Modified ribose cap analog and its use.

Description

1 / 120 Modified ribose cap analog and its use. TECHNICAL FIELD

[0001] This disclosure pertains to the technical field of chemical and biological engineering and relates to a modified ribose cap analogue and a use thereof. FUNDAMENTALS

[0002] The chemical nature of a cap structure is a special structure at the 5' end of mRNA formed by modification during mRNA transcription, namely an m7GPPPN structure, also known as a methylguanosine cap. It is formed under the cocatalysis of RNA triphosphatase, guanylyltransferase, mRNA (guanine-N7) methyltransferase, and mRNA (nucleoside-2') methyltransferase. Depending on the degree of methylation, three types of caps can be formed, namely CAP0, CAP1, and CAP2, which are m7G5'ppp5'Np, m7G5'ppp5'NmpNp, and m7G5'ppp5'NmpNmpNp, respectively.

[0003] The cap structure is necessary for the initiation of mRNA translation, which provides a signal for mRNA recognition by the ribosome, assists the ribosome in binding to the mRNA, and allows translation to begin from AUG. Meanwhile, the cap structure can increase mRNA stability and protect the mRNA from 5'^3' exonuclease attack.

[0004] Simply put, the cap structure is like a steel helmet for mRNA, which can not only protect the mRNA from being destroyed, but also performs the helmet imprint through chemical modification to facilitate recognition by other members. In addition to the natural cap structure, cap structure analogs are also used primarily to improve the stability of mRNA structures during in vitro transcription, with ARCA and Cap1 structure analogs being the most common.

[0005] Studies have shown that the cap structure of mRNA is significantly linked to mRNA quality control and the body's innate immunity. Therefore, the invention of a new cap analog is of great importance for increasing mRNA stability and improving mRNA translation efficiency. CONTENT OF THE PRESENT INVENTION Petition 870250022825, dated 03 / 24 / 2025, p. 13 / 331 2 / 120

[0006] In response to the deficiencies of the prior art, the present disclosure aims to provide a modified ribose cap analog and an use thereof. The modified ribose cap analog of the present disclosure can improve mRNA stability and / or mRNA translation efficiency.

[0007] To achieve the previous objective, this disclosure adopts the following technical solutions:

[0008] A first aspect of the present disclosure provides a modified ribose cap analogue, or a stereoisomer, a pharmaceutically acceptable salt or a solvate thereof, wherein the modified ribose cap analogue has a structure of formula (I):

[0010] where n is selected from 1 or 0; m is selected from 1 or 0;

[0011] Xi is selected from -C(OH)H- or a single bond; I

[0012] X2 is selected from -CH-, -O-, -CN, -C(O)-, N ,O or a single bond;

[0013] X3 is selected from -CH2, -CFH- or -O-;

[0014] Ri is selected from -OH or -NHAc;

[0015] R2 is selected from -CHs, -F, -H or absent;

[0016] R3 is selected from -OR6, -NHC(O)R7, -N(R8)2, -CN, -F, -C(O)N(R9)2, substituted or unsubstituted C1-C3 alkyl, -H or absent;

[0017] R4 is selected from -CH?, -F or -H;

[0018] R5 is selected from -OR10, -NHC(O)Rn, -N(Ri2)2, -CF2H, -F, -C(O)N(Ri3)2, alkyl C1-C3 replaced, not replaced, or missing;

[0019] Ró, R8, Rio, Rn, R12 and R13 are each independently selected from Petition 870250022825, dated 03 / 24 / 2025, p. 14 / 331 3 / 120 unsubstituted C1-C3 alkyl or -H;

[0020] R7 is selected from -OR14, -NHC(O)R15, -F, or substituted or unsubstituted C1-C3 alkyl;

[0021] R9 is selected from unsubstituted C2-C3 alkyl;

[0022] R14 and R15 are each independently selected from unsubstituted C1-C3 alkyl.

[0023] Compared with modified ribose cap analogs in the prior art, the modified ribose cap analogs of the present disclosure can result in a significant increase in in vitro mRNA transcription yield, capping rate, mRNA translation efficiency, and the amount and duration of protein expression per mRNA in mice, as well as a significant decrease in the decapping rate.

[0024] A second aspect of the present disclosure provides a use of the modified ribose cap analog, or stereoisomer, pharmaceutically acceptable salt or solvate thereof according to the first aspect of the present disclosure in the preparation of an in vitro cotranscription mRNA capping reagent.

[0025] A third aspect of the present disclosure provides an RNA molecule comprising the modified ribose cap analog, or stereoisomer, pharmaceutically acceptable salt or solvate thereof according to the first aspect of the present disclosure as a cap structure or a cap structure fragment.

[0026] A fourth aspect of the present disclosure provides a pharmaceutical composition comprising the RNA molecule according to the third aspect of the present disclosure.

[0027] A fifth aspect of the present disclosure provides a method for synthesizing an mRNA molecule for non-disease diagnostic and therapeutic purposes comprising the steps of: co-incubating the modified ribose cap analog, or stereoisomer, pharmaceutically acceptable salt or solvate thereof according to the first aspect of the present disclosure with a polynucleotide template for template transcription.

[0028] A sixth aspect of the present disclosure provides a capped mRNA transcription reaction system for non-disease diagnostic and therapeutic purposes, comprising: Petition 870250022825, dated 03 / 24 / 2025, page 15 / 331 4 / 120

[0029] (1) the modified ribose cap analogue, or the stereoisomer, the pharmaceutically acceptable salt or the solvate thereof according to the first aspect of this disclosure; and (2) a template of polynucleotide, NTPs (nucleoside triphosphates) and an RNA polymerase.

[0030] A seventh aspect of the present disclosure provides a kit comprising: (1) the modified ribose cap analogue, or stereoisomer, pharmaceutically acceptable salt or solvate thereof according to the first aspect of the present disclosure; and (2) a nucleotide triphosphate molecule and an RNA polymerase.

[0031] An eighth aspect of the present disclosure provides a method for improving the intracellular stability of an RNA, comprising incorporating the modified ribose cap analog, or stereoisomer, pharmaceutically acceptable salt or solvate thereof according to the first aspect of the present disclosure into the RNA.

[0032] A ninth aspect of the present disclosure provides a method for introducing RNA into a cell, comprising contacting the cell with the modified ribose cap analog, or the stereoisomer, the pharmaceutically acceptable salt or the solvate thereof according to the first aspect of the present disclosure, or the pharmaceutical composition according to the fourth aspect of the present disclosure.

[0033] A tenth aspect of the present disclosure provides a method for inhibiting RNA translation in a cell, comprising contacting the cell with the modified ribose cap analog, or the stereoisomer, the pharmaceutically acceptable salt or solvate thereof according to the first aspect of the present disclosure, or the pharmaceutical composition according to the fourth aspect of the present disclosure.

[0034] An eleventh aspect of the present disclosure provides for a use of the modified ribose cap analogue, or of the stereoisomer, of the pharmaceutically acceptable salt or of the solvate thereof according to the first aspect of the present disclosure, or of the pharmaceutical composition according to the fourth aspect of the present disclosure in the preparation of a vaccine.

[0035] In relation to the state of the art, the present disclosure has the following beneficial effects:

[0036] Compared to modified ribose cap analogs in the prior art, the modified ribose cap analogs of the present disclosure may result in an increase Petition 870250022825, dated 03 / 24 / 2025, page 16 / 331 5 / 120 significant in vitro mRNA transcription yield, capping rate, mRNA translation efficiency, and the amount and duration of protein expression per mRNA in mice, as well as a significant decrease in the decapping rate. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] FIG. 1 is a graph showing the results of in vitro mRNA transcription yield 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 cap analogs.

[0038] FIG. 2 is a graph showing the capping rate results 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 cap analogs.

[0039] FIG. Figure 3 shows the results of the cap mRNA relative fluorescence intensity assay 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 analogous cap.

[0040] FIG. 4 is a graph showing the results of the decapping rate of the DCP2 enzyme using YK-CAP-106, YK-CAP-107, YK-CAP-108, YK-CAP-109, YK-CAP-110, YK-CAP111, 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, YKCAP-105, 5227, CAP-2'O-ethyl, N-7113, compound 14, HN3002 and m6A as analogues of cap. DETAILED DESCRIPTION OF THE PREFERRED MODALITY

[0041] The technical solutions of this disclosure are further described below in specific embodiments. It should be understood by those skilled in the art that the examples Petition 870250022825, dated 03 / 24 / 2025, page 17 / 331 6 / 120 are for ease of understanding only and should not be construed as a specific limitation of this disclosure.

[0042] This disclosure may be incorporated into other specific forms without departing from the essential attributes of this disclosure. It should be understood that any and all embodiments of this disclosure may be combined with technical features in any other embodiment or a plurality of other embodiments to obtain additional embodiments under the premise of no conflict. This disclosure includes additional embodiments obtained from such combinations.

[0043] Except as exemplified or otherwise indicated, all numbers indicating quantitative properties, such as doses, in this disclosure shall be understood as modified in all cases by the term approximately. It shall also be understood that any numerical range cited in this disclosure is intended to include all subranges within the range and any combination of the various limits of the range or subranges.

[0044] Unless defined otherwise, all terms (including technical and scientific terms) used herein have the same meaning as generally understood by one skilled in the art to which the present invention pertains. Furthermore, terms as defined in commonly used dictionaries should be interpreted as having a 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 defined as such in this document.

[0045] As used herein, the term C1-C3 refers to a group with any integer number of carbon atoms within the range of 1 to 3 in the main chain, such as 1, 2, or 3 carbon atoms. The term C6-15 refers to a group with any integer number of carbon atoms within the range of 6 to 15, such as 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 carbon atoms. The limitation of other ranges of carbon atoms, and so forth, indicates that the limited number of carbon atoms in a group can be any integer value within the limited range.

[0046] As used herein, the term alkyl refers to a saturated aliphatic hydrocarbon group with a linear or branched chain; non-limiting examples include methyl, ethyl, propyl, isopropyl, etc. Petition 870250022825, dated 03 / 24 / 2025, p. 18 / 331 7 / 120

[0047] As used herein, the term salt refers to a corresponding salt of a modified nucleoside compound (or nucleotide compound) of this disclosure that can be conveniently or desirably prepared, purified and / or treated, such as a pharmaceutically acceptable salt. Unless otherwise indicated, references to a specific compound in this disclosure also include a salt form thereof.

[0048] As used herein, cap analog refers to a structure at the 5' end of a mature mRNA formed by post-transcriptional modification in eukaryotes, namely an m7GPPPN structure, also known as a methylguanosine cap. The structure can prevent mRNA degradation at the 5' end, help RNA transcripts pass through selective pores in the nuclear membrane and enter the cytoplasm, improve translation, and help complete the entire splicing process.

[0049] The words comprising, including or containing and similar words used in this disclosure mean that the element appearing before the word encompasses the elements listed after the word and their equivalents and does not exclude elements not recited. The term comprising or including (containing), as used in this document, may be open, semi-closed and closed. In other words, the term also includes consisting essentially of or consisting of.

[0050] The term pharmaceutically acceptable in this disclosure means that a compound or composition is chemically and / or toxicologically compatible with the other ingredients that make up the preparation and / or with the human or mammal in which it is used to prevent or treat a disease or condition.

[0051] The term solvate in this disclosure refers to a complex formed by combining a compound of formula (I) or a pharmaceutically acceptable salt thereof with a solvent (e.g., ethanol or water). It should be understood that any solvate of a compound of formula I for use in the treatment of a disease or condition may provide different properties (including pharmacokinetic properties), however, it will result in the compound of formula I after being absorbed in an individual, so that the use of the compound of formula I encompasses the use of any solvate of the compound of formula I, respectively.

[0052] It should also be understood that the compound of formula I or the pharmaceutically acceptable salt thereof can be isolated in the form of a solvate and therefore any solvate is Petition 870250022825, dated 03 / 24 / 2025, p. 19 / 331 8 / 120 included in the scope of this disclosure. For example, the compound of formula I or the pharmaceutically acceptable salt thereof may exist in a non-solvated form as well as a form solvated with a pharmaceutically acceptable solvent (e.g., water, ethanol).

[0053] This disclosure also includes a salt of the compound described herein, especially a pharmaceutically acceptable salt. The compounds in this disclosure having sufficiently acidic or sufficiently basic functional groups can react with a wide variety of bases or acids to form salts. Alternatively, compounds that are inherently charged (e.g., compounds with a quaternary nitrogen) can form salts with appropriate counter-ions (e.g., halide ions, such as bromide ions, chloride ions, or fluoride ions, especially bromide ions).

[0054] The pharmaceutically acceptable salt of the present disclosure may be, for example, an acid addition salt of the compound of the present disclosure that is sufficiently basic and contains a nitrogen atom in a chain or ring of the compound of formula (I), for example, an acid addition salt formed with an inorganic acid, such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, phosphoric acid or nitric acid, or an acid addition salt formed with an organic acid, such as formic acid, acetic acid, acetoacetic acid, pyruvic acid, trifluoroacetic acid, propionic acid, butyric acid, hexanoic acid, heptanoic acid, undecanoic acid, lauric acid, benzoic acid, salicylic acid, 2-(4-hydroxybenzoyl)benzoic acid, camphoric acid, cinnamic acid, cyclopentylpropionic acid, 3-hydroxy-2-naphthoic acid, nicotinic acid, pamoic acid, pectinic acid, persulfuric acid, 3-phenylpropionic acid, picric acid, pivalic acid,2-hydroxyethanesulfonic acid, itaconic acid, sulfamic acid, trifluoromethanesulfonic acid, dodecylsulfuric acid, ethanesulfonic acid, benzenesulfonic acid, ptoluenesulfonic acid, methanesulfonic acid, 2-naphthalenesulfonic acid, naphthalenedisulfonic acid, camphorsulfonic acid, citric acid, tartaric acid, stearic acid, lactic acid, oxalic acid, malonic acid, succinic acid, malic acid, adipic acid, alginic acid, maleic acid, fumaric acid, D-gluconic acid, mandelic acid, ascorbic acid, glucoheptanoic acid, glycerophosphoric acid, aspartic acid, sulfosalicylic acid, or thiocyanic acid. Petition 870250022825, dated 03 / 24 / 2025, page 20 / 331 9 / 120

[0055] In addition, another suitable pharmaceutically acceptable salt of the compound of the present disclosure that is sufficiently acidic 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 (for example, a salt formed with NH3 or ammonia water), or a salt formed with an organic base that provides a physiologically acceptable cation, for example, a salt formed with triethylamine, N-methylglucamine, dimethylglucamine, ethylglucamine, lysine, dicyclohexylamine, 1,6-hexanediamine, ethanolamine, glucosamine, sarcosine, serinol, tris(hydroxymethyl)aminomethane, aminopropanediol, 1-amino-2,3,4-butanetriol.Furthermore, groups containing basic nitrogen can be quaternized with the following reagents: lower alkyl halides, such as methyl, ethyl, propyl and butyl chlorides, bromides and iodides; dialkyl sulfates, such as dimethyl, diethyl, dibutyl and diamyl sulfates; long-chain halides, such as decyl, lauryl, myristyl and stearyl chlorides, bromides and iodides; aralkyl halides, such as benzyl and phenethyl bromides.

[0056] It will also be recognized by those skilled in the art that the acid addition salts of the compound of formula (I) of the present disclosure can be prepared by reacting the compound with a suitable inorganic or organic acid by any of the known methods. Alternatively, the base addition salts of the acid compound of the present disclosure are prepared by reacting the compound with a suitable base by various known methods.

[0057] This disclosure includes all possible salts of the compound of formula (I) of this disclosure, which may be a single salt or any mixture of the salt in any ratio.

[0058] Certain compounds of the present disclosure may exist as one or more stereoisomers. Stereoisomers include geometric isomers, diastereomers, and enantiomers. Consequently, the compound of formula (I) of the present disclosure also includes 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 lower side effects than other stereoisomers. Single stereoisomers and optically active mixtures may be obtained by methods such as chiral source synthesis, chiral catalysis, and chiral resolution. The racemate may be chirally resolved by chromatographic resolution or chemical resolution. For example, a chiral acid resolving reagent, Petition 870250022825, dated 03 / 24 / 2025, page 21 / 331 10 / 120, such as chiral tartaric acid and chiral malic acid, can be added to form a salt with the compound of the present disclosure, and the physicochemical properties of the product, such as the difference in solubility, can be used for separation.

[0059] In this disclosure, when the name of a compound is inconsistent with the structural formula, the structural formula shall prevail.

[0060] The descriptive report of this disclosure should be interpreted as consistent with the laws and principles of chemical bonding. In some cases, a hydrogen atom may be removed to accommodate a substituent at a given position.

[0061] It should be understood that the term compound of the present disclosure, as used herein, may include a compound of formula (I), a solvate thereof, a pharmaceutically acceptable salt thereof, a stereoisomer thereof or mixtures thereof, as the context requires.

[0062] A first aspect of the present disclosure provides a modified ribose cap analogue, or a stereoisomer, a pharmaceutically acceptable salt or a solvate thereof, wherein the modified ribose cap analogue has a structure of formula (I):

[0064] where n is selected from 1 or 0; m is selected from 1 or 0;

[0065] Xi is selected from -C(OH)H- or a single bond; I

[0066] X2 is selected from -CH-, -O-, -CN, -C(O)-, N ,O or a single bond;

[0067] X3 is selected from -CH2, -CFH- or -O-;

[0068] Ri is selected from -OH or -NHAc;

[0069] R2 is selected from -CHç -F, -H or absent; Petition 870250022825, dated 03 / 24 / 2025, p. 22 / 331 11 / 120

[0070] R3 is selected from -OR6, -NHC(O)R7, -N(R8)2, -CN, -F, -C(O)N(R9)2, substituted or unsubstituted C1-C3 alkyl, -H or absent;

[0071] R4 is selected from -CH3, -F or -H;

[0072] R5 is selected from -OR10, -NHC(O)Rn, -N(Ri2)2, -CF2H, -F, -C(O)N(Ri3)2, alkyl C1-C3 replaced, not replaced, or missing;

[0073] Rho, R8, Rio, Rn, Ri2 and R13 are each independently selected from unsubstituted C1-C3 alkyl or -H;

[0074] R7 is selected from -OR14, -NHC(O)Ris, -F, or substituted or unsubstituted C1-C3 alkyl;

[0075] R9 is selected from unsubstituted C2-C3 alkyl;

[0076] Rm and R15 are each independently selected from unsubstituted C1-C3 alkyl.

[0077] In one form, X2 is -O-.

[0078] In one embodiment, X2 is -O- and R2 is absent.

[0079] In one embodiment, X2 is -O- and R3 is -H.

[0080] In one embodiment, X2 is -O-, R2 is absent and R3 is -H.

[0081] In one form, X2 is -CH-.

[0082] In one form, X2 is -CH- and R2 is -H, -CH3 or -F.

[0083] In one embodiment, X2 is -CH- and R3 is -N(CH33)2, -C(O)CH3, -OCH3, -NHC(O)CH3, or -F.

[0084] In one embodiment, X2 is -CH-, R2 is -H, -CH3, or -F, and R3 is -N(CH3)2, -C(O)CH3, OCH3, -NHC(O)CH3, or -F.

[0085] In one form, X2 is -CN-.

[0086] In one modality, X2 is -CN and R2 is absent.

[0087] In one embodiment, X2 is -CN-, R2 is absent and R3 is absent.

[0088] In one embodiment, X2 is N

[0089] In one modality, X2 is

[0090] In one modality, X2 is I N—e R2 is missing. I N—e R3 is missing. Petition 870250022825, dated 03 / 24 / 2025, p. 23 / 331 12 / 120 I

[0091] In one embodiment, X2 is N, r2 is absent and R3 is absent.

[0092] In one embodiment, X2 is -C(O)-.

[0093] In one embodiment, X2 is -C(O)- and R2 is absent.

[0094] In one embodiment, X2 is -C(O)- and R3 is -N(CH2CH3)2 or -N(CH2CH2CH3)2.

[0095] In one embodiment, X2 is -C(O)-, R2 is absent and R3 is -N(CH2CH3)2 or N(CH2CH2CH3)2.

[0096] In one embodiment, X3 is -O-.

[0097] In one embodiment, X3 is -O- and R7 is -CH3 or -OCH3.

[0098] In one embodiment, X3 is -CH2-,

[0099] In one embodiment, X3 is -CH2- and R7 is -OCH3, -NHC(O)CH3, or -F.

[0100] In one modality, X3 is -CFH-.

[0101] In one embodiment, X3 is -CFH- and R7 is -F.

[0102] In one modality, m is 1.

[0103] In one embodiment, m is 1 and R5 is -OCH3, -F or -CF2H; preferably, m is 1 and R5 is -OCEE.

[0104] In one form, Rho is -H.

[0105] In one embodiment, the modified ribose cap analog is YK-CAP-101, YK-CAP102, 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 as shown below: Petition 870250022825, dated 03 / 24 / 2025, page 24 / 331 13 / 120 Oh oh

[0106] YK-CAP-101 Oh oh

[0107] YK-CAP-102 OH OH

[0108] YK-CAP-103 Petition 870250022825, dated 03 / 24 / 2025, page 25 / 331 14 / 120 OH OH

[0110] OH OH YK-CAP-105

[0112] OH OH YK-CAP-107 Petition 870250022825, dated 03 / 24 / 2025, page 26 / 331 15 / 120

[0113] YK-CAP-108 Oh oh

[0114] YK-CAP-109

[0115] OH OH YK-CAP-110

[0116] OH OH YK-CAP-111 Petition 870250022825, dated 03 / 24 / 2025, p. 27 / 331 16 / 120 Oh oh

[0117] YK-CAP-112

[0118] YK-CAP-113 OH ÕH YK-CAP-115 OH OH

[0120] Petition 870250022825, dated 03 / 24 / 2025, p. 28 / 331 17 / 120

[0121] YK-CAP-116 OH OH

[0122]

[0123] OH OH YK-CAP-117 OH OH YK-CAP-118 OH OH YK-CAP-119

[0124]

[0125] A second aspect of the present disclosure provides a use of the modified ribose cap analogue, or stereoisomer, of the pharmaceutically acceptable salt or solvate of Petition 870250022825, dated 03 / 24 / 2025, p. 29 / 331 18 / 120 same according to the first aspect of the present disclosure in the preparation of an in vitro cotranscription mRNA capping reagent.

[0126] A third aspect of the present disclosure provides an RNA molecule comprising the modified ribose cap analog, or stereoisomer, pharmaceutically acceptable salt or solvate thereof according to the first aspect of the present disclosure as a cap structure or a cap structure fragment.

[0127] A fourth aspect of the present disclosure provides a pharmaceutical composition comprising the RNA molecule according to the third aspect of the present disclosure.

[0128] In one embodiment, the pharmaceutical composition further comprises at least one RNA delivery agent.

[0129] The RNA delivery agent can be, for example, lipid nanoparticles (LNPs). Lipid nanoparticles are widely used in small molecule drugs and nucleic acid delivery. mRNA encapsulated by LNPs can be protected from extracellular ribonucleases and facilitates intracellular mRNA delivery. For lipid nanoparticles, see the review Chemistry of Lipid Nanoparticles for RNA Delivery. Acc Chem Res. 2022 Jan 4; 55(1): 2-12.

[0130] In one embodiment, at least one RNA delivery agent comprises at least one cationic lipid.

[0131] The term cationic lipid, as used herein, refers to a lipid that is positively charged at a selected pH value. For example, see cationic lipids disclosed in literature such as WO2023133946A1, CN115745820A and Chemistry of Lipid Nanoparticles for RNA Delivery. Acc Chem Res. 2022 Jan 4; 55(1): 2-12.

[0132] In one embodiment, the cationic lipid is selected from one or a combination of at least two of the following compounds:

[0133] (1) a compound of formula (II), or an N-oxide, a solvate, a pharmaceutically acceptable salt or a stereoisomer thereof, wherein G1 is C1-6 alkylene; G2 is C2-5 alkylene; G3 is C1-3 alkylene; L1 is C6-15 linear alkyl; L2 is C12-25 branched alkyl; Petition 870250022825, dated 03 / 24 / 2025, p. 30 / 331 19 / 120 OH I

[0134] O (II);

[0135] (2) a compound of formula (III), or an N-oxide, a solvate, a pharmaceutically acceptable salt or a stereoisomer thereof, wherein Gi is C2-8 alkylene; G2 is C2-8 alkylene; Li is -C(O)O- or -OC(O)-; L2 is -C(O)O- or -OC(O)-; Ri is linear or branched C-25 alkyl; R2 is linear or branched C-25 alkyl; G1 is HO(CH2)2- or HO(CH2)3-; G4 is HO(CH2)2- or HO(CH2)3-; L is (CH2)2-, -(CH2)3- or -(CH2)4-; g3 NG,—L,—R1 LZ N G2 l~2 R2

[0136] G4 (III);

[0137] (3) a compound of formula (IV), or an N-oxide, a solvate, a pharmaceutically acceptable salt or a stereoisomer thereof, wherein Gi is C1-6 alkylene; G2 is C2-8 alkylene; Ri is C2-20 linear or branched alkyl; R2 is C2-25 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-;

[0138] w(IV);

[0139] (4) a compound of formula (V), or an N-oxide, a solvate, a pharmaceutically acceptable salt or a stereoisomer thereof, wherein G1 is C1-8 alkylene; G2 is C2-8 alkylene; R1 is C1-25 linear or branched alkyl; R2 is C12-25 linear or branched alkyl; G3 is HO(CH2)2N(R3)CH2CH(OH)CH2-, wherein G1 is -CH3, -CH2CH3 or -CH2CH2OH; Petition 870250022825, dated 03 / 24 / 2025, page 31 / 331 20 / 120

[0140] G3xG N (V);

[0141] (5) a compound of formula (VI), or an N-oxide, a solvate, a pharmaceutically acceptable salt or a stereoisomer thereof, wherein G1 and G2 are each independently unsubstituted C1-C12 alkylene; G3 is 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 C1-C12 hydrocarbyl; and R5 is H or C1-C12 hydrocarbyl; R3. <G3 R1,

[0142] o 0 (VI);

[0143] (6) a compound of formula (VII), or an N-oxide, a solvate, a pharmaceutically acceptable salt or a stereoisomer thereof, 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)2Rs and heterocycle; n is 1, 2 or 3;

[0145] (7) a compound of formula (VIII), or an N-oxide, a solvate, a pharmaceutically acceptable salt or a stereoisomer thereof,

[0147] 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 or DLIN. Petition 870250022825, dated 03 / 24 / 2025, page 32 / 331 21 / 120 MC3-DMA:

[0149] YK-401

[0151]

[0152] I

[0153] DLIN-MC3-DMA

[0154] In a more preferred embodiment, the cationic lipid is YK-009. Petition 870250022825, dated 03 / 24 / 2025, page 33 / 331 22 / 120

[0155] In one embodiment, at least one RNA delivery agent additionally comprises a neutral lipid.

[0156] In the present disclosure, neutral lipid refers to an auxiliary lipid that is unloaded or exists in a zwitterionic form at a selected pH value. Neutral lipid can regulate the fluidity of nanoparticles in a lipid bilayer structure and improve efficiency by promoting lipid phase transition and can also affect target organ specificity.

[0157] In one embodiment, the neutral lipid includes one or a combination of at least two of phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, ceramide, sterol or their derivatives.

[0158] In one embodiment, the neutral lipid is selected from one or a combination of at least two of the following: 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoylsn-glycero-3-phosphocholine (DPPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-diundecanoyl-sn-glycero-3-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 Diether PC), 1-oleoyl-2cholesteryl-hemi succinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3 phosphocholine (C16 Lyso PC), 1,2-dilinolenoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl-sngglycero-3-phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-dioleoyl-snglycero-3-phosphoethanolamine (DOPE), 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME 16.0 PE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, sodium salt of 1,2-dioleoyl-sn-glycero-3-phosphorac-(1-glycerol) (DOPG), dipalmitoyl phosphatidylglycerol (DPPG), palmitoyl oleoyl phosphatidylethanolamine (POPE), distearoyl phosphatidyl ethanolamine (DSPE), dipalmitoyl phosphatidylethanolamine (DPPE), dimyristoyl phosphoethanolamine (DMPE), 1-stearoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (SOPE), 1-stearoyl-2-oleoyl-phosphatidylcholine (SOPC), sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyl oleoyl phosphatidylcholine, lysophosphatidylcholine or lysophosphatidylethanolamine (LPE).

[0159] In a preferred embodiment, the neutral lipid is DOPE and / or DSPC. Petition 870250022825, dated 03 / 24 / 2025, page 34 / 331 23 / 120

[0160] In one embodiment, at least one RNA delivery agent further comprises a structural lipid.

[0161] In this disclosure, structural lipid refers to a lipid that increases the stability of nanoparticles by filling the gaps between lipids.

[0162] In one embodiment, the structural lipid is selected from one or a combination of at least two of the following: cholesterol, non-sterol, sitosterol, ergosterol, campesterol, stigmasterol, brassinosterol, tomatine, ursolic acid, α-tocopherol, or corticosteroid.

[0163] In a preferred embodiment, the structural lipid is cholesterol.

[0164] In one embodiment, at least one RNA delivery agent further comprises a lipid conjugated to a polymer.

[0165] In this disclosure, the polymer-conjugated lipid refers primarily to a lipid modified with polyethylene glycol (PEG). The hydrophilic PEG stabilizes the lipid nanoparticles (LNPs), regulates the size of the nanoparticles by limiting lipid fusion, and increases the half-life of the nanoparticles by reducing non-specific interactions with macrophages.

[0166] In one embodiment, the lipid conjugated to the polymer is selected from one or a combination of at least two of the following: distearoyl phosphatidylethanolamine polyethylene glycol 2000 (DSPE-PEG2000), dimyristoylglycero-3-methoxypolyethylene glycol 2000 (DMG-PEG2000) or methoxypolyethylene glycol ditetradecylacetamide (ALC-0159).

[0167] In one embodiment, the RNA delivery agent comprises a neutral lipid, a structural lipid and a polymer-conjugated lipid, wherein the molar ratio of the cationic lipid, the neutral lipid, the structural lipid and the polymer-conjugated lipid is (25 to 75):(5 to 25):(15 to 65):(0.5 to 10), such as (35 to 49):(7.5 to 15):(35 to 55):(1 to 5).

[0168] In one embodiment, the pharmaceutical composition further comprises one or at least two cell-penetrating peptides.

[0169] A seventh aspect of the present disclosure provides a kit comprising: (1) the modified ribose cap analogue, or stereoisomer, pharmaceutically acceptable salt or solvate thereof according to the first aspect of the present disclosure; and (2) a nucleotide triphosphate molecule and an RNA polymerase.

[0170] In one embodiment, the kit further comprises one or a combination of at least two of an RNAase inhibitor, an inorganic pyrophosphatase, Mg2+, an agent of Petition 870250022825, dated 03 / 24 / 2025, page 35 / 331 24 / 120 crowding or a buffer.

[0171] This disclosure may be incorporated into other specific forms without departing from the essential attributes of this disclosure. It should be understood that any and all embodiments of this disclosure may be combined with technical features in any other embodiment or a plurality of other embodiments to obtain additional embodiments under the premise of no conflict. This disclosure includes additional embodiments obtained from such combinations.

[0172] The embodiments of this disclosure will be described in detail below in conjunction with examples, but it will be understood by those skilled in the art that the following examples are merely used to illustrate this disclosure and should not be considered as limiting the scope of this disclosure. Examples without indication of specific conditions follow conventional conditions or those recommended by the manufacturer. Reagents or instruments used without indication from the manufacturers are all conventional, commercially available products.

[0173] The following abbreviations represent the following reagents:

[0174] IBX: 2-iodoxybenzoic acid; BF3^Et?O: boron trifluoride diethyl ether; Allyltrimethylsilane: allyltrimethylsilane; TEA: triethylamine; Ac?O: acetic anhydride; HOAc: acetic acid; conc H2SO4: concentrated sulfuric acid; BSA: N,O-bis(trimethylsilyl)acetamide; TMSOTf: trimethylsilyl trifluoromethanesulfonate; Toluene: toluene; MeOH: methanol; Boc2O: di-tert-butyl dicarbonate; DIEA: N,N-diisopropylethylamine; DMAP: 4-dimethylaminopyridine; DMSO: dimethyl sulfoxide; HATU: 2-(7azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate; THF: tetrahydrofuran; TBSCl: tert-butyldimethylsilyl chloride; ImH: imidazole; DMF: N,N-dimethylformamide; TBAF: tetrabutylammonium fluoride; TBSOTf: tert-butyldimethylsilyl trifluoromethanesulfonate; NMO: N-methylmorpholine-N-oxide; m-CPBA: m-chloroperoxybenzoic acid; DIAD: diisopropyl azodicarboxylate; NCS: N-chlorosuccinimide; PO(MeO)3: trimethyl phosphate; PySSPy: 2,2'-dithiodipyridine; PPh3: triphenylphosphine;TEAP: triethylamine phosphate; TEAB: triethylamine bicarbonate; MTBE: tert-butylmethyl ether; DCM: dichloromethane; EA: ethyl acetate; DAST: aminoethyl sulfur trifluoride; AcSH: thioacetic acid.;

[0175] Example 1: Petition 870250022825, dated 03 / 24 / 2025, page 36 / 331 25 / 120

[0176] 1. Synthesis of the INT-I intermediate Ac2O DMAc, 160 °C

[0177] INT-I-PM1

[0178] Step 1: Synthesis of INT-I-PM1

[0179] 2-Amino-9J7-purin-6-ol (50.0 g, 0.33 mol) was dissolved in NA-dimethylacetamide (500 mL) and acetic anhydride (100 mL, 1.06 mol) was added to it. The mixture was heated to 160°C and stirred and reacted until clear, indicating that the reaction was complete. Heating was stopped and the reaction mixture was cooled naturally to room temperature to precipitate a large amount of solid, which was filtered. The filter cake was washed with ethanol until white to obtain INT-I-PM1 (60.0 g, 0.31 mol, 94.1%). C7H7N5O2, MS (ES): m / z (M+H+) 194.1.

[0180] Step 2: INT-I Synthesis

[0181] INT-I-PM1 (60.0 g, 0.31 mol) was dissolved in pyridine (200 mL), then N,N-diisopropylethylamine (120.2 g, 0.93 mol) was added and the mixture was cooled to 0 °C. Diphenylcarbamoyl chloride (86.2 g, 0.37 mol) was dissolved in pyridine (100 mL) and the mixture was added slowly dropwise to the above reaction system in an ice bath. After the dropwise addition was complete, the ice bath was removed. The reaction mixture was naturally heated to room temperature and stirred and reacted for 3 hours. LCMS monitored that there was no starting material remaining and the reaction was complete. The reaction system was quenched with 100 mL of water and evaporated to dryness by rotary evaporation under reduced pressure. The residue was added to 800 mL of a mixed solvent of ethanol and water (1:1, v / v) and heated under reflux for 2 hours.Heating was stopped and the reaction mixture was cooled naturally to room temperature to precipitate a large amount of solid, which was filtered. 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.

[0182] 2. Synthesis of the INT-II intermediate Petition 870250022825, dated 03 / 24 / 2025, pp. 37 / 331 26 / 120 ,N HO-PO ON TEA 0

[0183] nh2 N N** N NH NH; HO OH pA(2'-OMe)mpGTEA nh2 PySSPy. imidazole AN PPhj. TEA. DMF O=p. NaÓ o— N n , N NH NH2 HO OH INT-II

[0184] pA(2'-OMe) mpG TEA (300.1 mg, 0.37 mmol), imidazole (347.2 mg, 5.10 mmol), dithiodipyridine (1123.6 mg, 5.10 mmol) and triethylamine (516.1 mg, 5.10 mmol) were dissolved in 2.0 mL of ultradry Α,Α-dimethylformamide, then triphenylphosphamide (1337.7 mg, 5.10 mmol) was added under a nitrogen atmosphere and the mixture was reacted at 25°C for 4 hours. After the reaction was complete, the reaction mixture was slowly added to a pre-cooled acetone solution containing sodium iodide (598.1 mg, 3.99 mmol), crystallized at 25°C for minutes, and centrifuged to obtain INT-II (240.6 mg, 0.30 mmol, yield: 80.9%) as a white solid. C24H30N12O13P2, MS(ES): m / z (MH'): 755.2.

[0185] 3. Synthesis of YK-CAP-101 H 1 <'NVnN N^NH, AcO^O^ . ^OAcBSA TMSOTf ArfZ^^OAr toluene ÒAc α l. H^N NN<O mOH0Mj AcO*A'-'-''\)Ac OAc YK-CAP-101-PM1 O HNX H2N ^N N. O ru> oh POClj PO(M«O)j. 01'

[0186] PySSPy. imidazole PPhj. TEA. DMF. rt ΗΝ^γ^ ΗΛ^'ν^Ν o OsOÉNN1 . ON·^ OH YK-CAP-101-PM4 NH, OfNj^NNNPO\o N w' ON.\° / N0 Ο Ο- N>„uOP-nçJ j O»2'VOyN'eN NH, HO OH fNT4l ZnQj. DMSO. 37 V TEAP ZfíClj. DMF. rt r 9M> hn%n Ν' 'N· OH YK-CAP-101-PM2 HljT HO >H OH OH TEA Mel, DMF rt Õ O -oPOPOP O OH OH YK-CAP-W1-PM5 NH; Ν' Ή ' O ó O õ 0<NY JNH.0-PoCH)NL^oJ YK-CAP-101 ÕH ÓH O NH N^NH, OHNIa Η,Ν * N N O > 0. _o.p.0H°” OH^TE* YK-CAP-101PM3 THEIR KivSr*; oo Ο P Ο β OH , Ó OH Ο ζΥ TEA YK-CAP-101-PM6

[0187]

[0188] Step 1: Synthesis of YK-CAP-101-PM1 To a 250 mL single-neck flask, 2-amino-6-chloropurine (6.29 g, 37.09 mmol), toluene (50 mL), and BSA (15.09 g, 74.19 mmol) were added. The system was heated. Petition 870250022825, dated 03 / 24 / 2025, pp. 38 / 331 27 / 120 at 80°C, stirred until clear, and cooled naturally to room temperature. α-Pentaacetylglucose (10.00 g, 25.62 mmol) was dissolved in 20 mL of toluene, and the mixture was added to the system, which was stirred and reacted 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 system was cooled naturally to room temperature, 100 mL of saturated sodium bicarbonate solution and 100 mL of ethyl acetate were added, stirred for 5 minutes, and filtered through diatomaceous earth. The phases were separated, and the aqueous phase was extracted with 100 mL of ethyl acetate. The ethyl acetate phases were combined, washed with 100 mL of saturated brine, dried over anhydrous sodium sulfate, and filtered.The filtrate was evaporated to dryness by rotary evaporation under reduced pressure to obtain a viscous substance, which was purified by silica gel column chromatography (0 to 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%). C19H22QN5O9, MS (ES): m / z (M+H+) 500.1.

[0189] Step 2: Synthesis of YK-CAP-101-PM2

[0190] YK-CAP-101-PM1 (8.40 g, 16.80 mmol) and aqueous sodium hydroxide solution (1 M, 40 mL, 40 mmol) were added to a 250 mL single-neck flask. The system was heated and stirred and refluxed for 3 hours. The system was cooled to room temperature and evaporated to dryness by rotary evaporation under reduced pressure. The residue was suspended with 100 mL of (methanol:dichloromethane = 1:20) for 10 minutes and filtered to obtain 6.0 g of a brown solid, which was purified by high-performance 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.

[0191] YK-CAP-101-PM2:1H NMR(400 MHz, DiO)6 8.71 (s, 1H), 5.57 (d, J = 9.2 Hz, 1H), 3.99 (dd, J = 9.2, 9.2 Hz, 1H), 3.86 (dd, J = 12.4, 2.0 Hz, 1H), 3.67 - 3.55 (m, 4H).

[0192] Step 3: Synthesis of YK-CAP-101-PM3

[0193] YK-CAP-101-PM2 (300 mg, 0.96 mmol) was dissolved in 3 mL of trimethyl phosphate. The mixture was cooled to 0°C under a nitrogen atmosphere, then phosphorus oxychloride (450 mg, 2.93 mmol) was added slowly dropwise and the mixture was stirred and reacted at 0°C for about 3 hours. After the reaction was complete, the mixture was added with 5 mL Petition 870250022825, dated 03 / 24 / 2025, pp. 39 / 331 28 / 120 of water, heated to room temperature, stirred for about 1.5 hours, then washed with dichloromethane (10 mL) and left for phase separation. 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 (eluted with water and 1.5 M TEAB at a ratio of 10:1). The peak of the target product was collected, concentrated, and lyophilized to obtain YK-CAP-101-PM3 (triethylamine salt, 340 mg, 0.69 mmol, 71.6%) as a white solid. C11H16N5O9P, MS(ES): m / z (MH)392.1.

[0194] Step 4: Synthesis of YK-CAP-101-PM4

[0195] YK-CAP-101-PM3 (340 mg, 0.69 mmol), imidazole (706 mg, 10.38 mmol), 2,2'-dithiodipyridine (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 mixture was stirred and reacted at room temperature for approximately 4 hours under a nitrogen atmosphere. After the reaction was complete, the reaction mixture was poured into a solution of sodium iodide (1225 mg, 8.17 mmol) in acetone (6 mL), stirred at room temperature for 30 minutes, and centrifuged to obtain a crude precipitate. The crude product was washed with acetone, and the phases were separated. The lower precipitate was collected and lyophilized to obtain YK-CAP-101-PM4 (sodium salt, 309 mg, 0.66 mmol, 96.2%) as a white solid. C14H18N7O8P, MS(ES): m / z (MH-)442.1.

[0196] Step 5: Synthesis of YK-CAP-101-PM5

[0197] 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), then zinc chloride (215 mg, 1.58 mmol) was added and the mixture was stirred and reacted at room temperature for approximately 21 hours under a nitrogen atmosphere. After the reaction was complete, the system was added with MTBE (10 mL), washed with ultrasonic stirring, allowed to stand, and the supernatant was discarded. The procedure was repeated once. The bottom substance was collected and concentrated under reduced pressure. The residue was dissolved in water (80 mL) until clear and purified by gel column chromatography (eluted with water and 1.5 M TEAB at a ratio of 20:1). The peak of the target product was collected, concentrated, and lyophilized to obtain YKCAP-101-PM3 (triethylamine salt, 340 mg, 0.69 mmol, 71.6%) as a white solid. C11H17N5O12P2, MS(ES): m / z (M-H')472.0. Petition 870250022825, dated 03 / 24 / 2025, page 40 / 331 29 / 120

[0198] Step 6: Synthesis of YK-CAP-101-PM6

[0199] 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 and reacted in an oil bath at 37°C for approximately 23 hours. After the reaction was complete, the system was dissolved in water (5 mL) until clear, washed with EA (25 mL), and the phases were separated. The lower aqueous phase was collected and concentrated under reduced pressure. The residue was dissolved in water (50 mL) until clear and purified by gel column chromatography (eluted with water and 1.5 M TEAB at a ratio of 10:1). The peak of the target product was collected, concentrated, lyophilized, and further desalted by preparative high-performance liquid chromatography (TEAB 50 mM and methanol mobile phase system) to obtain YK-CAP-101PM6 (triethylamine salt, 95 mg, 0.16 mmol, 28.8%) as a white solid. C12H19N5O12P2, MS(ES): m / z (MH-)486.0.

[0200] Step 7: Synthesis of YK-CAP-101

[0201] 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), then zinc chloride (518 mg, 3.80 mmol) was added and the mixture was stirred and reacted in an oil bath at 37°C for about 3 days under a nitrogen atmosphere. After the reaction was complete, the mixture was dissolved in 0.25 M EDTA solution until clear, then 1.5 M TEAB was added to adjust the pH to 6-7 and purified by gel column chromatography (eluted with water and 1.5 M TEAB at a ratio of 10:1). The peak of the target product was collected, concentrated, lyophilized, and further purified by preparative high-performance liquid chromatography to obtain the final product YK-CAP-101 (20 mg, 16.30 pmol, 10.2%). C33H45N15O25P4, MS(ES): m / z (MH-)1174.2.

[0202] 1H NMR (400 MHz, DiO)6 8.43 (d, J = 1.3 Hz, 1H), 8.38 (s, 1H), 8.06 (d, J = 6.0 Hz, 2H), 6.01 (d, J = 5.4 Hz, J, 89 (1H), 5.81 (d, J = 4.5 Hz, 1H), 4.97 - 4.86 (m, 3H), 4.66 (t, J = 4.8 Hz, 1H), 4.52 - 4.35 (m, 4H), 4.32 (d, J = 31 H = 4.5 (H4), Hz (s, 2H), 3.97 (s, 3H), 3.78 (t, J = 7.0 Hz, 1H), 3.43 (s, 4H).31PNMR (D2O,162 MHz) δ 0.92 (s, 1P), −11.15.11 (d, 1.3 Hz), = 17.8 Hz, 1P), −22.23 (t, J = 17.8 Hz, 1P).

[0203] 4. Synthesis of YK-CAP-102 Petition 870250022825, dated 3 / 24 / 2025, p. 41 / 331 30 / 120

[0204] BSA.TMWIT Today N*Oil M) YK4AP· IO2-PMI YX-CAF· IO2FM2 S ·» su <> op oit JX <>U AcHP* ~ >,||ItA ΥΚ<ΑΙΜ02·ΡΜ] o HN4 S^SSIV mudazo] -t ------------- HN N * O -NPPPhi, TEA. DMLn -OPN THREE* AdlS ΛΐΗ OH YK-CAP· IO2-PM4 Nil· The .*<< 0Μ v*o Ο Ο- N * Of <>. CJ «ΚχΟ / 1N NH, HO OH rxiT-ir ZnCI.. [>MSO, J5TMC TEAR For PMF, rt O 1IXΧ-N.,. N ' K * N ft O OF Ο P 011IW,. (Mt (Min AcMN Yh OHηΛ YK'l ΛΡ ΙΟΪ ΡΜί O ι(ν^λ; H.K'Sr'-'1(which ,or -or-po->-OH j I o on AdIN(A,(1 nA YK-CAPIO2-PM6°UI,NIL· IINj YY t / 's »«Va· ο ο ο I .-. X () (,_p.(,_p.OX I 6 0 01'° ^llxYm ο ο -NVM1w ppotxyW, 'U«J TKXAP- YES HO HO

[0205] Stage 1: Synthesis of YK-CAP-102-PM1

[0206] According to the synthesis method of YK-CAP-101-PM 1, aD glucosamine penta acetate (10.0 g, 25.68 mmol) was used as the starting material to obtain YK-CAP102-PM1 (8.79 g, 61.62 mmol%) (17.62%). Ci9H23ClN6O8,MS (ES): m / z (M+H+) 499.1.

[0207] Step 2: Synthesis of YK-CAP-102-PM2

[0208] According to the synthesis method of YK-CAP-101-PM2, YK-CAP-102-PM1 (8.79 g, 17.62 mmol) was used as starting material to obtain YK-CAP-102-PM2 (2.37 g, 6.69 mmol, 37.96%). Ci3Hi8N6O6,MS (ES): m / z (M+H+) 355.2.

[0209] YK-CAP-102-PM2:1HNMR(400 MHz,DMSO-t / 6)ô 8.41 (s, 1H), 7.97 (d, J= 9.2 Hz, 1H), 7.65 (s, 1H), 6.76 (s, 2H), 5.32 (d, J= 10.2 Hz, 1H), 4.15 (q, J = 9.8 Hz, 1H), 3.67 (d, J = 11.9 Hz, 1H), 3.55 - 3.41 (m, 2H), 3.25 (s, 2H), 1.64 (s, 3H).

[0210] Step 3: Synthesis of YK-CAP-102-PM3

[0211] According to the synthesis route of YK-CAP-101-PM3, YK-CAP-102-PM2 (500 mg, 1.41 mmol) was used as starting material to obtain YK-CAP-102-PM3 (triethylamine salt, 702 mg, 1.31 mmol, 93.0%). Ci3Hi9N6O9P,MS(ES): m / z (M-H')433.1.

[0212] Step 4: Synthesis of the intermediate YK-CAP-102-PM4

[0213] According to the YK-CAP-101-PM4 synthesis route, YK-CAP-102-PM3 (702 mg, 1.31 mmol) was used as starting material to obtain YK-CAP-102-PM4 (sodium salt, Petition 870250022825, dated 03 / 24 / 2025, page 42 / 331 31 / 120 521 mg, 1.03 mmol, 78.5%). Ci6H2iNsOsP,MS(ES): m / z (M-H')483.1.

[0214] Step 5: Synthesis of the intermediate YK-CAP-102-PM5

[0215] According to the synthesis route of YK-CAP-101-PM5, YK-CAP-102-PM4 (521 mg, 1.03 mmol) was used as starting material to obtain YK-CAP-102-PM5 (triethylamine salt, 338 mg, 0.55 mmol, 53.3%). Ci3H2oN60i2P2,MS(ES): m / z (MH-)513.0.

[0216] Step 6: Synthesis of the intermediate YK-CAP-102-PM6

[0217] According to the synthesis route of YK-CAP-101-PM6, YK-CAP-102-PM5 (338 mg, 0.55 mmol) was used as starting material to obtain YK-CAP-102-PM6 (triethylamine salt, 158 mg, 0.25 mmol, 45.6%). C14H22N6O12P2,MS(ES): m / z (MH-)527.1.

[0218] Stage 7: Summary of YK-CAP-102

[0219] According to the synthesis of YK-CAP-101, YK-CAP-102-PM6 (158 mg, 0.25 mmol) was used as the starting material to obtain YK-CAP-102 (23.9 mg, 18.85 μmol, 7.5%). C35H48N16O25P4,MS(ES): m / z (MH-)1115.1.

[0220] 1H NMR (400 MHz, D2O) δ 8.42 (d, J = 1.3 Hz, 1H), 8.38 (s, 1H), 8.04 (d, J = 5.8 Hz, 2H), 6.00 (d, J = 5.4 Hz, 1H), 5.88 (d, J = 2.3 Hz, 1H), 5.81 (d, J = 4.4 Hz, 1H), 4.95 - 4.83 (m, 3H), 4.64 (t, J = 4.7 Hz, 1H), 4.53 - 4.36 (m, 4H), 4.30 (d, J = 4.4 Hz, 1H), 4.20 (s, 2H), 4.06 (s, 2H), 3.93 (s, 3H), 3.75 (t, J = 7.0 Hz, 3H), 3.62 (s, 3H), 1.67 (s, 3H).31P NMR (D2O, 162 MHz) δ -0.92 (s, 1P), -11.21 (d, J = 19.2 Hz, 1P), -11.55 (d, J = 17.3 Hz, 1P), -23.43 (t, J = 17.6 Hz, 1P).

[0221] 5. Summary of YK-CAP-103 Petition 870250022825, 03 / 24 / 2025, pág. 43 / 331 32 / 120

[0222] H,,r .. <> ho ><>_><> TBirsoVo °~ο <' _ IBDPSC1 .1 ACN. refluxo ·° imidazole. FEET <>() YK<AP I03 PMI YKCAP-IO3.PM2 DIBAL-II DCM YK4AP-I03-PM3 THREE TEA, IX Mot* TBDPSO l-HuOK. THF YK-CAP-1O3-PM4 YK<ΛΡ·101·ΜΜ5 TBDPSO K O IBDPSOV-O O MM)4. NMO KIOa I MBH«t. ' Oil - .. TIIF.HjO θ β THF. H-O °> O McíMI tbdpm) V ο °η ιμι>ρμ) \ ο <>r* — BC1.TFA, DAMP [ — ο5(54* Ο YK-CAP-IOJ-PMX YK-CAP-lOJPM» YK-CAP-103-PM6 YKCAP-IO3-PM7 N Cl UN N N : Ml;> LiH. DMSO YK-C AP-I03-PMH YK-CAP-I03-PMH ..'N'KMI' - H.N ' NX' N O PySSPy. inudazol11 N N PCMMcOh.OXπO P OH 011 PPh.rtA.DMI, the All Afi TFA YKCAPI0.VPMI2 Md DMF rt YK-C AP IO3-PMI Ml· ONX * \p>£x°-N*0o o?p° :-. ,|N- Ν οΛχΟ / · N NII; ll,N n N o o1,0o<>(*<>»» OH INI II <> OH -------------------------Ah Αιι TA / Mi. OMso r, YK-CAPIOJPMI5 MXxHA XsΟ O ItAPoOPOHHI ,,., _____ ' O»l OH ZtKl·. DMI.and 1 The OH TEA YK-CAP-I03-PMI4 (11. . N′-Ν· O Ah Ah N OO OI ο Ρ ο p Ο p <> I (> f* ooo ' o ,N INK. help N NH; YKAP.HEIGHT||O

[0223] Stage 1: Synthesis of YK-CAP-103-PM1

[0224] To a single-necked flask containing acetonitrile (200 mL) were added (4A,5A)-5-((R)-1,2-dihydroxyethyl)-2,2-dimethyl-1,3-dioxolane-4-carbaldehyde (10.00 g, 52.58 mmol) and methyl 2-(triphenyl-5-phosphoranylidene)acetate (21.6 g, 64.60 mmol). The mixture was heated to 90°C and stirred and reacted at 90°C for 10 hours. After completion of the reaction, the mixture was diluted with ethyl acetate (300 mL), washed with saturated brine, and the organic phase was dried over anhydrous sodium sulfate. The organic phase was evaporated by rotary evaporation under vacuum to remove the solvent to obtain a yellow oily compound (26.7 g), which was used directly in the next reaction step without purification.

[0225] Step 2: Synthesis of YK-CAP-103-PM2

[0226] To a single-necked flask containing dichloromethane (200 mL) were sequentially added 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), and the mixture was stirred and reacted at room temperature overnight. After the reaction was complete, the mixture Petition 870250022825, dated 03 / 24 / 2025, page 44 / 331 33 / 120 was diluted with dichloromethane (100 mL) and washed with saturated brine (200 mL x 3). The organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under vacuum. The residue was purified by silica gel chromatography (0 to 17% ethyl acetate / n-hexane) to obtain YK-CAP-103-PM2 (20.0 g, 41.27 mmol).

[0227] Step 3: Synthesis of YK-CAP-103-PM3

[0228] YK-CAP-103-PM2 (20.0 g, 41.27 mmol) was dissolved in a single-necked flask containing 150 mL of DCM. The mixture was cooled to -78°C and a solution of diisobutylaluminum hydride in toluene (1.5 M, 63.2 mL, 94.8 mmol) was slowly added dropwise. After the dropwise addition was complete, the mixture was heated to room temperature and stirred and reacted overnight. After the reaction was complete, the mixture was slowly added with 65 mL of methanol in an ice bath, followed by the generation of a white flocculant solid. The mixture was then added with sodium sulfate decahydrate, stirred for 20 minutes, and subjected to suction filtration to remove the solid. The filtrate was evaporated to dryness by rotary evaporation and then the residue was purified by silica gel chromatography (0 to 30% ethyl acetate / n-hexane) to obtain YK-CAP-103-PM3 (15.00 g, 32.85 mmol, 79.6%).

[0229] Step 4: Synthesis of YK-CAP-103-PM4

[0230] To a single-necked flask containing dichloromethane (60 mL) were sequentially added 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), and the mixture was stirred and reacted at room temperature overnight. After the reaction was complete, the mixture was diluted with dichloromethane (50 mL) and washed with saturated brine (100 mL x 2). The organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under vacuum. The residue was purified by silica gel chromatography (0 to 20% ethyl acetate / n-hexane) to obtain YK-CAP-103-PM4 (17.00 g, 27.83 mmol, 84.7%).

[0231] Step 5: Synthesis of YK-CAP-103-PM5

[0232] YK-CAP-103-PM4 (17.00 g, 27.83 mmol) was dissolved in a single-necked flask containing tetrahydrofuran (150 mL), and a solution of potassium tert-butoxide in tetrahydrofuran (1 M, 61.0 mL, 61.0 mmol) was slowly added dropwise at -40°C. After the dropwise addition was complete, the mixture was stirred and reacted at temperature. Petition 870250022825, dated 03 / 24 / 2025, page 45 / 331 34 / 120 ambient for 3 hours, diluted with ethyl acetate (200 mL) and washed with saturated brine (150 mL x 2). The organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under vacuum. The residue was purified by silica gel chromatography (0 to 11% ethyl acetate / n-hexane) to obtain YK-CAP-103-PM5 (3.29 g, 7.50 mmol, 27.0%).

[0233] Step 6: Synthesis of YK-CAP-103-PM6

[0234] 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 osmate dihydrate (140 mg, 0.38 mmol) and N-methylmorpholine N-oxide (1.05 g, 9.00 mmol) were sequentially added to the above mixed solution. The mixture was heated to 40°C and stirred and reacted at 40°C for 6 hours. After completion of the reaction, the mixture was diluted with ethyl acetate (100 mL) and washed with saturated aqueous sodium sulfite solution (80 mL x 2). The organic phase was dried over anhydrous sodium sulfate, filtered, and the solvent was removed under vacuum to obtain YK-CAP-103PM6 (3.48 g, 7.36 mmol, 98.2%).

[0235] Step 7: Synthesis of YK-CAP-103-PM7

[0236] 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 sequentially added to the above mixed solution. The mixture was heated to 40°C and stirred and reacted at 40°C for 6 hours. After completion of the reaction, the mixture was diluted with ethyl acetate (100 mL) and washed with saturated aqueous sodium sulfite solution (50 mL x 2). The organic phase was 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%).

[0237] Step 8: Synthesis of YK-CAP-103-PM8

[0238] YK-CAP-103-PM7 (5.19 g, 11.78 mmol) was dissolved in a single-neck flask containing methanol (100 mL), then sodium borohydride (0.54 g, 13.18 mmol) was added in batches in an ice bath and the mixture was stirred and reacted at room temperature for 3 hours. After the reaction was complete, the mixture was diluted with water (150 mL) and extracted with ethyl acetate (150 mL x 2). The organic phases were combined, dried with anhydrous sodium sulfate, filtered and concentrated under vacuum. The residue was purified by silica gel chromatography (0 to 13% ethyl acetate / n-hexane) to obtain YK-CAP-103-PM8 (3.30 g, 7.46 mmol, 63.3%). Petition 870250022825, dated 03 / 24 / 2025, page 46 / 331 35 / 120

[0239] Step 9: Synthesis of YK-CAP-103-PM9

[0240] According to the synthesis method of YK-CAP-103-PM4, YK-CAP-103-PM8 (3.30 g, 7.46 mmol) was used as starting material to obtain YK-CAP-103-PM9 (3.42 g, 5.73 mmol, 76.8%).

[0241] Step 10: Synthesis of YK-CAP-103-PM10

[0242] 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 the mixture was stirred and reacted at 90°C for 1 hour. A solution of YK-CAP-103-PM9 (3.42 g, 5.73 mmol) in DMSO (15 mL) was then added to it and the mixture was stirred and reacted for a further 5 hours. After the reaction was complete, the mixture was diluted with water (100 mL) and extracted with dichloromethane (150 mL x 2). The organic phases were combined, dried with anhydrous sodium sulfate, filtered and concentrated under vacuum. The residue was purified by silica gel chromatography (0 to 25% ethyl acetate / dichloromethane) to obtain YK-CAP-103-PM10 (1.90 g, 3.20 mmol, 55.9%). C30H36ClN5O4Si, MS (ES): m / z (M+H+) 595.2.

[0243] Step 11: Synthesis of YK-CAP-103-PM11

[0244] YK-CAP-103-PM10 (1.90 g, 3.20 mmol) was dissolved in tetrahydrofuran (40 mL), then 1 M HCl (80 mL) was added and the mixture was stirred and reacted at 90°C for 7 hours. After the reaction was complete, the solvent was removed under reduced pressure and the residue was purified by high-performance preparative liquid chromatography to obtain the compound YKCAP-103-PM11 (587 mg, 1.97 mmol, 61.7%) as a white solid. C11H15N5O5, MS (ES): m / z (M+H+) 298.2.

[0245] YK-CAP-103-PM11: 1H NMR (400 MHz,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).

[0246] Step 12: Synthesis of YK-CAP-103-PM12

[0247] According to the synthesis route of YK-CAP-101-PM3, YK-CAP-103-PM11 (300 mg, 1.01 mmol) was used as starting material to obtain YK-CAP-103-PM12 (triethylamine salt, 350 mg, 0.73 mmol, 72.5%). C11H16N5O8P, MS (ES): m / z (MH-) 376.1.

[0248] Step 13: Synthesis of YK-CAP-103-PM13

[0249] According to the synthesis pathway of YK-CAP-101-PM4, YK-CAP-103-PM12 (350 mg, Petition 870250022825, dated 03 / 24 / 2025, page 47 / 331 36 / 120 0.73 mmol) was used as starting material to obtain YK-CAP-103-PM13 (sodium salt, 320 mg, 0.71 mmol, 97.4%). C14H18N7O7P, MS (ES): m / z (MH) 426.1.

[0250] Step 14: Synthesis of YK-CAP-103-PM14

[0251] According to the synthesis route of YK-CAP-101-PM5, YK-CAP-103-PM13 (320 mg, 0.71 mmol) was used as starting material to obtain YK-CAP-103-PM14 (triethylamine salt, 250 mg, 0.45 mmol, 62.9%). C11H17N5O11P2, MS (ES): m / z (MH-) 456.0.

[0252] Step 15: Synthesis of YK-CAP-103-PM15

[0253] According to the synthesis route of YK-CAP-101-PM6, YK-CAP-103-PM14 (250 mg, 0.45 mmol) was used as starting material to obtain YK-CAP-103-PM15 (triethylamine salt, 90 mg, 0.16 mmol, 34.9%). C12H20N5O11P2, MS (ES): m / z (MH-) 470.1.

[0254] Step 16: Synthesis of YK-CAP-103

[0255] According to the YK-CAP-101 synthesis route, YK-CAP-103-PM15 (90 mg, 0.16 mmol) was used as starting material to obtain YK-CAP-103 (29 mg, 23.95 μmol, 15.0%). C33H45N15O24P4, MS (ES): m / z (MH-) 1158.2.

[0256] 1H NMR (400 MHz, D2O) δ 8.41 (d, J = 1.2 Hz, 1H), 8.33 (d, J = 2.3 Hz, 1H), 8.02 (d, J = 4.8 Hz, 2H), 6.5, J11 (H = 4), J = = 2.4 Hz, 1H), 5.71 (d, J = 4.6 Hz, 1H), 5.20 - 5.03 (m, 2H), 4.91 - 4.77 (m, 3H), 4.63 (t, J = 4.6 Hz, 1H - 4.3), 4.3 (m (d, J = 3.1 Hz, 1H), 4.10 (s, 3H), 4.02 (s, 2H), 3.92 (s, 3H), 3.54 (t, J = 7.0 Hz, 1H), 3.32 (s, 3H).391P NMR (D2OHz, 1H). -11.22 (d, J = 19.4 Hz, 1P), -11.33 (d, J = 17.2 Hz, 1P), -24.43 (t, J = 17.7 Hz, 1P).

[0257] 6. Synthesis of YK-CAP-104 Petition 870250022825, dated 3 / 24 / 2025, p. 48 / 331 37 / 120 HO TBDPSO TBDPSC1, Im0O DCM, OV-f t ho O ' IBX TBOPSO^0θ PhjPCH^r. meUjB0PS0^ O or TBAF. THE V° O MeCN.90°COO' THE. ·7ΒΤ wrong* . q· ' ' O YK-CAP-104-PM1 YKCAP-1O4-PM2 YK-CAP-104-PM3 YK-CAP-104-PM4 BiO NM0.K2Os01ho^0 ΤΗΡ / Η,Ο * L.O' H BzO N BiO PPh3,CBr4. and „ °0HO .,-- ° ° __. Br .- N MeCN7Py=1 / 1 c^CO3. MeCN. 70°C Sh ° OAST. DCM YK-CAP-104-PM5 YK-CAP-104-PM6 YK-CAP-104-PM7 YK-CAP-104-PM8

[0258] λ* λ BzO INT-IΝ*“*0CHjCOOH.AcjON*>«0O*6BSA TMSOTf, I ° 2eqconH^SO4[ OAc Tol.80 x to 110 X YK-CAP-104-PM9 YK-CAP-104-PM10 Ph ΟNΡΊ BiONoNH» / M<,OH N50C · N .NNHAc f OAc OHNI f OH YK-CAP-104-PM11 YK-CAP-104-PM12 O .1µ O POlMoOh.NO0^0” Ah f7 tea w O HN ~ PySSPy nmdazole 1 - h2n n PPh). DMF tea, rt ΥΚΧΑΡΊ04-ΡΜ13 YK-CAP-104-PM14 YK-CAP-KMPMIS O HN CHj N O 0 OPOP OH 0 OHRT FN TEA YK-CAP-1O4-PM16 HjN N Mel. DMF nh2 ONi NNPO\0 2* N 0Nay*N Ο 0 N OP oNH ONa\O>NN NH? HO OH INT-II ZnClj. DMSO. 371 THEIR HN HjN N NH,NN N- Ο Ο ON o -%°ro°' °1 No ΑηΓν 0 0Nnh OPO CHjNn3NH / Ο I HO HO YK.CAP.104

[0259] Step 1: Synthesis of YK-CAP-104-PM1

[0260] 1,2-O-isopropyl-AD-ribofuranose (20.0 g, 0.11 mol) was dissolved in dichloromethane. Imidazole (11.6 g, 0.17 mol) and TBDPSC1 (33.0 g, 0.12 mol) were added to the above system. The mixture was stirred and reacted at room temperature for 15 hours. The reaction mixture was added with saturated sodium bicarbonate solution and extracted with dichloromethane. The dichloromethane phase was then washed once with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was evaporated to dryness by rotary evaporation under reduced pressure. The residue was subjected to silica gel chromatography (0 to 17% ethyl acetate / hexane) to obtain YK-CAP-104-PM1 (33.5 g, 78.16 mmol, 71.1%).

[0261] Step 2: Synthesis of YK-CAP-104-PM2

[0262] YK-CAP-104-PM1 (33.5 g, 78.16 mmol) was dissolved in acetonitrile and 2-iodoxybenzoic acid (28.5 g, 101.8 mmol) was added to it. The mixture was heated to 90°C and stirred and reacted for 5 hours. The reaction mixture was filtered and the filtrate was evaporated to dryness by rotary evaporation under reduced pressure to obtain YK-CAP-104-PM2 (32.7 Petition 870250022825, dated 03 / 24 / 2025, page 49 / 331 38 / 120 g, 76.66 mmol, 98.1%).

[0263] Step 3: Synthesis of YK-CAP-104-PM3

[0264] (Bromomethyl)triphenylphosphonium bromide (60.3 g, 138.3 mmol) was dissolved in tetrahydrofuran (400 mL). The mixture 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 dropwise addition was complete, the reaction system was naturally heated to 0°C and stirred and reacted for 2 hours. The above reaction system was cooled again to -78°C and a solution of YK-CAP-104-PM2 (48.0 g, 112.52 mmol) in tetrahydrofuran (100 mL) was slowly added dropwise. After the dropwise addition was complete, the reaction system was heated to room temperature and stirred and reacted overnight. The reaction system was quenched with a saturated ammonium chloride solution (200 mL) and extracted with ethyl acetate (300 mL x 3).The organic phases were combined, washed with saturated aqueous NaCl solution, dried over anhydrous sodium sulfate, filtered, and evaporated to dryness by rotary evaporation. The residue was purified by silica gel chromatography (0 to 20% ethyl acetate / n-hexane) to obtain YK-CAP-104-PM3 (39.0 g, 91.85 mmol, 81.6%).

[0265] Step 4: Synthesis of YK-CAP-104-PM4

[0266] YK-CAP-104-PM3 (34.0 g, 80.07 mmol) was dissolved in tetrahydrofuran (200 mL), then tetrabutylammonium fluoride (52.0 g, 198.9 mmol) was added and the mixture was stirred and reacted at room temperature for 1 hour. The reaction system was then topped up with a saturated aqueous solution of ammonium chloride and extracted with ethyl acetate (200 mL x 3). The organic phases were combined, washed with a saturated aqueous solution of NaCl, dried over anhydrous sodium sulfate, filtered, and evaporated to dryness by rotary evaporation. The residue was purified by silica gel chromatography (0 to 60% ethyl acetate / n-hexane) to obtain YK-CAP-104-PM4 (13.5 g, 72.50 mmol, 90.5%).

[0267] Step 5: Synthesis of YK-CAP-104-PM5

[0268] 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 to it. The reaction system was cooled to 0°C and benzoyl chloride (11.2 g, 79.7 mmol) was added slowly dropwise. The reaction system was then heated to room temperature and stirred and reacted for 1 hour. After the reaction was complete, the reaction system was quenched with aqueous solution. Petition 870250022825, dated 03 / 24 / 2025, page 50 / 331 39 / 120 saturated sodium bicarbonate (100 mL) and extracted with dichloromethane (100 mL x 3). The organic phases were combined, washed with saturated aqueous NaCl solution, dried over anhydrous sodium sulfate, filtered, and evaporated to dryness by rotary evaporation. The residue was purified by silica gel chromatography (0 to 20% ethyl acetate / n-hexane) to obtain YK-CAP-104-PM5 (19.6 g, 67.51 mmol, 93.1%).

[0269] Step 6: Synthesis of YK-CAP-104-PM6

[0270] 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 osmate dihydrate (2.0 g, 6.4 mmol) were weighed sequentially and added to the mixture. The mixture was stirred and reacted at room temperature overnight. After the reaction was complete, the reaction system was quenched with a saturated aqueous solution of sodium sulfite and extracted with ethyl acetate (200 mL x 3). The organic phases were combined, washed with a saturated aqueous solution of NaCl, dried over anhydrous sodium sulfate, filtered, and evaporated to dryness by rotary evaporation. The residue was purified by silica gel chromatography (0 to 60% ethyl acetate / n-hexane) to obtain YK-CAP-104-PM6 (20.5 g, 63.21 mmol, 98.7%).

[0271] Step 7: Synthesis of YK-CAP-104-PM7

[0272] YK-CAP-104-PM6 (17.7 g, 54.57 mmol) as starting material 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 and added to the mixture. The above reaction system was heated to 70°C under a nitrogen atmosphere and stirred and reacted for 6 hours. Thin-layer chromatography (TLC) monitored that the reaction was complete. The reaction mixture was evaporated to dryness by rotary evaporation under vacuum. The residue was purified by silica gel chromatography (0 to 50% ethyl acetate / n-hexane) to obtain YK-CAP-104-PM7 (13.9 g, 35.90 mmol, 65.8%).

[0273] Step 8: Synthesis of YK-CAP-104-PM8

[0274] YK-CAP-104-PM7 (13.4 g, 34.60 mmol) was dissolved in acetonitrile (120 mL). Potassium carbonate (14.4 g, 104.2 mmol) and dimethylamine hydrochloride (3.4 g, 41.1 mmol) were sequentially added. The above system was heated to 70°C and reacted for 16 hours. TLC monitored that the reaction was complete. The system was cooled to Petition 870250022825, dated 03 / 24 / 2025, page 51 / 331 40 / 120 ambient temperature, filtered, and the organic phase was evaporated to dryness by rotary evaporation under reduced pressure. The residue was purified by silica gel chromatography (0 to 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.

[0275] Step 9: Synthesis of YK-CAP-104-PM9

[0276] YK-CAP-104-PM8 (8.5 g, 24.18 mmol) was dissolved in dichloromethane (160 mL). The mixture was cooled to -40°C and a solution of diethylamine trifluoride of sulfur (4.7 g, 29.0 mmol) in dichloromethane (10 mL) was slowly added dropwise. After the dropwise addition was complete, the mixture was slowly heated to 0°C and stirred and reacted for 4 hours. TLC monitored that the reaction was complete. The reaction system was quenched with saturated aqueous sodium bicarbonate solution (100 mL) and extracted with dichloromethane (100 mL x 3). The organic phases were combined, washed with saturated aqueous NaCl solution, dried over anhydrous sodium sulfate, filtered, and evaporated to dryness by rotary evaporation. The residue was purified by silica gel chromatography (0 to 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.

[0277] Step 10: Synthesis of YK-CAP-104-PM10

[0278] YK-CAP-104-PM9 (4.7 g, 13.30 mmol) was dissolved in acetic acid (100 mL), then acetic anhydride (16.4 g, 160.6 mmol) was added, and concentrated sulfuric acid (4.7 g, 26.8 mmol) was added slowly dropwise to the same. After the dropwise addition was complete, the mixture was slowly heated to 40°C and stirred and reacted for 16 hours. LCMS monitored that the reaction was complete. The reaction system was quenched with saturated aqueous sodium bicarbonate solution to adjust the pH to neutrality and extracted with ethyl acetate (200 mL x 3). The organic phases were combined, washed with saturated aqueous NaCl solution, dried over anhydrous sodium sulfate, filtered, and evaporated to dryness by rotary evaporation. The residue was purified by silica gel chromatography (0 to 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.

[0279] Step 11: Synthesis of YK-CAP-104-PM11

[0280] The intermediate INT-I (2.7 g, 6.9 mmol) was dissolved in 1,2-dichloroethane (50 mL) Petition 870250022825, dated 03 / 24 / 2025, page 52 / 331 41 / 120 and N,O-bis(trimethylsilyl)acetamide (2.8 g, 13.8 mmol) was added to it. The reaction system was heated to 80°C, stirred, and reacted for 2 hours and evaporated to dryness by rotary evaporation under reduced pressure. The residue was dissolved in toluene (30 mL), then a solution of YK-CAP-104-PM10 (2.5 g, 6.29 mmol) in toluene (20 mL) was added, and trimethylsilyl trifluoromethanesulfonate (1.5 g, 6.7 mmol) was added slowly dropwise to it. The reaction system was heated to 70°C, stirred, and reacted for 2 hours. TLC monitored that the reaction was complete. The reaction system was quenched with saturated aqueous sodium bicarbonate solution and extracted with ethyl acetate (50 mL x 3). The organic phases were combined, washed with a saturated aqueous NaCl solution, dried over anhydrous sodium sulfate, filtered, and evaporated to dryness by rotary evaporation under reduced pressure.The residue was purified by silica gel chromatography (0 to 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.

[0281] Step 12: Synthesis of YK-CAP-104-PM12

[0282] 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 above reaction system was heated to 50°C and stirred and reacted for 10 hours. LCMS monitored that the reaction was complete. The reaction mixture was evaporated to dryness by rotary evaporation to obtain 2.5 g of crude product, which was purified by high-performance preparative liquid chromatography to obtain YK-CAP-104PM12 (560 mg, 1.64 mmol, 44.0%). C13H19FN6O4, MS (ES): m / z (M+H+) 343.1.

[0283] YK-CAP-104-PM12: 1H NMR (400 MHz, DMSO-d6) δ 9.98 (s, 1H), 7.93 (s, 1H), 6.52 (s, 2H), 5.69 (d, J = 6.1 Hz, 1H), 5.51 (s, 1H), 4.97 - 4.85 (m, 1H), 4.37 (d, J = 6.4 Hz, 1H), 4.14 (d, J = 4.4 Hz, 1H), 3.77 (d, J = 12.1 Hz, 1H), 3.62 (s, 1H), 2.59 - 2.33 (m, 8H).

[0284] Step 13: Synthesis of YK-CAP-104-PM13

[0285] According to the synthesis route of YK-CAP-101-PM3, YK-CAP-104-PM12 (560 mg, 1.64 mmol) was used as starting material to obtain YK-CAP-104-PM13 (triethylamine salt, 557 mg, 1.06 mmol, 64.9%). C13H20FN6O7P, MS (ES): m / z (MH-) 421.1.

[0286] Step 14: Synthesis of YK-CAP-104-PM14

[0287] According to the YK-CAP-101-PM4 synthesis route, YK-CAP-104-PM13 (557 mg, 1.06 mmol) was used as starting material to obtain YK-CAP-104-PM14 (sodium salt, Petition 870250022825, dated 03 / 24 / 2025, page 53 / 331 42 / 120 414 mg, 0.84 mmol, 79.0%). C16H23FN8O6P, MS (ES): m / z (MH-) 471.1.

[0288] Step 15: Synthesis of YK-CAP-104-PM15

[0289] According to the synthesis route of YK-CAP-101-PM5, YK-CAP-104-PM14 (414 mg, 0.84 mmol) was used as starting material to obtain YK-CAP-104-PM15 (triethylamine salt, 300 mg, 0.50 mmol, 59.2%). C13H21FN6O10P2, MS (ES): m / z (MH-) 501.1.

[0290] Step 16: Synthesis of YK-CAP-104-PM16

[0291] According to the synthesis route of YK-CAP-101-PM6, YK-CAP-104-PM15 (300 mg, 0.50 mmol) was used as starting material to obtain YK-CAP-104-PM16 (triethylamine salt, 124 mg, 0.20 mmol, 40.2%). C14H24FN6O10P2, MS (ES): m / z (MH-) 515.1.

[0292] Step 17: Synthesis of YK-CAP-104

[0293] According to the YK-CAP-101 synthesis route, YK-CAP-104-PM16 (124 mg, 0.20 mmol) was used as starting material to obtain YK-CAP-104 (ammonium salt, 21 mg, 16.72 pmol, 8.4%). C35H49FN16O23P4, MS (ES): m / z (MH-) 1203.1.

[0294] 1H NMR (400 MHz, D2O) δ 8.52 (d, J = 1.2 Hz, 1H), 8.44 (d, J = 2.2 Hz, 1H), 7.86 (d, J = 4.4 Hz, 2H), 6.5, J 5.4 (H2), J = = 2.4 Hz, 1H), 5.81 (d, J = 4.6 Hz, 1H), 4.81 - 4.67 (m, 3H), 4.63 (t, J = 4.6 Hz, 1H), 4.53 - 4.35 (m, 5.1.3), J = 4.2 (s, 3H), 4.12 (s, 2H), 3.78 (s, 3H), 3.32 (t, J = 7.0 Hz, 1H), 3.12 (s, 3H), 2.55 - 2.35 (m, 8H), 31P NMR (D2-M0, 16, 16 -11.32 (d, J = 20.7 Hz, 1P), -12.33 (d, J = 17.5 Hz, 1P), -23.28 (t, J = 16.8 Hz, 1P).

[0295] 7. Synthesis of YK-CAP-105 Petition 870250022825, dated 3 / 24 / 2025, p. 54 / 331 43 / 120 HO AcO _ ° NHA* IXT4 Ac <MI. π -BSA.IMSOIÍ, Ad)MO CobaltCatalyst.fnulsdaoo MIA* Dioxane. F.tOH ' SHA* OA* NlhMcUH HO (HI POLY, crt**'* HX PySSPy unidazole HS TEA? HXo-OP OP (Ml Md. I)MI (TH OH Ah As ΠΛ „ Ah A· P OH (HI TEA IIX - Ο P Ο P (Ml You (HI HO (Ml ZM1.. DMMA A,, A (Hi THEIR A« Ax 1XH,

[0296] HO HO

[0297] Step 1: Synthesis of YK-CAP-105-PM1

[0298] YK-CAP-104-PM4 (5.76 g, 30.93 mmol) was dissolved in acetic acid (8 mL), then acetic anhydride (31.6 g, 310 mmol) and sulfuric acid (500 pL) were added and the mixture was stirred and reacted at room temperature for 4 hours. After the reaction was complete, the reaction mixture was added with aqueous NaHCL solution to adjust the pH to weak acidity and extracted twice with ethyl acetate. The organic phase was evaporated to dryness by rotary evaporation and the residue was purified by silica gel chromatography (0 to 80% ethyl acetate / / / -hexane) to obtain YK-CAP-105-PM1 (5.9 g, 21.67 mmol, 70.1%).

[0299] Step 2: Synthesis of YK-CAP-105-PM2

[0300] Intermediate INT-I (9.27 g, 23.87 mmol) and A,O-bis(trimethylsilyl)acetamide (9.72 g, 47.8 mmol) were dissolved in 1,2-dichloroethane (60 mL). The mixture was stirred at 80°C for 2 hours and evaporated to dryness by rotary evaporation to remove the solvent. YKCAP-105-PM1 (5.9 g, 21.67 mmol) was dissolved in toluene (80 mL) and added to the above residue. Trimethylsilyl trifluoromethanesulfonate (5.31 g, 23.9 mmol) was then added to the same, and the mixture was reacted at 70°C for 2 hours. ATLC monitored that the reaction was complete. After filtration, the filtrate was evaporated to dryness by rotary evaporation under reduced pressure. The residue was purified by silica gel column chromatography (0 to 80% ethyl acetate / / / -hexane) to obtain YK-CAP-105-PM2 (10.00 g, 16.65 mmol, 76.8%).

[0301] Step 3: Synthesis of YK-CAP-105-PM3

[0302] YK-CAP-105-PM2 (8.00 g, 13.32 mmol) and [A]A'-(1,1,2,2-tetramethylethane)bis(3,5-di Petition 870250022825, dated 03 / 24 / 2025, page 55 / 331 44 / 120 tert-butylsalicylideneimine)]cobalt(n) (403 mg, 0.67 mmol) were dissolved in 1,4-dioxane (30 mL), then benzenesulfonyl cyanide (72.30 g, 400 mmol) was added and the mixture was stirred and reacted at room temperature for 5 minutes. Phenylsilane (1.73 g, 16 mmol) was dissolved in anhydrous ethanol (60 mL) and added to the above mixture. The mixture was stirred at room temperature for 3 hours. After the reaction was complete, the reaction mixture was filtered and extracted twice with water and ethyl acetate. The organic phase was evaporated to dryness by rotary evaporation and the residue was purified by silica gel chromatography (0 to 70% ethyl acetate / n-hexane) to obtain YK-CAP-105-PM3 (4.50 g, 7.17 mmol, 53.8%).

[0303] Step 4: Synthesis of YK-CAP-105-PM4

[0304] YK-CAP-105-PM3 (4.50 g, 7.17 mmol) was dissolved in 7 M ammonia / methanol (50 mL) and the mixture was stirred and reacted at 50°C for 4 hours. After completion of the reaction, the reaction mixture was evaporated to dryness by rotary evaporation to remove the solvent and the residue was purified by high-performance preparative liquid chromatography to obtain YKCAP-105-PM4 (800 mg, 2.61 mmol, 36.4%). C12H14N6O4, MS (ES): m / z (M+H+) 307.11.

[0305] YK-CAP-105-PM4: 1H NMR (400 MHz, DMSO-d6) δ 10.66 (s, 1H), 7.97 (s, 1H), 6.54 (d, J = 5.2 Hz, 3H), 5.74 (d, J = 7.2 Hz, 1H), 5.40 (t, J = 4.4 Hz, 1H), 4.62 - 4.65 (m, 1H), 4.31 (t, J = 3.2 Hz, 1H), 3.59 - 3.69 (m, 2H), 1.53 (s, 3H).

[0306] Step 5: Synthesis of YK-CAP-105-PM5

[0307] According to the synthesis route of YK-CAP-101-PM3, YK-CAP-105-PM4 (800 mg, 2.61 mmol) was used as starting material to obtain YK-CAP-105-PM5 (triethylamine salt, 994 mg, 2.04 mmol, 78.1%). C12H15N6O7P, MS (ES): m / z (MH-) 385.1.

[0308] Step 6: Synthesis of YK-CAP-105-PM6

[0309] According to the synthesis route of YK-CAP-101-PM4, YK-CAP-105-PM5 (994 mg, 2.04 mmol) was used as starting material to obtain YK-CAP-105-PM6 (sodium salt, 661 mg, 1.44 mmol, 70.7%). C15H17N8O6P, MS (ES): m / z (MH-) 435.1.

[0310] Step 7: Synthesis of YK-CAP-105-PM7

[0311] According to the synthesis route of YK-CAP-101-PM5, YK-CAP-105-PM6 (661 mg, 1.44 mmol) was used as starting material to obtain YK-CAP-105-PM7 (triethylamine salt, 612 mg, 1.08 mmol, 74.8%). C12H16N6O10P2, MS (ES): m / z (MH-) 465.0.

[0312] Step 8: Synthesis of YK-CAP-105-PM8 Petition 870250022825, dated 03 / 24 / 2025, page 56 / 331 45 / 120

[0313] De acordo com a via de síntese de YK-CAP-101-PM6, YK-CAP-105-PM7 (612 mg, 1,08 mmol) foi usado como material de partida para obter YK-CAP-105-PM8 (sal de trietilamina, 325 mg, 0,56 mmol, 51,7%). C13H19N6O10P2, MS (ES): m / z (MH') 479,1.

[0314] Stage 9: Summary of YK-CAP-105

[0315] According to the synthesis of YK-CAP-101, YK-CAP-105-PM8 (325 mg, 0.56 mmol) was used as the starting material to obtain YK-CAP-105 (16 mg, 13.12 pmol, 2.3%). C34H44N16O23P4, MS (ES): m / z (MH) 1267.1.

[0316] 'H NMR. (400 MHz, D2O) δ 8.49 (d, J= 1.4 Hz, 1H), 8.44 (d, J = 2.5 Hz, 1H), 7.88 (d, J= 4.2 Hz, 2H), 6.22 (d, J= 4.7 Hz, 1H), 5.93 (d, J= 2.4 Hz, 1H), 5.80 (d, J= 4.5 Hz, 1H), 4.81 - 4.66 (m, 3H), 4.62 - 4.32 (m, 5H), 4.22 (d, J= 3.1 Hz, 1H), 4.18 (s, 2H), 4.10 (s, 2H), 3.77 (s, 3H), 3.31 (t, 7= 6.8 Hz, 1H), 3.11 (s, 3H), 1.53 (s, 3H).31PNMR(D2O, 162 MHz) δ -0.90 (s, IP), -11.33 (d, 7= 20.2 Hz, IP), -12.12 (d, 7= 17.1 Hz, IP), -22.34 (t, 7= 16.2 Hz, IP)·

[0317] 8. Synthesis of YK-CAP-106

[0318] AcO * OAc Ph ] OAPtl \.ov * Rainfall Switches. femlMhuw NHAc EtOH N| OAc Ph Ο κθ YK-CAP-IO5PM2 YK4AP1O6PMI HO ' SHiMcOH.II / ) HN OH OH N * NH; O foci,HN–IkN N POlMcOiuOX YK-C AP-IO6-PM4 ο 0 UNM SHN HN NNoo” OPOlO''1I'MEll.NA OH OH-OH NHAc TEA n YK-CAP-I06-PM7 Ph <> A p»> AcO ' O PPh,.H;O.TIIE x^O \ -S SNHAc H:N OAc YK-CAPI06.PM2 O NN. Or PySSPy. umdazol H;N N0θοΐθ^ PPh,. TEA. IJMF.n All *HAc TEA YK-CAP-IO6-PM5 nil * ν' AcO AcC IJI A.IX^M HN NNo <>”nN In 1, OH NHAc YK-CAP-I06-PM6 Ph OA I YK <AP.|06.PM3 TEAP ZnClj. DMF. rt CH, NKOOo-O P O P OH OH OH NHAcTFA YKCAPI06.PM8 NPOxq N ON·' * O <) OP n N' O * 'Ml HO OH INTII ZnCl₂ DMSO, 37°C THE HN ' H;NN CH, N 'N N- OOOv ·()OPOPO PO , 0 Is0 0 0 01'Mha' «Η. o?o%X(E ο I YK-CAP-106 HO HO SH;

[0319] Step 1: Synthesis of YK-CAP-106-PM1

[0320] To YK-CAP-105-PM2 (23.00 g, 38.30 mmol) were added β-toluenesulfonyl azide (211.46 g, 1.07 mol) and cobalt catalyst ([A,A'-(1,1,2,2-tetramethylethane)bis(3,5-di / erc-butylsalicylideneimine)]cobalt(II)) (696 mg, 1.15 mmol), and the mixture was stirred for 30 minutes under a nitrogen atmosphere. A phenylsilane solution (10.22 g, 45.96 mmol) in Petition 870250022825, dated 03 / 24 / 2025, page 57 / 331 46 / 120 anhydrous ethanol (40 mL) was added dropwise to the mixture at room temperature over 30 minutes. The mixture was stirred and reacted at room temperature for 2 hours. LC-MS monitored that the reaction was complete. The reaction was terminated. The reaction mixture was extracted with EA and saturated aqueous NaCl solution. The phases were separated, and the aqueous phase was extracted twice with EA. The organic phases were combined and evaporated by rotary evaporation to remove the solvent. The crude product was purified by flash column chromatography (EA / PE = 0 to 100%) to obtain YK-CAP-106-PM1 (4.90 g, 7.61 mmol, 19.9%). C30H29N9O8, MS (ES): m / z (M+H+) 644.3.

[0321] Step 2: Synthesis of YK-CAP-106-PM2

[0322] To YK-CAP-106-PM1 (4.90 g, 7.61 mmol) were added triphenylphosphine (2.78 g, 10.59 mmol), water (4.9 mL) and tetrahydrofuran (49 mL). The mixture was heated to 50°C and stirred and reacted for 8 hours. LC-MS monitored that the reaction was complete. The reaction was terminated and the reaction mixture was evaporated by rotary evaporation under reduced pressure to remove the solvent. The crude product was purified by flash column chromatography (MeOH / DCM = 0 to 10%) to obtain YK-CAP-106-PM2 (4.22 g, 6.83 mmol, 89.8%). C30H31N7O8, MS (ES): m / z (M+H+) 618.2.

[0323] Step 3: Synthesis of YK-CAP-106-PM3

[0324] 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 dropwise addition was complete, the mixture was heated to room temperature and reacted for 24 hours. LC-MS monitored that the reaction was complete. The reaction was terminated. The reaction mixture was poured into ice-cold water and stirred. The phases were separated and the aqueous phase was extracted twice with dichloromethane. The organic phases were combined and evaporated by rotary evaporation under reduced pressure to remove the solvent. The crude product was purified by flash column chromatography (MeOH / DCM = 0 to 10%) to obtain YK-CAP-106-PM3 (2.94 g, 4.46 mmol, 67.1%). C32H33N7O9, MS (ES): m / z (M+H+) 660.3.

[0325] Step 4: Synthesis of YK-CAP-106-PM4

[0326] To YK-CAP-106-PM3 (2.94 g, 4.46 mmol) were added 7 M ammonia / methanol solution (29 mL) and water (5.9 mL), and the mixture was stirred and reacted at temperature Petition 870250022825, dated 03 / 24 / 2025, page 58 / 331 47 / 120 ambient temperature for 24 hours. LC-MS monitored that the reaction was complete. The reaction was terminated and the reaction mixture was evaporated by rotary evaporation under reduced pressure to remove the solvent. The crude product was added with EA, stirred, and subjected to suction filtration to obtain YK-CAP-106-PM4 (1.36 g, 4.02 mmol, 90.1%). C13H18N6O5, MS (ES): m / z (M+H+) 339.3.

[0327] YK-CAP-106-PM4:1H NMR (400 MHz, MeOD) δ 7.99 (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).

[0328] Step 5: Synthesis of YK-CAP-106-PM5

[0329] According to the synthesis route of YK-CAP-101-PM3, YK-CAP-106-PM4 (500 mg, 1.48 mmol) was used as starting material to obtain YK-CAP-106-PM5 (triethylamine salt, 480 mg, 0.92 mmol, 62.4%). C13H19N6O8P, MS (ES): m / z (MH-) 417.2.

[0330] Step 6: Synthesis of YK-CAP-106-PM6

[0331] According to the synthesis route of YK-CAP-101-PM4, YK-CAP-106-PM5 (480 mg, 0.92 mmol) was used as starting material to obtain YK-CAP-106-PM6 (sodium salt, 410 mg, 0.84 mmol, 90.9%). C16H21N8O7P, MS (ES): m / z (MH-) 467.1.

[0332] Step 7: Synthesis of YK-CAP-106-PM7

[0333] According to the synthesis route of YK-CAP-101-PM5, YK-CAP-106-PM6 (410 mg, 0.84 mmol) was used as starting material to obtain YK-CAP-106-PM7 (triethylamine salt, 331 mg, 0.55 mmol, 65.7%). C13H20N6O11P2, MS (ES): m / z (MH-) 497.0.

[0334] Step 8: Synthesis of YK-CAP-106-PM8

[0335] De acordo com a rota de síntese de YK-CAP-101-PM6, YK-CAP-106-PM7 (331 mg, 0,55 mmol) foi usado como material de partida para obter YK-CAP-106-PM8 (sal de trietilamina, 150 mg, 0,24 mmol, 44,5%). C14H22N6O11P2, MS (ES): m / z (MH-) 511,1.

[0336] Stage 9: Summary of YK-CAP-106

[0337] According to the synthesis of YK-CAP-101, YK-CAP-106-PM8 (150 mg, 0.24 mmol) was used as the starting material to obtain YK-CAP-106 (32 mg, 25.56 μmol, 10.6%). C35H48N16O24P4, MS (ES): m / z (MH-) 1199.1.

[0338] 1H NMR (400 MHz, D2O) δ 8.50 (d, J = 1.4 Hz, 1H), 8.44 (d, J = 2.2 Hz, 1H), 7.78 (d, J = 4.2 Hz, 2H), 6.24 (d, J = 4.6 Hz, 1H), 5.94 (d, J = 2.3 Hz, 1H), 5.81 (d, J = 4.4 Hz, 1H), 4.86 - 4.76 (m, 3H), 4.61 - 4.55 (m, 4H), 4.52 (d, J = 3.6 Hz, 2H), 4.28 (s, 2H), 4.20 (s, 1H), Petition 870250022825, 03 / 24 / 2025, pág. 59 / 331 48 / 120 3.93 − 3.82 (m, 5H), 3.23 (t, J = 6.7 Hz, 1H), 3.12 (s, 2H), 2.03 (s, 3H), 1.62 (s, 3H).31P NMR(s1O, 162, IP.9) 2- J= 20.1 Hz, IP), −12.13 (d, J= 16.8 Hz, IP), -22.11 (t, J= 16.4 Hz, IP).

[0339] 9. Synthesis of YK-CAP-107 TBDPSO He / she ' TBDPSO BH>CLOSE * OH TBDPSO MgMeBr *> ° P TIF, 0 °C H- ° TBDPSO O (t 1-Truth. Mcl THF O °' YK-CAP-I04-PM3 YK-CAP.107.PM I YK-CAP-IO7-PM2 YK-CAP-I07-PM3 YK-CAP-I07-PM4 Ac.O. pictures. H,SO4Ai:O()Ac AcOII.H PLEASE _ .,' < ixit t AcO „ 'nN _______2ÍL·!_____., ; DBSA.IX Vol. NO OAp jN° iTMSOTf.Tol . 70 P HU…. 7M NHj in McOH -N Ρ(κ*|Λ HO O-: OllN.N PCXMeO^OX' - ' NEW YK-CAP-IO7-PM5 YK-CAP-I07.PM6 YK-CAP-IO7-PM7 The hunUn. H;NNN -O-P-CIR A 1 ° OH · TEA YK-CAP-I07-PM8 '1 UN Sr-NI . II n PySSPy. imidazole H,NNN -op ft PPh,, TEA. DMF.n I—IDN“ YK-CAP-I07-PM0 O 9 CH, νη\ν,hnν'ο 0 TEAP * Ο -θ θ ρ'θΗ MeLDMÉN^-0. <> ^'Η ------ ',__, OH Oil - 11° ° ZnCI,. OMF. π Λ1Π TEAηOil1„ TEA ° I YK-CAP-I07-PMI0 YK-CAP-I07-PMII NH, —, ON-NonL°·nnoo?o°nLη O&iXO,N N NIH2 HO OH INT-II OR Mb O O O μ J ORORORORO,()IΝ° ° P VA The ONf N the JNH, the=PDO CHwNNH, ZnCl·, DMSO, 37°C

[0340] YK-CAP-I07 HO HO

[0341] Stage 1: Synthesis of YK-CAP-107-PM1

[0342] A 1 M borane solution in tetrahydrofuran (325 mL, 0.325 mol) was added to a three-necked flask, and a solution of YK-CAP-104-PM3 (60.00 g, 0.14 mol) in tetrahydrofuran (180 mL) was slowly added dropwise at 0 °C. After the dropwise addition was complete, the mixture was stirred and reacted at room temperature for 2 hours. THF / H2O (1:1, 120 mL), 2 N NaOH (261 mL), and 30% hydrogen peroxide (271 mL) were then sequentially and slowly added dropwise at 0 °C. After the dropwise addition was complete, the mixture was stirred and reacted at room temperature overnight. TLC monitored that the starting material reacted completely. The reaction was terminated. The reaction mixture was extracted with water. The organic phase was sequentially washed with saturated aqueous sodium thiosulfate solution and saturated brine, dried over anhydrous sodium sulfate, filtered, and evaporated by rotary evaporation to remove the... Petition 870250022825, dated 03 / 24 / 2025, page 60 / 331 49 / 120 solvent. The crude product was purified by flash column chromatography (PE: EA = 0 at 25%) to obtain the compound YK-CAP-107-PM1 (48.80 g, 0.11 mol, 78.8%) as a colorless oil.

[0343] Step 2: Synthesis of YK-CAP-107-PM2

[0344] YK-CAP-107-PM1 (34.20 g, 77.27 mmol) was dissolved in acetonitrile and 2-iodoxybenzoic acid (28.10 g, 100.5 mmol) was added to the above system. The mixture was heated to 90°C and stirred and reacted for 5 hours. The reaction mixture was cooled to room temperature, filtered, and the filtrate was evaporated to dryness by rotary evaporation to obtain the crude product YK-CAP-107-PM2 (34.50 g) as a light yellow oily liquid, which was used directly in the next step without purification.

[0345] Step 3: Synthesis of YK-CAP-107-PM3

[0346] YK-CAP-107-PM2 (32.90 g, calculated as 74.67 mmol) was dissolved in tetrahydrofuran. The mixture was cooled to 0°C under a nitrogen atmosphere and a 1 M solution of methylmagnesium bromide in tetrahydrofuran (97.1 mL, 97.1 mmol) was slowly added dropwise to it. After the dropwise addition was complete, the mixture was stirred at room temperature for 3 hours. The reaction mixture was cooled to 0°C, quenched with saturated ammonium chloride, and extracted with ethyl acetate. The organic phase was dried and evaporated to dryness by rotary evaporation. The crude product was purified by flash column chromatography (PE: EA = 0 to 41%) to obtain YK-CAP-107-PM3 (16.80 g, 36.79 mmol, 49.3%) as a light yellow oily liquid.

[0347] Step 4: Synthesis of YK-CAP-107-PM4

[0348] YK-CAP-107-PM3 (16.80 g, 36.79 mmol) was dissolved in THF and the mixture was cooled to 0°C under a nitrogen atmosphere. A solution of sodium tert-butoxide (11.20 g, 116.7 mmol) in THF was slowly added to the above system. The mixture was stirred and reacted at room temperature for 1.5 hours, and iodomethane (27.60 g, 194.5 mmol) was added slowly dropwise to the above system. After the dropwise addition was complete, the mixture was stirred and reacted for a further 3 hours. TLC detected that the reaction was complete. The reaction mixture was quenched with saturated ammonium chloride solution and extracted with ethyl acetate. The organic phase was washed 3 to 5 times with saturated brine, dried, and evaporated to dryness by rotary evaporation. The crude product was purified by flash column chromatography (PE: EA = 0 to 12%) to obtain YK-CAP-107-PM4 (13.16 g, Petition 870250022825, dated 03 / 24 / 2025, page 61 / 331 50 / 120 27.96 mmol, 76.0%) as a light yellow oily liquid.

[0349] Step 5: Synthesis of YK-CAP-107-PM5

[0350] 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 mixture was stirred and reacted at room temperature for 4 hours. 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 by rotary evaporation to obtain YK-CAP-107-PM5 (11.38 g) as a yellow oily liquid, which was used directly in the next reaction step.

[0351] Step 6: Synthesis of YK-CAP-107-PM6

[0352] 2-acetamido-9H-purin-6-yldiphenylcarbamate (6.30 g, 16.22 mmol) was dissolved in 1,2-dichloroethane (100 mL) and N,O-bis(trimethylsilyl)acetamide (6.60 g, 32.4 mmol) was added. The mixture was heated to 80°C, stirred, and reacted for 1.5 hours and subjected to rotary evaporation to remove the reaction solvent. A solution of YK-CAP-107-PM5 (6.00 g, calculated as 18.85 mmol) in toluene (100 mL) and TMSOTf (3.6 g, 16.2 mmol) was then sequentially added at room temperature. The mixture was then heated to 70°C and stirred and reacted for a further 3.5 hours. The reaction system was diluted with ethyl acetate, washed once with a saturated sodium bicarbonate solution, filtered to remove the insoluble substance, and the filtrate phases were separated. The organic phase was dried and evaporated to dryness by rotary evaporation.The crude product was purified by flash column chromatography (DCM: EA = 0 to 52%) to obtain YK-CAP-107-PM6 (3.20 g, 4.95 mmol, 26.3%) as a yellow solid. C32H34N6O9, MS (ES): m / z (M+H+) 647.2.

[0353] Step 7: Synthesis of YK-CAP-107-PM7

[0354] YK-CAP-107-PM6 (3.20 g, 4.95 mmol) was dissolved in a 7 M ammonia / methanol solution and water (5:1, 24 mL). The mixture was heated to 50°C and stirred and reacted for 6 hours. The reaction mixture was evaporated to dryness by rotary evaporation, resuspended twice with EA, and subjected to suction filtration to collect the filter cake to obtain YK-CAP-107PM7 (1.38 g, 4.24 mmol, 85.7%) as a white solid. C13H19N5O5, MS (ES): m / z (M+H+) 326.2.

[0355] YK-CAP-107-PM7:1H NMR (400 MHz, MeOD) δ 8.07 (s, 1H), 5.82 (d, J = 2.6 Hz, Petition 870250022825, dated 03 / 24 / 2025, page 62 / 331 51 / 120 1H), 4.53 (dd, J = 6.1, 2.7 Hz, 1H), 4.36 (ddd, J = 8.3, 3.9, 2.6 Hz, 1H), 3.95 (dd, J = 12.1, 2.4 Hz, 1H), 3.78 - 3.67 (m, 2H), 3.34 (s, 3H), 2.11 (q, J = 7.4 Hz, 1H), 1.23 (d, J = 6.1 Hz, 3H).

[0356] Step 8: Synthesis of YK-CAP-107-PM8

[0357] According to the synthesis route of YK-CAP-101-PM3, YK-CAP-107-PM7 (1.20 g, 3.69 mmol) was used as starting material to obtain YK-CAP-107-PM8 (triethylamine salt, 644 mg, 1.27 mmol, 34.4%). C13H20N5O8P, MS (ES): m / z (M-H') 404.1.

[0358] Step 9: Synthesis of YK-CAP-107-PM9

[0359] According to the synthesis route of YK-CAP-101-PM4, YK-CAP-107-PM8 (644 mg, 1.27 mmol) was used as starting material to obtain YK-CAP-107-PM9 (sodium salt, 549 mg, 1.15 mmol, 90.6%). C16H22N7O7P, MS (ES): m / z (MH-) 454.2.

[0360] Step 10: Synthesis of YK-CAP-107-PM10

[0361] According to the synthesis route of YK-CAP-101-PM5, YK-CAP-107-PM9 (549 mg, 1.15 mmol) was used as starting material to obtain YK-CAP-107-PM10 (triethylamine salt, 531 mg, 0.91 mmol, 78.7%). C13H21N5O11P2, MS (ES): m / z (MH-) 484.0.

[0362] Step 11: Synthesis of YK-CAP-107-PM11

[0363] According to the synthesis route of YK-CAP-101-PM6, YK-CAP-107-PM10 (531 mg, 0.91 mmol) was used as starting material to obtain YK-CAP-107-PM11 (triethylamine salt, 163 mg, 0.27 mmol, 29.8%). C14H23N5O11P2, MS (ES): m / z (MH-) 498.1.

[0364] Step 12: Synthesis of YK-CAP-107

[0365] According to the YK-CAP-101 synthesis route, YK-CAP-107-PM11 (163 mg, 0.27 mmol) was used as starting material to obtain YK-CAP-107 (35 mg, 28.25 μmol, 10.5%). C35H49N15O24P4, MS (ES): m / z (MH-) 1186.1.

[0366] 1H NMR (400 MHz, D2O) δ 8.43 (d, J = 1.2 Hz, 1H), 8.34 (d, J = 2.1 Hz, 1H), 7.73 (d, J = 4.1 Hz, 2H), 6.5, 7 = 1.4 (H J = 2.3 Hz, 1H), 5.71 (d, J = 4.4 Hz, 1H), 4.76 - 4.61 (m, 3H), 4.51 - 4.45 (m, 4H), 4.42 (d, J = 3.5 Hz, H1), 4.3 (s). 3.95 – 3.92 (m, 1H), 3.90 – 3.84 (m, 2H), 3.78 – 3.67 (m, 2H), 3.13 (t, J = 6.7 Hz, 1H), 3.34 (s, 3H, 3.7 =H) (s Hz, 1H), 1.22 (d, J = 6.2 Hz, 3H).31P NMR (D2O, 161 MHz) δ −0.88 (s, 1P), −11.23 (d, J = 20.2 Hz, 1P), −12.22 (d, J = 16.6 Hz, 1P), −22.32 (t, J = 16.1 Hz, 1P).

[0367] 10. Synthesis of YK-CAP-108 Petition 870250022825, dated 3 / 24 / 2025, p. 63 / 331 52 / 120

[0368] TAX 0 o HO ' ; 'NH ' b PPh3, DEAD. THF YK-CAP-107-PM3 O YK-CAP- 10&-PM3 POO, AcO Ac?O.cat AcOH HN COST __ 0-° YK-CAP-106-PM1 OAc OAc The YK-CAP- 108-PM4 TBOPSO ΝΗ,ΝΗτ-Η,Ο °0 ΕΙΟΗ Η2Ν YK-CAP-106-PM2 DiEAAcO OCM * TBDPSO *^° O ..B 0 TBAF / THF YK-CAP-10ΒΡΜ2 o HNN HjN ΡΟ(ΜβΟ)).0Ρ o AhI THEIR Ο P OH OH nhactea YK-CAP-108-PM7 HN CH» N MH.DMFM?Nrt HNNO-NN NO NHAc MT-1 1)BSA.DCE ~2)TMS0Tf' Toi AcOΛ^0 ^n HN OA^ O ,NNO NHAc YK-CAP-106-PMS HERE M NHjjn MeOH HN ohn .NNHj YK-CAP-108PM6 PySSPy. imidazole H?N PPhj. TEA. DMF.fi POP OH 0 OH NHAc YK-CAP-10B-PM10 NHjNNN&?.'°'NN 0 ο Ο N1wu OP ONM 0Ne\O,Nn NH; I OH INT-II Zrdt. DMSO. 37 X o Ο PN ON· TEAP HN H,N1NHAc ZnOj. DMF.rt o POP OH OH OH NO NO! YK-CAP-106-PM8 YK-CAP-10B-PM9 HN H / 4 CH, N NH, OP Ο P Ο P Ο I 0 0 0' ' NHAc 3NH / NO N NH OPO CHjNN ΟΛI YK-CAP-108 NO

[0369] Stage 1: Synthesis of YK-CAP-108-PM1

[0370] 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 to it. The system was cooled to 0°C under a nitrogen atmosphere, and a solution of DEAD (9.3 g, 53.40 mmol) in THF (30 mL) was added dropwise. After the dropwise addition was complete, the mixture was reacted for 3 hours. ATLC monitored that the reaction was complete (DCM). After the reaction was complete, the system was added dropwise with 30 mL of purified water at 0°C to quench the reaction. The reaction mixture was extracted with EA (200 mL x 3). The organic phase was sequentially washed with saturated brine (400 mL), dried over anhydrous sodium sulfate, and evaporated to dryness by rotary evaporation. The residue was purified by normal-phase silica gel column chromatography (PE: EA = 0 to 40%) to obtain YK-CAP-108-PM1 (18.8 g, 32.09 mmol, 83.8%) as a yellow oil.

[0371] Step 2: Synthesis of YK-CAP-108-PM2

[0372] YK-CAP-108-PM1 (18.8 g, 32.09 mmol) was dissolved in 200 mL of ethanol, then 85% hydrazine hydrate (37.8 g, 0.64 mol) was added and the system was heated to 80°C. Petition 870250022825, dated 03 / 24 / 2025, page 64 / 331 53 / 120 and reacted for 1 hour. The TLC monitored that the reaction was complete (PE / EA = 3 / 1). The reaction was terminated. The system was cooled to room temperature, purified water (200 mL) and EA (200 mL) were added, it was stirred for 10 minutes, and the phases were separated. The aqueous phase was extracted with EA (200 mL x 2). The organic phase was sequentially washed with saturated brine (300 mL), dried over anhydrous sodium sulfate, and evaporated to dryness by rotary evaporation to obtain YK-CAP-108-PM2 (14.2 g, 31.16 mmol, 97.1%) as a light yellow oil.

[0373] Step 3: Synthesis of YK-CAP-108-PM3

[0374] 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 to it. The system was cooled to 0°C under a nitrogen atmosphere and a solution of acetyl chloride (2.9 g, 37.40 mmol) in DCM (20 mL) was added dropwise. After the dropwise addition was complete, the mixture was reacted at a controlled temperature of 0°C for 1 hour. The TLC monitored that the reaction was complete (PE / EA = 3 / 1). The reaction was terminated. The system was heated to room temperature, 200 mL of saturated sodium bicarbonate solution was added to quench the reaction, and the phases were separated. The aqueous phase was extracted with DCM (200 mL x 2). The organic phase was sequentially washed with saturated brine (300 mL), dried over anhydrous sodium sulfate, and evaporated to dryness by rotary evaporation.The residue was purified by normal-phase silica gel column chromatography (DCM: MeOH = 0 to 40%) to obtain YK-CAP-108-PM3 (12.5 g, 25.12 mmol, 80.6%) as a yellow oil.

[0375] Step 4: Synthesis of YK-CAP-108-PM4

[0376] 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 a nitrogen atmosphere and 1 M TBAF (37.7 mL) was added dropwise. After the dropwise addition was complete, the mixture was heated to room temperature and reacted for 2 hours. TLC monitored that the reaction was complete (PE / EA = 3 / 1). The reaction was terminated. The reaction mixture was evaporated directly to dryness by rotary evaporation and the residue was purified by normal-phase silica gel column chromatography (DCM: MeOH = 0 to 40%) to obtain YK-CAP-108-PM4 (5.8 g, 22.37 mmol, 89.0%) as a yellow oil.

[0377] Step 5: Synthesis of YK-CAP-108-PM5 Petition 870250022825, dated 03 / 24 / 2025, page 65 / 331 54 / 120

[0378] YK-CAP-108-PM4 (4.5 g, 17.35 mmol) was dissolved in 15 mL of acetic acid, then acetic anhydride (35.4 g, 34.71 mmol) and p-toluenesulfonic acid (1.5 g, 8.67 mmol) were added and the system was heated to 50 °C. The reaction was monitored by TLC. After the reaction was complete, the system was cooled to room temperature, 100 mL of purified water and 100 mL of EA were added, it was stirred for 10 minutes, and the phases were separated. The aqueous phase was extracted with EA (100 mL x 2). The organic phase was sequentially washed with saturated brine (500 mL), dried over anhydrous sodium sulfate, and evaporated to dryness by rotary evaporation. The residue was purified by normal-phase silica gel column chromatography (DCM: MeOH = 0 to 40%) to obtain YK-CAP-108-PM5 (5.5 g, 15.94 mmol, 91.8%) as a yellow oil.

[0379] Step 6: Synthesis of YK-CAP-108-PM6

[0380] The intermediate INT-I (6.8 g, 17.53 mmol) was dissolved in 1,2-dichloroethane (50 mL) and N,O-bis(trimethylsilyl)acetamide (7.1 g, 35.06 mmol) was added to it. The reaction system was heated to 80°C, stirred, and reacted for 2 hours and evaporated to dryness by rotary evaporation under reduced pressure. The residue was dissolved in toluene (30 mL), then a solution of YK-CAP-108-PM5 (5.5 g, 15.94 mmol) in toluene (20 mL) was added, and trimethylsilyl trifluoromethanesulfonate (3.9 g, 17.53 mmol) was added slowly dropwise to it. The reaction system was heated to 70°C, stirred, and reacted for 2 hours. TLC monitored that the reaction was complete. The reaction system was quenched with a saturated aqueous solution of sodium bicarbonate and extracted with ethyl acetate (100 mL x 3).The organic phases were combined, washed with saturated aqueous NaCl solution (300 mL), dried over anhydrous sodium sulfate, filtered, and evaporated to dryness by rotary evaporation under reduced pressure. The residue was purified by silica gel chromatography (DCM: MeOH = 0 to 100%) to obtain YK-CAP-108-PM6 (3.8 g, 5.64 mmol, 35.4%). C33H35N7O9, MS (ES): m / z (M+H+) 674.3.

[0381] Step 7: Synthesis of YK-CAP-108-PM7

[0382] YK-CAP-108-PM6 (3.8 g, 5.64 mmol) was dissolved in a mixed solvent of 4 MNH3 / MeOH (40 mL) and water (4 mL), and the mixture was stirred and reacted at room temperature overnight. LCMS monitored that the reaction was complete. The reaction mixture was evaporated to dryness by rotary evaporation and suspended with EA (100 mL x 2) at room temperature. Petition 870250022825, dated 03 / 24 / 2025, page 66 / 331 55 / 120 ambient temperature to obtain YK-CAP-108-PM7 (1.92 g, 5.45 mmol, 96.6%) as a whitish solid. C14H20N6O5, MS (ES): m / z (M+H+) 353.1.

[0383] YK-CAP-108-PM7:1H NMR (400 MHz, MeOD) δ 8.02 (s, 1H), 5.71 (d, J = 2.5 Hz, 1H), 4.43 (d, J = 2.4 Hz, 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).

[0384] Step 8: Synthesis of YK-CAP-108-PM8

[0385] According to the synthesis route of YK-CAP-101-PM3, YK-CAP-108-PM7 (1.50 g, 4.26 mmol) was used as starting material to obtain YK-CAP-108-PM8 (triethylamine salt, 1.11 g, 2.08 mmol, 48.8%). C14H21N6O8P, MS (ES): m / z (MH) 431.1.

[0386] Step 9: Synthesis of YK-CAP-108-PM9

[0387] According to the synthesis route of YK-CAP-101-PM4, YK-CAP-108-PM8 (1.11 g, 2.08 mmol) was used as starting material to obtain YK-CAP-108-PM9 (sodium salt, 778 mg, 1.54 mmol, 74.2%). C17H23N8O7P, MS (ES): m / z (MH) 481.1.

[0388] Step 10: Synthesis of YK-CAP-108-PM10

[0389] According to the synthesis route of YK-CAP-101-PM5, YK-CAP-108-PM9 (778 mg, 1.54 mmol) was used as starting material to obtain YK-CAP-108-PM10 (triethylamine salt, 512 mg, 0.83 mmol, 54.2%). C14H22N6O11P2, MS (ES): m / z (MH-) 511.0.

[0390] Step 11: Synthesis of YK-CAP-108-PM11

[0391] According to the synthesis route of YK-CAP-101-PM6, YK-CAP-108-PM10 (512 mg, 0.83 mmol) was used as starting material to obtain YK-CAP-108-PM11 (triethylamine salt, 117 mg, 0.19 mmol, 22.5%). C15H24N6O11P2, MS (ES): m / z (MH-) 525.1.

[0392] Step 12: Synthesis of YK-CAP-108

[0393] According to the YK-CAP-101 synthesis route, YK-CAP-108-PM11 (117 mg, 0.19 mmol) was used as starting material to obtain YK-CAP-108 (18 mg, 14.22 μmol, 7.5%). C36H50N16O24P4, MS (ES): m / z (MH-) 1213.1.

[0394] 1H NMR (400 MHz, D2O) δ 8.43 - 8.32 (m, 1H), 8.12 (d, J = 2.5 Hz, 1H), 7.73 - 7.54 (m, 2H), 6.22 (d, J = 4.5 Hz, 1H), 5.35 (d, J = 2.3 Hz, 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 = 6.2 Hz, 3H).31P NMR (D2O, 163 MHz) δ -0.87 (s, 1P), -11.21 (d, J = 20.1 Hz, 1P), -11.62 (d, J = 16.6 Hz, 1P), -21.67 (t, J Petição 870250022825, de 24 / 03 / 2025, pág. 67 / 331 56 / 120 = 17.2 Hz, IP).

[0395] 11. Síntese de YK-CAP-109

[0396] TBDPSOΛΟ OH°' YK-CAP-107-PM3 PO(MeO)v0°C YK-CAP-109-PM4 OH HN DAST THF θCH) Mel. DMF H2N N TBDPSO ) Q Ac.O. cat H;SO4 AcOH.rt YK-CAP-109-PM1 H-.N O UN III1o -OP OH ' OH .- TEA AcO F Λ OAc * \ Ν.θΥ INT-Y™* DBSA. DCE. 80 °C 2) TMSOTf. Tol.. 70 °C YK-CAP-I09-PM2 O PySSPy. imidazole Η2Ν PPhj. TEA. DMF.rt AcO »N. THEIR 7M NHj in McOH NHAc H2O YK-CAP-1O9-PM5 νη2ον->ν N-NSí?aW 'Νο Ν Ν ο -θ+-ΝΛϊ • ~· ÓNa YK-CAP-1O9-PM3 AM TEAR :N ZnCl·, DMF.rt YK-CAP-109-PM6 YES -Ο P OP-OH ' OH OH TEA YK-CAP-I09-PM7 TEA -OPO P OH O OH YK-CAP-109-PM8 o?o°- <Vnh ONayOfNV'NH, HO~OH INT-ll ZnCl2, DMSO. 37°C Ο CHt HN Ν. h2n N THEIR OP OPO PO O Ó Ó NH, N N 0 <Y ?*< οΨ-ό ch^Y Ν*νΗ· 3ΝΗ4· Q IΝ ΝΗ· YK-CAP-109 --HO HO

[0397] Step 1: Synthesis of YK-CAP-109-PM1

[0398] 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 added slowly dropwise. The reaction system was stirred and reacted at 0°C for 4 hours. The system was slowly added with saturated aqueous sodium bicarbonate solution to quench the reaction, then DCM was added, stirred, and the phases were separated. The organic phase was washed twice with saturated aqueous sodium bicarbonate solution and the phases were separated. The organic phase was washed two to three times with saturated brine, dried over anhydrous sodium sulfate, filtered, and evaporated to dryness by rotary evaporation. The residue was purified by silica gel column chromatography (0 to 30% ethyl acetate / w-hexane) to obtain YK-CAP-109-PM1 (6.1 g, 13.30 mmol, 60.7%).

[0399] Step 2: Synthesis of YK-CAP-109-PM2

[0400] YK-CAP-109-PM1 (6.1 g, 13.30 mmol) was dissolved in acetic acid, then acetic anhydride (27.2 g, 266.0 mmol) was added, and concentrated sulfuric acid (280 pL) was added slowly dropwise. After the dropwise addition was complete, the mixture was stirred at room temperature for 16 hours. The reaction mixture was added to 200 mL of water and extracted with ethyl acetate. The organic phase was washed three times with a saturated aqueous solution of sodium bicarbonate to adjust the pH to Petition 870250022825, dated 03 / 24 / 2025, page 68 / 331 57 / 120 alkalinity, then dried and evaporated to dryness by rotary evaporation to obtain the crude product YK-CAP-109-PM2 (5.80 g) as a yellow oily liquid, which was used directly in the next reaction step.

[0401] Step 3: Synthesis of YK-CAP-109-PM3

[0402] The intermediate INT-I (6.2 g, 15.96 mmol) was dissolved in 1,2-dichloroethane and N,O bis(trimethylsilyl)acetamide (8.1 g, 39.9 mmol) was added to it. The mixture was heated to 80°C, stirred and reacted for 2 hours and the reaction solvent was removed under reduced pressure. The mixture was redissolved in toluene and a solution of YK-CAP-109-PM2 (5.8 g, calculated as 13.30 mmol) in toluene and TMSOTf (4.4 g, 19.9 mmol) was sequentially added at room temperature. The mixture was then heated to 70°C and stirred and reacted for a further 2 hours. The reaction system was diluted with ethyl acetate, washed once with saturated sodium bicarbonate solution, filtered to remove the insoluble substance and the filtrate phases were separated. The organic phase was dried over anhydrous sodium sulfate and evaporated by rotary evaporation under reduced pressure to remove the solvent.The residue was purified by silica gel column chromatography (0 to 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.

[0403] Step 4: Synthesis of YK-CAP-109-PM4

[0404] YK-CAP-109-PM3 (3.1 g, 4.88 mmol) was dissolved in a 7 M ammonia / methanol and water solution (5:1), and the mixture was stirred and reacted at room temperature for 16 hours. The reaction mixture was evaporated to dryness by rotary evaporation to remove the solvent, and the crude product was recrystallized with ethyl acetate to obtain YK-CAP-109-PM4 (1.1 g, 3.51 mmol, 71.9%). C12H16FN5O4, MS (ES): m / z (M+H+) 314.1.

[0405] YK-CAP-109-PM4:1H NMR (400 MHz, DMSO-d6) δ 11.45 (s, 1H), 8.00 (s, 1H), δ 6.40 (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 = 6.2 Hz, 3H).

[0406] Step 5: Synthesis of YK-CAP-109-PM5

[0407] Phosphorus oxychloride (1.6 g, 10.5 mmol) was dissolved in 20 mL of trimethyl phosphate. The mixture was cooled to 0°C under a nitrogen atmosphere. The above reaction system was slowly added to YK-CAP-109-PM4 (1.1 g, 3.51 mmol) and stirred and reacted to Petition 870250022825, dated 03 / 24 / 2025, page 69 / 331 58 / 120 The reaction was heated to 0°C for approximately 4 hours. After the reaction was complete, the reaction mixture was added to 20 mL of ice-cold water and washed twice with ethyl acetate. The aqueous phase was added to ammonia water to adjust the pH to 3.5 and stored in a refrigerator overnight. The following day, the pH was continuously adjusted to 6.5 and the mixture was diluted to 400 mL for sample loading. The sample was purified by gel column chromatography (eluted with water and 1.5 M TEAB at a 1:4 ratio). The peak of the target product was collected, concentrated, and lyophilized to obtain YK-CAP-109-PM5 (triethylamine salt, 980 mg, 1.98 mmol, 56.5%) as a white solid. C12H17FN5O7P, MS (ES): m / z (MH) 392.1.

[0408] Step 6: Synthesis of YK-CAP-109-PM6

[0409] According to the synthesis route of YK-CAP-101-PM4, YK-CAP-109-PM5 (980 mg, 1.98 mmol) was used as starting material to obtain YK-CAP-109-PM6 (sodium salt, 900 mg, 1.93 mmol, 97.6%). C15H19FN7O6P, MS (ES): m / z (MH) 442.1.

[0410] Step 7: Synthesis of YK-CAP-109-PM7

[0411] According to the synthesis route of YK-CAP-101-PM5, YK-CAP-109-PM6 (900 mg, 1.93 mmol) was used as starting material to obtain YK-CAP-109-PM7 (triethylamine salt, 850 mg, 1.48 mmol, 76.7%). C12H18FN5O10P2, MS (ES): m / z (MH) 472.2.

[0412] Step 8: Synthesis of YK-CAP-109-PM8

[0413] According to the synthesis route of YK-CAP-101-PM6, YK-CAP-109-PM7 (850 mg, 1.48 mmol) was used as starting material to obtain YK-CAP-109-PM8 (triethylamine salt, 450 mg, 0.76 mmol, 51.7%). C13H20FN5O10P2, MS (ES): m / z (MH) 486.1.

[0414] Step 9: Synthesis of YK-CAP-109

[0415] According to the synthesis of YK-CAP-101, YK-CAP-109-PM8 (100 mg, 0.17 mmol) was used as the starting material to obtain YK-CAP-109 (35 mg, 28.53 μmol, 16.8%). C34H46FN15O23P4, MS (ES): m / z (MH-) 1174.2.

[0416] 1H NMR (400 MHz, D2O) δ 8.53 - 8.41 (m, 1H), 8.14 (d, J = 2.6 Hz, 1H), 7.63 - 7.55 (m, 2H), 6.17 (d, J = 4.4 Hz, 1H), 5.65 (d, J = 2.6 Hz, 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 = 6.1 Hz, 3H).31P NMR (D2O, 163 MHz) δ -0.89 (s, 1P), -11.21 (d, J = 20.8 Hz, 1P), -11.74 (d, J = 16.6 Hz, 1P), -22.12 (t, J = 18.0 Hz, 1P). Petition 870250022825, 03 / 24 / 2025, pág. 70 / 331 59 / 120

[0417] 12. Summary of YK-CAP-110 TBDPSO TBDPSO DAST < °>Q AcO Ac2O, cat. H2SO4* -OsOAc O' b DCM. 0*C AcOH. rt HfCNNHAc INT-I 1) BSA. DCE. 80 C 2) TMSOTf, Vol., 70 C AcO „ N F Οα£^Ν F NHAc NH3, MeOH YK-CAP-107-PM2 YK-CAP-110-PM1 YK-CAP-110-PM2 YK-CAP-110-PM3 HO N .Oh! Χ / Ή ΟΗΝγΝF NH2 POCI3 O hn\nη2νλ>ναν PO(MeOh, 0 C ... O o1O P OH OH PySSPy. imidazole . h2n Tf PPh3. TEA. DMF. rt0~N -OPNJ HE^ TEAP ZnCI2, DMF. rt OO −OP−OPOH w YK-CAP-110-PM4 YK-CAP-110-PM5 YK-CAP-110-PM6 YK-CAP-110-PM7

[0418] NH2.0NN O ΗΝΛ Mel. DMF h2N N rt ch3 N / > N· The ... -O-PO-p-OH °teT ? F YK-CAP-110-PM8 OPn°N.*NHn2ÓI$a\O,NNNHj HO OH INT-II ' TN NH H2N NN ZnCI2, DMSO. 37 C OOO nh2n?n -,o opopotfo-, or OOO \_J 0 . 1NH 3NH? OPO CHfl^N^NH. HO HO YK-CAP-110

[0419] Step 1: Synthesis of YK-CAP-110-PM1

[0420] 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 added slowly dropwise. The reaction system was stirred and reacted at 0°C for 4 hours. The system was slowly added with saturated aqueous sodium bicarbonate solution to quench the reaction, then DCM was added, stirred, and the phases were separated. The organic phase was washed twice with saturated aqueous sodium bicarbonate solution and the phases were separated. The organic phase was washed two to three times with saturated brine, dried over anhydrous sodium sulfate, filtered, and evaporated to dryness by rotary evaporation. The residue was purified by silica gel column chromatography (0 to 30% ethyl acetate / w-hexane) to obtain YK-CAP-110-PM1 (10.6 g, 22.91 mmol, 70.1%).

[0421] Step 2: Synthesis of YK-CAP-110-PM2

[0422] YK-CAP-110-PM1 (5.6 g, 12.11 mmol) was dissolved in acetic acid, then acetic anhydride (24.7 g, 242.1 mmol) was added, and concentrated sulfuric acid (280 pL) was added slowly dropwise to the same. After the dropwise addition was completed, the mixture was stirred at room temperature for 16 hours. The reaction mixture was added with 200 mL of water and extracted with ethyl acetate. The organic phase was washed three times with Petition 870250022825, dated 03 / 24 / 2025, page 71 / 331 60 / 120 saturated aqueous solution of sodium bicarbonate to adjust the pH to alkalinity, then dried and evaporated to dryness by rotary evaporation to obtain the crude product YK-CAP-110-PM2 (6.0 g) as a yellow oily liquid, which was used directly in the next reaction step.

[0423] Step 3: Synthesis of YK-CAP-110-PM3

[0424] The intermediate INT-I (5.2 g, 13.4 mmol) was dissolved in 1,2-dichloroethane and N,O bis(trimethylsilyl)acetamide (7.4 g, 36.3 mmol) was added to it. The mixture was heated to 80°C, stirred and reacted for 2 hours and the reaction solvent was removed under reduced pressure. The mixture was redissolved in toluene and a solution of YK-CAP-110-PM2 (6.0 g, calculated as 12.11 mmol) in toluene and TMSOTf (3.0 g, 13.4 mmol) was sequentially added to it at room temperature. The mixture was then heated to 70°C and stirred and reacted for a further 2 hours. The reaction system was diluted with ethyl acetate, washed once with a saturated sodium bicarbonate solution, filtered to remove the insoluble substance, and the filtrate phases were separated. The organic phase was dried over anhydrous sodium sulfate and evaporated by rotary evaporation under reduced pressure to remove the solvent.The residue was purified by silica gel column chromatography (0 to 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.

[0425] Step 4: Synthesis of YK-CAP-110-PM4

[0426] YK-CAP-110-PM3 (3.5 g, 5.48 mmol) was dissolved in a 4 M ammonia / methanol and water solution (5:1), and the mixture was stirred and reacted at room temperature for 16 hours. The reaction mixture was evaporated to dryness by rotary evaporation to remove the solvent, and the crude product was recrystallized with ethyl acetate to obtain YK-CAP-110-PM4 (1.3 g, 4.10 mmol, 74.7%). C11H13F2N5O4, MS (ES): m / z (M+H+) 318.1.

[0427] YK-CAP-110-PM4:1H NMR (400 MHz, DMSO-d6) δ 10.56 (s, 1H), 8.00 (s, 1H), δ 6.40 (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).

[0428] Step 5: Synthesis of YK-CAP-110-PM5

[0429] Phosphorus oxychloride (1.9 g, 12.3 mmol) was dissolved in 20 mL of trimethyl phosphate. The mixture was cooled to 0°C under a nitrogen atmosphere and YK-CAP-110-PM4 (1.3 g, 4.10 mmol) was slowly added to the above reaction system. The mixture was stirred and reacted at 0°C for about 4 hours. After the reaction was complete, the reaction mixture was Petition 870250022825, dated 03 / 24 / 2025, page 72 / 331 61 / 120 added with 20 mL of ice-cold water and washed twice with ethyl acetate. The aqueous phase was added with ammonia water to adjust the pH to 3.5 and stored in a refrigerator overnight. The following day, the pH was continuously adjusted to 6.5 and the mixture was diluted to 400 mL for sample loading. The sample was purified by gel column chromatography (eluted with water and 1.5 M TEAB at a 1:5 ratio). The peak of the target product was collected, concentrated, and lyophilized to obtain YK-CAP-110-PM5 (triethylamine salt, 1.1 g, 2.21 mmol, 53.8%) as a white solid. C11H14F2N5O7P, MS (ES): m / z (MH-) 396.1.

[0430] Step 6: Synthesis of YK-CAP-110-PM6

[0431] According to the synthesis route of YK-CAP-101-PM4, YK-CAP-110-PM5 (1.1 g, 2.21 mmol) was used as starting material to obtain YK-CAP-110-PM6 (sodium salt, 1.0 g, 2.13 mmol, 96.4%). C14H16F2N7O6P, MS (ES): m / z (MH) 446.1.

[0432] Step 7: Synthesis of YK-CAP-110-PM7

[0433] According to the synthesis route of YK-CAP-101-PM5, YK-CAP-110-PM6 (1.0 g, 2.13 mmol) was used as starting material to obtain YK-CAP-110-PM7 (triethylamine salt, 900 mg, 1.56 mmol, 73.1%). C11H15F2N5O10P2, MS (ES): m / z (MH) 476.1.

[0434] Step 8: Synthesis of YK-CAP-110-PM8

[0435] According to the synthesis route of YK-CAP-101-PM6, YK-CAP-110-PM7 (900 mg, 1.56 mmol) was used as starting material to obtain YK-CAP-110-PM8 (triethylamine salt, 500 mg, 0.84 mmol, 54.1%). C12H17F2N5O10P2, MS (ES): m / z (MH-) 490.2.

[0436] Step 9: Synthesis of YK-CAP-110

[0437] According to the YK-CAP-101 synthesis route, YK-CAP-110-PM8 (100 mg, 0.17 mmol) was used as starting material to obtain YK-CAP-110 (25 mg, 22.03 μmol, 13.0%). C33H43F2N15O23P4, MS (ES): m / z (MH-) 1178.1.

[0438] 1H NMR (400 MHz, D2O) δ 8.43 (d, J = 1.5 Hz, 1H), 8.36 (d, J = 2.6 Hz, 1H), 8.12 (d, J = 4.6 Hz, 2H), 6.5, 8 = 10 (H4), J J = 2.2 Hz, 1H), 5.61 (d, J = 4.4 Hz, 1H), 4.90 - 4.88 (m, 3H), 4.85 - 4.82 (m, 1H), 4.62 (t, J = 4.7 Hz. 1H), 1H - 4.5. (d, J = 3.0 Hz, 1H), 4.12 (s, 2H), 4.04 (s, 2H), 3.93 (s, 3H), 3.64 (t, J = 7.0 Hz, 1H), 3.41 (s, 3H), 3.28 (DH, m 3.12). MHz) δ −0.93 (s, 1P), −11.17 (d, J = 19.2 Hz, 1P), −11.35 (d, J = 17.1 Hz, 1P), −24.38 (t, J = 17.6 Hz, 1P).

[0439] 13. Synthesis of YK-CAP-111 Petition 870250022825, dated 3 / 24 / 2025, p. 73 / 331 62 / 120 TBDPSO <,0, TBDPSO YK-CAP.111.PM1 TBDPSO DEA HAW. DIEA. CH,CN YK.CAP-111-PM2 Oh?Oh, paint. H;SO< YK.CAP-111.PM3 •O O AcOH. rt AcO OAc INT-I 1) BSA, DCE. 80 *C 2) TMSOTf, Tol.. 70 X

[0440] YK-CAP-107-PM1 AcOHO- oV NH3. MeOH YK-CAP-111-PM5 ' b nhac HN' Ν' YK-CAP-111-PM9 O – OPOP – OH OH OH TEA Vo. °HN' ,OH POCis YK.CAP-111.PM4 O N-^0Ac' O o —Ο-Ρ-ΟΗ PySSPy, inudazoi TEAP YK-CAP-111-PM8 ZnClj, DMF. ft NH2 PO(M«O)j, or PPhj. TEA. DMF. in O' YK-CAP-111-PM6 YK-CAP-111-PM7 (Me)jSO4, H2O OH TEA NH ZnCI2, DMSO, 37Γ TEA YK-CAP-111 YK-CAP-111-PM100 0\ O'P Õ tH3N -OPOP–OH OH Oh OH

[0441] Step 1: Synthesis of YK-CAP-111-PM1

[0442] YK-CAP-107-PM1 (35.0 g, 79.07 mmol) was dissolved in acetonitrile and water (1:1, 280 mL), then (diacetoxyiodo)benzene (53.5 g, 166.03 mmol), sodium bicarbonate (9.96 g, 118.56 mmol) and TEMPO (1.85 g, 11.85 mmol) were sequentially added to it in an ice bath and the mixture was reacted at room temperature for 2 hours. TLC monitored that the starting material reacted completely. The reaction was terminated. The reaction mixture was quenched with saturated aqueous sodium thiosulfate solution, extracted with EA and the phases were separated. The organic phase was dried over anhydrous sodium sulfate, filtered and evaporated by rotary evaporation to remove the solvent to obtain the crude product YKCAP-111-PM1 (68.0 g) as a brown oil, which was used directly in the next reaction step without purification.

[0443] Step 2: Synthesis of YK-CAP-111-PM2

[0444] YK-CAP-111-PM1 (34.0 g, calculated as 39.54 mmol) was dissolved in acetonitrile and the mixture was 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 minutes, and then diethylamine (6.6 g, 90.24 mmol) was added. The mixture was heated to room temperature and stirred and reacted for 4 hours. The reaction mixture was concentrated under reduced pressure and the residue was purified by silica gel column chromatography (0 to 20% ethyl acetate / n-hexane) to obtain YK-CAP-111-PM2 (17.5 g, 34.20 mmol, 86.5%).

[0445] Step 3: Synthesis of YK-CAP-111-PM3

[0446] YK-CAP-111-PM2 (17.5 g, 34.20 mmol) was dissolved in tetrahydrofuran, then Petition 870250022825, dated 03 / 24 / 2025, page 74 / 331 63 / 120 TBAF (13.4 g, 51.25 mmol) was added and the mixture was stirred and reacted at room temperature for 2 hours. The reaction mixture was evaporated to dryness by rotary evaporation and the residue was purified by silica gel column chromatography (0 to 90% ethyl acetate / n-hexane) to obtain YK-CAP-111-PM3 (8.9 g, 32.56 mmol, 95.2%).

[0447] Step 4: Synthesis of YK-CAP-111-PM4

[0448] YK-CAP-111-PM3 (4.0 g, 14.63 mmol) was dissolved in acetic acid, then sulfuric acid (300 μL) was added and the mixture was stirred at room temperature for 30 minutes. Acetic anhydride (30.0 g, 293.86 mmol) was then added. The mixture was stirred at room temperature for 18 hours. The reaction mixture was added with 200 mL of water and extracted with ethyl acetate. The organic phase was washed three times with saturated aqueous sodium bicarbonate solution to adjust the pH to alkalinity, then dried and evaporated to dryness by rotary evaporation to obtain the crude product YK-CAP-111-PM4 (3.19 g) as a yellow oily liquid, which was used directly in the next reaction step.

[0449] Step 5: Synthesis of YK-CAP-111-PM5

[0450] The intermediate INT-I (3.79 g, 9.76 mmol) was dissolved in 1,2-dichloroethane and N,O bis(trimethylsilyl)acetamide (5.42 g, 26.64 mmol) was added to it. The mixture was heated to 80°C, stirred and reacted for 2 hours and the reaction solvent was removed under reduced pressure. A solution of YK-CAP-111-PM4 (3.19 g) in toluene and TMSOTf (2.96 g, 13.32 mmol) was sequentially added at room temperature. The mixture was then heated to 70°C and stirred and reacted for a further 2 hours. The reaction system was diluted with ethyl acetate, washed once with saturated sodium bicarbonate solution, filtered to remove the insoluble substance and the filtrate phases were separated. The organic phase was dried over anhydrous sodium sulfate and evaporated by rotary evaporation under reduced pressure to remove the solvent. The residue was purified by silica gel column chromatography (0 to 60% ethyl acetate / DCM) to obtain YK-CAP-111-PM5 (2.20 g, 3.20 mmol).

[0451] Step 6: Synthesis of YK-CAP-111-PM6

[0452] YK-CAP-111-PM5 (2.20 g, 3.20 mmol) was dissolved in a 4 M ammonia / methanol and water solution (5:1), and the mixture was stirred and reacted at room temperature for 16 hours. The reaction mixture was evaporated to dryness by rotary evaporation to remove the solvent, and the crude product was recrystallized with ethyl acetate to obtain YK-CAP-111-PM6 (890 Petition 870250022825, dated 03 / 24 / 2025, page 75 / 331 64 / 120 mg, 2.43 mmol, 75.9%). C15H22N6O5, MS (ES): m / z (M+H+) 367.1.

[0453] YK-CAP-111-PM6: 1H NMR (400 MHz, DMSO-d6) δ 11.40 (s, 1H), 8.31 (s, 1H), 6.38 (s, 2H), 6.16 (d, J = 4.3 Hz, 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 = 4.1 Hz, 4H), 2.65 - 2.62 (m, 1H), 1.17 - 1.15 (t, J = 4.1 Hz, 6H).

[0454] Step 7: Synthesis of YK-CAP-111-PM7

[0455] Phosphorus oxychloride (1.3 g, 8.48 mmol) was dissolved in 15 mL of trimethyl phosphate. The mixture was cooled to 0°C under a nitrogen atmosphere and YK-CAP-111-PM6 (880 mg, 2.42 mmol) was slowly added to the above reaction system. The mixture was stirred and reacted at 0°C for approximately 4 hours. After the reaction was complete, the reaction mixture was added with 20 mL of ice-cold water and washed twice with ethyl acetate. The aqueous phase was added with ammonia water to adjust the pH to 3.5 and refrigerated overnight. The pH was continuously adjusted to 6.5 and the mixture was diluted to 400 mL for sample loading. The sample was purified by gel column chromatography (eluted with water and 1.5 M TEAB at a ratio of 5:1). The peak of the target product was collected, concentrated, and lyophilized to obtain YK-CAP-111-PM7 (triethylamine salt, 900 mg, 1.64 mmol, 68.5%) as a white solid. C15H23N6O8P, MS (ES): m / z (MH) 445.1.

[0456] Step 8: Synthesis of YK-CAP-111-PM8

[0457] According to the synthesis route of YK-CAP-101-PM4, YK-CAP-111-PM7 (900 mg, 1.64 mmol) was used as starting material to obtain YK-CAP-111-PM8 (sodium salt, 730 mg, 1.41 mmol, 85.7%). C18H25N8O7P, MS (ES): m / z (MH) 495.1.

[0458] Step 9: Synthesis of YK-CAP-111-PM9

[0459] According to the synthesis route of YK-CAP-101-PM5, YK-CAP-111-PM8 (720 mg, 1.39 mmol) was used as starting material to obtain YK-CAP-111-PM9 (triethylamine salt, 750 mg, 1.20 mmol, 86.1%). C15H24N6O11P2, MS (ES): m / z (MH-) 525.1.

[0460] Step 10: Synthesis of YK-CAP-111-PM10

[0461] YK-CAP-111-PM9 (400 mg, 0.64 mmol) was dissolved in H2O (20 mL) and the mixture was added with glacial acetic acid to adjust the pH to 4.0. Dimethyl sulfate (800 μL, 8.45 mmol) was added to the same within 30 minutes. The reaction system was added with aqueous NaOH solution (0.1 M) to maintain the pH between 3.8 and 4.1 and stirred at temperature Petition 870250022825, dated 03 / 24 / 2025, page 76 / 331 65 / 120 ambient temperature for 5 hours. After the reaction was complete, the reaction mixture was extracted twice with dichloromethane. The pH of the aqueous phase was adjusted to 6.5 and the volume was fixed at 400 mL. The residue was purified by gel column chromatography (eluted with water and 1 M TEAB at a 3:2 ratio). The peak of the target product 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.

[0462] Step 11: Synthesis of YK-CAP-111

[0463] According to the synthesis route of YK-CAP-101, YK-CAP-111-PM10 (100 mg, 0.16 mmol) was used as the starting material to obtain YK-CAP-111 (ammonium salt, 50 mg, pmol%), 39.0 C37H52N16O24P4, MS (ES): m / z (MH') 1127.2.

[0464] 'HNMR (400 MHz, D2O) δ 8.43 (d, J= 1.3 Hz, 1H), 8.35 (s, 1H), 8.06 (d, J= 6.0 Hz, 2H), 6.01 (d, J= 5.6. Hz (3 Hz), J=8.1 1H), 5.82 (d, J= 4.5 Hz, 1H), 4.97 - 4.84 (m, 3H), 4.66 (t, J= 4.8 Hz, 1H), 4.51 - 4.35 (m, 4H), 4.32 (H, 5), J= 4, 4. 4.09 (s, 2H), 3.97 (s, 3H), 3.72 (t, J= 7.0 Hz, 1H), 3.35 - 3.21 (d, J= 4.1 Hz, 4H), 3.04 (s, 3H), 1.94-t , 1.14 (1.1 , 1.14). J= 4.1 Hz, 6H).31PNMR(D2O, 162 MHz) δ −0.92 (s, IP), −11.15 (d, J= 19.2 Hz, IP), −11.62 (d,J=17.1Hz, IP), −23.46 = 1 (t., J

[0465] 14. Synthesis of YK-CAP-112 TBDPSO DPA TBDPSO . o TBAF OH < °s O AcO Ac;O. paint. H2SO4\ 1)BSA. DCE. 80 C 2) TMSOTf, Td, 70 C NHAc INT-I AcO

[0466] HOΌ' O YK-CAP-111-PM1 YK-CAP-112-PM1 YK-CAP-112-PM2 AcOH. rt YK-CAP-112-PM3 °ύν OA*-, ° NHAc N HATU, DIEA. CH3CN YOUR N Ρ^Νν^< ' ohN -,N OH nh2 YK-CAP-112-PM4 YK-CAP-112-PM5 poa, -O- -'° POiMeOb 0C The lP-OH OH HjN PySSPy. imidazole AM PPh3, TEA, DMF, rt0OPN_j0ÓNanTFAP Ah E on ZnCI2. DMF, rt YK-CAP-112-PM6 YK-CAP-112-PM7 NH2λν o ηΛγΝ' HjN^NNO Oo-OP-O-POH % OH OH o CH3 hn-SÁ HjN (Me)2SO4. H2O N N_o... -o »-ff-OP-OH O OH no O?o°N'NH H ONa^O.#NN NH2H / i HO” OH INT-II CH3 OzN rt TEA Zfíty. DMSO. 37 C o Ah L AM OOO OPOPO-PO 0 ò 0 NH^ íV MM o á~oN NH 3NH / OPO CH,NO „ I HO HO YK-CAP-112-PM8 YK-CAP-112-PM9 YK-CAP-112 nh2

[0467] Step 1: Synthesis of YK-CAP- 112-PM1 Petition 870250022825, of 24 / 03 / 2025, p. 77 / 331 66 / 120

[0468] According to the synthesis route of YK-CAP-111-PM2, YK-CAP-111-PM1 (10.0 g, 21.90 mmol) and dipropylamine (5.5 g, 54.35 mmol) were used as starting materials to obtain YK-CAP-111-111-PM2 (11.01 g 20.38 mmol, 93.1%).

[0469] Step 2: Synthesis of YK-CAP-112-PM2

[0470] According to the YK-CAP-111-PM3 synthesis route, YK-CAP-112-PM1 (11.0 g, 20.38 mmol) was used as starting material to obtain YK-CAP-112-PM2 (6.0 g, 19.91 mmol, 97.7%).

[0471] Step 3: Synthesis of YK-CAP-112-PM3

[0472] According to the synthesis route of YK-CAP-111-PM4, YK-CAP-112-PM2 (6.0 g, 19.91 mmol) was used as starting material to obtain the crude product YK-CAP-112-PM3 (5.1 g) as a yellow oily liquid, which was used directly in the next reaction step.

[0473] Step 4: Synthesis of YK-CAP-112-PM4

[0474] According to the YK-CAP-111-PM5 synthesis route, YK-CAP-112-PM3 (5.1 g) was used as starting material to obtain YK-CAP-112-PM4 (3.9 g, 5.45 mmol). C36H41N7O9, MS (ES): m / z (M+H+) 716.3.

[0475] Step 5: Synthesis of YK-CAP-112-PM5

[0476] According to the synthetic route of YK-CAP-111-PM6, YK-CAP-112-PM4 (3.9 g, 5.45 mmol) were used as the starting material to obtain YK-CAP-112-PM5 (1.7 g, 4.31 mmol, 79.1%). C17H26N6O5, MS (ES): m / z (M+H+) 395.2.

[0477] YK-CAP-112-PM5:1H NMR (400 MHz, DMSO-d6) δ 11.37 (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), 3.99 - 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 = 1.8 Hz, 6H).

[0478] Stage 6: Summary of YK-CAP-112-PM6

[0479] De acordo com a rota de síntese de YK-CAP-111-PM7, YK-CAP-112-PM5 (1,7 g, 4,31 mmol) foi usado como material de partida para obter YK-CAP-112-PM6 (sal de trietilamina, 1,80 g, 3,13 mmol, 72,6%). C17H27N6O8P, MS (ES): m / z (MH) 473,1.

[0480] Stage 7: Summary of YK-CAP-112-PM7

[0481] According to the synthesis route of YK-CAP-101-PM4, YK-CAP-112-PM6 (1.8 g, 3.13 mmol) was used as starting material to obtain YK-CAP-112-PM7 (sodium salt, Petition 870250022825, dated 03 / 24 / 2025, page 78 / 331 67 / 120 1.47 g, 2.69 mmol, 86.0%). C20H29N8O7P, MS (ES): m / z (MH) 523.1.

[0482] Step 8: Synthesis of YK-CAP-112-PM8

[0483] According to the synthesis route of YK-CAP-101-PM5, YK-CAP-112-PM7 (1.47 g, 2.69 mmol) was used as starting material to obtain YK-CAP-112-PM8 (triethylamine salt, 1.30 g, 1.98 mmol, 73.7%). C17H28N6O11P2, MS (ES): m / z (MH-) 553.1.

[0484] Step 9: Synthesis of YK-CAP-112-PM9

[0485] According to the synthesis route of YK-CAP-111-PM10, YK-CAP-112-PM8 (600 mg, 0.92 mmol) was used as starting material to obtain YK-CAP-112-PM9 (triethylamine salt, 200 mg, 0.30 mmol, 32.6%). C18H30N6O11P2, MS (ES): m / z (MH-) 567.1.

[0486] Step 10: Synthesis of YK-CAP-112

[0487] In accordance with the synthesis route of YK-CAP-101, YK-CAP-112-PM9 (200 mg, 0.30 mmol) was used as the starting material to obtain YK-CAP-112 (ammonium salt, 50 mg, 38.23 pmol, 12.8%). C39H56N16O24P4, MS (ES): m / z (MH-) 1255.2.

[0488] 1HNMR (400 MHz, D2O) δ 8.43 (d, J = 1.3 Hz, 1H), 8.34 (s, 1H), 8.11 (d, J = 6.1 Hz, 2H), 6.05 (d, J = 5.3 Hz, 1H), 5.87 (d, J = 2.3 Hz, 1H), 5.81 (d, J = 4.4 Hz, 1H), 4.91 to 4.82 (m, 2H), 4.64 (t, J = 4.7), H (m, 5H), 4.30 (d, J = 4.6 Hz, 1H), 4.22 (s, 2H), 4.11 (s, 2H), 3.96 (s, 3H), 3.72 (t, J = 7.0 Hz, 1H), (s, 3H), 2.22 - 2.14 (m, 1H), 1.58 - 1.54 (m, 4H), 0.87 (t, J = 1.8 Hz, 6H).31P NMR (D2O, 162 MHz) δ -0.94 (s, 1P), -1. 1P), -11.52 (d, J = 17.2 Hz, 1P), -23.11 (t, J = 17.3 Hz, 1P).

[0489] 15. Synthesis of YK-CAP-113 Petition 870250022825, of 24 / 03 / 2025, p. 79 / 331 68 / 120 T1PPS<1 Pin the na Shots <> (Ml DeM-Deep Λ<*Ν.40Τ ? O ρι-,ρ οι,β· ,τιι>. -tv. Shoots i »-Rulλ π bolt T> *·ΒΒΝ.40Χ. bolt V°O ------------► n?DS \— / \*οο;ΙΙλμγ(.λ(, oh YK-C'AP-113-PMl YK-CAP-II3-PM2 YK-CAP-113-PM3 YK-CAMI3-PM4 SWI.Mcl ΠΠ Pindina OMTiCt YK-CAP II3-FM7 <) ο ηΆ * I1NH ONN HO- ,, x J.... OWTlO- <j X ’ χ \_J Η I \_i O Λ«Ο IM. ~ <> ' χ„ <> S-HHT. ACN,S°Sjj3 0.1 Μ I emTHF / Piiidine / Water . AcO <>Ac YK CAP-IB-PMIO OR4 AP-IB-PMII N PN— OH - DIEA. l-methyhmidazoL ACN 0.1 M I.em THF Pmdma Water MMhO.McOH YKCAP-IBPMU ÍH> PO. <M11 HO HÓ YKCAP-IB-PMB

[0490] <> <H, HX Hs\ X ·> <> -θ4 O>-N • O ON.N Ah Ah ZíUI. DMSO. IT-T ·« 'S.\ Η.Χ ' X ' X· OO OJ ojoloío.(>. * Oh oh Ah Ah ixh.- ó „ x.1..,, or <> ° I 7 O f X XH:< O I HO HO VK <AF-IIJ

[0491] Step 1: Synthesis of YK-CAP-113-PM1

[0492] Methyl-beta-D-ribofuranoside (100.0 g, 0.61 mol) was dissolved in 1 L of anhydrous pindine and TIPDSC1 (230.6 g, 0.73 mol) was added dropwise to the same in an ice water bath. After the dropwise addition was completed, the mixture was heated to room temperature, stirred, and reacted for 12 hours. The reaction mixture was concentrated under reduced pressure to remove a large amount of solvent, and the residue was purified by silica gel column chromatography (0 to 30% ethyl acetate / / / -hexane) to obtain YKCAP-113-PM1 (212.2 g, 0.52 mol, 85.7%).

[0493] Step 2: Synthesis of YK-CAP-113-PM2

[0494] YK-CAP-113-PM1 (212.2 g, 0.52 mol) was dissolved in 2 L of acetonitrile and Dess-Martin periodinane (485.1 g, 1.14 mol) was added to it. The mixture was heated to 40°C and stirred and reacted for 12 hours. The reaction mixture was cooled to the Petition 870250022825, dated 03 / 24 / 2025, page 80 / 331 69 / 120 room temperature, filtered and the filtrate was evaporated to dryness by rotary evaporation under reduced pressure to obtain YK-CAP-113-PM2 (208.8 g, 0.52 mol, 98.9%).

[0495] Step 3: Synthesis of YK-CAP-113-PM3

[0496] Bromomethyltriphenylphosphonium bromide (408.7 g, 1.14 mol) was dissolved in tetrahydrofuran (3000 mL). The mixture was cooled to -78°C and a 2.5 M n-butyllithium solution in tetrahydrofuran (456 mL, 1.14 mol) was slowly added dropwise to it. After the dropwise addition was complete, the reaction system was heated to 0°C and stirred and reacted for 2 hours. The above reaction system was cooled again to -78°C and a solution of YK-CAP-113-PM2 (208.8 g, 0.52 mol) in tetrahydrofuran (400 mL) was slowly added dropwise to it. After the dropwise addition was complete, the reaction system was heated to room temperature and stirred and reacted overnight. The reaction system was quenched with a saturated ammonium chloride solution (2000 mL) and extracted with ethyl acetate (2000 mL x 3).The organic phases were combined, washed with saturated aqueous NaCl solution, dried over anhydrous sodium sulfate, filtered, and evaporated to dryness by rotary evaporation. The residue was purified by silica gel chromatography (0 to 20% ethyl acetate / n-hexane) to obtain YK-CAP-113-PM3 (108.8 g, 0.27 mol, 52.4%).

[0497] Step 4: Synthesis of YK-CAP-113-PM4

[0498] A 1 M solution of 9-BBN in tetrahydrofuran (540 mL, 0.54 mmol) was added to a three-necked flask and the mixture was cooled to 0°C under a nitrogen atmosphere. A solution of YK-CAP-113-PM3 (108.8 g, 0.27 mol) in tetrahydrofuran was slowly added dropwise to the above system. After the dropwise addition was complete, the mixture was stirred and reacted at room temperature for 2 hours. The reaction system was cooled again to 0°C and water / tetrahydrofuran (1:1) (540 mL), 2 N sodium hydroxide solution (540 mL) and 30% hydrogen peroxide (460 mL) were sequentially and slowly added to it. After the addition was complete, the reaction system was heated to room temperature and stirred and reacted for a further 3 hours. The reaction system was diluted with ethyl acetate and the phases were separated. The aqueous phase was extracted again once more with ethyl acetate.The organic phases were combined, dried, filtered, and the filtrate was evaporated to dryness by rotary evaporation under reduced pressure. The residue was purified by silica gel column chromatography (0 to 17% ethyl acetate / n-hexane) to obtain... Petition 870250022825, dated 03 / 24 / 2025, page 81 / 331 70 / 120 YK-CAP-113-PM4 (98.0 g, 0.23 mol, 86.2%).

[0499] Step 5: Synthesis of YK-CAP-113-PM5

[0500] 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) and the mixture was cooled to 0 °C. Iodomethane (13.5 g, 95.11 mmol) was then added dropwise under a nitrogen atmosphere and the mixture was stirred and reacted for about 5 hours. After the reaction was complete, the system was quenched with water (10 mL) and extracted with EA (200 mL x 3). The organic phases were combined, washed with saturated aqueous NaCl solution, dried over anhydrous sodium sulfate, filtered, and evaporated to dryness by rotary evaporation. The residue was purified by silica gel chromatography (0 to 20% ethyl acetate / n-hexane) to obtain YK-CAP-113PM5 (17.8 g, 40.95 mmol, 86.1%).

[0501] Step 6: Synthesis of YK-CAP-113-PM6

[0502] Benzoyladenosine (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 under a nitrogen atmosphere, stirred for 12 hours, and evaporated to dryness by rotary evaporation to remove the solvent. YK-CAP-113-PM5 (17.8 g, 40.95 mmol) was dissolved in 1,2-dichloroethane (300 mL) and added to the above residue. Trimethylsilyl trifluoromethanesulfonate (10.1 g, 48.97 mmol) was then added, and the mixture was reacted at 80°C for 5 hours. TLC monitored that the reaction was complete. After filtration, the filtrate was evaporated to dryness by rotary evaporation under reduced pressure. The residue was purified by silica gel column chromatography (0 to 80% ethyl acetate / n-hexane) to obtain YK-CAP-113-PM6 (14.5 g, 22.59 mmol, 55.2%). C31H47N5O6Si2, MS (ES): m / z (M+H+) 642.3.

[0503] Step 7: Synthesis of YK-CAP-113-PM7

[0504] YK-CAP-113-PM6 (14.5 g, 22.59 mmol) was dissolved in tetrahydrofuran (100 mL), then tetrabutylammonium fluoride (23.6 g, 90.26 mmol) was added and the mixture was stirred and reacted at room temperature for 1 hour. The reaction system was then topped up with a saturated aqueous solution of ammonium chloride and extracted with ethyl acetate (200 mL x 3). The organic phases were combined, washed with a saturated aqueous solution of NaCl, dried over anhydrous sodium sulfate, filtered, and evaporated to dryness by rotary evaporation. The Petition 870250022825, dated 03 / 24 / 2025, page 82 / 331 71 / 120 residue was purified by silica gel chromatography (0 to 60% ethyl acetate / n-hexane) to obtain YK-CAP-113-PM7 (7.5 g, 18.78 mmol, 83.1%). C19H21N5O5, MS (ES): m / z (M+H+) 400.2.

[0505] Step 8: Synthesis of YK-CAP-113-PM8

[0506] YK-CAP-113-PM7 (7.5 g, 18.78 mmol) was dissolved in pyridine (50 mL), then 4,4'-dimethoxytritil chloride (10.2 g, 30.10 mmol) was added at room temperature and the mixture was stirred and reacted at room temperature for 3 hours. TLC monitored that the reaction was complete. The reaction mixture was quenched with 10 mL of methanol, stirred for 10 minutes and subjected to rotary evaporation until no solvent was evaporated to obtain a 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.

[0507] Step 9: Synthesis of YK-CAP-113-PM9

[0508] YK-CAP-113-PM8 (8.8 g, 12.54 mmol) was dissolved in acetonitrile (100 mL), then N-methylimidazole (1.1 g, 13.40 mmol) and bis(diisopropylamino)(2-cyanoethoxy)phosphine (11.3 g, 37.49 mmol) were added sequentially and the mixture was stirred and reacted for 6 hours under a nitrogen atmosphere. TLC monitored that the reaction was complete. The reaction mixture was diluted with ethyl acetate. The organic phase was washed with saturated aqueous sodium bicarbonate solution and the phases were separated. The organic phase was washed once with water and the phases were 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 subjected to rotary evaporation under reduced pressure until no solvent had evaporated to obtain a crude product. The crude product was purified by flash column chromatography.The product fraction was evaporated to remove the solvent, then dissolved in ethyl acetate, added dropwise with chilled n-hexane and stirred for 10 minutes. The residue was filtered to obtain YK-CAP113-PM9 (8.1 g, 8.98 mmol, 71.6%). C49H56N7O8P, MS (ES): m / z (MH-) 900.4.

[0509] Step 10: Synthesis of YK-CAP-113-PM10

[0510] YK-CAP-113-PM9 (4.0 g, 4.43 mmol), N-isobutyryl-2',3'-acetylguanosine (2.0 g, 4.57 mmol) and tetrazole (3.1 g, 44.25 mmol) were dissolved in acetonitrile (50 mL) and the mixture was Petition 870250022825, dated 03 / 24 / 2025, page 83 / 331 72 / 120 stirred and reacted at room temperature for 3 hours under a nitrogen atmosphere. The above reaction mixture was added to 0.1 M iodine solution (53 mL), stirred and reacted for a further 1 hour, diluted with 200 mL of brine and extracted with dichloromethane (200 mL x 3). The organic phases were combined, dried over anhydrous sodium sulfate, filtered and evaporated to dryness by rotary evaporation. The organic phase was subjected to rotary evaporation under reduced pressure until no solvent was evaporated to obtain YK-CAP113-PM10 (5.2 g, 4.15 mmol, 93.5%). C61H64N11O17P, MS (ES): m / z (MH) 1252.4.

[0511] Step 11: Synthesis of YK-CAP-113-PM11

[0512] YK-CAP-113-PM10 (5.2 g, 4.15 mmol) was dissolved in an 80% aqueous acetic acid solution (20 mL) and the mixture was stirred and reacted at room temperature for 2 hours under a nitrogen atmosphere. The reaction system was concentrated under reduced pressure to remove the acetic acid and subjected to rotary evaporation until no solvent evaporated to obtain a 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.

[0513] Step 12: Synthesis of YK-CAP-113-PM12

[0514] YK-CAP-113-PM11 (3.0 g, 3.15 mmol) was dissolved in acetonitrile (30 mL), then N-methylimidazole (0.5 g, 6.09 mmol) and bis(diisopropylamino)(2-cyanoethoxy)phosphine (2.8 g, 9.29 mmol) were sequentially added to it and the mixture was stirred and reacted for 2 hours under a nitrogen atmosphere. The above reaction mixture was added with 0.1 M iodine solution (40 mL), stirred and reacted for another 1 hour, diluted with 200 mL of brine and extracted with dichloromethane (200 mL x 3). The organic phases were combined, dried over anhydrous sodium sulfate, filtered and evaporated to dryness by rotary evaporation. The organic phase was subjected to rotary evaporation under reduced pressure until no solvent was evaporated to obtain YK-CAP-113-PM12 (1.8 g, 1.66 mmol, 52.6%). C43H50N12O18P2, MS (ES): m / z (MH-) 1083.3.

[0515] Step 13: Synthesis of YK-CAP-113-PM13

[0516] YK-CAP-113-PM12 (1.8 g, 1.66 mmol) was dissolved in a mixed solution of ammonia water (10 mL) and methanol (5 mL). The mixture was heated to 50°C under a nitrogen atmosphere and stirred and reacted for 24 hours. The reaction system was concentrated under Petition 870250022825, dated 03 / 24 / 2025, page 84 / 331 73 / 120 reduced pressure to remove solvent and subjected to rotary evaporation until no solvent was evaporated to obtain a crude product. The crude product was dissolved in water (50 mL) until clarified and purified by gel column chromatography (eluted with water and 1.5 M TEAB at a ratio of 10:1). The peak of the target product was collected, concentrated, lyophilized and further desalted by high-performance preparative liquid chromatography (50 mM TEAB and methanol mobile phase system) to obtain YK-CAP-113PM13 (triethylamine salt, 660 mg, 0.80 mmol, 48.4%) as a white solid. C22H30N10O14P2, MS (ES): m / z (MH-) 719.1.

[0517] Stage 14: Summary of YK-CAP-113

[0518] According to the synthesis method of YK-CAP-101, YK-CAP-113-PM11 (150 mg, 0.18 mmol) and Im-m7GDP (193 mg, 0.36 mmol) for use as part materials to obtain YK-CAP-113 (amônium salt, 40 mg, 33.04 pmol, 18.1%). C33H45N15O24P4, MS (ES): m / z (MH-) 1158.2.

[0519] 1HNMR (400 MHz, D2O) δ 8.44 (d, J = 1.3 Hz, 1H), 8.33 (s, 1H), 8.08 (d, J = 6.0 Hz, 2H), 6.02 (d, J = 5.4 Hz, 1H), 5.90 (d, J = 2.3 Hz, 1H), 5.82 (d, J = 4.4 Hz, 1H), 4.95 - 4.82 (m, 3H), 4.63 (t, J = 4.5 Hz, 1H), 4.53 - 4.31 (m, 4H), 4.26 (d, J = 4.5 Hz, 1H), 4.21 (s, 3H), 4.09 (s, 1H), 3.76 (t, J = 7.0 Hz, 1H), 3.43 (s, 3H), 3.35 (s, 3H), 3.21 - 3.11 (m, 2H), 2.42 2.35 (m, 1H).31PNMR (D2O,162 MHz) δ -0.94 (s, 1P), -11.34 (d, J = 19.2 Hz, 1P), -11.82 (d, J = 17.3 Hz, 1P), -23.11 (t, J = 17.3 Hz, 1P).

[0520] 16. Summary of YK-CAP-114 Petition 870250022825, 03 / 24 / 2025, pág. 85 / 331 74 / 120

[0521] IIPDS k- / O (HI YK-CAP-II3-PM4 ° <> c> ° ►rims kJ mp.iMAü.n <Hr( °5o S>HAH; <HI ° O TlA.ActXIMM * TIPDS k- / * o -XIInpos V— / o NHAc YK-CAP-II4-PMI YK-CAP-II4-PM2 YK-CAP-I 14-PMI (> HX ‘SN N^-B / THEM. fear. 130°C.2h; ° <> Xs *. TYPES kJ ixt. TMsoTi.wn .$h oM1A« nir ho οN. íHAI.Rl .bolt HO XHAc O MX · Pyridinasx * IAITiO os. . DMTfCI and * HO XHAc N: YK-CAP-I 14- PM4 YK-CAP-I I4-PM5 DMTfOo WATER. 1-Methylimidazole. ACN HOon o OP O XHA6 AcO OAc and HX XHAc YK4 AMI4-PM· NII O N II HODEA. 1-methylimidazoi. ACN 0.1 Mi, and THEPyridine / Water YK-CAP-114-PM9 <> HI: HX jj-N. HN XN* <> O Ο P Ο PNIO OXa All All ZflClj. DMSO. JTC YK-CAP-I I4-PM6 O II ONE N N Ac<) OAc Neil O N -N:· II 5-BBT. ACN 0.1 M I.em THF / Pyridine / Water -NHAN YK-CAP-I 14-l'M* N.H., H.O.McOH. NIIAK Sire HO PO (HI Art) OAc YK-CAP-I I4-PMI0 ch,Ml,,ISV NN HN X ''*· OOOJ <> I· <> p <> r <1 . „ . N () of ' All Ahjnho xnAe\ Nnh or ο II o ¥N Ml·· < <> I υκ<λγ-ιι4 ho ito Overall Xll; N . N __ $ XHAfJ ,JNil NOT Ο=ά OH <- J HO HO YK-CAP-114-Ι'ΜΙΙ XII.

[0522] Stage 1: Synthesis of YK-CAP-114-PM1

[0523] 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). The mixture was cooled to 0°C and stirred and reacted for approximately 5 hours under a nitrogen atmosphere. After the reaction was complete, the system was quenched with water (10 mL) and extracted with EA (200 mL x 3). The organic phases were combined, washed with saturated aqueous NaCl solution, dried over anhydrous sodium sulfate, filtered, and evaporated to dryness by rotary evaporation. The residue was purified by silica gel chromatography (0 to 20% ethyl acetate / w-hexane) to obtain YK-CAP-114-PM1 (15.9 g, 28.92 mmol, 91.5%). C27H43NO7Si2, MS (ES): m / z (M+H+) 550.3.

[0524] Step 2: Synthesis of YK-CAP-114-PM2

[0525] YK-CAP-114-PM1 (15.9 g, 28.92 mmol) was dissolved in 300 mL of ethanol and 50 mL Petition 870250022825, dated 03 / 24 / 2025, page 86 / 331 75 / 120 of hydrazine hydrate were added to the mixture. The mixture was heated to 80°C and stirred and reacted for 12 hours. After the reaction was complete, the system was quenched with water (10 mL) and extracted with EA (200 mL x 3). The organic phases were combined, washed with saturated aqueous NaCl solution, dried over anhydrous sodium sulfate, filtered, and evaporated to dryness by rotary evaporation to obtain YK-CAP-114-PM2 (12.0 g, 28.59 mmol, 98.9%). C19H41NO5Si2, MS (ES): m / z (M+H+) 420.3.

[0526] Step 3: Synthesis of YK-CAP-114-PM3

[0527] YK-CAP-114-PM2 (12.0 g, 28.59 mmol) and triethylamine (5.9 g, 58.31 mmol) were dissolved in dichloromethane (150 mL). The mixture was cooled to 0°C and acetyl chloride (3.3 g, 42.04 mmol) was added slowly dropwise. After the dropwise addition was complete, the reaction system was maintained at 0°C and stirred and reacted for 3 hours. After the reaction was complete, the system was quenched with water (20 mL) and extracted with DCM (200 mL x 3). The organic phases were combined, washed with saturated aqueous NaCl solution, dried over anhydrous sodium sulfate, filtered, and evaporated to dryness by rotary evaporation. The residue was purified by silica gel chromatography (0 to 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.

[0528] Step 4: Synthesis of YK-CAP-114-PM4

[0529] According to the synthesis method of YK-CAP-113-PM6, YK-CAP-114-PM3 (12.2 g, 26.42 mmol) was used as starting material to obtain YK-CAP-114-PM4 (8.5 g, 12.71 mmol, 48.1%). C32H48N6O6Si2, MS (ES): m / z (M+H+) 669.3.

[0530] Step 5: Synthesis of YK-CAP-114-PM5

[0531] According to the synthesis method of YK-CAP-113-PM7, YK-CAP-114-PM4 (8.5 g, 12.71 mmol) was used as starting material to obtain YK-CAP-114-PM5 (4.4 g, 10.32 mmol, 81.2%). C20H22N6O5, MS (ES): m / z (M+H+) 427.2.

[0532] Step 6: Synthesis of YK-CAP-114-PM6

[0533] According to the synthesis method of YK-CAP-113-PM8, YK-CAP-114-PM5 (4.4 g, 10.32 mmol) was used as starting material to obtain YK-CAP-114-PM6 (5.5 g, 7.55 mmol, 73.1%). C41H40N6O7, MS (ES): m / z (M+H+) 729.3.

[0534] Step 7: Synthesis of YK-CAP-114-PM7 Petition 870250022825, dated 03 / 24 / 2025, page 87 / 331 76 / 120

[0535] According to the synthesis method of YK-CAP-113-PM9, YK-CAP-114-PM6 (5.5 g, 7.55 mmol) was used as starting material to obtain YK-CAP-114-PM7 (5.0 g, 5.38 mmol, 71.3%). C50H57N8O8P, MS (ES): m / z (MH) 927.4.

[0536] Step 8: Synthesis of YK-CAP-114-PM8

[0537] According to the synthesis method of YK-CAP-113-PM10, YK-CAP-114-PM7 (5.0 g, 5.38 mmol) was used as starting material to obtain YK-CAP-114-PM8 (5.7 g, 4.45 mmol, 82.7%). C62H65N12O17P, MS (ES): m / z (MH) 1279.4.

[0538] Step 9: Synthesis of YK-CAP-114-PM9

[0539] According to the synthesis method of YK-CAP-113-PM11, YK-CAP-114-PM8 (5.7 g, 4.45 mmol) was used as starting material to obtain YK-CAP-114-PM9 (3.5 g, 3.58 mmol, 80.4%). C41H47N12O15P, MS (ES): m / z (MH) 977.3.

[0540] Step 10: Synthesis of YK-CAP-114-PM10

[0541] According to the synthesis method of YK-CAP-113-PM12, YK-CAP-114-PM9 (3.5 g, 3.58 mmol) was used as starting material to obtain YK-CAP-114-PM10 (2.0 g, 1.80 mmol, 50.3%). C44H51N13O18P2, MS (ES): m / z (MH) 1110.3.

[0542] Step 11: Synthesis of YK-CAP-114-PM11

[0543] According to the synthesis method of YK-CAP-113-PM13, YK-CAP-114-PM10 (2.0 g, 1.80 mmol) was used as starting material to obtain YK-CAP-114-PM11 (triethylamine salt, 800 mg, 0.94 mmol, 52.4%). C23H31N11O14P2, MS (ES): m / z (MH-) 746.2.

[0544] Step 12: Synthesis of YK-CAP-114

[0545] According to the synthesis method of YK-CAP-113, YK-CAP-114-PM11 (150 mg, 0.18 mmol) was used as starting material to obtain YK-CAP-114 (ammonium salt, 26 mg, 21.01 pmol, 11.9%). C34H46N16O24P4, MS (ES): m / z (MH-) 1185.2.

[0546] 1H NMR (400 MHz, D2O) δ 8.42 (d, J = 1.4 Hz, 1H), 8.32 (s, 1H), 8.11 (d, J = 6.0 Hz, 2H), 6.12 (d, J = 5.4 Hz, 1H), 5.92 (d, J = 2.3 Hz, 1H), 5.84 (d, J = 4.3 Hz, 1H), 4.92 - 4.86 (m, 3H), 4.61 (t, J = 4.5 Hz, 1H), 4.52 - 4.33 (m, 4H), 4.21 (d, J = 4.5 Hz, 1H), 4.16 (s, 3H), 4.03 (s, 1H), 3.72 (t, J = 6.3 Hz, 1H), 3.42 (s, 3H), 3.32 - 3.23 (m, 2H), 2.42 - 2.32 (m, 1H), 1.88 (s, 3H).31P NMR (D2O, 162 MHz) δ -0.88 (s, 1P), -11.12 (d, J = 19.1 Hz, 1P), -12.21 (d, J = 17.2 Hz, 1P), -23.55 (t, J = 17.1 Hz, 1P).

[0547] 17. Summary of YK-CAP-115 Petition 870250022825, 03 / 24 / 2025, pág. 88 / 331 77 / 120 INI ΗΝ

[0548] O o TIPOS V- / Ό DA.S1 \,-BzAJIMDS. Pyridine. 130eC. 2h; π if IO Pindina OH YKCAP IB PM4 VKX AP-II5-PMI HN (Kt, TMSOTf.m .Sh DMTrt)o T1PDS HN N,DMTfO—.o IK) VK-CAP-115-PM4 Ad )11 HS UN IK”— DIEA. 1-Methylinudazole. The ACN HN SH O NS II AcO OAc YK4AMIS-PM7 All All ONi More;. DMSO. J7'C C> YK4 AP-115-PM: HO−·, o Act) TBAFJtT. bolt YKCAP-II5PM3 HN DMTrCI NH O DMTrO II 04· o AcO OAc N'N . Η I YK-CAP-II5-PM6 YK4 AP IIS PM5 O 5BBT. ACS 0.1 M l of THFTyridine / Water ΝΗ,Η-O.McOII YK-t AP II5 PMX DIEA. 1-methylimidazole. ACN 0.1 M Glue THF / Pyridine / Water He ('ll, ii A HS ·N SH; ILN N· Ah Ah ASH* NH Ml. Nrc YK-CAP-II5 with HO NH; NK THEIR IK) P o « OH1 TEArtI OP oh’ HO HO N SH; YK4 APII5PSW

[0549] Step 1: Synthesis of YK-CAP-115-PM1

[0550] YK-CAP-113-PM4 (20.0 g, 47.54 mmol) was dissolved in dichloromethane (200 mL). The mixture was cooled to -40°C and a solution of diethylaminosulfur trifluoride (9.2 g, 57.08 mmol) in dichloromethane (20 mL) was slowly added dropwise. After the dropwise addition was complete, the mixture was slowly heated to 0°C and stirred and reacted for 4 hours. TLC monitored that the reaction was complete. The reaction system was quenched with a saturated aqueous solution of sodium bicarbonate (100 mL) and extracted with dichloromethane (200 mL x 3). The organic phases were combined, washed with a saturated aqueous solution of NaCl, dried over anhydrous sodium sulfate, filtered, and evaporated to dryness by rotary evaporation. The residue was purified by silica gel chromatography (0 to 40% ethyl acetate / / / -hexane) to obtain YK-CAP-115-PM1 (11.2 g, 26.50 mmol, 55.7%).

[0551] Step 2: Synthesis of YK-CAP-115-PM2

[0552] According to the synthesis method of YK-CAP-113-PM6, YK-CAP-115-PM1 (11.2 g, 26.50 mmol) was used as starting material to obtain YK-CAP-115-PM2 (8.6 g, 13.65 Petition 870250022825, dated 03 / 24 / 2025, page 89 / 331 78 / 120 mmol, 51.5%). C30H44FN5O5SÍ2, MS (ES): m / z (M+H+) 630.3.

[0553] Step 3: Synthesis of YK-CAP-115-PM3

[0554] According to the synthesis method of YK-CAP-113-PM7, YK-CAP-115-PM2 (8.6 g, 13.65 mmol) was used as starting material to obtain YK-CAP-115-PM3 (4.8 g, 12.39 mmol, 90.8%). C18H18FN5O4, MS (ES): m / z (MH-) 388.1.

[0555] Step 4: Synthesis of YK-CAP-115-PM4

[0556] According to the synthesis method of YK-CAP-113-PM8, YK-CAP-115-PM3 (4.8 g, 12.39 mmol) was used as starting material to obtain YK-CAP-115-PM4 (5.6 g, 8.12 mmol, 65.5%). C39H36FN5O6, MS (ES): m / z (MH) 690.3.

[0557] Step 5: Synthesis of YK-CAP-115-PM5

[0558] According to the synthesis method of YK-CAP-113-PM9, YK-CAP-115-PM4 (5.6 g, 8.12 mmol) was used as starting material to obtain YK-CAP-115-PM5 (5.0 g, 5.62 mmol, 69.2%). C48H53FN7O7P, MS (ES): m / z (MH-) 888.4.

[0559] Step 6: Synthesis of YK-CAP-115-PM6

[0560] According to the synthesis method of YK-CAP-113-PM10, YK-CAP-115-PM5 (5.0 g, 5.62 mmol) was used as starting material to obtain YK-CAP-115-PM6 (4.9 g, 3.94 mmol, 70.2%). C60H61FN11O16P, MS (ES): m / z (MH-) 1240.4.

[0561] Step 7: Synthesis of YK-CAP-115-PM7

[0562] According to the synthesis method of YK-CAP-113-PM11, YK-CAP-115-PM6 (4.9 g, 3.94 mmol) was used as starting material to obtain YK-CAP-115-PM7 (2.3 g, 2.45 mmol, 62.0%). C39H43FN11O14P, MS (ES): m / z (MH-) 938.3.

[0563] Step 8: Synthesis of YK-CAP-115-PM8

[0564] According to the synthesis method of YK-CAP-113-PM12, YK-CAP-115-PM7 (2.3 g, 2.45 mmol) was used as starting material to obtain YK-CAP-115-PM8 (2.0 g, 1.86 mmol, 76.2%). C42H47FN12O17P2, MS (ES): m / z (MH-) 1071.3.

[0565] Step 9: Synthesis of YK-CAP-115-PM9

[0566] According to the synthesis method of YK-CAP-113-PM13, YK-CAP-115-PM8 (2.0 g, 1.86 mmol) was used as starting material to obtain YK-CAP-115-PM9 (triethylamine salt, 720 mg, 0.89 mmol, 47.7%). C21H27FN10O13P2, MS (ES): m / z (MH-) 707.1.

[0567] Step 10: Synthesis of YK-CAP-115 Petition 870250022825, dated 03 / 24 / 2025, page 90 / 331 79 / 120

[0568] According to the synthesis method of YK-CAP-113, YK-CAP-115-PM9 (150 mg, 0.19 mmol) was used as starting material to obtain YK-CAP-115 (ammonium salt, 31 mg, 25.86 pmol, 14.0%). C32H42FN15O23P4, MS (ES): m / z (M-H') 1146.1.

[0569] 'HNMR (400 MHz, D2O) δ 8.44 (d, J= 1.4 Hz, 1H), 8.32 (s, 1H), 8.12 (d, J= 6.0 Hz, 2H), 6.12 (d, J= 5.2. 2.2 Hz, J=5. 1 1H), 5.82 (d, J= 4.2 Hz, 1H), 4.95 - 4.82 (m, 3H), 4.80 - 4.73 (m, 2H), 4.53 (t, J= 4.5 Hz, 1H), 4.41. - 1, J= 4.34 ( Hz, 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(D.2,8-s) 162 M (d, J= 19.1 Hz, IP), −12.41 (d, J= 17.2 Hz, IP), −22.42 (t,J= 17.0 Hz, IP).

[0570] 18. Synthesis of YK-CAP-116 O IIN1· or s1'svy0 IIIX'<? C / °l,AST lxMθ CyuN. BeA.HMIIS. Piridina. 13ff> C.2h; <>- <>N* ΠΙΙ A>' OilΤ,ΡΙΧ* Ο =oπΜ>5χιο' >-F DCt. TMSOTr.SOT_My TYPES )— / TBAFjn.pernoite F _·F YK-CATII3-PM4 ΥΚ-ΤΆΡ-ΙI6-PM1 YK-CAP-I16-PM2 YKCAP-I I6-PM3

[0571] I YK-CAP·!16-PM6 o nMl O OONNY - . The “ AcO OAc HHT. ATS 0.1 ΜI emTHF / Pyridine / Water YES- ow YES YK-CAP-II6-PM4 YKX AP-II6-PM7 N , N <» N1 1 The t OIOI on1; ' ÓfAftla o=p oir o K <> Pyridine DMTphil - v ' < p N / OPO or N;% O N=— * N DAY. 1-methymidazole. ACN 0.1 M LemTHF / Pyridine? Water VM AP Hfr-PMK Clh AcO OAc YK-CAP-IÍ6-PM9 The f HN i HjK ' N ' O In HN ΙΙΛ N HO HO MC-CAP-116-PMIO ZnCT. DMSO. 37T Ml. S N1 ' s \ OO O1 O ' ,· OOO 7 AllTAll jmv of-s NLo õr1 Ó JN SH. K <> I YK-CAP-II6 HO HO

[0572] Stage 1: Synthesis of YK-CAP-116-PM1

[0573] YK-CAP-113-PM4 (20.0 g, 47.54 mmol) was dissolved in acetonitrile (200 mL) and 2-iodoxybenzoic acid (16.0 g, 57.14 mmol) was added to it. The mixture was heated to 90°C and stirred and reacted for 4 hours. The reaction mixture was cooled to room temperature, filtered, and the filtrate was evaporated to dryness by rotary evaporation under pressure. Petition 870250022825, dated 03 / 24 / 2025, page 91 / 331 80 / 120 reduced to obtain YK-CAP-116-PM1 (18.4 g, 43.95 mmol, 92.4%).

[0574] Step 2: Synthesis of YK-CAP-116-PM2

[0575] 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 diethylaminosulfur trifluoride (21.3 g, 132.14 mmol) in dichloromethane (40 mL) was slowly added dropwise. After the dropwise addition was complete, the reaction system was slowly heated to 0°C and stirred and reacted for 4 hours. TLC monitored that the reaction was complete. The reaction system was quenched with saturated aqueous sodium bicarbonate solution (200 mL) and extracted with dichloromethane (300 mL x 3). The organic phases were combined, washed with saturated aqueous NaCl solution, dried over anhydrous sodium sulfate, filtered, and evaporated to dryness by rotary evaporation. The residue was purified by silica gel chromatography (0 to 40% ethyl acetate / n-hexane) to obtain YK-CAP-116-PM2 (12.8 g, 29.05 mmol, 66.1%).

[0576] Step 3: Synthesis of YK-CAP-116-PM3

[0577] According to the synthesis method of YK-CAP-113-PM6, YK-CAP-116-PM2 (12.8 g, 29.05 mmol) was used as starting material to obtain YK-CAP-116-PM3 (9.6 g, 14.82 mmol, 51.0%). C30H43F2N5O5Si2, MS (ES): m / z (M+H+) 648.3.

[0578] Step 4: Synthesis of YK-CAP-116-PM4

[0579] According to the synthesis method of YK-CAP-113-PM7, YK-CAP-116-PM3 (9.6 g, 14.82 mmol) was used as starting material to obtain YK-CAP-116-PM4 (5.2 g, 12.83 mmol, 86.6%). C18H17F2N5O4, MS (ES): m / z (M+H+) 406.1.

[0580] Step 5: Synthesis of YK-CAP-116-PM5

[0581] According to the synthesis method of YK-CAP-113-PM8, YK-CAP-116-PM4 (5.2 g, 12.83 mmol) was used as starting material to obtain YK-CAP-116-PM5 (6.4 g, 9.04 mmol, 70.5%). C39H35F2N5O6, MS (ES): m / z (M+H+) 708.3.

[0582] Step 6: Synthesis of YK-CAP-116-PM6

[0583] According to the synthesis method of YK-CAP-113-PM9, YK-CAP-116-PM5 (6.4 g, 9.04 mmol) was used as starting material to obtain YK-CAP-116-PM6 (5.5 g, 6.06 mmol, 67.0%). C48H52F2N7O7P, MS (ES): m / z (MH-) 906.4.

[0584] Step 7: Synthesis of YK-CAP-116-PM7

[0585] According to the synthesis method of YK-CAP-113-PM10, YK-CAP-116-PM6 (5,5 Petition 870250022825, dated 03 / 24 / 2025, page 92 / 331 81 / 120 g, 6.06 mmol) was used as starting material to obtain YK-CAP-116-PM7 (5.3 g, 4.21 mmol, 69.4%). C60H60F2N11O16P, MS (ES): m / z (MH) 1258.4.

[0586] Step 8: Synthesis of YK-CAP-116-PM8

[0587] According to the synthesis method of YK-CAP-113-PM11, YK-CAP-116-PM7 (5.3 g, 4.21 mmol) was used as starting material to obtain YK-CAP-116-PM8 (2.8 g, 2.92 mmol, 69.5%). C39H42F2N11O14P, MS (ES): m / z (MH-) 956.3.

[0588] Step 9: Synthesis of YK-CAP-116-PM9

[0589] According to the synthesis method of YK-CAP-113-PM12, YK-CAP-116-PM8 (2.8 g, 2.92 mmol) was used as starting material to obtain YK-CAP-116-PM9 (2.2 g, 2.02 mmol, 69.0%). C42H46F2N12O17P2, MS (ES): m / z (MH-) 1089.3.

[0590] Step 10: Synthesis of YK-CAP-116-PM10

[0591] According to the synthesis method of YK-CAP-113-PM13, YK-CAP-116-PM9 (2.2 g, 2.02 mmol) was used as starting material to obtain YK-CAP-116-PM10 (triethylamine salt, 580 mg, 0.70 mmol, 34.7%). C21H26F2N10O13P2, MS (ES): m / z (MH-) 725.1.

[0592] Step 11: Synthesis of YK-CAP-116

[0593] According to the synthesis method of YK-CAP-113, YK-CAP-116-PM10 (150 mg, 0.18 mmol) was used as starting material to obtain YK-CAP-116 (ammonium salt, 17 mg, 13.97 pmol, 7.7%). C32H41F2N15O23P4, MS (ES): m / z (MH-) 1164.1.

[0594] 1HNMR (400 MHz, D2O) δ 8.52 (d, J = 1.4 Hz, 1H), 8.45 (s, 1H), 8.25 (d, J = 6.0 Hz, 2H), 6.29 (d, J = 5.2 ( 5.2 Hz), J ,6 = 1 1H), 5.89 (d, J = 4.2 Hz, 1H), 4.91 - 4.81 (m, 3H), 4.59 (t, J = 4.6 Hz, 1H), 4.42 - 4.31 (m, 4H), 4.25 (H, 1 J 4.1), 4.25 (H, J 4.1). 4.02 (s, 1H), 3.86 - 3.73 (m, 2H), 3.63 (t, J = 6.3 Hz, 1H), 3.43 (s, 3H), 2.41 - 2.27 (m, 1H).31P NMR (D2O-M0, 18) 16 −11.65 (d, J = 19.1 Hz, 1P), −12.27 (d, J = 17.8 Hz, 1P), −23.88 (t, J = 17.3 Hz, 1P).

[0595] 19. Synthesis of YK-CAP-117 Petition 870250022825, dated 3 / 24 / 2025, p. 93 / 331 82 / 120

[0596] tfM) you— θ O t-HuSa. Mail on HO V---------BnO » !„,<>' AcO.nrf H«SO4IKK) —-----o«Ac AcOH.fi _ RnOOAfc' / Λ <^O it'll / b NIIAc INTI hBSA .DCE.8O V 2) TMSOTf. Round. 7(PC YK-CMMI7-PMI YK-CAP-II7-PM2 K>ΛN )KO^vS°i Λ IMKDC.MNN O ' - ------------. UoO OAc · NHAc YK-CA?ir-PM3 HO o O ; IK) ΥΚ4.ΆΡ-Ι I7-PM4 Mel. DM! HS-\*ΚΧΊ' oW o •n- O UH PUMeOh.OV OH Ah All*»ltA o PySSPy. imidazolH^ m, rtA.0Mi n o'·»' Ah Ah ° rN· N Ο O -OP OP-OH ' OH OH Ah Ah °TFA YK-CAP-inPMK YK-CAP-II7-PM5 YKX Ap.lirPMfc YK-C AP-II7-PM7 IK) OH HEADLINE CH. N N· <) Ah A N1N OO OJ o jNo ^OOO *' Y11° 1KU00 N JNH. oroCH.NN -.J ΥΚ4ΆΡ-Π7 HO IK) NH NH;

[0597] Stage 1: Synthesis of YK-CAP-117-PM1

[0598] According to the synthesis route of YK-CAP-107-PM4, 3-O-benzyl-4-C benzyloxymethyl-1,2-O-isopropylidene-AD-ribofuranose (10.00 g, 24.97 mmol) was used as starting material to obtain YK-CAP-117-PM1 (8.62 g, 20.80 mmol, 83.3%).

[0599] Step 2: Synthesis of YK-CAP-117-PM2

[0600] YK-CAP-117-PM1 (8.62 g, 20.80 mmol) was dissolved in acetic acid, then acetic anhydride (21.23 g, 207.95 mmol) was added, and concentrated sulfuric acid (380 pL) was added slowly dropwise to the same. After the dropwise addition was completed, the mixture was stirred at room temperature for 16 hours. The reaction mixture was added with 300 mL of water and extracted with ethyl acetate. The organic phase was washed three times with saturated aqueous sodium bicarbonate solution to adjust the pH to alkalinity, then dried and evaporated to dryness by rotary evaporation. The residue was purified by silica gel chromatography (0 to 40% ethyl acetate / w-hexane) to obtain YK-CAP-117-PM2 (8.13 g, 17.73 mmol, 85.3%) as a yellow oily liquid.

[0601] Step 3: Synthesis of YK-CAP-117-PM3

[0602] According to the synthesis route of YK-CAP-104-PM11, YK-CAP-117-PM2 (8.13 g, 17.73 mmol) was used as starting material to obtain YK-CAP-117-PM3 (6.24 g, 7.93 mmol, 44.7%). C43H42N6O9, MS (ES): m / z (M+H+) 787.3. Petition 870250022825, dated 03 / 24 / 2025, page 94 / 331 83 / 120

[0603] Step 4: Synthesis of YK-CAP-117-PM4

[0604] YK-CAP-117-PM3 (6.24 g, 7.93 mmol) was dissolved in dichloromethane (200 mL). The above 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 to it. After the dropwise addition was complete, the reaction system was slowly heated to 0°C and stirred at the same temperature for 3 hours. TLC showed that the reaction was complete. The reaction system was cooled again to -40°C, quenched with methanol, evaporated to dryness by rotary evaporation, left at room temperature for 24 hours, DCM was added dropwise to precipitate a solid, and filtered to obtain a brown crude product, which was purified by preparative HPLC to obtain YK-CAP117-PM4 (1.19 g, 3.64 mmol, 45.8%). C12H17N6O5, MS (ES): m / z (M+H+) 328.1.

[0605] YK-CAP-117-PM4:1H NMR (400 MHz, MeOD) δ 8.15 (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).

[0606] Step 5: Synthesis of YK-CAP-117-PM5

[0607] According to the synthesis route of YK-CAP-101-PM3, YK-CAP-117-PM4 (1.19 g, 3.64 mmol) was used as starting material to obtain YK-CAP-117-PM5 (triethylamine salt, 1.23 g, 2.42 mmol, 66.5%). C12H18N5O9P, MS (ES): m / z (MH-) 406.1.

[0608] Step 6: Synthesis of YK-CAP-117-PM6

[0609] According to the synthesis route of YK-CAP-101-PM4, YK-CAP-117-PM5 (1.23 g, 2.42 mmol) was used as starting material to obtain YK-CAP-117-PM6 (sodium salt, 993 mg, 2.07 mmol, 85.6%). C15H20N7O8P, MS (ES): m / z (MH-) 456.1.

[0610] Step 7: Synthesis of YK-CAP-117-PM7

[0611] According to the synthesis route of YK-CAP-101-PM5, YK-CAP-117-PM6 (993 mg, 2.07 mmol) was used as starting material to obtain YK-CAP-117-PM7 (triethylamine salt, 794 mg, 1.35 mmol, 65.1%). C12H19N5O12P2, MS (ES): m / z (MH-) 486.1.

[0612] Step 8: Synthesis of YK-CAP-117-PM8

[0613] According to the synthesis route of YK-CAP-101-PM6, YK-CAP-117-PM7 (794 mg, 1.35 mmol) was used as starting material to obtain YK-CAP-117-PM8 (triethylamine salt, 520 mg, 0.86 mmol, 64.0%). C13H21N5O12P2, MS (ES): m / z (MH-) 500.1.

[0614] Step 9: Synthesis of YK-CAP-117 Petition 870250022825, dated 03 / 24 / 2025, page 95 / 331 84 / 120

[0615] According to the YK-CAP-101 synthesis route, YK-CAP-117-PM8 (150 mg, 0.25 mmol) was used as starting material to obtain YK-CAP-117 (ammonium salt, 26 mg, 20.95 pmol, 8.4%). C34H47N15O25P4, MS (ES): m / z (M-H') 1188.1.

[0616] 'HNMR (400 MHz, D2O) δ 8.44 (d, J = 1.5 Hz, 1H), 8.26 (d, J = 2.5 Hz, 2H), 8.15 (d, 7= 4.5 Hz, 1H), 6.2=1, 51 (d. (d, 7= 2.4 Hz, 1H), 5.51 (d, 7= 4.6 Hz, 1H), 4.90 - 4.86 (m, 3H), 4.60 (t, 7= 4.7 Hz, 1H), 4.50 - 7.38, d 9 (m, 2 1H), 4.13 (s, 2H), 4.05 (s, 2H), 3.92 (s, 3H), 3.54 (t, 7= 7.0 Hz, 2H), 3.44 - 3.31 (m, 3H), 2.97 (s, MR, 3D(H).321P). (s, IP), −11.22 (d, 7= 19.1 Hz, IP),−11.32 (d, 7= 16.4 Hz, IP), −22.12 (t, 7= 17.4 Hz, IP).

[0617] 20. Synthesis of YK-CAP-118

[0618] BnO HOBnO HO . O DAST. DC M BnO O Ai'^O. fall IÇSO4 AvOII. rt OH YES,,NN NH; YK-CAP-11K-PM4 E.g. DMF HN H;NN POlMcOh, 0*C Clij N Ο. ΑίΓΑΐΙF YK-CAP-II8PMK BnO .0OAc BnO<)Av YK-CAP-II8-PM2 YK-CAP-1 IS-PMI POC1. BnO°' O OP Ol< OH PySSPy. imidazole PPh,. TEA. DMF. rt HN YK-CAP-I1K-PM5 .. o ύ NfO-t ο NNONa ' f NH. CH, Oil TLA Ο Ο N .. , »i'o ÍH;! YES. \.O,NN NH. HO (HI ΙΓΤΤ-ΙΙ ZnCL. DMSO. 17%' HN H;S -N· Sc -N IN / % OR N-. Ml Ac 1) RSA. DCE. MFC ;>TMSOTf. Round. wc0What: « PN 7 ON·· lie Oh oh F YK-CAJ'-IIH-PMft ΑιΓAhf 3NI / YK-CAP-1 IK BnO Oh. BCI,. DC M BnO OAc ' Ml Ac YK-CAP-1 IK-PM3 O HN ItAP HjN OO tPOP-OH ΖηΠ,. DMF. ft YK-CAP-1 IK-PM7 NH; Ó 0 , o-é-ò O<1N HEY I tõ HO NH N NH, TEA

[0619] Step 1: Synthesis of YK-CAP-118-PM1

[0620] According to the synthesis route of YK-CAP-109-PM1, 3-O-benzyl-4-C benzyloxymethyl-1,2-O-isopropylidene-AD-ribofuranose (10.00 g, 24.97 mmol) was used as starting material to obtain YK-CAP-118-PM1 (9.11 g, 22.64 mmol, 90.6%).

[0621] Step 2: Synthesis of YK-CAP-118-PM2

[0622] According to the YK-CAP-117-PM2 synthesis route, YK-CAP-118-PM1 (9.11 g, 22.64 mmol) was used as starting material to obtain YK-CAP-118-PM2 (7.18 g, 16.08 mmol, 71.0%).

[0623] Step 3: Synthesis of YK-CAP-118-PM3 Petition 870250022825, dated 03 / 24 / 2025, page 96 / 331 85 / 120

[0624] According to the YK-CAP-104-PM11 synthesis route, YK-CAP-118-PM2 (7.18 g, 16.08 mmol) was used as starting material to obtain YK-CAP-118-PM3 (6.64 g, 8.57 mmol, 53.3%). C42H39FN6O8, MS (ES): m / z (M+H+) 775.3.

[0625] Step 4: Synthesis of YK-CAP-118-PM4

[0626] According to the YK-CAP-117-PM4 synthesis route, YK-CAP-118-PM3 (6.64 g, 8.57 mmol) was used as starting material to obtain YK-CAP-118-PM4 (2.31 g, 7.33 mmol, 85.5%). C11H14FN5O5, MS (ES): m / z (M+H+) 316.1.

[0627] YK-CAP-118-PM4:1H NMR(400 MHz, MeOD) δ 8.12 (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).

[0628] Step 5: Synthesis of YK-CAP-118-PM5

[0629] According to the synthesis route of YK-CAP-101-PM3, YK-CAP-118-PM4 (1.30 g, 4.12 mmol) was used as starting material to obtain YK-CAP-118-PM5 (triethylamine salt, 1.15 g, 2.32 mmol, 56.2%). C11H15FN5O8P, MS (ES): m / z (MH-) 394.1.

[0630] Step 6: Synthesis of YK-CAP-118-PM6

[0631] According to the synthesis route of YK-CAP-101-PM4, YK-CAP-118-PM5 (1.15 g, 2.32 mmol) was used as starting material to obtain YK-CAP-118-PM6 (sodium salt, 870 mg, 1.86 mmol, 80.4%). C14H17FN7O7P, MS (ES): m / z (MH) 444.1.

[0632] Step 7: Synthesis of YK-CAP-118-PM7

[0633] According to the synthesis route of YK-CAP-101-PM5, YK-CAP-118-PM6 (870 mg, 1.86 mmol) was used as starting material to obtain YK-CAP-118-PM7 (triethylamine salt, 664 mg, 1.15 mmol, 61.9%). C11H16FN5O11P2, MS (ES): m / z (MH-) 474.1.

[0634] Step 8: Synthesis of YK-CAP-118-PM8

[0635] According to the synthesis route of YK-CAP-101-PM6, YK-CAP-118-PM7 (664 mg, 1.15 mmol) was used as starting material to obtain YK-CAP-118-PM8 (triethylamine salt, 419 mg, 0.71 mmol, 61.6%). C12H18FN5O11P2, MS (ES): m / z (MH-) 488.1.

[0636] Step 9: Synthesis of YK-CAP-118

[0637] According to the synthesis route of YK-CAP-101, YK-CAP-118-PM8 (150 mg, 0.25 mmol) was used as starting material to obtain YK-CAP-118 (ammonium salt, 33 mg, 7% pmol%), 26.166 C33H44FN15O24P4, MS (ES): m / z (MH-) 1176.1.

[0638] 1H NMR (400 MHz, D2O) δ 8.37 (d, J = 1.2 Hz, 1H), 8.33 − 8.21 (m, 1H), 8.14 (d, J Petition 870250022825, dated 3 / 24 / 2025, p. 97 / 331 86 / 120 = 4.5 Hz, 2H), 6.33 (d, J= 5.1 Hz, 1H), 5.61 (d, J= 2.3 Hz, 1H), 5.45 (d, J= 4.6 Hz, 1H), 4.92 - 4. 4.83 (m, 4 2H), 4.50 - 4.38 (m, 4H), 4.31 (d, J= 2.9 Hz, 1H), 4.11 (s, 2H), 4.01 (s, 2H), 3.90 - 3.76 (m, 3H), 3.52 - 4.6 (H0), J 3.34 (m, 3H).31PNMR (D2O, 162 MHz) δ −0.90 (s, IP), −11.23 (d, J= 19.1 Hz, IP), −11.59 (d, J= 16.0 Hz, IP), −22 J = 1 (t., IP).

[0639] 21. Synthesis of YK-CAP-119 hA or BnO s°HO A BnO IBX.CH«CN BnO χ,θ-x O DAST The BnO χθ O Ac_4>..nr H SO. BnO AcOH. π .OAc INTIFBnOOAcllBSA.DCE.KO <6 2) TMSOTf. Toll.. 70 « YK-C AP-II9-PMI YK-CAP-II9-PM2 YK4 AMI9-PM3 BnO <> N.. fbm) yes BCIj. DCM HO -NS OH POC1, HNl–N NHAc t >HN HOUMI NH; RKMcOh. re Ο P OH PySSPy. imidazole H?N . I'Ph,, NO. DMF. rt Oh Oh ftTEA YK-CAP-119-PM4 YK-CAP-II9-PMS YK-CAP-I19-PMb NII. 'N

[0640] O HN * .-N. HN ο O -Ο-Ρ-Ο-Ρ-α< Mel. 'X oil OH ---TEA YIU AP II9-PMK UN O 1. αιΟΝ,νγ Ο Ο- N of o ·. . . ΟΧ·\°-ΝN SH; CH, UN HN DMF HN NrO-PO-iOH ~ χ Ó OH Am Ah» ► nA YK-CAP-119-PM·» HO OH I NT H ZaCI.. DMSO. 37V HN -ο P oF o ^00 Oh oh. -° x ON· An Ah *NTtAP ZaCb. DMF. n VK-CAP-l 19-PM7 NH. N . f-Nil.opo°(1l,s°L_ OI YK-CAP-119 HOX HO Ml N Ml.·

[0641] Step 1: Synthesis of YK-CAP- 119-PM1

[0642] 3-O-benzyl-4-C-benzyloxymethyl-l,2-O-isopropylideno-AD-ribofuranose (10.0 g, A solution of 24.97 mmol) was dissolved in 50 mL of acetonitrile, and 2-iodoxybenzoic acid (10.49 g, 37.46 mmol) was added. The mixture was then heated to 70°C, stirred, and reacted for 2 hours. TLC monitored that the reaction was complete. The reaction was terminated. The reaction mixture was cooled to room temperature, then filtered through diatomaceous earth, and the filter cake was rinsed with acetonitrile (50 mL). The filtrate was evaporated to dryness by rotary evaporation under reduced pressure and dried under vacuum to obtain YK-CAP-119-PM1 (10.21 g) as a clear yellow liquid, which was used directly in the next step (the yield was calculated as 100%).

[0643] Step 2: Synthesis of YK-CAP-119-PM2

[0644] According to the YK-CAP-110-PM1 synthesis route, YK-CAP-119-PM1 (10.21 g, calculated as 24.97 mmol) was used as starting material to obtain YK-CAP-119-PM2 Petition 870250022825, dated 03 / 24 / 2025, page 98 / 331 87 / 120 (8.16 g, 19.41 mmol, 77.7%).

[0645] Step 3: Synthesis of YK-CAP-119-PM3

[0646] According to the YK-CAP-117-PM2 synthesis route, YK-CAP-119-PM2 (8.16 g, 19.41 mmol) was used as starting material to obtain YK-CAP-119-PM3 (6.55 g, 14.10 mmol, 72.7%).

[0647] Step 4: Synthesis of YK-CAP-119-PM4

[0648] According to the synthesis route of YK-CAP-104-PM11, YK-CAP-119-PM3 (6.55 g, 14.10 mmol) was used as starting material to obtain YK-CAP-119-PM4 (5.77 g, 7.28 mmol, 51.6%). C42H38F2N6O8, MS (ES): m / z (M+H+) 793.3.

[0649] Step 5: Synthesis of YK-CAP-119-PM5

[0650] According to the YK-CAP-117-PM4 synthesis route, YK-CAP-119-PM4 (5.77 g, 7.28 mmol) was used as starting material to obtain YK-CAP-119-PM5 (2.16 g, 6.48 mmol, 89.0%). C11H13F2N5O5, MS (ES): m / z (M+H+) 334.1.

[0651] YK-CAP-119-PM5:1H NMR (400 MHz, MeOD) δ 8.24 (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).

[0652] Step 6: Synthesis of YK-CAP-119-PM6

[0653] According to the synthesis route of YK-CAP-101-PM3, YK-CAP-119-PM5 (1.50 g, 4.50 mmol) was used as starting material to obtain YK-CAP-119-PM6 (triethylamine salt, 1.29 g, 2.51 mmol, 55.7%). C11H14F2N5O8P, MS (ES): m / z (MH-) 412.1.

[0654] Step 7: Synthesis of YK-CAP-119-PM7

[0655] According to the synthesis route of YK-CAP-101-PM4, YK-CAP-119-PM6 (1.29 g, 2.51 mmol) was used as starting material to obtain YK-CAP-119-PM7 (sodium salt, 991 mg, 2.04 mmol, 81.4%). C14H16F2N7O7P, MS (ES): m / z (MH-) 462.1.

[0656] Step 8: Synthesis of YK-CAP-119-PM8

[0657] According to the synthesis route of YK-CAP-101-PM5, YK-CAP-119-PM7 (991 mg, 2.04 mmol) was used as starting material to obtain YK-CAP-119-PM8 (triethylamine salt, 774 mg, 1.30 mmol, 63.8%). C11H15F2N5O11P2, MS (ES): m / z (MH-) 492.0.

[0658] Step 9: Synthesis of YK-CAP-119-PM9

[0659] According to the YK-CAP-101-PM6 synthesis route, YK-CAP-119-PM8 (774 mg, 1.30 mmol) was used as starting material to obtain YK-CAP-119-PM9 (salt of Petition 870250022825, dated 03 / 24 / 2025, page 99 / 331 88 / 120 triethylamine, 533 mg, 0.88 mmol, 67.3%). C12H17F2N5O11P2, MS (ES): m / z (M-H') 506.1.

[0660] Step 10: Synthesis of YK-CAP-119

[0661] According to the YK-CAP-101 synthesis route, YK-CAP-119-PM9 (150 mg, 0.25 mmol) was used as starting material to obtain YK-CAP-119 (ammonium salt, 32 mg, 25.67 pmol, 10.3%). C33H43F2N15O24P4, MS (ES): m / z (M-EF) 1194.1.

[0662] 'HNMR (400 MHz, D2O) δ 8.35 (d, J= 1.2 Hz, 1H), 8.30 - 8.19 (m, 1H), 8.12 (d, J = 4.4 Hz, 2H), 6.25 (d, J= 5.2 Hz, 1H), 5.65 (d, J= 2.2 Hz, 1H), 5.27 (d, J= 4.5 Hz, 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= 2.9 Hz, 1H), 4.04 (s, 2H), 3.88 - 3.75 (m, 3H), 3.42 (t, J= 7.0 Hz, 1H), 3.36 - 3.31 (m, 3H).31P NMR(D2O, 162 MHz) δ-0.93 (s, IP), -12.13 (d, J= 19.1 Hz, IP),-13.31 (d, J= 16.1 Hz, IP), -24.57 (t, 7= 17.4 Hz, IP).

[0663] 22. Síntese do composto 5227:

[0664] BnO O l-BuNa. Mcl HO . BnO ° THF BnO <^O 0 ’’O' BnO ° 5227-S IK) HO OH NIL <227·ΡΜ4 POC1 5227-PMI POtMcO)vO°C O Md. DMF N CH hl _O. Ah Ah 5227-PMK HN HN H0-O-0-OH , 0 OH TEA AcjO. water ll?SO4 AcOH. rt Ah Ah ' 5227 PM5 N .. BnO OAc BnOOAc 5227-PM2 TEA HN-ZL O NHAc INT-I BnO . THEIR DBSA.IXE.WT !)TMSOTf.Tol..70T AM PySSPy. imidazole H»NN PPhi.THA.DMf.rt Nil· po-\o ns ON,' *So O 0- N^amiop 0 .Ml ONa\O^NN NHj HO OH INT-II ZnCI2. DMSO. 37T ,<) O k. Ο P NN AllTAll 5227-PM6 BnO OAc f NHAc 5227-PM3 HN TRAP ZnCh. DMF. rt () ' 0 TEA BCh. DCM OOO 0 O 0 OH 5227-PM7 HN H,N N' nr ,-N Ah Ah NII 3NH / oo 0 -o-0 o-0 o-0 o->„0 0 0 0 o HO HO

[0665] According to the YK-CAP-117 synthesis method, 5227-S was used as starting material to obtain 35 mg of compound 5227.

[0666] 23. Synthesis of CAP-2'O-ethyl Petition 870250022825, dated 03 / 24 / 2025, page 100 / 331 89 / 120 CAP-2'O-ethyl - OH OH

[0667]

[0668] ethyl.

[0669]

[0670] According to the method in W02023025073A1, 47 mg of CAP-2'O were obtained. Example 2: In vitro transcription yield of mRNA and capping rate. I. Structural differences in capsular analogs Petition 870250022825, dated 03 / 24 / 2025, page 101 / 331 90 / 120

[0671]

[0672] Table 1: Structures of cap analogs Name Structure Observation YK-CAP-101 9 h, nh2hi / ιΓΛ z / N1i nh / V o 9 9 9 JN η ,_O_p_O_p_O_p-O ο IN ΙΊ ό ό ó W g H0 i ó 0H ιH ιH ù3<H o=p-õ ch,\AnANHj YK-CAP-101 ÓH ÕH Structure projected in the present disclosure, synthesized in Example 1 YK-CAP-102 O çh, nh2 hn |Τ\ / ''Τι N H'N N NLo _o-{J_O-4-O-ü-O. 0 Ϋ N í 1 ill Ο Jl OOO \ 7 ιι AcHN^Y^OH q / 'qf'NH OH 3NH4 ? - JI J o=Po CH3N^N^NH2 YK-CAP-102 OH ÒH Structure projected in the present disclosure, synthesized in Example 1 YK-CAP-103 I NH2hX1 / > H2N-^N ,N*· OOO \ JI Z II / II N^T II 3. ο-τ-ο-Ρ-ο-Ρ-ο-Ρ-ο-ι,ο-J o < 7 0 0 0 \ 7 JÍ OH OH 3NH4· Ó - Õ (z J| 0=P-0 CH, n- <n*<NH2 YK-CAP-103 ÕH 0H Estrutura projetada na presente divulgação, sintetizada no Exemplo 1 Petition 870250022825, of 24 / 03 / 2025, p. 102 / 331 91 / 120

[0673] YK-CAP-104 ff N-1, A <N~líS 2 ~ O-P-O—P-ο—P-O-1 Λ 1 N <_>222 \ 7 m -V 3NH,* YV / ÃA / O=pO LJ YK CAP· 104 ς----{ ÔH ÓH Structure designed in the present disclosure, summarized in Example 1 YK-CAP-105 fí P> NH, A ?Ίι Ϊ η,νλ\λ^ 0 0 ο • Λ ί—Ο-Ρ-Ο-Ρ-Ο-Ρ-Ο-· η 1 Ν ν°-> Α AA fl wXx YK-CAP.1M ÓH Structure designed in the present disclosure, summarized in Example 1 YK-CAP-106 Ο ΓΗ JÍ . J ΝΗ, XjX e n AL £ '> ?Ί| ? :----f OH OH Structure designed in this disclosure, summarized in Example 1 YK-CAP-108 U FH> NH, a ? yS JXL „ W υ°γ ό AA vU X Rn·^ Wh. · oMh, <XX, 1 YK-CAP‘06 ·---. OH ÓH Estrutura projetada na presente divulgação, sintetizada no Exemplo 1 Petition 870250022825, dated 03 / 24 / 2025, page 103 / 331 92 / 120

[0674] YK-CAP-109 I PH> NH, , 8 ? <x? ,N,- YK-CAP--W ÔH Õh Estrutura projetada na presente divulgação, sintetizada no Exemplo 1 YK-CAP-110 fl FK· NH, JaJQ o o o < = Λ j-O-P-O-^-O—P-O—। n I : ___' 1 1 1 L-D*^J G <_>OOO x 7 H XX õ _ô ;γγ rL SN-I. o=JO O 1 | ^oJ ÔH ÕH YK-CAP-110 The structure projected in the present disclosure, synthesized in Example 1 YK.-CAP-111 J? PH» NHj „Xa>. s , , ΜΛ N = Ϊ lí 11 o οηΛ.χ-χ 1Λ (ΪΎ Ο ·* 3HH,' O=pO CH, S--k's <iK'Nhi ÔH ÔH Υ«ΑΡ···1 Estrutura projetada na presente divulgação, sintetizada no Exemplo 1 YK-CAP-112 U ΡΗ· nh2 <'nyS = Λ —o—P-O-P-O-P-O-, n 1 eS A A A S õhX z\z j -Á (JJ1 Ο Ϋ 3NH<* O=*-O CniN^-N^lMH; YK-CAP-112 ÕH ÕH Estrutura projetada na presente divulgação, sintetizada no Exemplo 1 YK-CAP-113 u PH> NH_. HN |r\ N^Á η,νΑΑ^ sss <1J >—< - - - >—f 0 OH OH 3NHZ õ \ a=k-õ °x < Y1 V^OJ YK-CAP-113 >----f ÕH ÔH Structure designed in the present disclosure, not used in Example 1 Petition 870250022825, dated 03 / 24 / 2025, page 104 / 331 93 / 120

[0675] YK-CAP-114 YK-CAP-115 YK-CAP-116 YK-CAP-117 YK-CAP-118 The structure outlined in this publication is summarized in... Example 1 The structure outlined in this publication is summarized in... Example 1 The structure outlined in this publication is summarized in... Example 1 The structure outlined in this publication is summarized in... Example 1 The structure outlined in this publication is summarized in... Example 1 Petition 870250022825, dated 03 / 24 / 2025, page 105 / 331 94 / 120

[0676] YK.-CAP-119 j? NH, 0 0 0 tjj i „ —0—pO—PO—PO—» η 1 sinx OH OH cr . - A. <' Jj'* * O=7O CH, L^oJ ÒH ÕH YK-CAP-11» Structure designed in this disclosure, synthesized in Example 1 5227 fí PH» sh2 Xjj Uo^r°-r°-ro''oi <o-j 0 <_2>o ° ° ? 'C_z Π 0H 0H 1KIH * ? - % < Jü JL 4 OPO CH, NN NHj 1 ÕH ÕH 5227 Synthesized in Example 1, compound 5227 on page 124 of the descriptive report of WO2022051677A1 CAP-2 Ό-ethyl JJ P*3 NHj jl L / / 4i 7 0 0 0 5 Λ J OH OH 3»*.· ô Ã N-JLh o=po / <114 <j nh, 1 cap-ro-elhy=":" oh sintetizado no exemplo 1, tabela 6 na página 27 do relatório descritivo de w02023025073a1 n-7113 2 fh’ nh2 í? s 9 \a <j : ~ — o-p-o-p-o-p-o-i n n ç 7 000 k y 1—i · 0 smi,· õ õ n__jl „ ' - chj tf *h o="P-O" a 11 j n^n^nhj s. ^o~j n7113 \__z óh õh comprado jiangsu synthgene biotechnology co., ltd., composto descrito reivindicação 15 cn116751827bPetition 870250022825, dated 03 / 24 / 2025, p. 106 / 331 95 / 120 Compound 14

[0677] HN3OO2 móA Acquired from Jiangsu Synthgene Biotechnology Co., Ltd., component 14 on page 90 of CN115803333A Acquired from Guangzhou Henovcom Bioscience Co., Ltd., component 3 on page 9 of CN115260264B Acquired from Jiangsu Synthgene Biotechnology Co., Ltd., compound 31 on page 21 of WO2023 / 147352A1

[0678] As can be seen in Table 1, the chemical structures of compounds YK-CAP-101 to 119 in the present disclosure have some similarities and some significant differences compared with mRNA cap analogs disclosed in the prior art as shown below:

[0679] 1. The first sugar ring of compounds YK-CAP-101 and YK-CAP-102 in the present disclosure is a 6-membered ring, while the first sugar ring of N7113 is a 5-membered ring. The other structures are exactly identical.

[0680] 2. The group attached to the first sugar and guanine ring of compound YK-CAP-103 in the present disclosure is different from that of N7113, that is, there is one more methylene group in the Cl position. The other structures are exactly identical.

[0681] 3 The first sugar ring of compounds YK-CAP-104 to 106 in the present disclosure has two substituents at the C3 position, which is different from N7113, that is, the C3 substituents of N7113 are hydroxyl and hydrogen; the C3 substituents of YK-CAP-104 are dimethylaminomethyl and fluorine, respectively; the C3 substituents of YK-CAP-105 are cyano and Petition 870250022825, dated 03 / 24 / 2025, page 107 / 331 96 / 120 methyl, respectively; the C3 substituents of YK-CAP-106 are acetamido and methyl, respectively. The other structures are exactly identical.

[0682] 4. The substituents at the C3 position of the first sugar ring of compounds YK-CAP107 to 112 in the present disclosure are different from those of N7113 and HN3002, that is, the C3 substituents of N7113 and HN3002 are hydroxyl and methoxymethyl; the C3 substituents of YKCAP-107 to 112 are 1-methoxyethyl, 1-acetamidoethyl, 1-fluoroethyl, difluoromethyl, N,N-diacetamido and N,N-dipropionamido, respectively. The other structures are exactly identical.

[0683] 5. The substituents at the C2 position of the second sugar ring of compounds YK-CAP113 to 116 in the present disclosure 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; the C2 substituents of YK-CAP113 to 116 are methoxymethyl, acetamidomethyl, 1-fluoromethyl and difluoromethyl, respectively. The other structures are exactly identical.

[0684] 6. The substituents at the C4 position of the first sugar ring of compounds YK-CAP117 to 119 in the present disclosure are different from those of N7113 and 5227, that is, the C4 substituents of N7113 and 5227 are hydrogen and methoxy; the C4 substituents of YK-CAP-117 to 119 are methoxymethyl, 1-fluoromethyl and difluoromethyl, respectively. The other structures are exactly identical.

[0685] 7. Compounds YK-CAP-101 to 119 in this disclosure differ greatly in structure from compound 14 and m6A, specifically, the first sugar ring of compound 14 is a blocked nucleic acid sugar ring, i.e., there is a methylene bridge between 2'O and C4'; the second base of m6A, adenine, is methylated.

[0686] II. Measurement of in vitro mRNA transcription yield and capping rate

[0687] 1. Experimental methods

[0688] (1) Capping synthesis using cap analogs

[0689] The plasmid was first linearized with a plasmid linearization enzyme and then the linearized plasmid was purified.

[0690] (2) Transcription and in vitro synthesis of mRNA

[0691] YK-CAP-101 to 119, compound 5227 and CAP-2'O-ethyl synthesized in Example 1 were used respectively as analogs of cap. The reaction system is shown in Table 2: Petition 870250022825, dated 03 / 24 / 2025, page 108 / 331 97 / 120

[0692]

[0693]

[0694] Table 2: In vitro transcription reaction system System Quantity T7 RNA polymerase 50U 10X buffer 2pL lOOmMATP IpL lOOmMGTP IpL lOOmMCTP IpL lOOmMUTP IpL 100 mM IpL analog Nuclease inhibitor 20U Inorganic pyrophosphatase 0.05U Sterile water Supplemented to 20 pL DNA template Iμε During the experiment, the volume of materials required for the system was first calculated, and then the sample addition was conducted. The system was first added with enzyme-free sterile water, followed by the sequential addition of 10X buffer, NTPs, and cap analogs, thoroughly mixed and gently centrifuged. Nuclease inhibitors, inorganic pyrophosphatase, T7RNA polymerase, and linearized DNA templates were then added. The system was thoroughly mixed, gently centrifuged, and incubated at 37°C. After 2 hours of incubation, 1 U of DNase I was added and incubated at 37°C for a further 30 minutes. The mRNA precipitate was then washed with 75% ethanol, and after the ethanol was briefly evaporated to dryness, the mRNA was redissolved in enzyme-free sterile water.

[0695] (3) The transcription product was purified and the in vitro mRNA transcription yield was recorded.

[0696] (4) The obtained mRNA was subjected to an annealing reaction with a probe.

[0697] The annealing reaction was performed in a PCR instrument: 95°C for 5 minutes; 65°C for 2 minutes; 55°C for 2 minutes; 40°C for 2 minutes; 22°C for 2 minutes. Petition 870250022825, dated 03 / 24 / 2025, p. 109 / 331 98 / 120

[0698] (5) Magnetic bead pretreatment and probe binding: 100 μL of magnetic beads were placed in a magnetic frame for pretreatment. The magnetic bead solution was added with 120 μL of sample and incubated at room temperature for 30 minutes with slow mixing.

[0699] (6) mRNA splicing and obtaining the 5' single-stranded mRNA sequence bound to the probe

[0700] The mixture was added with 20 μL of RNase H (5 0μL) and incubated at 37°C for 3 hours, stirring every half hour. After incubation, the magnetic beads were washed and then added with 100 μL of 75% methanol heated to 80°C. The mixture was heated to 80°C on a heating plate, held for 3 minutes, then placed in a magnetic frame to aspirate the supernatant and dried at room temperature for 45 minutes to a volume of 10 μL using a centrifugal evaporator. The sample was then resuspended in 50 μL of 100 μM EDTA / 1% MeOH and ready for LC-MS analysis to determine RNA capping in the transcription reaction. Since there is a significant difference in molecular weight between capped and uncapped bases, the capping rate of mRNA transcription initiated by different cap analogs can be determined based on the difference in molecular mass.

[0701] 2. Experimental Results

[0702] Measurement results of in vitro mRNA transcription yield and capping rate show that the modified ribose cap analogs of the present disclosure have a significant difference in in vitro mRNA transcription yield and capping rate. Compared with the modified ribose cap analogs in the prior art, the modified ribose cap analogs of the present disclosure show a significant increase in both in vitro mRNA transcription yield and capping rate.

[0703] The specific in vitro transcription yield of mRNA and the capping rate are shown in Table 3. Petition 870250022825, dated 03 / 24 / 2025, page 110 / 331 99 / 120

[0704] Table 3: In vitro mRNA transcription yield and capping rate

[0705]

[0706] Name Yield per unit model Increase compared to 5227 Capping rate (%) Increase compared to 5227 YK-CAP-101 32.5 -75.8 42.8 -40.5 YK-CAP-102 44 1 -67 1 30.2 -580 YK-CAP-103 880 -344 71 1 -11 YK-CAP-104 73.2 -45.5 77.2 7.4 YK-CAP-105 823 -38.7 693 -3.6 YK-CAP-106 155.5 15.9 95.3 32.5 YK-CAP-107 170 7 27.2 973 35.3 YK-CAP-108 151 8 13 1 958 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-111 173.5 293 98 1 364 YK-CAP-112 169 2 26 1 952 32.4 YK-CAP-113 166 1 23 8 952 324 YK-CAP-114 161 2 20 1 952 324 YK-CAP-115 158 1 178 95.7 33 1 YK-CAP-116 159 4 188 964 34.1 YK-CAP-117 153.2 142 95 1 32.3 YK-CAP-118 172.2 28.3 97.2 352 YK-CAP-119 154.2 149 963 33 9 Composite 14 123 2 -8.2 783 8.9 5227 134 2 0 71 9 0 1) The modified ribose cap analogs of this disclosure show a significant difference in in vitro mRNA transcription yield and capping rate. YKCAP-106 to 119 have significantly higher in vitro mRNA transcription yield and capping rate than YK-CAP-101 to 105. YK-CAP-111 has the highest transcription yield and capping rate, with the transcription yield being 5.3 times that of YK-CAP-101 (the lowest) and the capping rate being 3.2 times that of YK-CAP-102 (the lowest).

[0707] As can be seen in Table 3, all modified ribose cap analogs in this disclosure are capable of transcribing mRNA. There is a significant difference in mRNA transcription activity between different modified ribose cap analogs. YKCAP-106 to 119 have very high in vitro mRNA transcription yields, all exceeding 150 pg. Specifically, YK-CAP-107, YK-CAP-110, YK-CAP-111, YK-CAP-117 and YK Petition 870250022825, dated 03 / 24 / 2025, page 111 / 331 100 / 120 CAP-118 has a yield of 170.7 μg, 172.1 μg, 173.5 μg, 153.2 μg, and 172.2 μg, respectively, with YK-CAP-111 having the highest yield of 173.5 μg (as shown in FIG. 1). YK-CAP-101 has the lowest in vitro mRNA transcription yield, which is only 32.5 μg. YK-CAP-102 to 105 also have a very low yield of 44.1 μg, 88.0 μg, 73.2 μg, and 82.3 μg, respectively. The transcription yield of YK-CAP-111 is 5.3 times, 3.9 times, 2.0 times, 2.4 times, and 2.1 times that of YK-CAP-101 at 105, respectively, showing a significant increase.

[0708] YK-CAP-106 to 119 have a very high capping rate, all above 95%. Specifically, YK-CAP-107, YK-CAP-110, YK-CAP-111, YK-CAP-117 and YK-CAP-118 have a capping rate of 97.3%, 97.4%, 98.1%, 95.1% and 97.2%, respectively, with YKCAP-111 having the highest capping rate of 98.1%.

[0709] YK-CAP-102 has the lowest capping rate, which is only 30.2%. YK-CAP-101 and YK-CAP-103 to 105 also have very low capping rates of 42.8%, 71.1%, 77.2% and 69.3%, respectively. The capping rate of YK-CAP-111 is 2.3 times, 3.2 times, 1.4 times, 1.3 times and 1.4 times higher than that of YK-CAP-101 to 105, respectively, showing a significant increase (as shown in FIG. 2).

[0710] Compared with modified ribose cap analogs in the prior art, the modified ribose cap analogs of the present disclosure show a significant increase in both in vitro mRNA transcription yield and capping rate. For example, the transcription yield of YK-CAP-111 is 40.8% higher than that of compound 14, and the capping rate of YK-CAP-111 is 36.4% higher than that of 5227.

[0711] The in vitro mRNA transcription yield and capping rate of compound 14 are 123.2 μg and 78.3%, respectively. The in vitro mRNA transcription yield and capping rate of YK-CAP-111 in the present disclosure are 40.8% and 25.3% higher than those of compound 14, respectively, showing a significant increase.

[0712] The in vitro mRNA transcription yield and capping rate of 5227 are 134.2 μg and 71.9%, respectively. The in vitro mRNA transcription yield and capping rate of YK-CAP-111 in the present disclosure are 29.3% and 36.4% higher than those of 5227, respectively, showing a significant increase.

[0713] 3) Analogs of modified ribose caps with similar structures vary widely Petition 870250022825, dated 03 / 24 / 2025, p. 112 / 331 101 / 120 in in vitro mRNA transcription yield and capping rate, making it impossible to predict in vitro mRNA transcription yield and capping rate based on structure.

[0714] The modified ribose cap analogs YK-CAP-117 to 119 designed in this disclosure have structures that are very similar. This series of compounds is also very similar in structure to 5227, but they vary greatly in in vitro mRNA transcription yield and capping rate.

[0715] For example, compounds YK-CAP-117 to 119 in the present disclosure differ from 5227 only in the substituent at the C4 position of the first sugar ring, namely, the C4 substituent of 5227 is methoxy; the C4 substituents of YK-CAP-117 to 119 are methoxymethyl, 1-fluoromethyl and difluoromethyl, respectively. The other structures are exactly identical. However, the in vitro mRNA transcription yield of YK-CAP-117 to 119 is 14.2%, 28.3% and 14.9% higher than that of 5227, respectively, and the capping rate of YK-CAP-117 to 119 is 32.3%, 35.2% and 33.9% higher than that of 5227, respectively, showing a significant increase.

[0716] It can be seen that modified ribose cap analogs with similar structures do not necessarily have similar mRNA transcription activities and capping rates. On the contrary, there may be a huge difference.

[0717] As can be seen from the in vitro mRNA transcription yield and capping rate, the modified ribose cap analogs YK-CAP-106 to 119 in the present disclosure show a significant increase in both in vitro mRNA transcription yield and capping rate compared with YK-CAP-101 to 105 in the present disclosure and compounds 14 and 5227 in the prior art, which demonstrates that the ribose modification of YK-CAP-106 to 119 shows excellent resistance to reverse transcription during in vitro mRNA transcription and can significantly increase the binding capacity of the cap structure to the capping enzyme, thus increasing the capping rate of mRNA transcription.

[0718] Example 3: Preparation and characterization of lipid nanoparticles

[0719] 1. Experimental methods

[0720] The cationic lipid YK-009 (Beijing Youcare Kechuang Pharmaceutical Technology Co., Ltd.), DSPC (AVT (Shanghai) Pharmaceutical Technology Co., Ltd.), cholesterol (AVT (Shanghai) Pharmaceutical Technology Co., Ltd.) and DMG-PEG2000 were dissolved in ethanol at a molar ratio of 49:10:39.5:1.5 and the mRNA was diluted in 50 mM citrate buffer. Petition 870250022825, dated 03 / 24 / 2025, page 113 / 331 102 / 120 (pH = 4). The ethanol lipid solution was mixed with aqueous Fluc-mRNA solution prepared from different cap structures at a volume ratio of 1:3 using a microfluidic device at a flow rate of 10 mL / min to prepare LNPs at a total lipid to mRNA weight ratio of approximately 15:1. The resulting liposomes were diluted to a 10-fold volume with PBS and then ultrafiltered with a 300 kDa ultrafiltration tube to remove the ethanol. The volume was then fixed to a certain volume with PBS. Finally, the LNPs were filtered through a sterile 0.2 μm filter to obtain an LNP preparation encapsulating Fluc-mRNA using YK-009 / DSPC / cholesterol / DMGPEG2000 (at a molar ratio of 49:10:39.5:1.5).

[0721] Particle size and polydispersity index (PDI) were determined by dynamic light scattering using a Malvern laser particle size analyzer. 10 μL of liposome solution were taken, diluted to 1 mL with RNase-free deionized water, and added to a sample set. Each sample was measured in triplicate. The measurement conditions were: a scattering angle of 90° and a temperature of 25°C. LNP encapsulation efficiency was determined using the RiboGreen Quant-iT RNA Quantification Kit (Thermo Fisher Scientific, UK) according to the manufacturer's instructions.

[0722] 2. Experimental Results

[0723] Specific characterization data for lipid nanoparticles are shown in Table 4. Petition 870250022825, dated 03 / 24 / 2025, page 114 / 331 103 / 120

[0724] Table 4: Characterization of lipid nanoparticles Name Particle size (nm) PDI EE (%) YK-CAP-101 75.45 0 043 934 YK-CAP-102 73.22 0055 953 YK-CAP-103 67.24 0053 949247 YK-CAP 95.7 YK-CAP-105 77.22 0 063 95.7 YK-CAP-106 6824 0 048 97 1 YK-CAP-107 78.42 0053 963 YK-CAP-108-1023 Y8603 13 -C 0035 97.2 YK-CAP-110 69 64 0 033 97.7 YK-CAP-111 78.33 0053 95 1 YK-CAP-112 83.24 0056 93.5 YK-CAP-112 83.24 005 79 YK-CAP-114 66 36 0 049 943 YK-CAP-115 68.35 0 034 95.3 YK-CAP-116 74.24 0 063 983 YK-CAP-117 72.56 941 -004 Y 0055 97.2 YK-CAP-119 82 45 0057 946 5227 83.34 0028 949 CAP-2'O-ethyl 8134 0035 963 N-7113 87.45 0078.318 942 HN3OO2 75.35 0058 95 1 m6A 75.76 0047 938

[0726] As can be seen in Table 4, good lipid nanoparticles can be prepared from Fluc-mRNA transcribed using the cap analogs YK-CAP-101 to 119 in the present disclosure and the cap analogs 5227, CAP-2'O-ethyl, N-7113, compound 14, HN3002 and m6A disclosed in the prior art. All lipid nanoparticles have a particle size between 66 and 88 nm, a PDI value between 0.023 and 0.078 and an encapsulation efficiency of 90% or more. Petition 870250022825, dated 03 / 24 / 2025, page 115 / 331 104 / 120

[0727] Example 4: Translation efficiency of different capped luciferase mRNAs

[0728] 1. Experimental methods

[0729] (1) HEK293T cells were cultured in DMEM medium containing 10% FBS and penicillin / streptomycin at 37°C with 5% CO2.

[0730] (2) The cells in the culture plate were digested and counted, then spread in a 96-well plate at 10,000 cells per well and cultured overnight until the cells adhered to the wall.

[0731] (3) When cell density reached approximately 80%, transfection was performed by adding 0.5 μg of mRNA sample and Lipofectamine MessengerMAX Transfection Reagent (Invitrogen) per well according to the manufacturer's instructions.

[0732] (4) After the transfected cells were cultured at 37°C with 5% CO2 for 24 hours, the growth medium was removed from the cells to be tested and the cells were rinsed with PBS. After centrifugation to remove PBS, 50 μL of 1x lysis buffer was added. The cells and all the liquid were transferred to a microcentrifuge tube and centrifuged.

[0733] ​​(5) 20 μL of sample were taken and added to 100 μL of pre-equilibrated Dual-Lumi™ II Luciferase Assay Reagent at room temperature and mixed appropriately.

[0734] (6) The mixture was incubated at room temperature (approximately 25°C) for minutes to stabilize the luminescence signal. Chemiluminescence was detected using a multifunctional microplate reader with a function to detect chemiluminescence, and the data were recorded.

[0735] 2. Experimental Results

[0736] Relative fluorescence readings of capped mRNAs are shown in Table 5. Relative fluorescence intensity is proportional to mRNA translation efficiency. Petition 870250022825, dated 03 / 24 / 2025, page 116 / 331 105 / 120

[0737]

[0738] Table 5: Relative fluorescence readings of capped mRNAs Name | Relative Fluorescence Intensity | 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 HN3OO2 1.13 3.0 m6A 0.38 1.0 Petition 870250022825, dated 03 / 24 / 2025, page 117 / 331 106 / 120

[0739] 1) The modified ribose cap analogs of the present disclosure show a significant difference in mRNA translation efficiency. YK-CAP-106 to 119 have significantly higher translation efficiency than YK-CAP-101 to 105. YK-CAP-111 has the highest translation efficiency, which is 9.6 times that of YK-CAP-101 (the lowest).

[0740] As can be seen in Table 5, the modified ribose cap analogs of the present disclosure show a significant difference in relative fluorescence intensity (corresponding to mRNA translation efficiency). YK-CAP-106 to YK-CAP-119 have high relative fluorescence intensity (between 1.4 and 2.2). Specifically, YK-CAP-107, YK-CAP-110, YK-CAP-111, YK-CAP-117 and YK-CAP-118 have a relative fluorescence intensity of 1.62, 1.95, 2.12, 1.82 and 1.65, respectively, with YK-CAP-111 having the highest relative fluorescence intensity of 2.12, followed by YK-CAP-110 with a relative fluorescence intensity of 1.95.

[0741] YK-CAP-101 has the lowest relative fluorescence intensity, which is only 0.22. YK-CAP-102 to YK-CAP-105 also have very low relative fluorescence intensities of 0.34, 0.77, 0.81 and 0.72, respectively. The relative fluorescence intensity of YK-CAP-111 is 9.6 times, 6.2 times, 2.8 times, 2.6 times and 2.9 times that of YK-CAP-101 to YK-CAP-105, respectively, while the relative fluorescence intensity of YK-CAP-110 is 8.9 times, 5.7 times, 2.5 times, 2.4 times and 2.7 times that of YK-CAP-101 to YK-CAP-105, respectively (as shown in FIG. 3).

[0742] 2) Compared with modified ribose cap analogs in the prior art, the modified ribose cap analogs of the present disclosure show a significant increase in mRNA translation efficiency. For example, the translation efficiency of YK-CAP-111 is 5.6 times that of m6A.

[0743] N-7113, HN3002, compound 14, and m6A have relative fluorescence intensities (corresponding to mRNA translation efficiency) of 1.00, 1.12, 1.13, and 0.38, respectively. The relative fluorescence intensity of YK-CAP-111 in this disclosure is 2.1 times, 1.9 times, 1.9 times, and 5.6 times that of N-7113, HN3002, compound 14, and m6A, respectively, while the relative fluorescence intensity of YK-CAP-110 in this disclosure is 1.9 times, 1.7 times, 1.7 times, and 5.1 times that of N-7113, HN3002, compound 14, and m6A, respectively. Petition 870250022825, dated 03 / 24 / 2025, p. 118 / 331 107 / 120

[0744] 3) Modified ribose cap analogs with similar structures vary widely in mRNA translation efficiency, making it impossible to predict mRNA translation efficiency.

[0745] The modified ribose cap analogs YK-CAP-117 to 119 designed in the present disclosure have structures that are very similar. This series of compounds is also very similar in structure to 5227, but they vary greatly in mRNA translation efficiency. For example, the YK-CAP-117 to 119 compounds in the present disclosure differ from 5227 only in the substituent at the C4 position of the first sugar ring, i.e., the C4 substituent of 5227 is methoxy; the C4 substituents of YK-CAP-117 to 119 are methoxymethyl, 1-fluoromethyl, and difluoromethyl, respectively. The other structures are exactly identical. However, the mRNA translation efficiency of YK-CAP-117 to 119 is 2.2 times, 2.0 times, and 1.8 times that of 5227, respectively, showing a significant increase.

[0746] Similarly, the modified ribose cap analogs YK-CAP-113 to 116 designed in the present disclosure have structures that are very similar. This series of compounds is also very similar in structure to CAP-2'O-ethyl, but they vary greatly in mRNA translation efficiency. For example, the compounds YK-CAP-113 to 116 in the present disclosure differ from CAP-2'O-ethyl only in the substituent at the C2 position of the second sugar ring, i.e., the C2 substituent of CAP-2'O-ethyl is ethoxy; the C2 substituents of YK-CAP-113 to 116 are methoxymethyl, acetamidomethyl, 1-fluoromethyl, and difluoromethyl, respectively. The other structures are exactly identical. However, the mRNA translation efficiency of YK-CAP-113 to 116 is 1.4 times, 1.3 times, 1.5 times, and 1.2 times that of CAP-2'O-ethyl, respectively, showing a significant increase.

[0747] It can be seen that modified ribose cap analogs with similar structures do not necessarily have translation efficiency similar to luciferase mRNA. On the contrary, there may be a huge difference.

[0748] As can be seen from the translation efficiency of different capped luciferase mRNAs, the ribose-modified cap analogs in this disclosure, including YK-CAP-106 to 119, show a significant increase in mRNA translation efficiency compared with both ribose-modified cap analogs with similar structures (including YK-CAP-101 to 105 in this disclosure, as well as 5227, CAP). Petition 870250022825, dated 03 / 24 / 2025, p. 119 / 331 108 / 120 2'O-ethyl, N-7113 and HN3002) and modified ribose cap analogs with very different structures (including compound 14 and m6A). It suggests that the modified ribose of YK-CAP-106 to 119 binds more readily to the cap-binding protein (EIF4E), which improves the translation efficiency of the target mRNA. Furthermore, modified ribose cap analogs with similar structures do not necessarily have similar luciferase mRNA translation efficiency. On the contrary, there may be a huge difference.

[0749] Example 5: Enzymatic stability test of pickling

[0750] 1. Experimental methods

[0751] 30 pmol of RNA purified by polyacrylamide gel electrophoresis (PAGE) were subjected to an enzymatic reaction with 50 U of mRNA decapping enzyme (New England Biolabs) and 1x MDE buffer at 37°C for 45 minutes. The enzymatic reagents were subjected to PAGE and stained with SYBR Green II (Lonza), followed by observation of the post-electrophoresis gel image on a Typhoon FLA 7000 instrument (GE Healthcare). The ratio of RNA capping to RNA decapping electrophoresis band intensity was counted using ImageQuant software (GE Healthcare), and the capping rate of the decapping enzyme was calculated.

[0752] 2. Experimental Results Petition 870250022825, dated 03 / 24 / 2025, pp. 120 / 331 109 / 120

[0753] Table 6: Decapping rate after treatment with decapping enzyme Name | Decapping Rate (%) | Decrease relative to N-7113 (%) | YK-CAP-101 | 324 | 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 | 339 | 9.9 | YK-CAP-106 | 17.3 | 265 | YK-CAP-107 | 11 | 5 | 32.3 | YK-CAP-108 | 163 | 27.5 | YK-CAP-109 | 17.3 | 265 | YK-CAP-110 | 102 | 33.6 | YK-CAP-111 | 11 | 5 | 32 | 3 | YK-CAP-112 | 119 | 31 | 9 YK-CAP-113 153 28.5 YK-CAP-114 142 296 YK-CAP-115 153 28.5 YK-CAP-116 143 29.5 YK-CAP-117 9.8 340 YK-CAP-118 11 9 31.9 YK-CAP-119 11 4 324 5227 23.2 206 CAP-2'O-ethyl 283 15.5 N-7113 43 8 0.0 Compound 14 268 17.0 HN3OO2 23.3 205 m6A 33 8 100

[0755] 1) The modified ribose cap analogs of the present disclosure show a significant difference in the decapping rate. YK-CAP-106 to 119 have a significantly lower decapping rate than YK-CAP-101 to 105. YK-CAP-117 has the lowest decapping rate, which is 36.3% lower than that of YK-CAP-104 (the highest).

[0756] As can be seen from the data in Table 6, the modified ribose cap analogs YK-CAP-101 to 119 in the present disclosure vary widely in pickling rate. YK-CAP-106 to 119 all have very low pickling rates. Specifically, YK-CAP107, YK-CAP-110, YK-CAP-111, YK-CAP-117 and YK-CAP-118 have a pickling rate Petition 870250022825, dated 03 / 24 / 2025, pp. 121 / 331 110 / 120 of 11.5%, 10.2%, 11.5%, 9.8% and 11.9%, respectively, with YK-CAP-117 having the lowest pickling rate of only 9.8%, followed by YK-CAP-110 with a pickling rate of 10.2%.

[0757] YK-CAP-104 has the highest pickling rate, which is 46.1%. YK-CAP-101, YK-CAP-102, YK-CAP-103 and YK-CAP-105 also have a high pickling rate of 32.4%, 41.2%, 36.5% and 33.9%, respectively. The decapping rate of YK-CAP-117 is 22.6%, 31.4%, 26.7%, 36.3%, and 24.1% lower than that of YK-CAP-101 to 105, respectively, while the decapping rate of YK-CAP-110 is 22.2%, 31.0%, 26.3%, 35.9%, and 23.7% lower than that of YK-CAP-101 to 105 (as shown in FIG. 4).

[0758] 2) Compared with modified ribose cap analogs with similar or very different structures in the prior art, the modified ribose cap analogs of the present disclosure show a significant decrease in the stripping rate. For example, the stripping rate of YK-CAP-117 is 34.0% lower than that of N-7113.

[0759] N-7113, compound 14, HN3002 and m6A have a pickling rate of 43.8%, 26.8%, 23.3% and 33.8%, respectively. The pickling rate of YK-CAP-117 in the present disclosure is 34.0%, 17.0%, 13.5% and 24.0% lower than that of N-7113, compound 14, HN3002 and m6A, respectively, while the pickling rate of YK-CAP-110 is 33.6%, 16.6%, 13.1% and 23.6% lower than that of N-7113, compound 14, HN3002 and m6A, respectively.

[0760] 3) Modified ribose cap analogs with similar structures vary widely in pickling rate, making it impossible to predict pickling rate based on structure.

[0761] The modified ribose cap analogs YK-CAP-117 to 119 designed in the present disclosure have structures that are very similar. This series of compounds is also very similar in structure to 5227, but they vary greatly in mRNA decapping rate. For example, the YK-CAP-117 to 119 compounds in the present disclosure differ from 5227 only in the substituent at the C4 position of the first sugar ring, i.e., the C4 substituent of 5227 is methoxy; the C4 substituents of YK-CAP-117 to 119 are methoxymethyl, 1-fluoromethyl, and difluoromethyl, respectively. The other structures are exactly identical. However, the mRNA decapping rate of YK-CAP-117 to 119 is 13.4%, 11.3%, and 11.8% lower than that of 5227, respectively, showing a significant decrease.

[0762] Similarly, the modified ribose cap analogs YK-CAP-113 to 116 Petition 870250022825, dated 03 / 24 / 2025, pp. 122 / 331 Compounds 111 / 120 projected in this disclosure have very similar structures. This series of compounds is also very similar in structure to CAP-2'O-ethyl, but they vary greatly in mRNA decapping rate. For example, compounds YK-CAP-113 to 116 in this disclosure differ from CAP-2'O-ethyl only in the substituent at the C2 position of the second sugar ring, i.e., the C2 substituent of CAP-2'O-ethyl is ethoxy; the C2 substituents of YK-CAP-113 to 116 are methoxymethyl, acetamidomethyl, 1-fluoromethyl, and difluoromethyl, respectively. The other structures are exactly identical. However, the mRNA decapping rate of YKCAP-113 to 116 is 13.0%, 14.1%, 13.0%, and 14.0% lower than that of CAP-2'O-ethyl, respectively, showing a significant decrease.

[0763] It can be seen that modified ribose cap analogs with similar structures do not necessarily have similar stripping rates. On the contrary, there can be a huge difference.

[0764] As can be seen from the DCP2 enzyme decapping rate, the modified ribose cap analogs in the present disclosure, including YK-CAP-106 to 119, show a significant decrease in the DCP2 enzyme decapping rate compared with both modified ribose cap analogs with similar structures (including YK-CAP-101 to 105 in the present disclosure, as well as 5227, CAP-2'O-ethyl, N-7113 and HN3002 in the prior art) and modified ribose cap analogs with very different structures (including compound 14 and m6A). Furthermore, modified ribose cap analogs with similar structures do not necessarily have similar decapping rates. On the contrary, there may be a huge difference.

[0765] Example 6: Experiments with animals

[0766] 1. Experimental methods

[0767] An LNP preparation containing 5 μg of Fluc-mRNA transcribed by a cap analog was injected intramuscularly into 4–6 week-old female BALB / C mice weighing 17–19 g. At specific time points after administration (6 hours, 12 hours, 24 hours, 48 ​​hours, 96 hours, and 168 hours), the mice were injected intraperitoneally with fluorescence imaging substrate. The mice were then allowed to move freely for 5 minutes, followed by detection of the total radiation intensity of the protein expressed by LNP-carrying mRNA in the mice using an imaging system. Petition 870250022825, dated 03 / 24 / 2025, pp. 123 / 331 112 / 120 in vivo small animal IVIS Spectrum (corresponding to the amount of protein expression).

[0768] 2. Experimental Results

[0769] The results are shown in Table 7. In in vivo imaging experiments in mice, the multiple of the total radiation intensity for each group of mice

[0770]

[0771] in relation to group m6A is shown in Table 8 (where the total radiation intensity is represented as the given value X 108p / s). Table 7: Experimental data from in vivo imaging in mice Name Total radiation intensity (* 108 p / s) 6h 12h 24h 48h 96h 168h YK-CAP-101 1 81 096 0.36 0 19 004 0.02 YK-CAP-106 5 88 685 1.76 0.53 0 11 0.03 YK-CAP-107 809 687 1 92 0.60 0 11 0.03 YK-CAP-108 642 603 1 70 1 04 0 19 002 YK-CAP-109 604 5 84 1 85 0.54 0 14 0.03 YK-CAP-110 952 1055 3 04 2 30 0.27 004 YK-CAP-111 861 934 2.75 1 95 0.27 0.03 YK-CAP-112 890 7.48 244 1 43 0.30 0.03 YK-CAP-113 607 7.63 2.02 1.29 0 31 004 YK-CAP-114 889 5 76 2.31 1.51 0.29 0.03 YK-CAP-115 620 5 42 1 85 1 21 0.25 0.02 YK-CAP-116 9 13 654 1 98 1 45 0.27 003 YK-CAP-117 899 800 208 1.73 0.35 0.02 38 145 0.53 048 007 002 CAP-2'O-ethyl 541 480 1.73 0.57 0.09 0.02 N-7113 5 10 433 1 49 049 0.05 0.02 m6A 2 35 2 12 066 0.52 008 0.02 Petition 870250022825, dated 03 / 24 / 2025, page 124 / 331 113 / 120

[0772] Table 8: Multiple of total radiation intensity in relation to m6A Nome 6h 12h 24h 48h 96h YK-CAP-101 08 0.5 0.5 04 1.5 YK-CAP-106 25 3.2 2.7 10 26 YK-CAP-107 3 4 3.2.8 YK 2.9 2.1 2.8 2.6 20 13 YK-CAP-109 26 2.8 2.8 10 2.7 YK-CAP-110 4 1 5.0 4.6 44 1.0 YK-CAP-111 3.7 4.4 4.11 7 Y K2 8 1.3 28 13 YK-CAP-113 26 3.6 3.1 2.5 1.2 YK-CAP-114 38 2.7 3.5 29 1.2 YK-CAP-115 26 2.6 2.8 23 1.2 YK-1.1.1 3 3.1 -116 YK-CAP-117 38 3.8 3.2 3 3 0.9 YK-CAP-118 30 30 3.1 26 1.2 YK-CAP-119 32 3.1 3.3 24 13 5227 10 CAPil 0.7 2.3 0.8 'O 2.3 2.6 11 2.4 N-7113 2.0 2.3 0.9 2.4 m6A 10 10 1.0 10 1.0

[0774] 1) The modified ribose cap analogs of the present disclosure show a significant difference in total radiation intensity and mRNA-expressed protein duration in mice. YK-CAP-106 to 119 have significantly higher total radiation intensity than YK-CAP-101 to 105. YK-CAP-110 has the highest total radiation intensity, which is 11.0 and 12.1 times that of YK-CAP-101 (the lowest) at 12 hours and 48 hours, respectively.

[0775] As can be seen from the data in Table 7, the different modified ribose cap analogs of the present disclosure vary greatly in the total radiation intensity of the mRNA-expressed protein in mice. YK-CAP-106 to 119 all have very high total radiation intensity from in vivo imaging in mice, among which YK-CAP-110 has the highest total radiation intensity, reaching 10.55 *108p / s at 12 hours and further reaching 2.30 *108p / s at 48 hours, while YK-CAP-111 has the second highest total radiation intensity, reaching 9.34 χ 108p / s at 12 hours and further reaching 1.95 x108p / s at 48 hours.

[0776] YK-CAP-101 has the lowest total radiation intensity, which is 0.96 χ108p / s at 12 hours and only 0.19 xl08p / s at 48 hours. The total radiation intensity of YK-CAP-110 is 11.0 times that of YK-CAP-101 at 12 hours and 12.1 times that of YK-CAP-101 at 48 hours. The total radiation intensity of YK-CAP-111 is 9.7 times that of YK-CAP-101 at 12 hours and Petition 870250022825, dated 03 / 24 / 2025, page 125 / 331 114 / 120 10.3 times that of YK-CAP-101 in 48 hours.

[0777] 2) Compared with modified ribose cap analogs with similar or very different structures in the prior art, the modified ribose cap analogs of the present disclosure show a significant increase in total radiation intensity and duration of mRNA-expressed protein in mice. For example, the total radiation intensity of YK-CAP-110 is 5.0 times that of m6A at 12 hours and 4.4 times that of m6A at 48 hours.

[0778] N-7113 and m6A have a total radiation intensity of 4.33 χ 108p / s 2.12 χ 108p / s in 12 hours, and 0.49 χ 108p / s 0.52 χ 108p / s in 48 hours.

[0779] The total radiation intensity of YK-CAP-110 in this disclosure is 2.4 times that of N-7113 and 5.0 times that of m6A at 12 hours and 4.7 times that of N-7113 and 4.4 times that of m6A at 48 hours.

[0780] The total radiation intensity of YK-CAP-111 in this disclosure is 2.2 times that of N-7113 and 4.4 times that of m6A at 12 hours and 4.0 times that of N-7113 and 3.8 times that of m6A at 48 hours.

[0781] 3) Modified ribose cap analogs with similar structures vary widely in total radiation intensity and duration of mRNA protein expression in mice, making it impossible to predict total radiation intensity and duration of mRNA protein expression in mice based on structure.

[0782] The modified ribose cap analogs YK-CAP-117 to 119 designed in the present disclosure have structures that are very similar. This series of compounds is also very similar in structure to 5227, but they vary greatly in the total radiation intensity of the protein expressed by mRNA in mice. For example, the YK-CAP-117 to 119 compounds in the present disclosure differ from 5227 only in the substituent at the C4 position of the first sugar ring, i.e., the C4 substituent of 5227 is methoxy; the C4 substituents of YK-CAP-117 to 119 are methoxymethyl, 1-fluoromethyl, and difluoromethyl, respectively. The other structures are exactly identical. However, the total radiation intensity of the protein expressed by mRNA in YK-CAP-117 to 119 mice is 3.8 times, 3.0 times, and 3.1 times that of 5227 at 6 hours, respectively, showing a significant increase.

[0783] Similarly, the modified ribose cap analogs YK-CAP-113 to 116 Petition 870250022825, dated 03 / 24 / 2025, pp. 126 / 331 Compounds 115 / 120 projected in this disclosure have very similar structures. This series of compounds is also very similar in structure to CAP-2'O-ethyl, but they vary greatly in the total radiation intensity of the protein expressed by mRNA in mice. For example, compounds YK-CAP-113 to 116 in this disclosure differ from CAP-2'O-ethyl only in the substituent at the C2 position of the second sugar ring, i.e., the C2 substituent of CAP-2'O-ethyl is ethoxy; the C2 substituents of YK-CAP-113 to 116 are methoxymethyl, acetamidomethyl, 1-fluoromethyl, and difluoromethyl, respectively. The other structures are exactly identical. However, the total radiation intensity of the protein expressed by mRNA in mice of YK-CAP-114 and YK-CAP-116 is 1.6 times and 1.7 times that of CAP-2'O-ethyl at 6 hours, respectively, showing a significant increase.

[0784] It can be seen that Fluc-mRNAs prepared from modified ribose cap analogs with similar structures do not necessarily have similar amounts and durations of protein expression in mice. On the contrary, there may be a huge difference.

[0785] As can be seen from animal experiments, modified ribose cap analogs in the present disclosure, such as YK-CAP-106 to 119, show a significant increase in the amount and duration of protein expression per mRNA in mice compared with both modified ribose cap analogs with similar structures (including YK-CAP-101 to 105 in the present disclosure, as well as 5227, N-7113 and CAP-2'O-ethyl in the prior art) and modified ribose cap analogs with very different structures (m6A).

[0786] In vivo experiments further demonstrate that mRNA transcribed using YK-CAP-106 to 119 in the present disclosure can be effectively distributed in the body by LNP delivery vectors and expressed efficiently and continuously. Furthermore, Fluc-mRNAs prepared from modified ribose cap analogs with similar structures do not necessarily have similar amounts and durations of protein expression in mice. On the contrary, there may be a huge difference.

[0787] In summary, the modified ribose cap analogs YK-CAP-106 to 119 in this disclosure show a significant increase in in vitro mRNA transcription yield, capping rate, mRNA translation efficiency, capping enzymatic stability and Petition 870250022825, dated 03 / 24 / 2025, page 127 / 331 116 / 120 quantity and duration of protein expression in animals compared with prior art modified ribose cap analogs (including 5227, CAP-2'O-ethyl, N-7113, compound 14, HN3002 and m6A). Indicates that the YK-CAP-106 to 119 cap structures provided by this disclosure can significantly increase the resistance of modified ribose structures to decapping enzymes, as well as their binding affinity to capping enzymes, providing a novel and efficient modified ribose cap structure for in vitro mRNA transcription.

[0788] 1. The compounds in this disclosure have very similar chemical structures. This series of compounds has some similarities and some significant differences in structure compared with mRNA cap analogs disclosed in the prior art.

[0789] 1) The first sugar ring of compounds YK-CAP-101 and YK-CAP-102 in the present disclosure is a 6-membered ring, while the first sugar ring of N7113 is a 5-membered ring. The other structures are exactly identical.

[0790] 2) The group attached to the first sugar and guanine ring of compound YK-CAP-103 in the present disclosure is different from that of N7113, that is, there is one more methylene group at the C1 position. The other structures are exactly identical.

[0791] 3) The first sugar ring of compounds YK-CAP-104 to 106 in the present disclosure has two substituents at the C3 position, which is different from N7113, that is, the C3 substituents of N7113 are hydroxyl and hydrogen; the C3 substituents of YK-CAP-104 are dimethylaminomethyl and fluorine, respectively; the C3 substituents of YK-CAP-105 are cyano and methyl, respectively; the C3 substituents of YK-CAP-106 are acetamido and methyl, respectively. The other structures are exactly identical.

[0792] 4) The substituents at the C3 position of the first sugar ring of compounds YK-CAP107 to 112 in the present disclosure are different from those of N7113 and HN3002, that is, the C3 substituents of N7113 and HN3002 are hydroxyl and methoxymethyl; the C3 substituents of YKCAP-107 to 112 are 1-methoxyethyl, 1-acetamidoethyl, 1-fluoroethyl, difluoromethyl, N,N-diacetamido and N,N-dipropionamido, respectively. The other structures are exactly identical.

[0793] 5) The substituents at the C2 position of the second sugar ring of compounds YK-CAP113 to 116 in the present disclosure are different from those of N7113 and CAP-2'O-ethyl, that is, the Petition 870250022825, dated 03 / 24 / 2025, pp. 128 / 331 117 / 120 The C2 substituents of N7113 and CAP-2'O-ethyl are methoxy and ethoxy; the C2 substituents of YK-CAP113 to 116 are methoxymethyl, acetamidomethyl, 1-fluoromethyl, and difluoromethyl, respectively. The other structures are exactly identical.

[0794] 6) The substituents at the C4 position of the first sugar ring of compounds YK-CAP117 to 119 in the present disclosure are different from those of N7113 and 5227, that is, the C4 substituents of N7113 and 5227 are hydrogen and methoxy; the C4 substituents of YK-CAP117 to 119 are methoxymethyl, 1-fluoromethyl and difluoromethyl, respectively. The other structures are exactly identical.

[0795] 7) Compounds YK-CAP-101 to 119 in this disclosure differ greatly in structure from compound 14 and m6A, specifically, the first sugar ring of compound 14 is a blocked nucleic acid sugar ring, i.e., there is a methylene bridge between 2'O and C4'; the second base of m6A, adenine, is methylated.

[0796] 2. The modified ribose cap analogs of the present disclosure show a significant difference in in vitro mRNA transcription yield and capping rate. Compared with the modified ribose cap analogs in the prior art, the modified ribose cap analogs of the present disclosure show a significant increase in both in vitro mRNA transcription yield and capping rate.

[0797] 1) The modified ribose cap analogs of the present disclosure show a significant difference in in vitro mRNA transcription yield and capping rate. YKCAP-106 to 119 have significantly higher in vitro mRNA transcription yield and capping rate than YK-CAP-101 to 105. YK-CAP-111 has the highest transcription yield and capping rate, with the transcription yield being 5.3 times that of YK-CAP101 (the lowest) and the capping rate being 3.2 times that of YK-CAP-102 (the lowest).

[0798] Compared with modified ribose cap analogs in the prior art, the modified ribose cap analogs of the present disclosure show a significant increase in both in vitro mRNA transcription yield and capping rate. For example, the transcription yield of YK-CAP-111 is 40.8% higher than that of compound 14, and the capping rate of YK-CAP-111 is 36.4% higher than that of 5227.

[0799] 3) Modified ribose cap analogs with similar structures vary widely in in vitro mRNA transcription yield and capping rate. For example, the yield Petition 870250022825, dated 03 / 24 / 2025, pp. 129 / 331 The in vitro transcription rate of YK-CAP-117 to 119 mRNA is 14.2%, 28.3%, and 14.9% higher than that of 5227, respectively, and the capping rate of YK-CAP-117 to 119 is 32.3%, 35.2%, and 33.9% higher than that of 5227, respectively.

[0800] 3. The modified ribose cap analogs of the present disclosure show a significant difference in mRNA translation efficiency. Compared with the modified ribose cap analogs in the prior art, the modified ribose cap analogs of the present disclosure show a significant increase in mRNA translation efficiency.

[0801] 1) The modified ribose cap analogs of the present disclosure show a significant difference in mRNA translation efficiency. YK-CAP-106 to 119 have significantly higher translation efficiency than YK-CAP-101 to 105. YK-CAP-111 has the highest translation efficiency, which is 9.6 times that of YK-CAP-101 (the lowest).

[0802] 2) Compared with modified ribose cap analogs of similar or very different structures in the state of the art, the modified ribose cap analogs of the present disclosure show a significant increase in mRNA translation efficiency. For example, the translation efficiency of YK-CAP-111 is 5.6 times that of m6A.

[0803] 3) Modified ribose cap analogs with similar structures vary widely in mRNA translation efficiency. For example, the mRNA translation efficiency of YKCAP-117 to 119 is 2.2 times, 2.0 times, and 1.8 times that of 5227, respectively.

[0804] 4. The modified ribose cap analogs of the present disclosure show a significant difference in the decapping rate. Compared with modified ribose cap analogs with similar or very different structures in the prior art, the modified ribose cap analogs of the present disclosure show a significant decrease in the decapping rate.

[0805] 1) The modified ribose cap analogs of the present disclosure show a significant difference in pickling rate. YK-CAP-106 to 119 have a significantly lower pickling rate than YK-CAP-101 to 105. YK-CAP-117 has the lowest pickling rate, which is 36.3% lower than that of YK-CAP-104 (the highest).

[0806] 2) Compared to modified ribose cap analogs with similar or very different structures in the prior art, the modified ribose cap analogs of the present disclosure show a significant decrease in the stripping rate. By Petition 870250022825, dated 03 / 24 / 2025, pp. 130 / 331 119 / 120 for example, the decapping rate of YK-CAP-117 is 34.0% lower than that of N-7113.

[0807] 3) Modified ribose cap analogs with similar structures vary widely in decapping rate. For example, the mRNA decapping rate of YK-CAP-113 to 116 is 13.0%, 14.1%, 13.0%, and 11.0% lower than that of CAP-2'O-ethyl, respectively.

[0808] 5. The modified ribose cap analogs of the present disclosure show a significant difference in total radiation intensity (corresponding to the amount of protein expression) and duration of protein expression per mRNA in mice. Compared with modified ribose cap analogs with similar or very different structures in the prior art, the modified ribose cap analogs of the present disclosure show a significant increase in the amount and duration of protein expression per mRNA in mice.

[0809] 1) The modified ribose cap analogs of the present disclosure show a significant difference in total radiation intensity and mRNA-expressed protein duration in mice. YK-CAP-106 to 119 have significantly higher total radiation intensity than YK-CAP-101 to 105. YK-CAP-110 has the highest total radiation intensity, which is 11.0 and 12.1 times that of YK-CAP-101 (the lowest) at 12 hours and 48 hours, respectively.

[0810] 2) Compared with modified ribose cap analogs with similar or very different structures in the prior art, the modified ribose cap analogs of the present disclosure show a significant increase in total radiation intensity and duration of mRNA protein expression in mice. For example, the total radiation intensity of YK-CAP-110 is 5.0 times that of m6A at 12 hours and 4.4 times that of m6A at 48 hours.

[0811] 3) Modified ribose cap analogs with similar structures vary widely in total radiation intensity and duration of mRNA protein expression in mice. For example, the total radiation intensity of mRNA protein expression in YK-CAP-117 to 119 mice is 3.8 times, 3.0 times, and 3.1 times that of 5227 at 6 hours, respectively.

[0812] The Applicant states that this disclosure illustrates the modified ribose cap analogs of this disclosure and their use through the examples above. However, this disclosure is not limited to these examples, which does not mean that the Petition 870250022825, dated 03 / 24 / 2025, pp. 131 / 331 120 / 120 This disclosure should be implemented depending on these examples. It should be understood by those skilled in the art that any improvements to this disclosure, equivalent substitutions of starting materials for the products of this disclosure, additions of auxiliary ingredients, selections of specific media, etc., all fall within the scope of protection and disclosure of this disclosure. Petition 870250022825, dated 03 / 24 / 2025, pp. 132 / 331< / j>

Claims

1 / 10 CLAIMS 1. Modified ribose cap analog or a pharmaceutically acceptable salt thereof, characterized in that the modified ribose cap analog has a structure of 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 as shown below: OH OH YK-CAP-106 OH OH YK-CAP-107 OH OH YK-CAP-108 Petition 870250022825, dated 03 / 24 / 2025, page 133 / 331 2 / 10 YK-CAP-109 OH OH YK-CAP-110 OH OH YK-CAP-111 OH OH YK-CAP-112 Petition 870250022825, dated 03 / 24 / 2025, page 134 / 331 3 / 10 YK-CAP-113 OH OH YK-CAP-114 OH OH YK-CAP-115 OH OH YK-CAP-116 OH OH Petition 870250022825, dated 03 / 24 / 2025, page 135 / 331 4 / 10 OH OH YK-CAP-117 OH OH YK-CAP-118 OH OH YK-CAP-119 2. Use of a modified ribose cap analog or a pharmaceutically acceptable salt thereof, according to claim 1, characterized in that it is used in the preparation of an in vitro cotranscription mRNA capping reagent.

3. RNA molecule, characterized in that it comprises a modified ribose cap analog or a pharmaceutically acceptable salt thereof, as defined in claim 1, as a cap structure or a cap structure fragment.

4. Pharmaceutical composition, characterized in that it comprises the RNA molecule as defined in claim 3. Petition 870250022825, dated 03 / 24 / 2025, page 136 / 331 5 / 10 5. Pharmaceutical composition according to claim 4, characterized in that the pharmaceutical composition further comprises at least one RNA delivery agent.

6. Pharmaceutical composition, according to claim 5, characterized in that at least one RNA delivery agent comprises at least one cationic lipid.

7. Pharmaceutical composition according to claim 6, characterized in that the cationic lipid is selected from one or more of the following compounds: (1) a compound of formula (II) or the pharmaceutically acceptable salt thereof, wherein G1 is C1-6 alkylene; G2 is C2-8 alkylene; G3 is C1-3 alkylene; Li is C6-25 linear alkyl; L2 is C12-25 branched alkyl; OH I 0 (Π) (2) a compound of formula (iii) or the pharmaceutically acceptable salt thereof, wherein Gi is C2-8 alkylene; G2 is C2-8 alkylene; LI is -C(O)O- or -OC(O)-; L2 is -C(O)O- or -OC(O); Ri is C6-25 linear or branched alkyl; R2 is C6-25 linear or branched alkyl; G / is HO(CH2)2- or HO(CH2)3-; G4 is HO(CH2)2- or HO(CH2)3-; L is (CH2)2-, -(CH2)3- or -(CH2)4.; g3 NG,—L,—Rt LZ N G2 1-2 R2 G4 (III) (3 ) a compound of formula (IV) or the pharmaceutically acceptable salt thereof, wherein Gi is C1-6 alkylene; G2 is C2-8 alkylene; Ri is C6-20 linear or branched alkyl; R2 is C1225 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-; Petition 870250022825, dated 2V03 / 2025, page 137 / 331 6 / 10 (IV) (4 ) a compound of formula (V) or the pharmaceutically acceptable salt thereof, wherein Gi is C1-8 alkylene; G2 is C2-8 alkylene; Ri is linear or branched C1-25 alkyl; R2 is linear or branched C1-25 alkyl; G3 is HO(CH2)2N(R3)CH2CH(OH)CH2-, wherein R3 is -CH3, -CH2CH3, or -CH2CH2OH; (V) (5) a compound of formula (VI) or the pharmaceutically acceptable salt thereof, wherein G1 and G2 are each independently unsubstituted C1-C12 alkylene; G3 is 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 C1-C12 hydrocarbyl; and R5 is H or C1-C1 hydrocarbyl;R2 ri I \ OO (VI) (6 ) a compound of formula (VII) or the pharmaceutically acceptable salt thereof, where R4 is selected from -(CH2)nQ and -(CH2)n CHQR; 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 heterocycle; n is 1, 2 or 3; Petition 870250022825, dated 24 / 03 / 2025, p. 138 / 331 7 / 10 (7) a compound of formula (VIII) or the pharmaceutically acceptable salt thereof; 8. Pharmaceutical composition, according to claim 7, characterized in that the cationic lipid is selected from one or a combination of at least two of YK009, YK-401, YK-305, ALC0315, SM102 or DLIN-MC3-DMA: YK-401 Petition 870250022825, dated 03 / 24 / 2025, page 139 / 331 8 / 10 DLIN-MC3-DMA 9. Pharmaceutical composition according to claim 8, characterized in that the cationic lipid is YK-009.

10. Pharmaceutical composition, according to claim 6, characterized in that at least one RNA delivery agent further comprises at least one neutral lipid.

11. Pharmaceutical composition, according to claim 10, characterized in that the neutral lipid comprises one or a combination of at least two of phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, ceramide or sterol.

12. Pharmaceutical composition, according to claim 11, characterized in that the neutral lipid is selected from one or a combination of at least two of the following: -3-phosphocholine, 1,2-dimyristoyl-sn-glycero-phosphocholine, l,2-dioleoyl-sn-glycero-3-phosphocholine, l,2-dipalmitoyl-sn-glycero-3-phosphocholine, l,2-distearoyl-sn-glycero-3-phosphocholine, 1,2-diundecanoyl-sn-glycero-phosphocholine, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine, l-oleoyl-2-cholesteryl-hemisuccinoyl-sn-glycero-3-phosphocholine, 1-hexadecyl-sn-glycero-3 -phosphocholine, 1,2-dilinolenoyl-sn-glycero-3-phosphocholine, l,2-diarachidonoyl-sn-glycero-3-phosphocholine, l,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, l,2-dioleoyl-sn-glycero-3-phosphoethanolamine, l,2-diphytanoyl-sn-glycero-3-phosphoethanolamine, 1,2distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2dilinolenoyl-sn-glycero-3-phosphoethanolamine, l,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, l,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, sodium salt of 1,2-dioleoyl-sn-glycero3-phosphorac-(l-glycerol), dipalmitoyl phosphatidylglycerol, palmitoyl oleoyl phosphatidylethanolamine, distearoyl-phosphatidyl-ethanolamine, dipalmitoyl phosphatidylethanolamine, dimyristol Petition 870250022825, dated 03 / 24 / 2025, page. 140 / 331 9 / 10 phosphoethanolamine, 1-stearoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine, 1-stearoyl-2-oleoylphosphatidylcholine, sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyl oleoyl phosphatidylcholine, lysophosphatidylcholine or lysophosphatidylethanolamine.

13. Pharmaceutical composition, according to claim 12, characterized in that the neutral lipid is DOPE and / or DSPC.

14. Pharmaceutical composition, according to claim 10, characterized in that at least one RNA delivery agent further comprises a structural lipid.

15. Pharmaceutical composition, according to claim 14, characterized in that the structural lipid is selected from one or a combination of at least two of the following: cholesterol, non-sterol, sitosterol, ergosterol, campesterol, stigmasterol, brassinosterol, tomatine, ursolic acid, α-tocopherol or corticosteroid.

16. Pharmaceutical composition, according to claim 15, characterized in that the structural lipid is cholesterol.

17. Pharmaceutical composition, according to claim 5, characterized in that at least one RNA delivery agent further comprises a lipid conjugated with a polymer.

18. Pharmaceutical composition, according to claim 17, characterized in that the lipid conjugated with polymer is selected from one or a combination of at least two of the following: distearoyl phosphatidylethanolamine polyethylene glycol 2000, dimyristoylglycero-3-methoxypolyethylene glycol 2000 or methoxypolyethylene glycol ditetradecylacetamide.

19. Pharmaceutical composition, according to any one of claims 4 to 18, characterized in that the pharmaceutical composition further comprises one or at least two cell-penetrating peptides.

20. Method for synthesizing an mRNA molecule for non-disease-related diagnostic and therapeutic purposes, characterized in that it comprises the steps of: coincubating the modified ribose cap analog, or stereoisomer, the pharmaceutically acceptable salt thereof, as defined in claim 1, with a template polynucleotide for transcription. Petition 870250022825, dated 03 / 24 / 2025, p. 141 / 331 10 / 10 21. Cap-based mRNA transcription reaction system for non-disease related diagnostic and therapeutic purposes, characterized in that it comprises: (1) the modified ribose cap analog, or the stereoisomer, the pharmaceutically acceptable salt thereof, as defined in claim 1; and (2) a polynucleotide template, NTPs and an RNA polymerase.

22. Kit, characterized in that it comprises: (1) the modified ribose cap analogue, or the stereoisomer, the pharmaceutically acceptable salt thereof, as defined in claim 1; and (2) a nucleotide triphosphate molecule and an RNA polymerase.

23. Kit according to claim 22, characterized in that the kit further comprises one or a combination of at least two of an RNAase inhibitor, an inorganic pyrophosphatase, Mg2+, a clumping agent or a buffer.

24. Method for introducing RNA into a cell, characterized in that it comprises contacting the cell with the RNA molecule, as defined in claim 3, or the pharmaceutical composition, as defined in any one of claims 4 to 19.

25. Use of the RNA molecule, as defined in claim 3, or of the pharmaceutical composition, as defined in any of claims 4 to 19, characterized in that it is in the preparation of a vaccine. Petition 870250022825, dated 03 / 24 / 2025, pp. 142 / 331

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