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 compound is introduced to the first position of the 5 '-tail end of the siRNA antisense strand, the activity of the siRNA can be remarkably improved, target gene mRNA can be silenced with higher selectivity, and the timeliness is longer. The druggability of the siRNA can be improved, so that related diseases caused by the target gene mRNA can be more effectively treated. # imgabs0 # can be more effectively treated.

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] However, siRNA therapy has not been rapidly developed in the clinic. Its in vivo stability and very limited bioavailability have become the main obstacles to the development of new oligonucleic acid drugs.

[0004] In order to comprehensively improve the activity, in vivo stability and pharmacokinetic properties of siRNA and enhance its bioavailability, technicians in this field often choose to chemically modify specific nucleotides in siRNA to improve the physicochemical properties of siRNA and enhance its in vivo bioavailability, thereby enhancing the silencing efficiency of siRNA on target gene mRNA.

[0005] The present invention synthesizes modified nucleotide monomers by medicinal chemistry means, and introduces them into the special position of siRNA - the first position of the 5'-end of the antisense chain, thereby improving the efficacy of siRNA. Therefore, the present invention is of great value to the development of siRNA drugs. The present invention synthesizes modified nucleotide monomers by medicinal chemistry means, and introduces them into the first position of the 5'-end of the antisense chain of siRNA by phosphodiester bond linkage. This modification of the siRNA sequence can improve the silencing effect of the siRNA target gene and prolong the timeliness of the siRNA action. Therefore, the present invention is of great value to 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 silencing effect of RNAi drug molecules on target gene silencing.

[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] X, X' are independently selected from O or S;

[0012] PG' is an alkyl group and an alkyl group derived therefrom, or an aryl group and an aryl group derived therefrom;

[0013] R 1 , R 2 may be independently selected to be H, F, or methyl;

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

[0015] Y is optionally 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 ;

[0016] R Y1 , R Y2 Each of them is independently selected from hydrogen, C1-C6 alkyl and cycloalkyl.

[0017] In some embodiments of the present invention, the compound of claim 1, wherein the unconventional base Bx is an unconventional purine, pyrimidine or nitrogen-containing heterocyclic base other than a conventional nucleoside base; preferably, it is one selected from the following structures:

[0018]

[0019] Wherein, R and R0 are selected from hydrogen, C1-C6 alkyl, and C3-C6 cycloalkyl.

[0020] In some embodiments of the present invention, the compound of claim 2 is characterized in that the specific structure of the unconventional base is as follows:

[0021]

[0022] In some embodiments of the present invention, the compound of any one of claim 1, characterized in that: Y is methoxy or methoxyethyl;

[0023] Z is

[0024] In some embodiments of the present invention, the compound according to claim 1 is specifically:

[0025]

[0026] PG is a protecting group for an amino group or a hydroxyl group, and is an alkylcarbonyl group / alkoxycarbonyl group, or an aryl group / aromaticalkoxycarbonyl group.

[0027] The present invention also discloses a compound for preparing a nucleotide monomer compound having a structure shown in Formula I in claim 1, and the specific structure is as follows:

[0028]

[0029] Wherein PG is a protecting group of an amino group or a hydroxyl group, which is an alkylcarbonyl group / alkoxycarbonyl group, or an aromatic group / aromaticalkoxycarbonyl group.

[0030] In some embodiments of the present invention, the nucleotide monomer compound of claim 1 is used as an intermediate in the preparation of an oligonucleotide; preferably, the oligonucleotide is siRNA.

[0031] In some embodiments of the present invention, the use described in claim 7 is characterized in that: the nucleotide monomer compound described in claim 1 is used as an intermediate in the preparation of siRNA antisense strands, specifically, the nucleotide monomer compound described in claim 1 of the present invention is located at the first position of the 5' end of the siRNA antisense strand.

[0032] The present invention also discloses 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 5' end of the antisense strand is a nucleotide having a structure shown in Formula VIa, and Site covalently linked to the rest of the siRNA:

[0033]

[0034] in:

[0035] X is independently selected from O or S;

[0036] 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 ;

[0037] R Y1 , RY2 Each is independently selected from hydrogen, C1-C6 alkyl, and cycloalkyl;

[0038] Bx is an unconventional base.

[0039] In some embodiments of the present invention, the siRNA according to claim 9 is characterized in that the specific structure of the unconventional base is as follows:

[0040]

[0041] Wherein, R and R0 represent hydrogen, C1-C6 alkyl, C3-C6 cycloalkyl.

[0042] In some embodiments of the present invention, the siRNA of claim 9 is characterized in that the specific structure of the unconventional base is as follows:

[0043]

[0044] In some embodiments of the present invention, the siRNA according to any one of claims 9 to 11 is characterized in that:

[0045] X is O or S;

[0046] Y is selected from methoxy or -OCH2CH2OCH3.

[0047] In some embodiments of the present invention, the siRNA according to any one of claims 9 to 12 is characterized in that: the structure represented by Formula VIa in the siRNA is specifically:

[0048]

[0049] The present invention also discloses a pharmaceutical composition comprising the siRNA according to any one of claims 9 to 13 and a pharmaceutically acceptable carrier.

[0050] The present invention also discloses a siRNA conjugate, which is formed by conjugating the siRNA described in any one of claims 9 to 13 with a conjugated molecule.

[0051] The present invention also discloses a kit comprising the siRNA according to any one of claims 9 to 15, and / or the pharmaceutical composition according to claim 14, and / or the siRNA conjugate according to claim 15.

[0052] In some embodiments of the present invention, the siRNA of any one of claims 9 to 13, and / or the pharmaceutical composition of claim 14, and / or the siRNA conjugate of claim 15, and / or the kit of claim 16 are used in the preparation of drugs.

[0053] In some embodiments of the present invention, the unconventional base Bx is selected from the following structures:

[0054]

[0055] 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.

[0056] The modified nucleotide of the present invention can improve the effect of RNAi drug molecules on silencing target genes and can enhance the drugability of siRNA, thereby more effectively treating related diseases caused by target gene mRNA.

[0057] 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.

[0058] 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.

[0059] 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.

[0060] 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).

[0061] 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.

[0062] 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.

[0063] In some embodiments, both the sense and antisense strands contain 1 to 4 phosphorothioate linkages.

[0064] 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.

[0065] 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.

[0066] 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.

[0067] 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.

[0068] 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.

[0069] 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).

[0070] 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.

[0071] 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.

[0072] 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.

[0073] 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.

[0074] 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.

[0075] 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.

[0076] The term "sequence" or "nucleotide sequence" refers to the order or sequence of nucleobases or nucleotides, expressed in alphabetical order using standard nucleotide nomenclature.

[0077] 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.

[0078] 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.

[0079] 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.

[0080] 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.

[0081] 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.

[0082] 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.

[0083] 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.

[0084] 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.

[0085] 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.

[0086] "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.

[0087] "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.

[0088] "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.

[0089] "Halogen" is fluorine, chlorine, bromine or iodine.

[0090] "Halo-substituted alkyl" refers to an alkyl group substituted with one or more halogens, wherein alkyl is as defined above.

[0091] "Ester" refers to a -C(O)O(alkyl) or -C(O)O(cycloalkyl) group wherein alkyl and cycloalkyl are as defined above.

[0092] "Acyl" refers to a compound containing a -C(O)R group, where R is alkyl, cycloalkyl, heterocyclyl, aryl or heteroaryl.

[0093] "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.

[0094] 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.

[0095] 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).

[0096] 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.

[0097] 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.

[0098] 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. BRIEF DESCRIPTION OF THE DRAWINGS

[0099] Figure 1 This is the H-NMR spectrum of monomer XY-EVP-006;

[0100] Figure 2 It is the XY-EVP-006P-NMR spectrum of monomer;

[0101] Figure 3 This is the MS spectrum of monomer XY-EVP-006;

[0102] Figure 4 This is the 1H-NMR spectrum of monomer XY-EVP-007;

[0103] Figure 5 It is the XY-EVP-007-1P-NMR spectrum of monomer;

[0104] Figure 6 This is the MS spectrum of monomer XY-EVP-007-1;

[0105] Figure 7 It is the 2H-NMR spectrum of monomer XY-EVP-007;

[0106] Figure 8 This is the XY-EVP-007-2P-NMR spectrum of the monomer;

[0107] Fig. 9 This is the MS spectrum of monomer XY-EVP-007-2;

[0108] Fig.10 A schematic diagram of the solid phase synthesis route for synthesizing siRNA sequences using a solid phase carrier;

[0109] Fig.11The structural diagram of the solid phase support (L96-PS) modified with GalNAc (L96);

[0110] Fig.12 The figure is a flow chart of the synthesis process of siRNA sequences using solid phase carriers. Specific implementation plan

[0111] 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.

[0112] 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.

[0113] The raw materials and equipment used in the present invention are all known products, which are obtained by purchasing commercially available products.

[0114] The compounds disclosed in the present invention can be prepared by the following synthetic methods.

[0115] Example

[0116] 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.

[0117] 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)).

[0118] Example 1. Synthesis of Compound XY-EVP-006 ((((E)-2-((2R,3R,4R,5R)-3-(((2-cyanoethoxy)(dipropylamino)phosphino)oxy)-4-methoxy-5-(6-(N-methylbenzamido)-9H-purin-9-yl)tetrahydrofuran-2-yl)vinyl)phosphoryl)bis(oxy))bis(methylene)bis(2,2-dimethylpropionate)

[0119]

[0120] Step 1. Synthesis of compound XY-EVP-006-2

[0121] Add the substrate XY-EVP-006-1 (5.0 g, 7.3 mmol) to a dry 250 mL single-mouth bottle, add anhydrous N, N-dimethylformamide (36 mL) to dissolve, then add tert-butyldimethylsilyl chloride (2.2 g, 14.5 mmol) and imidazole (1.2 g, 17.4 mmol) under nitrogen protection, and stir the reaction system at room temperature overnight. Then add 50 mL of dichloromethane to the reaction solution, wash with water (200 mL*2), and wash with saturated brine (200 mL), dry the organic phase with anhydrous Na2SO4, filter, and concentrate, and the residue is purified by column chromatography (petroleum ether: ethyl acetate = 1:2), and concentrated to obtain the product XY-EVP-006-2 (5.6 g, 96.1%), LCMS (ESI) m / z: 804.6 [M+H] + .

[0122] Step 2. Synthesis of compound XY-EVP-006-3

[0123] Add the substrate XY-EVP-006-2 (5.6 g, 7.0 mmol) to a dry 100 mL single-mouth bottle, add anhydrous N, N-dimethylformamide (25 mL) to dissolve, then add cesium carbonate (4.1 g, 12.5 mmol) and iodomethane (2.0 g, 13.9 mmol), and stir the reaction system at room temperature for 2 hours. Then add 30 mL of dichloromethane to the reaction solution, wash with water (100 mL*2), and wash with saturated brine (100 mL), dry the organic phase with anhydrous Na2SO4, filter, and concentrate, and the residue is purified by column chromatography (petroleum ether: ethyl acetate = 1:1-1:2), and concentrate to obtain the product XY-EVP-006-3 (3.2 g, 56.7%), LCMS (ESI) m / z: 818.6 [M+H] + .

[0124] Step 3. Synthesis of compound XY-EVP-006-4

[0125] Slowly add formic acid (21 mL) to a solution of substrate XY-EVP-006-3 (2.0 g, 2.4 mmol) in water (17 mL). After the addition, the reaction system is stirred at room temperature for 16 h. Then add ammonia water at 0°C until pH = 7-8, extract with ethyl acetate (200 mL*3), and wash with saturated brine (100 mL). The organic phase is dried over anhydrous Na2SO4, filtered, and concentrated. The residue is purified by column chromatography (ethyl acetate) and concentrated to obtain the product XY-EVP-006-4 (1.5 g, 89.4%). 1H NMR(400MHz,DMSO-d6)δ8.71(s,1H),8.58(s,1H),7.34-7.31(m,3H),7.28-7.17(m,2 H),6.86(m,1H),6.11(d,J=5.2Hz,1H),6.12-6.00(m,1H),5.65-5.54(m,4H),4.68(t ,J=4.4Hz,1H),4.59(t,J=4.9Hz,1H),4.54-4.51(m,1H),3.67(s,3H),3.27(s,3H),1 .10(d,J=6.4Hz,18H),0.90(s,9H),0.12(d,J=1.4Hz,6H).LCMS(ESI)m / z:704.6[M+H] + .

[0126] Step 4. Synthesis of compound XY-EVP-006

[0127] At room temperature, compound XY-EVP-006-4 (1.1 g, 1.5 mmol) was dissolved in toluene (15 mL*3) and concentrated three times, then dissolved in dry dichloromethane (11 mL), and then 4,5-dicyanoimidazole (158.6 mg, 1.3 mmol) and bis(diisopropylamino)(2-cyanoethoxy)phosphine (584.7 mg, 1.9 mmol) were added under nitrogen protection. After addition, the reaction system was stirred at room temperature under nitrogen protection for 1 h. Concentrated to obtain a crude product, which was purified by C18 (72% ACN in 0.05% NH4HCO3 / H2O), concentrated, and extracted with dichloromethane to obtain the product XY-EVP-006 (1.0 g, 76.4%).

[0128] 1 H NMR (400MHz, DMSO-d6) δ8.70(d,J=5.6Hz,1H),8.56(d,J=2.5Hz,1H),7.39-7.28(m,3H),7.27-7.14(m,2H),6.89(m,1H),6.20-5.98( m,2H),5.66-5.52(m,4H),4.81-4.62(m,3H),3.91-3.54(m,7H),3.34(d,3H),2.81(m,2H),1.22-1.13(m,12H),1.14-1.05(m,18H). See attached Figure 1 .

[0129] 31 P NMR (162MHz, DMSO-d6) δ149.64 (d, J = 129.0 Hz), 16.80 (d, J = 40.0 Hz). See attached Figure 2 .

[0130] LCMS (ESI) m / z: 904.6 [M+H] + . See attached Figure 3 .

[0131] Example 2: Synthesis of Compound XY-EVP-007-1

[0132]

[0133] Experimental operation

[0134] Synthesis of compound XY-EVP-007-1-2 :

[0135] Add the substrate XY-EVP-007-1-1 (2.4 g, 3.0 mol) dissolved in 3% DCA in DCM (24 mL) to a dry 100 mL single-mouth bottle, then add Et3SiH (12 mL) under nitrogen protection, and keep the reaction system at room temperature for 2 hours after addition. LCMS detected that the raw material disappeared completely, and the reaction was completed. Then add 100 mL of dichloromethane to the reaction solution, wash with water (1000 mL*3), and wash with saturated brine (1000 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 = 5:1), and concentrated to obtain a white solid product XY-EVP-007-1-2 (1.3 g, 2.6 mmol, 80% yield).

[0136] ESI-LCMS m / z 499.2[M+H] + . 1 H NMR (400MHz, DMSO-d6) δ11.14(s,2H),8.60(s,1H),8.05-8.04(m,2H),7.77-7.05(m,2H),7.63-7.51(m,3H),7.43-7.41(m,1H),6.70-6.69(m ,1H),6.32-6.31(m,1H),5.15-5.14(m,1H),4.49-4.48(m,1H),4.27-4 .25(m,1H),3.64-3.53(m,2H),3.23(s,3H),0.91(s,9H),0.12(s,6H).

[0137] Synthesis of compound XY-EVP-007-1-3 :

[0138] Add the substrate XY-EVP-007-1-2 (1.3 g, 2.6 mmol) dissolved in DMSO (15 mL) to a dry 100 mL single-mouth bottle, then add EDCI (1.5 g, 7.8 mmol) and pyridine (0.2 g, 2.6 mmol) under nitrogen protection, and slowly add TFA (0.1 g, 1.3 mmol) at 0 ° C. After the addition, the reaction system is kept stirring at room temperature for 2 hours. LCMS detected that the raw material completely disappeared and the reaction was completed. Then add 100 mL of ethyl acetate to the reaction solution, wash with water (500 mL*3), and wash with saturated brine (500 mL). The organic phase is dried over anhydrous Na2SO4, filtered, and concentrated to obtain a crude oily product XY-EVP-007-1-3 (2.0 g,).

[0139] ESI-LCMS: m / z 497.2[M+H] + .

[0140] Synthesis of compound XY-EVP-007-1-4 :

[0141] In a dry 100mL single-mouth bottle, add the substrate XY-EVP-007-1-3 (2.0g) dissolved in THF (20mL) mixture, then add POM seter (4.4g, 7.8mmol) and NaH (0.6g, 15.6mmol) under nitrogen protection, and keep the reaction system stirring at room temperature for 2 hours. LCMS detected that the raw material disappeared completely, and the reaction was completed. Then add 100mL of ethyl acetate to the reaction solution, wash with water (200mL*3), and wash with saturated brine (100mL), 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-EVP-007-1-4 (1.4g, 1.5mmol, 60% yield).

[0142] ESI-LCMS: m / z 917.3 [M+NH4] - ; 1 H NMR (400MHz, DMSO-d6) δ8.41(s,1H),7.80(s,1H),7.66-7.64(m,4H),7.46-7.43(m,2H),7.34-7.30(m,4H),6. 39-6.38(m,1H),6.18-6.17(m,1H),5.50-5.43(m,5H),3.14(s,3H),1.01(s,8H),0.96(s,19H),0.77(s,10H).

[0143] Synthesis of compound XY-EVP-007-1-5 :

[0144] Add the substrate XY-EVP-007-1-4 (1.4 g, 1.5 mmol) dissolved in 1:1 formic acid: water (20 mL) to a dry 100 mL single-mouth bottle, and keep the reaction system at room temperature and stir for 17 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 over 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 white solid product XY-EVP-007-1-5 (550 mg, 0.8 mmol, 60% yield).

[0145] ESI-LCMS: m / z 689.2[M+H] + ; 1 H NMR(400MHz,DMSO-d6)δ11.10(s,1H),8.54(s,1H),7.99-7.97(m,2H),,7. 58-7.44(m,4H),6.90-6.79(m,1H),6.65-6.64(m,1H),6.30-6.10(m,3H),6 .02-5.91(m,1H),5.59-5.50(m,8H),4.46-4.45(m,1H),4.29-4.27(m,2H) ,3.25(s,3H),2.80(s,1H),2.64(s,1H),1.07(s,13H),1.03-1.02(m,18H).

[0146] Synthesis of compound XY-EVP-007-1 :

[0147] In a dry 50mL single-mouth bottle, add the substrate XY-EVP-007-1-5 (550mg, 0.8mmol) dissolved in anhydrous DCM (10mL), then add DCI (84mg, 0.6mmol) and CEP [N (iPr) 2] 2 (288mg, 0.9mmol) under nitrogen protection. After the addition, the reaction system is kept stirring at room temperature for 2 hours. LCMS detected that the raw material completely disappeared and the reaction was completed. Then 100mL of dichloromethane was added to the reaction solution, washed with water (100mL * 3), and washed with saturated brine (100mL), 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-EVP-007-1 (530mg, 0.6mmol, 75% yield).

[0148] 1 H NMR(400MHz,DMSO-d6)δ11.20(s,1H),8.62(s,1H),8.08-8.06(m,2H),7.7 0-7.63(m,2H),7.57-7.54(m,2H),7.04-6.85(m,1H),6.75-6.74(m,1H),6 .14-6.07(m,1H),5.66-5.56(m,4H),4.75-4.59(m,3H),3.89-3.63(m,1H) ,3.39(s,3H),2.86-2.82(m,2H),2.23-2.16(m,12H),1.12-1.10(m,18H). See Figure 4 .

[0149] 31 P NMR (162MHz, DMSO-d6) δ 150.09, 149.27, 16.95, 16.69. Figure 5 .

[0150] ESI-LCMS: m / z 888.6[MH] - ;See Figure 6 .

[0151] Example 3: Synthesis of Compound XY-EVP-007-2

[0152]

[0153] Experimental operation

[0154] Synthesis of compound XY-EVP-007-2-9 :

[0155] Add substrate 8-P2 (700 mg, 1.8 mmol) to a dry 50 mL single-mouth bottle and dissolve it in anhydrous DMF (10 mL), then add EDCI (1.1 g, 5.8 mmol) and pyridine (371 mg, 1.9 mmol) under nitrogen protection. After the addition, the reaction system is cooled to 5 ° C and TFA (115 mg, 1.0 mmol) is slowly added dropwise. After the addition, the reaction system is kept stirring at room temperature for 2 hours. LCMS detection shows that the raw material disappears completely and the reaction is over. Then add 100 mL of ethyl acetate to the reaction solution, wash with saturated sodium bicarbonate aqueous solution and water (100 mL*3), and wash with saturated brine (100 mL). The organic phase is dried over anhydrous Na2SO4, filtered, and concentrated to obtain a crude yellow solid product XY-EVP-007-2-9 (700 mg, 1.8 mmol). ESI-LCMS m / z 412.3 [M+H2O] + .

[0156] Synthesis of compound XY-EVP-007-2-10 :

[0157] Add the substrate XY-EVP-007-2-9 (700 mg, 1.8 mmol) dissolved in anhydrous toluene (8 mL) to a dry 50 mL single-mouth bottle, then add POM ester (1.4 g, 2.2 mmol) and KOH (180 mg, 3.2 mmol) at room temperature. After the addition is complete, the reaction system is kept stirring at room temperature for 2 hours. LCMS detected that the raw material completely disappeared and the reaction was completed. Then 100 mL of ethyl acetate was added to the reaction solution, and the mixture was washed twice with water and saturated brine respectively. The organic phase was dried over anhydrous Na2SO4, filtered, and concentrated to obtain a crude product. The product was concentrated with reverse phase C18 (CH3CN / H2O (0.05% NH4HCO3) = 7 / 3) to obtain a white solid product XY-EVP-007-2-10 (750 mg, 1.1 mmol). ESI-LCMS m / z 700.1 [M+H2O] + . Compound XY-EVP-007-2-11 become :

[0158] Substrate XY-EVP-007-2-10 (750 mg, 1.1 mmol) was added to a dry 50 mL single-mouth bottle and dissolved in formic acid aqueous solution (formic acid / water = 1:1) (8 mL), and then the reaction system was kept at room temperature and stirred overnight. LCMS detected that the raw material completely disappeared and the reaction was completed. Then 100 mL of ethyl acetate was added to the reaction solution, washed with saturated sodium bicarbonate aqueous solution (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) = 7 / 3) was used to concentrate to obtain a white solid product XY-EVP-007-2-11 (400 mg, 0.7 mmol, 39% three-step yield).

[0159] ESI-LCMS m / z 586.5[M+H] + . 1 H NMR(400MHz,DMSO-d6)δ11.50(s,1H),7.92(s,1H),7.34-7.25(m,1H),6.98-6.79(m,1H),6.62-6.5 0(m,1H),6.22-5.94(m,2H),5.72-5.51(m,5H),4.56-4.15(m,3H),3.31(s,3H),1.20-1.03(m,18H). 31 P NMR (162 MHz, DMSO-d6) δ 17.30.

[0160] Synthesis of compound XY-EVP-007-2 :

[0161] Substrate XY-EVP-007-2-11 (400 mg, 0.7 mmol) was added to a dry 50 mL single-mouth bottle and dissolved in anhydrous DCM (5 mL), followed by addition of DCI (68 mg, 0.6 mmol) and CEP [N (iPr) 2] 2 (247 mg, 0.8 mmol) under nitrogen protection. After 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-EVP-007-2 (400 mg, 0.5 mmol, 71% yield).

[0162] 1 H NMR (400MHz, DMSO-d6) δ12.04(s,1H),7.91(s,1H),7.35-7.21(m,1H),7.02-6.78(m,1H),6.63-6.52(m,1H),6.22-5.97(m ,2H),5.68-5.51(m,4H),4.75-4.37(m,3H),3.90-3.55(m,4H),3.39-3.26(m,3H),2.87-2.77(m,2H),1.22-1.06(m,30H). See Figure 7 .

[0163] 31 P NMR (162MHz, DMSO-d6) δ 150.10, 149.28, 16.91, 16.64. Figure 8 .

[0164] ESI-LCMS: m / z 784.4 [M+H] - .See Fig. 9 .

[0165] Example 4: siRNA Synthesis

[0166] 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.10 The structure of the GalNAc(L96) modified solid phase carrier (L96-PS) is as follows Fig.11 :The Primer support 5G unylinker 350 carrier structure is as shown below:

[0167]

[0168] ●PS stands for polystyrene solid phase carrier

[0169] 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.10 ).

[0170] 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.

[0171] See the process flow Fig.12 .

[0172] Process Description:

[0173] 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.

[0174] (1) Synthesis procedure

[0175] The following units are included:

[0176] 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.

[0177] 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.

[0178] 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.

[0179] 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.

[0180] 5) VP protecting group removal and aminolysis

[0181] 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.

[0182] LC-MS detection after conjugate synthesis 006 007-1 007-2

[0183]

[0184] (3) Purification

[0185] 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.

[0186] Chromatographic column: PS-15Q10*250mm

[0187] Flow rate: 4ml / min

[0188] Detection wavelength: 260nm

[0189] Ion column preparation purification gradient program:

[0190] Mobile phase: Phase A: 10mM NaOH solution (PH=10) Phase B: 10mM NaOH solution (PH=10) + 2M NaCl

[0191]

[0192] After preparation, the samples were confirmed by mass spectrometry and purity.

[0193] (4) Ultrafiltration

[0194] 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.

[0195] (5) Annealing

[0196] 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.

[0197] A PC sequence known to have an inhibitory effect on the ANGPTL3 gene was used as a tool sequence to compare the effects before and after modification.

[0198] The PC sequence before modification is:

[0199]

[0200] The modified sequence is:

[0201]

[0202] Test conjugate information:

[0203]

[0204] Among them, C, G, U, A, and T represent the base composition of the nucleotide, 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 a thiophosphate group; L96 means that the linker-targeting ligand part L96 is conjugated thereto; mvpBx represents the base-modified 2'-methoxy nucleoside monomer described in this patent, and Bx represents modified bases such as I, mA, mA', X, B, U1, U2, U3, U4, U5, U6, B1, and B2; there is no symbol between directly adjacent nucleotides.

[0205] Example 6: In vitro activity detection

[0206] Cell culture and transfection

[0207] Cell culture: Hep3B cells (ATCC) were cultured in MEM complete medium (Gibco, supplemented with 10% FBS) at 37°C and 5% CO2 until nearly confluent, and then the cells were digested with trypsin and plated. 2.0×10 5 Hep3B cells and 0.09 mL MEM complete medium (Gibco, supplemented with 10% FBS) were cultured at 37° C. and 5% CO 2 for 16-24 h before transfection.

[0208] Cell transfection: 0.15 μL lipofectamine RNAiMax (Invitrogen) was added to 4.85 μL opti-MEM per well, and then 5.0 μL siRNA was added to mix, added to PCR tubes and incubated at room temperature for 5 minutes, and finally the siRNA mixture was added to the above cells and cultured for 24 hours before RNA extraction. Single-dose experiments were performed at 10nM and 0.1nM or 0.1nM and 0.01nM siRNA duplex concentrations. IC50 testing experiments were performed at 10nM, 1.0nM, 0.1nM, 0.01nM, 0.001nM, 0.0001nM, and 0.00001nM siRNA duplex concentrations.

[0209] RNA extraction

[0210] Use the total RNA isolation kit (Omega, cat: R6834-02): collect cells, wash with 1% PBS, then add 400 μL of lysis buffer (containing 2% β-mercaptoethanol) to lyse the cells, and follow the instructions of the RNA isolation kit for subsequent steps. Finally, add 30 μL of RNase-free water, let stand for 2 minutes, and then centrifuge at 14000g for 2 minutes to collect RNA.

[0211] cDNA synthesis

[0212] cDNA synthesis was performed using the gDNA removal cDNA synthesis kit of Quanshijin (Beijing Quanshijin Biotechnology Co., Ltd., Beijing, China Cat#AE311-03). 1 μg of total RNA was added to each sample, and cDNA synthesis was performed using a gradient thermal cycler (LongGene, A600) according to the instructions.

[0213] Real-time quantitative PCR

[0214] The synthesized cDNA and mixed master solution (including primers, qPCR premix and ultrapure water) were added to a 384-well plate (Biocon Media, Cat# PC-0040-9U) so that the final real-time fluorescence quantitative PCR system contained 0.25 μM of upstream and downstream primers of the target gene (ANGPTL3) or internal reference gene (GADPH) and 1×SYBR Green premix (Applied Biosystem).

[0215] Using the ΔΔCt assay in ABIQuantStudio TM Real-time fluorescence PCR was performed in a 6-well real-time fluorescence PCR system. Each duplex was tested for 3-4 independent transfections, and each transfection was tested in triplicate.

[0216] Conjugate Hep3B single dose test results

[0217]

[0218] Example 7 In vivo activity detection in wild-type mice

[0219] 6-8 week old male C57BL / 6J mice (Chengdu Weitonglihua Experimental Animal Technology Co., Ltd.). Blood was collected and serum was separated before grouping and administration, and used for ELISA to detect the level of mouse ANGPTL3 protein. Mice were randomly divided into 4 groups according to the level of ANGPTL3, defined as day0. On Day1, PBS, PC, conjugate 2, conjugate 3, and conjugate 4 were subcutaneously injected, respectively, with a drug dose of 1 mpk. After administration on day8, day15, and day22, serum was separated from the blood and used for ELISA to detect ANGPTL3. The serum protein concentration of each mouse at each time point was compared with the protein concentration of the mouse before administration (day0), and the detection results of different conjugates are shown in the table.

[0220] Changes in ANGPTL3 protein expression before and after administration of different siRNA modified duplexes

[0221]

[0222] Changes in ANGPTL3 protein expression before and after administration of different siRNA modified duplexes

[0223]

[0224] The above experimental results show that after the nucleotide monomer of the present invention is introduced at the first position of the 5'-end of the siRNA antisense chain, the activity of the siRNA can be significantly improved, thereby silencing the target gene mRNA with higher selectivity, reducing the dosage of siRNA, expanding the safety window of siRNA drugs, and improving the drugability of siRNA, thereby more effectively treating related diseases caused by the 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; X, X' are independently O or S; PG' is an alkyl group and an alkyl group derived therefrom, or an aryl group and an aryl group derived therefrom; R 1 , R 2 may be independently selected to be H, F, or methyl; Z is a phosphate moiety, a phosphorothioate moiety, or a phosphoramidite moiety. Y is optionally hydrogen, halogen, azido, C 1 -C 6 Alkyl, C 1 -C 6 Alkoxy, -O-(C 1 -C 6 Alkylene)-O-(C 1 -C 6 Alkyl), -O-(C 1 -C 6 Alkylene)-C(O)NR Y1 R Y2 ; R Y1 , R Y2 are independently selected from hydrogen, C 1 -C 6 Alkyl, cycloalkyl.

2. The compound according to claim 1, wherein the unconventional base Bx is an unconventional purine, pyrimidine or nitrogen-containing heterocyclic base other than a conventional nucleoside base; preferably, it is one selected from the following structures: in, R, R 0 Selected from hydrogen, C 1 -C 6 Alkyl, C 3 -C 6 Cycloalkyl.

3. The compound according to claim 2, It is characterized in that The specific structure of the unconventional base is as follows:

4. The compound according to any one of claim 1, Features: Y is methoxy or methoxyethyl; Z is 5. The compound according to claim 1, specifically: PG is a protecting group for an amino group or a hydroxyl group, and is an alkylcarbonyl group / alkoxycarbonyl group, or an aryl group / aromaticalkoxycarbonyl group.

6. A compound for preparing a nucleotide monomer compound having a structure as shown in Formula I of claim 1, wherein the specific structure is as follows: PG is a protecting group for an amino group or a hydroxyl group, and is an alkylcarbonyl group / alkoxycarbonyl group, or an aryl group / aromaticalkoxycarbonyl group.

7. Use of the nucleotide monomer compound according to claim 1 as an intermediate in the preparation of an oligonucleotide; preferably, the oligonucleotide is siRNA.

8. The use according to claim 7, Features: The use of the nucleotide monomer compound described in claim 1 as an intermediate in the preparation of siRNA antisense strand. Specifically, the nucleotide monomer compound described in claim 1 of the present invention is located at the first position of the 5' end of the siRNA antisense strand.

9. 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; in, The 5' end of the antisense strand is a nucleotide having the structure shown in Formula VIa, 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, C 1 -C 6 Alkyl, C 1 -C 6 Alkoxy, -O-(C 1 -C 6 Alkylene)-O-(C 1 -C 6 Alkyl), -O-(C 1 -C 6 Alkylene)-C(O)NR Y1 R Y2 ; R Y1 , R Y2 are independently selected from hydrogen, C 1 -C 6 Alkyl, cycloalkyl; Bx is an unconventional base.

10. The siRNA according to claim 9, It is characterized in that The specific structure of the unconventional base is as follows: Among them, R, R 0 Represents hydrogen, C 1 -C 6 Alkyl, C 3 -C 6 Cycloalkyl.

11. The siRNA according to claim 9, Features: The specific structure of the unconventional base is as follows:

12. The siRNA according to any one of claims 9 to 11, Features: X is O or S; Y is selected from methoxy or -OCH 2 CH 2 OCH 3 .

13. The siRNA according to any one of claims 9 to 12, Features: The structure shown in Formula VIa of the siRNA is specifically:

14. A pharmaceutical composition comprising the siRNA according to any one of claims 9 to 13 and a pharmaceutically acceptable carrier.

15. An siRNA conjugate, formed by conjugating the siRNA according to any one of claims 9 to 13 with a conjugated molecule. 16 . A kit comprising the siRNA according to any one of claims 9 to 15 and / or the pharmaceutical composition according to claim 14 and / or the siRNA conjugate according to claim 15 .

17. Use of the siRNA according to any one of claims 9 to 13, and / or the pharmaceutical composition according to claim 14, and / or the siRNA conjugate according to claim 15, and / or the kit according to claim 16 in the preparation of a drug.

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