific tautomer, but are intended to cover all tautomeric forms.

[0065] The compounds of the present invention may include one or more asymmetric centers and may therefore exist in a variety of stereoisomeric forms, for example, enantiomers and / or diastereomeric forms. For example, the compounds of the present invention may be individual enantiomers, diastereomers, or geometric isomers (e.g., cis and trans isomers), or may be in the form of mixtures of stereoisomers, including racemic mixtures and mixtures enriched in one or more stereoisomers. Isomers may be separated from the mixture by methods known to those skilled in the art, including chiral high pressure liquid chromatography (HPLC) and the formation and crystallization of chiral salts; or preferred isomers may be prepared by asymmetric synthesis.

[0066] The present invention also includes isotopically labeled compounds (isotopic variants) which are identical to those described in formula (I) but for which one or more atoms are replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number usually found in nature. Examples of isotopes that can be introduced into the compounds of the present invention include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine and chlorine, for example 2 H. 3 H. 13 C. 11 C. 14 C. 15 N. 18 O. 17 O. 31 P. 32 P. 35 S. 18 F and 36 Cl. Compounds of the present invention containing the above-mentioned isotopes and / or other isotopes of other atoms, their prodrugs and pharmaceutically acceptable salts of the compounds or prodrugs are within the scope of the present invention. Certain isotopically labeled compounds of the present invention, such as those in which radioactive isotopes (e.g. 3 H and 14 C) can be used in drug and / or substrate tissue distribution assays. 3 H and carbon-14, i.e. 14 C isotopes are particularly preferred because they are easy to prepare and detect. 2 H, because greater metabolic stability can provide therapeutic benefits, such as prolonged in vivo half-life or reduced dosage requirements, and thus may be preferred in some cases. Isotopically labeled compounds of formula (I) of the present invention and their prodrugs can generally be prepared by substituting readily available isotopically labeled reagents for non-isotopically labeled reagents when carrying out the processes disclosed in the following schemes and / or the Examples and Preparations.

[0067] Compounds of the present invention

[0068] The present invention particularly relates to oligonucleotides comprising one or more compounds of formula (I), or pharmaceutically acceptable salts, tautomers or stereoisomers thereof:

[0069] in,

[0070] *1 and *2 represent chiral centers, independently selected from (S) configuration or (R) configuration;

[0071] represents H, or indicates the position of phosphate or phosphorothioate linkage to the adjacent nucleotide;

[0072] R1 represents H, or represents the position of linkage to the phosphate or phosphorothioate of another adjacent nucleotide;

[0073] and R1 are not H at the same time;

[0074] L1 and L2 are independently selected from C 1-10 Alkylene, C 2-10 Alkenylene or C 2-10 Alkyne, wherein non-adjacent 1, 2, 3, 4 or 5 carbon atoms in the group may be replaced by heteroatoms selected from O, S and N, preferably C 1-10 alkylene;

[0075] The L1 and L2 are optionally substituted with 1, 2, 3, 4 or 5 R;

[0076] Rs is selected from H, D, halogen, C 1-6 Alkyl or C 1-6 alkyl halide;

[0077] R is selected from H, D, halogen, C 1-6 Alkyl or C 1-6 alkyl halide;

[0078] k is 1, 2, 3, 4, or 5;

[0079] Base is selected from H, modified or unmodified bases or leaving groups, preferably modified or unmodified A, U, T, G and C; preferably, Base is selected from

[0080] The present invention also relates to a compound of formula (I'), or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof:

[0081] wherein R1 and R2 are independently selected from H, a reactive phosphorus group, a hydroxyl protecting group or a solid support;

[0082] Base' is selected from H, modified or unmodified bases or leaving groups, preferably modified or unmodified A, U, T, G and C; preferably, Base' is selected from

[0083] *1, *2, L1, L2, Rs and k are as defined herein.

[0084] The present invention also relates to a double-stranded RNA having a sense strand and an antisense strand, each strand having 14 to 30 nucleotides, wherein the antisense strand comprises a sequence that is sufficiently complementary to the sense strand and the target mRNA, wherein the sense strand and / or the antisense strand comprises one or more compounds of formula (I) as described herein, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof:

[0085] Wherein, each variable is as defined herein.

[0086] *1 and *2

[0087] *1 and *2 represent chiral centers, independently selected from (S) configuration or (R) configuration.

[0088] In one embodiment, *1 is the (S) configuration; in another embodiment, *1 is the (R) configuration; in another embodiment, *1 is a mixture of the (S) configuration and the (R) configuration.

[0089] In one embodiment, *2 is the (S) configuration; in another embodiment, *2 is the (R) configuration; in another embodiment, *2 is a mixture of the (S) configuration and the (R) configuration.

[0090] R1 and R2

[0091] In one embodiment, represents H; in another embodiment, Indicates the position of the phosphate or phosphorothioate linkage to the adjacent nucleotide.

[0092] In one embodiment, R1 represents H; in another embodiment, R1 represents the position of attachment to the phosphate or phosphorothioate of another adjacent nucleotide.

[0093] In one embodiment, and R1 are not H at the same time.

[0094] In one embodiment, R1 represents a reactive phosphorus group, for example a phosphoramidite, an H-phosphonate, an alkyl-phosphonate, a phosphate or a phosphate mimetic, for example a natural phosphate, a phosphorothioate, a phosphorodithioate, a boranephosphate, a boranephosphorothioate, a phosphonate, a halogen-substituted phosphonate and a phosphate, a phosphoramidate, a phosphodiester, a phosphotriester, a phosphorothioate diester, a phosphorothioate triester, a diphosphate or a triphosphate, preferably -P(OCH2CH2CN)(N(iPr)2);

[0095] In another embodiment, R1 represents a hydroxy protecting group, such as trimethylsilyl (TMS), triethylsilyl (TES), dimethylisopropylsilyl (DMIPS), diethylisopropylsilyl (DEIPS), tert-butyldimethylsilyl (TBDMS), tert-butyldiphenylsilyl (TBDPS), triisopropylsilyl (TIPS), acetyl (Ac), chloroacetyl, dichloroacetyl, trichloroacetyl, trifluoroacetyl (TFA), benzoyl, p-methoxybenzoyl, 9-fluorenylmethoxycarbonyl (Fmoc), allyloxycarbonyl (Alloc), 2,2,2-trichloroethoxycarbonyl (Tr oc), benzyloxycarbonyl (Cbz), tert-butyloxycarbonyl (Boc), benzyl (Bn), p-methoxybenzyl (PMB), allyl, triphenylmethyl (Tr), bis-p-methoxytrityl (DMTr), methoxymethyl (MOM), phenoxymethyl (BOM), 2,2,2-trichloroethoxymethyl, 2-methoxyethoxymethyl (MEM), methylthiomethyl (MTM), p-methoxybenzyloxymethyl (PMBM), 4,4'-dimethoxytrityl, -P(OCH2CH2CN)(N(iPr)2) or -C(O)CH2CH2C(O)OH, preferably DMTr;

[0096] In another embodiment, R1 represents a solid support.

[0097] In one embodiment, R2 represents H;

[0098] In another embodiment, R2 represents a reactive phosphorus group, such as a phosphoramidite, an H-phosphonate, an alkyl-phosphonate, a phosphate or a phosphate mimetic, such as a natural phosphate, a phosphorothioate, a phosphorodithioate, a boranophosphate, a boranophosphothioate, a phosphonate, a halogen-substituted phosphonate and a phosphate, a phosphoramidate, a phosphodiester, a phosphotriester, a phosphorothioate diester, a phosphorothioate triester, a diphosphate or a triphosphate, preferably -P(OCH2CH2CN)(N(iPr)2);

[0099] In another embodiment, R2 represents a hydroxy protecting group, such as trimethylsilyl (TMS), triethylsilyl (TES), dimethylisopropylsilyl (DMIPS), diethylisopropylsilyl (DEIPS), tert-butyldimethylsilyl (TBDMS), tert-butyldiphenylsilyl (TBDPS), triisopropylsilyl (TIPS), acetyl (Ac), chloroacetyl, dichloroacetyl, trichloroacetyl, trifluoroacetyl (TFA), benzoyl, p-methoxybenzoyl, 9-fluorenylmethoxycarbonyl (Fmoc), allyloxycarbonyl (Alloc), 2,2,2-trichloroethoxycarbonyl (Tr oc), benzyloxycarbonyl (Cbz), tert-butyloxycarbonyl (Boc), benzyl (Bn), p-methoxybenzyl (PMB), allyl, triphenylmethyl (Tr), bis-p-methoxytrityl (DMTr), methoxymethyl (MOM), phenoxymethyl (BOM), 2,2,2-trichloroethoxymethyl, 2-methoxyethoxymethyl (MEM), methylthiomethyl (MTM), p-methoxybenzyloxymethyl (PMBM), 4,4'-dimethoxytrityl, -P(OCH2CH2CN)(N(iPr)2) or -C(O)CH2CH2C(O)OH, preferably DMTr;

[0100] In another embodiment, R1 represents a solid support.

[0101] L1 and L2

[0102] In one embodiment, L1 is C 1-10 Alkylene; in another embodiment, L1 is C 1-6 Alkylene; in another embodiment, L1 is C 1-4 Alkylene, preferably -CH2- or -CH2CH2-; in another embodiment, L1 is C 2-10 Alkenylene; in another embodiment, L1 is C 2-10 Alkyne; In another embodiment, the aforementioned C 1-10 Alkylene, C 1-6 Alkylene, C 1-4 Alkylene, -CH2-, -CH2CH2-, C 2-10 Alkenylene or C 2-10 Non-adjacent 1, 2, 3, 4 or 5 carbon atoms in the alkynylene group may be replaced by heteroatoms selected from O, S and N.

[0103] In one embodiment, L1 is unsubstituted; in another embodiment, L1 is substituted with 1 R; in another embodiment, L1 is substituted with 2 Rs; in another embodiment, L1 is substituted with 3 Rs; in another embodiment, L1 is substituted with 4 Rs; in another embodiment, L1 is substituted with 5 Rs.

[0104] In one embodiment, L2 is C 1-10 Alkylene; in another embodiment, L2 is C 1-6 Alkylene; in another embodiment, L2 is C 1-4 Alkylene, preferably -CH2- or -CH2CH2-; in another embodiment, L2 is C 2-10 Alkenylene; in another embodiment, L2 is C 2-10 Alkyne; In another embodiment, the aforementioned C 1-10 Alkylene, C 1-6 Alkylene, C 1-4 Alkylene, -CH2-, -CH2CH2-, C 2-10 Alkenylene or C 2-10 Non-adjacent 1, 2, 3, 4 or 5 carbon atoms in the alkynylene group may be replaced by heteroatoms selected from O, S and N.

[0105] In one embodiment, L2 is unsubstituted; in another embodiment, L2 is substituted with 1 R; in another embodiment, L2 is substituted with 2 Rs; in another embodiment, L2 is substituted with 3 Rs; in another embodiment, L2 is substituted with 4 Rs; in another embodiment, L2 is substituted with 5 Rs.

[0106] R

[0107] In one embodiment, R is H; in another embodiment, R is D; in another embodiment, R is halogen; in another embodiment, R is C 1-6 Alkyl; in another embodiment, R is C 1-4 Alkyl, such as methyl; in another embodiment, R is C 1-6 In another embodiment, R is C 1-4 Halogenated alkyl.

[0108] Rs

[0109] In one embodiment, Rs is H; in another embodiment, Rs is D; in another embodiment, Rs is halogen; in another embodiment, Rs is C 1-6 Alkyl; in another embodiment, Rs is C 1-4 Alkyl, such as methyl; in another embodiment, Rs is C 1-6 In another embodiment, Rs is C 1-4 Halogenated alkyl.

[0110] k

[0111] In one embodiment, k is 1, 2, 3, 4 or 5.

[0112] Base and Base'

[0113] In one embodiment, Base is H; in another embodiment, Base is a modified or unmodified base or a leaving group, such as, preferably, modified or unmodified A, U, T, G, and C.

[0114] In one embodiment, Base' is H; in another embodiment, Base' is a modified or unmodified base or a leaving group, such as, preferably, modified or unmodified A, U, T, G, and C.

[0115] In a more specific embodiment, Base is In another more specific embodiment, Base is In another more specific embodiment, Base is In another more specific embodiment, Base is In another more specific embodiment, Base is

[0116] In a more specific embodiment, Base' is In another more specific embodiment, Base' is In another more specific embodiment, Base' is In another more specific embodiment, Base' is In another more specific embodiment, Base' is

[0117] 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 R1 can be combined with R2, The present invention is intended to include any combination of the technical solutions of *1, *2, L1, L2, R, Rs, k, Base and Base', etc., which are not listed one by one due to space limitations.

[0118] The present invention also provides a vector comprising a nucleotide sequence encoding the siRNA of the present invention. The vector of the present invention is capable of amplifying or expressing the nucleotide sequence encoding the siRNA of the present invention linked thereto.

[0119] For example, siRNA targeting the PCSK9 gene can be expressed from a transcription unit inserted into a DNA or RNA vector. Expression can be short-lived (a few hours to a few weeks) or continuous (a few weeks to a few months or longer), depending on the specific construct and target tissue or cell type used. The coding nucleotides of the siRNA can be introduced into a linear construct, a circular plasmid or a viral vector. The nucleotides of the siRNA can be integrated into the cell genome for stable expression, or expressed in an extrachromosomal stable inheritance. In general, siRNA expression vectors are typically DNA plasmids or viral vectors.

[0120] Viral vector systems containing siRNA coding sequences include, but are not limited to: (a) adenoviral vectors; (b) retroviral vectors; (c) adeno-associated viral vectors; (d) herpes simplex virus vectors; (e) SV40 vectors; (f) polyoma virus vectors; (g) papilloma virus vectors; (h) picornavirus vectors; (i) poxvirus vectors; and (j) helper virus-dependent adenovirus or gut-free adenovirus.

[0121] The present invention also provides a cell containing the siRNA or vector of the present invention, wherein the siRNA or vector of the present invention can be transcribed in the cell.

[0122] The present invention specifically relates to the following technical solutions:

[0123] In some embodiments, the present invention relates to oligonucleotides comprising one or more compounds of formula (I), or pharmaceutically acceptable salts, tautomers or stereoisomers thereof:

[0124] in,

[0125] *1 and *2 represent chiral centers, independently selected from (S) configuration or (R) configuration;

[0126] represents H, or indicates the position of phosphate or phosphorothioate linkage to the adjacent nucleotide;

[0127] R1 represents H, or represents the position of linkage to the phosphate or phosphorothioate of another adjacent nucleotide;

[0128] and R1 are not H at the same time;

[0129] L1 and L2 are independently selected from C 1-10 Alkylene, C 2-10 Alkenylene or C 2-10 Alkyne, wherein non-adjacent 1, 2, 3, 4 or 5 carbon atoms in the group may be replaced by heteroatoms selected from O, S and N, preferably C 1-10alkylene;

[0130] The L1 and L2 are optionally substituted with 1, 2, 3, 4 or 5 R;

[0131] Rs is selected from H, D, halogen, C 1-6 Alkyl or C 1-6 alkyl halide;

[0132] R is selected from H, D, halogen, C 1-6 Alkyl or C 1-6 alkyl halide;

[0133] k is 1, 2, 3, 4, or 5;

[0134] Base is selected from H, modified or unmodified bases or leaving groups, preferably modified or unmodified A, U, T, G and C; preferably, Base is selected from

[0135] In some embodiments, the present invention relates to the above oligonucleotide, wherein L1 and L2 are independently selected from C 1-6 Alkylene, C 2-6 Alkenylene or C 2-6 Alkyne, preferably C 1-6 alkylene;

[0136] The L1 and L2 are optionally substituted with 1, 2, 3, 4 or 5 R;

[0137] R is selected from H, D, halogen, C 1-6 Alkyl or C 1-6 alkyl halide;

[0138] Preferably, L1 and L2 are independently C 1-4 Alkylene, preferably -CH2- or -CH2CH2-;

[0139] The L1 and L2 are optionally substituted with 1, 2 or 3 R;

[0140] R is selected from H, D, halogen or C 1-4 alkyl.

[0141] In some embodiments, the present invention relates to the aforementioned oligonucleotide, wherein:

[0142] *1 and *2 represent chiral centers, independently selected from (S) configuration or (R) configuration;

[0143] represents H, or indicates the position of phosphate or phosphorothioate linkage to the adjacent nucleotide;

[0144] R1 represents H, or represents the position of linkage to the phosphate or phosphorothioate of another adjacent nucleotide;

[0145] and R1 are not H at the same time;

[0146] L1 and L2 are independently selected from C 1-6 Alkylene, C 2-6 Alkenylene or C 2-6 Alkyne, preferably C 1-6 alkylene;

[0147] The L1 and L2 are optionally substituted with 1, 2, 3, 4 or 5 R;

[0148] Rs is selected from H, D, halogen, C 1-6 Alkyl or C 1-6 alkyl halide;

[0149] R is selected from H, D, halogen, C 1-6 Alkyl or C 1-6 alkyl halide;

[0150] k is 1, 2, or 3;

[0151] Base is selected from

[0152] In some embodiments, the present invention relates to the aforementioned oligonucleotide, wherein:

[0153] *1 and *2 represent chiral centers, independently selected from (S) configuration or (R) configuration;

[0154] represents H, or indicates the position of phosphate or phosphorothioate linkage to the adjacent nucleotide;

[0155] R1 represents H, or represents the position of linkage to the phosphate or phosphorothioate of another adjacent nucleotide;

[0156] and R1 are not H at the same time;

[0157] L1 and L2 are independently C 1-4 Alkylene, preferably -CH2-;

[0158] The L1 and L2 are optionally substituted with 1, 2 or 3 R;

[0159] Rs is selected from H, D, halogen or C 1-4 alkyl;

[0160] R is selected from H, D, halogen or C 1-4 alkyl;

[0161] k is 1, 2, or 3;

[0162] Base is selected from

[0163] In some embodiments, the present invention relates to the aforementioned oligonucleotide, wherein:

[0164] *1 and *2 represent chiral centers, independently selected from (S) configuration or (R) configuration;

[0165] represents H, or indicates the position of phosphate or phosphorothioate linkage to the adjacent nucleotide;

[0166] R1 represents H, or represents the position of linkage to the phosphate or phosphorothioate of another adjacent nucleotide;

[0167] and R1 are not H at the same time;

[0168] L1 and L2 are independently C 1-4 Alkylene, preferably -CH2-;

[0169] Rs is H or C 1-4 Alkyl, preferably H;

[0170] k is 1, 2, or 3;

[0171] Base is selected from

[0172] In some embodiments, the present invention relates to the above oligonucleotide, wherein the compound of formula (I) is selected from the compound of formula (II) or (III), or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof:

[0173] in,

[0174] The variables are as defined above;

[0175] In some specific embodiments, the compound of formula (I) is selected from the following compounds, or pharmaceutically acceptable salts, tautomers or stereoisomers thereof:

[0176] represents H, or indicates the position of phosphate or phosphorothioate linkage to the adjacent nucleotide;

[0177] R1 represents H, or represents the position of linkage to the phosphate or phosphorothioate of another adjacent nucleotide;

[0178] and R1 are not H at the same time.

[0179] In some embodiments, the present invention relates to the above-described oligonucleotide having 14 to 30 nucleotides.

[0180] In some embodiments, the present invention relates to the above oligonucleotide, which comprises a compound of formula (I) as described herein, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof at the 5' end.

[0181] In some embodiments, the present invention relates to the above oligonucleotide, which comprises a compound of formula (I) as described herein, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof at the 3' end.

[0182] In some embodiments, the present invention relates to the above oligonucleotide, which comprises a compound of formula (I) as described herein, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof at the 5' end and the 3' end, respectively.

[0183] In some embodiments, the present invention relates to the above-mentioned oligonucleotides, which comprise one or more compounds of formula (I) as described herein, or pharmaceutically acceptable salts, tautomers or stereoisomers thereof, within the oligonucleotide.

[0184] In some embodiments, the present invention relates to a compound of formula (I'), or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof:

[0185] wherein R1 and R2 are independently selected from H, a reactive phosphorus group, a hydroxyl protecting group or a solid support;

[0186] Base' is selected from H, modified or unmodified bases or leaving groups, preferably modified or unmodified A, U, T, G and C; preferably, Base' is selected from

[0187] *1, *2, L1, L2, Rs and k are as defined above.

[0188] In some embodiments, the present invention relates to compounds of the aforementioned formula (I'), wherein R1 and R2 are H.

[0189] In some embodiments, the present invention relates to compounds of formula (I') above, wherein R1 and R2 are selected from reactive phosphorus groups, preferably phosphoramidites, H-phosphonates, alkyl-phosphonates, phosphates or phosphate mimetics, such as natural phosphates, phosphorothioates, phosphorodithioates, boranephosphates, boranephosphorothioates, phosphonates, halogen-substituted phosphonates and phosphates, phosphoramidates, phosphodiesters, phosphotriesters, phosphorothioatediesters, phosphorothioate triesters, diphosphates or triphosphates, preferably -P(OCH2CH2CN)(N(iPr)2).

[0190] In some embodiments, the present invention relates to compounds of formula (I') above, wherein R1 and R2 are selected from protecting groups, preferably hydroxy protecting groups, such as trimethylsilyl (TMS), triethylsilyl (TES), dimethylisopropylsilyl (DMIPS), diethylisopropylsilyl (DEIPS), tert-butyldimethylsilyl (TBDMS), tert-butyldiphenylsilyl (TBDPS), triisopropylsilyl (TIPS), acetyl (Ac), chloroacetyl, dichloroacetyl, trichloroacetyl, trifluoroacetyl (TFA), benzoyl, p-methoxybenzoyl, 9-fluorenylmethoxycarbonyl (Fmoc), allyloxy Carbonyl (Alloc), 2,2,2-trichloroethoxycarbonyl (Troc), benzyloxycarbonyl (Cbz), tert-butyloxycarbonyl (Boc), benzyl (Bn), p-methoxybenzyl (PMB), allyl, triphenylmethyl (Tr), bis-p-methoxytrityl (DMTr), methoxymethyl (MOM), phenoxymethyl (BOM), 2,2,2-trichloroethoxymethyl, 2-methoxyethoxymethyl (MEM), methylthiomethyl (MTM), p-methoxybenzyloxymethyl (PMBM), -C(O)CH2CH2C(O)OH or 4,4'-dimethoxytrityl, preferably DMTr.

[0191] In some embodiments, the compound of formula (I') is selected from the compound of formula (II') or (III'), or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof:

[0192] in,

[0193] Base' is selected from

[0194] The other variables are as defined above.

[0195] In some specific embodiments, the compound of formula (II') or (III') is selected from the following compounds, or pharmaceutically acceptable salts, tautomers or stereoisomers thereof:

[0196] In some embodiments, the present invention relates to a double-stranded RNA having a sense strand and an antisense strand, each strand having 14 to 30 nucleotides, the antisense strand comprising a sequence sufficiently complementary to the sense strand and the target mRNA, wherein the sense strand and / or the antisense strand comprises one or more compounds of formula (I) as described herein, or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof:

[0197] Wherein, each variable is as defined herein.

[0198] In some embodiments, the compound of formula (I) is selected from a compound of formula (II) or formula (III), or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof:

[0199] Wherein, each variable is as defined in this article;

[0200] In some specific embodiments, the compound of formula (I) is selected from the following compounds, or pharmaceutically acceptable salts, tautomers or stereoisomers thereof:

[0201] represents H, or indicates the position of phosphate or phosphorothioate linkage to the adjacent nucleotide;

[0202] R1 represents H, or represents the position of linkage to the phosphate or phosphorothioate of another adjacent nucleotide;

[0203] and R1 are not H at the same time.

[0204] In some embodiments, the sense strand of the double-stranded RNA comprises a compound of Formula (I) as described herein, or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof, at the 5' end.

[0205] In some embodiments, the sense strand of the double-stranded RNA comprises a compound of Formula (I) as described herein, or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof, at the 3' end.

[0206] In some embodiments, the sense strand of the double-stranded RNA comprises a compound of Formula (I) as described herein, or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof, at each of the 5' and 3' ends.

[0207] In some embodiments, the sense strand of the double-stranded RNA comprises one or more compounds of Formula (I) as described herein, or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof, within the oligonucleotide.

[0208] In some embodiments, the antisense strand of the double-stranded RNA comprises a compound of Formula (I) as described herein, or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof, at the 5' end.

[0209] In some embodiments, the antisense strand of the double-stranded RNA comprises a compound of Formula (I) as described herein, or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof, at the 3' end.

[0210] In some embodiments, the antisense strand of the double-stranded RNA comprises a compound of Formula (I) as described herein, or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof, at each of the 5' and 3' ends.

[0211] In some embodiments, the antisense strand of the double-stranded RNA comprises one or more compounds of Formula (I) as described herein, or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof, within the oligonucleotide.

[0212] In some embodiments, the sense and antisense strands of the double-stranded RNA each have 20 to 25 nucleotides.

[0213] In some embodiments, one or more of the arbitrary sites of the double-stranded RNA may be a compound of formula (I), for example:

[0214] nucleotide 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 of the 5' end of the sense strand;

[0215] nucleotide 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 at the 5' end of the antisense strand.

[0216] In some embodiments, the present invention relates to a double-stranded RNA, wherein the nucleotides 2 and 14 at the 5' end of the antisense strand are not compounds of formula (I).

[0217] In some specific embodiments, one or more of the following sites of the double-stranded RNA is a compound of formula (I):

[0218] nucleotides 4, 6, 7, 9, 10, 13, 15, and 17 at the 5′ end of the sense strand;

[0219] Nucleotides 1, 4, 5, 6, 7, 12, 16, 18, 20, and 21 at the 5' end of the antisense strand.

[0220] In some specific embodiments, one or more of the following sites of the double-stranded RNA is a compound of formula (I):

[0221] nucleotides 4, 6, 7, 9, 10, 13, 15, and 17 at the 5′ end of the sense strand;

[0222] Nucleotides 1, 4, 6, 7, 16, 18, 20, and 21 at the 5' end of the antisense strand.

[0223] In some embodiments, the present invention relates to double-stranded RNA, wherein the compound of formula (I) is located at position 2-8, such as position 2, 3, 4, 5, 6, 7 or 8, of the 5' end of the antisense strand.

[0224] In some specific embodiments, the compound of formula (I) is located at position 5-7, such as position 5, position 6 or position 7, of the 5' end of the antisense strand.

[0225] In some specific embodiments, the compound of formula (I) is located at the 5th position of the 5' end of the antisense strand.

[0226] In some specific embodiments, the compound of formula (I) is located at position 7 of the 5' end of the antisense strand.

[0227] In some embodiments, the double-stranded RNA exhibits reduced off-target toxicity compared to a double-stranded RNA having the same sequence but not comprising a compound of Formula (I) as described herein.

[0228] In some embodiments, the antisense strand of the double-stranded RNA has a sequence that is sufficiently complementary to the sense strand and the target mRNA, and has the ability to induce degradation of the target mRNA.

[0229] In some embodiments, the present invention relates to a pharmaceutical composition comprising a double-stranded RNA molecule as described herein, and a pharmaceutically acceptable carrier or excipient.

[0230] In some embodiments, the present invention relates to a kit comprising a double-stranded RNA molecule as described herein.

[0231] In some embodiments, the present invention relates to a cell containing a double-stranded RNA molecule as described herein.

[0232] In some embodiments, the present invention relates to a method for inhibiting the expression of a target gene in a cell, comprising the step of introducing a double-stranded RNA molecule as described herein into the cell.

[0233] In some embodiments, the present invention relates to a method for inhibiting the expression of a target gene in a cell, comprising expressing a double-stranded RNA molecule as described herein in the cell.

[0234] In some embodiments, the present invention relates to a method for reducing off-target toxicity in a cell, comprising the step of introducing a double-stranded RNA molecule as described herein into the cell.

[0235] In some embodiments, the present invention relates to a method for reducing off-target toxicity in a cell, comprising expressing a double-stranded RNA molecule as described herein in the cell. Example

[0236] The following examples are intended to illustrate the present invention but are not intended to limit the scope of the present invention.

[0237] Example 1. Preparation of Compound CE1

[0238] 1. Preparation of Compound 1b

[0239] Compound 1a (3.00 g, 14.1 mmol) was dissolved in acetonitrile (60 mL), and IBX (5.94 g, 21.2 mmol) was added at 80°C. After stirring at 80°C for one hour, the reaction solution was filtered and the filtrate was concentrated under reduced pressure to obtain crude compound 1b (2.97 g, 14.1 mmol, 99.9%), which was used directly in the next reaction.

[0240] 2. Preparation of Compound 1c

[0241] Compound 1b (2.97 g, 14.1 mmol) was dissolved in 1,4-dioxane (57.0 mL), and aqueous formaldehyde (4.16 mL, 113 mmol) and aqueous NaOH (26.8 mL, 53.6) were added at 0°C. After stirring at 25°C for one hour, the reaction mixture was adjusted to pH 5-7 with 10% aqueous acetic acid and concentrated under reduced pressure. The resulting residue was purified by reverse-phase column chromatography (NH4HCO3 aqueous solution / acetonitrile) to afford compound 1c (570 mg, 16.7%).

[0242] 1 H NMR (400MHz, DMSO-d6) δ7.96(d,J=8.4Hz,1H),6.05-6.02(t,J=5.2Hz,1H),5.60(d,J=8.0Hz,1H),5.03-5.01(t,J=5.2Hz,1H) ,4.84-4.81(t,J=5.2Hz,1H),3.49-4.47(t,J=5.2Hz,2H),3.33-3.28(t,J=9.2Hz,2H),2.32-2.34(m,1H),1.93-1.86(m,3H).

[0243] 3. Preparation of Compounds 1d-P1 and 1d-P2

[0244] Compound 1c (2.5 g, 10.3 mmol) was dissolved in pyridine (16.5 mL), and DMTrCl (3.7 g, 10.8 mmol) was added at 0°C. After stirring at 0°C for one hour, the reaction solution was extracted with ethyl acetate (50.0 mL*2), washed with saturated aqueous sodium chloride (50.0 mL), and the organic phase was dried over Na2SO4, filtered, and concentrated under reduced pressure. The resulting residue was separated and purified by reverse-phase column chromatography (NH4HCO3 aqueous solution / acetonitrile) to afford compound 1d-P1 (1.90 g, 33.8%) and compound 1d-P2 (710 mg, 12.6%).

[0245] Compound 1d-P1: 1 H NMR (400MHz, CDCl3) δ8.55 (s, 1H), 7.66 (d, J = 8.4Hz, 1H), 7.34 (d, J = 7.2Hz, 2 H),7.27-7.05(m,7H),6.85-6.73(m,4H),6.04(t,J=6.0Hz,1H),5.66(dd,J= 2.0,8.0Hz,1H),3.78(d,J=11.6Hz,1H),3.72(s,6H),3.64(d,J=11.6Hz,1H) ,3.10-2.94(m,2H),2.45-2.28(m,1H),2.10-1.95(m,2H),1.91-1.82(m,1H).

[0246] Compound 1d-P2: 1 H NMR (400MHz, CDCl3) δ9.07 (br s,1H),7.59(d,J=8.4Hz,1H),7.32(d,J=7.6Hz,2H),7.24-7.14(m,7H),6.76(d,J=8.8Hz,4H),6.14(t,J=6.0Hz ,1H),5.28(d,J=8.0Hz,1H),3.72(s,6H),3.54-3.42(m,2H),3.25(q,J=10.0Hz,2H),2.50-2.40(m,1H),2.30(br s,1H),2.13-2.05(m,1H),2.00-1.93(m,1H).

[0247] 4. Preparation of Compound CE1-P1

[0248] Compound 1d-P1 (600 mg, 1.10 mmol) was dissolved in dichloromethane (4.20 mL), and compound 1e (0.70 mL, 2.20 mmol) and DCI (143 mg, 1.21 mmol) were added at 0°C. After stirring at 0°C for two hours, the reaction solution was directly purified on a silica gel column (petroleum ether / ethyl acetate / triethylamine) to obtain compound CE1-P1 (650 mg, yield: 79.2%).

[0249] 1 H NMR (400MHz, DMSO-d6) δ11.3(brs,1H),7.82-7.72(m,1H),7.38-7.24(m,9H),6.88(d,J=7.6Hz,4H), 6.14(d,J=4.0Hz,1H),5.62(d,J=6.0Hz ,1H),3.85-3.68(m,10H),3.50(d,J=1.2Hz,2H),3.02(s,2H),2.73-2 .70(m,2H),2.28-2.26(m,1H),2.07-1.92(m,3H),1.12-1.04(m,12H).

[0250] 5. Preparation of Compound CE1-P2

[0251] The synthesis steps of compound CE1-P2 were the same as those of compound CE1-P1 except for the starting compounds.

[0252] 1 H NMR(400MHz,DMSO-d6)δ11.3(brs,1H),7.60(d,J=5.60Hz,1H),7.36-7.24(m,9H),6.88(brs,4H),6.12(brs,1H),5.3 0(brs,1H),3.73-3.51(m,12H),3.24-3.16(m,2H),2.70(m,2H),2.50(s,1H),2.06-2.01(m,3H),1.11-1.04(m,12H).

[0253] Example 2. Preparation of Compounds CE2-CE4

[0254] According to the method of Example 1 above, the uracil base of compound 1a in Example 1 was replaced by the corresponding base as the starting compound to prepare the following compounds CE2-P1, CE2-P2, CE3-P1, CE3-P2, CE4-P1 and CE4-P2.

[0255] Example 3. Preparation of Compound E7

[0256] 1. Preparation of Intermediates 3-4

[0257] 1.1 Preparation of Compound 2

[0258] To compound 1 (300 g, 2.01 mol) in DCM (1.80 L) was slowly added benzyl (2,5-dioxopyrrolidin-1-yl) carbonate (600 g, 2.40 mol) at 15°C and TEA (203 g, 2.01 mol, 280 mL) was added dropwise. After the addition, the mixture was stirred at 25°C for 16 hours. TLC (dichloromethane:methanol=10:1) showed that reactant 1 (R f =0.32) is retained and an important new point is detected (R f =0.52). The reaction mixture was washed with saturated sodium bicarbonate solution (1.00 L x 2), and the organic phase was washed with brine (1.00 L), dried over anhydrous Na2SO4 and concentrated in vacuo. Without purification, compound 2 (about 385 g) was obtained as a yellow oil.

[0259] 1.2 Preparation of compound 2A

[0260] At 0-15 ℃, DMAP (19.8g, 162mmol) was added to a pyridine (1.75L) solution of compound 4 (350g, 1.62mol, HCl) and Ac2O (994g, 9.74mol, 912mL) at a disposable rate and TEA (164g, 1.62mol, 226mL) was added dropwise. The mixture was stirred at 25 ℃ for 16 hours. LCMS (product: RT=0.687min) showed that the starting reactants were completely consumed. EtOAc (1.40L) was added to the mixture at 25 ℃ and stirred for 30 minutes, then the mixture was filtered and the filter cake was washed with EtOAc (300mL). The filter cake was ground with water (1.45L) for 30 minutes at 25 ℃. The mixture was filtered and the filter cake was washed with water (175mL x 3), and the filter cake was collected to obtain compound 2A (about 580g) as a white solid.

[0261] 1.3 Preparation of compound 2B

[0262] Three reactions were performed in parallel.

[0263] To a solution of compound 2A (200 g, 514 mmol) in DCM (800 mL) was added TMSOTf (137 g, 616 mmol, 111 mL) dropwise at 10-15° C. over 0.5 h. The mixture was then stirred at 25° C. for 3 h. TLC (dichloromethane:methanol=20:1) showed that compound 2A (R f =0.54) is completely consumed and the new point (R f =0.24) was formed. The three reactions were combined. The mixture was cooled to 0-15°C and slowly poured into a solution of NaHCO3 (300 g dissolved in 3.00 L water) at 0-5°C. The organic phase was separated and the aqueous phase was extracted with DCM (1.00 L x 3). The organic layers were combined, dried over Na2SO4, filtered and concentrated in vacuo. Without purification, compound 2B (approximately 507 g) was obtained as a yellow oil and used in the next step.

[0264] 1.4 Preparation of compound 3

[0265] To a mixture of compound 2B (250 g, 759 mmol) and compound 2 (151 g, 531 mmol) in DCM (1.00 L) was added TMSOTf (84.4 g, 380 mmol, 69.0 mL) dropwise at 0-10° C. and the mixture was stirred at 20° C. for 12 hours. TLC (dichloromethane:methanol=20:1) showed that compound 2 (R f =0.33) is completely consumed and a new point is formed (R f =0.03). The combined reaction was cooled to 0-5°C and then poured into NaHCO (aqueous solution, 100 g dissolved in 1 L of water) and stirred at 5-10°C for 10 minutes. The phases were separated. The aqueous phase was extracted with DCM (500 mL x 2), the combined organic phases were dried over NaSO, filtered, and the filtrate was concentrated in vacuo. Without purification, compound 3 (approximately 360 g) was obtained as a yellow oil.

[0266] 1 H NMR: (400 MHz, DMSO).

[0267] δ=7.79-7.37(m,1H),7.35-7.26(m,5H),5.21-5.20(m,1H),5.00-4.95(m,3H),4.55-4.53(m,1H),4.03-3.86(m,3H),3.61-3.59(m,1H) ,3.59-3.57(m,1H),3.48-3.40(m,6H),3.39-3.31(m,2H),3.14-3.13(m,2H),2.09(s,3H),1.99(s,3H),1.88(s,3H),1.76-1.74(m,3H).

[0268] 1.5 Preparation of intermediate 3-4 (TEA salt)

[0269] Three reactions were performed in parallel.

[0270] To a mixture of Pd / C (18.0 g, 16.3 mmol, 10% content) in THF (1.80 L) was added compound 3 (180 g, 293 mmol) and TFA (33.5 g, 293 mmol, 21.8 mL) under an argon atmosphere. The suspension was vented and vented three times with hydrogen. The mixture was stirred at H2 (50 Psi) and 30 ° C for 2 hours. LCMS (product: RT = 0.697 min) showed that compound 3 was consumed and a product peak was detected. The three reactions were combined. The mixture was filtered through celite and the filtrate was concentrated under reduced pressure to remove the solvent. Intermediate 3-4 (TFA salt) (393 g, 660 mmol, 74.8% yield, 99.6% purity, TFA) was obtained as a yellow solid without purification.

[0271] 1 H NMR: (400 MHz, DMSO-d6)

[0272] δ=7.92(d,J=9.1Hz,4H),5.27-5.17(m,1H),5.03-4.91(m,1H),4.60-4.50(m,1H),4.09-3.97(m,4H),3.85(s ,2H),3.65-3.46(m,10H),3.04-2.92(m,2H),2.10(s,3H),2.00(s,3H),1.94-1.86(m,3H),1.82-1.71(m,4H).

[0273] 2. Preparation of Intermediates 3-5

[0274] 2.1 Preparation of compound 5

[0275] To a solution of compound 4B (10.0 g, 35.5 mmol, 1.00 eq) and compound 3-4 (46.3 g, 78.2 mmol, 2.20 eq, TFA) prepared above in DCM (1.00 L) was added DIEA (30.3 g, 234 mmol, 40.8 mL, 6.60 eq) in one portion at 25°C. The mixture was stirred for half an hour at 25°C. HBTU (30.3 g, 234 mmol, 40.8 mL, 6.60 eq) was added to the mixture. The mixture was stirred at 25°C for 16 hours. LCMS (product: RT = 0.681 mins) indicated the reaction was complete, and the mixture was concentrated in vacuo. At 20 ℃, 0.50N HCl (200mL x 2) was added to the mixture, and extracted with DCM (3x 500mL), the combined organic layers were washed with saturated NaHCO (3x 800mL) until pH=8, washed with brine (3x 500mL), and dried and concentrated in vacuo to purify. The residue was purified by column chromatography (SiO , DCM:MeOH=50:1-15:1). The residue was concentrated in vacuo at 40 ℃ and purified by preparative-MPLC (column: 800g Agela C18; mobile phase: [water-ACN]; 15-45% 25min; 45% 10min). Vacuum drying was used to obtain compound 5 (approximately 180g+75.0g+87.0g+40.0g+38.0g) as a yellow solid.

[0276] 417.0 g of compound 3-4 was converted into compound 5 through 9 batches.

[0277] 2.2 Preparation of intermediate 3-3

[0278] Compound 5 (73.0 g, 61.7 mmol, 1.00 eq) and TFA (7.04 g, 61.7 mmol, 4.57 mL, 1.00 eq) were added to a solution of Pd / C (3.00 g, 10% content) in THF (300 mL) under an argon atmosphere. The suspension was degassed and purged with hydrogen three times. The mixture was stirred at 20°C under H2 (20 Psi) for 16 hours. TLC (dichloromethane:methanol = 8:1, R f =0.0) indicated that the reaction was complete. The mixture was filtered through celite, and the filtrate was concentrated under pressure to remove the solvent to obtain compound 3-3 (about 33.4 g + 129 g + 75.0 g) as a white solid.

[0279] 1 H NMR: (400 MHz, DMSO)

[0280] δ=8.53(t,J=5.2Hz,1H),8.18(d,J=2.4Hz,3H),8.03(t,J=5.2Hz,1H),7.84(dd,J=3.6Hz,2H) ,5.22(d,J=3.2Hz,2H),4.96(dd,J=3.2Hz,2H),4.55(d,J=8.4Hz,2H),4.02(t,J=8.8Hz,6H), 3.77-3.59(m,5H),3.58-3.45(m,21H),3.40-3.20(m,4H),2.18(t,J=7.6Hz,2H), 2.17(d,J=8.0Hz,6H),2.10(s,6H),1.99(s,6H),1.90-1.80(m,8H),1.77(s,6H).

[0281] 3. Preparation of Compound E7

[0282] 3.1 Preparation of compound 3

[0283] Compound 1 (2.00 g, 1.87 mmol, prepared according to the method for Intermediate 3-3 above) was dissolved in DCM (20.0 mL) at room temperature. DIEA (0.135 mL, 0.814 mmol) and compound 2 (0.550 g, 0.814 mmol) were added sequentially to the solution, and the nitrogen atmosphere was replaced three times. The reaction mixture was stirred at 25°C for 16 hours. LC-MS / MS detected the product's MS response, and thin-layer chromatography (dichloromethane / methanol = 5 / 1) showed the disappearance of the starting material and the formation of new spots. The reaction solution was concentrated under reduced pressure, and the resulting crude product was purified by column chromatography (dichloromethane / methanol = 5 / 1) to afford compound 3 (approximately 780 mg) as a white solid.

[0284] 1 H NMR (400 MHz, CD3OD)

[0285] δ=7.28-7.42(m,5H),5.30-5.34(m,4H),5.04-5.14(m,6H),4.63-4.67( m,4H),4.36-4.44(m,2H),4.00-4.20(m,23H),3.91-3.95(m,4H),3.69-3 .77(m,9H),3.52-3.67(m,32H),3.34-3.43(m,9H),2.29-2.31(m,4H),2. 14(s,12H),2.03(s,12H),1.92-1.96(m,24H).LCMS:m / z=1221.6(M / 2+H) + .

[0286] 32 Preparation of compound 4

[0287] Compound 3 (1.10 g, 0.451 mmol) was dissolved in MeOH (10.0 mL) at room temperature. 10% wet Pd / C (0.050 g, 0.451 mmol) was added to the solution. The hydrogen atmosphere was replaced three times, and the reaction mixture was stirred at 25°C for 18 hours under a hydrogen atmosphere (14.696 psi). Liquid chromatography-mass spectrometry (LC-MS / MS) detected the product. Thin-layer chromatography (dichloromethane / methanol = 10 / 1, phosphomolybdic acid) revealed complete consumption of the starting material and the formation of new spots. The reaction mixture was filtered, and the filtrate was concentrated under reduced pressure to yield compound 4 (approximately 840 mg) as a white solid.

[0288] 1 H NMR (400 MHz, CD3OD)

[0289] δ=5.32-5.34(m 4H),5.06-5.10(m,4H),4.63-4.65(m,4H),4.38-4.40(m,2H),3.99-4.20(m,20H), 3.90-3.97(m,4H),3.69-3.76(m,6H),3.50-3.68(m,36H),3.35-3.44(m,11H),2.28-2 .38(m,4H),2.15(s,12H),2.03(s,12H),1.90-1.94(m,24H).LCMS:m / z=1154.7(M / 2+H) + .

[0290] 3.3 Preparation of compound 6

[0291] Compound 5 (232 mg, 0.364 mmol) was dissolved in DCM (10.0 mL) at room temperature. HBTU (207 mg, 0.546 mmol), DIEA (0.181 mL, 1.09 mmol), and compound 4 (840 mg, 0.364 mmol) were added sequentially to the solution, and the atmosphere was purged with nitrogen three times. The reaction mixture was stirred at 25°C for 1 hour. LC / MS analysis revealed the disappearance of the starting material, while thin-layer chromatography (dichloromethane / methanol = 5 / 1) showed the disappearance of the starting material and the formation of new spots. The reaction mixture was concentrated under reduced pressure, and the resulting crude product was purified by column chromatography (dichloromethane / methanol = 8 / 1 to 5 / 1) to afford compound 6 (approximately 620 mg) as a white solid.

[0292] 1 H NMR (400 MHz, CD3OD)

[0293] δ=7.41-7.43(m,2H),7.23-7.34(m,7H),6.83-6.90(m,4H),5.31-5.35(m,4H),5.01-5.12(m ,4H),4.63-4.65(m,4H),4.41-4.45(m,2H),4.31-4.33(m,1H),3.99-4.22(m,22H),3.87-3.9 7(m,6H),3.58-3.81(m,45H),3.34-3.43(m,10H),2.19-2.40(m,10H),2.14(s,12H),2.02(s ,12H),1.92-1.96(mz,24H),1.48-1.63(m,4H),1.28-1.38(m,8H).LCMS:m / z=1460.0(M / 2+H) + .

[0294] 4. Preparation of Compound E7

[0295] Compound 6 (300 mg, 0.103 mmol) was dissolved in DCM (10.0 mL) at room temperature. DIEA (0.102 mL, 0.618 mmol), compound 7 (10.3 mg, 0.103 mmol), and DMAP (12.6 mg, 0.103 mmol) were added sequentially to the solution, and the atmosphere was replaced with nitrogen three times. The reaction mixture was stirred at 25°C for 2 hours. LC / MS / MS analysis confirmed the disappearance of the starting material. The reaction solution was concentrated under reduced pressure, and the resulting crude product was separated by preparative HPLC (Preparative-HPLC, column: Waters Xbridge BEH C18 100*30 mm*10 μm; mobile phase: water-ACN; B%: 17%-57%, 5 min) to afford compound E7 (53.0 mg, yield 17.08%, purity 78.94%) as a white solid.

[0296] 1 H NMR (400 MHz, CD3OD)

[0297] δ=7.41-7.45(m,2H),7.17-7.34(m,7H),6.85-6.89(m,4H),5.32-5.36(m,4H),5.03-5.13(m,4H),4.63- 4.67(m,4H),4.38-4.47(m,2H),4.32-4.34(m,1H),4.01-4.26(m,22H),3.88-4.00(m,6H),3.77-3.81(m ,7H),3.49-3.76(m,45H),3.33-3.47(m,10H),2.56-2.62(m,2H),2.45-2.55(m,3H),2.21-2.38(m,7H), 2.14(s,12H),2.05-2.11(m,2H),2.02(s,12H),1.92-1.96(m,24H),1.47-1.68(m,4H),1.28-1.34(m,8H)

[0298] MS: m / z = 3022.36 (M+H) + .

[0299] Example 4 Preparation of siRNA

[0300] The siRNA of the present invention is prepared using the solid phase phosphoramidite method well known in the art. Specific methods can be found in, for example, PCT Publication Nos. WO2016081444 and WO2019105419, and are briefly described below.

[0301] 1. Preparation of siRNA with Sense Strand Linked to Ligand

[0302] 1.1 Ligand-CPG Carrier Connection

[0303] 1.1.1 Ligation of compound E7 to CPG carrier

[0304] Compound E7 (53 mg, 0.018 mmol) and HBTU (13.3 mg, 0.035 mmol) were mixed and added to acetonitrile (5 mL) and shaken to dissolve. DIEA (9.0 mg, 0.07 mmol) and DMAP (2.1 mg, 0.018 mmol) were then added and shaken to dissolve until the solution became clear. ) was added to the reaction solution, and the reaction was shaken overnight at 20°C. Samples were taken and monitored for TLC, which indicated the reaction was complete. The developing solvent was DCM / methanol = 4:1, and the color was developed with phosphomolybdic acid. Filtered using a fritted funnel, the filter cake was washed with anhydrous acetonitrile (20 mL x 5), and the filter cake was filtered under reduced pressure using an oil pump for 6 h to obtain 530 mg of an off-white solid.

[0305] Place 530 mg of the condensed product in a 50 mL round-bottom flask. Add Cap C (DMAP / acetonitrile), Cap B (N-methylimidazole / pyridine / acetonitrile), and Cap A (acetic anhydride / acetonitrile) in that order. Shake overnight at room temperature. Filter, wash the filter cake with acetonitrile (20 mL x 4). Remove the filter cake and filter under reduced pressure with an oil pump for 8 hours to obtain 200 mg of an off-white solid, which was used for solid-phase synthesis.

[0306] 1.1.2 Ligand L96 and CPG carrier connection

[0307] The ligand L96 was linked to the CPG carrier using methods known in the art.

[0308] 1.2 Synthesis of the positive sense chain (SS chain)

[0309] Using the solid-phase phosphoramidite synthesis method, the solid support prepared above is used as the starting cycle to link nucleoside monomers or nucleotide analog monomers (including compounds of formula (I) of the present invention) one by one in the 3'-5' direction according to the arrangement order of the positive strand nucleotides. Each linking of a nucleoside monomer involves four steps: deprotection, coupling, capping, and oxidation or thiolation. The conditions for the synthesis of oligonucleotides at a synthesis scale of 5 μmol are as follows:

[0310] The nucleoside monomer was provided in a 0.05 mol / L acetonitrile solution. The reaction conditions for each step were the same, i.e., the temperature was 25 degrees Celsius. Deprotection was performed three times using a 3% trichloroacetic acid-dichloromethane solution. The coupling reaction was performed twice using a 0.25 mol / L ETT-acetonitrile solution as the activator. Capping was performed twice using 10% acetic anhydride-acetonitrile and pyridine / N-methylimidazole / acetonitrile (10:14:76, v / v / v). Oxidation was performed twice using 0.05 mol / L iodine / tetrahydrofuran / pyridine / water (70 / 20 / 10, v / v / v). Thiolysis was performed twice using 0.2 mol / L PADS in acetonitrile / 3-methylpyridine (1 / 1, v / v).

[0311] 1.3 Synthesis of antisense strand (AS strand)

[0312] Using a solid-phase phosphoramidite synthesis method, a blank CPG solid support is used as the starting cycle. Nucleoside monomers or nucleotide analog monomers (including compounds of formula (I) of the present invention) are linked one by one in the 3'-5' direction according to the nucleotide arrangement order of the antisense strand. Each linking of a nucleoside monomer involves four steps: deprotection, coupling, capping, and oxidation or thiolation. The synthesis conditions for a 5 μmol oligonucleotide of the antisense strand are the same as those for the sense strand.

[0313] 1.4 Oligonucleotide purification and annealing

[0314] 1.4.1 Ammonolysis

[0315] Add the synthesized solid phase carrier (sense chain or antisense chain) to a 5 mL centrifuge tube, add 3% diethylamine / ammonia water (v / v), react in a constant temperature water bath at 35 degrees (or 55 degrees) for 16 hours (or 8 hours), filter, wash the solid phase carrier three times with ethanol / water, each time 1 mL, and centrifuge the filtrate to purify the crude product.

[0316] 1.4.2 Purification

[0317] Purification and desalination methods are well known in the art. For example, a column packed with a strong anion filler can be used, and a sodium chloride-sodium hydroxide system can be used for elution and purification. The product can be collected and pipetted, and desalination can be performed using a gel-filled purification column, and the elution system can be pure water.

[0318] 1.4.3 Annealing

[0319] According to Table 6, the sense strand (SS strand) and the antisense strand (AS strand) were mixed at a molar ratio (SS strand / AS strand = 1 / 1.05), heated in a water bath to 70-95 degrees, maintained for 3-5 minutes, naturally cooled to room temperature, and the system was lyophilized to obtain the product.

[0320] siRNA conjugated with L96 was obtained in a similar manner.

[0321] Table 1. siRNA sequences used in the present invention

[0322] In this article, the meanings of the abbreviations are as follows:

[0323] The A, U, G, and C distribution represents the natural adenine, uracil, guanine, and cytosine ribonucleotides.

[0324] m indicates that the nucleotide adjacent to its left is a 2'-OCH3 modified nucleotide. For example, Am, Um, Gm, and Cm represent 2'-OCH3 modified A, U, G, and C.

[0325] f indicates that the nucleotide adjacent to its left is a 2'-F modified nucleotide. For example, Af, Uf, Gf, and Cf represent 2'-F modified A, U, G, and C, respectively.

[0326] d indicates that the adjacent nucleotide to the right is a 2'-deoxyribonucleotide (i.e., natural DNA). For example, dA, dT, dG, and dC represent adenine deoxyribonucleotide, thymine deoxyribonucleotide, guanine deoxyribonucleotide, and cytosine deoxyribonucleotide.

[0327] "s" or "s-" indicates that the two adjacent nucleotides and / or delivery vectors are linked by phosphorothioate.

[0328] L96 represents a GalNAc delivery vector of the following structure well known in the art, wherein The position of the siRNA is indicated by a phosphate group or a phosphorothioate group, as described in, for example, PCT Publication Nos. WO2009073809 and WO2009082607.

[0329] GL6 represents a GalNAc delivery vector of the following structure, wherein Indicates the position where the phosphate group or phosphorothioate group is attached to the siRNA

[0330] The nucleotide analog monomers of the present invention are shown in Table 2.

[0331] In the order of 5'–>3' oligonucleotide chains: It means that it is connected to the 3' carbon atom of the previous nucleotide in the oligonucleotide chain through a backbone group (such as a phosphate group or a thiophosphate group). Indicates that the oligonucleotide is linked to the next nucleotide in the oligonucleotide chain through the backbone group and the 5' carbon atom of the next nucleotide.

[0332] Table 2. Nucleotide analog monomers of the present invention

[0333] Example 5. Activity screening experiment of primary hepatocytes (PMH) of C57BL / 6 wild-type mice

[0334] 1. Free intake

[0335] Isolate C57BL / 6 wild-type mouse liver primary cells, count, and plate in 24-well plates, 900 μL / well, 8×10 4 cells / well; 96-well plate, 100 μL / well, 1×10 4 cells / well. Then choose free uptake.

[0336] 10 μL of the diluted compound was added to 90 μL of Opti-MEM, mixed well, added to the corresponding wells, and cultured in a 37°C, 5% CO2 incubator for 24 hours. No siRNA was added to the control group.

[0337] 2. Fluorescence quantitative PCR

[0338] Total RNA was extracted using a high-throughput nucleic acid extraction instrument-magnetic bead method (Fanzhi Medical, FG0412; Hangzhou Aosheng, Auto-pure96), and reverse transcribed (PrimeScript TM II 1st Strand cDNA Synthesis Kit (Takara, 6210B) and then fluorescent quantitative PCR detection (TaqMan TM Fast Advanced Master Mix (ABI, 4444965)).

[0339] 3. Data Statistics

[0340] calculate The values ​​were converted into percentages to obtain the residual inhibition rate;

[0341] △△Ct=[(target gene of Ct experimental group-internal reference of Ct experimental group)-(target gene of Ct control group-internal reference of Ct control group)].

[0342] The target gene is mTTR and the control gene is mGAPDH.

[0343] 4. mTTR gene test results

[0344] In this experiment, C57BL / 6 wild-type mouse primary hepatocytes were selected. After plating the cells in 96-well plates, the starting concentration of the compound was selected to be 10 nM, and 5 concentration points (10 nM, 1 nM, 0.1 nM, 0.01 nM, 0.001 nM) were diluted 10-fold to perform a 5-point IC50 activity screening of C57BL / 6 wild-type mouse primary hepatocytes - free uptake. The experimental results are shown in Table 4.

[0345] Table 4. Results of the PMH 5-point IC50 activity screening experiment of siRNA compounds targeting the mTTR gene carrying the nucleotides of the present invention - free uptake:

[0346] Example 6. Activity screening of Huh7 cell lines

[0347] Cell transfection

[0348] On the first day, Huh7 cells were digested, resuspended, and counted. The cell suspension was plated into a 96-well plate at 100 μL / well, with 1×10 4 cells / well, and transfection was performed 18 h later.

[0349] On the second day, 20 μM siRNA stock solution was diluted with Opti-MEM. 198 μL Opti-MEM was added to 2 μL siRNA stock solution. The final siRNA concentration was as shown below. Pipet and mix thoroughly. Set aside.

[0350] The next day, dilute 0.9 μL RNAiMAX (Thermo, 13778150) with 14.1 μL Opti-MEM, gently pipette to mix, and let stand at room temperature for 5 minutes. Then, take 15 μL of the prepared RNAi-MAX mixture and 15 μL of the diluted siRNA and gently pipette to mix, avoiding the introduction of bubbles. Let stand at room temperature for 10 minutes, then add 10 μL / well to a 96-well plate. Incubate in a 37°C, 5% CO2 incubator for 24 hours (no siRNA was added to the control group).

[0351] RNA extraction

[0352] Cell RNA was extracted using a nucleic acid extractor (Auto-pure96, Hangzhou Aosheng) according to the operating instructions of the high-throughput cell RNA extraction kit (Fanzhi Medical, FG0417-L).

[0353] RNA reverse transcription

[0354] Denaturation reaction mixture preparation reference PrimeScript TM II 1st Strand cDNA Synthesis Kit (Takara, 6210B), single-well preparation volume: Oligo dT Primer 1 μL, dNTP Mixture 1 μL, template RNA 12.5 μL, incubate at 65°C in a conventional PCR instrument for 5 minutes, then quickly cool on ice for 2 minutes.

[0355] Reverse transcription reaction solution preparation reference PrimeScript TM II 1st Strand cDNA Synthesis Kit (Takara, 6210B). Each well contains 4 μL of 5× PrimeScript II Buffer, 0.5 μL of RNase Inhibitor, and 1 μL of PrimeScript II RTase.

[0356] Slowly mix 14.5 μL of the denatured reaction solution with the reverse transcription reaction solution, incubate at 42°C for 45 minutes for reverse transcription using a conventional PCR instrument, incubate at 95°C for 5 minutes to inactivate the enzyme, and cool the reverse transcription product (cDNA) at 4°C.

[0357] After inversion, 30 μL of DNase- and RNase-free distilled water was added to the cDNA sample in each well.

[0358] Fluorescence quantitative PCR

[0359] ReferenceTaqMan TMQuantitative PCR (ABI, QuantStudio3) was performed using 20 μL of Fast Advanced Master Mix (ABI, 4444965). The reaction schedule was: (50°C, 2 minutes) × 1 cycle; (95°C, 20 seconds) × 1 cycle; (95°C, 1 second; 60°C, 24 seconds) × 40 cycles.

[0360] Statistics

[0361] calculate The values ​​were converted into percentages to obtain the residual inhibition rate;

[0362] △△Ct=[(target gene of Ct experimental group-internal reference of Ct experimental group)-(target gene of Ct control group-internal reference of Ct control group)].

[0363] The target gene was hApoc3, and the internal reference was hACTB.

[0364] In this experiment, Huh7 cell line was selected, and the starting concentration of the compound was 10 nM, and 11 concentration points of 3-fold gradient dilution (10 nM, 3.33 nM, 1.11 nM, 0.37 nM, 0.123 nM, 0.041 nM, 0.0136 nM, 0.0045 nM, 0.00152 nM, 0.000508 nM, 0.000169 nM) were selected for cell line activity screening of the compounds of the present invention. The experimental screening results are shown in Table 6.

[0365] Example 7. Detection of off-target activity of siRNA compound psi-CHECK2

[0366] 1. Plasmid preparation:

[0367] Off-target plasmid: The corresponding antisense chain off-target plasmid was designed according to the compound sequence. The psiCHECK2 GSSM-5Hits recombinant plasmid was prepared by Sangon Biotech (Shanghai) Co., Ltd. and the recombinant plasmid was diluted to 1000 ng / μL for use.

[0368] 2. Cell transfection:

[0369] HEK293A cells (Nanjing Kebai, Cat. No. CBP60436) were plated in 96-well plates with 100 μL of cell resuspension. The cell number was 8 × 10 3 cells / well.

[0370] On the second day, the complete culture medium in the wells was first aspirated and discarded, and replaced with 80 μL / well of Opti-MEM culture medium, and the cells were starved for about 1.5 h.

[0371] Plasmid mixture: Single-well preparation volume: plasmid 0.01 μL / well, Opti-MEM 8.99 μL / well.

[0372] Lipo mix: dilute Lipo 2000 (Lipofectamine TM 2000 transfection reagent, Thermo, 11668019), incubate at room temperature for 5 minutes, and prepare the Lipo mixture in the following amounts: Lipo 0.2 μL / well, Opti-MEM 9.8 μL / well.

[0373] Aliquot 22 μL of the prepared Lipo mixture, 2.2 μL of the compound, and 19.8 μL of the plasmid mixture into corresponding wells (designated Well A). Mix thoroughly by pipetting and incubate at room temperature for 20 minutes before co-transfection. Finally, add 20 μL of the Well A mixture to each well of cells, adding 80 μL of Opti-MEM to the original volume, for a final volume of 100 μL per well. After incubation at 37°C in a 5% CO2 incubator for 4 hours, add 100 μL of DMEM supplemented with 20% fetal bovine serum to each well. Incubate at 37°C in a 5% CO2 incubator for 24 hours before testing.

[0374] 3. Result detection:

[0375] Before the experiment, mix the ( Luciferase Assay System, Promega, E2940) was reconstituted and after equilibration to room temperature, DMEM was added to each tube at a ratio of 1:1 to prepare substrate I for immediate use. Stop& Buffer was reconstituted and then equilibrated to room temperature. Stop& Prepared as substrate II at a ratio of 100:1 and used immediately.

[0376] The vacuum pump sucks away the original culture medium in the 96-well culture plate;

[0377] Add 150 μL of substrate I to each well and incubate on a shaker at room temperature for 10 min;

[0378] 120 μL of substrate I was transferred to a 96-well microplate, and the Firefly chemiluminescence value was read on a microplate reader (Tecan, Infinite 200);

[0379] Then, 60 μL of substrate II was added to each well, and the cells were incubated on a shaker at room temperature for 10 min. The Renilla chemiluminescence value was read on a microplate reader.

[0380] 4. Data Analysis and Processing

[0381] Fluorescence activity was measured by a microplate reader. The collected Renilla signal was normalized by the Firefly signal standard. The inhibitory effect of siRNA was calculated by comparing it with the untreated result (residual inhibitory activity). The calculation process is as follows:

[0382] Normalized Ren / Fir ratio: Ratio = Renilla (Renilla luciferase) / Firefly (Firefly luciferase).

[0383] Residual inhibition rate: 2 replicates (Ratio siRNA / Ratio control )*100% mean: where Ratio control The average value of Ratio of two replicate wells in the control well (without siNRA) was used to calculate the Ratio of two replicate wells. siRNA / Ratio control , and then take the mean value as the residual inhibition rate;

[0384] Graphing: Graphpad Prism

[0385] Half maximal inhibitory concentration (IC50): In this experiment, Top and Bottom plots were used. The IC50 value was calculated using the formula Y = Bottom + (Top - Bottom) / (1 + 10^((LogIC50 - X) * HillSlope)), where Y = 50 and X = log(concentration).

[0386] In this experiment, HEK293A cell line was selected, and the starting concentration of the compound was 40 nM, and 11 concentration points of 3-fold gradient dilution (40 nM, 13.3 nM, 4.44 nM, 1.48 nM, 0.493 nM, 0.164 nM, 0.0548 nM, 0.0182 nM, 0.00609 nM, 0.00203 nM, 0.000677 nM) were selected for screening of the off-target activity of the compound of the present invention against psiCHECK2 GSSM-5 Hits. The experimental screening results are shown in Table 7.

[0387] The above is a further detailed description of the present invention in conjunction with specific preferred embodiments, and the specific implementation of the present invention should not be considered to be limited to these descriptions. For those skilled in the art of the present invention, without departing from the concept of the present invention, several simple deductions or substitutions can be made, which should be considered to fall within the scope of protection of the present invention.

Claims

1. An oligonucleotide comprising one or more compounds of formula (I), or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof: in, *1 and *2 represent chiral centers, independently selected from (S) configuration or (R) configuration; represents H, or represents the position of phosphate or phosphorothioate linkage to the adjacent nucleotide; R1 represents H, or represents the position of linkage to the phosphate or phosphorothioate of another adjacent nucleotide; and R1 are not H at the same time; L1 and L2 are independently selected from C 1-10 Alkylene, C 2-10 Alkenylene or C 2-10 Alkyne, wherein 1, 2, 3, 4 or 5 non-adjacent carbon atoms in the group may be replaced by heteroatoms selected from O, S and N, preferably C 1-10 Alkylene; The L1 and L2 are optionally substituted with 1, 2, 3, 4 or 5 R; Rs is selected from H, D, halogen, C 1-6 Alkyl or C 1-6 Haloalkyl; R is selected from H, D, halogen, C 1-6 Alkyl or C 1-6 Haloalkyl; k is 1, 2, 3, 4 or 5; Base is selected from H, modified or unmodified bases or leaving groups, preferably modified or unmodified A, U, T, G and C; preferably, Base is selected from 2. The oligonucleotide of claim 1, wherein L1 and L2 are independently selected from C 1-6 Alkylene, C 2-6 Alkenylene or C 2-6 Alkyne, preferably C 1-6 Alkylene; The L1 and L2 are optionally substituted with 1, 2, 3, 4 or 5 R; R is selected from H, D, halogen, C 1-6 Alkyl or C 1-6 Haloalkyl; Preferably, L1 and L2 are independently C 1-4 Alkylene, preferably -CH2- or -CH2CH2-; The L1 and L2 are optionally substituted by 1, 2 or 3 R; R is selected from H, D, halogen or C 1-4 alkyl.

3. The oligonucleotide of claim 1 or 2, wherein: *1 and *2 represent chiral centers, independently selected from (S) configuration or (R) configuration; represents H, or represents the position of phosphate or phosphorothioate linkage to the adjacent nucleotide; R1 represents H, or represents the position of linkage to the phosphate or phosphorothioate of another adjacent nucleotide; and R1 are not H at the same time; L1 and L2 are independently selected from C 1-6 Alkylene, C 2-6 Alkenylene or C 2-6 Alkyne, preferably C 1-6 Alkylene; The L1 and L2 are optionally substituted with 1, 2, 3, 4 or 5 R; Rs is selected from H, D, halogen, C 1-6 Alkyl or C 1-6 Haloalkyl; R is selected from H, D, halogen, C 1-6 Alkyl or C 1-6 Haloalkyl; k is 1, 2, or 3; Base is selected from 4. The oligonucleotide of any one of claims 1 to 3, wherein: *1 and *2 represent chiral centers, independently selected from (S) configuration or (R) configuration; represents H, or represents the position of phosphate or phosphorothioate linkage to the adjacent nucleotide; R1 represents H, or represents the position of linkage to the phosphate or phosphorothioate of another adjacent nucleotide; and R1 are not H at the same time; L1 and L2 are independently C 1-4 Alkylene, preferably -CH2-; The L1 and L2 are optionally substituted by 1, 2 or 3 R; Rs is selected from H, D, halogen or C 1-4 alkyl; R is selected from H, D, halogen or C 1-4 alkyl; k is 1, 2 or 3; Base is selected from 5. The oligonucleotide of any one of claims 1 to 4, wherein: *1 and *2 represent chiral centers, independently selected from (S) configuration or (R) configuration; represents H, or represents the position of phosphate or phosphorothioate linkage to the adjacent nucleotide; R1 represents H, or represents the position of linkage to the phosphate or phosphorothioate of another adjacent nucleotide; and R1 are not H at the same time; L1 and L2 are independently C 1-4 Alkylene, preferably -CH2-; Rs is H or C 1-4 Alkyl, preferably H; k is 1, 2 or 3; Base is selected from 6. The oligonucleotide according to any one of claims 1 to 5, wherein The compound of formula (I) is selected from the compounds of formula (II) or (III), or pharmaceutically acceptable salts, tautomers or stereoisomers thereof: in, Each variable is as defined in any one of claims 1 to 5; Preferably, the compound of formula (I) is selected from the following compounds, or pharmaceutically acceptable salts, tautomers or stereoisomers thereof: represents H, or represents the position of phosphate or phosphorothioate linkage to the adjacent nucleotide; R1 represents H, or represents the position of linkage to the phosphate or phosphorothioate of another adjacent nucleotide; and R1 are not H at the same time.

7. The oligonucleotide of any one of claims 1 to 6, having 14 to 30 nucleotides.

8. An oligonucleotide according to any one of claims 1 to 7, comprising at the 5' end a compound of formula (I) according to any one of claims 1 to 6, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof.

9. The oligonucleotide according to any one of claims 1 to 8, comprising at the 3' end a compound of formula (I) according to any one of claims 1 to 6, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof.

10. The oligonucleotide according to any one of claims 1 to 9, which comprises one compound of formula (I) according to any one of claims 1 to 6 at each of the 5' end and the 3' end, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof.

11. An oligonucleotide according to any one of claims 1 to 10, comprising one or more compounds of formula (I) according to any one of claims 1 to 6, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, within the oligonucleotide.

12. A compound of formula (I'), or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof: in, R1 and R2 are independently selected from H, a reactive phosphorus group, a hydroxyl protecting group or a solid support; Base' is selected from H, a modified or unmodified base or a leaving group, preferably modified or unmodified A, U, T, G and C; preferably, Base' is selected from *1, *2, L1, L2, Rs and k are as defined in any one of claims 1 to 5.

13. The compound of claim 12, wherein R1 and R2 are H.

14. The compound of claim 12 or 13, wherein R1 and R2 are selected from reactive phosphorus groups, preferably phosphoramidites, H-phosphonates, alkyl-phosphonates, phosphates or phosphate mimetics, such as natural phosphates, phosphorothioates, phosphorodithioates, boranophosphates, boranophosphorothioates, phosphonates, halogen-substituted phosphonates and phosphates, phosphoramidates, phosphodiesters, phosphotriesters, phosphorothioate diesters, phosphorothioate triesters, diphosphates or triphosphates, preferably -P(OCH2CH2CN)(N(iPr)2).

15. The compound according to any one of claims 12 to 14, wherein R1 and R2 are selected from protecting groups, preferably hydroxy protecting groups, such as trimethylsilyl (TMS), triethylsilyl (TES), dimethylisopropylsilyl (DMIPS), diethylisopropylsilyl (DEIPS), tert-butyldimethylsilyl (TBDMS), tert-butyldiphenylsilyl (TBDPS), triisopropylsilyl (TIPS), acetyl (Ac), chloroacetyl, dichloroacetyl, trichloroacetyl, trifluoroacetyl (TFA), benzoyl, p-methoxybenzoyl, 9-fluorenylmethoxycarbonyl (Fmoc), allyloxycarbonyl (Alloc), 2,2, 2-trichloroethoxycarbonyl (Troc), benzyloxycarbonyl (Cbz), tert-butyloxycarbonyl (Boc), benzyl (Bn), p-methoxybenzyl (PMB), allyl, triphenylmethyl (Tr), di-p-methoxytrityl (DMTr), methoxymethyl (MOM), phenoxymethyl (BOM), 2,2,2-trichloroethoxymethyl, 2-methoxyethoxymethyl (MEM), methylthiomethyl (MTM), p-methoxybenzyloxymethyl (PMBM), -C(O)CH2CH2C(O)OH or 4,4'-dimethoxytrityl, preferably DMTr.

16. The compound of any one of claims 12 to 15, wherein the compound of formula (I') is selected from the compound of formula (II') or (III'), or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof: in, Base' is selected from The other variables are as defined in any one of claims 12-15. Preferably, the compound of formula (II') or (III') is selected from the following compounds, or pharmaceutically acceptable salts, tautomers or stereoisomers thereof:

17. A double-stranded RNA having a sense strand and an antisense strand, each strand having 14 to 30 nucleotides, wherein the antisense strand comprises a sequence that is sufficiently complementary to the sense strand and the target mRNA, wherein the sense strand and / or the antisense strand comprises one or more compounds of formula (I) as described in any one of claims 1 to 6, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof: in, The variables are as defined in any one of claims 1-5.

18. The double-stranded RNA of claim 17, wherein the compound of formula (I) is selected from a compound of formula (II) or formula (III), or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof: in, Each variable is as defined in any one of claims 1 to 5; Preferably, the compound of formula (I) is selected from the following compounds, or pharmaceutically acceptable salts, tautomers or stereoisomers thereof: represents H, or represents the position of phosphate or phosphorothioate linkage to the adjacent nucleotide; R1 represents H, or represents the position of linkage to the phosphate or phosphorothioate of another adjacent nucleotide; and R1 are not H at the same time.

19. The double-stranded RNA molecule of claim 17 or 18, wherein the nucleotides at positions 2 and 14 at the 5' end of the antisense strand are not compounds of formula (I); Preferably, One or more of the following sites are compounds of formula (I): nucleotides 4, 6, 7, 9, 10, 13, 15, and 17 at the 5′ end of the sense strand; nucleotides 1, 4, 5, 6, 7, 12, 16, 18, 20, and 21 at the 5′ end of the antisense strand; Preferably, One or more of the following sites are compounds of formula (I): nucleotides 4, 6, 7, 9, 10, 13, 15, and 17 at the 5′ end of the sense strand; Nucleotides 1, 4, 6, 7, 16, 18, 20, and 21 at the 5' end of the antisense strand.

20. The double-stranded RNA molecule according to any one of claims 17 to 19, wherein the compound of formula (I) is located at position 2-8, preferably position 5-7, such as position 5, position 6 or position 7, more preferably position 5 or position 7, of the 5' end of the antisense strand.

21. The double-stranded RNA molecule of any one of claims 17-20, wherein the double-stranded RNA exhibits reduced off-target toxicity compared to a compound of formula (I) having the same sequence but not comprising any one of claims 1-7.

22. The double-stranded RNA molecule according to any one of claims 17 to 21, wherein the antisense strand has a sequence that is sufficiently complementary to the sense strand and the target mRNA, and has the ability to induce degradation of the target mRNA.

23. A pharmaceutical composition comprising the double-stranded RNA molecule according to any one of claims 17 to 22, and a pharmaceutically acceptable carrier or excipient.

24. A kit comprising the double-stranded RNA molecule according to any one of claims 17 to 22.

25. A cell comprising the double-stranded RNA molecule of any one of claims 17 to 22.

26. A method for inhibiting the expression of a target gene in a cell, comprising the step of introducing the double-stranded RNA molecule according to any one of claims 17 to 22 into the cell.

27. A method for inhibiting the expression of a target gene in a cell, comprising expressing the double-stranded RNA molecule of any one of claims 17 to 22 in the cell.

28. A method for reducing off-target toxicity in a cell, comprising the step of introducing the double-stranded RNA molecule of any one of claims 17 to 22 into the cell.

29. A method for reducing off-target toxicity in a cell, comprising expressing the double-stranded RNA molecule of any one of claims 17-22 in the cell.