3'-deoxyadenosine compounds and their use in the preparation of antitumor drugs

Abstract

The application discloses a 3'-deoxyadenosine compound and application thereof in preparation of an antitumor drug. The 3'-deoxyadenosine compound and the pharmaceutical composition thereof have good antitumor proliferation effect. Compared with a parent drug, the 3'-deoxyadenosine compound has better affinity to a cell membrane, so that the half-life of in-vivo metabolism of the drug is longer, and the drug stays in the body for a longer time. Compared with other nucleoside antitumor drugs, the cordycepin derivative and the pharmaceutical composition thereof have a wider range of tumor types and effects, including excellent inhibition effect on gastric cancer, pancreatic cancer, liver cancer, small cell lung cancer, colorectal cancer, melanoma, ovarian cancer and the like, and the side effect is lower and the curative effect is better.

Description

[0001] This invention is a divisional application filed on July 12, 2022, with the designation 2023107401308, concerning a chemically modified compound with anticancer effects based on 3'-deoxyadenosine. Technical Field

[0002] This invention belongs to the field of biomedicine, specifically relating to 3'-deoxyadenosine compounds and their use in the preparation of antitumor drugs. Background Technology

[0003] With the accumulation of external environmental stimuli and cellular damage, the body's cellular adaptability changes. One outcome is the loss of cellular adaptability, eventually leading to aging; the other is an abnormal increase in cellular adaptability, transforming into cancer. Cancer is a common disease that threatens human life and health, with global cancer incidence and mortality rates consistently rising. Currently, treatment methods for malignant tumors mainly include surgery, radiotherapy, and chemotherapy, with chemotherapy primarily using synthetic drugs. Chemotherapy drugs have a proven effect in inhibiting tumors and are one of the more effective and widely used treatment methods for malignant tumors. However, their toxic side effects are widespread and severe, and drug resistance exists. Furthermore, chemotherapy drugs have poor selectivity for tumor cells and normal cells; while killing or inhibiting tumor cells, they can also damage the growth of normal cells and have a direct impact on the functions of the heart, liver, kidneys, and nervous system, exhibiting a certain degree of toxicity to the human body. Therefore, finding low-toxicity and highly effective anti-tumor drugs is essential in cancer treatment.

[0004] Nucleosides, as one of the most important endogenous compounds in the human body, play a crucial role in metabolic processes. Modification and derivatization of nucleoside compounds are a major pathway for preparing antitumor drugs. Currently available nucleoside antitumor drugs include furofibrate, fludarabine, cladribine, clofarabine, fludarabine phosphate, and trisatatabine. Cordycepin (3'-deoxyadenosine), the main active ingredient of Cordyceps sinensis, belongs to the nucleoside analogue family. During its metabolism in the body, it exhibits excellent effects on cellular adaptive changes (anti-aging, anti-cancer), immune regulation, and inflammation reduction.

[0005] The anticancer mechanism of cordycepin mainly involves inducing apoptosis, regulating the cell cycle, and interfering with the expression of matrix metalloproteinases (MMPs), thereby inhibiting tumor cell invasion and metastasis. The signaling pathways related to inducing tumor cell apoptosis include the NF-κB signaling pathway and the mitogen-activated protein kinase (MAPK) signaling pathway. Cell cycle regulation is mainly manifested in cancer cells by shortening the G1 phase and prolonging the G2 and M phases, arresting the cell cycle at the G2 / M phase and thus inhibiting cell proliferation. Interference with MMP expression is mainly achieved by inhibiting the NF-κB signaling pathway, ultimately inhibiting MMP-9 expression. However, nucleoside analogs have poor lipid solubility, are difficult to absorb, are easily inactivated by deaminase metabolism, have short half-lives, low targeting specificity, and are prone to developing resistance in some tumor cells or viruses, all of which significantly reduce the effectiveness of nucleoside analogs. Summary of the Invention

[0006] Purpose of the invention: The technical problem to be solved by the present invention is to address the shortcomings of existing nucleoside drugs and to provide a series of nucleoside analogs (cordycepin derivatives) with cordycepin as the parent nucleus and modified by chemical modification, by combining the natural pharmacological activity of cordycepin.

[0007] Another technical problem to be solved by the present invention is to provide a composition containing the above-mentioned nucleoside analogue.

[0008] Another technical problem to be solved by the present invention is to provide the application of the above-mentioned nucleoside analogs and their compositions in products for preventing and treating diseases related to the functional damage and variation of cells in mammals or humans.

[0009] The final technical problem to be solved by this invention is to provide a method for preparing the above-mentioned nucleoside analogues.

[0010] To solve the first technical problem mentioned above, the present invention discloses a cordycepin derivative as shown in Formula I, or a pharmaceutically acceptable salt, stereoisomer, tautomer, solvate, prodrug, or metabolite thereof;

[0011]

[0012] I

[0013] Wherein, R1 is selected from hydrogen, phosphoric acid group, substituted phosphate ester group, phosphonic acid group, substituted phosphonate ester group, alkyl alcohol group, amino acid alkyl ester group, amino acid alkyl alcohol ester group, alkyl acid alkyl ester group, or cycloalkyl polyol group; the substitution is any one or more functional groups selected from alkoxy, halogen-substituted alkoxy, aryloxy, amino acid ester amide group, alkyl ester group, and alkyl acid methyl ester oxy; R2 is selected from hydrogen or azide group; R3 is selected from hydrogen, fluorine, chlorine, or azide group; R4 is selected from hydroxyl, cyano, and β-amide. -γ-cyclosulfonyloxy, amino acid carboxylic acid ester group, amino acid alkyl ester phosphonic acid phenyl ester group, or amino acid alkyl ester phosphonic phosphate ester group; R5 is selected from hydrogen, bromovinyl, mercapto, methyl, fluorine, or chlorine; R6 is selected from amino, substituted formamide group; the substitution is any one or more functional groups selected from alkyl, aryl, cycloalkyl, furanyl, pyridyl; R7 is selected from hydrogen, or isopropylamino; and there is no case where R1, R2, R3, R5, R7 are selected from hydrogen, R4 is selected from hydroxyl, and R6 is selected from amino.

[0014] In some embodiments, R1 is selected from hydrogen, such as R1 1 -R1 25 The group shown.

[0015]

[0016] In some embodiments, R4 is selected from R4 1 -R4 7 The group shown.

[0017]

[0018] In some embodiments, R6 is selected from amino groups, or such as R6 1 -R6 16 The group shown.

[0019]

[0020] In some embodiments, the cordycepin derivative represented by Formula I is selected from any one of compounds 1 to 40.

[0021] Compound 1: Compound 2: Compound 3: Compound 4: Compound 5: Compound 6: Compound 7: Compound 8: Compound 9: Compound 10: Compound 11: Compound 12: Compound 13: Compound 14: Compound 15: Compound 16: Compound 17: Compound 18: Compound 19: Compound 20: Compound 21: Compound 22: Compound 23: Compound 24: Compound 25: Compound 26: Compound 27: Compound 28: Compound 29: Compound 30: Compound 31: Compound 32: Compound 33: Compound 34: Compound 35: Compound 36: Compound 37: Compound 38: Compound 39: Compound 40:

[0022] To address the second technical problem mentioned above, this invention discloses a pharmaceutical composition comprising at least one of the cordycepin derivatives described above, or a pharmaceutically acceptable salt, stereoisomer, tautomer, solvate, prodrug, or metabolite thereof; and at least one immune checkpoint inhibitor.

[0023] In some embodiments, the immune checkpoint inhibitor is selected from PD-1 and / or CTLA4 monoclonal antibodies.

[0024] In some embodiments, the cordycepin derivative, or its pharmaceutically acceptable salt, stereoisomer, tautomer, solvate, prodrug, or metabolite, is present in a mass ratio of 1:0.2 to 10 to an immune checkpoint inhibitor, in some embodiments 1:0.2 to 8, in some embodiments 1:0.2 to 5, in some embodiments 1:0.2 to 3, in some embodiments 1:0.2 to 2, in some embodiments 1:0.5 to 1.5, in some embodiments 1:0.8 to 1.2, and in some embodiments 1:1.

[0025] In some embodiments, the dosage form of the pharmaceutical composition is selected from tablets, pills, capsules, drops, syrups, disintegrants, injections, sustained-release formulations, or kits.

[0026] To address the third technical problem mentioned above, this invention discloses the use of the above-mentioned cordycepin derivatives, or pharmaceutically acceptable salts, stereoisomers, tautomers, solvates, prodrugs, or metabolites thereof, or combinations thereof, in the preparation of products for preventing and treating diseases related to functional damage and variation of cells in mammals or humans.

[0027] In some embodiments, the disease associated with the variation in cellular functional impairment is a tumor. In some embodiments, the tumor includes, but is not limited to, gastric cancer, pancreatic cancer, liver cancer, small cell lung cancer, non-small cell lung cancer, colorectal cancer, esophageal cancer, prostate cancer, melanoma, glioma, and ovarian cancer. In some embodiments, the tumor is any one of gastric cancer, pancreatic cancer, liver cancer, small cell lung cancer, colorectal cancer, melanoma, and ovarian cancer.

[0028] In some embodiments, the product includes, but is not limited to, a drug.

[0029] To address the fourth technical problem mentioned above, this invention discloses a method for preparing the aforementioned cordycepin derivatives. The preparation method is a synthetic approach corresponding to the modification method. Considering that other active groups in the cordycepin or modified cordycepin molecules will participate in the reaction during the reaction process, they should be appropriately protected. The protection methods involved in this invention are the protection and deprotection of hydroxyl and amino groups. The protection and deprotection methods are all conventional methods in the field. In addition, some reaction systems require the addition of protective gases for protection, which are all conventional methods in the experimental process in the field.

[0030] Specifically as follows:

[0031] A. Modify R1

[0032] When R1 in Formula I is selected from phosphate group, substituted phosphate ester group, phosphonic acid group, substituted phosphonate ester group, alkyl alcohol group, amino acid alkyl ester group, amino acid alkyl alcohol ester group, alkyl acid alkyl ester group, or cycloalkyl polyol group; and the substitution is any one or more functional groups selected from alkoxy, halogen-substituted alkoxy, aryloxy, amino acid ester amide group, alkyl ester group, and alkyl acid methyl ester oxy; the preparation method of the cordycepin derivative shown in Formula I is as follows: in an organic solvent, using compound IR-1 as a raw material, a chemical reaction is carried out to obtain the cordycepin derivative shown in Formula I.

[0033]

[0034] IR-1

[0035] In formula IR-1, R2~R7 are the same as R2~R7 in formula I, or are independently selected from protecting groups.

[0036] When R1 in Formula I is selected from phosphate groups, in some embodiments, the preparation method of the cordycepin derivative shown in Formula I is as follows: in trimethyl phosphite and / or triethyl phosphite, compound IR-1 is reacted with the modifier phosphorus oxychloride. In some embodiments, the ratio of compound IR-1, modifier and organic solvent is 1 mmol: 3~6 mmol: 15~30 mL. In some embodiments, the reaction temperature is -10~5 ℃. In some embodiments, the reaction time is 0.5~2 h.

[0037] When R1 in Formula I is selected from substituted phosphate ester groups, in some embodiments, the preparation method of the cordycepin derivative shown in Formula I is as follows: in anhydrous N,N-dimethylformamide and / or tetrahydrofuran, using tert-butylmagnesium chloride as a catalyst, compound IR-1 reacts with a modifier-substituted nitrobenzene phosphate, wherein the substitution is any one or more functional groups substituted by alkoxy, halogen-substituted alkoxy, aryloxy, amino acid ester amide, alkyl ester, or alkyl acid methyl ester oxy. In some embodiments, the ratio of compound IR-1, modifier, catalyst, and organic solvent is 1 mmol: 1~3 mmol: 1~2 mmol: 9~15 mL. In some embodiments, the reaction temperature is 20~40 °C. In some embodiments, the reaction time is 2~5 h.

[0038] When R1 in Formula I is selected from phosphonic acid group or substituted phosphonate group, in some embodiments, the preparation method of the cordycepin derivative shown in Formula I is as follows: in anhydrous N,N-dimethylformamide, with NaH as catalyst, compound IR-1 reacts with a modifier-substituted or unsubstituted p-toluenesulfonyloxymethyl phosphate, wherein the substitution is any one or more functional groups substituted by alkoxy, halogen-substituted alkoxy, aryloxy, amino acid ester amide, alkyl ester, or alkyl acid methyl ester oxy. In some embodiments, the molar ratio of compound IR-1, modifier, catalyst, and organic solvent is 0.1 mol: 0.1~0.15 mol: 0.2~0.03 mol: 100~200 mL, and in some embodiments it is 0.1 mol: 0.1~0.15 mol: 0.2~0.03 mol: 150 mL. In some embodiments, the reaction temperature is -20~0 °C, and in some embodiments the reaction time is 0.5-6 h.

[0039] When R1 in Formula I is selected from alkyl alcohol, amino acid alkyl ester, amino acid alkyl alcohol ester, alkyl acid alkyl ester, or cycloalkyl polyol, in some embodiments, the preparation method of the cordycepin derivative shown in Formula I is as follows: in butanone, with potassium carbonate as a catalyst, compound IR-1 reacts with a modifier, wherein the modifier is a halogen-substituted alkyl alcohol, amino acid alkyl ester, amino acid alkyl alcohol ester, alkyl acid alkyl ester, or cycloalkyl polyol. In some embodiments, the halogen is bromine or chlorine. In some embodiments, the ratio of compound IR-1, modifier, catalyst, and organic solvent is 1 mmol: 0.5~1.5 mmol: 1~3 mmol: 5~8 mL, and in some embodiments it is 1 mmol: 1 mmol: 1~3 mmol: 5~8 mL. In some embodiments, the reaction temperature is 40~100 °C, and in some embodiments, the reaction time is 8~20 h.

[0040] B. Modify R2

[0041] When R2 in Formula I is selected from azide group; the preparation method of the cordycepin derivative shown in Formula I is as follows: in an organic solvent, compound IR-2 is used as a raw material to carry out a cyclization reaction to obtain intermediate IR-2a, 5-(6-amino-9H-purin-9-yl)-1,4-dioxapyrrole[2.4]heptane-6-ol or its derivative; in an organic solvent, the obtained intermediate IR-2a is subjected to a ring-opening reaction to obtain the cordycepin derivative shown in Formula I;

[0042]

[0043] IR-2

[0044] In formula IR-2, R1 and R3~R7 are the same as R1 and R3~R7 in formula I, or are selected independently from the protecting group.

[0045] In some embodiments, the intermediate IR-2a is prepared by cyclizing compound IR-2 in dichloromethane under the catalysis of phosphorus pentoxide and m-chloroperoxybenzoic acid. In some embodiments, the ratio of compound IR-2, phosphorus pentoxide, m-chloroperoxybenzoic acid and dichloromethane is 1 mmol: 1.2~2 mmol: 2~3 mmol: 10~20 mL. In some embodiments, the cyclization reaction temperature is 20~60 °C. In some embodiments, the cyclization reaction time is 3~10 h.

[0046] In some embodiments, the preparation method of the cordycepin derivative shown in Formula I is as follows: the intermediate IR-2a obtained in anhydrous dimethylformamide undergoes a ring-opening reaction with sodium azide. In some embodiments, the ratio of the amount of compound IR-2a, sodium azide and dimethylformamide is 1 mmol: 4~5 mmol: 2~5 mL. In some embodiments, the temperature of the ring-opening reaction is 100~120 °C. In some embodiments, the time of the ring-opening reaction is 12~16 h.

[0047]

[0048] IR-2a

[0049] C. Modify R3

[0050] When R3 in Formula I is selected from fluorine, chlorine, or azide, the preparation method of the cordycepin derivative shown in Formula I is as follows: in an organic solvent, using compound IR-3 as a raw material, a chemical reaction is carried out to obtain the cordycepin derivative shown in Formula I.

[0051]

[0052] IR-3

[0053] In formula IR-3, R1, R2, and R5~R7 are the same as R1, R2, and R5~R7 in formula I, or are independently selected from protecting groups.

[0054] When R3 in Formula I is selected from fluorine or chlorine, in some embodiments, the preparation method of the cordycepin derivative shown in Formula I is as follows: Compound IR-3 is reacted with trifluoromethanesulfonic anhydride in pyridine and dichloromethane to obtain intermediate IR-3a, 5-(6-amino-9H-purin-9-yl)-4-hydroxy-2-(hydroxymethyl)tetrahydrofuran-3-yltrifluoromethanesulfonate or its derivative; intermediate IR-3a is subjected to a substitution reaction with hydrofluoric acid, sulfur trifluoride or hydrochloric acid in ethyl acetate to obtain the cordycepin derivative shown in Formula I; wherein, in the preparation method of intermediate IR-3a, in some embodiments, the ratio of compound IR-3, trifluoromethanesulfonic anhydride, pyridine and dichloromethane is 1 mmol: 1~1.5 mmol: 0.15~0.2 mL: 10~20 mL; in some embodiments, the reaction temperature is -5~5 °C; in some embodiments, it is 0 °C; and in some embodiments, the reaction time is 1~3 minutes. h; wherein, in the preparation method of the cordycepin derivative shown in Formula I, in some embodiments, the intermediate IR-3a is subjected to a substitution reaction with a triethylamine solution of 37% hydrofluoric acid, diethylaminotrifluoride, or hydrochloric acid; in some embodiments, the ratio of the intermediate IR-3a, the triethylamine solution of 37% hydrofluoric acid, diethylaminotrifluoride, or hydrochloric acid, to ethyl acetate is 1 mmol: 2~3 mmol: 4~10 mL; in some embodiments, the reaction temperature is 60~80 °C; and in some embodiments, the reaction time is 8~10 h.

[0055]

[0056] IR-3a IR-3b

[0057] When R3 in Formula I is selected from azide, in some embodiments, the preparation method of the cordycepin derivative shown in Formula I is as follows: In N,N-dimethylformamide, compound IR-3 is subjected to a cyclization reaction catalyzed by triphenylphosphine and diisopropyl azodicarbonate to obtain intermediate IR-3b, 4-(6-amino-9H-purin-9-yl)-3,6-dioxane[3.1.0]hexane-2-yl)methanol or its derivative; in dimethylformamide, the obtained intermediate IR-3b is subjected to a ring-opening reaction with sodium azide to obtain the cordycepin derivative shown in Formula I; wherein, in the preparation method of intermediate IR-3b, in some embodiments, the ratio of compound IR-3, triphenylphosphine, diisopropyl azodicarbonate and N,N-dimethylformamide is 10.0 mmol: 16~28 mmol: 1.6~2.8 mmol: 15~50 mL, and in some embodiments it is 10.0 mmol: 22 mL. mmol: 2.2 mmol: 15~50 mL, in some embodiments, the cyclization reaction temperature is 10~60 °C, in some embodiments, the cyclization reaction time is 1~5 h; in some embodiments, in the preparation method of the cordycepin derivative of Formula I, the ratio of intermediate IR-3b, sodium azide and dimethylformamide is 1 mmol: 4~5 mmol: 2~5 mL, in some embodiments, the ring-opening reaction temperature is 100~120 °C, in some embodiments, the ring-opening reaction time is 12~16 h.

[0058] D. Modify R4

[0059] When R4 in Formula I is selected from cyano, β-amide-γ-cyclosulfonyloxy, amino acid carboxylic acid ester, amino acid alkyl ester phosphonic acid phenyl ester, or amino acid alkyl ester phosphonic acid ester; the preparation method of the cordycepin derivative shown in Formula I is as follows: in an organic solvent, using compound IR-4 as a raw material, a chemical reaction is carried out to obtain the cordycepin derivative shown in Formula I.

[0060]

[0061] IR-4

[0062] In formula IR-4, R1~R3 ​​and R5~R7 are the same as R1~R3 ​​and R5~R7 in formula I, or are independently selected from protecting groups.

[0063] When R4 in Formula I is selected from cyano, in some embodiments, the preparation method of the cordycepin derivative shown in Formula I is as follows: In dichloromethane, compound IR-4, trifluoromethanesulfonic acid, and trimethylsilyl trifluoromethanesulfonate are first stirred at -50 to -30°C, and then reacted with trimethylnitrile silane and triethylamine; in some embodiments, the ratio of the amount of compound IR-4, trifluoromethanesulfonic acid, trimethylsilyl trifluoromethanesulfonate, and dichloromethane is 10 mmol: 0.8~1.8 mL: 2.2~3.2 mL: 90~110 mL, and in some embodiments, it is 10 mmol: 1.3 mL: 2.7 mL: 100 mL; in some embodiments, the stirring time is 20~40 min, and in some embodiments, it is 30 min; in some embodiments, the ratio of the amount of compound IR-4 to trimethylnitrile silane and triethylamine is 10 mmol: 3.4~4.3 g: 3~4 g. mL, in some embodiments the reaction temperature is 20~30°C, in some embodiments room temperature, and in some embodiments the reaction time is 2~4 h.

[0064] When R4 in Formula I is selected from β-amide-γ-cyclosulfonyloxy, in some embodiments, the preparation method of the cordycepin derivative shown in Formula I is as follows: Compound IR-4 is first oxidized to prepare intermediate IR-4a, 2-(6-amino-1,6-dihydro-9H-purin-9-yl)-5-(hydroxymethyl)dihydrofuran-3(2H)-one or its derivative, and then cyanided to prepare intermediate IR-4b, 2-(6-amino-1,6-dihydro-9H-purin-9-yl)-5-(hydroxymethyl)dihydrofuran-3(2H)-one or its derivative. -(hydroxymethyl)-3-isocyanatetetrahydrofuran-3-ol or its derivatives, followed by methanesulfonation to prepare intermediate IR-4c, 2-(6-amino-1,6-dihydro-9H-purin-9-yl)-5-(hydroxymethyl)-3-isocyanatetetrahydrofuran-3-ylmethanesulfonate or its derivatives, and finally cyclized to prepare the cordycepin derivative of formula I; in some embodiments, the intermediate IR-4a is prepared by: in acetone, compound IR-4 and Jones reagent 2.2 In the M-chromium trioxide reaction, in some embodiments, the ratio of compound IR-4, Jones' reagent, and acetone is 20 mmol: 5-8 mL: 50-100 mL; in some embodiments, the reaction temperature is 0-40 °C; and in some embodiments, the reaction time is 1-4 h. In some embodiments, the intermediate IR-4b is prepared by reacting intermediate IR-4a with trimethylsilyl cyanide and boron trifluoride diethyl ether in dichloromethane. In some embodiments, the ratio of intermediate IR-4a, trimethylsilyl cyanide, boron trifluoride diethyl ether, and dichloromethane is 20 mmol: 20-40 mmol: 10-30 mmol: 50-100 mL; in some embodiments, it is 20 mmol: 20-40 mmol: 20 mmol: 50-100 mL. In some embodiments, the reaction temperature is 0-40 °C; and in some embodiments, the reaction time is 1-4 h. h; In some embodiments, the intermediate IR-4c is prepared by reacting intermediate IR-4b with triethylamine and methanesulfonyl chloride in anhydrous dichloromethane. In some embodiments, the amounts of intermediate IR-4b, triethylamine, methanesulfonyl chloride, and anhydrous dichloromethane are 2.2 mmol: 10~15 mmol: 4~8 mmol: 5~10 mL. In some embodiments, the reaction temperature is -30~0 °C. In some embodiments, the reaction time is 2~4 h. In some embodiments, the cordycepin derivative of Formula I is prepared by reacting intermediate IR-4c with cesium carbonate in anhydrous acetonitrile. In some embodiments, the ratio of intermediate IR-4c, cesium carbonate, and anhydrous acetonitrile is 1 mmol: 0.5~2.5 mmol: 3~10 mL. In some embodiments, it is 1 mmol: 1.5 mmol: 3~10 mL; in some embodiments, the reaction temperature is 0~40 °C; in some embodiments, the reaction time is 2~4 h.

[0065]

[0066] IR-4a IR-4b IR-4c

[0067] When R4 in Formula I is selected from an amino acid carboxylic acid ester group, in some embodiments, the preparation method of the cordycepin derivative shown in Formula I is as follows: in anhydrous pyridine, compound IR-4 is reacted with amino acid acyl chloride to obtain the cordycepin derivative shown in Formula I; in some embodiments, the ratio of the amount of compound IR-4, amino acid acyl chloride and pyridine is 10 mmol: 5~15 mmol: 50~100 mL, in some embodiments it is 10 mmol: 10 mmol: 50~100 mL, in some embodiments the reaction temperature is 20~60 ℃, in some embodiments it is 40 ℃, and in some embodiments the reaction time is 6~20 h.

[0068] When R4 in Formula I is selected from amino acid alkyl ester phosphonate phenyl ester, in some embodiments, the preparation method of the cordycepin derivative shown in Formula I is as follows: in anhydrous N,N-dimethylformamide, with NaH as a catalyst, compound IR-4 reacts with the modifier amino acid alkyl ester-substituted p-toluenesulfonyloxymethyl phenyl phosphate. In some embodiments, the ratio of compound IR-4, modifier, NaH and anhydrous N,N-dimethylformamide is 0.1 mol: 0.1~0.15 mol: 0.2~0.03 mol: 100~200 mL, and in some embodiments it is 0.1 mol: 0.1~0.15 mol: 0.2~0.03 mol: 150 mL. In some embodiments, the reaction temperature is -20~0 °C, and in some embodiments, the reaction time is 0.5-6 h.

[0069] When R4 in Formula I is selected from amino acid alkyl ester phenyl phosphate, in some embodiments, the preparation method of the cordycepin derivative shown in Formula I is as follows: in anhydrous N,N-dimethylformamide and / or tetrahydrofuran, using tert-butylmagnesium chloride as a catalyst, compound IR-4 reacts with nitrobenzene phosphate substituted with a modifier, wherein the substitution is any one or more functional groups substituted with aryloxy or alkyl methyl ester oxy. In some embodiments, the ratio of compound IR-4, modifier, catalyst and organic solvent is 1 mmol: 1~3 mmol: 1~2 mmol: 9~15 mL. In some embodiments, the reaction temperature is 20~40 °C. In some embodiments, the reaction time is 2~5 h.

[0070] E. Modify R5

[0071] In Formula I, R5 is selected from bromovinyl, mercapto, methyl, fluorine, or chlorine; the preparation method of the cordycepin derivative shown in Formula I is as follows: in an organic solvent, using compound IR-5 as a raw material, a chemical reaction is carried out to obtain the cordycepin derivative shown in Formula I.

[0072]

[0073] IR-5

[0074] In formula IR-5, R1~R4 and R6~R7 are the same as R1~R4 and R6~R7 in formula I, or are independently selected from protecting groups.

[0075] When R5 in Formula I is selected from bromovinyl alcohol, in some embodiments, the preparation method of the cordycepin derivative shown in Formula I is as follows: Compound IR-5 is subjected to an iodination reaction to obtain intermediate IR-5a, 2-(6-amino-2-iodo-1,6-dihydro-9H-purin-9-yl)-5-(hydroxymethyl)tetrahydrofuran-3-ol or a derivative thereof; intermediate IR-5a is subjected to a methyl acrylate substitution reaction to obtain intermediate IR-5b, methyl(E)-3-(6-amino-3-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-6,9-dihydro-1H-purin-2-yl)acrylate or a derivative thereof; intermediate IR-5b is subjected to hydrolysis to obtain intermediate IR-5c, (E)-... -3-(6-amino-9-3-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-6,9-dihydro-1H-purin-2-yl)acrylic acid or a derivative thereof, intermediate IR-5c is subjected to bromination with N-bromosuccinimide to obtain the cordycepin derivative shown in Formula I; in some embodiments, the intermediate IR-5a is prepared by reacting compound IR-5 with elemental iodine in dilute nitric acid to prepare intermediate IR-5a. In some embodiments, the molar ratio of compound IR-5 to elemental iodine is 1 mmol: 0.5~0.8 mmol. In some embodiments, the reaction temperature is 100~120 °C, in some embodiments it is 110 °C, and in some embodiments the reaction time is 4~6 minutes. h; In some embodiments, the intermediate IR-5b is prepared by reacting intermediate IR-5a with methyl acrylate and triethylamine in 1,4-dioxane under the catalysis of palladium acetate and triphenylphosphine. In some embodiments, the molar ratio of intermediate IR-5a, methyl acrylate, triethylamine, palladium acetate, triphenylphosphine and 1,4-dioxane is 1 mmol: 3~4 mmol: 0.1~0.5 mL: 0.01~0.09 mmol: 0.05~0.15 mmol: 10~20 mL, and in some embodiments it is 1 mmol: 3~4 mmol: 0.1~0.5 mL: 0.05 mmol: 0.01 mmol: 10~20 mL. In some embodiments, the reaction temperature is 50~90 °C, and in some embodiments, the reaction time is 0.5~2 hours. h; In some embodiments, the intermediate IR-5c is prepared by hydrolyzing intermediate IR-5b with sodium hydroxide solution, wherein in some embodiments, the concentration of sodium hydroxide solution is 0.5~3.The concentration of the intermediate IR-5b is 5 mol / L, and in some embodiments it is 2 mol / L. In some embodiments, the ratio of the intermediate IR-5b to the sodium hydroxide solution is 1 g: 10-14 mL, and in some embodiments it is 1 g: 12 mL. In some embodiments, the reaction temperature is 20-30 °C, and in some embodiments it is room temperature. In some embodiments, the reaction time is 3-5 h. In some embodiments, the preparation method of the cordycepin derivative of Formula I is as follows: intermediate IR-5c reacts with N-bromosuccinimide in a mixed solvent of water and acetone under potassium carbonate catalysis. In some embodiments, the ratio of intermediate IR-5c, N-bromosuccinimide, potassium carbonate, and mixed solvent is 1 mmol: 1-3 mmol: 1-3 mmol: 15-30 mL. In some embodiments, the volume ratio of water to acetone is 1:4-8, and in some embodiments it is 1:6.

[0076]

[0077] IR-5a IR-5b IR-5c

[0078] In some embodiments, unmodified cordycepin is directly modified with vinyl bromide, and the reaction pathway is as follows:

[0079] .

[0080] When R5 in Formula I is selected from thiol, in some embodiments, the preparation method of the cordycepin derivative shown in Formula I is as follows: Compound IR-5 is reacted with hydrogen peroxide in acetic acid to obtain intermediate IR-5d, 1N-oxy-3'-deoxyadenosine or its derivative; intermediate IR-5d is heated under reflux in hydrochloric acid aqueous solution to obtain intermediate IR-5e, 5-amino-N'-hydroxy-3-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-1H-imidazol-4-carboxamide or its derivative; intermediate IR-5e is dissolved in water and reacted under hydrogen atmosphere with Raney nickel catalysis to obtain intermediate IR. -5f, 5-amino-3-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-1H-imidazol-4-carboxamide or a derivative thereof, intermediate IR-5f is reacted with methanol, pyridine and carbon disulfide to prepare the cordycepin derivative shown in Formula I; in some embodiments, the intermediate IR-5f is prepared by reacting compound IR-5 with acetic acid in hydrogen peroxide, in some embodiments the concentration of hydrogen peroxide is 20%~40%, in some embodiments it is 30%, and in some embodiments the ratio of compound IR-5, hydrogen peroxide and acetic acid is 1 mmol: 2~3 In some embodiments, the reaction temperature is 30-50 °C, and the reaction time is 2-4 days. In some embodiments, the intermediate IR-5e is prepared by heating intermediate IR-5d under reflux in an aqueous hydrochloric acid solution. In some embodiments, the concentration of the aqueous hydrochloric acid solution is 1-5 mol / L, and in some embodiments it is 3 mol / L. In some embodiments, the ratio of intermediate IR-5d to aqueous hydrochloric acid solution is 1 mmol: 3-5 mL. In some embodiments, the reflux time is 10-30 min. In the preparation method of intermediate IR-5f, in some embodiments, the ratio of intermediate IR-5e, Raney nickel, and water is 1 mmol: 0.08-0.2 g: 10-20 mL. In some embodiments, the reaction temperature is 50-70 °C. In some embodiments, the reaction time is 2-5 days; wherein, in the preparation method of the cordycepin derivative of Formula I, in some embodiments, the ratio of the intermediate IR-5f to methanol, pyridine and carbon disulfide is 1 mmol: 5-10 mL, in some embodiments, the volume ratio of methanol, pyridine and carbon disulfide is 4:3-7:0.5-3.5, in some embodiments it is 4:5:2, in some embodiments the reaction temperature is 30-50 °C, in some embodiments it is 40 °C, and in some embodiments the reaction time is 3-5 days.

[0081]

[0082] IR-5d IR-5e IR-5f

[0083] In some embodiments, unmodified cordycepin is directly modified with thiol groups, and the specific reaction method is as follows:

[0084] .

[0085] When R5 in Formula I is selected from methyl, in some embodiments, the preparation method of the cordycepin derivative shown in Formula I is as follows: in dichloromethane solvent, compound IR-5, iodomethane, and potassium carbonate react. In some embodiments, the ratio of the amount of compound IR-5, iodomethane, potassium carbonate, and dichloromethane is 1 mmol: 1~2 mmol: 1.5~3 mmol: 5~10 mL. In some embodiments, the reaction temperature is 20~50 °C. In some embodiments, the reaction time is 3~10 h.

[0086] When R5 in Formula I is selected from fluorine or chlorine, in some embodiments, the preparation method of the cordycepin derivative shown in Formula I is as follows: Compound IR-5 is subjected to a nitroderivatization reaction to obtain a nitro-terminal IR-5g, 2-(6-amino-2-nitro-1,6-dihydro-9H-purin-9-yl)-5-(hydroxymethyl)tetrahydrofuran-3-ol, and then subjected to a substitution reaction to obtain the cordycepin derivative shown in Formula I; in some embodiments, the intermediate IR-5g is prepared by reacting compound IR-5 with tetrabutylammonium nitrate in dichloromethane under the catalysis of trifluoroacetic anhydride. In some embodiments, the ratio of compound IR-5, tetrabutylammonium nitrate, trifluoroacetic acid, and dichloromethane is 1 mmol: 1.4~2 mmol: 1~2 mmol: 15~35 mL. In some embodiments, the reaction temperature is -10~10 ℃, and the reaction time is 0.5~20 minutes. h; In some embodiments, the method for preparing the cordycepin derivative of Formula I is as follows: in acetonitrile, the nitration intermediate IR-5g is reacted with tetrabutylammonium fluoride or tetrabutylammonium chloride. In some embodiments, the ratio of the nitration intermediate IR-5g, tetrabutylammonium fluoride or tetrabutylammonium chloride to acetonitrile is 1 mmol: 1.3~1.5 mmol: 30~50 mL. The reaction temperature is -5~5 ℃, and in some embodiments it is 0 ℃. In some embodiments, the reaction time is 20~30 min.

[0087]

[0088] IR-5g

[0089] F. Modification of R6

[0090] When R6 in Formula I is selected from a substituted formamide group; the substitution is any one or more functional groups selected from alkyl, aryl, cycloalkyl, furanyl, and pyridyl; the preparation method of the cordycepin derivative shown in Formula I is as follows: in an organic solvent, using compound IR-6 as a raw material, a chemical reaction is carried out to obtain the cordycepin derivative shown in Formula I.

[0091]

[0092] IR-6

[0093] In formula IR-6, R1~R5 and R7 are the same as R1~R5 and R7 in formula I, or are selected independently from the protecting group.

[0094] In some embodiments, the preparation method of the cordycepin derivative shown in Formula I is as follows: under anhydrous pyridine, compound IR-6 is reacted with a substituted acyl chloride, wherein the substitution is any one or more functional groups selected from alkyl, aryl, cycloalkyl, furanyl, and pyridyl. In some embodiments, the ratio of compound IR-6, substituted acyl chloride, and anhydrous pyridine is 1 mmol: 1~2 mmol: 5~10 mL. In some embodiments, the reaction temperature is 0~60 °C. In some embodiments, the reaction time is 2~20 h.

[0095] G. Modify R7

[0096] When R7 in Formula I is selected from isopropylamino, the preparation method of the cordycepin derivative shown in Formula I is as follows: in an organic solvent, using compound IR-7 as a raw material, a chemical reaction is carried out to obtain the cordycepin derivative shown in Formula I.

[0097]

[0098] IR-7

[0099] In formula IR-7, R1~R6 are the same as R1~R6 in formula I, or are independently selected from protecting groups.

[0100] In some embodiments, the preparation method of the cordycepin derivative shown in Formula I is as follows: in dioxane, compound IR-7 is subjected to a 2-propylamine substitution reaction. In some embodiments, the ratio of compound IR-7 to 2-propylamine and dioxane is 10 mmol: 10~30 mmol: 50~100 mL. In some embodiments, the reaction temperature is 60~120 °C. In some embodiments, the reaction time is 10~30 h.

[0101] In this invention, the protective base includes, but is not limited to, -OTBS, -OAc, -NHCbz, -OTBPS, and -OTBDPS; in some embodiments, R4 is the same as R4 in Formula I or is selected from -OTBS, -OAc, and -OTBDPS; in some embodiments, R6 is the same as R6 in Formula I or is selected from -NHCbz.

[0102] In this invention, the substituted nitrobenzene phosphate (the substitution is any one or more functional groups selected from alkoxy, halogen-substituted alkoxy, aryloxy, amino acid ester amide, alkyl ester, and alkyl acid methyl ester oxy) is prepared according to the following method or other methods in the prior art.

[0103] In anhydrous dichloromethane, phenyl chlorophosphate, p-nitrophenol, and their corresponding substituted alcohols or amines are prepared under the catalysis of triethylamine at 0–25 °C. The substituted groups are any one or more functional groups selected from alkoxy, halogen-substituted alkoxy, aryloxy, amino acid ester amide, alkyl ester, and alkyl acid methyl ester oxy. In some embodiments, the molar volume ratio of phenyl chlorophosphate, p-nitrophenol, the corresponding substituted product, triethylamine, and anhydrous dichloromethane is 1 mmol:1 mmol:1–2 mmol:2–5 mmol:5–10 mL.

[0104] In this invention, the substituted p-toluenesulfonyloxymethyl phosphate (the substitution is any one or more functional groups selected from alkoxy, halogen-substituted alkoxy, aryloxy, amino acid ester amide, alkyl ester, and alkyl acid methyl ester oxy) is prepared according to the following method or other methods in the prior art.

[0105] The substituted chlorophosphate, p-toluenesulfonyl chloride, and formaldehyde are prepared in toluene under triethylamine catalysis at 0–105 °C. The substituted chlorophosphate is a chlorophosphate substituted with any one or more functional groups, including alkoxy, halogen-substituted alkoxy, aryloxy, amino acid ester amide, alkyl ester, and alkyl acid methyl ester oxy. In some embodiments, the molar volume ratio of the substituted chlorophosphate, p-toluenesulfonyl chloride, formaldehyde, triethylamine, and toluene is 1 mol: 1 mol: 0.8–1.2 mol: 180–210 mL: 500–800 mL.

[0106] In this invention, the term "prevention" means administering the compound or preparation described in this application to prevent a disease or one or more symptoms associated with said disease, and includes: preventing the occurrence of a disease or disease state in mammals, particularly when these mammals are susceptible to related cancer symptoms.

[0107] The term "pharmaceutically acceptable" in this invention refers to compounds, materials, compositions, and / or dosage forms that, within the bounds of reliable medical judgment, are suitable for use in contact with human and animal tissues without excessive toxicity, irritation, allergic reactions, or other problems or complications, in proportion to a reasonable benefit / risk ratio.

[0108] The compounds of this invention can exist in specific geometric or stereoisomeric forms. This invention contemplates all such compounds, including cis and trans isomers, (-)- and (+)- enantiomers, (R)- and (S)- enantiomers, diastereomers, (D)- isomers, (L)- isomers, and racemic mixtures thereof, as well as other mixtures, such as mixtures enriched with enantiomers or diastereomers, all of which are within the scope of this invention. Additional asymmetric carbon atoms may be present in substituents such as alkyl groups. All such isomers and mixtures thereof are included within the scope of this invention.

[0109] Beneficial effects: Compared with the prior art, the present invention has the following advantages:

[0110] The cordycepin derivatives and pharmaceutical compositions provided by this invention exhibit excellent anti-tumor proliferation effects. Compared to the parent drug, the cordycepin derivatives have better affinity for cell membranes, resulting in a longer metabolic half-life and longer retention time in the body. Compared to other nucleoside antitumor drugs, the cordycepin derivatives and pharmaceutical compositions provided by this invention have a broader range of tumor types and effects, including excellent inhibitory effects on gastric cancer, pancreatic cancer, liver cancer, small cell lung cancer, colorectal cancer, melanoma, and ovarian cancer, with fewer side effects and better efficacy.

[0111] Instruction manual illustrations

[0112] Figure 1 The study compared the blank control group, cordycepin control group, and compound group to the antitumor effects on a mouse model of Hep-1-6 liver cancer cells transplanted into the mouse model.

[0113] Figure 2 The inhibitory effects of compound 16 and immune checkpoint inhibitors on a mouse model of colon cancer MC38 cell transplantation were investigated.

[0114] Figure 3 The study compared the antitumor effects of the control group and the compound group on a melanoma B16-F10 transplanted mouse model.

[0115] Figure 4 The inhibitory effects of compound 24 and immune checkpoint inhibitors on a mouse ID8 ovarian cancer allogeneic xenograft model were studied.

[0116] Figure 5The study compared the antitumor effects of the control group and the compound group on a mouse model of gastric cancer BGC-823 cell transplantation.

[0117] Figure 6 The study compared the antitumor effects of the control group and the compound group on a mouse model of pancreatic cancer Pan02-luc cell transplantation. Detailed Implementation

[0118] Unless otherwise specified, the experimental methods described in the following examples are conventional methods; unless otherwise specified, the reagents and materials are commercially available, and the drug evaluation experiments, including cell models and animal models, are commercially available unless otherwise specified.

[0119] The cordycepin described in this invention is obtained through a bio-fermentation route (CN 111117896B). The preparation method of cordycepin or its derivatives follows the method described in the specification. Post-processing mainly employs conventional organic experimental post-processing operations, including but not limited to filtration, quenching, extraction, rotary evaporation, recrystallization, and column chromatography. The preparation methods of the compounds mentioned in the examples are not exhaustive; for ease of explanation, only the preparation processes of a few representative compounds are listed here.

[0120] The cell and animal model experiments related to the anti-cancer effects of cordycepin or its derivatives in the embodiments are not all effective experimental results, but are only listed relevant experiments to illustrate the effects. Furthermore, the drug evaluation experiments, including cell model and animal model experiments, are conducted in accordance with ethical rules.

[0121] The drug evaluation described above, when used to house the mouse model, follows these guidelines: Five nude mice are housed in each cage in a laminar flow cabinet; water and feed are provided every three days; bedding is changed weekly; the number of mice per cage should not exceed five; mice in separate cages should be kept in groups as much as possible, avoiding single-cage placement; animal management must comply with national standards for animal husbandry and management, while also paying attention to the animals' behavioral needs to avoid unnecessary stress; the animals' normal physiological and behavioral needs must be met, such as defecation, urination, maintaining a constant body temperature, normal activity, posture adjustment, and reproduction; good ventilation should be provided to keep the animals dry; they should have free access to water and food, with easy replenishment, replacement, and cleaning; a sturdy and safe environment should be provided to prevent escape or limbs from getting stuck in gaps; sharp edges or protrusions should be avoided to prevent injury to the animals; and observation of the animals should not disturb them.

[0122] The zebrafish model used was a wild-type AB strain zebrafish obtained from the School of Biological and Pharmaceutical Engineering, Nanjing University of Technology. Reproduction was carried out through natural pair mating, with 30 fish per experimental group, aged 2 days post-flop (dpf). They were housed in aquarium water at 28℃ (water quality: 200 mg of readily soluble sea salt added per 1 L of reverse osmosis water; conductivity 480–510 uS / cm; pH 6.9–7.2; hardness 53.7–71.6 mg / L CaCO3), and the husbandry management met the requirements of the international AAALAC certification.

[0123] The method for determining the inhibitory activity of the compound on tumor cell proliferation using the MTT assay is as follows: Take one flask of cells in good exponential growth phase, add 0.25% trypsin digestion solution, digest to detach the adherent cells, and count 2-4 × 10⁻⁴ cells. 4 Cells were prepared at a concentration of 10 cells / mL to form a cell suspension. The cell suspension was seeded into 96-well plates at 90 μL / well and incubated in a CO2 incubator for 24 hours. The prepared compound was then added for testing at 10 μL / well, and the plates were incubated for 72 hours. MTT reagent was added to the 96-well plates at 10 μL / well, and the reaction was carried out in an incubator for 4 hours. The supernatant was then removed, and dimethyl sulfoxide (DMSO) was added at 100 μL / well. After the crystals dissolved, the absorbance of each well was measured at 570 nm using an ELISA reader, and the cell inhibition rate was calculated. An S-curve was plotted between the compound concentration and the corresponding inhibition rate. The IC50 of the corresponding compound was obtained. 50 .

[0124] Example 1

[0125] (1) Methods for hydroxyl protection and deprotection of cordycepin

[0126] The protection 1a and deprotection method of the hydroxyl groups at the R1 and R4 positions of cordycepin described herein:

[0127]

[0128] Protection: 0.251 g (1 mmol) of cordycepin, 0.3618 g (2.4 mmol) of TBSCl, and 0.3404 g (5 mmol) of imidazole were added to 10 mL of DMF. The mixture was stirred at room temperature for 10 h. After the reaction was complete, the mixture was extracted with water and ethyl acetate. The organic phase was concentrated to give 0.4607 g of 1a, with a yield of 96.03%. MSI-MS: 480.8 [M+H] + .

[0129] Deprotection: Prepare 10 ml of solvent (tetrabutylammonium fluoride: THF = 2:1), add 0.48 g (1 mmol) of 1a, stir at room temperature for 5 h, extract with water and ethyl acetate after the reaction is complete, concentrate the organic phase, recrystallize to obtain 0.246 g of cordycepin, yield 97.96%.

[0130] Protection 1b of the hydroxyl group at the R1 position of cordycepin and the deprotection method thereof:

[0131]

[0132] Protection: 0.251 g (1 mmol) of cordycepin, 0.3618 g (2.4 mmol) of TBSCl, and 0.3404 g (5 mmol) of imidazole were added to 10 mL of DMF. The mixture was stirred at room temperature for 10 h. After the reaction was complete, the mixture was extracted with water and ethyl acetate. The organic phase was concentrated to give 0.4607 g of 1a (96.03%). Separately, 0.48 g (1 mmol) of 1a was added to 10 mL of solvent (acetic acid:water:THF = 13:7:3). The mixture was stirred at room temperature for 15 h. After the reaction was complete, the mixture was extracted with water and ethyl acetate. The organic phase was concentrated and column chromatography was used to give 0.293 g of 1b (79.99%), MSI-MS: 366.5 [M+H]. + .

[0133] Deprotection: 0.366 g (1 mmol) of 1b was added to 10 mL of 0.1 M hydrochloric acid-methanol solution and stirred at room temperature for 10 h. After the reaction was completed, the pH was adjusted to neutral with 0.1 M sodium carbonate aqueous solution and extracted with ethyl acetate. The organic phase was concentrated and obtained by rotary evaporation or recrystallization, yielding 0.215 g of cordycepin with a yield of 85.56%.

[0134] The protection method of the hydroxyl group at the R4 position of cordycepin and the deprotection method thereof are as follows:

[0135]

[0136] Protection: 1.256 g (5 mmol) of cordycepin was added to 10 mL of dichloromethane and 10 mL of diisopropylethylamine under nitrogen protection, followed by dropwise addition of 0.451 g (1.6 mmol) of TBDPSCl. The mixture was stirred at room temperature for 2 h, concentrated, and purified by column chromatography to give 2.20 g of 1c, with a yield of 89.9%, MSI-MS: 366.5 [M+H]. + .

[0137] Deprotection: Prepare 10 mL of solvent (tetrabutylammonium fluoride: THF = 2:1), add 0.490 g (1 mmol) of 1c, stir at room temperature for 5 h, extract with water and ethyl acetate after the reaction, concentrate the organic phase, recrystallize to obtain 0.244 g of cordycepin, with a yield of 97.01%.

[0138] (2) Specific preparation method of compound 2-(6-amino-9H-purine-9-yl)-5-(2-hydroxyethoxy)methyl)tetrahydrofuran-3-ol

[0139] Using compound 1b as a starting material, 3.65 g (10 mmol) of 1b and 0.81 g (10 mmol) of 2-chloro-ethanol were placed in a round-bottom flask. 60 mL of butanone was added, and the reaction mixture was heated to dissolve. 2.07 g (15 mmol) of potassium carbonate was also added. The reaction was carried out at 80 °C for 8 h, and the reaction was monitored by TLC. After the reaction was complete, the mixture was filtered, the reaction solution was concentrated, and extracted twice with 60 mL of water and 60 mL of ethyl acetate. The organic phases were combined, and the resulting viscous liquid was obtained by rotary evaporation under reduced pressure. This viscous liquid was deprotected and subjected to column chromatography to obtain compound 1, weighing 2.24 g, with a yield of 76%. The detection results of the prepared compound 1 are as follows: 1 H NMR (400 MHz, DMSO-d6) δ 8.56 (s, 1H), 8.35 (s, 1H), 7.09 (s, 2H), 6.14 (d, 1H), 5.47-5.3 (d, 2H), 5.01 (m, 1H), 4.12 (m, 1H), 3.74 (m, 1H), 3.61-3.56 (m, 4H), 3.51-3.46 (m, 2H), 2.04-1.92 (m, 2H). 13 C NMR(100 MHz, DMSO-d6) δ 156.1, 152.4, 149.8, 140.1, 119.6, 95.2, 82.1, 75.2,74.7, 70.4, 61.1, 34.5. MSI-MS: 296.3 [M+H] + .

[0140] Example 2

[0141] Following the protection method in Example 1, 3.65 g (10 mmol) of compound 1b was added to 150 mL of triethyl phosphite and 4.62 g (30 mmol) of phosphorus oxychloride under ice bath conditions. The reaction was carried out at 0 °C for 2 h. After the reaction was completed, 1000 mL of water was added to quench the reaction in an ice bath. Dichloromethane was added for multiple extractions. The organic phases were combined, and the solvent was removed by rotary evaporation to obtain a viscous liquid. The prepared viscous liquid was purified by preparative liquid chromatography (C18 preparative column, Waters preparative HPLC, mobile phase was 30% acetonitrile aqueous solution, flow rate was 2 mL / min). The sample peak eluent was concentrated and weighed to obtain the phosphorylated compound, totaling 3.02 g, with a yield of 70%.

[0142] Compound 2 was prepared according to the deprotection scheme in Example 1b, and the detection results of the prepared compound 2 are as follows: 1 H NMR (400 MHz, DMSO-d6) δ 8.54 (s, 1H), 8.36 (s, 1H), 7.11 (s, 2H), 6.19 (d, 1H), 5.15 (d, 1H), 4.28-4.23 (m, 2H), 4.2 (s, 2H), 4.02 (m, 1H), 3.71 (m, 1H), 2.06-1.90 (m, 2H). 13 C NMR (100 MHz, DMSO-d6) δ 156.2, 152.7,149.5, 140.2, 119.3, 97.2, 76.1, 74.6, 69.5, 34.5. MSI-MS: 332.2 [M+H] + .

[0143] Example 3

[0144]

[0145] Compound 1b 36.5 g (100 mmol) was added to a 500 mL reaction flask along with 150 mL of DMF as solvent, and the mixture was heated to dissolve. After rapid cooling to room temperature, 80% NaH 6.4 g (267 mmol) was added in portions with stirring, and the mixture was kept at room temperature and stirred for 15 min. The reaction mixture was cooled to -10 °C in a cryogenic reactor, and 45.1 g (140 mmol) of p-toluenesulfonyloxymethyl phosphate was added dropwise. After the addition was complete, the mixture was stirred at this temperature for 1 h, and then gradually heated to room temperature and stirred for 4 h. After the reaction was complete, glacial acetic acid was added dropwise to adjust the pH to neutral. The reaction mixture was filtered, washed with dichloromethane, and the filtrate was extracted three times with water (60 mL). The dichloromethane phases were combined, the solvent was removed by vacuum distillation, and toluene was added to the residue for recrystallization. The mother liquor was filtered, and the filter cake was dried under reduced pressure at 50 °C to obtain a white powdery solid 3a, weighing 26.87 g, with a yield of 52%. MSI-MS: 540.6 [M+23]. + .

[0146] 5.17 g (10 mmol) of compound 3a was added to a 100 mL reaction flask, followed by 20 mL of n-butyronitrile. 6.52 g (60 mmol) of trimethylchlorosilane was then added dropwise at room temperature. After the addition was complete, the reaction mixture was refluxed for 24 h. After the reaction was complete, the solvent was concentrated to dryness by vacuum distillation. 20 mL of water was added to the residue, and 2 M sodium hydroxide was added to adjust the pH to approximately 8. The mixture was then extracted three times with 50 mL of ethyl acetate. The aqueous phase was neutralized to pH 3–4 with 1 M hydrochloric acid. The mixture was then heated to 70–80 °C for crystallization to obtain compound 3b, weighing 3.40 g, with a yield of 74%. MSI-MS: 460.6 [M+H] + .

[0147] Compound 3b was treated according to the deprotection scheme of Example 1b to obtain compound 3. The detection results of the prepared compound are as follows: 1 H NMR (400 MHz, DMSO-d6) δ 8.58 (s, 1H), 8.32 (s, 1H), 7.13(s, 2H), 6.12 (d, 1H), 5.39 (d, 1H), 4.81 (s, 2H), 4.02 (m, 1H), 3.98 (m,1H), 3.74 (d, 2H), 3.63-3.56 (m, 2H), 2.08-1.94 (m, 2H). 13C NMR (100 MHz, DMSO-d6) δ 156.7, 152.6, 149.1, 140.4, 119.5, 98.2, 81.1, 76.3, 74.3, 73.1,34.7. MSI-MS: 346.3 [M+H] + .

[0148] Example 4

[0149]

[0150] Compound 1b 3.65 g (10 mmol) was added to a 500 mL reaction flask and dissolved in 100 mL of anhydrous DMF. Diisobutyl phosphate (4-nitrophenyl) 4.96 g (15 mmol) was also added. Separately, tert-butylmagnesium chloride 1.17 g (10 mmol) was dissolved in 20 mL of THF and slowly added dropwise to the above reaction solution. The reaction mixture was gradually heated to room temperature and reacted for 2 h, monitored by TLC. After the reaction was complete, the resulting mixture was allowed to stand, diluted with 100 mL of ethyl acetate, washed and extracted three times with 50 mL of saturated sodium bicarbonate solution each time, followed by extraction with 50 mL of saturated sodium chloride solution. The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The resulting oil was purified by silica gel column chromatography (eluent: dichloro / methanol = 10:1) to give compound 4a, weighing 4.24 g, with a yield of 76%.

[0151] Compound 4 was obtained by treating 4a using the deprotection method described in Example 1b. The detection results of the prepared compound 4 are as follows: 1 H NMR (400 MHz, DMSO-d6) δ 8.52 (s, 1H), 8.31 (s, 1H), 7.11 (s, 2H), 6.15 (d, 1H), 5.33 (d, 1H), 4.24-4.05 (m,2H), 4.02 (m, 1H), 3.93 (m,4H), 3.77 (m, 1H), 2.08-1.83 (m, 2H), 1.33 (m, 2H), 0.90 (d, 12H). 13 C NMR(100 MHz, DMSO-d6) δ 154.3, 151.7, 148.4, 141.9, 119.2, 98.2, 74.1, 74.0,73.5, 68.1, 34.2, 28.6, 19.5. MSI-MS: 466.7 [M+Na] + .

[0152] Example 5

[0153]

[0154] Compound 5 was prepared according to the method for preparing 3a in Example 3 and combined with the deprotection method in Example 1b, wherein an equivalent amount of p-toluenesulfonyloxymethyl phosphate diethyl ester was replaced with (((toluoxy)methyl)phosphoryl)bis(oxy))bis(methylene)bis(2-methylpropionic acid), and the overall yield of compound 5 was 74%. The detection results of the prepared compound 5 are as follows: 1 H NMR (400 MHz, DMSO-d6) δ 8.57 (s, 1H), 8.33 (s, 1H), 7.11 (s, 2H), 6.84 (d, 4H), 6.18 (d, 1H), 5.46 (d, 1H), 4.12 (m, 1H), 3.94 (m, 1H), 3.81(d, 2H), 3.60-3.44 (m, 2H), 2.55 (m, 2H), 2.08-1.82 (m, 2H), 1.14 (d, 12H). 13 C NMR (100 MHz, DMSO-d6) δ 174.2, 155.7, 153.6, 149.5, 141.2, 119.5, 99.4,93.2, 77.4, 74.5, 74.1, 71.5, 34.2, 33.6, 19.3. MSI-MS: 546.5 [M+H] + .

[0155] Example 6

[0156]

[0157]

[0158] The preparation method of methyl((4-nitrophenoxy)(phenoxy)phosphoryl)-D-valine is as follows:

[0159] 1.17 g (10 mmol) of valine was dissolved in 50 mL of dichloromethane. The solution was cooled to approximately 0 °C, and 2.11 g (10 mmol) of phenyl phosphate dichloride was added, followed by the slow addition of 2.02 g (20 mmol) of triethylamine. The reaction was heated to room temperature and stirred for 80 min. 1.39 g (10 mmol) of p-nitrophenol was added, followed by the dropwise addition of 2.02 g (20 mmol) of triethylamine. The mixture was stirred at room temperature for 180 min. After the reaction was complete, the sample was washed with diethyl ether and filtered to remove the solids. The filtrate was concentrated using a rotary evaporator, and the resulting sample was purified by silica gel column chromatography (elution: n-hexane / ethyl acetate (1:1)). The chemical analysis results are as follows: 1 H NMR (400 MHz, DMSO-d6) δ 8.25 (d,2H), 7.40 (m, 2H), 7.35 (d, 2H), 7.20 (m, 3H), 3.68 (s, 1H), 3.65 (s, 3H),3.35 (m, 1H), 1.90 (m, 2H), 0.86 (m, 3H). 13 C NMR (100 MHz, DMSO-d6) δ 171.6,156.5, 150.5, 141.1, 130.2, 126.5, 121.7, 121.4, 120.4, 52.2, 51.9, 26.4,9.6. MSI-MS: 395.3 [M+H] + .

[0160] Compound 6 was prepared according to the method for preparing 4a in Example 4, combined with the deprotection method in Example 1b, wherein diisobutyl phosphate (4-nitrophenyl) was replaced by an equivalent amount of methyl ((4-nitrophenoxy)(phenoxy)phosphoryl)-D-valine. The yield of compound 6 was 68%. The detection results of the final prepared compound 6 are as follows: 1H NMR (400MHz, DMSO-d6) δ 8.58 (s, 1H), 8.33 (s, 1H), 7.43 (m, 2H), 7.20 (m, 3H) 7.08(s, 2H), 6.15 (d, 1H), 5.47 (d, 1H), 4.26-4.12 (m, 2H), 4.02 (m, 1H), 3.75(m, 1H), 3.67 (d, 1H), 3.62 (s, 3H), 3.28 (d, 1H), 2.41 (m, 1H), 2.08-1.96(m, 2H), 0.98 (d, 6H). 13 C NMR (100 MHz, DMSO-d6) δ 173.6, 154.9, 151.5,150.6, 149.8, 141.3, 132.7, 123.3, 120.8, 119.2, 98.2, 76.1, 74.5, 72.0,57.6, 52.3, 35.2, 32.4, 20.1. MSI-MS: 521.4 [M+H] + .

[0161] Example 7

[0162]

[0163] The preparation process of isopropyl (phenoxy((tolyloxy)methyl)phosphoryl)alanine ester is as follows:

[0164] 216.15 g (1.65 mol) of isopropyl alanine, 155 g (1.65 mol) of phenol, and 240 mL of chloroform were added to a 1000 mL reaction flask. Under ice-salt bath conditions, with stirring, 138.8 g (1 mol) of phosphorus trichloride was added dropwise at approximately 5 °C, maintaining the reaction temperature between 5 and 10 °C. After the addition of phosphorus trichloride was complete, stirring was continued for 30 min, the ice-salt bath was removed, and the temperature was gradually raised to room temperature. Stirring continued for 2 h, and then hydrogen chloride gas was removed under reduced pressure at 50 °C. The reaction was cooled to room temperature, and 80 mL of saturated sodium bicarbonate aqueous solution was slowly added. Then, sodium bicarbonate solid powder was added to adjust the pH of the reaction solution to 7–8. The precipitated salt was filtered off, and the filtrate was placed in a separatory funnel to separate the chloroform layer. The aqueous layer was extracted once with 100 mL of chloroform. The organic phases were combined and washed once each with saturated sodium bicarbonate aqueous solution and water. The chloroform layer was then distilled under reduced pressure, and the residue was the target product, phenyl chlorophosphate alanine isopropyl ester, with a yield of 87%.

[0165] 306 g (1 mol) of phenyl chlorophosphate alanine isopropyl ester, 40.6 g (1.35 mol) of paraformaldehyde, 14 mL of triethylamine, and 260 mL of toluene were added to a 1000 mL reaction flask. The reaction was slowly heated to 105 °C under nitrogen protection, at which point the reaction was vigorous, and refluxed for 3 h. After the reaction was completed, the mixture was cooled to 0 °C in an ice-salt bath, and 169.5 g (0.9 mol) of p-toluenesulfonyl chloride, 220 mL of toluene, and 177 mL of triethylamine were added dropwise. The reaction temperature was maintained at 0 °C with stirring for 2 h, and then gradually increased to room temperature with stirring for 12 h. After the reaction was completed, the mixture was filtered, and the filter cake was washed with toluene (50 mL × 3). The mother liquors were combined and washed successively with 5% sodium carbonate aqueous solution (200 mL × 2) and water (200 mL × 2), and the organic phase was obtained after separation. Vacuum distillation of the organic phase yielded a pale yellow oily substance, isopropyl(phenoxy((tolyloxy)methyl)phosphoryl)alanine ester, which was used in subsequent reactions. The weight was 291 g, with a yield of 64%. The chemical analysis results of the prepared product are as follows: 1 H NMR (400 MHz, DMSO-d6) δ 7.74 (d,2H), 7.45-7.39 (m, 4H), 7.22 (m, 3H), 4.95 (m, 1H), 3.91 (d, 2H), 3.65 (s,1H), 3.57 (m, 1H), 2.42 (s, 3H), 1.28 (d, 3H), 1.18 (d, 6H). 13 C NMR (100 MHz, DMSO-d6) δ 171.6, 150.4, 144.4, 140.3, 130.5, 130.1, 128.5, 121.4, 120.2, 69.6, 63.8, 50.5, 21.7, 21.5, 19.2. MSI-MS: 456.5 [M+H] + .

[0166] Compound 7 was prepared according to the method for preparing 3a in Example 3, combined with the deprotection method in Example 1b, wherein an equivalent amount of p-toluenesulfonyloxymethyl phosphate diethyl ester was replaced with isopropyl (phenoxy((toluoxy)methyl)phosphonyl)alanine ester. The yield of compound 7 was 52%. The detection results of the prepared compound 7 are as follows: 1H NMR(400 MHz, DMSO-d6) δ 8.59 (s, 1H), 8.37 (s, 1H), 7.45 (m, 2H), 7.24 (m, 3H), 7.12 (s, 2H), 6.16 (d, 1H), 5.33 (d, 1H), 4.98 (m, 1H), 4.08-3.99 (m, 2H), 3.84 (m, 1H), 3.77 (m, 2H), 3.58-3.42 (m, 3H), 2.08-1.96 (m, 2H), 1.29 (d,3H), 1.16 (d, 6H). 13 C NMR (100 MHz, DMSO-d6) δ 172.1, 156.3, 152.5, 150.4,149.8, 140.1, 130.2, 122.1, 120.5, 119.1, 98.2, 79.1, 74.8, 74.7, 73.1, 69.6,52.3, 34.5, 22.5, 18.8. MSI-MS: 535.6 [M+H] + .

[0167] Example 8

[0168]

[0169] Using compound 1b as a starting material, 3.65 g (10 mmol) of 1b and 2.10 g (10 mmol) of compound 2-chloro-3-hydroxypropylvaline were placed in a round-bottom flask. 60 mL of butanone was added, and the mixture was heated to dissolve the reactants. 2.07 g (15 mmol) of potassium carbonate was also added. The reaction was carried out at 80 °C for 10 h, and the reaction was monitored by TLC. After the reaction was complete, the mixture was filtered, and the reaction solution was concentrated. Extraction was performed twice with 60 mL of water and 60 mL of ethyl acetate. The organic phases were combined, and the resulting viscous liquid was obtained by rotary evaporation under reduced pressure. Column chromatography was performed to obtain compound 8a, weighing 3.24 g, with a yield of 52%. MSI-MS: 539.6 [M+H]. + .

[0170] Compound 8 was prepared from compound 8a as a substrate using the deprotection method described in Example 1b, with a yield of 91%. The detection results for the prepared compound 8 are as follows: 1H NMR (400 MHz, DMSO-d6) δ 8.88 (s,2H), 8.54 (s, 1H), 8.37 (s, 1H), 7.14 (s, 2H), 6.18 (d, 1H), 5.57 (d, 1H),4.33-4.11 (m, 3H) 4.03 (m, 1H), 3.96-3.89 (m, 3H), 3.61-3.36 (m, 4H), 2.37(m, 1H), 2.08-1.82 (m, 2H), 0.98 (d, 6H). 13 C NMR (100 MHz, DMSO-d6) δ 173.5,154.6, 153.5, 150.4, 141.3, 119.6, 99.5, 85.6, 75.7, 74.0, 73.1, 63.3, 62.5,58.7, 34.7, 30.8, 18.8. MSI-MS: 425.4 [M+H] + .

[0171] Example 9

[0172]

[0173] The reaction was carried out using compound 2 as a starting material. 9.00 g (27.2 mmol) of compound 2 and 4.88 g (27.2 mmol) of Cb2Cl were added to 38.5 mL of toluene and 38.5 mL of water, along with 4.70 g (34 mmol) of K2CO3. The mixture was stirred vigorously at temperatures below 25 °C. After stirring at room temperature for 3 h, 0.275 g (2.72 mmol) of triethylamine and 5.78 g of sodium chloride were added sequentially, and the mixture was stirred for another 30 min. The organic layer was separated and concentrated to obtain the desired oily product 9a, weighing 11.38 g, with a yield of 90%. MSI-MS: 488.7 [M+Na] + .

[0174] Compound 9a, 9.3 g (20 mmol), was dissolved in 60 mL of acetone at room temperature with stirring. 5.50 mL of Jones' reagent (2.2 M chromium trioxide, 12.1 mmol) was added dropwise over 2 h at room temperature. The resulting reaction mixture was stirred for another 1 h at room temperature and filtered. The solution was concentrated under reduced pressure to give an oily liquid. This liquid was dissolved in 30 mL of diethyl ether, washed once with 30 mL of saturated ammonium chloride solution, dried over anhydrous magnesium sulfate, concentrated under reduced pressure, and subjected to column chromatography to give compound 9b, weighing 3.98 g (43% yield), MSI-MS: 464.4 [M+H]. + .

[0175] 4 mL (30 mmol) of trimethylsilyl cyanide and 2.53 mL (20 mmol) of boron trifluoride diethyl ether were added to a 50 mL solution of 9b (9.26 g, 20 mmol). The mixture was stirred at room temperature for 2 hours, and the solvent was evaporated to dryness. The resulting residue was dissolved in 100 mL of ethyl acetate, washed twice with 50 mL of brine, and dried over Na₂SO₄. After filtration and solvent evaporation, the solution was purified by column chromatography (n-hexane / ethyl acetate, 1:2) to give a white compound 9c, weighing 7.45 g, in 76% yield, MSI-MS: 491.9 [M+H]. + .

[0176] 1.9 mL of Et3N (14 mmol) was added to 5 mL of anhydrous dichloromethane, and 1.08 g (2.2 mmol) of compound 9c was added to dissolve the solution. After cooling the mixture to -30 °C, 460 μL (6 mmol) of methanesulfonyl chloride was slowly added. The mixture was stirred at -20 °C for 1 h and then at 0 °C for 1 h. The mixture was then concentrated under reduced pressure to remove volatiles, and the residue was dissolved in 10 mL of ethyl acetate, followed by washing twice with 10 mL of water and 10 mL of brine. The organic phase (Na2SO4) was dried and concentrated under reduced pressure, and purified by column chromatography (n-hexane:ethyl acetate, 10:1) to give a white amorphous solid 9d, weighing 0.975 g, with a yield of 78%. The preparation results are as follows: 1 H NMR (400 MHz, DMSO-d6) δ 10.45 (s, 1H),8.36 (s, 1H), 8.20 (s, 1H), 7.34-7.32 (s, 5H), 6.12 (s, 1H), 4.65 (s, 2H),4.30-4.03 (m, 4H), 3.74 (m,1H), 3.15 (s, 3H), 2.41-2.16 (m, 2H). 13C NMR (100MHz, DMSO-d6) δ 153.6, 152.5, 151.7, 149.8, 140.2, 136.4, 128.9, 127.7,127.2, 123.5, 120.4, 97.4, 71.6, 69.2, 68.2, 66.9, 38.2, 37.8. MSI-MS: 569.4[M+H] + .

[0177] 490 mg (1.5 mmol) of cesium carbonate was added to a 3 mL suspension of 0.568 g (1 mmol) of 9d in anhydrous acetonitrile, and the mixture was stirred at room temperature for 2 h. The solvent was removed, and the resulting residue was dissolved in 20 mL of ethyl acetate and washed twice, successively with 10 mL of water and 10 mL of brine. The organic phase (Na₂SO₄) was dried, filtered, and then concentrated under reduced pressure. Purification by column chromatography (n-hexane / ethyl acetate, 3:1) gave 4.49 g of white solid 9e, in 79% yield, MSI-MS: 569.4 [M+H]. + .

[0178] 5.68 g of compound 9e (10 mmol) was dissolved in 200 mL of methanol. Then, 1.5 g of ammonium formate (30 mmol) and 0.75 g of 10% Pd-C were added, and the reaction mixture was stirred at room temperature for 10 min, followed by heating to reflux for 45 min. The mixture was filtered through diatomaceous earth, and the filtrate was evaporated to dryness to give 4.12 g of compound 9, with a yield of 95%. The detection results of the prepared compound 9 are as follows: 1 H NMR (400 MHz, DMSO-d6) δ 8.59 (s, 1H), 8.38 (s, 1H), 7.14 (s, 2H), 6.88 (s, 2H), 6.16 (d, 1H), 5.25 (s, 1H), 4.28 (m, 1H), 4.18(s, 2H), 4.02 (m, 1H), 3.74 (m, 1H), 2.09-1.96 (m, 2H). 13 C NMR (100 MHz, DMSO-d6) δ 168.5, 155.1, 151.4, 147.8, 141.1, 119.4, 98.2, 88.9, 86.1, 73.0,68.1, 37.9. MSI-MS: 435.3 [M+H] + .

[0179] Example 10

[0180]

[0181] 2.51 g (10 mmol) of cordycepin was added to 40.00 mL of anhydrous pyridine and 8.5 mL of acetic anhydride under ice bath conditions. The reaction was monitored by HPLC and ended after approximately 5 h. The solvent was removed to obtain a viscous liquid 10a, weighing 2.68 g, with a yield of 80%. MSI-MS: 358.6 [M+Na] + .

[0182] Compound 10a (3.35 g, 10 mmol) and octadecyl chloride (2.89 g, 10 mmol) were reacted in an ice bath with 60 mL of anhydrous pyridine. The reaction mixture was gradually heated to 40 °C and reacted for 10 h. The reaction was monitored by TLC. After the reaction was complete, water and ethyl acetate were added for extraction. The organic phase was back-extracted and collected by rotary evaporation to obtain an oily liquid, 10b. After purification by column chromatography, 5.40 g of purified 10b was obtained, with a yield of 90%. MSI-MS: 624.4 [M+Na] + .

[0183] 6.01 g (10 mmol) of compound 10b was dissolved in 450 mL of ammonia-methanol solution and reacted at room temperature with stirring. The reaction was monitored by thin-layer chromatography, and the reaction was stopped after 4 h. The solvent was removed to obtain the target product 10, and 4.67 g of the compound was weighed, with a yield of 90%.

[0184] Alternatively, the protection and deprotection process can be performed according to the preparation method of 1a in Example 1, wherein the protected 1a undergoes an acylation reaction in the second step described here, followed by deprotection to prepare compound 10. The detection results of the prepared compound 10 are as follows: 1 H NMR (400 MHz, DMSO-d6) δ 10.55 (s, 1H), 8.64 (s, 1H), 8.38 (s, 1H), 6.18 (d, 1H), 5.35 (d, 1H), 5.04 (m, 1H), 4.02 (m, 1H), 3.76 (m, 1H), 3.58(m, 1H), 3.52 (m,1H), 2.35 (m, 2H), 2.07-1.94 (m, 2H), 1.58 (m, 2H), 1.30-1.26 (m, 28H), 0.89 (m, 3H). 13C10 NMR (100 MHz, DMSO-d6) δ 173.5, 153.3, 152.5, 148.8, 141.0, 122.6, 99.2, 84.1, 74.3, 63.8, 38.5, 34.8, 31.5, 29.5, 28.8, 25.6, 22.4, 14.7. (Some alkyl peaks overlap) MSI-MS: 540.7 [M+Na] + .

[0185] Example 11

[0186] Compound 11 was prepared from cordycepin via protection, acylation, and deprotection steps, following the method of Example 10, wherein octadecyl chloride was replaced with cyclopentylformyl chloride, with an overall yield of 72%. The detection results of the prepared compound 11 are as follows: 1 HNMR (400 MHz, DMSO-d6) δ 10.52 (s, 1H), 8.66 (s, 1H), 8.38 (s, 1H), 6.16 (d,1H), 5.28 (d, 1H), 5.01 (m, 1H), 4.01 (m, 1H), 3.77 (m, 1H), 3.57-3.46 (m,2H), 2.46 (m, 1H), 2.08-1.88 (m, 2H), 1.80-1.55 (m, 8H). 13 C NMR (100 MHz, DMSO-d6) δ 172.5, 153.3, 151.5, 149.5, 139.8, 123.6, 98.4, 82.1, 74.9, 63.8,49.5, 34.5, 32.4, 24.5. MSI-MS: 348.2 [M+H] + .

[0187] Example 12

[0188] Compound 12 was prepared from cordycepin via protection, acylation, and deprotection steps, according to the method of Example 10, wherein octadecyl chloride was replaced with pyridine-3-carboxyl chloride, with an overall yield of 76%. The detection results of the prepared compound 12 are as follows: 1HNMR (400 MHz, DMSO-d6) δ 11.05 (s, 1H), 8.86 (d, 2H), 8.41 (s, 1H), 8.22 (s,1H), 8.01(d, 2H), 6.15 (d, 1H), 5.22 (d, 1H), 4.98 (m, 1H), 4.02 (m, 1H), 3.75 (m, 1H), 3.58-3.46 (m, 2H), 2.08-1.88 (m, 2H). 13 C NMR (100 MHz, DMSO-d6)δ 165.5, 152.3, 151.9, 149.8, 149.7, 140.9, 140.4, 123.4, 120.5, 99.4, 82.8,74.7, 63.8, 35.4. MSI-MS: 357.3 [M+H] + .

[0189] Example 13

[0190]

[0191] A nitration mixture was prepared by adding 193 µL (1.39 mmol) of 2,2,2-trifluoroacetic anhydride to a solution of 428 mg (1.40 mmol) of tetrabutylamine nitrate in 15 mL of dry dichloromethane at 0 °C. After reacting at 0 °C for 45 min, the solution was slowly added to a solution of 480 mg (1 mmol) of compound 1a in 15 mL of dry dichloromethane. After reacting at room temperature in the dark for 14 h, the reaction mixture was extracted by pouring it into a cold mixture of H₂O (50 mL), saturated NaHCO₃ (35 mL), and CH₂Cl₂:Et₂O (1:2, 30 mL). Extraction was performed twice with CH₂Cl₂:Et₂O (1:2, 30 mL). The organic extract was washed with brine, dried over anhydrous Na₂SO₄, and then dried under vacuum (keeping the temperature below 40 °C). The crude product was purified by column chromatography, eluting with CH2Cl2 followed by CH2Cl2:acetone (99:1 to 95:5) to give compound 13a, weighing 273 mg, in 52% yield. MSI-MS: 525.4 [M+H] + .

[0192] At 0 °C, TBAF (1.3 equivalents, 600 µL, 0.6 mmol) was added dropwise over 1 minute to a suspension of 236 mg (0.45 mmol) of 13a in 15 mL of dry acetonitrile. The mixture was stirred for 20 minutes, and the resulting solution was evaporated under vacuum without heating. The crude product was purified by column chromatography (CH₂Cl₂:acetone 100:0 to 90:10) to give compound 13b, weighing 78 mg, in 35% yield, MSI-MS: 498.8 [M+H]. + .

[0193] Compound 13 was prepared by phosphorylation according to the method in Example 2 and deprotection according to Example 1, with a yield of 65%. The detection results of the prepared compound 13 are as follows: 1 H NMR (400 MHz, DMSO-d6) δ 8.38 (s, 1H), 6.98 (s, 2H), 6.16 (d, 1H), 5.36 (d, 1H), 4.28 (m,1H), 4.21 (s, 2H), 4.02 (m, 1H), 3.95 (m, 1H), 3.74 (m, 1H), 2.02-1.82 (m,2H). 13 C NMR (100 MHz, DMSO-d6) δ 158.3, 157.2, 148.8, 141.5, 118.6, 97.2,74.9, 74.6, 67.1, 35.5. MSI-MS: 372.2 [M+Na] + .

[0194] Example 14

[0195]

[0196] Compound 1a (4.80 g, 10 mmol) was added to 60 mL of acetic acid and stirred at 40 °C until dissolved. After the solution cooled to room temperature, 5 mL of 30% hydrogen peroxide (25 mmol) was added. The solution was stirred at 40 °C for 3 days, filtered, and the solid was recrystallized in water to give compound 14a, weighing 3.37 g (68% yield), MSI-MS: 497.4 [M+H]. + .

[0197] 4.96 g (10 mmol) of 14a was added to 40 mL of 3 M hydrochloric acid aqueous solution, and the mixture was heated under reflux until the solid was completely dissolved. Reflux was continued for 10 min, then cooled to room temperature. After removing the solvent, 20 mL of ethanol was added. The precipitated crystals were filtered and dried to give compound 14b, weighing 2.92 g, with a yield of 62%. MSI-MS: 487.5 [M+H] + .

[0198] Compound 14b (4.86 g, 10 mmol) was dissolved in 100 mL of water. Activated Raney Ni (1.0 g) was added in portions, and hydrogen gas was introduced. The reaction mixture was stirred at 55 °C for 4 days. Hydrogen gas was then stopped, and the inorganic salts were removed by filtration. The solvent was then removed by vacuum distillation. The crude product was washed with ethanol and diethyl ether, and dried to give a gray solid 14c (3.30 g, yield 70%). MSI-MS: 495.8 [M+Na]. + .

[0199] Methanol, pyridine, and carbon disulfide were mixed in a volume ratio of 4:5:2 to obtain a solution of 50 mL. 4.72 g (10 mmol) of 14c was added, and a balloon was placed over the reflux condenser to prevent the carbon disulfide from evaporating. The reaction mixture was stirred at 40 °C for 4 days. After filtration, the crude product was heated under reflux in a 5% (w / w) sulfuric acid aqueous solution for 20 min, filtered while hot to remove impurities. After cooling to room temperature, it was filtered and dried to obtain gray crystals 14d, weighing 2.82 g, with a yield of 55%. Compound 14d was prepared according to the phosphorylation method in Example 2 and the deprotection method in Example 1 to obtain compound 14, with a yield of 71%. The detection results of the prepared compound 14 are as follows: 1 H NMR (400 MHz, DMSO-d6) δ 12.22 (s, 1H),8.36 (s, 1H), 6.99 (s, 2H), 6.16 (d, 1H), 5.33 (d, 1H), 4.30 (m, 1H),4.21 (s,2H), 4.02 (m, 1H), 3.94 (m, 1H), 3.71 (m, 1H), 2.06-1.88 (m, 2H). 13 C NMR (100MHz, DMSO-d6) δ 154.3, 152.1, 149.2, 139.8, 118.6, 99.2, 74.8, 74.3, 67.1,35.1. MSI-MS: 364.3[M+H] + .

[0200] Example 15

[0201]

[0202] In 10 mL of DMF, 2.67 g (10 mmol) of adenosine, 3.618 g (24 mmol) of TBSCl, and 3.95 g (50 mmol) of pyridine were added. The mixture was stirred at room temperature for 10 h. After the reaction was complete, the mixture was extracted with water and ethyl acetate. The concentrated organic phase was subjected to column chromatography to give 2.33 g of 15a, with a yield of 47%. MSI-MS: 497.7 [M+H] + .

[0203] Under nitrogen protection, a solution of 4.96 g (10 mmol) of 15a and 1.5 mL of pyridine in 100 mL of dichloromethane was stirred and cooled to -5 °C. 2.5 mL (15 mmol) of trifluoromethanesulfonic anhydride was added dropwise, and the reaction was continued at 0 °C for 2 h. After TLC showed complete reaction, the mixture was poured into 100 mL of ice water, stirred, and separated into layers. The aqueous phase was extracted with 100 mL of dichloromethane. The combined dichloromethane phases were washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure, and the residue was recrystallized with 40 mL of petroleum ether. After filtration and drying, 15b was obtained, weighing 5.65 g, with a yield of 90%. The preparation results are as follows: 1 HNMR (400 MHz, DMSO-d6) δ 8.59 (s, 1H), 8.35 (s, 1H), 7.11 (s, 2H), 6.15 (d ,1H), 5.05 (m, 1H), 4.55 (m, 1H), 4.12 (m, 1H), 4.02-3.79 (m, 2H), 0.99 (s,18H), 0.20 (s, 12H). 13 C NMR (100 MHz, DMSO-d6) δ 156.2, 152.5, 149.7, 140.2,119.5, 118.4, 96.5, 86.4, 83.5, 73.8, 63.2, 30.8, 30.6, 25.8, 0. MSI-MS:628.8 [M+H] + .

[0204] Under nitrogen protection, 6.28 g (10 mmol) of 15b was dissolved in ethyl acetate (40 mL), and 10 mL (22 mmol) of a 37% hydrofluoric acid triethylamine solution was added. The mixture was stirred and heated to 70 °C for approximately 8 h. After the reaction was complete as indicated by TLC, the mixture was cooled to room temperature, washed with saturated sodium bicarbonate solution until neutral, then washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was recrystallized from anhydrous methanol (30 mL) to give solid 15c, weighing 2.04 g, with a yield of 41%. Compound 15 was prepared by phosphorylation according to the method in Example 2 and deprotection according to Example 1, with a yield of 77%. The detection results of the prepared compound 15 are as follows: 1 H NMR (400 MHz, DMSO-d6) δ8.58 (s, 1H), 8.37 (s, 1H), 7.07 (s, 2H), 6.15 (d, 1H), 5.33 (d, 1H), 4.70(m, 1H), 4.44 (m, 1H), 4.28 (m, 1H), 4.18 (s, 2H), 4.02 (m, 1H) 3.55 (m, 1H). 13 C NMR (100 MHz, DMSO-d6) δ 155.3, 151.4, 148.9, 141.1, 119.6, 97.2, 90.1,79.0, 73.4, 61.5. MSI-MS: 350.2 [M+H] + .

[0205] Example 16

[0206]

[0207] Compound 7 was prepared according to the method of Example 7. Compound 16 was prepared from compound 7. 5.34 g (10 mmol) of compound 7 was dissolved in 100 mL of dichloromethane solution, and then 1.3 mL (1.50 g, 10 mmol) of trifluoromethanesulfonic acid was added. After stirring the reaction for 10 min, 2.7 mL of trimethylsilyl trifluoromethanesulfonate (10 mmol) was slowly added dropwise to the solution, and the resulting mixture was stirred for 30 min at -40 °C. Then, 3.96 g (40 mmol) of trimethylnitrile silane was slowly added, and the mixture was stirred for 2 h. Next, 3.5 mL of triethylamine was added dropwise, and the reaction mixture was heated to room temperature. Then, 5.5 g of solid sodium bicarbonate and 20.7 mL of water were added. The resulting mixture was stirred for 10 min. The mixture was then extracted with dichloromethane and water to obtain an organic extract, which was washed with brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude residue was purified by column chromatography to obtain a gray solid product, compound 16, weighing 1.71 g, with a yield of 32%. The detection results of compound 16 are as follows: 1 H NMR (400 MHz, DMSO-d6) δ 8.57 (s, 1H), 8.36 (s, 1H), 7.41 (m,2H), 7.22 (m, 3H), 7.09 (s, 2H), 6.17 (d, 1H), 4.99 (d, 1H), 3.97 (m, 1H),3.74-3.33 (m, 6H), 2.88 (m, 1H), 2.04-1.92 (m, 2H), 1.28-1.14 (d, 9H). 13 C NMR(100 MHz, DMSO-d6) δ 173.4, 155.3, 152.5, 150.2, 149.8, 141.3, 130.2, 121.6,120.5,118.2, 92.2, 77.4, 75.0, 73.1, 69.1, 51.2, 26.5, 25.8, 22.4, 20.1. MSI-MS: 544.5 [M+H] + .

[0208] Example 17

[0209]

[0210] Compound 13b was prepared according to the method of Example 13. Compound 17a was prepared from 13b using the protection method of 1b in Example 1. Compound 17 was prepared from 17a using the method for preparing 3a in Example 3, wherein an equivalent of diethyl p-toluylsulfoxymethylphosphonic acid was replaced with ((((toluoxy)methyl)phosphonyl)bis(oxy))bis(methylene)bis(2,2-dimethylpropionic acid), and an equivalent of 1b was replaced with 17a. The overall yield was 61%. The detection results of the prepared compound 17 are as follows: 1 H NMR (400 MHz, DMSO-d6) δ 8.33 (s, 1H), 6.99 (s, 2H), 6.88 (s, 4H), 6.16 (d, 1H), 5.37 (d, 1H), 4.02 (m, 1H), 3.95 (m, 1H), 3.85 (s, 2H),3.61-3.42 (m, 2H), 2.05-1.88 (m, 2H), 1.25 (s, 18H). 13 C NMR (100 MHz, DMSO-d6) δ 175.2, 158.3, 157.5, 148.6, 140.3, 119.2, 99.2, 92.2, 79.1, 74.8, 74.3,71.2, 38.5, 35.8 26.8. MSI-MS: 592.5 [M+H] + .

[0211] Example 18

[0212]

[0213] Compound 18a was prepared according to the method of Example 15 and the protection method of 1b in Example 1. Compound 18 was then prepared using compound 18a as a starting material according to the method for preparing 3a in Example 3, wherein an equivalent of diethyl p-toluylsulfoxymethylphosphonate was replaced with ((((toluoxy)methyl)phosphono)bis(oxy))bis(methylene)bis(2,2-dimethylpropionic acid), and an equivalent of 1b was replaced with 18a. The overall yield was 34%. The detection results of the prepared compound 18 are as follows: 1H NMR (400MHz, DMSO-d6) δ 8.59 (s, 1H), 8.32 (s, 1H), 7.09 (s, 2H), 6.84 (s, 4H), 6.15(d, 1H), 5.35 (d, 1H), 4.71-4.64 (m, 2H), 3.85 (m, 2H), 3.60-3.33 (m, 3H), 1.27 (s, 18H). 13 C NMR (100 MHz, DMSO-d6) δ 175.9, 157.3, 151.5, 149.8, 141.3,118.9, 98.2, 92.8, 91.5, 79.4, 73.3, 71.4, 70.5, 38.6, 27.8. MSI-MS: 592.4 [M+H] + .

[0214] Example 19

[0215]

[0216] Compound 14d was prepared according to the method of Example 14. Using 14d as a starting material, compound 19a was prepared according to the protection method of 1b in Example 1. Compound 19 was then prepared using compound 19a as a starting material according to the method for preparing 3a in Example 3, wherein an equivalent of diethyl p-toluylsulfoxymethylphosphonic acid was replaced with ((((toluoxy)methyl)phosphonyl)bis(oxy))bis(methylene)bis(2,2-dimethylpropionic acid), and an equivalent of 1b was replaced with 19a. The overall yield was 18%. The detection results of the prepared compound 19 are as follows: 1 H NMR (400 MHz, DMSO-d6) δ 12.12 (s, 1H), 8.34 (s, 1H), 7.01 (s, 2H), 6.84 (s, 4H), 6.12 (d, 1H), 5.35 (d, 1H), 4.01 (m, 1H), 3.93 (m,1H), 3.85 (s, 2H), 3.63-3.37 (m, 2H), 2.06-1.85 (m, 2H), 1.27 (s, 18H). 13 CNMR (100 MHz, DMSO-d6) δ 175.8, 155.4, 152.5, 149.7, 140.5, 118.9, 98.7,92.8, 78.1, 74.9, 74.7, 71.2, 38.7, 34.5, 27.5. MSI-MS: 606.6 [M+H]+ .

[0217] Example 20

[0218]

[0219] Compound 17a was prepared according to the methods of Examples 13 and 17, and compound 20 was prepared according to the method of Example 7, wherein diethyl p-toluylsulfoxymethylphosphonate was replaced by isopropyl (phenoxy((toluoxy)methyl)phosphonyl)alanine ester in an equivalent manner, and 1b was replaced by 17a, with an overall yield of 67%. The detection results of the prepared compound 20 are as follows: 1 H NMR (400 MHz, DMSO-d6) δ 8.37 (s, 1H), 7.40 (m, 2H), 7.20 (m, 3H), 7.02(s, 2H), 6.15 (d, 1H), 5.38 (s, 1H), 4.94 (m, 1H), 4.00 (m, 1H), 3.95 (m,1H), 3.71-3.53 (m, 5H), 3.34 (m, 1H), 2.06-1.82 (m, 2H), 1.28-1.14 (d, 9H). 13 C NMR (100 MHz, DMSO-d6) δ 172.1, 157.3, 156.8, 150.0, 149.8, 140.2, 121.3,120.6, 119.2, 97.4, 76.6, 75.0, 74.8, 72.1, 69.4, 50.1, 34.5, 21.7, 19.5.MSI-MS: 553.5 [M+H] + .

[0220] Example 21

[0221]

[0222] Compound 13b was prepared according to the method in Example 13. Using 13b as a starting material, compound 21a was prepared according to the method for preparing 10b in Example 10a. Using 21a as a starting material, compound 21 was prepared according to the deprotection method in Example 1a, with an overall yield of 74%. The detection results of the prepared compound 21 are as follows: 1H NMR (400 MHz, DMSO-d6) δ 10.53(s, 1H), 8.34 (s, 1H), 6.15 (d, 1H), 5.6 (d, 1H), 5.02 (m, 1H), 4.01 (m, 1H),3.78 (m, 1H), 3.57 (m, 1H), 3.51 (m,1H), 2.35 (m, 2H), 2.09-1.91 (m, 2H), 1.52 (m, 2H), 1.33-1.25 (m, 28H), 0.88 (m, 3H). 13 C10 NMR (100 MHz, DMSO-d6) δ 172.5, 153.5, 152.7, 148.6, 141.2, 122.4, 99.1, 84.5, 74.6, 63.4, 37.3, 34.2, 32.3, 29.4, 28.7, 25.7, 22.3, 14.5. (Some alkyl peaks overlap) MSI-MS: 536.6 [M+H] + .

[0223] Example 22

[0224]

[0225] 15b was prepared from adenosine using the method described in Example 15. Using 15b as a starting material, 6.28 g (10 mmol) of 15b was dissolved in ethyl acetate (40 mL) under nitrogen protection. 10 mL (22 mmol) of a 37% hydrochloric acid triethylamine solution was added, and the mixture was stirred and heated to 70 °C for approximately 8 h. After the reaction was complete as indicated by TLC, the mixture was cooled to room temperature, washed with saturated sodium bicarbonate solution until neutral, then washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure, and the residue was recrystallized from anhydrous methanol (30 mL) to give solid 22a, weighing 1.95 g, with a yield of 38%. MSI-MS: 515.2 [M+H]. + .

[0226] Using 22a as raw material, compound 22 was prepared according to the method for preparing 10b from 10a in Example 10, combined with the deprotection method from 1a in Example 1, with an overall yield of 79%. The detection results of the prepared compound 22 are as follows: 1H NMR (400 MHz, DMSO-d6) δ 10.60 (s, 1H), 8.67 (s, 1H), 8.35 (s, 1H), 6.15 (d, 1H), 5.33 (d,1H), 4.98 (m, 1H), 4.31 (m, 1H), 4.02 (m, 1H), 3.78 (m, 1H), 3.58 (m, 1H), 3.53 (m,1H), 2.32 (m, 2H), 1.51 (m, 2H), 1.34-1.21 (m, 28H), 0.87 (m, 3H). 13 CNMR (100 MHz, DMSO-d6) δ 171.4, 152.5, 151.6, 148.4, 140.5, 121.4, 98.7, 77.5, 74.3, 68.1, 59.4, 38.2, 33.7, 31.5, 28.1, 27.5, 25.4, 22.1, 14.6. (Some alkyl peaks overlap) MSI-MS: 553.2 [M+H] + .

[0227] Example 23

[0228]

[0229] Compound 23a was prepared from compound 12 using the protection method described in Example 1b. Compound 23 was then prepared from 23a using the process described in Example 17a. In the reaction, 17a was replaced by an equivalent amount of 23a, resulting in an overall yield of 81%. The detection results for the prepared compound 23 are as follows: 1 H NMR (400 MHz, DMSO-d6) δ 11.05,(s, 1H), 8,85 (d, 2H), 8.34 (s, 1H), 8.18 (s, 1H), 8.00(d, 2H), 6.84 (s, 4H),6.14 (d, 1H), 5.35 (d, 1H), 4.01 (m, 1H), 3.94 (m, 1H), 3.86 (s, 2H), 3.60-3.38 (m, 2H), 2.06-1.84 (m, 2H), 1.27 (s, 18H). 13C NMR (100 MHz, DMSO-d6) δ175.8, 165.1, 152.4, 151.5, 149.8, 149.6, 140.7, 140.2, 121.2, 120.1, 99.5,92.8, 78.1, 74.7, 74.5, 71.4, 38.7, 34.5, 27.2. MSI-MS: 679.6 [M+H] + .

[0230] Example 24

[0231]

[0232] Compound 23a was prepared according to the method in Example 23. Compound 24 was then prepared using 23a as a starting material according to the method in Example 7, wherein compound 1b was replaced by an equivalent amount of 23a, with an overall yield of 85%. The detection results of the prepared compound 24 are as follows: 1 H NMR (400 MHz, DMSO-d6) δ 11.08 (s, 1H), 8.81(d, 2H), 8.36 (s,1H), 8.18 (s, 1H), 8.02 (d ,2H), 7.42 (m, 2H), 7.21 (m, 3H), 6.16 (d, 1H),5.35 (s, 1H), 4.92 (m, 1H), 4.01-3.95 (m, 2H), 3.65-3.54 (m, 5H), 3.35 (m,1H), 2.04-1.84 (m, 2H), 1.25-1.18 (d, 9H). 13 C NMR (100 MHz, DMSO-d6) δ 171.1,165.2, 152.7, 151.8, 150.4, 149.8, 149.5, 140.5, 140.1, 130.4, 123.2, 121.8,121.4, 120.2, 99.3, 76.7, 74.8, 74.5, 72.5, 69.8, 50.4, 34.7, 21.5, 19.3.MSI-MS: 640.6 [M+H] + .

[0233] Example 25

[0234]

[0235] Under an argon atmosphere, 2.67 g (10.0 mmol) of adenosine and 35.78 g (22 mmol) of PPh were added to a round-bottom flask. Then, 18 mL of DMF was added, and the mixture was stirred at room temperature until the solid was completely dissolved. The solution was cooled to 10 °C, and 4.4 mL (2.2 mmol) of diisopropyl azodicarbonate (DIAD) was rapidly added dropwise. The reaction mixture was allowed to reach room temperature. The mixture was stirred at 40 °C for 2 hours, after which the DMF was evaporated under reduced pressure to obtain a viscous liquid. The mixture was partitioned between 150 mL of water and 100 mL of diethyl ether. The aqueous layer was collected and washed with 100 mL of diethyl ether. The organic phase was concentrated under reduced pressure, and the crude product was purified by column chromatography, eluting with a mixture of DCM and acetone (3:1 to 2:3). Compound 25a, a white solid, weighed 1.84 g, in 74% yield. The results of its preparation are as follows: 1 H NMR (400 MHz, DMSO-d6) δ 8.57(s, 1H), 8.36 (s, 1H), 7.10 (s ,2H), 6.37 (d, 1H), 4.59 (m, 1H), 3.95 (s,1H), 3.57-3.50 (m, 2H), 2.62-2.55 (m, 2H). 13 C NMR (100 MHz, DMSO-d6) δ 156.3,152.5, 149.9, 140.5, 119.5, 90.4, 82.6, 63.5, 60.8, 59.6. MSI-MS: 250.2 [M+H] + .

[0236] 2.49 g of compound 25a (10 mmol) and 2.6 g (40 mmol) of sodium azide were added to a flask, along with 20 mL of DMF as solvent. The reaction was carried out at 120 °C for 16 h, and the reaction progress was monitored by TLC. After the reaction was completed, water was added to quench the reaction. The aqueous phase was extracted three times with ethyl acetate (180 mL). The organic phases were combined, washed with water, and the solvent was removed under reduced pressure to obtain a foamy solid 25b, weighing 2.22 g, with a yield of 76%. MSI-MS: 293.2 [M+H] + .

[0237] Using 25b as a starting material, 25c was prepared according to the protective method for preparing 1b in Example 1. Using 25c as a starting material, compound 25 was prepared by replacing an equivalent amount of 1b with 25c according to the method in Example 2, with a yield of 76%. The detection results of the prepared compound 25 are as follows: 1H NMR (400 MHz, DMSO-d6) δ 8.57 (s, 1H), 8.38 (s, 1H), 7.09 (s, 2H), 6.17 (d, 1H), 5.35 (d, 1H), 4.27-4.24 (m, 2H), 4.19 (s, 2H), 4.02 (m, 1H), 3.81 (m, 1H), 1.75 (m, 1H). 13 C NMR (100 MHz, DMSO-d6) δ 158.2,152.4, 149.8, 140.5, 119.4, 101.2, 78.1, 73.2, 68.4, 57.5. MSI-MS: 373.2 [M+H] + .

[0238] Example 26

[0239]

[0240] Compound 25c was prepared according to the preparation method in Example 25. Compound 26a was prepared using 25c as a starting material according to the method for preparing compound 17a in Example 17. Compound 26 was then prepared using the deprotection method described in Example 1b, in which 25c was used to replace 17a in an equivalent amount, resulting in a total yield of 54%. The detection results for the prepared compound 26 are as follows: 1 H NMR (400 MHz, DMSO-d6) δ 8.58 (s, 1H), 8.34 (s, 1H), 7.04 (s, 2H), 6.82(s, 4H), 6.15 (d, 1H), 5.35 (d, 1H), 4.00 (m, 2H), 3.85 (s, 2H), 3.62-3.41(m, 2H), 1.84 (m, 1H), 1.26 (s, 18H). 13 C NMR (100 MHz, DMSO-d6) δ 175.4,157.3, 152.5, 149.7, 140.2, 119.5, 99.8, 92.8, 79.0, 72.3, 71.5, 71.1, 56.5,38.8 27.6. MSI-MS: 615.6 [M+H] + .

[0241] Example 27

[0242]

[0243] Compound 25c was prepared according to the preparation method in Example 25. Using 25c as a starting material, compound 27 was prepared by replacing 1b with an equivalent amount of 25c according to the preparation method in Example 7, with an overall yield of 43%. The detection results of the prepared compound 27 are as follows: 1 H NMR (400 MHz, DMSO-d6) δ 8.58 (s, 1H), 8.34 (s, 1H), 7.42(m, 2H), 7.21 (m, 3H), 7.07 (s, 2H), 6.15 (d, 1H), 5.34 (s ,1H), 4.95 (d,1H), 3.99 (m, 2H), 3.76-3.34 (m, 6H), 1.88 (m, 1H), 1.27-1.16 (d, 9H). 13 C NMR(100 MHz, DMSO-d6) δ 171.4, 156.4, 152.1, 150.1,149.5, 140.3, 130.5, 121.2,120.5,119.4, 100.5, 78.4, 72.8, 72.0, 71.5, 69.1, 57.2, 50.5, 21.5, 19.1.MSI-MS: 576.5 [M+H] + .

[0244] Example 28

[0245]

[0246] Using compound 27 as a starting material, compound 28a was prepared according to the protection method of 1a or 1b in Example 1. Using compound 28a as a starting material, compound 28a was prepared by replacing cordycepin or 1b with equivalent amounts of compound 28a according to the preparation method of Example 12, with an overall yield of 37%. The detection results of the prepared compound 28 are as follows: 1 H NMR(400 MHz, DMSO-d6) δ 11.07 (s, 1H), 8.83 (d, 2H), 8.35 (s, 1H), 8.20 (s, 1H), 8.01 (d, 2H), 7.41 (m, 2H), 7.21 (m, 3H), 6.15 (d, 1H), 5.35 (s,1H), 4.94(d, 1H), 4.00 (m, 2H), 3.72-3.32 (m, 6H), 1.82 (m, 1H), 1.24-1.17 (d, 9H). 13CNMR (100 MHz, DMSO-d6) δ 171.5, 164.5, 152.1, 151.6, 150.1, 149.8, 149.4,140.7, 140.3, 130.2, 122.2, 121.5, 121.1, 120.4, 100.4, 78.7, 72.5, 72.1,71.5, 69.2, 57.4, 50.8, 21.6, 19.2. MSI-MS: 681.6 [M+H] + .

[0247] Example 29

[0248]

[0249] Compound 1c was prepared according to the protected method for preparing 1c in Example 1. Using 1c as a starting material, the reaction was further carried out. 9.95 g (27.2 mmol) of compound 1c and 4.88 g (27.2 mmol) of Cb2Cl were added to 38.5 mL of toluene and 38.5 mL of water, along with 4.70 g (34 mmol) of K2CO3. The mixture was vigorously stirred at a temperature below 25 °C. After stirring at room temperature for 3 h, 0.275 g (2.72 mmol) of triethylamine and 5.78 g of sodium chloride were added sequentially, and the mixture was stirred for another 30 min. The organic layer was separated and concentrated to obtain the desired oily product 29a, weighing 13.1 g, with a yield of 90%. MSI-MS: 522.6 [M+Na]. + .

[0250] Compound 29a (4.99 g, 10 mmol) and valine acyl chloride (1.36 g, 10 mmol) were reacted in an ice bath with 60 mL of anhydrous pyridine. The reaction mixture was gradually heated to 40 °C and reacted for 10 h. The reaction was monitored by TLC. After the reaction was complete, water and ethyl acetate were added for extraction. The organic phase was back-extracted and collected by rotary evaporation to obtain an oily liquid, 10b. After purification by column chromatography, 5.03 g of purified 29b was obtained, with a yield of 84%. MSI-MS: 625.5 [M+Na]. + .

[0251] 5.99 g of compound 29b (10 mmol) was dissolved in 200 mL of methanol. Then, 1.5 g of ammonium formate (30 mmol) and 0.75 g of 10% Pd-C were added, and the reaction mixture was stirred at room temperature for 10 min, followed by heating to reflux for 45 min. The mixture was filtered through diatomaceous earth, and the filtrate was evaporated to dryness to give 4.41 g of compound 29c, with a yield of 95%. Using 29c as a starting material, compound 29 was prepared by deprotection according to the method in Example 1c, with a yield of 90%. The results of the analysis of the prepared compound 29 are as follows: 1 H NMR (400 MHz, DMSO-d6) δ 8.89 (s, 2H), 8.58 (s, 1H), 8.37 (s, 1H), 7.05 (s, 2H), 6.73 (d, 1H), 5.01 (m, 1H), 4.92 (s, 1H), 4.27 (m,1H), 3.75 (m, 1H), 3.56-3.47 (m, 2H), 2.38 (m, 1H), 2.13 (m, 1H), 1.89 (m,1H), 0.98 (d, 6H). 13 C NMR (100 MHz, DMSO-d6) δ 172.1, 156.0, 152.3, 149.5,140.1, 119.4, 102.2, 82.5, 74.4, 63.7, 59.5, 33.1, 30.5, 19.1. MSI-MS: 351.3[M+H] + .

[0252] Example 30

[0253]

[0254] Compound 30 was prepared by reacting compound 29 as a starting material according to the method in Example 2, wherein 1b was replaced by an equivalent amount of 29. Compound 30 was obtained with a yield of 84%. The detection results of the prepared compound 30 are as follows: 1 H NMR(400 MHz, DMSO-d6) δ 8.90 (s, 2H), 8.56 (s, 1H), 8.33 (s, 1H), 7.04 (s, 2H), 6.75 (d, 1H), 5.00 (m, 1H), 4.27-4.02 (m, 5H), 3.73 (m, 1H), 2.36 (m, 1H), 2.16 (m, 1H), 1.88 (m, 1H), 0.96 (d, 6H).13 C NMR (100 MHz, DMSO-d6) δ 172.4,156.3, 152.5, 149.8, 140.2, 119.5, 101.2, 76.1, 74.2, 68.7, 59.3, 32.1, 30.4,19.0. MSI-MS: 431.3 [M+H] + .

[0255] Example 31

[0256]

[0257] Using compound 30 as a starting material, compound 31 was prepared by replacing 1a with an equivalent amount of compound 30 according to the preparation method of Example 12, with a yield of 91%. The detection results of the prepared compound 31 are as follows: 1 H NMR (400 MHz, DMSO-d6) δ 11.08 (s, 1H), 8.92 (s, 2H), 8.82 (d, 2H), 8.34 (s, 1H), 8.20 (s,1H), 8.02(d, 2H), 6.73 (d, 1H), 5.03 (m, 1H), 4.29-4.01 (m, 5H), 3.75 (m,1H), 2.39 (m, 1H), 2.15 (m, 1H), 1.88 (m, 1H), 0.97 (d, 6H). 13 C NMR (100 MHz, DMSO-d6) δ 171.4, 164.5, 152.3, 151.1, 149.9, 149.7, 140.5, 140.1, 123.7,121.5, 101.7, 77.1, 74.5, 68.2, 59.8, 32.4, 30.8, 19.2. MSI-MS: 558.4 [M+Na] + .

[0258] Example 32

[0259]

[0260] Using compound 29 as a starting material, compound 32a was prepared by replacing an equivalent amount of compound 1b with compound 29 according to the preparation method in Example 3, with a yield of 77%. Using compound 32a as a substrate, compound 32 was prepared by replacing an equivalent amount of 1a with compound 32a according to the preparation method in Example 12, with a yield of 90%. The detection results of the prepared compound 32 are as follows: 1H NMR (400 MHz, DMSO-d6) δ 11.05 (s, 1H), 8.91 (s, 2H), 8.84 (d, 2H), 8.35 (s, 1H), 8.18 (s, 1H), 8.01(d, 2H), 6.75 (d, 1H), 5.01 (m, 0.96 (d, 6H). 13 C NMR (100 MHz, DMSO-d6) δ171.5, 165.5, 152.4, 151.1, 149.8, 140.7, 140.1, 123.5, 121.7, 102.7, 76.8,76.6, 75.5, 74.3, 59.5, 32.5, 30.5, 18.9. MSI-MS: 550.4 [M+H] + .

[0261] Example 33

[0262]

[0263] Using compound 1c as a starting material, and following the preparation method in Example 6, compound 1b was replaced by an equivalent amount of compound 1c to prepare compound 33a with a yield of 62%. Compound 33a was then reacted according to the deprotection scheme for 1c in Example 1 to prepare compound 33 with a yield of 90%. The detection results of the prepared compound 33 are as follows: 1 H NMR (400 MHz, DMSO-d6) δ8.58 (s, 1H), 8.33 (s, 1H), 7.42 (m, 2H), 7.21 (m, 3H), 7.08 (s, 2H), 6.15(d, 1H), 5.00 (m, 1H), 4.02 (m, 1H), 3.71-3.48 (m, 8H), 2.24-1.98 (m, 2H), 1.25 (d, 3H). 13C NMR (100 MHz, DMSO-d6) δ 172.1, 156.1, 152.4, 149.9, 149.6,140.3, 131.0, 121.5, 120.2, 119.6, 101.8, 82.1, 74.3, 63.4, 52.1, 46.2, 32.5,19.2. MSI-MS:439.4 [M+H] + .

[0264] Example 34

[0265]

[0266] Compound 33a was prepared according to the process of Example 33. Using 33a as a starting material, compound 34 was prepared by replacing 1a in an equivalent amount with compound 33a according to the preparation method of Example 12, with an overall yield of 83%. The detection results of the prepared compound 34 are as follows: 1 H NMR (400 MHz, DMSO-d6) δ 11.02 (s, 1H), 8.81 (d, 2H), 8.34 (s, 1H), 8.17 (s, 1H), 8.02 (d, 2H), 7.41 (m, 2H), 7.23 (m, 3H), 6.16(d, 1H), 4.98 (m, 1H), 4.01 (m, 1H), 3.78-3.47 (m, 8H), 2.25-1.97 (m, 2H), 1.27 (d, 3H). 13 C NMR (100 MHz, DMSO-d6) δ 171.4, 163.8, 152.1, 151.5, 150.4,149.8, 149.7, 140.8, 140.5, 130.2, 122.5, 121.7, 121.2, 119.9, 102.4, 82.5,74.5, 63.5, 51.7, 46.3, 32.7, 19.4. MSI-MS:598.5 [M+H] + .

[0267] Example 35

[0268]

[0269] 2.51 g (10 mmol) of cordycepin was dissolved in dilute nitric acid, heated to 110 °C, and 1.26 g (5 mmol) of elemental iodine was added. The reaction was monitored by TLC. After the reaction was complete, the mixture was extracted with petroleum ether, and the aqueous phase was collected. The organic phase was extracted with deionized water, and the combined aqueous phases were rotary evaporated to give compound 35a, weighing 2.72 g, with a yield of 72%. MSI-MS: 378.2 [M+H]. + .

[0270] 100 mL of anhydrous 1,4-dioxane was placed in a reactor under argon protection and heated to 70 °C. Palladium acetate (0.11 g, 0.5 mmol), triphenylphosphine (0.26 g, 1 mmol), and triethylamine (1.8 mL) were added sequentially, and the mixture was stirred for approximately 30 min. Then, compound 35a (3.77 g, 10 mmol) and methyl acrylate (2.59 g, 30 mmol) were added. The reaction was carried out at 80 °C for 2 h until the reaction was complete. The mixture was filtered, the filtrate was collected, allowed to stand, and then filtered again to obtain a white flocculent solid, compound 35b, weighing 2.41 g, with a yield of 72.7%. MSI-MS: 336.4 [M+H]. + .

[0271] 1.00 g of compound 35b was added dropwise to 12 mL of sodium hydroxide solution (2 mol / L). After stirring at room temperature for 3 h, concentrated hydrochloric acid was added dropwise under ice bath conditions until the pH reached 1, at which point a large amount of white precipitate formed. The precipitate was filtered to obtain a white solid 35c, weighing 0.88 g, with a yield of 92%. MSI-MS: 322.6 [M+H] + .

[0272] 3.21 g (10 mmol) of compound 35c was added to 120 mL of water and heated to 100 °C with stirring. Then, 2.07 g (15 mmol) of anhydrous potassium carbonate was added, and 2.67 g (15 mmol) of NBS was dissolved in 22.5 mL of acetone and 22.5 mL of water, respectively. The mixture was added dropwise to a flask over 135 min. The mixture was stirred for 3 h, the reaction was stopped, half of the solvent was removed, and the mixture was placed in a refrigerator overnight. A large amount of needle-like brown crystals precipitated. The crystals were filtered to give compound 35d, weighing 1.78 g, with a yield of 50%. MSI-MS: 357.3 [M+H] + .

[0273] 1.78 g (5 mmol) of compound 35d was added to 40.00 mL of anhydrous pyridine and 8.5 mL of acetic anhydride under ice bath conditions. The reaction was monitored by HPLC and ended after about 5 h. The solvent was removed to obtain a viscous liquid 35e, which weighed 1.76 g, yielding 80%. Compound 35e was used to prepare compound 35f using the method of Example 16, with a yield of 95%. Compound 35f was used to prepare compound 35 using the phosphorylation method of Example 2 and the deprotection method in Example 1, with a yield of 72%. The detection results of the prepared compound 35 are as follows: 1 H NMR (400 MHz, DMSO-d6) δ 11.05(s, 1H), 8.84 (d, 2H), 8.31 (s, 1H), 8.02 (d, 2H), 7.11 (d, 1H), 6.70 (d,1H),6.11 (d, 1H), 5.35 (s, 1H), 4.31-4.02 (m, 5H), 3.76 (m, 1H), 2.07-1.84 (m, 2H). 13 C NMR (100 MHz, DMSO-d6) δ 164.5, 152.6, 151.8, 149.8, 149.7, 140.9,140.3, 134.5, 124.3, 123.4, 121.5, 98.5, 74.5, 74.3, 68.5, 34.7. MSI-MS:542.2 [M+H] + .

[0274] Example 36

[0275]

[0276] Compound 17a was prepared according to the method of Example 17. Using 17a as a starting material, it was prepared according to the method of Example 8, except that 2-chloro-3-hydroxypropylvaline was replaced by an equivalent amount of 3-chloro-5-(hydroxymethyl)cyclopentane-1,2-diol to prepare compound 36a. After deprotection, compound 36 was obtained, with an overall yield of 41%. The detection results of compound 36 are as follows: 1H NMR (400 MHz, DMSO-d6) δ 8.37 (s, 1H), 7.01 (s, 2H), 6.15 (d,1H), 5.92 (s, 1H), 5.37 (d, 1H), 4.38 (s, 1H), 4.25 (s, 1H), 4.02 (m, 2H),3.84 (m, 1H), 3.65-3.31 (m, 6H), 2.07-1.82 (m, 2H), 1.70-1.44 (m, 3H). 13 C NMR(100 MHz, DMSO-d6) δ 156.1, 153.5, 149.8, 140.3, 119.5, 99.2, 83.5, 81.2,76.2, 75.7, 74.8, 73.1, 64.1, 34.9, 34.7, 31.5. MSI-MS: 416.8 [M+H] + .

[0277] Example 37

[0278]

[0279] Compound 1b 3.65 g (10 mmol) and m-chloroperoxybenzoic acid 3.44 g (20 mmol) were added to a reaction flask, dissolved in 100 mL of anhydrous dichloromethane, and then 2.13 g (15 mmol) of phosphorus pentoxide were added in an ice bath. The reaction was gradually heated to 40 °C and carried out for 3 h. After the reaction was completed, the mixture was filtered, and the reaction was quenched by adding 100 mL of saturated sodium bicarbonate. The mixture was washed twice with 50 mL of dichloromethane and back-extracted twice with 60 mL of saturated sodium bicarbonate. The organic phases were combined, concentrated under reduced pressure, and column chromatography was performed to obtain compound 37a, weighing 1.49 g, with a yield of 41%. The prepared compound was analyzed as follows: 1 H NMR (400 MHz, DMSO-d6) δ 8.56 (s, 1H), 8.36 (s, 1H), 7.10 (s, 2H), 6.16(d, 1H), 4.02 (s, 1H), 2.75-2.45 (m ,2H), 2.13 (d ,2H), 0.99 (s, 9H), 0.22 (s, 6H). 13C NMR (100 MHz, DMSO-d6) δ 156.5, 152.5, 149.8, 140.4, 119.6,102.5, 85.9, 69.3, 52.7, 42.5, 30.8, 25.8, 0. MSI-MS: 364.5 [M+H] + .

[0280] 3.63 g of compound 37a (10 mmol) and 2.6 g (40 mmol) of sodium azide were added to a flask, along with 20 mL of DMF as solvent. The reaction was carried out at 120 °C for 16 h, and the reaction progress was monitored by TLC. After the reaction was completed, water was added to quench the reaction. The aqueous phase was extracted three times with ethyl acetate (180 mL). The organic phases were combined, washed with water, and the solvent was removed under reduced pressure to obtain a foamy solid 37b, weighing 2.77 g, with a yield of 68%. Compound 37 was prepared from 37b using the method for preparing compound 17a in Example 17, with 37b replacing 17a in an equivalent amount, resulting in an overall yield of 50%. The detection results of the prepared compound 37 are as follows: 1 H NMR (400 MHz, DMSO-d6) δ 8.58 (s, 1H), 8.34 (s, 1H), 7.07 (s, 2H), 6.82(d, 4H), 6.15 (d, 1H), 5.34 (d, 1H), 4.02 (m, 1H), 3.84 (d,2H), 3.61-3.42 (m, 2H), 2.11-1.80 (m, 2H), 1.28(d, 18H). 13 C NMR (100 MHz, DMSO-d6) δ 175.2, 156.3, 152.6, 149.9, 140.2, 119.4, 100.3, 93.1, 90.2, 81.0,71.5, 68.8, 38.6, 36.2, 27.4. MSI-MS: 637.6 [M+Na] + .

[0281] Example 38

[0282]

[0283] Compound 38 was prepared from compound 2 using the method of Example 35, with an overall yield of 24%. The detection results of the prepared compound 38 are as follows: 1H NMR (400 MHz, DMSO-d6) δ 8.35 (s,1H), 7.09 (d, 1H), 6.95 (s, 2H), 6.68 (d, 1H), 6.15 (d, 1H), 5.35 (s, 1H),4.27-4.02 (m, 5H), 3.74 (m, 1H), 2.07-1.84(m, 2H). 13 C NMR (100 MHz, DMSO-d6)δ 156.1, 152.3, 149.8, 140.2, 134.9, 124.2, 119.5, 98.7, 74.9, 74.3, 68.1,34.5. MSI-MS: 458.3 [M+N] + .

[0284] Example 39

[0285]

[0286] Compound 39 was prepared using compound 1b as a starting material. First, 3.65 g (10 mmol) of compound 1b and 1.18 g (20 mmol) of 2-propylamine were added to a reaction flask, and 60 mL of dioxane was added as a solvent. The reaction was refluxed at 80 °C for 20 h, and the reaction was monitored by TLC. After the reaction was complete, the reaction solution was concentrated, and then extracted twice with 50 mL of water and 50 mL of ethyl acetate. The organic phases were combined and concentrated under reduced pressure to obtain an oily liquid. The oily liquid was purified by column chromatography to obtain a pale yellow oily liquid, compound 39a, weighing 1.44 g, with a yield of 34%.

[0287] Compound 39 was prepared from compound 39a using the method described in Example 2, with 1b replaced by an equivalent amount of 39a, resulting in a yield of 91%. The detection results of the prepared compound 39 are as follows: 1 H NMR (400 MHz, DMSO-d6) δ 8.15 (s, 1H), 7.05 (s, 2H), 6.15 (d, 1H), 5.98 (s, 1H), 5.38 (s,1H), 4.28-3.95 (m, 6H), 3.75 (m, 1H), 2.05-1.81 (m, 2H), 1.18 (d, 6H). 13C NMR(100 MHz, DMSO-d6) δ 153.1, 152.3, 151.5, 149.8, 118.5, 99.7, 74.8, 74.5,68.5, 46.5, 34.3, 23.5. MSI-MS: 389.3 [M+H] + .

[0288] Example 40

[0289]

[0290] Compound 1b was prepared according to the method described in Example 8, except that 2-chloro-3-hydroxypropylvaline was replaced by an equivalent amount of 3-chloro-5-(hydroxymethyl)cyclopentane-1,2-diol, yielding compound 40 with a total yield of 46%. The detection results of compound 40 are as follows: 1 H NMR (400 MHz, DMSO-d6) δ 8.58 (s,1H), 8.37 (s, 1H), 7.04 (s, 2H), 6.15 (d, 1H), 5.91 (s, 1H), 5.37 (d, 1H),4.37 (s, 1H), 4.25 (s, 1H), 4.00 (m, 2H), 3.82 (m, 1H), 3.63-3.32 (m, 6H), 2.08-1.81 (m, 2H), 1.71-1.45 (m, 3H). 13 C NMR (100 MHz, DMSO-d6) δ 156.3,153.4, 149.7, 140.2, 119.4, 99.5, 83.4, 81.1, 76.1, 75.5, 74.9, 73.6, 64.4,34.9, 34.7, 31.6. MSI-MS: 382.3 [M+H] + .

[0291] Example 41: Antitumor effect of cordycepin modified derivatives on liver cancer

[0292] Establishment of a mouse transplanted liver cancer model and drug evaluation. 2.5 × 10⁻⁶ cells were suspended in 100 μL of PBS. 5 One Hep-1-6 hepatocellular carcinoma cell was inoculated into the lateral thigh of the left hind leg of a C57BL / 6j nude mouse; one week later, the transplanted tumor reached approximately 100 mm. 3At the time of experimentation, mice were randomly divided into twelve groups of 10 mice each: a blank control group, a cordycepin control group, and a drug group (compounds 2, 3, 5, 7, 15, 24, 27, 31, 35, 40). The blank control group was administered corn steep liquor and other nutrients, along with DMSO as a solvent control, via gavage every other day. The cordycepin control group and the drug group were administered the compound drugs (500 μg / mouse / time) via gavage every other day for 14 consecutive days. Changes in liver cancer tumor size were observed in both groups. After the experiment, mice were sacrificed, and blood and corresponding tissue samples were collected. The tumors were immediately photographed and weighed. A portion of the tumor tissue was fixed in formalin solution for further analysis. The two vertical diameters (length and width) of the transplanted tumor were measured every two days using calipers to calculate the tumor size. The results of the tumor volume changes and the tumor treatment results of the blank control group, cordycepin control group, and drug group within 14 days are shown in the table below. Figure 1 Calculate the volume using the formula: Tumor volume (mm²) 3 = 1 / 2 × (length × width) 2 Experimental results showed that the drug groups, used alone, significantly inhibited tumor growth in mice, with effects superior to the cordycepin control group. Compound 24, in particular, exhibited excellent anti-liver cancer tumor activity (900 mm). 3 Reduced to <100 mm 3 (P<0.001) indicates that the compound drug group has the effect of killing liver cancer tumor cells or activating tumor immunity.

[0293] Example 42: Antitumor effect of cordycepin modified derivatives on small cell lung cancer

[0294] The MTT assay was used to analyze the dose-response relationship between cordycepin and compounds 1, 2, 4, 5, 7, 8, 10, 12, 16, 18, 21, 23, 24, 35, and 38 and cordycepin in small cell lung cancer cell lines H1048, H446, and H69. The half-maximal inhibitory concentration (IC50) was calculated. 50 The results are summarized in the table below. In vitro antitumor experiments showed that, compared with cordycepin, the modified cordycepin derivatives had a lower effective concentration against tumor cells. Compounds 35 and 38 exhibited strong in vitro killing activity against all three cell types, but also showed high toxicity, followed by compounds 18 and 21. Cordycepin, however, showed a lower IC50 concentration in small cell lung cancer cell lines H1048 and H446. 50 All concentrations are above 100 μM, indicating a high effective concentration. In practical applications, a large amount of drug may be required to achieve the desired effect.

[0295] Table 1: IC50 of the compound in three types of small cell lung cancer cells 50 (μM)

[0296]

[0297] Example 43: Determination of the maximum tolerated dose of cordycepin-modified derivatives in zebrafish in vivo.

[0298] Two hundred and forty wild-type AB strain zebrafish at 3 dpf were randomly selected and placed in eight-well plates, with 30 zebrafish per well and 3 mL of water per well. Cordycepin was diluted to the maximum lethal dose (MDT) and dissolved in DMSO. The solution was added to each well in a concentration gradient (MDT diluted 100-fold, with eight intermediate concentration gradients used for testing). A normal control group, a solvent control group G (5% glucose), and a solvent control group D (DMSO) were also included. During the experiment, zebrafish in each group were cultured at 35 °C. After the zebrafish reached 5 dpf, lethality and developmental deformities were investigated at different doses. The maximum tolerated dose (MTD) of the test substance in normal zebrafish was determined by a survival rate >90% and a teratogenicity rate <20%. Cordycepin was selected as the control group. Compounds 2, 5, 6, 7, 9, 11, 13, 20, 24, 25, 28, 35, 36, 37, and 39 were selected and tested for MTD in zebrafish using the method described above. The test results are shown in Table 2.

[0299] Table 2: Maximum Tolerable Limit (MTD) of Natural Products and Positive Control Drugs in Zebrafish Models

[0300]

[0301] Example 44: Inhibitory effect of cordycepin modified derivatives on small cell lung cancer in zebrafish

[0302] Zebrafish small cell lung cancer xenograft models were established using three cell lines: H69, H446, and H1048. 1×10⁻⁶ cells were prepared for each type of cell line. 6 Cell suspensions of H69, H446, and H1048 cells at a concentration of mL were placed in serum-free cell culture medium. 5 mL of red fluorescent dye (CM-DiI) cell labeling solution was added to each mL of cell suspension, and the mixture was gently mixed. The cells were incubated at 37 °C for 20 min, centrifuged at 1500 rpm for 5 min, and the supernatant was removed. The cells were resuspended in serum-free culture medium. This process was repeated twice to obtain the desired labeled cell suspension. Approximately 100 cells were transplanted into the yolk sac of zebrafish via microinjection to establish a zebrafish-human small cell lung cancer xenograft model. Zebrafish injected with ovarian cancer cells were then cultured at 35 °C for 3 days post-flop (dpf).

[0303] The effects of cordycepin and prepared compounds 2, 5, 6, 7, 9, 11, 13, 20, 24, 25, 28, 35, 36, 37, and 39 on the proliferation of small cell lung cancer in zebrafish were tested. When the tumor-bearing zebrafish model reached 3 days post-flop (dpf), zebrafish with good tumor homogeneity were selected under a microscope and randomly assigned to 6-well plates, 30 fish per well. At 3 dpf, the above compounds were diluted according to the MTD dosage and dissolved in DMSO, then added to each well. A normal control group, a model control group, and a solvent control group (DMSO) were also set up, with 3 mL of water per well. After the zebrafish in each experimental group were cultured at 35 ℃ for 5 dpf, 10 zebrafish from each group were randomly selected for observation, photography, and image saving under a fluorescence microscope. Image analysis was performed using Nikon NIS-Elements 13.10 advanced image processing software to calculate the fluorescence intensity (S) of the zebrafish xenograft. The inhibition results are shown in Table 3 below. The growth inhibitory effects of Xiaoyaping injection and paclitaxel alone on zebrafish-human ovarian cancer-sensitive strains were calculated based on overall fluorescence intensity, using the following formula:

[0304] Tumor growth inhibition rate (%) = S(model control group) - S(drug group) / S(model control group) × 100%

[0305] Table 3: Effects of each compound on the proliferation of three cell lines in zebrafish model

[0306]

[0307] Example 45: Inhibitory effect of cordycepin modified derivatives on colon cancer

[0308] Establishment of a mouse colon cancer transplantation model and drug evaluation. 2.5 × 10⁻⁶ cells were suspended in 100 μL of PBS. 6 One MC-38 colon cancer cell line was inoculated into the lateral thigh of the left hind leg of a C57BL / 6j nude mouse; one week later, the transplanted tumor reached approximately 100 mm. 3At the age of 18, the mice were randomly divided into seven groups of eight each: control group (IgG), compound 16 drug + IgG group, PD-1 antibody treatment group, CTLA4 monoclonal antibody treatment group, compound 16 drug + PD-1 combination treatment group, and compound 16 drug + CTLA4 monoclonal antibody combination treatment group. Control group IgG: Immunoglobulin G (IgG) (500 ug / time / animal) was administered by gavage daily for 28 consecutive days; Cordycepin + IgG: Immunoglobulin G (IgG) and cordycepin (both 500 ug / time / animal) were administered by gavage daily for 28 consecutive days; Compound 16 + IgG group: Immunoglobulin G (IgG) and Compound 16 (both 500 ug / time / animal) were administered by gavage daily for 28 consecutive days; Antibody treatment group: Antibody (500 ug / time / animal) was administered by gavage every 4 days for 28 consecutive days; Compound 16 + Antibody combined treatment group: Immunoglobulin Compound 16 (500 ug / time / animal) was administered by gavage daily, and antibody (500 ug / time / animal) was administered by gavage every 4 days for 28 consecutive days. Mice were euthanized after the experiment, and blood and corresponding tissue samples were collected. The tumor was immediately photographed and weighed. A portion of the tumor tissue was fixed in formalin solution for further testing. The two vertical diameters (length and width) of the transplanted tumor were measured every two days using calipers to calculate the size of the transplanted tumor and its volume using the formula: Tumor Volume (mm²) 3 = 1 / 2 × (length × width) 2 The changes in colon cancer tumor size in seven groups of mice were observed, and the results are as follows: Figure 2 As shown. After 28 days, the experimental results indicated that cordycepin had an anti-colon cancer proliferation effect (size 1300 mm). 3 Reduced to 730 mm 3 Compound 16, when used alone, exhibits significantly greater anti-tumor growth efficacy than cordycepin (size 1300 mm). 3 Reduced to 480 mm 3 (P<0.01), immune checkpoint inhibitors PD-1 and CTLA4, used alone, also showed significant anti-tumor growth effects (size 1300 mm). 3 Reduced to 448 mm 3 (p<0.01), when compound 16 and the immune checkpoint inhibitors PD-1 and CTLA4 are used simultaneously, their synergistic effect can significantly enhance the antitumor effect (size 1300 mm). 3 Reduced to 260 mm 3 about).

[0309] Example 46: Inhibitory effect of cordycepin modified derivatives on melanoma

[0310] Establishment of a mouse transplanted melanoma model and drug evaluation: 2.5 × 10⁻⁶ cells were suspended in 100 μL of PBS. 5 One B16-F10 melanoma cell was inoculated into the lateral thigh of the left hind leg of a C57BL / 6j nude mouse; one week later, the transplanted tumor reached approximately 100 mm. 3 During the experiment, mice were randomly divided into ten groups of 10 mice each: a blank control group, a cordycepin group, and groups containing compounds 13, 14, 17, 19, 22, 26, 29, and 34. The blank control group received corn steep liquor and other nutrients, along with DMSO as a solvent, via gavage daily for 24 days. The compound groups received the corresponding compounds via gavage every other day (500 μg / mouse per dose) for 24 days. Melanoma size changes were observed every 4 days initially and every 2 days later in the experiment. Mice were sacrificed at the end of the experiment, and blood and corresponding tissue samples were collected. Tumors were immediately photographed and weighed. A portion of the tumor tissue was fixed in formalin for further analysis. The two vertical diameters (length and width) of the transplanted tumor were measured every two days using calipers to calculate the tumor size and volume using the formula: Tumor Volume (mm²). 3 = 1 / 2 × (length × width) 2 The experimental results are shown in Figure 3 As shown. Experimental results indicate that cordycepin itself can inhibit the proliferation of melanoma (1760 mm). 3 Reduced to 600 mm 3 Cordycepin-modified derivatives are more effective than cordycepin (1760 mm). 3 Reduced to <560mm 3 Compounds 19 and 34, when used alone, significantly inhibited tumor growth in mice (1760 mm). 3 Reduced to approximately 240 mm 3 (P<0.001), the above results suggest that the synthesized product has the effect of killing tumor cells.

[0311] Example 47: Inhibitory effect of cordycepin modified derivatives on ovarian cancer (experimental study)

[0312] Establishment of a mouse transplanted ovarian cancer model and drug evaluation. 2.5 × 10⁻⁶ cells were suspended in 100 μL of PBS. 6 ID8 ovarian cancer cells were inoculated into the lateral thigh of the left hind leg of C57BL / 6j nude mice; approximately one week later, the transplanted tumor reached about 100 mm. 3At the age of 10, the mice were randomly divided into five groups of 10 each: control group, cordycepin group, compound 24 drug group, PD-1+TIM3 antibody treatment group, and compound 24 drug + PD-1+TIM3 antibody treatment group. Control group: Immunoglobulin G (IgG) (500 ug / time / animal) and PBS were administered by gavage daily for 15 consecutive days; Cordycepin group: Cordycepin (500 ug / time / animal) was administered by gavage daily and prepared with PBS for 15 consecutive days; Compound 24 drug group: Compound 24 (500 ug / time / animal) was administered by gavage daily and prepared with PBS for 15 consecutive days; PD-1+TIM3 antibody treatment group: Antibody PD-1+TIM3 (500 ug / time / animal) was administered by gavage every 4 days for 15 consecutive days; Compound 24 drug + PD-1+TIM3 antibody treatment group: Immunoglobulin compound 24 (500 ug / time / animal) was administered by gavage daily, and antibody PD-1+TIM3 (500 ug / time / animal) was administered by gavage every 4 days for 15 consecutive days. The size changes of ovarian tumors in the five groups of mice were observed every two days. After the experiment, the mice were sacrificed, and blood and corresponding tissue samples were collected. The tumors were immediately photographed and weighed. A portion of the tumor tissue was fixed in formalin solution for further testing. The two vertical diameters (length and width) of the transplanted tumor were measured every two days using calipers to calculate the size of the transplanted tumor and its volume according to the formula: Tumor volume (mm²) 3 = 1 / 2 × (length × width) 2 The experimental results are shown in Figure 4 As shown in the figure. Experimental results indicate that cordycepin has an anti-proliferative effect on ovarian cancer (size reduction of 1.5 times in 15 days). Compound 24 alone has a more significant anti-tumor growth effect than cordycepin (size reduction of approximately 2 times in 15 days). Immune checkpoint inhibitors PD-1 and CTLA4 alone also have significant anti-tumor growth effects (size reduction of more than 2 times in 15 days). When compound 24 and immune checkpoint inhibitors PD-1 and CTLA4 are used simultaneously, the anti-tumor effect is greatly enhanced (size reduction of approximately 4 times in 15 days).

[0313] Example 48: Inhibitory effect of cordycepin modified derivatives on gastric cancer cells in vitro

[0314] The MTT assay was used to analyze the dose-response relationship curves of cordycepin and forty prepared compounds in gastric cancer cell lines AGS and BGC-823. The half-maximal inhibitory concentration (IC50) was calculated. 50 The results are summarized in Table 4 below. In vitro antitumor experiments showed that, compared with cordycepin, the modified cordycepin derivative had a lower effective concentration against tumor cells.

[0315] Table 4: IC50 of cordycepin and cordycepin-modified compounds in gastric cancer cells50 (μM)

[0316]

[0317] Example 49: In vivo tumor inhibition effect of cordycepin modified derivatives on a gastric cancer-mouse model

[0318] Establishment of a mouse transplanted gastric cancer model and drug evaluation: 2×10⁻⁶ cells were suspended in 100 μL of PBS. 7 BGC-823 gastric cancer cells were inoculated into the anterior chest wall of C57BL / 6j nude mice after disinfection with 75% ethanol. The inoculation was performed by manually touching the point where the apex of the heart beat most strongly, approximately 3 mm to the left of the sternum in the second intercostal space. After about one week, the transplanted tumor reached approximately 100 mm. 3 At the time of experimentation, mice were randomly divided into five groups of 10 mice each: a control group, a cordycepin group, and drug groups containing compounds 16, 18, and 24. The control group received daily gavage with nutrients such as corn steep liquor and DMSO as a solvent. The cordycepin group received daily gavage with cordycepin (500 μg / mouse) prepared in DMSO. The drug groups received daily gavage with the compound (500 μg / mouse) prepared in DMSO. Administration continued for 18 days. Changes in gastric tumor size were observed every two days in all five groups. Mice were sacrificed at the end of the experiment, and blood and corresponding tissue samples were collected. The tumors were immediately photographed and weighed. The weighing results are shown below. Figure 5 As shown in the figure. The experimental results show that cordycepin can effectively inhibit the proliferation of gastric tumors, reducing the tumor growth rate by about 4 times compared to the control group (from 1.73 g to 0.46 g). The modified cordycepin has a more significant effect than cordycepin, with compound 24 reducing the tumor growth rate by about 12 times compared to the control group (from 1.73 g to 0.14 g).

[0319] Example 50: In vivo tumor inhibition effect of cordycepin modified derivatives on pancreatic cancer-mouse model

[0320] Establishment of a mouse model of pancreatic cancer transplantation and drug evaluation: Pancreatic cancer Pan02-luc cells were placed in DMEM medium containing 10% fetal bovine serum and incubated at 37 ℃ in a CO2 incubator. Cells were passaged by EDTA digestion every 2-3 days. When the desired cell count was reached, cells in the logarithmic growth phase were harvested and resuspended in culture medium to a concentration of 1×10⁻⁶ cells / mL. 7 / mL. C57BL / 6j nude mice were fed under pathogen-free conditions. When the mice reached 6 weeks of age, 200 μL of pancreatic cancer Pan02-uc cells were subcutaneously injected into the right dorsal side near the axilla of each mouse. The appearance of xenografts within about one week indicated successful establishment of the model. Mice were randomly divided into five groups of 10 mice each: control group, cordycepin group, and drug groups containing compounds 11, 16, and 24. Control group: administered corn steep liquor and other nutrients and DMSO as a solvent via gavage daily. Cordycepin group: administered cordycepin (500 μg / mouse / time) via gavage daily, prepared with DMSO. Drug groups: administered the compound (500 μg / mouse / time) via gavage daily, prepared with DMSO. Administration continued for 24 days. The survival rate of the mice in the five groups was then observed daily, and the median survival time was studied. The experimental results are as follows: Figure 6 As shown in the figure. The experimental results showed that cordycepin alone significantly prolonged the survival of tumor-bearing mice (from 41 days to 52 days). The compound groups further prolonged the survival of tumor-bearing mice (from 41 days to >58 days) on the basis of cordycepin alone. Among them, compound 24 had the best effect, prolonging the survival of tumor-bearing mice by nearly half (from 41 days to 76 days).

[0321] This invention provides a compound derived from cordycepin, its preparation method, and its application in the preparation of products for preventing and treating diseases related to functional cell damage and mutation. Many methods and approaches exist for implementing this technical solution; the above description is merely a preferred embodiment. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of this invention, and these improvements and modifications should also be considered within the scope of protection of this invention. All components not explicitly stated in this embodiment can be implemented using existing technologies.

Claims

1. A cordycepin derivative as shown in Formula I, or a pharmaceutically acceptable salt, stereoisomer, tautomer, solvate, prodrug, or metabolite thereof; I in, R1 is selected from R1 1 -R1 25 The indicated groups; R2 is selected from hydrogen or azide group; R3 is selected from hydrogen, fluorine, chlorine, or azide. R4 is selected from R4 1 -R4 7 The indicated groups; R5 is selected from hydrogen, bromovinyl, mercapto, methyl, fluorine, or chlorine; R6 is selected from amino groups, or such as R6 1 -R6 16 The indicated groups; R7 is selected from hydrogen or isopropylamino; Furthermore, there are no cases where R1, R2, R3, R5, and R7 are selected from hydrogen, R4 is selected from hydroxyl, and R6 is selected from amino; and there is no case where R1 is R1. 16 In the case shown, R2 is selected from hydrogen, R3 is selected from hydrogen, R4 is selected from hydroxyl, R5 is selected from hydrogen, fluorine, or chlorine, R6 is selected from pyridinecarboxamide or amino, and R7 is selected from hydrogen; 。 2. The cordycepin derivative according to claim 1, or its pharmaceutically acceptable salt, stereoisomer, tautomer, solvate, prodrug, or metabolite, characterized in that, R1 is selected from hydrogen or phosphate group; R2 is selected from hydrogen or azide group; R3 is selected from hydrogen; R4 is selected from R4 6 R4 7 Or R4 3 The indicated groups; R5 is selected from hydrogen, bromovinyl, mercapto, methyl, fluorine, or chlorine; R6 is selected from amino, or R6 15 The indicated group; R7 is selected from hydrogen or isopropylamino; Preferably, the cordycepin derivative shown in Formula I is compound 33 or compound 34; Compound 33, Compound 34, Compound 9.

3. The cordycepin derivative according to claim 1, or its pharmaceutically acceptable salt, stereoisomer, tautomer, solvate, prodrug, or metabolite, characterized in that, R1 is selected from R1 10 -R1 11 or R1 14 -R1 16 The indicated groups; R2 is selected from hydrogen or azide; R3 is selected from hydrogen; R4 is selected from hydroxyl; R5 is selected from mercapto; R6 is selected from amino; R7 is selected from hydrogen or isopropylamino; Preferably, the cordycepin derivative shown in Formula I is compound 19; Compound 19.

4. The cordycepin derivative according to claim 1, or its pharmaceutically acceptable salt, stereoisomer, tautomer, solvate, prodrug, or metabolite, characterized in that, R1 is selected from R1 10 -R1 11 or R1 14 -R1 16 The indicated groups; R2 is selected from hydrogen or azide; R3 is selected from hydrogen or fluorine; R4 is selected from hydroxyl; R5 is selected from hydrogen, fluorine, or chlorine; R6 is selected from amino; R7 is selected from hydrogen or isopropylamino; and R3 and R5 are not both hydrogen; Preferably, the cordycepin derivative shown in Formula I is compound 18 or compound 17; Compound 18. Compound 17.

5. The cordycepin derivative according to claim 1, or its pharmaceutically acceptable salt, stereoisomer, tautomer, solvate, prodrug, or metabolite, characterized in that, R1 is selected from R1 10 -R1 11 or R1 14 -R1 16 The indicated groups are: R2 is selected from hydrogen or azide; R3 is selected from azide; R4 is selected from hydroxyl; R5 is selected from hydrogen, bromovinyl, mercapto, methyl, fluorine, or chlorine; R6 is selected from amino or hydroxyl. 15 The indicated group; R7 is selected from hydrogen or isopropylamino; Preferably, the cordycepin derivative shown in Formula I is compound 26, compound 27, or compound 28; Compound 26, Compound 27, Compound 28.

6. The cordycepin derivative according to claim 1, or a pharmaceutically acceptable salt, stereoisomer, tautomer, solvate, prodrug, or metabolite thereof, characterized in that, R1 is selected from hydrogen, phosphate group, or phosphonate group; R2 is selected from hydrogen or azide group; R3 is selected from hydrogen; R4 is selected from R4 5 The indicated groups; R5 is selected from hydrogen, bromovinyl, mercapto, methyl, fluorine, or chlorine; R6 is selected from amino, or R6 15 The indicated group; R7 is selected from hydrogen or isopropylamino; Preferably, the cordycepin derivative shown in Formula I is compound 29, compound 30, compound 31, or compound 32; Compound 29, Compound 30, Compound 31, Compound 32.

7. The cordycepin derivative according to claim 1, or a pharmaceutically acceptable salt, stereoisomer, tautomer, solvate, prodrug, or metabolite thereof, characterized in that, R1 is selected from phosphate or phosphonic acid; R2 is selected from hydrogen or azide; R3 is selected from hydrogen; R4 is selected from hydroxyl; R5 is selected from bromovinyl; R6 is selected from amino or R6 15 The indicated group; R7 is selected from hydrogen or isopropylamino; Preferably, the cordycepin derivative shown in Formula I is compound 35 or compound 38; Compound 35. Compound 38.

8. The cordycepin derivative according to claim 1, or a pharmaceutically acceptable salt, stereoisomer, tautomer, solvate, prodrug, or metabolite thereof, characterized in that, R1 is selected from hydrogen; R2 is selected from hydrogen or azide group; R3 is selected from hydrogen or chlorine; R4 is selected from hydroxyl group; R5 is selected from hydrogen or fluorine group; R6 is selected from R6 5 The indicated group; R7 is selected from hydrogen or isopropylamino; and R3 and R5 are not both hydrogen; Preferably, the cordycepin derivative shown in Formula I is compound 21 or compound 22; Compound 21 Compound 22 Optionally, the cordycepin derivative shown in Formula I is selected from any of the following; 。 9. A pharmaceutical composition comprising at least one cordycepin derivative as described in claims 1 to 8, or a pharmaceutically acceptable salt, stereoisomer, tautomer, solvate, prodrug, or metabolite thereof; and at least one immune checkpoint inhibitor; preferably, the immune checkpoint inhibitor is selected from PD-1 and / or CTLA4 monoclonal antibodies; preferably, the mass ratio of the cordycepin derivative, or a pharmaceutically acceptable salt, stereoisomer, tautomer, solvate, prodrug, or metabolite thereof, to the immune checkpoint inhibitor is 1:0.2 to 10, preferably 1:0.2 to 8, preferably 1:0.2 to 5, preferably 1:0.2 to 3, preferably 1:0.2 to 2, preferably 1:0.5 to 1.5, preferably 1:0.8 to 1.2, preferably 1:1; preferably, the dosage form of the pharmaceutical composition is selected from tablets, pills, capsules, pellets, syrups, disintegrants, injections, sustained-release formulations, or kits.

10. The use of any cordycepin derivative of claims 1 to 8, or a pharmaceutically acceptable salt, stereoisomer, tautomer, solvate, prodrug, or metabolite thereof, or the pharmaceutical combination of claim 9, in the preparation of a product for the prevention and treatment of diseases related to cellular functional impairment in mammals or humans; preferably, the disease related to cellular functional impairment is a tumor; preferably, the tumor includes gastric cancer, pancreatic cancer, liver cancer, small cell lung cancer, non-small cell lung cancer, colorectal cancer, esophageal cancer, prostate cancer, melanoma, glioma, and ovarian cancer; preferably, the tumor is any one of gastric cancer, pancreatic cancer, liver cancer, small cell lung cancer, colorectal cancer, melanoma, and ovarian cancer.

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