MRNA (messenger ribonucleic acid) capping compound and application thereof

Abstract

The invention relates to an mRNA capping compound shown in a formula (I), wherein each group is defined in the specification. According to the capping compound, the stability of mRNA is improved, and the translation efficiency of mRNA is improved. # imgabs0 #

Description

Field of the Invention

[0001] The invention belongs to the field of biomedicine, and specifically relates to an mRNA capping compound and application thereof in mRNA capping. Background Art

[0002] The 5' capping compound is a critical structure of mRNA, which is crucial for mRNA stability, efficient translation and reducing the immunogenicity of mRNA.

[0003] The naturally structured capping compounds can be recognized and hydrolyzed by the decapping enzyme (DCP2), which reduces the stability of mRNA in the body and ultimately reduces the translation efficiency of the target mRNA.

[0004] The modified capping compound developed in the present application protects mRNA from the attack of 5'→3' exonuclease, that is, it has the effect of resisting the degradation of 5'-exonuclease, increases the stability of mRNA, and improves the translation efficiency of mRNA. Summary of the invention

[0005] In order to solve the above problems, the present application provides an mRNA capping compound, which not only has higher capping efficiency and translation efficiency, but also has a lower decapping rate after the capped mRNA is treated with a decapping enzyme, and has higher stability.

[0006] Specifically, the present invention provides, on one hand, a compound of formula (I), or a pharmaceutically acceptable salt, stereoisomer, tautomer or isotopic variant thereof:

[0007]

[0008] in,

[0009] X2 and X3 are independently selected from O, S, NH, CH2 or CH;

[0010] X1, X4 and X5 are independently selected from CH=CH, CH2CH2, CH2O, OCH2, CH2CH2O, OCH2CH2, CH2OCH2, CH2S or SCH2; and at least one, two or three of X1, X4 and X5 are CH=CH;

[0011] Y1, Y2, Y3 and Y4 are independently selected from O or S;

[0012] R1, R2, R3, R4, R5 and R6 are independently selected from H, OH, halogen, C 1-6 Alkyl, C 1-6 Alkoxy, bridged ring, C 1-6 Haloalkyl, C 1-6 Halogenated alkoxy, C2-6 Alkenyl, C 2-6 Alkynyl, C 3-10 Cycloalkyl, amino, azido, cyano, C 1-6 Alkylcarbonylamino, (C 1-6 alkyl)2-aminocarbonyl, C 1-6 Alkylcarbonyl, methylsulfonamide, phenylsulfonamide, 3-8 membered heterocyclic group, C 6-10 Aryl or 5-10 membered heteroaryl, wherein the C 1-6 Alkyl, C 1-6 Alkoxy, C 1-6 Haloalkyl, C 1-6 Halogenated alkoxy, C 2-6 Alkenyl, C 2-6 Alkynyl, C 3-10 Cycloalkyl, amino, C 1-6 Alkylcarbonylamino, 3-8 membered heterocyclic group, C 6-10 Aryl and 5-10 membered heteroaryl are unsubstituted or optionally substituted with 1, 2, 3 or more OH, halogen, azido, cyano, acetyl, C 1-6 Alkyl, C 1-6 Haloalkyl, C 1-6 Halogenated alkoxy, C 2-6 Alkenyl, C 2-6 Alkynyl, C 1-6 Alkoxy, C 1-6 Haloalkoxy, aminocarbonyl C 1-6 Alkoxy, (C 1-6 Alkyl)2AminoC 1-6 Alkoxy, C 2-6 Alkenyl C 1-6 Alkoxy, C 2-6 Alkynyl C 1-6 Alkoxy, phenyl C 1-6 Alkoxy, C 1-6 Halogenated alkylthiol, (C 1-6 alkyl)2-aminocarbonyl, C 1-6 Alkyl amino ester group, C 1-6 Alkylcarbonyl, aminocarbonyl, 3-8 membered heterocyclic group, C 1-6 Alkylcarbonylamino, aldehydeamino or phenylcarbonyl substitution;

[0013] R7 is selected from H, OH, halogen, C 1-6 Alkyl, C 1-6 Alkoxy, bridged ring, C 1-6 Haloalkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 3-10 Cycloalkyl, 3-8 membered heterocyclic group, C 6-10 Aryl or 5-10 membered heteroaryl, wherein the C1-6 Alkyl, C 1-6 Alkoxy, C 1-6 Haloalkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 3-10 Cycloalkyl, 3-8 membered heterocyclic group, C 6-10 Aryl and 5-10 membered heteroaryl are unsubstituted or optionally substituted with 1, 2, 3 or more halogen, C 1-6 Alkyl, C 1-6 Haloalkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 1-6 Alkoxy or C 1-6 Haloalkoxy substitution;

[0014] R8 and R9 are independently selected from OH, halogen or methoxy;

[0015] B1, B2 and B3 are independently selected from natural, modified or non-natural nucleobases.

[0016] In an optional embodiment, the compound of formula (I), or a pharmaceutically acceptable salt, stereoisomer, tautomer or isotopic variant thereof, wherein:

[0017] At least one of R1, R2, R3, R4, R5 and R6 is not OH or methoxy;

[0018] In an optional embodiment, the compound of formula (I), or a pharmaceutically acceptable salt, stereoisomer, tautomer or isotopic variant thereof, wherein:

[0019] X1 is selected from CH=CH, CH2CH2, CH2O, OCH2, CH2CH2O, OCH2CH2,

[0020] CH2OCH2, CH2S or SCH2, preferably CH=CH.

[0021] In an optional embodiment, the compound of formula (I), or a pharmaceutically acceptable salt, stereoisomer, tautomer or isotopic variant thereof, wherein:

[0022] X4 is selected from CH=CH, CH2CH2, CH2O, OCH2, CH2CH2O, OCH2CH2,

[0023] CH2OCH2, CH2S or SCH2, preferably CH=CH.

[0024] In an optional embodiment, the compound of formula (I), or a pharmaceutically acceptable salt, stereoisomer, tautomer or isotopic variant thereof, wherein:

[0025] X5 is selected from CH=CH, CH2CH2, CH2O, OCH2, CH2CH2O, OCH2CH2,

[0026] CH2OCH2, CH2S or SCH2, preferably CH=CH.

[0027] In an optional embodiment, the compound of formula (I), or a pharmaceutically acceptable salt, stereoisomer, tautomer or isotopic variant thereof, wherein:

[0028] R1 and R2 are independently selected from H, OH or F.

[0029] In an alternative embodiment, the compound of formula (I), or a pharmaceutically acceptable salt, stereoisomer, tautomer or isotopic variant thereof, wherein:

[0030] R3 and R4 are independently selected from H, OH, F, C 1-6 Alkyl, C 1-6 Alkoxy, bridged ring or C 1-6 A haloalkyl group, wherein C 1-6 Alkyl, C 1-6 Alkoxy or C 1-6 The haloalkyl group is unsubstituted or optionally substituted with 1, 2, 3 or more OH, F, C 1-6 Alkyl or C 1-6 Alkoxy substitution.

[0031] Further, R3 and R4 are independently selected from H, OH, F, C 1-3 Alkyl, C 1-3 Alkoxy, bridged ring or C 1-3 A haloalkyl group, wherein C 1-3 Alkyl, C 1-3 Alkoxy or C 1-3 The haloalkyl group is unsubstituted or optionally substituted with 1 or 2 OH, F, C 1-3 Alkyl or C 1-3 Alkoxy substitution.

[0032] Further, R3 and R4 are independently selected from H, OH, methoxy, F, -CF3, -CH2F, -CHF2, -CH(CH3)F, -CHFCH2F, -CH2CHF2, -CF2CF3, -CH2OH, -CH2CH2OH or -CH2CH2OCH3.

[0033] In an optional embodiment, the compound of formula (I), or a pharmaceutically acceptable salt, stereoisomer, tautomer or isotopic variant thereof, wherein:

[0034] R5 and R6 are independently selected from H, OH, F, C1-6 Alkyl, C 1-6 Alkoxy, bridged ring or C 1-6 A haloalkyl group, wherein C 1-6 Alkyl is unsubstituted or optionally substituted with 1, 2, 3 or more Fs.

[0035] Further, R5 and R6 are independently selected from H, OH, F, C 1-3 Alkyl, C 1-3 Alkoxy, bridged ring or C 1-3 A haloalkyl group, wherein C 1-3 The alkyl group is unsubstituted or optionally substituted with 1 or 2 F groups.

[0036] Further, R5 and R6 are independently selected from H, OH, methoxy, bridged ring, F, -CF3, -CH2F or -CHF2.

[0037] In an optional embodiment, the compound of formula (I), or a pharmaceutically acceptable salt, stereoisomer, tautomer or isotopic variant thereof, wherein:

[0038] R7 is selected from OH, F or methoxy.

[0039] In an optional embodiment, the compound of formula (I), or a pharmaceutically acceptable salt, stereoisomer, tautomer or isotopic variant thereof, wherein:

[0040] R7 is selected from

[0041] In an optional embodiment, any of the above compounds of formula (I), or pharmaceutically acceptable salts, stereoisomers, tautomers or isotopic variants thereof, wherein R8 and R9 are OH.

[0042] In an optional embodiment, any of the above compounds of formula (I), or a pharmaceutically acceptable salt, stereoisomer, tautomer or isotopic variant thereof, has the following general formula:

[0043]

[0044]

[0045] wherein each group is as defined above.

[0046] In an optional embodiment, the compound of formula (I), or a pharmaceutically acceptable salt, stereoisomer, tautomer or isotopic variant thereof, wherein the compound is selected from:

[0047]

[0048]

[0049]

[0050]

[0051]

[0052]

[0053]

[0054]

[0055]

[0056]

[0057]

[0058]

[0059]

[0060]

[0061]

[0062]

[0063]

[0064]

[0065] In another aspect, the present invention provides a compound of formula (V), or a pharmaceutically acceptable salt, stereoisomer, tautomer or isotopic variant thereof:

[0066]

[0067] in,

[0068] B4 and B5 are independently selected from natural, modified or unnatural nucleobases;

[0069] Z1 and Z2 are independently selected from H, halogen or C 1-6 Alkoxy, and at least one or both of Z1 and Z2 are halogen or C 1-6 Alkoxy;

[0070] R a , R b , R c and Rd are independently selected from H, OH, halogen, C 1-6 Alkyl, C 1-6 Alkoxy, bridged ring, C 1-6 Haloalkyl, azido, where C 1-6 Alkyl, C 1-6 Alkoxy, C 1-6 The haloalkyl group is unsubstituted or optionally substituted with 1, 2, 3 or more halogen, OH, C 1-6 Alkyl, C 1-6 Alkoxy, phenyl C 1-6 Alkoxy, C 1-6 Alkylcarbonylamino, azido substitution.

[0071] In an optional embodiment, the compound of formula (V), or a pharmaceutically acceptable salt, stereoisomer, tautomer or isotopic variant thereof, wherein:

[0072] Z1 is selected from H, halogen or C 1-6 Alkoxy, preferably halogen; more preferably, Z1 is F.

[0073] In an optional embodiment, the compound of formula (V), or a pharmaceutically acceptable salt, stereoisomer, tautomer or isotopic variant thereof, wherein:

[0074] Z2 is selected from H, halogen or C 1-6 Alkoxy, preferably halogen; more preferably, Z2 is F.

[0075] In an optional embodiment, the compound of formula (V), or a pharmaceutically acceptable salt, stereoisomer, tautomer or isotopic variant thereof, wherein:

[0076] R a Selected from OH, F or methoxy;

[0077] Preferably, R a Selected from OH.

[0078] In an optional embodiment, the compound of formula (V), or a pharmaceutically acceptable salt, stereoisomer, tautomer or isotopic variant thereof, wherein:

[0079] R b Selected from OH, F, C 1-6 Alkoxy;

[0080] Preferably, R b Selected from OH or methoxy.

[0081] In an optional embodiment, the compound of formula (V), or a pharmaceutically acceptable salt, stereoisomer, tautomer or isotopic variant thereof, wherein:

[0082] R c Selected from OH, F, C 1-6 The alkoxy group is preferably a methoxy group.

[0083] In an optional embodiment, the compound of formula (V), or a pharmaceutically acceptable salt, stereoisomer, tautomer or isotopic variant thereof, wherein:

[0084] R d Selected from OH, F, C 1-6 Alkoxy group, preferably OH.

[0085] In an optional embodiment, the compound of formula (V), or a pharmaceutically acceptable salt, stereoisomer, tautomer or isotopic variant thereof, wherein the compound is selected from:

[0086]

[0087]

[0088]

[0089] In another aspect, the present invention provides a compound, or a pharmaceutically acceptable salt, stereoisomer, tautomer or isotopic variant thereof, wherein the compound is selected from:

[0090]

[0091]

[0092]

[0093]

[0094]

[0095]

[0096]

[0097]

[0098]

[0099]

[0100]

[0101]

[0102]

[0103]

[0104] In another aspect, the present invention provides the use of a compound as an in vitro co-transcribed RNA capping agent, wherein the compound is any of the above-mentioned compounds of formula (I) or any of the above-mentioned compounds of formula (V), or a pharmaceutically acceptable salt, stereoisomer, tautomer or isotopic variant thereof.

[0105] In an alternative embodiment, the above use is used for mRNA capping under an in vitro T7 RNA polymerase system.

[0106] In another aspect, the present invention provides a method for synthesizing RNA, comprising incubating a compound of formula (I) or (V) of the present invention, or a pharmaceutically acceptable salt, stereoisomer, tautomer or isotopic variant thereof with a nucleotide template to perform template transcription.

[0107] In another aspect, the present invention provides a complex comprising a compound of formula (I) of the present invention, or a pharmaceutically acceptable salt, stereoisomer, tautomer or isotopic variant thereof and a DNA template, wherein the DNA template includes a promoter region containing a transcription start site, the transcription start site having a first nucleotide at nucleotide position +1 and a second nucleotide at nucleotide position +2; and B1 is complementary to the nucleoside base at transcription template position +1 on the DNA template, and B2 is complementary to the nucleoside base at transcription template position +2 on the DNA template.

[0108] In another aspect, the present invention provides a complex comprising a compound of formula (V) of the present invention, or a pharmaceutically acceptable salt, stereoisomer, tautomer or isotopic variant thereof and a DNA template, wherein the DNA template includes a promoter region containing a transcription start site, the transcription start site having a first nucleotide at nucleotide position +1 and a second nucleotide at nucleotide position +2; and B4 is complementary to the nucleoside base at transcription template position +1 on the DNA template, and B5 is complementary to the nucleoside base at transcription template position +2 on the DNA template.

[0109] Explanation of terms

[0110] Chemical Definition

[0111] Definitions of specific functional groups and chemical terms are described in more detail below.

[0112] When a numerical range is listed, it is intended to include every value and sub-range within the stated range. For example, "C 1-6 "Alkyl" includes C1, C2, C3, C4, C5, C6, C1-6 , C 1-5 , C 1-4 , C 1-3 , C 1-2 , C 2-6 , C 2-5 , C 2-4 , C 2-3 , C 3-6 , C 3-5 , C 3-4 , C 4-6 , C 4-5 and C 5-6 alkyl.

[0113] “C 1-6 "Alkyl" refers to a straight or branched chain saturated hydrocarbon group having 1 to 6 carbon atoms. In some embodiments, C 1-4 Alkyl is preferred. In some embodiments, C 1-3 Alkyl is preferred. In some embodiments, C 1-2 Alkyl groups are preferred. 1-6 Examples of alkyl groups include methyl (C1), ethyl (C2), n-propyl (C3), isopropyl (C3), n-butyl (C4), tert-butyl (C4), sec-butyl (C4), isobutyl (C4), n-pentyl (C5), 3-pentyl (C5), pentyl (C5), neopentyl (C5), 3-methyl-2-butyl (C5), tert-pentyl (C5), and n-hexyl (C6). The term “C 1-6 "Alkyl" also includes heteroalkyl groups in which one or more (e.g., 1, 2, 3 or 4) carbon atoms are replaced by heteroatoms (e.g., oxygen (O), sulfur (S), nitrogen (N), boron (B), silicon (Si), phosphorus (P)). The alkyl group may be optionally substituted by one or more substituents, for example, by 1 to 5 substituents, 1 to 3 substituents or 1 substituent. Conventional alkyl abbreviations include: Me (-CH3), Et (-CH2CH3), iPr (-CH(CH3)2), nPr (-CH2CH2CH3), n-Bu (-CH2CH2CH2CH3) or i-Bu (-CH2CH(CH3)2).

[0114] "Halo" or "halogen" refers to fluorine (F), chlorine (Cl), bromine (Br) and iodine (I).

[0115] Therefore, “C 1-6 "Haloalkyl" refers to the above-mentioned "C 1-6 "alkyl", which is substituted by one or more halogen groups. In some embodiments, C 1-3 Haloalkyl is particularly preferred, more preferably C 1-2Haloalkyl. Exemplary haloalkyls include, but are not limited to, -CF3, -CH2F, -CHF2, -CHFCH2F, -CH2CHF2, -CF2CF3, -CCl3, -CH2Cl, -CHCl2, 2,2,2-trifluoro-1,1-dimethyl-ethyl, and the like. The haloalkyl group may be substituted at any available point of attachment, for example, by 1 to 5 substituents, 1 to 3 substituents, or 1 substituent.

[0116] "Hydroxyalkyl" refers to an alkyl group substituted with at least one hydroxyl group. Exemplary hydroxyalkyl groups include -CH2OH, -CH2CH2OH, -C(H)(OH)C(OH)H2, and the like.

[0117] “C 2-6 "Alkenyl" refers to a straight or branched hydrocarbon group having 2 to 6 carbon atoms and one or more carbon-carbon double bonds (e.g., 1, 2, or 3 carbon-carbon double bonds). The one or more carbon-carbon double bonds can be internal (e.g., in 2-butenyl) or terminal (e.g., in 1-butenyl). In some embodiments, C 2-4 Alkenyl is particularly preferred. Examples of the alkenyl include, but are not limited to, vinyl (C2), 1-propenyl (C3), 2-propenyl (C3), 1-butenyl (C4), 2-butenyl (C4), butadienyl (C4), pentenyl (C5), pentadienyl (C5), hexenyl (C6), and the like. Regardless of whether the alkenyl is modified with "substituted", each of the alkenyl groups is optionally substituted independently, for example, 1 to 5 substituents, 1 to 3 substituents or 1 substituent, and suitable substituents are defined as follows.

[0118] “C 2-6 "Alkynyl" refers to a straight or branched chain hydrocarbon group having 2 to 6 carbon atoms, one or more carbon-carbon triple bonds (e.g., 1, 2, or 3 carbon-carbon triple bonds), and optionally one or more carbon-carbon double bonds (e.g., 1, 2, or 3 carbon-carbon double bonds). In some embodiments, C 2-4 Alkynyl is particularly preferred. In some embodiments, alkynyl does not contain any double bond. One or more carbon triple bonds can be inside (e.g., in 2-butynyl) or end (e.g., in 1-butynyl). Examples of the alkynyl include, but are not limited to, ethynyl (C2), 1-propynyl (C3), 2-propynyl (C3), 1-butynyl (C4), 2-butynyl (C4), pentynyl (C5), hexynyl (C6), etc. Regardless of whether the alkynyl is modified with "substituted", each of the alkynyls is independently optionally substituted, e.g., 1 to 5 substituents, 1 to 3 substituents or 1 substituent, and suitable substituents are defined as follows.

[0119] “C 1-6 Alkoxy" and "C1-6 "Haloalkoxy" refers to -OR, where R is as above "C 1-6 Alkyl" and "C 1-6 "Haloalkyl" is defined as

[0120] "Bridged ring" refers to a methylene bridge between the 2'0 and 4'C of a nucleotide monomer, or to a sugar analog, a nucleoside, a nucleotide monomer or a nucleic acid, each of which contains such a bridge, e.g. wait.

[0121] “C 3-10 "Cycloalkyl" refers to a non-aromatic cyclic hydrocarbon group having 3 to 10 ring carbon atoms and zero heteroatoms. In some embodiments, C 3-8 Cycloalkyl, C 4-6 Cycloalkyl is preferred, C 3-6 Cycloalkyl is particularly preferred, more preferably C 5-6 Cycloalkyl. Cycloalkyl also includes ring systems in which the above cycloalkyl ring is fused to one or more aryl or heteroaryl groups, wherein the point of attachment is on the cycloalkyl ring, and in such cases, the number of carbons continues to represent the number of carbons in the cycloalkyl system. Exemplary cycloalkyls include, but are not limited to, cyclopropyl (C3), cyclopropenyl (C3), cyclobutyl (C4), cyclobutenyl (C4), cyclopentyl (C5), cyclopentenyl (C5), cyclohexyl (C6), cyclohexenyl (C6), cyclohexadienyl (C6), cycloheptyl (C7), cycloheptenyl (C7), cycloheptadienyl (C7), cycloheptatrienyl (C7), cyclooctyl (C8), cyclooctenyl (C8), bicyclo[2.2.1]heptyl (C7), bicyclo[2.2.2]octyl (C8), cyclononyl (C9), cyclononenyl (C9), cyclodec ... 10 ), cyclodecenyl (C 10 ), octahydro-1H-indenyl (C9), decahydronaphthyl (C 10 ), spiro[4.5]decyl (C 10 ), bornyl, adamantyl, etc. Regardless of whether the cycloalkyl group is preceded by "substituted", each of the cycloalkyl group is independently optionally substituted, for example, 1 to 5 substituents, 1 to 3 substituents or 1 substituent, and suitable substituents are defined as follows.

[0122] "3-10 membered heterocyclyl" refers to a group of a 3- to 10-membered non-aromatic ring system having ring carbon atoms and 1 to 4 ring heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen, sulfur, boron, phosphorus and silicon. In heterocyclyl groups containing one or more nitrogen atoms, the point of attachment may be a carbon or nitrogen atom as long as the valence permits. In some embodiments, 4-10 membered heterocyclyl is preferred, which is a 4-10 membered non-aromatic ring system having ring carbon atoms and 1 to 4 ring heteroatoms; in some embodiments, 5-10 membered heterocyclyl is preferred, which is a 5-10 membered non-aromatic ring system having ring carbon atoms and 1 to 4 ring heteroatoms; in some embodiments, 3-8 membered heterocyclyl is preferred, which is a 3-8 membered non-aromatic ring system having ring carbon atoms and 1 to 3 ring heteroatoms; in some embodiments, 3-6 membered heterocyclyl is particularly preferred, which is a 3-6 membered non-aromatic ring system having ring carbon atoms and 1 to 3 ring heteroatoms; more preferably, 5-6 membered heterocyclyl is a 5-6 membered non-aromatic ring system having ring carbon atoms and 1 to 3 ring heteroatoms. Heterocyclyl also includes a ring system in which the above heterocyclyl ring is fused to one or more cycloalkyl, aryl or heteroaryl groups, wherein the point of attachment is on the heterocyclyl ring; and in such a case, the number of ring members continues to represent the number of ring members in the heterocyclyl ring system. Regardless of whether the heterocyclyl is preceded by "substituted", each of the heterocyclyl groups is independently optionally substituted, for example, 1 to 5 substituents, 1 to 3 substituents or 1 substituent, and suitable substituents are defined as follows.

[0123] Exemplary 3-membered heterocyclic groups containing one heteroatom include, but are not limited to, aziridine, oxirane, and thiorenyl. Exemplary 4-membered heterocyclic groups containing one heteroatom include, but are not limited to, azetidine, oxetane, and thietane. Exemplary 5-membered heterocyclic groups containing one heteroatom include, but are not limited to, tetrahydrofuranyl, dihydrofuranyl, tetrahydrothienyl, dihydrothienyl, pyrrolidinyl, dihydropyrrolyl, and pyrrolyl-2,5-dione. Exemplary 5-membered heterocyclic groups containing two heteroatoms include, but are not limited to, dioxolanyl, oxasulfuranyl, disulfuranyl, and oxazolidin-2-one. Exemplary 5-membered heterocyclic groups containing three heteroatoms include, but are not limited to, triazolinyl, oxadiazolinyl, and thiadiazolinyl. Exemplary 6-membered heterocyclic groups containing one heteroatom include, but are not limited to, piperidinyl, tetrahydropyranyl, dihydropyridinyl, and thianyl. Exemplary 6-membered heterocyclic groups containing two heteroatoms include, but are not limited to, piperazinyl, morpholinyl, dithianyl, dioxanyl. Exemplary 6-membered heterocyclic groups containing three heteroatoms include, but are not limited to, hexahydrotriazine (triazinanyl). Exemplary 7-membered heterocyclic groups containing one heteroatom include, but are not limited to, azepanyl, oxepane and thianyl. Exemplary 8-membered heterocyclic groups containing one heteroatom include, but are not limited to, azocanyl, oxepane and thianyl. Exemplary 5-membered heterocyclic groups fused to a C6 aryl ring (also referred to herein as 5,6-bicyclic heterocyclic groups) include, but are not limited to, dihydroindole, isoindole, dihydrobenzofuranyl, dihydrobenzothienyl, benzoxazolinone, and the like. Exemplary 6-membered heterocyclyl groups (also referred to herein as 6,6-bicyclic heterocyclyl groups) fused to a C6 aryl ring include, but are not limited to, tetrahydroquinolinyl, tetrahydroisoquinolinyl, and the like.

[0124] “C 6-10 "Aryl" refers to a monocyclic or polycyclic (e.g., bicyclic or tricyclic) 4n+2 aromatic ring system (e.g., having 6 or 10 π electrons shared in a cyclic arrangement) group having 6-10 ring carbon atoms and zero heteroatoms. In some embodiments, aryl has six ring carbon atoms ("C6 aryl"; e.g., phenyl). In some embodiments, aryl has ten ring carbon atoms ("C 10 "aryl"; for example, naphthyl, for example, 1-naphthyl and 2-naphthyl). In some embodiments, C 6-10Aryl is particularly preferred, more preferably C6 aryl. Aryl also includes a ring system in which the above-mentioned aryl ring is fused with one or more cycloalkyl or heterocyclic groups, and the connection point is on the aryl ring, in which case the number of carbon atoms continues to represent the number of carbon atoms in the aryl ring system. Regardless of whether the aryl is modified with "substituted", each of the aryl groups is independently optionally substituted, for example, 1 to 5 substituents, 1 to 3 substituents or 1 substituent, and suitable substituents are defined as follows.

[0125] "5-10 membered heteroaryl" refers to a group of a 5-10 membered monocyclic or bicyclic 4n+2 aromatic ring system (e.g., having 6 or 10 π electrons shared in a cyclic arrangement) having ring carbon atoms and 1-4 ring heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen and sulfur. In heteroaryl groups containing one or more nitrogen atoms, the point of attachment may be a carbon or nitrogen atom as long as the valence permits. Heteroaryl bicyclic ring systems may include one or more heteroatoms in one or both rings. Heteroaryl also includes a ring system in which the above-mentioned heteroaryl ring is fused to one or more cycloalkyl or heterocyclic groups, and the point of attachment is on the heteroaryl ring, in which case the number of carbon atoms continues to represent the number of carbon atoms in the heteroaryl ring system. In some embodiments, 5-6 membered heteroaryl groups are particularly preferred, which are 5-6 membered monocyclic or bicyclic 4n+2 aromatic ring systems having ring carbon atoms and 1-4 ring heteroatoms. In some embodiments, 5-membered heteroaryl is particularly preferred, which is a 5-membered monocyclic or bicyclic 4n+2 aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms. Regardless of whether the heteroaryl is preceded by "substituted", each of the heteroaryl groups is independently optionally substituted, for example, 1 to 5 substituents, 1 to 3 substituents or 1 substituent, and suitable substituents are defined as follows.

[0126] Exemplary 5-membered heteroaryl groups containing one heteroatom include, but are not limited to, pyrrolyl, furanyl, and thienyl. Exemplary 5-membered heteroaryl groups containing two heteroatoms include, but are not limited to, imidazolyl, pyrazolyl, oxazolyl, isoxazolyl, thiazolyl, and isothiazolyl. Exemplary 5-membered heteroaryl groups containing three heteroatoms include, but are not limited to, triazolyl, oxadiazolyl, and thiadiazolyl. Exemplary 5-membered heteroaryl groups containing four heteroatoms include, but are not limited to, tetrazolyl. Exemplary 6-membered heteroaryl groups containing one heteroatom include, but are not limited to, pyridyl. Exemplary 6-membered heteroaryl groups containing two heteroatoms include, but are not limited to, pyridazinyl, pyrimidinyl, and pyrazinyl. Exemplary 6-membered heteroaryl groups containing three or four heteroatoms include, but are not limited to, triazinyl and tetrazinyl, respectively. Exemplary 7-membered heteroaryl groups containing one heteroatom include, but are not limited to, azacycloheptatrienyl, oxacycloheptatrienyl, and thiacycloheptatrienyl. Exemplary 5,6-bicyclic heteroaryl groups include, but are not limited to, indolyl, isoindolyl, indazolyl, benzotriazolyl, benzothiophenyl, isobenzothiophenyl, benzofuranyl, benzisofuranyl, benzimidazolyl, benzoxazolyl, benzisoxazolyl, benzoxadiazolyl, benzothiazolyl, benzisothiazolyl, benzothiadiazolyl, indazinyl, and purinyl. Exemplary 6,6-bicyclic heteroaryl groups include, but are not limited to, naphthyridinyl, pteridinyl, quinolyl, isoquinolyl, cinnolinyl, quinoxalinyl, phthalazinyl, and quinazolinyl.

[0127] In this application, the term "optional" or "optionally" generally means that the subsequently described event or circumstance may but need not occur, and the description includes occasions 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 may include situations where the heterocyclic group is substituted with an alkyl group and situations where the heterocyclic group is not substituted with an alkyl group.

[0128] "Deuterated" or "deuterium" refers to a compound or group in which one or more hydrogens are replaced by deuterium; deuteration can be mono-, di-, poly- or full-substituted. The terms "one or more deuterated" and "one or more deuterated" are used interchangeably. The deuterium isotope content of deuterium at the deuterated position is at least greater than 0.015% of the natural deuterium isotope content, preferably greater than 30%, more preferably greater than 50%, more preferably greater than 75%, more preferably greater than 95%, and more preferably greater than 99%.

[0129] As used herein, the term "compound of the present application" refers to the compound of the present application. The term also includes various pharmaceutically acceptable salts, stereoisomers, enantiomers, diastereomers, meso-, racemic- or tautomers of the compound of the present application.

[0130] Biological definition

[0131] The "nucleoside base" in this application can be either a natural nucleoside base or a modified nucleoside base. Natural nucleoside bases include, but are not limited to, adenine (A), guanine (G), cytosine (C), uracil (U), thymine (T) and any one of their derivatives.

[0132] "Modified nucleoside base" refers to a substance obtained by replacing one or more hydrogen atoms of a natural nucleoside base, including but not limited to N6-methyladenine, N1-methyladenine, N6-2'-O-dimethyladenosine, pseudouridine, N1-methylpseudouridine, 5-iodouridine, 4-thiouridine, 2-thiouridine, 5-methyluridine, pseudoisocytosine, 5-methoxycytosine, 2-thiouridine, 5-hydroxycytosine, N1-methylcytosine, 5-hydroxymethylcytosine, hypoxanthine, N1-methylguanine, isoguanine, etc.

[0133] Other definitions

[0134] In the present application, the term "comprising" generally refers to including the features explicitly specified, but not excluding other elements. The terms "above" and "below" generally refer to the case where the number is inclusive.

[0135] As used herein, the term "pharmaceutically acceptable salt" means an acid addition salt or a base addition salt of the compounds of the invention which are suitable, within the scope of sound medical judgment, for use in contact with the tissues of patients without undue toxicity, irritation, allergic response, or the like, commensurate with a reasonable benefit / risk ratio, and effective for their intended use, including, where possible, zwitterionic forms of the compounds of the invention.

[0136] Pharmaceutically acceptable salts can be sulfates, pyrosulfates, bisulfates, sulfites, bisulfites, nitrates, borates, phosphates, monohydrogen phosphates, dihydrogen phosphates, metaphosphates, pyrophosphates, chlorides, bromides, iodides, etc. prepared from inorganic acids. Representative salts include hydrobromides, hydrochlorides, sulfates, bisulfates, nitrates, borates, and phosphates, etc. Salts can also be prepared from organic acids, such as aliphatic mono- and dicarboxylic acids, phenyl-substituted alkanoic acids, hydroxyalkanoic acids, alkanedioic acids, aromatic acids, aliphatic and aromatic sulfonic acids, etc. Representative salts include acetate, propionate, valerate, oleate, palmitate, stearate, laurate, octanoate, isobutyrate, oxalate, malonate, succinate, suberate, sebacate, fumarate, mandelate, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, naphthoate, benzenesulfonate, toluenesulfonate, phenylacetate, citrate, lactate, maleate, tartrate, methanesulfonate, glucoheptonate, lactobionate, laurylsulfonate, and isethionate, etc. Pharmaceutically acceptable salts may include alkali and alkaline earth metal based cations such as sodium, lithium, potassium, calcium, magnesium, etc., as well as non-toxic ammonium, quaternary ammonium, and amine cations, including but not limited to ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, ethylamine, etc. Also contemplated are salts of amino acids, such as argininate, gluconate, galacturonate, and the like (see, e.g., Berge S Metal., "Pharmaceutical Salts," J. Pharm. Sci., 1977; 66: 1-19, incorporated herein by reference).

[0137] The compounds of the present invention include one or more asymmetric centers and may therefore exist in a variety of stereoisomeric forms, for example, enantiomers and / or diastereoisomeric forms. For example, the compounds of the present invention may be individual enantiomers, diastereomers or geometric isomers (e.g., cis and trans isomers), or may be in the form of a mixture of stereoisomers, including racemic mixtures and mixtures enriched in one or more stereoisomers. Isomers may be separated from the mixture by methods known to those skilled in the art, including chiral high pressure liquid chromatography (HPLC) and the formation and crystallization of chiral salts; or preferred isomers may be prepared by asymmetric synthesis. Example

[0138] The specific embodiments of the present invention are described below by specific specific examples. People familiar with the technology can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. Without being limited by any theory, the following examples are only for illustrating the compounds, preparation methods and uses of the present invention, and are not used to limit the scope of the present invention.

[0139] Example 1: Synthesis of the ammonium salt of compound 12 using intermediates 12-10 and 12-14 as raw materials

[0140] Intermediate 12-10 (12.0 mmol) and intermediate 12-14 (10.0 mmol) were suspended in DMSO (150.0 mL), and zinc chloride (80.0 mmol) was added to the reaction solution under ice bath. The reaction was then stirred at room temperature for 18 hours, and then the reaction was terminated with 0.25 M EDTA-2Na (105.0 mmol) solution. The mixture was loaded onto a DEAE Sephadex column. The product was eluted with a gradient of 0-1.0 M aqueous ammonium bicarbonate solution. The components with a purity greater than 98% were collected and desalted by reverse phase preparative chromatography and concentrated to obtain the ammonium salt of compound 12. The reaction route flow is as follows:

[0141]

[0142] Among them, intermediate 12-10 is obtained by the following steps:

[0143] Weigh the intermediate 12-1 (50.0 g, 164.9 mmol) and dissolve it in pyridine (500 mL). Add 4,4'-dimethoxytrityl chloride (DMTrCl, 67.1 g, 197.9 mmol) in batches under an ice bath and stir overnight at room temperature. After the reaction is completed, concentrate under reduced pressure to remove the solvent, and purify by column chromatography to obtain the intermediate 12-2 (89.6 g, 89.7%).

[0144] Weigh the intermediate 12-2 (89.6 g, 147.9 mmol) and dissolve it in pyridine (900.0 mL). Add 4-dimethylaminopyridine (1.8 g, 14.8 mmol) and isobutyryl chloride (39.4 g, 369.7 mmol) to the reaction solution under ice bath, and heat to 80 ° C and stir overnight. After the reaction is completed, concentrate under reduced pressure to remove pyridine. Dissolve the obtained paste in ethyl acetate (900 mL), wash with water (600.0 mL), and concentrate the organic phase under reduced pressure. The crude product is purified by column chromatography to obtain intermediate 12-3 (96.5 g, 87.5%).

[0145] Weigh the intermediate 12-3 (96.5 g, 129.4 mmol) and dissolve it in dichloromethane (600.0 mL). Add a solution of trichloroacetic acid (42.3 g, 258.8 mmol) in dichloromethane (400.0 mL) dropwise to the reaction solution. Stir at room temperature for 2 hours. After the reaction is completed, wash the reaction solution with 10% sodium bicarbonate aqueous solution (500.0 mL). The organic phase is concentrated under reduced pressure and purified by column chromatography to obtain the intermediate 12-4 (52.6 g, 91.6%).

[0146] Weigh the intermediate 12-4 (52.6 g, 118.5 mmol) and dissolve it in DMF (550.0 mL). Add DMSO (55.6 g, 711.0 mmol) and EDCI (68.0 g, 355.5 mmol) to the reaction solution at room temperature, and then add pyridine (9.4 g, 118.5 mmol) and trifluoroacetic acid (113.5 g, 118.5 mmol) dropwise to the reaction solution. After reacting at room temperature for 5 hours, pour the reaction solution into water (1.5 L) and extract it with ethyl acetate (400.0 mL) three times. Combine the organic phases, wash them with saturated sodium bicarbonate aqueous solution (600.0 mL) and saturated sodium chloride aqueous solution (600.0 mL) respectively, and then concentrate under reduced pressure. The crude product is purified by column chromatography to obtain intermediate 12-5 (39.2 g, 74.9%).

[0147] Tetraethyl methylene diphosphate (38.3 g, 133.1 mmol) was added dropwise to a suspension of NaH (5.3 g, 60%, 133.1 mmol) in THF (200.0 mL) under an ice bath, followed by stirring at 0°C for 0.5 hours. A THF solution (300.0 mL) of intermediate 12-5 (39.2 g, 88.7 mmol) was slowly added dropwise to the reaction solution. Stir at room temperature overnight. After the reaction was completed, a saturated aqueous ammonium chloride solution (500.0 mL) was added dropwise to the reaction solution under an ice bath to quench the reaction. After extraction with ethyl acetate (300.0 mL) twice, the organic phases were combined, concentrated under reduced pressure, and purified by column chromatography to obtain intermediate 12-6 (40.9 g, 80.1%).

[0148] Weigh the intermediate 12-6 (40.9 g, 71.0 mmol) and dissolve it in dichloromethane (400.0 mL). Add trimethylsilyl bromide (108.7 g, 710.0 mmol) to the reaction solution at room temperature. Stir at room temperature overnight. After the reaction is completed, concentrate under reduced pressure to obtain the crude product of intermediate 12-7, which is directly used for the next step without purification.

[0149] The crude product of the intermediate 12-7 in the previous step was dissolved in concentrated aqueous ammonia (500.0 mL) and methanol (150.0 mL), heated to 40°C and stirred for 8 hours. After the reaction was completed, it was concentrated under reduced pressure and stripped twice with water (200.0 ml) to remove the solvent, then diluted with water, loaded on DEAE Sephadex, and eluted with a linear gradient of 0-1.0 M TEAB aqueous solution. The pure components were collected, concentrated, and freeze-dried to obtain intermediate 12-8 (27.9 g, containing 2 triethylamine salts, 67.7%, 2-step yield).

[0150] Weigh the intermediate 12-8 (27.9 g, 48.1 mmol) and dissolve it in purified water (300.0 mL). The reaction solution was cooled to 4 ° C. Dimethyl sulfate (35.4 g, 280.8 mmol) was slowly added dropwise. During the process, 2M sodium hydroxide was used to adjust the pH between 4 and 5. The reaction was monitored by HPLC. After the reaction was completed, it was diluted with water and loaded onto DEAE Sephadex. It was gradient eluted with 0-1.0 M TEAB aqueous solution. The pure components were collected, concentrated under reduced pressure, and then freeze-dried to obtain intermediate 12-9 (16.3 g, 1 triethylamine salt, 68.3%).

[0151] Weigh the intermediate 12-9 (16.3 g, 32.8 mmol) and suspend it in DMF (200.0 mL). Add triphenylphosphine (17.2 g, 65.6 mmol), 2,2'-disulfide dipyridine (14.4 g, 65.6 mmol), imidazole (17.9 g, 262.4 mmol) and triethylamine (3.3 g, 32.8 mmol) in turn at room temperature. Stir at room temperature for 10 hours under nitrogen atmosphere. After the reaction is completed, slowly add the reaction solution to an acetone solution (2.0 L) of sodium perchlorate (131.2 mmol). Filter the precipitated solid, wash the filter cake with acetone until it is white, and remove the solvent under reduced pressure to obtain the intermediate 12-10 (11.7 g, 80.3%).

[0152] The reaction process is as follows:

[0153]

[0154] Among them, intermediates 12-14 are obtained by the following steps:

[0155] Weigh 2'OEt-rA phosphoramidite monomer (91.0 g, 0.1 mol) and N2-isobutyryl-2',3'acetylguanosine (43.7 g, 0.1 mol) and dissolve in DCM (900.0 mL). Add tetrazole (12.6 g, 0.18 mol) under nitrogen atmosphere. Stir at room temperature for 2 hours. After the reaction is completed, add DDTT (61.6 g, 0.3 mol) to the reaction solution and continue to react at room temperature for 3 hours. Add trichloroacetic acid (49.0 g, 0.3 mol) in DCM (0.2 L) to the reaction solution and react at room temperature for 2 hours. Wash the reaction solution with 10% sodium bicarbonate aqueous solution and saturated brine, respectively, and concentrate the organic phase under reduced pressure and purify it by column chromatography to obtain intermediate 12-11 (59.4 g, 61.4%).

[0156] Take intermediate 12-11 (59.4 g, 61.4 mmol) and bis(2-cyanoethyl)-N,N-diisopropylphosphoramidite (25.0 g, 92.1 mmol) and dissolve them in DCM (600.0 mL). Add tetrazole (6.4 g, 92.1 mmol) under nitrogen atmosphere. Stir at room temperature for 2 hours. After the reaction is completed, add tert-butyl hydroperoxide aqueous solution (0.18 mol, 70%) dropwise to the reaction solution, and continue to react at room temperature for 1 hour. Wash the reaction solution with 10% sodium sulfite aqueous solution, 10% sodium bicarbonate aqueous solution, and saturated brine, respectively, and then concentrate the organic phase under reduced pressure and dissolve it in methanol (0.5 L) and ammonia water (1.0 L), and stir at room temperature overnight. After the solvent was removed by concentration, the mixture was diluted with water and loaded onto DEAE Sephadex. The mixture was eluted with a linear gradient of 0-1.0 M TEAB eluent and concentrated to obtain the triethylamine salt of intermediate 12-12 (32.0 g, 81.4%).

[0157] Weigh the intermediate 12-12 (32.0 g, 46 mmol) and suspend it in DMSO (350.0 mL). Add triphenylphosphine (24.1 g, 92 mmol), 2,2'-disulfide dipyridine (20.3 g, 92 mmol), imidazole (31.4 g, 460 mmol) and triethylamine (9.1 g, 92 mmol) in turn at room temperature. Stir at room temperature for 10 hours under nitrogen atmosphere. After the reaction is completed, slowly add the reaction solution to an acetone solution (1.5 L) of sodium perchlorate (18.4 mmol). Filter the precipitated solid, wash the filter cake with acetone until it is white, and remove the solvent under reduced pressure to obtain the intermediate 12-13 (31.0 g, 89.1%).

[0158] Intermediate 12-13 (31.0 g, 41 mmol) and tributylamine phosphate (46.4 g, 164 mmol) were weighed and suspended in DMF (350.0 mL). Zinc chloride (45.7 g, 328.7 mmol) was added to the mixture in batches after cooling to 0°C under a nitrogen atmosphere. Then it was stirred at room temperature for 5 hours. After the reaction was completed, it was diluted with water, loaded on DEAE Sephadex, and gradient eluted with 0-1.0 M TEAB aqueous solution. The pure components were collected, concentrated under reduced pressure, and freeze-dried to obtain the triethylamine salt of intermediate 12-14 (29 g, 64.5%).

[0159] The reaction process is as follows:

[0160]

[0161] Example 2: Synthesis of the ammonium salt of compound 15 using intermediate 15-10 and intermediate 12-14 as raw materials

[0162] Using intermediates 15-10 and 12-14 as raw materials, the ammonium salt of compound 15 was obtained by referring to the synthesis method of compound 12 in the target product in Example 1. The reaction scheme is as follows:

[0163]

[0164] Among them, intermediate 15-10 is obtained by referring to the synthesis method of intermediate 12-10 in Example 1. The reaction scheme is as follows:

[0165]

[0166] Example 3: Synthesis of the ammonium salt of compound 16 using intermediate 15-10 and intermediate 16-4 as raw materials

[0167] Using intermediate 15-10 and intermediate 16-4 as raw materials, the ammonium salt of compound 16 was obtained by referring to the synthesis method of compound 12 in the target product in Example 1. The reaction scheme is as follows:

[0168]

[0169] Among them, intermediate 16-4 is obtained by referring to the synthesis method of intermediate 12-14 in Example 1. The reaction scheme is as follows:

[0170]

[0171] Example 4: Synthesis of the ammonium salt of compound 21 using intermediate 21-15 and intermediate 12-14 as raw materials

[0172] Using intermediates 21-15 and 12-14 as raw materials, the ammonium salt of compound 21 was obtained by referring to the synthesis method of compound 12 in the target product in Example 1. The reaction scheme is as follows:

[0173]

[0174] Among them, intermediate 21-15 is obtained by the following steps:

[0175] A mixture of compound 21-1 (57.0 g, 0.3 mol), TBDPSCl (105.0 g, 0.37 mol) and imidazole (32.2 g, 0.6 mol) in DMF (500.0 mL) was stirred at room temperature for 3 hours. After the reaction was complete, the solvent was removed by concentration under reduced pressure. The residue was dissolved in ethyl acetate (1.0 L), washed with water (400.0 mL*2) and brine (300.0 mL), dried over anhydrous sodium sulfate, and concentrated to dryness under reduced pressure to obtain a crude product of intermediate 21-2 (129.1 g, 100%), which was directly used in the next step without purification.

[0176] Weigh the intermediate 21-2 (129 g, 0.3 mol) and dissolve it in acetonitrile (1.2 L). Add IBX (126 g, 0.45 mol) and heat to 90 ° C and stir overnight. After the reaction is completed, cool to room temperature, filter, wash the filter cake with a small amount of acetonitrile and concentrate the filtrate to dryness to obtain the crude intermediate 21-3 (125.2 g, 97.8%), which is directly used in the next step without purification.

[0177] To a solution of lithium diisopropylamide (2M in THF, 0.44mol) in THF (1.0L) was added methyl triphenylphosphonium bromide (113.9g, 0.32mol) in batches. After the reaction solution was heated to 50°C for half an hour, intermediate 21-3 (125.2g, 0.29mol) in THF (400.0mL) was added dropwise to the reaction solution. The reaction was maintained at 50°C for 1 hour. After the reaction was completed, the mixture was cooled to room temperature. The reaction solution was poured into 1.5L saturated aqueous ammonium chloride solution. After separation, the aqueous phase was extracted with ethyl acetate (300.0mL). The organic phases were combined, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by column chromatography to obtain intermediate 21-4 (99.6g, 80.9%).

[0178] A solution of intermediate 21-4 (99.6 g, 0.23 mol) in THF (800.0 mL) was slowly added dropwise to a BH3-THF solution (1 M, 0.46 mol) at 0°C. After the addition was completed, the reaction was stirred at room temperature for 1 h. After THF / H2O (1:1, 800.0 mL) was slowly added dropwise at 0°C, 2M NaOH aqueous solution (1.0 L) and 30% H2O2 (1.0 L, 4 eq) were added dropwise to the reaction solution respectively. After the addition was completed, the mixture was stirred at room temperature for 2 hours. After the reaction was completed, the reaction solution was poured into ice water. It was extracted with ethyl acetate (400.0 mL*3). The organic phases were combined, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by column chromatography to obtain intermediate 21-5 (75.2 g, 74.9%).

[0179] Dissolve the intermediate 21-5 (75.2 g, 0.17 mol) in dichloromethane (750.0 mL), cool to 0 ° C, and slowly add DAST (54.8 g, 0.34 mol) to the reaction solution. After the addition, stir at 0 ° C for 2 hours. After the reaction is completed, slowly add the reaction solution to saturated sodium bicarbonate (500.0 mL) for quenching, and extract with ethyl acetate (300.0 mL*2). Combine the organic phases, dry with anhydrous sodium sulfate, and concentrate under reduced pressure. After purification by column chromatography, intermediate 21-6 (51.6 g, 68.3%) is obtained.

[0180] Take intermediate 21-6 (51.6 g, 0.12 mol) and dissolve it in acetic acid (180.0 mL). Add acetic anhydride (57.2 g, 0.56 mol) at room temperature, then cool to below 10 ° C, and add concentrated sulfuric acid (5.2 mL) dropwise to the reaction solution. Stir overnight at room temperature. After the reaction solution is diluted with DCM (800.0 mL), it is washed with water, saturated sodium bicarbonate aqueous solution, and saturated brine in sequence, dried over anhydrous sodium sulfate, and the organic phase is concentrated under reduced pressure to obtain the crude intermediate 21-7, which is directly used in the next step without purification.

[0181] The crude compound 21-7 obtained in the previous step and isobutyryl-guanine (31.0 g, 0.14 mol) were suspended in DCE (600.0 mL), and BSA (56.9 g, 0.28 mol) was added to the reaction solution. After the reaction was heated to 70°C and stirred for 1 hour, the temperature was lowered to 0°C, and TMSOTf (77.8 g, 0.35 mol) was added dropwise to the reaction solution. After the addition was completed, the temperature was raised to 70°C and stirred for 3 hours. After cooling to room temperature, the reaction solution was washed with a saturated sodium bicarbonate aqueous solution, and the organic phase was concentrated under reduced pressure and purified by column chromatography to obtain intermediate 21-8 (50.1 g, 64.3%).

[0182] Take intermediate 21-8 (50.1 g, 77.2 mmol) and dissolve it in anhydrous DCM (550.0 mL), add 3HF-TEA (42.0 g, 0.26 mol) and stir at room temperature for 10 hours. After the reaction is completed, the reaction solution is washed with water, and the aqueous phase is extracted once with ethyl acetate. The organic phases are combined, dried over anhydrous sodium sulfate and concentrated under reduced pressure. After purification by column chromatography, intermediate 21-9 (27.7 g, 87.1%) is obtained.

[0183] Intermediates 21-10 to 21-15 were obtained by referring to the synthesis method of intermediates 12-5 to 12-10 in Example 1.

[0184] The reaction route is as follows:

[0185]

[0186] Example 5: Synthesis of the ammonium salt of compound 27 using intermediate 27-12 and intermediate 12-14 as raw materials

[0187] Using intermediate 27-12 and intermediate 12-14 as raw materials, the ammonium salt of compound 27 was obtained by referring to the synthesis method of compound 12 in the target product in Example 1. The reaction scheme is as follows:

[0188]

[0189] Among them, intermediate 27-12 is obtained by the following steps:

[0190] Weigh the intermediate 21-5 (30 g, 67.8 mmol) and dissolve it in acetonitrile (300.0 mL). Add IBX (37.9 g, 135.5 mmol) and heat to 90 °C and stir for 4 hours. After the reaction is completed, cool to room temperature, filter, wash the filter cake with a small amount of acetonitrile and concentrate the filtrate to dryness to obtain the crude intermediate 27-1 (29.9 g, 100%), which is directly used in the next step without purification.

[0191] Anhydrous cerium chloride (11.0 g, 44.7 mmol) was suspended in anhydrous tetrahydrofuran (100.0 ml) and stirred at room temperature for 15 minutes. Methylmagnesium bromide (1.0 M tetrahydrofuran solution, 88.1 mmol) was added dropwise to the reaction solution under ice bath. After stirring at 0°C for 1.5 hours, the reaction mixture was cooled to -78°C and a tetrahydrofuran solution (300.0 mL) of intermediate 27-1 (29.9 g, 67.8 mmol) was added. After stirring at -78°C for 2 hours, the reaction mixture was returned to room temperature and carefully quenched with a saturated ammonium chloride solution. Extracted with ethyl acetate (300.0 mL*3). Washed with 5% hydrochloric acid, saturated sodium bicarbonate and brine in sequence, dried over anhydrous sodium sulfate and concentrated under reduced pressure, the crude product was purified by silica gel column chromatography to obtain intermediate 27-2 (24.6 g, 79.5%).

[0192] Dissolve intermediate 27-2 (24.6 g, 53.9 mmol) in dichloromethane (250.0 mL), cool to 0 ° C, and slowly add DAST (17.4 g, 107.8 mmol) to the reaction solution. After the addition, stir at 0 ° C for 2 hours. After the reaction is completed, slowly add the reaction solution to saturated sodium bicarbonate (500.0 mL) for quenching, and extract with ethyl acetate (300.0 mL*2). Combine the organic phases, dry with anhydrous sodium sulfate, and concentrate under reduced pressure. After purification by column chromatography, intermediate 27-3 (16.7 g, 67.6%) is obtained.

[0193] Intermediates 27-4 to 27-12 were obtained by referring to the synthesis method of intermediates 21-7 to 21-15 in Example 4.

[0194] The reaction route is as follows:

[0195]

[0196] Example 6: Synthesis of the ammonium salt of compound 28 using intermediate 28-10 and intermediate 12-14 as raw materials

[0197] Using intermediate 28-10 and intermediate 12-14 as raw materials, the ammonium salt of compound 28 was obtained by referring to the synthesis method of compound 12 in the target product in Example 1. The reaction scheme is as follows:

[0198]

[0199] Among them, intermediate 28-10 was obtained by referring to the synthesis method of intermediate 27-12 in Example 5.

[0200] The reaction route is as follows:

[0201]

[0202] Example 7: Synthesis of the ammonium salt of compound 32 using intermediate 28-10 and intermediate 32-4 as raw materials

[0203] Using intermediate 28-10 and intermediate 32-4 as raw materials, the ammonium salt of compound 32 was obtained by referring to the synthesis method of compound 12 in the target product in Example 1. The reaction scheme is as follows:

[0204]

[0205] Among them, intermediate 32-4 was obtained by referring to the synthesis method of intermediate 16-4 in Example 3.

[0206] The reaction route is as follows:

[0207]

[0208] Example 8: Synthesis of the ammonium salt of compound 36 using intermediate 36-10 and intermediate 12-14 as raw materials

[0209] Using intermediate 36-10 and intermediate 12-14 as raw materials, the ammonium salt of compound 36 was obtained by referring to the synthesis method of compound 12 in the target product in Example 1. The reaction scheme is as follows:

[0210]

[0211] Among them, intermediate 36-10 is obtained by the following steps:

[0212] Weigh the intermediate 21-5 (30 g, 67.8 mmol) and dissolve it in dichloromethane (300.0 mL). Add triethylamine (13.7 g, 135.5 mmol) and cool to 0 ° C. Add acetyl chloride (6.9 g, 88.1 mmol). After the dropwise addition, return to room temperature and react overnight. The reaction solution is washed with a saturated sodium chloride aqueous solution, and the organic phase is dried over anhydrous sodium sulfate and concentrated under reduced pressure to a crude product. Purify by column chromatography to obtain the intermediate 36-1 (29.5 g, 89.9%).

[0213] Intermediates 36-2 to 36-10 were obtained by referring to the synthesis method of intermediates 28-2 to 28-10 in Example 6.

[0214] The reaction route is as follows:

[0215]

[0216] Example 9: Synthesis of the ammonium salt of compound 38 using intermediate 38-12 and intermediate 12-14 as raw materials

[0217] Using intermediate 38-12 and intermediate 12-14 as raw materials, the ammonium salt of compound 38 was obtained by referring to the synthesis method of compound 12 in the target product in Example 1. The reaction scheme is as follows:

[0218]

[0219] Among them, intermediates 38-1 to 38-2 were obtained by referring to the synthesis method of intermediates 21-4 to 21-5 in Example 4.

[0220] Intermediates 38-3 to 38-12 were obtained by referring to the synthesis method of intermediates 36-1 to 36-10 in Example 7.

[0221] The reaction route is as follows:

[0222]

[0223] Example 10: Synthesis of the ammonium salt of compound 41 using intermediate 41-10 and intermediate 12-14 as raw materials

[0224] Using intermediates 41-10 and 12-14 as raw materials, the ammonium salt of compound 41 was obtained by referring to the synthesis method of compound 12 in the target product in Example 1. The reaction scheme is as follows:

[0225]

[0226] Among them, intermediate 41-10 is obtained by the following steps:

[0227] Weigh the intermediate 38-2 (20 g, 43.8 mmol) and dissolve it in tetrahydrofuran (200.0 mL). Cool to 0 ° C, add sodium hydride (60%, 1.3 g, 65.7 mmol) to the reaction solution in batches. After the addition, return to room temperature and react for 1 hour. Then cool to 0 ° C, add iodomethane (9.3 g, 65.7 mmol) dropwise to the reaction solution. After the dropwise addition, return to room temperature and react for 3 hours. After the reaction is completed, quench with saturated ammonium chloride aqueous solution, separate the liquids and extract the aqueous phase with ethyl acetate (300.0 mL). After combining the organic phases, wash with saturated sodium chloride aqueous solution, dry over anhydrous sodium sulfate and concentrate under reduced pressure to a crude product. Purify by column chromatography to obtain intermediate 41-1 (16.7 g, 80.8%).

[0228] Intermediates 41-2 to 41-10 were obtained by referring to the synthesis method of intermediates 38-4 to 38-12 in Example 8.

[0229] The reaction route is as follows:

[0230]

[0231] Example 11: Synthesis of the ammonium salt of compound 80 using intermediate 80-10 and intermediate 12-14 as raw materials

[0232] Using intermediates 80-10 and 12-14 as raw materials, the ammonium salt of compound 80 was obtained by referring to the synthesis method of compound 12 in the target product in Example 1. The reaction scheme is as follows:

[0233]

[0234] Among them, intermediates 80-2 to 80-10 were obtained by referring to the synthesis method of intermediates 12-2 to 12-10 in Example 1.

[0235] The reaction route is as follows:

[0236]

[0237] Example 12: Synthesis of the ammonium salt of compound 92 using intermediate 92-7 and intermediate 92-11 as raw materials

[0238] Using intermediates 92-7 and 92-11 as raw materials, the ammonium salt of compound 92 was obtained by referring to the synthesis method of compound 12 in Example 1. The reaction scheme is as follows:

[0239]

[0240] Among them, intermediates 92-2 to 92-7 were obtained by referring to the synthesis method of intermediates 80-5 to 80-10 in Example 10.

[0241] The reaction route is as follows:

[0242]

[0243] Intermediates 92-8 to 92-11 were obtained by referring to the synthesis method of intermediates 12-11 to 12-14 in Example 1.

[0244] The reaction route is as follows:

[0245]

[0246] Example 13: Synthesis of the ammonium salt of compound 94 using intermediate 92-7 and intermediate 94-4 as raw materials

[0247] Using intermediate 92-7 and intermediate 94-4 as raw materials, the ammonium salt of compound 94 was obtained by referring to the synthesis method of compound 12 in the target product in Example 1. The reaction scheme is as follows:

[0248]

[0249] Among them, intermediates 94-1 to 94-4 were obtained by referring to the synthesis method of intermediates 92-8 to 92-11 in Example 11.

[0250] The reaction route is as follows:

[0251]

[0252] Example 14: Synthesis of the ammonium salt of compound 97 using intermediate 92-7 and intermediate 97-5 as raw materials

[0253] Using intermediate 92-7 and intermediate 97-5 as raw materials, the ammonium salt of compound 97 was obtained by referring to the synthesis method of compound 12 in the target product in Example 1. The reaction scheme is as follows:

[0254]

[0255] Intermediates 97-2 to 97-5 were obtained by referring to the synthesis method of intermediates 92-8 to 92-11 in Example 11.

[0256] The reaction route is as follows:

[0257]

[0258] Example 15: Synthesis of the ammonium salt of compound 102 using intermediate 92-7 and intermediate 102-5 as raw materials

[0259] Using intermediate 92-7 and intermediate 102-5 as raw materials, the ammonium salt of compound 102 was obtained by referring to the synthesis method of compound 12 in the target product in Example 1. The reaction scheme is as follows:

[0260]

[0261] Intermediates 102-2 to 102-5 were obtained by referring to the synthesis method of intermediates 97-2 to 97-5 in Example 14.

[0262] The reaction route is as follows:

[0263]

[0264] Example 16: Synthesis of the ammonium salt of compound 103 using intermediate 102-5 and intermediate 103-7 as raw materials

[0265] Using intermediate 103-7 and intermediate 102-5 as raw materials, the ammonium salt of compound 103 was obtained by referring to the synthesis method of compound 12 in the target product in Example 1. The reaction scheme is as follows:

[0266]

[0267] Intermediates 103-2 to 103-7 were obtained by referring to the synthesis method of intermediates 92-2 to 92-7 in Example 11.

[0268] The reaction route is as follows:

[0269]

[0270] Example 17: Synthesis of the ammonium salt of compound 128 using intermediate 92-7 and intermediate 128-4 as raw materials

[0271] Using intermediate 92-7 and intermediate 128-4 as raw materials, the ammonium salt of compound 128 was obtained by referring to the synthesis method of compound 12 in the target product in Example 1. The reaction scheme is as follows:

[0272]

[0273] Among them, intermediate 128-4 is obtained by the following steps:

[0274] Weigh the intermediate 12-11 (15 g, 16.0 mmol) and dissolve it in pyridine (300.0 mL). Add iodine (6.1 g, 24.0 mmol) and triphenylphosphine (6.3 g, 24.0 mmol) to the reaction solution at room temperature. After reacting at room temperature for 2 hours, add saturated sodium thiosulfate to the reaction solution until the reaction solution turns light yellow. After concentrating under reduced pressure to remove the solvent, dilute with dichloromethane (350.0 mL). The reaction solution is washed with water (300.0 mL) and saturated sodium chloride aqueous solution (300.0 mL) and separated. The organic phase is dried over anhydrous sodium sulfate and concentrated under reduced pressure. Purification by column chromatography gives the intermediate 128-1 (13.7, 81.7%).

[0275] Dissolve triethylamine thiophosphate (5.6 g, 26.2 mmol) and DBU (4.0 g, 26.2 mmol) in DMF (150.0 mL). Stir at room temperature for 5 minutes and add intermediate 128-1 (13.7, 13.1 mmol) to the reaction solution. Continue stirring at room temperature for 1 hour and add concentrated ammonia (200.0 mL) to the reaction solution. Heat to 50 ° C and react for 6 hours. Concentrate under reduced pressure to remove the solvent and dilute with water, load onto DEAE Sephadex, elute with a linear gradient of 0-1.0 M TEAB eluent, and concentrate to obtain the triethylamine salt of intermediate 128-2 (7.1 g, 52.8%).

[0276] Intermediates 128-3 and 128-4 were obtained by referring to the synthesis method of intermediates 12-13 and 12-14 in Example 1.

[0277] The reaction route is as follows:

[0278]

[0279] Example 18: Synthesis of the ammonium salt of compound 170 using intermediate 170-15 and intermediate 12-14 as raw materials

[0280] Using intermediate 170-15 and intermediate 12-14 as raw materials, the ammonium salt of compound 170 was obtained by referring to the synthesis method of compound 12 in the target product in Example 1. The reaction scheme is as follows:

[0281]

[0282] Among them, intermediate 170-15 is obtained by the following steps:

[0283] Weigh intermediate 21-2 (50 g, 116.6 mmol), 2-bromomethylnaphthalene (28.3 g, 128.3 mmol) and tetrabutylammonium bromide (3.8 g, 11.7 mmol) and dissolve them in DMF. Cool the reaction solution to 0°C, and add sodium hydride (60%, 7.0 g, 174.9 mmol) to the reaction solution in batches. Then stir at room temperature for 4 hours. After the reaction is completed, quench the reaction with saturated aqueous ammonium chloride solution. Extract with ethyl acetate (300.0 mL*2). Combine the organic phases, dry the organic phases with anhydrous sodium sulfate, and concentrate under reduced pressure. Purify by column chromatography to obtain intermediate 170-1 (59.3 g, 89.4%).

[0284] Weigh the intermediate 170-1 (59.3 g, 104.2 mmol) and dissolve it in THF (400.0), and add tetrabutylammonium fluoride (1.0 M in THF, 156.3 mmol) to the reaction solution at room temperature. After stirring at room temperature for 2 hours, the reaction is complete. The reaction solution is washed with 1 M hydrochloric acid aqueous solution, and the aqueous phase is extracted with ethyl acetate (300.0 mL). After combining the organic phases, concentrate under reduced pressure and purify by column chromatography to obtain the intermediate 170-2 (32.3 g, 93.8%).

[0285] Intermediate 170-2 (32.3 g, 97.7 mmol) was dissolved in acetonitrile (300.0 mL). IBX (41.0 g, 146.5 mmol) was added and the temperature was raised to 90 °C and stirred for 5 hours. After the reaction was completed, the temperature was lowered to room temperature and filtered. The filter cake was washed with a small amount of acetonitrile and the filtrate was concentrated to dryness to obtain the crude product of intermediate 170-3 (31.7 g, 98.8%), which was directly used in the next step without purification.

[0286] Weigh intermediates 170-3 (31.7 g, 96.5 mmol) and 170-4 (26.8 g, 96.5 mmol) and dissolve them in THF (300.0 mL). Add the solution dropwise to samarium iodide (0.1 M in THF, 231.6 mmol) at room temperature. React at room temperature overnight. After the reaction is completed, add 0.1 N hydrochloric acid solution (300.0 mL) to the reaction solution. Extract with ethyl acetate (300.0 mL*2). Combine the organic phases, wash the organic phase with saturated sodium thiosulfate (300.0 mL) and separate the liquids. Dry the organic phase with anhydrous sodium sulfate and concentrate under reduced pressure. Purify by column chromatography to obtain intermediate 170-5 (26.1 g, 56.4%).

[0287] Intermediate 170-5 (26.1 g, 54.4 mmol) was dissolved in acetonitrile (300.0 mL). IBX (30.4 g, 108.8 mmol) was added and the temperature was raised to 90 °C and stirred for 5 hours. After the reaction was completed, the temperature was lowered to room temperature and filtered. The filter cake was washed with a small amount of acetonitrile and the filtrate was concentrated to dryness to obtain the crude product of intermediate 170-3 (26.0 g, 99.8%), which was directly used in the next step without purification.

[0288] Dissolve the intermediate 170-3 (26.0 g, 54.3 mmol) in dichloromethane (300.0 mL), cool to 0°C, and slowly drop DAST (17.5 g, 108.6 mmol) into the reaction solution. After the addition, reflux and stir overnight. After the reaction is completed, cool to room temperature, slowly drop the reaction solution into saturated sodium bicarbonate (400.0 mL) for quenching, and extract with ethyl acetate (300.0 mL*2). Combine the organic phases, dry over anhydrous sodium sulfate, and concentrate under reduced pressure. Purify by column chromatography to obtain the intermediate 170-7 (13.1 g, 48.3%).

[0289] Weigh the intermediate 170-7 (13.1 g, 26.2 mmol) and dissolve it in THF (150.0 mL). Add potassium tert-butoxide (5.9 g, 52.4 mmol) to the reaction solution and stir it at room temperature overnight. After the reaction is completed, adjust the pH to neutral with 1M hydrochloric acid aqueous solution. After concentrating under reduced pressure, dilute with ethyl acetate (300.0 mL), wash with water (300.0 mL) and saturated sodium chloride aqueous solution (300.0 mL) respectively, and dry the organic phase with anhydrous sodium sulfate. After concentrating under reduced pressure, purify by column chromatography to obtain intermediate 170-8 (10.8 g, 86.1%).

[0290] Take intermediate 170-8 (10.8 g, 22.5 mmol) and dissolve it in acetic acid (100.0 mL). Add acetic anhydride (11.5 g, 112.5 mmol) at room temperature, cool to below 10 ° C, and add concentrated sulfuric acid (1.1 mL) dropwise to the reaction solution. Stir overnight at room temperature. After the reaction solution is diluted with DCM (400.0 mL), it is washed with water, saturated sodium bicarbonate aqueous solution, and saturated brine in sequence, dried over anhydrous sodium sulfate, and the organic phase is concentrated under reduced pressure to obtain the crude intermediate 170-9, which is directly used in the next step without purification.

[0291] The crude intermediate 170-9 and isobutyryl-guanine (6.0 g, 27.0 mmol) were suspended in DCE (150.0 mL), and BSA (13.7 g, 67.5 mmol) was added to the reaction solution. After the reaction was heated to 70°C and stirred for 1 hour, the temperature was lowered to 0°C, and TMSOTf (10.0 g, 45.0 mol) was added dropwise to the reaction solution. After the addition was complete, the temperature was raised to 70°C and stirred for 3 hours. After cooling to room temperature, the reaction solution was washed with a saturated sodium bicarbonate aqueous solution, and the organic phase was concentrated under reduced pressure and purified by column chromatography to obtain intermediate 170-10 (10.1 g, 65.7%).

[0292] Intermediate 170-10 (10.1 g, 14.8 mmol) was dissolved in a mixed solvent of dichloromethane and methanol (9:1, 100.0 mL). DDQ (10.1 g, 44.4 mmol) was added to the reaction solution in batches. The reaction solution was stirred at room temperature under nitrogen for 5 hours. After the reaction was complete, the reaction solution was poured into ethyl acetate (300.0 mL), washed with saturated thiosulfate (300.0 mL*2) and saturated sodium bicarbonate (300.0 mL*2) in sequence until the water layer was no longer yellow, and then washed with saturated sodium chloride (300.0 mL). The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure, and purified by column chromatography to obtain intermediate 170-11 (5.9 g, 73.6%).

[0293] Intermediates 170-12 to 170-15 were obtained by referring to the synthesis method of intermediates 12-7 to 12-10 in Example 1.

[0294] The reaction route is as follows:

[0295]

[0296] Example 19: Synthesis of the ammonium salt of compound 173 using intermediate 173-5 and intermediate 12-14 as raw materials

[0297] Using intermediate 173-5 and intermediate 12-14 as raw materials, the ammonium salt of compound 173 was obtained by referring to the synthesis method of compound 12 in the target product in Example 1. The reaction scheme is as follows:

[0298]

[0299] Intermediates 173-1 to 173-5 were obtained by referring to the synthesis method of intermediates 103-3 to 103-7 in Example 15. The reaction scheme is as follows:

[0300]

[0301] Comparative Example 1: The ammonium salt of Comparative Example 1 was synthesized using intermediate A and intermediates 12-14 as raw materials

[0302] Using intermediate A and intermediates 12-14 as raw materials, the ammonium salt of comparative example 1 was obtained by referring to the synthesis method of compound 12 in the target product of example 1. The reaction scheme is as follows:

[0303]

[0304] Comparative Example 2: The ammonium salt of Comparative Example 2 was synthesized using intermediate 103-7 and intermediate 12-14 as raw materials

[0305] Using intermediate 103-7 and intermediate 12-14 as raw materials, the ammonium salt of comparative example 2 was obtained by referring to the synthesis method of compound 12 in the target product of example 1. The reaction scheme is as follows:

[0306]

[0307] Test example:

[0308] Test Example 1: Determination of mRNA in vitro transcription yield and capping efficiency

[0309] The capping compound of the embodiment is used to perform the IVT reaction of mRNA (in vitro synthesis reaction of mRNA). First, calculate the volume of materials required for the system, and then add the sample (IVT reaction system is shown in Table 1). Sterile enzyme-free water is added to the system, followed by 10×buffer, NTPs, and cap analogs in sequence, and then centrifuged gently after mixing. Then, nuclease inhibitors, inorganic pyrophosphatase, T7 RNA polymerase, and linearized DNA template are added. After fully mixing, centrifugation is performed gently and incubated at 37 degrees Celsius. After 2 hours, 1U of DNaseI is added, and incubation at 37 degrees Celsius is continued for 30 minutes to remove the DNA template, and then the RNA is purified using a magnetic bead purification method. The purified mRNA is dissolved with sterile enzyme-free water, and then quantitatively detected using NanodropOne.

[0310] Table 1 IVT reaction system

[0311]

[0312]

[0313] Liquid chromatography mass spectrometry (LC-MS) was used to detect the IVT capping rate of mRNA with different starting cap analogs; first, a labeled DNA probe matching the starting base of the transcribed mRNA was designed, usually with a biotin label. After the streptavidin-labeled magnetic beads were washed, they were incubated with the synthetic DNA probe, mRNA and 10×RNaseH reaction buffer at room temperature for 30 minutes, slowly mixed during incubation, and then 20μlRNaseH (5U / μL) was added and incubated at 37 degrees for 3h, mixing every half an hour. After the incubation, the magnetic beads were washed, and 100μL of 75% methanol heated to 80 degrees Celsius was added to the washed magnetic beads. The mixture was heated to 80 degrees Celsius on a heating plate for 3 minutes, and then placed on a magnetic rack to absorb the supernatant, and dried to 10μl at room temperature for 45 minutes using an evaporating centrifuge. The sample was then resuspended in 50 μl of 100 μM EDTA / 1% MeOH and used for LC-MS analysis to determine the capping of RNA in the transcription reaction. Since capped and non-capped bases have obvious differences in molecular weight, the difference in molecular mass can be used to determine the capping rate of mRNA transcription initiated by different cap analogs. The specific results are shown in Table 2.

[0314] As can be seen from Table 2, when the capping compound of the present application is used to perform an IVT reaction of mRNA, the mRNA in vitro transcription yield and capping efficiency are better than those of Comparative Example 1 and Comparative Example 2, or are equivalent to those of Comparative Example 1 and Comparative Example 2.

[0315] Table 2 mRNA yield and capping efficiency

[0316]

[0317]

[0318]

[0319]

[0320]

[0321] Test Example 2: mRNA translation efficiency

[0322] The eGFP coding sequence was used as a DNA template and the cap analogs of the example were used as the starting point for in vitro transcription. The different mRNA products obtained were then used to transfect 293T cells. 293T cells were cultured at (0.5-1)×10 5Cells were plated (24-well plate). During transfection, the cell density is generally 60-80%, and 2 μg of mRNA is transfected per well. The transfection reagent is Lipofectamine MessengerMAX Transfection Reagent (Invitrogen) and the operation is performed with reference to its method of use. The transfected cells were placed in a 37-degree Celsius, CO2 incubator, and replaced with fresh complete medium 4-6 hours after transfection. After incubation in a 37-degree Celsius CO2 incubator for 72 hours, the fluorescence intensity of GFP was observed under a fluorescence microscope, and the fluorescence intensity ratio of the embodiment to the comparative example 1 was calculated based on the fluorescence intensity.

[0323] As shown in Table 3, when the cap analog of the present application is used as the starting point for in vitro transcription, after the transfected cells are incubated for a long time (72 h), the translation efficiency of the mRNA is higher than that of Comparative Example 1 and Comparative Example 2, respectively.

[0324] Table 3 mRNA translation efficiency

[0325]

[0326]

[0327]

[0328]

[0329]

[0330] Test Example 3: Study on the effect of decapping enzyme on the stability of cap structure

[0331] mRNA (200 μg) modified with different cap analogs was mixed with 1 μl mRNA decapping enzyme (New England Biolabs) and 1×MDE buffer and then subjected to enzymatic reaction at 37°C for 45 min. The enzymatic reaction was subjected to PAGE electrophoresis and SYBR Green II (Lonza) staining, and the gel image after electrophoresis was observed on a Typhoon FLA7000 (GE Healthcare) instrument. The ratio of the electrophoretic band intensity of capped RNA and decapped RNA was calculated using ImageQuant (GE Healthcare) software, and the decapping rate after treatment with decapping enzyme (DCP2 enzyme) was calculated. Statistical test was performed using the Dunnett test of KaleidaGraph (Synergy) software.

[0332] From the data in Table 4, it can be seen that after being treated with the decapping enzyme (DCP2 enzyme), the decapping rate of the capped compound of the present application is significantly lower than that of Comparative Example 1 and Comparative Example 2.

[0333] Table 4 Decapping rate after decapping enzyme treatment

[0334]

[0335]

[0336]

[0337]

[0338] The above contents are further detailed descriptions of the present invention in combination with specific preferred embodiments, and it cannot be determined that the specific implementation of the present invention is limited to these descriptions. For ordinary technicians in the technical field to which the present invention belongs, several simple deductions or substitutions can be made without departing from the concept of the present invention, which should be regarded as falling within the protection scope of the present invention.

Claims

1. A compound of formula (I), or a pharmaceutically acceptable salt, stereoisomer, tautomer or isotopic variant thereof: in, X2 and X3 are independently selected from O, S, NH, CH2 or CH; X1, X4 and X5 are independently selected from CH=CH, CH2CH2, CH2O, OCH2, CH2CH2O, OCH2CH2, CH2OCH2, CH2S or SCH2; and at least one, two or three of X1, X4 and X5 are CH=CH; Y1, Y2, Y3 and Y4 are independently selected from O or S; R1, R2, R3, R4, R5 and R6 are independently selected from H, OH, halogen, C 1-6 Alkyl, C 1-6 Alkoxy, bridged ring, C 1-6 Haloalkyl, C 1-6 Halogenated alkoxy, C 2-6 Alkenyl, C 2-6 Alkynyl, C 3-10 Cycloalkyl, amino, azido, cyano, C 1-6 Alkylcarbonylamino, (C 1-6 alkyl)2-aminocarbonyl, C 1-6 Alkylcarbonyl, methylsulfonamide, phenylsulfonamide, 3-8 membered heterocyclic group, C 6-10 Aryl or 5-10 membered heteroaryl, wherein the C 1-6 Alkyl, C 1-6 Alkoxy, C 1-6 Haloalkyl, C 1-6 Halogenated alkoxy, C 2-6 Alkenyl, C 2-6 Alkynyl, C 3-10 Cycloalkyl, amino, C 1-6 Alkylcarbonylamino, 3-8 membered heterocyclic group, C 6-10 Aryl and 5-10 membered heteroaryl are unsubstituted or optionally substituted with 1, 2, 3 or more OH, halogen, azido, cyano, acetyl, C 1-6 Alkyl, C 1-6 Haloalkyl, C 1-6 Halogenated alkoxy, C 2-6 Alkenyl, C 2-6 Alkynyl, C 1-6 Alkoxy, C 1-6 Haloalkoxy, aminocarbonyl C 1-6 Alkoxy, (C 1-6 Alkyl)2AminoC 1-6 Alkoxy, C 2-6 Alkenyl C 1-6 Alkoxy, C 2-6 Alkynyl C 1-6 Alkoxy, phenyl C 1-6 Alkoxy, C 1-6 Halogenated alkylthiol, (C 1-6 alkyl)2-aminocarbonyl, C 1-6 Alkyl amino ester group, C 1-6 Alkylcarbonyl, aminocarbonyl, 3-8 membered heterocyclic group, C 1-6 Alkylcarbonylamino, aldehydeamino or phenylcarbonyl substitution; R7 is selected from H, OH, halogen, C 1-6 Alkyl, C 1-6 Alkoxy, bridged ring, C 1-6 Haloalkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 3-10 Cycloalkyl, 3-8 membered heterocyclic group, C 6-10 Aryl or 5-10 membered heteroaryl, wherein the C 1-6 Alkyl, C 1-6 Alkoxy, C 1-6 Haloalkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 3-10 Cycloalkyl, 3-8 membered heterocyclic group, C 6-10 Aryl and 5-10 membered heteroaryl are unsubstituted or optionally substituted with 1, 2, 3 or more halogen, C 1-6 Alkyl, C 1-6 Haloalkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 1-6 Alkoxy or C 1-6 Haloalkoxy substitution; R8 and R9 are independently selected from OH, halogen or methoxy; B1, B2 and B3 are independently selected from natural, modified or non-natural nucleobases.

2. The compound of formula (I) according to claim 1, or a pharmaceutically acceptable salt, stereoisomer, tautomer or isotopic variant thereof, wherein: X1 is selected from CH=CH, CH2CH2, CH2O, OCH2, CH2CH2O, OCH2CH2, CH2OCH2, CH2S or SCH2, preferably CH=CH.

3. The compound of formula (I) according to claim 1, or a pharmaceutically acceptable salt, stereoisomer, tautomer or isotopic variant thereof, wherein: X4 is selected from CH=CH, CH2CH2, CH2O, OCH2, CH2CH2O, OCH2CH2, CH2OCH2, CH2S or SCH2, preferably CH=CH.

4. The compound of formula (I) according to claim 1, or a pharmaceutically acceptable salt, stereoisomer, tautomer or isotopic variant thereof, wherein: X5 is selected from CH=CH, CH2CH2, CH2O, OCH2, CH2CH2O, OCH2CH2, CH2OCH2, CH2S or SCH2, preferably CH=CH.

5. The compound of formula (I) according to claim 1, or a pharmaceutically acceptable salt, stereoisomer, tautomer or isotopic variant thereof, wherein: R1 and R2 are independently selected from H, OH or F.

6. The compound of formula (I) according to claim 1, or a pharmaceutically acceptable salt, stereoisomer, tautomer or isotopic variant thereof, wherein: R3 and R4 are independently selected from H, OH, F, C 1-6 Alkyl, C 1-6 Alkoxy, bridged ring or C 1-6 A haloalkyl group, wherein C 1-6 Alkyl, C 1-6 Alkoxy or C 1-6 The haloalkyl group is unsubstituted or optionally substituted with 1, 2, 3 or more OH, F, C 1-6 Alkyl or C 1-6 Alkoxy substitution.

7. The compound of formula (I) according to claim 1, or a pharmaceutically acceptable salt, stereoisomer, tautomer or isotopic variant thereof, wherein: R5 and R6 are independently selected from H, OH, F, C 1-6 Alkyl, C 1-6 Alkoxy, bridged ring or C 1-6 A haloalkyl group, wherein C 1-6 Alkyl is unsubstituted or optionally substituted with 1, 2, 3 or more Fs.

8. The compound of formula (I) according to claim 1, or a pharmaceutically acceptable salt, stereoisomer, tautomer or isotopic variant thereof, wherein: R7 is selected from OH, F or methoxy.

9. The compound of formula (I) according to claim 1, or a pharmaceutically acceptable salt, stereoisomer, tautomer or isotopic variant thereof, wherein: R7 is selected from 10. A compound of formula (I) according to any one of claims 1 to 9, or a pharmaceutically acceptable salt, stereoisomer, tautomer or isotopic variant thereof, wherein: R8 and R9 are OH.

11. A compound of formula (I) according to any one of claims 1 to 10, or a pharmaceutically acceptable salt, stereoisomer, tautomer or isotopic variant thereof, having the following general formula: wherein each group is as defined in any one of claims 1 to 10.

12. A compound of formula (I) according to any one of claims 1 to 11, or a pharmaceutically acceptable salt, stereoisomer, tautomer or isotopic variant thereof, wherein the compound is selected from:

13. A compound of formula (V), or a pharmaceutically acceptable salt, stereoisomer, tautomer or isotopic variant thereof: in, B4 and B5 are independently selected from natural, modified or unnatural nucleobases; Z1 and Z2 are independently selected from H, halogen or C 1-6 Alkoxy, and at least one or both of Z1 and Z2 are halogen or C 1-6 Alkoxy; R a , R b , R c and R d are independently selected from H, OH, halogen, C 1-6 Alkyl, C 1-6 Alkoxy, bridged ring, C 1-6 Haloalkyl, azido, where C 1-6 Alkyl, C 1-6 Alkoxy, C 1-6 The haloalkyl group is unsubstituted or optionally substituted with 1, 2, 3 or more halogen, OH, C 1-6 Alkyl, C 1-6 Alkoxy, phenyl C 1-6 Alkoxy, C 1-6 Alkylcarbonylamino, azido substitution.

14. The compound of formula (V) according to claim 13, or a pharmaceutically acceptable salt, stereoisomer, tautomer or isotopic variant thereof, wherein: Z1 is selected from H, halogen or C 1-6 Alkoxy, preferably halogen; more preferably, Z1 is F.

15. The compound of formula (V) according to claim 13, or a pharmaceutically acceptable salt, stereoisomer, tautomer or isotopic variant thereof, wherein: Z2 is selected from H, halogen or C 1-6 Alkoxy, preferably halogen; more preferably, Z2 is F.

16. The compound of formula (V) according to claim 13, or a pharmaceutically acceptable salt, stereoisomer, tautomer or isotopic variant thereof, wherein: R a Selected from OH, F or methoxy; Preferably, R a Selected from OH.

17. The compound of formula (V) according to claim 13, or a pharmaceutically acceptable salt, stereoisomer, tautomer or isotopic variant thereof, wherein: R b Selected from OH, F, C 1-6 Alkoxy; Preferably, R b Selected from OH or methoxy.

18. The compound of formula (V) according to claim 13, or a pharmaceutically acceptable salt, stereoisomer, tautomer or isotopic variant thereof, wherein: R c Selected from OH, F, C 1-6 The alkoxy group is preferably a methoxy group.

19. The compound of formula (V) according to claim 13, or a pharmaceutically acceptable salt, stereoisomer, tautomer or isotopic variant thereof, wherein: R d Selected from OH, F, C 1-6 Alkoxy group, preferably OH.

20. A compound of formula (V) according to any one of claims 13 to 19, or a pharmaceutically acceptable salt, stereoisomer, tautomer or isotopic variant thereof, wherein the compound is selected from:

21. Use of a compound as an in vitro co-transcribed RNA capping agent, wherein the compound is a compound of formula (I) according to any one of claims 1 to 12 or a compound of formula (V) according to any one of claims 13 to 20, or a pharmaceutically acceptable salt, stereoisomer, tautomer or isotopic variant thereof.

22. The use according to claim 21, which is used for mRNA capping under an in vitro T7 RNA polymerase system.

23. A method for synthesizing RNA, comprising incubating a compound of formula (I) according to any one of claims 1 to 12 or a compound of formula (V) according to any one of claims 13 to 20, or a pharmaceutically acceptable salt, stereoisomer, tautomer or isotopic variant thereof with a nucleotide template to perform template transcription.

24. A complex comprising a compound of formula (I) according to any one of claims 1 to 12, or a pharmaceutically acceptable salt, stereoisomer, tautomer or isotopic variant thereof and a DNA template, wherein the DNA template includes a promoter region containing a transcription start site having a first nucleotide at nucleotide position +1 and a second nucleotide at nucleotide position +2; and B1 is complementary to the nucleobase at transcription template position +1 on the DNA template, and B2 is complementary to the nucleobase at transcription template position +2 on the DNA template.

25. A complex comprising a compound of formula (V) according to any one of claims 13 to 20, or a pharmaceutically acceptable salt, stereoisomer, tautomer or isotopic variant thereof and a DNA template, wherein the DNA template includes a promoter region containing a transcription start site having a first nucleotide at nucleotide position +1 and a second nucleotide at nucleotide position +2; and B4 is complementary to the nucleobase at transcription template position +1 on the DNA template, and B5 is complementary to the nucleobase at transcription template position +2 on the DNA template.

Images