Cyclic phosphonate-modified nucleotide

Abstract

The present invention relates to cyclo-phosphonic acid-modified oligonucleotides. The oligonucleotides of the present invention exhibit one or more of improved stability, reduced off-target toxicity, and improved efficacy.

Description

Technical Field

[0001] The present invention belongs to the field of medicine, and particularly relates to double-stranded RNA having a cyclic phosphonate ester structure.

Background Art

[0002] RNA interference is an efficient and specific degradation phenomenon of target mRNA induced by double-stranded RNA (also known as dsRNA or siRNA).

[0003] The 5'-phosphate of the guide strand (antisense strand) of siRNA forms an electrostatic interaction with the cationic amino acid residues near the interface of the MID and PIWI structural domains of the Ago2 protein, thereby stabilizing the complex formed by the guide strand and Ago2. Thus, the 5'-phosphate of the siRNA guide strand is essential for RNAi-based gene silencing. However, the 5'-phosphate can be cleaved by endogenous phosphatases, losing the 5'-terminal phosphate and potentially causing a significant decrease in siRNA activity.

[0004] By introducing a phosphonate-modified nucleotide 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 enhanced. For example, WO2011139702A2 and WO2013033230A1 disclose nucleotides containing 5'-vinylphosphonate (referred to as E-VP or VP), where 5'-E-VP can replace 5'-phosphate for binding to Ago2 and is highly resistant to endogenous phosphatases, thereby improving the activity and / or stability of siRNA.

[0005] WO2017214112A1 discloses nucleotides containing 5'-cyclic phosphonate ester.

[0006] However, the inventors have found that the 5'-E-VP is still bound to the sugar ring via a C-C single bond that rotates freely, and as a result, the phosphonate can be in different positions relative to the sugar ring (see the figure below. The position of ap, i.e., the unmodified 5'-phosphate, is the conformation that binds to the Ago2 protein).

[0007]

Chemical Structure

[0008] A significant portion of these conformations (e.g., +sc or -sc) may be unfavorable for binding to the Ago2 cationic region. Therefore, in this field, there is a need to develop phosphonate ester modifications that can fix the Ago2-binding conformation.

Summary of the Invention

Means for Solving the Problems

[0009] The inventors unexpectedly discovered that restricting the rotation of the terminal phosphonic acid by a rigid ring structure and fixing it in the Ago2-binding conformation promotes further enhancement of siRNA activity.

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

Chemical Structure

Chemical Structure

[0011] X and Y are each independently CR a R b or (CRa R b ) 2. Preferably CR a R b is selected from the following.

[0012] Z is CR a .

[0013] R a and R b are independently selected from H, D, halogen, OH, NH2, CN, C 1-6 alkyl, C 1-6 haloalkyl, C 1-6 alkoxy, C 1-6 haloalkoxy, C 2-6 alkenyl or C 2-6 alkynyl, and the R a and R b are optionally further substituted with one, two, three, four or five independently selected R#. R1 is selected from O or S.

[0014] R2 and R3 are independently selected from OH, SH, NH2, C 1-6 alkyl, C 1-6 haloalkyl, C 1-6 alkoxy or C 1-6 haloalkyl, and the R2 and R3 are optionally further substituted with one, two, three, four or five independently selected R#. R# is selected from H, D, halogen, C 1-6 alkyl, C 1-6 haloalkyl, C 2-6 alkenyl or C 2-6 alkynyl. Here, the definition of each of the aforementioned groups is optionally substituted with one, two, three, four, five or more deuterium atoms until fully deuterated.

[0015] Without being bound by a particular theory, the structure of the present invention includes a four-membered carbon ring, which is a relatively rigid structure that allows the oligonucleotide of the present invention (especially the phosphonate ester group at its 5'-end) to bind better to the Ago2 protein, thereby increasing the activity of the oligonucleotide.

[0016] Through molecular simulation, the compound of the present invention can fix the phosphonic acid in an ap conformation very similar to that of unmodified 5'-phosphate-bound Ago2, in which the phosphonate completely matches Arg812, Lys570, Lys566 and Lys533, forming multiple salt bridge bonds and hydrogen bonds, and further forming hydrogen bonds with Tyr529 and Cys526 respectively. Furthermore, the base (uracil) of this cyclic phosphonic acid compound forms hydrogen bonds with the backbone amides of Thr526 and Gly524 respectively. Due to the combined effect of these structures, the binding of the compound of the present invention to the Ago2 protein is stabilized.

[0017] Through molecular simulation, similar to the spiro compound, due to the binding of a sugar ring-linked highly rigid aromatic ring (including a five-membered aromatic ring and a six-membered aromatic ring), the 5'-terminal phosphate is locked in a specific conformation that highly mimics the ap conformation of the natural 5'-phosphate, and the phosphonate completely matches Arg812, Lys570, Lys566 and Lys533, forming multiple salt bridge bonds and hydrogen bonds, and further forming hydrogen bonds with Tyr529 and Cys526 respectively. The base uracil on the sugar ring also forms hydrogen bonds with the backbone amides of Thr526 and Gly524 respectively. Due to the combined effect of these structures, the binding of the compound of the present invention to the Ago2 protein is stabilized.

[0018] Description of the Invention Definitions Chemical Definitions The definitions of specific functional groups and chemical terms will be described in detail below.

[0019] When enumerating a range of values, the established range includes each value and sub-ranges within that 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 , C 3-4 , C 4-6 , C 4-5 and C 5-6 alkyl.

[0020] "C 1-6 alkyl" means a straight-chain or branched-chain saturated hydrocarbon having 1 to 6 carbon atoms. In some embodiments, C 1-4 alkyl and C 1-2 alkyl are preferred. Examples of C 1-6 alkyl 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), n-hexyl (C6). The term "C 1-6 alkyl" also includes heteroalkyl 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). Alkyl may be optionally substituted with one or more substituents, e.g., 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).

[0021] "C 2-6"Alkenyl" means a straight-chain or branched-chain hydrocarbon having 2 to 6 carbon atoms and at least one carbon-carbon double bond. In some embodiments, C 2-4 alkenyl is preferred. C 2-6 Examples of alkenyl include vinyl (C2), 1-propenyl (C3), 2-propenyl (C3), 1-butenyl (C4), 2-butenyl (C4), butadiene (C4), pentenyl (C5), pentadienyl (C5), hexene (C6), and the like. The term "C 2-6 alkenyl" also includes heteroalkenyl in which one or more (e.g., 1, 2, 3, or 4) carbon atoms are substituted with heteroatoms (e.g., oxygen, sulfur, nitrogen, boron, silicon, phosphorus). Alkenyl may be optionally substituted with one or more substituents, such as 1 to 5 substituents, 1 to 3 substituents, or 1 substituent.

[0022] "C 2-6 alkynyl" means a straight-chain or branched-chain hydrocarbon having 2 to 6 carbons, 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. C 2-6 Examples of alkynyl include ethynyl (C2), 1-propynyl (C3), 2-propynyl (C3), 1-butyne (C4), 2-butyne (C4), pentynyl (C5), acetylene (C6), and the like, but are not limited thereto. The term "C 2-6 alkynyl" also includes heteroalkynyl in which one or more (e.g., 1, 2, 3, or 4) carbon atoms are substituted with heteroatoms (e.g., oxygen, sulfur, nitrogen, boron, silicon, phosphorus). Alkynyl may be optionally substituted with one or more substituents, such as 1 to 5 substituents, 1 to 3 substituents, or 1 substituent.

[0023] "Halogenated" or "halogen" means fluorine (F), chlorine (Cl), bromine (Br), iodine (I).

[0024] Thus, "C 1-6"Alkyl halide" means the above-mentioned "C" substituted with one or more halogens. 1-6 In some embodiments, C 1-4 alkyl halide is particularly preferred, more preferably C 1-2 alkyl halide. Exemplary alkyl halides include, but are not limited to, -CF3, -CH2F, -CHF2, -CHFCH2F, -CH2CHF2, -CF2CF3, -CCl3, -CH2Cl, -CHCl2, 2,2,2-trifluoro-1,1-dimethyl-ethyl, etc. The alkyl halide may be substituted with available bonding sites, such as 1 to 5 substituents, 1 to 3 substituents, or 1 substituent.

[0025] "C 1-6 alkoxy" means an -O-R group, where R is as defined for the above-mentioned "C 1-6 alkyl".

[0026] "C 1-6 halogenated alkoxy" means an -O-R group, where R is as defined for the above-mentioned "C 1-6 alkyl halide".

[0027] "C 5-6 cycloalkyl" means a non-aromatic cyclic hydrocarbon having 5 to 6 cyclic carbon atoms and zero heteroatoms. Cycloalkyl further includes a ring system in which the cycloalkyl ring is bonded to one or more aryl or heteroaryl groups, where the bonding point is on the cycloalkyl ring and the carbon number continues to represent the carbon number of the cycloalkyl system. Exemplary cycloalkyls include, but are not limited to, cyclopentyl (C5), cyclopentenyl (C5), cyclohexyl (C6), cyclohexenyl (C6), cyclohexadienyl (C6), etc. Cycloalkyl may be optionally substituted with one or more substituents, such as 1 to 5 substituents, 1 to 3 substituents, or 1 substituent.

[0028] The "5- or 6-membered heterocyclic ring" refers to a group of a 5- or 6-membered non-aromatic ring system having cyclic carbon atoms and 1 to 3 cyclic heteroatoms, and each heteroatom is independently selected from nitrogen, oxygen, sulfur, boron, phosphorus, and silicon. In a heterocyclic ring containing one or more nitrogen atoms, as long as the valence allows, the bonding point may be a carbon atom or a nitrogen atom. The heterocyclic ring further includes a ring system in which the above heterocyclic ring is bonded with one or more cycloalkyls, where the bonding point is on the cycloalkyl ring, or a ring system in which the above heterocyclic ring is bonded with one or more aryls or heteroaryls, where the bonding point is on the heterocyclic ring. In such a case, the number of ring members continues to indicate the number of ring members in the heterocyclic ring system. Exemplary 5-membered heterocyclic rings containing one heteroatom include, but are not limited to, tetrahydrofuran, dihydrofuran, tetrahydrothiophene, dihydrothiophene, pyrrolidine, dihydropyrrole, and pyrrole-2,5-dione. Exemplary 5-membered heterocyclic rings containing two heteroatoms include, but are not limited to, dioxolane alkyl, oxasulfuranyl, disulfuranyl, and oxazolidin-2-one. Exemplary 5-membered heterocyclic rings containing two heteroatoms include, but are not limited to, triazolyl, oxazoline, and thiazoline. Exemplary 6-membered heterocyclic rings containing one heteroatom include, but are not limited to, piperidyl, tetrahydropyran, dihydropyridinyl, and thianyl. Exemplary 6-membered heterocyclic rings containing two heteroatoms include, but are not limited to, piperazinyl, morpholine, dithiocyclohexyl, and dioxinyl. Exemplary 6-membered heterocyclic rings containing three heteroatoms include, but are not limited to, triazinanyl. The heterocyclic ring may be optionally substituted with one or more substituents, for example, 1 to 5 substituents, 1 to 3 substituents, or 1 substituent.

[0029] The alkyl, alkenyl, alkynyl, cycloalkyl, and heterocyclic ring defined in this article are optionally substituted groups.

[0030] Exemplary substituents on a carbon atom include halogen, -CN, -NO2, -N3, -SO2H, -SO3H, -OH, -OR aa , -ON(R bb )2, -N(R bb )2, -N(R bb )3 + X - , -N(OR cc )R bb , -SH, -SR aa , -SSR cc , -C(=O)R aa , -CO2H, -CHO, -C(OR cc )2, -CO2R aa , -OC(=O)R aa , -OCO2R aa , -C(=O)N(R bb )2, -OC(=O)N(R bb )2, -NR bb C(=O)R aa , -NR bb CO2R aa , -NR bb C(=O)N(R bb )2, -C(=NR bb )R aa , -C(=NR bb )OR aa , -OC(=NR bb )R aa , -OC(=NR bb )OR aa , -C(=NR bb )N(R bb )2, -OC(=NR bb )N(R bb )2, -NR bb C(=NR bb )N(R bb )2, -C(=O)NR bb SO2R aa , -NR bb SO2R aa , -SO2N(R bb )2, -SO2R aa , -SO2OR aa , -OSO2R aa , -S(=O)R aa , -OS(=O)R aa , -Si(R aa)3, -OSi(R aa )3, -C(=S)N(R bb )2, -C(=O)SR aa , -C(=S)SR aa , -SC(=S)SR aa , -SC(=O)SR aa , -OC(=O)SR aa , -SC(=O)OR aa , -SC(=O)R aa , -P(=O)2R aa , -OP(=O)2R aa , -P(=O)(R aa )2, -OP(=O)(R aa )2, -OP(=O)(OR cc )2, -P(=O)2N(R bb )2, -OP(=O)2N(R bb )2, -P(=O)(NR bb )2, -OP(=O)(NR bb )2, -NR bb P(=O)(OR cc )2, -NR bb P(=O)(NR bb )2, -P(R cc )2, -P(R cc )3, -OP(R cc )2, -OP(R cc )3, -B(R aa )2, -B(OR cc )2, -BR aa (OR cc ), alkyl, alkyl halide, alkenyl, alkynyl, cycloalkyl, heterocycle, aryl, and heteroaryl are exemplified, but not limited thereto. Here, each alkyl, alkenyl, alkynyl, cycloalkyl, heterocycle, aryl, and heteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5 R dd groups.

[0031] Or two hydrogen couplings on a carbon atom are =O, =S, =NN(R bb )2, =NNR bb C(=O)R aa , =NNR bb C(=O)OR aa , =NNRbb S(=O)2R aa 、=NR bb or =NOR cc and is substituted therewith.

[0032] R aa Each of which is independently selected from alkyl, halogenated alkyl, alkenyl, alkynyl, cycloalkyl, heterocycle, aryl and heteroaryl, or two R aa groups are bonded to form a heterocyclic or heteroaryl ring, and each alkyl, alkenyl, alkynyl, cycloalkyl, heterocycle, aryl and heteroaryl is independently substituted with 0, 1, 2, 3, 4 or 5 R dd groups.

[0033] R bb are each independently 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, halogenated alkyl, alkenyl, alkynyl, cycloalkyl, heterocycle, aryl and heteroaryl, or two R bbThe base is bonded to form a complex ring or a heteroaryl ring, where each alkyl, alkenyl, alkynyl, cycloalkyl, complex ring, aryl, and heteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5 R dd groups.

[0034] Each R cc is independently selected from hydrogen, alkyl, halogenated alkyl, alkenyl, alkynyl, cycloalkyl, complex ring, aryl and heteroaryl, or two R cc groups are bonded to form a complex ring or a heteroaryl ring, and each alkyl, alkenyl, alkynyl, cycloalkyl, complex ring, aryl and heteroaryl is independently substituted with 0, 1, 2, 3, 4 or 5 R dd groups.

[0035] Each R dd is independently halogen, -CN, -NO2, -N3, -SO2H, -SO3H, -OH, -OR ee , -ON(R ff )2, -N(R ff )2, -N(R ff )3 + X - , -N(OR ee )R ff , -SH, -SR ee , -SSR ee , -C(=O)R ee , -CO2H, -CO2R ee , -OC(=O)R ee , -OCO2R ee , -C(=O)N(R ff )2, -OC(=O)N(R ff )2, -NR ff C(=O)R ee , -NR ff CO2R ee , -NR ff C(=O)N(R ff )2, -C(=NR ff )OR ee , -OC(=NR ff )R ee , -OC(=NR ff )ORee , -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, halogenated alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, wherein each alkyl, alkenyl, alkynyl, cycloalkyl, heterocycle, aryl, heteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5 R gg groups, or two R dd substituents may be joined to form =O or =S.

[0036] R ee each of which is independently selected from alkyl, halogenated alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heterocycle, and heteroaryl, wherein each alkyl, alkenyl, alkynyl, cycloalkyl, heterocycle, aryl, and heteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5 R gg groups.

[0037] R ffEach of them is independently selected from hydrogen, alkyl, halogenated alkyl, alkenyl, alkynyl, cycloalkyl, heterocycle, aryl and heteroaryl, or two R ff groups are bonded to form a heterocyclic or heteroaryl ring, and each alkyl, alkenyl, alkynyl, cycloalkyl, heterocycle, aryl and heteroaryl is independently substituted with 0, 1, 2, 3, 4 or 5 R gg groups.

[0038] Each of R gg is independently halogen, -CN, -NO2, -N3, -SO2H, -SO3H, -OH, -OC 1-6 alkyl, -ON(C 1-6 alkyl)2, -N(C 1-6 alkyl)2, -N(C 1-6 alkyl)3 + X - , -NH(C 1-6 alkyl)2 + X - , -NH2(C 1-6 alkyl) + X - , -NH3 + X - , -N(OC 1-6 alkyl)(C 1-6 alkyl), -N(OH)(C 1-6 alkyl), -NH(OH), -SH, -SC 1-6 alkyl, -SS(C 1-6 alkyl), -C(=O)(C 1-6 alkyl), -CO2H, -CO2(C 1-6 alkyl), -OC(=O)(C 1-6 alkyl), -OCO2(C 1-6 alkyl), -C(=O)NH2, -C(=O)N(C 1-6 alkyl)2, -OC(=O)NH(C 1-6 alkyl), -NHC(=O)(C 1-6 alkyl), -N(C 1-6 alkyl)C(=O)(C 1-6 alkyl), -NHCO2(C 1-6 alkyl), -NHC(=O)N(C 1-6(alkyl)2, -NHC(=O)NH(C 1-6 (alkyl), -NHC(=O)NH2, -C(=NH)O(C 1-6 (alkyl), -OC(=NH)(C 1-6 (alkyl), -OC(=NH)OC 1-6 (alkyl, -C(=NH)N(C 1-6 (alkyl)2, -C(=NH)NH(C 1-6 (alkyl), -C(=NH)NH2, -OC(=NH)N(C 1-6 (alkyl)2, -OC(NH)NH(C 1-6 (alkyl), -OC(NH)NH2, -NHC(NH)N(C 1-6 (alkyl)2, -NHC(=NH)NH2, -NHSO2(C 1-6 (alkyl), -SO2N(C 1-6 (alkyl)2, -SO2NH(C 1-6 (alkyl), -SO2NH2, -SO2C 1-6 (alkyl), -SO2OC 1-6 (alkyl), -OSO2C 1-6 (alkyl), -SOC 1-6 (alkyl), -Si(C 1-6 (alkyl)3, -OSi(C 1-6 (alkyl)3, -C(=S)N(C 1-6 (alkyl)2, C(=S)NH(C 1-6 (alkyl), C(=S)NH2, -C(=O)S(C 1-6 (alkyl), -C(=S)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 haloalkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C7 cycloalkyl, C6-C 10 aryl, C3-C7 heterocycle, C5-C 10 heteroaryl. Or, two Rs ggA substituent may be bonded to form =O or =S. Here, X- is a counter ion.

[0039] Exemplary substituents on the nitrogen atom include hydrogen, -OH, -OR aa , -N(R cc )2, -CN, -C(=O)R aa , -C(=O)N(R cc )2, -CO2R aa , -SO2R aa , -C(=NR bb )R aa , -C(=NR cc )OR aa , -C(=NR cc )N(R cc )2, -SO2N(R cc )2, -SO2R cc , -SO2OR cc , -SOR aa , -C(=S)N(R cc )2, -C(=O)SR cc , -C(=S)SR cc , -P(=O)2R aa , -P(=O)(R aa )2, -P(=O)2N(R cc )2, -P(=O)(NR cc )2, alkyl, halogenated alkyl, alkenyl, alkynyl, cycloalkyl, heterocycle, aryl, and heteroaryl, or two R cc groups bonded to the nitrogen atom form a heterocycloalkyl or heteroaryl ring, where each alkyl, alkenyl, alkynyl, cycloalkyl, heterocycle, aryl, and heteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5 R dd groups, and R aa , R bb , R cc , and R dd are as defined above.

[0040] Other definitions In this manuscript, the term "siRNA" refers to a double-stranded RNA molecule that can mediate the silencing of a target RNA complementary thereto (e.g., mRNA, e.g., the transcription product of a gene encoding a protein). siRNA is usually a double strand containing an antisense strand complementary to the target RNA and a sense strand complementary to the antisense strand. For convenience, such an mRNA is also referred to herein as the mRNA to be silenced here. Such a gene is also called a target gene. Usually, the RNA to be silenced is an endogenous gene or a pathogen gene. Furthermore, RNAs other than mRNA (e.g., tRNA) and viral RNAs can also be targeted.

[0041] The "antisense strand" means the strand of siRNA containing a region that is completely, sufficiently, or substantially complementary to the target sequence. The term "sense strand" means the strand of siRNA containing a region that is completely, sufficiently, or substantially complementary to the region that is the antisense strand as defined by the terms in this manuscript.

[0042] The "complementary region" means the region on the antisense strand that is completely, sufficiently, or substantially complementary to the target mRNA sequence. If the complementary region is not completely complementary to the target sequence, the mismatch can be located in the internal or terminal region of the molecule. Generally, the most resistant mismatches are located within 5, 4, 3, 2, or 1 nucleotide of the terminal region, e.g., the 5' and / or 3' termini. The part of the antisense strand that is most sensitive to mismatches is called the "seed region". For example, in an siRNA with a 19nt strand, the 19th position (from 5' to 3') can tolerate some mismatches.

[0043] The term "complementary" refers to the ability of a first polynucleotide to hybridize with a second polynucleotide under certain conditions, such as stringent conditions. For example, stringent conditions can include 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA, 50 °C or 70 °C, for 12-16 hours. In terms of meeting the above requirements regarding the ability to hybridize, "complementary" sequences may include, or may consist solely of, 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 pairs or Hoogstein base pairs.

[0044] A polynucleotide that is "at least partially complementary", "sufficiently complementary", or "substantially complementary" to a messenger RNA (mRNA) means a polynucleotide that is substantially complementary to the binding portion of the target mRNA. For example, if a sequence is substantially complementary to a continuous portion of the mRNA encoding PCSK9, the polynucleotide is at least partially complementary to PCSK9 mRNA. In this article, the terms "complementary", "fully complementary", "sufficiently complementary", and "substantially complementary" are used for base pair formation between the sense strand and the antisense strand of an siRNA, or between the antisense strand of an siRNA reagent and the target sequence.

[0045] "Sufficiently complementary" means that the sense strand only needs to be complementary to the antisense strand in order to maintain the double-stranded characteristic of the whole molecule. In other words, normally complete complementarity is required, but in some cases, especially in the antisense strand, one or more, for example 6, 5, 4, 3, 2, or 1 mismatches may be included (with respect to the target mRNA), yet still the sense strand and the antisense strand can maintain the overall double-stranded characteristic of the molecule.

[0046] "shRNA" means short hairpin RNA. shRNA consists of two short inverted repeat sequences. The shRNA cloned into the shRNA expression vector consists of two short inverted repeat sequences separated by a loop sequence, forming a hairpin structure controlled by the polIII promoter. Subsequently, 5 to 6 Ts are ligated as the transcription terminator of RNA polymerase III.

[0047] "Nucleoside" is a compound composed of two substances, a purine base or a pyrimidine base and ribose or deoxyribose. "Nucleotide" is a compound composed of three substances, a purine base or a pyrimidine base, ribose or deoxyribose, and phosphoric acid. "Oligonucleotide" means, for example, a nucleic acid molecule (RNA or DNA) having a length of less than 100, 200, 300, or 400 nucleotides, which may be single-stranded or double-stranded.

[0048] "Nucleic acid base" is the basic unit for synthesizing nucleosides, nucleotides, and nucleic acids. Its constituent elements contain nitrogen and it is also called "nitrogenous base". In this article, unless otherwise specified, the capital letters A, U, T, G, and C represent the nucleic acid base compositions of the nucleotides adenine, uracil, thymine, guanine, and cytosine, respectively.

[0049] The "modifications" of the nucleotides described in this article include, but are not limited to, methoxy modification, fluorination modification, phosphorothioate bond, or protection with conventional protecting groups. For example, the fluorinated modified nucleotide refers to a nucleotide formed by substituting the hydroxyl group at the 2'-position of the ribose of the nucleotide with fluorine, and the methoxy modified nucleotide refers to a nucleotide formed by substituting the 2'-hydroxyl of the ribose with methoxy.

[0050] The "modified nucleotide" used in this manuscript includes, but is not limited to, 2'-O-methyl modified nucleotide, 2'-fluorinated modified nucleotide, 2'-deoxy-modified nucleotide, inosine modified ribonucleotide, deprotonated nucleotide, inverted abasic deoxyribonucleotide, nucleotide containing thiophosphate ester, vinylphosphonate modified nucleotide, locked nucleotide, 2'-amino-modified nucleotide, 2'-alkyl-modified nucleotide, morpholino nucleotide, phosphoramidate, unnatural base containing nucleotide, and terminal nucleotide, deoxyribonucleotide or conventional protecting group protection etc. bound to cholesterol-based derivatives or dodecanedioic acid dodecaneamide. For example, the 2'-fluorinated modified nucleotide refers to a nucleotide formed by substitution of the hydroxyl at the 2'-position of the ribose of the nucleotide with fluorine. The 2'-deoxy-modified nucleotide refers to a nucleotide formed by substitution of the 2'-hydroxyl of the ribose with methoxy.

[0051] The "reactive phosphate group" is a phosphorus-containing group contained in a nucleotide unit or a nucleotide analog unit, which can react with a hydroxyl or amino contained in another molecule, particularly another nucleotide unit or another nucleotide analog, by a nucleophilic substitution reaction. Typically, such a reaction generates a phosphodiester bond that binds the first nucleotide unit or the first nucleotide analog unit to the second nucleotide unit or the second nucleotide analog unit. The reactive phosphate group can be selected from phosphoramidite, H-phosphonate ester, alkyl-phosphonate ester, phosphate ester, or phosphate ester analog. It includes, but is not limited to, natural phosphate ester, thiophosphate, phosphorodithioate, borane phosphate ester, borane phosphorothioate, phosphonate ester, halogen-substituted phosphonate ester and phosphate ester, phosphoramidate, phosphodiester, triphosphate, dibasic phosphodiester, tribasic phosphate triester, bisphosphate, and phosphate tris. Preferably, it is -P(OCH2CH2CN)(N(iPr)2).

[0052] The "protecting group", also called "protecting atomic group", means an atom or atomic group added to a molecule to prevent unwanted chemical reactions of existing groups within the molecule. The "protecting group" may be an unstable chemical moiety known in the art and is used to protect reactive groups such as hydroxyl, amino, mercaptan groups, etc. to prevent unwanted or premature reactions during chemical synthesis. The protecting group is usually used selectively and / or orthogonally to protect that site during reaction with other reaction sites, and then the unprotected groups can either be left intact or removed so as to be available for further reactions.

[0053] Non-limiting lists of protecting groups include benzyl, benzyl substitution, alkylcarbonyl and alkoxycarbonyl (e.g., Tert-butoxycarbonyl (Boc), acetyl or isobutyryl), aryl-alkyl-carbonyl and alkoxycarbonyl (e.g., benzyloxycarbonyl), methyl ether substitution (e.g., methoxymethyl ether), ethyl ether substitution, benzyl ether substitution, tetrahydropyran ether, silyl (e.g., trimethylsilyl, triethylsilanol, triisopropylsilyl, Tert-butyldimethylsilyl, triisopropylsilyloxymethyl, [2-(trimethylsilyl)ethylene glycol mono]methyl or Tert-butyldiphenylsilyl), ester (e.g., benzoate), carbonate ester (e.g., methyl methoxymethyl carbonate), sulfonate (e.g., tosylate or methanesulfonate), acyclic ketone (e.g., dimethyl acetal), cyclic ketone (e.g., 1,3-dioxane, 1,3-dioxolane and those described herein), acyclic acetal, cyclic acetal (e.g., those described herein), acyclic hemiacetal, cyclic hemiacetal, dithioacetal (e.g., 1,3-dithiane or 1,3-dithiolane), orthoester (e.g., those described herein) and triarylmethyl (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, benzyloxyacetaldehyde dimethyl acetal (BOM), β - methoxyethoxymethyl ether (MEM), methoxymethyl ether (MOM), p - methoxybenzyl ether (PMB), methylthiomethyl ether, tert - butoxycarbonyl (Piv), tetrahydropyran (THP), trityl (Trt), methoxytrityl [(4 - methoxyphenol)diphenylmethyl](MMT), 4,4’ - dimethoxytrityl, bis(4 - methoxyphenyl)phenylmethanol (DMT), trimethylsilyl ether (TMS), tert - butyldimethylsilyl ether (TBDMS), triisopropylsilyloxymethyl ether (TOM), triisopropylsilyl ether (TIPS), methyl ether, ethylene glycol monoethyl ether (EE), N,N - dimethylformamidine and 2 - cyanoethyl (CE).

[0054] The term "hydroxyl protecting group" means a group that can protect hydroxyl from chemical reactions and can be removed under specific conditions to restore hydroxyl. It mainly includes silyl - type protecting groups, acyl - type protecting groups or ether - type protecting groups, preferably as follows.

[0055] Trimethylsilyl (TMS), triethylsilanol (TES), dimethylisopropylsilyl (DMIPS), diethylisopropylsilyl (DEIPS), tert-butyldimethylsilyl (TBDMS), tert-butyldiphenylsilyl (TBDPS), triisopropylsilyl (TIPS), acetyl (Ac), acetyl chloride, dichloroacetyl, trichloroacetyl, trifluoroacetyl (TFA), benzoyl, methoxybenzoyl, 9-fluorenylmethyloxycarbonyl (Fmoc), allyloxycarbonyl (Alloc), 2,2,2-trichloroethoxycarbonyl (Troc), benzyloxycarbonyl (Cbz), tert-butoxycarbonyl (Boc), benzyl (Bn), methoxybenzyl (PMB), allyl compound, trityl (Tr), 4,4-dimethoxytrityl (DMTr), methoxymethyl (MOM), phenoxymethyl (BOM), 2,2,2-trichloroethoxymethyl, 2-methoxyethoxymethyl (MEM), methylthiomethyl (MTM), 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.

[0056] As used herein, the term "pharmaceutically acceptable salt" refers to carboxylate salts and amino acid addition salts that are suitable for use in contact with patient tissues without undue toxicity, irritation, allergic reaction, etc. within the scope of sound medical judgment, are commensurate with a reasonable benefit / risk ratio, and (if possible) include the zwitterionic forms of the compounds of the present invention and are effective for the intended uses. The present invention includes tautomers, which are functional group isomers resulting from the rapid movement of atoms at two positions within a molecule. Compounds exist in different tautomeric forms, and the compounds are not limited to specific tautomers and are intended to cover all tautomeric forms.

[0057] The compounds of the present invention may contain one or more asymmetric centers and, as such, may exist in various stereoisomeric forms, such as enantiomers and / or diastereoisomers. For example, the compounds of the present invention may be in the form of individual enantiomers, diastereoisomers or geometric isomers (e.g., cis-trans isomers), or in the form of mixtures of stereoisomers including racemates and mixtures enriched in one or more stereoisomers. The isomers can be isolated from the mixtures by methods known to those skilled in the art, including chiral high performance liquid chromatography (HPLC) and the formation and crystallization of chiral salts. Alternatively or preferably, the isomers can be prepared by asymmetric synthesis.

[0058] The present invention also includes isotopically labeled compounds (isotope variants) which are equivalent to those described by formula (I) but in which one or more atoms are replaced by atoms having an atomic mass or mass number different from that generally found in nature. Examples of isotopes which can be incorporated into the compounds of the present invention include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine and chlorine, such as 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 isotopes and / or isotopes of other atoms, prodrugs thereof, and pharmaceutically acceptable salts of said compounds or said prodrugs are within the scope of the present invention. Compounds of the present invention labeled with certain isotopes, for example radioactive isotopes (e.g., 3 H and 14 C), can be used for the measurement of pharmaceuticals and / or basal tissue distribution. Tritium, i.e., 3 H and carbon-14, i.e., 14 C isotopes are particularly preferred because they are easy to prepare and detect. Furthermore, heavier isotopes, such as deuterium, i.e.,2 Replacement with H may be preferred in some cases because it can provide therapeutic benefits, such as an extended in vivo half-life or a reduced dose requirement, due to higher metabolic stability. The isotopically labeled compounds of formula (I) of the present invention and their prodrugs can generally be prepared by replacing non-isotopically labeled reagents with isotopically labeled reagents that are readily available, by performing the steps disclosed in the steps and / or examples and preparation examples listed below.

[0059] The compounds of the present invention The present invention relates to an oligonucleotide or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, wherein the 5'-end of the oligonucleotide contains a structure represented by formula (I), [Chemical formula] wherein each group is as defined in the context.

[0060] The present invention also relates to an oligonucleotide or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, wherein the 5'-end of the oligonucleotide contains a structure represented by formula (II), [Chemical formula] wherein each group is as defined in the context.

[0061] The present invention also relates to an oligonucleotide or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, wherein the 5'-end of the oligonucleotide contains a single nucleotide represented by formula (III), formula (IV) or formula (V), [Chemical formula] wherein each group is as defined in the context.

[0062] The present invention also relates to a compound represented by formula (VI), formula (VII) or formula (VIII) or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, [Chemical formula] Here, each group is as defined in the context.

[0063]

Chemical formula

Chemical formula

Chemical formula

[0064] X, Y, and Z In one embodiment, X is CR a R b , preferably CH2, and in another embodiment, X is (CR a R b )2.

[0065] In one embodiment, Y is CR a R b , preferably CH2, and in another embodiment, Y is (CR a R b )2.

[0066] In one embodiment, Z is CR a , preferably CH.

[0067] Here, X and Y are optionally substituted with one or two deuterium atoms, and Z is optionally substituted with one deuterium atom.

[0068] L1, L2, and L3 In one embodiment, L1 is a chemical bond, and in another embodiment, L1 is O, and in another embodiment, L1 is S, and in another embodiment, L1 is C 1-4 alkylene.

[0069] In one embodiment, L2 is C 1-4 alkylene, preferably C 1-2 alkylene.

[0070] In one embodiment, L3 is C 1-4 alkylene, preferably C 1-2 alkylene.

[0071] In one embodiment, L2 and L3 are connected and together with L1 and its adjacent carbon atoms form C 5-6 cycloalkyl, and in another embodiment, L2 and L3 are connected and together with L1 and its adjacent carbon atoms form a 5- to 6-membered heterocyclic ring, preferably U, L1, L2 together with L3 and its adjacent carbon atoms form ribose or deoxyribose, and is optionally substituted with R5.

[0072] In one embodiment, L1 is unsubstituted. In another embodiment, L1 is further substituted with one R#. In another embodiment, L1 is further substituted with two independently selected R#s. In another embodiment, L1 is further substituted with three independently selected R#s. In another embodiment, L1 is further substituted with four independently selected R#s. In another embodiment, L1 is further substituted with five independently selected R#s.

[0073] In one embodiment, L2 is unsubstituted. In another embodiment, L2 is further substituted with one R#. In another embodiment, L2 is further substituted with two independently selected R#s. In another embodiment, L2 is further substituted with three independently selected R#s. In another embodiment, L2 is further substituted with four independently selected R#s. In another embodiment, L2 is further substituted with five independently selected R#s.

[0074] In one embodiment, L3 is unsubstituted; in another embodiment, L3 is further substituted with one R#; in another embodiment, L3 is further substituted with two independently selected R#; in another embodiment, L3 is further substituted with three independently selected R#; in another embodiment, L3 is further substituted with four independently selected R#; in another embodiment, L3 is further substituted with five independently selected R#.

[0075] Here, the aforementioned L1, L2, and L3 are optionally substituted with one, two, three, four, five or more deuterium atoms until they are fully deuterated.

[0076] U In one embodiment, U is O; in another embodiment, U is S; in another embodiment, U is CR a R b , for example CH2; in another embodiment, U is NR c , for example NH.

[0077] Here, the aforementioned U is optionally substituted with one or more deuterium atoms until it is fully deuterated.

[0078] Q In one embodiment, Q is H; in another embodiment, Q is D; in another embodiment, Q is a halogen; in another embodiment, Q is OH; in another embodiment, Q is C 1-6 alkyl, for example C 1-4 alkyl; in another embodiment, Q is C 1-6 haloalkyl, for example C 1-4 haloalkyl; in another embodiment, Q is C 1-6 alkoxy, for example C 1-4 alkoxy; in another embodiment, Q is C 1-6 haloalkoxy, for example C 1-4 haloalkoxy; in another embodiment, Q is C 2-6 alkenyl, for example C 2-4is alkenyl, and in another embodiment, Q is C 2-6 alkynyl, for example C 2-4 is alkynyl.

[0079] Here, the aforementioned Q is optionally substituted with one, two, three, four, five or more deuterium atoms until it is fully deuterated.

[0080] R1 In one embodiment, R1 is O, and in another embodiment, R1 is S.

[0081] R2 and R3 In one embodiment, R2 is OH, in another embodiment, R2 is SH, in another embodiment, R2 is NH2, and in another embodiment, R2 is C 1-6 is alkyl, and in another embodiment, R2 is C 1-6 is halogenated alkyl, and in another embodiment, R2 is C 1-6 is alkoxy, and in another embodiment, R2 is C 1-6 is halogenated alkoxy.

[0082] In one embodiment, R2 is OR d and in another embodiment, R2 is OP1, and in another embodiment, R2 is SR d and in another embodiment, R2 is SP1, and in another embodiment, R2 is NR e R f and in another embodiment, R2 is unsubstituted, in another embodiment, R2 is further substituted with one R#, in another embodiment, R2 is further substituted with two independently selected R#, in another embodiment, R2 is further substituted with three independently selected R#, in another embodiment, R2 is further substituted with four independently selected R#, and in another embodiment, R2 is further substituted with five independently selected R#.

[0083]

[0084] ​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 haloalkyl; in another embodiment, R3 is C 1-6 alkoxy; in another embodiment, R3 is C 1-6 haloalkoxy.

[0085] 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 .

[0086] In one embodiment, R3 is unsubstituted; in another embodiment, R3 is further substituted with one R#; in another embodiment, R3 is further substituted with two independently selected R#s; in another embodiment, R3 is further substituted with three independently selected R#s; in another embodiment, R3 is further substituted with four independently selected R#s; in another embodiment, R3 is further substituted with five independently selected R#s.

[0087] Here, the aforementioned R2 and R3 are optionally substituted with one, two, three, four, five or more deuterium atoms until they are fully deuterated.

[0088] R4, R5, R6 and R7 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, such as C 1-4 alkyl; in another embodiment, R4 is C 1-6 haloalkyl, such as C 1-4is an alkyl halide, and in another embodiment, R4 is C 1-6 alkoxy, such as C 1-4 alkoxy, and in another embodiment, R4 is C 1-6 halogenated alkoxy, such as C 1-4 halogenated alkoxy.

[0089] In certain embodiments, 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, such as C 1-4 alkyl, and in another embodiment, R5 is C 1-6 halogenated alkyl, such as C 1-4 halogenated alkyl, and in another embodiment, R5 is C 1-6 alkoxy, such as C 1-4 alkoxy, and in another embodiment, R5 is C 1-6 halogenated alkoxy, such as C 1-4 halogenated alkoxy.

[0090] In certain embodiments, 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, such as C 1-4 alkyl, and in another embodiment, R6 is C 1-6 halogenated alkyl, such as C 1-4 halogenated alkyl, and in another embodiment, R6 is C 1-6 alkoxy, such as C 1-4 alkoxy, and in another embodiment, R6 is C 1-6 halogenated alkoxy, such as C 1-4 halogenated alkoxy.

[0091] In one embodiment, R7 is H; in another embodiment, R7 is D; in another embodiment, R7 is a halogen; in another embodiment, R7 is OH; in another embodiment, R7 is C 1-6 alkyl, such as C 1-4 alkyl; in another embodiment, R7 is C 1-6 haloalkyl, such as C 1-4 haloalkyl; in another embodiment, R7 is C 1-6 alkoxy, such as C 1-4 alkoxy; in another embodiment, R7 is C 1-6 haloalkoxy, such as C 1-4 haloalkoxy.

[0092] Here, the aforementioned R4, R5, R6, and R7 are optionally substituted with one, two, three, four, five, or more deuterium atoms until they are fully deuterated.

[0093] R a R b and R c In one embodiment, R a is H; in another embodiment, R a is D; in another embodiment, R a is a 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 is C 1-6 alkyl, such as C 1-4 alkyl; in another embodiment, R a is C 1-6 haloalkyl, such as C 1-4 haloalkyl; in another embodiment, R a is C 1-6 alkoxy, such as C 1-4 alkoxy; in another embodiment, R a is C 1-6 haloalkoxy, such as C1-4 is a halogenated alkoxy, and in another embodiment, R a is C 2-6 alkenyl, for example C 2-4 alkenyl, and in another embodiment, R a is C 2-6 alkynyl, for example C 2-4 alkynyl.

[0094] In one embodiment, R a is unsubstituted, and in another embodiment, R a is further substituted with one R#, and in another embodiment, R a is further substituted with two independently selected R#s, and in another embodiment, R a is further substituted with three independently selected R#s, and in another embodiment, R a is further substituted with four independently selected R#s, and in another embodiment, R a is further substituted with five independently selected R#s.

[0095] In one embodiment, R b is H, and in another embodiment, R b is D, and in another embodiment, R b is a halogen, and in another embodiment, R b is OH, and in another embodiment, R b is NH2, and in another embodiment, R b is CN, and in another embodiment, R b is C 1-6 alkyl, for example C 1-4 alkyl, and in another embodiment, R b is C 1-6 halogenated alkyl, for example C 1-4 halogenated alkyl, and in another embodiment, R b is C 1-6 alkoxy, for example C 1-4 alkoxy, and in another embodiment, R b is C 1-6 halogenated alkoxy, for example C 1-4is a halogenated alkoxy, and in another embodiment, R b is C 2-6 alkenyl, for example C 2-4 alkenyl, and in another embodiment, R b is C 2-6 alkynyl, for example C 2-4 alkynyl.

[0096] In one embodiment, R b is unsubstituted, and in another embodiment, R b is further substituted with one R#, and in another embodiment, R b is further substituted with two independently selected R#, and in another embodiment, R b is further substituted with three independently selected R#, and in another embodiment, R b is further substituted with four independently selected R#, and in another embodiment, R b is further substituted with five independently selected R#.

[0097] In one embodiment, R c is H, and in another embodiment, R c is C 1-6 alkyl, and in another embodiment, R c is C 1-6 halogenated alkyl, and in another embodiment, R c is C 2-6 alkenyl, and in another embodiment, R c is C 2-6 alkynyl.

[0098] Here, the aforementioned R a , R b and R c are optionally substituted with one, two, three, four, five or more deuterium atoms until fully deuterated.

[0099] R d , R e and R f In one embodiment, R d is H, and in another embodiment, Rd is C 1-6 alkyl, such as C 1-4 alkyl, and in another embodiment, R d is C 1-6 haloalkyl, such as C 1-4 haloalkyl, and in another embodiment, R d is C 2-6 alkenyl, and in another embodiment, R d is C 2-6 is alkynyl.

[0100] In one embodiment, R d is unsubstituted, and in another embodiment, R d is optionally substituted with one or more D until fully deuterated, and in another embodiment, R d is substituted with halogen, and in another embodiment, R d is C 1-6 alkyl substituted, and in another embodiment, R d is C 1-6 substituted with haloalkyl.

[0101] In one embodiment, R e is H, and in another embodiment, R e is C 1-6 alkyl, such as C 1-4 alkyl, and in another embodiment, R e is C 1-6 haloalkyl, such as C 1-4 haloalkyl.

[0102] In one embodiment, R e is unsubstituted, and in another embodiment, R e is optionally substituted with one or more D until fully deuterated, and in another embodiment, R e is substituted with halogen, and in another embodiment, R e is C 1-6 alkyl substituted, and in another embodiment, R e is C 1-6 substituted with haloalkyl.

[0103] In certain embodiments, R f is H, and in another embodiment, R f is C 1-6 alkyl, e.g., C 1-4 alkyl, and in another embodiment, R f is C 1-6 haloalkyl, e.g., C 1-4 haloalkyl.

[0104] In certain embodiments, R f is unsubstituted, and in another embodiment, R f is optionally substituted with one or more D until fully deuterated, and in another embodiment, R f is substituted with halogen, and in another embodiment, R f is C 1-6 alkyl substituted, and in another embodiment, R f is C 1-6 haloalkyl substituted.

[0105] Here, the aforementioned R d , R e and R f are optionally substituted with one, two, three, four, five or more deuterium atoms until fully deuterated.

[0106] P1 and P2 In certain embodiments, P1 is a protecting group, and in another embodiment, P1 is a hydroxyl protecting group.

[0107] In certain embodiments, P2 is a reactive phosphate group, e.g., -P(OCH2CH2CN)(N(iPr)2).

[0108] Base and Base’ In certain embodiments, Base is H, and in another embodiment, Base is a modified or unmodified base or leaving group, and in another embodiment, Base is a modified or unmodified A, U, T, G, and C, e.g.,

Chemical formula

[0109] In one embodiment, Base’ is H; in another embodiment, Base’ is an unmodified or modified base or leaving group; in another embodiment, Base’ is an unmodified or modified A, U, T, G, and C, for example

Chemical formula

[0110] R# In one embodiment, R# is H; in another embodiment, R# is D; in another embodiment, R# is a halogen; in another embodiment, R# is C 1-6 alkyl; in another embodiment, R# is C 1-6 halogenated alkyl; in another embodiment, R# is C 2-6 alkenyl; in another embodiment, R# is C 2-6 alkynyl, wherein the aforementioned R# is optionally substituted with one, two, three, four, five or more deuterium atoms until it is fully deuterated.

[0111] GalNAc In one embodiment, GalNAc is a linking group represented by formula (X),

Chemical formula

Chemical formula

Chemical formula

Chemical formula

[0112] In another embodiment, GalNAc is a linking group represented by formula (I’),

Chemical formula

Chemical formula

Chemical formula

[0113] In another embodiment, GalNAc is a linking group represented by formula (X), Q G is independently H,

Chemical formula

Chemical Structure

[0114] In another embodiment, GalNAc is a linking group represented by formula (X), Q G is independently H,

Chemical formula

Chemical formula

[0115] Any aspect or any combination of the foregoing specific embodiments can be combined with any aspect or any combination of other specific embodiments. For example, any aspect or any combination of X can be combined with any aspect or any combination of Y, Z, L1, L2, L3, R1, R2, R3, R4, R5, R6, R7, U, Q, P1, P2, Base, and Base’. The present invention is intended to include all combinations of aspects and will not be enumerated one by one for the sake of brevity.

[0116] The present invention further provides a vector comprising a nucleotide sequence encoding the siRNA of the present invention. The vectors of the present invention can bind to amplify or express the nucleotides encoding the siRNA of the present invention.

[0117] For example, siRNAs targeting the PCSK9 gene can be expressed from transcription units inserted into DNA or RNA vectors. Expression can be transient (from a few hours to a few weeks) or constitutive (from a few weeks to several months or longer) and depends on the particular construct used and the target tissue or cell type. The coding nucleotides of the siRNA can be introduced into a linear construct, circular plasmid, or viral vector. The nucleotides of the siRNA can be integrated into the cellular genome for stable expression or stably inherited and expressed extrachromosomally. Generally, siRNA expression vectors are usually DNA plasmids or viral vectors.

[0118] Examples of viral vector systems comprising the coding sequence of siRNA include, but are not limited to, (a) adenoviral vectors, (b) retroviral vectors, (c) adeno-associated viral vectors, (d) herpes simplex viral vectors, (e) SV40 vectors, (f) polyomavirus vectors, (g) papillomavirus vectors, (h) microRNA viral vectors, (i) poxvirus vectors, and (j) helper virus-dependent adenoviruses or gutless adenoviruses.

[0119] The present invention further provides a cell containing the siRNA or vector of the present invention, and the siRNA or vector of the present invention can be transcribed intracellularly.

[0120] 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 contains a structure represented by the following formula:

Chemical formula

Chemical formula

[0121] Specifically, the present invention relates to the following.

[0122] 1. An oligonucleotide or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, wherein the 5'-end of the oligonucleotide contains a structure represented by formula (I):

Chemical formula

Chemical formula

[0123] X and Y are each independently CR a R b or (CR a R b )2, preferably selected from CR a R b .

[0124] Z is CR a .

[0125] R a and R b each independently is selected from H, D, halogen, OH, NH2, CN, C 1-6 alkyl, C 1-6 haloalkyl, C 1-6 alkoxy, C 1-6 haloalkyl, C 2-6 alkenyl or C 2-6 alkynyl, and said R a and R b are optionally further substituted with one, two, three, four or five independently selected R#s, R1 is selected from O or S.

[0126] R2 and R3 each independently are selected from OH, SH, NH2, C 1-6 alkyl, C 1-6 haloalkyl, C 1-6 alkoxy or C 1-6 haloalkyl, and said R2 and R3 are optionally further substituted with one, two, three, four or five independently selected R#s, R# is selected from H, D, halogen, C 1-6 alkyl, C 1-6 haloalkyl, C 2-6 alkenyl or C 2-6 alkynyl, wherein the definition of each of the foregoing groups is optionally substituted with one, two, three, four, five or more deuterium atoms until fully deuterated.

[0127] 2. The oligonucleotide of embodiment 1 or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof,

Chemical formula

[0128] X and Y each independently are CR a R b or (CR a Rb ) 2. Preferably CR a R b is selected from the following.

[0129] Z is CR a is as follows.

[0130] R a and R b are 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 halogenated alkoxy, and R1 is selected from O or S.

[0131] R2 and R3 are independently selected from OH, SH, C 1-6 alkoxy or C 1-6 halogenated alkoxy, and said R2 and R3 are optionally further substituted with one, two or three independently selected R#s, wherein R# is selected from H, D, halogen, C 1-6 alkyl and C 1-6 halogenated alkyl.

[0132] 3. An oligonucleotide according to embodiment 1 or 2, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, wherein [Chemical formula] represents the attachment to the remaining part of the oligonucleotide, preferably a nucleoside which is unmodified or modified.

[0133] X and Y are each independently selected from CR a R b or (CR a R b )2, preferably CR a R b is selected from the following.

[0134] Z is CR a is as follows.

[0135] R a and R b are independently selected from H, D, halogen, C 1-4 alkyl or C 1-4 haloalkyl, and R1 is selected from O or S.

[0136] R2 and R3 are independently selected from OH or SH, preferably OH.

[0137] 4. An oligonucleotide according to any one of aspects 1 to 3 or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, wherein

Chem.

[0138] 5. An oligonucleotide according to aspect 1 or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, having the structure of formula (II), wherein

Chem.

Chem.

[0139] L1 is selected from a chemical bond, O, S or C 1-4 alkylene, and L2 and L3 are independently C1-4 selected from alkylene, wherein L1, L2 and L3 are each independently optionally substituted with one, two, three, four or five independently selected R#s, U is O, S, CR a R b or NR c selected from, R a and R b are each independently H, D, halogen, OH, NH2, CN, C 1-6 alkyl, C 1-6 haloalkyl, C 1-6 alkoxy, C 1-6 haloalkoxy, C 2-6 alkenyl or C 2-6 alkynyl selected from, R c is each independently H, C 1-6 alkyl, C 1-6 haloalkyl, C 2-6 alkenyl or C 2-6 alkynyl selected from, wherein said R a , R b and R c are each optionally further substituted with one, two, three, four or five independently selected R#s, alternatively, L2 and L3 are connected and together with L1 and its adjacent carbon atom form a C 5-6 cycloalkyl or 5- to 6-membered heterocyclic ring, preferably U, L1, L2 and L3 together with their adjacent carbon atoms form ribose or deoxyribose, optionally substituted with R5, R1 is selected from O or S.

[0140] R2 and R3 are each independently OH, SH, NH2, C 1-6 alkyl, C 1-6 haloalkyl, C 1-6 alkoxy or C 1-6 haloalkoxy selected from, and said R2 and R3 are each optionally substituted with one, two, three, four or five independently selected R#s, R5 is H, D, halogen, C1-6 Selected from alkoxy or C 1-6 halogenated alkoxy, R# is 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, a modified or unmodified base, wherein the definition of each of the foregoing groups is optionally substituted with one, two, three, four, five or more deuterium atoms until completely deuterated.

[0141] 6. An oligonucleotide according to aspect 5 or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof,

Chemical formula

[0142] R a and R b are 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 halogenated alkoxy, 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 independently optionally substituted with one, two or three independently selected R#s, U is selected from O, S, NH or CH2, alternatively, L2 and L3 are connected and together with L1 and its adjacent carbon atom form C 5-6Form a cycloalkyl or 5- to 6-membered heterocyclic ring. Preferably, U, L1, L2 and L3, together with their adjacent carbon atoms, form ribose or deoxyribose, optionally substituted with R5, R1 is selected from O or S.

[0143] R2 and R3 are independently OH, SH, C 1-6 alkoxy or C 1-6 halogenated alkoxy, and said R2 and R3 are optionally substituted with one, two or three independently selected R# R5 is H, D, C 1-6 alkyl, C 1-6 halogenated alkyl, C 1-6 alkoxy or C 1-6 halogenated alkoxy R# is H, D, halogen, C 1-6 alkyl and C 1-6 halogenated alkyl Base is selected from H, a modified or unmodified base.

[0144] 7. An oligonucleotide according to embodiment 5 or 6, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof,

Chemical formula

[0145] R a and R b are independently H, D, halogen, C 1-4 alkyl or C 1-4 halogenated alkyl L1 is selected from a chemical bond or C 1-4 alkylene L2 and L3 are independently C 1-4selected from alkylene, U is selected from O or S, R1 is selected from O or S.

[0146] R2 and R3 are independently selected from OH or SH, preferably OH, Base is,

Chemical formula

[0147] 8. An oligonucleotide according to any one of Aspects 5 to 7, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof,

Chemical formula

Chemical formula

[0148] 9. An oligonucleotide according to Aspect 1, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, wherein the 5'-end of the oligonucleotide contains a single nucleotide represented by formula (III), formula (IV) or formula (V),

Chemical formula

[0149] 10. An oligonucleotide of Aspect 9 or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, wherein Q is H, D, halogen, OH, C 1-6 alkyl, C 1-6 halogenated alkyl, C 1-6 alkoxy or C 1-6 halogenated alkoxy, and is selected from R4, R5, R6, and R7 are each independently H, D, halogen, C 1-6 alkyl, C 1-6 halogenated alkyl, C 2-6 alkenyl or C 2-6 alkynyl, and is selected from Base is H, a modified or unmodified base, preferably

Chemical Structure

[0150] 11. An oligonucleotide according to aspect 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 each independently selected from H, halogen, C 1-4 alkyl or C 1-4 halogenated alkyl, preferably H, Base is

Chemical formula

Chemical formula

[0151] 12. An oligonucleotide according to any of aspects 9 - 11, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, wherein the 5'-end of said oligonucleotide comprises a nucleotide monomer having the following structure

Chemical formula

[0152] 13. An oligonucleotide according to any of aspects 9 - 12, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, wherein the 5'-end of said oligonucleotide comprises a nucleotide monomer having the following structure

Chemical formula

Chemical formula

[0153] 14. A compound represented by formula (VI), formula (VII) or formula (VIII), or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, wherein

Chemical formula

[0154] R2 and R3 are independently OR d , OP1, SR d , SP1 or NR e R f selected from R d is H, C 1-6 alkyl, C 1-6 haloalkyl, C 2-6 alkenyl or C 2-6 alkynyl, and is optionally substituted with D, halogen, C 1-6 alkyl or C 1-6 haloalkyl until fully deuterated, R e and R f are independently selected from H, C 1-6 alkyl or C 1-6 haloalkyl, and said R e and R f may be optionally substituted with D, halogen, C 1-6 alkyl or C 1-6 haloalkyl until fully deuterated, P1 is selected from protecting groups, preferably a hydroxyl protecting group, P2 is selected from reactive phosphate 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 of aspects 5 - 11, Here, the definition of each of the aforementioned groups is optionally substituted with one, two, three, four, five or more deuterium atoms until completely deuterated.

[0155] 15. A compound according to embodiment 14 or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, R1 is selected from O or S.

[0156] R2 and R3 are independently OR d , OP1, SR d , SP1 or NR e R f selected from R d is H, C 1-4 alkyl or C 1-4 haloalkyl, optionally substituted with D or halogen until completely deuterated, R e and R f are independently H, C 1-4 alkyl or C 1-4 haloalkyl, said R e and R f may be optionally substituted with D or halogen until completely deuterated, P1 is selected from protecting groups, preferably a hydroxyl protecting group, P2 is selected from reactive phosphate groups, preferably -P(OCH2CH2CN)(N(iPr)2), Base’ is

Chemical formula

[0157] 16. A compound according to embodiment 14 or 15 or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, R1 is selected from O or S, preferably O, R2 and R3 are independently ORd or selected from OP1, R d is H, C 1-4 alkyl or C 1-4 haloalkyl, R e and R f are independently H, C 1-4 alkyl or C 1-4 haloalkyl, P1 is selected from protecting groups, preferably a hydroxyl protecting group, P2 is selected from reactive phosphate groups, preferably -P(OCH2CH2CN)(N(iPr)2), Base’ is

Chemical formula

Chemical formula

[0158] 17. A compound according to any of Aspects 14 to 16, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, wherein the compound is

Chemical formula

[0159] 18. An oligonucleotide according to any of Aspects 1 to 13, wherein the oligonucleotide is a single-stranded having 14 to 30 nucleotides.

[0160] 19. An oligonucleotide according to any of Aspects 1 to 13, wherein the oligonucleotide is a double-stranded RNA comprising a sense strand and an antisense strand, each strand having 14 to 30 nucleotides, and the antisense strand has a sequence sufficiently complementary to the sense strand and the target mRNA.

[0161] 20. The oligonucleotide of embodiment 19, wherein the double-stranded RNA comprises, in the antisense strand, the structure of formula (I), (II), (III), (IV), (V), (IIIa), (IIIb), (IVa), (IVb), (Va) or (Vb) described in any one of embodiments 1 to 12 or the nucleotide monomer described in embodiment 13.

[0162] 21. The oligonucleotide of any one of embodiments 19 to 20, wherein the double-stranded RNA is further coupled to a ligand, and preferably, the ligand comprises one or more GalNAc.

[0163] 22. A nucleic acid molecule, wherein the nucleotide sequence of the nucleic acid molecule comprises one or more nucleotide monomers described in any one of embodiments 9 to 13.

[0164] 23. The nucleic acid molecule of embodiment 22, wherein the nucleic acid is selected from DNA, RNA, and DNA / RNA heterojunctions.

[0165] 24. The nucleic acid molecule of embodiment 23, wherein the nucleic acid molecule is single-stranded or double-stranded.

[0166] 25. The nucleic acid molecule of any one of embodiments 22 to 24, wherein the nucleic acid molecule is selected from small interfering RNA (siRNA) and short hairpin RNA (shRNA).

[0167] 26. A vector comprising a nucleotide sequence encoding the double-stranded RNA described in any one of embodiments 19 to 21.

[0168] 27. A cell comprising the double-stranded RNA described in any one of embodiments 19 to 21 or the vector described in embodiment 24.

[0169] 28. A pharmaceutical composition comprising the double-stranded RNA molecule described in any one of embodiments 19 to 21 and a pharmaceutically acceptable carrier or excipient.

[0170] 29. A kit comprising the double-stranded RNA molecule according to any one of aspects 19 to 21.

[0171] 30. A method for inhibiting the expression of a target gene in a cell, the method comprising introducing the double-stranded RNA molecule according to any one of aspects 19 to 21 into the cell.

[0172] Without being bound by a particular theory, the configurations of formula (IIIa), formula (IVa) and formula (Va) enable the oligonucleotide of the present invention (especially the phosphonate ester group at its 5'-end) to bind better to the Ago2 protein, thereby increasing the activity of the oligonucleotide.

[0173] Examples The following examples are for illustrative purposes only and do not limit the present invention.

[0174] Example 1 Preparation of Compounds E1-1 and E1-2

Chemical formula

[0175] 1. Preparation of Compound 1b

Chemical formula

[0176] 11H NMR: 400 MHz CDCl3 δ 4.97 (s, 1H), 4.84 (d, J = 6.0 Hz, 1H), 4.59 (d, J = 6.0 Hz, 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).

[0177] 2. Preparation of Compound 1c

Chemical formula

[0178] 1 1H NMR: 400 MHz CDCl3 δ 4.98 (s, 1H), 4.69 (d, J = 6.0 Hz, 1H), 4.56 (d, J = 6.0 Hz, 1H), 4.39 - 4.35 (m, 1H), 3.30 (s, 3H), 3.24 - 3.20 (m, 1H), 3.09 (t, J = 10.0 Hz, 1H), 1.41 (s, 3H), 1.26 (s, 3H).

[0179] 3. Preparation of Compound 1d

Chem.

[0180] 1 H NMR: 400 MHz CDCl3 δ 5.11 (s, 1H), 5.02 (d, J = 5.6 Hz, 1H), 4.06 (d, J = 1.6 Hz, 1H), 4.50 (d, J = 5.6 Hz, 1H), 4.39 (d, J = 1.6 Hz, 1H), 3.41 (s, 3H), 3.24 - 3.20 (m, 1H), 3.09 (t, J = 10.0 Hz, 1H), 1.47 (s, 3H), 1.35 (s, 3H).

[0181] 4. Preparation of Compound 1e

Chem.

[0182] 1 1H NMR: 400 MHz in CDCl3, δ 5.12 (d, J = 5.6 Hz, 1H), 5.09 (s, 1H), 4.70 (d, J = 6.0 Hz, 1H), 3.70 - 3.55 (m, 2H), 3.54 (s, 3H), 1.45 (s, 3H), 1.36 (s, 3H).

[0183] 5. Preparation of Compound 1f

Chem.

[0184] 1 1H NMR: 400 MHz in CDCl3, δ 4.97 (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).

[0185] 6. Preparation of Compound 1g

Chem.

[0186] 1 H NMR: 400 MHz CDCl3 δ 5.27 (s, 1H), 5.01 (brs, 1H), 4.78 (s, 1H), 4.65 (d, J = 5.6 Hz, 1H), 4.49 (d, J = 6.0 Hz, 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).

[0187] 7. Preparation of Compound 1h

Chemical Structure

[0188] 11H NMR: 400 MHz CDCl3 δ 4.88 - 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).

[0189] 8. Preparation of Compound 1i

Chemical Structure

[0190] 1 1H NMR: 400 MHz CDCl3 δ 4.75 - 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).

[0191] 9. Preparation of Compound 1j

Chemical Structure

[0192] 10. Preparation of Compound 1k

Chemical formula

[0193] 1 H NMR: 400 MHz CDCl3 δ 5.38 - 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).

[0194] 11. Preparation of Compound 1l

Chemical formula

[0195] 1 H NMR: 400 MHz CDCl3 δ 6.11 (d, J = 2.8 Hz, 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).

[0196] 12. Preparation of Compound 1n

Chemical Structure

[0197] 1 1H NMR: 400 MHz CDCl3 δ 9.70 (brs, 0.46H), 9.60 (brs, 0.60H), 7.17 (d, J = 8.0 Hz, 0.60H), 7.12 (d, J = 8.0 Hz, 0.49H), 5.98 (d, J = 6.4 Hz, 0.43H), 5.93 (d, J = 5.2 Hz, 0.55H), 5.78 (dt, J1 = 8.0 Hz, J2 = 2.0 Hz, 1H), 5.50 - 5.45 (m, 2H), 4.45 - 4.02 (m, 5H), 2.77 - 2.42 (m, 6H), 2.18 (s, 1.22H), 2.16 (s, 1.74 H), 2.04 (s, 1.31H), 2.02 (s, 1.67H), 1.32 - 1.28 (m, 6H).

[0198] 13. Preparation of Compound 1o

Chem.

[0199] 11H NMR: 400 MHz CDCl3 δ 7.36 - 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).

[0200] 14. Preparation of Compound 1p

Chem.

[0201] 1 1H NMR: 400 MHz CDCl3 δ 7.34 - 7.06 (m, 5H), 5.83 (s, 0.52H), 5.71 (d, J = 8.4 Hz, 0.41H), 5.661 (d, J = 8.4 Hz, 0.53H), 5.60 (d, J = 2.0 Hz, 0.59H), 5.53 (d, J = 5.2 Hz, 0.41H), 5.42 - 5.36 (m, 2H), 4.62 (s, 2H), 4.51 (d, J = 3.6 Hz, 0.45H), 3.72 (d, J = 2.8 Hz, 0.36H), 3.54 (d, J = 2.0 Hz, 0.48H), 2.93 - 2.40 (m, 5H), 2.24 - 2.13 (m, 1H), 1.27 - 1.23 (m, 6H).

[0202] 15. Preparation of Compound 1q

Chem.

[0203] 1 H NMR: 400 MHz CDCl3 δ 5.11 (s, 1H), 5.02 (d, J = 5.6 Hz, 1H), 4.06 (d, J = 1.6 Hz, 1H), 4.50 (d, J = 5.6 Hz, 1H), 4.39 (d, J = 1.6 Hz, 1H), 3.41 (s, 3H), 3.24 - 3.20 (m, 1H), 3.09 (t, J = 10.0 Hz, 1H), 1.47 (s, 3H), 1.35 (s, 3H).

[0204] 16. Preparation of Compounds 1q-1 and 1q-2

Chemical Structure

[0205] m / z: ES+ [M+H]+ 525.2 m / z: ES+ [M+H]+ 525.2 HPLC: retention time 0.820 min (Column: XBridge C18 2.1*50mm, 5um; Mobile phase: A: 10mM NH4HCO3 aqueous solution B: Acetonitrile; Gradient: 0 - 0.01 min 5% B, 0.01 - 0.7 min 5 - 95% B, 0.7 - 1.16 min 95% B, 1.16 - 1.5min, 95% - 5% B; Flow rate: 1.5 mL / min; Column temp.: 40°C) 1 1H NMR: 400 MHz CDCl3 δ 7.37 - 7.08 (m, 5H), 7.09 (d, J = 8.0 Hz, 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 = 4.8 Hz, 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).

[0206] Compound 1q - 2: m / z: ES+ [M + H]+ 525.2 HPLC: retention time 0.836 min (Column: XBridge C18 2.1*50mm, 5um; Mobile phase: A: 10mM NH4HCO3 aqueous solution B: Acetonitrile; Gradient: 0 - 0.01 min 5% B, 0.01 - 0.7 min 5 - 95% B, 0.7 - 1.16 min 95% B, 1.16 - 1.5min, 95% - 5% B; Flow rate: 1.5 mL / min; Column temp.: 40°C) 11H NMR: 400 MHz CDCl3 δ 7.37 - 7.27 (m, 5H), 7.08 (d, J = 8.0 Hz, 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 = 4.8 Hz, 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).

[0207] 17. Preparation of Compound 1r-1

Chem.

[0208] m / z: ES+ [M+H]+ 405.3 11H NMR: 400 MHz CDCl3 δ 8.51 - 8.43 (m, 1H), 7.15 (d, J = 8.4 Hz, 1H), 5.78 (d, J = 3.2 Hz, 1H), 5.76 (dd, J1 = 8.0 Hz, J2 = 2.4 Hz, 1H), 4.16 - 4.07 (m, 5H), 3.94 (dd, J1 = 4.8 Hz, J2 = 3.2 Hz, 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).

[0209] 18. Preparation of Compound 1r - 2

Chemical Structure

[0210] 1 1H NMR: 400 MHz CDCl3 δ 9.16 - 8.81 (m, 1H), 7.15 (d, J = 8.0 Hz, 1H), 5.78 (d, J = 2.8 Hz, 1H), 5.76 (dd, J1 = 8.0 Hz, J2 = 2.0 Hz, 1H), 4.65 (brs, 1H), 4.18 - 4.08 (m, 5H), 3.96 (dd, J1 = 4.8 Hz, J2 = 3.2 Hz, 1H), 3.53 (s, 3H), 3.05 - 2.95 (m, 1H), 2.81 - 2.41 (m, 2H), 1.34 - 1.30 (m, 6H).

[0211] 19. Preparation of Compound E1 - 1

Chemical Structure

[0212] 1 H NMR: 400 MHz CDCl3 δ 9.17 (brs, 1H), 7.24 (d, J = 8.4 Hz, 1H), 5.83 - 5.79 (m, 1H), 5.64 - 5.61 (m, 1H), 4.43 - 4.33 (m, 1H), 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).

[0213] 20. Preparation of Compound E1-2

Chemical Structure

[0214] 11H NMR: 400 MHz CDCl3 δ 9.06 (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).

[0215] Example 2 Preparation of Compounds E2-1 and E2-2

Chemical Structure

[0216] 1. Preparation of Compound 2c

Chemical Structure

[0217] 11H NMR: 400 MHz DMSO-d6 δ 7.35 - 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).

[0218] 2. Preparation of Compound 2d

Chemical Structure

[0219] 1 1H NMR: 400 MHz CDCl3 δ 7.36 - 7.26 (m, 5H), 4.45 (s, 2H), 3.68 (s, 3H), 3.59 - 3.56 (m, 3H), 3.53 - 3.49 (m, 1H), 3.10 - 3.08 (m, 1H), 3.06 - 3.04 (m, 1H), 2.26 (t, J = 6.0 Hz, 2H).

[0220] 3. Preparation of Compound 2f

Chemical Structure

[0221] 1 H NMR: 400 MHz CDCl3 δ 7.34-7.26 (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).

[0222] 4. Preparation of compound 2g

Chemical formula

[0223] 5. Preparation of compound 2h

Chemical formula

[0224] 1 H NMR: 400 MHz CDCl3 δ 7.34 - 7.24 (m, 5H), 4.43 - 4.41 (m, 2H), 3.63 - 3.60 (m, 3H), 3.49 - 3.40 (m, 2H), 2.79 - 2.59 (m, 3H), 2.39 - 2.28 (m, 1H), 2.17 - 2.13 (m, 2H), 1.32 - 1.29 (m, 6H).

[0225] 6. Preparation of Compound 2i

Chemical Structure

[0226] 11H NMR: 400 MHz DMSO-d6 δ 7.35 - 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).

[0227] 7. Preparation of Compound 2j

Chemical Structure

[0228] 1 1H NMR: 400 MHz CDCl3 δ 8.03 - 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, 2H), 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).

[0229] 8. Preparation of Compound 2k

Chemical Structure

[0230] 1 H NMR: 400 MHz CDCl3 δ 8.06 - 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, 2H), 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).

[0231] 9. Preparation of Compound 2m

Chemical Structure

[0232] 1 H NMR: 400 MHz CDCl3 δ 8.06 - 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, 1H), 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).

[0233] 10. Preparation of compound 2n

Chemical Structure

[0234] 11H NMR: 400 MHz DMSO-d6 δ 11.21 (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 = 5.2 HZ, 1H), 2.75 - 2.55 (m, 1H), 1.93 - 1.87 (m, 6H), 1.22 - 1.18 (m, 6H).

[0235] 11. Preparation of Compounds 2n-1 and 2n-2

Chemical Structure

[0236] Compound 2n-1: HPLC: Retention time 3.065 min (Column: Chiralpak AD-3, 150×4.6 mm I.D., 3 μm; Mobile phase A: CO2, B: IPA (0.2% NH3 (7M 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) 11H NMR: 400 MHz DMSO-d6 δ 11.20 (brs, 1H), 7.64 (d, J = 8.0 Hz, 1H), 5.53 (d, J = 8.0 Hz, 1H), 4.78 (t, J = 5.2 Hz, 1H), 3.99 - 3.93 (m, 4H), 3.55 - 3.75 (m, 2H), 3.42 (d, J = 5.6 Hz, 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).

[0237] Compound 2n-2: HPLC: Retention time 3.626 min (Column: Chiralpak AD-3, 150×4.6 mm I.D., 3 μm; Mobile phase: A: CO2, B: IPA (0.2% NH3 (7M 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) 1 1H NMR: 400 MHz DMSO-d6 δ 11.22 (brs, 1H), 7.68 (d, J = 8.0 Hz, 1H), 5.56 - 5.53 (m, 1H), 4.68 (t, J = 5.2 Hz, 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).

[0238] 12. Preparation of Compound E2-1

Chem.

[0239] 1 H NMR: 400 MHz DMSO-d6 δ 11.21 (brs, 1H), 7.63 (d, J = 8.0 Hz, 1H), 5.55 (d, J = 8.0 Hz, 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).

[0240] 13. Preparation of Compound E2-2

Chemical formula

[0241] 11H NMR: 400 MHz DMSO-d6 δ 11.21 (brs, 1H), 7.66 (d, J = 8.0 HZ, 1H), 5.55 (d, J = 8.0 Hz, 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 = 7.2 Hz, 6H), 1.15 - 1.13 (m, 12H).

[0242] Example 3 Preparation of Compound E3

Chemical Structure

[0243] Example 4 Preparation of siRNA The siRNA of the present invention was prepared by a known solid-phase phosphoramidite method. For specific methods, reference can be made to, for example, PCT Publication Nos. WO2016081444 and WO2019105419, and the outline is as follows.

[0244] 1 Synthesis of sense strand (SS strand) By the solid-phase phosphoramidite synthesis method, starting from a blank CPG solid support or a solid support connected with L96 as the starting cycle, nucleoside monomers are connected one by one in the 3'-5' direction according to the sense strand nucleotide sequence order. Each time a nucleoside monomer is connected, it includes four process reactions: deprotection, coupling, capping, and oxidation or thiolation. The synthesis scale is that the synthesis conditions of 5 μmol of oligonucleic acid are as follows.

[0245] The nucleoside monomer provides a 0.05 mol / L acetonitrile solution. The reaction conditions for each step are the same, that is, the temperature is 25 °C. For deprotection, it is deprotected three times with a 3% trichloroacetic acid - dichloromethane solution. The activator used in the coupling reaction is a 0.25 mol / L ETT - acetonitrile solution, and it is coupled twice. For capping, it is capped twice with 10% acetic anhydride - acetonitrile and pyridine / N - methylimidazole / acetonitrile (10:14:76, v / v / v). For oxidation, it is oxidized twice with a 0.05 mol / L iodine / tetrahydrofuran / pyridine / water (70 / 20 / 10, v / v / v) solution. For thiolation, it is thiolated twice with a 0.2 mol / L PADS acetonitrile / 3 - methylpyridine (1 / 1, v / v) solution.

[0246] 2 Synthesis of the antisense strand (AS strand) By the solid - phase phosphoramidite synthesis method, starting from a blank CPG solid support as the starting cycle, the nucleoside monomers or the nucleotide dimers of the present invention are connected one by one in the 3’ - 5’ direction according to the antisense strand nucleotide sequence order. Each time a nucleoside monomer or a nucleotide dimer of the present invention is connected, it includes four step reactions: deprotection, coupling, capping, oxidation or thiolation. The synthesis conditions for 5 μmol of the oligonucleic acid of the antisense strand are the same as those of the sense strand.

[0247] 3 Purification and annealing of oligonucleotides 3.1 Aminolysis The synthesized solid support (sense strand or antisense strand) is put into a 5 mL centrifuge tube, 3% diethylamine / ammonia water (v / v) is added, and it is reacted for 16 hours (or 8 hours) under a constant temperature water bath at 35 °C (or 55 °C), filtered, the solid support is washed three times with ethanol / water, 1 mL each time, and after the filtrate is centrifugally concentrated, the crude product is purified.

[0248] 3.2 Purification The methods of purification and desalination are well-known to those skilled in the art. For example, it may be eluted and washed with a strong anion-exchange column and a sodium chloride-sodium hydroxide system, or the product may be collected and desalted with a gel-filtration purification column, and the elution system was pure water.

[0249] 3.3 Annealing According to the following table, 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 to 70-95 °C in a water bath, held for 3-5 min, naturally cooled to room temperature, and the system was lyophilized to obtain the product.

[0250] The sequences of the siRNAs used in the present invention are as follows.

Table 0

[0251] In this specification, the meanings of the respective abbreviations are as follows.

[0252] A, U, G, and C represent natural adenine ribonucleotide, uracil ribonucleotide, guanine ribonucleotide, and cytosine ribonucleotide, respectively.

[0253] m represents that the nucleotide adjacent to its left is a nucleotide modified with 2'-OCH3. For example, Am, Um, Gm, and Cm represent A, U, G, and C modified with 2'-OCH3, respectively.

[0254] f represents that the nucleotide adjacent to its left is a nucleotide modified with 2'-F. For example, Af, Uf, Gf, and Cf represent A, U, G, and C modified with 2'-F, respectively.

[0255] “s” or “s-” represents that the two nucleotides adjacent to its left and right and / or the introduced carrier are connected via a phosphorothioate ester.

[0256] L96 represents a GalNAc-introducing carrier with the following known structure, where [Chemical formula] represents the position where it binds to siRNA via a phosphate ester or thiophosphate ester. For example, reference can be made to PCT Publication Nos. WO2009073809 and WO2009082607. [Chemical formula]

[0257] VP represents a nucleotide in which the nucleotide adjacent to its right side is modified with vinylphosphonate. As is known, for example, reference can be made to PCT Publication Nos. WO2011139702, WO2013033230, and WO2019105419.

[0258] SCP1a, SCP1b, SCP2a, and SCP2b represent alternatives to the nucleotides of the aforementioned structure, where Base can be any base. For example, SCP1a-U represents that Base is uracil.

[0259] Example 5 Activity Screening Experiment of Primary Hepatocytes (PMH) from C57BL / 6 Wild-Type Mice 1. Free Uptake or Transfection Primary hepatocytes from C57BL / 6 wild-type mice were isolated, counted, plated in 24-well plates at 900 μL / well, 8×10 4 cells / well, and plated in 96-well plates at 100 μL / well, 1×10 4 cells / well. Then, free uptake or transfection was selected.

[0260] Free uptake: 10 μL of the diluted compound was added to 90 μL of Opti-MEM and mixed uniformly, then added to the corresponding wells and cultured in a 37°C, 5% CO2 incubator for 24 hours. No siRNA was added to the control group.

[0261] Transfection: 10 μL of the diluted compound was added to 40 μL of Opti-MEM and mixed uniformly. 3 μL of RNAiMAX was added to 47 μL of Opti-MEM and mixed uniformly. After incubating for 5 min, it was mixed uniformly with the diluted compound, left standing at room temperature for 10 min, added to the corresponding wells, and cultured in a 37°C, 5% CO2 incubator for 24 h. No siRNA was added to the control group.

[0262] 2. Real-time PCR Total RNA was extracted using a high-throughput nucleic acid extraction device - magnetic bead method (FANZHIMEDICAL, FG0412; Hangzhou Ausheng, Auto-pure96), and reverse transcription (PrimeScript TM II 1st Strand cDNA Synthesis Kit (Takara, 6210B)) was performed. Then, real-time PCR detection (TaqMan TM Fast Advanced Master Mix (ABI, 4444965)) was carried out.

[0263]

Table 1

[0264] 3. Data statistics 2 -△△Ct Values were calculated and converted to percentages to obtain the residual inhibition rate. △△Ct = [(Ct of target gene in experimental group - Ct of internal standard in experimental group) - (Ct of target gene in control group - Ct of internal standard in control group)]. Here, the target gene is mAPOB or mFXII, and the control gene is mGAPDH.

[0265] 4. Experimental results of mAPOB gene By the transfection method, primary hepatocytes of C57BL / 6 wild-type mice were selected. After plating the cells in 24-well plates, the initial concentration of the compound was set at 40 nM, and five concentration points (40 nM, 4 nM, 0.4 nM, 0.04 nM, 0.004 nM) were serially diluted 10-fold to perform a 5-point IC50 activity screening on primary hepatocytes of C57BL / 6 wild-type mice. The experimental results are shown in Table 2.

[0266]

Table 2

[0267] 5. Experimental results of mFXII gene By the free intake method, primary hepatocytes of C57BL / 6 wild-type mice were selected. After plating the cells in 96-well plates, the initial concentration of the compound was set at 100 nM, and five concentration points (100 nM, 10 nM, 1 nM, 0.1 nM, 0.01 nM) were serially diluted 10-fold to perform a 5-point IC50 activity screening - free intake on primary hepatocytes of C57BL / 6 wild-type mice. The experimental results are shown in Table 3.

[0268]

Table 3

[0269] Example 6 Verification of the drug efficacy of the compound in a C57BL / 6 mouse model C57BL / 6 mice (male, 18 - 21 g, 6 - 8 weeks old, Si Pei Fu (Suzhou) Biotechnology Co., Ltd.) were randomly grouped, with 9 mice in each group (the mice were euthanized on the 14th and 28th days, and then the livers were collected (N = 3 / group)). The dosage was calculated according to the body weight for each animal, and a single dose was administered by subcutaneous injection. The siRNA conjugate was provided to the CRO company as a 10 mg / mL solution (using 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 made up to the required solution concentration and volume, and the administration volume of physiological saline and siRNA conjugate was 5 mL / kg.

[0270] Blood samples were collected and detected respectively before administration (recorded as the second day before administration), grouped by LDL on the second day, and the remaining samples were retained for the detection of the target protein. And on the 7th, 14th, and 21st days after administration (recorded as the 0th day), blood was collected from the orbital venous plexus of the mice (before each blood collection, all were starved for 5 hours), and Suzhou Anling directly measured the serum LDL concentration (Max, low-density lipoprotein cholesterol detection reagent) by the direct method (Neusoft fully automatic biochemical analyzer, NT-1000) at each time point, and the remaining samples were retained for the detection of the target protein (Mouse ApoB ELISA Kit, Abcam, ab230932) by the ELISA method. Among them, 10 mg of liver (n = 3 / group / time point) was collected on the 14th, 28th, and 42nd days, put into RNAlater solution, frozen and stored at -80 °C, and sent by dry ice for use in the extraction and detection of liver mRNA expression (the detection primers are shown in Table 4 and need to be updated at subsequent time points). The experimental results are shown in Table 5.

[0271]

Table 4

[0272]

Table 5

[0273] Example 7 Activity Screening Experiment of Primary Hepatocytes (PMH) of C57BL / 6 Wild-Type Mice Using the method of Example 3, more compounds of the present invention were tested.

[0274]

Table 6

[0275]

Table 7

[0276] Example 8 Molecular Simulation Through molecular simulation, the compound of the present invention can immobilize phosphonic acid in an ap conformation very similar to unmodified 5'-phosphate-bound Ago2, in which the phosphonate perfectly matches Arg812, Lys570, Lys566 and Lys533, forms multiple salt bridge bonds and hydrogen bonds, and further forms hydrogen bonds with Tyr529 and Cys526 respectively. Furthermore, the base (uracil) of this cyclic phosphonic acid compound forms hydrogen bonds with the backbone amides of Thr526 and Gly524 respectively. Due to the combined effect of these structures, the binding of the compound of the present invention to Ago2 protein is stabilized.

[0277] Through molecular simulation, similar to the spiro compound, due to the binding of the sugar ring-linked highly rigid aromatic rings (including five-membered aromatic rings and six-membered aromatic rings), the 5'-terminal phosphate is locked in a specific conformation that highly mimics the ap conformation of the natural 5'-phosphate, the phosphonate perfectly matches Arg812, Lys570, Lys566 and Lys533, forms multiple salt bridge bonds and hydrogen bonds, and further forms hydrogen bonds with Tyr529 and Cys526 respectively. Similarly, the base uracil on the sugar ring also forms hydrogen bonds with the backbone amides of Thr526 and Gly524 respectively. Due to the combined effect of these structures, the binding of the compound of the present invention to Ago2 protein is stabilized.

Claims

1. An oligonucleotide or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, wherein the 5'-end of the oligonucleotide contains a structure represented by formula (I), 【Chemical Formula 1】 wherein, 【Chemical 2】 represents the remaining portion attached to the oligonucleotide, preferably attached to a modified or unmodified nucleoside X and Y are each independently CR ａ R ｂ or (CR ａ R ｂ ) ２ selected from, preferably CR ａ R ｂ and Z is CR ａ and R ａ and R ｂ are each independently selected from H, D, halogen, OH, NH ２ , CN, C １－６ alkyl, C １－６ haloalkyl, C １－６ alkoxy, C １－６ haloalkoxy, C ２－６ alkenyl or C ２－６ alkynyl, and said R ａ and R ｂ are each optionally further substituted with one, two, three, four or five independently selected R# R １ is selected from O or S, R ２ and R ３ are independently selected from OH, SH, NH ２ , C １－６ alkyl, C １－６ haloalkyl, C １－６ alkoxy or C １－６ haloalkoxy, and said R ２ and R ３ are optionally further substituted with one, two, three, four or five 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, and wherein the definition of each of the foregoing groups is optionally substituted with one, two, three, four, five or more deuterium atoms until fully deuterated, an oligonucleotide or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof.

2. 【Fig. 3】 represents the remaining portion attached to the oligonucleotide, preferably attached to a modified or unmodified nucleoside X and Y are each independently CR ａ R ｂ or (CR ａ R ｂ ) ２ selected from, preferably CR ａ R ｂ and Z is CR ａ wherein R ａ and R ｂ are independently selected from H, D, halogen, OH, NH ２ , CN, C １－６ alkyl, C １－６ haloalkyl, C １－６ alkoxy or C １－６ haloalkoxy, R １ is selected from O or S, R ２ and R ３ are independently selected from OH, SH, C １－６ alkoxy or C １－６ haloalkoxy, and said R ２ and R ３ are optionally further substituted with one, two or three independently selected R# R# is selected from H, D, halogen, C １－６ alkyl and C １－６ alkyl halide, the oligonucleotide according to claim 1 or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof.

3. 【Fig. 4】 represents the remaining portion attached to the oligonucleotide, preferably attached to a modified or unmodified nucleoside, X and Y are each independently CR ａ R ｂ or (CR ａ R ｂ ) ２ selected from, preferably CR ａ R ｂ and Z is CR ａ and R ａ and R ｂ are each independently selected from H, D, halogen, C １－４ alkyl or C １－４ haloalkyl, R １ is selected from O or S, R ２ and R ３ each independently is selected from OH or SH, preferably OH, the oligonucleotide according to claim 1 or 2 or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof.

4. 【Fig. 5】 represents the 5'-terminal group attached to the oligonucleotide, preferably attached to a modified or unmodified nucleotide, X and Y are CH ２ and Z is CH, R １ is O, R ２ and R ３ is OH, the oligonucleotide according to any one of claims 1 to 3 or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof.

5. having the structure of formula (II), 【Chemical Formula 6】 wherein, [Chemical 7] represents the remaining portion attached to the oligonucleotide, X and Y are CR ａ R ｂ and Z is CR ａ and L １ is selected from a chemical bond, O, S or C １－４ and alkylene L ２ and L ３ are independently selected from C １－４ alkylene, Said L １ , L ２ and L ３ are each independently optionally substituted with one, two, three, four or five independently selected R#s, U is selected from O, S, CR ａ R ｂ or NR ｃ and is selected from R ａ and R ｂ are independently selected from H, D, halogen, OH, NH ２ , CN, C １－６ alkyl, C １－６ haloalkyl, C １－６ alkoxy, C １－６ haloalkoxy, C ２－６ alkenyl or C ２－６ alkynyl, R ｃ is independently selected from H, C １－６ alkyl, C １－６ haloalkyl, C ２－６ alkenyl or C ２－６ alkynyl, and Said R ａ , R ｂ and R ｃ is further substituted by any one, two, three, four or five independently selected R# Alternatively, L ２ is connected to L ３ and together with L １ and its adjacent carbon atom forms a C ５－６ cycloalkyl or 5- to 6-membered heterocyclic ring, preferably U, L １ , L ２ and L ３ together with their adjacent carbon atoms form ribose or deoxyribose, optionally substituted with R ５ . R １ is selected from O or S, R ２ and R ３ are independently selected from OH, SH, NH ２ , C １－６ alkyl, C １－６ haloalkyl, C １－６ alkoxy or C １－６ haloalkoxy, and said R ２ and R ３ are optionally substituted with one, two, three, four or five independently selected R# R ５ is selected from H, D, halogen, C １－６ alkoxy or C １－６ halogenated alkoxy, and 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, a modified or unmodified base, wherein the definition of each of the foregoing groups is optionally substituted with one, two, three, four, five or more deuterium atoms until fully deuterated, the oligonucleotide according to claim 1 or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof.

6. 【Fig. 8】 represents the remaining portion attached to the oligonucleotide, X and Y are CR ａ R ｂ wherein Z is CR ａ is the case. R ａ and R ｂ are independently selected from H, D, halogen, OH, NH ２ , CN, C １－６ alkyl, C １－６ haloalkyl, C １－６ alkoxy or C １－６ haloalkoxy, L １ is selected from a chemical bond, O or C １－４ and alkylene L ２ and L ３ each independently selected from C １－４ alkylene, said L １ , L ２ and L ３ are each independently optionally substituted with one, two or three independently selected R# U is O, S, NH or CH ２ selected from Alternatively, L ２ and L ３ are connected, and together with L １ and its adjacent carbon atom form C ５－６ cycloalkyl or a 5- to 6-membered heterocyclic ring, preferably U, L １ , L ２ and L ３ together with their adjacent carbon atoms form ribose or deoxyribose, optionally substituted with R ５ ​ R １ is selected from O or S, R ２ and R ３ are independently selected from OH, SH, C １－６ alkoxy or C １－６ halogenated alkoxy, and said R ２ and R ３ are optionally substituted with one, two or three independently selected R# R ５ is selected from H, D, C １－６ alkyl, C １－６ halogenated alkyl, C １－６ alkoxy or C １－６ halogenated alkoxy, and R# is selected from H, D, halogen, C １－６ alkyl and C １－６ haloalkyl, Base is selected from H, a modified or unmodified base, the oligonucleotide according to claim 5 or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof.

7. 【Fig. 9】 represents the remaining portion attached to the oligonucleotide, X and Y are CR ａ R ｂ wherein Z is CR ａ and R ａ and R ｂ are each independently selected from H, D, halogen, C １－４ alkyl or C １－４ haloalkyl, L １ is selected from a chemical bond or C １－４ alkylene, L ２ and L ３ are independently selected from C １－４ alkylene, U is selected from O or S, R １ is selected from O or S, R ２ and R ３ is independently selected from OH or SH, preferably OH, Base is 【Chemical Formula 10】 selected from the oligonucleotide according to claim 5 or 6 or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof.

8. 【Fig. 11】 represents the remaining portion attached to the oligonucleotide, X and Y are CH ２ and Z is CH, L １ is a chemical bond, L ２ and L ３ each independently is selected from C １－２ alkylene U is O, R １ is O, R ２ and R ３ are OH, Base is 【Chemical Formula 12】 selected from the oligonucleotide according to any one of claims 5 to 7 or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof.

9. The 5'-end of the oligonucleotide contains a single nucleotide represented by formula (III), formula (IV) or formula (V), 【Chemical Formula 13】 Here, 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, and R ４ 、 R ５ 、 R ６ and R ７ are each independently selected from H, D, halogen, OH, C １－６ alkyl, C １－６ haloalkyl, C １－６ alkoxy, C １－６ haloalkoxy, C ２－６ alkenyl or C ２－６ alkynyl, Base is selected from H, a modified, or unmodified base, L １ 、L ３ 、X, Y, Z, R １ 、R ２ 、R ３ 、U are as defined in any one of claims 5 to 8, wherein the definition of each of the foregoing groups is optionally substituted with one, two, three, four, five or more deuterium atoms until fully deuterated, the oligonucleotide according to claim 1, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof.

10. 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 halogenated alkoxy, and R ４ 、 R ５ 、 R ６ and R ７ are independently selected from H, D, halogen, C １－６ alkyl, C １－６ haloalkyl, C ２－６ alkenyl or C ２－６ alkynyl, Base is selected from H, a modified, or unmodified base, preferably 【Chemical 14】 being, L １ 、L ３ 、X, Y, Z, R １ 、R ２ 、R ３ 、U is as defined in any one of claims 5 to 8, the oligonucleotide according to claim 9 or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof.

11. Q is selected from H, halogen or C １－４ alkoxy, preferably H, F or methoxy, R ４ 、 R ５ 、 R ６ and R ７ are each independently selected from H, halogen, C １－４ alkyl or C １－４ haloalkyl, preferably H, Base is 【Chemical 15】 selected from, preferably 【Chemical 16】 being, L １ 、L ３ 、X, Y, Z, R １ 、R ２ 、R ３ 、U is as defined in any of claims 5 to 8, the oligonucleotide according to claim 9 or 10 or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof.

12. The 5'-end of the oligonucleotide contains a single nucleotide having the following structure, 【Chemical 17】 wherein each group is as defined in any of claims 9 to 11, the oligonucleotide according to any of claims 9 to 11, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof.

13. The 5'-end of the oligonucleotide contains a single nucleotide having the following structure, 【Chemical 18】 wherein Base is 【Chemical Formula 19】 selected from, the oligonucleotide according to any of claims 9 to 12, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof.

14. A compound represented by formula (VI), formula (VII) or formula (VIII), or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, 【Chemical 20】 wherein, R １ is selected from O or S, R ２ and R ３ are independently selected from OR ｄ OP １ SR ｄ SP １ or NR ｅ R ｆ and are selected from R ｄ is selected from H, C １－６ alkyl, C １－６ halogenated alkyl, C ２－６ alkenyl or C ２－６ alkynyl, and is optionally substituted with D, halogen, C １－６ alkyl or C １－６ halogenated alkyl until it is completely deuterated, R ｅ and R ｆ are independently selected from H, C １－６ alkyl or C １－６ haloalkyl, and said R ｅ and R ｆ may be optionally substituted with D, halogen, C １－６ alkyl or C １－６ haloalkyl until fully deuterated, P １ is selected from protecting groups, preferably a hydroxyl protecting group, P ２ is selected from reactive phosphate groups and is preferably -P(OCH ２ CH ２ CN)(N(iPr) ２ ) and Base' is independently selected from H, a modified, or unmodified base or a leaving group, L １ 、L ３ 、X, Y, Z, U, Q, R ４ 、R ５ 、R ６ and R ７ are as defined in any of claims 5 to 11, wherein the definition of each of the foregoing groups is optionally substituted with one, two, three, four, five or more deuterium atoms until fully deuterated, the compound or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof.

15. R １ is selected from O or S, R ２ and R ３ are independently OR ｄ , OP １ , SR ｄ , SP １ or NR ｅ R ｆ selected from R ｄ is selected from H, C １－４ alkyl or C １－４ haloalkyl, is optionally substituted with D or halogen until fully deuterated, R ｅ and R ｆ are each independently selected from H, C １－４ alkyl or C １－４ haloalkyl, and said R ｅ and R ｆ may each optionally be substituted with D or halogen until fully deuterated P １ is selected from protecting groups, preferably a hydroxyl protecting group, P ２ is selected from reactive phosphate groups, preferably -P(OCH ２ CH ２ CN)(N(iPr) ２ ) and Base' is 【Chemical 21】 selected from, L １ 、L ３ 、X, Y, Z, U, Q, R ４ 、R ５ 、R ６ and R ７ is a compound or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof according to claim 14, as defined in any of claims 5 to 11.

16. R １ is selected from O or S, preferably O, R ２ and R ３ are each independently selected from OR ｄ or OP １ and are selected from R ｄ is selected from H, C １－４ alkyl or C １－４ halogenated alkyl, R ｅ and R ｆ are independently selected from H, C １－４ alkyl or C １－４ haloalkyl, P １ is selected from protecting groups, preferably a hydroxyl protecting group, P ２ is selected from reactive phosphate groups, preferably -P(OCH ２ CH ２ CN)(N(iPr) ２ ) and Base' is 【Chemical formula 22】 selected from, more preferably 【Chemical 23】 being, L １ 、L ３ 、X, Y, Z, U, Q, R ４ 、R ５ 、R ６ and R ７ is as defined in any of claims 5 to 11, a compound or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof according to claim 14 or 15.

17. The compound is 【Chemical 24】 selected from, the compound according to any of claims 14 to 16, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof.

18. The oligonucleotide is a single strand having 14 to 30 nucleotides, the oligonucleotide according to any of claims 1 to 13.

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

20. The double-stranded RNA contains 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. The oligonucleotide according to claim 19.

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

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

23. The nucleic acid is selected from DNA, RNA and DNA / RNA heterojunctions. The nucleic acid molecule according to claim 22.

24. The nucleic acid molecule is single-stranded or double-stranded. The nucleic acid molecule according to claim 23.

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

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

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

28. A kit containing 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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