Cyclic phosphonate modified nucleotides

Abstract

The present invention provides oligonucleotides with cyclic phosphonate modifications. The oligonucleotides of the invention exhibit one or more of enhanced stability, reduced off-target toxicity, and enhanced effectiveness.

Description

Cyclic phosphonate-modified nucleotides Field of the Invention

[0001] The present invention belongs to the field of medicine, and in particular relates to double-stranded RNA with a cyclic phosphonate structure. Background Art

[0002] RNA interference is a phenomenon in which double-stranded RNA (dsRNA, also known as siRNA) induces the efficient and specific degradation of target mRNA.

[0003] The 5'-phosphate of the guide strand (antisense strand) in siRNA stabilizes the complex formed by the guide strand and Ago2 by forming electrostatic interactions with cationic amino acid residues near the interface of the Ago2 protein MID and PIWI domain. Therefore, the 5'-phosphate of the siRNA guide strand is crucial for RNAi-based gene silencing. However, the 5'-phosphate can be cleaved by endogenous phosphatases, resulting in the loss of the 5' terminal phosphate group and a significant reduction in siRNA activity.

[0004] By introducing phosphonate-modified nucleotides at the end of small interfering RNA (siRNA), particularly at the 5' end of the antisense strand, the in vivo activity of siRNA can be significantly improved. For example, WO2011139702A2 and WO2013033230A1 disclose a nucleotide containing 5'-vinylphosphonate (abbreviated as E-VP or VP). 5'-E-VP can replace 5'-phosphate to bind to Ago2 and is highly resistant to endogenous phosphatases, thereby improving the activity and / or stability of siRNA.

[0005] WO2017214112A1 discloses a nucleotide comprising a 5'-cyclic-phosphonate.

[0006] However, the inventors found that 5'-E-VP is connected to the sugar ring through a CC single bond, and the CC single bond can still rotate freely, resulting in the phosphonic acid group being in different positions relative to the sugar ring (see the figure below, where the ap position is the conformation of the unmodified 5'-phosphate bound to the Ago2 protein).

[0007] A considerable portion of these conformations (e.g., +sc and -sc) may be unfavorable for binding to the Ago2 cationic region. Therefore, there is a need in the art to develop phosphonate modifications that can lock the Ago2 binding conformation.

[0008] Summary of the Invention

[0009] The inventors unexpectedly discovered that restricting the rotation of the terminal phosphonic acid through a rigid ring structure and locking it in the Ago2-binding conformation is beneficial to further improve the activity of siRNA.

[0010] In one aspect, the present invention relates to an oligonucleotide or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, wherein the 5' end of the oligonucleotide comprises a structure represented by formula (I):

[0011] in,

[0012] represents linkage to the remainder of the oligonucleotide, preferably to a modified or unmodified nucleoside;

[0013] X and Y are each independently selected from CR a R b or (CR a R b )2, preferably CR a R b ;

[0014] Z is CR a ;

[0015] R a and R b Independently selected from H, D, halogen, OH, NH2, CN, C 1-6 Alkyl, C 1-6 Halogenated alkyl, C 1-6 Alkoxy, C 1-6 Halogenated alkoxy, C 2-6 Alkenyl or C 2-6 Alkynyl; the R a and R b is optionally further substituted with 1, 2, 3, 4 or 5 independently selected R#;

[0016] R1 is selected from O or S;

[0017] R2 and R3 are independently selected from OH, SH, NH2, C 1-6 Alkyl, C 1-6 Halogenated alkyl, C 1-6 Alkoxy or C 1-6 Haloalkoxy; said R2 and R3 are optionally further substituted by 1, 2, 3, 4 or 5 independently selected R#;

[0018] R# is selected from H, D, halogen, C 1-6 Alkyl, C 1-6 Halogenated alkyl, C 2-6 Alkenyl or C 2-6 Alkynyl;

[0019] The above groups are optionally substituted by 1, 2, 3, 4, 5 or more deuterium atoms, up to full deuteration.

[0020] Without being bound by a particular theory, the structure of the present invention contains a four-membered carbon ring. This relatively rigid structure enables the oligonucleotide of the present invention (especially the phosphonate group at its 5' end) to better bind to the Ago2 protein, thereby improving the activity of the oligonucleotide.

[0021] Through molecular modeling, we observed that the compounds of the present invention can lock the phosphonic acid in an ap conformation that is highly similar to the unmodified 5'-phosphate bound to Ago2. The phosphonate group perfectly matches Arg812, Lys570, Lys566, and Lys533, forming multiple salt bridges and hydrogen bonds. It also forms a hydrogen bond with Tyr529 and Cys526. In addition, the base (uracil) in the spirocyclic phosphonic acid compound forms a hydrogen bond with the backbone amide of Thr526 and Gly524. These structural factors together stabilize the binding of the compounds of the present invention to the Ago2 protein.

[0022] Molecular simulations show that, similar to spiro compounds, after the sugar ring is fused with a highly rigid aromatic ring (including a five-membered aromatic ring and a six-membered aromatic ring), the 5'-terminal phosphonate is locked in a specific conformation that highly mimics the ap conformation of the natural 5'-phosphate. The phosphonate group perfectly matches Arg812, Lys570, Lys566, and Lys533, forming multiple salt bridge bonds and hydrogen bonds. It also forms a hydrogen bond with Tyr529 and Cys526. The base uracil on the sugar ring also forms a hydrogen bond with the backbone amide of Thr526 and Gly524. These structures together stabilize the binding of the compounds of the present invention to the Ago2 protein.

[0023] Detailed Description of the Invention

[0024] definition

[0025] Chemical definition

[0026] Definitions of specific functional groups and chemical terms are described in more detail below.

[0027] When a numerical range is listed, it is intended to include every value and sub-range within the stated range. For example, "C 1-6 "Alkyl" includes C1, C2, C3, C4, C5, C6, C 1-6 、C 1-5 、C 1-4 、C 1-3 、C 1-2 、C 2-6 、C 2-5 、C 2-4 、C 2-3 、C 3-6 、C 3-5 、C3-4 、C 4-6 、C 4-5 and C 5-6 alkyl.

[0028] “C 1-6 "Alkyl" refers to a straight or branched chain saturated hydrocarbon group having 1 to 6 carbon atoms. In some embodiments, C 1-4 Alkyl and C 1-2 Alkyl groups are preferred. 1-6 Examples of alkyl groups include: methyl (C1), ethyl (C2), n-propyl (C3), isopropyl (C3), n-butyl (C4), tert-butyl (C4), sec-butyl (C4), isobutyl (C4), n-pentyl (C5), 3-pentyl (C5), pentyl (C5), neopentyl (C5), 3-methyl-2-butyl (C5), tert-pentyl (C5), and n-hexyl (C6). The term “C 1-6 "Alkyl" also includes heteroalkyl groups in which one or more (e.g., 1, 2, 3, or 4) carbon atoms are replaced by heteroatoms (e.g., oxygen, sulfur, nitrogen, boron, silicon, phosphorus). The alkyl group may be optionally substituted 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).

[0029] “C 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 2-4 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.

[0030] “C 2-6 "Alkynyl" refers to a straight or branched chain hydrocarbon group having 2 to 6 carbon atoms, at least one carbon-carbon triple bond, and optionally one or more carbon-carbon double bonds. In some embodiments, C 2-4 Alkynyl is preferred. 2-6Examples of alkynyl groups include, but are not limited to, ethynyl (C2), 1-propynyl (C3), 2-propynyl (C3), 1-butynyl (C4), 2-butynyl (C4), 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.

[0031] "Halo" or "halogen" refers to fluorine (F), chlorine (Cl), bromine (Br) and iodine (I).

[0032] 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.

[0033] “C 1-6 "Alkoxy" refers to a -OR group, wherein R is as described above in "C 1-6 "alkyl" is defined.

[0034] “C 1-6 "Haloalkoxy" refers to a -OR group, wherein R is as described above in "C 1-6 "Haloalkyl" is defined.

[0035] “C 5-6 "Cycloalkyl" refers to a non-aromatic cyclic hydrocarbon group having 5 to 6 ring carbon atoms and zero heteroatoms. Cycloalkyl also includes ring systems in which the above-mentioned cycloalkyl ring is fused to one or more aryl or heteroaryl groups, wherein the point of attachment is on the cycloalkyl ring, and in such cases, the number of carbons continues to refer to the number of carbons in the cycloalkyl system. Exemplary cycloalkyl groups include, but are not limited to, cyclopentyl (C5), cyclopentenyl (C5), cyclohexyl (C6), cyclohexenyl (C6), cyclohexadienyl (C6), and the like. Cycloalkyl groups can be optionally substituted with one or more substituents, for example, with 1 to 5 substituents, 1 to 3 substituents, or 1 substituent.

[0036] "5-6 membered heterocyclyl" refers to a radical of a 5- to 6-membered non-aromatic ring system having ring carbon atoms and 1 to 3 ring heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen, sulfur, boron, phosphorus, and silicon. In heterocyclyl groups containing one or more nitrogen atoms, the point of attachment may be a carbon or nitrogen atom, as valence permits. Heterocyclyl also includes ring systems in which the above-mentioned heterocyclyl ring is fused to one or more cycloalkyl groups, where the point of attachment is on the cycloalkyl ring, or ring systems in which the above-mentioned heterocyclyl ring is fused to one or more aryl or heteroaryl groups, where the point of attachment is on the heterocyclyl ring; and in such cases, the number of ring members continues to represent the number of ring members in the heterocyclyl ring system. Exemplary 5-membered heterocyclyl groups containing one heteroatom include, but are not limited to, tetrahydrofuranyl, dihydrofuranyl, tetrahydrothienyl, dihydrothienyl, pyrrolidinyl, dihydropyrrolyl, and pyrrolyl-2,5-dione. Exemplary 5-membered heterocyclic groups containing two heteroatoms include, but are not limited to, dioxolanyl, oxasulfuranyl, disulfuranyl, and oxazolidin-2-one. Exemplary 5-membered heterocyclic groups containing three heteroatoms include, but are not limited to, triazolinyl, oxadiazolinyl, and thiadiazolinyl. Exemplary 6-membered heterocyclic groups containing one heteroatom include, but are not limited to, piperidinyl, tetrahydropyranyl, dihydropyridinyl, and thianyl. Exemplary 6-membered heterocyclic groups containing two heteroatoms include, but are not limited to, piperazinyl, morpholinyl, dithianyl, and dioxanyl. Exemplary 6-membered heterocyclic groups containing three heteroatoms include, but are not limited to, hexahydrotriazinyl. The heterocyclic group may be optionally substituted with one or more substituents, for example, with 1 to 5 substituents, 1 to 3 substituents, or 1 substituent.

[0037] Alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, etc., as defined herein, are optionally substituted groups.

[0038] 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)Raa 、-OCO2R aa 、-C(=O)N(R bb )2、-OC(=O)N(R bb )2、-NR bb C(=O)R aa 、-NR bb CO2R aa 、-NR bb C(=O)N(R bb )2、-C(=NR bb )R aa 、-C(=NR bb )OR aa 、-OC(=NR bb )R aa 、-OC(=NR bb )OR aa 、-C(=NR bb )N(R bb )2、-OC(=NR bb )N(R bb )2、-NR bb C(=NR bb )N(R bb )2、-C(=O)NR bb SO2R aa 、-NR bb SO2R aa 、-SO2N(R bb )2、-SO2R aa 、-SO2OR aa 、-OSO2R aa 、-S(=O)R aa 、-OS(=O)R aa 、-Si(R aa )3、-OSi(R aa )3、-C(=S)N(R bb )2、-C(=O)SR aa 、-C(=S)SR aa 、-SC(=S)SR aa 、-SC(=O)SR aa 、-OC(=O)SR aa 、-SC(=O)OR aa 、-SC(=O)R aa 、-P(=O)2R aa 、-OP(=O)2R aa 、-P(=O)(R aa )2、-OP(=O)(R aa )2、-OP(=O)(ORcc )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;

[0039] Or the two geminal hydrogen atoms on the carbon atom are replaced by groups =O, =S, =NN(R bb )2, =NNR bb C(=O)R aa 、=NNR bb C(=O)OR aa 、=NNR bb S(=O)2R aa 、=NR bb or = NOR cc replace;

[0040] 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;

[0041] 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;

[0042] R cc Each of R is independently selected from hydrogen, alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl and heteroaryl, or two R cc The groups are combined to form a heterocyclyl or heteroaryl ring, wherein each alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl and heteroaryl group is independently replaced by 0, 1, 2, 3, 4 or 5 R dd group substitution;

[0043] 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(Rff )2、-OC(=O)N(R ff )2、-NR ff C(=O)R ee 、-NR ff CO2R ee 、-NR ff C(=O)N(R ff )2、-C(=NR ff )OR ee 、-OC(=NR ff )R ee 、-OC(=NR ff )OR ee 、-C(=NR ff )N(R ff )2、-OC(=NR ff )N(R ff )2、-NR ff C(=NR ff )N(R ff )2、-NR ff SO2R ee 、-SO2N(R ff )2, -SO2R ee 、-SO2OR ee 、-OSO2R ee 、-S(=O)R ee 、-Si(R ee )3、-OSi(R ee )3、-C(=S)N(R ff )2, -C(=O)SR ee 、-C(=S)SR ee 、-SC(=S)SR ee 、-P(=O)2R ee 、-P(=O)(R ee )2、-OP(=O)(R ee )2、-OP(=O)(OR 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;

[0044] R eeEach 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;

[0045] 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;

[0046] 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)(C 1-6 Alkyl), -N(OH)(C 1-6 Alkyl), -NH(OH), -SH, -SC 1-6 Alkyl, -SS(C 1-6 alkyl), -C(=O)(C 1-6 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(C1-6 alkyl)2, -NHC(=O)NH(C 1-6 alkyl), -NHC(=O)NH2, -C(=NH)O(C 1-6 alkyl), -OC(=NH)(C 1-6 alkyl), -OC(=NH)OC 1-6 Alkyl, -C(=NH)N(C 1-6 Alkyl)2, -C(=NH)NH(C 1-6 alkyl), -C(=NH)NH2, -OC(=NH)N(C 1-6 Alkyl)2, -OC(NH)NH(C 1-6 alkyl), -OC(NH)NH2, -NHC(NH)N(C 1-6 Alkyl)2, -NHC(=NH)NH2, -NHSO2(C 1-6 Alkyl), -SO2N(C 1-6 Alkyl)2, -SO2NH(C 1-6 alkyl), -SO2NH2, -SO2C 1-6 Alkyl, -SO2OC 1-6 Alkyl, -OSO2C 1-6 Alkyl, -SOC 1-6 Alkyl, -Si(C 1-6 alkyl)3, -OSi(C 1-6 alkyl)3, -C(=S)N(C 1-6 alkyl)2、C(=S)NH(C 1-6 alkyl), C(=S)NH2, -C(=O)S(C 1-6 alkyl), -C(=S)SC 1-6 Alkyl, -SC(=S)SC 1-6 Alkyl, -P(=O)2(C 1-6 alkyl), -P(=O)(C 1-6 alkyl)2, -OP(=O)(C 1-6 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.

[0047] Exemplary substituents on nitrogen atoms include, but are not limited to, hydrogen, -OH, -ORaa 、-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 、R bb 、R cc and R dd As mentioned above.

[0048] Other definitions

[0049] 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.

[0050] 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.

[0051] 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.

[0052] 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.

[0053] A polynucleotide that is "at least partially complementary," "sufficiently complementary," or "substantially 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," "sufficiently 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.

[0054] "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.

[0055] "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.

[0056] "Nucleoside" is a compound composed of two substances: a purine base or a pyrimidine base and a ribose or deoxyribose sugar. "Nucleotide" is a compound composed of three substances: a purine base or a pyrimidine base, a ribose or deoxyribose sugar and a phosphate. "Oligonucleotide" refers to a nucleic acid molecule (RNA or DNA) with a length of, for example, less than 100, 200, 300 or 400 nucleotides, which can be single-stranded or double-stranded.

[0057] "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.

[0058] 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.

[0059] " 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.

[0060] " 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).

[0061] 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.

[0062] 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).

[0063] 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:

[0064] 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.

[0065] 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.

[0066] 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.

[0067] 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.

[0068] 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.

[0069] Compounds of the present invention

[0070] The present invention relates to an oligonucleotide or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, wherein the 5' end of the oligonucleotide comprises a structure represented by formula (I):

[0071] wherein each group is as defined above and below.

[0072] The present invention also relates to an oligonucleotide or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, wherein the 5' end of the oligonucleotide comprises a structure represented by formula (II):

[0073] wherein each group is as defined above and below.

[0074] The present invention also relates to an oligonucleotide or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, wherein the 5' end of the oligonucleotide comprises a nucleotide monomer represented by formula (III), formula (IV) or formula (V):

[0075] wherein each group is as defined above and below.

[0076] The present invention also relates to compounds represented by formula (VI), formula (VII) or formula (VIII) or pharmaceutically acceptable salts, tautomers or stereoisomers thereof:

[0077] wherein each group is as defined above and below.

[0078] In one embodiment, represents the remainder of the oligonucleotide; in another embodiment, Indicates attachment to modified or unmodified nucleosides.

[0079] X, Y, and Z

[0080] In one embodiment, X is CR a R b , preferably CH2; in another embodiment, X is (CR a R b )2.

[0081] In one embodiment, Y is CR a R b , preferably CH2; in another embodiment, Y is (CR a R b )2.

[0082] In one embodiment, Z is CR a , preferably CH.

[0083] wherein X and Y are optionally substituted by 1 or 2 deuterium atoms, and Z is optionally substituted by 1 deuterium atom.

[0084] L1, L2, and L3

[0085] In one embodiment, L1 is a chemical bond; in another embodiment, L1 is O; in another embodiment, L1 is S; in another embodiment, L1 is C 1-4 Alkylene.

[0086] In one embodiment, L2 is C 1-4 Alkylene, preferably C1-2 Alkylene.

[0087] In one embodiment, L3 is C 1-4 Alkylene, preferably C 1-2 Alkylene.

[0088] In one embodiment, L2 and L3 are linked to form a C 5-6 In another embodiment, L2 and L3 are linked to form a 5-6 membered heterocyclic group together with L1 and its adjacent carbon atoms, preferably U, L1, L2 and L3 and their adjacent carbon atoms together form ribose or deoxyribose, which is optionally substituted by R5.

[0089] In one embodiment, L1 is unsubstituted; in another embodiment, L1 is further substituted with 1 R#; in another embodiment, L1 is further substituted with 2 independently selected R#; in another embodiment, L1 is further substituted with 3 independently selected R#; in another embodiment, L1 is further substituted with 4 independently selected R#; in another embodiment, L1 is further substituted with 5 independently selected R#.

[0090] In one embodiment, L2 is unsubstituted; in another embodiment, L2 is further substituted with 1 R#; in another embodiment, L2 is further substituted with 2 independently selected R#; in another embodiment, L2 is further substituted with 3 independently selected R#; in another embodiment, L2 is further substituted with 4 independently selected R#; in another embodiment, L2 is further substituted with 5 independently selected R#.

[0091] In one embodiment, L3 is unsubstituted; in another embodiment, L3 is further substituted with 1 R#; in another embodiment, L3 is further substituted with 2 independently selected R#; in another embodiment, L3 is further substituted with 3 independently selected R#; in another embodiment, L3 is further substituted with 4 independently selected R#; in another embodiment, L3 is further substituted with 5 independently selected R#.

[0092] wherein the above L1, L2 and L3 are optionally substituted by 1, 2, 3, 4, 5 or more deuterium atoms, until fully deuterated.

[0093] U

[0094] In one embodiment, U is O; in another embodiment, U is S; in another embodiment, U is CR a R b , such as CH2; in another embodiment, U is NRc , such as NH.

[0095] wherein the above U is optionally substituted by one or more deuterium atoms until it is fully deuterated.

[0096] Q

[0097] In one embodiment, Q is H; in another embodiment, Q is D; in another embodiment, Q is halogen; in another embodiment, Q is OH; in another embodiment, Q is C 1-6 Alkyl groups, such as C 1-4 Alkyl; in another embodiment, Q is C 1-6 Haloalkyl, such as C 1-4 haloalkyl; in another embodiment, Q is C 1-6 Alkoxy, such as C 1-4 Alkoxy; in another embodiment, Q is C 1-6 Haloalkoxy, such as C 1-4 In another embodiment, Q is C 2-6 Alkenyl, such as C 2-4 In another embodiment, Q is C 2-6 Alkynyl, such as C 2-4 Alkynyl.

[0098] wherein the above Q is optionally substituted by 1, 2, 3, 4, 5 or more deuterium atoms, up to full deuteration.

[0099] R1

[0100] In one embodiment, R1 is O; in another embodiment, R1 is S.

[0101] R2 and R3

[0102] In one embodiment, R2 is OH; in another embodiment, R2 is SH; in another embodiment, R2 is NH2; in another embodiment, R2 is C 1-6 Alkyl; in another embodiment, R2 is C 1-6 In another embodiment, R2 is C 1-6 Alkoxy; in another embodiment, R2 is C 1-6 Halogenated alkoxy.

[0103] In one embodiment, R2 is OR d ; In another embodiment, R2 is OP1; In another embodiment, R2 is SR d ; In another embodiment, R2 is SP1; In another embodiment, R2 is NRe R f .

[0104] In one embodiment, R2 is unsubstituted; in another embodiment, R2 is further substituted with 1 R#; in another embodiment, R2 is further substituted with 2 independently selected R#; in another embodiment, R2 is further substituted with 3 independently selected R#; in another embodiment, R2 is further substituted with 4 independently selected R#; in another embodiment, R2 is further substituted with 5 independently selected R#.

[0105] In one embodiment, R3 is OH; in another embodiment, R3 is SH; in another embodiment, R3 is NH2; in another embodiment, R3 is C 1-6 Alkyl; in another embodiment, R3 is C 1-6 In another embodiment, R3 is C 1-6 Alkoxy; in another embodiment, R3 is C 1-6 Halogenated alkoxy.

[0106] In one embodiment, R3 is OR d ; In another embodiment, R3 is OP1; In another embodiment, R3 is SR d ; In another embodiment, R3 is SP1; In another embodiment, R3 is NR e R f .

[0107] In one embodiment, R3 is unsubstituted; in another embodiment, R3 is further substituted with 1 R#; in another embodiment, R3 is further substituted with 2 independently selected R#; in another embodiment, R3 is further substituted with 3 independently selected R#; in another embodiment, R3 is further substituted with 4 independently selected R#; in another embodiment, R3 is further substituted with 5 independently selected R#.

[0108] wherein the above R2 and R3 are optionally substituted by 1, 2, 3, 4, 5 or more deuterium atoms, until fully deuterated.

[0109] R4, R5, R6, and R7

[0110] In one embodiment, R4 is H; in another embodiment, R4 is D; in another embodiment, R4 is halogen; in another embodiment, R4 is OH; in another embodiment, R4 is C 1-6 Alkyl groups, such as C 1-4 Alkyl; in another embodiment, R4 is C1-6 Haloalkyl, such as C 1-4 In another embodiment, R4 is C 1-6 Alkoxy, such as C 1-4 Alkoxy; in another embodiment, R4 is C 1-6 Haloalkoxy, such as C 1-4 Halogenated alkoxy.

[0111] In one embodiment, R5 is H; in another embodiment, R5 is D; in another embodiment, R5 is halogen; in another embodiment, R5 is OH; in another embodiment, R5 is C 1-6 Alkyl groups, such as C 1-4 Alkyl; in another embodiment, R5 is C 1-6 Haloalkyl, such as C 1-4 In another embodiment, R5 is C 1-6 Alkoxy, such as C 1-4 Alkoxy; in another embodiment, R5 is C 1-6 Haloalkoxy, such as C 1-4 Halogenated alkoxy.

[0112] In one embodiment, R6 is H; in another embodiment, R6 is D; in another embodiment, R6 is halogen; in another embodiment, R6 is OH; in another embodiment, R6 is C 1-6 Alkyl groups, such as C 1-4 Alkyl; in another embodiment, R6 is C 1-6 Haloalkyl, such as C 1-4 In another embodiment, R6 is C 1-6 Alkoxy, such as C 1-4 Alkoxy; in another embodiment, R6 is C 1-6 Haloalkoxy, such as C 1-4 Halogenated alkoxy.

[0113] In one embodiment, R7 is H; in another embodiment, R7 is D; in another embodiment, R7 is halogen; in another embodiment, R7 is OH; in another embodiment, R7 is C 1-6 Alkyl groups, such as C 1-4 Alkyl; in another embodiment, R7 is C 1-6 Haloalkyl, such as C 1-4 In another embodiment, R7 is C 1-6 Alkoxy, such as C 1-4 Alkoxy; in another embodiment, R7 is C 1-6Haloalkoxy, such as C 1-4 Halogenated alkoxy.

[0114] wherein the above R4, R5, R6 and R7 are optionally substituted by 1, 2, 3, 4, 5 or more deuterium atoms, until fully deuterated.

[0115] R a 、R b and R c

[0116] In one embodiment, R a is H; in another embodiment, R a is D; in another embodiment, R a is halogen; in another embodiment, R a is OH; in another embodiment, R a is NH2; in another embodiment, R a is CN; in another embodiment, R a C 1-6 Alkyl groups, such as C 1-4 Alkyl; in another embodiment, R a C 1-6 Haloalkyl, such as C 1-4 haloalkyl; in another embodiment, R a C 1-6 Alkoxy, such as C 1-4 Alkoxy; in another embodiment, R a C 1-6 Haloalkoxy, such as C 1-4 Haloalkoxy; in another embodiment, R a C 2-6 Alkenyl, such as C 2-4 alkenyl; in another embodiment, R a C 2-6 Alkynyl, such as C 2-4 Alkynyl.

[0117] In one embodiment, R a is unsubstituted; in another embodiment, R a is further substituted with 1 R#; in another embodiment, R a is further substituted with 2 independently selected R#; in another embodiment, R a is further substituted with 3 independently selected R#; in another embodiment, R a is further substituted with 4 independently selected R#; in another embodiment, R a Further replaced by 5 independently selected R#.

[0118] In one embodiment, R b is H; in another embodiment, R b is D; in another embodiment, R b is halogen; in another embodiment, R b is OH; in another embodiment, R b is NH2; in another embodiment, R b is CN; In another embodiment, R b C 1-6 Alkyl groups, such as C 1-4 Alkyl; in another embodiment, R b C 1-6 Haloalkyl, such as C 1-4 haloalkyl; in another embodiment, R b C 1-6 Alkoxy, such as C 1-4 Alkoxy; in another embodiment, R b C 1-6 Haloalkoxy, such as C 1-4 Haloalkoxy; in another embodiment, R b C 2-6 Alkenyl, such as C 2-4 alkenyl; in another embodiment, R b C 2-6 Alkynyl, such as C 2-4 Alkynyl.

[0119] In one embodiment, R b is unsubstituted; in another embodiment, R b is further substituted with 1 R#; in another embodiment, R b is further substituted with 2 independently selected R#; in another embodiment, R b is further substituted with 3 independently selected R#; in another embodiment, R b is further substituted with 4 independently selected R#; in another embodiment, R b Further replaced by 5 independently selected R#.

[0120] In one embodiment, R c is H; in another embodiment, R c C 1-6 Alkyl; in another embodiment, R c C 1-6 haloalkyl; in another embodiment, R c C 2-6alkenyl; in another embodiment, R c C 2-6 Alkynyl.

[0121] The above R a 、R b and R c Optionally substituted with 1, 2, 3, 4, 5 or more deuterium atoms, up to full deuteration.

[0122] R d 、R e and R f

[0123] In one embodiment, R d is H; in another embodiment, R d C 1-6 Alkyl groups, such as C 1-4 Alkyl; in another embodiment, R d C 1-6 Haloalkyl, such as C 1-4 haloalkyl; in another embodiment, R d C 2-6 alkenyl; in another embodiment, R d C 2-6 Alkynyl.

[0124] In one embodiment, R d is unsubstituted; in another embodiment, R d is optionally substituted with one or more Ds, up to full deuteration; in another embodiment, R d is substituted with halogen; in another embodiment, R d C 1-6 Alkyl substituted; in another embodiment, R d C 1-6 Haloalkyl substitution.

[0125] In one embodiment, R e is H; in another embodiment, R e C 1-6 Alkyl groups, such as C 1-4 Alkyl; in another embodiment, R e C 1-6 Haloalkyl, such as C 1-4 Halogenated alkyl.

[0126] In one embodiment, R e is unsubstituted; in another embodiment, R e is optionally substituted with one or more Ds, up to full deuteration; in another embodiment, Re is substituted with halogen; in another embodiment, R e C 1-6 Alkyl substituted; in another embodiment, R e C 1-6 Haloalkyl substitution.

[0127] In one embodiment, R f is H; in another embodiment, R f C 1-6 Alkyl groups, such as C 1-4 Alkyl; in another embodiment, R f C 1-6 Haloalkyl, such as C 1-4 Halogenated alkyl.

[0128] In one embodiment, R f is unsubstituted; in another embodiment, R f is optionally substituted with one or more Ds, up to full deuteration; in another embodiment, R f is substituted with halogen; in another embodiment, R f C 1-6 Alkyl substituted; in another embodiment, R f C 1-6 Haloalkyl substitution.

[0129] The above R d 、R e and R f Optionally substituted with 1, 2, 3, 4, 5 or more deuterium atoms, up to full deuteration.

[0130] P1 and P2

[0131] In one embodiment, P1 is a protecting group; in another embodiment, P1 is a hydroxy protecting group.

[0132] In one embodiment, P2 is a reactive phosphorus group, such as -P(OCH2CH2CN)(N(iPr)2).

[0133] Base and Base'

[0134] In one embodiment, Base is H; in another embodiment, Base is a modified or unmodified base or a leaving group; in another embodiment, Base is selected from modified or unmodified A, U, T, G and C, for example

[0135] In one embodiment, Base' is H; in another embodiment, Base' is a modified or unmodified base or a leaving group; in another embodiment, Base' is selected from modified or unmodified A, U, T, G and C, for example

[0136] R#

[0137] 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 1-6 In another embodiment, R# is C 2-6 In another embodiment, R# is C 2-6 Alkynyl;

[0138] wherein the above R# is optionally substituted by 1, 2, 3, 4, 5 or more deuterium atoms, until fully deuterated.

[0139] GalNAc

[0140] In one embodiment, GalNAc is a conjugated group represented by formula (X):

[0141] in,

[0142] Indicates the location of attachment to a biomolecule;

[0143] Q G Independently H,

[0144] Among them L G1 is a chemical bond, -CH2-, -CH2CH2-, -C(O)-, -CH2O-, -CH2O-CH2CH2O-, or -NHC(O)-(CH2NHC(O)) a -;

[0145] L G2 is a chemical bond or -CH2CH2C(O)-;

[0146] L G3 For chemical bonds, -(NHCH2CH2) b -、-(NHCH2CH2CH2) b - or -C(O)CH2-;

[0147] L G4 -(OCH2CH2)c -、-(OCH2CH2CH2) c -、-(OCH2CH2CH2CH2) c -、-(OCH2CH2CH2CH2CH2) c -or-NHC(O)-(CH2) d -;

[0148] Where a = 0, 1, 2 or 3;

[0149] b = 1, 2, 3, 4, or 5;

[0150] c = 1, 2, 3, 4, or 5;

[0151] d = 1, 2, 3, 4, 5, 6, 7, or 8;

[0152] A is a chemical bond, -CH2O- or -NHC(O)-;

[0153] A' is a chemical bond, -C(O)NH-, -NHC(O)- or -O(CH2CH2O) e -;

[0154] Where e is 1, 2, 3, 4 or 5;

[0155] B is a chemical bond, -CH2-, -C(O)-, -M-, -CH2-M- or -C(O)-M-;

[0156] Where M is

[0157] R G1 and R G2 Together to form -CH2CH2O- or -CH2CH(R G )-O-, and R G3 is H;

[0158] or R G1 and R G3 Together they form -C 1-2 Alkylene-, and R G2 is H;

[0159] where R G For-OR G ', -CH2OR G 'or-CH2CH2OR G ', where R G ' is H, a hydroxyl protecting group or a solid phase support, wherein the hydroxyl protecting group is preferably -C(O)CH2CH2C(O)OH or 4,4'-dimethoxytrityl;

[0160] m1 = 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

[0161] n1=0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0162] In another embodiment, GalNAc is a conjugate group represented by formula (I'):

[0163] in,

[0164] Indicates the location of attachment to a biomolecule;

[0165] Q G Independently H,

[0166] Among them L G1 is a chemical bond, -CH2-, -CH2CH2-, -C(O)-, -CH2O-, -CH2O-CH2CH2O-, or -NHC(O)-(CH2NHC(O)) a -;

[0167] L G2 is a chemical bond or -CH2CH2C(O)-;

[0168] L G3 For chemical bonds, -(NHCH2CH2) b -、-(NHCH2CH2CH2) b - or -C(O)CH2-;

[0169] L G4 -(OCH2CH2) c -、-(OCH2CH2CH2) c -、-(OCH2CH2CH2CH2) c -、-(OCH2CH2CH2CH2CH2) c -or-NHC(O)-(CH2) d -;

[0170] Where a = 0, 1, 2 or 3;

[0171] b = 1, 2, 3, 4, or 5;

[0172] c = 1, 2, 3, 4, or 5;

[0173] d = 1, 2, 3, 4, 5, 6, 7, or 8;

[0174] A is -CH2O- or -NHC(O)-;

[0175] A' is a chemical bond, -C(O)NH- or -NHC(O)-;

[0176] R G1 and R G2 Together to form -CH2CH2O- or -CH2CH(R G )-O-, and R G3 is H;

[0177] or R G1 and R G3 Together they form -C 1-2 Alkylene-, and R G2 is H;

[0178] where R G For-OR G ', -CH2OR G 'or-CH2CH2OR G ', where R G ' is H, a hydroxyl protecting group or a solid phase support, wherein the hydroxyl protecting group is preferably -C(O)CH2CH2C(O)OH or 4,4'-dimethoxytrityl;

[0179] m1 = 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

[0180] n1=0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0181] In another embodiment, GalNAc is a conjugated group represented by formula (X), wherein,

[0182] Q G Independently H,

[0183] Among them L G1 is a chemical bond, -CH2-, -CH2CH2-, -C(O)-, -CH2O-, -CH2O-CH2CH2O-, or -NHC(O)-(CH2NHC(O)) a -;

[0184] L G2 is a chemical bond or -CH2CH2C(O)-;

[0185] L G3 For chemical bonds, -(NHCH2CH2) b -、-(NHCH2CH2CH2) b - or -C(O)CH2-;

[0186] LG4 -(OCH2CH2) c -、-(OCH2CH2CH2) c -、-(OCH2CH2CH2CH2) c -、-(OCH2CH2CH2CH2CH2) c -or-NHC(O)-(CH2) d -;

[0187] Where a = 0, 1, 2 or 3;

[0188] b = 1, 2, 3, 4, or 5;

[0189] c = 1, 2, 3, 4, or 5;

[0190] d = 1, 2, 3, 4, 5, 6, 7, or 8;

[0191] A is a chemical bond, -CH2O- or -NHC(O)-;

[0192] A' is a chemical bond, -C(O)NH-, -NHC(O)- or -O(CH2CH2O) e -;

[0193] Where e is 1, 2, 3, 4 or 5;

[0194] B is a chemical bond, -CH2-, -M-, -CH2-M- or -C(O)-M-;

[0195] Where M is

[0196] R G1 and R G2 Together to form -CH2CH2O- or -CH2CH(R G )-O-, and R G3 is H;

[0197] or R G1 and R G3 Together they form -C 1-2 Alkylene-, and R G2 is H;

[0198] where R G For-OR G ', -CH2OR G 'or-CH2CH2OR G ', where R G ' is H, a hydroxyl protecting group or a solid phase support, wherein the hydroxyl protecting group is preferably -C(O)CH2CH2C(O)OH or 4,4'-dimethoxytrityl;

[0199] m1 = 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

[0200] n1=0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0201] In another embodiment, GalNAc is a conjugated group represented by formula (X), wherein:

[0202] Q G Independently H,

[0203] Among them L G1 is a chemical bond, -CH2-, -CH2CH2-, -C(O)-, -CH2O-, -CH2O-CH2CH2O-, or -NHC(O)-(CH2NHC(O)) a -;

[0204] L G2 is a chemical bond or -CH2CH2C(O)-;

[0205] L G3 For chemical bonds, -(NHCH2CH2) b -、-(NHCH2CH2CH2) b - or -C(O)CH2-;

[0206] L G4 -(OCH2CH2) c -、-(OCH2CH2CH2) c -、-(OCH2CH2CH2CH2) c -、-(OCH2CH2CH2CH2CH2) c -or-NHC(O)-(CH2) d -;

[0207] Where a = 0, 1, 2 or 3;

[0208] b = 1, 2, 3, 4, or 5;

[0209] c = 1, 2, 3, 4, or 5;

[0210] d = 1, 2, 3, 4, 5, 6, 7, or 8;

[0211] A is a chemical bond, -CH2O- or -NHC(O)-;

[0212] A' is -O(CH2CH2O) e -;

[0213] Where e is 1, 2, 3, 4 or 5;

[0214] B is a chemical bond, -CH2-, -C(O)-, -M-, -CH2-M- or -C(O)-M-;

[0215] Where M is

[0216] R G1 and R G2 Together to form -CH2CH2O- or -CH2CH(R G )-O-, and R G3 is H;

[0217] or R G1 and R G3 Together they form -C 1-2 Alkylene-, and R G2 is H;

[0218] where R G For-OR G ', -CH2OR G 'or-CH2CH2OR G ', where R G ' is H, a hydroxyl protecting group or a solid phase support, wherein the hydroxyl protecting group is preferably -C(O)CH2CH2C(O)OH or 4,4'-dimethoxytrityl;

[0219] m1 = 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

[0220] n1=0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0221] Any technical solution or any combination thereof in any of the above specific embodiments may be combined with any technical solution or any combination thereof in any other specific embodiment. For example, any technical solution or any combination thereof of X may be combined with any technical solution or any combination thereof of Y, Z, L1, L2, L3, R1, R2, R3, R4, R5, R6, R7, U, Q, P1, P2, Base, and Base', etc., or any combination thereof. The present invention is intended to include all such combinations of technical solutions, which are not listed here one by one due to space limitations.

[0222] 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.

[0223] 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.

[0224] 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.

[0225] 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.

[0226] In another aspect, the present invention relates to an oligonucleotide or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, wherein the 5' end of the oligonucleotide comprises a structure represented by the following formula:

[0227] in indicates the remainder of the oligonucleotide and Base is as defined above and below.

[0228] The present invention specifically relates to the following technical solutions:

[0229] 1. An oligonucleotide or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, wherein the 5' end of the oligonucleotide comprises a structure represented by formula (I):

[0230] in,

[0231] represents linkage to the remainder of the oligonucleotide, preferably to a modified or unmodified nucleoside;

[0232] X and Y are each independently selected from CR a R b or (CR a R b )2, preferably CR a R b ;

[0233] Z is CR a ;

[0234] R a and R b Independently selected from H, D, halogen, OH, NH2, CN, C 1-6 Alkyl, C 1-6 Halogenated alkyl, C 1-6 Alkoxy, C 1-6 Halogenated alkoxy, C 2-6 Alkenyl or C 2-6 Alkynyl; the R a and R b is optionally further substituted with 1, 2, 3, 4 or 5 independently selected R#;

[0235] R1 is selected from O or S;

[0236] R2 and R3 are independently selected from OH, SH, NH2, C 1-6 Alkyl, C 1-6 Halogenated alkyl, C 1-6 Alkoxy or C 1-6 Haloalkoxy; said R2 and R3 are optionally further substituted by 1, 2, 3, 4 or 5 independently selected R#;

[0237] R# is selected from H, D, halogen, C 1-6 Alkyl, C 1-6 Halogenated alkyl, C 2-6 Alkenyl or C 2-6 Alkynyl;

[0238] The above groups are optionally substituted by 1, 2, 3, 4, 5 or more deuterium atoms, up to full deuteration.

[0239] 2. The oligonucleotide of technical solution 1 or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, wherein:

[0240] represents linkage to the remainder of the oligonucleotide, preferably to a modified or unmodified nucleoside;

[0241] X and Y are each independently selected from CR a R b or (CR a R b )2, preferably CR a R b ;

[0242] Z is CR a ;

[0243] Ra and R b Independently selected from H, D, halogen, OH, NH2, CN, C 1-6 Alkyl, C 1-6 Halogenated alkyl, C 1-6 Alkoxy or C 1-6 haloalkoxy;

[0244] R1 is selected from O or S;

[0245] R2 and R3 are independently selected from OH, SH, C 1-6 Alkoxy or C 1-6 Haloalkoxy; said R2 and R3 are optionally further substituted by 1, 2 or 3 independently selected R#;

[0246] R# is selected from H, D, halogen, C 1-6 Alkyl and C 1-6 Halogenated alkyl.

[0247] 3. The oligonucleotide of technical solution 1 or 2 or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, wherein:

[0248] represents linkage to the remainder of the oligonucleotide, preferably to a modified or unmodified nucleoside;

[0249] X and Y are each independently selected from CR a R b or (CR a R b )2, preferably CR a R b ;

[0250] Z is CR a ;

[0251] R a and R b Independently selected from H, D, halogen, C 1-4 Alkyl or C 1-4 alkyl halide;

[0252] R1 is selected from O or S;

[0253] R2 and R3 are independently selected from OH or SH, preferably OH.

[0254] 4. The oligonucleotide of any one of technical solutions 1 to 3 or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, wherein:

[0255] represents a 5' terminal group attached to the oligonucleotide, preferably to a modified or unmodified nucleoside;

[0256] X and Y are CH2;

[0257] Z is CH;

[0258] R1 is O;

[0259] R2 and R3 are OH.

[0260] 5. The oligonucleotide of technical solution 1 or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, which has a structure of formula (II):

[0261] in,

[0262] represents the remainder of the oligonucleotide attached;

[0263] X and Y are CR a R b ;

[0264] Z is CR a ;

[0265] L1 is selected from a chemical bond, O, S or C 1-4 alkylene;

[0266] L2 and L3 are independently selected from C 1-4 alkylene;

[0267] The L1, L2 and L3 are optionally independently substituted with 1, 2, 3, 4 or 5 independently selected R#;

[0268] U is selected from O, S, CR a R b or NR c ;

[0269] R a and R b Independently selected from H, D, halogen, OH, NH2, CN, C 1-6 Alkyl, C 1-6 Halogenated alkyl, C 1-6 Alkoxy, C 1-6 Halogenated alkoxy, C 2-6 Alkenyl or C 2-6 Alkynyl;

[0270] R c Independently selected from H, C 1-6 Alkyl, C 1-6 Halogenated alkyl, C 2-6 Alkenyl or C 2-6 Alkynyl;

[0271] The R a 、Rb and R c is optionally further substituted with 1, 2, 3, 4 or 5 independently selected R#;

[0272] Alternatively, L2 and L3 are connected to form a C 5-6 Cycloalkyl or 5-6 membered heterocyclic group, preferably U, L1, L2 and L3 and their adjacent carbon atoms together form ribose or deoxyribose, which is optionally substituted by R5;

[0273] R1 is selected from O or S;

[0274] R2 and R3 are independently selected from OH, SH, NH2, C 1-6 Alkyl, C 1-6 Halogenated alkyl, C 1-6 Alkoxy or C 1-6 Haloalkoxy; said R2 and R3 are optionally substituted by 1, 2, 3, 4 or 5 independently selected R#;

[0275] R5 is selected from H, D, halogen, C 1-6 Alkoxy or C 1-6 haloalkoxy;

[0276] R# is selected from H, D, halogen, C 1-6 Alkyl, C 1-6 Halogenated alkyl, C 2-6 Alkenyl or C 2-6 Alkynyl;

[0277] Base is selected from H, modified or unmodified base;

[0278] The above groups are optionally substituted by 1, 2, 3, 4, 5 or more deuterium atoms, up to full deuteration.

[0279] 6. The oligonucleotide of technical solution 5 or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, wherein:

[0280] represents the remainder of the oligonucleotide attached;

[0281] X and Y are CR a R b ;

[0282] Z is CR a ;

[0283] R a and R b Independently selected from H, D, halogen, OH, NH2, CN, C 1-6 Alkyl, C 1-6 Halogenated alkyl, C1-6 Alkoxy or C 1-6 haloalkoxy;

[0284] L1 is selected from a chemical bond, O or C 1-4 alkylene;

[0285] L2 and L3 are independently selected from C 1-4 alkylene;

[0286] Said L1, L2 and L3 are optionally independently substituted with 1, 2 or 3 independently selected R#;

[0287] U is selected from O, S, NH or CH2;

[0288] Alternatively, L2 and L3 are connected to form a C 5-6 Cycloalkyl or 5-6 membered heterocyclic group, preferably U, L1, L2 and L3 and their adjacent carbon atoms together form ribose or deoxyribose, which is optionally substituted by R5;

[0289] R1 is selected from O or S;

[0290] R2 and R3 are independently selected from OH, SH, C 1-6 Alkoxy or C 1-6 Haloalkoxy; said R2 and R3 are optionally substituted by 1, 2 or 3 independently selected R#;

[0291] R5 is selected from H, D, C 1-6 Alkyl, C 1-6 Halogenated alkyl, C 1-6 Alkoxy or C 1-6 haloalkoxy;

[0292] R# is selected from H, D, halogen, C 1-6 Alkyl and C 1-6 alkyl halide;

[0293] Base is selected from H, modified or unmodified bases.

[0294] 7. The oligonucleotide of claim 5 or 6, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, wherein:

[0295] represents the remainder of the oligonucleotide attached;

[0296] X and Y are CR a R b ;

[0297] Z is CR a ;

[0298] R a and Rb Independently selected from H, D, halogen, C 1-4 Alkyl or C 1-4 alkyl halide;

[0299] L1 is selected from a chemical bond or C 1-4 alkylene;

[0300] L2 and L3 are independently selected from C 1-4 alkylene;

[0301] U is selected from O or S;

[0302] R1 is selected from O or S;

[0303] R2 and R3 are independently selected from OH or SH, preferably OH;

[0304] Base is selected from

[0305] 8. The oligonucleotide of any one of technical solutions 5-7 or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, wherein:

[0306] represents the remainder of the oligonucleotide attached;

[0307] X and Y are CH2;

[0308] Z is CH;

[0309] L1 is a chemical bond;

[0310] L2 and L3 are independently selected from C 1-2 alkylene;

[0311] U is O;

[0312] R1 is O;

[0313] R2 and R3 are OH;

[0314] Base is selected from

[0315] 9. The oligonucleotide of technical solution 1 or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, wherein the 5' end of the oligonucleotide comprises a nucleotide monomer represented by formula (III), formula (IV) or formula (V):

[0316] in,

[0317] Q is selected from H, D, halogen, OH, C 1-6 Alkyl, C 1-6 Halogenated alkyl, C 1-6 Alkoxy, C 1-6Halogenated alkoxy, C 2-6 Alkenyl or C 2-6 Alkynyl;

[0318] R4, R5, R6 and R7 are independently selected from H, D, halogen, OH, C 1-6 Alkyl, C 1-6 Halogenated alkyl, C 1-6 Alkoxy, C 1-6 Halogenated alkoxy, C 2-6 Alkenyl or C 2-6 Alkynyl;

[0319] Base is selected from H, modified or unmodified base;

[0320] L1, L3, X, Y, Z, R1, R2, R3, and U are as defined in any one of technical solutions 5 to 8;

[0321] The above groups are optionally substituted by 1, 2, 3, 4, 5 or more deuterium atoms, up to full deuteration.

[0322] 10. The oligonucleotide of claim 9 or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, wherein:

[0323] Q is selected from H, D, halogen, OH, C 1-6 Alkyl, C 1-6 Halogenated alkyl, C 1-6 Alkoxy or C 1-6 haloalkoxy;

[0324] R4, R5, R6 and R7 are independently selected from H, D, halogen, C 1-6 Alkyl, C 1-6 Halogenated alkyl, C 2-6 Alkenyl or C 2-6 Alkynyl;

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

[0326] L1, L3, X, Y, Z, R1, R2, R3, and U are as defined in any one of technical solutions 5 to 8.

[0327] 11. The oligonucleotide of claim 9 or 10, or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof, wherein:

[0328] Q is selected from H, halogen or C 1-4 Alkoxy, preferably H, F or methoxy;

[0329] R4, R5, R6 and R7 are independently selected from H, halogen, C1-4 Alkyl or C 1-4 Haloalkyl, preferably H;

[0330] Base is selected from Preferably

[0331] L1, L3, X, Y, Z, R1, R2, R3, and U are as defined in any one of technical solutions 5 to 8.

[0332] 12. The oligonucleotide according to any one of technical solutions 9 to 11, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, wherein the 5' end of the oligonucleotide comprises a nucleotide monomer having the following structure:

[0333] Wherein, each group is defined as in any one of technical solutions 9-11.

[0334] 13. The oligonucleotide according to any one of technical solutions 9 to 12, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, wherein the 5' end of the oligonucleotide comprises a nucleotide monomer having the following structure:

[0335] Among them, Base is selected from

[0336] 14. A compound represented by formula (VI), formula (VII) or formula (VIII) or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof:

[0337] in,

[0338] R1 is selected from O or S;

[0339] R2 and R3 are independently selected from OR d , OP1, SR d , SP1 or NR e R f ;

[0340] R d Selected from H, C 1-6 Alkyl, C 1-6 Halogenated alkyl, C 2-6 Alkenyl or C 2-6 Alkynyl, which is optionally substituted by D, halogen, C 1-6 Alkyl or C 1-6 Haloalkyl substitution until complete deuteration;

[0341] R e and R f Independently selected from H, C 1-6 Alkyl or C1-6 haloalkyl, the R e and R f Optionally, D, halogen, C 1-6 Alkyl or C 1-6 Haloalkyl substitution until complete deuteration;

[0342] P1 is selected from a protecting group, preferably a hydroxy protecting group;

[0343] P2 is selected from reactive phosphorus groups, preferably -P(OCH2CH2CN)(N(iPr)2);

[0344] Base' is independently selected from H, a modified or unmodified base, or a leaving group;

[0345] L1, L3, X, Y, Z, U, Q, R4, R5, R6 and R7 are as defined in any one of technical solutions 5-11;

[0346] The above groups are optionally substituted by 1, 2, 3, 4, 5 or more deuterium atoms, up to full deuteration.

[0347] 15. The compound of claim 14 or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, wherein:

[0348] R1 is selected from O or S;

[0349] R2 and R3 are independently selected from OR d , OP1, SR d , SP1 or NR e R f ;

[0350] R d Selected from H, C 1-4 Alkyl or C 1-4 haloalkyl, optionally substituted with D or halogen, up to full deuteration;

[0351] R e and R f Independently selected from H, C 1-4 Alkyl or C 1-4 haloalkyl, the R e and R f may be optionally substituted with D or halogen up to full deuteration;

[0352] P1 is selected from a protecting group, preferably a hydroxy protecting group;

[0353] P2 is selected from reactive phosphorus groups, preferably -P(OCH2CH2CN)(N(iPr)2);

[0354] Base' is selected from

[0355] L1, L3, X, Y, Z, U, Q, R4, R5, R6 and R7 are as defined in any one of technical solutions 5-11.

[0356] 16. The compound of claim 14 or 15, or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof, wherein:

[0357] R1 is selected from O or S, preferably O;

[0358] R2 and R3 are independently selected from OR d or OP1;

[0359] R d Selected from H, C 1-4 Alkyl or C 1-4 alkyl halide;

[0360] R e and R f Independently selected from H, C 1-4 Alkyl or C 1-4 alkyl halide;

[0361] P1 is selected from a protecting group, preferably a hydroxy protecting group;

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

[0363] Base' is selected from More preferably

[0364] L1, L3, X, Y, Z, U, Q, R4, R5, R6 and R7 are as defined in any one of technical solutions 5-11.

[0365] 17. The compound according to any one of technical solutions 14 to 16, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, wherein the compound is selected from:

[0366] 18. The oligonucleotide of any one of technical solutions 1-13, wherein the oligonucleotide is single-stranded and has 14 to 30 nucleotides.

[0367] 19. The oligonucleotide of any one of technical solutions 1-13, wherein the oligonucleotide is a double-stranded RNA comprising a sense strand and an antisense strand, wherein each strand has 14 to 30 nucleotides, and the antisense strand has a sequence that is fully complementary to the sense strand and the target mRNA.

[0368] 20. The oligonucleotide of technical solution 19, wherein the double-stranded RNA comprises the structure of formula (I), (II), (III), (IV), (V), (IIIb), (IVa), (IVb), (Va) or (Vb) according to any one of technical solutions 1 to 12 or the nucleotide monomer described in technical solution 13 in the antisense strand.

[0369] 21. The oligonucleotide of any one of technical solutions 19-20, wherein the double-stranded RNA is further coupled to a ligand, preferably, the ligand comprises one or more GalNAc.

[0370] 22. A nucleic acid molecule, the nucleotide sequence of which contains one or more nucleotide monomers as described in any one of technical solutions 9 to 13.

[0371] 23. The nucleic acid molecule of technical solution 22, wherein the nucleic acid is selected from DNA, RNA and DNA / RNA hybrid.

[0372] 24. The nucleic acid molecule of technical solution 23, wherein the nucleic acid molecule is single-stranded or double-stranded.

[0373] 25. The nucleic acid molecule of any one of technical solutions 22-24, wherein the nucleic acid molecule is selected from small interfering RNA (siRNA) and short hairpin RNA (shRNA).

[0374] 26. A vector comprising a nucleotide sequence encoding the double-stranded RNA according to any one of the aforementioned technical solutions 19-21.

[0375] 27. A cell containing the double-stranded RNA according to any one of Technical Solutions 19 to 21 or the vector according to Technical Solution 24.

[0376] 28. A pharmaceutical composition comprising the double-stranded RNA molecule according to any one of technical solutions 19 to 21, and a pharmaceutically acceptable carrier or excipient.

[0377] 29. A kit comprising a double-stranded RNA molecule as described in any one of technical solutions 19-21.

[0378] 30. 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 technical solutions 19 to 21 into the cell.

[0379] Without being bound by a specific theory, the stereo configurations of Formula (IIIa), Formula (IVa) and Formula (Va) can enable the oligonucleotides of the present invention (especially the phosphonate group at the 5' end thereof) to better bind to the Ago2 protein, thereby improving the activity of the oligonucleotides. Example

[0380] The following examples are intended to illustrate the present invention but are not intended to limit the scope of the present invention.

[0381] Example 1 Preparation of Compounds E1-1 and E1-2

[0382] 1. Preparation of Compound 1b

[0383] Compound 1a (200 g, 1.33 mol, 1.00 eq) was suspended in a mixture of anhydrous acetone (1.00 L) and anhydrous methanol (1.00 L). Concentrated sulfuric acid (20.0 mL, 0.27 eq) was added dropwise and allowed to react at 25°C for 24 hours. The reaction solution was neutralized with saturated sodium bicarbonate and concentrated. The resulting residue was dissolved in ethyl acetate and washed three times with saturated brine (500 mL). The organic phase was dried over anhydrous sodium sulfate and concentrated to yield compound 1b (242 g, 88.9%).

[0384] 1 H NMR:400MHz CDCl3δ4.97(s,1H),4.84(d,J=6.0Hz,1H),4.59(d,J=6.0Hz,1H),4.44-4.43(m,1H),3.72-3.59(m,2H),3.44(s,3H),1.49(s,3H),1.32(s,3H).

[0385] 2. Preparation of Compound 1c

[0386] Compound 1b (310 g, 1.52 mol, 1.00 eq), imidazole (206 g, 3.02 mol, 2.00 eq), and triphenylphosphine (476 g, 1.82 mol, 1.20 eq) were dissolved in toluene (2.1 L). Elemental iodine (446 g, 1.76 mmol, 1.16 eq) was added portionwise at room temperature. After addition, the temperature was raised to 70°C and stirred for 1.5 hours. TLC indicated the reaction was complete. Methanol (60 mL) was added to the reaction solution to quench it, and the temperature was then lowered. A saturated aqueous sodium thiosulfate solution (2.1 L) was added. The organic phase was separated, collected, washed twice with saturated brine (1.5 L), dried over anhydrous sodium sulfate, filtered, and concentrated. The resulting residue was filtered through a slurry of methyl tert-butyl ether. The filtrate was concentrated, and the resulting crude product was purified by silica gel column chromatography (eluent: petroleum ether / ethyl acetate) to yield compound 1c (401 g, 1.27 mol, 91.1%).

[0387] 1H NMR:400MHz CDCl3δ4.98(s,1H),4.69(d,J=6.0Hz,1H),4.56(d,J=6.0Hz,1H),4.39-4.35(m,1H) ,3.30(s,3H),3.24-3.20(m,1H),3.09(t,J=10.0Hz,1H),1.41(s,3H),1.26(s,3H).

[0388] 3. Preparation of Compound 1d

[0389] Compound 1c (203 g, 646 mmol, 1.00 eq) was dissolved in tetrahydrofuran (2.03 L), and potassium tert-butoxide (145 g, 1.29 mol, 1.29 eq) was added portionwise at 0°C. Stir at 25°C for 16 hours. The mixture was cooled to 5°C and quenched with ice water (1.1 L). The mixture was extracted with methyl tert-butyl ether (2.0 L), washed with saturated brine (2.0 L), dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was purified by silica gel column chromatography (eluent: petroleum ether / ethyl acetate) to afford compound 1d (160 g, 91.3%).

[0390] 1 H NMR:400MHz CDCl3δ5.11(s,1H),5.02(d,J=5.6Hz,1H),4.06(d,J=1.6Hz,1H),4.50(d,J=5.6Hz,1H),4.39(d, J=1.6Hz,1H),3.41(s,3H),3.24-3.20(m,1H),3.09(t,J=10.0Hz,1H),1.47(s,3H),1.35(s,3H).

[0391] 4. Preparation of Compound 1e

[0392] A zinc copper reagent (90.0 g, 1.37 mol, 5.50 eq) was dispersed in diethyl ether (200 mL). Compound 1d (60 g, 241 mmol, 1.00 eq) was added at 25°C. A solution of trichloroacetyl chloride (61.5 g, 338 mmol, 1.40 eq) in diethyl ether (200 mL) was added dropwise, and stirring was continued for 1 hour. The mixture was filtered, and the filter cake was rinsed with methyl tert-butyl ether (500 mL). The filtrate was poured into saturated aqueous sodium bicarbonate solution (2.50 L), filtered, and washed three times with saturated brine (500 mL). The mixture was dried over anhydrous sodium sulfate, filtered, and concentrated to give crude compound 1e (71.8 g).

[0393] 1H NMR: 400MHz CDCl3δ5.12(d,J=5.6Hz,1H),5.09(s,1H),4.70(d,J=6.0Hz,1H),3.70-3.55(m,2H),3.54(s,3H),1.45(s,3H),1.36(s,3H).

[0394] 5. Preparation of Compound 1f

[0395] Compound 1e (71.8 g, 242 mmol, 1.00 eq) was dissolved in tetrahydrofuran (1.60 L), and glacial acetic acid (69.1 mL, 1.20 mol, 5.00 eq) was added. Zinc powder (142 g, 2.17 mol, 9.00 eq) was then added portionwise. The mixture was stirred at 25°C for 18 hours. After completion of the reaction, the mixture was filtered and the filtrate was concentrated to obtain a crude product. The crude product was dissolved in methyl tert-butyl ether and poured into a saturated aqueous sodium bicarbonate solution (2.0 L). The product was filtered and the filtrate was washed twice with saturated brine (500 mL), dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was purified by silica gel column chromatography (eluent: petroleum ether / ethyl acetate / dichloromethane) to obtain compound 1f (31 g, 56.3% yield over two steps).

[0396] 1 H NMR:400MHz CDCl3δ4.97(s,1H),4.70-4.67(m,2H),3.54-3.48(m,2H),3.37-3.31(m,4H),3.16-3.10(m,1H),3.09-3.03(m,1H),1.43(s,3H),1.34(s,3H).

[0397] 6. Preparation of Compound 1g

[0398] Compound 1f (50 g, 219 mmol, 1.00 eq) and diethyl phosphite (33.2 g, 240 mmol, 1.20 eq) were dissolved in dichloromethane (100 mL). 1,8-diazabicycloundec-7-ene (DBU, 6.67 g, 43.8 mmol, 0.20 eq) was added at 0°C and stirred at 25°C for 18 hours. The reaction solution was washed three times with saturated NH4Cl (100 mL) and once with saturated brine (100 mL), dried over anhydrous sodium sulfate, and concentrated. The crude product was purified by silica gel column chromatography (eluent: petroleum ether / dichloromethane) to obtain compound 1g (70 g, 87.2%).

[0399] 1H NMR: 400MHz CDCl3δ5.27(s,1H),5.01(brs,1H),4.78(s,1H),4.65(d,J=5.6Hz,1H),4.49(d, J=6.0Hz,1H),4.20-4.05(m,5H),3.31(s,1H),3.29-3.05(m,1H),2.75- 2.68(m,1H),2.50-2.40(m,1H),2.35-2.25(m,1H),1.36-1.25(m,12H).

[0400] 7. Preparation of Compound 1h

[0401] Compound 1g (80.2 g, 219 mmol, 1.00 eq) and DMAP (40.1 g, 328 mmol, 1.50 eq) were dissolved in acetonitrile (562 mL), and monomethyl oxalyl chloride (40.2 g, 328 mmol, 1.50 eq) was added at 5°C. The mixture was stirred at 25°C for half an hour. The reaction solution was concentrated, and the crude product was dissolved in ethyl acetate and washed five times with saturated ammonium chloride (300 mL), once with water (200 mL), and once with brine (200 mL). The mixture was dried over anhydrous sodium sulfate, filtered, and concentrated to obtain crude compound 1h (99 g).

[0402] 1 H NMR:400MHz CDCl3δ4.88-4.80(m,2H),4.67-4.53(m,2H),4.24-4.12(m,4H),3.89(s,3H),3.51-3.43(m,1H), 3.35-3.33(m,3H),3.15-3.07(m,1H),2.92-2.80(m,1H),2.69-2.61(m,1H),1.39-1.29(m,12H).

[0403] 8. Preparation of Compound 1i

[0404] Compound 1h (99 g, 218 mmol, 1.00 eq) was dissolved in anhydrous toluene (1.00 L). Tri-n-butyltin hydride (76.4 g, 262 mmol, 1.20 eq) and azobisisobutyronitrile (AIBN, 1.08 g, 6.56 mmol, 0.03 eq) were added. Refluxed for 2 hours. The reaction mixture was concentrated, and the crude product was purified by silica gel column chromatography (eluent: petroleum ether / ethyl acetate) to obtain crude compound 1i (146 g).

[0405] 1H NMR: 400MHz CDCl3δ4.75-4.49(m,3H),4.05-3.95(m,4H),3.26-3.24(m,3H),2.76-2.07(m,5H),1.39-1.29(m,12H).

[0406] 9. Preparation of Compound 1j

[0407] Compound 1i (141 g, 201 mmol, 1.00 eq) was dissolved in methanol (1.41 L) and aqueous HCl (704 mL, 1.40 mol, 2 M, 7.00 eq) was added. The reaction mixture was stirred at 60°C for 1 hour. The reaction mixture was extracted with a mixture of methyl tert-butyl ether and petroleum ether. The aqueous phase was removed and the pH was adjusted to 8 with saturated sodium bicarbonate. The aqueous phase was concentrated, and tetrahydrofuran was added to the resulting residue, which was filtered. The filtrate was concentrated to afford crude compound 1j (62.4 g, 201 mmol).

[0408] 10. Preparation of Compound 1k

[0409] Compound 1j (62.4 g, 201 mmol, 1.00 eq) was dissolved in pyridine (300 mL), and acetic anhydride (47.4 mL, 502 mmol, 2.50 eq) was added. The mixture was stirred at 25°C for 12 hours. The reaction solution was diluted with saturated sodium bicarbonate (1.00 L) and extracted twice with ethyl acetate (600 mL). The mixture was dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was purified by silica gel column chromatography (eluent: petroleum ether / ethyl acetate) to afford compound 1k (74 g, 93.3%).

[0410] 1 H NMR: 400MHz CDCl3δ5.38-5.10(m,3H),4.07-4.03(m,4H),3.36-3.32(m,3H),2.41-2.05(m,5H),2.04-1.98(m,9H),1.27-1.23(m,6H).

[0411] 11. Preparation of Compound 11

[0412] Compound 1k (79.3 g, 201 mmol, 1.00 eq) was dissolved in ethyl acetate (476 mL), and acetic anhydride (62.6 mL, 663 mmol, 3.30 eq) and concentrated sulfuric acid (5.38 mL, 100 mmol, 0.50 eq) were added. The mixture was stirred at 25°C for 3 hours. The reaction solution was neutralized with saturated aqueous sodium bicarbonate solution and extracted twice with ethyl acetate (1.00 L). The organic phases were combined, washed twice with saturated brine (500 mL), dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was purified by silica gel column chromatography to yield compound 1l (43 g, 50.6%).

[0413] 1 H NMR:400MHz CDC13δ6.11(d,J=2.8Hz,1H),5.44-5.39(m,1H),5.33-5.32(m,1H),4.10-4.05(m,5H),2.80-2.35(m,4H),2.12-2.02(m,9H),1.32-1.23(m,6H).

[0414] 12. Preparation of Compound 1n

[0415] Compound 1m (8.55 g, 76.2 mmol, 2.30 eq) and bis(trimethylsilyl)acetamide (BSA, 34.3 g, 169 mmol, 5.10 eq) were suspended in acetonitrile (200 mL) and stirred at 85°C for 1 hour. After cooling, a solution of compound 11 (14.0 g, 33.1 mmol, 1.00 eq) in acetonitrile (50.0 mL) was added, followed by tin tetrachloride (37.1 g, 142 mmol, 4.30 eq). The mixture was stirred at 25°C for 15 minutes and then in an oil bath at 85°C for 45 minutes. After cooling, the reaction solution was poured into a saturated aqueous sodium bicarbonate solution (1.50 L) and extracted three times with dichloromethane (700 mL). The organic phases were combined, washed with saturated brine (500 mL), dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was purified by silica gel column chromatography (eluent: dichloromethane / methanol) to obtain compound 1n (14.4 g, 84.3%).

[0416] 1H NMR:400MHz CDCl3δ9.70(brs,0.46H),9.60(brs,0.60H),7.17(d,J=8.0Hz,0.60H),7.12(d,J= 8.0Hz,0.49H),5.98(d,J=6.4Hz,0.43H),5.93(d,J=5.2Hz,0.55H),5.78(dt,J1=8 .0Hz, J2=2.0Hz,1H),5.50-5.45(m,2H),4.45-4.02(m,5H),2.77-2.42(m,6H),2.1 8(s,1.22H),2.16(s,1.74H),2.04(s,1.31H),2.02(s,1.67H),1.32-1.28(m,6H).

[0417] 13. Preparation of Compound 1o

[0418] Compound 1n (8.30 g, 17.4 mmol, 1.00 eq) was dissolved in DMF (40.0 mL), and DBU (2.93 g, 19.2 mmol, 1.10 eq) was added. After cooling, benzyloxymethyl chloride (BOMCl, 3.01 g, 19.2 mmol, 1.10 eq) was added. The mixture was stirred at 0-5°C for 2.5 hours. The reaction solution was diluted with saturated ammonium chloride (160 mL) and extracted twice with dichloromethane (80.0 mL). The combined organic phases were washed with saturated brine (80 mL), dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was purified by silica gel column chromatography (eluent: dichloromethane / methanol) to obtain compound 1o (12.0 g, 92.3%).

[0419] 1 H NMR:400MHz CDCl3δ7.36-7.24(m,5H),7.15-7.09(m,1H),5.96-5.92(m,1H),5.81-5.79(m,1H),5.52-5.42(m,4H),4. 67(s,2H),4.15-4.05(m,4H),2.80-2.45(m,5H),2.19-2.17(m,3H),2.05-2.03(m,3H),1.34-1.30(m,6H).

[0420] 14. Preparation of Compound 1p

[0421] Compound 1o (10.4 g, 17.4 mmol, 1.00 eq) was dissolved in methanolic ammonia (29.9 mL, 7 M) and stirred at 21°C for 1 hour. The reaction solution was concentrated to obtain a crude product, which was purified by silica gel column chromatography (eluent: dichloromethane / methanol) to obtain compound 1p (8.00 g, 62.7%).

[0422] 1 H NMR: 400 MHz CDCl3δ7.34-7.06(m,5H),5.83(s,0.52H),5.71(d,J=8.4Hz,0.41H),5.661(d, J=8.4Hz,0.53H),5.60(d,J=2.0Hz,0.59H),5.53(d,J=5.2Hz,0.41H),5.42-5.3 6(m,2H),4.62(s,2H),4.51(d,J=3.6Hz,0.45H),3.72(d,J=2.8Hz,0.36H),3.5 4(d,J=2.0Hz,0.48H),2.93-2.40(m,5H),2.24-2.13(m,1H),1.27-1.23(m,6H).

[0423] 15. Preparation of Compound 1q

[0424] Compound 1p (1 g, 1.95 mmol, 1.00 eq) was dissolved in acetone (19.6 mL), and silver oxide (3.63 g, 15.6 mmol, 10.0 eq) and iodomethane (1.22 mL, 19.5 mmol, 10.0 eq) were added. The mixture was stirred at 25°C for 24 hours. The reaction mixture was filtered and concentrated to obtain crude compound 1q (1.36 g).

[0425] 1 H NMR:400MHz CDCl3δ5.11(s,1H),5.02(d,J=5.6Hz,1H),4.06(d,J=1.6Hz,1H),4.50(d,J=5.6Hz,1H),4.39(d, J=1.6Hz,1H),3.41(s,3H),3.24-3.20(m,1H),3.09(t,J=10.0Hz,1H),1.47(s,3H),1.35(s,3H).

[0426] 16. Preparation of Compounds 1q-1 and 1q-2

[0427] The crude compound 1q (10.9 g, 20.7 mmol, 1.00 eq) was reacted with C 18The residue was purified by reverse phase column chromatography (aqueous ammonium bicarbonate / acetonitrile) to obtain compound 1q-1 (1.50 g, 11.5%) and compound 1q-2 (1.50 g, 11.5%).

[0428] Compound 1q-1:

[0429] m / z:ES+[M+H]+525.2

[0430] HPLC: Retention time 0.820min (Column: 95%B, 1.16-1.5min, 95%-5%B; Flow rate: 1.5mL / min; Column temp.: 40℃)

[0431] 1 H NMR:400MHz CDC13δ7.37-7.08(m,5H),7.09(d,J=8.0Hz,1H),5.76-5.74(m,2H),5.49-5.44(m,2H),4.73-4.67(m,2H),4.15-4.06(m,4H),4. 02(d,J=4.8Hz,1H),3.87-3.85(m,1H),3.56(s,3H),2.81-2.64(m,2H),2.50-2.39(m,2H),2.36-2.27(m,1H),1.34-1.30(m,6H).

[0432] Compound 1q-2:

[0433] m / z:ES+[M+H]+525.2

[0434] HPLC: retention time 0.836min (Column: 95%B, 1.16-1.5min, 95%-5%B; Flow rate: 1.5mL / min; Column temp.: 40℃)

[0435] 1 H NMR:400MHz CDC13δ7.37-7.27(m,5H),7.08(d,J=8.0Hz,1H),5.75-5.73(m,2H),5.50-5.45(m,2H),4.73-4.67(m,2H),4.18-4.08( m,4H),4.06(d,J=4.8Hz,1H),3.88-3.86(m,1H),3.56(s,3H),3.06-2.95(m,1H),2.83-2.41(m,5H),1.34-1.31(m,6H).

[0436] 17. Preparation of Compound 1r-1

[0437] Compound 1q-1 (1.00 g, 1.90 mmol, 1.00 eq) was dissolved in anhydrous dichloromethane (DCM) (20.0 mL) and cooled to -60°C. Boron trichloride (7.62 mL, 7.62 mmol, 1 M dichloromethane solution, 4.00 eq) was added and stirred at -20°C for 1 hour. The reaction solution was quenched with anhydrous ethanol (10.0 mL), neutralized with concentrated aqueous ammonia to a pH of approximately 7, and concentrated. The crude product was added to a mixed solution of dichloromethane and ethanol and filtered. The filtrate was concentrated and the resulting residue was purified by C 18 The residue was purified by reverse phase column chromatography (aqueous ammonium bicarbonate solution / acetonitrile) to obtain compound 1r-1 (400 mg, 51.9%).

[0438] m / z:ES+[M+H]+405.3

[0439] 1 H NMR:400MHz CDCl3δ8.51-8.43(m,1H),7.15(d,J=8.4Hz,1H),5.78(d,J=3.2Hz,1H),5.76(dd,J1=8.0Hz,J2=2.4Hz,1H),4.16-4.07(m,5H),3.94(dd , J1=4.8Hz, J2=3.2Hz,1H),3.55(s,3H),2.96-2.94(m,1H),2.79-2.66(m,2H),2.52-2.41(m,2H),2.35-2.26(m,1H),1.34-1.31(m,6H).

[0440] 18. Preparation of Compound 1r-2

[0441] Compound 1r-2 was obtained using the same synthetic method as compound 1r-1.

[0442] 1 H NMR:400MHz CDCl3δ9.16-8.81(m,1H),7.15(d,J=8.0Hz,1H),5.78(d,J=2.8Hz,1H),5.76(dd,J1=8.0Hz,J2=2.0Hz,1H),4.65(brs,1H),4 .18-4.08(m,5H),3.96(dd,J1=4.8Hz,J2=3.2Hz,1H),3.53(s,3H),3.05-2.95(m,1H),2.81-2.41(m,2H),1.34-1.30(m,6H).

[0443] 19. Preparation of Compound E1-1

[0444] Compound 1r-1 (500 mg, 1.23 mmol, 1.00 eq) was dissolved in dichloromethane (5.00 mL), and compound 1s (0.59 mL, 1.85 mmol, 1.50 eq) was added. The temperature was cooled to 0°C, 4,5-dicyanoimidazole (160 mg, 1.36 mmol, 1.10 eq) was added, and the mixture was stirred at 15°C for 4 hours. The reaction solution was quenched with saturated sodium bicarbonate (10.0 mL) and extracted twice with dichloromethane (10.0 mL). The crude product was concentrated to give a crude product, which was purified by silica gel column chromatography (eluent: dichloromethane / acetone) to give compound E1-1 (530 mg, 70.9%).

[0445] 1 H NMR:400MHz CDCl3δ9.17(brs,1H),7.24(d,J=8.4Hz,1H),5.83-5.79(m,1H),5.64-5.61(m,1H),4.43-4.33(m,1 H),4.09-3.62(m,9H),3.43-3.38(m,3H),2.86-2.66(m,3H),2.57-2.21(m,4H),1.28-1.18(m,18H).

[0446] 20. Preparation of Compound E1-2

[0447] Compound 1r-2 (500 mg, 1.23 mmol, 1.00 eq) was dissolved in dichloromethane (5.00 mL), and compound 1s (0.59 mL, 1.85 mmol, 1.50 eq) was added. The temperature was cooled to 0°C, 4,5-dicyanoimidazole (160 mg, 1.36 mmol, 1.10 eq) was added, and the mixture was stirred at 15°C for 4 hours. The reaction solution was quenched with saturated sodium bicarbonate (10.0 mL) and extracted twice with dichloromethane (10.0 mL). The crude product was concentrated and purified by silica gel column chromatography (eluent: dichloromethane / acetone) to obtain compound E1-2 (480 mg, 64.0%).

[0448] 1 H NMR:400MHz CDCl3δ9.06(brs,1H),7.33-7.31(m,1H),5.89-5.88(m,1H),5.66-5.64(m,1H),4.49-4. 40(m,1H),4.08-3.64(m,9H),3.40-3.36(m,3H),3.00-2.19(m,7H),1.29-1.18(m,18H).

[0449] Example 2 Preparation of Compounds E2-1 and E2-2

[0450] 1. Preparation of Compound 2c

[0451] Under a nitrogen atmosphere, a solution of compound 2a (86.0 g, 493 mmol) in THF (250 mL) was added dropwise to a solution of LDA in THF (271 mL, 543 mmol, 2 M) at -70 to -65°C. After 30 minutes, a solution of compound 2b (142 g, 543 mmol) in THF (250 mL) was added and stirred for 1 hour. After completion of the reaction, the reaction solution was poured into saturated aqueous ammonium chloride (2 L) and extracted three times with methyl tert-butyl ether. The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography (eluent: petroleum ether / ethyl acetate) to yield compound 2c (113 g, 70.6%).

[0452] 1H NMR:400MHz DMSO-d6δ7.35-7.31(m,2H),7.28-7.24(m,3H),4.36(s,2H),3.52(s,3H),3.39-3.36(m,2H),3.03(s,3H),3 .00(s,3H),2.46-2.45(m,1H),2.44-2.43(m,1H),2.12-2.11(m,1H),2.10-2.09(m,1H),2.03-2.00(m,2H).

[0453] 2. Preparation of Compound 2d

[0454] 1M hydrochloric acid (217 mL, 217 mmol) was added to a solution of compound 2c (13.4 g, 43.4 mmol) in THF (200 mL) at 15°C and stirred for 24 hours. The reaction mixture was extracted three times with ethyl acetate. The organic phases were combined, washed twice with saturated sodium bicarbonate solution and saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography (eluent: petroleum ether / ethyl acetate) to afford compound 2d (9.9 g, 76.7%).

[0455] 1 H NMR:400MHz CDCl3δ7.36-7.26(m,5H),4.45(s,2H),3.68(s,3H),3.59-3.56(m,3H),3.5 3-3.49(m,1H),3.10-3.08(m,1H),3.06-3.04(m,1H),2.26(t,J=6.0Hz,2H).

[0456] 3. Preparation of Compound 2f

[0457] DBU (1.15 g, 7.54 mmol) was added to a solution of compound 2d (9.90 g, 37.7 mmol) and compound 2e (6.25 g, 45.2 mmol) in dichloromethane (20 mL) at 0 to 5°C and stirred at 15°C for 24 hours. The reaction solution was concentrated under reduced pressure, and the resulting residue was purified by silica gel column chromatography (eluent: petroleum ether / ethyl acetate / dichloromethane) to give compound 2f (15.5 g, 92.3%).

[0458] 1H NMR:400MHz CDCl3δ7.34-7.26(m,5H),4.42-4.41(m,2H),4.20-4.11(m,5H),3.64-3.60(m,3H),3.49-3.45(m,1H),3.44- 3.41(m,1H),3.11-3.04(m,1H),2.70-2.56(m,2H),2.28-2.25(m,1H),2.20-2.04(m,2H),1.38-1.30(m,6H).

[0459] 4. Preparation of Compound 2g

[0460] Compound 2f (14.5 g, 36.2 mmol) and DMAP (6.64 g, 54.3 mmol) were dissolved in acetonitrile (100 mL), and monomethyl oxalyl chloride (5.00 mL, 54.3 mmol) was added at 5°C. The mixture was stirred at 15°C for half an hour. The reaction solution was concentrated, and the crude product was dissolved in ethyl acetate, washed with saturated ammonium chloride and brine in sequence, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain crude compound 2g (16.4 g).

[0461] 5. Preparation of Compound 2h

[0462] Compound 2g (17.5g, 35.9mmol) was dissolved in anhydrous toluene (175mL), and tri-n-butyltin hydride (12.5g, 43.1mmol) and azobisisobutyronitrile (AIBN, 0.30g, 1.79mmol) were added. Refluxed for 2 hours. The reaction mixture was concentrated, and the crude product was purified by silica gel column chromatography (eluent: petroleum ether / ethyl acetate) to obtain compound 2h (11.3g, 79.9% (two steps)).

[0463] 1 H NMR:400MHz CDCl3δ7.34-7.24(m,5H),4.43-4.41(m,2H),3.63-3.60(m,3H),3.49-3.40(m,2 H),2.79-2.59(m,3H),2.39-2.28(m,1H),2.17-2.13(m,2H),1.32-1.29(m,6H).

[0464] 6. Preparation of Compound 2i

[0465] Compound 2h (8.80 g, 22.8 mmol) was dissolved in THF (176 mL) and methanol (3.1 mL, 76 mmol). Lithium borohydride (2.49 g, 114 mmol) was added at 0°C and stirred at 15°C for 2 hours. Water (100 mL) was added to the reaction solution, and the pH was adjusted to 3 with 1M hydrochloric acid. The mixture was extracted three times with dichloromethane. The organic phases were combined, washed sequentially with saturated sodium bicarbonate solution and saturated brine, dried over anhydrous sodium sulfate, and concentrated. The crude product was purified by silica gel column chromatography (eluent: dichloromethane / methanol) to obtain compound 2i (7.86 g, 52.4%).

[0466] 1 H NMR:400MHz DMSO-d6δ7.35-7.25(m,5H),4.70-4.68(m,1H)4.44-4.42(m,2H),3.97-3.92(m, 4H),3.49-3.26(m,3H),2.60-2.50(m,1H),1.96-1.67(m,5H),1.21-1.18(m,6H).

[0467] 7. Preparation of Compound 2j

[0468] Compound 2i (7.86 g, 22.0 mmol) was dissolved in pyridine (47 mL), and benzoyl chloride (2.81 mL, 24.2 mmol) was added at 5°C. The mixture was stirred at 15°C for 1 hour. The reaction solution was concentrated, and the residue was dissolved in ethyl acetate (40 mL). The residue was washed sequentially with 2M hydrochloric acid, saturated aqueous sodium bicarbonate, and saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain crude compound 2j (10 g).

[0469] 1 H NMR:400MHz CDCl3δ8.03-7.98(m,2H),7.54-7.43(m,1H),7.42-7.39(m,2H),7.27-7.23(m,5H),4.45-4.44(m,2H),4.33-4.22(m,2 H),4.11-4.03(m,4H),3.56-3.51(m,2H),2.72-2.62(m,1H),2.45-2.05(m,4H),1.97-1.93(m,1H),1.30-1.27(m,6H).

[0470] 8. Preparation of Compound 2k

[0471] Compound 2j (8.00 g, 17.3 mmol) was dissolved in dichloromethane (160 mL). A 1 M solution of boron trichloride (69.4 mL, 69.4 mmol) in dichloromethane was added at -60°C. The mixture was allowed to react at -20°C for 1 hour. Ethanol (40.5 mL, 694 mmol) was added to the reaction solution, followed by saturated aqueous sodium bicarbonate solution. The organic phase was washed sequentially with saturated aqueous sodium bicarbonate solution and saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated. The resulting residue was purified by silica gel column chromatography (eluents: petroleum ether / ethyl acetate, then dichloromethane / methanol) to afford compound 2k (6.10 g, 85.3%).

[0472] 1 H NMR:400MHz CDCl3δ8.06-7.99(m,2H),7.59-7.53(m,1H),7.47-7.42(m,2H),4.35-4.28(m,2H),4.16-4.03(m,4H),3.83-3.74(m,2 H),2.78-2.65(m,1H),2.50-2.33(m,2H),2.21-2.06(m,2H),2.00-1.97(m,1H),1.86-1.82(m,1H),1.37-1.27(m,6H).

[0473] 9. Preparation of Compound 2m

[0474] Compound 2k (5.80 g, 15.6 mmol), compound 2l (3.55 g, 16.4 mmol), and triphenylphosphine (4.93 g, 18.7 mmol) were dissolved in tetrahydrofuran (58 mL). Diethyl azodicarboxylate (DEAD) (3.27 g, 18.7 mmol) was added at 5°C, and the reaction mixture was stirred at 15°C for 2 hours. Water was added to the reaction mixture, and the mixture was extracted twice with ethyl acetate. The combined organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The resulting residue was extracted with a mixture of methyl tert-butyl ether and petroleum ether. The aqueous phase was adjusted to pH 8 with saturated sodium bicarbonate, concentrated, and the resulting residue was purified by silica gel column chromatography (eluents: petroleum ether / ethyl acetate, then dichloromethane / methanol) to yield compound 2m (7.40 g, 61.9%).

[0475] 1H NMR:400MHz CDCl3δ8.06-8.00(m,2H),7.94-7.91(m,2H),7.66-7.51(m,2H),7.42-7.32(m,5H),7.32-7.29(m,1H),5.80-5.77(m,1 H),4.39-4.38(m,2H),4.12-4.02(m,5H),3.87-3.83(m,2H),2.79-2.67(m,1H),2.44-1.95(m,6H),1.33-1.25(m,6H).

[0476] 10. Preparation of Compound 2n

[0477] Compound 2m (3.00 g, 5.27 mmol) was dissolved in methanolic ammonia (150 mL, 7 M) and reacted at 60°C for 48 hours. The reaction solution was concentrated, and the residue was purified by silica gel column chromatography (eluent: dichloromethane / methanol) to obtain crude compound 2n (3.10 g).

[0478] 1 H NMR:400MHz DMSO-d6δ11.21(brs,1H),7.69-7.63(m,1H),5.56-5.52(m,1H),4.80-4.77(m,1H),3.98-3.93(m,4H ),3.60-3.69(m,2H),3.42(d,J=5.2HZ,1H),2.75-2.55(m,1H),1.93-1.87(m,6H),1.22-1.18(m,6H).

[0479] 11. Preparation of Compounds 2n-1 and 2n-2

[0480] Compound 2n (3.70 g, 10.2 mmol) was purified by SFC to give compound 2n-1 (2.00 g, 50.7%) and compound 2n-2 (1.00 g, 24.1%). Purification column: DAICEL CHIRALPAK AD (250 mm x 30 mm, 10 μm); mobile phase: [A: CO2; B: IPA]; B%: 40.00%-40.00%, 3.00 min.

[0481] Compound 2n-1:

[0482] HPLC: retention time 3.065 min (column: Chiralpak AD-3, 150×4.6 mm ID, 3 μm; mobile phase: A: CO 2 , B: IPA (0.2% NH 3 (7 M methanol solution), v / v); gradient: 0-0.5 min 10% B, 0.5-3.5 min 10-50% B, 3.5-4.5 min 50% B, 4.5-5.0 min, 50%-10% B; flow rate: 2.5 mL / min; column temperature: 35° C.)

[0483] 1 H NMR:400MHz DMSO-d6δ11.20(brs,1H),7.64(d,J=8.0Hz,1H),5.53(d,J=8.0Hz,1H),4.78(t,J=5.2Hz,1H),3.99-3.93(m,4H),3. 55-3.75(m,2H),3.42(d,J=5.6Hz,2H),2.70-2.60(m,1H),1.93-1.87(m,4H),1.68-1.67(m,2H),1.22-1.18(m,6H).

[0484] Compound 2n-2:

[0485] HPLC: retention time 3.626 min (Column: Chiralpak AD-3, 150×4.6 mm ID, 3 μm; Mobile phase: A: CO2, B: IPA (0.2% NH3 (7 M methanol solution), v / v); Gradient: 0-0.5 min 10% B, 0.5-3.5 min 10-50% B, 3.5-4.5 min 50% B, 4.5-5.0 min, 50%-10% B; Flow rate: 2.5 mL / min; Column temperature: 35°C)

[0486] 1 H NMR:400MHz DMSO-d6δ11.22(brs,1H),7.68(d,J=8.0Hz,1H),5.56-5.53(m,1H),4.68(t,J=5.2Hz,1H), 4.00-3.91(m,4H),3.68-3.64(m,2H),3.31-3.30(m,2H),2.74-2.62(m,1H),2.03-1.93(m, 2H),1.88-1.75(m,4H),1.22-1.19(m,6H).

[0487] 12. Preparation of Compound E2-1

[0488] Compound 2n-1 (1.00 g, 2.77 mmol) was dissolved in dichloromethane (10 mL), and compound 1s (1.32 mL, 4.16 mmol) was added. The temperature was cooled to 0°C, 4,5-dicyanoimidazole (DCI) (360 mg, 3.05 mmol) was added, and the mixture was stirred at 15°C for 1 hour. The reaction solution was quenched with saturated sodium bicarbonate (20.0 mL), extracted with dichloromethane (20.0 mL), washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated. The resulting crude product was purified by silica gel column chromatography (eluent: dichloromethane / acetone) to obtain compound E2-1 (1.41 g, 89.5%).

[0489] 1 H NMR:400MHz DMSO-d6δ11.21(brs,1H),7.63(d,J=8.0Hz,1H),5.55(d,J=8.0Hz,1H),3.98-3.94(m,4H),3. 80-3.62(m,8H),2.79-2.76(m,3H),2.06-1.93(m,4H),1.80-1.75(m,2H),1.21-1.14(m,18H).

[0490] 13. Preparation of Compound E2-2

[0491] Compound 2n-2 (780 mg, 2.16 mmol) was dissolved in dichloromethane (7.8 mL), and compound 1s (936 mg, 3.24 mmol) was added. The temperature was cooled to 0°C, 4,5-dicyanoimidazole (280 mg, 2.37 mmol) was added, and the mixture was stirred at 15°C for 1 hour. The reaction solution was quenched with saturated sodium bicarbonate (20.0 mL), extracted with dichloromethane (20.0 mL), washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated to obtain a crude product which was purified by silica gel column chromatography (eluent: dichloromethane / acetone) to obtain compound E2-2 (860 mg, 60.7%).

[0492] 1 H NMR:400MHz DMSO-d6δ11.21(brs,1H),7.66(d,J=8.0HZ,1H),5.55(d,J=8.0Hz,1H),4.03-3.90(m,4H),3.81-3.66(m, 4H),3.61-3.31(m,4H),2.80-2.67(m,3H),2.05-1.84(m,6H),1.21(t,J=7.2Hz,6H),1.15-1.13(m,12H).

[0493] Example 3 Preparation of Compound E3

[0494] Compound E3 was prepared according to the above steps.

[0495] Example 4 Preparation of siRNA

[0496] 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.

[0497] 1 Synthesis of positive sense chain (SS chain)

[0498] Using solid-phase phosphoramidite synthesis, a blank CPG solid support or a solid support connected to L96 is used as the starting cycle. Nucleoside monomers are attached one by one in the 3'-5' direction according to the order of the positive chain nucleotides. Each ligation of a nucleoside monomer involves four steps: deprotection, coupling, capping, and oxidation or thiolation. The synthesis conditions for a 5 μmol oligonucleotide are as follows:

[0499] 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°C, and 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).

[0500] 2 Synthesis of antisense strand (AS strand)

[0501] Using solid-phase phosphoramidite synthesis, a blank CPG solid support was used as the starting cycle. Nucleoside monomers or nucleotide duplexes of the present invention were linked one by one in the 3'-5' direction according to the order of the antisense nucleotides. Each ligation involved four steps: deprotection, coupling, capping, and oxidation or thiolation. The synthesis conditions for 5 μmol of the antisense oligonucleotide were the same as those for the sense strand.

[0502] 3. Oligonucleotide Purification and Annealing

[0503] 3.1 Ammonolysis

[0504] The synthesized solid phase carrier (sense chain or antisense chain) was added to a 5 mL centrifuge tube, 3% diethylamine / ammonia water (v / v) was added, and the reaction was carried out in a constant temperature water bath at 35°C (or 55°C) for 16 hours (or 8 hours). After filtering, the solid phase carrier was washed three times with ethanol / water, each time with 1 mL. The filtrate was centrifuged and concentrated, and the crude product was purified.

[0505] 3.2 Purification

[0506] 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, and the product can be collected and stored in a tube. A gel-filled purification column can be used for desalination, and the elution system can be pure water.

[0507] 3.3 Annealing

[0508] According to the table below, the sense strand (SS strand) and the antisense strand (AS strand) were mixed at a molar ratio (SS strand / AS strand = 1 / 1.05), heated in a water bath to 70-95°C for 3-5 minutes, cooled naturally to room temperature, and the system was lyophilized to obtain the product.

[0509] The siRNA sequences used in the present invention are as follows:

[0510] In this article, the meanings of the abbreviations are as follows:

[0511] The A, U, G, and C distribution represents the natural adenine, uracil, guanine, and cytosine ribonucleotides.

[0512] 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.

[0513] 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.

[0514] "s" or "s-" indicates that the two adjacent nucleotides and / or delivery vectors are linked by phosphorothioate.

[0515] 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.

[0516] VP indicates that the adjacent nucleotide on its right side is a vinylphosphonate-modified nucleotide, which is well known in the art, and can be found in, for example, PCT Publication Nos. WO2011139702, WO2013033230, and WO2019105419.

[0517] SCP1a, SCP1b, SCP2a and SCP2b represent nucleotide substitutes of the above-mentioned structure, wherein Base can be any base, for example, SCP1a-U represents that Base is uracil.

[0518] Example 5 C57BL / 6 wild-type mouse primary hepatocyte (PMH) activity screening experiment

[0519] 1. Free uptake or transfection

[0520] 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 or transfection.

[0521] Free uptake: Add 10 μL of diluted compound to 90 μL of Opti-MEM, mix well, add to corresponding wells, and incubate in a 37°C, 5% CO2 incubator for 24 hours. No siRNA was added to the control group.

[0522] Transfection: Add 10 μL of diluted compound to 40 μL of Opti-MEM and mix thoroughly. Add 3 μL of RNAiMAX to 47 μL of Opti-MEM and mix thoroughly. Incubate for 5 minutes, then mix with the diluted compound. Let stand at room temperature for 10 minutes, then add to the corresponding wells and incubate in a 37°C, 5% CO2 incubator for 24 hours. No siRNA was added to the control group.

[0523] 2. Fluorescence quantitative PCR

[0524] 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)).

[0525] Table 1 Primer information

[0526] 3. Data Statistics

[0527] Calculation 2 -△△Ct The values ​​were converted into percentages to obtain the residual inhibition rate;

[0528] △△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)].

[0529] The target gene is mAPOB or mFXII, and the control gene is mGAPDH.

[0530] 4. mAPOB gene experimental results

[0531] Using the transfection method, C57BL / 6 wild-type mouse primary hepatocytes were selected. After plating the cells in 24-well plates, the starting concentration of the compound was selected to be 40 nM, and 5 concentration points (40 nM, 4 nM, 0.4 nM, 0.04 nM, 0.004 nM) were diluted 10-fold to perform a 5-point IC50 activity screening of C57BL / 6 wild-type mouse primary hepatocytes. The experimental results are shown in Table 2.

[0532] Table 2 Results of the 5-point IC50 activity screening experiment of the siRNA compound PMH targeting the mAPOB gene carrying the nucleotides of the present invention

[0533] 5. mFXII gene experimental results

[0534] Using the free uptake method, C57BL / 6 wild-type mouse primary hepatocytes were selected, and after plating the cells in 96-well plates, the compound was selected with a starting concentration of 100 nM, and 5 concentration points (100 nM, 10 nM, 1 nM, 0.1 nM, 0.01 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 3.

[0535] Table 3 Results of the 5-point IC50 activity screening experiment of the siRNA compound PMH targeting the mFXII gene carrying the nucleotide of the present invention

[0536] Example 6 Verification of Compound Efficacy in C57BL / 6 Mouse Model

[0537] C57BL / 6 mice (male, 18-21 g, 6-8 weeks, Sibeifu (Suzhou) Biotechnology Co., Ltd.) were randomly divided into groups of 9 animals per group (mice were euthanized on the 14th and 28th days (N=3 / group) for liver removal). The dosage of each animal was calculated based on body weight, and a single subcutaneous injection was used for administration. The siRNA conjugate was provided to the CRO company as a 10 mg / mL solution (0.9% sodium chloride aqueous solution as the solvent); specifically, before the experiment, the siRNA conjugate was dissolved in 0.9% sodium chloride aqueous solution and diluted to the required solution concentration and volume. The administration volume of physiological saline and siRNA conjugate was 5 mL / kg.

[0538] Blood was collected before administration (designated as day -2) for testing. Groups were divided based on LDL levels on day 2, with the remaining samples retained for target protein detection. Blood was collected from the orbital venous plexus of mice on days 7, 14, and 21 after administration (designated as day 0) (starved for 5 hours before each blood draw). Serum LDL concentrations (Meikang brand, low-density lipoprotein cholesterol detection reagent) were measured by Suzhou Anling at each time point using a direct method (Neusoft fully automatic biochemical analyzer, NT-1000). The remaining samples were retained for ELISA detection of the target protein (Mouse ApoB ELISA Kit, Abcam, ab230932). On days 14, 28, and 42, 10 mg of liver (n = 3 / group / time point) was collected, placed in RNAlater solution, frozen at -80°C, and shipped on dry ice for extraction and detection of liver mRNA expression (see Table 4 for detection primers, which will be updated at subsequent time points). The experimental results are shown in Table 5.

[0539] Table 4 Primer information

[0540] Table 5 Pharmacological efficacy of three siRNA compounds in C57BL / 6 mouse model

[0541] Example 7 C57BL / 6 wild-type mouse primary hepatocyte (PMH) activity screening experiment

[0542] According to the method of Example 3, more compounds of the present invention were tested.

[0543] Table 6 Results of the 5-point IC50 activity screening experiment of the siRNA compound PMH targeting the mAPOB gene carrying the compound of the present invention

[0544] Table 7 Results of the 5-point IC50 activity screening experiment of the PMH siRNA compound targeting the mFXII gene carrying the compound of the present invention

[0545] Example 8 Molecular simulation

[0546] Through molecular modeling, we observed that the compounds of the present invention can lock the phosphonic acid in an ap conformation that is highly similar to the unmodified 5'-phosphate bound to Ago2. The phosphonate group perfectly matches Arg812, Lys570, Lys566, and Lys533, forming multiple salt bridges and hydrogen bonds. It also forms a hydrogen bond with Tyr529 and Cys526. In addition, the base (uracil) in the spirocyclic phosphonic acid compound forms a hydrogen bond with the backbone amide of Thr526 and Gly524. These structural factors together stabilize the binding of the compounds of the present invention to the Ago2 protein.

[0547] Molecular simulations show that, similar to spiro compounds, after the sugar ring is fused with a highly rigid aromatic ring (including a five-membered aromatic ring and a six-membered aromatic ring), the 5'-terminal phosphonate is locked in a specific conformation that highly mimics the ap conformation of the natural 5'-phosphate. The phosphonate group perfectly matches Arg812, Lys570, Lys566, and Lys533, forming multiple salt bridge bonds and hydrogen bonds. It also forms a hydrogen bond with Tyr529 and Cys526. The base uracil on the sugar ring also forms a hydrogen bond with the backbone amide of Thr526 and Gly524. These structures together stabilize the binding of the compounds of the present invention to the Ago2 protein.

Claims

1. An oligonucleotide or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, wherein the 5' end of the oligonucleotide comprises a structure represented by formula (I): in, represents linkage to the remainder of the oligonucleotide, preferably to a modified or unmodified nucleoside; X and Y are each independently selected from CR a R b or (CR a R b )2, preferably CR a R b ; Z is CR a ; R a and R b Independently selected from H, D, halogen, OH, NH2, CN, C 1-6 Alkyl, C 1-6 Halogenated alkyl, C 1-6 Alkoxy, C 1-6 Halogenated alkoxy, C 2-6 Alkenyl or C 2-6 Alkynyl; the R a and R b is optionally further substituted with 1, 2, 3, 4 or 5 independently selected R#; R1 is selected from O or S; R2 and R3 are independently selected from OH, SH, NH2, C 1-6 Alkyl, C 1-6 Halogenated alkyl, C 1-6 Alkoxy or C 1-6 Haloalkoxy; said R2 and R3 are optionally further substituted by 1, 2, 3, 4 or 5 independently selected R#; R# is selected from H, D, halogen, C 1-6 Alkyl, C 1-6 Halogenated alkyl, C 2-6 Alkenyl or C 2-6 Alkynyl; The above groups are optionally substituted by 1, 2, 3, 4, 5 or more deuterium atoms, up to full deuteration.

2. The oligonucleotide of claim 1 or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, wherein: represents linkage to the remainder of the oligonucleotide, preferably to a modified or unmodified nucleoside; X and Y are each independently selected from CR a R b or (CR a R b )2, preferably CR a R b ; Z is CR a ; R a and R b Independently selected from H, D, halogen, OH, NH2, CN, C 1-6 Alkyl, C 1-6 Halogenated alkyl, C 1-6 Alkoxy or C 1-6 haloalkoxy; R1 is selected from O or S; R2 and R3 are independently selected from OH, SH, C 1-6 Alkoxy or C 1-6 Haloalkoxy; said R2 and R3 are optionally further substituted by 1, 2 or 3 independently selected R#; R# is selected from H, D, halogen, C 1-6 Alkyl and C 1-6 Halogenated alkyl.

3. The oligonucleotide of claim 1 or 2, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, wherein: represents linkage to the remainder of the oligonucleotide, preferably to a modified or unmodified nucleoside; X and Y are each independently selected from CR a R b or (CR a R b )2, preferably CR a R b ; Z is CR a ; R a and R b Independently selected from H, D, halogen, C 1-4 Alkyl or C 1-4 alkyl halide; R1 is selected from O or S; R2 and R3 are independently selected from OH or SH, preferably OH.

4. The oligonucleotide of any one of claims 1 to 3, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, wherein: represents a 5' terminal group attached to the oligonucleotide, preferably to a modified or unmodified nucleoside; X and Y are CH2; Z is CH; R1 is O; R2 and R3 are OH.

5. The oligonucleotide of claim 1 or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, which has the structure of formula (II): in, represents the remainder of the oligonucleotide attached; X and Y are CR a R b ; Z is CR a ; L1 is selected from a chemical bond, O, S or C 1-4 alkylene; L2 and L3 are independently selected from C 1-4 alkylene; The L1, L2 and L3 are optionally independently substituted with 1, 2, 3, 4 or 5 independently selected R#; U is selected from O, S, CR a R b or NR c ; R a and R b Independently selected from H, D, halogen, OH, NH2, CN, C 1-6 Alkyl, C 1-6 Halogenated alkyl, C 1-6 Alkoxy, C 1-6 Halogenated alkoxy, C 2-6 Alkenyl or C 2-6 Alkynyl; R c Independently selected from H, C 1-6 Alkyl, C 1-6 Halogenated alkyl, C 2-6 Alkenyl or C 2-6 Alkynyl; The R a 、R b and R c is optionally further substituted with 1, 2, 3, 4 or 5 independently selected R#; Alternatively, L2 and L3 are connected to form a C 5-6 Cycloalkyl or 5-6 membered heterocyclic group, preferably U, L1, L2 and L3 and their adjacent carbon atoms together form ribose or deoxyribose, which is optionally substituted by R5; R1 is selected from O or S; R2 and R3 are independently selected from OH, SH, NH2, C 1-6 Alkyl, C 1-6 Halogenated alkyl, C 1-6 Alkoxy or C 1-6 Haloalkoxy; said R2 and R3 are optionally substituted by 1, 2, 3, 4 or 5 independently selected R#; R5 is selected from H, D, halogen, C 1-6 Alkoxy or C 1-6 haloalkoxy; R# is selected from H, D, halogen, C 1-6 Alkyl, C 1-6 Halogenated alkyl, C 2-6 Alkenyl or C 2-6 Alkynyl; Base is selected from H, modified or unmodified base; The above groups are optionally substituted by 1, 2, 3, 4, 5 or more deuterium atoms, up to full deuteration.

6. The oligonucleotide of claim 5 or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, wherein: represents the remainder of the oligonucleotide attached; X and Y are CR a R b ; Z is CR a ; R a and R b Independently selected from H, D, halogen, OH, NH2, CN, C 1-6 Alkyl, C 1-6 Halogenated alkyl, C 1-6 Alkoxy or C 1-6 haloalkoxy; L1 is selected from a chemical bond, O or C 1-4 alkylene; L2 and L3 are independently selected from C 1-4 alkylene; Said L1, L2 and L3 are optionally independently substituted with 1, 2 or 3 independently selected R#; U is selected from O, S, NH or CH2; Alternatively, L2 and L3 are connected to form a C 5-6 Cycloalkyl or 5-6 membered heterocyclic group, preferably U, L1, L2 and L3 and their adjacent carbon atoms together form ribose or deoxyribose, which is optionally substituted by R5; R1 is selected from O or S; R2 and R3 are independently selected from OH, SH, C 1-6 Alkoxy or C 1-6 Haloalkoxy; said R2 and R3 are optionally substituted by 1, 2 or 3 independently selected R#; R5 is selected from H, D, C 1-6 Alkyl, C 1-6 Halogenated alkyl, C 1-6 Alkoxy or C 1-6 haloalkoxy; R# is selected from H, D, halogen, C 1-6 Alkyl and C 1-6 alkyl halide; Base is selected from H, modified or unmodified bases.

7. The oligonucleotide of claim 5 or 6, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, wherein: represents the remainder of the oligonucleotide attached; X and Y are CR a R b ; Z is CR a ; R a and R b Independently selected from H, D, halogen, C 1-4 Alkyl or C 1-4 alkyl halide; L1 is selected from a chemical bond or C 1-4 alkylene; L2 and L3 are independently selected from C 1-4 alkylene; U is selected from O or S; R1 is selected from O or S; R2 and R3 are independently selected from OH or SH, preferably OH; Base is selected from 8. The oligonucleotide of any one of claims 5 to 7, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, wherein: represents the remainder of the oligonucleotide attached; X and Y are CH2; Z is CH; L1 is a chemical bond; L2 and L3 are independently selected from C 1-2 alkylene; U is O; R1 is O; R2 and R3 are OH; Base is selected from 9. The oligonucleotide of claim 1 or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, wherein the 5' end of the oligonucleotide comprises a nucleotide monomer represented by formula (III), formula (IV) or formula (V): in, Q is selected from H, D, halogen, OH, C 1-6 Alkyl, C 1-6 Halogenated alkyl, C 1-6 Alkoxy, C 1-6 Halogenated alkoxy, C 2-6 Alkenyl or C 2-6 Alkynyl; R4, R5, R6 and R7 are independently selected from H, D, halogen, OH, C 1-6 Alkyl, C 1-6 Halogenated alkyl, C 1-6 Alkoxy, C 1-6 Halogenated alkoxy, C 2-6 Alkenyl or C 2-6 Alkynyl; Base is selected from H, modified or unmodified base; L1, L3, X, Y, Z, R1, R2, R3, U are as defined in any one of claims 5 to 8; The above groups are optionally substituted by 1, 2, 3, 4, 5 or more deuterium atoms, up to full deuteration.

10. The oligonucleotide of claim 9 or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, wherein: Q is selected from H, D, halogen, OH, C 1-6 Alkyl, C 1-6 Halogenated alkyl, C 1-6 Alkoxy or C 1-6 haloalkoxy; R4, R5, R6 and R7 are independently selected from H, D, halogen, C 1-6 Alkyl, C 1-6 Halogenated alkyl, C 2-6 Alkenyl or C 2-6 Alkynyl; Base is selected from H, modified or unmodified bases, preferably L1, L3, X, Y, Z, R1, R2, R3, and U are as defined in any one of claims 5 to 8.

11. The oligonucleotide of claim 9 or 10, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, wherein: Q is selected from H, halogen or C 1-4 Alkoxy, preferably H, F or methoxy; R4, R5, R6 and R7 are independently selected from H, halogen, C 1-4 Alkyl or C 1-4 Haloalkyl, preferably H; Base is selected from Preferably L1, L3, X, Y, Z, R1, R2, R3, and U are as defined in any one of claims 5 to 8.

12. The oligonucleotide according to any one of claims 9 to 11, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, wherein: The 5' end of the oligonucleotide comprises a nucleotide monomer having the following structure: wherein each group is as defined in any one of claims 9 to 11.

13. The oligonucleotide according to any one of claims 9 to 12, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, wherein: The 5' end of the oligonucleotide comprises a nucleotide monomer having the following structure: Among them, Base is selected from 14. A compound represented by formula (VI), formula (VII) or formula (VIII) or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof: in, R1 is selected from O or S; R2 and R3 are independently selected from OR d , OP1, SR d , SP1 or NR e R f ; R d Selected from H, C 1-6 Alkyl, C 1-6 Halogenated alkyl, C 2-6 Alkenyl or C 2-6 Alkynyl, which is optionally substituted by D, halogen, C 1-6 Alkyl or C 1-6 Haloalkyl substitution until complete deuteration; R e and R f Independently selected from H, C 1-6 Alkyl or C 1-6 haloalkyl, the R e and R f Optionally, D, halogen, C 1-6 Alkyl or C 1-6 Haloalkyl substitution until complete deuteration; P1 is selected from a protecting group, preferably a hydroxy protecting group; P2 is selected from reactive phosphorus groups, preferably -P(OCH2CH2CN)(N(iPr)2); Base' is independently selected from H, a modified or unmodified base, or a leaving group; L1, L3, X, Y, Z, U, Q, R4, R5, R6 and R7 are as defined in any one of claims 5 to 11; The above groups are optionally substituted by 1, 2, 3, 4, 5 or more deuterium atoms, up to full deuteration.

15. The compound of claim 14 or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, wherein: R1 is selected from O or S; R2 and R3 are independently selected from OR d , OP1, SR d , SP1 or NR e R f ; R d Selected from H, C 1-4 Alkyl or C 1-4 haloalkyl, optionally substituted with D or halogen, up to full deuteration; R e and R f Independently selected from H, C 1-4 Alkyl or C 1-4 haloalkyl, the R e and R f may be optionally substituted with D or halogen up to full deuteration; P1 is selected from a protecting group, preferably a hydroxy protecting group; P2 is selected from reactive phosphorus groups, preferably -P(OCH2CH2CN)(N(iPr)2); Base' is selected from L1, L3, X, Y, Z, U, Q, R4, R5, R6 and R7 are as defined in any one of claims 5 to 11.

16. The compound of claim 14 or 15, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, wherein: R1 is selected from O or S, preferably O; R2 and R3 are independently selected from OR d or OP1; R d Selected from H, C 1-4 Alkyl or C 1-4 alkyl halide; R e and R f Independently selected from H, C 1-4 Alkyl or C 1-4 alkyl halide; P1 is selected from a protecting group, preferably a hydroxy protecting group; P2 is selected from reactive phosphorus groups, preferably -P(OCH2CH2CN)(N(iPr)2); Base' is selected from More preferably L1, L3, X, Y, Z, U, Q, R4, R5, R6 and R7 are as defined in any one of claims 5 to 11.

17. The compound according to any one of claims 14 to 16, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, wherein: The compound is selected from:

18. The oligonucleotide according to any one of claims 1 to 13, wherein The oligonucleotide is single-stranded and has 14 to 30 nucleotides.

19. The oligonucleotide according to any one of claims 1 to 13, wherein The oligonucleotide is a double-stranded RNA comprising a sense strand and an antisense strand, wherein each strand has 14 to 30 nucleotides, and the antisense strand has a sequence that is substantially complementary to the sense strand and the target mRNA.

20. The oligonucleotide of claim 19, wherein The double-stranded RNA comprises the structure of formula (I), (II), (III), (IV), (V), (IIIa), (IIIb), (IVa), (IVb), (Va) or (Vb) according to any one of claims 1 to 12 or the nucleotide monomer according to claim 13 in the antisense strand.

21. The oligonucleotide according to any one of claims 19 to 20, wherein the double-stranded RNA is further coupled to a ligand, preferably, the ligand comprises one or more GalNAc groups.

22. A nucleic acid molecule comprising one or more nucleotide monomers according to any one of claims 9 to 13 in its nucleotide sequence.

23. The nucleic acid molecule of claim 22, wherein the nucleic acid is selected from the group consisting of DNA, RNA, and a DNA / RNA hybrid.

24. The nucleic acid molecule of claim 23, which is single-stranded or double-stranded.

25. The nucleic acid molecule of any one of claims 22 to 24, wherein the nucleic acid molecule is selected from the group consisting of small interfering RNA (siRNA) and short hairpin RNA (shRNA).

26. A cell comprising the double-stranded RNA of any one of claims 19 to 21.

27. A pharmaceutical composition comprising the double-stranded RNA molecule according to any one of claims 19 to 21, and a pharmaceutically acceptable carrier or excipient.

28. A kit comprising the double-stranded RNA molecule according to any one of claims 19 to 21.

29. 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 19 to 21 into the cell.

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