Hypoxia activated prodrug derivative and synthesis method thereof

Abstract

The invention belongs to the technical field of drug synthesis, and particularly provides a hypoxia activated prodrug derivative and a synthesis method thereof. The method comprises the following steps: by taking benzimidazole as a raw material, carrying out ring opening, decarboxylation, esterification, iodination, bromine substitution, reduction and cyano substitution to obtain (1-methyl-1H-imidazole-2-nitrile-5-yl) methanol and (1-methyl-1H-imidazole-2-nitrile-4-yl) methanol; then, the N, N '-bis (2-bromoethyl) diamino phosphonic acid (1-methyl-1H-imidazole-2-nitrile-5-yl) methyl ester and bromoisophosphamide nitrogen mustard are subjected to condensation reaction to obtain N, N'-bis (2-bromoethyl) diamino phosphonic acid (1-methyl-1H-imidazole-2-nitrile-5-yl) methyl ester or N, N '-bis (2-bromoethyl) diamino phosphonic acid (1-methyl-1H-imidazole-2-nitrile-4-yl) methyl ester. Through the test on the cell inhibition rate of ovarian cancer cells, the inhibition rate of the N, N '-bis (2-bromoethyl) diamino phosphonic acid (1-methyl-1H-imidazole-2-nitrile-5-yl) methyl ester is equivalent to that of TH-302.

Description

Technical Field

[0001] The invention belongs to the technical field of drug synthesis, and specifically provides a hypoxia activated prodrug derivative and a synthesis method thereof. Background Art

[0002] The distribution of blood vessels in tumor tissue is different from that in normal tissue and is disordered, resulting in its microenvironment being scattered with hypoxic and normoxic areas. In normoxic areas where blood vessels are relatively abundant, tumor cells are generated rapidly and are extremely sensitive to traditional chemotherapy, while tumor cells in hypoxic areas are dormant and resistant to standard chemotherapy and radiotherapy, becoming the main obstacle to tumor treatment. In the process of adapting to the hypoxic tumor microenvironment, cancer cells acquire invasiveness and metastasis, and develop resistance to chemotherapy and radiotherapy. The development of hypoxia-activated anti-tumor drugs that can kill tumor cells in hypoxic microenvironments is currently a hot topic.

[0003] Hypoxia-activated prodrugs are selective hypoxia-activated DNA alkylating agents with high cytotoxicity, which can selectively deliver cytotoxic or cytostatic agents to hypoxic tumor cells and have low toxicity to normal tissues. TH-302 is one of the most effective and clinically studied hypoxia-activated prodrugs.

[0004]

[0005] The traditional synthetic route of hypoxia-activated prodrug TH-302 has the disadvantages of high raw material cost, complex route, and difficulty in modifying the groups on the nitrogen atom of the parent ring. In addition, long-term use of the same drug may cause tumor cells to develop certain drug resistance. Therefore, it is very necessary to modify the structure of TH-302 and develop new alternative molecules, which has broad application prospects. Summary of the invention

[0006] The purpose of the present invention is to provide a new hypoxia-activated prodrug derivative and a synthesis method thereof. The hypoxia-activated prodrug derivative provided by the present invention has the following general formula compounds I and II:

[0007]

[0008] The synthetic route of the hypoxia activated prodrug derivative is shown below:

[0009]

[0010] The synthesis method of the hypoxia activated prodrug derivative comprises the following specific steps:

[0011] (1) Ring opening: Add benzimidazole and a small amount of water to a reaction flask and stir, then drop concentrated sulfuric acid into it. After the dropwise addition, slowly add potassium dichromate (add a small amount of ice cubes during the process to prevent the system temperature from being too high), raise the temperature to 95°C and stir to react for 15 minutes. Pour the system into ice water while it is still hot, place it in a refrigerator to cool for several hours, filter it, wash the filter cake with water several times, and dry it to obtain a light green solid imidazole-4,5-dicarboxylic acid.

[0012] (2) Decarboxylation: Add imidazole-4,5-dicarboxylic acid and acetic anhydride to a reaction flask, heat under reflux and stir until the solution turns brown, cool to room temperature, filter, and spin-dry the filtrate to obtain a black viscous solid. Add water, stir at room temperature for 2 hours, then heat to 100°C and stir for 5 hours, then reduce to 60°C, add activated carbon and ethanol, heat under reflux for 16 hours, reduce to 70°C, filter while hot to obtain a light yellow liquid, place at room temperature for crystallization, then place at 0°C for crystallization, filter, wash the solid with acetone for several times, and dry to obtain a white solid 1H-imidazole-4-carboxylic acid.

[0013] (3) Esterification: 1H-imidazole-4-carboxylic acid, ethanol, concentrated sulfuric acid and ferric sulfate were added to a reaction flask, mixed and heated under reflux for 21 hours, the system was cooled to room temperature, 5N sodium hydroxide solution was added dropwise to adjust the pH value of the system to 7, the system was extracted with ethyl acetate, the organic phase was dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain white solid 1H-imidazole-4-carboxylic acid ethyl ester.

[0014] (4) Iodination: Add 1H-imidazole-4-carboxylic acid ethyl ester, potassium tert-butoxide and acetone to a reaction flask, slowly add iodomethane, react at room temperature for 16 hours, extract the system with ethyl acetate and water, dry the organic phase with anhydrous sodium sulfate, concentrate under reduced pressure, and separate by silica gel column chromatography to obtain 1-methyl-1H-imidazole-5-carboxylic acid ethyl ester and 1-methyl-1H-imidazole-4-carboxylic acid ethyl ester.

[0015] Among them, the molar ratio of 1H-imidazole-4-carboxylic acid ethyl ester, potassium tert-butoxide and iodomethane is 1:1.5:1.5.

[0016] (5) Bromine substitution: Add 1-methyl-1H-imidazole-5-carboxylic acid ethyl ester or 1-methyl-1H-imidazole-4-carboxylic acid ethyl ester, tetrahydrofuran and bromosuccinimide to a reaction flask. Heat under reflux and stir overnight, remove the solvent by distillation under reduced pressure, and separate by silica gel column chromatography to obtain 1-methyl-2-bromo-1H-imidazole-5-carboxylic acid ethyl ester or 1-methyl-2-bromo-1H-imidazole-4-carboxylic acid ethyl ester.

[0017] The molar ratio of 1-methyl-1H-imidazole-5-carboxylic acid ethyl ester or 1-methyl-1H-imidazole-4-carboxylic acid ethyl ester to bromosuccinimide is 1:1.

[0018] (6) Reduction: Under anhydrous and oxygen-free conditions, 1-methyl-2-bromo-1H-imidazole-5-carboxylic acid ethyl ester or 1-methyl-2-bromo-1H-imidazole-4-carboxylic acid ethyl ester, sodium borohydride, calcium chloride and anhydrous ethanol are added to a reaction flask, and the mixture is stirred at room temperature overnight. The solvent is removed under reduced pressure, and the system is dissolved in a saturated ammonium chloride solution. The mixture is extracted with ethyl acetate, and the organic layer is separated and dried over anhydrous sodium sulfate. The (1-methyl-2-bromo-1H-imidazole-5-yl)methanol or (1-methyl-2-bromo-1H-imidazole-4-yl)methanol is obtained by separation through silica gel column chromatography.

[0019] Wherein, the molar ratio of 1-methyl-2-bromo-1H-imidazole-5-carboxylic acid ethyl ester or 1-methyl-2-bromo-1H-imidazole-4-carboxylic acid ethyl ester, sodium borohydride and calcium chloride is 1:5:1.

[0020] (7) Cyano substitution: Under anhydrous and oxygen-free conditions, (1-methyl-2-bromo-1H-imidazol-5-yl)methanol or (1-methyl-2-bromo-1H-imidazol-4-yl)methanol, cuprous cyanide, potassium iodide and anhydrous dimethyl sulfoxide were added to a reaction flask, and the mixture was reacted at 100° C. for 48 hours. The mixture was cooled to room temperature, and the system was poured into a saturated aqueous sodium bicarbonate solution, and extracted with ethyl acetate. The organic phase was dried over anhydrous sodium sulfate, concentrated under reduced pressure, and separated by silica gel column chromatography to obtain (1-methyl-1H-imidazol-2-carbonitrile-5-yl)methanol or (1-methyl-1H-imidazol-2-carbonitrile-4-yl)methanol.

[0021] The molar ratio of (1-methyl-2-bromo-1H-imidazol-5-yl)methanol or (1-methyl-2-bromo-1H-imidazol-4-yl)methanol, cuprous cyanide and potassium iodide is 1:1.1:1.

[0022] (8) Synthesis of bromoisophosphoramide mustard (Br-IPM): 2-bromoethylamine hydrobromide and anhydrous dichloromethane were added to a reaction flask under anhydrous and anaerobic conditions, the reaction system temperature was cooled to -78°C, phosphorus oxychloride was slowly added dropwise to the reaction flask, after the addition was completed, a mixed solution of triethylamine and dichloromethane was added dropwise to the reaction flask, after the addition was completed, the temperature was maintained at -78°C and the reaction was continued with stirring for 1 hour, the temperature was raised to 25°C, and the reaction was continued with stirring for 2 hours; after the reaction was stopped, the reaction was filtered, the filtrate was concentrated, ethyl acetate was added, and the reaction was filtered again, the filtrate was concentrated under reduced pressure to a yellow viscous liquid, a mixed solution of tetrahydrofuran and water was added, the reaction was stirred at 25°C for 5 hours, and then the tetrahydrofuran was spin-dried and kept stirring at 0°C for 15 hours, and finally cooled to -20°C, frozen for crystallization for 2 hours, filtered to obtain a white solid, and dried naturally at room temperature for 48 hours to obtain N,N'-bis(2-bromoethyl)diaminophosphonic acid.

[0023] Wherein, the molar ratio of 2-bromoethylamine hydrobromide, phosphorus oxychloride and triethylamine is 2:1:4.

[0024] (9) Mitsunobu reaction: (1-methyl-1H-imidazole-2-carbonitrile-5-yl)methanol or (1-methyl-1H-imidazole-2-carbonitrile-4-yl)methanol, N,N'-bis(2-bromoethyl)diaminophosphonic acid, triphenylphosphine and anhydrous tetrahydrofuran were added to a reaction flask under nitrogen protection. After cooling to 0°C, diisopropyl azodicarboxylate was added dropwise to the reaction flask. The temperature was raised to 25°C and the reaction was allowed to proceed for 6 h. The reaction was stopped. After concentration under reduced pressure, the mixture was separated by silica gel column chromatography to obtain N,N'-bis(2-bromoethyl)diaminophosphonic acid (1-methyl-1H-imidazole-2-carbonitrile-5-yl) methyl ester or N,N'-bis(2-bromoethyl)diaminophosphonic acid (1-methyl-1H-imidazole-2-carbonitrile-4-yl) methyl ester.

[0025] Among them, the molar ratio of (1-methyl-1H-imidazole-2-carbonitrile-5-yl)methanol or (1-methyl-1H-imidazole-2-carbonitrile-4-yl)methanol, N,N'-bis(2-bromoethyl)diaminophosphonic acid, triphenylphosphine, and diisopropyl azodicarboxylate is 1:0.5:1:1.

[0026] Beneficial effects:

[0027] The present invention uses benzimidazole with lower cost as raw material to synthesize N,N'-bis(2-bromoethyl)diaminophosphonic acid (1-methyl-1H-imidazole-2-nitrile-5-yl) methyl ester and its isomer N,N'-bis(2-bromoethyl)diaminophosphonic acid (1-methyl-1H-imidazole-2-nitrile-4-yl) methyl ester, and the method synthesizes two new hypoxia-activated prodrug derivatives with a high yield. The cell inhibition rate activity test data of the drug show that compared with the inhibition rate of TH-302, the inhibition rate of compound I on ovarian cancer cell lines is better, and the inhibition rate of compound II on ovarian cancer cell lines is relatively low; for compounds I and II that are isomers of each other, the structure of compound I has a better inhibitory effect on ovarian cancer cell lines. It is inferred that the two characteristics of cyano group, conjugated electron-withdrawing and self-oxidation-reduction, can improve the drug activity of compound I to increase the anti-cancer efficacy of the drug. At the same time, the hypoxia-activated prodrug compound I, compound II, and TH-302 have different structures but similar effects, which can solve the problem of tumor resistance to this type of drug. DETAILED DESCRIPTION

[0028] In order to better understand the present invention, the present invention will be further described below by way of examples.

[0029] Example 1

[0030] Add benzimidazole (1 g, 8.5 mmol) and 20 ml of water to the reaction flask and stir, then drop 98% concentrated sulfuric acid (14 mL). After the drop is complete, add potassium dichromate (7.4 g, 25.2 mmol) within 15 minutes (10 g of ice cubes are added three times during the process to prevent the system temperature from being too high). Heat to 95 ° C and stir for 15 minutes. Pour the system into ice water while it is hot, place it in a refrigerator to cool for 3 hours, filter, wash the filter cake with water 3 times, and dry it to obtain a light green solid imidazole-4,5-dicarboxylic acid 2. Yield: 81%. 1 HNMR (300MHz, DMSO-d6) δ9.08 (s, 1H).

[0031] Example 2

[0032] Add imidazole-4,5-dicarboxylic acid (2 g, 12.7 mmol) and acetic anhydride (70 mL) to the reaction bottle, heat at 130 ° C, reflux and stir until the solution turns brown, cool to room temperature, filter, and spin-dry the filtrate to obtain a black viscous solid. Add water (30 mL), stir at room temperature for 2 hours, then raise to 100 ° C and stir for 5 hours, reduce to 60 ° C, add activated carbon (200 mg) and ethanol (30 mL), heat and reflux for 16 hours, reduce to 70 ° C, filter while hot to obtain a light yellow liquid, place at room temperature for crystallization for 2 hours, and then place at 0 ° C for crystallization for 24 hours, filter, wash the solid with acetone several times, and dry to obtain a white solid 1H-imidazole-4-carboxylic acid 3, Yield: 55%. 1 H NMR (300MHz, DMSO-d6) δ12.63(s,1H),7.76(s,1H),7.68(s,1H).

[0033] Example 3

[0034] 1H-imidazole-4-carboxylic acid (897 mg, 8 mmol), 10 ml of ethanol, 1 ml of concentrated sulfuric acid and ferric sulfate (430 mg, 1.1 mmol) were added to the reaction bottle, mixed and heated to 60°C and refluxed for 21 hours, the system was cooled to room temperature, 5N sodium hydroxide solution was added dropwise to adjust the pH value of the system to 7, the system was extracted with ethyl acetate, the organic phase was dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain white solid 1H-imidazole-4-carboxylic acid ethyl ester 4, Yield: 96%. 1 H NMR (400MHz, DMSO-d6) δ12.64(s,1H),7.84(s,1H),7.76(s,1H),4.20(q,J=6.8Hz,2H),1.26(t,J=7.0Hz,3H).

[0035] In addition, the effect of reaction time on the reaction was further studied:

[0036]

[0037]

[0038] When the amount of the raw material 1H-imidazole-4-carboxylic acid was 112.09 mg, 1.0 mmol, and the reflux temperature was 60°C, the reaction time of the reaction was investigated. When the initial reaction time was 12 hours, the yield was only 52%. When the reaction time was extended to 21 hours, the yield of the reaction reached 96%. The yield remained unchanged when the reaction time was further extended. Therefore, the optimal reaction time of the reaction was determined to be 21 hours, which can stably produce the target compound 1H-imidazole-4-carboxylic acid ethyl ester.

[0039] Example 4

[0040] 1H-imidazole-4-carboxylic acid ethyl ester (280 mg, 2 mmol), acetone (10 mL), potassium tert-butoxide (337 mg, 3 mmol), and iodomethane (1.5 eq.) were added to the reaction bottle and stirred at room temperature for 16 h. After stopping the reaction, the solvent was spin-dried, extracted with ethyl acetate and water, and the organic phase was dried over anhydrous sodium sulfate. After concentration under reduced pressure, it was separated and purified by silica gel column chromatography [eluent: V (petroleum ether) / V (ethyl acetate) = 1 / 1] to obtain 1-methyl-1H-imidazole-5-carboxylic acid ethyl ester 5. Yield: 38%. 1 H NMR (300 MHz, Chloroform-d) δ 7.70 (s, 1H), 7.52 (s, 1H), 4.30 (q, J = 7.1 Hz, 2H), 3.89 (s, 3H), 1.35 (t, J = 7.1 Hz, 3H), and 1-methyl-1H-imidazole-4-carboxylic acid ethyl ester 6, Yield: 37%. 1 H NMR (300MHz, Chloroform-d) δ7.67 (s, 1H), 7.42 (s, 1H), 4.16 (q, J = 7.1Hz, 2H), 3.69 (s, 3H), 1.15 (t, J = 7.1Hz, 3H).

[0041] In addition, the effects of the solvent, the type of base, the equivalent number of base, and the equivalent number of iodomethane on the reaction were further studied.

[0042] Table 2

[0043]

[0044] The initial reaction conditions were 1.0 equivalent of 1H-imidazole-4-carboxylic acid ethyl ester, N,N-dimethylformamide (DMF) as the reaction solvent, 1.0 equivalent of iodomethane under the action of 1.0 equivalent of potassium carbonate, stirring at room temperature for 16 hours, and the total yield was only 21%. First, the reaction solvent was screened, and when the solvent was acetone, the total yield could be increased to 36%; then the type of base was screened, and when potassium tert-butoxide was used in an amount of 1.5 equivalents, the reaction yield was greatly increased to 68%; finally, when the equivalent of iodomethane was increased to 1.5 equivalents, the total yield of the reaction increased to 75%. The final reaction conditions are: using 1H-imidazole-4-carboxylic acid ethyl ester as raw material, using 1.5 equivalents of potassium tert-butoxide and 1.5 equivalents of iodomethane under the action of acetone solution, stirring the reaction at room temperature for 16 hours, 1-methyl-1H-imidazole-5-carboxylic acid ethyl ester (38%) and 1-methyl-1H-imidazole-4-carboxylic acid ethyl ester (37%) can be obtained in a high yield, and the total yield reaches 75%.

[0045] Example 5

[0046] To the reaction flask, 1-methyl-1H-imidazole-5-carboxylic acid ethyl ester (231 mg, 1.5 mmol), bromosuccinimide (267 mg, 1.5 mmol), and tetrahydrofuran (15 mL) were added, and the mixture was heated under reflux with stirring overnight. The system was cooled to room temperature, and the solvent was removed under reduced pressure. The mixture was separated and purified by silica gel column chromatography [eluent: V (petroleum ether) / V (ethyl acetate) = 3 / 1] to obtain 2-bromo-1-methyl-1H-imidazole-5-carboxylic acid ethyl ester 7. Yield: 61%. 1 H NMR (300MHz, Chloroform-d) δ7.67 (s, 1H), 4.32 (q, J = 7.1Hz, 2H), 3.90 (s, 3H), 1.36 (t, J = 7.1Hz, 3H).

[0047] In addition, the source of bromine for this reaction was screened:

[0048] Table 3

[0049]

[0050] First, 1.0 equivalent of liquid bromine was selected as the bromine source, and the mixture was heated under reflux overnight in tetrahydrofuran as solvent, but the product was finally obtained with a yield of only 15%. Then, the bromine source was replaced with dibromohydantoin (DBDMH), and 2-bromo-1-methyl-1H-imidazole-5-carboxylic acid ethyl ester was finally obtained with a yield of 44%. Finally, when the bromine source was replaced with bromosuccinimide (NBS), the yield was increased to 61%.

[0051] Example 6

[0052] The 1-methyl-1H-imidazole-5-carboxylic acid ethyl ester in Example 5 was replaced with 1-methyl-1H-imidazole-4-carboxylic acid ethyl ester to obtain 2-bromo-1-methyl-1H-imidazole-4-carboxylic acid ethyl ester 8. Yield: 55%. 1 H NMR (300MHz, Chloroform-d) δ7.57 (s, 1H), 4.27 (q, J = 7.1Hz, 2H), 3.61 (s, 3H), 1.29 (t, J = 7.1Hz, 3H).

[0053] In addition, the reaction solvent and temperature conditions for this reaction were screened:

[0054] Table 4

[0055]

[0056] For the screening of solvents, three solvents were selected: acetonitrile, acetone and tetrahydrofuran, and tetrahydrofuran had the best effect. When the reaction temperature was lowered to 45°C, the yield decreased, and when the reaction temperature was raised to 85°C, the yield did not increase. Therefore, the final preferred condition is that 1-methyl-1H-imidazole-4-carboxylic acid ethyl ester is stirred overnight (12 hours) at 65°C under the action of tetrahydrofuran as a solvent, and 2-bromo-1-methyl-1H-imidazole-4-carboxylic acid ethyl ester is obtained with a yield of 55%.

[0057] Example 7

[0058] Under anhydrous and oxygen-free conditions, sodium borohydride (189 mg, 5.0 mmol), anhydrous calcium chloride (111 mg, 1.0 mmol) and ethyl 2-bromo-1-methyl-1H-imidazole-5-carboxylate (233 mg, 1.0 mmol) in 10 mL of anhydrous ethanol solution were added to the reaction bottle, stirred at room temperature overnight, the solvent was removed under reduced pressure, the system was dissolved in saturated ammonium chloride solution, extracted with ethyl acetate, the organic layer was dried over anhydrous sodium sulfate, concentrated under reduced pressure, and separated and purified by silica gel column chromatography [eluent: V (ethyl acetate) / V (methanol) = 30 / 1] to obtain (2-bromo-1-methyl-1H-imidazole-5-yl)methanol 9. Yield: 63%. 1 H NMR (300MHz, Chloroform-d) δ6.89 (s, 1H), 4.61 (s, 2H), 3.65 (s, 3H).

[0059] Example 8

[0060] The 2-bromo-1-methyl-1H-imidazole-5-carboxylic acid ethyl ester in Example 7 was replaced with 2-bromo-1-methyl-1H-imidazole-4-carboxylic acid ethyl ester to obtain (2-bromo-1-methyl-1H-imidazole-4-yl)methanol 10. Yield: 65%. 1 H NMR (300MHz, Chloroform-d) δ6.91 (s, 1H), 4.51 (s, 2H), 3.57 (s, 3H).

[0061] Example 9

[0062] Under anhydrous and oxygen-free conditions, (2-bromo-1-methyl-1H-imidazol-5-yl)methanol (95 mg,

[0063] 0.5mmol), cuprous cyanide (49mg, 0.55mmol), potassium iodide (83mg, 0.5mmol) were added to a mixture of 7mL of anhydrous dimethyl sulfoxide. The mixture was reacted at 100°C for 48h, cooled to room temperature, poured into a saturated sodium bicarbonate solution, extracted with ethyl acetate, and the organic phase was dried over anhydrous sodium sulfate, concentrated under reduced pressure, and separated and purified by silica gel column chromatography [eluent: V (ethyl acetate) / V (methanol) = 40 / 1] to obtain (1-methyl-1H-imidazole-2-carbonitrile-5-yl)methanol 11. Yield: 62%. 1 HNMR (300MHz, DMSO-d6) δ7.11 (s, 1H), 5.40 (t, J = 5.4Hz, 1H), 4.50 (d, J = 5.3Hz, 2H), 3.77 (s, 3H); 13 C NMR (75MHz, DMSO-d6) δ137.78,130.20,121.86,112.21,53.02,32.37.

[0064] Example 10

[0065] The (2-bromo-1-methyl-1H-imidazol-5-yl)methanol in Example 9 was replaced with (2-bromo-1-methyl-1H-imidazol-4-yl)methanol to obtain (1-methyl-1H-imidazol-2-carbonitrile-4-yl)methanol 12. Yield: 57%. 1 H NMR (300MHz, DMSO-d6) δ7.43 (s, 1H), 5.17 (t, J = 4.8Hz, 1H), 4.35 (d, J = 5.0Hz, 2H), 3.81 (s, 3H); 13 C NMR (75MHz, DMSO-d6) δ145.76, 123.76, 34.29.

[0066] Embodiment 11

[0067] Under nitrogen protection, 6.3 g (30.8 mmol, 2.0 eq.) of 2-bromoethylamine hydrobromide and anhydrous dichloromethane (40 mL) were added to the reaction flask, the reaction system temperature was cooled to -78 ° C, and 1.42 mL (15.4 mmol, 1.0 eq.) of phosphorus oxychloride was slowly added dropwise to the reaction flask. After the addition was completed, a mixed solution of triethylamine (9 mL) and dichloromethane (15 mL) was added dropwise to the reaction flask. After the addition was completed, the reaction was maintained at -78 ° C and stirred for 1 h. The temperature was raised. to 25°C, continue stirring to react for 2h; after stopping the reaction, filter, concentrate the filtrate, add ethyl acetate, filter again, vacuum concentrate the filtrate to a yellow viscous liquid, add tetrahydrofuran (2mL) and water (4mL), stir and react at 25°C for 5h, then spin dry tetrahydrofuran, keep stirring at 0°C for 15h, finally cool to -20°C, freeze crystallize for 2h, filter to obtain a white solid, and dry naturally at room temperature for 48h to obtain N,N'-bis(2-bromoethyl)diaminophosphonic acid 13. Yield 50%. 1 H NMR (300MHz, DMSO-d6) δ6.68 (s, 3H), 3.41 (t, J = 7.2Hz, 4H), 3.07 (dt, J = 12.5, 7.2Hz, 4H); 13 C NMR (75MHz, DMSO-d6) δ: 43.46, 34.43.

[0068] Example 12

[0069] To the reaction flask, 50 mg (0.28 mmol, 1.0 eq.) of (1-methyl-1H-imidazole-2-carbonitrile-5-yl)methanol, 44 mg (0.14 mmol, 0.5 eq.) of N,N'-bis(2-bromoethyl)diaminophosphonic acid, 74 mg (0.28 mmol, 1.0 eq.) of triphenylphosphine and anhydrous tetrahydrofuran (15 mL) were added, the reaction system was cooled to 0°C, 55 μL (0.28 mmol, 1.0 eq.) of DIAD was added dropwise to the reaction flask, the temperature was raised to 25°C and the reaction was carried out for 6 h. After the reaction was stopped, the mixture was concentrated under reduced pressure to obtain a crude product, which was purified by silica gel column chromatography [eluent: V (ethyl acetate) / V (methanol) = 20 / 1] to obtain the product N,N'-bis(2-bromoethyl)diaminophosphonic acid (1-methyl-1H-imidazole-2-carbonitrile-5-yl) methyl ester I. Yield: 62%. 1 H NMR (300MHz, Chloroform-d) δ7.20 (s, 1H), 5.01 (d, J = 8.2Hz, 2H), 3.85 (s, 3H), 3.42 (t, J = 5.7Hz, 6H), 3.29 (dt, J = 11.7, 5.8Hz, 4H); 13C NMR (75MHz, Chloroform-d) δ131.22, 130.49, 122.81, 109.78, 54.83, 41.88, 33.58, 31.29.

[0070] Embodiment 13

[0071] Replace the (1-methyl-1H-imidazole-2-carbonitrile-5-yl)methanol in Example 12 with (1-methyl-1H-imidazole-2-carbonitrile-4-yl)methanol to obtain N,N'-bis(2-bromoethyl)diaminophosphonic acid (1-methyl-1H-imidazole-2-carbonitrile-4-yl)methyl ester II. Yield: 45%. 1 H NMR (300MHz, Chloroform-d) δ7.19 (s, 1H), 4.93 (d, J = 10.2Hz, 2H), 3.85 (s, 3H), 3.44 (s, 6H), 3.36-3.29 (m, 4H); 13 C NMR (75MHz, Chloroform-d) δ139.59,122.55,121.37,109.46,59.44,41.90,33.66,33.25.

[0072] For the reactions of Example 12 and Example 13, first, at 0°C, under nitrogen protection, 1.0 equivalent of (1-methyl-1H-imidazole-2-carbonitrile-5-yl)methanol or (1-methyl-1H-imidazole-2-carbonitrile-4-yl)methanol was reacted with 1.0 equivalent of N,N'-bis(2-bromoethyl)diaminophosphonic acid to synthesize N,N'-bis(2-bromoethyl)diaminophosphonic acid (1-methyl-1H-imidazole- The reaction mixture was stirred for 24 hours and then stirred for 3 hours. The reaction mixture was stirred for 24 hours. The reaction mixture was stirred for 3 hours. The reaction mixture was stirred for 4 hours. The reaction mixture was stirred for 3 hours. The reaction mixture was stirred for 4 hours. The reaction mixture was stirred for 3 hours. The reaction mixture was stirred for 4 hours. The reaction mixture was stirred for 3 hours. The yield of N,N'-bis(2-bromoethyl)diaminophosphonic acid (1-methyl-1H-imidazole-2-carbonitrile-5-yl) methyl ester or N,N'-bis(2-bromoethyl)diaminophosphonic acid (1-methyl-1H-imidazole-2-carbonitrile-4-yl) methyl ester was significantly improved by increasing the amount of alcohol to four times that of N,N'-bis(2-bromoethyl)diaminophosphonic acid and raising the reaction temperature to 40 ℃ remained unchanged, so it was determined that the optimal synthesis conditions for this reaction were 25℃, when 2.0 equivalents of alcohol were reacted with N,N'-bis(2-bromoethyl)diaminophosphonic acid to obtain N,N'-bis(2-bromoethyl)diaminophosphonic acid (1-methyl-1H-imidazole-2-carbonitrile-5-yl) methyl ester in a yield of 62% and N,N'-bis(2-bromoethyl)diaminophosphonic acid (1-methyl-1H-imidazole-2-carbonitrile-4-yl) methyl ester in a yield of 45%.

[0073] Embodiment 14

[0074] Using the ovarian cancer cell line (SKOV3) as the experimental cells, the effects of compound I, compound II, and TH-302 on the inhibition rate of ovarian cancer cells were investigated at a concentration of 50 μM.

[0075] The cell inhibition rate test method is as follows:

[0076] The cell proliferation inhibition activity was determined by CCK-8 (Cell Counting Kit-8) colorimetric method. Cells in the logarithmic growth phase were collected and the cell culture concentration was 5×10 3cells / 0.1mL / well, 96-well plate. After the cells adhered, they were treated with 50μM of the test compound (Ⅰ, Ⅱ, TH-302). At the same time, a control group was set up, and an equal volume of solvent DMSO was added as a negative control. After 72 hours, the original culture medium was discarded, and the cells were incubated with a nutrient solution containing 10% CCK-8 for 1-4 hours. Finally, the absorbance values ​​(OD values) of the test wells (As), control wells (Ac) and blank wells (Ab) were read at a wavelength of 450nm on an enzyme-linked immunosorbent assay analyzer (Multiskan FC enzyme reader). The inhibition rate was calculated as follows: [(Ac-As) / (Ac-Ab)]×100%, and the results are shown in the following table:

[0077] Table 5

[0078]

[0079] The results showed that compound Ⅰ had a better inhibitory effect on ovarian cancer cell lines.

Claims

1. A hypoxia activated prodrug derivative, characterized in that: The structural formulas of the hypoxia activated prodrug derivatives are shown in Formulas Ⅰ and Ⅱ:

2. A method for synthesizing the hypoxia activated prodrug derivative according to claim 1, characterized in that: The synthesis method steps are as follows: (1) Ring opening: benzimidazole and water were added to a reaction flask and stirred, and concentrated sulfuric acid was then added dropwise. After the addition was complete, potassium dichromate was added, and the temperature was raised to 95°C and stirred for reaction for 15 minutes. The system was poured into ice water while still hot, and placed in a refrigerator for cooling. The system was filtered, the filter cake was washed with water, and dried to obtain a light green solid imidazole-4,5-dicarboxylic acid; (2) Decarboxylation: Add imidazole-4,5-dicarboxylic acid and acetic anhydride to a reaction flask, heat under reflux and stir until the solution turns brown, cool to room temperature, filter, and spin-dry the filtrate to obtain a black viscous solid. Add water, stir at room temperature for 2 hours, then heat to 100°C and stir for 5 hours, then reduce to 60°C, add activated carbon and ethanol, heat under reflux for 16 hours, reduce to 70°C, filter while hot to obtain a light yellow liquid, place at room temperature for crystallization, then place at 0°C for crystallization, filter, wash the solid with acetone, and dry to obtain a white solid 1H-imidazole-4-carboxylic acid; (3) Esterification: 1H-imidazole-4-carboxylic acid, ethanol, concentrated sulfuric acid and ferric sulfate were added to a reaction flask, mixed and heated under reflux for 21 hours, the system was cooled to room temperature, 5N sodium hydroxide solution was added dropwise to adjust the pH value of the system to 7, the system was extracted with ethyl acetate, the organic phase was dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain white solid 1H-imidazole-4-carboxylic acid ethyl ester; (4) Iodination: Add 1H-imidazole-4-carboxylic acid ethyl ester, potassium tert-butoxide and acetone to a reaction flask, then add iodomethane, react at room temperature for 16 hours, extract the system with ethyl acetate and water, dry the organic phase with anhydrous sodium sulfate, concentrate under reduced pressure, and separate by silica gel column chromatography to obtain 1-methyl-1H-imidazole-5-carboxylic acid ethyl ester and 1-methyl-1H-imidazole-4-carboxylic acid ethyl ester; (5) Bromine substitution: Add 1-methyl-1H-imidazole-5-carboxylic acid ethyl ester or 1-methyl-1H-imidazole-4-carboxylic acid ethyl ester, tetrahydrofuran and bromosuccinimide to a reaction flask; heat under reflux and stir overnight, remove the solvent by distillation under reduced pressure, and separate by silica gel column chromatography to obtain 1-methyl-2-bromo-1H-imidazole-5-carboxylic acid ethyl ester or 1-methyl-2-bromo-1H-imidazole-4-carboxylic acid ethyl ester; (6) Reduction: under anhydrous and oxygen-free conditions, add 1-methyl-2-bromo-1H-imidazole-5-carboxylic acid ethyl ester or 1-methyl-2-bromo-1H-imidazole-4-carboxylic acid ethyl ester, sodium borohydride, calcium chloride and anhydrous ethanol to a reaction flask, stir at room temperature overnight, remove the solvent under reduced pressure, dissolve the system in saturated ammonium chloride solution, extract with ethyl acetate, separate the organic layer, and dry with anhydrous sodium sulfate, and separate by silica gel column chromatography to obtain (1-methyl-2-bromo-1H-imidazole-5-yl)methanol or (1-methyl-2-bromo-1H-imidazole-4-yl)methanol; (7) Cyano substitution: under anhydrous and oxygen-free conditions, add (1-methyl-2-bromo-1H-imidazol-5-yl)methanol or (1-methyl-2-bromo-1H-imidazol-4-yl)methanol, cuprous cyanide, potassium iodide and anhydrous dimethyl sulfoxide to a reaction flask, react at 100°C for 48 hours, cool to room temperature, pour the system into a saturated sodium bicarbonate aqueous solution, extract with ethyl acetate, dry the organic phase with anhydrous sodium sulfate, concentrate under reduced pressure, and separate by silica gel column chromatography to obtain (1-methyl-1H-imidazol-2-carbonitrile-5-yl)methanol or (1-methyl-1H-imidazol-2-carbonitrile-4-yl)methanol; (8) Synthesis of bromoisophosphoramide mustard: 2-bromoethylamine hydrobromide and anhydrous dichloromethane were added to a reaction flask under anhydrous and anaerobic conditions, the reaction system temperature was cooled to -78°C, phosphorus oxychloride was added dropwise to the reaction flask, after the addition was completed, a mixed solution of triethylamine and dichloromethane was added dropwise to the reaction flask, after the addition was completed, the temperature was maintained at -78°C and stirred for 1 hour, the temperature was raised to 25°C, and the reaction was stirred for 2 hours; after stopping the reaction, the reaction was filtered, the filtrate was concentrated, ethyl acetate was added, and the reaction was filtered again, the filtrate was concentrated under reduced pressure to a yellow viscous liquid, a mixed solution of tetrahydrofuran and water was added, the reaction was stirred at 25°C for 5 hours, and then the tetrahydrofuran was spin-dried, the reaction was maintained at 0°C for 15 hours, and finally the reaction was cooled to -20°C, frozen for crystallization for 2 hours, filtered to obtain a white solid, and dried naturally at room temperature for 48 hours to obtain N,N'-bis(2-bromoethyl)diaminophosphonic acid; (9) Mitsunobu reaction: (1-methyl-1H-imidazole-2-carbonitrile-5-yl)methanol or (1-methyl-1H-imidazole-2-carbonitrile-4-yl)methanol, N,N'-bis(2-bromoethyl)diaminophosphonic acid, triphenylphosphine and anhydrous tetrahydrofuran were added to a reaction flask under nitrogen protection. After cooling to 0°C, diisopropyl azodicarboxylate was added dropwise to the reaction flask. The temperature was raised to 25°C and the reaction was allowed to proceed for 6 h. The reaction was stopped. After concentration under reduced pressure, the mixture was separated by silica gel column chromatography to obtain N,N'-bis(2-bromoethyl)diaminophosphonic acid (1-methyl-1H-imidazole-2-carbonitrile-5-yl) methyl ester or N,N'-bis(2-bromoethyl)diaminophosphonic acid (1-methyl-1H-imidazole-2-carbonitrile-4-yl) methyl ester.

3. The method for synthesizing the hypoxia activated prodrug derivative according to claim 2, characterized in that: In step (4), the molar ratio of ethyl 1H-imidazole-4-carboxylate, potassium tert-butoxide and methyl iodide is 1:1.5:1.

5.

4. The method for synthesizing the hypoxia activated prodrug derivative according to claim 2, characterized in that: In step (5), the molar ratio of 1-methyl-1H-imidazole-5-carboxylic acid ethyl ester or 1-methyl-1H-imidazole-4-carboxylic acid ethyl ester to bromosuccinimide is 1:

1.

5. The method for synthesizing the hypoxia activated prodrug derivative according to claim 2, characterized in that: In step (6), the molar ratio of 1-methyl-2-bromo-1H-imidazole-5-carboxylic acid ethyl ester or 1-methyl-2-bromo-1H-imidazole-4-carboxylic acid ethyl ester, sodium borohydride and calcium chloride is 1:5:

1.

6. The method for synthesizing the hypoxia activated prodrug derivative according to claim 2, characterized in that: In step (7), the molar ratio of (1-methyl-2-bromo-1H-imidazol-5-yl)methanol or (1-methyl-2-bromo-1H-imidazol-4-yl)methanol, cuprous cyanide and potassium iodide is 1:1.1:

1.

7. The method for synthesizing the hypoxia activated prodrug derivative according to claim 2, characterized in that: In step (8), the molar ratio of 2-bromoethylamine hydrobromide, phosphorus oxychloride and triethylamine is 2:1:

4.

8. The method for synthesizing the hypoxia activated prodrug derivative according to claim 2, characterized in that: In step (9), the molar ratio of (1-methyl-1H-imidazole-2-carbonitrile-5-yl)methanol or (1-methyl-1H-imidazole-2-carbonitrile-4-yl)methanol, N,N'-bis(2-bromoethyl)diaminophosphonic acid, triphenylphosphine and diisopropyl azodicarboxylate is 1:0.5:1:1.

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