WO2024191139A1 - Composition for prevention or treatment of kras mutant cancer - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for the prevention or treatment of KRAS G12 mutant cancer, comprising a triazolopyrimidinone derivative and an MEK inhibitor. The composition according to the present invention exhibits a significant synergistic effect on anticancer activity against KRAS G12 mutant cancer and inhibition of tumor formation, and thus may be useful as a therapeutic regimen or complex for KRAS G12 mutant cancer.

Description

Composition for prevention or treatment of KRAS mutant cancer

The present invention relates to a composition for preventing or treating KRAS mutant cancer, and more particularly, to a pharmaceutical composition for preventing or treating KRAS G12 mutant cancer comprising a triazolopyrimidinone derivative and a MEK inhibitor.

The KRAS protein encoded by the KRAS gene (Kirsten rat sarcoma viral oncogene homolog) belongs to the RAS protein family and is a small GTP-ase involved in intracellular signal transduction. The KRAS protein exists in the cell in an inactive state (KRAS-GDP) or an activated state (KRAS-GTP), and is involved in cell signaling pathways such as MAPK and PI3K that regulate cell survival, proliferation, and apoptosis.

KRAS mutations are caused by genetic alterations in the gene encoding KRAS, and they keep Ras signaling activated, causing cell proliferation and tumorigenesis. These mutations are known to increase resistance to apoptosis in pancreatic cancer cells, thereby causing anticancer drug resistance. KRAS mutations can be classified into various types depending on changes in location and sequence. Representative mutations include G12D, G12V, G12R, G12C, in which glycine, the amino acid residue at position 12, is substituted with aspartic acid, valine, arginine, or cysteine, and G13D, G13V, G13H, in which glycine, the amino acid residue at position 13, is substituted with aspartic acid, valine, or histidine. It has been reported that the oncological aspects differ depending on the type of KRAS mutation, the effectiveness of anticancer treatment can be determined, and consequently, there is a difference in the patient's survival rate.

Most KRAS mutations disrupt the ability of KRAS to hydrolyze GTP, resulting in functional changes in the KRAS protein and the subsequent transduction of growth signals to the nucleus, which promotes cell growth and division and contributes to oncogenesis. G12D, G12V, and G13D mutations in KRAS disrupt GAP activity, allowing KRAS to remain bound to GTP, locking KRAS in an activated state by tyrosine kinases, and persistently activating downstream signaling pathways (e.g., PI3K, RAF-MEK-ERK (MAPK), RAL-GEF, etc.).

Targeted therapies that block Ras-GTPase are being developed as treatments targeting KRAS, but the lack of a binding pocket in GTP-bound-KRAS makes it difficult to develop appropriate treatments because it directly interferes with the binding of small molecules that target KRAS. As for conventional KRAS G12C gene mutation treatments, clinical trials are being conducted in combination with inhibitors of PD-1, EGFR, and SHP2, such as Sotorasib and Adagrasib, for non-small cell lung cancer, colorectal cancer, and other solid cancers.

Mitogen-activated protein kinase (MEK) is frequently upregulated in various cancer cell types and plays a key role in activating the RAS/RAF/MEK/ERK signaling pathway, which regulates cell growth and affects tumorigenesis. MEK inhibitors, which selectively inhibit MEK protein, have shown significant anticancer effects in KRAS-mutant cancers through activation of the RAS/RAF/MEK pathway. However, inherently acquired resistance to MEK inhibitors has been frequently observed in clinical trials, and there is a need for the development of improved therapeutic regimens to overcome resistance to MEK inhibitors and exhibit effective anticancer activity.

Against this backdrop, the inventors of the present invention have confirmed that a triazolopyrimidinone derivative compound having tankyrase inhibitory activity, when administered in combination with a MEK inhibitor, has a potent anticancer effect on KRAS mutant cancer, thereby completing the present invention as a significant therapeutic agent capable of overcoming resistance to MEK inhibitors.

One object of the present invention is to provide a pharmaceutical composition for preventing or treating KRAS G12 mutant cancer, comprising a triazolopyrimidinone derivative compound or a pharmaceutically acceptable salt thereof; and a MEK inhibitor as active ingredients.

Another object of the present invention is to provide a method for preventing or treating KRAS G12 mutant cancer, comprising the step of administering a triazolopyrimidinone derivative compound or a pharmaceutically acceptable salt thereof; and a MEK inhibitor to a subject who has developed or is at risk of developing KRAS G12 mutant cancer.

Another object of the present invention is to provide a triazolopyrimidinone derivative compound or a pharmaceutically acceptable salt thereof; and a MEK inhibitor for the prevention or treatment of KRAS G12 mutant cancer.

This is explained specifically as follows. Meanwhile, each description and embodiment disclosed in the present invention can also be applied to each other description and embodiment. That is, all combinations of various elements disclosed in the present invention fall within the scope of the present invention. In addition, the scope of the present invention cannot be considered limited by the specific description described below.

One aspect of the present invention for achieving the above object provides a pharmaceutical composition for preventing or treating KRAS G12 mutant cancer, comprising a triazolopyrimidinone derivative compound of the following chemical formula 1 or a pharmaceutically acceptable salt thereof; and a MEK inhibitor as active ingredients:

[Chemical Formula 1]

.

The present inventors confirmed an excellent anticancer effect on KRAS G12 mutant cancer when a triazolopyrimidinone derivative compound of the above chemical formula 1 and a MEK inhibitor were administered together, and completed the present invention.

In the present invention, “KRAS” belongs to the RAS protein family and is a small GTP-ase involved in intracellular signal transduction. The KRAS gene is one of the Ras genes, which are oncogenes involved in tumorigenesis, and is a factor constituting part of the RAS/MAPK, a cell signal transduction pathway that regulates cell growth, cell maturation, and cell death. The KRAS protein may be a wild-type human KRAS protein represented by the amino acid sequence presented in Genbank Accession No. AGC09594, and may be composed of, for example, the amino acid sequence of SEQ ID NO: 1, but is not limited thereto.

In the present invention, “KRAS mutation” or “KARS mutation” may be a genetic modification of the KRAS gene in which some amino acids of the KRAS protein encoded therefrom are substituted, deleted or inserted. Specifically, the KARS mutation may be a substitution of the 12th amino acid (G12), the 13th amino acid (G13) or the 146th amino acid (A146) of the KRAS protein with another amino acid, and may be, for example, a KRAS G12C mutation, a KRAS G12D mutation, a KRAS G12V mutation, a KRAS G12R mutation, a KRAS G12S mutation, a KRAS G12A mutation, a KRAS G13C mutation, a KRAS G13D mutation, a KRAS G13V mutation, a KRAS G13H mutation, a KRAS A146T mutation and a KRAS A146V mutation, but is not limited thereto.

In the present invention, “genetic modification” means a change in the form or properties of a gene due to a structural change in DNA that may occur during the replication and division of the gene. A genetic modification may occur in a gene base sequence, and may occur in the form of a base transposition, a base inversion, a missense mutation, a termination mutation, a base duplication, a base deletion, a reversion mutation, and a suppressor mutation. The KRAS gene mutation may appear in the form of a missense mutation, which means a genetic mutation phenomenon involved in protein synthesis, in which one of the three base sequences forming an amino acid is changed to code for a different amino acid.

In the present invention, “amino acid” means an organic compound that constitutes a monomer unit of peptides including proteins. It is composed of carbon, hydrogen, oxygen, nitrogen, etc., and includes functional groups such as an amino group and a carboxyl group. There are more than 500 naturally occurring amino acids, and 20 of them have their own designated codons. In addition to their role as protein residues, amino acids are involved in various processes such as neurotransmitters and biosynthesis. The amino acids may be, but are not limited to, alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine.

Additionally, throughout this specification, conventional one-letter and three-letter codes for naturally occurring amino acids are used, and amino acids referred to herein by abbreviations are described according to the IUPAC-IUB nomenclature as follows:

Alanine: A, Arginine: R, Asparagine: N, Aspartic acid: D, Cysteine: C, Glutamic acid: E, Glutamine: Q, Glycine: G, Histidine: H, Isoleucine: I, Leucine: L, Lysine: K, Methionine: M, Phenylalanine: F, Proline: P, Serine: S, Threonine: T, Tryptophan: W, Tyrosine: Y, and Valine: V.

The above “KRAS G12 mutation” may be a substitution of glycine (G), an amino acid residue at the 12th position of the KRAS protein, with valine (V), aspartic acid (D), serine (S), alanine (A), arginine (R), or cysteine (C), and may specifically be, but is not limited to, G12V, G12D, G12S, G12A, G12R, or G12C.

Specifically, the KRAS G12 mutation,

(1) KRAS G12V mutation in which the amino acid residue glycine at position 12 in the amino acid sequence of sequence number 1 is substituted with valine;

(2) KRAS G12D mutation in which the amino acid residue glycine at position 12 in the amino acid sequence of sequence number 1 is substituted with aspartic acid;

(3) KRAS G12S mutation in which the amino acid residue glycine at position 12 in the amino acid sequence of sequence number 1 is substituted with serine;

(4) KRAS G12A mutation in which the amino acid residue glycine at position 12 in the amino acid sequence of sequence number 1 is substituted with alanine;

(5) KRAS G12R mutation in which the amino acid residue glycine at position 12 in the amino acid sequence of sequence number 1 is substituted with arginine, and

(6) It may be at least one selected from the group consisting of KRAS G12C mutations in which the amino acid residue glycine at position 12 in the amino acid sequence of sequence number 1 is substituted with cysteine.

In the present invention, cancer is a disease related to the regulation of cell death, and is a disease caused by excessive cell proliferation when the normal balance of cell death is disrupted, and has the same meaning as commonly used in the art. In the present invention, the cancer includes both malignant tumors and benign tumors.

The cancer may be a solid cancer, and specifically may be one or more solid cancers selected from the group consisting of colon cancer, lung cancer, colon cancer, gastric cancer, hepatocellular cancer, breast cancer, medulloblastoma, melanoma, pancreatic adenocarcinoma, thyroid cancer, and prostate cancer, but is not limited thereto.

The cancer may be a KRAS mutant cancer. KRAS mutant cancer refers to a cancer having a genetic mutation in the KRAS gene encoding a GTP hydrolase belonging to the RAS super protein family, and KRAS mutation is known to be involved in the carcinogenesis process by promoting cell growth and division. Specifically, the KRAS mutant cancer may be a KRAS G12 mutant cancer in which a KRAS G12 mutation appears. The composition for preventing or treating KRAS G12 mutant cancer of the present invention may have a therapeutic effect on all cancers exhibiting a KRAS G12 mutation.

The above KRAS G12 mutant cancer may be a MEK inhibitor-resistant cancer, and may refer to a cancer that has developed intrinsic resistance and/or acquired resistance to MEK inhibitors due to KRAS G12 mutations, etc. Intrinsic resistance means that the cancer does not respond to drug treatment with a decrease in the size of the cancer, and acquired resistance means that a cancer in a progressive disease state shows new metastatic lesions or a cancer that has previously been in remission grows again.

According to one embodiment of the present invention, it was confirmed that the composition of the present invention exhibits an excellent synergistic effect on the inhibition of cell death and tumor formation of KRAS mutant, particularly KRAS G12 mutant cancer cell lines.

In the present invention, the triazolopyrimidinone derivative compound may be a compound represented by the following chemical formula 1 or a pharmaceutically acceptable salt thereof.

[Chemical Formula 1]

The compound represented by the above chemical formula 1 may be referred to as 5-(4-(2,6-difluoro-4-(2-methoxyethoxy)phenyl)piperazin-1-yl)-3-methyl-3,6-dihydro-7H-[1,2,3]triazolo[4,5-d]pyrimidin-7-one.

The triazolopyrimidinone derivative compound of the above chemical formula 1 may inhibit the activity of Tankyrase enzyme, and specifically, may inhibit the activity of Tankyrase 1 (TNKS1), Tankyrase 2 (TNKS2), or both.

The triazolopyrimidinone derivative compound represented by the above chemical formula 1 or a pharmaceutically acceptable salt thereof can exhibit an effect of suppressing tumor formation by inhibiting tankyrase enzyme activity and regulating the WNT/β-catenin signaling pathway of cancer cells, thereby being useful for the prevention or treatment of KRAS G12 mutant cancer.

The triazolopyrimidinone derivative compound of the above chemical formula 1 may exist in the form of a pharmaceutically acceptable salt. The term "pharmaceutically acceptable salt" as used herein means any organic or inorganic addition salt of the above compound at a concentration that is relatively non-toxic and harmless to a patient and has an effective effect, and the side effects due to the salt do not reduce the beneficial efficacy of the compound represented by the chemical formula 1.

Acid addition salts are prepared by conventional methods, for example, by dissolving the compound in an excess of an aqueous acid solution and precipitating the salt using a water-miscible organic solvent, for example, methanol, ethanol, acetone or acetonitrile. Equimolar amounts of the compound and an acid or alcohol (e.g., glycol monomethyl ether) in water can be heated, and the mixture can then be evaporated to dryness, or the precipitated salt can be filtered off with suction.

At this time, organic acids and inorganic acids can be used as free acids, and inorganic acids such as hydrochloric acid, phosphoric acid, sulfuric acid, nitric acid, and tartaric acid can be used, and organic acids such as methanesulfonic acid, p-toluenesulfonic acid, acetic acid, trifluoroacetic acid, maleic acid, succinic acid, oxalic acid, benzoic acid, tartaric acid, fumaric acid, mandelic acid, propionic acid, citric acid, lactic acid, glycolic acid, gluconic acid, galacturonic acid, glutamic acid, glutaric acid, glucuronic acid, aspartic acid, ascorbic acid, carbonic acid, vanillic acid, and hydroiodic acid can be used, but are not limited thereto.

Pharmaceutically acceptable salts of the compounds of the present invention include salts of acidic or basic groups which may be present in the compounds of Formula 1, unless otherwise indicated. For example, pharmaceutically acceptable salts may include sodium, calcium and potassium salts of hydroxy groups, and other pharmaceutically acceptable salts of amino groups include hydrobromide, sulfate, hydrogen sulfate, phosphate, hydrogen phosphate, dihydrogen phosphate, acetate, succinate, citrate, tartrate, lactate, mandelate, methanesulfonate (mesylate) and p-toluenesulfonate (tosylate) salts, and the like, and can be prepared by methods for preparing salts known in the art.

As a salt of the triazolopyrimidinone derivative compound of the above chemical formula 1 of the present invention, any salt of the triazolopyrimidinone derivative of the above chemical formula 1 that is a pharmaceutically acceptable salt and exhibits inhibitory activity against tankyrase 1 and/or tankyrase 2 equivalent to that of the triazolopyrimidinone derivative compound of the above chemical formula 1 may be used without limitation.

In the present invention, the MEK inhibitor is a substance that inhibits the function of MEK (Mitogen-activated protein kinase), and may be a compound having an activity of inhibiting MEK1 and/or MEK2, or a pharmaceutically acceptable salt thereof.

The above MEK inhibitors are known to have single anticancer activity in a number of human cancer models, and provide an effect of inhibiting tumor cell proliferation through inhibition of MEK1 and MEK2.

Specifically, the MEK inhibitors include Trametinib, Selumetinib, Mirdametinib, Binimetinib, Cobimetinib, Pimasertib, Pelitinib, Refametinib, Zaphnometinib, Honokiol, U0126-EtOH, PD184352, PD58059, BIX-02189, BIX-02188, TAK-733, AZD8330, SL-327, GDC-0623, BI-847325, RO5126766, PD318088, PD184161, GW284543 and Any one or more selected from the group consisting of APS-2-79, but is not limited thereto.

In the present invention, "treatment" refers to an intervention to alter the natural course of an individual or cell having a disease, which may be performed during the progression of the pathological condition or to prevent it. The desired therapeutic effect includes preventing the occurrence or recurrence of the disease, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, alleviating or temporarily alleviating the disease condition, and reversing or improving the prognosis. In particular, the present invention includes all acts that improve the course of cancer by administering the pharmaceutical composition of the present invention.

In the present invention, “prevention” means any act of inhibiting or delaying the occurrence, spread, and recurrence of KRAS G12 mutant cancer by administering the composition of the present invention.

The composition of the present invention may additionally contain a pharmaceutically acceptable carrier, diluent or excipient, and may be formulated and used in various forms, such as oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups and aerosols, and injections of sterile injection solutions, according to conventional methods according to each intended use, and may be administered orally or through various routes, including intravenous, intraperitoneal, subcutaneous, rectal and topical administration. Examples of suitable carriers, excipients or diluents which may be included in such compositions include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. In addition, the compositions of the present invention may further comprise fillers, anticoagulants, lubricants, wetting agents, fragrances, emulsifiers, preservatives and the like.

Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid preparations are formulated by mixing at least one excipient, such as starch, calcium carbonate, sucrose, lactose, gelatin, etc., with the composition. In addition, in addition to simple excipients, lubricants such as magnesium stearate and talc may be used.

Oral liquid preparations include suspensions, solutions, emulsions, syrups, etc., and in addition to commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, flavoring agents, and preservatives may be included.

Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous solvents and suspensions may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. Suppository bases may include withepsol, macrogol, Tween 61, cacao butter, laurin butter, glycerogelatin, and the like. Meanwhile, injections may include conventional additives such as solubilizers, isotonic agents, suspending agents, emulsifiers, stabilizers, and preservatives.

In the present invention, the pharmaceutical composition can be administered to a subject in a pharmaceutically effective amount. In the present invention, administration refers to introducing the pharmaceutical composition of the present invention into a subject in any appropriate manner, and the administration route can be administered through various routes, such as oral or parenteral, as long as it can reach the target tissue. Examples of the administration route include, but are not limited to, oral, intramuscular, intravenous, intraarterial, subcutaneous, intraperitoneal, pulmonary, and nasal. It is important to administer an amount that can obtain the maximum effect with the minimum amount without side effects by considering all of the above factors, and this can be easily determined by those skilled in the art.

In the pharmaceutical composition of the present invention, the triazolopyrimidinone derivative compound and the MEK inhibitor may be administered simultaneously or at different times.

Another aspect of the present invention for achieving the above object provides a method for preventing or treating KRAS G12 mutant cancer, comprising the step of administering a triazolopyrimidinone derivative compound of the above chemical formula 1 or a pharmaceutically acceptable salt thereof; and a MEK inhibitor to a subject who has developed or is at risk of developing KRAS G12 mutant cancer.

For prophylactic use, the compositions of the present invention are administered to a subject suspected of having or at risk for developing a disease, disorder, or condition described herein. For therapeutic use, the compositions of the present invention are administered to a subject, such as a patient already suffering from a disorder described herein, in an amount sufficient to treat or at least partially arrest the symptoms of a disease, disorder, or condition described herein. Amounts effective for such use will depend on the severity and course of the disease, disorder, or condition, previous treatment, the subject's health status and responsiveness to the drugs, and the judgment of the physician or veterinarian.

In the preventive or therapeutic method of the present invention, the triazolopyrimidinone derivative compound of the chemical formula 1 and the MEK inhibitor can be administered to a subject in a pharmaceutically effective amount.

The term “subject” in the present invention means any animal, including mammals such as humans, that has developed or can develop a cancer disease including solid cancer, specifically KRAS G12 mutant cancer, and typically may be a human or an animal other than a human that can exhibit a beneficial effect by treatment using the composition of the present invention, but includes without limitation any subject that has symptoms of a cancer disease or is likely to have such symptoms. As described above, by administering the pharmaceutical composition of the present invention to a subject, KRAS G12 mutant cancer can be effectively prevented, improved, or treated.

In the present invention, the term “administration” means introducing a given substance into an animal, including a human, by any appropriate method, and the route of administration of the composition of the present invention may be oral or parenteral administration through any general route as long as it can reach the target tissue. In addition, the composition of the present invention may be administered by any device through which the active ingredient can move to the target cell.

The term “pharmaceutically effective amount” in the present invention means an amount sufficient to prevent and/or treat a disease at a reasonable benefit/risk ratio applicable to medical use. An appropriate dosage and frequency of administration can be selected according to a method known in the art, and the dosage and frequency of administration of the pharmaceutical composition of the present invention actually administered can be appropriately determined by various factors such as the type of symptom to be treated, administration route, sex, health condition, diet, age and weight of the subject, and the severity of the disease, and this can be easily determined by a person skilled in the art.

The pharmaceutical composition of the present invention may further comprise a known anticancer agent in addition to a triazolopyrimidinone derivative compound or a pharmaceutically acceptable salt thereof and a MEK inhibitor, and may be used in combination with other treatments known to treat KRAS G12 mutant cancer. Other treatments include, but are not limited to, chemotherapy, radiation therapy, hormone therapy, bone marrow transplantation, stem-cell replacement therapy, other biological therapy, immunotherapy, and the like.

The matters mentioned in the pharmaceutical composition and preventive/therapeutic method of the present invention are equally applicable unless they are contradictory to each other.

Another aspect of the present invention for achieving the above object provides a use of a triazolopyrimidinone derivative compound of the above chemical formula 1 or a pharmaceutically acceptable salt thereof; and a MEK inhibitor for preventing or treating KRAS G12 mutant cancer.

In addition, the present invention provides a use of a triazolopyrimidinone derivative compound of the above formula 1 or a pharmaceutically acceptable salt thereof; and a MEK inhibitor for preparing a medicament for preventing or treating KRAS G12 mutant cancer.

The matters mentioned in the pharmaceutical composition and use of the present invention are equally applicable unless they are contradictory to each other.

The embodiments of the present invention can be modified in various different forms, and the scope of the present invention is not limited to the embodiments described below. In addition, the embodiments of the present invention are provided so that a person having average knowledge in the relevant technical field can more completely explain the present invention. Furthermore, throughout the specification, the term "including" a certain component does not exclude other components unless specifically stated to the contrary, but rather means that other components can be further included.

Combination therapy of a triazolopyrimidinone derivative compound according to the present invention and a MEK inhibitor can exhibit a remarkable synergistic effect on anticancer activity and tumorigenesis inhibition against KRAS G12 mutant cancer.

Figure 1 is a graph showing the change in tumor volume over 4 weeks after single or combined administration of a triazolopyrimidinone derivative and a MEK inhibitor (trametinib) in a KRAS G12 mutant xenograft animal model.

The present invention will be described in more detail through the following examples. However, the following examples are only intended to concretize the content of the present invention and the present invention is not limited thereto.

Materials and Methods

1. Preparation of triazolopyrimidinone derivatives

As an intermediate for preparing triazolopyrimidinone derivatives, 7-isopropoxy-3-methyl-5-(methylsulfonyl)-3H-[1,2,3]triazolo[4,5-d]pyrimidine (300 mg, 1.1 mmol) and 1-(2,6-difluoro-4-(2-(methoxyethoxy)phenyl)piperazine hydrochloride (409 mg, 1.32 mmol) were added to a sealed tube along with 2 ml of EtOH and 0.29 ml (1.65 mmol) of DIPEA, and the mixture was stirred under reflux for 2 hours. After the reaction was completed, the mixture was concentrated under reduced pressure and diluted with EA. The organic layer was washed with purified water, dried over Na ₂ SO ₄ , filtered, concentrated under reduced pressure, and separated by column chromatography to obtain a white solid compound.

427 mg (0.921 mmol) of the obtained solid compound was dissolved in 6 ml of AcOH and 1.5 ml of a 35% HCl aqueous solution, and stirred at 60 to 70°C for 2 hours. After completion of the reaction, the mixture was concentrated under reduced pressure, and 2 N NaOH aqueous solution was added dropwise to adjust the pH to 5 to 6. The formed crystals were filtered and separated by column chromatography to obtain a triazolopyrimidinone derivative compound represented by the chemical formula 1 (Compound A).

2. Preparation of MEK inhibitor

Trametinib was purchased from TargetMol Chemicals, and cobimetinib, selumetinib, mirdametinib, and binimetinib were purchased and used from Selleck Chemicals.

3. Preparation of cell lines

SW480 (CCL-228), SW620 (CCL-227), COLO320DM (CCL-220), A549 (CCL-185), H358 (CRL-5807), DLD-1 (CCL-221), HCT-15 (CCL-225), LS-1034 (CRL-2158), KM12C (CVCL-9547), and COLO205 (CCL-222) cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA), and cell line identification was authenticated using Short Tandem Repeat (STR) analysis according to the ANSI/ATCC ASN-0002-2012 guidelines.

4. Calculation of synergy effect according to anticancer effect

Synergistic interactions due to drug combinations were calculated by comparing the response of the drug alone to the response from the combination against a dose-additive reference model with the drug itself. The deviation from the dose-additive value can be evaluated numerically using the Combination Index (CI), which quantifies the overall strength of the combination effect.

Specifically, the degree of cancer cell death according to drug treatment was measured through absorbance, and the CI value was derived from the FA value (Fraction-Affected value) of each combination according to the Chou-Talalay method using CompuSyn software (ComboSyn, Inc., Paramus, NJ, USA).

Based on the derived CI values, the synergistic effect of the combination of compound A and MEK inhibitor was evaluated according to the following numerical range distinction:

0.1-0.3 , strong synergy; 0.3-0.7 , synergy; 0.7-0.9 , weak synergy; 0.9-1.1 , additive effect; 1.1-1.5 , weak antagonism; greater than 1.5 , antagonism.

Example 1. Confirmation of synergistic anticancer effects in combination with various MEK inhibitors in KRAS mutant cancer cell lines

To confirm the synergistic effect of the triazolopyrimidinone derivative according to the present invention on KRAS mutant cancer, the combination index (CI) for cancer cell death following co-administration with various MEK inhibitors in cancer cell lines expressing KRAS mutations was measured.

Specifically, the colon cancer cell line SW480 expressing KRAS G12V mutation was seeded in 96-well plates at a density of 3,000 cells per well in triplicate per group. MEK inhibitors trametinib, selumetinib, mirdametinib, binimetinib, or cobimetinib (0-50 nM; 0, 3.125, 6.25, 12.5, 25, and 50 nM), individually or in combination with compound A (0-20 uM; 0, 1.25, 2.5, 5, 10, and 20 uM), were added to the wells. Cell viability was determined by adding WST-8 reagent (CYT3000; LPS solution) 72 hours after treatment, and measuring the absorbance at 450 nm using a VersaMax microplate meter (Molecular Device). The measured values were calculated using the combination index calculation method, and the results are shown in Table 1.

MEK inhibitor	Trametinib	Selumetinib	Mirdametinib	Binimetinib	Cobimetinib
Combination Index (CI)	0.22	0.26	0.32	0.3	0.34

As confirmed in Table 1 above, the combination index in all experimental groups was less than 0.7, confirming that combined treatment with compound A and a MEK inhibitor exhibited a synergistic anticancer effect on KRAS G12 mutant cancer.

Example 2. Confirmation of anticancer effect according to KRAS mutation type

KRAS mutations exist in various types depending on changes in location and sequence, and the oncological aspects and anticancer effects may vary depending on the type of KRAS mutation. Therefore, the anticancer effects of the combination therapy of the present invention were compared according to the type of KRAS mutation.

Specifically, SW480 and SW620 (G12V), COLO320DM (G12D), A549 (G12S), H358 (G12C) were used as KRAS G12 mutant cancer cell lines; DLD-1 and HCT-15 (G13D) were used as KRAS G13 mutant cancer cell lines; LS-1034 (A146T) was used as KRAS A146 mutant cancer cell line, and KM12C, COLO205, and H358 were used as wild-type cell lines without mutations.

The above KRAS cancer cell lines were treated with MEK inhibitor (trametinib) and compound A in the same manner as in Example 1, and the absorbance was measured to calculate the combination index, and the results are shown in Tables 2 and 3.

KRAS mutation	G12V		G12D	G12S	G12C
Cell line	SW480	SW620	COLO320DM	A549	H358
Combination Index (CI)	0.22	0.16	0.41	0.52	0.15

KRAS mutation	G13D		A146T	Wild type
Cell line	DLD-1	HCT-15	LS-1034	KM12C	COLO205	H358
Combination Index (CI)	0.92	1.3	1.2	1.1	1.35	1.94

As confirmed in Tables 2 and 3 above, the combination index in KRAS G12 mutant cancer cell lines was all less than 0.7, indicating a synergistic anticancer effect, whereas in KRAS G13D and KRAS A146T mutants and wild-type cancer cell lines, it was confirmed that an additive effect or rather an antagonistic effect with a reduced effect was shown.

These results indicate that the combination therapy according to the present invention exhibits excellent anticancer activity specifically against KRAS G12 mutant cancers among cancers in which KRAS mutations appear, and therefore, the combination therapy according to the present invention can be expected to exhibit excellent anticancer effects in patients with KRAS G12 mutations.

Example 3. Confirmation of synergistic anticancer effect using KRAS G12 mutant xenograft animal model

To confirm the synergistic anticancer effect of combined administration of triazolopyrimidinone derivatives and MEK inhibitors, tumor volumes were measured in xenograft animal models.

Specifically, BALB/c Nude mice (strain: Mus Musculus, age: 6-8 weeks, sex: female, body weight: 18-22 g, supplier: Zhejiang Vital River Laboratory Animal Technology Co., Ltd.) were subcutaneously implanted with SW480 tumor cells (5 × 10 ⁶ ) into the flank of each mouse. On day 6 after tumor implantation, when the average tumor volume reached 124 mm ^{3 ,} the animals were grouped for drug administration. Compound A and/or trametinib were orally administered once daily for 28 days at 10, 30 mg/kg and/or 0.2 mg/kg, respectively. Before the start of drug administration, the tumor sizes of the mice were measured in two dimensions using a caliper, and measured twice a week after drug administration, and the results of the changes in tumor volumes were compared among the groups, and the results are shown in Fig. 1.

As confirmed in Figure 1, it was confirmed that combined administration of a triazolopyrimidinone derivative and a MEK inhibitor according to the present invention showed an excellent synergistic effect in the tumor formation inhibition effect compared to the monoadministration group.

In conclusion, it was found that combined administration of a triazolopyrimidinone derivative and a MEK inhibitor showed a remarkable synergistic effect on anticancer activity and tumor formation inhibition against KRAS G12 mutant cancer compared to single administration, and it was confirmed that the combined administration therapy according to the present invention can be utilized as a therapeutic agent showing superior efficacy compared to conventional treatment therapy in KRAS G12 mutant cancer.

Claims (10)

1. A pharmaceutical composition for preventing or treating KRAS G12 mutant cancer, comprising a triazolopyrimidinone derivative compound of the following chemical formula 1 or a pharmaceutically acceptable salt thereof; and a MEK inhibitor as active ingredients:

   [Chemical Formula 1]

   

   .
2. In the first paragraph,

   A pharmaceutical composition for preventing or treating KRAS G12 mutant cancer, wherein the KRAS G12 mutation is at least one mutation selected from the group consisting of G12V, G12D, G12S, G12A, G12R, and G12C.
3. In the second paragraph,

   The above KRAS G12 mutation is,

   (1) KRAS G12V mutation in which the amino acid residue glycine at position 12 in the amino acid sequence of sequence number 1 is substituted with valine;

   (2) KRAS G12D mutation in which the amino acid residue glycine at position 12 in the amino acid sequence of sequence number 1 is substituted with aspartic acid;

   (3) KRAS G12S mutation in which the amino acid residue glycine at position 12 in the amino acid sequence of sequence number 1 is substituted with serine;

   (4) KRAS G12A mutation in which the amino acid residue glycine at position 12 in the amino acid sequence of sequence number 1 is substituted with alanine;

   (5) KRAS G12R mutation in which the amino acid residue glycine at position 12 in the amino acid sequence of sequence number 1 is substituted with arginine; and

   (6) A pharmaceutical composition for preventing or treating KRAS G12 mutant cancer, wherein at least one KRAS G12C mutation is selected from the group consisting of KRAS G12 mutations in which the amino acid residue glycine at position 12 in the amino acid sequence of sequence number 1 is substituted with cysteine.
4. In the first paragraph,

   A pharmaceutical composition for preventing or treating KRAS G12 mutant cancer, wherein the KRAS G12 mutant cancer is a MEK inhibitor-resistant cancer.
5. In the first paragraph,

   A pharmaceutical composition for the prevention or treatment of KRAS G12 mutant cancer, wherein the cancer is a solid cancer selected from the group consisting of colorectal cancer, lung cancer, colon cancer, gastric cancer, hepatocellular cancer, breast cancer, medulloblastoma, melanoma, pancreatic adenocarcinoma, thyroid cancer and prostate cancer.
6. In the first paragraph,

   A pharmaceutical composition for preventing or treating KRAS G12 mutant cancer, wherein the triazolopyrimidinone derivative compound of the above chemical formula 1 or a pharmaceutically acceptable salt thereof inhibits Tankyrase enzyme activity.
7. In the first paragraph,

   The above MEK inhibitors include Trametinib, Selumetinib, Mirdametinib, Binimetinib, Cobimetinib, Pimasertib, Pelitinib, Refametinib, Zafnometinib, Honokiol, U0126-EtOH, PD184352, PD58059, BIX-02189, BIX-02188, TAK-733, AZD8330, SL-327, GDC-0623, BI-847325, RO5126766, PD318088, PD184161, GW284543 and A pharmaceutical composition for the prevention or treatment of KRAS G12 mutant cancer, wherein the composition comprises at least one compound selected from the group consisting of APS-2-79.
8. In the first paragraph,

   A pharmaceutical composition for preventing or treating KRAS G12 mutant cancer, wherein the composition further comprises a pharmaceutically acceptable carrier, diluent or excipient.
9. A method for preventing or treating KRAS G12 mutant cancer, comprising administering to a subject having developed or at risk of developing KRAS G12 mutant cancer a triazolopyrimidinone derivative compound of the following chemical formula 1 or a pharmaceutically acceptable salt thereof; and a MEK inhibitor:

   [Chemical Formula 1]

   

   .
10. A triazolopyrimidinone derivative compound of the following chemical formula 1 or a pharmaceutically acceptable salt thereof; and a MEK inhibitor for use in preventing or treating KRAS G12 mutant cancer:

    [Chemical Formula 1]

    

    .

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