Cordycepin derivatives with antitumor effects

Abstract

The present invention discloses a compound having an antitumor effect by derivatization of cordycepin, the structure of which is represented by formula I. The cordycepin derivative and its pharmaceutical composition according to the present invention have excellent antitumor growth effect. Compared with the parent drug, the cordycepin derivative has excellent affinity to cell membranes, so that the half-life during which the drug is metabolized in the body is longer, and the time during which the drug remains in the body is longer. Compared with other nucleoside antitumor drugs, the cordycepin derivative and its pharmaceutical composition according to the present invention have a wider range of tumor types and action, and have excellent inhibitory effect against gastric cancer, pancreatic cancer, liver cancer, small cell lung cancer, colorectal cancer, melanoma, ovarian cancer, etc., with reduced side effects and excellent therapeutic effect. JPEG2025511448000109.jpg42166

Description

Technical Field

[0001] The present invention belongs to the field of biopharmaceuticals, and specifically relates to a compound obtained by derivatizing cordycepin, a preparation method thereof, and use in the preparation of products for preventing and treating related diseases caused by mutations in cell function disorders.

Background Art

[0002] With the stimulation of the external environment and the accumulation of cell damage, the cell compatibility in the living body changes. One is that cell compatibility is lost and eventually develops into aging, and the other is that cell compatibility is abnormally enhanced and changes into cancer. Cancer is a common disease threatening human life and health, and the cancer incidence rate and mortality rate are on the rise worldwide. Currently, the treatment methods for malignant tumors mainly include surgical treatment, radiotherapy, chemotherapy, etc. Chemotherapy mainly uses synthetic drugs. The inhibitory effect of chemotherapeutic drugs on tumors is worthy of affirmation. It is effective against malignant tumors and is also one of the commonly used treatment methods. However, its toxic side effects are extensive and serious, and there is a problem of drug resistance. In addition, chemotherapeutic drugs have low selectivity for tumor cells and normal cells. While killing or inhibiting tumor cells, they also damage the growth of normal cells, directly affect the functions of the heart, liver, kidneys and nervous system, and give a certain toxicity to the human body. Therefore, in tumor treatment, it is very necessary to find anti-tumor drugs with low toxicity and high efficiency.

[0003] As one of the most important endogenous compounds in the human body, nucleosides play an important role in the metabolic process of the living body. The modification and derivatization of nucleoside compounds are one of the main methods for preparing anti-tumor drugs. Currently, commercially available nucleoside anti-tumor drugs include fludarabine, cladribine, clofarabine, phosphorylated fludarabine, troxacitabine, etc. Cordycepin (3'-deoxyadenosine) is the main active ingredient of Cordyceps sinensis, belongs to nucleoside analogs, and has excellent effects in cell compatibility changes (anti-aging, anti-cancer), immune regulation, inflammation removal, etc. in the in vivo metabolic process.

[0004] The mechanism of the anti-cancer effect of cordycepin mainly induces apoptosis, regulates the cell cycle, and inhibits the invasion and metastasis of tumor cells by interfering with the expression of matrix metalloproteinase (MMP). The signal transduction pathways related to the induction of apoptosis in tumor cells include the NF-κB signal transduction pathway and the mitogen-activated protein kinase (MAPK) signal transduction pathway. The regulation of the cell cycle is mainly that in cancer cells, cordycepin shortens the G1 phase during the cell cycle, prolongs the G2 and M phases, arrests the cell cycle at the G2 / M phase, and inhibits cell proliferation. The process of interfering with the expression of matrix metalloproteinase mainly inhibits the NF-κB signal transduction pathway and finally inhibits the expression of MMP-9. However, nucleoside analogs have poor lipid solubility, are difficult to absorb, are easily metabolically inactivated by deaminase, have a short half-life, weak targeting, and some tumor cells and viruses are prone to expressing drug resistance, all of which significantly reduce the efficacy of nucleoside drugs.

Summary of the Invention

Problems to be Solved by the Invention

[0005] The object of the present invention: In view of the deficiencies of conventional nucleoside drugs, the technical problem to be solved by the present invention is to provide a series of nucleoside analogs (cordycepin derivatives) chemically modified with cordycepin as the parent nucleus in combination with the natural pharmacological activity of cordycepin.

[0006] Furthermore, the technical problem to be solved by the present invention is to provide a composition containing the above nucleoside analogs.

[0007] Furthermore, the technical problem to be solved by the present invention is to provide the use of the above nucleoside analogs and their compositions in products for preventing and treating related diseases caused by mutations in cell dysfunction in mammals or humans.

[0008] Finally, the technical problem to be solved by the present invention is to provide a method for preparing the above nucleoside analogs.

Means for Solving the Problems

[0009] In order to solve the above first technical problem, the present invention discloses a cordycepin derivative represented by formula I, or a pharmaceutically acceptable salt, stereoisomer, tautomer, solvate, prodrug or metabolite thereof.

Chemical formula

[0010] In the formula, R 1 is selected from hydrogen, 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 functional groups selected from an alkoxy group, an alkoxy group substituted with a halogen, an aryloxy group, an amido amino acid group, an alkyl ester group, and an alkyl acid methyl ester oxy group. R 2 is selected from hydrogen or an azide group. R 3 is selected from hydrogen, fluorine, chlorine, or an azide group. R 4 is selected from a hydroxyl group, a cyano group, a β-amido-γ-cyclosulfonyloxy group, an amino acid carboxylic acid ester group, an amino acid alkyl ester phosphonic acid phenyl ester group, or an amino acid alkyl ester phosphate phenyl ester group. R 5 is selected from hydrogen, a bromovinyl group, a mercapto group, a methyl group, fluorine, or chlorine. R 6 is selected from an amino group and a substituted carboxamide group, and the substitution is any one or more functional groups selected from an alkyl group, an aryl group, a cycloalkyl group, a furyl group, and a pyridyl group. R 7 is selected from hydrogen or an isopropylamino group, and R 1 R 2 R 3 R5 , R 7 is selected from hydrogen, R 4 is selected from hydroxyl groups, R 6 does not exist when it is selected from amino groups.

[0011] In some embodiments, R 1 is hydrogen, R 1 1 -R 1 7 represents a phosphate group, diethyl phosphate group, di-n-propyl phosphate, diisopropyl phosphate, diisobutyl phosphate, di-n-butyl phosphate, or diisobutyryloxymethoxy phosphate, R 1 8 -R 1 11 represents a phosphonic acid group, ethyl trifluorophosphonate, diisobutyryloxymethoxyphosphonic acid, or pivaloyloxymethoxyphosphonic acid group, or R 1 12 -R 1 13 represents a cyclophosphonotrifluoroglycol ester glycol ester group, or a cyclophosphonoethylene glycol ester group, R 1 14 -R 1 16 represents (R)-phosphorylisoleucine methyl ester phenyl ester, (S)-phosphorylisoleucine methyl ester phenyl ester, or phosphoalanine isopropyl ester phenyl ester, R 1 17 -R 1 22 represents hydroxyethyl, 1,3-dihydroxy-2-propyl, alanine ethyl ester, isoleucine ethyl ester, isoleucine-1,3-propanediol ester, or glycine ethyl ester, or R 1 23 -R 1 25Dimethyl dicarbonate-propylene glycol, 2-isopropoxyhexahydrocyclopentan[d][1,3,2]dioxolin-7-ol, or 1-hydroxymethyl-2,3-dihydroxy-4-cyclopentyl, selected from, and in some embodiments, R 1 is hydrogen, or R 1 1 、R 1 5 、R 1 8 、R 1 10 、R 1 15 、R 1 16 、R 1 17 、R 1 21 selected from the structures represented by any of.

Chemical formula

[0012] In some embodiments, R 4 is selected from a hydroxyl group, a cyano group, β-amido-γ-cyclosulfonyloxy, 2-amino-propionyloxy, 2-amino-3-methyl-butyryloxy, phosphoryloxyalanine methyl ester phenyl ester, or phosphonamidoisoleucine methyl ester phenyl ester represented by R 4 1 -R 4 7 and in some embodiments, R 4 is a cyano group, a cyano group, or R 4 3 、R 4 5 、or R 4 6 selected from the structures represented by any of.

Chemical formula

[0013] In some embodiments, R 5is selected from hydrogen, a bromovinyl group, a mercapto group, fluorine, or chlorine.

[0014] In some embodiments, R 6 is an amino group, or R 6 1 -R 6 16 is selected from an acetamide group, a butylamide group, an octylamide group, a dodecylamide group, an octadecylamide group, an isopropionamide group, an isobutylamide group, a pivalamide group, a 2-ethyl n-butylamide group, a 3,3-dimethyl-butylamide group, a cyclohexylcarboxamide group, a cyclopentylcarboxamide group, a benzamide group, a furancarboxamide group, a pyridinecarboxamide group, or a hexadecylamide group represented by, and in some embodiments, R 6 is an amino group, or R 6 5 , R 6 12 , R 6 13 , or R 6 15 is selected from the structures represented by any of.

Chemical formula

[0015] In some embodiments, it is a cordycepin derivative represented by formula I, or a pharmaceutically acceptable salt thereof, wherein R 1 is a group represented by R 1 16 , R 2 is selected from hydrogen, R 3 is selected from hydrogen, R 4 is selected from a hydroxyl group, R 5 is selected from hydrogen, fluorine, or chlorine, R 6 is selected from pyridinecarboxamide or an amino group, R 7 is selected from hydrogen, and in some embodiments, the cordycepin derivative represented by formula I is selected from the following compound 24, the following compound 7, or the following compound 20.

[0016] In some embodiments, the colchicine derivative represented by Formula I is selected from any one of Compounds 1 to 40.

Chemical formula

Chemical formula

[0017] In order to solve the above-mentioned second technical problem, the present invention discloses a pharmaceutical composition comprising at least one of the above-mentioned colchicine derivatives, or a pharmaceutically acceptable salt, stereoisomer, tautomer, solvate, prodrug or metabolite thereof, and at least one immune checkpoint inhibitor.

[0018] In some embodiments, the immune checkpoint inhibitor is selected from PD-1 and / or CTLA4 monoclonal antibodies.

[0019] In some embodiments, the mass ratio of the colchicine derivative, or a pharmaceutically acceptable salt, stereoisomer, tautomer, solvate, prodrug or metabolite thereof, to the immune checkpoint inhibitor is 1:0.2 to 10, 1:0.2 to 8 in some embodiments, 1:0.2 to 5 in some embodiments, 1:0.2 to 3 in some embodiments, 1:0.2 to 2 in some embodiments, 1:0.5 to 1.5 in some embodiments, 1:0.8 to 1.2 in some embodiments, and 1:1 in some embodiments.

[0020] In some embodiments, the dosage form of the pharmaceutical composition is selected from tablets, pellets, capsules, pills, syrups, disintegrants, injections, sustained-release agents, or kits.

[0021] The present invention discloses the use of the above-mentioned curdione 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 related diseases caused by mutations in cell dysfunction in mammals or humans.

[0022] In some embodiments, the related disease caused by the mutation of the cell dysfunction is a tumor. In some embodiments, the tumors include, but are 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.

[0023] In some embodiments, the product includes, but is not limited to, a drug.

[0024] In order to solve the above-mentioned fourth technical problem, the present invention discloses a preparation method of the above-mentioned curdione derivative. The preparation method is a synthetic means corresponding to the modification method. When reacting, since other active groups in curdione or the modified curdione molecule participate in the progress of the reaction, it is necessary to appropriately protect the active groups. Considering this, the protection method according to the present invention is the protection and deprotection of hydroxyl groups and amino groups. Both the protection and deprotection means are conventional means in the art. Some other reaction systems need to add a protective gas for protection, and these are all conventional means in the experimental process in the art.

[0025] Specifically, it is as follows.

[0026] A.R 1 To modify In formula I, R 1is 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 when the substitution is by any one or more functional groups of an alkoxy group, an alkoxy group substituted with a halogen, an aryloxy group, an amido amino acid group, an alkyl ester group, or a methyl ester oxy group of an alkyl acid, the method for preparing a cordycepin derivative represented by formula I is to carry out a chemical reaction using compound I-R-1 as a raw material in an organic solvent to obtain a cordycepin derivative represented by formula I.

Chemical formula

[0027] In formula I-R-1, R 2 ~R 7 is the same as R 2 ~R 7 in formula I or is independently selected from protecting groups.

[0028] When R 1 in formula I is selected from a phosphate group, in some embodiments, the method for preparing a cordycepin derivative represented by formula I is to react compound I-R-1 with phosphorus oxychloride as a modifier in trimethyl phosphite and / or triethyl phosphite. In some embodiments, the ratio of the amounts used of the compound I-R-1, the modifier, and the organic solvent is 1 mmol: 3 to 6 mmol: 15 to 30 mL. In some embodiments, the reaction temperature is -10 to 5 °C. In some embodiments, the reaction time is 0.5 to 2 hours.

[0029] When R 1When selected from substituted phosphate ester groups, in some embodiments, the method for preparing the cordycepin derivative represented by formula I is to react compound I-R-1 with nitrophenyl phosphate substituted with a modifier in anhydrous N,N-dimethylformamide and / or tetrahydrofuran using tert-butylmagnesium chloride as a catalyst. The substitution is substituted with any one or more functional groups of an alkoxy group, an alkoxy group substituted with a halogen, an aryloxy group, an amido amino acid group, an alkyl ester group, and a methyloxy alkyl acid group. In some embodiments, the ratio of the amounts of use of the compound I-R-1, the modifier, the catalyst, and the 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 hours.

[0030] R in formula I 1 When selected from a phosphonic acid group or a substituted phosphonic acid ester group, in some embodiments, the method for preparing the cordycepin derivative represented by formula I is to react compound I-R-1 with substituted or unsubstituted p-toluenesulfonyloxymethyl phosphate in anhydrous N,N-dimethylformamide using NaH as a catalyst. The substitution is substituted with any one or more functional groups of an alkoxy group, an alkoxy group substituted with a halogen, an aryloxy group, an amido amino acid group, an alkyl ester group, and a methyloxy alkyl acid group. In some embodiments, the ratio of the amounts of use of the compound I-R-1, the modifier, the catalyst, and the organic solvent is 0.1 mol: 0.1-0.15 mol: 0.2-0.03 mol: 100-200 mL. Depending on the embodiment, it is 0.1 mol: 0.1-0.15 mol: 0.2-0.03 mol: 150 mL. In some embodiments, the reaction temperature is -20-0 °C. In some embodiments, the reaction time is 0.5-6 hours.

[0031] R in formula I 1When it is selected from 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, in some embodiments, the method for preparing the cordycepin derivative represented by formula I is to use potassium carbonate as a catalyst in methyl ethyl ketone and react a modifier which is an alkyl alcohol, an amino acid alkyl ester, an amino acid alkyl alcohol ester, an alkyl acid alkyl ester or a cycloalkyl polyol substituted with a halogen with compound I-R-1. In some embodiments, the halogen is bromine or chlorine. In some embodiments, the ratio of the amounts used of the compound I-R-1, the modifier, the catalyst and the organic solvent is 1 mmol: 0.5-1.5 mmol: 1-3 mmol: 5-8 mL, and in some embodiments according to the examples, it is 1 mmol: 1 mmol: 1-3 mmol: 5-8 mL. In some embodiments, the reaction temperature is 40-100 °C, and in some embodiments, the reaction time is 8-20 hours.

[0032] B.R 2 to modify In formula I, R 2 When it is selected from an azide group, the method for preparing the cordycepin derivative represented by formula I is to carry out a cyclization reaction with compound I-R-2 as a raw material in an organic solvent to obtain intermediate I-R-2a, 5-(6-amino-9H-purin-9-yl)-1,4-dioxapyrrole[2.4]heptan-6-ol or its derivative, and then carry out a ring-opening reaction on the obtained intermediate I-R-2a in an organic solvent to obtain the cordycepin derivative represented by formula I.

Chemical formula

[0033] In formula I-R-2, R 1 , R 3 ~R 7 is the same as R 1 , R 3 ~R 7 in formula I, or each is independently selected from protecting groups.

[0034] In some embodiments, the method for preparing the intermediate I-R-2a involves subjecting compound I-R-2 to a cyclization reaction in dichloromethane under the catalysis of phosphorus pentoxide and metachloroperbenzoic acid. In some embodiments, the ratio of the amounts of compound I-R-2, phosphorus pentoxide, metachloroperbenzoic acid, and dichloromethane used is 1 mmol: 1.2 - 2 mmol: 2 - 3 mmol: 10 - 20 mL. In some embodiments, the cyclization reaction temperature is 20°C - 60°C. In some embodiments, the cyclization reaction time is 3 hours - 10 hours.

[0035] In some embodiments, the method for preparing the cordycepin derivative represented by formula I involves subjecting the obtained intermediate I-R-2a and sodium azide to a ring-opening reaction in anhydrous dimethylformamide. In some embodiments, the ratio of the amounts of compound I-R-2a, sodium azide, and dimethylformamide used is 1 mmol: 4 - 5 mmol: 2 - 5 mL. In some embodiments, the ring-opening reaction temperature is 100 - 120°C. In some embodiments, the ring-opening reaction time is 12 - 16 hours.

[0036] C.R 3 to modify In formula I, when R 3 is selected from fluorine, chlorine, or an azide group, the method for preparing the cordycepin derivative represented by formula I involves carrying out a chemical reaction using compound I-R-3 as a raw material in an organic solvent to obtain the cordycepin derivative represented by formula I.

Chemical formula

[0037] In formula I-R-3, R 1 , R 2 , R 5 ~R 7 are the same as R 1 , R 2 , R 5 ~R 7 in formula I, or are each independently selected from protecting groups.

[0038] In formula I, when R 3 is selected from fluorine or chlorine, in some embodiments, the method for preparing the cordycepin derivative represented by formula I is to react compound I-R-3 with trifluoromethanesulfonic anhydride in pyridine and dichloromethane to obtain intermediate I-R-3a, 5-(6-amino-9H-purin-9-yl)-4-hydroxy-2-(hydroxymethyl)tetrahydrofuran-3-yl trifluoromethanesulfonate or a derivative thereof, and in ethyl acetate, react intermediate I-R-3a with hydrofluoric acid, sulfur trifluoride or hydrochloric acid by substitution reaction to obtain the cordycepin derivative represented by formula I. In the method for preparing the intermediate I-R-3a, in some embodiments, the ratio of the amounts of the compound I-R-3, trifluoromethanesulfonic anhydride, pyridine and dichloromethane used is 1 mmol: 1-1.5 mmol: 0.15-0.2 mL: 10-20 mL. In some embodiments, the reaction temperature is -5 to 5 °C. In some embodiments, it is 0 °C. In some embodiments, the reaction time is 1 to 3 hours. The method for preparing the cordycepin derivative represented by formula I is, in some embodiments, to carry out a substitution reaction between intermediate I-R-3a and 37% hydrofluoric acid or a triethylamine solution of diethylaminosulfur trifluoride or hydrochloric acid. In some embodiments, the ratio of the amounts of the intermediate I-R-3a, 37% hydrofluoric acid or a triethylamine solution of diethylaminosulfur trifluoride or hydrochloric acid, and ethyl acetate used is 1 mmol: 2-3 mmol: 4-10 mL. In some embodiments, the reaction temperature is 60 to 80 °C. In some embodiments, the reaction time is 8 to 10 hours.

[0039] In formula I, when R 3When it is selected from azide groups, in some embodiments, the method for preparing the cordycepin derivative represented by Formula I is to cyclize compound I-R-3 in N,N-dimethylformamide under the catalysis of triphenylphosphine and diisopropyl azodicarboxylate to obtain intermediate I-R-3b, 4-(6-amino-9H-purin-9-yl)-3,6-dioxane[3.1.0]hexan-2-yl)methanol or its derivative. In N,N-dimethylformamide, the obtained intermediate I-R-3b is subjected to a ring-opening reaction with sodium azide to obtain the cordycepin derivative represented by Formula I. In the method for preparing the intermediate I-R-3b, in some embodiments, the ratio of the usage amounts 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, it is 10.0 mmol:22 mmol:2.2 mmol:15 - 50 mL. In some embodiments, the cyclization reaction temperature is 10 - 60 °C. In some embodiments, the cyclization reaction time is 1 - 5 hours. In some embodiments, in the method for preparing the cordycepin derivative represented by Formula I, the ratio of the usage amounts of the intermediate I-R-3b, sodium azide, and dimethylformamide is 1 mmol:4 - 5 mmol:2 - 5 mL. In some embodiments, the ring-opening reaction temperature is 100 - 120 °C. In some embodiments, the ring-opening reaction time is 12 - 16 hours.

[0040] D.R 4 to modify In Formula I, R 4 When it is selected from a cyano group, a β-amide-γ-cyclosulfonyloxy group, an amino acid carboxylic acid ester group, an amino acid alkyl ester phosphonic acid phenyl ester group, or an amino acid alkyl ester phosphoric acid phenyl ester group, the method for preparing the cordycepin derivative represented by Formula I is to carry out a chemical reaction using compound I-R-4 as a raw material in an organic solvent to obtain the cordycepin derivative represented by Formula I.

Chemical formula

[0041] In formula I-R-4, R 1 ~R 3 、R 5 ~R 7 is the same as R 1 ~R 3 、R 5 ~R 7 in formula I, or is independently selected from protecting groups respectively.

[0042] In formula I, when R 4 is selected from cyano groups, in some embodiments, the preparation method of the cordycepin derivative represented by formula I is to stir compound I-R-4, trifluoromethanesulfonic acid, trimethylsilyl triflate in dichloromethane at an environment of -50 to -30 °C, and then react with trimethylsilyl cyanide and triethylamine. In some embodiments, the ratio of the usage amounts of the compound I-R-4, trifluoromethanesulfonic acid, trimethylsilyl triflate, and dichloromethane is 10 mmol: 0.8 to 1.8 mL: 2.2 to 3.2 mL: 90 to 110 mL, and in some embodiments according to the examples, it is 10 mmol: 1.3 mL: 2.7 mL: 100 mL. In some embodiments, the stirring time is 20 to 40 minutes, and in some embodiments, it is 30 minutes. In some embodiments, the ratio of the usage amounts of the compound I-R-4, trimethylsilyl cyanide, and triethylamine is 10 mmol: 3.4 to 4.3 g: 3 to 4 mL. In some embodiments, the reaction temperature is 20 to 30 °C, and in some embodiments according to the examples, it is room temperature. In some embodiments, the reaction time is 2 to 4 hours.

[0043] In formula I, R 4When it is selected from a β-amide-γ-cyclosulfonyloxy group, in some embodiments, the method for preparing the cordycepin derivative represented by Formula I is to oxidize Compound I-R-4 to obtain Intermediate I-R-4a, 2-(6-amino-1,6-dihydro-9H-purin-9-yl)-5-(hydroxymethyl)dihydrofuran-3(2H)-one or a derivative thereof, and then further subject it to a cyanation reaction to obtain Intermediate I-R-4b, 2-(6-amino-1,6-dihydro-9H-purin-9-yl)-5-(hydroxymethyl)-3-isocyanatotetrahydrofuran-3-ol or a derivative thereof, and then further subject it to methylsulfonic acid esterification to obtain Intermediate I-R-4c, 2-(6-amino-1,6-dihydro-9H-purin-9-yl)-5-(hydroxymethyl)-3-isocyanatotetrahydrofuran-3-yl methanesulfonate or a derivative thereof, and finally subject it to a ring-closure reaction to obtain the cordycepin derivative represented by Formula I. In some embodiments, the method for preparing the Intermediate I-R-4a is to react Compound I-R-4 with chromium trioxide of Jones reagent (2.2M) in acetone. In some embodiments, the ratio of the usage amounts of the Compound I-R-4, Jones reagent, and acetone is 20 mmol: 5-8 mL: 50-100 mL. In some embodiments, the reaction temperature is 0-40 °C. In some embodiments, the reaction time is 1-4 hours. In some embodiments, the method for preparing the Intermediate I-R-4b is to react Intermediate I-R-4a with trimethylsilyl cyanide and boron trifluoride diethyl ether in dichloromethane. In some embodiments, the ratio of the usage amounts of the Intermediate I-R-4a, trimethylsilyl cyanide, boron trifluoride diethyl ether, and dichloromethane is 20 mmol: 20-40 mmol: 10-30 mmol: 50-100 mL. In some embodiments, it is 20 mmol: 20-40 mmol: 20 mmol: 50-100 mL. In some embodiments, the reaction temperature is 0-40 °C. In some embodiments, the reaction time is 1-4 hours.In some embodiments, the method for preparing the intermediate I-R-4c is to react the intermediate I-R-4b with triethylamine and methanesulfonyl chloride in anhydrous dichloromethane. In some embodiments, the ratio of the amounts of the intermediate I-R-4b, triethylamine, methanesulfonyl chloride, and anhydrous dichloromethane used is 2.2 mmol: 10 - 15 mmol: 4 - 8 mmol: 5 - 10 mL. In some embodiments, the reaction temperature is -30 to 0 °C. In some embodiments, the reaction time is 2 - 4 hours. In some embodiments, the method for preparing the cordycepin derivative represented by Formula I is to react the intermediate I-R-4c with cesium carbonate in anhydrous acetonitrile. In some embodiments, the ratio of the amounts of the intermediate I-R-4c, cesium carbonate, and anhydrous acetonitrile used is 1 mmol: 0.5 - 2.5 mmol: 3 - 10 mL. In some embodiments, it is 1 mmol: 1.5 mmol: 3 - 10 mL. In some embodiments, the reaction temperature is 0 to 40 °C. In some embodiments, the reaction time is 2 - 4 hours.

[0044] In Formula I, R 4 When it is selected from amino acid carboxylic acid ester groups, in some embodiments, the method for preparing the cordycepin derivative represented by Formula I is to react Compound I-R-4 with an amino acid chloride in anhydrous pyridine to obtain the cordycepin derivative represented by Formula I. In some embodiments, the ratio of the amounts of Compound I-R-4, amino acid chloride, and pyridine used is 10 mmol: 5 - 15 mmol: 50 - 100 mL. In some embodiments, it is 10 mmol: 10 mmol: 50 - 100 mL. In some embodiments, the reaction temperature is 20 - 60 °C. In some embodiments, it is 40 °C. In some embodiments, the reaction time is 6 - 20 hours.

[0045] In Formula I, R 4When selected from amino acid alkyl ester phosphonic acid phenyl ester groups, in some embodiments, the method for preparing the cordycepin derivative represented by formula I is to use NaH as a catalyst in anhydrous N,N-dimethylformamide to react compound I-R-4 with p-toluenesulfonyloxymethyl phenyl phosphate substituted with an amino acid alkyl ester as a modifier. In some embodiments, the ratio of the amounts of use of the compound I-R-4, modifier, NaH, and anhydrous N,N-dimethylformamide is 0.1 mol:0.1 - 0.15 mol:0.2 - 0.03 mol:100 - 200 mL, and in some embodiments according to the examples, it is 0.1 mol:0.1 - 0.15 mol:0.2 - 0.03 mol:150 mL. In some embodiments, the reaction temperature is -20 to 0 °C, and in some embodiments, the reaction time is 0.5 to 6 hours.

[0046] In formula I, R 4 When selected from amino acid alkyl ester phosphoric acid phenyl ester groups, in some embodiments, the method for preparing the cordycepin derivative represented by formula I is to use tert-butylmagnesium chloride as a catalyst in anhydrous N,N-dimethylformamide and / or tetrahydrofuran to react compound I-R-4 with nitrophenyl phosphate substituted with a modifier, and the substitution is with one or more functional groups of an aryloxy group or a methyloxy group of an alkyl acid. In some embodiments, the ratio of the amounts of use of the compound I-R-4, modifier, catalyst, and organic solvent is 1 mmol:1 - 3 mmol:1 - 2 mmol:9 - 15 mL. In some embodiments, the reaction temperature is 20 to 40 °C, and in some embodiments, the reaction time is 2 to 5 hours.

[0047] E.R 5 To modify In formula I, R 5 When selected from a bromovinyl group, a mercapto group, a methyl group, fluorine, or chlorine, the method for preparing the cordycepin derivative represented by formula I is to carry out a chemical reaction using compound I-R-5 as a raw material in an organic solvent to obtain the cordycepin derivative represented by formula I. [Chemical formula]

[0048] In formula I-R-5, R 1 ~R 4 、R 6 ~R 7 is the same as R 1 ~R 4 、R 6 ~R 7 in formula I, or is each independently selected from protecting groups.

[0049] In formula I, R 5When it is selected from bromovinyl groups, in some embodiments, the method for preparing the cordycepin derivative represented by formula I is to subject compound I-R-5 to an iodination reaction to obtain intermediate I-R-5a, 2-(6-amino-2-iodo-1,6-dihydro-9H-purin-9-yl)-5-(hydroxymethyl)tetrahydrofuran-3-ol or a derivative thereof, subject intermediate I-R-5a to a substitution reaction with methyl acrylate to obtain 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 a derivative thereof, hydrolyze intermediate I-R-5b to obtain intermediate I-R-5c, (E)-3-(6-amino-9-3-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-6,9-dihydro-1H-purin-2-yl)acrylic acid or a derivative thereof, subject intermediate I-R-5c to a bromination reaction with N-bromosuccinimide to obtain the cordycepin derivative represented by formula I. In some embodiments, the method for preparing the intermediate I-R-5a is to subject compound I-R-5 and elemental iodine to an iodination reaction in dilute nitric acid to obtain intermediate I-R-5a. In some embodiments, the ratio of the usage amounts of compound I-R-5 and elemental iodine is 1 mmol:0.5 to 0.8 mmol. In some embodiments, the reaction temperature is 100 to 120 °C. In some embodiments, it is 110 °C. In some embodiments, the reaction time is 4 to 6 hours. In some embodiments, the method for preparing the intermediate I-R-5b is to react intermediate I-R-5a with methyl acrylate and triethylamine in 1,4-dioxane under the catalysis of palladium acetate and triphenylphosphine.In some embodiments, the ratio of the usage amounts of the intermediate I-R-5a, methyl acrylate, triethylamine, palladium acetate, triphenylphosphine, and 1,4-dioxane is 1 mmol: 3 to 4 mmol: 0.1 to 0.5 mL: 0.01 to 0.09 mmol: 0.05 to 0.15 mmol: 10 to 20 mL. Depending on the embodiments, it is 1 mmol: 3 to 4 mmol: 0.1 to 0.5 mL: 0.05 mmol: 0.01 mmol: 10 to 20 mL. In some embodiments, the reaction temperature is 50 to 90 °C. In some embodiments, the reaction time is 0.5 to 2 hours. In some embodiments, the preparation method of the intermediate I-R-5c is to carry out a hydrolysis reaction between the intermediate I-R-5b and a sodium hydroxide solution. In some embodiments, the concentration of the sodium hydroxide solution is 0.5 to 3.5 mol / L. Depending on the embodiments, it is 2 mol / L. In some embodiments, the ratio of the usage amounts of the intermediate I-R-5b and the sodium hydroxide solution is 1 g: 10 to 14 mL. In some embodiments, it is 1 g: 12 mL. In some embodiments, the reaction temperature is 20 to 30 °C. Depending on the embodiments, it is room temperature. In some embodiments, the reaction time is 3 to 5 hours. In some embodiments, the production method of the cordycepin derivative represented by formula I is to react the intermediate I-R-5c and N-bromosuccinimide in a mixed solvent of water and acetone under a potassium carbonate catalyst. In some embodiments, the ratio of the usage amounts of the intermediate I-R-5c, N-bromosuccinimide, potassium carbonate, and the mixed solvent is 1 mmol: 1 to 3 mmol: 1 to 3 mmol: 15 to 30 mL. In some embodiments, the volume ratio of water to acetone is 1: 4 to 8. Depending on the embodiments, it is 1: 6.

Chemical formula

[0050] In some embodiments, directly modify unmodified cordycepin with bromovinyl, and the reaction pathway is

Chemical formula

[0051] In formula I, R 5When selected from a mercapto group, in some embodiments, the method for preparing the cordycepin derivative represented by Formula I is to react Compound I-R-5 with hydrogen peroxide in acetic acid to obtain Intermediate I-R-5d, 1N-oxide-3'-deoxyadenosine or a derivative thereof, heat and reflux Intermediate I-R-5d in an aqueous hydrochloric acid solution to obtain Intermediate I-R-5e, 5-amino-N'-hydroxy-3-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-1H-imidazole-4-carboxamide or a derivative thereof, dissolve Intermediate I-R-5e in water, react it with a Raney nickel catalyst under a hydrogen atmosphere to obtain Intermediate I-R-5f, 5-amino-3-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-1H-imidazole-4-carboxamide or a derivative thereof, and react Intermediate I-R-5f with methanol, pyridine and carbon disulfide to obtain the cordycepin derivative represented by Formula I. In some embodiments, the method for preparing the Intermediate I-R-5d is to react Compound I-R-5 with acetic acid in hydrogen peroxide water. In some embodiments, the concentration of the hydrogen peroxide water is 20% to 40%, in some embodiments the concentration of the hydrogen peroxide water is 30%, in some embodiments, the ratio of the amounts of Compound I-R-5, hydrogen peroxide water and acetic acid used is 1 mmol: 2 to 3 mol: 1 to 6 mL, in some embodiments, the reaction temperature is 30 to 50 °C, and in some embodiments, the reaction time is 2 to 4 days. In some embodiments, the method for preparing the Intermediate I-R-5e is to heat and reflux Intermediate I-R-5d in an aqueous hydrochloric acid solution. In some embodiments, the concentration of the aqueous hydrochloric acid solution is 1 to 5 mol / L, and in some embodiments it is 3 mol / L depending on the example. In some embodiments, the ratio of the amounts of Intermediate I-R-5d and the aqueous hydrochloric acid solution used is 1 mmol: 3 to 5 mL, and in some embodiments, the heat reflux time is 10 to 30 minutes. In the method for preparing the Intermediate I-R-5f, in some embodiments, the ratio of the amounts of Intermediate I-R-5e, Raney nickel and water used is 1 mmol: 0.08 to 0.2 g: 10 to 20 mL, in some embodiments, the reaction temperature is 50 to 70 °C, and in some embodiments, the reaction time is 2 to 5 days.In the preparation method of the corcidin derivative represented by formula I, in some embodiments, the ratio of the usage amounts of the intermediate I-R-5f, methanol, pyridine and carbon disulfide is 1 mmol: 5-10 mL. In some embodiments, the ratio of the usage amounts of the methanol, pyridine and carbon disulfide is 4: 3-7: 0.5-3.5, and in some embodiments, it is 4: 5: 2. In some embodiments, the reaction temperature is 30-50 ° C, and in some embodiments, it is 40 ° C. In some embodiments, the reaction time is 3-5 days.

Chemical formula

[0052] In some embodiments, unmodified corcidin is directly modified with a mercapto group. The specific reaction method is

Chemical formula

[0053] In formula I, when R 5 is selected from a methyl group, in some embodiments, the preparation method of the corcidin derivative represented by formula I is to react compound I-R-5, iodomethane and potassium carbonate in a dichloromethane solvent. In some embodiments, the ratio of the usage amounts 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. In some embodiments, the reaction time is 3-10 hours.

[0054] In formula I, R 5When it is selected from fluorine or chlorine, in some embodiments, the method for preparing the cordycepin derivative represented by formula I is to nitro-derivatize compound I-R-5 to obtain a nitration intermediate I-R-5g, 2-(6-amino-2-nitro-1,6-dihydro-9H-purin-9-yl)-5-(hydroxymethyl)tetrahydrofuran-3-ol, and then further carry out a substitution reaction to obtain the cordycepin derivative represented by formula I. In some embodiments, the method for preparing the intermediate I-R-5g is to react compound I-R-5 with tetrabutylammonium nitrate in dichloromethane in the presence of anhydrous trifluoroacetic acid as a catalyst. In some embodiments, the ratio of the amounts of use 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 to 10 °C. In some embodiments, the reaction time is 0.5 to 20 hours. In some embodiments, the method for preparing the cordycepin derivative represented by formula I is to react the nitration intermediate I-R-5g with tetrabutylammonium fluoride or tetrabutylammonium chloride in acetonitrile. In some embodiments, the ratio of the amounts of use of the nitration 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 to 5 °C, and in some examples it is 0 °C. In some embodiments, the reaction time is 20 to 30 minutes.

Chemical formula

[0055] F.R 6 to modify In formula I, R 6is selected from the carboxamide groups to be replaced, and when the replacement is substituted with any one or more functional groups of an alkyl group, an aryl group, a cycloalkyl group, a furyl group, or a pyridyl group, the method for preparing the cordycepin derivative represented by Formula I is to carry out a chemical reaction using Compound I-R-6 as a raw material in an organic solvent to obtain the cordycepin derivative represented by Formula I.

Chemical formula

[0056] In Formula I-R-6, R 1 ~R 5 、R 7 is the same as R 1 ~R 5 、R 7 in Formula I, or is independently selected from protecting groups.

[0057] In some embodiments, the method for preparing the cordycepin derivative represented by Formula I is to react Compound I-R-6 with a substituted acid chloride in anhydrous pyridine. The substitution is with any one or more functional groups of an alkyl group, an aryl group, a cycloalkyl group, a furyl group, or a pyridyl group. In some embodiments, the ratio of the amounts of Compound I-R-6, the substituted acid chloride, and anhydrous pyridine used is 1 mmol: 1-2 mmol: 5-10 mL. In some embodiments, the reaction temperature is 0-60 °C. In some embodiments, the reaction time is 2-20 hours.

[0058] G.R 7 for modification In Formula I, when R 7 is selected from isopropylamino groups, the method for preparing the cordycepin derivative represented by Formula I is to carry out a chemical reaction using Compound I-R-7 as a raw material in an organic solvent to obtain the cordycepin derivative represented by Formula I.

Chemical formula

[0059] In Formula I-R-7, R1 ~R 6 is R in formula I 1 ~R 6 is the same as or is independently selected from protecting groups

[0060] In some embodiments, the method for preparing the corjesepine derivative represented by formula I is to react compound I-R-7 with 2-propylamine in dioxane. In some embodiments, the ratio of the amounts of use of the 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 °C. In some embodiments, the reaction time is 10-30 hours.

[0061] In the present invention, the protecting groups include, but are not limited to, -OTBS, -OAc, -NHCbz, -OTBPS, -OTBDPS. In some embodiments, R 4 is R in formula I 4 is the same as or is selected from -OTBS, -OAc, -OTBDPS. In some embodiments, R 6 is R in formula I 6 is the same as or is selected from -NHCbz

[0062] In the present invention, the substituted nitrophenyl phosphate (the substitution is by any one or more functional groups of an alkoxy group, an alkoxy group substituted with a halogen, an aryloxy group, an amido amino acid group, an alkyl ester group, a methyl oxyalkyl acid group) is obtained by the following method or other methods of the prior art.

[0063] In anhydrous dichloromethane, phenyl chlorophosphate, p-nitrophenol and the corresponding substituted alcohol or amine are prepared with triethylamine as a catalyst in an environment of 0 to 25 °C, and the substitution is any one or more functional groups selected from an alkoxy group, an alkoxy group substituted with a halogen, an aryloxy group, an amido amino acid group, an alkyl ester group, and a methyloxy alkyl acid group. In some embodiments, the molar volume ratio of the phenyl chlorophosphate, p-nitrophenol, the corresponding substituent, triethylamine and anhydrous dichloromethane is 1 mmol: 1 mmol: 1 to 2 mmol: 2 to 5 mmol: 5 to 10 mL.

[0064] In the present invention, the substituted p-toluenesulfonyloxymethyl phosphate (the substitution is any one or more functional groups selected from an alkoxy group, an alkoxy group substituted with a halogen, an aryloxy group, an amido amino acid group, an alkyl ester group, and a methyloxy alkyl acid group) is obtained by the following method or other methods of the prior art.

[0065] The corresponding substituted chlorophosphate, p-toluenesulfonyl chloride, and formaldehyde are prepared with triethylamine as a catalyst in toluene in an environment of 0 to 105 °C. The substituted chlorophosphate is a chlorophosphate substituted with any one or more functional groups selected from an alkoxy group, an alkoxy group substituted with a halogen, an aryloxy group, an amido amino acid group, an alkyl ester group, and a methyloxy alkyl acid group. 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 to 1.2 mol: 180 to 210 mL: 500 to 800 mL.

[0066] As can be easily understood by those skilled in the art, the present invention is to modify the group of R 1 ~R 7 However, R 1 is hydrogen, R 2 is hydrogen, R3 is hydrogen, and R 5 is hydrogen, and R 7 is hydrogen, and R 4 is a hydroxyl group, or when R 6 is an amino group, there is no need to modify them. In the preparation method of the present invention, each of the above substituents can be modified according to the priority order of the stability of the prepared intermediate without going against the common knowledge in the technical field, so as to obtain each example of the present invention. For example, modifications that do not contain an ester group, a phosphate group, or a phosphonic acid group (for example, modifications such as -CN, -N 3 , -F, -SH, etc.) are carried out first, then the modification of the ester group is carried out, and finally the modification of the phosphate group or the phosphonic acid group can be carried out.

[0067] The term "prevention" in the present invention means administering the compounds or formulations described herein to prevent a disease or one or more symptoms associated with the disease, and preventing the occurrence of a disease or disease state in a mammal, particularly a cancer symptom that these mammals are prone to induce.

[0068] The term "pharmaceutically acceptable" in the present invention means a compound, material, composition and / or dosage form suitable for contact use with human and animal tissues within the scope of reliable medical judgment without excessive toxicity, irritation, anaphylaxis or other problems or complications commensurate with a reasonable benefit / risk ratio.

[0069] The compounds of the present invention may exist in specific geometric forms 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, and racemic mixtures and other mixtures, such as mixtures enriched in enantiomers or diastereomers, and all of these mixtures are within the scope of the present invention. Substituents such as alkyl groups may have additional asymmetric carbon atoms. All of these isomers and their mixtures are included within the scope of the present invention.

Advantages of the Invention

[0070] Compared with the prior art, the beneficial effects of the present invention are as follows. The colchicine derivatives and pharmaceutical compositions thereof according to the present invention have excellent anti-tumor growth effects. Compared with the parent drug, the colchicine derivatives have excellent affinity for cell membranes, so the half-life of the drug in the body is prolonged, and the residence time in the body is longer. Compared with other nucleoside anti-tumor drugs, the colchicine derivatives and pharmaceutical compositions thereof according to the present invention have a wider range of tumor types and action ranges, and have excellent inhibitory effects on gastric cancer, pancreatic cancer, liver cancer, small cell lung cancer, colorectal cancer, melanoma, ovarian cancer, etc., with reduced side effects and excellent therapeutic effects.

Brief Description of the Drawings

[0071]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Modes for Carrying Out the Invention

[0072] Unless otherwise specified, all the experimental methods described in the following examples are conventional methods. Unless otherwise specified, all reagents and materials are commercially available. Unless otherwise specified, drug evaluation experiments, including cell models and animal models, are commercially available.

[0073] The cordycepin described in the present invention is obtained from the preparation of the biotransformation pathway (specification of Chinese Patent No. 111117896). The preparation method of cordycepin or its derivatives is prepared based on the method in the specification, and the post-treatment method mainly adopts the operation methods of post-treatment of conventional organic experiments such as filtration, quenching, extraction, rotary evaporation, recrystallization, column chromatography, etc., but is not limited thereto. The preparation methods of the compounds described in the examples are not for all compounds. For the sake of convenience of explanation, only the preparation processes of representative compounds are listed in this specification.

[0074] The experiments on cells related to the anti-cancer effect of cordycepin or its derivatives and animal model experiments in the examples are not all the results of effective experiments, but only related experiments cited for the purpose of explaining the effect. And drug evaluation experiments including cell models and animal models comply with ethical rules.

[0075] Regarding the breeding of mouse models in drug evaluation, the mice are bred in a laminar flow cabinet, with 5 nude mice bred per cage. Drinking water and feed are added once every 3 days, and the bedding is changed once a week. The number of mice bred in each cage is 5 or less. As much as possible, the mice are not bred in a single cage, but are grouped together in a single cage. Animal management must comply with national standards regarding the breeding and management of animals. In order to avoid the occurrence of unnecessary stress, attention is paid to the behavior of the animals, satisfying the normal physiological and behavioral desires of the animals, such as defecation, urination, maintaining a constant body temperature, normal activities, adjusting posture, and reproduction. The ventilation is good, the animals are kept in a dry state, they can freely ingest drinking water and food, and the replenishment, replacement operation, and cleaning are easy. In order to avoid accidents such as the escape of animals and being pinched into the gaps between the limbs, a strong and safe environment is provided, avoiding the harm caused by sharp ends or protrusions to the animals, and observing without disturbing the animals, all in accordance with the above.

[0076] The zebrafish model is a wild-type AB strain zebrafish derived from the College of Biotechnology and Pharmaceutical Engineering of Nanjing Tech University, and is carried out by natural mating and breeding. There are 30 in each experimental group, and the age is 2 dpf. It is bred in fish culture water at 28 °C (water quality: adding 200 mg of quick-soluble sea salt per liter of reverse osmosis water, with a conductivity of 480 - 510 μS / cm, a pH of 6.9 - 7.2, and a hardness of 53.7 - 71.6 mg / L CaCO 3 is). The breeding management meets the requirements of international AAALAC accreditation.

[0077] The method for measuring the inhibitory activity of a compound on tumor cell proliferation by the MTT method is to take one tube of cells in a good state of logarithmic growth phase, add 0.25% trypsin digestive solution, digest to make the adherent cells fall off, and count to 2 - 4×10 4 cells / mL to make a cell suspension. The cell suspension is inoculated into a 96-well plate at 90 μL / well, and incubated in a constant temperature CO 2It was cultured in an incubator for 24 hours. The obtained compound was added and tested, and then cultured at 10 μL / well for 72 hours. 10 μL / well of MTT reagent was added to a 96-well plate, reacted in an incubator for 4 hours, the supernatant was aspirated, 100 μL / well of dimethyl sulfoxide was added, and after the crystals were dissolved, the absorbance of each well was measured at a wavelength of 570 nm using a microplate reader, and the cell inhibition rate was calculated. An S-shaped curve was created with the compound concentration and the corresponding inhibition rate. The IC 50 was obtained.

[0078] (Example 1) Compound: Preparation of 2-(6-amino-9H-purin-9-yl)-5-(2-hydroxyethoxymethyl)tetrahydrofuran-3-ol

[0079] (1) Method for protecting and deprotecting the hydroxyl group of cordycepin The R 1 and R 4 methods for protecting and deprotecting the hydroxyl groups at the positions

Chemical formula

[0080] Protection: To 10 mL of DMF, 0.251 g (1 mmol) of cordycepin, 0.3618 g (2.4 mmol) of TBSCl, and 0.3404 g (5 mmol) of imidazole were added, stirred at room temperature for 10 hours. After the reaction was completed, it was extracted with water and ethyl acetate, and the organic phase was concentrated to obtain 1a (0.4607 g, yield 96.03%, MSI-MS: 480.8 [M+H] + ) was obtained.

[0081] Deprotection: 10 ml of solvent (tetrabutylammonium fluoride:THF = 2:1) was prepared, 0.48 g (1 mmol) of 1a was added, stirred at room temperature for 5 hours. After the reaction was completed, it was extracted with water and ethyl acetate, and the organic phase was concentrated and recrystallized to obtain cordycepin (0.246 g, yield 97.96%).

[0082] The R of cordycepin1 Method for Protecting and Deprotecting Hydroxyl Group at a Certain Position 1b

Chemical Structure

[0083] Protection: To 10 mL of DMF, add 0.251 g (1 mmol) of cordycepic acid, 0.3618 g (2.4 mmol) of TBSCl, and 0.3404 g (5 mmol) of imidazole, stir at room temperature for 10 hours. After the reaction is completed, extract with water and ethyl acetate, concentrate the organic phase to obtain 0.4607 g of 1a (yield 96.03%). Further, prepare 10 ml of solvent (acetic acid: water: THF = 13:7:3), add 0.48 g (1 mmol) of 1a, stir at room temperature for 15 hours. After the reaction is completed, extract with water and ethyl acetate, concentrate the organic phase, and obtain 1b (0.293 g, yield 79.99%, MSI-MS: 366.5[M+H] + ) by column chromatography.

[0084] Deprotection: Add 0.366 g (1 mmol) of 1b to 10 mL of 0.1 M hydrochloric acid methanol solution, stir at room temperature for 10 hours. After the reaction is completed, adjust the pH to neutral with 0.1 M aqueous sodium carbonate solution, extract with ethyl acetate, concentrate the organic phase, and obtain cordycepic acid (0.215 g, yield 85.56%) by rotary evaporation or recrystallization.

[0085] R of cordycepic acid 4 Method for Protecting and Deprotecting Hydroxyl Group at a Certain Position 1c

Chemical Structure

[0086] Protection: Under a nitrogen atmosphere, add 1.256 g (5 mmol) of cordycepic acid to 10 mL of dichloromethane and 10 mL of diisopropylethylamine solvent, then dropwise add 0.451 g (1.6 mmol) of TBDPSCl, stir at room temperature for 2 hours, concentrate, and purify by column chromatography to obtain 1c (2.20 g, yield 89.9%, MSI-MS: 366.5[M+H] + ) by column chromatography.

[0087] Deprotection: Prepare 10 ml of a solvent (tetrabutylammonium fluoride: THF = 2:1), add 0.490 g (1 mmol) of 1c, stir at room temperature for 5 hours. After the reaction is completed, extract with water and ethyl acetate, concentrate the organic phase, and recrystallize to obtain cordycepin (0.244 g, yield 97.01%).

[0088] (2) Specific preparation method of compound 2-(6-amino-9H-purin-9-yl)-5-(2-hydroxyethoxy)methyl)tetrahydrofuran-3-ol Using compound 1b as a raw material, add 3.65 g (10 mmol) of 1b and 0.81 g (10 mmol) of 2-chloro-ethanol to a round-bottom flask, add 60 mL of methyl ethyl ketone to the round-bottom flask to dissolve the reactants by heating, and further add 2.07 g (15 mmol) of potassium carbonate to the round-bottom flask. The reaction was carried out at 80 °C for 8 hours, and the progress of the reaction was monitored by TLC. After the reaction was completed, filter, concentrate the reaction solution, add 60 mL of water and 60 mL of ethyl acetate and extract twice, combine the organic phases, perform rotary evaporation under reduced pressure to obtain a viscous liquid, deprotect the viscous liquid, and obtain compound 1 (weight 2.24 g, yield 76%) by column chromatography. The detection results of the obtained compound 1 were 1 HNMR(400MHz,DMSO-d6)δ8.56(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 CNMR(100MHz,DMSO-d6)δ156.1,152.4,149.8,140.1,119.6,95.2,82.1,75.2,74.7,70.4,61.1,34.5.MSI-MS:296.3[M+H] + as follows.

[0089] (Example 2) Preparation of compound: Methyl dihydrogen phosphate of (5-(6-amino-9H-purin-9-yl)-4-hydroxytetrahydrofuran-2-yl) According to the protection method of Example 1, 3.65 g (10 mmol) of compound 1b was taken, and 150 mL of triethyl phosphite and 4.62 g (30 mmol) of phosphorus oxychloride were added under ice bath conditions. The reaction was carried out at 0 °C for 2 hours. After the reaction was completed, 1000 mL of water was added in an ice bath environment to quench the reaction, and dichloromethane was added for extraction multiple times. The organic phases were combined, and the solvent was removed by rotary evaporation to obtain a viscous liquid. Liquid phase separation purification was performed on the obtained viscous liquid (C18 preparative column, Waters liquid phase preparation, mobile phase: 30% aqueous acetonitrile solution, flow rate: 2 mL / min). After concentrating the effluent of the sample peak, the phosphorylated compound (total weight 3.02 g, yield 70%) was obtained.

[0090] According to the deprotection method of 1b in Example 1, compound 2 was obtained. The detection results of the obtained compound 2 were 1 HNMR(400MHz,DMSO-d6)δ8.54(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(100MHz,DMSO-d6)δ156.2,152.7,149.5,140.2,119.3,97.2,76.1,74.6,69.5,34.5.MSI-MS:332.2[M+H] + as follows.

[0091] (Example 3) ((((5-(6-amino-9H-purin-9-yl)-4-hydroxytetrahydrofuran-2-yl)methoxy)methyl)phosphonic acid

Chemical Structure

[0092] 36.5 g (100 mmol) of Compound 1b was added to a 500 mL reaction flask, and 150 mL of DMF was added as a solvent and heated to dissolve. After rapid cooling to room temperature, 6.4 g (267 mmol) of 80% NaH was added portionwise with stirring, and the mixture was stirred at room temperature for 15 minutes. The reaction solution was cooled to -10 °C in a low-temperature reactor, and 45.1 g (140 mmol) of diethyl p-toluenesulfonyloxymethylphosphonate was added dropwise. After the addition was complete, the reaction was stirred at this temperature for 1 hour, gradually warmed to room temperature and stirred for 4 hours. 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, the filtrate was extracted three times with water (60 mL), the dichloromethane phases were combined, the solvent was distilled off under reduced pressure, and toluene was added to the residue for recrystallization. The mother liquor of the crystals was filtered, and the filter cake was dried under reduced pressure at 50 °C to obtain a white powdery solid 3a (weight 26.87 g, yield 52%, MSI-MS: 540.6[M+23] + ) was obtained.

[0093] 5.17 g (10 mmol) of Compound 3a was taken and added to a 100 mL reaction flask, 20 mL of n-butyronitrile was added, and 6.52 g (60 mmol) of trimethylchlorosilane was added dropwise at room temperature. After the addition was complete, the temperature of the reaction solution was raised and refluxed for 24 hours. After the reaction was completed, the solvent was distilled off under reduced pressure to dryness by concentration. 20 mL of water was added to the residue, and 2M sodium hydroxide was further added to adjust the pH to ≒8. Then, it was extracted three times with ethyl acetate (50 mL), and the aqueous phase was neutralized with 1M hydrochloric acid until the pH = 3 - 4. Then, it was heated to 70 - 80 °C to crystallize to obtain Compound 3b (weight 3.40 g, yield 74%, MSI-MS: 460.6[M+H] + ) was obtained.

[0094] According to the deprotection method of Example 1b, Compound 3b was treated to obtain Compound 3. The detection results of the prepared compound were 1 HNMR(400MHz,DMSO-d6)δ8.58(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 CNMR(100MHz, DMSO-d6) δ 156.7, 152.6, 149.1, 140.4, 119.5, 98.2, 81.1, 76.3, 74.3, 73.1, 34.7. MSI-MS: 346.3 [M+H] + It was.

[0095] (Example 4) Preparation of compound: Diisobutyl ((5-(6-amino-9H-purin-9-yl)-4-hydroxytetrahydrofuran-2-yl)methyl) phosphate [Chemical formula]

[0096] 3.65 g (10 mmol) of compound 1b was added to a 500 mL reaction flask, dissolved in 100 mL of anhydrous DMF, and 4.96 g (15 mmol) of diisobutyl phosphate (4-nitrophenyl) was further added. Further, 1.17 g (10 mmol) of tert-butylmagnesium chloride was dissolved in 20 mL of THF, and the solution was gradually added dropwise to the above reaction solution. The reaction solution was gradually heated to room temperature and reacted for 2 hours, and the reaction was monitored with a TLC plate. After completion of the reaction, the reacted mixture was allowed to stand, diluted with 100 mL of ethyl acetate, washed and extracted three times with 50 mL each of saturated aqueous sodium hydrogen carbonate solution, and then extracted with 50 mL of saturated aqueous sodium chloride solution. The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The obtained oil was purified by silica gel column chromatography (eluent: dichloromethane / methanol = 10:1) to obtain compound 4a (weight 4.24 g, yield 76%).

[0097] According to the deprotection means of 1b in Example 1, 4a was treated to obtain compound 4, and the detection results of the prepared compound 4 were 1HNMR(400MHz, DMSO-d6) δ 8.52 (s, 1H), 8.31 (s, 1H), 7.11 (s, 2H), 6.15 (d, 1H), 5.33 (d, 1H), 4.24 - 4.05 (m, 2H), 4.02 (m, 1H), 3.93 (m, 4H), 3.77 (m, 1H), 2.08 - 1.83 (m, 2H), 1.33 (m, 2H), 0.90 (d, 12H). 13 CNMR(100MHz, DMSO-d6) δ 154.3, 151.7, 148.4, 141.9, 119.2, 98.2, 74.1, 74.0, 73.5, 68.1, 34.2, 28.6, 19.5. MSI-MS: 466.7[M+Na] + It was

[0098] (Example 5) Compound: Preparation of ((((5-(6-amino-9H-purin-9-yl)-4-hydroxytetrahydrofuran-2-yl)methoxy)methyl)phosphoryl)bis(oxy)bis(methylene)bis(2-methylpropionic acid) [Chemical formula]

[0099] According to the preparation method of 3a in Example 3, in combination with the deprotection method of 1b in Example 1, Compound 5 was prepared, and diethyl p-toluenesulfonyloxymethylphosphonate was replaced with an equivalent amount in ((((toluyloxy)methyl)phosphoryl)bis(oxy))bis(methylene)bis(2-methylpropionic acid) to obtain Compound 5 (total yield 74%). The detection results of the obtained Compound 5 were 1 HNMR(400MHz, 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). 13CNMR (100 MHz, DMSO-d6) δ 174.2, 155.7, 153.6, 149.5, 141.2, 119.5, 99.4, 93.2, 77.4, 74.5, 74.1, 71.5, 34.2, 33.6, 19.3. MSI-MS: 546.5 [M+H] + It was

[0100] (Example 6) Preparation of methyl ((5-(6-amino-9H-purin-9-yl)-4-hydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)-D-valine, and the preparation process is as follows. [Chemical formula]

[0101] The preparation method of methyl ((4-nitrophenoxy)(phenoxy)phosphoryl)-D-valine is as follows.

[0102] 1.17 g (10 mmol) of valine was dissolved in 50 mL of dichloromethane. The solution was cooled to about 0 °C, 2.11 g (10 mmol) of phenyl dichlorophosphate was added, and then 2.02 g (20 mmol) of triethylamine was slowly added dropwise. The reaction mixture was warmed to room temperature and stirred for 80 minutes. 1.39 g (10 mmol) of p-nitrophenol was added to the reaction mixture, and then 2.02 g (20 mmol) of triethylamine was added dropwise, followed by stirring at room temperature for 180 minutes. After completion of the reaction, it was washed with diethyl ether, and the obtained solid was removed by filtration. The filtrate was concentrated using a rotary evaporator, and the obtained sample was purified by silica gel column chromatography (eluent: n-hexane / ethyl acetate (1:1)). The detection results of the obtained compound are 1 HNMR (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). 13CNMR (100 MHz, DMSO-d6) δ 171.6, 156.5, 150.5, 141.1, 130.2, 126.5, 121.7, 121.4, 120.4, 52.2, 51.9, 26.4, 9.6. MSI-MS: 395.3 [M+H] + It was

[0103] According to the preparation method of 4a in Example 4, in combination with the deprotection method of 1b in Example 1, diisobutyl phosphate (4-nitrophenyl) was equivalently substituted with methyl ((4-nitrophenoxy)(phenoxy)phosphoryl)-D-valine to obtain Compound 6 (yield 68%). The detection results of the finally obtained Compound 6 were 1 HNMR (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 CNMR (100 MHz, DMSO-d6) δ 173.6, 154.9, 151.5, 150.6, 149.8, 141.3, 132.7, 123.3, 120.8, 119.2, 98.2, 76.1, 74.5, 72.0, 57.6, 52.3, 35.2, 32.4, 20.1. MSI-MS: 521.4 [M+H] + It was

[0104] (Example 7) Compound: Preparation of isopropyl ((((5-(6-amino-9H-purin-9-yl)-4-hydroxytetrahydrofuran-2-yl)methoxy)methyl)(phenoxy)phosphoryl)alanine ester, and the preparation process is as follows. [Chemical formula]

[0105] The preparation method of isopropyl (phenoxy((toloxy)methyl)phosphoryl)alanine ester is as follows.

[0106] Into a 1000 mL reaction flask, add 216.15 g (1.65 mol) of isopropyl alanine ester, 155 g (1.65 mol) of phenol, and 240 mL of chloroform. While stirring under ice-salt bath conditions, dropwise add 138.8 g (1 mol) of phosphorus trichloride starting from around 5 °C, and control the reaction temperature between 5 and 10 °C. After the dropwise addition of phosphorus trichloride is completed, continue stirring for 30 minutes, remove the ice-salt bath, gradually warm up to room temperature, continue stirring for 2 hours, then reduce the pressure at 50 °C to remove hydrogen chloride gas. Cool the reaction product to room temperature, slowly add 80 mL of saturated sodium bicarbonate aqueous solution, then add solid sodium bicarbonate powder to adjust the pH of the reaction solution to 7 - 8. Remove the precipitated salt by filtration, put the filtrate into a separatory funnel to separate the chloroform layer, extract the aqueous layer once with 100 mL of chloroform, combine the organic phases, wash them once each with saturated sodium bicarbonate aqueous solution and water, take the chloroform layer, and perform vacuum distillation. The residue is the target product, phenylalanine isopropyl chlorophosphate, and the yield is 87%.

[0107] Into a 1000 mL reaction flask, 306 g (1 mol) of phenylalanine isopropyl chlorophosphate, 40.6 g (1.35 mol) of paraformaldehyde, 14 mL of triethylamine, and 260 mL of toluene were added. The reaction was gradually heated to 105 °C under a nitrogen atmosphere. At this time, the reaction was intense, and it was refluxed for 3 hours. After the reaction was completed, it was cooled to 0 °C in an ice-salt bath, 169.5 g (0.9 mol) of p-toluenesulfonyl chloride was added, 220 mL of toluene was added, and then 177 mL of triethylamine was added dropwise. It was stirred for 2 hours while maintaining the reaction temperature at 0 °C, gradually heated to room temperature, and stirred for 12 hours. After the reaction was completed, it was filtered, the filter cake was washed with toluene (50 mL × 3), and the mother liquor was combined and washed successively with 5% aqueous sodium carbonate solution (200 mL × 2) and water (200 mL × 2), and then the layers were separated to obtain an organic phase. The organic phase was distilled under reduced pressure to obtain isopropyl (phenoxy((toloxy)methyl)phosphoryl)alanine ester as a pale yellow oily substance, which was used in the subsequent reaction. The weight was 291 g, the yield was 64%, and the detection results of the obtained compound were, 1 HNMR(400MHz,DMSO-d6)δ7.74(d,2H),7.45-7.39(m,4H),7.22(m,3H),4.95(m,1H),3.91(d,2H),3.65(s,1H),3.57(m,1H),2.42(s,3H),1.28(d,3H),1.18(d,6H). 13 CNMR(100MHz,DMSO-d6)δ171.6,150.4,144.4,140.3,130.5,130.1,128.5,121.4,120.2,69.6,63.8,50.5,21.7,21.5,19.2.MSI-MS:456.5[M+H] + as follows.

[0108] According to the preparation method of 3a in Example 3, in combination with the deprotection method of 1b in Example 1, Compound 7 was prepared, and diethyl p-toluenesulfonyloxymethylphosphonate was replaced with isopropyl (phenoxy((toloxy)methyl)phosphoryl)alanine ester in an equivalent amount to obtain Compound 7 (yield 52%). The detection results of the obtained Compound 7 were, 1HNMR(400MHz, 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 CNMR(100MHz, DMSO-d6) δ 172.1, 156.3, 152.5, 150.4, 149.8, 140.1, 130.2, 122.1, 120.5, 119.1, 98.2, 79.1, 74.8, 74.7, 73.1, 69.6, 52.3, 34.5, 22.5, 18.8. MSI-MS: 535.6[M+H] + It was as follows.

[0109] (Example 8) 2-(-5-(6-Amino-9H-purin-9-yl)-4-hydroxytetrahydrofuran-2-yl)methoxy)-3-hydroxypropyl valine salt [Chemical formula]

[0110] Using compound 1b as a raw material, 3.65 g (10 mmol) of 1b and 2.10 g (10 mmol) of 2-chloro-3-hydroxypropyl valine were added to a round-bottom flask. 60 mL of methyl ethyl ketone was added to the round-bottom flask to dissolve the reactants by heating. Further, 2.07 g (15 mmol) of potassium carbonate was added to the round-bottom flask. The reaction was carried out at 80 °C for 10 hours, and the progress of the reaction was monitored by TLC. After the reaction was completed, filtration was performed, the reaction solution was concentrated, 60 mL of water and 60 mL of ethyl acetate were added, and extraction was carried out twice. The organic phases were combined and rotary evaporated under reduced pressure to obtain a viscous liquid. The viscous liquid was subjected to column chromatography to obtain compound 8a (weight 3.24 g, yield 52%, MSI-MS: 539.6[M+H] + ) was obtained.

[0111] Using compound 8a as a substrate, it was prepared according to the deprotection method of 1b in Example 1 to obtain compound 8 (yield 91%). The detection results of the obtained compound 8 are as follows: 1 HNMR(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 CNMR(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. MSI-MS: 425.4[M + H] + It was as follows.

[0112] (Example 9) The preparation of methyl (4-amino-6-(6-amino-9H-purin-9-yl)-2,2-dioxide-1,7-dioxa-2-azaspiro[4.4]non-3-en-8-yl) dihydrogen phosphate and the preparation method are as follows. [Chemical formula]

[0113] Reacting compound 2 as a raw material, 9.00 g (27.2 mmol) of compound 2 and 4.88 g (27.2 mmol) of CbzCl were added to 38.5 mL of toluene and 38.5 mL of water, and further 4.70 g (34 mmol) of K 2 CO 3 was added. The mixture was vigorously stirred at a temperature of 25°C or lower. Then, after stirring at room temperature for 3 hours, 0.275 g (2.72 mmol) of triethylamine and 5.78 g of sodium chloride were sequentially added, and the mixture was stirred for another 30 minutes. The organic layer was separated and concentrated to obtain the desired oily product 9a (weight 11.38 g, yield 90%, MSI-MS: 488.7[M + Na] + ).

[0114] At room temperature, 9.3 g (20 mmol) of compound 9a was stirred and dissolved in 60 mL of acetone. 5.50 mL (2.2 M chromium(III) oxide, 12.1 mmol) of Jones reagent was added to the solution and added dropwise at room temperature for 2 hours. The resulting reaction mixture was further stirred at room temperature for 1 hour, filtered, and concentrated under reduced pressure to obtain an oily liquid. This liquid was dissolved in 30 mL of diethyl ether, washed once with 30 mL of saturated ammonium chloride solution, dried over anhydrous magnesium sulfate, concentrated under reduced pressure, and then subjected to column chromatography to obtain compound 9b (weight 3.98 g, yield 43%, MSI-MS: 464.4 [M+H] + ) was obtained.

[0115] 4 mL (30 mmol) of trimethylsilyl cyanide and 2.53 mL (20 mmol) of boron trifluoride diethyl etherate were added to 50 mL of a dichloromethane solution of 9.26 g (20 mmol) of 9b. The mixture was stirred at room temperature for 2 hours, and the solvent was evaporated until dry. The resulting residue was dissolved in 100 mL of ethyl acetate, washed twice with 50 mL of brine, and dried (Na 2 SO 4 ). After filtering and evaporating the solvent, it was purified by column chromatography (n-hexane / ethyl acetate, 1:2) to obtain white compound 9c (weight 7.45 g, yield 76%, MSI-MS: 491.9 [M+H] + ) was obtained.

[0116] Et 3 1.9 mL of N (14 mmol) was added to 5 mL of anhydrous dichloromethane, and 1.08 g (2.2 mmol) of compound 9c was added to the solution and dissolved. After cooling the mixture to -30 °C, 460 μL (6 mmol) of methanesulfonyl chloride was slowly added. The mixture was continuously stirred at -20 °C for 1 hour and continuously stirred at 0 °C for 1 hour. Then, the volatiles were removed by concentration under reduced pressure, and the residue was dissolved in 10 mL of ethyl acetate and then washed twice successively with 10 mL of water and 10 mL of brine. The organic phase (Na 2 SO 4) was dried, concentrated under reduced pressure, and purified by column chromatography (n-hexane:ethyl acetate, 10:1) to obtain a white amorphous solid 9d (weight 0.975 g, yield 78%). The detection results of the preparation were 1 HNMR(400MHz,DMSO-d6)δ10.45(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 CNMR(100MHz,DMSO-d6)δ153.6,152.5,151.7,149.8,140.2,136.4,128.9,127.7,127.2,123.5,120.4,97.4,71.6,69.2,68.2,66.9,38.2,37.8.MSI-MS:569.4[M+H] + as follows.

[0117] 490 mg (1.5 mmol) of cesium carbonate was added to a 3 mL anhydrous acetonitrile suspension of 0.568 g (1 mmol) of 9d, and the mixture was stirred at room temperature for 2 hours. The solvent was removed, and the resulting residue was dissolved in 20 mL of ethyl acetate and washed twice successively with 10 mL of water and 10 mL of brine. The organic phase (Na 2 SO 4 ) was dried, filtered, and then concentrated under reduced pressure. It was purified by column chromatography (n-hexane / ethyl acetate, 3:1) to obtain a white solid 9e (weight 4.49 g, yield 79%, MSI-MS:569.4[M+H] + ).

[0118] 5.68 g of compound 9e (10 mmol) was dissolved in 200 mL of methanol. Then, 1.5 g of ammonium formate (30 mmol) and 0.75 g of 10% Pd-C were added, and the reaction mixture was stirred at room temperature for 10 minutes and then heated to reflux for 45 minutes. The mixture was filtered through diatomaceous earth, and the filtrate was evaporated to dryness to obtain compound 9 (4.12 g, yield 95%). The detection results of the obtained compound 9 were 1HNMR(400MHz, 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 CNMR(100MHz, 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] + It was.

[0119] (Example 10) Preparation of compound: N-(3-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-9H-purin-6-yl) octadecanamide [Chemical formula]

[0120] 2.51 g (10 mmol) of cordycepin was taken, and 40.00 mL of anhydrous pyridine and 8.5 mL of acetic anhydride were added under ice bath conditions. The reaction was monitored by HPLC liquid phase, and the reaction was terminated after about 5 hours. The solvent was removed to obtain a viscous liquid 10a (weight 2.68 g, yield 80%, MSI-MS: 358.6 [M + Na] + )

[0121] 3.35 g (10 mmol) of compound 10a and 2.89 g (10 mmol) of octadecanoic acid chloride were taken, 60 mL of anhydrous pyridine was added under ice bath conditions, and the temperature was gradually raised to 40 °C and reacted for 10 hours. The progress of the reaction was monitored by TLC. After the reaction was completed, water and ethyl acetate were added for extraction, the organic phase was back-extracted, the organic phase was recovered and rotary evaporated to obtain an oily liquid 10b, which was purified by column chromatography. The purified product 10b (5.40 g, yield 90%, MSI-MS: 624.4 [M + Na] + )

[0122] 6.01 g (10 mmol) of compound 10b was taken, dissolved in 450 mL of methanol ammonia solution and reacted, stirred at room temperature, and the reaction was detected by thin layer chromatography. The reaction was stopped after 4 hours. The solvent was removed to obtain the target product 10 (weight 4.67 g, yield 90%).

[0123] Alternatively, with reference to the preparation method of 1a in Example 1 for protection and deprotection, after acylating the protected 1a by this second step, deprotection was carried out to obtain compound 10. The detection results of the obtained compound 10 were 1 HNMR (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 CNMR (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 peak overlap) MSI-MS: 540.7 [M+Na] + as follows.

[0124] (Example 11) N-(3-Hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-9H-purin-6-yl)cyclopentanecarboxamide According to the method of Example 10, compound 11 (total yield 72%) was obtained by the steps of protecting, acylating, and deprotecting cordycepin, and octadecanoic acid chloride was replaced with cyclopentanecarbonyl chloride. The detection results of the obtained compound 11 were 1HNMR(400MHz, 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 CNMR(100MHz, DMSO-d6) δ 172.5, 153.3, 151.5, 149.5, 139.8, 123.6, 98.4, 82.1, 74.9, 63.8, 49.5, 34.5, 32.4, 24.5. MSI-MS: 348.2[M + H] + It was as follows.

[0125] (Example 12) N-(3-Hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-9H-purin-6-yl)isonicotinamide According to the method of Example 10, compound 12 (total yield 76%) was obtained by the steps of protecting, acylating, and deprotecting cordycepin, and octadecanoic acid chloride was replaced with pyridine-3-carboxylic acid chloride. The detection results of the obtained compound 12 were 1 HNMR(400MHz, 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 CNMR(100MHz, 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] + It was as follows.

[0126] (Example 13) (5-(6-Amino-2-fluoro-9H-purin-9-yl)-4-hydroxytetrahydrofuran-2-yl)methyl dihydrogen phosphate [Chemical formula]

[0127] Under the environment of 0 °C, 193 μL (1.39 mmol) of trifluoroacetic anhydride was added to a solution of 428 mg (1.40 mmol) of tetrabutylammonium nitrate in dry dichloromethane (15 mL) to obtain a nitration mixture. After reacting at 0 °C for 45 minutes, the solution was slowly added to 480 mg (1 mmol) of compound 1a in dry dichloromethane (15 mL). After reacting under light shielding at room temperature for 14 hours, the reaction mixture was poured into a cold mixture of H 2 O (50 mL), saturated NaHCO 3 (35 mL) and CH 2 Cl 2 :Et 2 O (1:2, 30 mL) for extraction. It was extracted twice with CH 2 Cl 2 :Et 2 O (1:2, 30 mL). The organic extract was washed with brine and dried over anhydrous Na 2 SO 4 , and then dried under vacuum (maintaining the temperature below 40 °C). The crude product was purified by column chromatography, eluted with CH 2 Cl 2 , and then purified with CH 2 Cl 2 :acetone (99:1 - 95:5) to obtain compound 13a (weight 273 mg, yield 52%, MSI-MS: 525.4[M + H] + )

[0128] To a suspension of 236 mg (0.45 mmol) of 13a in dry acetonitrile (15 mL), TBAF (1.3 equivalents, 600 μL, 0.6 mmol) was added dropwise at 0 °C over 1 minute. The mixture was stirred for 20 minutes, and the resulting solution was evaporated under vacuum without heating. The crude product was purified by column chromatography (CH 2 Cl 2 :acetone (100:0 - 90:10)) to obtain compound 13b (weight 78 mg, yield 35%, MSI-MS: 498.8[M + H]+ ) was obtained.

[0129] The obtained compound 13b was prepared according to the phosphorylation method of Example 2 and the deprotection method of Example 1 to obtain compound 13 (yield 65%). The detection results of the obtained compound 13 were 1 HNMR (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 CNMR (100 MHz, DMSO-d6) δ 158.3, 157.2, 148.8, 141.5, 118.6, 97.2, 74.9, 74.6, 67.1, 35.5. MSI-MS: 372.2 [M+Na] + as follows.

[0130] (Example 14) (Methyl dihydrogen phosphate (5-(6-amino-2-mercapto-9H-purin-9-yl)-4-hydroxytetrahydrofuran-2-yl)) [Chemical formula]

[0131] 4.80 g (10 mmol) of compound 1a was added to 60 mL of acetic acid and stirred at 40 °C until dissolved. After cooling the solution to room temperature, 5 mL (25 mmol) of 30% hydrogen peroxide aqueous solution was added. The solution was stirred in an environment of 40 °C for 3 days, filtered, and the solid was recrystallized in water to obtain compound 14a (weight 3.37 g, yield 68%, MSI-MS: 497.4 [M+H] + ) was obtained.

[0132] 4.96 g (10 mmol) of 14a was added to 40 mL of 3M hydrochloric acid aqueous solution, and the mixture was heated to reflux until the solid was completely dissolved. After continuing to reflux for 10 minutes, it was cooled to room temperature. After removing the solvent, 20 mL of ethanol was added. The precipitated crystals were filtered and dried to obtain compound 14b (weight 2.92 g, yield 62%, MSI-MS: 487.5 [M+H] +) was obtained.

[0133] 4.86 g (10 mmol) of Compound 14b was dissolved in 100 mL of water, 1.0 g of activated Raney Ni was added in portions, and hydrogen gas was introduced. The reaction mixture was stirred at 55 °C for 4 days. Introduction of hydrogen gas was stopped, inorganic salts were removed by filtration, the solvent was removed by distillation under reduced pressure, the crude product was washed with ethanol and diethyl ether respectively, and after drying, gray solid 14c (weight 3.30 g, yield 70%, MSI-MS: 495.8 [M+Na] + ) was obtained.

[0134] Methanol, pyridine and carbon disulfide were mixed at a volume ratio of 4:5:2 to obtain 50 mL of a solution. 4.72 g (10 mmol) of 14c was added, and a balloon was attached to the reflux condenser to prevent the volatilization of carbon disulfide. The reaction mixture was stirred at 40 °C for 4 days. It was filtered, the crude product was heated to reflux in a 5% aqueous sulfuric acid solution by mass for 20 minutes, filtered while heating to remove impurities. It was cooled to room temperature, filtered, and dried to obtain gray crystals 14d (weight 2.82 g, yield 55%). The obtained Compound 14d was prepared according to the phosphorylation method of Example 2 and the deprotection method of Example 1 to obtain Compound 14 (yield 71%). The detection results of the obtained Compound 14 were 1 HNMR(400MHz,DMSO-d6)δ12.22(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 CNMR(100MHz,DMSO-d6)δ154.3,152.1,149.2,139.8,118.6,99.2,74.8,74.3,67.1,35.1.MSI-MS:364.3[M+H] + as follows.

[0135] (Example 15) (Methyl dihydrogen phosphate of (5-(6-amino-9H-purin-9-yl)-3-fluoro-4-hydroxytetrahydrofuran-2-yl)) [Chemical formula]

[0136] In 10 mL of DMF, 2.67 g (10 mmol) of adenosine, 3.618 g (24 mmol) of TBSCl, and 3.95 g (50 mmol) of pyridine were added, and the mixture was stirred at room temperature for 10 hours. After completion of the reaction, it was extracted with water and ethyl acetate, and the organic phase was concentrated and purified by column chromatography to obtain 15a (2.33 g, yield 47%, MSI-MS: 497.7 [M+H] + ) was obtained.

[0137] A solution of 4.96 g (10 mmol) of 15a and pyridine (1.5 mL) in dichloromethane (100 mL) was stirred under a nitrogen atmosphere and cooled to -5 °C. 2.5 mL (15 mmol) of trifluoromethanesulfonic anhydride was added dropwise, and the reaction was continued at 0 °C for 2 hours. After TLC indicated completion of the reaction, it was poured into ice water (100 mL), stirred, separated, and the aqueous phase was extracted with dichloromethane (100 mL). The dichloromethane phases were combined, washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, the filtrate was concentrated under reduced pressure, petroleum ether (40 mL) was added to the residue for recrystallization, and after filtration, it was dried to obtain 15b (weight 5.65 g, yield 90%). The detection results of the preparation were 1 HNMR(400MHz,DMSO-d6)δ8.59(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 CNMR(100MHz,DMSO-d6)δ156.2,152.5,149.7,140.2,119.5,118.4,96.5,86.4,83.5,73.8,63.2,30.8,30.6,25.8,0.MSI-MS:628.8[M+H] + as follows.

[0138] 15b 6.28 g (10 mmol) was dissolved in ethyl acetate (40 mL) under a nitrogen gas atmosphere, 10 mL (22 mmol) of a triethylamine solution of 37% hydrofluoric acid was added, and the mixture was stirred and heated to 70 °C for reaction for about 8 hours. After TLC indicated the completion of the reaction, it was cooled to room temperature, washed with saturated sodium hydrogen carbonate solution until neutral, washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, the filtrate was concentrated under reduced pressure, and the residue was recrystallized from anhydrous methanol (30 mL) to obtain solid 15c (weight 2.04 g, yield 41%). The obtained compound 15c was prepared according to the phosphorylation method of Example 2 and the deprotection method of Example 1 to obtain compound 15 (yield 77%). The detection results of the obtained compound 15 were 1 HNMR(400MHz,DMSO-d6)δ8.58(s,1H),8.37(s,1H),7.07(s,2H),6.15(d,1H),5.33(d,1H),4.70(m,1H),4.44(m,1H),4.28(m,1H),4.18(s,2H),4.02(m,1H)3.55(m,1H). 13 CNMR(100MHz,DMSO-d6)δ155.3,151.4,148.9,141.1,119.6,97.2,90.1,79.0,73.4,61.5.MSI-MS:350.2[M+H] + as follows.

[0139] (Example 16) Isopropyl ((((5-(6-amino-9H-purin-9-yl)-4-cyanotetrahydrofuran-2-yl)methoxy)methyl)(phenoxy)phosphoryl)alanine ester [Chemical formula]

[0140] Compound 7 was obtained according to the method of Example 7, and compound 16 was prepared using compound 7 as a raw material. 5.34 g (10 mmol) of compound 7 was taken and dissolved in 100 mL of dichloromethane solution, and 1.3 mL (1.50 g, 10 mmol) of trifluoromethanesulfonic acid was further added. After the reaction mixture was stirred for 10 minutes, 2.7 mL of trimethylsilyl triflate (10 mmol) was slowly added dropwise to the solution, and the resulting mixture was stirred in an environment of -40 °C for 30 minutes. Then, 3.96 g (40 mmol) of trimethylsilyl cyanide was slowly added, and the mixture was stirred for 2 hours. Thereafter, 3.5 mL of triethylamine was added dropwise, and the reaction mixture was heated to room temperature. Then, 5.5 g of solid sodium bicarbonate and 20.7 mL of water were added. The resulting mixture was stirred for 10 minutes. 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 (weight 1.71 g, yield 32%). The detection results of the obtained compound 16 were 1 HNMR(400MHz,DMSO-d6)δ8.57(s,1H),8.36(s,1H),7.41(m,2H),7.22(m,3H),7.09(s,2H),6.17(d,1H),4.99(d,1H),3.97(m,1H),3.74-3.33(m,6H),2.88(m,1H),2.04-1.92(m,2H),1.28-1.14(d,9H). 13 CNMR(100MHz,DMSO-d6)δ173.4,155.3,152.5,150.2,149.8,141.3,130.2,121.6,120.5,118.2,92.2,77.4,75.0,73.1,69.1,51.2,26.5,25.8,22.4,20.1.MSI-MS:544.5[M+H] + as follows.

[0141] (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-dimethylpropionic acid)

Chem.

[0142] Compound 13b was obtained according to the method of Example 13. Using 13b as the starting material, compound 17a was obtained according to the protection method of 1b in Example 1. Using compound 17a as the starting material, compound 17 was obtained according to the method for preparing 3a in Example 3. Diethyl (p-toluenesulfonyloxymethyl)phosphonate was replaced in an equivalent amount with (((triyloxy)methyl)phosphoryl)bis(oxy))bis(methylene)bis(2,2-dimethylpropionic acid), and 1b was replaced in an equivalent amount with 17a, with an overall yield of 61%. The detection results of the obtained compound 17 were 1 HNMR(400MHz,DMSO-d6)δ8.33(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 CNMR(100MHz,DMSO-d6)δ175.2,158.3,157.5,148.6,140.3,119.2,99.2,92.2,79.1,74.8,74.3,71.2,38.5,35.8,26.8.MSI-MS:592.5[M + H] + as follows.

[0143] (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-dimethylpropionic acid)

Chem.

[0144] Referring to the method of Example 15 and the protection method of 1b in Example 1, compound 18a was obtained, and compound 18 was obtained according to the method of preparing 3a in Example 3 using compound 18a as the raw material. Diethyl (p-toluenesulfonyloxymethyl)phosphonate was substituted in an equivalent amount for (((triyloxy)methyl)phosphoryl)bis(oxy))bis(methylene)bis(2,2-dimethylpropionic acid), and 1b was substituted in an equivalent amount for 18a, with an overall yield of 34%. The detection results of the obtained compound 18 are 1 HNMR(400MHz,DMSO-d6)δ8.59(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(100MHz,DMSO-d6)δ175.9,157.3,151.5,149.8,141.3,118.9,98.2,92.8,91.5,79.4,73.3,71.4,70.5,38.6,27.8.MSI-MS:592.4[M+H] + as follows.

[0145] (Example 19) ((((5-(6-Amino-2-mercapto-9H-purin-9-yl)-4-hydroxytetrahydrofuran-2-yl)methoxy)methyl)phosphoryl)bis(oxy)bis(methylene)bis(2,2-dimethylpropionic acid) [Chemical formula]

[0146] Compound 14d was obtained with reference to the method of Example 14. Using 14d as a raw material, compound 19a was obtained according to the protection method of 1b in Example 1. Using compound 19a as a raw material, compound 19 was obtained according to the method for preparing 3a in Example 3. Diethyl (p-toluenesulfonyloxymethyl)phosphonate was substituted in an equivalent amount for (((toluyloxy)methyl)phosphoryl)bis(oxy))bis(methylene)bis(2,2-dimethylpropionic acid), and 1b was substituted in an equivalent amount for 19a, with an overall yield of 18%. The detection results of the obtained compound 19 were 1 HNMR(400MHz,DMSO-d6)δ12.12(s,1H),8.34(s,1H),7.01(s,2H),6.84(s,4H),6.12(d,1H),5.35(d,1H),4.01(m,1H),3.93(m,1H),3.85(s,2H),3.63 - 3.37(m,2H),2.06 - 1.85(m,2H),1.27(s,18H). 13 CNMR(100MHz,DMSO-d6)δ175.8,155.4,152.5,149.7,140.5,118.9,98.7,92.8,78.1,74.9,74.7,71.2,38.7,34.5,27.5.MSI-MS:606.6[M + H] + as follows.

[0147] (Example 20) Isopropyl ((((5-(6-amino-2-fluoro-9H-purin-9-yl)-4-hydroxytetrahydrofuran-2-yl)methoxy)methyl)(phenoxy)phosphoryl)alanine ester [Chemical formula]

[0148] Compound 17a was obtained according to the methods of Examples 13 and 17, and compound 20 was prepared with reference to the method of Example 7. Diethyl (p-toluenesulfonyloxymethyl)phosphonate was substituted in an equivalent amount for isopropyl (phenoxy((toluyloxy)methyl)phosphoryl)alanine ester, and 1b was substituted for 17a, with an overall yield of 67%. The detection results of the obtained compound 20 were 1HNMR(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 CNMR(100 MHz, DMSO-d6) δ 172.1, 157.3, 156.8, 150.0, 149.8, 140.2, 121.3, 120.6, 119.2, 97.4, 76.6, 75.0, 74.8, 72.1, 69.4, 50.1, 34.5, 21.7, 19.5. MSI-MS: 553.5 [M+H] + It was as follows.

[0149] (Example 21) N-(2-Fluoro-3-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-9H-purin-6-yl) octadecanamide [Chemical formula]

[0150] Compound 13b was obtained with reference to the method of Example 13. Using 13b as a starting material, compound 21a was prepared with reference to the method for preparing 10b from 10a in Example 10. Using 21a as a starting material, compound 21 was prepared with reference to the deprotection method of 1a in Example 1, and the total yield was 74%. The detection results of the obtained compound 21 were 1 HNMR(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). 13CNMR (100 MHz, DMSO-d6) δ 172.5, 153.5, 152.7, 148.6, 141.2, 122.4, 99.1, 84.5, 74.6, 63.4, 37.3, 34.2, 32.3, 29.4, 28.7, 25.7, 22.3, 14.5. (Some alkyl peaks are overlapped) MSI-MS: 536.6 [M+H] + It was.

[0151] (Example 22) N-(4-Chloro-3-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-9H-purin-6-yl) octadecamide [Chemical formula]

[0152] Using adenosine as a raw material, 15b was obtained with reference to the method of Example 15. Using 15b as a raw material, 6.28 g (10 mmol) of 15b was dissolved in ethyl acetate (40 mL) under a nitrogen gas atmosphere, 10 mL (22 mmol) of a triethylamine solution of 37% hydrochloric acid was added, and the mixture was stirred and heated to 70 °C for about 8 hours for reaction. After TLC indicated the completion of the reaction, it was cooled to room temperature, washed with saturated sodium bicarbonate solution until neutral, washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, the filtrate was concentrated under reduced pressure, and the residue was recrystallized from anhydrous methanol (30 mL) to obtain solid 22a (weight 1.95 g, yield 38%, MSI-MS: 515.2 [M+H] + ) was obtained.

[0153] Using 22a as the raw material, compound 22 was prepared in combination with the deprotection method of 1a in Example 1 according to the method for preparing 10a to 10b in Example 10, and the total yield was 79%. The detection results of the obtained compound 22 were as follows: HNMR(400MHz,DMSO-d6)δ10.60(s,1H),8.67(s,1H),8.35(s,1H),6.15(d,1H),5.33(d,1H),4.98(m,1H),4.31(m,1H),4.02(m,1H),3.78(m,1H),3.58(m,1H),3.53(m,1H),2.32(m,2H),1.51(m,2H),1.34-1.21(m,28H),0.87(m,3H). 13 CNMR(100MHz,DMSO-d6)δ171.4,152.5,151.6,148.4,140.5,121.4,98.7,77.5,74.3,68.1,59.4,38.2,33.7,31.5,28.1,27.5,25.4,22.1,14.6.(Partial alkyl peak overlap) MSI-MS:553.2[M+H] + It was as follows.

[0154] (Example 23) ((((4-Hydroxy-5-(6-(isonicotinamido)-9H-purin-9-yl)tetrahydrofuran-2-yl)methoxy)methyl)phosphoryl)bis(oxy))bis(methylene)bis(2,2-dimethylpropionic acid)

Chemical Structure

[0155] Using compound 12 as the raw material, compound 23a was prepared according to the protection method of 1b in Example 1. Using 23a as the raw material, compound 23 was prepared according to the process of adjusting 17a to 17 in Example 17. 17a during the reaction was replaced with an equivalent amount of 23a, and the total yield of the reaction was 81%. The detection results of the obtained compound 23 were as follows: 1HNMR (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). 13 CNMR (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. MSI-MS: 679.6 [M+H] + It was as follows.

[0156] (Example 24) Isopropyl ((((4-hydroxy-5-(6-(isonicotinamido)-9H-purin-9-yl)tetrahydrofuran-2-yl)methoxy)methyl)(phenoxy)phosphoryl)alanine ester [Chemical formula]

[0157] Compound 23a was obtained according to the method of Example 23, and compound 24 was obtained according to the method of Example 7 using 23a as a raw material. Compound 1b was replaced with 23a in an equimolar amount, and the total yield was 85%. The detection results of the obtained compound 24 were 1 HNMR (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). 13CNMR(100MHz, DMSO-d6) δ 171.1, 165.2, 152.7, 151.8, 150.4, 149.8, 149.5, 140.5, 140.1, 130.4, 123.2, 121.8, 121.4, 120.2, 99.3, 76.7, 74.8, 74.5, 72.5, 69.8, 50.4, 34.7, 21.5, 19.3. MSI-MS: 640.6 [M+H] + was obtained.

[0158] (Example 25) (5-(6-Amino-9H-purin-9-yl)-3-azido-4-hydroxytetrahydrofuran-2-yl)methyl dihydrogen phosphate [Chemical formula]

[0159] Under an argon gas atmosphere, 2.67 g (10.0 mmol) of adenosine and 5.78 g (22 mmol) of PPh 3 were added to a round-bottom flask. Then, 18 mL of DMF was added, and the resulting mixture was stirred at room temperature until the solid was completely dissolved. This solution was cooled to 10 °C, and 4.4 mL (2.2 mmol) of diisopropyl azodicarboxylate (DIAD) was rapidly added dropwise. The reaction mixture was brought to room temperature. After stirring at 40 °C for 2 hours, the DMF was evaporated under reduced pressure to obtain a viscous liquid, and 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 and eluted with a DCM and acetone solvent mixture (3:1 to 2:3). A white solid compound 25a (weight 1.84 g, yield 74%) was obtained. The detection results of the obtained compound were 1 HNMR(400MHz, 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). 13CNMR (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] + It was.

[0160] 2.49 g (10 mmol) of compound 25a and 2.6 g (40 mmol) of sodium azide were added to a flask, and further 20 mL of DMF was added as a solvent. The reaction was carried out at 120 °C for 16 hours, and the progress of the reaction was monitored with a TLC plate. After the reaction was completed, water was added for quenching, and the aqueous phase was extracted 3 times with ethyl acetate (180 mL). The organic phases were combined, washed with water, and the solvent was removed under reduced pressure to obtain a foamy solid 25b (weight 2.22 g, yield 76%, MSI-MS: 293.2 [M+H] + ) was obtained.

[0161] Using 25b as a raw material, 25c was obtained according to the protection method for preparing 1b in Example 1. Using 25c as a raw material, according to the method of Example 2, 1b was replaced with an equivalent amount of 25c to obtain compound 25 (yield 76%). The detection results of the obtained compound 25 were 1 HNMR (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 CNMR (100 MHz, DMSO-d6) δ 158.2, 152.4, 149.8, 140.5, 119.4, 101.2, 78.1, 73.2, 68.4, 57.5. MSI-MS: 373.2 [M+H] + It was.

[0162] (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-dimethylpropionic acid) [Chemical formula]

[0163] Compound 25c was obtained according to the preparation method of Example 25. Using 25c as the starting material, compound 26a was obtained according to the method for preparing compound 17 from compound 17a in Example 17. Compound 26 was obtained by the deprotection method of 1b in Example 1. 25c was replaced with an equivalent amount of 17a, and the total yield was 54%. The detection results of the obtained compound 26 were 1 HNMR(400MHz,DMSO-d6)δ8.58(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 CNMR(100MHz,DMSO-d6)δ175.4,157.3,152.5,149.7,140.2,119.5,99.8,92.8,79.0,72.3,71.5,71.1,56.5,38.8,27.6.MSI-MS:615.6[M + H] + as follows.

[0164] (Example 27) Isopropyl ((((5-(6-amino-9H-purin-9-yl)-3-azido-4-hydroxytetrahydrofuran-2-yl)methoxy)methyl)(phenoxy)phosphoryl)alaninate [Chemical formula]

[0165] Compound 25c was obtained according to the preparation method of Example 25. Using 25c as the starting material, according to the preparation method of Example 7, 1b was replaced with an equivalent amount of 25c to obtain compound 27 (total yield 43%). The detection results of the obtained compound 27 were 1HNMR(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 CNMR(100 MHz, DMSO-d6) δ 171.4, 156.4, 152.1, 150.1, 149.5, 140.3, 130.5, 121.2, 120.5, 119.4, 100.5, 78.4, 72.8, 72.0, 71.5, 69.1, 57.2, 50.5, 21.5, 19.1. MSI-MS: 576.5 [M + H] + It was.

[0166] (Example 28) Isopropyl ((((3 - azido - 4 - hydroxy - 5 - (6 - (isonicotinamido) - 9H - purin - 9 - yl) tetrahydrofuran - 2 - yl) methoxy) methyl) (phenoxy) phosphoryl) alanine ester [Chemical formula]

[0167] Using the obtained compound 27 as a raw material, compound 28a was obtained with reference to the protection method of 1a or 1b in Example 1. Using compound 28a as a raw material, with reference to the preparation method of Example 12, cordycepin or 1b was substituted in an equimolar amount for compound 28a to obtain compound 28 (total yield 37%). The detection results of the obtained compound 28 are 1 HNMR(400 MHz, DMSO-d6) δ 11.07 (s, 1H), 8.83 (d, 2H), 8.35 (s, 1H), 8.20 (s, 1H), 8.01 (d, 2H), 7.41 (m, 2H), 7.21 (m, 3H), 6.15 (d, 1H), 5.35 (s, 1H), 4.94 (d, 1H), 4.00 (m, 2H), 3.72 - 3.32 (m, 6H), 1.82 (m, 1H), 1.24 - 1.17 (d, 9H). 13CNMR (100 MHz, DMSO-d6) δ 171.5, 164.5, 152.1, 151.6, 150.1, 149.8, 149.4, 140.7, 140.3, 130.2, 122.2, 121.5, 121.1, 120.4, 100.4, 78.7, 72.5, 72.1, 71.5, 69.2, 57.4, 50.8, 21.6, 19.2. MSI-MS: 681.6 [M+H] + It was.

[0168] (Example 29) 2-(6-Amino-9H-purin-9-yl)-5-(hydroxymethyl)tetrahydrofuran-3-yl valine salt [Chemical formula]

[0169] According to the protection method for preparing 1c in Example 1, compound 1c was obtained. Using 1c as a raw material, further reaction was carried out. 9.95 g (27.2 mmol) of compound 1c and 4.88 g (27.2 mmol) of CbzCl were added to 38.5 mL of toluene and 38.5 mL of water. Further, 4.70 g (34 mmol) of K 2 CO 3 was added, and the mixture was vigorously stirred at a temperature of 25°C or lower. Then, after stirring at room temperature for 3 hours, 0.275 g (2.72 mmol) of triethylamine and 5.78 g of sodium chloride were sequentially added, and the mixture was further stirred for 30 minutes. The organic layer was separated and concentrated to obtain the desired oily product 29a (weight 13.1 g, yield 90%, MSI-MS: 522.6 [M+Na] + ).

[0170] 4.99 g (10 mmol) of compound 29a and 1.36 g (10 mmol) of valeric acid chloride were taken, 60 mL of anhydrous pyridine was added under an ice bath environment, the temperature was gradually raised to 40 °C and reacted for 10 hours, and the progress of the reaction was monitored by TLC. After the reaction was completed, water and ethyl acetate were added for extraction, the organic phase was back-extracted, the organic phase was recovered and rotary evaporated to obtain an oily liquid 10b, which was purified by column chromatography. The purified product 29b (5.03 g, yield 84%, MSI-MS: 625.5 [M+Na] + ) was obtained.

[0171] 5.99 g of compound 29b (10 mmol) was dissolved in 200 mL of methanol. Then, 1.5 g of ammonium formate (30 mmol) and 0.75 g of 10% Pd-C were added, and the reaction mixture was stirred at room temperature for 10 minutes and then heated to reflux for 45 minutes. The mixture was filtered through diatomaceous earth, and the filtrate was evaporated until dry to obtain compound 29c (4.41 g, yield 95%). Using 29c as the raw material, deprotection was carried out according to the method of 1c in Example 1 to obtain compound 29 (yield 90%). The detection results of the obtained compound 29 were 1 HNMR(400MHz,DMSO-d6)δ8.89(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 CNMR(100MHz,DMSO-d6)δ172.1,156.0,152.3,149.5,140.1,119.4,102.2,82.5,74.4,63.7,59.5,33.1,30.5,19.1.MSI-MS:351.3[M+H] + as follows.

[0172] (Example 30) 2-(6-Amino-9H-purin-9-yl)-5-(phosphonooxy)methyl)tetrahydrofuran-3-yl valerate salt

Chemical Structure

[0173] Using compound 29 as a raw material, the reaction was carried out according to the method of Example 2 to obtain compound 30. 1b was replaced with 29 in an equivalent amount to obtain compound 30 (yield 84%). The detection results of the obtained compound 30 are 1 HNMR(400MHz, DMSO-d6) δ8.90(s, 2H), 8.56(s, 1H), 8.33(s, 1H), 7.04(s, 2H), 6.75(d, 1H), 5.00(m, 1H), 4.27 - 4.02(m, 5H), 3.73(m, 1H), 2.36(m, 1H), 2.16(m, 1H), 1.88(m, 1H), 0.96(d, 6H). 13 CNMR(100MHz, DMSO-d6) δ172.4, 156.3, 152.5, 149.8, 140.2, 119.5, 101.2, 76.1, 74.2, 68.7, 59.3, 32.1, 30.4, 19.0. MSI-MS: 431.3[M + H] + as follows.

[0174] (Example 31) 2-(6-(Isonicotinamido)-9H-purin-9-yl)-5-(phosphono)methyl)tetrahydrofuran-3-yl valine salt [Chemical formula]

[0175] Using compound 30 as a raw material, according to the preparation method of Example 12, 1a was replaced with compound 30 in an equivalent amount to obtain compound 31 (yield 91%). The detection results of the obtained compound 31 are 1 HNMR(400MHz, DMSO-d6) δ11.08(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). 13CNMR (100 MHz, DMSO-d6) δ 171.4, 164.5, 152.3, 151.1, 149.9, 149.7, 140.5, 140.1, 123.7, 121.5, 101.7, 77.1, 74.5, 68.2, 59.8, 32.4, 30.8, 19.2. MSI-MS: 558.4 [M+Na] + It was.

[0176] (Example 32) (((5-(6-(Isonicotinamide)-9H-purin-9-yl)-4-(pentyloxy)tetrahydrofuran-2-yl)methoxy)methyl)phosphonic acid [Chemical formula]

[0177] Using compound 29 as a raw material, according to the preparation method of Example 3, compound 1b was replaced with an equivalent amount in compound 29 to obtain compound 32a (yield 77%). Using compound 32a as a substrate, according to the preparation method of Example 12, 1a was replaced with an equivalent amount in compound 32a to obtain compound 32 (yield 90%). The detection results of the obtained compound 32 were 1 HNMR (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 CNMR (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] + It was.

[0178] (Example 33) Methyl (((2-(6-amino-9H-purin-9-yl)-5-(hydroxymethyl)tetrahydrofuran-3-yl)oxy)(phenoxy)phosphoryl)alaninate

Chem.

[0179] Using compound 1c as a raw material, according to the preparation method of Example 6, compound 1b was replaced with an equivalent amount of compound 1c to obtain compound 33a (yield 62%). Then, according to the deprotection method of 1c in Example 1, 33a was reacted to obtain compound 33 (yield 90%). The detection results of the obtained compound 33 were 1 HNMR(400MHz, DMSO-d6) δ8.58(s, 1H), 8.33(s, 1H), 7.42(m, 2H), 7.21(m, 3H), 7.08(s, 2H), 6.15(d, 1H), 5.00(m, 1H), 4.02(m, 1H), 3.71 - 3.48(m, 8H), 2.24 - 1.98(m, 2H), 1.25(d, 3H). 13 CNMR(100MHz, DMSO-d6) δ172.1, 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] + as follows.

[0180] (Example 34) Methyl (((5-(hydroxymethyl)-2-(6-(isonicotinamido)-9H-purin-9-yl)tetrahydrofuran-3-yl)oxy(phenoxy)phosphoryl)alaninate

Chem.

[0181] According to the steps of Example 33, compound 33a was obtained. Using compound 33a as a raw material, according to the preparation method of Example 12, 1a was replaced with an equivalent amount of compound 33a to obtain compound 34 (total yield 83%). The detection results of the obtained compound 34 were1 HNMR (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 CNMR (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] + It was.

[0182] (Example 35) (Methyl (5-(2-((E)-2-bromocarbonyl)-6-(isonicotinamido)-9H-purin-9-yl)-4-hydroxytetrahydrofuran-2-yl)dihydrogen phosphate)

Chemical formula

[0183] Dissolve 2.51 g (10 mmol) of cordycepin in dilute nitric acid, heat to 110 °C, add 1.26 g (5 mmol) of elemental iodine, react for 4 hours, and monitor the reaction by TLC method. After completion of the reaction, extract with petroleum ether, recover the aqueous phase, extract the organic phase solution with deionized water, combine the aqueous phases and perform rotary evaporation to obtain compound 35a (weight 2.72 g, yield 72%, MSI-MS: 378.2 [M + H] + ) was obtained.

[0184] Put 100 mL of anhydrous 1,4-dioxane into a reactor, introduce argon gas for protection, heat to 70 °C, and sequentially add 0.11 g (0.5 mmol) of palladium acetate, 0.26 g (1 mmol) of triphenylphosphine, and 1.8 mL of triethylamine, and stir for about 30 minutes. Add 3.77 g (10 mmol) of compound 35a and 2.59 g (30 mmol) of methyl acrylate, react for 2 hours under an 80 °C environment, then terminate the reaction, filter, collect the filtrate, let it stand, and filter again to obtain compound 35b as a white cotton-like solid (weight 2.41 g, yield 72.7%, MSI-MS: 336.4 [M+H] + ) was obtained.

[0185] Take 1.00 g of compound 35b, dropwise add 12 mL of sodium hydroxide solution (2 mol / L), stir at room temperature for 3 hours, then dropwise add concentrated hydrochloric acid until the pH reaches 1 under ice bath conditions, and a large amount of white precipitate was formed. Filter by suction to obtain white solid 35c (weight 0.88 g, yield 92%, MSI-MS: 322.6 [M+H] + ) was obtained.

[0186] Take 3.21 g (10 mmol) of compound 35c, add it to 120 mL of water, heat to 100 °C while stirring, then add 2.07 g (15 mmol) of anhydrous potassium carbonate, dissolve 2.67 g (15 mmol) of NBS in 22.5 mL of acetone and 22.5 mL of water respectively, drop the mixed solution into the flask, and complete the dropping within 135 minutes. Stir for 3 hours, stop the reaction, remove half of the solvent, and place it in the refrigerator overnight. A large amount of needle-like brown crystals were precipitated, and filtered by suction to obtain compound 35d (weight 1.78 g, yield 50%, MSI-MS: 357.3 [M+H] + ) was obtained.

[0187] 1.78 g (5 mmol) of compound 35d was taken, and 40.00 mL of anhydrous pyridine and 8.5 mL of acetic anhydride were added under ice bath conditions. The reaction was monitored by HPLC liquid phase, and the reaction was terminated after about 5 hours. The solvent was removed to obtain a viscous liquid 35e (weight 1.76 g, yield 80%). Compound 35f (yield 95%) was prepared using the method of Example 16 with compound 35e, and compound 35 (yield 72%) was obtained using the phosphorylation method of Example 2 and the deprotection method of Example 1. The detection results of the obtained compound 35 were 1 HNMR (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 CNMR (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] + as follows.

[0188] (Example 36) 3 - ((5 - (6 - Amino - 2 - fluoro - 9H - purin - 9 - yl) - 4 - hydroxytetrahydrofuran - 2 - yl)methoxy) - 5 - (hydroxymethyl)cyclopentane - 1,2 - diol [Chemical formula]

[0189] Compound 17a was obtained according to the method of Example 17. Using 17a as the raw material, it was prepared according to the preparation method of Example 8. 2 - Chloro - 3 - hydroxypropyl valine was replaced with 3 - chloro - 5 - (hydroxymethyl)cyclopentane - 1,2 - diol in an equivalent amount to obtain compound 36a. After deprotection, compound 36 (total yield 41%) was obtained. The detection results of the obtained compound 36 were 1HNMR(400MHz, 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 CNMR(100MHz, 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] + was as follows.

[0190] (Example 37) Compound: Preparation of ((((5-(6-Amino-9H-purin-9-yl)-2-azido-4-hydroxytetrahydrofuran-2-yl)methoxy)methyl)phosphoryl)bis(oxy)bis(methylene)bis(2,2-dimethylpropionic acid) [Chemical formula]

[0191] 3.65 g (10 mmol) of Compound 1b and 3.44 g (20 mmol) of metachloroperbenzoic acid were added to a reaction flask, 100 mL of anhydrous dichloromethane was added and dissolved, 2.13 g (15 mmol) of phosphorus pentoxide was separately added under an ice bath environment, the temperature was gradually raised to 40 °C and reacted for 3 hours. After the reaction was completed, filtration was carried out, 100 mL of saturated sodium bicarbonate was added to quench the reaction, and it was further washed twice with 50 mL of dichloromethane and back-extracted twice with 60 mL of saturated sodium bicarbonate. The organic phases were combined, concentrated under reduced pressure, and column chromatography was carried out to obtain Compound 37a (weight 1.49 g, yield 41%). The detection results of the obtained compound were 1HNMR (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] + It was.

[0192] 3.63 g (10 mmol) of compound 37a and 2.6 g (40 mmol) of sodium azide were added to a flask, and further 20 mL of DMF was added as a solvent. The reaction was carried out at 120 °C for 16 hours, and the progress of the reaction was monitored with a TLC plate. After the reaction was completed, water was added to quench it, and the aqueous phase was extracted 3 times with ethyl acetate (180 mL). The organic phases were combined, washed with water, and the solvent was removed under reduced pressure to obtain a foamy solid 37b (weight 2.77 g, yield 68%). Compound 37 was obtained according to the method for preparing compound 17 from compound 17a of Example 17 using 37b as a raw material. 37b was replaced with an equivalent amount of 17a, and the total yield was 50%. The detection results of the obtained compound 37 were 1 HNMR (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 CNMR (100 MHz, DMSO-d6) δ 175.2, 156.3, 152.6, 149.9, 140.2, 119.4, 100.3, 93.1, 90.2, 81.0, 71.5, 68.8, 38.6, 36.2, 27.4. MSI-MS: 637.6 [M+Na] + It was.

[0193] (Example 38) Preparation of compound: methyl dihydrogen phosphate (5-(6-amino-2-((E)-2-bromocarbonyl-9H-purin-9-yl)-4-hydroxytetrahydrofuran-2-yl)). The reaction steps are as follows.

Chemical formula

[0194] Using compound 2 as the raw material and the method of Example 35, compound 38 (overall yield 24%) was obtained from compound 2. The detection results of the obtained compound 38 were 1 HNMR(400MHz,DMSO-d6)δ8.35(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 CNMR(100MHz,DMSO-d6)δ156.1,152.3,149.8,140.2,134.9,124.2,119.5,98.7,74.9,74.3,68.1,34.5.MSI-MS:458.3[M + N] + as follows.

[0195] (Example 39) Preparation of compound: methyl dihydrogen phosphate (5-(6-amino-8-(isopropylamino)-9H-purin-9-yl)-4-hydroxytetrahydrofuran-2-yl). The preparation steps are as follows.

Chemical formula

[0196] Compound 39 was prepared using compound 1b as a raw material. First, 3.65 g (10 mmol) of compound 1b and 1.18 g (20 mmol) of 2-propylamine were added to a reaction flask, 60 mL of dioxane was added as a solvent, and the mixture was refluxed at 80 °C for 20 hours. The progress of the reaction was monitored by TLC. After the reaction was completed, the reaction solution was concentrated, then 50 mL of water and 50 mL of ethyl acetate were added and extracted twice. The organic phases were combined and concentrated under reduced pressure to obtain an oily liquid, which was purified by column chromatography to obtain a pale yellow oily liquid as compound 39a (weight 1.44 g, yield 34%).

[0197] Using compound 39a as a raw material, compound 39 was prepared according to the preparation method in Example 2, with 1b replaced by an equivalent amount of 39a, and the yield was 91%. The detection results of the obtained compound 39 were 1 HNMR(400MHz,DMSO-d6)δ8.15(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 CNMR(100MHz,DMSO-d6)δ153.1,152.3,151.5,149.8,118.5,99.7,74.8,74.5,68.5,46.5,34.3,23.5.MSI-MS:389.3[M+H] + as follows.

[0198] (Example 40) Preparation of the compound: 3-((5-(6-amino-9H-purin-9-yl)-4-hydroxytetrahydrofuran-2-yl)methoxy)-5-(hydroxymethyl)cyclopentane-1,2-diol. The preparation process is as follows.

Chemical formula

[0199] Using compound 1b as a raw material, it was prepared according to the preparation method of Example 8. 2-Chloro-3-hydroxypropyl valine was substituted with 3-chloro-5-(hydroxymethyl)cyclopentane-1,2-diol in an equivalent amount to obtain compound 40 (total yield 46%). The detection results of the obtained compound 40 are 1 HNMR(400MHz,DMSO-d6)δ8.58(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 CNMR(100MHz,DMSO-d6)δ156.3,153.4,149.7,140.2,119.4,99.5,83.4,81.1,76.1,75.5,74.9,73.6,64.4,34.9,34.7,31.6.MSI-MS:382.3[M+H] + as follows.

[0200] (Example 41) Experimental antitumor effect of modified derivatives of cordycepin against liver cancer Establishment of a mouse liver cancer transplantation model and drug evaluation. 2.5×10 5 individual liver cancer cells Hep-1-6 were suspended in 100 μL of PBS and inoculated on the outer side of the thigh of the left lower limb of C57BL / 6j nude mice. The transplanted tumor was about 100 mm after 1 week 3When the size was reached, 10 mice were randomly assigned to 12 groups, which were the blank group, the colchicine control group, and the drug groups (compounds 2, 3, 5, 7, 15, 24, 27, 31, 35, 40), respectively. The blank group was force-fed with nutrients such as corn syrup and DMSO as a solvent control every other day, and the colchicine control group and the drug groups were force-fed with compound drugs (500 μg / dose / mouse) every other day, respectively, and observed continuously for 14 days. The changes in the size of hepatocellular carcinoma tumors in two groups of mice were observed. After the experiment, the mice were sacrificed, blood and corresponding tissue specimens were collected, the tumor body was immediately imaged and weighed, and a part of the tumor tissue was collected and fixed with formalin solution for further examination. The two vertical diameters (length and width) of the transplanted tumor were measured every two days using vernier calipers, the size of the transplanted tumor was calculated, and the experimental results of the volume change of the tumor and the results of the tumors treated with the blank group, the colchicine control group, and the drug groups within 14 days are shown in Figure 1. Formula: Tumor volume (mm 3 ) = 1 / 2 × (length × width) 2 The volume was calculated according to. From the experimental results, when the drug groups were used alone, they could significantly inhibit the growth of mouse tumors, and the effects were all superior to those of the colchicine control group. Among them, compound 24 had an excellent anti-hepatocellular carcinoma tumor effect (shrinking from 900 mm 3 to less than 100 mm 3 ) (P < 0.001), indicating that the compound drug group had an effect of killing hepatocellular carcinoma tumor cells or activating tumor immunity.

[0201] (Example 42) Experimental antitumor effects of modified derivatives of colchicine on small cell lung cancer The dose-effect curves of the antitumor effects of colchicine and compounds 1, 2, 4, 5, 7, 8, 10, 12, 16, 18, 21, 23, 24, 35, 38 in small cell lung cancer cell lines H1048, H446, and H69 were analyzed using the MTT assay. 50% inhibitory concentration (IC 50) was calculated and the results were summarized in the following table. From the results of in vitro antitumor experiments, compared with colchicine, the effective concentration of the modified colchicine derivatives against tumor cells decreased. Among them, compounds 35 and 38 had a strong in vitro killing effect on three types of cells, but their toxicity was also high, indicating that compounds 18 and 21 followed. The IC 50 of colchicine in small cell lung cancer cell lines H1048 and H446 were both higher than 100 μM, the effective concentration was relatively high, and a large amount of drug might be required to exert an effect during practical use.

[0202]

Table 1

[0203] (Example 43) Measurement of the maximum tolerated dose of colchicine-modified derivatives in zebrafish in vivo experiments A total of 240 3dpf wild-type AB zebrafish were randomly selected at 30 per well and collected into an 8-well plate with a volume of 3 mL of fish-raising water per well. Colchicine was diluted according to the maximum lethal dose, dissolved in DMSO, and added to each well at a concentration gradient (the maximum lethal dose was diluted 100-fold and tested with 8 intermediate concentration gradient values), and a normal control group, a solvent control group G (5% glucose), and a solvent control group D (DMSO) were set. During the experimental period, the zebrafish in each group were cultured in an environment at 35 °C. After treating the zebrafish with the test article until 5 dpf, the lethality and developmental malformations of the zebrafish at different doses were examined, and the maximum tolerated dose (MTD) of the test article for normal zebrafish was determined at a concentration with a survival rate of more than 90% and a teratogenic rate of less than 20%. Colchicine was selected as the control group. Furthermore, compounds 2, 5, 6, 7, 9, 11, 13, 20, 24, 25, 28, 35, 36, 37, and 39 were selected to test the MTD in zebrafish according to the above method, and the test results are shown in Table 2.

[0204]

Table 2

[0205] (Example 44) Inhibitory effect of a modified derivative of curdione on small cell lung cancer in zebrafish in vivo Zebrafish xenograft tumor models of small cell lung cancer with three types of H69, H446, and H1048 cells were established respectively: H69, H446, and H1048 cell suspensions at a concentration of 1×10 6 mL were prepared in serum-free cell culture medium. 5 mL of a red fluorescent dye (CM-DiI) cell labeling solution was added per 1 mL of the cell suspension, gently mixed, incubated at 37 °C for 20 minutes, centrifuged at 1500 rpm for 5 minutes, the supernatant was removed, and after adding serum-free medium again and resuspending, the above steps were repeated twice to obtain the desired labeled cell suspension, which was transplanted into the zebrafish yolk sac by microinjection, about 100 cells were transplanted per fish, a zebrafish-human small cell lung cancer sensitive strain xenograft tumor model was established, and zebrafish injected with ovarian cancer cells were cultured at 35 °C until 3 dpf.

[0206] Test the effects of colchicine and the prepared compounds 2, 5, 6, 7, 9, 11, 13, 20, 24, 25, 28, 35, 36, 37, 39 on the growth of small cell lung cancer in zebrafish in vivo. When the tumor zebrafish model was inhibited until 3 dpf, zebrafish with good consistency of transplanted tumors were selected under a microscope and randomly assigned to 6-well plates with 30 fish per well. At 3 dpf, the above compounds were diluted at the MTD dose, dissolved in DMSO and added to each well, and a normal control group, a model control group and a solvent control group (DMSO) were set at a volume of 3 mL of fish culture water per well. After culturing the zebrafish in each experimental group at 35 °C until 5 dpf, 10 tear zebrafish per experimental group were randomly selected and observed under a fluorescence microscope, photographed and saved, and image analysis was performed using Nikon NIS-Elements l3.10 advanced image processing software to calculate the fluorescence intensity (S) of zebrafish transplanted tumors. The inhibition results are shown in Table 3 below. Among them, the inhibition results of the transplanted zebrafish models of H446 small cell lung cancer in the control group, colchicine group, compound 24 and compound 35 groups are shown in Figure 2. Calculate the growth inhibition effects of Tongguan Teng injection and paclitaxel alone on zebrafish-human ovarian cancer sensitive strains in terms of total fluorescence intensity. The formula is as follows. Tumor growth inhibition effect (%) = [S (model control group) - S (drug group)] / S (model control group) × 100%

[0207]

Table 3

[0208] (Example 45) Experimental inhibitory effect of modified derivatives of colchicine on colorectal cancer Establishment of a mouse colorectal cancer transplantation model and drug evaluation. Suspend 2.5×10 6 individual MC-38 colorectal cancer cells in 100 μL of PBS and inoculate them on the outer side of the thigh of the left lower limb of C57BL / 6j nude mice. After one week, the transplanted tumor is about 100 mm 3When the size reached, the mice were randomly assigned into 7 groups, with 8 mice in each group, and they were respectively the control group IgG, the compound 16 drug + IgG group, the PD-1 antibody treatment group, the CTLA4 monoclonal antibody treatment group, the compound 16 drug + PD-1 combination treatment group, and the compound 16 drug + CTLA4 monoclonal antibody combination treatment group. Control group IgG: Immunoglobulin G (IgG) (500 μg / mouse / time) was continuously administered by forced gastric gavage daily for 28 days. Cordycepin + IgG: Immunoglobulin G (IgG) and cordycepin (both 500 μg / mouse / time) were continuously administered by forced gastric gavage daily for 28 days. Compound 16 drug + IgG: Immunoglobulin G (IgG) and compound 16 drug (both 500 μg / mouse / time) were continuously administered by forced gastric gavage daily for 28 days. Antibody treatment group: The antibody (500 μg / mouse / time) was administered by gastric gavage every 4 days continuously for 28 days. Compound 16 drug + antibody combination treatment group: Immunoglobulin (500 μg / mouse / time) was continuously administered by forced gastric gavage daily for 28 days, and compound 16 drug (500 μg / mouse / time) was administered by gastric gavage every 4 days continuously for 28 days. After the experiment ended, the mice were sacrificed, blood and corresponding tissue specimens were collected, the tumor body was immediately imaged and weighed, a part of the tumor tissue was collected and fixed with formalin solution for further examination. Using calipers, the two perpendicular diameters (length and width) of the transplanted tumor were measured every 2 days, 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 results of observing the size changes of colorectal cancer tumors in 7 groups of mice are shown in Figure 3. After 28 days, from the experimental results, cordycepin had an anti-proliferative effect on colorectal cancer (the size shrank from 1300 mm 3 to 730 mm 3 ), when compound 16 was used alone, it had a more significant anti-tumor proliferative effect than cordycepin (the size shrank from 1300 mm 3 to 480 mm 3 ) (P < 0.01), when the immune checkpoint inhibitors PD-1 and CTLA4 were used alone, they had a significant anti-tumor proliferative effect (the size shrank from 1300 mm 3 to 448 mm 3Reduced to (P < 0.01), when Compound 16 was combined with the immune checkpoint inhibitors PD-1 and CTLA4 respectively, the antitumor effect was significantly enhanced by their synergistic effect (the size was reduced from 1300 mm 3 to 260 mm 3 ).

[0209] (Example 46) Experimental inhibitory effect of modified derivatives of cordycepin on melanoma Establishment of a mouse melanoma transplantation model and drug evaluation: 2.5×10 5 individual B16-F10 melanoma cells were suspended in 100 μL of PBS and inoculated into the outer side of the thigh of the left lower limb of C57BL / 6j nude mice. One week later, when the transplanted tumor reached about 100 mm 3 in size, the mice were randomly assigned to 10 groups, with 10 mice in each group, and were respectively used as the 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 was continuously force-fed with nutrients such as corn syrup and DMSO daily for 24 days. The compound groups were force-fed with the corresponding compounds (500 μg / mouse / time) every other day for 24 days continuously. In the first stage, the size changes of melanoma in 10 groups of mice were observed every 4 days, and in the later stage, every 2 days. After the experiment, the mice were sacrificed, blood and corresponding tissue specimens were collected, the tumor body was immediately imaged and weighed, a part of the tumor tissue was collected and fixed with formalin solution for further examination. The two vertical diameters (length and width) of the transplanted tumor were measured every 2 days using calipers, the size of the transplanted tumor was calculated, and the volume was calculated using the formula: tumor volume (mm 3 ) = 1 / 2×(length × width) 2 , and the experimental results are shown in Figure 4. From the experimental results, cordycepin itself inhibited the growth of melanoma (reduced from 1760 mm 3 to 600 mm 3 ), and the modified derivatives of cordycepin were more effective than cordycepin (reduced from 1760 mm 3 to 560 mm 3When compound 19 and 34 were used alone (reduced to less than), the growth of mouse tumors was significantly inhibited (from 1760 mm 3 to approximately 240 mm 3 and reduced) (P < 0.001). The above results indicated that the synthetic compound had the effect of killing tumor cells.

[0210] (Example 46-1) Compound 27 is used to inhibit melanoma by the above method having the same level of inhibitory effect as compound 26.

[0211] (Example 47) Experimental inhibitory effect of modified derivatives of cordycepin on ovarian cancer Establishment of a mouse ovarian cancer transplantation model and drug evaluation. 2.5×10 6 ID8 ovarian cancer cells were suspended in 100 μL of PBS and inoculated into the outer thigh of the left lower limb of C57BL / 6j nude mice. Approximately one week later, the transplanted tumor was approximately 100 mm 3When the size reached, the mice were randomly divided into 5 groups, with 10 mice in each group, which were the control group, the colchicine group, the compound 24 drug group, the PD-1+TIM3 antibody treatment group, and the compound 24 drug+PD-1+TIM3 antibody treatment group, respectively. Control group: Immunoglobulin G (IgG) (500 μg / time / mouse) and PBS were continuously administered intragastrically by force every day for 15 days. Colchicine group: Colchicine (500 μg / time / mouse) was formulated with PBS and continuously administered intragastrically by force every day for 15 days. Compound 24 drug group: Compound 24 (500 μg / time / mouse) was formulated with PBS and continuously administered intragastrically by force every day for 15 days. PD-1+TIM3 antibody treatment group: Antibodies PD-1+TIM3 (500 μg / time / mouse each) were administered intragastrically every 4 days for 15 consecutive days. Compound 24 drug+PD-1+TIM3 antibody treatment group: Immunoglobulin and compound 24 drug (500 μg / time / mouse) were continuously administered intragastrically by force every day for 15 days, and antibodies PD-1+TIM3 (500 μg / time / mouse each) were administered intragastrically every 4 days for 15 consecutive days. The size change of ovarian tumors in the 5 groups of mice was observed every 2 days. After the experiment, the mice were sacrificed, blood and corresponding tissue specimens were collected, the tumor body was immediately imaged and weighed, and a part of the tumor tissue was collected and fixed with formalin solution for further examination. The two vertical diameters (length and width) of the transplanted tumor were measured every 2 days using calipers, the size of the transplanted tumor was calculated, and the volume was calculated by the formula: tumor volume (mm 3 ) = 1 / 2×(length×width) 2 , and the experimental results are shown in Figure 5. From the experimental results, it was shown that colchicine had an anti-proliferative effect on ovarian cancer (the size was reduced by 1.5 times in 15 days). When compound 24 was used alone, it had a more significant anti-tumor proliferative effect than colchicine (the size was reduced by about 2 times in 15 days). When the immune checkpoint inhibitors PD-1 and CTLA4 were used alone, they had a significant anti-tumor proliferative effect (the size was reduced by more than about 2 times in 15 days). When compound 24, the immune checkpoint inhibitor PD-1, and CTLA4 were used in combination respectively, the anti-tumor effect was significantly enhanced (the size was reduced by about 4 times in 15 days).

[0212] (Example 48) Experimental inhibitory effect of modified derivatives of cordycepin on in vitro gastric cancer cells The antitumor dose - effect curves of cordycepin and 40 obtained compounds in gastric cancer cell lines AGS and BGC - 823 were analyzed using the MTT assay. The 50% inhibitory concentration (IC 50 ) was calculated, and the results were summarized in Table 4 below. The results of the in vitro antitumor experiment showed that the effective concentration of the modified cordycepin derivatives against tumor cells decreased compared with cordycepin.

[0213] [Table 4]

[0214] (Example 49) Experimental inhibitory effect of modified derivatives of cordycepin on in vivo tumors of gastric cancer mouse models Establishment of a mouse gastric cancer transplantation model and drug evaluation: 2×10 7 individual BGC - 823 gastric cancer cells were suspended in 100 μL of PBS. The anterior chest wall of C57BL / 6j nude mice was disinfected with 75% ethanol, and the most obvious location of the apical pulsation was touched by hand. The cells were inoculated into the left ventricle about 3 mm from the left edge of the sternum at the second intercostal space. After about one week, the transplanted tumor was about 100 mm 3When the size reached [specific size], the mice were randomly assigned into 5 groups, with 10 mice in each group, which were respectively the control group, the cordycepin group, and the drug groups of compound 16, 18, and 24. Control group: Nutrients such as corn syrup and DMSO were used as the solvent control group and were force-fed intragastrically every day. Cordycepin group: Cordycepin (500 μg / mouse / time) was formulated with DMSO and force-fed intragastrically every day. Compound drug groups: The compound (500 μg / mouse / time) was formulated with DMSO and force-fed intragastrically every day. Administration was carried out continuously for 18 days. The size changes of gastric tumors in the 5 groups of mice were observed every 2 days. After the experiment ended, the mice were sacrificed, blood and corresponding tissue specimens were collected, the tumor body was immediately imaged and weighed, and the weighing experimental results are shown in Figure 6. From the experimental results, cordycepin can effectively inhibit the growth of gastric tumors, shrinking by about 4 times compared with the control group (shrinking from 1.73 g to 0.46 g), and the modified cordycepin has a more significant effect than cordycepin. Among them, the compound 24 group was found to have shrunk by about 12 times compared with the control group (shrinking from 1.73 g to 0.14 g).

[0215] (Example 50) Experimental inhibitory effect of modified derivatives of cordycepin on in vivo tumors of pancreatic cancer mouse models Establishment of a mouse pancreatic cancer transplantation model and drug evaluation: Pancreatic cancer Pan02-luc cells were placed in DMEM medium containing 10% fetal bovine serum and placed in a CO 2 incubator at 37°C. Pancreatin ED-TA was digested and passaged every 2 - 3 days. When the cells reached the desired number, cells in the logarithmic growth phase were collected and resuspended in the medium at a density of 1×10 7Resuspended in / mL. C57BL / 6j nude mice were bred under specific pathogen-free conditions. After the mice grew to 6 weeks old, 200 μL of pancreatic cancer Pan02-uc cells were subcutaneously injected near the axilla on the right back of each mouse. The appearance of transplanted tumors within about 1 week indicated the successful establishment of the model. The mice were randomly assigned to 5 groups, with 10 mice in each group, which were the control group, the colchicine group, and the compound 11, 16, and 24 drug groups, respectively. Control group: Nutrients such as corn syrup and DMSO were used as the solvent control group and were force-fed intragastrically daily. Colchicine group: Colchicine (500 μg / mouse / time) was formulated with DMSO and force-fed intragastrically daily. Compound drug group: The compound (500 μg / mouse / time) was formulated with DMSO and force-fed intragastrically daily. The administration was continued for 24 consecutive days. Subsequently, the survival rates of the 5 groups of mice were observed daily, and the median survival time was examined. The results are shown in Figure 7. From the experimental results, by administering colchicine alone, the survival period of tumor-bearing mice was significantly extended (increased from 41 days to 52 days). The compound group further extended the survival period of tumor-bearing mice based on colchicine (increased from 41 days to more than 58 days). Compound 24 was the most effective, indicating that it extended nearly half of the survival period of tumor-bearing mice (increased from 41 days to 76 days).

[0216] The above is merely a preferred embodiment of the present invention. Those skilled in the art can make various improvements and modifications without departing from the spirit of the present invention, and these improvements and modifications should also be regarded as within the protection scope of the present invention. It should be noted that each component not clarified in this example can be realized using the prior art.

[0217] (Appendix) (Appendix 1) A colchicine derivative represented by formula I, or a pharmaceutically acceptable salt, stereoisomer, tautomer, solvate, prodrug, or metabolite thereof. [Chemical formula] (In the formula, R 1 is R 1 10 -R1 11 、 or R 1 14 -R 1 16 selected from the groups represented by, R 2 is selected from hydrogen or an azide group, R 3 is selected from hydrogen, R 4 is selected from a cyano group, R 5 is selected from hydrogen, a bromovinyl group, a mercapto group, a methyl group, fluorine, or chlorine, R 6 is selected from an amino group, R 7 is selected from hydrogen or an isopropylamino group.)

[0218] (Appendix 2) The cordycepin derivative represented by formula I is the compound 16, the cordycepin derivative represented by formula I described in Appendix 1, or a pharmaceutically acceptable salt, stereoisomer, tautomer, solvate, prodrug or metabolite thereof. [Chemical formula]

[0219] (Appendix 3) The cordycepin derivative represented by formula I, or a pharmaceutically acceptable salt, stereoisomer, tautomer, solvate, prodrug or metabolite thereof. [Chemical formula] (In the formula, R 1 is R 1 10 -R 1 11 、 or R 1 14 -R 1 16 selected from the groups represented by, R 2 is selected from hydrogen or an azide group, R 3 is selected from hydrogen, R 4 is selected from a hydroxyl group, R 5 is selected from hydrogen, a bromovinyl group, a mercapto group, a methyl group, fluorine, or chlorine, R6 is selected from the groups represented by R 6 15 and R 7 is selected from hydrogen or an isopropylamino group.)

[0220] (Appendix 4) The cordycepin derivative represented by formula I is the cordycepin derivative represented by formula I described in Appendix 3, which is compound 24 or compound 23, or a pharmaceutically acceptable salt, stereoisomer, tautomer, solvate, prodrug or metabolite thereof. [Chemical formula]

[0221] (Appendix 5) The cordycepin derivative represented by formula I, or a pharmaceutically acceptable salt, stereoisomer, tautomer, solvate, prodrug or metabolite thereof. [Chemical formula] (In the formula, R 1 is selected from hydrogen, R 2 is selected from hydrogen or an azide group, R 3 is selected from hydrogen, R 4 is R 4 6 or R 4 7 and is selected from the groups represented by R 5 is selected from hydrogen, a bromovinyl group, a mercapto group, a methyl group, fluorine or chlorine, and R 6 is R 6 15 and is selected from the groups represented by R 7 is selected from hydrogen or an isopropylamino group.)

[0222] (Appendix 6) The cordycepin derivative represented by formula I is the cordycepin derivative represented by formula I described in Appendix 5, which is compound 34, or a pharmaceutically acceptable salt, stereoisomer, tautomer, solvate, prodrug or metabolite thereof. [Chemical formula]

[0223] (Appendix 7) The kordsepine derivative represented by formula I, or a pharmaceutically acceptable salt, stereoisomer, tautomer, solvate, prodrug or metabolite thereof. [Chemical formula] (In the formula, R 1 is R 1 10 -R 1 11 , or R 1 14 -R 1 16 selected from the groups represented by, R 2 is selected from hydrogen or an azide group, R 3 is selected from hydrogen, R 4 is selected from a hydroxyl group, R 5 is selected from a mercapto group, R 6 is selected from an amino group, R 7 is selected from hydrogen or an isopropylamino group.)

[0224] (Appendix 8) The kordsepine derivative represented by formula I according to Appendix 7, characterized in that the kordsepine derivative represented by formula I is compound 19, or a pharmaceutically acceptable salt, stereoisomer, tautomer, solvate, prodrug or metabolite thereof. [Chemical formula]

[0225] (Appendix 9) The kordsepine derivative represented by formula I, or a pharmaceutically acceptable salt, stereoisomer, tautomer, solvate, prodrug or metabolite thereof. [Chemical formula] (In the formula, R 1 is R1 10 -R 1 11 、 or R 1 14 -R 1 16 selected from the groups represented by, R 2 is selected from hydrogen or an azide group, R 3 is selected from hydrogen, R 4 is selected from a hydroxyl group, R 5 is selected from fluorine, R 6 is selected from an amino group, R 7 is selected from hydrogen or an isopropylamino group.)

[0226] (Appendix 10) The cordycepin derivative represented by formula I is the cordycepin derivative represented by formula I described in Appendix 9, which is compound 20 or compound 17, or a pharmaceutically acceptable salt, stereoisomer, tautomer, solvate, prodrug or metabolite thereof.

Chemical formula

[0227] (Appendix 11) The cordycepin derivative represented by formula I, or a pharmaceutically acceptable salt, stereoisomer, tautomer, solvate, prodrug or metabolite thereof.

Chemical formula

[0228] (Appendix 12) The kordsepine derivative represented by formula I is the compound 18, or a pharmaceutically acceptable salt, stereoisomer, tautomer, solvate, prodrug or metabolite thereof, of the kordsepine derivative represented by formula I described in Appendix 11. [Chemical formula]

[0229] (Appendix 13) The kordsepine derivative represented by formula I, or a pharmaceutically acceptable salt, stereoisomer, tautomer, solvate, prodrug or metabolite thereof. [Chemical formula] (In the formula, R 1 is R 1 10 -R 1 11 , or R 1 14 -R 1 16 selected from the groups represented by, R 2 is selected from hydrogen, or an azide group, R 3 is selected from an azide group, R 4 is selected from a hydroxyl group, R 5 is selected from hydrogen, a bromovinyl group, a mercapto group, a methyl group, fluorine, or chlorine, R 6 is selected from an amino group, R 7 is selected from hydrogen, or an isopropylamino group.)

[0230] (Appendix 14) The cordycepin derivative represented by Formula I is the compound 26, or the compound 27, and is the cordycepin derivative represented by Formula I described in Appendix 13, or a pharmaceutically acceptable salt, stereoisomer, tautomer, solvate, prodrug or metabolite thereof.

Chem.

[0231] (Appendix 15) The cordycepin derivative represented by Formula I, or a pharmaceutically acceptable salt, stereoisomer, tautomer, solvate, prodrug or metabolite thereof.

Chem.

[0232] (Appendix 16) The cordycepin derivative represented by Formula I is the compound 29, and is the cordycepin derivative represented by Formula I described in Appendix 15, or a pharmaceutically acceptable salt, stereoisomer, tautomer, solvate, prodrug or metabolite thereof.

Chem.

[0233] (Appendix 17) The pharmaceutical composition comprises a colchicine derivative as described in any one of Supplementary Notes 1 to 16, or a pharmaceutically acceptable salt, stereoisomer, tautomer, solvate, prodrug or metabolite thereof, and at least one immune checkpoint inhibitor. Preferably, the immune checkpoint inhibitor is selected from PD-1 and / or CTLA4 monoclonal antibodies. Preferably, the mass ratio of the colchicine derivative, or a pharmaceutically acceptable salt, stereoisomer, tautomer, solvate, prodrug or metabolite thereof, to the immune checkpoint inhibitor is 1:0.2 to 10, preferably 1:0.2 to 8, more preferably 1:0.2 to 5, even more preferably 1:0.2 to 3, still more preferably 1:0.2 to 2, most preferably 1:0.5 to 1.5, and particularly preferably 1:0.8 to 1.2, and most particularly preferably 1:1.

[0234] (Supplementary Note 18) The pharmaceutical composition according to Supplementary Note 17, wherein the dosage form of the pharmaceutical composition is selected from tablets, pellets, capsules, pills, syrups, disintegrants, injections, sustained-release agents, or kits.

[0235] (Supplementary Note 19) Use of a colchicine derivative as described in any one of Supplementary Notes 1 to 16, or a pharmaceutically acceptable salt, stereoisomer, tautomer, solvate, prodrug or metabolite thereof, or a pharmaceutical composition as described in Supplementary Note 17 or 18, in the preparation of a product for preventing and treating related diseases caused by mutations in cell dysfunction in mammals or humans, wherein the related diseases caused by mutations in cell dysfunction are preferably tumors, and the tumors preferably include 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, and the tumor is preferably any one of gastric cancer, pancreatic cancer, liver cancer, small cell lung cancer, colorectal cancer, melanoma and ovarian cancer.

[0236] (Supplementary Note 20) A method for preparing a colchicine derivative as described in any one of Supplementary Notes 1 to 16, In formula I, R1 is R 1 10 -R 1 11 or R 1 14 -R 1 16 When selected from the groups represented by, the method for preparing the kordsepiline derivative represented by formula I is to carry out a chemical reaction with compound I-R-1 as a raw material in an organic solvent to obtain the kordsepiline derivative represented by formula I.

Chemical formula

[0237] (Appendix 21) A method for preparing a colchicine derivative according to any one of Appendices 1 to 16, In formula I, R 3 When selected from fluorine or an azide group, the method for preparing a colchicine derivative represented by formula I is to carry out a chemical reaction using compound I-R-3 as a raw material in an organic solvent to obtain a colchicine derivative represented by formula I, [Chemical formula] (In formula I-R-3, R 1 , R 2 , R 5 ~R 7 is the same as R 1 , R 2 , R 5 ~R 7 in formula I, or each is independently selected from protecting groups.) In formula I, R 3When R is selected from fluorine, preferably, the method for preparing the cordycepin derivative represented by Formula I is to react Compound I-R-3 with trifluoromethanesulfonic anhydride in pyridine and dichloromethane to obtain Intermediate I-R-3a, 5-(6-amino-9H-purin-9-yl)-4-hydroxy-2-(hydroxymethyl)tetrahydrofuran-3-yl trifluoromethanesulfonate or a derivative thereof. In ethyl acetate, Intermediate I-R-3a is subjected to a substitution reaction with hydrofluoric acid and sulfur trifluoride to obtain the cordycepin derivative represented by Formula I. In the method for preparing the Intermediate I-R-3a, preferably, the ratio of the amounts of use of the Compound I-R-3, trifluoromethanesulfonic anhydride, pyridine and dichloromethane is 1 mmol: 1 to 1.5 mmol: 0.15 to 0.2 mL: 10 to 20 mL. Preferably, the reaction temperature is -5 to 5 °C, preferably 0 °C, and preferably the reaction time is 1 to 3 hours. In the method for preparing the cordycepin derivative represented by Formula I, preferably, Intermediate I-R-3a is subjected to a substitution reaction with 37% hydrofluoric acid or diethylaminosulfur trifluoride or a triethylamine solution of hydrochloric acid. Preferably, the ratio of the amounts of use of the Intermediate I-R-3a, 37% hydrofluoric acid or diethylaminosulfur trifluoride or a triethylamine solution of hydrochloric acid and ethyl acetate is 1 mmol: 2 to 3 mmol: 4 to 10 mL. Preferably, the reaction temperature is 60 to 80 °C, and preferably the reaction time is 8 to 10 hours. In Formula I, R 3When R is selected from azide groups, preferably, the method for preparing the cordycepin derivative represented by formula I is to cyclize compound I-R-3 in N,N-dimethylformamide under the catalysis of triphenylphosphine and diisopropyl azodicarboxylate to obtain intermediate I-R-3b, 4-(6-amino-9H-purin-9-yl)-3,6-dioxane[3.1.0]hexan-2-yl)methanol or its derivative, and in dimethylformamide, react the obtained intermediate I-R-3b with sodium azide by a ring-opening reaction to obtain the cordycepin derivative represented by formula I. In the method for preparing the intermediate I-R-3b, preferably, the ratio of the amounts of the compound I-R-3, triphenylphosphine, diisopropyl azodicarboxylate and N,N-dimethylformamide used is 10.0 mmol:16 - 28 mmol:1.6 - 2.8 mmol:15 - 50 mL, preferably 10.0 mmol:22 mmol:2.2 mmol:15 - 50 mL. Preferably, the cyclization reaction temperature is 10 - 60 °C, preferably, the cyclization reaction time is 1 - 5 hours. Preferably, in the method for preparing the cordycepin derivative represented by formula I, the ratio of the amounts of the intermediate I-R-3b, sodium azide and dimethylformamide used is 1 mmol:4 - 5 mmol:2 - 5 mL. Preferably, the ring-opening reaction temperature is 100 - 120 °C, preferably, the ring-opening reaction time is 12 - 16 hours, characterized in that Preparation method.

[0238] (Appendix 22) A method for preparing the cordycepin derivative according to any one of Appendices 1 to 16, In formula I, R 4 is a cyano group, R 4 5 ~R 4 7 When selected from the groups represented by, the method for preparing the cordycepin derivative represented by formula I is to carry out a chemical reaction using compound I-R-4 as a raw material in an organic solvent to obtain the cordycepin derivative represented by formula I.

Chemical formula

[0239] (Appendix 23) A method for preparing a kordsepiline derivative according to any one of Appendices 1 to 16, In formula I, R 5 When selected from a mercapto group or fluorine, the method for preparing a kordsepiline derivative represented by formula I is to carry out a chemical reaction using compound I-R-5 as a raw material in an organic solvent to obtain a kordsepiline derivative represented by formula I.

Chemical formula

Chemical formula

Chemical formula

[0240] (Supplementary Note 24) A method for preparing a colchicine derivative according to any one of Supplementary Notes 1 to 16, In formula I, R 6 is R 6 15 When selected from the groups represented by, the method for preparing a colchicine derivative represented by formula I is to carry out a chemical reaction using compound I-R-6 as a raw material in an organic solvent to obtain a colchicine derivative represented by formula I, [Chemical formula] (In formula I-R-6, R 1 ~R 5 , R 7 is the same as R 1 ~R 5 , R 7 in formula I or is each independently selected from protecting groups.) The method for preparing a colchicine derivative represented by formula I preferably reacts compound I-R-6 with a substituted acid chloride in anhydrous pyridine, wherein the substitution is by a pyridyl group. Preferably, the ratio of the amounts used of compound I-R-6, the substituted acid chloride, and anhydrous pyridine is 1 mmol: 1-2 mmol: 5-10 mL. Preferably, the reaction temperature is 0-60 °C. Preferably, the reaction time is 2-20 hours, characterized by preparation method.

Claims

1. A cordycepin derivative represented by formula I, or a pharma- ceutically acceptable salt thereof. 【Chemistry 1】 (In the formula, R １ is R １ １６ is a group represented by R ２ is hydrogen, R ３ is hydrogen, R ４ is a hydroxyl group, R ５ is selected from hydrogen, fluorine, or chlorine; R ６ is selected from pyridine carboxamide or an amino group; R ７ is hydrogen.)

2. 2. The cordycepin derivative of formula I according to claim 1, or a pharma- ceutically acceptable salt thereof, wherein the cordycepin derivative of formula I is selected from the following compounds: 【Chemistry 2】

3. A pharmaceutical composition comprising the cordycepin derivative according to claim 1 or 2, or a pharma- ceutically acceptable salt thereof, and at least one immune checkpoint inhibitor, wherein the immune checkpoint inhibitor is selected from PD-1 and / or CTLA4 monoclonal antibodies.

4. The pharmaceutical composition described in claim 3, wherein the mass ratio of the cordycepin derivative, or a pharma- ceutical acceptable salt thereof, to the immune checkpoint inhibitor is 1:0.2-5.

5. The pharmaceutical composition described in claim 3, wherein the mass ratio of the cordycepin derivative, or a pharma- ceutically acceptable salt thereof, to the immune checkpoint inhibitor is 1:0.2-2.

6. The pharmaceutical composition described in claim 3, wherein the mass ratio of the cordycepin derivative, or a pharma- ceutical acceptable salt thereof, to the immune checkpoint inhibitor is 1:0.5-1.

5.

7. The pharmaceutical composition described in claim 3, wherein the mass ratio of the cordycepin derivative, or a pharma- ceutically acceptable salt thereof, to the immune checkpoint inhibitor is 1:

1.

8. The pharmaceutical composition according to claim 3, wherein the dosage form of the pharmaceutical composition is selected from the group consisting of tablets, pellets, capsules, pills, syrups, disintegrants, injections, sustained release agents, and kits.

9. 2. Use of the cordycepin derivative or its pharma- ceutically acceptable salt according to claim 1 in the preparation of a product for preventing and treating a disease associated with a mutation in cellular dysfunction in humans, wherein the disease associated with a mutation in cellular dysfunction is a tumor.

10. The use of claim 9, wherein the tumor comprises 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.

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