Combination therapy for treating gastrointestinal stromal tumor

Abstract

To provide a method for treating a tumor with c-KIT mutation.SOLUTION: A method for treating a tumor with one or a plurality of c-KIT mutations in a patient in need of the treatment, which includes administering an effective amount of 1-[4- bromo-5-[1-ethyl-7-(methylamino)-2-oxo-1,2-dihydro-1,6-naphthyridine-3-yl]-2-fluorophenyl]-3-phenylurea, or a pharmaceutical acceptable salt thereof, and an effective amount of one or a plurality of MAPKAP kinase inhibitors to the patient.SELECTED DRAWING: Figure 1-1

Description

Background Art

[0001] Cross-reference to Related Applications This application claims priority to U.S. Patent Application No. 62 / 624,448, filed on January 31, 2018, which is hereby incorporated by reference in its entirety.

[0002] c-KIT (also known as KIT, CD117, and stem cell factor receptor) is a 145 kDa transmembrane tyrosine kinase protein that acts as a type III receptor. The c-KIT proto-oncogene, located on chromosome 4q11-21, encodes the c-KIT receptor, and its ligand is stem cell factor (SCF, hematopoietic stem cell factor, kit ligand, mast cell growth factor). The receptor has tyrosine protein kinase activity, and binding of the ligand SCF leads to autophosphorylation of c-KIT and its association with substrates such as phosphatidylinositol 3-kinase (PI3K). Tyrosine phosphorylation by protein tyrosine kinases is particularly important in cell signaling and can mediate signals for major intracellular processes such as proliferation, survival, differentiation, apoptosis, adhesion, invasion, and migration.

[0003] The role of c-KIT expression and activity has been studied in hematological malignancies and solid tumors such as acute leukemia and gastrointestinal stromal tumors (GISTs). Most GISTs are associated with primary activating mutations in the genes encoding c-KIT (75–80% of GISTs), a closely related RTK, or PDGFRα (8% of non-c-KIT mutant GISTs), and the acquisition of gain-of-function mutations and expression of constitutively phosphorylated c-KIT in the c-KIT gene are seen in many GISTs. Most of the c-KIT primary mutations causing GISTs affect the juxtamembrane (JM) region of the protein encoded by exon 11 and consist of in-frame deletions or insertions, or missense mutations (i.e., V560D). Exon 11 mutations of c-KIT have been identified as primary mutations in approximately 65% of GISTs. Such JM domain mutations disrupt the autoinhibitory mechanism of the c-KIT kinase and lead to constitutive kinase activity and cellular transformation events in GISTs. Other c-KIT primary mutations causing GISTs are located in exon 9 (AY501–502 duplication / insertion, 8%), exon 13 (mutation, 1%), and exon 17 (mutation, 1%).

[0004] The clinical importance of c-KIT expression in malignant tumors was demonstrated in a study using Gleevec® (imatinib mesylate, STI571 (signal transduction inhibitor number 571), Novartis Pharma AG, Basel, Switzerland), which specifically inhibits the tyrosine kinase receptor. Furthermore, a clinically relevant breakthrough was the finding of the antitumor effect of this compound in GIST, a group of tumors generally regarded as resistant to conventional chemotherapy. However, while a major response is seen after first-line treatment of GIST with the c-KIT inhibitor, Gleevec®, and a significant number of patients with metastatic and / or inoperable GIST benefit from treatment with Gleevec®, complete tumor remission is rare, and approximately 50% of patients experience disease recurrence within 2 years of treatment. It has also been reported that the combination of the c-KIT inhibitor imatinib and the MEK kinase inhibitor MEK162 results in increased in vitro growth inhibition and in vivo tumor regression in various GIST cancer cell lines compared to treatment with either single agent.

[0005] GISTs most often become resistant to Gleevec® and Molecular target small molecule therapies targeting c-KIT secondary mutations remain elusive. GIST patients who relapse after treatment with Gleevec® or Sutent® still have diseases caused by c-KIT mutations. These secondary mutations occur on the same allele as the primary mutation in the JM region and thus represent a more invasive activated form of c-KIT than the original primary mutation. These c-KIT secondary mutations identified in GIST lead to acquired drug resistance. Secondary mutations are seen in the ATP binding pocket (exon 13, i.e., K642E, V654A; exon 14, i.e., T670I), and the activation loop (exon 17, i.e., N822K, D816H, D816V, D820A; exon 18 A829P). These various c-KIT secondary mutations have been reported as follows: sunitinib malate (Sutent®, Pfizer) is an inhibitor of multiple RTKs, particularly c-KIT and PDGFRα in this context, and has been shown to be effective against certain imatinib-resistant c-KIT mutants, such as the ATP-binding pocket mutants V654A and T670I. Certain Gleevec®-resistant mutants are also resistant to sunitinib, such as D816H and D816V, which are located in the activation loop of the c-KIT catalytic domain encoded by exon 17. The average survival period after progression due to Gleevec® resistance remains relatively short.

[0006] Within individual patients, it has been demonstrated that complex, multiple c-KIT secondary mutations occur and change, and such changes in the c-KIT mutation status have been demonstrated by biopsy samples obtained from various progressing metastases within each patient. The heterogeneous complexity of c-KIT mutations within individual patients highlights the unmet medical need to identify c-KIT kinase inhibitors that are effective over a broad range of primary and secondary c-KIT mutations. Furthermore, as opposed to being merely cytotoxic, there is a desire to identify therapies that are cytotoxic or cytocidal to c-KIT-mediated GIST and that consequently result in disease remission and / or a reduction in disease recurrence.

Summary of the Invention

[0007] The present disclosure is directed to the combined use of Compound A of a c-KIT inhibitor or Compound B of a c-KIT inhibitor with an inhibitor of the MAPKAP kinase signaling pathway. As used herein, the MAPKAP pathway is defined as signal transduction via the kinases RAF→MEK→ERK. The combined use of Compound A or Compound B of a c-KIT inhibitor with a MEK inhibitor containing trametinib, an ERK inhibitor containing ulixertinib, or a RAF inhibitor containing LY3009120 unexpectedly demonstrated that it leads to cell death, apoptosis or long-term cell quiescence of GIST cells, enhanced GIST tumor regression in vivo, or eradication of GIST cells in a colony formation assay, as compared to the combined use of imatinib with a MEK inhibitor or compared to monotherapy with Compound A, imatinib or a MEK inhibitor. Furthermore, it has been demonstrated that the combined use of Compound A of a c-KIT inhibitor with a MEK inhibitor leads to cell death or apoptosis and enhanced eradication of GIST cancer cell lines that are resistant to imatinib in combination with a MEK inhibitor. In a colony growth assay of GIST cells, Compound A exhibited excellent synergistic effects in combination with a MEK inhibitor as compared to imatinib in combination with a MEK inhibitor. The present disclosure relates, in part, to a method of treating a tumor in a patient using Compound A described herein or a pharmaceutically acceptable salt thereof.

[0008] For example, a method of treating a tumor with one or more c-KIT mutations in a patient in need thereof, comprising administering to the patient an effective amount of 1-[4-bromo-5-[1-ethyl-7-(methylamino)-2-oxo-1,2-dihydro-1,6-naphthyridin-3-yl]-2-fluorophenyl]-3-phenylurea or a pharmaceutically acceptable salt thereof, and an effective amount of a mitogen-activated protein kinase inhibitor (MEK inhibitor) and / or or an effective amount of an extracellular signal-regulated kinase inhibitor (ERK inhibitor).

[0009] The present disclosure also provides a method of treating a solid tumor in a patient resistant to imatinib, comprising administering to the patient an effective amount of 1-[4-bromo-5-[1-ethyl-7-(methylamino)-2-oxo-1,2-dihydro-1,6-naphthyridin-3-yl]-2-fluorophenyl]-3-phenylurea or a pharmaceutically acceptable salt thereof, and administering to the patient an effective amount of an MEK inhibitor or an ERK inhibitor selected from the group consisting of trametinib, binimetinib, cobimetinib, and ulixertinib, wherein the solid tumor is selected from the group consisting of lung adenocarcinoma, lung squamous cell carcinoma, glioblastoma, pediatric glioma, astrocytoma, sarcoma, gastrointestinal stromal tumor (GIST), and melanoma.

[0010] Also contemplated herein is a method of treating an imatinib-resistant gastrointestinal stromal tumor or an imatinib-resistant melanoma in a patient in need thereof, which comprises administering to the patient an effective amount of 1-[4-bromo-5-[1-ethyl-7-(methylamino)-2-oxo-1,2-dihydro-1,6-naphthyridin-3-yl]-2-fluorophenyl]-3-phenylurea, or a pharmaceutically acceptable salt thereof, and administering to the patient an effective amount of an MEK or ERK inhibitor selected from the group consisting of trametinib, binimetinib, cobimetinib, and ulixertinib.

[0011] The present disclosure also provides a method for treating solid tumors in a patient in need thereof, comprising administering to the patient an effective amount of 1-[4-bromo-5-[1-ethyl-7-(methylamino)-2-oxo-1,2-dihydro-1,6-naphthyridin-3-yl]-2-fluorophenyl]-3-phenylurea or a pharmaceutically acceptable salt thereof, and administering to the patient an effective amount of a MEK inhibitor or an ERK inhibitor selected from the group consisting of trametinib, binimetinib, cobimetinib, and ulixertinib, wherein the solid tumor is selected from the group consisting of lung adenocarcinoma, lung squamous cell carcinoma, glioblastoma, pediatric glioma, astrocytoma, sarcoma, gastrointestinal stromal tumor (GIST), and melanoma.

[0012] Also contemplated herein is a method for treating a gastrointestinal stromal tumor or melanoma in a patient in need thereof, which comprises administering to the patient an effective amount of 1-[4-bromo-5-[1-ethyl-7-(methylamino)-2-oxo-1,2-dihydro-1,6-naphthyridin-3-yl]-2-fluorophenyl]-3-phenylurea, or a pharmaceutically acceptable salt thereof, and administering to the patient an effective amount of a MEK or ERK inhibitor selected from the group consisting of trametinib, binimetinib, cobimetinib, and ulixertinib.

[0013] Further contemplated herein is the above-described method for treating solid tumors in a patient in need thereof, which comprises administering to the patient an effective amount of 1-[4-bromo-5-[1-ethyl-7-(methylamino)-2-oxo-1,2-dihydro-1,6-naphthyridin-3-yl]-2-fluorophenyl]-3-phenylurea, or a pharmaceutically acceptable salt thereof, and administering to the patient an effective amount of a RAF inhibitor. BRIEF DESCRIPTION OF THE DRAWINGS

[0014]

Figure 1-1

[0015]

Figure 1-2

[0016]

Figure 1-3

[0017] Figure 1D is a matrix diagram of synergism based on the combination index method for various treatments with compound A and trametinib for 48 hours in GIST-T1 cells. The combination index plot shows synergism graphed with the combination index (CI) on the y-axis and the fraction affected (Fa) on the x-axis.

[0018]

Figure 1-4

[0019] Figure 1F is a matrix diagram of synergism based on the combination index method for various treatments with compound A and trametinib for 24 hours in GIST-T1 / D816E imatinib-resistant cells. The combination index plot shows synergism graphed with the combination index (CI) on the y-axis and the fraction affected (Fa) on the x-axis.

[0020]

Figure 1-5

[0021] Figure 1H is a matrix diagram of synergism based on the combination index method for various treatments over 24 hours with compound A and trametinib in GIST-T1 / T670I imatinib-resistant cells. The combination index plot shows synergism graphed with the combination index (CI) on the y-axis and the fraction affected (Fa) on the x-axis.

[0022]

Figure 2-1

[0023] Figure 2B is a matrix diagram of synergism based on the combination index method for various treatments over 24 hours with compound B and trametinib in GIST-T1 cells. The combination index plot shows synergism graphed with the combination index (CI) on the y-axis and the fraction affected (Fa) on the x-axis.

[0024]

Figure 2-2

[0025] Figure 2D is a matrix diagram of synergism based on the combination index method for various treatments over 24 hours with compound B and trametinib in GIST-T1 / D816E imatinib-resistant cells. The combination index plot shows synergism graphed with the combination index (CI) on the y-axis and the fraction affected (Fa) on the x-axis.

[0026]

Figure 2-3

[0027] Figure 2F is a matrix diagram of synergism based on the combination index method for various treatments over 24 hours with compound B and trametinib in GIST-T1 / T670I imatinib-resistant cells. The combination index plot shows synergism graphed with the y-axis as the combination index (CI) and the x-axis as the fraction affected (Fa).

[0028]

Figure 3-1

[0029] Figure 3B is a matrix diagram of synergism based on the combination index method for various treatments over 24 hours with compound A and binimetinib in GIST-T1 cells. The combination index plot shows synergism graphed with the y-axis as the combination index (CI) and the x-axis as the fraction affected (Fa).

[0030]

Figure 3-2

[0031] Figure 3D is a matrix diagram of synergism based on the combination index method for various treatments over 24 hours with compound A and binimetinib in GIST-T1 / D816E imatinib-resistant cells. The combination index plot shows synergism graphed with the y-axis as the combination index (CI) and the x-axis as the fraction affected (Fa).

[0032]

Figure 3-3

[0033] Figure 3F is a matrix diagram of synergism based on the combination index method for various treatments over 24 hours with compound A and vinitumetinib in GIST-T1 / T670I imatinib-resistant cells. The combination index plot shows synergism graphed with the combination index (CI) on the y-axis and the fraction affected (Fa) on the x-axis.

[0034]

Figure 4-1

[0035] Figure 4B is a matrix diagram of synergism based on the combination index method for various treatments over 24 hours with compound B and vinitumetinib in GIST-T1 cells. The combination index plot shows synergism graphed with the combination index (CI) on the y-axis and the fraction affected (Fa) on the x-axis.

[0036]

Figure 4-2

[0037] Figure 4D is a matrix diagram of synergism based on the combination index method for various treatments over 24 hours with compound B and vinitumetinib in GIST-T1 / D816E imatinib-resistant cells. The combination index plot shows synergism graphed with the combination index (CI) on the y-axis and the fraction affected (Fa) on the x-axis.

[0038]

Figure 4-3

[0039] Figure 4F is a matrix diagram of synergism based on the combination index method for various treatments over 24 hours with compound B and vinitumetinib in GIST-T1 / T670I imatinib-resistant cells. The combination index plot shows synergism graphed with the combination index (CI) on the y-axis and the fraction affected (Fa) on the x-axis.

[0040]

Figure 5-1

[0041] Figure 5B is a matrix diagram of synergism based on the combination index method for various treatments over 24 hours with compound A and cobimetinib in GIST-T1 cells. The combination index plot shows synergism graphed with the combination index (CI) on the y-axis and the fraction affected (Fa) on the x-axis.

[0042]

Figure 5-2

[0043] Figure 5D is a matrix diagram of synergism based on the combination index method for various treatments over 24 hours with compound A and cobimetinib in GIST-T1 / D816E imatinib-resistant cells. The combination index plot shows synergism graphed with the combination index (CI) on the y-axis and the fraction affected (Fa) on the x-axis.

[0044]

Figure 5-3

[0045] Figure 5F is a matrix diagram of synergism based on the combination index method for various treatments over 24 hours with compound A and cobimetinib in GIST-T1 / T670I imatinib-resistant cells. The combination index plot shows synergism graphed with the y-axis as the combination index (CI) and the x-axis as the fraction affected (Fa).

[0046]

Figure 6-1

[0047] Figure 6B is a matrix diagram of synergism based on the combination index method for various treatments over 24 hours with compound B and cobimetinib in GIST-T1 cells. The combination index plot shows synergism graphed with the y-axis as the combination index (CI) and the x-axis as the fraction affected (Fa).

[0048]

Figure 6-2

[0049] Figure 6D is a matrix diagram of synergism based on the combination index method for various treatments over 24 hours with compound B and cobimetinib in GIST-T1 / D816E imatinib-resistant cells. The combination index plot shows synergism graphed with the y-axis as the combination index (CI) and the x-axis as the fraction affected ( Fa) and shows synergism graphed with the y-axis as the combination index (CI) and the x-axis as the fraction affected (Fa).

[0050]

Figure 6-3

[0051] Figure 6F is a matrix diagram of synergism based on the combination index method for various treatments over 24 hours with compound B and cobimetinib in GIST-T1 / T670I imatinib-resistant cells. The combination index plot shows synergism graphed with the combination index (CI) on the y-axis and the fraction affected (Fa) on the x-axis.

[0052]

Figure 7-1

[0053] Figure 7B is a matrix diagram of synergism based on the combination index method for various treatments over 24 hours with compound A and ulixertinib in GIST-T1 cells. The combination index plot shows synergism graphed with the combination index (CI) on the y-axis and the fraction affected (Fa) on the x-axis.

[0054]

Figure 7-2

[0055] Figure 7D is a matrix diagram of synergism based on the combination index method for various treatments over 24 hours with compound A and ulixertinib in GIST-T1 / T670I imatinib-resistant cells.

[0056]

Figure 8-1

[0057]

Figure 8-2

[0058]

Figure 8-3

[0059] Figure 8D shows an image of a representative culture plate and a graphical representation of the number of GIST-T1 / T670I colonies counted after various treatments with Compound A, imatinib, and trametinib over a two-week period followed by a ten-day recovery period.

[0060]

Figure 9-1

[0061]

Figure 9-2

[0062]

Figure 9-3

[0063]

Figure 10-1

[0064]

Figure 10-2

[0065]

Figure 10-3

[0066]

Figure 11-1

[0067]

Figure 11-2

[0068]

Figure 11-3

[0069]

Figure 12-1

[0070]

Figure 12-2

[0071]

Figure 12-3

[0072]

Figure 13-1

[0073]

Figure 13-2

[0074]

Figure 13-3

[0075]

Figure 14-1

[0076]

Figure 14-2

[0077]

Figure 14-3

[0078]

Figure 15

[0079]

Figure 16-1

[0080] Figure 16B shows images of representative culture plates of GIST-T1 colonies transfected with a vector control (Figure 16B.1) or N-ras G12D (Figure 16B.2) after various treatments with compound A, imatinib, and trametinib followed by a 10-day recovery period.

[0081]

Figure 16-2

[0082] Figure 16D shows an image of a representative culture plate of GIST-T1 colonies transfected with N-ras G12D after various treatments with compound A and trametinib followed by an extended 21 day recovery period.

[0083]

Figure 17

[0084] Figure 17B is a graphical representation of Ba / F3 V560D KIT cell proliferation with a c-KIT secondary mutation at T670I, K807E, or D816V in saturation mutagenesis, or cell proliferation retaining only the original c-KIT V560D mutation combined with additional non-c-KIT resistance mechanisms, following combination treatment with either imatinib and trametinib (left panel) or compound A and trametinib (right panel).

[0085]

Figure 18

[0086] Figure 18B is an extension of the graphical representation from Figure 18A, showing the restoration of the effect on tumor regression following treatment with single-agent compound A, single-agent trametinib, or a combination of compound A and trametinib.

MODE FOR CARRYING OUT THE INVENTION

[0087] 〔00087〕1-[4-Bromo-5-[1-ethyl-7-(methylamino)-2-oxo -1,2-dihydro-1,6-naphthyridin-3-yl]-2-fluorophenyl]-3-phenylurea (Compound A) in combination with a MAPKAP kinase pathway inhibitor such as trametinib unexpectedly exhibits a synergistic effect, leading to cell death, apoptosis, or long-term cell quiescence of GIST cells, inducing eradication of tumor cells, inducing tumor regression, reducing tumor volume, inhibiting tumor regrowth, and / or enhancing cell death, apoptosis, or cell quiescence or eradication of GIST cancer cell lines resistant to imatinib when combined with a MEK inhibitor in the attached examples. Additionally, the combination therapies disclosed herein appear to be cytotoxic, as opposed to merely cytostatic.

[0088] While not wishing to be bound by any particular theory, many c-KIT inhibitors are thought to inhibit only specific mutant forms of c-KIT, such as the prominent exon 11 mutations observed in GIST. Other mutant forms of c-KIT are resistant to many c-KIT inhibitors and often arise as secondary mutations in exons 13, 14, 17, and 18, giving rise to tumors that are resistant to treatment with c-KIT inhibitors. The present disclosure provides a method of treating tumors such as c-KIT-mediated tumors like GIST by inhibiting both c-KIT and the MAPKAP pathway kinase using a c-KIT inhibitor disclosed herein as Compound A or a pharmaceutically acceptable salt thereof or Compound B or a pharmaceutically acceptable salt thereof. Surprisingly, Compound A and Compound B (and their pharmaceutically acceptable salts) exhibit a synergistic effect with a MEK inhibitor, an ERK inhibitor, or a RAF inhibitor, leading to cell death, apoptosis, or long-term cell quiescence of GIST cells, inducing eradication of tumor cells to the limit of detection, reducing tumor volume, inhibiting tumor regrowth, and / or enhancing cell death, apoptosis, cell quiescence, or eradication to the limit of detection of GIST cancer cell lines resistant to imatinib when combined with a MAPKAP kinase inhibitor. Compound A is c-KIT resistant In GIST cells containing mutations, it shows excellent efficacy and synergy when combined with MEK inhibition, compared to imatinib combined with MEK inhibition. Furthermore, the level of efficacy and synergy of compound A combined with MEK inhibition, as well as the degree of long-term cell stasis or eradication of GIST tumor cells, is superior to that of imatinib combined with MEK inhibition, even for cell lines known to be sensitive to imatinib. Without wishing to be bound by any particular theory again, compound A may act through a mechanism involving inhibition of drug efflux pumps, including the BCRP efflux pump, in tumor cells such as GIST cells. It is considered that compound A can inhibit a wider range of mutant forms of c-KIT than previous c-KIT inhibitors, including imatinib, in GIST cells. Imatinib is a substrate of the BCRP efflux pump, leading to a decrease in intracellular concentration in tumor cells where this efflux pump is present (Eechoute, K, et al, Clin Cancer Res. 2015, 17, 406 - 15). GIST tumors have been demonstrated to have overexpression of the BCRP efflux pump in 93% (42 / 45) of the GIST patient tumors evaluated (Feldman, R, et al. J Clin Oncol. 2015, 33, 58). Compound A is a potent inhibitor of the BCRP efflux transporter, and the IC 50 value shows 40 nM.

[0089] Accordingly, in certain embodiments, the present disclosure provides a method for inducing long-term cell quiescence, inducing cell death, inducing apoptosis of tumor cells, inducing eradication of tumor cells, inducing tumor regression, reducing tumor volume, inhibiting tumor regrowth, or inhibiting the growth of drug-resistant tumor cells, the method comprising administering to a patient in need thereof an effective amount of: (i) 1-[4-bromo-5-[1-ethyl-7-(methylamino)-2-oxo-1,2-dihydro-1,6-naphthyridin-3-yl]-2-fluorophenyl]-3-phenylurea or a pharmaceutically acceptable salt thereof, or 1-(5-(7-amino-1-ethyl-2-oxo-1,2-dihydro-1,6-naphthyridin-3-yl)-4-bromo-2-fluorophenyl)-3-phenylurea or a pharmaceutically acceptable salt thereof; and (ii) a MAPKAP kinase inhibitor, such as trametinib, binimetinib or cobimetinib, which are MEK inhibitors, ulixertinib, which is an ERK inhibitor, or a RAF inhibitor. In certain embodiments of any of the methods disclosed herein, the tumor is a c-KIT-mediated solid tumor, such as c-KIT-mediated GIST or melanoma. Definitions

[0090] 〔00090〕As used herein, Compounds A and B refer to 1-[4 -bromo-5-[1-ethyl-7-(methylamino)-2-oxo-1,2-dihydro-1,6-naphthyridin-3-yl]-2-fluorophenyl]-3-phenylurea, and 1-(5-(7-amino-1-ethyl-2-oxo-1,2-dihydro-1,6-naphthyridin-3-yl)-4-bromo-2-fluorophenyl)-3-phenylurea, respectively. Pharmaceutically acceptable salts, tautomers, hydrates, and solvates of Compounds A and B are also contemplated in the present disclosure. The structures of Compounds A and B are shown below:

Chemical formula

Chem.

[0091] The methods for preparing Compound A and Compound B are disclosed in U.S. Patent No. 8,461,179 B1, the content of which is incorporated herein by reference.

[0092] Exemplary methods and materials are described herein. In the specification and the appended claims, unless the context clearly dictates otherwise, the singular forms also include the plural forms. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains.

[0093] Throughout this disclosure, various patents, patent applications, and published publications are referenced. The disclosures of these patents, patent applications, and published publications are hereby incorporated by reference in their entirety to more particularly describe the state of the art known to those of ordinary skill in the art as of the date of this disclosure. In the event of any inconsistency between those patents, patent applications, and published publications and this disclosure, this disclosure shall control.

[0094] For convenience, the specific terms employed in this specification, examples, and claims are summarized herein. Unless otherwise defined, all technical and scientific terms used in this disclosure have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. The first definition presented for a group or term presented in this disclosure applies to that group or term individually, or as part of another group, throughout this disclosure unless otherwise indicated.

[0095]

[00095] "Pharmaceutically acceptable carrier, diluent, or excipient" includes any of the following: The term "adjuvant," "carrier," "excipient," "glidant," "sweetener," "diluent," "preservative," "dye / colorant," "flavor enhancer," "surfactant," "wetting agent," "dispersing agent," "suspending agent," "stabilizer," "isotonic agent," "solvent," or "emulsifier" may be used interchangeably with "adjuvant," "carrier," "excipient," "glidant," "sweetener," "diluent," "preservative," "dye / colorant," "flavor enhancer," "surfactant," "wetting agent," "dispersing agent," "suspending agent," "stabilizer," "isotonic agent," "solvent," or "emulsifier" or may be used interchangeably.

[0096] "Pharmaceutically acceptable salts" include acid addition salts.

[0097] "Pharmaceutically acceptable acid addition salts" refers to salts that retain the biological effectiveness and properties of the free bases, which are not biologically or otherwise undesirable, and include, for example, but not limited to, inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like; and for example, but not limited to, acetic acid, 2,2-dichloroacetic acid, adipic acid, alginic acid, ascorbic acid, aspartic acid, benzenesulfonic acid, benzoic acid, 4-acetamidobenzoic acid, camphoric acid, camphor-10-sulfonic acid, capric acid, caproic acid, caprylic acid, carbonic acid, cinnamic acid, citric acid, cyclamic acid, dodecyl sulfuric acid, ethane-1,2-disulfonic acid, ethanesulfonic acid, 2-hydroxyethanesulfonic acid, It is formed of organic acids such as formic acid, fumaric acid, galactaric acid, gentisic acid, glucoheptonic acid, gluconic acid, glucuronic acid, glutamic acid, glutaric acid, 2-oxo-glutaric acid, glycerophosphoric acid, glycolic acid, hippuric acid, isobutyric acid, lactic acid, lactobionic acid, lauric acid, maleic acid, malic acid, malonic acid, mandelic acid, methanesulfonic acid, mucic acid, naphthalene-1,5-disulfonic acid, naphthalene-2-sulfonic acid, 1-hydroxy-2-naphthoic acid, nicotinic acid, oleic acid, orotic acid, oxalic acid, palmitic acid, pamoic acid, propionic acid, pyroglutamic acid, pyruvic acid, salicylic acid, 4-aminosalicylic acid, sebacic acid, stearic acid, succinic acid, tartaric acid, thiocyanic acid, p-toluenesulfonic acid, trifluoroacetic acid, undecylenic acid, etc.

[0098] "Pharmaceutical composition" refers to a formulation of a compound described herein, such as compound A or a pharmaceutically acceptable salt thereof, and a biologically active compound with a medium generally acceptable in the art for delivery to a mammal, such as a human. Such media include all pharmaceutically acceptable carriers, diluents, or excipients therefor.

[0099] For example, in the combination therapy of the present disclosure which is compound A combined with a MEK inhibitor, patients or subjects "in need of treatment" include patients having diseases and / or symptoms that can be treated with the combinations disclosed herein to achieve beneficial treatment outcomes, such as GIST patients. Beneficial outcomes include objective response, increased progression-free survival, increased overall survival, long-term stable disease, and / or reduction in symptom severity or delay in symptom onset. In certain embodiments, the patient in need of treatment is troubled by tumor growth or tumor progression, and the patient is troubled by, but not limited to, lung adenocarcinoma, lung squamous cell carcinoma, glioblastoma, pediatric glioma, astrocytoma, sarcoma, melanoma, or gastrointestinal stromal tumor.

[0100] 〔00100〕When used in connection with the compounds or other therapeutic agents disclosed herein, " The term "effective amount" refers to an amount of a therapeutic agent, e.g., Compound A or a MEK inhibitor, alone or in combination, that is useful for treating or preventing a disease or disorder. The effective amount of a therapeutic agent used in combination therapy is an amount of each therapeutic agent that is useful for treating or preventing a disease or disorder when used in combination therapy, even if the amount of one or both of the therapeutic agents in the absence of the other agent is not effective in treating or preventing the disease or disorder. In certain embodiments, the effective amount results in long-term cytostasis of GIST cells, cytocidal GIST cell death, apoptosis of GIST cells, eradication of GIST cells, regression of GIST, reduction of GIST tumor volume, inhibition of GIST regrowth, and / or long-term cytostasis, cell death, apoptosis, or eradication to the limit of detection of GIST cancer cell lines resistant to imatinib in combination with a MEK inhibitor, and / or leads to beneficial clinical outcomes for the symptoms treated with this compound as compared to not being treated. The "effective amount" can vary depending on the form of administration, the particular location of the disease or disorder, and the age, weight, and general health of the subject. The amount of the compound to be administered will depend on the degree, severity, and type of the disease or condition, the amount of treatment desired, and the release characteristics of the pharmaceutical formulation. It will also depend on the health status, size, weight, age, gender, and tolerance to the agent of the subject. Typically, the compound is administered for a period of time sufficient to achieve the desired therapeutic effect.

[0101] The terms "treat", "treating", and "treatment" refer to inducing long-term cytostasis of GIST cells, inducing cytocidal GIST cell death, inducing apoptosis of GIST cells, inducing eradication of GIST tumor cells to the limit of visual detection as determined by stereomicroscopy at 5x magnification, causing regression of GIST tumors in a patient, reducing GIST tumor volume, inhibiting GIST regrowth, and / or alleviating, delaying, or regressing, e.g., one or more symptoms as described herein Administering the combination therapy disclosed herein, etc., inhibiting the growth of resistant GIST cells in a given treatment, and inducing regression of GIST even if the GIST cannot actually be removed, which means including the entire scope of intervention in patients suffering from "cancer" with the intention of doing so. In some embodiments, treatment includes, for example, completely removing the disease or disorder, such as GIST. Treating may also be curing, improving, or at least partially alleviating the disorder.

[0102] As used herein, "cancer" refers to a neoplasm that has the ability to invade surrounding tissues and metastasize (spread to other organs), and ultimately, if untreated, can cause death to the patient. In certain embodiments, the cancer can be a solid tumor.

[0103] As used herein, "tumor" refers to a neoplasm. This is a term that may refer to a benign (generally harmless) or malignant (cancerous) growth. Malignant growths can originate from solid organs or the bone marrow.

[0104] "Tumor growth" as defined herein refers to the growth of tumors caused by genomic mutations of the c-KIT gene, which can alter the expression and / or activity of the c-KIT protein.

[0105] 〔00105〕"Tumor progression" as defined herein refers to, for example, the growth of existing c-KIT-dependent tumors such as GIST, where such growth of the existing tumor can be caused by further genomic alterations of c-KIT that confer resistance to treatment. KIT-dependent tumors, in which case such growth of the existing tumor can be caused by further genomic alterations of c-KIT that confer resistance to treatment.

[0106] "Tumor regression", "complete remission", and "partial response" as defined herein refer to a decrease in tumor size determined by weight or volume as determined by the RECIST 1.1 or Choi criteria.

[0107] The eradication of an existing c-KIT-mediated tumor, such as a c-KIT-mediated GIST, is defined as "complete cytocidal cell death" of the tumor to the limit of detection as determined by solid microscopy at 5-fold for in vitro evaluation, or as complete remission as determined by RECIST 1.1 or Choi's criteria for in vivo preclinical or clinical evaluation without the possibility of tumor regrowth under preclinical or clinical conditions. Thus, "eradication of a c-KIT-mediated tumor" indicates that all cells of the c-KIT-mediated tumor are killed or removed to the limit of detection without the possibility of c-KIT-mediated tumor regrowth.

[0108] As used herein, "tumor regrowth" refers to the growth of a tumor that has previously ceased or regressed in growth after treatment, such as with Gleevec® or Sutent®. In certain embodiments, tumor regrowth occurs due to the introduction of a secondary c-Kit mutation in the tumor cells. In other embodiments, tumor regrowth occurs due to the activation or mutation of a different signaling pathway, including but not limited to the activation of the MAPKAP signaling pathway that includes signal transduction via the MEK kinase.

[0109] As used herein, "cell stasis" refers to cells that have stopped dividing and remain in a quiescent, non-replicating state.

[0110] 〔00110〕As used herein, "apoptosis" refers to programmed cell death Characteristics of apoptosis detectable by histological and histochemical methods include cell shrinkage, increased membrane permeability, condensation of the nucleus and cytoplasm, endolytic cleavage of nuclear DNA into oligonucleosomal fragments, and by macrophages It includes the final formation of apoptotic bodies that are engulfed and removed. Apoptosis is mainly mediated by caspases, which are aspartic acid-specific serine proteases. Apoptosis can be induced through an essential genetic programming in response to various conditions, such as DNA damage or the removal of growth factors, or apoptosis can be induced by external factors such as irradiation-induced damage to cellular DNA and some cytotoxic drugs used in cancer treatment. It can be inhibited by endogenous factors (such as cytokines) and some drugs (such as protease inhibitors). Apoptosis typically does not occur or is impaired in malignant cells. In certain embodiments, apoptosis refers to programmed cell death determined by an increase in cleaved and activated caspases 3 and 7.

[0111] "Combination therapy" is a treatment that includes the administration of two or more therapeutic agents, such as compound A and a MEK inhibitor, to a patient. The two or more therapeutic agents may be delivered simultaneously, for example, in separate pharmaceutical compositions or in the same pharmaceutical composition, or the two or more therapeutic agents may be delivered at different times. For example, the two or more therapeutic agents may be delivered simultaneously or during overlapping times, and / or one therapeutic agent may be delivered before or after the other therapeutic agent. Treatment involving the combination of a KIT inhibitor, such as compound A, and a MEK inhibitor may include treatment with either single agent, where the period of concurrent treatment with both agents precedes or follows. However, during some period, an effective amount of two or more therapeutic agents is intended to be present in the patient's body.

[0112] The "MAPKAP pathway inhibitor" is an inhibitor of the MAP kinase signaling pathway. Inhibitors of this pathway include RAS inhibitors, RAF inhibitors (e.g., dabrafenib, vemurafenib, LY3009120), MEK inhibitors (e.g., trametinib, binimetinib, cobimetinib), and ERK inhibitors (e.g., ulixertinib). The terms "MAPKAP pathway inhibitor" and "MAPKAP kinase inhibitor" are used interchangeably herein. Treatment method

[0113] The compounds and compositions described herein can be used to treat the tumors of patients in need thereof. For example, provided is a method of treating a tumor with one or more c-KIT mutations in a patient in need thereof, the method comprising administering to the patient an effective amount of 1-[4-bromo-5-[1-ethyl-7-(methylamino)-2-oxo-1,2-dihydro-1,6-naphthyridin-3-yl]-2-fluorophenyl]-3-phenylurea, or a pharmaceutically acceptable salt thereof, and an effective amount of one or more MAPKAP kinase inhibitors. In one embodiment, the MAPKAP kinase inhibitor is selected from the group consisting of a mitogen-activated protein kinase inhibitor (MEK inhibitor) and an effective amount of an extracellular signal-regulated kinase inhibitor (ERK inhibitor).

[0114] The c-KIT mutation can be a primary mutation in exon 9, exon 11, exon 13, or exon 17 of the c-KIT gene. In another embodiment, the c-KIT mutation is a deletion mutation.

[0115]

[00115] Further, the tumor is one or more resistant secondary mutations in the c-KIT gene It may be accompanied by differences. In some embodiments, the resistant secondary mutation is in exon 13, exon 14, exon 17, or exon 18 of the c-KIT gene. In some embodiments, the resistant secondary mutation is in exon 17 of the c-KIT gene. In some embodiments, the resistant secondary mutation is a substitution of aspartic acid at codon 816, or a substitution of asparagine at codon 822. In some embodiments, the resistant secondary mutation is one of D816V, D816E, D816H, D8 20A, T670I, or N822V. In some embodiments, the resistant secondary mutation is acquired after prior administration of imatinib, sunitinib or regorafenib, or a pharmaceutically acceptable salt thereof, to the patient.

[0116] Such disclosed methods further include determining whether the tumor is accompanied by a c-KIT secondary mutation. In some embodiments, determining whether the tumor is accompanied by a c-KIT secondary mutation includes identifying the mutation in the DNA extracted from the tumor sample. In some embodiments, determining whether the tumor is accompanied by a c-KIT secondary mutation includes identifying the mutation in the circulating tumor DNA. In another embodiment, the tumor showed resistance to treatment with imatinib mesylate, sunitinib malate, or regorafenib.

[0117] Furthermore, the tumor can be selected from the group consisting of lung adenocarcinoma, lung squamous cell carcinoma, glioblastoma, pediatric glioma, astrocytoma, sarcoma, gastrointestinal stromal tumor (GIST), and melanoma. In some embodiments, the tumor is melanoma. In some embodiments, the tumor is GIST.

[0118] This method may further include administering to the patient a cancer targeted therapeutic agent, a cancer targeted biologic agent, an immune checkpoint inhibitor, and / or a chemotherapeutic agent. The method may also further include administering to the patient a RAF inhibitor.

[0119] In another embodiment, 1-[4-bromo-5-[1-ethyl-7-(methylamino)-2-oxo-1,2-dihydro-1,6-naphthyridin-3-yl]-2-fluorophenyl-3-phenylurea, or a pharmaceutically acceptable salt thereof, and the MAPKAP kinase inhibitor are administered substantially simultaneously or sequentially.

[0120]

[00120] The MEK inhibitors in the methods of this disclosure include trametinib, selumetinib, co- In some embodiments, the MEK inhibitor is selected from the group consisting of bimetinib and binimetinib. In some embodiments, the MEK inhibitor is binimetinib. In some embodiments, the MEK inhibitor is trametinib. In some embodiments, the ERK inhibitor is selected from the group consisting of ulixertinib, SCH772984, and LY3214996.

[0121] Administration according to these disclosed methods for two or more weeks can result in a patient experiencing at least a 30% partial reduction in tumor volume. In some embodiments, treatment results in a complete reduction in tumor volume.

[0122] The disclosed methods may further include determining whether the tumor or tumor cells contain a primary mutation in the c-KIT gene. In some embodiments, the primary mutation is in exon 11 of the c-KIT gene. In some embodiments, the primary mutation is in exon 9 of the c-KIT gene. In some embodiments, the primary mutation is a deletion mutation. In some embodiments, the primary mutation is V560D. In other embodiments, one or more additional secondary c-KIT mutations are present.

[0123] A method for treating solid tumors in patients resistant to imatinib, comprising administering to a patient an effective amount of 1-[4-bromo-5-[1-ethyl-7-(methylamino)-2-oxo-1,2-dihydro-1,6-naphthyridin-3-yl]-2-fluorophenyl]-3-phenylurea or a pharmaceutically acceptable salt thereof, and administering to the patient an effective amount of a MAPKAP kinase inhibitor selected from the group consisting of trametinib, binimetinib, cobimetinib, and ulixertinib. The solid tumor is selected from the group consisting of lung adenocarcinoma, lung squamous cell carcinoma, glioblastoma, pediatric glioma, astrocytoma, sarcoma, gastrointestinal stromal tumor (GIST), and melanoma. In some embodiments, the method further comprises administering a RAF inhibitor. In some embodiments, the RAF inhibitor is a pan-RAF inhibitor. Also provided is the above method comprising administering an effective amount of a MAPKAP kinase inhibitor selected from the group consisting of trametinib, binimetinib, cobimetinib, and ulixertinib. The solid tumor is selected from the group consisting of lung adenocarcinoma, lung squamous cell carcinoma, glioblastoma, pediatric glioma, astrocytoma, sarcoma, gastrointestinal stromal tumor (GIST), and melanoma. In some embodiments, the method further comprises administering a RAF inhibitor. In some embodiments, the RAF inhibitor is a pan-RAF inhibitor.

[0124] Also provided herein is a method for treating imatinib-resistant gastrointestinal stromal tumor or imatinib-resistant melanoma in a patient in need thereof, comprising administering to the patient an effective amount of 1-[4-bromo-5-[1-ethyl-7-(methylamino)-2-oxo-1,2-dihydro-1,6-naphthyridin-3-yl]-2-fluorophenyl]-3-phenylurea, or a pharmaceutically acceptable salt thereof, and administering to the patient an effective amount of a MAPKAP kinase inhibitor selected from the group consisting of trametinib, binimetinib, cobimetinib, and ulixertinib.

[0125] 〔00125〕In some embodiments, the method is accompanied by a mutation in the c-KIT gene Further comprising determining whether or not. In some embodiments, the mutation is in exon 17 of the c-KIT gene. In some embodiments, the c-KIT mutation is a substitution of aspartic acid at codon 816, or a substitution of asparagine at codon 822. In some embodiments, the mutation is one of D816V, D816E, D816H, D820A, T670I, or N822V.

[0126] Furthermore, provided is a method of treating solid tumors in a patient in need thereof, comprising administering to the patient an effective amount of 1-[4-bromo-5-[1-ethyl-7-(methylamino)-2-oxo-1,2-dihydro-1,6-naphthyridin-3-yl]-2-fluorophenyl]-3-phenylurea, or a pharmaceutically acceptable salt thereof, and administering to the patient an effective amount of a RAF inhibitor.

[0127] In such disclosed methods, the solid tumor can be selected from the group consisting of lung adenocarcinoma, lung squamous cell carcinoma, GIST, and melanoma. In some embodiments, the solid tumor is associated with one or more mutations of the c-KIT gene.

[0128] Furthermore, the RAF inhibitor can be a pan-RAF inhibitor. In another embodiment, the RAF inhibitor is dabrafenib, vemurafenib, or LY3009120.

[0129] A method for treating a solid tumor in a patient in need thereof, the method comprising administering to the patient an effective amount of 1-[4-bromo-5-[1-ethyl-7-(methylamino)-2-oxo-1,2-dihydro-1,6-naphthyridin-3-yl]-2-fluorophenyl]-3-phenylurea or a pharmaceutically acceptable salt thereof, and administering to the patient an effective amount of a MAPKAP kinase inhibitor selected from the group consisting of trametinib, binimetinib, cobimetinib, and ulixertinib, wherein the solid tumor is selected from the group consisting of lung adenocarcinoma, lung squamous cell carcinoma, glioblastoma, pediatric glioma, astrocytoma, sarcoma, gastrointestinal stromal tumor (GIST), and melanoma. In some embodiments, the method further comprises administering a RAF inhibitor. In some embodiments, the RAF inhibitor is a pan-RAF inhibitor.

[0130]

[00130] As used herein, a method for treating a gastrointestinal stromal tumor or melanoma in a patient in need thereof, the method comprising administering to the patient an effective amount of 1-[4-bromo-5-[1-ethyl-7-(methylamino)-2-oxo-1,2-dihydro-1,6-naphthyridin-3-yl]-2-fluorophenyl]-3-phenylurea, or a pharmaceutically acceptable salt thereof, and administering to the patient an effective amount of a MAPKAP kinase inhibitor selected from the group consisting of trametinib, binimetinib, cobimetinib, and ulixertinib.

[0131] In some embodiments, the method further comprises determining whether the tumor is associated with a mutation in the c-KIT gene. In some embodiments, the mutation is in exon 17 of the c-KIT gene. In some embodiments, the c-KIT mutation is a substitution of aspartic acid at codon 816, or a substitution of asparagine at codon 822. In some embodiments, the mutation is one of D816V, D816E, D816H, D820A, T670I, or N822V.

[0132] In one embodiment, the present disclosure provides a method for treating or preventing a tumor, optionally a c-KIT mediated tumor, in a patient, such as a GIST, the method comprising administering to a patient in need thereof, an effective amount of Compound A or a pharmaceutically acceptable salt thereof, in combination with an effective amount of a MEK inhibitor, such as trametinib. In related embodiments, the present disclosure provides a method for treating or preventing a tumor, optionally a c-KIT mediated tumor, in a patient, such as a GIST, the method comprising administering to a patient in need thereof, an effective amount of Compound B or a pharmaceutically acceptable salt thereof, in combination with an effective amount of a MEK inhibitor, such as trametinib.

[0133] In certain embodiments, these methods include methods related to, for example, inducing long-term quiescence of tumor cells such as GIST cells, killing tumor cells such as GIST cells, inducing apoptosis of tumor cells such as GIST cells, inducing eradication of tumor cells to the limit of detection such as GIST cells, inducing regression of tumor cells such as regression of GIST, decreasing tumor volume such as GIST tumor volume, inhibiting tumor regrowth such as regrowth of GIST. In another specific embodiment, these methods include methods of inducing long-term quiescence of tumor cells such as GIST cells. In another specific embodiment, these methods include methods of killing tumor cells such as GIST cells. In another specific embodiment, these methods include methods of inducing apoptosis of tumor cells such as GIST cells. In another specific embodiment, these methods include methods of inducing eradication of tumor cells to the limit of detection such as GIST cells. In another specific embodiment, these methods include methods of inducing tumor regression such as regression of GIST. In another specific embodiment, these methods include methods of decreasing tumor volume such as GIST tumor volume. In another specific embodiment, these methods include methods of inhibiting tumor regrowth such as regrowth of GIST. In another specific embodiment, these methods include methods of inhibiting the growth of drug-resistant tumor cells such as drug-resistant GIST cells. In certain embodiments, this method includes a method of eradicating tumors to the limit of detection, such as GIST, in a subject. In any particular embodiment of the methods disclosed herein, tumor growth or tumor progression in a patient is caused by c-KIT overexpression, structural phosphorylation of c-KIT, increased c-KIT activity, oncogenic c-KIT missense mutations, oncogenic deletion c-KIT mutations, oncogenic nucleotide duplications / insertions, oncogenic c-KIT gene rearrangements leading to c-KIT fusion proteins, in-frame deletions within the c-KIT gene, and / or oncogenic c-KIT gene amplifications. In one embodiment, tumor growth or tumor progression is caused by structural phosphorylation of c-KIT.In certain embodiments, the tumor comprises one or more of the c-KIT primary activation mutations and / or c-KIT secondary mutations disclosed herein. In another particular embodiment, the tumor comprises one or more mutations in genes other than c-KIT that cause tumor growth by signaling through the MAPKAP pathway involved in RAF, MEK or ERK kinase activation.

[0134] When the methods described herein refer to treatment with compound A or a pharmaceutically acceptable salt thereof, or with compound B or a pharmaceutically acceptable salt thereof, it means that only one of compound A or a pharmaceutically acceptable salt thereof, or compound B or a pharmaceutically acceptable salt thereof is required. However, it is understood that these methods include administering both compound A or a pharmaceutically acceptable salt thereof, and compound B or a pharmaceutically acceptable salt thereof, to a patient in combination with a MEK inhibitor, an ERK inhibitor or a RAF inhibitor. Furthermore, when compound A is administered to a subject in combination with a MEK inhibitor, an ERK inhibitor or a RAF inhibitor, it is understood that a portion of the amount of compound A is metabolized in vivo to compound B, and that the in vivo mixture of compound A and compound B can also be used in combination with a MEK inhibitor, an ERK inhibitor or a RAF inhibitor to effectively treat a subject.

[0135] 〔00135〕Exemplary MEK inhibitors that can be used in accordance with the methods and compositions of the present disclosure include, but are not limited to, trametinib, selumetinib, cobimetinib, and binimetinib.

[0136] Exemplary ERK inhibitors that can be used in accordance with the methods and compositions of the present disclosure include, but are not limited to, ulixertinib, SCH772984, LY3214996, larotrectinib, and VX-11e.

[0137] ​​Exemplary RAF inhibitors that can be used in accordance with the methods and compositions of the present disclosure include, but are not limited to, LY3009120, dabrafenib, and vemurafenib.

[0138] In one embodiment, Compound A or a pharmaceutically acceptable salt thereof and a MEK inhibitor, such as trametinib, are administered to a patient suffering from a c-KIT-mediated tumor, such as GIST. In another embodiment, Compound B or a pharmaceutically acceptable salt thereof and a MEK inhibitor, such as trametinib, are administered to a patient suffering from a c-KIT-mediated tumor, such as GIST.

[0139] In related embodiments, Compound A or a pharmaceutically acceptable salt thereof and a MEK inhibitor, such as trametinib, are administered to a patient suffering from a tumor, such as a patient having GIST, wherein tumor growth or tumor progression is caused by a c-KIT primary activating mutation and / or a c-KIT secondary mutation. In another embodiment, Compound B or a pharmaceutically acceptable salt thereof and a MEK inhibitor, such as trametinib, are administered to a patient suffering from a tumor, such as a patient having GIST, wherein tumor growth or tumor progression is caused by a c-KIT primary activating mutation and / or a c-KIT secondary mutation. In certain embodiments, the c-KIT primary activating mutation is an exon 11 mutation (e.g., 57 base pair exon 11 deletion). In certain embodiments, the c-KIT primary activating mutation is a duplication of A~Y502~503 in exon 9. In certain embodiments, the c-KIT primary activating mutation is an exon 13 mutation. In certain embodiments, the c-KIT primary activating mutation is an exon 17 mutation. In certain embodiments, the c-KIT secondary mutation is any resistance mutation disclosed herein, such as the T670I mutation or the D816E mutation. In certain embodiments, multiple c-KIT secondary resistance mutations are present simultaneously in the subject.

[0140]

[00140] In certain embodiments, compound A or a pharmaceutically acceptable salt thereof and In certain embodiments, Compound B or a pharmaceutically acceptable salt thereof and a MEK inhibitor, such as trametinib, are administered to a cancer patient. In certain embodiments, Compound B or a pharmaceutically acceptable salt thereof and a MEK inhibitor, such as trametinib, are administered to a cancer patient. In embodiments, the tumor or cancer is lung adenocarcinoma, lung squamous cell carcinoma, glioblastoma, pediatric glioma, astrocytoma, sarcoma, melanoma, or gastrointestinal stromal tumor (GIST). In one embodiment, the cancer is melanoma. In another embodiment, the tumor or cancer is gastrointestinal stromal tumor (GIST). In certain embodiments of any of these methods, the tumor or cancer is a c-KIT-mediated cancer, e.g., c-KIT-mediated GIST or melanoma.

[0141] Treatment by combination of Compound A or a pharmaceutically acceptable salt thereof, or Compound B or a pharmaceutically acceptable salt thereof, with a MEK inhibitor such as trametinib involves administering Compound A or a pharmaceutically acceptable salt thereof or Compound B or a pharmaceutically acceptable salt thereof before, after, simultaneously with or during a period overlapping with the administration of the MEK inhibitor. It is understood that the effective amount of any of Compound A or a pharmaceutically acceptable salt thereof, Compound B or a pharmaceutically acceptable salt thereof, or a MEK inhibitor such as trametinib may vary when used in the combination disclosed herein as compared to when any of these agents is used alone for the same purpose, for example, for treating or preventing a tumor. In certain embodiments, the effective amount of Compound A or a pharmaceutically acceptable salt thereof, or Compound B or a pharmaceutically acceptable salt thereof, is less when administered as a combination therapy with a MEK inhibitor such as trametinib than when administered as a monotherapy, for example, for treating or preventing GIST. In certain embodiments, the effective amount of a MEK inhibitor such as trametinib is less when administered in combination therapy with Compound A or a pharmaceutically acceptable salt thereof or in combination therapy with Compound B or a pharmaceutically acceptable salt thereof, for example, for treating or preventing GIST.

[0142] Any of the methods disclosed herein may further include determining whether the tumor to be treated is associated with one or more c-KIT gene mutations. Such determination can be made by conventional methods for determining the presence of gene mutations in a biological sample, such as a tumor sample, blood sample, or plasma sample, obtained from a patient. Further, such determination can be made by reviewing the results of a test performed to determine the presence of one or more c-KIT gene mutations in a biological sample, such as a tumor sample, blood sample, or plasma sample, obtained from a patient. In certain embodiments of any of the methods disclosed herein, these methods are performed on patients identified as having tumors associated with one or more c-KIT gene mutations. The c-KIT gene mutations include, but are not limited to, any of those specifically described herein.

[0143] In various aspects of any of the methods disclosed herein, treatment with either compound A or a pharmaceutically acceptable salt thereof, or compound B or a pharmaceutically acceptable salt thereof, in combination with, for example, a MEK inhibitor such as trametinib, induces long-term cytostasis of tumor cells, such as GIST cells, induces death of tumor cells, such as GIST cells, induces apoptosis of tumor cells, such as GIST cells, induces eradication of tumor cells to the limit of detection, such as GIST cells, induces regression of tumor cells, such as GIST cells, decreases the weight or volume of a tumor, such as a GIST tumor, and inhibits regrowth of a tumor, such as a GIST tumor. In another aspect of any of the methods disclosed herein, treatment with either compound A or a pharmaceutically acceptable salt thereof, or compound B or a pharmaceutically acceptable salt thereof, in combination with, for example, a MEK inhibitor such as trametinib, induces long-term cytostasis of tumor cells, such as GIST cells, induces death of tumor cells, such as GIST cells, induces apoptosis of tumor cells, such as GIST cells, induces eradication of tumor cells to the limit of detection, such as GIST cells, induces regression of tumor cells, such as GIST cells, decreases the weight or volume of a tumor, such as a GIST tumor, and inhibits regrowth of a tumor, such as a drug-resistant GIST tumor, in a drug-resistant tumor, such as a drug-resistant GIST tumor. Another aspect of any of the methods disclosed herein is that treatment with either compound A or a pharmaceutically acceptable salt thereof, or compound B or a pharmaceutically acceptable salt thereof, in combination with, for example, a MEK inhibitor such as trametinib, eradicates tumors to the limit of detection in a treated patient, such as a GIST patient. Methods for measuring or determining the amount of tumor cell stasis, tumor cell death, tumor cell apoptosis, tumor regression, tumor weight or volume, tumor regrowth, growth of resistant tumor cells, and tumor eradication are known in the art and include any method described herein.

[0144] In certain embodiments, the treatment is by the following combination: compound A or a pharmaceutically acceptable salt thereof, or compound B or a pharmaceutically acceptable salt thereof; and an MEK inhibitor such as trametinib, which results in an increase in the amount of tumor cell quiescence, tumor cell death, or tumor cell apoptosis, for example in GIST cells, as compared to the amount of tumor cell quiescence, cell death, or apoptosis of the same type of or the same tumor type of tumor cells that are treated or not treated by, for example, the MEK inhibitor alone such as trametinib or by a c-KIT inhibitor alone such as imatinib or by the MEK inhibitor such as trametinib in combination with the c-KIT inhibitor imatinib. For example, the cell quiescence, cell death, or apoptosis can increase by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 10-fold, or at least 20-fold. In certain embodiments, the amount of apoptosis is determined by measuring the caspase activity of the tumor cells.

[0145] 〔00145〕In certain embodiments, the treatment is by the following combination: compound A or its pharmaceutically acceptable salt, or compound B or its pharmaceutically acceptable salt; and an MEK inhibitor such as trametinib, which results in an increase in tumor regression or a decrease in tumor size or volume (e.g., GIST), as compared to the size, e.g., weight or mass, of the same type of tumor or the same tumor that is treated or not treated by, for example, the MEK inhibitor alone such as trametinib or by a c-KIT inhibitor alone such as imatinib. For example, the tumor weight or volume can decrease by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%.

[0146] In certain embodiments, the treatment is by the following combination: compound A or a pharmaceutically acceptable salt thereof, or compound B or a pharmaceutically acceptable salt thereof; and a MEK inhibitor such as trametinib, which inhibits the amount of tumor growth or regrowth, such as GIST growth or regrowth, more significantly compared to the amount of growth or regrowth of the same type or same tumor that is either treated or untreated with only the MEK inhibitor such as trametinib or only the c-KIT inhibitor such as imatinib. For example, tumor growth or regrowth can be inhibited by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%.

[0147] In certain embodiments, the treatment is by the following combination: compound A or a pharmaceutically acceptable salt thereof, or compound B or a pharmaceutically acceptable salt thereof; and a MEK inhibitor such as trametinib, which, compared to the amount of growth of resistant tumor cells of the same type or same tumor that is either treated or untreated with only the MEK inhibitor such as trametinib or only the c-KIT inhibitor such as imatinib or a combination of the MEK inhibitor such as trametinib and the c-KIT inhibitor such as imatinib, more significantly inhibits the growth of resistant tumor cells, such as resistant GIST cells. It inhibits proliferation. For example, the amount or number of resistant tumor cell proliferation can be inhibited by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%. In certain embodiments, the resistant tumor cells are resistant to treatment with a c-KIT inhibitor such as imatinib and / or a MEK inhibitor such as trametinib. In certain embodiments, the resistant tumor cells contain a c-KIT secondary mutation. In certain embodiments, the c-KIT secondary mutation is a mutation in any of the following amino acid residues of c-KIT: V654, N655, T670, L783, D816, D820, N822, Y823, A829, and / or T847, and includes but is not limited to any of the amino acid substitutions shown in the accompanying figures. In certain embodiments, the resistant tumor cells contain an activated MAPKAP kinase pathway, and in certain embodiments, they contain mutations in the RAS gene such as the N-RAS or K-RAS gene, the fibroblast growth factor receptor (FGFR) gene, and / or the neurofibromin-1 (NF1) gene. In certain embodiments, the mutation is the N-RAS G12D mutation.

[0148] In certain embodiments, treatment with a combination of a MEK inhibitor, such as trametinib, with either Compound A or a pharmaceutically acceptable salt thereof, or Compound B or a pharmaceutically acceptable salt thereof, results in eradication of tumors to the limit of detection, such as in GIST. In certain embodiments, eradication of tumors means that no tumors detectable in the patient remain to the limit of detection. In certain embodiments, after eradication of tumors, such as GIST tumors, by the combination therapies disclosed herein, no tumors detectable in the patient are present for at least 6 months, at least 1 year, at least 2 years, at least 5 years, or at least 10 years. Eradication of tumors can be determined by positron emission tomography (PET), CT scan, absence of cell-free DNA (cfDNA) in the blood containing the c-KIT mutation, absence of circulating tumor cells (CTC) present in the vasculature of the subject, or absence of cancer cell biomarkers in the vasculature of the subject's circulating blood.

[0149] In various aspects of any of the methods disclosed herein, treatment with either compound A or a pharmaceutically acceptable salt thereof, or compound B or a pharmaceutically acceptable salt thereof, in combination with an ERK inhibitor such as, for example, vx-680, induces long-term cell quiescence of tumor cells such as, for example, GIST cells, induces death of tumor cells such as, for example, GIST cells, induces apoptosis of tumor cells such as, for example, GIST cells, induces eradication of tumor cells up to the limit of detection such as, for example, GIST cells, induces regression of tumors such as, for example, GIST tumors, decreases the weight or volume of tumors such as, for example, GIST tumors, and inhibits regrowth of tumors such as, for example, GIST tumors. In another aspect of any of the methods disclosed herein, treatment with either compound A or a pharmaceutically acceptable salt thereof, or compound B or a pharmaceutically acceptable salt thereof, in combination with an ERK inhibitor such as, for example, vx-680, induces long-term cell quiescence of tumor cells such as, for example, GIST cells, induces death of tumor cells such as, for example, GIST cells, induces apoptosis of tumor cells such as, for example, GIST cells, induces eradication of tumor cells up to the limit of detection such as, for example, GIST cells, induces regression of tumors such as, for example, GIST tumors, decreases the weight or volume of tumors such as, for example, GIST tumors, and inhibits regrowth of tumors such as, for example, GIST tumors in drug-resistant tumors such as, for example, drug-resistant GIST. In another aspect of any of the methods disclosed herein, treatment with either compound A or a pharmaceutically acceptable salt thereof, or compound B or a pharmaceutically acceptable salt thereof, in combination with an ERK inhibitor such as, for example, vx-680, eradicates tumors up to the limit of detection in a patient being treated such as, for example, a GIST patient. Methods of measuring or determining the amount of tumor cell quiescence, tumor cell death, tumor cell apoptosis, tumor regression, tumor weight or volume, tumor regrowth, growth of resistant tumor cells, and tumor eradication are known in the art and include any method described herein.

[0150] 〔00150〕In certain embodiments, treatment by the following combination: compound A or its A pharmaceutically acceptable salt, or Compound B or a pharmaceutically acceptable salt thereof; and an ERK inhibitor such as sunitinib, alone or in combination with a c-KIT inhibitor such as imatinib, results in an increase in the amount of tumor cell quiescence, cell death, or apoptosis, compared to the amount of tumor cell quiescence, cell death, or apoptosis of the same type or the same tumor type of tumor cells that are either treated or not treated with, for example, only the ERK inhibitor sunitinib or only the c-KIT inhibitor imatinib. For example, cell quiescence, cell death, or apoptosis can increase by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 10-fold, or at least 20-fold. In certain embodiments, the amount of apoptosis is determined by measuring the caspase activity of the tumor cells.

[0151] In certain embodiments, treatment is by the following combination: Compound A or a pharmaceutically acceptable salt thereof, or Compound B or a pharmaceutically acceptable salt thereof; and an ERK inhibitor such as sunitinib, alone or in combination with a c-KIT inhibitor such as imatinib, results in an increase in tumor regression or a decrease in tumor size or volume (e.g., GIST), compared to the size of the tumor, e.g., weight or volume, of the same type or the same tumor that is either treated or not treated with, for example, only the ERK inhibitor imatinib or only the c-KIT inhibitor imatinib. For example, the tumor weight or volume can decrease by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%.

[0152] In certain embodiments, the treatment is by the following combination: compound A or a pharmaceutically acceptable salt thereof, or compound B or a pharmaceutically acceptable salt thereof; and an ERK inhibitor such as nilotinib, which inhibits the amount of tumor growth or regrowth, such as GIST growth or regrowth, more significantly compared to the amount of growth or regrowth of the same type or same tumor that is either treated or untreated with only the ERK inhibitor such as nilotinib or only a c-KIT inhibitor such as imatinib. For example, the tumor growth or regrowth can be inhibited by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%.

[0153] In certain embodiments, the treatment is by the following combination: compound A or a pharmaceutically acceptable salt thereof, or compound B or a pharmaceutically acceptable salt thereof; and an ERK inhibitor such as nilotinib, which inhibits the growth of resistant tumor cells of the same type or same tumor, which is either treated or untreated with only the ERK inhibitor such as nilotinib or only a c-KIT inhibitor such as imatinib or a combination of the ERK inhibitor such as nilotinib and the c-KIT inhibitor such as imatinib, more significantly compared to the amount of growth of the resistant tumor cells. For example, the amount or number of resistant tumor cell growth can be inhibited by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%. In certain embodiments, the resistant tumor cells are resistant to treatment with a c-KIT inhibitor such as imatinib and / or an ERK inhibitor such as nilotinib. In certain embodiments, the resistant tumor cells contain a c-KIT secondary mutation. In certain embodiments, the c-KIT secondary mutation is any of the following c-KIT amino acid residues Any of the mutations: V654, N655, T670, L783, D816, D820, N822, Y823, A829, and / or T847, including but not limited to any of the amino acid substitutions shown in the accompanying figures. In certain embodiments, the resistant tumor cells include an activated MAPKAP kinase pathway, and in certain embodiments, they include mutations in mutations in genes such as the RAS gene, for example the N-RAS or K-RAS gene, the fibroblast growth factor receptor (FGFR) gene, and / or the neurofibromin-1 (NF1) gene. In certain embodiments, the mutation is the N-RAS G12D mutation.

[0154] In certain embodiments, treatment with a combination of compound A or a pharmaceutically acceptable salt thereof, or compound B or a pharmaceutically acceptable salt thereof, in combination with an ERK inhibitor, for example ulixertinib, results in eradication of the tumor to the limit of detection, for example in GIST. In certain embodiments, eradication of the tumor means that no tumor detectable in the patient remains up to the limit of detection. In certain embodiments, after eradication of a tumor, for example a GIST tumor, by the combination therapies disclosed herein, no tumor detectable in the patient is present for at least 6 months, at least 1 year, at least 2 years, at least 5 years, or at least 10 years. Eradication of the tumor can be determined by positron emission tomography (PET), CT scan, absence of cell-free DNA (cfDNA) containing the c-KIT mutation in the blood, absence of circulating tumor cells (CTC) in the vasculature of the subject, or absence of cancer cell biomarkers in the vasculature of the subject's circulating blood.

[0155] 〔00155〕In various aspects of any of the methods disclosed herein, for example dabrafenib Treatment with compound A or a pharmaceutically acceptable salt thereof, or compound B or a pharmaceutically acceptable salt thereof, in combination with a RAF inhibitor that is a nib, induces long-term cell quiescence of tumor cells, such as GIST cells, induces death of tumor cells, such as GIST cells, induces apoptosis of tumor cells, such as GIST cells, induces eradication of tumor cells to the limit of detection, such as GIST cells, induces regression of tumors, such as GIST tumors, reduces the weight or volume of tumors, such as GIST tumors, and inhibits regrowth of tumors, such as GIST tumors. In another aspect of any of the methods disclosed herein, treatment with either compound A or a pharmaceutically acceptable salt thereof, or compound B or a pharmaceutically acceptable salt thereof, in combination with a RAF inhibitor that is, for example, dabrafenib, induces long-term cell quiescence of tumor cells, such as GIST cells, induces death of tumor cells, such as GIST cells, induces apoptosis of tumor cells, such as GIST cells, induces eradication of tumor cells to the limit of detection, such as GIST cells, induces regression of tumors, such as GIST tumors, reduces the weight or volume of tumors, such as GIST tumors, and inhibits regrowth of tumors, such as drug-resistant GIST tumors in drug-resistant tumors that are, for example, drug-resistant GIST. In another aspect of any of the methods disclosed herein, treatment with either compound A or a pharmaceutically acceptable salt thereof, or compound B or a pharmaceutically acceptable salt thereof, in combination with a RAF inhibitor that is, for example, dabrafenib, eradicates tumors to the limit of detection in treated patients, such as GIST patients. Methods for measuring or determining the amounts of tumor cell quiescence, tumor cell death, tumor cell apoptosis, tumor regression, tumor weight or volume, tumor regrowth, growth of resistant tumor cells, and tumor eradication are known in the art and include any of the methods described herein.

[0156] In certain embodiments, the treatment is by the following combination: compound A or a pharmaceutically acceptable salt thereof, or compound B or a pharmaceutically acceptable salt thereof; and a RAF inhibitor such as dabrafenib, which results in an increase in tumor cell retention, tumor cell death, or apoptosis of tumor cells, such as GIST cells, compared to the amount of tumor cell retention, cell death, or apoptosis of the same type or the same tumor type that is either treated or not treated by the RAF inhibitor alone such as dabrafenib or by the c-KIT inhibitor imatinib alone or by the combination of the RAF inhibitor dabrafenib and the c-KIT inhibitor imatinib. For example, cell stasis, cell death, or apoptosis can increase by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 10-fold, or at least 20-fold. In certain embodiments, the amount of apoptosis is determined by measuring the caspase activity of the tumor cells. Compared to the amount of tumor cell retention, cell death, or apoptosis of the same type or the same tumor type that is either treated or not treated by the RAF inhibitor alone such as dabrafenib or by the c-KIT inhibitor imatinib alone or by the combination of the RAF inhibitor dabrafenib and the c-KIT inhibitor imatinib, the treatment by the combination of compound A or a pharmaceutically acceptable salt thereof, or compound B or a pharmaceutically acceptable salt thereof, and a RAF inhibitor such as dabrafenib results in an increase in tumor cell retention, tumor cell death, or apoptosis of tumor cells, such as GIST cells. For example, cell stasis, cell death, or apoptosis can increase by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 10-fold, or at least 20-fold. In certain embodiments, the amount of apoptosis is determined by measuring the caspase activity of the tumor cells.

[0157] In certain embodiments, the treatment is by the following combination: compound A or a pharmaceutically acceptable salt thereof, or compound B or a pharmaceutically acceptable salt thereof; and a RAF inhibitor such as dabrafenib, which results in an increase in tumor regression or a decrease in tumor size or volume (e.g., GIST) compared to the tumor size, e.g., weight or volume, of the same type or the same tumor that is either treated or not treated by the RAF inhibitor alone such as dabrafenib or by the c-KIT inhibitor imatinib alone. For example, the tumor weight or volume can decrease by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%.

[0158] In certain embodiments, the treatment is by the following combination: compound A or a pharmaceutically acceptable salt thereof, or compound B or a pharmaceutically acceptable salt thereof; and a RAF inhibitor, such as dabrafenib, inhibits the amount of tumor growth or regrowth, such as GIST growth or regrowth, more significantly compared to the amount of growth or regrowth of the same type or the same tumor that is either treated or not treated by only the RAF inhibitor, such as dabrafenib, or only by a c-KIT inhibitor, such as imatinib. For example, the tumor growth or regrowth can be inhibited by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%.

[0159] In certain embodiments, the treatment is by the following combination: compound A or a pharmaceutically acceptable salt thereof, or compound B or a pharmaceutically acceptable salt thereof, and; an RAF inhibitor such as dabrafenib, which inhibits the growth of resistant tumor cells of the same type or the same tumor, which may or may not have been treated, more significantly compared to the amount of growth of resistant tumor cells treated only with an RAF inhibitor such as dabrafenib or only with a c-KIT inhibitor such as imatinib or in combination with an RAF inhibitor such as dabrafenib and a c-KIT inhibitor such as imatinib. For example, the amount or number of resistant tumor cell growth can be inhibited by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%. In certain embodiments, the resistant tumor cells are resistant to treatment with a c-KIT inhibitor such as imatinib and / or an RAF inhibitor such as dabrafenib. In certain embodiments, the resistant tumor cells contain a c-KIT secondary mutation. In certain embodiments, the c-KIT secondary mutation is a mutation in any of the following amino acid residues of c-KIT: V654, N655, T670, L783, D816, D820, N822, Y823, A829, and / or T847, including but not limited to any of the amino acid substitutions shown in the accompanying figures. In certain embodiments, the resistant tumor cells contain an activated MAPKAP kinase pathway, and in certain embodiments, they contain mutations in the RAS gene such as the N-RAS or K-RAS gene, the fibroblast growth factor receptor (FGFR) gene, and / or the neurofibromin-1 (NF1) gene including mutations in the mutations in the mutations. In certain embodiments, the mutation is the N-RAS G12D mutation.

[0160] 〔00160〕In certain embodiments, in combination with an RAF inhibitor such as dabrafenib Treatment with a combination of compound A or a pharmaceutically acceptable salt thereof, or compound B or a pharmaceutically acceptable salt thereof, results in eradication of tumors to the limit of detection, for example in GIST. In certain embodiments, eradication of tumors means that there are no longer any tumors detectable in the patient to the limit of detection. In certain embodiments, after eradication of tumors, for example in GIST, by the combination therapies disclosed herein, there are no detectable tumors in the patient for at least 6 months, at least 1 year, at least 2 years, at least 5 years, or at least 10 years. Eradication of tumors can be determined by positron emission tomography (PET), CT scan, absence of cell-free DNA (cfDNA) in the blood containing c-KIT mutations, absence of circulating tumor cells (CTCs) in the vasculature of the subject, or absence of cancer cell biomarkers in the vasculature of the subject's circulating blood.

[0161] The present disclosure describes combination therapies involving administration of compound A or a pharmaceutically acceptable salt thereof, or compound B or a pharmaceutically acceptable salt thereof, with one or more MAPKAP kinase inhibitors, for example a MEK inhibitor, an ERK inhibitor or a RAF inhibitor. The combination therapies described herein can be used by themselves or in further combination with one or more additional therapeutic agents (for example, one or more of the additional therapeutic agents described below). For example, either compound A or a pharmaceutically acceptable salt thereof, or compound B or a pharmaceutically acceptable salt thereof, and a MEK inhibitor can be administered together with a cancer target therapeutic agent, a cancer target biological agent, an immune checkpoint inhibitor, or a chemotherapeutic agent. In another embodiment, compound A or compound B and the MEK inhibitor are administered without other therapeutic agents. The therapeutic agents can be administered together with or sequentially with another therapeutic agent described herein in the combination therapy.

[0162] Combination therapy is the administration of two or more therapeutic agents, which can be achieved by administering each of these therapeutic agents separately by prescription or by administering two or more therapeutic agents in a single formulation. Other combinations are also included in combination therapy. The two or more therapeutic agents in combination therapy can be administered simultaneously, but this is not essential. For example, the administration of the first agent (or combination of agents) can precede the administration of the second agent (or combination of agents) by only a few minutes, hours, days, or weeks. Thus, the two or more agents can be administered within a few minutes of each other, or within 1, 2, 3, 6, 9, 12, 15, 18, or 24 hours of each other, or within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14 days of each other, or within 2, 3, 4, 5, 6, 7, 8, 9 weeks or more of each other. In some cases, even longer intervals are possible. In many cases, it is desirable but not essential for the two or more agents used in combination therapy to be present in the patient's body simultaneously.

[0163] Combination therapy can also include the administration of one or more agents two or more times using the component agents in different orders. For example, when using agents X and Y in combination, they can be administered one or more times in any combination, continuously, for example, in the order of X-Y-X, X-X-Y, Y-X-Y, Y-Y-X, X-X-Y-Y, etc.

[0164] One or more additional therapeutic agents that can be administered according to the present disclosure include cytotoxic agents, cisplatin, doxorubicin, etoposide, irinotecan, topotecan, paclitaxel, docetaxel, epothilone, tamoxifen, 5-fluorouracil, methotrexate, temozolomide, cyclophosphamide, lonafarnib, tipifarnib, 4-((5 -((4-(3-Chlorophenyl)-3-oxopiperazin-1-yl)methyl)-1H-imidazol-1-yl)methyl)benzonitrile hydrochloride, (R)-1-((1H-imidazol-5-yl)methyl)-3-benzyl-4-(thiophen-2-ylsulfonyl)-2,3,4,5-Tetrahydro-1H-benzodiazepine-7-carbonitrile, cetuximab, imatinib, interferon alpha-2b, pegylated interferon alpha-2b, aromatase combination, gemcitabine, uracil mustard, chloromethine, ifosfamide, melphalan, chlorambucil, pipobroman, triethylenemelamine, triethylenethiophosphoramine, busulfan, carmustine, lomustine, streptozocin, dacarbazine, floxuridine, cytarabine, 6-mercaptopurine, 6-thioguanine, fludarabine phosphate, leucovorin, oxaliplatin, pentostatin, vinblastine, vincristine, vindesine, bleomycin, dactinomycin, daunorubicin, epirubicin, idarubicin, mitomycin, deoxycoformycin, mitomycin-C, L-asparaginase, teniposide 17α-ethinyl estradiol, diethylstilbestrol, testosterone, prednisone, fluoxymesterone, drostanolone propionate, testolactone, megestrol acetate, methylprednisolone, methyltestosterone, prednisolone, triamcinolone, chlorotrianisene, 17α-hydroxyprogesterone, aminoglutethimide, estramustine, medroxyprogesterone acetate, leuprolide acetate, flutamide, tamoxifen citrate, goserelin acetate, carboplatin, hydroxyurea, amsacrine, procarbazine, mitotane, mitoxantrone, levamisole, vinorelbine, anastrozole, letrozole, capecitabine, raloxifene, droloxafine, hexamethylmelamine, bevacizumab, trastuzumab, tositumomab, bortezomib, ibritumomab tiuxetan, arsenic trioxide, porfimer sodium, cetuximab, thiotepa, altretamine, fulvestrant, exemestane, rituximab, alemtuzumab, dexamethasone, bicalutamide, chlorambucil, and valrubicin, among others, but not limited to these.,

[0165] As one or more additional therapeutic agents that can be administered, without limitation there can be mentioned AKT inhibitors, alkylating agents, all-trans retinoic acid, anti-androgens, azacitidine, BCL2 inhibitors, BCL-XL inhibitors, BCR-ABL inhibitors, BTK inhibitors, BTK / LCK / LYN inhibitors, CDK1 / 2 / 4 / 6 / 7 / 9 inhibitors, CDK4 / 6 inhibitors, CDK9 inhibitors, CBP / p300 inhibitors, EGFR inhibitors, endothelin receptor antagonists, RAF inhibitors, MEK inhibitors, ERK inhibitors, farnesyltransferase inhibitors, FLT3 inhibitors, glucocorticoid receptor agonists, HDM2 inhibitors, histone deacetylase inhibitors, IKKβ inhibitors, immunomodulatory agents (IMiD), ingenol, ITK inhibitors, JAK1 / JAK2 / JAK3 / TYK2 inhibitors, mTOR inhibitors, PI3 kinase inhibitors, dual PI3 kinase / mTOR inhibitors, proteasome inhibitors, protein kinase C agonists, SUV39H1 inhibitors, TRAIL, VEGFR2 inhibitors, Wnt / β-catenin signaling inhibitors, decitabine, and anti-CD20 monoclonal antibodies.

[0166] In certain embodiments, the additional therapeutic agent is, for example, ipilimumab and tremelimumab but not limited to CTLA4 inhibitors, for example pembrolizumab and nivolumab but not limited to PD1 inhibitors, for example atezolizumab (formerly MPDL3280A), durvalumab (formerly MEDI4736), avelumab, PDR001 but not limited to PDL1 inhibitors, for example urelumab and PF-05082566 but not limited to 4-1BB or 4-1BB ligand inhibitors, for example MEDI6469 but not limited to OX40 ligand agonists, for example TRX518 but not limited to GITR agents, for example balstilimab but not limited CD27 inhibitors that cannot be, TNFRSF25 or TL1A inhibitors, such as, but not limited to, CD40 agonists such as CP-870893, HVEM or LIGHT or LTA or BTLA or CD160 inhibitors, such as, but not limited to, LAG3 inhibitors such as BMS-986016, TIM3 inhibitors, Siglecs inhibitors, ICOS or ICOS ligand agonists, such as, but not limited to, B7 such as MGA271 An immunomodulatory agent selected from the group consisting of H3 inhibitors, B7 H4 inhibitors, VISTA inhibitors, HHLA2 or TMIGD2 inhibitors, butyrophilin inhibitors including BTNL2 inhibitors, CD244 or CD48 inhibitors, inhibitors of TIGIT and PVR family members, such as KIR inhibitors including but not limited to lirilumab, inhibitors of ILT and LIR, such as NKG2D and NKG2A inhibitors including but not limited to IPH2201, MICA and MICB inhibitors, CD244 inhibitors such as emactuzumab, cabiralizumab, pexidartinib, ARRY382, BLZ945, CSF1R inhibitors including but not limited to these, IDO inhibitors including but not limited to INCB024360, thalidomide, lenalidomide, TGFβ inhibitors including but not limited to galunisertib, adenosine, or CD39 or CD73 inhibitors, such as ulocuplumab and (3S,6S,9S,12R,17R,20S,23S,26S,29S,34aS)-N-((S)-1-amino-5-guanidino-1-oxopentan-2-yl)-26,29-bis(4-aminobutyl)-17-((S)-2-((S)-2-((S)-2-(4-fluorobenzamide)-5-guanidinopentanamide)-5-guanidinopentanamide)-3-(naphthalen-2-yl)propanamide)-6-(3-guanidinopropyl)-3,20-bis(4-hydroxybenzyl)-1,4,7,10,18,21,24,27,30-nonaoxo-9,23-bis(3-ureidopropyl)triacontahydro-1H,16H-pyrrolo[2,1-p][1,2]dithia[5,8,11,14,17,20,23,26,29]nonaazacyclodotriacontine-12-carboxamide BKT140, CXCR4 or CXCL12 inhibitors including but not limited to, for example, babiximab, phosphatidylserine inhibitors including but not limited to, for example, CC-90002, SIRPA or CD47, such as VEGF inhibitors including but not limited to bevacizumab, and neuropilin inhibitors including but not limited to, for example, MNRP1685A.

[0167] Pharmaceutical composition Aspects of the present disclosure are directed to methods of treatment that include administration of a combination of the compounds disclosed herein, or one or more pharmaceutical compositions comprising such compounds and a pharmaceutically acceptable diluent, excipient or carrier. In certain embodiments, the methods disclosed herein involve administering a first pharmaceutical composition comprising either compound A or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable diluent, excipient or carrier, and a second pharmaceutical composition comprising, for example, a MEK inhibitor such as trametinib, an ERK inhibitor such as ulixertinib, a RAF inhibitor such as LY3009120 or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable diluent, excipient or carrier. In certain embodiments, the methods disclosed herein involve administering a first pharmaceutical composition comprising compound B or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable diluent, excipient or carrier, and a second pharmaceutical composition comprising, for example, a MEK inhibitor such as trametinib, an ERK inhibitor such as ulixertinib, a RAF inhibitor such as LY3009120 or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable diluent, excipient or carrier. In certain embodiments, the methods disclosed herein involve administering a pharmaceutical composition comprising compound A or a pharmaceutically acceptable salt thereof, a MEK inhibitor such as trametinib, and a pharmaceutically acceptable diluent, excipient or carrier. In certain embodiments, the methods disclosed herein involve administering a pharmaceutical composition comprising compound B or a pharmaceutically acceptable salt thereof, a MEK inhibitor such as trametinib, and a pharmaceutically acceptable diluent, excipient or carrier. Involved in administering the pharmaceutical composition.

[0168] In certain embodiments, the methods disclosed herein involve administering a pharmaceutical composition comprising Compound A or a pharmaceutically acceptable salt thereof, an ERK inhibitor such as ulixertinib, and a pharmaceutically acceptable diluent, excipient, or carrier. In certain embodiments, the methods disclosed herein involve administering a pharmaceutical composition comprising Compound B or a pharmaceutically acceptable salt thereof, an ERK inhibitor such as ulixertinib, and a pharmaceutically acceptable diluent, excipient, or carrier.

[0169] In certain embodiments, the methods disclosed herein involve administering a pharmaceutical composition comprising Compound A or a pharmaceutically acceptable salt thereof, a RAF inhibitor such as LY3009120, and a pharmaceutically acceptable diluent, excipient, or carrier. In certain embodiments, the methods disclosed herein involve administering a pharmaceutical composition comprising Compound B or a pharmaceutically acceptable salt thereof, a RAF inhibitor such as LY3001290, and a pharmaceutically acceptable diluent, excipient, or carrier.

[0170] 〔00170〕In the use of the pharmaceutical compositions of the compounds described herein, the pharmaceutically acceptable carrier can be either solid or liquid. Solid forms include powders, tablets, dispersible granules, capsules, cachets, and suppositories. Powders and tablets may consist of from about 5 to about 95 percent active ingredient. Suitable solid carriers are known in the art and include, for example, magnesium carbonate, magnesium stearate, talc, sugar, or lactose. Tablets, powders, cachets, and capsules can be used as solid dosage forms suitable for oral administration. Examples of pharmaceutically acceptable carriers and examples of methods for manufacturing various compositions can be found in A. Gennaro (ed.), Remington’s Pharmaceutical Sciences, 18th Edition, (1990), Mack Publishing Co., Easton, Pa, which is hereby incorporated by reference in its entirety.

[0171] Liquid form preparations include solution, suspension and emulsion.For example, for parenteral injection, water or water-propylene glycol solution, or for oral solution, suspension and emulsion, sweetener and opacifier are added.Liquid form preparations also include the solution for intranasal administration.

[0172] Liquid compositions, particularly injectable ones, can be prepared, for example, by dissolving, dispersing, etc. For example, the disclosed compounds are dissolved or mixed with a pharmaceutically acceptable solvent, such as, for example, water, saline, aqueous dextrose, glycerin, ethanol, etc., to form an injectable isotonic solution or suspension. Proteins such as albumin, chylomicron particles, or serum proteins can be used to solubilize the disclosed compounds.

[0173] Parenteral injectable administration is generally used for subcutaneous, intramuscular, or intravenous injections and infusions. Injectables can be prepared in conventional forms, either as liquid solutions or suspensions, or as solid forms suitable for dissolving in liquid prior to injection.

[0174] Aerosol formulations suitable for inhalation may also be used. These formulations may include solutions and solids in powder form, which may be in combination with a pharmaceutically acceptable carrier, such as an inert compressed gas, for example nitrogen.

[0175]

[00175] Also, regarding use, liquid forms for either oral or parenteral administration are Also contemplated are solid form preparations which are intended to be converted shortly before use into liquid preparations. Forms include solutions, suspensions and emulsions.

[0176] dose In some embodiments, for a treatment protocol, when compound A or compound B (or a pharmaceutically acceptable salt thereof) is used in combination with, for example, a MEK inhibitor (e.g., trametinib), the two therapeutic agents may be administered together or in a "dual regimen" where the two therapeutic agents are dosed and administered separately. When compound A or B (or a pharmaceutically acceptable salt thereof) and the MEK inhibitor are dosed separately, typical dosages of compound A or compound B (or a pharmaceutically acceptable salt thereof) administered to a subject in need of treatment are typically about 5 mg / day to about 5000 mg / day, and in other embodiments about 50 mg / day to about 1000 mg / day. Other dosages may be from about 10 mmol up to about 250 mmol / day, about 20 mmol to about 70 mmol / day, or about 30 mmol to about 60 mmol / day. The effective dosage of the disclosed compounds, when used with respect to the indicated effect, ranges from about 0.5 mg to about 5000 mg of the disclosed compound required for the treatment of the condition. Compositions for in vivo or in vitro use can contain about 0.5, 5, 20, 50, 75, 100, 150, 250, 500, 750, 1000, 1250, 2500, 3500, or 5000 mg of the disclosed compound, or can contain the disclosed compound in ranges from one amount to another amount listed in the dosage listings. With respect to oral administration, typical recommended daily dosing regimens can range from about 1 mg / day to about 500 mg / day or 1 mg / day to 200 mg / day, in a single dose or in divided doses of 2 to 4. In one embodiment, a typical daily oral dosage regimen is 150 mg.

[0177] In certain embodiments, the dosage of the MEK inhibitor is consistent with the dosages disclosed previously and / or the dosages approved for use by the Food and Drug Administration. In other embodiments, the dosage of the MEK inhibitor is less than the previously approved dosage, for example, about 20%, about 50%, or about 80% of the approved dosage. In certain embodiments, the dosage of trametinib is about 0.5 mg to 20 mg orally per day, for example, about 1 mg per day, or about 2 mg per day. In certain embodiments, the dosage of cobimetinib is about 10 mg to 200 mg per day, for example, about 30 mg per day, or about 60 mg per day. In certain embodiments, the dosage of binimetinib is about 10 mg to 200 mg twice a day, for example, about 25 mg or about 45 mg twice a day. In certain embodiments, the dosage of selumetinib is about 10 mg to 200 mg per day, for example, about 30 mg or about 75 mg twice a day.

[0178] The amounts and frequencies of administration of the compounds and / or their pharmaceutically acceptable salts and other therapeutic agents described herein will be adjusted according to the judgment of the attending physician, taking into account factors such as the age, symptoms and size of the patient, and the severity of the condition being treated.

[0179] The compounds of the present disclosure (e.g., Compound A or Compound B (and their pharmaceutically acceptable salts), MEK inhibitors, and other therapeutic agents) can be administered by any suitable route. The compounds can be administered orally (e.g., with food) in the form of capsules, suspensions, tablets, pills, dragees, solutions, gels, syrups, slurries, etc. Methods for encapsulating the compositions (such as within a coating of hard gelatin or cyclodextran) are known in the art (Baker, et al., “Controlled Release of Biological, which is hereby incorporated by reference in its entirety). "Active Agents", John Wiley and Sons, 1986). The compounds can be administered to a subject in conjunction with a pharmaceutically acceptable carrier as part of a pharmaceutical composition. The dosage form of the pharmaceutical composition will vary according to the route of administration selected. Suitable pharmaceutical carriers may include inert ingredients that do not interact with the compounds. The carrier is biocompatible , i.e., non-toxic, non-inflammatory, non-immunogenic, and does not cause other undesirable reactions at the site of administration.

[0180] 〔00180〕Exemplary pharmaceutical compositions include the compounds described herein and pharmaceutically acceptable carriers A solid, for example, a diluent which is a) for example, purified water, a triglyceride oil such as a hydrogenated or partially hydrogenated vegetable oil or a mixture thereof, corn oil, olive oil, sunflower oil, safflower oil, a fish oil such as EPA or DHA or an ester or triglyceride or a mixture thereof, an omega-3 fatty acid or a derivative thereof, lactose, dextrose, sucrose, mannitol, sorbitol, cellulose, sodium, saccharin, glucose and / or glycine; b) a lubricant which is for example silica, talc, stearic acid, its magnesium or calcium salt, sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride and / or polyethylene glycol, also for tablets; c) a binder which is for example magnesium aluminum silicate, starch paste, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose, magnesium carbonate, natural sugars such as glucose or beta-lactose, corn sweeteners, natural and synthetic rubbers such as acacia, tragacanth or sodium alginate, waxes and / or polyvinylpyrrolidone, if desired; d) a disintegrant which is for example starch, agar, methylcellulose, bentonite, xanthan gum, alginic acid or its sodium salt, or a foaming mixture; e) an absorbent, a coloring agent, a flavoring agent and a sweetening agent; f) an emulsifier or a dispersant which is for example Tween80, Labrasol, HPMC, DOSS, Caproyl 909, Labrafac, Labrafil, Peceol, Transcutol, CAPMUL MCM, CAPMUL PG-12, Captex 355, Gelucire, vitamin E TGPS or other acceptable emulsifiers; and / or g) an agent for enhancing the absorption of a compound which is for example cyclodextrin, hydroxypropyl cyclodextrin, PEG400, PEG200, etc., and tablets and gelatin capsules containing the above carriers.

[0181] If prepared as a fixed dose, such combinations will employ the compounds described herein within the dosage ranges described herein or known to those of skill in the art.

[0182] Because the compounds described herein (e.g., Compound A and Compound B, and MAPKAP kinase inhibitors, including MEK inhibitors) are intended for use in pharmaceutical compositions, those skilled in the art will appreciate that they can be provided in substantially pure form, e.g., at least 60% pure, at least 75% pure, at least 85% pure, and at least 98% pure (w / w). Pharmaceutical formulations may be in unit dosage form. In such formulations, they are prepared by dividing them into suitably sized unit doses containing an appropriate amount of Compound A or Compound B, e.g., an effective amount to achieve a desired purpose as described herein. [Example]

[0183] It has been found that treatment of c-KIT-mediated tumor cells with either Compound A or a pharmaceutically acceptable salt thereof in combination with a MEK inhibitor, an ERK inhibitor, or a RAF inhibitor unexpectedly and synergistically induces tumor cell apoptosis. Furthermore, this combination therapy inhibits tumor cell proliferation, including tumor cells with secondary mutations that confer resistance to other c-KIT inhibitors and / or MEK, ERK, or RAF inhibitors. Furthermore, the combination therapy disclosed herein appears to have a long-term effect on tumor cell stasis, as opposed to rapid tumor regrowth without drug combination treatment. Furthermore, the combination therapy disclosed herein appears to have a cytotoxic effect on tumor cells, as opposed to a purely cytoplasmic effect. Furthermore, the combination therapy disclosed herein is most The combination therapy appeared to eradicate GIST tumor cells to the limit of detection, with no tumor cell colony growth after removal of the combination therapy, including a 21-day drug-free recovery period. This unexpected finding was confirmed in biochemical and cellular assays, including those described herein.

[0184] The present disclosure will be further described by the following examples, which are not to be construed as limiting the present disclosure in scope or spirit to the specific procedures described herein. It is to be understood that the examples are presented to illustrate particular embodiments and are not intended to limit the scope of the present disclosure. Further, it is to be understood that various other embodiments, variations, and equivalents that may be suggested to those skilled in the art can be contemplated without departing from the spirit of the present disclosure and / or the scope of the appended claims.

[0185] Example 1. The combined treatment of Compound A with trametinib induces apoptosis in imatinib-resistant cells of GIST-T1, GIST-T1 / D816E, and GIST-T1 / T670I 〔00185〕To demonstrate that the combined treatment with Compound A and trametinib induces apoptosis in GIST-T1 (57bp exon 11 deletion) imatinib-sensitive cells, GIST-T1 / D816E imatinib-resistant cells, and GIST-T1 / T670I) imatinib-resistant cells, assays were performed in 96-well plates seeded with 10,000 cells per well of GIST-T1, GIST-T1 / D816E, or GIST-T1 / T670I. Cells were treated with vehicle control, Compound A, trametinib, or combinations thereof at various concentrations and grown for 24 and 48 hours in the presence of the drug treatment. Apoptosis was evaluated by measuring caspase 3 / 7 activity.

[0186] Figures 1A and 1C are graphical representations showing the relative percentages of caspase activity determined for various treatments of GIST-T1 cells (compared to a vehicle control set at 100%). Figures 1B and 1D are matrix diagrams and combination index plots of synergism based on the combination index (CI) method described by Chou and Talalay (1984) and the computer software of Chou and Martin (2005). CI < 1 indicates synergism, CI = 1 indicates additivity, and CI > 1 indicates antagonism. A strong synergistic effect in inducing apoptosis in GIST-T1 cells was shown by the combination treatment with compound A and trametinib for 24 hours (Figures 1A, B) and 48 hours (Figures 1C, D).

[0187] Figure 1E is a graphical representation showing caspase activity from various treatments of GIST-T1 / D816E imatinib-resistant cells. Figure 1F is a matrix diagram and combination index plot of synergism based on the combination index (CI) method described by Chou and Talalay (1984) and the computer software of Chou and Martin (2005). CI < 1 indicates synergism, CI = 1 indicates additivity, and CI > 1 indicates antagonism. A strong synergistic effect in inducing apoptosis in GIST-T1 / D816E imatinib-resistant cells was shown by the combination treatment with compound A and trametinib for 24 hours.

[0188] Figure 1G is a graphical representation showing caspase activity from various treatments of GIST-T1 / T670I imatinib-resistant cells. Figure 1H is a matrix diagram and combination index plot of synergism based on the combination index (CI) method described by Chou and Talalay (1984) and the computer software of Chou and Martin (2005). CI < 1 indicates synergism, CI = 1 indicates additivity, and CI > 1 indicates antagonism. A strong synergistic effect in inducing apoptosis in GI ST-T1 / T670I imatinib-resistant cells was shown by the combination treatment with compound A and trametinib for 24 hours.

[0189] Example 2. The combined treatment of Compound B with trametinib induces apoptosis in imatinib-resistant GIST-T1, GIST-T1 / D816E, and GIST-T1 / T670I cells Figure 2A is a graphical representation showing the relative percentage of caspase activity determined for various treatments of GIST-T1 cells (compared to a vehicle control set at 100%). Figure 2B is a matrix diagram and combination index plot of the synergistic effect as described in Example 1, which is a 24-hour combined treatment with Compound B and trametinib (Figures 2A, B), showing a strong synergistic effect in inducing apoptosis in GIST-T1 cells.

[0190] 〔00190〕Figure 2C is a graphical representation showing caspase activity from various treatments of GIST-T1 / D816E imatinib-resistant cells Figure 2D is a matrix diagram and combination index plot. A strong synergistic effect in inducing apoptosis in GIST-T1 / D816E imatinib-resistant cells was shown by the 24-hour combined treatment with Compound B and trametinib.

[0191] Figure 2E is a graphical representation showing caspase activity from various treatments of GIST-T1 / T670I imatinib-resistant cells. Figure 2F is a matrix diagram and combination index plot. A strong synergistic effect in inducing apoptosis in GIST-T1 / T670I imatinib-resistant cells was shown by the 24-hour combined treatment with Compound B and trametinib.

[0192] Example 3. The combined treatment of Compound A with binimetinib induces apoptosis in imatinib-resistant GIST-T1, GIST-T1 / D816E, and GIST-T1 / T670I cells Figure 3A is a graphical display showing the relative percentages of caspase activity determined for various treatments of GIST-T1 cells (compared to a vehicle control set at 100%). Figure 3B shows a matrix diagram of synergism and a combination index plot based on the combination index (CI) method described in Example 1. A strong synergistic effect inducing apoptosis in GIST-T1 cells was shown by the 24-hour combination treatment with Compound A and binimetinib (Figure 3A, 3B).

[0193] Figure 3C is a graphical display showing caspase activity from various treatments of GIST-T1 / D816E imatinib-resistant cells. Figure 3D is a matrix diagram of synergism and a combination index plot based on the combination index (CI) method described in Example 1. A strong synergistic effect inducing apoptosis in GIST-T1 / D816E imatinib-resistant cells was shown by the 24-hour combination treatment with Compound A and binimetinib.

[0194] Figure 3E is a graphical display showing caspase activity from various treatments of GIST-T1 / T670I imatinib-resistant cells. Figure 3F is a matrix diagram of synergism and a combination index plot. A strong synergistic effect inducing apoptosis in GIST-T1 / T670I imatinib-resistant cells was shown by the 24-hour combination treatment with Compound A and binimetinib.

[0195] Example 4. Combination treatment of Compound B with binimetinib induces apoptosis in GIST-T1, imatinib-resistant cells of GIST-T1 / D816E, and imatinib-resistant cells of GIST-T1 / T670I 〔00195〕Figure 4A shows caspases determined for various treatments of GIST-T1 cells It is a graphical display showing the relative ratio of activity (compared to the vehicle control set at 100%). Figure 4B is a matrix diagram and combination index plot of synergism based on the combination index (CI) method described by Chou and Talalay (1984). The combination treatment for 24 hours (Figure 4A, B) with compound B and vemurafenib showed a strong synergistic effect in inducing apoptosis in GIST-T1 cells.

[0196] Figure 4C is a graphical display showing caspase activity from various treatments of GIST-T1 / D816E imatinib-resistant cells. Figure 4D is a matrix diagram and combination index plot of synergism. The combination treatment for 24 hours with compound B and vemurafenib showed a strong synergistic effect in inducing apoptosis in GIST-T1 / D816E imatinib-resistant cells.

[0197] Figure 4E is a graphical display showing caspase activity from various treatments of GIST-T1 / T670I imatinib-resistant cells. Figure 4F is a matrix diagram and combination index plot of synergism. The combination treatment for 24 hours with compound B and vemurafenib showed a strong synergistic effect in inducing apoptosis in GIST-T1 / T670I imatinib-resistant cells.

[0198] Example 5. The combination treatment of compound A with cobimetinib induces apoptosis in GIST-T1 imatinib-sensitive cells, GIST-T1 / D816E imatinib-resistant cells, and GIST-T1 / T670I imatinib-resistant cells Figure 5A is a graphical display showing the relative ratio of caspase activity (compared to the vehicle control set at 100%) determined for various treatments of GIST-T1 cells. Figure 5B shows a matrix diagram and combination index plot of synergism based on the combination index (CI) method described in Example 1. The combination treatment for 24 hours (Figure 5A, 5B) with compound A and cobimetinib showed a strong synergistic effect in inducing apoptosis in GIST-T1 cells.

[0199] Figure 5C is a graphical representation showing caspase activity from various treatments of GIST-T1 / D816E imatinib-resistant cells. Figure 5D is a matrix diagram and combination index plot of synergism based on the combination index (CI) method described in Example 1. A strong synergistic effect inducing apoptosis of GIST-T1 / D816E imatinib-resistant cells was shown by the 24-hour combination treatment with compound A and cobimetinib.

[0200] 〔00200〕Figure 5E is a graphical representation showing caspase activity from various treatments of GIST-T1 / T670I imatinib-resistant cells. Figure 5F is a matrix diagram and combination index plot of synergism based on the combination index (CI) method described in Example 1. A strong synergistic effect inducing apoptosis of GIST-T1 / T670I imatinib-resistant cells was shown by the 24-hour combination treatment with compound A and cobimetinib.

[0201] Example 6. Combination treatment of compound B with cobimetinib induces apoptosis in GIST-T1, imatinib-resistant cells of GIST-T1 / D816E, and imatinib-resistant cells of GIST-T1 / T670I Figure 6A is a graphical representation showing the relative percentage of caspase activity (compared to the vehicle control set at 100%) determined for various treatments of GIST-T1 cells. Figure 6B is a matrix diagram and combination index plot of synergism. A strong synergistic effect inducing apoptosis in GIST-T1 cells was shown by the 24-hour (Figure 6A, 6B) combination treatment with compound B and cobimetinib.

[0202] Figure 6C is a graphical representation showing caspase activity from various treatments of GIST-T1 / D816E imatinib-resistant cells. Figure 6D is a matrix diagram and combination index plot of synergism. A strong synergistic effect inducing apoptosis of GIST-T1 / D816E imatinib-resistant cells was shown by the 24-hour combination treatment with compound B and cobimetinib.

[0203] Figure 6E is a graphical representation showing caspase activity from various treatments of GIST-T1 / T670I imatinib-resistant cells. Figure 6F is a matrix diagram of synergistic effects and a combination index plot. A strong synergistic effect was shown to induce apoptosis in GIST-T1 / T670I imatinib-resistant cells by the 24-hour combined treatment with compound B and cobimetinib.

[0204] Example 7. Combined treatment of compound A with ulixertinib (BVD-523) induces apoptosis in GIST-T1 and GIST-T1 / T670I imatinib-resistant cells Figure 7A is a graphical representation showing the relative percentage of caspase activity (compared to the vehicle control set at 100%) determined for various treatments of GIST-T1 cells. Figure 7B shows a matrix diagram of synergistic effects and a combination index plot based on the combination index (CI) method described in Example 1. The 24-hour combined treatment with compound A and ulixertinib (Figures 7A, 7B) showed a strong synergistic effect to induce apoptosis in GIST-T1 cells at higher concentrations.

[0205] 〔00205〕Figure 7C is a graphical representation showing caspase activity from various treatments of GIST-T1 / T670I imatinib-resistant cells. Figure 7D is a matrix diagram of synergistic effects and a combination index plot based on the combination index (CI) method described in Example 1. A strong synergistic effect was shown to induce apoptosis in GIST-T1 / T670I imatinib-resistant cells by the 24-hour combined treatment with compound A and ulixertinib.

[0206] Example 8. Combined treatment of compound B with ulixertinib (Bvd-523) induces apoptosis in GIST-T1 and GIST-T1 / T670I imatinib-resistant cells Using the figure of the synergistic effect of caspase activity and the combination index plot, the synergistic effect regarding the combination of Compound B and nilotinib can be shown when inducing apoptosis in GIST-T1, GIST-T1 / D816E imatinib-resistant cells, and GIST-T1 / T670I imatinib-resistant cells.

[0207] Example 9. The combined treatment of Compound A with SCH772984 induces apoptosis in GIST-T1, GIST-T1 / D816E imatinib-resistant cells, and GIST-T1 / T670I imatinib-resistant cells. Using the figure of the synergistic effect of caspase activity and the combination index plot, the synergistic effect regarding the combination of Compound A and SCH772984 can be shown when inducing apoptosis in GIST-T1, GIST-T1 / D816E imatinib-resistant cells, and GIST-T1 / T670I imatinib-resistant cells.

[0208] Example 10. The combined treatment of Compound B with SCH772984 induces apoptosis in GIST-T1, GIST-T1 / D816E imatinib-resistant cells, and GIST-T1 / T670I imatinib-resistant cells. Using the figure of the synergistic effect of caspase activity and the combination index plot, the synergistic effect regarding the combination of Compound B and SCH77298 4 can be shown.

[0209] Example 11. The combined treatment of Compound A with LY3009120 induces apoptosis in GIST-T1, GIST-T1 / D816E imatinib-resistant cells, and GIST-T1 / T670I imatinib-resistant cells. Using the figure of the synergistic effect of caspase activity and the combination index plot, the synergistic effect regarding the combination of Compound A and LY3009120 can be shown when inducing apoptosis in GIST-T1, GIST-T1 / D816E imatinib-resistant cells, and GIST-T1 / T670I imatinib-resistant cells.

[0210] Example 12. The combined treatment of Compound B with LY3009120 induces apoptosis in GIST-T1, imatinib-resistant cells of GIST-T1 / D816E, and imatinib-resistant cells of GIST-T1 / T670I 〔00210〕Using the figure of the synergistic effect of caspase activity and the combination index plot, GIS T-T1, the synergistic effect regarding the combination of Compound B and LY3009120 can be shown when inducing apoptosis in GIST-T1 / D816E imatinib-resistant cells and GIST-T1 / T670I imatinib-resistant cells.

[0211] Example 13. The combined treatment of Compound A with dabrafenib induces apoptosis in GIST-T1, imatinib-resistant cells of GIST-T1 / D816E, and imatinib-resistant cells of GIST-T1 / T670I Using the figure of the synergistic effect of caspase activity and the combination index plot, the synergistic effect regarding the combination of Compound A and dabrafenib can be shown when inducing apoptosis in GIST-T1, GIST-T1 / D816E imatinib-resistant cells, and GIST-T1 / T670I imatinib-resistant cells. .

[0212] Example 14. The combined treatment of Compound B with dabrafenib induces apoptosis in GIST-T1, imatinib-resistant cells of GIST-T1 / D816E, and imatinib-resistant cells of GIST-T1 / T670I Using the figure of the synergistic effect of caspase activity and the combination index plot, the synergistic effect regarding the combination of Compound B and dabrafenib can be shown when inducing apoptosis in GIST-T1, GIST-T1 / D816E imatinib-resistant cells, and GIST-T1 / T670I imatinib-resistant cells.

[0213] Example 15. Combination treatment prevents colony growth in imatinib-resistant cells of GIST-T1, GIST-T1 / D816E, and GIST-T1 / T670I A test was conducted to demonstrate that combination treatment with Compound A and trametinib prevents cell growth in GIST-T1 (57bp exon 11 deletion) imatinib-sensitive cells, GIST-T1 / D816E imatinib-resistant cells, and GIST-T1 / T670I imatinib-resistant cells. The assay was performed in 6-well plates seeded with 100 cells per well. The cells were treated with vehicle control, Compound A, trametinib, imatinib (IM), or combinations thereof at various concentrations and cultured for 2 weeks. After treatment, the drugs were washed off and the cells were cultured in normal medium for 1 - 3 weeks. The growing cell colonies were stained with crystal violet and counted.

[0214] Figure 8A shows an image of a representative culture plate and a graphical representation of the number of GIST-T1 colonies counted from various treatments. GIST T1 cells are sensitive to imatinib and Compound A as single agents. Each of imatinib and Compound A as single agents shows a similar decrease in GIST T1 colony growth to 23 - 30% of the vehicle control. It should be noted that two-week combined treatment with 50 nM of Compound A and either 50 nM or 100 nM of trametinib unexpectedly led to complete cell stasis or eradication of GIST T1 colony growth to the limit of detection as visualized by stereomicroscopy at 5-fold, and no colonies were detected 9 days after removal of the combined treatment (indicated by the arrow in Figure 8A). In contrast, two-week combined treatment with 100 nM of imatinib and either 50 nM or 100 nM of trametinib did not lead to complete tumor cell stasis or eradication 9 days after removal of the combined treatment.

[0215] [

[00215] ]Figure 8B shows an image of a representative culture plate and the GI counted from various treatments The graph shows the colony number of ST-T1 / D816E. Note that each of imatinib (500 nM) and compound A (100 nM or 250 nM) as a single agent was accompanied by colony growth up to about 61 - 72% of the vehicle control, indicating a similar lack of cytocidal efficacy against GIST T1 / D816E (Figure 8B). Two-week combination treatment with compound A (100 nM or 250 nM) and trametinib (100 nM) led to almost complete cytostasis by compound A (100 nM), and after 10 days of recovery, complete cytostasis or eradication of colony growth in GIST-T1 / D816E cells by the combination of trametinib (100 nM) and compound A (250 nM) was achieved up to the detection limit visualized by stereomicroscopy at 5-fold magnification (see arrows in Figure 8B). On the other hand, two-week combination treatment with imatinib (500 nM) and trametinib (50 nM or 100 nM) did not lead to complete cytostasis or eradication of tumor cells (see graph in Figure 8B). This was prominent when cells were cultured for an additional 10 days without drugs, and about 20 - 25 colonies grew. Figure 8C shows images of representative culture plates when the concentration of compound A was further decreased to 25 nM, 50 nM, or 100 nM and evaluated in combination with 50 nM trametinib. Complete tumor cell stasis or eradication up to the detection limit visualized by stereomicroscopy at 5-fold magnification of tumor colony growth was achieved with 100 nM compound A in combination with trametinib after 10 days of recovery (see arrows in Figure 8C), almost complete tumor cell stasis or substantial eradication (1% of the vehicle control) was achieved with 50 nM compound A in combination with trametinib (see arrows in Figure 8C), and significant tumor cell stasis or death was achieved with 25 nM compound A (11% of the vehicle control). In contrast, the combination of 100 nM imatinib with 50 nM trametinib did not eradicate tumor colony growth and achieved mild tumor cell stasis or death (60% of the vehicle control) after 10 days of recovery.

[0216] Figure 8D shows an image of a representative culture plate and a graphical representation of the number of GIST-T1 / T670I colonies counted from various treatments. It should be noted that each of imatinib (500 nM) and compound A (250 nM or 500 nM) as single agents showed a similar decrease in GIST T1 / T670I colony growth to approximately 44 - 49% of the vehicle control. Two-week treatment with 250 nM or 500 nM of compound A in combination with either 50 nM or 100 nM of trametinib led to complete cell stasis or eradication of GIST T1 / T670I colony growth to the limit of detection as visualized by stereomicroscopy at 5-fold, and no colonies were detected 10 days after removal of the combination treatment (indicated by the arrow in Figure 8D). In contrast, two-week treatment with 500 nM of imatinib in combination with either 50 nM or 100 nM of trametinib did not lead to complete tumor cell stasis or eradication of tumor cells 9 days after removal of the combination treatment.

[0217] Example 16. Combination treatment of compound B with trametinib prevents colony growth in imatinib-resistant cells of GIST-T1, GIST-T1 / D816E, and GIST-T1 / T670I The experiments described in Example 8 were also performed with combination treatment of compound B and trametinib in three GIST cell lines.

[0218] Figure 9A shows an image of a representative culture plate and a graphical display of the number of GIST-T1 colonies counted from various treatments. GIST T1 cells were sensitive to compound B as a single agent, showing a 42 - 54% decrease in GIST T1 colony growth compared to the vehicle control. Two-week combination treatment with 50 nM or 100 nM of compound B and either 50 nM or 100 nM of trametinib led to significant cytostasis with almost no colony growth. On the other hand, combination treatment of 250 nM of compound A with either 50 nM or 100 nM of trametinib led to complete cytostasis or eradication of GIST T1 colony growth to the limit of detection as visualized by 5-fold magnification stereomicroscopy, and no colonies were detected 10 days after removal of the combination treatment (indicated by the arrow in Figure 9A). Colony growth was prevented even after an extended long-term recovery of 20 days in total.

[0219] Figure 9B shows an image of a representative culture plate and a graphical display of the number of GIST-T1 / D816E colonies counted from various treatments. Note that compound B as a single agent (50 nM, 100 nM, or 250 nM) showed a cytocidal efficacy against GIST T1 / D816E with colony growth at approximately 59 - 84% of the vehicle control (Figure 9B). Two-week combination treatment with compound B (250 nM) and trametinib (50 nM) or compound B (100 nM or 250 nM) and trametinib (100 nM) led to >90% cytostasis or eradication of colony growth in GIST-T1 / D816E cells as visualized by 5-fold magnification stereomicroscopy after 10 days of recovery (see arrow in Figure 9B), and treatment with compound B (250 nM) maintained cytostasis or cell death in combination with trametinib (100 nM) even after an extended long period of 20 days.

[0220] 〔00220〕Figure 9C shows an image of a representative culture plate and GI counted from various treatments The graph shows the colony number of ST-T1 / T670I. Note that compound B as a single agent (50 nM, 100 nM or 250 nM) showed GIST T1 / T670I colony growth to about 75 - 78% of the vehicle control. Two-week treatment with either 100 nM or 250 nM of compound B in combination with either 50 nM or 100 nM of trametinib led to complete cell stasis or eradication of GIST T1 / T670I colony growth to the limit of detection as visualized by stereomicroscopy at 5-fold, and no colonies were detected 10 days after removal of the combination treatment (indicated by the arrow in Fig. 9C). Inhibition of growth was maintained even after 20 days of extended culture without drugs.

[0221] Example 17. Combination treatment of compound A with binimetinib prevents colony growth in imatinib-resistant cells of GIST-T1, GIST-T1 / D816E and GIST-T1 / T670I A study was conducted to demonstrate that combination treatment of compound A and binimetinib prevents colony growth in three GIST cell lines as described in Example 15.

[0222] Figure 10A shows an image of a representative culture plate and a graph showing the colony number of GIST-T1 counted from various treatments. Note that each of imatinib and compound A as single agents showed a similar decrease in GIST T1 colony growth to 36 - 41% of the vehicle control. Two-week combination treatments of 100 nM or 250 nM of compound A with 500 nM, 1 μM, 2 μM or 3 μM of either binimetinib were evaluated. The combination of compound A (100 nM or 250 nM) with binimetinib (2 μM or 3 μM) led to complete cell stasis or eradication of GIST T1 colony growth to the limit of detection as visualized by stereomicroscopy at 5-fold, and no colonies were detected 10 days after removal of the combination treatment (indicated by the arrow in Fig. 10A). In contrast, 500 nM of imatinib Combined treatment with bosutinib and 2 weeks of any of 500 nM, 1 μM, 2 μM, or 3 μM of vemurafenib did not result in complete tumor cell stasis or eradication 10 days after removal of the combined treatment. The effect was clearer after incubation for an extended period without the drug, with about 10 - 15 colonies visible with imatinib and no colony growth observed with compound A.

[0223] Figure 10B shows images of representative culture plates and a graphical representation of the number of GIST - T1 / D816E colonies counted from various treatments. Note that each of imatinib (500 nM) and compound A (100 nM or 250 nM) as single agents was associated with colony growth that was about 60 - 95% of the vehicle control, indicating a lack of cytocidal efficacy against GIST T1 / D816E (Figure 10B). The combined treatment of compound A (100 nM or 250 nM) with vemurafenib (3 μM) led to complete cell stasis or eradication of colony growth in GIST - T1 / D816E cells to the limit of detection as visualized by stereomicroscopy at 5 - fold magnification 10 days after recovery (see arrow in Figure 10B), while the 2 - week combined treatment with imatinib (500 nM) and vemurafenib (500 nM, 1 μM, 2 μM, or 3 μM) did not lead to complete cell stasis or eradication of tumor cells. The effect was clearer after incubation for an extended period, with complete inhibition not induced by treatment with imatinib, while with compound A, cell stasis or cell death was maintained even after 20 days.

[0224] Figure 10C shows an image of a representative culture plate and a graphical display of the number of colonies of GIST-T1 / T670I) counted from various treatments. It should be noted that imatinib (500 nM) as a single agent showed no decrease in colonies, while compound A (100 nM or 250 nM) as a single agent showed a dose-dependent decrease in colony growth up to about 78 - 89% of the vehicle control. Treatment with 250 nM of compound A in combination with 1 μM, 2 μM, or 3 μM of binimetinib for 2 weeks led to complete cell stasis or eradication of GIST T1 / T670I colony growth to the limit of detection as visualized by 5-fold magnification stereomicroscopy, and no colonies were detected 10 days after removal of the combination treatment (indicated by the arrow in Figure 10C). Treatment with 100 nM of compound A in combination with 3 μM of binimetinib for 2 weeks led to complete cell stasis or eradication of GIST T1 / T670I colony growth to the limit of detection as visualized by 5-fold magnification stereomicroscopy, and no colonies were detected 10 days after removal of the combination treatment (indicated by the arrow in Figure 10C). Cell stasis was maintained even after an extended 20-day period after removal of the drug. In contrast, treatment with 500 nM of imatinib in combination with 500 nM, 1 μM, 2 μM, or 3 μM of binimetinib for 2 weeks did not lead to complete tumor cell stasis or eradication of tumor cells 10 days after removal of the combination treatment.

[0225] Example 18. Combination treatment of compound B with binimetinib prevents colony growth in imatinib-resistant cells of GIST-T1, GIST-T1 / D816E, and GIST-T1 / T670I 〔00225〕To demonstrate that combination treatment of compound B and binimetinib prevents colony growth in the three GIST cell lines as described in Example 15 a test was conducted.

[0226] Figure 11A shows an image of a representative culture plate and a graphical display of the number of GIST-T1 colonies counted from various treatments. Note that each concentration of Compound B as a single agent shows a similar decrease in GIST T1 colony growth, up to 27 - 31% of the vehicle control. The 2-week combination treatment with 250 nM of Compound B and 2 μM or 3 μM of vimenetinib led to complete cytostasis or eradication of GIST T1 colony growth to the limit of detection as visualized by 5-fold magnification with a stereomicroscope, and no colonies were detected 10 days after removal of the combination treatment (indicated by the arrow in Figure 11A), and significant cytostasis or cell death of GIST-T1 cells was maintained even after a 20-day extended long-term recovery period (upper right panel of Figure 11A). The 2-week combination treatment with 100 nM of Compound B and 3 μM of vimenetinib led to complete cytostasis or eradication of GIST T1 colony growth to the limit of detection as visualized by 5-fold magnification with a stereomicroscope, and no colonies were detected 10 days after removal of the combination treatment (indicated by the arrow in Figure 11A). The 2-week combination treatment led to complete cytostasis or eradication of GIST T1 colony growth to the limit of detection as visualized by 5-fold magnification with a stereomicroscope, and no colonies were detected 10 days after removal of the combination treatment (indicated by the arrow in Figure 11A).

[0227] Figure 11B shows an image of a representative culture plate and a graphical display of the number of GIST-T1 / D816E colonies counted from various treatments. Note that Compound B (100 nM or 250 nM) as a single agent was associated with colony growth of approximately 74 - 83% of the vehicle control, showing a similar lack of cytocidal efficacy against GIST T1 / D816E (Figure 11B). The 2-week combination treatment with 100 nM or 250 nM of Compound B and either 2 μM or 3 μM of vimenetinib led to complete cytostasis or eradication of GIST-T1 / D816E cell colony growth to the limit of detection as visualized by 5-fold magnification with a stereomicroscope 10 days after recovery (see arrow in Figure 11B). Cytostasis was maintained even after an extended 20-day period with higher concentrations of Compound B.

[0228] Figure 11C shows an image of a representative culture plate and a graphical representation of the number of GIST-T1 / T670I colonies counted from various treatments. Note that the compound B as a single agent (100 nM or 250 nM) showed a decrease in GIST T1 / T670I colony growth to about 72 - 78% of the vehicle control. Treatment with either 100 nM or 250 nM of compound B and 3 μM of vemurafenib for 2 weeks unexpectedly led to complete cytostasis or eradication of GIST T1 / T670I colony growth to the limit of detection as visualized by stereomicroscopy at 5x, and no colonies were detected 10 days after removal of the combination treatment (indicated by the arrow in Figure 11C). Treatment with 250 nM of compound B and 2 μM of vemurafenib for 2 weeks unexpectedly led to complete cytostasis or eradication of GIST T1 / T670I colony growth to the limit of detection as visualized by stereomicroscopy at 5x, and no colonies were detected 10 days after removal of the combination treatment (indicated by the arrow in Figure 11C). The cytostasis was maintained even after an extended 20-day period after removal of the drug.

[0229] Example 19. Combination treatment of compound A with cobimetinib prevents colony growth in imatinib-resistant cells of GIST-T1, GIST-T1 / D816E, and GIST-T1 / T670I To demonstrate that combination treatment of compound A and cobimetinib prevents colony growth in three GIST cell lines as described in Example 15, a test was conducted.

[0230] 〔00230〕Figure 12A shows an image of a representative culture plate and G counted from various treatments Shows the graphical representation of the number of colonies of IST-T1. Note that each of imatinib and compound A as single agents shows a similar decrease in GIST T1 colony growth up to 18 - 23% of the vehicle control. Two-week combination treatment with 250 nM of compound A and cobimetinib at either 100 nM, 200 nM or 500 nM led to complete cytostasis or eradication of GIST T1 colony growth to the limit of detection as visualized by 5-fold magnified stereomicroscopy, and no colonies were detected 10 days after removal of the combination treatment (indicated by the arrow in Fig. 12A). Two-week combination treatment with 100 nM of compound A and 500 nM of cobimetinib led to complete cytostasis or eradication of GIST T1 colony growth to the limit of detection as visualized by 5-fold magnified stereomicroscopy, and no colonies were detected 10 days after removal of the combination treatment (indicated by the arrow in Fig. 12A). In contrast, two-week combination treatment with 500 nM of imatinib and cobimetinib at 100 nM, 200 nM or 500 nM did not lead to complete tumor cell cytostasis or eradication 10 days after removal of the combination treatment.

[0231] The effect was clearer after an extended period of incubation, with approximately 10 - 15 colonies growing with 500 nM of imatinib, and no colony growth observed with 500 nM of compound A and 500 nM of cobimetinib.

[0232] Figure 12B shows an image of a representative culture plate and a graphical representation of the number of GIST-T1 / D816E colonies counted from various treatments. It should be noted that each of imatinib (500 nM) and compound A (100 nM or 250 nM) as single agents was associated with colony growth of approximately 65 - 74% of the vehicle control and showed a nearly similar lack of cytocidal efficacy against GIST T1 / D816E (Figure 12B). Two-week combination treatment with compound A (250 nM) and cobimetinib (100 nM, 200 nM or 500 nM) led to complete cytostasis or eradication of colony growth in GIST-T1 / D816E cells to the limit of detection as visualized by stereomicroscopy at 5-fold after 10 days of recovery (see arrows in Figure 12B), while two-week combination treatment with imatinib (500 nM) and cobimetinib (100 nM, 200 nM or 500 nM) did not lead to complete cytostasis or eradication of tumor cells. The effect was more evident after an extended period of incubation, treatment with imatinib did not lead to complete inhibition, while with compound A, significant cytostasis or maintenance of cell death was seen even 20 days after removal of the drug.

[0233] Figure 12C shows an image of a representative culture plate and a graphical representation of the number of GIST-T1 / T670I colonies counted from various treatments. Two-week treatment with either 50 nM or 100 nM of compound A in combination with cobimetinib (200 nM or 500 nM) unexpectedly led to >99% inhibition of GIST T1 / T670I colony growth as visualized by stereomicroscopy at 5-fold (indicated by arrows in Figure 12C). Cytostasis was maintained even after an extended 20-day period after removal of the drug. In contrast, two-week treatment with up to 500 nM of cobimetinib in combination with 500 nM of imatinib did not lead to robust cell stasis or eradication of cells 10 days after removal of the combination treatment.

[0234] Example 20. The combined treatment of compound B with cobimetinib prevents colony growth in imatinib-resistant cells of GIST-T1, GIST-T1 / D816E, and GIST-T1 / T670I. To demonstrate that the combined treatment of compound B and cobimetinib prevents colony growth in three GIST cell lines as described in Example 15, a test was conducted.

[0235] 〔00235〕Figure 13A shows an image of a representative culture plate and a graphical representation of the number of colonies of G IST-T1 counted from various treatments. Note that compound B as a single agent shows a similar reduction in GIST T1 colony growth to 42 - 54% of the vehicle control. Two-week combined treatment with 50 nM, 100 nM, or 250 nM of compound B and either 200 nM or 500 nM of cobimetinib led to complete or near-complete cytostasis or eradication of GIST T1 colony growth to the limit of detection as visualized by stereomicroscopy at 5-fold magnification, and no colonies were detected 10 days after removal of the combined treatment (indicated by the arrow in Figure 13A). Two-week combined treatment with 100 nM or 250 nM of compound B and either 200 nM or 500 nM of cobimetinib resulted in significant cytostasis or cell death of GIST-T1 cells being maintained even after a prolonged 20-day recovery period.

[0236] Figure 13B shows an image of a representative culture plate and a graphical display of the number of GIST-T1 / D816E colonies counted from various treatments. Note that compound B as a single agent (100 nM or 250 nM) was associated with colony growth of approximately 58 - 84% of the vehicle control, indicating a lack of cytocidal efficacy against GIST T1 / D816E (Figure 13B). Two-week combination treatment with 50 nM, 100 nM, or 250 nM of compound B and either 200 nM or 500 nM of cobimetinib led to >90 inhibition of colony growth in GIST-T1 / D816E cells as visualized by 5-fold stereomicroscopy after 10 days of recovery (see arrow in Figure 13B). Cytostasis was significantly maintained even after an extended period of 20 days with compound B at higher concentrations.

[0237] Figure 13C shows an image of a representative culture plate and a graphical display of the number of GIST-T1 / T670I colonies counted from various treatments. Note that compound B as a single agent (100 nM or 250 nM) showed a decrease in GIST T1 / T670I colony growth up to approximately 75 - 78% of the vehicle control. Two-week treatment with either 50 nM, 100 nM, or 250 nM of compound B and 200 nM or 500 nM of cobimetinib led to complete cytostasis or eradication of GIST T1 / T670I colony growth to the limit of detection as visualized by 5-fold stereomicroscopy, and no colonies were detected 10 days after removal of the combination treatment (indicated by the arrow in Figure 13C). Cytostasis was maintained even after an extended 20-day period after removal of the drug.

[0238] Example 21. Combination treatment of compound A with ulixertinib prevents colony growth in imatinib-resistant cells of GIST-T1, GIST-T1 / D816E, and GIST-T1 / T670I A study was conducted to demonstrate that combination treatment of compound A and ulixertinib prevents colony growth in three GIST cell lines as described in Example 15.

[0239] Figure 14A shows an image of a representative culture plate and a graphical display of the number of GIST-T1 colonies counted from various treatments. Note that compound A as a single agent showed a nearly similar decrease in GIST T1 colony growth up to 37 - 41% of the vehicle control. Two-week combination treatment with compound A at 50 nM, 100 nM, or 250 nM and either 1 μM, 2 μM, or 3 μM of vatalanib led to a significant decrease in GIST T1 colony growth as visualized by 5-fold stereomicroscopy (indicated by the arrows in Figure 14A).

[0240] 〔00240〕Figure 14B shows an image of a representative culture plate and a graphical display of the number of G IST-T1 / D816E colonies counted from various treatments. Note that compound A (50 nM, 100 nM, or 250 nM) as a single agent was associated with colony growth of approximately 81 - 93% of the vehicle control and showed a nearly similar lack of cytocidal efficacy against GIST T1 / D816E (Figure 14B). Two-week combination treatment with compound A (250 nM) and vatalanib (2 μM or 3 μM) led to complete cytostasis or eradication of colony growth in GIST-T1 / D816E cells to the limit of detection as visualized by 5-fold stereomicroscopy 10 days after recovery (see the arrows in Figure 14B).

[0241] Figure 14C shows an image of a representative culture plate and a graphical display of the number of GIST-T1 / T670I colonies counted from various treatments. Treatment for two weeks with either 100 nM or 250 nM of compound A and vatalanib (2 μM or 3 μM) unexpectedly led to complete cytostasis or eradication of GIST T1 / T670I colony growth to the limit of detection as visualized by 5-fold stereomicroscopy, and no colonies were detected 10 days after removal of the combination treatment (indicated by the arrows in Figure 14C). Cytostasis was maintained even after an extended 20-day period after removal of the drug.

[0242] ​Example 22. The combined treatment of compound B with nilotinib prevents colony growth in imatinib-resistant GIST-T1 / D816E cells Figure 15 shows an image of a representative culture plate and a graphical representation of the number of GIST-T1 / D816E colonies counted from various treatments. Note that compound B as a single agent (50 nM, 100 nM, or 250 nM) was associated with colony growth of approximately 52 - 95% of the vehicle control, showing a similar lack of a cell-destructive effect on GIST T1 / D816E (Figure 15). Two weeks of combined treatment with compound B (250 nM) and nilotinib (3 μM) led to complete cell stasis or eradication of GIST T1 / D816E colony growth to the limit of detection as visualized by stereomicroscopy at 5-fold magnification, and no colonies were detected 10 days after removal of the combined treatment (indicated by the arrow in Figure 15).

[0243] Example 23. The combined treatment of compound A and the ERK inhibitor SCH772984 prevents colony growth in imatinib-resistant GIST-T1 / D816E cells Using the protocol outlined in Example 15, synergy can be shown for the combination of compound A and SCH772984 in preventing colony growth in imatinib-resistant GIST-T1, GIST-T1 / T670I, and GIST-T1 / D816E cells.

[0244] Example 24. The combined treatment of compound B and the ERK inhibitor SCH772984 prevents colony growth in imatinib-resistant GIST-T1 / D816E cells Using the protocol outlined in Example 15, synergy can be shown for the combination of compound B and SCH772984 in preventing colony growth in imatinib-resistant GIST-T1, GIST-T1 / T670I, and GIST-T1 / D816E cells.

[0245] Example 25. The combined treatment of compound A and the RAF inhibitor LY3009120 prevents colony growth in imatinib-resistant cells of GIST-T1 / D816E

[00245] Using the protocol outlined in Example 15, when preventing colony growth in imatinib-resistant cells of GIST-T1, GIST-T 1 / T670I and GIST-T1 / D816E, a synergistic effect can be shown for the combined use of compound A and LY3009120.

[0246] Example 26. The combined treatment of compound B and the RAF inhibitor LY3009120 prevents colony growth in imatinib-resistant cells of GIST-T1 / D816E Using the protocol outlined in Example 15, when preventing colony growth in imatinib-resistant cells of GIST-T1, GIST-T1 / T670I and GIST-T1 / D816E, a synergistic effect can be shown for the combined use of compound B and LY3009120.

[0247] Example 27. The combined treatment of compound A and the RAF inhibitor dabrafenib prevents colony growth in imatinib-resistant cells of GIST-T1 / D816E Using the protocol outlined in Example 15, when preventing colony growth in imatinib-resistant cells of GIST-T1, GIST-T1 / T670I and GIST-T1 / D816E, a synergistic effect can be shown for the combined use of compound A and dabrafenib can be shown.

[0248] Example 28. The combined treatment of compound B and the RAF inhibitor dabrafenib prevents colony growth in imatinib-resistant cells of GIST-T1 / D816E Using the protocol outlined in Example 15, when preventing colony growth in imatinib-resistant cells of GIST-T1, GIST-T1 / T670I and GIST-T1 / D816E, a synergistic effect can be shown for the combined use of compound B and dabrafenib.

[0249] Example 29. The combination treatment induces apoptosis in GIST-T1 cells transfected with N-ras G12D A study was conducted to demonstrate that the combination treatment of Compound A and trametinib induces apoptosis in GIST-T1 cells transfected with an empty vector control (EV) and the mutant N-ras G12D. For GIST-T1 cells transfected with the vector control or N-ras G12D, assays were performed in 96-well plates seeded with 10,000 cells per well. The cells were treated with vehicle control, Compound A, trametinib, or their combination at various concentrations and allowed to grow for 48 hours. Apoptosis was evaluated by measuring caspase 3 / 7 activity.

[0250] 〔00250〕Figure 16A provides a graphical representation of caspase activity measurements after various treatments. A 48-hour combination treatment with 50 nM of Compound A and trametinib (50 nM or 100 nM) induced an increase in apoptosis in GIST-T1 cells transfected with the mutant N-ras G12D compared to cells treated with either single-agent Compound A or trametinib.

[0251] Example 30. The combination treatment prevents colony growth in GIST-T1 cells transfected with N-ras G12D A study was conducted to demonstrate that the combination treatment of Compound A and trametinib prevents resistant colony growth in GIST-T1 cells transfected with an empty vector control and the mutant N-ras G12D. Assays were performed in 6-well plates seeded with 100 cells per well. The cells were treated with vehicle control, 5 nM of Compound A, 50 nM or 100 nM of trametinib, or their combination and cultured for 2 weeks. In the same experiment, the cells were treated with vehicle control, 100 nM of imatinib, 50 nM or 100 nM of trametinib, or their combination. After 2 weeks, the drugs were washed out and the cells were cultured in normal medium for 1-3 weeks. The colonies were stained with crystal violet and counted.

[0252] Figure 16B shows an image of a representative culture plate, and Figure 16C shows a graphical representation of the number of GIST-T1 colonies transfected with vector control (Figure 16B.1) and mutant N-ras G12D (Figure 16B.2) counted after various treatments. Quantification of colony growth in GIST T-1 cells transfected with vector control and N-ras G12D is shown in Figures 16C.1 and 16C.2, respectively. Combination treatment with 100 nM imatinib and 50 nM trametinib resulted in colony growth (35% of vehicle control), and combination of 100 nM imatinib with 100 nM trametinib also resulted in colony growth (19% of vehicle). In contrast, combination of compound A with trametinib unexpectedly resulted in superior cell stasis or cell death compared to combination with imatinib. Two-week combination treatment with 50 nM compound A and 50 nM trametinib led to nearly complete cell stasis or cell death (2% of vehicle control), and combination of 50 nM compound A with 100 nM trametinib led to complete (0% of vehicle control colony growth) cell stasis or cell death to the limit of detection visualized by stereomicroscopy 5-fold after 10-day drug washout and recovery (see arrow in Figure 16C.2). control).

[0253] Figure 16D shows an image of a representative culture plate of the number of GIST-T1 colonies transfected with mutant N-ras G12D counted after an extended drug-free recovery period. Two-week combination treatment with 100 nM compound A and 50 nM or 100 nM trametinib led to nearly complete inhibition of colony growth in GIST-T1 cells transfected with N-ras G12D after a 21-day extended long-term recovery period.

[0254] Example 32. Combination treatment prevents colony growth of drug-resistant GIST cells In Ba / F3 cells transfected with the oncogenic KIT V560D mutant, a saturation mutagenesis assay was performed. DNA nicking was induced with N-ethyl-N-nitrosourea (ENU) for 18 hours to randomly generate additional mutations in the KIT gene or other genes. The assay was performed in 6-well plates seeded with 100 cells per well. After washing the ENU, the wells were incubated with 10 nM trametinib, 25 nM or 100 nM or 250 nM compound A, or 100 nM or 250 nM or 500 nM imatinib in combination with 10 nM trametinib and 25 nM, 100 nM, or 250 nM compound A. Wells showing resistance to drug treatment showed growth of Ba / F3 cells. These cells were subjected to PCR and sequencing of the KIT gene to determine the presence of resistant secondary mutations induced by ENU treatment.

[0255]

[00255] Figure 17A shows the growth of Ba / F3 colonies resistant to imatinib It is a graph display. The imatinib-resistant KIT secondary mutants of T670I, K807E, and / or D816V were identified by PCR and DNA sequencing of genomic DNA in Ba / F3 cells exposed to 100 nM, 250 nM, or 500 nM imatinib as a single agent (Figure 17A, left panel). Figure 17A (right panel) is a graph display of a saturation mutagenesis test of Ba / F3 cells using Compound A. Monotherapy with 25 nM, 100 nM, or 250 nM of Compound A did not lead to the growth of new resistant secondary mutations as determined by PCR and DNA sequencing. Only Ba / F3 cells containing the original V560D (parent) KIT mutant grew after exposure to Compound A, presumably reflecting mutations in genes other than KIT (Figure 17A, right panel). Figure 17B is a graph display showing Ba / F3 cell colony growth with imatinib in the presence of trametinib or Compound A in the presence of trametinib. The combination of 250 nM or 500 nM imatinib with 10 nM trametinib did not lead to the growth of new resistant secondary mutations, but led to the growth of the original KIT V560D (parent) cells, presumably reflecting mutations in genes other than KIT (Figure 17B, left panel). Significantly, in contrast to the combination test of imatinib with trametinib, the combination of 25, 100, or 250 nM of Compound A with 10 nM trametinib led to complete cell stasis or cell death without any cell growth up to the limit of detection as determined by visual inspection of any wells (Figure 17B, right panel).

[0256] Example 33. In Vivo Xenograft Test of Compound A Combined with Trametinib The GIST T1 xenograft model was conducted in accordance with all laws, regulations, and guidelines of the US National Institutes of Health (NIH), approval by the Animal Care AND Use Committee of MI Bioresearch (Ann Arbor, Michigan, USA), and approval of an AAALAC-accredited facility. All mice were fed ad libitum with diet and water. All mice were observed for clinical signs at least once a day. Immediately below the right axilla of female nude mice from Envigo (HsdCrl: Nude-Foxn1nu athymic, 6 - 7 weeks old), 5 million cells in Dulbecco's phosphate-buffered saline mixed with the same amount of Matrigel were subcutaneously inoculated with a 27-gauge needle and syringe. When the tumor volume reached an average of 117 mm 3 over 10 days, the mice were randomly assigned to each group so that the average tumor volume of all groups was within 10% of the overall average tumor volume for the test population. The groups were treated as follows from days 10 - 27: vehicle control diet (n = 10); compound A was formulated in the mice's diet to achieve approximately 100 mg / kg / day (n = 10); or compound A was formulated in the mice's diet to achieve approximately 25 mg / kg / day (n = 10), trametinib was orally administered at 0.5 mg / kg BID and given a vehicle control diet (n = 10), trametinib was orally administered at 0.5 mg / kg BID and given a diet formulated with compound A (to achieve treatment with compound A at approximately 100 mg / kg / day) (n = 10), or trametinib was orally administered at 0.5 mg / kg BID and given a diet formulated with compound A (to achieve treatment with compound A at approximately 25 mg / kg / day (n = 10). On day 27, all animals were placed on a control diet and tumor regrowth was observed. Tumor volume and body weight were measured three times a week. Tumor mass (mg) was estimated from caliper measurements using the formula: Tumor mass (mg = mm 3 ) = (length × width 2 ) / 2.

[0257] Figures 18A and 18B are graphical representations showing the inhibition of tumor growth compared to vehicle control. Figure 18B is the same data as Figure 18A but is expanded to show the differences between the cohorts treated with Compound A or Compound A / trametinib. Treatment with trametinib led to a slight inhibition of tumor growth compared to vehicle control. At high dose of Compound A (about 100 mg / kg / day), 6 / 10 mice had complete tumor regression and the remaining 4 / 10 mice had partial tumor regression during the dosing period. At low dose of Compound A (about 25 mg / kg / day), 2 / 10 mice had complete tumor regression and 6 / 10 had partial tumor regression. At high dose of Compound A (about 100 mg / kg / day) in combination with trametinib, 10 / 10 mice had complete tumor regression during the dosing period. At low dose of Compound A (about 25 mg / kg / day) in combination with trametinib, 5 / 10 mice had complete tumor regression and 5 / 10 had partial tumor regression. Further, after the dosing period, all cohorts treated with Compound A had tumor regrowth slower than the initial tumor growth of the vehicle control, indicating a long-term effect on tumor cell growth until the end of the 66-day study. At high dose of Compound A (about 100 mg / kg / day), 1 / 10 mice remained with partial tumor regression at the end of the study. At low dose of Compound A (about 25 mg / kg / day), 2 / 10 mice remained with partial tumor regression at the end of the study. At high dose of Compound A (about 100 mg / kg / day) in combination with trametinib, 1 / 10 mice remained with complete tumor regression and 4 / 10 mice remained with partial tumor regression at the end of the study. At low dose of Compound A (about 25 mg / kg / day) in combination with trametinib, 2 / 10 mice remained with partial tumor regression at the end of the study. These data indicate that the combination of Compound A and trametinib induces cell death and / or completes a long cell stasis of at least 40 days after dosing.

[0258] Example 34. Compound A is a potent inhibitor of the BCRP efflux transporter To investigate the inhibition of the BCRP drug efflux transporter by Compound A, a vesicular transport inhibition assay was performed using inside-out membrane vesicles prepared from BCRP-expressing cells in the presence of a low permeability probe substrate and ATP. The potential of Compound A to modify the uptake of the probe substrate into transporter-containing vesicles was measured.

[0259] The in vitro interaction potential of Compound A with the human efflux transporter BCRP was investigated at seven concentrations in a vesicular transport inhibition assay. Compound A effectively inhibited the transport of the BCRP probe substrate, with 44% inhibition observed at the lowest concentration tested (0.04 μM). The IC50 value was estimated to be approximately 0.04 μM.

[0260] Equivalents 〔00260〕One of ordinary skill in the art will recognize, or be able to ascertain using no more than routine experimentation, numerous equivalents to the specific embodiments specifically described herein. Such equivalents are intended to be encompassed by the following claims.

Claims

1. 1. A medicament for use in treating a tumor with one or more c-KIT mutations in a patient in need thereof, comprising: an effective amount 【Chemical 1】 or a pharmaceutically acceptable salt thereof; A pharmaceutical comprising an effective amount of one or more MAPKAP kinase inhibitors in combination.

2. The pharmaceutical composition of claim 1, wherein the MAPKAP kinase inhibitor is selected from the group consisting of a mitogen-activated protein kinase inhibitor (MEK inhibitor) and an effective amount of an extracellular signal-regulated kinase inhibitor (ERK inhibitor).

3. The method of claim 1, wherein the c-KIT mutation is a primary mutation in exon 9, exon 11, exon 13, or exon 17 of the c-KIT gene.

4. The method according to any one of claims 1 to 3, wherein the tumor is associated with one or more secondary resistance mutations in the c-KIT gene.

5. The pharmaceutical agent according to claim 4, wherein the secondary resistance mutation is in exon 13, exon 14, exon 17, or exon 18 of the c-KIT gene.

6. The pharmaceutical agent according to claim 5, wherein the secondary resistance mutation is in exon 17 of the c-KIT gene.

7. The method of claim 4, wherein the secondary resistance mutation is a substitution of aspartic acid at codon 816 or an asparagine at codon 822.

8. The medicament of claim 4, wherein the secondary resistance mutation is one of D816V, D816E, D816H, D820A, T670I, or N822V.

9. The method of claim 4, wherein the secondary resistance mutation is one of V654A or T670I.

10. The method of claim 1, wherein the c-KIT mutation is a deletion mutation.

11. The pharmaceutical composition of claim 4, wherein the secondary resistance mutation was acquired after prior administration of imatinib, sunitinib or regorafenib, or a pharmaceutically acceptable salt thereof, to the patient.

12. The method of claim 4, wherein the treatment further comprises determining whether the tumor is associated with a c-KIT resistance secondary mutation.

13. The method of claim 12, wherein determining whether the tumor is associated with a secondary resistance mutation in c-KIT comprises identifying the mutation in DNA extracted from the tumor sample.

14. The method of claim 12, wherein determining whether the tumor is associated with a c-KIT resistance secondary mutation comprises identifying the mutation in circulating tumor DNA.

15. The pharmaceutical composition of claim 4, wherein the tumor has been resistant to treatment with imatinib mesylate, sunitinib malate, or regorafenib.

16. The pharmaceutical composition of claim 1, wherein the tumor is selected from the group consisting of lung adenocarcinoma, lung squamous cell carcinoma, glioblastoma, pediatric glioma, astrocytoma, sarcoma, gastrointestinal stromal tumor (GIST), and melanoma.

17. The pharmaceutical composition of claim 16, wherein the tumor is melanoma.

18. The pharmaceutical composition of claim 16, wherein the tumor is a GIST.

19. 10. The pharmaceutical of claim 1, further comprising a cancer targeted therapeutic agent, a cancer targeted biologic, an immune checkpoint inhibitor, an immunomodulatory agent, and / or a chemotherapeutic agent.

20. The pharmaceutical composition of claim 1 further comprising a RAF inhibitor.

21. The aforementioned 【Chemistry 2】 3. The pharmaceutical composition according to claim 2, wherein the compound or a pharmaceutically acceptable salt thereof and the MAPKAP kinase inhibitor are administered substantially simultaneously or sequentially.

22. The pharmaceutical composition of claim 2, wherein the MEK inhibitor is selected from the group consisting of trametinib, selumetinib, cobimetinib, and binimetinib.

23. The pharmaceutical composition of claim 2, wherein the ERK inhibitor is selected from the group consisting of ulixertinib, SCH772984, and LY3214996.

24. The method of claim 2, wherein the patient experiences a partial reduction of at least 30% in tumor volume after use of the medicament for two weeks or more.

25. The pharmaceutical of claim 1, which results in a complete reduction in tumor volume.

26. The method of claim 1, wherein the treatment further comprises determining whether the tumor or tumor cells contain a primary mutation in the c-KIT gene.

27. The method of claim 26, wherein the primary mutation is in exon 11 of the c-KIT gene.

28. The method of claim 26, wherein the primary mutation is in exon 9 of the c-KIT gene.

29. The method of claim 26, wherein the primary mutation is a deletion mutation.

30. The method of any one of claims 26 to 28, wherein the primary mutation is V560D.

31. The method of claim 1, wherein one or more additional secondary mutations in c-KIT are present.

32. for use in the treatment of solid tumors in patients resistant to imatinib; an effective amount 【Chemistry 3】 or a pharmaceutically acceptable salt thereof; A pharmaceutical composition comprising an effective amount of a MAPKAP kinase inhibitor selected from the group consisting of trametinib, binimetinib, cobimetinib, and ulixertinib, in combination, The solid tumor is selected from the group consisting of lung adenocarcinoma, lung squamous cell carcinoma, glioblastoma, pediatric glioma, astrocytoma, sarcoma, gastrointestinal stromal tumor (GIST), and melanoma.

33. The pharmaceutical composition of claim 32, further comprising a RAF inhibitor.

34. The pharmaceutical composition of claim 33, wherein the RAF inhibitor is a pan-RAF inhibitor.

35. For use in the treatment of imatinib-resistant gastrointestinal stromal tumors or imatinib-resistant melanoma in a patient in need thereof. an effective amount 【Chemistry 4】 or a pharmaceutically acceptable salt thereof; A pharmaceutical comprising in combination an effective amount of a MAPKAP kinase inhibitor selected from the group consisting of trametinib, binimetinib, cobimetinib, and ulixertinib.

36. The method of claim 35, wherein the treatment further comprises determining whether the tumor is associated with a mutation in the c-KIT gene.

37. The method of claim 36, wherein the mutation is in exon 17 of the c-KIT gene.

38. The method of claim 36 or 37, wherein the c-KIT mutation is a substitution of aspartic acid at codon 816 or an asparagine at codon 822.

39. 38. The pharmaceutical of claim 36 or 37, wherein the mutation is one of D816V, D816E, D816H, D820A, T670I, or N822V.

40. The pharmaceutical composition of claim 36, wherein the mutation is a V654A mutation in exon 13 or a T670I mutation in exon 14.

41. 1. A medicament for use in the treatment of a solid tumor in a patient in need thereof, comprising: an effective amount 【Chemistry 5】 or a pharmaceutically acceptable salt thereof in combination with an effective amount of a MAPKAP kinase inhibitor.

42. 42. The pharmaceutical composition of claim 41, further comprising a RAF inhibitor.

43. The pharmaceutical composition of claim 42, wherein the RAF inhibitor is a pan-RAF inhibitor.

44. The method of claim 41, wherein the treatment further comprises determining whether the tumor is associated with a mutation in the c-KIT gene.

45. The method of claim 44, wherein the mutation is in exon 17 of the c-KIT gene.

46. The method of claim 44 or 45, wherein the c-KIT mutation is a substitution of aspartic acid at codon 816 or an asparagine at codon 822.

47. 46. ​​The pharmaceutical of claim 44 or 45, wherein the mutation is one of D816V, D816E, D816H, D820A, T670I, or N822V.

48. The pharmaceutical composition of claim 44, wherein the mutation is a V654A mutation in exon 13 or a T670I mutation in exon 14.

49. The pharmaceutical composition of any one of claims 41 to 45, wherein the solid tumor is selected from the group consisting of lung adenocarcinoma, lung squamous cell carcinoma, glioblastoma, GIST, and melanoma.

50. The pharmaceutical agent according to any one of claims 41 to 49, wherein the solid tumor is associated with one or more mutations in the c-KIT gene.

51. The pharmaceutical composition of claim 42, wherein the RAF inhibitor is dabrafenib or LY3009120.

52. 1. A medicament for use in the treatment of a solid tumor in a patient in need thereof, comprising: an effective amount 【Chemistry 6】 or a pharmaceutically acceptable salt thereof; in combination with an effective amount of a MAPKAP kinase inhibitor selected from the group consisting of trametinib, binimetinib, cobimetinib, and ulixertinib; The solid tumor is selected from the group consisting of lung adenocarcinoma, lung squamous cell carcinoma, glioblastoma, pediatric glioma, astrocytoma, sarcoma, gastrointestinal stromal tumor (GIST), and melanoma.

53. 53. The pharmaceutical composition of claim 52, further comprising a RAF inhibitor.

54. The pharmaceutical agent of claim 53, wherein the RAF inhibitor is a pan-RAF inhibitor.

55. 1. A medicament for use in treating gastrointestinal stromal tumor or melanoma in a patient in need thereof, comprising: an effective amount 【Chemistry 7】 or a pharmaceutically acceptable salt thereof; A medicine comprising in combination an effective amount of a MAPKAP kinase inhibitor selected from the group consisting of trametinib, binimetinib, cobimetinib, and ulixertinib.

56. The pharmaceutical agent of claim 55, wherein the treatment further comprises determining whether the tumor is associated with a mutation in the c-KIT gene.

57. The pharmaceutical agent of claim 56, wherein the mutation is a V654A mutation in exon 13 or a T670I mutation in exon 14.

Images