Use of MAPK12 inhibitor and derivative thereof in preparation of Anti-tumor drug

Abstract

The present invention provides use of an MAPK12 inhibitor and a derivative thereof in the preparation of an anti-tumor drug, and particularly relates to the technical field of medicines. The MAPK12 inhibitor (i.e., MIM2) can effectively inhibit the in vitro proliferation ability of colorectal cancer, gastric cancer, pancreatic cancer, and breast cancer cells. MIM2 can effectively inhibit the in vivo proliferation ability of colorectal cancer in NSG mice, and is expected to be used as an anti-tumor small molecule drug in the treatment of colorectal cancer, gastric cancer, pancreatic cancer, breast cancer, and other tumors.

Description

Application of a MAPK12 inhibitor and its derivatives in the preparation of anti-tumor drugs Technical Field

[0001] The present application relates to the field of medical technology, and in particular to the use of a MAPK12 inhibitor and its derivatives in the preparation of anti-tumor drugs. Background Art

[0002] Colorectal, gastric, and pancreatic cancers are among the most lethal gastrointestinal malignancies, with breast cancer being the top three malignant solid tumors in terms of both incidence and mortality. Early symptoms of these cancers are often subtle; for example, most colorectal and gastric cancer patients have already metastasized and advanced at the time of initial diagnosis, presenting significant challenges for clinical diagnosis and treatment. Currently, the primary treatment strategy for these solid tumors is still radical surgery, supplemented by radiotherapy and chemotherapy. However, due to the complexities of malignant solid tumors, such as rapid progression, difficulty in detection, and incomplete surgical resection, these tumors contribute to their spread and metastasis. Chemotherapy is often used as the primary treatment modality. Chemotherapeutic agents include a wide range of drugs, such as alkylating agents, antimetabolites, and antitumor antibiotics. Because chemotherapeutic drugs are cytotoxic, they can kill both tumor cells and normal cells, resulting in toxic side effects that limit their use. Therefore, identifying effective and safe treatments is crucial for prolonging survival and improving quality of life for cancer patients.

[0003] Numerous studies have demonstrated that protein kinases play a key role in the development and progression of human tumors, representing the largest group of genes identified in cancer gene surveys. Kinase- and phosphatase-mediated reversible protein phosphorylation plays a crucial role in regulating cellular functions such as cell proliferation, apoptosis, subcellular translocation, inflammation, and metabolism. Kinase modulators have become a major focus in cancer treatment. Since the approval of imatinib in 2001, over 70 kinase inhibitors have been approved. However, the current development of kinase inhibitors remains challenging. Kinase inhibitors can slow tumor progression but cannot cure cancer, as advanced tumors develop evasion pathways to escape target inhibition, leading to drug resistance. This represents another pressing challenge facing kinase inhibitors, in addition to selectivity. Given the crucial role of kinases in cancer treatment, they warrant the rational design of novel kinase inhibitors as anti-cancer drug targets, particularly for colorectal, gastric, pancreatic, and breast cancers, with the goal of identifying small molecule compounds with improved safety and efficacy. Summary of the Invention

[0004] The purpose of this application is to overcome the deficiencies of the above-mentioned prior art and to provide a use of a MAPK12 inhibitor and its derivatives in the preparation of anti-tumor drugs.

[0005] To achieve the above objectives, the technical solutions adopted in this application are:

[0006] The present application provides a use of a MAPK12 inhibitor and its derivatives in the preparation of anti-tumor drugs. The molecular formula of the MAPK12 inhibitor is C 42 H 50 N2O6, the structural formula is shown in formula (I);

[0007] Formula (I).

[0008] The molecular formula of the MAPK12 inhibitor of the present application is C 42 H 50 N2O6, with a structural formula as shown in formula (I), is a small molecule derived from MolPort, with a compound number of Molport-044-810-679.

[0009] The small molecule compound of the present application was screened from the ZINC15 chemical database and is named ZINC000824519333. The functions of this small molecule in vivo and in vitro are currently unknown and it is referred to as "MIM2" in this specification.

[0010] Through experiments, this application shows that MIM2 can effectively inhibit the proliferation of tumors in vitro and in vivo, and is expected to be used as an anti-tumor small molecule drug in the treatment of tumors (including at least one of colorectal cancer, gastric cancer, pancreatic cancer, and breast cancer).

[0011] As a preferred embodiment of the application described in this application, the mass concentration of the MAPK12 inhibitor is ≥1 μM.

[0012] The MAPK12 inhibitors at the concentrations of this application showed a dose-dependent killing effect on colorectal cancer, gastric cancer, pancreatic cancer, and breast cancer cell lines. 50 Calculation of the values ​​revealed that MIM2 had a weak killing effect on normal cells, indicating that MIM2 has a high specificity and a significant killing effect on tumor cells. Furthermore, over time, colorectal cancer, gastric cancer, pancreatic cancer, and breast cancer cell lines gradually died under MIM2 treatment, while cells in the control group continued to grow normally.

[0013] As a preferred embodiment of the application described in this application, the mass concentration of the MAPK12 inhibitor is 1~200 μM.

[0014] As a preferred embodiment of the application described in the present application, the derivative of the MAPK12 inhibitor includes at least one of a pharmaceutically acceptable salt of a MAPK12 inhibitor, a pharmaceutically acceptable ester of a MAPK12 inhibitor, a pharmaceutically acceptable ether of a MAPK12 inhibitor, a pharmaceutically acceptable amide of a MAPK12 inhibitor, and a pharmaceutically acceptable glycosylation of a MAPK12 inhibitor.

[0015] The derivatives of the MAPK12 inhibitors of the present application are MIM2 derivatives obtained by in vivo hydrolysis and having the same or similar pharmaceutical activity as MIM2.

[0016] "Pharmaceutically acceptable salts" include acid addition salts and base addition salts.

[0017] "Pharmaceutically acceptable acid addition salts" refers to salts that retain the biological effectiveness and properties of the free bases, are not biologically or otherwise undesirable, and are formed with inorganic and organic acids such as, but not limited to, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like, and organic acids such as, but not limited to, acetic acid, adipic acid, alginic acid, ascorbic acid, aspartic acid, benzenesulfonic acid, benzoic acid, camphoric acid, camphor-10sulfonic acid, capric acid, hexanoic acid, caprylic acid, carbonic acid, cinnamic acid, citric acid, cyclamic acid, dodecylsulfuric acid, ethane 1,2-disulfonic acid, ethanesulfonic acid, 2-hydroxyethanesulfonic acid, fumaric acid, galactaric acid, gentisic acid, glucoheptanoic acid, gluconic acid, glucuronic acid, glutamic acid, glutaric acid, 2-oxoglutaric acid, glycerophosphoric acid, glycolic acid, hippuric acid, isobutyric acid, lactic acid, lactobionic acid, lauric acid, maleic acid, malic acid, malonic acid, mandelic acid, methanesulfonic acid, and the like.

[0018] "Pharmaceutically acceptable base addition salts" refers to salts that retain the biological effectiveness and properties of the free acids and are not biologically or otherwise undesirable. These salts are prepared by the addition of inorganic or organic bases to the free acids. Salts derived from inorganic bases include, but are not limited to, sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum, and the like. Preferred inorganic salts are ammonium, sodium, potassium, calcium, and magnesium salts. Salts derived from organic bases include, but are not limited to, salts of primary, secondary, and tertiary amines, substituted amines (including naturally occurring substituted amines), cyclic amines, and basic ion exchange resins, such as ammonia, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, diethanolamine, ethanolamine, dicyclohexylamine, lysine, arginine, histidine, caffeine, ethylenediamine, glucamine, methylglucamine, theobromine, triethanolamine, tromethamine, purines, piperazine, piperidine, N-ethylpiperidine, polyamine resins, and the like. Particularly preferred organic bases are isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline, and caffeine.

[0019] As a preferred embodiment of the application described in this application, the tumor is cancer.

[0020] As a preferred embodiment of the application described in the present application, the cancer includes at least one of colorectal cancer, gastric cancer, pancreatic cancer, and breast cancer.

[0021] The MIM2 of the present application can effectively inhibit the in vitro proliferation ability of colorectal cancer, gastric cancer, pancreatic cancer, and breast cancer cells. In NSG mice, MIM2 can effectively inhibit the in vivo proliferation ability of colorectal cancer. It is expected to be used as an anti-tumor small molecule drug in the treatment of colorectal cancer, gastric cancer, pancreatic cancer, breast cancer and other tumors.

[0022] The colorectal cancer includes DLD1 cell line or HCT116 cell line; the gastric cancer includes NCI-N87 cell line or SNU668 cell line; the pancreatic cancer includes ASPC-1 cell line or HPAC cell line; the breast cancer includes SKBR3 cell line or MDA-MB-231 cell line.

[0023] The present application provides a pharmaceutical composition for treating tumors, which comprises the above-mentioned MAPK12 inhibitor and its derivatives.

[0024] The pharmaceutical composition of the present application can effectively treat tumors such as colorectal cancer, gastric cancer, pancreatic cancer, and breast cancer, and inhibit the proliferation of colorectal cancer, gastric cancer, pancreatic cancer, and breast cancer cells in vitro. MIM2 can effectively inhibit the in vivo proliferation ability of colorectal cancer in NSG mice.

[0025] As a preferred embodiment of the pharmaceutical composition described in the present application, the pharmaceutical composition further comprises excipients.

[0026] In addition, it should be noted that the products or drugs mentioned in this application may be combined with commonly acceptable carriers, diluents or excipients as needed, including but not limited to any adjuvants, carriers, excipients, glidants, sweeteners, diluents, preservatives, dyes / colorants, flavorings, surfactants, wetting agents, dispersants, suspending agents, stabilizers, isotonic agents, solvents or emulsifiers.

[0027] As a preferred embodiment of the pharmaceutical composition described in the present application, the pharmaceutical composition is prepared into an external preparation, and the external preparation includes a suppository or an enema.

[0028] The products or drugs mentioned in this application can also be prepared into small-dose injections, lyophilized powder injections, large-volume injections, ordinary tablets, ordinary capsules, soft capsules, pills, micropills, granules, sustained-release tablets, lozenges, orally rapidly disintegrating tablets, dispersible tablets, effervescent tablets, enteric-coated tablets, enteric-coated capsules, delayed-release tablets, timed / positioned-release tablets, sustained-release capsules, controlled-release capsules, capsules containing micropills or small tablets, pH-dependent capsules containing micropills or small tablets, granules, oral liquids, films, patches, gels, ointments and other dosage forms as needed.

[0029] In some embodiments, the term "treat" refers to an approach for obtaining beneficial or desired results (including, but not limited to, a therapeutic benefit and / or a prophylactic benefit) with respect to a disease, disorder, or medical condition. A therapeutic benefit is defined as the elimination or amelioration of the underlying disorder being treated. Additionally, a therapeutic benefit is achieved by the elimination or amelioration of one or more physiological symptoms associated with the underlying disorder, such that improvement is observed in the individual, notwithstanding the potential for the individual to still be suffering from the underlying disorder.

[0030] Compared with the prior art, the present invention has the following advantages:

[0031] The present application provides an application of a MAPK12 inhibitor and its derivatives in the preparation of anti-tumor drugs. The MAPK12 inhibitor (i.e., MIM2) of the present application can effectively inhibit the in vitro proliferation ability of colorectal cancer, gastric cancer, pancreatic cancer, and breast cancer cells. In NSG mice, MIM2 can effectively inhibit the in vivo proliferation ability of colorectal cancer. It is expected to be used as an anti-tumor small molecule drug in the treatment of tumors such as colorectal cancer, gastric cancer, pancreatic cancer, and breast cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 shows the inhibitory effects of different concentrations of MIM2 on different cells (Figure 1-A shows the inhibitory effects of different concentrations of MIM2 on colorectal cancer cell lines and normal intestinal epithelial cells; Figure 1-B shows the inhibitory effects of different concentrations of MIM2 on gastric cancer cell lines; Figure 1-C shows the inhibitory effects of different concentrations of MIM2 on pancreatic cancer cell lines; Figure 1-D shows the inhibitory effects of different concentrations of MIM2 on breast cancer cell lines)

[0033] FIG2 is a graph showing the inhibition of MIM2 on colorectal cancer cells, gastric cancer cells, pancreatic cancer cells, and breast cancer cells under time gradient;

[0034] FIG3 shows the effect of MIM2 under concentration gradient on the colony-forming ability of colorectal cancer cells;

[0035] FIG4 shows the effect of MIM2 under concentration gradient on the colony-forming ability of gastric cancer cells;

[0036] FIG5 shows the effect of MIM2 under concentration gradient on the colony-forming ability of pancreatic cancer cells;

[0037] Figure 6 shows the effect of MIM2 concentration gradient on the colony formation ability of breast cancer cells;

[0038] Figure 7 shows the changes in tumor volume and mouse body weight after MIM2 treatment;

[0039] FIG8 shows the affinity detection of MIM2 and its target MAPK12. DETAILED DESCRIPTION

[0040] To better illustrate the purpose, technical solutions, and advantages of this application, the application will be further described below in conjunction with specific implementation examples and accompanying drawings, but the implementation examples do not limit this application in any form. Unless otherwise specified, the reagents, methods, and equipment used in this application are conventional reagents, methods, and equipment in this technical field. Unless otherwise specified, the reagents and materials used in this application are commercially available.

[0041] The colorectal cancer, gastric cancer, pancreatic cancer, and breast cancer cell lines mentioned in this application are derived from existing cell lines in our laboratory and can also be purchased commercially. The culture medium used to culture the cells was purchased from Gibco, and fetal bovine serum was purchased from ExCell Bio. The cell lines were cultured in RPMI-1640, DMEM, and McCoy's 5A medium containing 10% fetal bovine serum in an incubator at 37°C and 5% CO2. Cells in good growth condition were selected for subsequent experiments.

[0042] Example 1. Cell proliferation experiment - concentration gradient

[0043] MIM2 was dissolved in DMSO to a 10 mM stock solution. After the cells reached 80% confluence, they were digested with 0.05% trypsin, resuspended in complete RPMI-1640 medium, counted, and seeded into a 96-well plate at 7,000 cells per well. 100 μl of culture medium was used for incubation. After the cell monolayer adhered, MIM2 was added to final concentrations of 0.001, 0.01, 0.1, 1, 10, 100, and 200 μM. Blank wells and wells containing 100% cell culture medium were also set up as controls. Three replicates were set up for each drug concentration. The plates were returned to a 37°C, 5% CO2 incubator. After 24 hours of incubation, 10 μl of MTT solution was added, and the cells were incubated in the incubator for another 4 hours. The supernatant was aspirated, and 150 μl of DMSO was added. The cells were incubated in the incubator for 10 minutes. The absorbance (OD) was measured at 490 nm using an automated microplate reader. The OD value of each well was determined. The inhibition rate of MIM2 on cells was calculated using the following formula: inhibition rate % = [(absorbance of control well - absorbance of experimental well) / (absorbance of control well - absorbance of blank well)] × 100%, cell survival rate % = 1 - inhibition rate %.

[0044] The experimental results, shown in Figure 1 (Figure 1-A, Figure 1-B, Figure 1-C, Figure 1-D) and Table 1, show that MIM2 exhibits a dose-dependent cytotoxicity against colorectal, gastric, pancreatic, and breast cancer cell lines. Calculation of IC50 values ​​revealed that MIM2 exhibited a weak cytotoxicity against normal NCM460 cells, indicating that MIM2 has a high specificity and a significant cytotoxic effect against tumor cells. The IC50 values ​​for MIM2 against different cell types were calculated (Table 1).

[0045] Table 1 IC50 of MIM2 against different cell lines

[0046]

[0047] Example 2: Cell proliferation experiment - time gradient

[0048] MIM2 was dissolved in DMSO to a 10 mM stock solution. Colorectal, gastric, pancreatic, and breast cancer cell lines were grown to 80% confluence, digested with 0.05% trypsin, resuspended in complete RPMI-1640 medium, counted, and seeded into 96-well plates at 3,000 cells per well in 100 μl of culture medium. After the cell monolayers adhered, MIM2 was added at concentrations corresponding to the IC50 values ​​of the different cell lines, resulting in final concentrations of 1 μM, 5 μM, 10 μM, 30 μM, 50 μM, and 100 μM, respectively. Blank wells and wells containing 100% cell culture medium were also established as controls. Three replicates were performed for each drug concentration. The plates were returned to a 37°C, 5% CO2 incubator. After incubation for 0, 24, 48, 72, and 96 hours, 10 μl of MTT solution was added and incubated in the incubator for another 4 hours. The supernatant was aspirated, and 150 μl of DMSO was added. The cells were incubated in the incubator for 10 minutes. The absorbance (OD) was measured at 490 nm using an automatic microplate reader. The inhibition rate of MIM2 on cells was calculated based on the OD value of each well using the following formula: % inhibition rate = [(absorbance of control well - absorbance of experimental well) / (absorbance of control well - absorbance of blank well)] × 100%. % cell viability = 1 - % inhibition rate.

[0049] The experimental results are shown in Figure 2. Over time, colorectal cancer, gastric cancer, pancreatic cancer, and breast cancer cell lines gradually died under MIM2 treatment, while cells in the control group could grow normally.

[0050] Example 3: Plate colony formation experiment

[0051] MIM2 was dissolved in DMSO to a 10 mM stock solution. Colorectal, gastric, pancreatic, and breast cancer cell lines were grown to 80% confluence, digested with 0.05% trypsin, resuspended in complete RPMI-1640 medium, counted, and plated in 24-well plates at 300 cells per well in 1 ml of culture medium. MIM2 was added at corresponding concentrations based on the IC50 values ​​of the different cell lines, resulting in final concentrations of 1 μM, 5 μM, 10 μM, 30 μM, 50 μM, and 100 μM, respectively. Each concentration gradient was replicated in triplicate and cultured in a 37°C, 5% CO2 incubator for 14 days. Once colonies were visible, the 24-well plates were removed, the medium was discarded, and the cells were washed three times with PBS and dried at room temperature. Cells were fixed with methanol for 30 minutes, stained with Giemsa for 10 minutes, and rinsed slowly with running water. After complete drying, cell colonies were counted under a microscope (≥50 cells were considered a colony). Plate colony formation rate = number of colonies formed / number of seeded cells × 100%. The above experiment was repeated 3 times.

[0052] The experimental results, as shown in Figures 3-6, show that over time, the growth of colorectal, gastric, pancreatic, and breast cancer cell lines was significantly inhibited under MIM2 treatment, while cells in the control group continued to grow normally. Therefore, MIM2 can inhibit the colony-forming ability of tumor cells.

[0053] Example 4: Subcutaneous tumor inhibition experiment in NSG mice

[0054] Ten female NSG mice aged 3-4 weeks were selected and 1×10 6 HCT116 cells were added, and subcutaneous tumors were observed in some NSG mice after about 5 days. After all 10 NSG mice had tumors, the NSG mice were randomly divided into 2 groups, a control group and a medication group, with 5 NSG mice in each group. Since MIM2 is dissolved by DMSO, the control group was given an intraperitoneal injection of 10 mg / kg of DMSO per day according to the body weight of the NSG mice, and the medication group was given an intraperitoneal injection of 10 mg / kg of MIM2 per day according to the body weight of the NSG mice. Since the time sequence of tumor formation in the 10 NSG mice was different, this application starts drug administration when all 10 NSG mice have subcutaneous tumors, and starts when the subcutaneous tumor of any NSG mouse grows to 1000 mm. 3 The tumor size was measured with a vernier caliper every other day. The length (L) and width (W) of the tumor were measured and the volume (mm) was calculated according to the formula 3 )=LW 2Tumor volume was calculated using a 400-μg / 200 μg / μl ratio. NSG mice were weighed every other day for a total of five measurements. The tumor volumes of each group of NSG mice were averaged, with the first measurement serving as the control group and each measurement after intraperitoneal injection serving as the experimental group. Tumor growth rate was calculated using the formula (experimental group - control group) / control group × 100%, and growth rate curves were plotted.

[0055] The experimental results, shown in Figure 7, showed that after 12 days of treatment, the NSG mice's lifestyle remained unaffected, with normal diet and activity. Calculation of tumor volume in each group revealed that the tumor growth rate in the treated group was significantly lower than that in the control group, demonstrating that MIM2 inhibited subcutaneous tumor growth in NSG mice. Furthermore, measurement of the average body weight of NSG mice in each group revealed no significant change with increasing treatment duration, further demonstrating that MIM2 had no significant toxic side effects on NSG mice.

[0056] Example 5: Target affinity detection

[0057] A His-tagged MAPK12 fusion protein was expressed in prokaryotes and purified via affinity and molecular sieve purification. The binding affinity of MIM2 to MAPK12 was then determined using isothermal titration calorimetry (ITC). In the ITC experiment, 200 μM MIM2 was titrated into a MAPK12 protein solution at a constant temperature, and the heat of binding (ΔH) was recorded in real time. With multiple titrations, the heat released or absorbed by the molecules upon binding is directly proportional to the number of bound molecules. When the system reaches saturation, only the heat of dilution is observed. The heat generated by each titration is plotted against the molar ratio of the titrant to the titrant molecule to generate a binding curve.

[0058] The experimental results are shown in Figure 8. The dissociation coefficient between MIM2 and MAPK12 is 694×10 -9 M, which is 694 nM, and the dissociation coefficient error is 147×10 -9 M, that is, 147 nM. According to the ITC fitting curve, the binding constant (KD) value is at the nM level, indicating that MIM2 binds strongly to MAPK12.

[0059] MIM2 has a significant inhibitory effect on colorectal cancer, gastric cancer, pancreatic cancer, and breast cancer, and has certain tumor specificity. It can be used as an anti-tumor small molecule compound in the treatment of various tumors.

[0060] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present application and are not intended to limit the scope of protection of the present application. Although the present application has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present application may be modified or replaced by equivalents without departing from the essence and scope of the technical solutions of the present application.

Claims

1. Use of a MAPK12 inhibitor and its derivatives in the preparation of anti-tumor drugs, characterized in that: The molecular formula of the MAPK12 inhibitor is C 42 H 50 N2O6, the structural formula is shown in formula (I); Formula (I).

2. The use according to claim 1, characterized in that The mass concentration of the MAPK12 inhibitor is ≥1 μM.

3. The use according to claim 2, characterized in that The mass concentration of the MAPK12 inhibitor is 1-200 μM.

4. The use according to claim 1, wherein The derivative of the MAPK12 inhibitor includes at least one of a pharmaceutically acceptable salt of the MAPK12 inhibitor, a pharmaceutically acceptable ester of the MAPK12 inhibitor, a pharmaceutically acceptable ether of the MAPK12 inhibitor, a pharmaceutically acceptable amide of the MAPK12 inhibitor, and a pharmaceutically acceptable glycosylation of the MAPK12 inhibitor.

5. The use according to claim 1, characterized in that The tumor is cancer.

6. The use according to claim 5, characterized in that The cancer includes at least one of colorectal cancer, gastric cancer, pancreatic cancer, and breast cancer.

7. The use according to claim 6, characterized in that The colorectal cancer includes DLD1 cell line or HCT116 cell line; the gastric cancer includes NCI-N87 cell line or SNU668 cell line; the pancreatic cancer includes ASPC-1 cell line or HPAC cell line; the breast cancer includes SKBR3 cell line or MDA-MB-231 cell line.

8. A pharmaceutical composition for treating tumors, characterized in that: The pharmaceutical composition comprises the MAPK12 inhibitor and its derivatives according to claim 1.

9. The pharmaceutical composition according to claim 8, wherein The pharmaceutical composition further comprises excipients.

10. The use according to claim 9, characterized in that The pharmaceutical composition is prepared into an external preparation, which includes a suppository or an enema.

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