A compound based on cordycepin having an anti-tumor effect through derivatization

Abstract

The application discloses a compound with an anti-tumor effect based on cordycepin and derived by derivatization, and a structure of the compound is shown as formula I. The cordycepin derivative and the pharmaceutical composition thereof have good anti-tumor proliferation effects. Compared with a parent drug, the cordycepin derivative has better affinity to a cell membrane, so that the half-life of metabolism of the drug in the body is longer, and the drug stays in the body for a longer time. Compared with other nucleoside anti-tumor drugs, the cordycepin derivative and the pharmaceutical composition thereof have a wider range of tumor types and effects, and have excellent inhibition effects on gastric cancer, pancreatic cancer, liver cancer, small cell lung cancer, colorectal cancer, melanoma, ovarian cancer and the like, and have lower side effects and better curative effects.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biological medicine, and particularly relates to a compound based on cordycepin and derived therefrom, a preparation method thereof, and application of the compound in preparation of a product for preventing and treating a disease caused by cell functional damage variation. BACKGROUND

[0002] With external environmental stimulation and accumulation of cell damage, the adaptability of cells in the body changes, one is the loss of cell adaptability, which eventually evolves into aging, and the other is the abnormal enhancement of cell adaptability, which turns into cancer. Cancer is a common disease that endangers human life and health, and the incidence and mortality of cancer have been increasing worldwide. At present, the treatment methods for malignant tumors mainly include surgery, radiotherapy and chemotherapy, among which chemotherapy mainly uses synthetic drugs. The inhibitory effect of chemotherapy drugs on tumors is worthy of affirmation, and it is also one of the more effective and commonly used treatment methods for malignant tumors, but its toxic and side effects are widespread and serious, and there is a problem of drug resistance. In addition, the selectivity of chemotherapy drugs for tumor cells and normal cells is poor, and while killing or inhibiting tumor cells, it can also damage the growth of normal cells, and will have a direct impact on the function of heart, liver, kidney and nervous system, and will produce certain toxicity to the human body. Therefore, it is necessary to find low-toxicity and high-efficiency antitumor drugs in tumor treatment.

[0003] Nucleosides are one of the most important endogenous compounds in the human body, and play an important role in the metabolic process of the body. One of the main ways to prepare antitumor drugs is to modify and derivatize nucleoside compounds. The nucleoside antitumor drugs on the market currently include furtulacil, fludarabine, cladribine, clofarabine, fludarabine phosphate, and troxacitabine. Cordycepin (3'-deoxyadenosine) is the main active ingredient of Cordyceps sinensis, and belongs to nucleoside analogs. It has excellent effects on cell adaptability changes (anti-aging, anti-carcinogenesis), immune regulation, and inflammation elimination in the metabolic process of the body.

[0004] The mechanism of the anticancer effect of cordycepin is mainly inducing apoptosis, regulating cell cycle, and interfering with matrix metalloproteinase (MMP) expression, thereby inhibiting tumor cell invasion and metastasis. The signal pathways related to the induction of tumor cell apoptosis include the NF-κB signal pathway and the mitogen-activated protein kinase (MAPK) signal pathway. The regulation of cell cycle mainly manifests that in cancer cells, cordycepin shortens the G1 phase, prolongs the G2 phase and the M phase, and makes the cell cycle arrest at the G2 / M phase, thereby inhibiting cell proliferation. The process of interfering with matrix metalloproteinase expression mainly occurs through inhibiting the NF-κB signal pathway, and finally inhibiting the expression of MMP-9. However, nucleoside analogs have poor fat solubility, are difficult to absorb, are easy to be inactivated by deaminase metabolism, have a short half-life, have low targeting, and some tumor cells or viruses are easy to produce drug resistance, which greatly reduces the use effect of nucleoside drugs. SUMMARY

[0005] The technical problem to be solved by the present application is to provide a series of nucleoside analogs (cordycepin derivatives) taking cordycepin as a mother nucleus and being chemically modified in combination with the natural pharmacological activity of cordycepin in view of the deficiencies of existing nucleoside drugs.

[0006] The present application also aims to provide a composition containing the above-mentioned nucleoside analog.

[0007] The present application also aims to provide the application of the above-mentioned nucleoside analog and the composition thereof in the prevention and treatment of diseases related to the functional damage of cells in mammals or human bodies.

[0008] The present application also aims to provide a preparation method of the above-mentioned nucleoside analog.

[0009] In order to solve the first technical problem, the present application discloses a cordycepin derivative as shown in formula I, or a pharmaceutically acceptable salt, stereoisomer, tautomer, solvate, prodrug, or metabolite thereof.

[0010]

[0011] wherein R1is selected from hydrogen, phosphoric acid, substituted phosphoric acid ester, phosphonic acid, substituted phosphonic acid ester, alkyl alcohol, amino acid alkyl ester, amino acid alkyl alcohol ester, alkyl acid alkyl ester, or cycloalkyl polyol; the substitution is any one or more functional groups of alkoxy, halogen substituted alkoxy, aryloxy, amino acid ester amide, alkyl ester, alkyl acid methyl ester oxy; R2is selected from hydrogen, or azido; R3is selected from hydrogen, fluoro, chloro, or azido; R4is selected from hydroxyl, cyano, beta-amide-gamma-cyclodisulfonatoxy, amino acid carboxylic acid ester, amino acid alkyl ester phosphonic acid phenyl ester, or amino acid alkyl ester phosphonic acid phenyl ester; R5is selected from hydrogen, bromo vinyl, mercapto, methyl, fluoro, or chloro; R6is selected from amino, substituted formamide; the substitution is any one or more functional groups of alkyl, aryl, cycloalkyl, furanyl, pyridinyl; R7is selected from hydrogen, or isopropyl amino; and the absence of R1, R2, R3, R5, R7is selected from hydrogen, R4is selected from hydroxyl, R6is selected from amino.

[0012] In some embodiments, R1is selected from hydrogen, as R1 1 -R1 7 phosphoric acid, diethyl phosphoric acid ester, di-n-propyl phosphoric acid ester, di-i-propyl phosphoric acid ester, di-i-butyl phosphoric acid ester, di-n-butyl phosphoric acid ester, or di-i-butyl phosphoric acid acyloxy methoxy, as R1 8 -R1 11 phosphonic acid, trifluoroethyl phosphonic acid ester, di-i-butyl phosphonic acid acyloxy methoxy, or neopentyl phosphonic acid acyloxy methoxy, as R1 12 -R1 13 cyclophosphonate trifluoroethanol glycol ester, or cyclophosphonate glycol ester, as R1 14 -R1 16 (R)-phosphoryl isoleucine methyl phenyl ester, (S)-phosphoryl isoleucine methyl phenyl ester, or phosphoryl alanine isopropyl phenyl ester, as R1 17 -R1 22 hydroxyethyl, 1,3-dihydroxy-2-propyl, alanine ethyl ester, isoleucine ethyl ester, isoleucine-1,3-propanediol ester, or glycine ethyl ester, or as R1 23 -R1 25 dimethyl propanediol dicarbonate ester, 2-i-propoxy hexahydrocyclopenta[d][1,3,2]dioxaphosphorin-7-ol, or 1-hydroxymethyl-2,3-dihydroxy-4-cyclopentyl; in some embodiments, R1is selected from hydrogen, or as R1 1 , R1 5 , R1 8 , R1 10 , R1 15 , R1 16 , R1 17R1 21 The structure shown in any one of them.

[0013]

[0014] In some embodiments, R4 is selected from R4 1 -R4 7 The indicated hydroxyl, cyano, β-amide-γ-cyclosulfonyloxy, 2-amino-propionyloxy, 2-amino-3-methyl-butyryloxy, phosphoryloxyalanine methyl ester phenyl ester, or phosphatidyl isoleucine methyl ester phenyl ester; in some embodiments, R4 is selected from cyano, cyano, or as shown by R4. 3 R4 5 or R4 6 The structure shown in any one of them.

[0015]

[0016] In some embodiments, R5 is selected from hydrogen, bromovinyl, mercapto, fluorine, or chlorine.

[0017] In some embodiments, R6 is selected from amino groups, or such as R6 1 -R6 16 The indicated groups are acetamido, butyramido, octamido, dodecylamido, octadecylamido, isopropylamido, isobutyramido, neopentylamido, 2-ethyl-n-butyramido, 3,3-dimethyl-butyramido, cyclohexylformamido, cyclopentylformamido, benzamidedo, furanamidedo, pyridineformamido, or hexadecylamido; in some embodiments, R6 is selected from amino groups, or as shown by R6. 5 R6 12 R6 13 or R6 15 The structure shown in any one of them.

[0018]

[0019] In some embodiments, a cordycepin derivative as shown in Formula I, or a pharmaceutically acceptable salt thereof; wherein R1 is R1 16 The groups shown are: 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; in some embodiments, the cordycepin derivatives shown in Formula I are selected from compound 24, compound 7 or compound 20 below.

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

[0021]

[0022]

[0023]

[0024] To solve the above-mentioned second technical problem, the present application discloses a pharmaceutical composition comprising at least one of the above-mentioned cordycepin derivatives, or a pharmaceutically acceptable salt, stereoisomer, tautomer, solvate, prodrug, or metabolite thereof; and at least one immune checkpoint inhibitor.

[0025] In some embodiments, the immune checkpoint inhibitor is selected from a PD-1 and / or CTLA4 monoclonal antibody.

[0026] In some embodiments, 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 1:0.2-10, in some embodiments 1:0.2-8, in some embodiments 1:0.2-5, in some embodiments 1:0.2-3, in some embodiments 1:0.2-2, in some embodiments 1:0.5-1.5, in some embodiments 1:0.8-1.2, and in some embodiments 1:1.

[0027] In some embodiments, the dosage form of the pharmaceutical composition is selected from a tablet, a pill, a capsule, a dripping pill, a syrup, a disintegrant, an injection, a sustained-release agent, or a kit.

[0028] To solve the above-mentioned third technical problem, the present application discloses the use of the above-mentioned cordycepin derivative, or a pharmaceutically acceptable salt, stereoisomer, tautomer, solvate, prodrug, or metabolite thereof, or the above-mentioned pharmaceutical composition in the preparation of a product for preventing and treating a disease related to the variation of cell functional damage in mammals or humans.

[0029] In some embodiments, the disease related to the variation of cell functional damage is a tumor, and 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.

[0030] In some embodiments, the product includes but is not limited to a drug.

[0031] To solve the fourth technical problem, the application discloses a preparation method of the cordycepin derivative. The preparation method is a corresponding modification method. In the reaction process, other active groups in the cordycepin or modified cordycepin molecules may participate in the reaction, and thus the active groups should be properly protected. The protection method disclosed in the application is the protection and deprotection of hydroxyl and amino groups. The protection and deprotection methods are conventional methods in the field. In addition, some reaction systems need to be protected by adding a protective gas. These are conventional methods in the experimental process in the field.

[0032] Specifically, the following steps are included:

[0033] A. modifying R1

[0034] When R1 in the formula I is selected from a phosphate group, a substituted phosphate ester group, a phosphonic acid group, a substituted phosphonic acid ester group, an alkyl alcohol group, an amino acid alkyl ester group, an amino acid alkyl alcohol ester group, an alkyl acid alkyl ester group, or a cycloalkyl polyol group, and the substitution is any one or more of the following functional groups: an alkoxy group, a halogen-substituted alkoxy group, an aryloxy group, an amino acid ester amide group, an alkyl ester group, and an alkyl acid methyl ester oxy group, the preparation method of the cordycepin derivative shown in the formula I is as follows: using compound I-R-1 as raw material, a chemical reaction is performed in an organic solvent to obtain the cordycepin derivative shown in the formula I.

[0035]

[0036] In the formula I-R-1, R2-R7 are the same as R2-R7 in the formula I, or are independently selected from a protecting group.

[0037] When R1 in the formula I is selected from a phosphate group, in some embodiments, the preparation method of the cordycepin derivative shown in the formula I is as follows: in trimethyl phosphite and / or triethyl phosphite, compound I-R-1 is reacted with a modifier trichloro phosphorus. In some embodiments, the amount ratio of the compound I-R-1, the modifier, and the 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.

[0038] When R1in Formula I is selected from substituted phosphate groups, in some embodiments, the method for preparing the cordycepin derivative of Formula I is reacting compound I-R-1 with a modifying agent substituted nitrophenyl phosphate, the substitution being any one or more of alkoxy, halogen-substituted alkoxy, aryloxy, amino acid ester amide, alkyl ester, alkyl acid methyl ester, in anhydrous N,N-dimethylformamide and / or tetrahydrofuran, with tert-butyl magnesium chloride as a catalyst, in some embodiments, the ratio of compound I-R-1, modifying agent, 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.

[0039] When R1in Formula I is selected from phosphonic acid groups, or substituted phosphonate groups, in some embodiments, the method for preparing the cordycepin derivative of Formula I is reacting compound I-R-1 with a modifying agent substituted or unsubstituted p-toluenesulfonyloxymethyl phosphate, the substitution being any one or more of alkoxy, halogen-substituted alkoxy, aryloxy, amino acid ester amide, alkyl ester, alkyl acid methyl ester, in anhydrous N,N-dimethylformamide, with NaH as a catalyst, in some embodiments, the ratio of compound I-R-1, modifying agent, catalyst, and organic solvent is 0.1 mol: 0.1-0.15 mol: 0.2-0.03 mol: 100-200 mL, in some embodiments, 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, in some embodiments, the reaction time is 0.5-6 h.

[0040] When R1in Formula I is selected from alkyl alcohol groups, amino acid alkyl ester groups, amino acid alkyl alcohol ester groups, alkyl acid alkyl ester groups, or cycloalkyl polyol groups, in some embodiments, the method for preparing the cordycepin derivative of Formula I is reacting compound I-R-1 with a modifying agent halogen-substituted alkyl alcohol, amino acid alkyl ester, amino acid alkyl alcohol ester, alkyl acid alkyl ester, or cycloalkyl polyol, in butanone, with potassium carbonate as a catalyst, in some embodiments, the halogen is bromine or chlorine, in some embodiments, the ratio of compound I-R-1, modifying agent, catalyst, and organic solvent is 1 mmol: 0.5-1.5 mmol: 1-3 mmol: 5-8 mL, in some embodiments, 1 mmol: 1 mmol: 1-3 mmol: 5-8 mL, in some embodiments, the reaction temperature is 40-100 °C, in some embodiments, the reaction time is 8-20 h.

[0041] B. Modification of R2

[0042] When R2in Formula I is selected from azido; the method for preparing the pteroptyl derivatives of Formula I is: performing a cyclization reaction on compound I-R-2 in an organic solvent to prepare intermediate I-R-2a, 5-(6-amino-9H-purin-9-yl)-1,4-dioxopyrrolo[2.4]heptan-6-ol or derivatives thereof; performing a ring-opening reaction on the obtained intermediate I-R-2a in an organic solvent to prepare the pteroptyl derivatives of Formula I;

[0043]

[0044] In Formula I-R-2, R1, R3-R7 are the same as R1, R3-R7 in Formula I, or are each independently selected from a protecting group.

[0045] In some embodiments, the method for preparing intermediate I-R-2a is: performing a cyclization reaction on compound I-R-2 in dichloromethane under catalysis of phosphorus pentoxide and meta-chloroperoxybenzoic acid, and in some embodiments, the use amount ratio of compound I-R-2, phosphorus pentoxide, meta-chloroperoxybenzoic acid and dichloromethane is 1 mmol: 1.2-2 mmol: 2-3 mmol: 10-20 mL, and in some embodiments, the temperature of the cyclization reaction is 20-60°C, and in some embodiments, the time of the cyclization reaction is 3-10 h.

[0046] In some embodiments, the method for preparing the pteroptyl derivatives of Formula I is: performing a ring-opening reaction on the obtained intermediate I-R-2a and sodium azide in anhydrous dimethylformamide, and in some embodiments, the use amount ratio of compound I-R-2a, sodium azide and dimethylformamide is 1 mmol: 4-5 mmol: 2-5 mL, and in some embodiments, the temperature of the ring-opening reaction is 100-120°C, and in some embodiments, the time of the ring-opening reaction is 12-16 h.

[0047] C. modifying R3

[0048] When R3in Formula I is selected from fluorine, chlorine, or azido; the method for preparing the pteroptyl derivatives of Formula I is: performing a chemical reaction on compound I-R-3 in an organic solvent to prepare the pteroptyl derivatives of Formula I;

[0049]

[0050] In Formula I-R-3, R1, R2, R5-R7 are the same as R1, R2, R5-R7 in Formula I, or are each independently selected from a protecting group.

[0051] When R3in formula I is selected from fluorine, or chlorine, in some embodiments, the method for preparing the pterolin derivative shown in formula I is: reacting compound I-R-3 with triflic anhydride in pyridine and dichloromethane to prepare intermediate I-R-3a, 5-(6-amino-9H-purin-9-yl)-4-hydroxy-2-(hydroxymethyl)tetrahydrofuran-3-yl trifluoromethanesulfonate or derivatives thereof; and reacting intermediate I-R-3a with hydrofluoric acid, sulfur trioxide or hydrochloric acid in ethyl acetate to prepare the pterolin derivative shown in formula I; wherein in the preparation of intermediate I-R-3a, in some embodiments, the amount ratio of compound I-R-3, triflic 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, 0°C, and in some embodiments, the reaction time is 1-3 h; wherein in the preparation of the pterolin derivative shown in formula I, in some embodiments, intermediate I-R-3a is reacted with 37% hydrofluoric acid or diethylaminosulfur trifluoride or hydrochloric acid solution in triethylamine, in some embodiments, the amount ratio of intermediate I-R-3a, 37% hydrofluoric acid or diethylaminosulfur trifluoride or hydrochloric acid solution in triethylamine, and 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.

[0052] When R3 in formula I is selected from azido group, in some embodiments, the method for preparing the cordycepin derivative shown in formula I is: cyclization reaction of compound I-R-3 in N,N-dimethylformamide under catalysis of triphenylphosphine and diisopropyl azodicarboxylate to prepare intermediate I-R-3b, 4-(6-amino-9H-purin-9-yl)-3,6-dioxabicyclo[3.1.0]hexan-2-yl)methanol or its derivative; ring-opening reaction of the obtained intermediate I-R-3b with sodium azide in dimethylformamide to prepare the cordycepin derivative shown in formula I; wherein, in the preparation method of the intermediate I-R-3b, in some embodiments, the ratio of the use amount of the compound I-R-3, triphenylphosphine, diisopropyl azodicarboxylate and N,N-dimethylformamide is 10.0 mmol: 16-28 mmol: 1.6-2.8 mmol: 15-50 mL, in some embodiments, 10.0 mmol: 22 mmol: 2.2 mmol: 15-50 mL, in some embodiments, the temperature of the cyclization reaction is 10-60°C, and in some embodiments, the time of the cyclization reaction is 1-5 h; in some embodiments, in the preparation method of the cordycepin derivative shown in formula I, the ratio of the use amount of the intermediate I-R-3b, 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, and in some embodiments, the time of the ring-opening reaction is 12-16 h.

[0053] D. Modification of R4

[0054] When R4 in formula I is selected from cyano group, β-amide-γ-cyclamoyloxy group, amino acid carboxylate group, amino acid alkyl ester phosphonate phenyl group, or amino acid alkyl ester phosphonate phenyl group; the method for preparing the cordycepin derivative shown in formula I is: chemical reaction of compound I-R-4 as raw material in an organic solvent to prepare the cordycepin derivative shown in formula I;

[0055]

[0056] In formula I-R-4, R1-R3, R5-R7 are the same as R1-R3, R5-R7 in formula I, or are independently selected from protecting groups.

[0057] When R4in formula I is selected from cyano, in some embodiments, the method for preparing the cordycepin derivative shown in formula I is: stirring compound I-R-4, trifluoromethanesulfonic acid, trimethylsilyl trifluoromethanesulfonate in dichloromethane at -50 to -30 °C, and then reacting with trimethylsilyl cyanide and triethylamine; in some embodiments, the amount ratio of compound I-R-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, 10 mmol: 1.3 mL: 2.7 mL: 100 mL; in some embodiments, the stirring time is 20-40 min, and in some embodiments, 30 min; in some embodiments, the amount ratio of compound I-R-4, trimethylsilyl cyanide, and triethylamine is 10 mmol: 3.4-4.3 g: 3-4 mL, in some embodiments, the reaction temperature is 20-30 °C, and in some embodiments, room temperature, and in some embodiments, the reaction time is 2-4 h.

[0058] When R4in Formula I is selected from β-amide-γ-cyclamoyloxy, in some embodiments, the method for preparing the cordycepin derivative shown in Formula I is: compound I-R-4 is first subjected to oxidation reaction to prepare intermediate I-R-4a, 2-(6-amino-1,6-dihydro-9H-purin-9-yl)-5-(hydroxymethyl)dihydrofuran-3(2H)-one or its derivative, then subjected to cyanation reaction to prepare intermediate I-R-4b, 2-(6-amino-1,6-dihydro-9H-purin-9-yl)-5-(hydroxymethyl)-3-isocyantetrahydrofuran-3-ol or its derivative, then subjected to methyl sulfonate to prepare intermediate I-R-4c, 2-(6-amino-1,6-dihydro-9H-purin-9-yl)-5-(hydroxymethyl)-3-isocyantetrahydrofuran-3-yl methane sulfonate or its derivative, and finally subjected to cyclization reaction to prepare the cordycepin derivative shown in Formula I; in some embodiments, the method for preparing the intermediate I-R-4a is: compound I-R-4 is reacted with Jones reagent 2.2M chromium trioxide in acetone, in some embodiments, the use amount ratio of compound I-R-4, Jones reagent and acetone is 20mmol:5-8mL:50-100mL, in some embodiments, the reaction temperature is 0-40℃, and in some embodiments, the reaction time is 1-4h; in some embodiments, the method for preparing the intermediate I-R-4b is: intermediate I-R-4a is reacted with trimethylsilyl cyanide, boron trifluoride ether in dichloromethane, in some embodiments, the use amount ratio of intermediate I-R-4a, trimethylsilyl cyanide, boron trifluoride ether and dichloromethane is 20mmol:20-40mmol:10-30mmol:50-100mL, in some embodiments, 20mmol:20-40mmol:20mmol:50-100mL, in some embodiments, the reaction temperature is 0-40℃, and in some embodiments, the reaction time is 1-4h; in some embodiments, the method for preparing the intermediate I-R-4c is: intermediate I-R-4b is reacted with triethylamine, methanesulfonyl chloride in anhydrous dichloromethane, in some embodiments, the use amount of intermediate I-R-4b, triethylamine, methanesulfonyl chloride and anhydrous dichloromethane is 2.2mmol:10-15mmol:4-8mmol:5-10mL, in some embodiments, the reaction temperature is -30-0℃, and in some embodiments, the reaction time is 2-4h; in some embodiments, the method for preparing the cordycepin derivative shown in Formula I is: intermediate I-R-4c is reacted with cesium carbonate in anhydrous acetonitrile, in some embodiments, the use amount ratio of intermediate I-R-4c, cesium carbonate and anhydrous acetonitrile is 1mmol:0.5-2.5mmol:3-10mL, in some embodiments, 1mmol:1.5 mmol: 3-10 mL, in some embodiments, the reaction temperature is 0-40 °C, and in some embodiments, the reaction time is 2-4 h.

[0059] When R4in Formula I is selected from the group consisting of amino acid carboxylate, in some embodiments, the method for preparing the pachymexin derivative of Formula I is reacting compound I-R-4 with amino acid acid chloride in anhydrous pyridine to produce the pachymexin derivative of Formula I; in some embodiments, the ratio of compound I-R-4, amino acid acid chloride, and pyridine is 10 mmol: 5-15 mmol: 50-100 mL, in some embodiments, 10 mmol: 10 mmol: 50-100 mL, in some embodiments, the reaction temperature is 20-60 °C, in some embodiments, 40 °C, and in some embodiments, the reaction time is 6-20 h.

[0060] When R4in Formula I is selected from the group consisting of amino acid alkyl ester phosphonate phenyl ester, in some embodiments, the method for preparing the pachymexin derivative of Formula I is reacting compound I-R-4 with modified amino acid alkyl ester substituted p-toluenesulfonyloxymethyl phosphonate phenyl ester in anhydrous N,N-dimethylformamide with NaH as catalyst, in some embodiments, the ratio of compound I-R-4, modified agent, NaH, and anhydrous N,N-dimethylformamide is 0.1 mol: 0.1-0.15 mol: 0.2-0.03 mol: 100-200 mL, in some embodiments, 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.

[0061] When R4in Formula I is selected from the group consisting of amino acid alkyl ester phosphonate phenyl ester, in some embodiments, the method for preparing the pachymexin derivative of Formula I is reacting compound I-R-4 with modified phosphonate nitrophenyl ester substituted with any one or more functional groups of aryloxy, alkyl acid methyl ester oxy in anhydrous N,N-dimethylformamide and / or tetrahydrofuran with tert-butyl magnesium chloride as catalyst, in some embodiments, the ratio of compound I-R-4, modified agent, 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, and in some embodiments, the reaction time is 2-5 h.

[0062] E. modifying R5

[0063] When R5 in formula I is selected from bromo-vinyl, thiol, methyl, fluoro, or chloro, the method for preparing the cordycepin derivative shown in formula I is: using compound I-R-5 as raw material, preparing the cordycepin derivative shown in formula I through chemical reaction in an organic solvent.

[0064]

[0065] In formula I-R-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.

[0066] When R5in formula I is selected from bromo vinyl, in some embodiments, the method for preparing the cordycepin derivative shown in formula I is: compound I-R-5 is subjected to iodination reaction to prepare intermediate I-R-5a, 2-(6-amino-2-iodo-1,6-dihydro-9H-purin-9-yl)-5-(hydroxymethyl)tetrahydrofuran-3-ol or its derivative, intermediate I-R-5a is subjected to methyl acrylate substitution reaction to prepare intermediate I-R-5b, methyl (E)-3-(6-amino-3-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-6,9-dihydro-1H-purin-2-yl) acrylate or its derivative, intermediate I-R-5b is subjected to hydrolysis to prepare intermediate I-R-5c, (E)-3-(6-amino-9-3-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-6,9-dihydro-1H-purin-2-yl) acrylate or its derivative, and intermediate I-R-5c is subjected to bromination reaction with N-bromosuccinimide to prepare the cordycepin derivative shown in formula I; in some embodiments, the method for preparing intermediate I-R-5a is: compound I-R-5 is subjected to iodination reaction with iodine in dilute nitric acid to prepare intermediate I-R-5a, in some embodiments, the amount ratio of compound I-R-5 to iodine is 1 mmol: 0.5-0.8 mmol, in some embodiments, the reaction temperature is 100-120°C, in some embodiments, the reaction temperature is 110°C, in some embodiments, the reaction time is 4-6 h; in some embodiments, the method for preparing intermediate I-R-5b is: intermediate I-R-5a is subjected to reaction with methyl acrylate and triethylamine in 1,4-dioxane in the presence of palladium acetate and triphenylphosphine, in some embodiments, the amount ratio of intermediate I-R-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, in some embodiments, the amount ratio of intermediate I-R-5a, methyl acrylate, triethylamine, palladium acetate, triphenylphosphine and 1,4-dioxane 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, in some embodiments, the reaction time is 0.5-2 h; in some embodiments, the method for preparing intermediate I-R-5c is: intermediate I-R-5b is subjected to hydrolysis reaction with sodium hydroxide solution, in some embodiments, the concentration of sodium hydroxide solution is 0.5-3.5 mol / L, in some embodiments, 2 mol / L, in some embodiments, the ratio of the amount of use of the intermediate I-R-5b and the sodium hydroxide solution is 1 g: 10-14 mL, in some embodiments, 1 g: 12 mL, in some embodiments, the reaction temperature is 20-30 °C, in some embodiments, 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 that the intermediate I-R-5c is reacted with N-bromosuccinimide under the catalysis of potassium carbonate in a mixed solvent of water and acetone, in some embodiments, the ratio of the amount of use of the intermediate I-R-5c, N-bromosuccinimide, potassium carbonate, and the 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, in some embodiments, 1: 6.

[0067]

[0068] In some embodiments, the bromo-vinyl modification is directly performed on the unmodified cordycepin, and the reaction path is as follows:

[0069]

[0070] When R5in formula I is selected from the group consisting of thiol, in some embodiments, the preparation method of the cordycepin derivative shown in formula I is that compound I-R-5 is reacted with hydrogen peroxide in acetic acid to prepare intermediate I-R-5d, 1N-oxidized-3'-deoxyadenosine or its derivative, intermediate I-R-5d is heated to reflux in hydrochloric acid aqueous solution to prepare intermediate I-R-5e, 5-amino-N'-hydroxy-3-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-1H-imidazole-4-carboxamide or its derivative, intermediate I-R-5e is dissolved in water and reacted under the catalysis of Raney nickel in a hydrogen environment to prepare intermediate I-R-5f, 5-amino-3-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-1H-imidazole-4-carboxamide or its derivative, and intermediate I-R-5f is reacted with methanol, pyridine and carbon disulfide to prepare the cordycepin derivative shown in formula I; in some embodiments, the preparation method of the intermediate I-R-5d is that compound I-R-5 is reacted with acetic acid in hydrogen peroxide, in some embodiments, the concentration of the hydrogen peroxide is 20% to 40%, in some embodiments, it is 30%, in some embodiments, the use amount ratio of the compound I-R-5, hydrogen peroxide and acetic acid is 1 mmol: 2 to 3 mol: 1 to 6 mL, in some embodiments, the reaction temperature is 30 to 50°C, in some embodiments, the reaction time is 2 to 4 days; in some embodiments, the preparation method of the intermediate I-R-5e is that intermediate I-R-5d is heated to reflux in hydrochloric acid aqueous solution, in some embodiments, the concentration of the hydrochloric acid aqueous solution is 1 to 5 mol / L, in some embodiments, it is 3 mol / L, in some embodiments, the use amount ratio of the intermediate I-R-5d and the hydrochloric acid aqueous solution is 1 mmol: 3 to 5 mL, in some embodiments, the heating reflux time is 10 to 30 min; in the preparation method of the intermediate I-R-5f, in some embodiments, the use amount ratio of the intermediate I-R-5e, Raney nickel and water is 1 mmol: 0.08 to 0.2 g: 10 to 20 mL, in some embodiments, the reaction temperature is 50 to 70°C, in some embodiments, the reaction time is 2 to 5 days; in the preparation method of the cordycepin derivative shown in formula I, in some embodiments, the use amount ratio of the intermediate I-R-5f, methanol, pyridine and carbon disulfide is 1 mmol: 5 to 10 mL, in some embodiments, the volume use amount ratio of the methanol, pyridine and carbon disulfide is 4: 3 to 7: 0.5 to 3.5, in some embodiments, it is 4: 5: 2, in some embodiments, the reaction temperature is 30 to 50°C, in some embodiments, it is 40°C, in some embodiments, the reaction time is 3 to 5 days.

[0071]

[0072] In some embodiments, the thiol modification is directly performed on unmodified cordycepin, and the reaction is performed as follows:

[0073]

[0074] When R5in Formula I is selected from methyl, in some embodiments, the method for preparing the cordycepin derivative shown in Formula I is that compound I-R-5, iodomethane, potassium carbonate are reacted in dichloromethane, and in some embodiments, the amount ratio of the compound I-R-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, and in some embodiments, the reaction time is 3-10 h.

[0075] When R5in Formula I is selected from fluorine or chlorine, in some embodiments, the method for preparing the cordycepin derivative shown in Formula I is that compound I-R-5 is subjected to nitro derivatization reaction to obtain nitro intermediate I-R-5g, 2-(6-amino-2-nitro-1,6-dihydro-9H-purin-9-yl)-5-(hydroxymethyl)tetrahydrofuran-3-ol, and then substitution reaction is performed to obtain the cordycepin derivative shown in Formula I; in some embodiments, the method for preparing the intermediate I-R-5g is that compound I-R-5 is reacted with tetrabutylammonium nitrate in dichloromethane under catalysis of trifluoroacetic anhydride, and in some embodiments, the amount ratio of the compound I-R-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 °C, and in some embodiments, the reaction time is 0.5-20 h; in some embodiments, the method for preparing the cordycepin derivative shown in Formula I is that nitro intermediate I-R-5g is reacted with tetrabutylammonium fluoride or tetrabutylammonium chloride in acetonitrile, and in some embodiments, the amount ratio of the nitro intermediate I-R-5g, tetrabutylammonium fluoride or tetrabutylammonium chloride and acetonitrile is 1 mmol: 1.3-1.5 mmol: 30-50 mL, the reaction temperature is -5-5 °C, in some embodiments, 0 °C, and in some embodiments, the reaction time is 20-30 min.

[0076]

[0077]

[0078] F. Modification of R6

[0079] When R6 in formula I is selected from substituted formamide group; the substitution is any one or more functional groups of alkyl, aryl, cycloalkyl, furanyl, pyridyl; the preparation method of the cordycepin derivative shown in formula I is: using compound I-R-6 as raw material, chemical reaction in an organic solvent, to prepare the cordycepin derivative shown in formula I;

[0080]

[0081] In formula I-R-6, R1~R5, R7 are the same as R1~R5, R7 in formula I, or are independently selected from a protecting group.

[0082] In some embodiments, the preparation method of the cordycepin derivative shown in formula I is: reacting compound I-R-6 with substituted acyl chloride in anhydrous pyridine, the substitution is any one or more functional groups of alkyl, aryl, cycloalkyl, furanyl, pyridyl, in some embodiments, the dosage ratio of compound I-R-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℃, in some embodiments, the reaction time is 2~20 h.

[0083] G. modifying R7

[0084] When R7 in formula I is selected from isopropylamino; the preparation method of the cordycepin derivative shown in formula I is: using compound I-R-7 as raw material, chemical reaction in an organic solvent, to prepare the cordycepin derivative shown in formula I;

[0085]

[0086] In formula I-R-7, R1~R6 are the same as R1~R6 in formula I, or are independently selected from a protecting group.

[0087] In some embodiments, the preparation method of the cordycepin derivative shown in formula I is: reacting compound I-R-7 with 2-propylamine in dioxane, in some embodiments, the dosage ratio of compound I-R-7, 2-propylamine and dioxane is 10 mmol: 10~30 mmol: 50~100 mL, in some embodiments, the reaction temperature is 60~120℃, in some embodiments, the reaction time is 10~30 h.

[0088] In the present application, the protecting group includes but is not limited to -OTBS, -OAc, -NHCbz, -OTBPS, -OTBDPS; in some embodiments, R4 is the same as R4 in formula I or is selected from -OTBS, -OAc, -OTBDPS; in some embodiments, R6 is the same as R6 in formula I or is selected from -NHCbz.

[0089] In the present application, the substituted nitrophenyl phosphate (the substitution is any one or more functional groups of alkoxy, halogen-substituted alkoxy, aryloxy, amino acid ester amido group, alkyl ester group, alkyl acid methyl ester oxy) is prepared by the following method or other methods in the prior art.

[0090] In anhydrous dichloromethane, phenyl chlorophosphate, p-nitrophenol and the corresponding substituted alcohol or amine are prepared at 0-25℃ under the catalysis of triethylamine, and the substitution is any one or more functional groups of alkoxy, halogen-substituted alkoxy, aryloxy, amino acid ester amido group, alkyl ester group, alkyl acid methyl ester oxy. In some embodiments, the molar volume ratio of the phenyl chlorophosphate, p-nitrophenol, the corresponding substitution, triethylamine and anhydrous dichloromethane is 1 mmol: 1 mmol: 1-2 mmol: 2-5 mmol: 5-10 mL.

[0091] In the present application, the substituted p-toluenesulfonyloxymethyl phosphate (the substitution is any one or more functional groups of alkoxy, halogen-substituted alkoxy, aryloxy, amino acid ester amido group, alkyl ester group, alkyl acid methyl ester oxy) is prepared by the following method or other methods in the prior art.

[0092] In the corresponding substituted chlorophosphate, p-toluenesulfonyl chloride, formaldehyde in toluene, under the catalysis of triethylamine at 0-105℃, the substituted chlorophosphate is any one or more functional groups of alkoxy, halogen-substituted alkoxy, aryloxy, amino acid ester amido group, alkyl ester group, alkyl acid methyl ester oxy. In some embodiments, the molar volume ratio of the corresponding 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.

[0093] As can be easily understood by those skilled in the art, the present application is based on cordycepin, and R1-R7 are modified. When R1 is hydrogen, R2 is hydrogen, R3 is hydrogen, R5 is hydrogen, R7 is hydrogen, R4 is hydroxyl, or R6 is amino, no modification is needed. In the preparation method of the present application, the above-mentioned substitutions can be modified according to the priority order of the stability of the prepared intermediates without violating the common sense in the art, i.e. each example of the present application is obtained, such as first modifying without ester group, phosphoric acid group, phosphonic acid group, such as modifying-CN, -N3, -F, -SH, etc., then modifying the ester group, and finally modifying the phosphoric acid group or the phosphonic acid group.

[0094] The term "prevention" in the present invention means that the compounds or formulations described in the present application are administered to prevent a disease or one or more symptoms associated with the disease, and includes: preventing the occurrence of a disease or disease state in a mammal, particularly when these mammals are susceptible to developing symptoms associated with the cancer.

[0095] The term "pharmaceutically acceptable" in the present invention refers to those compounds, materials, compositions, and / or dosage forms that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0096] The compounds of the present invention can exist in specific geometric or stereoisomeric forms. The present invention contemplates all such compounds, including cis- and trans-isomers, (-)- and (+)-enantiomers, (R)- and (S)-enantiomers, diastereomers, (D)- isomers, (L)-isomers, as well as the racemic mixtures and other mixtures thereof, such as for example, mixtures of enantiomers or diastereomers, all of which are intended to be within the scope of the present invention. Additional asymmetric carbon atoms can be present in a substituent group. All such isomers, as well as mixtures thereof, are included within the scope of the present invention.

[0097] Advantages: Compared with the prior art, the present invention has the following advantages:

[0098] The cordycepin derivative and the pharmaceutical composition thereof provided by the present invention have good anti-tumor proliferation effect. Compared with the parent drug, the cordycepin derivative has better affinity to the cell membrane, so that the half-life of the drug metabolism in the body is longer, and the drug stays in the body for a longer time. Compared with other nucleoside anti-tumor drugs, the cordycepin derivative and the pharmaceutical composition thereof provided by the present invention 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, lower side effects and better curative effect.

[0099] Drawings of the specification

[0100] Figure 1 Anti-tumor effect of the blank group, the cordycepin control group and the compound group on the liver cancer cell Hep-1-6 transplanted mouse model.

[0101] Figure 2 Inhibition effect of the control group and each compound group on the small cell lung cancer H446 cell tumor-bearing zebrafish model.

[0102] Figure 3 Inhibition effect of compound 16 and immune checkpoint inhibitors on the colon cancer MC38 cell transplanted mouse model.

[0103] Figure 4 The anti-tumor effects of the control group and the compound group on the B16-F10 melanoma transplanted mouse model.

[0104] Figure 5 The inhibitory effects of compound 24 and immune checkpoint inhibitors on the ID8 ovarian cancer syngeneic tumor model in mice.

[0105] Figure 6 The anti-tumor effects of the control group and the compound group on the BGC-823 gastric cancer cell transplanted mouse model.

[0106] Figure 7 The anti-tumor effects of the control group and the compound group on the Pan02-luc pancreatic cancer cell transplanted mouse model. DETAILED DESCRIPTION

[0107] The experimental methods described in the following examples are all conventional methods unless otherwise specified. The reagents and materials described herein can be obtained from commercial sources unless otherwise specified. The drug evaluation experiments described herein include cell models and animal models, which can be obtained from commercial sources unless otherwise specified.

[0108] The cordycepin in the present application is obtained from a biological fermentation pathway (CN 111117896B). The preparation method of cordycepin or its derivatives is prepared according to the method in the specification. The post-treatment method mainly adopts conventional organic experimental post-treatment operation methods including but not limited to filtration, quenching, extraction, rotary evaporation, recrystallization, column chromatography, etc. The preparation methods of the compounds mentioned in the examples are not all the compounds. In order to facilitate the description, only the preparation processes of several representative compounds are listed here.

[0109] The anti-cancer related cell and animal model experiments of cordycepin or its derivatives in the examples are not all effective experimental results, but only related experiments listed to illustrate the effects. The drug evaluation experiments described herein include cell models and animal models, which follow the ethical rules.

[0110] The drug evaluation of the mouse model is raised according to the following: raising and treating the mice in a laminar flow cabinet, raising 5 nude mice per cage, adding water and feed once every 3 days, and replacing the bedding once a week; the number of mice raised in each cage is not more than 5, and the mice in different cages are as far as possible to be grouped, and the mice are as far as possible not to be raised in single cages; the animal management must comply with the relevant standards of the state on animal raising and management, and attention must be paid to the behavioral needs of the animals to avoid unnecessary stress; the normal physiological and behavioral needs of the animals are met, such as defecation, urination, maintaining constant body temperature, normal activity, adjusting posture, and reproduction; good ventilation is provided to keep the animals dry; the animals can freely obtain water and food, and the operation of supplementing and replacing and cleaning is easy; a solid and safe environment is provided to avoid accidents such as the animals escaping or their limbs being trapped in the gap; the animals are protected from being hurt by sharp edges or protrusions; and the animals are not disturbed when being observed.

[0111] The zebrafish model is wild-type AB strain zebrafish from the School of Biology and Pharmaceutical Engineering of Nanjing University of Technology, and is bred by natural pair mating, with 30 fish per experimental group and an age of 2 dpf. The fish are raised in fish water at 28°C (water quality: 200 mg of instant sea salt is added to 1 L of reverse osmosis water, the conductivity is 480-510 uS / cm, the pH is 6.9-7.2, and the hardness is 53.7-71.6 mg / L CaCO3), and the raising management complies with the requirements of international AAALAC certification.

[0112] The method for determining the tumor cell proliferation inhibition activity of the compound by the MTT method is as follows: a bottle of cells in good condition in the exponential growth phase is taken, 0.25% trypsin digestion solution is added, and the adherent cells are detached by digestion, and counted to be 2-4 x 10 4 The cell suspension is inoculated on a 96-well plate, 90 μL / well, and placed in a constant-temperature CO2 incubator for 24 hours. The prepared compound is added for testing, 10 μL / well, and cultured for 72 hours. The MTT reagent is added to the 96-well plate, 10 μL / well, and reacted in the incubator for 4 hours. The supernatant is removed, dimethyl sulfoxide is added, 100 μL / well, and after the crystals are dissolved, the absorbance of each well is determined at a wavelength of 570 nm by an enzyme-linked immunoassay instrument, and the cell inhibition rate is calculated. The compound concentration and the corresponding inhibition rate are used to draw an S curve. The IC 50 .

[0113] Example 1: Preparation of the compound: 2-(6-amino-9H-purin-9-yl)-5-(2-hydroxyethoxy) methyl)tetrahydrofuran-3-ol;

[0114] (1) Method for protecting and deprotecting the hydroxyl group in cordycepin

[0115] Protection of the hydroxyl group at R1 of cordycepin 1a and deprotection method:

[0116]

[0117] Protection: Cordycepin 0.251 g (1 mmol), TBSCl 0.3618 g (2.4 mmol) and imidazole 0.3404 g (5 mmol) were added in 10 mL DMF and stirred at room temperature for 10 h. After the reaction was completed, the product was extracted with water and ethyl acetate. The organic phase was concentrated to obtain 1a 0.4607 g with a yield of 96.03%, and MS I-MS: 480.8 [M+H] + .

[0118] Deprotection: 10 ml (tetrabutylammonium fluoride: THF = 2:1) solvent was configured, 1a 0.48 g (1 mmol) was added, and stirred at room temperature for 5 h. After the reaction was completed, the product was extracted with water and ethyl acetate. The organic phase was concentrated and recrystallized to obtain cordycepin 0.246 g with a yield of 97.96%.

[0119] Protection of the hydroxyl group at R1 of cordycepin 1a and deprotection method:

[0120]

[0121] Protection: Cordycepin 0.251 g (1 mmol), TBSCl 0.3618 g (2.4 mmol) and imidazole 0.3404 g (5 mmol) were added in 10 mL DMF and stirred at room temperature for 10 h. After the reaction was completed, the product was extracted with water and ethyl acetate. The organic phase was concentrated to obtain 1a 0.4607 g with a yield of 96.03%, and MS I-MS: 480.8 [M+H] + .

[0122] Deprotection: 10 mL of 0.1 M methanolic hydrochloric acid solution was added to 1b 0.366 g (1 mmol), and stirred at room temperature for 10 h. After the reaction was completed, the product was extracted with 0.1 M aqueous sodium carbonate solution, adjusted to neutral pH, and extracted with ethyl acetate. The organic phase was concentrated by rotary evaporation or recrystallization to obtain cordycepin 0.215 g with a yield of 85.56%.

[0123] Protection of the hydroxyl group at R1 of cordycepin 1a and deprotection method:

[0124]

[0125] deprotection: configure 10 mL (tetra butyl ammonium fluoride: THF = 2: 1) solvent, add 1c 0.490 g (1 mmol), stir at room temperature for 5 h, after the reaction is completed, extract with water and ethyl acetate, concentrate the organic phase, recrystallize to obtain cordycepin 0.244 g, the yield is 97.01%. + .

[0126] deprotection: configure 10 mL (tetra butyl ammonium fluoride: THF = 2: 1) solvent, add 1c 0.490 g (1 mmol), stir at room temperature for 5 h, after the reaction is completed, extract with water and ethyl acetate, concentrate the organic phase, recrystallize to obtain cordycepin 0.244 g, the yield is 97.01%.

[0127] (2) Specific preparation method of compound 2-(6-amino-9H-purin-9-yl)-5-(2- hydroxyethoxy) methyl) tetrahydrofuran-3-ol

[0128] Take 1b 3.65 g (10 mmol) and compound 2-chloro-ethanol 0.81 g (10 mmol) in a round bottom flask, add 60 mL butanone to dissolve the reactants, and then add potassium carbonate 2.07 g (15 mmol) to the mixture. The reaction was carried out at 80°C for 8 h, and the reaction progress was monitored by TLC. After the reaction was completed, the reaction liquid was filtered, concentrated, and extracted with 60 mL water and 60 mL ethyl acetate twice. The organic phase was combined and concentrated under reduced pressure to obtain a viscous liquid. The compound 1 was obtained by deprotection and column chromatography, and the weight was 2.24 g, the yield was 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. MS I-MS: 296.3 [M+H] + .

[0129] Example 2: Preparation of compound (5-(6-amino-9H-purin-9-yl)-4- hydroxytetrahydrofuran-2-yl)methyl dihydrogen phosphate

[0130] According to the protection method in Example 1, compound 1b 3.65 g (10 mmol) was added to triethyl phosphite 150 mL, phosphorus oxychloride 4.62 g (30 mmol) under ice bath conditions. The reaction was reacted at 0°C for 2h, after the reaction was completed, 1000 mL of water was added under ice bath conditions to quench the reaction, dichloromethane was added for multiple extractions, the organic phase was combined, the solvent was removed by rotary evaporation to obtain a viscous liquid, and the viscous liquid obtained was purified by preparative liquid phase separation (C18 preparative column, Waters preparative liquid phase, mobile phase was 30% acetonitrile aqueous solution, flow rate was 2 mL / min), the sample peak segment effluent was concentrated and weighed to obtain the phosphated compound, a total of 3.02 g, yield 70%.

[0131] According to the deprotection scheme of 1b in Example 1, compound 2 was prepared, and the detection results of the prepared compound 2 were 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 CNMR (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] + .

[0132] Example 3: (((((5-(6-amino-9H-purin-9-yl)-4-hydroxytetrahydrofuran-2-yl)methoxy)methyl)phosphonic acid

[0133]

[0134] To a 500 mL reaction flask was added compound 1b 36.5 g (100 mmol), 150 mL DMF was added as solvent, and heated to dissolve. After rapid cooling to room temperature, 80% NaH 6.4 g (267 mmol) was added in portions with stirring, and maintained at room temperature for 15 min. The reaction solution was cooled to -10 °C under a low temperature reactor, and diethyl p-toluenesulfonyloxymethyl phosphate 45.1 g (140 mmol) was added dropwise. After the dropwise addition was completed, the reaction was stirred at this temperature for 1 h, and then gradually warmed to room temperature for 4 h. After the reaction was completed, glacial acetic acid was added dropwise to adjust the pH to neutral, the reaction solution was filtered, washed with dichloromethane, and the filtrate was extracted with water (60 mL) three times. The dichloromethane phases were combined, and the solvent was removed under reduced pressure. Toluene was added to the residue, and recrystallization was performed. The crystallization mother liquor was filtered, and the filter cake was dried at 50 °C under reduced pressure to obtain white powdery solid 3a. The weight was 26.87 g, and the yield was 52%, MS I-MS: 540.6 [M+23] + .

[0135] Compound 3a 5.17 g (10 mmol) was taken into a 100 mL reaction flask, 20 mL n-butyl cyanide was added, and trimethylchlorosilane 6.52 g (60 mmol) was added dropwise at room temperature. After the dropwise addition was completed, the reaction solution was warmed to reflux for 24 h. After the reaction was completed, the solvent was distilled off under reduced pressure, and the residue was concentrated to dryness. Water 20 mL was added to the residue, 2 M sodium hydroxide was added to adjust the pH to about 8, and then extracted with ethyl acetate (50 mL) three times. The aqueous phase was neutralized to pH = 3-4 with 1 M hydrochloric acid. Then, crystallization was performed by heating to 70-80 °C to obtain compound 3b. The weight was 3.40 g, and the yield was 74%, MS I-MS: 460.6 [M+H] + .

[0136] Compound 3b was treated according to the deprotection scheme of Example 1b to obtain compound 3, and the detection results of the prepared compound were 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). 13 C 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. MS I-MS: 346.3 [M+H] + .

[0137] Example 4: Preparation of compound (5-(6-amino-9H-purin-9-yl)-4-hydroxytetrahydrofuran-2-yl)methylphosphodiisobutyl ester

[0138]

[0139] 3.65 g (10 mmol) of compound 1b was added to a 500 mL reaction flask and dissolved in 100 mL of anhydrous DMF. 4.96 g (15 mmol) of diisobutyl phosphate (4-nitrophenyl) was also added. 1.17 g (10 mmol) of tert-butylmagnesium chloride 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, and then extracted 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%.

[0140] 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(400MHz,DMSO-d6)δ8.52(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 (100MHz, DMSO-d6) δ154.3,151.7,148.4,141.9,119.2,98.2,74.1,74.0,73.5,68.1,34.2,28.6,19.5.MSI-MS:466.7[M+Na] + .

[0141] Example 5: Preparation of compound: (((((((((5-(6-amino-9H-purin-9-yl)-4-hydroxytetrahydrofuran-2-yl)methoxy)methyl)phosphoryl)bis(oxy)bis(methylene)bis(2-methylpropionic acid)

[0142]

[0143] Compound 5 was prepared according to the procedure for the preparation of 3a in Example 3 and in conjunction with the deprotection procedure for lb in Example 1, wherein the equivalent of diethyl ((p-tolylsulfonyloxy)methyl)phosphonate was replaced with (((toluoyloxy)methyl)phosphonoxy))bis(methylene)bis(2-methylpropanoic acid) to give compound 5 in a total yield of 74%. The compound 5 prepared therein was tested and the results 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. MS-MS: 546.5 [M+H] + .

[0144] Example 6: Preparation of compound: methyl ((5-(6-amino-9H-purin-9-yl)-4- hydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)-D-valine, wherein the preparation was carried out as follows:

[0145]

[0146]

[0147] Methyl ((4-nitrophenoxy)(phenoxy)phosphoryl)-D-valine was prepared as follows:

[0148] Valine 1.17 g (10 mmol) was dissolved in 50 mL of dichloromethane. The solution was cooled to about 0 °C and phenyl dichlorophosphate 2.11 g (10 mmol) was added, followed by the slow dropwise addition of triethylamine 2.02 g (20 mmol), the reaction was allowed to warm to room temperature and stirred for 80 min. The reactant p-nitrophenol 1.39 g (10 mmol) was added, followed by the dropwise addition of triethylamine 2.02 g (20 mmol), and stirred at room temperature for 180 min. After the reaction was completed, it was washed with diethyl ether and filtered to remove the solid produced. The filtrate was concentrated on a rotary evaporator and the sample obtained was purified by column chromatography on silica gel (eluent: n-hexane / ethyl acetate (1 : 1)) to give compound 5, which was tested and the results are as follows: 1H 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. MS-MS: 395.3 [M+H] + .

[0149] Compound 6 was prepared following the procedure for the preparation of 4a in Example 4 and in combination with the deprotection procedure for lb in Example 1, wherein diisobutyl phosphate (4-nitrophenyl) was replaced as an equivalent of methyl ((4-nitrophenoxy)(phenoxy)phosphoryl)-D-valinate, with a yield of 68% for the preparation of compound 6. The results of the testing of the final prepared compound 6 were as follows: 1 H NMR (400 MHz, 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. MS-MS: 521.4 [M+H] + .

[0150] Example 7: Preparation of compound: isopropyl(((((((((5-(6-amino-9H-purin-9-yl)-4- hydroxytetrahydrofuran-2-yl)methoxy)methyl)(phenoxy)phosphoryl)proline ester, prepared as follows:

[0151]

[0152] The preparation process of isopropyl(phenoxy((toluoyloxy)methyl) phosphoryl) alaninate is as follows:

[0153] 1000 mL reaction bottle is added isopropyl alaninate 216.15 g (1.65 mol), phenol 155 g (1.65 mol), chloroform 240 mL, and stirred under ice salt bath condition, and phosphorus trichloride 138.8 g (1 mol) is added dropwise at about 5°C, and the reaction temperature is controlled at 5-10°C. After the dropwise addition of phosphorus trichloride is completed, continue to stir for 30 min, remove the ice salt bath, gradually warm to room temperature, continue to stir for 2 h, and then remove hydrogen chloride gas at 50°C under reduced pressure. The reaction is cooled to room temperature, 80 mL of saturated sodium bicarbonate aqueous solution is slowly added, then sodium bicarbonate solid powder is added to adjust the pH of the reaction solution to 7-8. The precipitated salt is filtered off, the filtrate is placed in a separatory funnel, and the chloroform layer is separated, the water layer is extracted with 100 mL of chloroform once, the organic phases are combined, and then washed with saturated sodium bicarbonate aqueous solution and water respectively, and then the chloroform layer is distilled under reduced pressure. The remaining material is the target product phenyl chlorophosphonate isopropyl alaninate, and the yield is 87%.

[0154] 1000 mL reaction bottle is added phenyl chlorophosphonate isopropyl alaninate 306 g (1 mol), paraformaldehyde 40.6 g (1.35 mol) and triethylamine 14 mL, and toluene 260 mL. The reaction is slowly warmed to 105°C under nitrogen protection, at this time the reaction is vigorous, and refluxed for 3 h. After the reaction is completed, the ice salt bath is cooled to 0°C, p-toluenesulfonyl chloride 169.5 g (0.9 mol) is added, toluene 220 mL is added, triethylamine 177 mL is added dropwise, and the reaction is stirred at 0°C for 2 h and then gradually warmed to room temperature and stirred for 12 h. After the reaction is completed, the filter cake is washed with toluene (50 mL x 3), the mother liquor is combined, and then washed with 5% sodium carbonate aqueous solution (200 mL x 2) and water (200 mL x 2) in sequence, and then the organic phase is separated. The organic phase is distilled under reduced pressure to obtain a light yellow oil, which is isopropyl(phenoxy((toluoyloxy)methyl) phosphoryl) alaninate, and the weight is 291 g, and the yield is 64%. The preparation of the product is 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). 13C 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. MS-MS: 456.5 [M+H] + .

[0155] Compound 7 was prepared according to the procedure for the preparation of 3a in Example 3 and in combination with the deprotection procedure for lb in Example 1, wherein diethyl p-toluenesulfonyloxymethylphosphonate was replaced as an equivalent of isopropyl (phenoxy((tolueneoxy)methyl)phosphoryl)alaninate, in a yield of 52% for the preparation of compound 7. The results of the testing of compound 7 prepared therein were as follows: 1 HNMR (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. MS-MS: 535.6 [M+H] + .

[0156] Example 8: 2-(-5-(6-amino-9H-purin-9-yl)-4-hydroxytetrahydrofuran-2-yl)methoxy)-3- hydroxypropyl valinate salt

[0157]

[0158] To compound 1b as raw material, 1b 3.65g (10mmol) and compound 2-chloro-3-hydroxypropyl valine 2.10g (10mmol) were taken in a round-bottom flask, 60mL butanone was added, the reactants were heated and dissolved, and 2.07g (15mmol) of potassium carbonate was added. The reaction was carried out at 80°C for 10h, and the reaction progress was monitored by TLC. After the reaction was completed, filtration was carried out, the reaction liquid was concentrated, 60mL water and 60mL ethyl acetate were added for extraction twice, the organic phase was combined, and rotary evaporation was carried out under reduced pressure to obtain a viscous liquid, which was subjected to column chromatography to obtain compound 8a, weighing 3.24g, with a yield of 52%, MS I-MS: 539.6 [M+H] + .

[0159] Compound 8a was used as a substrate, and compound 8 was prepared according to the deprotection method of 1b in Example 1, with a yield of 91%. The detection results of compound 8 prepared therein are as follows: 1 H NMR (400MHz, 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 (100MHz, 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. MS I-MS: 425.4 [M+H] + .

[0160] Example 9: Preparation of compound: (4-amino-6-(6-amino-9H-purin-9-yl)-2,2-dioxide-1,7-dioxa-2-thiaspiro[4.4]non-3-ene-8-yl) dihydrogen phosphate methyl ester, the preparation method is as follows:

[0161]

[0162] Reaction with compound 2 as starting material, compound 2 9.00 g (27.2 mmol) and CbzCl 4.88 g (27.2 mmol) were added to 38.5 mL of toluene and 38.5 mL of water, further K2CO3 4.70 g (34 mmol) was added, the mixture was stirred vigorously at a temperature below 25 °C. After stirring at room temperature for 3 h, triethylamine 0.275 g (2.72 mmol) and 5.78 g of sodium chloride were added sequentially, the mixture was stirred for another 30 min. The organic layer was separated and concentrated to obtain the desired product as an oil 9a, 11.38 g in yield of 90%, MS I-MS: 488.7 [M+Na] + .

[0163] Compound 9a 9.3 g (20 mmol) was stirred and dissolved in 60 mL of acetone at room temperature, to the solution Jones reagent 5.50 mL (2.2 M chromium trioxide, 12.1 mmol) solution was added dropwise at room temperature for 2 h. The resulting reaction mixture was stirred for another 1 h at room temperature and filtered, after concentration under reduced pressure an oily liquid was obtained. 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 column chromatography to obtain compound 9b, 3.98 g in yield of 43%, MS I-MS: 464.4 [M+H] + .

[0164] Trimethylsilyl cyanide 4 mL (30 mmol) and boron trifluoride diethyl ether 2.53 mL (20 mmol) were added to a solution of 9b 9.26 g (20 mmol) in 50 mL of dichloromethane. The mixture was stirred at room temperature for 2 hours and the solvent was evaporated to dryness. The residue thus obtained was dissolved in 100 mL of ethyl acetate, washed twice with 50 mL of brine and dried (Na2SO4). After filtration and evaporation of the solvent, purification by column chromatography (n-hexane / ethyl acetate, 1:2) gave white compound 9c, 7.45 g in yield of 76%, MS I-MS: 491.9 [M+H] + .

[0165] To 5 mL of dry dichloromethane was added 1.9 mL of Et3N (14 mmol) and to the solution was dissolved 1.08 g (2.2 mmol) of compound 9c. After the mixture was cooled to -30 °C, methanesulfonyl chloride 460 uL (6 mmol) was slowly added. The mixture was stirred at -20 °C for 1 h and at 0 °C for 1 h. After that, the volatiles were removed by concentration under reduced pressure and the residue was dissolved in 10 mL of ethyl acetate and then washed twice with 10 mL of water and 10 mL of brine, respectively. The organic phase was dried (Na2S04) and concentrated under reduced pressure and purified by column chromatography (n-hexane: ethyl acetate, 10:1) to obtain 9d as a white amorphous solid, 0.975 g, yield 78%, the results of the preparation of which were tested 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). 13 C NMR (100 MHz, 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. MS I-MS: 569.4 [M+H] + .

[0166] Cesium carbonate 490 mg (1.5 mmol) was added to a suspension of 9d 0.568 g (1 mmol) in 3 mL of dry acetonitrile and the mixture was stirred at room temperature for 2 h. The solvent was removed and the residue thus obtained was dissolved in 20 mL of ethyl acetate and washed twice with 10 mL of water and 10 mL of brine, respectively. The organic phase was dried (Na2S04) and filtered and then concentrated under reduced pressure. Purification by column chromatography (n-hexane / ethyl acetate, 3:1) gave 9e as a white solid, 4.49 g, yield 79%, MSI-MS: 569.4 [M+H] + .

[0167] Compound 5.68 g of 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 and then heated to reflux for 45 min. The mixture was filtered through celite and the filtrate was evaporated to dryness to obtain 4.12 g of compound 9, yield 95%. The results of the preparation of compound 9 were tested 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] + .

[0168] Example 10: Preparation of compound: N-(3-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-9H-purin-6-yl)octadecanamide

[0169]

[0170] Take cordycepin 2.51 g (10 mmol) in an ice bath, add anhydrous pyridine 40.00 mL, acetic anhydride 8.5 mL. Monitor the reaction by HPLC liquid phase, the reaction is about 5 h after the reaction is completed. Remove the solvent to obtain a viscous liquid 10a, weighing 2.68 g, yield 80%, MSI-MS: 358.6 [M+Na] + .

[0171] Take compound 10a 3.35 g (10 mmol) and octadecanoyl chloride 2.89 g (10 mmol) in an ice bath, add anhydrous pyridine 60 mL, the reaction is gradually warmed to 40°C for 10 h, monitor the reaction by TLC, after the reaction is completed, add water and ethyl acetate extraction, organic phase is back extracted, the organic phase is collected and rotary evaporated to obtain an oily liquid 10b, after column chromatography purification, 10b purified product is 5.40 g, yield is 90%, MSI-MS: 624.4 [M+Na] + .

[0172] Take compound 10b 6.01 g (10 mmol), dissolve with ammonia methanol solution 450 mL and react, stir at room temperature, detect the reaction by thin layer chromatography, stop the reaction after 4 h. Remove the solvent to obtain the target product 10, weighing 4.67 g, yield is 90%.

[0173] or by referring to the preparation method of 1a in Example 1, wherein the protected 1a is acylated by the second step here, and then deprotected 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). 13 C 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. (partial alkyl peaks overlap) MS I-MS: 540.7 [M+Na] + .

[0174] Example 11: N-(3-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-9H-purin-6-yl)cyclopentanecarboxamide

[0175] According to the method of Example 10, compound 11 is prepared from cordycepin by the steps of protection, acylation and deprotection, wherein octadecanoyl chloride is replaced by cyclopentanecarbonyl chloride, and the total yield is 72%. The detection results of the prepared compound 11 are as follows: 1 H NMR (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. MS I-MS: 348.2 [M+H] + .

[0176] Example 12: N-(3-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-9H-purin-6-yl)isonicotinamide

[0177] Compound 12 was prepared from the protected, acylated, deprotected steps of Cordycepin following the procedure of Example 10, wherein octadecanoyl chloride was replaced by pyridine-3-carbonyl chloride, in a total yield of 76%. The results of the tests performed on Compound 12 prepared therein are as follows: 1 HNMR (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] + .

[0178] Example 13: (5-(6-amino-2-fluoro-9H-purin-9-yl)-4-hydroxytetrahydrofuran-2-yl)methyl dihydrogen phosphate

[0179]

[0180] A nitration mixture was prepared by adding 2,2,2-trifluoroacetic anhydride 193 μL (1.39 mmol) to a solution of tetra-n-butylammonium nitrate 428 mg (1.40 mmol) in dry dichloromethane (15 mL) at 0 °C. After 45 min at 0 °C, the solution was slowly added to compound la 480 mg (1 mmol) in dry dichloromethane (15 mL). After 14 h at room temperature in the dark, the reaction mixture was poured into a cold mixture of H2O (50 mL), saturated NaHCO3(35 mL) and CH2Cl2:Et2O (1 :2, 30 mL) for extraction. Two extractions were performed with CH2Cl2:Et2O (1 :2, 30 mL). The organic extract was washed with brine, dried over anhydrous Na2SO4and oven dried under vacuum (keeping the temperature below 40 °C). The crude was purified by column chromatography eluting with CH2Cl2and then CH2Cl2:acetone (99:1 to 95:5) to give compound 13a, 273 mg, yield 52%, MSI-MS: 525.4 [M+H] + .

[0181] TBAF (1.3 eq, 600 μΐ^, 0.6 mmol) was added dropwise to a suspension of 13a 236 mg (0.45 mmol) in dry acetonitrile (15 mL) at 0°C over 1 min. The mixture was stirred for 20 min and the resulting solution was evaporated under vacuum without heating. The crude product was purified by column chromatography (CH2CI2: acetone 100:0 to 90:10) to give compound 13b, 78 mg, 35% yield, MS I-MS: 498.8 [M+H] + .

[0182] Compound 13 was prepared from compound 13b following the phosphorylation method of example 2 and the deprotection method of example 1 with a 65% yield. The results of the tests performed on compound 13 prepared 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. MS I-MS: 372.2 [M+Na] + .

[0183] Example 14: (5-(6-amino-2-mercapto-9H-purin-9-yl)-4-hydroxytetrahydrofuran-2- yl) dihydrogen phosphate

[0184]

[0185] Compound 1a 4.80 g (10 mmol) was added in acetic acid 60 mL and stirred at 40°C until dissolution. After the solution was cooled to room temperature, 30% hydrogen peroxide 5 mL (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, 3.37 g, 68% yield, MS I-MS: 497.4 [M+H] + .

[0186] To 14a 4.96 g (10 mmol) in 40 mL of 3M aqueous hydrochloric acid, heat to reflux until the solid is completely dissolved. Continue refluxing for 10 min, then cool to room temperature. Remove the solvent and add 20 mL of ethanol. Crystals precipitate, which are filtered and dried to give compound 14b, 2.92 g, 62% yield, MS I-MS: 487.5 [M+H] + .

[0187] Dissolve compound 14b 4.86 g (10 mmol) in 100 mL of water and add activated Raney Ni 1.0 g in portions with hydrogen gas bubbling. Stir the reaction mixture at 55 °C for 4 days. Stop the hydrogen gas bubbling and filter to remove the inorganic salts. Remove the solvent under reduced pressure and wash the crude product with ethanol and diethyl ether, respectively, and dry to give gray solid 14c, 3.30 g, 70% yield, MS I-MS: 495.8 [M+Na] + .

[0188] Mix methanol, pyridine and carbon disulfide in a volume ratio of 4:5:2 to give 50 mL of solution, and add 14c 4.72 g (10 mmol). Fit a balloon on the reflux condenser to prevent carbon disulfide from evaporating. Stir the reaction mixture at 40 °C for 4 days. Filter and heat the crude product in 5% aqueous sulfuric acid at reflux for 20 min. Filter while hot to remove the impurities. Cool to room temperature, filter and dry to give gray crystals 14d, 2.82 g, 55% yield. Prepare compound 14 from the prepared compound 14d according to the phosphorylation method of Example 2 and the deprotection method of Example 1, with a yield of 71%. The test 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 (100 MHz, DMSO-d6) δ 154.3, 152.1, 149.2, 139.8, 118.6, 99.2, 74.8, 74.3, 67.1, 35.1. MS I-MS: 364.3 [M+H] + .

[0189] Example 15: (5-(6-amino-9H-purin-9-yl)-3-fluoro-4-hydroxytetrahydrofuran-2-yl) dihydrogen phosphate

[0190]

[0191] In 10 mL of DMF, add adenosine 2.67 g (10 mmol), TBSCl 3.618 g (24 mmol) and pyridine 3.95 g (50 mmol), stir at room temperature for 10 h, after the reaction is completed, extract with water and ethyl acetate, concentrate the organic phase and purify by column chromatography to obtain 15a 2.33 g, with a yield of 47%, MS I-MS: 497.7 [M+H] + .

[0192] Stir 15a 4.96 g (10 mmol) and pyridine (1.5 mL) in dichloromethane (100 mL) under nitrogen protection and cool to -5 °C, drop trifluoromethanesulfonic anhydride 2.5 mL (15 mmol), continue to react at 0 °C for 2 h, TLC shows that the reaction is complete, pour into ice water (100 mL), stir and separate the layers, extract the aqueous phase with dichloromethane (100 mL). Wash the combined dichloromethane phases with saturated sodium chloride solution, dry over anhydrous sodium sulfate, filter, concentrate the filtrate under reduced pressure, add petroleum ether (40 mL) to the residue and recrystallize, filter and dry to obtain 15b, weighing 5.65 g, with a yield of 90%, the preparation results of which are as follows: 1 H NMR (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. MS I-MS: 628.8 [M+H] + .

[0193] Dissolve 15b 6.28 g (10 mmol) in ethyl acetate (40 mL) under nitrogen protection, add 37% hydrofluoric acid in triethylamine solution 10 mL (22 mmol), stir and warm to 70 °C for about 8 h. After the reaction is complete, cool to room temperature, wash with saturated sodium bicarbonate solution until neutral, then wash with saturated sodium chloride solution, dry over anhydrous sodium sulfate, filter, concentrate the filtrate under reduced pressure, recrystallize the residue with anhydrous methanol (30 mL) to obtain solid 15c, weighing 2.04 g, with a yield of 41%. Prepare compound 15 from the prepared compound 15c according to the phosphorylation method in Example 2 and the deprotection method in 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) δ 8.58 (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. MS-MS: 350.2 [M+H] + .

[0194] Example 16: Isopropyl((((((((5-(6-amino-9H-purin-9-yl)-4-cyanotetrahydrofuran-2- yl)methoxy)methyl)(phenoxy)phosphoryl)alaninate

[0195]

[0196] Compound 7 was prepared according to the method of Example 7, and used as a starting material to prepare compound 16. 5.34 g (10 mmol) of compound 7 was dissolved in 100 mL of dichloromethane solution, and 1.3 mL (1.50 g, 10 mmol) of trifluoromethylsulfonic acid was added. After stirring the reaction for 10 minutes, 2.7 mL of trimethylsilyl triflate (10 mmol) was slowly added dropwise to the solution, and the resulting mixture was stirred for 30 min in an environment of -40°C. Then 3.96 g (40 mmol) of trimethylsilyl cyanide was slowly added, and the mixture was stirred for 2 h. Thereafter, 3.5 mL of triethylamine was added dropwise, and the reaction mixture was allowed to warm 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. Then extraction was performed 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 the product compound 16 as a gray solid, which weighed 1.71 g, and the yield was 32%. The test results of the prepared compound 16 were 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). 13C 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. MS-MS: 544.5 [M+H] + .

[0197] Example 17: (((( (((((5-(6-amino-2-fluoro-9H-purin-9-yl)-4-hydroxytetrahydrofuran-2-yl)methoxy)methyl)phosphoryl)bis(oxy)bis(methylene)bis(2,2-dimethylpropanoic acid

[0198]

[0199] Compound 13b was prepared according to the method of Example 13, and compound 17 was prepared according to the method of Example 1 for the protection of lb using compound 13b as the starting material, according to the method of Example 3 for the preparation of 3a using compound 17a as the starting material, wherein diethyl p-tolylsulfonyloxymethylphosphonate was replaced by ((( (toluoxyl)methyl)phosphoryl)bis(oxy))bis(methylene)bis(2,2-dimethylpropanoic acid) in equivalent amount, lb was replaced by 17a in equivalent amount, and the overall yield was 61%. The detection results of compound 17 prepared in the process 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. MS-MS: 592.5 [M+H] + .

[0200] Example 18: (((( ((((5-(6-amino-9H-purin-9-yl)-3-fluoro-4-hydroxytetrahydrofuran-2-yl)methoxy)methyl)phosphoryl)bis(oxy))bis(methylene)bis(2,2-dimethylpropanoic acid)

[0201]

[0202] Compound 18 was prepared according to the procedure of Example 15, and the deprotection procedure of 1b in Example 1, using compound 18a as starting material, according to the procedure of Example 3 to prepare 3a, using diethyl p-tolylsulfonyloxymethylphosphonate in equivalent amount instead of (((toluoyloxy)methyl)phosphonoxy)bis(oxy)bis(methylene)bis(2,2-dimethylpropanoic acid), and 1b in equivalent amount instead of 18a, with an overall yield of 34%. Compound 18 prepared therein was characterized as follows: 1 H NMR (400 MHz, 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 CNMR (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. MS-MS: 592.4 [M+H] + .

[0203] Example 19: ((((((5-(6-amino-2-mercapto-9H-purin-9-yl)-4-hydroxytetrahydrofuran-2- yl)methoxy)methyl)phosphonoxy)bis(oxy)bis(methylene)bis(2,2-dimethylpropanoic acid)

[0204]

[0205] Compound 19 was prepared according to the procedure of Example 14, and the deprotection procedure of 1b in Example 1, using compound 19a as starting material, according to the procedure of Example 3 to prepare 3a, using diethyl p-tolylsulfonyloxymethylphosphonate in equivalent amount instead of (((toluoyloxy)methyl)phosphonoxy)bis(oxy)bis(methylene)bis(2,2-dimethylpropanoic acid), and 1b in equivalent amount instead of 19a, with an overall yield of 18%. Compound 19 prepared therein was characterized as follows: 1H 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 C NMR (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. MS-MS: 606.6 [M+H] + .

[0206] Example 20: Isopropyl((((5-(6-amino-2-fluoro-9H-purin-9-yl)-4- hydroxytetrahydrofuran-2-yl)methoxy)methyl)(phenoxy)phosphoryl)alaninate

[0207]

[0208] Compound 17a was prepared according to the procedure of Example 13 and Example 17, and compound 20 was prepared according to the procedure of Example 7, wherein diethyl p-tolylsulfonyloxymethylphosphonate was replaced with an equivalent of isopropyl(phenoxy((toluoyloxy)methyl)phosphoryl)alaninate, 1b was replaced with 17a, and the overall yield was 67%. The results of the testing of compound 20 prepared therein 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. MS-MS: 553.5 [M+H] + .

[0209] Example 21: N-(2-fluoro-3-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-9H-purin-6- yl)octadecanamide

[0210]

[0211] Compound 13b was prepared according to the method of Example 13, and compound 21a was prepared from 13b according to the method of Example 10 for the preparation of 10b from 10a, and compound 21 was prepared from 21a according to the deprotection method of Example 1 for the preparation of 1a, with an overall yield of 74%. The test results of compound 21 prepared are as follows: 1 H 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 C 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. (Partial overlap of alkyl peaks) MS I-MS: 536.6 [M+H] + .

[0212] Example 22: N-(4-chloro-3-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-9H-purin-6- yl)octadecanamide

[0213]

[0214] Compound 15b was prepared according to the method of Example 15 from adenosine, and 6.28 g (10 mmol) of 15b was dissolved in ethyl acetate (40 mL) under nitrogen protection, 10 mL (22 mmol) of 37% hydrochloric acid solution in triethylamine was added, and the mixture was stirred and heated to 70°C for about 8 h. TLC showed that the reaction was complete, and then the mixture was cooled to room temperature, washed with saturated sodium bicarbonate solution until neutral, and then washed with saturated sodium chloride solution. After drying over anhydrous sodium sulfate, the mixture was filtered, and the filtrate was concentrated under reduced pressure. The residue was recrystallized from anhydrous methanol (30 mL) to obtain solid 22a, which weighed 1.95 g with a yield of 38%, and MS I-MS: 515.2 [M+H] + .

[0215] Compound 22 was prepared from 22a following the procedure of Example 10 for the preparation of 10b from 10a in combination with the deprotection procedure of Example 1 for 1a in overall yield of 79%. The compound 22 prepared was characterized by the following results: 1 H 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 C NMR (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. (Partial overlap of alkyl peaks) MS I-MS: 553.2 [M+H] + .

[0216] Example 23: (((((4-Hydroxy-5-(6-(isonicotinamido)-9H-purin-9-yl)tetrahydrofuran-2- yl)methoxy)methyl)oxy)bis(oxy))bis(methylene)bis(2,2-dimethylpropanoic acid)

[0217]

[0218] Compound 23 was prepared from compound 12 following the protection procedure of Example 1 for the preparation of 1b from 1a in combination with the procedure of Example 17 for the preparation of 17 from 17a, replacing 17a with 23a in equivalent amount in the reaction. The overall yield of the reaction was 81%. The compound 23 prepared was characterized by the following results: 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) δ 175.8, 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. MS-MS: 679.6 [M+H] + .

[0219] Example 24: Isopropyl((((4-hydroxy-5-(6-(isonicotinamido)-9H-purin-9-yl)tetrahydrofuran-2- yl)methoxy)methyl)(phenoxy)phosphoryl)alaninate

[0220]

[0221] Compound 23a was prepared according to the procedure in Example 23, and compound 24 was prepared according to the procedure in Example 7 using 23a instead of 1b in equal equivalent, with an overall yield of 85%. The detection results of compound 24 prepared 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. MS-MS: 640.6 [M+H] + .

[0222] Example 25: (5-(6-amino-9H-purin-9-yl)-3-azido-4-hydroxytetrahydrofuran-2-yl) dimethyl phosphate

[0223]

[0224] To a round bottom flask under argon atmosphere was added adenosine 2.67 g (10.0 mmol) and PPh3 5.78 g (22 mmol). Then DMF 18 mL was added and the resulting mixture was stirred at room temperature until the solid was completely dissolved. The solution was cooled to 10 °C and diisopropyl azodicarboxylate (DIAD) 4.4 mL (2.2 mmol) was added dropwise rapidly. The reaction mixture was allowed to reach room temperature. After 2 hours of stirring at 40 °C, 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 was obtained as a white solid, 1.84 g, yield 74%, the results of the preparation of compound 25a were 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] + .

[0225] To a round bottom flask under argon atmosphere was added adenosine 2.67 g (10.0 mmol) and PPh3 5.78 g (22 mmol). Then DMF 18 mL was added and the resulting mixture was stirred at room temperature until the solid was completely dissolved. The solution was cooled to 10 °C and diisopropyl azodicarboxylate (DIAD) 4.4 mL (2.2 mmol) was added dropwise rapidly. The reaction mixture was allowed to reach room temperature. After 2 hours of stirring at 40 °C, 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 was obtained as a white solid, 1.84 g, yield 74%, the results of the preparation of compound 25a were as follows: + .

[0226] To a round bottom flask under argon atmosphere was added adenosine 2.67 g (10.0 mmol) and PPh3 5.78 g (22 mmol). Then DMF 18 mL was added and the resulting mixture was stirred at room temperature until the solid was completely dissolved. The solution was cooled to 10 °C and diisopropyl azodicarboxylate (DIAD) 4.4 mL (2.2 mmol) was added dropwise rapidly. The reaction mixture was allowed to reach room temperature. After 2 hours of stirring at 40 °C, 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 was obtained as a white solid, 1.84 g, yield 74%, the results of the preparation of compound 25a were 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. MS-MS: 373.2 [M+H] + .

[0227] Example 26: (((((5-(6-amino-9H-purin-9-yl)-3-azido-4-hydroxytetrahydrofuran-2- yl)methoxy)methyl)phosphoryl)bis(oxy))bis(methylene)bis(2,2-dimethylpropanoic acid)

[0228]

[0229] Compound 25c was prepared according to the preparation method in Example 25, and compound 26a was prepared according to the preparation method of compound 17a in Example 17 using 25c as raw material, wherein 25c was used to replace 17a in equivalent amount, and compound 26 was prepared by the deprotection method of 1b in Example 1, with a total yield of 54%. The detection results of compound 26 prepared 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. MS-MS: 615.6 [M+H] + .

[0230] Example 27: isopropyl((((5-(6-amino-9H-purin-9-yl)-3-azido-4-hydroxytetrahydrofuran-2- yl)methoxy)methyl)(phenoxy)phosphoryl)alaninate

[0231]

[0232] Compound 25c was prepared according to the preparation method in Example 25. Compound 27 was prepared according to the preparation method in Example 7, using 25c as the raw material, and replacing 1b with 25c in equivalent amount, with a total 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. MS-MS: 576.5 [M+H] + .

[0233] Example 28: Isopropyl((((3-azido-4-hydroxy-5-(6-(isonicotinamido)-9H-purin-9-yl)tetrahydrofuran-2-yl)methoxy)methyl)(phenoxy)phosphoryl)alaninate

[0234]

[0235] Compound 28a was prepared according to the protection method of 1a or 1b in Example 1, using the prepared compound 27 as the raw material. Compound 28 was prepared according to the preparation method in Example 12, using compound 28a as the raw material, and replacing cordycepin or 1b with compound 28a in equivalent amount, with a total 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). 13C NMR (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] + .

[0236] Example 29: 2-(6-amino-9H-purin-9-yl)-5-(hydroxymethyl)tetrahydrofuran-3-yl valinate salt

[0237]

[0238] Compound 1c was prepared according to the protection method described in example 1c. Compound 1c 9.95 g (27.2 mmol) and CbzCl 4.88 g (27.2 mmol) were added to 38.5 mL of toluene and 38.5 mL of water, and K2CO3 4.70 g (34 mmol) was added, and the mixture was stirred vigorously at a temperature below 25 °C. After stirring at room temperature for 3 h, triethylamine 0.275 g (2.72 mmol) 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 product 29a as an oil, 13.1 g, in a yield of 90%, MSI-MS: 522.6 [M+Na] + .

[0239] Compound 29a 4.99 g (10 mmol) and valine acid chloride 1.36 g (10 mmol) were added to 60 mL of anhydrous pyridine in an ice bath environment, and the reaction was gradually warmed to 40 °C for 10 h. The reaction was monitored by TLC, and after the reaction was completed, water was added and extracted with ethyl acetate, the organic phase was back-extracted, and the organic phase was collected and rotary evaporated to obtain 10b as an oily liquid, which was purified by column chromatography to obtain 29b as a purified product, 5.03 g, in a yield of 84%, MSI-MS: 625.5 [M+Na] + .

[0240] 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 and then heated to reflux for 45 min. The mixture was filtered through celite and the filtrate was evaporated to dryness to give 4.41 g of compound 29c in 95% yield. Compound 29 was prepared by deprotection of 29c following the procedure of lc in Example 1 in 90% yield. The compound 29 prepared therein was characterized 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. MS-MS: 351.3 [M+H] + .

[0241] Example 30: 2-(6-amino-9H-purin-9-yl)-5-(phosphonooxy)methyl)tetrahydrofuran-3- yl valinate salt

[0242]

[0243] Compound 30 was prepared by following the procedure of Example 2 using compound 29 as starting material, wherein lb was replaced by 29 in equivalent amount, to give compound 30 in 84% yield. The compound 30 prepared therein was characterized 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). 13C 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. MS-MS: 558.4 [M+Na] + .

[0244] Example 31: 2-(6-(Isonicotinamido)-9H-purin-9-yl)-5-(phosphonomethyl)tetrahydrofuran-3-yl valinate

[0245]

[0246] Using compound 30 as raw material, according to the preparation method of example 12, replace compound 1a with compound 30 in equivalent amount to prepare compound 31, the yield is 91%. The detection results of compound 31 prepared therein 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. MS-MS: 558.4 [M+Na] + .

[0247] Example 32 (((5-(6-(Isonicotinamido)-9H-purin-9-yl)-4-(pentyloxy)tetrahydrofuran-2-yl)methoxy)methyl) phosphonic acid

[0248]

[0249] Using compound 29 as raw material, according to the preparation method in example 3, replace compound 1b with compound 29 in equivalent amount to prepare compound 32a, the yield is 77%. Using compound 32a as substrate, according to the preparation method of example 12, replace compound 1a with compound 32a in equivalent amount to prepare compound 32, the yield is 90%. The detection results of compound 32 prepared therein 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, 1H), 4.81 (s, 2H), 4.24 (m, 1H), 3.95 (m, 1H), 3.73 (d, 2H), 3.60-3.35 (m, 2H), 2.37 (m, 1H), 2.14 (m, 1H), 1.89 (m, 1H), 0.96 (d, 6H). 13 C NMR (100 MHz, DMSO-d6) δ 171.5, 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] + .

[0250] Example 33: methyl (((2-(6-amino-9H-purin-9-yl)-5-(hydroxymethyl)tetrahydrofuran-3-yl)oxy)(phenoxy)phosphoryl)alaninate

[0251]

[0252] Using compound 1c as starting material, compound 33a was prepared according to the preparation method in Example 6, replacing compound 1b with compound 1c in equivalent amount, with a yield of 62%. Compound 33 was prepared by reacting 33a according to the deprotection scheme of 1c in Example 1, with a yield of 90%. The detection results of compound 33 prepared are as follows: 1 H NMR (400 MHz, DMSO-d6) δ 8.58 (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). 13 C 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] + .

[0253] Example 34: methyl (((5-(hydroxymethyl)-2-(6-(isonicotinamido)-9H-purin-9-yl)tetrahydrofuran-3-yl)oxy)(phenoxy)phosphoryl)alaninate

[0254]

[0255] Compound 33a was prepared according to the procedure of Example 33. Compound 34 was prepared according to the procedure of Example 12, using compound 33a instead of 1a in an equivalent amount, with an overall yield of 83%. The detection results of compound 34 prepared 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] + .

[0256] Example 35: (5-(2-((E)-2-bromovinyl)-6-(isonicotinamido)-9H-purin-9-yl)-4- hydroxytetrahydrofuran-2-yl) dihydrogen phosphate

[0257]

[0258] Verdinsin 2.51 g (10 mmol) was dissolved in dilute nitric acid, heated to 110°C, and 1.26 g (5 mmol) of elemental iodine was added and reacted for 4 h, and the reaction was monitored by TLC method. After the reaction was completed, the organic phase solution was extracted with petroleum ether, and the water phase was collected. The water phase was extracted with deionized water, and the water phase was combined and rotary evaporated to obtain compound 35a weighing 2.72 g with a yield of 72%, MSI-MS: 378.2 [M+H] + .

[0259] Take 100 mL of anhydrous 1,4-dioxane in the reactor, argon protection, heated to 70℃, sequentially add palladium acetate 0.11 g (0.5 mmol), triphenylphosphine 0.26 g (1 mmol), triethylamine 1.8 mL, stirring for about 30 min. Add compound 35a 3.77 g (10 mmol), methyl acrylate 2.59 g (30 mmol), the reaction is carried out at 80℃ for 2 h, then the reaction is completed, filtered, collected the filtrate, stand, and then filter, to obtain white flocculent solid, compound 35b, weighing 2.41 g, yield 72.7%, MS I-MS: 336.4 [M+H] + .

[0260] Take compound 35b 1.00 g dropwise into 12 mL of sodium hydroxide solution (2 mol / L), stirring at room temperature for 3 h, then dropwise add concentrated hydrochloric acid under ice bath conditions until pH is 1, at this time a large amount of white precipitate is generated. Filter to obtain white solid 35c, weighing 0.88 g, yield 92%, MS I-MS: 322.6 [M+H] + .

[0261] Take compound 35c 3.21 g (10 mmol) and add 120 mL of water and heat to 100℃ and stir, then add anhydrous potassium carbonate 2.07 g (15 mmol), take NBS 2.67 g (15 mmol) and dissolve in 22.5 mL of acetone and 22.5 mL of water, and dropwise add the mixture to the flask, dropwise add for 135 min. Stir for 3 h, stop the reaction, remove half of the solvent, and place in the refrigerator overnight. A large amount of needle-shaped brown crystals are precipitated, and are filtered to obtain compound 35d, weighing 1.78 g, yield 50%, MS I-MS: 357.3 [M+H] + .

[0262] Take compound 35d 1.78 g (5 mmol) and add anhydrous pyridine 40.00 mL under ice bath conditions, and add acetic anhydride 8.5 mL. Monitor the reaction by HPLC, and the reaction is completed after about 5 h. Remove the solvent to obtain viscous liquid 35e, weighing 1.76 g, yield 80%. Compound 35e is used to prepare compound 35f by the method of Example 16, yield 95%; compound 35f is used to prepare compound 35 by the phosphorylation method of Example 2 and the deprotection method of Example 1, yield 72%, and the detection results of compound 35 prepared are as follows: 1H 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] + .

[0263] Example 36: 3-((5-(6-amino-2-fluoro-9H-purin-9-yl)-4-hydroxytetrahydrofuran-2- yl)methoxy)-5-(hydroxymethyl)cyclopentane-1,2-diol

[0264]

[0265] Compound 17a was prepared according to the method of Example 17, and 17a was used as a starting material to prepare according to the method of Example 8, wherein 2-chloro-3-hydroxypropyl valine was replaced by 3-chloro-5-(hydroxymethyl)cyclopentane-1,2-diol in equivalent amount, to prepare compound 36a, and after deprotection, compound 36 was prepared with a total yield of 41%. The test results of the prepared compound 36 are as follows: 1 H 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] + .

[0266] Example 37: Preparation of compound: ((((((5-(6-amino-9H-purin-9-yl)-2-azido-4- hydroxytetrahydrofuran-2-yl)methoxy)methyl)phosphoryl)bis(oxy)bis(methylene)bis(2,2- dimethylpropanoic acid), which was prepared as follows:

[0267]

[0268] Compound 1b 3.65 g (10 mmol) and m-chloroperbenzoic acid 3.44 g (20 mmol) were added to a reaction flask, dissolved in anhydrous dichloromethane 100 mL, and phosphorus pentoxide 2.13 g (15 mmol) was added to the reaction flask in an ice bath environment, and the reaction was gradually warmed to 40 °C for 3 h. After the reaction was completed, it was filtered, quenched with 100 mL of saturated sodium bicarbonate, washed twice with 50 mL of dichloromethane, and back-extracted twice with 60 mL of saturated sodium bicarbonate. The organic phase was combined and concentrated under reduced pressure, and compound 37a was obtained by column chromatography, weighing 1.49 g, with a yield of 41%. The prepared compound was detected 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). 13 CNMR (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] + .

[0269] Compound 37a 3.63 g (10 mmol) and sodium azide 2.6 g (40 mmol) were added to a flask, and 20 mL of DMF was added as a solvent. The reaction was carried out at 120 °C for 16 h, and the reaction progress was monitored by TLC plate. After the reaction was completed, it was quenched with water, and the aqueous phase was extracted with ethyl acetate (180 mL) three times. The organic phase was 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 according to the method of Example 17 for the preparation of compound 17a using 37b as the starting material, replacing 17a with an equivalent amount of 37b, with a total yield of 50%. The detection results of the prepared compound 37 are as follows: 1H 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. MS-MS: 637.6 [M+Na] + .

[0270] Example 38: Preparation of compound: (5-(6-amino-2-((E)-2-bromovinyl)-9H-purin-9-yl)- 4-hydroxytetrahydrofuran-2-yl) dihydrogen phosphate, the reaction process is as follows:

[0271]

[0272] Compound 38 was prepared from compound 2 by using the method of Example 35 with compound 2 as raw material, the overall yield was 24%, and the test results of prepared compound 38 were as follows: 1 H 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. MS-MS: 458.3 [M+N] + .

[0273] Example 39: Preparation of compound: (5-(6-amino-8-(isopropylamino)-9H-purin-9-yl)-4- hydroxytetrahydrofuran-2-yl) dihydrogen phosphate, the preparation process is as follows:

[0274]

[0275] Compound 39 was prepared using compound 1b as starting material, compound 1b 3.65g (10mmol) and 2-propylamine 1.18g (20mmol) were taken in a reaction flask, to this 60 mL of dioxane was added as solvent, the reaction was refluxed at 80 °C for 20 h, the progress of the reaction was monitored by TLC. After completion of the reaction, the reaction mixture was concentrated, to this 50 mL of water and 50 mL of ethyl acetate was added and extracted twice with ethyl acetate, the organic layers were combined and concentrated under reduced pressure to get an oily liquid, which was purified by column chromatography to get compound 39a as a light yellow oily liquid, 1.44g in yield 34%.

[0276] Compound 39 was prepared using compound 39a as starting material, which was prepared as per the procedure given in example 2, in which 1b was replaced with 39a in equimolar quantity, the yield was 91%. The compound 39 prepared was found to have the following results: 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). 13 C 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. MS-MS: 389.3 [M+H] + .

[0277] Example 40: Preparation of compound: 3-((5-(6-amino-9H-purin-9-yl)-4- hydroxytetrahydrofuran-2-yl)methoxy)-5-(hydroxymethyl)cyclopentane-1,2-diol, which was prepared as follows:

[0278]

[0279] Compound 40 was prepared using compound 1b as starting material, which was prepared as per the procedure given in example 8, in which 2-chloro-3- hydroxypropyl valine was replaced with 3-chloro-5-(hydroxymethyl)cyclopentane-1,2- diol in equimolar quantity, the yield was 46%. The compound 40 prepared was found to have the following results: 1H 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. MS-MS: 382.3 [M+H] + .

[0280] Example 41: Anti-tumor effect of cordycepin modified derivatives on liver cancer

[0281] Establishment of mouse transplanted liver cancer model and drug evaluation. 2.5 x 10 5 Hep-1-6 liver cancer cells were suspended in 100 μL of PBS and inoculated into the left lower limb of the lateral thigh of C57BL / 6j nude mice; after 1 week, when the transplanted tumor reached about 100 mm 3 in size, the mice were randomly divided into twelve groups, with 10 mice in each group, namely, a blank group, a cordycepin control group, and drug groups (compounds 2, 3, 5, 7, 15, 24, 27, 31, 35, 40); the blank group was given corn syrup and DSO as a solvent control every other day, and the cordycepin control group and the drug groups were given the drug compound (500 ug / time / mouse) every other day; continuous observation was conducted for 14 days; the changes in the size of liver cancer tumors in the two groups of mice were observed, the mice were sacrificed after the experiment, blood and corresponding tissue samples were collected, the tumor was immediately photographed and weighed, part of the tumor tissue was fixed in a formaldehyde solution for further detection, and the transplanted tumor size was calculated every two days by measuring the two perpendicular diameters (length and width) of the transplanted tumor with a caliper, and the results of the tumor volume change experiment and the results of the tumors treated in the blank group, the cordycepin control group, and the drug groups within 14 days are shown in Figure 1 . The volume was calculated according to the formula: tumor volume (mm 3 ) = 1 / 2 x (length x width) 2 The experimental results showed that the drug groups alone could significantly inhibit the growth of mouse tumors, and the effect was better than that of the cordycepin control group, among which compound 24 had excellent anti-liver cancer tumor effect (900 mm 3 was reduced to <100 mm 3(P<0.001), indicating that the compound drug group has the efficacy of killing liver cancer tumor cells or activating tumor immunity.

[0282] Example 42: Anti-tumor experimental effect of cordycepin modified derivatives on small cell lung cancer

[0283] The MTT method was used to analyze the anti-tumor dose-effect relationship curves of cordycepin and compounds 1, 2, 4, 5, 7, 8, 10, 12, 16, 18, 21, 23, 24, 35, 38 and cordycepin in small cell lung cancer cell lines H1048, H446 and H69. The results of calculating the half inhibitory concentration (IC 50 ) are shown in the following table. The results of in vitro anti-tumor experiments show that compared with cordycepin, the effective concentration of modified cordycepin derivatives on tumor cells is reduced, among which compounds 35 and 38 have strong in vitro killing effect on the three cells, but their toxicity is also large, followed by compounds 18 and 21. Among them, the IC 50 of cordycepin in small cell lung cancer cell lines H1048 and H446 is higher than 100 μM, and the effective concentration is high, so a large amount of drug may be needed in actual application.

[0284] Table 1: IC 50 (μM) of compounds in three small cell lung cancer cells

[0285]

[0286]

[0287] Example 43: Determination of maximum tolerated dose of cordycepin modified derivatives in zebrafish in vivo experiment

[0288] Randomly selected 240 wild type AB strain zebrafish of 3 dpf in 8-hole plate, 30 in each hole, and the fish water capacity of each hole is 3 mL. Cordycepin is diluted according to the maximum lethal dose, dissolved in DMSO and added to each hole according to the concentration gradient (100 times dilution of the maximum lethal dose, 8 concentration gradient values are taken in the middle for testing). At the same time, normal control group, solvent control group G (5% glucose) and solvent control group D (DMSO) are set. During the experiment, the zebrafish in each group is cultured at 35°C environment. After the zebrafish treated with test product is 5 dpf, the mortality and developmental malformation of zebrafish under different doses are investigated respectively. The maximum tolerance dose (MTD) of the test product to normal zebrafish is determined when the survival rate is > 90% and the teratogenic rate is < 20%. Cordycepin is selected as the control group, and other compounds 2, 5, 6, 7, 9, 11, 13, 20, 24, 25, 28, 35, 36, 37 and 39 are tested for MTD according to the above method. The test results are shown in Table 2.

[0289] Table 2: Maximum tolerance dose (MTD) of natural products and positive control drugs in zebrafish model

[0290] Compound No. MTD Compound No. MTD Cordycepin 995 μM 20 120 nM 2 845 μM 24 5 μM 5 525 μM 25 12 μM 6 384 μM 28 5 μM 7 62 μM 35 25 nM 9 14 μM 36 20 nM 11 75 μM 37 170 nM 13 50 nM 39 28 μM

[0291] Example 44: Inhibitory effect of cordycepin modified derivatives on small cell lung cancer in zebrafish in vivo

[0292] Three kinds of H69, H446 and H1048 cell zebrafish small cell lung cancer xenograft models are established respectively: 1×10 6 The H69, H446 and H1048 cell suspensions with a concentration of 1×10

[0293] Test the effect of cordycepin and the prepared compounds 2, 5, 6, 7, 9, 11, 13, 20, 24, 25, 28, 35, 36, 37, 39 on the proliferation of small cell lung cancer in zebrafish: at 3dpf, select zebrafish with consistent tumor transplants under a microscope, and randomly distribute them into 6-well plates, with 30 fish per well. At 3dpf, dilute the above compounds according to the MTD dose, dissolve them in DMSO, and add them to each well. At the same time, set up normal control group, model control group and solvent control group (DMSO), with 3mL of fish water per well. Continue to cultivate the zebrafish in each experimental group at 35°C to 5dpf, then randomly select 10 zebrafish from each experimental group, observe, photograph and save the pictures under a fluorescence microscope, analyze the images using Nikon NIS-Elements l3.10 advanced image processing software, calculate the fluorescence intensity (S) of the zebrafish transplanted tumors, and the inhibition results are shown in Table 3 below. The inhibition results of the control group, the cordycepin group, compound 24 and compound 35 group on the H446 small cell lung cancer transplanted zebrafish model are shown in Table 3 below. Figure 2 The growth inhibition effect of Xian'ailing injection and paclitaxel alone on zebrafish-human ovarian cancer sensitive strain was calculated by the total fluorescence intensity, and the formula is as follows:

[0294] Tumor growth inhibition (%) = S (model control group) - S (drug group) / S (model control group) x 100%

[0295] Table 3: Effect of each compound on the proliferation of three cell lines in zebrafish model

[0296]

[0297] Example 45: Inhibition experiment effect of cordycepin modified derivatives on colon cancer

[0298] Establishment of mouse transplanted colon cancer model and drug evaluation. 2.5x10 6 MC-38 colon cancer cells were suspended in 100μL of PBS and inoculated into the left lower limb of C57BL / 6j nude mice; after 1 week, the transplanted tumors reached about 100mm 3Size, mice were randomly divided into seven groups, 8 in each group, respectively, control group IgG, compound 16 drug + IgG group, PD-1 antibody treatment group, CTLA4 monoclonal antibody treatment group, compound 16 drug + PD-1 combination therapy group, compound 16 drug + CTLA4 monoclonal antibody combination therapy group. Control group IgG: intragastric administration of immunoglobulin G (IgG) (500 ug / time / each), 28 days continuously; Cordycepin + IgG: intragastric administration of immunoglobulin G (IgG) and cordycepin (both are 500 ug / time / each), 28 days continuously; Compound 16 drug + IgG group: intragastric administration of immunoglobulin G (IgG) and compound 16 drug (both are 500 ug / time / each), 28 days continuously; Antibody treatment group: intragastric administration of antibody (500 ug / time / each), 28 days continuously; Compound 16 drug + antibody combination therapy group: intragastric administration of immunoglobulin compound 16 drug (500 ug / time / each), intragastric administration of antibody (500 ug / time / each) every 4 days, 28 days continuously. After the end of the experiment, the mice were sacrificed, the blood and the corresponding tissue specimens were collected, the tumor body was immediately photographed and weighed, part of the tumor tissue was fixed in formalin solution, and further detection was carried out, the two perpendicular diameters (length and width) of the transplanted tumor were measured every two days with a caliper, the size of the transplanted tumor was calculated, and the volume was calculated according to the formula: tumor volume (mm 3 ) = 1 / 2 × (length × width) 2 , the size change of colon cancer tumor in the seven groups of mice was observed, and the results are shown in Figure 3 . After 28 days, the experimental results show that cordycepin has an anti-colon cancer proliferation effect (size 1300mm 3 reduced to 730mm 3 ), compound 16 alone has a significant anti-tumor growth effect (size 1300mm 3 reduced to 480mm 3 ) (P<0.01), immune checkpoint inhibitors PD-1 and CTLA4 alone also have a significant anti-tumor growth effect (size 1300mm 3 reduced to about 448mm 3 ) (P<0.01), and the use of compound 16 and immune checkpoint inhibitors PD-1 and CTLA4 at the same time can greatly improve the anti-tumor effect (size 1300mm 3 reduced to about 260mm 3 ).

[0299] Example 46: Inhibition effect of cordycepin modified derivative on melanoma

[0300] Establishment of mouse transplanted melanoma model and drug evaluation: 2.5 × 10 5A B16-F10 melanoma cell is inoculated on the left lower limb of the lateral thigh of a C57BL / 6j nude mouse; after 1 week, the transplanted tumor reaches about 100mm 3 When the size is appropriate, the mice are randomly divided into ten groups, 10 mice in each group, which are blank control group, cordycepin group, compound 13 group, compound 14 group, compound 17 group, compound 19 group, compound 22 group, compound 26 group, compound 29 group, and compound 34 group. The blank control group is given corn syrup and DMSO as a solvent every day for 24 consecutive days; the compound group is given the corresponding compound (500ug / time / mouse) every other day for 24 consecutive days; the size change of melanoma in the ten groups of mice is observed every 4 days in the early stage and every two days in the later stage, and the mice are sacrificed after the experiment, blood and corresponding tissue specimens are collected, the tumor is immediately photographed and weighed, part of the tumor tissue is fixed in formalin solution for further detection, and the two perpendicular diameters (length and width) of the transplanted tumor are measured every two days with a caliper, the size of the transplanted tumor is calculated, and the volume is calculated according to the formula: tumor volume (mm 3 ) = 1 / 2 × (length × width) 2 , and the experimental results are shown in Figure 4 . The experimental results show that cordycepin itself can inhibit the proliferation of melanoma (1760mm 3 is reduced to 600mm 3 , and cordycepin modified derivatives are better than cordycepin (1760mm 3 is reduced to <560mm 3 , and compounds 19 and 34 can significantly inhibit the growth of mouse tumors (1760mm 3 is reduced to about 240mm 3 ) (P<0.001), which indicates that the synthesized compounds have the effect of killing tumor cells.

[0301] Example 46-1, compound 27 is used to inhibit melanoma in the above manner, which has the same level of inhibitory effect as compound 26.

[0302] Mouse ovarian cancer model establishment and drug evaluation. 2.5×10 6 ID8 ovarian cancer cells are suspended in 100μL of PBS and inoculated on the left lower limb of the lateral thigh of a C57BL / 6j nude mouse; about one week later, the transplanted tumor reaches about 100mm 3When the size is appropriate, the mice are randomly divided into five groups, 10 mice in each group, namely the control group, the cordycepin group, the compound 24 drug group, the PD-1+TIM3 antibody treatment group, and the compound 24 drug+PD-1+TIM3 antibody treatment group. The control group: intragastrically administered immunoglobulin G (IgG) (500 ug / time / mouse) and PBS every day for 15 consecutive days; the cordycepin group: intragastrically administered cordycepin (500 ug / time / mouse) and PBS every day for 15 consecutive days; the compound 24 drug group: intragastrically administered compound 24 (500 ug / time / mouse) and PBS every day for 15 consecutive days; the PD-1+TIM3 antibody treatment group: intragastrically administered antibodies PD-1+TIM3 (500 ug / time / mouse, respectively) every 4 days for 15 consecutive days; the compound 24 drug+PD-1+TIM3 antibody treatment group: intragastrically administered immunoglobulin compound 24 drug (500 ug / time / mouse) every day and intragastrically administered antibodies PD-1+TIM3 (500 ug / time / mouse, respectively) every 4 days for 15 consecutive days. The size change of the ovarian tumor in the five groups of mice is observed every two days, and the mice are sacrificed after the experiment, and blood and corresponding tissue specimens are collected. The tumor body is immediately photographed and weighed, and part of the tumor tissue is fixed in a formaldehyde solution for further detection. The two perpendicular diameters (length and width) of the transplanted tumor are measured every two days with a caliper, the size of the transplanted tumor is calculated, and the volume is calculated according to the formula: tumor volume (mm 3 ) = 1 / 2 x (length x width) 2 , and the experimental results are shown in Figure 5 . The experimental results show that cordycepin has an anti-ovarian cancer proliferation effect (15-day size reduction of 1.5 times), compound 24 alone has a significantly anti-tumor growth effect (15-day size reduction of about 2 times) than cordycepin, and immune checkpoint inhibitors PD-1 and CTLA4 alone also have a significantly anti-tumor growth effect (15-day size reduction of more than about 2 times), and compound 24 and immune checkpoint inhibitors PD-1 and CTLA4 are used at the same time, which can greatly improve the anti-tumor effect (15-day size reduction of about 4 times).

[0303] Example 48: Inhibition effect of cordycepin modified derivatives on in vitro-gastric cancer cells

[0304] The MTT experiment method is used to analyze the anti-tumor dose-effect relationship curves of cordycepin and forty kinds of prepared compounds in gastric cancer cell lines AGS and BGC-823, respectively. The results of calculating the half inhibitory concentration (IC 50 ) are summarized in Table 4. The in vitro anti-tumor experiment results show that the effective concentration of the modified cordycepin derivatives on tumor cells is reduced compared with cordycepin.

[0305] Table 4: IC 50(μM)

[0306]

[0307]

[0308] Example 49: In vivo tumor inhibition effect of cordycepin modified derivatives on a gastric cancer-mouse model

[0309] 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 preparation (500 μg / mouse). 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 6 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 1.73g to 0.46g). 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 1.73g to 0.14g).

[0310] Example 50: In vivo tumor inhibition effect of cordycepin modified derivatives on pancreatic cancer-mouse model

[0311] 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°C in a CO2 incubator. Cells were passaged using 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 raised in a pathogen-free environment, and when the mice grew to 6 weeks, 200 uL of pancreatic cancer Pan02-uc cell line was injected subcutaneously in the right dorsal near the axillary of each mouse, and the appearance of tumor transplantation within about one week indicated that the model was successfully established. The mice were randomly divided into five groups, 10 in each group, namely the control group, the cordycepin group, the compound 11, 16, 24 drug groups. The control group: corn syrup and DMSO as a solvent were given intragastrically every day; the cordycepin group: cordycepin (500 ug / time / each) was given intragastrically every day and prepared with DMSO; the compound drug group: the compound (500 ug / time / each) was given intragastrically every day and prepared with DMSO. Continuous administration for 24 days. Then the survival rate of the five groups of mice was observed every day, and the median survival time was studied, and the experimental results are shown in Table 1. Figure 7 The experimental results show that cordycepin alone has a significant effect of prolonging the survival period of tumor-bearing mice (from 41 days to 52 days), and the compound group further prolongs the survival period of tumor-bearing mice (from 41 days to >58 days) on the basis of cordycepin, among which compound 24 has the best effect, prolonging the survival period of tumor-bearing mice by nearly half (from 41 days to 76 days).

[0312] The above only describes the preferred embodiments of the present application, and it should be noted that for ordinary skilled persons in the art, several improvements and refinements can be made without departing from the principles of the present application, and these improvements and refinements should also be considered as the protection scope of the present application. The components not explicitly described in the embodiments can be realized by the existing technology.

Claims

1. A cordycepin derivative as shown in Formula I, or a pharmaceutically acceptable salt or stereoisomer thereof; in, R1 is selected from R1 10 -R1 11 or R1 14 -R1 16 The indicated groups; R2 is selected from hydrogen; 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.

2. The cordycepin derivative of Formula I according to claim 1, or a pharmaceutically acceptable salt or stereoisomer thereof, characterized in that, The cordycepin derivative shown in Formula I is compound 19; Compound 19.

3. A cordycepin derivative as shown in Formula I, or a pharmaceutically acceptable salt or stereoisomer thereof; in, R1 is selected from R1 10 -R1 11 or R1 14 -R1 15 The indicated groups; R2 is selected from hydrogen; R3 is selected from hydrogen; R4 is selected from hydroxyl; R5 is selected from fluorine; R6 is selected from amino; R7 is selected from hydrogen.

4. The cordycepin derivative of Formula I according to claim 3, or a pharmaceutically acceptable salt or stereoisomer thereof, characterized in that, The cordycepin derivative shown in Formula I is compound 17; Compound 17.

5. A cordycepin derivative as shown in Formula I, or a pharmaceutically acceptable salt or stereoisomer thereof; in, R1 is selected from R1 10 -R1 11 or R1 14 -R1 16 The indicated groups are: R2 is selected from hydrogen; R3 is selected from fluorine; R4 is selected from hydroxyl; R5 is selected from hydrogen, mercapto, fluorine; R6 is selected from amino; and R7 is selected from hydrogen.

6. The cordycepin derivative of Formula I according to claim 5, or a pharmaceutically acceptable salt or stereoisomer thereof, characterized in that, The cordycepin derivative shown in Formula I is compound 18; Compound 18.

7. A cordycepin derivative as shown in Formula I, or a pharmaceutically acceptable salt or stereoisomer thereof; in, R1 is selected from R1 10 -R1 11 or R1 14 -R1 16 The indicated groups are: R2 is selected from hydrogen; 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; R7 is selected from hydrogen.

8. The cordycepin derivative of Formula I according to claim 7, or a pharmaceutically acceptable salt or stereoisomer thereof, characterized in that, The cordycepin derivative shown in Formula I is compound 26 or compound 27; Compound 26 Compound 27.

9. A pharmaceutical composition comprising at least one cordycepin derivative as described in claims 1 to 8, or a pharmaceutically acceptable salt or stereoisomer thereof; and at least one immune checkpoint inhibitor.

10. The composition according to claim 9, characterized in that, The immune checkpoint inhibitor is selected from PD-1 and / or CTLA4 monoclonal antibodies.

11. The composition according to claim 9, characterized in that, The cordycepin derivative, or its pharmaceutically acceptable salt or stereoisomer, is present in a mass ratio of 1:0.2 to 10 with the immune checkpoint inhibitor.

12. The composition according to claim 9, characterized in that, The cordycepin derivative, or its pharmaceutically acceptable salt or stereoisomer, is present in a mass ratio of 1:0.2 to 8 with an immune checkpoint inhibitor.

13. The composition according to claim 9, characterized in that, The cordycepin derivative, or its pharmaceutically acceptable salt or stereoisomer, is present in a mass ratio of 1:0.2 to 5 to the immune checkpoint inhibitor.

14. The composition according to claim 9, characterized in that, The cordycepin derivative, or its pharmaceutically acceptable salt or stereoisomer, is present in a mass ratio of 1:0.2 to 3 to the immune checkpoint inhibitor.

15. The composition according to claim 9, characterized in that, The cordycepin derivative, or its pharmaceutically acceptable salt or stereoisomer, is present in a mass ratio of 1:0.2 to 2 to the immune checkpoint inhibitor.

16. The composition according to claim 9, characterized in that, The cordycepin derivative, or its pharmaceutically acceptable salt or stereoisomer, is present in a mass ratio of 1:0.5 to 1.5 to the immune checkpoint inhibitor.

17. The composition according to claim 9, characterized in that, The cordycepin derivative, or its pharmaceutically acceptable salt or stereoisomer, is present in a mass ratio of 1:0.8 to 1.2 to the immune checkpoint inhibitor.

18. The composition according to claim 9, characterized in that, The cordycepin derivative, or its pharmaceutically acceptable salt or stereoisomer, is present in a mass ratio of 1:1 to the immune checkpoint inhibitor.

19. The pharmaceutical composition according to claim 9, characterized in that, The dosage form of the pharmaceutical composition is selected from tablets, pills, capsules, drop pills, syrups, disintegrants, injections, sustained-release formulations, or kits.

20. The use of any cordycepin derivative of claims 1 to 8, or a pharmaceutically acceptable salt or stereoisomer thereof, or the pharmaceutical composition of any one of claims 9 to 19, in the preparation of products for the prevention and treatment of diseases related to functional damage of cells in mammals or humans.

21. The application according to claim 20, characterized in that, The diseases associated with the aforementioned cellular functional impairment and alterations are tumors.

22. The application according to claim 21, characterized in that, The tumor is any one of the following: 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.

23. The method for preparing the cordycepin derivative according to any one of claims 1 to 8, characterized in that, In equation I, R1 is selected from R1 10 -R1 11 or R1 14 - R1 16 When the group shown is present; 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; IR-1; In formula IR-1, R2~R7 are the same as R2~R7 in formula I, or are independently selected from protecting groups; When R1 in equation I is selected from R1 14 - R1 15 When the group shown is present, the preparation method of the cordycepin derivative shown in Formula I is as follows: in anhydrous N,N-dimethylformamide and / or tetrahydrofuran, with tert-butylmagnesium chloride as catalyst, compound IR-1 is reacted with nitrobenzene phosphate substituted by a modifier, wherein the substitution is aryloxy group and any one or more functional groups among amino acid ester amide group; When R1 in equation I is selected from R1 10 -R1 11 or R1 16 When the group shown is present, 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-1 is reacted with p-toluenesulfonyloxymethyl phosphate substituted by a modifier, wherein the substitution is aryloxy or any one or more functional groups among amino acid ester amide groups.

24. The preparation method according to claim 23, characterized in that, When R1 in equation I is selected from R1 14 - R1 15 When the indicated group is used, the ratio of compound IR-1, modifier, catalyst and organic solvent is 1 mmol: 1~3 mmol: 1~2 mmol: 9~15 mL, and the reaction temperature is 20~40 °C; When R1 in equation I is selected from R1 10 -R1 11 or R1 16 When the indicated group is used, the 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 the reaction temperature is -20~0 ℃.

25. The method for preparing the cordycepin derivative according to any one of claims 1 to 8, characterized in that, When R3 in Formula I is selected from fluorine or azide group; 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. IR-3 IR-3a IR-3b; 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; In formula IR-3a, R1, R2, R4~R7 are the same as R1, R2, R4~R7 in formula I, or are independently selected from protecting groups; In formula IR-3b, R1, R2, and R5~R7 are the same as R1, R2, and R5~R7 in formula I, or are independently selected from protecting groups; When R3 in Formula I is selected from fluorine, the preparation method of the cordycepin derivative shown in Formula I is as follows: in pyridine and dichloromethane, compound IR-3 is reacted with trifluoromethanesulfonic anhydride to obtain intermediate IR-3a; in ethyl acetate, intermediate IR-3a is reacted with hydrofluoric acid and sulfur trifluoride to obtain the cordycepin derivative shown in Formula I. When R3 in Formula I is selected from azide, 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 under the catalysis of 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 derivatives; 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.

26. The preparation method according to claim 25, characterized in that, When R3 in Formula I is selected from fluorine, in the preparation method of the intermediate IR-3a, the ratio of the amount of compound IR-3, trifluoromethanesulfonic anhydride, pyridine and dichloromethane is 1 mmol: 1~1.5 mmol: 0.15~0.2 mL: 10~20 mL, and the reaction temperature is -5~5 ℃; wherein, in the preparation method of the cordycepin derivative shown in Formula I, the intermediate IR-3a is subjected to a substitution reaction with a 37% hydrofluoric acid or triethylamine solution of diethylaminotrifluoride or hydrochloric acid, and the ratio of the amount of intermediate IR-3a, 37% hydrofluoric acid or triethylamine solution of diethylaminotrifluoride or hydrochloric acid to ethyl acetate is 1 mmol: 2~3 mmol: 4~10 mL, and the reaction temperature is 60~80 ℃; When R3 in Formula I is selected from azide, in the preparation method of the intermediate IR-3b, the ratio of the amount 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, the cyclization reaction temperature is 10~60 °C, and the cyclization reaction time is 1~5 h; in the preparation method of the cordycepin derivative of Formula I, the ratio of the amount of intermediate IR-3b, sodium azide and dimethylformamide is 1 mmol: 4~5 mmol: 2~5 mL, and the ring-opening reaction temperature is 100~120 °C.

27. The method for preparing the cordycepin derivative according to any one of claims 1 to 8, characterized in that, In Formula I, R5 is selected from mercapto or fluorine; 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. IR-5; 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; When R5 in Formula I is selected from mercapto, the preparation method of the cordycepin derivative shown in Formula I is as follows: In acetic acid, compound IR-5 is reacted with hydrogen peroxide to obtain intermediate IR-5d; intermediate IR-5d is heated under reflux in hydrochloric acid aqueous solution to obtain intermediate IR-5e; intermediate IR-5e is dissolved in water and reacted under hydrogen atmosphere with Raney nickel catalysis to obtain intermediate IR-5f; intermediate IR-5f is reacted with methanol, pyridine and carbon disulfide to obtain the cordycepin derivative shown in Formula I. IR-5d, IR-5e, IR-5f; In formulas IR-5d, IR-5e, and IR-5f, R1~R4 and R6~R7 are the same as R1~R4 and R6~R7 in formula I, or are independently selected from protecting groups; When R5 in Formula I is selected from fluorine, the preparation method of the cordycepin derivative shown in Formula I is as follows: the compound IR-5 is subjected to a nitro derivatization reaction to obtain the nitro intermediate IR-5g, and then a substitution reaction is carried out to obtain the cordycepin derivative shown in Formula I. IR-5g.

28. The preparation method according to claim 27, characterized in that, When R5 in Formula I is selected from mercapto, the intermediate IR-5d is prepared by reacting compound IR-5 with acetic acid in hydrogen peroxide; wherein the concentration of hydrogen peroxide is 20%~40%, the ratio of compound IR-5, hydrogen peroxide and acetic acid is 1 mmol: 2~3 mol: 1~6 mL, and the reaction temperature is 30~50 °C. The preparation method of the intermediate IR-5e is as follows: the intermediate IR-5d is heated under reflux in hydrochloric acid aqueous solution; wherein the concentration of the hydrochloric acid aqueous solution is 1~5 mol / L, and the volume ratio of the intermediate IR-5d to the hydrochloric acid aqueous solution is 1 mmol: 3~5 mL; In the preparation method of the intermediate IR-5f, the ratio of intermediate IR-5e, Raney nickel, and water is 1 mmol: 0.08~0.2 g: 10~20 mL, and the reaction temperature is 50~70 °C. In the preparation method of the cordycepin derivative of Formula I, the ratio of the intermediate IR-5f to methanol, pyridine and carbon disulfide is 1 mmol: 5~10 mL, the volume ratio of methanol, pyridine and carbon disulfide is 4:3~7:0.5~3.5, and the reaction temperature is 30~50 °C.

29. The preparation method according to claim 27, characterized in that, When R5 in Formula I is selected from fluorine, the intermediate IR-5g is prepared by reacting compound IR-5 with tetrabutylammonium nitrate in dichloromethane under the catalysis of trifluoroacetic anhydride; the ratio of the amount of compound IR-5, tetrabutylammonium nitrate, trifluoroacetic acid and dichloromethane is 1 mmol: 1.4~2 mmol: 1~2 mmol: 15~35 mL; wherein, the cordycepin derivative of Formula I is prepared by reacting nitrated intermediate IR-5g with tetrabutylammonium fluoride in acetonitrile, the ratio of the amount of nitrated intermediate IR-5g, tetrabutylammonium fluoride and acetonitrile is 1 mmol: 1.3~1.5 mmol: 30~50 mL, and the reaction temperature is -5~5 ℃.

30. The method for preparing the cordycepin derivative according to any one of claims 1 to 8, characterized in that, In equation I, R6 is selected from R6 15 The group shown; 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; IR-6; 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.

31. The method according to claim 30, characterized in that, 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 pyridyl substitution, and the ratio of compound IR-6, substituted acyl chloride and anhydrous pyridine is 1 mmol: 1~2 mmol: 5~10 mL, and the reaction temperature is 0~60 ℃.

Images