Combination Therapies for the Treatment of Cancer

Abstract

The present disclosure provides methods for treating cancer by combination therapy with an SHP2 inhibitor, such as a compound of Formula I, an FGFR inhibitor, a B-Raf inhibitor, a MEK inhibitor, or a MET inhibitor.

Description

Technical Field

[0001] Cross-reference This application claims the benefit of U.S. Provisional Patent Application No. 63 / 124,663, filed December 11, 2020, U.S. Provisional Patent Application No. 63 / 124,667, filed December 11, 2020, U.S. Provisional Patent Application No. 63 / 124,671, filed December 11, 2020, and U.S. Provisional Patent Application No. 63 / 124,674, filed December 11, 2020, and each application is incorporated herein by reference in its entirety.

Background Art

[0002] Src homology-2 phosphatase (SHP2) is a non-receptor protein phosphatase that is ubiquitously expressed in various tissues and cell types (see reviews: Tajan M et al., Eur J Med Genet 2016 58(10):509-25; Grossmann KS et al., Adv Cancer Res 2010 106:53-89). SHP2 consists of two Src homology 2 (N-SH2 and C-SH2) domains at its NH2 terminus, a catalytic PTP (protein-tyrosine phosphatase) domain, and a C-terminal tail with regulatory properties. In the basal state, the intermolecular interaction between the SH2 domain and the PTP domain impedes the access of the substrate to the catalytic pocket and maintains SHP2 in a closed auto-inhibited conformation. In response to stimulation, SHP2-activating proteins with phosphotyrosine motifs bind to the SH2 domain, resulting in the exposure of the active site and the enzymatic activation of SHP2.

Summary of the Invention

[0003] The embodiments disclosed herein generally relate to compositions and methods for treating cancer, including using an SHP2 inhibitor in combination with an FGFR inhibitor, a B-Raf inhibitor, a MEK inhibitor or a MET inhibitor to provide an unexpected synergistic effect.

[0004] SHP2 plays a crucial role in fundamental cellular functions, including proliferation, differentiation, cell cycle maintenance, and motility. By dephosphorylating its associated signaling molecules, SHP2 modulates multiple intracellular signaling pathways in response to a wide range of growth factors, cytokines, and hormones. Cellular signaling processes involving SHP2 include the RAS-MAPK (mitogen-activated protein kinase), PI3K (phosphoinositol 3-kinase)-AKT, and JAK-STAT pathways.

[0005] SHP2 also plays a signal-enhancing role in this pathway, acting downstream of RTKs and upstream of RAS. One common mechanism of resistance involves RTK activation, which promotes the reactivation of MAPK signaling. RTK activation recruits SHP2 via direct binding and through adapter proteins. These interactions result in the conversion of SHP2 from a closed (inactive) conformation to an open (active) conformation. SHP2 is a key promoter of RAS signaling reactivation, evading pharmacological inhibition in both primary and secondary resistance. Inhibition of SHP2 often results in an overall attenuation of upstream RTK signaling that drives oncogenic signaling and adaptive tumor escape (see Prahallad, A. et al. Cell Reports 12, 1978-1985 (2015); Chen YN, Nature 535, 148-152 (2016)), and all teachings, including but not limited to all methods, compounds, compositions, data, etc., for use in conjunction with any of the embodiments and disclosures herein, are incorporated herein by reference in their entirety.

[0006] Fibroblast growth factor receptors (FGFRs) bind to members of the fibroblast growth factor family of proteins and also influence the RAS-MAPK signaling pathway upstream of the RAS. The opportunity to target signaling pathways from multiple directions and potentially improve the feedback loop upstream of Ras via SHP2 provides an opportunity to develop ways to use combination therapies. This disclosure provides such methods while offering unexpected synergistic effects.

[0007] The RAS-MAPK signaling pathway involves the Raf family of proteins. This family consists of three related kinases (A-, B-, and C-Raf) that act as downstream effectors of Ras. In particular, B-Raf is a serine / threonine protein kinase that activates the MAP kinase / ERK signaling pathway. Constitutively active B-Raf mutants are generally known to cause cancer by excessively signaling cell proliferation. For example, the activated B-Raf V600E kinase mutant occurs in about 7% of human malignancies and about 50-60% of melanomas.

[0008] The RAS-MAPK signaling pathway also includes MEK1 and MEK2. MEK1 and MEK2 are dual-function serine / threonine and tyrosine protein kinases, also known as MAP kinase kinases. MEK plays a pivotal role in the RAS-regulated RAF-MEK-ERK signaling pathway, which transmits signals from growth factor receptors to the nucleus and regulates, among other things, cell proliferation, differentiation, survival, and invasion.

[0009] Finally, the pivotal protein tyrosine kinase, extracellular MET (or c-MET), acts upstream of the RAS-MAPK signaling pathway. The opportunity to target signaling pathways from multiple angles and potentially improve the upstream feedback loop of Ras via SHP2 provides an opportunity to develop ways to use combination therapies. Embodiments disclosed herein do so while providing unexpected synergies.

[0010] In a first aspect, the disclosure relates to a method for treating a subject having cancer, wherein the subject is given a therapeutically effective amount of a compound of formula I or a pharmaceutically acceptable salt thereof.

[0011] [ka] The present invention provides a method comprising the step of administering in combination with an FGFR inhibitor. In some embodiments, FGFR in the subject is constitutively active. In some embodiments, the cancer is lung cancer. In some embodiments, the cancer is hepatocellular carcinoma. In some embodiments, the cancer is cholangiocarcinoma. In some embodiments, the cancer is pancreatic ductal adenocarcinoma (PDAC). In some embodiments, the inhibitor is selected from the group consisting of erdafitinib, AZD4547, Ly2874455, CH5183284, NVP-BGJ398, INCB054828, logaratinib, PRN1371, TAS-120, BLU-554, H3B-6527, and FGF401. In some embodiments, the FGFR inhibitor is erdafitinib. In some embodiments, the FGFR inhibitor is pemigatinib, infiglatinib, dovitinib, ponatinib, nintedanib, and fisogatinib. In some embodiments, the method includes the step of administering a third MAPK pathway inhibitor. In some embodiments, the administration is orally. In some embodiments, the dose of the compound of formula I ranges from 20 mg to 400 mg per day. In some embodiments, the dose of the FGFR inhibitor ranges from 1 mg to 500 mg per day.

[0012] In a second aspect, the disclosure relates to a therapeutically effective amount of a compound of formula I or a pharmaceutically acceptable salt thereof.

[0013] [ka] The present invention provides a method for treating liver cancer in a subject, comprising the step of orally administering to the subject a compound of formula I in combination with erdafitinib. In some embodiments, the compound of formula I is administered once or twice daily. In some embodiments, erdafitinib is administered once or twice daily. In some embodiments, the subject is human.

[0014] In a third embodiment, the disclosure provides a kit comprising a compound of formula I or a pharmaceutically acceptable salt thereof and an FGFR inhibitor. In some embodiments, the compound of formula I and the FGFR inhibitor are contained in separate packages. In some embodiments, the kit further includes instructions for administering the contents of the kit to a target for the treatment of cancer. In some embodiments, the FGFR inhibitor is one or more of erdafitinib, AZD4547, Ly2874455, CH5183284, NVP-BGJ398, INCB054828, rogaratinib, PRN1371, TAS-120, BLU-554, H3B-6527, FGF401, pemigatinib, infiglatinib, dovitinib, ponatinib, nintedanib, and fisogatinib.

[0015] In a fourth aspect, the disclosure relates to a method for treating a subject having cancer, wherein the subject is given a therapeutically effective amount of a compound of formula I or a pharmaceutically acceptable salt thereof.

[0016] [ka] The present invention provides a method comprising the step of administering a compound in combination with an inhibitor of the B-Raf protein having a class 1 mutation. In some embodiments, the class 1 mutation is V600E. In some embodiments, the cancer is colorectal cancer. In some embodiments, the cancer is melanoma. In some embodiments, the cancer is thyroid cancer. In some embodiments, the cancer is pancreatic ductal adenocarcinoma (PDAC). In some embodiments, the inhibitor is selected from the group consisting of encorafenib, vemurafenib, dabrafenib, sorafenib, and regorafenib. In some embodiments, the inhibitor is encorafenib. In some embodiments, the inhibitor is vemurafenib. In some embodiments, the inhibitor is dabrafenib. In some embodiments, the inhibitor is sorafenib. In some embodiments, the inhibitor is regorafenib. In some embodiments, the method comprises the step of administering a third MAPK pathway inhibitor. In some embodiments, the administration is orally. In some embodiments, the dose of the compound of formula I ranges from 20 mg to 400 mg daily. In some embodiments, the dose of the B-Raf inhibitor ranges from 1 mg to 500 mg.

[0017] In another embodiment, the Disclosure relates to a therapeutically effective amount of a compound of formula I or a pharmaceutically acceptable salt thereof.

[0018] [ka] The present invention provides a method for treating colorectal cancer in a subject, comprising the step of orally administering to the subject a compound of formula I in combination with the B-Raf inhibitor erdafitinib. In some embodiments, the compound of formula I is administered once or twice daily. In some embodiments, erdafitinib is administered once or twice daily. In some embodiments, the subject is human.

[0019] In another aspect, the disclosure relates to a method for treating a subject having cancer, wherein the subject is given a therapeutically effective amount of a compound of formula I or a pharmaceutically acceptable salt thereof.

[0020] [ka] The present invention provides a method that includes the step of administering in combination with encorafenib.

[0021] In another aspect, the disclosure relates to a method for treating a subject having cancer, wherein the subject is given a therapeutically effective amount of a compound of formula I or a pharmaceutically acceptable salt thereof.

[0022] [ka] The present invention provides a method that includes the step of administering in combination with vemurafenib.

[0023] In another aspect, the disclosure relates to a method for treating a subject having cancer, wherein the subject is given a therapeutically effective amount of a compound of formula I or a pharmaceutically acceptable salt thereof.

[0024] [ka] The present invention provides a method that includes the step of administering in combination with dabrafenib.

[0025] In another aspect, the disclosure relates to a method for treating a subject having cancer, wherein the subject is given a therapeutically effective amount of a compound of formula I or a pharmaceutically acceptable salt thereof.

[0026] [ka] The present invention provides a method that includes the step of administering in combination with sorafenib.

[0027] In another aspect, the disclosure relates to a method for treating a subject having cancer, wherein the subject is given a therapeutically effective amount of a compound of formula I or a pharmaceutically acceptable salt thereof.

[0028] [ka] The present invention provides a method comprising the step of administering regorafenib in combination with encorafenib.

[0029] In various embodiments of the methods described herein, the cancer is colorectal cancer. In some embodiments, the cancer is thyroid cancer. In some embodiments, the cancer is melanoma. In some embodiments, the cancer is adenocarcinoma of the pancreatic ducts (PDAC). In some embodiments, the dose of the B-Raf inhibitor is less than the dose required for monotherapy with the B-Raf inhibitor. In some embodiments, the dose of the compound of formula I is less than the dose required for monotherapy with the compound of formula I.

[0030] In another aspect, the present disclosure provides a method for inhibiting ERK1 / 2 phosphorylation in a cell population, wherein the cell population is subjected to a compound of formula I combined with regorafenib or a pharmaceutically acceptable salt thereof.

[0031] [ka] The present invention provides a method comprising the step of contacting with a substance. In some embodiments, the concentration of the compound of formula I is in the range of 1 nM to 500 nM. In some embodiments, the concentration of encorafenib is in the range of 10 nM to 20 nM.

[0032] In another embodiment, the disclosure provides a kit comprising a compound of formula I or a pharmaceutically acceptable salt thereof, and a B-Raf inhibitor. In some embodiments, the compound of formula I and the B-Raf inhibitor are contained in separate packages. In some embodiments, the kit further includes instructions for administering the contents of the kit to a target for the treatment of cancer. In some embodiments, the B-Raf inhibitor is one or more of encorafenib, vemurafenib, dabrafenib, sorafenib, and regorafenib.

[0033] In another aspect, the disclosure relates to a method for treating a subject having cancer, wherein the subject is given a therapeutically effective amount of a compound of formula I or a pharmaceutically acceptable salt thereof.

[0034] [ka] The present invention provides a method comprising the step of administering in combination with a MEK inhibitor. In some embodiments, the MEK inhibitor selectively inhibits MEK1 or MEK2, or both MEK1 and MEK2. In some embodiments, the cancer is metastatic. In some embodiments, the cancer is colorectal cancer. In some embodiments, the cancer is melanoma. In some embodiments, the cancer is lung cancer. In some embodiments, the cancer is pancreatic cancer. In some embodiments, the cancer is breast cancer. In some embodiments, the cancer is adenocarcinoma of the pancreatic ducts (PDAC). In some embodiments, the MEK inhibitor is selected from the group consisting of trametinib, cobimetinib, binimetinib, PD-0325901, selumetinib, and CI-1040. In some embodiments, the MEK inhibitor is trametinib. In some embodiments, the MEK inhibitor is cobimetinib. In some embodiments, the MEK inhibitor is binimetinib. In some embodiments, the MEK inhibitor is PD-325901. In some embodiments, the MEK inhibitor is CI-1040. In some embodiments, the method includes the step of administering an additional MAPK pathway inhibitor. In some embodiments, the administration is orally. In some embodiments, the dose of the compound of formula I ranges from 20 mg to 400 mg per day. In some embodiments, the dose of the MEK inhibitor ranges from 1 mg to 500 mg per day.

[0035] In another embodiment, the Disclosure relates to a therapeutically effective amount of a compound of formula I or a pharmaceutically acceptable salt thereof.

[0036] [ka] The present invention provides a method for treating cancer in a subject, comprising the step of orally administering to the subject a compound of formula I in combination with the MEK inhibitor binimetinib or trametinib. In some embodiments, the compound of formula I is administered once or twice daily. In some embodiments, binimetinib or trametinib is administered once or twice daily. In some embodiments, the subject is human.

[0037] In another aspect, the disclosure relates to a method for treating a subject having cancer, wherein the subject is given a therapeutically effective amount of a compound of formula I or a pharmaceutically acceptable salt thereof.

[0038] [ka] The present invention provides a method that includes the step of administering in combination with binimetinib.

[0039] In another aspect, the disclosure relates to a method for treating a subject having cancer, wherein the subject is given a therapeutically effective amount of a compound of formula I or a pharmaceutically acceptable salt thereof.

[0040] [ka] The present invention provides a method that includes the step of administering in combination with trametinib.

[0041] In various embodiments of the methods described herein, the cancer is colorectal cancer. In some embodiments, the cancer is lung cancer. In some embodiments, the cancer is melanoma. In some embodiments, the dose of the MEK inhibitor is less than the dose required for monotherapy with the MEK inhibitor. In some embodiments, the dose of the compound of formula I is less than the dose required for monotherapy with the compound of formula I.

[0042] In another aspect, the present disclosure provides a method for inhibiting ERK1 / 2 phosphorylation in a cell population, comprising a compound of formula I combined with binimetinib or trametinib, or a pharmaceutically acceptable salt thereof.

[0043] [ka] The present invention provides a method comprising the step of contacting with a substance. In some embodiments, the concentration of the compound of formula I is in the range of 1 nM to 1,000 nM. In some embodiments, the concentration of the MEK inhibitor is in the range of 10 nM to 500 nM.

[0044] In another embodiment, the disclosure provides a kit comprising a compound of formula I or a pharmaceutically acceptable salt thereof and a MEK inhibitor. In some embodiments, the compound of formula I and the MEK inhibitor are contained in separate packages. In some embodiments, the kit further includes instructions for administering the contents of the kit to a target for the treatment of cancer. In some embodiments, the MEK inhibitor is one or more of trametinib or binimetinib.

[0045] In another aspect, the disclosure relates to a method for treating a subject having cancer, wherein the subject is given a therapeutically effective amount of a compound of formula I or a pharmaceutically acceptable salt thereof.

[0046] [ka] The present invention provides a method comprising the step of administering a compound in combination with a MET inhibitor. In some embodiments, the MET inhibitor is also an ALK inhibitor, a ROS1 inhibitor, or both. In some embodiments, the cancer is non-small cell lung cancer. In some embodiments, the cancer is gastric cancer. In some embodiments, the cancer is gastric adenocarcinoma. In some embodiments, the cancer is pancreatic ductal adenocarcinoma (PDAC). In some embodiments, the MET inhibitor is selected from the group consisting of crizotinib, tepotinib, savolitinib, cabozantinib, and tivantinib. In some embodiments, the MET inhibitor is crizotinib. In some embodiments, the MET inhibitor is tepotinib. In some embodiments, the inhibitor is savolitinib. In some embodiments, the inhibitor is cabozantinib. In some embodiments, the inhibitor is tivantinib. In some embodiments, the method comprises the step of administering a third MAPK pathway inhibitor. In some embodiments, the administration is orally. In some embodiments, the dose of the compound of formula I ranges from 10 mg to 500 mg daily. In some embodiments, the dosage of the inhibitor ranges from 20 mg to 400 mg per day.

[0047] In another embodiment, the Disclosure relates to a therapeutically effective amount of a compound of formula I or a pharmaceutically acceptable salt thereof.

[0048] [ka] The present invention provides a method for treating gastric cancer in a subject, comprising the step of orally administering to the subject a compound of formula I in combination with crizotinib. In some embodiments, the compound of formula I is administered once or twice daily. In some embodiments, crizotinib is administered once or twice daily. In some embodiments, the subject is human.

[0049] In the last embodiment, the disclosure provides a kit comprising a compound of formula I or a pharmaceutically acceptable salt thereof and a MET inhibitor. In some embodiments, the compound of formula I and the MET inhibitor are contained in separate packages. In some embodiments, the kit further includes instructions for administering the contents of the kit to a target for the treatment of cancer. In some embodiments, the MET inhibitor is one or more of crizotinib, tepotinib, savolitinib, cabozantinib, and tivantinib.

[0050] In some embodiments, the compound of formula I or a pharmaceutically acceptable salt thereof is formulated as a pharmaceutical composition.

[0051] In some embodiments, the compound of formula I or a pharmaceutically acceptable salt thereof is formulated as an oral composition.

[0052] In some embodiments, the compound of formula I or a pharmaceutically acceptable salt thereof is administered once or twice daily.

[0053] In some embodiments, the compound of formula I or a pharmaceutically acceptable salt thereof is administered in a continuous 28-day cycle.

[0054] In some embodiments, the compound of formula I or a pharmaceutically acceptable salt thereof is administered once daily in an amount of about 10 mg to about 140 mg.

[0055] In some embodiments, the compound or a pharmaceutically acceptable salt thereof is administered once daily in a three-week cycle consisting of two weeks of administration of the compound followed by one week of non-administration of the compound.

[0056] In some embodiments, the compound of formula I or a pharmaceutically acceptable salt thereof is administered once daily in a four-week cycle consisting of three weeks of administration of the compound followed by one week of non-administration of the compound.

[0057] In some embodiments, the compound of formula I or a pharmaceutically acceptable salt thereof is administered over a period of 6 weeks.

[0058] In some embodiments, the compound of formula I or a pharmaceutically acceptable salt thereof is administered over a period of 8 weeks.

[0059] In some embodiments, the compound of formula I or a pharmaceutically acceptable salt thereof is administered three times a week.

[0060] In some embodiments, the compound of formula I or a pharmaceutically acceptable salt thereof is administered on days 1, 3, and 5 of the week.

[0061] In some embodiments, the compound of formula I or a pharmaceutically acceptable salt thereof is administered four times a week.

[0062] In some embodiments, the compound of formula I or a pharmaceutically acceptable salt thereof is administered in a three-week cycle consisting of two weeks of administration of the compound followed by one week of non-administration of the compound.

[0063] In some embodiments, the compound of formula I or a pharmaceutically acceptable salt thereof is administered in a four-week cycle consisting of three weeks of administration of the compound followed by one week of non-administration of the compound.

[0064] In some embodiments, the compound of formula I or a pharmaceutically acceptable salt thereof is administered twice daily, two days a week.

[0065] In some embodiments, the compound of formula I or a pharmaceutically acceptable salt thereof is administered over a period of 8 weeks.

[0066] In some embodiments, the compound of formula I or a pharmaceutically acceptable salt thereof is administered on the first and second days of each week.

[0067] In some embodiments, the cancer is selected from lung cancer, gastric cancer, liver cancer, colon cancer, kidney cancer, breast cancer, pancreatic cancer, pancreatic ductal adenocarcinoma (PDAC), juvenile myelomonocytic leukemia, neuroblastoma, melanoma, and acute myeloid leukemia. [Brief explanation of the drawing]

[0068] Novel features of the present invention are described in detail in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by referring to the following detailed description and appended drawings which describe exemplary embodiments in which the principles of the present invention are utilized.

[0069] [Figure 1A] The data shown demonstrates that the combination of compound I and the FGFR inhibitor erdafitinib exhibits a synergistic effect in vitro. This data indicates a significant degree of synergy in the combination of compound I and erdafitinib. [Figure 1B] This shows synergistic effect data in the Hep3B cancer cell line using the compound of formula I in combination with erdafitinib. This data indicates a significant synergistic effect with the combination of the compound of formula I and erdafitinib. [Figure 2] The graphs show tumor volume over the treatment period in the hepatocellular carcinoma CDX model KATO III, with treatment using compound I alone, erdafitinib alone, and a combination of compound I and erdafitinib. [Figure 3] The graphs show the tumor volume over the treatment period for FGFR2-amplifying gastric cancer CDX SNU-16, treated with compound I alone, erdafitinib alone, and a combination of compound I and erdafitinib. [Figure 4] The graphs show tumor volume over treatment periods in the FGF19-FGFR4-dependent hepatocellular carcinoma CDX model Huh-7, with treatment using compound I alone, erdafitinib alone, and a combination of compound I and erdafitinib. [Figure 5]The data shows that the combination of the compound in formula I and encorafenib exerts a synergistic effect across multiple BRAF V600E mutant cells. [Figure 6] This shows synergistic effect data in the RKO BRAFV600E CRC cell line using the compound of formula I in combination with the BRAF inhibitor encorafenib. This data indicates a significant synergistic effect with the combination of the compound of formula I and encorafenib. [Figure 7] This shows synergistic effect data in the WiDr BRAFV600E CRC cell line using the compound of formula I in combination with the BRAF inhibitor encorafenib. This data indicates a significant synergistic effect with the combination of the compound of formula I and encorafenib. [Figure 8] This shows synergistic effect data in the HT29 BRAFV600E CRC cell line using the compound of formula I in combination with the BRAF inhibitor encorafenib. This data indicates a significant synergistic effect with the combination of the compound of formula I and encorafenib. [Figure 9A] This gel shows synergistic inhibition of ERK1 / 2 phosphorylation in the RKO colorectal cancer cell line. Figure 9A shows robust reduction of pERK1 / 2 using a combination of the compound of formula I and encorafenib. [Figure 9B] This gel shows potent inhibition of ERK1 / 2 phosphorylation in the WiDr colorectal cancer cell line. Figure 9B shows robust reduction of pERK1 / 2 using the combination of the compound of formula I with encorafenib. [Figure 9C] Figure 9C shows plots of the antiproliferative activity of the compound of formula I alone or in combination with encorafenib in the RKO colorectal cancer cell line. The combination of the compound of formula I and encorafenib suggests that the inhibitory activity of the compound of formula I was increased. [Figure 9D]Figure 9D shows plots of the antiproliferative activity of the compound of formula I or the compound of formula I in combination with encorafenib in the WiDr colorectal cancer cell line. The combination of the compound of formula I and encorafenib suggests that the inhibitory activity of the compound of formula I was increased. [Figure 10A] This gel compares the synergistic inhibition of ERK1 / 2 phosphorylation in RKO colorectal cancer cell lines with the following combinations: compound of formula I + encorafenib, TNO155 + encorafenib, and RMC-4550 + encorafenib. It indicates that inhibition of ERK1 / 2 phosphorylation is most effective with the combination of the SHP2 inhibitor compound of formula I and encorafenib. [Figure 10B] The bar graph shows pERK as a percentage of the control, with 1. control, 2. (compound of formula I), 3. encorafenib, and 4. (compound of formula I) + encorafenib, indicating that inhibition of ERK1 / 2 phosphorylation is most effective with the combination of the SHP2 inhibitor compound of formula I and encorafenib. [Figure 10C] The bar graph shows pERK as a percentage of the control, with 1. control, 2. TNO155, 3. encorafenib, and 4. TNO155 + encorafenib, indicating that inhibition of ERK1 / 2 phosphorylation is most effective with the combination of a formula I SHP2 inhibitor compound and encorafenib. [Figure 10D] The bar graph shows pERK as a percentage of the control, with 1. control, 2. RMC-4550, 3. encorafenib, and 4. RMC-4550 + encorafenib, indicating that inhibition of ERK1 / 2 phosphorylation is most effective with the combination of a formula I SHP2 inhibitor compound and encorafenib. [Figure 11] The graphs show tumor volume over the treatment period in the BRAFV600E mutant CRC PDX model CR0029, with treatment using compound I alone, encorafenib alone, and a combination of compound I and encorafenib. [Figure 12]The graphs show tumor volume over treatment periods in the BRAFV600E mutant CRC PDX model CR004, with treatment using compound I alone, encorafenib alone, and a combination of compound I and encorafenib. [Figure 13] The graphs show tumor volume over the treatment period in the BRAFV600E mutant CRC CDX model WiDr, with treatment using compound I alone, encorafenib alone, and a combination of compound I and encorafenib. [Figure 14] The graphs show tumor volume over the treatment period in the BRAFV600E mutant CRC CDX model HT-29, with treatment using compound I alone, encorafenib alone, and a combination of compound I and encorafenib. [Figure 15] The graphs show tumor volume over the treatment period in the BRAFV600E mutant thyroid cancer CDX model BHT-101, with treatment using compound I alone, encorafenib alone, and a combination of compound I and encorafenib. [Figure 16] The graphs show tumor volume over the treatment period in the BRAFV600E mutant CRC CDX model RKO, with treatment using compound I alone, encorafenib alone, and a combination of compound I and encorafenib. [Figure 17A] This shows synergistic effect data in the NCI-H508 cancer cell line using a combination of the compound of formula I and trametinib. [Figure 17B] This shows synergistic effect data in the NCI-H508 cancer cell line using a combination of the compound of formula I and binimethinib. [Figure 17C] The graphs and tables show the synergistic effects of the combination of compound I and trametinib in the NCI-H1666 cancer cell line. [Figure 17D] This shows synergistic effect data in the NCI-H1666 cancer cell line using a combination of the compound of formula I and binimetinib. [Figure 18A] This shows synergistic effect data in the MeWo cancer cell line using a combination of the compound of formula I and trametinib. [Figure 18B] This shows synergistic effect data in the MeWo cancer cell line using a combination of the compound of formula I and binimethinib. [Figure 18C] The synergistic effect data of the compound of formula I combined with trametinib in the NCI-H1838 cancer cell line is shown. [Figure 18D] The synergistic effect data of the compound of formula I combined with binimetinib in the NCI-H1838 cancer cell line is shown. [Figure 19A] The table shows the percentage activity versus inhibitor concentration (log M) in NCI-H508 cells treated with the compound of formula I alone and in combination with binimetinib. The table shows the IC50 data for NCI-H508 cells treated with the compound of formula I alone and in combination with binimetinib. [Figure 19B] The table shows the percentage activity versus inhibitor concentration (logM) in MeWo cells treated with the compound of formula I alone and in combination with binimetinib. The table shows the IC50 data in MeWo cells treated with the compound of formula I alone and in combination with binimetinib. [Figure 20A] This shows a Western blot gel demonstrating synergistic inhibition of ERK1 / 2 phosphorylation in the NCI-H508 cancer cell line. [Figure 20B] Figure 20A shows a quantitative bar graph of the Western blot. [Figure 20C] This shows a Western blot gel demonstrating synergistic inhibition of ERK1 / 2 phosphorylation in the MeWo(NF1 LoF) cancer cell line. [Figure 20D] Figure 20C shows the quantitative analysis of the Western blot bar graph. [Figure 21A] This shows synergistic effect data in the NCI-H2009 (KRAS G12A) cancer cell line using a combination of the compound of formula I and trametinib. [Figure 21B] This shows synergistic effect data in the LS513 (KRAS G12D) cancer cell line using a combination of the compound of formula I and trametinib. [Figure 21C]This shows synergistic effect data in the A549 (KRAS G12S) cancer cell line using a combination of the compound of formula I and trametinib. [Figure 21D] This shows synergistic effect data in the NCI-H727 (KRAS G12V) cancer cell line using a combination of the compound of formula I and trametinib. [Figure 22A] This shows synergistic effect data in the NCI-H2009 (KRAS G12A) cancer cell line using a combination of the compound of formula I and binimetinib. [Figure 22B] This shows synergistic effect data in the LS513 (KRAS G12D) cancer cell line using a combination of the compound of formula I and binimetinib. [Figure 22C] This shows synergistic effect data in the A549 (KRAS G12S) cancer cell line using a combination of the compound of formula I and binimetinib. [Figure 22D] This shows synergistic effect data in the NCI-H727 (KRAS G12V) cancer cell line using a combination of the compound of formula I and binimetinib. [Figure 23A] The graph shows the percentage of activity versus the inhibitor concentration (logM) in LS513 (KRAS G12D) cells treated with the compound of formula I alone and in combination with trametinib. [Figure 23B] The table shows plots of activity percentage versus inhibitor concentration (logM) in NCI-H2009 (KRAS G12D) cells treated with the compound of formula I alone and in combination with trametinib. The table shows data for NCI-H508 cells treated with the compound of formula I alone and in combination with trametinib. [Figure 23C] The bar graph shows the percentage of CTG activity, indicating that formula I alone or trametinib alone have the least effect on cell viability. In summary, these data demonstrate that the combination of a formula I compound with a MEK inhibitor provides synergistic inhibition of cancer cell viability in BRAF class III, NF1 LoF, and KRAS G12X mutant cancers. [Figure 24]The graph shows tumor volume over treatment periods in the NF1 LoF mutant melanoma CDX model MeWo, with treatment using compound I alone, trametinib alone, and a combination of compound I and trametinib. [Figure 25] The graphs show tumor volume over time in the NF1 LoF mutant melanoma CDX model MeWo, with treatment of compound I alone, binimetinib alone, and a combination of compound I and binimetinib. [Figure 26] The graphs show tumor volume over treatment periods in the BRAF class III mutant CRC CDX model NCI-H508, treated with formula I compound alone, trametinib alone, and a combination of formula I compound and trametinib. [Figure 27] The graphs show tumor volume over treatment periods in the NF1 LoF mutant NSCLC CDX model NCI-H1838, with treatment using compound I alone, trametinib alone, and a combination of compound I and trametinib. [Figure 28A] This shows synergistic effect data in the Hs746T cancer cell line using a combination of the compound of formula I and crizotinib. [Figure 28B] This shows synergistic effect data in the MKN-45 cancer cell line using a combination of the compound of formula I and crizotinib. [Figure 28C] This shows synergistic effect data in the EBC-1 cancer cell line using a combination of the compound of formula I and crizotinib. [Figure 29] The graphs show tumor volume over treatment periods in the c-MET amplified gastric cancer CDX model SNU-5, with treatment using compound I alone, crizotinib alone, and a combination of compound I and crizotinib. [Figure 30] The graphs show tumor volume over treatment periods in the c-MET amplified NSCLC CDX model NCI-H1993, with treatment using compound I alone, crizotinib alone, and a combination of compound I and crizotinib. [Modes for carrying out the invention]

[0070] I. General This disclosure relates to a method for treating a subject having cancer, wherein the subject is given a therapeutically effective amount of a compound of formula I or a pharmaceutically acceptable salt thereof.

[0071] [ka] The present invention provides a method comprising the step of administering a compound of formula I in combination with an FGFR inhibitor. The following examples demonstrate unexpected synergistic effects for the combination. The combination therapies disclosed herein using the compound of formula I or a pharmaceutically acceptable salt thereof may show superior results compared to alternative combinations of SHP2 inhibitors used in combination with FGFR inhibitors. Furthermore, the combination of the SHP2 inhibitor of formula I and the FGFR inhibitor provides a method that allows for the use of lower doses than those of either drug used alone in monotherapy, thereby helping to reduce potential side effects. In particular, the combination therapy may be effective in cancer cells expressing mutations including, but not limited to, FGFR4 mutations and amplified FGFR expression.

[0072] In a first aspect, the disclosure relates to a method for treating a subject having cancer, wherein the subject is given a therapeutically effective amount of a compound of formula I or a pharmaceutically acceptable salt thereof.

[0073] [ka] The present invention provides a method comprising the step of administering a substance. In some embodiments, FGFR in the subject is constitutively active. In some embodiments, the cancer is lung cancer. In some embodiments, the cancer is hepatocellular carcinoma. In some embodiments, the cancer is cholangiocarcinoma. In some embodiments, the cancer is pancreatic ductal adenocarcinoma (PDAC). In some embodiments, the inhibitor is selected from the group consisting of erdafitinib, AZD4547, Ly2874455, CH5183284, NVP-BGJ398, INCB054828, logaratinib, PRN1371, TAS-120, BLU-554, H3B-6527, and FGF401. In some embodiments, the FGFR inhibitor is erdafitinib. In some embodiments, the FGFR inhibitor is pemigatinib, infiglatinib, dovitinib, ponatinib, nintedanib, and fisogatinib. In some embodiments, the method includes the step of administering a third MAPK pathway inhibitor. In some embodiments, the administration is orally. In some embodiments, the dose of the compound of formula I ranges from 20 mg to 400 mg per day. In some embodiments, the dose of the FGFR inhibitor ranges from 1 mg to 500 mg per day.

[0074] In a second aspect, the disclosure relates to a therapeutically effective amount of a compound of formula I or a pharmaceutically acceptable salt thereof.

[0075] [ka] The present invention provides a method for treating liver cancer in a subject, comprising the step of orally administering to the subject a compound of formula I in combination with erdafitinib. In some embodiments, the compound of formula I is administered once or twice daily. In some embodiments, erdafitinib is administered once or twice daily. In some embodiments, the subject is human.

[0076] In a third embodiment, the disclosure provides a kit comprising a compound of formula I or a pharmaceutically acceptable salt thereof and an FGFR inhibitor. In some embodiments, the compound of formula I and the FGFR inhibitor are contained in separate packages. In some embodiments, the kit further includes instructions for administering the contents of the kit to a target for the treatment of cancer. In some embodiments, the FGFR inhibitor is one or more of erdafitinib, AZD4547, Ly2874455, CH5183284, NVP-BGJ398, INCB054828, rogalatinib, PRN1371, TAS-120, BLU-554, H3B-6527, FGF401, pemigatinib, infiglatinib, dovitinib, ponatinib, nintedanib, and fisogatinib.

[0077] In a fourth aspect, the disclosure relates to a method for treating a subject having cancer, wherein the subject is given a therapeutically effective amount of a compound of formula I or a pharmaceutically acceptable salt thereof.

[0078] [ka] The present invention provides a method comprising the step of administering a compound in combination with an inhibitor of the B-Raf protein having a class 1 mutation. In some embodiments, the class 1 mutation is V600E. In some embodiments, the cancer is colorectal cancer. In some embodiments, the cancer is melanoma. In some embodiments, the cancer is thyroid cancer. In some embodiments, the cancer is pancreatic ductal adenocarcinoma (PDAC). In some embodiments, the inhibitor is selected from the group consisting of encorafenib, vemurafenib, dabrafenib, sorafenib, and regorafenib. In some embodiments, the inhibitor is encorafenib. In some embodiments, the inhibitor is vemurafenib. In some embodiments, the inhibitor is dabrafenib. In some embodiments, the inhibitor is sorafenib. In some embodiments, the inhibitor is regorafenib. In some embodiments, the method comprises the step of administering a third MAPK pathway inhibitor. In some embodiments, the administration is orally. In some embodiments, the dose of the compound of formula I ranges from 20 mg to 400 mg daily. In some embodiments, the dose of the B-Raf inhibitor ranges from 1 mg to 500 mg.

[0079] This disclosure relates to a method for treating a subject having cancer, wherein the subject is given a therapeutically effective amount of a compound of formula I or a pharmaceutically acceptable salt thereof.

[0080] [ka] The present invention provides a method comprising the step of administering in combination with a class 1 variant B-Ra inhibitor. The following examples demonstrate the unexpected significant synergistic effect of the combination. The combination therapies disclosed herein using the compound of formula I or a pharmaceutically acceptable salt thereof can show superior results compared to alternative combinations of SHP2 inhibitors used in combination with class 1 variant B-Raf inhibitors. Furthermore, the combination of the SHP2 inhibitor of formula I and the class 1 variant B-Raf inhibitor provides a method that allows for the use of lower doses than those of either drug used alone in monotherapy, thereby helping to reduce potential side effects. In particular, the combination therapy may be effective against cancer cells expressing the BRAF V600E mutation.

[0081] In a fourth aspect, the disclosure relates to a method for treating a subject having cancer, wherein the subject is given a therapeutically effective amount of a compound of formula I or a pharmaceutically acceptable salt thereof.

[0082] [ka] The present invention provides a method comprising the step of administering a compound in combination with an inhibitor of the B-Raf protein having a class 1 mutation. In some embodiments, the class 1 mutation is V600E. In some embodiments, the cancer is colorectal cancer. In some embodiments, the cancer is melanoma. In some embodiments, the cancer is thyroid cancer. In some embodiments, the cancer is pancreatic ductal adenocarcinoma (PDAC). In some embodiments, the inhibitor is selected from the group consisting of encorafenib, vemurafenib, dabrafenib, sorafenib, and regorafenib. In some embodiments, the inhibitor is encorafenib. In some embodiments, the inhibitor is vemurafenib. In some embodiments, the inhibitor is dabrafenib. In some embodiments, the inhibitor is sorafenib. In some embodiments, the inhibitor is regorafenib. In some embodiments, the method comprises the step of administering a third MAPK pathway inhibitor. In some embodiments, the administration is orally. In some embodiments, the dose of the compound of formula I ranges from 20 mg to 400 mg daily. In some embodiments, the dose of the B-Raf inhibitor ranges from 1 mg to 500 mg.

[0083] In another embodiment, the Disclosure relates to a therapeutically effective amount of a compound of formula I or a pharmaceutically acceptable salt thereof.

[0084] [ka] The present invention provides a method for treating colorectal cancer in a subject, comprising the step of orally administering to the subject a compound of formula I in combination with the B-Raf inhibitor erdafitinib. In some embodiments, the compound of formula I is administered once or twice daily. In some embodiments, erdafitinib is administered once or twice daily. In some embodiments, the subject is human.

[0085] In another aspect, the disclosure relates to a method for treating a subject having cancer, wherein the subject is given a therapeutically effective amount of a compound of formula I or a pharmaceutically acceptable salt thereof.

[0086] [ka] The present invention provides a method that includes the step of administering in combination with encorafenib.

[0087] In another aspect, the disclosure relates to a method for treating a subject having cancer, wherein the subject is given a therapeutically effective amount of a compound of formula I or a pharmaceutically acceptable salt thereof.

[0088] [ka] The present invention provides a method that includes the step of administering in combination with vemurafenib.

[0089] In another aspect, the disclosure relates to a method for treating a subject having cancer, wherein the subject is given a therapeutically effective amount of a compound of formula I or a pharmaceutically acceptable salt thereof.

[0090] [ka] The present invention provides a method that includes the step of administering in combination with dabrafenib.

[0091] In another aspect, the disclosure relates to a method for treating a subject having cancer, wherein the subject is given a therapeutically effective amount of a compound of formula I or a pharmaceutically acceptable salt thereof.

[0092] [ka] The present invention provides a method that includes the step of administering in combination with sorafenib.

[0093] In another aspect, the disclosure relates to a method for treating a subject having cancer, wherein the subject is given a therapeutically effective amount of a compound of formula I or a pharmaceutically acceptable salt thereof.

[0094] [ka] The present invention provides a method that includes the step of administering in combination with regorafenib.

[0095] In various embodiments of the methods described herein, the cancer is colorectal cancer. In some embodiments, the cancer is thyroid cancer. In some embodiments, the cancer is melanoma. In some embodiments, the cancer is adenocarcinoma of the pancreatic ducts (PDAC). In some embodiments, the dose of the B-Raf inhibitor is less than the dose required for monotherapy with the B-Raf inhibitor. In some embodiments, the dose of the compound of formula I is less than the dose required for monotherapy with the compound of formula I.

[0096] In another aspect, the present disclosure provides a method for inhibiting ERK1 / 2 phosphorylation in a cell population, wherein the cell population is subjected to a compound of formula I combined with regorafenib or a pharmaceutically acceptable salt thereof.

[0097] [ka] The present invention provides a method comprising the step of contacting with a substance. In some embodiments, the concentration of the compound of formula I is in the range of 1 nM to 500 nM. In some embodiments, the concentration of encorafenib is in the range of 10 nM to 20 nM.

[0098] In another embodiment, the disclosure provides a kit comprising a compound of formula I or a pharmaceutically acceptable salt thereof, and a B-Raf inhibitor. In some embodiments, the compound of formula I and the B-Raf inhibitor are contained in separate packages. In some embodiments, the kit further includes instructions for administering the contents of the kit to a target for the treatment of cancer. In some embodiments, the B-Raf inhibitor is one or more of encorafenib, vemurafenib, dabrafenib, sorafenib, and regorafenib.

[0099] This embodiment is a method for treating a subject with cancer, comprising administering a therapeutically effective amount of the compound of formula I or a pharmaceutically acceptable salt thereof to the subject.

[0100] [ka] The present invention provides a method comprising the step of administering in combination with a MEK inhibitor. The following example demonstrates the unexpected synergistic effect of the combination. The combination therapies disclosed herein using the compound of formula I or a pharmaceutically acceptable salt thereof may show superior results compared to alternative combinations of SHP2 inhibitors used in combination with MEK inhibitors. Furthermore, the combination of the SHP2 inhibitor of formula I and the MEK inhibitor provides a method that allows for the use of lower doses than those of either drug used alone in monotherapy, which may help reduce potential side effects. In particular, the combination therapy may be effective in cancer cells expressing mutations including, but not limited to, FGFR4 mutations and KRAS G12X mutations.

[0101] In another aspect, the disclosure relates to a method for treating a subject having cancer, wherein the subject is given a therapeutically effective amount of a compound of formula I or a pharmaceutically acceptable salt thereof.

[0102] [ka] The present invention provides a method comprising the step of administering in combination with a MEK inhibitor. In some embodiments, the MEK inhibitor selectively inhibits MEK1 or MEK2, or both MEK1 and MEK2. In some embodiments, the cancer is metastatic. In some embodiments, the cancer is colorectal cancer. In some embodiments, the cancer is melanoma. In some embodiments, the cancer is lung cancer. In some embodiments, the cancer is pancreatic cancer. In some embodiments, the cancer is breast cancer. In some embodiments, the cancer is adenocarcinoma of the pancreatic ducts (PDAC). In some embodiments, the MEK inhibitor is selected from the group consisting of trametinib, cobimetinib, binimetinib, PD-0325901, selumetinib, and CI-1040. In some embodiments, the MEK inhibitor is trametinib. In some embodiments, the MEK inhibitor is cobimetinib. In some embodiments, the MEK inhibitor is binimetinib. In some embodiments, the MEK inhibitor is PD-325901. In some embodiments, the MEK inhibitor is CI-1040. In some embodiments, the method includes the step of administering an additional MAPK pathway inhibitor. In some embodiments, the administration is orally. In some embodiments, the dose of the compound of formula I ranges from 20 mg to 400 mg per day. In some embodiments, the dose of the MEK inhibitor ranges from 1 mg to 500 mg per day.

[0103] In another embodiment, the Disclosure relates to a therapeutically effective amount of a compound of formula I or a pharmaceutically acceptable salt thereof.

[0104] [ka] The present invention provides a method for treating cancer in a subject, comprising the step of orally administering to the subject a compound of formula I in combination with the MEK inhibitor binimetinib or trametinib. In some embodiments, the compound of formula I is administered once or twice daily. In some embodiments, binimetinib or trametinib is administered once or twice daily. In some embodiments, the subject is human.

[0105] In another aspect, the disclosure relates to a method for treating a subject having cancer, wherein the subject is given a therapeutically effective amount of a compound of formula I or a pharmaceutically acceptable salt thereof.

[0106] [ka] The present invention provides a method that includes the step of administering in combination with binimetinib.

[0107] In another aspect, the disclosure relates to a method for treating a subject having cancer, wherein the subject is given a therapeutically effective amount of a compound of formula I or a pharmaceutically acceptable salt thereof.

[0108] [ka] The present invention provides a method that includes the step of administering in combination with trametinib.

[0109] In various embodiments of the methods described herein, the cancer is colorectal cancer. In some embodiments, the cancer is lung cancer. In some embodiments, the cancer is melanoma. In some embodiments, the cancer is pancreatic ductal adenocarcinoma (PDAC). In some embodiments, the dose of the MEK inhibitor is less than the dose required for monotherapy with the MEK inhibitor. In some embodiments, the dose of the compound of formula I is less than the dose required for monotherapy with the compound of formula I.

[0110] In another aspect, the present disclosure provides a method for inhibiting ERK1 / 2 phosphorylation in a cell population, comprising a compound of formula I combined with binimetinib or trametinib, or a pharmaceutically acceptable salt thereof.

[0111] [ka] The present invention provides a method comprising the step of contacting with a substance. In some embodiments, the concentration of the compound of formula I is in the range of 1 nM to 1,000 nM. In some embodiments, the concentration of the MEK inhibitor is in the range of 10 nM to 500 nM.

[0112] In another embodiment, the disclosure provides a kit comprising a compound of formula I or a pharmaceutically acceptable salt thereof and a MEK inhibitor. In some embodiments, the compound of formula I and the MEK inhibitor are contained in separate packages. In some embodiments, the kit further includes instructions for administering the contents of the kit to a target for the treatment of cancer. In some embodiments, the MEK inhibitor is one or more of trametinib or binimetinib.

[0113] This disclosure relates to a method for treating a subject having cancer, wherein the subject is given a therapeutically effective amount of a compound of formula I or a pharmaceutically acceptable salt thereof.

[0114] [ka] The present invention provides a method comprising the step of administering a compound of formula I in combination with a MET inhibitor. The following examples demonstrate unexpected synergistic effects for the combination. The combination therapies disclosed herein using the compound of formula I or a pharmaceutically acceptable salt thereof can show superior results compared to alternative combinations of SHP2 inhibitors used in combination with MET inhibitors. Furthermore, the combination of the SHP2 inhibitor of formula I and the MET inhibitor provides a method that allows for the use of lower doses than those used in monotherapy of either drug alone, which may help reduce potential side effects. In particular, the combination therapy may be effective against cancer cells expressing abnormal mutations in MET.

[0115] In another aspect, the disclosure relates to a method for treating a subject having cancer, wherein the subject is given a therapeutically effective amount of a compound of formula I or a pharmaceutically acceptable salt thereof.

[0116] [ka] The present invention provides a method comprising the step of administering a compound in combination with a MET inhibitor. In some embodiments, the MET inhibitor is also an ALK inhibitor, a ROS1 inhibitor, or both. In some embodiments, the cancer is non-small cell lung cancer. In some embodiments, the cancer is gastric cancer. In some embodiments, the cancer is gastric adenocarcinoma. In some embodiments, the cancer is adenocarcinoma of the pancreatic ducts (PDAC). In some embodiments, the MET inhibitor is selected from the group consisting of crizotinib, tepotinib, savolitinib, cabozantinib, and tivantinib. In some embodiments, the MET inhibitor is crizotinib. In some embodiments, the MET inhibitor is tepotinib. In some embodiments, the inhibitor is savolitinib. In some embodiments, the inhibitor is cabozantinib. In some embodiments, the inhibitor is tivantinib. In some embodiments, the method comprises the step of administering a third MAPK pathway inhibitor. In some embodiments, the administration is orally. In some embodiments, the dose of the compound of formula I ranges from 10 mg to 500 mg daily. In some embodiments, the dosage of the inhibitor ranges from 20 mg to 400 mg per day.

[0117] In another embodiment, the Disclosure relates to a therapeutically effective amount of a compound of formula I or a pharmaceutically acceptable salt thereof.

[0118] [ka] The present invention provides a method for treating gastric cancer in a subject, comprising the step of orally administering to the subject a compound of formula I in combination with crizotinib. In some embodiments, the compound of formula I is administered once or twice daily. In some embodiments, crizotinib is administered once or twice daily. In some embodiments, the subject is human.

[0119] In the last embodiment, the disclosure provides a kit comprising a compound of formula I or a pharmaceutically acceptable salt thereof and a MET inhibitor. In some embodiments, the compound of formula I and the MET inhibitor are contained in separate packages. In some embodiments, the kit further includes instructions for administering the contents of the kit to a target for the treatment of cancer. In some embodiments, the MET inhibitor is one or more of crizotinib, tepotinib, savolitinib, cabozantinib, and tivantinib.

[0120] Therefore, such procedures are suitable for the use of companion diagnostics to support appropriate patient population selection. These, and other, advantages will be recognized by those skilled in the art.

[0121] II. Definition Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art, as the embodiments described herein cover. Furthermore, any method or material similar to or equivalent to the methods or materials described herein may be used in the implementation of the embodiments herein. For the purposes of the embodiments disclosed herein, the following terms are defined:

[0122] As used herein, “a,” “an,” or “the” includes not only embodiments having one component but also embodiments having multiple components. For example, the singular forms “a,” “an,” and “the” include multiple referents unless the context explicitly indicates otherwise. Thus, for example, a reference to “cells” includes multiple such cells, and a reference to “drugs” includes one or more drugs known to those skilled in the art.

[0123] "Pharmacologically acceptable excipients" refer to substances that assist in the administration and absorption of an activator to a subject. Useful pharmaceutical excipients in this embodiment include, but are not limited to, binders, fillers, disintegrants, lubricants, surfactants, coatings, sweeteners, flavorings, and colorants. Those skilled in the art will recognize that other pharmaceutical excipients may be useful in this embodiment.

[0124] "To treat," "to treat," and "treatment" refer to any sign of success in treating or improving an injury, injury, injury, or condition, including any objective or subjective parameters such as remission, sedation, reduction of symptoms, or making the injury, condition, or state more tolerable to the patient, slowing the rate of degeneration or deterioration, reducing the debilitation at the end of degeneration, or improving the patient's physical or mental health. Treatment or improvement of symptoms may be based on objective or subjective parameters, including the results of a physical examination, neuropsychiatric examination, and / or psychiatric evaluation.

[0125] "Administration" refers to oral administration, suppository administration, topical contact, parenteral administration, intravenous, intraperitoneal, intramuscular, intrafocal, intranasal or subcutaneous administration, intrathecal administration, or implantation of a sustained-release device to the subject, such as a mini osmotic pump. In the context of the combination therapies disclosed herein, administration may occur at separate times, simultaneously, or substantially simultaneously.

[0126] The "therapeutic dose" refers to the amount administered that produces a therapeutic effect. The exact dose depends on the purpose of the treatment and can be determined by those skilled in the art using known techniques (see, for example, Lieberman, Pharmaceutical Dosage Forms (vols. 1 3, 1992), Lloyd, The Art, Science and Technology of Pharmaceutical Compounding (1999), Pickar, Dosage Calculations (1999), and Remington: The Science and Practice of Pharmacy, 20th Edition, 2003, Gennaro, Ed., Lippincott, Williams & Wilkins). In sensitized cells, the therapeutic dose may often be lower than the conventional therapeutic dose for unsensitized cells.

[0127] "Inhibition," "to inhibit," and "inhibitor" refer to compounds or methods that partially or completely block or interfere with a particular action or function.

[0128] "Cancer" refers to all types of cancer, neoplasms, or malignant tumors found in mammals (e.g., humans), including but not limited to leukemia, lymphoma, carcinoma, and sarcoma. Exemplary cancers that can be treated with the compounds or methods provided herein include brain cancer, glioma, glioblastoma, prostate cancer, colorectal cancer, pancreatic cancer, medulloblastoma, melanoma, cervical cancer, gastric cancer, ovarian cancer, lung cancer, head cancer, Hodgkin's disease, and non-Hodgkin lymphoma. Exemplary cancers that can be treated with the compounds or methods provided herein include cancers of the thyroid, endocrine system, brain, breast, cervix, colon, head and neck, liver, kidney, lung, ovary, pancreas, rectum, stomach, and uterus. Further examples include thyroid cancer, bile duct cancer, pancreatic adenocarcinoma, cutaneous melanoma, colon adenocarcinoma, rectal adenocarcinoma, gastric adenocarcinoma, esophageal cancer, head and neck squamous cell carcinoma, invasive breast cancer, lung adenocarcinoma, lung squamous cell carcinoma, non-small cell lung cancer, mesothelioma, multiple myeloma, neuroblastoma, glioma, glioblastoma multiforme, ovarian cancer, rhabdomyosarcoma, primary thrombocythemia, primary macroglobulinemia, primary brain tumor, malignant pancreatic insulanoma, malignant carcinoid, bladder cancer, pre-malignant cutaneous lesions, testicular cancer, thyroid cancer, neuroblastoma, esophageal cancer, urogenital tract cancer, malignant hypercalcemia, endometrial cancer, adrenocortical carcinoma, neoplasms of the endocrine or exocrine pancreas, thyroid carcinoma, melanoma, colorectal cancer, papillary thyroid carcinoma, hepatocellular carcinoma, pancreatic ductal adenocarcinoma (PDAC), or prostate cancer.

[0129] "FGFR inhibitors" refer to any inhibitor of wild-type FGFR or FGFR variants. FGFR variants include, but are not limited to, single nucleotide polymorphisms, exon insertions and deletions, and polysomy. Specific examples of mutations and inhibitors include, but are not limited to, FGFR1 gene copy gain, FGFR1 gene amplification, FGFR2 gene copy gain, FGFR2 gene amplification, FGFR3 gene copy gain, FGFR3 gene amplification, FGFR4 gene copy gain, FGFR4 gene amplification, FGFR1 T141R, FGFR1 R445W, FGFR1 N546K, FGFR1 K656E, FGFR1 G818R, FGFR2 S252W, FGFR2 P253R, FGFR2 A315T, FGFR2 D336N, FGFR2 Y375C, FGFR2 C382R, FGFR2 V395D, FGFR2 D471N, FGFR2 I547V, FGFR2 N549K, FGFR2 N549Y, FGFR2 K659E, FGFR3 SI 3 IL, FGFR3 R248C, FGFR3 S249C, FGFR3 G370C, FGFR3 S371C, FGFR3 Y373C, FGFR3 G380R, FGFR3 R399C, FGFR3 E627K, FGFR3 K650E, FGFR3 K650M, FGFR3 V677I, FGFR3 D785Y, FGFR4 R183 S, FGFR4 R394Q, FGFR4 D425N, FGFR4 V510L, FGFR4 R61 OH, and FGFR fusions (e.g., FGFR3-TACC3, FGFR2-TACC3, FGFR2-NPM1, FGFR2-TACC2, FGFR2- Examples include BICC1, FGFR2-C10orf68, FGFR3-JAKMIPl, FGFR2-KIAA1598, FGFR2-NCALD, FGFR2-NOL4, FGFR1-NTM, FGFR2-PPAPDC1 A, FGFR3-TNIP2, and FGFR3-WHSCI). In some embodiments, but not limited to, one or more of the above-listed variants may be specifically excluded from embodiments including any methods, kits, and compositions described herein.Inhibitors include, but are not limited to, erdafitinib, pemigatinib, infiglatinib, dovitinib, ponatinib, nintedanib, and fisogatinib. In some embodiments, one or more of the variants listed above may be specifically excluded from any embodiment, including but not limited to the methods, kits, and compositions described herein.

[0130] "Class 1 mutant B-Raf" or "B-Raf protein having a Class 1 mutation" generally refers to any mutation that deviates from the wild-type B-Raf protein at V600 (valine 600). In particular, such mutant B-Raf proteins include mutations containing the V600E mutation. Other Class 1 BRAF mutations include, but are not limited to, V600K, V600D, V600L, V600M, and V600R. In some embodiments, one or more of the mutations listed above may be specifically excluded from any embodiments, including but not limited to, the methods, kits, and compositions described herein.

[0131] The term "MEK inhibitor" generally refers to any inhibitor that selectively inhibits MEK1 or MEK2, or both MEK1 and MEK2. Examples of inhibitors include, but are not limited to, trametinib, cobimetinib, binimetinib, PD-0325901, selumetinib, and CI-1040.

[0132] "MET inhibitors" refer to any inhibitor of wild-type MET or MET variants. MET variants include, but are not limited to, single nucleotide polymorphisms, exon insertions and deletions, and polysomy. Specific examples of mutations and inhibitors include MET gene copy gain, MET gene amplification, MET E34K, MET H150Y, MET E168D, MET L269V, MET L299F, MET S323G, MET M362T, MET N375S, MET C385Y, MET R970C, MET R988C, MET P1009S, MET T1010I, MET S1058P, MET exon 14 skipping mutation, MET exon 14 splice variant, MET Al 108 S, MET VI 1101, MET Hl 112R, MET Hl 112L, MET Hl 1121, MET HJ1124D, MET G1137 V, MET Ml 149T, MET T11911, MET V1206L, MET L1213V, MET D1228V, MET Examples of inhibitors include, but are not limited to, Y1230C, MET Y1230H, MET Y1230D, MET Y1235D, MET V1238I, MET D1246N, MET Y1248C, MET Y1248D, MET Y1248H, MET K1262R, MET M1268T, MET M1268I, and MET V13121. In some embodiments, one or more of the variants listed in this paragraph and elsewhere in this specification may be specifically excluded from any embodiment, including but not limited to, the methods, kits, and compositions described herein. Examples of inhibitors include, but are not limited to, crizotinib, capatinib, tepotinib, savolitinib, tivantinib, cabozantinib, foretinib, amibantamab, onarutuzumab, emibetuzumab, and ficratuzumab. In some embodiments, one or more of the inhibitors listed in this paragraph and elsewhere in this specification may be specifically excluded from any embodiment, including but not limited to the methods, kits, and compositions described herein.

[0133] "Subject" refers to an organism suffering from or prone to a disease or condition that can be treated by administration of the pharmaceutical compositions provided herein. Non-limiting examples include humans, other mammals, cattle, rats, mice, dogs, monkeys, goats, sheep, cows, deer, horses, and other non-mammals. In some embodiments, the patient is human.

[0134] III. Method of Administration In some embodiments, the compound of formula I or a pharmaceutically acceptable salt thereof is formulated as a pharmaceutical composition. In some embodiments, the compound of formula I or a pharmaceutically acceptable salt thereof is formulated as an oral composition.

[0135] In some embodiments, the compound of formula I or a pharmaceutically acceptable salt thereof is administered once or twice daily. In some embodiments, the compound of formula I or a pharmaceutically acceptable salt thereof is administered once daily. In some embodiments, the compound of formula I or a pharmaceutically acceptable salt thereof is administered twice daily. In some embodiments, the compound of formula I or a pharmaceutically acceptable salt thereof is administered in a continuous 28-day cycle.

[0136] In some embodiments, the compound of formula I or a pharmaceutically acceptable salt thereof is administered once daily in an amount of about 10 mg to about 140 mg.

[0137] In some embodiments, the compound of formula I or a pharmaceutically acceptable salt thereof is administered once daily in a three-week cycle consisting of two weeks of administration of the compound followed by one week of non-administration of the compound.

[0138] In some embodiments, the compound of formula I or a pharmaceutically acceptable salt thereof is administered once daily in a four-week cycle consisting of three weeks of administration of the compound followed by one week of non-administration of the compound.

[0139] In some embodiments, the compound of formula I or a pharmaceutically acceptable salt thereof is administered over a period of 6 weeks. In some embodiments, the compound of formula I or a pharmaceutically acceptable salt thereof is administered over a period of 8 weeks.

[0140] In some embodiments, the compound of formula I, or a pharmaceutically acceptable salt thereof, is administered three times a week. In some embodiments, the compound of formula I, or a pharmaceutically acceptable salt thereof, is administered on the first, third, and fifth days of the week.

[0141] In some embodiments, the compound of formula I, or a pharmaceutically acceptable salt thereof, is administered four times a week.

[0142] In some embodiments, the compound of formula I or a pharmaceutically acceptable salt thereof is administered in a three-week cycle consisting of two weeks of administration of the compound followed by one week of non-administration of the compound.

[0143] In some embodiments, the compound of formula I or a pharmaceutically acceptable salt thereof is administered in a four-week cycle consisting of three weeks of administration of the compound followed by one week of non-administration of the compound.

[0144] In some embodiments, the compound of formula I or a pharmaceutically acceptable salt thereof is administered twice daily for two days a week. In some embodiments, the compound of formula I or a pharmaceutically acceptable salt thereof is administered over eight weeks. In some embodiments, the compound of formula I or a pharmaceutically acceptable salt thereof is administered on the first and second days of each week.

[0145] In some embodiments, the cancer is selected from lung cancer, gastric cancer, liver cancer, colon cancer, kidney cancer, breast cancer, pancreatic cancer, juvenile myelomonocytic leukemia, neuroblastoma, melanoma, and acute myeloid leukemia. In some embodiments, the cancer is pancreatic ductal adenocarcinoma (PDAC).

[0146] IV. Combination Methods In some embodiments, the method includes the step of administering a third MAPK pathway inhibitor. While not theoretically bound, suppression of MAPK signaling in cancer cells may result in downregulation of PD-L1 expression, increasing the likelihood of cancer cells being detected by the immune system. Such third MAPK pathway inhibitors may be based on other mutations in proteins in the MAPK pathway. In some embodiments, any MAPK pathway inhibitor can be employed, including those targeting K-Ras, N-Ras, H-Ras, PDGFRA, PDGFRB, MET, FGFR, ALK, ROS1, TRKA, TRKB, TRKC, EGFR, IGF1R, GRB2, SOS, ARAF, BRAF, RAFI, MEK1, MEK2, c-Myc, CDK4, CDK6, CDK2, ERK1, and ERK2. Non-exclusive examples of MEK inhibitors include trametinib, cobimetinib, binimetinib, PD-0325901, selumetinib, and CI-1040. Exemplary MAPK pathway inhibitors, but not limited to, include afatinib, osimertinib, erlotinib, gefitinib, lapatinib, neratinib, dacomitinib, vandetanib, cetuximab, panitumumab, nimotuzumab, necitumumab, trametinib, binimetinib, cobimetinib, selumetinib, urixertinib, LTT462, and LY3214996. In some embodiments, one or more inhibitors listed in this paragraph and elsewhere in this specification may be specifically excluded from any embodiment, including but not limited to the methods, kits, and compositions described herein.

[0147] The methods disclosed herein can be combined with other chemotherapeutic agents. Examples of such agents can be found in *Cancer Principles and Practice of Oncology* by VT Devita and S. Hellman (editors), 6th edition (February 15, 2001), Lippincott Williams & Wilkins Publishers, and all of its teachings, including, but not limited to, all methods, compounds, compositions, data, etc., are incorporated herein by reference for use in conjunction with any of the embodiments and disclosures herein. Those skilled in the art will be able to identify which combinations of agents are useful based on the specific characteristics of the drugs and the diseases involved.

[0148] In some embodiments, the method may include the simultaneous administration of at least one cytotoxic agent. The term “cytotoxic agent,” as used herein, refers to a substance that inhibits or interferes with cellular function and / or causes cell death or destruction. Cytotoxic agents include, but are not limited to, radioisotopes (e.g., At211, I131, I125, Y90, Re186, Re188, Sm153, Bi212, P32, Pb212, and radioisotopes of Lu), chemotherapeutic agents, growth inhibitors, enzymes such as nucleases and their fragments, and toxins, including fragments and / or variants thereof, of small molecule toxins or enzymatically active toxins of bacterial, fungal, plant, or animal origin.

[0149] Examples of cytotoxic agents may be selected from antimicrotubule agents, platinum coordination complexes, alkylating agents, antibiotics, topoisomerase II inhibitors, antimetabolites, topoisomerase I inhibitors, hormones and hormone analogs, signaling pathway inhibitors, non-receptor tyrosine kinase angiogenesis inhibitors, immunotherapeutic agents, apoptosis promoters, LDH-A inhibitors, fatty acid biosynthesis inhibitors, cell cycle signaling inhibitors, HDAC inhibitors, proteasome inhibitors, and cancer metabolism inhibitors.

[0150] Chemotherapy agents include chemical compounds useful for treating cancer. Examples of chemotherapeutic agents include erlotinib (TARCEVA®, Genentech / OSIPharm.), bortezomib (VELCADE®, Millennium Pharm.), disulfiram, epigallocatechin gallate, salinosporamide A, carfilzomib, 17-AAG (geldanamycin), radicicol, lactate dehydrogenase A (LDH-A), fulvestrant (FASLODEX®, AstraZeneca), sunitinib (SUTENT®, Pfizer / Sugen), and letrozole (FEMARA®, Novartis). Imatinib mesylate (GLEEVEC®, Novartis), financiate (VATALANIB®, Novartis), oxaliplatin (ELOXATIN®, Sanofi), 5-FU (5-fluorouracil), leucovorin, rapamycin (sirolimus, RAPAMUNE®, Wyeth), lapatinib (TYKERB®, GSK572016, Glaxo Alkylating agents such as Smith Kline, ronafamib (SCH66336), sorafenib (NEXAVAR®, Bayer Labs), gefitinib (IRESSA®, AstraZeneca), AG1478, thiotepa, and CYTOXAN® cyclophosphamide; alkyl sulfonates such as busulfan, improsulfan, and pigosulfan; aziridines such as benzodopa, carbocone, metsuredopa, and uredopa; ethylenimines and methylamelamines, including altoretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylomellamine;Acetogenins (especially bratacin and bratacinone), camptothecin (including topotecan and irinotecan), bryostatin, calcistatin, CC1065 (including its adzeresin, karzeresin, and bizeresin synthetic analogs), cryptophycin (especially cryptophycin 1 and cryptophycin 8), corticosteroids (including prednisone and prednisolone), cyproterone acetate, 5-α-reductase (including finasteride and dutasteride), vorinostat, romidepsin, panobinostat, valproic acid, mosetinostat, dorastatin; aldesleukin, talc duocalmycin (including synthetic analogs, kW-2189 and CB1-TM1); eleutherobin; pancratistatin; sarcodictiin (a sarcodictyin; spongistatin; chlorambutyl, chlornafadin, chlorophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, nobembitin, phenesterine, prednimustine, trophosphamide, uracil mustard and other nitrogen mustards; nitrosourea such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; engineeric antibiotics (e.g., calicheamicin, especially calicheamicin γ1L and calicheamicin CO2) (Angew Chem. Intl. Ed. Engl. 1994) Antibiotics such as 33:183-186); dynemycin including dynemycin A; bisphosphonates such as clodronate; esperamycin;(and neocardinostatin chromophores and related chromoprotein engine antibiotics), aclasinomycins, actinomycin, ausramycin, azaserin, bleomycin, kactinomycin, carabicin, caminomycin, cardinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, ADRIAMYCIN® (doxorubicin), morpholino Xorubicin, cyanomorpholinodoxorubicin, 2-pyrrolinodoxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcelomycin, mitomycin such as mitomycin C, mycophenolate and nogaramycin, olibomycins, peplomycin, porphyromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidine, ubenimex, dinostatin, zorubicin; methotrexate and antimetabolites such as 5-fluorouracil (5-FU); folate analogs such as denopterin, methotrexate, pteropterin, and trimethrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, and thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, and phloxuridine; androgen analogs such as carsterone, dromostanolone propionate, epithiostanol, mepitiostane, and testactone. Gen; anti-adrenal agents such as aminoglutethimide, mitotane, and trilostane; folic acid supplements such as frolinic acid; acegraton; aldofamide glycoside; aminolevulinic acid; enyluracil; amsacrin; bestrabusil; bisantren; edatraxate; defofamine; demecoltin; diazicon; elfomithine; eriptinium acetate; epotilon; etogluside; gallium nitrate; hydroxyurea; lentinan;Lonidaininine; mytansinoids such as mytansin and anthamitosin; mitogwazone; mitoxantrone; mopidamnol; nitraerine; pentostatin; fenamet; pirarubicin; losoxantrone; podophyllic acid; 2-ethylhydrazide; procarbazine; PSKR polysaccharide complex (JHS Natural Products, Eugene, Oreg.); Lazoxane; Rhizoxin; Schizofuran; Spirogermanium; Tenuazonic acid; Triadicone; 2,2',2”-Trichlorotriethylamine; Trichothecene (especially T-2 toxin, Veraculine A, Loridine A, and Angidin); Urethane; Vindesine; Dacarbazine; Mannomustine; Mitobronitol; Mitractol; Pipobroman; Gacytosine; Arabinoside ("Ara-C"); Cyclophosphamide; Thiotepa; Taxoids, e.g., TAXOL (Paclitaxel; Bristol-Myers Squibb Oncology, Princeton, NJ), Abraxane (registered trademark) (excluding Cremofol), Albumin-modified nanoparticle formulations of Paclitaxel (American Pharmaceutical Partners, Schaumberg, III.), and Taxotere® (docetaxel, doxetaxel); Sanofi-Aventis); chlorambucil; GEMZAR® (gemcitabine); 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin and carboplatin; vinblastine; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; NAVELBINE® (vinorelbine); novantrone; teniposide; edatrexate; daunomycin, aminopterin, capecitabine (XELODA®), ibandronate, CPT-11, topoisomerase inhibitor RFS 2000 difluoromethylornithine (DMFO), retinoids such as retinoic acid, and any of the above pharmaceutically acceptable salts, acids, and derivatives.

[0151] Chemotherapy agents also include (i) anti-hormone agents that act to modulate or inhibit the hormonal effects on tumors, such as anti-estrogens and selective estrogen receptor modulators (SERMs), including, for example, tamoxifen (NOLVADEX®; tamoxifen citrate), raloxifene, droloxifene, iodoxifene, 4-hydroxytamoxifen, trioxyfen, keoxyfen, LY117018, onapristone, and FARESTON® (toremifene citrate); and (ii) Aromatase inhibitors that inhibit aromatase, an enzyme that regulates estrogen production in the adrenal gland, such as 4(5)-imidazole, aminoglutethimide, MEGASE® (megestrol acetate), AROMASIN® (exemestane; Pfizer), formestany, fadrozol, RIVISOR® (borozol), FEMARA® (letrozole; Novartis), and ARIMIIDEX® (anastrozole; AstraZeneca); (iii) (iv) Protein kinase inhibitors; (v) Lipid kinase inhibitors; (vi) Antisense oligonucleotides such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; (vii) (viii) Ribozymes such as VEGF expression inhibitors (e.g., ANGIOZYME®) and HER2 expression inhibitors; (viii) Vaccines such as gene therapy vaccines, e.g., ALLOVECTIN®, LEUVECTIN®, and VAXID®; PROLEUKIN®, rIL-2; Topoisomerase 1 inhibitors such as LURTOTECAN®; ABARELIX® rmRH;and (ix) comprising any pharmaceutically acceptable salt, acid, and derivative thereof;

[0152] The chemotherapy agents also include antibodies such as alemtuzumab (Campath) and bevacizumab (AVASTIN®, Genentech); cetuximab (ERBITUX®, Imclone); panitumumab (VECTIBIX®, Amgen); rituximab (RITUXAN®, Genentech / Biogen Idee); pertuzumab (OMNITARG®, 2C4, Genentech); trastuzumab (HERCEPTIN®, Genentech); tositumomab (Bexxar, Corixia); and the antibody-drug conjugate gemtuzumab ozogamicin (MYLOTARG®, Wyeth).Further humanized monoclonal antibodies with therapeutic potential as drugs in combination with the compounds of the present invention include apolizumab, aselizumab, atlizumab, bapinetuzumab, vibatuzumab meltansine, cantuzumab meltansine, sedelizumab, certolizumab pegol, sidofcituzumab, sidotuzumab, daclizumab, eculizumab, efalizumab, epratuzumab, erulizumab, felbizumab, phonolizumab, gemtuzumab ozogamicin, inotuzumab ozogamicin, ipilimumab, rabetuzumab, lintuzumab, matsuzumab, mepolizumab, and motabizumab (motoviz). umab), natalizumab, nimotuzumab, nolovizumab, numavizumab, ocrelizumab, omalizumab, palivizumab, pascolizumab, pectuzumab, pectuzumab, pexelizumab, ralivizumab, ranibizumab, reslivizumab, resyvizumab, loberizumab, luprizumab, cibrotuzumab, cyprizumab, sontuzumab, acatuzumab tetraxetan This includes tetraxetan, tadocizumab, talizumab, tefivazumab, tocilizumab, tralizumab, tucotuzumab celmoleukin, tucusituzumab, umabizumab, urtoxazumab, ustekinumab, bicilizumab, and interleukin-12 antibody (ABT-874 / J695, Wyeth Research and Abbott Laboratories), which is a full-length IgG1 antibody that is exclusively a human sequence recombinant and genetically modified to recognize the p40 protein.

[0153] Chemotherapy agents also include "EGFR inhibitors," which refer to compounds that bind to EGFR or its variant forms, or otherwise directly interact with it, preventing or reducing its signaling activity; these are also called "EGFR antagonists." Examples of such drugs include antibodies and small molecules that bind to EGFR. Examples of antibodies that bind to EGFR include MAb579 (ATCC CRLHB8506), MAb455 (ATCC CRLHB8507), MAb225 (ATCC CRL8508), MAb528 (ATCC CRL8509) (see U.S. Patent 4,943,533, Mendelsohn el al.), and their variants (chimeric 225 (C225 or cetuximab, ERBUTIX®) and remodeled human 225 (H225) (WO 96 / 40210, Imclone Systems) (See Inc., etc.); IMC-11F8 (Imclone), a fully human EGFR target antibody; antibodies that bind to type II mutant EGFR (US Patent No. 5,212,290); humanized and chimeric antibodies that bind to EGFR as described in US Patent No. 5,891,996; and human antibodies that bind to EGFR (such as ABX-EGF or panitumumab (See WO98 / 50433, Abgenix / Amgen)); EMD55900 (Stragliotto et al. Eur. J. Cancer 32A:636-640(1996)); EMD7200 (matsuzumab) (EMD / Merck), a humanized EGFR antibody directed towards EGFR and competing with both EGF and TGF-α for EGFR binding; human EGFR antibody, HuMax-EGFR (GenMab); known as E1.1, E2.4, E2.5, E6.2, E6.4, E2.11, E6.3 and E7.6.3, and a fully human antibody described in US6,235,883; MDX-447 (Medarex Inc); and mAb806 or humanized mAb806 (Johns et al., J. Biol. Chem. Examples include 279(29):30375-30384(2004). Anti-EGFR antibodies can be conjugated with cytotoxic agents, thus generating immunoconjugates (see, for example, EP659,439A2, Merck Patent GmbH).EGFR antagonists are covered by U.S. Patent Nos. 5,616,582, 5,457,105, 5,475,001, 5,654,307, 5,679,683, 6,084,095, 6,265,410, 6,455,534, 6,521,620, 6,596,726, 6,713,484, 5,770,599, 6,140,332, 5,866,572, 6,399,602, 6,344,459, 6,602,863, 6,391,874, 6,344,455, 5,760,041, 6,002,008, and This includes 5,747,498, as well as small molecules such as compounds described in PCT publications WO98 / 14451, W098 / 50038, W099 / 09016, and WO99 / 24037.Specific small molecule EGFR antagonists include OSI-774 (CP-358774, erlotinib, TARCEVA®, Genentech / OSI Pharmaceuticals); PD183805 (CI 1033, 2-propenamide, N-[4-[(3-chloro-4-fluorophenyl)amino]-7-[3-(4-morpholinyl)propoxy]-6-quinazolinyl]-, dihydrochloride, Pfizer Inc.); ZD1839, gefitinib (IRESSA®) 4-(3'-chloro-4'-fluoroanilino)-7-methoxy-6-(3-morpholinopropoxy)quinazoline, AstraZeneca); ZM 105180((6-amino-4-(3-methylphenyl-amino)-quinazoline, Zeneca); BIBX-1382(N8-(3-chloro-4-fluorophenyl)-N2-(1-methyl-piperidine-4-yl)-pyrimido[5,4-d]pyrimidine-2,8-diamine, Boehringer Ingelheim); PKI-166((R)-4-[4-[(1-phenylethyl)amino]-1H-pyrrolo[2,3-d]pyrimidine-6-yl]-phenol); (R)-6-(4-hydroxyphenyl)-4-[(1-phenylethyl)amino]-7H-pyrrolo[2,3-d]pyrimidine); CL-387785(N-[4-[(3-bromophenyl)amino]-6-quinazolinyl]-2-butinamide); EKB Examples include -569(N-[4-[(3-chloro-4-fluorophenyl)amino]-3-cyano-7-ethoxy-6-quinolinyl]-4-(dimethylamino)-2-butinamide)(Wyeth);AG1478(Pfizer);AG1571(SU5271;Pfizer); and dual EGFR / HER2 tyrosine kinase inhibitors such as lapatinib ((TYKERB®, GSK572016 or N-[3-chloro-4-[(3-fluorophenyl)methoxy]phenyl]-6[5[[[2methylsulfonyl)ethyl]amino]methyl]-2-furanyl]-4-quinazolinamine). Each of the above references is incorporated herein by reference in its entirety, with respect to all teachings, including, but not limited to, all methods, compounds, compositions, data, etc., for use in conjunction with any of the embodiments and disclosures herein.

[0154] Chemotherapy agents also include "tyrosine kinase inhibitors," including the EGFR-targeting drugs mentioned in the previous paragraph; small molecule HER2 tyrosine kinase inhibitors such as TAK165 available from Takeda; oral selective inhibitors of ErbB2 receptor tyrosine kinase (CP-724, 714, Pfizer and OSI); dual HER inhibitors such as EKB-569 (available from Wyeth), which preferentially binds to EGFR but inhibits both HER2 and EGFR overexpressing cells; lapatinib (GSK572016; available from Glaxo-SmithKline), an oral HER2 and EGFR tyrosine kinase inhibitor; PKI-166 (available from Novartis); pan-HER inhibitors such as canertinib (CI-1033; Pharmacia); Raf-1 inhibitors, which are antisense agents that inhibit Raf-1 signaling, such as ISIS-5132 available from ISIS Pharmaceuticals; imatinib mesylate (GLEEVEC®, Glaxo Non-HER-targeted TK inhibitors such as (available from SmithKline); multi-targeted tyrosine kinase inhibitors such as sunitinib (SUTENT®, available from Pfizer); VEGF receptor tyrosine kinase inhibitors such as batalanib (PTK787 / ZK222584, available from Novartis / Schering AG); MAPK extracellular regulatory kinase I inhibitor CI-1040 (available from Pharmacia); quinazolins such as PD153035,4-(3-chloroanilino)quinazoline; pyridopyrimidines; pyridopyrimidines; pyrrolopyrimidines such as CGP59326, CGP60261, and CGP62706; pyrazolopyrimidines, 4-(phenylamino)-7H-pyrrolo[2,3-d]pyrimidine; curcumin (diferloylmethane, 4,5-Bis(4-fluoroanilino)phthalimide; Tylphostine containing the nitrothiophene moiety; PD-0183805 (Warner-Lamber); Antisense molecules (e.g., those that bind to nucleic acids encoding HER); Quinoxaline (US Patent 5,804,396); Triphostine (US Patent 5,804,396); ZD6474 (Astra Zeneca); PTK-787 (Novartis / Schering AG);Examples include pan-HER inhibitors such as CI-1033 (Pfizer); Affinitac (ISIS 3521; ISIS / Lilly); Imatinib mesylate (GLEEVEC®); PKI166 (Novartis); GW2016 (Glaxo SmithKline); CI-1033 (Pfizer); EKB-569 (Wyeth); Semaxinib (Pfizer); ZD6474 (AstraZeneca); PTK-787 (Novartis / Schering AG); INC-1C11 (Imclone); Rapamycin (Sirolimus, RAPAMUNE®), or the following patent publications: U.S. Patent 5,804,396; WO1999 / 09016 (American Cyanamid); WO1998 / 43960 (American Cyanamid); WO1997 / 38983 (Warner As described in WO1999 / 06378 (Warner Lambert); WO1999 / 06396 (Warner Lambert); WO1996 / 30347 (Pfizer, Inc); WO1996 / 33978 (Zeneca); WO1996 / 3397 (Zeneca) and WO1996 / 33980 (Zeneca). Each of the above references is incorporated herein by reference in its entirety, with respect to all teachings, including, but not limited to, all methods, compounds, compositions, data, etc., for use in conjunction with any of the embodiments and disclosures herein.

[0155] Chemotherapy agents also include dexamethasone, interferon, colchicine, methoprine, cyclosporine, amphotericin, metronidazole, alemtuzumab, alitretinoin, allopurinol, amifostine, arsenic trioxide, asparaginase, BCG Live, bevacuzimab, besarotene, cladribine, clofarabine, darbepoetin alfa, denileukin, dexazoxane, epoetin alfa, erotinib, filgrastim, histrelin acetate, ibritumomab, interferon alfa-2a, and interferon. This includes alpha-ene, cyclosporine, nandrolone, nelarabine, nofetumomab, oprelbequine, parifermin, pamidronate, pegademase, pegaspargase, pegfilgrastim, pemetrexeddisodium, plicamycin, porfimer sodium, quinacrine, rasburicase, salglamostim, temozolomide, VM-26, 6-TG, toremifene, tretinoin, ATRA, barulurubicin, zoledronate, and zoledronic acid, as well as pharmaceutically acceptable salts thereof.

[0156] Chemotherapy agents also include hydrocortisone, hydrocortisone acetate, cortisone acetate, thixocortol pivalate, triamcinolone acetonide, triamcinolone alcohol, mometasone, amcinonide, budesonide, desonide, fluocinonide, fluocinolone acetonide, betamethasone, betamethasone sodium phosphate, dexamethasone, dexamethasone sodium phosphate, fluocortone, hydrocortisone-17-br Chilate, hydrocortisone-17-valerate, acromethasone dipropionate, betamethasone valerate, betamethasone dipropionate, prednicarbate, clobetasone-17-butyrate, clobetasol-17-propionate, fluocortone caproate, fluocortone pivalate, and flupredniden acetate; phenylalanine-glutamine-glycine (FEG) and its D-isomer (feG) (IMULAN Immunoselective anti-inflammatory peptides (ImSAIDs) such as BioTherapeutics, LLC; antirheumatic drugs such as azathioprine, cyclosporine (cyclosporine A), D-penicillamine, gold salt, hydroxychloroquine, leflunomidocycline, and sulfasalazine; tumor necrosis factor α (TNFα) blockers such as etanercept (Enbrel), infliximab (Remicade), adalimumab (Humira), certolizumab pegol (Cimzia), and golimumab (Simponi); and interleukin-1 such as anakinra (Kineret). (IL-1) blockers, T-cell costimulatory blockers such as abatacept (Orencia), interleukin-6 (IL-6) blockers such as tocilizumab (ACTEMERA®); interleukin-13 (IL-13) blockers such as lebrikizumab; interferon-alpha (IFN) blockers such as lonthalizumab; beta-7 integrin blockers such as rhuMAb beta-7; IgE pathway blockers such as anti-M1 prime; secretory homotrimer LTa3 and membrane-bound heterotrimer LTa1 / β2 blockers such as anti-lymphotoxin α (LTa); radioisotopes (e.g., At 211 , I 131 , I 125 , Y 90 Re 186 Re188 、Sm 153 、Bi 212 、P 32 、Pb 212Radioactive isotopes of Lu; other research agents such as thioplatin, PS-341, phenylbutyrate, ET-18-OCH3, or farnesyltransferase inhibitors (L-739749, L-744832); polyphenols such as quercetin, resveratrol, piceatannol, epigallocatechin gallate, theaflavins, flavanols, procyanidins, betulinic acid, and their derivatives; autophagy inhibitors such as chloroquine; δ-9-tetrahydrocannabinol (dronabinol, MARINOL (registered trademark)). )); β-Lapacon; Lapachol; Colchicine; Betulic acid; Acetylcamptothecin, Scopolectin, and 9-aminocamptothecin); Podophyllotoxin; Tegafur (UFTORAL®); Bexarotene (TARGRETIN®); Clodronate (e.g., BONEFOS® or OSTAC®), Etidronate (DIDROCAL®), NE-58095, Zoledronic acid / Zoledrone (ZOMETA®), Alendronate (FOSAMAX®), Bisphosphonates such as pamidronate (AREDIA®), tildronate (SKELID®), or risedronate (ACTONEL®); and epidermal growth factor receptor (EGF-R); vaccines such as THERATOPE® vaccine; perifosin, COX-2 inhibitors (e.g., celecoxib or etoricoxib), proteosome inhibitors (e.g., PS341); CCI-779; tipifarnib (R11577); olafenib, ABT510; oblimersen sodium (GENASE This includes combinations of two or more of the above, such as Bcl-2 inhibitors like NSE(registered trademark); pixantrone; farnesyltransferase inhibitors like ronafarnib (SCH6636, SARASAR(trademark)); and pharmaceutically acceptable salts, acids, or derivatives of any of the above; CHOP, an abbreviation for combination therapy of cyclophosphamide, doxorubicin, vincristine, and prednisolone; and FOLFOX, an abbreviation for treatment regimens with oxaliplatin (ELOXATIN(trademark)) in combination with 5-FU and leucovorin.

[0157] Chemotherapy agents also include nonsteroidal anti-inflammatory drugs (NSAIDs) that have analgesic, antipyretic, and anti-inflammatory effects. NSAIDs include non-selective inhibitors of the enzyme cyclooxygenase. Specific examples of NSAIDs include aspirin, propionic acid derivatives such as ibuprofen, fenoprofen, ketoprofen, flurbiprofen, oxaprozin, and naproxen, acetate derivatives such as indomethacin, sulindac, etodolac, and diclofenac, enolic acid derivatives such as piroxicam, meloxicam, tenoxicam, droxicam, romoxicam, and isoxicam, fenamic acid derivatives such as mefenamic acid, meclofenamic acid, flufenamic acid, and tolfenamic acid, and COX-2 inhibitors such as celecoxib, etoricoxib, lumiracoxib, parecoxib, rofecoxib, and valdecoxib. NSAIDs may be indicated for the relief of symptoms of conditions such as rheumatoid arthritis, osteoarthritis, inflammatory arthritis, ankylosing spondylitis, psoriatic arthritis, Reiter's syndrome, acute gout, dysmenorrhea, metastatic bone pain, headaches and migraines, postoperative pain, mild to moderate pain due to inflammation and tissue damage, fever, ileus, and colitis.

[0158] In some embodiments, chemotherapeutic agents include, but are not limited to, doxorubicin, dexamethasone, vincristine, cyclophosphamide, fluorouracil, topotecan, interferon, platinum derivatives, taxanes (e.g., paclitaxel, docetaxel), vinca alkaloids (e.g., vinblastine), anthracyclines (e.g., doxorubicin), epipodophilotoxin (e.g., etoposide), cisplatin, in particular, mTOR inhibitors (e.g., rapamycin), methotrexate, actinomycin D, drastatin 10, colchicine, trimethrexate, methoprine, cyclosporine, daunorubicin, teniposide, amphotericin, alkylating agents (e.g., chlorambucil), 5-fluorouracil, camptothecin, cisplatin, metronidazole, and imatinib mesylate. In other embodiments, the compounds disclosed herein are administered in combination with a biological agent such as bevacizumab or panitumumab.

[0159] In certain embodiments, the compounds disclosed herein, or pharmaceutically acceptable compositions thereof, include avalerix, aldesleukin, alemtuzumab, alitretinoin, allopurinol, altretamine, amiphostine, anastrozole, arsenic trioxide, asparaginase, azacitidine, and BCG.live, bevacuzimab, fluorouracil, bexarotene, bleomycin, bortezomib, busulfan, carsterone, capecitabine, camptothecin, carboplatin, carmustine, cetuximab, chlorambucil, cladribine, clofarabine, cyclophosphamide, cytarabine, dactinomycin, darbepoetin alfa, daunorubicin, denileukin, dexazoxane, docetaxel, doxorubicin (neutral), doxorubicin hydrochloride, dromostanolone propionate, epirubicin Epoetin alfa, erotinib, estramustine, etoposide phosphate, etoposide, exemestane, filgrastim, floxuridine, fludarabine, fulvestrant, gefitinib, gemcitabine, mesilumab, gemstimab acetate, gemsererin acetate, fluvestinyl phosphate, interferon α-2a, interferon α-2b, irinotecan, lenalidomide, letrozole, leucovorin, leuprolide acetate, levamizole, lomustine, megestrol acetate, melfa Lan, mercaptopurine, 6-MP, mesna, methotrexate, methoxsalen, mitomycin C, mitotane, mitoxantrone, nandrolone, nelarabine, nofetumomab, oprelbequin, oxaliplatin, paclitaxel, parifermin, pamidronate, pegademase, pegaspargase, pegfilgrastim, pemetrexed disodium pentostatin, pipobromane, plicamycin, porfimer sodium, procarbazine, quinacrine, rasburicase, rituximab, salguar It is administered in combination with any one or more antiproliferative agents or chemotherapeutic agents selected from ramostim, sorafenib, streptozosin, sunitinib maleate, talc, tamoxifen, temozolomide, teniposide, VM-26, testolactone, thioguanine, 6-TG, thiotepa, topotecan, toremifene, tocitumomab, trastuzumab, tretinoin, ATRA, uracil mustard, barururubicin, vinblastine, vincristine, vinorelbine, zoledronate, or zoledronic acid.

[0160] In some embodiments, the dose of the compound of formula I may be any suitable amount for treating cancer. For example, the dose may be a daily dose between 1 mg and 500 mg by weight. As a further example, the daily dose may be in the range of about 20 mg to 400 mg (or any lower range or lower value, including endpoints) in between. In some embodiments, the dose range of the compound of formula I may be 10 mg to 300 mg. In some embodiments, the dose range of the compound of formula I may be 10 mg to 100 mg. In some embodiments, the dose range of the compound of formula I may be 5 mg to 50 mg. The daily dose can be achieved by a single dose (e.g., QD) or over a day (e.g., BID, TID, QID, etc.) through multiple doses to provide a total daily dose. In some embodiments, the dose of the MEK inhibitor is any suitable amount. For example, it may be an amount in the range of 1 mg to 500 mg per day (or any lower range or lower value (including endpoints)) in between. The dosage of a MEK inhibitor may be the same as or less than the approved dosage for any given MEK inhibitor and may depend on the given indication. In some embodiments, trametinib may be administered once daily in doses ranging from about 1 mg to about 10 mg. For example, trametinib is approved at 2 mg once daily. Dose reductions such as 1.5 mg QD and 1 mg QD are also approved. In some embodiments, binimetinib may be administered in doses ranging from about 30 mg to about 100 mg. For example, binimetinib is approved at a dose of 45 mg twice daily. Dose reductions such as about 30 mg BID are also approved. It will be understood that each of the above-listed ranges may include any subrange or partial point within it, including the endpoint. The typical dosage range for adults is generally 5 mg to 2 g / day.Tablets or other forms of presentation provided in discrete units may conveniently contain units containing one or more compounds in amounts effective as such doses or multiples thereof, e.g., 5 mg to 500 mg, typically about 10 mg to 200 mg. The amount of active ingredient that can be combined with a carrier material to produce a single-dose form depends on the host being treated and the specific mode of administration. In some embodiments, administration is orally.

[0161] In some embodiments, a method is provided for treating colorectal cancer and NSCLC cancer in a subject, comprising the step of orally administering to the subject a therapeutically effective amount of a compound of formula I or a pharmaceutically acceptable salt thereof in combination with trametinib or binimetinib. In some embodiments, the compound of formula I is administered once or twice daily. In some embodiments, trametinib or binimetinib may be administered once or twice daily. The drugs may be administered concurrently, for example, as described herein.

[0162] In some embodiments, the subject is human. In some embodiments, the subject is a non-human mammal such as a primate, rodent, dog, cat, or other small animal.

[0163] In some embodiments, a method for inhibiting ERK1 / 2 phosphorylation is provided, comprising contacting a cell population with formula I or a pharmaceutically acceptable salt thereof combined with trametinib or binimetinib. In some embodiments, the concentration of the compound of formula I is in the range of 1 nm to 1 micromol, or 1 nm to 500 nM, or 1 nM to 20 nM. In some embodiments, the concentration of trametinib or binimetinib is in the range of 10 nM to 1 micromol, or 10 nM to 500 nM.

[0164] composition The compounds of formula I disclosed herein may exist as salts. This embodiment includes such salts that may be pharmaceutically acceptable. Examples of applicable salt forms include hydrochlorides, hydrobroms, sulfates, methanesulfons, nitrates, maleates, acetates, citrates, fumarates, tartrates (e.g., (+)-tartrates, (-)-tartrates, or mixtures thereof including racemic mixtures), succinates, benzoates, and salts with amino acids such as glutamic acid. These salts can be prepared by methods known to those skilled in the art. Also included are base addition salts such as sodium, potassium, calcium, and ammonium, organic amino or magnesium salts, or similar salts. If the compounds of this embodiment contain relatively basic functional groups, acid addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of the desired acid, either raw or in a suitable inert solvent. Examples of acceptable acid addition salts include those derived from inorganic acids such as hydrochloric acid, hydrobromic acid, nitric acid, carbonic acid, monohydrogenic acid, phosphoric acid, monohydrogenic acid, dihydrogenic acid, sulfuric acid, monohydrogenic acid, hydroiodic acid, or phosphorous acid, as well as salts derived from organic acids such as acetic acid, propionic acid, isobutyric acid, maleic acid, malonic acid, benzoic acid, succinic acid, suberic acid, fumaric acid, lactic acid, mandelic acid, phthalic acid, benzenesulfonic acid, p-tolylsulfonic acid, citric acid, tartaric acid, methanesulfonic acid, etc. Also included are salts of amino acids such as alginates, and salts of organic acids such as glucuronic acid or galacturonic acid. Certain specific compounds in this embodiment possess both basic and acidic functionalities, allowing the compound to be converted into either a base or an acid addition salt.

[0165] Other salts include salts of acids or bases of the compounds used in the methods of this embodiment. Exemplary examples of pharmaceutically acceptable salts are mineral acid salts (e.g., hydrochloric acid, hydrobromic acid, phosphoric acid), organic acid salts (e.g., acetic acid, propionic acid, glutamic acid, citrate), and quaternary ammonium salts (e.g., methyl iodide, ethyl iodide). It will be understood that pharmaceutically acceptable salts are nontoxic. Further information regarding appropriate pharmaceutically acceptable salts can be found in Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, Pa., 1985, which incorporates, by reference, all of its teachings, including but not limited to all methods, compounds, compositions, data, etc., for use with any of the embodiments and disclosures herein.

[0166] Pharmaceutically acceptable salts include salts of the active compound prepared with relatively non-toxic acids or bases, depending on the specific substituents found on the compounds described herein. If the compounds of this embodiment contain relatively acidic functional groups, base addition salts can be obtained by contacting the neutral form of such compound with a sufficient amount of the desired base, either raw or in a suitable inert solvent. Examples of pharmaceutically acceptable base addition salts include sodium, potassium, calcium, ammonium, organic amino, or magnesium salts, or similar salts. If the compounds of this embodiment contain relatively basic functional groups, acid addition salts can be obtained by contacting the neutral form of such compound with a sufficient amount of the desired acid, either raw or in a suitable inert solvent. Examples of pharmaceutically acceptable acid addition salts include those derived from inorganic acids such as hydrochloric acid, hydrobromic acid, nitric acid, carbonic acid, monohydrogenic acid, phosphoric acid, monohydrogenic acid, dihydrogenic acid, sulfuric acid, monohydrogenic acid, hydroiodic acid, or phosphorous acid, as well as salts derived from relatively non-toxic organic acids such as acetic acid, propionic acid, isobutyric acid, maleic acid, malonic acid, benzoic acid, succinic acid, suberic acid, fumaric acid, lactic acid, mandelic acid, phthalic acid, benzenesulfonic acid, p-tolylsulfonic acid, citric acid, tartaric acid, and methanesulfonic acid. Also included are salts of amino acids such as alginates, and salts of organic acids such as glucuronic acid or galacturonic acid (see, for example, Berge et al., “Pharmaceutical Salts”, Journal of Pharmaceutical Science, 1977, 66, 1-19), and all teachings, including but not limited to all methods, compounds, compositions, data, etc., for use with any of the embodiments and disclosures herein are incorporated herein by reference in their entirety. Certain compounds in these embodiments contain both basic and acidic functional groups that enable the conversion of the compound into either a base-addition salt or an acid-addition salt.

[0167] The neutral form of the compound is preferably regenerated by contacting the salt with a base or acid and isolating the parent compound in the conventional manner. The parent form of the compound differs from the various salt forms in certain physical properties, such as solubility in polar solvents.

[0168] Certain compounds of this embodiment may exist in a non-solvated form and in a solvated form, including a hydrated form. Generally, the solvated forms are equivalent to the non-solvated forms and are included within the scope of this embodiment. Certain compounds of this embodiment may exist in multiple crystalline or amorphous forms. Generally, all physical forms are equivalent to the uses intended by this embodiment and are intended to be within the scope of this embodiment.

[0169] Certain compounds in this embodiment have an asymmetric carbon atom (optical center) or a double bond; the forms of enantiomers, racemates, diastereomers, tautomers, geometric isomers, and stereoisomers may be defined with respect to absolute stereochemistry as (R)- or (S)- or (D)- or (L)- for amino acids, and individual isomers are encompassed within the scope of this embodiment. The compounds of this embodiment do not include those known to be too unstable in the art to be synthesized and / or separated. This embodiment is intended to include compounds in racemic and optically pure forms. Optically active (R)- and (S)-, or (D)- and (L)- isomers may be prepared using chiral synthons or chiral reagents, or separated using conventional techniques.

[0170] Unless otherwise indicated, the compounds of this embodiment may contain isotopes of atoms in an unnatural ratio in one or more of the atoms constituting such a compound. For example, the compounds of the present invention may contain, for example, deuterium ( 2 H), tritium ( 3 H), Iodine-125( 125 I), Fluorine-18( 18 F), Nitrogen-15( 15 N), oxygen-17( 17 O), oxygen-18( 18 O), carbon-13 ( 13C), or carbon-14 ( 14 They can be labeled with radioactive or stable isotopes such as C). All isotopic changes of the compounds in the embodiments of the present invention, whether radioactive or not, are included within the scope of the embodiments of the present invention.

[0171] In addition to salt forms, this embodiment provides compounds in prodrug form. The prodrugs of the compounds described herein are compounds that readily undergo chemical changes under physiological conditions for providing the compounds of this embodiment. Furthermore, the prodrugs can be converted to the compounds of the embodiments of the present invention by chemical or biochemical methods in an ex vivo environment. For example, the prodrug can be slowly converted to the compounds of the embodiments of the present invention when placed in a transdermal patch reservoir with a suitable enzyme or chemical reagent.

[0172] In some embodiments, a pharmaceutical composition comprising a compound of formula I and a pharmaceutically acceptable excipient is provided. In some embodiments, the pharmaceutical composition is configured as an oral tablet.

[0173] The compounds of the embodiments of the present invention can be prepared and administered in a wide variety of oral, parenteral, and topical dosage forms. Oral formulations include tablets, pills, powders, sugar-coated tablets, capsules, liquids, lozenges, gels, syrups, slurries, and suspensions suitable for patient intake. The compounds of the embodiments can also be administered by injection, i.e., intravenously, intramuscularly, intradermally, subcutaneously, intradurally, or intraperitoneally. Furthermore, the compounds described herein can be administered by inhalation, for example, intranasally. In addition, the compounds of the embodiments of the present invention can be administered percutaneously. The compounds of formula I disclosed herein may also be administered via intraocular, intravaginal, and intrarectal routes, including suppositories, inhalants, powders, and aerosol formulations (see Rohatagi, J. Clin. Pharmacol. 35:1187-1193, 1995; Tjwa, Ann. Allergy Asthma Immunol. 75:107-111, 1995 for examples of inhaled steroids), and all teachings, including but not limited to all methods, compounds, compositions, data, etc., for use with any of the embodiments and disclosures herein are incorporated herein by reference. Accordingly, these embodiments also provide pharmaceutical compositions comprising one or more pharmaceutically acceptable carriers and / or excipients and either a compound of formula I or a pharmaceutically acceptable salt of a compound of formula I.

[0174] For preparing pharmaceutical compositions from the compounds of embodiments of the present invention, pharmaceutically acceptable carriers may be either solid or liquid. Solid preparations include powders, tablets, pills, capsules, cachets, suppositories, and dispersible granules. The solid carrier may be one or more substances that also act as diluents, flavoring agents, surfactants, binders, preservatives, tablet disintegrants, or encapsulating materials. Further details regarding the techniques for formulation and administration are described in detail in scientific literature and patent documents, for example, with reference to the latest edition of Remington's Pharmaceutical Sciences, Maack Publishing Co, Easton PA ("Remington's"), and all teachings therein, including but not limited to all methods, compounds, compositions, data, etc., for use with any of the embodiments and disclosures herein, are incorporated herein by reference.

[0175] In powders, the carrier is a finely ground solid in a mixture with the finely ground active ingredient. In tablets, the active ingredient is mixed in appropriate proportions with a carrier having the required binding properties and, if necessary, additional excipients, and then compressed into the desired shape and size.

[0176] Powders, capsules, and tablets preferably contain 5% or 10% to 70% of the active compound. Suitable carriers include magnesium carbonate, magnesium stearate, talc, sugars, lactose, pectin, dextrin, starch, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose, low-melting-point waxes, and cocoa butter. The term “preparation” is intended to include formulations of the active compound with encapsulating material as a carrier, providing capsules in which the active ingredient is surrounded by a carrier, with or without other excipients, and thus associated with the carrier. Similarly, cachets and lozenges are included. Tablets, powders, capsules, pills, cachets, and lozenges may be used as solid dosage forms suitable for oral administration.

[0177] Suitable solid excipients include, but are not limited to, sugars such as lactose, sucrose, mannitol, or sorbitol, starches from corn, wheat, rice, potato, or other plants, celluloses such as methylcellulose, hydroxypropylmethylcellulose, or sodium carboxymethylcellulose, and gums including gum arabic and tragacanth gum, as well as carbohydrate or protein fillers including proteins such as gelatin and collagen. If desired, disintegrants or solubilizing agents such as crosslinked polyvinylpyrrolidone, agar, alginic acid, or sodium alginate, and their salts may be added.

[0178] The sugar core is provided with a suitable coating such as a concentrated sugar solution, and the coating may further include gum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and / or titanium dioxide, lacquer solution, and a suitable organic solvent or solvent mixture. Dyes or pigments may be added to the coating of the tablets or dragees for product identification or to characterize the amount (i.e., dosage) of the active compound. The pharmaceutical preparations disclosed herein can be used orally, for example, using push-fit capsules made of gelatin, as well as soft sealed capsules made with a coating of gelatin and glycerol or sorbitol. The push-fit capsules can contain a compound of formula I mixed with a filler or binder such as lactose or starch, a lubricant such as talc or magnesium stearate, and optionally a stabilizer. In the soft capsules, the compound of formula I can be dissolved or suspended in a suitable liquid such as fatty oil, liquid paraffin, or liquid polyethylene glycol, with or without a stabilizer.

[0179] Examples of liquid preparations include solutions, suspensions, and emulsions, such as water or water / propylene glycol solutions. For parenteral injection, the liquid preparation can be formulated in a solution in an aqueous polyethylene glycol solution.

[0180] An aqueous solution suitable for oral use can be prepared by dissolving the active ingredient in water and adding the desired suitable coloring agent, flavoring agent, stabilizer, and thickening agent. An aqueous suspension suitable for oral use can be prepared by dispersing the micronized active ingredient in water together with a viscous material such as natural or synthetic rubber, resin, methylcellulose, sodium carboxymethylcellulose, hydroxypropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, tragacanth gum, and acacia gum, and a dispersant or wetting agent such as a naturally occurring phosphatide (e.g., lecithin), a condensation product of an alkylene oxide and a fatty acid (e.g., polyoxyethylene stearate), a condensation product of ethylene oxide and a long-chain aliphatic alcohol (e.g., heptadecaethyleneoxy cetanol), a condensation product of ethylene oxide and a partial ester derived from a fatty acid and a hexitol (e.g., polyoxyethylene sorbitol mono-oleate), or a condensation product of ethylene oxide and a partial ester derived from a fatty acid and a hexitol anhydride (e.g., polyoxyethylene sorbitan mono-oleate). The aqueous suspension may also contain one or more preservatives, such as ethyl or n-propyl p-hydroxybenzoate, one or more coloring agents, one or more flavoring agents, and one or more sweetening agents, such as sucrose, aspartame, or saccharin. The formulation can be adjusted with respect to the volumetric osmolality.

[0181] Solid form preparations intended to be converted into liquid form preparations for oral administration immediately before use are also included. Such liquid forms include solutions, suspensions, and emulsions. These preparations may contain, in addition to the active ingredient, coloring agents, fragrances, stabilizers, buffering agents, artificial and natural sweeteners, dispersants, thickening agents, solubilizing agents, and the like.

[0182] Oily suspensions can be formulated by suspending the compound of formula I in a vegetable oil such as peanut oil, olive oil, sesame oil, or coconut oil, or a mineral oil such as liquid paraffin, or a mixture thereof. Oily suspensions may contain thickeners such as beeswax, solid paraffin, or cetyl alcohol. Sweeteners such as glycerol, sorbitol, or sucrose can be added to provide an oral preparation with a pleasant mouthfeel. These formulations can be preserved by adding antioxidants such as ascorbic acid. An example of an oil vehicle for injection can be found in Minto, J. Pharmacol. Exp. Ther. 281:93-102, 1997, which is incorporated herein by reference in its entirety with respect to all teachings, including, but not limited to, all methods, compounds, compositions, data, etc., for use in conjunction with any of the embodiments and disclosures herein. The pharmaceutical formulations disclosed herein may also be in the form of oil-in-water emulsions. The oil phase may be the vegetable oil or mineral oil described above, or a mixture thereof. Suitable emulsifiers include naturally occurring gums such as acacia gum and tragacanth gum, naturally occurring phosphatides such as soy lecithin, esters or partial esters derived from fatty acids such as sorbitan mono-oleate and hexitol anhydride, and condensation products of the above partial esters such as polyoxyethylene sorbitan mono-oleate with ethylene oxide. Emulsions may also contain sweeteners and flavorings, as in the case of syrup and elixir formulations. Such formulations may also contain lubricants, preservatives, or colorants.

[0183] Pharmaceutical formulations of the compounds of formula I disclosed herein may be provided as salts and may be formed by comprising a base, i.e., a cationic salt such as an alkali, an alkali metal salt such as sodium, lithium, potassium, calcium, magnesium, and an ammonium salt such as ammonium, trimethylammonium, diethylammonium, and tris-(hydroxymethyl)-methylammonium salt.

[0184] Pharmaceutical preparations are preferably in unit dosage forms. In such forms, the preparation is subdivided into unit doses containing an appropriate amount of the active ingredient. The unit dosage form may be a packaged preparation, and the packaging contains separate amounts of the preparation, such as packaged tablets, capsules, and powders in vials or ampoules. Alternatively, the unit dosage form may be the capsule, tablet, cachet, or lozenge itself, or any of these in an appropriate number of packaged forms.

[0185] The amount of the active ingredient in a unit dose preparation may vary or be adjusted, depending on the specific use and the potency of the active ingredient, from 0.1 mg to 10,000 mg, more typically from 1.0 mg to 1,000 mg, and most typically from 10 mg to 500 mg. The composition may also contain other suitable therapeutic agents, if desired.

[0186] The administration regimen should also take into account pharmacokinetic parameters well known in the art, namely absorption rate, bioavailability, metabolism, and clearance (see, for example, Hidalgo-Aragones (1996) J. Steroid Biochem. Mol. Biol. 58:611-617; Groning (1996) Pharmazie 51:337-341; Fotherby (1996) Contraception 54:59-69; Johnson (1995) J. Pharm. Sci. 84:1144-1146; Rohatagi (1995) Pharmazie 50:610-613; Brophy (1983) Eur. J. Clin. Pharmacol. 24: 103-108; The most recent Remington's (each of which is incorporated herein by reference in its entirety, with respect to all of its teachings, including, but not limited to, all methods, compounds, compositions, data, etc., for use in any of the embodiments and disclosures herein). State-of-the-art technology enables clinicians to determine the dosage regimen for each individual patient, GR and / or MR modulator, and each disease or condition being treated.

[0187] A single or multiple administration of a compound formulation of formula I may be administered according to the required and acceptable dosage and frequency for the patient. The formulation must provide a sufficient amount of the active agent to effectively treat the disease condition. Accordingly, in one embodiment, a pharmaceutical formulation for oral administration of a compound of formula I is a daily dose between about 0.5 mg / kg body weight / day and about 30 mg / kg body weight / day, including all subranges and subvalues ​​within that, including the endpoint. In alternative embodiments, doses of about 1 mg to about 20 mg / kg body weight / patient / day are used. Lower doses may be used in particular when administering the drug to an anatomically occluded site such as the cerebrospinal fluid (CSF) space, into the bloodstream, into a body cavity, or into the lumen of an organ, in contrast to oral administration. Substantially higher doses may be used for topical administration. Practical methods for preparing formulations containing a compound of formula I for parenteral administration are known or evident to those skilled in the art and are described in detail in publications such as Remington's mentioned above. Furthermore, Nieman, In “Receptor Mediated Antisteroid Action,” Agarwal, et al., eds., De Gruyter, New York (1987) is also referenced, and all of its teachings, including but not limited to all methods, compounds, compositions, data, etc., for use in conjunction with any of the embodiments and disclosures herein, are incorporated herein by reference in their entirety.

[0188] In some embodiments, simultaneous administration includes administering one activator within 0.5, 1, 2, 4, 6, 8, 10, 12, 16, 20, or 24 hours (or any lower range or value within 24 hours) of the second activator. Simultaneous administration includes administering two activators simultaneously, nearly simultaneously (e.g., within approximately 1, 5, 10, 15, 20, or 30 minutes of each other (or, e.g., any lower range of time or a lower value of time between 0 and 30 minutes)), or sequentially in any order. In some embodiments, simultaneous administration can be achieved by simultaneous formulation, i.e., by preparing a single pharmaceutical composition containing both activators. In some embodiments, the activators may be formulated separately. In some embodiments, the activators and / or adjuvants may be linked or conjugated to each other. At least one dose of the drugs may be administered, for example, simultaneously. At least one dose of the drugs may be administered, for example, within a few minutes or less than one hour from each other. At least one dose of the drug may be administered, for example, at different times on the same day, or on different days.

[0189] Pharmaceutical compositions comprising compounds of formula I disclosed herein are formulated in one or more acceptable carriers, then placed in appropriate containers and labeled for the treatment of the desired condition. Such labels for the administration of compounds of formula I include, for example, instructions regarding the amount, frequency, and method of administration.

[0190] Pharmaceutical administration The administration regimens of the compounds described herein will naturally vary depending on known factors such as the pharmacodynamic characteristics of the particular drug and its mode and route of administration; the recipient's species, age, sex, health status, medical condition, and weight; the nature and severity of symptoms; the type of concurrent treatment; the frequency of treatment; the route of administration, the patient's renal and hepatic function, and the desired effect. Clinicians can determine and prescribe the effective dose of the drug needed to prevent, inhibit, or halt the progression of a disease or disorder.

[0191] As a general guideline, the daily oral dose of each active ingredient, when used for the desired effect, is in the range of about 0.001 to about 1000 mg / kg body weight, preferably about 0.01 to about 100 mg / kg body weight / day, and most preferably about 0.1 to about 20 mg / kg / day. In some embodiments, the compound of formula I is administered in doses between about 10 mg / day and about 200 mg / day. In some embodiments, the compound of formula (I) may be administered in doses of approximately 10 mg / day, 20 mg / day, 30 mg / day, 40 mg / day, 50 mg / day, 60 mg / day, 70 mg / day, 80 mg / day, 90 mg / day, 100 mg / day, 110 mg / day, 120 mg / day, 130 mg / day, 140 mg / day, 150 mg / day, 160 mg / day, 170 mg / day, 180 mg / day, 190 mg / day, or 200 mg / day. The dose may be any value or sub-range within the listed range.

[0192] Depending on the patient's condition and the intended therapeutic effect, the frequency of administration of the therapeutic agent may vary, for example, from once a day to six times a day. That is, the administration frequency may be QD, i.e., once a day; BID, i.e., twice a day; TID, i.e., three times a day; QID, i.e., four times a day, five times a day, or six times a day. In another embodiment, the administration frequency may be BIW, i.e., twice a week; TIW, i.e., three times a week; or QIW, i.e., four times a week.

[0193] Depending on the patient's condition and the intended therapeutic effect, the treatment cycle may include periods during which the therapeutic agent is not administered. As used herein, “interval administration” refers to administration of the therapeutic agent and the subsequent gap of days or weeks. For example, a treatment cycle may be three weeks, comprising two weeks of administration of the therapeutic agent followed by one week without administration of the therapeutic agent. In some embodiments, the treatment cycle is four weeks, comprising three weeks of administration followed by one week without administration of the therapeutic agent.

[0194] As used herein, the term "treatment cycle" refers to a predetermined period for the administration of a therapeutic agent. Typically, the patient is examined at the end of each treatment cycle to assess the effectiveness of the therapy.

[0195] In one embodiment, each treatment cycle has a duration of approximately 3 days or more. In another embodiment, each treatment cycle has a duration of approximately 3 to 60 days. In another embodiment, each treatment cycle has a duration of approximately 5 to 50 days. In another embodiment, each treatment cycle has a duration of approximately 7 to 28 days. In another embodiment, each treatment cycle has a duration of 28 days. In one embodiment, each treatment cycle has a duration of approximately 29 days. In another embodiment, each treatment cycle has a duration of approximately 30 days. In another embodiment, each treatment cycle has a duration of approximately 31 days. In another embodiment, the treatment cycle has a duration of approximately 1 month. In another embodiment, the treatment cycle is any duration from 3 to 8 weeks. In another embodiment, the treatment cycle is any duration from 3 to 6 weeks. In yet another embodiment, the treatment is 3 weeks long. In another embodiment, the treatment cycle is 1 month long. In another embodiment, each treatment cycle is 4 weeks long. In another embodiment, each treatment cycle is 5 weeks long. In another embodiment, each treatment cycle is 6 weeks long. In another embodiment, each treatment cycle is 7 weeks long. In another embodiment, each treatment cycle is 8 weeks long. The duration of a treatment cycle may include any value or sub-range within the enumerated range, including the endpoint.

[0196] As used herein, the terms “concurrent administration” or “simultaneous administration” mean the administration of (a) an additional therapeutic agent and (b) a compound of formula (I) or its salts, solvates, esters, and / or prodrugs together in a coordinated manner. For example, concurrent administration may be simultaneous administration, sequential administration, overlapping administration, interval administration, consecutive administration, or a combination thereof.

[0197] In some embodiments, the administration regimen for the compound of formula (I) is once daily over a continuous 28-day cycle. In some embodiments, the once-daily administration regimen for the compound of formula (I) may be, but is not limited to, 20 mg / day, 30 mg / day, 40 mg / day, 50 mg / day, or 60 mg / day. The compound of formula (I) may be administered once daily in any amount from 20 mg to 60 mg. The dose may be any value within the enumerated range or a sub-range.

[0198] In some embodiments, the administration regimen of the compound of formula (I) is twice daily over a continuous 28-day cycle. In some embodiments, the twice-daily administration regimen of the compound of formula (I) may be, but are not limited to, 10 mg / day, 20 mg / day, 30 mg / day, 40 mg / day, 50 mg / day, 60 mg / day, 70 mg / day, 80 mg / day, 90 mg / day, or 100 mg / day. The compound of formula (I) may be administered twice daily in any amount from 20 mg to 80 mg. In some embodiments, the compound of formula (I) may be administered in any amount from 10 mg / day to 100 mg / day. The dose may be any value within or below the listed range.

[0199] In some embodiments, the administration regimen for the compound of formula (I) may be a dose of some amount from 20 mg to 60 mg once daily for two weeks, followed by a one-week rest period (e.g., two weeks on, one week off) for six weeks. In some embodiments, the administration regimen for the compound of formula (I) may be a dose of some amount from 10 mg to 100 mg twice daily for two weeks, followed by a one-week rest period (e.g., two weeks on, one week off) for six weeks.

[0200] In some embodiments, the dosing regimen for the compound of formula (I) may be for 8 weeks, with a certain amount of administration from 20 mg to 60 mg once daily for 3 weeks, followed by a 1-week drug holiday (e.g., 3 weeks on, 1 week off). In some embodiments, the dosing regimen for the compound of formula (I) may be for 8 weeks, with a certain amount of administration from 10 mg to 100 mg twice daily for 3 weeks, followed by a 1-week drug holiday (e.g., 8 weeks on, 1 week off).

[0201] In some embodiments, the dosing regimen for the compound of formula (I) can be twice a day on the 1st and 2nd day of each week for 8 weeks. In some embodiments, the dosage of the compound of formula (I) can be, but is not limited to, 10 mg, 20 mg, 30 mg, 40 mg, 50 mg, 60 mg, 70 mg, 80 mg, 90 mg, 100 mg.

[0202] In some embodiments, the compound of formula I or a pharmaceutically acceptable salt thereof is administered once a day in a 3-week cycle that includes 2 weeks of administration of the compound and then 1 week of non-administration of the compound.

[0203] In some embodiments, the compound of formula I or a pharmaceutically acceptable salt thereof is administered once a day in a 4-week cycle that includes 3 weeks of administration of the compound and then 1 week of non-administration of the compound.

[0204] In some embodiments, the compound of formula I, or a pharmaceutically acceptable salt thereof, is administered for 6 weeks. In some embodiments, the compound of formula I or a pharmaceutically acceptable salt thereof is administered for 8 weeks.

[0205] In some embodiments, the compound of formula I or a pharmaceutically acceptable salt thereof is administered three times a week. In some embodiments, the compound of formula I or a pharmaceutically acceptable salt thereof is administered on the 1st, 3rd, and 5th days of the week.

[0206] In some embodiments, the compound of formula I or a pharmaceutically acceptable salt thereof is administered four times a week.

[0207] In some embodiments, the compound of formula I or a pharmaceutically acceptable salt thereof is administered in a three-week cycle consisting of two weeks of administration of the compound followed by one week of non-administration of the compound.

[0208] In some embodiments, the compound of formula I or a pharmaceutically acceptable salt thereof is administered in a four-week cycle comprising three weeks of administration of the compound followed by one week of non-administration of the compound.

[0209] In some embodiments, the compound of formula I or a pharmaceutically acceptable salt thereof is administered twice daily for two days a week. In some embodiments, the compound of formula I or a pharmaceutically acceptable salt thereof is administered over a period of eight weeks. In some embodiments, the compound of formula I or a pharmaceutically acceptable salt thereof is administered on the first and second days of each week.

[0210] If the compound of formula I is administered multiple times a week, the dose may be administered on any day or combination of days within the week. For example, a three-times-a-week administration may include administration on days 1, 3, and 5; administration on days 1, 2, and 3; and administration on days 1, 3, and 5. A two-days-a-week administration may include administration on days 1 and 2; days 1 and 3; days 1 and 4; days 1 and 5; and days 1 and 7.

[0211] Kits and products Some embodiments of this disclosure relate to kits and products comprising a compound of formula I and / or at least one FGFR inhibitor. For example, a kit or product may comprise a package or container containing the compound of formula I. Such kits and products may further comprise an insert or label containing approved drug administration and indication information, including how the compound of formula I is used in combination with a separately provided FGFR inhibitor. The kits can be used in methods for treating cancer as described herein.

[0212] In some embodiments, a kit or product may include both a compound of formula I and at least one FGFR inhibitor. In some embodiments, the FGFR inhibitor is, for example, erdafitinib. Such a kit may include one or more containers or packages, one or more containers or packages containing one or both combination drugs together in a single container and / or package, or in separate packages / containers. In some cases, the two drugs are packaged separately but contained in a single package, container or box. Such kits and products may further include a product insert or label containing approved drug administration and indication information, including how to use the compound of formula I in combination with the FGFR inhibitor. The kit may be used in methods of treating cancer as described herein.

[0213] Some embodiments of this disclosure relate to kits and products comprising a compound of Formula 1 and / or at least one B-Raf inhibitor. For example, a kit or product may comprise a package or container containing the compound of Formula 1. Such kits and products may further comprise an insert or label containing approved drug administration and indication information, including how to use the compound of Formula 1 in combination with the separately provided B-Raf inhibitor. The kits can be used in methods of treating cancer as described herein.

[0214] In some embodiments, the kit or product may include both the compound of Formula 1 and at least one B-Raf inhibitor. In some embodiments, the B-Raf inhibitor is, for example, encorafenib. Such a kit may include one or more containers or packages, one or more containers or packages containing one or both combination drugs together in a single container and / or package, or in separate packages / containers. In some examples, the two drugs are packaged separately but contained in a single package, container or box. Such kits and products may further include inserts or labels with approved drug administration and indication information, including how to use the compound of Formula 1 in combination with the B-Raf inhibitor. The kit may be used in methods of treating cancer as described herein.

[0215] Some embodiments of this disclosure relate to kits and products comprising a compound of formula I and / or at least one MEK inhibitor. For example, a kit or product may comprise a package or container containing the compound of formula I. Such kits and products may further comprise an insert or label containing approved drug administration and indication information, including how to use the compound of formula I in combination with the separately provided MEK inhibitor. The kits can be used in methods of treating cancer as described herein.

[0216] In some embodiments, the kit or product may include both the compound of Formula 1 and at least one MEK inhibitor. In some embodiments, the MEK inhibitor may be, for example, trametinib or binimetinib. Such a kit may include one or more containers or packages, one or more of which may contain one or both combination drugs together in a single container and / or package, or in separate packages / containers. In some examples, the two drugs may be packaged separately but contained in a single package, container, or box. Such kits and products may further include inserts or labels containing approved drug administration and indication information, including how the compound of Formula 1 is used in combination with the MEK inhibitor. The kit may be used in methods of treating cancer as described herein.

[0217] Some embodiments of this disclosure relate to kits and products comprising a compound of formula I and / or at least one MET inhibitor. For example, a kit or product may comprise a package or container containing the compound of formula I. Such kits and products may further comprise an insert or label containing approved drug administration and indication information, including how to use the compound of formula I in combination with the separately provided MET inhibitor. The kit can be used in methods of treating cancer as described herein.

[0218] In some embodiments, the kit or product may include both a compound of Formula 1 and at least one MET inhibitor. In some embodiments, the MET inhibitor may be, for example, crizotinib, tepotinib, savolitinib, cabozantinib, or tivantinib. Such a kit may include one or more containers or packages, one or more of which may contain one or both combination drugs together in a single container and / or package, or in separate packages / containers. In some examples, the two drugs may be packaged separately but contained in a single package, container, or box. Such a kit and result may further include a result insert or label with approved drug administration and indication information, including a method of using the compound of Formula I in combination with a MET inhibitor. The kit may be used in a method of treating cancer as described herein. [Examples]

[0219] Example 1 - Synergistic combination of compound I and an FGFR inhibitor This embodiment demonstrates a synergistic combination of the compound of formula I and an FGFR inhibitor.

[0220] Combined cell proliferation assay Cells (2000 cells per well) were seeded in 100 μl of cell culture medium on a 96-well plate. Using the Tecan D300e Digital Dispenser's combination matrix protocol, cells were treated with erdafitinib and compound I at concentrations ranging from 0 to 10 μM. On day 5, 50 μl of CellTiter-Glo (CTG) reagent (Promega) was added, and the plate was incubated for 10 minutes with gentle shaking. After 10 minutes of incubation, the luminescence signal was determined according to the manufacturer's instructions (Promega), and combination data was generated using the Combinefit software with a standard HSA model. Synergistic effects of combinations are represented by positive numbers in the results table. Negative numbers represent antagonistic effects of combinations.

[0221] result Figures 1A and 1B show data demonstrating in vitro synergistic effects when the compound of formula I is combined with the FGFR inhibitor erdafitinib. Figure 1A shows 3D graphic synergistic effect data in the Hep3B cancer cell line using the combination of the compound of formula I and erdafitinib. Figure IB shows 3D graphic synergistic effect data in the JHH-7 cancer cell line using the combination of the compound of formula I and erdafitinib. Figures 1A and 1B show data demonstrating in vitro synergistic effects when the compound of formula I is combined with the FGFR inhibitor erdafitinib. Figure 1A shows synergistic effect data in the Hep3B cancer cell line using the combination of the compound of formula I and erdafitinib. Figure 1B shows synergistic effect data in the JHH-7 cancer cell line using the combination of the compound of formula I and erdafitinib.

[0222] Example 2 - Combination therapy of compound I and erdafitinib in the FGFR2-amplified hepatocellular carcinoma CDX model KATO III material Acetic acid in 100 mM water was prepared for the vehicle / control, with the pH adjusted to 4.8-5.0. This was then stored under ambient conditions for 28 days during administration to mice.

[0223] Each week, a fresh sample of the compound of formula I was prepared in a 100 mM acetate buffer vehicle and stored under ambient conditions. The concomitant agent, erdafitinib, was prepared in a 20% HP-P-CD vehicle and stored at 2–8°C.

[0224] Female Balb / c nude mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. The mice were 6-8 weeks old at the time of transplantation. The mice were housed in a specially sourced pathogen-free (SPF) environment and allowed to acclimate to the new environment for at least 3 days before the start of any experiment, according to the IACUC protocol.

[0225] All procedures related to the handling, care, and treatment of animals in this study were carried out in accordance with guidelines approved by the WuXiAppTec Animal Care Committee (IACUC). During the study, animal care and use were carried out in accordance with the regulations of the International Association for Accreditation and Evaluation of Laboratory Animal Care (AAALAC). In addition, all parts of this study conducted at WuXiAppTec adhered to the research protocol made and approved by the principal investigator and applicable standard operating procedures (SOPs).

[0226] Preparation of xenograft models The KATO-III cell line was a human hepatoma cell line amplified by FGFR. The KATO-III cell line was purchased from ATCC (ATCC® HTB-103®). It was mixed with 50% Matrigel in a 10x10⁶ cell line. 6 A 200 μL cell suspension containing tumor cells was subcutaneously transplanted into the right flank of mice using a syringe. After subcutaneous transplantation, the average tumor volume was 220 mm³. 3 When the condition was met, mice with tumors were randomized into different groups, each containing eight mice. The randomization day is denoted as treatment day 0.

[0227] treatment Treatment was initiated on a day following randomization. The treatment initiation day was denoted as Treatment Day 1. Mice were orally administered vehicle control solution, compound I alone at 10 mg / kg BID, and erdafitinib alone at 10 mg / kg QD. Another group received combination treatment with compound I and erdafitinib, with compound I administered at 10 mg / kg BID and erdafitinib at 10 mg / kg QD. The administration volume was 5 mL / kg, and the interval between BID regimens was 8 hours. In the combination group, erdafitinib was administered 1 hour after the first BID administration of compound I. This study was concluded on Treatment Day 28 as defined in the study protocol.

[0228] result Figure 2 shows graphs of tumor volume over the treatment period in the hepatocellular carcinoma CDX model KATO III, with treatment using compound I alone, erdafitinib alone, and a combination of compound I and erdafitinib. No significant changes in body weight were observed in either the control or treatment group.

[0229] conclusion As shown in Figure 2, the combination of compound I and erdafitinib demonstrated superior tumor growth inhibition in the FGFR2-amplified hepatocellular carcinoma CDX model KATO III compared to treatment with compound I alone or erdafitinib alone.

[0230] Example 3 - Combination therapy of compound I and erdafitinib in FGFR2-amplified gastric cancer CDXSNU-16 material Acetic acid in 100 mM water was prepared for the vehicle / control, with the pH adjusted to 4.8-5.0. This was then stored under ambient conditions for 28 days during administration to mice.

[0231] Each week, a fresh sample of the compound of formula I was prepared in a 100 mM acetate buffer vehicle and stored under ambient conditions. Each week, the concomitant agent erdafitinib was prepared in a 20% HP-P-CD vehicle and stored at 2–8°C.

[0232] Female Balb / c nude mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. The mice were 6-8 weeks old at the time of transplantation. The mice were housed in a specially sourced pathogen-free (SPF) environment and allowed to acclimate to the new environment for at least 3 days before the start of any experiment, according to the IACUC protocol.

[0233] All procedures related to the handling, care, and treatment of animals in this study were carried out in accordance with guidelines approved by the WuXiAppTec Animal Care Committee (IACUC). During the study, animal care and use were carried out in accordance with the regulations of the International Association for Accreditation and Evaluation of Laboratory Animal Care (AAALAC). In addition, all parts of this study conducted at WuXiAppTec adhered to the research protocol approved by the principal investigator and applicable standard operating procedures (SOPs).

[0234] Preparation of xenograft models The SNU16 cell line was a human gastric cancer cell line exhibiting FGFR amplification. The SNU16 cell line was purchased from ATCC (ATCC® CRL-1420®). It was mixed with 50% Matrigel in a 10x10⁶ cell line. 6 A 200 μL cell suspension containing tumor cells was subcutaneously transplanted into the right flank of mice using a syringe. After subcutaneous transplantation, the average tumor volume was 180 mm². 3 When the condition was met, mice with tumors were randomized into different groups, each containing eight mice. The randomization day is denoted as treatment day 0.

[0235] treatment Treatment began on the day of randomization. The treatment start day is denoted as Treatment Day 0. Mice were orally administered vehicle control solution, compound I of formula 1 alone at 10 mg / kg BID, compound I of formula 1 alone at 30 mg / kg QD, and erdafitinib at 10 mg / kg QD. Two additional groups received combination treatment with compound I of formula 1 and erdafitinib: one group received compound I of formula 1 at 10 mg / kg BID and erdafitinib at 10 mg / kg QD, and the other group received compound I of formula 1 at 30 mg / kg QD and erdafitinib at 10 mg / kg QD. The administration volume of each compound was 5 mL / kg, and the interval between BID regimens was 8 hours. In the combination groups, erdafitinib was administered once after the QD administration of compound I of formula 1, or after the first BID administration of compound I of formula 1. This study was concluded on day 28 of treatment, as defined in the study protocol.

[0236] result Figure 3 shows graphs of tumor volume over treatment time in the FGFR2-amplified gastric cancer CDX model SNU-16, with treatment of compound I alone, erdafitinib alone, and a combination of compound I and erdafitinib. No significant changes in body weight were observed in either the control or treatment groups.

[0237] conclusion As shown in Figure 3, the combination of the compound of formula I and erdafitinib demonstrated superior tumor growth inhibition in the FGFR2-amplified gastric cancer CDX model SNLT-16 compared to treatment with the compound of formula I alone or with erdafitinib alone.

[0238] Example 4 - Combination therapy of compound I and erdafitinib in FGF19-FDFR4-dependent amplified hepatocellular carcinoma CDX model Huh-7 material Each week, a fresh sample of the compound of formula I was prepared in a 100 mM acetate buffer vehicle and stored under ambient conditions. Each week, the concomitant agent erdafitinib was freshly prepared in a 20% HP-P-CD vehicle and stored at 2–8°C.

[0239] Female Balb / c nude mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. The mice were 6-8 weeks old at the time of transplantation. The mice were housed in a specially sourced pathogen-free (SPF) environment and allowed to acclimate to the new environment for at least 3 days before the start of any experiment, according to the IACUC protocol.

[0240] Preparation of xenograft models The Huh-7 cell line was a human hepatocellular carcinoma cell line exhibiting FGFR overexpression. The Huh-7 cell line was purchased from the Japanese Collection of Research Bioresources Cell Bank (JCRB Cell Bank (JCRB0403)). Huh-7 cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) containing 10% fetal bovine serum (FBS) and 1% antibiotic-antifungal agent (AA) at 37°C under a 5% CO2 atmosphere in air. The medium was refreshed every 2-3 days, and tumor cells were routinely subcultured at 80-90% confluence using trypsin-EDTA. Cells growing in the exponential growth phase were collected and counted for inoculation. Huh-7 tumor cells were subcutaneously transplanted into mice. 10 × 10⁶ cells were mixed with 50% Matrigel. 6 A 200 μL cell suspension containing tumor cells was subcutaneously transplanted into the right flank using a syringe. The tumor volume was 500-1000 mm³. 3 When it reaches the area, tumor fragments (15-30 mm) 3 The tumor was collected and then subcutaneously transplanted into the right flank of mice using an 18g trocar needle. The animals' health and tumor growth were monitored daily. Tumor volume was measured twice weekly with a caliper if the tumor was palpable and measurable. After subcutaneous transplantation, the average tumor volume was 146 mm². 3 When the condition was met, mice with tumors were randomized into different groups, each containing eight mice. The randomization day is denoted as treatment day 0.

[0241] treatment Treatment was initiated on a day following randomization. The treatment initiation day was denoted as Treatment Day 1. In the monotherapy groups, mice were orally administered vehicle control solution, compound I alone at 10 mg / kg BID, compound I alone at 30 mg / kg QD, and erdafitinib at 10 mg / kg QD. Two additional groups received combination treatment with compound I and erdafitinib: one group received compound I at 10 mg / kg BID and erdafitinib at 10 mg / kg QD, and the other group received compound I at 30 mg / kg QD and erdafitinib at 10 mg / kg QD. The administration volume of each compound was 5 mL / kg, and the interval between BID regimens was 8 hours. In the combination therapy group, erdafitinib was administered 1 hour after the QD dose of compound I of formula 1, or 1 hour after the first BID dose of compound I of formula 1. The study was completed on day 21 of treatment.

[0242] result Figure 4 shows graphs of tumor volume over treatment periods in the FGF19-FGFR4-dependent hepatocellular carcinoma CDX model Huh-7, with treatment using compound I alone, erdafitinib alone, and a combination of compound I and erdafitinib. No significant weight changes were observed in either the control or treatment groups.

[0243] conclusion As shown in Figure 4, the combination of the compound of formula I and erdafitinib demonstrated superior tumor growth inhibition in the FGF19-FGFR4-dependent hepatocellular carcinoma CDX model Huh-7 compared to treatment with the compound of formula I alone or with erdafitinib alone.

[0244] Example 5 - In vitro synergistic combination of compound I and an inhibitor of class 1 mutant B-Raf protein. Cell proliferation assay Cells (2000 cells per well) were seeded in 100 μl of cell culture medium on a 96-well plate and treated with compound I alone or with compound I in combination with a constant concentration of encorafenib. On day 5, 50 μl of CellTiter-Glo (CTG) reagent (Promega) was added, and the plate was incubated for 10 minutes with gentle shaking. After 10 minutes of incubation, the luminescence signal was determined according to the manufacturer's instructions (Promega), and the graph was plotted using PrismGraphPad.

[0245] Combined cell proliferation assay Cells (2000 cells per well) were seeded in 100 μl of cell culture medium on a 96-well plate. Cells were treated with the compound of formula I and encorafenib at concentrations ranging from 0 to 10 μM using the Tecan D300e Digital Dispenser's combination matrix protocol. On day 5, 50 μl of CellTiter-Glo (CTG) reagent (Promega) was added, and the plate was incubated for 10 minutes with gentle shaking. After 10 minutes of incubation, the luminescence signal was determined according to the manufacturer's instructions (Promega), and combination data was generated using the standard HSA model with Combinenefit software. Synergistic effects of combinations are represented by positive numbers in the results table. Negative numbers represent antagonistic effects of combinations.

[0246] Western blotting on pERK and ERK Cells were incubated with the compound for 4 hours. After treatment, cells were lysed on ice for 10 minutes using Thermo Fisher RIPA lysis buffer containing protease and phosphatase inhibitors. Cells were centrifuged using a microcentrifuge at 4°C for 10 minutes. The supernatant was transferred to a pre-cooled microcentrifuge tube, and the protein concentration of the lysate was measured using the BCA method. An equal volume of protein from the cell lysate supernatant was used for immunoblotting against pERK and ERK.

[0247] result Figure 5 shows data demonstrating that the combination of the compound of formula I and encorafenib exhibits synergistic effects across multiple BRAF V600E mutant cells.

[0248] Figure 6 shows RKO BRAF using a combination of the compound of formula I and the BRAF inhibitor encorafenib. V600E The synergistic effect data in CRC cell lines is shown. This data indicates a significant synergistic effect when the compound of formula I is combined with encorafenib.

[0249] Figure 7 shows WiDr BRAF using a combination of the compound of formula I and the BRAF inhibitor encorafenib. V600E The synergistic effect data in CRC cell lines is shown. This data indicates a significant synergistic effect when the compound of formula I is combined with encorafenib.

[0250] Figure 8 shows HT29 BRAF using a combination of the compound of formula I and the BRAF inhibitor encorafenib. V600E The synergistic effect data in CRC cell lines is shown. This data indicates a significant synergistic effect when the compound of formula I is combined with encorafenib.

[0251] Figure 9A shows a gel illustrating robust inhibition of ERK1 / 2 phosphorylation in the RKO colorectal cancer cell line. Figure 9BA shows a gel illustrating robust inhibition of ERK1 / 2 phosphorylation in the WiDr colorectal cancer cell line. Figure 9C shows plots of the antiproliferative activity of compound I, alone or in combination with encorafenib, in the RKO colorectal cancer cell line. Figure 9D shows plots of the antiproliferative activity of compound I, or in combination with encorafenib, in the WiDr colorectal cancer cell line. Figures 9A-9B indicate robust inhibition of pERK1 / 2 using the combination of compound I and encorafenib. Figures 9C-9D suggest that the combination of compound I and encorafenib increased the inhibitory activity of compound I.

[0252] Figures 10A-10D show comparative studies of the efficacy of SHP2 inhibitors and encorafenib combinations in RKO colorectal cancer cell lines. Figure 10A shows a gel comparing the inhibition of ERK1 / 2 phosphorylation in RKO colorectal cancer cell lines with the compound of formula I + encorafenib, TNO155 + encorafenib, and RMC-4550 + encorafenib combinations. Figure 10B shows a bar graph of pERK as a percentage of the control, with 1. control, 2. (compound of formula I), 3. encorafenib, and 4. (compound of formula I) + encorafenib. Figure 10C shows a bar graph of pERK as a percentage of the target, with 1. control, 2. TNO155, 3. encorafenib, and 4. TNO155 + encorafenib. Figure 10D shows a bar graph of pERK as a percentage of the control, with 1. control, 2. RMC-4550, 3. encorafenib, and 4. RMC-4550 + encorafenib. As shown in Figures 10A to 10D, inhibition of ERK1 / 2 phosphorylation is most effective with the combination of the SHP2 inhibitor compound of formula I and encorafenib.

[0253] Example 6 - BRAF V600E Combination therapy of compound I and encorafenib in mutant CRC PDX model CR0029 material Vehicle / control and 100 mM acetic acid in deionized water were prepared by adjusting the pH to 4.8-5.0, and stored under ambient conditions for 28 days during administration to mice.

[0254] Each week, a fresh sample of the compound of formula I was prepared in a 100 mM acetate buffer vehicle and stored under ambient conditions. Each week, the concomitant agent encorafenib was prepared in a 0.5% CMC and 0.5% Tween 80 vehicle and stored at 2–8°C.

[0255] Female Balb / c nude mice were purchased from SPF (Beijing) Laboratory Animal Technology Co., Ltd. The mice were 7-9 weeks old at the time of transplantation. The mice were housed in a special pathogen-free (SPF) environment and allowed to acclimate to the new environment for at least 3 days before the start of each experiment, according to the IACUC protocol.

[0256] All procedures related to the handling, care, and treatment of animals in this study were conducted in accordance with guidelines approved by the Institutional Animal Care and Use Committee (IACUC) of Crown Bioscience (Taicang, China). During the study, animal care and use were carried out in accordance with the regulations of the International Association for Accreditation and Evaluation of Laboratory Animal Care (AAALAC). In addition, all parts of this study conducted at Crown Bioscience (Taicang, China) adhered to research protocols approved by the principal investigator and applicable standard operating procedures (SOPs).

[0257] Preparation of xenograft models The CR0029 PDX model was established for preclinical efficacy studies in CrownBio. This PDX model was derived from a Chinese female CRC patient. BRAF in PDX model CR0029 V600E Mutations were confirmed by both RNA sequencing and exome sequencing. Mouse skin was cleaned with appropriate surgical scrub and iodine on the right flank. Tumor fragments (2-3 mm in diameter) taken from the PDX model were subcutaneously transplanted into the right flank of female Balb / c nude mice using an 18g trocar needle.

[0258] The animals' health and tumor growth were monitored daily. Tumor volume was measured twice a week using a caliper if the tumor was palpable and measurable. The average tumor volume was 141 mm². 3 (110~176mm 3When the tumor reached a certain level, mice with tumors were randomized into seven different groups, each containing eight mice. The randomization day is denoted as treatment day 0.

[0259] treatment Treatment began on the day of randomization. The treatment start day is denoted as Treatment Day 0. Mice were orally administered vehicle control solution, compound I alone at 10 mg / kg BID, and encorafenib alone at 90 mg / kg QD. Another group received combination treatment, with compound I administered at 10 mg / kg BID and encorafenib administered at 90 mg / kg QD. The administration volume of each compound was 5 mL / kg, and the interval between BID regimens was 8 hours. In the combination group, encorafenib was administered 1 hour after administration of compound I. This study concluded on Treatment Day 28 as defined in the study protocol.

[0260] result Figure 11 shows BRAF V600E The graphs show tumor volume over the treatment period in the mutant CRC PDX model CR0029, with treatment using compound I alone, encorafenib alone, and a combination of compound I and encorafenib. No significant weight changes were observed in either the control or treatment group.

[0261] conclusion As shown in Figure 11, the combination of the compound of formula I and encorafenib produces BRAF V600E In the mutant CRC PDX model CR0029, superior tumor growth inhibition was demonstrated compared to treatment with formula I compound alone or encorafenib alone.

[0262] Example 7-BRAF V600E Combination therapy material of compound I and encorafenib in mutant CRC PDX model CR004 Acetic acid in 100 mM water was prepared for the vehicle / control, with the pH adjusted to 4.8-5.0. This was then stored under ambient conditions for 28 days during administration to mice.

[0263] Each week, a fresh sample of the compound of formula I was prepared in a 100 mM acetate buffer vehicle and stored under ambient conditions. Each week, the concomitant agent encorafenib was prepared in a 0.5% CMC and 0.5% Tween 80 vehicle and stored at 2–8°C.

[0264] Female Balb / c nude mice were purchased from SPF (Beijing) Laboratory Animal Technology Co., Ltd. The mice were 9-11 weeks old at the time of transplantation. The mice were housed in a special pathogen-free (SPF) environment and allowed to acclimate to the new environment for at least 3 days before the start of each experiment, according to the IACUC protocol.

[0265] All procedures related to the handling, care, and treatment of animals in this study were carried out in accordance with guidelines approved by the Animal Care Committee (IACUC) of Crown Bioscience (Beijing, China). During the study, animal care and use were carried out in accordance with the regulations of the International Association for Accreditation and Evaluation of Laboratory Animal Care (AAALAC). In addition, all parts of this study conducted at Crown Bioscience (Beijing, China) adhered to research protocols that were made and approved by the principal investigator and applicable standard operating procedures (SOPs).

[0266] Preparation of PDX The CR0004 PDX model was established for preclinical efficacy studies in CrownBio. This PDX model was derived from a 73-year-old Chinese male CRC patient. BRAF in PDX model CR0004 V600E Mutations were confirmed by both RNA sequencing and exome sequencing. Mouse skin was cleaned with appropriate surgical scrub and iodine on the right flank. Tumor fragments (2-3 mm in diameter) taken from the PDX model were subcutaneously transplanted into the right flank of female Balb / c nude mice using an 18g trocar needle. The average tumor volume was 141 mm². 3 (121~180mm 3When the tumor reached a certain level, the mice with tumors were randomly divided into six test groups, each containing eight mice.

[0267] treatment Treatment began on the day of randomization. The treatment start day is denoted as Treatment Day 0. Mice were orally administered vehicle control solution, compound I alone at 10 mg / kg BID, and encorafenib alone at 90 mg / kg QD. Another group received combination treatment, with compound I administered at 10 mg / kg BID and encorafenib administered at 90 mg / kg QD. The administration volume of each compound was 5 mL / kg, and the interval between BID regimens was 8 hours. In the combination group, encorafenib was administered 1 hour after administration of compound I. This study concluded on Treatment Day 28 as defined in the study protocol.

[0268] result Figure 12 shows BRAF V600E The graphs show tumor volume over the treatment period in the mutant CRC PDX model CR004, with treatment using compound I alone, encorafenib alone, and a combination of compound I and encorafenib. No significant weight changes were observed in either the control or treatment group.

[0269] conclusion As shown in Figure 12, the combination of the compound of formula I and encorafenib produces BRAF V600E In the mutant CRC PDX model CR004, superior inhibition of tumor growth was demonstrated compared to treatment with formula I compound alone or encorafenib alone.

[0270] Example 8-BRAF V600E Combination therapy of compound I and encorafenib in the mutant CRC CDX model WiDr material Acetic acid in 100 mM water was prepared for the vehicle / control, with the pH adjusted to 4.8-5.0. This was then stored under ambient conditions for 28 days during administration to mice.

[0271] Each week, a fresh sample of the compound of formula I was prepared in a 100 mM acetate buffer vehicle and stored under ambient conditions. Each week, the concomitant agent encorafenib was prepared in a 0.5% CMC and 0.5% Tween 80 vehicle and stored at 2–8°C.

[0272] Female Balb / c nude mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. The mice were 6-8 weeks old at the time of transplantation. The mice were housed in a specially sourced pathogen-free (SPF) environment and allowed to acclimate to the new environment for at least 3 days before the start of any experiment, according to the IACUC protocol.

[0273] All procedures related to the handling, care, and treatment of animals in this study were carried out in accordance with guidelines approved by the WuXiAppTec Animal Care Committee (IACUC). During the study, animal care and use were carried out in accordance with the regulations of the International Association for Accreditation and Evaluation of Laboratory Animal Care (AAALAC). In addition, all parts of this study conducted at WuXiAppTec adhered to the research protocol made and approved by the principal investigator and applicable standard operating procedures (SOPs).

[0274] Preparation of xenograft models WiDr is BRAF V600E The cell line was a human CRC tumor cell line with a mutation. The WiDr cell line was purchased from the European Collection of Authenticated Cell Cultures (EC ACC, 85111501). WiDr cells were cultured at 37°C in an atmosphere of 5% CO2 in air in a medium containing EMEM (EBSS) + 10% fetal bovine serum (FBS), 2 mM glutamine, and supplemented with 1% non-essential amino acids (NEAA). The medium was changed every 2-3 days, and the tumor cells were routinely subcultured at 80-90% confluence using trypsin-EDTA. Cells growing in the exponential growth phase were collected and counted for inoculation.

[0275] WiDr tumor cells were subcutaneously transplanted into mice. A 200 μL cell suspension containing 5 × 10⁶ tumor cells was subcutaneously transplanted into the right flank of the mice using a syringe. The animals' health and tumor growth were monitored daily. Tumor volume was measured twice weekly using a caliper if the tumor was palpable and measurable. The average tumor volume was 189 mm². 3 (139~240mm 3 When the tumor reached a certain level, mice with tumors were randomized into different groups of 8 mice each. The randomization day is denoted as treatment day 0.

[0276] treatment Treatment began on the day of randomization. The treatment start day is denoted as Treatment Day 0. Mice were orally administered vehicle control solution, compound I alone at 10 mg / kg BID, compound I alone at 30 mg / kg QD, and encorafenib alone at 90 mg / kg QD. Two additional groups received combination treatment with compound I and encorafenib. The first group received compound I at 10 mg / kg BID and encorafenib at 90 mg / kg QD, while the second group received compound I at 30 mg / kg QD and encorafenib at 90 mg / kg QD. The administration volume of compound I and encorafenib was 5 mL / kg, and the interval between BID regimens was 8 hours. In the combination groups, encorafenib was administered 1 hour after the QD dose of compound I. This study was concluded on day 28 of treatment, as defined in the study protocol.

[0277] result Figure 13 shows BRAF V600E The graphs show tumor volume over the treatment period in the mutant CRC CDX model WiDr, with treatment using compound I alone, encorafenib alone, and a combination of compound I and encorafenib. No significant weight changes were observed in either the control or treatment group.

[0278] conclusion As shown in Figure 13, the combination of the compound of formula I and encorafenib produces BRAFV600E In the mutant CRC CDX model WiDr, superior inhibition of tumor growth was demonstrated compared to treatment with formula I compound alone or encorafenib alone.

[0279] Example 9 - BRAF V600E Combination therapy of compound I and encorafenib in the mutant CRC CDX model HT-29 material Acetic acid in 100 mM water was prepared for the vehicle / control, with the pH adjusted to 4.8-5.0. This was then stored under ambient conditions for 28 days during administration to mice.

[0280] Each week, the test substance of formula I was freshly prepared in a 100 mM acetate buffer vehicle and stored under ambient conditions. Each week, the concomitant agent encorafenib was freshly prepared in a 0.5% CMC and 0.5% Tween 80 vehicle and stored at 2–8°C.

[0281] Female Balb / c nude mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. The mice were housed in a specially sourced pathogen-free (SPF) environment and allowed to acclimate to the new environment for at least three days before the start of each experiment. The mice were 6–8 weeks old at the time of transplantation.

[0282] All procedures related to the handling, care, and treatment of animals in this study were carried out in accordance with protocols and guidelines approved by GenenDesign's Animal Care Committee (IACUC). The animal facilities and programs operated under the standards of the Guidelines for the Management and Use of Laboratory Animals (NRC, 2011) and were accredited by the International Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC). Specifically, all parts of this study conducted at GenenDesign adhered to research protocols reviewed and approved by the IACUC and applicable standard operating procedures (SOPs).

[0283] Preparation of xenograft models HT-29 is BRAF V600E The HT-29 cell line was a mutant human CRC tumor cell line. The HT-29 cell line was purchased from the American Type Culture Collection (ATCC® CRL-2577®). HT-29 cells were cultured in McCoy's 5a medium + 10% fetal bovine serum (FBS) at 37°C under a 5% CO2 atmosphere in air. The medium was changed every 2-3 days, and the tumor cells were routinely subcultured at 80-90% confluence with trypsin-EDTA. Cells growing in the exponential growth phase were collected and counted for inoculation.

[0284] HT-29 tumor cells were subcutaneously transplanted into mice. 2 × 10⁶ cells were mixed with 50% Matrigel. 6 A 200 μL cell suspension containing tumor cells was subcutaneously transplanted into the right flank of mice using a syringe. The animals' health and tumor growth were monitored daily. Tumor volume was measured twice weekly using a caliper if the tumor was palpable and measurable. The average tumor volume was 200 mm³. 3 (146~259mm 3 When the tumor reached a certain level, mice with tumors were randomized into different groups of 8 mice each. The randomization day is denoted as treatment day 0.

[0285] treatment Treatment began on the day of randomization. The treatment start day is denoted as Treatment Day 0. Mice were orally administered vehicle control solution, compound I alone at 10 mg / kg BID, compound I alone at 30 mg / kg QD, and encorafenib alone at 90 mg / kg QD. Two additional groups received combination treatment with compound I and encorafenib. The first group received compound I at 10 mg / kg BID and encorafenib at 90 mg / kg QD, while the second group received compound I at 30 mg / kg QD and encorafenib at 90 mg / kg QD. The administration volume of each compound was 5 mL / kg, and the interval between BID regimens was 8 hours. In the combination groups, encorafenib was administered 1 hour after administration of compound I. This study was concluded on day 28 of treatment, as defined in the study protocol.

[0286] result Figure 14 shows BRAF V600E The graphs show tumor volume over the treatment period in the mutant CRC CDX model HT-29, treated with formula I compound alone, encorafenib alone, and a combination of formula I compound and encorafenib. No significant weight changes were observed in either the control or treatment group.

[0287] conclusion As shown in Figure 14, the combination of the compound of formula I and encorafenib produces BRAF V600E In the mutant CRC CDX model HT-29, superior tumor growth inhibition was demonstrated compared to treatment with formula I compound alone or encorafenib alone.

[0288] Example 10-BRAF V600E Combination therapy of compound I and encorafenib in the variant thyroid cancer CDX model BHT-101 material Acetic acid in 100 mM water was prepared for the vehicle / control, with the pH adjusted to 4.8-5.0. This was then stored under ambient conditions for 20 days during administration to mice.

[0289] Each week, a fresh sample of the compound of formula I was prepared in a vehicle of 100 mM acetate buffer and stored under ambient conditions. Each week, the concomitant agent encorafenib was prepared in a vehicle of 0.5% CMC and 0.5% Tween 80 and stored at 2–8°C.

[0290] Female Balb / c nude mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. The mice were housed in a specially sourced pathogen-free (SPF) environment and allowed to acclimate to the new environment for at least three days before the start of each experiment. The mice were 6–8 weeks old at the time of transplantation.

[0291] All procedures related to the handling, care, and treatment of animals in this study were carried out in accordance with protocols and guidelines approved by GenenDesign's Animal Care Committee (IACUC). The animal facilities and programs operated under the standards of the Guidelines for the Management and Use of Laboratory Animals (NRC, 2011) and were accredited by the International Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC). Specifically, all parts of this study conducted at GenenDesign adhered to research protocols reviewed and approved by the IACUC and applicable standard operating procedures (SOPs).

[0292] Preparation of xenograft models BHT-101 is BRAF V600EThe cell line was a mutant human thyroid cancer cell line. The BHT-101 cell line was purchased from the Cell Bank of Chinese Academy of Sciences (originally from DSMZ-German Collection of Microorganisms and Cell Cultures GmbH). BHT-101 cells were cultured at 37°C in a 5% CO2 atmosphere in air in DMEM medium containing 20% ​​fetal bovine serum (FBS) and supplemented with 1×Glutamax solution and 1 mM sodium pyruvate. The medium was changed every 2-3 days, and tumor cells were routinely subcultured at 80-90% confluence using trypsin-EDTA. Cells growing in the exponential growth phase were collected and counted for inoculation.

[0293] Huh-7101 tumor cells were subcutaneously transplanted into mice. 2 × 10⁶ cells were mixed with 50% Matrigel. 6 A 200 μL cell suspension containing tumor cells was subcutaneously transplanted into the right flank of mice using a syringe. The animals' health and tumor growth were monitored daily. Tumor volume was measured twice weekly using a caliper if the tumor was palpable and measurable. The average tumor volume was 190 mm². 3 (146~258mm 3 When the tumor reached a certain level, mice with tumors were randomized into different groups of 8 mice each. The randomization day is denoted as treatment day 0.

[0294] treatment Treatment began on the day of randomization. The treatment start day is denoted as Treatment Day 0. Mice were orally administered vehicle control solution, compound I alone at 10 mg / kg BID, compound I alone at 30 mg / kg QD, and encorafenib alone at 90 mg / kg QD. Two additional groups received combination treatment with compound I and encorafenib. The first group received compound I at 10 mg / kg BID and encorafenib at 90 mg / kg QD, while the second group received compound I at 30 mg / kg QD and encorafenib at 90 mg / kg QD. The administration volume of each compound was 5 mL / kg, and the interval between BID regimens was 8 hours. In the combination groups, encorafenib was administered 1 hour after administration of compound I. The study was terminated on day 20 of treatment, earlier than the original termination date defined in the study protocol due to rapid tumor growth. Half of the tumors in the vehicle control group reached the tumor volume threshold (2,000 mm) according to the IACUC protocol on day 20 of treatment. 3 It exceeded ).

[0295] result Figure 15 shows BRAF V600E The graphs show tumor volume over the treatment period in the mutant thyroid cancer CDX model BHT-101, with treatment using compound I alone, encorafenib alone, and a combination of compound I and encorafenib. No significant weight changes were observed in either the control or treatment group.

[0296] conclusion As shown in Figure 15, the combination of the compound of formula I and encorafenib produces BRAF V600E In the mutant thyroid cancer CDX model BHT-101, superior tumor growth inhibition was demonstrated compared to treatment with formula I compound alone or encorafenib alone.

[0297] Example 11-BRAF V600E Combination therapy of compound I and encorafenib in the mutant CRC CDX model RKO material Acetic acid in 100 mM water was prepared for the vehicle / control, with the pH adjusted to 4.8-5.0. This was then stored under ambient conditions for 16 days during administration to mice.

[0298] Each week, the test substance of formula I was freshly prepared in a 100 mM acetate buffer vehicle and stored under ambient conditions. Each week, the concomitant agent encorafenib was freshly prepared in a 0.5% CMC and 0.5% Tween 80 vehicle and stored at 2–8°C.

[0299] Female Balb / c nude mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. The mice were housed in a specially sourced pathogen-free (SPF) environment and allowed to acclimate to the new environment for at least three days before the start of each experiment. The mice were 6–8 weeks old at the time of transplantation.

[0300] All procedures related to the handling, care, and treatment of animals in this study were carried out in accordance with protocols and guidelines approved by GenenDesign's Animal Care Committee (IACUC). The animal facilities and programs operated under the standards of the Guidelines for the Management and Use of Laboratory Animals (NRC, 2011) and were accredited by the International Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC). Specifically, all parts of this study conducted at GenenDesign adhered to research protocols reviewed and approved by the IACUC and applicable standard operating procedures (SOPs).

[0301] Preparation of xenograft models RKO is BRAF V600EThe RKO cell line was a mutant human CRC tumor cell line. The RKO cell line was purchased from the American Cell Culture and Cell Line Preservation Service (ATCC® CRL-2577®). RKO cells were cultured at 37°C in an atmosphere of 5% CO2 in air in a medium containing MEM + 10% fetal bovine serum (FBS) supplemented with non-essential amino acids. The medium was changed every 2-3 days, and the tumor cells were routinely subcultured at 80-90% confluence using trypsin-EDTA. Cells growing in the exponential growth phase were collected and counted for inoculation.

[0302] RKO tumor cells were subcutaneously transplanted into mice. 2 × 10⁶ cells were mixed with 50% Matrigel. 6 A 200 μL cell suspension containing tumor cells was subcutaneously transplanted into the right flank of mice using a syringe. The animals' health and tumor growth were monitored daily. Tumor volume was measured twice weekly using a caliper if the tumor was palpable and measurable. The average tumor volume was 217 mm². 3 (163~262mm 3 When the tumor reached a certain level, mice with tumors were randomized into different groups of 8 mice each. The randomization day is denoted as treatment day 0.

[0303] treatment Treatment began on the day of randomization. The treatment start day is denoted as Treatment Day 0. Mice were orally administered vehicle control solution, compound I alone at 10 mg / kg BID, compound I alone at 30 mg / kg QD, and encorafenib alone at 90 mg / kg QD. Two additional groups received combination treatment with compound I: the first group received compound I at 10 mg / kg BID and encorafenib at 90 mg / kg QD, and the second group received compound I at 30 mg / kg QD and encorafenib at 90 mg / kg QD. The administration volume of each compound was 5 mL / kg, and the interval between BID regimens was 8 hours. In the combination groups, encorafenib was administered 1 hour after the administration of compound I QD. The study was terminated on day 16 of treatment, earlier than the original termination date defined in the study protocol due to rapid tumor growth. The majority of tumors in the vehicle control group reached the tumor volume threshold (2,000 mm) according to the IACUC protocol on day 16 of treatment. 3 It exceeded ).

[0304] result Figure 16 shows BRAF V600E The graphs show tumor volume over the treatment period in the mutant CRC CDX model RKO, with treatment using compound I alone, encorafenib alone, and a combination of compound I and encorafenib. No significant weight changes were observed in either the control or treatment group.

[0305] conclusion As shown in Figure 16, the combination of the compound of formula I and encorafenib produces BRAF V600E In the mutant CRC CDX model RKO, superior tumor growth inhibition was demonstrated compared to treatment with formula I compound alone or encorafenib alone.

[0306] Example 12 - Synergistic combination of compound I and MEK inhibitor This embodiment demonstrates a synergistic combination of the compound of formula I and an inhibitor of MEK.

[0307] Cell proliferation assay Cells (2000 cells per well) were seeded in 100 μl of cell culture medium on a 96-well plate and treated with compound I alone or with compound I and a constant concentration of trametinib or binimetinib. On day 5, 50 μl of CellTiter-Glo (CTG) reagent (Promega) was added, and the plate was incubated for 10 minutes with gentle shaking. After 10 minutes of incubation, the luminescence signal was determined according to the manufacturer's instructions (Promega), and the graph was plotted using PrismGraphPad.

[0308] Combined cell proliferation assay Cells (2000 cells per well) were seeded in 100 μl of cell culture medium on a 96-well plate. Cells were treated with the compound of formula I and trametinib or binimetinib at concentrations ranging from 0 to 10 μM using the combination matrix protocol of the Tecan D300e Digital Dispenser. On day 5, 50 μl of CellTiter-Glo (CTG) reagent (Promega) was added, and the plate was incubated for 10 minutes with gentle shaking. After 10 minutes of incubation, the luminescence signal was determined according to the manufacturer's instructions (Promega), and combination data was generated using the standard HSA model with Combinenefit software. Synergistic effects of combinations are represented by positive numbers in the results table. Negative numbers represent antagonistic effects of combinations.

[0309] Western blotting on pERK and ERK NCI-H508 cells were treated with a compound for 4 hours. After treatment, the cells were lysed on ice for 10 minutes using Thermo Fisher RIPA lysis buffer containing protease and phosphatase inhibitors. The cells were centrifuged using a microcentrifuge at 4°C for 10 minutes. The supernatant was transferred to a pre-cooled microcentrifuge tube, and the protein concentration of the lysate was measured using the BCA method. An equal volume of protein from the cell lysate supernatant was used for immunoblotting against pERK and ERK.

[0310] MeWo cells were treated with a compound for 4 hours. After treatment, the cells were lysed on ice for 10 minutes using Thermo Fisher RIPA lysis buffer containing protease and phosphatase inhibitors. The cells were centrifuged using a microcentrifuge at 4°C for 10 minutes. The supernatant was transferred to a pre-cooled microcentrifuge tube, and the protein concentration of the lysate was measured using the BCA method. An equal volume of protein from the cell lysate supernatant was used for immunoblotting against pERK and ERK.

[0311] result Figure 17A shows synergistic effect data in the NCI-H508 cancer cell line using the combination of compound I and trametinib. Figure 17B shows synergistic effect data in the NCI-H508 cancer cell line using the combination of compound I and binimetinib. Figure 17C shows graph and table data of synergistic effect in the NCI-H1666 cancer cell line using the combination of compound I and trametinib. Figure 17D shows synergistic effect data in the NCI-H1666 cancer cell line using the combination of compound I and binimetinib.

[0312] Figure 18A shows synergistic effect data in the MeWo cancer cell line using the compound of formula I in combination with trametinib. Figure 18B shows synergistic effect data in the MeWo cancer cell line using the compound of formula I in combination with binimetinib. Figure 18C shows synergistic effect data in the NCI-H1838 cancer cell line using the compound of formula I in combination with trametinib. Figure 18D shows synergistic effect data in the NCI-H1838 cancer cell line using the compound of formula I in combination with binimetinib.

[0313] Figure 19A shows plots of activity percentage versus inhibitor concentration (logM) in NCI-H508 cells treated with compound I alone and in combination with binimetinib. The table shows the IC50 data for NCI-H508 cells treated with compound I alone and in combination with binimetinib. Figure 19B shows plots of activity percentage versus inhibitor concentration (logM) in MeWo cells treated with compound I alone and in combination with binimetinib. The IC50 data in the table are for MeWo cells treated with compound I alone and in combination with binimetinib.

[0314] Figure 20A shows a Western blot gel demonstrating synergistic inhibition of ERK1 / 2 phosphorylation in the NCI-H508 cancer cell line. Figure 20B shows a quantitative bar graph of the Western blot in Figure 20A. Figure 20C shows a Western blot gel demonstrating synergistic inhibition of ERK1 / 2 phosphorylation in the MeWo(NF1 LoF) cancer cell line. Figure 20D shows a quantitative bar graph of the Western blot in Figure 20C.

[0315] Figure 21A shows synergistic effect data in the NCI-H2009 (KRAS G12A) cancer cell line using the combination of compound I and trametinib. Figure 2IB shows synergistic effect data in the LS513 (KRAS G12D) cancer cell line using the combination of compound I and trametinib. Figure 21C shows synergistic effect data in the A549 (KRAS G12S) cancer cell line using the combination of compound I and trametinib. Figure 21D shows synergistic effect data in the NCI-H727 (KRAS G12V) cancer cell line using the combination of compound I and trametinib.

[0316] Figure 22A shows synergistic effect data in the NCI-H2009 (KRAS G12AA) cancer cell line using the combination of compound I and binimetinib. Figure 22B shows synergistic effect data in the LS513 (KRAS G12D) cancer cell line using the combination of compound I and binimetinib. Figure 22C shows synergistic effect data in the A549 (KRAS G12S) cancer cell line using the combination of compound I and binimetinib. Figure 22D shows synergistic effect data in the NCI-H727 (KRAS G12V) cancer cell line using the combination of compound I and binimetinib.

[0317] Figure 23A shows a plot of activity percentage versus inhibitor concentration (logM) in LS513 (KRAS G12D) cells treated with the compound of formula I alone and in combination with trametinib. Figure 23B shows a plot of activity percentage versus inhibitor concentration (logM) in NCI-H2009 (KRAS G12D) cells treated with the compound of formula I alone and in combination with trametinib. The table shows data for NCI-H508 cells treated with the compound of formula I alone and in combination with trametinib. Figure 23C shows a bar graph of CTG activity percentage, showing that formula I alone or trametinib alone have the least effect on cell viability. In summary, these data indicate that the combination of the compound of formula I and a MEK inhibitor provides synergistic inhibition of cancer cell viability in BRAF class III, NF1 LoF, and KRAS G12X mutant cancers.

[0318] Example 13 - Combination therapy of compound I and trametinib in the NF1 LoF mutant melanoma CDX model MeWo

[0319] material For the vehicle / control, 100 mM acetic acid in deionized water was prepared by adjusting the pH to 4.8-5.0 and stored under ambient conditions for 28 days of administration in mice. Weekly, the test substance of the compound of formula I was freshly prepared in a 100 mM acetate buffer vehicle and stored under ambient conditions. Weekly, the concomitant agent trametinib was prepared in a 0.5% HPMC and 0.2% Tween 80 vehicle and stored under ambient conditions.

[0320] Female Balb / c nude mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. The mice were 6-8 weeks old at the time of transplantation. The mice were housed in a specially sourced pathogen-free (SPF) environment and allowed to acclimate to the new environment for at least 3 days before the start of any experiment, according to the IACUC protocol.

[0321] All procedures related to the handling, care, and treatment of animals in this study were carried out in accordance with guidelines approved by the WuXiAppTec Animal Care Committee (IACUC). During the study, animal care and use were carried out in accordance with the regulations of the International Association for Accreditation and Evaluation of Laboratory Animal Care (AAALAC). In addition, all parts of this study conducted at WuXiAppTec adhered to the research protocol made and approved by the principal investigator and applicable standard operating procedures (SOPs).

[0322] Preparation of xenograft models MeWo was a human melanoma cell line carrying the NFl Q1336* mutation. The MeWo cell line was purchased from the American Cell Culture and Cell Line Preservation Service (ATCC® HTB-65®). MeWo cells were cultured in Minimum Essential Media (MEM) containing 10% fetal bovine serum (FBS) and 1% non-essential amino acids (NEAA) at 37°C under a 5% CO2 atmosphere in air. The medium was changed every 2-3 days, and tumor cells were routinely subcultured at 80-90% confluence using trypsin-EDTA. Cells growing in the exponential growth phase were collected and counted for inoculation.

[0323] MeWo tumor cells were subcutaneously transplanted into mice. 5 × 10⁶ cells were mixed with 50% Matrigel. 6 A 200 μL cell suspension containing tumor cells was subcutaneously transplanted into the right flank of mice using a syringe. The animals' health and tumor growth were monitored daily. Tumor volume was measured twice weekly using a caliper if the tumor was palpable and measurable. The average tumor volume was 191 mm². 3 (150~242mm 3 When the tumor reached a certain level, mice with tumors were randomized into different groups of 8 mice each. The randomization day is denoted as treatment day 0.

[0324] treatment Treatment was initiated on a day after randomization. The treatment initiation day is denoted as Treatment Day 1. Mice were orally administered vehicle control solution, compound I alone at 10 mg / kg / dose BID, compound I alone at 30 mg / kg QD, and trametinib at 0.4 mg / kg QD. Two groups received combination treatment with compound I and trametinib: one group received compound I at 10 mg / kg / dose BID and trametinib at mg / kg QD, and the other group received compound I at 30 mg / kg QD and trametinib at 10 mg / kg QD. Both combination groups received trametinib at 0.4 mg / kg QD. The administration volume was 5 mL / kg, and the interval between BID regimens was 8 hours. In the combination groups, trametinib was administered 1 hour after the BID or QD administration schedule of compound I.

[0325] result Figure 24 shows graphs of tumor volume over treatment periods in the NF1 LoF mutation melanoma CDX model MeWo, with treatment using compound I alone, trametinib alone, and a combination of compound I and trametinib. No significant weight changes were observed in the control or treatment groups.

[0326] conclusion As shown in Figure 24, the combination of compound I and trametinib demonstrated superior tumor growth inhibition in the NF1 LoF mutant melanoma CDX model MeWo compared to treatment with compound I alone or trametinib alone.

[0327] Example 14 - Combination therapy of compound I and binimetinib in the NF1 LoF mutant melanoma CDX model MeWo material Acetic acid in 100 mM water was prepared for the vehicle / control, with the pH adjusted to 4.8-5.0. This was then stored under ambient conditions for 28 days during administration to mice.

[0328] Test samples of the compound of formula I were freshly prepared weekly in a vehicle of 100 mM acetate buffer and stored under ambient conditions. The combination agent, vinimetinib, was freshly prepared weekly in a vehicle of 1.0% MC and 0.5% Tween 80 and stored at 2–8°C.

[0329] Female Balb / c nude mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. The mice were 6-8 weeks old at the time of transplantation. The mice were housed in a specially sourced pathogen-free (SPF) environment and allowed to acclimate to the new environment for at least 3 days before the start of any experiment, according to the IACUC protocol.

[0330] All procedures related to the handling, care, and treatment of animals in this study were carried out in accordance with guidelines approved by GenenDesign's Animal Care Committee (IACUC). During the study, animal care and use were carried out in accordance with the regulations of the International Association for Accreditation and Evaluation of Laboratory Animal Care (AAALAC). In addition, all parts of this study conducted at GenenDesign adhered to the research protocol made and approved by the principal investigator and applicable standard operating procedures (SOPs).

[0331] Preparation of xenograft models MeWo was a human melanoma cell line carrying the NFl Q1336* mutation. The MeWo cell line was purchased from the American Cell Culture and Cell Line Preservation Service (ATCC® HTB-65®). MeWo cells were cultured in Minimum Essential Media (MEM) containing 10% fetal bovine serum (FBS) and 1% non-essential amino acids (NEAA) at 37°C under a 5% CO2 atmosphere in air. The medium was changed every 2-3 days, and tumor cells were routinely subcultured at 80-90% confluence using trypsin-EDTA. Cells growing in the exponential growth phase were collected and counted for inoculation.

[0332] MeWo tumor cells were subcutaneously transplanted into mice. Briefly, 5 × 10⁶ cells were mixed with 50% Matrigel. 6 A 200 μL cell suspension containing tumor cells was subcutaneously transplanted into the right flank of mice using a syringe. The animals' health and tumor growth were monitored daily. Tumor volume was measured twice weekly using a caliper if the tumor was palpable and measurable. When the tumor volume reached an average of 195 mm³ (ranging from 141 to 267 mm³), the mice with tumors were randomized into different groups of 8 mice each. The randomization day is denoted as treatment day 0.

[0333] treatment Treatment was initiated on a day following randomization. The treatment initiation day is denoted as Treatment Day 1. Mice were orally administered vehicle control solution, compound I alone at 15 mg / kg QD, compound I alone at 30 mg / kg QD, binimetinib alone at 6 mg / kg QD, and binimetinib alone at 9 mg / kg / BID. Two additional groups received combination treatment with compound I and binimetinib: one group received compound I at 15 mg / kg QD and binimetinib at 6 mg / kg BID, and the other group received compound I at 15 mg / kg QD and binimetinib at 9 mg / kg / BID. The administration volume was 5 mL / kg, and the interval between BID regimens was 8 hours. In the combination groups, binimetinib was administered 1 hour after the administration of compound I QD. This study was concluded on day 28 of treatment, as defined in the study protocol.

[0334] result Figure 25 shows graphs of tumor volume over time in the NF1 LoF mutation melanoma CDX model MeWo, with treatment of compound I alone, binimetinib alone, and a combination of compound I and binimetinib. No significant weight changes were observed in the control or treatment groups.

[0335] conclusion As shown in Figure 25, the combination of compound I and binimetinib demonstrated superior tumor growth inhibition in the NF1 LoF mutant melanoma CDX model MeWo compared to treatment with compound I alone or binimetinib alone.

[0336] Example 15 - Combination therapy of compound I and trametinib in BRAFV class III mutant CRC CDX model NCI-H508 material Acetic acid in 100 mM water was prepared for the vehicle / control, with the pH adjusted to 4.8-5.0. This was then stored under ambient conditions for 28 days during administration to mice.

[0337] Each week, a fresh sample of the compound of formula I was prepared in a 100 mM acetate buffer vehicle and stored under ambient conditions. Each week, the concomitant agent trametinib was prepared in a 0.5% HPMC and 0.2% Tween 80 vehicle and stored under ambient conditions.

[0338] Female Balb / c nude mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. The mice were 6-8 weeks old at the time of transplantation. The mice were housed in a specially sourced pathogen-free (SPF) environment and allowed to acclimate to the new environment for at least 3 days before the start of any experiment, according to the IACUC protocol.

[0339] All procedures related to the handling, care, and treatment of animals in this study were carried out in accordance with guidelines approved by the WuXiAppTec Animal Care Committee (IACUC). During the study, animal care and use were carried out in accordance with the regulations of the International Association for Accreditation and Evaluation of Laboratory Animal Care (AAALAC). In addition, all parts of this study conducted at WuXiAppTec adhered to the research protocol made and approved by the principal investigator and applicable standard operating procedures (SOPs).

[0340] Preparation of xenograft models NCLH508 was a human CRC cell line carrying the BRAF class III mutation (BRAF G596R). The NCI-H508 cell line was purchased from the American Type Culture Collection (ATCC® CCL-253®). NCI-H508 cells were cultured at 37°C in a medium containing RPMI-1640 + 10% fetal bovine serum (FBS) and 1% antibiotic-antifungal agent (AA) under a 5% CO2 atmosphere in air. The medium was changed every 2-3 days, and tumor cells were routinely subcultured at 80-90% confluence with trypsin-EDTA. Cells growing in the exponential growth phase were collected and counted for inoculation.

[0341] NCLH508 tumor cells were subcutaneously injected into mice. Briefly, 5 × 10⁶ cells were mixed with 50% Matrigel. 6 A 200 μL cell suspension containing tumor cells was subcutaneously transplanted into the right flank of mice using a syringe. The animals' health and tumor growth were monitored daily. Tumor volume was measured twice weekly using a caliper if the tumor was palpable and measurable. When the tumor volume reached an average of 182 mm³ (ranging from 108 to 287 mm³), the tumor-bearing mice were randomized into different groups of 8 mice each. The randomization day is denoted as treatment day 0.

[0342] treatment Treatment was initiated on a day following randomization. The treatment initiation day was denoted as Treatment Day 1. Mice were orally administered vehicle control solution, compound I alone at 10 mg / kg BID, compound I alone at 30 mg / kg QD, and trametinib alone at 0.4 mg / kg QD. Two groups received combination treatment with compound I and trametinib: one group received compound I at 10 mg / kg BID and trametinib at 0.4 mg / kg QD, and the other group received compound I at 0.4 mg / kg QD and trametinib at 30 mg / kg QD. The administration volume was 5 mL / kg, and the interval between BID regimens was 8 hours. In the combination group, trametinib was administered 1 hour after the BID or QD administration schedule of the compound.

[0343] result Figure 26 shows graphs of tumor volume over treatment periods in the BRAF class III mutant CRC CDX model NCI-H508, treated with compound I alone, trametinib alone, and a combination of compound I and trametinib. No significant weight changes were observed in either the control or treatment groups.

[0344] conclusion As shown in Figure 26, the combination of the compound of formula I with trametinib demonstrated superior tumor growth inhibition in the BRAF class III mutant CRC CDX model NCI-H508 compared to treatment with the compound of formula I alone or with trametinib alone.

[0345] Example 16 - Combination therapy of compound I and trametinib in NF1 LoF mutant NSCLC CDX model NCI-H1838 material Acetic acid in 100 mM water was prepared for the vehicle / control, with the pH adjusted to 4.8-5.0. This was then stored under ambient conditions for 28 days during administration to mice.

[0346] Each week, a fresh sample of the compound of formula I was prepared in a 100 mM acetate buffer vehicle and stored under ambient conditions. Each week, the concomitant agent trametinib was prepared in a 0.5% HPMC and 0.2% Tween 80 vehicle and stored under ambient conditions.

[0347] Female SCID Beige mice (Cat#405) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. The mice were 6-8 weeks old at the time of transplantation. The mice were housed in a specially sourced pathogen-free (SPF) environment and allowed to acclimate to the new environment for at least 3 days before the start of each experiment, according to the IACUC protocol.

[0348] All procedures related to the handling, care, and treatment of animals in this study were carried out in accordance with guidelines approved by the WuXiAppTec Animal Care Committee (IACUC). During the study, animal care and use were carried out in accordance with the regulations of the International Association for Accreditation and Evaluation of Laboratory Animal Care (AAALAC). In addition, all parts of this study conducted at WuXiAppTec adhered to the research protocol approved by the principal investigator and applicable standard operating procedures (SOPs).

[0349] Preparation of xenograft models NCLH1838 was a human lung adenocarcinoma cell line carrying the NF1 LOF mutation (NF1 N184fs). The NCI-H1838 cell line was purchased from the American Cell Culture and Cell Line Preservation Service (ATCC® CRL-5899®). NCI-H1838 cells were cultured at 37°C in a 5% CO2 atmosphere in air in a medium containing RPMI-1640 + 10% fetal bovine serum (FBS) and 1% antibiotic-antifungal agent (AA). The medium was changed every 2-3 days, and tumor cells were routinely subcultured at 80-90% confluence with trypsin-EDTA. Cells growing in the exponential growth phase were collected and counted for inoculation.

[0350] NCI-H1838 tumor cells were subcutaneously transplanted into mice. Briefly, 5 × 10⁶ cells were mixed with 50% Matrigel. 6 A 200 μL cell suspension containing tumor cells was subcutaneously transplanted into the right flank of mice using a syringe. The animals' health and tumor growth were monitored daily. Tumor volume was measured twice weekly using a caliper if the tumor was palpable and measurable. When the tumor volume reached an average of 254 mm³ (ranging from 149 to 503 mm³), the mice with tumors were randomized into different groups of 8 mice each. The randomization day is denoted as treatment day 0.

[0351] treatment Treatment was initiated on a day following randomization. The treatment initiation day was denoted as Treatment Day 1. Mice were orally administered vehicle control solution, compound I alone at 10 mg / kg BID, compound I alone at 30 mg / kg QD, and trametinib alone at 0.4 mg / kg QD. Two groups received combination treatment with compound I and trametinib: one group received compound I at 10 mg / kg BID and trametinib at 0.4 mg / kg QD, and the other group received compound I at 30 mg / kg QD and trametinib at 0.4 mg / kg QD. The administration volume was 5 mL / kg, and the interval between BID regimens was 8 hours. In the combination group, trametinib was administered 1 hour after the administration of compound I or the QD administration schedule.

[0352] result Figure 27 shows graphs of tumor volume over treatment periods in the NF1 LoF mutant NSCLC CDX model NCI-H1838, with treatment using compound I alone, trametinib alone, and a combination of compound I and trametinib. No significant changes in body weight were observed in either the control or treatment groups.

[0353] conclusion As shown in Figure 27, the combination of the compound of formula I with trametinib demonstrated superior inhibition of tumor growth in the NF1 LoF mutant NSCLC CDX model NCI-H1838 compared to treatment with the compound of formula I alone or with trametinib alone.

[0354] Example 17 - Synergistic combination of compound I and MET inhibitor Combined cell proliferation assay Cells (2000 cells per well) were seeded in 100 μl of cell culture medium on a 96-well plate. Using the Tecan D300e Digital Dispenser's combination matrix protocol, cells were treated with crizotinib and compound I at concentrations ranging from 0 to 10 μM. On day 5, 50 μl of CellTiter-Glo (CTG) reagent (Promega) was added, and the plate was incubated for 10 minutes with gentle shaking. After 10 minutes of incubation, the luminescence signal was determined according to the provider's instructions (Promega), and combination data was generated using the standard HSA model with Combinefit software. Synergistic effects of combinations are represented by positive numbers in the results table. Negative numbers represent antagonistic effects of combinations.

[0355] result Figure 28A shows synergistic effect data in the Hs746T cancer cell line using the combination of compound I and crizotinib. Figure 28B shows synergistic effect data in the MKN-45 cancer cell line using the combination of compound I and crizotinib. Figure 28C shows synergistic effect data in the EBC-1 cancer cell line using the combination of compound I and crizotinib.

[0356] Example 18 - Combination therapy of compound I and crizotinib in cMET-amplified gastric cancer CDX model SNU-5 material Acetic acid in 100 mM water was prepared for the vehicle / control, with the pH adjusted to 4.8-5.0. This was then stored under ambient conditions for 28 days during administration to mice.

[0357] Each week, a fresh sample of the compound of formula I was prepared in a 100 mM acetate buffer vehicle and stored under ambient conditions. The concomitant agent, crizotinib, was prepared in a 0.5% methylcellulose vehicle and stored at 2–8°C.

[0358] Female Balb / c nude mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. The mice were 6-8 weeks old at the time of transplantation. The mice were housed in a specially sourced pathogen-free (SPF) environment and allowed to acclimate to their new environment for at least 3 days before the start of any experiment, according to the IACUC protocol. All procedures related to the handling, care, and treatment of animals in this study were carried out in accordance with guidelines approved by the WuXiAppTec Animal Experimentation Committee (IACUC). During the study, animal care and use were carried out in accordance with the regulations of the International Association for Accreditation and Evaluation of Laboratory Animal Care (AAALAC). In addition, all parts of this study conducted at WuXiAppTec adhered to the research protocol made and approved by the principal investigator and applicable standard operating procedures (SOPs).

[0359] Preparation of xenograft models SNU-5 was a c-MET amplified gastric cancer cell line. The SNU-5 cell line was purchased from the American Cell Culture and Cell Line Preservation Service (ATCC® CRL-SNU-5®). SNU-5 cells were cultured at 37°C in an atmosphere of 5% CO2 in air in a medium containing IMDM (Iscove's Dulbecco modified medium) + 20% fetal bovine serum (FBS) and 1% antibiotic-antifungal agent (AA). The medium was changed every 2-3 days, and tumor cells were routinely passaged at 80-90% confluence with trypsin-EDTA. Cells growing in the exponential growth phase were collected and counted for inoculation. SNU-5 tumor cells (passage 13) were subcutaneously transplanted into mice. 10 × 10 6 A 200 μL cell suspension containing individual tumor cells was subcutaneously transplanted into the right flank of mice using a syringe. The animals' health and tumor growth were monitored daily. Tumor volume was measured twice weekly using a caliper if the tumor was palpable and measurable. 34 days after subcutaneous transplantation, the average tumor volume was 227 mm³. 3 When the condition was met, mice with tumors were randomized into different groups, each containing eight mice. The randomization day is denoted as treatment day 0.

[0360] treatment Treatment was initiated on a day following randomization. The treatment initiation day was denoted as Treatment Day 1. Mice were orally administered vehicle control solution, compound I alone at 10 mg / kg BID, compound I alone at 30 mg / kg QD, and crizotinib alone at 50 mg / kg BID. Two additional groups received combination treatment with compound I and crizotinib: one group received compound I at 5 mg / kg BID and crizotinib at 50 mg / kg BID, and the other group received compound I at 15 mg / kg QD and crizotinib at 50 mg / kg BID. The administration volume was 5 mL / kg, and the interval between BID regimens was 8 hours. In the combination groups, crizotinib was administered 1 hour after the QD administration of compound I, or 1 hour after the first dose of the BID administration schedule. This study was concluded on day 28 of treatment, as defined in the study protocol.

[0361] result Figure 29 shows graphs of tumor volume over the treatment period for the compound of formula I alone, crizotinib alone, and a combination of the compound of formula I and crizotinib in the c-MET amplified gastric cancer CDX model SNU-5. No significant changes in body weight were observed in the control group or the treatment group.

[0362] conclusion As shown in Figure 29, the combination of compound I and crizotinib demonstrated superior tumor growth inhibition in the c-MET-amplified gastric cancer CDX model SNU-5 compared to treatment with compound I alone or crizotinib alone.

[0363] Example 19-c - Combination therapy of compound I and crizotinib in the MET-amplified NSCLC CDX model NCI-H1993 material Acetic acid in 100 mM water was prepared for the vehicle / control, with the pH adjusted to 4.8-5.0. This was then stored under ambient conditions for 28 days during administration to mice.

[0364] Each week, a fresh sample of the compound of formula I was prepared in a 100 mM acetate buffer vehicle and stored under ambient conditions. The concomitant agent, crizotinib, was prepared in a 0.5% methylcellulose vehicle and stored at 2–8°C.

[0365] Female Balb / c nude mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. The mice were 6-8 weeks old at the time of transplantation. The mice were housed in a specially sourced pathogen-free (SPF) environment and allowed to acclimate to their new environment for at least 3 days before the start of any experiment, according to the IACUC protocol. All procedures related to the handling, care, and treatment of animals in this study were carried out in accordance with guidelines approved by the WuXiAppTec Animal Experimentation Committee (IACUC). During the study, animal care and use were carried out in accordance with the regulations of the International Association for Accreditation and Evaluation of Laboratory Animal Care (AAALAC). In addition, all parts of this study conducted at WuXiAppTec adhered to the research protocol made and approved by the principal investigator and applicable standard operating procedures (SOPs).

[0366] Preparation of xenograft models NCI-H1993 was a c-Affi-amplified NSCLC cell line. The NCI-H1993 cell line was purchased from the American Cell Culture Lineage Preservation Service (ATCC® CRL-5909®). NCI-H1993 cells were cultured at 37°C in a 5% CO2 atmosphere in air in a medium containing RPMI-1640 + 10% fetal bovine serum (FBS) and 1% antibiotic-antifungal agent (AA). The medium was changed every 2-3 days, and tumor cells were routinely passaged with trypsin-EDTA at 80-90% confluence. Cells growing in the exponential growth phase were collected and counted for inoculation. NCI-H1993 tumor cells (passage 13) were subcutaneously injected into mice. 5 × 10⁶ cells mixed with 50% Matrigel were administered. 6 A 200 μL cell suspension containing tumor cells was subcutaneously transplanted into the right flank of mice using a syringe. The animals' health and tumor growth were monitored daily. Tumor volume was measured twice weekly using a caliper if the tumor was palpable and measurable. Ten days after subcutaneous transplantation, the average tumor volume was 201 mm³. 3 When the condition was met, mice with tumors were randomized into different groups, each containing eight mice. The randomization day is denoted as treatment day 0.

[0367] treatment Treatment began on a day after randomization. The treatment start day is denoted as Treatment Day 1. Mice were orally administered vehicle control solution, compound I alone at 10 mg / kg BID, compound I alone at 30 mg / kg QD, and crizotinib at 50 mg / kg BID. Another group received combination treatment, with compound I at 5 mg / kg BID and crizotinib at 50 mg / kg BID. The administration volume was 5 mL / kg, and the interval between BID regimens was 8 hours. In the combination group, crizotinib was administered 1 hour after the first BID dose of compound I. This study was completed on Treatment Day 28 as defined in the study protocol.

[0368] result Figure 30 shows graphs of tumor volume over treatment periods in the c-MET amplified NSCLC CDX model NCI-H1993, with treatment using compound I alone, crizotinib alone, and a combination of compound I and crizotinib. No significant weight changes were observed in either the control or treatment groups.

[0369] conclusion As shown in Figure 30, the combination of compound I and crizotinib demonstrated superior tumor growth inhibition in the c-MET-amplified NSCLC CDX model NCI-H1993 compared to treatment with compound I alone or crizotinib alone.

[0370] The embodiments described above are explained in some detail by examples and embodiments for the purpose of clarity of understanding, but those skilled in the art will understand that certain changes and modifications may be made within the scope of the appended claims. Furthermore, each reference provided herein is incorporated by reference to the same extent as each reference would be incorporated by reference individually. In the event of any conflict between this application and the references provided herein, this application shall prevail.

Claims

1. A composition for use in a method of treating a subject having cancer, the composition comprising a compound of formula I: 【Chemistry 1】 or a pharma- ceutically acceptable salt thereof, wherein the method comprises administering said composition in combination with a MEK inhibitor.

2. The composition described in claim 1, wherein the MEK inhibitor selectively inhibits MEK1, selectively inhibits MEK2, or selectively inhibits both MEK1 and MEK2.

3. The composition of claim 1, wherein the cancer is metastatic.

4. The composition described in claim 1, wherein the cancer is colon cancer.

5. The composition of claim 1, wherein the cancer is melanoma.

6. The composition described in claim 1, wherein the cancer is lung cancer.

7. The composition described in claim 1, wherein the cancer is pancreatic cancer.

8. The composition described in claim 1, wherein the cancer is breast cancer.

9. The composition of claim 1, wherein the cancer is pancreatic ductal adenocarcinoma (PDAC).

10. The composition of claim 1, wherein the MEK inhibitor is selected from the group consisting of trametinib, cobimetinib, binimetinib, PD-0325901, selumetinib, and C1-1040.

11. The composition described in claim 1, wherein the MEK inhibitor is trametinib.

12. The composition of claim 1, wherein the method comprises administering a further MAPK pathway inhibitor.

13. The composition described in claim 1, wherein the administration is oral.

14. The composition of claim 1, wherein the dosage of the compound of formula I is in the range of 20 mg daily to 400 mg daily.

15. The composition of claim 1, wherein the dosage of the MEK inhibitor ranges from 1 mg daily to 500 mg daily.

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