Application of N-[(3-chlorphenyl) methyl]-5-methyl-4-[(morpholine-4-yl) methyl]-1, 2-azole-3-formamide in preparation of anti-melanoma drugs

Abstract

The invention relates to the technical field of biological pharmacy, in particular to an application of N-[(3-chlorphenyl) methyl]-5-methyl-4-[(morpholine-4-yl) methyl]-1, 2-azole-3-formamide in preparation of an anti-melanoma drug and a preparation method of the N-[(3-chlorphenyl) methyl]-5-methyl-4-[(morpholine-4-yl) methyl]-1, 2-azole-3-formamide. According to the invention, specific pharmacophore and hydrophobic / hydrophilic modification are introduced in structure, so that the combination selectivity and stability of the compound and a target spot are improved; on the action mechanism, the compound can effectively interfere with a Bcl-3 signal channel. In-vivo experiments prove that the compound disclosed by the invention has a good tumor inhibition effect in an animal model, meanwhile, no obvious systemic toxicity is observed, and a relatively high therapeutic index is shown. In addition, the compound has good physicochemical properties and synthesis process feasibility, is convenient for industrial production, and is expected to become a novel candidate drug for clinical melanoma resistance.

Description

Technical Field

[0001] The present invention relates to the technical field of biopharmaceuticals, and in particular to the use of N-[(3-chlorophenyl)methyl]-5-methyl-4-[(morpholin-4-yl)methyl]-1,2-oxazole-3-carboxamide in the preparation of anti-melanoma drugs. Background Art

[0002] Melanoma is a highly malignant tumor that originates from melanocytes in the epidermis and is highly invasive and metastatic. Although surgical resection can offer a good prognosis for early-stage melanoma, current treatment options for advanced or metastatic melanoma remain significantly limited, and survival rates remain low. Existing treatment options primarily include surgery, radiotherapy, chemotherapy, targeted therapies, and immunotherapy. In recent years, the clinical application of targeted drugs targeting BRAF and MEK mutations (such as BRAF inhibitors and MEK inhibitors) and immune checkpoint inhibitors (such as PD-1 and CTLA-4 antibodies) has significantly improved prognosis for some patients. However, due to the development of drug resistance, variable response to treatment, and immune-related adverse reactions, existing therapies still cannot meet the clinical needs of all patients. Small molecule compounds, due to their well-defined structures, flexible synthesis, and strong cell membrane penetration, hold broad application prospects in cancer treatment. Therefore, the development of new small molecule drugs for melanoma has significant clinical significance and market value. Summary of the Invention

[0003] To solve the above problems, the present invention provides the use of N-[(3-chlorophenyl)methyl]-5-methyl-4-[(morpholin-4-yl)methyl]-1,2-oxazole-3-carboxamide in the preparation of anti-melanoma drugs.

[0004] The present invention is achieved through the following technical solutions: Use of N-[(3-chlorophenyl)methyl]-5-methyl-4-[(morpholin-4-yl)methyl]-1,2-oxazole-3-carboxamide in the preparation of anti-melanoma drugs, wherein the structural formula of the N-[(3-chlorophenyl)methyl]-5-methyl-4-[(morpholin-4-yl)methyl]-1,2-oxazole-3-carboxamide is shown below: .

[0005] Preferably, the drug contains N-[(3-chlorophenyl)methyl]-5-methyl-4-[(morpholin-4-yl)methyl]-1,2-oxazole-3-carboxamide as the only active ingredient.

[0006] Preferably, the drug further comprises a pharmaceutically acceptable carrier.

[0007] Preferably, the carrier comprises any one or more of glycerol, lecithin and cholesterol.

[0008] Preferably, the effective concentration of the N-[(3-chlorophenyl)methyl]-5-methyl-4-[(morpholin-4-yl)methyl]-1,2-oxazole-3-carboxamide in the drug is 5 mg / kg to 40 mg / kg.

[0009] Preferably, the medicine includes oral dosage form, injection dosage form and inhalation dosage form.

[0010] Preferably, the medicine is in the form of a solution, tablet or granule.

[0011] Preferably, the drug is a solution, and the solvents in the solution are DMSO, polyethylene glycol 300, Tween-80 and normal saline; the volume ratio of DMSO, polyethylene glycol 300, Tween-80 and normal saline is 1:4:5:4.5.

[0012] The effective concentration of N-[(3-chlorophenyl)methyl]-5-methyl-4-[(morpholin-4-yl)methyl]-1,2-oxazole-3-carboxamide in the drug is 5 mg / kg to 40 mg / kg.

[0013] Preferably, the physiological saline is a sodium chloride aqueous solution with a mass concentration of 0.9%.

[0014] Compared with the prior art, the present invention has the following beneficial effects: The present invention provides the use of N-[(3-chlorophenyl)methyl]-5-methyl-4-[(morpholin-4-yl)methyl]-1,2-oxazole-3-carboxamide in the preparation of an anti-melanoma drug. Research results of the present invention demonstrate that N-[(3-chlorophenyl)methyl]-5-methyl-4-[(morpholin-4-yl)methyl]-1,2-oxazole-3-carboxamide significantly inhibits melanoma growth in animals, reducing melanoma volume by more than 80%. This suggests that N-[(3-chlorophenyl)methyl]-5-methyl-4-[(morpholin-4-yl)methyl]-1,2-oxazole-3-carboxamide has significant clinical application potential. The compound exhibits minimal toxicity in mice; even at a daily dose of 40 mg / kg for one month, the mice exhibit no significant changes in body weight, demonstrating its safety and low cytotoxicity. When the compound was injected intraperitoneally into mice at a dose of 20 mg / kg, the size of melanoma in the mice was inhibited by more than 80%, indicating that it has a highly effective anti-melanoma effect.

[0015] Compared with the prior art, the small molecule compound provided by the application has significant advantages in structural design, targeting mechanism and anti-tumor activity. Although the existing drugs for treating melanoma such as BRAF or MEK inhibitors have certain curative effects, they generally have problems such as rapid development of drug resistance, significant side effects, narrow treatment window and limited effect on some patient groups.

[0016] The application improves the binding selectivity and stability of the compound to the target by introducing specific pharmacophores and hydrophobic / hydrophilic modifications in structure; in the mechanism of action, the compound can effectively interfere with the Bcl-3 signal pathway. Through in vivo experiments, the compound of the application has good tumor inhibition effect in animal models, and no obvious systemic toxicity is observed, showing a high therapeutic index. In addition, the compound has good physicochemical properties and synthesis process feasibility, is convenient for industrialized production, and is expected to become a new candidate drug for clinical anti-melanoma. BRIEF DESCRIPTION OF DRAWINGS

[0017] In order to more clearly illustrate the technical solutions in the embodiments of the application or the prior art, the following will briefly introduce the drawings needed to be used in the embodiments or the prior art description. Obviously, the drawings in the following description are only some embodiments of the application, and other drawings can be obtained by those skilled in the art without creative labor.

[0018] Figure 1 The body weight change result graph of the application within 30 days of different concentration administration.

[0019] Figure 2 The result graph of the in vivo significant anti-melanoma effect of N-[(3-chlorophenyl)methyl]-5-methyl-4-[(morpholin-4-yl)methyl]-1,2-oxazole-3-carboxamide; Figure 2 In the figure, A is the melanoma solid tumor growth result graph, and four parallel samples are set for the control group and N-[(3-chlorophenyl)methyl]-5-methyl-4-[(morpholin-4-yl)methyl]-1,2-oxazole-3-carboxamide 20mg / kg administration; B is the result graph of statistical analysis of tumor weight data of each group. DETAILED DESCRIPTION

[0020] In order to facilitate the understanding of the application, the application will be described more fully below, and preferred embodiments of the application are given. However, the application can be realized in many different forms, and is not limited to the embodiments described in the application. On the contrary, the purpose of providing these embodiments is to make the disclosure of the application more thorough and comprehensive.

[0021] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0022] The present invention will be described in detail below with reference to the accompanying drawings and specific examples, but they should not be construed as limiting the present invention. Unless otherwise specified, the technical means used in the following examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained from commercial sources.

[0023] Currently, anti-melanoma drug evaluation models are mainly divided into in vitro models (invitromodel) and in vivo models (invivomodel).

[0024] Among them, in vitro models mainly use various cell lines to evaluate drugs. Their advantages are that they can provide a large number of cells with the same genetic characteristics as research objects, are easy to operate, can eliminate the influence of other external factors, and can detect drug toxicity, effective concentration, etc., providing more basis for later mechanism research.

[0025] The advantage of in vivo models is that they allow for a realistic and systematic evaluation of the effects of candidate drugs in vivo.

[0026] The anti-melanoma drug evaluation model used in the present invention is an in vivo model. Specifically, B16 melanoma cells are injected into C57B6N mice to construct a melanoma mouse model to evaluate the in vivo anti-melanoma effect of N-[(3-chlorophenyl)methyl]-5-methyl-4-[(morpholin-4-yl)methyl]-1,2-oxazole-3-carboxamide.

[0027] The experimental materials used in the present invention are as follows: experimental animals Strain and number: C57BL / 6N mice (female), approximately 6 weeks old, weighing 16-18 g.

[0028] Rearing conditions: SPF environment, constant temperature (22±2℃), humidity (50±10%), 12 h light / dark cycle, free access to food and water.

[0029] cell lines Melanoma cells: B16-F10 (mouse melanoma cell line), sourced from ATCC.

[0030] Culture conditions: DMEM high-glucose medium (containing 10% FBS and 1% penicillin-streptomycin), 37°C, 5% CO2 incubator.

[0031] The drugs required for the experiment of the present invention are as follows: N-[(3-chlorophenyl)methyl]-5-methyl-4-[(morpholin-4-yl)methyl]-1,2-oxazole-3-carboxamide, also known as N-[(3-chlorophenyl)methyl]-5-methyl-4-[(morpholin-4-yl)methyl]-1,2-oxazole-3-carboxamide, was purchased from Shanghai Taoshu Biotechnology Co., Ltd. The structural formula of N-[(3-chlorophenyl)methyl]-5-methyl-4-[(morpholin-4-yl)methyl]-1,2-oxazole-3-carboxamide is shown below:

[0032] Example 1: Toxicity test of N-[(3-chlorophenyl)methyl]-5-methyl-4-[(morpholin-4-yl)methyl]-1,2-oxazole-3-carboxamide in mice To comprehensively evaluate the potential toxicity of the compound N-[(3-chlorophenyl)methyl]-5-methyl-4-[(morpholin-4-yl)methyl]-1,2-oxazole-3-carboxamide in mice and determine its appropriate dosage in mice, the present invention designed and conducted a 30-day subacute toxicity study. This study used standard C57BL / 6N mice as the experimental animal model to ensure the reliability and universality of the experimental results.

[0033] First, a dosing solution for the compound was prepared. Since the target compound is a lipid-soluble small molecule, a commonly used lipid-soluble drug solvent system was selected for dissolution. The specific solvent ratio is: DMSO + 40% polyethylene glycol 300 + 5% Tween-80 + 45% saline (Saline). The volume ratio of DMSO, polyethylene glycol 300, Tween-80, and saline is 1:4:5:4.5; saline is a 0.9% sodium chloride aqueous solution. This solvent system effectively improves the compound's solubility while exhibiting good biocompatibility. It is commonly used in in vivo mouse dosing experiments and does not cause significant toxicity to the mice themselves.

[0034] In terms of animal grouping and dosing, healthy adult C57BL / 6N mice were randomly divided into five groups, each containing an equal number of mice. A dose gradient was established: 0 mg / kg (blank control), 5 mg / kg, 10 mg / kg, 20 mg / kg, and 40 mg / kg. The 0 mg / kg group received only the same volume of the dosing solvent as a control to eliminate any potential effects of the solvent itself. All experimental groups received the drug via intraperitoneal injection once daily for 30 days.

[0035] Throughout the dosing cycle, the physiological status of the mice was closely monitored and recorded. This included weighing the mice at a fixed time daily, observing their food intake, water intake, activity level, coat gloss, mental state, and any abnormal reactions such as diarrhea, skin redness, swelling, or surface lesions. Weight change is considered an important indicator for assessing systemic drug toxicity, and sustained weight loss often indicates potential toxic side effects, such as metabolic disorders, gastrointestinal dysfunction, or organ damage.

[0036] The experimental results showed that the mice in all dose groups were in good overall condition throughout the 30-day observation period. It is particularly noteworthy that even in the high-dose group with the maximum dose of 40 mg / kg, the weight change curve of all mice was almost exactly the same as that of the control group, with no significant weight loss or fluctuation (see Figure 1 Furthermore, no significant abnormalities were observed in the mice's behavior, mobility, food and water intake, or mental state. Compared to the control group, the mice in the experimental group were more active in their daily activities and showed no signs of stress or discomfort, such as sluggishness, lethargy, hunched backs, or erect piloerection.

[0037] This result fully demonstrates that under the conditions of intraperitoneal injection for 30 consecutive days, N-[(3-chlorophenyl)methyl]-5-methyl-4-[(morpholin-4-yl)methyl]-1,2-oxazole-3-carboxamide did not cause abnormal changes in mouse body weight even at the highest dose of 40 mg / kg, indicating that the compound has extremely low systemic toxicity within this dose range and has good tolerability and biosafety.

[0038] In summary, the results of the subacute toxicity experiment show that N-[(3-chlorophenyl)methyl]-5-methyl-4-[(morpholin-4-yl)methyl]-1,2-oxazole-3-carboxamide exhibits high safety in C57BL / 6N mice. During the 30-day dosing cycle, no obvious adverse reactions or toxicity were observed at either the low dose (10 mg / kg) or the high dose (40 mg / kg). Therefore, this compound can be safely used in in vivo pharmacodynamic studies of melanoma or other tumor models within this dose range. It also provides solid data support for future chronic toxicity evaluations and preclinical safety assessments at higher doses and longer periods. Example 2: N-[(3-chlorophenyl)methyl]-5-methyl-4-[(morpholin-4-yl)methyl]-1,2-oxazole-3-carboxamide has significant in vivo anti-melanoma effects.

[0039] To further systematically evaluate the anti-tumor activity of the compound N-[(3-chlorophenyl)methyl]-5-methyl-4-[(morpholin-4-yl)methyl]-1,2-oxazole-3-carboxamide against melanoma in vivo, the present application constructed a classic C57BL / 6N mouse melanoma model. In the specific experimental process, first, the mice were anesthetized and injected with melanoma cells under sterile conditions. Each C57BL / 6N mouse was subcutaneously inoculated with 1 x 10 6 B16 melanoma cells to rapidly induce the formation of melanoma. B16 melanoma cells are a commonly used murine tumor cell line used in biomedical research for modeling human skin cancer. The B16 cell line was developed by the Jackson Laboratory in the United States in 1954, and was taken from tumor cells in the ears of C57BL / 6 mice. B16 cells are an important model for studying tumor metastasis and solid tumor formation, and are one of the earliest tools used to study tumor metastasis, grow rapidly, and can form observable solid tumors in a short period of time, with good experimental consistency and repeatability.

[0040] Three days after melanoma cell inoculation, to ensure that the tumor is fully established and enters the rapid growth phase, the present experiment began to administer the target compound by intraperitoneal injection. The experimental group mice were intraperitoneally injected with 20 mg / kg of N-[(3-chlorophenyl)methyl]-5-methyl-4-[(morpholin-4-yl)methyl]-1,2-oxazole-3-carboxamide daily, and the administration was continued until the 15th day of the experiment. The control group mice were only injected with the same volume of normal saline or the corresponding administration solvent. During the administration process, the mice were checked daily for health status, including mental state, food and water intake, and changes in subcutaneous tumor volume.

[0041] At the end of the 15th day experiment, 4 mice from each group were randomly selected for dissection, and the subcutaneously growing melanoma solid tumors were completely removed, with careful removal of excess connective tissue and adipose tissue around them. Subsequently, the wet weight of each tumor was measured using a high-precision electronic balance, and the tumor weight data of each group was statistically analyzed.

[0042] The experimental results are shown in Figure 2 The melanoma in the control group mice grew rapidly, with an average tumor weight of up to 2.41 grams, and some individuals even showed ulceration, necrosis, and other phenomena, indicating a heavy tumor burden. In mice treated with 20 mg / kg of N-[(3-chlorophenyl)methyl]-5-methyl-4-[(morpholin-4-yl)methyl]-1,2-oxazole-3-carboxamide, the growth of melanoma was significantly inhibited, with an average tumor weight of only 0.38 grams, a reduction of about 84.3% compared to the control group. This significant tumor inhibition effect indicates that the target compound has very strong anti-melanoma activity in vivo.

[0043] Furthermore, no significant toxic side effects were observed at this dose of the target compound during the experiment. All mice maintained stable weight changes, with no significant decrease, and maintained good activity, suggesting that the compound exhibited good biosafety and tolerability at this dose.

[0044] In summary, the results of this study fully demonstrate that N-[(3-chlorophenyl)methyl]-5-methyl-4-[(morpholin-4-yl)methyl]-1,2-oxazole-3-carboxamide not only exhibits excellent anti-melanoma activity in vitro but also effectively inhibits melanoma growth in mice, demonstrating favorable in vivo efficacy. Therefore, this compound holds promise as a potential candidate for the treatment of melanoma and warrants further investigation into its mechanism of action, as well as pharmacokinetic and toxicological evaluations.

[0045] The technical features of the above-mentioned embodiments can be combined arbitrarily. In order to make the description concise, not all possible combinations of the technical features in the above-mentioned embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0046] The above-described embodiments merely represent several implementation methods of the present invention, and their descriptions are relatively specific and detailed, but they should not be construed as limiting the scope of the patent. A person skilled in the art would be able to make various modifications and improvements without departing from the spirit of the present invention, and these modifications and improvements fall within the scope of protection of the present invention. Therefore, the scope of protection of the patent for this invention shall be based on the appended claims.

Claims

1. Use of N-[(3-chlorophenyl)methyl]-5-methyl-4-[(morpholin-4-yl)methyl]-1,2-oxazole-3-carboxamide in the preparation of anti-melanoma drugs, characterized in that: The structural formula of the N-[(3-chlorophenyl)methyl]-5-methyl-4-[(morpholin-4-yl)methyl]-1,2-oxazole-3-carboxamide is shown below: 。 2. The use according to claim 1, characterized in that The medicine uses N-[(3-chlorophenyl)methyl]-5-methyl-4-[(morpholin-4-yl)methyl]-1,2-oxazole-3-carboxamide as the only active ingredient.

3. The use according to claim 2, characterized in that The drug further includes a pharmaceutically acceptable carrier.

4. The use according to claim 3, characterized in that The carrier includes any one or more of glycerol, lecithin and cholesterol.

5. The use according to claim 2, characterized in that The effective concentration of N-[(3-chlorophenyl)methyl]-5-methyl-4-[(morpholin-4-yl)methyl]-1,2-oxazole-3-carboxamide in the drug is 5 mg / kg to 40 mg / kg.

6. The use according to claim 1, characterized in that The medicine includes oral dosage form, injection dosage form and inhalation dosage form.

7. The use according to claim 1, characterized in that The medicine is in the form of solution, tablet or granule.

8. The use according to claim 7, characterized in that The drug is a solution, and the solvents in the solution are DMSO, polyethylene glycol 300, Tween-80 and normal saline; the volume ratio of DMSO, polyethylene glycol 300, Tween-80 and normal saline is 1:4:5:4.5; The effective concentration of N-[(3-chlorophenyl)methyl]-5-methyl-4-[(morpholin-4-yl)methyl]-1,2-oxazole-3-carboxamide in the drug is 5 mg / kg to 40 mg / kg.

9. The use according to claim 8, characterized in that The physiological saline solution is a sodium chloride aqueous solution with a mass concentration of 0.9%.

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