A hypoxia-activated prodrug derivative and its synthesis method

Abstract

The present invention belongs to the field of drug synthesis technology, and specifically provides a hypoxia-activated prodrug derivative and a synthesis method thereof. Using benzimidazole as raw material, (1-methyl-1H-imidazole-2-nitrile-5-yl)methanol and (1-methyl-1H-imidazole-2-nitrile-4-yl)methanol are obtained by ring opening, decarboxylation, esterification, iodination, bromine substitution, reduction, and cyano substitution; then it is condensed with bromoisophosphoramide nitrogen mustard to obtain N, N'-bis (2-bromoethyl) diaminophosphonic acid (1-methyl-1H-imidazole-2-nitrile-5-yl) methyl ester or N, N'-bis (2-bromoethyl) diaminophosphonic acid (1-methyl-1H-imidazole-2-nitrile-4-yl) methyl ester. After testing the inhibition rate of ovarian cancer cells, the inhibition rate of N, N'-bis (2-bromoethyl) diaminophosphonic acid (1-methyl-1H-imidazole-2-nitrile-5-yl) methyl ester of the present invention is comparable to that of TH-302.

Description

Technical Field

[0001] The present 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 rapidly generated and are extremely sensitive to traditional chemotherapy, while tumor cells in hypoxic areas are dormant and resistant to standard chemotherapy and radiotherapy, becoming a major 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. They can selectively deliver cytotoxic or cytostatic agents to hypoxic tumor cells while maintaining low toxicity to normal tissues. TH-302 is one of the most effective and clinically studied hypoxia-activated prodrugs currently available.

[0004]

[0005] The traditional synthetic route for the hypoxia-activated prodrug TH-302 suffers from high raw material costs, complex routes, and difficulty modifying groups on the parent ring nitrogen atom. Furthermore, long-term use of the same drug can lead to the development of drug resistance in tumor cells. Therefore, structural modification of TH-302 and the development of novel alternative molecules are highly desirable and have 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 method for synthesizing the same. The hypoxia-activated prodrug derivative provided by the present invention has the following general formulas: 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 addition is complete, slowly add potassium dichromate (add a small amount of ice cubes continuously during the process to prevent the system temperature from being too high), heat to 95°C, stir and 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, 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 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, and 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, and the organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain 1H-imidazole-4-carboxylic acid ethyl ester as a white solid.

[0014] (4) Iodination: 1H-imidazole-4-carboxylic acid ethyl ester, potassium tert-butoxide and acetone were added to the reaction flask, and iodomethane was slowly added. The reaction was carried out at room temperature for 16 hours. The system was extracted with ethyl acetate and water. The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure. After separation by silica gel column chromatography, 1-methyl-1H-imidazole-5-carboxylic acid ethyl ester and 1-methyl-1H-imidazole-4-carboxylic acid ethyl ester were obtained.

[0015] 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 with stirring overnight. Distill under reduced pressure to remove the solvent, and separate the mixture 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 were added to a reaction flask, stirred at room temperature overnight, and the solvent was removed under reduced pressure. The system was dissolved in saturated ammonium chloride solution, extracted with ethyl acetate, and the organic layer was 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 was separated by silica gel column chromatography.

[0019] 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, extracted with ethyl acetate, and the organic phase was dried over anhydrous sodium sulfate. After concentration under reduced pressure, the organic phase was 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 stopping the reaction, the reaction was filtered, the filtrate was concentrated, ethyl acetate was added, and the filtrate 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, and filtered to obtain a white solid, which was naturally dried 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, and the mixture was concentrated under reduced pressure and 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] 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] This invention uses the lower-cost benzimidazole as a raw material to synthesize N,N'-bis(2-bromoethyl)diaminophosphonic acid (1-methyl-1H-imidazol-2-carbonitrile-5-yl) methyl ester and its isomer, N,N'-bis(2-bromoethyl)diaminophosphonic acid (1-methyl-1H-imidazol-2-carbonitrile-4-yl) methyl ester. This method yields two new hypoxia-activated prodrug derivatives in high yield. Cytostatic activity testing of the drugs demonstrates that, compared with TH-302, compound I exhibits a superior inhibitory rate against ovarian cancer cell lines, while compound II exhibits a relatively low inhibitory rate. Of the isomers of compounds I and II, compound I exhibits a superior inhibitory effect against ovarian cancer cell lines. It is speculated that the conjugated electron-withdrawing and self-redox properties of the cyano group enhance compound I's pharmacological activity, thereby increasing its anticancer efficacy. 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 with reference to the following examples.

[0029] Example 1

[0030] Benzimidazole (1 g, 8.5 mmol) and 20 ml of water were added to the reaction flask and stirred. 98% concentrated sulfuric acid (14 mL) was then added dropwise. Potassium dichromate (7.4 g, 25.2 mmol) was added within 15 minutes (10 g of ice cubes were added three times during the process to prevent the system temperature from being too high). The temperature was raised to 95°C and stirred for 15 minutes. The system was poured into ice water while hot and placed in a refrigerator for cooling for 3 hours. The system was filtered, the filter cake was washed with water three times, and dried 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] Imidazole-4,5-dicarboxylic acid (2 g, 12.7 mmol) and acetic anhydride (70 mL) were added to the reaction flask, heated at 130 ° C and refluxed with stirring until the solution turned brown, cooled to room temperature, filtered, and the filtrate was spin-dried to obtain a black viscous solid. Water (30 mL) was added and stirred at room temperature for 2 hours, then raised to 100 ° C and stirred for 5 hours, then lowered to 60 ° C, activated carbon (200 mg) and ethanol (30 mL) were added, heated to reflux for 16 hours, lowered to 70 ° C, and filtered while hot to obtain a light yellow liquid. It was placed at room temperature for crystallization for 2 hours, then placed at 0 ° C for crystallization for 24 hours, filtered, and the solid was washed with acetone several times and dried 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 flask, mixed and heated to 60 ° C. and refluxed for 21 hours. The system was cooled to room temperature and 5N sodium hydroxide solution was added dropwise to adjust the pH value of the system to 7. The system was extracted with ethyl acetate, and 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 reaction time was investigated using 112.09 mg (1.0 mmol) of the starting material 1H-imidazole-4-carboxylic acid at a reflux temperature of 60°C, the initial reaction time of 12 hours resulted in a yield of only 52%. However, extending the reaction time to 21 hours resulted in a yield of 96%, and further extensions of the reaction time maintained the yield. Therefore, the optimal reaction time for this reaction was determined to be 21 hours, which consistently produces the target compound, ethyl 1H-imidazole-4-carboxylate.

[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 flask and stirred at room temperature for 16 h. After stopping the reaction, the solvent was dried and the mixture was extracted with ethyl acetate and water. The organic phase was dried over anhydrous sodium sulfate, concentrated under reduced pressure, and then 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 four conditions, namely solvent, type of base, equivalent number of base, and equivalent number of iodomethane, on the reaction were further studied.

[0042] Table 2

[0043]

[0044] The initial reaction conditions were: 1.0 equivalent of ethyl 1H-imidazole-4-carboxylate in N,N-dimethylformamide (DMF) as the reaction solvent, 1.0 equivalent of methyl iodide in the presence of 1.0 equivalent of potassium carbonate, and stirring at room temperature for 16 hours, resulting in a total yield of only 21%. First, the reaction solvent was screened, and when acetone was used, the total yield increased to 36%. Next, the base was screened, and when potassium tert-butoxide was used at 1.5 equivalents, the yield increased significantly to 68%. Finally, increasing the number of equivalents of methyl iodide to 1.5 equivalents increased the total yield to 75%. The final reaction conditions are as follows: 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 in the presence 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, ethyl 1-methyl-1H-imidazole-5-carboxylate (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 product was separated and purified by silica gel column chromatography [eluent: V (petroleum ether) / V (ethyl acetate) = 3 / 1] to obtain ethyl 2-bromo-1-methyl-1H-imidazole-5-carboxylate 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 used as the bromine source, and heating under reflux overnight in tetrahydrofuran as the solvent resulted in only a 15% yield of the product. Subsequently, the bromine source was replaced with dibromohydantoin (DBDMH), resulting in a 44% yield of ethyl 2-bromo-1-methyl-1H-imidazole-5-carboxylate. Finally, when the bromine source was replaced with bromosuccinimide (NBS), the yield 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 conditions were that 1-methyl-1H-imidazole-4-carboxylic acid ethyl ester was stirred at 65°C under the action of tetrahydrofuran as a solvent overnight (12 hours), and 2-bromo-1-methyl-1H-imidazole-4-carboxylic acid ethyl ester was 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 were added to the reaction flask and stirred at room temperature overnight. The solvent was removed under reduced pressure and the system was dissolved in saturated ammonium chloride solution and extracted with ethyl acetate. The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The product was 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] 7 mL of anhydrous dimethyl sulfoxide was added to a mixture of 1,4-dimethoxy-2-nitropropane (47 mg, 0.5 mmol), cuprous cyanide (49 mg, 0.55 mmol), and potassium iodide (83 mg, 0.5 mmol). The mixture was reacted at 100°C for 48 h, cooled to room temperature, poured into a saturated sodium bicarbonate solution, and extracted with ethyl acetate. 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] Example 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 dropwise addition was completed, a mixed solution of triethylamine (9 mL) and dichloromethane (15 mL) was added dropwise to the reaction flask. After the dropwise addition was completed, the reaction was continued at -78 ° C with stirring for 1 h, and the temperature was raised. To 25 ° C, continue stirring to react for 2 hours; after stopping the reaction, filter, concentrate the filtrate, add ethyl acetate, filter again, and vacuum concentrate the filtrate to a yellow viscous liquid. Add tetrahydrofuran (2 mL) and water (4 mL). Stir and react at 25 ° C for 5 hours, then spin dry the tetrahydrofuran, keep stirring at 0 ° C for 15 hours, and finally cool to -20 ° C, freeze crystallize for 2 hours, filter to obtain a white solid, and dry it at room temperature for 48 hours 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-imidazol-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, and 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 allowed to proceed for 6 h. After stopping the reaction, 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-imidazol-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] Example 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 Examples 12 and 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- 2-nitrile-5-yl) methyl ester or N,N'-bis(2-bromoethyl)diaminophosphonic acid (1-methyl-1H-imidazole-2-nitrile-4-yl) methyl ester, TLC monitoring showed that no new substance was generated; the reaction temperature was raised to 25 ° C, and the target product was found by TLC monitoring, but the yield was not ideal; the amount of (1-methyl-1H-imidazole-2-nitrile-5-yl) methanol or (1-methyl-1H-imidazole-2-nitrile-4-yl) methanol was increased to N,N'-bis(2-bromoethyl)diaminophosphonic acid (1-methyl-1H-imidazole-2-nitrile-4-yl) methyl ester. The yield of N,N'-bis(2-bromoethyl)diaminophosphonic acid (1-methyl-1H-imidazol-2-carbonitrile-5-yl) methyl ester or N,N'-bis(2-bromoethyl)diaminophosphonic acid (1-methyl-1H-imidazol-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 the optimal synthesis conditions for this reaction were determined to be 25℃. When 2.0 equivalents of alcohol were reacted with N,N'-bis(2-bromoethyl)diaminophosphonic acid, N,N'-bis(2-bromoethyl)diaminophosphonic acid (1-methyl-1H-imidazole-2-carbonitrile-5-yl) methyl ester was obtained in a yield of 62%, and N,N'-bis(2-bromoethyl)diaminophosphonic acid (1-methyl-1H-imidazole-2-carbonitrile-4-yl) methyl ester was obtained in a yield of 45%.

[0073] Example 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 inhibitory activity was determined by CCK-8 (Cell Counting Kit-8) colorimetric method. Cells in the logarithmic growth phase were collected and cultured at a concentration of 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: Add benzimidazole and water to a reaction flask and stir, then add concentrated sulfuric acid dropwise. After the addition is complete, add potassium dichromate, heat to 95°C, and stir to react for 15 minutes. Pour the system into ice water while it is still hot, cool in a refrigerator, filter, wash the filter cake with water, and dry 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 cool to 60°C, add activated carbon and ethanol, heat under reflux for 16 hours, cool 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, and 5N sodium hydroxide solution was added dropwise to adjust the pH value of the system to 7. The system was extracted with ethyl acetate, and the organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain 1H-imidazole-4-carboxylic acid ethyl ester as a white solid. (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 with stirring 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, 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 were added to a reaction flask, stirred at room temperature overnight, and the solvent was removed under reduced pressure. The system was dissolved in saturated ammonium chloride solution, extracted with ethyl acetate, and the organic layer was separated and dried over anhydrous sodium sulfate. (1-methyl-2-bromo-1H-imidazole-5-yl)methanol or (1-methyl-2-bromo-1H-imidazole-4-yl)methanol was obtained by separation by silica gel column chromatography; (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 mixture was poured into a saturated aqueous sodium bicarbonate solution, extracted with ethyl acetate, and the organic phase was dried over anhydrous sodium sulfate. After concentration under reduced pressure, the organic phase was 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. (8) Synthesis of bromoisophosphoramide mustard: 2-bromoethylamine hydrobromide and anhydrous dichloromethane were added to a reaction flask under anhydrous and oxygen-free conditions, the reaction system temperature was cooled to -78°C, phosphorus oxychloride was added dropwise to the reaction flask, after the addition was complete, a mixed solution of triethylamine and dichloromethane was added dropwise to the reaction flask, after the addition was complete, 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 stopping the reaction, the reaction was filtered, the filtrate was concentrated, ethyl acetate was added, and the filtrate 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, the tetrahydrofuran was then dried, the reaction was maintained 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; (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, and the mixture was concentrated under reduced pressure and 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 1H-imidazole-4-carboxylic acid ethyl ester, potassium tert-butoxide and iodomethane is 1:1.5:1.

5.

4. The method for synthesizing the hypoxia-activated prodrug derivative according to claim 2, wherein: 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 and bromosuccinimide is 1:

1.

5. The method for synthesizing the hypoxia-activated prodrug derivative according to claim 2, wherein: 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, wherein: 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, wherein: 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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