C6'-substituted locked nucleic acid-modified cap analog and use thereof

Abstract

To provide a C6'-substituted locked nucleic acid-modified cap analog and a method of use thereof.SOLUTION: The present disclosure provides a compound represented by a formula (I), or a stereoisomer, a pharmaceutically acceptable salt or a solvate thereof. In the formula, X is O, S, or the like; R1 to R6 are each H, substituted / unsubstituted C1-C6 alkyl, C2-C6 alkenyl, or the like; Y1a to Y1f are each O, S, CH2, CCl2, or the like; Y2a to Y2d are each =O or =S; Y3a to Y3d are each OH or SH; and B1 and B2 are each a natural / modified pyrimidine nucleotide base or natural / modified purine nucleotide base.SELECTED DRAWING: Figure 4

Description

Technical Field

[0001] The present invention relates to the technical fields of chemistry and biotechnology, and particularly to C6'-substituted locked nucleic acid modified cap analogs and their use.

Background Art

[0002] The chemical nature of the cap structure is a special structure located at the 5'-end of mRNA formed by modification during the transcription process of mRNA, that is, the m7GPPPN structure, also referred to as the methylguanosine cap. This is formed under the co-catalysis of RNA triphosphatase, guanylyltransferase, mRNA (guanine-N7) methyltransferase, and mRNA (nucleoside-2') methyltransferase. Depending on the degree of methylation, three types of caps, namely CAP 0 type, CAP 1 type, and CAP 2 type, can be formed, which are m7G5'ppp5'Np, m7G5'ppp5'NmpNp, and m7G5'ppp5'NmpNmpNp respectively.

[0003] The cap structure is a structure necessary for the initiation of mRNA translation, provides a signal for the ribosome to recognize mRNA, and helps the ribosome bind to mRNA so that translation starts from AUG. At the same time, the cap structure enhances the stability of mRNA and can protect mRNA from the attack of 5'→3' exonucleases.

[0004] In summary, the cap structure plays a role like a steel helmet in mRNA, can not only protect mRNA from being damaged, but also be easily recognized by other members by adding an imprint to the steel helmet through chemical modification. In addition to the natural cap structure, cap structure analogs are also widely used in the in vitro transcription process to improve the stability of the mRNA structure. Common ones include ARCA and Cap 1 structure analogs, etc.

[0005] According to research, it has been shown that the cap structure of mRNA has an important relationship with mRNA quality control and the innate immunity of organisms. Therefore, inventing new cap analogs is of great significance for enhancing the stability of mRNA and improving the efficiency of mRNA translation.

Summary of the Invention

[0006] In order to improve the stability of mRNA and / or the translation efficiency of mRNA, the present invention provides a novel modified locked nucleoside mRNA capping analog and its use.

[0007] Specifically, the first aspect of the present invention provides a compound represented by formula (I) or its stereoisomer, pharmaceutically acceptable salt or solvate.

[0008]

Chemical Formula

[0009] The second aspect of the present invention provides the use of a compound represented by formula (I) according to the first aspect of the present invention or a stereoisomer, pharmaceutically acceptable salt or solvate thereof in the application for the manufacture of an in vitro co-transcribed mRNA capping reagent.

[0010] The third aspect of the present invention provides an RNA molecule comprising, as a cap structure or a cap structure fragment, a compound represented by formula (I) according to the first aspect of the present invention or a stereoisomer thereof, a pharmaceutically acceptable salt or solvate.

[0011] The fourth aspect of the present invention provides a pharmaceutical composition comprising the RNA molecule according to the third aspect of the present invention.

[0012] The fifth aspect of the present invention provides a method for synthesizing an mRNA molecule for purposes other than the diagnosis and treatment of diseases, comprising culturing a compound represented by formula (I) according to the first aspect of the present invention or a stereoisomer thereof, a pharmaceutically acceptable salt or solvate together with a polynucleotide template to perform template transcription.

[0013] The sixth aspect of the present invention is (1) a compound represented by formula (I) according to the first aspect of the present invention or a stereoisomer thereof, a pharmaceutically acceptable salt or solvate, and (2) a polynucleotide template, NTPs and RNA polymerase to provide a capping mRNA transcription reaction system for purposes other than the diagnosis and treatment of diseases.

[0014] The seventh aspect of the present invention is (1) a compound represented by formula (I) according to the first aspect of the present invention or a stereoisomer thereof, a pharmaceutically acceptable salt or solvate, and (2) nucleotide triphosphate molecules, and RNA polymerase to provide a kit.

[0015] The eighth aspect of the present invention provides a method for improving the intracellular stability of RNA, comprising mixing a compound represented by formula (I) according to the first aspect of the present invention or a stereoisomer thereof, a pharmaceutically acceptable salt or solvate with the RNA.

[0016] The ninth aspect of the present invention provides a method for introducing RNA into cells, comprising contacting the cells with the pharmaceutical composition according to the fourth aspect of the present invention.

[0017] The tenth aspect of the present invention provides a method for performing RNA translation inhibition in cells, which includes the step of contacting cells with the pharmaceutical composition according to the fourth aspect of the present invention.

[0018] The eleventh aspect of the present invention provides the use of the pharmaceutical composition according to the fourth aspect of the present invention in the manufacture of a vaccine.

Brief Description of the Drawings

[0019] To more clearly illustrate the specific embodiments of the present invention or the technical solutions according to the prior art, hereinafter, the drawings necessary for the description of the specific embodiments or the existing technology will be briefly introduced. It is self-evident that the drawings described below are some embodiments of the present invention, and those skilled in the art can obtain other drawings based on these drawings on the premise of not making creative efforts.

[0020]

Figure 1

[0021]

Figure 2

[0022]

Figure 3

[0023]

Figure 4

[0024]

Figure 5

Mode for Carrying Out the Invention

[0025] To make the objectives, technical solutions, and advantages of the embodiments of the present disclosure clearer, the technical solutions of the embodiments of the present disclosure will be clearly and completely described below in combination with the drawings of the embodiments of the present disclosure. Obviously, the described embodiments are only a part of the embodiments of the present disclosure, not all of the embodiments. Based on the described embodiments of the present disclosure, all other embodiments obtained by those skilled in the art without creative efforts are included within the protection scope of the present invention.

[0026] The present invention can be embodied in other specific forms without departing from the essential characteristics of the present invention. It should be understood that, without contradiction, any and all embodiments of the present invention can obtain additional embodiments by combining the technical features of any other embodiment or a plurality of other embodiments. The present invention includes such additional embodiments obtained from such combinations.

[0027] All publications and patents mentioned in the present disclosure are hereby incorporated by reference in their entirety into the present disclosure. In case of any conflict between the uses or terms used in any incorporated publication or patent and the uses or terms used in the present disclosure, the uses or terms used in the present disclosure shall prevail.

[0028] The section titles used in this specification are used only for the purpose of organizing the article and should not be construed as limiting the categories described.

[0029] Unless otherwise defined, all technical and scientific terms used in this specification have the ordinary meaning in the technical field to which the claimed subject matter belongs. If there are multiple definitions for a term, the definition described in the specification shall prevail.

[0030] Unless there are working examples or other instructions, all numerical values listing quantitative characteristics such as dosages in the specification and claims should be understood as being modified by the term "about" in all cases. Furthermore, any numerical range recited in this disclosure should be understood to include all sub-ranges within that range, and any combination of the corresponding ranges or endpoints of the sub-ranges.

[0031] Unless otherwise defined, all terms (including technical and scientific terms) used in the present invention have the same meaning as commonly understood by those skilled in the technical field to which the present invention belongs. Terms defined as in commonly used dictionaries should be interpreted as having a meaning consistent with their meaning in the context of the related art, and should not be interpreted as having an idealized or overly formal meaning unless explicitly defined in the present invention.

[0032] As used in the present invention, the term "C1-C6" refers to a carbon atom having any integer value within the range of 1 to 6 in the main chain of the group, for example, 1, 2, 3, 4, 5, 6 carbon atoms. Similarly, the term "C2-C6" refers to a carbon atom having any integer value within the range of 2 to 6 in the main chain of the group, for example, 2, 3, 4, 5, 6 carbon atoms.

[0033] As used in the present invention, the term "alkyl" refers to a saturated aliphatic hydrocarbon group having a straight or branched chain, and non-limiting examples thereof include methyl, ethyl, propyl, n-butyl, tert-butyl, pentyl, hexyl and the like. As used in the present invention, the term "alkenyl" refers to a hydrocarbon group having at least one carbon-carbon double bond at one or more positions along the carbon chain of alkyl, and non-limiting examples thereof include vinyl, propenyl, butenyl and the like.

[0034] As used in the present invention, the term "alkynyl" means a hydrocarbon group having at least one carbon-carbon triple bond at one or more positions along the carbon chain of alkyl, and non-limiting examples thereof include ethynyl, propynyl and the like. As used in the present invention, the term "aryl" means a group containing a carbocyclic aromatic system, and non-limiting examples thereof include phenyl, naphthyl, anthracenyl, phenanthrenyl, pyrenyl and the like. When aryl contains a plurality of rings, each ring can be condensed with each other. As used in the present invention, the term "heteroaryl" refers to a group having a carbocyclic aromatic system containing at least one heteroatom selected from N, O, Si, P and S as ring-forming atoms, and non-limiting examples thereof include pyridinyl, pyrimidinyl, pyrazinyl, pyridazinyl, triazinyl, quinolyl, isoquinolinyl and the like. When heteroaryl contains a plurality of rings, each ring can be condensed with each other.

[0035] As used in the present invention, the terms "heteroalkyl" and "heteroaryl" refer to alkyl containing at least one heteroatom selected from N, O, Si, P and S.

[0036] As used in the present invention, the term "salt" refers to the corresponding salt of a modified nucleotide compound (or nucleotide compound) of the present invention that is convenient or desirable for manufacture, purification and / or treatment, for example, a pharmaceutically acceptable salt. Unless otherwise specified, references to a particular compound in the present invention include its salt forms.

[0037] As used in the present invention, a "capping analog" is the structure at the 5' end of mature mRNA formed by post-transcriptional modification in eukaryotes, i.e., the m7GPPPN structure also known as the methylguanosine cap. This structure can have effects such as preventing the degradation of mRNA at the 5' end, allowing the RNA transcript to enter the cytoplasm through the selective pores of the nuclear membrane, enhancing translation, and assisting in the completion of the overall splicing process. As used in the present invention, the portion between the wavy lines of the nucleic acid polymer is a structure embedded in the nucleic acid polymer sequence, and the outer portion of the wavy lines represents other sequences of the nucleic acid polymer.

[0038] Bases, also called nucleobases, refer to the heterocyclic base portion of a nucleoside. The nucleobases may be naturally occurring or modified, for example, natural or modified pyrimidine nucleotide bases, or natural or modified purine nucleotide bases. The bases include, but are not limited to, cytosine, guanine, adenine, thymine, uracil, hypoxanthine, xanthine, 7-methylguanine, pseudouracil, thiouracil, 5,6-dihydrouracil, 5-bromouracil, 5-iodouracil, 5-methylcytosine, and 5-hydroxymethylcytosine.

[0039] Similar terms such as "comprising", "containing", or "including" as used in the present disclosure mean that the element appearing before the term includes the elements listed after the term and their equivalents without excluding elements not listed. The terms "containing" or "comprising (including)" as used herein may be open-ended, semi-closed, or closed. In other words, the terms also include "consisting essentially of..." or "consisting of...".

[0040] The term "optionally" refers to the situation described in the present disclosure may or may not occur. For example, being optionally substituted means that it may be either substituted or unsubstituted. Being substituted means that at least one hydrogen of the group is substituted by another chemical group such as methyl, ethyl, methoxy, ethoxy, halogen, hydroxyl, mercapto, amino, nitro, -CN, etc.

[0041] The term "pharmaceutically acceptable" in the present disclosure refers to a compound or composition being chemically and / or toxicologically compatible with other components constituting the formulation and / or a human or mammal using it to prevent or treat a disease or disorder.

[0042] The term "solvate" in the present disclosure refers to a complex formed by combining a compound represented by formula (I) or a pharmaceutically acceptable salt thereof with a solvent (such as ethanol or water). Any solvate of the compound represented by formula (I) used in the treatment of a disease or disorder can provide different properties (including pharmacokinetic properties), but since the compound represented by formula (I) is obtained when absorbed by a subject, the use of the compound represented by formula (I) should be understood to include the use of any solvate of the compound represented by formula (I).

[0043] The term "hydrate" refers to the solvent in the above "solvate" being water.

[0044] Furthermore, it should be understood that the compound represented by formula (I) or a pharmaceutically acceptable salt thereof can be separated in the form of a solvate, and thus any of the above solvates are all included within the scope of the present invention. For example, the compound represented by formula (I) or a pharmaceutically acceptable salt thereof can exist in a non-solvated form, as well as in a solvated form formed with a pharmaceutically acceptable solvent (such as water, ethanol, etc.).

[0045] The present invention also includes salts of the compounds described herein, particularly pharmaceutically acceptable salts. Compounds of the present invention having sufficiently acidic or sufficiently alkaline functional groups can react with many bases or acids to form salts. Alternatively, compounds that are essentially charged (e.g., compounds having a quaternary nitrogen) can form salts with appropriate counterions (e.g., halogen ions such as bromide ion, chloride ion or fluoride ion, particularly bromide ion).

[0046] The term "pharmaceutically acceptable salt" means a relatively non-toxic addition salt of a compound of the present disclosure. See, for example, "Pharmaceutical Salts" by S. M. Berge et al., J. Pharm. Sci. 1977, 66, 1-19.

[0047] Suitable pharmaceutically acceptable salts of the compounds of the present disclosure may be, for example, acid addition salts of the compounds of the present disclosure that are sufficiently alkaline and have a nitrogen atom in the chain or ring, for example, acid addition salts formed with inorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, phosphoric acid or nitric acid, or, for example, formic acid, acetic acid, acetoacetic acid, pyruvic acid, trifluoroacetic acid, propionic acid, butyric acid, caproic acid, enanthic 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, dodecyl sulfate, 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, glucoheptonic acid, glycerophosphoric acid, aspartic acid, sulfosalicylic acid or thiocyanic acid and the like.

[0048] Furthermore, another suitable pharmaceutically acceptable salt of the compound of the present invention that is sufficiently acidic 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., a salt formed with NH3 or aqueous ammonia), or a salt formed with an organic base that provides a physiologically acceptable cation, for example, a salt formed with substances such as triethylamine, N-methylglucamine, dimethylglucamine, ethylglucamine, lysine, dicyclohexylamine, 1,6-hexanediamine, ethanolamine, glucosamine, sarcosine, serinol, tris(hydroxymethyl)aminomethane, aminopropylene glycol, 1-amino-2,3,4-butanetriol. Alternatively, the basic nitrogen-containing group can be quaternized with reagents such as lower alkyl halides such as methyl, ethyl, propyl, butyl chlorides, bromides, and iodides, dialkyl sulfates such as dimethyl sulfate, diethyl sulfate, dibutyl sulfate, and diamyl sulfate, long-chain halides such as decyl, lauryl, myristyl, stearyl chlorides, bromides, and iodides, and aralkyl halides such as benzyl and phenethyl bromide.

[0049] One skilled in the art can also recognize that the acid addition salts of the compound to be protected may be prepared by reacting the compound 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 it with a suitable base by various known methods.

[0050] The present invention includes all possible salts of the compounds of the present disclosure, which may be a single salt or any mixture of the salts in any ratio.

[0051] The specific compounds of the present disclosure can exist in the form of one or more stereoisomers. Stereoisomers include geometric isomers, diastereomers, and enantiomers. Therefore, the compounds claimed in the present disclosure further include racemic mixtures, single stereoisomers, and mixtures having optical activity. Those skilled in the art should understand that one stereoisomer may have better effects and / or fewer side effects than other stereoisomers. Single stereoisomers and mixtures having optical activity can be obtained by methods such as chiral-derived synthesis, chiral catalysts, and chiral resolution. Racemates can be chiral-resolved by chromatographic resolution or chemical resolution. For example, chiral acid resolving reagents such as chiral tartaric acid and chiral malic acid can be added to form salts with the compounds of the present disclosure, and the physical and chemical properties of the products such as differences in solubility can also be utilized.

[0052] Alkyl (or alkylene) may be unsubstituted, or alkyl (or alkylene) may be substituted, meaning that at least one hydrogen is substituted with other chemical groups such as methyl, ethyl, methoxy, ethoxy, halogen, hydroxyl, mercapto, amino, nitro, -CN, etc.

[0053] In the present disclosure, when the compound name does not match the structural formula, the structural formula shall take precedence.

[0054] The specification of the present disclosure should be interpreted as conforming to the laws and principles of chemical bonding. In some cases, a hydrogen atom can be removed to accommodate a substituent at a given position.

[0055] It should be understood that the term "compounds of the present disclosure" used in the present disclosure includes compounds represented by formula (I) according to the context, their solvates, their pharmaceutically acceptable salts, their stereoisomers, and mixtures thereof.

[0056] Modified locked nucleoside mRNA capping analog The present invention provides a compound represented by formula (I), or a stereoisomer, pharmaceutically acceptable salt or solvate thereof.

[0057]

Chemical formula

[0058] In one embodiment, X is absent. When X is absent, R4 is, for example, -CF2H, -N(CH3)2 or -CN.

[0059] In one embodiment, X is -C(=O)-. When X is -C(=O)-, R4 is, for example, -N(CH3)2.

[0060] In one embodiment, X is -S(=O)2-. When X is -S(=O)2-, R4 is, for example, -N(CH3)2 or -CH3.

[0061] In one embodiment, R1 is a hydrogen atom or methyl, preferably a hydrogen atom.

[0062] In one embodiment, R2 is a hydrogen atom or methyl, preferably methyl.

[0063] In one embodiment, R3 is a hydrogen atom or methyl, preferably a hydrogen atom.

[0064] In one embodiment, R5 is a hydrogen atom or methyl, preferably methyl.

[0065] In one embodiment, R6 is a hydrogen atom or methyl, preferably a hydrogen atom.

[0066] In one embodiment, Y 1a is -O-.

[0067] In one embodiment, Y 1b is -O-.

[0068] In one embodiment, Y 1c is -O-.

[0069] In one embodiment, Y 1d is -O-.

[0070] In one embodiment, Y 1e is -O-.

[0071] In one embodiment, Y 1f is -O-.

[0072] In one embodiment, Y 2a is =O.

[0073] In one embodiment, Y 2b is =O.

[0074] In one embodiment, Y 2c is =O.

[0075] In one embodiment, Y 2d is =O.

[0076] In one embodiment, Y 3a is -OH.

[0077] In one embodiment, Y 3b is -OH.

[0078] In one embodiment, Y 3c is -OH.

[0079] In one embodiment, Y 3d is -OH.

[0080] In one embodiment,

[0081]

Chemical formula

[0082] In one embodiment, B1 and B2 are independently selected from substituted or unsubstituted adenine, guanine, thymine, cytosine, uracil, or pharmaceutically acceptable salts thereof. For example, B1 and B2 are independently selected from cytosine, guanine, adenine, thymine, uracil, hypoxanthine, xanthine, 7-methylguanine, pseudouracil, thiouracil, 5,6-dihydrouracil, 5-bromouracil, 5-iodouracil, 5-methylcytosine, 5-hydroxymethylcytosine, or pharmaceutically acceptable salts thereof.

[0083] In one embodiment, B1 is adenine.

[0084] In one embodiment, B2 is 7-methylguanine or guanine, preferably guanine.

[0085] For example, the compound represented by formula (I) is YK-CAP-001, YK-CAP-002, YK-CAP-003, YK-CAP-004, YK-CAP-005 or YK-CAP-006, the structure of which is represented as follows.

[0086] [Chemical formula] JPEG2025107988000006.jpg216158

[0087] Compared with similar compounds in the prior art, the modified locked nucleoside capping analogs of the present application have the following advantages. 1) It has a completely different structure from similar compounds in the prior art and is a completely new compound.

[0088] 2) Compared with similar compounds in the prior art, the in vitro transcription yield of mRNA of the modified locked nucleoside cap analogs of the present application is significantly improved, the capping rate is significantly improved, the translation efficiency of the target mRNA is significantly improved, the decapping rate is significantly decreased, and both the amount and duration of the protein expressed by the mRNA in the mouse body are significantly improved.

[0089] For example, the in vitro transcription yield and capping rate of mRNA of YK-CAP-004 are improved by 35.0% and 18.8% respectively compared with compound 14, the translation efficiency reaches 4 times that of m6A, the decapping rate is decreased by 38.3% compared with N-7413, and the average radiation intensity (equivalent to the protein expression level) reaches 2.1 times and 3.4 times that of m6A at 6 hours and 96 hours respectively.

[0090] Furthermore, the present application discovers that it is very likely that there are significant differences in the in vitro transcription yield of mRNA, capping rate, translation efficiency of the target mRNA, decapping rate, amount of protein expressed by the mRNA in the mouse body and duration among the modified locked nucleoside capping analogs having the same structure.

[0091] For example, when comparing YK-CAP-001 and YK-CAP-002, which have very similar structures, with Compound 14, the in vitro transcription yield of mRNA of YK-CAP-004 was improved by 82.8% compared to YK-CAP-001, the capping rate was improved by 26.1% compared to YK-CAP-002, the mRNA translation efficiency was 2.0 times that of YK-CAP-002, the decapping rate was decreased by 18.5% compared to Compound 14, and the average radiation intensity of the protein expressed by the mRNA in the mouse body was 2.6 times and 5.5 times that of YK-CAP-002 at 6 hours and 96 hours, respectively.

[0092] Therefore, the activity level cannot be estimated based on the structure of the modified locked nucleoside cap analog.

[0093] The RNA delivery agent may be, for example, lipid nanoparticles (LNPs). Lipid nanoparticles are widely used for the delivery of small molecule drugs and nucleic acids, and mRNA coated with LNPs can be protected from the influence of extracellular ribonucleases and is useful for intracellular mRNA delivery. See the review article Chemistry of Lipid Nanoparticles for RNA Delivery. Acc Chem Res. 2022 Jan 4; 55(1):2-12.

[0094] Cationic lipid Lipid nanoparticles generally contain cationic lipids.

[0095] As used herein, the term "cationic lipid" refers to a lipid that carries a positive charge at a selected pH value. For example, reference can be made to the cationic lipids disclosed in WO2023133946A1, CN115745820A, Chemistry of Lipid Nanoparticles for RNA Delivery. Acc Chem Res. 2022 Jan 4;55(1):2-12, etc.

[0096] In one embodiment, the cationic lipid is one or more selected from the following compounds. (1) A compound represented by formula (II), or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer 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 linear alkyl, L2 is C 12~25 branched alkyl,

[0097]

Chemical Structure

[0098]

Chemical Structure

[0099] [Chemical formula] (4) is a compound represented by formula (V), or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof, wherein G1 is C 1~8 alkylene, G2 is C 2~8 alkylene, R1 is C 6~25 a straight-chain or branched-chain alkyl, R2 is C 12~25 a straight-chain or branched-chain alkyl, G3 is HO(CH2)2N(R3)CH2CH(OH)CH2-, where R3 is -CH3 or -CH2CH3 or -CH2CH2OH,

[0100] [Chemical formula] (5) is a compound represented by formula (VI), or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof, wherein G 1 and G 2 are each independently unsubstituted C6-C 10 alkylene, G 3 is unsubstituted 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, -C(=O)OR 4 , -OC(=O)R 4 or -NR 5 C(=O)R4 and R 4 is a C1-C 12 hydrocarbon group, and R 5 is H or a C1-C6 hydrocarbon group,

[0101]

Chem.

[0102]

Chem.

[0103]

Chem.

[0104] In one preferred embodiment, the cationic lipid is selected from one or more of YK-009, YK-401, YK-305, ALC0315, SM102, DLIN-MC3-DMA.

[0105]

Chem.

[0106] In one more preferred embodiment, the cationic lipid is YK-009.

[0107] Neutral lipid The RNA delivery agent containing the cationic lipid may also contain a neutral lipid. In the present disclosure, the neutral lipid refers to a lipid that plays an auxiliary role of not being charged or existing in an amphoteric ion form at a selected pH value. The neutral lipid can promote lipid phase transition, adjust the fluidity of the nanoparticles to a lipid bilayer structure to improve efficiency, and can also affect target organ specificity.

[0108] In one embodiment, the molar ratio of the cationic lipid to the neutral lipid is about 1:1 to 15:1, for example, about 14:1, 13:1, 12:1, 11:1, 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1 and 2:1. In one preferred embodiment, the molar ratio of the cationic lipid to the neutral lipid is about 4.5:1. In another preferred embodiment, the molar ratio of the cationic lipid to the neutral lipid is about 4.9:1.

[0109] For example, the neutral lipid may include one or more of phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, ceramide, sterol and its derivatives.

[0110] The RNA delivery agent containing the cationic lipid may include one or more neutral lipid-phospholipids such as one or more (poly)unsaturated lipids. The phospholipid can form one or more lipid bilayers. Generally, the phospholipid may include a phospholipid moiety and one or more fatty acid moieties.

[0111] The neutral lipid moiety can be selected from the non-limiting group consisting of phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylserine, phosphatidic acid, 2-lyso-phosphatidylcholine, and sphingomyelin. The fatty acid moiety can be selected from the non-limiting group consisting of lauric acid, myristic acid, myristoleic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleic acid, α-linolenic acid, erucic acid, phytic acid, arachidic acid, arachidonic acid, eicosapentaenoic acid, behenic acid, docosapentaenoic acid, and docosahexaenoic acid. Also included are non-natural species including natural species having modifications and substitutions including branching, oxidation, cyclization, and alkynes. For example, the phospholipid may be functionalized with one or more alkynes (e.g., alkenyls in which one or more double bonds are replaced with triple bonds) or cross-linked with said one or more alkynes. Under appropriate reaction conditions, the alkynyl may undergo a copper-catalyzed cycloaddition reaction upon exposure to azide. Such reactions can be used to functionalize the lipid bilayer of the composition to promote membrane permeability or cell recognition, or to couple the composition with useful components such as targeting or imaging moieties (e.g., dyes).

[0112] Neutral lipids that can be used in such compositions are 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-doundecanoyl-sn-glycero-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 Diether PC), 1-oleoyl-2-cholesteryl hemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), 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 (ME 16.0 PE), 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 (DOPG), dipalmitoyl phosphatidylglycerol (DPPG), palmitoyl oleoyl phosphatidylethanolamine (POPE), distearoyl-phosphatidylethanolamine (DSPE), dipalmitoyl phosphatidylethanolamine (DPPE), dimyristoyl phosphatidylethanolamine (DMPE), 1-stearoyl-2-oleoyl-stearoylethanolamine (SOPE), 1-stearoyl-2-oleoyl-phosphatidylcholine (SOPC), sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyl oleoyl phosphatidylcholine, lysophosphatidylcholine, lysophosphatidylethanolamine (LPE) and mixtures thereof can be selected from the non-limiting group consisting of.

[0113] In some embodiments, the neutral lipid comprises DSPC. In some embodiments, the neutral lipid comprises DOPE. In some embodiments, the neutral lipid comprises both DSPC and DOPE.

[0114] Structural lipid The RNA delivery agent containing a cationic lipid may further contain one or more structural lipids. In the present disclosure, the structural lipid refers to a lipid that fills the space between lipids to improve the stability of the nanoparticles.

[0115] In one embodiment, the molar ratio of the cationic lipid to the structural lipid is about 0.6:1 to 3:1, for example, about 1.0:1, 1.1:1, 1.2:1, 1.3:1, 1.4:1, 1.5:1, 1.6:1, 1.7:1, 1.8:1, 1.9:1, 2.0:1.

[0116] The structural lipid can be selected from the group consisting of cholesterol, non-sterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatine, ursolic acid, α-tocopherol, corticosteroid and mixtures thereof, but is not limited thereto. In some embodiments, the structural lipid is cholesterol. In some embodiments, the structural lipid comprises cholesterol and corticosteroid (e.g., prednisolone, dexamethasone, prednisone and hydrocortisone) or a combination thereof.

[0117] Polymer-conjugated lipid The RNA delivery agent containing the cationic lipid may further contain one or more polymer-conjugated lipids. The polymer-conjugated lipid mainly refers to polyethylene glycol (PEG)-modified lipid. The hydrophilic PEG stabilizes the LNP, limits lipid fusion to adjust the nanoparticle size, and reduces non-specific interaction with macrophages, thereby increasing the nanoparticle half-life.

[0118] In one embodiment, the polymer-conjugated lipid is one or more selected from PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, PEG-modified dialkylglycerol. The molecular weight of the PEG-modified PEG is generally 350 - 5000 Da.

[0119] For example, the polymer-conjugated lipid is one or more selected from distearoyl phosphatidylethanolamine polyethylene glycol 2000 (DSPE-PEG2000), 1,2-dimyristoyl-rac-glycero-methoxypolyethylene glycol-2000 (DMG-PEG2000), and methoxypolyethylene glycol ditetradecylacetamide (ALC-0159).

[0120] In one embodiment, the polymer-conjugated lipid is DMG-PEG2000.

[0121] In one embodiment, the RNA delivery agent includes a neutral lipid, a structural lipid, and a polymer-conjugated lipid, and the molar ratio of the cationic lipid, the neutral lipid, the structural lipid, and the polymer-conjugated lipid is (25-75):(5-25):(15-65):(0.5-10), for example, (35-49):(7.5-15):(35-55):(1-5).

[0122] In one embodiment, the RNA delivery agent includes a neutral lipid, a structural lipid, and a polymer-conjugated lipid, and the molar ratio of the cationic lipid, the neutral lipid, the structural lipid, and the polymer-conjugated lipid is 49:10:39.5:1.5.

[0123] The embodiments of the present invention will be described in detail in combination with the following examples. Those skilled in the art should understand that the following examples are only for explaining the present invention and should not be regarded as limiting the scope of the present invention. When specific conditions are not specified in the examples, they are carried out according to normal conditions or the conditions recommended by the manufacturer. When the manufacturer of the reagents or equipment used is not specified, all are common products that can be purchased commercially.

[0124] The following abbreviations represent the following reagents respectively. IBX: 2-Iodoxybenzoic acid; BF3·Et2O: Boron trifluoride diethyl etherate; Allyltrimethylsilane: Allyltrimethylsilane; TEA: Triethylamine; Ac2O: Acetic anhydride; HOAc: Acetic acid; conc H2SO4: Concentrated sulfuric acid; BSA: N,O-Bis(trimethylsilyl)acetamide; TMSOTf: Trimethylsilyl trifluoromethanesulfonate; Toluene: Toluene; MeOH: Methanol; Boc2O: Di-tert-butyl dicarbonate; DIEA: N,N-Diisopropylethylamine; DMAP: 4-Dimethylaminopyridine; DMSO: Dimethyl sulfoxide; HATU: 2-(7-Azabenzotriazol-1-yl)-N,N,N’,N’-tetramethyluronium hexafluorophosphate; THF: Tetrahydrofuran; TBSCl: tert-Butyldimethylsilyl chloride; Imidazole: Imidazole; DMF: N,N-Dimethylformamide; TBAF: Tetrabutylammonium fluoride; TBSOTf: tert-Butyldimethylsilyl trifluoromethanesulfonate; NMO: N-Methylmorpholine-N-oxide; m-CPBA: m-Chloroperbenzoic acid; DIAD: Diisopropyl azodicarboxylate; NCS: N-Chlorosuccinimide; PO(MeO)3: Trimethyl phosphate; PySSPy: 2,2’-Dithiodipyridine; imidazole: Imidazole; PPh3: Triphenylphosphine; TEAP: Triethylamine phosphate; TEAB: Triethylamine bicarbonate; MTBE: Methyl tert-butyl ether; DCM: Dichloromethane; EA: Ethyl acetate; DAST: Diethylaminosulfur trifluoride; AcSH: Thioacetic acid.

[0125] Example 1: 1. Synthesis of Intermediate INT-I

[0126]

Chemical formula

[0127] Step 1: Synthesis of INT-I-PM1 3 - O - Benzyl - 4 - C - benzyloxymethyl - 1,2 - O - isopropylidene - α - D - ribofuranose (100.0 g, 0.25 mol) was dissolved in 500 mL of acetonitrile, 2 - iodoxybenzoic acid (104.9 g, 0.37 mol) was added, the temperature was raised to 70 °C, and the mixture was stirred and reacted for 2 hours, and the completion of the reaction was monitored by TLC. The reaction was stopped, and after the reaction system was cooled to room temperature, it was filtered through diatomaceous earth, the cake was washed with acetonitrile (500 mL), the filtrate was spin - dried under reduced pressure and then vacuum - dried to obtain a pale yellow liquid INT - I - PM1 (102.1 g), which was directly used in the next step.

[0128] Step 2: Synthesis of INT - I - PM2 INT - I - PM1 (102.1 g, 0.25 mol) was dissolved in 400 mL of dichloromethane, stirred and cooled to - 40 °C, boron trifluoride diethyl etherate solution (50.82 g, 0.35 mol) was slowly added, and the mixture was stirred and reacted for 5 minutes. Allyltrimethylsilane (52.9 mL, 0.33 mol) was added dropwise, and the mixture was stirred and reacted for 2 hours, and the completion of the reaction was monitored by TLC. The reaction solution was slowly poured into 1000 mL of saturated aqueous sodium bicarbonate solution for quenching, separated, the aqueous phase was extracted with dichloromethane (500 mL×2), the organic phase was washed with saturated brine (500 mL×2), dried over anhydrous sodium sulfate, spin - dried, and the residue was purified by normal - phase silica gel column chromatography to obtain a pale yellow liquid INT - I - PM2 (103.0 g, 0.23 mol).

[0129] Step 3: Synthesis of INT - I - PM3 INT-I-PM2 (103.0 g, 0.23 mol) was dissolved in 500 mL of dichloromethane, triethylamine (70.9 g, 0.70 mol) was added, and ethanesulfonyl chloride (60.4 g, 0.47 mol) was added dropwise under an ice bath. After the addition was complete, the ice bath was removed, and the temperature was allowed to rise naturally to room temperature. The mixture was stirred for 2 hours to react, and the completion of the reaction was monitored by TLC. After the reaction was complete, the reaction solution was poured into 1000 mL of saturated aqueous sodium bicarbonate solution to quench and separate the reaction system. The aqueous phase was extracted with dichloromethane (500 mL × 2), the organic phases were combined, washed with saturated brine (1000 mL), dried over anhydrous sodium sulfate, and spin-dried to obtain a brown viscous substance. The residue was purified by normal-phase silica gel column chromatography to obtain a pale yellow liquid INT-I-PM3 (106.0 g, 0.20 mol, yield: 86.5%).

[0130] Step 4: Synthesis of INT-I-PM4 INT-I-PM3 (106.0 g, 0.20 mol) was dissolved in 200 mL of acetic acid, acetic anhydride (125.6 g, 1.23 mol) was added, 2.4 mL of concentrated sulfuric acid was added dropwise, and the mixture was stirred at room temperature for 1 hour to react, and the completion of the reaction was monitored by TLC. After the reaction was complete, ethyl acetate (800 mL) and water (800 mL) were added, and the mixture was separated. The aqueous phase was extracted once with ethyl acetate (800 mL), the organic phases were combined, washed with water (800 mL × 3), washed with saturated aqueous sodium bicarbonate solution, washed twice with saturated brine, dried over anhydrous sodium sulfate, and spin-dried to obtain a pale yellow liquid INT-I-PM4 (86.5 g, 0.15 mol, yield: 75.0%).

[0131] Step 5: Synthesis of INT-I-PM5 2-Amino-6-chloropurine (30.50 g, 0.18 mol) and 250 mL of toluene were added to a 1000 mL reaction flask. Subsequently, N,O-bis(trimethylsilyl)acetamide (61.00 g, 0.30 mol) was added. The mixture was heated to 80 °C, stirred until dissolved, and then cooled to room temperature. INT-I-PM4 (86.5 g, 0.15 mol) dissolved in 150 mL of toluene was added, and the mixture was stirred for 5 minutes. Trimethylsilyl trifluoromethanesulfonate (40.00 g, 0.18 mol) dissolved in 50 mL of toluene was added dropwise. The temperature was rapidly raised to 110 °C, and the reaction was carried out for 3 hours while monitoring the completion of the reaction by TLC. The reaction was stopped. After the reaction system was cooled to room temperature, ethyl acetate (300 mL) was added for extraction. The mixture was washed with a saturated aqueous sodium bicarbonate solution (500 mL). The solid precipitated on diatomaceous earth was filtered, and the filtrate was separated. The aqueous phase was back-extracted with ethyl acetate (500 mL). The organic phases were combined, dried over anhydrous Na2SO4, the solvent was spin-dried, and the residue was purified by normal-phase silica gel column chromatography to obtain INT-I-PM5 (103.02 g, 0.15 mol, yield: 100%). C 32 H 36 ClN5O8S, MS(ES): m / z(M+H + ) 686.2.

[0132] Step 6: Synthesis of INT-I-PM6 INT-I-PM5 (103.00 g, 0.15 mol) and 500 mL of methanol were added to a 1000 mL reaction flask. The mixture was stirred until dissolved, potassium carbonate (103.73 g, 0.75 mol) was added, and the reaction was carried out at room temperature for 23 hours while monitoring the completion of the reaction by TLC. The reaction was stopped. The mixture was filtered through diatomaceous earth, and the solvent was removed by spin-evaporation under reduced pressure. The residue was extracted with ethyl acetate (800 mL) and water (500 mL), and separated. The aqueous phase was back-extracted with ethyl acetate (300 mL). The organic phases were combined, washed once with a saturated aqueous sodium chloride solution (600 mL), dried over anhydrous sodium sulfate, the solvent was spin-dried to obtain a crude product, and INT-I-PM6 (44.03 g, 0.083 mol, yield: 55.4%) was obtained by normal-phase silica gel column chromatography. C 29 H31 N5O5, MS(ES): m / z(M+H + ) 530.2。

[0133] Step 7: Synthesis of INT-I INT-I-PM6 (44.00 g, 0.083 mol) was dissolved in dichloromethane (500 mL), and N,N-diisopropylethylamine (32.15 g, 0.24 mol), 4-dimethylaminopyridine (1.01 g, 0.01 mol), and di-tert-butyl dicarbonate (54.45 g, 0.24 mol) were sequentially added. The above reaction system was stirred overnight at 30 °C. It was confirmed by TLC that the reaction was almost complete, and the solvent was evaporated under reduced pressure. Purification by silica gel chromatography gave INT-I (49.81 g, 68.25 mmol, yield: 82.2%). C 39 H 47 N5O9, MS(ES): m / z(M+H + ) 730.2。

[0134] 2. Synthesis of Intermediate INT-II

[0135]

Chemical Structure

[0136] pA(2’-OMe)mpG·TEA (300.1 mg, 0.37 mmol), imidazole (347.2 mg, 5.10 mmol), dithiodipyridine (1123.6 mg, 5.10 mmol), and triethylamine (516.1 mg, 5.10 mmol) were dissolved in 2.0 mL of ultra-dry N,N-dimethylformamide. Triphenylphosphine (1337.7 mg, 5.10 mmol) was added under the protection of nitrogen gas, and the reaction was carried out at 25 °C for 4 hours. After the reaction was completed, the reaction solution was slowly added to an acetone solution containing pre-cooled sodium iodide (598.1 mg, 3.99 mmol), and crystallization was carried out at 25 °C for 30 minutes. After centrifugation, white solid INT-II (240.6 mg, 0.30 mmol, yield: 80.9%) was obtained. C 24 H 30 N 12 O13 P2, MS(ES): m / z(M-H - ): 755.15。

[0137] 3. Synthesis of YK-CAP-001

[0138]

Chemical formula

[0139] Step 1: Synthesis of YK-CAP-001-PM1 INT-I (49.81 g, 68.25 mmol) was dissolved in tetrahydrofuran (400 mL) and water (100 mL), and N-methylmorpholine N-oxide (12.00 g, 102.37 mmol) and potassium osmate dihydrate (1.26 g, 3.41 mmol) were sequentially added to the above mixed solution. The reaction system was stirred at room temperature for 5 hours, and TLC indicated that the reaction was complete. The reaction system was quenched with saturated sodium sulfite, extracted with ethyl acetate (500 mL × 2), and the organic phase was washed with 200 mL of saturated sodium chloride solution. Finally, the organic phase was dried over anhydrous sodium sulfate, filtered, and spin-dried to obtain YK-CAP-001-PM1 (52.00 g, 68.08 mmol, yield: 99.8%). C 39 H 49 N5O 11 , MS(ES): m / z(M+H + ) 764.3。

[0140] Step 2: Synthesis of YK-CAP-001-PM2 YK-CAP-001-PM1 (52.00 g, 68.08 mmol) was dissolved in tetrahydrofuran (500 mL) and water (125 mL), sodium periodate (21.84 g, 102.11 mmol) was weighed and added, the reaction system was heated to 30 °C overnight, and TLC indicated that the reaction was complete. The reaction system was quenched with saturated sodium sulfite, extracted with ethyl acetate (500 mL × 3), the organic phases were combined, the organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and spin-dried. YK-CAP-001-PM2 (45.5 g, 62.2 mmol, yield: 91.3%) was obtained. C 38 H 45 N5O 10 , MS(ES): m / z(M+H + ) 732.3。

[0141] Step 3: Synthesis of YK-CAP-001-PM3 YK-CAP-001-PM2 (15.30 g, 20.90 mmol) and dimethylamine hydrochloride (8.52 g, 104.50 mmol) were dissolved in dichloroethane (300 mL), sodium triacetoxyborohydride (22.16 g, 104.50 mmol) was slowly added at room temperature, and the mixture was stirred at room temperature for 15 hours to react. LCMS confirmed that the reaction was complete. The reaction system was diluted with 200 mL of dichloromethane, quenched by adding saturated ammonium chloride aqueous solution, separated, the aqueous phase was extracted with dichloromethane (200 mL × 2), the organic phases were combined, the organic phase was washed with 100 mL of saturated sodium chloride aqueous solution, dried over anhydrous sodium sulfate, filtered, and spin-dried. Purification by silica gel chromatography gave YK-CAP-001-PM3 (10.80 g, 14.19 mmol, yield: 67.9%). C 40 H 52 N6O9, MS(ES): m / z(M+H + ) 761.3。

[0142] Step 4: Synthesis of YK-CAP-001-PM4 Using YK-CAP-001-PM3 (10.60 g, 13.93 mmol) as a raw material, it was dissolved in dichloromethane (300 mL). The reaction system was cooled to -40 °C under a nitrogen gas atmosphere, and a dichloromethane solution of boron trichloride (111.4 mL, 111.4 mmol) was slowly added dropwise. After the addition was complete, the reaction system was slowly warmed to 0 °C, and the reaction system was stirred at this temperature for 3 hours. TLC indicated that the reaction was complete. The temperature was cooled to -40 °C again, and the reaction system was quenched with methanol, spin-dried, and left at room temperature for 24 hours to obtain 5.5 g of a brown crude product. 1.5 g was purified by preparative HPLC to obtain YK-CAP-001-PM4 (508.47 mg, 1.39 mmol). C 15 H 22 N6O5, MS(ES): m / z(M+H + ) 367.1.

[0143] 1 H NMR (400 MHz, MeOD) δ 8.16 (s, 1H), 4.51 (s, 1H), 4.41 (s, 1H), 4.29 - 4.27 (m, 1H), 3.91 (s, 2H), 3.47 - 3.34 (m, 2H), 2.95 (s, 6H), 2.86 - 2.85 (m, 1H), 2.25 - 2.20 (m, 2H).

[0144] Step 5: Synthesis of YK-CAP-001-PM5 Intermediate The YK-CAP-001-PM4 intermediate (380.0 mg, 1.04 mmol) was dissolved in 4.0 mL of trimethyl phosphate, placed in an ice bath, and phosphorus oxychloride (1.69 g, 10.37 mmol) was slowly added dropwise under the protection of nitrogen gas, and the reaction was carried out at 0 °C for 4 hours. After the reaction was completed, 4.0 mL of purified water was slowly added under the ice bath, and the mixture was stirred in the ice bath for 1 hour. Next, dichloromethane (4 mL × 3) was added and washed 3 times. The aqueous phase was taken, concentrated under reduced pressure to remove the organic solvent, the residue was dissolved in 200 mL of purified water, and purified by gel column chromatography to obtain the white solid YK-CAP-001-PM5 triethylamine salt (260.0 mg, 0.47 mmol, yield: 45.8%). C15 H 23 N6O8P, MS(ES): m / z(M-H - ) 445.13。

[0145] Step 6: Synthesis of YK-CAP-001-PM6 Intermediate The YK-CAP-001-PM5 triethylamine salt (260.0 mg, 0.47 mmol), imidazole (384.0 mg, 5.64 mmol), dithenyldipyridine (1242.5 mg, 5.64 mmol), and triethylamine (570.7 mg, 5.64 mmol) were dissolved in 2.0 mL of ultra-dry N,N-dimethylformamide. Under the protection of nitrogen gas, triphenylphosphine (1479.3 mg, 5.64 mmol) was added, and the reaction was carried out at 25 °C for 4 hours. After the reaction was completed, the reaction solution was slowly added to an acetone solution containing pre-cooled sodium iodide (704.5 mg, 4.7 mmol), and crystallization was carried out at 25 °C for 30 minutes. After repeated centrifugation, a white solid YK-CAP-001-PM6 (165 mg, 0.32 mmol, yield: 67.0%) was obtained. C 18 H 25 N8O7P, MS(ES): m / z(M-H - ) 495.16。

[0146] Step 7: Synthesis of YK-CAP-001-PM7 Intermediate The YK-CAP-001-PM6 (165 mg, 0.32 mmol), triethylamine phosphate (197.2 mg, 0.99 mmol), and zinc chloride (90.0 mg, 0.66 mmol) were dissolved in 2.0 mL of ultra-dry N,N-dimethylformamide, and the reaction was carried out at 25 °C for 16 hours. After the reaction was completed, methyl tert-butyl ether was added and stirred for 10 minutes, then allowed to stand, and the supernatant was removed. The lower-layer turbid liquid was taken. The solvent was removed by concentration under reduced pressure, and the residue was dissolved in 100 mL of purified water and purified by gel column chromatography to obtain a white solid YK-CAP-001-PM7 triethylamine salt (150.5 mg, 0.24 mmol, yield: 75.3%). C 15 H 24 N6O 11 P2, MS(ES): m / z(M-H- ) 525.10。

[0147] Step 8: Synthesis of YK-CAP-001-PM8 Intermediate The YK-CAP-001-PM7 triethylamine salt (150.5 mg, 0.24 mmol) and methyl iodide (136.3 mg, 0.96 mmol) were dissolved in 1.5 mL of N,N-dimethylformamide and reacted at 40 °C for 16 hours. After completion of the reaction, 20 mL of purified water was added, and the aqueous phase was washed three times with dichloromethane (20 mL × 3). Then the aqueous phase was taken, concentrated under reduced pressure to remove the solvent, the residue was dissolved in 100 mL of purified water, and purified by gel column chromatography to obtain 320 mg of a white crude product. The obtained crude product was further purified by preparative liquid chromatography to obtain the white solid YK-CAP-001-PM8 triethylamine salt (75 mg, 0.12 mmol, yield: 48.7%). C 16 H 26 N6O 11 P2, MS(ES): m / z(M-H - ) 539.11。

[0148] Step 9: Synthesis of YK-CAP-001 The YK-CAP-001-PM8 triethylamine salt (75 mg, 0.12 mmol), INT-II (136.2 mg, 0.18 mmol) and ZnCl2 (163.6 mg, 1.2 mmol) were dissolved in 0.4 mL of DMSO and reacted at 37 °C for 48 hours. After completion of the reaction, 0.1 M EDTA was added until the solution became transparent, water was added to dilute to 100 mL, and purified by gel column chromatography to obtain a white crude product (200.6 mg). The obtained crude product was further purified by preparative liquid chromatography to obtain the white solid YK-CAP-001 (16.9 mg, 13.20, yield: 11.3%). C 37 H 52 N 16 O 24 P4, MS(ES): m / z(M-H - ) 1227.21。

[0149] 11H NMR (400 MHz, D2O) δ 8.28 (s, 1H), 7.92 (s, 1H), 7.83 (s, 1H), 5.86 (d, J = 6.0 Hz, 1H), 5.74 (d, J = 6.0 Hz, 1H), 5.54 (s, 1H), 4.84 - 4.83 (m, 1H), 4.43 -4.42 (m, 4H), 4.33 - 4.31 (m, 2H), 4.22 -4.20 (m, 4H), 4.10 - 4.09 (m, 3H), 4.00 - 3.99 (m, 1H), 3.93 -.3.92 (m, 1H), 3.91 (s, 3H), 3.29 (s, 3H), 3.13 - 3.03 (m, 2H), 2.93 (s, 6H), 2.24 - 2.20 (m, 2H); 31 31P NMR (D2O, 162 MHz) δ -0.92 (s, 1P), -11.11 (d, J = 19.4 Hz, 1P), -11.59 (d, J = 17.8 Hz, 1P), -22.91 (t, J = 17.8 Hz, 1P).

[0150] 4. Synthesis of YK-CAP-002

[0151]

Chemical Structure

[0152] Step 1: Synthesis of YK-CAP-002-PM1 YK-CAP-001-PM2 (22.0 g, 30.06 mmol) was dissolved in 440 mL of tetrahydrofuran, cooled to -10 °C, and 26% aqueous ammonia (250 mL) was added to the reaction system. Under the condition of -10 °C, the mixture was stirred and reacted for 5 minutes. Next, elemental iodine (23.0 g, 90.62 mmol) was added, and the reaction was continued by stirring for 2 hours at this temperature. Then, the temperature was raised to room temperature and the reaction was continued by stirring for another 15 minutes. The reaction solution was quenched with saturated sodium thiosulfate solution, extracted with ethyl acetate, the organic phase was dried, spin-dried, and purified by silica gel chromatography to obtain a pale yellow oily liquid YK-CAP-002-PM1 (14.7 g, 20.17 mmol, yield: 67.1%). C 38 H 44 N6O9, MS(ES): m / z(M+H+) 729.42.

[0153] Step 2: Synthesis of YK-CAP-002-PM2 YK-CAP-002-PM1 (4.0 g, 5.49 mmol) was dissolved in dichloromethane, cooled to -40 °C, and a 1 M solution of boron trichloride in dichloromethane (27.5 mL) was slowly added dropwise under the protection of nitrogen gas. After the addition was complete and the temperature reached from room temperature to 0 °C, the reaction was continued for 5 hours. Next, after the reaction system was cooled to -40 °C again, methanol was added to quench the reaction system. The reaction solution was concentrated under reduced pressure and spin-dried to obtain a crude product, and the crude product was purified by preparative high performance liquid chromatography to obtain YK-CAP-002-PM2 (490 mg, 1.47 mmol, yield: 26.8%). C 13 H 14 N6O5, MS(ES): m / z(M+H + ) 335.18.

[0154] 1 H NMR (400 MHz, MeOD) δ 8.16 (s, 1H), 5.92 (s, 1H), 4.54 (s, 1H), 4.48 - 4.42 (m, 2H), 3.92 (s, 2H), 2.94 - 2.92 (m, 2H).

[0155] Step 3: Synthesis of YK-CAP-002-PM3 Intermediate Using YK-CAP-002-PM2 (490 mg, 1.47 mmol) as a raw material, according to the synthesis route of YK-CAP-001-PM5, YK-CAP-002-PM3 triethylamine salt (1.07 g, 2.08 mmol) was obtained. C 13 H 15 N6O8P, MS(ES): m / z(M-H - ) 413.07.

[0156] Step 4: Synthesis of YK-CAP-002-PM4 Intermediate Using YK-CAP-002-PM3 triethylamine salt (1.07 g, 2.08 mmol) as a raw material, according to the synthesis route of YK-CAP-001-PM6, YK-CAP-002-PM4 (578.5 mg, 1.19 mmol) was obtained. C 16 H 17 N8O7P, MS(ES): m / z(M-H - ) 463.10.

[0157] Step 5: Synthesis of YK-CAP-002-PM5 Intermediate Using YK-CAP-002-PM4 (578.5 mg, 1.19 mmol) as a raw material, according to the synthesis route of YK-CAP-001-PM7, YK-CAP-002-PM5 (391.9 mg, 0.66 mmol, yield: 55.3%) was obtained. C 13 H 16 N6O 11 P2, MS(ES): m / z(M-H - )493.04.

[0158] Step 6: Synthesis of YK-CAP-002-PM6 Intermediate Using YK-CAP-002-PM5 (391.9 mg, 0.66 mmol) as a raw material, according to the synthesis route of YK-CAP-001-PM8, YK-CAP-002-PM6 (58 mg, 0.1 mmol, yield: 14.5%) was obtained. C 14 H 18 N6O 11 P2, MS(ES): m / z(M-H- ) 507.05。

[0159] Step 7: Synthesis of YK-CAP-002 Using YK-CAP-002-PM6 (58 mg, 0.1 mmol) as the raw material, according to the synthesis route of YK-CAP-001, YK-CAP-002 (17.5 mg, 14.02 μmol, yield: 14.7%) was obtained. C 35 H 44 N 16 O 24 P4, MS(ES): m / z(M-H - ) 1195.14。

[0160] 1 1H NMR (400 MHz, D2O) δ 8.27 (s, 1H), 7.94 (s, 1H), 7.85 (s, 1H), 5.87 (d, J = 6.0 Hz, 1H), 5.73 (d, J = 6.0 Hz, 1H), 5.55 (s, 1H), 4.85 - 4.83 (m, 1H), 4.44 - 4.40 (m, 4H), 4.31 - 4.30 (m, 2H), 4.25 - 4.23 (m, 4H), 4.10 - 4.08 (m, 3H), 3.99 - 3.97 (m, 1H), 3.94 - 3.92 (m, 4H), 3.29 (s, 3H), 2.95 - 2.90 (m, 2H); 31 31P NMR (D2O, 162 MHz) δ -0.93 (s, 1P), -11.14 (d, J = 19.6 Hz, 1P), -11.60 (d, J = 17.8 Hz, 1P), -22.93 (t, J = 17.8 Hz, 1P).

[0161] 5. Synthesis of YK-CAP-003

[0162]

Chemical formula

[0163] Step 1: Synthesis of YK-CAP-003-PM1 YK-CAP-001-PM1 (22.00 g, 30.14 mmol) was dissolved in a mixed solution of dichloromethane (220 mL), acetonitrile (220 mL), and water (330 mL). Sodium periodate (52.06 g, 241.12 mmol) and ruthenium(III) chloride (250 mg, 1.21 mmol) were added to the above solution, and the mixture was stirred at 30 °C for 48 hours to react. After the reaction was completed, the solvent was spin-dried, ethyl acetate (400 mL) was added to dissolve it, and it was washed three times with purified water (3 × 120 mL). The aqueous phase was back-extracted once with 200 mL of ethyl acetate. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was spin-dried under vacuum. The residue was purified by silica gel chromatography to obtain YK-CAP-003-PM1 (19.87 g, 26.58 mmol, yield: 88.2%). C 38 H 45 N5O 11 , MS(ES): m / z(M+H + )748.3。

[0164] Step 2: Synthesis of YK-CAP-003-PM2 YK-CAP-003-PM1 (19.87 g, 26.58 mmol) was dissolved in tetrahydrofuran (200 mL). Dimethylamine hydrochloride (2.38 g, 29.23 mmol), 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (12.13 g, 31.90 mmol), and N,N-diisopropylethylamine (10.31 g, 79.74 mmol) were sequentially added to the above solution, and the mixture was stirred overnight at room temperature to react. After the reaction was completed, the solvent was removed by spinning, ethyl acetate (400 mL) was added to dissolve it, and it was washed with purified water (3 × 120 mL). The aqueous phase was back-extracted with 200 mL of ethyl acetate. The organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under vacuum. The residue was purified by silica gel chromatography to obtain YK-CAP-003-PM2 (13.10 g, 16.91 mmol, yield: 63.6%). C 40 H 50 N6O 10 , MS(ES): m / z(M+H +)775.3。

[0165] Step 3: Synthesis of YK-CAP-003-PM3 YK-CAP-003-PM3 (13.0 g, 16.78 mmol) was dissolved in dichloromethane (195 mL), and a 1 M boron trichloride solution in dichloromethane (134 mL, 134 mmol) was slowly added dropwise at -40 °C. After the addition was complete, the temperature was raised to 0 °C, and the mixture was stirred and reacted for 6 hours. After the reaction was complete, it was cooled to -40 °C, methanol (200 mL) was added to quench the reaction system, the reaction solution was spin-evaporated under vacuum to remove the solvent, and the YK-CAP-003-PM3 crude product was obtained by placing it in a sealed environment at room temperature for 24 hours. It was added dropwise to 500 mL of dichloromethane to precipitate a solid, which was filtered to obtain a total of 7.89 g of cake. 2.0 g was taken and purified by HPLC to obtain pure YK-CAP-003-PM3 (956.04 mg, 2.51 mmol). C 15 H 20 N6O6, MS(ES): m / z(M+H + )381.32。

[0166] 1 H NMR (400 MHz, CD3OD) δ 7.97 (s, 1H), 5.84 (s, 1H), 4.70 - 4.63 (m, 1H), 4.45 - 4.38 (m, 2H), 3.91 - 3.75 (m, 2H), 3.15 (s, 3H), 2.99 - 2.70 (m, 5H).

[0167] Step 4: Synthesis of YK-CAP-003-PM4 Intermediate Using YK-CAP-003-PM3 (956.04 mg, 2.51 mmol) as the raw material, following the synthesis route of YK-CAP-001-PM5, YK-CAP-003-PM4 triethylamine salt (921.3 mg, 1.64 mmol, yield: 65.3%) was obtained. C 15 H 21 N6O9P, MS(ES): m / z(M-H - )459.11。

[0168] Step 5: Synthesis of YK-CAP-003-PM5 Intermediate Using YK-CAP-003-PM4 triethylamine salt (921.3 mg, 1.64 mmol) as a raw material, according to the synthesis route of YK-CAP-001-PM6, YK-CAP-003-PM5 (510.3 mg, 0.96 mmol, yield: 58.4%) was obtained. C 18 H 23 N8O8P, MS(ES): m / z(M-H - ) 509.12。

[0169] Step 6: Synthesis of YK-CAP-003-PM6 Intermediate Using YK-CAP-003-PM5 (510.3 mg, 0.96 mmol) as a raw material, according to the synthesis route of YK-CAP-001-PM7, YK-CAP-003-PM6 (853.8 mg, 1.33 mmol) was obtained. C 15 H 22 N6O 12 P2, MS(ES): m / z(M-H - ) 539.08。

[0170] Step 7: Synthesis of YK-CAP-003-PM7 Intermediate Using YK-CAP-003-PM6 (853.8 mg, 1.33 mmol) as a raw material, according to the synthesis route of YK-CAP-001-PM8, YK-CAP-003-PM7 (170.0 mg, 0.26 mmol) was obtained. C 16 H 24 N6O 12 P2, MS(ES): m / z(M-H - ) 553.09。

[0171] Step 8: Synthesis of YK-CAP-003 Using YK-CAP-003-PM7 (170.0 mg, 0.26 mmol) as a raw material, according to the synthesis route of YK-CAP-001, YK-CAP-003 (20.3 mg, 15.7 μmol, yield: 6.0%) was obtained. C 37 H 50 N 16 O 25P4, MS(ES): m / z(M-H - ) 1241.19.

[0172] 1 H NMR (400 MHz, D2O) δ 8.31 (s, 1H), 7.97 (s, 1H), 7.89 (s, 1H), 5.91 (d, J = 6.2 Hz, 1H), 5.77 (d, J = 5.8 Hz, 1H), 5.56 (s, 1H), 4.87 - 4.85 (m, 1H), 4.46 - 4.43 (m, 4H), 4.33 - 4.31 (m, 2H), 4.23 - 4.20 (m, 4H), 4.13 - 4.10 (m, 3H), 4.05 - 4.03 (m, 1H), 3.96 - 3.93 (m, 1H), 3.91 - 3.74 (m, 5H), 3.31 (s, 3H), 3.15 (s, 3H), 2.96 (s, 3H); 31 P NMR (D2O, 162 MHz) δ -0.96 (s, 1P), -11.15 (d, J = 19.4 Hz, 1P), -11.63 (d, J = 17.7 Hz, 1P), -22.97 (t, J = 17.8 Hz, 1P).

[0173] 6. Synthesis of YK-CAP-004

[0174]

Chemical formula

[0175] Step 1: Synthesis of YK-CAP-004-PM1 YK-CAP-001-PM2 (12.65 g, 17.29 mmol) was dissolved in dichloromethane (70 mL), and the reaction system was cooled to -40 °C. A dichloromethane solution of diethylaminosulfur trifluoride (8.36 g, 51.86 mmol) was slowly added dropwise. After the addition was complete, the reaction system was returned to 0 °C and reacted for 4 hours. The completion of the reaction was monitored by LCMS. The reaction system was quenched with a saturated aqueous sodium bicarbonate solution and extracted with dichloromethane (200 mL × 3). The organic phases were combined, washed with 200 mL of a saturated aqueous sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and spin-dried. Purification by silica gel chromatography gave YK-CAP-004-PM1 (7.92 g, 10.51 mmol, yield: 60.8%). C 38 H 45 F2N5O9, MS(ES): m / z(M+H + ) 754.3.

[0176] Step 2: Synthesis of YK-CAP-004-PM2 Using YK-CAP-004-PM1 (4.00 g, 5.31 mmol) as a raw material, it was dissolved in dichloromethane (30 mL). The above reaction system was cooled to -40 °C under a nitrogen gas atmosphere, and a dichloromethane solution of 1 M boron trichloride (26.53 mL, 26.53 mmol) was slowly added dropwise. After the addition was complete, the reaction system was slowly warmed to 0 °C, and the reaction system was stirred at this temperature for 4 hours. The completion of the reaction was monitored by TLC. The temperature was cooled to -40 °C again, the reaction system was quenched with methanol, spin-dried, left at room temperature for 24 hours, 200 mL of dichloromethane was added, a solid was precipitated, and filtration gave 2.0 g of a crude product. Purification by preparative HPLC gave YK-CAP-004-PM2 (926.34 mg, 2.58 mmol, yield: 48.8%). C 13 H 15 F2N5O5, MS(ES): m / z(M+H + ) 360.1.

[0177] 11H NMR (400 MHz, MeOD) δ 7.94 (s, 1H), 6.34 - 5.94 (m, 1H), 5.86 (s, 1H), 4.62 (s, 1H), 4.44 (dd, J = 24.2, 8.7 Hz, 3H), 3.99 - 3.84 (m, 2H), 2.48 - 2.08 (m, 2H).

[0178] Step 3: Synthesis of YK-CAP-004-PM3 Intermediate Using YK-CAP-004-PM2 (926.34 mg, 2.58 mmol) as a raw material, following the synthesis route of YK-CAP-001-PM5, YK-CAP-004-PM3 triethylamine salt (1.48 g, 2.74 mmol) was obtained. C 13 H 16 F2N5O8P, MS(ES): m / z(M-H - ) 438.07。

[0179] Step 4: Synthesis of YK-CAP-004-PM4 Intermediate Using YK-CAP-002-PM3 triethylamine salt (1.48 g, 2.74 mmol) as a raw material, following the synthesis route of YK-CAP-001-PM6, YK-CAP-004-PM4 (771.8 mg, 1.51 mmol) was obtained. C 16 H 18 F2N7O7P, MS(ES): m / z(M-H - ) 488.08。

[0180] Step 5: Synthesis of YK-CAP-004-PM5 Intermediate Using YK-CAP-004-PM4 (771.8 mg, 1.51 mmol) as a raw material, following the synthesis route of YK-CAP-001-PM7, YK-CAP-004-PM5 (1.22 g, 1.97 mmol) was obtained. C 13 H 17 F2N5O 11 P2, MS(ES): m / z(M-H - )518.04。

[0181] Step 6: Synthesis of YK-CAP-004-PM6 Intermediate Using YK-CAP-004-PM5 (1.22 g, 1.97 mmol) as the raw material, following the synthetic route of YK-CAP-001-PM8, YK-CAP-004-PM6 (172.6 mg, 0.27 mmol) was obtained. C 14 H 19 F2N5O 11 P2, MS(ES): m / z(M-H - ) 532.05.

[0182] Step 7: Synthesis of YK-CAP-004 Using YK-CAP-004-PM6 (172.6 mg, 0.27 mmol) as the raw material, following the synthetic route of YK-CAP-001, YK-CAP-004 (22.3 mg, 17.5 μmol, yield: 6.4%) was obtained. C 35 H 42 F2N 15 O 24 P4, MS(ES): m / z(M-H - )1220.15.

[0183] 1 1H NMR (400 MHz, D2O) δ 8.32 (s, 1H), 7.98 (s, 1H), 7.87 (s, 1H), 6.35 - 5.95 (m, 1H), 5.91 (d, J = 6.0 Hz, 1H), 5.76 (d, J = 6.0 Hz, 1H), 5.57 (s, 1H), 4.85 - 4.82 (m, 1H), 4.47 - 4.45 (m, 4H), 4.38 - 4.32 (m, 2H), 4.36 - 4.25 (m, 4H), 4.24 - 4.15 (m, 3H), 4.01 - 3.99 (m, 1H), 3.94 (s, 2H), 3.31 (s, 3H), 2.48 - 2.08 (m, 2H); 31P NMR (D2O, 162 MHz) δ -0.91 (s, 1P), -11.06 (d, J = 19.1 Hz, 1P), -11.60 (d, J = 18.3 Hz, 1P), -22.88 (t, J = 17.8 Hz, 1P).

[0184] 7. Synthesis of YK-CAP-005

[0185] [Chemical formula]

[0186] Step 1: Synthesis of YK-CAP-005-PM1 Compound INT-PM2 (44.0 g, 0.10 mol) was dissolved in 450 mL of dichloromethane. Under the protection of nitrogen gas, it was stirred and cooled to 0 °C. Pyridine (23.9 g, 0.30 mol) was added, and then tert-butyldimethylsilyl trifluoromethanesulfonate (40.0 g, 0.15 mol) was added dropwise. After the addition was complete, the temperature was raised to room temperature for reaction. The completion of the reaction was monitored by TLC. The reaction system was directly spin-dried under reduced pressure and purified by FLASH to obtain a colorless liquid YK-CAP-005-PM1 (47.0 g, 84.72 mmol, yield: 84.82%).

[0187] Step 2: Synthesis of YK-CAP-005-PM2 Compound YK-CAP-005-PM1 (47.0 g, 84.72 mmol) was dissolved in a mixed solvent of 470 mL of tetrahydrofuran and 47 mL of water. Sequentially, N-methylmorpholine-N-oxide (14.9 g, 0.13 mol) and potassium osmate (1.3 g, 0.003 mol) were added, and the reaction was carried out at room temperature for 5 hours. The completion of the reaction was monitored by TLC. The reaction solution was slowly poured into 500 mL of saturated aqueous sodium sulfite solution, separated, and the aqueous phase was extracted with ethyl acetate (300 mL × 2). The organic phases were combined, washed successively with saturated brine (300 mL), dried over anhydrous sodium sulfate, concentrated under reduced pressure and spin-dried to obtain a pale yellow liquid, the crude product of YK-CAP-005-PM2 (53.2 g, 90.35 mmol).

[0188] Step 3: Synthesis of YK-CAP-005-PM3 The crude product of YK-CAP-005-PM2 (53.2 g, 90.35 mmol) was dissolved in a mixed solvent of 500 mL of tetrahydrofuran and 100 mL of water. Sodium periodate (29.0 g, 0.14 mol) was added again, and the reaction was carried out at room temperature for 3 hours. The completion of the reaction was monitored by TLC. The reaction solution was slowly poured into 500 mL of saturated aqueous sodium sulfite solution, separated, and the aqueous phase was extracted with ethyl acetate (400 mL × 2). The organic phases were combined, washed successively with saturated brine (400 mL), dried over anhydrous sodium sulfate, concentrated under reduced pressure and spin-dried to obtain a yellow oil, YK-CAP-005-PM3 (47.4 g, 85.13 mmol).

[0189] Step 4: Synthesis of YK-CAP-005-PM4 Compound YK-CAP-005-PM3 (47.4 g, 85.13 mmol) was dissolved in 500 mL of MeOH, stirred and cooled to 0 °C under the protection of nitrogen gas, and sodium borohydride (3.86 g, 0.10 mol) was added in one batch. After the addition was completed, the reaction was continued for 3 hours. The completion of the reaction was monitored by TLC. The reaction system was cooled to 0 °C, saturated ammonium chloride aqueous solution (300 mL) was added to quench the reaction system, and it was further extracted with ethyl acetate (400 mL × 3). The organic phases were combined, washed successively with saturated brine (400 mL), dried over anhydrous sodium sulfate, concentrated under reduced pressure and spin-dried to obtain a yellow oily crude product of YK-CAP-005-PM4 (49.0 g, 0.088 mol).

[0190] Step 5: Synthesis of YK-CAP-005-PM5 The crude product of YK-CAP-005-PM4 (49.0 g, 0.088 mol) was dissolved in 500 mL of dichloromethane, and triethylamine (26.6 g, 0.26 mol) was further added. It was stirred and cooled to 0 °C under the protection of nitrogen gas, and ethanesulfonyl chloride (22.5 g, 0.18 mol) was added dropwise. After the dropwise addition was completed, the temperature was raised to room temperature and the reaction was carried out for 3 hours until the raw materials disappeared. The completion of the reaction was monitored by TLC. 500 mL of saturated sodium bicarbonate aqueous solution was added to the reaction system, separated, the aqueous phase was extracted with dichloromethane (400 mL × 2), the organic phase was washed with saturated brine (500 mL), dried over anhydrous sodium sulfate, concentrated under reduced pressure and spin-dried to obtain a pale yellow oily crude product of YK-CAP-005-PM5 (58.0 g, 0.089 mol).

[0191] Step 6: Synthesis of YK-CAP-005-PM6 The crude product YK-CAP-005-PM5 (58.0 g, 0.089 mol) was dissolved in 400 mL of N,N-dimethylformamide, sodium thiomethoxide (15.6 g, 0.22 mol) was added, and the mixture was reacted at room temperature for 4 hours to complete the reaction. The completion of the reaction was monitored by TLC. The reaction solution was slowly poured into 1500 mL of water, and the reaction system was extracted with ethyl acetate (500 mL × 3). The organic phases were combined, washed successively with saturated brine (1500 mL), dried over anhydrous sodium sulfate, concentrated under reduced pressure and spin-dried to obtain a brown oily substance YK-CAP-005-PM6 (50.0 g, 0.085 mol).

[0192] Step 7: Synthesis of YK-CAP-005-PM7 YK-CAP-005-PM6 (50.0 g, 0.085 mol) was dissolved in 500 mL of N,N-dimethylformamide, stirred under the protection of nitrogen gas and cooled to 0 °C, and m-chloroperoxybenzoic acid (33.7 g, 0.19 mol) was added in one portion. After the addition was completed, the temperature was raised to room temperature and the reaction was continued for 3 hours until the raw materials disappeared. The completion of the reaction was monitored by TLC. The reaction solution was poured into an aqueous solution of saturated sodium bicarbonate (1500 mL), extracted with ethyl acetate (500 mL × 3), the organic phase was washed with saturated brine (1500 mL), dried over anhydrous sodium sulfate, concentrated under reduced pressure and spin-dried to obtain a yellow oily substance YK-CAP-005-PM7 (52.6 g, 84.72 mmol, yield: 99.8%).

[0193] Step 8: Synthesis of YK-CAP-005-PM8 YK-CAP-005-PM7 (52.6 g, 84.72 mmol) was dissolved in 400 mL of N,N-dimethylformamide, 1 M tetrabutylammonium fluoride (127 mL, 127 mmol) was added, and the reaction was carried out at room temperature for 1 hour. The completion of the reaction was monitored by TLC. 1000 mL of purified water and 800 mL of ethyl acetate were added to the reaction system, stirred for 5 minutes for separation, the aqueous phase was extracted with ethyl acetate (500 mL × 2), the organic phases were combined, washed successively with saturated brine (1000 mL), dried over anhydrous sodium sulfate, concentrated under reduced pressure and spin-dried, and purified by FLASH to obtain a yellow oily substance YK-CAP-005-PM8 (43.2 g).

[0194] Step 9: Synthesis of YK-CAP-005-PM9 YK-CAP-005-PM8 (18.7 g, 36.91 mmol) was dissolved in 200 mL of dichloromethane, triethylamine (11.2 g, 0.11 mol) was further added, cooled to 0 °C with stirring under the protection of nitrogen gas, and ethanesulfonyl chloride (22.5 g, 0.18 mol) was added dropwise. After the addition was completed, the temperature was raised to room temperature and the reaction was carried out for 3 hours. The completion of the reaction was monitored by TLC. 400 mL of saturated aqueous sodium bicarbonate solution was added to the reaction system for separation, the aqueous phase was extracted with dichloromethane (200 mL × 2), the organic phase was washed with saturated brine (400 mL), dried over anhydrous sodium sulfate, concentrated under reduced pressure and spin-dried, and purified by FLASH to obtain a pale yellow oily substance YK-CAP-005-PM9 (13.4 g, 22.38 mmol, yield: 60.6%).

[0195] Step 10: Synthesis of YK-CAP-005-PM10 Compound YK-CAP-005-PM9 (13.4 g, 22.38 mmol) was dissolved in 130 mL of acetic acid, and acetic anhydride (13.7 g, 0.134 mol) was further added. Concentrated sulfuric acid (0.27 mL) was added dropwise. After the addition was completed, the reaction was carried out at room temperature for 2 hours until the raw materials disappeared. The completion of the reaction was monitored by TLC. 400 mL of purified water and 400 mL of ethyl acetate were added to the reaction system, stirred for 5 minutes for separation, the aqueous phase was extracted with ethyl acetate (300 mL × 2), the organic phases were combined, adjusted to alkaline pH with saturated aqueous sodium bicarbonate solution in sequence, further washed with saturated brine (1.0 L), dried over anhydrous sodium sulfate, dried by spin-drying under reduced pressure, and yellow oily substance YK-CAP-005-PM10 (13.4 g, 20.85 mmol, yield: 93.18%) was obtained.

[0196] Step 11: Synthesis of YK-CAP-005-PM11 2-Amino-6-chloropurine (5.1 g, 30.08 mmol) was dissolved in toluene (100 mL), N,O-bis(trimethylsilyl)acetamide (12.2 g, 0.06 mol) was added, heated to 80 °C to dissolve the reaction system transparently, the heating was removed, cooled to room temperature, and a toluene solution (50 mL) of compound YK-CAP-005-PM10 (12.8 g, 19.92 mmol) was added with stirring. Trimethylsilyl trifluoromethanesulfonate (6.6 g, 0.03 mol) was added dropwise, heated until refluxing, stirred and reacted for 2 hours until the raw materials disappeared. The completion of the reaction was monitored by TLC. The reaction system was cooled to room temperature, quenched by adding saturated aqueous sodium bicarbonate solution (300 mL), then ethyl acetate (300 mL) was added, filtered through diatomaceous earth for separation, the aqueous phase was extracted with ethyl acetate (200 mL × 2), the organic phases were combined, washed with saturated brine (300 mL × 1) in sequence, dried over anhydrous sodium sulfate, and spin-dried to obtain yellow viscous substance YK-CAP-005-PM11 (15.5 g, 20.60 mmol).

[0197] Step 12: Synthesis of YK-CAP-005-PM12 Compound YK-CAP-005-PM11 (15.5 g, 20.60 mmol) was dissolved in 200 mL of anhydrous methanol, and anhydrous potassium carbonate (14.2 g, 0.10 mol) was further added, followed by reaction at room temperature. The reaction was monitored by TLC. After completion of the reaction, the reaction system was directly concentrated under reduced pressure and spin-dried, and purified by FLASH to obtain a yellow solid YK-CAP-005-PM12 (11.8 g, 19.81 mmol). C 29 H 33 N5O7S, MS(ES): m / z(M+H + )596.38。

[0198] Step 13: Synthesis of YK-CAP-005-PM13 Compound YK-CAP-005-PM12 (11.8 g, 19.81 mmol) was dissolved in 200 mL of dichloromethane, cooled to -40 °C under the protection of nitrogen gas, and a 1 M boron trichloride solution in dichloromethane (167 mL) was added dropwise. After the addition was complete, the reaction system was warmed to 0 °C and allowed to react. The reaction was monitored by LC-MS. After completion of the reaction, the reaction system was cooled to -40 °C, methanol (200 mL) was added dropwise to quench the reaction system, and it was directly concentrated under reduced pressure and spin-dried. A 120 mL solution of 4 M hydrochloric acid in 1,4-dioxane was added, and the temperature was raised to 60 °C until the reaction was complete. After completion of the reaction, the reaction system was concentrated under reduced pressure and spin-dried. Methanol (40 mL) was added to dissolve it, and then it was added dropwise to 800 mL of dichloromethane to precipitate a solid, which was suction filtered and dried to obtain a yellow solid YK-CAP-005-PM13 (7.0 g, 17.44 mmol, yield: 88.1%). C 14 H 19 N5O7S,MS(ES): m / z(M+H + )402.18。

[0199] 11H NMR (400 MHz, DMSO-d6) δ 10.93 (s, 1H), 8.15 (s, 1H), 6.79 (s, 1H), 5.71 (s, 1H), 4.28 (s, 2H), 4.16 (dd, J = 9.0, 4.2 Hz, 1H), 3.75 (d, J = 2.3 Hz, 2H), 3.36 - 3.14 (m, 2H), 3.02 (s, 3H), 2.11 - 1.85 (m, 2H).

[0200] Step 14: Synthesis of YK-CAP-005-PM14 Intermediate Using YK-CAP-005-PM13 (3.4 g, 8.47 mmol) as the raw material, following the synthetic route of YK-CAP-001-PM5, YK-CAP-005-PM14 triethylamine salt (639.8 mg, 1.10 mmol, yield: 13.0%) was obtained. C 14 H 20 N5O 10 PS, MS(ES): m / z(M-H - ) 480.07.

[0201] Step 15: Synthesis of YK-CAP-005-PM15 Intermediate Using YK-CAP-005-PM14 triethylamine salt (639.8 mg, 1.10 mmol) as the raw material, following the synthetic route of YK-CAP-001-PM6, YK-CAP-004-PM15 (333.4 mg, 0.60 mmol, yield: 54.9%) was obtained. C 17 H 22 N7O9PS, MS(ES): m / z(M-H - )530.08.

[0202] Step 16: Synthesis of YK-CAP-005-PM16 Intermediate Using YK-CAP-005-PM15 (333.4 mg, 0.60 mmol) as the raw material, following the synthetic route of YK-CAP-001-PM7, YK-CAP-005-PM16 (528 mg, 0.80 mmol) was obtained. C 14 H 21 N5O 13P2S, MS(ES): m / z(M-H - ) 560.03。

[0203] Step 17: Synthesis of YK-CAP-005-PM17 Intermediate Using YK-CAP-005-PM16 (528 mg, 0.80 mmol) as a raw material, following the synthesis route of YK-CAP-001-PM8, YK-CAP-005-PM17 (74.7 mg, 0.11 mmol) was obtained. C 15 H 23 N5O 13 P2S, MS(ES): m / z(M-H - ) 574.05。

[0204] Step 18: Synthesis of YK-CAP-005 Using YK-CAP-005-PM17 (74.7 mg, 0.11 mmol) as a raw material, following the synthesis route of YK-CAP-001, YK-CAP-005 (19.3 mg, 14.7 μmol, 13.3%) was obtained. C 36 H 49 N 15 O 26 P4S, MS(ES): m / z(M-H - ) 1262.14。

[0205] 1 1H NMR (400 MHz, D2O) δ 8.33 (s, 1H), 7.98 (s, 1H), 7.87 (s, 1H), 5.88 (d, J = 6.1 Hz, 1H), 5.77 (d, J = 6.3 Hz, 1H), 5.57 (s, 1H), 4.86 - 4.84 (m, 1H), 4.48 - 4.41 (m, 4H), 4.35 - 4.32 (m, 2H), 4.25 - 4.23 (m, 5H), 4.11 - 4.09 (m, 3H), 4.06 - 4.03 (m, 1H), 3.96 (s, 3H), 3.36 - 3.14 (m, 2H), 3.07 (s, 3H), 2.87 (s, 3H), 2.11 - 1.84 (m, 2H); 31P NMR (D2O, 162 MHz) δ -0.94 (s, 1P), -11.13 (d, J = 19.2 Hz, 1P), -11.64 (d, J = 18.5 Hz, 1P), -22.95 (t, J = 18.0 Hz, 1P).

[0206] 8. Synthesis of YK-CAP-006

[0207]

Chemical Structure

[0208] Step 1: Synthesis of YK-CAP-006-PM1 Dissolve triphenylphosphine (11.27 g, 42.98 mmol) in 150 mL of tetrahydrofuran, cool the reaction system to 0 °C, add diisopropyl azodicarboxylate (14.48 g, 71.64 mmol), stir for 10 minutes, add a tetrahydrofuran solution of compound YK-CAP-005-PM4 (20.0 g, 35.79 mmol), and slowly add thioacetic acid (5.45 g, 71.64 mmol) dropwise. After the addition is complete, slowly return to room temperature and stir for reaction for 16 hours, and monitor by TLC that the reaction is complete. After the reaction is complete, quench the reaction system with water, extract the aqueous phase with ethyl acetate (200 mL × 2), combine the organic phases, wash with saturated sodium chloride, dry over anhydrous sodium sulfate, filter, and spin dry. Purify by FLASH to obtain a colorless oily liquid YK-CAP-006-PM1 (14.4 g, 23.34 mmol, yield: 65.2%).

[0209] Step 2: Synthesis of YK-CAP-006-PM2 Compound N-chlorosuccinimide (12.48 g, 93.48 mmol) was dissolved in 100 mL of acetonitrile and 20 mL of 2N hydrochloric acid aqueous solution. A solution of compound YK-CAP-006-PM1 (14.4 g, 23.34 mmol) in acetonitrile was slowly added dropwise, and the reaction was carried out for 10 minutes. The completion of the reaction was monitored by LCMS. 30 mL of water was added to the reaction system to quench the reaction. The aqueous phase was extracted with ethyl acetate (100 mL × 2). The organic phases were combined, washed with saturated sodium chloride, dried over anhydrous sodium sulfate, filtered, and spin-dried to obtain a colorless oily liquid YK-CAP-006-PM2 (13.4 g, 20.90 mmol, yield: 89.5%).

[0210] Step 3: Synthesis of YK-CAP-006-PM3 Dimethylamine hydrochloride (2.05 g, 25.12 mmol) was weighed and dissolved in 100 mL of dichloromethane. Triethylamine (6.35 g, 62.79 mmol) was weighed and added. The reaction system was cooled to 0 °C. A solution of compound YK-CAP-006-PM2 (13.40 g, 20.90 mmol) in dichloromethane was slowly added dropwise. After the addition was completed, the reaction was carried out for 30 minutes under the condition of 0 °C. The completion of the reaction was monitored by LCMS. The reaction system was directly spin-dried under reduced pressure, dissolved in a small amount of dichloromethane, and purified by FLASH to obtain a colorless liquid YK-CAP-006-PM3 (6.0 g, 9.23 mmol, yield: 44.2%).

[0211] Step 4: Synthesis of YK-CAP-006-PM4 Compound YK-CAP-006-PM3 (6.0 g, 9.23 mmol) was dissolved in 400 mL of tetrahydrofuran, tetrabutylammonium fluoride / tetrahydrofuran (11.09 mL, 11.09 mmol) was slowly added, and the reaction was carried out at room temperature for 1 hour. The completion of the reaction was monitored by TLC. The reaction solution was slowly poured into 100 mL of saturated aqueous sodium bicarbonate solution, separated, the aqueous phase was extracted with ethyl acetate (100 mL × 2), the organic phases were combined, washed successively with saturated brine (50 mL), dried over anhydrous sodium sulfate, concentrated under reduced pressure and spin-dried, and purified by FLASH to obtain colorless liquid YK-CAP-006-PM4 (4.0 g, 7.47 mmol, yield: 80.8%).

[0212] Step 5: Synthesis of YK-CAP-006-PM5 Compound YK-CAP-006-PM4 (4.0 g, 7.47 mmol) was dissolved in 200 mL of dichloromethane solution, triethylamine (2.27 g, 22.41 mmol) was added, the reaction system was cooled to 0 °C, and a dichloromethane solution of ethanesulfonyl chloride (1.15 g, 8.96 mmol) was slowly added dropwise. After the addition was complete, the reaction system was reacted at 0 °C for 2 hours. The completion of the reaction was monitored by TLC. The reaction solution was slowly poured into 50 mL of saturated aqueous sodium bicarbonate solution to quench the reaction system, ethyl acetate was added for separation, and the mixture was extracted with ethyl acetate (150 mL × 2). The organic phases were combined, washed successively with saturated brine (500 mL), dried over anhydrous sodium sulfate, concentrated under reduced pressure and spin-dried, and purified by FLASH to obtain colorless liquid YK-CAP-006-PM5 (4.4 g, 7.01 mmol, yield: 93.9%).

[0213] Step 6: Synthesis of YK-CAP-006-PM6 Compound YK-CAP-006-PM5 (4.4 g, 7.01 mmol) was dissolved in 30 mL of acetic acid, acetic anhydride (4.3 g, 42.12 mmol) was added, and under the condition of stirring at room temperature, an acetic acid solution of concentrated sulfuric acid (44 μL) was slowly added dropwise, and the mixture was stirred at room temperature for 1 hour. The completion of the reaction was monitored by TLC. The reaction solution was slowly added dropwise to 500 mL of saturated aqueous sodium hydrogen carbonate solution to adjust the pH to neutral, extracted with ethyl acetate (300 mL × 3), the organic phases were combined, washed with 50 mL of saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure and spin-dried to obtain a colorless oil, YK-CAP-006-PM6 (3.1 g, 4.61 mmol, yield: 65.8%).

[0214] Step 7: Synthesis of YK-CAP-006-PM7 2-Amino-6-chloroguanine (862.6 mg, 5.08 mmol) was dissolved in 30 mL of toluene, N,O-bis(trimethylsilyl)acetamide (2.07 g, 10.16 mmol) was weighed and added, and under the protection of nitrogen gas, the reaction system was heated to 80 °C and stirred until the solution became transparent, then the heating was stopped and the temperature was returned to room temperature. A toluene solution of compound YK-CAP-006-PM6 (3.1 g, 4.61 mmol) was slowly added, and then trimethylsilyl trifluoromethanesulfonate (1.13 g, 5.08 mmol) was added. Immediately, the temperature was raised to 110 °C and the reaction was carried out for 2 hours. The completion of the reaction was monitored by LCMS. Water (15 mL) and ethyl acetate (20 mL) were added to the reaction system, a solid was precipitated from the reaction system, diatomaceous earth was added and filtered, and the solution was extracted with ethyl acetate (80 mL × 2). The organic phase was washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, concentrated under reduced pressure and spin-dried, and purified by FLASH to obtain a colorless oil, YK-CAP-006-PM7 (3.0 g, 3.84 mmol, yield: 83.1%).

[0215] Step 8: Synthesis of YK-CAP-006-PM8 Compound YK-CAP-006-PM7 (3.0 g, 3.84 mmol) was dissolved in 20 mL of methanol, potassium carbonate (15.6 g, 23.10 mmol) was added, and the reaction was carried out at room temperature for 24 hours. The completion of the reaction was monitored by LCMS. After filtration, the filtrate was stirred and purified by FLASH to obtain a colorless oil, YK-CAP-006-PM8 (2.1 g, 3.36 mmol, yield: 87.5%).

[0216] Step 9: Synthesis of YK-CAP-006-PM9 YK-CAP-006-PM8 (2.1 g, 3.36 mmol) was dissolved in 25 mL of dichloromethane. Under the protection of nitrogen gas, the solution was stirred and cooled to 0 °C, and boron trichloride (3.94 g, 33.60 mmol) was slowly added dropwise. After the addition was complete, the reaction was continued at 0 °C for 6 hours, and the disappearance of the raw material was monitored by LCMS. The reaction system was cooled to -40 °C and quenched with methanol. After spin-drying, the target compound was monitored by LCMS. The product was dissolved in 20 mL of methanol and slowly added dropwise to 150 mL of stirred dichloromethane to precipitate a white solid, and 1.7 g of the crude product was obtained. The crude product was purified by preparative high performance liquid chromatography to obtain a white solid, YK-CAP-006-PM9 (1.012 g, 2.35 mmol, yield: 69.94%).

[0217] 1 H NMR (400 MHz, DMSO) δ 10.70 (s, 1H), 7.89 (s, 1H), 6.63 (s, 2H), 5.68 (s, 1H), 4.29 (d, J = 18.9 Hz, 2H), 4.17 (dd, J = 9.4, 3.5 Hz, 1H), 3.77 (d, J = 13.2 Hz, 3H), 3.30 - 3.03 (m, 2H), 2.81 (s, 6H), 2.12 - 1.85 (m, 2H).

[0218] Step 10: Synthesis of YK-CAP-006-PM10 YK-CAP-006-PM9 (1.01 g, 2.35 mmol) was used as a raw material, and according to the synthetic route of YK-CAP-001-PM5, YK-CAP-006-PM10 triethylamine salt (1.05 g, 1.72 mmol, yield: 73.2%) was obtained. C 15 H 23 N6O 10 PS, MS(ES): m / z(M-H - ) 509.09。

[0219] Step 11: Synthesis of YK-CAP-006-PM11 YK-CAP-006-PM10 triethylamine salt (1.05 g, 1.72 mmol) was used as a raw material, and according to the synthetic route of YK-CAP-001-PM6, YK-CAP-006-PM11 (809.2 mg, 1.39 mmol, yield: 80.9%) was obtained. C 18 H 25 N8O9PS, MS(ES): m / z(M-H - ) 559.10。

[0220] Step 12: Synthesis of YK-CAP-006-PM12 YK-CAP-006-PM11 (809.2 mg, 1.39 mmol) was used as a raw material, and according to the synthetic route of YK-CAP-001-PM7, YK-CAP-006-PM12 triethylamine salt (475.5 mg, 0.69 mmol, yield: 49.5%) was obtained. C 15 H 24 N6O 13 P2S, MS(ES): m / z(M-H - ) 589.06。

[0221] Step 13: Synthesis of YK-CAP-006-PM13 YK-CAP-006-PM12 triethylamine salt (475.5 mg, 0.69 mmol) was used as a raw material, and according to the synthetic route of YK-CAP-001-PM8, YK-CAP-006-PM13 triethylamine salt (235 mg, 0.33 mmol, yield: 48.4%) was obtained. C 16 H 26 N6O13 P2S, MS(ES): m / z(M-H - ) 603.08。

[0222] Step 14: Synthesis of YK-CAP-006 Using YK-CAP-006-PM13 (235 mg, 0.33 mmol) as a raw material, according to the synthesis route of YK-CAP-001, YK-CAP-006 (32.0 mg, 23.8 μmol, yield: 7.1%) was obtained. C 37 H 52 N 16 O 26 P4S, MS(ES): m / z(M-H - ) 1291.17。

[0223] 1 1H NMR (400 MHz, D2O) δ 8.33 (s, 1H), 7.97 (s, 1H), 7.90 (s, 1H), 5.89 (d, J = 6.1 Hz, 1H), 5.79 (d, J = 6.3 Hz, 1H), 5.59 (s, 1H), 4.90 - 4.84 (m, 1H), 4.47 - 4.43 (m, 4H), 4.35 - 4.33 (m, 2H), 4.29 - 4.26 (m, 4H), 4.14 - 4.08 (m, 4H), 3.98 - 3.95 (m, 1H), 3.93 (s, 3H), 3.36 (s, 3H), 3.33 - 3.00 (m, 2H), 2.83 (s, 6H), 2.00 - 1.93 (m, 2H); 31 31P NMR (D2O, 202 MHz) δ -0.91 (s, 1P), -11.47 (m, 2P), -22.80 (t, J = 17.8 Hz, 1P).

[0224] Example 2: mRNA in vitro transcription yield and capping rate The chemical structures of the compounds YK-CAP-001 to 006 of the present application are very similar. Compared with the mRNA capping analogs disclosed in the prior art, the structures of some of these series of compounds are similar to some, and there are very large differences from some.

[0225] The modified locked nucleoside capping analogs of the present application showed significant differences in the in vitro transcription yield and capping rate of mRNA. For example, the transcription yield of YK-CAP-004 was 82.8% higher than that of YK-CAP-001, and the capping rate was 26.1% higher than that of YK-CAP-002. Compared with the modified locked nucleoside capping analogs of the prior art, the modified locked nucleoside capping analogs of the present application showed remarkable improvements in both the mRNA in vitro transcription yield and the capping rate. For example, the transcription yield and capping rate of YK-CAP-004 were 35.0% and 18.8% higher than those of Compound 14, respectively.

[0226] I. Structural differences of capping analogs

[0227]

Table 1

[0228] As can be seen from Table 1, the chemical structures of Compounds YK-CAP-001 to 006 of the present application are very similar. Compared with the mRNA capping analogs disclosed in the prior art, some of these series of compounds have similar structures, while some have very large differences, specifically as shown below. 1. The structures of the compounds YK-CAP-001, YK-CAP-002, YK-CAP-003, YK-CAP-004, YK-CAP-005, and YK-CAP-006 in this application are very similar. Only the groups linked to the methylene bridge connecting the 2'-O and C4' of the ribose ring, that is, the substituents on C6', are different. YK-CAP-001 is dimethylaminoethyl, YK-CAP-002 is cyanomethyl, YK-CAP-003 is dimethylaminocarbonylmethyl, YK-CAP-004 is 2,2-difluoroethyl, YK-CAP-005 is methylsulfonylethyl, and YK-CAP-006 is dimethylaminosulfonylethyl. The other structures are exactly the same.

[0229] 2. The compounds YK-CAP-001 to 006 in this application are similar in structure to compound 14, but the difference is only that there is no substituent on the methylene bridge connecting the 2'-O and C4' of the ribose ring of compound 14, that is, there is no substituent on C6'.

[0230] 3. The compounds YK-CAP-001 to 006 in this application have a greater structural difference from N-7413, HN3002, and m6A. There is no methylene bridge between the 2'-O and C4' of the ribose ring of these three compounds. Furthermore, the second base adenine in m6A is methylated.

[0231] II. Measurement of mRNA in vitro transcription yield and capping rate 1. Experimental method (1) Capping synthesis using capping analogs First, the plasmid was linearized using a plasmid linearization enzyme, and then the linearized plasmid was purified.

[0232] (2) In vitro transcription synthesis of mRNA YK-CAP-001 to 006 and compound 14 in Table 1 were each used as capping analogs, and the reaction system was as shown in Table 2.

[0233]

Table 2

[0234] During the experiment process, first, the volumes of the materials required for the reaction system were calculated, and then the samples were added. First, sterile distilled water was added to the reaction system, and then 10× buffer, NTPs, and capping analogs were sequentially added and uniformly mixed. After gentle centrifugation, next, nuclease inhibitor, inorganic pyrophosphatase, T7 RNA polymerase, and linearized DNA template were added, and after complete mixing, gentle centrifugation was performed, and then cultured at 37°C. After 2 hours, 1 U of DNase I was added, and after continuous culturing at 37°C for 30 minutes, the mRNA precipitate was washed with 75% ethanol. After allowing the ethanol to volatilize and dry for a while, the mRNA was redissolved with sterile distilled water.

[0235] (3) The transcription products were purified, and the mRNA in vitro transcription yield was recorded.

[0236] (4) The obtained mRNA was subjected to an annealing reaction with the probe.

[0237] Using a PCR device, annealing was performed at 95°C for 5 minutes, 65°C for 2 minutes, 55°C for 2 minutes, 40°C for 2 minutes, and 22°C for 2 minutes.

[0238] (5) Magnetic bead pretreatment and probe binding: For pretreatment, 100 μL of magnetic beads were taken and placed on a magnetic stand. 120 μL of the sample and the magnetic bead solution were added, cultured at room temperature for 30 minutes, and gently and uniformly mixed while culturing.

[0239] (6) The mRNA was cleaved to obtain the single-stranded sequence of the mRNA 5 bound to the probe. , A single-stranded sequence was obtained.

[0240] 20 μL of Rnase H (5 U / μL) was added, and the mixture was cultured at 37 °C for 3 hours with mixing once every 30 minutes. After the culture was completed, the magnetic beads were washed. 100 μL of 75% methanol heated to 80 °C was added to the washed magnetic beads. The mixture was heated to 80 °C on a heating plate and maintained for 3 minutes. Then, it was left on a magnetic stand, the supernatant was aspirated, and it was dried at room temperature to 10 μL using an evaporative centrifuge for 45 minutes. Next, the sample was resuspended in 50 μL of 100 μM EDTA / 1% MeOH, and thus it could be used for LC-MS analysis to determine the capping status of RNA during the transcription reaction. Since there are differences in the molecular weights of capping and non-capping bases, the difference in molecular weights was used to determine the capping rate of mRNA transcription initiated by different capping analogs.

[0241] 2. Experimental Results According to the measurement results of the in vitro transcription yield and capping rate of mRNA, it was shown that there were significant differences in the in vitro transcription yield and capping rate of mRNA for the modified locked nucleoside capping analogs of this application. For example, the transcription yield of YK-CAP-004 was 82.8% higher than that of YK-CAP-001, and the capping rate was 26.1% higher than that of YK-CAP-002. Compared with the modified locked nucleoside capping analogs of the prior art, the modified locked nucleoside capping analogs of this application had significantly improved both the in vitro transcription yield and capping rate of mRNA. For example, the transcription yield and capping rate of YK-CAP-004 were 35.0% and 18.8% higher than those of Compound 14, respectively.

[0242] The specific in vitro transcription yield and capping rate of mRNA are shown in Table 3.

[0243]

Table 3

[0244] 1) The modified locked nucleoside capping analogs of this application showed significant differences in the in vitro transcription yield and capping rate of mRNA. Both the transcription yield and capping rate of YK-CAP-004 were the highest. The transcription yield was improved by 82.8% compared to YK-CAP-001 with the lowest transcription yield, and the capping rate was improved by 26.1% compared to YK-CAP-002 with the lowest capping rate.

[0245] As can be seen from Table 3, all the modified locked nucleoside capping analogs of this application were able to transcribe mRNA. The differences in the mRNA transcription activities of different modified locked nucleoside capping analogs were very prominent. The highest mRNA in vitro transcription yield was 163.2 μg for YK-CAP-004, and the yields of YK-CAP-003, YK-CAP-005, and YK-CAP-006 were 151.0 μg, 152.6 μg, and 153.1 μg respectively, all of which were very high, exceeding 150 μg.

[0246] The lowest mRNA in vitro transcription yield was 89.3 μg for YK-CAP-001, and the yield of YK-CAP-002 was only 103.1 μg, which was very low. The transcription yield of YK-CAP-004 was improved by 82.8% compared to YK-CAP-001 and by 58.3% compared to YK-CAP-002, showing a significant improvement. (Figure 1) The highest capping rate was 96.3% for YK-CAP-004, and the capping rates of YK-CAP-003, YK-CAP-005, and YK-CAP-006 were 94.8%, 95.2%, and 95.1% respectively, all of which were very high, exceeding 90%.

[0247] The lowest capping rate was only 70.2% for YK-CAP-002, and the capping rate of YK-CAP-001 was only 73.1%, which was very low. The capping rate of YK-CAP-004 was improved by 26.1% compared to YK-CAP-002 and by 23.2% compared to YK-CAP-001, showing a significant improvement. (Figure 2) 2) The modified locked nucleoside capping analogs of the present application showed significantly improved mRNA in vitro transcription yields and capping rates compared to the modified locked nucleoside capping analogs of the prior art. For example, the transcription yield and capping rate of YK-CAP-004 were improved by 35.0% and 18.8% respectively compared to Compound 14.

[0248] The mRNA in vitro transcription yield of Compound 14 was 120.9 μg. Among the present application, YK-CAP-004, YK-CAP-003, YK-CAP-005 and YK-CAP-006 were improved by 35.0%, 24.9%, 26.2% and 26.6% respectively compared to Compound 14, showing significant improvement.

[0249] The capping rate of Compound 14 was 77.5%. YK-CAP-004, YK-CAP-003, YK-CAP-005 and YK-CAP-006 were improved by 18.8%, 17.3%, 17.7% and 17.6% respectively compared to Compound 14, showing significant improvement.

[0250] 3) Modified locked nucleoside capping analogs with similar structures had very large differences in mRNA in vitro transcription yields and capping rates.

[0251] The structures among the modified locked nucleoside capping analogs designed in the present application were very similar, and such a series of compounds were very similar to the structure of Compound 14. However, there were very large differences in mRNA in vitro transcription yields and capping rates.

[0252] For example, compared with YK-CAP-001, YK-CAP-002, and Compound 14, YK-CAP-004 only differs in the group attached to the methylene bridge connecting the 2'-O and C4' of the ribose ring, i.e., only the substituent at C6' is different. YK-CAP-004 is 2,2-difluoroethyl, YK-CAP-001 is dimethylaminoethyl, and YK-CAP-002 is cyanomethyl, while Compound 14 has no substituent and the other structures are exactly the same. However, the in vitro mRNA transcription yield of YK-CAP-004 was improved by 82.8%, 58.3%, and 35.0% compared with YK-CAP-001, YK-CAP-002, and Compound 14, respectively, and the capping rate was improved by 23.2%, 26.1%, and 18.8%, respectively, showing a significant improvement.

[0253] From this, it was found that modified locked nucleoside capping analogs with similar structures do not necessarily have similar mRNA transcription activities and capping rates, and conversely, there is a very high possibility of having a very large difference.

[0254] As can be seen from the in vitro mRNA transcription yield and capping rate, the modified locked nucleoside capping analogs YK-CAP-004, YK-CAP-003, YK-CAP-005, and YK-CAP-006 of the present application showed a significant improvement in both the in vitro mRNA transcription yield and capping rate compared with YK-CAP-001 and YK-CAP-002 of the present application, or compared with Compound 14 of the prior art.

[0255] The modified locked nucleoside capping analogs YK-CAP-004, YK-CAP-003, YK-CAP-005, and YK-CAP-006 of the present application replace the original five-membered sugar ring structure with a bridged ring structure, and each has 2,2-difluoroethyl (YK-CAP-004), dimethylaminocarbonylmethyl (YK-CAP-003), methylsulfonylethyl (YK-CAP-005), and dimethylaminosulfonylethyl (YK-CAP-006) introduced into the methylene bridge (i.e., C6') connecting the 2'-O and 4C' of the ribose ring. Since the bridged ring structure cannot be used as the transcription start site, it has an excellent reverse transcription prevention effect during in vitro mRNA transcription, greatly increases the binding ability of the cap structure and the capping enzyme, and increases the capping rate of the transcribed mRNA.

[0256] Furthermore, it was found that modified locked nucleoside capping analogs with similar structures do not necessarily have similar mRNA transcription activities and capping rates, and conversely, there is a very high possibility of having a very large difference.

[0257] Example 3: Preparation and Characterization of Lipid Nanoparticles 1. Experimental Method The cationic lipid YK-009 (Beijing Youcare Kechuang Pharmaceutical Technology Co., Ltd.), DSPC (AVT (Shanghai) Pharmaceutical Technology Co., Ltd.), cholesterol (AVT (Shanghai) Pharmaceutical Technology Co., Ltd.) and DMG-PEG2000 were dissolved in ethanol at a molar ratio of 49:10:39.5:1.5, and the mRNA was diluted to pH = 4 with 50 mM citrate buffer. Using a microfluidic device, the ethanol lipid solution and the aqueous Fluc mRNA solution prepared with a different capping structure were mixed at a flow rate of 10 mL / min at a volume ratio of 1:3 to produce LNP with a weight ratio of total lipid to mRNA of about 15:1. The obtained liposomes were diluted 10-fold with PBS and then ultrafiltered through a 300 KDa ultrafiltration tube to remove ethanol. Next, it was fixed to a predetermined volume with PBS, and finally, the lipid nanoparticles were filtered through a 0.2 μm sterile filter to obtain an LNP formulation encapsulating Fluc-mRNA with YK-009 / DSPC / cholesterol / DMG-PEG2000 (molar ratio 49:10:39.5:1.5).

[0258] The polydispersity index (PDI) was measured using a Malvern laser analyzer with dynamic light scattering. 10 μL of the liposome solution was taken, diluted to 1 mL with deionized water without RNase, added to the sample pool, and each sample was measured 3 times repeatedly. The measurement conditions were a scattering angle of 90° and 25 °C. According to the instructions provided by the manufacturer, the encapsulation efficiency of the lipid nanoparticles was determined using the Quant it Ribogreen RNA quantification measurement kit (Thermo Fisher Scientific, UK).

[0259] 2. Experimental Results Specific characteristic data of the lipid nanoparticles are shown in Table 4.

[0260]

Table 4

[0261] As can be seen from Table 4, the capping analogs YK-CAP-001 to 006 of the present application, the capping analog N-7413, compound 14, HN3002 disclosed in the prior art, and the Fluc mRNA transcribed from m6A can all produce good lipid nanoparticles. The particle sizes of all lipid nanoparticles were 82 to 103 nm, the PDI values were 0.018 to 0.065, and the encapsulation efficiencies were all over 90%.

[0262] Example 4: Translation efficiency of different capping luciferase mRNAs There were significant differences in the translation efficiency of the modified locked nucleoside capping analogs of the present application for mRNA. The translation efficiency of YK-CAP-004 was the highest, which was twice that of YK-CAP-002 with the lowest translation efficiency. Compared with the modified locked nucleoside capping analogs in the prior art with similar or very different structures, the mRNA translation efficiency of the modified locked nucleoside capping analogs of the present application was significantly improved. For example, the translation efficiency of YK-CAP-004 reached 4 times that of m6A.

[0263] 1. Experimental method (1) HEK293T cells were cultured in DMEM medium containing 10% FBS and penicillin / streptomycin at 37 °C under 5% CO2.

[0264] (2) The cells were digested in a culture dish, counted, and seeded in a 96-well plate at 10,000 cells / well and cultured overnight until the cells adhered to the wall.

[0265] (3) When the cell density reached about 80%, transfection was performed. 0.5 μg of mRNA sample and Lipofectamine MessengerMAX Transfection Reagent (Invitrogen) reagent were added to each well, and the transfection steps in the instruction manual were followed.

[0266] (4) The transfected cells were placed at 37 °C and 5% CO2 and continuously cultured for 24 hours. The growth medium was removed from the test cells, and the cells were washed with PBS. After centrifugation to remove PBS, 50 μL of 1× lysis buffer was added. After transferring the cells and all the liquid to a microcentrifuge tube, centrifugation was performed.

[0267] (5) 20 μL of the sample was collected and 100 μL of Dual-Lumi TM II firefly luciferase detection reagent equilibrated to room temperature was added and mixed uniformly as appropriate.

[0268] (6) The cells were cultured at room temperature (about 25 °C) for 5 minutes to stabilize the luminescence signal. Chemiluminescence detection was performed using a multifunctional microplate reader equipped with a chemiluminescence detection function, and the data were recorded. The relative fluorescence reading values of the specific capping mRNA are shown in Table 5.

[0269] 2. Experimental Results The relative fluorescence reading values of the capping mRNA are shown in Table 5, and the relative fluorescence intensity was directly proportional to the translation efficiency of the mRNA.

[0270]

Table 5

[0271] 1) There was a significant difference in the translation efficiency of the modified locked nucleoside capping analogs of the present application. The translation efficiency of YK-CAP-004 was the highest, which was twice that of YK-CAP-002 with the lowest translation efficiency.

[0272] As can be seen from Table 5, there was a significant difference in the relative fluorescence intensity (corresponding to the translation efficiency of the mRNA) of the modified locked nucleoside capping analogs of the present application. The highest intensity was YK-CAP-004 with the relative fluorescence intensity reaching 1.41. The relative fluorescence intensities of YK-CAP-003, YK-CAP-005, and YK-CAP-006 also reached 1.23, 1.28, and 1.31 respectively, all of which were relatively high.

[0273] The relative fluorescence intensity was the lowest for YK-CAP-002 at only 0.69. The relative fluorescence intensity of YK-CAP-001 was also very low at only 0.78. The relative fluorescence intensities of YK-CAP-004, YK-CAP-003, YK-CAP-005, and YK-CAP-006 were 2.0 times, 1.8 times, 1.9 times, and 1.9 times that of YK-CAP-002, and 1.8 times, 1.6 times, 1.6 times, and 1.7 times that of YK-CAP-001, respectively. (Figure 3) 2) The modified locked nucleoside capping analogs of the present application showed a remarkable improvement in both mRNA translation efficiencies compared to the modified locked nucleoside capping analogs of the prior art. For example, the translation efficiency of YK-CAP-004 reached 4 times that of m6A.

[0274] The relative fluorescence intensities (corresponding to the mRNA translation efficiency) of N-7413, HN3002, and m6A were 1.00, 1.13, and 0.35, respectively. The relative fluorescence intensities of YK-CAP-004, YK-CAP-003, YK-CAP-005, and YK-CAP-006 in the present application were 1.4 times, 1.2 times, 1.3 times, and 1.3 times that of N-7413, 1.2 times, 1.1 times, 1.1 times, and 1.2 times that of HN3002, and 4.0 times, 3.5 times, 3.7 times, and 3.7 times that of m6A, respectively.

[0275] 3) Modified locked nucleoside capping analogs with similar structures showed a very large difference in the mRNA translation efficiency.

[0276] The structures among the modified locked nucleoside capping analogs designed in the present application were very similar, but there was a very large difference in the mRNA translation efficiency.

[0277] For example, compared with YK-CAP-001 and YK-CAP-002, YK-CAP-004 only differs in the group attached to the methylene bridge connecting the 2'-O and C4' of the ribose ring, that is, only the substituent at C6' is different. YK-CAP-004 is 2,2-difluoroethyl, YK-CAP-001 is dimethylaminoethyl, and YK-CAP-002 is cyanomethyl, and the other structures are exactly the same. However, the translation efficiency of YK-CAP-004 mRNA was 1.8 times and 2.0 times that of YK-CAP-001 and YK-CAP-002 respectively, showing a significant improvement.

[0278] From this, it was found that the translation efficiency of luciferase mRNA of modified locked nucleoside capping analogs with similar structures is not necessarily similar, and conversely, there is a very high possibility of having a very large difference.

[0279] As can be seen from the translation efficiency of different capping luciferase mRNAs, the modified locked nucleoside capping analogs of the present application, including YK-CAP-004, YK-CAP-003, YK-CAP-005 and YK-CAP-006, have significantly improved mRNA translation efficiency compared with modified locked nucleoside capping analogs with similar structures (including YK-CAP-001 and YK-CAP-002 of the present application) or modified locked nucleoside capping analogs with large structural differences (including N-7413, HN3002 and m6A). This indicates that the bridge ring nucleotide structures with substituents of 2,2-difluoroethyl (YK-CAP-004), dimethylaminocarbonylmethyl (YK-CAP-003), methylsulfonylethyl (YK-CAP-005) and dimethylaminosulfonylethyl (YK-CAP-006) can more stabilize the structure of the 5-membered sugar ring, be easily bound by the cap-binding protein (EIF4E), and improve the translation efficiency of the target mRNA.

[0280] Furthermore, it was found that the translation efficiencies of luciferase mRNAs with modified locked nucleoside capping analogs having similar structures are not necessarily similar, and conversely, there is a very high possibility of having a very large difference.

[0281] Example 5. Decapping Enzyme Stability Test There was a significant difference in the decapping rate of the modified locked nucleoside capping analog of the present application. The decapping rate of YK-CAP-004 was the lowest, decreasing by 16.0% compared to YK-CAP-001 with the highest rate. Compared with modified locked nucleoside capping analogs with similar structures in the prior art or having very large differences, the decapping rate of the compounds of the present application significantly decreased. For example, the decapping rate of YK-CAP-004 decreased by 38.3% compared to N-7413.

[0282] 1. Experimental Method 30 pmol of RNA purified by polyacrylamide gel electrophoresis PAGE was subjected to an enzymatic reaction at 37°C for 45 minutes with 50 U of mRNA decapping enzyme (New England Biolabs) and 1×MDE buffer. After staining the enzymatic reaction product with PAGE electrophoresis SYBR Green II (Lonza), the gel pattern after electrophoresis was observed with a Typhoon FLA 7000 (GE Healthcare) instrument. Using Image Quant (GE Healthcare) software, the ratio of the electrophoretic band intensities of capping RNA and decapping-removed RNA was statistically analyzed, and the decapping rate of the decapping enzyme was calculated (see Table 6).

[0283] 2. Experimental Results

[0284]

Table 6

[0285] 1) The modified locked nucleoside capping analogs of this application showed a significant difference in the decapping rate. The decapping rate of YK-CAP-004 was the lowest, decreasing by 16.0% compared to the highest YK-CAP-001.

[0286] As can be seen from the data in Table 6, the modified locked nucleic acid cap analogs YK-CAP-001, YK-CAP-002, YK-CAP-003, YK-CAP-004, YK-CAP-005, and YK-CAP-006 of this application had a very large difference in the decapping rate. The decapping rate of YK-CAP-004 was only 10.3%, the lowest, and the decapping rates of YK-CAP-003, YK-CAP-005, and YK-CAP-006 were also very low at 11.8%, 12.8%, and 11.2% respectively.

[0287] The decapping rate of YK-CAP-001 reached 26.3%, the highest, and the decapping rate of YK-CAP-002 also reached 24.6%, which was relatively high. The decapping rates of YK-CAP-004, YK-CAP-003, YK-CAP-005, and YK-CAP-006 decreased by 16.0%, 14.5%, 13.5%, and 15.1% respectively compared to YK-CAP-001, and by 14.3%, 12.8%, 11.8%, and 13.4% respectively compared to YK-CAP-002.

[0288] 2) Compared with the modified locked nucleoside capping analogs with similar structures in the prior art or with very large differences, the decapping rate of the modified locked nucleoside capping analogs of this application decreased significantly. For example, the decapping rate of YK-CAP-004 decreased by 38.3% compared to N-7413.

[0289] The decapping rates of N-7413, Compound 14, HN3002, and m6A were 48.6%, 28.8%, 23.2%, and 27.5%, respectively. The decapping rates of YK-CAP-004, YK-CAP-003, YK-CAP-005, and YK-CAP-006 of this application were 38.3%, 36.8%, 35.8%, and 37.4% lower than that of N-7413, 18.5%, 17.0%, 16.0%, and 17.6% lower than that of Compound 14, 12.9%, 11.4%, 10.4%, and 12.0% lower than that of HN3002, and 17.2%, 15.7%, 14.7%, and 16.3% lower than that of m6A, respectively.

[0290] 3) Modified locked nucleoside capping analogs with similar structures had very large differences in decapping rates.

[0291] The structures among the modified locked nucleoside capping analogs designed in this application were very similar. Such a series of compounds was very similar to the structure of Compound 14, but there were very large differences in decapping rates.

[0292] For example, compared with YK-CAP-001, YK-CAP-002, and Compound 14, YK-CAP-004 differed only in the group bonded to the methylene bridge connecting the 2'-O and C4' of the ribose ring, that is, the substituent at C6'. YK-CAP-004 was 2,2-difluoroethyl, YK-CAP-001 was dimethylaminoethyl, YK-CAP-002 was cyanomethyl, while Compound 14 had no substituent and the other structures were exactly the same. However, the mRNA decapping rate of YK-CAP-004 decreased by 16.0%, 14.3%, and 18.5% compared with YK-CAP-001, YK-CAP-002, and Compound 14, respectively, showing a significant decrease.

[0293] It was found that modified locked nucleoside capping analogs with similar structures did not necessarily have similar m decapping rates, and conversely, there was a very high possibility of having very large differences.

[0294] As can be seen from the DCP2 enzyme decapping rate, the modified locked nucleoside capping analogs of the present application, including YK-CAP-004, YK-CAP-003, YK-CAP-005 and YK-CAP-006, are either structurally similar modified locked nucleoside capping analogs (including YK-CAP-001 and YK-CAP-002 of the present application, and compound 14 of the prior art) or modified locked nucleoside capping analogs with a large structural difference (including N-7413, HN3002 and m6A). Compared with them, both of the DCP2 enzyme decapping rates decreased significantly.

[0295] Furthermore, it was found that modified locked nucleoside capping analogs with similar structures do not necessarily have similar m decapping rates, and conversely, there is a very high possibility of having a very large difference.

[0296] Example 6: Animal Experiment Furthermore, the protein expression level and duration in the mouse body of Fluc mRNA produced from different modified locked nucleic acid cap analogs were considered. According to the results, it was shown that there were significant differences in the average radiation intensity (corresponding to the protein expression level) and duration of the proteins expressed by the modified locked nucleoside capping analogs of the present application in the mouse body of mRNA. The fluorescence expression intensity of YK-CAP-004 was the highest, which was 2.6 times and 5.5 times that of YK-CAP-002 with the lowest at 6 hours and 96 hours respectively. Compared with the modified locked nucleoside capping analogs of the prior art with similar or very different structures, the modified locked nucleoside capping analogs of the present application significantly improved the protein expression level and duration of mRNA in the mouse body. For example, the average radiation intensity of YK-CAP-004 was 2.1 times that of m6A at 6 hours and 3.4 times at 96 hours.

[0297] 1. Experimental Method An LNP formulation containing 5 μg of the capping analogs YK-CAP-001 to 006 of the present application, the capping analog N-7413 of the prior art, compound 14, and m6A-transcribed Fluc mRNA was intramuscularly injected into female BALB / C mice aged 4 to 6 weeks and weighing 17 to 19 g. At specific time points (6 hours, 12 hours, 24 hours, 48 hours, 96 hours, and 168 hours) after administration, a fluorescent imaging substrate was intraperitoneally injected into the mice. After allowing the mice to move freely for 5 minutes, the average radiation intensity (corresponding to the protein expression level) of the protein expressed by the mRNA carried by the LNP in the mice was detected using an IVIS Spectrum small animal live imager.

[0298] 2. Experimental Results The detection results are shown in Table 7 and Figure 5.

[0299] [Table 7]

[0300] 1) It was shown that there were significant differences in the average radiation intensity and duration of the protein expressed by the mRNA in the mice for the modified locked nucleoside capping analogs of the present application. The average radiation intensity of YK-CAP-004 was the highest, being 2.6 times and 5.5 times that of YK-CAP-002, which was the lowest at 6 hours and 96 hours, respectively.

[0301] As can be seen from the data in Table 7, there were very large differences in the average radiation intensity of the protein expressed by the mRNA in the mice for different modified locked nucleic acid cap analogs of the present application. The average radiation intensity of YK-CAP-004 was the highest, reaching 3,856,100 at 6 hours and 114,933 at 96 hours. The average radiation intensities of live imaging of mice with YK-CAP-003, YK-CAP-005, and YK-CAP-006 were also very high, being 3,212,750, 3,171,000, and 3,303,900 at 6 hours, and 57,893, 110,595, and 75,840 at 96 hours, respectively.

[0302] The average radiation intensity of YK-CAP-002 was 1466925 in 6 hours, and only 20893 in 96 hours, which was the lowest. The average radiation intensity of YK-CAP-001 was 1728300 in 6 hours and 43485 in 96 hours, which was also lower.

[0303] The average radiation intensity of YK-CAP-004 was 2.6 times that of YK-CAP-002 in 6 hours and 5.5 times in 96 hours. The average radiation intensity of YK-CAP-003 was 2.2 times that of YK-CAP-002 in 6 hours and 2.8 times in 96 hours. The average radiation intensity of YK-CAP-005 was 2.2 times that of YK-CAP-002 in 6 hours and 5.3 times in 96 hours. The average radiation intensity of YK-CAP-006 was 2.3 times that of YK-CAP-002 in 6 hours and 3.6 times in 96 hours.

[0304] The average radiation intensity of YK-CAP-004 was 2.2 times that of YK-CAP-001 in 6 hours and 2.6 times in 96 hours. The average radiation intensity of YK-CAP-003 was 1.9 times that of YK-CAP-001 in 6 hours and 1.3 times in 96 hours. The average radiation intensity of YK-CAP-005 was 1.8 times that of YK-CAP-001 in 6 hours and 2.5 times in 96 hours. The average radiation intensity of YK-CAP-006 was 1.9 times that of YK-CAP-001 in 6 hours and 1.7 times in 96 hours.

[0305] 2) Compared with modified locked nucleoside capping analogs with similar structures or very different structures in the prior art, the average radiation intensity and duration of the proteins expressed by the mRNA of the modified locked nucleoside capping analogs of the present application were significantly improved. For example, the average radiation intensity of YK-CAP-004 was 2.1 times that of m6A in 6 hours and 3.4 times in 96 hours.

[0306] The average radiation intensities of N-7413, Compound 14, and m6A were 2777750, 2589500, and 1801200 respectively in 6 hours, and 68400, 57270, and 33586 respectively in 96 hours.

[0307] The average radiation intensity of YK-CAP-004 of this application was 1.4 times, 1.5 times and 2.1 times that of N-7413, Compound 14 and m6A respectively in 6 hours, and 1.7 times, 2.0 times and 3.4 times respectively in 96 hours.

[0308] The average radiation intensity of YK-CAP-003 was 1.2 times, 1.2 times and 1.8 times that of N-7413, Compound 14 and m6A respectively in 6 hours, and 0.8 times, 1.0 times and 1.7 times respectively in 96 hours.

[0309] The average radiation intensity of YK-CAP-005 was 1.1 times, 1.2 times and 1.8 times that of N-7413, Compound 14 and m6A respectively in 6 hours, and 1.6 times, 1.9 times and 3.3 times respectively in 96 hours.

[0310] The average radiation intensity of YK-CAP-006 was 1.2 times, 1.3 times and 1.8 times that of N-7413, Compound 14 and m6A respectively in 6 hours, and 1.1 times, 1.3 times and 2.3 times respectively in 96 hours.

[0311] 3) Modified locked nucleoside capping analogs with similar structures had very large differences in the average radiation intensity and duration of the proteins expressed in the mouse body of mRNA.

[0312] The structures among the modified locked nucleoside capping analogs designed in this application are very similar, and such a series of compounds are very similar to the structure of Compound 14, but there are very large differences in the average radiation intensity and duration of the proteins expressed in the mouse body of mRNA.

[0313] For example, compared with YK-CAP-001, YK-CAP-002 and Compound 14, YK-CAP-004 only differs in the group attached to the methylene bridge connecting the 2'-O and C4' of the ribose ring, that is, only the substituent at C6' is different. YK-CAP-004 is 2,2-difluoroethyl, YK-CAP-001 is dimethylaminoethyl, and YK-CAP-002 is cyanomethyl, while Compound 14 has no substituent and the other structures are exactly the same. However, the average radiation intensity of YK-CAP-004 is 2.2 times, 2.6 times and 1.5 times that of YK-CAP-001, YK-CAP-002 and Compound 14 at 6 hours, and 2.6 times, 5.5 times and 2.0 times that of YK-CAP-001, YK-CAP-002 and Compound 14 at 96 hours, showing a significant improvement.

[0314] As can be seen from animal experiments, for example, the modified locked nucleoside capping analogs of the present application such as YK-CAP-004, YK-CAP-003, YK-CAP-005 and YK-CAP-006 have significantly improved both the protein expression level and duration in the mouse body compared with structurally similar modified locked nucleoside capping analogs (including YK-CAP-001 and YK-CAP-002 of the present application and Compound 14 of the prior art) or modified locked nucleoside capping analogs with large structural differences (including N-7413 and m6A).

[0315] In vivo experiments further demonstrated that the mRNA transcribed by YK-CAP-003, YK-CAP-004, YK-CAP-005 and YK-CAP-006 of the present application can be effectively delivered into the body by the LNP delivery vector and can be expressed efficiently and continuously.

[0316] Furthermore, the protein expression level and duration of Fluc mRNA produced by modified locked nucleoside capping analogs with similar structures in the mouse body are not necessarily similar, and on the contrary, there is a very high possibility of having a very large difference.

[0317] In summary, the modified locked nucleoside capping analogs YK-CAP-004, YK-CAP-003, YK-CAP-005 and YK-CAP-006 of the present application have all shown remarkable improvements in mRNA in vitro transcription yield, capping rate, mRNA translation efficiency, stability of the decapping enzyme, and protein expression level and duration in vivo compared to the modified locked nucleoside capping analogs of the prior art (including N-7413, Compound 14, HN3002 and m6A). This indicates that the capping structures of YK-CAP-004, YK-CAP-003, YK-CAP-005 and YK-CAP-006 provided by the present invention have significantly improved the resistance of the locked nucleic acid structure to the decapping enzyme and the binding affinity with the capping enzyme, and can provide a novel highly efficient modified locked nucleic acid capping structure for in vitro transcription of mRNA.

[0318] 1. The chemical structures of the compounds of the present application are very similar. Compared with the mRNA capping analogs disclosed in the prior art, some of these series of compounds have similar structures, while some have very large differences.

[0319] 1) Although the structures of the compounds of the present application are very similar, it has been shown that only the substituents at C6', that is, the groups linked to the methylene bridge connecting 2'-O and C4' of the ribose ring, are different.

[0320] 2) The compounds of the present application are similar in structure to Compound 14, but the difference is only that there is no substituent on the methylene bridge (i.e., C6') connecting 2'-O and C4' of the ribose ring of Compound 14.

[0321] 3) The compounds of the present application have a greater structural difference from N-7413, HN3002 and m6A. There is no methylene bridge between 2'-O and C4' of the ribose ring of these three compounds. Furthermore, the second base adenine of m6A is methylated.

[0322] 2. The modified locked nucleoside capping analogs of the present application showed significant differences in the in vitro transcription yield and capping rate of mRNA. Compared with the modified locked nucleoside capping analogs of the prior art, the modified locked nucleoside capping analogs of the present application showed remarkable improvements in both the in vitro transcription yield and capping rate of mRNA.

[0323] 1) The modified locked nucleoside capping analogs of the present application showed significant differences in the in vitro transcription yield and capping rate of mRNA. The transcription yield and capping rate of YK-CAP-004 were both the highest, with the transcription yield being 82.8% higher than that of YK-CAP-001 with the lowest transcription yield, and the capping rate being 26.1% higher than that of YK-CAP-002 with the lowest capping rate.

[0324] 2) The modified locked nucleoside capping analogs of the present application showed remarkable improvements in both the in vitro transcription yield and capping rate of mRNA compared with the modified locked nucleoside capping analogs of the prior art. For example, the transcription yield and capping rate of YK-CAP-004 were 35.0% and 18.8% higher than those of Compound 14, respectively.

[0325] 3) Modified locked nucleoside capping analogs with similar structures showed very large differences in the in vitro transcription yield and capping rate of mRNA. For example, the in vitro transcription yield of YK-CAP-004 was 82.8%, 58.3% and 35.0% higher than those of YK-CAP-001, YK-CAP-002 and Compound 14, respectively, and the capping rate was 23.2%, 26.1% and 18.8% higher, respectively.

[0326] 3. The modified locked nucleoside capping analogs of the present application showed significant differences in the translation efficiency of mRNA. Compared with the modified locked nucleoside capping analogs of the prior art, the mRNA translation efficiency of the modified locked nucleoside capping analogs of the present application was remarkably improved.

[0327] 1) The modified locked nucleoside capping analogs of this application showed significant differences in the translation efficiency of mRNA. The translation efficiency of YK-CAP-004 was the highest, being twice that of YK-CAP-002 with the lowest efficiency.

[0328] 2) Compared with modified locked nucleoside capping analogs with similar structures in the prior art or those with very large differences, the mRNA translation efficiency of the modified locked nucleoside capping analogs of this application was significantly improved. For example, the translation efficiency of YK-CAP-004 reached four times that of m6A.

[0329] 3) Modified locked nucleoside capping analogs with similar structures showed very large differences in the translation efficiency of mRNA. For example, the translation efficiency of YK-CAP-004 mRNA was 1.8 times and 2.0 times that of YK-CAP-001 and YK-CAP-002 respectively.

[0330] 4. The modified locked nucleoside capping analogs of this application showed significant differences in the decapping rate. Compared with modified locked nucleoside capping analogs with similar structures in the prior art or those with very large differences, the decapping rates of the modified locked nucleoside capping analogs of this application all significantly decreased.

[0331] 1) The modified locked nucleoside capping analogs of this application showed significant differences in the decapping rate. The decapping rate of YK-CAP-004 was the lowest, decreasing by 16.0% compared to YK-CAP-001 with the highest rate.

[0332] 2) Compared with modified locked nucleoside capping analogs with similar structures in the prior art or those with very large differences, the decapping rates of the modified locked nucleoside capping analogs of this application all significantly decreased. For example, the decapping rate of YK-CAP-004 decreased by 38.3% compared to N-7413.

[0333] 3) Modified locked nucleoside capping analogs with similar structures had a very large difference in the decapping rate. For example, the mRNA decapping rate of YK-CAP-004 decreased by 16.0%, 14.3%, and 18.5% compared to YK-CAP-001, YK-CAP-002, and Compound 14, respectively.

[0334] 5. It was shown that the modified locked nucleoside capping analogs of the present application had a significant difference in the average radiation intensity (corresponding to the protein expression level) and duration of the protein expressed in the mouse body of mRNA. Compared with modified locked nucleoside capping analogs with similar or very different structures in the prior art, the modified locked nucleoside capping analogs of the present application significantly improved both the protein expression level and duration of mRNA in the mouse body.

[0335] 1) It was shown that the modified locked nucleoside capping analogs of the present application had a significant difference in the average radiation intensity and duration of the protein expressed in the mouse body of mRNA. The fluorescence expression intensity of YK-CAP-004 was the highest, being 2.6 times and 5.5 times that of YK-CAP-002, which was the lowest at 6 hours and 96 hours, respectively.

[0336] 2) Compared with modified locked nucleoside capping analogs with similar or very different structures in the prior art, both the average radiation intensity and duration of the protein expressed in the mouse body of mRNA of the modified locked nucleoside capping analogs of the present application were significantly improved. For example, the average radiation intensity of YK-CAP-004 was 2.1 times that of m6A at 6 hours and 3.4 times that of m6A at 96 hours.

[0337] 3) Modified lock nucleoside capping analogs with similar structures had very large differences in the average radiation intensity and duration of the proteins expressed in mouse bodies of mRNA. For example, the average radiation intensity of YK-CAP-004 was 2.2 times, 2.6 times, and 1.5 times that of YK-CAP-001, YK-CAP-002, and compound 14 at 6 hours, and 2.6 times, 5.5 times, and 2.0 times that of YK-CAP-001, YK-CAP-002, and compound 14 at 96 hours.

[0338] Finally, each of the above examples is only for explaining the technical solution means of the present invention and is not limited thereto. Although the present invention has been described in detail with reference to the above examples, those skilled in the art can still modify the technical solution means described in each of the above examples, or equivalently replace some or all of its technical features. It should be noted that such modifications or replacements do not deviate from the essence of the corresponding technical solution means from the scope of the technical solution means of each example of the present invention.

Claims

1. A compound represented by formula (I), or a stereoisomer, pharmaceutically acceptable salt or solvate thereof. 【Chemical 1】 (However, X is selected from -O-, -S-, -N(R １ ), -C(=O)-, -C(=O)N(R １ ), -NR １ C(=O)-, -C(=O)O-, -OC(=O)-, -S(=O) ２ -, or does not exist, n is selected from integers of 1 to 3, R １ 、 R ２ 、 R ３ 、 R ５ 、 R ６ are independently selected from a hydrogen atom, a substituted or unsubstituted C １ -C ６ alkyl, C ２ -C ６ alkenyl, C ２ -C ６ alkynyl, C ３ -C ６ cycloalkyl or benzyl, R ４ is selected from halogen, -NR １ R ２ , a hydrogen atom, -CN, substituted or unsubstituted C １ ~C ６ alkyl, C ２ ~C ６ alkenyl or C ２ ~C ６ alkynyl, and R １ and R ２ each time it appears, independently represents a hydrogen atom, a substituted or unsubstituted C １ to C ６ alkyl, C ２ to C ６ alkenyl, C ２ to C ６ alkynyl, C ３ to C ６ cycloalkyl, phenyl or benzyl, and is selected from Y １ａ 、Y １ｂ 、Y １ｃ 、Y １ｄ 、Y １ｅ 、Y １ｆ is independently selected from -O-, -S-, -CH ２ -, -CCl ２ -, -CF ２ - or -NH- and Y ２ａ 、 Y ２ｂ 、 Y ２ｃ 、 Y ２ｄ is independently selected from =O or =S, Y ３ａ , Y ３ｂ , Y ３ｃ , Y ３ｄ is independently selected from -OH or -SH, B １ and B ２ are each independently selected from a natural or modified pyrimidine nucleotide base, a natural or modified purine nucleotide base, or a pharmaceutically acceptable salt of each of these.)

2. (1) X is selected from -O-, -S-, -NH-, -N(CH ３ ), -C(=O)-, -C(=O)N(CH ３ ), -N(CH ３ ), -C(=O)NH-, -NH-C(=O)-, -C(=O)O-, -OC(=O)-, -S(=O) ２ -, or is absent, (2) X is selected from -C(=O)-, -S(=O)- or does not exist, ２ under the condition that it is selected or does not exist, (3) The condition that n is 1 or 2, (4) R １ , R ２ , R ３ , R ５ , R ６ are independently hydrogen atoms or methyl under the condition that (5) R ４ is halogen, -N(CH ３ )H, -NH ２ , -N(CH ３ ) ２ , a hydrogen atom, -CN, -CH ３ , -CF ２ H, -CCl ２ H, -CFH ２ , -CClH ２ , -CH=CH ２ or C ２ selected from alkynyl, (6) R ４ is -CH ３ -, -CF ２ H, -N(CH ３ ) ２ or a condition selected from -CN, (7) R １ and R ２ are independently selected from a hydrogen atom and a methyl group each time they appear The compound represented by formula (I) according to claim 1, or a stereoisomer, pharmaceutically acceptable salt or solvate thereof, which satisfies one or more of the above.

3. The compound represented by formula (I) according to claim 1, or a stereoisomer, pharmaceutically acceptable salt or solvate thereof, wherein X does not exist.

4. R ４ is -CF ２ H, -N(CH ３ ) ２ or -CN, a compound represented by formula (I) according to claim 3, or a stereoisomer, pharmaceutically acceptable salt or solvate thereof.

5. The compound represented by formula (I) according to claim 1, or a stereoisomer, pharmaceutically acceptable salt or solvate thereof, wherein X is -C(=O)-.

6. R ４ is, the compound represented by formula (I) according to claim 5 or a stereoisomer, pharmaceutically acceptable salt or solvate thereof, which is -N(CH ３ ). ２ ​

7. X is -S(=O)- ２ The compound represented by formula (I) according to claim 1, or a stereoisomer, pharmaceutically acceptable salt or solvate thereof, wherein X is -S(=O)-

8. R ４ is -N(CH ３ ) ２ or -CH ３ and is a compound represented by formula (I) according to claim 7, or a stereoisomer, pharmaceutically acceptable salt or solvate thereof.

9. R １ is a hydrogen atom or methyl, a compound represented by formula (I) according to any one of claims 1 to 8, or a stereoisomer, pharmaceutically acceptable salt or solvate thereof.

10. R １ is a hydrogen atom, a compound represented by formula (I) according to claim 9, or a stereoisomer, pharmaceutically acceptable salt or solvate thereof.

11. R ２ is a hydrogen atom or methyl, a compound represented by formula (I) according to any one of claims 1 to 8, or a stereoisomer, pharmaceutically acceptable salt or solvate thereof.

12. R ２ is methyl, a compound represented by formula (I) according to claim 11, or a stereoisomer, pharmaceutically acceptable salt or solvate thereof.

13. R ３ is a hydrogen atom or methyl, a compound represented by formula (I) according to any one of claims 1 to 8, or a stereoisomer, pharmaceutically acceptable salt or solvate thereof.

14. R ３ is a hydrogen atom, a compound represented by formula (I) according to claim 13, or a stereoisomer, pharmaceutically acceptable salt or solvate thereof.

15. R ５ is a hydrogen atom or methyl, a compound represented by formula (I) according to any one of claims 1 to 8, or a stereoisomer, pharmaceutically acceptable salt or solvate thereof.

16. R ５ is methyl, the compound represented by formula (I) according to claim 15, or a stereoisomer, pharmaceutically acceptable salt or solvate thereof.

17. R ６ is a hydrogen atom or methyl, a compound represented by formula (I) according to any one of claims 1 to 8, or a stereoisomer, pharmaceutically acceptable salt or solvate thereof.

18. R ６ is a hydrogen atom, a compound represented by formula (I) according to claim 17, or a stereoisomer, pharmaceutically acceptable salt or solvate thereof.

19. Y １ａ is —O—, a compound represented by formula (I) according to any one of claims 1 to 8, or a stereoisomer, pharmaceutically acceptable salt or solvate thereof.

20. Y １ｂ is —O—, a compound represented by formula (I) according to any one of claims 1 to 8, or a stereoisomer, pharmaceutically acceptable salt or solvate thereof.

21. Y １ｃ is —O—, a compound represented by formula (I) according to any one of claims 1 to 8 or a stereoisomer, pharmaceutically acceptable salt or solvate thereof.

22. Y １ｄ is —O—, a compound represented by formula (I) according to any one of claims 1 to 8, or a stereoisomer, pharmaceutically acceptable salt or solvate thereof.

23. Y １ｅ is —O—, a compound represented by formula (I) according to any one of claims 1 to 8, or a stereoisomer, pharmaceutically acceptable salt or solvate thereof.

24. Y １ｆ is —O—, a compound represented by formula (I) according to any one of claims 1 to 8 or a stereoisomer, pharmaceutically acceptable salt or solvate thereof.

25. Y ２ａ is a compound represented by formula (I) according to any one of claims 1 to 8, or a stereoisomer, pharmaceutically acceptable salt or solvate thereof, which is =O.

26. Y ２ｂ is a compound represented by the formula (I) according to any one of claims 1 to 8, or a stereoisomer, pharmaceutically acceptable salt or solvate thereof, which is =O.

27. Y ２ｃ is a compound represented by formula (I) according to any one of claims 1 to 8, or a stereoisomer, pharmaceutically acceptable salt or solvate thereof, which is =O.

28. Y ２ｄ is a compound represented by the formula (I) according to any one of claims 1 to 8, or a stereoisomer, pharmaceutically acceptable salt or solvate thereof, which is =O.

29. Y ３ａ is a compound represented by formula (I) according to any one of claims 1 to 8, or a stereoisomer, pharmaceutically acceptable salt or solvate thereof, which is -OH.

30. Y ３ｂ The compound of formula (I) according to any one of claims 1 to 8, wherein Y is -OH.

31. Y ３ｃ is —OH, a compound represented by formula (I) according to any one of claims 1 to 8, or a stereoisomer, pharmaceutically acceptable salt or solvate thereof.

32. Y ３ｄ is a compound represented by formula (I) according to any one of claims 1 to 8, or a stereoisomer, pharmaceutically acceptable salt or solvate thereof, which is -OH.

33. 【Fig. 2】 The compound represented by formula (I) according to any one of claims 1 to 8, or a stereoisomer, pharmaceutically acceptable salt or solvate thereof, wherein the group is in the R configuration, S configuration or (R+S) configuration.

34. 【Fig. 3】 The compound represented by formula (I) according to claim 33, or a stereoisomer, pharmaceutically acceptable salt or solvate thereof, wherein the group is in the R configuration.

35. B １ and B ２ is independently selected from substituted or unsubstituted adenine, guanine, thymine, cytosine, uracil, or a pharmaceutically acceptable salt of each thereof, a compound of formula (I) according to any one of claims 1 to 8, or a stereoisomer, pharmaceutically acceptable salt or solvate thereof.

36. B １ and B ２ each independently is selected from cytosine, guanine, adenine, thymine, uracil, hypoxanthine, xanthine, 7-methylguanine, pseudouracil, thiouracil, 5,6-dihydrouracil, 5-bromouracil, 5-iodouracil, 5-methylcytosine, 5-hydroxymethylcytosine, or a pharmaceutically acceptable salt thereof, a compound represented by formula (I) according to any one of claims 1 to 8 or a stereoisomer, pharmaceutically acceptable salt or solvate thereof.

37. B １ is adenine, a compound represented by formula (I) according to claim 36, or a stereoisomer, pharmaceutically acceptable salt or solvate thereof.

38. B ２ is the compound represented by formula (I) according to any one of claims 1 to 8, or a stereoisomer, pharmaceutically acceptable salt or solvate thereof, which is 7-methylguanine or guanine.

39. B ２ is guanine, a compound represented by formula (I) according to claim 38, or a stereoisomer, pharmaceutically acceptable salt or solvate thereof.

40. The compound represented by formula (I) according to claim 1, or a stereoisomer, pharmaceutically acceptable salt or solvate thereof, wherein the structure of the compound represented by formula (I) is YK-CAP-001, YK-CAP-002, YK-CAP-003, YK-CAP-004, YK-CAP-005 or YK-CAP-006 represented by the following formula. [Chemical Formula 4] [Chemical]

41. Use of the compound represented by formula (I) according to any one of claims 1 to 40, or a stereoisomer, pharmaceutically acceptable salt or solvate thereof, in the manufacture of an in vitro co-transcriptional mRNA capping reagent.

42. An RNA molecule comprising the compound represented by formula (I) according to any one of claims 1 to 40, or a stereoisomer, pharmaceutically acceptable salt or solvate thereof, as a cap structure or cap structure fragment.

43. A pharmaceutical composition comprising the RNA molecule according to claim 42.

44. The pharmaceutical composition according to claim 43, further comprising at least one RNA delivery agent.

45. The pharmaceutical composition according to claim 44, wherein the at least one RNA delivery agent comprises at least one cationic lipid.

46. The pharmaceutical composition according to claim 44, wherein the at least one RNA delivery agent further comprises at least one neutral lipid.

47. The cationic lipid is (1) A compound represented by formula (II), or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof, 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, 【Chemical Formula 5】 The compound represented by formula (III), or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof, wherein G １ is 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 - and G ４ is HO(CH ２ ), ２ - or HO(CH ２ ), ３ - and L is (CH ２ ), ２ - or -(CH ２ ), ３ - or -(CH ２ ), ４ - and 【Chemical Formula 6】 a compound represented by formula (IV), or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof, 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 ３ O(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 - and 【Chemical Formula 7】 The compound represented by formula (V), or its N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer, wherein G １ is C １～８ alkylene, G ２ is C ２～８ alkylene, R １ is C ６～２５ linear or branched alkyl, R ２ is C １２～２５ linear or branched alkyl, G ３ is HO(CH ２ ) ２ N(R ３ )CH ２ CH(OH)CH ２ -, wherein R ３ is -CH ３ or -CH ２ CH ３ or -CH ２ CH ２ OH, 【Chemical 8】 a compound represented by formula (VI), or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof, wherein G １ and G ２ are each independently unsubstituted C ６ to C １０ alkylene, G ３ is unsubstituted C １ to C １２ alkylene, R １ and R ２ are each independently C ６ to C ２４ alkyl or C ６ to C ２４ alkenyl, R ３ is OR ５ , N, -C(=O)OR ４ , -OC(=O)R ４ or -NR ５ C(=O)R ４ wherein R ４ is a C １ to C １２ hydrocarbon group, and R ５ is H or a C １ to C ６ hydrocarbon group, [Chemical Formula 9] The compound represented by formula (VII), or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof, wherein R ４ is selected from - (CH ２ ) ｎ Q and - (CH ２ ) ｎ CHQR, 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 ８ and a heterocyclic ring, n is 1, 2 or 3, 【Chemical Formula 10】 (7) a compound represented by formula (VIII), or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof The pharmaceutical composition according to claim 45, which is selected from one or more of the compounds. 【Chemical Formula 11】

48. The composition according to claim 47, wherein the cationic lipid is selected from one or more of YK-009, YK-401, YK-305, ALC0315, SM102, DLIN-MC3-DMA. 【Chemical Formula 12】

49. The pharmaceutical composition according to claim 48, wherein the cationic lipid is YK-009.

50. The pharmaceutical composition according to claim 46, wherein the neutral lipid comprises one or more of phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, ceramide, sterol and derivatives thereof.

51. The neutral lipid is 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-didodecanoyl-sn-glycero-phosphocholine (DUPPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 Diether PC), 1-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), 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 (ME 16.0 PE), 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,The pharmaceutical composition according to claim 50, which is selected from one or more of 2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG), dipalmitoyl phosphatidylglycerol (DPPG), palmitoyl oleoyl phosphatidylethanolamine (POPE), distearoyl-phosphatidyl-ethanolamine (DSPE), dipalmitoyl phosphatidylethanolamine (DPPE), dimyristoyl phosphatidylethanolamine (DMPE), 1-stearoyl-2-oleoyl-stearoylethanolamine (SOPE), 1-stearoyl-2-oleoyl-phosphatidylcholine (SOPC), sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyl oleoyl phosphatidylcholine, lysophosphatidylcholine, lysophosphatidylethanolamine (LPE), and mixtures thereof.

52. The pharmaceutical composition according to claim 51, wherein the neutral lipid is DOPE and / or DSPC.

53. The pharmaceutical composition according to claim 44, wherein the at least one RNA delivery agent further comprises a structural lipid.

54. The pharmaceutical composition according to claim 53, wherein the structural lipid is selected from one or more of cholesterol, non-sterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatine, ursolic acid, α-tocopherol, corticosteroid.

55. The pharmaceutical composition according to claim 54, wherein the structural lipid is cholesterol.

56. The pharmaceutical composition according to claim 44, wherein the at least one RNA delivery agent further comprises a polymer-conjugated lipid.

57. The pharmaceutical composition according to claim 56, wherein the polymeric conjugated lipid is selected from one or more of distearoyl phosphatidylethanolamine polyethylene glycol 2000 (DSPE-PEG2000), 1,2-dimyristoyl-rac-glycero-methoxypolyethylene glycol-2000 (DMG-PEG2000), and methoxypolyethylene glycol ditetradecylacetamide (ALC-0159).

58. The pharmaceutical composition according to any one of claims 44 to 57, further comprising one or more cell-penetrating peptides.

59. A method for synthesizing mRNA molecules for purposes other than the diagnosis and treatment of diseases, comprising culturing a compound represented by formula (I) according to any one of claims 1 to 40 or a stereoisomer, pharmaceutically acceptable salt or solvate thereof together with a polynucleotide template to perform template transcription.

60. (1) A compound represented by formula (I) according to any one of claims 1 to 40 or a stereoisomer, pharmaceutically acceptable salt or solvate thereof, and (2) A polynucleotide template, NTPs and RNA polymerase A capping mRNA transcription reaction system for purposes other than the diagnosis and treatment of diseases.

61. (1) A compound represented by formula (I) according to any one of claims 1 to 40 or a stereoisomer, pharmaceutically acceptable salt or solvate thereof, and (2) Nucleotide triphosphate molecules and RNA polymerase A kit comprising.

62. RNA enzyme inhibitor, inorganic pyrophosphatase, Mg ２＋ The kit according to claim 61, further comprising one or more of a crowding agent, a buffer, or any combination thereof.

63. A method for improving the intracellular stability of RNA, comprising mixing a compound represented by formula (I) according to any one of claims 1 to 40 or a stereoisomer, pharmaceutically acceptable salt or solvate thereof with the RNA.

64. A method for introducing RNA into cells, comprising contacting the cells with the pharmaceutical composition according to any one of claims 43 to 58.

65. A method for performing RNA translation inhibition in cells, comprising contacting the cells with the pharmaceutical composition according to any one of claims 43 to 58.

66. Use of the pharmaceutical composition according to any one of claims 43 to 58 in the manufacture of a vaccine.

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