C6'-substituted locked nucleic acid modified cap analogs and uses 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 chemical and biotechnology, and in particular to C6'-substituted locked nucleic acid modified cap analogs and their uses. [Background technology]

[0002] The chemical essence of the cap structure is a special structure located at the 5' end of mRNA, formed by modification during the mRNA transcription process, i.e., the m7GPPPN structure, also known as the methylguanosine cap. It is formed under the cooperative catalytic action of RNA triphosphatase, guanylyltransferase, mRNA (guanine-N7) methyltransferase, and mRNA (nucleoside-2') methyltransferase. Depending on the degree of methylation, three types of caps can be formed: CAP 0, CAP 1, and CAP 2, which are m7G5'ppp5'Np, m7G5'ppp5'NmpNp, and m7G5'ppp5'NmpNmpNp, respectively.

[0003] The cap structure is essential for the initiation of mRNA translation, providing a signal for ribosomes to recognize the mRNA and helping ribosomes bind to the mRNA so that translation begins at the AUG. At the same time, the cap structure enhances mRNA stability and protects the mRNA from attack by 5'→3' exonucleases.

[0004] In summary, the cap structure acts like a steel helmet on mRNA, protecting it from damage and allowing it to be easily recognized by other members of the mRNA community by adding a mark to the steel helmet through chemical modification. In addition to the native cap structure, cap structure analogs are often used to improve the stability of mRNA structure during in vitro transcription, including ARCA and Cap1 analogs.

[0005] Research has shown that the mRNA cap structure is important for mRNA quality control and the innate immunity of living organisms, so the development of novel cap analogs is of great significance for enhancing mRNA stability and improving the efficiency of mRNA translation. Summary of the Invention

[0006] To improve mRNA stability and / or mRNA translation efficiency, the present invention provides novel modified locked nucleoside mRNA capping analogs and uses thereof.

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

[0008] [ka] however, X is -O-, -S-, -N(R 1 )-, -C(=O)-, -C(=O)N(R 1 )-, -NR 1 X is preferably selected from —O—, —S—, —NH—, —N(CH3)—, —C(═O)—, —C(═O)N(CH3)—, —N(CH3)C(═O)—, —C(═O)NH—, —NHC(═O)—, —C(═O)O—, —OC(═O)—, —S(═O)— or is absent, and X is more preferably selected from —C(═O)—, —S(═O)— or is absent; n is selected from an integer of 1 to 3, preferably n is 1 or 2; R1, R2, R3, R5, and R6 are independently selected from a hydrogen atom, substituted or unsubstituted C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C6 cycloalkyl, and benzyl, and preferably independently a hydrogen atom or methyl; R4 is halogen, -NR 1 R 2, a hydrogen atom, —CN, substituted or unsubstituted C1-C6 alkyl, C2-C6 alkenyl or C2-C6 alkynyl, preferably selected from halogen, —N(CH3)H, —NH2, —N(CH3)2, a hydrogen atom, —CN, —CH3, —CF2H, —CCl2H, —CFH2, —CClH2, —CH═CH2 or C2 alkynyl, more preferably selected from —CH3, —CF2H, —N(CH3)2 or —CN; R 1 and R 2 is independently selected at each occurrence from a hydrogen atom, substituted or unsubstituted C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C6 cycloalkyl, phenyl, or benzyl, preferably R 1 and R 2 is independently selected at each occurrence from a hydrogen atom and methyl; Y 1a , Y 1b , Y 1c , Y 1d , Y 1e , Y 1f are independently selected from —O—, —S—, —CH—, —CCl—, —CF—, or —NH—; Y 2a , Y 2b , Y 2c , Y 2d are independently selected from =O or =S; Y 3a , Y 3b , Y 3c , Y 3d are independently selected from -OH or -SH; B1 and B2 are independently selected from natural or modified pyrimidine nucleotide bases, natural or modified purine nucleotide bases, or pharmaceutically acceptable salts thereof.

[0009] A second aspect of the present invention provides the use of a compound of formula (I) according to the first aspect of the present invention or a stereoisomer, pharmaceutically acceptable salt or solvate thereof in the preparation of an in vitro co-transcriptional mRNA capping reagent.

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

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

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

[0013] A 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, pharmaceutically acceptable salt or solvate thereof, and (2) Polynucleotide template, NTPs, and RNA polymerase The present invention provides a capping mRNA transcription reaction system for purposes other than disease diagnosis and treatment, comprising:

[0014] A 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, pharmaceutically acceptable salt or solvate thereof, and (2) Nucleotide triphosphate molecules and RNA polymerase A kit is provided, comprising:

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

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

[0017] A tenth aspect of the invention provides a method for effecting RNA translation inhibition in a cell, comprising the step of contacting the cell with a pharmaceutical composition according to the fourth aspect of the invention.

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

[0019] In order to more clearly describe the specific embodiments of the present invention or the technical solutions of the prior art, the following will briefly introduce drawings necessary for describing the specific embodiments or the existing technology. It is obvious 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 without making any creative efforts.

[0020] [Figure 1] 1 shows the mRNA in vitro transcription yields of modified locked nucleoside capping analogs YK-CAP-004, YK-CAP-003, YK-CAP-005, YK-CAP-006, YK-CAP-001, YK-CAP-002, and Compound 14.

[0021] [Figure 2] The capping ratios of modified locked nucleoside capping analogs YK-CAP-004, YK-CAP-003, YK-CAP-005, YK-CAP-006, YK-CAP-001, YK-CAP-002 and Compound 14 are shown.

[0022] [Figure 3] 1 shows the relative fluorescence intensity of modified locked nucleoside capping analogs YK-CAP-004, YK-CAP-003, YK-CAP-005, YK-CAP-006, YK-CAP-001, YK-CAP-002, N-7413, HN3002, and m6A.

[0023] [Figure 4] Figure 1 shows the DCP2 enzyme decapping rates of modified locked nucleoside capping analogs YK-CAP-004, YK-CAP-003, YK-CAP-005, YK-CAP-006, YK-CAP-001, YK-CAP-002, N-7413, compound 14, HN3002, and m6A.

[0024] [Figure 5] Figure 1 shows the average radiation intensity of proteins expressed in mice using modified locked nucleoside capping analogs YK-CAP-004, YK-CAP-003, YK-CAP-002, compound 14, and m6A-transcribed Fluc mRNA loaded in LNPs. DETAILED DESCRIPTION OF THE INVENTION

[0025] In order to make the objectives, technical solutions and advantages of the embodiments of the present disclosure clearer, the following will clearly and completely describe the technical solutions of the embodiments of the present disclosure 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, but 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 fall within the scope of protection of the present invention.

[0026] The present invention may be embodied in other specific forms without departing from the essential characteristics of the invention. It is to be understood that, without contradiction, any and all embodiments of the present invention may be combined with technical features of any other embodiment or embodiments to form additional embodiments. The present invention includes additional embodiments resulting from such combinations.

[0027] All publications and patents mentioned in this disclosure are incorporated by reference in their entirety. If the usage or terminology used in any publication or patent incorporated by reference conflicts with the usage or terminology used in this disclosure, the usage or terminology used in this disclosure shall control.

[0028] The section titles used herein are for organizational purposes only and should not be construed as limiting the categories described.

[0029] Unless otherwise defined, all technical and scientific terms used herein have their ordinary meaning within the art to which the claimed subject matter belongs. In the event that there are multiple definitions for a term, those set forth in the specification prevail.

[0030] Except in the working examples or where otherwise indicated, all numerical values ​​reciting quantitative properties, such as dosage, in the specification and claims should be understood as being modified in all instances by the term "about." Furthermore, any numerical range recited in this disclosure should be understood to include all subranges within that range, and any combination of the endpoints of the corresponding ranges or subranges.

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

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

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

[0034] As used herein, the term "alkynyl" refers to a hydrocarbon group having at least one carbon-carbon triple bond at one or more positions along the alkyl carbon chain, non-limiting examples of which include ethynyl, propynyl, etc. As used herein, the term "aryl" refers to a group containing a carbocyclic aromatic system, non-limiting examples of which include phenyl, naphthyl, anthracenyl, phenanthrenyl, pyrenyl, etc., and when an aryl contains multiple rings, the rings can be fused to each other. As used herein, 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 a ring-forming atom, non-limiting examples of which include pyridinyl, pyrimidinyl, pyrazinyl, pyridazinyl, triazinyl, quinolyl, isoquinolinyl, etc., and when a heteroaryl contains multiple rings, the rings can be fused to each other.

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

[0036] As used herein, 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 preparation, purification, and / or handling, e.g., a pharmaceutically acceptable salt. Unless otherwise specified, a reference to a particular compound in the present invention also includes its salt forms.

[0037] As used herein, a "capping analog" refers to a 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 mRNA degradation at the 5' end, allowing RNA transcripts to pass through selective pores in the nuclear membrane and enter the cytoplasm, enhancing translation, and helping to complete the entire shearing process. As used herein, the portion of a nucleic acid polymer between the wavy lines represents a structure embedded in the nucleic acid polymer sequence, while the portion outside the wavy lines represents other sequences in the nucleic acid polymer.

[0038] Bases, also referred to as nucleobases, refer to the heterocyclic base moiety of a nucleoside. Nucleobases may be naturally occurring or modified, such as natural or modified pyrimidine nucleotide bases or natural or modified purine nucleotide bases. 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] As used in this disclosure, terms such as "comprise," "contain," or "comprise," or similar terms, mean that the elements appearing before the term include the elements listed after the term and equivalents thereof, without excluding elements not listed. As used herein, the terms "comprise" or "comprise" may be open, semi-closed, and closed. In other words, the terms also include "consisting essentially of" or "consisting of."

[0040] The term "optionally" refers to the situation described in this disclosure that may or may not occur. For example, optionally substituted refers to the possibility of being substituted or unsubstituted. Substituted refers to the replacement of at least one hydrogen of the group with another chemical group such as methyl, ethyl, methoxy, ethoxy, halogen, hydroxyl, mercapto, amino, nitro, -CN, etc.

[0041] The term "pharmaceutically acceptable" in this disclosure refers to a compound or composition that is chemically and / or toxicologically compatible with the other ingredients that make up the formulation and / or with the human or mammalian organism that uses it to prevent or treat a disease or condition.

[0042] The term "solvate," as used in the present disclosure, refers to a complex formed by combining a compound of formula (I) or a pharmaceutically acceptable salt thereof with a solvent (e.g., ethanol or water). Any solvate of a compound of formula (I) used to treat a disease or condition may provide different properties (including pharmacokinetic properties), but when absorbed by a subject, yields a compound of formula (I), and therefore, each use of a compound of formula (I) should be understood to include the use of any solvate of a compound of formula (I).

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

[0044] Furthermore, it should be understood that the compounds of formula (I) or pharmaceutically acceptable salts thereof can be isolated in the form of solvates, and therefore, any such solvates are all included within the scope of the present invention. For example, the compounds of formula (I) or pharmaceutically acceptable salts thereof can exist in unsolvated forms as well as solvated forms formed with pharmaceutically acceptable solvents (e.g., 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 inherently charged (e.g., compounds having a quaternary nitrogen) can form salts with a suitable counterion (e.g., a halide ion such as bromide, chloride, or fluoride, especially bromide).

[0046] The term "pharmaceutically acceptable salts" refers to relatively non-toxic addition salts of compounds of the present disclosure. See, for example, SM Berge et al., "Pharmaceutical Salts," 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 compounds of the present disclosure that are sufficiently alkaline having 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 with acids such as 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, pectic acid, persulfate, 3-phenylpropionic acid, and the like. Examples of suitable acid addition salts include those formed with organic acids such as acetic 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, glycerophosphate, aspartic acid, sulfosalicylic acid, and thiocyanic acid.

[0048] Furthermore, other suitable pharmaceutically acceptable salts of compounds of the invention that are sufficiently acidic are alkali metal salts such as sodium or potassium salts, alkaline earth metal salts such as calcium or magnesium salts, ammonium salts (e.g., salts formed with NH or aqueous ammonia), or salts formed with organic bases that provide physiologically acceptable cations, such as salts formed with substances such as triethylamine, N-methylglucamine, dimethylglucamine, ethylglucamine, lysine, dicyclohexylamine, 1,6-hexanediamine, ethanolamine, glucosamine, sarcosine, serinol, trihydroxymethylaminomethane, aminopropylene glycol, 1-amino-2,3,4-butanetriol, and the like. Alternatively, alkaline nitrogen-containing groups can be quaternized with reagents such as lower alkyl halides, such as methyl, ethyl, propyl, and butyl chlorides, bromides, and iodides; dialkyl sulfates, such as dimethyl sulfate, diethyl sulfate, dibutyl sulfate, and diamyl sulfate; long chain halides, such as decyl, lauryl, myristyl, and stearyl chlorides, bromides, and iodides; and aralkyl halides, such as benzyl and phenethyl bromide.

[0049] Those skilled in the art will also recognize that acid addition salts of the compounds to be protected may be prepared by reacting said compounds with the appropriate inorganic or organic acid by any of the known methods. Alternatively, base addition salts of acidic compounds of the present disclosure are prepared by reacting them with the appropriate base by a variety of 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 said salts in any ratio.

[0051] Certain compounds of the present disclosure can exist in one or more stereoisomeric forms. Stereoisomers include geometric isomers, diastereomers, and enantiomers. Accordingly, compounds claimed herein also include racemic mixtures, single stereoisomers, and optically active mixtures. Those skilled in the art will appreciate that one stereoisomer may have superior efficacy and / or fewer side effects than other stereoisomers. Single stereoisomers and optically active mixtures can be obtained by methods such as chiral synthesis, chiral catalysis, and chiral resolution. Racemates can be chirally resolved by chromatographic or chemical resolution. For example, chiral acid resolving agents, such as chiral tartaric acid or chiral malic acid, can be added to form salts with the compounds of the present disclosure, and the physical and chemical properties of the resulting salts, such as differences in solubility, can also be exploited.

[0052] Alkyl (or alkylene) can be unsubstituted, or the alkyl (or alkylene) can be substituted, meaning that at least one hydrogen has been replaced with another chemical group such as methyl, ethyl, methoxy, ethoxy, halogen, hydroxyl, mercapto, amino, nitro, -CN, etc.

[0053] In this disclosure, when a compound name and a structural formula are inconsistent, the structural formula shall take precedence.

[0054] The specifications of the present disclosure are to be construed in accordance with the rules and principles of chemical bonding. In some cases, hydrogen atoms may be removed to accommodate substituents at a given position.

[0055] The term "compounds of the present disclosure," as used in this disclosure, should be understood to include compounds represented by formula (I), solvates thereof, pharmaceutically acceptable salts thereof, stereoisomers thereof, and mixtures thereof, depending on the context.

[0056] mRNA-capping analogues of modified locked nucleosides The present invention provides a compound represented by formula (I) or a stereoisomer, pharmaceutically acceptable salt or solvate thereof.

[0057] [ka] however, X is -O-, -S-, -N(R 1 )-, -C(=O)-, -C(=O)N(R 1 )-, -NR 1 X is preferably selected from —O—, —S—, —NH—, —N(CH3)—, —C(═O)—, —C(═O)N(CH3)—, —N(CH3)C(═O)—, —C(═O)NH—, —NHC(═O)—, —C(═O)O—, —OC(═O)—, —S(═O)— or is absent, and X is more preferably selected from —C(═O)—, —S(═O)— or is absent; n is selected from an integer of 1 to 3, preferably n is 1 or 2; R1, R2, R3, R5, and R6 are independently selected from a hydrogen atom, substituted or unsubstituted C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C6 cycloalkyl, and benzyl, and preferably independently a hydrogen atom or methyl; R4 is halogen, -NR 1 R 2 , a hydrogen atom, —CN, substituted or unsubstituted C1-C6 alkyl, C2-C6 alkenyl or C2-C6 alkynyl, preferably selected from halogen, —N(CH3)H, —NH2, —N(CH3)2, a hydrogen atom, —CN, —CH3, —CF2H, —CCl2H, —CFH2, —CClH2, —CH═CH2 or C2 alkynyl, more preferably selected from —CH3, —CF2H, —N(CH3)2 or —CN; R 1 and R 2is independently selected at each occurrence from a hydrogen atom, substituted or unsubstituted C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C6 cycloalkyl, phenyl, or benzyl, preferably R 1 and R 2 is independently selected at each occurrence from a hydrogen atom and methyl; Y 1a , Y 1b , Y 1c , Y 1d , Y 1e , Y 1f are independently selected from —O—, —S—, —CH—, —CCl—, —CF—, or —NH—; Y 2a , Y 2b , Y 2c , Y 2d are independently selected from =O or =S; Y 3a , Y 3b , Y 3c , Y 3d are independently selected from -OH or -SH; B1 and B2 are independently selected from natural or modified pyrimidine nucleotide bases, natural or modified purine nucleotide bases, or pharmaceutically acceptable salts thereof.

[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] [ka] The group may be in the R, S or (R+S) configuration, preferably in the R configuration.

[0082] In one embodiment, B1 and B2 are independently selected from substituted or unsubstituted adenine, guanine, thymine, cytosine, uracil, or a pharmaceutically acceptable salt 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 a pharmaceutically acceptable salt 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, whose structure is represented below.

[0086] [ka] JPEG0007788017000005.jpg216158

[0087] Compared with similar compounds of the prior art, the modified locked nucleoside capping analogs of the present application have the following advantages: 1) Its structure was completely different from similar compounds in the prior art, making it a completely novel compound.

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

[0089] For example, the mRNA in vitro transcription yield and capping rate of YK-CAP-004 were 35.0% and 18.8% higher than those of compound 14, respectively, the translation efficiency was four times that of m6A, the decapping rate was 38.3% lower than that of N-7413, and the average radiation intensity (corresponding to protein expression level) was 2.1 and 3.4 times that of m6A at 6 and 96 hours, respectively.

[0090] Furthermore, the present application has discovered that modified locked nucleoside capping analogs with similar structures are highly likely to have significant differences in the in vitro transcription yield of mRNA, capping rate, translation efficiency of target mRNA, decapping rate, and the amount and duration of protein expressed by mRNA in mice.

[0091] For example, when YK-CAP-001 and YK-CAP-002, which have very similar structures, were compared with compound 14, the mRNA in vitro transcription yield of YK-CAP-004 was 82.8% higher than that of YK-CAP-001, the capping rate was 26.1% higher than that of YK-CAP-002, the mRNA translation efficiency was 2.0-fold higher than that of YK-CAP-002, and the decapping rate was 18.5% lower than that of compound 14. The average radiation intensity of the protein expressed in the mRNA of mice was 2.6-fold and 5.5-fold higher than that of YK-CAP-002 at 6 and 96 hours, respectively.

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

[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 LNP-coated mRNA can be protected from the effects of extracellular ribonucleases, which is useful for intracellular mRNA delivery. For lipid nanoparticles, see the comprehensive article Chemistry of Lipid Nanoparticles for RNA Delivery. Acc Chem Res. 2022 Jan 4; 55(1):2-12.

[0094] cationic lipids Lipid nanoparticles generally comprise cationic lipids.

[0095] The term cationic lipid used herein refers to the lipid that is positively charged at selected pH value.For example, refer to the cationic lipid disclosed in WO2023133946A1, CN115745820A, Chemistry of Lipid Nanoparticles for RNA Delivery.According to 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, and G2 is C 2~8 alkylene, and G3 is C 1~3 alkylene, and L1 is C 6~15 is a straight chain alkyl, and L2 is C 12~25 is a branched alkyl;

[0097] [ka] (2) A compound represented by formula (III), or an N-oxide, solvate, pharmaceutically acceptable salt, or stereoisomer thereof, wherein G1 is C 2~8 alkylene, and G2 is C 2~8 alkylene, L1 is -C(O)O- or -OC(O)-, L2 is -C(O)O- or -OC(O)-, and R1 is C 6~25 is a straight or branched chain alkyl; R2 is C 6~25 is a straight or branched chain alkyl, G3 is HO(CH2)2- or HO(CH2)3-, G4 is HO(CH2)2- or HO(CH2)3-, and L is (CH2)2- or -(CH2)3- or -(CH2)4-;

[0098] [ka] (3) A compound represented by formula (IV), or an N-oxide, solvate, pharmaceutically acceptable salt, or stereoisomer thereof, wherein G1 is C 1~6 alkylene, and G2 is C 2~8 alkylene, and R1 is C 6~20 is a straight or branched chain alkyl; R2 is C 12~25is a branched alkyl, and G3 is HO(CH2)2N(CH3)(CH2)2-, HO(CH2)2N(CH2CH3)(CH2)2-, (HO(CH2)2)2N(CH2)2-, CHO(CH2)2N(CH3)(CH2)2-, (CH3)2N(CH2)3SC(O)O(CH2)2-, (CH3)2N(CH2)3SC(O)-, CH3NH(CH2)2N(CH3)(CH2)2- or CH3CH2NH(CH2)2-;

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

[0100] [ka] (5) A compound represented by formula (VI), or an N-oxide, solvate, pharmaceutically acceptable salt, or stereoisomer thereof, wherein: 1 and G 2 are each independently unsubstituted C6 to C 10 alkylene, and G 3 is unsubstituted C1 to C 12 alkylene, and R 1 and R 2 are independently C6 to C 24 Alkyl or C6-C 24 alkenyl, and R 3 is OR 5 , N, -C(=O)OR 4 , -OC(=O)R 4 or -NR 5 C(=O)R4 and R 4 is C1~C 12 is a hydrocarbon group, and R 5 is H or a C1-C6 hydrocarbon group,

[0101] [ka] (6) A compound represented by formula (VII), or an N-oxide, solvate, pharmaceutically acceptable salt, or stereoisomer thereof, wherein R4 is -(CH2) n Q and -(CH2) n CHQR, where Q is -OR, -OH, -O(CH2) n n is selected from the group consisting of N(R), -OC(O)R, -CX, -CN, -N(R)C(O)R, -N(H)C(O)R, -N(R)S(O)R, -N(H)S(O)R, -N(R)C(O)N(R), -N(H)C(O)N(R), -N(H)C(O)N(H)(R), -N(R)C(S)N(R), -N(H)C(S)N(R), -N(R)S(O)R and heterocycle;

[0102] [ka] (7) A compound represented by formula (VIII), or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof.

[0103] [ka]

[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] [ka]

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

[0107] neutral lipid RNA delivery agents containing cationic lipids may also contain neutral lipids.In the present disclosure, neutral lipids refer to lipids that play an auxiliary role and are uncharged and exist in zwitterionic form at selected pH values.The neutral lipids can promote lipid phase transition, adjust the fluidity of nanoparticles to lipid bilayer structure, improve efficiency, and 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, such as 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 comprise one or more of phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, ceramide, sterol, and derivatives thereof.

[0110] The RNA delivery agent containing cationic lipid may contain one or more neutral lipids-phospholipids, such as one or more (poly)unsaturated lipids.The phospholipid can be formed into one or more lipid bilayers.Generally, the phospholipid may contain 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-lysophosphatidylcholine, 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, phytanic acid, arachidic acid, arachidonic acid, eicosapentaenoic acid, behenic acid, docosapentaenoic acid, and docosahexaenoic acid. Also included are non-naturally occurring species, including naturally occurring species with modifications and substitutions, including branching, oxidation, cyclization, and alkynes. For example, the phospholipids can be functionalized with one or more alkynes (e.g., alkenyl in which one or more double bonds are replaced with triple bonds) or cross-linked with one or more alkynes. Under appropriate reaction conditions, alkynyls can undergo copper-catalyzed cycloaddition reactions when exposed to azides. Such reactions can be used to functionalize the lipid bilayer of the composition to promote membrane permeability or cellular recognition, or to couple the composition to useful components such as targeting or imaging moieties (e.g., dyes).

[0112] Neutral lipids that can be used in such compositions include 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-diundecanoyl-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-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, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG), dipalmitoylphosphatidylglycerol (DPPG), palmitoyloleoylphosphatidylethanolamine (POPE), distearoyl-phosphatidyl The phosphatidylcholine may be selected from the non-limiting group consisting of 1-stearoyl-2-oleoyl-ethanolamine (DSPE), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphatidylethanolamine (DMPE), 1-stearoyl-2-oleoyl-stearoylethanolamine (SOPE), 1-stearoyl-2-oleoyl-phosphatidylcholine (SOPC), sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoylphosphatidylcholine, lysophosphatidylcholine, lysophosphatidylethanolamine (LPE), and mixtures thereof.

[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] structured lipids An RNA delivery agent comprising a cationic lipid may further comprise one or more structural lipids, which in the present disclosure refer to lipids that fill the spaces 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 structured lipid can be selected from the group consisting of, but not limited to, cholesterol, non-sterols, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatine, ursolic acid, α-tocopherol, corticosteroids, and mixtures thereof. In some embodiments, the structured lipid is cholesterol. In some embodiments, the structured lipid includes cholesterol and a corticosteroid (e.g., prednisolone, dexamethasone, prednisone, and hydrocortisone), or a combination thereof.

[0117] Polymer-conjugated lipids The cationic lipid-containing RNA delivery agent may further contain one or more polymer-conjugated lipids. Polymer-conjugated lipids primarily refer to polyethylene glycol (PEG)-modified lipids. Hydrophilic PEG stabilizes LNPs, restricts lipid fusion, regulates nanoparticle size, and reduces nonspecific interactions with macrophages, thereby increasing nanoparticle half-life.

[0118] In one embodiment, the polymer-conjugated lipid is one or more selected from the group consisting of PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, and PEG-modified dialkylglycerol. The molecular weight of the PEG-modified PEG is generally 350 to 5,000 Da.

[0119] For example, the polymer-conjugated lipid is one or more selected from distearoylphosphatidylethanolamine 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 comprises a neutral lipid, a structured lipid, and a polymer-conjugated lipid, and the molar ratio of the cationic lipid, the neutral lipid, the structured 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 comprises a neutral lipid, a structured lipid, and a polymer-conjugated lipid, wherein the molar ratio of the cationic lipid, the neutral lipid, the structured lipid, and the polymer-conjugated lipid is 49:10:39.5:1.5.

[0123] The following examples are provided to illustrate the present invention in detail, but those skilled in the art should understand that the following examples are merely illustrative of the present invention and should not be construed as limiting the scope of the present invention. Unless specific conditions are specified in the examples, they are carried out according to standard conditions or manufacturer's recommended conditions. Unless the manufacturers of the reagents or instruments used are specified, they are all commercially available general products.

[0124] The following abbreviations represent the following reagents: 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-butyldicarbonate; 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; TBA F: 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] [ka]

[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, and 2-iodoxybenzoic acid (104.9 g, 0.37 mol) was added. The mixture was heated to 70 °C and stirred for 2 h. The reaction was monitored for completion by TLC. The reaction was quenched, cooled to room temperature, filtered through diatomaceous earth, and the cake was rinsed with acetonitrile (500 mL). The filtrate was spun dry under reduced pressure and dried in vacuo to give INT-I-PM1 (102.1 g), a pale yellow liquid, which was used directly 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 stirred for 5 min. Allyltrimethylsilane (52.9 mL, 0.33 mol) was added dropwise and stirred for 2 h. The reaction was monitored for completion by TLC. The reaction solution was slowly poured into 1000 mL of saturated aqueous sodium bicarbonate solution to quench the reaction. The aqueous phase was extracted with dichloromethane (500 mL x 2). The organic phase was washed with saturated brine (500 mL x 2), dried over anhydrous sodium sulfate, and spin-dried. The residue was purified by normal-phase silica gel column chromatography to give INT-I-PM2 (103.0 g, 0.23 mol) as a pale yellow liquid.

[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, the mixture was allowed to warm to room temperature, and the reaction was stirred for 2 hours. The reaction completion was monitored by TLC. After completion of the reaction, the reaction solution was poured into 1000 mL of saturated aqueous sodium bicarbonate to quench the reaction. The reaction was separated, and the aqueous phase was extracted with dichloromethane (500 mL x 2). The combined organic phases were washed with saturated brine (1000 mL), dried over anhydrous sodium sulfate, and spin-dried to give a brown viscous material. The residue was purified by normal-phase silica gel column chromatography to give INT-I-PM3 (106.0 g, 0.20 mol, yield: 86.5%) as a pale yellow liquid.

[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, and 2.4 mL of concentrated sulfuric acid was added dropwise. The mixture was stirred at room temperature for 1 h and monitored for completion by TLC. After completion of the reaction, 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 combined organic phases were washed with water (800 mL x 3), saturated aqueous sodium bicarbonate, and saturated brine twice. The organic phases were dried over anhydrous sodium sulfate and spin-dried to give INT-I-PM4 (86.5 g, 0.15 mol, 75.0% yield) as a pale yellow liquid.

[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, followed by N,O-bis(trimethylsilyl)acetamide (61.00 g, 0.30 mol). The mixture was heated to 80°C and stirred to dissolve. After cooling to room temperature, INT-I-PM4 (86.5 g, 0.15 mol) dissolved in 150 mL of toluene was added and stirred for 5 minutes. Trimethylsilyl trifluoromethanesulfonate (40.00 g, 0.18 mol) dissolved in 50 mL of toluene was added dropwise, and the mixture was rapidly heated to 110°C and reacted for 3 hours. The reaction was monitored for completion by TLC. The reaction was stopped and the reaction mixture was cooled to room temperature. Then, ethyl acetate (300 mL) was added for extraction, washed with saturated aqueous sodium bicarbonate (500 mL), and the precipitated solid was filtered through diatomaceous earth. The filtrate was separated, and the aqueous phase was back-extracted with ethyl acetate (500 mL). The organic phases were combined, dried over anhydrous Na2SO4, and the solvent was spin-dried. The residue was purified by normal-phase silica gel column chromatography to give INT-I-PM5 (103.02 g, 0.15 mol, yield: 100%). 32 H 36 ClNOS, 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 and stirred to dissolve. Potassium carbonate (103.73 g, 0.75 mol) was added and the reaction was allowed to proceed at room temperature for 23 hours. The reaction was monitored for completion by TLC. The reaction was then quenched, filtered through diatomaceous earth, and the solvent was removed by spin-drying under reduced pressure. The mixture was extracted with ethyl acetate (800 mL) and water (500 mL), separated, and the aqueous phase was back-extracted with ethyl acetate (300 mL). The combined organic phases were washed once with saturated aqueous sodium chloride (600 mL), dried over anhydrous sodium sulfate, and the solvent was removed by spin-drying to give the crude product. INT-I-PM6 (44.03 g, 0.083 mol, 55.4% yield) was obtained by normal-phase silica gel column chromatography. 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 added sequentially. The reaction mixture was stirred overnight at 30 °C. TLC confirmed that the reaction was nearly complete, and the solvent was evaporated under reduced pressure. Purification by silica gel chromatography afforded INT-I (49.81 g, 68.25 mmol, 82.2% yield). 39 H 47 N5O9, MS(ES): m / z(M+H + )730.2.

[0134] 2. Synthesis of intermediate INT-II

[0135] [ka]

[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 nitrogen gas protection and reacted at 25 °C for 4 h. After reaction completion, the reaction solution was slowly added to a pre-cooled acetone solution containing sodium iodide (598.1 mg, 3.99 mmol) and crystallized at 25 °C for 30 min. After centrifugation, a white solid, INT-II (240.6 mg, 0.30 mmol, 80.9% yield), was obtained. 24 H 30 N 12 O13 P2, MS(ES): m / z(MH - ): 755.15.

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

[0138] [ka]

[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). N-methylmorpholine oxide (12.00 g, 102.37 mmol) and potassium osmate dihydrate (1.26 g, 3.41 mmol) were added to the mixture. The reaction was stirred at room temperature for 5 hours, and TLC showed the reaction was complete. The reaction was quenched with saturated sodium sulfite, extracted with ethyl acetate (500 mL x 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 give YK-CAP-001-PM1 (52.00 g, 68.08 mmol, yield: 99.8%). 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), and sodium periodate (21.84 g, 102.11 mmol) was weighed and added. The reaction was warmed to 30°C overnight, and TLC showed the reaction was complete. The reaction was quenched with saturated sodium sulfite and extracted with ethyl acetate (500 mL x 3). The organic phases were combined, 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. 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. The reaction was stirred at room temperature for 15 hours, and LCMS confirmed the reaction was complete. The reaction was diluted with 200 mL of dichloromethane, quenched with saturated aqueous ammonium chloride, separated, and the aqueous phase was extracted with dichloromethane (200 mL x 2). The combined organic phase was washed with 100 mL of saturated aqueous sodium chloride, dried over anhydrous sodium sulfate, filtered, and spin-dried. Purification by silica gel chromatography afforded YK-CAP-001-PM3 (10.80 g, 14.19 mmol, 67.9% yield). 40 H 52 N6O9, MS(ES): m / z(M+H + )761.3.

[0142] Step 4: Synthesis of YK-CAP-001-PM4 YK-CAP-001-PM3 (10.60 g, 13.93 mmol) was used as the starting material and dissolved in dichloromethane (300 mL). The reaction mixture was cooled to -40°C under a nitrogen 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 mixture was slowly warmed to 0°C and stirred at that temperature for 3 hours. TLC showed the reaction was complete, and the mixture was cooled again to -40°C. The reaction mixture was quenched with methanol, spin-dried, and left at room temperature for 24 hours to give 5.5 g of a brown crude product. 1.5 g was purified by preparative HPLC to give YK-CAP-001-PM4 (508.47 mg, 1.39 mmol). 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 under nitrogen gas protection, phosphorus oxychloride (1.69 g, 10.37 mmol) was slowly added dropwise. The reaction was allowed to proceed at 0°C for 4 hours. After the reaction was complete, 4.0 mL of purified water was slowly added in an ice bath and the mixture was stirred for 1 hour. Next, the mixture was washed three times with dichloromethane (4 mL x 3). The aqueous phase was separated and 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 a white solid, YK-CAP-001-PM5 triethylamine salt (260.0 mg, 0.47 mmol, yield: 45.8%).15 H 23 N6O8P, MS(ES): m / z(MH - ) 445.13.

[0145] Step 6: Synthesis of YK-CAP-001-PM6 intermediate YK-CAP-001-PM5 triethylamine salt (260.0 mg, 0.47 mmol), imidazole (384.0 mg, 5.64 mmol), dithiodipyridine (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. Triphenylphosphine (1479.3 mg, 5.64 mmol) was added under nitrogen gas protection and reacted at 25 °C for 4 hours. After completion of the reaction, the reaction solution was slowly added to a pre-cooled acetone solution containing sodium iodide (704.5 mg, 4.7 mmol) and crystallized at 25 °C for 30 minutes. After repeated centrifugation, a white solid, YK-CAP-001-PM6 (165 mg, 0.32 mmol, 67.0% yield), was obtained. 18 H 25 N8O7P, MS(ES): m / z(MH - ) 495.16.

[0146] Step 7: Synthesis of YK-CAP-001-PM7 intermediate 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 reacted at 25°C for 16 hours. After the reaction was complete, methyl tert-butyl ether was added and the mixture was stirred for 10 minutes. The mixture was then allowed to stand, the supernatant was removed, and the cloudy liquid at the bottom was collected. 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%). 15 H 24 NO 11 P2, MS(ES): m / z(MH- ) 525.10.

[0147] Step 8: Synthesis of YK-CAP-001-PM8 intermediate 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 the reaction was completed, 20 mL of purified water was added, and the aqueous phase was washed three times with dichloromethane (20 mL x 3). The aqueous phase was then separated and 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 resulting crude product was further purified by preparative liquid chromatography to obtain a white solid, YK-CAP-001-PM8 triethylamine salt (75 mg, 0.12 mmol, yield: 48.7%). 16 H 26 NO 11 P2, MS(ES): m / z(MH - ) 539.11.

[0148] Step 9: Synthesis of YK-CAP-001 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 the reaction was completed, 0.1 M EDTA was added until the solution became clear, and the mixture was diluted to 100 mL with water. The mixture was purified by gel column chromatography to obtain a white crude product (200.6 mg). The resulting crude product was further purified by preparative liquid chromatography to obtain a white solid, YK-CAP-001 (16.9 mg, 13.2 g, yield: 11.3%). 37 H 52 N 16 O 24 P4, MS(ES): m / z(MH - ) 1227.21.

[0149] 1H 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 P 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]

change

[0152] Synthesis of ステップ1:YK-CAP-002-PM1 YK-CAP-001-PM2 (22.0 g, 30.06 mmol) was dissolved in 440 mL of tetrahydrofuran and cooled to -10°C. 26% aqueous ammonia (250 mL) was added to the reaction mixture. The mixture was stirred at -10°C for 5 minutes, and then iodine element (23.0 g, 90.62 mmol) was added. The mixture was stirred at this temperature for 2 hours, then warmed to room temperature and stirred for an additional 15 minutes. The reaction mixture was quenched with saturated sodium thiosulfate solution, extracted with ethyl acetate, and 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%). 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 and cooled to -40°C. Under nitrogen gas protection, 1 M boron trichloride in dichloromethane (27.5 mL) was slowly added dropwise. After the addition was complete, the temperature was allowed to rise from room temperature to 0°C and the reaction was continued for 5 hours. Next, the reaction system was cooled again to -40°C and quenched by adding methanol. The reaction solution was spun dry under reduced pressure to obtain a crude product, which was purified by preparative high-pressure liquid chromatography to obtain YK-CAP-002-PM2 (490 mg, 1.47 mmol, yield: 26.8%). 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, the synthetic route of YK-CAP-001-PM5 was followed to obtain YK-CAP-002-PM3 triethylamine salt (1.07 g, 2.08 mmol). 13 H 15 N6O8P, MS(ES): m / z(MH - ) 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, YK-CAP-002-PM4 (578.5 mg, 1.19 mmol) was obtained according to the synthetic route of YK-CAP-001-PM6. 16 H 17 N8O7P, MS(ES): m / z(MH - ) 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, YK-CAP-002-PM5 (391.9 mg, 0.66 mmol, yield: 55.3%) was obtained according to the synthetic route of YK-CAP-001-PM7. 13 H 16 NO 11 P2, MS(ES): m / z(MH - )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, YK-CAP-002-PM6 (58 mg, 0.1 mmol, yield: 14.5%) was obtained according to the synthetic route of YK-CAP-001-PM8. 14 H 18 NO 11 P2, MS(ES): m / z(MH- ) 507.05.

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

[0160] 1 H 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 P 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] [ka]

[0163] Step 1: Synthesis of YK-CAP-003-PM1 YK-CAP-001-PM1 (22.00 g, 30.14 mmol) was dissolved in a mixture of dichloromethane (220 mL), acetonitrile (220 mL), and water (330 mL). Sodium periodate (52.06 g, 241.12 mmol) and ruthenium trichloride (250 mg, 1.21 mmol) were added to the solution and stirred at 30 °C for 48 hours. After the reaction was completed, the solvent was spin-dried, and ethyl acetate (400 mL) was added to dissolve the residue. The mixture 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%). 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 added to the solution, and the mixture was stirred overnight at room temperature. After completion of the reaction, the solvent was removed by spinning, and the mixture was dissolved in ethyl acetate (400 mL). The mixture was washed with purified water (3 x 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 give YK-CAP-003-PM2 (13.10 g, 16.91 mmol, yield: 63.6%). 40 H 50 NO 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 1 M boron trichloride dichloromethane solution (134 mL, 134 mmol) was slowly added dropwise at -40°C. After the addition was complete, the mixture was warmed to 0°C and stirred for 6 hours. After the reaction was complete, the mixture was cooled to -40°C and methanol (200 mL) was added to quench the reaction. The reaction solution was spun under vacuum to remove the solvent, and the mixture was left in a sealed environment at room temperature for 24 hours to obtain crude YK-CAP-003-PM3. This crude product was added dropwise to 500 mL of dichloromethane, precipitated, and filtered to obtain a total of 7.89 g of cake. 2.0 g was purified by HPLC to obtain pure YK-CAP-003-PM3 (956.04 mg, 2.51 mmol). 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 a raw material, YK-CAP-003-PM4 triethylamine salt (921.3 mg, 1.64 mmol, yield: 65.3%) was obtained according to the synthetic route of YK-CAP-001-PM5. 15 H 21 N6O9P, MS(ES): m / z(MH - )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, YK-CAP-003-PM5 (510.3 mg, 0.96 mmol, yield: 58.4%) was obtained according to the synthetic route of YK-CAP-001-PM6. 18 H 23 N8O8P, MS(ES): m / z(MH - ) 509.12.

[0169] Step 6: Synthesis of YK-CAP-003-PM6 intermediate YK-CAP-003-PM5 (510.3 mg, 0.96 mmol) was used as a raw material and YK-CAP-003-PM6 (853.8 mg, 1.33 mmol) was obtained according to the synthetic route of YK-CAP-001-PM7. 15 H 22 NO 12 P2, MS(ES): m / z(MH - ) 539.08.

[0170] Step 7: Synthesis of YK-CAP-003-PM7 intermediate YK-CAP-003-PM6 (853.8 mg, 1.33 mmol) was used as a raw material and YK-CAP-003-PM7 (170.0 mg, 0.26 mmol) was obtained according to the synthetic route of YK-CAP-001-PM8. 16 H 24 NO 12 P2, MS(ES): m / z(MH - ) 553.09.

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

change

[0175] Synthesis of ステップ1:YK-CAP-004-PM1 YK-CAP-001-PM2 (12.65 g, 17.29 mmol) was dissolved in dichloromethane (70 mL), the reaction mixture was cooled to -40 °C, and a dichloromethane solution of diethylaminosulfur trifluoride (8.36 g, 51.86 mmol) was slowly added dropwise. After the addition was complete, the reaction mixture was returned to 0 °C and reacted for 4 hours. Completion of the reaction was monitored by LCMS. The reaction mixture was quenched with saturated aqueous sodium bicarbonate solution and extracted with dichloromethane (200 mL x 3). The organic phases were combined and washed with 200 mL of saturated aqueous sodium chloride solution. The organic phase was 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%). 38 H 45 F2N5O9, MS(ES): m / z(M+H + )754.3.

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

[0177] 1H 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, YK-CAP-004-PM3 triethylamine salt (1.48 g, 2.74 mmol) was obtained according to the synthetic route of YK-CAP-001-PM5. 13 H 16 F2N5O8P, MS(ES): m / z(MH - ) 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, YK-CAP-004-PM4 (771.8 mg, 1.51 mmol) was obtained according to the synthetic route of YK-CAP-001-PM6. 16 H 18 F2N7O7P, MS(ES): m / z(MH - ) 488.08.

[0180] Step 5: Synthesis of YK-CAP-004-PM5 intermediate YK-CAP-004-PM4 (771.8 mg, 1.51 mmol) was used as a raw material and YK-CAP-004-PM5 (1.22 g, 1.97 mmol) was obtained according to the synthetic route of YK-CAP-001-PM7. 13 H 17 F2N5O 11 P2, MS(ES): m / z(MH - )518.04.

[0181] Step 6: Synthesis of YK-CAP-004-PM6 intermediate YK-CAP-004-PM5 (1.22 g, 1.97 mmol) was used as a raw material and YK-CAP-004-PM6 (172.6 mg, 0.27 mmol) was obtained according to the synthetic route of YK-CAP-001-PM8. 14 H 19 F2N5O 11 P2, MS(ES): m / z(MH - ) 532.05.

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

[0183] 1 H 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] [ka]

[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 and cooled to 0 °C under nitrogen gas protection with stirring. Pyridine (23.9 g, 0.30 mol) was added, followed by dropwise addition of tert-butyldimethylsilyl trifluoromethanesulfonate (40.0 g, 0.15 mol). After the addition was complete, the mixture was warmed to room temperature and allowed to react. The completion of the reaction was monitored by TLC. The reaction mixture was directly spun dry 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 470 mL of a tetrahydrofuran / 47 mL water mixture, followed by the addition of N-methylmorpholine-N-oxide (14.9 g, 0.13 mol) and potassium osmate (1.3 g, 0.003 mol). The mixture was allowed to react at room temperature for 5 hours. The reaction was monitored for completion 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 x 2). The combined organic phases were washed sequentially with saturated brine (300 mL), dried over anhydrous sodium sulfate, and spun to dryness under reduced pressure to obtain crude YK-CAP-005-PM2 (53.2 g, 90.35 mmol) as a pale yellow liquid.

[0188] Step 3: Synthesis of YK-CAP-005-PM3 The crude YK-CAP-005-PM2 product (53.2 g, 90.35 mmol) was dissolved in 500 mL of a tetrahydrofuran / 100 mL water mixture, and sodium periodate (29.0 g, 0.14 mol) was added again. The reaction was allowed to proceed 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 x 2). The combined organic phases were washed sequentially with saturated brine (400 mL), dried over anhydrous sodium sulfate, and spun to dryness under reduced pressure 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 and cooled to 0 °C under nitrogen gas protection with stirring. Sodium borohydride (3.86 g, 0.10 mol) was added in batches. After the addition was complete, the reaction was continued for 3 hours. The completion of the reaction was monitored by TLC. The reaction was cooled to 0 °C and quenched by adding saturated aqueous ammonium chloride solution (300 mL). The reaction was further extracted with ethyl acetate (400 mL × 3). The combined organic phases were washed successively with saturated brine (400 mL), dried over anhydrous sodium sulfate, and spun to dryness under reduced pressure to obtain crude product YK-CAP-005-PM4 (49.0 g, 0.088 mol) as a yellow oil.

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

[0191] Step 6: Synthesis of YK-CAP-005-PM6 The crude YK-CAP-005-PM5 product (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 reaction was allowed to proceed at room temperature for 4 hours until completion. The completion of the reaction was monitored by TLC. The reaction solution was slowly poured into 1500 mL of water, and the reaction mixture was extracted with ethyl acetate (500 mL x 3). The combined organic phases were washed sequentially with saturated brine (1500 mL), dried over anhydrous sodium sulfate, and spun to dryness under reduced pressure to yield a brown oil, 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 and cooled to 0 °C under nitrogen gas protection with stirring. m-Chloroperoxybenzoic acid (33.7 g, 0.19 mol) was added in batches. After the addition was complete, the mixture was warmed to room temperature and allowed to react for 3 h until the starting material disappeared. The reaction was monitored by TLC for completion. The reaction solution was poured into saturated aqueous sodium bicarbonate (1500 mL) and extracted with ethyl acetate (500 mL x 3). The organic phase was washed with saturated brine (1500 mL), dried over anhydrous sodium sulfate, and spin-dried under reduced pressure to give a yellow oil, YK-CAP-005-PM7 (52.6 g, 84.72 mmol, 99.8% yield).

[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 mixture was allowed to react at room temperature for 1 hour. The reaction was monitored for completion by TLC. 1000 mL of purified water and 800 mL of ethyl acetate were added to the reaction mixture, stirred for 5 minutes, and the mixture was separated. The aqueous phase was extracted with ethyl acetate (500 mL x 2). The combined organic phases were washed sequentially with saturated brine (1000 mL), dried over anhydrous sodium sulfate, and spin-dried under reduced pressure. The mixture was purified by FLASH to yield a yellow oil, 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 and triethylamine (11.2 g, 0.11 mol) was added. Under nitrogen gas protection, the mixture was stirred and cooled to 0 °C, and ethanesulfonyl chloride (22.5 g, 0.18 mol) was added dropwise. After the addition was complete, the mixture was warmed to room temperature and reacted for 3 hours. The reaction was monitored for completion by TLC. 400 mL of saturated aqueous sodium bicarbonate solution was added to the reaction mixture, and the mixture was separated. The aqueous phase was extracted with dichloromethane (200 mL x 2). The organic phase was washed with saturated brine (400 mL), dried over anhydrous sodium sulfate, and spin-dried under reduced pressure. The mixture was purified by FLASH to give YK-CAP-005-PM9 (13.4 g, 22.38 mmol, 60.6% yield) as a pale yellow oil.

[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 added. Concentrated sulfuric acid (0.27 mL) was then added dropwise. After the addition was complete, the mixture was allowed to react at room temperature for 2 hours until the starting materials had disappeared. The reaction was monitored by TLC. 400 mL of purified water and 400 mL of ethyl acetate were added to the reaction mixture, and the mixture was stirred for 5 minutes and allowed to separate. The aqueous phase was extracted with ethyl acetate (300 mL x 2). The combined organic phases were then adjusted to alkaline pH with saturated aqueous sodium bicarbonate, washed with saturated brine (1.0 L), dried over anhydrous sodium sulfate, and spun dry under reduced pressure to obtain a yellow oil, YK-CAP-005-PM10 (13.4 g, 20.85 mmol, 93.18% yield).

[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, and the mixture was heated to 80 °C until the reaction mixture became clear and dissolved. The heat was removed, the mixture was cooled to room temperature, and a toluene solution (50 mL) of compound YK-CAP-005-PM10 (12.8 g, 19.92 mmol) was added under stirring. Trimethylsilyl trifluoromethanesulfonate (6.6 g, 0.03 mol) was added dropwise, the mixture was heated to reflux, and the mixture was stirred for 2 hours until the raw materials had disappeared. The reaction was monitored by TLC. The reaction was cooled to room temperature and quenched by the addition of saturated sodium bicarbonate solution (300 mL), followed by the addition of ethyl acetate (300 mL), filtered through diatomaceous earth, separated, the aqueous phase extracted with ethyl acetate (200 mL × 2), the combined organic phase washed sequentially with saturated brine (300 mL × 1), dried over anhydrous sodium sulfate, and spin-dried to give a 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 added and reacted at room temperature. The completion of the reaction was monitored by TLC. After completion of the reaction, the reaction system was directly spin-dried under reduced pressure and purified by FLASH to obtain a yellow solid, YK-CAP-005-PM12 (11.8 g, 19.81 mmol). 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 and cooled to -40 °C under nitrogen gas protection. 1 M boron trichloride in dichloromethane (167 mL) was added dropwise. After the addition was complete, the reaction mixture was warmed to 0 °C and reacted. The reaction was monitored by LC-MS. After the reaction was complete, the reaction mixture was cooled to -40 °C and quenched by adding methanol (200 mL). The mixture was then directly spun dry under reduced pressure. 120 mL of 4 M hydrochloric acid in 1,4-dioxane was added and the mixture was heated to 60 °C until the reaction was complete. After the reaction was completed, the reaction system was spun dry under reduced pressure, and methanol (40 mL) was added to dissolve the product. The solid was then added dropwise to 800 mL of dichloromethane to precipitate the product. The product was then suction filtered and dried to obtain a yellow solid, YK-CAP-005-PM13 (7.0 g, 17.44 mmol, yield: 88.1%). 14 H 19 N5O7S, MS(ES): m / z(M+H + )402.18.

[0199] 1H 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 a raw material, YK-CAP-005-PM14 triethylamine salt (639.8 mg, 1.10 mmol, yield: 13.0%) was obtained according to the synthetic route of YK-CAP-001-PM5. 14 H 20 N5O 10 PS, MS(ES): m / z(MH - ) 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 a raw material, YK-CAP-004-PM15 (333.4 mg, 0.60 mmol, yield: 54.9%) was obtained according to the synthetic route of YK-CAP-001-PM6. 17 H 22 N7O9PS, MS(ES): m / z(MH - )530.08.

[0202] Step 16: Synthesis of YK-CAP-005-PM16 intermediate YK-CAP-005-PM15 (333.4 mg, 0.60 mmol) was used as a raw material and YK-CAP-005-PM16 (528 mg, 0.80 mmol) was obtained according to the synthetic route of YK-CAP-001-PM7. 14 H 21 N5O 13P2S, MS(ES): m / z(MH - )560.03.

[0203] Step 17: Synthesis of YK-CAP-005-PM17 intermediate YK-CAP-005-PM16 (528 mg, 0.80 mmol) was used as a raw material and YK-CAP-005-PM17 (74.7 mg, 0.11 mmol) was obtained according to the synthetic route of YK-CAP-001-PM8. 15 H 23 N5O 13 P2S, MS(ES): m / z(MH - ) 574.05.

[0204] Step 18: Synthesis of YK-CAP-005 YK-CAP-005-PM17 (74.7 mg, 0.11 mmol) was used as a starting material and YK-CAP-005 (19.3 mg, 14.7 μmol, 13.3%) was obtained according to the synthetic route of YK-CAP-001. 36 H 49 N 15 O 26 P4S, MS(ES): m / z(MH - ) 1262.14.

[0205] 1 H 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] [ka]

[0208] Step 1: Synthesis of YK-CAP-006-PM1 Triphenylphosphine (11.27 g, 42.98 mmol) was dissolved in 150 mL of tetrahydrofuran, the reaction mixture was cooled to 0 °C, diisopropyl azodicarboxylate (14.48 g, 71.64 mmol) was added, and the mixture was stirred for 10 minutes. A solution of compound YK-CAP-005-PM4 (20.0 g, 35.79 mmol) in tetrahydrofuran was added, and thioacetic acid (5.45 g, 71.64 mmol) was slowly added dropwise. After the addition was complete, the mixture was slowly returned to room temperature and stirred for 16 hours. The reaction was monitored for completion by TLC. After completion of the reaction, the reaction mixture was quenched with water, and the aqueous phase was extracted with ethyl acetate (200 mL × 2). The combined organic phases were washed with saturated sodium chloride, dried over anhydrous sodium sulfate, filtered, and spun dry. Purification by FLASH gave 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 aqueous hydrochloric acid. Compound YK-CAP-006-PM1 (14.4 g, 23.34 mmol) in acetonitrile was slowly added dropwise and allowed to react for 10 minutes. The reaction was monitored for completion by LCMS. 30 mL of water was added to the reaction mixture to quench the reaction. The aqueous phase was extracted with ethyl acetate (100 mL x 2). The combined organic phases were washed with saturated sodium chloride, dried over anhydrous sodium sulfate, filtered, and spin-dried. A colorless oily liquid, YK-CAP-006-PM2 (13.4 g, 20.90 mmol, yield: 89.5%), was obtained.

[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, and triethylamine (6.35 g, 62.79 mmol) was weighed and added. The reaction mixture was cooled to 0 ° C., and a dichloromethane solution of compound YK-CAP-006-PM2 (13.40 g, 20.90 mmol) was slowly added dropwise. After the addition was complete, the reaction was carried out at 0 ° C. for 30 minutes, and the completion of the reaction was monitored by LCMS. The reaction mixture 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, and tetrabutylammonium fluoride / tetrahydrofuran (11.09 mL, 11.09 mmol) was slowly added. The reaction was allowed to proceed 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, and the aqueous phase was extracted with ethyl acetate (100 mL x 2). The combined organic phases were washed sequentially with saturated brine (50 mL), dried over anhydrous sodium sulfate, and spin-dried under reduced pressure. The mixture was 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, triethylamine (2.27 g, 22.41 mmol) was added, the reaction mixture 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 mixture was incubated at 0 °C for 2 hours. The completion of the reaction was monitored by TLC. The reaction mixture was quenched by slowly pouring the reaction mixture into 50 mL of saturated aqueous sodium bicarbonate solution. Ethyl acetate was added to separate the mixture, and the mixture was extracted with ethyl acetate (150 mL x 2). The combined organic phases were washed sequentially with saturated brine (500 mL), dried over anhydrous sodium sulfate, and spun dry under reduced pressure. The mixture was 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, and acetic anhydride (4.3 g, 42.12 mmol) was added. A solution of concentrated sulfuric acid (44 μL) in acetic acid was slowly added dropwise at room temperature under stirring conditions. 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 bicarbonate solution to adjust the pH to neutral, and extracted with ethyl acetate (300 mL x 3). The combined organic phases were washed with 50 mL of saturated brine, dried over anhydrous sodium sulfate, filtered, and spun to dryness under reduced pressure to obtain 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, and N,O-bis(trimethylsilyl)acetamide (2.07 g, 10.16 mmol) was added. Under nitrogen gas protection, the reaction mixture was heated to 80 °C and stirred until the solution became clear. The heating was stopped and the mixture was returned to room temperature. A toluene solution of compound YK-CAP-006-PM6 (3.1 g, 4.61 mmol) was slowly added, followed by trimethylsilyl trifluoromethanesulfonate (1.13 g, 5.08 mmol). The mixture was immediately heated to 110 °C and reacted for 2 h. Completion was monitored by LCMS. Water (15 mL) and ethyl acetate (20 mL) were added to the reaction mixture. A solid precipitated from the reaction mixture. Diatomaceous earth was added, and the mixture was filtered. 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, spin-dried under reduced pressure, 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 allowed to proceed at room temperature for 24 hours. The reaction was monitored for completion by LCMS. After filtration, the filtrate was stirred and purified by FLASH to give colorless oil YK-CAP-006-PM8 (2.1 g, 3.36 mmol, 87.5% yield).

[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 and cooled to 0 °C under nitrogen gas protection with stirring. Boron trichloride (3.94 g, 33.60 mmol) was added dropwise slowly. 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 was cooled to -40 °C and quenched with methanol. The mixture was spun dry and the target compound was monitored by LCMS. The product was dissolved in 20 mL of methanol and added dropwise slowly to 150 mL of stirred dichloromethane. A white solid precipitated, yielding 1.7 g of crude product. The crude product was purified by preparative high-pressure liquid chromatography to yield 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 Using YK-CAP-006-PM9 (1.01 g, 2.35 mmol) as a raw material, YK-CAP-006-PM10 triethylamine salt (1.05 g, 1.72 mmol, yield: 73.2%) was obtained according to the synthetic route of YK-CAP-001-PM5. 15 H 23 NO 10 PS, MS(ES): m / z(MH - ) 509.09.

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

[0220] Step 12: Synthesis of YK-CAP-006-PM12 Using YK-CAP-006-PM11 (809.2 mg, 1.39 mmol) as a raw material, YK-CAP-006-PM12 triethylamine salt (475.5 mg, 0.69 mmol, yield: 49.5%) was obtained according to the synthetic route of YK-CAP-001-PM7. 15 H 24 NO 13 P2S, MS(ES): m / z(MH - ) 589.06.

[0221] Step 13: Synthesis of YK-CAP-006-PM13 Using YK-CAP-006-PM12 triethylamine salt (475.5 mg, 0.69 mmol) as a raw material, YK-CAP-006-PM13 triethylamine salt (235 mg, 0.33 mmol, yield: 48.4%) was obtained according to the synthetic route of YK-CAP-001-PM8. 16 H 26 NO13 P2S, MS(ES): m / z(MH - ) 603.08.

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

[0223] 1 H 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 P 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, and compared with the mRNA capping analogs disclosed in the prior art, some of these compounds in this series have similar structures and some have very significant differences.

[0225] The modified locked nucleoside capping analogs of the present application showed significant differences in mRNA in vitro transcription yield and capping rate. 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 significant improvements in both mRNA in vitro transcription yield and capping rate. For example, the transcription yield and capping rate of YK-CAP-004 were 35.0% and 18.8%, respectively, higher than those of compound 14.

[0226] I. Structural Differences of Capping Analogues

[0227] [Table 1] TIFF0007788017000023.tif197159TIFF0007788017000024.tif150159

[0228] As can be seen from Table 1, the chemical structures of the compounds YK-CAP-001 to 006 of the present application are very similar, and compared with the mRNA capping analogs disclosed in the prior art, some of these compounds in this series have similar structures and some have very significant differences, as shown below. 1. The structures of compounds YK-CAP-001, YK-CAP-002, YK-CAP-003, YK-CAP-004, YK-CAP-005 and YK-CAP-006 of the present application are very similar, with the only difference being the group linked to the methylene bridge connecting the 2'-O of the ribose ring to C4', i.e., the substituent on C6': 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, while the other structures are exactly the same.

[0229] 2. Compounds YK-CAP-001 to 006 of the present application are structurally similar to compound 14, but the only difference is that compound 14 has no substituent on the methylene bridge connecting the 2'-O and C4' of the ribose ring, i.e., no substituent on C6'.

[0230] 3. The compounds YK-CAP-001 to 006 of the present application have larger structural differences from N-7413, HN3002 and m6A, as these three compounds lack a methylene bridge between the 2'-O and C4' of the ribose ring, and 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 analogues First, the plasmid was linearized using a plasmid linearizing 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 used as capping analogs, and the reaction system was as shown in Table 2.

[0233] [Table 2]

[0234] During the experiment, the volume of materials required for the reaction was calculated and then the sample was added. Sterile water was added to the reaction system, followed by 10x buffer, NTPs, and capping analogs. After uniform mixing, the mixture was gently centrifuged. Nuclease inhibitors, inorganic pyrophosphatase, T7 RNA polymerase, and linearized DNA template were then added. After thorough mixing, the mixture was gently centrifuged and incubated at 37°C. After 2 hours, 1 U of DNase I was added. The mixture was then incubated at 37°C for 30 minutes. The mRNA precipitate was then washed with 75% ethanol, allowed to evaporate, and allowed to dry. The mRNA was then redissolved in sterile 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 a probe.

[0237] Annealing was carried out in a PCR machine at 95°C for 5 min, 65°C for 2 min, 55°C for 2 min, 40°C for 2 min, and 22°C for 2 min.

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

[0239] (6) mRNA is cleaved and the resulting mRNA 5 is bound to the probe. , The single-stranded sequence was obtained.

[0240] 20 μL of RNase H (5 U / μL) was added and the mixture was incubated at 37°C for 3 hours, mixing every 30 minutes. After incubation, the magnetic beads were washed. 100 μL of 75% methanol preheated to 80°C was added to the washed beads. The mixture was heated to 80°C on a heating plate for 3 minutes, then placed on a magnetic stand. The supernatant was aspirated and dried to 10 μL using an evaporative centrifuge at room temperature for 45 minutes. The sample was then resuspended in 50 μL of 100 μM EDTA / 1% MeOH and used for LC-MS analysis to determine the capping status of RNA during the transcription reaction. Because capping and non-capping bases have different molecular weights, the molecular weight difference could be used to determine the capping rate of mRNA transcription initiated by different capping analogs.

[0241] 2. Experimental Results Measurement results of mRNA in vitro transcription yield and capping rate showed that the modified locked nucleoside capping analogs of the present application had significant differences in mRNA in vitro transcription yield and capping rate. 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 exhibited significantly improved mRNA in vitro transcription yield and capping rate. For example, the transcription yield and capping rate of YK-CAP-004 were 35.0% and 18.8%, respectively, higher than those of compound 14.

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

[0243] [Table 3]

[0244] 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. YK-CAP-004 had the highest transcription yield and capping rate, with the transcription yield being 82.8% higher than the lowest YK-CAP-001 and the capping rate being 26.1% higher than the lowest YK-CAP-002.

[0245] As can be seen from Table 3, all of the modified locked nucleoside capping analogs of the present application were able to transcribe mRNA. The difference in mRNA transcription activity between different modified locked nucleoside capping analogs was quite significant, with YK-CAP-004 having the highest in vitro mRNA transcription yield, reaching 163.2 μg, 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 exceeding 150 μg, all of which were very high.

[0246] The lowest mRNA in vitro transcription yield was YK-CAP-001, at only 89.3 μg, and YK-CAP-002 also had a very low yield of only 103.1 μg. The transcription yield of YK-CAP-004 was significantly higher than that of YK-CAP-001, at 82.8% and 58.3%, respectively (Figure 1). The highest capping rate was YK-CAP-004, reaching 96.3%, followed by YK-CAP-003, YK-CAP-005, and YK-CAP-006, which were 94.8%, 95.2%, and 95.1%, respectively, all exceeding 90%, making them extremely high.

[0247] The lowest capping rate was YK-CAP-002 at just 70.2%, and the capping rate of YK-CAP-001 was also very low at just 73.1%. The capping rates of YK-CAP-004 were significantly higher than those of YK-CAP-002 and YK-CAP-001, at 26.1% and 23.2%, respectively (Figure 2). 2) The modified locked nucleoside capping analogs of the present application significantly improved both the mRNA in vitro transcription yield and capping rate 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 35.0% and 18.8%, respectively, higher than those of compound 14.

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

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

[0250] 3) Modified locked nucleoside capping analogs with similar structures showed large differences in mRNA in vitro transcription yield and capping rate.

[0251] The structures of each modified locked nucleoside capping analog designed in this application were very similar, and this series of compounds was very similar to the structure of compound 14. However, there were significant differences in the mRNA in vitro transcription yield and capping rate.

[0252] For example, compared with YK-CAP-001, YK-CAP-002, and compound 14, YK-CAP-004 differs only in the group attached to the methylene bridge connecting the 2'-O of the ribose ring to the C4', i.e., the substituent at C6': YK-CAP-004 is 2,2-difluoroethyl, YK-CAP-001 is dimethylaminoethyl, and YK-CAP-002 is cyanomethyl, whereas compound 14 has no substituent and the other structures are identical. However, the mRNA in vitro 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 significantly improved by 23.2%, 26.1%, and 18.8%, respectively.

[0253] This indicates that modified locked nucleoside capping analogs with similar structures do not necessarily have similar mRNA transcription activity and capping rate, and that there is a very high possibility that there may be significant differences.

[0254] As can be seen from the mRNA in vitro 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 exhibited significantly improved mRNA in vitro transcription yield and capping rate compared to YK-CAP-001 and YK-CAP-002 of the present application or to 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, introducing 2,2-difluoroethyl (YK-CAP-004), dimethylaminocarbonylmethyl (YK-CAP-003), methylsulfonylethyl (YK-CAP-005), and dimethylaminosulfonylethyl (YK-CAP-006), respectively, into the methylene bridge (i.e., C6') connecting the 2'-O and 4'-C of the ribose ring. Since the bridged ring structure cannot be used as the initiation site of transcription, they have excellent reverse transcription prevention effects during in vitro mRNA transcription, significantly increase the binding ability of the cap structure with the capping enzyme, and increase 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 activity and capping rate, and on the contrary, there is a very high possibility that there will be very large differences.

[0257] Example 3: Preparation and characterization of lipid nanoparticles 1. Experimental Method 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. The mRNA was diluted to pH 4 with 50 mM citrate buffer. The ethanolic lipid solution and the aqueous Fluc mRNA solution prepared with the different capping structures were mixed at a volume ratio of 1:3 using a microfluidic device at a flow rate of 10 mL / min to produce LNPs with a total lipid to mRNA weight ratio of approximately 15:1. The resulting liposomes were diluted 10 times with PBS and then ultrafiltered using a 300 kDa ultrafiltration tube to remove the ethanol. Next, the mixture 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 in YK-009 / DSPC / cholesterol / DMG-PEG2000 (molar ratio 49:10:39.5:1.5).

[0258] Polydispersity index (PDI) was measured using dynamic light scattering with a Malvern laser analyzer. 10 μL of liposome solution was diluted to 1 mL with RNAse-free deionized water and added to the sample pool. Each sample was measured in triplicate. Measurement conditions were a 90° scattering angle and 25°C. Lipid nanoparticle encapsulation efficiency was determined using the Quantit Ribogreen RNA Quantification Kit (Thermo Fisher Scientific, UK) according to the manufacturer's instructions.

[0259] 2. Experimental Results Specific property data of 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 YK-CAP-006 of the present application and the Fluc mRNA transcribed from the capping analogs N-7413, compound 14, HN3002, and m6A disclosed in the prior art all produced good lipid nanoparticles. The particle size of all lipid nanoparticles was 82 to 103 nm, the PDI value was 0.018 to 0.065, and the encapsulation efficiency was all above 90%.

[0262] Example 4: Translation efficiency of differently capped luciferase mRNAs The modified locked nucleoside capping analogs of the present application showed significant differences in mRNA translation efficiency. YK-CAP-004 had the highest translation efficiency, twice that of YK-CAP-002, which had the lowest. Compared with modified locked nucleoside capping analogs of the prior art that have similar or significantly 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 was four times that of m6A.

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

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

[0265] (3) When the cell density reached approximately 80%, transfection was performed by adding 0.5 μg of mRNA sample and Lipofectamine MessengerMAX Transfection Reagent (Invitrogen) to each well and following the transfection steps in the manufacturer's instructions.

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

[0267] (5) Take 20 μL of sample and add 100 μL of Dual-LumiNova solution equilibrated to room temperature. TM II firefly luciferase detection reagent was added and mixed appropriately to homogeneity.

[0268] (6) The plate was incubated at room temperature (approximately 25°C) for 5 minutes to stabilize the luminescence signal. Chemiluminescence detection was performed using a multifunctional microplate reader equipped with chemiluminescence detection capabilities, and the data were recorded. The relative fluorescence readings for specific capped mRNAs are shown in Table 5.

[0269] 2. Experimental Results The relative fluorescence readings of the capped 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) The modified locked nucleoside capping analogs of the present application showed significant differences in mRNA translation efficiency. YK-CAP-004 had the highest translation efficiency, which was twice as high as that of YK-CAP-002, the lowest.

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

[0273] The lowest relative fluorescence intensity was YK-CAP-002, at just 0.69. The relative fluorescence intensity of YK-CAP-001 was also very low, at just 0.78. The relative fluorescence intensities of YK-CAP-004, YK-CAP-003, YK-CAP-005, and YK-CAP-006 were 2.0-fold, 1.8-fold, 1.9-fold, and 1.9-fold that of YK-CAP-002, and 1.8-fold, 1.6-fold, 1.6-fold, and 1.7-fold that of YK-CAP-001, respectively (Figure 3). 2) The modified locked nucleoside capping analogs of the present application significantly improved both mRNA translation efficiency and mRNA translation efficiency compared to conventional modified locked nucleoside capping analogs. For example, the translation efficiency of YK-CAP-004 was four times higher than that of m6A.

[0274] The relative fluorescence intensities (corresponding to 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-fold, 1.2-fold, 1.3-fold, and 1.3-fold that of N-7413, 1.2-fold, 1.1-fold, 1.1-fold, and 1.2-fold that of HN3002, and 4.0-fold, 3.5-fold, 3.7-fold, and 3.7-fold that of m6A, respectively.

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

[0276] Although the modified locked nucleoside capping analogs designed in this application are very similar in structure, there were significant differences in mRNA translation efficiency.

[0277] For example, compared with YK-CAP-001 and YK-CAP-002, YK-CAP-004 differs only in the group attached to the methylene bridge connecting the 2'-O of the ribose ring to the C4', i.e., the C6' substituent, which is 2,2-difluoroethyl in YK-CAP-004, dimethylaminoethyl in YK-CAP-001, and cyanomethyl in YK-CAP-002, while the other structures are identical. However, the translation efficiency of YK-CAP-004 mRNA was significantly improved, being 1.8- and 2.0-fold higher than that of YK-CAP-001 and YK-CAP-002, respectively.

[0278] This indicates that the luciferase mRNA translation efficiencies of modified locked nucleoside capping analogs with similar structures are not necessarily similar, and there is a very high possibility that there may be significant differences.

[0279] As can be seen from the translation efficiency of differently capped 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, all showed significantly improved mRNA translation efficiency compared to structurally similar modified locked nucleoside capping analogs (including YK-CAP-001 and YK-CAP-002 of the present application) or structurally significantly different modified locked nucleoside capping analogs (including N-7413, HN3002, and m6A). This indicates that bridged 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 five-membered sugar ring structure, more easily bind to the cap-binding protein (EIF4E), and improve the translation efficiency of target mRNA.

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

[0281] Example 5. Decapping enzyme stability test The modified locked nucleoside capping analogs of the present application had significantly different decapping rates. YK-CAP-004 had the lowest decapping rate, 16.0% lower than the highest YK-CAP-001. Compared with modified locked nucleoside capping analogs of the prior art with similar or significantly different structures, the decapping rates of the compounds of the present application were significantly lower. For example, the decapping rate of YK-CAP-004 was 38.3% lower than that of N-7413.

[0282] 1. Experimental Method Thirty pmol of PAGE-purified RNA was subjected to enzyme-catalyzed reaction with 50 U mRNA Decapping Enzyme (New England Biolabs) in 1x MDE buffer at 37°C for 45 minutes. The enzyme-catalyzed reaction mixture was stained with SYBR Green II (Lonza) for PAGE electrophoresis, and the gel pattern after electrophoresis was observed on a Typhoon FLA 7000 (GE Healthcare) instrument. The electrophoretic band intensity ratio of capped RNA to uncapped RNA was calculated using Image Quant (GE Healthcare) software, 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 the present application had significant differences in decapping rates. YK-CAP-004 had the lowest decapping rate, 16.0% lower than the highest rate of YK-CAP-001.

[0286] As can be seen from the data in Table 6, the modified locked nucleic acid cap analogs of the present application, YK-CAP-001, YK-CAP-002, YK-CAP-003, YK-CAP-004, YK-CAP-005, and YK-CAP-006, had very different decapping rates. The lowest decapping rate was YK-CAP-004, at only 10.3%, while 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 was 26.3%, the highest, and that of YK-CAP-002 was also relatively high at 24.6%. The decapping rates of YK-CAP-004, YK-CAP-003, YK-CAP-005, and YK-CAP-006 were 16.0%, 14.5%, 13.5%, and 15.1% lower than that of YK-CAP-001, and 14.3%, 12.8%, 11.8%, and 13.4% lower than that of YK-CAP-002, respectively.

[0288] 2) Compared with modified locked nucleoside capping analogs of the prior art with similar or significantly different structures, the decapping rate of the modified locked nucleoside capping analogs of the present application was significantly reduced, for example, the decapping rate of YK-CAP-004 was 38.3% lower than that of 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 the present application were reduced by 38.3%, 36.8%, 35.8%, and 37.4% compared to N-7413, 18.5%, 17.0%, 16.0%, and 17.6% compared to compound 14, 12.9%, 11.4%, 10.4%, and 12.0% compared to HN3002, and 17.2%, 15.7%, 14.7%, and 16.3% compared to m6A, respectively.

[0290] 3) The decapping rates of modified locked nucleoside capping analogs with similar structures were significantly different.

[0291] Although the structures of each modified locked nucleoside capping analog designed in this application are very similar, and this series of compounds is very similar to the structure of compound 14, there were very large differences in the decapping rates.

[0292] For example, compared with YK-CAP-001, YK-CAP-002, and compound 14, YK-CAP-004 differs only in the group attached to the methylene bridge connecting the 2'-O of the ribose ring to C4', i.e., the substituent at C6': YK-CAP-004 is 2,2-difluoroethyl, YK-CAP-001 is dimethylaminoethyl, and YK-CAP-002 is cyanomethyl, whereas compound 14 has no substituent and the other structures are identical. However, the mRNA decapping rate of YK-CAP-004 was significantly reduced by 16.0%, 14.3%, and 18.5% compared to YK-CAP-001, YK-CAP-002, and compound 14, respectively.

[0293] This indicates that modified locked nucleoside capping analogs with similar structures do not necessarily have similar m decapping rates, and that there is a very high possibility that there may be 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, showed a marked decrease in both DCP2 enzyme decapping rate and DCP2 enzyme decapping rate, compared to 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 significantly different structures (including N-7413, HN3002, and m6A).

[0295] Furthermore, it was found that modified locked nucleoside capping analogs with similar structures do not necessarily have similar m decapping rates, but rather are very likely to have very large differences.

[0296] Example 6: Animal Experiments Furthermore, we investigated the in vivo protein expression level and duration of Fluc mRNA produced from different modified locked nucleoside cap analogs in mice. The results showed that the modified locked nucleoside cap analogs of the present application had significant differences in the average radiation intensity (corresponding to protein expression level) and duration of protein expression in mice. YK-CAP-004 had the highest fluorescence expression intensity, 2.6-fold and 5.5-fold higher than the lowest YK-CAP-002 at 6 and 96 hours, respectively. Compared with modified locked nucleoside cap analogs of the prior art with similar or significantly different structures, the modified locked nucleoside cap analogs of the present application showed significantly improved in vivo protein expression level and duration of mRNA expression in mice. For example, the average radiation intensity of YK-CAP-004 was 2.1-fold higher than that of m6A at 6 hours and 3.4-fold higher at 96 hours.

[0297] 1. Experimental Method 5 μg of an LNP formulation containing the capping analogs YK-CAP-001 to 006 of the present application, the conventional capping analog N-7413, 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, 12, 24, 48, 96, and 168 hours) after administration, the mice were intraperitoneally injected with a fluorescent imaging substrate. 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 encapsulated in the LNPs 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 FIG.

[0299] [Table 7]

[0300] 1) The present invention demonstrated that the modified locked nucleoside capping analogs significantly differed in the mean radiation intensity and duration of protein expression in mice. YK-CAP-004 had the highest mean radiation intensity, 2.6-fold and 5.5-fold higher than the lowest intensity, YK-CAP-002, at 6 and 96 hours, respectively.

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

[0302] The mean radiation intensity of YK-CAP-002 was 1,466,925 at 6 hours and only 20,893 at 96 hours, the lowest, while the mean radiation intensity of YK-CAP-001 was also lower, at 1,728,300 at 6 hours and 43,485 at 96 hours.

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

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

[0305] 2) Compared with modified locked nucleoside capping analogs of the prior art with similar or significantly different structures, the average radiation intensity and duration of protein expression in mice from 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.

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

[0307] The mean radiation intensities of YK-CAP-004 of the present application were 1.4-fold, 1.5-fold, and 2.1-fold those of N-7413, compound 14, and m6A, respectively, at 6 hours, and 1.7-fold, 2.0-fold, and 3.4-fold, respectively, at 96 hours.

[0308] The mean radiation intensities of YK-CAP-003 were 1.2-fold, 1.2-fold, and 1.8-fold those of N-7413, compound 14, and m6A, respectively, at 6 hours, and 0.8-fold, 1.0-fold, and 1.7-fold, respectively, at 96 hours.

[0309] The mean radiation intensities of YK-CAP-005 were 1.1-fold, 1.2-fold, and 1.8-fold higher than those of N-7413, compound 14, and m6A, respectively, at 6 hours, and 1.6-fold, 1.9-fold, and 3.3-fold higher than those of N-7413, compound 14, and m6A, respectively, at 96 hours.

[0310] The mean radiation intensities of YK-CAP-006 were 1.2-fold, 1.3-fold, and 1.8-fold higher than those of N-7413, compound 14, and m6A, respectively, at 6 hours, and 1.1-fold, 1.3-fold, and 2.3-fold higher than those of N-7413, compound 14, and m6A, respectively, at 96 hours.

[0311] 3) The structurally similar modified locked nucleoside capping analogs showed very large differences in the average radiation intensity and duration of mRNA expression of proteins in mice.

[0312] The structures of each modified locked nucleoside capping analog designed in this application are very similar, and this series of compounds is very similar in structure to compound 14, but there were very large differences in the average radiation intensity and duration of protein expression in the mouse mRNA.

[0313] For example, compared with YK-CAP-001, YK-CAP-002, and compound 14, YK-CAP-004 differs only in the group attached to the methylene bridge connecting the 2'-O of the ribose ring to the C4', i.e., the substituent at C6': YK-CAP-004 is 2,2-difluoroethyl, YK-CAP-001 is dimethylaminoethyl, and YK-CAP-002 is cyanomethyl, whereas compound 14 has no substituent and the other structures are identical. However, the average radiation intensity of YK-CAP-004 was 2.2-fold, 2.6-fold, and 1.5-fold higher than those of YK-CAP-001, YK-CAP-002, and compound 14 at 6 hours, and 2.6-fold, 5.5-fold, and 2.0-fold higher than those of YK-CAP-001, YK-CAP-002, and compound 14 at 96 hours, respectively, demonstrating significant improvements.

[0314] As can be seen from animal experiments, 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, significantly improved both the amount and duration of mRNA protein expression in mice 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 structurally different modified locked nucleoside capping analogs (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 expressed highly efficiently and continuously.

[0316] Furthermore, the amount and duration of protein expression in mice of Fluc mRNA produced by modified locked nucleoside capping analogs with similar structures were not necessarily similar, and there was a very high possibility that there were significant differences.

[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 significantly improved the mRNA in vitro transcription yield, capping rate, mRNA translation efficiency, decapping enzyme stability, and in vivo protein expression level and duration compared to conventional modified locked nucleoside capping analogs (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 significantly improved the resistance of locked nucleic acid structures to decapping enzymes and their binding affinity with capping enzymes, providing novel, highly efficient modified locked nucleic acid capping structures for in vitro mRNA transcription.

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

[0319] 1) It has been shown that the structures of the compounds of the present application are very similar, but the only difference is the substituent at C6', i.e., the group attached to the methylene bridge connecting 2'-O and C4' of the ribose ring.

[0320] 2) The compound of the present application is structurally similar to compound 14, with the only difference being that compound 14 has no substituent on the methylene bridge (i.e., C6') connecting the 2'-O and C4' of the ribose ring.

[0321] 3) The compounds of the present application have a greater structural difference from N-7413, HN3002 and m6A, in that these three compounds lack a methylene bridge between the 2'-O and C4' of the ribose ring, and 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 significant 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. YK-CAP-004 had the highest transcription yield and capping rate, with the transcription yield being 82.8% higher than the lowest YK-CAP-001 and the capping rate being 26.1% higher than the lowest YK-CAP-002.

[0324] 2) The modified locked nucleoside capping analogs of the present application significantly improved both the mRNA in vitro transcription yield and capping rate 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 35.0% and 18.8%, respectively, higher than those of compound 14.

[0325] 3) Modified locked nucleoside capping analogs with similar structures showed significant differences in mRNA in vitro transcription yield and capping rate. For example, the mRNA in vitro 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.

[0326] 3. The modified locked nucleoside capping analogs of the present application showed a significant difference in mRNA translation efficiency. 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 significantly improved.

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

[0328] 2) Compared with the modified locked nucleoside capping analogs of the prior art that have similar or significantly different structures, the mRNA translation efficiency of the modified locked nucleoside capping analogs of the present application is significantly improved. For example, the translation efficiency of YK-CAP-004 is four times that of m6A.

[0329] 3) Structurally similar modified locked nucleoside capping analogs showed large differences in mRNA translation efficiency, for example, the translation efficiency of YK-CAP-004 mRNA was 1.8-fold and 2.0-fold higher than that of YK-CAP-001 and YK-CAP-002, respectively.

[0330] 4. The modified locked nucleoside capping analogs of the present application had significantly different decapping rates. Compared with modified locked nucleoside capping analogs of the prior art that have similar or significantly different structures, the decapping rates of the modified locked nucleoside capping analogs of the present application were all significantly reduced.

[0331] 1) The modified locked nucleoside capping analogs of the present application had significant differences in decapping rates. YK-CAP-004 had the lowest decapping rate, 16.0% lower than the highest rate of YK-CAP-001.

[0332] 2) Compared with the modified locked nucleoside capping analogs of the prior art that have similar or significantly different structures, the decapping rates of the modified locked nucleoside capping analogs of the present invention are all significantly reduced, for example, the decapping rate of YK-CAP-004 is 38.3% lower than that of N-7413.

[0333] 3) The decapping rates of structurally similar modified locked nucleoside capping analogs varied significantly. For example, the mRNA decapping rates of YK-CAP-004 were reduced by 16.0%, 14.3%, and 18.5% compared with those of YK-CAP-001, YK-CAP-002, and compound 14, respectively.

[0334] 5. The modified locked nucleoside capping analogs of the present application were shown to have significant differences in the average radiation intensity (corresponding to the amount of protein expressed) and duration of protein expression in mice from mRNA. Compared with modified locked nucleoside capping analogs of the prior art that have similar or significantly different structures, the modified locked nucleoside capping analogs of the present application showed significant improvements in both the amount and duration of protein expression in mice from mRNA.

[0335] 1) The modified locked nucleoside capping analogs of this application showed significant differences in the mean radiation intensity and duration of protein expression in mice. YK-CAP-004 had the highest fluorescence intensity, 2.6-fold and 5.5-fold higher than the lowest intensity, YK-CAP-002, at 6 and 96 hours, respectively.

[0336] 2) Compared with modified locked nucleoside capping analogs of the prior art with similar or significantly different structures, the mean radiation intensity and duration of protein expression in mice from mRNA of the modified locked nucleoside capping analogs of the present application were significantly improved. For example, the mean 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) Structurally similar modified locked nucleoside capping analogs showed significant differences in the mean radiation intensity and duration of mRNA expression in mice. For example, the mean radiation intensity of YK-CAP-004 was 2.2-fold, 2.6-fold, and 1.5-fold higher than that of YK-CAP-001, YK-CAP-002, and Compound 14 at 6 hours, and 2.6-fold, 5.5-fold, and 2.0-fold higher than that of YK-CAP-001, YK-CAP-002, and Compound 14 at 96 hours.

[0338] Finally, it should be noted that the above embodiments are only for illustrating the technical solutions of the present invention and are not limited thereto. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art may still modify the technical solutions described in the above embodiments or equally replace some or all of the technical features thereof, and such modification or replacement does not mean that the essence of the corresponding technical solutions deviates from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A compound or a pharmaceutically acceptable salt thereof, wherein the compound is YK-CAP-003, YK-CAP-004, YK-CAP-005, or YK-CAP-006, having a structure represented by the following formula: 【Chemistry 1】 【change】

2. An RNA molecule comprising the compound of claim 1 or a pharmaceutically acceptable salt thereof as a cap structure or cap structure fragment.

3. A pharmaceutical composition comprising the RNA molecule of claim 2.

4. The pharmaceutical composition of claim 3 , further comprising at least one RNA delivery agent.

5. The pharmaceutical composition of claim 4 , wherein the at least one RNA delivery agent comprises at least one cationic lipid.

6. The pharmaceutical composition of claim 4 , wherein the at least one RNA delivery agent further comprises at least one neutral lipid.

7. The cationic lipid is selected from one or more of the following compounds: (1) A compound represented by formula (II) or a pharmaceutically acceptable salt thereof, wherein G １ is C １～６ alkylene, and G ２ is C ２～８ alkylene, and G ３ is C １～３ alkylene, and L １ is C ６～１５ is a linear alkyl; L ２ is C １２～２５ is a branched alkyl 【Chemistry 2】 (2) A compound represented by formula (III) or a pharmaceutically acceptable salt thereof, wherein: １ is C ２～８ alkylene, and G ２ is C ２～８ alkylene, and L １ is —C(O)O— or —OC(O)—, and L ２ is —C(O)O— or —OC(O)—, and R １ is C ６～２５ is a straight or branched chain alkyl; R ２ is C ６～２５ is a straight or branched chain 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 -is, 【Transformation 3】 (3) A compound represented by formula (IV) or a pharmaceutically acceptable salt thereof, wherein: １ is C １～６ alkylene, and G ２ is C ２～８ alkylene, and R １ is C ６～２０ is a straight or branched chain alkyl; R ２ is C １２～２５ is a 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 -is, 【Chemistry 4】 (4) A compound represented by formula (V) or a pharmaceutically acceptable salt thereof, wherein G １ is C １～８ alkylene, and G ２ is C ２～８ alkylene, and R １ is C ６～２５ is a straight or branched chain alkyl; R ２ is C １２～２５ is a straight or branched chain alkyl; G ３ is HO(CH ２ ) ２ N (R ３ ) CH ２ CH(OH)CH ２ -, where R ３ is -CH ３ or -CH ２ CH ３ or -CH ２ CH ２ OH, 【Transformation 5】 (5) A compound represented by formula (VI) or a pharmaceutically acceptable salt thereof, wherein G １ and G ２ are each independently an unsubstituted C ６ ~C １０ alkylene, and G ３ is unsubstituted C １ ~C １２ alkylene, and R １ and R ２ are each independently C ６ ~C ２４ Alkyl or C ６ ~C ２４ alkenyl, and R ３ is -OR ５ , -C(=O)OR ４ , —OC(═O)R ４ or -NR ５ C(=O)R ４ and R ４ is C １ ~C １２ is a hydrocarbon group, and R ５ is H or C １ ~C ６ is a hydrocarbon group, 【Transformation 6】 (6) A compound represented by formula (VII) or a pharmaceutically acceptable salt thereof, wherein R ４ is -(CH ２ ) ｎ Q, where Q is —OH or —CN, and n is 1, 2, or 3; 【Transformation 7】 (7) The pharmaceutical composition according to claim 5, which is a compound represented by formula (VIII) or a pharmaceutically acceptable salt thereof. 【Transformation 8】

8. 8. The pharmaceutical composition of claim 7, wherein the cationic lipid is selected from one or more of YK-009, YK-401, YK-305, ALC-0315, SM-102, DLIN-MC3-DMA. 【Chemistry 9】

9. 9. The pharmaceutical composition of claim 8, wherein the cationic lipid is YK-009.

10. 7. The pharmaceutical composition of claim 6, wherein the neutral lipid comprises one or more of phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, ceramide, and sterol.

11. The neutral lipids include 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-diundecanoyl-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-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, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG), dipalmitoylphosphatidylglycerol (DPPG), palmitoyloleoylphosphatidylethanolamine (POPE), distearoyl-phosphatidylethanolamine (DSPE), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphatidylethanolamine (DMPE), 1-stearoyl-2-oleoyl-stearate 11. The pharmaceutical composition of claim 10, wherein the phosphatidylcholine is selected from one or more of: 1-stearoyl-2-oleoyl-phosphatidylcholine (SOPE), 1-stearoyl-2-oleoyl-phosphatidylcholine (SOPC), sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoylphosphatidylcholine, lysophosphatidylcholine, lysophosphatidylethanolamine (LPE), and mixtures thereof.

12. The pharmaceutical composition according to claim 11, wherein the neutral lipid is DOPE and / or DSPC.

13. The pharmaceutical composition of claim 4 , wherein the at least one RNA delivery agent further comprises a structured lipid.

14. 14. The pharmaceutical composition of claim 13, wherein the structured lipid is selected from one or more of cholesterol, non-sterols, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatine, ursolic acid, α-tocopherol, corticosteroids.

15. 15. The pharmaceutical composition of claim 14, wherein the structured lipid is cholesterol.

16. The pharmaceutical composition of claim 4 , wherein the at least one RNA delivery agent further comprises a polymer-conjugated lipid.

17. 17. The pharmaceutical composition of claim 16, wherein the polymer-conjugated lipid is selected from one or more of distearoylphosphatidylethanolamine polyethylene glycol 2000 (DSPE-PEG2000), 1,2-dimyristoyl-rac-glycero-methoxypolyethylene glycol-2000 (DMG-PEG2000), and methoxypolyethylene glycol ditetradecylacetamide (ALC-0159).

18. The pharmaceutical composition according to any one of claims 4 to 17, further comprising one or more cell-penetrating peptides.

19. (1) The compound according to claim 1 or a pharmaceutically acceptable salt thereof, and (2) Nucleotide triphosphate molecules and RNA polymerase Includes a kit.

20. RNA enzyme inhibitor, inorganic pyrophosphatase, Mg ２＋ 20. The kit of claim 19, further comprising one or more of a crowding agent, a buffering agent, or any combination thereof.

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