Ribose-modified cap analog and use thereof

Abstract

This invention provides a ribose-modified capped analogue and its application. This invention belongs to the field of chemical and biological engineering technology. The ribose-modified capped analogue has the structure shown in formula (I). The ribose-modified capped analogue of this invention can improve the stability of mRNA and / or the mRNA translation efficiency.

Description

Technical Field

[0001] This invention belongs to the field of chemical and biological engineering technology, and relates to a ribose-modified capped analogue and its application.

[0002] This application claims priority to Chinese Patent Application No. 2024105145551, filed on April 26, 2024, and Chinese Patent Application No. 2024108141919, filed on June 24, 2024. The full text of the aforementioned Chinese patent applications is incorporated herein by reference. Prior Technology

[0003] The chemical essence of the cap structure is a special structure located at the 5' end of mRNA, namely the m7GPPPN structure, also known as the methylguanosine cap, formed during mRNA transcription. It is formed under the combined catalysis of RNA triphosphatase, guanylate transferase, mRNA (guanine-N7) methyltransferase, and mRNA (nucleoside-2') methyltransferase. Different degrees of methylation can form three types of caps: CAP0, CAP1, and CAP2, namely m7G5'ppp5'Np, m7G5'ppp5'NmpNp, and m7G5'ppp5'NmpNmpNp, respectively.

[0004] The cap structure is essential for the initiation of mRNA translation. It provides a signal for ribosomes to recognize mRNA, assists in the binding of ribosomes to mRNA, and enables translation to begin from AUG. Simultaneously, the cap structure increases the stability of mRNA, protecting it from attack by 5'→3' exonucleases.

[0005] In simple terms, the cap structure acts like a helmet for mRNA, protecting it from damage and allowing it to be chemically modified to be recognized by other molecules. Besides the natural cap structure, cap structure analogs are frequently used during in vitro transcription to improve mRNA structural stability; common examples include ARCA and Cap1 analogs.

[0006] Studies have shown that the cap structure of mRNA is closely related to mRNA quality control and the body's innate immunity. Therefore, inventing a novel cap analogue is of great significance for increasing mRNA stability and improving mRNA translation efficiency. Summary of the Invention

[0007] To address the shortcomings of existing technologies, the present invention aims to provide a ribose-modified capped analogue and its applications. The ribose-modified capped analogue described in this invention can improve mRNA stability and / or mRNA translation efficiency.

[0008] To achieve this objective, the present invention adopts the following technical solution: In a first aspect, the present invention provides a ribose-modified capped analogue or its stereoisomer, a pharmaceutically acceptable salt or solvate, wherein the ribose-modified capped analogue has the structure shown in formula (I): Where n is selected from 1 or 0; m is selected from 1 or 0; X1 is selected from -C(OH)H- or a single bond; X2 is selected from -CH-, -O-, -CN, -C(O)-, Or a single key; X3 is selected from -CH2, -CFH-, or -O-; R1 is selected from -OH or -NHAc; R2 is selected from -CH3, -F, -H, or does not exist; R3 is selected from -OR6, -NHC(O)R7, -N(R8)2, -CN, -F, -C(O)N(R9)2, substituted or unsubstituted C1-C3 alkyl groups, -H, or is absent; R4 is selected from -CH3, -F, or -H; R5 is selected from -OR10, -NHC(O)R11, -N(R12)2, -CF2H, -F, -C(O)N(R13)2, substituted or unsubstituted C1-C3 alkyl groups, or is absent; R6, R8, R10, R11, R12, and R13 are each independently selected from unsubstituted C1-C3 alkyl groups or -H groups; R7 is selected from -OR14, -NHC(O)R15, -F, or substituted or unsubstituted C1-C3 alkyl groups; R9 is selected from unsubstituted C2-C3 alkyl groups; R14 and R15 are each independently selected from unsubstituted C1-C3 alkyl groups.

[0009] Compared with existing ribose-modified capping analogs, the ribose-modified capping analogs of the present invention can significantly improve the in vitro transcription yield of mRNA, the capping rate, the mRNA translation efficiency, the protein expression level and duration of mRNA in mice, and significantly reduce the uncapping rate.

[0010] In a second aspect, the present invention provides the use of the ribose-modified capping analogues or their stereoisomers, pharmaceutically acceptable salts or solvates described in the first aspect of the present invention, in the preparation of capping reagents for in vitro co-transcriptionalized mRNA.

[0011] Thirdly, the present invention provides an RNA molecule comprising, as described in the first aspect of the present invention, a ribose-modified capped analogue or its stereoisomer, a pharmaceutically acceptable salt or solvate as a cap structure or a cap structure fragment.

[0012] Fourthly, the present invention provides a pharmaceutical composition comprising the RNA molecule of the third aspect of the present invention.

[0013] Fifthly, the present invention provides a method for synthesizing mRNA molecules for non-disease diagnosis and treatment purposes, comprising the following steps: co-incubating a ribose-modified capped analogue or its stereoisomer, a pharmaceutically acceptable salt or solvate, as described in the first aspect of the present invention, with a polynucleotide template to perform template transcription.

[0014] Sixthly, the present invention provides a capped mRNA transcription reaction system for non-disease diagnosis and treatment purposes, comprising: (1) the ribose-modified capped analogue or its stereoisomer, pharmaceutically acceptable salt or solvate of the first aspect of the present invention; and (2) a polynucleotide template, NTPs (nucleoside triphosphates) and RNA polymerase.

[0015] In a seventh aspect, the present invention provides a kit comprising: (1) the ribose-modified capped analogue or its stereoisomer as described in the first aspect of the present invention, a pharmaceutically acceptable salt or solvate; and (2) a nucleotide triphosphate molecule, and an RNA polymerase.

[0016] Eighthly, the present invention provides a method for improving the intracellular stability of RNA, comprising incorporating the ribose-modified capped analogue or its stereoisomer, a pharmaceutically acceptable salt or solvate of the first aspect of the present invention into the RNA.

[0017] In a ninth aspect, the present invention provides a method for introducing RNA into a cell, the method comprising contacting the cell with a ribose-modified capped analogue or stereoisomer thereof as described in the first aspect of the present invention, a pharmaceutically acceptable salt or solvate, or a pharmaceutical composition as described in the fourth aspect of the present invention.

[0018] In a tenth aspect, the present invention provides a method for performing RNA translation inhibition in cells, the method comprising contacting the cells with a ribose-modified capped analogue or stereoisomer thereof as described in the first aspect of the present invention, a pharmaceutically acceptable salt or solvate, or a pharmaceutical composition as described in the fourth aspect of the present invention.

[0019] In one aspect, the present invention provides the use of the ribose-modified capped analogues or stereoisomers thereof described in the first aspect of the present invention, pharmaceutically acceptable salts or solvates, or pharmaceutical compositions described in the fourth aspect of the present invention in the preparation of vaccines.

[0020] Compared with the prior art, the present invention has the following beneficial effects: Compared with existing ribose-modified capping analogs, the ribose-modified capping analogs of the present invention can significantly improve the in vitro transcription yield of mRNA, the capping rate, the mRNA translation efficiency, the protein expression level and duration of mRNA in mice, and significantly reduce the uncapping rate. Simple Explanation of the Diagram

[0021] Figure 1 shows the in vitro mRNA transcription yield results using YK-CAP-106, YK-CAP-107, YK-CAP-108, YK-CAP-109, YK-CAP-110, YK-CAP-111, YK-CAP-112, YK-CAP-113, YK-CAP-114, YK-CAP-115, YK-CAP-116, YK-CAP-117, YK-CAP-118, YK-CAP-119, YK-CAP-101, YK-CAP-102, YK-CAP-103, YK-CAP-104, YK-CAP-105, compound 14, and 5227 as capped analogs. Figure 2 shows the capping rate of mRNA transcription initiated by YK-CAP-106, YK-CAP-107, YK-CAP-108, YK-CAP-109, YK-CAP-110, YK-CAP-111, YK-CAP-112, YK-CAP-113, YK-CAP-114, YK-CAP-115, YK-CAP-116, YK-CAP-117, YK-CAP-118, YK-CAP-119, YK-CAP-101, YK-CAP-102, YK-CAP-103, YK-CAP-104, YK-CAP-105, compound 14, and 5227 as capping analogs. Figure 3 shows the relative fluorescence intensity test results of capped mRNAs using YK-CAP-106, YK-CAP-107, YK-CAP-108, YK-CAP-109, YK-CAP-110, YK-CAP-111, YK-CAP-112, YK-CAP-113, YK-CAP-114, YK-CAP-115, YK-CAP-116, YK-CAP-117, YK-CAP-118, YK-CAP-119, YK-CAP-101, YK-CAP-102, YK-CAP-103, YK-CAP-104, YK-CAP-105, 5227, CAP-2'O-ethyl, N-7113, compound 14, HN3002, and m6A as capped analogs. Figure 4 shows the decapping rate of DCP2 enzyme using YK-CAP-106, YK-CAP-107, YK-CAP-108, YK-CAP-109, YK-CAP-110, YK-CAP-111, YK-CAP-112, YK-CAP-113, YK-CAP-114, YK-CAP-115, YK-CAP-116, YK-CAP-117, YK-CAP-118, YK-CAP-119, YK-CAP-101, YK-CAP-102, YK-CAP-103, YK-CAP-104, YK-CAP-105, 5227, CAP-2'O-ethyl, N-7113, compound 14, HN3002, and m6A as capping analogs. Implementation

[0022] The technical solution of the present invention will be further illustrated below through specific embodiments. Those skilled in the art should understand that the embodiments described are merely illustrative of the invention and should not be construed as limiting the invention.

[0023] This invention may be implemented in other specific forms without departing from its essential attributes. It should be understood that, without conflict, any and all embodiments of this invention can be combined with technical features of any or more other embodiments to obtain further embodiments. This invention includes such further embodiments obtained through combinations.

[0024] Unless otherwise indicated in the embodiments or elsewhere, all numerical figures for quantitative properties, such as dosage, stated in this invention should be understood to be modified by the term "about" in all cases. It should also be understood that any numerical ranges enumerated in this invention are intended to include all subranges within that range and any combination of the endpoints of that range or subranges.

[0025] Unless otherwise defined, all terms used in this invention (including technical and scientific terms) shall have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Terms, such as those defined in common dictionaries, shall be interpreted as having the meaning consistent with their meaning in the context of the relevant art, and shall not be interpreted in an idealized or overly formal sense, unless expressly so defined in this invention.

[0026] As used in this invention, the term "C1~C3" refers to a group having any integer number of carbon atoms in the main chain, ranging from 1 to 3, such as 1, 2, or 3 carbon atoms. The term "C6~15" refers to a group having any integer number of carbon atoms in the range of 6 to 15, such as 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 carbon atoms. Other carbon atom ranges are defined similarly, indicating that the number of carbon atoms in the defined group can be any integer value within the defined range.

[0027] As used in this invention, the term "alkyl" refers to a saturated aliphatic hydrocarbon group having a straight chain or branches; non-limiting examples include methyl, ethyl, propyl, isopropyl, etc.

[0028] As used herein, the term "salt" refers to a salt that allows for the convenient or desirable preparation, purification, and / or treatment of the modified nucleoside compounds (or nucleotide compounds) of this invention, for example, a pharmaceutically acceptable salt. Unless otherwise stated, references to specific compounds in this invention also include their salt forms.

[0029] As used in this invention, "capped analogue" refers to a structure at the 5' end of mature mRNA formed through post-transcriptional modification in eukaryotes, namely the m7GPPPN structure, also known as a methylguanosine cap. This structure can prevent mRNA degradation at the 5' end, help RNA transcripts cross selective pores of the nuclear membrane into the cytoplasm, enhance translation, and assist in completing the entire cleavage process.

[0030] As used herein, the terms "comprising," "containing," or "including" mean that the element preceding the word encompasses the elements listed following the word and their equivalents, without excluding elements not described. The terms "containing" or "comprising (including)" as used herein can be open-ended, semi-closed, or closed-ended. In other words, the terms also include "consistently composed of" or "composed of."

[0031] In this invention, the term "pharmaceutically acceptable" means that a compound or composition is chemically and / or toxicologically compatible with other components constituting the preparation and / or with humans or mammals for the prevention or treatment of diseases or conditions.

[0032] In this invention, the term "solvent" refers to a complex formed by combining a compound of formula (I) or a pharmaceutically acceptable salt thereof with a solvent (e.g., ethanol or water). It should be understood that any solvate of a compound of formula (I) used in the treatment of a disease or condition, although it may provide different properties (including pharmacokinetic properties), will yield the compound of formula (I) once absorbed into the subject, such that the use of the compound of formula (I) respectively encompasses the use of any solvate of the compound of formula (I).

[0033] It should be further understood that compounds of formula (I) or pharmaceutically acceptable salts thereof can be isolated as solvates, and therefore any such solvates are included within the scope of this invention. For example, compounds of formula (I) or pharmaceutically acceptable salts thereof may exist in unsolvated forms as well as in solvated forms formed with pharmaceutically acceptable solvents (such as water, ethanol, etc.).

[0034] This invention also includes salts of the compounds described herein, particularly pharmaceutically acceptable salts. Compounds of this invention having sufficiently acidic or sufficiently basic functional groups can react with many bases or acids to form salts. Alternatively, compounds that are inherently charged (e.g., compounds having a quaternary nitrogen atom) can form salts with suitable counterions (e.g., halide ions such as bromide, chloride, or fluoride ions, especially bromide ions).

[0035] The pharmaceutically acceptable salts described in this invention can be, for example, acid addition salts of compounds of this invention that carry nitrogen atoms in the chain or ring of a compound with the structure shown in formula (I) and have sufficient basicity, such as acid addition salts formed with inorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, phosphoric acid, or nitric acid, or acid addition salts formed with organic acids such as formic acid, acetic acid, acetic acid, pyruvic acid, trifluoroacetic acid, propionic acid, butyric acid, hexanoic acid, heptanoic acid, undecanoic acid, lauric acid, benzoic acid, salicylic acid, 2-(4-hydroxybenzoyl)benzoic acid, camphoric acid, cinnamic acid, and cyclopentane. Propionic acid, 3-hydroxy-2-naphtholic acid, nicotinic acid, pyruvic acid, pectinic acid, persulfate, 3-phenylpropionic acid, picric acid, pentyl acid, 2-hydroxyethanesulfonic acid, itaconic acid, aminosulfonic acid, trifluoromethanesulfonic acid, dodecyl sulfuric 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, glucohepanoic acid, glycerophosphate, aspartic acid, sulfosalicylic acid, or thiocyanate.

[0036] Alternatively, another suitable pharmaceutically acceptable salt of the compounds of the present invention having sufficient acidity is an alkali metal salt, such as a sodium or potassium salt, an alkaline earth metal salt such as a calcium or magnesium salt, an ammonium salt (e.g., a salt formed with NH3 or ammonia), or a salt formed with an organic base that provides a physiologically acceptable cation, such as a salt formed with triethylamine, N-methylglucosamine, dimethylglucosamine, ethylglucosamine, lysine, dicyclohexylamine, 1,6-hexanediamine, ethanolamine, glucosamine, sarcosine, serine, trihydroxymethylaminomethane, aminopropylene glycol, 1-amino-2,3,4-butanetriol. In addition, basic nitrogen-containing groups can be quaternarily ammonized using the following reagents: lower alkyl halides, such as methyl, ethyl, propyl and butyl chlorides, bromides and iodides; dialkyl sulfates, such as dimethyl sulfate, diethyl sulfate, dibutyl sulfate and dipentyl sulfate; long-chain halides, such as decyl, lauryl, myristyl and stearyl chlorides, bromides and iodides; aralkyl halides such as benzyl and phenethyl bromides, etc.

[0037] Those skilled in the art will also recognize that the acid addition salts of the compounds of formula (I) described herein can be prepared by reacting the compounds with a suitable inorganic or organic acid using any of the known methods. Alternatively, the base addition salts of the acidic compounds of this disclosure can be prepared by reacting them with a suitable base using various known methods.

[0038] This invention includes all possible salts of the compounds of formula (I) described herein, which may be a single salt or any mixture of said salts in any proportion.

[0039] Some compounds of the present invention can exist in the form of one or more stereoisomers. Stereoisomers include geometric isomers, diastereomers, and enantiomers. Therefore, the compounds of formula (I) of the present invention also include racemic mixtures, single stereoisomers, and optically active mixtures. It should be understood by those skilled in the art that one stereoisomer may have better efficacy and / or fewer side effects than other stereoisomers. Single stereoisomers and optically active mixtures can be obtained by chiral source synthesis, chiral catalysis, chiral resolution, etc. Racemates can be chirally resolved by chromatographic resolution or chemical resolution. For example, the compounds of the present invention can be separated by adding chiral acid resolving reagents such as chiral tartaric acid or chiral malic acid to form salts with them, and by utilizing the physicochemical properties of the products, such as differences in solubility.

[0040] In this invention, when the name of a compound is inconsistent with its structural formula, the structural formula shall prevail.

[0041] This invention should be interpreted as consistent with the laws and principles of chemical bonding. In some cases, hydrogen atoms may be removed to accommodate substituents at a given position.

[0042] It should be understood that the term "compound of the present invention" as used in this invention may, depending on the context, include: compounds of formula (I), their solvates, their pharmaceutically acceptable salts, their stereoisomers, and mixtures thereof.

[0043] In a first aspect, the present invention provides a ribose-modified capped analogue or its stereoisomer, a pharmaceutically acceptable salt or solvate, wherein the ribose-modified capped analogue has the structure shown in formula (I): Where n is selected from 1 or 0; m is selected from 1 or 0; X1 is selected from -C(OH)H- or a single bond; X2 is selected from -CH-, -O-, -CN, -C(O)-, Or a single key; X3 is selected from -CH2, -CFH-, or -O-; R1 is selected from -OH or -NHAc; R2 is selected from -CH3, -F, -H, or does not exist; R3 is selected from -OR6, -NHC(O)R7, -N(R8)2, -CN, -F, -C(O)N(R9)2, substituted or unsubstituted C1-C3 alkyl groups, -H, or is absent; R4 is selected from -CH3, -F, or -H; R5 is selected from -OR10, -NHC(O)R11, -N(R12)2, -CF2H, -F, -C(O)N(R13)2, substituted or unsubstituted C1-C3 alkyl groups, or is absent; R6, R8, R10, R11, R12, and R13 are each independently selected from unsubstituted C1-C3 alkyl groups or -H groups; R7 is selected from -OR14, -NHC(O)R15, -F, or substituted or unsubstituted C1-C3 alkyl groups; R9 is selected from unsubstituted C2-C3 alkyl groups; R14 and R15 are each independently selected from unsubstituted C1-C3 alkyl groups.

[0044] In one implementation, X2 is -O-.

[0045] In one implementation, X2 is -O-, and R2 does not exist.

[0046] In one implementation, X2 is -O- and R3 is -H.

[0047] In one implementation, X2 is -O-, R2 is non-existent, and R3 is -H.

[0048] In one implementation, X2 is -CH-.

[0049] In one implementation, X2 is -CH- and R2 is -H, -CH3, or -F.

[0050] In one embodiment, X2 is -CH-, and R3 is -N(CH3)2, -C(O)CH3, -OCH3, -NHC(O)CH3, or -F.

[0051] In one embodiment, X2 is -CH-, R2 is -H, -CH3 or -F, R3 is -F, and R3 is -N(CH3)2, -C(O)CH3, -OCH3, -NHC(O)CH3 or -F.

[0052] In one implementation, X2 is -CN.

[0053] In one implementation, X2 is -CN, and R2 does not exist.

[0054] In one implementation, X2 is -CN, R2 does not exist, and R3 does not exist.

[0055] In one implementation, X2 is... .

[0056] In one implementation, X2 is... R2 does not exist.

[0057] In one implementation, X2 is... R3 does not exist.

[0058] In one implementation, X2 is... R2 does not exist, and R3 does not exist.

[0059] In one implementation, X2 is -C(O)-.

[0060] In one implementation, X2 is -C(O)-, and R2 does not exist.

[0061] In one embodiment, X2 is -C(O)- and R3 is -N(CH2CH3)2 or -N(CH2CH2CH3)2.

[0062] In one implementation, X2 is -C(O)-, R2 is absent, and R3 is -N(CH2CH3)2 or -N(CH2CH2CH3)2.

[0063] In one implementation, X3 is -O-.

[0064] In one implementation, X3 is -O- and R7 is -CH3 or -OCH3.

[0065] In one implementation, X3 is -CH2-.

[0066] In one embodiment, X3 is -CH2- and R7 is -OCH3, -NHC(O)CH3, or -F.

[0067] In one implementation, X3 is -CFH-.

[0068] In one implementation, X3 is -CFH- and R7 is -F.

[0069] In one implementation, m is 1.

[0070] In one embodiment, m is 1, and R5 is -OCH3, -F, or -CF2H; preferably, m is 1 and R5 is -OCH3.

[0071] In one implementation, R6 is -H.

[0072] In one embodiment, the ribose-modified capped analogue is one of the following: YK-CAP-101, YK-CAP-102, YK-CAP-103, YK-CAP-104, YK-CAP-105, YK-CAP-106, YK-CAP-107, YK-CAP-108, YK-CAP-109, YK-CAP-110, YK-CAP-111, YK-CAP-112, YK-CAP-113, YK-CAP-114, YK-CAP-115, YK-CAP-116, YK-CAP-117, YK-CAP-118, or YK-CAP-119: ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; .

[0073] In a second aspect, the present invention provides the use of the ribose-modified capping analogues or their stereoisomers, pharmaceutically acceptable salts or solvates described in the first aspect of the present invention, in the preparation of capping reagents for in vitro co-transcriptionalized mRNA.

[0074] Thirdly, the present invention provides an RNA molecule comprising, as described in the first aspect of the present invention, a ribose-modified capped analogue or its stereoisomer, a pharmaceutically acceptable salt or solvate as a cap structure or a cap structure fragment.

[0075] Fourthly, the present invention provides a pharmaceutical composition comprising the RNA molecule of the third aspect of the present invention.

[0076] In one embodiment, the pharmaceutical composition further comprises at least one RNA delivery agent.

[0077] RNA delivery agents can be, for example, lipid nanoparticles (LNPs). Lipid nanoparticles are widely used in the delivery of small molecule drugs and nucleic acids. mRNA coated with LNPs can be protected from the effects of extracellular ribonucleases and facilitate the delivery of intracellular mRNA. For more information on lipid nanoparticles, please refer to the review article Chemistry of Lipid Nanoparticles for RNA Delivery. Acc Chem Res. 2022 Jan 4;55(1):2-12.

[0078] In one embodiment, the at least one RNA delivery agent comprises at least one cationic lipid.

[0079] The term cationic lipids used in this article refers to lipids that carry a positive charge at a selected pH value. For example, see the 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.

[0080] In one embodiment, the cationic lipid is selected from one or a combination of at least two of the following compounds: (1) The compound represented by formula (II), or its N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer, wherein G1 is a C1-6 alkylene; G2 is a C2-8 alkylene; G3 is a C1-3 alkylene; L1 is a C6-15 straight-chain alkyl; L2 is a C12-25 branched-chain alkyl; (II); (2) The compound shown in formula (III), or its N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer, wherein G1 is a C2-8 alkylene group; G2 is a C2-8 alkylene group; L1 is -C(O)O- or -OC(O)-; L2 is -C(O)O- or -OC(O)-; R1 is a C6-25 straight-chain or branched alkyl group; R2 is a C6-25 straight-chain or branched alkyl group; 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-; (III); (3) The compound shown in formula (IV), or its N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer, wherein: G1 is a C1-6 alkylene; G2 is a C2-8 alkylene; R1 is a C6-20 straight-chain or branched alkyl; R2 is a C12-25 branched alkyl; G3 is: HO(CH2)2N(CH3)(CH2)2-, HO(CH2)2N(CH2CH3)(CH2)2-, (HO(CH2)2)2N(CH2)2-, CH3O(CH2)2N(CH3)(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) The compound shown in formula (V), or its N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer, wherein G1 is a C1-8 alkylene; G2 is a C2-8 alkylene; R1 is a C6-25 straight-chain or branched alkyl; R2 is a C12-25 straight-chain or branched alkyl; G3 is: HO(CH2)2N(R3)CH2CH(OH)CH2-, wherein R3 is -CH3 or -CH2CH3 or -CH2CH2OH; (V); (5) The compound represented by formula (VI), or its N-oxide, solvate, pharmaceutically acceptable salt, or stereoisomer, wherein G1 and G2 are each independently unsubstituted C6-C10 alkylene; G3 is an unsubstituted C1-C12 alkylene; R1 and R2 are each independently C6-C24 alkyl or C6-C24 alkenyl; R3 is OR5, N, -C(=O)OR4, -OC(=O)R4, or -NR5C(=O)R4; R4 is a C1-C12 hydrocarbon; and R5 is H or a C1-C6 hydrocarbon. (VI); (6) The compound shown in formula (VII), or its N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer, wherein R4 is selected from -(CH2)nQ and -(CH2)nCHQR; Q is selected from the group consisting of: -OR, -OH, -O(CH2)nN(R)2, -OC(O)R, -CX3, -CN, -N(R)C(O)R, -N(H)C(O)R -N(R)S(O)2R, -N(H)S(O)2R, -N(R)C(O)N(R)2, -N(H)C(O)N(R)2, -N(H)C(O)N(H)(R), -N(R)C(S)N(R)2, -N(H)C(S)N(R)2, -N(H)C(S)N(H)(R), -N(R)S(O)2R8 and heterocycles; n is 1, 2 or 3; (VII); (7) The compound represented by formula (VIII), or its N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer, (VIII).

[0081] In a preferred embodiment, the cationic lipid is selected from one or a combination of at least two of YK-009, YK-401, YK-305, ALC0315, SM102, and DLIN-MC3-DMA. , , , , , .

[0082] In a more preferred embodiment, the cationic lipid is YK-009.

[0083] In one embodiment, the at least one RNA delivery agent further comprises at least one neutral lipid.

[0084] In this invention, neutral lipids refer to lipids that are uncharged at a selected pH value or exist in a zwitterionic form and play an auxiliary role. These neutral lipids may regulate the fluidity of nanoparticles to form lipid bilayer structures and improve efficiency by promoting lipid phase transitions, and may also affect the specificity of target organs.

[0085] In one embodiment, the neutral lipids include one or a combination of at least two of phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, ceramide, sterols and their derivatives.

[0086] In one embodiment, the neutral lipid is selected from one or a combination of at least two of the following: 1,2-dilinoleyl-sn-glycerol-3-phosphate choline (DLPC), 1,2-dimyristyl-sn-glycerol-3-phosphate choline (DMPC), 1,2-dilinoleyl-sn-glycerol-3-phosphate choline (DOPC), 1,2-dispalmitoyl-sn-glycerol-3-phosphate choline (DPPC), 1,2-distearateyl-sn-glycerol-3-phosphate choline (DSPC), 1,2-diundecanoyl-sn-glycerol-3-phosphate choline (DUPC), 1-palmitoyl-2-oleyl-sn-glycerol-3-phosphate choline (POPC), 1,2-di-O-octadecenyl-sn-glycerol-3-phosphate choline (18:0 Diether) 1,2-Olecaprylyl-2-cholesterolylhemisuccinyl-sn-glycerol-3-phosphate choline (OChemsPC), 1-hexadecyl-sn-glycerol-3-phosphate choline (C16 Lyso PC), 1,2-dilinalinyl-sn-glycerol-3-phosphate choline, 1,2-diarachidonicyl-sn-glycerol-3-phosphate choline, 1,2-bis(docosahexaenoenyl)-sn-glycerol-3-phosphate choline, 1,2-diolecaprylyl-sn-glycerol-3-phosphate ethanolamine (DOPE), 1,2-diphydanyl-sn-glycerol-3-phosphate ethanolamine (ME 16.0) PE), 1,2-distearate-sn-glycerol-3-phosphate ethanolamine, 1,2-dilinoleyl-sn-glycerol-3-phosphate ethanolamine, 1,2-dilininyl-sn-glycerol-3-phosphate ethanolamine, 1,2-diarachidonicyl-sn-glycerol-3-phosphate ethanolamine, 1,2-bis(docosahexaenoyl)-sn-glycerol-3-phosphate ethanolamine, 1,2-dioleyl-sn-glycerol-3-phosphate-rac-(1-glycerol) sodium salt (DOPG), dipalmitoylphosphatidylglycerol (DPPG), palmitoyl oleyl Phosphatidylethanolamine (POPE), distearyl-phosphatidylethanolamine (DSPE), dipalmitoylphosphatidylethanolamine (DPPE), dimyristylphosphatidylethanolamine (DMPE), 1-stearyl-2-oleoyl-SN-glycerol-3-phosphatidylethanolamine (SOPE), 1-stearyl-2-oleoylphosphatidylcholine (SOPC), sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoylphosphatidylcholine, lysophosphatidylcholine, lysophosphatidylethanolamine (LPE).

[0087] In a preferred embodiment, the neutral lipid is DOPE and / or DSPC.

[0088] In one embodiment, the at least one RNA delivery agent further comprises a structural lipid.

[0089] In this invention, structural lipids refer to lipids that enhance the stability of nanoparticles by filling the gaps between lipids.

[0090] In one embodiment, the structural lipid is selected from one or a combination of at least two of the following: cholesterol, non-steroidal, sitosterol, ergosterol, campesterol, stigmasterol, brassinosteroid, tomatine, ursolic acid, α-tocopherol, or corticosteroids.

[0091] In a preferred embodiment, the structural lipid is cholesterol.

[0092] In one embodiment, the at least one RNA delivery agent further comprises a polymeric conjugated lipid.

[0093] In this invention, polymer-conjugated lipids mainly refer to lipids modified with polyethylene glycol (PEG). Hydrophilic PEG-stabilized lipid nanoparticles (LNPs) regulate nanoparticle size by restricting lipid fusion and increase nanoparticle half-life by reducing non-specific interactions with macrophages.

[0094] In one embodiment, the polymeric conjugated lipid is selected from one or a combination of at least two of the following: distearate phospholipid ethanolamine polyethylene glycol 2000 (DSPE-PEG2000), dimyristyl glycerin-3-methoxy polyethylene glycol 2000 (DMG-PEG2000), and methoxy polyethylene glycol bis(tetradecyl acetylamine) (ALC-0159).

[0095] In one embodiment, the RNA delivery agent comprises neutral lipids, structural lipids, and polymeric conjugated lipids, wherein the molar ratio of the cationic lipids, the neutral lipids, the structural lipids, and the polymeric conjugated lipids is (25~75):(5~25):(15~65):(0.5~10), for example (35~49):(7.5~15):(35~55):(1~5).

[0096] In one embodiment, the pharmaceutical composition further includes one or at least two cell-penetrating peptides.

[0097] The present invention provides a kit comprising: (1) a ribose-modified capped analogue or its stereoisomer as described in the first aspect of the present invention, a pharmaceutically acceptable salt or solvate; and (2) a nucleotide triphosphate molecule, and an RNA polymerase.

[0098] In one embodiment, the kit further comprises one or a combination of at least two of the following substances: RNase inhibitor, inorganic pyrophosphatase, Mg2+, crowding agent, or buffer.

[0099] This invention may be implemented in other specific forms without departing from its essential attributes. It should be understood that, without conflict, any and all embodiments of this invention can be combined with technical features of any or more other embodiments to obtain further embodiments. This invention includes such further embodiments obtained through combinations.

[0100] The embodiments of the present invention will be described in detail below with reference to examples. However, those skilled in the art will understand that the following examples are for illustrative purposes only and should not be considered as limiting the scope of the invention. Unless otherwise specified in the examples, conventional conditions or conditions recommended by the manufacturer are followed. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0101] The following abbreviations represent the following reagents: IBX: 2-Iodobenzoic acid; BF3·Et2O: Boron trifluoride ether; 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: dibutyl dicarbonate; DIEA: N,N-diisopropylethylamine; DMAP: 4-dimethylaminopyridine; DMSO: dimethyl sulfoxide; HATU: 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate; THF: tetrahydrofuran; TBSCl: tributyldimethylchlorosilane; Imidazole: imidazole; DMF: N,N-dimethylmethamide; TBAF: Tetrabutylammonium fluoride; TBSOTf: Tributyldimethylsilyltrifluoromethanesulfonate; NMO: N-methylmorpholine-N-oxide; m-CPBA: m-chloroperoxybenzoic acid; DIAD: Diisopropyl azodicarbonate; NCS: N-chlorobutyldioxane-imide; PO(MeO)3: Trimethyl phosphate; PySSPy: 2,2'-disulfide dipyridine; PPh3: Triphenylphosphine; TEAP: Triethylamine phosphate; TEAB: Triethylamine bicarbonate; MTBE: Methyl tributyl ether; DCM: Dichloromethane; EA: Ethyl acetate; DAST: Diethylaminosulfur trifluoride; AcSH: Thioacetic acid.

[0102] Example 1: 1. Synthesis of intermediate INT-I

[0103] Step 1: Synthesis of INT-I-PM1

[0104] 2-Amino-9H-purine-6-ol (50.0 g, 0.33 mol) was dissolved in N,N-dimethylacetamide (500 mL), and acetic anhydride (100 mL, 1.06 mol) was added. The mixture was heated to 160 °C and stirred until the solution became clear, indicating that the reaction was complete. Heating was stopped, and the mixture was allowed to cool naturally to room temperature. A large amount of solid precipitated out. The solid was filtered, and the filter cake was washed with ethanol until it turned white, yielding INT-I-PM1 (60.0 g, 0.31 mol, 94.1%). C7H7N5O2, MS (ES): m / z (M+H+) 194.1.

[0105] Step 2: Synthesis of INT-I

[0106] INT-I-PM1 (60.0 g, 0.31 mol) was dissolved in pyridine (200 mL), and N,N-diisopropylethylamine (120.2 g, 0.93 mol) was added and the mixture was cooled to 0 °C. Diphenylaminomethylchlorophenoxyacetate (86.2 g, 0.37 mol) was dissolved in pyridine (100 mL) and slowly added dropwise to the above reaction system under ice bath conditions. After the addition was complete, the ice bath was removed, the mixture was allowed to warm naturally to room temperature, and the reaction was stirred for 3 h. LC-MS monitoring showed no reactants remaining, indicating the reaction was complete. 100 mL of water was added to the reaction system to quench the reaction, and the mixture was evaporated to dryness under reduced pressure. The residue was added to a mixture of 800 mL of ethanol and water (1:1 v / v), heated to reflux for 2 h, heating was stopped, and the mixture was allowed to cool naturally to room temperature. A large amount of solid precipitated out. The solid was filtered, and the filter cake was washed with ethanol to obtain INT-I (59.7 g, 0.15 mol, 49.6%). C20H16N6O3, MS(ES): m / z(M+H+)389.1.

[0107] 2. Synthesis of intermediate INT-II

[0108] pA(2'-OMe)mpG·TEA (300.1 mg, 0.37 mmol), imidazole (347.2 mg, 5.10 mmol), dithiopyridine (1123.6 mg, 5.10 mmol), and triethylamine (516.1 mg, 5.10 mmol) were dissolved in 2.0 mL of ultradry N,N-dimethylformamide. Triphenylphosphine (1337.7 mg, 5.10 mmol) was added under nitrogen protection, and the mixture was reacted at 25 °C for 4 h. After the reaction was complete, the reaction solution was slowly added to a pre-cooled acetone solution containing sodium iodide (598.1 mg, 3.99 mmol), and crystallization was carried out at 25 °C for 30 min. Centrifugation yielded a white solid INT-II (240.6 mg, 0.30 mmol, yield 80.9%). C24H30N12O13P2, MS (ES): m / z (MH-): 755.2.

[0109] 3. Synthesis of YK-CAP-101

[0110] Step 1: Synthesis of YK-CAP-101-PM1

[0111] 2-Amino-6-chloropurine (6.29 g, 37.09 mmol), toluene (50 mL), and BSA (15.09 g, 74.19 mmol) were added to a 250 mL single-necked flask. The system was heated to 80°C and stirred until clear, then allowed to cool naturally to room temperature. α-Pentaacetylglucose (10.00 g, 25.62 mmol) was dissolved in 20 mL of toluene and added to the system. The mixture was stirred at room temperature for 5 minutes. TMSOTf (8.24 g, 37.09 mmol) was added at room temperature, and the system was heated to 110°C and reacted at 110°C for 3 hours. After the reaction was complete, the mixture was allowed to cool naturally to room temperature. 100 mL of saturated sodium bicarbonate solution and 100 mL of ethyl acetate were added, and the mixture was stirred for 5 minutes. The mixture was filtered through diatomaceous earth, and the aqueous phase was extracted with 100 mL of ethyl acetate. The combined ethyl acetate phases were washed with 100 mL of saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was evaporated to dryness under reduced pressure to obtain a viscous substance. This substance was purified by silica gel column chromatography (0-100% ethyl acetate / n-hexane). The product was collected and concentrated to obtain YK-CAP-101-PM1 (8.40 g, 16.80 mmol, 65.6%). C19H22ClN5O9, MS (ES): m / z (M+H+) 500.1.

[0112] Step 2: Synthesis of YK-CAP-101-PM2

[0113] YK-CAP-101-PM1 (8.40 g, 16.80 mmol) and sodium hydroxide aqueous solution (1 M, 40 mL, 40 mmol) were added to a 250 mL single-necked flask, and the system was heated and refluxed with stirring for 3 h. The system was cooled to room temperature, evaporated to dryness under reduced pressure, and the residue was slurried with 100 mL of methanol:dichloromethane = 1:20 for 10 minutes. After filtration, 6.0 g of brown solid was obtained, which was purified by high-pressure preparative liquid chromatography to obtain YK-CAP-101-PM2 (2.13 g, 6.80 mmol, 40.5%). C11H15N5O6, MS (ES): m / z (M+H+) 314.1. YK-CAP-101-PM2: 1HNMR(400MHz,D2O)δ8.71(s,1H),5.57(d,J=9.2Hz,1H),3.99(dd,J=9.2,9.2Hz,1H),3.86(dd,J=12.4,2.0Hz,1H),3.67–3.55(m,4H).

[0114] Step 3: Synthesis of YK-CAP-101-PM3

[0115] YK-CAP-101-PM2 (300 mg, 0.96 mmol) was dissolved in 3 mL of trimethyl phosphate. Under nitrogen protection, the temperature was lowered to 0 °C, and phosphorus oxychloride (450 mg, 2.93 mmol) was slowly added dropwise. The reaction was stirred at 0 °C for approximately 3 h. After the reaction was complete, 5 mL of water was added, and the mixture was transferred to a container and stirred for approximately 1.5 h. Then, 10 mL of dichloromethane was added for washing. The mixture was allowed to stand and separated, and the upper aqueous phase was collected and concentrated under reduced pressure. The concentrated mixture was diluted with water to 180 mL and purified by gel column chromatography (water: 1.5 MTEAB = 10:1 elution). The target product peak was collected, concentrated, and lyophilized to obtain a white solid, YK-CAP-101-PM3 (triethylamine salt, 340 mg, 0.69 mmol, 71.6%), C11H16N5O9P, MS (ES): m / z (MH-) 392.1.

[0116] Step 4: Synthesis of YK-CAP-101-PM4

[0117] YK-CAP-101-PM3 (340 mg, 0.69 mmol), imidazole (706 mg, 10.38 mmol), 2,2'-dithiopyridine (2287 mg, 10.38 mmol), triethylamine (1050 mg, 10.38 mmol), and triphenylphosphine (2723 mg, 10.38 mmol) were dissolved in 4 mL of dry N,N-dimethylformamide. The reaction was carried out under nitrogen protection with stirring at room temperature for about 4 hours. After the reaction was completed, the reaction mixture was poured into a solution of sodium iodide (1225 mg, 8.17 mmol) in acetone (6 mL). After stirring at room temperature for 30 min, the mixture was centrifuged to obtain a crude precipitate. The precipitate was washed with acetone, separated, and the lower precipitate was collected and lyophilized to obtain a white solid YK-CAP-101-PM4 (sodium salt, 309 mg, 0.66 mmol, 96.2%), C14H18N7O8P, MS (ES): m / z (MH-) 442.1.

[0118] Step 5: Synthesis of YK-CAP-101-PM5

[0119] YK-CAP-101-PM4 (309 mg, 0.66 mmol) and TEAP (458 mg, 2.30 mmol) were dissolved in dry N,N-dimethylformamide (5 mL), and zinc chloride (215 mg, 1.58 mmol) was added. The mixture was stirred at rt under nitrogen protection for approximately 21 h. After the reaction was complete, MTBE (10 mL) was added to the system, and the mixture was ultrasonically stirred and washed. After standing, the supernatant was discarded, and the reaction was repeated once. The bottom layer was collected and concentrated under reduced pressure. Water (80 mL) was added to dissolve the solid, and the mixture was purified by gel column chromatography (elution with water and 1.5 MTEAB = 20:1). The target product peak was collected, concentrated, and lyophilized to obtain a white solid, YK-CAP-101-PM5 (triethylamine salt, 320 mg, 0.56 mmol, 84.4%), C11H17N5O12P2, MS (ES): m / z (MH-) 472.0.

[0120] Step 6: Synthesis of YK-CAP-101-PM6

[0121] YK-CAP-101-PM5 (320 mg, 0.56 mmol) and iodomethane (960 mg, 6.76 mmol) were dissolved in dry N,N-dimethylformamide (4 mL), and the mixture was stirred in an oil bath at 37 °C for approximately 23 h. After the reaction was complete, water (5 mL) was added to the system to dissolve the residue, and the mixture was washed with EA (25 mL). The mixture was separated, and the lower aqueous layer was collected and concentrated under reduced pressure. Water (50 mL) was added to dissolve the residue, and the mixture was purified by gel column chromatography (elution with water and 1.5 MTEAB = 10:1). The target product peak was collected, concentrated, lyophilized, and desalted again by high-performance liquid chromatography (50 mMTEAB and methanol mobile phase) to obtain YK-CAP-101-PM6 (triethylamine salt, 95 mg, 0.16 mmol, 28.8%) as a white solid, C12H19N5O12P2, MS (ES): m / z (MH-) 486.0.

[0122] Step 7: Synthesis of YK-CAP-101

[0123] YK-CAP-101-PM6 (95 mg, 0.16 mmol) and INT-II (219 mg, 0.27 mmol) were dissolved in dry dimethyl sulfoxide (1.2 mL), and zinc chloride (518 mg, 3.80 mmol) was added. The mixture was stirred in an oil bath at 37 °C under nitrogen protection for approximately 3 days. After the reaction was complete, 0.25 MEDTA solution was added to dissolve the product, and the pH was adjusted to 6-7 with 1.5 MTEAB. The product was purified by gel column chromatography (eluting with water and 1.5 MTEAB = 10:1), and the target product peak was collected, concentrated, and lyophilized. Further purification by high-performance liquid chromatography yielded the final product YK-CAP-101 (20 mg, 16.30 µmol, 10.2%), C33H45N15O25P4, MS (ES): m / z (MH-) 1174.2. 1HNMR(400MHz,D2O)δ8.43(d,J=1.3Hz,1H),8.38(s,1H),8.06(d,J=6.0Hz,2H),6.01(d,J=5.4Hz,1H),5. 89(d,J=2.3Hz,1H),5.81(d,J=4.5Hz,1H),4.97–4.86(m,3H),4.66(t,J=4.8Hz,1H),4.52–4.35(m,4H),4. 32(d,J=4.5Hz,1H),4.21(s,3H),4.09(s,2H),3.97(s,3H),3.78(t,J=7.0Hz,1H),3.43(s,4H).31PNMR(D 2O,162MHz)δ-0.92(s,1P),-11.11(d,J=19.3Hz,1P),-11.50(d,J=17.8Hz,1P),-22.23(t,J=17.8Hz,1P).

[0124] 4. Synthesis of YK-CAP-102

[0125] Step 1: Synthesis of YK-CAP-102-PM1

[0126] YK-CAP-102-PM1 (8.79 g, 17.62 mmol, 68.61%) was synthesized from α-D-aminoglucose pentaacetate (10.0 g, 25.68 mmol) using the same method as YK-CAP-101-PM1. C19H23ClN6O8, MS (ES): m / z (M+H+) 499.1.

[0127] Step 2: Synthesis of YK-CAP-102-PM2

[0128] Using YK-CAP-102-PM1 (8.79 g, 17.62 mmol) as the starting material, YK-CAP-101-PM2 (2.37 g, 6.69 mmol, 37.96%) was synthesized according to the same method as YK-CAP-101-PM2. C13H18N6O6, MS (ES): m / z (M+H+) 355.2. YK-CAP-102-PM2:1HNMR(400MHz,DMSO-d 6)δ8.41(s,1H),7.97(d,J=9.2Hz,1H),7.65(s,1H),6.76(s,2H),5.32(d,J=10.2Hz,1H),4 .15(q,J=9.8Hz,1H),3.67(d,J=11.9Hz,1H),3.55–3.41(m,2H),3.25(s,2H),1.64(s,3H).

[0129] Step 3: Synthesis of YK-CAP-102-PM3

[0130] Using YK-CAP-102-PM2 (500 mg, 1.41 mmol) as a starting material, YK-CAP-101-PM3 (triethylamine salt, 702 mg, 1.31 mmol, 93.0%) was synthesized via the same route as YK-CAP-101-PM3. C13H19N6O9P, MS (ES): m / z (MH-) 433.1.

[0131] Step 4: Synthesis of YK-CAP-102-PM4 intermediate

[0132] Using YK-CAP-102-PM3 (702 mg, 1.31 mmol) as a starting material, YK-CAP-101-PM4 (sodium salt, 521 mg, 1.03 mmol, 78.5%) was synthesized via the same route as YK-CAP-101-PM4. C16H21N8O8P, MS (ES): m / z (MH-) 483.1.

[0133] Step 5: Synthesis of YK-CAP-102-PM5 intermediate

[0134] Using YK-CAP-102-PM4 (521 mg, 1.03 mmol) as a starting material, YK-CAP-101-PM5 (triethylamine salt, 338 mg, 0.55 mmol, 53.3%) was synthesized via the same route as YK-CAP-101-PM5. C13H20N6O12P2, MS (ES): m / z (MH-) 513.0.

[0135] Step 6: Synthesis of YK-CAP-102-PM6 intermediate

[0136] Using YK-CAP-102-PM5 (338 mg, 0.55 mmol) as a starting material, YK-CAP-101-PM6 (triethylamine salt, 158 mg, 0.25 mmol, 45.6%) was synthesized via the same route as YK-CAP-101-PM6. C14H22N6O12P2, MS (ES): m / z (MH-) 527.1.

[0137] Step 7: Synthesis of YK-CAP-102

[0138] YK-CAP-102 (23.9 mg, 18.85 μmol, 7.5%) was obtained from YK-CAP-101 via the same synthetic route as YK-CAP-101. C35H48N16O25P4, MS (ES): m / z (MH-) 1115.1. 1HNMR(400MHz,D2O)δ8.42(d,J=1.3Hz,1H),8.38(s,1H),8.04(d,J=5.8Hz,2H),6.00(d,J=5.4Hz,1H),5.88( d,J=2.3Hz,1H),5.81(d,J=4.4Hz,1H),4.95–4.83(m,3H),4.64(t,J=4.7Hz,1H),4.53–4.36(m,4H),4.30(d, J=4.4Hz,1H),4.20(s,2H),4.06(s,2H),3.93(s,3H),3.75(t,J=7.0Hz,3H),3.62(s,3H),1.67(s,3H).31PNM R(D2O,162MHz)δ-0.92(s,1P),-11.21(d,J=19.2Hz,1P),-11.55(d,J=17.3Hz,1P),-23.43(t,J=17.6Hz,1P).

[0139] 5. Synthesis of YK-CAP-103

[0140] Step 1: Synthesis of YK-CAP-103-PM1

[0141] (4R,5R)-5-((R)-1,2-dihydroxyethyl)-2,2-dimethyl-1,3-dioxolane-4-carboxaldehyde (10.00 g, 52.58 mmol) and methyl 2-(triphenyl-5-phosphatidylinide)acetate (21.6 g, 64.60 mmol) were added to a single-necked flask containing acetonitrile (200 mL). The mixture was heated and stirred at 90 °C for 10 hours. After the reaction was complete, ethyl acetate (300 mL) was added to dilute the mixture. The mixture was washed with saturated brine and the organic phase was dried over anhydrous sodium sulfate. The solvent was removed by vacuum compression to obtain a yellow oily compound (26.7 g), which did not require purification and was used directly in the next reaction.

[0142] Step 2: Synthesis of YK-CAP-103-PM2

[0143] YK-CAP-103-PM1 (12.8 g, calculated as 25.21 mmol), tert-butyldiphenylchlorosilane (17.2 g, 62.42 mmol), and imidazole (5.31 g, 78.03 mmol) were sequentially added to a single-necked flask containing dichloromethane (200 mL), and the mixture was stirred overnight at room temperature. After the reaction was complete, the mixture was diluted with dichloromethane (100 mL), washed with saturated brine (3 × 200 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under vacuum. The residue was purified by silica gel chromatography (0-17% ethyl acetate / n-hexane) to obtain YK-CAP-103-PM2 (20.0 g, 41.27 mmol).

[0144] Step 3: Synthesis of YK-CAP-103-PM3

[0145] YK-CAP-103-PM2 (20.0 g, 41.27 mmol) was dissolved in a single-necked flask containing 150 mL of LCM. The solution was cooled to -78 °C, and diisobutylaluminum hydride toluene solution (1.5 M, 63.2 mL, 94.8 mmol) was slowly added dropwise. After the addition was complete, the mixture was brought to room temperature and stirred overnight. After the reaction was complete, 65 mL of methanol was slowly added under ice bath conditions, followed by the formation of a white flocculent solid. Sodium sulfate decahydrate was then added, and the mixture was stirred for 20 minutes. The solid was removed by filtration, and the filtrate was evaporated to dryness. The residue was purified by silica gel chromatography (0-30% ethyl acetate / n-hexane) to obtain YK-CAP-103-PM3 (15.00 g, 32.85 mmol, 79.6%).

[0146] Step 4: Synthesis of YK-CAP-103-PM4

[0147] YK-CAP-103-PM3 (15.00 g, 32.85 mmol), p-toluenesulfonyl chloride (7.5 g, 39.40 mmol), and triethylamine (5.0 g, 49.30 mmol) were added sequentially to a single-necked flask containing dichloromethane (60 mL). The mixture was stirred overnight at room temperature. After the reaction was complete, the mixture was diluted with dichloromethane (50 mL), washed with saturated brine (2 × 100 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under vacuum. The residue was purified by silica gel chromatography (0-20% ethyl acetate / n-hexane) to obtain YK-CAP-103-PM4 (17.00 g, 27.83 mmol, 84.7%).

[0148] Step 5: Synthesis of YK-CAP-103-PM5

[0149] YK-CAP-103-PM4 (17.00 g, 27.83 mmol) was dissolved in a single-necked flask containing tetrahydrofuran (150 mL). A solution of potassium butoxide in tetrahydrofuran (1 M, 61.0 mL, 61.0 mmol) was slowly added dropwise at -40 °C. After the addition was complete, the mixture was stirred at room temperature for 3 hours. The mixture was then diluted with ethyl acetate (200 mL), washed with saturated brine (2 × 150 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under vacuum. The residue was purified by silica gel chromatography (0-11% ethyl acetate / n-hexane) to obtain YK-CAP-103-PM5 (3.29 g, 7.50 mmol, 27.0%).

[0150] Step 6: Synthesis of YK-CAP-103-PM6

[0151] YK-CAP-103-PM5 (3.29 g, 7.50 mmol) was dissolved in a mixed solution of tetrahydrofuran (25 mL) and water (5 mL). Potassium osmium tetroxide dihydrate (140 mg, 0.38 mmol) and N-methyl-N-morpholine oxide (1.05 g, 9.00 mmol) were added sequentially to the mixed solution, and the mixture was heated and stirred at 40 °C for 6 hours. After the reaction was complete, the mixture was diluted with ethyl acetate (100 mL), washed with saturated sodium sulfite aqueous solution (2 × 80 mL), dried over anhydrous sodium sulfate, filtered, and the solvent was removed under vacuum to obtain YK-CAP-103-PM6 (3.48 g, 7.36 mmol, 98.2%).

[0152] Step 7: Synthesis of YK-CAP-103-PM7

[0153] YK-CAP-103-PM6 (3.48 g, 7.36 mmol) was dissolved in a mixed solution of tetrahydrofuran (25 mL) and water (5 mL). Potassium periodate (2.54 g, 11.04 mmol) was added sequentially to the mixed solution, and the mixture was heated and stirred at 40 °C for 6 hours. After the reaction was complete, the mixture was diluted with ethyl acetate (100 mL), washed with saturated sodium sulfite aqueous solution (2 × 50 mL), dried over anhydrous sodium sulfate, filtered, and the solvent was removed under vacuum to obtain YK-CAP-103-PM7 (3.14 g, 7.13 mmol, 96.9%).

[0154] Step 8: Synthesis of YK-CAP-103-PM8

[0155] YK-CAP-103-PM7 (5.19 g, 11.78 mmol) was dissolved in a single-necked flask containing methanol (100 mL). Sodium borohydride (0.54 g, 13.18 mmol) was added in portions under ice bath conditions. The mixture was then stirred at room temperature for 3 hours. After the reaction was complete, the mixture was diluted with water (150 mL) and extracted with ethyl acetate (2 × 150 mL). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum. The residue was purified by silica gel chromatography (0-13% ethyl acetate / n-hexane) to obtain YK-CAP-103-PM8 (3.30 g, 7.46 mmol, 63.3%).

[0156] Step 9: Synthesis of YK-CAP-103-PM9

[0157] Using YK-CAP-103-PM8 (3.30 g, 7.46 mmol) as the raw material, YK-CAP-103-PM9 (3.42 g, 5.73 mmol, 76.8%) was obtained by following the synthesis method of YK-CAP-103-PM4.

[0158] Step 10: Synthesis of YK-CAP-103-PM10

[0159] 2-Amino-6-chloroguanine (1.17 g, 6.90 mmol) and lithium hydride (55 mg, 6.88 mmol) were dissolved in 30 mL of DMSO and reacted at 90 °C for 1 hour. Then, a DMSO solution of YK-CAP-103-PM9 (3.42 g, 5.73 mmol) (15 mL) was added, and the reaction was continued for 5 hours. After the reaction was complete, the solution was diluted with water (100 mL), extracted with dichloromethane (2 × 150 mL), and the organic phases were combined. The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum. The residue was purified by silica gel chromatography (0-25% ethyl acetate / dichloromethane) to give YK-CAP-103-PM10 (1.90 g, 3.20 mmol, 55.9%). C30H36ClN5O4Si, MS (ES): m / z (M+H+) 595.2.

[0160] Step 11: Synthesis of YK-CAP-103-PM11

[0161] YK-CAP-103-PM10 (1.90 g, 3.20 mmol) was dissolved in tetrahydrofuran (40 mL), followed by the addition of 1 M HCl (80 mL). The mixture was stirred at 90 °C for 7 hours. After the reaction was complete, the solvent was removed under reduced pressure, and the residue was purified by pre-HPLC to give a white solid compound YK-CAP-103-PM11 (587 mg, 1.97 mmol, 61.7%). C11H15N5O5, MS (ES): m / z (M+H+) 298.2. YK-CAP-103-PM11:1HNMR(400MHz,DMSO-d6)δ10.57(s,1H),7.68(s,1H),6.46(s,2H),5.00 -4.53(m,2H),4.17-4.09(m,1H),4.00-3.87(m,2H),3.77-3.61(m,3H),3.46-3.32(m,3H).

[0162] Step 12: Synthesis of YK-CAP-103-PM12

[0163] Using YK-CAP-103-PM11 (300 mg, 1.01 mmol) as a starting material, YK-CAP-101-PM12 (triethylamine salt, 350 mg, 0.73 mmol, 72.5%) was obtained via the synthetic route of YK-CAP-101-PM3. C11H16N5O8P, MS (ES): m / z (MH-) 376.1.

[0164] Step 13: Synthesis of YK-CAP-103-PM13

[0165] Using YK-CAP-103-PM12 (350 mg, 0.73 mmol) as a starting material, YK-CAP-101-PM4 was synthesized to obtain YK-CAP-103-PM13 (sodium salt, 320 mg, 0.71 mmol, 97.4%). C14H18N7O7P, MS (ES): m / z (MH-) 426.1.

[0166] Step 14: Synthesis of YK-CAP-103-PM14

[0167] Using YK-CAP-103-PM13 (320 mg, 0.71 mmol) as a starting material, YK-CAP-101-PM5 was synthesized to obtain YK-CAP-103-PM14 (triethylamine salt, 250 mg, 0.45 mmol, 62.9%). C11H17N5O11P2, MS (ES): m / z (MH-) 456.0.

[0168] Step 15: Synthesis of YK-CAP-103-PM15

[0169] Using YK-CAP-103-PM14 (250 mg, 0.45 mmol) as a starting material, YK-CAP-101-PM6 was synthesized to obtain YK-CAP-103-PM15 (triethylamine salt, 90 mg, 0.16 mmol, 34.9%). C12H20N5O11P2, MS (ES): m / z (MH-) 470.1.

[0170] Step 16: Synthesis of YK-CAP-103

[0171] YK-CAP-103 (29 mg, 23.95 µmol, 15.0%) was obtained from YK-CAP-101 via the same synthetic route as YK-CAP-101. C33H45N15O24P4, MS (ES): m / z (MH-) 1158.2. 1HNMR(400MHz,D2O)δ8.41(d,J=1.2Hz,1H),8.33(d,J=2.3Hz,1H),8.02(d,J=4.8Hz,2H),6.11(d,J=5.4Hz,1H), 5.91(d,J=2.4Hz,1H),5.71(d,J=4.6Hz,1H),5.20–5.03(m,2H),4.91–4.77(m,3H),4.63(t,J=4.7Hz,1H),4.53–4 .35(m,4H),4.26(d,J=3.1Hz,1H),4.10(s,3H),4.02(s,2H),3.92(s,3H),3.54(t,J=7.0Hz,1H),3.32(s,3H).31 PNMR(D2O,162MHz)δ-0.91(s,1P),-11.22(d,J=19.4Hz,1P),-11.33(d,J=17.2Hz,1P),-24.43(t,J=17.7Hz,1P).

[0172] 6. Synthesis of YK-CAP-104

[0173] Step 1: Synthesis of YK-CAP-104-PM1

[0174] 1,2-O-isopropyl-A-D-ribofuranosyl furanose (20.0 g, 0.11 mol) was dissolved in dichloromethane, and imidazole (11.6 g, 0.17 mol) and TBDPSCl (33.0 g, 0.12 mol) were added to the above system. The reaction mixture was stirred for 15 hours at room temperature. The reaction solution was added to a saturated sodium bicarbonate solution and extracted with dichloromethane. The dichloromethane phase was washed once with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was evaporated to dryness under reduced pressure. The residue was subjected to silica gel chromatography (0-17% ethyl acetate / n-hexane) to obtain YK-CAP-104-PM1 (33.5 g, 78.16 mmol, 71.1%).

[0175] Step 2: Synthesis of YK-CAP-104-PM2

[0176] YK-CAP-104-PM1 (33.5 g, 78.16 mmol) was dissolved in acetonitrile, and 2-iodobenzoic acid (28.5 g, 101.8 mmol) was added. The mixture was heated to 90 °C and stirred for 5 hours. The reaction solution was filtered, and the filtrate was evaporated to dryness under reduced pressure to obtain YK-CAP-104-PM2 (32.7 g, 76.66 mmol, 98.1%).

[0177] Step 3: Synthesis of YK-CAP-104-PM3

[0178] 60.3 g (138.3 mmol) of bromomethyltriphenylphosphine bromide was dissolved in 400 mL of tetrahydrofuran. The solution was cooled to -78 °C, and a 2.5 M solution of n-butyllithium in tetrahydrofuran (76 mL, 190 mmol) was slowly added dropwise. After the addition was complete, the reaction system was heated to 0 °C and stirred for 2 hours. The reaction system was then cooled back to -78 °C, and a 100 mL solution of YK-CAP-104-PM2 (48.0 g, 112.52 mmol) in tetrahydrofuran was slowly added dropwise. After the addition was complete, the reaction system was heated to room temperature and stirred overnight. The reaction system was quenched with 200 mL of saturated ammonium chloride solution, extracted with 300 mL × 3 ethyl acetate, and the organic phases were combined. The organic phases were washed with saturated NaCl aqueous solution, dried over anhydrous sodium sulfate, filtered, and evaporated to dryness. The residue was purified by silica gel chromatography (0-20% ethyl acetate / n-hexane) to obtain YK-CAP-104-PM3 (39.0g, 91.85mmol, 81.6%).

[0179] Step 4: Synthesis of YK-CAP-104-PM4

[0180] YK-CAP-104-PM3 (34.0 g, 80.07 mmol) was dissolved in tetrahydrofuran (200 mL), and tetrabutylammonium fluoride (52.0 g, 198.9 mmol) was added. The mixture was stirred at room temperature for 1 hour. A saturated aqueous solution of ammonium chloride was added to the reaction mixture, and the mixture was extracted with ethyl acetate (200 mL × 3). The organic phases were combined, washed with a saturated aqueous solution of NaCl, dried over anhydrous sodium sulfate, filtered, and evaporated to dryness. The residue was purified by silica gel chromatography (0-60% ethyl acetate / n-hexane) to give YK-CAP-104-PM4 (13.5 g, 72.50 mmol, 90.5%).

[0181] Step 5: Synthesis of YK-CAP-104-PM5

[0182] YK-CAP-104-PM4 (13.5 g, 72.50 mmol) was dissolved in dichloromethane (150 mL), and triethylamine (22.0 g, 217.4 mmol) was added. The reaction system was cooled to 0 °C, and benzoyl chloride (11.2 g, 79.7 mmol) was slowly added dropwise. The mixture was then naturally heated to room temperature and stirred for 1 h. After the reaction was complete, the reaction system was quenched with saturated sodium bicarbonate aqueous solution (100 mL), extracted with dichloromethane (100 mL × 3), and the organic phases were combined. The organic phases were washed with saturated NaCl aqueous solution, dried over anhydrous sodium sulfate, filtered, and evaporated to dryness. The residue was purified by silica gel chromatography (0-20% ethyl acetate / n-hexane) to obtain YK-CAP-104-PM5 (19.6 g, 67.51 mmol, 93.1%).

[0183] Step 6: Synthesis of YK-CAP-104-PM6

[0184] YK-CAP-104-PM5 (18.6 g, 64.07 mmol) was dissolved in a mixed solvent of tetrahydrofuran (160 mL) and water (40 mL). N-methylmorpholine oxide (11.3 g, 96.5 mmol) and potassium osmium tetroxide dihydrate (2.0 g, 6.4 mmol) were then added sequentially, and the mixture was stirred overnight at room temperature. After the reaction was complete, the solution was quenched with a saturated aqueous sodium sulfite solution, extracted with ethyl acetate (200 mL × 3), and the organic phases were combined. The organic phases were washed with a saturated aqueous sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and evaporated to dryness. The residue was purified by silica gel chromatography (0-60% ethyl acetate / n-hexane) to obtain YK-CAP-104-PM6 (20.5 g, 63.21 mmol, 98.7%).

[0185] Step 7: Synthesis of YK-CAP-104-PM7

[0186] Using YK-CAP-104-PM6 (17.7 g, 54.57 mmol) as a starting material, it was dissolved in acetonitrile (140 mL) and pyridine (140 mL). Imidazole (11.1 g, 163.8 mmol), triphenylphosphine (21.5 g, 81.9 mmol), and carbon tetrabromide (27.1 g, 81.9 mmol) were weighed sequentially. The reaction system was heated to 70 °C under a nitrogen atmosphere and stirred for 6 h. The reaction was monitored for completeness by thin-layer chromatography, and the product was evaporated to dryness under vacuum. The residue was purified by silica gel chromatography (0-50% ethyl acetate / n-hexane) to obtain YK-CAP-104-PM7 (13.9 g, 35.90 mmol, 65.8%).

[0187] Step 8: Synthesis of YK-CAP-104-PM8

[0188] YK-CAP-104-PM7 (13.4 g, 34.60 mmol) was dissolved in acetonitrile (120 mL), followed by the addition of potassium carbonate (14.4 g, 104.2 mmol) and dimethylamine hydrochloride (3.4 g, 41.1 mmol). The mixture was heated to 70 °C and reacted for 16 h, with thin-layer chromatography monitoring for complete reaction. After cooling to room temperature, the mixture was filtered, and the organic phase was evaporated to dryness under reduced pressure. The residue was purified by silica gel chromatography (0-50% ethyl acetate / n-hexane) to obtain YK-CAP-104-PM8 (8.5 g, 24.18 mmol, 69.9%). C18H25NO6, MS (ES): m / z (M+H+) 352.1.

[0189] Step 9: Synthesis of YK-CAP-104-PM9

[0190] YK-CAP-104-PM8 (8.5 g, 24.18 mmol) was dissolved in dichloromethane (160 mL), cooled to -40 °C, and a solution of diethylaminotrifluoride (4.7 g, 29.0 mmol) in dichloromethane (10 mL) was slowly added dropwise. After the addition was complete, the temperature was slowly raised to 0 °C, and the reaction was stirred for 4 h. The reaction was monitored for completeness by thin-layer chromatography. The reaction system was quenched with saturated sodium bicarbonate aqueous solution (100 mL), extracted with dichloromethane (100 mL × 3), and the organic phases were combined, washed with saturated NaCl aqueous solution, dried over anhydrous sodium sulfate, filtered, and evaporated to dryness. The residue was purified by silica gel chromatography (0-40% ethyl acetate / n-hexane) to obtain YK-CAP-104-PM9 (5.2 g, 14.71 mmol, 60.8%). C18H24FNO5, MS(ES): m / z(M+H+)354.2.

[0191] Step 10: Synthesis of YK-CAP-104-PM10

[0192] YK-CAP-104-PM9 (4.7 g, 13.30 mmol) was dissolved in acetic acid (100 mL), and acetic anhydride (16.4 g, 160.6 mmol) was added. Concentrated sulfuric acid (4.7 g, 26.8 mmol) was then slowly added dropwise. After the addition was complete, the temperature was slowly raised to 40 °C, and the reaction was stirred for 16 h. The reaction was monitored for completeness by LC-MS. The reaction system was quenched with saturated sodium bicarbonate aqueous solution, and the pH was adjusted to neutral. Extraction was performed with ethyl acetate (200 mL × 3). The organic phases were combined, washed with saturated NaCl aqueous solution, dried over anhydrous sodium sulfate, filtered, and evaporated to dryness. The residue was purified by silica gel chromatography (0-40% ethyl acetate / n-hexane) to obtain YK-CAP-104-PM10 (2.5 g, 6.29 mmol, 47.3%). C19H24FNO7, MS (ES): m / z (M+H+) 398.1.

[0193] Step 11: Synthesis of YK-CAP-104-PM11

[0194] Intermediate INT-I (2.7 g, 6.9 mmol) was dissolved in 1,2-dichloroethane (50 mL), and N,O-bis(trimethylsilylacetamide) (2.8 g, 13.8 mmol) was added. The reaction mixture was heated to 80 °C and stirred for 2 h, then evaporated to dryness under reduced pressure. The residue was dissolved in toluene (30 mL), and a toluene (20 mL) solution of YK-CAP-104-PM10 (2.5 g, 6.29 mmol) was added. Trimethylsilyl trifluoromethanesulfonate (1.5 g, 6.7 mmol) was slowly added dropwise. The reaction mixture was heated to 70 °C and stirred for 2 h. Thin-layer chromatography was used to monitor the completeness of the reaction. The reaction mixture was quenched with saturated sodium bicarbonate aqueous solution, extracted with ethyl acetate (50 mL × 3), and the organic phases were combined. The organic phases were washed with saturated NaCl aqueous solution, dried over anhydrous sodium sulfate, filtered, and evaporated to dryness under reduced pressure. The residue was purified by silica gel chromatography (0-70% ethyl acetate / dichloromethane) to obtain YK-CAP-104-PM11 (2.7 g, 3.72 mmol, 59.1%). C37H36FN7O8, MS (ES): m / z (M+H+) 726.2.

[0195] Step 12: Synthesis of YK-CAP-104-PM12

[0196] YK-CAP-104-PM11 (2.7 g, 3.72 mmol) was dissolved in a mixed solvent of NH3 / MeOH (20 mL) and water (4 mL). The reaction system was heated to 50 °C and stirred for 10 h. LC-MS monitoring showed that the reaction was complete. The product was evaporated to dryness to obtain 2.5 g of crude product, which was purified by high-pressure preparative liquid chromatography to obtain YK-CAP-104-PM12 (560 mg, 1.64 mmol, 44.0%). C13H19FN6O4, MS (ES): m / z (M+H+) 343.1. YK-CAP-104-PM12:1HNMR(400MHz,DMSO-d 6) δ9.98(s,1H),7.93(s,1H),6.52(s,2H),5.69(d,J=6.1Hz,1H),5.51(s,1H),4.97-4.85(m,1H),4 .37(d,J=6.4Hz,1H),4.14(d,J=4.4Hz,1H),3.77(d,J=12.1Hz,1H),3.62(s,1H),2.59–2.33(m,8H).

[0197] Step 13: Synthesis of YK-CAP-104-PM13

[0198] Using YK-CAP-104-PM12 (560 mg, 1.64 mmol) as a starting material, YK-CAP-101-PM13 (triethylamine salt, 557 mg, 1.06 mmol, 64.9%) was synthesized via the same route as YK-CAP-101-PM3. C13H20FN6O7P, MS (ES): m / z (MH-) 421.1.

[0199] Step 14: Synthesis of YK-CAP-104-PM14

[0200] Using YK-CAP-104-PM13 (557 mg, 1.06 mmol) as a starting material, YK-CAP-101-PM14 (sodium salt, 414 mg, 0.84 mmol, 79.0%) was synthesized via the same route as YK-CAP-101-PM4. C16H23FN8O6P, MS (ES): m / z (MH-) 471.1.

[0201] Step 15: Synthesis of YK-CAP-104-PM15

[0202] Using YK-CAP-104-PM14 (414 mg, 0.84 mmol) as a starting material, YK-CAP-101-PM15 (triethylamine salt, 300 mg, 0.50 mmol, 59.2%) was obtained via the synthetic route of YK-CAP-101-PM5. C13H21FN6O10P2, MS (ES): m / z (MH-) 501.1.

[0203] Step 16: Synthesis of YK-CAP-104-PM16

[0204] Using YK-CAP-104-PM15 (300 mg, 0.50 mmol) as a starting material, YK-CAP-101-PM6 was synthesized via the same route as YK-CAP-101-PM6 to obtain YK-CAP-104-PM16 (triethylamine salt, 124 mg, 0.20 mmol, 40.2%). C14H24FN6O10P2, MS (ES): m / z (MH-) 515.1.

[0205] Step 17: Synthesis of YK-CAP-104

[0206] YK-CAP-104-PM16 (124 mg, 0.20 mmol) was used as a starting material, and YK-CAP-104 (ammonium salt, 21 mg, 16.72 µmol, 8.4%) was obtained by following the synthetic route of YK-CAP-101. C35H49FN16O23P4, MS (ES): m / z (MH-) 1203.1. 1HNMR(400MHz,D2O)δ8.52(d,J=1.2Hz,1H),8.44(d,J=2.2Hz,1H),7.86(d,J=4.4Hz,2H),6.14(d,J=5.2Hz,1H), 5.94(d,J=2.4Hz,1H),5.81(d,J=4.6Hz,1H),4.81–4.67(m,3H),4.63(t,J=4.6Hz,1H),4.53–4.35(m,5H),4.23(d ,J=3.1Hz,1H),4.19(s,3H),4.12(s,2H),3.78(s,3H),3.32(t,J=7.0Hz,1H),3.12(s,3H),2.55–2.35(m,8H).31 PNMR(D2O,162MHz)δ-0.92(s,1P),-11.32(d,J=20.7Hz,1P),-12.33(d,J=17.5Hz,1P),-23.28(t,J=16.8Hz,1P).

[0207] 7. Synthesis of YK-CAP-105

[0208] Step 1: Synthesis of YK-CAP-105-PM1

[0209] YK-CAP-104-PM4 (5.76 g, 30.93 mmol) was dissolved in acetic acid (8 mL). Acetic anhydride (31.6 g, 310 mmol) and sulfuric acid (500 μL) were added to the mixture, and the mixture was stirred at room temperature for 4 hours. After the reaction was complete, the pH was adjusted to weakly acidic with NaHCO3 aqueous solution. The mixture was extracted twice with ethyl acetate, and the organic phase was evaporated to dryness. The residue was purified by silica gel chromatography (0-80% ethyl acetate / n-hexane) to obtain YK-CAP-105-PM1 (5.9 g, 21.67 mmol, 70.1%).

[0210] Step 2: Synthesis of YK-CAP-105-PM2

[0211] Intermediate INT-I (9.27 g, 23.87 mmol) and N,O-bis(trimethylsilylacetamide) (9.72 g, 47.8 mmol) were dissolved in 1,2-dichloroethane (60 mL), stirred at 80 °C for 2 hours, and the solvent was evaporated to dryness. YK-CAP-105-PM1 (5.9 g, 21.67 mmol) was dissolved in toluene (80 mL) and added to the above residue, followed by trimethylsilyl trifluoromethanesulfonate (5.31 g, 23.9 mmol). The reaction was carried out at 70 °C for 2 hours, and the reaction was monitored for completeness by thin-layer chromatography. The mixture was filtered, and the filtrate was evaporated to dryness under reduced pressure. The residue was purified by silica gel column chromatography (0-80% ethyl acetate / n-hexane) to obtain YK-CAP-105-PM2 (10.00 g, 16.65 mmol, 76.8%).

[0212] Step 3: Synthesis of YK-CAP-105-PM3

[0213] YK-CAP-105-PM2 (8.00 g, 13.32 mmol) and [N,N'-(1,1,2,2-tetramethylethane)bis(3,5-di-tertiary-butylsalicylic acid imine)]cobalt(II) (403 mg, 0.67 mmol) were dissolved in 1,4-dioxane (30 mL). Benzenesulfonyl cyanide (72.30 g, 400 mmol) was added to the mixture, and the mixture was stirred at room temperature for 5 min. Benzenesilane (1.73 g, 16 mmol) was dissolved in anhydrous ethanol (60 mL) and added to the mixture. The mixture was stirred at room temperature for 3 hours. After the reaction was complete, the mixture was filtered, extracted twice with water and ethyl acetate, and the organic phase was evaporated to dryness. The residue was purified by silica gel chromatography (0-70% ethyl acetate / n-hexane) to obtain YK-CAP-105-PM3 (4.50 g, 7.17 mmol, 53.8%).

[0214] Step 4: Synthesis of YK-CAP-105-PM4

[0215] YK-CAP-105-PM3 (4.50 g, 7.17 mmol) was dissolved in 7 M ammonia-methanol (50 mL), and the mixture was stirred at 50 °C for 4 hours. After the reaction was complete, the solvent was evaporated under reduced pressure, and the residue was purified by pre-HPLC to obtain YK-CAP-105-PM4 (800 mg, 2.61 mmol, 36.4%). C12H14N6O4, MS (ES): m / z (M+H+) 307.11. YK-CAP-105-PM4:1HNMR(400MHz,DMSO-d 6) δ10.66(s,1H),7.97(s,1H),6.54(d,J=5.2Hz,3H),5.74(d,J=7.2Hz,1H),5.40(t,J =4.4Hz,1H),4.62-4.65(m,1H),4.31(t,J=3.2Hz,1H),3.59-3.69(m,2H),1.53(s,3H).

[0216] Step 5: Synthesis of YK-CAP-105-PM5

[0217] Using YK-CAP-105-PM4 (800 mg, 2.61 mmol) as a starting material, YK-CAP-101-PM5 (triethylamine salt, 994 mg, 2.04 mmol, 78.1%) was synthesized via the same route as YK-CAP-101-PM3. C12H15N6O7P, MS (ES): m / z (MH-) 385.1.

[0218] Step 6: Synthesis of YK-CAP-105-PM6

[0219] Using YK-CAP-105-PM5 (994 mg, 2.04 mmol) as a starting material, YK-CAP-101-PM4 was synthesized to obtain YK-CAP-105-PM6 (sodium salt, 661 mg, 1.44 mmol, 70.7%). C15H17N8O6P, MS (ES): m / z (MH-) 435.1.

[0220] Step 7: Synthesis of YK-CAP-105-PM7

[0221] Using YK-CAP-105-PM6 (661 mg, 1.44 mmol) as a starting material, YK-CAP-101-PM5 was synthesized via the same route to obtain YK-CAP-105-PM7 (triethylamine salt, 612 mg, 1.08 mmol, 74.8%). C12H16N6O10P2, MS (ES): m / z (MH-) 465.0.

[0222] Step 8: Synthesis of YK-CAP-105-PM8

[0223] Using YK-CAP-105-PM7 (612 mg, 1.08 mmol) as a starting material, YK-CAP-101-PM6 was synthesized to obtain YK-CAP-105-PM8 (triethylamine salt, 325 mg, 0.56 mmol, 51.7%). C13H19N6O10P2, MS (ES): m / z (MH-) 479.1.

[0224] Step 9: Synthesis of YK-CAP-105

[0225] YK-CAP-105 (16 mg, 13.12 µmol, 2.3%) was obtained from YK-CAP-101 via the synthetic route of YK-CAP-101. C34H44N16O23P4, MS (ES): m / z (MH-) 1267.1. 1HNMR(400MHz,D2O)δ8.49(d,J=1.4Hz,1H),8.44(d,J=2.5Hz,1H),7.88(d,J=4.2Hz,2H),6.22(d,J=4.7H z,1H),5.93(d,J=2.4Hz,1H),5.80(d,J=4.5Hz,1H),4.81–4.66(m,3H),4.62–4.32(m,5H),4.22(d,J=3.1H z,1H),4.18(s,2H),4.10(s,2H),3.77(s,3H),3.31(t,J=6.8Hz,1H),3.11(s,3H),1.53(s,3H).31PNMR(D 2O,162MHz)δ-0.90(s,1P),-11.33(d,J=20.2Hz,1P),-12.12(d,J=17.1Hz,1P),-22.34(t,J=16.2Hz,1P).

[0226] 8. Synthesis of YK-CAP-106

[0227] Step 1: Synthesis of YK-CAP-106-PM1

[0228] p-Toluenesulfonyl azide (211.46 g, 1.07 mol) and cobalt catalyst ([N,N'-(1,1,2,2-tetramethylethane)bis(3,5-di-tertiary-butylsalicylic acid)]cobalt(II)) (696 mg, 1.15 mmol) were added to YK-CAP-105-PM2 (23.00 g, 38.30 mmol) and stirred for 30 min under nitrogen protection. Then, styrene silane (10.22 g, 45.96 mmol) dissolved in anhydrous ethanol (40 mL) was added dropwise at room temperature over 30 min, and the reaction was stirred at room temperature for 2 h. The reaction was monitored by LC-MS until complete. The reaction was then stopped, and the reaction mixture was extracted with EA and saturated sodium chloride aqueous solution. The mixture was separated, and the aqueous phase was back-extracted twice with EA. The organic phases were combined, the solvent was removed by rotary evaporation, and the crude product was purified by Flash column chromatography (EA / PE = 0-100%) to obtain YK-CAP-106-PM1 (4.90 g, 7.61 mmol, 19.9%). C30H29N9O8, MS (ES): m / z (M+H+) 644.3.

[0229] Step 2: Synthesis of YK-CAP-106-PM2

[0230] Triphenylphosphine (2.78 g, 10.59 mmol), water (4.9 mL), and tetrahydrofuran (49 mL) were added to YK-CAP-106-PM1 (4.90 g, 7.61 mmol), and the mixture was heated to 50 °C and stirred for 8 hours. The reaction was monitored by LC-MS until complete. The reaction was then stopped, and the solvent was removed from the reaction solution under reduced pressure. The crude product was purified by flash column chromatography (MeOH / DCM = 0-10%) to obtain YK-CAP-106-PM2 (4.22 g, 6.83 mmol, 89.8%). C30H31N7O8, MS (ES): m / z (M+H+) 618.2.

[0231] Step 3: Synthesis of YK-CAP-106-PM3

[0232] Triethylamine (5.04 g, 49.84 mmol) and dichloromethane (82 mL) were added to YK-CAP-106-PM2 (4.10 g, 6.64 mmol). The mixture was cooled to 0 °C, and acetyl chloride (1.68 g, 21.36 mmol) was added dropwise. After the addition was complete, the mixture was allowed to react at room temperature for 24 hours. The reaction was monitored by LC-MS until complete. The reaction was then stopped, and the reaction solution was poured into ice water and stirred. The mixture was separated into liquid and liquid phases. The aqueous phase was back-extracted twice with dichloromethane. The organic phases were combined, and the solvent was removed under reduced pressure. The crude product was purified by Flash column chromatography (MeOH / DCM = 0-10%) to obtain YK-CAP-106-PM3 (2.94 g, 4.46 mmol, 67.1%). C32H33N7O9, MS (ES): m / z (M+H+) 660.3.

[0233] Step 4: Synthesis of YK-CAP-106-PM4

[0234] 29 mL of 7 M ammonia-methanol solution and 5.9 mL of water were added to YK-CAP-106-PM3 (2.94 g, 4.46 mmol), and the mixture was stirred at room temperature for 24 hours. The reaction was monitored by LC-MS until complete. The reaction was then stopped, and the solvent was removed by rotary evaporation under reduced pressure. The crude product was added to EA solution and stirred. The mixture was then filtered to obtain a yellow solid, YK-CAP-106-PM4 (1.36 g, 4.02 mmol, 90.1%). C13H18N6O5, MS (ES): m / z (M+H+) 339.3. YK-CAP-106-PM4: 1HNMR(400MHz,MeOD)δ7.99(s,1H),5.84(d,1H),4.77(s,1H),4.66(d,1H),3.93-3.82(m,2H),2.06(s,3H),1.63(s,3H).

[0235] Step 5: Synthesis of YK-CAP-106-PM5

[0236] Using YK-CAP-106-PM4 (500 mg, 1.48 mmol) as a starting material, YK-CAP-101-PM5 (triethylamine salt, 480 mg, 0.92 mmol, 62.4%) was obtained via the synthetic route of YK-CAP-106-PM3. C13H19N6O8P, MS (ES): m / z (MH-) 417.2.

[0237] Step 6: Synthesis of YK-CAP-106-PM6

[0238] Using YK-CAP-106-PM5 (480 mg, 0.92 mmol) as a starting material, YK-CAP-101-PM4 was synthesized to obtain YK-CAP-106-PM6 (sodium salt, 410 mg, 0.84 mmol, 90.9%). C16H21N8O7P, MS (ES): m / z (MH-) 467.1.

[0239] Step 7: Synthesis of YK-CAP-106-PM7

[0240] Using YK-CAP-106-PM6 (410 mg, 0.84 mmol) as a starting material, YK-CAP-101-PM5 was synthesized to obtain YK-CAP-106-PM7 (triethylamine salt, 331 mg, 0.55 mmol, 65.7%). C13H20N6O11P2, MS (ES): m / z (MH-) 497.0.

[0241] Step 8: Synthesis of YK-CAP-106-PM8

[0242] Using YK-CAP-106-PM7 (331 mg, 0.55 mmol) as a starting material, YK-CAP-101-PM6 was synthesized via the same route as YK-CAP-106-PM8 to obtain YK-CAP-106-PM8 (triethylamine salt, 150 mg, 0.24 mmol, 44.5%). C14H22N6O11P2, MS (ES): m / z (MH-) 511.1.

[0243] Step 9: Synthesis of YK-CAP-106

[0244] YK-CAP-106 (32 mg, 25.56 µmol, 10.6%) was obtained from YK-CAP-101 via the synthetic route of YK-CAP-101. C35H48N16O24P4, MS (ES): m / z (MH-) 1199.1. 1HNMR(400MHz,D2O)δ8.50(d,J=1.4Hz,1H),8.44(d,J=2.2Hz,1H),7.78(d,J=4.2Hz,2H),6.24(d,J=4.6Hz,1H ),5.94(d,J=2.3Hz,1H),5.81(d,J=4.4Hz,1H),4.86–4.76(m,3H),4.61–4.55(m,4H),4.52(d,J=3.6Hz,2H),4. 28(s,2H),4.20(s,1H),3.93-3.82(m,5H),3.23(t,J=6.7Hz,1H),3.12(s,2H),2.03(s,3H),1.62(s,3H).31PN MR(D2O,162MHz)δ-0.92(s,1P),-11.33(d,J=20.1Hz,1P),-12.13(d,J=16.8Hz,1P),-22.11(t,J=16.4Hz,1P).

[0245] 9. Synthesis of YK-CAP-107

[0246] Step 1: Synthesis of YK-CAP-107-PM1

[0247] Add 1M borohydride tetrahydrofuran solution (325 mL, 0.325 mol) to a three-necked flask, and slowly add 180 mL of tetrahydrofuran solution of YK-CAP-104-PM3 (60.00 g, 0.14 mol) at 0 °C. After the addition is complete, stir the reaction at room temperature for 2 hours. Then, slowly add THF / H2O (1:1, 120 mL), 2NNaOH (261 mL), and 30% hydrogen peroxide (271 mL) at 0 °C. After the addition was complete, the mixture was stirred overnight at room temperature. The reaction was monitored by TLC until the reactants were fully reacted, and then the reaction was stopped. Water was added for extraction. The organic phase was washed successively with saturated sodium thiosulfate aqueous solution and saturated brine. The organic phase was dried with anhydrous sodium sulfate, filtered, and the solvent was removed from the organic phase by rotary evaporation. The crude product was purified by flash column chromatography (PE:EA = 0~25%) to obtain a colorless oily compound YK-CAP-107-PM1 (48.80 g, 0.11 mol, 78.8%).

[0248] Step 2: Synthesis of YK-CAP-107-PM2

[0249] YK-CAP-107-PM1 (34.20 g, 77.27 mmol) was dissolved in acetonitrile, and 2-iodobenzoic acid (28.10 g, 100.5 mmol) was added to the above system. The mixture was heated to 90 °C and stirred for 5 h. After cooling to room temperature, the reaction solution was filtered, and the filtrate was evaporated to dryness to obtain crude YK-CAP-107-PM2 (34.50 g), a pale yellow oily liquid. No purification was required, and it was used directly in the next step.

[0250] Step 3: Synthesis of YK-CAP-107-PM3

[0251] YK-CAP-107-PM2 (32.90 g, calculated as 74.67 mmol) was dissolved in tetrahydrofuran. Under nitrogen protection, the mixture was cooled to 0 °C, and 1 M methyl magnesium bromide tetrahydrofuran solution (97.1 mL, 97.1 mmol) was slowly added dropwise. After the addition was complete, the mixture was stirred at room temperature for 3 h. The reaction solution was cooled to 0 °C, the reaction was quenched with saturated ammonium chloride, extracted with ethyl acetate, the organic phase was dried under rotary evaporation, and the crude product was purified by Flash column chromatography (PE:EA = 0~41%) to obtain a pale yellow oily liquid YK-CAP-107-PM3 (16.80 g, 36.79 mmol, 49.3%).

[0252] Step 4: Synthesis of YK-CAP-107-PM4

[0253] YK-CAP-107-PM3 (16.80 g, 36.79 mmol) was dissolved in THF. Under nitrogen protection, the mixture was cooled to 0°C, and a THF solution of sodium butoxide (11.20 g, 116.7 mmol) was slowly added to the system. After stirring at room temperature for 1.5 h, methyl iodoform (27.60 g, 194.5 mmol) was slowly added dropwise to the system. After the addition was complete, the reaction was stirred for another 3 h. The reaction was confirmed by TLC, and saturated ammonium chloride solution was added to quench the reaction. The mixture was extracted with ethyl acetate, and the organic phase was washed 3-5 times with saturated brine. The organic phase was dried and then evaporated to dryness. The crude product was purified by Flash column chromatography (PE:EA = 0-12%) to obtain a pale yellow oily liquid YK-CAP-107-PM4 (13.16 g, 27.96 mmol, 76.0%).

[0254] Step 5: Synthesis of YK-CAP-107-PM5

[0255] YK-CAP-107-PM4 (13.16 g, 27.96 mmol) was dissolved in glacial acetic acid (130 mL). Acetic anhydride (17.13 g, 167.8 mmol) and concentrated sulfuric acid (0.52 mL) were added sequentially to the above system. The reaction mixture was stirred at room temperature for 4 h. The reaction system was diluted with ethyl acetate, washed once with water, and then washed three times with saturated sodium bicarbonate solution. The organic phase was dried and evaporated to dryness to obtain a yellow oily liquid YK-CAP-107-PM5 (11.38 g), which was directly used in the next reaction step.

[0256] Step 6: Synthesis of YK-CAP-107-PM6

[0257] 2-Acetamino-9H-purine-6-yldiphenylaminocarbamate (6.30 g, 16.22 mmol) was dissolved in 1,2-dichloroethane (100 mL), and N,O-bis(trimethylsilylacetamide) (6.60 g, 32.4 mmol) was added. The mixture was heated to 80 °C and stirred for 1.5 hours. The reaction solvent was then removed by rotary evaporation. At room temperature, a toluene solution (100 mL) of YK-CAP-107-PM5 (6.00 g, calculated as 18.85 mmol) and TMSOTf were added sequentially. (3.6 g, 16.2 mmol), then heated to 70 °C and stirred for another 3.5 h. The reaction mixture was diluted with ethyl acetate, washed once with saturated sodium bicarbonate solution, filtered to remove insoluble matter, separated from the filtrate, dried the organic phase, evaporated to dryness, and purified by Flash column chromatography (DCM:EA = 0~52%) to obtain a yellow solid YK-CAP-107-PM6 (3.20 g, 4.95 mmol, 26.3%). C32H34N6O9, MS (ES): m / z (M+H+) 647.2.

[0258] Step 7: Synthesis of YK-CAP-107-PM7

[0259] YK-CAP-107-PM6 (3.20 g, 4.95 mmol) was dissolved in 7 M ammonia-methanol solution and water (5:1, 24 mL). The mixture was heated to 50 °C and stirred for 6 hours. The reaction mixture was evaporated to dryness, EA was added and the mixture was stirred twice, filtered, and the filter cake was collected to obtain a white solid YK-CAP-107-PM7 (1.38 g, 4.24 mmol, 85.7%). C13H19N5O5, MS (ES): m / z (M+H+) 326.2. YK-CAP-107-PM7: 1HNMR(400MHz,MeOD)δ8.07(s,1H),5.82(d,J=2.6Hz,1H),4.53(dd,J=6.1,2.7Hz,1H),4.36(ddd,J=8.3, 3.9,2.6Hz,1H),3.95(dd,J=12.1,2.4Hz,1H),3.78–3.67(m,2H),3.34(s,3H),2.11(q,J=7.4Hz,1H),1.23(d,J=6.1Hz,3H).

[0260] Step 8: Synthesis of YK-CAP-107-PM8

[0261] Using YK-CAP-107-PM7 (1.20 g, 3.69 mmol) as a starting material, YK-CAP-107-PM8 (triethylamine salt, 644 mg, 1.27 mmol, 34.4%) was synthesized via the same route as YK-CAP-101-PM3. C13H20N5O8P, MS (ES): m / z (MH-) 404.1.

[0262] Step 9: Synthesis of YK-CAP-107-PM9

[0263] Using YK-CAP-107-PM8 (644 mg, 1.27 mmol) as a starting material, YK-CAP-101-PM4 was synthesized to obtain YK-CAP-107-PM9 (sodium salt, 549 mg, 1.15 mmol, 90.6%). C16H22N7O7P, MS (ES): m / z (MH-) 454.2.

[0264] Step 10: Synthesis of YK-CAP-107-PM10

[0265] Using YK-CAP-107-PM9 (549 mg, 1.15 mmol) as a starting material, YK-CAP-101-PM5 was synthesized to obtain YK-CAP-107-PM10 (triethylamine salt, 531 mg, 0.91 mmol, 78.7%). C13H21N5O11P2, MS (ES): m / z (MH-) 484.0.

[0266] Step 11: Synthesis of YK-CAP-107-PM11

[0267] Using YK-CAP-107-PM10 (531 mg, 0.91 mmol) as a starting material, YK-CAP-101-PM6 was synthesized to obtain YK-CAP-107-PM11 (triethylamine salt, 163 mg, 0.27 mmol, 29.8%). C14H23N5O11P2, MS (ES): m / z (MH-) 498.1.

[0268] Step 12: Synthesis of YK-CAP-107

[0269] YK-CAP-107 (35 mg, 28.25 µmol, 10.5%) was obtained from YK-CAP-107-PM11 (163 mg, 0.27 mmol) via the synthetic route of YK-CAP-101. C35H49N15O24P4, MS (ES): m / z (MH-) 1186.1. 1HNMR(400MHz,D2O)δ8.43(d,J=1.2Hz,1H),8.34(d,J=2.1Hz,1H),7.73(d,J=4.1Hz,2H),6.21(d,J =4.5Hz,1H),5.74(d,J=2.3Hz,1H),5.71(d,J=4.4Hz,1H),4.76–4.61(m,3H),4.51–4.45(m,4H),4. 42(d,J=3.5Hz,2H),4.31(s,2H),4.10(s,2H),3.95–3.92(m,1H),3.90–3.84(m,2H),3.78–3.67(m, 2H),3.13(t,J=6.7Hz,1H),3.34(s,3H),3.10(s,2H),2.07(q,J=7.4Hz,1H),1.22(d,J=6.2Hz,3H). 31PNMR(D2O,161MHz)δ-0.88(s,1P),-11.23(d,J=20.2Hz,1P),-12.22(d,J=16.6Hz,1P),-22.32(t,J=16.1Hz,1P).

[0270] 10. Synthesis of YK-CAP-108

[0271] Step 1: Synthesis of YK-CAP-108-PM1

[0272] YK-CAP-107-PM3 (17.5 g, 38.32 mmol) was dissolved in 300 mL of THF, and triphenylphosphine (12.1 g, 46.00 mmol) and phthalimide (6.76 g, 46.00 mmol) were added. Under nitrogen protection, the system was cooled to 0 °C, and a THF solution of DEAD (9.3 g, 53.40 mmol) in 30 mL was added dropwise. After the addition was complete, the reaction was continued for 3 h, and the reaction was monitored by TLC to ensure complete reaction (DCM). After the reaction was complete, 30 mL of purified water was added dropwise to quench the reaction at 0 °C. The mixture was extracted with EA (200 mL × 3), and the organic phase was dried successively by saturated brine (400 mL) and anhydrous sodium sulfate. The residue was purified by normal-phase silica column chromatography (PE:EA = 0~40%) to obtain a yellow oily substance YK-CAP-108-PM1 (18.8 g, 32.09 mmol, 83.8%).

[0273] Step 2: Synthesis of YK-CAP-108-PM2

[0274] YK-CAP-108-PM1 (18.8 g, 32.09 mmol) was dissolved in 200 mL of ethanol, and 85% hydrazine hydrate (37.8 g, 0.64 mol) was added. The system was heated to 80 °C and reacted for 1 h. The reaction was monitored by TLC until it was complete (PE / EA = 3 / 1). The reaction was stopped, the system was cooled to room temperature, 200 mL of purified water and EA (200 mL) were added, and the mixture was stirred for 10 min. The mixture was separated into two phases. The aqueous phase was extracted with EA (200 mL × 2), and the organic phase was washed successively with saturated brine (300 mL), dried over anhydrous sodium sulfate, and evaporated to dryness to obtain a pale yellow oily substance YK-CAP-108-PM2 (14.2 g, 31.16 mmol, 97.1%).

[0275] Step 3: Synthesis of YK-CAP-108-PM3

[0276] YK-CAP-108-PM2 (14.2 g, 31.16 mmol) was dissolved in 150 mL of DCM, and DIEA (12.1 g, 93.49 mmol) was added. Under nitrogen protection, the system was cooled to 0 °C, and 20 mL of DCM solution containing acetyl chloride (2.9 g, 37.40 mmol) was added dropwise. After the addition was complete, the reaction was maintained at 0 °C for 1 h, and the reaction was monitored by TLC to ensure complete reaction (PE / EA = 3 / 1). The reaction was stopped, the system was brought to room temperature, and 200 mL of saturated sodium bicarbonate solution was added to quench the reaction. The mixture was separated; the aqueous phase was extracted with DCM (200 mL × 2), and the organic phase was washed successively with saturated brine (300 mL), dried over anhydrous sodium sulfate, and evaporated to dryness. The residue was purified by normal-phase silica gel column chromatography (DCM:MeOH = 0~40%) to obtain a yellow oily substance YK-CAP-108-PM3 (12.5 g, 25.12 mmol, 80.6%).

[0277] Step 4: Synthesis of YK-CAP-108-PM4

[0278] YK-CAP-108-PM3 (12.5 g, 25.12 mmol) was dissolved in 60 mL of THF. The system was cooled to 0 °C under nitrogen protection, and 1 MTBAF (37.7 mL) was added dropwise. After the addition was complete, the mixture was brought back to room temperature and reacted for 2 h. The reaction was monitored by TLC until it was complete (PE / EA = 3 / 1). The reaction was stopped, and the reaction solution was directly evaporated to dryness. The residue was purified by normal-phase silica gel column chromatography (DCM: MeOH = 0~40%) to obtain a yellow oily substance YK-CAP-108-PM4 (5.8 g, 22.37 mmol, 89.0%).

[0279] Step 5: Synthesis of YK-CAP-108-PM5

[0280] YK-CAP-108-PM4 (4.5 g, 17.35 mmol) was dissolved in 15 mL of acetic acid, and acetic anhydride (35.4 g, 34.71 mmol) and p-toluenesulfonic acid (1.5 g, 8.67 mmol) were added. The system was heated to 50 °C, and the reaction was monitored by LC-MS. After the reaction was completed, the system was cooled to room temperature, and 100 mL of purified water and 100 mL of EA were added. The mixture was stirred for 10 min and separated. The aqueous phase was extracted with EA (100 mL × 2), and the organic phase was washed successively with saturated brine (500 mL), dried over anhydrous sodium sulfate, and evaporated to dryness. The residue was purified by normal-phase silica gel column chromatography (DCM: MeOH = 0~40%) to obtain a yellow oily substance YK-CAP-108-PM5 (5.5 g, 15.94 mmol, 91.8%).

[0281] Step 6: Synthesis of YK-CAP-108-PM6

[0282] The intermediate INT-I (6.8 g, 17.53 mmol) was dissolved in 1,2-dichloroethane (50 mL), and N,O-bis(trimethylsilylacetamide) (7.1 g, 35.06 mmol) was added. The reaction system was heated to 80 °C, stirred for 2 h, and then evaporated to dryness under reduced pressure. The residue was dissolved in toluene (30 mL), and a solution of YK-CAP-108-PM5 (5.5 g, 15.94 mmol) in toluene (20 mL) was added. Trimethyl trifluoromethanesulfonate (3.9 g, 17.53 mmol) was slowly added dropwise. The reaction system was heated to 70 °C and stirred for 2 h. The reaction was monitored for completeness by thin-layer chromatography. The reaction system was quenched with saturated sodium bicarbonate aqueous solution and extracted with ethyl acetate (100 mL × 3). The organic phases were combined, washed with saturated NaCl aqueous solution (300 mL), dried over anhydrous sodium sulfate, filtered, and evaporated to dryness under reduced pressure. The residue was purified by silica gel chromatography (DCM: MeOH = 0~100%) to obtain YK-CAP-108-PM6 (3.8 g, 5.64 mmol, 35.4%). C33H35N7O9, MS (ES): m / z (M+H+) 674.3.

[0283] Step 7: Synthesis of YK-CAP-108-PM7

[0284] YK-CAP-108-PM6 (3.8 g, 5.64 mmol) was dissolved in a mixed solvent of 4 M NH3 / MeOH (40 mL) and water (4 mL). The mixture was stirred overnight at room temperature. LC-MS monitoring showed that the reaction was complete. The solution was evaporated to dryness and then slurried at room temperature with EA (100 mL × 2) to obtain a white solid YK-CAP-108-PM7 (1.92 g, 5.45 mmol, 96.6%). C14H20N6O5, MS (ES): m / z (M+H+) 353.1. YK-CAP-108-PM7: 1HNMR(400MHz,MeOD)δ8.02(s,1H),5.71(d,J=2.5Hz,1H),4.43(d,J=2.4Hz,1H),4 .31–4.20(m,1H),3.93–3.82(m,1H),3.78–3.67(m,2H),2.34–2.25(m,1H),2.06(s,3H),1.63(s,3H).

[0285] Step 8: Synthesis of YK-CAP-108-PM8

[0286] Using YK-CAP-108-PM7 (1.50 g, 4.26 mmol) as a starting material, YK-CAP-101-PM3 was synthesized via the same route to obtain YK-CAP-108-PM8 (triethylamine salt, 1.11 g, 2.08 mmol, 48.8%). C14H21N6O8P, MS (ES): m / z (MH-) 431.1.

[0287] Step 9: Synthesis of YK-CAP-108-PM9

[0288] Using YK-CAP-108-PM8 (1.11 g, 2.08 mmol) as a starting material, YK-CAP-101-PM4 was synthesized via the same route to obtain YK-CAP-108-PM9 (sodium salt, 778 mg, 1.54 mmol, 74.2%). C17H23N8O7P, MS (ES): m / z (MH-) 481.1.

[0289] Step 10: Synthesis of YK-CAP-108-PM10

[0290] Using YK-CAP-108-PM9 (778 mg, 1.54 mmol) as a starting material, YK-CAP-101-PM5 was synthesized to obtain YK-CAP-108-PM10 (triethylamine salt, 512 mg, 0.83 mmol, 54.2%). C14H22N6O11P2, MS (ES): m / z (MH-) 511.0.

[0291] Step 11: Synthesis of YK-CAP-108-PM11

[0292] Using YK-CAP-108-PM10 (512 mg, 0.83 mmol) as a starting material, YK-CAP-101-PM6 was synthesized to obtain YK-CAP-108-PM11 (triethylamine salt, 117 mg, 0.19 mmol, 22.5%). C15H24N6O11P2, MS (ES): m / z (MH-) 525.1.

[0293] Step 12: Synthesis of YK-CAP-108

[0294] YK-CAP-108 (18 mg, 14.22 µmol, 7.5%) was obtained from YK-CAP-101 via the synthetic route of YK-CAP-101. C36H50N16O24P4, MS (ES): m / z (MH-) 1213.1. 1HNMR(400MHz,D2O) δ8.43–8.32(m,1H), 8.12(d,J=2.5Hz,1H), 7.73-7.54(m,2H), 6.22(d,J=4.5Hz,1H), 5.35(d,J=2.3Hz,1H), 5.22–5.11(m,1H), 4.72-4.51(m,3H), 4.41-4.33(m,6H), 4.21(s,2H), 4.13(s,2H), 3.90-3.84(m,3H), 3.13-3.11(m,3H), 3.10(s,2H), 2.32–2.26(m,1H), 2.17(s,3H), 1.42(d,J=6.2Hz,3H). 31PNMR(D2O,163MHz)δ-0.87(s,1P),-11.21(d,J=20.1Hz,1P),-11.62(d,J=16.6Hz,1P),-21.67(t,J=17.2Hz,1P).

[0295] 11. Synthesis of YK-CAP-109

[0296] Step 1: Synthesis of YK-CAP-109-PM1

[0297] YK-CAP-107-PM3 (10.0 g, 21.90 mmol) was dissolved in dichloromethane. The reaction system was cooled to 0 °C, and DAST (7.1 g, 43.80 mmol) was slowly added dropwise. The reaction was stirred at 0 °C for 4 hours. The reaction was quenched by slowly adding saturated sodium bicarbonate aqueous solution. DCM was added, stirred, and separated. The organic phase was washed twice with saturated sodium bicarbonate aqueous solution and separated. The organic phase was washed 2-3 times with saturated brine, dried over anhydrous sodium sulfate, filtered, and evaporated to dryness. The residue was purified by silica gel column chromatography (0-30% ethyl acetate / n-hexane) to obtain YK-CAP-109-PM1 (6.1 g, 13.30 mmol, 60.7%).

[0298] Step 2: Synthesis of YK-CAP-109-PM2

[0299] YK-CAP-109-PM1 (6.1 g, 13.30 mmol) was dissolved in acetic acid, and acetic anhydride (27.2 g, 266.0 mmol) was added. Concentrated sulfuric acid (280 μl) was then slowly added dropwise. After the addition was complete, the mixture was stirred at room temperature for 16 hours. 200 mL of water was added to the reaction mixture, and the solution was extracted with ethyl acetate. The organic phase was washed three times with a saturated sodium bicarbonate aqueous solution until the pH was alkaline. The organic phase was dried and evaporated to dryness to obtain crude YK-CAP-109-PM2 (5.80 g), a yellow oily liquid, which was directly used for the next reaction.

[0300] Step 3: Synthesis of YK-CAP-109-PM3

[0301] Intermediate INT-I (6.2 g, 15.96 mmol) was dissolved in 1,2-dichloroethane, and N,O-bis(trimethylsilylacetamide) (8.1 g, 39.9 mmol) was added. The mixture was heated to 80 °C and stirred for 2 hours. The solvent was then removed under reduced pressure, and the solution was dissolved again in toluene. At room temperature, a toluene solution of YK-CAP-109-PM2 (5.8 g, calculated as 13.30 mmol) and TMSOTf (4.4 g, 19.9 mmol) were added sequentially. The mixture was then heated to 70 °C and stirred for another 2 hours. The reaction mixture was diluted with ethyl acetate, washed once with saturated sodium bicarbonate solution, and the insoluble matter was filtered off. The filtrate was separated, dried over anhydrous sodium sulfate on the organic phase, and the solvent was removed under reduced pressure. The residue was purified by silica gel column chromatography (0-30% ethyl acetate / DCM) to obtain YK-CAP-109-PM3 (3.1 g, 4.88 mmol). C31H31FN6O8,MS(ES):m / z(M+H+)635.2.

[0302] Step 4: Synthesis of YK-CAP-109-PM4

[0303] YK-CAP-109-PM3 (3.1 g, 4.88 mmol) was dissolved in 7 M ammonia-methanol solution and water (5:1), and the mixture was stirred at room temperature for 16 hours. The solvent was evaporated under reduced pressure, and the crude product was recrystallized from ethyl acetate to give YK-CAP-109-PM4 (1.1 g, 3.51 mmol, 71.9%). C12H16FN5O4, MS (ES): m / z (M+H+) 314.1. YK-CAP-109-PM4:1HNMR(400MHz,DMSO-d 6)δ11.45(s,1H),8.00(s,1H),δ6.40(s,2H),5.73–5.56(m,2H),4.83–4.81(m,1H),4.31–4.29(m,1H),4.17 -4.13(m,1H),4.60(s,1H),3.76–3.72(m,1H),3.63-3.50(m,1H),2.34–2.26(m,1H),1.42(d,J=6.2Hz,3H).

[0304] Step 5: Synthesis of YK-CAP-109-PM5

[0305] Phosphorus oxychloride (1.6 g, 10.5 mmol) was dissolved in 20 mL of trimethyl phosphate. Under nitrogen protection, the mixture was cooled to 0 °C, and YK-CAP-109-PM4 (1.1 g, 3.51 mmol) was slowly added to the reaction system and stirred at 0 °C for approximately 4 h. After the reaction was complete, 20 mL of ice water was added, and the mixture was washed twice with ethyl acetate. The pH of the aqueous phase was adjusted to 3.5 with ammonia, and the mixture was placed in a refrigerator overnight. The pH was further adjusted to 6.5 the next day, and the solution was diluted to 400 mL for loading. The solution was purified by gel column chromatography (water: 1.5 MTEAB = 1:4 elution), the target product peak was collected, concentrated, and lyophilized to obtain a white solid, YK-CAP-109-PM5 (triethylamine salt, 980 mg, 1.98 mmol, 56.5%), C12H17FN5O7P, MS (ES): m / z (MH-) 392.1.

[0306] Step 6: Synthesis of YK-CAP-109-PM6

[0307] Using YK-CAP-109-PM5 (980 mg, 1.98 mmol) as a raw material, YK-CAP-101-PM4 was synthesized to obtain YK-CAP-109-PM6 (sodium salt, 900 mg, 1.93 mmol, 97.6%), C15H19FN7O6P, MS (ES): m / z (MH-) 442.1.

[0308] Step 7: Synthesis of YK-CAP-109-PM7

[0309] Using YK-CAP-109-PM6 (900 mg, 1.93 mmol) as a starting material, YK-CAP-101-PM5 was synthesized to obtain YK-CAP-109-PM7 (triethylamine salt, 850 mg, 1.48 mmol, 76.7%), C12H18FN5O10P2, MS (ES): m / z (MH-) 472.2.

[0310] Step 8: Synthesis of YK-CAP-109-PM8

[0311] Using YK-CAP-109-PM7 (850 mg, 1.48 mmol) as the starting material, YK-CAP-101-PM6 was synthesized to obtain YK-CAP-109-PM8 (triethylamine salt, 450 mg, 0.76 mmol, 51.7%), C13H20FN5O10P2, MS (ES): m / z (MH-) 486.1.

[0312] Step 9: Synthesis of YK-CAP-109

[0313] Using YK-CAP-109-PM8 (100 mg, 0.17 mmol) as the starting material, YK-CAP-109 (35 mg, 28.53 µmol, 16.8%) was obtained by following the synthetic route of YK-CAP-101. The chromatogram was C34H46FN15O23P4. The MS (ES) value was m / z (MH-) 1174.2. 1HNMR(400MHz,D2O)δ8.53-8.41(m,1H),8.14(d,J=2.6Hz,1H),7.63-7.55(m,2H ),6.17(d,J=4.4Hz,1H),5.65(d,J=2.6Hz,1H),5.32-5.21(m,1H),4.62-4.42(m, 3H),4.36–4.33(m,4H),4.22-4.15(m,3H),4.11(s,2H),4.03(s,1H),3.91-3.82( m,3H),3.21-3.15(m,3H),3.11(s,2H),2.31-2.22(m,1H),1.52(d,J=6.1Hz,3H). 31PNMR(D2O,163MHz)δ-0.89(s,1P),-11.21(d,J=20.8Hz,1P),-11.74(d,J=16.6Hz,1P),-22.12(t,J=18.0Hz,1P).

[0314] 12. Synthesis of YK-CAP-110

[0315] Step 1: Synthesis of YK-CAP-110-PM1

[0316] YK-CAP-107-PM2 (14.4 g, 32.68 mmol) was dissolved in dichloromethane. The reaction system was cooled to 0°C, and DAST (16.7 g, 103.5 mmol) was slowly added dropwise. The reaction was stirred for 4 hours at 0°C. The reaction was quenched by slowly adding saturated sodium bicarbonate solution. DCM was added, and the mixture was stirred and separated. The organic phase was washed twice with saturated sodium bicarbonate solution and separated. The organic phase was washed 2-3 times with saturated brine, dried over anhydrous sodium sulfate, filtered, and evaporated to dryness. The residue was purified by silica gel column chromatography (0-30% ethyl acetate / n-hexane) to obtain YK-CAP-110-PM1 (10.6g, 22.91mmol, 70.1%).

[0317] Step 2: Synthesis of YK-CAP-110-PM2

[0318] YK-CAP-110-PM1 (5.6 g, 12.11 mmol) was dissolved in acetic acid, and acetic anhydride (24.7 g, 242.1 mmol) was added. Concentrated sulfuric acid (280 μl) was then slowly added dropwise. After the addition was complete, the mixture was stirred at room temperature for 16 hours. 200 mL of water was added to the reaction mixture, and the solution was extracted with ethyl acetate. The organic phase was washed three times with a saturated sodium bicarbonate aqueous solution until the pH was alkaline. The organic phase was dried and evaporated to dryness to obtain crude YK-CAP-110-PM2 (6.0 g), a yellow oily liquid, which was directly used for the next reaction.

[0319] Step 3: Synthesis of YK-CAP-110-PM3

[0320] Intermediate INT-I (5.2 g, 13.4 mmol) was dissolved in 1,2-dichloroethane, and N,O-bis(trimethylsilylacetamide) (7.4 g, 36.3 mmol) was added. The mixture was heated to 80 °C and stirred for 2 hours. The solvent was then removed under reduced pressure, and the mixture was dissolved again in toluene. At room temperature, a toluene solution of YK-CAP-110-PM2 (6.0 g, calculated as 12.11 mmol) and TMSOTf (3.0 g, 13.4 mmol) were added sequentially. The mixture was then heated to 70 °C and stirred for another 2 hours. The reaction mixture was diluted with ethyl acetate, washed once with saturated sodium bicarbonate solution, and the insoluble matter was filtered off. The filtrate was separated, dried over anhydrous sodium sulfate on the organic phase, and the solvent was removed under reduced pressure. The residue was purified by silica gel column chromatography (0-30% ethyl acetate / DCM) to obtain YK-CAP-110-PM3 (3.5 g, 5.48 mmol). C30H28F2N6O8,MS(ES):m / z(M+H+)639.1.

[0321] Step 4: Synthesis of YK-CAP-110-PM4

[0322] YK-CAP-110-PM3 (3.5 g, 5.48 mmol) was dissolved in 4 M ammonia-methanol solution and water (5:1), and the mixture was stirred at room temperature for 16 hours. The solvent was evaporated under reduced pressure, and the crude product was recrystallized from ethyl acetate to give YK-CAP-110-PM4 (1.3 g, 4.10 mmol, 74.7%). C11H13F2N5O4, MS (ES): m / z (M+H+) 318.1. YK-CAP-110-PM4:1HNMR(400MHz,DMSO-d 6)δ10.56(s,1H),8.00(s,1H),δ6.40(s,2H),5.71–5.56(m,2H),4.86–4.83(m,1H),4.36–4.32( m,1H),4.19-4.15(m,1H),4.62(s,1H),3.75–3.73(m,1H),3.62-3.49(m,1H),3.27–3.22(m,1H).

[0323] Step 5: Synthesis of YK-CAP-110-PM5

[0324] Phosphorus oxychloride (1.9 g, 12.3 mmol) was dissolved in 20 mL of trimethyl phosphate. Under nitrogen protection, the temperature was lowered to 0 °C, and YK-CAP-110-PM4 (1.3 g, 4.10 mmol) was slowly added to the reaction system and stirred at 0 °C for approximately 4 h. After the reaction was complete, 20 mL of ice water was added, and the mixture was washed twice with ethyl acetate. The pH of the aqueous phase was adjusted to 3.5 with ammonia, and the mixture was placed in a refrigerator overnight. The pH was further adjusted to 6.5 the next day, and the solution was diluted to 400 mL for loading. The solution was purified by gel column chromatography (water: 1.5 MTEAB = 1:5 elution), the target product peak was collected, concentrated, and lyophilized to obtain a white solid, YK-CAP-110-PM5 (triethylamine salt, 1.1 g, 2.21 mmol, 53.8%), C11H14F2N5O7P, MS (ES): m / z (MH-) 396.1.

[0325] Step 6: Synthesis of YK-CAP-110-PM6

[0326] Using YK-CAP-110-PM5 (1.1 g, 2.21 mmol) as a starting material, YK-CAP-101-PM6 (sodium salt, 1.0 g, 2.13 mmol, 96.4%) was obtained by following the synthetic route of YK-CAP-101-PM4, C14H16F2N7O6P, MS (ES): m / z (MH-) 446.1.

[0327] Step 7: Synthesis of YK-CAP-110-PM7

[0328] Using YK-CAP-110-PM6 (1.0 g, 2.13 mmol) as the starting material, YK-CAP-101-PM7 (triethylamine salt, 900 mg, 1.56 mmol, 73.1%) was obtained by following the synthetic route of YK-CAP-101-PM5, C11H15F2N5O10P2, MS (ES): m / z (MH-) 476.1.

[0329] Step 8: Synthesis of YK-CAP-110-PM8

[0330] Using YK-CAP-110-PM7 (900 mg, 1.56 mmol) as the starting material, YK-CAP-101-PM8 (triethylamine salt, 500 mg, 0.84 mmol, 54.1%) was obtained by following the synthetic route of YK-CAP-101-PM6, C12H17F2N5O10P2, MS (ES): m / z (MH-) 490.2.

[0331] Step 9: Synthesis of YK-CAP-110

[0332] Using YK-CAP-110-PM8 (100 mg, 0.17 mmol) as the starting material, YK-CAP-110 (25 mg, 22.03 µmol, 13.0%) was obtained by following the synthetic route of YK-CAP-101, C33H43F2N15O23P4, MS (ES): m / z (MH-) 1178.1. 1HNMR(400MHz,D2O)δ8.43(d,J=1.5Hz,1H),8.36(d,J=2.6Hz,1H),8.12(d,J=4.6Hz,2H),6.10(d,J=5.4Hz,1H),5.88 (d,J=2.2Hz,1H),5.61(d,J=4.4Hz,1H),4.90–4.88(m,3H),4.85–4.82(m,1H),4.62(t,J=4.7Hz,1H),4.51–4.38(m,4H ),4.23(d,J=3.0Hz,1H),4.12(s,2H),4.04(s,2H),3.93(s,3H),3.64(t,J=7.0Hz,1H),3.41(s,3H),3.28–3.22(m,1H ).31PNMR(D2O,162MHz)δ-0.93(s,1P),-11.17(d,J=19.2Hz,1P),-11.35(d,J=17.1Hz,1P),-24.38(t,J=17.6Hz,1P).

[0333] 13. Synthesis of YK-CAP-111

[0334] Step 1: Synthesis of YK-CAP-111-PM1

[0335] YK-CAP-107-PM1 (35.0 g, 79.07 mmol) was dissolved in acetonitrile and water (1:1, 280 mL). Iodobenzene acetate (53.5 g, 166.03 mmol), sodium bicarbonate (9.96 g, 118.56 mmol), and TEMPO (1.85 g, 11.85 mmol) were added sequentially under ice bath conditions. The reaction was carried out at room temperature for 2 hours. The reaction was monitored by TLC until the reactants were completely reacted. The reaction was stopped, and a saturated sodium thiosulfate aqueous solution was added to the reaction solution to quench the reaction. The solution was extracted with EA, separated, and the organic phase was dried with anhydrous sodium sulfate. After filtration, the solvent was removed from the organic phase by rotary vortexing to obtain 68.0 g of crude brown oily product YK-CAP-111-PM1, which was not purified and directly proceeded to the next reaction.

[0336] Step 2: Synthesis of YK-CAP-111-PM2

[0337] YK-CAP-111-PM1 (34.0 g, calculated as 39.54 mmol) was dissolved in acetonitrile and cooled to 0 °C. DIEA (14.0 g, 108.33 mmol) and HATU (17.86 g, 46.97 mmol) were added. The mixture was stirred for 20 min, and then diethylamine (6.6 g, 90.24 mmol) was added. The mixture was heated to room temperature and stirred for 4 hours. The reaction solution was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (0-20% ethyl acetate / n-hexane) to obtain YK-CAP-111-PM2 (17.5 g, 34.20 mmol, 86.5%).

[0338] Step 3: Synthesis of YK-CAP-111-PM3

[0339] YK-CAP-111-PM2 (17.5 g, 34.20 mmol) was dissolved in tetrahydrofuran, and TBAF (13.4 g, 51.25 mmol) was added. The mixture was stirred at room temperature for 2 hours. The reaction solution was evaporated to dryness, and the residue was purified by silica gel column chromatography (0-90% ethyl acetate / n-hexane) to obtain YK-CAP-111-PM3 (8.9 g, 32.56 mmol, 95.2%).

[0340] Step 4: Synthesis of YK-CAP-111-PM4

[0341] YK-CAP-111-PM3 (4.0 g, 14.63 mmol) was dissolved in acetic acid, and sulfuric acid (300 μL) was added. The mixture was stirred at room temperature for 30 min. Then, acetic anhydride (30.0 g, 293.86 mmol) was added. The mixture was stirred at room temperature for 18 h. 200 mL of water was added to the reaction mixture, and the solution was extracted with ethyl acetate. The organic phase was washed three times with saturated sodium bicarbonate aqueous solution until the pH was alkaline. The organic phase was dried and evaporated to dryness to obtain crude YK-CAP-111-PM4 (3.19 g), a yellow oily liquid, which was directly used for the next reaction.

[0342] Step 5: Synthesis of YK-CAP-111-PM5

[0343] Intermediate INT-I (3.79 g, 9.76 mmol) was dissolved in 1,2-dichloroethane, and N,O-bis(trimethylsilylacetamide) (5.42 g, 26.64 mmol) was added. The mixture was heated to 80 °C and stirred for 2 hours. The reaction solvent was then removed under reduced pressure. At room temperature, a toluene solution of YK-CAP-111-PM4 (3.19 g) and TMSOTf (2.96 g, 13.32 mmol) were added sequentially. The mixture was then heated to 70 °C and stirred for another 2 hours. The reaction mixture was diluted with ethyl acetate, washed once with saturated sodium bicarbonate solution, and the insoluble matter was filtered off. The filtrate was separated, and the organic phase was dried over anhydrous sodium sulfate. The solvent was removed under reduced pressure, and the residue was purified by silica gel column chromatography (0-60% ethyl acetate / DCM) to obtain YK-CAP-111-PM5 (2.20 g, 3.20 mmol).

[0344] Step 6: Synthesis of YK-CAP-111-PM6

[0345] YK-CAP-111-PM5 (2.20 g, 3.20 mmol) was dissolved in 4 M ammonia-methanol solution and water (5:1), and the reaction was stirred at room temperature for 16 hours. The solvent was evaporated under reduced pressure, and the crude product was recrystallized from ethyl acetate to give YK-CAP-111-PM6 (890 mg, 2.43 mmol, 75.9%). C15H22N6O5, MS (ES): m / z (M+H+) 367.1. YK-CAP-111-PM6:1HNMR(400MHz,DMSO-d 6) δ11.40(s,1H),8.31(s,1H),6.38(s,2H),6.16(d,J=4.3Hz,1H),5.38-5.41(m,1H),4.84-4.91(m,1H),4.28-4.31(m, 1H),3.98-4.02(m,1H),3.45-3.49(m,2H),3.33-3.21(q,J=4.1Hz,4H),2.65-2.62(m,1H),1.17-1.15(t,J=4.1Hz,6H).

[0346] Step 7: Synthesis of YK-CAP-111-PM7

[0347] Phosphorus oxychloride (1.3 g, 8.48 mmol) was dissolved in 15 mL of trimethyl phosphate. Under nitrogen protection, the mixture was cooled to 0 °C, and YK-CAP-111-PM6 (880 mg, 2.42 mmol) was slowly added to the reaction system. The mixture was stirred at 0 °C for approximately 4 h. After the reaction was complete, the mixture was added to 20 mL of ice water, washed twice with ethyl acetate, and the pH of the aqueous phase was adjusted to 3.5 with ammonia and refrigerated overnight. The pH was further adjusted to 6.5, and the mixture was diluted to 400 mL for loading. The mixture was purified by gel column chromatography (water: 1.5 MTEAB = 5:1 elution), the target product peak was collected, concentrated, and lyophilized to obtain a white solid, YK-CAP-111-PM7 (triethylamine salt, 900 mg, 1.64 mmol, 68.5%), C15H23N6O8P, MS (ES): m / z (MH-) 445.1.

[0348] Step 8: Synthesis of YK-CAP-111-PM8

[0349] Using YK-CAP-111-PM7 (900 mg, 1.64 mmol) as a starting material, YK-CAP-101-PM8 (sodium salt, 730 mg, 1.41 mmol, 85.7%) was obtained by following the synthetic route of YK-CAP-101-PM4, C18H25N8O7P, MS (ES): m / z (MH-) 495.1.

[0350] Step 9: Synthesis of YK-CAP-111-PM9

[0351] Using YK-CAP-111-PM8 (720 mg, 1.39 mmol) as the starting material, YK-CAP-101-PM9 (triethylamine salt, 750 mg, 1.20 mmol, 86.1%) was obtained by following the synthetic route of YK-CAP-101-PM5, C15H24N6O11P2, MS (ES): m / z (MH-) 525.1.

[0352] Step 10: Synthesis of YK-CAP-111-PM10

[0353] YK-CAP-111-PM9 (400 mg, 0.64 mmol) was dissolved in 20 mL of H2O, and the pH was adjusted to 4.0 with glacial acetic acid. Dimethyl sulfate (800 μL, 8.45 mmol) was added over 30 min. The pH of the reaction system was maintained between 3.8 and 4.1 with 0.1 M NaOH aqueous solution, and the mixture was stirred at room temperature for 5 hours. After the reaction was completed, the reaction solution was extracted twice with dichloromethane. The aqueous phase was adjusted to pH 6.5, and the volume was brought to 400 mL. The solution was purified by gel column chromatography (elution with water and 1 MTEAB = 3:2), the target product peak was collected, concentrated, and lyophilized to obtain YK-CAP-111-PM10 (triethylamine salt, 350 mg, 0.55 mmol, 85.6%) as a white solid, C16H26N6O11P2, MS (ES): m / z (MH-) 539.1.

[0354] Step 11: Synthesis of YK-CAP-111

[0355] Using YK-CAP-111-PM10 (100 mg, 0.16 mmol) as the starting material, YK-CAP-111 (ammonium salt, 50 mg, 39.07 μmmol, 25.1%) was obtained by following the synthetic route of YK-CAP-101, C37H52N16O24P4, MS (ES): m / z (MH-) 1127.2. 1HNMR(400MHz,D2O)δ8.43(d,J=1.3Hz,1H),8.35(s,1H),8.06(d,J=6.0Hz,2H),6.01(d,J=5.4Hz,1H),5.86(d,J=2.3Hz,1H) ,5.82(d,J=4.5Hz,1H),4.97–4.84(m,3H),4.66(t,J=4.8Hz,1H),4.51–4.35(m,4H),4.32(d,J=4.5Hz,1H),4.21(s,2H),4.09 (s,2H),3.97(s,3H),3.72(t,J=7.0Hz,1H),3.35–3.21(d,J=4.1Hz,4H),3.04(s,3H),1.94-1.87(m,1H),1.21–1.14(t,J=4. 1Hz,6H).31PNMR(D2O,162MHz)δ-0.92(s,1P),-11.15(d,J=19.2Hz,1P),-11.62(d,J=17.1Hz,1P),-23.46(t,J=17.5Hz,1P).

[0356] 14. Synthesis of YK-CAP-112

[0357] Step 1: Synthesis of YK-CAP-112-PM1

[0358] Using YK-CAP-111-PM1 (10.0 g, 21.90 mmol) and dipropylamine (5.5 g, 54.35 mmol) as raw materials, YK-CAP-112-PM1 (11.0 g, 20.38 mmol, 93.1%) was obtained by following the synthetic route of YK-CAP-111-PM2.

[0359] Step 2: Synthesis of YK-CAP-112-PM2

[0360] Using YK–CAP-112-PM1 (11.0 g, 20.38 mmol) as the starting material, YK-CAP-112-PM2 (6.0 g, 19.91 mmol, 97.7%) was obtained by following the synthetic route of YK-CAP-111-PM3.

[0361] Step 3: Synthesis of YK-CAP-112-PM3

[0362] Using YK-CAP-112-PM2 (6.0 g, 19.91 mmol) as the starting material, crude YK-CAP-112-PM3 (5.1 g) was obtained by following the synthetic route of YK-CAP-111-PM4. It was a yellow oily liquid and was directly used for the next reaction.

[0363] Step 4: Synthesis of YK-CAP-112-PM4

[0364] YK-CAP-112-PM4 (3.9 g, 5.45 mmol) was obtained from YK-CAP-111-PM5 via the synthetic route of YK-CAP-112-PM3 (5.1 g). C36H41N7O9, MS (ES): m / z (M+H+) 716.3.

[0365] Step 5: Synthesis of YK-CAP-112-PM5

[0366] YK-CAP-112-PM5 (1.7g, 4.31mmol, 79.1%) was obtained from YK-CAP-111-PM6 via the synthetic route of YK-CAP-112-PM4 (3.9g, 5.45mmol). C17H26N6O5, MS (ES): m / z (M+H+) 395.2. YK-CAP-112-PM5:1HNMR(400MHz,DMSO-d 6) δ11.37(s,1H),8.41(s,1H),6.28(s,2H),6.19(d,J=4.1,1H),5.46(s,1H),5.01(s,1H),4.25-4.28(m,1H),3.9 9-4.01(m,1H),3.41-3.43(m,2H),3.21-3.24(m,4H),2.56-2.58(m,1H),1.57-1.54(m,4H),0.87(t,J=1.8Hz,6H).

[0367] Step 6: Synthesis of YK-CAP-112-PM6

[0368] Using YK-CAP-112-PM5 (1.7 g, 4.31 mmol) as the starting material, YK-CAP-111-PM6 (triethylamine salt, 1.80 g, 3.13 mmol, 72.6%) was obtained by following the synthetic route of YK-CAP-111-PM7, C17H27N6O8P, MS (ES): m / z (MH-) 473.1.

[0369] Step 7: Synthesis of YK-CAP-112-PM7

[0370] Using YK-CAP-112-PM6 (1.8 g, 3.13 mmol) as a raw material, YK-CAP-101-PM7 (sodium salt, 1.47 g, 2.69 mmol, 86.0%), C20H29N8O7P, was obtained via the synthetic route of YK-CAP-101-PM4. MS (ES): m / z (MH-) 523.1.

[0371] Step 8: Synthesis of YK-CAP-112-PM8

[0372] Using YK-CAP-112-PM7 (1.47 g, 2.69 mmol) as the starting material, YK-CAP-101-PM5 was synthesized to obtain YK-CAP-112-PM8 (triethylamine salt, 1.30 g, 1.98 mmol, 73.7%), C17H28N6O11P2, MS (ES): m / z (MH-) 553.1.

[0373] Step 9: Synthesis of YK-CAP-112-PM9

[0374] Using YK-CAP-112-PM8 (600 mg, 0.92 mmol) as the starting material, YK-CAP-111-PM9 (triethylamine salt, 200 mg, 0.30 mmol, 32.6%) was obtained by following the synthetic route of YK-CAP-111-PM10, C18H30N6O11P2, MS (ES): m / z (MH-) 567.1.

[0375] Step 10: Synthesis of YK-CAP-112

[0376] Using YK-CAP-112-PM9 (200 mg, 0.30 mmol) as the starting material, YK-CAP-112 (ammonium salt, 50 mg, 38.23 μmol, 12.8%) was obtained by following the synthetic route of YK-CAP-101, C39H56N16O24P4, MS (ES): m / z (MH-) 1255.2. 1HNMR(400MHz,D2O)δ8.43(d,J=1.3Hz,1H),8.34(s,1H),8.11(d,J=6.1Hz,2H),6.05(d,J=5.3Hz,1H),5.87(d,J=2.3Hz,1H),5. 81(d,J=4.4Hz,1H),4.91–4.82(m,2H),4.64(t,J=4.7Hz,1H),4.48–4.37(m,5H),4.30(d,J=4.6Hz,1H),4.22(s,2H),4.11(s,2H ),3.96(s,3H),3.72(t,J=7.0Hz,1H),3.33–3.26(t,J=4.1Hz,4H),3.11(s,3H),2.22–2.14(m,1H),1.58-1.54(m,4H),0.87(t,J =1.8Hz,6H).31PNMR(D2O,162MHz)δ-0.94(s,1P),-11.22(d,J=19.1Hz,1P),-11.52(d,J=17.2Hz,1P),-23.11(t,J=17.3Hz,1P).

[0377] 15. Synthesis of YK-CAP-113

[0378] Step 1: Synthesis of YK-CAP-113-PM1

[0379] Methyl-beta-D-furanoside (100.0 g, 0.61 mol) was dissolved in 1 L of anhydrous pyridine, and TIPDSCl (230.6 g, 0.73 mol) was added dropwise under ice-water bath conditions. After the addition was complete, the mixture was brought to room temperature and stirred for 12 hours. After depressurization to remove a large amount of solvent, the residue was purified by silica gel column chromatography (0-30% ethyl acetate / n-hexane) to obtain YK-CAP-113-PM1 (212.2 g, 0.52 mol, 85.7%).

[0380] Step 2: Synthesis of YK-CAP-113-PM2

[0381] YK-CAP-113-PM1 (212.2 g, 0.52 mol) was dissolved in 2 L of acetonitrile, and Dess-Martin reagent (485.1 g, 1.14 mol) was added. The mixture was heated to 40 °C and stirred for 12 hours. After cooling to room temperature, the reaction solution was filtered, and the filtrate was evaporated to dryness under reduced pressure to obtain YK-CAP-113-PM2 (208.8 g, 0.52 mol, 98.9%).

[0382] Step 3: Synthesis of YK-CAP-113-PM3

[0383] 408.7 g (1.14 mol) of bromomethyltriphenylphosphine bromide was dissolved in 3000 mL of tetrahydrofuran. The solution was cooled to -78 °C, and a 2.5 M solution of n-butyllithium in tetrahydrofuran (456 mL, 1.14 mol) was slowly added dropwise. After the addition was complete, the reaction system was heated to 0 °C and stirred for 2 hours. The reaction system was then cooled back to -78 °C, and a 400 mL solution of YK-CAP-113-PM2 (208.8 g, 0.52 mol) in tetrahydrofuran was slowly added dropwise. After the addition was complete, the reaction system was heated to room temperature and stirred overnight. The reaction system was quenched with 2000 mL of saturated ammonium chloride solution, extracted with ethyl acetate (2000 mL × 3), and the organic phases were combined. The organic phases were washed with saturated NaCl aqueous solution, dried over anhydrous sodium sulfate, filtered, and evaporated to dryness. The residue was purified by silica gel chromatography (0-20% ethyl acetate / n-hexane) to obtain YK-CAP-113-PM3 (108.8 g, 0.27 mol, 52.4%).

[0384] Step 4: Synthesis of YK-CAP-113-PM4

[0385] 1M 9-BBN tetrahydrofuran (540 mL, 0.54 mmol) was added to a three-necked flask. Under nitrogen protection, the mixture was cooled to 0°C. Then, a tetrahydrofuran solution of YK-CAP-113-PM3 (108.8 g, 0.27 mol) was slowly added dropwise to the system. After the addition was complete, the mixture was stirred at room temperature for 2 hours. The reaction system was then cooled back to 0°C. Subsequently, 540 mL of water / tetrahydrofuran (1:1), 540 mL of 2N sodium hydroxide solution, and 460 mL of 30% hydrogen peroxide were slowly added sequentially. After the addition was complete, the mixture was brought to room temperature and stirred for another 3 hours. The reaction system was diluted with ethyl acetate, separated, and the aqueous phase was back-extracted once with ethyl acetate. The organic phases were combined, dried, filtered, and the filtrate was evaporated to dryness under reduced pressure. The residue was purified by silica gel column chromatography (0-17% ethyl acetate / n-hexane) to obtain YK-CAP-113-PM4 (98.0 g, 0.23 mol, 86.2%).

[0386] Step 5: Synthesis of YK-CAP-113-PM5

[0387] YK-CAP-113-PM4 (20.0 g, 47.54 mmol) and sodium hydride (1369 mg, 57.04 mmol) were dissolved in dry tetrahydrofuran (100 mL). After cooling to 0 °C, iodomethane (13.5 g, 95.11 mmol) was added dropwise under nitrogen protection, and the reaction was stirred for about 5 h. After the reaction was completed, water (10 mL) was added to quench the reaction, and the mixture was extracted with EA (200 mL × 3). The organic phases were combined, washed with saturated NaCl aqueous solution, dried over anhydrous sodium sulfate, filtered, and evaporated to dryness. The residue was purified by silica gel chromatography (0-20% ethyl acetate / n-hexane) to obtain YK-CAP-113-PM5 (17.8 g, 40.95 mmol, 86.1%).

[0388] Step 6: Synthesis of YK-CAP-113-PM6

[0389] Benzoyl adenosine (39.2 g, 163.85 mmol) was dissolved in hexamethyldisilazane (500 mL), and a catalytic amount of ammonium sulfate was added. The mixture was heated to 130 °C and stirred for 12 hours under nitrogen protection, and the solvent was evaporated to dryness. YK-CAP-113-PM5 (17.8 g, 40.95 mmol) was dissolved in 1,2-dichloroethane (300 mL) and added to the above residue. Then, trimethylsilyl trifluoromethanesulfonate (10.1 g, 48.97 mmol) was added, and the mixture was reacted at 80 °C for 5 hours. The reaction was monitored for completeness by thin-layer chromatography. Filter, evaporate the filtrate to dryness under reduced pressure, and purify the residue by silica gel column chromatography (0-80% ethyl acetate / n-hexane) to obtain YK-CAP-113-PM6 (14.5g, 22.59mmol, 55.2%), C31H47N5O6Si2, MS (ES): m / z (M+H+) 642.3.

[0390] Step 7: Synthesis of YK-CAP-113-PM7

[0391] YK-CAP-113-PM6 (14.5 g, 22.59 mmol) was dissolved in tetrahydrofuran (100 mL), and tetrabutylammonium fluoride (23.6 g, 90.26 mmol) was added. The mixture was stirred at room temperature for 1 hour. A saturated aqueous solution of ammonium chloride was added to the reaction mixture, and the mixture was extracted with ethyl acetate (200 mL × 3). The organic phases were combined, washed with a saturated aqueous solution of NaCl, dried over anhydrous sodium sulfate, filtered, and evaporated to dryness. The residue was purified by silica gel chromatography (0-60% ethyl acetate / n-hexane) to give YK-CAP-113-PM7 (7.5 g, 18.78 mmol, 83.1%), C19H21N5O5, MS (ES): m / z (M+H+) 400.2.

[0392] Step 8: Synthesis of YK-CAP-113-PM8

[0393] YK-CAP-113-PM7 (7.5 g, 18.78 mmol) was dissolved in pyridine (50 mL), and 4,4'-dimethoxytriphenylchloromethane (10.2 g, 30.10 mmol) was added at room temperature. The mixture was stirred at room temperature for 3 h. The reaction was monitored by TLC until complete. 10 mL of methanol was added to the reaction solution to quench the reaction, and the mixture was stirred for 10 min. The mixture was then rotary evaporated until no solvent was distilled off to obtain the crude product. The crude product was purified by flash column chromatography to obtain YK-CAP-113-PM8 (8.8 g, 12.54 mmol, 66.8%), C40H39N5O7, MS (ES): m / z (M+H+) 702.3.

[0394] Step 9: Synthesis of YK-CAP-113-PM9

[0395] YK-CAP-113-PM8 (8.8 g, 12.54 mmol) was dissolved in acetonitrile (100 mL), and N-methylimidazole (1.1 g, 13.40 mmol) and bis(diisopropylamino)(2-cyanoethoxy)phosphine (11.3 g, 37.49 mmol) were added sequentially. The mixture was stirred for 6 h under nitrogen protection. The reaction was monitored by TLC until complete. The reaction solution was diluted with ethyl acetate, and the organic phase was washed with saturated sodium bicarbonate aqueous solution and separated. The organic phase was washed once with water and separated. The aqueous phases were combined and back-extracted once with ethyl acetate. The organic phases were combined, dried over anhydrous sodium sulfate, and filtered. The organic phase was rotary evaporated under reduced pressure until no solvent was distilled off to obtain the crude product. The crude product was purified by flash column chromatography. After the solvent was removed from the product fraction, it was dissolved in ethyl acetate and added dropwise to ice-cold n-hexane. The mixture was stirred for 10 min. Filtration yielded YK-CAP-113-PM9 (8.1g, 8.98mmol, 71.6%), C49H56N7O8P, MS (ES): m / z (MH-) 900.4.

[0396] Step 10: Synthesis of YK-CAP-113-PM10

[0397] YK-CAP-113-PM9 (4.0 g, 4.43 mmol), N-isobutyl-2′,3′-acetylglucosinolate (2.0 g, 4.57 mmol), and tetrazolium (3.1 g, 44.25 mmol) were dissolved in acetonitrile (50 mL) and reacted under nitrogen atmosphere at room temperature with stirring for 3 h. 0.1 M iodine solution (53 mL) was added to the reaction solution, and the reaction was stirred for another 1 h. The solution was then diluted with 200 mL of brine, extracted with dichloromethane (200 mL × 3), and the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and evaporated to dryness. The organic phase was evaporated under reduced pressure until no solvent was removed, yielding YK-CAP-113-PM10 (5.2 g, 4.15 mmol, 93.5%), C61H64N11O17P, MS (ES): m / z (MH-) 1252.4.

[0398] Step 11: Synthesis of YK-CAP-113-PM11

[0399] YK-CAP-113-PM10 (5.2 g, 4.15 mmol) was dissolved in 20 mL of 80% acetic acid aqueous solution and reacted under nitrogen atmosphere at room temperature with stirring for 2 h. The reaction system was concentrated under reduced pressure to remove acetic acid, and the mixture was rotary evaporated until no solvent was distilled off to obtain the crude product. The crude product was purified by flash column chromatography to obtain YK-CAP-113-PM11 (3.0 g, 3.15 mmol, 76.0%), C40H46N11O15P, MS (ES): m / z (MH-) 950.3.

[0400] Step 12: Synthesis of YK-CAP-113-PM12

[0401] YK-CAP-113-PM11 (3.0 g, 3.15 mmol) was dissolved in acetonitrile (30 mL), and N-methylimidazole (0.5 g, 6.09 mmol) and bis(diisopropylamino)(2-cyanoethoxy)phosphine (2.8 g, 9.29 mmol) were added sequentially. The mixture was stirred for 2 h under nitrogen protection. 0.1 M iodine solution (40 mL) was added to the reaction solution, and the mixture was stirred for another 1 h. The solution was diluted with 200 mL of brine, extracted with dichloromethane (200 mL × 3), and the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and evaporated to dryness. The organic phase was evaporated under reduced pressure until no solvent was removed, yielding YK-CAP-113-PM12 (1.8 g, 1.66 mmol, 52.6%), C43H50N12O18P2, MS (ES): m / z (MH-) 1083.3.

[0402] Step 13: Synthesis of YK-CAP-113-PM13

[0403] YK-CAP-113-PM12 (1.8 g, 1.66 mmol) was dissolved in a mixed solution of ammonia (10 mL) and methanol (5 mL). The mixture was heated to 50 °C and stirred for 24 h under nitrogen protection. The reaction system was concentrated under reduced pressure to remove the solvent, and the crude product was obtained by rotary evaporation until no solvent was distilled off. Water (50 mL) was added to dissolve the crude product, and the product was purified by gel column chromatography (elution with water and 1.5 MTEAB = 10:1). The target product peak was collected, concentrated, lyophilized, and desalted again by high-performance liquid chromatography (50 mM MTEAB and methanol mobile phase) to obtain YK-CAP-113-PM13 (triethylamine salt, 660 mg, 0.80 mmol, 48.4%), a white solid, C22H30N10O14P2, MS (ES): m / z (MH-) 719.1.

[0404] Step 14: Synthesis of YK-CAP-113

[0405] Using YK-CAP-113-PM11 (150 mg, 0.18 mmol) and Im-m7GDP (193 mg, 0.36 mmol) as raw materials, YK-CAP-113 (ammonium salt, 40 mg, 33.04 µmol, 18.1%) was obtained according to the synthesis method of YK-CAP-101. C33H45N15O24P4, MS (ES): m / z (MH-) 1158.2. 1HNMR(400MHz,D2O)δ8.44(d,J=1.3Hz,1H),8.33(s,1H),8.08(d,J=6.0Hz,2H),6.02(d,J=5.4Hz,1H),5.90(d,J=2 .3Hz,1H),5.82(d,J=4.4Hz,1H),4.95–4.82(m,3H),4.63(t,J=4.5Hz,1H),4.53–4.31(m,4H),4.26(d,J=4.5Hz,1H) ,4.21(s,3H),4.09(s,1H),3.76(t,J=7.0Hz,1H),3.43(s,3H),3.35(s,3H),3.21-3.11(m,2H),2.42–2.35(m,1H). 31PNMR(D2O,162MHz)δ-0.94(s,1P),-11.34(d,J=19.2Hz,1P),-11.82(d,J=17.3Hz,1P),-23.11(t,J=17.3Hz,1P).

[0406] 16. Synthesis of YK-CAP-114

[0407] Step 1: Synthesis of YK-CAP-114-PM1

[0408] YK-CAP-113-PM4 (13.3 g, 31.61 mmol), phthalimide (5.6 g, 38.06 mmol), triphenylphosphine (16.6 g, 63.29 mmol), and DIAD (7.7 g, 38.08 mmol) were dissolved in dry tetrahydrofuran (150 mL). After cooling to 0 °C, the mixture was stirred under nitrogen protection for approximately 5 h. After the reaction was complete, water (10 mL) was added to quench the reaction, and the mixture was extracted with EA (200 mL × 3). The organic phases were combined, washed with saturated NaCl aqueous solution, dried over anhydrous sodium sulfate, filtered, and evaporated to dryness. The residue was purified by silica gel chromatography (0-20% ethyl acetate / n-hexane) to obtain YK-CAP-114-PM1 (15.9 g, 28.92 mmol, 91.5%). C27H43NO7Si2, MS (ES): m / z (M+H+) 550.3.

[0409] Step 2: Synthesis of YK-CAP-114-PM2

[0410] YK-CAP-114-PM1 (15.9 g, 28.92 mmol) was dissolved in 300 mL of ethanol, and 50 mL of hydrazine hydrate was added. The mixture was heated to 80 °C and stirred for 12 hours. After the reaction was complete, water (10 mL) was added to quench the reaction, and the mixture was extracted with EA (200 mL × 3). The organic phases were combined, washed with saturated NaCl aqueous solution, dried over anhydrous sodium sulfate, filtered, and evaporated to dryness. YK-CAP-114-PM2 (12.0 g, 28.59 mmol, 98.9%) was obtained. C19H41NO5Si2, MS (ES): m / z (M+H+) 420.3.

[0411] Step 3: Synthesis of YK-CAP-114-PM3

[0412] YK-CAP-114-PM2 (12.0 g, 28.59 mmol) and triethylamine (5.9 g, 58.31 mmol) were dissolved in dichloromethane (150 mL), cooled to 0 °C, and acetyl chloride (3.3 g, 42.04 mmol) was slowly added dropwise. After the addition was complete, the reaction system was maintained at 0 °C and stirred for 3 hours. After the reaction was completed, water (20 mL) was added to quench the reaction, and the mixture was extracted with DCM (200 mL × 3). The organic phases were combined, washed with saturated NaCl aqueous solution, dried over anhydrous sodium sulfate, filtered, and evaporated to dryness. The residue was purified by silica gel chromatography (0-20% ethyl acetate / n-hexane) to obtain YK-CAP-114-PM3 (12.2 g, 26.42 mmol, 92.4%). C21H43NO6Si2, MS (ES): m / z (M+H+) 462.3.

[0413] Step 4: Synthesis of YK-CAP-114-PM4

[0414] Using YK-CAP-114-PM3 (12.2 g, 26.42 mmol) as the starting material, YK-CAP-113-PM6 was synthesized using the same method as YK-CAP-114-PM4 (8.5 g, 12.71 mmol, 48.1%). C32H48N6O6Si2, MS (ES): m / z (M+H+) 669.3.

[0415] Step 5: Synthesis of YK-CAP-114-PM5

[0416] YK-CAP-114-PM5 (4.4 g, 10.32 mmol, 81.2%) was obtained from YK-CAP-113-PM7 using YK-CAP-114-PM4 (8.5 g, 12.71 mmol) as the starting material. The synthesis method was the same as that used for YK-CAP-113-PM7. The MS (ES) assay was performed using C20H22N6O5, with m / z (M+H+) 427.2.

[0417] Step 6: Synthesis of YK-CAP-114-PM6

[0418] YK-CAP-114-PM6 (5.5g, 7.55mmol, 73.1%) was obtained from YK-CAP-113-PM8 using YK-CAP-114-PM5 (4.4g, 10.32mmol) as the starting material. The synthesis method was the same as that used for YK-CAP-113-PM8. The MS (ES) values ​​were: C41H40N6O7, m / z (M+H+) 729.3.

[0419] Step 7: Synthesis of YK-CAP-114-PM7

[0420] YK-CAP-114-PM7 (5.0 g, 5.38 mmol, 71.3%) was obtained from YK-CAP-113-PM9 using YK-CAP-114-PM6 (5.5 g, 7.55 mmol) as the starting material. C50H57N8O8P, MS (ES): m / z (MH-) 927.4.

[0421] Step 8: Synthesis of YK-CAP-114-PM8

[0422] YK-CAP-114-PM8 (5.7g, 4.45mmol, 82.7%) was obtained from YK-CAP-113-PM10 using the same synthesis method as YK-CAP-114-PM7 (5.0g, 5.38mmol). C62H65N12O17P, MS (ES): m / z (MH-) 1279.4.

[0423] Step 9: Synthesis of YK-CAP-114-PM9

[0424] YK-CAP-114-PM9 (3.5g, 3.58mmol, 80.4%) was obtained from YK-CAP-113-PM11 using YK-CAP-114-PM8 (5.7g, 4.45mmol) as the starting material. C41H47N12O15P, MS (ES): m / z (MH-) 977.3.

[0425] Step 10: Synthesis of YK-CAP-114-PM10

[0426] YK-CAP-114-PM10 (2.0 g, 1.80 mmol, 50.3%) was obtained from YK-CAP-114-PM9 (3.5 g, 3.58 mmol) using the same synthesis method as YK-CAP-113-PM12. C44H51N13O18P2, MS (ES): m / z (MH-) 1110.3.

[0427] Step 11: Synthesis of YK-CAP-114-PM11

[0428] YK-CAP-114-PM11 (triethylamine salt, 800 mg, 0.94 mmol, 52.4%) was obtained from YK-CAP-114-PM10 (2.0 g, 1.80 mmol) using the same synthesis method as YK-CAP-113-PM13. C23H31N11O14P2, MS (ES): m / z (MH-) 746.2.

[0429] Step 12: Synthesis of YK-CAP-114

[0430] YK-CAP-114-PM11 (150 mg, 0.18 mmol) was used as a starting material, and YK-CAP-114 (ammonium salt, 26 mg, 21.01 µmol, 11.9%) was obtained by following the synthesis method of YK-CAP-113. C34H46N16O24P4, MS (ES): m / z (MH-) 1185.2. 1HNMR(400MHz,D2O)δ8.42(d,J=1.4Hz,1H),8.32(s,1H),8.11(d,J=6.0Hz,2H),6.12(d,J=5.4Hz,1H),5.92(d,J=2 .3Hz,1H),5.84(d,J=4.3Hz,1H),4.92–4.86(m,3H),4.61(t,J=4.5Hz,1H),4.52–4.33(m,4H),4.21(d,J=4.5Hz,1H) ,4.16(s,3H),4.03(s,1H),3.72(t,J=6.3Hz,1H),3.42(s,3H),3.32–3.23(m,2H),2.42–2.32(m,1H),1.88(s,3H). 31PNMR(D2O,162MHz)δ-0.88(s,1P),-11.12(d,J=19.1Hz,1P),-12.21(d,J=17.2Hz,1P),-23.55(t,J=17.1Hz,1P).

[0431] 17. Synthesis of YK-CAP-115

[0432] Step 1: Synthesis of YK-CAP-115-PM1

[0433] YK-CAP-113-PM4 (20.0 g, 47.54 mmol) was dissolved in dichloromethane (200 mL), cooled to -40 °C, and a solution of diethylaminotrifluoride (9.2 g, 57.08 mmol) in dichloromethane (20 mL) was slowly added dropwise. After the addition was complete, the temperature was slowly raised to 0 °C, and the reaction was stirred for 4 h. The reaction was monitored for completeness by thin-layer chromatography. The reaction system was quenched with saturated sodium bicarbonate aqueous solution (100 mL), extracted with dichloromethane (200 mL × 3), and the organic phases were combined, washed with saturated NaCl aqueous solution, dried over anhydrous sodium sulfate, filtered, and evaporated to dryness. The residue was purified by silica gel chromatography (0-40% ethyl acetate / n-hexane) to obtain YK-CAP-115-PM1 (11.2 g, 26.50 mmol, 55.7%).

[0434] Step 2: Synthesis of YK-CAP-115-PM2

[0435] YK-CAP-115-PM2 (8.6 g, 13.65 mmol, 51.5%) was obtained from YK-CAP-115-PM1 (11.2 g, 26.50 mmol) using the same synthesis method as YK-CAP-113-PM6. C30H44FN5O5Si2, MS (ES): m / z (M+H+) 630.3.

[0436] Step 3: Synthesis of YK-CAP-115-PM3

[0437] YK-CAP-115-PM3 (4.8 g, 12.39 mmol, 90.8%) was obtained from YK-CAP-115-PM2 (8.6 g, 13.65 mmol) using the same synthesis method as YK-CAP-113-PM7. C18H18FN5O4, MS (ES): m / z (MH-) 388.1.

[0438] Step 4: Synthesis of YK-CAP-115-PM4

[0439] YK-CAP-115-PM4 (5.6 g, 8.12 mmol, 65.5%) was obtained from YK-CAP-113-PM8 using YK-CAP-115-PM3 (4.8 g, 12.39 mmol) as the starting material. The synthesis method was the same as that used for YK-CAP-113-PM8. The chromatogram (C39H36FN5O6) was analyzed by MS (ES): m / z (MH-) 690.3.

[0440] Step 5: Synthesis of YK-CAP-115-PM5

[0441] YK-CAP-115-PM5 (5.0 g, 5.62 mmol, 69.2%) was obtained from YK-CAP-113-PM9 using YK-CAP-115-PM4 (5.6 g, 8.12 mmol) as the starting material. C48H53FN7O7P, MS (ES): m / z (MH-) 888.4.

[0442] Step 6: Synthesis of YK-CAP-115-PM6

[0443] YK-CAP-115-PM6 (4.9 g, 3.94 mmol, 70.2%) was obtained from YK-CAP-113-PM10 using YK-CAP-115-PM5 (5.0 g, 5.62 mmol) as the starting material. C60H61FN11O16P, MS (ES): m / z (MH-) 1240.4.

[0444] Step 7: Synthesis of YK-CAP-115-PM7

[0445] YK-CAP-115-PM7 (2.3 g, 2.45 mmol, 62.0%) was obtained from YK-CAP-113-PM11 using YK-CAP-115-PM6 (4.9 g, 3.94 mmol) as the starting material. C39H43FN11O14P, MS (ES): m / z (MH-) 938.3.

[0446] Step 8: Synthesis of YK-CAP-115-PM8

[0447] YK-CAP-115-PM8 (2.0 g, 1.86 mmol, 76.2%) was obtained from YK-CAP-113-PM12 using the same synthesis method as YK-CAP-115-PM7. C42H47FN12O17P2, MS (ES): m / z (MH-) 1071.3.

[0448] Step 9: Synthesis of YK-CAP-115-PM9

[0449] YK-CAP-115-PM9 (triethylamine salt, 720 mg, 0.89 mmol, 47.7%) was obtained from YK-CAP-115-PM8 (2.0 g, 1.86 mmol) using the same synthesis method as YK-CAP-113-PM13. C21H27FN10O13P2, MS (ES): m / z (MH-) 707.1.

[0450] Step 10: Synthesis of YK-CAP-115

[0451] Using YK-CAP-115-PM9 (150 mg, 0.19 mmol) as a starting material, YK-CAP-115 (ammonium salt, 31 mg, 25.86 µmol, 14.0%) was obtained by following the synthesis method of YK-CAP-113. C32H42FN15O23P4, MS (ES): m / z (MH-) 1146.1. 1HNMR(400MHz,D2O)δ8.44(d,J=1.4Hz,1H),8.32(s,1H),8.12(d,J=6.0Hz,2H),6.12(d,J=5.2Hz,1H),5.95(d, J=2.2Hz,1H),5.82(d,J=4.2Hz,1H),4.95–4.82(m,3H),4.80–4.73(m,2H),4.53(t,J=4.5Hz,1H),4.41–4.34(m, 4H),4.21(d,J=4.1Hz,1H),4.13(s,3H),4.04(s,1H),3.61(t,J=6.3Hz,1H),3.42(s,3H),2.92–2.81(m,1H).31P NMR(D2O,162MHz)δ-0.86(s,1P),-11.62(d,J=19.1Hz,1P),-12.41(d,J=17.2Hz,1P),-22.42(t,J=17.0Hz,1P).

[0452] 18. Synthesis of YK-CAP-116

[0453] Step 1: Synthesis of YK-CAP-116-PM1

[0454] YK-CAP-113-PM4 (20.0 g, 47.54 mmol) was dissolved in acetonitrile (200 mL), and 2-iodobenzoic acid (16.0 g, 57.14 mmol) was added. The mixture was heated to 90 °C and stirred for 4 hours. After cooling to room temperature, the reaction solution was filtered, and the filtrate was evaporated to dryness under reduced pressure to obtain YK-CAP-116-PM1 (18.4 g, 43.95 mmol, 92.4%).

[0455] Step 2: Synthesis of YK-CAP-116-PM2

[0456] YK-CAP-116-PM1 (18.4 g, 43.95 mmol) was dissolved in dichloromethane (200 mL). The reaction system was cooled to -40 °C, and a solution of diethylaminotrifluoride (21.3 g, 132.14 mmol) in dichloromethane (40 mL) was slowly added dropwise. After the addition was complete, the temperature was slowly raised to 0 °C, and the reaction was stirred for 4 h. The reaction was monitored for completeness by thin-layer chromatography. The reaction system was quenched with saturated sodium bicarbonate aqueous solution (200 mL), extracted with dichloromethane (300 mL × 3), and the organic phases were combined, washed with saturated NaCl aqueous solution, dried over anhydrous sodium sulfate, filtered, and evaporated to dryness. The residue was purified by silica gel chromatography (0-40% ethyl acetate / n-hexane) to obtain YK-CAP-116-PM2 (12.8 g, 29.05 mmol, 66.1%).

[0457] Step 3: Synthesis of YK-CAP-116-PM3

[0458] YK-CAP-116-PM3 (9.6 g, 14.82 mmol, 51.0%) was obtained from YK-CAP-116-PM2 (12.8 g, 29.05 mmol) using the same synthesis method as YK-CAP-113-PM6. C30H43F2N5O5Si2, MS (ES): m / z (M+H+) 648.3.

[0459] Step 4: Synthesis of YK-CAP-116-PM4

[0460] YK-CAP-116-PM4 (5.2 g, 12.83 mmol, 86.6%) was obtained from YK-CAP-113-PM7 using the same synthesis method as YK-CAP-116-PM3. C18H17F2N5O4, MS (ES): m / z (M+H+) 406.1.

[0461] Step 5: Synthesis of YK-CAP-116-PM5

[0462] YK-CAP-116-PM5 (6.4 g, 9.04 mmol, 70.5%) was obtained from YK-CAP-113-PM8 using YK-CAP-116-PM4 (5.2 g, 12.83 mmol) as the starting material. The synthesis method was the same as that used for YK-CAP-113-PM8. The chromatogram (C39H35F2N5O6) was analyzed by MS (ES): m / z (M+H+) 708.3.

[0463] Step 6: Synthesis of YK-CAP-116-PM6

[0464] YK-CAP-116-PM6 (5.5 g, 6.06 mmol, 67.0%) was obtained from YK-CAP-113-PM9 using YK-CAP-116-PM5 (6.4 g, 9.04 mmol) as the starting material. C48H52F2N7O7P, MS (ES): m / z (MH-) 906.4.

[0465] Step 7: Synthesis of YK-CAP-116-PM7

[0466] YK-CAP-116-PM7 (5.3 g, 4.21 mmol, 69.4%) was obtained from YK-CAP-113-PM10 using YK-CAP-116-PM6 (5.5 g, 6.06 mmol) as the starting material. C60H60F2N11O16P, MS (ES): m / z (MH-) 1258.4.

[0467] Step 8: Synthesis of YK-CAP-116-PM8

[0468] YK-CAP-116-PM8 (2.8 g, 2.92 mmol, 69.5%) was obtained from YK-CAP-116-PM7 (5.3 g, 4.21 mmol) using the same synthesis method as YK-CAP-113-PM11. C39H42F2N11O14P, MS (ES): m / z (MH-) 956.3.

[0469] Step 9: Synthesis of YK-CAP-116-PM9

[0470] YK-CAP-116-PM9 (2.2 g, 2.02 mmol, 69.0%) was obtained from YK-CAP-116-PM8 (2.8 g, 2.92 mmol) using the same synthesis method as YK-CAP-113-PM12. C42H46F2N12O17P2, MS (ES): m / z (MH-) 1089.3.

[0471] Step 10: Synthesis of YK-CAP-116-PM10

[0472] YK-CAP-116-PM10 (triethylamine salt, 580 mg, 0.70 mmol, 34.7%) was obtained from YK-CAP-116-PM9 (2.2 g, 2.02 mmol) using the same synthesis method as YK-CAP-113-PM13. C21H26F2N10O13P2, MS (ES): m / z (MH-) 725.1.

[0473] Step 11: Synthesis of YK-CAP-116

[0474] YK-CAP-116 (ammonium salt, 17 mg, 13.97 µmol, 7.7%) was obtained from YK-CAP-116-PM10 (150 mg, 0.18 mmol) using the same synthesis method as YK-CAP-113. C32H41F2N15O23P4, MS (ES): m / z (MH-) 1164.1. 1HNMR(400MHz,D2O)δ8.52(d,J=1.4Hz,1H),8.45(s,1H),8.25(d,J=6.0Hz,2H),6.29(d,J=5.2Hz,1H),6.11(d, J=2.1Hz,1H),5.89(d,J=4.2Hz,1H),4.91–4.81(m,3H),4.59(t,J=4.6Hz,1H),4.42–4.31(m,4H),4.25(d,J=4.1 Hz,1H),4.11(s,2H),4.02(s,1H),3.86–3.73(m,2H),3.63(t,J=6.3Hz,1H),3.43(s,3H),2.41–2.27(m,1H).31P NMR(D2O,162MHz)δ-0.82(s,1P),-11.65(d,J=19.1Hz,1P),-12.27(d,J=17.8Hz,1P),-23.88(t,J=17.3Hz,1P).

[0475] 19. Synthesis of YK-CAP-117

[0476] Step 1: Synthesis of YK-CAP-117-PM1

[0477] Using 3-O-benzyl-4-C-benzyloxymethyl-1,2-O-isopropylidene-A-D-ribofuranosyl (10.00 g, 24.97 mmol) as a starting material, YK-CAP-117-PM1 (8.62 g, 20.80 mmol, 83.3%) was obtained by following the synthetic route of YK-CAP-107-PM4.

[0478] Step 2: Synthesis of YK-CAP-117-PM2

[0479] YK-CAP-117-PM1 (8.62 g, 20.80 mmol) was dissolved in acetic acid, and acetic anhydride (21.23 g, 207.95 mmol) was added. Concentrated sulfuric acid (380 μl) was then slowly added dropwise. After the addition was complete, the mixture was stirred at room temperature for 16 hours. 300 mL of water was added to the reaction mixture, and the solution was extracted with ethyl acetate. The organic phase was washed three times with saturated sodium bicarbonate aqueous solution until the pH was alkaline. The organic phase was dried and evaporated to dryness. The residue was purified by silica gel chromatography (0-40% ethyl acetate / n-hexane) to obtain a yellow oily liquid, YK-CAP-117-PM2 (8.13 g, 17.73 mmol, 85.3%).

[0480] Step 3: Synthesis of YK-CAP-117-PM3

[0481] Using YK-CAP-117-PM2 (8.13 g, 17.73 mmol) as a starting material, YK-CAP-104-PM11 was synthesized to obtain YK-CAP-117-PM3 (6.24 g, 7.93 mmol, 44.7%). C43H42N6O9, MS (ES): m / z (M+H+) 787.3.

[0482] Step 4: Synthesis of YK-CAP-117-PM4

[0483] YK-CAP-117-PM3 (6.24 g, 7.93 mmol) was dissolved in dichloromethane (200 mL). The reaction system was cooled to -40 °C under a nitrogen atmosphere, and a 1 M boron trichloride solution (79.3 mL, 79.30 mmol) in dichloromethane 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 h. TLC showed that the reaction was complete. The system was then cooled back to -40 °C, the reaction was quenched with methanol, evaporated to dryness, and allowed to stand at room temperature for 24 h. The solid was then added dropwise to DCM, filtered to obtain a brown crude product, and purified by HPLC to obtain YK-CAP-117-PM4 (1.19 g, 3.64 mmol, 45.8%). C12H17N6O5, MS (ES): m / z (M+H+) 328.1. YK-CAP-117-PM4: 1HNMR(400MHz,MeOD)δ8.15(s,1H),4.51(s,1H),4.41(s,1H),4.21-4.17(m,1H),3.81(s,2H),3.44-3.31(m,2H),2.97(s,3H).

[0484] Step 5: Synthesis of YK-CAP-117-PM5

[0485] YK-CAP-117-PM5 (triethylamine salt, 1.23 g, 2.42 mmol, 66.5%) was obtained from YK-CAP-117-PM4 (1.19 g, 3.64 mmol) via the synthetic route of YK-CAP-101-PM3. C12H18N5O9P, MS (ES): m / z (MH-) 406.1.

[0486] Step 6: Synthesis of YK-CAP-117-PM6

[0487] Using YK-CAP-117-PM5 (1.23 g, 2.42 mmol) as the starting material, YK-CAP-101-PM6 (sodium salt, 993 mg, 2.07 mmol, 85.6%) was obtained by following the synthetic route of YK-CAP-101-PM4, C15H20N7O8P, MS (ES): m / z (MH-) 456.1.

[0488] Step 7: Synthesis of YK-CAP-117-PM7

[0489] Using YK-CAP-117-PM6 (993 mg, 2.07 mmol) as a starting material, YK-CAP-101-PM7 (triethylamine salt, 794 mg, 1.35 mmol, 65.1%) was obtained by following the synthetic route of YK-CAP-101-PM5, C12H19N5O12P2, MS (ES): m / z (MH-) 486.1.

[0490] Step 8: Synthesis of YK-CAP-117-PM8

[0491] Using YK-CAP-117-PM7 (794 mg, 1.35 mmol) as the starting material, YK-CAP-101-PM6 was synthesized to obtain YK-CAP-117-PM8 (triethylamine salt, 520 mg, 0.86 mmol, 64.0%), C13H21N5O12P2, MS (ES): m / z (MH-) 500.1.

[0492] Step 9: Synthesis of YK-CAP-117

[0493] Using YK-CAP-117-PM8 (150 mg, 0.25 mmol) as the starting material, YK-CAP-117 (ammonium salt, 26 mg, 20.95 µmol, 8.4%) was obtained by following the synthetic route of YK-CAP-101, C34H47N15O25P4, MS (ES): m / z (MH-) 1188.1. 1HNMR(400MHz,D2O)δ8.44(d,J=1.5Hz,1H),8.26(d,J=2.5Hz,2H),8.15(d,J=4.5Hz,1H),6.31(d,J=5.2Hz,1H), 5.72(d,J=2.4Hz,1H),5.51(d,J=4.6Hz,1H),4.90–4.86(m,3H),4.60(t,J=4.7Hz,1H),4.50–4.38(m,5H),4.22(d ,J=2.9Hz,1H),4.13(s,2H),4.05(s,2H),3.92(s,3H),3.54(t,J=7.0Hz,2H),3.44–3.31(m,3H),2.97(s,3H).31 PNMR(D2O,162MHz)δ-0.91(s,1P),-11.22(d,J=19.1Hz,1P),-11.32(d,J=16.4Hz,1P),-22.12(t,J=17.4Hz,1P).

[0494] 20. Synthesis of YK-CAP-118

[0495] Step 1: Synthesis of YK-CAP-118-PM1

[0496] Using 3-O-benzyl-4-C-benzyloxymethyl-1,2-O-isopropylidene-A-D-ribofuranosyl (10.00 g, 24.97 mmol) as a starting material, YK-CAP-118-PM1 (9.11 g, 22.64 mmol, 90.6%) was synthesized via the YK-CAP-109-PM1 route.

[0497] Step 2: Synthesis of YK-CAP-118-PM2

[0498] Using YK-CAP-118-PM1 (9.11 g, 22.64 mmol) as the starting material, YK-CAP-117-PM2 (7.18 g, 16.08 mmol, 71.0%) was obtained by following the synthetic route of YK-CAP-117-PM2.

[0499] Step 3: Synthesis of YK-CAP-118-PM3

[0500] YK-CAP-118-PM3 (6.64 g, 8.57 mmol, 53.3%) was obtained from YK-CAP-104-PM11 via the synthetic route of YK-CAP-118-PM2 (7.18 g, 16.08 mmol). C42H39FN6O8, MS (ES): m / z (M+H+) 775.3.

[0501] Step 4: Synthesis of YK-CAP-118-PM4

[0502] Using YK-CAP-118-PM3 (6.64 g, 8.57 mmol) as a starting material, YK-CAP-117-PM4 (2.31 g, 7.33 mmol, 85.5%) was synthesized via the same route as YK-CAP-117-PM4. C11H14FN5O5, MS (ES): m / z (M+H+) 316.1. YK-CAP-118-PM4: 1HNMR(400MHz,MeOD)δ8.12(s,1H),4.51(s,1H),4.40(s,1H),4.27-4.22(m,1H),3.88(s,2H),3.72-3.62(m,2H).

[0503] Step 5: Synthesis of YK-CAP-118-PM5

[0504] YK-CAP-118-PM5 (triethylamine salt, 1.15 g, 2.32 mmol, 56.2%) was obtained from YK-CAP-118-PM4 (1.30 g, 4.12 mmol) via the synthetic route of YK-CAP-101-PM3. C11H15FN5O8P, MS (ES): m / z (MH-) 394.1.

[0505] Step 6: Synthesis of YK-CAP-118-PM6

[0506] Using YK-CAP-118-PM5 (1.15 g, 2.32 mmol) as the starting material, YK-CAP-101-PM6 (sodium salt, 870 mg, 1.86 mmol, 80.4%) was obtained by following the synthetic route of YK-CAP-101-PM4, C14H17FN7O7P, MS (ES): m / z (MH-) 444.1.

[0507] Step 7: Synthesis of YK-CAP-118-PM7

[0508] Using YK-CAP-118-PM6 (870 mg, 1.86 mmol) as a starting material, YK-CAP-101-PM7 (triethylamine salt, 664 mg, 1.15 mmol, 61.9%) was obtained by following the synthetic route of YK-CAP-101-PM5, C11H16FN5O11P2, MS (ES): m / z (MH-) 474.1.

[0509] Step 8: Synthesis of YK-CAP-118-PM8

[0510] Using YK-CAP-118-PM7 (664 mg, 1.15 mmol) as the starting material, YK-CAP-101-PM6 was synthesized via the same route as YK-CAP-101-PM6 to obtain YK-CAP-118-PM8 (triethylamine salt, 419 mg, 0.71 mmol, 61.6%), C12H18FN5O11P2, MS (ES): m / z (MH-) 488.1.

[0511] Step 9: Synthesis of YK-CAP-118

[0512] Using YK-CAP-118-PM8 (150 mg, 0.25 mmol) as the starting material, YK-CAP-118 (ammonium salt, 33 mg, 26.86 µmol, 10.7%) was obtained by following the synthetic route of YK-CAP-101, C33H44FN15O24P4, MS (ES): m / z (MH-) 1176.1. 1HNMR(400MHz,D2O)δ8.37(d,J=1.2Hz,1H),8.33–8.21(m,1H),8.14(d,J=4.5Hz,2H),6.33(d,J=5.1Hz,1H), 5.61(d,J=2.3Hz,1H),5.45(d,J=4.6Hz,1H),4.92–4.83(m,3H),4.63–4.55(m,2H),4.50–4.38(m,4H),4.31(d ,J=2.9Hz,1H),4.11(s,2H),4.01(s,2H),3.90-3.76(m,3H),3.52(t,J=7.0Hz,2H),3.46-3.34(m,3H).31PNM R(D2O,162MHz)δ-0.90(s,1P),-11.23(d,J=19.1Hz,1P),-11.59(d,J=16.0Hz,1P),-22.33(t,J=17.2Hz,1P).

[0513] 21. Synthesis of YK-CAP-119

[0514] Step 1: Synthesis of YK-CAP-119-PM1

[0515] 3-O-benzyl-4-C-benzyloxymethyl-1,2-O-isopropylidene-A-D-ribofuranoside (10.0 g, 24.97 mmol) was dissolved in 50 mL of acetonitrile, and 2-iodobenzoic acid (10.49 g, 37.46 mmol) was added. The mixture was heated to 70 °C and stirred for 2 h. The reaction was monitored by TLC until complete. The reaction was stopped and allowed to cool to room temperature. The mixture was then filtered through diatomaceous earth, washed with acetonitrile (50 mL) from the filter cake, and the filtrate was evaporated under reduced pressure and dried under vacuum to obtain a pale yellow liquid YK-CAP-119-PM1 (10.21 g), which was directly used in the next step (yield calculated as 100%).

[0516] Step 2: Synthesis of YK-CAP-119-PM2

[0517] Using YK-CAP-119-PM1 (10.21 g, calculated as 24.97 mmol) as the raw material, YK-CAP-119-PM2 (8.16 g, 19.41 mmol, 77.7%) was obtained by following the synthetic route of YK-CAP-110-PM1.

[0518] Step 3: Synthesis of YK-CAP-119-PM3

[0519] Using YK-CAP-119-PM2 (8.16 g, 19.41 mmol) as a raw material, YK-CAP-117-PM2 was synthesized via the same route to obtain YK-CAP-119-PM3 (6.55 g, 14.10 mmol, 72.7%).

[0520] Step 4: Synthesis of YK-CAP-119-PM4

[0521] YK-CAP-119-PM4 (5.77 g, 7.28 mmol, 51.6%) was synthesized from YK-CAP-104-PM11 via the same route as YK-CAP-119-PM3 (6.55 g, 14.10 mmol). The synthesis was carried out using C42H38F2N6O8, and MS (ES) showed m / z (M+H+) 793.3.

[0522] Step 5: Synthesis of YK-CAP-119-PM5

[0523] Using YK-CAP-119-PM4 (5.77 g, 7.28 mmol) as a starting material, YK-CAP-117-PM5 (2.16 g, 6.48 mmol, 89.0%) was synthesized via the same route as YK-CAP-117-PM4. C11H13F2N5O5, MS (ES): m / z (M+H+) 334.1. YK-CAP-119-PM5: 1HNMR(400MHz,MeOD)δ8.24(s,1H),4.77(s,1H),4.56(s,1H),4.51(s,1H),4.27-4.22(m,1H),3.72-3.62(m,2H).

[0524] Step 6: Synthesis of YK-CAP-119-PM6

[0525] YK-CAP-119-PM6 (triethylamine salt, 1.29 g, 2.51 mmol, 55.7%) was synthesized from YK-CAP-101-PM3 via the same route. C11H14F2N5O8P, MS (ES): m / z (MH-) 412.1.

[0526] Step 7: Synthesis of YK-CAP-119-PM7

[0527] Using YK-CAP-119-PM6 (1.29 g, 2.51 mmol) as the starting material, YK-CAP-101-PM7 (sodium salt, 991 mg, 2.04 mmol, 81.4%) was obtained by following the synthetic route of YK-CAP-101-PM4, C14H16F2N7O7P, MS (ES): m / z (MH-) 462.1.

[0528] Step 8: Synthesis of YK-CAP-119-PM8

[0529] Using YK-CAP-119-PM7 (991 mg, 2.04 mmol) as the starting material, YK-CAP-101-PM5 was synthesized to obtain YK-CAP-119-PM8 (triethylamine salt, 774 mg, 1.30 mmol, 63.8%), C11H15F2N5O11P2, MS (ES): m / z (MH-) 492.0.

[0530] Step 9: Synthesis of YK-CAP-119-PM9

[0531] Using YK-CAP-119-PM8 (774 mg, 1.30 mmol) as the starting material, YK-CAP-101-PM6 was synthesized to obtain YK-CAP-119-PM9 (triethylamine salt, 533 mg, 0.88 mmol, 67.3%), C12H17F2N5O11P2, MS(ES): m / z(MH-) 506.1.

[0532] Step 10: Synthesis of YK-CAP-119

[0533] Using YK-CAP-119-PM9 (150 mg, 0.25 mmol) as the starting material, YK-CAP-119 (ammonium salt, 32 mg, 25.67 µmol, 10.3%) was obtained by following the synthetic route of YK-CAP-101, C33H43F2N15O24P4, MS (ES): m / z (MH-) 1194.1. 1HNMR(400MHz,D2O)δ8.35(d,J=1.2Hz,1H), 8.30 -8.19(m,1H), 8.12(d,J=4.4Hz,2H), 6.25(d,J=5.2Hz,1H), 5.65(d,J=2.2Hz,1H), 5.27(d,J=4.5Hz,1H), 4.93–4.88(m,3H), 4.83-4.79(s,1H), 4.66–4.57(m,2H), 4.51-4.42(m,5H), 4.22(d,J=2.9Hz,1H), 4.04(s,2H), 3.88-3.75(m,3H), 3.42(t,J=7.0Hz,1H), 3.36–3.31(m,3H). 31PNMR(D2O,162MHz)δ-0.93(s,1P),-12.13(d,J=19.1Hz,1P),-13.31(d,J=16.1Hz,1P),-24.57(t,J=17.4Hz,1P).

[0534] 22. Synthesis of compound 5227:

[0535] Using 5227-S as the starting material, 35 mg of compound 5227 was obtained according to the synthesis method of YK-CAP-117.

[0536] 23. Synthesis of CAP-2'O-ethyl

[0537] Following the method in WO2023025073A1, 47 mg of CAP-2'O-ethyl was obtained.

[0538] Example 2: mRNA in vitro transcription yield and capping rate I. Structural differences of capped analogues Table 1. Structure of capped analogues name structure Remark YK-CAP-101 The design structure of this application, synthesized in Example 1 YK-CAP-102 The design structure of this application, synthesized in Example 1 YK-CAP-103 The design structure of this application, synthesized in Example 1 YK-CAP-104 The design structure of this application, synthesized in Example 1 YK-CAP-105 The design structure of this application, synthesized in Example 1 YK-CAP-106 The design structure of this application, synthesized in Example 1 YK-CAP-107 The design structure of this application, synthesized in Example 1 YK-CAP-108 The design structure of this application, synthesized in Example 1 YK-CAP-109 The design structure of this application, synthesized in Example 1 YK-CAP-110 The design structure of this application, synthesized in Example 1 YK-CAP-111 The design structure of this application, synthesized in Example 1 YK-CAP-112 The design structure of this application, synthesized in Example 1 YK-CAP-113 The design structure of this application, synthesized in Example 1 YK-CAP-114 The design structure of this application, synthesized in Example 1 YK-CAP-115 The design structure of this application, synthesized in Example 1 YK-CAP-116 The design structure of this application, synthesized in Example 1 YK-CAP-117 The design structure of this application, synthesized in Example 1 YK-CAP-118 The design structure of this application, synthesized in Example 1 YK-CAP-119 The design structure of this application, synthesized in Example 1 5227 Compound 5227, synthesized in Example 1, is described on page 124 of the specification in WO2022051677A1. CAP-2'O-ethyl The synthesis in Example 1 is described in Table 6 on page 27 of the instruction manual WO2023025073A1. N-7113 The compound described in claim 15 of the patent application on page 2 of CN116751827B (purchased from Jiangsu Shenji Biotechnology Co., Ltd.) is an externally sourced compound. Compound 14 Purchased from Jiangsu Shenji Biotechnology Co., Ltd., compound 14 on page 90 of CN115803333A. HN3002 Compound 3, purchased from Guangzhou Hengnuokang Pharmaceutical Technology Co., Ltd., is listed on page 9 of CN115260264B. m6A Purchased from Jiangsu Shenji Biotechnology Co., Ltd., WO2023 / 147352Al, compound 31 on page 21.

[0539] As shown in Table 1, the chemical structures of compounds YK-CAP-101~119 in this application are similar to those of existing mRNA capped analogs, but differ significantly from others, as detailed below: 1. In this application, compounds YK-CAP-101 and YK-CAP-102 have a six-membered sugar ring as the first sugar ring, and N7113 has a five-membered sugar ring; the other structures are completely identical. 2. The first sugar ring and the guanine-linked group of compound YK-CAP-103 in this application are different from those of N7113, that is, there is an extra methylene group at the C1 position, but the other structures are exactly the same. 3. In this application, compounds YK-CAP-104~106 have two substituents on the first sugar ring at C3, which is different from N7113. Specifically, the C3 substituents of N7113 are hydroxyl and hydrogen; the C3 substituents of YK-CAP-104 are dimethylamine methyl and fluorine, the C3 substituents of YK-CAP-105 are cyano and methyl, and the C3 substituents of YK-CAP-106 are acetaminophen and methyl. The other structures are completely the same. 4. The C3 substituent on the first sugar ring of compound YK-CAP-107~112 in this application is different from that of N7113 and HN3002. That is, the C3 substituent of N7113 and HN3002 is hydroxyl and methoxymethyl; the C3 substituent of YK-CAP-107~112 is 1-methoxyethyl, 1-acetaminoethyl, 1-fluoroethyl, difluoromethyl, N,N-diacetamino, N,N-di-n-propylacetamino, and the other structures are exactly the same. 5. The C2 substituents on the second sugar ring of compounds YK-CAP-113~116 in this application are different from those of N7113 and CAP-2'O-ethyl. That is, the C2 substituents of N7113 and CAP-2'O-ethyl are methoxy and ethoxy, respectively; the C2 substituents of YK-CAP-113~116 are methoxymethyl, acetaminomethyl, 1-fluoromethyl, and difluoromethyl, respectively, and the other structures are completely the same. 6. The C4 substituents on the first sugar ring of compounds YK-CAP-117~119 in this application are different from those of N7113 and 5227. That is, the C4 substituents of N7113 and 5227 are hydrogen and methoxy, respectively; the C4 substituents of YK-CAP-117~119 are methoxymethyl, 1-fluoromethyl and difluoromethyl, respectively, and the other structures are exactly the same. 7. The compounds YK-CAP-101~119 of this application have significant structural differences from compounds 14 and m6A. The first sugar ring of compound 14 is a locked nucleic acid sugar ring, that is, there is a methylene bridge between 2'-O and C4'; the second base of m6A, adenine, is modified by methylation.

[0540] II. Determination of mRNA in vitro transcription yield and capping rate 1. Experimental Methods (1) Capping synthesis using capping analogues First, the plasmids are linearized using a plasmid linearization enzyme, and then the linearized plasmids are purified. (2) In vitro transcription to synthesize mRNA YK-CAP-101~119, compound 5227, and CAP-2'O-ethyl synthesized in Example 1 were used as capped analogs, and the reaction systems are shown in Table 2: Table 2 In vitro transcription reaction system system Dosage T7 RNA polymerase 50U 10X buffer 2μL 100mM ATP 1μL 100mMGTP 1μL 100mMCTP 1μL 100mMUTP 1μL 100mM capped analogue 1μL nuclease inhibitors 20U Inorganic pyrophosphatase 0.05U sterile enzyme-free water Make up to 20 μL DNA template 1μg In the experiment, the required volume of materials for the system was first calculated, and then samples were added. First, sterile, enzyme-free water was added to the system, followed by 10X buffer, NTPs, and a capping analogue. After mixing, the mixture was gently centrifuged. Then, nuclease inhibitors, inorganic pyrophosphatase, T7 RNA polymerase, and linearized DNA template were added, mixed thoroughly, and gently centrifuged again. The mixture was incubated at 37°C for 2 hours. After 2 hours, 1 U of DNase I was added, and the mixture was incubated at 37°C for another 30 minutes. The mRNA precipitate was then washed with 75% ethanol, and after briefly evaporating and drying the ethanol, the mRNA was reconstituted with sterile, enzyme-free water. (3) Purify the transcription products and record the in vitro transcription yield of mRNA. (4) Anneal the obtained mRNA with the probe. Annealing was performed in a PCR instrument at 95℃ for 5 min, 65℃ for 2 min, 55℃ for 2 min, 40℃ for 2 min, and 22℃ for 2 min. (5) Pretreatment of magnetic beads and binding to probe: Place 100 μL of magnetic beads on a magnetic rack for pretreatment. Add 120 μL of sample and magnetic bead solution and incubate at room temperature for 30 min, mixing slowly while incubating. (6) Cut the mRNA and obtain the 5' single-stranded sequence of the mRNA that binds to the probe. Add 20 μL Lnase H (5 U / μL) and incubate at 37°C for 3 hours, mixing every half hour. After incubation, wash the magnetic beads. Add 100 μL of 75% methanol heated to 80°C to the washed beads. Heat the mixture to 80°C on a hot plate for 3 minutes, then place it on a magnetic rack and aspirate the supernatant. Dry the supernatant at room temperature for 45 minutes to 10 μL using an evaporative centrifuge. Resuspend the sample in 50 μL of 100 μM EDTA / 1% MeOH for LC-MS analysis to determine the capping status of RNA during transcription. Since capped and uncapped bases have significant molecular weight differences, the capping rate of mRNA transcription initiated by different capping analogs can be determined by using the molecular weight difference. 2. Experimental Results The results of the in vitro transcription yield and capping rate measurements of mRNA showed that the ribose-modified capping analogues of this invention exhibited significant differences in both mRNA transcription yield and capping rate. Compared with existing ribose-modified capping analogues, the ribose-modified capping analogues of this invention significantly improved both mRNA transcription yield and capping rate in vitro. The specific mRNA in vitro transcription yield and capping rate are shown in Table 3. Table 3. mRNA in vitro transcription yield and capping rate name Unit template yield (μg) An increase (%) compared to 5227 Hat-on rate (%) An increase (%) compared to 5227 YK-CAP-101 32.5 -75.8 42.8 -40.5 YK-CAP-102 44.1 -67.1 30.2 -58.0 YK-CAP-103 88.0 -34.4 71.1 -1.1 YK-CAP-104 73.2 -45.5 77.2 7.4 YK-CAP-105 82.3 -38.7 69.3 -3.6 YK-CAP-106 155.5 15.9 95.3 32.5 YK-CAP-107 170.7 27.2 97.3 35.3 YK-CAP-108 151.8 13.1 95.8 33.2 YK-CAP-109 168.1 25.3 95.2 32.4 YK-CAP-110 172.1 28.2 97.4 35.5 YK-CAP-111 173.5 29.3 98.1 36.4 YK-CAP-112 169.2 26.1 95.2 32.4 YK-CAP-113 166.1 23.8 95.2 32.4 YK-CAP-114 161.2 20.1 95.2 32.4 YK-CAP-115 158.1 17.8 95.7 33.1 YK-CAP-116 159.4 18.8 96.4 34.1 YK-CAP-117 153.2 14.2 95.1 32.3 YK-CAP-118 172.2 28.3 97.2 35.2 YK-CAP-119 154.2 14.9 96.3 33.9 Compound 14 123.2 -8.2 78.3 8.9 5227 134.2 0 71.9 0 1) The ribose-modified capping analogs in this application showed significant differences in in vitro mRNA transcription yield and capping rate. The in vitro transcription yield and capping rate of YK-CAP-106~119 mRNA were significantly higher than those of YK-CAP-101~105. YK-CAP-111 had the highest transcription yield and capping rate, with a transcription yield 5.3 times that of the lowest YK-CAP-101 and a capping rate 3.2 times that of the lowest YK-CAP-102. As shown in Table 3, all the ribose-modified capped analogs in this application can transcribe mRNA. The mRNA transcription activity of different ribose-modified capped analogs varies significantly. The in vitro transcription yields of YK-CAP-106–119 mRNAs are all very high, exceeding 150 μg. Among them, YK-CAP-107, YK-CAP-110, YK-CAP-111, YK-CAP-117, and YK-CAP-118 yielded 170.7 μg, 172.1 μg, 173.5 μg, 153.2 μg, and 172.2 μg, respectively, with YK-CAP-111 achieving the highest yield of 173.5 μg (Figure 1). The lowest mRNA transcription yield was achieved by YK-CAP-101, at only 32.5 μg. YK-CAP-102–105 also showed low yields, at 44.1 μg, 88.0 μg, 73.2 μg, and 82.3 μg, respectively. YK-CAP-111, however, showed significantly higher transcription yields, being 5.3-fold, 3.9-fold, 2.0-fold, 2.4-fold, and 2.1-fold higher than YK-CAP-101–105. The capping rates of YK-CAP-106~119 are all very high, all exceeding 95%. Among them, the capping rates of YK-CAP-107, YK-CAP-110, YK-CAP-111, YK-CAP-117 and YK-CAP-118 are 97.3%, 97.4%, 98.1%, 95.1% and 97.2% respectively, with YK-CAP-111 having the highest capping rate at 98.1%. The lowest capping rate was achieved by YK-CAP-102, at only 30.2%. YK-CAP-101 and YK-CAP-103~105 also had low capping rates, at 42.8%, 71.1%, 77.2%, and 69.3%, respectively. YK-CAP-111 showed a significant improvement in capping rates, increasing by 2.3 times, 3.2 times, 1.4 times, 1.3 times, and 1.4 times compared to YK-CAP-101~105 (Figure 2). 2) The ribose-modified capping analogues of this application significantly improve both the in vitro transcription yield and capping rate of mRNA compared to existing ribose-modified capping analogues. For example, the transcription yield of YK-CAP-111 is 40.8% higher than that of compound 14, and the capping rate is 36.4% higher than that of 5227. The in vitro transcription yield and capping rate of compound 14 were 123.2 μg and 78.3%, respectively. In this application, the in vitro transcription yield and capping rate of YK-CAP-111 were 40.8% and 25.3% higher than those of compound 14, respectively, which is a significant improvement. The in vitro transcription yield and capping rate of mRNA of compound 5227 were 134.2 μg and 71.9%, respectively. In this application, the in vitro transcription yield and capping rate of mRNA of YK-CAP-111 were increased by 29.3% and 36.4% respectively compared with compound 5227, which is a significant improvement. 3) Riboside-modified capping analogs with similar structures have huge differences in mRNA in vitro transcription yield and capping rate, making it impossible to infer the mRNA in vitro transcription yield and capping rate based on structure. The ribose-modified capping analogs YK-CAP-117~119 designed in this application have very similar structures. This series of compounds is also very similar in structure to 5227, but the in vitro transcription yield and capping rate of mRNA differ greatly. For example, compounds YK-CAP-117~119 in this application differ from 5227 only in the substituent at C4 of the first sugar ring; that is, the C4 substituent in 5227 is methoxy, while the C4 substituents in YK-CAP-117~119 are methoxymethyl, 1-fluoromethyl, and difluoromethyl, respectively, with the other structures being identical. However, the in vitro transcription yield of YK-CAP-117~119 mRNA increased by 14.2%, 28.3%, and 14.9% compared to 5227, respectively, and the capping rate increased by 32.3%, 35.2%, and 33.9%, respectively, showing a significant improvement. Therefore, it can be seen that ribose-modified capping analogs with similar structures do not necessarily have similar mRNA transcription activities and capping rates; on the contrary, they are very likely to be very different. As can be seen from the in vitro transcription yield and capping rate of mRNA, the ribose-modified capping analogs YK-CAP-106~119 in this application significantly improve the in vitro transcription yield and capping rate of mRNA compared with YK-CAP-101~105 in this application, as well as with compounds 14 and 5227 in the prior art. This proves that the ribose modification of YK-CAP-106~119 has a good anti-reverse transcription effect during in vitro mRNA transcription, and can greatly increase the binding ability of the cap structure to the capping enzyme, thereby increasing the capping rate of transcribed mRNA.

[0541] Example 3: Preparation and characterization of lipid nanoparticles 1. Experimental Methods Cationic lipids YK-009 (Beijing Yuekang Kechuang Pharmaceutical Technology Co., Ltd.), DSPC (Aivert (Shanghai) Pharmaceutical Technology Co., Ltd.), cholesterol (Aivert (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 was mixed with the above-prepared Fluc mRNA aqueous solution with different capping structures 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 resulting liposomes were diluted 10 times with PBS and then ultrafiltered in 300 kDa ultrafiltration tubes to remove ethanol. The mixture was then brought to a certain volume with PBS. Finally, the lipid nanoparticles were filtered through a 0.2 μm sterile filter to obtain an LNP formulation of Fluc-mRNA encapsulated with YK-009 / DSPC / cholesterol / DMG-PEG2000 (molar percentage of 49:10:39.5:1.5). Particle size and polydispersity index (PDI) were determined using a Malvern laser particle size analyzer based on dynamic light scattering. 10 μL of liposome solution was diluted to 1 mL with RNase-free deionized water and added to the sample cell; each sample was measured three times. Measurement conditions were: 90° scattering angle, 25°C. The encapsulation efficiency of the lipid nanoparticles was determined using the Quant-it Ribogreen RNA Quantification Kit (Thermo Fisher Scientific, UK) according to the manufacturer's instructions. 2. Experimental Results The specific characterization data of the lipid nanoparticles are shown in Table 4. Table 4 Characterization of lipid nanoparticles name Particle size (nm) PDI Encapsulation efficiency (%) YK-CAP-101 75.45 0.043 93.4 YK-CAP-102 73.22 0.055 95.3 YK-CAP-103 67.24 0.053 94.7 YK-CAP-104 69.24 0.047 95.7 YK-CAP-105 77.22 0.063 95.7 YK-CAP-106 68.24 0.048 97.1 YK-CAP-107 78.42 0.053 96.3 YK-CAP-108 83.13 0.023 96.4 YK-CAP-109 86.35 0.035 97.2 YK-CAP-110 69.64 0.033 97.7 YK-CAP-111 78.33 0.053 95.1 YK-CAP-112 83.24 0.056 93.5 YK-CAP-113 79.34 0.043 96.1 YK-CAP-114 66.36 0.049 94.3 YK-CAP-115 68.35 0.034 95.3 YK-CAP-116 74.24 0.063 98.3 YK-CAP-117 72.56 0.044 94.3 YK-CAP-118 75.84 0.055 97.2 YK-CAP-119 82.45 0.057 94.6 5227 83.34 0.028 94.9 CAP-2'O-ethyl 81.34 0.035 96.3 N-7113 87.45 0.078 96.1 Compound 14 79.35 0.073 94.2 HN3002 75.35 0.058 95.1 m6A 75.76 0.047 93.8 As shown in Table 4, good lipid nanoparticles can be prepared from Fluc mRNA transcribed from capped analogs YK-CAP-101~119 in this application and from capped analogs 5227, CAP-2'O-ethyl, N-7113, compound 14, HN3002, and m6A disclosed in the prior art. All lipid nanoparticles have particle sizes between 66 and 88 nm, PDI values ​​between 0.023 and 0.078, and encapsulation efficiencies exceeding 90%.

[0542] Example 4: Translation efficiency of different capped luciferase mRNAs 1. Experimental Methods (1) HEK293T cells were cultured in DMEM medium containing 10% FBS and penicillin / streptomycin at 37°C and 5% CO2. (2) Digest and count the cells in the culture dish, and spread 10,000 cells per well into a 96-well plate and culture overnight until the cells adhere. (3) When the cell density is about 80%, perform transfection by adding 0.5 μg of mRNA sample and Lipofectamine Messenger MAX Transfection Reagent (Invitrogen) to each well. Follow the instructions for transfection steps. (4) After transfecting the cells, they were cultured at 37°C and 5% CO2 for 24 hours. The growth medium was then removed from the cells and the cells were washed with PBS. After centrifugation to remove the PBS, 50 μL of 1x lysis buffer was added. The cells and all liquids were then transferred to a microcentrifuge tube and centrifuged. (5) Take 20 μL of sample, add 100 μL of Dual-Lumi™ II firefly luciferase detection reagent that has been equilibrated to room temperature, and mix well. (6) Incubate at room temperature (approximately 25ºC) for 5 minutes to allow the luminescence signal to stabilize. Perform chemiluminescence detection using a multi-functional microplate reader with chemiluminescence detection function and record the data. 2. Experimental Results The relative fluorescence readings of capped mRNAs are shown in Table 5. The relative fluorescence intensity is directly proportional to the translation efficiency of the mRNA. Table 5. Relative fluorescence readings of capped mRNA name Relative fluorescence intensity It is a multiple of m6A YK-CAP-101 0.22 0.6 YK-CAP-102 0.34 0.9 YK-CAP-103 0.77 2.0 YK-CAP-104 0.81 2.1 YK-CAP-105 0.72 1.9 YK-CAP-106 1.53 4.0 YK-CAP-107 1.62 4.3 YK-CAP-108 1.43 3.8 YK-CAP-109 1.53 4.0 YK-CAP-110 1.95 5.1 YK-CAP-111 2.12 5.6 YK-CAP-112 1.74 4.6 YK-CAP-113 1.63 4.3 YK-CAP-114 1.45 3.8 YK-CAP-115 1.69 4.4 YK-CAP-116 1.39 3.7 YK-CAP-117 1.82 4.8 YK-CAP-118 1.65 4.3 YK-CAP-119 1.45 3.8 5227 0.82 2.2 CAP-2'O-ethyl 1.13 3.0 N-7113 1.00 2.6 Compound 14 1.12 2.9 HN3002 1.13 3.0 m6A 0.38 1.0 1) The ribose-modified capped analogues in this application exhibit significant differences in mRNA translation efficiency, with YK-CAP-106~119 showing significantly higher translation efficiency than YK-CAP-101~105. YK-CAP-111 demonstrates the strongest translation efficiency, being 9.6 times that of the lowest, YK-CAP-101. As shown in Table 5, the relative fluorescence intensities (corresponding to mRNA translation efficiency) of the ribose-modified capped analogs in this application differed significantly. YK-CAP-106–119 all exhibited relatively high relative fluorescence intensities (between 1.4 and 2.2), with YK-CAP-107, YK-CAP-110, YK-CAP-111, YK-CAP-117, and YK-CAP-118 showing relative fluorescence intensities of 1.62, 1.95, 2.12, 1.82, and 1.65, respectively. YK-CAP-111 had the highest intensity at 2.12, followed by YK-CAP-110 at 1.95. The lowest relative fluorescence intensity was found in YK-CAP-101, at only 0.22. The relative fluorescence intensities of YK-CAP-102 to 105 were also very low, at 0.34, 0.77, 0.81, and 0.72, respectively. The relative fluorescence intensities of YK-CAP-111 were 9.6 times, 6.2 times, 2.8 times, 2.6 times, and 2.9 times that of YK-CAP-001 to 105, respectively, while the relative fluorescence intensities of YK-CAP-110 were 8.9 times, 5.7 times, 2.5 times, 2.4 times, and 2.7 times that of YK-CAP-001 to 105, respectively (Figure 3). 2) The ribose-modified capped analogues of this application exhibit significantly improved mRNA translation efficiency compared to existing ribose-modified capped analogues. For example, the translation efficiency of YK-CAP-111 reached 5.6 times that of m6A. The relative fluorescence intensities (corresponding to mRNA translation efficiency) of N-7113, HN3002, compound 14, and m6A were 1.00, 1.12, 1.13, and 0.38, respectively. The relative fluorescence intensities of YK-CAP-111 in this application were 2.1 times, 1.9 times, 1.9 times, and 5.6 times that of N-7113, HN3002, compound 14, and m6A, respectively. The relative fluorescence intensities of YK-CAP-110 in this application were 1.9 times, 1.7 times, 1.7 times, and 5.1 times that of N-7113, HN3002, compound 14, and m6A, respectively. 3) Riboso-modified capped analogs with similar structures have vastly different mRNA translation efficiencies, making it impossible to infer mRNA translation efficiency based on structure. The structures of the ribose-modified capped analogs designed in this application, YK-CAP-117~119, are very similar, and this series of compounds is also very similar to 5227. However, their mRNA translation efficiencies differ significantly. For example, the only difference between compounds YK-CAP-117~119 and 5227 is the substituent at C4 on the first sugar ring; that is, the C4 substituent in 5227 is methoxy, while the C4 substituents in YK-CAP-117~119 are methoxymethyl, 1-fluoromethyl, and difluoromethyl, respectively, with the other structures being identical. However, the mRNA translation efficiencies of YK-CAP-117~119 are 2.2 times, 2.0 times, and 1.8 times that of 5227, respectively, showing a significant improvement. Similarly, the structures of the ribose-modified capped analogs designed in this application, YK-CAP-113~116, are very similar, and this series of compounds is also very similar to the structure of CAP-2'O-ethyl. However, their mRNA translation efficiencies differ greatly. For example, the C2 substituent on the second sugar ring of compounds YK-CAP-113~116 in this application differs from that of CAP-2'O-ethyl only in that the C2 substituent of CAP-2'O-ethyl is ethoxy; the C2 substituents of YK-CAP-113~116 are methoxymethyl, acetaminomethyl, 1-fluoromethyl, and difluoromethyl, respectively, while the other structures are completely identical. However, the mRNA translation efficiencies of YK-CAP-113~116 are 1.4 times, 1.3 times, 1.5 times, and 1.2 times that of CAP-2'O-ethyl, respectively, showing a significant improvement. Therefore, it can be seen that the translation efficiency of luciferase mRNAs with similar structures and ribose modifications is not necessarily similar; on the contrary, they are very likely to differ greatly. The translation efficiency of different capped luciferase mRNAs shows that the ribose-modified capped analogs in this application, including YK-CAP-106~119, exhibit significantly improved mRNA translation efficiency compared to structurally similar ribose-modified capped analogs (including YK-CAP-101~105, 5227, CAP-2'O-ethyl, N-7113, and HN3002) and structurally different ribose-modified capped analogs (including compounds 14 and m6A). This indicates that the modified ribose of YK-CAP-106~119 binds more readily to the cap-binding protein (EIF4E), thereby improving the translation efficiency of the target mRNA. Furthermore, it is not necessarily true that structurally similar ribose-modified capped analog luciferase mRNAs have similar translation efficiencies; on the contrary, they are highly likely to differ significantly.

[0543] Example 5. Stability test of uncapped enzyme 1. Experimental Methods 30 pmol RNAs purified by polyacrylamide gel electrophoresis (PAGE) were reacted with 50 U mRNA uncapping enzyme (New England Biolabs) and 1×MDE buffer at 37°C for 45 min. The reactants were then subjected to PAGE electrophoresis and stained with SYBR Green II (Lonza). The gel images were observed on a Typhoon FLA7000 (GE Healthcare) instrument. The ratio of capped RNA to uncapped RNA band intensity was calculated using ImageQuant (GE Healthcare) software to determine the uncapping rate of the uncapping enzyme. 2. Experimental Results Table 6. Decapping rate after decapping enzyme treatment name DCP2 enzyme uncapping rate (%) Reduction (%) compared to N-7113 YK-CAP-101 32.4 11.4 YK-CAP-102 41.2 2.6 YK-CAP-103 36.5 7.3 YK-CAP-104 46.1 -2.3 YK-CAP-105 33.9 9.9 YK-CAP-106 17.3 26.5 YK-CAP-107 11.5 32.3 YK-CAP-108 16.3 27.5 YK-CAP-109 17.3 26.5 YK-CAP-110 10.2 33.6 YK-CAP-111 11.5 32.3 YK-CAP-112 11.9 31.9 YK-CAP-113 15.3 28.5 YK-CAP-114 14.2 29.6 YK-CAP-115 15.3 28.5 YK-CAP-116 14.3 29.5 YK-CAP-117 9.8 34.0 YK-CAP-118 11.9 31.9 YK-CAP-119 11.4 32.4 5227 23.2 20.6 CAP-2'O-ethyl 28.3 15.5 N-7113 43.8 0.0 Compound 14 26.8 17.0 HN3002 23.3 20.5 m6A 33.8 10.0 1) The ribose-modified capped analogues in this application show significant differences in capping rates. The capping rates of YK-CAP-106~119 are significantly lower than those of YK-CAP-101~105. YK-CAP-117 has the lowest capping rate, which is 36.3% lower than that of YK-CAP-104, the highest rate. As shown in Table 6, the decapping rates of the ribose-modified cap analogs YK-CAP-101~119 in this application vary greatly. The decapping rates of YK-CAP-106~119 are all very low. Among them, the decapping rates of YK-CAP-107, YK-CAP-110, YK-CAP-111, YK-CAP-117 and YK-CAP-118 are 11.5%, 10.2%, 11.5%, 9.8% and 11.9%, respectively. The lowest decapping rate is that of YK-CAP-117, at only 9.8%, followed by YK-CAP-110 at 10.2%. YK-CAP-104 had the highest decapsulation rate at 46.1%, followed by YK-CAP-101, YK-CAP-102, YK-CAP-103, and YK-CAP-105 at 32.4%, 41.2%, 36.5%, and 33.9%, respectively. YK-CAP-117 showed a decapsulation rate 22.6%, 31.4%, 26.7%, 36.3%, and 24.1% lower than YK-CAP-101–105, respectively, while YK-CAP-110 showed a decapsulation rate 22.2%, 31.0%, 26.3%, 35.9%, and 23.7% lower than YK-CAP-101–105 (Figure 4). 2) The ribose-modified capped analogues of this application have a significantly lower decapping rate compared to ribose-modified capped analogues with similar or significantly different structures in the prior art. For example, the decapping rate of YK-CAP-117 is 34.0% lower than that of N-7113. The decapping rates of N-7113, Compound 14, HN3002, and m6A were 43.8%, 26.8%, 23.3%, and 33.8%, respectively. In this application, the decapping rate of YK-CAP-117 was reduced by 34.0%, 17.0%, 13.5%, and 24.0% compared to N-7113, Compound 14, HN3002, and m6A, respectively, and the decapping rate of YK-CAP-110 was reduced by 33.6%, 16.6%, 13.1%, and 23.6% compared to N-7113, Compound 14, HN3002, and m6A, respectively. 3) Riboside-modified capping analogs with similar structures have vastly different uncapping rates, making it impossible to infer the mRNA capping rate based on their structure. The structures of the ribose-modified capped analogs designed in this application, YK-CAP-117~119, are very similar, and this series of compounds is also very similar to 5227. However, their mRNA uncapping rates differ significantly. For example, the only difference between compounds YK-CAP-117~119 and 5227 is the substituent at C4 on the first sugar ring; that is, the C4 substituent in 5227 is methoxy, while the C4 substituents in YK-CAP-117~119 are methoxymethyl, 1-fluoromethyl, and difluoromethyl, respectively, with the other structures being identical. However, the mRNA uncapping rates of YK-CAP-117~119 are significantly lower than those of 5227, decreasing by 13.4%, 11.3%, and 11.8%, respectively. Similarly, the structures of the ribose-modified capped analogs designed in this application, YK-CAP-113~116, are very similar, and this series of compounds is also very similar to the structure of CAP-2'O-ethyl. However, the mRNA uncapping rates differ greatly. For example, the C2 substituent on the second sugar ring of compounds YK-CAP-113~116 in this application differs from that of CAP-2'O-ethyl only in that the C2 substituent of CAP-2'O-ethyl is ethoxy; the C2 substituents of YK-CAP-113~116 are methoxymethyl, acetaminomethyl, 1-fluoromethyl, and difluoromethyl, respectively, while the other structures are completely identical. However, the mRNA uncapping rates of YK-CAP-113~116 are significantly lower than those of CAP-2'O-ethyl, decreasing by 13.0%, 14.1%, 13.0%, and 14.0%, respectively. Therefore, it can be seen that ribose-modified capped analogs with similar structures do not necessarily have similar capping rates; on the contrary, they are very likely to have huge differences. As can be seen from the decapping rate of the DCP2 enzyme, the ribose-modified capped analogs in this application, including YK-CAP-106~119, show significantly lower decapping rates compared to structurally similar ribose-modified capped analogs (including YK-CAP-101~105 in this application, as well as 5227, CAP-2'O-ethyl, N-7113, and HN3002 in the prior art) and ribose-modified capped analogs with significantly different structures (including compounds 14 and m6A). Furthermore, structurally similar ribose-modified capped analogs do not necessarily have similar decapping rates; on the contrary, they are very likely to differ greatly.

[0544] Example 6: Animal Experiment 1. Experimental Methods LNP formulation containing 5 μg of Fluc-mRNA transcribed from a capped analog was intramuscularly injected into 4-6 week old female BALB / c mice weighing 17-19 g. At specific time points after administration (6 h, 12 h, 24 h, 48 h, 96 h, and 168 h), mice were intraperitoneally injected with a fluorescence imaging substrate. The mice were allowed to move freely for 5 min. The total radiation intensity (corresponding to the protein expression level) of the protein expressed by the LNP-carried mRNA in the mice was then detected using an IVIS Spectrum small animal in vivo imaging system. 2. Experimental Results The test results are shown in Table 7. In the mouse in vivo imaging experiment, the multiples of the total radiation intensity of each group of mice relative to the m6A group are shown in Table 8 (where the total radiation intensity is the value mentioned × 10⁸ p / s). Table 7. Mouse in vivo imaging experimental data name Total radiation intensity (×10⁸ p / s) 6h 12h 24h 48h 96h 168h YK-CAP-101 1.81 0.96 0.36 0.19 0.04 0.02 YK-CAP-106 5.88 6.85 1.76 0.53 0.11 0.03 YK-CAP-107 8.09 6.87 1.92 0.60 0.11 0.03 YK-CAP-108 6.42 6.03 1.70 1.04 0.19 0.02 YK-CAP-109 6.04 5.84 1.85 0.54 0.14 0.03 YK-CAP-110 9.52 10.55 3.04 2.30 0.27 0.04 YK-CAP-111 8.61 9.34 2.75 1.95 0.27 0.03 YK-CAP-112 8.90 7.48 2.44 1.43 0.30 0.03 YK-CAP-113 6.07 7.63 2.02 1.29 0.31 0.04 YK-CAP-114 8.89 5.76 2.31 1.51 0.29 0.03 YK-CAP-115 6.20 5.42 1.85 1.21 0.25 0.02 YK-CAP-116 9.13 6.54 1.98 1.45 0.27 0.03 YK-CAP-117 8.99 8.00 2.08 1.73 0.35 0.02 YK-CAP-118 7.06 6.28 2.03 1.33 0.35 0.02 YK-CAP-119 7.47 6.65 2.15 1.26 0.25 0.02 5227 2.38 1.45 0.53 0.48 0.07 0.02 CAP-2'O-ethyl 5.41 4.80 1.73 0.57 0.09 0.02 N-7113 5.10 4.33 1.49 0.49 0.05 0.02 m6A 2.35 2.12 0.66 0.52 0.08 0.02 Table 8. Total radiation intensity relative to m6A multiples name 6h 12h 24h 48h 96h YK-CAP-101 0.8 0.5 0.5 0.4 1.5 YK-CAP-106 2.5 3.2 2.7 1.0 2.6 YK-CAP-107 3.4 3.2 2.9 1.2 2.5 YK-CAP-108 2.7 2.8 2.6 2.0 1.3 YK-CAP-109 2.6 2.8 2.8 1.0 2.7 YK-CAP-110 4.1 5.0 4.6 4.4 1.0 YK-CAP-111 3.7 4.4 4.2 3.8 1.1 YK-CAP-112 3.8 3.5 3.7 2.8 1.3 YK-CAP-113 2.6 3.6 3.1 2.5 1.2 YK-CAP-114 3.8 2.7 3.5 2.9 1.2 YK-CAP-115 2.6 2.6 2.8 2.3 1.2 YK-CAP-116 3.9 3.1 3.0 2.8 1.1 YK-CAP-117 3.8 3.8 3.2 3.3 0.9 YK-CAP-118 3.0 3.0 3.1 2.6 1.2 YK-CAP-119 3.2 3.1 3.3 2.4 1.3 5227 1.0 0.7 0.8 0.9 0.9 CAP-2'O-ethyl 2.3 2.3 2.6 1.1 2.4 N-7113 2.2 2.0 2.3 0.9 2.4 m6A 1.0 1.0 1.0 1.0 1.0 1) The ribose-modified capped analogues in this application showed significant differences in the total radiation intensity and duration of the proteins expressed by their mRNAs in mice, with YK-CAP-106~119 being significantly higher than YK-CAP-101~105. YK-CAP-110 had the highest total radiation intensity, which was 11.0 times and 12.1 times higher than the lowest, YK-CAP-101, at 12h and 48h, respectively. As can be seen from the data in Table 7, the total radiation intensity of proteins expressed by different ribose-modified cap analogs mRNAs in mice varied greatly. The total radiation intensity in in vivo imaging of mice with YK-CAP-106~119 mice was very high, with YK-CAP-110 having the highest total radiation intensity, reaching 10.55×10⁸ p / s at 12h and still reaching 2.30×10⁸ p / s at 48h. YK-CAP-111 was the second highest, reaching 9.34×10⁸ p / s at 12h and still reaching 1.95×10⁸ p / s at 48h. YK-CAP-101 has the lowest total radiation intensity, at 0.96 × 10⁸ p / s over 12 hours and only 0.19 × 10⁸ p / s over 48 hours. YK-CAP-110's total radiation intensity is 11.0 times that of YK-CAP-101 over 12 hours and 12.1 times over 48 hours. YK-CAP-111's total radiation intensity is 9.7 times that of YK-CAP-101 over 12 hours and 10.3 times over 48 hours. 2) The ribose-modified capped analogue of this application significantly increases the total radiation intensity and duration of the protein expressed by the mRNA in mice compared with existing ribose-modified capped analogues that are structurally similar or significantly different. For example, the total radiation intensity of YK-CAP-110 is 5.0 times that of m6A at 12h and 4.4 times that at 48h. The total radiation intensities of N-7113 and m6A were 4.33×10⁸ p / s and 2.12×10⁸ p / s, respectively, over 12 hours, and 0.49×10⁸ p / s and 0.52×10⁸ p / s, respectively, over 48 hours. The total radiation intensity of YK-CAP-110 in this application is 2.4 times and 5.0 times that of N-7113 and m6A, respectively, at 12h, and 4.7 times and 4.4 times, respectively, at 48h. The total radiation intensity of YK-CAP-111 in this application is 2.2 times and 4.4 times that of N-7113 and m6A, respectively, at 12h, and 4.0 times and 3.8 times, respectively, at 48h. 3) Ribosaccharide-modified analogs with similar structures exhibit vastly different total radiation intensity and duration of protein expression in mice, making it impossible to infer the total radiation intensity and duration of protein expression in mice based on their structures. The structures of the ribose-modified capped analogs designed in this application, YK-CAP-117~119, are very similar, and this series of compounds is also very similar to 5227. However, the total radiation intensity of the proteins expressed by the mRNA in mice varies greatly. For example, the compounds YK-CAP-117~119 in this application differ from 5227 only in the substituent group at C4 on the first sugar ring; that is, the C4 substituent of 5227 is methoxy, while the C4 substituents of YK-CAP-117~119 are methoxymethyl, 1-fluoromethyl, and difluoromethyl, respectively, with the other structures being completely identical. However, the total radiation intensity of the proteins expressed by YK-CAP-117~119 mRNA in mice after 6 hours is 3.8 times, 3.0 times, and 3.1 times that of 5227, respectively, showing a significant increase. Similarly, the structures of the ribose-modified capped analogs designed in this application, YK-CAP-113~116, are very similar, and this series of compounds is also very similar to the structure of CAP-2'O-ethyl. However, the total radiation intensity of the proteins expressed by the mRNA in mice varies greatly. For example, the C2 substituent on the second sugar ring of compounds YK-CAP-113~116 in this application differs from that of CAP-2'O-ethyl only in that the C2 substituent of CAP-2'O-ethyl is ethoxy; the C2 substituents of YK-CAP-113~116 are methoxymethyl, acetaminomethyl, 1-fluoromethyl, and difluoromethyl, respectively, while the other structures are completely identical. However, the total radiation intensity of the proteins expressed by YK-CAP-114 and YK-CAP-116 mRNA in mice after 6 hours is 1.6 times and 1.7 times that of CAP-2'O-ethyl, respectively, which is significantly higher. Therefore, it can be seen that Fluc mRNA prepared from structurally similar ribose-modified capped analogs does not necessarily have similar protein expression levels and durations in mice; on the contrary, they are very likely to differ greatly. Animal experiments show that the ribonucleotide-modified capped analogs in this application, such as YK-CAP-106~119, significantly increase the protein expression level and duration of mRNA in mice, whether compared with structurally similar ribonucleotide-modified capped analogs (including YK-CAP-101~105 in this application, as well as 5227, N-7113 and CAP-2'O-ethyl in the prior art) or with structurally different ribonucleotide-modified capped analogs (m6A). In vivo experiments further demonstrated that the mRNA transcribed from YK-CAP-106~119 in this application can be effectively delivered into the body by the LNP delivery vector and expressed efficiently and sustainably. Furthermore, Fluc mRNA prepared from structurally similar ribose-modified capped analogs does not necessarily have similar protein expression levels and durations in mice; on the contrary, they are very likely to differ significantly. In summary, the ribose-modified capping analogs YK-CAP-106~119 of this application significantly improve mRNA in vitro transcription yield, capping rate, mRNA translation efficiency, uncapping enzyme stability, and protein expression levels and duration in animals compared to existing ribose-modified capping analogs (including 5227, CAP-2'O-ethyl, N-7113, compound 14, HN3002, and m6A). This indicates that the YK-CAP-106~119 capping structure provided by this invention can significantly improve the tolerance of modified ribose structures to uncapping enzymes and their binding affinity to capping enzymes, providing a novel and efficient ribose-modified capping structure for in vitro mRNA transcription. 1. The chemical structures of the compounds in this application are very similar. Compared with the mRNA capping analogs disclosed in the prior art, some of these compounds are similar in structure, while others are very different. 1) In this application, the first sugar ring of compounds YK-CAP-101 and YK-CAP-102 is a six-membered ring, and N7113 is a five-membered sugar ring, and the other structures are completely the same. 2) The first sugar ring and the guanine-linked group of compound YK-CAP-103 in this application are different from those of N7113, that is, there is an extra methylene group at the C1 position, but the other structures are exactly the same. 3) In this application, compounds YK-CAP-104~106 have two substituents on the first sugar ring at C3, which is different from N7113. Specifically, the C3 substituents of N7113 are hydroxyl and hydrogen; the C3 substituents of YK-CAP-104 are dimethylamine methyl and fluorine, the C3 substituents of YK-CAP-105 are cyano and methyl, and the C3 substituents of YK-CAP-106 are acetaminophen and methyl. The other structures are completely the same. 4) The C3 substituent on the first sugar ring of compound YK-CAP-107~112 in this application is different from that of N7113 and HN3002. That is, the C3 substituent of N7113 and HN3002 is hydroxyl and methoxymethyl; the C3 substituent of YK-CAP-107~112 is 1-methoxyethyl, 1-acetaminoethyl, 1-fluoroethyl, difluoromethyl, N,N-diacetamino, N,N-di-n-propylacetamino, and the other structures are exactly the same. 5) The C2 substituents on the second sugar ring of compounds YK-CAP-113~116 in this application are different from those of N7113 and CAP-2'O-ethyl. That is, the C2 substituents of N7113 and CAP-2'O-ethyl are methoxy and ethoxy, respectively; the C2 substituents of YK-CAP-113~116 are methoxymethyl, acetaminomethyl, 1-fluoromethyl, and difluoromethyl, respectively, and the other structures are completely the same. 6) The C4 substituents on the first sugar ring of compounds YK-CAP-117~119 in this application are different from those of N7113 and 5227. That is, the C4 substituents of N7113 and 5227 are hydrogen and methoxy, respectively; the C4 substituents of YK-CAP-117~119 are methoxymethyl, 1-fluoromethyl and difluoromethyl, respectively, and the other structures are exactly the same. 7) The compounds YK-CAP-101~119 of this application have significant structural differences from compounds 14 and m6A. The first sugar ring of compound 14 is a locked nucleic acid sugar ring, that is, there is a methylene bridge between 2'-O and C4'; the second base of m6A, adenine, is modified by methylation. 2. The ribose-modified capping analogues in this application exhibit significant differences in mRNA in vitro transcription yield and capping rate. Compared with existing ribose-modified capping analogues, the ribose-modified capping analogues in this application show significantly improved mRNA in vitro transcription yield and capping rate. 1) The ribose-modified capping analogs in this application showed significant differences in in vitro mRNA transcription yield and capping rate. The in vitro transcription yield and capping rate of YK-CAP-106~119 mRNA were significantly higher than those of YK-CAP-101~105. YK-CAP-111 had the highest transcription yield and capping rate, with a transcription yield 5.3 times that of the lowest YK-CAP-101 and a capping rate 3.2 times that of the lowest YK-CAP-102. 2) The ribose-modified capping analogues in this application significantly improve both the in vitro transcription yield and capping rate of mRNA compared to existing ribose-modified capping analogues. For example, the transcription yield of YK-CAP-111 is 40.8% higher than that of compound 14, and the capping rate is 36.4% higher than that of 5227. 3) Riboside-modified capping analogs with similar structures exhibit significant differences in mRNA in vitro transcription yield and capping rate. For example, the in vitro transcription yield of YK-CAP-117~119 mRNA increased by 14.2%, 28.3%, and 14.9% compared to 5227, respectively, while the capping rate increased by 32.3%, 35.2%, and 33.9%, respectively. 3. The ribose-modified capped analogues in this application exhibit significantly different mRNA translation efficiency. Compared with existing ribose-modified capped analogues, the ribose-modified capped analogues in this application demonstrate significantly improved mRNA translation efficiency. 1) The ribose-modified capped analogues in this application exhibit significant differences in mRNA translation efficiency, with YK-CAP-106~119 showing significantly higher translation efficiencies than YK-CAP-101~105. YK-CAP-111 has the highest translation efficiency, being 9.6 times that of the lowest, YK-CAP-101. 2) The ribose-modified capped analogues of this application exhibit significantly improved mRNA translation efficiency compared to existing ribose-modified capped analogues with similar or significantly different structures. For example, the translation efficiency of YK-CAP-111 is 5.6 times that of m6A. 3) Riboside-modified capped analogs with similar structures exhibit vastly different mRNA translation efficiencies. For example, the translation efficiencies of YK-CAP-117~119 mRNAs are 2.2 times, 2.0 times, and 1.8 times that of 5227, respectively. 4. The ribose-modified capped analogs in this application exhibit significantly different decapping rates. Compared with ribose-modified capped analogs with similar or significantly different structures in the prior art, the decapping rates of the ribose-modified capped analogs in this application are all significantly reduced. 1) The ribose-modified capped analogues in this application exhibit significant differences in decapping rates. The decapping rates of YK-CAP-106~119 are significantly lower than those of YK-CAP-101~105. YK-CAP-117 has the lowest decapping rate, which is 36.3% lower than that of YK-CAP-104, which has the highest rate. 2) The ribose-modified capped analogues of this application exhibit significantly lower decapping rates compared to ribose-modified capped analogues with similar or significantly different structures in the prior art. For example, the decapping rate of YK-CAP-117 is 34.0% lower than that of N-7113. 3) Ribosyl-modified capping analogs with similar structures exhibit vastly different uncapping rates. For example, the uncapping rates of YK-CAP-113~116 mRNA were reduced by 13.0%, 14.1%, 13.0%, and 11.0% compared to CAP-2'O-ethyl, respectively. 5. The ribose-modified capped analogues of this application exhibit significant differences in the total radiation intensity (corresponding to protein expression level) and duration of mRNA expression in mice. Compared with existing ribose-modified capped analogues with similar or significantly different structures, the ribose-modified capped analogues of this application significantly enhance both the mRNA expression level and duration in mice. 1) The ribose-modified capped analogues in this application showed significant differences in the total radiation intensity and duration of the proteins expressed by their mRNAs in mice, with YK-CAP-106~119 being significantly higher than YK-CAP-101~105. YK-CAP-110 had the highest total radiation intensity, which was 11.0 times and 12.1 times higher than the lowest, YK-CAP-101, at 12h and 48h, respectively. 2) Compared with existing riboglycolic-modified capped analogs, the riboglycolic-modified capped analogs of this application significantly increase the total radiation intensity and duration of protein expression in mice. For example, the total radiation intensity of YK-CAP-110 is 5.0 times that of m6A at 12h and 4.4 times that at 48h. 3) Ribosaccharide-modified analogues with similar structures exhibit significant differences in the total radiation intensity and duration of proteins expressed by their mRNAs in mice. For example, the total radiation intensity of proteins expressed by YK-CAP-117~119 mRNAs in mice after 6 hours was 3.8, 3.0, and 3.1 times that of 5227, respectively.

[0545] The applicant declares that this invention illustrates the ribose-modified capped analogues and their applications through the above embodiments, but this invention is not limited to the above embodiments, that is, it does not mean that this invention must rely on the above embodiments to be implemented. Those skilled in the art should understand that any improvements to this invention, equivalent substitutions of the raw materials of the product, addition of auxiliary components, and selection of specific methods, etc., all fall within the protection and disclosure scope of this invention.

Claims

1. A ribose-modified capped analogue or a pharmaceutically acceptable salt thereof, wherein, The ribose-modified capped analogues are those with the following structures: YK-CAP-106, YK-CAP-107, YK-CAP-108, YK-CAP-109, YK-CAP-110, YK-CAP-111, YK-CAP-112, YK-CAP-113, YK-CAP-114, YK-CAP-115, YK-CAP-116, YK-CAP-117, YK-CAP-118, or YK-CAP-119.

2. Use of a ribose-modified capping analogue as described in claim 1 or a pharmaceutically acceptable salt thereof in the preparation of an in vitro co-transcribed mRNA capping reagent.

3. An RNA molecule, wherein, It comprises a ribose-modified capped analogue as described in claim 1 or a pharmaceutically acceptable salt thereof as a cap structure or a cap structure fragment.

4. A pharmaceutical composition, wherein, It contains RNA molecules as described in claim 3.

5. The pharmaceutical composition as claimed in claim 4, wherein, The pharmaceutical composition further includes at least one RNA delivery agent.

6. The pharmaceutical composition as claimed in claim 5, wherein, The at least one RNA delivery agent includes at least one cationic lipid.

7. The pharmaceutical composition as claimed in claim 6, wherein, The cationic lipid is selected from one or more of the following compounds: (1) a compound represented by formula (II), or a pharmaceutically acceptable salt thereof, wherein G1 is a C1-6 alkylene; G2 is a C2-8 alkylene; G3 is a C1-3 alkylene; L1 is a C6-15 straight-chain alkyl; L2 is a C12-25 branched-chain alkyl; (II) (2) The compound shown in formula (III), or a pharmaceutically acceptable salt thereof, wherein G1 is a C2-8 alkylene group; G2 is a C2-8 alkylene group; L1 is -C(O)O- or -OC(O)-; L2 is -C(O)O- or -OC(O)-; R1 is a C6-25 straight-chain or branched alkyl group; R2 is a C6-25 straight-chain or branched alkyl group; 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-; (III) (3) The compound shown in formula (IV), or a pharmaceutically acceptable salt thereof, wherein: G1 is a C1-6 alkylene; G2 is a C2-8 alkylene; R1 is a C6-20 straight-chain or branched alkyl; R2 is a C12-25 branched alkyl; G3 is: HO(CH2)2N(CH3)(CH2)2-, HO(CH2)2N(CH2CH3)(CH2)2-, (HO(CH2)2)2N(CH2)2-, CH3O(CH2)2N(CH3)(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) The compound shown in formula (V), or a pharmaceutically acceptable salt thereof, wherein G1 is a C1-8 alkylene group; G2 is a C2-8 alkylene group; R1 is a C6-25 straight-chain or branched alkyl group; R2 is a C12-25 straight-chain or branched alkyl group; G3 is: HO(CH2)2N(R3)CH2CH(OH)CH2-, wherein R3 is -CH3 or -CH2CH3 or -CH2CH2OH; (V) (5) The compound shown in formula (VI), or a pharmaceutically acceptable salt thereof, wherein G1 and G2 are each independently unsubstituted C6-C10 alkylene; G3 is an unsubstituted C1-C12 alkylene; R1 and R2 are each independently C6-C24 alkyl or C6-C24 alkenyl; R3 is -OR5, -C(=O)OR4, -OC(=O)R4 or -NR5C(=O)R4; R4 is a C1-C12 hydrocarbon; and R5 is H or a C1-C6 hydrocarbon; (VI) (6) The compound shown in formula (VII), or a pharmaceutically acceptable salt thereof, wherein R4 is selected from -(CH2)nQ; Q is selected from the group consisting of -OH or -CN; n is 1, 2 or 3;(VII) (7) The compound represented by formula (VIII), or a pharmaceutically acceptable salt thereof, (VIII).

8. The pharmaceutical composition of claim 7, wherein the cationic lipid is selected from one or a combination of at least two of YK-009, YK-401, YK-305, ALC-0315, SM-102, and DLIN-MC3-DMA: , , , , , .

9. The pharmaceutical composition as claimed in claim 8, wherein, The cationic lipid is YK-009.

10. The pharmaceutical composition as claimed in claim 6, wherein, The at least one RNA delivery agent further comprises at least one neutral lipid.

11. The pharmaceutical composition as claimed in claim 10, wherein, The neutral lipids include one or a combination of at least two of phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, ceramide, or sterols.

12. The pharmaceutical composition as described in claim 11, wherein, The neutral lipids are selected from one or a combination of at least two of the following: 1,2-dilinyl-sn-glycerol-3-phosphocholine, 1,2-dimyristyl-sn-glycerol-3-phosphocholine, 1,2-dilinyl-sn-glycerol-3-phosphocholine, 1,2-dipalmityl-sn-glycerol-3-phosphocholine, 1,2-distearatelyl-sn-glycerol-3-phosphocholine, 1,2-diundecylyl-sn-glycerol-3-phosphocholine, 1-palmityl-2-oleyl-sn-glycerol-3-phosphocholine. - Phosphocholine, 1,2-di-O-octadecenyl-sn-glycerol-3-phosphate choline, 1-oleyl-2-cholesterolylhemisuccinyl-sn-glycerol-3-phosphate choline, 1-hexadecyl-sn-glycerol-3-phosphate choline, 1,2-dilininyl-sn-glycerol-3-phosphate choline, 1,2-diarachidonicyl-sn-glycerol-3-phosphate choline, 1,2-bis(docosahexaenoyl)-sn-glycerol-3-phosphate choline, 1,2-dioleyl-sn-glycerol-3-phosphate choline Alkylamines, 1,2-diphydanyl-sn-glycerol-3-phosphate ethanolamine, 1,2-distearatelyl-sn-glycerol-3-phosphate ethanolamine, 1,2-dilinylyl-sn-glycerol-3-phosphate ethanolamine, 1,2-dilininyl-sn-glycerol-3-phosphate ethanolamine, 1,2-diarachidonicyl-sn-glycerol-3-phosphate ethanolamine, 1,2-bis(docosahexaenoyl)yl-sn-glycerol-3-phosphate ethanolamine, 1,2-dioleyl-sn-glycerol-3-phosphate-rac-( Sodium 1-glycerol, dipalmitoylphosphatidylglycerol, palmitoyloleoylphosphatidylethanolamine, dipalmitoylphosphatidylethanolamine, dipalmitoylphosphatidylethanolamine, dimyristylphosphoethanolamine, 1-stearoyl-2-oleoyl-SN-glycerol-3-phosphatidylethanolamine, 1-stearoyl-2-oleoylphosphatidylcholine, sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoylphosphatidylcholine, lysophosphatidylcholine, lysophosphatidylethanolamine.

13. The pharmaceutical composition as described in claim 12, wherein, The neutral lipids are DOPE and / or DSPC.

14. The pharmaceutical composition as claimed in claim 10, wherein, The at least one RNA delivery agent further includes structural lipids.

15. The pharmaceutical composition as claimed in claim 14, wherein, The structural lipids are selected from one or a combination of at least two of the following: cholesterol, non-steroidal, sitosterol, ergosterol, campesterol, stigmasterol, brassinosteroid, tomatine, ursolic acid, α-tocopherol, and corticosteroids.

16. The pharmaceutical composition as claimed in claim 15, wherein, The structural lipid in question is cholesterol.

17. The pharmaceutical composition of claim 5, wherein, The at least one RNA delivery agent further comprises a polymeric conjugated lipid.

18. The pharmaceutical composition as claimed in claim 17, wherein, The polymeric conjugated lipid is selected from one or a combination of at least two of the following: distearate phospholipid ethanolamine polyethylene glycol 2000, dimyristyl glycerin-3-methoxy polyethylene glycol 2000, and methoxy polyethylene glycol ditetradecyl acetamide.

19. The pharmaceutical composition as claimed in any one of claims 4-18, wherein, The pharmaceutical composition also includes one or at least two cell-penetrating peptides.

20. A method for synthesizing mRNA molecules for non-disease diagnosis and treatment purposes, wherein, The procedure includes the following steps: co-incubating a ribose-modified capped analogue or its stereoisomer, or a pharmaceutically acceptable salt, as described in claim 1, with a polynucleotide template to perform template transcription.

21. A capped mRNA transcription reaction system for non-disease diagnosis and treatment purposes, wherein, include: (1) Ribosyl-modified capped analogues or stereoisomers thereof as described in claim 1, or pharmaceutically acceptable salts; (2) Polynucleotide templates, NTPs and RNA polymerase.

22. A reagent kit, wherein, include: (1) Ribosyl-modified capped analogues or stereoisomers thereof as described in claim 1, or pharmaceutically acceptable salts; (2) Nucleotide triphosphate molecules, and RNA polymerase.

23. The kit as claimed in claim 22, wherein, The kit further comprises one or a combination of at least two of the following: RNase inhibitor, inorganic pyrophosphatase, Mg2+, crowding agent, and buffer.

24. A method for improving the in vitro and intracellular stability of RNA, wherein, The method includes incorporating a ribose-modified capped analogue as described in claim 1 or a pharmaceutically acceptable salt thereof into RNA.

25. A method for introducing RNA into cells in vitro, wherein, The method includes contacting the cells with an RNA molecule as described in claim 3 or a pharmaceutical composition as described in any one of claims 4-19.

26. Use of an RNA molecule as described in claim 3 or a pharmaceutical composition as described in any one of claims 4-19 in the preparation of a vaccine.

Images