Composition for preventing, alleviating or treating cancer comprising compound inhibiting activity of adamts4 and adamts5 proteins

Abstract

The present invention relates to a composition for preventing or treating cancer, comprising a compound represented by chemical formula 1 or chemical formula 2 or a pharmaceutically acceptable salt thereof. The composition has inhibitory activity specific to ADAMTS4 or ADAMTS5 protein, increases infiltration of lymphocytes and M1 macrophages into tumors, and inhibits growth or migration of cancer cells by inhibiting CXCL16 secreted from tumor-associated macrophages, exhibiting an effect of reducing the size of tumors, and thus may be used for preventing, alleviating or treating cancer.

Description

Composition for the prevention, improvement, or treatment of cancer comprising compounds that inhibit the activity of ADAMTS4 and ADAMTS5 proteins

[0001] The present invention relates to a composition for the prevention or treatment of cancer comprising a compound represented by Formula 1 or Formula 2 or a pharmaceutically acceptable salt thereof.

[0002]

[0003] ADAMTS4 (aggrecanase-1) and ADAMTS5 (aggrecanase-2) are proteases that degrade aggrecan proteins, which are major components of the extracellular matrix (ECM). The degradation of the ECM plays a crucial role in cell migration, growth, differentiation, and tissue remodeling, and is known to have a significant impact on cancer cell invasion and metastasis.

[0004] Activation of ADAMTS4 / 5 proteins influences tumor growth in relation to the degradation of the extracellular matrix (ECM). ADAMTS4 / 5 proteins are characterized by increased ECM remodeling and invasiveness, such as by degrading the ECM to facilitate the proliferation of tumor cells into surrounding tissues. Furthermore, the degradation of the ECM promotes the formation of new blood vessels, which supply the nutrients and oxygen necessary for tumor growth; ADAMTS4 / 5 and tumor-associated macrophages (TAMs) contribute to tumor growth by promoting this angiogenesis. In addition, tumor-associated macrophages (TAMs) evade immune surveillance by secreting immunosuppressive cytokines, and ADAMTS4 / 5 reconstruct the ECM to hinder the infiltration of immune cells, which helps tumors avoid attacks by the immune system (Matrix Biol, 44-46, 77-85; Cellular Physiology and Biochemistry, 46(4), 1693-1703; and Gastric Cancer, 22(2), 287-301).

[0005] The ECM-degrading ability of ADAMTS4 / 5 and the immunosuppressive and tumor-promoting activities of tumor-associated macrophages are important subjects of research in tumor biology, and therapeutic strategies that inhibit their interactions could be a significant breakthrough in cancer treatment.

[0006] Accordingly, the inventors completed the present invention by confirming the anticancer effect of a compound that inhibits or suppresses the activity of ADAMTS4 / 5 proteins.

[0007]

[0008] The object of the present invention is to provide a pharmaceutical composition for the prevention or treatment of cancer comprising a compound represented by the following formula 1 or formula 2 or a pharmaceutically acceptable salt thereof.

[0009] [Chemical Formula 1]

[0010]

[0011] [Chemical Formula 2]

[0012]

[0013] Another objective of the present invention is to provide a food composition for the prevention or improvement of cancer comprising a compound represented by Formula 1 or Formula 2 or a food-grade acceptable salt thereof.

[0014] Another object of the present invention is to provide a method for preventing or treating cancer comprising the step of administering to an individual a compound represented by Formula 1 or Formula 2 or a pharmaceutically acceptable salt thereof.

[0015] Another object of the present invention is to provide a method for inhibiting the activity of ADAMTS4 protein or ADAMTS5 protein, comprising the step of administering to an individual a compound represented by Formula 1 or Formula 2 or a pharmaceutically acceptable salt thereof.

[0016]

[0017] To achieve the above objective, the present invention provides a pharmaceutical composition for the prevention or treatment of cancer comprising a compound represented by the following formula 1 or formula 2 or a pharmaceutically acceptable salt thereof.

[0018] [Chemical Formula 1]

[0019]

[0020] [Chemical Formula 2]

[0021]

[0022] In one embodiment of the present invention, the composition may have specific inhibitory activity on a protein selected from the group consisting of ADAMTS4 (aggrecanase-1) protein and ADAMTS5 (aggrecanase-2) protein.

[0023] In one embodiment of the present invention, the composition may inhibit the growth or migration of cancer cells.

[0024] In one embodiment of the present invention, the composition may reduce the size of the tumor.

[0025] In one embodiment of the present invention, the composition may increase the infiltration of lymphocytes into a tumor, and the lymphocytes are CD4 + T cells and CD8 + It may be selected from a group of T cells.

[0026] In one embodiment of the present invention, the composition may increase the infiltration of M1 macrophages into a tumor.

[0027] In one embodiment of the present invention, the composition may inhibit the concentration of the soluble form of CXCL16 secreted from tumor-associated macrophages.

[0028] In one embodiment of the present invention, the cancer may be selected from the group consisting of breast cancer, uterine cancer, esophageal cancer, stomach cancer, brain cancer, rectal cancer, colorectal cancer, lung cancer, skin cancer, ovarian cancer, cervical cancer, kidney cancer, blood cancer, pancreatic cancer, prostate cancer, testicular cancer, laryngeal cancer, oral cancer, head and neck cancer, thyroid cancer, liver cancer, bladder cancer, osteosarcoma, lymphoma, leukemia, thymoma, thymic cancer, squamous cell carcinoma, adenocarcinoma, brain tumor, spinal cord tumor, pituitary tumor, hemangioma, epidermal tumor, colloid cyst, glioma, oligodendrocyte glioma, lymphoma and glioblastoma, but is not limited thereto.

[0029] In addition, the present invention provides a food composition for the prevention or improvement of cancer comprising a compound represented by Formula 1 or Formula 2 or a food-grade acceptable salt thereof.

[0030] In addition, the present invention provides a method for preventing or treating cancer comprising the step of administering to an individual a compound represented by Formula 1 or Formula 2 or a pharmaceutically acceptable salt thereof.

[0031] In addition, the present invention provides a method for inhibiting the activity of ADAMTS4 protein or ADAMTS5 protein, comprising the step of administering to an individual a compound represented by Formula 1 or Formula 2 or a pharmaceutically acceptable salt thereof.

[0032]

[0033] The composition according to the present invention has specific inhibitory activity on ADAMTS4 or ADAMTS5 proteins, increases the infiltration of lymphocytes and M1 macrophages into the tumor, and inhibits the growth or migration of cancer cells and reduces the size of the tumor by inhibiting CXCL16 secreted by tumor-associated macrophages, so it can be usefully used for the prevention, improvement, or treatment of cancer.

[0034]

[0035] Figure 1 shows the experimental process of administering compound 1 or compound 2 once a day (QD) to an allogeneic mouse cancer model.

[0036] Figure 2A shows the results of measuring the tumor size after administering compound 1 or compound 2 to a mouse cancer model, and Figure 2B shows the results of measuring the body weight of the mouse.

[0037] Figure 3 shows the results of analyzing the degree (percentage, %) of T cells infiltrated into the tumor after administering compound 1 or compound 2 to a mouse cancer model.

[0038] Figure 4A shows the results of analyzing the ratio of M1 / M2 macrophages in a tumor after administering compound 1 or compound 2 to a mouse cancer model, and Figure 4B shows the results of analyzing the degree (percentage, %) of M1 macrophages infiltrated into the tumor.

[0039] Figure 5 shows the results of analyzing the concentration of the soluble form of CXCL16 secreted by tumor-associated macrophages after treating human breast cancer cell line MDA-MB-157 with compound 1 (A) or compound 2 (B) and stimulating them with IFN-γ and TNF-α.

[0040] Figure 6 shows the results of measuring the migration area of ​​the cell lines as a relative gap area (%) after co-culturing human breast cancer cell line MDA-MB-157 and human monocyte cell line THP-1 cells, treating them with compound 1 (A) or compound 2 (B), and making a scratch.

[0041]

[0042] The present invention will be described in detail below.

[0043] The terms used in this invention have been selected based on currently widely used general terms whenever possible, taking into account the functions of the invention; however, these terms may vary depending on the intent of those skilled in the art or the emergence of new technologies. Additionally, in specific cases, terms may be selected arbitrarily, and in such cases, their meanings will be described in detail in the description section of the relevant embodiments. Therefore, the terms used in this invention should be defined not merely by their names, but based on their meanings and the overall content of the invention.

[0044] In the present invention, when a component or step is described as "comprising," this means that, unless specifically stated otherwise, it does not exclude other components or steps but may include additional components or steps.

[0045]

[0046] The present invention provides a pharmaceutical composition for the prevention or treatment of cancer comprising a compound represented by the following chemical formula 1 or chemical formula 2 or a pharmaceutically acceptable salt thereof.

[0047] [Chemical Formula 1]

[0048]

[0049] [Chemical Formula 2]

[0050]

[0051]

[0052] The compound represented by the above chemical formula 1 is 5-cyclopropyl-5-(3-oxo-3-(1,3,4,9-tetrahydro-2H-pyrido[3,4-b]indole-2-yl)propyl)imidazolidin-2,4-dione, and the compound represented by the above chemical formula 2 is N-((4-cyclopropyl-2,5-dioxomidazolidin-4-yl)methyl)-1,3,4,9-tetrahydro-2H-pyrido[3,4-b]indole-2-carboxamide.

[0053] The term "pharmaceuticalally acceptable salt" in this invention refers to a salt prepared according to methods conventional in the art, and such methods are known to those skilled in the art. Specifically, the pharmaceutically acceptable salt includes, but is not limited to, salts derived from the following inorganic acids, organic acids, and bases that are pharmacologically or physiologically acceptable. Examples of suitable acids may include hydrochloric acid, bromic acid, hydrobromide, sulfuric acid, nitric acid, nitrous acid, phosphoric acid, perchloric acid, fumaric acid, maleic acid, phosphoric acid, glycolic acid, lactic acid, salicylic acid, succinic acid, methanesulfonic acid, toluene-p-sulfonic acid, tartaric acid, acetic acid, trifluoroacetic acid, citric acid, methanesulfonic acid, formic acid, benzoic acid, malonic acid, naphthalene-2-sulfonic acid, benzenesulfonic acid, succinic acid, oxalic acid, benzoic acid, tartaric acid, fumaric acid, manderic acid, propionic acid, citric acid, lactic acid, glycolic acid, gluconic acid, galacturonic acid, glutamic acid, glutaric acid, glucuronic acid, aspartic acid, ascorbic acid, carboxylic acid, vanillic acid, hydroiodide, etc. In addition, salts derived from suitable bases may include, but are not limited to, alkali metals, e.g., sodium, potassium, or calcium, and alkaline earth metals, e.g., magnesium.

[0054] In the present invention, the composition may have specific inhibitory activity on a protein selected from the group consisting of ADAMTS4 (aggrecanase-1) protein and ADAMTS5 (aggrecanase-2) protein. Inhibition of the activity of ADAMTS4 (aggrecanase-1) protein and ADAMTS5 (aggrecanase-2) protein may be considered effective for the prevention, improvement, or treatment of cancer.

[0055] The terms of the present invention, "ADAMTS4 (aggrecanase-1) and ADAMTS5 (aggrecanase-2) proteins," refer to proteolytic enzymes that degrade aggrecan proteins, which are major components of the extracellular matrix (ECM).

[0056] The term "prevention" above refers to any action that suppresses or delays the occurrence, spread, and recurrence of a disease.

[0057] The term "treatment" above refers to any act that improves or beneficially alters the symptoms of a disease.

[0058] In the present invention, it was confirmed that the above composition has the effect of inhibiting the growth or migration of cancer cells and reducing the size of tumors. In addition, the above composition [induces] lymphocytes, i.e., CD4, into the tumor + T cells and CD8 + It was confirmed that the composition has the effect of increasing the infiltration of selected lymphocytes from a group of T cells and the effect of increasing the infiltration of M1 macrophages into the tumor. In addition, it was confirmed that the composition is effective in preventing, improving, or treating cancer by inhibiting the concentration of the soluble form of CXCL16 secreted by tumor-associated macrophages.

[0059] The term "tumor-associated macrophages (TAMs)" in this invention refers to immune cells found in the tumor microenvironment that exert significant influence on the growth and development of tumors. Tumor-associated macrophages can be classified into M1 macrophages and M2 macrophages; M1 macrophages exhibit anti-tumor effects, while M2 macrophages possess tumor-promoting and immunosuppressive functions. In most tumors, tumor-associated macrophages differentiate into the M2 type, promoting tumor growth, angiogenesis, invasion, and metastasis.

[0060] In the present invention, the cancer may be selected from the group consisting of breast cancer, uterine cancer, esophageal cancer, stomach cancer, brain cancer, rectal cancer, colorectal cancer, lung cancer, skin cancer, ovarian cancer, cervical cancer, kidney cancer, blood cancer, pancreatic cancer, prostate cancer, testicular cancer, laryngeal cancer, oral cancer, head and neck cancer, thyroid cancer, liver cancer, bladder cancer, osteosarcoma, lymphoma, leukemia, thymoma, thymic cancer, squamous cell carcinoma, adenocarcinoma, brain tumor, spinal cord tumor, pituitary tumor, hemangioma, epidermal tumor, colloid cyst, glioma, oligodendrocyte glioma, lymphoma and glioblastoma, but is not limited thereto.

[0061] The pharmaceutical composition of the present invention may further comprise a pharmaceutically acceptable carrier. In the present invention, the term "pharmaceutically acceptable" means exhibiting properties that are non-toxic to cells or humans exposed to the composition. The carrier may be used without limitation as long as it is known in the art, such as buffers, preservatives, analgesics, solubilizers, isotonic agents, stabilizers, bases, excipients, lubricants, etc.

[0062] In addition, the pharmaceutical composition of the present invention may be formulated and used in the form of oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, and aerosols, as well as external preparations, suppositories, and sterile injectable solutions, according to conventional methods. Furthermore, it may be used in the form of external skin preparations such as ointments, lotions, sprays, patches, creams, powders, suspensions, gels, or gels. Carriers, excipients, and diluents that may be included in the composition of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil. When formulating, the composition is prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants.

[0063] Solid dosage forms for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid dosage forms are prepared by mixing at least one excipient, such as starch, calcium carbonate, sucrose or lactose, gelatin, etc., with the above-mentioned Psychotria rubra extract. In addition to simple excipients, lubricants such as magnesium styrate and talc are also used. Liquid dosage forms for oral administration include suspensions, oral liquids, emulsions, syrups, etc., and may contain various excipients, such as humectants, sweeteners, flavorings, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, and suppositories. As non-aqueous solvents and suspending agents, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate may be used. As bases for suppositories, Witepsol, Macrogol, Tween 61, cocoa paste, laurin paste, glycerogelatin, etc. may be used.

[0064] In addition, the present invention provides a food composition for the prevention or improvement of cancer comprising a compound represented by Formula 1 or Formula 2 or a food-grade acceptable salt thereof.

[0065] The above term, "improvement," refers to any action that at least reduces parameters related to the condition being treated, such as the severity of symptoms.

[0066] The above food-grade acceptable salts may be obtained by reacting with inorganic acids such as hydrochloric acid, bromic acid, sulfuric acid, nitric acid, and phosphoric acid; sulfonic acids such as methanesulfonic acid, ethanesulfonic acid, and p-toluenesulfonic acid; and organic carboxylic acids such as tartaric acid, formic acid, citric acid, acetic acid, trichloroacetic acid, trifluoroacetic acid, capric acid, isobutanoic acid, malonic acid, succinic acid, phthalic acid, gluconic acid, benzoic acid, lactic acid, fumaric acid, maleic acid, and salicylic acid. Additionally, the compounds of the present invention may be obtained by reacting them with a base to form salts such as ammonium salts, alkali metal salts such as sodium or potassium salts, alkaline earth metal salts such as calcium or magnesium salts, salts of organic bases such as dicyclohexylamine, N-methyl-D-glucarmine, and tris(hydroxymethyl)methylamine, and amino acid salts such as arginine and lysine, but are not limited thereto.

[0067] The food composition of the present invention may be used interchangeably with terms known in the art, such as functional food and health functional food.

[0068] The term "functional food" in the present invention refers to a food manufactured and processed using raw materials or ingredients having functional properties useful to the human body as defined in Act No. 6727 of the Health Functional Foods Act, and "functionality" means consuming for the purpose of obtaining useful effects for health purposes, such as regulating nutrients or physiological actions on the structure and function of the human body.

[0069] The term "health functional food" in the present invention refers to a food manufactured or processed by methods such as extraction, concentration, purification, or mixing, using a specific component as a raw material or a specific component contained in a food raw material for the purpose of health supplementation, and is a food designed and processed to sufficiently exert biological regulatory functions on the body, such as biological defense, regulation of biological rhythms, and prevention and recovery of disease, through said component, and the composition for the health food can perform functions related to the prevention of disease and recovery of disease.

[0070] There are no restrictions on the types of food in which the composition of the present invention can be used. In addition, the composition of the present invention may be prepared by mixing other suitable auxiliary ingredients that may be included in food and known additives, depending on the choice of a person skilled in the art. Examples of foods to which it can be added include meat, sausage, bread, chocolate, candies, snacks, confectionery, pizza, ramen, other noodles, chewing gum, dairy products including ice cream, various soups, beverages, tea, drinks, alcoholic beverages, and vitamin complexes, and it may be prepared by adding it to juices, teas, jellies, and juices prepared using the extract and fractions thereof according to the present invention as main ingredients.

[0071] In addition, the foods to which the present invention can be applied may include all foods, such as special nutritional foods (e.g., infant formula, baby food, etc.), processed meat products, fish products, tofu products, jelly products, noodles (e.g., ramen, noodles, etc.), health supplements, seasoning foods (e.g., soy sauce, soybean paste, red pepper paste, mixed sauce, etc.), sauces, confectionery products (e.g., snacks), dairy products (e.g., fermented milk, cheese, etc.), other processed foods, kimchi, pickled foods (various types of kimchi, pickled vegetables, etc.), beverages (e.g., fruit and vegetable beverages, soy milk, fermented beverages, etc.), and natural seasonings (e.g., ramen soup, etc.).

[0072] When the health functional food composition of the present invention is used in the form of a beverage, it may contain various sweeteners, flavorings, or natural carbohydrates as additional ingredients, as in conventional beverages. In addition to the above, the health functional food composition of the present invention may contain various nutritional supplements, vitamins, electrolytes, flavorings, colorings, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc. Furthermore, it may contain fruit pulp for the production of natural fruit juices, fruit juice beverages, and vegetable beverages.

[0073] In addition, the present invention provides a method for preventing or treating cancer comprising the step of administering to an individual a compound represented by Formula 1 or Formula 2 or a pharmaceutically acceptable salt thereof.

[0074] The pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount. The term "administration" in the present invention means introducing a specific substance into an individual by an appropriate method, and the route of administration of said composition may be any general route as long as it can reach the target tissue. It may be administered intraperitoneally, intravenously, intramuscularly, subcutaneously, intradermally, orally, topically, intranasally, intrapulmonaryly, or rectally, but is not limited thereto.

[0075] The term "individual" above refers to all animals, including humans, rats, mice, and livestock. Preferably, the animal may be not only humans but also mammals such as cattle, horses, sheep, pigs, goats, camels, antelopes, dogs, and cats that require treatment for similar symptoms, but is not limited thereto.

[0076] The term "pharmaceuticalally effective dose" above refers to an amount sufficient to treat a disease with a reasonable benefit / risk ratio applicable to medical treatment and that does not cause adverse effects. The effective dose level can be readily determined by a person skilled in the art based on factors including the patient's gender, age, weight, health status, type and severity of the disease, drug activity, sensitivity to the drug, method of administration, time of administration, route of administration, and elimination rate, duration of treatment, drugs used in combination or simultaneously, and other factors well known in the medical field. The above recommended dose may be administered once a day or divided into several doses.

[0077] In addition, the present invention provides a method for inhibiting the activity of ADAMTS4 protein or ADAMTS5 protein, comprising the step of administering to an individual a compound represented by Formula 1 or Formula 2 or a pharmaceutically acceptable salt thereof.

[0078]

[0079] The present invention will be explained in more detail below through examples. These examples are intended to explain the invention more specifically, and the scope of the invention is not limited to these examples.

[0080]

[0081] Example 1. Confirmation of ADMATS4 and ADMATS5 protein inhibitory activity by a compound

[0082] Experiments were conducted to determine whether compound 1 or compound 2 of the present invention has inhibitory activity against ADMATS4 and ADMATS5 proteins. First, compound 1, compound 2, or the control compound GLPG1972 were treated with ADAMTS5, ADAMTS4, ADAM10, and ADAM17 proteins, and then a Fluorescence Resonance Energy Transfer (FRET) assay was performed.

[0083] Specifically, the fluorescent substrate used in FRET [peptide, Anaspec (sequence information: Abz-Thr-Glu-Gly-Glu-Ala-Arg-Gly-Ser-Val-Ile-Dap(Dnp)-Lys-Lys-NH2)] was prepared as a 1 mM stock of ADAMTS4 (Aggrecanase-1) and ADAMTS5 (Aggrecanase-2) of the ADAMTS protease family, and a 5 μM stock of human ADAMTS5 recombinant protein (R&D systems, Ser262-Pro622) was prepared. 44 μL of FRET analysis buffer (phosphate buffered saline based, pH 7.4) was dispensed into each well of a 96-well plate. Then, 250 nM of human recombinant ADAMTS5 (rhADAMTS5) and various concentrations (0.01–100 μM) of synthetic compounds or DMSO (control) were added, and the mixture was reacted at room temperature for 15 minutes. Subsequently, 40 μL of buffer and 10 μL of FRET substrate (final concentration, 100 μM) were added to each well, shaken well for 5 seconds, and the baseline fluorescence (Ex / Em = 340 / 420 nm) was measured using a microplate reader (THERMO, Varioskan LUX Multimode Microplate Reader). Afterward, the plate was sealed, the light was blocked, and the mixture was reacted for 2 hours. Finally, the fluorescence (Ex / Em = 340 / 420 nm) was measured using the microplate reader.

[0084] As a result, the IC of Compound 1 and Compound 2 for ADAMTS5 50 The values ​​were 0.019 μM and 0.035 μM, respectively, for the control compound GLPG1972 (IC 50 It was confirmed that the inhibitory activity on ADAMTS5 was superior compared to (= 0.043 μM) (Table 1). In addition, the IC5 of Compounds 1 and 2 against ADAMTS4 50The values ​​were 0.019 μM and 0.100 μM, respectively, confirming that the ADAMTS4 inhibitory activity was also excellent (Table 1).

[0085] In addition, additional FRET assays were performed on ADAM10 and ADAM17. As a result, neither compound 1 nor compound 2 showed any significant inhibitory activity against ADAM10 and ADAM17 (Table 1). GI254023X, known as an ADAM10 inhibitor, and TAPI-0, known to inhibit ADAM17, inhibited ADAM10 and ADAM17 at low concentrations as previously known, confirming that there were no issues with the FRET system.

[0086] From the above results, it was confirmed that compound 1 or compound 2 of the present invention has the effect of specifically inhibiting ADAMTS4 and ADAMTS5 proteins.

[0087]

[0088] Inhibitor(IC 50 , μM)NameADAMTS5ADAMTS4ADAM10ADAM17ADAMTS4 / 5Compound 10.0190.00314.64016.190ADAMTS4 / 5Compound 20.0350.100-21.110ADAMTS5GLPG19720.0430.019--ADAM10GI254023X--0.016-ADAM17TAPI-0---0.007

[0089]

[0090] Example 2. Confirmation of cancer cell growth inhibitory efficacy in a triple-negative breast cancer mouse model

[0091] To verify the in vivo inhibitory efficacy of Compound 1 or Compound 2 on cancer cell growth, experiments were conducted using an allogeneic mouse cancer model. The allogeneic mouse cancer model was constructed by transplanting the 4T1 cell line, a mouse triple-negative breast cancer cell, into Balb / c mice (Fig. 1). Starting 14 days after transplantation, 20 mg / kg of Compound 1 or Compound 2 was administered once daily (QD) to confirm the inhibitory efficacy on cancer cell growth for 20 days. Tumor size was measured at 2-day intervals, and comparative analysis was performed using the average values ​​for all individuals in each group.

[0092] As a result, it was confirmed that the tumor volume in the groups administered compound 1 or compound 2 decreased by 36.40% and 27.84%, respectively, compared to the control group (Vehicle) (Fig. 2A). However, no significant difference was observed in the body weight of the mice (Fig. 2B).

[0093] From the above results, it was confirmed that compound 1 or compound 2 of the present invention has an inhibitory effect on the growth of cancer cells.

[0094]

[0095] Example 3. Measurement of tumor-infiltrating lymphocyte infiltration in a triple-negative breast cancer mouse model

[0096] Experiments were conducted to determine whether there was a difference in the degree of infiltration of tumor-infiltrating lymphocytes by compound 1 or compound 2 in vivo. Briefly, tumors were isolated from mice, minced, and isolated as single cells, and the percentage of T cells was analyzed using a flow cytometer.

[0097] As a result, in the group administered compound 1 or compound 2, CD4 into the tumor compared to the control group (Vehicle) + T cells and CD8 + T cell infiltration significantly increased (Fig. 3).

[0098] From the above results, Compound 1 or Compound 2 of the present invention is introduced into the tumor CD4 + T cells and CD8 + It was confirmed that it has an effect of inhibiting tumor cell growth by increasing T cell infiltration.

[0099]

[0100] Example 4. Measurement of M1 macrophage infiltration and M2 macrophage inhibitory ability in a triple-negative breast cancer mouse model

[0101] Experiments were conducted to determine whether there were differences in the degree of M1 macrophage infiltration and the M1 / M2 macrophage infiltration ratio caused by Compound 1 or Compound 2 in vivo. Briefly, tumors were isolated from mice, minced, and isolated as single cells, and the M1 / M2 macrophage ratio and the degree of M1 macrophages (percentage, %) were analyzed using a flow cytometer.

[0102] As a result, in the group administered compound 1 or compound 2, the infiltration of M1 macrophages into the tumor was significantly increased compared to the control group (Vehicle), and the ratio of M1 / M2 macrophages within the tumor also significantly increased with the decrease in M2 macrophages (Fig. 4).

[0103] From the above results, it was confirmed that compound 1 or compound 2 of the present invention has the effect of inhibiting the growth of tumor cells by increasing the infiltration of M1 macrophages into the tumor.

[0104]

[0105] Example 5. Measurement of the ability to inhibit the secretion of chemokines secreted by tumor-associated macrophages in a triple-negative breast cancer mouse model

[0106] Experiments were conducted to determine whether Compound 1 or Compound 2 has the ability to inhibit the secretion of CXCL16, a chemokine secreted by tumor-associated macrophages. CXCL16 is characterized by existing on the cell membrane surface and being secreted in a soluble form by proteolytic enzymes, and the soluble form of CXCL16 on the cell membrane increases when treated with specific cytokines such as IFN-γ or TNF-α. Accordingly, in the present invention, it was determined whether the soluble form of CXCL16 is inhibited by Compound 1 or Compound 2 in the presence of specific cytokines.

[0107] Specifically, human breast cancer cell line MDA-MB-157 was placed in each well of a 12-well plate at a rate of 1.5 × 10⁶ 5 Cells were seeded and cultured for 24 hours. Afterward, they were washed with phosphate-buffered saline (PBS) and treated with various concentrations (10 μM or 20 μM) of the compounds or DMSO (control) in serum-free medium. After 4 hours, cancer cell lines were stimulated for 24 hours by treating them with the cytokines IFN-γ (20 ng / ml) and TNF-α (20 ng / ml). After 24 hours, the cell culture medium was centrifuged at 300×g for 10 minutes to remove cell debris, and then filtered through a 0.22-µm size filter. Subsequently, the cells were washed with cold phosphate-buffered saline (PBS), lysed in RIPA buffer (25 mM Tris-HCl pH 7.6, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS, and a protease inhibitor cocktail) at 4°C for 30 minutes, and centrifuged at 14,000×g for 15 minutes. The obtained supernatant was analyzed together with the cell culture medium using ELISA (R&D Systems, Minneapolis, MN, USA) according to the manufacturer's instructions. The analysis results were corrected to protein concentrations quantified by BCA (Thermo, Rockford, Illinois, USA).

[0108] As a result, it was confirmed that the concentration of the soluble form of CXCL16 secreted by tumor-associated macrophages was significantly inhibited in the groups treated with Compound 1 or Compound 2 compared to the control group (DMSO) (Fig. 5). However, GLPG1972, a previously known selective ADAMTS5 inhibitor, did not have an effect on inhibiting the secretion of CXCL16 (data not shown).

[0109] From the above results, it was confirmed that compound 1 or compound 2 of the present invention has the effect of inhibiting the growth of tumor cells by significantly inhibiting the soluble form of CXCL16 secreted by tumor-associated macrophages.

[0110]

[0111] Example 6. Confirmation of inhibition of cancer cell migration ability

[0112] A wound healing assay was performed to determine the effect of Compound 1 or Compound 2 on the migration of cancer cells. Specifically, the human breast cancer cell line MDA-MB-157 was placed in each well of a 24-well plate at a rate of 7 × 10⁶ 4 The cells were inoculated and cultured for 24 hours. Subsequently, 1.4 × 10⁶ human mononuclear cell lines, THP-1, were added to each well of the cultured cancer cells. 5Additional cells were dispensed and co-cultured. To convert THP-1 cells into tumor-associated macrophages, 5 μM of PMA (phorbol 12-myristate 13-acetate) was added, and the cells were cultured for 24 hours with the medium containing compounds or DMSO at various concentrations (1–10 μM). Subsequently, straight scratches of the same size and area were created using an AutoScratch Wound Making Tool (BioTek, Winooski, VT, USA). Detached cells were washed with medium to remove them, and then cultured again in medium containing the compounds. After 16–20 hours, the extent to which the scratched areas were filled with cancer cells was observed under a microscope; then, the cells were fixed with 100% methanol for 5 minutes and stained with 1% crystal violet reagent for 20 minutes. After washing and drying the plates, images were taken and the migration area was measured using the EVOS M7000 imaging system (Thermo Fisher Scientific). The migration area of ​​cancer cells / macrophages co-culture compared to cancer cell-only culture was set to 100%, and the degree of migration inhibition by the synthetic compound was expressed as a percentage (%).

[0113] As a result, in the group co-cultured with cancer cell lines and tumor-associated macrophages, cell migration was significantly increased compared to the control group without tumor-associated macrophages, whereas in the groups treated with Compound 1 or Compound 2, cancer cell migration was inhibited by 63.8% and 65.6%, respectively (Fig. 6). However, GLPG1972, a previously known selective ADAMTS5 inhibitor, did not have the ability to inhibit cancer cell migration (data not shown).

[0114] From the above results, it was confirmed that compound 1 or compound 2 of the present invention exhibits an anticancer effect by inhibiting tumor-associated macrophages and thereby inhibiting the migration of tumor cells.

[0115]

[0116] The foregoing description of the present invention is for illustrative purposes only, and those skilled in the art will understand that other specific forms can be easily modified without altering the technical spirit or essential features of the present invention. Therefore, the embodiments described above should be understood as illustrative in all respects and not restrictive. For example, each component described as a single unit may be implemented in a distributed manner, and components described as distributed may likewise be implemented in a combined form.

[0117] The scope of the present invention is defined by the claims set forth below, and all modifications or variations derived from the meaning and scope of the claims and equivalent concepts thereof should be interpreted as being included within the scope of the present invention.

Claims

1. A pharmaceutical composition for the prevention or treatment of cancer comprising a compound represented by the following Chemical Formula 1 or Chemical Formula 2 or a pharmaceutically acceptable salt thereof: [Chemical Formula 1] [Chemical Formula 2] .

2. In Paragraph 1, The above composition is a pharmaceutical composition having specific inhibitory activity on a protein selected from the group consisting of ADAMTS4 (aggrecanase-1) protein and ADAMTS5 (aggrecanase-2) protein.

3. In Paragraph 1, The above composition is a pharmaceutical composition that inhibits the growth or migration of cancer cells.

4. In Paragraph 1, The above composition is a pharmaceutical composition that reduces the size of a tumor.

5. In Paragraph 1, The above composition is a pharmaceutical composition that increases the infiltration of lymphocytes into a tumor.

6. In Paragraph 5, The above lymphocytes are CD4 + T cells and CD8 + A pharmaceutical composition selected from a group of T cells.

7. In Paragraph 1, The above composition is a pharmaceutical composition that increases the infiltration of M1 macrophages into a tumor.

8. In Paragraph 1, The above composition is a pharmaceutical composition that inhibits the concentration of the soluble form of CXCL16 secreted by tumor-associated macrophages.

9. In Paragraph 1, A pharmaceutical composition wherein the above cancer is selected from the group consisting of breast cancer, uterine cancer, esophageal cancer, stomach cancer, brain cancer, rectal cancer, colorectal cancer, lung cancer, skin cancer, ovarian cancer, cervical cancer, kidney cancer, blood cancer, pancreatic cancer, prostate cancer, testicular cancer, laryngeal cancer, oral cancer, head and neck cancer, thyroid cancer, liver cancer, bladder cancer, osteosarcoma, lymphoma, leukemia, thymoma, thymic cancer, squamous cell carcinoma, adenocarcinoma, brain tumor, spinal cord tumor, pituitary tumor, hemangioma, epidermal tumor, colloid cyst, glioma, oligodendrocyte glioma, lymphoma, and glioblastoma.

10. A food composition for the prevention or improvement of cancer comprising a compound represented by the following Chemical Formula 1 or Chemical Formula 2 or a food-grade acceptable salt thereof: [Chemical Formula 1] [Chemical Formula 2] .

11. A method for the prevention or treatment of cancer comprising the step of administering to an individual a compound represented by the following Chemical Formula 1 or Chemical Formula 2 or a pharmaceutically acceptable salt thereof: [Chemical Formula 1] [Chemical Formula 2] .

12. A method for inhibiting the activity of ADAMTS4 protein or ADAMTS5 protein, comprising the step of administering to an individual a compound represented by the following Chemical Formula 1 or Chemical Formula 2 or a pharmaceutically acceptable salt thereof: [Chemical Formula 1] [Chemical Formula 2] .

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