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

Abstract

The present invention provides a C6'-substituted lock nucleic acid modified capping analog and its uses, said capping analog improves the stability of mRNA and / or the translation efficiency of mRNA.

Description

Technology Field

[0001] The present invention relates to the field of chemical and bioengineering technology, and in particular to C6'-substituted lock nucleic acid modified capping analogs and their uses. Background Technology

[0002] The chemical nature of the cap structure is a special structure located at the 5' end of mRNA formed by modification during the transcription process of mRNA, namely the m7GPPPN structure, also known as the methylguanosine cap. This is formed under the co-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 a structure necessary 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 can begin from AUG. At the same time, the cap structure can increase the stability of the mRNA and protect it from attack by 5'→3' exonucleases.

[0004] In summary, the cap structure acts like a steel helmet for mRNA, protecting the mRNA from damage and allowing it to be easily recognized by other members by imprinting on the steel helmet through chemical modification. In addition to natural cap structures, cap structure analogs are widely used to enhance the stability of mRNA structures during in vitro transcription processes, including common ARCA and Cap 1 structure analogs.

[0005] According to research, the cap structure of mRNA has been shown to be significantly related to mRNA quality control and the organism's intrinsic immunity. Therefore, inventing new cap analogs is of great significance in increasing mRNA stability and improving the efficiency of mRNA translation. The problem to be solved

[0006] To improve the stability of mRNA and / or the translation efficiency of mRNA, the present invention provides a novel modified lock nucleoside mRNA capping analog and its uses. means of solving the problem

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

[0008]

[0009] In the above formula,

[0010] X is -O-, -S-, -N(R 1 )-, -C(=O)-, -C(=O)N(R 1 )-, -NR 1 X is selected from C(=O)-, -C(=O)O-, -OC(=O)-, -S(=O)2- or a component, and 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)2- or a component, and X is more preferably selected from -C(=O)-, -S(=O)2- or a component, and

[0011] n is selected from integers 1 to 3, preferably n is 1 or 2, and

[0012] R1, R2, R3, R5, and R6 are independently selected from hydrogen atoms, substituted or unsubstituted C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C6 cycloalkyl, or benzyl, preferably independently hydrogen atoms or methyl, and

[0013] R4 is a halogen, -NR 1 R 2 , hydrogen atom, -CN, selected from substituted or unsubstituted C1-C6 alkyl, C2-C6 alkenyl or C2-C6 alkynyl, preferably selected from halogen, -N(CH3)H, -NH2, -N(CH3)2, hydrogen atom, -CN, -CH3, -CF2H, -CCl2H, -CFH2, -CClH2, -CH=CH2 or C2 alkynyl, more preferably selected from -CH3, -CF2H, -N(CH3)2 or -CN,

[0014] R 1 and R 2 Each time it appears, it is independently selected from hydrogen atoms, 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 from hydrogen atoms and methyl whenever it appears,

[0015] Y 1a , Y 1b , Y 1c , Y 1d , Y 1e , Y 1f is independently selected from -O-, -S-, -CH2-, -CCl2-, -CF2-, or -NH-, and

[0016] Y 2a , Y 2b , Y 2c , Y 2d is independently selected from =O or =S, and

[0017] Y 3a , Y 3b , Y 3c , Y3d is independently selected from -OH or -SH, and

[0018] B1 and B2 are independently selected from natural or modified pyrimidine nucleotide bases, natural or modified purine nucleotide bases, or pharmaceutically acceptable salts of each of these.

[0019] A second aspect of the present invention provides the use of a compound represented by formula (I) according to the first aspect of the present invention, or a stereoisomer thereof, a pharmaceutically acceptable salt or solvate, in the application of the preparation of an in vitro co-transcribed mRNA capping reagent.

[0020] A third aspect of the present invention provides an RNA molecule comprising a compound represented by formula (I) according to the first aspect of the present invention or a stereoisomer thereof, a pharmaceutically acceptable salt or solvate as a cap structure or a cap structure fragment.

[0021] A fourth aspect of the present invention provides a pharmaceutical composition comprising an RNA molecule according to a third aspect of the present invention.

[0022] A fifth aspect of the present invention provides a method for synthesizing mRNA molecules for non-disease diagnostic and therapeutic purposes, comprising the step of performing template transcription by incubating a compound represented by formula (I) according to the first aspect of the present invention or a stereoisomer thereof, a pharmaceutically acceptable salt or solvate, together with a polynucleotide template.

[0023] A sixth aspect of the present invention provides a capping mRNA transcription reaction system for non-disease diagnosis and treatment purposes, which,

[0024] (1) A compound represented by formula (I) according to the first aspect of the present invention or a stereoisomer thereof, a pharmaceutically acceptable salt or solvate, and

[0025] (2) Polynucleotide templates, NTPs, and RNA polymerase

[0026] Includes

[0027] The seventh aspect of the present invention provides a kit, which,

[0028] (1) A compound represented by formula (I) according to the first aspect of the present invention or a stereoisomer thereof, a pharmaceutically acceptable salt or solvate, and

[0029] (2) Nucleotide triphosphate molecule and RNA polymerase

[0030] Includes

[0031] The 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 thereof, a pharmaceutically acceptable salt or solvate, with the RNA.

[0032] A ninth aspect of the present invention provides a method for introducing RNA into a cell, comprising the step of bringing the cell into contact with a pharmaceutical composition according to the fourth aspect of the present invention.

[0033] The tenth aspect of the present invention provides a method for performing RNA translation inhibition in a cell, comprising the step of contacting the cell with a pharmaceutical composition according to the fourth aspect of the present invention.

[0034] The eleventh aspect of the present invention provides a use of a pharmaceutical composition according to the fourth aspect of the present invention in the manufacture of a vaccine. Brief explanation of the drawing

[0035] In order to more clearly explain specific embodiments of the present invention or technical solutions according to prior art, the drawings to be used in describing specific embodiments or prior art are briefly introduced below. It is obvious that the drawings described below represent some embodiments of the present invention, and that those skilled in the art may obtain other drawings from these drawings without striving to create an inventive step. Figure 1 shows the mRNA in vitro transcription yields of modified lock nucleoside capping analogs YK-CAP-004, YK-CAP-003, YK-CAP-005, YK-CAP-006, YK-CAP-001, YK-CAP-002 and compound 14. Figure 2 shows the capping rates of modified lock nucleoside capping analogs YK-CAP-004, YK-CAP-003, YK-CAP-005, YK-CAP-006, YK-CAP-001, YK-CAP-002 and compound 14. Figure 3 shows the relative fluorescence intensity of modified lock 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. Figure 4 shows the DCP2 enzyme decapping rates of modified lock 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. Figure 5 shows the average radiation intensity of proteins expressed in vivo in mice, including mRNA carried by LNPs of Fluc mRNA transcribed by modified lock nucleoside capping analogs YK-CAP-004, YK-CAP-003, YK-CAP-002, compound 14, and m6A. Specific details for implementing the invention

[0036] To make the purpose, technical solution, and advantages of the embodiments of the present disclosure clearer, the technical solution of the embodiments of the present disclosure is described below in conjunction with the drawings of the embodiments of the present disclosure in a clear and complete manner. It is obvious that the described embodiments are not all but some of the embodiments of the present disclosure. Based on the embodiments of the present disclosure, all other embodiments obtained by a person skilled in the art without striving to create an inventive step fall within the scope of protection of the present invention.

[0037] The present invention may be embodied in other specific forms without departing from the essential attributes of the present invention. It should be understood that, without conflict, any and all embodiments of the present invention may be combined with any other embodiments or the technical features of a plurality of other embodiments to obtain additional embodiments. The present invention includes additional embodiments obtained by such combinations.

[0038] All publications and patents mentioned in this disclosure are incorporated by reference in their entirety. In the event that a use or term used in any publication or patent incorporated by reference conflicts with a use or term used in this disclosure, the use or term used in this disclosure shall prevail.

[0039] Section titles used in the text are for the purpose of structuring sentences only and should not be interpreted as a limitation on the topics described.

[0040] Unless otherwise defined, all technical and scientific terms used in this text have their ordinary meanings in the field to which the claimed subject matter belongs. If multiple definitions exist for a particular term, the definition in this text shall prevail.

[0041] Except in working examples or as otherwise specified, all numbers representing quantitative characteristics, such as dosages specified in the specification and claims, should be understood to be modified in all cases by the term "about." Additionally, any numerical range listed in this application should be understood to include all sub-ranges within such range and any combination of each endpoint of such range or sub-range.

[0042] Unless otherwise explicitly defined, all terms used in this invention (including technical and scientific terms) have the same meaning as generally understood by a person skilled in the art to which this invention pertains. Terms such as those defined in commonly used dictionaries should be interpreted in a sense consistent with their meaning in the context of the relevant technology, and should not be interpreted in an idealized or overly formal sense unless explicitly defined in this invention.

[0043] As used in the present invention, the term "C1-C6" refers to carbon atoms having any integer value within the range of 1 to 6 in the main chain of the group, for example, 1, 2, 3, 4, 5, or 6 carbon atoms. Similarly, the term "C2-C6" refers to carbon atoms having any integer value within the range of 2 to 6 in the main chain of the group, for example, 2, 3, 4, 5, or 6 carbon atoms.

[0044] As used in the present invention, the term "alkyl" means a saturated aliphatic hydrocarbon group having a straight or branched chain, and non-limiting examples thereof include methyl, ethyl, propyl, n-butyl, tert-butyl, pentyl, hexyl, etc.

[0045] As used in the present invention, the term "alkenyl" means a hydrocarbon group having at least one carbon-carbon double bond at one or more positions along the carbon chain of an alkyl group, and non-limiting examples thereof include vinyl, propenyl, butenyl, etc.

[0046] As used in the present invention, the term "alkynyl" refers to a hydrocarbon group having at least one carbon-carbon triple bond at one or more positions along the carbon chain of an alkyl group, and non-limiting examples thereof include ethinyl, propynyl, etc. As used in the present invention, the term "aryl" refers to a group comprising a carbon ring aromatic system, and non-limiting examples thereof include phenyl, naphthyl, anthracenyl, phenanthrenyl, pyrenyl, etc., and when the aryl comprises multiple rings, each ring may be fused with each other. As used in the present invention, the term "heteroaryl" means a group having a carbon ring aromatic system comprising at least one heteroatom selected from N, O, Si, P and S as a ring-forming atom, and non-limiting examples include pyridinyl, pyrimidinyl, pyrazinyl, pyridazinyl, triazinyl, quinolyl, isoquinolinyl, etc., and when the heteroaryl comprises a plurality of rings, each ring may be fused with each other.

[0047] As used in the present invention, the terms "heteroalkyl" and "heteroaryl" mean an alkyl group comprising at least one heteroatom selected from N, O, Si, P, and S.

[0048] As used in the present invention, the term “salt” means a corresponding salt of a modified nucleoside compound (or nucleotide compound) of the present invention that may be convenient or desirable for manufacturing, purifying, and / or processing, such as a pharmaceutically acceptable salt. Unless otherwise specified, references to specific compounds in the present invention also include their salt forms.

[0049] As used in the present invention, the "capping analog" is a structure at the 5' end of mature mRNA formed by post-transcriptional modification in eukaryotes, namely the m7GPPPN structure also known as the methylguanosine cap. This structure can have effects such as preventing the degradation of mRNA at the 5' end, enabling RNA transcription products to pass through selective channels in the nuclear membrane into the cytoplasm, enhancing translation, and helping to complete the entire shearing process. As used in the present invention, the portion between the wavy lines of the nucleic acid polymer is a structure embedded in the nucleic acid polymer sequence, and the portion outside the wavy lines represents a different sequence of the nucleic acid polymer.

[0050] Bases, also called nucleobases, refer to the heterocyclic base portion of a nucleoside. Nucleobases may be naturally occurring or modified, for example, 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.

[0051] As used in this disclosure, words such as “include,” “contain,” and “include” mean that the element preceding such word includes the element listed following such word and its equivalents, and does not exclude any unlisted elements. The terms “contain” or “include (include)” as used in the text may be open, semi-closed, and closed. That is, the terms also include “essentially composed of …” or “composed of …”.

[0052] The term “optionally” means that the situation described in the present disclosure may or may not occur. For example, being optionally substituted means that it may be substituted or unsubstituted. Being substituted means that at least one hydrogen of the group is replaced by another chemical group such as methyl, ethyl, methoxy, ethoxy, halogen, hydroxyl, mercapto, amino, nitro, -CN, etc.

[0053] In this application, the term "pharmaceuticalally acceptable" means that the compound or composition is chemically and / or toxicologically compatible with other components constituting the formulation and / or with humans or mammals using it to prevent or treat a disease or illness.

[0054] In this application, the term "solvent" refers to a complex formed by combining the compound of Formula (I) or a pharmaceutically acceptable salt thereof with a solvent (e.g., ethanol or water). Any solvent of the compound of Formula (I) used for the treatment of a disease or illness may provide different properties (including pharmacokinetic properties), but since the compound of Formula (I) is obtained upon absorption by a subject, the use of the compound of Formula (I) should be understood to include the use of any solvent of the compound of Formula (I).

[0055] The term "hydrate" refers to the case where the solvent is water among the above terms "solvent."

[0056] Additionally, the compound of formula (I) or a pharmaceutically acceptable salt thereof may be separated into solvates, and thus any such solvates should be understood to be included within the scope of the present invention. For example, the compound of formula (I) or a pharmaceutically acceptable salt thereof may exist in a nonsolvent form as well as in a solvated form formed with a pharmaceutically acceptable solvent (e.g., water, ethanol, etc.).

[0057] The present invention also comprises salts of compounds according to the text, particularly pharmaceutically acceptable salts. Compounds of the present invention having functional groups that are sufficiently acidic or sufficiently alkaline may react with many bases or acids to form salts. Alternatively, compounds that are inherently charged (e.g., compounds having quaternary nitrogen) may form salts with suitable counterions (e.g., halogenated ions such as bromide ions, chloride ions, or fluoride ions, particularly bromide ions).

[0058] The term "pharmaceuticalally acceptable salt" refers to a relatively non-toxic addition salt of the compounds of the present disclosure. For example, SM Berge et al. "Pharmaceutical Salts," J. Pharm. Sci. See 1977, 66, 1-19.

[0059] A suitable pharmaceutically acceptable salt of the compounds of the present disclosure may be, for example, an acid addition salt of the compounds of the present disclosure that is sufficiently alkaline and has a nitrogen atom in the chain or ring, an acid addition salt formed with an inorganic acid such as, for example, hydrochloric acid, hydrobromide, hydroiodide, sulfuric acid, phosphoric acid, or nitric acid, or, for example, formic acid, acetic acid, acetoacetic acid, pyruvate, trifluoroacetic acid, propionic acid, butyric acid, hexanoic acid, heptanoic acid, undecanic acid, lauric acid, benzoic acid, salicylic acid, 2-(4-hydroxybenzoyl)benzoic acid, camphor acid, cinnamic acid, cyclopentylpropionic acid, 3-hydroxy-2-naphthoic acid, nicotinic acid, parmoic acid, pectinic acid, persulfate, 3-phenylpropionic acid, picric acid, pivalic acid, 2-hydroxyethanesulfonic acid, itaconic acid, It is an acid addition salt formed with organic acids such as aminosulfonic acid, trifluoromethanesulfonic acid, dodecylsulfic acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, methanesulfonic acid, 2-naphthalenesulfonic acid, naphthalenedisulfonic acid, camphorsulfonic acid, citric acid, tartaric acid, stearic acid, lactic acid, oxalic acid, malonic acid, succinic acid, malic acid, adipic acid, alginic acid, maleic acid, fumaric acid, D-gluconic acid, mandelic acid, ascorbic acid, glucoheptanoic acid, glycerophosphate, aspartic acid, sulfosalicylic acid, or sulfocyanic acid.

[0060] In addition, another suitable pharmaceutically acceptable salt of the compound of the present invention that is sufficiently acidic is a basic metal salt such as a sodium or potassium salt, a basic earth metal salt such as a calcium or magnesium salt, an ammonium salt (e.g., a salt formed with NH3 or water ammonia), or a salt formed with an organic base providing a physiologically acceptable cation, for example, a salt formed with a substance 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; or, the alkaline nitrogen-containing group is a lower alkyl halide 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 chloride, bromide, and iodide; and aralkyl halides such as benzyl and phenethyl boromide can be quaternized with reagents.

[0061] Those skilled in the art will also recognize that an acid addition salt of the compound to be protected may be prepared by reacting said compound with a suitable inorganic or organic acid by any of the known methods. Alternatively, a base addition salt of the acidic compound of the present disclosure is prepared by reacting it with a suitable base by various known methods.

[0062] The present invention comprises all possible salts of the compounds of the present disclosure, which may be a single salt or any mixture of said salts in any proportion.

[0063] Specific compounds of the present disclosure may exist as one or more stereoisomers. Stereoisomers include geometric isomers, diastereomers, and enantiomers. Accordingly, the compounds claiming protection under the present disclosure further comprise racemic mixtures, monostereoisomers, and mixtures having optical activity. Those skilled in the art should understand that one stereoisomer may have better efficacy and / or fewer side effects than another stereoisomer. Monostereoisomers and optically active mixtures may be obtained through methods such as chiral synthesis, chiral catalysis, and chiral splitting. Racemic mixtures may be chiral split through chromatographic splitting or chemical splitting. For example, chiral acid splitting reagents such as chiral tartaric acid or chiral malic acid may be added to form salts with the compounds of the present disclosure, and can be separated using physical and chemical properties of the products, such as differences in solubility.

[0064] The alkyl (or alkylene) may not be substituted, or the alkyl (or alkylene) may be substituted, meaning that at least one hydrogen is replaced by another chemical group such as methyl, ethyl, methoxy, ethoxy, halogen, hydroxyl, mercapto, amino, nitro, -CN, etc.

[0065] In the present application, if the name of a compound does not match its structural formula, the structural formula shall be used as the basis.

[0066] The specification of this disclosure should be interpreted as consistent with the laws and principles of chemical bonding. In some cases, hydrogen atoms may be removed to fit into a substituent at a given position.

[0067] It should be understood that the term “compound of the present disclosure” as used in this application may include, depending on the context, the compound of formula (I), its solvate, its pharmaceutically acceptable salt, its stereoisomer, and mixtures thereof.

[0068] mRNA capping analogs of modified lock nucleosides

[0069] The present invention provides a compound represented by formula (I) or a stereoisomer thereof, a pharmaceutically acceptable salt or solvate, and

[0070]

[0071] In the above formula,

[0072] X is -O-, -S-, -N(R 1 )-, -C(=O)-, -C(=O)N(R 1 )-, -NR 1 X is selected from C(=O)-, -C(=O)O-, -OC(=O)-, -S(=O)2- or a component, and 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)2- or a component, and X is more preferably selected from -C(=O)-, -S(=O)2- or a component, and

[0073] n is selected from integers 1 to 3, preferably n is 1 or 2, and

[0074] R1, R2, R3, R5, and R6 are independently selected from hydrogen atoms, substituted or unsubstituted C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C6 cycloalkyl, or benzyl, preferably independently hydrogen atoms or methyl, and

[0075] R4 is a halogen, -NR 1 R 2, hydrogen atom, -CN, selected from substituted or unsubstituted C1-C6 alkyl, C2-C6 alkenyl or C2-C6 alkynyl, preferably selected from halogen, -N(CH3)H, -NH2, -N(CH3)2, hydrogen atom, -CN, -CH3, -CF2H, -CCl2H, -CFH2, -CClH2, -CH=CH2 or C2 alkynyl, more preferably selected from -CH3, -CF2H, -N(CH3)2 or -CN,

[0076] R 1 and R 2 Each time it appears, it is independently selected from hydrogen atoms, 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 from hydrogen atoms and methyl whenever it appears,

[0077] Y 1a , Y 1b , Y 1c , Y 1d , Y 1e , Y 1f is independently selected from -O-, -S-, -CH2-, -CCl2-, -CF2-, or -NH-, and

[0078] Y 2a , Y 2b , Y 2c , Y 2d is independently selected from =O or =S, and

[0079] Y 3a , Y 3b , Y 3c , Y 3d is independently selected from -OH or -SH, and

[0080] B1 and B2 are independently selected from natural or modified pyrimidine nucleotide bases, natural or modified purine nucleotide bases, or pharmaceutically acceptable salts of each of these.

[0081] In one embodiment, X is a component. If X is a component, R4 is, for example, -CF2H, -N(CH3)2, or -CN.

[0082] In one embodiment, X is -C(=O)-. If X is -C(=O)-, R4 is, for example, -N(CH3)2.

[0083] In one embodiment, X is -S(=O)2-. If X is -S(=O)2-, R4 is, for example, -N(CH3)2 or -CH3.

[0084] In one embodiment, R1 is a hydrogen atom or methyl, preferably a hydrogen atom.

[0085] In one embodiment, R2 is a hydrogen atom or methyl, preferably methyl.

[0086] In one embodiment, R3 is a hydrogen atom or methyl, preferably a hydrogen atom.

[0087] In one embodiment, R5 is a hydrogen atom or methyl, preferably methyl.

[0088] In one embodiment, R6 is a hydrogen atom or methyl, preferably a hydrogen atom.

[0089] In one embodiment, Y 1a is -O-.

[0090] In one embodiment, Y 1b is -O-.

[0091] In one embodiment, Y 1c is -O-.

[0092] In one embodiment, Y 1d is -O-.

[0093] In one embodiment, Y 1e is -O-.

[0094] In one embodiment, Y 1f is -O-.

[0095] In one embodiment, Y 2a is =O.

[0096] In one embodiment, Y 2b is =O.

[0097] In one embodiment, Y 2c is =O.

[0098] In one embodiment, Y 2d is =O.

[0099] In one embodiment, Y 3a is -OH.

[0100] In one embodiment, Y 3b is -OH.

[0101] In one embodiment, Y 3c is -OH.

[0102] In one embodiment, Y 3d is -OH.

[0103] In one embodiment, The R array or S array or ( R + S It is an arrangement, and preferably R It is an array.

[0104] In one embodiment, B1 and B2 are independently selected from substituted or unsubstituted adenine, guanine, thymine, cytosine, uracil, or pharmaceutically acceptable salts of each thereof. For example, B1 and B2 are independently selected from cytosine, guanine, adenine, thymine, uracil, hypoxanthine, xanthine, 7-methylguanine, pseudouracil, thiouracil, 5,6-dihydrouracil, 5-bromouracil, 5-iodouracil, 5-methylcytosine, 5-hydroxymethylcytosine, or pharmaceutically acceptable salts of each thereof.

[0105] In one embodiment, B1 is adenine.

[0106] In one embodiment, B2 is 7-methylguanine or guanine, preferably guanine.

[0107] For example, the compound represented by formula (I) is a structure represented as YK-CAP-001, YK-CAP-002, YK-CAP-003, YK-CAP-004, YK-CAP-005, or YK-CAP-006.

[0108]

[0109]

[0110]

[0111]

[0112]

[0113] .

[0114] Compared to similar compounds of existing technology, the modified lock nucleoside capping analog of the present application has the following advantages.

[0115] 1) It is a completely new compound with a structure that is completely different from similar compounds in existing technology.

[0116] 2) Compared to similar compounds of existing technology, the mRNA in vitro transcription yield of the modified lock nucleoside cap analog of the present application is significantly improved, the capping rate is significantly improved, the translation efficiency of the target mRNA is significantly improved, the decapping rate is significantly reduced, and both the amount and duration of protein expression of the mRNA in vivo in mice are significantly improved.

[0117] For example, the in vitro mRNA transcription yield and capping rate of YK-CAP-004 are improved by 35.0% and 18.8%, respectively, compared to compound 14, the translation efficiency reaches 4 times that of m6A, the decapping rate decreases by 38.3% compared to N-7413, and the average radiation intensity (corresponding to protein expression amount) reaches 2.1 times and 3.4 times that of m6A at 6 hours and 96 hours, respectively.

[0118] In addition, the present application has discovered that modified lock nucleoside capping analogs having 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 expression of mRNA in vivo in mice.

[0119] For example, compared to YK-CAP-001, YK-CAP-002, and compound 14, which have very similar structures, the in vitro mRNA transcription yield of YK-CAP-004 is 82.8% higher than that of YK-CAP-001, the capping rate is 26.1% higher than that of YK-CAP-002, the mRNA translation efficiency is 2.0 times that of YK-CAP-002, the decapping rate is 18.5% lower than that of compound 14, and the average radiation intensity of the protein expressed by the mRNA in vivo in mice is 2.6 times and 5.5 times that of YK-CAP-002 at 6 hours and 96 hours, respectively.

[0120] Therefore, it is impossible to infer the activity level of the modified lock nucleoside cap analog based on its structure.

[0121] RNA delivery agents can be, for example, lipid nanoparticles (LNPs). Lipid nanoparticles are widely used for the delivery of small molecule drugs and nucleic acids, and mRNA coated with LNPs can be protected from the influence of extracellular ribonucleases and aid in the delivery of intracellular mRNA. For lipid nanoparticles, refer to the comprehensive paper *Chemistry of Lipid Nanoparticles for RNA Delivery*. *Act Chem Res. 2022 Jan 4; 55(1):2-12*.

[0122] cationic lipids

[0123] Lipid nanoparticles generally contain cationic lipids.

[0124] As used in this text, the term cationic lipid refers to a lipid that carries a positive charge at a selected pH value. For example, reference may be made to cationic lipids disclosed in the literature, such as WO2023133946A1, CN115745820A, Chemistry of Lipid Nanoparticles for RNA Delivery. Acc Chem Res. 2022 Jan 4;55(1):2-12.

[0125] In one embodiment, the cationic lipid is one or more selected from the following compounds.

[0126] (1) A compound represented by formula (II), or its N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer, where G1 is C 1-6 It is an alkylene, and G2 is C 2-8 It is an alkylene, and G3 is C 1-3 It is an alkylene, and L1 is C 6-15 It is a straight-chain alkyl, and L2 is C 12-25 It is a branched-chain alkyl, and

[0127] (II)

[0128] (2) A compound represented by formula (III), or its N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer, where G1 is C 2-8 It is an alkylene, and G2 is C 2-8 It is an alkylene, L1 is -C(O)O- or -OC(O)-, L2 is -C(O)O- or -OC(O)-, and R1 is C 6-25 It is a straight-chain or branched-chain alkyl, and R2 is C 6-25 It is a straight-chain or branched-chain alkyl, G3 is HO(CH2)2- or HO(CH2)3-, G4 is HO(CH2)2- or HO(CH2)3-, L is -(CH2)2- or -(CH2)3- or -(CH2)4-, and

[0129] (III)

[0130] (3) A compound represented by formula (IV), or its N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer, where G1 is C 1-6 It is an alkylene, and G2 is C 2-8 It is an alkylene, and R1 is C 6-20 It is a straight-chain or branched-chain alkyl, and R2 is C 12-25 is branched chain alkyl, G3 is HO(CH2)2N(CH3)(CH2)2-, HO(CH2)2N(CH2CH3)(CH2)2-, (HO(CH2)2)2N(CH2)2-, CH3O(CH2)2N(CH3)(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-,

[0131] (IV)

[0132] (4) A compound represented by formula (V), or its N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer, where G1 is C 1-8 It is an alkylene, and G2 is C 2-8 It is an alkylene, and R1 is C 6-25It is a straight-chain or branched-chain alkyl, and R2 is C 12-25 It is a straight-chain or branched-chain alkyl, and G3 is HO(CH2)2N(R3)CH2CH(OH)CH2-, where R3 is -CH3 or -CH2CH3 or -CH2CH2OH,

[0133] (V)

[0134] (5) A compound represented by formula (VI), or its N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer, where G 1 and G 2 Each is independently an unsubstituted C6-C 10 It is an alkylene, and G 3 Unsubstituted C1-C 12 It is an alkylene, and R 1 and R 2 Each independently C6-C 24 alkyl or C6-C 24 It is alkenyl, and R 3 -OR 5 , -C(=O)OR 4 , -OC(=O)R 4 or -NR 5 C(=O)R 4 and R 4 is C1-C 12 It is a hydrocarbon group, and also R 5 is H or a C1-C6 hydrocarbon group, and

[0135] (VI)

[0136] (6) A compound represented by formula (VII), or its N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer, where R4 is -(CH2) n Selected from Q, where Q is -OH or -CN, and n is 1, 2 or 3, and

[0137] (VII)

[0138] (7) A compound represented by formula (VIII), or its N-oxide, solvate, pharmaceutically acceptable salt, or stereoisomer.

[0139] (VIII)

[0140] In a preferred embodiment, the cationic lipid is one or more selected from YK-009, YK-401, YK-305, ALC0315, SM102, and DLIN-MC3-DMA.

[0141] ,

[0142] ,

[0143] ,

[0144] ,

[0145] ,

[0146] .

[0147] In a more preferred embodiment, the cationic lipid is YK-009.

[0148] Neutral lipids

[0149] RNA delivery systems containing cationic lipids may also contain neutral lipids. In this disclosure, neutral lipids refer to lipids that play an auxiliary role, existing in a non-charged or positively charged form at selected pH values. The neutral lipids can promote lipid phase transitions to control the fluidity of nanoparticles into a lipid bilayer structure and improve efficiency, and may also affect target organ specificity.

[0150] In one embodiment, the molar ratio of the cationic lipid to the neutral lipid is about 1:1 to 15:1, for example, about 14:1, 13:1, 12:1, 11:1, 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, and 2:1. In a 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.

[0151] For example, the neutral lipid may include one or more of phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, ceramide, sterol, and derivatives thereof.

[0152] RNA delivery agents containing cationic lipids may include one or more neutral lipid-phospholipids, such as one or more (poly)unsaturated lipids. Phospholipids may be assembled into one or more lipid bilayers. Generally, phospholipids may include a phospholipid portion and one or more fatty acid portions.

[0153] The neutral lipid portion may be selected from a non-limiting group consisting of phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylserine, phosphatidic acid, 2-lysophosphatidylcholine, and sphingomyelin. The fatty acid portion may be selected from a non-limiting group consisting of lauric acid, myristic acid, myristoleic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleic acid, α-linolenic acid, erucic acid, phytic acid, arachidic acid, arachidonic acid, eicosapentaenoic acid, behenic acid, docosapentaenoic acid, and docosahexaenoic acid. Additionally, non-natural species are included, including natural species having modifications and substitutions including branching, oxidation, cyclization, and alkynes. For example, phospholipids may be functionalized into one or more alkynes (e.g., alkenyls in which one or more double bonds are substituted for triple bonds) or cross-linked with said one or more alkynes. Under appropriate reaction conditions, alkynes may undergo a cyclization addition reaction catalytically by a copper catalyst when exposed to azide. Such reactions can be used to functionalize the lipid bilayer of the composition to promote membrane permeability or cell recognition, or to couple the composition with useful components such as targeting or imaging parts (e.g., dyes).

[0154] Neutral lipids that can be used in such compositions are 1,2-dilinoleoy-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 1,2-dioleoy-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-diundecanoy-sn-glycero-phosphocholine (DUPC), 1-palmitoyl-2-oleoy-sn-glycero-3-phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 Diether PC), 1-oleoyl-2-cholesteryl hemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 LysoPC), 1,2-dilinolenoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dipitanoyl-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-Diarachidonoyyl-sn-glycero-3-phosphoethanolamine, 1,2-Didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2-Dioleoyl-sn-glycero-3-phosphate-rac-(1-glycerol)sodium salt (DOPG), Dipalmitoylphosphatidylglycerol (DPPG), Palmitoyloleylphosphatidylethanolamine (POPE), Distearoyl-phosphatidyl-ethanolamine (DSPE), It may be selected from a non-limiting group consisting of 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.

[0155] In some embodiments, the neutral lipid comprises DSPC. In certain embodiments, the neutral lipid comprises DOPE. In some embodiments, the neutral lipid comprises both DSPC and DOPE.

[0156] Structural geology

[0157] RNA delivery agents comprising cationic lipids may also comprise one or more structural lipids. In this disclosure, structural lipids refer to lipids that fill the gaps between lipids to improve the stability of nanoparticles.

[0158] 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.

[0159] Structural lipids may be selected from, but are not limited to, the group consisting of cholesterol, nonsterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatin, ursolic acid, α-tocopherol, corticosteroids, and mixtures thereof. In some embodiments, the structural lipid is cholesterol. In some embodiments, the structural lipid comprises cholesterol and corticosteroids (e.g., prednisolone, dexamethasone, prednisone, and hydrocortisone) or combinations thereof.

[0160] Polymer conjugated lipids

[0161] RNA delivery systems containing cationic lipids may also contain one or more polymer conjugated lipids. Polymer conjugated lipids primarily refer to polyethylene glycol (PEG) modified lipids. Hydrophilic PEG stabilizes LNPs, controls nanoparticle size by limiting lipid fusion, and increases nanoparticle half-life by reducing non-specific interactions with macrophages.

[0162] In one embodiment, the polymer conjugated lipid is one or more selected from PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, and PEG-modified dialkylglycerol. The molecular weight of the PEG-modified PEG is generally 350 to 5000 Da.

[0163] For example, the polymer conjugated lipid is one or more selected from distearoyl phosphatidyl ethanolamine polyethylene glycol 2000 (DSPE-PEG2000), 1,2-dimyritoyl-rac-glycero-3-methoxypolyethyleneglycol-2000 (DMG-PEG2000), or methoxypoly(ethylene glycol) ditetradecylacetamide (ALC-0159).

[0164] In one embodiment, the polymer conjugated lipid is DMG-PEG2000.

[0165] In one embodiment, the RNA delivery agent comprises a neutral lipid, a structural lipid, and a polymer conjugated lipid, and the molar ratio of the cationic lipid, the neutral lipid, the structural lipid, and the polymer conjugated lipid is (25 to 75):(5 to 25):(15 to 65):(0.5 to 10), and for example, (35 to 49):(7.5 to 15):(35 to 55):(1 to 5).

[0166] In one embodiment, the RNA delivery agent comprises a neutral lipid, a structural lipid, and a polymer conjugated lipid, and the molar ratio of the cationic lipid, the neutral lipid, the structural lipid, and the polymer conjugated lipid is 49:10:39.5:1.5.

[0167] Embodiments of the present invention are described in detail below by combining examples; however, those skilled in the art will understand that the following examples are merely for illustrating the invention and should not be construed as limiting the scope of the invention. Where specific conditions are not specified in the examples, they are performed under ordinary conditions or conditions recommended by the manufacturer. Where the manufacturer of the reagents or equipment used is not specified, they are all general products available on the market.

[0168] The following abbreviations each represent the reagents listed below.

[0169] IBX: 2-iodooxybenzoic acid, BF3·Et2O: boron trifluoride diethyl etherate, Allyltrimethylsilane: allyltrimethylsilane, TEA: triethylamine, Ac2O: acetic anhydride, HOAc: acetic acid, conc H2SO4: concentrated sulfuric acid, BSA: N,O-bis(trimethylsilyl)acetamide, TMSOTf: trimethylsilyl trifluoromethanesulfonate, Toluene: toluene, MeOH: methanol, Boc2O: di-tert-butyl dicarbonate, DIEA: N,N-diisopropylethylamine, DMAP: 4-dimethylaminopyridine, DMSO: dimethyl sulfoxide, HATU: 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate, THF: tetrahydrofuran, TBSCl: tert-butyldimethylsilyl chloride, Imidazole: Imidazole, DMF: N,N-dimethylformamide, TBAF: tetrabutylammonium fluoride, TBSOTf: tert-butyldimethylsilyl trifluoromethanesulfonate, NMO: 4-methylmorpholine-4-oxide, m-CPBA: 3-chloroperoxybenzoic acid, DIAD: diisopropyl azodicarboxylate, NCS: N-chlorosuccinimide, PO(MeO)3: trimethylphosphate, PySSPy: 2,2'-dithiodipyridine, imidazole: Imidazole, PPh3: triphenylphosphine, TEAP: triethylammonium phosphate, TEAB: triethylammonium formate, MTBE: methyl tert-butyl ether, DCM: dichloromethane, EA: ethyl Acetate, DAST: diethylamine sulfur trifluoride, AcSH: thioacetic acid.

[0170] Example 1:

[0171] 1. Synthesis of the intermediate INT-I

[0172]

[0173] Step 1: Synthesis of INT-I-PM1

[0174] 3-O-Benzyl-4-C-Benzyloxymethyl-1,2-O-Isopropylidene-A-D-Ribofuranose (100.0 g, 0.25 mol) was dissolved in 500 mL of acetonitrile, 2-iodooxybenzoic acid (104.9 g, 0.37 mol) was added, the temperature was raised to 70°C, the reaction system was stirred for 2 hours, and the completion of the reaction was monitored by TLC. After stopping the reaction and cooling the reaction system to room temperature, the mixture was filtered through diatomaceous earth, the cake was rinsed with acetonitrile (500 mL), the filtrate was rotary dried under reduced pressure and vacuum dried to obtain a pale yellow liquid, INT-I-PM1 (102.1 g), which was used directly in the next step.

[0175] Step 2: Synthesis of INT-I-PM2

[0176] INT-I-PM1 (102.1 g, 0.25 mol) was dissolved in 400 mL of dichloromethane and stirred, then cooled to -40°C. A boron trifluoride ether solution (50.82 g, 0.35 mol) was slowly added, and the reaction system was stirred for 5 minutes. Allyltrimethylsilane (52.9 mL, 0.33 mol) was added dropwise, and the reaction system was stirred for 2 hours. The completion of the reaction was monitored by TLC. The reaction solution was slowly poured into 1000 mL of saturated sodium bicarbonate aqueous solution, quenched, and separated. The aqueous phase was extracted with dichloromethane (500 mL × 2), the organic phase was washed with saturated saline solution (500 mL × 2), dried with anhydrous sodium sulfate, and rotary dried. The residue was purified by normal-phase silica gel column chromatography to obtain INT-I-PM2 (103.0 g, 0.23 mol), a pale yellow liquid.

[0177] Step 3: Synthesis of INT-I-PM3

[0178] 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 ethylsulfonyl chloride (60.4 g, 0.47 mol) was added dropwise under an ice bath. After the dropwise addition was completed, the ice bath was removed and the temperature was naturally raised to room temperature. The reaction system was stirred for 2 hours, and the completion of the reaction was monitored by TLC. After the reaction was completed, the reaction solution was poured into 1000 mL of saturated sodium bicarbonate aqueous solution to quench the reaction and separate the phases. The aqueous phase was extracted with dichloromethane (500 mL × 2), the organic phase was combined, washed with saturated saline solution (1000 mL), dried with anhydrous sodium sulfate, and rotary dried to obtain a brown viscous substance. The residue was purified by normal-phase silica gel column chromatography to obtain a pale yellow liquid, INT-I-PM3 (106.0 g, 0.20 mol, yield 86.5%).

[0179] Step 4: Synthesis of INT-I-PM4

[0180] 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 reaction system was stirred at room temperature for 1 hour, and the completion of the reaction was monitored by TLC. After the reaction was completed, ethyl acetate (800 mL) and water (800 mL) were added and the liquids were separated. The aqueous phase was extracted once with ethyl acetate (800 mL), the organic phase was combined, washed with water (800 mL × 3), washed with a saturated sodium bicarbonate aqueous solution, washed twice with saturated saline solution, dried with anhydrous sodium sulfate, and rotary dried to obtain a pale yellow liquid, INT-I-PM4 (86.5 g, 0.15 mol, yield 75.0%).

[0181] Step 5: Synthesis of INT-I-PM5

[0182] 2-amino-6-chloropurine (30.50 g, 0.18 mol) and 250 mL of toluene were added to a 1000 mL reaction flask, N,O-bis(trimethylsilyl)acetamide (61.00 g, 0.30 mol) was added, heated to 80 °C and stirred to dissolve, then cooled 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 trifluorosulfonate (40.00 g, 0.18 mol) dissolved in 50 mL of toluene was added dropwise, the temperature was rapidly raised to 110 °C and reacted for 3 hours, and the completion of the reaction was monitored by TLC. The reaction was stopped, and once the reaction cooled to room temperature, ethyl acetate (300 mL) was added for extraction, washed with an aqueous solution of saturated sodium bicarbonate (500 mL), the solid precipitated as diatomite was filtered, the filtrate was separated, the aqueous phase was back-extracted with ethyl acetate (500 mL), the organic phase was combined, dried with anhydrous Na2SO4, the solvent was rotary-dried, and the residue was purified by normal-phase silica gel column chromatography to obtain INT-I-PM5 (103.02 g, 0.15 mol, yield 100%). C 32 H 36 ClN5O8S, MS(ES): m / z(M+H + )686.2.

[0183] Step 6: Synthesis of INT-I-PM6

[0184] INT-I-PM5 (103.00g, 0.15mol) and 500mL of methanol were added to a 1000mL reaction flask and stirred to dissolve, potassium carbonate (103.73g, 0.75mol) was added and the mixture was reacted at room temperature for 23 hours, and the completion of the reaction was monitored by TLC. The reaction was stopped, filtered with diatomaceous earth, and the solvent was removed by rotating under reduced pressure. Ethyl acetate (800 mL) and water (500 mL) were added for extraction and separation. The aqueous phase was back-extracted with ethyl acetate (300 mL), the organic phase was combined, washed once with a saturated sodium chloride aqueous solution (600 mL), dried with anhydrous sodium sulfate, and the solvent was rotary-dried. The crude product obtained was subjected to normal-phase silica gel column chromatography to obtain INT-I-PM6 (44.03 g, 0.083 mol, yield 55.4%). C 29 H 31 N5O5, MS(ES): m / z(M+H + )530.2.

[0185] Step 7: Synthesis of INT-I

[0186] 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 system was stirred overnight at 30°C. The near completion of the reaction was monitored by TLC, and the solvent was removed by evaporation under reduced pressure. INT-I (49.81 g, 68.25 mmol, yield 82.2%) was obtained by purification using silica gel chromatography. C 39 H 47 N5O9, MS(ES): m / z(M+H + )730.2.

[0187] 2. Synthesis of Intermediate INT-II

[0188]

[0189] 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, and triphenylphosphine (1337.7 mg, 5.10 mmol) was added under the protection of nitrogen gas, followed by a reaction at 25°C for 4 hours. After the reaction was completed, 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 minutes. INT-II (240.6 mg, 0.30 mmol, yield 80.9%), a white solid, was obtained by centrifugation. C 24 H 30 N 12 O 13 P2, MS(ES): m / z(MH - ): 755.15.

[0190] 3. Synthesis of YK-CAP-001

[0191]

[0192] Step 1: Synthesis of YK-CAP-001-PM1

[0193] INT-I (49.81 g, 68.25 mmol) was dissolved in tetrahydrofuran (400 mL) and water (100 mL), and N-methylmorpholine oxide (12.00 g, 102.37 mmol) and potassium osmate dihydrate (1.26 g, 3.41 mmol) were sequentially added to the mixture. The reaction system was stirred at room temperature for 5 hours, and TLC indicated that the reaction was complete. The reaction was quenched with saturated sodium sulfite, extracted with ethyl acetate (500 mL × 2), and the organic phase was washed with 200 mL of saturated sodium chloride solution. Finally, the organic phase was dried with anhydrous sodium sulfate, filtered, and rotary dried to obtain YK-CAP-001-PM1 (52.00 g, 68.08 mmol, yield 99.8%). C 39 H 49 N5O 11 , MS(ES): m / z(M+H + )764.3.

[0194] Step 2: Synthesis of YK-CAP-001-PM2

[0195] YK-CAP-001-PM1 (52.00 g, 68.08 mmol) was dissolved in tetrahydrofuran (500 mL) and water (125 mL), sodium periodate (21.84 g, 102.11 mmol) was weighed and added, the reaction system was heated to 30°C and left overnight, and TLC indicated that the reaction was complete. The reaction system was quenched with saturated sodium sulfite, extracted with ethyl acetate (500 mL × 3), the organic phases were combined, washed with saturated saline, dried with anhydrous sodium sulfate, filtered, and rotary dried. YK-CAP-001-PM2 (45.5 g, 62.2 mmol, yield 91.3%) was obtained. C 38 H 45 N5O 10 , MS(ES): m / z(M+H + )732.3.

[0196] Step 3: Synthesis of YK-CAP-001-PM3

[0197] 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 borohydrotriacetate (22.16 g, 104.50 mmol) was slowly added at room temperature, and the reaction system was stirred at room temperature for 15 hours and monitored by LCMS to indicate that the reaction was complete. The reaction system was diluted with 200 mL of dichloromethane, quenched with a saturated aqueous ammonium chloride solution, and separated. The aqueous phase was extracted with dichloromethane (200 mL × 2), the organic phase was combined, the organic phase was washed with 100 mL of a saturated aqueous sodium chloride solution, dried with anhydrous sodium sulfate, filtered, and rotary dried. YK-CAP-001-PM3 (10.80 g, 14.19 mmol, yield 67.9%) was obtained by purification by silica gel chromatography. C 40 H 52 N6O9, MS(ES): m / z(M+H + )761.3.

[0198] Step 4: Synthesis of YK-CAP-001-PM4

[0199] YK-CAP-001-PM3 (10.60 g, 13.93 mmol) was used as a raw material and dissolved in dichloromethane (300 mL). The reaction system was cooled to -40°C under a nitrogen gas atmosphere, and a dichloromethane solution of boron trichloride (111.4 mL, 111.4 mmol) was slowly added dropwise. After the addition was complete, the reaction system was slowly heated to 0°C and stirred at this temperature for 3 hours. TLC indicated that the reaction was complete; the system was then cooled back to -40°C, the reaction was quenched with methanol, rotary dried, and left at room temperature for 24 hours to obtain 5.5 g of a brown crude product. 1.5 g was taken and purified by preparative HPLC to obtain YK-CAP-001-PM4 (508.47 mg, 1.39 mmol). C 15 H 22 N6O5, MS(ES): m / z(M+H +)367.1.

[0200] 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).

[0201] Step 5: Synthesis of YK-CAP-001-PM5 Intermediate

[0202] YK-CAP-001-PM4 intermediate (380.0 mg, 1.04 mmol) was dissolved in 4.0 mL of trimethylphosphate and placed in an ice bath. Under the protection of nitrogen gas, phosphorus oxychloride (1.69 g, 10.37 mmol) was slowly added dropwise, and the mixture was reacted at 0°C for 4 hours. After the reaction was complete, 4.0 mL of purified water was slowly added to the ice bath, and the mixture was stirred for 1 hour. Then, dichloromethane (4 mL × 3) was added to wash the mixture three times. The aqueous phase was collected and concentrated under reduced pressure to remove the organic solvent. The residue was dissolved in 200 mL of purified water and purified using a gel column to obtain the white solid YK-CAP-001-PM5 triethylamine salt (260.0 mg, 0.47 mmol, yield 45.8%). C 15 H 23 N6O8P, MS(ES): m / z(MH - ) 445.13.

[0203] Step 6: Synthesis of YK-CAP-001-PM6 Intermediate

[0204] 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, and triphenylphosphine (1479.3 mg, 5.64 mmol) was added under the protection of nitrogen gas, followed by a reaction at 25°C for 4 hours. After the reaction was completed, 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. The white solid YK-CAP-001-PM6 (165 mg, 0.32 mmol, yield 67.0%) was obtained by repeated centrifugation. C 18 H 25 N8O7P, MS(ES): m / z(MH - ) 495.16.

[0205] Step 7: Synthesis of YK-CAP-001-PM7 Intermediate

[0206] YK-CAP-001-PM6 (165 mg, 0.32 mmol), triethylammonium 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, stirred for 10 minutes, and left to stand; subsequently, the supernatant was removed, and the turbidity in the lower layer was removed. After removing the organic solvent by vacuum concentration, the residue was dissolved in 100 mL of purified water and purified using a gel column to obtain the white solid YK-CAP-001-PM7 triethylamine salt (150.5 mg, 0.24 mmol, yield 75.3%). C 15 H 24 N6O 11 P2, MS(ES): m / z(MH - ) 525.10.

[0207] Step 8: Synthesis of YK-CAP-001-PM8 Intermediate

[0208] 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 complete, 20 mL of purified water was added, and the aqueous phase was washed three times with dichloromethane (20 mL × 3). The aqueous phase was then collected and concentrated under reduced pressure to remove the solvent. The residue was dissolved in 100 mL of purified water and purified using a gel column to obtain 320 mg of a white crude product. The obtained crude product was further purified into a preparative liquid phase to obtain a white solid, YK-CAP-001-PM8 triethylamine salt (75 mg, 0.12 mmol, yield 48.7%). C 16 H 26 N6O 11 P2, MS(ES): m / z(MH - ) 539.11.

[0209] Step 9: Synthesis of YK-CAP-001

[0210] 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 complete, 0.1 M EDTA was added until the solution dissolved clearly, followed by the addition of water to dilute to 100 mL. The solution was then purified using a gel column to obtain a white crude product (200.6 mg). The obtained crude product was further purified into a preparative liquid phase to obtain the white solid YK-CAP-001 (16.9 mg, 13.20 μmol, yield 11.3%). C 37 H 52 N 16 O 24 P4, MS(ES): m / z(MH - ) 1227.21.

[0211] 1 H NMR (400 MHz, DO) 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 (DO, 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).

[0212] 4. Synthesis of YK-CAP-002

[0213]

[0214] Step 1: Synthesis of YK-CAP-002-PM1

[0215] YK-CAP-001-PM2 (22.0 g, 30.06 mmol) was dissolved in 440 mL of tetrahydrofuran and cooled to -10°C, after which 26% ammonia water (250 mL) was added to the reaction system. The reaction system was stirred for 5 minutes at -10°C, followed by the addition of elemental iodine (23.0 g, 90.62 mmol). The reaction system was stirred continuously at the above temperature for 2 hours, then raised to room temperature, and stirred for 15 minutes. The reaction solution was quenched with a saturated sodium thiosulfate solution, extracted with ethyl acetate, dried the organic phase, and rotary dried. After purification by silica gel chromatography, YK-CAP-002-PM1 (14.7 g, 20.17 mmol, yield 67.1%), a pale yellow oily liquid, was obtained. C 38 H 44 N6O9, MS(ES): m / z(M+H+)729.42.

[0216] Step 2: Synthesis of YK-CAP-002-PM2

[0217] YK-CAP-002-PM1 (4.0 g, 5.49 mmol) was dissolved in dichloromethane and cooled to -40°C. Under nitrogen protection, 27.5 mL of a 1 M dichloromethane solution of boron trichloride was slowly added dropwise. After the addition was complete, the temperature was lowered from room temperature to 0°C and the reaction was continued for 5 hours. Subsequently, the reaction system was cooled again to -40°C, and methanol was added to quench the reaction system. The reaction solution was rotary-dried under reduced pressure, and the resulting crude product was purified by preparative high-pressure liquid chromatography to obtain YK-CAP-002-PM2 (490 mg, 1.47 mmol, yield 26.8%). C 13 H 14 N6O5, MS(ES): m / z(M+H + ) 335.18.

[0218] 1H 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).

[0219] Step 3: Synthesis of YK-CAP-002-PM3 Intermediate

[0220] YK-CAP-002-PM3 triethylamine salt (1.07 g, 2.08 mmol) was obtained using YK-CAP-002-PM2 (490 mg, 1.47 mmol) as a raw material according to the synthesis route of YK-CAP-001-PM5. C 13 H 15 N6O8P, MS(ES): m / z(MH - ) 413.07.

[0221] Step 4: Synthesis of YK-CAP-002-PM4 Intermediate

[0222] YK-CAP-002-PM4 (578.5 mg, 1.19 mmol) was obtained using YK-CAP-002-PM3 triethylamine salt (1.07 g, 2.08 mmol) as a raw material according to the synthesis route of YK-CAP-001-PM6. C 16 H 17 N8O7P, MS(ES): m / z(MH - ) 463.10.

[0223] Step 5: Synthesis of YK-CAP-002-PM5 Intermediate

[0224] YK-CAP-002-PM5 (391.9 mg, 0.66 mmol, yield 55.3%) was obtained using YK-CAP-002-PM4 (578.5 mg, 1.19 mmol) as a raw material and following the synthesis route of YK-CAP-001-PM7. C 13 H 16 N6O 11 P2, MS(ES): m / z(MH - )493.04.

[0225] Step 6: Synthesis of YK-CAP-002-PM6 Intermediate

[0226] YK-CAP-002-PM6 (58 mg, 0.1 mmol, yield 14.5%) was obtained using YK-CAP-002-PM5 (391.9 mg, 0.66 mmol) as a raw material and following the synthesis route of YK-CAP-001-PM8. C 14 H 18 N6O 11 P2, MS(ES): m / z(MH - ) 507.05.

[0227] Step 7: Synthesis of YK-CAP-002

[0228] YK-CAP-002 (17.5 mg, 14.02 μmol, yield 14.7%) was obtained using YK-CAP-002-PM6 (58 mg, 0.1 mmol) as a raw material according to the synthesis route of YK-CAP-001. C 35 H 44 N 16 O 24 P4, MS(ES): m / z(MH - ) 1195.14.

[0229] 1 H NMR (400 MHz, DO) 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 (DO, 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).

[0230] 5. Synthesis of YK-CAP-003

[0231]

[0232] Step 1: Synthesis of YK-CAP-003-PM1

[0233] YK-CAP-001-PM1 (22.00g, 30.14mmol) was dissolved in a mixed solution of dichloromethane (220mL), acetonitrile (220mL), and water (330mL), sodium periodate (52.06g, 241.12mmol) and ruthenium trichloride (250mg, 1.21mmol) were added to the solution, and the reaction system was stirred at 30℃ for 48 hours. After the reaction was completed, the solvent was rotary-dried, ethyl acetate (400 mL) was added to dissolve it, and it was washed three times with purified water (3 × 120 mL). The aqueous phase was back-extracted once with 200 mL of ethyl acetate, the organic phase was combined, the organic phase was dried with anhydrous sodium sulfate, filtered, and the filtrate was rotary-dried under vacuum and reduced pressure conditions. The residue was purified by silica gel chromatography to obtain YK-CAP-003-PM1 (19.87 g, 26.58 mmol, yield 88.2%). C 38 H 45 N5O 11 , MS(ES): m / z(M+H + )748.3.

[0234] Step 2: Synthesis of YK-CAP-003-PM2

[0235] YK-CAP-003-PM1 (19.87 g, 26.58 mmol) was dissolved in tetrahydrofuran (200 mL), and dimethylamine hydrochloride (2.38 g, 29.23 mmol), 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (12.13 g, 31.90 mmol), and N,N-diisopropylethylamine (10.31 g, 79.74 mmol) were sequentially added to the solution, and the reaction system was stirred overnight at room temperature. After the reaction was completed, the solvent was removed by rotating, ethyl acetate (400 mL) was added to dissolve it, and it was washed with purified water (3 × 120 mL). The aqueous phase was back-extracted once with 200 mL of ethyl acetate, the organic phase was dried with anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure under vacuum. The residue was purified by silica gel chromatography to obtain YK-CAP-003-PM2 (13.10 g, 16.91 mmol, 63.6%). C 40 H 50 N6O 10 , MS(ES): m / z(M+H + )775.3.

[0236] Step 3: Synthesis of YK-CAP-003-PM3

[0237] YK-CAP-003-PM2 (13.0 g, 16.78 mmol) was dissolved in dichloromethane (195 mL), and a 1 M dichloromethane solution of boron trichloride (134 mL, 134 mmol) was slowly added dropwise at -40°C. After the addition was complete, the temperature was raised to 0°C and the reaction system was stirred for 6 hours. After the reaction was complete, the solution was cooled to -40°C, and 200 mL of methanol was added to quench the reaction. The reaction solution was then rotated under vacuum and reduced pressure conditions to remove the solvent, and the solution was sealed and left at room temperature for 24 hours to obtain the crude product YK-CAP-003-PM3. This was added dropwise to 500 mL of dichloromethane, and the solid precipitated. The mixture was filtered to obtain a total of 7.89 g of cake. 2.0 g of this cake was taken and purified by HPLC to obtain pure YK-CAP-003-PM3 (956.04 mg, 2.51 mmol). C 15 H 20 N6O6, MS(ES): m / z(M+H + )381.32.

[0238] 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).

[0239] Step 4: Synthesis of YK-CAP-003-PM4 Intermediate

[0240] YK-CAP-003-PM4 triethylamine salt (921.3 mg, 1.64 mmol, yield 65.3%) was obtained using YK-CAP-003-PM3 (956.04 mg, 2.51 mmol) as a raw material according to the synthesis route of YK-CAP-001-PM5. C 15 H 21 N6O9P, MS(ES): m / z(MH - )459.11.

[0241] Step 5: Synthesis of YK-CAP-003-PM5 Intermediate

[0242] YK-CAP-003-PM5 (510.3 mg, 0.96 mmol, yield 58.4%) was obtained using YK-CAP-003-PM4 triethylamine salt (921.3 mg, 1.64 mmol) as a raw material according to the synthesis route of YK-CAP-001-PM6. C 18 H 23 N8O8P, MS(ES): m / z(MH - ) 509.12.

[0243] Step 6: Synthesis of YK-CAP-003-PM6 Intermediate

[0244] YK-CAP-003-PM6 (853.8 mg, 1.33 mmol) was obtained using YK-CAP-003-PM5 (510.3 mg, 0.96 mmol) as a raw material and following the synthesis route of YK-CAP-001-PM7. C 15 H 22 N6O 12 P2, MS(ES): m / z(MH - ) 539.08.

[0245] Step 7: Synthesis of YK-CAP-003-PM7 Intermediate

[0246] YK-CAP-003-PM7 (170.0 mg, 0.26 mmol) was obtained using YK-CAP-003-PM6 (853.8 mg, 1.33 mmol) as a raw material and following the synthesis route of YK-CAP-001-PM8. C 16 H 24 N6O 12 P2, MS(ES): m / z(MH - ) 553.09.

[0247] Step 8: Synthesis of YK-CAP-003

[0248] YK-CAP-003 (20.3 mg, 15.7 μmol, yield 6.0%) was obtained using YK-CAP-003-PM7 (170.0 mg, 0.26 mmol) as a raw material according to the synthesis route of YK-CAP-001. C 37 H 50 N16 O 25 P4, MS(ES): m / z(MH - )1241.19.

[0249] 1 H NMR (400 MHz, DO) 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 (DO, 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).

[0250] 6. Synthesis of YK-CAP-004

[0251]

[0252] Step 1: Synthesis of YK-CAP-004-PM1

[0253] YK-CAP-001-PM2 (12.65 g, 17.29 mmol) was dissolved in dichloromethane (70 mL), the reaction system was cooled to -40°C, and diethylamine sulfur trifluoride (8.36 g, 51.86 mmol) was slowly added dropwise to the dichloromethane solution. After the addition was complete, the reaction system was returned to 0°C and reacted for 4 hours. The reaction was monitored by LCMS to indicate completion. The reaction system was quenched with a saturated aqueous sodium bicarbonate solution, extracted with dichloromethane (200 mL × 3), the organic phase was combined, washed with 200 mL of a saturated aqueous sodium chloride solution, the organic phase was dried with anhydrous sodium sulfate, filtered, and rotary dried. Purified by silica gel chromatography, YK-CAP-004-PM1 (7.92 g, 10.51 mmol, yield 60.8%) was obtained. C 38 H 45 F2N5O9, MS(ES): m / z(M+H + )754.3.

[0254] Step 2: Synthesis of YK-CAP-004-PM2

[0255] YK-CAP-004-PM1 (4.00g, 5.31mmol) was used as a raw material and dissolved in dichloromethane (30mL). The reaction system was cooled to -40°C under a nitrogen gas atmosphere, and a 1M dichloromethane solution of boron trichloride (26.53mL, 26.53mmol) was slowly added dropwise. After the dropwise addition was complete, the reaction system was slowly heated to 0°C and stirred at the above temperature for 4 hours. After monitoring the completion of the reaction by TLC, the system was cooled back to -40°C, the reaction was quenched with methanol, rotary dried, left at room temperature for 24 hours, 200mL of dichloromethane was added, and the precipitated solid was filtered to obtain 2.0g of crude product. YK-CAP-004-PM2 (926.34 mg, 2.58 mmol, yield 48.8%) was obtained by purification using preparative HPLC. C 13 H 15 F2N5O5, MS(ES): m / z(M+H +)360.1.

[0256] 1 H 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).

[0257] Step 3: Synthesis of YK-CAP-004-PM3 Intermediate

[0258] YK-CAP-004-PM3 triethylamine salt (1.48 g, 2.74 mmol) was obtained according to the synthesis route of YK-CAP-001-PM5 using YK-CAP-004-PM2 (926.34 mg, 2.5 8 mmol) as a raw material. C 13 H 16 F2N5O8P, MS(ES): m / z(MH - ) 438.07.

[0259] Step 4: Synthesis of YK-CAP-004-PM4 Intermediate

[0260] YK-CAP-004-PM4 (771.8 mg, 1.51 mmol) was obtained using YK-CAP-004-PM3 triethylamine salt (1.48 g, 2.74 mmol) as a raw material according to the synthesis route of YK-CAP-001-PM6. C 16 H 18 F2N7O7P, MS(ES): m / z(MH - ) 488.08.

[0261] Step 5: Synthesis of YK-CAP-004-PM5 Intermediate

[0262] YK-CAP-004-PM5 (1.22 g, 1.97 mmol) was obtained using YK-CAP-004-PM4 (771.8 mg, 1.51 mmol) as a raw material according to the synthesis route of YK-CAP-001-PM7. C 13 H 17 F2N5O 11P2, MS(ES): m / z(MH - )518.04.

[0263] Step 6: Synthesis of YK-CAP-004-PM6 Intermediate

[0264] YK-CAP-004-PM6 (172.6 mg, 0.27 mmol) was obtained using YK-CAP-004-PM5 (1.22 g, 1.97 mmol) as a raw material according to the synthesis route of YK-CAP-001-PM8. C 14 H 19 F2N5O 11 P2, MS(ES): m / z(MH - ) 532.05.

[0265] Step 7: Synthesis of YK-CAP-004

[0266] YK-CAP-004 (22.3 mg, 17.5 μmol, yield 6.4%) was obtained using YK-CAP-004-PM6 (172.6 mg, 0.27 mmol) as a raw material according to the synthesis route of YK-CAP-001. C 35 H 42 F2N 15 O 24 P4, MS(ES): m / z(MH - )1220.15.

[0267] 1 H NMR (400 MHz, DO) 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 (DO, 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).

[0268] 7. Synthesis of YK-CAP-005

[0269]

[0270] Step 1: Synthesis of YK-CAP-005-PM1

[0271] Compound INT-PM2 (44.0 g, 0.10 mol) was dissolved in 450 mL of dichloromethane, protected by nitrogen gas, stirred, cooled to 0°C, pyridine (23.9 g, 0.30 mol) was added, followed by the dropwise addition of tert-butyldimethylsilyl trifluoromethanesulfonate (40.0 g, 0.15 mol). After the dropwise addition was completed, the temperature was raised to room temperature and the reaction was carried out. The completion of the reaction was monitored by TLC. The reaction system was subjected to direct vacuum, rotary dried, and purified with FLASH to obtain the colorless liquid YK-CAP-005-PM1 (47.0 g, 84.72 mmol, yield 84.82%).

[0272] Step 2: Synthesis of YK-CAP-005-PM2

[0273] Compound YK-CAP-005-PM1 (47.0 g, 84.72 mmol) was dissolved in 470 mL of tetrahydrofuran / 47 mL of water mixed solvent, 4-methylmorpholine-4-oxide (14.9 g, 0.13 mol) and potassium osmate (1.3 g, 0.003 mol) were added sequentially, and the mixture was reacted at room temperature for 5 hours. The completion of the reaction was monitored by TLC. The reaction solution was slowly poured into 500 mL of saturated sodium sulfite aqueous solution and separated. The aqueous phase was extracted with ethyl acetate (300 mL × 2), the organic phase was combined, washed sequentially with saturated saline (300 mL), dried with anhydrous sodium sulfate, and then vacuum-dried to obtain the crude product YK-CAP-005-PM2 (53.2 g, 90.35 mmol), which is a pale yellow liquid.

[0274] Step 3: Synthesis of YK-CAP-005-PM3

[0275] The crude product YK-CAP-005-PM2 (53.2 g, 90.35 mmol) was dissolved in 500 mL of tetrahydrofuran / 100 mL of water mixed solvent, sodium periodate (29.0 g, 0.14 mol) was added, and the reaction was carried out at room temperature for 3 hours. The completion of the reaction was monitored by TLC. The reaction solution was slowly poured into 500 mL of saturated sodium sulfite aqueous solution and separated. The aqueous phase was extracted with ethyl acetate (400 mL × 2), the organic phase was combined, washed sequentially with saturated saline (400 mL), dried with anhydrous sodium sulfate, and then vacuum-dried to obtain the yellow oily substance YK-CAP-005-PM3 (47.4 g, 85.13 mmol).

[0276] Step 4: Synthesis of YK-CAP-005-PM4

[0277] Compound YK-CAP-005-PM3 (47.4 g, 85.13 mmol) was dissolved in 500 mL of MeOH, protected by nitrogen gas, stirred, cooled to 0°C, and sodium borohydride (3.86 g, 0.10 mol) was added in batches. After the addition was completed, the reaction was continued for 3 hours. The completion of the reaction was monitored by TLC. The reaction system was cooled to 0°C, and a saturated aqueous ammonium chloride solution (300 mL) was added to quench the reaction. Then, the reaction was extracted with ethyl acetate (400 mL × 3), the organic phase was combined, washed sequentially with saturated saline solution (400 mL), dried with anhydrous sodium sulfate, and rotary dried under reduced pressure to obtain a crude product, YK-CAP-005-PM4 (49.0 g, 0.088 mol), which is a yellow oily substance.

[0278] Step 5: Synthesis of YK-CAP-005-PM5

[0279] The crude product YK-CAP-005-PM4 (49.0 g, 0.088 mol) was dissolved in 500 mL of dichloromethane, then triethylamine (26.6 g, 0.26 mol) was added, protected by nitrogen gas, stirred, cooled to 0°C, and ethylsulfonyl chloride (22.5 g, 0.18 mol) was added dropwise. After the dropwise addition was completed, the temperature was raised to room temperature and the reaction was carried out for 3 hours until the raw materials were consumed. The completion of the reaction was monitored by TLC. 500 mL of saturated sodium bicarbonate aqueous solution was added to the reaction system and separated. The aqueous phase was extracted with dichloromethane (400 mL × 2), the organic phase was washed with saturated saline solution (500 mL), dried with anhydrous sodium sulfate, and then vacuum-dried to obtain a crude product, YK-CAP-005-PM5 (58.0 g, 0.089 mol), which is a pale yellow oily substance.

[0280] Step 6: Synthesis of YK-CAP-005-PM6

[0281] The crude product YK-CAP-005-PM5 (58.0 g, 0.089 mol) was dissolved in 400 mL of N,N-dimethylformamide, sodium thiomethoxide (15.6 g, 0.22 mol) was added, and the reaction was carried out at room temperature for 4 hours until the reaction was complete. The completion of the reaction was monitored by TLC. The reaction solution was slowly poured into 1500 mL of water, the reaction system was extracted with ethyl acetate (500 mL × 3), the organic phase was combined, washed sequentially with saturated brine (1500 mL), dried with anhydrous sodium sulfate, and then vacuum-dried to obtain the brown oily substance YK-CAP-005-PM6 (50.0 g, 0.085 mol).

[0282] Step 7: Synthesis of YK-CAP-005-PM7

[0283] YK-CAP-005-PM6 (50.0 g, 0.085 mol) was dissolved in 500 mL of N,N-dimethylformamide, protected by nitrogen gas, stirred, cooled to 0°C, and m-chloroperoxybenzoic acid (33.7 g, 0.19 mol) was added in batches. After the dropwise addition was completed, the temperature was raised to room temperature and the reaction was continued for 3 hours until the raw materials disappeared. The completion of the reaction was monitored by TLC. The reaction solution was poured into a saturated sodium bicarbonate aqueous solution (1500 mL) and extracted with ethyl acetate (500 mL × 3). The organic phase was washed with saturated saline solution (1500 mL), dried with anhydrous sodium sulfate, and then vacuum-dried to obtain the yellow oily substance YK-CAP-005-PM7 (52.6 g, 84.72 mmol, yield 99.8%).

[0284] Step 8: Synthesis of YK-CAP-005-PM8

[0285] 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 reacted at room temperature for 1 hour. The completion of the reaction was monitored by TLC. 1000 mL of purified water and 800 mL of ethyl acetate were added to the reaction system, stirred for 5 minutes, and separated. The aqueous phase was extracted with ethyl acetate (500 mL × 2), the organic phase was combined, washed sequentially with saturated brine (1000 mL), dried with anhydrous sodium sulfate, vacuum-rotary dried, and purified by FLASH to obtain the yellow oily substance YK-CAP-005-PM8 (43.2 g).

[0286] Step 9: Synthesis of YK-CAP-005-PM9

[0287] YK-CAP-005-PM8 (18.7 g, 36.91 mmol) was dissolved in 200 mL of dichloromethane, then triethylamine (11.2 g, 0.11 mol) was added, the mixture was protected by nitrogen gas and stirred, cooled to 0°C, and ethylsulfonyl chloride (22.5 g, 0.18 mol) was added dropwise. After the addition was complete, the temperature was raised to room temperature and the reaction was carried out for 3 hours. The completion of the reaction was monitored by TLC. 400 mL of saturated sodium bicarbonate aqueous solution was added to the reaction system and separated. The aqueous phase was extracted with dichloromethane (200 mL × 2), the organic phase was washed with saturated saline (400 mL), dried with anhydrous sodium sulfate, vacuum-rotary dried, and purified with FLASH to obtain the pale yellow oily substance YK-CAP-005-PM9 (13.4 g, 22.38 mmol, yield 60.6%).

[0288] Step 10: Synthesis of YK-CAP-005-PM10

[0289] Compound YK-CAP-005-PM9 (13.4 g, 22.38 mmol) was dissolved in 130 mL of acetic acid, then acetic anhydride (13.7 g, 0.134 mol) was added, and concentrated sulfuric acid (0.27 mL) was added dropwise. After the addition was complete, the reaction was carried out at room temperature for 2 hours until the raw materials disappeared. The completion of the reaction was monitored by TLC. 400 mL of purified water and 400 mL of ethyl acetate were added to the reaction system, stirred for 5 minutes, and separated. The aqueous phase was extracted with ethyl acetate (300 mL × 2), the organic phase was combined, and the pH was sequentially adjusted to alkaline with a saturated sodium bicarbonate aqueous solution. Then, the mixture was washed with saturated brine (1.0 L), dried with anhydrous sodium sulfate, subjected to reduced pressure, and rotary dried to obtain the yellow oily substance YK-CAP-005-PM10 (13.4 g, 20.85 mmol, yield 93.18%).

[0290] Step 11: Synthesis of YK-CAP-005-PM11

[0291] 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 reaction system was heated to 80°C to clarify the solution. The heat was removed, and the mixture was cooled to room temperature. While stirring, a toluene solution (50 mL) of compound YK-CAP-005-PM10 (12.8 g, 19.92 mmol) was added, followed by the dropwise addition of trimethylsilyl trifluoromethanesulfonate (6.6 g, 0.03 mol). The mixture was heated until reflux occurred, and the reaction system was stirred for 2 hours until the raw materials were consumed. The completion of the reaction was monitored by TLC. The reaction system was cooled to room temperature, saturated sodium bicarbonate solution (300 mL) was added and quenched, then ethyl acetate (300 mL) was added, filtered with diatomaceous earth, and separated. The aqueous phase was extracted with ethyl acetate (200 mL × 2), the organic phase was combined, washed sequentially with saturated brine (300 mL × 1), dried with anhydrous sodium sulfate, and rotary dried to obtain the yellow viscous substance YK-CAP-005-PM11 (15.5 g, 20.60 mmol).

[0292] Step 12: Synthesis of YK-CAP-005-PM12

[0293] Compound YK-CAP-005-PM11 (15.5 g, 20.60 mmol) was dissolved in 200 mL of anhydrous methanol, and then 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 the reaction was complete, the reaction system was subjected to direct reduced pressure and rotary dried, and purified by flash to obtain the yellow solid YK-CAP-005-PM12 (11.8 g, 19.81 mmol). C 29 H 33 N5O7S, MS(ES): m / z(M+H + )596.38.

[0294] Step 13: Synthesis of YK-CAP-005-PM13

[0295] Compound YK-CAP-005-PM12 (11.8g, 19.81mmol) was dissolved in 200mL of dichloromethane, protected by nitrogen gas, cooled to -40℃, and added dropwise to a 1M dichloromethane solution of boron trichloride (167mL). After the dropwise addition was completed, the reaction system was heated to 0℃ and the reaction was carried out. The completion of the reaction was monitored by LC-MS. After the reaction was completed, the reaction system was cooled to -40℃, methanol (200mL) was added dropwise to quench the reaction, and the mixture was subjected to direct vacuum and rotary drying. Then, 120mL of a 1,4-dioxane solution of 4M hydrochloric acid was added, and the temperature was raised to 60℃ until the reaction was complete. After the reaction was completed, the reaction system was depressurized and rotary dried. Methanol (40 mL) was added to dissolve the mixture, which was then added dropwise to 800 mL of dichloromethane to precipitate a solid. The mixture was filtered by suction and dried to obtain the yellow solid YK-CAP-005-PM13 (7.0 g, 17.44 mmol, yield 88.1%). C 14 H 19 N5O7S, MS(ES): m / z(M+H + )402.18.

[0296] 1 ¹H NMR (400 MHz, DMSO- d 6) 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).

[0297] Step 14: Synthesis of YK-CAP-005-PM14 Intermediate

[0298] YK-CAP-005-PM14 triethylamine salt (639.8 mg, 1.10 mmol, yield 13.0%) was obtained using YK-CAP-005-PM13 (3.4 g, 8.47 mmol) as a raw material and following the synthesis route of YK-CAP-001-PM5. C 14 H 20 N5O 10 PS, MS(ES): m / z(MH - ) 480.07.

[0299] Step 15: Synthesis of YK-CAP-005-PM15 Intermediate

[0300] YK-CAP-005-PM15 (333.4 mg, 0.60 mmol, yield 54.9%) was obtained by using YK-CAP-005-PM14 triethylamine salt (639.8 mg, 1.10 mmol) as a raw material and following the synthesis route of YK-CAP-001-PM6.C 17 H 22 N7O9PS, MS(ES): m / z(MH - )530.08.

[0301] Step 16: Synthesis of YK-CAP-005-PM16 Intermediate

[0302] YK-CAP-005-PM16 (528 mg, 0.80 mmol) was obtained using YK-CAP-005-PM15 (333.4 mg, 0.60 mmol) as a raw material according to the synthesis route of YK-CAP-001-PM7. C 14 H 21 N5O 13 P2S, MS(ES): m / z(MH - )560.03.

[0303] Step 17: Synthesis of YK-CAP-005-PM17 Intermediate

[0304] YK-CAP-005-PM17 (74.7 mg, 0.11 mmol) was obtained using YK-CAP-005-PM16 (528 mg, 0.80 mmol) as a raw material and following the synthesis route of YK-CAP-001-PM8. C 15 H 23N5O 13 P2S, MS(ES): m / z(MH - ) 574.05.

[0305] Step 18: Synthesis of YK-CAP-005

[0306] YK-CAP-005 (19.3 mg, 14.7 μmol, 13.3%) was obtained using YK-CAP-005-PM17 (74.7 mg, 0.11 mmol) as a raw material and following the synthesis route of YK-CAP-001. C 36 H 49 N 15 O 26 P4S, MS(ES): m / z(MH - ) 1262.14.

[0307] 1 H NMR (400 MHz, DO) 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); 31 P NMR (DO, 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).

[0308] 8. Synthesis of YK-CAP-006

[0309]

[0310] Step 1: Synthesis of YK-CAP-006-PM1

[0311] Triphenylphosphine (11.27 g, 42.98 mmol) was dissolved in 150 mL of tetrahydrofuran, the reaction system was cooled to 0°C, diisopropyl azodicarboxylate (14.48 g, 71.64 mmol) was added and stirred for 10 minutes, a tetrahydrofuran solution of compound YK-CAP-005-PM4 (20.0 g, 35.79 mmol) was added, followed by the slow dropwise addition of thioacetic acid (5.45 g, 71.64 mmol). After the dropwise addition was complete, the system was slowly returned to room temperature, and the reaction system was stirred for 16 hours while monitoring the completion of the reaction by TLC. After the reaction was complete, the reaction was quenched with water, the aqueous phase was extracted with ethyl acetate (200 mL × 2), the organic phase was combined, washed with saturated sodium chloride, dried with anhydrous sodium sulfate, filtered, and rotary dried. YK-CAP-006-PM1 (14.4g, 23.34mmol, yield 65.2%), a colorless oily liquid, was obtained by purification with FLASH.

[0312] Step 2: Synthesis of YK-CAP-006-PM2

[0313] Compound N-chlorosuccinimide (12.48 g, 93.48 mmol) was dissolved in 100 mL of acetonitrile and 20 mL of 2N aqueous hydrochloric acid solution, and the solution was slowly added dropwise to an acetonitrile solution of compound YK-CAP-006-PM1 (14.4 g, 23.34 mmol) and reacted for 10 minutes, and the completion of the reaction was monitored by LCMS. 30 mL of water was added to the reaction system to quench the reaction, the aqueous phase was extracted with ethyl acetate (100 mL × 2), the organic phase was combined, washed with saturated sodium chloride, dried with anhydrous sodium sulfate, filtered, and rotary dried. A colorless oily liquid, YK-CAP-006-PM2 (13.4 g, 20.90 mmol, yield 89.5%), was obtained.

[0314] Step 3: Synthesis of YK-CAP-006-PM3

[0315] 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. After cooling the reaction system to 0°C, 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 for 30 minutes at 0°C, and the completion of the reaction was monitored using LCMS. The reaction system was subjected to direct vacuum and rotary drying, dissolved in a small amount of dichloromethane, and purified by FLASH to obtain the colorless liquid YK-CAP-006-PM3 (6.0 g, 9.23 mmol, yield 44.2%).

[0316] Step 4: Synthesis of YK-CAP-006-PM4

[0317] 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 and reacted 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 sodium bicarbonate aqueous solution, separated, the aqueous phase was extracted with ethyl acetate (100 mL × 2), the organic phase was combined, washed sequentially with saturated saline (50 mL), dried with anhydrous sodium sulfate, vacuum-rotary dried, and purified by FLASH to obtain the colorless liquid YK-CAP-006-PM4 (4.0 g, 7.47 mmol, yield 80.8%).

[0318] Step 5: Synthesis of YK-CAP-006-PM5

[0319] Compound YK-CAP-006-PM4 (4.0 g, 7.47 mmol) was dissolved in 200 mL of dichloromethane solution, triethylamine (2.27 g, 22.41 mmol) was added, the reaction system was cooled to 0°C, and a dichloromethane solution of ethylsulfonyl chloride (1.15 g, 8.96 mmol) was slowly added dropwise. After the addition was complete, the reaction system was reacted at 0°C for 2 hours. The completion of the reaction was monitored by TLC. The reaction solution was slowly poured into 50 mL of saturated sodium bicarbonate aqueous solution, the reaction was quenched, ethyl acetate was added to separate the liquids, the mixture was extracted with ethyl acetate (150 mL × 2), the organic phase was combined, the mixture was sequentially washed with saturated brine (500 mL), dried with anhydrous sodium sulfate, vacuum-rotary dried, and purified with FLASH to obtain the colorless liquid YK-CAP-006-PM5 (4.4 g, 7.01 mmol, yield 93.9%).

[0320] Step 6: Synthesis of YK-CAP-006-PM6

[0321] Compound YK-CAP-006-PM5 (4.4g, 7.01mmol) was dissolved in 30mL of acetic acid, acetic anhydride (4.3g, 42.12mmol) was added, and an acetic acid solution of concentrated sulfuric acid (44μL) was slowly added dropwise under conditions of stirring at room temperature, and the mixture was stirred for 1 hour at room temperature. The completion of the reaction was monitored by TLC. The reaction solution was slowly added dropwise to 500mL of saturated sodium bicarbonate aqueous solution, the pH was adjusted to neutral, and then extracted with ethyl acetate (300mL × 3). The organic phase was combined, washed with 50mL of saturated saline solution, dried with anhydrous sodium sulfate, filtered, and rotary dried under reduced pressure to obtain the colorless oily substance YK-CAP-006-PM6 (3.1g, 4.61mmol, yield 65.8%).

[0322] Step 7: Synthesis of YK-CAP-006-PM7

[0323] 2-amino-6-chloroguanine (862.6 mg, 5.08 mmol) was dissolved in 30 mL of toluene, N,O-bis(trimethylsilyl)acetamide (2.07 g, 10.16 mmol) was weighed and added, the reaction system was heated to 80°C under nitrogen gas protection and stirred until the solution dissolved clearly, then heating was stopped and returned to room temperature, then a toluene solution of compound YK-CAP-006-PM6 (3.1 g, 4.61 mmol) was slowly added, followed by the addition of trimethylsilyl trifluoromethanesulfonate (1.13 g, 5.08 mmol), the temperature was immediately raised to 110°C and the reaction was carried out for 2 hours, and the reaction was monitored by LCMS to indicate completion. Water (15 mL) and ethyl acetate (20 mL) were added to the reaction system, and a solid was precipitated in the reaction system. Diatomaceous earth was added and filtered, and the solution was extracted with ethyl acetate (80 mL × 2). The organic phase was washed with saturated brine (20 mL), dried with anhydrous sodium sulfate, vacuum-dried, rotary dried, and purified with FLASH to obtain the colorless oily substance YK-CAP-006-PM7 (3.0 g, 3.84 mmol, yield 83.1%).

[0324] Step 8: Synthesis of YK-CAP-006-PM8

[0325] Compound YK-CAP-006-PM7 (3.0 g, 3.84 mmol) was dissolved in 20 mL of methanol, potassium carbonate (15.6 g, 23.10 mmol) was added, and the reaction was carried out at room temperature for 24 hours, and the completion of the reaction was monitored by LCMS. The mixture was filtered, the filtrate was stirred, and purified with FLASH to obtain the colorless oily substance YK-CAP-006-PM8 (2.1 g, 3.36 mmol, yield 87.5%).

[0326] Step 9: Synthesis of YK-CAP-006-PM9

[0327] YK-CAP-006-PM8 (2.1 g, 3.36 mmol) was dissolved in 25 mL of dichloromethane, protected by nitrogen gas, stirred, cooled to 0°C, and boron trichloride (3.94 g, 33.60 mmol) was slowly added dropwise. After the addition was complete, the reaction was continued at 0°C for 6 hours, and the disappearance of the raw material was monitored using LCMS. The mixture was cooled to -40°C, and methanol was added to quench the reaction. The mixture was rotary dried, and the target compound was monitored using LCMS. The product was dissolved in 20 mL of methanol and slowly added dropwise to 150 mL of stirred dichloromethane, resulting in the precipitation of a white solid and yielding 1.7 g of crude product. The crude product was purified by preparative high-pressure liquid chromatography to obtain the solid white YK-CAP-006-PM9 (1.012 g, 2.35 mmol, yield 69.94%).

[0328] 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).

[0329] Step 10: Synthesis of YK-CAP-006-PM10

[0330] YK-CAP-006-PM10 triethylamine salt (1.05 g, 1.72 mmol, yield 73.2%) was obtained using YK-CAP-006-PM9 (1.01 g, 2.35 mmol) as a raw material according to the synthesis route of YK-CAP-001-PM5. C 15 H 23 N6O 10 PS, MS(ES): m / z(MH - ) 509.09.

[0331] Step 11: Synthesis of YK-CAP-006-PM11

[0332] YK-CAP-006-PM11 (809.2 mg, 1.39 mmol, yield 80.9%) was obtained using YK-CAP-006-PM10 triethylamine salt (1.05 g, 1.72 mmol) as a raw material and following the synthesis route of YK-CAP-001-PM6. C 18 H 25 N8O9PS, MS(ES): m / z(MH - ) 559.10.

[0333] Step 12: Synthesis of YK-CAP-006-PM12

[0334] YK-CAP-006-PM12 triethylamine salt (475.5 mg, 0.69 mmol, yield 49.5%) was obtained using YK-CAP-006-PM11 (809.2 mg, 1.39 mmol) as a raw material and following the synthesis route of YK-CAP-001-PM7. C 15 H 24 N6O 13 P2S, MS(ES): m / z(MH - ) 589.06.

[0335] Step 13: Synthesis of YK-CAP-006-PM13

[0336] YK-CAP-006-PM13 triethylamine salt (235 mg, 0.33 mmol, yield 48.4%) was obtained using YK-CAP-006-PM12 triethylamine salt (475.5 mg, 0.69 mmol) as a raw material according to the synthesis route of YK-CAP-001-PM8. C 16 H 26 N6O 13 P2S, MS(ES): m / z(MH - ) 603.08.

[0337] Step 14: Synthesis of YK-CAP-006

[0338] YK-CAP-006 (32.0 mg, 23.8 μmol, yield 7.1%) was obtained using YK-CAP-006-PM13 (235 mg, 0.33 mmol) as a raw material according to the synthesis route of YK-CAP-001. C 37 H 52 N 16 O 26 P4S, MS(ES): m / z(MH - ) 1291.17.

[0339] 1 H NMR (400 MHz, DO) 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 (DO, 202 MHz) δ -0.91 (s, 1P), -11.47 (m, 2P), -22.80 (t, J = 17.8 Hz, 1P).

[0340] Example 2: mRNA in vitro transcription yield and capping rate

[0341] The chemical structures of compounds YK-CAP-001 to 006 of the present application are very similar, and compared to mRNA capping analogs disclosed in the prior art, some of these series of compounds have similar structures and some have very large differences.

[0342] The modified locking nucleoside capping analog of the present application showed significant differences in the in vitro transcription yield and capping rate of mRNA. For example, the transcription yield of YK-CAP-004 was improved by 82.8% compared to YK-CAP-001, and the capping rate was improved by 26.1% compared to YK-CAP-002. Compared to modified locking nucleoside capping analogs of the prior art, the modified locking nucleoside capping analog of the present application showed significant improvements in both the in vitro transcription yield and capping rate of mRNA. For example, the transcription yield and capping rate of YK-CAP-004 were improved by 35.0% and 18.8%, respectively, compared to Compound 14.

[0343] I. Structural Differences in Capping Analogues

[0344]

[0345]

[0346]

[0347] As can be seen from Table 1, the chemical structures of compounds YK-CAP-001 to 006 of the present application are very similar, and compared to mRNA capping analogs disclosed in the prior art, some of these compounds have similar structures while others have very large differences, specifically as follows.

[0348] 1. The structures of the compounds YK-CAP-001, YK-CAP-002, YK-CAP-003, YK-CAP-004, YK-CAP-005, and YK-CAP-006 of the present application are very similar, differing only in the substituent at C6', which is the group connected to the methylene bridge connecting the 2'-O and C4' of the ribose ring; 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 other structures are completely identical.

[0349] 2. Compounds YK-CAP-001 to 006 of the present application are similar in structure to compound 14, but in that there are no substituents on the methylene bridge connecting the 2'-O and C4' of the ribose ring of compound 14, that is, there are no substituents on C6'.

[0350] 3. The compounds YK-CAP-001 to 006 of the present application differ relatively significantly in structure from N-7413, HN3002 and m6A, and there is no methylene bridge between the 2'-O and C4' of the ribose ring of these three compounds, and the second base adenine of m6A is modified by methylation.

[0351] II. Measurement of mRNA In Vitro Transcription Yield and Capping Rate

[0352] 1. Experimental Method

[0353] (1) Capping synthesis using capping analogs

[0354] First, the plasmid was linearized using a plasmid linearization enzyme, and then the linearized plasmid was purified.

[0355] (2) In vitro transcription synthesis of mRNA

[0356] YK-CAP-001 to 006 and compound 14 of Table 1 were each used as capping analogs, and the reaction system is as shown in Table 2.

[0357]

[0358] In the experimental procedure, the volume of materials required for the reaction system was calculated first, and then the samples were added. First, enzyme-free sterile water was added to the reaction system, followed by the sequential addition of 10× buffer, NTPs, and capping analogs. After uniform mixing and gentle centrifugation, nuclease inhibitors, inorganic pyrophosphatase, T7 RNA polymerase, and linearized DNA templates were added and thoroughly mixed, followed by gentle centrifugation and incubation at 37°C. After 2 hours, 1U of DNase I was added, and the mixture was incubated at 37°C for 30 minutes. Subsequently, the mRNA precipitate was washed with 75% ethanol, the ethanol was briefly evaporated to dry the precipitate, and the mRNA was redissolved with enzyme-free sterile water.

[0359] (3) The transcription product was purified and the mRNA in vitro transcription yield was recorded.

[0360] (4) The obtained mRNA was subjected to an annealing reaction with a probe.

[0361] Annealing was performed in a PCR machine at 95°C for 5 minutes, 65°C for 2 minutes, 55°C for 2 minutes, 40°C for 2 minutes, and 22°C for 2 minutes.

[0362] (5) Magnetic bead pretreatment and probe binding: For pretreatment, 100 μL of magnetic beads were taken and placed on a magnetic stand. 120 μL of sample and magnetic bead solution were added and incubated at room temperature for 30 minutes, and mixed slowly and uniformly during incubation.

[0363] (6) Cut the mRNA and bind the mRNA to the probe 5 ， A single-strand sequence was obtained.

[0364] 20 μL of Rnase H (5 U / μL) was added, and the mixture was incubated at 37°C for 3 hours and mixed once every 30 minutes. After incubation, the magnetic beads were washed, and 100 μL of 75% methanol heated to 80°C was added to the washed beads. The mixture was heated to 80°C on a heating plate and maintained for 3 minutes, then left on a magnetic stand. The supernatant was aspirated, and the mixture was 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, which is suitable for LC-MS analysis, to determine the RNA capping status during the transcription reaction. Since there is a distinct difference in molecular weight between capping and non-capping bases, the difference in molecular mass was used to determine the capping rate of mRNA transcription initiated by different capping analogs.

[0365] 2. Experimental Results

[0366] According to the measurement results of the in vitro transcription yield and capping rate of mRNA, the modified locking nucleoside capping analog of the present application was found to have a significant difference in the in vitro transcription yield and capping rate of mRNA. For example, the transcription yield of YK-CAP-004 was improved by 82.8% compared to YK-CAP-001, and the capping rate was improved by 26.1% compared to YK-CAP-002. Compared to modified locking nucleoside capping analogs of the prior art, the modified locking nucleoside capping analog of the present application showed a significant improvement in both the in vitro transcription yield and capping rate of mRNA. For example, the transcription yield and capping rate of YK-CAP-004 were improved by 35.0% and 18.8%, respectively, compared to Compound 14.

[0367] Specific mRNA in vitro transcription yield and capping rate are shown in Table 3.

[0368]

[0369] 1) The modified lock nucleoside capping analogs of the present application showed significant differences in the in vitro transcription yield and capping rate of mRNA. Both the transcription yield and capping rate of YK-CAP-004 were the highest, with the transcription yield improving by 82.8% compared to the lowest YK-CAP-001 and the capping rate improving by 26.1% compared to the lowest YK-CAP-002.

[0370] As can be seen in Table 3, all modified lock nucleoside capping analogs of the present application were able to transcribe mRNA. The differences in mRNA transcription activity among the different modified lock nucleoside capping analogs were very significant, and the highest in vitro mRNA transcription yield was YK-CAP-004, reaching 163.2 μg, while the yields of YK-CAP-003, YK-CAP-005, and YK-CAP-006 were also very high, exceeding 150 μg with 151.0 μg, 152.6 μg, and 153.1 μg, respectively.

[0371] The lowest in vitro transcription yield of mRNA was found in YK-CAP-001 at only 89.3 μg, and the yield of YK-CAP-002 was also very low at only 103.1 μg. The transcription yield of YK-CAP-004 was significantly improved, increasing by 82.8% compared to YK-CAP-001 and by 58.3% compared to YK-CAP-002, respectively. (Fig. 1)

[0372] The capping rate was highest for YK-CAP-004, reaching 96.3%, and YK-CAP-003, YK-CAP-005, and YK-CAP-006 were also very high, exceeding 90% with 94.8%, 95.2%, and 95.1%, respectively.

[0373] The lowest capping rate was found in YK-CAP-002 at only 70.2%, and the capping rate of YK-CAP-001 was also very low at only 73.1%. The capping rate of YK-CAP-004 was significantly improved, increasing by 26.1% compared to YK-CAP-002 and by 23.2% compared to YK-CAP-001, respectively. (Fig. 2)

[0374] 2) Compared to modified lock nucleoside capping analogs of the present application, both mRNA in vitro transcription yield and capping rate were significantly improved. For example, the transcription yield and capping rate of YK-CAP-004 were improved by 35.0% and 18.8%, respectively, compared to compound 14.

[0375] The in vitro mRNA transcription yield of compound 14 is 120.9 μg, and YK-CAP-004, YK-CAP-003, YK-CAP-005, and YK-CAP-006 in this application were significantly improved by 35.0%, 24.9%, 26.2%, and 26.6%, respectively, compared to compound 14.

[0376] The capping rate of compound 14 was 77.5%, and 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.

[0377] 3) Structurally similar modified lock nucleoside capping analogs showed very large differences in mRNA transcription yield and capping rate.

[0378] The structures between each modified lock nucleoside capping analog designed in this application are very similar, and this series of compounds was also very similar to the structure of compound 14. However, there were very large differences in mRNA in vitro transcription yield and capping rate.

[0379] For example, compared to YK-CAP-001, YK-CAP-002, and Compound 14, YK-CAP-004 differed only in the substituent at C6', which is the group connected to the methylene bridge connecting the 2'-O and C4' of the ribose ring; YK-CAP-004 is 2,2-difluoroethyl, YK-CAP-001 is dimethylaminoethyl, and YK-CAP-002 is cyanomethyl, whereas Compound 14 has no substituents, and other structures were completely identical. However, the mRNA in vitro transcription yield of YK-CAP-004 was significantly improved by 82.8%, 58.3%, and 35.0% compared to 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.

[0380] From this, it was found that modified lock nucleoside capping analogs with similar structures do not necessarily have similar mRNA transcription activity and capping rates, but on the contrary, there is a very high possibility that they have very large differences.

[0381] As can be seen from the mRNA in vitro transcription yield and capping rate, the modified lock nucleoside capping analogs YK-CAP-004, YK-CAP-003, YK-CAP-005, and YK-CAP-006 of the present application both showed 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.

[0382] The modified locking nucleoside capping analogs of the present application, YK-CAP-004, YK-CAP-003, YK-CAP-005, and YK-CAP-006, replace the original pentagonal sugar ring structure with a bridge ring structure and introduce 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 4C' of the ribose ring. Since the bridge ring structure cannot be used as a transcription start site, they have an excellent anti-reverse transcription effect during mRNA in vitro transcription and significantly increase the binding ability of the cap structure to the capping enzyme, thereby increasing the capping rate of the transcribed mRNA.

[0383] In addition, it was found that modified lock nucleoside capping analogs with similar structures do not necessarily have similar mRNA transcriptional activity and capping rates, and on the contrary, are highly likely to have very large differences.

[0384] Example 3: Preparation and Characterization of Lipid Nanoparticles

[0385] 1. Experimental Method

[0386] Cationic lipid YK-009 (Beijing Youcare Kechuang Pharmaceutical Technology Co., Ltd.), DSPC (AVT (Shanghai) Pharmaceutical Technology Co., Ltd.), cholesterol (AVT (Shanghai) Pharmaceutical Technology Co., Ltd.), and DMG-PEG2000 were dissolved in ethanol at a molar ratio of 49:10:39.5:1.5, and mRNA was diluted in 50 mM citrate buffer at pH=4. Using a microfluidic device, the ethanol lipid solution and the aqueous Fluc mRNA solution prepared with the above different capping structures were mixed at a volume ratio of 1:3 at a flow rate of 10 mL / min to prepare LNPs with a total lipid to mRNA weight ratio of approximately 15:1. The obtained liposomes were diluted to a 10-fold volume with PBS, and then ultrafiltered through a 300 kDa ultrafiltration tube to remove ethanol. Next, the lipid nanoparticles were fixed to a constant volume with PBS, and finally, the lipid nanoparticles were filtered through a 0.2 μm sterile filter to obtain an LNP preparation in which Fluc-mRNA was encapsulated with YK-009 / DSPC / cholesterol / DMG-PEG2000 (molar ratio 49:10:39.5:1.5).

[0387] The polydispersity index (PDI) was measured using a Malvern laser particle size analyzer based on dynamic light scattering. 10 μL of liposome solution was taken, diluted to 1 mL with RNA enzyme-free deionized water, added to the sample pool, and each sample was measured three times. Measurement conditions: 90° scattering angle, 25°C. The encapsulation efficiency of lipid nanoparticles was determined using the Quant-it Ribogreen RNA quantification kit (Thermo Fisher Scientific, UK) according to the manufacturer's instructions.

[0388] 2. Experimental Results

[0389] Specific characterization data of lipid nanoparticles are shown in Table 4.

[0390]

[0391] As can be seen in Table 4, Fluc mRNA transcribed from the capping analogs YK-CAP-001 to 006 of the present application and the capping analogs N-7413, Compound 14, HN3002, and m6A disclosed in the prior art were all capable of producing good lipid nanoparticles. The particle size of all lipid nanoparticles was between 82 and 103 nm, the PDI value was between 0.018 and 0.065, and the encapsulation efficiency was all 90% or higher.

[0392] Example 4: Translation efficiency of different capping luciferase mRNAs

[0393] The modified locking nucleoside capping analogs of the present application showed significant differences in mRNA translation efficiency. YK-CAP-004 had the highest translation efficiency, which is twice that of the lowest, YK-CAP-002. Compared to modified locking nucleoside capping analogs of the existing technology that have similar or very different structures, the mRNA translation efficiency of the modified locking nucleoside capping analogs of the present application was significantly improved. For example, the translation efficiency of YK-CAP-004 reached four times that of m6A.

[0394] 1. Experimental Method

[0395] (1) HEK293T cells were cultured in DMEM medium containing 10% FBS and penicillin / streptomycin at 37°C under 5% CO2 conditions.

[0396] (2) Digest and count the cells in the culture dish, plate 10,000 cells per well in a 96-well plate, and culture overnight until the cells attached to the wall.

[0397] (3) Transfection was performed when the cell density was about 80%, and 0.5 μg of mRNA sample and Lipofectamine Messenger MAX Transfection Reagent (Invitrogen) were added to each well, and the transfection step was performed by referring to the guidelines.

[0398] (4) Transfected cells were cultured continuously for 24 hours at 37°C in 5% CO2, after which the growth medium was removed from the cells to be tested and the cells were rinsed with PBS. After removing the PBS by centrifugation, 50 μL of 1× lysis buffer was added, and the cells and all liquid were transferred to a microcentrifuge tube and centrifuged.

[0399] (5) 20 μL of sample was taken and 100 μL of Dual-Lumi™ II firefly luciferase detection reagent equilibrated at room temperature was added and mixed appropriately and uniformly.

[0400] (6) The luminescence signal was stabilized by incubating at room temperature (approx. 25°C) for 5 minutes. Chemiluminescence detection was performed using a multifunctional microplate reader equipped with a chemiluminescence detection function, and the data was recorded. The relative fluorescence readings of specific capping mRNAs are shown in Table 5.

[0401] 2. Experimental Results

[0402] The relative fluorescence readings of capping mRNA are shown in Table 5, and the relative fluorescence intensity was directly proportional to the translation efficiency of the mRNA.

[0403]

[0404] 1) The modified lock nucleoside capping analogs of the present application showed significant differences in mRNA translation efficiency. YK-CAP-004 had the strongest translation efficiency, which was twice that of the lowest YK-CAP-002.

[0405] As can be seen in Table 5, there were significant differences in the relative fluorescence intensity (corresponding to the translation efficiency of mRNA) of the modified lock nucleoside capping analogs of the present application. The highest intensity was found in YK-CAP-004, which reached a relative fluorescence intensity of 1.41, while the relative fluorescence intensities of YK-CAP-003, YK-CAP-005, and YK-CAP-006 also reached 1.23, 1.28, and 1.31, respectively, all of which were relatively high.

[0406] The lowest relative fluorescence intensity was found in YK-CAP-002, which was only 0.69. The relative fluorescence intensity of YK-CAP-001 was also very low, at only 0.78. The relative fluorescence intensities of YK-CAP-004, YK-CAP-003, YK-CAP-005, and YK-CAP-006 were 2.0, 1.8, 1.9, and 1.9 times that of YK-CAP-002, respectively, and 1.8, 1.6, 1.6, and 1.7 times that of YK-CAP-001. (Fig. 3)

[0407] 2) Compared to modified lock nucleoside capping analogs of the present application, the mRNA translation efficiency of all modified lock nucleoside capping analogs of the prior art was significantly improved. For example, the translation efficiency of YK-CAP-004 reached four times that of m6A.

[0408] The relative fluorescence intensities (corresponding to the translation efficiency of mRNA) of N-7413, HN3002, and m6A were 1.00, 1.13, and 0.35, respectively. In the present application, the relative fluorescence intensities of YK-CAP-004, YK-CAP-003, YK-CAP-005, and YK-CAP-006 were 1.4 times, 1.2 times, 1.3 times, and 1.3 times of N-7413, 1.2 times, 1.1 times, 1.1 times, and 1.2 times of HN3002, and 4.0 times, 3.5 times, 3.7 times, and 3.7 times of m6A, respectively.

[0409] 3) Structurally similar modified lock nucleoside capping analogs showed very large differences in mRNA translation efficiency.

[0410] Although the structures of the modified lock nucleoside capping analogs designed in this application are very similar, there was a very large difference in mRNA translation efficiency.

[0411] For example, compared to YK-CAP-001 and YK-CAP-002, YK-CAP-004 differs only in the substituent at C6', which is the group connected to the methylene bridge linking the 2'-O and C4' of the ribose ring; YK-CAP-004 is 2,2-difluoroethyl, YK-CAP-001 is dimethylaminoethyl, and YK-CAP-002 is cyanomethyl, while the other structures are completely identical. However, the translation efficiency of YK-CAP-004 mRNA was significantly improved by 1.8 times and 2.0 times compared to YK-CAP-001 and YK-CAP-002, respectively.

[0412] From this, it was found that the translation efficiency of luciferase mRNA of modified lock nucleoside capping analogs with similar structures is not necessarily similar, and on the contrary, it is highly likely that there is a very large difference.

[0413] As can be seen from the translation efficiency of different capping luciferase mRNAs, the modified lock 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 translation efficiency of mRNA compared to modified lock nucleoside capping analogs with similar structures (including YK-CAP-001 and YK-CAP-002 of the present application) or modified lock nucleoside capping analogs with large structural differences (including N-7413, HN3002, and m6A). This indicates that the bridge ring nucleoside structures of the substituents 2,2-difluoroethyl (YK-CAP-004), dimethylaminocarbonylmethyl (YK-CAP-003), methylsulfonylethyl (YK-CAP-005), and dimethylaminosulfonylethyl (YK-CAP-006) can further stabilize the shape of the pentaneous sugar ring and bind more easily to the cap-binding protein (EIF4E), thereby improving the translation efficiency of the target mRNA.

[0414] In addition, it was found that the translation efficiency of luciferase mRNA of modified lock nucleoside capping analogs with similar structures is not necessarily similar, and on the contrary, it is highly likely to have a very large difference.

[0415] Example 5. Decapping enzyme stability test

[0416] The modified lock nucleoside capping analogs of the present application showed significant differences in decapping rates. The decapping rate of YK-CAP-004 was the lowest, decreasing by 16.0% compared to the highest, YK-CAP-001. Compared to modified lock nucleoside capping analogs of the prior art that have similar or very different structures, the decapping rates of the compounds of the present application were significantly reduced. For example, the decapping rate of YK-CAP-004 was reduced by 38.3% compared to N-7413.

[0417] 1. Experimental Method

[0418] 30 pmol RNAs purified by polyacrylamide gel electrophoresis (PAGE) were enzyme-catalyzed with 50 U mRNA decapsulase (New England Biolabs) and 1× MDE buffer at 37°C for 45 minutes. The enzyme-catalyzed reaction mixture was stained with PAGE electrophoresis SYBR Green II (Lonza), and the gel plots were observed after electrophoresis using a Typhoon FLA 7000 (GE Healthcare) instrument. The ratio of electrophoretic band intensities between capping RNA and decapsulase RNA was statistically analyzed using Image Quant (GE Healthcare) software, and the decapping rate of the decapsulase was calculated (see Table 6).

[0419] 2. Experimental Results

[0420]

[0421] 1) The modified lock nucleoside capping analogs of the present application showed significant differences in decapping rates. The decapping rate of YK-CAP-004 was the lowest, decreasing by 16.0% compared to the highest rate of YK-CAP-001.

[0422] As can be seen from the data in Table 6, the modified lock nucleoside capping analogs of the present application, YK-CAP-001, YK-CAP-002, YK-CAP-003, YK-CAP-004, YK-CAP-005, and YK-CAP-006, showed very large differences in decapping rates. The lowest decapping rate was for YK-CAP-004 at only 10.3%, and the decapping rates of YK-CAP-003, YK-CAP-005, and YK-CAP-006 were also very low at 11.8%, 12.8%, and 11.2%, respectively.

[0423] The decapping rate of YK-CAP-001 reached 26.3%, which was the highest, and the decapping rate of YK-CAP-002 also reached 24.6%, which was relatively high. The decapping rates of YK-CAP-004, YK-CAP-003, YK-CAP-005, and YK-CAP-006 decreased by 16.0%, 14.5%, 13.5%, and 15.1% compared to YK-CAP-001, and by 14.3%, 12.8%, 11.8%, and 13.4% compared to YK-CAP-002, respectively.

[0424] 2) Compared to modified lock nucleoside capping analogs of existing technology that have structures similar or very different, the decapping rate of the modified lock nucleoside capping analog of the present application has been significantly reduced. For example, the decapping rate of YK-CAP-004 has been reduced by 38.3% compared to N-7413.

[0425] 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, by 18.5%, 17.0%, 16.0%, and 17.6% compared to Compound 14, by 12.9%, 11.4%, 10.4%, and 12.0% compared to HN3002, and by 17.2%, 15.7%, 14.7%, and 16.3% compared to m6A, respectively.

[0426] 3) Modified lock nucleoside capping analogs with similar structures showed very large differences in decapping rates.

[0427] The structures between each modified lock nucleoside capping analog designed in this application are very similar, and this series of compounds is also very similar to the structure of compound 14, but the decapping rates were very different.

[0428] For example, compared to YK-CAP-001, YK-CAP-002, and Compound 14, YK-CAP-004 differs only in the substituent at C6', which is the group connected to the methylene bridge linking the 2'-O and C4' of the ribose ring; YK-CAP-004 is 2,2-difluoroethyl, YK-CAP-001 is dimethylaminoethyl, and YK-CAP-002 is cyanomethyl, whereas Compound 14 has no substituents, and the other structures are completely 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.

[0429] From this, it was found that modified lock nucleoside capping analogs with similar structures do not necessarily have similar decapping rates, and on the contrary, are highly likely to have very large differences.

[0430] As can be seen from the DCP2 enzyme decapping rate, the modified lock nucleoside capping analogs of the present application, including YK-CAP-004, YK-CAP-003, YK-CAP-005, and YK-CAP-006, all showed a significantly reduced DCP2 enzyme decapping rate compared to modified lock nucleoside capping analogs with similar structures (including YK-CAP-001 and YK-CAP-002 of the present application, and Compound 14 of the prior art) or modified lock nucleoside capping analogs with large structural differences (including N-7413, HN3002, and m6A).

[0431] In addition, it was found that modified lock nucleoside capping analogs with similar structures do not necessarily have similar decapping rates, and on the contrary, are highly likely to have very large differences.

[0432] Example 6: Animal experiment

[0433] In addition, the protein expression levels and durations in mice of Fluc mRNA prepared from different modified lock nucleoside capping analogs were examined. According to the results, the modified lock nucleoside capping analogs of the present application showed significant differences in the average radiation intensity (corresponding to protein expression levels) and duration of protein expression in mice. YK-CAP-004 exhibited the highest fluorescence expression intensity, which was 2.6 times and 5.5 times that of the lowest YK-CAP-002 at 6 and 96 hours, respectively. Compared to modified lock nucleoside capping analogs of the prior art that have similar or significantly different structures, the modified lock nucleoside capping analogs of the present application showed significantly improved protein expression levels and durations in mice of mRNA. For example, the average radiation intensity of YK-CAP-004 was 2.1 times that of m6A at 6 hours and 3.4 times at 96 hours.

[0434] 1. Experimental Method

[0435] 5 μg of the capping analogs of the present application YK-CAP-001 to 006 and the capping analog of the prior art N-7413, compound 14, and an LNP preparation containing m6A transcribed Fluc-mRNA were intramuscularly injected into female BALB / C mice aged 4 to 6 weeks and weighing 17 to 19 g. After administration, a fluorescent imaging substrate was injected intraperitoneally into the mice at specific time points (6 h, 12 h, 24 h, 48 h, 96 h, and 168 h). After allowing the mice to move freely for 5 minutes, the average radiation intensity of the protein expressed by the mRNA carried by the LNP in the mice (corresponding to the amount of protein expression) was detected using an IVIS Spectrum small animal live imager.

[0436] 2. Experimental Results

[0437] The detection results are as shown in Table 7 and Figure 5.

[0438]

[0439] 1) The modified lock nucleoside capping analogs of the present application were found to have significant differences in the average radiation intensity and duration of proteins expressed in mRNA within mice. The average radiation intensity of YK-CAP-004 was the highest, and at 6 hours and 96 hours, it was 2.6 times and 5.5 times that of the lowest YK-CAP-002, respectively.

[0440] As can be seen from the data in Table 7, the different modified lock nucleic acid cap analogs of the present application showed very large differences in the average radiation intensity of proteins expressed in the mRNA of mice. The average radiation intensity of YK-CAP-004 was the highest, reaching 3856100 at 6 hours and 114933 at 96 hours. The average radiation intensity of in vivo mouse imaging for YK-CAP-003, YK-CAP-005, and YK-CAP-006 was also very high, at 3212750, 3171000, and 3303900 at 6 hours, respectively, and at 57893, 110595, and 75840 at 96 hours, respectively.

[0441] The average radiation intensity of YK-CAP-002 was the lowest at 1,466,925 at 6 hours and 20,893 at 96 hours, and the average radiation intensity of YK-CAP-001 was also relatively low at 1,728,300 at 6 hours and 43,485 at 96 hours.

[0442] The average radiation intensity of YK-CAP-004 was 2.6 times that of YK-CAP-002 at 6 hours and 5.5 times that of YK-CAP-002 at 96 hours. The average radiation intensity of YK-CAP-003 was 2.2 times that of YK-CAP-002 at 6 hours and 2.8 times that of YK-CAP-002 at 96 hours. The average radiation intensity of YK-CAP-005 was 2.2 times that of YK-CAP-002 at 6 hours and 5.3 times that of YK-CAP-002 at 96 hours. The average radiation intensity of YK-CAP-006 was 2.3 times that of YK-CAP-002 at 6 hours and 3.6 times that of YK-CAP-002 at 96 hours.

[0443] The average radiation intensity of YK-CAP-004 was 2.2 times that of YK-CAP-001 at 6 hours and 2.6 times that of YK-CAP-001 at 96 hours. The average radiation intensity of YK-CAP-003 was 1.9 times that of YK-CAP-001 at 6 hours and 1.3 times that of YK-CAP-001 at 96 hours. The average radiation intensity of YK-CAP-005 was 1.8 times that of YK-CAP-001 at 6 hours and 2.5 times that of YK-CAP-001 at 96 hours. The average radiation intensity of YK-CAP-006 was 1.9 times that of YK-CAP-001 at 6 hours and 1.7 times that of YK-CAP-001 at 96 hours.

[0444] 2) Compared to modified lock nucleoside capping analogs of existing technology that have structures similar or very different, the average radiation intensity and duration of proteins expressed in mRNA mice of the modified lock nucleoside capping analog of the present application were significantly improved. For example, the average radiation intensity of YK-CAP-004 was 2.1 times m6A at 6 hours and 3.4 times at 96 hours.

[0445] The average 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.

[0446] The average radiation intensity of YK-CAP-004 of the present application was 1.4 times, 1.5 times, and 2.1 times that of N-7413, Compound 14, and m6A, respectively, at 6 hours, and 1.7 times, 2.0 times, and 3.4 times that of N-7413, Compound 14, and m6A, respectively, at 96 hours.

[0447] The average radiation intensity of YK-CAP-003 was 1.2 times, 1.2 times, and 1.8 times that of N-7413, Compound 14, and m6A at 6 hours, respectively, and 0.8 times, 1.0 times, and 1.7 times that of N-7413, Compound 14, and m6A at 96 hours, respectively.

[0448] The average radiation intensity of YK-CAP-005 was 1.1 times, 1.2 times, and 1.8 times that of N-7413, Compound 14, and m6A at 6 hours, respectively, and 1.6 times, 1.9 times, and 3.3 times that of N-7413, Compound 14, and m6A at 96 hours, respectively.

[0449] The average radiation intensity of YK-CAP-006 was 1.2 times, 1.3 times, and 1.8 times that of N-7413, Compound 14, and m6A at 6 hours, respectively, and 1.1 times, 1.3 times, and 2.3 times that of N-7413, Compound 14, and m6A at 96 hours, respectively.

[0450] 3) It was found that structurally similar modified lock nucleoside capping analogs showed very large differences in the average radiation intensity and duration of proteins expressed in the mouse mRNA.

[0451] The structures between each modified lock nucleoside capping analog designed in this application are very similar, and this series of compounds is also very similar to the structure of compound 14, but there were very large differences in the average radiation intensity and duration of the protein expressed in the mouse mRNA.

[0452] For example, compared to YK-CAP-001, YK-CAP-002 and Compound 14, YK-CAP-004 differs only in the substituent at C6', which is the group connected to the methylene bridge connecting the 2'-O and C4' of the ribose ring; 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 completely identical. However, the average radiation intensity of YK-CAP-004 was 2.2 times, 2.6 times, and 1.5 times that of YK-CAP-001, YK-CAP-002, and Compound 14 at 6 hours, and 2.6 times, 5.5 times, and 2.0 times that of YK-CAP-001, YK-CAP-002, and Compound 14 at 96 hours, and was significantly improved.

[0453] As can be seen from animal experiments, modified lock nucleoside capping analogs of the present application, such as YK-CAP-004, YK-CAP-003, YK-CAP-005, and YK-CAP-006, showed significantly improved protein expression levels and durations of mRNA in mice compared to modified lock nucleoside capping analogs with similar structures (including YK-CAP-001 and YK-CAP-002 of the present application, and Compound 14 of the prior art) or modified lock nucleoside capping analogs with large structural differences (including N-7413 and m6A).

[0454] In vivo experiments further demonstrated that 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 via an LNP delivery vector and expressed efficiently and continuously.

[0455] In addition, it was found that the in vivo protein expression levels and durations of Fluc mRNA produced by modified lock nucleoside capping analogs with similar structures in mice are not necessarily similar, and on the contrary, there is a very high possibility that they will have very large differences.

[0456] In summary, the modified lock nucleoside capping analogs YK-CAP-004, YK-CAP-003, YK-CAP-005, and YK-CAP-006 of the present application significantly improved mRNA in vitro transcription yield, capping rate, mRNA translation efficiency, and decapping enzyme stability, as well as protein expression levels and duration in animals, compared to modified lock nucleoside capping analogs of the prior art (including N-7413, Compound 14, HN3002, and m6A). This indicates that the capping structures of YK-CAP-004, YK-CAP-003, YK-CAP-005, and YK-CAP-006 provided by the present invention significantly improve the resistance of the lock nucleic acid structure to the decapping enzyme and the binding affinity with the capping enzyme, and can provide a novel, highly efficient modified lock nucleic acid capping structure for the in vitro transcription of mRNA.

[0457] 1. The chemical structures of the compounds of the present application are very similar, and compared to mRNA capping analogs disclosed in the prior art, some of these series of compounds have similar structures and some have very large differences.

[0458] 1) Although the structure of the compound of the present application is very similar, it was found that only the substituent at the group connected to the methylene bridge connecting the 2'-O and C4' of the ribose ring, i.e., C6', is different.

[0459] 2) The compound of the present application is similar in structure to compound 14, but lacks substituents only on the methylene bridge (i.e., C6') connecting the 2'-O and C4' of the ribose ring of compound 14.

[0460] 3) The compound of the present application has a relatively large structural difference from N-7413, HN3002 and m6A, and there is no methylene bridge between the 2'-O and C4' of the ribose ring of these three compounds, and also the second base adenine of m6A is modified by methylation.

[0461] 2. The modified locking nucleoside capping analog of the present application showed a significant difference in mRNA in vitro transcription yield and capping rate. Compared to the modified locking nucleoside capping analog of the prior art, the modified locking nucleoside capping analog of the present application showed a significant improvement in both mRNA in vitro transcription yield and capping rate.

[0462] 1) The modified lock nucleoside capping analogs of the present application showed significant differences in the in vitro transcription yield and capping rate of mRNA. Both the transcription yield and capping rate of YK-CAP-004 were the highest, with the transcription yield improving by 82.8% compared to the lowest YK-CAP-001 and the capping rate improving by 26.1% compared to the lowest YK-CAP-002.

[0463] 2) Compared to modified locking nucleoside capping analogs of the present application, both mRNA in vitro transcription yield and capping rate were significantly improved. For example, the transcription yield and capping rate of YK-CAP-004 were improved by 35.0% and 18.8%, respectively, compared to compound 14.

[0464] 3) The structurally similar modified lock nucleoside capping analogs showed very large differences in the in vitro transcription yield and capping rate of mRNA. For example, the in vitro transcription yield of mRNA of YK-CAP-004 was improved by 82.8%, 58.3%, and 35.0% compared to 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.

[0465] 3. The modified locking nucleoside capping analog of the present application showed a significant difference in mRNA translation efficiency. Compared to the modified locking nucleoside capping analog of the prior art, the mRNA translation efficiency of the modified locking nucleoside capping analog of the present application was significantly improved.

[0466] 1) The modified lock 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 that of the lowest YK-CAP-002.

[0467] 2) Compared to modified lock nucleoside capping analogs of existing technology that have structures similar or very different, the mRNA translation efficiency of the modified lock nucleoside capping analog of the present application has been significantly improved. For example, the translation efficiency of YK-CAP-004 reached four times that of m6A.

[0468] 3) The structurally similar modified lock nucleoside capping analogs showed very large differences in mRNA translation efficiency. For example, the translation efficiency of YK-CAP-004 mRNA was 1.8 times and 2.0 times that of YK-CAP-001 and YK-CAP-002, respectively.

[0469] 4. The modified locking nucleoside capping analogs of the present application showed a significant difference in decapping rate. Compared to modified locking nucleoside capping analogs of the existing technology that have similar or very different structures, the decapping rates of the modified locking nucleoside capping analogs of the present application were all significantly reduced.

[0470] 1) The modified lock nucleoside capping analogs of the present application showed significant differences in decapping rates. The decapping rate of YK-CAP-004 was the lowest, decreasing by 16.0% compared to the highest rate of YK-CAP-001.

[0471] 2) Compared to modified lock nucleoside capping analogs of existing technology that have structures similar or very different, the decapping rates of the modified lock nucleoside capping analogs of the present application were all significantly reduced. For example, the decapping rate of YK-CAP-004 was reduced by 38.3% compared to N-7413.

[0472] 3) The structurally similar modified lock nucleoside capping analogs showed very large differences in decapping rates. For example, the mRNA decapping rate of YK-CAP-004 was reduced by 16.0%, 14.3%, and 18.5% compared to YK-CAP-001, YK-CAP-002, and compound 14, respectively.

[0473] 5. The modified locking nucleoside capping analog of the present application showed significant differences in the average radiation intensity (corresponding to the amount of protein expression) and duration of proteins expressed in mRNA within mice. Compared to modified locking nucleoside capping analogs of the prior art that have similar or very different structures, the modified locking nucleoside capping analog of the present application showed significant improvements in both the amount of protein expression and the duration of mRNA within mice.

[0474] 1) The modified lock nucleoside capping analogs of the present application were found to have significant differences in the average radiation intensity and duration of proteins expressed in the body of mRNA in mice. The fluorescence expression intensity of YK-CAP-004 was the highest, and at 6 hours and 96 hours, it was 2.6 times and 5.5 times that of YK-CAP-002, which had the lowest intensity.

[0475] 2) Compared to modified lock nucleoside capping analogs of existing technology that have structures similar or very different, the average radiation intensity and duration of proteins expressed in mRNA mice of the modified lock nucleoside capping analog of the present application were both significantly improved. For example, the average radiation intensity of YK-CAP-004 was 2.1 times m6A at 6 hours and 3.4 times m6A at 96 hours.

[0476] 3) Structurally similar modified lock nucleoside capping analogs showed very large differences in the average radiation intensity and duration of proteins expressed in the mice mRNA. For example, the average radiation intensity of YK-CAP-004 was 2.2 times, 2.6 times, and 1.5 times that of YK-CAP-001, YK-CAP-002, and Compound 14 at 6 hours, and 2.6 times, 5.5 times, and 2.0 times that of YK-CAP-001, YK-CAP-002, and Compound 14 at 96 hours.

[0477] Finally, each of the above embodiments is merely for illustrating the technical solution of the present invention and is not intended to be limiting. Although the present invention has been described in detail with reference to each of the aforementioned embodiments, those skilled in the art may still modify the technical solution described in each of the aforementioned embodiments or replace some or all of the technical features therewith with equivalents. It should be noted that such modification or replacement does not cause the essence of the corresponding technical solution to fall outside the scope of the technical solution of each embodiment of the present invention.

Claims

Claim 1 A compound that is YK-CAP-003, YK-CAP-004, YK-CAP-005, or YK-CAP-006 having the structure shown below, or a pharmaceutically acceptable salt thereof. Claim 2 An RNA molecule comprising a compound according to claim 1 or a pharmaceutically acceptable salt thereof as a cap structure or a cap structure fragment. Claim 3 A pharmaceutical composition comprising an RNA molecule according to paragraph 2. Claim 4 A pharmaceutical composition according to paragraph 3, further comprising at least one RNA delivery agent. Claim 5 In paragraph 4, the above-mentioned at least one RNA delivery agent is a pharmaceutical composition comprising at least one cationic lipid. Claim 6 In paragraph 4, the above-mentioned at least one RNA delivery agent is a pharmaceutical composition further comprising at least one neutral lipid. Claim 7 In claim 5, the above cationic lipid is a pharmaceutical composition selected from one or more of the following compounds: (1) a compound represented by formula (II) or a pharmaceutically acceptable salt thereof, wherein G1 is C 1-6 It is an alkylene, and G2 is C 2-8 It is an alkylene, and G3 is C 1-3 It is an alkylene, and L1 is C 6-15 It is a straight-chain alkyl, and L2 is C 12-25 It is a branched-chain alkyl, and (II)(2) A compound represented by formula (III), or a pharmaceutically acceptable salt thereof, wherein G1 is C 2-8 It is an alkylene, and G2 is C 2-8 It is an alkylene, L1 is -C(O)O- or -OC(O)-, L2 is -C(O)O- or -OC(O)-, and R1 is C 6-25 It is a straight-chain or branched-chain alkyl, and R2 is C 6-25 It is a straight-chain or branched-chain alkyl, G3 is HO(CH2)2- or HO(CH2)3-, G4 is HO(CH2)2- or HO(CH2)3-, L is -(CH2)2- or -(CH2)3- or -(CH2)4-, and (III)(3) A compound represented by formula (IV), or a pharmaceutically acceptable salt thereof, wherein G1 is C 1-6 It is an alkylene, and G2 is C 2-8 It is an alkylene, and R1 is C 6-20 It is a straight-chain or branched-chain alkyl, and R2 is C 12-25 is branched chain alkyl, G3 is HO(CH2)2N(CH3)(CH2)2-, HO(CH2)2N(CH2CH3)(CH2)2-, (HO(CH2)2)2N(CH2)2-, CH3O(CH2)2N(CH3)(CH2)2-, (CH3)2N(CH2)3SC(O)O(CH2)2-, (CH3)2N(CH2)3SC(O)-, CH3NH(CH2)2N(CH3)(CH2)2-, or CH3CH2NH(CH2)2-, (IV)(4) A compound represented by formula (V), or a pharmaceutically acceptable salt thereof, wherein G1 is C 1-8 It is an alkylene, and G2 is C 2-8 It is an alkylene, and R1 is C 6-25 It is a straight-chain or branched-chain alkyl, and R2 is C 12-25 It is a straight-chain or branched-chain alkyl, and G3 is HO(CH2)2N(R3)CH2CH(OH)CH2-, where R3 is -CH3 or -CH2CH3 or -CH2CH2OH, and (V)(5) A compound represented by formula (VI), or a pharmaceutically acceptable salt thereof, where G 1 and G 2 Each is independently an unsubstituted C6-C 10 It is an alkylene, and G 3 Unsubstituted C1-C 12 It is an alkylene, and R 1 and R 2 Each independently C6-C 24 alkyl or C6-C 24 It is alkenyl, and R 3 -OR 5 , -C(=O)OR 4 , -OC(=O)R 4 or -NR 5 C(=O)R 4 and R 4 is C1-C 12 It is a hydrocarbon group, and also R 5 is H or a C1-C6 hydrocarbon group, and (VI)(6) A compound represented by formula (VII), or a pharmaceutically acceptable salt thereof, where R4 is -(CH2) n Selected from Q, where Q is -OH or -CN, n is 1, 2 or 3, and (VII)(7) A compound represented by formula (VIII), or a pharmaceutically acceptable salt thereof. (VIII) Claim 8 A pharmaceutical composition according to claim 7, wherein the cationic lipid is one or more selected from YK-009, YK-401, YK-305, ALC-0315, SM-102, or DLIN-MC3-DMA: , , , , , . Claim 9 In claim 8, the above-mentioned cationic lipid is a pharmaceutical composition in which YK-009. Claim 10 In claim 6, the neutral lipid is a pharmaceutical composition comprising one or more of phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, ceramide, and sterol. Claim 11 In claim 10, the neutral lipids are 1,2-dilinoleoy-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 1,2-dioleoy-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-diundecanoy-sn-glycero-phosphocholine (DUPC), 1-palmitoyl-2-oleoy-sn-glycero-3-phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 Diether PC), 1-oleoyl-2-cholesteryl hemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 LysoPC), 1,2-dilinolenoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dipitanoyl-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-Diarachidonoyyl-sn-glycero-3-phosphoethanolamine, 1,2-Didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2-Dioleoyl-sn-glycero-3-phosphate-rac-(1-glycerol)sodium salt (DOPG), Dipalmitoylphosphatidylglycerol (DPPG), Palmitoyloleylphosphatidylethanolamine (POPE), Distearoyl-phosphatidyl-ethanolamine (DSPE), A pharmaceutical composition comprising one or more selected from 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. Claim 12 In claim 11, the above neutral lipid is a pharmaceutical composition in which DOPE and / or DSPC. Claim 13 In paragraph 4, the above-mentioned at least one RNA delivery agent is a pharmaceutical composition further comprising a structural lipid. Claim 14 A pharmaceutical composition according to claim 13, wherein the structural lipid is one or more selected from cholesterol, nonsterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatin, ursolic acid, α-tocopherol, and corticosteroid. Claim 15 A pharmaceutical composition in which the structural lipid of claim 14 is cholesterol. Claim 16 In paragraph 4, the above-mentioned at least one RNA delivery agent is a pharmaceutical composition further comprising a polymer conjugated lipid. Claim 17 A pharmaceutical composition according to claim 16, wherein the polymer conjugated lipid is one or more selected from distearoyl phosphatidyl ethanolamine polyethylene glycol 2000 (DSPE-PEG2000), 1,2-dimyritoyl-rac-glycero-3-methoxypolyethyleneglycol-2000 (DMG-PEG2000), and methoxypoly(ethylene glycol) ditetradecylacetamide (ALC-0159). Claim 18 (1) A compound according to claim 1 or a pharmaceutically acceptable salt thereof, and (2) a kit comprising a nucleotide triphosphate molecule and RNA polymerase. Claim 19 A kit according to claim 18, further comprising one or more of an RNA enzyme inhibitor, an inorganic pyrophosphatase, a buffer, or any combination thereof. Claim 20 delete

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