Application of antitumor compound C11

Abstract

The invention belongs to the field of medicines, and relates to application of an anti-tumor compound C11. Specifically, the invention relates to application of a compound C11 and a crystal form, a pharmaceutically acceptable salt, an ester, a hydrate, a solvate or a prodrug thereof in preparation of an NME4 protein inhibitor, and the structural formula of the compound C11 is shown as a formula I. In-vitro experiment results show that C11 has a good inhibition effect on a plurality of colorectal cancer cell lines, and the action target of C11 is NME4; animal experiments show that C11 can effectively inhibit growth of colorectal cancer of nude mice. The results show that the compound C11 can be used as an NME4 inhibitor, can be used as a drug for NME4 high-expression tumors, and especially can be used as a drug for clinical treatment of colorectal cancer. The invention provides a new therapeutic drug for colorectal cancer, also provides a new way for overcoming the drug resistance of the colorectal cancer, and also provides a new therapeutic strategy for clinical treatment of other types of tumors. # imgabs0 #

Description

Technical Field

[0001] The present invention belongs to the field of medicine, and in particular, relates to a new use of an anti-tumor compound C11. Background Art

[0002] Colorectal cancer is a digestive tract tumor that occurs in people of all ages. According to global cancer research reports, the incidence of colorectal cancer ranks third in the world, and the mortality rate ranks second. In my country's cancer research report, the incidence of colorectal cancer in my country ranks second among all tumors, and the mortality rate ranks fourth. Colorectal cancer patients often show clinical symptoms such as gastrointestinal bleeding and obstruction, and terminal patients often die of multiple organ metastasis and failure. Unfortunately, there is currently no means to completely cure colorectal cancer, so limiting the growth of colorectal cancer is still the focus of colorectal cancer drug development.

[0003] Nucleoside diphosphate kinase 4 (NME4) is located on the inner membrane of mitochondria and is mainly involved in the mutual conversion of ATP / ADP and GTP / GDP through the transfer of high-energy phosphate groups. Previous research results suggest that NME4 is highly associated with cystic fibrosis and Barth syndrome. However, current studies have suggested that NME4 is elevated in gastric cancer and colorectal cancer, but there has been no relevant research on its relationship with the pathogenesis of colorectal cancer. Summary of the invention

[0004] The purpose of the present invention is to provide a new use of compound C11.

[0005] Compound C11 (CAS No: 31980-12-8, also referred to herein as C11) is recorded in the pubchem compound library, with the chemical structural formula C9H 10 N2O2; Molecular weight: English name 2,2-Dimethyl-2H-benzimidazole 1,3-dioxide. The chemical structure is shown in Figure I:

[0006]

[0007] Compound C11 was first synthesized by VA Samsonov and LB Volodarskii of the Novosibirsk Organic Chemistry Society. The synthesis method is to react cyclohexanedione and cycloheptanedione 1,2-dioxime with acetone, cyclopentanone, and methyl ethyl ketone to obtain the corresponding 2H-imidazole 1,3-dioxide. There is no record of the use of compound C11 as a drug.

[0008] Through computer virtual screening, the applicant discovered that compound C11 binds to NME4 and can enter the active pocket, which led to the completion of the present invention.

[0009] In order to achieve the above objectives, the first aspect of the present invention provides the use of compound C11 and its crystal form, pharmaceutically acceptable salt, ester, hydrate, solvate or prodrug in the preparation of NME4 protein inhibitors.

[0010] The second aspect of the present invention provides the use of antitumor compound C11 and its crystal form, pharmaceutically acceptable salt, ester, hydrate, solvate or prodrug in the preparation of NME4 high-expressing tumor cell proliferation inhibitor. The compound C11 targets NME4 protein and inhibits the activity of NME4.

[0011] According to a preferred embodiment of the present invention, the NME4-high expressing tumor cells are colorectal cancer cells.

[0012] The examples of the present invention demonstrate that compound C11 has killing activity against multiple colorectal cancer cell lines, including but not limited to HCT116, SW620, DLD-1, MC38, CT26 or HROC374 cell lines.

[0013] The third aspect of the present invention provides the use of anti-tumor compound C11 and its crystal form, pharmaceutically acceptable salt, ester, hydrate, solvate or prodrug in the preparation of a drug or pharmaceutical composition for preventing and / or treating NME4-overexpressing tumors. Compound 11 inhibits tumor cell proliferation by targeting the NME4 gene or protein, thereby achieving the purpose of preventing and treating tumors.

[0014] According to a preferred embodiment of the present invention, the NME4-high expressing tumor is colorectal cancer.

[0015] The anti-tumor compound C11 and its crystal form, pharmaceutically acceptable salt, ester, hydrate, solvate or prodrug are used as active ingredients in the drug and as one of the active ingredients in the pharmaceutical composition, and the pharmaceutical composition also contains at least one other drug. The other drug is a drug with a synergistic anti-tumor effect, such as a drug for preventing and / or treating colorectal cancer.

[0016] In the present invention, the term "prodrug" refers to an agent that is converted into a prototype drug in vivo. Prodrugs are usually drug precursors that are converted into active substances through administration and absorption, or are changed into more active species through some processes, such as conversion through metabolic pathways. Some prodrugs have chemical groups that make them less active and / or have altered solubility or some other properties compared to the prototype drug. Once the chemical groups of the prodrug are removed and / or modified, the active drug is obtained.

[0017] In the present invention, the drug and pharmaceutical composition may be in solid or liquid form, and the dosage form may be tablets, dispersible tablets, lozenges, orodisintegrating tablets, sustained-release agents, capsules, soft capsules, dripping pills, granules, injections, powder injections or aerosols, etc. When the compound of the present invention is used for the above purposes, it may be mixed with one or more pharmaceutically acceptable carriers or excipients, such as solvents, sustained-release agents, etc., and may be administered orally in the following forms: tablets, pills, capsules, dispersible powders, granules or suspensions (containing, for example, 0.05-5% suspending agents), syrups (containing, for example, about 10-50% sugar), and elixirs (containing 20-50% ethanol), or administered externally in the form of ointments, gels, medicated adhesive plasters, etc., or parenterally in the form of sterile injectable solutions or suspensions (containing about 0.05-5% suspending agents in isotonic media). Suitable routes of administration include, but are not limited to, oral, intravenous, rectal, aerosol, parenteral, ocular, pulmonary, transdermal, vaginal, otic, nasal, and topical.

[0018] The technical effects of the present invention are:

[0019] The present invention provides a new anti-tumor use of compound C11 based on the inhibitory activity of NME4 of compound C11. The results of in vitro experiments show that C11 has a good inhibitory effect on multiple colorectal cancer cell lines, and its target is NME4; animal experiments show that C11 can effectively inhibit the growth of colorectal cancer in nude mice. The above results show that compound C11 can be used as an inhibitor of NME4, as a drug for tumors with high NME4 expression, and especially as a drug for the clinical treatment of colorectal cancer. The present invention provides a new therapeutic drug for colorectal cancer, a new way to overcome the drug resistance of the disease, and a new therapeutic strategy for the treatment of other types of clinical tumors.

[0020] Other features and advantages of the present invention will be described in detail in the following detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] The above and other objects, features and advantages of the present invention will become more apparent through a more detailed description of exemplary embodiments of the present invention with reference to the accompanying drawings.

[0022] Figure 1-4 These are the inhibition curves of compound C11 on colorectal cancer cells HCT116, SW620, DLD-1, and MC38, respectively.

[0023] Figure 5-9 These are the animal experimental results of compound C11 inhibiting colorectal cancer.

[0024] Fig.10 This is the binding result of compound C11 to the NME4 drug target. DETAILED DESCRIPTION

[0025] The preferred embodiments of the present invention will be described in more detail below. Although the preferred embodiments of the present invention are described below, it should be understood that the present invention can be implemented in various forms and should not be limited to the embodiments set forth herein.

[0026] If the specific conditions are not specified in the examples, all the experiments were carried out under conventional conditions or the conditions recommended by the manufacturer. If the manufacturers of the reagents or instruments are not specified, all of them are conventional products that can be purchased from the market.

[0027] Example 1 Cytotoxicity of Compound C11 against Colorectal Cancer Cells

[0028] 1. Experimental materials:

[0029] (1) Cell lines: Human or mouse colorectal cancer cells (HCT116 cells, DLD-1 cells, SW620 cells, MC38 cells) were cultured in the corresponding culture medium containing 10% fetal bovine serum.

[0030] (2) Main reagents: RPMI 1640 medium (Gbico, USA), DMEM medium (Gbico, USA), C11 compound, Cell counting kit 8 (CCK8, Yisheng Company, China).

[0031] (3) Main instruments: CO2 incubator (Thermo Forma, USA) and automatic microplate reader (Tecan, Switzerland).

[0032] 2. Experimental methods:

[0033] HCT116, DLD-1, SW620, and MC38 cell lines are adherent culture cells and are routinely cultured in the corresponding culture medium containing 10% fetal bovine serum at 37°C and 5% CO2. HCT116 and DLD-1 cell lines in the logarithmic growth phase were inoculated in sterile 96-well plates at a density of 1×10 3 / well, culture system 99μL, add 1μL to each well, so that the final drug concentration is 1, 5, 10, 25, 50μM. Set up 6 replicates for each sample, and set up positive control and blank control. After 72 hours of action, add CCK8 solution, 10μM / well, act at 37℃ for 1 hour, measure the OD value at 450nm with an enzyme reader, and calculate the cell proliferation inhibition rate (IR%) according to the following formula: [(control OD-blank OD)-(sample OD-blank OD)] / (control-blank)×100%

[0034] 3. Experimental results

[0035] Figure 1-4The killing activity of C11 compound on HCT116, SW620, DLD-1, and MC38 cell lines is shown, and the IC50 is 21.43 μM, 2.201 μM, 126.8 μM, and 5.679 μM, respectively. It can be seen that C11 compound has obvious killing activity on multiple colorectal cancer cell lines of humans and mice.

[0036] Example 2: Animal experiment on colorectal cancer

[0037] 1. Experimental materials:

[0038] (1) Cell lines: Mouse colon cancer cell line CT26 and human primary colorectal cancer cell line HROC374 were cultured in RPMI 1640 medium and DMEM / F12 medium (Gbico, USA) containing 10% fetal bovine serum, respectively.

[0039] (2) Experimental animals: Female BALB / C mice (inoculated with CT26 cells) and female BALB / C nude mice (inoculated with HROC374 cells) were housed in an SPF environment.

[0040] 2. Experimental methods:

[0041] (1) Cell culture, same as in Example 1.

[0042] (2) Containing 3×10 5 CT26 cells and 2×10 6 Phosphate buffered saline (PBS) of HROC374 cells was injected into the subcutaneous left armpit of BALB / C mice and BALB / C nude mice, and the mice were randomly divided into control group and treatment group when the tumor grew and could be measured. The mice in the treatment group were intraperitoneally injected with 25 mg / kg and 50 mg / kg of C11 compound every day, and the mice in the control group were injected with the same volume of solvent (2% DMSO + 40% PEG + 58% sterile water). The tumor size was measured every two days (the length and width of the tumor were measured, and the volume = 1 / 2 x length x width). 2 ). Wait until the first mouse reaches the ethical limit (2500m 3 ) and the mice were killed and photographed.

[0043] 3. Experimental results

[0044] The results of inoculation of CT26 cells are shown in Figure 5-7 shown. Figure 5 The tumor photos of the mice in the treatment group inoculated with CT26 cells and the mice in the control group are shown. It can be seen that compared with the mice in the control group, the mice in the two treatment groups showed a significant reduction in tumor volume. Figure 6 The curve of tumor volume changing with time also confirms that the tumor volume of mice in the drug-treated group is significantly different from that in the control group. Figure 7The tumor weight of each group is shown. It can be seen that the tumor weight of the control group mice is significantly higher than that of the drug-treated group, and there is no significant difference between the two drug-treated groups.

[0045] The results of inoculating HROC374 cells are as follows Figure 8-9 shown. Figure 8 The tumor photos of the mice in the drug-treated group and the control group inoculated with HROC374 cells are shown. It can be seen that the tumor volume of the mice in the drug-treated group was significantly reduced compared with the control group. Fig. 9 The curve of tumor volume changing with time also confirms that the tumor volume of mice in the drug-treated group is significantly different from that in the control group.

[0046] The above animal experiments show that C11 compound can effectively inhibit the growth of colorectal cancer in nude mice.

[0047] Example 3: Binding experiment of C11 compound to target NME4

[0048] 1. Experimental materials:

[0049] (1) Cell line: human colon cancer cell line HCT116.

[0050] (2) Main reagents: 5X western loading buffer, phosphate buffered saline (PBS), mouse anti-NME4 monoclonal antibody (TA50110, Aorui Dongyuan, China), mouse anti-ACTB monoclonal antibody (TA811000, Aorui Dongyuan, China), horseradish peroxidase-labeled goat anti-mouse IgG (H+L) (A0216, Beyotime, China), and protease inhibitor (P0016, Beyotime, China).

[0051] (3) Main instruments: metal bath heater, liquid nitrogen, 37°C constant temperature water bath, electrophoresis apparatus, electric shaker, exposure apparatus (Tanon, China)

[0052] 2. Experimental method: C11 compound (final concentration 25 μM) was added to the HCT116 cell culture dish for 6 hours, and an equal amount of DMSO solvent was used as a control. After washing with PBS, 1 mL of PBS containing protease inhibitors was added again, and the cells were collected with a cell scraper and divided into 6 portions, each of which was 100 μL. The temperature gradient was set to 42°C, 48°C, 55°C, 62°C, 67°C, and 75°C. Each sample was placed in a metal bath of the corresponding temperature, heated for 3 minutes, and then the cells were lysed by circulating liquid nitrogen at -37°C for 3 times. The sample was centrifuged at 20,000g and 4°C for 10 minutes, and the supernatant was collected. The supernatant sample was added with 1 / 4 sample volume of loading buffer and mixed well, and then heated in a 95°C metal bath for 10 minutes. The sample was then subjected to electrophoresis-transfer-blocking-antibody incubation according to the standard western blot process, and the band was finally exposed and preserved.

[0053] 3. Experimental results

[0054] The experimental results are as follows Fig.10 shown. Fig.10 The left picture is a WB electrophoresis diagram, and the right picture is a curve chart made based on the band darkness and temperature of the DMSO-NME4 group and C11-NME4 in the WB electrophoresis diagram.

[0055] Depend on Fig.10 It can be seen that the protein content of NME4 in the C11 compound group is significantly higher than that in the solvent DMSO group under the same temperature conditions, indicating that the C11 compound binds to the NME4 protein and maintains its stability, making it not easily degraded by high temperature.

[0056] The embodiments of the present invention have been described above, and the above description is exemplary, not exhaustive, and is not limited to the disclosed embodiments. Many modifications and changes will be apparent to those skilled in the art without departing from the scope and spirit of the described embodiments.

Claims

1. Use of compound C11 and its crystal form, pharmaceutically acceptable salt, ester, hydrate, solvate or prodrug in the preparation of NME4 protein inhibitors, wherein the structural formula of compound C11 is shown in formula I:

2. Use of the antitumor compound C11 and its crystal forms, pharmaceutically acceptable salts, esters, hydrates, solvates or prodrugs in the preparation of NME4 overexpressing tumor cell proliferation inhibitors.

3. The use according to claim 2, wherein: The NME4 high-expressing tumor cells are colorectal cancer cells.

4. The use according to claim 3, wherein: The colorectal cancer cells are HCT116, SW620, DLD-1, MC38, CT26 or HROC374 cell lines.

5. Use of the anti-tumor compound C11 and its crystal forms, pharmaceutically acceptable salts, esters, hydrates, solvates or prodrugs in the preparation of drugs or pharmaceutical compositions for preventing and / or treating NME4-overexpressing tumors.

6. The use according to claim 5, wherein: The NME4 high-expressing tumor is colorectal cancer.

7. The use according to claim 5, wherein: The medicament or pharmaceutical composition comprises one or more pharmaceutically acceptable carriers or excipients.

8. The use according to claim 5, wherein: The active component of the drug is the anti-tumor compound C11 as claimed in claim 1 and its crystal form, pharmaceutically acceptable salt, ester, hydrate, solvate or prodrug; The active components of the pharmaceutical composition include the anti-tumor compound C11 and its crystal form, pharmaceutically acceptable salt, ester, hydrate, solvate or prodrug, and at least one other drug.

9. The use according to claim 5, wherein: The other drugs are drugs for preventing and / or treating colorectal cancer.

10. The use according to claim 5, wherein: The dosage form of the medicine or pharmaceutical composition is tablet, dispersible tablet, lozenge, orodisintegrating tablet, sustained-release agent, capsule, soft capsule, dropping pill, granule, injection, powder injection or aerosol.

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