Use of pyrido[3,4-d]pyrimidin-8-one derivative for preventing, ameliorating or treating lung cancer

Abstract

The present invention relates to the use of a pyrido[3,4-d]pyrimidin-8-one derivative in preventing, ameliorating or treating lung cancer. Specifically, the present invention is not limited to a specific KRAS mutation (G12C), and exhibited potent cell proliferation inhibitory activity at nM levels in a wide range of RAS / RAF-driven tumor cell lines, such as KRAS G12V, G12S, and Q61H mutations and an NRAS Q61K mutation. Therefore, the pan-RAF inhibitory mechanism of the present invention can effectively block various RAS / RAF downstream signaling pathways, thereby providing excellent anticancer efficacy against a wide range of KRAS / NRAS mutant cancers. In addition, the present invention demonstrated superior efficacy in overcoming complex acquired resistance mechanisms that arise after treatment with a KRAS G12C inhibitor (G12Ci), in the following two aspects. Specifically, the present invention exhibited significantly superior inhibitory efficacy than existing G12C inhibitors (G12Ci) in a KRAS G12C-Y96D model including a secondary mutation at a drug-binding site. In addition, the present invention exhibited superior or similar anticancer activity as compared to G12C inhibitors (G12Ci) in signal bypass models, such as CCDC6-RET fusion. These results indicate that the simultaneous inhibitory activity of DDRs by PHI-501 neutralizes acquired resistance by blocking bypass-activated signaling pathways, thereby overcoming acquired resistance to existing G12C inhibitor (G12Ci) therapeutic agents. In addition, the present invention demonstrated the simultaneous blockade of two pathways critical to the survival of KRAS-driven cancer cells, that is, the MAPK pathway and the PI3K / AKT bypass pathway, at the molecular level. Thus, the phosphorylation of p-ERK (a marker for MAPK activity), p-AKT (a marker for PI3K / AKT activity), and pDDR1 was significantly and simultaneously reduced in both in-vitro and in-vivo models, demonstrating that the dual mechanism of action functions effect

Description

Uses of pyrido[3,4-D]pyrimidin-8-one derivatives for the prevention, improvement, or treatment of lung cancer

[0001] The present invention relates to the use of pyrido[3,4-D]pyrimidin-8-one derivatives for the prevention, improvement, or treatment of lung cancer.

[0002] The KRAS gene is one of the most frequently found mutant oncogenes in human cancer. Oncogenic KRAS mutations cause cancer by maintaining the KRAS protein in a persistently active state (GTP-bound state), thereby promoting cell proliferation, growth, and survival by abnormally activating downstream signaling pathways such as the MAPK signaling pathway (RAF-MEK-ERK) and the PI3K signaling pathway (PI3K-AKT-mTOR).

[0003] Recently, G12C inhibitors (G12Ci, e.g., sotorasib, adagrasib) that directly target G12C, a specific mutation of the KRAS protein, have been developed, presenting new treatment possibilities for patients with KRAS-mutated lung cancer. However, despite the initial response, limitations have been reported in which resistance develops in most patients within a few months of drug administration, leading to a recurrence of the disease.

[0004] The aforementioned drug resistance mechanisms exhibit very complex and heterogeneous characteristics. First, there are resistance mechanisms in which secondary mutations occur in the KRAS gene itself to interfere with drug binding. This includes modifications of codons other than G12C (e.g., 13, 61) or mutations in the drug binding site (e.g., Y96D). Second, there are resistance mechanisms in which KRAS downstream or bypass signaling pathways are activated to neutralize KRAS inhibition. This includes activating mutations in downstream genes such as NRAS or BRAF, the formation of novel oncogenic gene combinations such as RET fusions, or the reactivation of the PI3K / AKT pathway through the activation of receptor tyrosine kinases (RTKs) such as discoidin domain receptors (DDRs).

[0005] Therefore, there is a very high medical demand for the development of a new mechanism of action that is effective against various KRAS mutations, not limited to G12C mutations, and can simultaneously overcome multifaceted resistance mechanisms.

[0006] One object of the present invention is to provide a pharmaceutical composition for the treatment or prevention of lung cancer, comprising as an active ingredient a compound represented by the following chemical formula 1 or a pharmaceutically acceptable salt thereof:

[0007] [Chemical Formula 1]

[0008] .

[0009] Another objective of the present invention is to provide a food composition for preventing or improving lung cancer, comprising as an active ingredient:

[0010] [Chemical Formula 1]

[0011] .

[0012] Another object of the present invention is to provide a method for treating lung cancer in mammals, comprising the step of administering an effective amount of a compound of Formula 1 or a pharmaceutically acceptable salt thereof, and a use of the compound of Formula 1 or a pharmaceutically acceptable salt thereof for the manufacture of a drug for treating or preventing lung cancer:

[0013] [Chemical Formula 1]

[0014] .

[0015] One aspect of the present invention provides a pharmaceutical composition for the treatment or prevention of lung cancer, comprising as an active ingredient a compound represented by the following chemical formula 1 or a pharmaceutically acceptable salt thereof:

[0016] [Chemical Formula 1]

[0017] .

[0018] In one embodiment of the present invention, the compound may inhibit pan-RAF.

[0019] In one embodiment of the present invention, the lung cancer may be a KRAS mutation lung cancer.

[0020] In one embodiment of the present invention, the KRAS variant may include one or more variants of G12C, G12V, G12S, and Q61H.

[0021] In one embodiment of the present invention, the compound may inhibit DDR1 or DDR2.

[0022] In one embodiment of the present invention, the lung cancer may be highly expressing DDR1.

[0023] In one embodiment of the present invention, the lung cancer may have acquired resistance to a KRAS G12C inhibitor due to a mutation of any one of Y96C, Y96D, Y96S, R68S, H95D, H95Q, and H95R.

[0024] In one embodiment of the present invention, the compound may simultaneously inhibit the MAPK pathway and the PI3K pathway.

[0025] Another aspect of the present invention provides a food composition for preventing or improving lung cancer, comprising as an active ingredient a compound represented by the following chemical formula 1 or a pharmaceutically acceptable salt thereof:

[0026] [Chemical Formula 1]

[0027] .

[0028] Another aspect of the present invention discloses a method for treating lung cancer in mammals, comprising the step of administering an effective amount of a compound of Formula 1 or a pharmaceutically acceptable salt thereof, and a use of the compound of Formula 1 or a pharmaceutically acceptable salt thereof for the manufacture of a drug for treating or preventing lung cancer.

[0029] [Chemical Formula 1]

[0030] .

[0031] A composition comprising a pyrido[3,4-D]pyrimidin-8-one derivative compound according to one embodiment of the present invention can be usefully utilized for the prevention, improvement, or treatment of lung cancer.

[0032] Specifically, the compound of the present invention simultaneously blocks the MAPK pathway and the PI3K / AKT bypass pathway, which are key survival pathways in lung cancer, through a dual mechanism of action that simultaneously inhibits pan-RAF and DDRs. Furthermore, it exhibited potent cell proliferation inhibitory activity at the nM level in a wide range of RAS / RAF-driven tumor cell lines, including KRAS G12V, G12S, Q61H mutations, and NRAS Q61K mutations, not limited to specific KRAS mutations (G12C). Therefore, the dual inhibitory mechanism of the compound of the present invention effectively blocks various RAS / RAF downstream signaling pathways, thereby providing excellent anticancer efficacy against a wide range of KRAS / NRAS-mutated cancers. Additionally, regarding the complex acquired resistance mechanisms that occur after treatment with KRAS G12C inhibitors (G12Ci), the compound of the present invention demonstrated superior efficacy in overcoming these resistances in the following two aspects. Specifically, in the KRAS G12C-Y96D model, a secondary mutation in the drug binding site, it showed significantly superior inhibitory efficacy compared to existing G12C inhibitors (G12Ci). In addition, in signal bypass models such as CCDC6-RET fusion, it showed superior or similar anticancer activity compared to existing G12C inhibitors (G12Ci). This indicates that the simultaneous inhibition of DDRs by the compound of the present invention can overcome acquired resistance by blocking bypass-activated signaling pathways, and through these results, it is possible to overcome acquired resistance to existing G12C inhibitors (G12Ci).

[0033] In addition, it was confirmed that the compound of the present invention simultaneously blocks two pathways critical to the survival of KRAS cancer cells—namely, the MAPK pathway and the PI3K / AKT bypass pathway—at the molecular level through the dual inhibition of pan-RAF / DDRs. Specifically, it significantly reduced the phosphorylation of p-ERK (MAPK activity indicator), p-AKT (PI3K / AKT activity indicator), and pDDR1 simultaneously in in vitro and in vivo models, and enhanced anticancer efficacy was confirmed in cell lines co-expressing DDR1, thereby demonstrating that dual inhibition induces a potent tumor growth inhibitory effect through synergy.

[0034] By confirming these powerful in vivo antitumor efficacy and clinical applicability, it can be seen that the compound of the present invention has very high clinical applicability as an innovative treatment that can overcome the limitations of existing treatments and address the unmet needs of patients with KRAS-mutated lung cancer.

[0035] Figure 1 shows the results of Example 1, which are the results of the evaluation of the selectivity and activity of PHI-501 in a Ba / F3 cell line into which the target gene was inserted.

[0036] Figures 2 to 4 show the results of Example 2, evaluating the anticancer efficacy of the compound of the present invention alone in NSCLC cells.

[0037] Figures 5 and 6 show the results of Example 3, Figure 5 shows the efficacy of the compound of the present invention in overcoming acquired resistance to KRAS G12C inhibitors (G12Ci), and Figure 6 shows the results of confirming the cell proliferation inhibitory ability by PHI-501 by introducing seven types of KRAS mutations with confirmed acquired drug resistance to G12C inhibitors (G12Ci) into MIAPACA-2 cancer cells.

[0038] Figures 7 and 8 show the results of Example 4, confirming the simultaneous blocking effect of the compound of the present invention on In Vitro MAPK and PI3K signals.

[0039] Figure 9 shows the results of Example 5, which confirm the in vivo antitumor efficacy and simultaneous inhibition of MAPK / PI3K of the compound of the present invention.

[0040] The present invention will be described in more detail below with reference to the drawings.

[0041]

[0042] Figure 1 shows the results of Example 1, which evaluate the selectivity and activity of PHI-501 in a Ba / F3 cell line into which a target gene is inserted. Figures 2 to 4 show the results of Example 2, which evaluate the anticancer efficacy of the compound of the present invention alone in NSCLC cells, and Figures 5 and 6 show the results of Example 3, where Figure 5 shows the efficacy of the compound of the present invention in overcoming acquired resistance to KRAS G12C inhibitors (G12Ci), and Figure 6 shows the results of confirming the cell proliferation inhibitory ability of PHI-501 by introducing seven types of KRAS mutations with confirmed acquired drug resistance to G12C inhibitors (G12Ci) into MIAPACA-2 cancer cells. Figures 7 and 8 show the results of Example 4, confirming the simultaneous blocking effect of the compound of the present invention on In Vitro MAPK and PI3K signals, and Figure 9 shows the results of Example 5, confirming the in vivo results of the antitumor efficacy and simultaneous inhibition of MAPK / PI3K of the compound of the present invention.

[0043]

[0044] One aspect of the present invention provides a pharmaceutical composition for the treatment or prevention of lung cancer, comprising as an active ingredient a compound represented by the following chemical formula 1 or a pharmaceutically acceptable salt thereof:

[0045] [Chemical Formula 1]

[0046] .

[0047] The compound represented by Formula 1, included as an active ingredient in the pharmaceutical composition of the present invention, is PHI-501, which is N-(4-methyl-3-(7-methyl-2-((6-methylpyridin-3-yl)amino)-8-oxo-7,8-dihydropyrido[3,4-d]pyrimidin-6-yl)phenyl)-3-(trifluoromethyl)benzamide (N-[4-methyl-3-[7-methyl-2-[(6-methylpyridin-3-yl)amino]-8-oxo-7,8-dihydropyrido[3,4-d]pyrimidin-6-yl]phenyl]-3-(trifluoromethyl)benzamide).

[0048] PHI-501, a compound represented by Chemical Formula 1 of the present invention, is a dual kinase inhibitor applicable to both pan-RAF and DDR and can be utilized as an excellent composition for the prevention and treatment of lung cancer.

[0049] In one embodiment of the present invention, the compound may inhibit pan-RAF. The RAF is an upstream regulator of the MAPK family, which is a key downstream pathway of KRAS-driven tumors, and PHI-501 of the present invention provides effective efficacy against a wide range of RAS / RAF-mutated cancers by inhibiting ARAF, BRAF, and CRAF. Specifically, the pan-RAF inhibitory efficacy of PHI-501 leads to broad anticancer activity, exhibiting potent cell proliferation inhibitory activity in multiple RAS / RAF-driven tumor cell lines, including KRAS G12V, G12S, Q61H mutations and NRAS Q61K mutations, not limited to G12C mutations (Example 2). Furthermore, in a drug binding site secondary mutation model (KRAS G12C-Y96D secondary mutation) against KRAS G12C inhibitors (G12Ci), PHI-501 demonstrated significantly superior inhibitory efficacy compared to the existing G12C inhibitor (G12Ci) (Example 3). This result demonstrates that the pan-RAF inhibitory mode of PHI-501 is not affected by KRAS binding site mutations, thereby proving that it possesses a superior mechanism for effectively overcoming resistance to the existing G12C inhibitor (G12Ci).

[0050] In one embodiment of the present invention, the lung cancer may be a KRAS mutation lung cancer. The KRAS mutation is one of the mutation types found in about 25% of patients with non-small cell lung cancer (NSCLC) and is a field with a very large clinical unmet need due to the low response rate to existing treatments.

[0051] It has been confirmed that the compounds of the present invention provide excellent therapeutic effects against KRAS-mutated lung cancer. Specifically, PHI-501 showed potent cell proliferation inhibitory activity at the nM level in various KRAS-mutated NSCLC cell lines, including the A549 cell line with the KRAS G12S mutation (Example 2), and further demonstrated significant and potent antitumor efficacy with a TGI of 76% when orally administered to an A549 xenograft mouse model (Example 5). This demonstrates that the compounds of the present invention effectively inhibit the growth of KRAS-mutated lung cancer in vivo. In addition, KRAS-driven lung cancer cells acquire survival and resistance through the PI3K / AKT bypass pathway in addition to the MAPK pathway. It was confirmed that PHI-501 effectively neutralizes two major survival pathways by significantly reducing the phosphorylation of p-ERK, a MAPK activity indicator, and p-AKT, a PI3K / AKT activity indicator, simultaneously at in vitro and in vivo levels (Examples 4 and 5). Therefore, the compounds of the present invention target KRAS-mutated lung cancer and can be an innovative therapeutic option that provides superior antitumor activity and mechanism of action against this clinically important and aggressive cancer type.

[0052] In one embodiment of the present invention, the KRAS mutation may include one or more mutations of G12C, G12V, G12S, and Q61H. KRAS mutations exist as various hotspot mutations within cancer types, and existing treatment methods limited to G12C mutations had limitations in failing to address the unmet medical needs of patients with other KRAS mutations such as G12V, G12S, and Q61H.

[0053] PHI-501, a compound of the present invention, provides superior efficacy against these wide range of KRAS mutations through a pan-RAF inhibitory mechanism. Specifically, PHI-501 demonstrated significantly superior or similar levels of cell proliferation inhibitory activity compared to existing KRAS G12C inhibitors (G12Ci) in various tumor cell lines harboring KRAS G12V, G12S, and Q61H mutations (Example 2). These results clearly demonstrate that PHI-501 can provide an innovative treatment option for a clinically important population of cancer patients with various KRAS mutations by effectively blocking the downstream RAF-MEK-ERK signaling pathway of KRAS regardless of the specific KRAS mutation.

[0054] In one embodiment of the present invention, the compound may inhibit DDR1 or DDR2. The DDRs (Discoid Domain Receptors) are receptor tyrosine kinase (RTK) family members involved in the tumor microenvironment and cell survival, and play an important role in the activation of bypass signals that induce acquired resistance to existing therapeutic agents in KRAS-mutated cancers.

[0055] It was confirmed that PHI-501, a compound of the present invention, possesses a dual mechanism of action that simultaneously inhibits DDR1 and DDR2 in addition to inhibiting pan-RAF (Example 1). Regarding RTK-based off-target bypass signaling activation models, such as CCDC6-RET fusion occurring after treatment with G12C inhibitors (G12Ci), PHI-501 demonstrated superior or similar anticancer activity compared to existing G12C inhibitors (G12Ci) due to this mechanism of simultaneous inhibition of DDRs (Example 3). These results demonstrate that the simultaneous inhibition of DDRs effectively blocks RTK bypass activation signaling pathways, thereby neutralizing acquired resistance. Furthermore, PHI-501 significantly and simultaneously reduced the phosphorylation of p-ERK, an indicator of MAPK pathway activity, and p-AKT, an indicator of PI3K / AKT bypass pathway activity, at the molecular level, and enhanced anticancer efficacy in cell lines co-expressing DDR1 (Example 4). This clearly demonstrates that DDRs inhibition induces a potent tumor growth inhibitory effect through synergy with pan-RAF inhibition by blocking bypass survival signals such as the PI3K / AKT pathway.

[0056] In one embodiment of the present invention, the lung cancer may be highly expressive of DDR1. The compound of the present invention provides a particularly superior therapeutic effect in a group of lung cancer patients highly expressing such DDR1. DDR1 is a type of receptor tyrosine kinase (RTK) that is involved in the survival and proliferation of tumor cells through collagen binding and is often highly expressed in KRAS / NRAS mutant cancer cells.

[0057] PHI-501 has a dual mechanism of action that simultaneously inhibits DDR1 in addition to its pan-RAF inhibitory mechanism, thereby inducing a powerful synergistic effect in cancer cells that highly express DDR1. Specifically, in cell lines co-expressing DDR1, treatment with PHI-501 was confirmed to show a significant enhancement in anticancer efficacy beyond the level of simple RAF inhibition (Example 4). These results indicate that the DDR1 inhibitory action of PHI-501 neutralizes bypass pathway activation and resistance mechanisms through synergy with the blockade of the MAPK signaling pathway downstream of KRAS.

[0058] In one embodiment of the present invention, the lung cancer may have acquired resistance to a KRAS G12C inhibitor (G12Ci) due to a mutation of any one of Y96C, Y96D, Y96S, R68S, H95D, H95Q, and H95R. The compound of the present invention offers an innovative therapeutic alternative for a group of cancer patients resistant to such mutations. Although G12C inhibitors (G12Ci) have brought significant progress in the treatment of cancer with KRAS G12C mutations, the occurrence of acquired resistance due to secondary mutations or off-target bypass signaling activation (such as RTK fusion) after treatment poses a serious impediment to the maintenance of clinical efficacy.

[0059] PHI-501, a compound of the present invention, overcame acquired resistance to G12C inhibitors (G12Ci) through a dual mechanism of action involving the simultaneous inhibition of pan-RAF and DDRs. Specifically, in a KRAS G12C-Y96D secondary mutation model in which resistance to G12C inhibitors (G12Ci) was induced, PHI-501 demonstrated significantly superior inhibitory efficacy compared to the existing G12C inhibitor (G12Ci) (Example 3). This demonstrates that PHI-501 can effectively treat resistant cancers because it directly inhibits RAF and is not affected by mutations in the KRAS drug binding site. Furthermore, regarding RTK-based bypass signaling activation models, such as CCDC6-RET fusion, which represent another resistance mechanism, PHI-501 exhibited superior or similar anticancer activity compared to the G12C inhibitor (G12Ci) (Example 3). This implies that the simultaneous inhibition of DDRs by PHI-501 neutralizes acquired resistance by blocking the RTK bypass activation signaling pathway, and can effectively control the entire resistance expression pathway of KRAS-mutated cancers. Furthermore, in addition to the acquired resistance to KRAS G12C inhibitors (G12Ci) caused by the aforementioned Y96D mutation, the compound PHI-501 of the present invention can provide an equivalent level of cell proliferation inhibitory ability against acquired resistance to KRAS G12C inhibitors (G12Ci) caused by any one of the mutations among Y96C, Y96S, R68S, H95D, H95Q, and H95R (Example 3, Table 3).

[0060] In one embodiment of the present invention, the compound may simultaneously inhibit the MAPK pathway and the PI3K pathway. KRAS-mutated cancer cells primarily rely on the MAPK pathway composed of RAF-MEK-ERK for survival and proliferation, while simultaneously activating bypass pathways such as PI3K / AKT, thereby tending to acquire resistance to therapeutic agents. Therefore, simultaneously blocking these two key survival pathways is essential for the therapeutic efficacy of KRAS-mutated cancer. Accordingly, PHI-501, a compound of the present invention, possesses a dual mechanism of action that effectively blocks the MAPK pathway through pan-RAF inhibition and bypass survival signals such as the PI3K / AKT pathway through DDRs inhibition. Specifically, it was confirmed that after treatment with PHI-501, the phosphorylation of p-ERK, a MAPK activity indicator, and p-AKT, a PI3K / AKT activity indicator, was significantly and simultaneously reduced in both in vitro and in vivo models in KRAS-driven cancer cells (Examples 4 and 5). Simultaneous blockade of these two key survival pathways induces a powerful synergistic effect, and enhancement of anticancer efficacy was confirmed in DDR1 co-expressing cell lines (Example 4). Consequently, PHI-501 overcomes the limitations of existing therapies that block only a single pathway and provides a superior and sustained antitumor effect against KRAS-mutated cancers by fundamentally neutralizing the survival strategies of cancer cells.

[0061] The pharmaceutical composition of the present invention may include a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier included in the pharmaceutical composition of the present invention is one commonly used in the manufacture of pharmaceuticals and includes, but is not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil. In addition to the above components, the pharmaceutical composition of the present invention may further include lubricants, humectants, sweeteners, flavorings, emulsifiers, suspending agents, preservatives, etc. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington: The Science and Practice of Pharmacy, 22nd edition (2013).

[0062] The pharmaceutical composition of the present invention may include various bases and / or additives that are necessary and appropriate for the formulation of the formulation, and may be prepared by further including known compounds such as nonionic surfactants, silicone polymers, extender pigments, fragrances, preservatives, fungicides, oxidation stabilizers, organic solvents, ionic or nonionic thickeners, emollients, antioxidants, free radical destroyers, opacifiers, stabilizers, emollients, silicones, α-hydroxy acids, defoaming agents, moisturizers, vitamins, insect repellents, fragrances, preservatives, surfactants, anti-inflammatory agents, substance P antagonists, fillers, polymers, propellants, basicizing or acidifying agents, or coloring agents, to the extent that the effect is not reduced.

[0063] Suitable dosages of the pharmaceutical composition of the present invention may be prescribed in various ways depending on factors such as the formulation method, mode of administration, patient's age, body weight, sex, pathological condition, food, time of administration, route of administration, excretion rate, and response sensitivity. The dosage of the pharmaceutical composition of the present invention may be 0.001 to 1000 mg / kg based on an adult.

[0064] The pharmaceutical composition of the present invention can be administered orally or parenterally.

[0065] The pharmaceutical composition of the present invention may be administered in various formulations when administered orally, such as tablets, pills, hard / soft capsules, liquids, suspensions, emulsifiers, syrups, granules, elixirs, troches, etc., and may further include various excipients, such as humectants, sweeteners, flavorings, preservatives, etc. Specifically, when the composition of the present invention is formulated into an oral administration formulation, it may further include suitable carriers, excipients, and diluents commonly used in its manufacture. The above carrier, excipient, and diluent may include, but are not limited to, 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 / or mineral oil. Additionally, it may be prepared by including diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, and surfactants that are commonly used in formulations, and may further include a lubricant such as magnesium stearate or talc in addition to the above excipients.

[0066] The pharmaceutical composition of the present invention may be administered parenterally, and may be administered, for example, by subcutaneous injection, intravenous injection, or intramuscular injection, but is not limited thereto.

[0067] Formulation into a parenteral administration formulation may, for example, be prepared by mixing the pharmaceutical composition of the present invention with water together with a stabilizer or a buffer to form a solution or suspension, and then preparing the composition in ampoule or vial units. Additionally, the composition may be sterile and may further include adjuvants such as preservatives, stabilizers, hydrating agents or emulsification promoters, salts and buffers for osmotic pressure regulation, and other therapeutically useful substances, and may be formulated by conventional methods.

[0068] Another aspect of the present invention provides a method for treating lung cancer comprising the step of administering a composition containing a compound represented by Formula 1 as an active ingredient to a lung cancer patient.

[0069] A pharmaceutical composition according to one embodiment of the present invention comprises a compound represented by Formula 1 as an active ingredient and exhibits an excellent anticancer effect against lung cancer. Accordingly, before administering the pharmaceutical composition of the present invention, a companion diagnosis step may be further included to select a group of patients who show an effect to the compound represented by Formula 1.

[0070] The term "companion diagnosis" as used in this invention refers to a diagnosis for predicting a patient's responsiveness to a specific drug treatment in advance.

[0071] Another aspect of the present invention provides a food composition for preventing or improving lung cancer, comprising as an active ingredient a compound represented by the following chemical formula 1 or a pharmaceutically acceptable salt thereof:

[0072] [Chemical Formula 1]

[0073] .

[0074] The food composition of the present invention can be prepared by adding raw materials and ingredients that are commonly added in the art, and in addition to containing a compound represented by Chemical Formula 1 as an active ingredient, it may contain various flavoring agents or natural carbohydrates as additional ingredients, as in conventional food compositions.

[0075] According to one embodiment of the present invention, the natural carbohydrate may be a conventional sugar such as a monosaccharide (e.g., glucose, fructose, etc.), a disaccharide (e.g., maltose, sucrose, etc.), and a polysaccharide (e.g., dextrin, cyclodextrin, etc.), and a sugar alcohol such as xylitol, sorbitol, erythritol, etc. The flavoring agent may include a natural flavoring agent (thaumatin), a stevia extract (e.g., rebaudioside A, glycyrrhizin, etc.), and / or a synthetic flavoring agent (saccharin, aspartame, etc.).

[0076] The food composition of the present invention may be formulated as a food composition by additionally including one or more carriers that are food- or pharmaceutically acceptable in addition to the active ingredients described above. The formulation form of the food composition may be a tablet, capsule, powder, granule, liquid, pill, liquid, syrup, juice, suspension, emulsion, or drops. For example, for formulation in the form of a tablet or capsule, the active ingredient may be combined with an oral, non-toxic, pharmaceutically acceptable inert carrier such as ethanol, glycerol, or water.

[0077] The food composition of the present invention may include a vitamin mixture consisting of vitamin A acetate, vitamin E, vitamin B1, vitamin B2, vitamin B6, vitamin B12, vitamin C, biotin, nicotinamide, folic acid, and calcium pantothenate, and one or more minerals that can be conventionally added in the industry, such as ferrous sulfate, zinc oxide, magnesium carbonate, monopotassium phosphate, disodium phosphate, potassium citrate, calcium carbonate, and magnesium chloride.

[0078] If necessary, suitable binders, lubricants, disintegrants, and colorants may also be included in the mixture. Suitable binders may include natural sugars such as starch, gelatin, glucose, or beta-lactose, corn sweeteners, natural and synthetic gums such as acacia, trackercanth, or sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride, etc. Disintegrants may include starch, methyl cellulose, agar, bentonite, xanthan gum, etc.

[0079] These ingredients may be used independently or in combination, and the proportion of these additives may be selected in the range of more than 0 and less than or equal to 20 parts by weight per 100 parts by weight of the food composition of the present invention, but is not limited thereto.

[0080] Meanwhile, various foods can be manufactured by applying methods for manufacturing various formulations known to a person skilled in the art to the food composition of the present invention. For example, the food composition of the present invention may be manufactured into conventional health functional food formulations such as beverages, pills, or powders, but is not limited thereto.

[0081] The food composition of the present invention has particularly excellent ability to inhibit the proliferation of lung cancer cell lines, so it can be usefully utilized for the prevention or improvement of lung cancer.

[0082]

[0083] Another aspect of the present invention discloses a method for treating lung cancer in mammals, comprising the step of administering an effective amount of a compound of Formula 1 or a pharmaceutically acceptable salt thereof, and a use of the compound of Formula 1 or a pharmaceutically acceptable salt thereof for the manufacture of a drug for treating or preventing lung cancer.

[0084] [Chemical Formula 1]

[0085] .

[0086]

[0087] The present invention will be explained in more detail below through one or more embodiments. However, these embodiments are intended to illustrate the invention and the scope of the invention is not limited to these embodiments.

[0088]

[0089] Example 1: Evaluation of Selectivity and Activity of PHI-501 in Ba / F3 Cell Lines with Inserted Target Gene

[0090] The efficacy and selectivity of PHI-501, a pan-RAF / DDR dual-target inhibitor, were evaluated using Ba / F3 cell lines with inserted target genes.

[0091] Specifically, to measure the cell proliferation inhibitory ability of PHI-501 according to the BRAF V600E mutation and DDR1 expression, Ba / F3-BRAF V600E and Ba / F3-BRAF V600E Experiments were performed using / DDR1 and Ba / F3 cell lines. Ba / F3-BRAF V600E Cells were used in RPMI-1640 medium containing 5% FBS, and Ba / F3-BRAF V600E / DDR1 cells were prepared by adding 100 µg / ml Collagen I (Sigma Aldrich) to RPMI-1640 medium containing 5% FBS. Ba / F3 cells were prepared by adding 10 ng / mg mouse IL-3 (R&D systems) to RPMI-1640 medium containing 5% FBS. For each cell line, 90 μL of a suspension containing 3,000 cells was dispensed into each 96-well plate and incubated in a cell culture incubator at 37°C under 5% CO2. The following day, 10 μL of the example compounds and DMSO control, diluted in DMSO at seven concentration ranges, were dispensed into 96-well plates and treated with the cells for 72 hours. After compound treatment, 10 μL of reagent was added to each 96-well plate using the D-Plus® CCK cell viability assay kit, and the plates were incubated in a 5% CO2, 37°C cell culture incubator for the specified reaction time. The 96-well plates were inserted into an Envision plate reader (PerkinElmer), and absorbance was measured. IC50, which indicates the ability to inhibit cancer cell proliferation 50 The values ​​were calculated by fitting dose-response curves using Graphpad software.

[0092] As a result, as confirmed in Figure 1, PHI-501 exhibited μM-level cell growth inhibitory activity in Ba / F3 cell lines without target gene insertion, whereas in Ba / F3-BRAF cells with single and dual target genes inserted V600E In the +DDR1 cell line, PHI-501 inhibited cell growth at the nM level. When compared to the Ba / F3 cell line without the inserted target gene, PHI-501 inhibited BRAF V600E It showed more than 100-fold selectivity against Ba / F3 cell lines in which the mutation and DDR1 protein were simultaneously overexpressed.

[0093]

[0094] Example 2: Evaluation of Growth Inhibitory Activity Against Broad-spectrum KRAS / NRAS Mutant NSCLC Cells

[0095] We evaluated whether PHI-501 has broad anticancer activity against various KRAS and NRAS variant NSCLCs not limited to G12C mutations, and whether there is a correlation with DDR1 expression.

[0096] Specifically, to measure the inhibitory effect of PHI-501 on the proliferation of KRAS and NRAS mutant NSCLC cells, experiments were performed using cell lines H23, H358, H441, A549, H460, and H1299. For each cell line, 90 μL of a suspension containing 3,000 or 2,000 cells was dispensed into 96-well plates using RPMI-1640 medium containing 10% FBS, and the cells were incubated in a cell culture incubator at 37°C under 5% CO2. The following day, 10 μL of each compound of the example (PHI-501, RAF inhibitors (Encorafenib, avutometinib), and KRAS inhibitors (sotarasib, adagrasib)), dissolved and diluted in DMSO at nine different concentration ranges, and a DMSO control were dispensed into 96-well plates and treated with the cells for 72 hours. After compound treatment, 100 μL of CellTiter-Glo® (Promega) reagent was added to each 96-well plate and mixed, then incubated at room temperature for 10 minutes. The 96-well plate was inserted into an Envision plate reader (PerkinElmer), and the luminescence signal was measured. IC50, which indicates the ability to inhibit cancer cell proliferation, was used. 50 The values ​​were calculated by fitting dose-response curves using Graphpad software.

[0097] As a result, as confirmed in Figures 2 to 4 and Table 1, PHI-501 demonstrated potent cell growth inhibitory activity in a total of six NSCLC cell lines, including KRAS G12V, G12S, Q61H, and NRAS Q61K mutant cell lines. In particular, lower IC50s were observed in cell lines with high DDR1 expression (NCI-H23, A549, etc.). 50 Values ​​were observed (Figs. 3, 4). In addition, a lower IC50 compared to encorafenib. 50 Display the value (Figs. 2, 3).

[0098] TypeCell lineDDR1 protein levelsKRAS statusPHI-501 IC 50 (μM)NSCLCH23+++G12C0.094NSCLCH358+G12C0.086NSCLCH441+G12V0.191NSCLCA549+++G12S0.068NSCLCH460-Q61H0.161NSCLCH1299-WT NRAS Q61K 0.086

[0099]

[0100] These results show that PHI-501 is effective against various KRAS / NRAS mutations not limited to G12C, and that pan-RAF / DDR dual inhibition is effective against a wide range of KRAS-driven tumors (comparison with RAF inhibitors (Fig. 4), comparison with KRAS inhibitors (Fig. 5)). In addition, it can be seen that this efficacy is correlated with DDR1 expression levels.

[0101]

[0102] Example 3: Demonstration of Efficacy of KRAS G12C Inhibitor (G12Ci) in Overcoming Acquired Resistance

[0103] The efficacy of PHI-501 in overcoming major acquired resistance mechanisms that occur after treatment with KRAS G12C inhibitors (G12Ci), namely secondary mutations and off-target signal bypass, was evaluated by comparison with existing G12Ci drugs.

[0104] Specifically, to measure the inhibitory effect of PHI-501 on KRAS G12C mutations and resistance-related cell proliferation, experiments were conducted using Ba / F3-KRAS G12C-Y96D and Ba / F3-CCDC6-RET cell lines. For each cell line, 90 μL of a suspension containing 3,000 cells was dispensed into 96-well plates using RPMI-1640 medium containing 10% FBS, and the cells were incubated in a cell culture incubator at 37°C under 5% CO2. The following day, 10 μL of each compound of the example (PHI-501, Encorafenib, Sotorasib, Adagrasib), dissolved and diluted in DMSO at nine different concentrations, and a DMSO control were dispensed into 96-well plates, and the cells were treated for 72 hours. After compound treatment, 100 μL of CellCounting-Lite 2.0 Luminescent Cell Viability Assay (Vazyme) Reagent was added to each 96-well plate and mixed, then incubated at room temperature for 20 minutes. The 96-well plate was inserted into a plate reader, and the luminescence signal was measured. The IC50, which indicates the ability to inhibit cancer cell proliferation, was measured. 50 The values ​​were calculated by fitting dose-response curves using Graphpad software.

[0105] As a result, as confirmed in Figure 5 and Table 2, PHI-501 demonstrated stronger inhibitory efficacy compared to G12C inhibitors (G12Ci) in the KRAS G12C-Y96D (drug binding site secondary mutation) cell line. Additionally, in the CCDC6-RET fusion (signal bypass) cell line, PHI-501 demonstrated superior or similar inhibitory efficacy compared to G12C inhibitors (G12Ci).

[0106] CompoundBa / F3-KRAS-G12C-Y96D IC 50 (μM)Ba / F3-CCDC6-RET IC 50(μM)PHI-5010.0950.280Encorafenib2.53.2Sotorasib8.6>10Adagrasib0.7950.730

[0107]

[0108] Through this, it was confirmed that the pan-RAF / DDR inhibitory mode of PHI-501 against the Y96D mutation induces stronger cell growth inhibition compared to existing BRAF inhibitors and KRAS inhibitors (Ba / F3-KRAS-G12C-Y96D IC 50 Results). Furthermore, regarding the efficacy against signal bypass induced by RET fusion, it can be confirmed that the dual inhibition of pan-RAF and DDRs by PHI-501 effectively blocks bypassed signaling pathways compared to three existing therapies, thereby overcoming off-target resistance of G12C inhibitors (G12Ci) (Ba / F3-CCDC6-RET IC 50 result).

[0109] Experiments were conducted to measure the cell proliferation inhibitory ability of PHI-501 by introducing seven types of KRAS mutations, confirmed to have acquired drug resistance to existing therapeutic agents such as G12C inhibitors (G12Ci), into MIAPACA-2 cancer cells. For each cell line, 90 μL of a suspension containing the set number of cells was dispensed into 96-well plates using DMEM medium containing 10% FBS, and the cells were incubated in a cell culture incubator at 37°C under 5% CO2. The following day, 10X dilutions of the control substance (Sotorasib), PHI-501, and DMSO control were prepared at nine concentration ranges, 10 μL of each were dispensed into 96-well plates, and the cells were treated for 72 hours. After compound treatment, 50 μL of CellTiter-Glo® Luminescent Cell Viability Assay (Promega) Reagent was added to each 96-well plate, mixed, shaken for 2 minutes, and then incubated at room temperature for 10 minutes. The 96-well plate was inserted into a plate reader, and the luminescence signal was measured. The absolute IC50, indicating the cancer cell proliferation inhibitory ability, was calculated. 50 The values ​​were calculated by fitting dose-response curves using Graphpad software.

[0110] As a result, as confirmed in Figure 6 and Table 3, the absolute IC50 of sotorasib in MIAPACA-2 cell lines into which seven types of KRAS mutations (Y96C, Y96D, Y96S, R68S, H95D, H95Q, H95R) were respectively introduced 50 All values ​​were confirmed to be 10 uM or higher, but the absolute IC of PHI-501 50 All values ​​were confirmed to be at the nM level, demonstrating superior cell growth inhibitory ability compared to the existing G12C inhibitor (G12Ci), sotorasib.

[0111]

[0112]

[0113] Example 4: Verification of Simultaneous Blocking of In Vitro MAPK and PI3K Signals

[0114] To investigate the effects of the dual inhibitory action of PHI-501 on intracellular signaling pathways, changes in the phosphorylation of key signaling proteins following EGF stimulation were analyzed using Western blot in A549 cells after PHI-501 treatment.

[0115] Specifically, using A549 lung cancer cells, we investigated whether PHI-501 inhibits intracellular changes in DDR1 and EGFR protein phosphorylation and downstream MAPK and PI3K signaling. A549 cells were inoculated with 3 ml of A549 cell suspension into 60 mm cell culture dishes using RPMI-1640 medium containing 10% FBS, and then incubated overnight in a cell culture incubator at 37°C under 5% CO2. The next day, cells for testing changes in DDR1 and EGFR protein phosphorylation levels were treated with 50 and 100 ng / ml EGF (R&D systems) for 30 minutes; after removing the medium and washing the cells with PBS, 40 μL of RIPA buffer for cell lysis was added to each cell to collect the cell lysates. For cells confirming changes in DDR1 and EGFR protein phosphorylation and downstream MAPK and PI3K signaling, A549 cells were treated with the example compounds and a DMSO control, and treated for 4 hours at 37°C under 5% CO2. After the compound treatment was completed, 50 ng / ml EGF (R&D systems) was additionally treated for 30 minutes. The medium was removed, and the cells were washed with PBS; cell lysates were collected by adding 40 μL of RIPA buffer for cell lysis to each cell. The protein concentration of the cell lysates was quantified to 30 μg using Pierce 660nm Protein Assay Reagent, and loading samples for Western blotting were prepared. After electrophoresis using 4-15% precast gels (Bio-rad, Mini-PROTEAN® TGX™ Precast Protein Gels), proteins were transferred to PDVF membranes using the Trans-Blot Turbo Transfer System.After attaching primary antibodies pDDR1 (Y792) (1:1000, Cell signaling technology), DDR1 (1:1000, Cell signaling technology), pEGFR (Y1068) (1:1000, Cell signaling technology), EGFR (1:1000, Cell signaling technology), pAKT (S473) (1:1000, Cell signaling technology), AKT (1:1000, Cell signaling technology), pERK (T202 / Y204) (1:1000, Cell signaling technology), ERK (1:1000, Cell signaling technology), and GAPDH (1:1000, Cell signaling technology) to the membrane, the membranes were labeled using a secondary antibody (1:10000, GenDEPOT), and then signals were detected using ChemiDoc™ Imaging Systems (Bio-rad).

[0116] As a result, as confirmed in Figures 7 and 8, the phosphorylation levels of p-ERK, an activator of the MAPK pathway, and p-AKT, an activator of the PI3K pathway, were significantly reduced simultaneously compared to the control group despite EGF stimulation through PHI-501 treatment. It was also confirmed that the phosphorylation of DDR1 (pDDR1) was reduced.

[0117] Through this, the reduction of p-ERK implies the blockade of the MAPK pathway through RAF inhibition, while the reduction of p-AKT and pDDR1 implies the blockade of the PI3K / AKT bypass pathway through DDR1 inhibition. These results directly demonstrate that the dual inhibition mechanism of Pan-RAF / DDRs by PHI-501 effectively neutralizes two major survival pathways of KRAS-mutated cancers simultaneously at the molecular level.

[0118]

[0119] Example 5: In Vivo Confirmation of Antitumor Efficacy and Simultaneous MAPK / PI3K Inhibition

[0120] The antitumor efficacy and the degree of inhibition of intratumoral signaling pathways by oral administration (po) of PHI-501 were finally evaluated in an A549 (KRAS G12S) xenograft mouse model.

[0121] Specifically, 5-week-old CAnN.Cg-Foxn1 nu / CrlOri female mice, after an acclimatization period, in an A549 cell suspension supplemented with PBS and Matrigel in a 1:1 ratio (2.5*10 7 Cells / head) were filled into disposable syringes and transplanted by administering 0.2 mL subcutaneously to the right dorsal region of mice. For animals in good health, the tumor volume was measured to be 80–120 mm² 3Animals that reached [the target] were selected and evenly separated into groups. The excipient control group and the test group for Compound 8 of the Example were orally administered at a dose of 40 mg / kg once daily. During the 28-day drug administration period, tumor volume was measured twice a week using calipers, and body weight was measured twice a week starting from the start of administration and on the day of tumor excision; if body weight was measured on the day of administration, it was performed prior to administration. On the last day of the drug administration period, animals were anesthetized with inhalation isoflulane, tumors were excised, and photographs were taken on a group basis. The tumor growth inhibition rate was calculated by substituting the tumor weight into the following formula (1 - mean tumor weight of the test substance group and positive control group / mean tumor weight of the negative control group). All data obtained from the experiment were analyzed using SAS (SAS Institute INC., USA). After testing for homogeneity of variances using the Bartlett test on all data, if homogeneity of variances was confirmed, a one-way analysis of variance (ANOVA) was performed. If significance (significance level: 0.05) was observed, a multiple t-test of Dunnett's was conducted to confirm the significance between the test groups and the excipient control group. If homogeneity of variances was rejected, a Kruskal-Wallis test was performed. If significance (significance level: 0.05) was observed, a multiple t-test of DSCF (Dwass-steel-Critchlow-Fligner) was conducted to confirm the significance between the test groups and the excipient control group. As a result, as shown in Figure 9, the PHI-501 (30 mg / kg BID) administration group showed a significant tumor growth inhibitory effect of TGI=76% compared to the control group. In addition, analysis of tumor tissue collected after administration confirmed that the expression of p-ERK, p-AKT, and pDDR1 was significantly reduced compared to the control group.

[0122] Through this, the simultaneous inhibition of p-ERK and p-AKT confirmed in vitro was successfully achieved in an in vivo tumor environment, and it was finally proven that this is the key mechanism inducing the potent antitumor efficacy of PHI-501.

[0123] The present invention has been described above with reference to its embodiments. Those skilled in the art will understand that the present invention may be embodied in modified forms without departing from the essential characteristics of the invention. Therefore, the disclosed embodiments should be considered in an illustrative rather than a restrictive sense. The scope of the invention is defined by the claims, not by the foregoing description, and all variations within the scope of equivalents should be interpreted as being included in the invention.

Claims

1. A pharmaceutical composition for the treatment or prevention of lung cancer comprising, as an active ingredient, a compound represented by the following chemical formula 1 or a pharmaceutically acceptable salt thereof: [Chemical Formula 1] .

2. In Paragraph 1, The above compound is a pharmaceutical composition for the treatment or prevention of lung cancer that inhibits pan-RAF.

3. In Paragraph 1, The above-mentioned lung cancer is a pharmaceutical composition for the treatment or prevention of lung cancer, wherein the lung cancer is KRAS-mutated lung cancer.

4. In Paragraph 3, A pharmaceutical composition for the treatment or prevention of lung cancer, wherein the above KRAS mutation comprises one or more of G12C, G12V, G12S, and Q61H mutations.

5. In Paragraph 1, The above compound is a pharmaceutical composition for the treatment or prevention of lung cancer that inhibits DDR1 or DDR2.

6. In Paragraph 1, A pharmaceutical composition for the treatment or prevention of lung cancer, wherein the above lung cancer is one that highly expresses DDR1.

7. In Paragraph 1, A pharmaceutical composition for the treatment or prevention of lung cancer having acquired resistance to a KRAS G12C inhibitor due to a mutation in any one of Y96C, Y96D, Y96S, R68S, H95D, H95Q, and H95R.

8. In Paragraph 1, The above compound is a pharmaceutical composition for the treatment or prevention of lung cancer that simultaneously inhibits the MAPK pathway and the PI3K pathway.

9. A food composition for preventing or improving lung cancer comprising, as an active ingredient, a compound represented by the following chemical formula 1 or a pharmaceutically acceptable salt thereof: [Chemical Formula 1] .

10. A method for treating lung cancer in mammals, comprising the step of administering an effective amount of a compound of Formula 1 below or a pharmaceutically acceptable salt thereof: [Chemical Formula 1] .

11. Use of the compound of Chemical Formula 1 or its pharmaceutically acceptable salt for the manufacture of a drug for the treatment or prevention of lung cancer: [Chemical Formula 1] .

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