A ribose-modified cap2 capping analog and uses thereof

Abstract

The application discloses a Cap2-capped analog with ribose modification and application thereof, and the Cap2-capped analog can improve unit template yield of mRNA and / or mRNA translation efficiency.

Description

TECHNICAL FIELD

[0001] The present disclosure belongs to the field of chemical and bioengineering technology, and relates to a ribose-modified Cap2 capping analogue and application thereof. BACKGROUND

[0002] The mRNA cap is a highly methylated modification at the 5' end of mRNA, which can protect mRNA from degradation, recruit complexes involved in mRNA processing, and mark cellular mRNA to avoid recognition by the immune system. In vitro synthesized RNA needs to complete the addition of 5' end cap structure through a certain synthesis process, so as to form a complete mRNA structure. The integrity of mRNA ensures the accurate translation of protein, and the 5'-Cap cap structure modification can protect mRNA from degradation by 5'-exonuclease activity, effectively promote the initiation of translation, and reduce immunogenicity.

[0003] In mammals, the main 5' cap structure is an inverted 7-methylguanosine nucleotide, which is connected to the first transcribed nucleotide by a 5'-5' triphosphate bond. The 7-methylguanosine is methylated at its 7 nitrogen position, which can be referred to as m7G or 7mG. This cap-like structure can be represented as 5'm7GpppN1(pN) X , wherein N is any nucleotide, and x is 0 or any number.

[0004] Under the action of guanine N-7 methyltransferase, the N7 position of guanine is methylated to form the cap structure Cap0 (m7GpppNpNp). Then under the action of 5'-O ribose methyltransferase, the 2'-O position of the first nucleotide of mRNA is methylated to form the cap structure Cap1, and the structure can contain m7G5'pppN12'-OMe (pN) X from 5' end to 3' end, wherein N is any nucleotide, and x can be any integer. The 2'-O positions of the first and second nucleotides of mRNA are both methylated to form the cap structure Cap2, and the structure can contain m7G5'pppN12'-OMepN22'-OMe (pN) X from 5' end to 3' end, wherein N is any nucleotide, and x can be any integer.

[0005] Cap2 can exist on all mRNAs, mRNA is slowly converted from Cap1 to Cap2 in the cytoplasm as it "ages", Cap2 is significantly enriched on mRNAs with longer half-lives, the increase of Cap1 level can activate the RIG-I mediated immune response, but the conversion of Cap1 to Cap2 can significantly reduce the ability of RNA binding and activation of RIG-I, the process of slow conversion of Cap1 to Cap2 in cells can enable the body to better cope with viruses. Therefore, the Cap2 cap structure analog can effectively improve the disadvantage of high immunogenicity of the cap structure analog in the existing mRNA synthesis system, and the Cap2 capping analog is chemically modified in the application, so as to further reduce the immunogenicity of mRNA while enhancing the translation efficiency. SUMMARY

[0006] In view of the deficiencies of the prior art, the purpose of the present application is to provide a ribose-modified Cap2 capping analog and its application. The ribose-modified Cap2 capping analog disclosed in the present application can improve the unit template yield of mRNA and / or the translation efficiency of mRNA.

[0007] The present disclosure provides a compound represented by formula (I) or a stereoisomer, a pharmaceutically acceptable salt or a solvate thereof,

[0008] ,

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

[0010] when R4 is -OR 4a or halogen, R5 is -OR 5a ;

[0011] when R4 is -OR 4a , halogen or -H, R5 is -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)O-R 5l , -OC(O)-R 5m or -S(O)2-R 5n ;

[0012] R 4a , R 5a , R 5b , R 5c , R 5d , R5e , R 5h , R 5i , R 5j , R 5k , R 5l , R 5m and R 5n are independently -H, C1-C3alkyl or halogenated C1-C3alkyl;

[0013] R 5f and R 5g are independently halogen, C1-C3alkyl, -OC1-C3alkyl, halogenated C1-C3alkyl or -NR 5-1 C(O)R 5-2 ;

[0014] R 5-1 and R 5-2 are independently -H or C1-C3alkyl;

[0015] R1, R2and R3are each independently -H, C1-C6alkyl, C 2- C6alkenyl, C2-C6alkynyl, C3-C6cycloalkyl or benzyl;

[0016] R6and R7are independently C1-C6alkyl, C 2- C6alkenyl, C2-C6alkynyl, C3-C6cycloalkyl or benzyl;

[0017] R8is C1-C3alkyl or -H;

[0018] X1, X2, X3and X4are one O — and the other three are -OH.

[0019] In some embodiments of the compound of Formula (I) or a stereoisomer, pharmaceutically acceptable salt or solvate thereof, the definitions of the moieties are as described below, and the definitions of the remaining moieties are as described in any other embodiment (hereinafter "in some embodiments").

[0020] In some embodiments, when R4is -OC1-C3alkyl or halogen, R5is -OH.

[0021] In some embodiments, when R4is -OC1-C3alkyl or halogen, and n is 1, 2 or 3, R5is -OH.

[0022] In some embodiments, when R4is -OCH3or F, R5is -OH.

[0023] In some embodiments, when R4is -OC1-C3alkyl, halogen or -H, R5is or -C(O)NR5j R 5k .

[0024] In some embodiments, when R4 is -H, R5 is or -C(O)NR 5j R 5k .

[0025] In some embodiments, when R4 is -H and n is 0, R5 is or -C(O)NR 5j R 5k .

[0026] In some embodiments, R 5f and R 5g are independently halogen, C1-C3 alkyl, or -OC1-C3 alkyl; R 5j and R 5k are independently C1-C3 alkyl.

[0027] In some embodiments, when R4 is -H, R5 is , , or .

[0028] In some embodiments, when R4 is -H, R5 is -CH(C1-C3 alkyl)(-OC1-C3 alkyl) or -CHF2.

[0029] In some embodiments, when R4 is -H, R5 is or .

[0030] In some embodiments, R1, R2, and R3 are each independently -H or C1-C6 alkyl.

[0031] In some embodiments, R1 is -H.

[0032] In some embodiments, R2 is C1-C6 alkyl, preferably C1-C3 alkyl.

[0033] In some embodiments, R2 is -CH3.

[0034] In some embodiments, R3 is -H.

[0035] In some embodiments, R6 and R7 are independently C1-C6 alkyl, preferably C1-C3 alkyl.

[0036] In some embodiments, R6 is -CH3.

[0037] In some embodiments, R7 is -CH3.

[0038] In some embodiments, R8 is -H.

[0039] In some embodiments, when R4 is -OC1-C3alkyl or halogen, and n is 1, R5 is -OH; when R4 is -H, and n is 0, R5 is -CH(C1-C3alkyl)(-OC1-C3alkyl) or -CHF2;

[0040] R1 and R3 are -H; R2 is C1-C3alkyl;

[0041] R6 and R7 are independently C1-C3alkyl;

[0042] R8 is -H;

[0043] one of X1, X2, X3 and X4 is O — and the remaining three are -OH.

[0044] In some embodiments, the pharmaceutically acceptable salt of the compound of Formula (I) is any one of the following structures:

[0045] , , , , or .

[0046] The present disclosure provides use of a compound of Formula (I) as described above, or a stereoisomer, a pharmaceutically acceptable salt, or a solvate thereof, in the preparation of an in vitro co-transcriptional mRNA capping reagent.

[0047] The present disclosure provides an RNA molecule comprising a compound of Formula (I) as described above, or a stereoisomer, a pharmaceutically acceptable salt, or a solvate thereof, as a cap structure or a cap structure fragment.

[0048] The present disclosure provides a pharmaceutical composition comprising an RNA molecule as described above.

[0049] In some embodiments, the pharmaceutical composition further comprises at least one delivery agent.

[0050] In some embodiments, the at least one delivery agent comprises at least one cationic lipid.

[0051] In some embodiments, the cationic lipid is one or at least two of the following compounds:

[0052] (i) a compound of Formula (II), a stereoisomer thereof, an N-oxide thereof, a solvate thereof, or a pharmaceutically acceptable salt thereof, wherein G1 is C1-6 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 alkyl groups;

[0053] (II)

[0054] (ii) The compound represented by formula (III), its stereoisomer, its N-oxide, its solvate or its pharmaceutically acceptable salt, 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-chain or branched alkyl; R2 is C 6-25 Straight-chain 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-;

[0055] (III)

[0056] (iii) The compound shown in formula (IV), its stereoisomer, its N-oxide, its solvate or its pharmaceutically acceptable salt, wherein: G1 is C 1-6 Alkylene; G2 is C 2-8 Alkylene; R1 is C 6-20 Straight-chain 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-;

[0057] (IV)

[0058] (iv) The compound represented by formula (V), its stereoisomers, its N-oxides, its solvates or pharmaceutically acceptable salts thereof, wherein G1 is C 1-8 Alkylene; G2 is C 2-8 Alkylene; R1 is C 6-25straight or branched alkyl; R2is C 12-25 straight or branched alkyl; G3is: HO(CH2)2N(R3)CH2CH(OH)CH2- wherein R3is -CH3, -CH2CH3, or -CH2CH2OH ;

[0059] (V)

[0060] (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 are each independently C6-C 10 alkylene; G 3 is C1-C 12 alkylene; R 1 and R 2 are each independently C6-C 24 alkyl or C6-C 24 alkenyl; R 3 is -OR 5 , -N(R 5 )-, -C(=O)OR 4 , -OC(=O)R 4 , or -NR 5 C(=O)R 4 ; R 4 is C1-C 12 alkyl; R 5 is H or C1-C6alkyl;

[0061] (VI)

[0062] (vi) a compound represented by Formula (VII), a stereoisomer thereof, an N-oxide thereof, a solvate thereof, or a pharmaceutically acceptable salt thereof, wherein R4is -(CH2) n Q; Q is -OR, -OH, -O(CH2) n N(R)2, -OC(O)R, -CX3, -CN, -N(R)C(O)R, -N(H)C(O)R, -N(R)S(O)2R, -N(H)S(O)2R, -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)2R, or a heterocycle; n is 1, 2, or 3; R is C 1-8 alkyl; X is H or C 1-8 alkyl;

[0063] (VII)

[0064] (vii) a compound represented by Formula (VIII), a stereoisomer thereof, an N-oxide thereof, a solvate thereof, or a pharmaceutically acceptable salt thereof,

[0065] (VIII).

[0066] In some embodiments, the cationic lipid is one or at least two of YK-009, YK-201, YK-407, YK-305, ALC-0315, SM-102, and DLIN-MC3-DMA:

[0067] , ,

[0068] , , , .

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

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

[0071] In some embodiments, the neutral lipid is one or at least two of phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, ceramide, and sterol.

[0072] In some embodiments, the neutral lipid is one or 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-distearyl-sn-glycero-3-phosphocholine, 1, 2-didodecanoyl-sn-glycero-phosphocholine, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, 1, 2-di-O-octadecenyl-sn-glycero-3-phosphocholine, 1-oleoyl-2-cholesterylhemisuccinoyl-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, 1, 2-diphytanoyl-sn-glycero-3-phosphoethanolamine, 1, 2-distearyl-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-phosphate-rac-(1-glycerol) sodium salt, dipalmitoyl phosphatidylglycerol, palmitoyloleoyl phosphatidyl ethanolamine, distearoyl-phosphatidyl-ethanolamine, dipalmitoyl phosphatidyl ethanolamine, dimyristyl phosphoethanolamine, 1-stearoyl-2-oleoyl-stearoylethanolamine, 1-stearoyl-2-oleoyl-phosphatidylcholine, sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoyl phosphatidylcholine, lysophosphatidylcholine, and lysophosphatidylethanolamine.

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

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

[0075] In some embodiments, the structural lipid is one or at least two of cholesterol, non- sterols, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatine, ursolic acid, a-tocopherol, and corticosteroids.

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

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

[0078] In some embodiments, the polymeric conjugated lipid is one or at least two of distearoylphosphatidylethanolamine polyethylene glycol 2000, dimyristoylglycerol-3- methoxypolyethylene glycol 2000, and methoxypolyethylene glycol bimatododecylacetamide.

[0079] In some embodiments, the polymeric conjugated lipid is dimyristoylglycerol-3- methoxypolyethylene glycol 2000 (DMG-PEG 2000).

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

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

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

[0083] The present disclosure also provides a method for synthesizing an mRNA molecule for non- disease diagnostic therapeutic purposes, comprising the following steps: co-incubating the compound of formula (I) as described above or a stereoisomer, a pharmaceutically acceptable salt, or a solvate thereof, with a polynucleotide template, and performing template transcription.

[0084] The present disclosure also provides a capped mRNA transcription reaction system for non-disease diagnostic therapeutic purposes, comprising: (1) the compound of formula (I) as described above or a stereoisomer, a pharmaceutically acceptable salt, or a solvate thereof; and (2) a polynucleotide template, NTPs, and an RNA polymerase.

[0085] The present disclosure also provides a kit comprising: (1) the compound of formula (I) as described above or a stereoisomer, a pharmaceutically acceptable salt, or a solvate thereof; and (2) nucleotide triphosphate molecules, and an RNA polymerase.

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

[0087] The present disclosure also provides a method of increasing stability of an RNA, the method comprising incorporating into the RNA a compound of Formula (I) as described above, or a stereoisomer, a pharmaceutically acceptable salt, or a solvate thereof.

[0088] The present disclosure also provides a method for introducing an RNA into a cell, the method comprising contacting the cell with an RNA molecule as described above or a pharmaceutical composition as described above.

[0089] The present disclosure also provides use of an RNA molecule as described above or a pharmaceutical composition as described above in the manufacture of a vaccine.

[0090] Definitions of Terms

[0091] All publications and patents mentioned in the present disclosure are hereby incorporated by reference in their entirety into the present disclosure. To the extent that any publication or patent incorporated by reference contradicts any disclosure herein, this present disclosure will control.

[0092] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0093] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of ordinary skill in the art to which this claimed subject matter belongs. If there is an inconsistency between the definitions of terms used in different sections of this disclosure, the term as defined in this section will control.

[0094] Except in the Examples, or where otherwise indicated, all numbers expressing quantities of doses in the specification are to be understood as being modified in all instances by the term "about." It should also be understood that the presentation of a range of values herein is intended to serve as a disclosure of all

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

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

[0097] As used herein, “ ” in a structural fragment means that the structural fragment is attached to the remainder of the molecule through the bond. For example, means that it is attached to the remainder of the molecule through the “ ” bond.

[0098] As used herein, the term “C1-C3” means that the backbone of the group has any integer value in the range of 1 to 3 carbon atoms, e.g., 1, 2, or 3 carbon atoms. The term “C 6-15 ” means that the number of carbon atoms in the group is any integer value in the range of 6 to 15, e.g., 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 carbon atoms. Limitations of other ranges of carbon atoms are interpreted similarly to mean that the number of carbon atoms in the defined group can be any integer value in the defined range.

[0099] As used herein, the term “halogen” means fluorine, chlorine, bromine, or iodine.

[0100] As used herein, the term “alkyl” means a saturated aliphatic hydrocarbon radical having a straight or branched chain; non-limiting examples of which include methyl, ethyl, propyl, isopropyl, and the like.

[0101] As used herein, the term “alkenyl” means a straight or branched chain hydrocarbon radical having at least one double bond, consisting solely of carbon and hydrogen atoms, having, for example, 2 to 6 (also for example, 2, 3, 4, 5) carbon atoms, and being attached to the rest of the molecule by a single bond. Alkenyl groups include, but are not limited to, ethenyl, propenyl, isopropenyl, , , and the like.

[0102] As used herein, the term “alkynyl” means a straight or branched chain hydrocarbon radical having at least one triple bond, consisting solely of carbon and hydrogen atoms, having, for example, 2 to 6 (also for example, 2, 3, 4, 5) carbon atoms, and being attached to the rest of the molecule by a single bond. Alkynyl groups include, but are not limited to, ethynyl, 1-propynyl, but-1-ynyl, but-2-ynyl, and the like.

[0103] As used herein, the term "cycloalkyl" refers to saturated monocyclic ring-like groups consisting only of carbon atoms having the specified number of carbon atoms (e.g., C3-C6). Cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and the like.

[0104] As used herein, the term "salt" refers to a corresponding salt that can be readily prepared, purified and / or handled of the modified nucleoside compounds (or nucleotide compounds) of the disclosure, e.g., a pharmaceutically acceptable salt. Unless otherwise specified, reference to a particular compound herein is also meant to include salt forms of the compound.

[0105] As used herein, "capped analog" refers to a structure at the 5' end of mature RNA formed upon post-transcriptional modification in eukaryotes, i.e. m7 the GpppN structure, also known as the methylguanosine cap. This structure can have the function of protecting RNA from degradation at the 5' end, helping the RNA transcript product to pass through the selective pore of nuclear membrane into cytoplasm, enhancing translation, helping to complete the process of splicing, and the like.

[0106] The use of the terms "including", "containing", or "comprising" and the like in the present disclosure means that the elements following the word are encompassed by the subject matter of the word, and that no other elements are excluded. The terms "containing" or "comprising" as used herein can be open, semi-closed and closed. In other words, the terms also include "consisting essentially of" or "consisting of".

[0107] The term "pharmaceutically acceptable" in the present disclosure means that the compound or composition is chemically and / or toxicologically compatible with the other ingredients constituting a formulation and / or with humans or mammals for which the prevention or treatment of a disease or condition is intended.

[0108] The term "solvate" in the present disclosure refers to a complex formed by combination of a compound of Formula (I) or a pharmaceutically acceptable salt thereof and a solvent (e.g., ethanol or water). It is understood that any solvate of a compound of Formula (I) used in the treatment of a disease or condition, although potentially providing different properties (including pharmacokinetic properties), will yield the compound of Formula (I) once absorbed into a subject, such that use of a compound of Formula (I) encompasses use of any solvate of a compound of Formula (I) respectively.

[0109] It is further understood that a compound of Formula (I) or a pharmaceutically acceptable salt thereof can be isolated in solvate form, and thus any such solvate is included within the scope of the present disclosure. For example, a compound of Formula (I) or a pharmaceutically acceptable salt thereof can exist in unsolvated as well as solvated forms with pharmaceutically acceptable solvents such as water, ethanol, and the like.

[0110] The present disclosure also includes salts, particularly pharmaceutically acceptable salts, of the compounds described herein. Compounds of the present disclosure having a sufficiently acidic or a sufficiently basic functional group can react with any of a number of inorganic or organic bases, and inorganic or organic acids, to form a salt. Alternatively, a compound that is itself a zwitterion (e.g., a compound having a quaternary nitrogen) can form a salt with an appropriate counterion such as, for example, a halide ion such as bromide, chloride, or fluoride, and particularly bromide.

[0111] A pharmaceutically acceptable salt of the present disclosure can be, for example, an acid addition salt of a compound of the present disclosure having a sufficiently basic functional group, for example a nitrogen atom, in the chain or ring of the compound of Formula (I), for example an acid addition salt with an inorganic acid such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, phosphoric acid, or nitric acid, or with an organic acid such as 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-hydroxyphenyl)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, sulfaminic acid, trifluoromethanesulfonic acid, dodecylsulfonic acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, methanesulfonic acid, 2-naphthalenesulfonic acid, naphthalene- disulfonic 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 a sufficiently acidic group is an alkali metal salt, for example a sodium or potassium salt, an alkaline earth metal salt, for example a calcium or magnesium salt, an ammonium salt (for example a salt with NH3 or aqueous ammonia), or a salt with an organic base which affords a physiologically acceptable cation, for example a salt with triethylamine, N-methylglucamine, dimethylglucamine, ethylglucamine, lysine, dicyclohexylamine, 1,6-hexanediamine, ethanolamine, glucosamine, sarcosine, serinol, tris-hydroxymethylaminomethane, aminopropanediol, 1-amino-2,3,4-butantriol. In addition, a basic nitrogen-containing group can be quaternized with such agents as lower alkyl halides, such as methyl, ethyl, propyl, and butyl chlorides, bromides, and iodides; dialkyl sulfates like dimethyl, diethyl, dibutyl, and diamyl sulfates; long chain halides such as decyl, lauryl, myristyl, and stearyl chlorides, bromides, and iodides; aralkyl halides like benzyl and phenethyl bromides and others.

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

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

[0115] Certain compounds of the present disclosure can exist in one or more stereoisomers. Stereoisomers include geometric isomers, diastereomers, and enantiomers. Thus, the compounds of formula (I) of the present disclosure also include racemic mixtures, single stereoisomers, and mixtures of optically active forms. Those skilled in the art will appreciate that one stereoisomer can have better efficacy and / or lower side effects than the other stereoisomer. Single stereoisomers and mixtures of optically active forms can be obtained by chiral source synthesis, chiral catalysis, chiral resolution, etc. Racemates can be chiral resolved by chromatographic resolution or chemical resolution. For example, a chiral resolving agent such as chiral tartaric acid, chiral malic acid, etc. can be added to form a salt of the compound of the present disclosure, and the product can be separated by taking advantage of the different physical and chemical properties such as solubility.

[0116] In the present disclosure, when the name of a compound is not consistent with the structural formula, the structural formula is correct.

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

[0118] It should be understood that the term "compound of the present disclosure", "compound" used in the present disclosure can include: the compound of formula (I), solvate thereof, pharmaceutically acceptable salt thereof, stereoisomer thereof, and mixtures thereof, according to the context.

[0119] On the basis of not violating the common sense of the art, the above-mentioned preferred conditions can be combined arbitrarily, i.e. to obtain each preferred example of the present disclosure.

[0120] The reagents and raw materials used in the present disclosure are commercially available.

[0121] The positive progress effect of the present disclosure is that: compared with the Cap2 capping analogs in the prior art, the ribose-modified Cap2 capping analogs of the present disclosure can significantly improve the mRNA in vitro transcription yield, capping rate, mRNA translation efficiency, mouse in vivo protein expression amount, and expression duration. BRIEF DESCRIPTION OF DRAWINGS

[0122] In order to more clearly illustrate the technical solutions in the specific embodiments of the present disclosure or the prior art, the drawings needed to be used in the description of the specific embodiments or the prior art will be briefly introduced. Obviously, the drawings in the following description are some embodiments of the present disclosure, and various changes made within the spirit and scope of the present disclosure by those of ordinary skill in the art can also be considered as the scope intended by the drawings of the present disclosure.

[0123] Figure 1 Figure 1 is a graph showing the results of mRNA in vitro transcription yield using YK-CAP-301~306, Comparative Example 1 and Comparative Example 2 as capping analogs.

[0124] Figure 2 Figure 2 is a graph showing the results of capping mRNA translation efficiency using YK-CAP-301~306, Comparative Example 1 and Comparative Example 2 as capping analogs.

[0125] Figure 3 Figure 3 is a graph showing the results of capping mRNA expression in mice using YK-CAP-301~306, Comparative Example 1 and Comparative Example 2 as capping analogs. DETAILED DESCRIPTION

[0126] In order to make the objects, technical solutions and advantages of the embodiments of the present disclosure clearer, the technical solutions of the embodiments of the present disclosure will be described clearly and completely below with reference to the drawings of the embodiments of the present disclosure. Obviously, the described embodiments are part of the embodiments of the present disclosure, rather than all the embodiments. It should be understood that the detailed description and specific embodiments (although representing the specific embodiments of the present disclosure) are given for illustrative purposes only, and various changes and modifications made within the spirit and scope of the present disclosure will become apparent to those skilled in the art after reading the detailed description.

[0127] The present disclosure can be implemented in other specific forms without departing from the essential attributes of the present disclosure. It should be understood that any and all embodiments of the present disclosure can be combined with the technical features in any other embodiment or multiple other embodiments to obtain additional embodiments, without conflict. The present disclosure includes the additional embodiments obtained by such combination.

[0128] The present disclosure will be further described below in conjunction with the embodiments. The implementation conditions used in the embodiments can be further adjusted according to different requirements of specific use, and the implementation conditions not specified are the conventional conditions in the industry. In the specific embodiments of the present disclosure, the raw materials used can be obtained by market purchase. Unless otherwise specified, all temperatures are given in degrees Celsius. The technical features involved in each embodiment of the present disclosure can be combined with each other as long as there is no conflict between them.

[0129] The following abbreviations letters represent the following reagents, respectively:

[0130] TEA: triethylamine; DMF: N, N-dimethylformamide; PySSPy: 2, 2'-dithiodipyridine; PPh3: triphenylphosphine; TEAB: triethylammonium bicarbonate; EDTA: ethylenediaminetetraacetic acid; DMSO: dimethyl sulfoxide; DSPC: 1, 2-distearoyl-sn-glycero-3-phosphocholine; DMG-PEG 2000: dimyristylglycero-3-methoxypolyethyleneglycol 2000.

[0131] Example 1: Synthesis of ribose-modified Cap2 type capping analog

[0132] 1. Synthesis of intermediate Im-PAmPGm

[0133]

[0134] PAmPGm (1.92 g, 2.66 mmol) was dissolved in DMF (25 mL), 2, 2-dithiodipyridine (1.17 g, 5.32 mmol), imidazole (1.45 g, 21.31 mmol), triphenylphosphine (1.39 g, 5.32 mmol) and triethylamine (0.27 g, 2.66 mmol) were added successively, and stirred at room temperature for 18 hours under N2protection. After the reaction was completed, the reaction solution was added dropwise to a solution of NaI (3.5 g) in acetonitrile (100 mL) at 0 °C, and stirred at room temperature for 40 minutes, then centrifuged and the supernatant was poured out. Acetonitrile was added to the obtained residue, and ultrasonic centrifugation was performed. This was repeated three times. The obtained residue was concentrated under reduced pressure to obtain the final product Im-PAmPGm (1.07 g, 1.39 mmol, 52.1%), C 25 H 32 N 12 O 13 P2, MS (ES): m / z (M-H - ) 769.5.

[0135] 2. Synthesis of YK-CAP-301

[0136]

[0137] YK-CAP-107-PM11 (120 mg, 0.24 mmol, synthesized according to CN 118389495 B) and Im-PAmPGm (185 mg, 0.24 mmol) were dissolved in DMSO, MgCl2was added at 0 °C. Stirring at 30 °C for 18 h under N2protection. After the reaction was completed, the reaction solution was added dropwise into ice water and stirred for 30 min. Purification by gel column and high-performance preparative liquid chromatography to obtain the final product YK-CAP-301 (32 mg, 26.6 μmol, 11.1%), C 36 H 51 N 15 O 24 P4, MS (ES): m / z (M-H - ) 1200.1.

[0138] 1 H NMR (400 MHz, D2O) δ 9.09 (s, 1H), 8.41 (s, 1H), 8.14 (s, 1H), 7.88 (s, 1H), 6.04 (d, J = 4.6 Hz, 1H), 5.70 (d, J = 5.5 Hz, 1H), 5.63 (s, 1H), 4.86 (m, 1H), 4.67 (m, 2H), 4.51 - 4.23 (m, 14H), 4.18 - 4.08 (m, 4H), 3.91 (s, 3H), 3.62 (m, 1H), 3.41 (s, 6H), 3.23 (s, 3H), 2.25 (m, 1H), 1.11 (d, J = 6.2 Hz, 3H). 31 P NMR (162 MHz, D2O) δ -0.88, -11.43, -11.57, -22.55 (4P).

[0139] 3. Synthesis of YK-CAP-302

[0140]

[0141] YK-CAP-109-PM8 (80 mg, 0.16 mmol, synthesized according to CN 118389495 B) as the raw material, according to the synthesis route of YK-CAP-301, to obtain the final product YK-CAP-302 (20 mg, 17 μmol, 10.2%), C 35 H 48 FN 15 O 23P4, MS (ES): m / z (M-H - ) 1188.9.

[0142] 1 H NMR (400 MHz, D2O) δ 9.04 (s, 1H), 8.15 (d, J = 2.5 Hz, 1H), 7.66 - 7.58 (m, 2H), 6.19 (d, J = 4.4 Hz, 1H), 5.68 (d, J = 2.6 Hz, 1H), 5.36 - 5.25 (m, 1H), 4.65 - 4.44 (m, 3H), 4.37 - 4.34 (m, 4H), 4.24 - 4.16 (m, 3H), 4.14 (s, 2H), 4.06 (s, 1H), 3.94 - 3.85 (m, 3H), 3.44 (s, 6H), 3.13 (s, 2H), 2.35 - 2.25 (m, 1H), 1.56 (d, J = 6.1 Hz, 3H). 31 P NMR (162 MHz, D2O) δ -0.89, -11.23, -11.65, -22.15 (4P).

[0143] 4. Synthesis of YK-CAP-303

[0144]

[0145] YK-CAP-303 (52 mg, 44 μmol, 17.9%) was obtained from YK-CAP-110-PM8 (150 mg, 0.31 mmol, synthesized according to CN 118389495 B) following the synthetic route of YK-CAP-301, C 34 H 45 F2N 15 O 23 P4, MS (ES): m / z (M-H - ) 1192.8.

[0146] 1H NMR (400 MHz, D20) δ 9.02 (s, 1H), 8.34 (s, 1H), 8.07 (s, 1H), 7.87 (s, 1H), 5.97 (d, J = 5.3 Hz, 1H), 5.77 - 5.72 (m, 2H), 4.89 - 4.78 (m, 2H), 4.65 (t, J = 4.7 Hz, 1H), 4.45 - 4.24 (m, 16H), 4.19 - 4.05 (m, 4H), 3.96 (s, 3H), 3.40 (s, 6H), 3.05 - 2.86 (m, 1H). 31 P NMR (162 MHz, D20) δ -0.89, -11.51, -11.64, -22.79 (4P).

[0147] 5. Synthesis of YK-CAP-304

[0148]

[0149] YK-CAP-304 (30 mg, 24 μmol, 16.3%) was obtained from YK-CAP-111-PM10 (80 mg, 0.15 mmol, synthesized according to CN 118389495 B) following the synthetic route of YK-CAP-301, C 38 H 54 N 16 O 24 P4, MS (ES): m / z (M-H - ) 1241.6.

[0150] 1H NMR (400 MHz, D20) δ 9.10 (s, 1H), 8.81 (s, 1H), 8.60 (s, 1H), 8.34 (s, 1H), 6.09 (d, J = 4.8 Hz, 1H), 5.80 (d, J = 3.5 Hz, 1H), 4.88 - 4.79 (m, 3H), 4.62 (m, 1H), 4.48 - 4.39 (m, 10H), 4.31 - 4.14 (m, 6H), 4.06 - 4.02 (m, 2H), 3.97 (s, 3H), 3.73 (t, J = 6.6 Hz, 1H), 3.44 (s, 6H), 3.36 - 3.26 (m, 4H), 1.08 - 1.00 (dt, J = 21.1, 7.1 Hz, 6H). 31 P NMR (162 MHz, D20) δ -0.91, 11.49, -11.74, -22.55 (4P).

[0151] 6. Synthesis of YK-CAP-305

[0152]

[0153] YK-CAP-305 (75 mg, 63 μmol, 25.7%) was obtained from YK-CAP-118-PM8 (120 mg, 0.25 mmol, synthesized according to CN 118389495 B) following the synthetic route of YK-CAP-301, C 34 H 46 FN 15 O 24 P4, MS (ES): m / z (M-H - ) 1190.2.

[0154] 1H NMR (400 MHz, D20) δ 8.89 (s, 1H), 8.73 (s, 1H), 8.15 (d, J = 4.5 Hz, 2H), 6.35 (d, J = 5.1 Hz, 1H), 5.63 (d, J = 2.3 Hz, 1H), 5.46 (d, J = 4.6 Hz, 1H), 4.93 - 4.84 (m, 3H), 4.65 - 4.57 (m, 2H), 4.52 - 4.39 (m, 4H), 4.33 (d, J = 2.9 Hz, 1H), 4.12 (s, 2H), 4.02 (s, 2H), 3.92-3.78 (m, 3H), 3.55 (t, J = 7.0 Hz, 2H), 3.44 (s, 6H). 31 P NMR (162 MHz, D20) δ -0.90, 11.26, -11.67, -22.45 (4P).

[0155] 7. Synthesis of YK-CAP-306

[0156]

[0157] YK-CAP-306 (85 mg, 71 μmol, 22.1%), C 35 H 49 N 15 O 25 P4, MS (ES): m / z (M-H - ) 1202.2.

[0158] 1H NMR (400 MHz, D20) δ 9.03 (s, 1H), 8.37 (s, 1H), 8.11 (s, 1H), 7.89(s, 1H), 5.96 (d, J = 5.3 Hz, 1H), 5.87 (d, J = 5.0 Hz, 1H), 5.74 (d, J = 5.6Hz, 1H), 4.85 (m, 1H), 4.53 - 4.33 (m, 13H), 4.29 - 4.24 (m, 2H), 4.21 - 4.09(m, 6H), 3.96 (s, 3H), 3.72 (d, J = 10.9 Hz, 1H), 3.59 (d, J = 10.9 Hz, 1H),3.39 (s, 6H), 3.36 (s, 3H). 31 P NMR (162 MHz, D20) δ -0.91, -11.46, -11.58, -22.78 (4P).

[0159] 8. Synthesis of Comparative Example 1 and Comparative Example 2

[0160]

[0161]

[0162] Comparative Example 1 (62 mg, 53 μmol) and Comparative Example 2 (66 mg, 56 μmol) were synthesized following the method disclosed in US20180105551A1.

[0163] 9. Synthesis of Comparative Example 3

[0164]

[0165] Comparative Example 3 (50 mg, 43 μmol) was synthesized following the method disclosed in CN118373866A.

[0166] 10. Synthesis of Comparative Example 4

[0167]

[0168] Comparative Example 4 (57 mg, 48 μmol) was synthesized following the method disclosed in CN115260264A.

[0169] 11. Synthesis of Comparative Example 5

[0170]

[0171] Synthesis of Comparative Example 5 (71 mg, 60 μmol) according to the method disclosed in CN118389495B.

[0172] 12. Synthesis of Comparative Example 6

[0173]

[0174] Synthesis of Comparative Example 6 (65 mg, 55 μmol) according to the method disclosed in CN118389495B.

[0175] Example 2: mRNA in vitro transcription yield and capping rate

[0176] I. Structural differences of capping analogs

[0177] Table 1. Structures of capping analogs

[0178]

[0179]

[0180]

[0181]

[0182] As can be seen from Table 1, the compounds YK-CAP-301 ~ YK-CAP-306 of the present application are similar in chemical structure, and these compounds are also similar in structure to Comparative Example 1, Comparative Example 2, Comparative Example 3, Comparative Example 4, Comparative Example 5 and Comparative Example 6, specifically as follows:

[0183] 1. The compounds YK-CAP-301 ~ YK-CAP-306 of the present application are only different in the substituent group at C3 and / or the substituent group at C4 of the first sugar ring, and the other structures are completely the same.

[0184] 2. The substituent group at C3 of the first sugar ring of the compounds YK-CAP-301 ~ YK-CAP-304 in the present application is different from that of Comparative Example 1, Comparative Example 2 and Comparative Example 4, i.e. the C3 substituent of Comparative Example 1 is hydroxyl, the C3 substituent of Comparative Example 2 is methoxy, and the C3 substituent of Comparative Example 4 is methoxymethyl; the C3 substituent of YK-CAP-301 ~ YK-CAP-304 is 1-methoxyethyl, 1-fluoroethyl, difluoromethyl and N, N-diethylaminoacyl, respectively, and the other structures are completely the same.

[0185] 3. The substituent group of C4 in the first sugar ring of compounds YK-CAP-305 and YK-CAP-306 in the present application is different from that of Comparative Example 1 and Comparative Example 3, i.e. the substituent group of C4 in Comparative Example 1 is hydrogen atom, and that in Comparative Example 3 is a bridged ring structure formed by C4 and C2; the substituent group of C4 in YK-CAP-305 and YK-CAP-306 is 1-fluoromethyl and methoxymethyl, respectively, and the other structures are completely the same.

[0186] 4. The substituent group of C2 in the third sugar ring of compounds YK-CAP-301 and YK-CAP-305 in the present application is different from that of Comparative Example 5 and Comparative Example 6, i.e. the substituent group of C2 in Comparative Example 5 and Comparative Example 6 is hydroxyl; the substituent group of C2 in YK-CAP-301 and YK-CAP-305 is methoxyl, and the other structures are completely the same.

[0187] II. In vitro transcription yield of mRNA and capping rate

[0188] 1. Experimental method

[0189] (1) Preparation of DNA template

[0190] 1) Luciferase protein CDS was constructed on pVAX1 vector (purchased from Thermo Fisher Scientific) by EcoRV enzyme digestion;

[0191] 2) The constructed plasmid on the pVAX1 vector in step 1) was mixed with 50 μL of E. coli competent cells Stbl2 (purchased from Thermo Fisher Scientific) and then subjected to ice bath for 30 minutes, 42 ℃ heat shock for 90 seconds, and then immediately placed back on ice for 2 minutes;

[0192] 3) 400 μL of LB medium (purchased from Yixing Biotechnology Co., Ltd.) was added, and the culture was slowly shaken and incubated at 30 ℃ for 45-60 minutes;

[0193] 4) 50-100 μL of bacterial solution was spread on LB solid medium containing kanamycin antibiotic (100 μg / mL, purchased from Yixing Biotechnology Co., Ltd.), and incubated at 37 ℃ overnight;

[0194] 5) The obtained monoclonal colonies were subjected to sequencing to verify their correctness, and the monoclonal colonies with correct sequencing were selected and incubated at 30 ℃ overnight;

[0195] 6) Endotoxin-free large-scale plasmid extraction kit (purchased from Yixing Biotechnology Co., Ltd.) was used for plasmid extraction;

[0196] 7) The obtained plasmid was linearized by restriction enzyme digestion and used as a transcription template, and the specific enzyme digestion process steps are shown in steps ①-③.

[0197] Step 1: 1 mg luciferase circular plasmid was linearized (BspQ I enzyme, purchased from Yixing Biotechnology Co., Ltd.) at 37 °C for 4 hours to obtain linearized DNA transcription template (enzyme digestion system is shown in Table 2).

[0198] Table 2 Enzyme digestion reaction system

[0199]

[0200] Step 2: After the reaction was completed, anhydrous ethanol and sodium acetate were added in sequence, and the volume ratio of anhydrous ethanol and 3 M sodium acetate was 1:3:1. 酶切反应产物 :V 无水乙醇 :V 3M醋酸钠 =1:3:1, and placed at -20 °C for 1 hour, and then centrifuged at 12000 rpm to reserve the precipitate.

[0201] Step 3: The precipitate of step 2 was washed with 70% ethanol for 2 times, and the material after centrifugation was placed at 55 °C for 10 minutes, and then 1.7 mL water for injection was added for dissolution.

[0202] The concentration of linearized plasmid in the dissolution solution was 500 ng / µL, the linearization ratio was more than 90%, and the purification recovery efficiency was 85%.

[0203] (2) In vitro transcription synthesis of mRNA

[0204] 1) Co-transcription capping reaction:

[0205] YK-CAP-301 ~ YK-CAP-306 synthesized in Example 1 and Comparative Example 1, Comparative Example 2, Comparative Example 3, Comparative Example 4, Comparative Example 5 and Comparative Example 6 were used as capping analogs, Fluc DNA prepared in (1) was used as a template, and NTP solution (NTPs) was used as a starting material to synthesize mRNA by T7 RNA polymerase. The specific reaction system is shown in Table 3, and the prepared reaction system was placed in a 37 °C incubator for 3 h.

[0206] Table 3 Co-transcription capping reaction system

[0207]

[0208] Note: The reagents used are purchased from Yixing Biotechnology Co., Ltd.

[0209] 2) Digestion of template DNA:

[0210] DNase I (purchased from Yixing Biotechnology Co., Ltd.) was added to the co-transcription capping reaction system after the completion of step 1) to a final concentration of 1 U / μg linearized plasmid, mixed, centrifuged, and placed at 37 ℃ for 1 hour to obtain the co-transcription capping product.

[0211] 3) Purification by lithium chloride precipitation method:

[0212] The co-transcription capping product obtained in step 2) was purified by lithium chloride precipitation method as follows:

[0213] Step 1) Add lithium chloride: lithium chloride solution (purchased from Thermo Fisher Scientific) was added to the product of step 2) to a final concentration of 2.8 M, and precipitated at low temperature for 2 hours.

[0214] Step 2) Precipitation: high-speed centrifugation at 12000 rpm for 15 minutes, and the precipitate was retained.

[0215] Step 3) Washing: washed twice with 75% ethanol, and dissolved with water for injection to obtain an mRNA solution. The purified mRNA solution was stored at -80 ℃.

[0216] (3) Annealing reaction of the obtained mRNA and the probe.

[0217] Annealing in a PCR instrument: 95 ºC for 5 min; 65 ºC for 2 min; 55 ºC for 2 min; 40 ºC for 2 min; 22 ºC for 2 min.

[0218] (4) Magnetic bead pretreatment and probe binding: take 100 μL magnetic beads and place them on a magnetic stand for pretreatment. Add 120 μL sample and magnetic bead solution, and incubate at room temperature for 30 min, mixing slowly during incubation.

[0219] (5) Cleavage of mRNA and acquisition of mRNA 5' single-stranded sequence bound to the probe.

[0220] Add 20 μL RnaseH (5 U / μL), and incubate at 37 ºC for 3 h, mixing every half hour. After incubation, wash the magnetic beads, and add 100 μL 75% methanol heated to 80 ºC to the washed magnetic beads. Heat the mixture to 80 ºC on a heating plate for 3 min, then place it on a magnetic stand to absorb the supernatant. Dry it at room temperature for 45 min to 10 μL using an evaporation centrifuge. Then resuspend the sample in 50 μL 100 μM EDTA / 1% MeOH, which can be used for LC-MS analysis to determine the capping of RNA in the transcription reaction. Since the bases of capped and uncapped have a significant difference in molecular weight, the capping rate of mRNA transcription starting from different capped analogs can be determined by using the difference in molecular mass.

[0221] (6) mRNA integrity evaluation was performed by Agilent 5200 Fragment Analyzer capillary electrophoresis system.

[0222] 1) Sample preparation: according to the sample concentration determined by the previous ultramicro spectrophotometer, each sample was diluted to 20 ~ 100 ng / μL by different multiples with enzyme-free water, and the sample and the pre-packaged Ladder were mixed and placed in a metal bath, denatured at 70 ºC for 2 min, and immediately placed on ice.

[0223] 2) Capillary reagent preparation:

[0224] Table 4 Preparation of capillary reagent

[0225]

[0226] 3) Sample running program setting and sample running on machine: sample injection voltage 5 kV, running time 4 s, separation voltage 8 kV, running time 45 min.

[0227] 4) Data analysis: Agilent Fragment Analyzer comes with ProSize analysis software, users can calculate the percentage of qualified RNA fragments or the percentage of degraded RNA by Smear analysis.

[0228] 2. Experimental results

[0229] The results of mRNA in vitro transcription yield, integrity and capping rate show that the mRNA in vitro transcription yield, integrity and capping rate of the Cap2 capping analogs in the disclosure are at a very high level. Compared with Comparative Example 1 and Comparative Example 2, the mRNA in vitro transcription yield of the Cap2 capping analogs of the disclosure is mostly improved, and the integrity and capping rate are at a very high level. The specific mRNA in vitro transcription yield, integrity and capping rate are shown in Table 5.

[0230] Table 5 Cotranscriptional capping reaction system

[0231]

[0232] From Table 5 and Figure 1 It can be seen that the ribose-modified Cap2 capping analogs in the present application can efficiently transcribe mRNA. Compared with the Cap2 or Cap1 capping analogs in the prior art, the mRNA in vitro transcription yield is significantly improved.

[0233] The mRNA in vitro transcription yields of Comparative Example 1, Comparative Example 2, Comparative Example 3, Comparative Example 4, Comparative Example 5 and Comparative Example 6 were 130.2 μg, 128.9 μg, 132.5 μg, 125.3 μg, 146.8 μg and 142.2 μg, respectively, and the mRNA in vitro transcription yields of YK-CAP-306 in the present application were increased by 30.0%, 31.3%, 27.7%, 35.0%, 15.3% and 19.0% compared with Comparative Example 1, Comparative Example 2, Comparative Example 3, Comparative Example 4, Comparative Example 5 and Comparative Example 6, respectively. It can be seen from the mRNA in vitro transcription yields that the mRNA in vitro transcription yields of the ribose-modified Cap2 capping analogs of the present application were significantly improved compared with the prior art Comparative Example 1, Comparative Example 2, Comparative Example 3, Comparative Example 4, Comparative Example 5 and Comparative Example 6, indicating that the ribose-modified Cap2 capping analogs of the present application have excellent anti-reverse transcription effect when used for in vitro transcription of mRNA, and can increase the binding ability of the cap structure to the capping enzyme, thereby increasing the in vitro transcription yield of the transcribed mRNA.

[0234] Example 3: Preparation and characterization of lipid nanoparticles

[0235] 1. Experimental methods

[0236] The cationic lipid YK-009 (Beijing Yekang Kekai Pharmaceutical Technology Co., Ltd.), DSPC (Avanti (Shanghai) Pharmaceutical Technology Co., Ltd.), cholesterol (Avanti (Shanghai) Pharmaceutical Technology Co., Ltd.) and DMG-PEG 2000 were dissolved in ethanol at a molar ratio of 49:10:39.5:1.5, and the mRNA was diluted in 50 mM citrate buffer, pH = 4. The ethanol lipid solution was mixed with the above-mentioned FLuc mRNA aqueous solution prepared from different capping structures at a volume ratio of 1:3 by using a microfluidic device at a flow rate of 10 mL / min to prepare LNP at a total lipid to mRNA weight ratio of about 15:1. The obtained liposomes were diluted with PBS to a total volume of 10 times, and then ultrafiltrated with a 300 KDa ultrafiltration tube to remove ethanol. After being diluted with PBS to a certain volume, finally, the lipid nanoparticles were filtered through a 0.2 μm sterile filter to obtain the LNP preparation of YK-009 / DSPC / cholesterol / DMG-PEG2000 (molar percentage 49:10:39.5:1.5) encapsulating self-replicating FLuc-mRNA.

[0237] The structure of YK-009 is .

[0238] Particle size and polydispersity index (PDI) were determined using dynamic light scattering with a Malvern laser particle sizer. 25 μL of liposome solution was diluted to 125 μL with physiological saline and added to the sample cell. Each sample was measured 3 times. The measurement conditions were: 90º scattering angle, 25ºC. The encapsulation efficiency of the lipid nanoparticles was determined using the Quant-it Ribogreen RNA quantitation assay kit (ThermoFisher Scientific, UK) according to the manufacturer's instructions.

[0239] 2. Experimental results

[0240] The characterization data of specific lipid nanoparticles are shown in Table 6.

[0241] Table 6 Characterization of lipid nanoparticles

[0242]

[0243] As can be seen from Table 6, the FLuc-mRNA transcribed from the capped analogs YK-CAP-301~YK-CAP-306 and Comparative Example 1, Comparative Example 2, Comparative Example 3, Comparative Example 4, Comparative Example 5 and Comparative Example 6 can all be prepared into good lipid nanoparticles. The particle size of all the lipid nanoparticles is between 75~80 nm, the PDI value is between 0.01~0.05, and the encapsulation efficiency is all above 95%.

[0244] Example 4: Translation efficiency of different capped luciferase mRNA

[0245] 1. Experimental method

[0246] (1) HEK293T cells were cultured in DMEM medium containing 10% FBS and penicillin / streptomycin at 37ºC under 5% CO2 conditions.

[0247] (2) The cells in the culture dish were digested and counted, and 10,000 cells per well were plated in a 96-well plate and cultured overnight until the cells adhered.

[0248] (3) When the cell density was about 80%, transfection was performed, 50 ng of mRNA sample was added to each well with Lipofectamine Messenger MAX Transfection Reagent (Invitrogen) reagent, and the transfection steps were performed according to the instructions.

[0249] (4) After the transfected cells are placed at 37 °C in 5% CO2 for continuous culture for 24 h, the growth medium is removed from the cells to be tested, and the cells are rinsed with PBS. After the PBS is removed by centrifugation, 50 μL of lx lysis buffer is added, and the cells and all the liquid are transferred to a microcentrifuge tube, which is then centrifuged.

[0250] (5) 20 μL of the sample is taken, 100 μL of Dual-Lumi™ II luciferase detection reagent, which has been equilibrated to room temperature, is added, and the mixture is mixed well.

[0251] (6) Incubation is performed at room temperature (about 25 °C) for 5 min to allow the luminescent signal to stabilize. Chemiluminescence detection is performed using a multifunctional microplate reader with chemiluminescence detection function, and the data are recorded (see Table 7 and Figure 2 ).

[0252] 2. Experimental results

[0253] The relative fluorescence readings of the capped mRNA are shown in Table 7 and Figure 2 The relative fluorescence intensity is proportional to the translation efficiency of the mRNA.

[0254] Table 7 Relative fluorescence intensity of capped mRNA

[0255]

[0256] It can be seen that the ribose-modified Cap2 capping analogs of the present application have significantly improved mRNA translation efficiency compared to the Cap2 or Cap1 capping analogs in the prior art. The relative fluorescence intensity (corresponding to the translation efficiency of the mRNA) of Comparative Example 1 is 0.9. The average fluorescence intensity of YK-CAP-301, YK-CAP-303, YK-CAP-305, and YK-CAP-306 is 2.8 times, 3.2 times, 2.1 times, and 2.5 times that of Comparative Example 1, 2.5 times, 2.9 times, 1.9 times, and 2.3 times that of Comparative Example 2, 2.1 times, 2.4 times, 1.6 times, and 1.9 times that of Comparative Example 3, 2.1 times, 2.4 times, 1.6 times, and 1.9 times that of Comparative Example 4, 1.4 times, 1.6 times, 1.1 times, and 1.3 times that of Comparative Example 5, and 1.7 times, 1.9 times, 1.3 times, and 1.5 times that of Comparative Example 6. This indicates that the use of the capping analogs of the present application to modify ribose can more easily bind to the cap-binding protein (EIF4E), thereby improving the translation efficiency of the target mRNA.

[0257] Example 5: Animal experiments

[0258] 1. Experimental method

[0259] LNP preparation containing 5 μg capped analogs-transcribed FLuc-mRNA was injected intramuscularly into 4-6-week-old female BALB / c mice weighing 17-19 g, and at specific time nodes (6 h, 24 h, 48 h, 96 h) after administration, the mice were injected intraperitoneally with a fluorescent imaging substrate. The mice were allowed to move freely for 5 min, and then the total radiation intensity of the protein expressed in the mice by the mRNA carried by the LNP was detected by the IVIS Spectrum small animal live imaging instrument.

[0260] 2. Experimental results

[0261] The detection results are shown in Table 8 and Figure 3 . In the mouse live imaging experiment, the fold of the total radiation intensity of the mice in each group relative to the control product Comparative Example 1 is shown in Table 9 (where the total radiation intensity is the numerical value × 10 8 p / s).

[0262] Table 8. Data of mouse live imaging experiment

[0263]

[0264] Table 9. Fold of total radiation intensity relative to Comparative Example 1

[0265]

[0266] Compared with Cap2 or Cap1 capping analogs in the prior art, the total radiation intensity and duration of the protein expressed in vivo in mice of the mRNA transcribed by the ribose-modified Cap2 capping analogs of the application are significantly improved. For example, the corresponding total radiation intensity of YK-CAP-303 is 2.53 times, 3.24 times, 1.96 times and 1.68 times that of the corresponding total radiation intensity of Comparative Example 1 at 6h, 24h, 48h and 72h, respectively, 2.43 times, 3.27 times, 1.90 times and 1.65 times that of Comparative Example 2, 2.43 times, 2.93 times, 2.16 times and 1.99 times that of Comparative Example 3, 2.42 times, 3.15 times, 1.63 times and 1.74 times that of Comparative Example 4, 2.37 times, 2.25 times, 1.15 times and 1.13 times that of Comparative Example 5, and 2.24 times, 2.62 times, 1.30 times and 1.34 times that of Comparative Example 6. The total radiation intensity of YK-CAP-306 at the corresponding time is 1.47 times, 1.71 times, 2.43 times and 2.03 times that of Comparative Example 1, respectively, 1.41 times, 1.73 times, 2.36 times and 2.00 times that of Comparative Example 2, 1.41 times, 1.55 times, 2.68 times and 2.41 times that of Comparative Example 3, 1.40 times, 1.67 times, 2.02 times and 2.11 times that of Comparative Example 4, 1.38 times, 1.19 times, 1.43 times and 1.37 times that of Comparative Example 5, and 1.30 times, 1.38 times, 1.61 times and 1.62 times that of Comparative Example 6.

[0267] As can be seen from the animal experiments, compared with the capping analogs of Comparative Example 1, Comparative Example 2, Comparative Example 3, Comparative Example 4, Comparative Example 5 and Comparative Example 6 in the prior art, the protein expression amount and duration in vivo of the mRNA transcribed by the ribose-modified Cap2 capping analogs of the application are significantly improved. The in vivo experiments further prove that the mRNA transcribed by YK-CAP-301, YK-CAP-303, YK-CAP-305 and YK-CAP-306 in the application can be effectively delivered to the body by the LNP delivery carrier and expressed efficiently and continuously.

[0268] Example 6: Immunogenicity study

[0269] 1. Experimental method

[0270] (1) The PBMC cells were cultured to 10 6 cells per well, 5 μg of mRNA sample and Lipofectamine Messenger MAX Transfection Reagent (Invitrogen) reagent were added to each well for transfection, and the transfection steps were carried out according to the instructions. After 24 h, the cells were collected by trypsin digestion and resuspended in PBS.

[0271] (2) Add fixative to the cells, and terminate fixation after 15 min. Collect the cells by centrifugation. Resuspend the cells by adding lysis solution, and incubate on ice for 30 min. Then, centrifuge at 4 °C, 2400 g for 10 min, and collect the supernatant.

[0272] (3) Add antibodies of TLR-3, TLR-7, TLR-8 and RIG-1 to the supernatant, and gently stir at 4 °C for 2 h. Add protein A / G magnetic beads, and continue to gently stir for 1 h.

[0273] (4) Centrifuge at 2500 rpm for 30 s to remove the supernatant. Resuspend the magnetic beads by adding RIP buffer, and then centrifuge at 2500 rpm for 30 s again to remove the supernatant. Repeat the washing three times, and then wash once with PBS.

[0274] (5) Resuspend the magnetic beads in TRIzol RNA extraction reagent, and separate the co-precipitated RNA. Elute the RNA with sterile enzyme-free water.

[0275] (6) Reverse transcribe the RNA into cDNA, and perform PCR quantitative analysis.

[0276] 2. Experimental results

[0277] The experimental results are shown in Table 10. The immunogenicity of mRNA is proportional to the copy number of the mRNA precipitated by the magnetic beads. The data in the table are relative values to Comparative Example 1.

[0278] Table 10 Relative immunogenicity of capped mRNA

[0279]

[0280] Compared with Cap2 or Cap1 capped analogs in the prior art, the immunogenicity of the mRNA transcribed from the ribose-modified Cap2 capped analogs of the application is significantly reduced. For example, the relative immunogenicity of YK-CAP-301, YK-CAP-303, YK-CAP-305 and YK-CAP-306 is 0.7 times, 0.6 times, 0.6 times and 0.5 times that of Comparative Example 1, 0.6 times, 0.5 times, 0.5 times and 0.5 times that of Comparative Example 2, 0.9 times, 0.8 times, 0.8 times and 0.6 times that of Comparative Example 3, 0.9 times, 0.8 times, 0.8 times and 0.6 times that of Comparative Example 4, 0.6 times, 0.5 times, 0.5 times and 0.5 times that of Comparative Example 5, and 0.6 times, 0.5 times, 0.5 times and 0.5 times that of Comparative Example 6. This indicates that the use of the capped analogs of the application to modify ribose can significantly reduce the immunogenicity of mRNA after transcription, and especially relative to Cap1 type capped analogs (Comparative Example 5 and Comparative Example 6), the safety of the vaccine is improved.

[0281] In summary, the Cap2-capped analogs YK-CAP-301, YK-CAP-303, YK-CAP-305 and YK-CAP-306 of the present application, their corresponding mRNA in vitro transcription yield, mRNA translation efficiency, and protein expression level and duration in animals are significantly improved compared with the Cap2-capped analogs Comparative Example 1, Comparative Example 2, Comparative Example 3, Comparative Example 4, Comparative Example 5 and Comparative Example 6 in the prior art. It is shown that the capped analogs provided by the present disclosure can significantly improve the affinity for binding with the capping enzyme, and provide a CAP2 cap structure that can be used for mRNA with high efficiency for in vitro transcription of mRNA. Details are as follows:

[0282] 1. Compared with the Cap2 or Cap1-capped analogs in the prior art, the Cap2-capped analogs of the present application have significantly improved mRNA in vitro transcription yield. For example, the mRNA in vitro transcription yield of Comparative Example 1, Comparative Example 2, Comparative Example 3, Comparative Example 4, Comparative Example 5 and Comparative Example 6 is 130.2 μg, 128.9 μg, 132.5 μg, 125.3 μg, 146.8 μg and 142.2 μg, respectively, and the mRNA in vitro transcription yield of YK-CAP-306 of the present application is increased by 30.0%, 31.3%, 27.7%, 35.0%, 15.3% and 19.0% compared with Comparative Example 1, Comparative Example 2, Comparative Example 3, Comparative Example 4, Comparative Example 5 and Comparative Example 6, respectively.

[0283] 2. Compared with the Cap2 or Cap1-capped analogs in the prior art, the Cap2-capped analogs of the present application have significantly improved mRNA translation efficiency. For example, the relative fluorescence intensity of YK-CAP-301 and YK-CAP-303 is 2.8 times and 3.2 times that of Comparative Example 1, 2.5 times and 2.9 times that of Comparative Example 2, 2.1 times and 2.4 times that of Comparative Example 3, 2.1 times and 2.4 times that of Comparative Example 4, 1.4 times and 1.6 times that of Comparative Example 5, and 1.7 times and 1.9 times that of Comparative Example 6.

[0284] 3. Compared with Cap2 or Cap1 capping analogues in the prior art, the total intensity and duration of protein expression in vivo of the mRNA transcribed by the ribose-modified Cap2 capping analogues of the present application are significantly improved. For example, the corresponding total intensity of YK-CAP-303 is 2.53 times, 3.24 times that of Comparative Example 1, 2.43 times, 3.27 times that of Comparative Example 2, 2.43 times, 2.93 times that of Comparative Example 3, 2.42 times, 3.15 times that of Comparative Example 4, 2.37 times, 2.25 times that of Comparative Example 5, and 2.24 times, 2.62 times that of Comparative Example 6 at 6h, 24h, respectively. The total intensity of YK-CAP-306 is 2.43 times and 2.03 times that of Comparative Example 1, 2.36 times and 2.00 times that of Comparative Example 2, 2.68 times and 2.41 times that of Comparative Example 3, 2.02 times and 2.11 times that of Comparative Example 4, 1.43 times and 1.37 times that of Comparative Example 5, and 1.61 times and 1.62 times that of Comparative Example 6 at 48h, 96h, respectively.

[0285] 4. Compared with Cap2 or Cap1 capping analogues in the prior art, the immunogenicity of the mRNA transcribed by the ribose-modified Cap2 capping analogues of the present application is significantly reduced. For example, the relative immunogenicity of YK-CAP-305, YK-CAP-306 is 0.6 times and 0.5 times that of Comparative Example 1, 0.5 times and 0.5 times that of Comparative Example 2, 0.8 times and 0.6 times that of Comparative Example 3, 0.8 times and 0.6 times that of Comparative Example 4, 0.5 times and 0.5 times that of Comparative Example 5, and 0.5 times and 0.5 times that of Comparative Example 6, respectively.

[0286] The above embodiments are only used to illustrate the technical solutions of the present disclosure, rather than limit them; although the present disclosure has been described in detail with reference to the foregoing embodiments, it should be understood by those skilled in the art that the technical solutions recorded in the foregoing embodiments can still be modified, or some or all of the technical features can be replaced equivalently. Any modification, equivalent replacement and improvement made within the spirit and principle of the present disclosure shall be included in the protection scope of the claims of the present disclosure.

Claims

1. A compound, characterized in that, The compound is any one of the following structures: , , or .

2. Use of a compound of claim 1 in the preparation of an in vitro co-transcriptional mRNA capping reagent.

3. An RNA molecule, characterized in that, which comprises the compound of claim 1 as a cap structure or cap structure fragment.

4. A pharmaceutical composition, characterized by, which comprises the RNA molecule of claim 3.

5. The pharmaceutical composition of claim 4, wherein, The pharmaceutical composition further comprises at least one delivery agent.

6. The pharmaceutical composition of claim 5, wherein, The at least one delivery agent comprises one or more of a cationic lipid, a neutral lipid, a structural lipid, and a polymer-conjugated lipid.

7. The pharmaceutical composition of claim 6, wherein, The pharmaceutical composition meets one or more of the following conditions: (1) the cationic lipid is one or at least two of the following compounds: (i) a compound represented by Formula (II), a stereoisomer thereof, an N-oxide thereof, a solvate thereof, or a pharmaceutically acceptable salt thereof, wherein G1is C 1-6 alkylene; G2is C 2-8 alkylene; G3is C 1-3 alkylene; L1is C 6-15 straight chain alkyl; L2is C 12-25 branched chain alkyl; (I) (ii) a compound represented by Formula (III), a stereoisomer thereof, an N-oxide thereof, a solvate thereof, or a pharmaceutically acceptable salt thereof, wherein G1is C 2-8 alkylene; G2is C 2~8 alkylene; L1is -C(O)O- or -OC(O)-; L2is -C(O)O- or -OC(O)-; R1is C 6-25 linear or branched alkyl; R2is C 6-25 linear or branched alkyl; G3is HO(CH2)2- or HO(CH2)3-; G4is 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: G1is C 1-6 alkylene; G2is C 2-8 alkylene; R1is C 6-20 linear or branched alkyl; R2is C 12-25 branched alkyl; G3is: HO(CH2)2N(CH3)(CH2)2-, HO(CH2)2N(CH2CH3)(CH2)2-, (HO(CH2)2)2N(CH2)2-, CH3O(CH2)2N(CH3)(CH2)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 G1is C 1-8 alkylene; G2is C 2-8 alkylene; R1is C 6-25 linear or branched alkyl; R2is C 12-25 linear or branched alkyl; G3is: HO(CH2)2N(R3)CH2CH(OH)CH2-, wherein R3is -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 is C1-C 12 alkylene; R 1 and R 2 each independently is C6-C 24 alkyl or C6-C 24 alkenyl; R 3 is -OR 5 , -N(R 5 )-, -C(=O)OR 4 , -OC(=O)R 4 or -NR 5 C(=O)R 4 ; R 4 is C1-C 12 alkyl; R 5 is H or C1-C6 alkyl; (VI) (vi) a compound represented by Formula (VII), a stereoisomer thereof, an N-oxide thereof, a solvate thereof, or a pharmaceutically acceptable salt thereof, wherein R4is -(CH2) n Q; Q is -OR, -OH, -O(CH2) n N(R)2, -OC(O)R, -CX3, -CN, -N(R)C(O)R, -N(H)C(O)R, -N(R)S(O)2R, -N(H)S(O)2R, -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)2R, or a heterocycle; n is 1, 2, or 3; R is C 1-8 alkyl; X is 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 one or at least two of phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, ceramide, and sterol; (3) the structural lipid is one or at least two of cholesterol, non-sterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, ursolic acid, a-tocopherol, and corticosteroid; and (4) the polymer-conjugated lipid is one or at least two of distearoylphosphatidylethanolamine polyethylene glycol 2000, 1,2-dimyristoyl-sn-glycero-3-methoxypolyethylene glycol 2000, and methoxypolyethylene glycol bismyristyl acetyl amide.

8. The pharmaceutical composition of claim 7, wherein, The pharmaceutical composition meets one or more of the following conditions: (1) the cationic lipid is one or at least two of YK-009, YK-201, YK-407, YK-305, ALC-0315, SM-102, and DLIN-MC3-DMA: 、 、 、 、 、 ； (2) the neutral lipid is one or 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, 1, 2-didodecanoyl-sn-glycero-phosphocholine, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, 1, 2-di-O-octadecenyl-sn-glycero-3-phosphocholine, 1-oleoyl-2-cholesterylhemisuccinoyl-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-didocosahexaoyl-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-dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1, 2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1, 2-didocosahexaoyl-sn-glycero-3-phosphoethanolamine, 1, 2-dioleoyl-sn-glycero-3-phosphate-rac-(1-glycerol) sodium salt, dipalmitoyl phosphatidylglycerol, palmitoyloleoyl phosphatidyl ethanolamine, distearoyl-phosphatidyl-ethanolamine, dipalmitoyl phosphatidyl ethanolamine, dimyristoyl phosphoethanolamine, 1-stearoyl-2-oleoyl-stearoylethanolamine, 1-stearoyl-2-oleoyl-phosphatidylcholine, sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoyl phosphatidylcholine, lysophosphatidylcholine, and lysophosphatidylethanolamine; (3) the structural lipid is cholesterol; and (4) the polymeric conjugated lipid is dimyristyl glycerol-3-methoxypolyethylene glycol 2000.

9. The pharmaceutical composition of claim 8, wherein, the cationic lipid is the YK-009; or, the neutral lipid is 1, 2-dioleoyl-sn-glycero-3-phosphoethanolamine and / or 1, 2-distearoyl-sn-glycero-3-phosphocholine.

10. The pharmaceutical composition of claim 6, wherein, The at least one delivery agent comprises YK-009, 1, 2-distearoyl-sn-glycero-3-phosphocholine, cholesterol, and dimyristoylglycero-3-methoxypolyethyleneglycol 2000; the YK-009 is .

11. The pharmaceutical composition of claim 10, wherein, the molar ratio of the YK-009, 1, 2-distearoyl-sn-glycero-3-phosphocholine, cholesterol, and dimyristyl glycerol-3-methoxypolyethylene glycol 2000 is 49:10:39.5:1.

5.

12. The pharmaceutical composition according to any one of claims 4-11, characterized in that, the pharmaceutical composition further comprises one or at least two cell-penetrating peptides.

13. A method of synthesizing mRNA molecules for non-disease diagnostic therapeutic purposes, characterized in that, which comprises the following steps: incubating the compound of claim 1 with a polynucleotide template for template transcription.

14. A capped mRNA transcription reaction system for non-disease diagnostic therapeutic purposes, characterized by, which comprises: (1) the compound of claim 1 ; and (2) a polynucleotide template, NTPs, and an RNA polymerase.

15. A kit comprising, comprising: (1) the compound of claim 1 ; and (2) nucleotide triphosphate molecules, and an RNA polymerase.

16. The kit of claim 15, wherein The kit further comprises one or at least two of an RNase inhibitor, an inorganic pyrophosphatase, Mg 2+ , a crowding agent and a buffer.

17. A method for enhancing the stability of RNA for non-disease diagnostic therapeutic purposes, characterized by, The method comprises incorporating the compound of claim 1 into an RNA.

18. A method for introducing RNA into a cell for non-disease diagnostic therapeutic purposes, characterized by, The method comprises contacting the cell with the RNA molecule of claim 3 or the pharmaceutical composition of any one of claims 4-11.

19. Use of the RNA molecule of claim 3 or the pharmaceutical composition of any one of claims 4-11 in the manufacture of a vaccine.

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