N, Napos; preparation method of-bis (2-bromo / chloroethyl) diamino phosphonic acid (1-methyl-4-nitro-1H-imidazole-2-cyanogen) methyl ester

Abstract

The invention belongs to the technical field of chemical synthesis, and particularly relates to a preparation method of N, N '-bis (2-bromine / chloroethyl) diamino phosphonic acid (1-methyl-4-nitro-1H-imidazole-2-cyanogen) methyl ester, 4-methylimidazole is used as a raw material, and (1-methyl-4-nitro-1H-imidazole-2-cyanogen) methanol is synthesized through nitration, methyl substitution, bromine substitution, bromomethyl substitution, hydrolysis and substitution; the preparation method comprises the following steps: reacting phosphorus oxychloride with ethyl amine salt to synthesize N, N '-bis (2-bromoethyl) diamino phosphonic acid and N, N'-bis (2-chloroethyl) diamino phosphonic acid, and finally condensing to obtain N, N '-bis (2-bromoethyl) diamino phosphonic acid (1-methyl-4-nitro-1H-imidazole-2-cyanogen) methyl ester and N, N'-bis (2-chloroethyl) diamino phosphonic acid (1-methyl-4-nitro-1H-imidazole-2-cyanogen) methyl ester. The compound can selectively inhibit ovarian cancer cell proliferation under a hypoxia condition.

Description

Technical Field

[0001] The invention belongs to the technical field of chemical synthesis, and specifically relates to a method for preparing N,N′-bis(2-bromo / chloroethyl)diaminophosphonic acid (1-methyl-4-nitro-1H-imidazole-2-cyano) methyl ester. Background Art

[0002] TH-302, a hypoxia-activated anticancer prodrug of nitroimidazole, utilizes the cytotoxic properties of bromoisophosphoramide mustard. It is composed of a 2-nitroimidazole moiety attached to bromoisophosphoramide mustard (Br-IPM). Nitroimidazoles undergo hypoxia-selective biochemical reduction in cells and in vivo systems, and are therefore widely used in the development of bioreductive prodrugs, including anaerobic antibacterial therapy and anticancer drugs. Bromoisophosphoramide mustard exerts its cytotoxic effects by binding to DNA and releasing cytotoxins, and it has been shown in clinical practice to have significant therapeutic effects against tumors. Based on the bioreduction mechanism of nitroimidazoles and the cytotoxic properties of bromoisophosphoramide mustard, TH-302 was developed as an anticancer therapeutic drug.

[0003] The traditional synthesis method of the intermediate compound (1-methyl-4-nitro-1H-imidazole-2-cyano)methanol is as follows:

[0004]

[0005] The synthesis method is complicated and requires harsh reaction conditions such as light, and the yield is not high. Therefore, the design and optimization of the reaction route for this compound is equally important.

[0006] In addition, regarding the modification of phosphoramide structure, Zhang and his team successfully designed and synthesized a new type of 2-nitroimidazole phosphoramide nitrogen mustard (2-NIPAM), which is based on TH-302 as a model and conjugated with other groups through azide-alkyne cycloaddition reaction to couple hypoxia selective drugs to imaging parts and other structures, thereby obtaining a series of derivatives. The synthesis method of 2-NIPAM is as follows:

[0007]

[0008] 2-NIPAM exhibits similar cytotoxicity and hypoxic selectivity as TH-302, indicating that the introduction of alkyne function does not significantly affect the activity of the preparation. In addition, phosphorus trichloride is highly toxic, the synthesis of 1-2 requires eight steps, and the docking method of 1-3 with the parent ring is complicated, with a yield of only 7.4%. Therefore, for the modification of phosphoramide nitrogen mustard, it is necessary to select and optimize the docking method between phosphoramide and the parent ring. Summary of the invention

[0009] The present invention aims to reduce the synthesis cost and steps of the compound and reduce the complicated processes in the traditional route; secondly, to improve the anti-tumor activity and reduce the toxic side effects by changing the molecular structure and chemical properties, thereby providing important technical support for the further development and application of ivophosphamide.

[0010] The present invention provides a method for synthesizing N,N'-bis(2-bromo / chloroethyl)diaminophosphonic acid (1-methyl-4-nitro-1H-imidazole-2-cyano) methyl ester. The specific synthesis method is mainly divided into three parts: the first part is the synthesis of (1-methyl-4-nitro-1H-imidazole-2-cyano) methanol, the second part is the synthesis of bromoisophosphoramide nitrogen mustard and chloroisophosphoramide nitrogen mustard, and the third part is to dock the compounds of the first two parts to obtain the final product N,N'-bis(2-bromo / chloroethyl)diaminophosphonic acid (1-methyl-4-nitro-1H-imidazole-2-cyano) methyl ester. The specific synthesis scheme is as follows:

[0011] 1. Preparation of (1-methyl-4-nitro-1H-imidazole-2-cyano)methanol:

[0012]

[0013] (1) Nitration: At 0°C, concentrated nitric acid and concentrated sulfuric acid were mixed in a volume ratio of 3:1, 4-methylimidazole was added to the mixed solution, and the mixture was heated to 80°C and refluxed for 48 hours. After the reaction was stopped, the mixture was cooled to room temperature, ice was poured into the mixture, and the pH was adjusted to 7 with a saturated sodium hydroxide solution to generate a yellow precipitate, which was filtered and washed with cold water to obtain a white solid, which was dried to obtain 4-nitro-5-methyl-1H-imidazole.

[0014] (2) Methyl substitution: Add 4-nitro-5-methyl-1H-imidazole, potassium tert-butoxide and acetone to the reaction flask in sequence and stir to dissolve. Cool the system to 0°C and slowly add iodomethane solution. After the addition is complete, heat to room temperature and react for 6 hours. Stop the reaction, cool and filter. After the filtrate is concentrated, the residue is separated and purified by silica gel column chromatography [eluent: petroleum ether: ethyl acetate = 1:4] to obtain 1,5-dimethyl-4-nitro-1H-imidazole.

[0015] The molar ratio of 4-nitro-5-methyl-1H-imidazole, potassium tert-butoxide and methyl iodide is 1:2:2.

[0016] (3) Bromination: Add 1,5-dimethyl-4-nitro-1H-imidazole and chloroform to a reaction flask, stir to dissolve, add liquid bromine, keep stirring at room temperature, and react for 36 hours. After stopping the reaction, add water to mix, distill to remove chloroform, filter and collect the resulting precipitate, and then wash with water to obtain a light yellow solid 2-bromo-1,5-dimethyl-4-nitro-1H-imidazole.

[0017] The molar ratio of 1,5-dimethyl-4-nitro-1H-imidazole and liquid bromine is 1:4.

[0018] (4) Bromo(methyl bromide): Add 2-bromo-1,5-dimethyl-4-nitro-1H-imidazole, N-bromosuccinimide and diisobutyl azodicarbonate to a reaction flask, add anhydrous acetonitrile under the protection of nitrogen, stir to dissolve, and heat to reflux at 80°C for 48 hours. After stopping the reaction, concentrate half of the solvent under reduced pressure, add the same volume of water and mix, and continue to concentrate under reduced pressure to obtain an off-white solid. Collect the precipitate by filtration, wash with water, and vacuum dry to obtain an off-white solid 2-bromo-5-bromomethyl-1-methyl-4-nitro-1H-imidazole.

[0019] The molar ratio of 2-bromo-1,5-dimethyl-4-nitro-1H-imidazole and N-bromosuccinimide is 1:3, and the amount of azobisisobutyl ester is 10% of the molar amount of 2-bromo-1,5-dimethyl-4-nitro-1H-imidazole.

[0020] (5) Hydrolysis reaction: 2-bromo-5-bromomethyl-1-methyl-4-nitro-1H-imidazole, calcium carbonate and 1,4-dioxane / water (volume ratio of 3:2) were added to a reaction flask, and the mixture was heated and stirred at 100° C. for 24 h. After the reaction was stopped, the mixture was extracted with ethyl acetate, the organic phase was dried over anhydrous sodium sulfate, the organic phases were combined, and the crude product was obtained by concentrating under reduced pressure to obtain 2-bromo-5-hydroxymethyl-1-methyl-4-nitro-1H-imidazole as a white solid.

[0021] The molar ratio of 2-bromo-5-bromomethyl-1-methyl-4-nitro-1H-imidazole and calcium carbonate is 1:5.

[0022] (6) Substitution reaction: 2-bromo-5-hydroxymethyl-1-methyl-4-nitro-1H-imidazole, potassium iodide, cuprous cyanide and anhydrous N-methylpyrrolidone were added to a reaction flask, and the mixture was heated and stirred at 100°C for 72 hours. After the reaction was stopped, the mixture was extracted with ethyl acetate, and the organic phase was dried over anhydrous sodium sulfate, combined and concentrated under reduced pressure to obtain a crude product, and an off-white solid (1-methyl-4-nitro-1H-imidazole-2-cyano)methanol was obtained.

[0023] The molar ratio of 2-bromo-5-hydroxymethyl-1-methyl-4-nitro-1H-imidazole, potassium iodide and cuprous cyanide is 1:2:2.

[0024] 2. Preparation of N,N'-bis(2-bromoethyl)diaminophosphonic acid and N,N'-bis(2-chloroethyl)diaminophosphonic acid.

[0025]

[0026] (1) Synthesis of N,N'-bis(2-bromoethyl)diaminophosphonic acid: 2-bromoethylamine hydrobromide and anhydrous dichloromethane were added to a reaction flask under nitrogen protection, 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 stirred for 1 hour, the temperature was raised to 25°C, and the reaction was stirred for 2 hours; after the reaction was stopped, the reaction was filtered, ethyl acetate was added after the filtrate was concentrated, and the reaction was filtered again, the filtrate was vacuum concentrated 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 -1°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.

[0027] The molar ratio of 2-bromoethylamine bromate, phosphorus oxychloride and triethylamine is 2.5:1:2.

[0028] (2) Synthesis of N,N'-bis(2-chloroethyl)diaminophosphonic acid: 2-chloroethylamine hydrochloride and anhydrous dichloromethane were added to a reaction flask under nitrogen protection, 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 stirred for 1 hour, the temperature was raised to 25°C, and the reaction was stirred 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 vacuum concentrated 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-chloroethyl)diaminophosphonic acid.

[0029] The molar ratio of 2-chloroethylamine bromate, phosphorus oxychloride and triethylamine is 2.5:1:2.

[0030] 3. Preparation of N,N'-bis(2-bromoethyl)diaminophosphonic acid (1-methyl-4-nitro-1H-imidazole-2-cyano) methyl ester or N,N'-bis(2-chloroethyl)diaminophosphonic acid (1-methyl-4-nitro-1H-imidazole-2-cyano) methyl ester.

[0031]

[0032] Condensation reaction: add (1-methyl-4-nitro-1H-imidazole-2-cyano)methanol, N,N'-bis(2-bromoethyl)diaminophosphonic acid or N,N'-bis(2-chloroethyl)diaminophosphonic acid, triphenylphosphine and anhydrous tetrahydrofuran to the reaction flask, cool the reaction system to 0°C, dropwise add diisopropyl azodicarboxylate (DIAD) to the reaction flask, raise the temperature to 25°C and react for 5h. After stopping the reaction, concentrate under reduced pressure to obtain a crude product, which is purified by silica gel column chromatography [eluent: methanol: ethyl acetate = 1:4] to obtain the product N,N'-bis(2-bromoethyl)diaminophosphonic acid (1-methyl-4-nitro-1H-imidazole-2-cyano) methyl ester or N,N'-bis(2-chloroethyl)diaminophosphonic acid (1-methyl-4-nitro-1H-imidazole-2-cyano) methyl ester, whose structure is as follows:

[0033] The chemical structure is shown below:

[0034]

[0035] The molar ratio of (1-methyl-4-nitro-1H-imidazole-2-cyano)methanol, N,N'-bis(2-bromoethyl)diaminophosphonic acid or N,N'-bis(2-chloroethyl)diaminophosphonic acid, triphenylphosphine and diisopropyl azodicarboxylate (DIAD) is 2:1:2:2.

[0036] Beneficial effects of the present invention:

[0037] 1. The present invention uses 4-methylimidazole as a raw material, and synthesizes a parent ring (1-methyl-4-nitro-1H-imidazole-2-cyano) methanol through nitration, methyl substitution, bromination, hydrolysis, and substitution reactions. The parent ring retains a nitro group that has a cytotoxic effect, and two isophosphoramide nitrogen mustards with different electron-withdrawing groups are synthesized. Finally, through alcohol-alcohol condensation, the parent ring is docked with the two isophosphoramide nitrogen mustards respectively to obtain the final product N, N'-bis (2-bromoethyl) diaminophosphonic acid (1-methyl-4-nitro-1H-imidazole-2-cyano) methyl ester or N, N'-bis (2-chloroethyl) diaminophosphonic acid (1-methyl-4-nitro-1H-imidazole-2-cyano) methyl ester.

[0038] 2. This method has low raw material cost and simple synthesis process, and is an innovative route that is very suitable for industrial production. In addition to methyl substitution, other alkyl groups can also be used, which provides more selectivity for nitrogen-containing parent ring modification.

[0039] 3. The results of the activity test on ovarian cancer cells showed that compound I had a better inhibition rate than compound II, and the inhibition rates of these two compounds on ovarian cancer cells were better than TH-302. The inhibitory effects of these two compounds on tumors can provide an important reference for the design of more effective anti-tumor drugs in the future. DETAILED DESCRIPTION

[0040] In order to better understand the present invention, the present invention will be further described below by way of examples.

[0041] Example 1

[0042] Step 1: Synthesis of 4-nitro-5-methyl-1H-imidazole

[0043]

[0044] At 0°C, concentrated nitric acid (24 mL) and concentrated sulfuric acid (8 mL) were mixed, 3.20 g (36.70 mmol) of 4-methylimidazole was added to the mixed solution, and the mixture was heated to 80°C and refluxed for 48 h. After the reaction was stopped, the mixture was cooled to room temperature, ice was poured into the mixture, and the pH was adjusted to 7 with a saturated sodium hydroxide solution to generate a yellow precipitate, which was filtered, washed with cold water, and the filter cake was dried to obtain 4-nitro-5-methyl-1H-imidazole 2.

[0045] Step 2: Synthesis of 1,5-dimethyl-4-nitro-1H-imidazole

[0046]

[0047] 2.00 g (15.70 mmol) of 4-nitro-5-methyl-1H-imidazole, 3.52 g (31.40 mmol) of potassium tert-butoxide and 30 mL of acetone were added to the reaction flask in sequence and stirred to dissolve. 1.47 mL (31.40 mmol) of iodomethane solution was added dropwise at 0°C. After the addition was completed, the mixture was heated to room temperature and reacted for 6 h. The reaction was stopped, cooled and filtered, and the filtrate was concentrated. The residue was separated and purified by silica gel column chromatography (eluent: petroleum ether: ethyl acetate = 1:4) to obtain 1,5-dimethyl-4-nitro-1H-imidazole 3.

[0048] Step 3: Synthesis of 2-bromo-1,5-dimethyl-4-nitro-1H-imidazole

[0049]

[0050] Add 2.00 g (14.18 mmol) of 1,5-dimethyl-4-nitro-1H-imidazole and chloroform (14 mL) to the reaction bottle, stir to dissolve, add 2.90 mL (56.72 mmol) of liquid bromine, keep stirring at room temperature, and react for 36 hours. After stopping the reaction, add water (18 mL) to mix, distill to remove chloroform, filter and collect the resulting precipitate, and then wash with water to obtain 2-bromo-1,5-dimethyl-4-nitro-1H-imidazole 4.

[0051] Step 4: Synthesis of 2-bromo-5-bromomethyl-1-methyl-4-nitro-1H-imidazole

[0052]

[0053] 2.00 g (9.17 mmol) of 2-bromo-1,5-dimethyl-4-nitro-1H-imidazole, 4.89 g (27.51 mmol) of N-bromosuccinimide and 0.15 g (10 mmol%) of azobisisobutyl ester were added to the reaction flask, and anhydrous acetonitrile (30 mL) was added under the protection of nitrogen, and the mixture was stirred to dissolve, and heated to reflux at 80°C for 48 h. After stopping the reaction, half of the solvent was concentrated under reduced pressure, and the same volume of water was added to mix, and the mixture was further concentrated under reduced pressure to obtain an off-white solid, which was collected by filtration, washed with water, and dried under vacuum to obtain 2-bromo-5-bromomethyl-1-methyl-4-nitro-1H-imidazole 5.

[0054] Step 5: Synthesis of 2-bromo-5-hydroxymethyl-1-methyl-4-nitro-1H-imidazole

[0055]

[0056] 1.50 g (3.35 mmol) of 2-bromo-5-bromomethyl-1-methyl-4-nitro-1H-imidazole, 1.68 g (16.75 mmol) of calcium carbonate and 1,4-dioxane / water (3:2 v / v, 60 mL) were added to the reaction bottle, and the mixture was heated and stirred at 100°C for 24 h. After the reaction was stopped, it was extracted with ethyl acetate (3×30 mL), the organic phase was dried over anhydrous sodium sulfate, the organic phases were combined, and the crude product was obtained by concentrating under reduced pressure to obtain 2-bromo-5-hydroxymethyl-1-methyl-4-nitro-1H-imidazole 6.

[0057] Using 2-bromo-5-bromomethyl-1-methyl-4-nitro-1H-imidazole as raw material and 1,4-dioxane: water (2:3) as solvent, the effect of calcium carbonate (CaCO3) dosage on the reaction was also investigated.

[0058]

[0059] Then, 2-bromo-5-bromomethyl-1-methyl-4-nitro-1H-imidazole was used as the raw material, calcium carbonate (5.0 eq.) was added, and the effect of the volume ratio of 1,4-dioxane to water on the reaction was investigated.

[0060]

[0061] After screening, the optimal conditions for the reaction were determined: 2-bromo-5-bromomethyl-1-methyl-4-nitro-1H-imidazole 5 (1.0 eq.); calcium carbonate (5.0 eq.); 1,4-dioxane and water (volume ratio 3:2) as solvents, with an addition amount of 60 mL; react at 100°C for 24 hours. Under the optimal reaction conditions, the target product 6 was obtained with a yield of 50%.

[0062] Step 6: Synthesis of (1-methyl-4-nitro-1H-imidazole-2-cyano)methanol

[0063]

[0064] 0.50 g (4.22 mmol) of 2-bromo-5-hydroxymethyl-1-methyl-4-nitro-1H-imidazole, 1.40 g (8.44 mmol) of potassium iodide and 0.82 g (8.44 mmol) of cuprous cyanide and anhydrous N-methylpyrrolidone (20 mL) were added to the reaction flask, and the mixture was heated and stirred at 100°C for 72 h. After stopping the reaction, the mixture was extracted with ethyl acetate (3×30 mL), and the organic phase was dried over anhydrous sodium sulfate, combined with the organic phases, and concentrated under reduced pressure to obtain a crude product, namely (1-methyl-4-nitro-1H-imidazole-2-cyano)methanol 7.

[0065] 2-Bromo-5-hydroxymethyl-1-methyl-4-nitro-1H-imidazole 6 was used as the raw material of this experiment, potassium iodide (2.0 eq.) was added, and the reaction was carried out at 100°C for 72 hours. The amount of cuprous cyanide was screened.

[0066]

[0067] Then the effect of solvent on the reaction was investigated.

[0068]

[0069] In summary, the optimal conditions for the reaction were determined: (2-bromo-1-methyl-4-nitro-1H-imidazol-5-yl)methanol (1.0 eq.); potassium iodide (2.0 eq.); cuprous cyanide (2.0 eq.); anhydrous N-methylpyrrolidone as solvent, added in an amount of 2.0 mL; reacted at 100°C for 72 h. Under the optimal reaction conditions, the target product 7 was obtained with a yield of 30%.

[0070] Step 7: Synthesis of N,N'-bis(2-bromoethyl)diaminophosphonic acid

[0071]

[0072] Under nitrogen protection, 6.30 g (30.70 mmol, 2.0 eq.) of 2-bromoethylamine hydrobromide and anhydrous dichloromethane (50 mL) were added to the reaction flask, and the temperature of the reaction system was cooled to -78 ° C. Within 10 minutes, 1.00 mL (12.30 mmol, 0.8 eq.) of phosphorus oxychloride was added dropwise to the reaction flask. After the addition was completed, a mixed solution of 4 mL (24.60 mmol, 1.6 eq.) of triethylamine and dichloromethane (6 mL) was added dropwise to the reaction flask. After the addition was completed, the temperature was maintained at -78 ° C and the reaction mixture was stirred for 2 hours. The reaction was stirred for 1 h, the temperature was raised to 25°C, and the reaction was continued to be stirred for 2 h. 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 in vacuo to a yellow viscous liquid, and a mixed solution of tetrahydrofuran (8 mL) and water (12 mL) was added. The reaction was stirred at 25°C for 5 h, and then the tetrahydrofuran was dried by spin drying, and the reaction was maintained at -1°C for 15 h. Finally, the reaction was cooled to -20°C, and the mixture was frozen for crystallization for 2 h. The white solid was filtered and dried at room temperature for 48 h to obtain N,N'-bis(2-bromoethyl)diaminophosphonic acid 8.

[0073] Step 8: Synthesis of N,N'-bis(2-chloroethyl)diaminophosphonic acid

[0074]

[0075] Under nitrogen protection, 3.60 g (31 mmol, 2.0 eq.) of 2-chloroethylamine hydrochloride and anhydrous dichloromethane (50 mL) were added to the reaction flask, and the temperature of the reaction system was cooled to -78 ° C. Within 10 minutes, 1.06 mL (12.40 mmol, 0.8 eq.) of phosphorus oxychloride was added dropwise to the reaction flask. After the addition was completed, a mixed solution of 4 mL (24.80 mmol, 1.6 eq.) of triethylamine and dichloromethane (8 mL) was added dropwise to the reaction flask. After the addition was completed, the mixture was kept at -78 ° C and stirred. The mixture was stirred for 1 h, heated to 25°C, and stirred for 2 h. After the reaction was stopped, the mixture was filtered, the filtrate was concentrated, ethyl acetate was added, and the mixture was filtered again. The filtrate was concentrated in vacuo to a yellow viscous liquid. A mixed solution of tetrahydrofuran (8 mL) and water (12 mL) was added, and the mixture was stirred for 5 h at 25°C. The tetrahydrofuran was then dried and stirred at 0°C for 15 h. The mixture was finally cooled to -20°C, frozen for crystallization for 2 h, and filtered to obtain a white solid. The solid was dried at room temperature for 48 h to obtain N,N'-bis(2-chloroethyl)diaminophosphonic acid 9.

[0076] Step 9: Synthesis of N,N'-bis(2-bromoethyl)diaminophosphonic acid (1-methyl-4-nitro-1H-imidazole-2-cyano) methyl ester or N,N'-bis(2-chloroethyl)diaminophosphonic acid (1-methyl-4-nitro-1H-imidazole-2-cyano) methyl ester

[0077]

[0078] Add 50 mg (0.28 mmol, 1.0 eq.) of (1-methyl-4-nitro-1H-imidazole-2-cyano)methanol, N,N'-bis(2-bromoethyl)diaminophosphonic acid or N,N'-bis(2-chloroethyl)diaminophosphonic acid (0.14 mmol, 0.5 eq.), 74 mg (0.28 mmol, 1.0 eq.) of triphenylphosphine and anhydrous tetrahydrofuran (15 mL) to the reaction flask, cool the reaction system to 0°C, and dropwise add diisopropyl azodicarboxylate (DI AD) 55 μL (0.28 mmol, 1.0 eq.), heated to 25 ° C for 5 h, after stopping the reaction, concentrated under reduced pressure to obtain a crude product, which was purified by silica gel column chromatography [eluent: V (methanol) / V (ethyl acetate) = 1 / 4] to obtain the product N, N'-bis (2-bromoethyl) diaminophosphonic acid (1-methyl-4-nitro-1H-imidazole-2-cyano) methyl ester 10 or N, N'-bis (2-chloroethyl) diaminophosphonic acid (1-methyl-4-nitro-1H-imidazole-2-cyano) methyl ester 11.

[0079] The specific products of the present invention and the characterization results are shown in the table below.

[0080] Table 1

[0081]

[0082]

[0083] Experimental example

[0084] Ovarian cancer cell line (SKOV3) was selected as experimental cells, and compound I, compound II and TH-302 were tested for in vitro antitumor activity. CCK-8 (Cell Counting Kit-8) colorimetric method was used to determine the cell proliferation inhibitory activity. 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 microplate reader). Specifically: cells in the logarithmic growth phase were collected. After the cells adhered to the wall, the cells were treated with 5μM of the test compound, and a control group was set up at the same time, with a final volume of 100μL. A solvent DMSO with an equal volume to the compound 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. The inhibition rate was calculated using the formula: [(Ac-As) / (Ac-Ab)]×100% [the absorbance values ​​(OD values) of the test well (As), control well (Ac) and blank well (Ab) were read at a wavelength of 450 nm on an enzyme-linked immunosorbent assay analyzer].

[0085] The results of the test on ovarian cancer cell lines are shown in the following table:

[0086] Table 2

[0087]

[0088] The cell inhibition rates of compound I and compound II on SKOV3 under the condition of 5 μM administration were both greater than those of TH-302, proving that the application of the present invention achieved a good therapeutic effect. Based on the nitro group contained in TH-302, the substitution of the electron-withdrawing group-cyano group also obtained better tumor cell toxicity for compounds I and II. In addition, compound I has a strong leaving group bromophosphoramide mustard, and compound II has chlorophosphoramide mustard. Obviously, the electron-withdrawing group bromine can show better activity and corresponding cytotoxicity than chlorine.

Claims

1. A method for preparing N,N'-bis(2-bromo / chloroethyl)diaminophosphonic acid (1-methyl-4-nitro-1H-imidazole-2-cyano) methyl ester, characterized in that: The preparation method steps are as follows: (1) Nitration: at 0°C, concentrated nitric acid and concentrated sulfuric acid were mixed, and 4-methylimidazole was added. The mixture was heated to 80°C and refluxed for 48 hours. The mixture was cooled to room temperature, and ice was poured into the mixture. The pH was adjusted to 7 with saturated sodium hydroxide solution to generate a yellow precipitate. The mixture was filtered and washed with cold water to obtain a white solid. The filter cake was dried to obtain 4-nitro-5-methyl-1H-imidazole. (2) Methyl substitution: 4-nitro-5-methyl-1H-imidazole, potassium tert-butoxide and acetone were added in sequence and stirred to dissolve. At 0°C, iodomethane solution was added dropwise. After the addition was completed, the mixture was heated to room temperature and reacted for 6 hours. The mixture was cooled and filtered. The filtrate was concentrated and purified by silica gel column chromatography to obtain 1,5-dimethyl-4-nitro-1H-imidazole. (3) Bromination: Add 1,5-dimethyl-4-nitro-1H-imidazole and chloroform, stir to dissolve, add liquid bromine, keep stirring at room temperature, react for 36 hours, add water to mix, distill to remove chloroform, collect the resulting precipitate by filtration, and wash with water to obtain a light yellow solid 2-bromo-1,5-dimethyl-4-nitro-1H-imidazole; (4) Bromo(methyl bromide): add 2-bromo-1,5-dimethyl-4-nitro-1H-imidazole, N-bromosuccinimide and diisobutyl azodicarbonate, add anhydrous acetonitrile under nitrogen protection, stir to dissolve, heat under reflux at 80°C for 48 hours, concentrate half of the solvent under reduced pressure, add the same volume of water and mix, continue to concentrate under reduced pressure to obtain an off-white solid, collect the precipitate by filtration, wash with water, and vacuum dry to obtain an off-white solid 2-bromo-5-bromomethyl-1-methyl-4-nitro-1H-imidazole; (5) Hydrolysis reaction: 2-bromo-5-bromomethyl-1-methyl-4-nitro-1H-imidazole, calcium carbonate and 1,4-dioxane / water were added, and the mixture was heated and stirred at 100° C. for 24 h, and then extracted with ethyl acetate. The organic phase was dried over anhydrous sodium sulfate, combined and concentrated under reduced pressure to obtain a crude white solid 2-bromo-5-hydroxymethyl-1-methyl-4-nitro-1H-imidazole; (6) Substitution reaction: 2-bromo-5-hydroxymethyl-1-methyl-4-nitro-1H-imidazole, potassium iodide and cuprous cyanide, anhydrous N-methylpyrrolidone were added, the mixture was heated and stirred at 100°C, reacted for 72 hours, extracted with ethyl acetate, the organic phase was dried over anhydrous sodium sulfate, the organic phases were combined, and concentrated under reduced pressure to obtain a crude off-white solid (1-methyl-4-nitro-1H-imidazole-2-cyano)methanol; (7) Synthesis of N,N'-bis(2-bromo / chloroethyl)diaminophosphonic acid: 2-bromoethylamine hydrobromide / 2-chloroethylamine hydrochloride and anhydrous dichloromethane were added to a reaction flask under nitrogen protection, 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 reaction was continued with stirring at -78°C 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, Filter, add ethyl acetate after the filtrate is concentrated, filter again, vacuum concentrate the filtrate to a yellow viscous liquid, add a mixed solution of tetrahydrofuran and water, stir and react at 25°C for 5h, then spin dry the tetrahydrofuran, keep stirring at -1°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 or N,N'-bis(2-chloroethyl)diaminophosphonic acid; (8) Condensation reaction: add (1-methyl-4-nitro-1H-imidazole-2-cyano)methanol, N,N'-bis(2-bromoethyl)diaminophosphonic acid or N,N'-bis(2-chloroethyl)diaminophosphonic acid, triphenylphosphine and anhydrous tetrahydrofuran, cool the reaction system to 0°C, then add diisopropyl azodicarboxylate DIAD dropwise, heat to 25°C and react for 5 hours, then concentrate under reduced pressure to obtain a crude product, which is purified by silica gel column chromatography to obtain the product N,N'-bis(2-bromoethyl)diaminophosphonic acid (1-methyl-4-nitro-1H-imidazole-2-cyano) methyl ester or N,N'-bis(2-chloroethyl)diaminophosphonic acid (1-methyl-4-nitro-1H-imidazole-2-cyano) methyl ester.

2. The method for preparing N,N'-bis(2-bromo / chloroethyl)diaminophosphonic acid (1-methyl-4-nitro-1H-imidazole-2-cyano)methyl ester according to claim 1, characterized in that: In step (1), the volume ratio of concentrated nitric acid to concentrated sulfuric acid is 3:

1.

3. The method for preparing N,N'-bis(2-bromo / chloroethyl)diaminophosphonic acid (1-methyl-4-nitro-1H-imidazole-2-cyano)methyl ester according to claim 1, characterized in that: In step (2), the molar ratio of 4-nitro-5-methyl-1H-imidazole, potassium tert-butoxide and methyl iodide is 1:2:

2.

4. The method for preparing N,N'-bis(2-bromo / chloroethyl)diaminophosphonic acid (1-methyl-4-nitro-1H-imidazole-2-cyano) methyl ester according to claim 1, characterized in that: In step (3), the molar ratio of 1,5-dimethyl-4-nitro-1H-imidazole to liquid bromine is 1:

4.

5. The method for preparing N,N'-bis(2-bromo / chloroethyl)diaminophosphonic acid (1-methyl-4-nitro-1H-imidazole-2-cyano)methyl ester according to claim 1, characterized in that: In step (4), the molar ratio of 2-bromo-1,5-dimethyl-4-nitro-1H-imidazole to N-bromosuccinimide is 1:3, and the amount of azobisisobutyl ester is 10% of 2-bromo-1,5-dimethyl-4-nitro-1H-imidazole.

6. The method for preparing N,N'-bis(2-bromo / chloroethyl)diaminophosphonic acid (1-methyl-4-nitro-1H-imidazole-2-cyano)methyl ester according to claim 1, characterized in that: In step (5), the molar ratio of 2-bromo-5-bromomethyl-1-methyl-4-nitro-1H-imidazole and calcium carbonate is 1:5, and the volume ratio of 1,4-dioxane to water is 3:

2.

7. The method for preparing N,N'-bis(2-bromo / chloroethyl)diaminophosphonic acid (1-methyl-4-nitro-1H-imidazole-2-cyano)methyl ester according to claim 1, characterized in that: In step (6), the molar ratio of 2-bromo-5-hydroxymethyl-1-methyl-4-nitro-1H-imidazole, potassium iodide and cuprous cyanide is 1:2:

2.

8. The method for preparing N,N'-bis(2-bromo / chloroethyl)diaminophosphonic acid (1-methyl-4-nitro-1H-imidazole-2-cyano)methyl ester according to claim 1, characterized in that: In step (7), the molar ratio of 2-bromoethylamine hydrobromide or 2-chloroethylamine hydrochloride, phosphorus oxychloride and triethylamine is 2.5:1:

2.

9. The method for preparing N,N'-bis(2-bromo / chloroethyl)diaminophosphonic acid (1-methyl-4-nitro-1H-imidazole-2-cyano)methyl ester according to claim 1, characterized in that: In step (9), the molar ratio of (1-methyl-4-nitro-1H-imidazole-2-cyano)methanol, N,N'-bis(2-bromoethyl)diaminophosphonic acid or N,N'-bis(2-chloroethyl)diaminophosphonic acid, triphenylphosphine and diisopropyl azodicarboxylate (DIAD) is 2:1:2:

2.

10. A N,N'-bis(2-bromo / chloroethyl)diaminophosphonic acid (1-methyl-4-nitro-1H-imidazole-2-cyano) methyl ester compound prepared according to any one of claims 1 to 9, characterized in that: The chemical structural formula of the compound is shown below:

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