Compound for modifying nucleotide monomer and application of compound in RNAi (Ribonucleic Acid Interference) medicine

Abstract

The invention relates to an application of a modified nucleotide monomer in nucleic acid interference (RNAi) drug molecules. The RNAi molecule (or oligomeric nucleic acid) carrying the nucleotide monomer disclosed by the invention is a double-stranded siRNA molecule. According to the present invention, the nucleotide monomer is introduced into the antisense strand seed region of siRNA through the specific 2 ', 5'-phosphate linking, such that the potential off-target effect of the antisense strand seed region can be significantly reduced, such that the siRNA can highly selectively silence the target gene mRNA, the siRNA treatment safety window is improved, and the related diseases caused by the target gene mRNA can be effectively treated.

Description

Technical Field

[0001] The present invention relates to the field of biochemistry, and in particular to the application of a bicyclic nucleoside analog (modified nucleotide monomer) in nucleic acid interference (RNAi) drug molecules. Background Art

[0002] RNA interference was first discovered and named in 1998. It refers to the double-stranded RNA (dsRNA) that specifically induces the degradation of homologous mRNA in cells, thereby downregulating the expression of the corresponding gene and causing the silencing of the corresponding gene after transcription.

[0003] From a genetic level, RNAi can treat almost all diseases, including tumors, infectious diseases, hereditary diseases, etc. Therefore, RNAi has become the hottest field of biomedical research and the most promising direction for new drug development in the future. Currently, 9 antisense oligonucleotide (ASO) drugs and 6 siRNA drugs have entered clinical application. They play a unique role in anti-infection, metabolism and genetic treatment.

[0004] The RNA interference (RNAi) phenomenon is a way of regulating gene expression that organisms have themselves. It is used to describe that double-stranded RNAi (dsRNAi) can block gene expression. The siRNA response mechanism is an RNA interference method mediated by a nuclease complex, which is usually called RNA-induced silencing complex (RISC). RISC degrades the target gene mRNA by combining the complementary pairing of the antisense strand of siRNA with the target gene mRNA.

[0005] As a new drug treatment mechanism, the advantage of siRNA is that it can target target genes in the pathogenic genome, has a strong and lasting therapeutic effect, can significantly reduce the frequency of dosing, and improve patient compliance with medication, thereby achieving better therapeutic effects and better safety.

[0006] However, siRNA therapy has also encountered unique challenges in its development. Targeted delivery of oligonucleic acid substances, potential off-target effects, and in vivo stability have been the three major challenges in the development of RNAi drugs. With the iterative development of GalNAc technology and modification technology over the past decade, liver-targeted delivery and in vivo stability issues have been solved and improved, but the potential safety risks of off-target bands affect the development of siRNA drugs.

[0007] Existing reports show that one of the main reasons for RNAi off-target effects is that some fragments of the siRNA sequence that binds to the target mRNA may function like microRNA (miRNA), which can bind to the 3'-untranscribed region (3'-UTR) of other mRNAs to inhibit the translation and stability of mRNA. While miRNA mainly recognizes target genes through base pairing between the seed region (position 2-8 at the 5'-end) and the target mRNA, the off-target effects caused by siRNA mainly come from the base complementarity between the antisense strand seed region of siRNA and one or more mRNAs. The sequence of the seed region can affect the expression of multiple genes and become the main factor for the side effects of RNAi drugs.

[0008] In order to effectively solve the off-target effect of the seed region, Alnylam found a new type of nucleoside monomer (glycerol nucleic acid, GNA for short) by modifying the nucleotide monomers located in the seed region. After introducing it into the seed region of the siRNA antisense strand, it can reduce the off-target effect of RNAi and improve the safety of the drug. See, for example, PCT Publication No. WO2018098328A1.

[0009] The present invention requires the development of a new nucleotide analog that helps reduce off-target toxicity after being incorporated into double-stranded RNA. Summary of the invention

[0010] The present invention solves the above problems by providing a novel nucleotide analog.

[0011] The novel modified nucleotides involved in the present invention can improve the selectivity of RNAi drug molecules for target gene silencing and reduce off-target effects.

[0012] The modified nucleotides of the present invention can improve the drugability of RNAi drug molecules. In some embodiments, the modified nucleotides of the present invention can enhance the selectivity of the antisense strand in the siRNA molecule to degrade the target gene. In some embodiments, the modified nucleotides of the present invention are introduced into the siRNA molecule without reducing the drug efficacy. Therefore, the modified nucleotides of the present invention can enhance the safety of the siRNA silencing and the drug efficacy.

[0013] the term:

[0014] As used herein, the term "C 1-6"Alkyl" refers to a saturated aliphatic hydrocarbon group, which is an alkyl group containing 1 to 6 carbon atoms. Non-limiting examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, n-hexyl, etc. The alkyl group can be substituted or unsubstituted. When substituted, the substituent can be substituted at any available point of attachment, and the substituent is preferably one or more of the following groups, which are independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkyloxy, heterocycloalkyloxy, cycloalkylthio, heterocycloalkylthio, oxo, carboxyl or carboxylate.

[0015] As used herein, the term "haloalkyl" refers to an alkyl group substituted with one or more halogens, wherein alkyl is as defined above.

[0016] As used herein, the term "C 1-6 "Alkoxy" refers to -O-(alkyl) or -O-(haloalkyl), wherein alkyl is as defined above. Non-limiting examples of alkoxy include: methoxy, ethoxy, propoxy, butoxy, cyclopropyloxy, cyclobutyloxy, cyclopentyloxy, cyclohexyloxy. Alkoxy may be optionally substituted or unsubstituted, and when substituted, the substituents are preferably one or more of the following groups independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxy, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkyloxy, heterocycloalkyloxy, cycloalkylthio, heterocycloalkylthio, carboxyl or carboxylate.

[0017] As used herein, the term "C 2-6 "Alkenyl" refers to a straight or branched hydrocarbon group having 2 to 6 carbon atoms and at least one carbon-carbon double bond. It includes: vinyl (C2), 1-propenyl (C3), 2-propenyl (C3), 1-butenyl (C4), 2-butenyl (C4), butadienyl (C4), pentenyl (C5), pentadienyl (C5), hexenyl (C6) and heteroalkenyl (one or more carbon atoms are replaced by heteroatoms, such as boron, nitrogen, oxygen, silicon, phosphorus, sulfur). The alkenyl group may be optionally substituted by one or more substituents.

[0018] “C 2-6 "Alkynyl" refers to a straight or branched hydrocarbon group having 2 to 6 carbon atoms and at least one carbon-carbon triple bond and optionally one or more carbon-carbon double bonds. It includes: ethynyl (C2), 1-propynyl (C3), 2-propynyl (C3), 1-butynyl (C4), 2-butynyl (C4), pentynyl (C5), hexynyl (C6) and heteroalkynyl (one or more carbon atoms are replaced by heteroatoms, such as boron, nitrogen, oxygen, silicon, phosphorus, sulfur). Alkynyl groups can be optionally substituted by one or more substituents.

[0019] The term "aryl" refers to a 6- to 14-membered all-carbon monocyclic or fused polycyclic (i.e., rings that share adjacent pairs of carbon atoms) group having a conjugated π electron system, preferably 6- to 10-membered, such as phenyl and naphthyl. The aryl group may be substituted or unsubstituted, and when substituted, the substituent is preferably one or more of the following groups independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkyloxy, heterocycloalkyloxy, cycloalkylthio, heterocycloalkylthio, carboxyl or carboxylate.

[0020] The term "hydroxy" refers to an -OH group.

[0021] The term "halogen" refers to fluorine, chlorine, bromine or iodine.

[0022] The term "amino" refers to -NH2.

[0023] The term "cyano" refers to -CN.

[0024] The term "nitro" refers to -NO2.

[0025] The term "carbonyl" refers to =0.

[0026] The term "carboxy" refers to -C(O)OH.

[0027] The term "thiol" refers to -SH.

[0028] The term "ester group" refers to -C(O)O(alkyl) or -C(O)O(cycloalkyl), wherein alkyl and cycloalkyl are as defined above.

[0029] The term "acyl" refers to a compound containing a -C(O)R group, wherein R is alkyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl.

[0030] The term "optional" or "optionally" means that the subsequently described event or circumstance may but need not occur, and the description includes instances where the event or circumstance occurs or does not occur. For example, "a heterocyclic group optionally substituted with an alkyl group" means that the alkyl group may but need not be present, and the description includes instances where the heterocyclic group is substituted with an alkyl group and instances where the heterocyclic group is not substituted with an alkyl group.

[0031] The term "substituted" means that one or more hydrogen atoms, preferably up to 5, more preferably 1 to 3 hydrogen atoms in a group are replaced independently of each other by a corresponding number of substituents. It goes without saying that the substituents are only in their possible chemical positions, which the skilled person can determine by means of the prior art and experimental conditions (by experiment or theory) as possible or impossible substitutions. For example, an amino or hydroxyl group with free hydrogen may be unstable when combined with a carbon atom with an unsaturated (e.g. olefinic) bond.

[0032] As used in the present invention, "small nucleic acid" is an oligonucleotide sequence containing about 10-50 single-stranded nucleotides or double-stranded nucleotide base pairs. In some embodiments, the small nucleic acid has a core base sequence that is at least partially complementary to the core sequence of the target gene expressed in the cell. In some embodiments, the oligonucleotide can regulate the expression of the corresponding target gene after being delivered to the cell expressing the gene. Target gene expression can be regulated in vitro or in vivo. "Small nucleic acid" includes, but is not limited to: oligonucleotides, single-stranded oligonucleotides, single-stranded antisense oligonucleotides, short interfering RNA (siRNA), double-stranded RNA (dsRNA), microRNA (miRNA), short hairpin RNA (shRNA), ribozymes, interfering RNA molecules and dicer enzyme substrates.

[0033] As used herein, the terms "oligonucleotide" and "polynucleotide" refer to a polymer of linked nucleosides, wherein each nucleoside may independently be modified or unmodified.

[0034] As used herein, "RNAi drug" refers to an oligonucleotide molecule containing RNA or RNA-like (eg, chemically modified RNA) that can reduce or inhibit the translation of messenger RNA (mRNA) in a sequence-specific manner.

[0035] As used in the present invention, RNAi drugs can act through RNA interference mechanisms (e.g., by inducing mRNA degradation through interaction with the mRNA interference pathway mechanism of mammalian cells (RNA-induced silencing complex RISC)), or other arbitrary mechanisms or pathways. Although it is believed that the term RNAi drug used in the present invention mainly acts through the RNA interference mechanism, the RNAi drug is not limited to or confined to any specific pathway or mechanism of action. RNAi drugs include, but are not limited to, single-stranded oligonucleotides, single-stranded antisense oligonucleotides, short interfering RNA (siRNA), double-stranded RNA (dsRNA), microRNA (miRNA), short hairpin RNA (shRNA) and dicer enzyme substrates. The RNAi drug of the present invention is composed of an oligonucleotide chain that is at least partially complementary to the mRNA as a target. In some embodiments, the RNAi drug of the present invention is double-stranded and is composed of an antisense chain and a sense chain that is at least partially complementary to the antisense chain.

[0036] As used herein, the term "antisense strand" refers to a strand of an siRNA that includes a region that is completely, fully or substantially complementary to a target sequence. The term "positive strand" refers to a strand of an siRNA that includes a region that is completely, fully or substantially complementary to a region as the term antisense strand as defined herein.

[0037] As used in the present invention, the term "complementary region" refers to a region on the antisense strand that is completely, fully or substantially complementary to the target mRNA sequence. In the case where the complementary region is not completely complementary to the target sequence, mispairing can be located in the interior or terminal region of the molecule. Typically, the most tolerated mispairing is located in the terminal region, for example, within 5, 4, 3, 2 or 1 nucleotides at the 5'- and / or 3'-ends. The antisense strand portion that is most sensitive to mispairing is referred to as a "seed region". For example, in a siRNA comprising a 19nt chain, the 19th position (from 5' to 3') can tolerate some mispairings.

[0038] As used in the present invention, the term "complementary" is used to describe the relationship between a first nucleotide sequence (e.g., RNAi drug sense strand or target mRNA) and a second nucleotide sequence (e.g., single-stranded antisense oligonucleotide or double-stranded RNAi drug antisense strand), and refers to the ability of an oligonucleotide or poly-oligonucleotide comprising a first nucleotide sequence to hybridize with an oligonucleotide or poly-oligonucleotide comprising a second nucleotide sequence under certain conditions (under mammalian physiological conditions or similar in vitro conditions) to form base pairing and form a duplex or double helix structure. Complementary sequences include Watson-Crick base pairing or non-Watson-Crick base pairing, and include natural or modified nucleotides or nucleotide analogs to at least the extent that they meet the above hybridization requirements. For example, for the purpose of determining identity or complementarity, monomers a and Af are complementary to U (or T) and are equivalent to A.

[0039] As used in the present invention, the terms "silencing", "reducing", "inhibiting", "down-regulating", or "knockdown" refer to the expression of a given gene, when the RNAi drug molecules of the present invention are used to treat cells, tissues, organs or animals for direct administration, compared with cells, tissues, organs or animals that have not been treated in this way, The level of expression of the gene is reduced or decreased.

[0040] As used herein, the term "sequence" or "nucleotide sequence" refers to the order or sequence of nucleobases or nucleotides, expressed in alphabetical order using standard nucleotide nomenclature.

[0041] As used in the present invention, the term "nucleoside" is a compound composed of two substances, a nucleotide base or a nucleobase, and ribose or deoxyribose; the term "nucleotide" is a compound composed of three substances, a nucleotide base or a nucleobase, ribose or deoxyribose, and phosphate; and "oligonucleotide" refers to a nucleic acid molecule of, for example, less than 100 nucleotides in length.

[0042] As used herein, "nucleotide base" or "nucleobase" is a heterocyclic pyrimidine or purine compound, a standard component of conventional nucleic acids, including adenine (A), guanine (G), cytosine (C), thymine (T) and uracil (U) conventional nucleotide bases. Nucleobases can be modified or replaced by novel unconventional nucleobases.

[0043] As used herein, the term "heterocyclic base moiety" is a novel nucleobase or modified nucleobase as defined herein. In some embodiments, the heterocyclic base moiety is a pyrimidine, a substituted pyrimidine, a purine or a substituted purine, and a nitrogen-containing heterocycle. In some embodiments, the heterocyclic base moiety is an unconventional purine or a substituted purine. In some embodiments, the heterocyclic base moiety is an unconventional pyrimidine or a substituted pyrimidine. In some embodiments, the heterocyclic base moiety contains a non-natural five-membered nitrogen-containing heterocycle. In some embodiments, especially when the 5'-modified nucleotides of the present invention are phosphoramidite compounds, the heterocyclic base moiety may contain one or more protecting groups.

[0044] As used in the present invention, "ribosyl replacement group" refers to a structure that can replace the five-membered furanose ring of naturally occurring ribonucleotides, such as morpholinyl, cyclohexenyl, cyclohexanehexol, or an open ring structure.

[0045] As used in the present invention, "reactive phosphorus group" refers to an active phosphorus-containing group contained in a nucleotide unit or a nucleotide analog unit, which can react with a hydroxyl or an amine group in another molecule, especially in another nucleotide unit or in another nucleotide analog by a nucleophilic attack reaction. Typically, such a reaction produces an ester-type internucleoside bond connecting the first nucleotide unit or the first nucleotide analog unit to the second nucleotide unit or the second nucleotide analog unit. The reactive phosphorus group can be selected from phosphoramidites, H-phosphonates, alkyl-phosphonates, phosphates or phosphate mimetics, including but not limited to: natural phosphates, thiophosphates, dithiophosphates, borane phosphates, borane thiophosphates, phosphonates, halogen-substituted phosphonates and phosphates, phosphoramidates, phosphodiesters, phosphotriesters, thiophosphate triesters, diphosphates and triphosphates, preferably -P(OCH2CH2CN)(N(iPr)2).

[0046] As used in the present invention, "protecting group" refers to an unstable chemical moiety known in the art for preventing reactive groups (e.g., hydroxyl, amino, carboxyl and sulfhydryl) from reacting undesirably during synthesis. Protecting groups are typically selectively and / or orthogonally used to protect sites at other reactive sites in the reaction, and are subsequently removed to release the unprotected group so that it can be used for further reactions. In some embodiments, a "substituted" group or substituent comprises a protecting group.

[0047] "Hydroxy protecting group" refers to a group that can prevent the hydroxyl group from chemical reaction and can be removed under specific conditions to restore the hydroxyl group. It mainly includes silane type protecting groups, acyl type protecting groups or ether type protecting groups, preferably as follows: trimethylsilyl (TMS), triethylsilyl (TES), dimethylisopropylsilyl (DMIPS), diethylisopropylsilyl (DEIPS), tert-butyldimethylsilyl (TBDMS), tert-butyldiphenylsilyl (TBDPS), triisopropylsilyl (TIPS), acetyl (Ac), chloroacetyl, dichloroacetyl, trichloroacetyl, trifluoroacetyl (TFA), benzoyl (Bz), p-methoxybenzoyl, 9-fluorenylmethoxycarbonyl (Fmoc) , allyloxycarbonyl (Alloc), 2,2,2-trichloroethoxycarbonyl (Troc), benzyloxycarbonyl (Cbz), tert-butyloxycarbonyl (Boc), benzyl (Bn), p-methoxybenzyl (PMB), allyl, triphenylmethyl (Tr), p-methoxytriphenylmethyl (MMTr), bis-p-methoxytriphenylmethyl (DMTr), methoxymethyl (MOM), phenoxymethyl (BOM), 2,2,2-trichloroethoxycarbonyl, 2-methoxyethoxymethyl (MEM), methylthiomethyl (MTM), p-methoxybenzyloxymethyl (PMBM).

[0048] When the compounds of the present invention contain double bonds (e.g., in olefins or imines), it is intended to mean that the compound contains both E and Z geometric isomers or cis and trans isomers. Similarly, it is also intended to include all tautomeric forms, which are functional group isomers produced by the rapid movement of an atom in two positions in a molecule. Compounds that exist in different tautomeric forms, a compound is not limited to any specific tautomer, but is intended to cover all tautomeric forms. The configuration diagram of any bond that appears in the present invention is selected only for convenience and is not intended to limit a specific configuration, unless otherwise specified in the text.

[0049] The compounds of the present invention may include one or more asymmetric centers and may therefore exist in a variety of stereoisomeric forms, such as enantiomers and / or diastereomers. For example, the compounds of the present invention may be individual enantiomers, diastereomers or geometric isomers (such as cis and trans isomers), or may be in the form of a mixture of stereoisomers, including racemic mixtures and mixtures enriched in one or more stereoisomers. Isomers may be separated from the mixture by methods known to those skilled in the art, including chiral high pressure liquid chromatography (HPLC) and the formation and crystallization of chiral salts; or preferred isomers may be prepared by asymmetric synthesis.

[0050] The compounds and compositions described herein may have certain atoms (e.g., N, O, or S atoms) in a protonated or deprotonated state, depending on the environment in which the compound or composition is located. Thus, as used herein, the structures described herein take into account that certain functional groups, such as OH, SH, or NH, may be protonated or deprotonated. The disclosure of the present invention is intended to encompass the above-described compounds and compositions, regardless of their protonation state based on the ambient pH, as will be readily appreciated by those of ordinary skill in the art.

[0051] The term "pharmaceutical composition" refers to a mixture containing one or more compounds of the present invention or their pharmaceutically acceptable salts or prodrugs under physiological conditions and other chemical components, as well as other components such as pharmaceutically acceptable carriers and excipients under physiological conditions. The purpose of a pharmaceutical composition is to facilitate administration to an organism, facilitate the absorption of the active ingredients, and thus exert biological activity.

[0052] The present invention also includes isotopically labeled compounds (isotopic variants) which are identical to those described in formula (I) but in which one or more atoms are replaced by atoms having an atomic mass or mass number different from the atomic mass or mass number commonly found in nature. Examples of isotopes that can be introduced into the compounds of the present invention include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine and chlorine, for example 2 H. 3 H. 13 C. 11 C. 14 C. 15 N. 18 O. 17 O. 31 P. 32 P. 35 S. 18 F. 36 Cl. Compounds of the present invention containing the above isotopes and / or other isotopes of other atoms, their prodrugs and pharmaceutically acceptable salts of the compounds or prodrugs are within the scope of the present invention. Certain isotopically labeled compounds of the present invention, such as those in which a radioactive isotope (e.g. 3 H and 14 C) can be used for drug and / or substrate tissue distribution assays. 3 H and carbon-14, i.e. 14 C isotopes are preferred, as they are easy to prepare and detect. 2H(D) is preferred in some cases because of its higher metabolic stability, which can provide therapeutic benefits, such as prolonged half-life in vivo or reduced dosage requirements. Isotope-labeled compounds of formula (I) of the present invention and their prodrugs can generally be prepared by replacing non-isotope-labeled reagents with readily available isotope-labeled reagents when carrying out the processes disclosed in the following schemes and / or Examples and Preparation Examples.

[0053] In one aspect, the present invention provides an application of a compound as a modified nucleotide monomer, wherein the application is to modify RNAi molecules;

[0054] The compound has the structure of general formula I:

[0055]

[0056] in:

[0057] X is selected from O or S or -CH2-, preferably O;

[0058] Y is selected from O or S or -CH2-, preferably O or -CH2-;

[0059] R1 and R2 are independently selected from H, D, halogen, -CN, C 1-6 Alkyl, C 1-6 Haloalkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, -OR a 、-C(O)R a 、-C(O)OR a 、-OC(O)R a 、-NR a R b 、-C(O)NR a R b 、-NR a C(O)R b 、-NR a C(O)OR b ;

[0060] R a and R b Independently selected from H, D, C 1-6 Alkyl, C 1-6 Haloalkyl, C 2-6 Alkenyl, C 2-6 Alkynyl;

[0061] B1 is H or P1, or a chemical bond to the phosphorus phosphate atom of another nucleotide or oligonucleotide ribose 2' or 3' end, preferably P1;

[0062] P1 is a hydroxyl protecting group, for example: trimethylsilyl (TMS), triethylsilyl (TES), dimethylisopropylsilyl (DMIPS), diethylisopropylsilyl (DEIPS), tert-butyldimethylsilyl (TBDMS), tert-butyldiphenylsilyl (TBDPS), triisopropylsilyl (TIPS), acetyl (Ac), chloroacetyl, dichloroacetyl, trichloroacetyl, trifluoroacetyl (TFA), benzoyl (Bz), p-methoxybenzoyl, 9-fluorenylmethoxycarbonyl (Fmoc), allyloxycarbonyl (Al loc), 2,2,2-trichloroethoxycarbonyl (Troc), benzyloxycarbonyl (Cbz), tert-butyloxycarbonyl (Boc), benzyl (Bn), p-methoxybenzyl (PMB), allyl, triphenylmethyl (Tr), p-methoxytriphenylmethyl (MMTr), bis-p-methoxytriphenylmethyl (DMTr), methoxymethyl (MOM), phenoxymethyl (BOM), 2,2,2-trichloroethoxycarbonyl, 2-methoxyethoxymethyl (MEM), methylthiomethyl (MTM), p-methoxybenzyloxymethyl (PMBM). Preferably, it is 4,4'-dimethoxytrityl (DMTr);

[0063] B2 is H or P2, or a chemical bond to the phosphorus phosphate atom of the 2' or 3' end of the ribose of another nucleotide or oligonucleotide, preferably P2;

[0064] P2 is a reactive phosphorus group, such as a phosphoramidite, an H-phosphonate, an alkyl-phosphonate, a phosphate or a phosphate mimetic, such as natural phosphates, phosphothioates, dithiophosphates, boranophosphates, boranothiophosphates, phosphonates, halogen-substituted phosphonates and phosphates, phosphoramidates, phosphodiesters, phosphotriesters, thiophosphate triesters, diphosphates and triphosphates, preferably -P(OCH2CH2CN)(N(iPr)2).

[0065] Base is selected from H, a modified or unmodified heterocyclic base or a leaving group, preferably modified or unmodified uracil (U), thymine (T), adenine (A), guanine (G) and cytosine (C);

[0066] n is independently selected from 0-2; preferably 0 or 1;

[0067] A double-stranded RNA molecule or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, comprising a sense strand and an antisense strand, wherein each strand has 14 to 30 nucleotides, and a specific position of the antisense strand contains one or more nucleotide monomers represented by formula (IV) or formula (V):

[0068]

[0069] in,

[0070] The nucleotide monomers are shown in the order 5'→3' from to --- connect;

[0071] Each group is as defined in any one of claims 1 to 9;

[0072] Preferably, the nucleotide monomer is selected from:

[0073]

[0074] Among them, Base is selected from

[0075] X

[0076] In one embodiment, X is O; in another embodiment, X is S; in another embodiment, X is -CH2-.

[0077] Y

[0078] In one embodiment, Y is O; in another embodiment, Y is S; in another embodiment, Y is -CH2-.

[0079] R1

[0080] In one embodiment, R1 is H; in another embodiment, R1 is D; in another embodiment, R1 is halogen; in another embodiment, R1 is -CN; in another embodiment, R1 is C 1-6 Alkyl; in another embodiment, R1 is C 1-6 haloalkyl; in another embodiment, R1 is C 2-6 In another embodiment, R1 is C 2-6 Alkynyl; in another embodiment, R1 is -OR a In another embodiment, R1 is -C(O)R a In another embodiment, R1 is -C(O)OR a In another embodiment, R1 is -OC(O)R a 、-NR a R b In another embodiment, R1 is -C(O)NR a R b In another embodiment, R1 is -NR a C(O)R b In another embodiment, R1 is -NR a C(O)OR b ;

[0081] Each R aIndependently selected from H, D, C 1-6 Alkyl, C 1-6 Haloalkyl, C 2-6 Alkenyl, C 2-6 Alkynyl;

[0082] Each R b Independently selected from H, D, C 1-6 Alkyl, C 1-6 Haloalkyl, C 2-6 Alkenyl, C 2-6 Alkynyl;

[0083] R2

[0084] In one embodiment, R2 is H; in another embodiment, R2 is D; in another embodiment, R2 is halogen; in another embodiment, R2 is -CN; in another embodiment, R2 is C 1-6 Alkyl; in another embodiment, R2 is C 1-6 haloalkyl; in another embodiment, R2 is C 2-6 In another embodiment, R2 is C 2-6 Alkynyl; in another embodiment, R2 is -OR a In another embodiment, R2 is -C(O)R a In another embodiment, R2 is -C(O)OR a In another embodiment, R2 is -OC(O)R a 、-NR a R b In another embodiment, R2 is -C(O)NR a R b In another embodiment, R2 is -NR a C(O)R b In another embodiment, R2 is -NR a C(O)OR b ;

[0085] Each R a Independently selected from H, D, C 1-6 Alkyl, C 1-6 Haloalkyl, C 2-6 Alkenyl, C 2-6 Alkynyl;

[0086] Each R b Independently selected from H, D, C 1-6 Alkyl, C 1-6 Haloalkyl, C 2-6 Alkenyl, C 2-6 Alkynyl;

[0087] B1 and B2

[0088] In one embodiment, B1 is H; in another embodiment, B1 is P1; in another embodiment, B1 is a chemical bond to the phosphorus phosphate atom of the 2' or 3' end of the ribose of another nucleotide or oligonucleotide;

[0089] In one embodiment, B2 is H; in another embodiment, B2 is P2; in another embodiment, B2 is a chemical bond to the phosphorus phosphate atom of the 2' or 3' end of the ribose of another nucleotide or oligonucleotide;

[0090] P1 and P2

[0091] In one embodiment, P1 is a protecting group; in another embodiment, P1 is a hydroxy protecting group, such as trimethylsilyl (TMS), triethylsilyl (TES), dimethylisopropylsilyl (DMIPS), diethylisopropylsilyl (DEIPS), tert-butyldimethylsilyl (TBDMS), tert-butyldiphenylsilyl (TBDPS), triisopropylsilyl (TIPS), acetyl (Ac), chloroacetyl, dichloroacetyl, trichloroacetyl, trifluoroacetyl (TFA), benzoyl (Bz), p-methoxybenzoyl, 9-fluorenylmethoxycarbonyl (Fmoc), allyloxycarbonyl (Allo c), 2,2,2-trichloroethoxycarbonyl (Troc), benzyloxycarbonyl (Cbz), tert-butyloxycarbonyl (Boc), benzyl (Bn), p-methoxybenzyl (PMB), allyl, triphenylmethyl (Tr), p-methoxytriphenylmethyl (MMTr), bis-p-methoxytriphenylmethyl (DMTr), methoxymethyl (MOM), phenoxymethyl (BOM), 2,2,2-trichloroethoxycarbonyl, 2-methoxyethoxymethyl (MEM), methylthiomethyl (MTM), p-methoxybenzyloxymethyl (PMBM), -P(OCH2CH2CN)(N(iPr)2), preferably DMTr.

[0092] In one embodiment, P2 is a reactive phosphorus group, such as a phosphoramidite, an H-phosphonate, an alkyl-phosphonate, a phosphate or a phosphate mimetic, such as natural phosphates, phosphothioates, dithiophosphates, boranophosphates, boranothiophosphates, phosphonates, halogen-substituted phosphonates and phosphates, phosphoramidates, phosphodiesters, phosphotriesters, thiophosphate triesters, diphosphates and triphosphates, preferably -P(OCH2CH2CN)(N(iPr)2).

[0093] Base

[0094] In one embodiment, Base is H; in another embodiment, Base is a modified or unmodified heterocyclic base or a leaving group, such as, preferably, modified or unmodified U, T, A, G, C.

[0095] In a more specific embodiment, Base is In another more specific embodiment, Base is In another more specific embodiment, Base is In another more specific embodiment, Base is In another more specific embodiment, Base is In another more specific embodiment, Base is In another more specific embodiment, Base is In another more specific embodiment, Base is

[0096] n

[0097] In one embodiment, n is 0; in another embodiment, n is 1; in another embodiment, n is 2.

[0098] Any technical solution or any combination thereof in any of the above specific embodiments may be combined with any technical solution or any combination thereof in other specific embodiments. The present invention is intended to include all combinations of these technical solutions.

[0099] The present invention specifically relates to the following technical solutions:

[0100] 1. Use of a compound as a modified nucleotide monomer, characterized in that:

[0101] Used to modify RNAi molecules;

[0102] The compound has the structure of general formula I:

[0103]

[0104] in:

[0105] X is selected from O or S or -CH2-, preferably O;

[0106] Y is selected from O or S or -CH2-, preferably O or -CH2-;

[0107] R1 and R2 are independently selected from H, D, halogen, -CN, C 1-6 Alkyl, C 1-6 Haloalkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, -ORa 、-C(O)R a 、-C(O)OR a 、-OC(O)R a 、-NR a R b 、-C(O)NR a R b 、-NR a C(O)R b 、-NR a C(O)OR b ;

[0108] R a and R b Independently selected from H, D, C 1-6 Alkyl, C 1-6 Haloalkyl, C 2-6 Alkenyl, C 2-6 Alkynyl;

[0109] B1 is H or P1, or a chemical bond to the phosphorus phosphate atom of another nucleotide or oligonucleotide ribose 2' or 3' end, preferably P1;

[0110] P1 is a hydroxyl protecting group, preferably 4,4'-dimethoxytrityl (DMTr);

[0111] B2 is H or P2, or a chemical bond to the phosphorus phosphate atom of the 2' or 3' end of the ribose of another nucleotide or oligonucleotide, preferably P2;

[0112] P2 is a reactive phosphorus group, preferably -P(OCH2CH2CN)(N(iPr)2);

[0113] Base is selected from H, a modified or unmodified heterocyclic base or a leaving group, preferably modified or unmodified uracil (U), thymine (T), adenine (A), guanine (G) and cytosine (C);

[0114] n is independently selected from 0-2; preferably 0 or 1;

[0115] 2. The use described in technical solution 1, which is a structure of formula (II) or formula (III) or a stereoisomer thereof

[0116]

[0117] Wherein, each group is as defined in claim 1;

[0118] 3. In the compound described in the technical solution, X is selected from O or S or -CH2-, preferably O;

[0119] 4. In the compound described in technical solution 2-3, Y is selected from O or S or -CH2-, preferably O or -CH2-;

[0120] 5. The compound described in technical solution 2-4, wherein n is independently selected from 0-2; preferably 0 or 1;

[0121] 6. The compound described in technical solution 2-5, R1 is selected from H, D, halogen, -CN, C 1-6 Alkyl, C 1-6 Haloalkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, -OR a 、-C(O)R a 、-C(O)OR a 、-OC(O)R a 、-NR a R b 、-C(O)NR a R b 、-NR a C(O)R b 、-NR a C(O)OR b ;

[0122] R a and R b Independently selected from H, D, C 1-6 Alkyl, C 1-6 Haloalkyl, C 2-6 Alkenyl, C 2-6 Alkynyl;

[0123] Preferably, R1 is H;

[0124] 7. The compound described in technical solution 2-6, R2 is selected from H, D, halogen, -CN, C 1-6 Alkyl, C 1-6 Haloalkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, -OR a 、-C(O)R a 、-C(O)OR a 、-OC(O)R a 、-NR a R b 、-C(O)NR a R b 、-NR a C(O)R b 、-NR a C(O)OR b ;

[0125] R a and Rb Independently selected from H, D, C 1-6 Alkyl, C 1-6 Haloalkyl, C 2-6 Alkenyl, C 2-6 Alkynyl;

[0126] Preferably, R2 is H;

[0127] 8. The compound described in technical solution 2-7, wherein Base is H, a modified or unmodified heterocyclic base or a leaving group.

[0128] Preferably,

[0129] Base is selected from Wherein PG is an amino protecting group;

[0130] More preferably, Base is selected from

[0131] 9. The compound described in technical solutions 1-8 is selected from:

[0132]

[0133] Wherein, Base is as defined in any one of technical solutions 1-8, preferably

[0134] 10. A double-stranded RNA molecule or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, comprising a sense strand and an antisense strand, wherein each strand has 14 to 30 nucleotides, and a specific position of the antisense strand contains one or more nucleotide monomers represented by formula (IV) or formula (V):

[0135]

[0136] in,

[0137] The nucleotide monomers are shown in the order 5'→3' from to --- connect;

[0138] Each group is as defined in any one of technical solutions 1-9;

[0139] Preferably, the nucleotide monomer is selected from:

[0140]

[0141] Among them, Base is selected from

[0142] 11. The double-stranded RNA molecule of technical solution 10, wherein the sense strand and the antisense strand each have 19 to 25 nucleotides.

[0143] 12. The double-stranded RNA molecule of technical solutions 10-11, wherein the nucleotide monomer is located at positions 2-8, preferably positions 5-8, more preferably positions 6 or 7, and more preferably positions 7, of the 5'-end of the antisense strand.

[0144] 13. A double-stranded RNA molecule according to any one of technical solutions 10-12, wherein the double-stranded RNA shows reduced thermodynamic stability compared to a double-stranded RNA molecule having the same sequence but not comprising the nucleotide monomer described in claim 10.

[0145] 14. A double-stranded RNA molecule according to any one of technical solutions 10-13, wherein the double-stranded RNA shows reduced off-target toxicity compared to a double-stranded RNA molecule having the same sequence but not comprising the nucleotide monomer described in claim 10.

[0146] 15. The double-stranded RNA molecule of any one of technical solutions 10-14, wherein the double-stranded RNA shows enhanced effectiveness compared to a double-stranded RNA molecule having the same sequence but not comprising the nucleotide monomer described in claim 10.

[0147] 16. A double-stranded RNA molecule according to any one of technical solutions 10-15, wherein the antisense strand has a sequence that is completely complementary to the sense strand and the target mRNA, and has the ability to bind to AGO2 to form a silencing complex.

[0148] 17. A double-stranded RNA molecule according to any one of technical solutions 10-16, wherein the antisense strand has a sequence that is completely complementary to the sense strand and the target mRNA, and has the ability to induce degradation of the sense strand and the target mRNA.

[0149] 18. The double-stranded RNA molecule of any one of technical solutions 10-17, wherein the sense strand and the antisense strand contain 5'- and / or 3'-overhangs.

[0150] 19. The double-stranded RNA molecule of any one of technical solutions 10-18, wherein the double-stranded RNA is coupled to a ligand, preferably, the ligand comprises one or more GalNAc.

[0151] 20. A nucleic acid molecule, the nucleotide sequence of which comprises one or more nucleotide monomers according to claim 10.

[0152] 21. The nucleic acid molecule of technical solution 20, wherein the nucleic acid molecule is single-stranded or double-stranded.

[0153] 22. The nucleic acid molecule of technical solution 20, wherein the nucleic acid molecule is linear or circular.

[0154] 23. The nucleic acid molecule of technical solution 20, wherein the nucleic acid is selected from DNA, RNA or a DNA / RNA hybrid.

[0155] 24. The nucleic acid molecule of any one of technical solutions 20-23, wherein the nucleic acid molecule is selected from small interfering RNA (siRNA) or short hairpin RNA (shRNA).

[0156] 25. A pharmaceutical combination comprising the double-stranded RNA molecule described in technical solutions 10-19, and a pharmaceutically acceptable carrier or excipient.

[0157] 26. A method for inhibiting the expression of a target gene in a cell, comprising the double-stranded RNA molecule according to any one of claims 10 to 19 and the step of introducing the double-stranded RNA molecule into a cell.

[0158] 27. A method for reducing off-target toxicity in cells, comprising the double-stranded RNA molecule according to any one of claims 10 to 19 and the step of introducing the double-stranded RNA molecule into cells.

[0159] Compared with the prior art, the present invention has the following beneficial effects:

[0160] The present invention introduces the nucleotide monomers into the antisense chain seed region of siRNA through specific 2', 5'-phosphate linkages, significantly reducing the potential off-target effects caused by the antisense chain seed region, thereby enabling siRNA to silence target gene mRNA with higher selectivity and a larger safety window for siRNA treatment, thereby effectively treating related diseases caused by target gene mRNA. BRIEF DESCRIPTION OF THE DRAWINGS

[0161] Figure 1 The process flow for the synthesis of double-stranded nucleic acid sequences.

[0162] Figure 2 Schematic diagram of solid phase synthesis.

[0163] Figure 3 These are the relative luciferase activity results for sequences NPD011-171 and NPD013s-213.

[0164] Figure 4 Schematic diagram of siRNA structure. The modified nucleotide monomer of the present invention is located at position 5 to 8 from the 5'-end of the antisense strand, preferably at position 6 or 7, more preferably at position 7. DETAILED DESCRIPTION

[0165] The present application is further described below through detailed examples. It should be understood that the following examples are only used to illustrate the present application and do not limit the content of the invention.

[0166] Further understand the compounds of the present invention and their preparation by examples, which illustrate some methods of preparing or using the compounds. However, it is to be understood that these examples are not limited to the present invention. Now known or further developed content of the present invention is considered to fall within the scope of the invention described in the present invention and claimed.

[0167] The compounds of the present invention are prepared using convenient starting materials and general experimental methods and steps. The present invention provides typical or preferred reaction conditions, such as reaction temperature, time, solvent, pressure, and molar ratio of reactants. However, unless otherwise specified, other reaction conditions can also be adopted. The optimized conditions may change with the use of specific reactants or solvents, but in general, the reaction optimized steps and conditions can be determined.

[0168] The present invention will be further described in detail below in conjunction with specific examples. The following examples are not intended to limit the present invention, but are only intended to illustrate the present invention. The experimental methods used in the following examples are generally conventional, unless otherwise specified, and the materials, reagents, etc. used in the following examples are commercially available, unless otherwise specified.

[0169] The instruments, equipment and reagents used in the examples can be obtained through various channels, such as commercial purchase or laboratory preparation.

[0170] In addition, some protecting groups may be used in the present invention to protect certain functional groups from unnecessary reactions. Protecting groups suitable for various functional groups and their protection or deprotection conditions are widely known to those skilled in the art. For example, TW Greene and GM Wuts's "Protective Groups in Organic Preparations" (3rd edition, Wiley, New York, 1999 and references therein) describes in detail the protection or deprotection of a large number of protecting groups.

[0171] The separation and purification of compounds and intermediates can be carried out by appropriate methods and steps according to specific needs, such as filtration, extraction, distillation, crystallization, column chromatography, preparative thin layer plate chromatography, preparative high performance liquid chromatography, or a combination of the above methods. The specific use method can refer to the examples described in the present invention. Of course, other similar separation and purification means can also be adopted. Conventional methods (including physical constants and spectral data) can be used to characterize them.

[0172] The structures of the compounds were determined by nuclear magnetic resonance (NMR) and / or mass spectrometry (MS). -6The unit of (ppm) is given. NMR measurements were performed using an Agent Pro Pulse 500 MHz nuclear magnetic resonance spectrometer, with deuterated dimethyl sulfoxide (DMSO-d6) and deuterated chloroform (CDCl3) as the measuring solvent and tetramethylsilane (TMS) as the internal standard.

[0173] Thermo Ultimate 3000-ISQ (manufacturer: Thermo) was used for MS measurement.

[0174] Thermo Ultimate 3000 (analytical column: DNApac RP, 4 um 3.0×100 mm) (manufacturer: Thermo) was used as a high performance liquid chromatograph.

[0175] The preparative liquid phase was analyzed using LC-100 high performance liquid chromatography (preparative column: PS-15Q, 10×250 mm) (manufacturer: Jiangsu Zhirun Technology).

[0176] As a nucleic acid synthesizer, Dr. Oligo 48 (manufacturer: BIOLYTIC) was used.

[0177] The thin layer chromatography silica gel plate used was Qingdao Ocean Chemical GF254 silica gel plate. The silica gel plate used in thin layer chromatography (TLC) had a specification of 0.15 mm to 0.2 mm, and the specification used for thin layer chromatography separation and purification products was 0.4 mm to 0.5 mm.

[0178] Column chromatography generally uses Qingdao Marine Silica Gel 100-200 mesh and 200-300 mesh silica gel as the carrier.

[0179] The known starting materials of the present invention can be synthesized by methods known in the art, or can be purchased from online shopping malls, Beijing Coupling, Sigma, J&K, Shanghai Aladdin, Shanghai Bid, and other companies.

[0180] Unless otherwise specified in the examples, the reactions can be carried out under an argon atmosphere or a nitrogen atmosphere.

[0181] Argon atmosphere or nitrogen atmosphere means that the reaction bottle is connected to an argon or nitrogen balloon with a capacity of about 1L.

[0182] The reaction solvent, organic solvent or inert solvent is expressed as the solvent used that does not participate in the reaction under the described reaction conditions, including, for example, benzene, toluene, acetonitrile, tetrahydrofuran (THF), dimethylformamide (DMF), chloroform, dichloromethane, ether, methanol, N-methylpyrrolidone (NMP), pyridine, etc. Unless otherwise specified in the examples, the solution refers to an aqueous solution.

[0183] The chemical reactions described in the present invention are generally carried out under normal pressure. The reaction temperature is between -78°C and 200°C. The reaction time and conditions are, for example, between -78°C and 200°C at one atmosphere, and are completed within about 1 to 24 hours. If the reaction is left overnight, the reaction time is generally 16 hours. In the embodiments, unless otherwise specified, the reaction temperature is room temperature, which is 20°C to 30°C.

[0184] The reaction progress in the embodiment is monitored by thin layer chromatography (TLC), and the developing solvent systems used in the reaction are: A: dichloromethane and methanol system, B: n-hexane and ethyl acetate system, C: petroleum ether and ethyl acetate system, D: acetone, and the volume ratio of the solvent is adjusted according to the polarity of the compound.

[0185] The eluent system of column chromatography and the developing solvent system of thin layer chromatography used for purifying compounds include: A: dichloromethane and methanol system, B: n-hexane and ethyl acetate system, C: petroleum ether and ethyl acetate system. The volume ratio of the solvent is adjusted according to the polarity of the compound, and a small amount of alkaline or acidic reagents such as triethylamine and acetic acid can also be added for adjustment.

[0186] In addition, the terms "first" and "second" are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features. Therefore, the features defined as "first" and "second" may explicitly or implicitly include at least one of the features. In the description of the present invention, the meaning of "plurality" is at least two, such as two, three, etc., unless otherwise clearly and specifically defined.

[0187] In the present invention, the term "optionally" is only used for descriptive purposes and cannot be understood as indicating or implying relative importance. Thus, a feature defined as "optionally" may explicitly or implicitly include or exclude the feature.

[0188] In the present invention, the term "sense strand" refers to the strand on the RNA molecule that carries the nucleotide sequence encoding protein amino acid information, which is called the sense strand, also known as the coding strand, meaningful strand or positive strand, and the other nucleotide sequence complementary to it is the antisense strand.

[0189] The instruments, equipment and reagents used in the examples can be obtained through various channels, such as purchased or prepared.

[0190] Unless otherwise defined, all professional and scientific terms used herein have the same meaning as those familiar to those skilled in the art. In addition, any method and material similar or equivalent to the described content can be applied to the method of the present invention.

[0191] Abbreviations

[0192] ℃ = degrees Celsius

[0193] BCl3 = Boron trichloride

[0194] DMTrCl=4,4'-dimethoxytrityl chloride

[0195] DCM = dichloromethane

[0196] ETT = 5-ethylthiotetrazolyl

[0197] EA = Ethyl acetate

[0198] ESI = Electrospray Ionization

[0199] g = grams

[0200] LC-MS = Liquid chromatography-mass spectrometry

[0201] M = moles per liter

[0202] [M+H] + = mass of parent compound + one proton

[0203] [M+Na] + = mass of parent compound + sodium ion

[0204] mg = milligrams

[0205] mL = milliliters

[0206] mmol = millimole

[0207] MS = Mass Spectrometry

[0208] m / z = mass-to-charge ratio

[0209] nM = nanomolar

[0210] PE = Petroleum Ether

[0211] Py = Pyridine

[0212] RP-HPLC = Reverse Phase High Performance Liquid Chromatography

[0213] TEA = triethylamine

[0214] THF = Tetrahydrofuran

[0215] TMSOTf = trimethylsilyl trifluoromethanesulfonate

[0216] The compounds disclosed in the present invention can be prepared by the following synthetic methods.

[0217] Example 1: Preparation of [(2R,3R,3As,6S,6aR)-6-(bis(4-methoxyphenyl)(phenyl)methoxy)-2-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)hexahydrofuro[3,2-b]furan-3-yl(2-cyanoethyl)diisopropylphosphoramidite] (OBC04-U-PN, this nucleoside is referred to as OBC04U in the sequence listing)

[0218]

[0219] The specific synthesis method is as follows:

[0220] Step 1: Preparation of [(2R,3R,3aS,6S,6aR)-6-benzyloxy-2-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)hexahydrofuro[3,2-b]furan-3-yl acetate] (OBC04-U1)

[0221] At room temperature, uracil (1.12 g, 10 mmol) was added to HMDS (10 mL, 50 mmol), and ammonium sulfate (0.33 g, 2.5 mmol) was added. The mixture was sealed, replaced with nitrogen three times, and heated to 130°C for overnight reaction. The solvent was removed by concentration under reduced pressure, and 20 mL of anhydrous dichloromethane was added to dissolve the mixture. [(3R, 3aS, 6S, 6aR)-6-(benzyloxy)hexahydrofurano[3,2-b]furan-2,3-diyl diacetate] (OBC04-1) (3.83 g, 3.9 mmol) was dissolved in 10 mL of dichloromethane and added to the reaction system. The mixture was cooled to 0°C, and TMSOTf (2.2 g, 10 mmol) was added. The mixture was heated to room temperature for overnight reaction. Saturated sodium bicarbonate solution (50 mL) was added to the reaction mixture to quench the mixture, and ethyl acetate (100 mL) was added to dilute and extract the mixture. The organic phase was washed with a saturated sodium chloride solution (50 mL × 2), dried over anhydrous sodium sulfate, and then filtered, and the filtrate was concentrated under reduced pressure. The crude product was purified by column chromatography using silica gel column chromatography (PE: EA = 2: 1 to 1: 1), and all target components were collected and concentrated to obtain [(2R, 3R, 3aS, 6S, 6aR)-6-benzyloxy-2-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)hexahydrofuro[3,2-b]furan-3-yl acetate] (OBC04-U1) (1.2 g, off-white foamy solid, molar yield: 66%).

[0222] LC-MS (ESI): m / z 411.11 [M+Na] + .

[0223] Step 2: Preparation of [(2R,3R,3aS,6S,6aR)-2-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-6-hydroxyhexahydrofuro[3,2-b]furan-3-yl acetate] (OBC04-U2)

[0224] At -20°C, add [(2R,3R,3aS,6S,6aR)-6-benzyloxy-2-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)hexahydrofuro[3,2-b]furan-3-yl acetate] (OBC04-U1) (1.2 g, 3.0 mmol) to a reaction bottle containing 20 mL of anhydrous dichloromethane, and slowly add a dichloromethane solution of boron trichloride (4.8 mL, 4.8 mmol, concentration of 1 M). After stirring for 2 hours, warm to room temperature and react for 3 hours. The crude product was directly purified by silica gel column chromatography (MeOH in DCM = 0% → 5%), and all target components were collected and concentrated to give [(2R, 3R, 3aS, 6S, 6aR)-2-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-6-hydroxyhexahydrofurano[3,2-b]furan-3-yl acetate] (OBC04-U2) (0.51 g, 1.71 mmol, white solid, 57%).

[0225] LC-MS (ESI): m / z 299.18 [M+H] + .

[0226] Step 3: Preparation of [(2R,3R,3aS,6S,6aR)-6-(bis(4-methoxyphenyl)(phenyl)methoxy)-2-(2,4-dioxo-3,4-dihydropyrimidin-1-(2H)-yl)hexahydrofuro[3,2-b]furan-3-yl acetate] (OBC04-U3):

[0227] At room temperature, add [(2R,3R,3aS,6S,6aR)-2-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-6-hydroxyhexahydrofurano[3,2-b]furan-3-yl acetate] (OBC04-U2) (440 mg, 1.47 mmol) and 4,4'-dimethoxytriphenylmethane chloride (600 mg, 1.77 mmol) to a reaction bottle containing 10 mL of anhydrous pyridine. Seal and replace with nitrogen three times. React at room temperature overnight. After the reaction is complete, concentrate the reaction solution under reduced pressure. Add ethyl acetate (50 mL) to redissolve and wash with saturated sodium bicarbonate solution (50 mL×3). Dry the organic phase with anhydrous sodium sulfate, filter, and concentrate the filtrate under reduced pressure. The crude product [(2R,3R,3aS,6S,6aR)-6-(bis(4-methoxyphenyl)(phenyl)methoxy)-2-(2,4-dioxo-3,4-dihydropyrimidin-1-(2H)-yl)hexahydrofuro[3,2-b]furan-3-yl acetate] (OBC04-U3) (0.8 g) was obtained.

[0228] LC-MS (ESI): m / z 601.22 [M+H] + .

[0229] Step 3: Preparation of [1-((2R,3R,3aR,6S,6aR)-6-(bis(4-methoxyphenyl)(phenyl)methoxy)-3-hydroxyhexahydrofuro[3,2-b]furan-2-yl)pyrimidine-2,4-(1H,3H)-dione] (OBC04-U4):

[0230] The crude product [(2R,3R,3aS,6S,6aR)-6-(bis(4-methoxyphenyl)(phenyl)methoxy)-2-(2,4-dioxo-3,4-dihydropyrimidin-1-(2H)-yl)hexahydrofuro[3,2-b]furan-3-yl acetate] (OBC04-U3) (800 mg) was dissolved in methanol (5 mL) at room temperature, and NH3 / MeOH (10 mL, 7 M) was added and stirred for 2 hours. After the reaction was complete, the reaction solution was concentrated under reduced pressure. The crude product was directly purified by silica gel column chromatography (PE:EA=2:1 to 1:1) to obtain [1-((2R,3R,3aR,6S,6aR)-6-(bis(4-methoxyphenyl)(phenyl)methoxy)-3-hydroxyhexahydrofuro[3,2-b]furan-2-yl)pyrimidine-2,4-(1H,3H)-dione] (OBC04-U4) (460 mg, 0.82 mmol, two-step molar yield: 56%).

[0231] LC-MS (ESI): m / z 559.22 [M+H] + .

[0232] Step 5: Preparation of [(2R,3R,3As,6S,6aR)-6-(bis(4-methoxyphenyl)(phenyl)methoxy)-2-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)hexahydrofuro[3,2-b]furan-3-yl(2-cyanoethyl)diisopropylphosphoramidite] (OBC04-U-PN)

[0233] Under argon protection, [1-((2R,3R,3aR,6S,6aR)-6-(bis(4-methoxyphenyl)(phenyl)methoxy)-3-hydroxyhexahydrofurano[3,2-b]furan-2-yl)pyrimidine-2,4-(1H,3H)-dione](OBC04-U4)(450mg, 0.80mmol) was co-rotated three times with anhydrous acetonitrile. At 0°C, 10mL of anhydrous dichloromethane was added to dissolve. 2-cyanoethyl-N,N,N',N'-tetraisopropylphosphorodiamidite (365mg, 1.2mmol) and 5-ethylthiotetrazolyl (105mg, 0.80mmol) were added to the above reaction system. Slowly warm to room temperature and continue stirring for 3 hours. After the reaction is completed, the reaction solution is concentrated under reduced pressure. The crude product was purified by flash silica gel column chromatography (EA:PE=1:2+0.5% Et3N), the eluent containing all target components was collected and concentrated under reduced pressure to give [(2R,3R,3As,6S,6aR)-6-(bis(4-methoxyphenyl)(phenyl)methoxy)-2-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)hexahydrofuran[3,2-b]furan-3-yl(2-cyanoethyl)diisopropylphosphoramidite](OBC04-U-PN) (500 mg, 0.66 mmol, off-white solid, 82%).

[0234] LC-MS (ESI): m / z 759.35 [M+H] + .

[0235] Example 2: Preparation of OBC04-A-PN (this nucleoside is referred to as OBC04A in the sequence listing)

[0236]

[0237]

[0238] Compound OBC04-A-PN was prepared according to the above steps.

[0239] Example 3: Preparation of OBC04-C-PN (this nucleoside is referred to as OBC04C in the sequence listing)

[0240]

[0241]

[0242] Compound OBC04-A-PN was prepared according to the above steps.

[0243] Example 4: Preparation of OBC04-G-PN (this nucleoside is referred to as OBC04G in the sequence listing)

[0244]

[0245]

[0246] Compound OBC04-G-PN was prepared according to the above steps.

[0247] Example 5: Preparation of BC04-U-PN (this nucleoside is referred to as BC04U in the sequence listing)

[0248]

[0249]

[0250] Compound BC04-U-PN was prepared according to the above steps.

[0251] Example 6: Synthesis of double-stranded nucleic acid sequences

[0252] Process flow chart Figure 1 shown.

[0253] Process Description:

[0254] Oligo synthesis starts with Universal-CPG, and uses 3'-O-(2-cyanoethyl)phosphoramidite / 4,4'dimethoxytrityl (dimethoxytrityl, DMT) group protection method to assemble oligonucleotide chains on a solid support (controlled pore glass (CPG). Each synthesis cycle includes hydroxyl deprotection, coupling, capping and oxidation (thiolation). Each coupling reaction is carried out by activating the appropriate phosphoramidite monomer and reacting with the protected nucleotide or free hydroxyl group of the oligonucleotide fixed on the support. According to the sequence information, the corresponding crude oligonucleotide single-stranded CPG-Oligo is synthesized cyclically, and the crude oligonucleotide single-strand is cleaved from the solid support and the relevant protecting groups are removed. Then the final product is obtained through preparative chromatography purification, ultrafiltration desalting, annealing, and freeze-drying steps. See. Figure 2 .

[0255] (1) Synthesis procedure

[0256] The following units are included:

[0257] 1) De-blocking: The 5'-OH end of the ribonucleotide is protected with a DMT group (dimethoxytrityl). In the first step of the synthesis, trichloroacetic acid (TCA) is used to remove the DMT protecting group from the solid phase support and the ribonucleotide, so that the 5'-OH of the naked ribonucleotide can be coupled with a new base.

[0258] 2) Coupling: Nucleotide monomers are mixed with activation reagents and react with CPG-Oligo in the synthesis column. The activation reagent provides a proton to the N atom of diisopropylamide on the 3'-phosphoric acid to form a phosphoramidite tetrazole active intermediate. When phosphoramidite tetrazole comes into contact with CPG-Oligo, it undergoes a nucleophilic reaction with its 5'-hydroxyl group, coupling occurs and tetrazole is removed, extending a nucleotide.

[0259] 3) Capping: Since the coupling efficiency cannot reach 100%, in order to prevent the uncoupled CPG-Oligo from continuing to the next step of coupling, the 5' hydroxyl group of CPG-Oligo is capped with a Cap reagent.

[0260] 4) Oxidation or thiolation: After the coupling reaction, the nucleotide is connected to the oligonucleotide on the CPG through a phosphite bond (trivalent phosphorus). This phosphite bond is unstable and easily hydrolyzed by acid and alkali. The trivalent phosphorus here is oxidized to pentavalent phosphorus by an oxidizing agent. Thiolation refers to the reaction of the trivalent phosphorus of the phosphite bond to form a phosphorus-sulfur bond under weak alkaline conditions by a thiolation agent.

[0261] 5) A series of syntheses were performed according to standard oligo solid phase synthesis. The synthesis sequence is as follows:

[0262]

[0263] The expression of the modification of nucleoside monomer is as follows:

[0264] a represents 2'-O-methyladenosine, u represents 2'-O-methyluridine, g represents 2'-O-methylguanosine, c represents 2'-O-methylcytosine, Af represents 2'-fluoroadenosine, Uf represents 2'-fluorouridine, Gf represents 2'-fluoroguanosine, Cf represents 2'-fluorocytosine, * represents connection with phosphorothioate bond, there is no symbol between adjacent nucleotides with direct phosphate bond.

[0265] The raw materials (phosphoramidite monomers) and auxiliary materials required for the synthesis of the above nucleic acid chains were purchased from the following companies:

[0266]

[0267]

[0268] The structure of 1048 is as follows, prepared by referring to the method described in patent CN202110008013.3:

[0269]

[0270] (2) Aminolysis

[0271] The synthesized CPG-oligo was transferred from the synthesis column to a 3 mL centrifuge tube, and 3% ethylenediamine / ammonia solution was added at 1 mL / 4 μmol, and aminolysis was performed at 65° C. for 5 h. After the aminolysis was completed, vacuum centrifugation was performed to remove the ammonia solution and purify the solution.

[0272] (3) Purification

[0273] After aminolysis, the sample was purified by anion column exchange chromatography, and the liquid phase system adopted high performance liquid chromatography conditions as follows.

[0274] Chromatographic column: PS-15Q 10×250mm

[0275] Flow rate: 4mL / min

[0276] Detection wavelength: 260nm

[0277] Ion column preparation purification gradient program:

[0278] Mobile phase: Phase A: 10 mM NaOH solution (PH = 10)

[0279] Phase B: 10 mM NaOH solution (PH = 10) + 2 M NaCl

[0280] Time (min) Mobile phase A (%) Mobile phase B (%) 0 100 0 5 80 20 15 78 22 50 60 40 80 50 50

[0281] After preparation, the samples were confirmed by mass spectrometry and purity.

[0282] (5) Step 4: Ultrafiltration

[0283] After purification, add 4 mL of PBS to dissolve the sample, transfer it to a 15 mL 1K ultrafiltration tube, centrifuge it at 5000 r / min for 45 min, and then use Nanodrop to detect whether the liquid in the centrifuge tube contains the sample. If the sample is detected at a wavelength of 260 nm, it means that the ultrafiltration membrane is damaged and the ultrafiltration tube needs to be replaced and ultrafiltration is repeated. If the sample is not detected at a wavelength of 260 nm, add 4 mL of RNase water and ultrafiltration again. Ultrafiltration is performed 3 times, each time for 45 min.

[0284] (6) Step 5: Annealing

[0285] After ultrafiltration of the sense and antisense chains, dilute the samples at a concentration of 3 mg / mL, and mix the sense and antisense chains at a molar ratio of 1:1. Heat the water bath to 90°C, place the sense and antisense chains mixed in proportion in the water bath, and anneal for 30 minutes. Turn off the water bath and let it cool to room temperature naturally. Take 10 μL of the sample for HPLC detection.

[0286] Example 7 siRNA silencing efficiency test

[0287] Experimental methods:

[0288] For the test sequence, recombinant plasmids for detecting on-target activity and off-target activity in the seed region (GSM) were constructed respectively. The inserted fragment of the on-target activity detection plasmid is the target gene sequence. The inserted sequence unit of the seed region off-target activity detection plasmid is completely complementary to the seed region of the antisense chain of the tested siRNA (positions 2 to 8 from the 5'-end), and the sequences at other positions are completely non-complementary (A:C; T:G; G:T; C:A). Five repeating sequence units are inserted into each recombinant plasmid, and 4 nucleotides are spaced between sequence units. The gene sequence was ordered from BGI, and the target sequence was cloned into psiCHECK TM -2 plasmid Xho1 / Not 1 site.

[0289] The silencing efficiency of the sequence on the target gene was evaluated in HEK293 cells using a fluorescent reporter enzyme system. HEK293 (ATCC) cells were cultured in DMEM (Gibco) medium supplemented with 10% FBS (Gibco). The cells were seeded in a 96-well white plate at a density of 15,000 cells per well the day before transfection and transfected after adherence. The transfection process was based on the standard procedure in the instruction manual of Lipofectemine2000 (Thermofisher). The transfection system included 0.4 μL Lipofectemine2000 / 25 ng plasmid / well and a selected dose of siRNA test substance, with 3 parallel wells set up for each transfection condition. The final concentration of siRNA was 10 nM. 24 hours after transfection, the Dual-Glo@Luciferase Assay System (Promega E2940) was used according to the standard procedure in the instruction manual, and the dual luciferase activity was detected using a full-spectrum microplate reader (Biotek, Synergy H1). The test results were compared with the single transfection of psiCHECK TM -2 recombinant plasmid without siRNA was used as the control group, and the "Renilla fluorescence luminescence value / firefly fluorescence luminescence value" of this group was set to 100%. The "Renilla fluorescence luminescence value / firefly fluorescence luminescence value" of other concentration groups were compared with the control group data to obtain relative luminescence values.

[0290] Experimental results:

[0291]

[0292] in conclusion:

[0293] Experimental results showed that BC04U could significantly reduce the off-target activity of the antisense strand seed region of the sequence while maintaining the on-target activity of the sequence.

[0294] The above contents are only used to illustrate the technical solution of the present invention, rather than to limit the protection scope of the present invention. Simple modifications or equivalent substitutions of the technical solution of the present invention by those skilled in the art do not deviate from the essence and scope of the technical solution of the present invention.

Claims

1. Use of a compound as a modified nucleotide monomer, characterized in that: Used to modify RNAi molecules; The compound has the structure of general formula I: in: X is selected from O or S or -CH2-, preferably O; Y is selected from O or S or -CH2-, preferably O or -CH2-; R1 and R2 are independently selected from H, D, halogen, -CN, C 1-6 Alkyl, C 1-6 Haloalkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, -OR a 、-C(O)R a 、-C(O)OR a 、-OC(O)R a 、-NR a R b 、-C(O)NR a R b 、-NR a C(O)R b 、-NR a C(O)OR b ; R a and R b Independently selected from H, D, C 1-6 Alkyl, C 1-6 Haloalkyl, C 2-6 Alkenyl, C 2-6 Alkynyl; B1 is H or P1, or a chemical bond to the phosphorus phosphate atom of another nucleotide or oligonucleotide ribose 2' or 3' end, preferably P1; P1 is a hydroxyl protecting group, preferably 4,4'-dimethoxytrityl (DMTr); B2 is H or P2, or a chemical bond to the phosphorus phosphate atom of the 2' or 3' end of the ribose of another nucleotide or oligonucleotide, preferably P2; P2 is a reactive phosphorus group, preferably -P(OCH2CH2CN)(N(iPr)2); Base is selected from H, a modified or unmodified heterocyclic base or a leaving group, preferably modified or unmodified uracil (U), thymine (T), adenine (A), guanine (G) and cytosine (C); n is independently selected from 0-2; preferably 0 or 1.

2. The compound for modifying a nucleotide monomer as claimed in claim 1, wherein the compound has a structure of the general formula II or the general formula III or a stereoisomer thereof in, Each group is as defined in claim 1.

3. The compound according to claim 2, characterized in that In the compound, X is selected from O or S or -CH2-, preferably O.

4. The compound according to claim 2, characterized in that The Y is selected from O or S or -CH2-, preferably O or -CH2-.

5. The compound according to claim, characterized in that Said n is independently selected from 0-2; preferably 0 or 1.

6. The compound according to claim 2, characterized in that R1 is selected from H, D, halogen, -CN, C 1-6 Alkyl, C 1-6 Haloalkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, -OR a 、-C(O)R a 、-C(O)OR a 、-OC(O)R a 、-NR a R b 、-C(O)NR a R b 、-NR a C(O)R b 、-NR a C(O)OR b ; R a and R b Independently selected from H, D, C 1-6 Alkyl, C 1-6 Haloalkyl, C 2-6 Alkenyl, C 2-6 Alkynyl; Preferably, R1 is H.

7. The compound according to claim 2, characterized in that R2 is selected from H, D, halogen, -CN, C 1-6 Alkyl, C 1-6 Haloalkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, -OR a 、-C(O)R a 、-C(O)OR a 、-OC(O)R a 、-NR a R b 、-C(O)NR a R b 、-NR a C(O)R b 、-NR a C(O)OR b ; R a and R b Independently selected from H, D, C 1-6 Alkyl, C 1-6 Haloalkyl, C 2-6 Alkenyl, C 2-6 Alkynyl; Preferably, R2 is H.

8. The compound according to claim 2, characterized in that The Base is H, a modified or unmodified heterocyclic base or a leaving group, Preferably, Base is selected from Wherein PG is an amino protecting group; More preferably, Base is selected from 9. The compound according to claim 2, selected from: in, Base is as defined in claim 8, preferably 10. A double-stranded RNA molecule or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, comprising a sense strand and an antisense strand, wherein each strand has 14 to 30 nucleotides, and a specific position of the antisense strand contains one or more nucleotide monomers represented by formula (IV) or formula (V): in, The nucleotide monomers are shown in the order 5'→3' from arrive connect; Each group is as defined in any one of claims 1 to 9; Preferably, the nucleotide monomer is selected from: Among them, Base is selected from 11. The double-stranded RNA molecule according to claim 10 or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, characterized in that: The sense and antisense strands each have 19 to 25 nucleotides.

12. The double-stranded RNA molecule according to claim 10 or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, characterized in that: The nucleotide monomer is located at position 2-8, preferably position 5-8, more preferably position 6 or 7, and more preferably position 7, of the 5'-end of the antisense strand.

13. The double-stranded RNA molecule according to claim 10 or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, characterized in that: The double-stranded RNA exhibits reduced thermodynamic stability compared to a double-stranded RNA molecule having the same sequence but not comprising the nucleotide monomer of claim 10.

14. The double-stranded RNA molecule according to claim 10 or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, characterized in that: The double-stranded RNA shows reduced off-target toxicity compared to a double-stranded RNA molecule having the same sequence but not comprising the nucleotide monomer of claim 10.

15. The double-stranded RNA molecule according to claim 10 or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, characterized in that: The double-stranded RNA shows enhanced effectiveness compared to a double-stranded RNA molecule having the same sequence but not comprising the nucleotide monomer of claim 10.

16. The double-stranded RNA molecule according to claim 10 or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, characterized in that: The antisense strand has a sequence that is completely complementary to the sense strand and the target mRNA, and has the ability to bind to AGO2 to form a silencing complex.

17. The double-stranded RNA molecule according to claim 10 or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, characterized in that: The antisense strand has a sequence that is completely complementary to the sense strand and the target mRNA, and has the ability to induce degradation of the sense strand and the target mRNA.

18. The double-stranded RNA molecule according to claim 10 or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, characterized in that: The sense strand and antisense strand contain 5'- and / or 3'-overhangs.

19. The double-stranded RNA molecule according to claim 10 or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, characterized in that: The double-stranded RNA is coupled to a ligand, preferably, the ligand comprises one or more GalNAc.

20. A nucleic acid molecule, the nucleotide sequence of which comprises one or more nucleotide monomers according to claim 10.

21. The nucleic acid molecule according to claim 20, characterized in that The nucleic acid molecule is single-stranded or double-stranded.

22. The nucleic acid molecule according to claim 20, characterized in that The nucleic acid molecule is linear or circular.

23. The nucleic acid molecule according to claim 20, characterized in that The nucleic acid is selected from DNA, RNA or a DNA / RNA hybrid.

24. The nucleic acid molecule according to any one of claims 20 to 23, characterized in that The nucleic acid molecule is selected from small interfering RNA (siRNA) or short hairpin RNA (shRNA).

25. A pharmaceutical composition comprising the double-stranded RNA molecule of claims 10-19, and a pharmaceutically acceptable carrier or excipient.

26. A method for inhibiting the expression of a target gene in a cell, characterized in that: The expression method comprises the double-stranded RNA molecule according to any one of claims 10 to 19 and the step of introducing the double-stranded RNA molecule into cells.

27. A method for reducing off-target toxicity in cells, characterized in that The method comprises the double-stranded RNA molecule according to any one of claims 10 to 19 and the step of introducing the double-stranded RNA molecule into a cell.

Images