Capping compound, and application thereof in mRNA capping

Abstract

The present invention provides a capping compound, and an application thereof in mRNA capping. The capping compound is a compound which conforms to the structure shown in the following formula, or a stereoisomer, pharmaceutically acceptable salt or solvate thereof. Since a 3 to 6 membered cycloalkyl group or a 3 to 6 membered heterocyclic group is provided, the capping compound of the present invention can improve mRNA stability, is not easily hydrolyzed by a decapping enzyme, and can improve an mRNA translation effect. (I)

Description

A capping compound and its application in mRNA capping

[0001] This application claims priority to the Chinese patent application filed with the China Patent Office on September 26, 2023, with application number 202311243069.2 and invention name “A capping compound and its application in mRNA capping”, the entire contents of which are incorporated by reference into this application. Technical Field

[0002] The present invention relates to the field of biomedicine technology, and in particular to a capping compound and its application in mRNA capping. Background Art

[0003] One of the main technical problems that mRNA needs to solve is how to improve mRNA stability and translation efficiency. The 5'UTR and 3'UTR located on both sides of the protein coding sequence directly affect the translation efficiency and stability of mRNA and determine the effectiveness of mRNA vaccines or drugs. Generally, the stability of mRNA vaccines or drugs is enhanced by adding a 5' cap structure (Cap) and a polyadenylic acid Poly (A) tail structure, so that it can maintain full biological activity for a longer period of time under different conditions.

[0004] The 5' cap structures currently on the market generally have disadvantages such as low capping efficiency and low mRNA translation efficiency. This application has developed a cyclic modified 5' cap structure that can not only improve the capping efficiency and mRNA translation efficiency at the cellular level, but also improve the mRNA translation efficiency in mice.

[0005] Summary of the Invention

[0006] In order to solve the problems of low capping efficiency of existing capping analogs and low mRNA translation efficiency, the present application provides a capping compound and its application in mRNA capping.

[0007] The present invention provides a capping compound, which is a compound having a structure according to formula (I), or a stereoisomer, a pharmaceutically acceptable salt, or a solvate thereof.

[0008] in,

[0009] Indicates a single key or none;

[0010] represents a single bond or a double bond;

[0011] R1 and R2 are independently H, OH, halogen, alkyl, alkoxy, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, heterocyclyl, heteroaryl, alkylthio, or alkoxy forming a bridged ring with the carbon atom at 4'; wherein the alkyl, alkoxy, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, heterocyclyl, heteroaryl, and alkylthio are unsubstituted or substituted with one or more R5, and R5 is any one of alkyl, alkoxy, substituted or unsubstituted acetamido;

[0012] R3 is OH, halogen, alkoxy, or an alkoxy group forms a bridged ring with the carbon on 4'; wherein the alkoxy group is unsubstituted or substituted with one or more R6, and R6 is any one of an alkyl group and an alkoxy group;

[0013] R4 is OH, alkyl, or alkoxy; wherein the alkyl or alkoxy group is unsubstituted or substituted by one or more R7, and R7 is any one of alkyl or alkoxy;

[0014] Ra, Rb and Rc are each independently a 3- to 6-membered cycloalkyl group, a 3- to 6-membered heterocyclyl group, CH2, CH or O, wherein Ra and X1 are not O at the same time, Rb and X4 are not O at the same time, and Rc and X5 are not O at the same time;

[0015] and at least one of Ra, Rb and Rc is a 3- to 6-membered cycloalkyl group or a 3- to 6-membered heterocyclic group;

[0016] X1, X2, X3, X4 and X5 are independently selected from O, S, CH2, CH, NH or none;

[0017] Y 1a 、Y 2a 、Y 3a and Y 4a are independently OH, SH or BH3;

[0018] Y 1b 、Y 2b 、Y 3b and Y 4b are independently O or S;

[0019] m is 0, 1, 2, or 3;

[0020] B1 and B2 are each independently a natural or modified nucleobase.

[0021] The pharmaceutically acceptable salt may be a sodium salt, potassium salt, ammonium salt, organic amine salt or the like, which are well known to those skilled in the art.

[0022] The capped analogues of the above general structural formula can improve the stability of mRNA, are not easily hydrolyzed by decapping enzymes, and can ultimately improve the translation effect of mRNA.

[0023] In an alternative embodiment, one, two or three of Ra, Rb and Rc are 3 to 6 membered cycloalkyl or 3 to 6 membered heterocyclyl.

[0024] In a preferred embodiment, any one or two of Ra, Rb and Rc are 3- to 6-membered cycloalkyl or 3- to 6-membered heterocyclic groups.

[0025] Furthermore, any one or two of Ra, Rb and Rc are three-membered or four-membered cycloalkyl groups, the structure of which is selected from: And there is no adjacent X1, X4 or X5.

[0026] Preferably, the capping compound of the present invention has a structure as shown in formula (II), or a stereoisomer, a pharmaceutically acceptable salt, or a solvate thereof.

[0027] Among them, Ra is selected from It has excellent performance in capping efficiency and mRNA translation efficiency.

[0028] More preferably: R3 is H, OH, C 1-6 Alkoxy or LNA, where C 1-6 The alkoxy group is unsubstituted or substituted by one or more R6, R6 is C 1-6 Alkyl or C 1-6 Alkoxy.

[0029] More preferably, R3 is H, OH or C 1-4 Alkoxy, R4 is H, OH or C 1-4 Alkoxy.

[0030] In addition, R1 and R2 are each independently H, OH, halogen, C 1-6 Alkyl, C 1-6 Alkoxy or LNA; wherein C 1-6 Alkyl, C 1-6 Alkoxy is unsubstituted or substituted by one or more R5, R5 is C 1-6 Alkyl, C 1-6 Any of an alkoxy group and a substituted or unsubstituted acetamido group.

[0031] As the above preferred compound, its stereoisomer, pharmaceutically acceptable salt, or solvate thereof, R1 and R2 are independently OH or C 1-4 Alkoxy; wherein C 1-4 The alkoxy group is unsubstituted or substituted by one or two R5, R5 is C 1-4 Alkyl, C 1-4 Alkoxy.

[0032] Structurally, it is further preferred that the capping compound of the present invention has a structure as shown in formula (III), or a stereoisomer, a pharmaceutically acceptable salt, or a solvate thereof.

[0033] More preferably, R3 is selected from H, OH, -O-methyl, -O-ethyl, -O-propyl, -O-isopropyl, -O-methylene-O-methyl, -O-ethylene-O-methyl, -O-methylene-O-ethyl, -O-ethylene-O-ethyl, and LNA.

[0034] More preferably, R4 is hydroxy or methoxy.

[0035] Further preferably, R1 and R2 are independently selected from H, OH, F, Cl, -methylene-acetamido, methyl, ethyl, n-propyl, isopropyl, -O-methyl, -O-ethyl, -O-n-propyl, -O-isopropyl, -methylene-O-methyl, -ethylidene-O-methyl, -methylene-O-ethyl, -ethylidene-O-ethyl, -O-methylene-O-methyl, -O-ethylidene-O-methyl, -O-methylene-O-ethyl, -O-ethylidene-O-ethyl, -methylene-acetamido, -ethyl-acetamido, -n-propyl-acetamido, -isopropyl-acetamido, -n-butyl-acetamido, -isobutyl-acetamido.

[0036] More preferably, B1 and B2 are each independently adenine (A), guanine (G), cytosine (C), uracil (U) or thymine (T).

[0037] Alternatively, any one or two of Ra, Rb, and Rc may be a three-membered or four-membered cycloalkyl group, the structure of which is selected from: And the adjacent X1, X4 or X5 is O.

[0038] Preferably, the capping compound of the present invention has a structure as shown in formula (IV), or a stereoisomer, a pharmaceutically acceptable salt, or a solvate thereof.

[0039] R1 and R2 are independently OH or C 1-4 Alkoxy; wherein C 1-4 Alkoxy is unsubstituted or substituted by one or more R5, R5 is C 1-4 Alkyl, C 1-4 alkoxy;

[0040] R3 is C 1-6 Alkoxy, where C 1-6 Alkoxy is unsubstituted or substituted by one or more R6, R6 is C 1-6 Alkyl or C 1-6 alkoxy;

[0041] R4 is H, OH or C 1-4 Alkoxy.

[0042] More preferably, R3 is selected from H, OH, -O-methyl, -O-ethyl, -O-n-propyl, -O-isopropyl, -O-methylene-O-methyl, -O-ethylene-O-methyl, -O-methylene-O-ethyl, -O-ethylene-O-ethyl, and LNA.

[0043] More preferably, R4 is hydroxy or methoxy.

[0044] Further preferably, R1 and R2 are independently selected from H, OH, F, Cl, -methylene-acetamido, methyl, ethyl, n-propyl, isopropyl, -O-methyl, -O-ethyl, -O-n-propyl, -O-isopropyl, -methylene-O-methyl, -ethylidene-O-methyl, -methylene-O-ethyl, -ethylidene-O-ethyl, -O-methylene-O-methyl, -O-ethylidene-O-methyl, -O-methylene-O-ethyl, -O-ethylidene-O-ethyl, -methylene-acetamido, -ethyl-acetamido, -n-propyl-acetamido, -isopropyl-acetamido, -n-butyl-acetamido, -isobutyl-acetamido.

[0045] More preferably, B1 and B2 are each independently adenine (A), guanine (G), cytosine (C), uracil (U) or thymine (T).

[0046] The capping compound of the present invention can be specifically selected from any of the following compounds, stereoisomers, or pharmaceutically acceptable salts or solvates:

[0047] Alternatively, the following compounds can also be used as the capping compound for mRNA capping.

[0048] The capping compound of the present invention, because it has a three-membered to six-membered cycloalkyl or heterocyclic group, can improve the stability of mRNA, is not easily hydrolyzed by decapping enzymes, and ultimately can improve the translation effect of mRNA.

[0049] The capping compound is used in the preparation of an in vitro co-transcribed RNA capping reagent. The capping analog structural fragment exists in mRNA and plays a role, and is used to prepare a pharmaceutical composition.

[0050] The present invention also includes a complex comprising the capping compound of the present invention, wherein the DNA template includes a promoter region comprising 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 position +1 of the DNA template, and B2 is complementary to the nucleobase at position +2 of the transcription template on the DNA template.

[0051] The present invention also includes RNA molecules having the capping compounds described herein.

[0052] The present invention also includes a pharmaceutical composition comprising the aforementioned RNA molecule and a pharmaceutically acceptable carrier. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] Figure 1: In vivo translation efficiency of mRNA of compounds 1, 2, 31, and 32 in mice.

[0054] Figure 2: Graph showing the in vivo translation efficiency of mRNA in mice of Comparative Example 1. DETAILED DESCRIPTION

[0055] The definitions of specific functional groups and chemical terms are as follows:

[0056] The term "alkyl" refers to a saturated aliphatic hydrocarbon group, which is a straight or branched chain group containing 1 to 20 carbon atoms, preferably an alkyl group containing 1 to 6 (e.g., 1, 2, 3, 4, 5 and 6) carbon atoms, and more preferably an alkyl group containing 1 to 4 carbon atoms.

[0057] The term "C 1-6 "Alkyl" refers to a straight or branched chain saturated hydrocarbon group having 1 to 6 carbon atoms. 1-6 Examples of alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, sec-butyl, isobutyl, n-pentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2-methylpropyl, 1,1,2-trimethylpropyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 2,2-dimethylbutyl, 1,3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2,3-dimethylbutyl, and the like.

[0058] The term "C 1-4 "Alkyl" refers to a straight or branched chain saturated hydrocarbon group having 1 to 4 carbon atoms. 1-4 Examples of the alkyl group include methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, sec-butyl, isobutyl and the like.

[0059] The term "C1-6 "Alkoxy" refers to a -OR group, wherein R is as described above in "C 1-6 "alkyl" is defined.

[0060] The term "C 1-4 "Alkoxy" refers to a -OR group, wherein R is as described above in "C 1-4 "alkyl" is defined.

[0061] "Halo" or "halogen" refers to fluorine (F), chlorine (Cl), bromine (Br) and iodine (I).

[0062] The term "cycloalkyl" includes any stable monocyclic or bicyclic ring having a specified number of carbon atoms, wherein any carbon atom may be saturated or unsaturated. For example, a 3- to 6-membered cycloalkyl is intended to include monocyclic or bicyclic rings having 3, 4, 5, or 6 carbon atoms. Examples of 3- to 6-membered cycloalkyls include, but are not limited to, cyclopropyl, cyclobutyl, cyclobutenyl, cyclopentyl, cyclopentenyl, and cyclohexyl.

[0063] The term "3- to 6-membered heterocyclyl" refers to a saturated or unsaturated monocyclic ring containing 3-6 ring atoms, wherein at least one ring atom is selected from nitrogen, sulfur and oxygen atoms.

[0064] "LNA", "locked nucleic acid unit", "locked nucleoside" or "locked nucleic acid" may be used interchangeably to include, but not limited to, a methylene bridge between the 2'O and 4'C of a nucleomonomer, or to refer to a sugar analog, a nucleoside, a nucleomonomer or a nucleic acid, each containing such a bridge, e.g. wait.

[0065] "Substituted" means that one or more hydrogen atoms, preferably up to 5, more preferably 1 to 3 hydrogen atoms in the group are replaced independently of one another by a corresponding number of substituents. It goes without saying that the substituents are only in their possible chemical positions, and a person skilled in the art can determine (by experiment or theory) without undue effort which substitutions are possible or impossible.

[0066] In this application, "base" and "nucleobase" are used interchangeably and may refer to either natural nucleobases or modified nucleobases. Natural nucleobases include, but are not limited to, adenine (A), guanine (G), cytosine (C), uracil (U), thymine (T), and any of their derivatives.

[0067] "Modified base" and "modified nucleoside base" can be used interchangeably and refer to substances obtained by replacing one or more hydrogen atoms of a natural nucleoside base, such as but not limited to N6-methyladenine. In this embodiment, the modifying group is selected from 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, N1-methylguanine, and isoguanine.

[0068] "Stereoisomers" are compounds that have the same chemical structure but differ in the way the atoms or groups are arranged in space. Stereoisomers include enantiomers, diastereomers, conformers (rotamers), geometric isomers (cis / trans), and atropisomers.

[0069] Example 1: Synthesis of the ammonium salt of compound 1 using intermediates A and B as raw materials

[0070] Intermediate A (15.0 mmol) and intermediate B (12.0 mmol) were suspended in DMF (90.0 mL), and ZnCl2 (120.0 mmol) was added to the reaction solution under ice bath. After stirring at room temperature for 24 hours, the reaction was terminated with 0.25 M EDTA-2Na (144.0 mmol) solution. The mixture was loaded onto a DEAE Sephadex column. The product was eluted using a linear gradient of 0-1.0 M aqueous ammonium bicarbonate solution. The fractions with a purity greater than 98% were collected, desalted by nanofiltration, and concentrated to obtain the ammonium salt of compound 1. The reaction process is as follows:

[0071] Wherein, compound A is obtained through the following steps.

[0072] Intermediate A1 (0.86 mol) was dissolved in DMF (5.0 L). DMSO (5.1 mol) and EDCI (2.6 mol) were added to the reaction mixture, followed by the dropwise addition of pyridine (0.86 mol) and trifluoroacetic acid (0.86 mol). After 5 hours of reaction at room temperature, the reaction mixture was diluted with ethyl acetate. The organic phase was washed with saturated sodium bicarbonate and water, then concentrated and purified by column chromatography to yield Intermediate A2.

[0073] Under nitrogen atmosphere, tetraethylmethylene diphosphate (0.78 mol) was added dropwise to a suspension of NaH (60%, 1.03 mol) in THF (1.0 L) under ice-bath, followed by stirring at 0°C for 0.5 hour. A solution of intermediate A2 (0.52 mol) in THF (2.0 L) was slowly added dropwise to the reaction solution. Stir at room temperature overnight. The reaction was quenched with saturated aqueous ammonium chloride under ice-bath and extracted three times with ethyl acetate (480.0 mL). The combined organic phases were washed twice with saturated sodium chloride (300.0 mL each time), concentrated under reduced pressure, and purified by column chromatography to obtain intermediate A3.

[0074] Under a nitrogen atmosphere, trimethylsulfoxide iodide (1.06 mol) was added to a suspension of NaH (60%, 1.88 mol) in DMSO (1.2 L) under ice-bath, followed by stirring at 0°C for 1 hour. A solution of intermediate A3 (0.47 mol) in DMSO (2.0 L) was slowly added dropwise to the reaction solution. Stir at room temperature for 6 hours. The reaction was quenched with saturated aqueous ammonium chloride under ice-bath and extracted three times with ethyl acetate (450.0 mL). The combined organic phases were washed twice with saturated sodium chloride, 300.0 mL each time, and the organic phases were concentrated under reduced pressure and purified by column chromatography to obtain intermediate A4.

[0075] Intermediate A4 (0.21 mol) was dissolved in 1.5 L of acetonitrile, and triethylsilyl bromide (2.1 mol) was added to the reaction solution. The mixture was stirred at room temperature overnight and concentrated to obtain a crude intermediate A5, which was used directly in the next reaction without purification.

[0076] The crude intermediate A5 was dissolved in THF (1.5 L) and TBAF (0.6 mol) was added at room temperature. The mixture was stirred at room temperature for 3 hours and concentrated under reduced pressure to obtain the crude intermediate A6, which was used directly in the next reaction without purification.

[0077] The crude intermediate A6 was dissolved in methanol (300.0 mL), and concentrated aqueous ammonia (1.3 L) was added to the reaction solution, which was stirred at room temperature overnight. After the reaction was completed, the solution was concentrated under reduced pressure and purified by reverse phase chromatography to obtain intermediate A7.

[0078] Intermediate A7 (0.1 mol), triphenylphosphine (0.2 mol), 2,2'-disulfide dipyridine (0.2 mol), imidazole (0.8 mol) and triethylamine (0.1 mol) were dissolved in DMF (380.0 mL) and stirred under nitrogen atmosphere for 10 hours. After the reaction, 4 M sodium perchlorate (0.4 mol) was added to acetone solution to precipitate the product. The filter cake was thoroughly washed with acetone to obtain intermediate A8.

[0079] Intermediate A8 (90.0 mmol) was dissolved in DMF (370.0 mL). Tributylamine phosphate (0.27 mol) and zinc chloride (0.72 mol) were added, and the mixture was stirred at room temperature for 5 hours. After the reaction, the mixture was diluted with water, loaded onto DEAE Sephadex, and eluted with a linear gradient of 0-1.0 M TEAB. The mixture was concentrated to yield Intermediate A9.

[0080] Intermediate A9 (70.0 mmol) was dissolved in purified water (560.0 mL). The reaction solution was cooled to 4°C and dimethyl sulfate (0.4 mol) was slowly added dropwise. During the process, the pH was adjusted to no more than 5 with 2 M sodium hydroxide. The reaction was monitored by HPLC. After completion of the reaction, the product was loaded onto DEAE Sephadex and eluted with a linear gradient of 0-1.0 M TEAB. The product was concentrated to obtain intermediate A10.

[0081] Intermediate A10 (53.0 mmol), triphenylphosphine (106.0 mmol), 2,2'-disulfide dipyridine (106.0 mmol), imidazole (4.2 mol) and triethylamine (53.0 mmol) were dissolved in DMF (350.0 mL) and stirred under nitrogen atmosphere for 10 hours. After the reaction, 4 M sodium perchlorate (0.16 mol) in acetone solution was added to precipitate the product. The filter cake was thoroughly washed with acetone to obtain intermediate A.

[0082] Wherein, compound B is obtained by the following steps:

[0083] 2'OMe-rA phosphoramidite monomer (0.23 mol) and N2-isobutyryl-2',3'acetylguanosine (0.23 mol) were weighed and dissolved in DCM (2.0 L). Tetrazole (0.5 mol) was added under a nitrogen atmosphere. Stir at room temperature for 2 hours. After the reaction, tert-butyl hydroperoxide (0.69 mol, 70%) was added dropwise to the reaction mixture, and the reaction continued at room temperature for 1 hour. Trichloroacetic acid (0.69 mol) in DCM (0.4 L) was added dropwise to the reaction mixture, and the reaction continued at room temperature for 2 hours. The reaction mixture was washed with 10% aqueous sodium sulfite, 10% aqueous sodium bicarbonate, and saturated brine, respectively. The organic phase was concentrated under reduced pressure and purified by column chromatography to obtain Intermediate B1.

[0084] Take intermediate B1 (0.18 mol) and bis(2-cyanoethyl)-N,N-diisopropylphosphoramidite (0.25 mol) and dissolve them in DCM (1.7 L). Tetrazole (0.39 mol) was added under a nitrogen atmosphere. Stir at room temperature for 2 hours. After the reaction, tert-butyl hydroperoxide aqueous solution (0.51 mol, 70%) was added dropwise to the reaction solution, and the reaction was continued at room temperature for 1 hour. The reaction solution was washed with 10% sodium sulfite aqueous solution, 10% sodium bicarbonate aqueous solution, and saturated brine, respectively. The organic phase was concentrated under reduced pressure and dissolved in methanol (0.5 L) and ammonia water (1.0 L), and stirred at room temperature overnight. After concentration to remove the solvent, it was diluted with water, loaded onto DEAE Sephadex, and eluted with a linear gradient of 0-1.0 M TEAB eluent, and concentrated to obtain intermediate B. Reaction route flow:

[0085] Example 2: Synthesis of the ammonium salt of compound 2 using intermediates B and C as raw materials

[0086] Using intermediates B and C as raw materials, the ammonium salt of compound 2 was obtained by referring to the synthesis method of compound 1 in Example 1. Reaction route:

[0087] Compound C is obtained by the following steps: Intermediate C2-C is obtained by following the synthesis method of intermediate A2-A in reference example 1. Reaction route:

[0088] Example 3: Synthesis of the ammonium salt of compound 5 using intermediates B and D as raw materials

[0089] Using intermediates B and D as raw materials, the ammonium salt of compound 5 was obtained by referring to the synthesis method of compound 1 in Example 1. Reaction route:

[0090] Wherein, compound D is obtained by the following steps:

[0091] Compound D1 (0.18 mol) and imidazole (0.36 mol) were dissolved in DMF (3.5 L). Tert-butyldiphenylsilyl chloride (0.2 mol) was added to the reaction mixture under an ice bath. The mixture was stirred at room temperature for 5 hours. The reaction mixture was poured into ice water and extracted twice with 500.0 mL of ethyl acetate each time. The organic phase was washed with saturated sodium chloride, dried, and concentrated under reduced pressure to yield intermediate D2, which was used directly in the next reaction without purification.

[0092] Acetic anhydride (0.45 mol) and pyridine (0.8 mol) were slowly added to a suspension of CrO3 (0.45 mol) in dichloromethane (900.0 mL) under an ice-salt bath. After stirring at room temperature for 15 minutes, Intermediate D2 (0.15 mol) was added to the reaction mixture. After stirring at room temperature for 1 hour, the reaction mixture was poured into ice-cold ethyl acetate and filtered. The filtrate was concentrated under reduced pressure and purified by column chromatography to obtain Intermediate D3.

[0093] Methyltriphenylphosphonium bromide (0.15 mol) was added dropwise to a suspension of NaH (60%, 0.12 mol) in THF (500.0 mL) under ice-cooling and stirred at 0°C for 0.5 hours. A solution of Intermediate D3 (0.12 mol) in THF (300.0 mL) was slowly added dropwise to the reaction mixture. Stir at room temperature overnight. Saturated aqueous ammonium chloride was added dropwise to the reaction mixture under ice-cooling to quench the reaction. The mixture was extracted with ethyl acetate, concentrated, and purified by column chromatography to obtain Intermediate D4.

[0094] Intermediate D4 (0.1 mol) was dissolved in anhydrous THF (420.0 mL), the temperature was controlled at -5 to 0°C, and the mixture was added dropwise to a borane dimethyl sulfide solution (0.2 mol, 10 M) under a nitrogen atmosphere. After the addition was complete, the mixture was stirred for 6 hours. Subsequently, an aqueous sodium hydroxide solution (0.7 mol, 350.0 mL) and hydrogen peroxide (80.0 mL, 30% aqueous solution) were added dropwise to the reaction solution. The mixture was returned to room temperature and stirred for 2 hours. Ethyl acetate (200.0 mL x 2) was added for extraction. The organic phase was washed with water and saturated brine in sequence, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by column chromatography to obtain intermediate D5.

[0095] A solution of Intermediate D5 (85.0 mmol) in THF (200.0 mL) was added dropwise to a suspension of NaH (60%, 95.0 mmol) in THF (170.0 mL) under ice-cooling, and stirred at 0°C for 0.5 hours. Iodomethane (100.0 mmol) was added dropwise to the reaction mixture. The mixture was stirred at room temperature for 3 hours. Saturated aqueous ammonium chloride was added dropwise to the reaction mixture under ice-cooling to quench the reaction. The mixture was extracted with ethyl acetate, concentrated, and purified by column chromatography to obtain Intermediate D6.

[0096] Intermediate D6 (70.0 mmol) was dissolved in anhydrous THF (300.0 mL). A 1 M solution of TBAF in THF (0.3 mol) was added and stirred at room temperature for 6 hours. The reaction mixture was washed with water, and the aqueous phase was extracted once with ethyl acetate. The organic phases were combined, dried, and concentrated. Purification by column chromatography afforded Intermediate D7.

[0097] Intermediate D7 (60.0 mmol) was dissolved in DMF (130.0 mL). DMSO (0.36 mol) and EDCI (0.18 mol) were added to the reaction solution, followed by the dropwise addition of pyridine (60.0 mmol) and trifluoroacetic acid (60.0 mmol). After reacting at room temperature for 5 hours, the reaction solution was diluted with ethyl acetate. The organic phase was washed with saturated sodium bicarbonate aqueous solution and water, then concentrated and purified by column chromatography to yield Intermediate D8.

[0098] Tetraethylmethylene diphosphate (80.0 mmol) was added dropwise to a suspension of NaH (60%, 53.0 mmol) in THF (50.0 mL) under ice-cooling, followed by stirring at 0°C for 0.5 hours. A solution of Intermediate D8 (53.0 mmol) in THF (70.0 mL) was slowly added dropwise to the reaction mixture. The mixture was stirred at room temperature overnight. Saturated aqueous ammonium chloride was added dropwise to the reaction mixture under ice-cooling to quench the reaction. The mixture was extracted with ethyl acetate, concentrated, and purified by column chromatography to afford Intermediate D9.

[0099] Intermediate D9 (44.0 mmol) was dissolved in acetic acid (80.0 mL). Acetic anhydride (132.0 mmol) was added at room temperature, and the temperature was cooled to below 10°C. Concentrated sulfuric acid (6.6 mmol) was added dropwise to the reaction mixture. Stirred overnight at room temperature. The reaction mixture was diluted with DCM (150.0 mL) and washed sequentially with water, saturated sodium bicarbonate aqueous solution, and saturated brine. It was dried over anhydrous sodium sulfate, and the organic phase was concentrated under reduced pressure to obtain intermediate D10, which was used directly in the next reaction without purification.

[0100] Compound D10 (40.0 mmol) and compound D11 (40.0 mmol) were weighed and suspended in DCE (300.0 mL). BSA (0.1 mol) was added to the reaction mixture. The reaction mixture was heated to 70°C and stirred for 1 hour. The temperature was then lowered to 0°C, and TMSOTf (0.12 mol) was added dropwise to the reaction mixture. After the addition was complete, the temperature was raised to 70°C and stirred for 3 hours. After cooling to room temperature, the reaction mixture was washed with saturated sodium bicarbonate solution. The organic phase was concentrated under reduced pressure and purified by column chromatography to yield intermediate D12.

[0101] Intermediate D13 was obtained by referring to the synthetic method of intermediate C4.

[0102] Intermediate D14 was obtained by referring to the synthetic method of intermediate C5.

[0103] Intermediate D15 was obtained by referring to the synthetic method of intermediate C7.

[0104] Intermediate D16 was obtained by referring to the synthetic method of intermediate C8.

[0105] Intermediate D17 was obtained by referring to the synthetic method of intermediate C9.

[0106] Intermediate D18 was obtained by referring to the synthetic method of intermediate C10.

[0107] Intermediate D was obtained by referring to the synthetic method of intermediate C.

[0108] Example 4: Synthesis of the ammonium salt of compound 9 using intermediates B and E as raw materials

[0109] Using intermediates B and E as raw materials, the ammonium salt of compound 9 was obtained by referring to the synthesis method of compound 1 in Example 1. The reaction scheme is as follows:

[0110] Compound E is obtained by the following steps: Intermediate E2-E is obtained by following the synthesis method of intermediate C2-C in Reference Example 2. Reaction route:

[0111] Example 5: Synthesis of the ammonium salt of compound 10 using intermediates B and F as raw materials

[0112] Using intermediates B and F as raw materials, the ammonium salt of compound 10 was obtained by referring to the synthesis method of compound 1 in Example 1. Reaction scheme:

[0113] Wherein, compound F is obtained by the following steps:

[0114] Dissolve intermediate D5 (0.33 mol), triphenylphosphine (0.66 mol), and DIAD (0.66 mol) in THF (1.5 L). Add DPPA (0.66 mol) dropwise to the reaction mixture. Stir at room temperature overnight. Concentrate under reduced pressure to remove the solvent. Purify by column chromatography to yield intermediate F1.

[0115] Intermediate F2 was obtained by referring to the synthesis method of intermediate D7 in Example 3.

[0116] Intermediate F3 was obtained by the synthesis method of intermediate D8 in Reference Example 3.

[0117] Intermediate F4 was obtained by referring to the synthesis method of intermediate D9 in Example 3.

[0118] Intermediate F5 was obtained by referring to the synthesis method of intermediate D10 in Example 3.

[0119] Intermediate F6 was obtained by the synthesis method of intermediate D12 in Reference Example 3.

[0120] Intermediate F7 was obtained by referring to the synthesis method of intermediate D13 in Example 3.

[0121] Intermediate F8 was obtained by referring to the synthesis method of intermediate D15 in Example 3.

[0122] Intermediate F8 (125.0 mmol) was dissolved in THF (1.0 L). Water (100.0 mL) and triphenylphosphine (187.5 mmol) were added, and the mixture was heated to 50°C and stirred overnight. The solvent was removed by concentration. The crude product was purified by column chromatography to obtain Intermediate F9.

[0123] Intermediate F9 (108.0 mmol) was dissolved in DCM (940.0 mL). After adding TEA (143.0 mmol), benzoyl chloride (130.0 mmol) was added dropwise to the reaction mixture under an ice bath. After returning to room temperature, the mixture was stirred for 2 hours. The reaction mixture was washed sequentially with water and saturated brine, dried over anhydrous sodium sulfate, and the organic phase was concentrated under reduced pressure and purified by column chromatography to obtain Intermediate F10.

[0124] Intermediate F11 was obtained by the synthesis method of intermediate D14 in Reference Example 3.

[0125] Intermediate F12 was obtained by referring to the synthesis method of intermediate D16 in Example 3.

[0126] Intermediate F13 was obtained by the synthesis method of intermediate D17 in Reference Example 3.

[0127] Intermediate F14 was obtained by the synthesis method of intermediate D18 in Reference Example 3.

[0128] Intermediate F was obtained by the synthesis method of Intermediate D in Reference Example 3.

[0129] Example 6: Synthesis of the ammonium salt of compound 14 using intermediates A and G as raw materials

[0130] Using intermediates A and G as raw materials, the ammonium salt of compound 14 was obtained by referring to the synthesis method of compound 1 in Example 1. Reaction route:

[0131] Wherein, compound G is obtained by the following steps:

[0132] Take intermediate B1 (0.18 mol) and bis(2-cyanoethyl)-N,N-diisopropylphosphoramidite (0.25 mol) and dissolve them in DCM (1.7 L). Add tetrazole (0.39 mol) under a nitrogen atmosphere. Stir at room temperature for 2 hours. After the reaction is completed, DDTT (0.51 mol) is added to the reaction solution and the reaction is continued at room temperature for 1 hour. The reaction solution is washed with 10% sodium sulfite aqueous solution, 10% sodium bicarbonate aqueous solution, and saturated brine, respectively. The organic phase is concentrated under reduced pressure and dissolved in methanol (0.5 L) and ammonia water (1.0 L), and stirred at room temperature overnight. After concentration to remove the solvent, dilute with water, load onto DEAE Sephadex, elute with a linear gradient of 0-1.0 M TEAB eluent, and concentrate to obtain intermediate G. Reaction route:

[0133] Example 7: Synthesis of the ammonium salt of compound 21 using intermediates H and I as raw materials

[0134] Using intermediates H and I as raw materials, the ammonium salt of compound 11 was obtained by referring to the synthesis method of compound 1 in Example 1. Reaction scheme:

[0135] Compound H is obtained by the following steps: Intermediates Y1 to Y are obtained respectively by referring to the synthesis methods of intermediates F14 and F in Example 5. Reaction route:

[0136] Compound I is obtained by the following steps:

[0137] Intermediate B1 (0.3 mol) was dissolved in DMF (1.2 L). DMSO (1.8 mol) and EDCI (0.9 mol) were added to the reaction solution, followed by the dropwise addition of pyridine (0.3 mol) and trifluoroacetic acid (0.3 mol). After 5 hours of reaction at room temperature, the reaction solution was diluted with ethyl acetate. The organic phase was washed with saturated sodium bicarbonate solution and water, then concentrated and purified by column chromatography to yield Intermediate I1.

[0138] Tetraethylmethylene diphosphate (0.24 mol) was added dropwise to a suspension of NaH (60%, 0.16 mol) in THF (500.0 mL) under ice-cooling, followed by stirring at 0°C for 0.5 hours. A solution of intermediate I1 (0.16 mol) in THF (400.0 mL) was slowly added dropwise to the reaction mixture. The mixture was stirred at room temperature overnight. Saturated aqueous ammonium chloride was added dropwise to the reaction mixture under ice-cooling to quench the reaction. The mixture was extracted with ethyl acetate, concentrated, and purified by column chromatography to yield intermediate I2.

[0139] Intermediate I3 was obtained by referring to the synthesis method of intermediate F7 in Example 5.

[0140] Intermediate I4 was obtained by referring to the synthesis method of intermediate F11 in Example 5.

[0141] Intermediate I was obtained by the synthesis method of Intermediate G in Reference Example 6.

[0142] Reaction route:

[0143] Example 8: Synthesis of the ammonium salt of compound 22 using intermediates H and J as raw materials

[0144] Using intermediates H and J as raw materials, the ammonium salt of compound 22 was obtained by referring to the synthesis method of compound 1 in Example 1. Reaction route:

[0145] Wherein, compound J is obtained by the following steps:

[0146] Intermediate A5 (0.21 mol) and DMT-2'OMe-A(Bz) (0.25 mol) were dissolved in DMF (1.5 L). DCC (0.5 mol) was added to the reaction mixture and allowed to stand overnight at room temperature. After completion, the reaction was quenched with water (4.5 L) and extracted with ethyl acetate three times, each time using 500.0 mL. The combined organic phases were concentrated under reduced pressure and purified by column chromatography to yield Intermediate J1.

[0147] Intermediate J1 (0.11 mol) was dissolved in 80% aqueous acetic acid solution (1.6 L) and stirred at room temperature overnight. After the reaction was completed, the solvent was removed by concentration under reduced pressure and purified by column chromatography to obtain intermediate J2.

[0148] Intermediate J was obtained by the synthesis method of intermediate B in Reference Example 1.

[0149] Example 9: Synthesis of the ammonium salt of compound 31 using intermediates B and K as raw materials

[0150] Using intermediates B and K as raw materials, refer to the ammonium salt of compound 31 of compound 1 in Example 1. Reaction route:

[0151] Wherein, compound K is obtained by the following steps:

[0152] Intermediate A2 (0.19 mol) was dissolved in acetonitrile (1.1 L). Aqueous sodium dihydrogen phosphate (0.2 M, 38.2 mmol) and hydrogen peroxide (30 wt.%, 0.21 mol) were added to the reaction mixture. The temperature was cooled to 0°C, and sodium chlorite (0.75 M, 0.29 mol) was added dropwise to the reaction mixture. After the addition was complete, the mixture was returned to room temperature and stirred for 1 hour. Aqueous sodium sulfite was added to the reaction mixture to quench the reaction. The reaction mixture was concentrated to obtain the crude product of intermediate K1.

[0153] Dissolve the crude intermediate K1 in DMF (1.0 L). Cool to 0°C, then add NaHCO₃ (1.35 mol) and iodomethane (0.34 mol) to the reaction mixture. Stir overnight at room temperature. Dilute the reaction mixture with water and extract twice with 500.0 mL of ethyl acetate. Combine the organic phases, concentrate under reduced pressure, and purify by column chromatography to yield intermediate K2.

[0154] Intermediate K2 (0.1 mol) was dissolved in anhydrous THF (610.0 mL), cooled to 0°C, and tetraisopropyl titanate (0.1 mol) and ethylmagnesium bromide (2.0 M in THF, 0.5 mol) were added under a nitrogen atmosphere. The mixture was returned to room temperature and stirred for 6 hours. Saturated aqueous ammonium chloride was added to the reaction mixture, the layers were separated, and the aqueous phase was filtered and extracted with ethyl acetate. The organic phases were combined and concentrated under reduced pressure. The crude product was purified by column chromatography to obtain intermediate K3.

[0155] Intermediate K4 was obtained by referring to the synthesis method of intermediate E6 in Example 4.

[0156] Intermediate K5 was obtained by referring to the synthesis method of intermediate E7 in Example 4.

[0157] Intermediate A3 (0.55 mol) was dissolved in trimethyl phosphate (200.0 ml). After cooling to -0°C, phosphorus oxychloride (0.1 mol) was added dropwise to the reaction mixture. After stirring at -0°C for 3 hours, TEAB solution was added dropwise to quench the reaction mixture. Purification by reverse-phase preparative chromatography afforded Intermediate K5.

[0158] Intermediate K7 was obtained by referring to the synthesis method of intermediate E8 in Example 4.

[0159] Intermediate K8 was obtained by referring to the synthesis method of intermediate E9 in Example 4.

[0160] Intermediate K9 was obtained by referring to the synthesis method of intermediate E10 in Example 4.

[0161] Intermediate K was obtained by the synthesis method of Intermediate E in Reference Example 4.

[0162] Example 10: Synthesis of the ammonium salt of compound 32 using intermediates B and L as raw materials

[0163] Using intermediates B and L as raw materials, the ammonium salt of compound 32 was obtained by referring to the synthesis method of compound 1 in Example 1. Reaction route:

[0164] Wherein, compound L is obtained by the following steps: intermediate L1-L is obtained by the synthesis method of intermediate K1-K in reference example 9.

[0165] Example 11: Synthesis of the ammonium salt of compound 37 using intermediates K and M as raw materials

[0166] Using intermediates K and M as raw materials, the ammonium salt of compound 37 was obtained by referring to the synthesis method of compound 1 in Example 1. Reaction scheme:

[0167] Wherein, compound M is obtained by the following steps: intermediate M is obtained by referring to the synthesis method of intermediate B in Example 1.

[0168] Example 12: Synthesis of the ammonium salt of compound 38 using intermediates A and M as raw materials

[0169] Using intermediates A and M as raw materials, the ammonium salt of compound 38 was obtained by referring to the synthesis method of compound 1 in Example 1. Reaction scheme:

[0170] Comparative Example 1: Synthesis of the ammonium salt of compound 43 using intermediates H and B as raw materials

[0171] Using intermediates H and B as raw materials, the ammonium salt of compound 43 was obtained by referring to the synthesis method of compound 1 in Example 1. Reaction route:

[0172] Biological activity detection part:

[0173] Test Example 1: Determination of mRNA in vitro transcription yield and capping efficiency

[0174] The IVT reaction of mRNA was performed using the cap analogs of the embodiment. 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 was added to the system, followed by 10× buffer, NTPs, and cap analogs in sequence, mixed and centrifuged gently, then nuclease inhibitors, inorganic pyrophosphatase, T7 RNA polymerase, and linearized DNA template were added, mixed thoroughly and centrifuged gently, and incubated at 37 degrees Celsius. After 2 hours, DNaseI1U was added and incubated at 37 degrees Celsius for 30 minutes to remove the DNA template, and then the RNA was purified using a magnetic bead purification method. The purified mRNA was dissolved with sterile enzyme-free water and then quantitatively detected using NanodropOne.

[0175] Table 1 IVT reaction system

[0176] Liquid chromatography-mass spectrometry (LC-MS) is used to detect the IVT capping rate of mRNA with different start cap analogs. First, a labeled DNA probe that matches the start base of the transcript mRNA needs to be designed. The label is usually a biotin label. After washing the streptavidin-labeled magnetic beads, they are incubated with the synthetic DNA probe, mRNA, and 10×RNaseH reaction buffer at room temperature for 30 minutes. Slowly mix while incubating. Then, 20ul RNaseH (5U / μL) is added and incubated at 37 degrees for 3h, mixing every half hour. After the incubation, the magnetic beads are washed and 100μL of 75% methanol heated to 80 degrees Celsius is added to the washed magnetic beads. The mixture is heated to 80 degrees Celsius on a hot plate and maintained for 3 minutes. Then, the supernatant is aspirated and dried to 10μl using an evaporating centrifuge at room temperature for 45 minutes. The sample is then resuspended in 50 μl of 100 μM EDTA / 1% MeOH and used for LC-MS analysis to determine the capping status of the RNA during the transcription reaction. Because capped and non-capped bases have distinct molecular weight differences, this molecular mass difference can be used to determine the capping rate of mRNA transcripts initiated by different cap analogs.

[0177] See Table 2 for specific results.

[0178] Table 2 mRNA yield and capping efficiency

[0179] The experimental results show that, compared with Comparative Example 1, the in vitro transcription yield and capping efficiency of mRNA were significantly improved when the cap analogs of the present application were used for the IVT reaction of mRNA.

[0180] Test Example 2: mRNA translation efficiency

[0181] Test method: Using the eGFP coding sequence as a DNA template, the cap analogs of Example and Comparative Example 1 were used as the starting point for in vitro transcription. The different mRNA products obtained were then transfected into 293T cells. 293T cells were transfected with (0.5-1)×10 5Cells were plated (24-well plates). During transfection, the cell density was generally 60-80%, and 2 μg of mRNA was transfected per well. The transfection reagent used was Lipofectamine Messenger MAX Transfection Reagent (Invitrogen) and the operation was performed according to its instructions. The transfected cells were placed in a 37-degree Celsius CO2 incubator. After 4-6 hours of transfection, fresh complete culture medium was replaced. After incubation in a 37-degree Celsius CO2 incubator for 24 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.

[0182] Table 3 mRNA translation efficiency

[0183] The experimental results show that after in vitro transcription starting with the cap analogue of the present application and incubating the transfected cells for 24 hours, the translation efficiency of the mRNA is higher than that of comparative example 1. The above results show that the cap analogue of the present application has higher translation efficiency at the cellular level.

[0184] Test Example 3: mRNA translation efficiency in mice

[0185] Experimental steps:

[0186] The mouse's tail was gently wiped with an alcohol cotton ball, and 1 μg of LNP-mRNA drug was injected into the tail vein using an insulin syringe. 24 hours after the mouse's tail vein administration, 10% chloral hydrate was injected intraperitoneally (50-60 μl / 20g, depending on the mouse's weight for anesthesia). After the mouse was anesthetized, 150 μl of the prepared luminescent substrate (luminescent substrate concentration 15 mg / ml, product number 2109GR001, brand biofroxx) was injected intraperitoneally. 5 minutes after the mouse luminescent substrate injection, the mouse was placed in a small animal in vivo imaging instrument, and the corresponding luciferase channel was selected for in vivo imaging of the mouse; the image was saved. Repeat this step at other time points, and the exposure parameters must be kept consistent for comparison. The effect of mRNA translation efficiency in mice is shown in Figure 1.

[0187] As shown in Figure 1, after tail vein administration, compounds 1, 2, 31, and 32 were concentrated in the liver, as was Comparative Example 1. 24 hours after administration, the protein expression levels of the modified mRNAs of compounds 1, 2, 31, and 32 in mice were higher than that of Comparative Example 1.

[0188] While embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions, and variations may be made to these embodiments without departing from the principles and spirit of the invention, and that the scope of the invention is defined by the appended claims and their equivalents.

Claims

1. A capping compound, which is a compound of the structure represented by formula (I), its stereoisomers, pharmaceutically acceptable salts, solvates, in, represents a single key or none; represents a single bond or a double bond; R1 and R2 are independently H, OH, halogen, alkyl, alkoxy, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, heterocyclyl, heteroaryl, alkylthio, or alkoxy forms a bridged ring with the carbon on 4'; wherein the alkyl, alkoxy, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, heterocyclyl, heteroaryl, alkylthio is unsubstituted or substituted with one or more R5, and R5 is any one of alkyl, alkoxy, substituted or unsubstituted acetamido; R3 is H, OH, halogen, alkoxy, or an alkoxy group and the carbon on 4' form a bridged ring; wherein the alkoxy group is unsubstituted or substituted by one or more R6, and R6 is any one of an alkyl group and an alkoxy group; R4 is H, OH, alkyl, or alkoxy; wherein the alkyl or alkoxy group is unsubstituted or substituted by one or more R7, and R7 is any one of alkyl or alkoxy; Ra, Rb and Rc are independently 3-6 membered cycloalkyl, 3-6 membered heterocyclyl, CH2, CH or O, wherein Ra and X1 are not O at the same time, Rb and X4 are not O at the same time, and Rc and X5 are not O at the same time; and at least one of Ra, Rb and Rc is 3-6 membered cycloalkyl or 3-6 membered heterocyclyl; X1, X2, X3, X4 and X5 are independently selected from O, S, CH2, CH, NH or none; Y 1a , Y 2a , Y 3a and Y 4a are independently OH, SH or BH3; Y 1b , Y 2b , Y 3b and Y 4b are independently O or S; m is 0, 1, 2 or 3; B1 and B2 are each independently a natural or modified nucleoside base.

2. The capping compound according to claim 1, characterized in that: Any one or both of Ra, Rb and Rc are 3- to 6-membered cycloalkyl groups or 3- to 6-membered heterocyclic groups.

3. The capping compound according to claim 2, characterized in that: Any one or two of Ra, Rb and Rc are three-membered cycloalkyl or four-membered cycloalkyl, and the structure thereof is selected from: And there is no adjacent X1, X4 or X5.

4. The capping compound according to claim 3, characterized in that is a compound having a structure as shown in formula (II), or a stereoisomer, a pharmaceutically acceptable salt, or a solvate thereof, Ra is selected from 5. The capping compound according to claim 1, characterized in that: R3 is H, OH, C 1-6 Alkoxy or LNA, where C 1-6 The alkoxy group is unsubstituted or substituted by one or more R6, R6 is C 1-6 Alkyl or C 1-6 Alkoxy.

6. The capping compound according to claim 5, characterized in that: R3 is H, OH or C 1-4 Alkoxy, R4 is H, OH or C 1-4 Alkoxy.

7. The capping compound according to claim 1, characterized in that: R1 and R2 are independently H, OH, halogen, C 1-6 Alkyl, C 1-6 Alkoxy or LNA; wherein C 1-6 Alkyl, C 1-6 The alkoxy group is unsubstituted or substituted by one or more R5, R5 is C 1-6 Alkyl, C 1-6 Any of an alkoxy group and a substituted or unsubstituted acetamide group.

8. The capping compound according to claim 7, characterized in that: R1 and R2 are independently OH or C 1-4 Alkoxy; wherein C 1-4 The alkoxy group is unsubstituted or substituted by one or two R5, R5 is C 1-4 Alkyl, C 1-4 Alkoxy.

9. The capping compound according to claim 8, characterized in that is a compound represented by formula (III), or a stereoisomer, a pharmaceutically acceptable salt, or a solvate thereof, 10. The capping compound according to claim 1, characterized in that: Any one or two of Ra, Rb and Rc are three-membered cycloalkyl or four-membered cycloalkyl, and the structure thereof is selected from: And the adjacent X1, X2 or X5 is O.

11. The capping compound according to claim 9, characterized in that: is a compound having a structure as shown in formula (IV), or a stereoisomer, a pharmaceutically acceptable salt, or a solvate thereof, R1 and R2 are independently OH or C 1-4 Alkoxy; wherein C 1-4 The alkoxy group is unsubstituted or substituted by one or more R5, R5 is C 1-4 Alkyl, C 1-4 Alkoxy; R3 is C 1-6 Alkoxy, where C 1-6 The alkoxy group is unsubstituted or substituted by one or more R6, R6 is C 1-6 alkyl or C 1-6 Alkoxy; R4 is H, OH or C 1-4 Alkoxy.

12. The capping compound according to any one of claims 1 to 11, characterized in that It is selected from any of the following compounds, or its stereoisomers, pharmaceutically acceptable salts, solvates, 13. Use of the capping compound according to any one of claims 1 to 12 in the preparation of an in vitro co-transcribed RNA capping reagent.

14. A complex comprising the capping compound of any one of claims 1 to 12 and a DNA template, wherein the DNA template includes a promoter region comprising 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 position +1 of the DNA template, and B2 is complementary to the nucleobase at position +2 of the transcription template on the DNA template.

15. An RNA molecule, characterized in that Comprising the capping compound according to any one of claims 1 to 12.

16. A pharmaceutical composition, characterized in that Comprising the RNA molecule of claim 15, and a pharmaceutically acceptable carrier.

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