AQP9 inhibitor and its preparation method and application

Abstract

The present invention proposes an AQP9 inhibitor and a preparation method and application thereof, belonging to the technical field of liver cancer tumor immunotherapy in the technical field of medical technology. The AQP9 inhibitor provided by the present invention is 1-(benzo[d][1,2,3]thiadiazole-6-yl)-3-(4-fluorophenyl)urea, which is synthesized for the first time by the present invention and can be used alone for screening or preparing drugs for treating solid tumors, especially for screening or preparing drugs for treating liver cancer, and can also be used in combination with a PD-1 blocker for screening or preparing drugs for treating solid tumors, especially for screening or preparing drugs for treating liver cancer.

Description

Technical Field

[0001] The present invention belongs to the technical field of liver cancer tumor immunotherapy in the technical field of medicine, and in particular relates to an AQP9 inhibitor and a preparation method and application thereof. Background Art

[0002] Systemic chemotherapy is the basic treatment for tumors, but chemotherapy drugs lack targeting. Secondly, most chemotherapy drugs have poor therapeutic effects on liver cancer. Currently, the more effective drugs for systemic chemotherapy of liver cancer are doxorubicin, 5-fluorouracil and cisplatin. However, these drugs have limited effects on the relief of liver cancer. There are many abnormal gene expressions in tumor cells, which may be closely related to the development and metastasis of tumors. The discovery of tumor targeted drugs can improve the current situation of poor targeting of chemotherapy drugs. Currently, sorafenib, regoratinib, lenvatinib, etc. are commonly used in liver cancer. However, after more than ten years of clinical use, the overall efficacy is low and the efficacy is poor.

[0003] In recent years, studies have found that changes in immune cells in the tumor microenvironment play a key role in the occurrence and development of liver cancer, making liver cancer immunotherapy gradually become a current research hotspot. The most widely used is PD-1 blocker, but the number of liver cancer patients with immune response is limited. In order to achieve better treatment effects, combined immunotherapy has become a new treatment strategy. However, liver cancer tends to be immune tolerant, which also brings challenges to the combined immunotherapy of liver cancer. This may be related to the abnormal expression of certain important genes. Therefore, it is crucial to find important immune-related key genes in liver cancer and develop new specific targeted therapeutic drugs for combined immunotherapy of liver cancer. Summary of the invention

[0004] In order to solve the above technical problems, the present invention proposes an AQP9 inhibitor and its preparation method and application. The AQP9 inhibitor of the present invention can not only be used alone to screen or prepare drugs for treating solid tumors, especially for screening or preparing drugs for treating liver cancer, but can also be used in combination with PD-1 blockers for screening or preparing drugs for treating solid tumors, especially for screening or preparing drugs for treating liver cancer.

[0005] To achieve the above object, the present invention provides the following technical solutions:

[0006] The present invention provides an AQP9 inhibitor, which is 1-(benzo[d][1,2,3]thiadiazole-6-yl)-3-(4-fluorophenyl)urea, and its structural formula is

[0007] The present invention provides a method for preparing the AQP9 inhibitor. The AQP9 inhibitor is prepared by condensing benzo[d][1,2,3]thiadiazole-6-amine and p-fluorophenyl isocyanate.

[0008] Furthermore, the preparation method of the AQP9 inhibitor comprises the following steps:

[0009] Benzo[d][1,2,3]thiadiazole-6-amine and anhydrous dichloromethane are mixed, and p-fluorophenyl isocyanate is added dropwise under nitrogen protection, and stirred to obtain a suspension, which is concentrated and the solid is washed to obtain a crude product. The crude product is dissolved in tetrahydrofuran, evaporated and concentrated, and subjected to column chromatography to obtain a solid which is the AQP9 inhibitor.

[0010] Furthermore, in the preparation method of the AQP9 inhibitor, the molar ratio of the benzo[d][1,2,3]thiadiazole-6-amine to p-fluorophenyl isocyanate is 11:12.

[0011] Furthermore, the preparation method of the AQP9 inhibitor is as follows:

[0012] Benzo[d][1,2,3]thiadiazole-6-amine (500 mg, 3.3 mmol) and anhydrous dichloromethane (15 mL) were added to a 100 mL dry round-bottom flask, and p-fluorophenyl isocyanate (0.41 mL, 3.6 mmol) was added dropwise at room temperature under nitrogen protection, and stirring was continued at room temperature for 24 hours to obtain a suspension. The liquid was concentrated, and the solid was washed 8 to 10 times with a mixed solution of petroleum ether: ethyl acetate = 2: 1 (volume ratio). After stirring and standing for a while during each washing, the supernatant was sucked off with a syringe. The final solid was a crude product with less impurities, and then the crude product was dissolved with tetrahydrofuran, mixed with silica gel and concentrated with a rotary evaporator, and dry-loaded for column chromatography. The eluent selected was petroleum ether: tetrahydrofuran = 3: 1 to 5: 1 (volume ratio) to obtain a white solid, which was the AQP9 inhibitor.

[0013] The present invention also provides the use of the AQP9 inhibitor in screening drugs for treating solid tumors.

[0014] The present invention also provides the use of the AQP9 inhibitor in preparing a drug for treating solid tumors.

[0015] The present invention also provides the use of the AQP9 inhibitor in screening drugs for treating liver cancer.

[0016] The present invention also provides the use of the AQP9 inhibitor in preparing a drug for treating liver cancer.

[0017] The present invention also provides the use of the AQP9 inhibitor combined with the PD-1 blocker in screening drugs for treating liver cancer.

[0018] The present invention also provides the use of the AQP9 inhibitor combined with a PD-1 blocker in the preparation of a drug for treating liver cancer.

[0019] Compared with the prior art, the present invention has the following advantages and technical effects:

[0020] 1. The 1-(benzo[d][1,2,3]thiadiazol-6-yl)-3-(4-fluorophenyl)urea synthesized for the first time in the present invention can be used as an AQP9 inhibitor and can significantly inhibit tumor proliferation;

[0021] 2. The AQP9 inhibitor of the present invention can increase the number of immune cells in the tumor microenvironment and can affect the peripheral immune system, thereby achieving an anti-tumor therapeutic effect;

[0022] 3. The present invention uses AQP9 inhibitors in combination with PD-1 blockers to treat liver cancer, and finds that AQP9 inhibitors can significantly enhance the therapeutic efficacy of PD-1 blockers and have obvious anti-tumor efficacy. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] The drawings constituting a part of the present application are used to provide a further understanding of the present application. The illustrative embodiments and descriptions of the present application are used to explain the present application and do not constitute an improper limitation on the present application. In the drawings:

[0024] Figure 1 The single-cell sequencing data of 21 liver cancer patient samples, where A is the flow chart for obtaining the single-cell sequencing data of 21 HCC patient samples, B is the different cell classifications obtained based on the single-cell sequencing data analysis, and C is the CD4 + T infiltration level analysis, D is CD8 + T is the analysis of infiltration level, E is the analysis of AQP9 expression level in malignant cells;

[0025] Figure 2 Where A is the structural formula of 1-(benzo[d][1,2,3]thiadiazol-6-yl)-3-(4-fluorophenyl)urea, and B is the spectrum of 1-(benzo[d][1,2,3]thiadiazol-6-yl)-3-(4-fluorophenyl)urea;

[0026] Figure 3 The effect of AQP9 inhibitor on the growth of liver cancer and tumor immune microenvironment in mice, where A is the effect of 20 mg / kg single-dose group and 40 mg / kg single-dose group on tumor growth rate, and B is the CD45 in the tumor of mice in the control group and 40 mg / kg single-dose group. + Changes in the number of cells, C is the CD8 + The number of T cells in the control group and the 40 mg / kg single-dose group is CD4 +The changes in the number of T cells, E is the CD8 + Changes in the number of T cells;

[0027] Figure 4 The figure shows the effects of AQP9 inhibitor combined with anti-PD-1 antibody on the growth and immune microenvironment of mouse liver cancer, wherein A is a flowchart of mouse dosing, B is the effect of the control group, 40 mg / kg single-dose group, anti-PD-1 antibody group and combined treatment group on tumor growth rate, C is the statistical results of mouse tumor weight at the end of treatment in the control group, 40 mg / kg single-dose group, anti-PD-1 antibody group and combined treatment group, D is the results of photographic records of tumors in the control group, 40 mg / kg single-dose group, anti-PD-1 antibody group and combined treatment group at the end of treatment, E is the statistical results of the number of cells in the draining lymph nodes (dLN) of the mice in the control group, 40 mg / kg single-dose group, anti-PD-1 antibody group and combined treatment group at the end of treatment, and F is the results of photographic records of the draining lymph nodes of the mice in the control group, 40 mg / kg single-dose group, anti-PD-1 antibody group and combined treatment group at the end of treatment. DETAILED DESCRIPTION

[0028] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0029] It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0030] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0031] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0032] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0033] The room temperature in the embodiments of the present invention refers to 25±2°C.

[0034] All raw materials used in the examples of the present invention are commercially available.

[0035] Single cell sequencing data analysis of 21 liver cancer samples used in the examples of the present invention:

[0036] Figure 1 Zhong A analyzed the single-cell sequencing data of 21 liver cancer patient samples and used clustering analysis and cell type recognition algorithms to perform cell classification and subtype recognition on the single-cell expression matrix. Figure 1 In B, cells are classified into the following types as shown in the figure: malignant cells, T cells, B cells, tumor-associated macrophages (TAM, tumor-associated macrophage), etc. Figure 1 Center C analyzes the CD4+T infiltration level in malignant cells. The blue area represents low CD4+T infiltration, and the red area represents high CD4+T infiltration. Figure 1 D shows the level of CD8+T infiltration in malignant cells. The blue area represents low CD8+T infiltration, and the red area represents high CD8+T infiltration. Figure 1 Middle E analyzes the expression level of AQP9 in malignant cells, and the red area represents high AQP9 expression level.

[0037] The technical solution of the present invention is further illustrated by the following embodiments.

[0038] Example 1

[0039] like Figure 2 As shown, according to the structural characteristics of AQP9 protein, the present invention uses benzothiadiazole amine and p-fluorophenyl isocyanate to undergo a condensation reaction to obtain an AQP9 inhibitor drug molecule containing a urea structure, namely 1-(benzo[d][1,2,3]thiadiazole-6-yl)-3-(4-fluorophenyl)urea, and the specific method is as follows:

[0040] In a 100 ml dry round-bottom flask, add benzo[d][1,2,3]thiadiazole-6-amine (500 mg, 3.3 mmol) and anhydrous dichloromethane (15 mL). At room temperature and under nitrogen protection, add p-fluorophenyl isocyanate (0.41 mL, 3.6 mmol) dropwise. Continue stirring at room temperature for 24 hours to obtain a suspension. The liquid was concentrated, and the solid was washed 8 to 10 times with a mixed solution of petroleum ether: ethyl acetate = 2: 1 (volume ratio). After stirring and standing for a while during each washing, the supernatant was removed with a syringe. The final solid was a crude product with fewer impurities. The crude product was then dissolved with tetrahydrofuran, mixed with silica gel and concentrated with a rotary evaporator. The dry sample was loaded for column chromatography, and petroleum ether: tetrahydrofuran = 4: 1 (volume ratio) was selected as the eluent to obtain 579 mg of a white solid. The H NMR spectrum confirmed that it was the product 1-(benzo[d][1,2,3]thiadiazol-6-yl)-3-(4-fluorophenyl)urea (AQP9 inhibitor), with a yield of 61% and a purity of more than 98%. 1 H NMR (600 MHz, DMSO-d6) δ9.32 (s, 1H), 8.91 (s, 1H), 8.59-8.55 (m, 2H), 7.68 (dd, J = 9.0, 1.2 Hz, 1H), 7.53-7.47 (m, 2H), 7.15 (t, J = 9.0 Hz, 2H). The specific spectrum is shown in Figure 2 As shown in B.

[0041] In the following test method, the test substance is the AQP9 inhibitor synthesized in Example 1, ie, 1-(benzo[d][1,2,3]thiadiazol-6-yl)-3-(4-fluorophenyl)urea.

[0042] 1. Anti-tumor activity detection of AQP9 inhibitors

[0043] Experimental cells: mouse liver cancer cells Hepa1-6, mice are clean grade C57BL / 6J mice.

[0044] Test methods

[0045] Male C57BL / 6J mice weighing 18-22 g were subcutaneously injected with Hepa1-6 on Day 0 to establish a mouse liver cancer model. The average size of the mouse tumor was 200 mm on Day 6. 3Around 100 μg / kg, the mice were divided into 3 groups according to the size of the tumor: control group (Ctrl), 20 mg / kg single-dose group and 40 mg / kg single-dose group; starting from Day 6, according to the grouping, the 20 mg / kg single-dose group was intraperitoneally injected with 20 mg / kg of the inhibitor, and the frequency of administration was once a day. The 40 mg / kg single-dose group was intraperitoneally injected with 40 mg / kg of the inhibitor, and the frequency of administration was once a day. Each mouse in the control group was intraperitoneally injected with the same volume of solvent as a control.

[0046] The solution configuration is as follows:

[0047] Inhibitor stock solution: 40 mg of drug powder was dissolved in 250 μL DMSO (dimethyl sulfoxide) to a concentration of 160 mg / mL.

[0048] Control group: 10 μL DMSO plus 100 μL PEG300 + 90 μL sterile water.

[0049] 20 mg / kg single-dose group: 5 μL inhibitor stock solution plus 100 μL PEG300 + 95 μL sterile water.

[0050] 40 mg / kg single-dose group: 10 μL inhibitor stock solution plus 100 μL PEG300 + 90 μL sterile water.

[0051] After drug treatment, the growth status of the tumor was detected every day, and the status of the mice receiving drug treatment was observed. During the entire experiment, the tumor volume of the mice was measured every day for statistical analysis, such as Figure 3 As shown in A, the tumor growth rate of mice treated with 20 mg / kg or 40 mg / kg of the drug was significantly slowed down, proving that this inhibitor can significantly inhibit tumor growth.

[0052] 2. Flow cytometric analysis of the effects of AQP9 inhibitors on the immune microenvironment in tumors

[0053] On the 10th day, all mice in the above 1. Antitumor activity test of drugs were killed by cervical dislocation, sprayed with alcohol, and then the subcutaneous tumors and draining lymph nodes were isolated.

[0054] The mouse tumor sample was cut into pieces with sterile scissors, and digested with a digestion solution supplemented with Collegase IV (purchased from Worthington, LS004188-1gm) and DNase I (purchased from Roche, 11284932001). The sample was placed in a 3D shaker at 37°C and rotated 360° for 45 minutes. The single-cell suspension was prepared by filtering through a 70μm filter. The single-cell suspension was transferred to a new centrifuge tube, and PBS solution was added. After centrifugation, the supernatant was discarded, and the sample was washed with PBS and centrifuged again. Resuspended in PBS, 100μL was taken into a flow tube, and the corresponding flow antibody was added for incubation and staining on ice for 30 minutes. The sample was washed with PBS and centrifuged again. Resuspended in 200μL PBS solution for flow cytometry analysis. Figure 3 As shown in BD, the number of immune cells in the tumors of the control group and the 40 mg / kg single-dose group was counted. Figure 3 Middle B shows that the number of CD45+T cells in the tumors of mice treated with 40 mg / kg of the drug was significantly increased. Figure 3 Middle C shows that the number of CD8+ T cells in the tumors of mice treated with 40 mg / kg of the drug was significantly increased. Figure 3 Medium D and Figure 3 Middle E shows that the number of CD4+T and CD8+T cells in the draining lymph nodes of mice treated with 40 mg / kg of the drug was significantly increased.

[0055] 3. Effects of AQP9 inhibitors (i.e., 1-(benzo[d][1,2,3]thiadiazol-6-yl)-3-(4-fluorophenyl)urea) combined with PD1 blockers on tumor proliferation

[0056] Male C57BL / 6J mice weighing 18-22 g were used to establish the Hepa1-6 subcutaneous liver cancer model in C57BL / 6J mice from Day 0. The average tumor size of the mice was 200 mm on Day 7. 3 They were divided into 4 groups according to the size of the tumor: control group (Ctrl), 40 mg / kg single-dose group (40 mg / kg), anti-PD-1 antibody group (Anti-PD1), and combined treatment group (Combo).

[0057] in:

[0058] 40 mg / kg single-dose group: Each mouse was intraperitoneally injected with 40 mg / kg of the drug, and the dosing frequency was once a day.

[0059] Anti-PD-1 antibody group: Each mouse was intraperitoneally injected with a single dose of 0.5 mg / kg PD-1 blocker, and the frequency of administration was once every two days.

[0060] Combined treatment group: Each mouse was intraperitoneally injected with 40 mg / kg of the drug, and the dosing frequency was once a day; a single dose of 0.5 mg / kg of PD-1 blocker was intraperitoneally injected, and the dosing frequency was once every two days.

[0061] Control group: Each mouse was intraperitoneally injected with PBS (200 μL / mouse / time) as a control, and the administration frequency was once a day.

[0062] After drug treatment, the growth status of the tumor was detected every day, and the living status of the mice receiving drug treatment was observed. After the start of drug administration, the tumor volume of the mice was measured every day.

[0063] like Figure 4 As shown in Figures B, C, and D, the tumors in the anti-PD-1 antibody group and the 40 mg / kg single-dose group were significantly suppressed compared to the control group, proving that both the PD-1 blocker and the 40 mg / kg single-dose alone can inhibit tumor proliferation. However, the tumor inhibition effect of the combined treatment group was more obvious than that of the two single-dose groups, proving that combined treatment can have a stronger ability to inhibit tumor proliferation. Figure 4 In E and F, the number and volume of cells in the draining lymph nodes of mice in the combined treatment group increased significantly, proving that the AQP9 inhibitor can mobilize the peripheral immune system, enhance the anti-tumor immunity level of mice, and thus inhibit tumor proliferation.

[0064] The above results prove that:

[0065] The 1-(benzo[d][1,2,3]thiadiazol-6-yl)-3-(4-fluorophenyl)urea synthesized for the first time in the present invention can be used as an AQP9 inhibitor, can be used in screening or preparing drugs for treating solid tumors, can significantly increase the number of immune cells in the tumor microenvironment, and can affect the peripheral immune system.

[0066] The 1-(benzo[d][1,2,3]thiadiazol-6-yl)-3-(4-fluorophenyl)urea synthesized for the first time in the present invention can be used as an AQP9 inhibitor and can be used in screening or preparing drugs for treating liver cancer.

[0067] The 1-(benzo[d][1,2,3]thiadiazol-6-yl)-3-(4-fluorophenyl)urea synthesized for the first time in the present invention can be used as an AQP9 inhibitor, and can be used in combination with a PD-1 blocker for screening or preparing drugs for treating liver cancer. The 1-(benzo[d][1,2,3]thiadiazol-6-yl)-3-(4-fluorophenyl)urea can significantly enhance the therapeutic efficacy of the PD-1 blocker and has obvious anti-tumor efficacy.

[0068] The above are only preferred specific implementations of the present application, but the protection scope of the present application is not limited thereto. Any changes or substitutions that can be easily thought of by a person skilled in the art within the technical scope disclosed in the present application should be included in the protection scope of the present application. Therefore, the protection scope of the present application should be based on the protection scope of the claims.

Claims

1. Use of an AQP9 inhibitor in the preparation of a drug for treating HCC liver cancer, characterized in that: The number of CD8+T cells increased significantly, and the AQP9 inhibitor was 1-(benzo[d][1,2,3]thiadiazol-6-yl)-3-(4-fluorophenyl)urea; The preparation method of the AQP9 inhibitor comprises the following steps: Mix benzo[d][1,2,3]thiadiazole-6-amine and anhydrous dichloromethane, add p-fluorophenyl isocyanate dropwise under nitrogen protection, stir to obtain a suspension, concentrate, wash the solid to obtain a crude product, dissolve the crude product in tetrahydrofuran, evaporate and concentrate, and perform column chromatography to obtain a solid which is the AQP9 inhibitor; The molar ratio of the benzo[d][1,2,3]thiadiazole-6-amine to p-fluorophenyl isocyanate is 11:12.

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