Capped analogs for self-replicating mRNA and uses thereof

Abstract

The invention provides a capped analogue for self-replicating mRNA and application of the capped analogue. The capped analogue for the self-replicating mRNA can improve the stability of the self-replicating mRNA and / or the translation efficiency of the self-replicating mRNA.

Description

Technical Field

[0001] The present disclosure belongs to the technical fields of chemistry and bioengineering, and particularly relates to capping analogs for self-replicating mRNA and their applications. Background Art

[0002] The cap structure is an essential element for RNA to exert its biological functions in vivo. Its presence helps RNA evade the innate immune response of cells and achieve efficient in vivo protein translation, and is the primary condition for stimulating the body's response. The chemical nature of the cap structure is a special structure located at the 5'-end of RNA formed by modification during the RNA transcription process, namely the m7GPPPN structure, also known as the methylguanosine cap. It is formed under the combined catalytic action of RNA triphosphatase, guanylyltransferase, RNA (guanine-N7) methyltransferase, and RNA (nucleoside-2') methyltransferase.

[0003] Self-replicating RNA (srRNA), also often referred to as self-amplifying RNA (saRNA), is a special type of RNA molecule that has the ability to self-replicate. Compared with traditional mRNA, the important difference of self-replicating mRNA is that it can use its own RNA sequence as a template for self-replication. This self-replicating property can achieve a higher protein expression effect with a lower vaccine dosage, improving the efficacy of the vaccine.

[0004] Similar to traditional mRNA, self-replicating mRNA also requires a cap structure in its structure to improve its stability and translation efficiency in cells. Self-replicating mRNA is usually designed based on the genomes of positive-sense RNA viruses, such as Venezuelan equine encephalitis virus (VEEV), Semliki Forest virus (SFV), and Sindbis virus (SIN), etc. The genomes of these positive-sense RNA viruses start with a 5'-AU sequence, and capping analogs of the m7GpppApU type can match this starting sequence well, thus better simulating the natural infection process of the virus.

[0005] Due to differences in sequence length and cap structure type, the structural modification methods applicable to the m7GpppApG capping analog used in the traditional mRNA preparation process may not be applicable to the development of self-replicating mRNA capping analogs. Therefore, it is crucial to provide capping analogs suitable for self-replicating mRNA. Summary of the Invention

[0006] In view of the deficiencies of the prior art, the purpose of the present disclosure is to provide a capping analog for self-replicating mRNA and its applications. The capping analog for self-replicating mRNA described in the present disclosure can improve the stability of self-replicating mRNA and / or the translation efficiency of self-replicating mRNA.

[0007] The present invention provides a compound represented by formula (I), its stereoisomers or its pharmaceutically acceptable salts:

[0008] ,

[0009] wherein n is 0, 1, 2 or 3;

[0010] R 5 is -OR 5a , -SR 5b , -NR 5c R 5d , -C(O)R 5e , , -NHC(O)-R 5h , -N(CH 3 )C(O)-R 5i , -C(O)NR 5j R 5k , -C(O)O-R 5l , -OC(O)-R 5m or -S(O) 2 -R 5n ;

[0011] R 4a , R 5a , R 5b , R 5c , R 5d , R 5e , R 5h , R 5i , R 5j , R 5k , R 5l , R 5m and R 5n are independently -H, C 1 -C 3 alkyl or halo C 1 -C 3 alkyl;

[0012] R 5f and R 5g are independently -H, halogen, C 1 -C 3 alkyl, -OC 1 -C 3 alkyl, halo C 1 -C3 alkyl or -NR 5-1 C(O)R 5-2 ;

[0013] R 5-1 and R 5-2 are independently -H or C 1 -C 3 alkyl;

[0014] R 1 、R 2 、R 3 、R 6 and R 7 each independently is -H, C 1 -C 6 alkyl, C 2- C 6 alkenyl, C 2 -C 6 alkynyl, C 3 -C 6 cycloalkyl or benzyl;

[0015] R 4 is -OR 4a , halogen or -H;

[0016] R 8 is -H or C 1 -C 3 alkyl.

[0017] In some embodiments, in the compounds of formula (I), their stereoisomers or their pharmaceutically acceptable salts, the definitions of some groups are as described below, and the definitions of the remaining groups are as described in any other embodiment (hereinafter referred to as "in some embodiments"):

[0018] n is 0 or 1.

[0019] In some embodiments, R 4 is -H, -OC 1 -C 3 alkyl or halogen.

[0020] In some embodiments, R 4 is -H or halogen.

[0021] In some embodiments, R 4 is -H, -F or -OCH 3 .

[0022] In some embodiments, R 5 is -OH, or -C(O)NR 5j R 5k ; R5f and R 5g are independently halogen, C 1 -C 3 alkyl, -OC 1 -C 3 alkyl or -NR 5-1 C(O)R 5-2 ; R 5j and R 5k are independently C 1 -C 3 alkyl; R 5-1 is H; R 5-2 is C 1 -C 3 alkyl.

[0023] In some embodiments, R 5 is -OH, or -C(O)NR 5j R 5k ; R 5f is C 1 -C 3 alkyl or halogen; R 5g is halogen, C 1 -C 3 alkyl or -OC 1 -C 3 alkyl; R 5j and R 5k are independently C 1 -C 3 alkyl.

[0024] In some embodiments, R 5 is -OH, , , , or .

[0025] In some embodiments, R 5 is -OH, , , or .

[0026] In some embodiments, R 1 is -H.

[0027] In some embodiments, R 2 is C 1 -C 3 alkyl.

[0028] In some embodiments, R 2 is -CH 3 .

[0029] In some embodiments, R 3 is -H.

[0030] In some embodiments, R 6 is C 1 -C 3 alkyl.

[0031] In some embodiments, R 6 is -CH 3 .

[0032] In some embodiments, R 7 is -H.

[0033] In some embodiments, R 8 is -H.

[0034] In some embodiments, n is 0 or 1; R 4 is -H or halogen;

[0035] R 5 is -OH, or -C(O)NR 5j R 5k ; R 5f and R 5g are independently halogen, C 1 -C 3 alkyl or -OC 1 -C 3 alkyl; R 5j and R 5k are independently C 1 -C 3 alkyl;

[0036] R 1 is -H; R 2 is C 1 -C 3 alkyl; R 3 is -H;

[0037] R 6 is C 1 -C 3 alkyl; R 7 is -H;

[0038] R 8 is -H.

[0039] In some embodiments, the pharmaceutically acceptable salts of the compounds of formula (I) are any of the following structures:

[0040] ,

[0041] ,

[0042] ,

[0043] ,

[0044] ,

[0045] or

[0046] .

[0047] The present invention provides the use of a compound represented by formula (I) as described above, its stereoisomers or its pharmaceutically acceptable salts in the preparation of an in vitro co-transcriptional mRNA capping reagent.

[0048] The present invention also provides an RNA molecule comprising a compound represented by formula (I) as described above, its stereoisomers or its pharmaceutically acceptable salts as a cap structure or a cap structure fragment.

[0049] The present invention also provides a pharmaceutical composition comprising the RNA molecule as described above.

[0050] In some embodiments, the pharmaceutical composition further comprises at least one delivery agent.

[0051] In some embodiments, the at least one delivery agent comprises at least one cationic lipid.

[0052] In some embodiments, the cationic lipid is any one of the following or a combination of at least two of them:

[0053] (i) A compound represented by formula (II), its stereoisomers, its N-oxides, its solvates or its pharmaceutically acceptable salts, wherein G 1 is C 1-6 alkylene; G 2 is C 2-8 alkylene; G 3 is C 1-3 alkylene; L 1 is C 6-15 linear alkyl; L 2 is C 12-25 branched alkyl;

[0054] (II)

[0055] (ii) A compound represented by formula (III), its stereoisomers, its N-oxides, its solvates or its pharmaceutically acceptable salts, wherein G 1is C 2-8 alkylene; G 2 is C 2-8 alkylene; L 1 is -C(O)O- or -OC(O)-; L 2 is -C(O)O- or -OC(O)-; R 1 is C 6-25 linear or branched alkyl; R 2 is C 6-25 linear or branched alkyl; G 3 is HO(CH 2 ) 2 - or HO(CH 2 ) 3 -; G 4 is HO(CH 2 ) 2 - or HO(CH 2 ) 3 -; L is (CH 2 ) 2 -、-(CH 2 ) 3 - or -(CH 2 ) 4 -;

[0056] (III)

[0057] (iii) a compound represented by formula (IV), its stereoisomers, its N-oxides, its solvates or its pharmaceutically acceptable salts, wherein: G 1 is C 1-6 alkylene; G 2 is C 2-8 alkylene; R 1 is C 6-20 linear or branched alkyl; R 2 is C 12-25 branched alkyl; G 3 is: HO(CH 2 ) 2 N(CH 3 )(CH 2 ) 2 -、HO(CH 2 ) 2 N(CH 2 CH 3 )(CH 2 ) 2 -、(HO(CH 2 ) 2 ) 2 N(CH 2 ) 2 -、CH 3O(CH 2 ) 2 N(CH 3 )(CH 2 ) 2 -、(CH 3 ) 2 N(CH 2 ) 3 SC(O)O(CH 2 ) 2 -、(CH 3 ) 2 N(CH 2 ) 3 SC(O)-、CH 3 NH(CH 2 ) 2 N(CH 3 )(CH 2 ) 2 - or CH 3 CH 2 NH(CH 2 ) 2 -;

[0058] (IV)

[0059] (iv) A compound represented by formula (V), a stereoisomer thereof, an N-oxide thereof, a solvate thereof or a pharmaceutically acceptable salt thereof, wherein G 1 C 1-8 Alkylene; G 2 C 2-8 Alkylene; R 1 C 6-25 Straight or branched alkyl; R 2 C 12-25 Straight or branched alkyl; G 3 HO(CH 2 ) 2 N(R 3 )CH 2 CH(OH)CH 2 -, where R 3 -CH 3 、-CH 2 CH 3 or -CH 2 CH 2 OH ;

[0060] (V)

[0061] (v) A compound represented by formula (VI), a stereoisomer thereof, an N-oxide thereof, a solvate thereof, or a pharmaceutically acceptable salt thereof, wherein G1 and G 2 are each independently C 6 -C 10 alkylene; G 3 is C 1 -C 12 alkylene; R 1 and R 2 are each independently C 6 -C 24 alkyl or C 6 -C 24 alkenyl; R 3 is OR 5 , N(R 5 ) 2 , -C(=O)OR 4 , -OC(=O)R 4 or -NR 5 C(=O)R 4 ; R 4 is C 1 -C 12 hydrocarbyl; R 5 is H or C 1 -C 6 hydrocarbyl;

[0062] (VI)

[0063] (vi) a compound represented by formula (VII), its stereoisomers, its N-oxides, its solvates or its pharmaceutically acceptable salts, wherein R 4 is -(CH 2 ) n Q; Q is -OR, -OH, -O(CH 2 ) n N(R) 2 , -OC(O)R, -CX 3 , -CN, -N(R)C(O)R, -N(H)C(O)R, -N(R)S(O) 2 R, -N(H)S(O) 2 R, -N(R)C(O)N(R) 2 , -N(H)C(O)N(R) 2 , -N(H)C(O)N(H)(R), -N(R)C(S)N(R) 2 , -N(H)C(S)N(R) 2 , -N(H)C(S)N(H)(R), -N(R)S(O) 2 R or a heterocycle; n is 1, 2 or 3; R is independently C 1-8 alkyl; X is independently H or C 1-8 alkyl;

[0064] (VII)

[0065] (vii) A compound represented by formula (VIII), its stereoisomers, its N-oxides, its solvates or its pharmaceutically acceptable salts,

[0066] (VIII).

[0067] In some embodiments, the cationic lipid is selected from any one or a combination of at least two of the group consisting of YK-009, YK-201, YK-407, YK-305, ALC-0315, SM-102 and DLIN-MC3-DMA:

[0068] , , , , , , .

[0069] In some embodiments, the cationic lipid is the YK-009.

[0070] In some embodiments, the at least one delivery agent further comprises at least one neutral lipid.

[0071] In some embodiments, the neutral lipid is selected from any one or a combination of at least two of the group consisting of phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, ceramide and sterol.

[0072] In some embodiments, the neutral lipid is selected from any one or a combination of at least two of 1,2-dilinoleoyl-sn-glycero-3-phosphocholine, 1,2-dimyristoyl-sn-glycero-phosphocholine, 1,2-dioleoyl-sn-glycero-3-phosphocholine, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine, 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dielaidoyl-sn-glycero-phosphocholine, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, 1,2-di-O-octadecenoyl-sn-glycero-3-phosphocholine, 1-oleoyl-2-cholesteryl succinyl-sn-glycero-3-phosphocholine, 1-hexadecyl-sn-glycero-3-phosphocholine, 1,2-dilinolenoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt, dipalmitoyl phosphatidylglycerol, palmitoyl oleoyl phosphatidylethanolamine, distearoyl phosphatidylethanolamine, dipalmitoyl phosphatidylethanolamine, dimyristoyl phosphoethanolamine, 1-stearoyl-2-oleoyl-stearoylethanolamine, 1-stearoyl-2-oleoyl-phosphatidylcholine, sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyl oleoyl phosphatidylcholine, lysophosphatidylcholine, and lysophosphatidylethanolamine.

[0073] In some embodiments, the neutral lipid is 1,2-distearoyl-sn-glycero-3-phosphocholine and / or 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine.

[0074] In some embodiments, the at least one delivery agent further comprises a structural lipid.

[0075] In some embodiments, the structural lipid is selected from any one or a combination of at least two of the group consisting of cholesterol, non-sterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatine, ursolic acid, α-tocopherol, and corticosteroid.

[0076] In some embodiments, the structural lipid is cholesterol.

[0077] In some embodiments, the at least one delivery agent further comprises a polymer-conjugated lipid.

[0078] In some embodiments, the polymer-conjugated lipid is selected from any one or a combination of at least two of the group consisting of distearoyl phosphatidylethanolamine polyethylene glycol 2000, 1,2-dimyristoyl-sn-glycero-3-methoxypolyethylene glycol 2000 (DMG-PEG2000), and methoxypolyethylene glycol ditetradecylacetamide.

[0079] In some embodiments, the polymer-conjugated lipid is 1,2-dimyristoyl-sn-glycero-3-methoxypolyethylene glycol 2000.

[0080] In some embodiments, the at least one delivery agent comprises YK-009, 1,2-distearoyl-sn-glycero-3-phosphocholine, cholesterol, and 1,2-dimyristoyl-sn-glycero-3-methoxypolyethylene glycol 2000; the YK-009 is 。

[0081] In some embodiments, the molar ratio of YK-009, 1,2-distearoyl-sn-glycero-3-phosphocholine, cholesterol, and 1,2-dimyristoyl-sn-glycero-3-methoxypolyethylene glycol 2000 is preferably 49:10:39.5:1.5.

[0082] In some embodiments, the pharmaceutical composition further comprises one or at least two cell-penetrating peptides (CPP).

[0083] The present invention provides a method for synthesizing an mRNA molecule for non-diagnostic and therapeutic purposes, which comprises the following steps: co-incubating a compound represented by formula (I) as described above, its stereoisomer, or its pharmaceutically acceptable salt with a polynucleotide template, and transcribing the polynucleotide template.

[0084] The present invention provides a capped mRNA transcription reaction system for non-diagnostic and non-therapeutic purposes, which comprises: (1) a compound represented by formula (I) as described above, its stereoisomers or its pharmaceutically acceptable salts; and (2) a polynucleotide template, NTPs and RNA polymerase.

[0085] The present invention provides a kit, which comprises: (1) a compound represented by formula (I) as described above, its stereoisomers or its pharmaceutically acceptable salts; and (2) nucleotide triphosphate molecules, and RNA polymerase.

[0086] In some embodiments, the kit further comprises any one or a combination of at least two of the group consisting of RNase inhibitor, inorganic pyrophosphatase, Mg 2+ , crowding agent and buffer.

[0087] The present invention provides a method for improving the stability of RNA, which comprises incorporating a compound represented by formula (I) as described above, its stereoisomers or its pharmaceutically acceptable salts into RNA.

[0088] The present invention provides a method for introducing RNA into cells, which comprises contacting the cells with the RNA molecule as described above or the pharmaceutical composition as described above.

[0089] The present invention provides the use of the RNA molecule as described above or the pharmaceutical composition as described above in the preparation of vaccines.

[0090] Term Definitions

[0091] All publications and patents mentioned in this disclosure are hereby incorporated by reference in their entirety into this disclosure. If there are conflicts between the uses or terms used in any of the publications and patents incorporated by reference and the uses or terms used in this disclosure, then the uses and terms of this disclosure shall prevail.

[0092] The section headings used herein are for the purpose of organizing the article only and should not be construed as limiting the subject matter.

[0093] Unless otherwise specified, all technical terms and scientific terms used herein have the ordinary meanings in the field to which the claimed subject matter belongs. If there are multiple definitions for a certain term, the definition herein shall prevail.

[0094] Except in the examples or otherwise indicated, all numbers representing quantitative properties such as dosages stated in this disclosure should be understood to be modified in all instances by the term "about". It should also be understood that any numerical ranges recited in this disclosure are intended to include all sub-ranges within that range and any combination of the endpoints of that range or sub-ranges.

[0095] In addition, when referring to a number or a numerical range, the term "about" means that the number or numerical range being referred to is an approximation within the typical tolerances in the art, within experimental variability, or within statistical experimental error, and thus the number or numerical range can vary, for example, between 1% and 15% of the stated number or numerical range. For example, "about" can be understood as being about 2 standard deviations from the mean value. When "about" is present before a series of numbers or a range, it should be understood that "about" can modify each number in the series or range.

[0096] Unless otherwise defined, all terms used in this disclosure (including technical and scientific terms) have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. Terms, such as those defined in a common dictionary, shall be interpreted as having a meaning consistent with their meaning in the context of the relevant art and shall not be interpreted in an idealized or overly formal sense unless explicitly so defined in this disclosure.

[0097] In this disclosure, " " in a structural fragment means that this structural fragment is connected to the rest of the molecule through this bond. For example, means that it is connected to the rest of the molecule through " ".

[0098] As used in this disclosure, the term "C 1 -C 3 " means a group having any integer value within the range of 1 to 3 carbon atoms in the main chain of the group, such as 1, 2, or 3 carbon atoms. The term "C 6-15 " means a group having any integer value within the range of 6 to 15 carbon atoms in the group, such as 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 carbon atoms. Limitations of other carbon atom ranges are to be understood analogously to mean that the number of carbon atoms in the defined group can be any integer value within the defined range.

[0099] As used in this disclosure, the term "alkyl" means a saturated aliphatic hydrocarbon group having a straight or branched chain; non-limiting examples thereof include methyl, ethyl, propyl, isopropyl, etc.

[0100] In this invention, the term "alkoxy" means a group R Y -O-, where the definition of R Y is the same as the term "alkyl". Alkoxy groups include, but are not limited to: methoxy, ethoxy, n-propoxy, isopropoxy, etc.

[0101] The term "alkenyl" refers to a straight-chain or branched-chain hydrocarbon group having at least one double bond, consisting only of carbon and hydrogen atoms, having, for example, 2 to 6 (such as 2, 3, 4, 5, or 6) carbon atoms, and being connected to the rest of the molecule by a single bond. Alkenyl includes, but is not limited to, vinyl, , , , , etc.

[0102] The term "alkynyl" refers to a straight-chain or branched-chain hydrocarbon group having at least one triple bond, consisting only of carbon and hydrogen atoms, having, for example, 2 to 6 (such as 2, 3, 4, 5, or 6) carbon atoms, and being connected to the rest of the molecule by a single bond. Alkynyl includes, but is not limited to, ethynyl, 1-propynyl, but-1-ynyl, but-2-ynyl, etc.

[0103] The term "halogen" refers to fluorine, chlorine, bromine, or iodine.

[0104] The term "cycloalkyl" refers to a saturated monocyclic group consisting only of carbon atoms having a specified number of carbon atoms (such as C 3 -C 6 ). Cycloalkyl includes, but is not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, etc.

[0105] As used in this disclosure, the term "salt" refers to the corresponding salts that can be conveniently or desirably prepared, purified, and / or processed for the modified nucleoside compounds (or nucleotide compounds) in this disclosure, for example, pharmaceutically acceptable salts. Unless otherwise specified, the reference to a particular compound in this disclosure also includes its salt form.

[0106] As used in this disclosure, "capping analogue" refers to a structure at the 5'-end of a mature RNA formed by post-transcriptional modification in eukaryotes, i.e., the m7GPPPN structure, also known as the methylguanosine cap. This structure can prevent RNA degradation at the 5'-end, help the RNA transcript pass through the selective pores of the nuclear membrane and enter the cytoplasm, enhance translation, and help complete the entire splicing process, etc.

[0107] The use of words such as "comprising", "containing", or "including" in this disclosure means that the elements appearing before the word cover the elements listed after the word and their equivalents, without excluding unrecorded elements. The terms "containing" or "including (comprising)" used herein can be open-ended, semi-closed, and closed-ended. In other words, the said terms also include "consisting essentially of...", or "consisting of...".

[0108] As used herein, the term "pharmaceutically acceptable" means that a compound or composition is chemically and / or toxicologically compatible with the other ingredients of the formulation and / or with the human or mammal to which it is administered for the prevention or treatment of a disease or disorder.

[0109] As used herein, the term "polynucleotide template" means a polynucleotide chain upon which other nucleic acid molecules are synthesized.

[0110] The present disclosure also encompasses salts of the compounds described herein, particularly pharmaceutically acceptable salts. Compounds of the present disclosure having functional groups with sufficient acidity or sufficient basicity can react with many bases or acids to form salts. Alternatively, compounds that are themselves charged (e.g., compounds having a quaternary nitrogen) can form salts with appropriate counterions (e.g., halide ions such as bromide, chloride, or fluoride ions, particularly bromide ions).

[0111] The pharmaceutically acceptable salts of the present disclosure can be, for example, acid addition salts of compounds of the present disclosure having sufficient basicity, where the nitrogen atom is carried in the chain or ring of the compound of the structure shown in formula (I), for example, acid addition salts formed with the following inorganic acids: for example, hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, phosphoric acid, or nitric acid, or acid addition salts formed with the following organic acids: for example, formic acid, acetic acid, acetoacetic acid, pyruvic acid, trifluoroacetic acid, propionic acid, butyric acid, hexanoic acid, heptanoic acid, undecanoic acid, lauric acid, benzoic acid, salicylic acid, 2-(4-hydroxybenzoyl)benzoic acid, camphoric acid, cinnamic acid, cyclopentanepropionic acid, 3-hydroxy-2-naphthoic acid, nicotinic acid, pamoic acid, pectinic acid, persulfuric acid, 3-phenylpropionic acid, picric acid, pivalic acid, 2-hydroxyethanesulfonic acid, itaconic acid, amidosulfonic acid, trifluoromethanesulfonic acid, dodecylsulfuric acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, methanesulfonic acid, 2-naphthalenesulfonic acid, naphthalenedisulfonic acid, camphorsulfonic acid, citric acid, tartaric acid, stearic acid, lactic acid, oxalic acid, malonic acid, succinic acid, malic acid, adipic acid, alginic acid, maleic acid, fumaric acid, D-gluconic acid, mandelic acid, ascorbic acid, glucoheptanoic acid, glycerophosphoric acid, aspartic acid, sulfosalicylic acid, or thiocyanic acid.

[0112] In addition, another suitable pharmaceutically acceptable salt of a compound of the present disclosure having sufficient acidity is an alkali metal salt, such as a sodium salt or a potassium salt, an alkaline earth metal salt such as a calcium salt or a magnesium salt, an ammonium salt (e.g., with NH 3or a salt formed with ammonia water), or a salt formed with an organic base that provides a physiologically acceptable cation, such as a salt formed with: triethylamine, N-methylglucosamine, dimethylglucosamine, ethylglucosamine, lysine, dicyclohexylamine, 1,6-hexanediamine, ethanolamine, glucosamine, sarcosine, serinol, tris(hydroxymethyl)aminomethane, aminopropanediol, 1-amino-2,3,4-butanetriol. Additionally, the basic nitrogen-containing groups can be quaternized with the following reagents: lower alkyl halides, such as methyl, ethyl, propyl, and butyl chlorides, bromides, and iodides; dialkyl sulfates, such as dimethyl sulfate, diethyl sulfate, dibutyl sulfate, and dipentyl sulfate; long-chain halides such as decyl, lauryl, myristyl, and stearyl chlorides, bromides, and iodides; aralkyl halides such as benzyl and phenethyl bromides, etc.

[0113] Those skilled in the art will also recognize that the acid addition salts of the compounds of formula (I) described in the present disclosure can be prepared by reacting the compounds with a suitable inorganic or organic acid by any of the known methods. Alternatively, the base addition salts of the acidic compounds of the present disclosure are prepared by reacting them with a suitable base by various known methods.

[0114] The present disclosure includes all possible salts of the compounds of formula (I) described in the present disclosure, which can be a single salt or any mixture of the salts in any proportion.

[0115] Some compounds of the present disclosure can exist in the form of one or more stereoisomers. Stereoisomers include geometric isomers, diastereoisomers, and enantiomers. Therefore, the compounds of formula (I) described in the present disclosure also include racemic mixtures, single stereoisomers, and optically active mixtures. Those skilled in the art should understand that one stereoisomer may have better efficacy and / or lower side effects than other stereoisomers. Single stereoisomers and optically active mixtures can be obtained by methods such as chiral source synthesis, chiral catalysis, and chiral resolution. Racemates can be subjected to chiral resolution by chromatographic resolution or chemical resolution. For example, chiral acid resolving agents such as chiral tartaric acid and chiral malic acid can be added to form salts with the compounds of the present disclosure, and separation can be carried out using the physical and chemical properties of the products, such as different solubilities.

[0116] In the present disclosure, when the name of a compound is inconsistent with the structural formula, the structural formula shall prevail.

[0117] The specification of the present disclosure should be interpreted in accordance with the laws and principles of chemical bonds. In some cases, hydrogen atoms may be removed in order to accommodate substituents at a given position.

[0118] It should be understood that the terms "compounds of the present disclosure" and "compounds" used in the present disclosure may, depending on the context, refer to: compounds of formula (I), their solvates, their pharmaceutically acceptable salts, their stereoisomers, or mixtures thereof.

[0119] On the basis of conforming to the common general knowledge in the art, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred examples of the present invention.

[0120] Unless otherwise specified, the reagents and raw materials used in the present disclosure can be obtained through commercial channels.

[0121] The positive and progressive effects of the present disclosure are as follows: The compounds represented by formula (I) provided in the present disclosure are mRNA capping analogs for self-replication and have at least one of the following advantages:

[0122] (1) For the mRNA capping analog of the present application, both the in vitro transcription yield and integrity of mRNA are significantly improved;

[0123] (2) For the mRNA capping analog of the present application, the translation efficiency of mRNA is significantly improved. BRIEF DESCRIPTION OF THE DRAWINGS

[0124] In order to more clearly illustrate the specific embodiments of the present disclosure or the technical solutions in the prior art, the following will briefly introduce the drawings required for use in the description of the specific embodiments or the prior art. Obviously, the drawings in the following description are some exemplary embodiments of the present disclosure, rather than limitations on the present disclosure. For those of ordinary skill in the art, various changes made within the spirit and scope of the present disclosure can also be regarded as the scope intended by the drawings of the present disclosure.

[0125] Figure 1 It is a graph showing the results of in vitro transcription yield of mRNA with YK-CAP-201, YK-CAP-202, YK-CAP-203, YK-CAP-204, YK-CAP-205, YK-CAP-206, YK-CAP-207 and N-7114 as capping analogs.

[0126] Figure 2 It is a graph showing the integrity results of mRNA transcription starting with YK-CAP-201, YK-CAP-202, YK-CAP-203, YK-CAP-204, YK-CAP-205, YK-CAP-206, YK-CAP-207 and N-7114 as capping analogs.

[0127] Figure 3The figure of capping rate of mRNA transcription initiated by YK-CAP-201, YK-CAP-202, YK-CAP-203, YK-CAP-204, YK-CAP-205, YK-CAP-206, YK-CAP-207 and N-7114 as capping analogs.

[0128] Figure 4 The figure of translation efficiency of capped mRNA with YK-CAP-201, YK-CAP-202, YK-CAP-203, YK-CAP-204, YK-CAP-205, YK-CAP-206, YK-CAP-207 and N-7114 as capping analogs.

[0129] Figure 5 The figure of in vivo expression of capped mRNA with YK-CAP-201, YK-CAP-202, YK-CAP-203, YK-CAP-204, YK-CAP-205, YK-CAP-206, YK-CAP-207 and N-7114 as capping analogs. Detailed implementation manners

[0130] The present disclosure will be further described below in conjunction with embodiments, but the present disclosure is not limited to the following embodiments. The implementation conditions adopted in the embodiments can be further adjusted according to different requirements of specific use, and the implementation conditions not specifically specified are conventional conditions in the art. In the specific embodiments of the present disclosure, the raw materials used can all be obtained through commercial channels. Unless otherwise specified, all temperatures are given in degrees Celsius. The technical features involved in each implementation manner of the present disclosure can be combined with each other as long as they do not conflict with each other.

[0131] The following abbreviated letters represent the following reagents respectively:

[0132] TEA: Triethylamine; DMF: N, N-Dimethylformamide; PySSPy: 2, 2'-Dithiopyridine; PPh 3 : Triphenylphosphine; TEAB: Triethylamine bicarbonate; EDTA: Ethylenediaminetetraacetic acid; DSPC: 1, 2-Distearoyl-sn-glycero-3-phosphocholine; DMG-PEG 2000: 1,2-Dimyristoyl-sn-glycero-3-methoxypolyethylene glycol 2000.

[0133] Example 1: Synthesis of capping analogs for self-replicating mRNA

[0134] 1. Synthesis of intermediate INT-I

[0135]

[0136] Dissolve pA(2'-OMe)mpU•TEA (3.10 g, 4.64 mmol), imidazole (1.58 g, 23.22 mmol), 2,2'-dithiopyridine (2.04 g, 9.28 mmol), triethylamine (0.47 g, 4.64 mmol) and triphenylphosphine (2.43 g, 9.28 mmol) in 31 mL of ultradry N,N-dimethylformamide, and place it at 25 o °C under nitrogen protection for 4 h. After the reaction is completed, slowly add the reaction solution to a pre-cooled acetone (150 mL) solution containing sodium iodide (5.08 g, 33.89 mmol), and crystallize at 25 o °C for 30 min, centrifuge, and lyophilize to obtain white solid INT-I (2.50 g, 3.28 mmol, yield 75.0%). C 23 H 29 N 9 O 14 P 2， MS(ES): m / z (M-H) - 716.1。

[0137] 2. Synthesis of YK-CAP-201

[0138]

[0139] Dissolve YK-CAP-201-PM1 (synthesized according to the synthesis route of YK-CAP-107-PM11 in reference patent CN118389495B) (130 mg, 0.26 mmol) and INT-I (513 mg, 0.72 mmol) in DMF, add ZnCl 2 ,N 2 Protect and stir at 37°C for 72 hours. After the reaction is completed, pour the reaction solution into an aqueous solution of EDTA, stir for 20 min, adjust the pH to 6.5 with ammonia water, purify by gel column (elute with water and 1 M TEAB = 3:1), collect the target product peak, concentrate, and lyophilize. Further purify by high-performance preparative liquid phase to obtain the final product YK-CAP-201 (54 mg, 47 μmol, 18.1%), C 34 H 49 N 12 O 25 P 4 ,MS(ES): m / z (M-H) - 1147.2. 1 H NMR (400 MHz, D 2O) δ 9.09 (s, 1H), 8.43 (s, 1H), 8.17 (s, 1H), 7.41 (d, J = 2.4 Hz, 1H), 6.03 (d, J = 4.4 Hz, 1H), 5.71 (d, J = 5.6 Hz, 1H), 5.47 (d, J = 2.4 Hz, 1H), 4.90 - 4.83 (m, 1H), 4.67 (t, J = 5.4 Hz, 2H), 4.52 (d, J = 5.2 Hz, 1H), 4.47 - 4.43 (m, 2H), 4.42 - 4.38 (m, 2H), 4.34 (t, J = 4.8 Hz, 1H), 4.31 - 4.23 (m, 2H), 4.21 - 4.15 (m, 1H), 4.14 - 4.11 (m, 2H), 4.10 - 4.06 (m, 1H), 3.93 (s, 3H), 3.66 - 3.55 (m, 1H), 3.42 (s, 3H), 3.25 (s, 3H), 2.33 - 2.24 (m, 1H), 1.12 (d, J = 6.0 Hz, 3H). 31 P NMR(162 MHz, D 2 O) δ -0.88, -11.46, -11.60, -22.76 (4P).

[0140] 3. Synthesis of YK-CAP-202

[0141]

[0142] Using YK-CAP-202-PM1 (synthesized according to the synthesis route of YK-CAP-108-PM10 in the reference patent CN118389495B) (100 mg, 0.19 mmol) and INT-I (179 mg, 0.25 mmol) as raw materials, following the synthesis route of YK-CAP-201, the final product YK-CAP-202 (20 mg, 17 µmol, 9.0%) was obtained, C 35 H 49 N 13 O 25 P 4 , MS(ES): m / z (M-H) - 1174.1. 1 H NMR (400 MHz, D 2O) δ 9.06 (s, 1H), 8.36 (s, 1H), 8.12 (s, 1H), 7.36(d, J = 2.4 Hz, 1H), 6.01 (d, J = 4.4 Hz, 1H), 5.68 (d, J = 5.6 Hz, 1H), 5.44(d, J = 2.4 Hz, 1H), 4.90 - 4.80 (m, 1H), 4.64 (t, J = 5.4 Hz, 2H), 4.48 (d,J = 5.2 Hz, 1H), 4.44 - 4.40 (m, 2H), 4.36 - 4.32 (m, 2H), 4.29 (t, J = 4.8Hz, 1H), 4.26 - 4.19 (m, 2H), 4.18 - 4.12 (m, 1H), 4.10 - 4.08 (m, 2H), 4.06- 4.01 (m, 1H), 3.88 (s, 3H), 3.63 - 3.51 (m, 1H), 3.40 (s, 3H), 2.29 - 2.20(m, 1H), 1.90 (s, 3H), 1.08 (d, J = 6.0 Hz, 3H). 31 P NMR (162 MHz, D 2 O) δ -0.89, -11.47, -11.63, -22.77 (4P).

[0143] 4. Synthesis of YK-CAP-203

[0144]

[0145] Using YK-CAP-203-PM1 (synthesized according to the synthesis route of YK-CAP-109-PM8 in the reference patent CN118389495B) (100 mg, 0.17 mmol) and INT-I (179 mg, 0.25 mmol) as raw materials, following the synthesis route of YK-CAP-201, the final product YK-CAP-203 (15 mg, 13 µmol, 6.5%) was obtained, C 33 H 45 FN 12 O 24 P 4 , MS(ES): m / z (M-H) - 1135.1. 1 HNMR (400 MHz, D 2O) δ 9.08 (s, 1H), 8.42 (s, 1H), 8.15 (s, 1H), 7.40 (d, J = 2.4 Hz, 1H), 6.17 (d, J = 4.4 Hz, 1H), 5.65 (d, J = 2.6 Hz, 1H), 5.47 (d, J = 2.4 Hz, 1H), 5.32 - 5.21 (m, 1H), 4.62 - 4.42 (m, 3H), 4.36 - 4.33 (m, 4H), 4.22 - 4.15 (m, 3H), 4.11 (s, 2H), 4.03 (s, 1H), 3.91 - 3.82 (m, 3H), 3.21 - 3.15 (m, 3H), 3.11 (s, 2H), 2.31 - 2.22 (m, 1H), 1.52 (d, J = 6.1 Hz, 3H). 31 PNMR (163MHz, D 2 O) δ -0.89, -11.21, -11.74, -22.12 (4P).

[0146] 5. Synthesis of YK-CAP-204

[0147]

[0148] Using YK-CAP-204-PM1 (synthesized according to the synthesis route of YK-CAP-110-PM8 in the reference patent CN118389495B) (180 mg, 0.31 mmol) and INT-I (470 mg, 0.62 mmol) as raw materials, following the synthesis route of YK-CAP-201, the final product YK-CAP-204 (71 mg, 49 µmol, 15.8%) was obtained, C 32 H 39 F 2 N 12 O 24 P 4 , MS(ES): m / z (M-H) - 1138.62. 1 H NMR (400 MHz, D 2O) δ 9.01 (s, 1H), 8.33 (s, 1H), 8.06 (s, 1H), 7.35 (d, J = 2.4 Hz, 1H), 6.30 - 5.99 (m, 1H), 5.96 (d, J = 5.2 Hz, 1H), 5.75 - 5.71 (m, 2H), 5.40 (d, J = 2.4 Hz, 1H), 4.90 - 4.82 (m, 1H), 4.81 - 4.76 (m, 1H), 4.68 - 4.61 (m, 4H), 4.45 - 4.31 (m, 6H), 4.30 - 4.21 (m, 3H), 4.20 - 4.10 (m, 4H), 4.07 - 4.00 (m, 2H), 3.93 (s, 4H), 3.38 (s, 4H), 2.97 - 2.85 (m, 1H). 31 P NMR (162 MHz, D 2 O) δ -0.88, -11.50, -11.64, -22.76 (4P).

[0149] 6. Synthesis of YK-CAP-205

[0150]

[0151] Using YK-CAP-205-PM1 (synthesized according to the synthesis route of YK-CAP-111-PM10 in the reference patent CN118389495B) (120 mg, 0.22 mmol) and INT-I (477 mg, 0.67 mmol) as raw materials, following the synthesis route of YK-CAP-201, the final product YK-CAP-205 (17 mg, 14 μmol, 6.5%) was obtained, C 36 H 52 N 13 O 25 P 4 , MS(ES): m / z (M-H) - 1188.2. 1 H NMR (400 MHz, D 2O) δ 9.11 (s, 1H), 8.82 (s, 1H), 8.60 (s, 1H), 7.51(d, J = 2.4 Hz, 1H), 6.09 (d, J = 4.8 Hz, 1H), 5.82 (d, J = 3.6 Hz, 2H), 5.53(d, J = 2.4 Hz, 1H), 4.90 - 4.84 (m, 2H), 4.82 - 4.78 (m, 1H), 4.62 (t, J =4.4 Hz, 1H), 4.52 - 4.36 (m, 5H), 4.34 - 4.09 (m, 6H), 4.08 - 4.00 (m, 1H),3.95 (s, 3H), 3.72 (t, J = 6.4 Hz, 1H), 3.43 (s, 3H), 3.39 - 3.21 (m, 5H),1.05 (dt, J = 21.2, 7.2 Hz, 7H). 31 P NMR (162 MHz, D 2 O) δ -0.92, 11.48, -11.73, -22.57 (4P).

[0152] 7. Synthesis of YK-CAP-206

[0153]

[0154] Using YK-CAP-206-PM1 (synthesized according to the synthesis route of YK-CAP-118-PM8 in the reference patent CN118389495B) (200 mg, 0.41 mmol) and INT-I (954 mg, 1.34 mmol) as raw materials, following the synthesis route of YK-CAP-201, the final product YK-CAP-206 (44 mg, 39 μmol, 9.4%) was obtained, C 34 H 44 N 12 O 25 P 4 , MS(ES): m / z (M-H) - 1137.2. 1 H NMR (400 MHz, D 2O) δ 9.01 (s, 1H), 8.36 (s, 1H), 8.09 (s, 1H), 7.35(d, J = 2.4 Hz, 1H), 5.95 (d, J = 5.2 Hz, 1H), 5.86 (d, J = 5.2 Hz, 1H), 5.73(d, J = 5.6 Hz, 1H), 5.40 (d, J = 2.4 Hz, 1H), 4.87 - 4.81 (m, 1H), 4.68 -4.64 (m, 2H), 4.53 (d, J = 5.6 Hz, 1H), 4.47 - 4.35 (m, 4H), 4.33 (t, J = 5.2Hz, 2H), 4.28 - 4.21 (m, 2H), 4.14 - 4.09 (m, 3H), 3.95 (s, 3H), 3.69 (d, J =10.8 Hz, 1H), 3.59 (d, J = 10.8 Hz, 1H), 3.35 (s, 3H). 31 P NMR (162 MHz, D 2 O)δ -0.90, -11.41, -11.48, -22.68 (4P).

[0155] 8. Synthesis of YK-CAP-207

[0156]

[0157] Using YK-CAP-207-PM1 (synthesized according to the synthesis route of YK-CAP-117-PM8 in the reference patent CN118389495B) (180 mg, 0.35 mmol) and INT-I (514 mg, 0.71 mmol) as raw materials, following the synthesis route of YK-CAP-201, the final product YK-CAP-207 (21 mg, 18 µmol, 5.0%) was obtained, C 33 H 47 N 12 O 26 P 4 , MS(ES): m / z (M-H) - 1149.18. 1 H NMR (400 MHz, D 2O) δ 9.01 (s, 1H), 8.36 (s, 1H), 8.09 (s, 1H), 7.35 (d, J = 2.4 Hz, 1H), 5.95 (d, J = 5.2 Hz, 1H), 5.86 (d, J = 5.2 Hz, 1H), 5.73 (d, J = 5.6 Hz, 1H), 5.40 (d, J = 2.4 Hz, 1H), 4.87 - 4.81 (m, 1H), 4.68 - 4.64 (m, 2H), 4.53 (d, J = 5.6 Hz, 1H), 4.44 - 4.39 (m, 2H), 4.33 (t, J = 5.2 Hz, 2H), 4.28 - 4.21 (m, 2H), 4.19 - 4.15 (m, 2H), 4.14 - 4.09 (m, 3H), 3.95 (s, 3H), 3.69 (d, J = 10.8 Hz, 1H), 3.59 (d, J = 10.8 Hz, 1H), 3.38 (s, 3H), 3.35 (s, 3H). 31 P NMR (162 MHz, D 2 O) δ -0.92, -11.44, -11.55, -22.74(4P).

[0158] Example 2: mRNA in vitro transcription yield and capping rate

[0159] I. Structural differences of capping analogs

[0160] Table 1 Structures of capping analogs

[0161]

[0162]

[0163]

[0164] As can be seen from Table 1, the chemical structures of the compounds YK-CAP-201 to YK-CAP-207 of the present application are similar to each other, specifically as follows:

[0165] 1. For the compounds YK-CAP-201 to YK-CAP-207 of the present application, only the substituents at C3 and / or C4 on the first sugar ring are different.

[0166] 2. The substituent at C3 on the first sugar ring of the compounds YK-CAP-201 to YK-CAP-205 in this application is different from that of N-7114: the C3 substituent of N-7114 is a hydroxyl group; the C3 substituents of YK-CAP-201 to YK-CAP-205 are 1-methoxyethyl, 1-acetamidoethyl, 1-fluoroethyl, difluoromethyl, and N,N-diethylacetamido, respectively.

[0167] 3. The substituent at C4 on the first sugar ring of the compounds YK-CAP-206 to YK-CAP-207 in this application is different from that of N-7114: the C4 substituent of N-7114 is a hydrogen atom; the C4 substituents of YK-CAP-206 to YK-CAP-207 are 1-fluoromethyl and methoxymethyl, respectively.

[0168] II. mRNA in vitro transcription yield and capping rate

[0169] 1. Experimental method

[0170] 1.1 Preparation of DNA template

[0171] 1) The luciferase (Luciferase protein CDS) was digested with EcoRV and ligated to the pVAX1 vector (Thermo Fisher Scientific).

[0172] 2) The plasmid constructed on the pVAX1 vector in step 1) was mixed with 50 μL of Escherichia coli competent cells Stbl2 (Thermo Fisher Scientific) and incubated on ice for 30 minutes, heat-shocked at 42 °C for 90 seconds, immediately placed back on ice, and incubated on ice for 2 minutes.

[0173] 3) 400 μL of LB medium (Thermo Fisher Scientific) was added, and the mixture was gently shaken and incubated on a shaker at 30 °C for 45 - 60 minutes.

[0174] 4) 50 - 100 μL of the bacterial solution was spread on an LB solid medium containing kanamycin antibiotic (100 μg / mL, Yeasen Biotech Co., Ltd.) and incubated upside down at 37 °C overnight.

[0175] 5) The resulting monoclonal colony plate was sequenced to verify its correctness, and the monoclonal colony with correct sequencing was picked and incubated on a shaker at 30 °C overnight.

[0176] 6) A plasmid extraction kit without endotoxin (MolPure® Endo-free Plasmid Maxi Kit, Yeasen Biotech Co., Ltd., product number: 19036ES10) was used for plasmid extraction.

[0177] 7) The extracted plasmid was digested with a restriction endonuclease to obtain a linearized plasmid, which was used as a transcription template. The specific steps of the digestion process are described in steps ① to ③ below.

[0178] Step ①: Take 1 mg of luciferase circular plasmid and digest it at 37 °C for 4 hours with BspQ I enzyme (purchased from Yeasen Biotech Co., Ltd.) to obtain a linearized DNA transcription template (the digestion reaction system is shown in Table 2).

[0179] Table 2 Digestion reaction system

[0180]

[0181] Step ②: After the reaction is completed, in the digestion reaction product solution, add anhydrous ethanol and 3 M sodium acetate in a volume ratio of V 酶切反应产物 :V 无水乙醇 :V 3M醋酸钠 = 1:3:1 in sequence, place it at -20 °C for precipitation for 1 hour, and then centrifuge at 12000 rmp to retain the precipitate.

[0182] Step ③: Wash the precipitate obtained in step ② twice with 70% ethanol and centrifuge. The centrifuged material is placed in an oven at 55 °C for drying for 10 minutes, and then 1.7 mL of injection water is added for dissolution.

[0183] The concentration of the linearized plasmid in the dissolution solution is 500 ng / µL, the linearization ratio is more than 90%, and the purification and recovery efficiency is 85%.

[0184] 1.2 In vitro transcription and synthesis of mRNA

[0185] 1) Co-transcriptional capping reaction:

[0186] Use YK-CAP-201 ~ YK-CAP-207 and N-7114 synthesized in Example 1 as capping analogs respectively. Using the Fluc DNA prepared in "1.1 Preparation of DNA template" above as a template and NTP solution (NTPs) as the starting material, mRNA is transcribed and synthesized by T7 RNA polymerase. The specific reaction system is shown in Table 3. The prepared reaction system is placed in a constant temperature incubator at 37 °C and oscillated for reaction for 3 h.

[0187] Table 3 Co-transcriptional capping reaction system

[0188]

[0189] Note: N-7114 in the capping analog was purchased from Cangzhou Weikexin Biochemical Technology Co., Ltd., and other capping analogs were synthesized with reference to the foregoing content of this application; other reagents except the capping analogs were purchased from Yeasen Biotech Co., Ltd.

[0190] 2) Digest the template DNA:

[0191] Add DNase I (Yeasen Biotechnology Co., Ltd.) to the co-transcriptional capping reaction system after the completion of the reaction in step 1) above, so that the final concentration of DNase I is equivalent to 1 U of DNase I per μg of linearized plasmid in the reaction solution. After mixing, centrifuge and incubate at 37 °C for 1 hour to obtain the co-transcriptional capping product.

[0192] 3) Purify by lithium chloride precipitation method:

[0193] Purify the co-transcriptional capping product obtained in step 2) above by the lithium chloride precipitation method as follows:

[0194] Step ① Add lithium chloride: Add lithium chloride solution (Thermo Fisher Scientific) to the product of step 2) above, with a final concentration of 2.8 M, and precipitate at low temperature for 2 hours;

[0195] Step ② Precipitation: Centrifuge at 12000 rmp for 15 minutes and retain the precipitate;

[0196] Step ③ Washing: Wash twice with 75% ethanol and dissolve with injection water to obtain the mRNA solution. Store the purified mRNA solution at -80 °C.

[0197] 1.3 Anneal the obtained mRNA with the probe

[0198] Perform annealing in a PCR instrument: 95 o °C for 5 min; 65 o °C for 2 min; 55 o °C for 2 min; 40 o °C for 2 min; 22 o °C for 2 min.

[0199] 1.4 Pretreat the magnetic beads and bind with the probe: Take 100 μL of magnetic beads and place them on a magnetic rack for pretreatment. Add 120 μL of the sample and incubate with the magnetic bead solution at room temperature for 30 min, mixing slowly during incubation.

[0200] 1.5 Shear the mRNA and obtain the 5' single-stranded sequence of the mRNA bound to the probe

[0201] Add 20 μL of RnaseH (5 U / μL), incubate at 37 o °C for 3 h, mixing once every half hour. After incubation, wash the magnetic beads. Add 100 μL of 75% methanol heated to 80 o °C to the washed magnetic beads, and heat the obtained mixture to 80 oIncubate at

[0202] (6)Use an Agilent 5200 Fragment Analyzer capillary electrophoresis system to evaluate the integrity of mRNA.

[0203] 1) Sample preparation: According to the sample concentration measured by a NanoDrop spectrophotometer in the early stage, dilute each sample to 20 - 100 ng / μL at different multiples with RNase-free water. Mix the samples and pre-aliquoted Ladder, then place them in a metal bath device (Hangzhou Youning, MiniH-100D) at 70 o °C for 2 min for denaturation, and immediately place on ice after taking out.

[0204] 2) Preparation of capillary reagents:

[0205] Table 4 Preparation of capillary reagents

[0206]

[0207] 3) Set the sample running program and load the sample for measurement: Set the injection voltage to 5 kV, running time to 4 s, separation voltage to 8 kV, and running time to 45 min.

[0208] 4) Data analysis: The Agilent Fragment Analyzer comes with ProSize analysis software. Users can batch calculate the percentage of RNA fragments that meet the requirements or the percentage of degraded RNA through Smear analysis.

[0209] 2. Experimental results

[0210] The measurement results of the in vitro transcription yield, integrity, and capping rate of mRNA show that the capping rates are basically the same and are at a very high level. Compared with the capping analogs for self-replicating mRNA in the prior art, the in vitro transcription yield and integrity of mRNA of the capping analogs for self-replicating mRNA of the present disclosure are significantly improved, and the capping rates are at a very high level. The specific in vitro transcription yield and capping rate of mRNA are shown in Table 5.

[0211] Table 5 Measurement results of the co-transcriptional capping reaction system

[0212]

[0213] As can be seen from Table 5, the capping analogs for self-replicating mRNA in the present application can all efficiently transcribe mRNA ( Figure 1 ). Compared with the capping analogs for self-replicating mRNA in the prior art, both the in vitro transcription yield and integrity of mRNA are significantly improved.

[0214] The in vitro transcription yield and integrity of mRNA of N-7114 ( Figure 2 ) are 175.3 μg and 60.1% respectively. The in vitro transcription yield and integrity of mRNA of YK-CAP-206 of the present application are increased by 18.6% and 18.1% respectively compared with N-7114.

[0215] From the in vitro transcription yield and integrity of mRNA, it can be seen that compared with N-7114 of the prior art, both the in vitro transcription yield and integrity of mRNA of the capping analogs for self-replicating mRNA of the present application are significantly improved, indicating that the capping analogs of the present application have excellent anti-retroviral effects when used for self-replicating mRNA, and can increase the binding ability between the cap structure and the capping enzyme, thereby increasing the capping rate of transcribed mRNA.

[0216] Example 3: Preparation and Characterization of Lipid Nanoparticles

[0217] 1. Experimental Method

[0218] Dissolve cationic lipid YK-009 (Beijing Yuekang Kechuang Pharmaceutical Technology Co., Ltd.), DSPC (A.V.T. (Shanghai) Pharmaceutical Technology Co., Ltd.), cholesterol (A.V.T. (Shanghai) Pharmaceutical Technology Co., Ltd.) and DMG-PEG 2000 in ethanol at a molar ratio of 49:10:39.5:1.5, and dilute the mRNA in 50 mM citrate buffer with pH = 4. Use a microfluidic device to mix the ethanol lipid solution with the aqueous solution of FLuc mRNA prepared from different cap structures at a flow rate of 10 mL / min at a volume ratio of 1:3 to prepare LNP with a mass ratio of total lipid to mRNA of about 15:1. After diluting the obtained liposome to 10 times its volume with PBS, ultrafiltration is carried out with a 300 KDa ultrafiltration tube to remove ethanol. Then, it is made up to a certain volume with PBS. Finally, the lipid nanoparticles are filtered through a 0.2 μm sterile filter to obtain an LNP preparation of YK-009 / DSPC / cholesterol / DMG-PEG2000 (molar ratio of 49:10:39.5:1.5) encapsulating self-replicating FLuc-mRNA.

[0219] The structural formula of YK-009 is as follows: .

[0220] The particle size and polydispersity index (PDI) were determined using a Malvern laser particle size analyzer by dynamic light scattering. 25 μL of the liposome solution was diluted to 125 μL with normal saline and added to the sample cell, and each sample was measured 3 times. The measurement conditions were: 90 o scattering angle, 25 o °C. According to the manufacturer's instructions, the encapsulation efficiency of the lipid nanoparticles was determined using the Quant-it Ribogreen RNA quantification assay kit (ThermoFisher Scientific, UK).

[0221] 2. Experimental results

[0222] The characterization data of the specific lipid nanoparticles are shown in Table 6.

[0223] Table 6 Characterization of lipid nanoparticles

[0224]

[0225] As can be seen from Table 6, self-replicating FLuc-mRNAs transcribed from the capping analogs YK-CAP-201 to YK-CAP-207 in this application and the capping analog N-7114 disclosed in the prior art can all be used to prepare good lipid nanoparticles. The particle sizes of all lipid nanoparticles are between 70 and 85 nm, the PDI values are between 0.031 and 0.096, and the encapsulation rates are all above 90%.

[0226] Example 4: Translation efficiency of luciferase mRNAs containing different cap structures

[0227] 1. Experimental method

[0228] (1) HEK293T cells were cultured in DMEM medium containing 10% FBS and penicillin / streptomycin at 37 o °C in 5% CO2.

[0229] (2) The cells in the culture dish were digested and counted, and 10,000 cells per well were seeded in a 96-well plate and cultured overnight until the cells adhered to the wall.

[0230] (3) When the cell density was approximately 80%, transfection was performed. 50 ng of the mRNA sample and Lipofectamine Messenger MAX Transfection Reagent (Invitrogen) were added to each well, and the transfection procedure was carried out according to the instructions.

[0231] (4) The transfected cells were placed at 37 o °C in 5% CO2 After continued incubation for 24 h, the growth medium was removed from the cells to be tested, and the cells were rinsed with PBS. After centrifuging to remove the PBS, 50 μL of 1x lysis buffer was added, the cells and all the liquid were transferred to a microcentrifuge tube, and then centrifuged.

[0232] (5) Take 20 μL of the sample, add 100 μL of Dual-Lumi™ II firefly luciferase detection reagent that has been equilibrated to room temperature, and mix well appropriately.

[0233] (6) Incubate at room temperature (about 25 o °C) for 5 min to stabilize the luminescence signal. Perform chemiluminescence detection using a multifunctional microplate reader with chemiluminescence detection function, and record the data (see Table 7 and Figure 4 ).

[0234] 2. Experimental results

[0235] The relative fluorescence readings of the capped mRNA are shown in Table 7, and the relative fluorescence intensity is proportional to the translation efficiency of the mRNA.

[0236] Table 7 Average fluorescence readings of capped mRNA

[0237]

[0238] It can be seen that the capped analog for self-replicating mRNA of the present application significantly improves the mRNA translation efficiency compared with the capped analogs for self-replicating mRNA in the prior art.

[0239] The average fluorescence intensity of N-7114 (corresponding to the translation efficiency of the mRNA) is 13241. The average fluorescence intensity of YK-CAP-206 is 3.0 times that of N-7114, and the average fluorescence intensity of YK-CAP-203 is 2.1 times that of N-7114.

[0240] The substituent at C3 on the first sugar ring of YK-CAP-201 to YK-CAP-205 of the present application is different from that of N-7114: the C3 substituent of N-7114 is a hydroxyl group; the C3 substituents of YK-CAP-201 to YK-CAP-205 are 1-methoxyethyl, 1-acetamidoethyl, 1-fluoroethyl, difluoromethyl, and N, N-diethylamide, respectively. However, the translation efficiencies of the corresponding mRNAs of YK-CAP-201, YK-CAP-203, YK-CAP-204, and YK-CAP-205 are 1.9 times, 2.1 times, 2.0 times, and 1.6 times that of N-7114, respectively.

[0241] From the translation efficiency of different capped luciferase mRNAs, it can be seen that the capping analogs for self-replicating mRNAs in this application, compared with the capping analog N-7114 for self-replicating mRNAs in the prior art, significantly improve the translation efficiency of mRNAs. This indicates that using the capping analogs of this application to modify ribose can more easily bind to the cap-binding protein (EIF4E), thereby improving the translation efficiency of the target mRNA.

[0242] Example 5: Determination of cell viability

[0243] 1. Experimental method

[0244] Add the LNP preparation containing 1 μg of self-replicating FLuc-mRNA prepared according to Example 3 to the cell culture medium in a 96-well plate. After culturing for 24 hours, add 10 μL of CCK-8 solution to each well. After incubating the culture plate in an incubator for 1 hour, measure the absorbance at 450 nm with an enzyme-linked immunosorbent assay (ELISA) reader. The results are shown in Table 8.

[0245] Cell viability can represent the toxicity of the transcribed self-replicating FLuc-mRNA to cells. The higher the cell viability, the lower the toxicity to cells.

[0246] 2. Experimental results

[0247] Table 8 Cell viability

[0248]

[0249] It can be seen that the self-replicating FLuc-mRNAs transcribed from the capping analogs YK-CAP-201 to YK-CAP-207 of this application and the capping analog N-7114 of the prior art all have low cytotoxicity, and the cell viability is greater than 90%.

[0250] Example 6: Animal experiment

[0251] 1. Experimental method

[0252] Inject the LNP preparation containing 5 μg of self-replicating FLuc-mRNA transcribed from the capping analog intramuscularly into female BALB / c mice aged 4 - 6 weeks and weighing 17 - 19 g, and intraperitoneally inject the fluorescence imaging substrate into the mice at 24 h, 48 h, 72 h, 120 h, and 192 h after administration. The mice are allowed to move freely for 5 minutes, and then the total radiation intensity of the protein expressed by the mRNA carried by the LNP in the mice is detected by an IVIS Spectrum small animal in vivo imaging system (which can reflect the expression level of the protein).

[0253] 2. Experimental results

[0254] The detection results are shown in Table 9 and Figure 5 . In the in vivo imaging experiment of mice, the multiple of the total radiation intensity of each group of mice relative to N-7114 is shown in Table 10 (where the total radiation intensity is the said value × 10 7 p / s).

[0255] Table 9 In vivo imaging experiment data of mice

[0256]

[0257] Table 10 Multiple of total radiation intensity relative to N-7114

[0258]

[0259] Compared with the capping analogs in the prior art, the total radiation intensity and duration of the proteins expressed by the mRNA transcribed from the capping analogs of the present application in mice are significantly improved. For example, the corresponding total radiation intensities of YK-CAP-206 are 2.5 times, 2.1 times, 2.4 times, 2.2 times and 2.3 times that of N-7114 at 24 h, 48 h, 72 h, 120 h and 192 h, respectively. The total radiation intensities of YK-CAP-205 at the corresponding times are 3.4 times, 2.0 times, 2.0 times, 1.7 times and 1.7 times that of N-7114, respectively.

[0260] In the present application, the substituent at C3 on the first sugar ring of compounds YK-CAP-201 to 205 is different from that of N-7114, that is, the C3 substituent of N-7114 is a hydroxyl group; the C3 substituents of YK-CAP-201 to 205 are 1-methoxyethyl, 1-acetamidoethyl, 1-fluoroethyl, difluoromethyl, N, N-diethylamide group, respectively. However, the total radiation intensities of the proteins expressed by YK-CAP-201, YK-CAP-203, YK-CAP-204 and YK-CAP-205 in mice at 24 h are 1.3 times, 2.4 times, 1.3 times and 3.4 times that of N-7114, respectively, and are 3.4 times, 6.3 times, 3.5 times and 8.8 times that of YK-CAP-202, respectively.

[0261] It can be seen from the animal experiments that, compared with the capping analog N-7114 in the prior art, the protein expression level and duration in mice of the mRNA transcribed from the capping analogs of the present application are significantly improved. The in vivo experiments further prove that the mRNA transcribed by YK-CAP-201, YK-CAP-203 to YK-CAP-206 in the present application can be effectively delivered into the body by the LNP delivery vector and expressed efficiently and continuously.

[0262] In summary, for the self-replicating mRNA capping analogs YK-CAP-201, YK-CAP-203 to YK-CAP-206 used for self-replicating RNA in this application, their corresponding in vitro transcription yields, integrity, capping rates, mRNA translation efficiencies of mRNA, as well as the protein expression levels and durations in animals are all significantly improved compared with the self-replicating mRNA capping analog N-7114 in the prior art. It shows that the capping analogs provided by this disclosure can significantly improve the affinity for binding to the capping enzyme, and provide a cap structure that can be highly effectively used for self-replicating mRNA for the in vitro transcription of mRNA.

[0263] 1. Compared with the capping analogs for self-replicating mRNA in the prior art, the capping analogs for self-replicating mRNA in this application have significantly improved in vitro transcription yields and integrity of mRNA. For example, the in vitro transcription yield and integrity of mRNA of N-7114 are 175.3 μg and 60.1% respectively, and the in vitro transcription yield and integrity of mRNA of YK-CAP-206 in this application are increased by 18.6% and 18.1% respectively compared with N-7114.

[0264] 2. Compared with the capping analogs for self-replicating mRNA in the prior art, the capping analogs for self-replicating mRNA in this application have significantly improved mRNA translation efficiency. For example, the translation efficiency of YK-CAP-206 in this application reaches 3.0 times that of N-7114.

[0265] The above embodiments are only used to illustrate the technical solutions of this disclosure, rather than to limit them; although this disclosure has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions recorded in the foregoing embodiments, or perform equivalent replacements for some or all of the technical features. Any modifications, equivalent replacements, and improvements made within the spirit and principle of this disclosure shall be included within the protection scope of the claims of this disclosure.

Claims

1. A compound represented by formula (I), a stereoisomer thereof or a pharmaceutically acceptable salt thereof; ， in, n is 0, 1, 2 or 3; R5 is -OR 5a 、-SR 5b 、-NR 5c R 5d 、-C(O)R 5e , 、-NHC(O)-R 5h 、-N(CH3)C(O)-R 5i 、-C(O)NR 5j R 5k 、-C(O)OR 5l 、-OC(O)-R 5m or -S(O)2-R 5n ; R 4a , R 5a , R 5b , R 5c , R 5d , R 5e , R 5h , R 5i , R 5j , R 5k , R 5l , R 5m and R 5n are independently -H, C1-C3 alkyl or halogenated C1-C3 alkyl; R 5f and R 5g are independently -H, halogen, C1-C3 alkyl, -OC1-C3 alkyl, halogenated C1-C3 alkyl or -NR 5-1 C(O)R 5-2 ; R 5-1 and R 5-2 are independently -H or C1-C3 alkyl; R1, R2, R3, R6 and R7 are each independently -H, C1-C6 alkyl, C 2- C6 alkenyl, C2-C6 alkynyl, C3-C6 cycloalkyl or benzyl; R4 is -OR 4a , -F, or -H; R8 is C1-C3 alkyl or -H.

2. The compound of formula (I) according to claim 1, its stereoisomer or a pharmaceutically acceptable salt thereof, characterized in that: The compound represented by formula (I) satisfies any one or a combination of at least two selected from the group consisting of the following conditions (1) to (4): (1) R4 is -H, -OC1-C3 alkyl or halogen; (2) R5 is -OH, or -C(O)NR 5j R 5k ; R 5f and R 5g are independently halogen, C1-C3 alkyl, -OC1-C3 alkyl or -NR 5-1 C(O)R 5-2 ; R 5j and R 5k are independently C1-C3 alkyl; R 5-1 H; R 5-2 is a C1-C3 alkyl group; (3) R2 is C1-C3 alkyl; and (4) R6 is a C1-C3 alkyl group.

3. The compound of formula (I) according to claim 1, its stereoisomer or a pharmaceutically acceptable salt thereof, characterized in that: The compound represented by formula (I) satisfies any one or a combination of at least two selected from the group consisting of the following conditions (1) to (9): (1) R4 is -H, -F or -OCH3; (2) R5 is -OH, , , , or ; (3) R1 is -H; (4) R2 is -CH3; (5) R3 is -H; (6) R6 is -CH3; (7) R7 is -H; (8) R8 is -H; and (9)n is 0 or 1.

4. The compound of formula (I) according to claim 1, its stereoisomer or a pharmaceutically acceptable salt thereof, characterized in that: n is 0 or 1; R4 is -H or halogen; R5 is -OH, or -C(O)NR 5j R 5k ; R 5f and R 5g R is independently halogen, C1-C3 alkyl or -OC1-C3 alkyl; 5j and R 5k are independently C1-C3 alkyl; R1 is -H; R2 is C1-C3 alkyl; R3 is -H; R6 is C1-C3 alkyl; R7 is -H; R8 is -H.

5. The compound of formula (I) according to claim 1, its stereoisomer or a pharmaceutically acceptable salt thereof, characterized in that: The pharmaceutically acceptable salt of the compound represented by formula (I) is any of the following structures: 、 、 、 、 、 or 。 6. Use of the compound of formula (I) as described in any one of claims 1 to 5, its stereoisomer or a pharmaceutically acceptable salt thereof in the preparation of an in vitro co-transcribed mRNA capping agent.

7. An RNA molecule, characterized in that It comprises the compound represented by formula (I) as claimed in claim 1, its stereoisomer or a pharmaceutically acceptable salt thereof as a cap structure or a cap structure fragment.

8. A pharmaceutical composition, characterized in that It comprises the RNA molecule according to claim 7.

9. The pharmaceutical composition according to claim 8, characterized in that The pharmaceutical composition further comprises at least one delivery agent.

10. The pharmaceutical composition according to claim 9, characterized in that The at least one delivery agent is selected from any one or a combination of at least two of the group consisting of cationic lipids, neutral lipids, structured lipids and polymer-conjugated lipids.

11. The pharmaceutical composition according to claim 10, characterized in that The pharmaceutical composition satisfies any one or a combination of at least two selected from the group consisting of the following conditions (1) to (4): (1) The cationic lipid is any one of the following or a combination of at least two of them: (i) A compound represented by formula (II), a stereoisomer thereof, an N-oxide thereof, a solvate thereof or a pharmaceutically acceptable salt thereof, wherein G1 is C 1-6 Alkylene; G2 is C 2-8 Alkylene; G3 is C 1-3 Alkylene; L1 is C 6-15 Straight chain alkyl; L2 is C 12-25 Branched chain alkyl; (II) (ii) A compound represented by formula (III), a stereoisomer thereof, an N-oxide thereof, a solvate thereof or a pharmaceutically acceptable salt thereof, wherein G1 is C 2-8 Alkylene; G2 is C 2-8 Alkylene; L1 is -C(O)O- or -OC(O)-; L2 is -C(O)O- or -OC(O)-; R1 is C 6-25 Straight or branched alkyl; R2 is C 6-25 Straight or branched alkyl; G3 is HO(CH2)2- or HO(CH2)3-; G4 is HO(CH2)2- or HO(CH2)3-; L is (CH2)2-, -(CH2)3- or -(CH2)4-; (III) (iii) A compound represented by formula (IV), a stereoisomer thereof, an N-oxide thereof, a solvate thereof or a pharmaceutically acceptable salt thereof, wherein: G1 is C 1-6 Alkylene; G2 is C 2-8 Alkylene; R1 is C 6-20 Straight or branched alkyl; R2 is C 12-25 Branched alkyl; G3 is HO(CH2)2N(CH3)(CH2)2-, HO(CH2)2N(CH2CH3)(CH2)2-, (HO(CH2)2)2N(CH2)2-, CH3O(CH2)2N(CH3)(C H2)2-, (CH3)2N(CH2)3SC(O)O(CH2)2-, (CH3)2N(CH2)3SC(O)-, CH3NH(CH2)2N(CH3)(CH2)2- or CH3CH2NH(CH2)2-; (IV) (iv) A compound represented by formula (V), a stereoisomer thereof, an N-oxide thereof, a solvate thereof or a pharmaceutically acceptable salt thereof, wherein G1 is C 1-8 Alkylene; G2 is C 2-8 Alkylene; R1 is C 6-25 Straight or branched alkyl; R2 is C 12-25 Straight or branched alkyl; G3 is: HO(CH2)2N(R3)CH2CH(OH)CH2-, wherein R3 is -CH3, -CH2CH3 or -CH2CH2OH; (V) (v) A compound represented by formula (VI), a stereoisomer thereof, an N-oxide thereof, a solvate thereof, or a pharmaceutically acceptable salt thereof, wherein G 1 and G 2 Each independently is C6-C 10 Alkylene; G 3 C1-C 12 Alkylene; R 1 and R 2 Each independently is C6-C 24 Alkyl or C6-C 24 Alkenyl; R 3 OR 5 、N(R 5 )2, -C(=O)OR 4 、-OC(=O)R 4 or -NR 5 C(=O)R 4 ; R 4 C1-C 12 Hydrocarbon; R 5 is H or a C1-C6 hydrocarbon group; (WE) (vi) A compound represented by formula (VII), a stereoisomer thereof, an N-oxide thereof, a solvate thereof, or a pharmaceutically acceptable salt thereof, wherein R4 is -(CH2) n Q; Q is -OR, -OH, -O(CH2) n wherein n is 1, 2 or 3; R is independently C(S) or C(O)R; wherein n is 1, 2 or 3; wherein R is independently C(O)N(R); wherein n is 1, 2 or 3; wherein R is independently C(O)N(R); wherein n is 1, 2 or 3; wherein R is independently C(O)N(R); wherein n is 1, 2 or 3; wherein R is independently C(O)N(R); wherein n is 1, 2 or 3; wherein R is independently C(O)N(R); wherein n is 1, 2 or 3; wherein R is independently C(O)N(R); wherein n is 1, 2 or 3; wherein R is independently C(O) 1-8 Alkyl; X is independently H or C 1-8 alkyl; (VII) (vii) a compound represented by formula (VIII), a stereoisomer thereof, an N-oxide thereof, a solvate thereof or a pharmaceutically acceptable salt thereof, (VIII); (2) the neutral lipid is selected from any one of the group consisting of phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, ceramide and sterol, or a combination of at least two thereof; (3) the structured lipid is selected from any one or a combination of at least two of the group consisting of cholesterol, non-sterols, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatine, ursolic acid, α-tocopherol and corticosteroids; and (4) The polymer conjugated lipid is any one selected from the group consisting of distearoylphosphatidylethanolamine polyethylene glycol 2000, 1,2-dimyristoyl-sn-glycero-3-methoxy polyethylene glycol 2000 and methoxy polyethylene glycol ditetradecanoyl acetamide, or a combination of at least two thereof.

12. The pharmaceutical composition according to claim 11, characterized in that The pharmaceutical composition satisfies any one or a combination of at least two selected from the group consisting of the following conditions (1) to (4): (1) The cationic lipid is selected from any one of the group consisting of YK-009, YK-201, YK-407, YK-305, ALC-0315, SM-102 and DLIN-MC3-DMA, or a combination of at least two of them: 、 、 、 、 、 、 ； (2) The neutral lipid is selected from 1, 2-dilinoleoyl-sn-glycero-3-phosphocholine, 1, 2-dimyristoyl-sn-glycero-phosphocholine, 1, 2-dioleoyl-sn-glycero-3-phosphocholine, 1, 2-dipalmitoyl-sn-glycero-3-phosphocholine, 1, 2-distearoyl-sn-glycero-3-phosphocholine, 1, 2-diondecanoyl-sn-glycero-phosphocholine, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, 1, 2-di-O-octadecenyl-sn-glycero-3-phosphocholine, 1-oleoyl-2-cholesteryl hemisuccinyl-sn-glycero-3-phosphocholine, 1-hexadecyl-sn-glycero-3-phosphocholine, 1, 2-Dilinolenoyl-sn-glycero-3-phosphocholine, 1, 2-Diarachidonoyl-sn-glycero-3-phosphocholine, 1, 2-Bisdocosahexaenoyl-sn-glycero-3-phosphocholine, 1, 2-Dioleoyl-sn-glycero-3-phosphoethanolamine, 1, 2-Diphytanoyl-sn-glycero-3-phosphoethanolamine, 1, 2-Distearoyl-sn-glycero-3-phosphoethanolamine, 1, 2-Dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1, 2-Dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1, 2-Diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1, 2-Bisdocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1, 2- 2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt, dipalmitoylphosphatidylglycerol, palmitoyloleoylphosphatidylethanolamine, distearoyl-phosphatidyl-ethanolamine, dipalmitoylphosphatidylethanolamine, dimyristoylphosphoethanolamine, 1-stearoyl-2-oleoyl-stearoylethanolamine, 1-stearoyl-2-oleoyl-phosphatidylcholine, sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoylphosphatidylcholine, lysophosphatidylcholine and lysophosphatidylethanolamine, or a combination of at least two of the following: (3) The structural lipid is cholesterol; and (4) The polymer conjugated lipid is 1,2-dimyristoyl-sn-glycero-3-methoxypolyethylene glycol 2000.

13. The pharmaceutical composition according to claim 12, characterized in that The cationic lipid is YK-009; and / or the neutral lipid is 1, 2-dioleoyl-sn-glycero-3-phosphoethanolamine and / or 1, 2-distearoyl-sn-glycero-3-phosphocholine.

14. The pharmaceutical composition according to claim 10, characterized in that The at least one delivery agent comprises YK-009, 1,2-distearoyl-sn-glycero-3-phosphocholine, cholesterol and 1,2-dimyristoyl-sn-glycero-3-methoxypolyethylene glycol 2000; the YK-009 is .

15. The pharmaceutical composition according to claim 14, characterized in that The molar ratio of YK-009, 1,2-distearoyl-sn-glycero-3-phosphocholine, cholesterol and 1,2-dimyristoyl-sn-glycero-3-methoxypolyethylene glycol 2000 is 49:10:39.5:1.

5.

16. The pharmaceutical composition according to any one of claims 8 to 15, characterized in that The pharmaceutical composition further comprises one or at least two cell penetrating peptides.

17. A method for synthesizing mRNA molecules for purposes other than disease diagnosis and treatment, characterized in that: The following steps are involved: The compound represented by formula (I) according to any one of claims 1 to 5, its stereoisomer, its pharmaceutically acceptable salt or its solvate is co-incubated with a polynucleotide template to transcribe the polynucleotide template.

18. A capped mRNA transcription reaction system for non-disease diagnosis and treatment purposes, characterized in that: include: (1) A compound of formula (I) according to any one of claims 1 to 5, a stereoisomer thereof or a pharmaceutically acceptable salt thereof; and (2) polynucleotide template, NTPs, and RNA polymerase.

19. A kit, characterized in that: include: (1) A compound of formula (I) according to any one of claims 1 to 5, a stereoisomer thereof or a pharmaceutically acceptable salt thereof; and (2) nucleotide triphosphate molecules, and RNA polymerase.

20. The kit according to claim 19, characterized in that The kit further comprises an RNase inhibitor, an inorganic pyrophosphatase, Mg 2+ , any one of the group consisting of a crowding agent and a buffer, or a combination of at least two of them.

21. A method for improving the stability of RNA, characterized in that: The method comprises incorporating the compound represented by formula (I) according to any one of claims 1 to 5, its stereoisomer or a pharmaceutically acceptable salt thereof into RNA.

22. A method for introducing RNA into a cell, characterized in that The method comprises contacting the cell with the RNA molecule of claim 7 or the pharmaceutical composition of any one of claims 8-15.

23. Use of the RNA molecule according to claim 7 or the pharmaceutical composition according to any one of claims 8 to 15 in the preparation of a vaccine.

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