MEK immune oncology inhibitors and therapeutic uses thereof

Abstract

Methods of treating cancers with a certain dual-MEK inhibitor (a pharmaceutically acceptable salt thereof) with a short half-life are provided, comprising the administration of a therapeutically effective amount of the dual-MEK inhibitor alone or in combination with another therapeutic agent. In some embodiments, the short half-life dual-MEK inhibitor is orally administered twice daily (BID) to treat a RAS-mutated or RAF-mutated form of cancer.

Description

MEK IMMUNE ONCOLOGY INHIBITORS AND THERAPEUTIC USES THEREOFRELATED APPLICATIONS

[0001] This patent application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 511,789, filed July 3, 2023; U.S. Provisional Patent Application No. 63 / 511,790, filed July 3, 2023; U.S. Provisional Patent Application No. 63 / 589,778, filed October 12, 2023; PCT / US2023 / 079156, filed November 8, 2023; and U.S. Provisional Patent Application No. 63 / 644,898, filed May 9, 2024; each of which is incorporated by reference in its entirety.TECHNICAL FIELD

[0002] The present disclosure relates to the fields of chemistry and medicine. More particularly, the present disclosure relates to methods of treatment of certain forms of cancer with MEK inhibitor therapies.BACKGROUND

[0003] Activating mutations of RAS in the MAPK pathway are observed in approximately 30% of all cancer patients, while inappropriate activation of the MAPK pathway is observed in up to 50% of all tumors and represents one of the most highly utilized signaling pathways in cancer. In aggressive solid tumors of the pancreas, skin, lungs, and colon, mutations in RAS and / or RAF are even more common. For example, approximately 40% of lung cancers and approximately 95% of pancreatic cancers are due to RAS and / or RAF mutations, and approximately 50% of melanomas, 40% of thyroid and 30% of ovarian tumors display activation mutations, mainly, in BRAF but also in ARAF and CRAF (RAFI). To date, mitogen-activated extracellular signal-regulated kinase (MEK) inhibitors approved by the Food and Drug Administration (FDA) have been ineffective at treating RAS mutant tumors likely attributable to well-known mechanisms of resistance, including C-isomer of rapidly accelerated fibrosarcoma (CRAF)-mediated MEK (re)activation, or CRAF-bypass.

[0004] First-generation MEK inhibitors were designed to prevent phosphorylation of ERK (i.e., a surrogate for activation) by MEK through therapeutic engagement of an allosteric pocket adjacent to the ATP binding site, but first-generation compounds fail tocontrol MEK phosphorylation at the RAF activation domain (e.g., Serine 218 and Serine 222 on MEK1), which is the initial step of MEK activation. Following treatment of RAS mutant cells, an increased accumulation of cytoplasmic pMEK contributes, in part, to the dogma that MEK inhibitors must sustain chronic target occupancy in order to inhibit the MAPK pathway effectively. However, chronic MEK inhibition leads to narrow therapeutic windows, significant class-effect toxicities and acquired or adaptive resistance of tumor cells. All of the first generation MEK inhibitors are designed for prolonged systemic circulation, either through longer half-life (e.g., up to 2 to 4 days), or moderate half-life (e.g., 3 to 6 hours) combined with increased dosing frequency. In either case, the prolonged systemic circulation contributes to high rates of adverse events by harming healthy normal cells, which also rely on the MAPK pathway. While significant advances have been made to better understand the underlying biology of the MAPK pathway, drugs designed to counteract these perturbations in the signaling pathway continue to be hindered by excess drug-related toxicity, short-lived durability of clinical response, and limited numbers of biomarker positive patients for mutation- specific drugs.

[0005] There remains an unmet medical need for methods of treating certain forms of cancer activated through the MAPK pathway, including methods of treatment to cyclically disrupt the ERK and MEK with a MEK inhibiting compound having a shorter half-life than prior “first generation” MEK inhibitor compounds such as trametinib, cobimetinib, binimetinib, and selumetinib.SUMMARY OF THE DISCLOSURE

[0006] In some embodiments, methods provided herein comprise the use of and / or a method of treatment comprising administering Compound B or a pharmaceutically acceptable salt thereof to a subject in need thereof,Compound B

[0007] Compound B is a dual-MEK inhibitor useful for the treatment of certain forms of cancer. In some embodiments, a method of treating a patient with a RAS-mutatcd or RAF-mutated cancer, comprises administering a therapeutically effective amount of Compound B to a patient in need thereof. In some embodiments, a method of treating a patient with a RAS-mutated or RAF-mutated cancer, comprises orally (p.o.) administering a therapeutically effective amount of Compound B to a patient in need thereof. In some embodiments, a method of treating a patient with a RAS-mutated or RAF mutated cancer, comprises orally (p.o.) administering a therapeutically effective amount of Compound B twice per day (BID) to a patient in need thereof.

[0008] In some embodiments, a method of treating a patient diagnosed with a cancer, comprising administering a compound having a structure of Compound B:Compound B, or a pharmaceutically acceptable salt thereof, twice per day (BID) to the patient in need thereof at a total daily dose of 40 mg - 640 mg, to treat the cancer. In some embodiments, the cancer is pancreatic adenocarcinoma, or a RAS- or RAF-mutated solid tumor. In some embodiments, the cancer is pancreatic adenocarcinoma, RAS-mutated cutaneous melanoma, RAF-mutated cutaneous melanoma, RAS-mutated NSCEC, a RAS-mutated GI solid tumor other than colorectal cancer, or a RAF-mutated solid tumor. In some embodiments, the cancer is pancreatic adenocarcinoma optionally comprising a RAS- or RAF- mutation. In some embodiments, the cancer is a KRAS mutated pancreatic adenocarcinoma. In some embodiments, the cancer is a RAS-mutated cutaneous melanoma, or a RAF-mutated cutaneous melanoma. In some embodiments, the cancer is a NRAS mutated melanoma. In some embodiments, the cancer is a BRAF mutated melanoma. In some embodiments, the cancer is RAS-mutated NSCFC. In some embodiments, the cancer is a KRAS-mutated NSCFC. In some embodiments, the cancer is a RAS-mutated GI solid tumor other than colorectal cancer. In some embodiments, the cancer is a RAF-mutated solid tumor.In some embodiments, 120 mg or 160 mg of Compound B is administered twice per day (BID) to the patient in need thereof, as Compound B of a pharmaceutically acceptable salt thereof. In some embodiments, 120 mg of Compound B is orally administered twice per day (BID) to the patient in need thereof, as Compound B of a pharmaceutically acceptable salt thereof. In some embodiments, 160 mg of Compound B is orally administered twice per day (BID) to the patient in need thereof, as Compound B of a pharmaceutically acceptable salt thereof.In some embodiments, the cancer has a mutation selected from the group consisting of: (a) a KRAS mutation selected from the group consisting of G12C, G12S, G12R, G12F, G12L, G12N, G12A, G12D, G12V, G13C, G13S, G13D, G13V, GBP, S17G, P34S, A59E, A59G, A59T, Q61K, Q61L, Q61R, and Q61H; (b) a NRAS mutation selected from the group consisting of: Q61R, Q61L, Q61K, Q61V; and (c) a BRAF mutation selected from the group consisting of: V600D, V600E, V600K, V600R.BRIEF DESCRIPTION OF THE DRAWINGS

[0009] Figure 1 illustrates graphs depicting compound B with a short plasma PK half-life in vivo.

[0010] Figure 2 illustrates graphs depicting compound B with a short plasma PK half-life in vivo.

[0011] Figure 3 illustrates graphs depicting compound B with a short plasma and tumor PK half-life in vivo.

[0012] Figure 4A-1 is a graph of data showing the efficacy of monotherapy of Compound B on the tumor growth rate of subcutaneous (SC) pre-established A375 Human malignant melanoma model for each respective treatment doses (90 - 180 mg / kg p.o. BID).

[0013] Figure 4A-2 is a graph of data showing the efficacy of monotherapy of Compound B on the tumor growth rate of subcutaneous (SC) pre-established A375 Human malignant melanoma model for each respective treatment doses (90 - 180 mg / kg p.o. BID).

[0014] Figure 4B is a graph of data showing the efficacy of monotherapy of binimetinib on the tumor growth rate of SC pre-established A375 Human malignant melanoma model for the treatment dose (3.5 mg / kg p.o. BID).

[0015] Figure 4C is a graph of data showing the efficacy of monotherapy of cncorafcnib on the tumor growth rate of SC pre-established A375 Human malignant melanoma model for the treatment dose (60 mg / kg p.o. BID).

[0016] Figure 4D is a graph of data showing the tolerability of monotherapy of Compound B as the percent body weight change for each respective treatment doses of SC pre- established A375 Human malignant melanoma model (90 - 180 mg / kg p.o. BID).

[0017] Figure 4E is a graph of data showing the tolerability of monotherapy of binimetinib as the percent body weight change for SC pre-established A375 Human malignant melanoma model for the treatment dose (3.5 mg / kg p.o. BID).

[0018] Figure 4F is a graph of data showing the tolerability of monotherapy of encorafenib as the percent body weight change for SC pre-established A375 Human malignant melanoma model for the treatment dose (60 mg / kg p.o. BID).

[0019] Figure 5A-1 is a graph of data, showing the anti-tumor effect of a combination of binimetinib at 3.5 mg / kg BID + encorafenib at 60 mg / kg on the tumor growth rate of a SC pre-established A375 human malignant melanoma model.

[0020] Figure 5A-2 is a graph of data showing the anti-tumor effect of a combination of binimetinib at 3.5 mg / kg BID + encorafenib at 60 mg / kg on the tumor growth rate of a SC pre-established A375 human malignant melanoma model.

[0021] Figure 5B-1 is a graph of data, showing the tolerability of a combination of binimetinib at 3.5 mg / kg BID + encorafenib at 60 mg / kg on the percent body weight change from a SC pre-established A375 human malignant melanoma model.

[0022] Figure 5C is a graph of data showing the anti-tumor effect of a combination of Compound B (90- 180 BID PO) and encorafenib at 60 mg / kg QD PO on the tumor growth rate of a SC pre-established A375 human malignant melanoma model.

[0023] Figure 6A-1 is a graph of data showing the efficacy of monotherapies of Compound B on the tumor growth rate of SC pre-established HT-29 Human colorectal cancer model for each respective treatment doses (90 - 180 mg / kg p.o. BID).

[0024] Figure 6A-2 is a graph of data showing the efficacy of monotherapies of Compound B on the tumor growth rate of SC pre-established HT-29 Human colorectal cancer model for each respective treatment doses (90 - 180 mg / kg p.o. BID).

[0025] Figure 6B is a graph of data showing the tolerability of monotherapy of Compound B as the percent body weight change for each respective treatment doses of SC pre- established HT-29 Human colorectal cancer model (90 to 180 mg / kg p.o. BID).

[0026] Figure 6C is a graph of data showing the efficacy of monotherapies of Binimetinib on the tumor growth rate of SC pre-established HT-29 Human colorectal cancer model for each respective treatment doses (3.5 mg / kg, p.o., BID).

[0027] Figure 6D is a graph of data showing the tolerability of monotherapy of Binimetinib as the percent body weight change for each respective treatment doses of SC pre- established HT-29 Human colorectal cancer model (3.5 mg / kg, p.o., BID).

[0028] Figure 6E is a graph of data showing the efficacy of monotherapies of Encorafenib on the tumor growth rate of SC pre-established HT-29 Human colorectal cancer model for each respective treatment doses (60 mg / kg, p.o, QD).

[0029] Figure 6F is a graph of data showing the tolerability of monotherapy of Encorafenib as the percent body weight change for each respective treatment doses of SC pre- established HT-29 Human colorectal cancer model (60 mg / kg, p.o, QD).

[0030] Figure 7 A illustrates a graph depicting MEKi, a-PD- 1 , a-CTLA-4 alone and combinations in CT-26 syngeneic mouse tumor model.

[0031] Figure 7B illustrates a graph depicting MEKi, a-PD- 1 , a-CTLA-4 alone and combinations in CT-26 syngeneic mouse tumor model.

[0032] Figure 8A illustrates graphs depicting Compound B, encorafenib alone and combinations in A-375 xenograft melanoma human tumor mouse model.

[0033] Figure 8B illustrates graphs depicting binimetinib, encorafenib alone and combinations in A-375 xenograft melanoma human tumor mouse model.

[0034] Figure 8C illustrates graphs depicting Compound B, binimetinib, and encorafenib alone and in various combinations in A-375 xenograft melanoma human tumor mouse model.

[0035] Figure 8D illustrates graphs depicting administration of a combination of Compound B and encorafenib after a period of dosing a binimetinib and encorafenib in A-375 xenograft melanoma human tumor mouse model.

[0036] Figure 9A-1 illustrates graphs depicting Compound B, encorafenib alone and combinations in the HT-29 xenograft colon human tumor mouse model.

[0037] Figure 9A-2 illustrates graphs depicting Compound B, encorafenib alone and combinations in the HT-29 xenograft colon human tumor mouse model.

[0038] Figure 9B-1 illustrates graphs depicting binimetinib, encorafenib alone and combinations in the HT-29 xenograft colon human tumor mouse model.

[0039] Figure 9B-2 illustrates graphs depicting binimetinib, encorafenib alone and combinations in the HT-29 xenograft colon human tumor mouse model.

[0040] Figure 9C illustrates graphs depicting tolerability of Compound B, binimetinib, encorafenib alone and combinations in the HT-29 xenograft colon human tumor mouse model.

[0041] Figure 9D illustrates graphs depicting tolerability Compound B, binimetinib, encorafenib alone and combinations in the HT-29 xenograft colon human tumor mouse model.

[0042] Figure 10A is a graph showing Efficacy of Compound B and encorafenib in A375 BRAFV600EHuman Melanoma Tumor Model.

[0043] Figure 1 OB is a graph showing Efficacy of binimetinib and encorafenib in A375 BRAFV600EHuman Melanoma Tumor Model.

[0044] Figure 10C is a graph showing tolerability by weight change with treatment with Compound B and encorafenib in A375 BRAFV600EHuman Melanoma Tumor Model.

[0045] Figure 10D is a graph showing tolerability by weight change with treatment with binimetinib and encorafenib in A375 BRAFV600EHuman Melanoma Tumor Model.

[0046] Figure 11A is a graph showing 3D-TGA Compound B dose responses calculated for sensitive tumor models.

[0047] Figure 11B is a graph showing 3D-TGA Compound B dose responses calculated for intermediate tumor models.

[0048] Figure 11C is a graph showing 3D-TGA Compound B dose responses calculated for resistant tumor models.

[0049] Figure 12A is a biomarker profile diagram of data from melanoma patients profiled in GENIE® database.

[0050] Figure 12B is a biomarker profile diagram of data from colorectal cancer patients profiled in GENIE® database.

[0051] Figure 12C is a biomarker profile diagram of data from thyroid cancer patients profiled in GENIE® database.

[0052] Figure 12D is a biomarker profile diagram of data from lung cancer patients profiled in GENIE® database.

[0053] Figure 13A is a graph showing humanized 3D-TGA of Compound B with encorafenib.

[0054] Figure 13B is a graph of the Zero Interaction Potency (ZIP) model synergy score for the combination of Compound B with encorafenib.

[0055] Figure 14A illustrates a graph of pERK:total ERK ratio (100 nM drug).

[0056] Figure 14B illustrates pMEK:total MEK ratio (100 nM drug).

[0057] Figure 15A is a graph of data showing the efficacy of monotherapy of Compound B on the tumor growth rate of SC pre-established Colon-26 mouse carcinoma model for each respective treatment doses (25 - 200 mg / kg p.o. BID).

[0058] Figure 15B is a graph of data showing the tolerability of monotherapy of Compound B as the percent body weight change for each respective treatment doses of SC pre- established Colon-26 mouse carcinoma model (25 - 200 mg / kg p.o. BID).

[0059] Figure 15C is a graph of data showing the efficacy of monotherapy of Compound B on the tumor growth rate of SC pre-established Colon-26 mouse carcinoma model for a dose of 150 mg / kg p.o. BID.

[0060] Figure 15D is a graph of data showing the efficacy of monotherapy of Compound B on the tumor growth rate of SC pre-established Colon-26 mouse carcinoma model for a dose of 175 mg / kg p.o. BID.

[0061] Figure 15E is a graph of data showing the efficacy of monotherapy of Compound B on the tumor growth rate of SC pre-established Colon-26 mouse carcinoma model for each respective treatment doses (25 - 200 mg / kg p.o. QD).

[0062] Figure 16A is a graph of data showing the efficacy of monotherapy of Compound B on the tumor growth rate of SC pre-established A549 human lung adenocarcinoma model for each respective treatment doses (60 - 180 mg / kg p.o. BID).

[0063] Figure 16B is a graph of data showing the tolerability of monotherapy of Compound B as the percent body weight change for each respective treatment doses SC pre- established A549 human lung adenocarcinoma model (60 - 180 mg / kg p.o. BID).

[0064] Figure 16C is a graph of data showing the efficacy of monotherapy of Compound B on the tumor growth rate of SC pre-established A549 human lung adenocarcinoma model for a dose of 150 mg / kg p.o. BID.

[0065] Figure 16D is a graph of data showing the efficacy of monotherapy of Compound B on the tumor growth rate of SC pre-established A549 human lung adenocarcinoma model for a dose of 180 mg / kg p.o. BID.

[0066] Figure 17A is a graph showing data obtained from an evaluation of Compound B as compared to vehicle using an A375 xenograft tumor model (tumor volume).

[0067] Figure 17B is a graph showing data obtained from an evaluation of Compound B as compared to vehicle using an HT-29 xenograft tumor model (tumor volume).

[0068] Figure 17C is a graph showing data obtained from an evaluation of Compound B as compared to vehicle using an A549 Xenograft Tumor Model (Tumor Volume).

[0069] Figure 17D is a graph showing data obtained from an evaluation of Compound B as compared to vehicle using a Colon-26 Xenograft Tumor Model (Tumor Volume).

[0070] Figure 17E is a graph showing results obtained from an evaluation of Compound B as compared to vehicle using a MIA PaCa-2 Xenograft Tumor Model (Tumor Volume).

[0071] Figure 17F is a graph showing data obtained from an evaluation of Compound B as compared to vehicle using a SK-MEL-2 xenograft tumor model (Tumor Volume).

[0072] Figure 18 is a graph showing the pharmacokinetics (PK) for Compound B in a human patient (Patient 1), at cycle 1, day 1 after administration of the initial 40 mg dose of Compound B administered orally (po). Patient 1 is diagnosed with pancreatic cancer having a KRAS G12V mutation and received 40 mg Compound B administered orally (po) twice daily (BID).

[0073] Figure 19A is a graph showing the pharmacokinetics (PK) for Compound B in a human patient (Patient 2), at cycle 1, day 1 after administration of the initial 40 mg dose of Compound B administered orally (po). Figure 19B is a graph showing PK7PD (pERK) for Compound B from patient 2 after receiving the initial 40 mg dose of Compound B administeredorally (po) twice daily (BID) (cycle 1 , day 15). Patient 2 is diagnosed with pancreatic cancer having a KRAS G12P mutation and received 40 mg Compound B administered orally (po) twice daily (BID).

[0074] Figure 20 is a graph showing the pharmacokinetics (PK) for Compound B in a human patient (Patient 4), at cycle 1, day 1 after administration of the initial 80 mg dose of Compound B administered orally (po). Patient 4 was diagnosed with colorectal cancer having a KRAS G12D mutation and received 80 mg Compound B administered orally (po) twice daily (BID).DETAILED DESCRIPTION

[0075] In some embodiments, methods provided herein include the use of and / or a method of treatment comprising administering Compound B or a pharmaceutically acceptable salt thereof to a subject in need thereof,Compound B.

[0076] In some embodiments, Compound B can be in free base form. The full chemical name of Compound B is 4-((dimethylamino)methyl)-3-(2-chloro-3-((N- methylsulfamoyl)amino)benzyl)-2-oxo-2H-chromen-7-yl dimethylcarbamate. Compound B is an off-white powder, with a molecular formula of C23H27CIN4O6S and a molecular weight of 523.00 g / mol. Compound B has a pKa of 9.1 and 8.9 (dimethylamine, acid [NH] moieties, respectively). The preparation of Compound B is described in Example 1.

[0077] Without being limited to any particular theory, Compound B was designed to resist MAPK pathway reactivation by reducing phosphorylation (activation) of MEK, thereby enhancing its ability to target RAS mutant cells, where CRAF-bypass is particularly active. Compound B’s unique pharmacokinetic profile allows it to disrupt the MAPK pathway in a deep but cyclic cadence. This deep cyclic inhibition is a novel strategy for targeting MEK, exploiting the fact that tumor cells with activating MAPK mutations are addicted to constant MAPK pathway signaling, while healthy cells do not. This approach aims to prevent tumorcell s from sustaining proliferative signaling, a hallmark of cancer, while improving overall tolerability by reducing the impact on healthy cells relative to chronic ablation that is foundational in first and second generation MEK inhibitors. Further, Compound B aims to exploit tumor addiction to MAPK signaling while sparing healthy cells that use, but are not dependent on, the same pathway.

[0078] In some embodiments, Compound B may act as a potent, non-adenosine 5'- triphosphate (ATP) competitive selective dual MEK inhibitor. Its biochemical inhibitory half- maximal inhibitory concentration (IC50) for activated ERK ranging from about 44.4 to about 153 nM across several cancer types. The most notable cell lines may include KRAS mutant lung adenocarcinoma and BRAF mutant colon carcinoma cancer cell lines. Corresponding reductions in pMEK were observed in RAS-mutant cells treated with about 100 nM Compound B, indicating that Compound B was not sensitive to the MAPK pathway feedback reactivation events, such as CRAF-bypass, which are known limitations of the first generation MEK inhibitors currently in clinical use. In vitro testing has shown that Compound B effectively inhibits cell proliferation, with IC50 values identifying 46.4% of the 28 cell lines tested in humanized three-dimensional (3D) cultures as sensitive (ICso < 3 pM), 21.4% as intermediate (IC50 range 3 to 10 pM), and 32.1% as resistant (ICso> 10 pM) to the inhibitory effects.

[0079] Compound B showed an immune-normalizing effect in vitro in two cell surface markers from 5 different human cancer cell lines with either KRAS, NRAS, or BRAF activating mutations. Compound B increased the levels of human leukocyte antigens A, B, and C (HLA-ABC) expression and decreased PD-L1 expression in a dose-dependent manner in all models tested, in both the presence and absence of interferon gamma (IFN-y).

[0080] Compound B has demonstrated in vitro and in vivo anti-tumor activity in the 3D tumor growth assay (TGA) and in mouse syngeneic and human xenograft models of activating MAPK pathway mutations including KRAS, NRAS, HRAS, BRAF, neurofibromatosis type 1 (NF1), and KSR. The minimal effective dose (MED) ranged from 60 to 180 mg / kg BID after oral administration, depending on the model. When combined with anti-PD-1 or anti-CTLA-4 checkpoint inhibitors at well-tolerated, sub-maximum tolerated dose (MTD) and sub-therapeutic dose levels, significant survival benefit was observed in immune-competent mouse tumor models.

[0081] Compound B has been evaluated in a wide range of human and murine solid tumor models, including those with activating MAPK pathway mutations in KRAS, NRAS, HRAS, BRAF, NF1, and KSR. Compound B inhibited the growth of RAF and RAS mutant tumors as a monotherapy across multiple human and murine solid tumor models. Compound B was shown to inhibit the growth of several pre-established subcutaneous (SC) syngeneic and human xenograft tumors in immunocompetent and immunodeficient mice. Tumor growth inhibition of over 90% was observed in the mouse syngeneic Colon-26 (KRASG12D) colon carcinoma allograft, as well as the human A549 (KRASG12S) lung adenocarcinoma xenograft after oral administration at 175 mg / kg and 150 mg / kg BID, respectively. Similar results were observed in the human A-375 (BRAFv600E) malignant melanoma xenograft in animals administered 180 mg / kg BID PO. Defining the MED as a dose / schedule of Compound B having a tumor growth inhibition (TGI) > 50%, the MED observed was a range from 60 mg / kg BID PO to 180 mg / kg BID PO, depending on the cell line used. Significant tumor reduction was observed at all doses and in all models.

[0082] In some embodiments, methods of treatment provide methods of administering a dual-MEK inhibitor having a high Cmax and a short plasma drug half-life at sequential doses that are administered at a dose interval (i.e., time between doses) that is greater than the plasma drug half-life. For example, in some embodiments, a method of treatment comprises administering a dual-MEK inhibitor on a dosing schedule providing a Cmax that achieves sufficiently high pharmacodynamic (PD) inhibition (e.g. 90% or greater) with sequential doses of the dual-MEK inhibitor being administered at intervals that are about 7 to about 18 times the duration of the plasma drug half-life.

[0083] In some embodiments, methods of treatment comprise the administration of a dual-MEK inhibitor to achieve deep cyclic inhibition of the MAPK pathway. Deep cyclic inhibition is a novel therapeutic approach for treatment of certain forms of cancer that aims to deprive tumor cells of the sustained proliferative signaling required for rapid growth, while sparing healthy cells through a cadenced, normalized level of signaling. In some embodiments, deep cyclic inhibition can be achieved by administering a dual-MEK inhibitor having sufficiently high pharmacodynamic inhibition of pERK (e.g., 90%+) and sufficiently short plasma half-life (e.g., 2 hours) at sequential dose intervals that are about seven or more timesthe plasma half-life (e.g., once or twice daily). In some embodiments, the dual-MEK inhibitor is Compound B, or a pharmaceutically acceptable salt thereof:Compound B.

[0084] In some embodiments, Compound B is a dual-MEK inhibitor useful for the treatment of solid tumors. In some embodiments, Compound B may be useful in treating solid tumors in patients with inappropriate activation of the mitogen-activated protein kinase (MAPK) pathway through mutations in the rat sarcoma (RAS) and / or rapidly accelerated fibrosarcoma (RAF) genes. In some embodiments, the solid tumors include melanoma, pancreatic, lung, thyroid and colorectal solid tumors.

[0085] In some embodiments, Compound B may achieve universal-MAPK activity with an accelerated twice-daily oral dosing cadence. In some embodiments, Compound B may also achieve deep cyclic inhibition of the MAPK pathway. In some embodiments, Compound B may act as an allosteric MEK inhibitor that drives deep cyclic inhibition of the MAPK pathway with a relatively shorter plasma half-life and accelerated pharmacokinetic cadence. In some embodiments, Compound B may act as an inhibitor of the growth of RAF and RAS mutant tumors as a monotherapy across multiple human and murine solid tumor models. In some embodiments, Compound B may target MEK in a way that disrupts the MAPK pathway at ERK through reduction of MEK activation. Compound B has a unique PK and pharmacodynamic, or PD, profile useful for treating distinct solid tumors, potentially including a broad range of MAPK-driven tumors as a monotherapy, as well as for normalizing the patient’s immune response and promoting maximal antitumor responses when administered in combination with select immune modulators.

[0086] In some embodiments, Compound B may engage MEK in a way that prioritizes disruption of MEK activation (lowering pMEK) itself, by preventing CRAF or other family members from activating MEK and subsequently limiting MEKs ability to phosphorylate or activate ERK (i.e., a so-called dual MEK mechanism). This approach affords Compound B the ability to prevent MAPK pathway reactivation at the level of RAF-MEKinstead of only MEK-ERK, like the first generation MEK inhibitors. Unlike first generation MEK inhibitors, cellular pools of pMEK do not expand after treatment with Compound B in RAS mutant cells. VS-6766 (CH5126766 or RO5126766), an investigational dual RAF / MEK inhibitor and Compound B are resistant to CRAF-mediated bypass of ERK suppression. VS- 6766 is representative of a class of drugs that could be considered second generation MEK inhibitors, as they share with first generation MEK inhibitors a longer plasma half-life (sustained drug trough levels with narrow therapeutic index).

[0087] Compound B is classified herein as a third-generation MEK inhibitor due to its dual MEK mechanism that prevents pathway reactivation in RAS mutant settings and its short drug half-life, which achieves nearly zero drug trough levels for deep, cyclic inhibition. Compound B exploits the allosteric pocket in MEK1 and MEK2 (MEK) to disrupt activation of MEK (pMEK) and its downstream targets, ERK1 and ERK2 (ERK). This contrasts with first generation MEK inhibitors, which engage the same allosteric pocket and disrupt the ability of MEK to activate ERK (pERK) but fail to regulate pMEK in certain settings. Second generation compounds with the dual MEK mechanism can better resist MAPK pathway activation events, including CRAF bypass, deeply impacting RAS or RAF mutant tumors by reducing cellular pools of both pMEK andpERK. However, second generation MEK inhibitors have faced the same tolerability challenges as first generation MEK inhibitors because they were all designed with long half-lives and dose schedules that drive prolonged systemic circulation. Compound B represents a third generation MEK inhibitor designed with both the ability to resist pathway reactivation and a short half-life which has negligible drug trough levels while dosing in 12h or 24h intervals. This pharmacokinetic (PK) profile was designed to potentially improve safety and tolerability by inhibiting MAPK signaling cyclically rather than ablating it chronically.

[0088] In primary pharmacodynamic assays, as described in the Example section below, Compound B inhibited pERK across multiple tumor cells harboring activating KRAS or BRAF mutations with half maximal inhibitory concentrations (IC50) values ranging from about44.4 nM to about 153.0 nM, with the most sensitive models being predominantly cancer cell lines that harbor activating MAPK pathway mutations. Corresponding reductions in pMEK were observed with treatment of cells with 100 nM Compound B, confirming that Compound B was not sensitive to the MAPK pathway feedback reactivation events in RASmutant models, such as CRAF-bypass. Compound B is a selective non-ATP competitive, allosteric inhibitor of MEK1 and MEK2 at doses up to 1 pM, with high affinity for MEK1.

[0089] Compound B was evaluated against vehicle in an aggressive murine colorectal tumor model (i.e., Colon-26), which expresses mutant KRASG12D. It was observed that Compound B demonstrated robust tumor growth inhibition at top effective doses of 150 and 175 mg / kg BID p.o., with multiple mice experiencing tumor regression during the first 7 days of dosing (as depicted below), with good tolerability, and evidenced limited (-8.61% to to +3.34% vs. vehicle group) changes in median BWL at doses in the range of 25 to 175 mg / kg BID p.o. Compound B displayed a plasma half-life in mice of only a 0.3 to 0.4 hours (0.5 to 0.7 in non-human primates).

[0090] In the early maximum tolerated dose, or MTD, studies, it was observed that oral administration of Compound B twice a day of up to 175 to 180 mg / kg / dose was well- tolerated in mice. In other preclinical studies, it was observed that the maximum therapeutic effect of Compound B was reached when administered orally twice a day between 150 and 180 mg / kg / dose.

[0091] In some embodiments, a RAF-mutated cancer refers to a type of cancer that includes a mutation in the RAF gene. In some embodiments, the RAF-mutated cancer includes a BRAF mutation. In some embodiments, the BRAF mutation includes a BRAF V600D mutation, BRAF V600E mutation, BRAF V600K mutation, or a combination thereof. In some embodiments, the RAF-mutated cancer includes, but is not limited to, colorectal, thyroid, nonsmall cell lung cancer, melanoma, pancreatic, lung, or a combination thereof. In some embodiments, the RAF-mutated cancer is a tumor.

[0092] In some embodiments, the dual-MEK inhibitor is Compound B administered to the patient once daily in a total dose amount of about 40 to about 640 mg to treat RAS or RAF mutated cancer. In some embodiments, the dual-MEK inhibitor is Compound B administered to the patient once daily in a total dose amount of 40 mg to treat RAS or RAF mutated cancer. In some embodiments, the dual-MEK inhibitor is Compound B administered to the patient once daily in a total dose amount of 60 mg to treat RAS or RAF mutated cancer. In some embodiments, the dual-MEK inhibitor is Compound B administered to the patient once daily in a total dose amount of 80 mg to treat RAS or RAF mutated cancer. In some embodiments, the dual-MEK inhibitor is Compound B administered to the patientonce daily in a total dose amount of 100 mg to treat RAS or RAF mutated cancer. In some embodiments, the dual-MEK inhibitor is Compound B administered to the patient once daily in a total dose amount of 120 mg to treat RAS or RAF mutated cancer. In some embodiments, the dual-MEK inhibitor is Compound B administered to the patient once daily in a total dose amount of 140 mg to treat RAS or RAF mutated cancer. In some embodiments, the dual-MEK inhibitor is Compound B administered to the patient once daily in a total dose amount of 160 mg to treat RAS or RAF mutated cancer. In some embodiments, the dual-MEK inhibitor is Compound B administered to the patient once daily in a total dose amount of 180 mg to treat RAS or RAF mutated cancer. In some embodiments, the dual-MEK inhibitor is Compound B administered to the patient once daily in a total dose amount of 200 mg to treat RAS or RAF mutated cancer. In some embodiments, the dual-MEK inhibitor is Compound B administered to the patient once daily in a total dose amount of 240 mg to treat RAS or RAF mutated cancer. In some embodiments, the dual-MEK inhibitor is Compound B administered to the patient once daily in a total dose amount of 260 mg to treat RAS or RAF mutated cancer. In some embodiments, the dual-MEK inhibitor is Compound B administered to the patient once daily in a total dose amount of 280 mg to treat RAS or RAF mutated cancer. In some embodiments, the dual-MEK inhibitor is Compound B administered to the patient once daily in a total dose amount of 300 mg to treat RAS or RAF mutated cancer. In some embodiments, the dual-MEK inhibitor is Compound B administered to the patient once daily in a total dose amount of 320 mg to treat RAS or RAF mutated cancer.

[0093] In some embodiments, the dual-MEK inhibitor is Compound B administered to the patient twice daily in a dose amount of 20 mg to treat RAS or RAF mutated cancer. In some embodiments, the dual-MEK inhibitor is Compound B administered to the patient twice daily in a dose amount of 40 mg to treat RAS or RAF mutated cancer. In some embodiments, the dual-MEK inhibitor is Compound B administered to the patient twice daily in a dose amount of 60 mg to treat RAS or RAF mutated cancer. In some embodiments, the dual-MEK inhibitor is Compound B administered to the patient twice daily in a dose amount of 80 mg to treat RAS or RAF mutated cancer. In some embodiments, the dual-MEK inhibitor is Compound B administered to the patient twice daily in a dose amount of 100 mg to treat RAS or RAF mutated cancer. In some embodiments, the dual-MEK inhibitor is Compound B administered to the patient twice daily in a dose amount of 120 mg to treat RAS or RAFmutated cancer. Tn some embodiments, the dual-MEK inhibitor is Compound B administered to the patient twice daily in a dose amount of 140 mg to treat RAS or RAF mutated cancer. In some embodiments, the dual-MEK inhibitor is Compound B administered to the patient twice daily in a dose amount of 160 mg to treat RAS or RAF mutated cancer. In some embodiments, the dual-MEK inhibitor is Compound B administered to the patient twice daily in a dose of about 20 mg, 40 mg, 50 mg, 60 mg, 70 mg, 80 mg, 90 mg, 100 mg, 110 mg 120 mg, 130 mg, 140 mg, 150 mg, 160 mg, 170 mg, 180 mg, 190 mg, 200 mg, 210 mg, 220 mg, 230 mg, 240 mg, 250 mg, 260 mg, 270 mg, 280 mg, 290 mg, 300 mg, 310 mg, 320 mg, or ranges including and / or spanning the aforementioned values.

[0094] In some embodiments, the dual-MEK inhibitor is Compound B administered to the patient once daily in a dose of about 20 mg, 40 mg, 50 mg, 60 mg, 70 mg, 80 mg, 90 mg, 100 mg, 110 mg 120 mg, 130 mg, 140 mg, 150 mg, 160 mg, 170 mg, 180 mg, 190 mg, 200 mg, 210 mg, 220 mg, 230 mg, 240 mg, 250 mg, 260 mg, 270 mg, 280 mg, 290 mg, 300 mg, 310 mg, 320 mg, or ranges including and / or spanning the aforementioned values. In some embodiments, the dual-MEK inhibitor is Compound B administered to the patient once daily in a total dose amount of about 40 to about 640 mg to treat RAS or RAF mutated cancers. In some embodiments, the dual-MEK inhibitor is Compound B administered to the patient once daily in a total dose amount of 40 mg to treat RAS or RAF mutated cancers. In some embodiments, the dual-MEK inhibitor is Compound B administered to the patient once daily in a total dose amount of 60 mg to treat RAS or RAF mutated cancers. In some embodiments, the dual-MEK inhibitor is Compound B administered to the patient once daily in a total dose amount of 80 mg to treat RAS or RAF mutated cancers. In some embodiments, the dual-MEK inhibitor is Compound B administered to the patient once daily in a total dose amount of 100 mg to treat RAS or RAF mutated cancers. In some embodiments, the dual- MEK inhibitor is Compound B administered to the patient once daily in a total dose amount of 120 mg to treat RAS or RAF mutated cancers. In some embodiments, the dual-MEK inhibitor is Compound B administered to the patient once daily in a total dose amount of 140 mg to treat RAS or RAF mutated cancers. In some embodiments, the dual-MEK inhibitor is Compound B administered to the patient once daily in a total dose amount of 160 mg to treat RAS or RAF mutated cancers. In some embodiments, the dual-MEK inhibitor is Compound B administered to the patient once daily in a total dose amount of 180 mg to treat RAS or RAFmutated cancers. In some embodiments, the dual-MEK inhibitor is Compound B administered to the patient once daily in a total dose amount of 200 mg to treat RAS or RAF mutated cancers. In some embodiments, the dual-MEK inhibitor is Compound B administered to the patient once daily in a total dose amount of 240 mg to treat RAS or RAF mutated cancers. In some embodiments, the dual-MEK inhibitor is Compound B administered to the patient once daily in a total dose amount of 260 mg to treat RAS or RAF mutated cancers. In some embodiments, the dual-MEK inhibitor is Compound B administered to the patient once daily in a total dose amount of 280 mg to treat RAS or RAF mutated cancers. In some embodiments, the dual- MEK inhibitor is Compound B administered to the patient once daily in a total dose amount of 300 mg to treat RAS or RAF mutated cancers. In some embodiments, the dual-MEK inhibitor is Compound B administered to the patient once daily in a total dose amount of 320 mg to treat RAS or RAF mutated cancers.Pharmaceutical Compositions

[0095] In another aspect, pharmaceutical compositions are disclosed; these compositions include an active pharmaceutical ingredient. In certain embodiments, the pharmaceutical composition includes Compound B as disclosed herein as the active pharmaceutical ingredient (API), and a pharmaceutically acceptable carrier comprising one or more excipients. In some embodiments, the pharmaceutical composition optionally further comprises an additional therapeutic compound (i.e., agent) with the pharmaceu tic ally acceptable carrier. The pharmaceutical composition can be a medicament.

[0096] In some embodiments, Compound B may be formulated as a pharmaceutical composition using a pharmaceutically acceptable excipients and administered by a variety of routes. In some embodiments, pharmaceutical composition may be formulated for oral administration. Such pharmaceutical compositions and processes for preparing them are well known in the art. (See, e.g., Remington: The Science and Practice of Pharmacy, L. V. Allen, Editor, 22nd Edition, Pharmaceutical Press, 2012).

[0097] Pharmaceutically acceptable carriers include those known in the art. The choice of a pharmaceutically acceptable carrier can depend, for example, on the desired route of administration of the composition. A pharmaceutical composition (preparation) can be administered to a subject by any of a number of routes of administration including, forexample, parenteral administration (e.g., intravenously, subcutaneously, or intramuscularly), oral administration (for example, tablets, and capsules); absorption through the oral mucosa (e.g., sublingually) or transdermally (for example as a patch applied to the skin) or topically (for example, as a cream, ointment or spray applied to the skin).

[0098] In certain embodiments, the present application is directed to a pharmaceutical composition comprising an active pharmaceutical ingredient. In certain embodiments, the pharmaceutical composition includes Compound B, as disclosed herein as the active pharmaceutical ingredient (API), and a pharmaceutically acceptable carrier comprising one or more excipients. In some embodiments, the pharmaceutical composition optionally further comprises an additional therapeutic compound (i.e., agent) with the pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition can be a medicament.

[0099] In some embodiments, pharmaceutical compositions comprising Compound B or pharmaceutically acceptable salts thereof can be formulated for oral administration. For example, a compound provided herein can be combined with suitable compendial excipients to form an oral unit dosage form, such as a capsule or tablet, containing a target dose of Compound B. The drug product can be prepared by first manufacturing Compound B as an active pharmaceutical ingredient (API), followed by roller compaction / milling with intragranular excipients and blending with extra granular excipients. A Drug Product can contain Compound B as the API and excipient components in a tablet in a desired dosage strength of Compound B. The blended material can be compressed to form tablets and then film coated. The excipients can be selected from materials appropriate for inclusion in a pharmaceutical composition for an intended purpose and route of delivery including providing a desired manufacturing and stability properties and / or desired in vivo characteristics or other properties to the pharmaceutical composition. In some embodiments, the pharmaceutical composition can include Compound B as the API in combination with a filler (e.g., a form of microcrystalline cellulose), a dry binder or disintegrant (e.g., a crosslinked polymer), a glidant (e.g., colloidal silicon dioxide) and / or a lubricant (e.g., magnesium stearate). In some embodiments, the pharmaceutical composition can comprise a material such as an extended release or disintegrant involved in carrying or transporting the API pharmaceutical agent from one organ, or portion of the body, to another organ, or portion ofthe body of a subject, including materials to desirable control the absorption of the API in the intestine.

[0100] The formulations may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy. The amount of active ingredient which can be combined with a carrier material to produce a single dosage form will vary depending upon the host being treated, the particular mode of administration. The amount of active ingredient that can be combined with a carrier material to produce a single dosage form will generally be that amount of the compound which produces a therapeutic effect. For use in the methods of this disclosure, active compounds can be given per se or as a pharmaceutical composition containing, for example, 0.1 to 99.5% (or, for example, 0.5 to 90%) of an active ingredient in combination with a pharmaceutically acceptable carrier.

[0101] Methods of preparing these formulations or compositions include the step of bringing into association an active compound, such as a compound of the disclosure, with the carrier and, optionally, one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association a compound of the present disclosure with liquid carriers, or finely divided solid carriers, or both, and then, if necessary, shaping the product.

[0102] To prepare solid dosage forms for oral administration, the active ingredient is mixed with one or more pharmaceutically acceptable carriers, such as sodium citrate or dicalcium phosphate, and / or any of the following: (1) fillers or extenders, (2) binders, (3) humectants, (4) disintegrating agents, (5) solution retarding agents, (6) absorption accelerators, (7) wetting agents, (8) absorbents, (9) lubricants, (10) complexing agents, and (11) coloring agents. In the case of capsules (including sprinkle capsules and gelatin capsules), tablets and pills, the pharmaceutical compositions may also comprise buffering agents. Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using suitable excipients. The pharmaceutical compositions according to the present disclosure may contain conventional pharmaceutical carriers and / or auxiliary agents. In some embodiments, he pharmaceutical compositions according to the present disclosure may contain conventional carrier agents including a binder, a lubricant and / or a glidant selected from those products and materials generally used in pharmaceutical industry for preparation of pharmaceutical compositions for an intended route of administration.

[0103] A tablet may be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets may be prepared using binder (for example, gelatin or hydroxypropylmethyl cellulose), lubricant, inert diluent, preservative, disintegrant (for example, sodium starch glycolate or cross-linked sodium carboxymethyl cellulose), surface-active or dispersing agent. Molded tablets may be made by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent.

[0104] In some embodiments, an oral dosage form of Compound B is provided, such as the oral dosage form disclosed herein. In some embodiments, an oral dosage form such as a tablet or capsule can include Compound B or a pharmaceutically acceptable salt thereof as the API and one or more excipients such as a dry binder, tablet disintegrant, an absorbent, filler or diluent, a lubricant, anti- adherent, binder, thickening agent, viscosityincreasing agent, coating agent and film former. In some embodiments, an oral dosage form such as a tablet or capsule can include about 20 mg to about 160 mg of Compound B or a pharmaceutically acceptable salt thereof as the API and one or more intra-granulation excipients such as a dry binder, tablet disintegrant, an absorbent, filler or diluent, a lubricant, anti-adherent, binder, thickening agent, viscosity-increasing agent, coating agent and film former. In some embodiments, an oral dosage form such as a tablet or capsule can include 20 mg, 120 mg or 160 mg of Compound B or a pharmaceutically acceptable salt thereof as the API and one or more intra-granulation excipients such as a dry binder, tablet disintegrant, an absorbent, filler or diluent, a lubricant, anti- adherent, binder, thickening agent, viscosityincreasing agent, coating agent and film former and one or more extra granulation excipients such as additional Compound B API granulate and a lubricant or film forming agent.

[0105] In some embodiments, an oral dosage form such as a tablet or capsule can include 20 mg, 120 mg or 160 mg of Compound B or a pharmaceutically acceptable salt thereof as the API and one or more intra-granulation excipients such as microcrystalline cellulose, crospovidone Type A, and hydroxypropyl cellulose, and one or more extra-granulation excipients such as API granulate of Compound B and magnesium stearate. In some embodiments, an oral dosage form such as a tablet or capsule can include about 20% w / w of Compound B or a pharmaceutically acceptable salt thereof as the API and about 60 to about 70% w / w microcrystalline cellulose, about 5% w / w crospovidone Type A, and about 10% w / w hydroxypropyl cellulose, and one or more extra-granulation excipients such as API granulateof Compound B (about 95 to about 99.5% w / w) and magnesium stearate (about 0.5 to about 5% w / w).

[0106] Techniques and compositions for making dosage forms useful in the methods described herein are described in the following references, all incorporated by reference herein: Modern Pharmaceutics, 4th Ed., Chapters 9 and 10 (Banker & Rhodes, editors, 2002); Lieberman et al., Pharmaceutical Dosage Forms: Tablets (1989); and Ansel, Introduction to Pharmaceutical Dosage Forms 8th Edition (2004).

[0107] Compound B may be formulated as a pharmaceutical composition using a pharmaceutically acceptable excipients and administered by a variety of routes. In some embodiments, such pharmaceutical compositions are for oral administration. Such pharmaceutical compositions and processes for preparing them arc well known in the ail. (See, e.g., Remington: The Science and Practice of Pharmacy, L. V. Allen, Editor, 22nd Edition, Pharmaceutical Press, 2012).

[0108] In some embodiments, the oral dosage form is about 20 mg to about 160 mg strength tablet for oral administration. For example, Compound B may be supplied as a 20 mg strength and 160 mg strength tablets for oral administration according to the formulation of Table 1. In some embodiments, the total tablet weight at 20 mg strength can be 100 mg and at 160 mg strength can be 800 mg.

[0109] An oral dosage form of Compound B can be prepared by combining Compound B as the active pharmaceutical ingredient (API) with pharmaceutically acceptable excipients to form a tablet. The intra granulation excipients can include microcrystalline cellulose (e.g., CEOLUS™ UF-711), crospovidone Type A (e.g., Polyplasdone™ XL), hydroxypropyl Cellulose HPC-SL, hyroxypropyl cellulose-Low Substituted L-HPC LH-21, and purified water. The extra granulation excipients can include Compound B and magnesium stearate (e.g., hyqual vegetable source).

[0110] For example, a tablet dosage form is described in the Table 1A below.Table 1A

[0111] In some embodiments, an oral dosage form of Compound B can be prepared by combining Compound B as the active pharmaceutical ingredient (API) with pharmaceutically acceptable excipients to form a tablet. In some embodiments, Compound B may be provided as 20 mg strength and 160 mg strength tablets for oral administration, containing excipients lactose monohydrate (e.g., 200 mesh or 200M), microcrystalline cellulose (MCC PH101, MCC PH102), croscarmellose sodium, hydroxypropyl cellulose (HPC) grade SL, and magnesium stearate. In some embodiments, the tablet is prepared using an intra-granulation composition comprising Compound B as the API, and an extra-granulation mixture. In some embodiments, the intra granulation excipients can include a fine milled powder of a-lactose monohydrate (e.g., lactose monohydrate 200M), a microcrystalline cellulose (e.g., MCC PH101), an internally cross-linked sodium carboxymethylcellulose (e.g., Croscarmellose Sodium), and a hydroxypropyl cellulose (e.g., Hydroxypropyl Cellulose HPC- SL). In some embodiments, the extra granulation excipients can include a microcrystalline cellulose (e.g., MCC PH102), an internally cross-linked sodium carboxymethylcellulose (e.g., Croscarmellose Sodium), and magnesium stearate (e.g., hyqual vegetable source). The tablets can be optionally coated with a film coating (Opadry® QX 321A80015). For example, a tablet dosage form is described in the Table IB below.Table IBMethods of Treatment / Uses

[0112] In certain aspects, therapeutic methods or uses are provided herein for treating, preventing, or ameliorating a disease or condition in a subject. Some embodiments relate to the use of a compound or composition described herein to treat, prevent, or ameliorate cancer associated with an oncogenic RAS mutation. Some embodiments relate to the use of a compound or composition as described herein to treat cancer characterized by expressing a mutant form of RAS protein in a subject. Some embodiments relate to the use of a compound or composition as described herein to inhibit the proliferation of a cell having a RAS mutation. Some embodiments relate to the use of a compound or composition as described herein to induce apoptosis in a cell having a RAS mutation. Some embodiments relate to the use of a compound or composition as described herein to inhibit the progression of cancer that is characterized by expressing a mutant form of RAS protein in a subject. In some embodiments, the RAS protein is a KRAS protein. In some embodiments, the RAS protein is an NRAS protein. In some embodiments, the RAS protein is an HRAS protein.

[0113] The MAPK pathway is inappropriately activated in approximately half of cancer patients cither directly through activation mutations in the pathway or indirectly through upstream alterations such as receptor tyrosine kinases (RTK). The RAS-RAF-MEK-ERK cascade is one of four distinct MAPK pathways and is the one most often exploited in cancer. Gain of function mutations in RAS (KRAS, NRAS, HRAS) or RAF (ARAF, BRAF, CRAF / RAF1) are commonly observed, and an additional fraction of human cancer display somatic mutations in RAF (i.e., ARAF, BRAF and CRAF (RAFI), where oncogenic activation of BRAF is most commonly observed. Further, RAS mutant tumors often carry a negative prognosis when compared to their RAS wild type counterparts. Although RAS mutations can be found in most tumor types, RAS variants are especially enriched in tumors that arise from the pancreas (up to 95% [KRAS]), colon (up to 45% in colorectal cancer (CRC) [KRAS]), lung (up to 40% in non-small cell lung cancer (NSCLC) [KRAS]), skin (up to 20 to 30% in melanoma [NRAS]), myeloid progenitors (up to 12% in acute myeloid leukemia (AML) [NRAS]) and bladder (up to 12% [HRAS]) among others (Figure 1). RAF and RAS mutations are mostly non-overlapping, but there are common tumor tissue types that display oncogenic RAF mutations, including lung cancer, melanoma and colorectal. These clinical observations have prompted extensive drug research and development aimed at identifying drugs that can disrupt mutant RAS, RAF or downstream kinases in the MAPK cascade, including MEK, and ERK. Several challenges have contributed to the limited clinical success for many new drug candidates and marketed agents. These include lack of broad targetability (e.g., drugs that are mainly limited to RAF-mutant disease rather than RAS mutant disease), limited biomarker population size (e.g., drugs specific to BRAFV600E / K or KRASG12C), pathway reactivation mechanisms (e.g., drugs susceptible to CRAF-bypass or adaptive resistance) and toxicities that limit clinical utility (e.g., drugs with poor tolerability requiring frequent dose modifications, shorter treatment durations and limited drug-drug combination options).

[0114] In some embodiments, methods of treating a cancer include providing Compound B in a subject in need thereof. In some embodiments, Compound B is orally administered to the patient in need thereof twice per day (BID). In some embodiments, Compound B is administered to the patient in need thereof in a daily dose of 240 mg - 320 mg. In some embodiments, the daily dose is 240 mg of Compound B administered as 120 mg of Compound B twice per day (BID). In some embodiments, the daily dose is 320 mg ofCompound B administered as 160 mg of Compound B twice per day (BID). In some embodiments, the dose can be administered orally. In some embodiments, the dose can be administered as two equal daily doses. In some embodiments, the subject is administered Compound B for at least one month, or at least three months, or at least six months. Compound B can be administered as a pharmaceutically acceptable salt of Compound B, in an amount providing a total of 240 mg to 320 mg of Compound B per day, such as twice daily (BID) doses of 120 mg or 160 mg of Compound B to a subject in need thereof. In some embodiments, a method of treating cancer comprises orally administering Compound B to a patient in need thereof an amount of Compound B or a pharmaceutically acceptable salt thereof providing a total of 120 mg of Compound B twice per day (BID). In some embodiments, a method of treating cancer comprises orally administering Compound B to a patient in need thereof an amount of Compound B or a pharmaceutically acceptable salt thereof providing a total of 160 mg of Compound B twice per day (BID).Combination of Compound B with Kinase Inhibitors

[0115] In some embodiments, one or more kinase inhibitors may be coadministered with Compound B. In some embodiments, Compound B is administered in combination with encorafenib. In some embodiments, Compound B is administered in combination with binimetinib, for the treatment of patients with unresectable or metastatic melanoma with a BRAF V600E or V600K mutation. In some embodiments, Compound B is administered in combination with cetuximab, for the treatment of adult patients with metastatic colorectal cancer (CRC) with a BRAF V600E mutation after prior therapy. In some embodiments, Compound B is administered in combination with encorafenib, wherein the encorafinib is administered at a dose of about 300 to about 450 mg orally once daily.

[0116] In some embodiments, Compound B is administered with a kinase inhibitor, such as encorafenib, in a method of treating patients with unresectable or metastatic melanoma with a BRAF V600E or V600K mutation.

[0117] In some embodiments, Compound B is administered with a kinase inhibitor, such as encorafenib, in a method of treating patients with metastatic colorectal cancer (CRC) with a BRAF V600E mutation after prior therapy.

[0118] Encorafenib is a kinase inhibitor. The chemical name is methyl N-{(2S)-1 - [(4-{ 3-[5-chloro-2-fluoro-3- (mcthancsulfonamido)phcnyl]- l-(propan-2-yl)- lH-pyrazol-4- yl}pyrimidin-2-yl)amino]propan-2- yl}carbamate. The molecular formula is C22H27CIFN7O4S and the molecular weight is 540 daltons. The chemical structure of encorafenib is shown below:

[0119] Encorafenib is a white to almost white powder. In aqueous media, encorafenib is slightly soluble at pH 1, very slightly soluble at pH 2, and insoluble at pH 3 and higher. Encorafenib capsules for oral use contain 75 mg of encorafenib with the following inactive ingredients: copovidone, poloxamer 188, microcrystalline cellulose, succinic acid, crospovidone, colloidal silicon dioxide, magnesium stearate (vegetable origin). The capsule shell contains gelatin, titanium dioxide, iron oxide red, iron oxide yellow, ferrosoferric oxide, monogramming ink (pharmaceutical glaze, ferrosoferric oxide, propylene glycol).

[0120] Encorafenib is a kinase inhibitor that targets BRAF V600E, as well as wildtype BRAF and CRAF in in vitro cell-free assays with ICso values of 0.35, 0.47, and 0.3 nM, respectively. Mutations in the BRAF gene, such as BRAF V600E, can result in constitutively activated BRAF kinases that may stimulate tumor cell growth. Encorafenib was also able to bind to other kinases in vitro including JNK1, JNK2, JNK3, LIMK1, LIMK2, MEK4, and STK36 and reduce ligand binding to these kinases at clinically achievable concentrations (<0.9 pM). Encorafenib inhibited in vitro growth of tumor cell lines expressing BRAF V600 E, D, and K mutations. In mice implanted with tumor cells expressing BRAF V600E, encorafenib induced tumor regressions associated with RAF / MEK / ERK pathway suppression.

[0121] In some embodiments, Compound B is administered in combination with binimctinib. Binimctinib is a reversible inhibitor of mitogen-activated extracellular signal regulated kinase 1 (MEK1) and MEK2 activity. MEK proteins are upstream regulators of the extracellular signal-related kinase (ERK) pathway. In vitro, binimetinib inhibited extracellular signal-related kinase (ERK) phosphorylation in cell- free assays as well as viability and MEK- dependent phosphorylation of BRAF-mutant human melanoma cell lines. Binimetinib also inhibited in vivo ERK phosphorylation and tumor growth in BRAF-mutant murine xenograft models.

[0122] In some embodiments, Compound B is administered in combination with a pharmaceutically acceptable salt of binimetinib. In some embodiments, Compound B is administered in combination with a mesylate salt of binimetinib. Binimetinib is a kinase inhibitor. The chemical name is 5-[(4-bromo-2-fluorophenyl)amino]-4-fluoro-N-(2 hydroxyethoxy)-l-methyl-lH-benzimidazole-6-carboxamide. The molecular formula is Ci7HisBrF2N4O3 and the molecular weight is 441.2 daltons. The chemical structure of binimetinib is shown below:(binimetinib).

[0123] Binimetinib can be administered as tablets for oral use, each containing 15 mg of binimetinib with the following inactive ingredients: lactose monohydrate, microcrystalline cellulose, croscarmellose sodium, magnesium stearate (vegetable source), and colloidal silicon dioxide. The coating contains polyvinyl alcohol, polyethylene glycol, titanium dioxide, talc, ferric oxide yellow, and ferrosoferric oxide.

[0124] In some embodiments, vemurafenib can be administered at a dose equivalent to 45 mg of binimetinib free base orally twice daily. In some embodiments, binimctinib can be administered orally twice daily in combination with Compound B at a doseof binimetinib or a pharmaceutically acceptable salt thereof that is equivalent to only 45 mg binimctinib free base. In some embodiments, a dose equivalent to 45 mg binimetinib free base can be orally administered twice daily, in combination with Compound B at a dose of about 20 to about 320 mg orally twice daily. In some embodiments, a dose equivalent to about 45 mg binimetinib free base can be orally administered twice daily, in combination with Compound B at a dose of about 120 to about 160 mg orally twice daily. In some embodiments, a dose equivalent to 45 mg binimetinib free base can be orally administered twice daily, in combination with Compound B at a dose of 120 mg orally twice daily. In some embodiments, a dose equivalent to 45 mg binimetinib free base can be orally administered twice daily, in combination with Compound B at a dose of 160 mg orally twice daily.

[0125] Binimetinib is FDA approved in combination with encorafenib, for the treatment of patients with unresectable or metastatic melanoma with a BRAF V600E or V600K mutation. Binimetinib and encorafenib target two different kinases in the RAS / RAF / MEK7ERK pathway. Compared to either drug alone, coadministration of encorafenib and binimetinib resulted in greater anti-proliferative activity in vitro in BRAF mutation-positive cell lines and greater anti-tumor activity with respect to tumor growth inhibition in BRAF V600E mutant human melanoma xenograft studies in mice. Additionally, the combination of binimetinib and encorafenib delayed the emergence of resistance in BRAF V600E mutant human melanoma xenografts in mice compared to either drug alone.

[0126] In some embodiments, one or more BRAF inhibitors may be coadministered with Compound B for the treatment of patients diagnosed with certain forms of RAF-mutated cancer. In some embodiments, one or more BRAF inhibitors may be coadministered with Compound B for the treatment of patients diagnosed with a BRAF V600- mutated cancer. In some embodiments, one or more BRAF inhibitors may be co-administered with Compound B for the treatment of patients diagnosed with a BRAF V600E mutated cancer. In some embodiments, one or more BRAF inhibitors may be co-administered with Compound B for the treatment of patients diagnosed with a BRAF V600K mutated cancer. In some embodiments, one or more BRAF inhibitors may be co-administered with Compound B for the treatment of patients diagnosed with a BRAF V600R mutated cancer.

[0127] In some embodiments, Compound B is administered in combination with dabrafenib. In some embodiments, Compound B is administered in combination with apharmaceutically acceptable salt of dabrafenib. In some embodiments, Compound B is administered in combination with a mesylate salt of dabrafenib. Dabrafenib mesylate has the following chemical structure:(dabrafenib mesylate).

[0128] In some embodiments, dabrafenib is administered as dabrafenib mesylate in capsules supplied as 50-mg or 75-mg capsules for oral administration. Each 50-mg capsule contains 59.25 mg dabrafenib mesylate equivalent to 50 mg of dabrafenib free base. Each 75- mg capsule contains 88.88 mg dabrafenib mesylate equivalent to 75 mg of dabrafenib free base. The dabrafenib mesylate capsules can contain inactive ingredients such as: colloidal silicon dioxide, magnesium stearate, and microcrystalline cellulose. The capsule shells can contain hypromellose, red iron oxide (El 72), and titanium dioxide (E 171 ).

[0129] In some embodiments, dabrafenib or a pharmaceutically acceptable salt thereof can be administered at a dose equivalent to 2 to 150 mg of dabrafenib free base orally twice daily. In some embodiments, dabrafenib can be administered orally once daily in combination with Compound B at a dose of dabrafenib or a pharmaceutically acceptable salt thereof that is equivalent to only 2 mg dabrafenib free base. In some embodiments, a dose equivalent to 2 mg dabrafenib free base can be orally administered once daily, in combination with Compound B at a dose of 20 to about 320 mg orally twice daily. In some embodiments, a dose equivalent to 2 mg dabrafenib free base can be orally administered once daily, in combination with Compound B at a dose of about 120 to about 160 mg orally twice daily. In some embodiments, a dose equivalent to 2 mg dabrafenib free base can be orally administered once daily, in combination with Compound B at a dose of 120 mg orally twice daily. In someembodiments, a dose equivalent to 2 mg dabrafenib free base can be orally administered once daily, in combination with Compound B at a dose equivalent to 160 mg dabrafenib free base orally twice daily. In some embodiments, methods of treatment comprise administering a therapeutically effective amount of Compound B in combination with dabrafenib to treat a patient diagnosed with a BRAF V600E mutation. In some embodiments, methods of treatment comprise administering a therapeutically effective amount of Compound B in combination with dabrafenib to treat a patient diagnosed with melanoma having a BRAF V600 mutation. In some embodiments, methods of treatment comprise administering a therapeutically effective amount of Compound B in combination with dabrafenib to treat a patient diagnosed with unresectable or metastatic melanoma having a BRAF V600E mutation. In some embodiments, methods of treatment comprise administering a therapeutically effective amount of Compound B in combination with dabrafenib to treat a patient diagnosed with unresectable or metastatic melanoma with a BRAF V600K. In some embodiments, methods of treatment comprise administering a therapeutically effective amount of Compound B in combination with dabrafenib to treat a patient diagnosed with unresectable or metastatic melanoma with a BRAF V600D.

[0130] In some embodiments, Compound B is administered in combination with vemurafenib. In some embodiments, Compound B is administered in combination with a pharmaceutically acceptable salt of vemurafenib. In some embodiments, Compound B is administered in combination with a mesylate salt of vemurafenib. Vemurafenib is a kinase inhibitor indicated for the treatment of patients with unresectable or metastatic melanoma with BRAF V600E mutation. Vemurafenib has the following chemical structure:(vemurafenib)

[0131] Vemurafenib is a low molecular weight, orally available inhibitor of some mutated forms of BRAF serine- threonine kinase, including BRAF V600E. Vemurafenib also inhibits other kinases in vitro such as CRAF, ARAF, wild-type BRAF, SRMS, ACK1, MAP4K5, and FGR at similar concentrations. Some mutations in the BRAF gene including V600E result in constitutively activated BRAF proteins, which can cause cell proliferation in the absence of growth factors that would normally be required for proliferation. Vemurafenib has anti-tumor effects in cellular and animal models of melanomas with mutated BRAF V600E.

[0132] In some embodiments, vemurafenib is administered in tablets containing 240 mg of vemurafenib, for oral administration. In some embodiments, the vemurafenib is administered in tablets containing the inactive ingredients forming a tablet core (e.g., hypromellose acetate succinate, croscarmellose sodium, colloidal silicon dioxide, magnesium stearate, and hydroxypropyl cellulose) and a tablet coating (poly (vinyl alcohol), titanium dioxide, polyethylene glycol 3350, talc, and iron oxide red).

[0133] In some embodiments, vemurafenib can be administered at a dose equivalent to 960 mg of vemurafenib free base orally twice daily. In some embodiments, vemurafenib can be administered orally twice daily in combination with Compound B at a dose of vemurafenib or a pharmaceutically acceptable salt thereof that is equivalent to only 960 mg vemurafenib free base. In some embodiments, a dose equivalent to 960 mg vemurafenib free base can be orally administered twice daily, in combination with Compound B at a dose of 20- 320 mg orally twice daily. In some embodiments, a dose equivalent to 960 mg vemurafenib free base can be orally administered twice daily, in combination with Compound B at a dose of 120-160 mg orally twice daily. In some embodiments, a dose equivalent to 960 mg vemurafenib free base can be orally administered twice daily, in combination with Compound B at a dose of 120 mg orally twice daily. In some embodiments, a dose equivalent to 960 mg vemurafenib free base can be orally administered twice daily, in combination with Compound B at a dose of 160 mg orally twice daily.Immune Checkpoint Inhibitors

[0134] In some embodiments, one or more immune checkpoint inhibitor may be co-administered with Compound B. A review describing immune checkpoint pathways andthe blockade of such pathways with immune checkpoint inhibitor compounds is provided by Pardoll in Nature Reviews Cancer (April 2012), pages 252-264, which is incorporated herein by reference in its entirety. Immune check point inhibitor compounds display anti-tumor activity by blocking one or more of the endogenous immune checkpoint pathways that downregulate an anti-tumor immune response. The inhibition or blockade of an immune checkpoint pathway typically involves inhibiting a checkpoint receptor and ligand interaction with an immune checkpoint inhibitor compound to reduce or eliminate the down regulation signal and resulting diminishment of the anti-tumor response.

[0135] In some embodiments of the present disclosure, the immune checkpoint inhibitor compound inhibits the signaling interaction between an immune checkpoint receptor and the corresponding ligand of the immune checkpoint receptor. The immune checkpoint inhibitor compound can act by blocking activation of the immune checkpoint pathway by inhibition (antagonism) of an immune checkpoint receptor (some examples of receptors include CTLA-4, PD-1, LAG-3, TIM-3, BTLA, and KIR) or by inhibition of a ligand of an immune checkpoint receptor (some examples of ligands include PD-L1 and PD-L2). In such embodiments, the effect of the immune checkpoint inhibitor compound is to reduce or eliminate down regulation of certain aspects of the immune system anti-tumor response in the tumor microenvironment.

[0136] The Programmed Death 1 (PD-1) protein is an inhibitory member of the extended CD28 / CTLA-4 family of T cell regulators (Okazaki et al. (2002) Curr Opin Immunol 14: 391779-82; Bennett et al. (2003) J. Immunol. 170:711-8; which are incorporated herein by reference in their entirety). Other members of the CD28 family include CD28, CTLA-4, ICOS and BTLA. PD-1 is suggested to exist as a monomer, lacking the unpaired cysteine residue characteristic of other CD28 family members. PD-1 is expressed on activated B cells, T cells, and monocytes.

[0137] The PD-1 gene encodes a 55 kDa type I transmembrane protein (Agata et al. (1996) Int Immunol. 8:765-72, which is incorporated herein by reference in its entirety). Although structurally similar to CTLA-4, PD-1 lacks the MYPPY motif that is important for B7-1 and B7-2 binding. Two ligands for PD-1 have been identified, PD-L1 (B7-H1) and PD- L2 (B7-DC), that have been shown to downregulate T cell activation upon binding to PD-1 (Freeman et al. (2000) J. Exp. Med. 192: 1027-34; Carter et al. (2002) Eur. J. Immunol. 32:634-43; which are incorporated herein by reference in their entirety). Both PD-L1 and PD-L2 are B7 homologs that bind to PD-1, but do not bind to other CD28 family members. PD-L1 is abundant in a variety of human cancers (Dong et al. (2002) Nat. Med. 8:787-9, which is incorporated herein by reference in its entirety).

[0138] PD-1 is known as an immunoinhibitory protein that negatively regulates TCR signals (Ishida, Y. et al. (1992) EMBO J. 11:3887-3895; Blank, C. et al. (Epub 2006 Dec. 29) Immunol. Immunother. 56(5):739-745; which are incorporated herein by reference in their entirety). The interaction between PD-1 and PD-L1 can act as an immune checkpoint, which can lead to, e.g., a decrease in tumor infiltrating lymphocytes, a decrease in T-cell receptor mediated proliferation, and / or immune evasion by cancerous cells (Dong et al. (2003) J. Mol. Med. 81:281-7; Blank et al. (2005) Cancer Immunol, Immunothcr. 54:307-314; Konishi et al. (2004) Clin. Cancer Res. 10:5094-100; which are incorporated herein by reference in their entirety). Immune suppression can be reversed by inhibiting the local interaction of PD- 1 with PD-L1 or PD-L2; the effect is additive when the interaction of PD-1 with PD-L2 is blocked as well (Iwai et al. (2002) Proc. Nat'l. Acad. Sci. USA 99:12293-7; Brown et al. (2003) J. Immunol. 170:1257-66; which are incorporated herein by reference in their entirety).

[0139] The immune checkpoint receptor cytotoxic T-lymphocyte associated antigen 4 (CTLA-4) is expressed on T-cells and is involved in signaling pathways that reduce the level of T-cell activation. It is believed that CTLA-4 can downregulate T-cell activation through competitive binding and sequestration of CD80 and CD86. In addition, CTLA-4 has been shown to be involved in enhancing the immunosuppressive activity of TReg cells.

[0140] The immune checkpoint receptor programmed death 1 (PD-1) is expressed by activated T-cells upon extended exposure to antigen. Engagement of PD-1 with its known binding ligands, PD-L1 and PD-L2, occurs primarily within the tumor microenvironment and results in downregulation of anti-tumor specific T-cell responses. Both PD-L1 and PD-L2 are known to be expressed on tumor cells. The expression of PD-L1 and PD-L2 on tumors has been correlated with decreased survival outcomes.

[0141] The immune checkpoint receptor T cell membrane protein 3 (TIM-3) is expressed on Thl and Tel cells, but not other T-cells. Interaction of TIM-3 with its ligand, galectin-9, produces a Thl cell death signal. TIM-3 has been reported to play a role inmaintaining T-cell exhaustion and blockade of TIM-3 has been shown to restore activity to exhausted T-cclls.

[0142] The immune checkpoint receptor B- and T-lymphocyte attenuator (BTLA) receptor is expressed on both resting and activated B-cells and T-cells. Activation of BTLA when combined with its ligand HVEM (herpes virus entry mediator) results in downregulation of both T-cell activation and proliferation. HVEM is expressed by certain tumors (e.g., melanoma) and tumor- associated endothelial cells.

[0143] The immune checkpoint receptors known as killer cell immunoglobulin- like receptors (KIR) are a polymorphic family of receptors expressed on NK cells and some T- cells and function as regulators of immune tolerance associated with natural killer (NK) cells. Blocking certain KIR receptors with inhibitor compounds can facilitate the destruction of tumors through the increased activity of NK cells.

[0144] In some embodiments of the present disclosure, the immune checkpoint inhibitor compound is a small organic molecule (molecular weight less than 1000 daltons), a peptide, a polypeptide, a protein, an antibody, an antibody fragment, or an antibody derivative. In some embodiments, the immune checkpoint inhibitor compound is an antibody. In some embodiments, the antibody is a monoclonal antibody, specifically a human or a humanized monoclonal antibody.

[0145] Monoclonal antibodies, antibody fragments, and antibody derivatives for blocking immune checkpoint pathways can be prepared by any of several methods known to those of ordinary skill in the art, including but not limited to, somatic cell hybridization techniques and hybridoma, methods. Hybridoma generation is described in Antibodies, A Laboratory Manual, Harlow and Lane, 1988, Cold Spring Harbor Publications, New York, which is incorporated herein by reference in its entirety. Human monoclonal antibodies can be identified and isolated by screening phage display libraries of human immunoglobulin genes by methods described for example in U.S. Pat. Nos. 5,223,409, 5,403,484, 5,571,698, 6,582,915, and 6,593,081, which are incorporated herein by reference in their entirety. Monoclonal antibodies can be prepared using the general methods described in U.S. Pat. No. 6,331,415 (Cabilly), which is incorporated herein by reference in its entirety.

[0146] As an example, human monoclonal antibodies can be prepared using a XenoMouse™ (Abgenix, Freemont, Calif.) or hybridomas of B cells from a XenoMouse. AXenoMouse is a murine host having functional human immunoglobulin genes as described in U.S. Pat. No. 6,162,963 (Kuchcrlapati), which is incorporated herein by reference in its entirety.

[0147] Methods for the preparation and use of immune checkpoint antibodies are described in the following illustrative publications. The preparation and therapeutic uses of anti-CTLA-4 antibodies are described in U.S. Pat. No. 7,229,628 (Allison), U.S. Pat. No. 7,311,910 (Linsley), and U.S. Pat. No. 8,017,144 (Korman), which are incorporated herein by reference in their entirety. The preparation and therapeutic uses of anti-PD-1 antibodies are described in U.S. Pat. No. 8,008,449 (Korman) and U.S. Patent Application No. 2011 / 0271358 (Freeman), which are incorporated herein by reference in their entirety. The preparation and therapeutic uses of anti-PD-Ll antibodies are described in U.S. Pat. No. 7,943,743 (Korman), which is incorporated herein by reference in its entirety. The preparation and therapeutic uses of anti-TIM-3 antibodies are described in U.S. Pat. No. 8,101,176 (Kuchroo) and U.S. Pat. No. 8,552,156 (Tagayanagi), which are incorporated herein by reference in their entirety. The preparation and therapeutic uses of anti-LAG-3 antibodies are described in U.S. Patent Application No. 2011 / 0150892 (Thudium) and International Publication Number W02014 / 008218 (Lonberg), which are incorporated herein by reference in their entirety. The preparation and therapeutic uses of anti-KIR antibodies are described in U.S. Pat. No. 8,119,775 (Moretta), which is incorporated herein by reference in its entirety. The preparation of antibodies that block BTLA regulated inhibitory pathways (anti-BTLA antibodies) are described in U.S. Pat. No. 8,563,694 (Mataraza), which is incorporated herein by reference in its entirety.

[0148] In some embodiments, the one or more immune checkpoint inhibitor is an inhibitor of PD-1, PD-L1, or CTLA-4. In some embodiments, the immune checkpoint inhibitor is a PD- 1 inhibitor. In some embodiments, the immune checkpoint inhibitor is a binding ligand of PD-L1. In some embodiments, the immune checkpoint inhibitor is a PD-L1 inhibitor. In some embodiments, the immune checkpoint inhibitor is a CTLA-4 inhibitor.

[0149] In some embodiments, the one or more immune checkpoint inhibitor as described herein includes a first immune checkpoint inhibitor and a second immune checkpoint inhibitor, wherein the first immune checkpoint inhibitor is different from the second immune checkpoint inhibitor. In some embodiments, the first and the second immune checkpointinhibitor are independently an inhibitor of PD-1 , PD-L1 or CTLA-4. In some embodiments, the first immune checkpoint inhibitor is a PD- 1 inhibitor, and the second immune checkpoint inhibitor is a CTLA-4 inhibitor.

[0150] In some embodiments, the immune checkpoint inhibitor is pembrolizumab, nivolumab, cemiplimab, atezolizumab, avelumab, pembrolizumab, pidilizumab, ipilimumab, BMS 936559, durvalumab, or any combinations thereof. In some embodiments, the one or more immune checkpoint inhibitor may include an anti-PD-1 HuMAbs can be selected from 17D8, 2D3, 4H1, 5C4 (also referred to herein as nivolumab), 4A1 1, 7D3 and 5F4, all of which are described in U.S. Pat. No. 8,008,449, which is incorporated herein by reference in its entirety. In some embodiments, the anti-PD-1 HuMAbs can be selected from 3G10, 12A4 (also referred to herein as BMS-936559), 10A5, 5F8, 10H10, 1B12, 7H1, 1 1E6, 12B7, and 13G4, all of which are described in U.S. Pat. No. 7,943,743, which is incorporated herein by reference in its entirety.

[0151] In some embodiments, the one or more immune checkpoint inhibitor may be incorporated in a pharmaceutically acceptable formulation. In some embodiments, the one or more immune checkpoint inhibitor is incorporated in a pharmaceutically acceptable aqueous formulation. Examples of acceptable aqueous formulations include isotonic buffered and pH 4.5-8 adjusted saline solutions such as Lactated Ringer's Solution and the like.

[0152] In some embodiments, the immune checkpoint inhibitor compound is incorporated in a pharmaceutically acceptable liposome formulation, wherein the formulation is a passive or targeted liposome formulation. Examples of methods for the preparation of suitable liposome formulations of antibodies are described U.S. Pat. No. 5,399,331 (Loughrey), U.S. Pat. No. 8,304,565 (Wu) and U.S. Pat. No. 7,780,882 (Chang), which are incorporated herein by reference in their entirety.

[0153] In some embodiments, the one or more immune checkpoint inhibitor may be an antibody. In some embodiments, the antibody is a dry, lyophilized solid that is reconstituted with an aqueous reconstitution solvent prior to use. In some embodiments, the antibody is incorporated in a pharmaceutically acceptable formulation and the pharmaceutically acceptable formulation is injected directly into a tumor. In some embodiments, the immune checkpoint inhibitor antibody is incorporated in a pharmaceutically acceptable formulation and the pharmaceutically acceptable formulation is injected into theperitumoral region surrounding a tumor. The peritumoral region may contain antitumor immune cells. In some embodiments, the antibody is incorporated in a pharmaceutically acceptable formulation and the pharmaceutically acceptable formulation is administered by intravenous injection or infusion. In some embodiments, the immune checkpoint inhibitor antibody is incorporated in a pharmaceutically acceptable formulation and the pharmaceutically acceptable formulation is administered by subcutaneous injection or intradermal injection. In some embodiments, the antibody is incorporated in a pharmaceutically acceptable formulation and the pharmaceutically acceptable formulation is administered by intraperitoneal injection or lavage.

[0154] The precise amount of immune checkpoint inhibitor compound incorporated in a particular method or therapeutic combination of the disclosure may vary according to factors known in art such as for example, the physical and clinical status of the subject, the method of administration, the content of the formulation, the physical and chemical nature of the immune checkpoint inhibitor compound, the intended dosing regimen or sequence. Those of ordinary skill in the art, however, can readily determine the appropriate amount with due consideration of such factors.

[0155] Aspects disclosed herein relate to administering to a subject in need an effective amount of Compound B or a pharmaceutically acceptable salt thereof), or a pharmaceutical composition that includes one or more compounds as described herein (such as Compound B or a pharmaceutically acceptable salt thereof).

[0156] As disclosed elsewhere herein, some embodiments pertain to treating a disease or condition, such as cancer, through administration of a compound or composition as disclosed herein. A subject in need of receiving a compound or composition as disclosed herein to improve the subject’ s health need not always be identified prior to receiving a first treatment with the compound or composition. For example, a subject may be predetermined that they will develop a disease or condition, such as cancer, prior to showing any signs of the disease or condition. Alternatively, the subject may receive treatment prophylactic ally if he or she is at risk or not developing a disease or condition, such as cancer, (e.g., once a patient shows symptoms of another disease or condition associated with a cancer). Accordingly, in some embodiments, the compound or composition may be administered to the subject after the subject receives an early-stage diagnosis. In some embodiments, not every subject is acandidate for such administration and identification of treatment subjects may be desirable. It is understood that patient selection depends upon a number of factors within the skill of the ordinarily skilled physician. Thus, some embodiments disclosed herein further comprise identifying a subject as one that will benefit from administering an effective amount of at least one compound or composition to increase longevity, increase survival time or increase life span.

[0157] In other aspects, the present disclosure is directed to a method for the treatment, prevention or prophylaxis of cancer can include administering to a subject in need thereof an effective amount of one or more compound described herein (such as Compound B, or a pharmaceutically acceptable salt thereof), or a pharmaceutical composition that includes a compound described herein (such as Compound B, or a pharmaceutically acceptable salt thereof). In certain embodiments, the cancer may be selected from brain cancer, breast cancer, lung cancer, ovarian cancer, pancreatic cancer, stomach cancer, prostate cancer, renal cancer, colorectal cancer or leukemia. In further or additional embodiments, the cancer is brain cancer or adrenocortical carcinoma. In further or additional embodiments, the cancer is breast cancer. In further or additional embodiments, the cancer is ovarian cancer. In further or additional embodiments, the cancer is pancreatic cancer. In further or additional embodiments, the cancer is stomach cancer. In further or additional embodiments, the cancer is prostate cancer. In further or additional embodiments, the cancer is renal cancer. In further or additional embodiments, the cancer is colorectal cancer. In further or additional embodiments, the cancer is myeloid leukemia. In further or additional embodiments, the cancer is glioblastoma. In further or additional embodiments, the cancer is follicular lymphoma. In further or additional embodiments, the cancer is pre-B acute leukemia. In further or additional embodiments, the cancer is chronic lymphocytic B-leukemia. In further or additional embodiments, the cancer is mesothelioma. In further or additional embodiments, the cancer is small cell line cancer.

[0158] Some embodiments relate to a method of inhibiting proliferation of a cell having a RAS mutation, comprising administering Compound B, or a pharmaceutically acceptable salt thereof). In some embodiments, the cancer may be associated with a RAS mutation. Some embodiments relate to a method of inducing apoptosis in a cell in a cell having a RAS mutation, comprising administering Compound B, or a pharmaceutically acceptable salt thereof). Some embodiments relate to a method of inhibiting proliferation of a cell having aKRAS mutation, comprising administering Compound B, or a pharmaceutically acceptable salt thereof). In some embodiments, the cancer may be associated with a KRAS mutation. Some embodiments relate to a method of inducing apoptosis in a cell in a cell having a KRAS mutation, comprising administering Compound B or a pharmaceutically acceptable salt thereof). Some embodiments relate to a method of inhibiting proliferation of a cell having a NRAS mutation, comprising administering Compound B or a pharmaceutically acceptable salt thereof). In some embodiments, the cancer may be associated with a NRAS mutation. Some embodiments relate to a method of inducing apoptosis in a cell in a cell having a RAS mutation, comprising administering Compound B, or a pharmaceutically acceptable salt thereof). In some embodiments, the KRAS mutation is at codons 12, 13, 59, 61 and / or 146. In some embodiments, the mutant form of the KRAS protein has one or more amino acid substitutions selected from the group consisting of G12C, G12S, G12R, G12F, G12L, G12N, G12A, G12D, G12V, G13C, G13S, G13D, G13V, GBP, S17G, P34S, A59E, A59G, A59T, Q61K, Q61L, Q61R, and Q61H. In some embodiments, the mutant form of the KRAS protein has one or more amino acid substitutions selected from the group consisting of G12C, GI2R, G12S, G12A, G12D, G12V, G13C, GBR, G13S, G13A, G13D, G13V, A59E, A59G, A59T, Q61K, Q61L, Q61R, Q61H, KI 17N, KI 17R, KI 17E, A146P, A146T and A146V.

[0159] In certain embodiments, the cancer is resistant to treatment of a MEK protein kinase inhibitor. In other embodiments, the cancer is resistant to treatment of a RAF protein kinase inhibitor. In still further embodiments, the resistance is acquired resistance. In other embodiments, the resistance is de novo resistance. In further or additional embodiments, the cancer is resistant to an anticancer agent.

[0160] In some aspects, provided herein are compounds or pharmaceutical compositions and methods for treating cancer comprising a therapeutically effective amount of a dual-RAF / MEK protein kinase inhibitor. In some embodiments, the administration of the dual-RAF / MEK protein kinase inhibitor provides an increase in the area under the serum concentration time curve (AUC) of the dual-RAF / MEK protein kinase inhibitor. In some embodiments, the cancer is resistant to treatment of a RAF protein kinase inhibitor. In further embodiments, the cancer is resistant to a RAF protein kinase inhibitor and the RAF protein kinase inhibitor comprises an A-RAF inhibitor, a B-RAF inhibitor, or a C-RAF inhibitor. Infurther embodiments, the cancer is resistant to a RAF protein kinase inhibitor, and the RAF protein kinase inhibitor comprises a B-RAF inhibitor.

[0161] In some embodiments, the resistant cancer is pancreatic, melanoma, colon, lung, or stomach cancer. In further embodiments, the resistant cancer is pancreatic. In additional embodiments, the resistant cancer is stomach. In alternative embodiments, provided are pharmaceutical combinations and methods for resensitizing cancer cells to treatment in a patient having or suspected of having a cancer resistant to an anticancer agent, comprising the step of administering to the patient a therapeutically effective amount of a dual-MEK / RAF inhibitor as disclosed herein.

[0162] In some embodiments, Compound B, or a pharmaceutically acceptable salt thereof, is co-administered with a CTLA-4 receptor inhibitor compound. In some embodiments, Compound B is co-administered a PD- 1 or PD-L1 receptor inhibitor compound.

[0163] In some embodiments, the method comprises treating a subject by coadministering a therapeutically effective amount of Compound B, or a pharmaceutically acceptable salt thereof, and a LAG-3 receptor inhibitor compound. In some embodiments, the method comprises treating a subject by co-administering a therapeutically effective amount of Compound B and a TIM-3 receptor inhibitor compound. In some embodiments, the method comprises treating a subject by co-administering a therapeutically effective amount of Compound B or a pharmaceutically acceptable salt thereof, and a BTLA receptor inhibitor compound. In some embodiments, the method comprises treating a subject by co-administering a therapeutically effective amount of Compound B, or a pharmaceutically acceptable salt thereof, and a KIR receptor inhibitor compound. In some embodiments, the method comprises treating a subject by co-administering a therapeutically effective amount of Compound B or a pharmaceutically acceptable salt thereof, and a PD-L1 inhibitor compound. In some embodiments, the method comprises treating a subject by co-administering a therapeutically effective amount of Compound B or a pharmaceutically acceptable salt thereof, and a PD-L2 inhibitor compound.

[0164] In some embodiments of the present disclosure, the method comprises treating a subject by co-administering a therapeutically effective amount of Compound B or a pharmaceutically acceptable salt thereof, and a blocking antibody of an immune checkpoint pathway. In some embodiments, the method comprises treating asubject by co-administering a therapeutically effective amount of Compound B, or a pharmaceutically acceptable salt thereof, and an anti-CTLA-4 receptor antibody. In some embodiments, the method comprises treating a subject by co-administering a therapeutically effective amount of Compound B or a pharmaceutically acceptable salt thereof, and an anti- PD-1 receptor antibody.

[0165] In some embodiments, the method comprises co-administering to a subject having a tumor a therapeutically effective amount of Compound B or a pharmaceutically acceptable salt thereof, and an anti-LAG-3 receptor antibody. In some embodiments, the method comprises co-administering to a subject having a tumor a therapeutically effective amount of Compound B, or a pharmaceutically acceptable salt thereof, and an anti-TIM-3 receptor antibody. In some embodiments, the method comprises co-administering to a subject having a tumor a therapeutically effective amount of Compound B, or a pharmaceutically acceptable salt thereof, and an anti-BTLA receptor antibody. In some embodiments, the method comprises co-administering to a subject having a tumor a therapeutically effective amount of Compound B or a pharmaceutically acceptable salt thereof, and an anti-KIR receptor antibody. In some embodiments, the anti-KIR receptor antibody is lirilumab. In some embodiments, the method comprises co-administering to a subject having a tumor a therapeutically effective amount of Compound B or a pharmaceutically acceptable salt thereof, and an anti-PD-1 antibody. In some embodiments the anti-PD-1 antibody is lambrolizumab, pidilizumab, or nivolumab. In some embodiments, the method comprises co-administering to a subject having a tumor a therapeutically effective amount of Compound B or a pharmaceutically acceptable salt thereof, and an anti-PD-Ll antibody. In some embodiments, the method comprises co-administering to a subject having a tumor a therapeutically effective amount of Compound B, or a pharmaceutically acceptable salt thereof, and an anti-PD-L2 antibody. In some embodiments, the method comprises co-administering to a subject having a tumor a therapeutically effective amount of Compound B or a pharmaceutically acceptable salt thereof, and an anti-CTLA-4 antibody. In some embodiments, the anti-CTLA-4 antibody is ipilimumab or tremelimumab.

[0166] In some embodiments, a method comprises co-administering Compound B or a pharmaceutically acceptable salt thereof, and an immune checkpoint inhibitor for treating, preventing, or ameliorating a cancer or tumor in a subject by administering Compound B or apharmaceutically acceptable salt thereof, and an immune checkpoint inhibitor. In some embodiments, the subject was resistant to prior treatment with only an immune checkpoint inhibitor.

[0167] In some embodiments, a method for treating a subject with a cancer or tumor comprises administering Compound B or a pharmaceutically acceptable salt thereof, and a PD-1 inhibitor. In some embodiments, a method for treating a subject with a cancer or tumor comprises administering Compound B or a pharmaceutically acceptable salt thereof, and a PD- L1 inhibitor. In some embodiments, a method for treating a subject with a cancer or tumor comprises administering Compound B or a pharmaceutically acceptable salt thereof, and a PD- L2 inhibitor. In some embodiments, a method for treating a subject with a cancer or tumor comprises administering Compound B or a pharmaceutically acceptable salt thereof, and a CTLA-4 inhibitor. In some embodiments, a method for treating a subject with a cancer or tumor comprises administering Compound B , a PD-1 inhibitor and a CTLA-4 inhibitor. In some embodiments, a method for treating a subject with a cancer or tumor comprises administering Compound B and a LAG-3 inhibitor. In some embodiments, a method for treating a subject with a cancer or tumor comprises administering Compound B and a KIR inhibitor. In some embodiments, a method for treating a subject with a cancer or tumor comprises administering Compound B and a TIM-3 inhibitor. In some embodiments, a method for treating a subject with a cancer or tumor comprises administering Compound B and a BTLA inhibitor.

[0168] In some embodiments, a method for treating a subject comprises treating a subject having exhibited resistance to a PD-1 inhibitor by administering a therapeutically effective amount of Compound B. In some embodiments, a method comprises treating a subject having exhibited resistance to a PD-L1 inhibitor by administering a therapeutically effective amount of Compound B. In some embodiments, a method comprises treating a subject having exhibited resistance to a PD-L2 inhibitor by administering a therapeutically effective amount of Compound B. In some embodiments, a method comprises treating a subject having exhibited resistance to a CTLA-4 inhibitor by administering a therapeutically effective amount of Compound B. In some embodiments, the method includes administering Compound B when the subject has exhibited resistance to two different immune checkpoint inhibitors. The two different immune inhibitors can be selectedfrom a CTLA-4 receptor inhibitor, a PD-1 receptor inhibitor, a LAG-3 receptor inhibitor, a TIM-3 receptor inhibitor, a BTLA receptor inhibitor, a KIR receptor inhibitor a PD- L1 inhibitor or a PD-L2 inhibitor. In some embodiments, a method comprises treating a subject having exhibited resistance to a PD- 1 inhibitor and CTLA-4 inhibitor by administering a therapeutically effective amount of Compound B. In some embodiments, a method comprises treating a subject having exhibited resistance to a PD-L1 inhibitor and a CTLA-4 inhibitor by administering a therapeutically effective amount of a compound of Formula (I). In some embodiments, a method comprises treating a subject having exhibited resistance to a PD-1 inhibitor, a PD-L1 inhibitor, and a CTLA-4 inhibitor by administering a therapeutically effective amount of Compound B, or a pharmaceutically acceptable salt thereof.

[0169] Some embodiments disclosed herein relate to a method of treating a mammal having a disease that can include administering to a subject in need thereof an effective amount of one or more compound described herein (such as Compound B or a pharmaceutically acceptable salt thereof), or a pharmaceutical composition that includes a compound described herein (such as Compound B or a pharmaceutically acceptable salt thereof). Other embodiments disclosed herein relate to a method of treating a subject with cancer cachexia that can include administering to a subject an effective amount of one or more compounds described herein (such as Compound B or a pharmaceutically acceptable salt thereof), or a pharmaceutically acceptable salt of any of the foregoing), or a pharmaceutical composition that includes a compound described herein such as Compound B or a pharmaceutically acceptable salt thereof).

[0170] Some embodiments described herein relate to using one or more compounds described herein (such as Compound B, or a pharmaceutically acceptable salt thereof), in the manufacture of a medicament for ameliorating and / or treating cancer or conditions of cancer, such as cancer cachexia, that can include administering to a subject an effective amount of one or more compounds described herein (such as Compound B or a pharmaceutically acceptable salt thereof). Still other embodiments described herein relate to one or more compounds described herein (such as Compound B or a pharmaceutically acceptable salt thereof) that can be used for ameliorating and / or treating cancer or conditions of cancer, such as cancer cachexia, by administering to a subject an effective amount of one or more compounds described herein, or a pharmaceutically acceptable salt thereof.

[0171] Some embodiments disclosed herein relate to methods of ameliorating and / or treating cancer that can include contacting a cancerous cell an effective amount of one or more compounds described herein (such as Compound B or a pharmaceutically acceptable salt thereof), or a pharmaceutical composition that includes one or more compounds described herein (such as Compound B or a pharmaceutically acceptable salt thereof). In some embodiments, Compound B or a pharmaceutically acceptable salt thereof, can act as an inhibitor of MEK. In some embodiments, Compound B or a pharmaceutically acceptable salt thereof, can act as an inhibitor of ERK. In some embodiments, Compound B or a pharmaceutically acceptable salt thereof, may act as a mitoSTAT3 inhibitor. In some embodiments, Compound B or a pharmaceutically acceptable salt thereof, may reduce inflammatory cachexia and muscle wasting.

[0172] In some embodiments, Compound B or a pharmaceutically acceptable salt thereof, may be administered in a single dose, once daily. In some embodiments, Compound B or a pharmaceutically acceptable salt thereof, may be administered in multiple doses, more than once per day. In some embodiments, Compound B or a pharmaceutically acceptable salt thereof, may be administered once a day. In some embodiments, Compound B or a pharmaceutically acceptable salt thereof, may be administered twice a day. In some embodiments, Compound B or a pharmaceutically acceptable salt thereof, may be administered trice a day. In some embodiments, Compound B or a pharmaceutically acceptable salt thereof, may be administered four times a day.

[0173] In some aspects, a Compound B or a pharmaceutically acceptable salt thereof, may inhibit abnormal cell growth. In some embodiments, the abnormal cell growth occurs in a mammal.

[0174] Methods for inhibiting abnormal cell growth may comprise administering an effective amount of Compound B or a pharmaceutically acceptable salt thereof, wherein abnormal cell growth is inhibited. Methods for inhibiting abnormal cell growth in a mammal may comprise administering to the mammal Compound B or a pharmaceutically acceptable salt thereof, wherein the amount of the compound is effective in inhibiting abnormal cell growth in the mammal.

[0175] In other aspects, the present disclosure is directed to a method for degrading, inhibiting the growth of or killing a cancer cell comprising contacting said cell with CompoundB or a pharmaceutically acceptable salt thereof, effective to degrade, inhibit the growth of or to kill said cell. In some embodiments, the cancer cells comprise brain, breast, lung, ovarian, pancreatic, stomach, prostate, renal, or colorectal cancer cells.

[0176] In some embodiments, the cancer cells are degraded. In some embodiments, 1 % of the cancer cells are degraded. In further or additional embodiments, 2% of the cancer cells are degraded. In further or additional embodiments, 3% of the cancer cells are degraded. In further or additional embodiments, 4% of the cancer cells are degraded. In further or additional embodiments, 5% of the cancer cells are degraded. In further or additional embodiments, 10% of the cancer cells are degraded. In further or additional embodiments, 20% of the cancer cells are degraded. In further or additional embodiments, 25% of the cancer cells are degraded. In further or additional embodiments, 30% of the cancer cells are degraded. In further or additional embodiments, 40% of the cancer cells are degraded. In further or additional embodiments, 50% of the cancer cells are degraded. In further or additional embodiments, 60% of the cancer cells are degraded. In further or additional embodiments, 70% of the cancer cells are degraded. In further or additional embodiments, 75% of the cancer cells are degraded. In further or additional embodiments, 80% of the cancer cells are degraded. In further or additional embodiments, 90% of the cancer cells are degraded. In further or additional embodiments, 100% of the cancer cells are degraded. In further or additional embodiments, essentially all of the cancer cells are degraded.

[0177] In some embodiments, the cancer cells are killed. In further or additional embodiments, 1% of the cancer cells are killed. In further or additional embodiments, 2% of the cancer cells are killed. In further or additional embodiments, 3% of the cancer cells are killed. In further or additional embodiments, 4% of the cancer cells are killed. In further or additional embodiments, 5% of the cancer cells are killed. In further or additional embodiments, 1.0% of the cancer cells are killed. In further or additional embodiments, 20% of the cancer cells are killed. In further or additional embodiments, 25% of the cancer cells are killed. In further or additional embodiments, 30% of the cancer cells are killed. In further or = Page 70 -additional embodiments, 40% of the cancer cells are killed. In further or additional embodiments, 50% of the cancer cells are killed. In further or additional embodiments, 60% of the cancer cells are killed. In further or additional embodiments, 70% of the cancer cells are killed. In further or additional embodiments, 75% of the cancer cells are killed. In further oradditional embodiments, 80% of the cancer cells are killed. In further or additional embodiments, 90% of the cancer cells arc killed. In further or additional embodiments, 100% of the cancer cells are killed. In farther or additional embodiments, essentially all of the cancer cells are killed.

[0178] In further or additional embodiments, the growth of the cancer cells is inhibited. In further or additional embodiments, the growth of the cancer cells is about 1% inhibited. In further or additional embodiments, the growth of the cancer cells is about 2% inhibited. In further or additional embodiments, the growth of the cancer cells is about 3% inhibited. In further or additional embodiments, the growth of the cancer cells is about 4% inhibited. In further or additional embodiments, the growth of the cancer cells is about 5% inhibited. In further or additional embodiments, the growth of the cancer cells is about 10% inhibited. In further or additional embodiments, the growth of the cancer cells is about 20% inhibited. In further or additional embodiments, the growth of the cancer cells is about 25% inhibited. In further or additional embodiments, the growth of the cancer cells is about 30% inhibited, hi further or additional embodiments, the growth of the cancer cells is about 40% inhibited. In further or additional embodiments, the growth of the cancer cells is about 50% inhibited. In further or additional embodiments, the growth of the cancer cells is about 60% inhibited. In further or additional embodiments, the growth of the cancer cells is about 70% inhibited. In further or additional embodiments, the growth of the cancer cells is about 75% inhibited. In further or additional embodiments, the growth of the cancer cells is about 80% inhibited. In further or additional embodiments, the growth of the cancer cells is about 90% inhibited. In further or additional embodiments, the growth of the cancer cells is about 100% inhibited.

[0179] In some embodiments, the size of a tumor is reduced by administering a therapeutically effective amount of Compound B or a pharmaceutically acceptable salt thereof. In further or additional embodiments, the size of a tumor is reduced by at least 1%. In further or additional embodiments, the size of a tumor is reduced by at least 2%. In further or additional embodiments, the size of a tumor is reduced by at least 3%. In further or additional embodiments, the size of a tumor is reduced by at least 4%. In further or additional embodiments, the size of a tumor is reduced by at least 5%. In further or additional embodiments, the size of a tumor is reduced by at least 10%. In further or additionalembodiments, the size of a tumor is reduced by at least 20%. In further or additional embodiments, the size of a tumor is reduced by at least 25%. In further or additional embodiments, the size of a tumor is reduced by at least 30%. In further or additional embodiments, the size of a tumor is reduced by at least 40%. In further or additional embodiments, the size of a tumor is reduced by at least 50%. In further or additional embodiments, the size of a tumor is reduced by at least 60%. In further or additional embodiments, the size of a tumor is reduced by at least 70%. In further or additional embodiments, the size of a tumor is reduced by at least 75%. In further or additional embodiments, the size of a tumor is reduced by at least 80%. In further or additional embodiments, the size of a tumor is reduced by at least 85%. In further or additional embodiments, the size of a tumor is reduced by at least 90%. In further or additional embodiments, the size of a tumor is reduced by at least 95%. In further or additional embodiments, the tumor is eradicated. In some embodiments, the size of a tumor does not increase.

[0180] In some embodiments, tumor proliferation is reduced by administering Compound B or a pharmaceutically acceptable salt thereof. In some embodiments, tumor proliferation is reduced by at least 1 %. In some embodiments, tumor proliferation is reduced by at least 2 %. In some embodiments, tumor proliferation is reduced by at least 3 %. In some embodiments, tumor proliferation is reduced by at least 4 %. In some embodiments, tumor proliferation is reduced by at least 5 %. In some embodiments, tumor proliferation is reduced by at least 10 %. In some embodiments, tumor proliferation is reduced by at least 20 %. In some embodiments, tumor proliferation is reduced by at least 25 %. In some embodiments, tumor proliferation is reduced by at least 30 %. In some embodiments, tumor proliferation is reduced by at least 40 %. In some embodiments, tumor proliferation is reduced by at least 50 %. In some embodiments, tumor proliferation is reduced by at least 60 %. In some embodiments, tumor proliferation is reduced by at least 70 %. In some embodiments, tumor proliferation is reduced by at least 75 %. In some embodiments, tumor proliferation is reduced by at least 75 %. In some embodiments, tumor proliferation is reduced by at least 80 %. In some embodiments, tumor proliferation is reduced by at least 90 %. In some embodiments, tumor proliferation is reduced by at least 95 %. In some embodiments, tumor proliferation is prevented.Detecting RAS-mutated cancer

[0181] In some embodiments, patients are selected for treatment with Compound B based on the presence of RAS mutation such as a KRAS G12D or KRAS G12S mutation in plasma or tumor specimens. If no mutation is detected in a plasma specimen, test tumor tissue. Additional information on FDA-approved tests for the detection of a KRAS G12C mutation is available at: https: / / www.fda.gov / CompanionDiagnostics. FDA approved diagnostics for identifying patients with a RAS-mutated cancer include the companion diagnostics provided in Table 2 below.Table 2.

[0182] In some embodiments, RAS mutations can be detected by next generation sequencing (NGS) techniques. For example, molecular diagnostics of HRAS / KRAS and NRAS targeted gene panels can be obtained by NGS techniques. Next generation sequencing of amplification products can be performed on the Ion Torrent Personal Genome Machine and analyzed with the Torrent Suite Software. Reference DNA sequences used for these genes can be found at https: / / www.ncbi.nlm.nih.gov / refseq / rsg / . This mutation panel is designed to detect targeted mutations only. Confirmation of these mutations is performed by traditional or real time PCR followed by Sanger sequencing, fluorescent melting curve analysis and / or pyro sequencing (CPT codes: 81210 (BRAF), 81275 (KRAS codons 12 and 13 in exon 2), 81276 (KRAS codon 61 in exon 3), 81403 (HRAS), 81311 (NRAS)).Definitions

[0183] The term “pharmaceutical composition,” as used herein, refers to a mixture of a compound disclosed herein with other chemical components, such as diluents or carriers. The pharmaceutical composition facilitates administration of the compound to an organism. Multiple techniques of administering a compound exist in the art including, but not limited to, oral, injection, aerosol, parenteral, and topical administration. Pharmaceutical compositions can also be obtained by reacting compounds with inorganic or organic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid and the like.

[0184] The term “carrier,” as used herein, refers to a chemical compound that facilitates the incorporation of a compound into cells or tissues. For example dimethyl sulfoxide (DMSO) is a commonly utilized carrier as it facilitates the uptake of many organic compounds into the cells or tissues of an organism.

[0185] The term “immune checkpoint inhibitor” as used herein refers to a molecule (e.g., small molecule, peptide, polypeptide, protein, antibody, antibody fragment and the like) that acts as an inhibitor (antagonist) of an immune checkpoint pathway. Inhibition of a pathway can include blockade of the pathway through binding to a receptor or signaling molecule that is part of the immune checkpoint pathway.

[0186] The term “diluent,” as used herein, refers to chemical compounds diluted in water that will dissolve the compound of interest as well as stabilize the biologically active form of the compound. Salts dissolved in buffered solutions are utilized as diluents in the art. One commonly used buffered solution is phosphate buffered saline because it mimics the salt conditions of human blood. Since buffer salts can control the pH of a solution at low concentrations, a buffered diluent rarely modifies the biological activity of a compound.

[0187] The term “physiologically acceptable,” as used herein, refers to a carrier or diluent that does not abrogate the biological activity and properties of the compound.

[0188] As used herein, an “excipient” refers to an inert substance that is added to a pharmaceutical composition to provide, without limitation, bulk, consistency, stability, binding ability, lubrication, disintegrating ability etc., to the composition. A “diluent” is a type of excipient.

[0189] The term “about,” as used herein, refers to a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length that varies by as much as 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1% to a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length. When a value is preceded by the term about, the component is not intended to be limited strictly to that value, but it is intended to include amounts that vary from the value.

[0190] The term “pharmaceutically acceptable carrier” or “pharmaceutically acceptable excipient,” as used herein, includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic compositions is contemplated. In addition, various adjuvants such as are commonly used in the art may be included. Considerations for the inclusion of various components in pharmaceutical compositions are described, e.g., in Gilman et al. (Eds.) (1990); Goodman and Gilman’s: The Pharmacological Basis of Therapeutics, 8th Ed., Pergamon Press, which is incorporated herein by reference in its entirety.

[0191] The term “pharmaceutically acceptable salt,” as used herein, refers to a salt of a compound that does not cause significant irritation to an organism to which it is administered and does not abrogate the biological activity and properties of the compound. In some embodiments, the salt is an acid addition salt of the compound. Pharmaceutical salts can be obtained by reacting a compound with inorganic acids such as hydrohalic acid (e.g., hydrochloric acid or hydrobromic acid), sulfuric acid, nitric acid, phosphoric acid and the like. Pharmaceutical salts can also be obtained by reacting a compound with an organic acid such as aliphatic or aromatic carboxylic or sulfonic acids, for example acetic, succinic, lactic, malic, tartaric, citric, ascorbic, nicotinic, methanesulfonic, ethanesulfonic, p-toluensulfonic, salicylic or naphthalenesulfonic acid. Pharmaceutical salts can also be obtained by reacting a compound with a base to form a salt such as an ammonium salt, an alkali metal salt, such as a sodium or a potassium salt, an alkaline earth metal salt, such as a calcium or a magnesium salt, a salt of organic bases such as dicyclohexylamine, N-methyl-D-glucamine, tris(hydroxymethyl)methylamine, C1-C7 alkylamine, cyclohexylamine, triethanolamine, ethylenediamine, and salts with amino acids such as arginine, lysine, and the like.

[0192] The term “subject,” as used herein, refers to a human or a non-human mammal, c.g., a dog, a cat, a mouse, a rat, a cow, a sheep, a pig, a goat, a non-human primate or a bird, e.g., a chicken, as well as any other vertebrate or invertebrate.Additional Embodiments

[0193] The following additional numbered embodiments, and combinations thereof, are also provided:

[0194] 1. A method of treating a patient with a RAS- or RAF- mutated cancer, comprising administering a therapeutically effective amount of a compound having a structure of Compound B:Compound B or a pharmaceutically acceptable salt thereof, in combination with a kinase inhibitor to the patient receiving to treat the RAS- or RAF- mutated cancer.

[0195] 2. The method of embodiment 1, wherein the kinase inhibitor is a BRAF inhibitor.

[0196] 3. The method of embodiment 2, wherein the BRAF inhibitor is dabrafenib, binimetinib, vemurafenib, encorafenib or a pharmaceutically acceptable salt thereof.

[0197] 4. A method of treating a patient with a RAS- or RAF- mutated cancer, comprising administering a therapeutically effective amount of a compound having a structure of Compound B:Compound Bor a pharmaceutically acceptable salt thereof, in combination with encorafenib to treat the RAS- or RAF- mutated cancer.

[0198] 5. The method of any one of embodiments 3 to 4, wherein the encorafenib is administered at a dose of about 300 mg to about 450 mg once daily.

[0199] 6. The method of any one of embodiments 3 to 4, wherein the encorafenib is administered at a dose of about 300 mg once daily.

[0200] 7. The method of any one of embodiments 3 to 4, wherein the encorafenib is administered at a dose of about 450 mg once daily.

[0201] 8. The method of any one of embodiments 5 to 7, wherein cancer has aBRAF V600D mutation.

[0202] 9. The method of any one of embodiments 5 to 7, wherein cancer has aBRAF V600E mutation.

[0203] 10. The method of any one of embodiments 5 to 7, wherein cancer has aBRAF V600K mutation.

[0204] 11. The method of any one of embodiments 8 to 10, wherein the cancer is unresectable or metastatic melanoma.

[0205] 12. The method of embodiment 9, wherein the cancer is metastatic colorectal cancer (CRC).

[0206] 13. The method of any one of embodiments 1 to 12, wherein the CompoundB is administered to the patient (a) in a dose amount that achieves a Cmax effective to inhibit pERK and pMEK and (b) with an interval between consecutive doses that is at least about seven times the plasma half to life of Compound B.

[0207] 14. A method of treating a patient with a melanoma cancer with a BRAFV600D, V600E or V600K mutation, the method comprising administering to the patient in need thereof 450 mg of encorafenib in combination with a therapeutically effective amount a compound having a structure of Compound B:Compound Bor a pharmaceutically acceptable salt thereof, to the patient in need thereof.

[0208] 15. The method of embodiment 14, wherein the cancer expresses a BRAFV600D mutation.

[0209] 16. The method of embodiment 1 , wherein the cancer expresses a BRAFV600E mutation.

[0210] 17. The method of embodiment 14, wherein the cancer expresses a BRAFV600K mutation.

[0211] 18. The method of any one of embodiments 15 to 17, wherein the cancer is unresectable or metastatic melanoma.

[0212] 19. A method of treating a patient with a colorectal cancer with a BRAFV600E mutation the method comprising administering to the patient in need thereof 300 mg of encorafenib in combination with a therapeutically effective amount a compound having a structure of Compound B:Compound B or a pharmaceutically acceptable salt thereof, to the patient in need thereof.

[0213] 20. The method of embodiment 19, wherein the cancer is metastatic colorectal cancer.

[0214] 21. A method of treating a patient with a RAS- or RAF- mutated cancer, comprising administering a therapeutically effective amount of an immune checkpoint inhibitor in combination with a compound having a structure of Compound B:

[0215] (Compound B)

[0216] or a pharmaceutically acceptable salt thereof, to the patient in need thereof.

[0217] 22. The method of embodiment 21, wherein the immune checkpoint inhibitor is an inhibitor of PD-1, PD- LI, PD- L2, PD- L3, PD- L4, CTLA- 4, LAG3, B7-H3, B7- H4, KIR or TIM3.

[0218] 23. The method of embodiment 21, wherein the immune checkpoint inhibitor is a PD- 1 inhibitor.

[0219] 24. The method of embodiment 21 , wherein the immune checkpoint inhibitor is a PD- L2 inhibitor.

[0220] 25. The method of embodiment 21, wherein the immune checkpoint inhibitor is a CTLA-4 inhibitor.

[0221] 26. The method of embodiment 21, wherein the immune checkpoint inhibitor is nivolumab, pembrolizumab, pidilizumab, ipilimumab, BMS 936559, tremelimumab, relatlimab, atezolizumab, avelumab, cemiplimab, durvalumab, tislelizumab, spartalizumab, or any combinations thereof.

[0222] 27. The method of any one of embodiments 21 to 26, wherein the cancer is selected from the group consisting of brain cancer, breast cancer, lung cancer, non to small cell lung cancer, ovarian cancer, pancreatic cancer, stomach cancer, prostate cancer, renal cancer, colorectal cancer or leukemia.

[0223] 28. The method of any one of embodiments 21 to 27, wherein the cancer is associated with a RAS mutation.

[0224] 29. The method of any one of embodiments 1 to 28, wherein the cancer has a RAS mutation that is a KRAS mutation selected from the group consisting of G12C, G12S, G12R, G12F, G12L, G12N, G12A, G12D, G12V, G13C, G13S, G13D, G13V, G13P, S17G, P34S, A59E, A59G, A59T, Q61K, Q61L, Q61R, and Q61H.

[0225] 30. The method of any one of embodiments 1 to 29, wherein Compound B is administered orally to the patient in need thereof.

[0226] 31. The method of any one of embodiments 1 to 30, wherein Compound B is administered only twice per day to the patient in need thereof.

[0227] 32. The method of any one of embodiments 1 to 31, wherein the therapeutically effective amount is a total of 40 mg to 640 mg of Compound B per day administered to the patient in need thereof.

[0228] 33. The method of any one of embodiments 1 to 32, wherein the therapeutically effective amount is a dose of 120 mg of Compound B per day administered twice per day (BID) to the patient in need thereof.

[0229] 34. The method of any one of embodiments 1 to 32, wherein the therapeutically effective amount is a dose of 140 mg of Compound B per day administered twice per day (BID) to the patient in need thereof.

[0230] 35. The method of any one of embodiments 1 to 32, wherein the therapeutically effective amount is a dose of 160 mg of Compound B per day administered twice per day (BID) to the patient in need thereof.

[0231] 36. A method of treating a patient with a cancer with a BRAF V600 mutation, the method comprising administering to the patient in need thereof a therapeutically effective amount of dabrafenib in combination with a therapeutically effective amount a compound having the structure of Compound B:Compound B or a pharmaceutically acceptable salt thereof, to the patient in need thereof.

[0232] 37. The method of embodiment 36, wherein the dabrafenib is orally administered in a dose of 2 mg to 150 mg once or twice daily.

[0233] 38. The method of embodiment 36, wherein dabrafenib is orally administered in a dose of 2 mg orally once daily.

[0234] 39. The method of any one of embodiments 36 to 38, wherein CompoundB is administered in a dose of 20 mg to 320 mg twice daily (BID).

[0235] 40. The method of any one of embodiments 36 to 38, wherein CompoundB is administered in a dose of 120 mg BID.

[0236] 41. The method of any one of embodiments 36 to 38, wherein CompoundB is administered in a dose of 160 mg BID.

[0237] 42. The method of any one of embodiments 36 to 41, wherein dabrafenib is administered as a dabrafenib mesylate salt, and the dose of debrafenib is calculated based on the weight of dabrafenib free base.

[0238] 43. The method of any one of embodiments 36 to 42, wherein the cancer has a BRAF V600D mutation.

[0239] 44. The method of any one of embodiments 36 to 42, wherein the cancer has a BRAF V600E mutation.

[0240] 45. The method of any one of embodiments 36 to 42, wherein the cancer has a BRAF V600K mutation.

[0241] 46. The method of any one of embodiments 36 to 42, wherein the cancer is unresectable or metastatic melanoma with a BRAF V600D, V600E or V600K mutation.

[0242] 47. A method of treating a patient with a cancer with a BRAF V600 mutation, the method comprising administering to the patient in need thereof a therapeutically effective amount of vemurafenib in combination with a therapeutically effective amount a compound having the structure of Compound B:Compound B or a pharmaceutically acceptable salt thereof, to the patient in need thereof.

[0243] 48. The method of embodiment 47, wherein the vemurafenib is orally administered in a dose of 960 mg twice daily.

[0244] 49. The method of any one of embodiments 47 to 48, wherein CompoundB is administered in a dose of 20 mg to 320 mg twice daily (BID).

[0245] 50. The method of any one of embodiments 47 to 48, wherein CompoundB is administered in a dose of 120 mg BID.

[0246] 51. The method of any one of embodiments 47 to 48, wherein CompoundB is administered in a dose of 160 mg BID.

[0247] 52. The method of any one of embodiments 47 to 51, wherein the cancer has a BRAF V600E mutation.

[0248] 53. The method of any one of embodiments 47 to 51, wherein the cancer is unresectable or metastatic melanoma with a BRAF V600D, V600E or V600K mutation.

[0249] 54. A method of treating a patient with a RAF to mutated cancer, comprising administering a therapeutically effective amount of a compound having the structure of Compound B:Compound B or a pharmaceutically acceptable salt thereof, in combination with a kinase inhibitor to a patient receiving to treat the RAF to mutated cancer.

[0250] 55. The method of embodiment 54, wherein the RAF to mutated cancer has a BRAF mutation.

[0251] 56. The method of embodiment 55, wherein the RAF to mutated cancer has a BRAF V600 mutation.

[0252] 57. The method of embodiment 56, wherein the RAF to mutated cancer has a BRAF V600E mutation.

[0253] 58. The method of embodiment 56, wherein the RAF to mutated cancer has a BRAF V600K mutation.

[0254] 59. The method of embodiment 56, wherein the RAF to mutated cancer has a BRAF V600D mutation.

[0255] 60. The method of embodiment 56, wherein the RAF to mutated cancer has a BRAF V600R mutation.

[0256] 61. The method of any one of embodiments 54 to 60, wherein the kinase inhibitor is a BRAF inhibitor.

[0257] 62. The method of embodiment 61, wherein the BRAF inhibitor is dabrafenib, or a pharmaceutically acceptable salt thereof.

[0258] 63. The method of embodiment 62, wherein the BRAF inhibitor is dabrafenib mesylate.

[0259] 64. The method of any one of embodiments 62 to 63, wherein the dabrafenib is orally administered at a dose equivalent to 2 mg of dabrafenib free base once daily.

[0260] 65. The method of embodiment 61 wherein the BRAF inhibitor is vemurafenib.

[0261] 66. The method of embodiment 65, wherein the vemurafenib is administered at a dose of 960 mg vemurafenib orally twice daily.

[0262] 67. The method of embodiment 61, wherein the BRAF inhibitor is binimetinib.

[0263] 68. The method of embodiment 67, wherein the binimetinib is administered orally at a dose of 45 mg binimetinib twice daily.

[0264] 69. A method of treating a patient with a RAS to mutated cancer, comprising administering a therapeutically effective amount of a compound having the structure of Compound B:Compound B or a pharmaceutically acceptable salt thereof, in combination with a kinase inhibitor to a patient receiving to treat the RAS to mutated cancer.

[0265] 70. The method of embodiment 69, wherein the RAS to mutated cancer has a KRAS mutation.

[0266] 71. The method of embodiment 70, wherein the KRAS mutation is a G12 mutation.

[0267] 72. The method of embodiment 70, wherein the KRAS mutation is a G12D mutation

[0268] 73. The method of embodiment 70, wherein the KRAS mutation is a G12S mutation.

[0269] 74. The method of embodiment 69, wherein the RAS to mutated cancer has a HR AS mutation.

[0270] 75. The method of embodiment 74, wherein the HRAS mutation is a G13 mutation.

[0271] 76. The method of embodiment 74, wherein the HRAS mutation is a G13D mutation.

[0272] 77. The method of embodiment 69, wherein the RAS to mutated cancer has a NR AS mutation.

[0273] 78. The method of embodiment 77, wherein the NRAS mutation is a Q61 mutation.

[0274] 79. The method of embodiment 77, wherein the NRAS mutation is a Q61R mutation.

[0275] 80. The method of embodiment 77, wherein the NRAS mutation is a Q61L mutation.

[0276] 81. The method of embodiment 77, wherein the NRAS mutation is a Q61K mutation.

[0277] 82. The method of embodiment 77, wherein the NRAS mutation is a Q61V mutation.

[0278] 83. The method of any one of embodiments 69 to 82, wherein the kinase inhibitor is a BRAF inhibitor.

[0279] 84. The method of embodiment 83, wherein the BRAF inhibitor is dabrafenib, or a pharmaceutically acceptable salt thereof.

[0280] 85. The method of embodiment 84, wherein the BRAF inhibitor is dabrafenib mesylate.

[0281] 86. The method of any one of embodiments 84 to 85, wherein the dabrafenib is orally administered at a dose equivalent to 2 mg of dabrafenib free base once daily.

[0282] 87. The method of embodiment 83, wherein the BRAF inhibitor is vemurafenib.

[0283] 88. The method of embodiment 87, wherein the vemurafenib is administered at a dose of 960 mg vemurafenib orally twice daily.

[0284] 89. The method of embodiment 83, wherein the BRAF inhibitor is binimetinib.

[0285] 90. The method of embodiment 89, wherein the binimetinib is administered orally at a dose of 45 mg binimetinib twice daily.

[0286] The following additional numbered embodiments, and combinations thereof, are also provided:

[0287] 1 . A method of treating a patient with a RAS- or RAF- mutated cancer, comprising administering a therapeutically effective amount of a compound having the structure of Compound B:Compound B or a pharmaceutically acceptable salt thereof, to the patient in need thereof.

[0288] 2. The method of embodiment 1, wherein Compound B is administered to the patient (a) in a dose amount that achieves a Cmax effective to inhibit pERK and pMEK and (b) with an interval between consecutive doses that is at least about seven times the plasma half to life of Compound B.

[0289] 3. The method of any one of embodiments 1 or 2, wherein the method comprises administering Compound B to the patient as a monotherapy to treat the cancer.

[0290] 4. The method of any one of embodiments lor 2, wherein the method comprises administering Compound B to the patient, wherein the patient is not receiving a compound for treating a BRAF mutated cancer or a checkpoint inhibitor compound to treat the RAF to mutated cancer.

[0291] 5. The method of any one of embodiments lor 2, wherein the method comprises administering Compound B to the patient without administering binimetinib or encorafenib to the patient to treat the cancer.

[0292] 6. The method of any one of embodiments lor 2, wherein the method comprises administering Compound B to the patient without administering binimetinib to the patient to treat the cancer.

[0293] 7. The method of any one of embodiments 1 or 2, wherein the method comprises administering Compound B to the patient without administering encorafenib to the patient to treat the cancer.

[0294] 8. The method of any one of embodiments 1 to 7, wherein the RAS to mutated cancer is a BRAF mutated cancer.

[0295] 9. The method of any one of embodiments 1 to 7, wherein the RAS to mutated cancer is a BRAF to V600E or V600K mutated cancer.

[0296] 10. The method of any one of embodiments 1 to 7, wherein the RAS to mutated cancer is selected from the group consisting of: RAS to mutated melanoma, RAS to mutated PDAC, RAS to mutated NSCLC and RAS to mutated CRC, where RAS is any activation mutation in KRAS, NRAS or HRAS.

[0297] 11. A method of treating a patient with a RAS- or RAF- mutated cancer, comprising administering a therapeutically effective amount of a compound having the structure of Compound B:Compound B or a pharmaceutically acceptable salt thereof, to the patient in need thereof, wherein the cancer expresses a KRAS mutation.

[0298] 12. The method of embodiment 11, wherein the cancer expresses a KRAS to G12D mutation.

[0299] 13. The method of embodiment 12, wherein the cancer is colorectal cancer.

[0300] 14. The method of embodiment 11, wherein the cancer expresses a KRAS to G12S mutation.

[0301] 15. The method of embodiment 14, wherein the cancer is lung cancer.

[0302] 16. The method of embodiment 14, wherein the cancer is NSCLC.

[0303] 17. A method of treating a patient with a RAS- or RAF- mutated cancer, comprising administering a therapeutically effective amount of a compound having the structure of Compound B:Compound B or a pharmaceutically acceptable salt thereof, to the patient in need thereof, wherein the cancer expresses a RAF mutation.

[0304] 18. The method of embodiment 17, wherein the RAS- mutated cancer has a BRAF mutation.

[0305] 19. The method of embodiment 17, wherein the RAS- mutated cancer has a BRAF to V600E mutation.

[0306] 20. The method of any one of embodiments 17 to 19, wherein the cancer is a melanoma cancer.

[0307] 21. The method of any one of embodiments 17 to 19, wherein the cancer is a colorectal cancer.

[0308] 22. The method of any one of embodiments 1 to 21, wherein Compound B is administered orally to the patient in need thereof.

[0309] 23. The method of any one of embodiments 1 to 22, wherein Compound B is administered once or twice per day to the patient in need thereof.

[0310] 24. The method of any one of embodiments 1 to 23, wherein the therapeutically effective amount is a total of 40 mg to 640 mg of Compound B per day administered to the patient in need thereof.

[0311] 25. The method of any one of embodiments 1 to 24, wherein the therapeutically effective amount is a dose of 120 mg of Compound B per day administered once (QD) or twice (BID) per day to the patient in need thereof.

[0312] 26. The method of any one of embodiments 1 to 24, wherein the therapeutically effective amount is a dose of 140 mg of Compound B per day administered once (QD) or twice (BID) per day to the patient in need thereof.

[0313] 27. The method of any one of embodiments 1 to 24, wherein the therapeutically effective amount is a dose of 160 mg of Compound B per day administered once (QD) or twice (BID) per day to the patient in need thereof.

[0314] 28. A method of treating a patient with a RAS- or RAF- mutated cancer, comprising administering a dose of 20 to 320 mg once (QD) or twice (BID) per day of a compound having the structure of Compound B:Compound B or a pharmaceutically acceptable salt thereof, to the patient in need thereof.

[0315] 29. The method of embodiment 28, wherein Compound B is orally administered (p.o.) to the patient.

[0316] 30. The method of any one of embodiments 28 to 29, wherein CompoundB is administered as a monotherapy to treat the patient.

[0317] 31. The method of any one of embodiments 28 to 29, wherein CompoundB is administered to a patient who is not being treated with any other compound for treatment of a BRAF to mutated cancer.

[0318] 32. The method of any one of embodiments 28 to 29, wherein CompoundB is administered to a patient who is not being treated with binimetinib or encorafenib.

[0319] 33. The method of any one of embodiments 28 to 32, wherein the cancer expresses a KRAS mutation.

[0320] 34. The method of embodiment 33, wherein the cancer expresses a KRASG12S or G12D mutation.

[0321] 35. The method of any one of embodiments 28 to 32, wherein the cancer is a RAS- or RAF- mutated cancer selected from the group consisting of colorectal cancer, lung cancer and melanoma cancer.

[0322] 36. A method of treating a patient with a BRAF to mutated lung cancer, the method comprising administering a dose of 120 mg once (QD) or twice (BID) per day p.o. of a compound having the structure of Compound B:Compound B,as free base or as a pharmaceutically acceptable salt thereof, to the patient in need thereof.

[0323] 37. A method of treating a patient with a BRAF to mutated lung cancer, the method comprising administering a dose of 140 mg once (QD) or twice (BID) per day p.o. of a compound having the structure of Compound B:Compound B, as free base or as a pharmaceutically acceptable salt thereof, to the patient in need thereof.

[0324] 38. A method of treating a patient with a BRAF to mutated lung cancer, the method comprising administering a dose of 160 mg once (QD) or twice (BID) per day p.o. of a compound having the structure of Compound B:Compound B, as free base or as a pharmaceutically acceptable salt thereof, to the patient in need thereof.

[0325] 39. The method of any one of embodiments 36 to 38, wherein the patient is diagnosed with NSCLC.

[0326] 40. A method of treating a patient with a RAS to mutated or a RAF to mutated melanoma cancer, the method comprising administering a dose of 120 mg once (QD) or twice (BID) per day p.o. of a compound having the structure of Compound B:Compound B, as free base or as a pharmaceutically acceptable salt thereof, to the patient in need thereof.

[0327] 41. A method of treating a patient with a RAS to mutated or a RAF to mutated melanoma cancer, the method comprising administering a dose of 140 mg once (QD) or twice (BID) per day p.o. of a compound having the structure of Compound B:Compound B, as free base or as a pharmaceutically acceptable salt thereof, to the patient in need thereof.

[0328] 42. A method of treating a patient with a RAS to mutated or RAF to mutated melanoma cancer, the method comprising administering a dose of 160 mg once (QD) or twice (BID) per day p.o. of a compound having the structure of Compound B:Compound B, as free base or as a pharmaceutically acceptable salt thereof, to the patient in need thereof.

[0329] 43. The method of any one of embodiments 40 to 42, wherein the RAS to mutated melanoma cancer has a NRAS mutation.

[0330] 44. The method of embodiment 43, wherein the NRAS mutation is selected from the group consisting of: Q61R, G13R, Q61L, and G12V.

[0331] 45. A method of treating a patient with a RAS to mutated pancreatic ductal adenocarcinoma (PDAC), comprising administering a therapeutically effective amount of a compound having the structure of Compound B:Compound Bor a pharmaceutically acceptable salt thereof, to the patient in need thereof, wherein the PDAC cancer expresses a KRAS mutation.

[0332] 46. The method of embodiment 45, wherein Compound B is administered orally to the patient in need thereof twice per day (p.o., BID).

[0333] 47. The method of embodiment 46, wherein a total dose of 20 mg to320 mg of Compound B is administered to the patient p.o., BID.

[0334] 48. The method of embodiment 46, wherein a total dose of 120 mg to 160 mg of Compound B is administered to the patient p.o., BID.

[0335] 49. The method of embodiment 46, wherein a total dose of 120 mg ofCompound B is administered to the patient p.o., BID.

[0336] 50. The method of embodiment 46, wherein a total dose of 160 mg ofCompound B is administered to the patient p.o., BID.

[0337] 51. The method of any one of embodiments 45 to 50, wherein CompoundB is administered as a second line of therapy to the patient who has previously been treated for PDAC.

[0338] 52. A method of treating a patient with a RAS to mutated melanoma, comprising administering a therapeutically effective amount of a compound having the structure of Compound B:Compound B or a pharmaceutically acceptable salt thereof, to the patient in need thereof, wherein the melanoma cancer expresses a HRAS, KRAS or NRAS mutation.

[0339] 53. The method of embodiment 52, wherein Compound B is administered orally to the patient in need thereof twice per day (p.o., BID).

[0340] 54. The method of embodiment 53, wherein a total dose of 20 mg to 320 mg of Compound B is administered to the patient p.o., BID.

[0341] 55. The method of embodiment 53, wherein a total dose of 120 mg to 160 mg of Compound B is administered to the patient p.o., BID.

[0342] 56. The method of embodiment 53, wherein a total dose of 120 mg ofCompound B is administered to the patient p.o., BID.

[0343] 57. The method of embodiment 53, wherein a total dose of 160 mg ofCompound B is administered to the patient p.o., BID.

[0344] 58. The method of any one of embodiments 52 to 57, wherein the melanoma expresses a HRAS mutation.

[0345] 59. The method of embodiment 58, wherein the HRAS mutation is G13D.

[0346] 60. The method of any one of embodiments 52 to 57, wherein the melanoma expresses a KRAS mutation.

[0347] 61. The method of embodiment 58, wherein the KRAS mutation is G13D.

[0348] 62. The method of any one of embodiments 52 to 57, wherein the melanoma expresses a NRAS mutation.

[0349] 63. The method of embodiment 62, wherein the NRAS mutation is a Q61 mutation.

[0350] 64. The method of embodiment 62, wherein the NRAS mutation is Q61R.

[0351] 65. The method of embodiment 62, wherein the NRAS mutation is Q61L.

[0352] 66. The method of embodiment 62, wherein the NRAS mutation is Q61K.

[0353] 67. The method of embodiment 62, wherein the NRAS mutation is Q61V.

[0354] 68. A method of treating a patient with a RAF to mutated melanoma, comprising administering a therapeutically effective amount of a compound having the structure of Compound B:Compound B or a pharmaceutically acceptable salt thereof, to the patient in need thereof, wherein the melanoma cancer expresses a BRAF mutation.

[0355] 69. The method of embodiment 68, wherein Compound B is administered orally to the patient in need thereof twice per day (p.o., BID).

[0356] 70. The method of embodiment 69, wherein a total dose of 20 mg to 320 mg of Compound B is administered to the patient p.o., BID.

[0357] 71. The method of embodiment 69, wherein a total dose of 120 mg to 160 mg of Compound B is administered to the patient p.o., BID.

[0358] 72. The method of embodiment 69, wherein a total dose of 120 mg ofCompound B is administered to the patient p.o., BID.

[0359] 73. The method of embodiment 69, wherein a total dose of 160 mg ofCompound B is administered to the patient p.o., BID.

[0360] 74. The method of any one of embodiments 68 to 73, wherein the melanoma expresses a BRAF V600E mutation.

[0361] 75. A method of treating a patient with a RAS to mutated lung cancer, comprising administering a therapeutically effective amount of a compound having the structure of Compound B:Compound B or a pharmaceutically acceptable salt thereof, to the patient in need thereof, wherein the lung cancer expresses a HRAS, KRAS or NRAS mutation.

[0362] 76. The method of embodiment 75, wherein Compound B is administered orally to the patient in need thereof twice per day (p.o., BID).

[0363] 77. The method of embodiment 76, wherein a total dose of 20 mg - 320 mg of Compound B is administered to the patient p.o., BID.

[0364] 78. The method of embodiment 76, wherein a total dose of 120 mg - 160 mg of Compound B is administered to the patient p.o., BID.

[0365] 79. The method of embodiment 76, wherein a total dose of 120 mg ofCompound B is administered to the patient p.o., BID.

[0366] 80. The method of embodiment 76, wherein a total dose of 160 mg ofCompound B is administered to the patient p.o., BID.

[0367] 81. The method of any one of embodiments 75 to 80, wherein the lung cancer is non to small cell lung cancer (NSCLC).

[0368] 82. The method of any one of embodiments 75 to 81, wherein the lung cancer has a KRAS mutation.

[0369] 83. The method of any one of embodiments 75 to 82, wherein the lung cancer has a KRAS G12 mutation.

[0370] 84. The method of any one of embodiments 75 to 83, wherein the lung cancer has a KRAS G12S mutation.

[0371] 85. A method of treating a patient with a RAS to mutated colorectal cancer, comprising administering a therapeutically effective amount of a compound having the structure of Compound B:Compound B or a pharmaceutically acceptable salt thereof, to the patient in need thereof, wherein the colorectal cancer expresses a HRAS, KRAS or NRAS mutation.

[0372] 86. The method of embodiment 85, wherein Compound B is administered orally to the patient in need thereof twice per day (p.o., BID).

[0373] 87. The method of embodiment 86, wherein a total dose of 20 mg to 320 mg of Compound B is administered to the patient p.o., BID.

[0374] 88. The method of embodiment 86, wherein a total dose of 120 mg to 160 mg of Compound B is administered to the patient p.o., BID.

[0375] 89. The method of embodiment 86, wherein a total dose of 120 mg ofCompound B is administered to the patient p.o., BID.

[0376] 90. The method of embodiment 86, wherein a total dose of 160 mg ofCompound B is administered to the patient p.o., BID.

[0377] 91. The method of any one of embodiments 85 to 90, wherein the colorectal cancer has a NRAS mutation.

[0378] 92. The method of any one of embodiments 85 to 91 , wherein the colorectal cancer has a KRAS mutation.

[0379] 93. The method of any one of embodiments 85 to 92, wherein the colorectal cancer has a KRAS G12 mutation.

[0380] 94. The method of any one of embodiments 85 to 93, wherein the colorectal cancer has a KRAS G12D mutation.

[0381] 95. A method of treating a patient with a RAF to mutated colorectal cancer, comprising administering a therapeutically effective amount of a compound having the structure of Compound B:Compound B or a pharmaceutically acceptable salt thereof, to the patient in need thereof, wherein the colorectal cancer expresses a BRAF mutation.

[0382] 96. The method of embodiment 95, wherein Compound B is administered orally to the patient in need thereof twice per day (p.o., BID).

[0383] 97. The method of embodiment 96, wherein a total dose of 20 mg to 320 mg of Compound B is administered to the patient p.o., BID.

[0384] 98. The method of embodiment 96, wherein a total dose of 120 mg to 160 mg of Compound B is administered to the patient p.o., BID.

[0385] 99. The method of embodiment 96, wherein a total dose of 120 mg ofCompound B is administered to the patient p.o., BID.

[0386] 100. The method of embodiment 96, wherein a total dose of 160 mg ofCompound B is administered to the patient p.o., BID.

[0387] 101. The method of any one of embodiments 95 to 100, wherein the colorectal cancer expresses a BRAF V600E mutation.

[0388] 102. A method of treating a patient with a RAS to mutated cancer, comprising orally administering (p.o.) 20 mg to 320 mg of a compound having the structure of Compound B:Compound B or a pharmaceutically acceptable salt thereof, twice per day (BID) to the patient in need thereof.

[0389] 103. The method of embodiment 102, wherein a total dose of 120 mg to 160 mg of Compound B is administered to the patient p.o., BID.

[0390] 104. The method of embodiment 103, wherein a total dose of 120 mg ofCompound B is administered to the patient p.o., BID.

[0391] 105. The method of embodiment 103, wherein a total dose of 160 mg ofCompound B is administered to the patient p.o., BID.

[0392] 106. The method of any one of embodiments 102 to 105, wherein the RAS to mutated cancer has a G12 mutation.

[0393] 107. The method of any one of embodiments 102 to 105, wherein the RAS to mutated cancer has a G13 mutation.

[0394] 108. The method of any one of embodiments 102 to 105, wherein the RAS to mutated cancer has a Q61 mutation.

[0395] 109. The method of any one of embodiments 102 to 105, wherein the cancer has a KRAS mutation.

[0396] 110. The method of any one of embodiments 102 to 109, wherein the cancer is a pancreatic, colorectal or lung adenocarcinoma.

[0397] 111. The method of any one of embodiments 102 to 105, wherein the cancer has a NRAS or HRAS mutation.

[0398] 112. The method of any one of embodiments 102 to 108 or 111, wherein the cancer is a hematologic, melanoma or thyroid cancer.

[0399] 113. The method of any one of embodiments 102 to 105 or 109 wherein the cancer has a non to G12 KRAS mutation.

[0400] 114. The method of embodiment 113, wherein the cancer is colorectal cancer.EXAMPLES

[0401] Additional embodiments are disclosed in further detail in the following examples, which are not in any way intended to limit the scope of the claims. It is recognized that the skilled artisan in the art of organic chemistry can readily carry out manipulations without further direction, that is, it is well within the scope and practice of the skilled artisan to carry out these manipulations. These include reduction of carbonyl compounds to their corresponding alcohols, oxidations, acylations, aromatic substitutions, both electrophilic and nucleophilic, etherifications, esterification and saponification and the like. These manipulations are discussed in standard texts such as March’s Advanced Organic Chemistry (Wiley), Carey and Sundberg, Advanced Organic Chemistry (incorporated herein by reference in their entirety) and the like.

[0402] The skilled artisan will readily appreciate that certain reactions are best carried out when other functionality is masked or protected in the molecule, thus avoiding any undesirable side reactions and / or increasing the yield of the reaction. Often the skilled artisan utilizes protecting groups to accomplish such increased yields or to avoid the undesired reactions. These reactions are found in the literature and are also well within the scope of the skilled artisan. Examples of many of these manipulations can be found for example in T. Greene and P. Wuts Protecting Groups in Organic Synthesis, 4th Ed., John Wiley & Sons (2007), incorporated herein by reference in its entirety.

[0403] The following example schemes are provided for the guidance of the reader and represent preferred methods for making the compounds exemplified herein. These methods are not limiting, and it will be apparent that other routes may be employed to prepare these compounds. Such methods specifically include solid phase-based chemistries, including combinatorial chemistry. The skilled artisan is thoroughly equipped to prepare these compounds by those methods given the literature and this disclosure. The compound numberings used in the synthetic schemes depicted below are meant for those specific schemes only, and should not be construed as or confused with same numberings in other sections of the application.

[0404] The following abbreviations are used in the Examples:BID = bis in die (twice daily);BWL = body weight loss;• PO = per os (oral).Materials & Methods for Examples 6-9

[0405] Media components, reagents and buffers for Western Blot: All cell culture media components were obtained from ThermoFisher Scientific. Cell lysis / Protein Extraction Reagent (Cell Signal Technology, Cat No: 9803). 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM NaiEDTA, 1 mM EGTA, 1% Triton, 2.5 mM sodium pyrophosphate, 1 mM beta-glycerophosphate, 1 mM NaaVCE, 1 g / ml leupeptin, Protease inhibitors (Roche, Cat no.11873580001), Phosphatase inhibitors (Cell Signaling Technologies, Cat No. 5870). Coomassie protein assay reagent (ThermoFisher Scientific, Cat. No. 1856209). Laemmli sample loading 4X buffer (ThermoFisher Scientific, Cat No. NP0007). MOPS / SDS electrophoresis running buffer (GenScript, Cat No. MOO138). Tris-buffered saline with Tween 20 (TBST buffer): 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.1% Tween 20 NuPAGE gels, 4-12% (ThermoFisher Scientific, Cat No. NP0322BOX). iBLOT nitrocellulose transfer kit (ThermoFisher Scientific Cat No. IB301002). Blocking Buffer (LICOR Cat No. 927-50000).

[0406] Antibodies: Phospho-STAT3 (S727), mouse polyclonal antibodies were obtained from BD Biosciences (Cat No. 612542), following 5 antibodies were obtained from Cell Signaling Technologies. Anti-STAT3, rabbit monoclonal antibodies (Cat No. 12640), Anti- phospho-MEKl / 2 (S218 / S222), rabbit polyclonal antibodies (Cat No: 9121), Anti MEK- 1 / 2, rabbit monoclonal antibodies (Cat No: 9122), Anti-ERK, mouse monoclonal antibodies (Cat No: 9107), and Anti-phospho-ERK, rabbit monoclonal antibodies (Cat No. 4377).

[0407] Secondary antibodies: IRDye 800CW goat anti-rabbit antibodies (LICOR Cat No. 926-32211), IRDye 680RD goat anti-rabbit antibodies (LICOR Cat No. 926-68071), IRDye 800CW goat anti-mouse antibodies (LICOR Cat No. 926-32210) and IRDye 680RD goat anti-mouse antibodies (LICOR Cat No. 926-68070).

[0408] Tumor Cell Lines: Cell Lines and Tissue Culture conditions: The A549 (Cat No. CCL-185) cell line was obtained from American Type Culture Collection (ATCC) and grown in T75 flasks in DMEM containing 10% FBS and Pen-Strep at 37°C in a humidified, 5% CO2 incubator.

[0409] The Colon26 syngeneic adenocarcinoma cell line was obtained from the National Cancer Institute. Colon26 tumor cells were maintained as exponentially growing cultures in RPMI-1640 medium containing 10% fetal bovine scrum, 2 mM glutamine, 100 units / mL penicillin G sodium, 100 pg / mL streptomycin sulfate, 25 pg / mL gentamicin, 10 mM HEPES, and 0.075% sodium bicarbonate. The tumor cells were grown in tissue culture flasks in a humidified incubator at 37 °C, in an atmosphere of 5% CO2 and 95% air.

[0410] Subculture conditions: Adherent cells were grown to approximately 90% confluency, culture medium was aspirated, and the cell layer was rinsed with PBS. Two mL trypsin solution (0.25%) was added to the flask and observed under an inverted microscope until cell layer is dispersed. Eight mL media was added, cells were spun down at 1000 x g for 5 minutes. Cell pellet was re-suspended in 10 mL media and an appropriate volume was inoculated into a new culture flask.

[0411] Compound (drug) treatment: Cells were plated in a 6-well plate at a density of 250,000 - 300,000 cells / well in 3mL media and incubated 37°C in a humidified, 5% CO2 incubator. Next day, lOmM stock solutions of compounds were diluted 10- and 100-fold in DMSO to yield 100 and 10 pM solutions, respectively. These solutions were added to the cells (3 pL / well), mixed by swirling the plate, and incubated at 37oC in a humidified, 5% CO2 incubator for 2 hours.

[0412] Cell lysis and protein estimation: Cells were washed with PBS, and scraped in 50 pL of lysis buffer containing protease and phosphatase inhibitors. Cell lysates were stored at -20°C. Cell lysates were thawed and spun at 12,000 rpm for one minute, 3 pl of the supernatant was added to 500 pL of Coomassie blue reagent following by 500 pL of water. Absorbance was read at 595 nm after 10 minutes of incubation. Protein standards were used (0 - 20 mg / mL) to calculate protein concentrations of test samples.

[0413] Western Blotting: For electrophoresis 20 ug of protein was mixed with 5 pl of 4X Laemmle’s sample buffer and 1 pl of 0.4 M DTT in a volume of 20 pl made up with lysis buffer. All samples were heated at 95°C for 5 minutes, cooled to room temperature and spun down. Protein samples were loaded onto 4-12% polyacrylamide gels and run at 100V for approximately 1.5 hours till the blue dye reached the bottom. After the run, gel was removed and protein transfer was done using iBlot for 7 minutes, as per manufacturer’s recommendations. After the transfer, nitrocellulose membrane was incubated on a shaker in 5mL of blocking buffer at room temperature for Ihr. The blot was then incubated overnight on a shaker in 5 ml of blocking buffer containing 0.2% Twccn-20 and primary antibody, at room temperature. Anti-phospho-STAT3 antibody was used at a dilution of 1:500, the other 3 primary antibodies were used at a dilution of 1:1000.

[0414] Next day, the blot was washed 3 times for 10 min each with 10 mL of TBST followed by incubation on a shaker in 5 ml of blocking buffer containing 0.2% Tween-20 and 0.5 pl of the IRDye labeled secondary antibodies, diluted 1:10000, at room temperature for Ihr. The blot was then washed 3 times for 10 min each with 10 mL of TBST and dried between sheets of paper towels. Imaging was done using LICOR’s Odyssey imaging system, quantitation was done using their software, Image Studio version 3.1.

[0415] Animal Studies: Female BALB / c mice (BALB / c Crl, Charles River) were eleven weeks old on Day 1 of the study and had a body weight (BW) range of 15.8 to 21.4 g. The animals were fed ad libitum water (reverse osmosis, 1 ppm Cl) and NIH 31 Modified and Irradiated Lab Diet® consisting of 18.0% crude protein, 5.0% crude fat, and 5.0% crude fiber. The mice were housed on irradiated Enrich-o’cobs™ bedding in static microisolators on a 12- hour light cycle at 20-22 °C (68-72 °F) and 40-60% humidity. Charles River Discovery Services North Carolina (CR Discovery Services) specifically complies with the recommendations of the Guide for Care and Use of Laboratory Animals with respect to restraint, husbandry, surgical procedures, feed and fluid regulation, and veterinary care. The animal care and use program at CR Discovery Services is accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC), which assures compliance with accepted standards for the care and use of laboratory animals.

[0416] In Vivo Implantation and Growth: Colon26 tumor cells used for implantation were harvested during log phase growth and re-suspended at a concentration of 1 x 107cells / mL in cold phosphate buffered saline (PBS). Each mouse was injected subcutaneously in the right flank with 1 x 106tumor cells (in a 0.1 mL cell suspension). The tumors were measured with a caliper in two dimensions to monitor size as the mean volume approached the desired 80 to 120 mm3range. Tumor size was calculated using the formula: Tumor volume (mm3) = (w2-l ) / 2, where w = width and 1 = length, in mm, of a tumor. Tumor weight may be estimated with the assumption that 1 mg is equivalent to 1 mm3 of tumor volume. Tumors were measured with a caliper twice weekly for the duration of the study.

[0417] Treatment: Tumor bearing BALB / c mice were randomized into treatment groups once target range was reached (n=5 per group). All treatments were administered orally (p.o.) once a day for fourteen days (QD x 14) in a volume of 10 mL / kg (0.2 mL per 20 g mouse), adjusted to the BW of each animal.

[0418] Sampling for Pharmacokinetic analysis: Blood, skeletal muscle, livers and tumors were collected from three animals each from the designated groups two hours after animals received a single dose. Full blood volume was collected by terminal cardiac puncture under isoflurane anesthesia, processed for plasma and the presence of K2EDTA anti-coagulant and stored at -80 °C. Skeletal muscle groups composed of right and left gastrocnemius, tibialis and soleus muscles were collected as a unit, snap frozen and stored at -80 °C. Livers were collected were snap frozen and shipped to CRL-Worcester for bioanalytical analysis. Sample inventories are appended.

[0419] Data Analysis: Tumors were measured using calipers twice per week with the data being expressed as either median + / - interquartile range or as individual plots in days. Tumor growth inhibition (TGI) was calculated as follows: %TGI =1 - (T / C) x 100, where: T = median Tumor volume for a treatment group, and C = median Tumor volume for the designated control group.EXAMPLE 1Synthesis of Compound B

[0420] Compound B was prepared in 1 step:

[0421] Step 1: 3-(3-bromo-2-fluorobenzyl)-4-((dimethylamino)methyl)-2-oxo- 2H-chromen-7-yl dimethylcarbamate (50 mg, 0.105 mmol, 1.0 eq.) was dissolved in Toluene (dry) (0.2 M) and flushed with argon. Then, Pd2(dba)3 (4.80 mg, 5.24 pmol, 0.05 eq.), t- BuXPhos (8.90 mg, 0.021 mmol, 0.2 eq.), K2CO3 (29.0 mg, 0.209 mmol, 2.0 eq.) and 2- methylpropane-2-sulfinamide (25.4 mg, 0.209 mmol, 2.0 eq.) were added. The formed reaction mixture was stirred for 17 hours at 50 °C. The reaction mixture was flushed with argon andPd2(dba)3 (4.80 mg, 5.24 pmol, 0.05 eq.) and t-BuXPhos (8.90 mg, 0.021 mmol, 0.2 eq.) were added. The reaction mixture was stirred for 48 hours at 50 °C. The impure product was purified by prep, basic to obtain the title compound (40 mg, 0.076 mmol, yield: 72.7%) as a white solid. Yield: The title compound was isolated as a white solid (73% over 1 step).

[0422] 3-(2-chloro-3-((N-methylsulfamoyl)amino)benzyl)-4- ((dimethylamino)methyl)-2-oxo-2H-chromen-7-yl dimethylcarbamate Analysis: LCMS (Method T): tR - 1.70 min; m / z calculated for [M+H]+ = 518.2, found = 518.2; 1H NMR (400 MHz, DMSO) 5 8.08 (d, J = 8.8 Hz, 1H), 7.61 (s, 1H), 7.22 (d, J = 2.3 Hz, 1H), 7.17 - 7.06 (m, 2H), 6.97 (t, J = 7.8 Hz, 1H), 6.76 (t, J = 7.0 Hz, 1H), 4.04 (s, 2H), 3.65 (s, 2H), 3.06 (s, 3H), 2.93 (s, 3H), 2.18 (s, 6H), 1.25 (s, 9H).EXAMPLE 2In Vitro Tarkget Engagement and Inhibition of pERK and pMEK

[0423] During a selectivity scan with more than 468 kinases, Compound B at 1 and 10 pM demonstrated high selectivity for MEK1 with 1.3% and 0.1% activity compared to control (98.7% and 99.9% inhibition) and MEK2 with 3.8% and 0.3% activity compared to control (96.2% and 99.7% inhibition), respectively.

[0424] In primary pharmacodynamic assays, Compound B inhibited pERK across a large panel of tumor cell models harboring either a KRASG12Sor a BRAFv600Emutation with IC50 (for pERK inhibition) values ranging from 44.4 nM to 153 nM. Potency was measured in cell-based assays (values arc the mean of different experiments, n=2 or n=3).Thc most sensitive cell lines were a KRASG12Smutant lung adenocarcinoma A549, and two BRAFv600Emutant colon carcinoma lines, HT-29 and CL-34 (Table 3).Table 3.

[0425] Corresponding reductions or maintenance in pMEK levels were observed within 2 hours following treatment of cells with 100 nM Compound B, compared to the induction of pMEK levels observed after a 2-hour treatment with first-generation MEK inhibitors, confirming that Compound B was not sensitive to the MAPK pathway feedback reactivation events, such as CRAF-bypass, that are a known limitation of first-generation MEK inhibitors currently in clinical use.EXAMPLE 3AEfficacy of Compound B, Binimetinib and Encorafenib inA375 BRAF V600E Human Melanoma Tumor Model as Monotherapy

[0426] Compound B was evaluated in a head-to-head against two FDA-approved MEK inhibitors to assess their control of pMEK, pERK and CRAF-bypass in a NRAS, HRAS, NF1 and BRAF mutant melanoma tumor models. The tumor cells were exposed with 100 nM of each drug for 2 hours and evaluated MEK and ERK activation levels. It was observed that Compound B reduced overall activity of the MAPK pathway at ERK and pathway reactivation at MEK through a decrease in MEK and ERK activation, resulting in CRAF-bypass resistance in RAS-mutant tumors. In contrast, it was observed that both FDA-approved MEK inhibitors displayed an increase in activated MEK, resulting in CRAF-bypass in RAS and NF1 mutant tumors (as depicted in the Examples).

[0427] Compound B was evaluated head-to-head against binimetinib and encorafenib monotherapy (Figures 4A-4F). The anti-tumor effect of monotherapy of Compound B was evaluated in a BRAF mutant melanoma tumor model (A) compared to binimetinib (B) and encorafenib (C) on the tumor growth rate of SC pre-established A375 Human malignant melanoma model. Also shown is the percent body weight change for each respective treatment (Figures 4D, 4E, 4F, respectively). Tumor volumes presented as the median tumor volume (Median + individual, mm3) of each group, with body weights expressed as median % baseline (Median + individual, %). Figure 4A-1 and Figure 4A-2 are graphs ofdata from Example 2A showing the efficacy of monotherapy of Compound B on the tumor growth rate of SC pre-established A375 Human malignant melanoma model for each respective treatment doses (90 - 180 mg / kg p.o. BID). Figure 4D is a graph of data from Example 2A showing the tolerability of monotherapy of Compound B as the percent body weight change for each respective treatment doses of SC pre-established A375 Human malignant melanoma model (90 - 180 mg / kg p.o. BID). Figure 4B is a graph of data from Example 2A showing the comparative efficacy of monotherapy of binimetinib on the tumor growth rate of SC pre-established A375 Human malignant melanoma model for the treatment dose (3.5 mg / kg p.o. BID). Figure 4E is a graph of data from Example 2A showing the efficacy of monotherapy of binimetinib as the percent body weight change for SC pre-established A375 Human malignant melanoma model for the treatment dose (3.5 mg / kg p.o. BID). Figure 4C is a graph of data from Example 2A showing the comparative efficacy of monotherapy of encorafenib on the tumor growth rate of SC pre-established A375 Human malignant melanoma model for the treatment dose (60 mg / kg p.o. BID). Figure 4F is a graph of data from Example 2A showing the efficacy of monotherapy of encorafenib as the percent body weight change for SC pre-established A375 Human malignant melanoma model for the treatment dose (60 mg / kg p.o. BID).

[0428] Compound B was next evaluated head-to-head against the combination of binimetinib with encorafenib (Figure 5A-5B-1) in the A375 BRAFV600E human melanoma mouse tumor xenograft model. The administered combination of binimetinib and encorafenib for BRAF mutant melanoma, such as BRAFV600E / K, is an FDA-approved combination (CDER Review NDA #210498). In addition, the drug doses and schedules used for binimetinib and encorafenib in these studies were aligned with human-relevant doses provided in the NDAs submitted to the FDA (CDER Review NDA #210498). The anti-tumor effect of combination of binimetinib at 3.5 mg / kg BID and encorafenib at 60 mg / kg on the tumor growth rate of SC pre-established A375 Human malignant melanoma model (Figure 5A-1 and 5A-2). Also shown is the percent body weight change for each respective treatment (Figure 5B-1). Tumor volumes presented as the median tumor volume (Median + individual, mm3) of each group, with body weights expressed as median % baseline (Median + individual, %).

[0429] When comparing Compound B alone (Figure 4A- 1 and Figure 4A-2) to the combination of binimetinib and encorafenib (Figure 5A), we observed that the combinationtherapy had greater overall tumor growth inhibition on day 21 (Table 4). Notably, the clinical challenge for MEK inhibitors or MEK + RAF inhibitors is poor overall tolerability, which often leads to dose reductions or termination of treatment. When Compound B was compared to binimetinib alone, Compound B displayed superior TGI (Figure 4A-1 vs. Figure 4B) at any of the dose levels tested. Given the greater single agent activity of Compound B observed over binimetinib alone, head-to-head, one can envision expanding Compound B into combinations with other MAPK pathway inhibitors, such as encorafenib, dabrafenib or vemurafenib, to treat BRAFV600E / K mutant cancers.

[0430] Table 4 provides the percentage Tumor Growth Inhibition (%TGI) on Day 21 in A375 BRAFV600EMelanoma Tumor Model.Table 4EXAMPLE 3BEfficacy of Compound B, Binimetinib and Encorafenib in A375 BRAF V600E Human Melanoma Tumor Model as Combination Therapy

[0431] Figures 8 A and 8B illustrates graphs depicting Compound B, binimetinib, encorafenib alone and combinations in A-375 xenograft melanoma human tumor mouse model.Cell Culture

[0432] The A-375 tumor cells were maintained in vitro with DMEM medium supplemented with 10% fetal bovine serum at 37°C in an atmosphere of 5% CO2 in air. The cells in exponential growth phase were harvested and quantitated before tumor inoculation. Tumor Inoculation

[0433] Each mouse was inoculated subcutaneously in the right upper flank region with 5xl06tumor cells in 0.1 ml of PBS with matrigel (1:1) for tumor development. Randomization

[0434] The randomization started when the mean tumor size reached approximately 147.05 mm3. 174 mice were enrolled in the study. All animals were randomly allocated to 14 study groups, 12 mice in groups 1-14 and 6 mice in group 15. Randomization was performed based on “Matched distribution” method (StudyDirector™ software, version 3.1.399.19). The date of randomization was denoted as day 0.Test Article Administration

[0435] The treatment was initiated on the same day of randomization (day 0) per study design.Observation and Data Collection

[0436] After tumor cells inoculation, the animals were checked daily for morbidity and mortality. During routine monitoring, the animals were checked for any effects of tumor growth and treatments on behavior such as mobility, food and water consumption, body weight gain / loss (Body weights were measured twice per week after randomization), eye / hair matting and any other abnormalities. Mortality and observed clinical signs were recorded for individualanimals in detail. Tumor volumes were measured twice per week after randomization in two dimensions using a caliper, and the volume was expressed in mm3 using the formula: “V = (L x W x W) / 2, where V was tumor volume, L was tumor length (the longest tumor dimension) and W was tumor width (the longest tumor dimension perpendicular to L). Dosing as well as tumor and body weight measurements were conducted in a Laminar Flow Cabinet.

[0437] The body weights and tumor volumes were measured by using StudyDirector™ software (version 3.1.399.19).Discussion / Conclusions

[0438] Here, the anti-tumor activity of Compound B as single agent was assessed in human A-375 BRAFv600Emutant melanoma tumor-bearing mice in the specified doses. Single agent activity of Compound B was compared to binimetinib and encorafenib as single agents, as well as in combination with encorafenib compared to binimetinib with encorafenib (FDA approved combination for BRAF mutant cancers; CDER Review NDA #210498).

[0439] As the vehicle treatment groups were euthanized due to reaching ethical endpoints, the TGI values were calculated on day 21. Compound B treatment alone administered at 90 mg / kg, 120 mg / kg and 180 mg / kg PO, BID showed a dose-dependent antitumor response with 38.5%, 52.6% and 87.4% of TGI value on day 21 respectively (P = 0.0924, 0.0027 and <0.0001 on day 21), compared to the vehicle group (Figures 8A and 8B; showing only 180 mg / kg BID PO). Encorafenib treatment alone administered at 60 mg / kg QD PO showed major anti-tumor response with 102.4% TGI on day 21 (P < 0.0001) compared to the vehicle group (Figures 8A and 8B). Binimetinib treatment alone administered at 3.5 mg / kg BID PO showed anti-tumor response with 26.1% TGI on day 21 (P = 0.9114) compared to the vehicle group (Figures 8 A and 8B). Co-administration of encorafenib (60 mg / kg, QD PO) and binimetinib (3.5 mg / kg, BID PO) showed anti-tumor efficacy with TGI of 103.9% on day 21 (P < 0.0001) when compared to the vehicle group (Figures 8A and 8B). Co-administration of Compound B (90 mg / kg, 120 mg / kg and 180 mg / kg BID PO) with encorafenib (60 mg / kg QD PO) showed major anti-tumor response with 103.7%, 104.6% and 104.9% TGI on day 21 respectively (P < 0.0001) compared to the vehicle group (Figures 8A and 8B). When animals that had not reached the ethical limit continued treatment to day 35, median tumor volume of the encorafenib and binimetinib treated group was 95.94 mm3and increasing. Treatment withCompound B (180 mg / kg) and encorafenib (60 mg / kg) showed a median tumor volume of 15.78 mm3on day 35, but differences were not significant.

[0440] When comparing Compound B alone (Figure 4A-1) to the combination of binimetinib and encorafenib (Figure 10B), it was observed that the combination therapy had greater overall tumor growth inhibition on day 21 (Table 4). Notably, the clinical challenge for MEK inhibitors or MEK + RAF inhibitors is poor overall tolerability, which often leads to dose reductions or termination of treatment. When Compound B was compared to binimetinib alone, Compound B displayed superior TGI (Figure 4A-1 vs. Figure 4B) at any of the dose levels tested. Also, when Compound B was dosed in combination with encorafenib, it displayed higher TGI to that of the encorafenib and binimetinib combination, particularly clear when tumor volume is observed on the log scale (Figure 8B). Greater single agent activity of Compound B over binimetinib alone, head-to-head, suggests a potential opportunity to expand Compound B into combinations with other MAPK pathway inhibitors, such as encorafenib, dabrafenib or vemurafenib, to treat BRAFV600E / Kmutant cancers.

[0441] Figures 10A-10D are graphs showing the anti-tumor effect of combination of Compound B at the specified doses + encorafenib at 60 mg / kg (A) or binimetinib at 3.5 mg / kg BID + encorafenib at 60 mg / kg (B) on the tumor growth rate of SC pre-established A375 Human malignant melanoma model. Also shown is the percent body weight change for each respective treatment (C, D). Tumor volumes presented as the median tumor volume (Median + individual, mm3) of each group, with body weights expressed as median % baseline (Median + individual, %).

[0442] In summary, Compound B was very well tolerated in this model. Compound B as a single agent produced dose dependent anti-tumor activities with statistical significance. The anti-tumor response of Compound B’s highest dose (180 mg / kg PO BID) was comparable to that of 150 mg / kg PO BID Compound B, 87.4% versus 93.0 % TGI. Compound B in combination with encorafenib resulted in tumor regressions at all doses and was comparable or superior to the combination of binimetinib and encorafenib.EXAMPLE 4AEfficacy of Compound B, Binimetinib and Encorafenib inHT-29 BRAFV600EHuman Colorectal Cancer Tumor Model as Monotherapy

[0443] After observing strong in vivo activity for Compound B in three tumor models (i.e., KRAS G12D mutant Colon-26, KRAS G12S mutant A549, and BRAFV600Emutant A375), additional in vivo studies were selected to evaluate the broader therapeutic and translational potential of Compound B.

[0444] The efficacy of Compound B was evaluated in HT-29, a BRAFV600Ehuman colorectal cancer (CRC) xenograft tumor model, as single agent, and compared head-to-head with binimetinib, and encorafenib. Figure 6A-1 and 6A-2 are graphs of data showing the efficacy of monotherapy of Compound B on the tumor growth rate of SC pre-established HT- 29 Human colorectal cancer model for each respective treatment doses (90 - 180 mg / kg p.o. BID). Figure 6B is a graph of data from Example 5 showing the tolerability of monotherapy of Compound B as the percent body weight change for each respective treatment doses of SC pre-established HT-29 Human colorectal cancer model (90 - 180 mg / kg p.o. BID). Tumor volumes presented as the median tumor volume (Median + individual, mm3) of each group, with body weights expressed as median % baseline (Median + individual, %).

[0445] Compound B showed a dose-dependent tumor growth inhibition, reaching 73.3% TGI at 180 mg / kg BID PO, and outperforming the commercially available MEK and RAF inhibitors as single agents (3.5 mg / kg BID PO binimetinib TGI = 26%; 60 mg / kg QD PO encorafenib TGI = 59.6%), while maintaining body weight (Figure 6A-1, 6A-2, B, C for tumor volume; Figures 6D, E, F for body weight change). Remarkably, Compound B as a single agent showed comparable tumor growth inhibition than the encorafenib and binimetinib combination (180 mg / kg BID PO Compound B TGI = 73.3% vs 3.5 mg / kg BID PO binimetinib + 60 mg / kg QD PO encorafenib TGI = 74%; Figure 6A-1 / 6A-2 vs. 9B-1 / 9B-2). This data in the HT-29 human colorectal cancer model further supports Compound B as a good candidate to treat B RAFv600E / Kmutant cancers.Cell Culture

[0446] The HT29 tumor cells were maintained in vitro with McCoy's 5a medium supplemented with 10% fetal bovine serum at 37°C in an atmosphere of 5% CO2 in air. The cells in exponential growth phase were harvested and quantitated before tumor inoculation.Tumor Inoculation

[0447] Each mouse was inoculated subcutaneously in the right upper flank region with 3xl06tumor cells in 0.1 mL of PBS for tumor development.Randomization

[0448] The randomization started when the mean tumor size reached approximately 134.16 mm3. 234 mice were enrolled in the study. All animals were randomly allocated to 20 study groups, 12 mice in groups 1-19 and 6 mice in group 20. Randomization was performed based on “Matched distribution” method (StudyDirector™ software, version 3.1.399.19).Test Article Administration

[0449] The treatment was initiated on the same day of randomization (day 0) per study design.Observation and Data Collection

[0450] After tumor cells inoculation, the animals were checked daily for morbidity and mortality. During routine monitoring, the animals were checked for any effects of tumor growth and treatments on behavior such as mobility, food and water consumption, body weight gain / loss (Body weights were measured twice per week after randomization), eye / hair matting and any other abnormalities. Mortality and observed clinical signs were recorded for individual animals in detail. Tumor volumes were measured twice per week after randomization in two dimensions using a caliper, and the volume was expressed in mm3using the formula: “V = (L x W x W) / 2, where V was tumor volume, L was tumor length (the longest tumor dimension) and W was tumor width (the longest tumor dimension perpendicular to L). Dosing as well as tumor and body weight measurements were conducted in a Laminar Flow Cabinet.

[0451] The body weights and tumor volumes were measured by using StudyDirector™ software (version 3.1.399.19).Discussion / Conclusions

[0452] Here, the anti-tumor activity of Compound B as single agent and in combination with encorafenib was assessed in HT-29 BRAFv600Emutant tumor-bearing mice in the specified doses. Compound B activity with and without encorafenib, was compared to that of binimetinib with and without encorafenib.

[0453] Single agent Compound B, at 90 mg / kg, 120 mg / kg and 180 mg / kg BID PO treatment showed clear dosc-dcpcndcnt trends with 12.8% (P>0.05), 45.5% (P<0.01) and 73.3% (PcO.OOOl) of TGI value on day 28 compared with the vehicle group, respectively (Figure 9A-1 and 9A-2). Binimetinib and encorafenib as single agents in this model were much less efficacious, with TGI values of 26.0% and 59.6%, respectively (Figure 6B and Figure 6C). Doses of 90 mg / kg, 120 mg / kg and 180 mg / kg Compound B BID PO combined with 60 mg / kg encorafenib QD PO produced significant anti-tumor responses with 84.6% (PcO.OOOl), 91.6% (PcO.OOOl) and 94.6% (PcO.OOOl) of TGI value on day 28 compared with the vehicle group, respectively (Figure 9A-1 and 9A-2). All were significantly better than the binimetinib and encorafenib combination, having a TGI value of 74.0% on day 28 (Figure 9B- 1 and 9B-2).

[0454] In summary, Compound B was tolerated in this model. Compound B in combination with encorafenib produced dose dependent anti-tumor activities with statistical significance. The anti-tumor response of Compound B at 180 mg / kg PO BID in combination with encorafenib at 60 mg / kg PO BID exceeded that of binimetinib in combination with encorafenib.EXAMPLE 4BEfficacy of Compound B, Binimetinib and Encorafenib inHT-29 BRAFV600EHuman Colorectal Cancer Tumor Model as Combination Therapy

[0455] Anti-tumor effect of combination of Compound B with encorafenib (Figure 9A-1 and 9A-2) or binimetinib with encorafenib (Figure 9B-1 and 9B-2). Also shown is the percent body weight change for each respective treatment (Figure 9C, Figure 9D). Tumor volumes presented as the median tumor volume (Median + individual, mm3) of each group, with body weights expressed as median % baseline (Median + individual, %).

[0456] The efficacy of Compound B was interrogated in HT-29, a BRAFV600E human colorectal cancer (CRC) xenograft tumor model, in combination with encorafenib, and compared head-to-head with binimetinib, encorafenib alone or in combination. Compound B showed a dose-dependent tumor growth inhibition, reaching 73.3% TGI at 180 mg / mk BID PO, and outperforming the commercially available MEK and RAF inhibitors as single agents (3.5 mg / kg BID PO binimetinib TGI = 26%; 60 mg / kg QD PO encorafenib TGI = 59.6%),while maintaining body weight (Figures 6A, 6B, 6C for tumor volume; Figures 6D, 6E, 6F for Body weight change). Remarkably, Compound B as a single agent showed comparable tumor growth inhibition than the encorafenib and binimetinib combination (180 mg / kg BID PO Compound B TGI = 73.3% vs 3.5 mg / kg BID PO binimetinib + 60 mg / kg QD PO encorafenib TGI = 74%). When dosed in combination with encorafenib, Compound B in all doses tested showed stronger tumor growth inhibition than the commercially available MEK+RAF inhibitors combination, reaching a 94.6% TGI at 180 mg / kg BID PO Compound B + 60 mg / kg encorafenib, and still maintaining body weight (see Figures 9A-D). Compound B inhibited tumor growth over 90% at their highest dose (180 mg / kg BID PO Compound B) in combination with 60 mg / kg QD PO encorafenib (TGI = 94.6% vs 94.2%, respectively). This data in the HT-29 human colorectal cancer model further supports Compound B as a good candidate to develop effective combination therapies with other MAPK pathway inhibitors, such as encorafenib, dabrafenib or vemurafenib, to treat B RAF V600E / Kmutant cancers.EXAMPLE 5Pharmacokinetic Properties of Compound B, binimetinib and selumetinib

[0457] The drug plasma half-life for selumetinib in mice has been reported at 2.3 hours at a steady state dose of 10 mg / kg PO (Denton CL, Gustafson DL. Pharmacokinetics and pharmacodynamics of AZD6244 (ARRY- 142886) in tumor-bearing nude mice. Cancer Chemother Pharmacol. 2011 Feb;67(2):349-60.) and binimetinib at 2.1 hours after a dose of 5 mg / kg PO (Stewart E, Honnel V, Ocarz M, Caufield W, Freeman B, Wang L. Screening plasma, tumor, and brain pharmacokinetics of binimetinib in female CD-I nu / nu mice bearing MAST38 RHB orthotopic xenografts after a single oral dose. 2018 June 6; RPT.109060- 1119338). Longer half-life (ti / 2) of existing MEK inhibitors, combined with the BID dosing schedule, resulting in more elevated drag trough levels especially in humans, may contribute to the observed discrepancy in tolerability between mice and man (i.e., binimetinib and selumetinib would predictably sustain more elevated drag trough levels in humans due to longer plasma ti / 2).

[0458] In addition to its novel mechanism of action that is resistant to CRAF bypass and other MAPK (re)activation events, Compound B has a short half-life (t 1 / 2) relative to other MEK inhibitors, which when combined with high Cmax with negligible trough levels, drivesdeep, cyclic inhibition of the MAPK pathway instead of chronic ablation, which is known to drive on-target toxicitics for this drug class. The plasma half-life of Compound B in mice was 1.3 hours, and the time to maximal plasma concentration (tmax) was 0.083 hour. With BID dosing, these kinetic properties produce deep inhibition of the MAPK pathway at drug concentration peaks alternating with negligible drug troughs that allow restoration of MAPK pathway signaling, as determined by the pERK levels.Table 5.Example 6Cell-based pERK Dose Response Study for pERK:total ERK Ratio and pMEK:total MEK Ratio for Compound B

[0459] Compound B was evaluated at 100 nM doses for impact on pERK:tERK and pMEK:tMEK levels across a panel of 9 melaoma tumor models compared to Selumetinib and Binimetinib.

[0460] Materials and Methods'. A549 or A375 cells were seeded in 6-well plates at an appropriate seeding density on day 0. On day 2 or 3, after checking the health and confluency of cells, the media was aspirated and replaced with 1 mL media containing a predetermined concentration of compound and incubated for 2 hours. After 2 hours, the media was aspirated, and the cells washed 2 times with cold PBS. Cells were lysed on ice, with 50 pL IX CST® lysis buffer +lmM PMSF for 5 minutes. After 5 minutes, cells were removed using a scraper and transferred to cold 1.5 mL tubes, and centrifuged for 10 min, 4°C, 14,000 x g. Supernatant was gently removed and snap frozen in liquid nitrogen. Protein concentration of the lysate was determined using Bradford Reagent (analyzed using SpectraMax® M2E) and diluted to 1 mg / mL or pERK or 1.5 mg / mL for pMEK analysis. Cell lysates were then analyzed for phosphor-ERK / total-ERK levels on a Jess system (ProteinSimple; Cat # JS3346) using the following antibodies: tERKl / 2 (CST 4696; 1:50) and pERKl / 2 (CST 4377; 1:50). For pMEK / tMEK, the following antibodies were used: tMEKl / 2 (CST 4694; 1:15) and pMEKl / 2 (CST 9154; 1:400). All dilutions were in Milk-free antibody diluent.Mouse & Human Microsome (ti / 2 min), Mouse & Human Clint ( l / min / mg)

[0461] Test compounds were dissolved in DMSO to a concentration of 10 mM and further diluted to 100 pM using acetonitrile. Liver microsomes from selected species were incubated in duplicate with the test compound at a final concentration of 1 pM in 0.1 M potassium phosphate buffer (pH 7.4) containing 3.3 mM MgCh, 0.5 mg / ml microsomal protein, in the presence or absence of NADPH (1 mM). Incubations were performed at 37 °C in a total volume of 500 pl. Control incubations with reference substances were included for each experiment.

[0462] At different time points (t = 0, 5, 15, 30, 45 min), 50 pl of the incubation mixture was transferred into a quench plate containing acetonitrile and internal standard (200 nM labetalol) cooled to 4°C. After the last time point, the quench plates were mixed thoroughlyand centrifuged for 15 minutes at 3700 rpm and 10°C (Eppendorf 5804R). The supernatant was transferred to new 96 well plates and subjected to LC-MS analysis. The disappearance of the parent compound was determined.

[0463] All sample analysis was performed using a Vanquish Horizon UHPLC- system equipped with an autosampler, a binary pump, a column compartment and a diode array detector coupled to a Q Exactive focus hybrid quadrupole- Orbitrap mass spectrometer (Thermo Fisher Scientific) equipped with heated electrospray ion source.

[0464] The percentage of test compound remaining was defined as the ratio of test compound peak area at a specific time point and the peak area in the t = 0 min samples multiplied by 100%.

[0465] The metabolic stability was evaluated by plotting the natural logarithm of the percentage test compound remaining versus time and performing linear regression. Using this graph, the following parameters were calculated: elimination constant k (min-1) = -slope, in vitro half-life (tl / 2) = ln(2) / k, in vitro intrinsic clearance Clint (in pl / min / mg protein) = [ln(2) x incubation volume in pl / mg protein] I tl / 2.Kinetic Solubility (PBS pH - 7.4; 4 hr)

[0466] Test compounds were dissolved in DMSO to a concentration of 10 mM and further diluted to 100 pM in buffer (10 mM PBS, pH 7.4) in a 96 well plate at a final DMSO concentration of 1%. The plates were shaken for 4 h at room temperature in an Eppendorf Thermomixer®. After incubation, the plates were centrifuged for 20 minutes at 4680 rpm. From the supernatant, 150 pL was transferred to a new 96 well plate and 50 pL DMSO was added to ensure continued dissolution. Samples were measured on LC-UV at injection volumes of 1 and 8 pL. Peak areas were determined and compared to peak areas obtained using calibration curves of the test compounds in DMSO. All sample analysis was performed using an Agilent 1290 HPLC-system equipped with an autosampler, a binary pump, a column compartment and a diode array detector. eLogD (lipophilicity; pH = 7.4)

[0467] Test compounds were dissolved in DMSO at a concentration of 10 mM and further diluted with methanol: water 1:1. Samples were analyzed using gradient HPLC with three different isocratic mobile phases of 0.25% octanol in methanol (60, 65 and 70%) and 20 mM MOPS buffer (pH 7.4) with decylamine. If needed, (for low ElogDoct compounds) theisocratic mobile phases were adjusted to e.g. 40, 45 and 50% methanol. Peaks were detected using a diode array at absorbance 220-320 nm.

[0468] Capacity factors data (k'= (tr — tO) / tO) obtained at various amounts of methanol were extrapolated to 0% methanol and k'w values are determined using a linear procedure. ElogDoct (7.4) is calculated using a series of reference standards with known LogD values. Each experiment was performed in triplicate.

[0469] All sample analysis was performed using an Agilent HPLC-system equipped with an autosampler, a binary pump, a column compartment and a diode array detector.PAMPA (pH = 7.4; Papp 10-6 cm / s)

[0470] PAMPA studies were conducted using the PAMPA Explorer kit (pION Inc.) and the double sink protocol (Avdeef, 2005). Stock solutions of all test compounds were dissolved in DMSO to a concentration of 10 mM. Each stock solution was diluted to 50 pM in pH 7.4 Prisma HT buffer (pION) and 200 pl was added to each well of the donor plate in triplicate. The polyvinylidene fluoride (PVDF, 0.45 pm) filter membrane on the acceptor plate was coated with 5 pl gastrointestinal tract lipid formulation (GIT-0, pION) and to each well of the acceptor plate, 200 pl of acceptor sink buffer (pION) was added. The acceptor filter plate was then carefully placed on top of the donor plate to form a sandwich. The sandwich was incubated at 25 °C for 4 h without stirring. UV-vis spectra of the solutions in the blank, reference, acceptor and donor plates were measured using a microplate reader (Tecan Infinite® 200PRO M Nano Plus). Permeability values were calculated using the PAMPA explorer software v. 3.8.0.2 (pION). Control incubations with ketoprofen (low permeability) and verapamil (high permeability) were included in each experiment.

[0471] Compound B was evaluated at 100 nM doses for impact on pERK:tERK and pMEK:tMEK levels across a panel of 9 melanoma tumor models compared to Selumetinib and Binimetinib. The nine models are described in Table 6A. Materials and methods are described for herein with the tumor models detailed in Table 6A.Table 6A

[0472] Tumor cell lines were acquired from ATCC, ECACC and DSMZ. 3D- Tumor Growth Assay (3D-TGA) sensitivity (green) defined as IC50 < lOpM in 72-hour ECM- based assay with %EdU readout, and IC50 >10pM considered resistant. Cell-based 2D in vitro molecular assays were performed to assess cellular levels of phosphorylated and total ERK and MEK across 9 melanoma models (100 nM drag for 2-hours followed by quantitative Western blot analysis for pERK, total ERK, pMEK and total MEK); Binimetinib & selumetinib were commercially purchased; n.t. = not yet tested. The results show that Compound B is a dual-MEK inhibitor due to the reductions in both pERK and pMEK observed across RAS mutant and NF-1 loss of function (LoF) tumor models.Table 6B. pERK to Total-ERK Ratio [100 nM Drug]Table 6C. pMEK to Total-MEK Ratio [100 nM Drug]EXAMPLE 7Mouse Pharmacokinetics (PK)

[0473] Female mice (BALC / c) received a single dose (3 mice / treatment) of vehicle (10% IN HC1 : 90% [20% Captisol in saline pH adjusted to 5.0 ± 0.1] adjusted to pH 3.0 ± 0.1) containing 5x compounds at 10 mg / kg by gavage (p.o.) and at 0.5, 1, 2, 4, 8, 12 and 24- hour post dose humanely euthanized.

[0474] Blood was obtained through cardiac puncture into K+EDTA tubes, mixed by inversion and centrifuged to obtain the plasma. Blood will be kept on ice or at approximately 4°C prior. Centrifugation will take place within 30 minutes of blood collection affording plasma samples. Plasma samples will be frozen as soon as possible after centrifugation and stored at -80°C until analysis. All samples will be analyzed by means of LC-MS / MS. Method development for bioanalysis will be performed by pharmacokinetics Department of Crown Bioscience (Taicang) Inc. using LC-MS / MS analysis (using Waters™ HSS T3 2.1x50mm (1.8um) LC column, and an API-4000 Electrospray MS unit). The results of a kinetic solubility (free base) study are represented in Table 7A.Table 7A

[0475] The results of the pharmacokinetics (oral cassette dosing) study are represented in Tabic 7B.Table 7BBALB / c immunocompetent mice 0.083, 0.5, 1, 2, 4, 6, 12, 24 hour after 5 mg / kg p.o. doseEXAMPLE 8Cyclic disruption of the MAPK pathway by dual MEK inhibitor by Compound B

[0476] In this study, compounds described herein were tested to determine possible cyclic disruption of the mitogen-activated protein kinase (MAPK) pathway by a dual MEK inhibitors, and the enhancement of PD-1 and CTLA-4 checkpoint blockade in RAS mutant tumors.

[0477] Mutations affecting the MAPK pathway have been, and continue to be, an appealing target for drug developers. First generation MEK inhibitors such as trametinib, cobimetinib, binimetinib, and selumetinib have been approved by Health Authorities most often in combination with approved BRAF inhibitors for the treatment of BRAF-mutated lung cancer and melanoma. These MEK inhibitors prevent activation of ERK and, in tandem with BRAF inhibitors, can effectively disrupt the aberrant signaling pathway. However, to induce cytoreduction in the malignant clones, they must sustain the effective target occupancy across the dosing interval, and this chronic pathway ablation is associated with significant on-target toxicides in normal cells. These class effect toxicides, in turn, can limit a patient’s therapeutic index, as dose interruptions and reductions are frequent and can allow the cancer under suboptimal treatment periods to re- establish the aberrant signaling and malignant behavior. Further, chronic suppression or ablation of the MAPK pathway at a single point can encourage the tumor to escape via acquired mutations or utilization of parallel pathways that bypass MAPK pathway addiction.

[0478] KRAS is the most frequently altered RAS gene (-85%) and is often mutated in pancreatic ductal adenocarcinoma (PDAC; 95%), non-small cell lung cancer (NSCLC; 40%) and colorectal cancer (CRC; 45%). KRAS-G12C inhibitors (sotorasib / adagrasib) have demonstrated single-agent activity in all three tumor types. However, acquired resistance and limited biomarker positive patients (e.g., only 1-3% of PDAC and CRC) limit broader access and overall response to G12C inhibitors, prompting evaluation of combination partners including immune therapies. In contrast to G12C-mutant focused KRAS inhibitors, MEK inhibitors could broaden the potential for immune therapy in RAS-mutant tumors, but they have been largely ineffective in this setting as monotherapy. Here, we demonstrate that the short-lived Dual-MEK inhibitor, Compound B, is active across multiple MAPK-driven tumor models both as a single agent and in combination with checkpoint inhibitors (CPI).

[0479] Currently approved MEK inhibitors have shown to be largely ineffective in RAS- mutated disease. For example, while data from the Phase 3 NEMO study evaluating binimetinib versus dacarbazine in NRAS-mutated melanoma showed that binimetinib offered a statistically significant advantage over dacarbazine in ORR (15% vs 7%) and PFS (2.8 months vs 1.5 months), it failed to demonstrate a benefit over dacarbazine in OS (11 months vs 10.1 months). Additionally, patients randomized to the binimetinib arm reported -50% more SAEs and -50% more patients on the binimetinib arm required dose modifications. Current MEK inhibitors’ limited efficacy in RAS-mutated disease is largely thought to be related to feedback mechanisms including CRAF-bypass. Mutated RAS preferentially selects RAFI, commonly referred to as CRAF, to trigger the downstream phosphorylation cascade in the MAPK pathway. Under normal cell function, phosphorylated ERK (pERK) serves to maintain CRAF in an inactive state through a negative feedback loop. In RAS-mutated disease, when the cellular pools of pERK are depleted via inhibition of MEK by one of the current MEK inhibitors, this negative feedback is disabled, resulting in elevated CRAF activation and subsequent reactivation of the MAPK pathway through MEK. It is believed that for a MEK inhibitor to be effective in RAS- mutated disease, it must block RAF activation of MEK, as well as MEK phosphorylation of ERK.

[0480] Until recently, there were no approved therapies for RAS-mutated disease, and targeting RAS directly was thought to be impossible given the lack of binding sites on the RAS protein. However, with the 2021 FDA approval of sotorasib, the first KRASG12Cinhibitor,for patients with NSCLC harboring KRASG12Cmutations, that obstacle was breached and has opened the door for the development of other KRAS inhibitors. And while this advancement has provided a viable targeted treatment option for these patients, sotorasib is only effective against mutations in the specific G12C codon, which represent approximately 15% of the overall KRAS mutation landscape. Further, a myriad of escape mechanisms has already been identified. As such, targets downstream of RAS, such as MEK, are still worth pursuing as they can remain agnostic to codon-specific RAS mutations.

[0481] First generation MEK inhibitors counteract pathway reactivation by sustaining drug occupancy in the allosteric pocket of MEK throughout the dosing cycle (i.e., chronic inhibition). This is generally achieved by endowing drugs with long half-lives and dosing at regular intervals or creating drugs with moderate half-lives and dosing at higher frequencies. Both approaches lead to chronic suppression of the MAPK pathway by sustaining active steady state drug trough levels. Second generation MEK inhibitors resist pathway reactivation by engaging the allosteric pocket of MEK in a unique way that prevents RAF activation of MEK itself but still have long half-lives, leading to chronic pathway ablation. Continuous disruption of this core biologic pathway creates at least 3 well-documented challenges for first and second generation MEK inhibitors: (1) tolerability, clinically limiting, class effect safety issues, (2) acquired / adaptive resistance: selective pressure for escape mutations, and (3) clinical utility: reduced drug-drug combination potential due to limitations on drug-related safety and toxicity. Table 8 illustrates differentiating characteristics of 1st, 2nd, and 3rd generation MEK inhibitors.Table 8.

[0482] Compound B is a third generation MEK inhibitor compound. Unlike previous MEK inhibitors, Compound B rapidly reaches maximal drug exposure while achieving a near- zero drug trough within 12-hour cycles. This allows Compound B to be dosed on a twice daily (BID) schedule to drive deep, cyclic inhibition of the MAPK pathway.

[0483] Methods: Compound B is a novel, third-generation Dual MEK inhibitor that reduces both pMEK and pERK in RAS and RAF-mutant tumor models at sub- 100 nM potencies. Compound B was evaluated in a series of preclinical in vitro and in vivo models enriched for activation mutations that increase MAPK pathway signaling. Cell-based 2D biochemical and 3D pharmacologic assays were performed along with multiple in vivo studies in RAS mutant and wildtype models: (1.) Colon 26, a KRASG12D CRC syngeneic model, (2.) A549, a KRASG12S NSCLC xenograft model, (3.) CT-26, a KRASG12D syngeneic model and (4.) MC38, a RAS wild-type syngeneic model. CT-26 (BALB / c) and MC38 (C57BL / 6) in vivo studies evaluated single-agent Compound B, PD-1 and CTLA-4 versus Compound B + CPI combinations.

[0484] Results: Figure 1 illustrates graphs depicting Compound B with a short plasma and tumor PK half-life in vivo. Figure 2 illustrates graphs depicting Compound B with a short plasma and tumor PK half-life in vivo. Figure 3 illustrates graphs depicting combined plasma and tumor pharmacokinetics at 2 mg / kg i.v. and 10 mg / kg p.o..

[0485] Colon 26 tumor-bearing syngeneic BALB / c mice Pharmacokinetics (PK) of Compound B in Colon 26 tumor bearing syngeneic BALB / c mice (timepoints: 0.083, 0.5, 1, 2, 8 hours after 10 mg / kg p.o. or 2 mg / kg i.v. dose); Standard Deviation (n=3 per timepoint); t non-human primate (NHP) dosed at 5 and 25 mg / kg p.o. and 1 mg / kg i.v. for PK assessment (PK data not shown). Table 9 provides data from this portion of the study.Table 9

[0486] Tumor Growth Inhibition (TGI) % = [1 - (Ti - T0) / (Ci - C0)]xl00%; Maximum Antitumor Effective Dose Range for Compound B in mice is 150 mg / kg to 175-180 mg / kg BID p.o. Table 10 provides data from this portion of the study.Table 10.

[0487] Based on the data from these studies, Compound B reduced pERK and pMEK across all RAS mutant models tested. Humanized 3D tumor models revealed a promising sensitivity profile for Compound B in RAF- and RAS-mutant models. The maximum tolerated dose (MTD) for BID dosing of Compound B was 175 to 180 mg / kg BID PO based on Colon 26 (96.4% TGI) and A549 (93.9% TGI) studies, yet enhanced MEKio + CPI combinations were identified at only 120 mg / kg BID PO Compound B. At 28 days treatment, 33% (4 / 12) CT-26 mice remained on study in the (10 mg / kg BIW IP) anti-PD-1 or anti-CTLA-4 alone treated groups, whereas 58% (7 / 12) mice remained in the Compound B treatment arm at 120 mg / kg BID PO. However, 92% (11 / 12) and 83% (10 / 12) mice remained in Compound B plus anti-PD-1 or anti-CTLA-4 combination at the same doses.

[0488] Elevated MAPK pathway signaling can promote deleterious effects on antitumor immunity, which has prompted multiple MEKi plus CPI combination trials. However, MEKi class effect toxicities have limited clinical utility of MEKi combinations. Instead of chronic MAPK pathway ablation, Compound B was designed to drive short bursts of Cmax driven inhibition of MEK. Compound B displayed activity across multiple RAS and RAF-mutant tumor models. These data suggest that moderated, cyclic inhibition of MEK in combination with CPIs may improve survival times versus monotherapy in MAPK-activated tumors. Antitumor responses with Compound B + / - CPIs in an immune compromised CT-26 model at the same doses, combinations and schedules were not observed, suggesting that moderated, cyclic disruption of the MAPK pathway can enhance CPI-dependent adaptive antitumor immunity and improve overall MEKio combination tolerability.EXAMPLE 9Head-to-Head Comparison of Compound B against Two FDA-Approved MEK Inhibitors Using a Panel of Nine Melanoma Tumor Models

[0489] Compound B was evaluated head-to-head against two FDA-approved MEK inhibitors to assess their control of pMEK, pERK and CRAF-bypass in a NRAS, HRAS, NF1 and BRAF mutant melanoma tumor models. The tumor cells were exposed with 100 nM of each drug for 2 hours and evaluated MEK and ERK activation levels. It was observed Compound B reduced overall activity of the MAPK pathway at ERK and pathway reactivation at MEK through a decrease in MEK and ERK activation, resulting in CRAF-bypass resistance.In contrast, it was observed that both FDA-approved MEK inhibitors displayed an increase in activated MEK, resulting in CRAF-bypass in RAS and NF1 mutant tumors. It was observed activity of Compound B in RAS and RAF mutant tumors; Compound B prevented downstream activation of ERK (| pERK) and inhibited activation of MEK (| pMEK).

[0490] Tumor cell lines were acquired from ATCC, ECACC and DSMZ. 3D- Tumor Growth Assay (3D-TGA) sensitivity (green fill) defined as IC50 < lOu M in 72-hour ECM-based assay with %EdU readout, and ICso >lOpM considered resistant. Cell-based 2D in vitro molecular' assays were performed to assess cellular levels of phosphorylated and total ERK and MEK across 9 melanoma models (100 nM drug for 2-hours followed by quantitative Western blot analysis for pERK, total ERK, pMEK and total MEK); Binimetinib & selumetinib were commercially purchased (MAPK pathway reactivation observed, pMEK); n.t. = not yet tested.EXAMPLE 10Cyclic disruption of the MAPK pathway by dual MEK inhibitor

[0491] In this study, compounds described herein were tested to determine possible cyclic disruption of the mitogen-activated protein kinase (MAPK) pathway by a dual MEK inhibitors, and the enhancement of PD-1 and CTLA-4 checkpoint blockade in RAS mutant tumors.

[0492] KRAS is the most frequently altered RAS gene (-85%) and is often mutated in pancreatic ductal adenocarcinoma (PDAC; 95%), non-small cell lung cancer (NSCLC; 40%) and colorectal cancer (CRC; 45%). KRAS-G12C inhibitors (sotorasib / adagrasib) have demonstrated single-agent activity in all three tumor types. However, acquired resistance and limited biomarker positive patients (e.g., only 1-3% of PDAC and CRC) limit broader access and overall response to G12C inhibitors, prompting evaluation of combination partners including immune therapies. In contrast to G12C-mutant focused KRAS inhibitors, MEK inhibitors could broaden the potential for immune therapy in RAS-mutant tumors, but they have been largely ineffective in this setting as monotherapy. Here, we demonstrate that the short-lived DuaLMEK inhibitor, Compound B, is active across multiple MAPK-driven tumor models both as a single agent and in combination with checkpoint inhibitors (CPI).Material and methods - CT26 and MC38

[0493] Figures 7A and 7B illustrate graphs depicting MEKi, a-PD-1, a-CTLA-4 alone and combinations in CT-26 / MC38 syngeneic mouse tumor models.

[0494] Figures 7 A illustrates graphs depicting MEKi, a-PD-1, a-CTLA-4 alone and combinations in CT-26 syngeneic mouse tumor model. Figures 7B illustrates graphs depicting MEKi, a-PD-1, a-CTLA-4 alone and combinations in MC38 syngeneic mouse tumor model.

[0495] Survival curves in the CT-26 syngeneic mouse tumor model are shown in Figure 7A (combination with anti-PDl ; top panel) and Figure 7A (combined with anti-CTLA4; bottom panel), with defined event (death) was assessed by time to tumor volume reaching 2000 mm3. The data were analyzed using Kaplan-Meier survival curves.

[0496] Median survival time of the vehicle groups were 16, and 13 and days, respectively for BID and QD. Median survival time of 30 mg / kg, 60 mg / kg and 120 mg / kg Compound B alone dosed twice per day were 16 (P>0.05, vs. BID vehicle group), 19 days (P<0.05, vs. BID vehicle group) and undefined (P<0.001, vs. BID vehicle group ), respectively.

[0497] Median survival time (MST) of IgG2a isotype control for anti-PD-1 group was 16 days. MST of anti-PD-1 treatment alone administered at 10 mg / kg, i.p., BIW was 22.5 days (P<0.05, vs. isotype control).

[0498] Less than half of the mice died (TV>2000 mm3) in 30 mg / kg, 60 mg / kg and 120 mg / kg Compound B, BID administration combined with 10 mg / kg anti-PD-1 treated groups. Only the 30 mg / kg BID group had an MST of 27 days (P<0.01), whereas the 60 and 120 mg / kg BID groups were all undefined (P<0.001, vs. BID vehicle group).

[0499] MST of IgG2b isotype control group for anti-CTLA-4 was 15 days. MST of anti-CTLA-4 treatment alone administered at 10 mg / kg, i.p., BIW was 26 days (P<0.001, vs. isotype control.

[0500] Less than half of the mice died (TV>2000 mm3) in 30 mg / kg, 60 mg / kg and 120 mg / kg Compound B, BID administration combined with 10 mg / kg anti-CTLA-4 treated groups. Only the 30 mg / kg QD group had a measured MST of 28 days (P<0.01 vs. BID vehicle group), with the 60 and 120 mg / kg QD groups having MST being undefined (P<0.001, vs. BID vehicle group).

[0501] Survival curves in the MC38 syngeneic mouse tumor model are shown in Figure 7B (combination with anti-PDl; upper panel) and (combined with anti-CTLA4; lower panel), with defined event (death) was assessed by time to tumor volume reaching 2000 mm3. The data were analyzed using Kaplan-Meier survival curves.

[0502] Median survival time (MST) of the vehicle group were 12.5 and 14 days for BID and QD, respectively. Median survival time of 30 mg / kg, 60 mg / kg and 120 mg / kg Compound B alone dosed twice per day were 15 (P>0.05, vs. BID vehicle group), 14 days (P<0.05, vs. BID vehicle group) and 17 (P<0.05, vs. BID vehicle group), respectively.

[0503] MST of IgG2a isotype control group for anti-PD-1 was 11 days. MST of anti-PD-1 treatment alone administered at 10 mg / kg, i.p., BIW was 29 days (P<0.001, vs control isotype).

[0504] MST of 30 mg / kg, 60 mg / kg and 120 mg / kg Compound B, BID administration combined with 10 mg / kg anti-PD-1 treated groups were 29 (PcO.001, vs. BID vehicle group), 26.5 (P<0.001, vs. BID vehicle group) and 31 days (P<0.001, vs. BID vehicle group), respectively.

[0505] MST of IgG2b isotype control for anti-CTLA-4 was 12.5 days. MST of anti- CTLA-4 treatment alone administered at 10 mg / kg, i.p., BIW was 22 days (PcO.001, vs. control isotype).

[0506] MST 30 mg / kg, 60 mg / kg and 120 mg / kg Compound B, BID administration combined with 10 mg / kg anti-CTLA-4 treated groups were 18 (P<0.001, vs. vehicle group 1), 18 (P<0.001, vs. BID vehicle group) and 21 days (P<0.001, vs. BID vehicle group), respectively.Cell Culture

[0507] The CT26 tumor cells were maintained in vitro with RPMI1640 medium supplemented with 10% fetal bovine serum at 37°C in an atmosphere of 5% CO2 in air. The cells in exponential growth phase were harvested and quantitated before tumor inoculation. The MC38 tumor cells were maintained in vitro with DMEM medium supplemented with 10% fetal bovine serum at 37 °C in an atmosphere of 5% CO2 in air. The cells in exponential growth phase were harvested and quantitated before tumor inoculation.Tumor Inoculation

[0508] Each mouse was inoculated subcutaneously in the right flank region withCT26 tumor cells (5 x 105) or MC38 tumor cells (l x 106) in 0.1 mL of PBS for tumor development.Randomization

[0509] The randomization was started when the mean tumor size reached approximately 81.07 mm3for CT-26 tumor bearing mice, and 90.43 mm3for MC38 tumor bearing mice. A total of 288 mice for each cell line were enrolled in the study and allocated into 24 groups, with 12 mice per group. Randomization was performed based on “Matched distribution” method (StudyDirector™ software, version 3.1.399.19).Test Article Administration

[0510] The treatment was initiated on the same day of randomization (day 0) per study design.Observation and Data Collection

[0511] After tumor cells inoculation, the animals were checked daily for morbidity and mortality. During routine monitoring, the animals were checked for any effects of tumor growth and treatments on behavior such as mobility, food and water consumption, body weight gain / loss (body weights would be measured three times per week after randomization), eye / hair matting and any other abnormalities. Mortality and observed clinical signs were recorded for individual animals in detail.

[0512] Tumor volumes were measured three times per week after randomization in two dimensions using a caliper, and the volume was expressed in mm3using the formula: V = (L x W x W) / 2, where V is tumor volume, L is tumor length (the longest tumor dimension) and W is tumor width (the longest tumor dimension perpendicular to L). Dosing as well as tumor and body weight measurements were conducted in a Laminar Flow Cabinet.

[0513] The body weights and tumor volumes were measured by using StudyDirector™ software (version 3.1.399.19).Discussion / Conclusions

[0514] KRAS is the most frequently altered RAS gene (-85%) and is often mutated in pancreatic ductal adenocarcinoma (PDAC; 95%), non-small cell lung cancer (NSCLC; 40%) and colorectal cancer (CRC; 45%) 1. KRAS-G12C inhibitors (sotorasib / adagrasib) havedemonstrated single-agent activity in all three tumor types. However, acquired resistance and limited biomarkcr positive patients (c.g., only 1-3% of PDAC and CRC) limit broader access and overall response to G12C inhibitors, prompting evaluation of combination partners including immune therapies. In contrast to G12C-mutant focused KRAS inhibitors, MEK inhibitors could broaden the potential for immune therapy in RAS-mutant tumors, but they have been largely ineffective in this setting as monotherapy. We demonstrated that the shortlived Dual-MEK inhibitor, Compound B, is active across multiple MAPK-driven tumor models both as a single agent and in combination with checkpoint inhibitors (CPI).

[0515] Compound B is a novel, third-generation Dual MEK inhibitor that reduces both pMEK and pERK in RAS- and RAF-mutant tumor models at sub-100 nM potencies. Compound B was evaluated in a series of preclinical in vitro and in vivo models enriched for activation mutations that increase MAPK pathway signaling. Cell-based 2D biochemical and 3D pharmacologic assays were performed along with multiple in vivo studies in RAS mutant and wildtype models: (1.) Colon 26, a KRASG12D CRC syngeneic model, (2.) A549, a KRASG12S NSCLC xenograft model, (3.) A375, a BRAFV600E melanoma xenograft model, (4.) HT29, a BRAFV600E CRC xenograft model, (5.) CT-26, a KRASG12D CRC syngeneic model and (6.) MC38, a RAS wild-type CRC syngeneic model. CT-26 (BALB / c) and MC38 (C57BL / 6) in vivo studies evaluated single-agent Compound B, PD-1 and CTLA-4 versus Compound B + CPI combinations.

[0516] Compound B reduced pERK and pMEK across all RAS mutant models tested. Humanized 3D tumor models revealed a promising sensitivity profile for Compound B in RAF- and RAS-mutant models. The maximum tolerated dose (MTD) for BID dosing of Compound B was 175 to 180 mg / kg BID PO (note: 200 mg / kg for QD PO dosing) based on Colon 26 (96.4% TGI) and A549 (93.9% TGI) studies, yet enhanced MEKio + CPI combinations were identified at 120 mg / kg or lower Compound B. At 28 days treatment, 33% (4 / 12) CT-26 mice remained on study in the (10 mg / kg BIW IP) anti-PD-1 or anti-CTLA-4 alone treated groups, whereas 58% (7 / 12) mice remained in the Compound B treatment arm at 120 mg / kg BID PO. However, 92% (11 / 12) and 83% (10 / 12) mice remained in the Compound B plus anti-PD-1 or anti-CTLA-4 combination at the same doses.

[0517] Elevated MAPK pathway signaling can promote deleterious effects on antitumor immunity, which has prompted multiple MEKi plus CPI combination trials2.However, MEKi class effect toxicities have limited clinical utility of MEKi combinations.!. Instead of chronic MAPK pathway ablation, Compound B was designed to drive short bursts of Cmax driven inhibition of MEK. Compound B displayed activity across multiple RAS- and RAF-mutant tumor models, and when combined with PD-1 or CTLA-4 checkpoint inhibitors at well-tolerated, sub-MTD dose levels, significant survival benefit was observed (p-values: <0.05 to <0.0001; CT-26). These data suggest that moderated, cyclic inhibition of MEK in combination with CPIs may improve survival times versus monotherapy in MAPK-activated tumors. Antitumor responses with Compound B + / - CPIs in an immune compromised CT-26 model at the same doses, combinations and schedules were not observed, suggesting that moderated, cyclic disruption of the MAPK pathway can enhance CPI-dependent adaptive antitumor immunity and improve overall MEKio combination tolerability.

[0518] Methods: Compound B is a novel, third-generation Dual MEK inhibitor that reduces both pMEK and pERK in RAS and RAF-mutant tumor models at sub- 100 nM potencies. Compound B was evaluated in a series of preclinical in vitro and in vivo models enriched for activation mutations that increase MAPK pathway signaling. Cell-based 2D biochemical and 3D pharmacologic assays were performed along with multiple in vivo studies in RAS mutant and wildtype models: (1.) Colon 26, a KRASG12D CRC syngeneic model, (2.) A549, a KRASG12S NSCLC xenograft model, (3.) CT-26, a KRASG12D syngeneic model and (4.) MC38, a RAS wild-type syngeneic model. CT-26 (BALB / c) and MC38 (C57BL / 6) in vivo studies evaluated single-agent Compound B, PD-1 and CTLA-4 versus Compound B + CPI combinations.

[0519] Results: Figure 1 illustrates graphs depicting Compound B with a short plasma and tumor PK half-life in vivo. Figure 2 illustrates graphs depicting Compound B with a short plasma and tumor PK half-life in vivo. Colon 26 tumor-bearing syngeneic BALB / c mice Pharmacokinetics (PK) of Compound B in Colon 26 tumor bearing syngeneic BALB / c mice (timepoints: 0.083, 0.5, 1, 2, 8 hours after 10 mg / kg p.o. or 2 mg / kg i.v. dose); Standard Deviation (n=3 per timepoint); f non-human primate (NHP) dosed at 5 and 25 mg / kg p.o. and 1 mg / kg i.v. for PK assessment (PK data not shown). Table 11 provides data from this portion of the study.Table 11

[0520] Table 12 illustrates differentiating characteristics of 1st, 2nd, and 3rd generation MEK inhibitors.Table 12

[0521] Figures 7A and 7B illustrate graphs depicting MEKi, a-PD-1, a-CTLA-4 alone and combinations in CT-26 / MC38. Figure 7A: CT-26 (KRASG12D) syngeneic colorectal tumor model in immune competent BALB / c mice (note: monotherapy and combinations were inactive in athymic nude CT-26 model - data not shown). Figure 7B: MC38 (RASwild-type) syngeneic colorectal tumor model in immune competent C57BL / 6mice (note: immune compromised model not evaluated). Tables 13 and 14 provides data from this portion of the study.Table 13. Compound B checkpoint inhibitor in KRASG12DCT-26Number of BALB / c mice (out of 12) with tumors through Day 28 with volumes lower than 2,000 mm3.* Monotherapy treated alive at Day 28 # Combination > 3 AdvantageTable 14. Compound B checkpoint inhibitor in RASwlld'typeMC38Number of C47BL / 6 mice (out of 12) with tumors through Day 28 with volumes lower than 2,000 mm3.* Monotherapy treated alive at Day 28 # Combination > 3 Advantage

[0522] Based on the data from these studies, Compound B reduced pERK and pMEK across all RAS mutant models tested. Humanized 3D tumor models revealed a promising sensitivity profile for Compound B in RAF- and RAS-mutant models. The maximum tolerated dose (MTD) for BID dosing of Compound B was 175 to 180 mg / kg BID PO based on Colon 26 (96.4% TGI) and A549 (93.9% TGI) studies, yet enhanced MEKio + CPI combinations were identified at only 120 mg / kg BID PO Compound B. At 28 days treatment, 33% (4 / 12) CT-26 mice remained on study in the (10 mg / kg BIW IP) anti-PD-1 or anti-CTLA-4 alone treated groups, whereas 58% (7 / 12) mice remained in the Compound B treatment arm at 120 mg / kg BID PO. However, 92% (11 / 12) and 83% (10 / 12) mice remained in Compound B plus anti-PD-1 or anti-CTLA-4 combination at the same doses.

[0523] Elevated MAPK pathway signaling can promote deleterious effects on antitumor immunity, which has prompted multiple MEKi plus CPI combination trials. However, MEKi class effect toxicities have limited clinical utility of MEKi combinations. Instead of chronic MAPK pathway ablation, Compound B was designed to drive short bursts of Cmax driven inhibition of MEK. Compound B displayed activity across multiple RAS and RAF-mutant tumor models, and when combined with PD-1 or CTLA-4 checkpoint inhibitors at well tolerated, sub-MED dose levels, significant survival benefit was observed (p-values: <0.05 to <0.0001; CT-26). These data suggest that moderated, cyclic inhibition of MEK in combination with CPIs may improve survival times versus monotherapy in MAPK-activated tumors. Antitumor responses with Compound B + / - CPIs in an immune compromised CT-26 model at the same doses, combinations and schedules were not observed, suggesting that moderated, cyclic disruption of the MAPK pathway can enhance CPI-dependent adaptive antitumor immunity and improve overall MEKio combination tolerability.EXAMPLE 11Deep Cyclic Inhibition (DCI) of the MAPK pathway with Compound B, alone and in combination with encorafenib demonstrates anti-tumor activity and tolerability in RAF mutant tumors in vivo

[0524] A significant proportion of cancer patients have tumors addicted to uncontrolled MAPK signaling. Activating mutations in RAS or RAF are often directlyresponsible and have been observed in over 20% of human tumors. Because MEK is downstream of RAS and RAF, it is an appealing drug target. However, MEK inhibitors have historically suffered from poor clinical durability, high toxicity and a susceptibility to pathway reactivation that has limited monotherapy activity in the RAS mutant setting. Unlike other MEK inhibitors, Compound B is designed with distinctive features including both (1.) a unique target engagement mechanism that helps resist MAPK pathway reactivation and (2.) a pharmacokinetic (PK) profile that enables fast cadence deep cyclic inhibition (DCI). DCI drives pulsatile targeted inhibition that deprives tumor cells of sustained oncogenic pathway signaling. Additionally, the goal of DCI, by achieving an adequately low drug trough, is to improve drug tolerability by allowing normal healthy cells an opportunity to maintain homeostatic signaling between doses.Experimental Procedures

[0525] Compound B has demonstrated promising activity in RAS-mutant xenograft tumor models. Here, the antitumor activity of Compound B was evaluated in over 60 humanized 3D tumor growth assays (3D-TGA), which included 30 BRAF class I-mutant tumor models. The humanized 3D-TGA better predicts in vivo tumor responses versus 2D culture and more accurately replicates human tumor biology (See Figures 11A, 1 IB, and 11C; and Figures 13A and 13B for 3D TGA data).Benchmarking 3D-TGA Response to Compound B

[0526] As monotherapy, Compound B demonstrated antitumor activity in over 50% (34 of 66) of the 3D-TGA models tested, including 30 BRAF-mutant preclinical models in which 19 (63%) showed activity. Similar to IMM-1-104, resistant models either lacked obvious MAPK pathway driver mutations or displayed parallel oncogenic pathway activation events. Likewise, sensitive and intermediate responses were strongly enriched for models harboring activation mutations in either RAS or RAF. Figures 11 A, 1 IB and 11C show results from 3D-TGA Compound B dose responses (n=66 models; 11 tumor indications); cell lines were tested in the humanized 3D-TGA (N=66) and assigned response of sensitive (IC50 < 3pM), intermediate (IC50 > 3p M and < 1 Op M), and resistant otherwise. The dark line on each plot represents the median of the individual curves.-IrBiomarkcr Profile of Patients

[0527] Using the GENIE database, a targetable population was selected on the basis of BRAF class Eli mutations. This population was then assessed for co-occurrence of mutations in genes that may partially contribute to resistance mechanisms with intersecting sets indicating concurrent mutations in patients. The bar charts detail co-occurrence frequencies in OncoTree5cohorts: (A) SKCM, MEL; (B) COAD, READ, COADREAD; (C) THPA; and (D) LU AD. Figure 12A is a biomarker profile diagram of data from melanoma patients profiled in GENIE® database. Figure 12B is a biomarker profile diagram of data from colorectal cancer patients profiled in GENIE® database. Figure 12C is a biomarker profile diagram of data from thyroid cancer patients profiled in GENIE® database. Figure 12D is a biomarker profile diagram of data from lung cancer patients profiled in GENIE® database.Compound B in A-375 (melanoma) and HT-29 (colorectal) BRAFV600E tumor models

[0528] Additionally, multiple drug-drug combinations have been explored, including vertical drug combinations with BRAF inhibitors. Compound B, binimetinib (MEK inhibitor) and encorafenib (BRAF inhibitor) were tested head-to-head as single agents and in combination with encorafenib in BRAFv600Emelanoma and colorectal subcutaneous tumor xenograft models in female BALB / c nude mice.

[0529] Compound B was evaluated as a monotherapy and as part of a combination therapy using the experimental procedures of Example 3A above. Both monotherapy and combination activity of Compound B were further explored in the A-375 (melanoma) and HT- 29 (colorectal) BRAFv600Etumor models. Monotherapy treatment with encorafenib or Compound B displayed superior tumor growth inhibition (TGI) when compared to binimetinib. The combination of Compound B plus encorafenib prompted greater TGI with superior durability of response when tested head-to-head against the combination of binimetinib plus encorafenib in vivo at reported human equivalent doses for registered dings (Figures 4A-2, 5 A- 2, 5C, 8C and 8D for A375 cells; and Figures 6A-2, 9A-2 and 9B-2 in HT29 cells).

[0530] Monotherapy evaluation of Compound B was performed to obtain data shown in Figure 4A-2 (for A375 cells) and Figure 6A-2 (for HT-29 cells). Figure 4A-2 is a graph of data showing the efficacy of monotherapy of Compound B (90 to 180 mg / kg BID PO)on the tumor growth rate of subcutaneous (SC) pre-established A375 Human malignant melanoma model for each respective treatment doses (90 to 180 mg / kg p.o. BID).

[0531] Combination of Compound B (I) and encorafenib (E) was evaluated in A- 375 (melanoma) and HT-29 (colorectal) BRAFv600Etumor models, to obtain data shown in Figure 5C (A375) and in Figures 9A2 (HT-29). Figure 5C is a graph of data showing the antitumor effect of a combination of Compound B (90 to 180 BID PO) and encorafenib at 60 mg / kg QD PO on the tumor growth rate of a SC pre-established A375 human malignant melanoma model. For comparison, Figure 5A-2 is a graph of data showing the anti-tumor effect of a combination of binimetinib at 3.5 mg / kg BID + encorafenib at 60 mg / kg on the tumor growth rate of a SC pre-established A375 human malignant melanoma model.

[0532] Figures 8C and 8D expand data shown in the Figures 9A2 and 9B2. Figure 8C illustrates graphs depicting Compound B, binimetinib, and encorafenib alone and in various combinations in A-375 xenograft melanoma human tumor mouse model. Figure 8D illustrates graphs depicting administration of a combination of Compound B and encorafenib after a period of dosing a binimetinib and encorafenib in A-375 xenograft melanoma human tumor mouse model.

[0533] Figure 6A-2 is a graph of data showing the efficacy of monotherapies of Compound B on the tumor growth rate of SC pre-established HT-29 Human colorectal cancer model for each respective treatment doses (90 - 180 mg / kg p.o. BID).

[0534] Figure 9A-2 illustrates graphs depicting Compound B, encorafenib alone and combinations in the HT-29 xenograft colon human tumor mouse model. Figure 9B-2 illustrates graphs depicting binimetinib, encorafenib alone and combinations in the HT-29 xenograft colon human tumor mouse model.

[0535] The synergy evaluation of Compund B with encorafenib in A-375 model was evaluated in 3D-TGA. Figure 13A is a graph showing humanized 3D-TGA of Compound B with encorafenib. Figure 13B is a graph of the Zero Interaction Potency (ZIP) model synergy score for the combination of Compound B with encorafenib. Human A375 BRAF V600E melanoma cells were embedded in 3D cultures and treated with encorafenib, or Compound B, alone or in combination for 72h. Percent EdU positive cells were quantified by high content imaging and normalized to DMSO. Transformed, normalized, nonlinear fit was graphed in R and IC50 values for encorafenib and Compound B were determined. SynergyFinder plus Rpackage was utilized to generate plots of each 2-drug combination found in this experiment. 3D surface ZIP synergy response (C) for cncorafcnib and Compound B (Figure 13B).

[0536] Compound B is a short-lived dual MEK inhibitor (MEKi) that works through Deep Cyclic Inhibition (DCI) and a fast pulsatile cadence (short half-life) that is distinctive from chronic MEKi. Compound B was optimized for twice daily oral dosing and has displayed promising antitumor activity and tolerability across a growing number of RAS and RAF mutant models (Universal MAPK activity). Building on earlier RAS mutant studies2, we found that Compound B (pulsatile DCI MEKi) plus encorafenib achieved superior TGI and durability in vivo versus binimetinib (sustained MEKi) plus encorafenib in BRAF mutant colorectal cancer and melanoma models. These data are consistent with the thesis that MEK DCI can outperform chronic MEKi as monotherapy and in vertical drug combinations.

[0537] Compound B demonstrated promising activity and tolerability in preclinical models alone and in combination with encorafenib. In combination with encorafenib, Compound B achieved a greater TGI in vivo than the combination of encorafenib plus binimetinib in BRAFv600Ecolorectal cancer and melanoma tumor models, suggesting an opportunity for Compound B as monotherapy or in combination in BRAF mutant tumors.

[0538] After promising in vitro results, follow-up in vivo studies were completed, where Compound B was evaluated as monotherapy in BALB / c mice tumor models with RAS and RAF mutations, including Examples 12-16.EXAMPLE 12Compound B inA375 (BRAF V600E) Melanoma Xenograft Tumor Model

[0539] Figure 17A is a graph showing data of Compound B using an A375 xenograft tumor model (tumor volume). A375 (BRAFV600E) Melanoma Xenograft Tumor Model in Athymic Nude Mice. Tumor Growth Inhibition; (TGI)% = [l-(Ti-To) / (Ci-Co)]xlOO; T = treatment groups; C = control groups; expanded TGI formula vs. previously l-[T / C]xl00% method.EXAMPLE 13Compound. B in HT-29 (BRAF V600E) Colorectal Xenograft Tumor Model

[0540] Compound B was evaluated in a BRAF V600E human colorectal xenograft model. When comparing Compound B to vehicle, we observed that the Compound B had greater tumor growth inhibition (Figure 17B). Figure 17B shows data of Compound B using a HT-29 xenograft tumor model (tumor volume). HT-29 (BRAFv600E) colorectal Xenograft Tumor Model in Athymic Nude Mice. Tumor Growth Inhibition; (TGI)% = [l-(Ti-To) / (Ci- Co)]xlOO; T = treatment groups; C = control groups; expanded TGI formula vs. previously 1- [T / C]xl00% method.EXAMPLE 14Compound B in A549 NSCLC Xenograft Tumor Model

[0541] Compound B was evaluated as a monotherapy in a KRASG12Shuman nonsmall cell lung cancer (NSCLC) xenograft model. Compound B was observed to have strong tumor growth inhibition (Figure 17C). Figure 17C shows data of Compound B using an A549 xenograft tumor model (tumor volume). A549 (KRASG12S) NSCLC Xenograft Tumor Model in Athymic Nude Mice. Tumor Growth Inhibition; (TGI)% = [l-(Ti-To) / (Ci-Co)]xlOO; T = treatment groups; C = control groups; expanded TGI formula vs. previously l-[T / C]xl00% method.EXAMPLE 15Compound B in Colon-26 Xenograft Tumor Model

[0542] Compound B was evaluated as a monotherapy in a KRASG12Dmouse xenograft model. Compound B was observed to have strong tumor growth inhibition (Figure 17D). Figure 17D shows data of Compound B using a Colon-26 xenograft tumor model (tumor volume). Colon-26 (KRASG12D) colorectal Xenograft Tumor Model in Syngeneic Mice. Tumor Growth Inhibition; (TGI)% = [l-(Ti-To) / (Ci-Co)]xlOO; T = treatment groups; C = control groups; expanded TGI formula vs. previously l-[T / C]xl00% method.EXAMPLE 16Compound B in MIA PaCa-2 (KRAS G12C) Pancreatic Cancer Xenograft Tumor Models

[0543] Compound B was evaluated for 28 days in the KRASG12Cxenograft model (z.e., MIA PaCa-2 (KRAS) Pancreatic Xenograft Tumor Model in Athymic Nude Mice). Compound B demonstrated tumor regression, which indicates activity, durability and tolerability of Compound B against a KRASG12Cmutant pancreatic cancer model. Figurel7E shows data of Compound B using a MIA PaCa-2 Xenograft Tumor Model: Tumor Volume. In Figure 17E: Tumor Growth Inhibition (TGI)% = [l-(Ti-To) / (Ci-Co)]xlOO; Expanded TGI formula vs. previous l-[T / C]xl00% method.EXAMPLE 17Compound B in SK-MEL-2 (NRAS Q61R) Melanoma Xenograft Tumor Model

[0544] Compound B was evaluated as a monotherapy for 28 days in the NRASQ61R mutant tumor model (i.e., SK-MEL-2). Figure 17F shows results of tumor volume measured with Compound B against vehicle in a NRAS-Q61R SK-MEL-2 melanoma xenograft study. Compound B as monotherapy showed tumor regressions at 150 and 180 mg / kg PO BID, which demonstrated activity and durability of Compound B against an NRAS Q61R mutant melanoma cancer model (Figure 17F).EXAMPLE 18Oral Unit Dosage Forms of Compound B

[0545] Compound B can be provided as 20 mg strength and 160 mg strength tablets for oral administration, containing excipients lactose monohydrate 200M, MCC PH101, MCC PH102, croscarmellose sodium, hydroxypropyl cellulose (HPC) grade SL, and magnesium stearate. Table 15 provides an example of an oral dosage tablet formulation of Compound B. Total tablet weight at 20 mg strength can be 100 mg and at 160 mg strength is 800 mg. Only the 160 mg strength tablets will be coated using Opadry QX 321A80015.Table 15.EXAMPLE 19A Phase l / 2a Human Clinical Trial Study of Compound B in Participants with Advanced or Metastatic Malignancies Harboring RAS or RAF Oncogenic Mutations

[0546] Compound B is a novel, oral, dual MEK1 / 2 inhibitor with a unique PK profile that affects signaling dynamics positioned to achieve optimal efficacy and safety.

[0547] This was a first in human Phase l / 2a multicenter, open-label, ascending dose study to characterize the safety, tolerability, optimal dose and preliminary anti-tumor activity of Compound B in participants with advanced or metastatic solid tumors harboring RAS or RAF oncogenic mutations. This study characterized the safety and anti-tumor activity of Compound B in participants with advanced or metastatic RAS- or RAF-mutated solid tumors. This study had 2 parts: (1) Dose Exploration featuring all-comer solid tumor dose escalation (Part la) and dose optimization (Part lb) stages and (2) featuring multiple POC generating expansion cohorts enriched for RAS- and RAF-mutated malignancies of interest. The dose study administered Compound B as monotherapy in Phase 2a tumor-specific cohorts.

[0548] Patients self-administered Compound B on a daily basis for up to 16 cycles (21-day cycles). During the first 2 cycles, PK and PD were assessed. Solid tumor types with RAS / RAF mutations were eligible.

[0549] Compound B was administered in a twice daily (BID) regimen for up to sixteen 21-day cycles (48 weeks). The treated patients received a prespecified planned dose of Compound B in the range 40 mg BID to 640 mg BID. The cohort dose escalation levels were as follows: 40, 80, 160, 320, 480 and 640 mg of Compound B twice per day (BID). The preferred dose of Compound B was the dose that best exhibited pharmacological characteristics of deep cyclic inhibition (DCI) of the MAPK pathway and provided adequate anti-tumor activity while not exceeding the MTD (if reached during dose escalation). Compound B was administered PO BID approximately 12 hours apart. Participants took their study drug with approximately 8 oz (240 mL) of water at around the same time each morning and evening on an empty stomach (either 1 hour before or 2 hours after food intake). If a participant had trouble swallowing the tablets, an additional 4 oz (120 mL) of water was allowed. Study drug tablets must not be cut, crushed, dissolved in liquid, or altered in any other way. On visit days where PK samples were collected, participants must have fasted for at least 2 hours, and waited to take their morning study drug until they were at the clinic and the predose sample had been collected; participants fasted for 1 additional hour after taking the study drug before being allowed to eat.

[0550] Treatment with the study drug continued for up to 16 cycles or until one of the treatment discontinuation criteria were met. On C1D1, participants were given a supply of study drug to cover the cycle interval including the visit window and instructions to continue intake at home. The study drug was re-supplied at each subsequent cycle on Day 1 (CXD1) throughout the participant’s treatment period. Unscheduled re-supplies were also permitted as needed.

[0551] The starting dose of Compound B was 40 mg administered PO BID. The planned maximum dose was 640 mg BID. During Part 1, dose escalation cohort dose levels were as follows: 40, 80, 160, 320, 480 and 640 mg administered BID. During Part 2, the dose of Compound B was the optimal dose determined by the Sponsor based on a composite review of safety, tolerability, PK, PD, and anti-tumor activity data accumulated during Phase I dose escalation and dose expansion.

[0552] Apart from when the study drug was taken at the Investigative Site (e.g., when PK samples arc collected), participants sclf-administcrcd Compound B at home according to the dosing instructions provided.

[0553] Participants received study drug for up to 16 cycles (48 weeks) or until one of the treatment discontinuation criteria were met. It was estimated that the study duration for most participants would last approximately 8 months.

[0554] The patient participants 18 years or older had advanced, unresectable, or metastatic solid tumors harboring RAS or RAF mutations and had received at least 1 prior line of systemic therapy for their advanced / metastatic disease. Participants with any advanced, unresectable, or metastatic RAS- or RAF-mutated solid tumors are allowed in Part 1, while Part 2 focuses on the following tumor type cohorts: Pancreatic Adenocarcinoma (PDAC), RAS-mutated cutaneous melanoma, RAF-mutated cutaneous melanoma, RAS-mutated NSCLC, Other RAS-mutated GI solid tumors (except CRC), or any other RAF-mutated solid tumor.

[0555] As noted above, this example describes a first-in-human (FIH), ascending dose study designed to evaluate the anti-tumor activity of Compound B in participants with advanced or metastatic solid tumors harboring RAS or RAF oncogenic mutations. Participants self-administered Compound B daily for up to 16 cycles, each lasting 21 days. During the first two cycles, pharmacokinetics (PK) and pharmacodynamics (PD) were assessed. Eligible participants included those with solid tumor types exhibiting RAS / RAF mutations. Inclusion criteria

[0556] For this study, participants were required to be aged 18 or older with a life expectancy greater than 16 weeks. For Part 1, participants had to have a histologically or cytologically confirmed diagnosis of a locally advanced unresectable or metastatic solid tumor malignancy harboring RAS (NRAS, KRAS, or HRAS) or RAF (ARAF, BRAF, RAFI) activating mutations, as documented by genomic analysis. The results of the mutation analysis had to be available prior to enrollment, and a prior genomics report from archival tissues or liquid biopsy demonstrating the mutation was acceptable.

[0557] For Part 2, participants were required a histologically or cytologically confirmed diagnosis of one of the following locally advanced unresectable or metastatic solid tumor malignancies: pancreatic adenocarcinoma, RAS-mutated melanoma, Class I BRAF-mutated melanoma, RAS-mutated non-small cell lung cancer (NSCLC), other RAS-mutated gastrointestinal (GI) cancers (excluding colorectal cancer), or any other RAF-mutated solid tumor, as documented by genomic analysis. The mutation analysis results had to be available prior to enrollment, and a prior genomics report from archival tissues or liquid biopsy demonstrating the mutation was acceptable.

[0558] Participants received at least one line of systemic standard-of-care treatment for their advanced or metastatic disease and, in the assessment of the investigator, would be unlikely to tolerate or derive clinically meaningful benefit from other treatment options. Participants previously treated with codon- specific inhibitors of KRAS (including investigational agents) were eligible. KRASG12C mutant participants had to have received prior treatment with a KRASG12C inhibitor for any approved indication. Participants also were required to have radiologic evidence of measurable disease (i.e., at least one target lesion) according to RECIST 1.1 criteria, an ECOG performance status of 0 or 1 , and adequate organ function.Exclusion criteria

[0559] For this study, exclusion criteria included the inability to swallow oral medications, symptomatic untreated or actively progressing known central nervous system metastases, uncontrolled pleural or pericardial effusion or ascites requiring repeated drainage more than once every 28 days (indwelling catheters were allowed), ahistory of severe COVID- 19 infection resulting in the current need for supplemental 02 therapy to maintain resting oxygen saturations of >90%, the presence of ongoing toxicities related to prior anticancer therapy that had not resolved to Grade <1 and were not otherwise allowed, impaired cardiac function or clinically significant cardiac disease, uncontrolled intercurrent illness (including but not limited to poorly controlled diabetes), or any medical condition determined by the investigator to be a risk.

[0560] Additional exclusions included a history or concurrent evidence of retinal vein occlusion (RVO) or current risk factors for RVO, a history of clinically significant serous retinopathy, central serous chorioretinopathy, or retinal edema, and a history of rhabdomyolysis within three months prior to Study Day 1. HIV-infected participants had to be on anti-retroviral therapy and have well-controlled HIV infection / disease. Participants with a history of HBV infection no longer requiring treatment were eligible, as were participants witha history of HCV infection if the HCV viral load was undetectable at screening. Females who were pregnant, breastfeeding, or planning to become pregnant and males who planned to father a child while enrolled in this study were excluded.

[0561] Patients in the clinical trial received Compound B, and pharmacokinetic measurements of Compound B were collected. Blood samples for evaluating the pharmacokinetic profile of Compound B for cycle 1 day 1 of treatment were taken after the initial oral dose of Compound B, at 10 minutes, 20 minutes, 40 minutes, 1 hour, 1.5 hours, 2 hours, 4 hours, 6 hours, 8, hours, 10-12 hours, and 24 hour time points. Pharmacokinetic curves from patients 1, 2, and 4 are shown in Figure 18, Figure 19A and Figure 20, respectively. Figure 18 is a graph showing the pharmacokinetics (PK) for Compound B in a human patient (Patient 1), at cycle 1, day 1 after administration of the initial 40 mg dose of Compound B administered orally (po). Patient 1 is diagnosed with pancreatic cancer having a KRAS G12V mutation and received 40 mg Compound B administered orally (po) twice daily (BID). Figure 19A is a graph showing the pharmacokinetics (PK) for Compound B in a human patient (Patient 2), at cycle 1, day 1 after administration of 40 mg of Compound B administered orally (po). Figure 20 is a graph showing the pharmacokinetics (PK) for Compound B in a human patient (Patient 4), at cycle 1, day 1 after administration of the initial 80 mg of Compound B administered orally (po). Patient 4 was diagnosed with colorectal cancer having a KRAS G12D mutation and received 80 mg Compound B administered orally (po) twice daily (BID).

[0562] Figure 19B is a graph showing pharmacodynamic profile (PD) (pERK) for Compound B from patient 2 after receiving 40 mg Compound B administered orally (po) twice daily (BID) (cycle 1, day 15).

[0563] The following patients were treated with Compound B monotherapy as part of the clinical trial study of Example 19 as outlined in Table 16.Table 16

[0564] In some embodiments, a dose of 120 mg Compound B BID po is administered to a patient in need thereof, including as a monotherapy to patients diagnosed with a cancer having a RAS or RAF mutation.

[0565] Although the foregoing has been described in some detail by way of illustrations and examples for purposes of clarity and understanding, it will be understood by those of skill in the art that numerous and various modifications can be made without departing from the spirit of the present disclosure. Therefore, it should be clearly understood that the forms disclosed herein are illustrative only and are not intended to limit the scope of the present disclosure, but rather to also cover all modification and alternatives coming with the true scope and spirit of the disclosure.

Claims

WHAT IS CLAIMED IS:

1. A method of treating a patient diagnosed with a cancer, comprising administering a compound having a structure of Compound B:Compound B, or a pharmaceutically acceptable salt thereof, twice per day (BID) to the patient in need thereof at a total daily dose of about 40 mg to about 640 mg, to treat the cancer.

2. The method of claim 1 , wherein the cancer is pancreatic adenocarcinoma, or a RAS- or RAF-mutated solid tumor.

3. The method of claim 2, wherein the cancer is a pancreatic adenocarcinoma, RAS- mutated cutaneous melanoma, RAF-mutated cutaneous melanoma, RAS-mutated NSCLC, a RAS-mutated GI solid tumor, or a RAF-mutated solid tumor.

4. The method of claim 3, wherein the cancer is a pancreatic adenocarcinoma optionally comprising a RAS- or RAF- mutation.

5. The method of claim 4, wherein the cancer is a KRAS mutated pancreatic adenocarcinoma.

6. The method of claim 3, wherein the cancer is a RAS-mutated cutaneous melanoma, or a RAF-mutated cutaneous melanoma.

7. The method of claim 6, wherein the cancer is a NRAS mutated or BRAF mutated melanoma.

8. The method of claim 3, wherein the cancer is a RAS-mutated NSCLC.

9. The method of claim 3, wherein the cancer is a KRAS-mutated NSCLC.

10. The method of claim 3, wherein the cancer is a RAS-mutated GI solid tumor other than colorectal cancer.

11. The method of claim 3, wherein the cancer is a RAF-mutated solid tumor.

12. The method of any one of claims 1 -1 1 , wherein Compound B or a pharmaceutically acceptable salt thereof is administered twice per day (BID) to the patient in need thereof, in a dose providing 120 mg or 160 mg of Compound B BID.

13. The method of claim 12, wherein 120 mg of Compound B free base is orally administered twice per day (BID) to the patient in need thereof, as Compound B of a pharmaceutically acceptable salt thereof.

14. The method of claim 12, wherein 160 mg of Compound B free base is orally administered twice per day (BID) to the patient in need thereof, as Compound B of a pharmaceutically acceptable salt thereof.

15. The method of claim 12, wherein the cancer has a mutation selected from the group consisting of:(a) a KRAS mutation selected from the group consisting of G12C, G12S, G12R, G12F, G12L, G12N, G12A, G12D, G12V, G13C, G13S, G13D, G13V, G13P, S17G, P34S, A59E, A59G, A59T, Q61K, Q61L, Q61R, and Q61H;(b) a NRAS mutation selected from the group consisting of: Q61R, Q61L, Q61K, Q61V; and(c) a BRAF mutation selected from the group consisting of: V600D, V600E, V600K, V600R.

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