Modified nucleotide monomers and uses thereof

Abstract

The invention relates to a nucleotide monomer compound and application thereof in nucleic acid interference (RNAi) drug molecules. After the nucleotide monomer is introduced into the siRNA antisense strand seed region, the potential off-target effect of the antisense strand seed region can be remarkably reduced, and the activity of siRNA can be maintained or improved, so that target gene mRNA is silenced with higher selectivity, the safety window of siRNA drugs is expanded, the druggability of siRNA can be improved, and the siRNA activity can be improved. Therefore, related diseases caused by the target gene mRNA can be treated more effectively. # imgabs0 #

Description

Technical Field

[0001] The present invention relates to the field of biochemistry, and in particular to a modified nucleotide monomer and application thereof in siRNA molecules. Background Art

[0002] In recent years, considerable progress has been made in the development of siRNA drugs. siRNA as an active ingredient in drugs is already known to the public. The corresponding siRNA mechanism of action is an RNA interference method mediated by a characteristic endonuclease complex. From a genetic level, siRNA can treat almost all diseases, including tumors, infectious diseases, genetic diseases, etc., so siRNA has become the hottest field of biomedical research. In theory, siRNA therapy can target target genes in any 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.

[0003] Studies have shown that a major factor in RNAi off-target effects is that some fragments of the siRNA sequence that binds to the target mRNA may function like microRNA (miRNA). They can bind to the 3'-untranscribed region (3'-UTR) of other mRNAs to inhibit the translation and stability of mRNA. MiRNA mainly recognizes target genes through base pairing between the seed region (position 2-8 at the 5'-end) and the target mRNA. Studies have also shown that the off-target effects caused by siRNA mainly come from the base complementarity between the antisense chain seed region of siRNA and one or more mRNAs. Therefore, the sequence of the antisense chain seed region may affect the expression of multiple genes and become the main factor in the side effects of RNAi drugs.

[0004] In order to effectively solve the off-target effect of the antisense chain 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 siRNA antisense chain seed region, it can reduce the off-target effect of RNAi and improve the safety of the drug. Therefore, the off-target effect of the antisense chain seed region can be reasonably solved by modifying the nucleoside monomers in its region.

[0005] The present invention synthesizes modified nucleotide monomers by means of medicinal chemistry, and introduces them into specific positions of the seed region of the siRNA antisense chain by means of phosphodiester bonds. This modification of the siRNA sequence can significantly reduce the off-target effect of siRNA while not affecting the silencing effect of the siRNA target gene, thereby expanding the safety window of the siRNA drug molecule. Therefore, the present invention has very important value for the development and clinical application of siRNA drugs. Summary of the invention

[0006] To solve the above problems, the present invention aims to provide a novel modified nucleotide that can improve the selectivity of RNAi drug molecules for target gene silencing and reduce off-target effects.

[0007] In one aspect, the present invention provides a nucleotide monomer compound having a structure as shown in Formula I, or a deuterated compound thereof, or a fluorinated compound thereof, or a usable salt thereof:

[0008]

[0009] in:

[0010] Bx is an unconventional base;

[0011] R is a hydroxyl protecting group; preferably, R is selected from 4,4'-bismethoxytrityl, 4-methoxytrityl;

[0012] Sug is a ribose ring or a ribose replacement ring;

[0013] n is 0 to 3; m is 0 to 3;

[0014] Z is a phosphate moiety, a phosphorothioate moiety, or a phosphoramidite moiety.

[0015] Further,

[0016] Sug is selected from the following structures:

[0017]

[0018] Among them, the aa end is connected to the Bx end, the bb end is connected to the OR end, and the cc end is connected to the Z end;

[0019] X is selected from O or S;

[0020] Y is selected from hydrogen, halogen, azido, C1-C6 alkyl, C1-C6 alkoxy, -O-(C1-C6 alkylene)-O-(C1-C6 alkyl), -O-(C1-C6 alkylene)-C(O)NR Y1 R Y2 ;

[0021] R Y1, R Y2 Each of them is independently selected from hydrogen, C1-C6 alkyl and cycloalkyl.

[0022] In some embodiments of the present invention, the compound represented by formula I is represented by formula II or formula III:

[0023]

[0024] in,

[0025] Bx is an unconventional base;

[0026] X is selected from O or S;

[0027] n is 0 to 3; m is 0 to 3;

[0028] R is a hydroxyl protecting group; preferably, R is selected from 4,4'-bismethoxytrityl, 4-methoxytrityl;

[0029] Y is selected from hydrogen, halogen, azido, C1-C6 alkyl, C1-C6 alkoxy, -O-(C1-C6 alkylene)-O-(C1-C6 alkyl), -O-(C1-C6 alkylene)-C(O)NR Y1 R Y2 ;

[0030] R Y1 , R Y2 Each of them is independently selected from hydrogen, C1-C6 alkyl and cycloalkyl.

[0031] Z is a phosphate moiety, a phosphorothioate moiety, or a phosphoramidite moiety.

[0032] In some embodiments of the present invention, the compound represented by formula I is represented by formula IV:

[0033]

[0034] Bx is an unconventional base;

[0035] R is a hydroxyl protecting group; preferably, R is selected from 4,4'-bismethoxytrityl, 4-methoxytrityl;

[0036] n is 0 to 3; m is 0 to 3;

[0037] Z is a phosphate moiety, a phosphorothioate moiety, or a phosphoramidite moiety.

[0038] In some embodiments of the present invention, the unconventional base is an unconventional purine, pyrimidine or nitrogen-containing heterocyclic base other than a conventional nucleoside base;

[0039] Preferably,

[0040] The unconventional base is selected from the A ring, the B ring, or a ring formed by mutual fusion or bridging of the A ring and the B ring;

[0041] Ring A and Ring B are independently selected from a five-membered, six-membered or seven-membered heterocyclic ring;

[0042] The ring formed by the mutual fusion and bridging of ring A and ring B is shown in formula V:

[0043]

[0044] in,

[0045] Ring A and Ring B are independently five-membered, six-membered or seven-membered heterocyclic rings;

[0046] The heterocyclic ring contains 0 to 3 O, S or N atoms;

[0047] The A ring, the B ring or the ring formed by fusion or bridging of the A ring and the B ring is optionally substituted by a C1-C6 alkyl or cycloalkyl group, a phenyl group, a phenyl group substituted by a C1-C3 alkyl group or a halogenated phenyl group.

[0048] In some embodiments of the present invention, the specific structure of the unconventional base is as follows:

[0049] ssdd; wherein R and R0 are selected from hydrogen, C1-C6 alkyl, and C3-C6 cycloalkyl.

[0050] In some embodiments of the present invention, the specific structure of the unconventional base is as follows:

[0051]

[0052] In some embodiments of the present invention, the specific structure of the unconventional base is as follows:

[0053]

[0054]

[0055] In some embodiments of the present invention,

[0056] R is selected from hydrogen or DMT;

[0057] m is 0; n is 0 or 1;

[0058] Y is a methoxy group;

[0059] Z is In some specific embodiments of the present invention, the compound is specifically:

[0060]

[0061]

[0062] The present invention also provides the use of any of the above-mentioned nucleotide monomer compounds as an intermediate in the preparation of oligonucleotides; preferably, the oligonucleotide is siRNA.

[0063] In some embodiments of the present invention, there is provided a use of the nucleotide monomer compound as an intermediate in preparing a siRNA antisense strand seed region.

[0064] On the other hand, the present invention also provides an siRNA, which comprises a sense strand and an antisense strand; the sense strand and the antisense strand each comprise 15 to 45 modified or unmodified nucleotides, and the sense strand and the antisense strand are partially complementary to form a double-stranded region; wherein the seed region of the antisense strand contains at least one nucleotide having a structure shown in Formula VIa or Formula VIb, and Site covalently linked to the rest of the siRNA:

[0065]

[0066] in:

[0067] X is independently selected from O or S;

[0068] Y is selected from hydrogen, halogen, azido, C1-C6 alkyl, C1-C6 alkoxy, -O-(C1-C6 alkylene)-O-(C1-C6 alkyl), -O-(C1-C6 alkylene)-C(O)NR Y1 R Y2 ;

[0069] R Y1 , R Y2 Each is independently selected from hydrogen, C1-C6 alkyl, and cycloalkyl;

[0070] Bx is an unconventional base;

[0071] n is 0 to 3.

[0072] Preferably, the nucleotides of the structure represented by Formula VIa or Formula VIb are located at positions 2, 3, 4, 5, 6, 7, and 8 of the antisense strand.

[0073] In another aspect, the present invention further provides an siRNA comprising a sense strand and an antisense strand; the sense strand and the antisense strand each comprise 15 to 45 modified or unmodified nucleotides, and the sense strand and the antisense strand are partially complementary to form a double-stranded region; wherein the seed region of the antisense strand contains at least one nucleotide having a structure as shown in Formula VIc, and Site covalently linked to the rest of the siRNA:

[0074]

[0075] in,

[0076] X is independently selected from O or S;

[0077] Bx is an unconventional base;

[0078] n is 0 to 3.

[0079] Preferably, the nucleotides of the structure represented by Formula VIc are located at positions 2, 3, 4, 5, 6, 7, and 8 of the antisense strand.

[0080] In some embodiments of the present invention, the unconventional base is an unconventional purine, pyrimidine or nitrogen-containing heterocyclic base other than a conventional nucleoside base;

[0081] Preferably,

[0082] The unconventional base is selected from the A ring, the B ring, or a ring formed by mutual fusion or bridging of the A ring and the B ring;

[0083] Ring A and Ring B are independently selected from a five-membered, six-membered or seven-membered heterocyclic ring;

[0084] The ring formed by the mutual fusion and bridging of ring A and ring B is shown in formula V:

[0085]

[0086] in,

[0087] Ring A and Ring B are independently five-membered, six-membered or seven-membered heterocyclic rings;

[0088] The heterocyclic ring contains 0 to 3 O, S or N atoms;

[0089] The A ring, the B ring or the ring formed by fusion or bridging of the A ring and the B ring is optionally substituted by a C1-C6 alkyl or cycloalkyl group, a phenyl group, a phenyl group substituted by a C1-C3 alkyl group or a halogenated phenyl group.

[0090] In some embodiments of the present invention, the specific structure of the unconventional base is as follows:

[0091]

[0092] Wherein, R and R0 represent hydrogen, C1-C6 alkyl, C3-C6 cycloalkyl.

[0093] In some embodiments of the present invention, the specific structure of the unconventional base is as follows:

[0094]

[0095] In some embodiments of the present invention,

[0096] X is O or S;

[0097] n is 0 or 1;

[0098] Y is selected from methoxy or -OCH2CH2OCH3.

[0099] In some embodiments of the present invention, the base of the base-modified nucleotide is selected from the following structures:

[0100] In some specific embodiments of the present invention, the structure of Formula VIa, Formula VIb or Formula VIc in the siRNA is specifically:

[0101]

[0102]

[0103] The present invention also provides a pharmaceutical composition comprising any of the above siRNAs and a pharmaceutically acceptable carrier.

[0104] The present invention also provides a siRNA conjugate, which is formed by conjugating any of the above siRNAs and a conjugated molecule.

[0105] The present invention also provides a kit comprising any of the above siRNAs, and / or the above pharmaceutical compositions and / or the above siRNA conjugates.

[0106] The present invention also provides the use of any of the above siRNAs, and / or the above pharmaceutical compositions, and / or the above siRNA conjugates, and / or the above kits in the preparation of drugs.

[0107] In another aspect, the present invention also provides a method for inhibiting the expression level of a target gene in a cell, the method comprising contacting an effective amount of the siRNA, pharmaceutical composition and / or siRNA conjugate of the present invention with the cell.

[0108] The modified nucleotides of the present invention can improve the selectivity of RNAi drug molecules for target gene silencing and reduce off-target effects.

[0109] After the nucleotide monomer of the present invention is introduced into the antisense chain seed region of siRNA, the potential off-target effect of the antisense chain seed region can be significantly reduced, and the activity of siRNA can be maintained or improved, thereby silencing the target gene mRNA with higher selectivity, expanding the safety window of siRNA drugs, and improving the drugability of siRNA, thereby more effectively treating related diseases caused by target gene mRNA.

[0110] As used herein, the "ribosyl replacement group" refers to a structure that can replace the five-membered furanose ring of a naturally occurring ribonucleotide, such as a morpholinyl group, a cyclohexenyl group, a cyclohexanehexol group, or an open ring structure.

[0111] The modified nucleotides of the present invention can improve the drugability of siRNA drug molecules. In some embodiments, the modified nucleotides of the present invention can enhance the effect of siRNA in degrading target genes. Therefore, the modified nucleotides of the present invention can enhance the pharmacodynamic effect of siRNA silencing.

[0112] The siRNA conjugate provided by the present invention is formed by conjugating siRNA and a conjugation molecule, and comprises the siRNA of the present invention and a conjugation group conjugated to the siRNA, wherein the conjugation group comprises a linker and a pharmaceutically acceptable targeting group and / or a delivery auxiliary group, and the siRNA, the linker and the targeting group or the delivery auxiliary group are sequentially covalently or non-covalently connected, each of the targeting groups is selected from a ligand capable of binding to a cell surface receptor, and each of the delivery auxiliary groups is selected from a group capable of increasing the biocompatibility of the siRNA conjugate in the target organ or tissue for delivery.

[0113] In some embodiments, the siRNA drug comprising the modified nucleotides of the present invention is double-stranded. With respect to double-stranded siRNA drugs, the length of the sense and antisense strands of the siRNA drug is independently 15-45 nucleotides. In some embodiments, the double-stranded siRNA drug comprises a sense strand and an antisense strand that are at least partially complementary to each other (at least 70% complementary). The antisense strand comprises a region having a sequence that is perfectly complementary (100% complementary) or at least partially complementary (at least 85% complementary) to a sequence in the target mRNA. The sense and antisense strands can be the same length or different lengths.

[0114] In some embodiments, the sense strand is about 19 nucleotides in length, while the antisense strand is about 21 nucleotides in length. In some embodiments, the sense strand is about 21 nucleotides in length, while the antisense strand is about 23 nucleotides in length. The perfect or partially complementary region between the sense strand and the antisense strand is typically 15-25 nucleotides in length and is located at or near the 5'-end of the antisense strand (e.g., the region is 1, 2, 3, or 4 nucleotides away from the 5'-end of the antisense strand, which is imperfect or partially complementary).

[0115] For double-stranded siRNA drugs, if there are other sense strand nucleotides, they can be identical or different from the corresponding sequence in the target mRNA. If there are other antisense strand nucleotides, they can be complementary or non-complementary to other nucleotides (if present) of the corresponding sense strand.

[0116] In some embodiments, the sense strand and antisense strand of the double-stranded siRNA drug comprising the modified nucleotides of the present invention contain the same number of nucleotides. In some embodiments, the sense strand and antisense strand of the siRNA drug of the present invention contain different numbers of nucleotides.

[0117] In some embodiments, both the sense and antisense strands contain 1 to 4 phosphorothioate linkages.

[0118] In some embodiments, in the siRNA of the present invention, each nucleotide is a modified or unmodified nucleotide. In the context of the present disclosure, the term "modified nucleotide" used refers to a nucleotide or nucleotide analog formed by replacing the 2' hydroxyl group of the ribose group of the nucleotide with other groups, or a nucleotide in which the base on the nucleotide is a modified base. The modified nucleotide will not cause the function of siRNA to regulate gene expression to be significantly weakened or lost. For example, the modified nucleotide disclosed in JK Watts, GF Deleavey, and MJ Damha, Chemically modified siRNA: tools and applications. Drug Discov. Today, 2008, 13 (19-20): 842-55 can be selected.

[0119] In some embodiments, the siRNA drug comprising the modified nucleotide inhibits the expression of the target mRNA in cells, tissues or in vivo. In some embodiments, a therapeutically effective amount of the siRNA drug comprising the modified nucleotide of the present invention is administered into the body, thereby inhibiting the expression of the target mRNA in the body.

[0120] In some embodiments, the siRNA drug is used to treat, prevent or control clinical manifestations related to the expression of the target mRNA. In some embodiments, a therapeutically or prophylactically effective amount of one or more siRNA drugs is administered to a subject in need of such treatment, prevention or control.

[0121] The siRNA medicine comprising the modified nucleotides of the present invention and the composition comprising the siRNA medicine of the present invention can be delivered to cells, tumors, tissues or objects using oligonucleotide delivery techniques known in the art. In general, any method suitable for delivering nucleic acid molecules (in vitro or in vivo) in the art can be applied to the siRNA medicine comprising one or more modified nucleotides of the present invention.

[0122] In some embodiments where the siRNA drug is double-stranded, a targeting ligand or targeting group, a linking group or a delivery vehicle is covalently linked to the sense strand to form a conjugate. In some embodiments, the targeting ligand, linking group and / or delivery vehicle is directly or indirectly linked to the 3' or 5' end of the sense strand via a linker.

[0123] In some embodiments, the targeting ligand or targeting group contains two to four terminal N-acetylgalactosamines (GalNAc), which are linked to a siRNA drug comprising one or more of the modified nucleotides. It is known that the triantennary (GalNAc) binds to ASGPR with greater affinity than the diantennary or monoantennary structures (Connolly et al., J. Biol. Chem., 1982, 257, 939-945).

[0124] Definitions of terms used in the present invention: Unless otherwise stated, the initial definitions provided for groups or terms in this document apply to the groups or terms throughout the specification; for terms that are not specifically defined in this document, the meaning that a person skilled in the art can give them should be given based on the disclosure and context.

[0125] In the present invention, unless otherwise specified, capital letters C, G, U, A, and T represent the base composition of nucleotides, including modified and unmodified nucleotides; m represents that a nucleotide adjacent to the right side of the mark m is a 2'-methoxy nucleotide; f represents that a nucleotide adjacent to the right side of the mark f is a 2'-fluoro nucleotide; d represents that a nucleotide adjacent to the right side of the mark d is a 2'-deoxy nucleotide; gn represents that a nucleotide adjacent to the right side of the mark gn is a glycerol nucleotide (GNA); the mark * represents that the two nucleotides adjacent to the left and right of the mark * (or between the nucleotide and the linker-targeting ligand part) are connected by thiophosphate groups; eVP represents that a nucleotide adjacent to the right side is a (E)-vinyl phosphate-modified nucleotide; and iab represents a reverse abasic residue.

[0126] In the present invention, "oligonucleotide" refers to a polymer of linked nucleosides, each of which may be independently modified or unmodified, and comprises an oligonucleotide sequence of about 10-50 single-stranded nucleotides or double-stranded nucleotide base pairs. In some embodiments, the oligonucleotide has a core base sequence that is at least partially complementary to a target gene core sequence expressed in a cell. In some embodiments, the oligonucleotide can regulate the expression of the corresponding target gene after being delivered to a cell expressing the gene. Target gene expression can be regulated in vitro or in vivo. "Oligonucleotide" includes, but is not limited to: single-stranded antisense oligonucleotides, short interfering RNA (siRNA), double-stranded RNA (dsRNA), micro RNA (miRNA), short hairpin RNA (shRNA), ribozymes, interfering RNA molecules, and dicer enzyme substrates.

[0127] In the present invention, "siRNA" 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.

[0128] In the present invention, the "seed region" refers to a specific region from the second base to the eighth base from the 5'-end in the oligonucleotide sequence.

[0129] siRNA can act through RNA interference mechanism (for example, 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 siRNA drug used in the present invention mainly acts through the RNA interference mechanism, the siRNA drug is not limited to or confined to any specific pathway or mechanism of action. siRNA drugs include, but are not limited to: single-stranded antisense oligonucleotides, short interfering RNA (siRNA), double-stranded RNA (dsRNA), microRNA (miRNA), short hairpin RNA (shRNA) and dicer enzyme substrates. The siRNA 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 siRNA drug of the present invention is double-stranded and consists of an antisense chain and a sense chain that is at least partially complementary to the antisense chain.

[0130] The terms "silencing", "reducing", "inhibiting", "down-regulating", or "knockdown" refer to the expression of a given gene, when the siRNA drug molecules described in 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.

[0131] The term "sequence" or "nucleotide sequence" refers to the order or sequence of nucleobases or nucleotides, expressed in alphabetical order using standard nucleotide nomenclature.

[0132] The term "heterocyclic base" is a 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, the heterocyclic base moiety may include one or more protecting groups.

[0133] In the present invention, "nucleotide base" or "nucleobase" is a heterocyclic pyrimidine or purine compound, wherein "conventional nucleoside bases" include bases of conventional nucleotides of adenine (A), guanine (G), cytosine (C), thymine (T) and uracil (U). Nucleobases can be modified or replaced by novel unconventional nucleobases. Nucleobases include naturally occurring nucleotide bases and non-naturally occurring nucleotide bases. It should be obvious to those skilled in the art that various nucleotide bases that were previously considered to be "non-naturally occurring" have been found in nature later. Thus, "nucleotide bases" include not only known purine and pyrimidine heterocycles, but also heterocyclic analogs and tautomers thereof. Exemplary nucleotide bases include adenine, guanine, thymine, cytosine, uracil, purine, xanthine, diaminopurine, 8-oxo-N6-methyladenine, 7-diazaxanthine, 7-diazaguanine, N4, N4-ethanocytosine, N6, N6-ethano-2,6-diaminopurine, 5-methylcytosine, 5-(C3-C6)-alkynylcytosine, 5-fluorouracil, 5-bromouracil, pseudoisocytosine, 2-hydroxy-5-methyl-4-triazolopyridine, isocytosine, isoguanine, inosine, and the "non-naturally occurring" nucleotide bases described in U.S. Pat. No. 5,432,272 to Benner et al. The term "nucleotide base" includes each and all of these examples and their analogs and tautomers. Particularly important nucleotide bases are adenine, guanine, thymine, cytosine and uracil, which are considered to be the naturally occurring nucleotide bases relevant to human therapeutic and diagnostic applications.

[0134] The term "complementary" is used to describe the relationship between a first nucleotide sequence (e.g., a siRNA drug sense strand or a target mRNA) and a second nucleotide sequence (e.g., a single-stranded antisense oligonucleotide or a double-stranded siRNA drug antisense strand), and refers to the ability of an oligonucleotide or polynucleotides comprising a first nucleotide sequence to hybridize with an oligonucleotide or polynucleotides 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 contain Watson-Crick base pairing or non-Watson-Crick base pairing, and contain 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.

[0135] 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, sense strand or positive strand, and the other nucleotide sequence that is complementary to it is the antisense strand.

[0136] The term "antisense strand" refers to a nucleotide sequence in mRNA expressed by a target gene that is substantially reverse complementary or essentially reverse complementary to the antisense strand and has the same length as the antisense strand.

[0137] 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.

[0138] 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, and those skilled in the art can determine (by experiment or theory) possible or impossible substitutions by means of the prior art and experimental conditions. 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.

[0139] The minimum and maximum carbon atom content in a hydrocarbon group is indicated by a prefix, for example, the prefix C a～ C b Alkyl refers to any alkyl group containing from "a" to "b" carbon atoms. Thus, for example, C 1～6 The alkyl group refers to a straight chain or branched chain alkyl group containing 1 to 6 carbon atoms.

[0140] Alkyl refers to a straight or branched hydrocarbon group in an alkane molecule, such as methyl-CH3, ethyl-CH2CH3, methylene-CH2-; the alkyl group may also be part of other groups, such as C1-C6 alkoxy, C1-C6 alkylamino. Non-limiting examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, n-hexyl, etc. The alkyl group may be substituted or unsubstituted, and when substituted, the substituent may be substituted at any available connection point, 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, oxy, carboxyl or carboxylate.

[0141] "Alkenyl" refers to a straight or branched chain hydrocarbon group having at least 2 carbon atoms and at least one site of vinyl unsaturation (>C=C<). a-b Alkenyl refers to an alkenyl group having from a to b carbon atoms and is intended to include, for example, ethenyl, propenyl, isopropenyl, 1,3-butadienyl, and the like.

[0142] "Alkynyl" refers to a straight chain monovalent hydrocarbon radical or a branched monovalent hydrocarbon radical containing at least one triple bond. The term "alkynyl" is also intended to include those hydrocarbon radicals having one triple bond and one double bond. For example, C 2-6 Alkynyl is meant to include ethynyl, propynyl, and the like.

[0143] "Alkoxy" refers to-O-(alkyl) or-O-(cycloalkyl), wherein the definition of alkyl is as described above. Non-limiting examples of alkoxy include: methoxy, ethoxy, propoxy, butoxy, cyclopropyloxy, cyclobutyloxy, cyclopentyloxy, cyclohexyloxy. Alkoxy can be optionally substituted or unsubstituted, and when substituted, substituents are preferably one or more of the following groups, which are independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, sulfhydryl, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkyloxy, heterocycloalkyloxy, cycloalkylthio, heterocycloalkylthio, carboxyl or carboxylate.

[0144] "Halogen" is fluorine, chlorine, bromine or iodine.

[0145] "Halo-substituted alkyl" refers to an alkyl group substituted with one or more halogens, wherein alkyl is as defined above.

[0146] "Ester" refers to a -C(O)O(alkyl) or -C(O)O(cycloalkyl) group wherein alkyl and cycloalkyl are as defined above.

[0147] "Acyl" refers to a compound containing a -C(O)R group, where R is alkyl, cycloalkyl, heterocyclyl, aryl or heteroaryl.

[0148] "Heteroatoms" Heteroatoms refer to atoms other than carbon and hydrogen in organic compounds. Generally, they refer to atoms that replace carbon in the molecular skeleton (especially the ring system). Nitrogen, oxygen, sulfur, phosphorus, boron, chlorine, bromine, iodine, etc. are all common heteroatoms. If there is a ring containing heteroatoms in an organic compound, it is called a heterocyclic compound.

[0149] 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 used selectively and / or orthogonally to protect sites at other reactive sites in the reaction, and are subsequently removed to release the unprotected group, enabling it to be used in further reactions. In some embodiments, a "substituted" group or substituent comprises a protecting group.

[0150] Representative hydroxyl protecting groups in the present invention are disclosed in Beaucage et al., Tetrahedron 1992, 48, 2223-2311, and Greene and Wuts, Protective Groups in Organic Synthesis, Chapter 2, 2d ed, John Wiley & Sons, New York, 1991, each of which is incorporated herein by reference in its entirety. In some embodiments, the protecting group is stable under alkaline conditions, but can be removed under acidic conditions. In some embodiments, non-exclusive examples of hydroxyl protecting groups that can be used in the present invention include dimethoxytrityl (DMT), monomethoxytrityl, 9-phenyloxanthene-9-yl (Pixyl) and 9-(p-methoxyphenyl)oxanthene-9-yl (Mox). In some embodiments, non-exclusive examples of hydroxy protecting groups that may be used in the present invention include Tr (trityl), MMTr (4-methoxytrityl), DMTr (4,4'-dimethoxytrityl), and TMTr (4,4',4"-trimethoxytrityl).

[0151] 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.

[0152] The terms "salts" and "usable salts" refer to acidic and / or basic salts of the above-mentioned compounds or their stereoisomers, formed with inorganic and / or organic acids and bases, and also include zwitterionic salts (inner salts), and also include quaternary ammonium salts, such as alkylammonium salts. These salts can be obtained directly in the final separation and purification of the compounds. They can also be obtained by mixing the above-mentioned compounds, or their stereoisomers, with a certain amount of acid or base appropriately (e.g., equivalent amounts). These salts may be precipitated in the solution and collected by filtration, or recovered after solvent evaporation, or obtained by freeze drying after reaction in an aqueous medium. BRIEF DESCRIPTION OF THE DRAWINGS

[0153] Figure 1 This is the MS spectrum of monomer XY-007-2;

[0154] Figure 2 This is the 2H-NMR spectrum of monomer XY-007;

[0155] Figure 3This is the monomer XY-007-2P-NMR spectrum;

[0156] Figure 4 This is the MS spectrum of monomer XY-007-3;

[0157] Figure 5 This is the 3H-NMR spectrum of monomer XY-007;

[0158] Figure 6 This is the monomer XY-007-3P-NMR spectrum;

[0159] Figure 7 This is the MS spectrum of monomer XY-007-5;

[0160] Figure 8 This is the 5H-NMR spectrum of monomer XY-007;

[0161] Fig. 9 This is the P-NMR spectrum of monomer XY-007-5;

[0162] Fig.10 This is the MS spectrum of monomer XY-007-6;

[0163] Fig.11 This is the 6H-NMR spectrum of monomer XY-007;

[0164] Fig.12 This is the P-NMR spectrum of monomer XY-007-6;

[0165] Fig.13 A schematic diagram of the solid phase synthesis route for synthesizing siRNA sequences using a solid phase carrier;

[0166] Fig.14 The structural diagram of the solid phase support (L96-PS) modified with GalNAc (L96);

[0167] Fig.15 The figure is a flow chart of the synthesis process of siRNA sequences using solid phase carriers. Specific implementation plan

[0168] Obviously, according to the above contents of the present invention, in accordance with common technical knowledge and customary means in the art, without departing from the above basic technical ideas of the present invention, other various forms of modification, replacement or change may be made.

[0169] The above contents of the present invention are further described in detail below through specific implementation methods in the form of embodiments. However, this should not be understood as the scope of the above subject matter of the present invention being limited to the following examples. All technologies realized based on the above contents of the present invention belong to the scope of the present invention.

[0170] The raw materials and equipment used in the present invention are all known products, which are obtained by purchasing commercially available products.

[0171] The compounds disclosed in the present invention can be prepared by the following synthetic methods.

[0172] Example

[0173] Hereinafter, in order to help understand the present disclosure, exemplary embodiments will be provided. However, the following embodiments are provided only to make it easier to understand the present disclosure and are not intended to limit the present disclosure.

[0174] Unless otherwise specified, the materials and reagents used in the examples are commercially available. The nucleic acid electrophoresis, real-time PCR and other operations used are all performed according to conventional protocols. For example, they can be performed according to the procedures described in Molecular Cloning (Cold Spring Harbor Laboratory Press (1989)).

[0175] Example 1: Chemical synthesis of monomer XY-007-2

[0176]

[0177] Synthesis of compound XY-007-2-2 :

[0178] Substrate XY-007-2-1 (134 g, 0.27 mol) was added to a dry 1000 mL single-mouth bottle and dissolved in anhydrous acetonitrile (150 mL). Then 1a (44.7 g, 0.29 mol), BSA (8.0 g, 53.2 mmol), TMSOTf (62 g, 0.28 mol) were added under nitrogen protection. After the addition, the reaction system was kept at 80 degrees and stirred for 2 hours. LCMS detected that the raw material completely disappeared and the reaction was completed. Then 2000 mL of ethyl acetate was added to the reaction solution, washed with water (1000 mL*3), and washed with saturated brine (1000 mL). The organic phase was dried over anhydrous Na2SO4, filtered, and concentrated to obtain a crude product, which was purified by column chromatography (petroleum ether: ethyl acetate = 5:1) and concentrated to obtain a white solid product XY-007-2-2 (44.0 g, 73.7 mmol, 30% yield).

[0179] ESI-LCMS m / z 598.1[M+H]. 1H NMR(400MHz,DMSO-d6)δ8.60(s,1H),8.06(s,1H),7.66-7.53(m,4H),7.40-7.38(m,2H),7.31-7.22(m,8H),6.88-6.8 6(m,4H),6.68-6.67(m,1H),5.56-5.55(m,1H),4.68-4.66(m,1H),4.12-4.10(m,1H),3.96-3.94(m,1H),3.73(s,6H).

[0180] Synthesis of compound XY-007-2-3 :

[0181] Add the substrate XY-007-2-2 (44.0 g, 73.7 mmol) dissolved in methanol (500 mL) to a dry 1000 mL single-mouth bottle, then add K2CO3 (2.1 g, 14.7 mmol) under nitrogen protection, and keep the reaction system at room temperature for 2 hours after the addition. The reaction is complete when the raw material disappears by LCMS. Then add 1000 mL of ethyl acetate to the reaction solution, wash with water (500 mL*3), and wash with saturated brine (500 mL), dry the organic phase with anhydrous Na2SO4, filter, and concentrate to obtain a crude product, which is purified by column chromatography (petroleum ether: ethyl acetate = 1:5), and concentrated to obtain a white solid product XY-007-2-3 (15.5 g, 54.4 mmol, 74% yield).

[0182] ESI-LCMS: m / z 284.2[MH] - ; 1 H NMR(400MHz,DMSO-d6)8.67(s,1H),8.02-8.01(s,1H),6.76-6.75(m,1H),6.22-6.20(m,1H),5.42-5.41(m,1H),5 .21-5.20(m,1H),5.08-5.06(m,1H),4.44-4.40(m,1H),4.14-4.05(m,1H),3.96-3.93(m,1H),3.68-3.54(m,2H).

[0183] Synthesis of compound XY-007-2-4 :

[0184] In a dry 500mL single-mouth bottle, add the substrate XY-007-2-3 (15.5g, 54.4mmol) dissolved in pyridine (200mL), then add DMTrCl (22.1g, 65.3mmol) under nitrogen protection, and keep the reaction system at room temperature for 2 hours. LCMS detected that the raw material completely disappeared and the reaction was completed. Then add 500mL of ethyl acetate to the reaction solution, wash with water (200mL*3), and wash with saturated brine (200mL), dry the organic phase with anhydrous Na2SO4, filter, and concentrate to obtain a crude product, which is purified by column chromatography (petroleum ether: ethyl acetate = 1:1), and concentrated to obtain a white solid product XY-007-2-4 (18.0g, 30.6mmol, 60% yield).

[0185] ESI-LCMS: m / z 586.2[MH] - ; 1 H NMR(400MHz,DMSO-d6)8.65(s,1H),7.84-7.83(m,1H),7.38-7.36(m,2H),7.29-7.21(m,7H),6.86-6.83(m,2H),6.72-6 .71(m,1H),6.21-6.20(m,1H),5.54-5.53(m,1H),5.25-5.24(m,1H),4.53-4.51(m,1H),4.23-4.22(m,1H),3.73(s,6H).

[0186] Synthesis of compound XY-007-2-5 :

[0187] Add the substrate XY-007-2-4 (18.0 g, 30.6 mmol) dissolved in DMF (200 mL) to a dry 500 mL single-mouth bottle, then add imidazole (3.7 g, 61.2 mmol) and TBSCl (4.6 g, 30.6 mmol) under nitrogen protection, and keep the reaction system at room temperature for 2 hours after the addition. LCMS detected that the raw material completely disappeared, and the reaction was completed. Then add 500 mL of ethyl acetate to the reaction solution, wash with water (100 mL*3), and wash with saturated brine (100 mL), dry the organic phase with anhydrous Na2SO4, filter, and concentrate to obtain a crude product, which is purified by column chromatography (petroleum ether: ethyl acetate = 3:1), and concentrated to obtain a mixture of white solid products XY-007-2-5 and XY-007-2-5' (19.8 g, 28.2 mmol, 90% yield).

[0188] ESI-LCMS: m / z 700.3[MH] - ; 1H NMR(400MHz,DMSO-d6)8.83(s,1H),8.04-8.03(m,1H),7.61-7.43(m,9H),,7.08-7.06(m,4H),6.94-6.93(m,1H),6.45 -6.43(m,1H),5.38-5.37(m,1H),4.83-4.80(m,1H),4.37-4.31(m,2H),3.94(s,6H),0.91(s,9H),0.20--0.10(m,6H).

[0189] Synthesis of compound XY-007-2-6 :

[0190] Add the substrate XY-007-2-5 (19.8 g, 28.2 mmol) dissolved in CH3I (200 mL) to a dry 500 mL single-mouth bottle, then add Ag2O (36 g, 155 mmol) under nitrogen protection, and stir the reaction system at 80 degrees for 2 hours. LCMS detected that the raw material disappeared completely, and the reaction was completed. Then filter and concentrate the reaction solution to obtain a crude product, which was purified by column chromatography (petroleum ether: ethyl acetate = 3:1) and concentrated to obtain a mixture of white solid products XY-007-2-6 and XY-007-2-6' (10.3 g, 14.4 mmol, 50% yield). ESI-LCMS: m / z 716.3 [M+H] + ;

[0191] Synthesis of compound XY-007-2-7 :

[0192] Substrate XY-007-2-6 (10.3 g, 14.4 mmol) dissolved in NH3 methanol solution (200 mL) was added to a dry 500 mL single-mouth bottle, and the reaction system was kept at 140 degrees and sealed and stirred for 5 hours. LCMS detected that the raw material disappeared by 70%, and the reaction was completed. Then the reaction solution was concentrated to obtain a crude product, which was purified by column chromatography (petroleum ether: ethyl acetate = 2: 1), and concentrated to obtain a mixture of white solid products XY-007-2-7 and XY-007-2-7', and SFC was separated to obtain white solid products XY-007-2-7 (700 mg, 1.0 mmol), XY-007-2-7' (710 mg, 1.0 mmol), and unresolved mixture (2.3 g, 3.2 mmol) with a total yield of 40%.

[0193] XY-007-2-7:ESI-LCMS:m / z 697.2[M+H] + ; 1H NMR(400MHz,DMSO-d6)8.26(s,1H),7.66-7.64(m,2H),7.56-7.64(m,9H), 7.42-7.38(m,2H),7.25(s,2H),7.13-7.11(m,4H),6.82-6.81(m,1H),6.31 -6.30(m,1H),4.98-4.96(m,1H),4.34-4.33(m,1H),4.05-4.03(m,1H),3.9 8-3.96(m,7H),3.60(s,3H),2.53(s,1H),0.96(s,8H),0.22--0.05(m,6H).

[0194] XY-007-2-7':ESI-LCMS:m / z 697.2[M+H] + ; 1 H NMR(400MHz,DMSO-d6)8.00(s,1H),7.32-7.30(m,2H),7.26-7.14(m,9H),6.99(s,2H),6.81-6.79(m,5H),6.56-6.53(m,1H),6.08-6.07(m,1H) ,4.47-4.45(m,1H),4.17-4.14(m,1H),3.90-3.86(m,1H),3.68-3.67(m ,7H),3.23(s,3H),3.07-3.03(m,7H),0.76(s,10H),0.00--0.05(m,6H).

[0195] Synthesis of compound XY-007-2-8 :

[0196] Add the substrate XY-007-2-7 (700 mg, 1.1 mmol) dissolved in pyridine (10 mL) to a dry 100 mL single-mouth bottle, then add BzCl (310 mg, 2.2 mmol) under nitrogen protection, and keep the reaction system at room temperature for 2 hours after the addition. LCMS detected the disappearance of the raw material, then add 100 mL of ethyl acetate to the reaction solution, wash with water (100 mL*3), and wash with saturated brine (100 mL), dry the organic phase over anhydrous Na2SO4, filter, and concentrate to obtain a crude product, which is purified by column chromatography (petroleum ether: ethyl acetate = 1:1), and concentrated to obtain a white solid product XY-007-2-8 (630 mg, 0.8 mmol, 72% yield). ESI-LCMS: m / z 801.3 [M+H] + ;

[0197] Synthesis of compound XY-007-2-9 :

[0198] Substrate XY-007-2-8 (630 mg, 0.8 mmol) was added to a dry 100 mL single-mouth bottle and dissolved in THF (10 mL), and then 3HF.TEA (644 mg, 4.0 mmol) and TEA (565 mg, 5.6 mmol) were added under nitrogen protection. After the addition, the reaction system was kept at 50 ° C and stirred overnight. LCMS detected that the raw material disappeared, and then 100 mL of ethyl acetate was added to the reaction solution, washed with water (100 mL*3), and washed with saturated brine (100 mL). The organic phase was dried over anhydrous Na2SO4, filtered, and concentrated to obtain a crude product, which was purified by column chromatography (petroleum ether: ethyl acetate = 1:2) and concentrated to obtain a white solid product XY-007-2-9 (490 mg, 0.7 mmol, 85% yield).

[0199] ESI-LCMS: m / z 687.7 [M+H] + ; 1 H NMR(400MHz,DMSO-d6)8.61(s,1H),8.08-8.05(m,2H),8.02-7.99(m,2H),7.76-7.53(m,7H),7.42-7.21(m,10H),6.88-6 .84(m,4H),6.72-6.71(m,1H),6.60-6.59(m,1H),4.57-4.55(m,1H),4.29-4.26(m,1H),3.73(s,6H),3.40-3.35(m,4H).

[0200] Synthesis of compound XY-007-2 :

[0201] Substrate XY-007-2-9 (490 mg, 0.7 mmol) was added to a dry 50 mL single-mouth bottle and dissolved in anhydrous DCM (10 mL), and then DCI (74 mg, 0.6 mmol) and CEP [N (iPr) 2] 2 (253 mg, 0.8 mmol) were added under nitrogen protection. After the addition, the reaction system was kept stirring at room temperature for 2 hours. LCMS detected that the raw material disappeared completely and the reaction was completed. Then 100 mL of dichloromethane was added to the reaction solution, washed with water (100 mL * 3), and washed with saturated brine (100 mL), the organic phase was dried over anhydrous Na2SO4, filtered, and concentrated to obtain a crude product with reverse phase C18 (CH3CN / H2O (0.05% NH4HCO3) = 1 / 0), and concentrated to obtain a white solid product XY-007-2 (460 mg, 0.5 mmol, 75% yield).

[0202] ESI-LCMS Figure 1):m / z 887.1[M+H] + ; 1 H NMR ( Figure 2 )(400MHz, DMSO-d6)δ8.71(s,1H),8.53(s,1H),7.99-7.98(m,2H),7.63-7.59(m,1H),7.55-7.42(m,6H ),7.36-7.20(m,9H),7.03-7.01(m,1H),6.84-6.80(m,4H),6.52-6.49(m,1H),4.68-4.55(m,1H),4.37 -4.35(m,1H),4.31-4.23(m,1H),3.99-3.85(m,1H),3.79-3.78(m,7H),3.67-3.50(m,4H),3.49-3.41( m,4H),3.35-3.31(m,1H),2.69-2.65(m,1H),2.39-2.35(m,1H),1.20-1.17(m,10H),1.05-1.03(m,4H). 31 P NMR ( Figure 3 )(162MHz,DMSO-d6)δ150.90,150.20.

[0203] Example 2: Chemical synthesis of monomer XY-007-3

[0204]

[0205] Synthesis of compound XY-007-3-1 :

[0206] Substrate XY-007-2-7' (700 mg, 0.9 mmol) was added to a dry 100 mL single-mouth bottle and dissolved in THF (10 mL), and then 3HF.TEA (704 mg, 4.3 mmol) and TEA (618 mg, 6.1 mmol) were added under nitrogen protection. After the addition, the reaction system was kept at 50 ° C and stirred overnight. LCMS detected that the raw material disappeared, and then 100 mL of ethyl acetate was added to the reaction solution, washed with water (100 mL*3), and washed with saturated brine (100 mL). The organic phase was dried over anhydrous Na2SO4, filtered, and concentrated to obtain a crude product, which was purified by column chromatography (petroleum ether: ethyl acetate = 1:2) and concentrated to obtain a white solid product XY-007-3-1 (480 mg, 0.7 mmol, 80% yield).

[0207] ESI-LC MS: m / z 687.7[M+H] + ; 1H NMR(400MHz,DMSO-d6)δ8.61(s,1H),8.08-8.05(m,2H),8.02-7.99(m,2H),7.76-7.53(m,7H),7.42-7.21(m,10H),6.88- 6.84(m,4H),6.72-6.71(m,1H),6.60-6.59(m,1H),4.57-4.55(m,1H),4.29-4.26(m,1H),3.73(s,6H),3.40-3.35(m,4H)

[0208] Synthesis of compound XY-007-3:

[0209] Substrate XY-007-3-1 (480 mg, 0.7 mmol) was added to a dry 50 mL single-mouth bottle and dissolved in anhydrous DCM (10 mL), and then DCI (74 mg, 0.6 mmol) and CEP [N (iPr) 2] 2 (253 mg, 0.8 mmol) were added under nitrogen protection. After the addition, the reaction system was kept stirring at room temperature for 2 hours. LCMS detected that the raw material disappeared completely and the reaction was completed. Then 100 mL of dichloromethane was added to the reaction solution, washed with water (100 mL * 3), and washed with saturated brine (100 mL), the organic phase was dried over anhydrous Na2SO4, filtered, and concentrated to obtain a crude product with reverse phase C18 (CH3CN / H2O (0.05% NH4HCO3) = 1 / 0), and concentrated to obtain a white solid product XY-007-3 (410 mg, 0.5 mmol, 70% yield).

[0210] ESI-LCMS Figure 4 ):m / z 887.4[M+H] + ; 1 H NMR ( Figure 5)(400MHz, DMSO-d6)δ8.71(s,1H),8.53(s,1H),7.99-7.98(m,2H),7.63-7.59(m,1H),7.55-7.42(m,6H ),7.36-7.20(m,9H),7.03-7.01(m,1H),6.84-6.80(m,4H),6.52-6.49(m,1H),4.68-4.55(m,1H),4.37 -4.35(m,1H),4.31-4.23(m,1H),3.99-3.85(m,1H),3.79-3.78(m,7H),3.67-3.50(m,4H),3.49-3.41( m,4H),3.35-3.31(m,1H),2.69-2.65(m,1H),2.39-2.35(m,1H),1.20-1.17(m,10H),1.05-1.03(m,4H), 31 P NMR ( Figure 6 )(600MHz,DMSO-d6)δ151.54,150.86.

[0211] Example 3: Chemical synthesis of monomer XY-007-5

[0212]

[0213] Synthesis of compound XY-007-5-2 :

[0214] Substrate XY-007-5-1 (170.0 g, 340.0 mmol) was added to a dry 3000 mL single-mouth bottle and dissolved in anhydrous acetonitrile (1.5 L), and then 1a (56.7 g, 370.0 mmol) and BSA (75.4 g, 0.37 mmol) were added under nitrogen protection. After the addition, the reaction system was kept at 50 ° C and stirred for 0.5 hours until the system was clear. The reaction system was cooled to 0 ° C, and TMSOTf (78.6 g, 350.0 mmol) was slowly added under nitrogen protection. After the addition, the reaction system was kept at 80 ° C and stirred for 2 hours. LCMS detected that the raw material completely disappeared, and the reaction was completed. Then add 2000mL of ethyl acetate to the reaction solution, wash with water (1000mL*3), and wash with saturated brine (1000mL), dry the organic phase over anhydrous Na2SO4, filter and concentrate to obtain the crude product, which is purified by column chromatography (petroleum ether: ethyl acetate = 5:1) and concentrated to obtain a white solid product XY-007-5-2 (53.5g, 89.6mmol, 30% yield).

[0215] ESI-LCMS m / z 598.1[M+H]. 1H NMR(400MHz,DMSO-d6)δ8.60(s,1H),8.06(s,1H),7.66-7.53(m,4H),7.40-7.38(m,2H),7.31-7.22(m,8H),6.88-6.8 6(m,4H),6.68-6.67(m,1H),5.56-5.55(m,1H),4.68-4.66(m,1H),4.12-4.10(m,1H),3.96-3.94(m,1H),3.73(s,6H).

[0216] Synthesis of compound XY-007-5-3 :

[0217] Add the substrate XY-007-5-2 (53.5 g, 89.6 mmol) dissolved in 1M MeONa methanol solution (500 mL) to a dry 1000 mL single-mouth bottle. After the addition, the reaction system was kept stirring at 60 ° C for 2 hours. LCMS detected that the raw material completely disappeared and the reaction was completed. Then the reaction solution was directly concentrated to obtain a crude product, which was purified by column chromatography (methanol: dichloromethane = 1:1) to obtain a white solid product XY-007-5-3 (19.0 g, 67.6 mmol, 75% yield). ESI-LCMS: m / z 282.1 [M+H] + ;

[0218] Synthesis of compound XY-007-5-4 :

[0219] In a dry 500mL single-mouth bottle, add the substrate XY-007-5-3 (19.0g, 67.6mmol) dissolved in pyridine (200mL), then add DMTrCl (25.2g, 74.4mmol) under nitrogen protection, and keep the reaction system at room temperature for 2 hours. LCMS detected that the raw material completely disappeared, and the reaction was completed. Then add 500mL of ethyl acetate to the reaction solution, wash with water (200mL*3), and wash with saturated brine (200mL), dry the organic phase with anhydrous Na2SO4, filter, and concentrate to obtain a crude product, which is purified by column chromatography (petroleum ether: ethyl acetate = 1:1), and concentrated to obtain a white solid product XY-007-5-4 (31.0g, 53.2mmol, 78% yield).

[0220] ESI-LCMS: m / z 582.3[MH] - ; 1H NMR(400MHz,DMSO-d6)δ8.65(s,1H),7.84-7.83(m,1H),7.38-7.36(m,2H),7.29-7.21(m,7H),6.86-6.83(m,2H),6.72-6 .71(m,1H),6.21-6.20(m,1H),5.54-5.53(m,1H),5.25-5.24(m,1H),4.53-4.51(m,1H),4.23-4.22(m,1H),3.73(s,6H).

[0221] Synthesis of compound XY-007-5-5 :

[0222] Add substrate XY-007-5-4 (31.0 g, 53.2 mmol) dissolved in DMF (300 mL) to a dry 500 mL single-mouth bottle, then add imidazole (4.7 g, 79.8 mmol) and TBSCl (7.2 g, 47.9 mmol) under nitrogen protection, and keep the reaction system at room temperature for 2 hours after addition. LCMS detected that the raw material completely disappeared, and the reaction was completed. Then add 500 mL of ethyl acetate to the reaction solution, wash with water (100 mL*3), and wash with saturated brine (100 mL), dry the organic phase with anhydrous Na2SO4, filter, and concentrate to obtain a crude product, which is purified by column chromatography (petroleum ether: ethyl acetate = 3:1), and concentrated to obtain a mixture of white solid products XY-007-5-5 and XY-007-5-5' (29.0 g, 41.6 mmol, 78% yield).

[0223] ESI-LCMS: m / z 696.4[MH] - ; 1 H NMR(400MHz,DMSO-d6)δ8.48-8.47(m,1H),7.64-7.59(m,1H),7.49-7.42(m,2H),7 .38-7.26(m,7H),6.95-6.90(m,4H),6.66-6.65(m,1H),6.29-6.18(m,1H),5.42-5 .15(m,1H),4.69-4.15(m,1H),4.43-4.12(m,1H),4.16-4.04(m,4H),3.81-3.80(m ,6H),3.35-3.32(m,1H),3.19-3.15(m,1H),0.91-0.78(m,9H),0.20--0.20(m,6H).

[0224] Synthesis of compound XY-007-5-6 :

[0225] In a dry 500mL single-mouth bottle, add a mixture of substrates XY-007-5-5 and XY-007-5-5' (29.0g, 41.6mmol) dissolved in CH3I (200mL), then add Ag2O (52.9g, 229mmol) under nitrogen protection, and stir the reaction system at 80 degrees for 2 hours. LCMS detected that the raw material completely disappeared and the reaction was completed. Then filter and concentrate the reaction solution to obtain a crude product, which was purified by column chromatography (petroleum ether: ethyl acetate = 3:1) and concentrated to obtain a mixture of white solid products XY-007-5-6 and XY-007-5-6' (16.5g, 23.2mmol, 55% yield). ESI-LCMS: m / z 712.3[M+H] + ;

[0226] Synthesis of compound XY-007-5-7:

[0227] In a dry 500mL single-necked bottle, a mixture of substrates XY-007-5-6 and XY-007-5-6' (8.0 g, 11.2 mmol) was added and dissolved in DCM (100 mL), and then 1M BBr3 DCM solution (56.2 mL, 56.2 mmol) was added under nitrogen protection. After the addition, the reaction system was stirred at room temperature for 2 hours. The reaction was completed when the raw material disappeared by LCMS. Then add 100 mL of ethyl acetate to the reaction solution, wash with sodium bicarbonate water (100 mL*3), and wash with saturated brine (100 mL), dry the organic phase over anhydrous Na2SO4, filter and concentrate to obtain a crude product which is purified by column chromatography (petroleum ether: ethyl acetate = 2:1) and concentrated to obtain white solid products XY-007-5-7 (680 mg, 1.7 mmol, 15% yield) and XY-007-5-7' (480 mg, 1.2 mmol, 30% yield).

[0228] XY-007-5-7:ESI-LCMS:m / z 396.1[M+H] + ; 1 H NMR(400MHz,DMSO-d6)δ11.87(s,1H),7.81(s,1H),7.31-7.30(m,1H),6.44-6.43(m,1H),6.05-5.99(m,1H),5.01-4.98(m,1H) ),4.34-4.32(m,1H),4.06-4.03(m,1H),3.78-3.76(m,1H),3.51-3.39(m,2H),3.10(s,3H),0.78(s,9H),0.10--0.10(m,6H).

[0229] XY-007-5-7':ESI-LCMS:m / z 396.1[M+H] + ; 1 H NMR(400MHz,DMSO-d6)δ12.05(s,1H),7.99(s,1H),7.49-7.48(m,1H),6.63-6.62(m,1H),5.28-5.26(m,1H),4.71-4.6 8(m,1H),4.12-4.08(m,1H),3.88-3.86(m,1H),3.76-3.63(m,2H),3.51(s,3H),1.00-0.72(m,9H),0.21--0.23(m,6H).

[0230] Synthesis of compound XY-007-5-8 :

[0231] Substrate XY-007-5-7 (460 mg, 1.1 mmol) was added to a dry 100 mL single-mouth bottle and dissolved in pyridine (10 mL), and then DMTrCl (472 mg, 1.4 mmol) was added under nitrogen protection. After the addition, the reaction system was kept at room temperature and stirred for 2 hours. LCMS detected the disappearance of the raw material, and then 100 mL of ethyl acetate was added to the reaction solution, washed with water (100 mL*3), and washed with saturated brine (100 mL). The organic phase was dried over anhydrous Na2SO4, filtered, and concentrated to obtain a crude product, which was purified by column chromatography (petroleum ether: ethyl acetate = 1:1), and concentrated to obtain a white solid product XY-007-5-8 (620 mg, 0.9 mmol, 81% yield).

[0232] ESI-LCMS: m / z 698.3[M+H] + ; 1 H NMR(400MHz,DMSO-d6)δ12.01(s,1H),7.92-7.91(m,1H),7.46-7.27(m,12H),6.96-6.92(m,5H),6.55-6.57(m,1H),6.0 9-6.07(m,1H),4.71-4.68(m,1H),4.17-4.14(m,1H),3.82-3.76(m,8H),3.41(s,3H),0.77(s,9H),0.00--0.10(m,6H).

[0233] Synthesis of compound XY-007-5-9 :

[0234] Substrate XY-007-5-8 (620 mg, 0.9 mmol) was added to a dry 100 mL single-mouth bottle and dissolved in THF (10 mL), and then 3HF.TEA (716 mg, 4.4 mmol) and TEA (628 mg, 6.2 mmol) were added under nitrogen protection. After the addition, the reaction system was kept at 50 ° C and stirred overnight. LCMS detected that the raw material disappeared, and then 100 mL of ethyl acetate was added to the reaction solution, washed with water (100 mL*3), and washed with saturated brine (100 mL). The organic phase was dried over anhydrous Na2SO4, filtered, and concentrated to obtain a crude product, which was purified by column chromatography (petroleum ether: ethyl acetate = 1:2) and concentrated to obtain a white solid product XY-007-5-9 (460 mg, 0.8 mmol, 85% yield).

[0235] ESI-LCMS: m / z 582.3 [M+H] + ; 1 H NMR(400MHz,DMSO-d6)δ12.01(s,1H),7.91(s,1H),7.40-7.39(m,2H),7.31-7.21(m,8H),6.88-6.86(m,2H),6.53-6.5 2(m,1H),6.16-6.15(m,1H),5.27-5.26(m,1H),4.29-4.26(m,1H),4.13-4.10(m,1H),4.04-4.00(m,1H),3.74(s,6H).

[0236] Synthesis of compound XY-007-5 :

[0237] Substrate XY-007-5-9 (460 mg, 0.8 mmol) was added to a dry 50 mL single-mouth bottle and dissolved in anhydrous DCM (10 mL), and then DCI (84 mg, 0.7 mmol) and CEP [N (iPr) 2] 2 (286 mg, 1.0 mmol) were added under nitrogen protection. After the addition, the reaction system was kept stirring at room temperature for 2 hours. LCMS detected that the raw material disappeared completely and the reaction was completed. Then 100 mL of dichloromethane was added to the reaction solution, washed with water (100 mL * 3), and washed with saturated brine (100 mL), the organic phase was dried over anhydrous Na2SO4, filtered, and concentrated to obtain a crude product. Reverse phase C18 (CH3CN / H2O (0.05% NH4HCO3) = 1 / 0) was used to concentrate to obtain a white solid product XY-007-5 (420 mg, 0.5 mmol, 70% yield).

[0238] ESI-LC MS ( Figure 7 ):m / z 782.1[MH] -; 1 H NMR ( Figure 8 )(400MHz, DMSO-d6)δ12.03(s,1H),7.90-7.89(m,1H),7.41-7.37(m,2H),7.32-7.20( m,8H),6.89-6.85(m,4H),6.55-6.54(m,1H),6.16-6.13(m,1H),4.51-4.46(m,1H),4. 37-4.30(m,1H),4.20-4.12(m,1H),3.84-3.73(m,7H),3.69-3.51(m,3H),3.34-3.20( m,10H),2.80-2.88(m,1H),2.61-2.58(m,1H),1.15-1.12(m,10H),1.02-0.90(m,3H), 31 P NMR ( Fig. 9 )(162MHz,DMSO-d6)δ149.55,149.27.

[0239] Example 4: Chemical synthesis of monomer XY-007-6

[0240]

[0241] Synthesis of compound XY-007-6-2 :

[0242] Substrate XY-007-6-1 (470 mg, 1.1 mmol) was added to a dry 100 mL single-mouth bottle and dissolved in pyridine (10 mL), and then DMTrCl (483 mg, 1.4 mmol) was added under nitrogen protection. After the addition, the reaction system was kept at room temperature and stirred for 2 hours. LCMS detected the disappearance of the raw material, and then 100 mL of ethyl acetate was added to the reaction solution, washed with water (100 mL*3), and washed with saturated brine (100 mL). The organic phase was dried over anhydrous Na2SO4, filtered, and concentrated to obtain a crude product, which was purified by column chromatography (petroleum ether: ethyl acetate = 1:1), and concentrated to obtain a white solid product XY-007-6-2 (630 mg, 0.9 mmol, 80% yield).

[0243] ESI-LCMS: m / z 698.3[M+H] + ; 1H NMR(400MHz,DMSO-d6)δ12.01(s,1H),7.92-7.91(m,1H),7.46-7.27(m,12H),6.96-6.92(m,5H),6.55-6.57(m,1H),6.0 9-6.07(m,1H),4.71-4.68(m,1H),4.17-4.14(m,1H),3.82-3.76(m,8H),3.41(s,3H),0.77(s,9H),0.10--0.25(m,6H).

[0244] Synthesis of compound XY-007-6-3 :

[0245] Substrate XY-007-6-2 (630 mg, 0.9 mmol) was added to a dry 100 mL single-mouth bottle and dissolved in THF (10 mL), and then 3HF.TEA (716 mg, 4.4 mmol) and TEA (628 mg, 6.2 mmol) were added under nitrogen protection. After the addition, the reaction system was kept at 50 ° C and stirred overnight. LCMS detected that the raw material disappeared, and then 100 mL of ethyl acetate was added to the reaction solution, washed with water (100 mL*3), and washed with saturated brine (100 mL). The organic phase was dried over anhydrous Na2SO4, filtered, and concentrated to obtain a crude product, which was purified by column chromatography (petroleum ether: ethyl acetate = 1:2) and concentrated to obtain a white solid product XY-007-6-3 (450 mg, 0.8 mmol, 80% yield).

[0246] ESI-LCMS: m / z 582.3 [M+H] + ; 1 H NMR(400MHz,DMSO-d6)δ12.01(s,1H),7.91(s,1H),7.40-7.39(m,2H),7.31-7.21(m,8H),6.88-6.86(m,2H),6.53-6.5 2(m,1H),6.16-6.15(m,1H),5.27-5.26(m,1H),4.29-4.26(m,1H),4.13-4.10(m,1H),4.04-4.00(m,1H),3.74(s,6H).

[0247] Synthesis of compound XY-007-6 :

[0248] Substrate XY-007-6-3 (450 mg, 0.8 mmol) was added to a dry 50 mL single-mouth bottle and dissolved in anhydrous DCM (10 mL), and then DCI (82 mg, 0.7 mmol) and CEP [N (iPr) 2] 2 (279 mg, 1.0 mmol) were added under nitrogen protection. After the addition, the reaction system was kept stirring at room temperature for 2 hours. LCMS detected that the raw material disappeared completely and the reaction was completed. Then 100 mL of dichloromethane was added to the reaction solution, washed with water (100 mL * 3), and washed with saturated brine (100 mL), the organic phase was dried over anhydrous Na2SO4, filtered, and concentrated to obtain a crude product with reverse phase C18 (CH3CN / H2O (0.05% NH4HCO3) = 1 / 0), and concentrated to obtain a white solid product XY-007-6 (350 mg, 0.4 mmol, 60% yield).

[0249] ESI-LCMS Fig.10 ):m / z 782.1[MH] - ; 1 H NMR ( Fig.11 )(400MHz, DMSO-d6)δ12.03(s,1H),7.90-7.89(m,1H),7.41-7.37(m,2H),7.32-7.20( m,8H),6.89-6.85(m,4H),6.55-6.54(m,1H),6.16-6.13(m,1H),4.51-4.46(m,1H),4. 37-4.30(m,1H),4.20-4.12(m,1H),3.84-3.73(m,7H),3.69-3.51(m,3H),3.34-3.20( m,10H),2.80-2.88(m,1H),2.61-2.58(m,1H),1.15-1.12(m,10H),1.02-2.00(m,3H). 31 P NMR ( Fig.12 )(600MHz,DMSO-d6)δ150.53,150.48.

[0250] Example 5: siRNA Synthesis

[0251] For the sense and antisense strands of the siRNA sequence of the present invention and the sense and antisense strands of the modified duplex, a solid phase support was used to start the chain synthesis; a solid phase support modified with GalNAc (L96) was used as the starting cycle of the sense chain synthesis, and a universal solid phase support (Primer support 5G unylinker 350) was used as the starting cycle of the antisense chain synthesis. Fig.13The structure of the GalNAc(L96) modified solid phase carrier (L96-PS) is as follows Fig.14 :The Primer support 5G unylinker 350 carrier structure is as shown below:

[0252]

[0253] ●PS stands for polystyrene solid phase carrier

[0254] For the sense and antisense strands of the siRNA sequence of the present invention and the sense and antisense strands of the modified duplex, PS was used as a solid phase carrier; a GalNAc-modified PS solid phase was used as the starting cycle for the synthesis of the sense strand, and a universal PS was used as the starting cycle for the antisense strand ( Fig.13 ).

[0255] Using a YB-192S synthesizer and phosphoramidite triester solid phase synthesis method, starting with a solid phase carrier, nucleoside monomers were sequentially connected in the 3'-5' direction to perform sequence synthesis with a synthesis scale of 0.2umol.

[0256] See the process flow Fig.15 .

[0257] Process Description:

[0258] Oligo synthesis starts with Universal-PS, and uses 3'-0-(2-cyanoethyl)phosphoramidite / 4,4'dimethoxytrityl (dimethoxytrityl, DMT) group protection to assemble oligonucleotide chains on the solid phase support PS. Each synthesis cycle includes 5'-hydroxyl deprotection, coupling, capping and oxidation (thiolation). Each coupling reaction is carried out by activating the appropriate phosphoramidite monomer and reacting with the free 5'-hydroxyl of the protected nucleotide or oligonucleotide fixed on the carrier. According to the sequence information, the corresponding crude oligonucleotide single-strand PS-Oligo is synthesized cyclically, and the crude oligonucleotide single-strand is cleaved from the solid phase 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.

[0259] (1) Synthesis procedure

[0260] The following units are included:

[0261] 1) De-blocking: The 5'-OH end of the ribonucleotide is protected by 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.

[0262] 2) Coupling: Nucleotide monomers are mixed with activation reagents and react with PS-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 PS-Oligo, it undergoes a nucleophilic reaction with its 5'-hydroxyl group, coupling occurs and tetrazole is removed, extending a nucleotide.

[0263] 3) Capping: Since the coupling efficiency cannot reach 100%, in order to prevent the uncoupled PS-Oligo from continuing to the next step of coupling, the 5'-hydroxyl group of PS-Oligo is capped with a Cap reagent.

[0264] 4) Oxidation or thiolation: After the coupling reaction, the nucleotide is connected to the oligonucleotide on the PS 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.

[0265] 5) VP protecting group removal and aminolysis

[0266] Transfer the synthesized PS-oligo from the synthesis column to a centrifuge tube, prepare a removal reagent according to the volume ratio of 3:2:100 = TMS-I: pyridine: DCM, and add the removal reagent to the centrifuge tube for reaction for 30 minutes. Prepare TEA / acetonitrile = 1:1 solution, add 2-mercaptoethanol (final concentration 2M), mix well, and add to the reaction to terminate the reaction. Remove the supernatant and wash the PS-oligo for 5min×2 times. After washing, add aminolysis solution (2-mercaptoethanol (2M) / 28% ammonia water) for aminolysis for 10h. After the aminolysis is completed, vacuum centrifuge to remove the ammonia water and purify.

[0267] LC-MS detection after conjugate synthesis

[0268]

[0269] (3) Purification

[0270] The sample diluted by ammonolysis was purified by anion column exchange chromatography, and the liquid phase system adopted high performance liquid chromatography. The chromatographic conditions were as follows.

[0271] Chromatographic column: PS-15Q10*250mm

[0272] Flow rate: 4ml / min

[0273] Detection wavelength: 260nm

[0274] Ion column preparation purification gradient program:

[0275] Mobile phase: Phase A: 10mM NaOH solution (PH=10) Phase B: 10mM NaOH solution (PH=10) + 2M NaCl

[0276]

[0277] After preparation, the samples were confirmed by mass spectrometry and purity.

[0278] (4) Ultrafiltration

[0279] After purification, add 4 ml PBS to dissolve the sample, transfer it to a 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 RNase water and ultrafiltration again. Ultrafiltration is performed 3 times, each time for 45 min.

[0280] (5) Annealing

[0281] 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 for 16 hours. Take 10 μL of the sample for HPLC detection.

[0282] PC sequences known to have inhibitory effects on genes such as TTR, HAO, and HBV were used as tool sequences to compare the effects before and after modification.

[0283] List of tested siRNA conjugates

[0284]

[0285]

[0286]

[0287] Among them, C, G, U, A, and T represent the base composition of nucleotides, including modified and unmodified nucleotides; m represents that the nucleotide adjacent to the right side of the mark m is a 2'-methoxy nucleotide; f represents that the nucleotide adjacent to the right side of the mark f is a 2'-fluoro nucleotide; the mark * represents that the two nucleotides adjacent to the left and right of the mark * (or between the nucleotide and the linker-targeting ligand part) are connected by thiophosphate groups; GalNAc (L96) means that the linker-targeting ligand part GalNAc (L96) is conjugated thereto; there is no symbol between directly adjacent nucleotides.

[0288] The unconventional base nucleotide monomer structure in the siRNA of the present invention is as follows:

[0289]

[0290] Example 6: Evaluation of off-target ability of siRNA seed region

[0291] Sequences for detecting on-target activity and off-target activity in the seed region were constructed for siRNA sequences. The on-target detection sequence is a sequence that completely matches the siRNA to be tested, and the off-target detection in the seed region is a sequence that completely matches the antisense strand seed region (5' end 2-8) of the siRNA to be tested, and the sequences in other positions are completely mismatched. Five on-target or off-target detection sequences in the seed region are inserted into each psiCHECK plasmid.

[0292] 293T cells were seeded into 96-well plates at 20,000 cells / well; siRNA to be tested was co-transfected with 25 ng of psiCHECK plasmid constructed in the target or seed region by lipofectamine 2000. After culturing for 24 h at 37°C and 5% CO2, the fluorescence value was determined by dual-luciferase reporter using Dual-Glo luciferase assay system (Promega, E2920). The psiCHECK plasmid without siRNA molecules was transfected alone as a control group, and each duplex was tested for 3-4 independent transfections. The transfection concentration of each siRNA was 50 nM, 10 nM, 2.0 nM, 0.4 nM, 0.08 nM, 0.016 nM, and 0.0032 nM. The target inhibition effect was determined by measuring Renilla luciferase normalized to the level of constitutively expressed firefly luciferase, and the IC50 of the siRNA in the target or seed region off-target activity was calculated. The off-target risk of the siRNA seed region was evaluated by the ratio of off-target activity IC50 of the seed region / on-target activity IC50. The test results are shown in the table below.

[0293] Off-target risk assessment of the conjugate seed region

[0294] serial number On target IC50(nM) Off target IC50(nM) Ratio Conjugate 1 0.001217 >50 >41084.6 Conjugate 2 0.0009985 0.3299 330.4 Conjugate 3 0.001448 >50 >34530.3 Conjugate 4 0.001384 >50 >36127.1 Conjugate 5 0.00503 >50 >9940.3 Conjugate 6 0.001359 0.4832 355.6 Conjugate 11 0.007037 0.7272 103.3 Conjugate 12 0.0882 0.2096 2.4 Conjugate 13 0.5061 >50 >98.8 Conjugate 14 0.009281 >50 >5387.4 Conjugate 15 0.08061 1.068 13.2 Conjugate 16 0.0622 0.231 3.7 Conjugate 21 0.06914 >50 >723.2 Conjugate 22 0.07488 0.5347 7.1 Conjugate 23 0.2879 >50 >173.7 Conjugate 24 0.2107 >50 >237.3 Conjugate 25 0.1517 >50 >329.6 Conjugate 26 0.1117 >50 >447.6 Conjugate 31 0.006334 2.729 433 Conjugate 32 0.02944 0.1539 5.17 Conjugate 33 0.007973 >50 >6250 Conjugate 34 0.01244 >50 >4019 Conjugate 35 0.01063 >50 >4703 Conjugate 36 0.02727 >50 >1834

[0295] The experimental results show that after the nucleotide monomer of the present invention is introduced into the antisense chain seed region of siRNA, the potential off-target effect of the antisense chain seed region can be significantly reduced, and the activity of siRNA can be maintained or improved, thereby silencing the target gene mRNA with higher selectivity, expanding the safety window of siRNA drugs, and improving the drugability of siRNA, thereby more effectively treating related diseases caused by target gene mRNA.

Claims

1. A nucleotide monomer compound having a structure as shown in Formula I, or a deuterated compound thereof, or a fluorinated compound thereof, or a usable salt thereof: in: Bx is an unconventional base; R is a hydroxyl protecting group; preferably, R is selected from 4,4'-bismethoxytrityl, 4-methoxytrityl; Sug is a ribose ring or a ribose replacement ring; n is 0 to 3; m is 0 to 3; Z is a phosphate moiety, a phosphorothioate moiety, or a phosphoramidite moiety.

2. The compound according to claim 1, characterized in that: Sug is selected from the following structures: Among them, the aa end is connected to the Bx end, the bb end is connected to the OR end, and the cc end is connected to the Z end; X is selected from O or S; Y is selected from hydrogen, halogen, azido, C1-C6 alkyl, C1-C6 alkoxy, -O-(C1-C6 alkylene)-O-(C1-C6 alkyl), -O-(C1-C6 alkylene)-C(O)NR Y1 R Y2 ; R Y1 , R Y2 Each of them is independently selected from hydrogen, C1-C6 alkyl and cycloalkyl.

3. The compound according to claim 1 or 2, characterized in that: The compound shown in formula I is shown in formula II or formula III: in, Bx is an unconventional base; X is selected from O or S; n is 0 to 3; m is 0 to 3; R is a hydroxyl protecting group; preferably, R is selected from 4,4'-bismethoxytrityl, 4-methoxytrityl; Y is selected from hydrogen, halogen, azido, C1-C6 alkyl, C1-C6 alkoxy, -O-(C1-C6 alkylene)-O-(C1-C6 alkyl), -O-(C1-C6 alkylene)-C(O)NR Y1 R Y2 ; R Y1 , R Y2 Each of them is independently selected from hydrogen, C1-C6 alkyl and cycloalkyl. Z is a phosphate moiety, a phosphorothioate moiety, or a phosphoramidite moiety.

4. The compound according to claim 1 or 2, characterized in that: The compound shown in formula I is shown in formula IV: Bx is an unconventional base; R is a hydroxyl protecting group; preferably, R is selected from 4,4'-bismethoxytrityl, 4-methoxytrityl; n is 0 to 3; m is 0 to 3; Z is a phosphate moiety, a phosphorothioate moiety, or a phosphoramidite moiety.

5. The compound according to any one of claims 1 to 4, characterized in that The unconventional base is an unconventional purine, pyrimidine or nitrogen-containing heterocyclic base other than conventional nucleoside bases; Preferably, The unconventional base is selected from the A ring, the B ring, or a ring formed by mutual fusion or bridging of the A ring and the B ring; Ring A and Ring B are independently selected from a five-membered, six-membered or seven-membered heterocyclic ring; The ring formed by the mutual fusion and bridging of ring A and ring B is shown in formula V: in, Ring A and Ring B are independently five-membered, six-membered or seven-membered heterocyclic rings; The heterocyclic ring contains 0 to 3 O, S or N atoms; The A ring, the B ring or the ring formed by fusion or bridging of the A ring and the B ring is optionally substituted by a C1-C6 alkyl or cycloalkyl group, a phenyl group, a phenyl group substituted by a C1-C3 alkyl group or a halogenated phenyl group.

6. The compound according to claim 5, characterized in that: The specific structure of the unconventional base is as follows: Wherein, R and R0 are selected from hydrogen, C1-C6 alkyl, and C3-C6 cycloalkyl.

7. The compound according to claim 5, characterized in that , the specific structure of the unconventional base is as follows:

8. The compound according to any one of claims 1 to 7, characterized in that: R is selected from hydrogen or DMT; m is 0; n is 0 or 1; Y is a methoxy group; Z is 9. The compound according to any one of claims 1 to 8, characterized in that The compound is specifically:

10. Use of the nucleotide monomer compound according to any one of claims 1 to 9 as an intermediate in the preparation of an oligonucleotide; preferably, the oligonucleotide is siRNA.

11. The use according to claim 10, characterized in that: Use of the nucleotide monomer compound according to any one of claims 1 to 8 as an intermediate in the preparation of siRNA antisense chain seed region.

12. An siRNA comprising a sense strand and an antisense strand; the sense strand and the antisense strand each comprise 15 to 45 modified or unmodified nucleotides, and the sense strand and the antisense strand are partially complementary to form a double-stranded region; wherein, The seed region of the antisense strand contains at least one nucleotide having a structure shown in Formula VIa or Formula VIb, and Site covalently linked to the rest of the siRNA: in: X is independently selected from O or S; Y is selected from hydrogen, halogen, azido, C1-C6 alkyl, C1-C6 alkoxy, -O-(C1-C6 alkylene)-O-(C1-C6 alkyl), -O-(C1-C6 alkylene)-C(O)NR Y1 R Y2 ; R Y1 , R Y2 Each is independently selected from hydrogen, C1-C6 alkyl, and cycloalkyl; Bx is an unconventional base; n is 0 to 3.

13. An siRNA comprising a sense strand and an antisense strand; the sense strand and the antisense strand each comprise 15 to 45 modified or unmodified nucleotides, and the sense strand and the antisense strand are partially complementary to form a double-stranded region; wherein, The seed region of the antisense strand contains at least one nucleotide having a structure shown in Formula VIc, and Site covalently linked to the rest of the siRNA: in, X is independently selected from O or S; Bx is an unconventional base; n is 0 to 3.

14. The siRNA according to claim 12 or 13, characterized in that: The unconventional base is an unconventional purine, pyrimidine or nitrogen-containing heterocyclic base other than conventional nucleoside bases; Preferably, The unconventional base is selected from the A ring, the B ring, or a ring formed by mutual fusion or bridging of the A ring and the B ring; Ring A and Ring B are independently selected from a five-membered, six-membered or seven-membered heterocyclic ring; The ring formed by the mutual fusion and bridging of ring A and ring B is shown in formula V: in, Ring A and Ring B are independently five-membered, six-membered or seven-membered heterocyclic rings; The heterocyclic ring contains 0 to 3 O, S or N atoms; The A ring, the B ring or the ring formed by fusion or bridging of the A ring and the B ring is optionally substituted by a C1-C6 alkyl or cycloalkyl group, a phenyl group, a phenyl group substituted by a C1-C3 alkyl group or a halogenated phenyl group.

15. The siRNA according to claim 14, characterized in that: The specific structure of the unconventional base is as follows: Wherein, R and R0 represent hydrogen, C1-C6 alkyl, C3-C6 cycloalkyl.

16. The siRNA according to claim 14, characterized in that: The specific structure of the unconventional base is as follows:

17. The siRNA according to any one of claims 11 to 16, characterized in that: X is O or S; n is 0 or 1; Y is selected from methoxy or -OCH2CH2OCH3.

18. The siRNA according to any one of claims 11 to 17, characterized in that: The structure shown in Formula VIa, Formula VIb or Formula VIc in the siRNA is specifically:

19. A pharmaceutical composition comprising the siRNA according to any one of claims 11 to 18 and a pharmaceutically acceptable carrier.

20. An siRNA conjugate, formed by conjugating the siRNA according to any one of claims 11 to 18 with a conjugated molecule.

21. A kit comprising the siRNA according to any one of claims 11 to 18, and / or the pharmaceutical composition according to claim 18, and / or the siRNA conjugate according to claim 19.

22. Use of the siRNA according to any one of claims 11 to 18, and / or the pharmaceutical composition according to claim 19, and / or the siRNA conjugate according to claim 20, and / or the kit according to claim 21 in the preparation of a drug.

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