A radiotherapy sensitizer, its preparation method and application

Abstract

The present invention discloses a radiosensitizer and its preparation method and application. In the present invention, a PARP inhibitor (PARPi) and a nitro derivative are covalently bonded to obtain a radiosensitizer. During radiotherapy, the radiosensitizer can not only fix DNA damage in tumor cells through the action principle of nitroimidazole drugs, but also inhibit the repair function of PARP, further amplifying the cell damage caused by radiation, thereby achieving the effect of radiotherapy sensitization and improving the efficacy of radiotherapy.

Description

Technical Field

[0001] The present invention relates to the field of radiotherapy sensitizers, and particularly to a radiotherapy sensitizer, a preparation method thereof and an application thereof. Background Art

[0002] Radiotherapy (RT), as one of the three major means of tumor treatment, has the advantages of non-invasiveness and relatively low systemic toxic and side effects. Clinically, about 60% - 70% of malignant tumor patients have received radiotherapy. Radiotherapy acts on tumor cell DNA through high-energy rays (such as X-rays), causing single-strand breaks (SSBs) and double-strand breaks (DSBs) of DNA, ultimately leading to cell growth arrest or death. In addition, high-energy rays can also stimulate water molecules in tumor cells to generate reactive oxygen species (ROS) or free radicals, inducing cell death through oxidative damage. However, the radiotherapy effect of most tumors in clinical practice is not ideal. The main reason is that tumor cells have complex and powerful DNA damage repair programs, including mismatch repair (MMR), base excision repair (BER), and nucleotide excision repair (NER), etc., which often lead to a reduction in radiation damage. For example, when DNA is damaged, poly-ADP-ribose polymerase (PARP) can quickly bind to the damaged site, activate the histones for DNA repair, excise the damaged bases, and finally repair the DNA damage. In recent years, PARP inhibitors, anti-tumor drugs targeting PARP, have gradually received extensive attention. By inhibiting PARP from losing its enzymatic activity, they prevent PARP protein from detaching from the DNA break damage site, block SSB repair, and continuously accumulate to cause DSB damage. Clinical data show that PARP inhibitors have relatively significant effects on tumors with homologous recombination deficiency (HRD), such as breast cancer, ovarian cancer, prostate cancer, and primary peritoneal cancer with BRCA mutations. In addition, there is evidence that PARP inhibitors (such as olaparib) show certain synergistic therapeutic effects when used in combination with radiotherapy, with potential radiotherapy sensitization effects (Radiother. Oncol., 2015, 116, 358 - 365).

[0003] On the other hand, the partial pressure of oxygen in tumor tissues is highly heterogeneous, with obvious hypoxic regions. For example, the average partial pressure of oxygen in solid tumor tissues such as cervical cancer, head and neck malignancies, and breast cancer is only 10 mmHg. Hypoxia is one of the most common characteristics of solid tumors. Tumor cells mainly regulate hypoxia-inducible factor-1α (HIF-1α) to adapt to the hypoxic environment. It is currently generally believed that the tumor hypoxic microenvironment can mediate the radioresistance of malignant tumors (Nat. Rev. Cancer, 2008, 8, 180-192). Compared with the normal oxygen environment, the radiation resistance of tumor cells in the hypoxic microenvironment can be increased by 2-3 times (Radiother. Oncol., 2017, 124, 533-540). In addition, the hypoxic microenvironment not only limits the formation of reactive oxygen species and free radicals during radiotherapy, but also the highly expressed thiol molecules can significantly reduce the DNA damage effect of high-energy rays, thereby leading to tumor radiotherapy resistance. Therefore, how to effectively improve the sensitivity of tumor radiotherapy and enhance the DNA damage effect of tumors is the key to improving the current poor radiotherapy effect of tumors.

[0004] Currently, radiotherapy sensitizers such as nitro compounds and nitrogen oxides developed still lack targeting. Clinical studies have found that when nitroimidazole drugs such as misonidazole and etanidazole are combined with fractionated radiotherapy, delayed peripheral neuropathy will occur, thus limiting their clinical application (Int. J. Radiat. Oncol. Biol. Phys. 1999, 44, 513-517). Recent studies have also found that nitroimidazole drugs can cause neurotoxicity. In addition, the treatment with nitroimidazole drugs has no significant effect on local control and overall survival in women receiving radiotherapy for cervical cancer (Br. J. Radiol. 2005, 78, 777-782). Therefore, the design and synthesis of targeted nitroimidazole drugs for radiotherapy sensitization have become the key. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a targeted therapeutic nitro compound, which during radiotherapy can not only fix the DNA damage of tumor cells through the action mechanism of nitroimidazole drugs, but also inhibit the repair effect of PARP, further amplifying the cell damage of rays, thereby achieving the radiotherapy sensitization effect and ultimately improving the radiotherapy efficacy.

[0006] To solve the above technical problem, in the first aspect, the present invention provides a radiotherapy sensitizer, and the structural general formula of the radiotherapy sensitizer is as follows:

[0007]

[0008] Wherein, R is

[0009] They are named as Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, and Compound 6 in sequence.

[0010] The second aspect of the present invention is to provide a preparation method of the radiosensitizer described in the first aspect, including the following steps:

[0011] Add 4-(4-fluoro-3-(piperazine-1-carbonyl)benzyl)phthalazin-1(2H)-one, a nitro compound, and an amide condensing agent into an organic solvent, and react in an inert gas environment to obtain the radiosensitizer.

[0012] Furthermore, the nitro compound is 3,5-dinitrobenzoic acid, 2-(5-methyl-2-nitro-1H-imidazol-1-yl)ethyl (4-nitrophenyl) carbonate, N,N-bis[(2-methyl-5-nitro-1H-imidazol-1-yl)-ethoxycarbonylmethyl]glycine, 5-nitrothiophene-2-carboxylic acid, 5-nitrofuran-2-carboxylic acid, or m-nitrobenzoic acid.

[0013] Furthermore, the amide condensing agent is selected from one or more of dicyclohexylcarbodiimide, diisopropylcarbodiimide, 1-ethyl-(3-dimethylaminopropyl)carbodiimide, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, and N,N-diisopropylethylamine.

[0014] Furthermore, the organic solvent is selected from one or more of N,N-dimethylformamide, tetrahydrofuran, and acetonitrile.

[0015] Furthermore, the molar ratio of 4-(4-fluoro-3-(piperazine-1-carbonyl)benzyl)phthalazin-1(2H)-one: nitro compound: amide condensing agent is 1:(2-6):(4-15).

[0016] Furthermore, the reaction time is 8-24 h; preferably 8-12 h; more preferably 12 h.

[0017] Furthermore, the mass ratio of the nitro compound to the organic solvent is 1:20-200; preferably 1:50-100; for example, 1:50, 1:60, 1:70, 1:80, 1:90, and 1:100, etc., including but not limited to the above-listed mass ratios.

[0018] The third aspect of the present invention is to provide the application of the radiosensitizer prepared by the method described in the second aspect in the preparation of drugs for treating tumors.

[0019] Furthermore, the tumor is esophageal cancer, head and neck cancer, nasopharyngeal cancer, ovarian cancer, lung cancer, gastric cancer, breast cancer, liver cancer, pancreatic cancer, colorectal cancer, skin cancer, malignant melanoma, cholangiocarcinoma, bladder cancer, or renal cancer; preferably breast cancer.

[0020] Advantages of the present invention:

[0021] The present invention enhances the radiotherapy effect through different mechanisms, improves the sensitivity of tumor cells to radiotherapy, and at the same time utilizes the synthetic lethality effect to improve the treatment effect on specific tumor cells and reduce the occurrence of drug resistance. The present invention prepares radiotherapy sensitizers containing different nitro groups, and the preparation method is simple, the cost is low, and it is easy to mass-produce. The introduction of these nitro groups not only enhances the targeting of the molecule to tumor cells, but also improves the activity of the compound in the hypoxic tumor microenvironment, thus exerting a more significant sensitization effect during radiotherapy. In addition, by optimizing the structure of the radiotherapy sensitizer, potential toxic and side effects are reduced, making the combined treatment safer and more effective. Combining PARPi with nitro derivatives, during radiotherapy, the radiotherapy sensitizing drug can not only fix the DNA damage of tumor cells through the action principle of nitroimidazole drugs, but also inhibit the repair effect of PARP, further amplifying the cell damage caused by radiation, thereby achieving the radiotherapy sensitization effect and improving the radiotherapy efficacy. Description of the drawings

[0022] Figure 1 are the nuclear magnetic resonance hydrogen spectrum and mass spectrum of Compound 1 prepared in Example 1 of the present invention;

[0023] Figure 2 are the nuclear magnetic resonance hydrogen spectrum and mass spectrum of Compound 2 prepared in Example 4 of the present invention;

[0024] Figure 3 are the nuclear magnetic resonance hydrogen spectrum and mass spectrum of Compound 3 prepared in Example 7 of the present invention;

[0025] Figure 4 are the nuclear magnetic resonance hydrogen spectrum and mass spectrum of Compound 4 prepared in Example 10 of the present invention;

[0026] Figure 5 are the nuclear magnetic resonance hydrogen spectrum and mass spectrum of Compound 5 prepared in Example 13 of the present invention;

[0027] Figure 6 are the nuclear magnetic resonance hydrogen spectrum and mass spectrum of Compound 6 prepared in Example 16 of the present invention;

[0028] Figure 7 is the toxicity test of Compounds 1-6 prepared by the present invention and olaparib on 4T1 cells at different concentrations;

[0029] Figure 8 is the toxicity test of Compounds 1-6 prepared by the present invention and olaparib on 4T1 cells after irradiation with 4 Gy of radiation at different concentrations;

[0030] Figure 9Confocal laser scanning microscopy images showing the competitive binding of compounds 1-6 prepared in the present invention to the PARP site in the cell nucleus with PARP-FL.

[0031] Figure 10 Results of the colony formation assay of compound 1 prepared in the present invention on 4T1 cells.

[0032] Figure 11 Results of the colony formation assay of compound 2 prepared in the present invention on 4T1 cells.

[0033] Figure 12 Results of the colony formation assay of compound 3 prepared in the present invention on 4T1 cells.

[0034] Figure 13 Results of the colony formation assay of compound 4 prepared in the present invention on 4T1 cells.

[0035] Figure 14 Results of the colony formation assay of compound 5 prepared in the present invention on 4T1 cells.

[0036] Figure 15 Results of the colony formation assay of compound 6 prepared in the present invention on 4T1 cells.

[0037] Figure 16 Results of the colony formation assay of olaparib on 4T1 cells. Detailed Description of the Invention

[0038] The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments, so that those skilled in the art can better understand the present invention and be able to implement it, but the embodiments given are not intended to limit the present invention.

[0039] Example 1 Preparation of Compound 1

[0040] This example relates to a method for preparing a radiosensitizer, and the specific operation method is as follows:

[0041] 4-(4-Fluoro-3-(piperazine-1-carbonyl)benzyl)phthalazin-1(2H)-one, 3,5-dinitrobenzoic acid and dicyclohexylcarbodiimide (DDC) were added to a reaction vessel in a molar ratio of 1:2:4, and 100 times the weight of 3,5-dinitrobenzoic acid of tetrahydrofuran (THF) was added as a solvent, and the reaction was carried out at room temperature for 12 hours under nitrogen protection. After the reaction was completed, it was extracted and purified by column chromatography (SiO 2 ; the eluent was dichloromethane / ethyl acetate), and a white solid product was obtained with a yield of 40%.

[0042] Example 2 Preparation of Compound 1

[0043] This example relates to a preparation method of a radiotherapy sensitizer, and the specific operation method is as follows:

[0044] 4-(4-Fluoro-3-(piperazine-1-carbonyl)benzyl)phthalazin-1(2H)-one, 3,5-dinitrobenzoic acid and diisopropylcarbodiimide (DIC) were added to a reaction vessel in a molar ratio of 1:2:4, and 100 times the weight of 3,5-dinitrobenzoic acid of tetrahydrofuran (THF) was added as a solvent, and the reaction was carried out at room temperature for 12 hours under nitrogen protection. After the reaction was completed, it was extracted and purified by column chromatography (SiO 2 ; the eluent was dichloromethane / ethyl acetate), and a white solid product was obtained with a yield of 48%.

[0045] Preparation of Compound 1 in Example 3

[0046] This example relates to a preparation method of a radiotherapy sensitizer, and the specific operation method is as follows:

[0047] 4-(4-Fluoro-3-(piperazine-1-carbonyl)benzyl)phthalazin-1(2H)-one, 3,5-dinitrobenzoic acid and diisopropylcarbodiimide (DIC) were added to a reaction vessel in a molar ratio of 1:4:8, and 100 times the weight of 3,5-dinitrobenzoic acid of acetonitrile (CH 3 CN) was added as a solvent, and the reaction was carried out at room temperature for 12 hours under nitrogen protection. After the reaction was completed, it was extracted and purified by column chromatography (SiO 2 ; the eluent was dichloromethane / ethyl acetate), and a white solid product was obtained with a yield of 60%.

[0048] Preparation of Compound 2 in Example 4

[0049] This example relates to a preparation method of a radiotherapy sensitizer, and the specific operation method is as follows:

[0050] 4-(4-Fluoro-3-(piperazine-1-carbonyl)benzyl)phthalazin-1(2H)-one, 2-(5-Methyl-2-nitro-1H-imidazol-1-yl)ethyl (4-nitrophenyl) carbonate, dicyclohexylcarbodiimide (DDC) and 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) were added to a reaction vessel in a molar ratio of 1:2:2:4, and 100 times the weight of 2-(5-Methyl-2-nitro-1H-imidazol-1-yl)ethyl (4-nitrophenyl) carbonate of tetrahydrofuran (THF) was added as a solvent, and the reaction was carried out at room temperature for 12 hours under nitrogen protection. After the reaction was completed, it was extracted and purified by column chromatography (SiO 2 ; the eluent was dichloromethane / ethyl acetate), and a white solid product was obtained with a yield of 30%.

[0051] Preparation of Compound 2 in Example 5

[0052] This example relates to a preparation method of a radiosensitizer, and the specific operation method is as follows:

[0053] 4-(4-Fluoro-3-(piperazine-1-carbonyl)benzyl)phthalazin-1(2H)-one, 2-(5-methyl-2-nitro-1H-imidazol-1-yl)ethyl (4-nitrophenyl) carbonate, dicyclohexylcarbodiimide (DDC) and 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) are added to a reaction vessel in a molar ratio of 1:4:4:6, and 100 times the weight of 2-(5-methyl-2-nitro-1H-imidazol-1-yl)ethyl (4-nitrophenyl) carbonate of tetrahydrofuran (THF) is added as a solvent, and the reaction is carried out at room temperature for 12 hours under nitrogen protection. After the reaction is completed, it is extracted and purified by column chromatography (SiO 2 ; the eluent is dichloromethane / ethyl acetate), and a white solid product is obtained with a yield of 46%.

[0054] Preparation of Compound 2 in Example 6

[0055] This example relates to a preparation method of a radiosensitizer, and the specific operation method is as follows:

[0056] 4-(4-Fluoro-3-(piperazine-1-carbonyl)benzyl)phthalazin-1(2H)-one, 2-(5-methyl-2-nitro-1H-imidazol-1-yl)ethyl (4-nitrophenyl) carbonate, dicyclohexylcarbodiimide (DDC) and 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) are added to a reaction vessel in a molar ratio of 1:2:2:4, and 100 times the weight of 2-(5-methyl-2-nitro-1H-imidazol-1-yl)ethyl (4-nitrophenyl) carbonate of acetonitrile (CH 3 CN) is added as a solvent, and the reaction is carried out at room temperature for 12 hours under nitrogen protection. After the reaction is completed, it is extracted and purified by column chromatography (SiO 2 ; the eluent is dichloromethane / ethyl acetate), and a white solid product is obtained with a yield of 55%.

[0057] Preparation of Compound 3 in Example 7

[0058] This example relates to a preparation method of a radiosensitizer, and the specific operation method is as follows:

[0059] 4-(4-Fluoro-3-(piperazine-1-carbonyl)benzyl)phthalazin-1(2H)-one, N,N-bis[(2-methyl-5-nitro-1H-imidazol-1-yl)-ethoxycarbonylmethyl]glycine, dicyclohexylcarbodiimide (DDC) and 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) were added to a reaction vessel in a molar ratio of 1:2:2:4. Tetrahydrofuran (THF) 100 times the weight of N,N-bis[(2-methyl-5-nitro-1H-imidazol-1-yl)-ethoxycarbonylmethyl]glycine was added as a solvent, and the reaction was carried out at room temperature for 12 hours under nitrogen protection. After the reaction was completed, it was extracted and purified by column chromatography (SiO 2 ; the eluent was dichloromethane / ethyl acetate), and a white solid product was obtained with a yield of 25%.

[0060] Preparation of Compound 3 in Example 8

[0061] This example relates to a preparation method of a radiosensitizer, and the specific operation method is as follows:

[0062] 4-(4-Fluoro-3-(piperazine-1-carbonyl)benzyl)phthalazin-1(2H)-one, N,N-bis[(2-methyl-5-nitro-1H-imidazol-1-yl)-ethoxycarbonylmethyl]glycine, dicyclohexylcarbodiimide (DDC) and 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) were added to a reaction vessel in a molar ratio of 1:2:4:6. Tetrahydrofuran (THF) 100 times the weight of N,N-bis[(2-methyl-5-nitro-1H-imidazol-1-yl)-ethoxycarbonylmethyl]glycine was added as a solvent, and the reaction was carried out at room temperature for 12 hours under nitrogen protection. After the reaction was completed, it was extracted and purified by column chromatography (SiO 2 ; the eluent was dichloromethane / ethyl acetate), and a white solid product was obtained with a yield of 40%.

[0063] Preparation of Compound 3 in Example 9

[0064] This example relates to a preparation method of a radiosensitizer, and the specific operation method is as follows:

[0065] 4-(4-Fluoro-3-(piperazine-1-carbonyl)benzyl)phthalazin-1(2H)-one, N,N-bis[(2-methyl-5-nitro-1H-imidazol-1-yl)-ethoxycarbonylmethyl]glycine, dicyclohexylcarbodiimide (DDC) and 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) were added to a reaction vessel in a molar ratio of 1:2:4:6. A solution of N,N-dimethylformamide (DMF) 100 times the weight of N,N-bis[(2-methyl-5-nitro-1H-imidazol-1-yl)-ethoxycarbonylmethyl]glycine was added as a solvent, and the reaction was carried out at room temperature for 12 hours under nitrogen protection. After the reaction was completed, it was extracted and purified by column chromatography (SiO 2 ; the eluent was dichloromethane / ethyl acetate), and a white solid product was obtained with a yield of 65%.

[0066] Preparation of Compound 4 in Example 10

[0067] This example relates to a preparation method of a radiosensitizer, and the specific operation method is as follows:

[0068] 4-(4-Fluoro-3-(piperazine-1-carbonyl)benzyl)phthalazin-1(2H)-one, 5-nitrothiophene-2-carboxylic acid, dicyclohexylcarbodiimide (DDC) and 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) were added to a reaction vessel in a molar ratio of 1:2:2:4. Tetrahydrofuran (THF) 100 times the weight of 5-nitrothiophene-2-carboxylic acid was added as a solvent, and the reaction was carried out at room temperature for 12 hours under nitrogen protection. After the reaction was completed, it was extracted and purified by column chromatography (SiO 2 ; the eluent was dichloromethane / ethyl acetate), and a white solid product was obtained with a yield of 49%.

[0069] Preparation of Compound 4 in Example 11

[0070] This example relates to a preparation method of a radiosensitizer, and the specific operation method is as follows:

[0071] 4-(4-Fluoro-3-(piperazine-1-carbonyl)benzyl)phthalazin-1(2H)-one, 5-nitrothiophene-2-carboxylic acid, dicyclohexylcarbodiimide (DDC) and 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) were added to a reaction vessel in a molar ratio of 1:4:4:6. Tetrahydrofuran (THF) 100 times the weight of 5-nitrothiophene-2-carboxylic acid was added as a solvent, and the reaction was carried out at room temperature for 12 hours under nitrogen protection. After the reaction was completed, it was extracted and purified by column chromatography (SiO 2 ; the eluent was dichloromethane / ethyl acetate), and a white solid product was obtained with a yield of 55%.

[0072] Preparation of Compound 4 in Example 12

[0073] This example relates to a preparation method of a radiosensitizer, and the specific operation method is as follows:

[0074] 4-(4-Fluoro-3-(piperazine-1-carbonyl)benzyl)phthalazin-1(2H)-one, 5-nitrothiophene-2-carboxylic acid, dicyclohexylcarbodiimide (DDC) and 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) are added to a reaction vessel in a molar ratio of 1:4:4:6, and acetonitrile (CH 3 CN) which is 100 times the weight of 5-nitrothiophene-2-carboxylic acid is added as a solvent, and the reaction is carried out at room temperature for 15 hours under nitrogen protection. After the reaction is completed, it is extracted and purified by column chromatography (SiO 2 ; the eluent is dichloromethane / ethyl acetate) to obtain a white solid product with a yield of 50%.

[0075] Preparation of Compound 5 in Example 13

[0076] This example relates to a preparation method of a radiosensitizer, and the specific operation method is as follows:

[0077] 4-(4-Fluoro-3-(piperazine-1-carbonyl)benzyl)phthalazin-1(2H)-one, 5-nitrofuran-2-carboxylic acid, dicyclohexylcarbodiimide (DDC) and 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) are added to a reaction vessel in a molar ratio of 1:4:4:6, and acetonitrile (CH 3 CN) which is 100 times the weight of 5-nitrofuran-2-carboxylic acid is added as a solvent, and the reaction is carried out at room temperature for 15 hours under nitrogen protection. After the reaction is completed, it is extracted and purified by column chromatography (SiO 2 ; the eluent is dichloromethane / ethyl acetate) to obtain a white solid product with a yield of 45%.

[0078] Preparation of Compound 5 in Example 14

[0079] This example relates to a preparation method of a radiosensitizer, and the specific operation method is as follows:

[0080] 4-(4-Fluoro-3-(piperazine-1-carbonyl)benzyl)phthalazin-1(2H)-one, 5-nitrofuran-2-carboxylic acid, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCl) and N,N-diisopropylethylamine (DIPEA) are added to a reaction vessel in a molar ratio of 1:5:5:10, and tetrahydrofuran (THF) which is 100 times the weight of 5-nitrofuran-2-carboxylic acid is added as a solvent, and the reaction is carried out at room temperature for 8 hours under nitrogen protection. After the reaction is completed, it is extracted and purified by column chromatography (SiO 2 ; the eluent is dichloromethane / ethyl acetate) to obtain a white solid product with a yield of 52%.

[0081] Preparation of Compound 5 in Example 15

[0082] This example relates to a preparation method of a radiosensitizer. The specific operation method is as follows:

[0083] 4-(4-Fluoro-3-(piperazine-1-carbonyl)benzyl)phthalazin-1(2H)-one, 5-nitrofurane-2-carboxylic acid, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCl), and N,N-diisopropylethylamine (DIPEA) were added to a reaction vessel in a molar ratio of 1:5:5:10. A solution of N,N-dimethylformamide (DMF) 50 times the weight of 5-nitrofurane-2-carboxylic acid was added as a solvent, and the reaction was carried out at room temperature for 8 hours under nitrogen protection. After the reaction was completed, it was extracted and purified by column chromatography (SiO 2 ; the eluent was dichloromethane / ethyl acetate), and a white solid product was obtained with a yield of 40%.

[0084] Preparation of Compound 6 in Example 16

[0085] This example relates to a preparation method of a radiosensitizer. The specific operation method is as follows:

[0086] 4-(4-Fluoro-3-(piperazine-1-carbonyl)benzyl)phthalazin-1(2H)-one, m-nitrobenzoic acid, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCl), and N,N-diisopropylethylamine (DIPEA) were added to a reaction vessel in a molar ratio of 1:2:2:4. Tetrahydrofuran (THF) 100 times the weight of m-nitrobenzoic acid was added as a solvent, and the reaction was carried out at room temperature for 24 hours under nitrogen protection. After the reaction was completed, it was extracted and purified by column chromatography (SiO 2 ; the eluent was dichloromethane / ethyl acetate), and a white solid product was obtained with a yield of 50%.

[0087] Preparation of Compound 6 in Example 17

[0088] This example relates to a preparation method of a radiosensitizer. The specific operation method is as follows:

[0089] 4-(4-Fluoro-3-(piperazine-1-carbonyl)benzyl)phthalazin-1(2H)-one, m-nitrobenzoic acid, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCl), and N,N-diisopropylethylamine (DIPEA) were added to a reaction vessel in a molar ratio of 1:6:4:8. A solution of N,N-dimethylformamide (DMF) 100 times the weight of m-nitrobenzoic acid was added as a solvent, and the reaction was carried out at room temperature for 15 hours under nitrogen protection. After the reaction was completed, it was extracted and purified by column chromatography (SiO 2; The eluent was dichloromethane / ethyl acetate), and a white solid product was obtained with a yield of 58%.

[0090] Preparation of Compound 6 in Example 18

[0091] This example relates to a preparation method of a radiosensitizer. The specific operation method is as follows:

[0092] 4-(4-Fluoro-3-(piperazine-1-carbonyl)benzyl)phthalazin-1(2H)-one, m-nitrobenzoic acid, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCl), and N,N-diisopropylethylamine (DIPEA) were added to a reaction vessel in a molar ratio of 1:5:5:10. N,N-dimethylformamide solution (DMF) 50 times the weight of m-nitrobenzoic acid was added as a solvent, and the reaction was carried out at room temperature for 12 hours under nitrogen protection. After the reaction was completed, it was extracted and purified by column chromatography (SiO 2 ; The eluent was dichloromethane / ethyl acetate), and a white solid product was obtained with a yield of 65%.

[0093] Comparative Example 1

[0094] This comparative example relates to a preparation method of a radiosensitizer. The specific operation steps are as follows:

[0095] 4-(4-Fluoro-3-(piperazine-1-carbonyl)benzyl)phthalazin-1(2H)-one, 3,5-dinitrobenzoic acid, and dicyclohexylcarbodiimide (DDC) were added to a reaction vessel in a molar ratio of 1:1:1. Tetrahydrofuran (THF) 100 times the weight of 3,5-dinitrobenzoic acid was added as a solvent, and the reaction was carried out at room temperature under nitrogen protection. The reaction process was monitored by TLC, and it was observed by spotting that the reaction did not proceed.

[0096] Test Example

[0097] 1. In vitro cytotoxicity test

[0098] The in vitro toxicity of a series of compounds was detected in mouse (mammary gland) cancer cells (4T1): A radiation irradiation group and a group not given radiation irradiation were set up respectively; the specific operation steps are as follows:

[0099] Take 4T1 cells in the logarithmic growth phase and place them in a 96-well plate (3×10 3Cells / well, with 5 replicate wells in a group, and a total of 7 groups were set) and cultured for 24 h. Observe that the cells adhered to the wall, had normal morphology and appropriate density. Prepare a drug stock solution with a concentration of 20 mM using DMSO and filter it through a 0.22 μm organic filter membrane. Dilute the stock solution to different concentrations with fresh medium: 25 μM, 50 μM, 100 μM, 200 μM, 400 μM, 800 μM. Discard the medium in the replicate wells of the 96-well plate and wash it 3 times with phosphate buffer. The first group of replicate wells is the control group, and fresh medium containing the same volume of DMSO is added. The replicate wells of other groups are added with the prepared medium containing the drug at the corresponding concentration (100 μL / well).

[0100] The cells in the X-ray irradiation group were irradiated with 4 Gy of X-rays 1 h after drug administration, and then the drug and the cells were co-incubated for 24 h (the group not given X-ray irradiation directly co-incubated the drug and the cells for 24 h). Then, wash again and replace the medium with fresh medium and continue to culture for 24 h. Finally, use the CCK8 reagent to detect the cell viability, determine the cell survival situation by detecting the absorbance at a wavelength of 450 nm, and then calculate the cell survival rate.

[0101] As Figure 7 , Figure 8 shown, after irradiation with X-rays, the IC 50 concentration of the compound decreased, indicating that the introduction of the nitro group increased the cytotoxicity of the compound. To achieve the same therapeutic effect, less radiation dose can be used, thereby reducing the damage to normal tissues and improving the quality of life and treatment tolerance of patients.

[0102] 2. Intranuclear PARP inhibitor competitive binding experiment

[0103] Detect the ability of a series of compounds to competitively bind to PARP inhibitors in murine (mammary gland) cancer cells (4T1) (intranuclear competitive binding experiment): Take 4T1 cells in the logarithmic growth phase, plate the cells in a confocal dish, 2×10 4Cells per dish, and the cells were cultured in a constant-temperature cell incubator for 24 h. The cells were observed to adhere to the wall, with normal morphology and uniform dispersion. After gently washing once with phosphate buffer solution, the medium was replaced with a medium containing different drugs at the same concentration (25 μM, 1 mL / dish) and cultured for 2 h. The PBS group was added with a medium containing the same volume of PBS. Then, it was washed three times again with phosphate buffer solution, and the medium was replaced with a fresh medium containing PARP-FL (5 μM, 1 mL / dish) and cultured for 1 h. Then, it was washed three times repeatedly with phosphate buffer solution, and the medium was replaced with a fresh medium containing the dye Hoechst33342 (5 μM, 1 mL / dish) and cultured for 10 min to stain the cell nuclei. Finally, after washing, it was replaced with a serum-free medium, and the co-localization of PARP-FL in the cell nuclei was observed and recorded using a confocal microscope, and the co-localization coefficient was calculated. The lower the co-localization coefficient, the higher the degree of binding of this drug to the PARP binding site in the cell nucleus, and the stronger the PARP inhibitor effect.

[0104] The results are as Figure 9 shown, and all of the prepared compounds 1-6 showed PARP inhibitor activity.

[0105] 3. Colony formation assay

[0106] The in vitro radiosensitization ability of a series of compounds was detected in murine (mammary gland) cancer cells (4T1) (colony formation assay): Logarithmic growth phase 4T1 cells were plated in 6-well plates. Different initial cell numbers corresponded to different radiation doses: 100 cells / well in the control group, 200 cells / well in the 2 Gy group, 500 cells / well in the 4 Gy group, 1×10 3 cells / well in the 6 Gy group, and 2×10 3 cells / well in the 8 Gy group. The cells were cultured in a constant-temperature cell incubator for 24 h. The cells were observed to adhere to the wall, with normal morphology and uniform dispersion. After gently washing once with phosphate buffer solution, the medium was replaced with a medium containing different drugs at the same concentration (1.25 μM, 2 mL / well) and cultured for 1 h. The control group was added with a medium containing the same volume of DMSO. Different doses of X-rays (2, 4, 6, 8 Gy) were irradiated, and then it was returned to the incubator and cultured for another 24 h. It was gently washed three times with phosphate buffer solution, and the medium was replaced with a fresh medium and cultured for 7 days. Cell colonies were observed under the microscope (the number of cells in a single colony > 50). Finally, the cell colonies were stained with crystal violet dye, then photographed and counted, and the mean lethal dose (D 0 ) and radiosensitization ratio (SER) of various drugs were calculated. The results are shown in Table 1, where SER represents the magnitude of the radiosensitization effect of this drug, and the SER of ordinary X-rays was defaulted to 1.

[0107] Table 1

[0108]

[0109] As Figures 10 - 16 shown, the addition of the nitro group makes the sensitization effect of the prepared compound significantly better than that of olaparib, because the introduction of the nitro group provides an electrophilic group for the compound, enhancing the damage of the compound to cells through redox reactions under radiation.

[0110] The above-described embodiments are only preferred embodiments given to fully illustrate the present invention, and the protection scope of the present invention is not limited thereto. Equivalent substitutions or transformations made by those skilled in the art on the basis of the present invention are all within the protection scope of the present invention. The protection scope of the present invention shall be subject to the claims.

Claims

1. A radiotherapy sensitizer, characterized in that: The general structural formula of the radiosensitizer is as follows: Among them, R is 2. A method for preparing the radiosensitizer according to claim 1, characterized in that: The following steps are involved: 4-(4-fluoro-3-(piperazine-1-carbonyl)benzyl)phthalazine-1(2H)-one, a nitro compound and an amide condensation agent are added into an organic solvent and reacted in an inert gas environment to obtain the radiotherapy sensitizer.

3. The preparation method according to claim 2, characterized in that: The nitro compound is 3,5-dinitrobenzoic acid, 2-(5-methyl-2-nitro-1H-imidazol-1-yl)ethyl(4-nitrophenyl)carbonate, N,N-bis[(2-methyl-5-nitro-1H-imidazol-1-yl)-ethoxycarbonylmethyl]glycine, 5-nitrothiophene-2-carboxylic acid, 5-nitrofuran-2-carboxylic acid or m-nitrobenzoic acid.

4. The preparation method according to claim 2, characterized in that: The amide condensation agent is selected from one or more of dicyclohexylcarbodiimide, diisopropylcarbodiimide, 1-ethyl-(3-dimethylaminopropyl)carbodiimide, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N,N-diisopropylethylamine.

5. The preparation method according to claim 2, characterized in that: The organic solvent is selected from one or more of N,N-dimethylformamide, tetrahydrofuran and acetonitrile.

6. The preparation method according to claim 2, characterized in that: The molar ratio of 4-(4-fluoro-3-(piperazine-1-carbonyl)benzyl)phthalazin-1(2H)-one:nitro compound:amide condensation agent is 1:(2-6):(4-15).

7. The preparation method according to claim 2, characterized in that: The reaction time is 8-24h.

8. The preparation method according to claim 2, characterized in that: The mass ratio of the nitro compound to the organic solvent is 1:20-200.

9. Use of the radiosensitizer prepared by the method according to any one of claims 2 to 8 in the preparation of drugs for treating tumors.

10. The use according to claim 9, characterized in that The tumor is esophageal cancer, head and neck cancer, nasopharyngeal cancer, ovarian cancer, lung cancer, gastric cancer, breast cancer, liver cancer, pancreatic cancer, colorectal cancer, skin cancer, malignant melanoma, bile duct cancer, bladder cancer or kidney cancer.

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