WO2019183385A1 - Mapk/erk inhibition for ovarian and other cancers - Google Patents

Abstract

Provided herein, are compounds, compositions and methods of treatment of cancers using an inhibitor of MAPK/ERK Kinase (MEK), in particular hydrazine derivatives.

Description

MAPK/ERK INHIBITION FOR OVARIAN AND OTHER CANCERS

RELATED APPLICATIONS

This application claims benefit of and priority to provisional patent application U.S.S.N. 62/646,823, filed on March 22, 2018, the contents of which are hereby incorporated by reference in its entirety.

FIELD OF INVENTION

This invention relates to cancer therapy.

BACKGROUND

Ovarian cancer is the 9th most common cancer afflicting American women, with an estimated 22,280 new cases in 2016. It is most often diagnosed at advanced stages, making it the 5th leading cause of cancer deaths among women, with approximately 14,240 deaths per year. Clear cell ovarian cancer (CCOC) is a high-grade subtype that comprises about 10% of all ovarian cancers. CCOC tends to be more chemotherapy resistant and as such, portends a worse prognosis. Despite poor response rates, the standard adjuvant first-line treatment for CCOC is chemotherapy. Due to the lack of current effective treatments, there is an urgent need for novel CCOC therapies.

SUMMARY

The invention provides a solution to long standing problems in treating cancers such as chemotherapy resistant cancers.

Accordingly, provided herein are compositions and methods of treating a cancer in a subject, comprising administering to the subject an inhibitor of MAPK/ERK Kinase (MEK), e.g., an ovarian cancer such as clear cell ovarian cancer or a pancreatic cancer or melanoma. For example, a composition comprising an inhibitor of MAPK/ERK Kinase (MEK) for use in treating cancer or other proliferative disorders is also within the invention.

The invention features a composition comprising a selective inhibitor of MAPK/ERK Kinase (MEK), wherein the inhibitor reduces activity of MEK1 and/or MEK2. For example, the composition comprises a compound with the following structure:

or a pharmaceutically acceptable salt thereof.

In preferred examples, the inhibitor of MEK does not substantially reduce activity of MEK3 and/or

MEK5.

The invention includes a compound of Formula I:

or a pharmaceutically acceptable salt thereof,

wherein

m is 0, 1, or 2;

n is 0, 1, or 2;

p is 0 or 1;

X is H or Ci-C₆ alkyl;

Y is F, Cl, Br, I, N0₂, CH , or CF₃; and

R in each instance is independently F, Cl, Br, I, N0₂, CFf, or CF₃.

For example, X is H or CH₃. In some example, R in each instance comprises F, Cl, Br, or I.

The invention also encompasses a pharmaceutical composition comprising at least one of the MEK-inhibiting compounds described herein, and a pharmaceutically acceptable excipient.

In some examples, the pharmaceutical composition further comprises an additional therapeutic agent, e.g., an anticancer agent. Exemplary anticancer agent include one or more platinum analogues.

An exemplary method of treating a cancer in a subject in need thereof is carried out by administering to the subject a therapeutically effective amount of at least one of the MEK- inhibiting compounds, or a pharmaceutically acceptable salt thereof. The subject is characterized as, e.g., diagnosed as, comprising a cancer such as ovarian cancer, pancreatic cancer, melanoma, medulloblastoma, or neuroblastoma. For example, the cancer is clear cell ovarian cancer. In preferred embodiments, the compound is administered orally. For example, at least one of the MEK-inhibiting compounds described herein, e.g., URML-3881, is administered at a dosage of about 0.001 mg/kg to about 1000 mg/kg, about 0.01 mg/kg to about 1000 mg/kg, about 0.1 mg/kg to about 1000 mg/kg, about 1 mg/kg to about 1000 mg/kg, about 1 mg/kg to about 500 mg/kg, about 1 mg/kg to about 300 mg/kg, about 1 mg/kg to about 100 mg/kg, about 1 mg/kg to about 50 mg/kg, about 10 mg/kg to about 300 mg/kg, about 10 mg/kg to about 100 mg/kg, about 10 mg/kg to about 50 mg/kg, or other dosages determined suitable, for a human subject.

Compounds described herein can be administered as needed for treatment, e.g., daily, weekly, monthly, bi-monthly, or as indicated by the condition of the subject. An additional anticancer agent is optionally administered. For example, the additional anticancer agent is a platinum analogue. The method may encompass treatment comprising URML-3881 and a platinum analogue such as cisplatin.

Generally, the method of treating a cancer in a subject comprises administering to a subject, e.g., a cancer patient, an inhibitor of MAPK/ERK Kinase (MEK), wherein the inhibitor reduces activity of MEK1 and/or MEK2. Preferably, the inhibitor does not substantially reduce activity of MEK3 and/or MEK5. In some examples, the inhibitor does not comprise trametinib or cobinetinib.

An exemplary subject to be treated is one comprising a cancer. For example, the cancer comprises ovarian cancer such as an epithelial ovarian cancer. The subject to be treated may be characterized as suffering from or diagnosed as comprising a clear cell ovarian cancer (CCOC). In other examples, the subject to be treated is characterized as suffering from or diagnosed as comprising ovarian cancer, pancreatic cancer, melanoma, medulloblastoma, or neuroblastoma.

The inhibitor of MAPK/ERK Kinase is selective, e.g., is specific for MEK1 and/or MEK2. In preferred embodiments, the inhibitor does not bind to MEK3 or MEK5. Optionally, the method further comprises administering to the subject platinum chemotherapy or

PI3K/mTOR inhibitory therapy. Abbreviations used herein include the following terms: MAPK, mitogen-activated protein kinase; MEK, mitogen-activated protein kinase; HGF, hepatocyte growth factor; EGFR, epidermal growth factor receptor; VEGF, vascular endothelial growth factor; KSR1, kinase suppressor of Ras 1.

Throughout this specification, various patents, patent applications and other types of publications (e.g., journal articles, electronic database entries, etc.) are referenced. The disclosure of all patents, patent applications, and other publications cited herein are hereby incorporated by reference in their entirety for all purposes.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. l is a series of photomicrographs showing that MAPK pathway expression in human CCOC is heterogeneously elevated. Representative pictures for p-ERK (green) staining from patient samples of either CCOC, serous ovarian cancer or adjacent normal ovarian tissue are shown. Images are overlaid to also show cell nuclei in blue (DAPI).

FIG 2A is a depiction of the chemical structure of URML-3881, a MEK inhibitor, showing its chemical structure, chemical formula, and exact mass (calculated exact mass obtained by summing the masses of the individual isotopes of the molecule).

FIG. 2B is a bar graph showing percent kinase activity in the presence of URML-3881 (30 nM), compared to kinase activity at baseline.

FIG. 2C is a photograph showing the results of a western blot assay. CCOC cell lines (OVMANA and OVTOKO) were cultured in the presence of increasing concentrations of ETRML-3881 for 24 hrs. Cell lysates were then obtained and p-ERK, ERK, p-MEK and MEK protein expression was determined by western blot using corresponding antibodies.

FIG. 2D is a diagram of the MAPK pathway at baseline, with MEK inhibition, when over-activated (as in CCOC), and when over-activated with MEK and RAF inhibition.

FIGS. 2A-D show that EIRML-3881 is a MEK inhibitor that reduces MAPK pathway activity in CCOC. ** = p<0.0l. FIG. 3 A is a bar graph showing viability of cancer cells treated with URML-3881. Six CCOC cell lines were cultured in the presence of 20 mM URML-3881 for 24 hrs and viability was determined as percentage of control tumor cell growth (DMSO as control).

FIG. 3B is a series of line graphs showing cancer cell viability and dose response to URML-3881. The same six cells lines shown in FIG. 3 A were cultured in serial dilutions of URML-3881 for 24 hrs and viability was determined as percentage of control tumor cell growth.

FIG. 3C is a photograph showing the results of a western blot assay. Six clear cell ovarian cancer cell lines were lysed and assessed for r-REA-15 and PEA- 15 protein expression (per 10 pg of protein from cell lysate) by western blot.

FIG. 3D is a bar graph showing cancer cell proliferation in response to treatment with EIRML-3881. OVMANA cells were cultured in the presence of serial dilutions of EIRML-3881 for 48 hrs and proliferation was assessed by BrdET incorporation. Proliferation rate is expressed as a percentage with DMSO (control) treated cells used as a reference.

FIG. 3E is a bar graph showing cancer cell apoptosis in in response to treatment with EIRML-3881. OVMANA cells were cultured with ETRML-3881 (5 and 20 pM) for 24 hrs and apoptotic status of cells was assessed by flow cytometry. 7-AAD uptake = dead cells. AnnexinV staining = actively dying or dead cells.

FIGS. 3A-E show that ETRML-3881 causes reduced CCOC viability due to induction of tumor cell apoptosis and inhibition of proliferation. * = p<0.05, ** = p<0.0l, *** = p<0.00l.

FIG 4A is a photograph of the results of a western blot assay. An art-recognized humanized mouse model for human cancer in which human tumor tissue is engrafted into immunodeficient NOD SCID gamma (NSG mice) were used. An in vivo mouse model of human cancer in which lxlO⁶ OVMANA cells were injected subcutaneously into the flank of NSG mice and allowed 8 weeks to establish into tumors was used to generate the data described herein.

NSG mice with established OVMANA xenografts were treated with one dose of oral ETRML- 3881 at 30 mg/kg or control and sacrificed 2.5 hrs later. Protein was isolated from the tumors and p-ERK and ERK were analyzed by western blot. NSG mice with established OVMANA xenografts were treated with one dose of oral EIRML-3881 at 30 mg/kg or control and sacrificed 2.5 hrs later. Protein was isolated from the tumors and p-ERK and ERK were analyzed by western blot.

FIG. 4B is a line graph showing animal weight in response to treatment with EIRML- 3881. NSG mice with established OVMANA xenografts were treated daily with ETRML-3881 (10 mg/kg or 30 mg/kg) or control by oral gavage for 21 days, animal weight was measured weekly throughout treatment

FIG. 4C is a line graph showing tumor volume in animals in response to treatment with EIRML-3881. Tumor volume was measured weekly throughout treatment and for 5 weeks afterwards. FIGS. 4A-C show that EIRML-3881 inhibits the MAPK pathway in CCOC in vivo , but a higher dose and/or increased frequency of dosing may be necessary to achieve desirable in vivo activity as a single agent.

FIG. 5A is a photograph of the results of a western blot assay. Alkylating agents are a class of chemotherapeutics that are known to induce ovarian cancer cell death by the inhibition of proliferation and the induction of apoptosis. Six CCOC cells lines were treated with an exemplary platinum-based chemotherapeutic agent with such activity, cisplatin. Cisplatin, was tested at 10 or 30 pM for 24 hrs, cell lysates were then collected and assessed for p-ERK/ERK expression.

FIG. 5B is a photograph of the results of a western blot assay. OVMANA cells were pretreated with ETRML-3881 or control (DMSO) one day before exposure to cisplatin chemotherapy and MAPK pathway and PI3k/AKT pathway activity was determined by western blot.

FIG. 5C is a bar graph showing cancer cell viability in response to URML-3881 treatment. OVMANA cells were pretreated with URML-3881 (2.5 or 10 mM) or control (DMSO) one day before exposure to chemotherapy (cisplatin 10 or 30 pM) for 24 hrs. Cell viability was then determined and reported as percentage compared to control.

FIG. 5D is a depiction of a model treatment scheme. Model animals, NSG mice, with established OVMANA xenografts were subjected to the model treatment scheme. Starting 8 weeks after tumor implantation, the animals were treated daily with oral URML-3881 (30mg/kg) or control (PBS) by oral gavage for 21 days starting on D-l and treated with 3 doses of weekly intraperitoneal cisplatin (4mg/kg) or control (PBS) starting on DO (arrows).

FIG. 5E is a line graph showing the effect on animal weight with treatment using URML- 3881. Animal weight was measured weekly throughout treatment and for 5 weeks afterwards. Error bars = standard deviation. Bottom bars removed from E for clarity.

FIG. 5F is a line graph showing the effect on tumor volume with treatment using URML- 3881. Tumor volume was measured weekly throughout treatment and for 5 weeks afterwards. Error bars = standard deviation. Bottom bars removed from E for clarity.

FIGS. 5A-F show that treatment with EIRML-3881 abrogates cisplatin-induced prosurvival MAPK signaling in CCOC, resulting in durable and dramatic tumor regression in vivo. * = p<0.05, ** = p<0.0l, and *** = p<0.00l.

FIG. 6A is a diagram showing a method of synthesis for EIRML-3881.

FIG. 6B is a photograph of the results of a western blot assay. MEK inhibitors URML- 3881 and AS703026 decrease p-ERK expression but result in enhanced p-MEK. OVMANA cells were cultured in the presence of URML-3881, AS703026, sorafenib or R05126766 for 24 hours at the indicated doses. Cell lysates were then obtained and p-ERK, ERK, p-MEK and MEK protein expression was determined by western blot.

FIG. 7A is a bar graph showing the results of a cell viability assay. This figure shows results using OVMANA, an ovarian cancer.

FIG. 7B is a bar graph showing the results of a cell viability assay. This figure shows results using A2058, a melanoma cancer.

FIG. 7C is a bar graph showing the results of a cell viability assay. This figure shows results using BXPC3, a pancreatic cancer.

FIG. 7D is a bar graph showing the results of a cell viability assay. This figure shows results using D283, a medulloblastoma cancer. FIG. 8 is a bar graph showing the results of an assay using IMR32 cells (neuroblastoma cancer cell type).

FIG. 9 is a bar graph showing the results of an assay using SMSKCNR cells

(neuroblastoma cancer cell type).

FIG. 10 is a bar graph showing the results of an assay using ES-2 cells (ovarian cancer cell type).

FIG. 11 is a bar graph showing the results of an assay using A2780 cells (ovarian cancer cell type).

DETAILED DESCRIPTION

The invention described herein provides compounds, compositions and methods of treatment for ovarian and other cancers.

The mitogen-activated protein kinase (MAPK) pathway, also known as the

Ras/Raf/MEK/ERK pathway, is vital to the survival of tumor cells. It is an intracellular signaling cascade by which activation of receptor tyrosine kinases at the cell surface can be transmitted to gene expression pathways. MAPK signaling is known to play a role in the survival, proliferation, migration and angiogenesis of tumor cells. In CCOC, vascular endothelial growth factor (VEGF), epidermal growth factor receptor (EGFR) and hepatocyte growth factor receptor (MET) are known to be overexpressed and all signal through the MAPK pathway. In addition, prosurvival signaling through this pathway is known to confer a chemotherapy resistant phenotype to tumor cells. The ability of the MAPK pathway to encourage tumorigenesis and contribute to cytotoxic escape make it an appealing therapeutic target in the treatment of CCOC. In fact, the inhibition of MEK, which is a MAP family kinase, has already shown some success in pre-clinical models of CCOC, and in early phase clinical trials of low-grade serous ovarian cancer, another chemotherapy resistant EOC subset. While the success of MAPK inhibition is dependent on activating BRAF and KRAS mutations in some cancer types, the mutational status does not seem to correlate to response in ovarian cancer.

Potent MEK inhibitors, e.g., a compound of Formula I, such as EIRML-3881, have been synthesized, developed and studied as described herein. In some embodiments, the compounds target, i.e., are selective for, MEK1 and 2 isoforms over other MEK isoforms and/or other kinases.

Chemotherapeutic Drugs

Chemotherapeutic agent such as an alkylating agent or alkylating-like agent that induces cancer cell death by apoptosis. Chemotherapeutics include platinum-based anti -neoplastic agents or drugs.

For example, platinum-based antineoplastic drugs (a.k.a., platins) are chemotherapeutic agents used to treat cancer. These chemotherapeutic drugs are complexes comprising platinum. Exemplary platinum-based drugs such as cisplatin, oxaliplatin, and carboplatin are shown below

Oxaliplatin

Nedaplatin

Phenanthriplatin

Picoplatin

Satraplatin

Anti-neoplastic drugs (chemotherapeutic drugs) useful in the invention include paclitaxel, doxorubicine, topotecan, EGFR inhibitors, PI-3K and AKT inhibitors as well as rapamycin and its analogs for treatment regimens exemplify the preferred compounds for combination therapy for treatment of malignancies with URML-3881.

Definitions

The terms "treating" and "treatment" as used herein refer to the administration of an agent or formulation to a clinically symptomatic individual afflicted with an adverse condition, disorder, or disease, so as to effect a reduction in severity and/or frequency of symptoms, eliminate the symptoms and/or their underlying cause, and/or facilitate improvement or remediation of damage.

An "effective amount" or "therapeutically effective amount" refers to an amount of therapeutic compound, such as a small molecule, an oligonucleotide, small molecule, antibody, or any other anticancer therapy, administered to a subject, either as a single dose or as part of a series of doses, which is effective to produce a desired therapeutic effect.

“Purified chemical compound” (such as a small molecule chemical compound) as used herein means the compound is sufficiently free of chemical contaminants resulting from its isolation or chemical synthesis to distinguish the chemical compound from the contaminants.

“Pharmaceutically acceptable excipient” includes without limitation any adjuvant, carrier, excipient, glidant, sweetening agent, diluent, preservative, dye/colorant, flavor enhancer, surfactant, wetting agent, dispersing agent, suspending agent, stabilizer, isotonic agent, solvent, or emulsifier which has been approved by the United States Food and Drug Administration as being acceptable for use in humans or domestic animals.

A“selective” inhibitor of a kinase means that a compound has at least a 3 -fold IC50 difference, e.g., at least lO-fold, 20-fold, 30-fold, 50-fold, lOO-fold, 300-fold, lOOO-fold, or at least 10000-fold IC50 difference, of one kinase over another kinase. For example, a compound selective for MEK1 over MEK3 has at least a 3 -fold lower MEK1 IC50 as compared to its MEK3 IC50.

Unless defined otherwise herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary⁷ skill in the art to which this invention pertains.

As used herein, the singular terms“a,”“an,” and“the” include the plural reference unless the context clearly indicates otherwise.

Compounds

In one embodiment, provided herein is a compound of Formula I:

or a pharmaceutically acceptable salt thereof, wherein

m is 0, 1, or 2;

n is 0, 1, or 2;

p is 0 or 1;

X is H or Ci-C₆ alkyl;

Y is F, Cl, Br, I, N0₂, CH , or CF₃; and

R in each instance is independently F, Cl, Br, I, N0₂, CH₃, or CF₃.

In some embodiments, m is 1 or 2. For example, m can be 2.

In some embodiments, n is 1 or 2. For example, n can be 2.

In some embodiments, p is 0. In some embodiments, p is 1.

In some embodiments, X is H or Ci-C₃ alkyl. In some embodiments, X is H or CH₃. For example, X can be H.

In some embodiments, Y is F, Cl, Br, I, CH₃, or CF₃. In some embodiments, Y is F, Cl, Br, I, or CH₃. In some embodiments, Y is F, Cl, Br, or I. In some embodiments, Y is F or I.

In some embodiments, R in each instance is independently F, Cl, Br, I, CH₃, or CF₃. In some embodiments, R in each instance is independently F, Cl, Br, I, or CH₃. In some embodiments, R in each instance is independently F, Cl, Br, or I. In some embodiments, R in each instance is independently F or I.

In certain embodiments, the compound of Formula I is URML-3881 or a

pharmaceutically acceptable salt thereof. Exemplary MEK inhibitor EIRML-3881 (also known as N-(l, l-dioxidothiomorpholino)-3-((2-fluoro-4-iodophenyl)amino)isonicotinamide) has the structure:

URML-3881 has physicochemical properties comparable to known MEK inhibitors trametinib and cobimetinib as shown in the table below. The predicted values were calculated using ChemDraw software (PerkinElmer Informatics, Inc.).

ETnlike other MEK inhibitors such as Trametinib or Cobimetinib, EIRML-3881 is a chemically a di-substituted hydrazine. An amino group linked to thiazane ring distinguishes EIRML-3881 from other MEK inhibitors. N-amino group spares the thiazane ring nitrogen in basic form whereas a similar structure of other MEK inhibitors carry an amidic nitrogen that has no basic character left. The basic nitrogen present in ETRML-3881 enables it to be converted into a variety of salt forms whereas this feature is absent in the MEK inhibitors described by others. ETRML-3881 has higher polar surface area and lower cLogP than the compound shown below.

Log P: 1.47

tPSA: 78.84

CLogP: 2.40669

CMR: 10.0978

LogS: -5.074

_.

LogS: -5.103

Physicochemical Properties of MEK inhibitor Compounds

The compound of Formula I is selective for MEK1 and/or MEK2. In some embodiments, the compound is selective over other MEK isoforms, e.g., MEK3 and/or MEK5. In some embodiments, the compound is selective over other kinases, e.g., one or more of EGFR, ERK1, VEGFR2, RAF1, MAPK14, and KSR1.

The compounds as described herein may be prepared and/or formulated as

pharmaceutically acceptable salts or when appropriate as a free base. Pharmaceutically acceptable salts are non-toxic salts of a free base form of a compound that possesses the desired pharmacological activity of the free base. These salts may be derived from inorganic or organic acids. For example, a compound that contains a basic nitrogen may be prepared as a

pharmaceutically acceptable salt by contacting the compound with an inorganic or organic acid. Non-limiting examples of pharmaceutically acceptable salts include sulfates, pyrosulfates, bisulfates, sulfites, bisulfites, phosphates, monohydrogen-phosphates, dihydrogenphosphates, metaphosphates, pyrophosphates, chlorides, bromides, iodides, acetates, propionates, decanoates, caprylates, acrylates, formates, isobutyrates, caproates, heptanoates, propiolates, oxalates, malonates, succinates, suberates, sebacates, fumarates, maleates, butyne-l,4-dioates, hexyne-l,6- dioates, benzoates, chlorobenzoates, methylbenzoates, dinitrobenzoates, hydroxybenzoates, methoxybenzoates, phthalates, sulfonates, methyl sulfonates, propylsulfonates, besylates, xylenesulfonates, naphthalene- 1 -sulfonates, naphthalene-2-sulfonates, phenylacetates, phenylpropionates, phenylbutyrates, citrates, lactates, g-hydroxybutyrates, glycolates, tartrates, and mandelates. Lists of other suitable pharmaceutically acceptable salts are found in

Remington: The Science and Practice of Pharmacy, 2 I^st Edition, Lippincott Wiliams and

Wilkins, Philadelphia, Pa., 2006.

Pharmaceutical compositions

Pharmaceutical compositions comprising the compounds disclosed herein, or

pharmaceutically acceptable salts thereof, may be prepared with one or more pharmaceutically acceptable excipients which may be selected in accord with ordinary practice. For example, the compound can be administered as salt of inorganic and organic acids, such as hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, methanesulphonic acid,

trifluoromethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid (tosylate salt), 1- naphthalenesulfonic acid, 2-naphthalenesulfonic acid, acetic acid, trifluoroacetic acid, malic acid, tartaric acid, citric acid, lactic acid, oxalic acid, succinic acid, fumaric acid, maleic acid, benzoic acid, salicylic acid, phenylacetic acid, and mandelic acid lysine, phosphate, camphor sulphonic acid (R and S and racemic mixture) along with the other non-toxic and non-active expecients such as Pharmaceutically acceptable excipients according to the invention are for example disintegrants, binders, lubricants, fillers, plasticizers, surfactants and wetting agents, film forming agents and coating materials, and coloring agents for example pigments. Disintegrants include, but are not limited to croscarmellose sodium, crospovidone, alginic acid, carboxymethylcellulose calcium, carboxymethyl cellulose sodium, microcrystalline cellulose, hydroxypropyl cellulose, low substituted hydroxypropyl cellulose, polacrillin potassium, cross- linked polyvinylpyrrolidone, sodium alginate, sodium starch glycollate, partially hydrolysed starch, sodium carboxymethyl starch and starch. Preference is given to croscarmellose sodium and/or cross-linked polyvinylpyrrolidone, more preference is given to croscarmellose sodium. The amount of the disintegrant contained in the pharmaceutical composition of can be from 0 to 15%, preferably from 5 to 12% by the total weight of the composition. Binders include, but are not limited to hydroxypropyl cellulose, hypromellose (hydroxypropyl methylcellulose, HPMC), microcrystalline cellulose, acacia, alginic acid, carboxymethylcellulose, ethylcellulose, methylcellulose, hydroxaethylcellulose, ethylhydroxyethylcellulose, polyvinyl alcohol, polyacrylates, carboxymethylcellulose calcium, carboxymethylcellulose sodium, compressible sugar, ethylcellulose, gelatin, liquid glucose, methylcellulose, polyvinyl pyrrolidone and pregelatinized starch. Preference is given to a hydrophilic binder which are soluble in the granulation liquid, more preference is given to hypromellose (hydroxypropyl methylcellulose, HPMC) and/or polyvinylpyrrolidone. In some preferred examples hypromellose is used.

A significant advantage of the invention is that the compound is useful and suitable for oral delivery. The compound itself is basic and a simple salt, e.g., phosphate salt form, renders the compound soluble in aqueous media enabling it to be suitable for oral administration to cancer patients.

Tablets may contain excipients including glidants, fillers, binders and the like. Aqueous compositions may be prepared in sterile form, and when intended for delivery by other than oral administration generally may be isotonic. All compositions may optionally contain excipients such as those set forth in the Rowe et al, Handbook of Pharmaceutical Excipients, 6^th edition, American Pharmacists Association, 2009. Excipients can include ascorbic acid and other antioxidants, chelating agents such as EDTA, carbohydrates such as dextrin,

hydroxyalkylcellulose, hydroxyalkylmethylcellulose, stearic acid and the like. In certain embodiments, the composition is provided as a solid dosage form, including a solid oral dosage form.

The compositions include those suitable for various administration routes, including oral administration. The compositions may be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. Such methods include the step of bringing into association the active ingredient ( e.g ., a compound of the present disclosure or a pharmaceutical salt thereof) with one or more pharmaceutically acceptable excipients. The compositions may be prepared by uniformly and intimately bringing into association the active ingredient with liquid excipients or finely divided solid excipients or both, and then, if necessary, shaping the product. Techniques and formulations generally are found in Remington: The Science and Practice of Pharmacy, 2 I^st Edition, Lippincott Wiliams and Wilkins, Philadelphia, Pa., 2006.

Compositions described herein that are suitable for oral administration may be presented as discrete units (a unit dosage form) including but not limited to capsules, cachets or tablets each containing a predetermined amount of the active ingredient. In one embodiment, the pharmaceutical composition is a tablet.

Pharmaceutical compositions disclosed herein comprise one or more compounds disclosed herein, or a pharmaceutically acceptable salt thereof, together with a pharmaceutically acceptable excipient and optionally other therapeutic agents. Pharmaceutical compositions containing the active ingredient may be in any form suitable for the intended method of administration. When used for oral use for example, tablets, troches, lozenges, aqueous or oil suspensions, dispersible powders or granules, emulsions, hard or soft capsules, syrups or elixirs may be prepared. Compositions intended for oral use may be prepared according to any method known to the art for the manufacture of pharmaceutical compositions and such compositions may contain one or more excipients including sweetening agents, flavoring agents, coloring agents and preserving agents, in order to provide a palatable preparation. Tablets containing the active ingredient in admixture with non-toxic pharmaceutically acceptable excipients which are suitable for manufacture of tablets are acceptable. These excipients may be, for example, inert diluents, such as calcium or sodium carbonate, lactose, lactose monohydrate, croscarmellose sodium, povidone, calcium or sodium phosphate; granulating and disintegrating agents, such as maize starch, or alginic acid; binding agents, such as cellulose, microcrystalline cellulose, starch, gelatin or acacia; and lubricating agents, such as magnesium stearate, stearic acid or talc. Tablets may be uncoated or may be coated by known techniques including microencapsulation to delay disintegration and adsorption in the gastrointestinal tract and thereby provide a sustained action over a longer period. For example, a time delay material such as glyceryl monostearate or glyceryl distearate alone or with a wax may be employed.

The amount of active ingredient that may be combined with the inactive ingredients to produce a dosage form may vary depending upon the intended treatment subject and the particular mode of administration. For example, in some embodiments, a dosage form for oral administration to humans may contain approximately 1 to 1000 mg of active material formulated with an appropriate and convenient amount of a pharmaceutically acceptable excipient. In certain embodiments, the pharmaceutically acceptable excipient varies from about 5 to about 95% of the total compositions (weightweight).

Methods of Treatment

Provided herein is a method of treating or reducing the severity of a cancerous condition, comprising administering to a human in need thereof a therapeutically effective amount of a compound of Formula I, or a pharmaceutically acceptable salt thereof. The disease or disorder is an ovarian cancer, e.g., an epithelial ovarian cancer such as CCOC.

Cancers that can be treated or prevented by the methods of the disclosure include solid tumors and lymphomas, including but not limited to adrenal cancer, bladder cancer, bone cancer, brain cancer, breast cancer, colon cancer, colorectal cancer, eye cancer, head-and-neck cancer, kidney cancer such as renal cell carcinoma, liver cancer, lung cancer such as non-small cell lung cancer, ovarian cancer, pancreatic cancer, prostate cancer, skin cancer such as squamous cell carcinoma and melanoma, thyroid cancer, uterine cancer, vaginal cancer, and myeloma such as multiple myeloma. The cancer can be naive, or relapsed and/or refractory.

In some examples, the compound of the disclosure may be employed with other therapeutic methods of cancer treatment. Preferably, combination therapy with

chemotherapeutic, hormonal, antibody, surgical and/or radiation treatments are used.

Examples of further cancer medicaments include intercalating substances such as anthracycline, doxorubicin, idarubicin, epirubicin,and daunorubicin; topoisomerase inhibitors such as irinotecan, topotecan, camptothecin, lamellarin D, etoposide, teniposide, mitoxantrone, amsacrine, ellipticines and aurintricarboxylic acid; nitrosourea compounds such as carmustine (BCNU), lomustine (CCNU), and streptozocin; nitrogen mustards such as cyclophosphamide, mechlorethamine, uramustine, bendamustine, melphalan, chlorambucil, mafosfamide, trofosfamid and ifosfamide; alkyl sulfonates such as busulfan and treosulfan; alkylating agents such as procarbazin, dacarbazin, temozolomid and thiotepa; platinum analogues such as cisplatin, carboplatin, nedaplatin, oxaliplatin, satraplatin, and triplatin tetranitrate; microtubule disruptive drugs such as vinblastine, colcemid and nocodazole; antifolates like methotrexate, aminopterin, dichloromethotrexat, pemetrexed, raltitrexed and pralatrexate: purine analogues like azathioprine, mercaptopurine, thioguanine, fludarabine, fludarabine phosphate, pentostatin and cladribine; pyrimidine analogues like 5-fluorouracil, floxuridine, cytarabine, 6-azauracil, gemcitabine; steroids such as gestagene, androgene, glucocorticoids, dexamethasone, prednisolone, and prednisone; anti -cancer antibodies such as monoclonal antibodies, e.g., alemtuzumab, apolizumab, cetuximab, epratuzumab, galiximab, gemtuzumab, ipilimumab, labetuzumab, panitumumab, rituximab, trastuzumab, nimotuzumab, mapatumumab, matuzumab, rhMab ICR62 and pertuzumab, radioactively labeled antibodies and antibody-drug conjugates; anti-cancer peptides such as radioactively labeled peptides and peptide-drug conjugates; and taxane and taxane analogues such as paclitaxel and docetaxel. In certain embodiments, a compound of Formula I described herein can be combined with a platinum analogue to treat ovarian cancer. For example, URML-3881 is combined with a platinum analogue such as cisplatin to treat ovarian cancer, e.g., clear cell ovarian cancer.

Example 1 : Synthesis of URML-3881

ORML-388!

3((2-fluoro-4-iodophenyl)amino)isonicotinic acid [1] (Ark Pharm Inc) (O.lmM) was coupled with 4-Aminothiomorpholine l,l-Dioxide [2] (TCI Inc.) (O.lmM) in dry

dichloromethane (lOmL) using DCC (Sigma Aldrich) (0. l2mM). Isonicotinic acid [2] was stirred with DCC at 0 °C in argon atmosphere for 10 minutes whereupon a white suspension was formed. Amine [2] was added and a catalytic amount of dimethyl aminopyridine (DMAP)

(Sigma Aldrich) was added. The reaction mixture was stirred overnight over a temperature range of 0 °C to room temperature. The precipitated product was filtered and washed repeatedly with hexane (4x5mL) and dried under vacuum. The final product, URML-3881, was synthesized multiple times in over 60% yields and was characterized by mass spectrometry.

Example 2: MEK inhibition by URML-3881 in cancer cells

The mitogen activated protein kinase (MAPK) pathway is vita! to the survival of tumor cells and is an appealing potential target in cancer therapeutics. It is a signaling cascade initiated by the activation of receptor tyrosine kinases at the cell surface. After this initial step, intracellular kinases are activated sequentially leading to a phosphorylated kinase entering the nucleus where it regulates several processes including transcription and proliferation. The classical, and best characterized, MAPK pathway involves Ras, Raf, MEK, and ERK and is known to play a role in tumor cell survival, proliferation, migration and angiogenesis. The role of MAPK pathway signaling in CCQC is poorly defined. Vascular endothelial growth factor (VEGF), epidermal growth factor receptor (EGFR) and hepatocyte growth factor receptor (MET) are over-expressed in this cancer type and all signal through the MAPK pathway. In the last decade four MAPK inhibitors have been approved by the FDA and are now in clinical use in melanoma and lung cancers with activating mutations. While CCQCs rarely cany activating mutations in the MAPK pathway, such mutations are not predictive of response to MAPK inhibition in ovarian cancer.

In addition to the tumor-promoting features of MAPK activation discussed above, this pathway is known to confer a chemotherapy resistant phenotype to tumor cells by mediating pro- survival signaling. Interestingly, MEK inhibition has been shown to overcome platinum- chemotherapy resistance in several cancer cell types. The ability of the MAPK pathway to contribute to cytotoxic escape makes it a candidate for combination treatment with chemotherapy in CCGC.

The role of the MAPK pathway in CCQC viability and chemotherapy resistance was studied to assess its activity as a therapeutic target. Inhibition of the kinase MEK is of particular interest since it represents an important bottle-neck in the pathway and directly activates p-ERK, the master regulator of downstream nuclear events. Additionally, attempts at blocking further upstream have led to paradoxical pathway overactivity in RAF wild-type tumor cells. A MEK inhibitor, named URML-3881, was made and its activity evaluated. The results indicate efficacy of MEK inhibition with URML-3881 in CCOC, both as a single-agent and as an adjunct to pi ati n um c hem oth er ap y .

The following materials and methods w¾re used to generate the data described herein.

Cell culture

Patient-derived clear cell ovarian cancer cell lines, OVMANA, OVAS, OVTOKO, HCH-

1 and RMG-1 were studied. ES-2 was purchased from ATCC (CRL-1978). ES-2 was grown in

FBS supplemented McCoy media (Gibco, 16600-082) while all other cell lines were grown in FBS supplemented RPMI (Gibco, 22400-089).

Western blot

Western blot assays were carried out using standard methods. OV ANA cells w'ere plated (5xl0⁵), after adherence media was replaced with fresh media containing either DMSG (control), URML-3881 at varying concentrations, R05126766 at 10 m.M, AS703026 at 10 mM or Sorafenib at 10 mM (all drugs except URML-3881 were obtained from Selleck Chemicals). Cell lysis buffer (Cell Signaling, 9803 S) with 1 mM PMSF was utilized for protein extraction.

Animal tumor tissue was snap frozen and stored in liquid nitrogen. It was then thawed and mechanically homogenized in ceil lysis buffer. Protein concentrations were determined using the DC Protein Assay (Bio-Rad, 500011 1). Western blot analysis using methods well known in the art. Primary antibodies against p-ERK (4370), ERK (9102), p-MEK (2338), MEK 1/2 (4694), p- AKT (9271), AKT (9272), pPEA-15 (2776) and PEA-15 (2780) were all obtained from Cell Signaling Technology and used at the recommended dilutions.

URML-3881 synthesis

The method of synthesis of URML-3881 is briefly described in Example 1, above. A schematic of synthesis is shown in FIG. 6A. 3((2-fluoro-4~iodopheny!)amino)isonicotinic acid [1] [Ark Pharm Inc, Catalog No: AK118913] (0.1 mM) was coupled with 4- Aminothiomorpholine 1,1-Dioxide [2] [TCI Inc, A1798] (0.1 mM) in dry dichioromethane (10 mL) using DCC (Sigma Aldrich, Cat No: 36650), (0.12mM). Isonicotinic acid [2] was stirred with DCC at 0°C in argon atmosphere for 10 minutes, a white suspension was formed. Amine [2] was added and a catalytic amount of dimethyl aminopyridine (DMAP) (Sigma Aldrich, 39405) was added. The reaction mixture was stirred overnight over a temperature range of OoC to room temperature. The precipitated product was filtered and washed repeatedly with hexane (4x5niL) and dried under vacuum. The final product, URML-3881, was synthesized multiple times in over 60% yields and was characterized by mass spectrometry.

In silico prediction of drug-like qualities

The polar surface area, molar mass, LogP, eLogP and LogS of drugs were predicted using the property prediction feature of ChemDraw software.

Cell free kinase assay

Kinase inhibition studies were performed by Reaction Biology Corp. In brief, purified kinases were incubated with known substrate, radiolabeled ATP and test drug or a predetermined reference compound. Enzyme activity was then determined by the presence of radiolabeled- phosphorylated substrate and calculated as a percentage of control. hn m unohi stochem i stry

A human ovarian tumor array was purchased from US Biomax (OV801 1). The paraffin embedded slides were baked, deparaffmized in Americlear (Adwin Scientific, 72060044), and rehydrated in decreasing concentrations of ethanol. Antigen retrieval was performed by- incubation in heated Dako target retrieval solution. Slides w¾re then permeabilized by adding Triton-X100 (0.15%) to wash solution, blocked in 50% horse serum, and incubated overnight with anti-p-ERK primary antibody (Cell Signaling, 4370) or isotype control per manufacturer’s recommendation. Slides were washed and incubated with an anti-rabbit secondary antibody conjugated to Dylight 488 (Invitrogen, 35552). Tissue sections on the slide were immersed in Vectashield with DAPI and eoverslipped. Slides w^?ere imaged at 60X on an Olympus BX43F fluorescence microscope utilizing cell Sens software, using the same exposure times for each image. Cell viability assays

The viability of cells (COCC cell lines) such as OVMANA OVAS, OVTOKO, HCH-1, RMG-1, and ES-2 was determined after drag treatment using the 96^® Aqueous-One-Solution Assay (Promega, G3580). Briefly, 5x10^s cancer cells were exposed to serial dilutions of URML- 3881 and incubated at 37°C for 24 hours. Media was then replaced with MTS reagent (1 : 10 dilution) and incubated for 2 hours. Absorbance was measured at 490 nm on a Bio-Rad iMARK microplate reader. Experiments were performed in triplicate; data are expressed as the mean percentage (± standard deviation) compared to control (DM SO) treated ceils in cells that were treated with both URML-3881 and cisplatin, URML-3881 was added 24 hours before cisplatin, and viability was determined 24 hours after the addition of cisplatin.

Apoptosis assays

Annexin V Apoptosis Detection Kit (eBioscience, 88-8007) was used. In brief, 5 x 10^s OVMANA cells/well were plated and allowed to adhere, then the media was replace with fresh complete media containing URML-3881 (5 or 20 mM) or DMSO control, and incubated for 24 hours at 37°C. in order to avoid losing dead/dying cells, the media was collected and centrifuged and the adherent cells were collected by trypsinization and combined with the nonadherent cells. Cells were stained with annexin-V-APC, followed by 7- A AD according to the manufacturer’s instructions. Cells were then immediately analyzed by flow cytometry_' on a BD LSR Fortessa cytometer.

Proliferation assay

Proliferation of cells was determined by BrdU incorporation using a Ceil Proliferation Assay Kit (Cell Signaling Technology, 6813). 5x10^s w^?ere plated into a 96 well flat bottom plate and allowed to adhere, serial dilutions of URML-3881 or DMSO control, and BrdU were added to wells. Cells were incubated for 48 hours at 37°C, and then washed, fixed, denatured and stained with anti-BrdU-HRP according to manufacturer’s instructions. TMB substrate exposure time was 30 minutes, then stop solution was added and the absorbance was measured at 450 nM on a Bio-Rad iMARK microplate reader. Experiments were performed in replicates of 9. Data are expressed as the mean percentage (± standard deviation) compared to control (DMSO) treated cells.

Xenograft animal model

NOD scid gamma (NSG) mice were injected subcutaneously in the right flank with !xlO⁶ QVMANA cells in 100 mΐ of matrigel (Coming, 356235). Tumors were allowed to establish for 8 weeks until they averaged approximately 100 mm3 in volume and animals were randomized. Treatment with URML-3881 was delivered daily via oral gavage for 21 days at either 10 mg/kg dosing or 30 mg/kg dosing (PBS control). When administered in the combination groups, cispiatin chemotherapy or control (PBS) was started 24 hours after URML-3881 treatment and was given intraperitoneally every 7 days (4 mg/kg) for 3 doses. Animals were monitored for signs of distress at least twice weekly throughout the course of the experiment. Tumor volume and animal weight was recorded weekly.

Statistical Analysis

Kinase inhibition, proliferation and cell viability were evaluated using a one-way

ANQVA with multiple comparisons. Apoptosis assay and animal studies were compared using a two-way ANOVA (GraphPad Prism 5.0 Software). P values <0.05 were considered statistically significant.

MAPK activity is elevated in CCQC

MAPK pathway overexpression in cancer is known to enhance proliferation and angiogenesis, thereby promoting tumor growth. The importance of the MAPK pathway has been shown in other HOC subtypes, such as low-grade serous carcinomas which often cany a KRAS mutation. However, the importance of this pathway in clear cell ovarian cancer has not been heavily studied. While RAS/RAF activating mutations are rare in CCOC, overexpression of MET, VEGF and EGFR, all which signal through the MAPK pathway, is frequently identified [4] ERK, is the substrate of MEK, and when activated by phosphorylation, it is considered to be a crucial effector in the MAPK pathway. Consequently, phosphorylated-ERK (pERK) levels are commonly used to quantitate MAPK pathway activity. Patient CCOC, high-grade serous ovarian cancer, and adjacent normal ovarian tissues were stained for p-ERK. While there is clearly variability between samples, the CCOC specimens expressed higher p-ERK than control ovary or high-grade serous cancer. These data suggest that MAPK over-activity is a previously unrecognized feature of CCQC.

URML- is a selective MEK 1/2 inhibitor

A MEK inhibitor URML-3881 (Fig. 2A), as selective, e.g., specific MEK1/2 inhibitor, was developed as described herein. This class of drugs has been plagued by issues such as poor solubility and high molecular weight which lead to suboptimal

pharmacokinetics/pharmacodynamics. In fact, the most commonly used MEK inhibitor, trametinib, requires the potentially toxic molecule dimethyl sulfoxide (DMSO) as a solvate, a feature that is unusual amongst drugs in clinical use. URML-3881 is a disubstituted hydrazine, carries a tertiary_' nitrogen that is basic, and can be salted variously to produce a salt form that is characterized by enhanced solubility and serum availability characteristics. Other MEK inhibitors carry a metabolically less stable side chains.

It is unclear to what degree oral DMSO is enhancing the side effect profile of trametinib, but intravenously delivered DMSO can cause a variety of serious and potentially life-threatening side effects. URML-3881 has been designed to possess optimized drug-like qualities compared to existing MEK inhibitors in an effort to enhance pharmacokinetics and pharmacodynamics. These qualities are associated with significant advantages including including improved predicted solubility and a smaller molar mass. Additionally, URML-3881 structurally incorporates a tertiary nitrogen (Fig. 2A), that when converted to salt forms enhances its aqueous solubility. Preferred salts, as mentioned above are: hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, methanesuiphonic acid, trifluoromethanesuifonic acid, benzenesulfonic acid, p-toluenesul tonic acid (tosylate salt), 1 -naphtha! enesu!fonic acid, 2-naphthalenesulfonic acid, acetic acid, triflu oroacetic acid, malic acid, tartaric acid, citric acid, lactic acid, oxalic acid, succinic acid, fumaric acid, maleic acid, benzoic acid, salicylic acid, phenyl acetic acid, and mandelic acid, lysine, phosphate, and/or camphor sulphonic acid (R and S and racemic mixture). A comparison of the predicted pharmacologic properties of URML-3881 in comparison to other MEK inhibitors show that it possesses more favorable drug-like characteristics in terms of molar mass, LogP, CLogP and Log S (see Table, above) URML-3881 is a potent inhibitor of MEK 1/2 (Fig. 2B), with an IC50 of 30 tiM in a cell-free kinase inhibition assay. This assay also established the drug’s specificity for MEK 1 and 2; showing negligible or no detectable inhibitory function amongst a panel of other kinases including MEK 3 and MEK 5, which are activated in alternative MAPK signaling pathways.

URML-3881 inhibits CCOC MEK activity in vitro

Two human-derived CCOC cell lines, OVMANA and OVTOKO, were subjected to increasing concentrations of URML-3881 and MAPK activity was determined by ERK phosphorylation. Western blot analyses revealed that increasing concentrations of MEK inhibitor resulted in less p-ERK expression by CCOC (Fig. 2C), while p-MEK conversely increased with URML-3881 concentration. Increased upstream MAPK activity confirms URML- 3881 as a specific functional MEK inhibitor in vitro, since p-MEK would not be increased if the drug was working further upstream. This upstream MAPK compensatory over-activity occurs due to the loss of negative feedback regulatory mechanisms (Fig. 2D). P-ERK inhibition and p- MEK accumulation was confirmed with another known specific MEK inhibitor (AS703026), while a combination MEK/BRAF inhibitor (R05126766) resulted in reduction of both p-ERK and p-MEK. Sorafemh, a Raf inhibitor with no known activity against MEK 1/2, did not cause a reduction in either p-ERK or p-MEK, and actually increased the levels of both phosphory!ated kinases. This finding is consistent with the recognized paradoxical activation of the MAPK pathway by Raf inhibitors in non-RAF mutant cancer cells, depicted in Fig. 2D.

URML-3881 inhibits CCOC viability both by induction of apoptosis and inhibition of proliferation

The ability of URML-3881 to inhibit tumor cell viability was determined. 20 mM of drug resulted in a significant reduction in the viability of 6 human-derived CCOC cell lines compared to a vehicle/excipient, e.g., a DMSO control (Fig. 3 A). This inhibition of CCOC viability was determined to be dose-dependent in 48 hour cell co-cultures with serial drug dilutions (Fig. 3B), with some cell lines showing a significant reduction in viability at concentrations as low as 1.25 mM. pPEA-15 regulates the ability of ovarian cancer ceils to respond to MEK inhibition. pPEA- 15 levels were assessed in various CCOC cell lines (isolated from different patients), and there was no clear correlation to MEK inhibitor response (Fig. 3C). For example OVMANA was the most sensitive cell line and did not have higher pPEA-15 levels. Since the MAPK pathway is involved in both ceil survival and proliferation, experiments were carried out to determine which process was most impacted by URML-3881. Proliferation was determined by BrdU incorporation and all doses of drug tested (from 0.625 mM to 20 mM) caused a comparable modest reduction in tumor cell proliferation of between 15 and 30%, in a manner that did not seem to be dose-dependent (Fig. 3D), i.e., the inhibition at the lowest concentration (0.625 and 1.25 mM was not significantly different than the inhibition at 20 mM). An apoptosis analysis revealed that 7-AAD and Annexin V were markedly increased in tumor cells at 20 mM of URML-3881 to 19% and 51% respectively, with a non-significant increase being seen at a lower dose of 5 mM (Fig. 3E). Therefore, inhibition of proliferation and induction of apoptosis are both mechanisms of URML-388 l’s inhibition of cell viability, with apoptosis occurring at an increased rate at higher doses.

URML-3881 as a single-agent

In clear cell ovarian cancer, URML-3881 is useful in combination therapy and may be useful/effective as a single-agent at certain doses. URML-3881 is useful in other cancer types as a single-agent as well as in combination with other chemotherapeutic drugs. URML-3881 (as a single-agent) shows in vitro responses against cell -lines derived from melanoma,

medulloblastoma and pancreatic cancer. Optionally, a combination of URML-3881 with cisplalin, paclitaxel, doxorubicin, topotecan, cyclophosphamide and/or other standard chemotherapies is useful against these and other cancers.

For in vivo experiments, CCOC tumor bearing animals were treated with a single-bolus of 30 mg/kg URML-3881 and sacrificed 2.5 hours later. This timepoint was chosen based on the known short Tmax values of other MEK inhibitor compounds . Tumors were harvested and p- ERK level was determined by western blot. As expected, treatment with URML-3881 reduced p- ERK in tumors (Fig. 4A), thus confirming in vivo efficacy as a MEK inhibitor. Animals were then treated with URML-3881 at doses of either 10 mg/kg or 30 mg/kg daily (by oral gavage) for 21 days and tumor volumes were measured throughout. While URML-3881 was well tolerated by the animals at both doses, as evidenced by stable weight (Fig. 4B), it was not observed to induce tumor regression or delaying tumor growth at the doses testedfFig. 4C).

Platinum chemotherapy increases MARK pathway activity in CCOC, but URML-3881 can inhibit this pro-survival signaling and enhance tumor cell death

Platinum-based chemotherapy, a staple in ovarian cancer treatment regimens increase ovarian cancer's reliance on the pro-survival MAPK pathway. The role of URML-3881 as a combinatorial agent with platinum chemotherapy was evaluated. There is an increase in MAPK pathway activity in CCOC upon exposure to platinum chemotherapy (10 and 30 mM), 5/6 cell lines (Fig. 5A). The baseline p-ERK in OVAS was very faint and did not clearly change with chemotherapy. Dual drug culture experiments were performed in which cells were pre-treated with URML-3881 for 24 hours and then exposed to cisplatin chemotherapy . URML-3881 was able to abrogate chemotherapy-induced MAPK signaling (Fig. 5B) and reduce tumor cell viability (Fig. 5C) in vitro. The URML-3881 10 mM combination treatments also caused a decrease in ERK expression in the unphosphorylated form. AKT w^?as run on the same blot (shown in the bottom panel of Fig. 5B) and confirms that there were consistent amounts of protein loaded into each lane. CCOC is characterized by overactivity of the PI3K/AKT pathway due to mutations in ARID 1 A and PTEN. The PI3K/AKT pathway is another intracellular signaling pathway that is known to enhance tumors genesis, and has extensive cross-talk with the MAPK pathway. p-AKT levels in CCOC were assessed as a measure of PI3K activity, and a compensatory increase was found when the MAPK pathway was inhibited with URML- 3881 (Fig. 5B). This PI3K upregulation upon MAPK inhibition was still apparent in the presence of 10 mM of cisplatin but was less apparent at the 30 mM cisplatin concentration.

Combination Cisplatin and URML-3881 reduces tumor size of CCOC in vivo and is well tolerated

To further validate combination MEK inhibition with URML-3881 and cisplatin as a treatment strategy for CCOC, NSG mice bearing established OVMANA CCOC xenografts were engrafted with URML-3881 daily (starting on day -I) and cisplatin at 4 mg/kg weekly for 3 weeks (starting on day 0) (Fig 5D). A significant and prolonged reduction in tumor volume was seen with combination URML-3881 and cisplatin compared to both control (p^::::0.02) and URML-3881 alone (p=0.0003) (Fig 5E). URML-3881 + cisplatin was not significantly different from cisplatin alone (p^:::Q.09), likely due to variability within the cisplatin group. Animal weight was recorded weekly and all treatment categories were well -tolerated with no significant changes between groups (Fig. 5F). There was a non-significant trend towards weight loss at day 21 in the URML-3881 + cisplatin group that improved weekly thereafter. Animals in all treatment groups retained baseline activity levels and showed no outward signs of distress.

Specific MEK1/2 inhibitors with improve solubility for cancer therapy

Specific MEK inhibition is useful as a treatment strategy in clear cell ovarian cancer and other cancer types such as MAPK over-expressing cancer types including pancreatic, lung, colon, and skin cancer. While MEK inhibition with URML-3881 showed promising in vitro activity as a single-agent, in vivo activity as a single-agent was not observed at the tested doses and dose schedule. When combined with cisplatin, URML-3881 was shown to inhibit prosurvival MAPK signaling and result in significant and long-lasting tumor regression, while cisplatin alone appeared to have no effect. Therefore, MEK inhibition with URML-3881 as a chemotherapy is useful for sensitizing adjunct to clear cell ovarian cancer treatment and represents a superior and advantageous method compared to earlier protocols.

Due to poor chemotherapy response rates and the lack of treatment alternatives, advanced clear cell ovarian cancer has carried a significantly worse prognosis when compared to other EOC subtypes. An agent that can increase chemotherapy susceptibility in this resistant disease has the potential to substantially prolong patients’ lives and enhance cure rates. The combination of MEK inhibition with URML-3881 and cisplatin w^?as weli tolerated by animals.

The MAPK and PBK/mTOR pathways cross-talk and co-regulate one another.

Additionally, CCQCs typically possesses heightened baseline activity of the PBK/mTOR pathway due to a high frequency of AR ID la and PTEN mutations. One possible reason for the lack of observed efficacy of single-agent URML-3881 in vivo is compensatory upregulation of RΪ3K activity. An increase in PI3K activity was observed after MAPK inhibition with URML- 3881 in vitro , and in the chemotherapy combination group, the addition of high dose cisplatin also caused PI3K upregulation. These findings provide the rationale for alternative combinatorial treatment strategies in CCOC, such as MEK inhibition with PBK/mTOR inhibition, platinum chemotherapy with PBK/mTOR inhibition, or all three agents together.

The data described herein provides evidence that combination MEK inhibition and cispiatin is effective against chemotherapy-resistant clear cell ovarian cancer. This treatment strategy is associated with significant advantages for a disease that has limited therapeutic options and carries very poor survival rates in the advanced or recurrent setting. These findings are applicable to the treatment of thousands of patients with many other cancer subtypes including cancers that have a high frequency of MAP K pathway activating mutations such as melanoma, lung cancer, ovarian cancer, colon cancer and pancreatic cancer. Additionally, MAPK overactivity and chemotherapy resistance are prominent features amongst highly fatal malignancies across cancer types.

OTHER EMBODIMENTS

While the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and

modifications are within the scope of the following claims.

The patent and scientific literature referred to herein establishes the knowledge that is available to those with skill in the art. All United States patents and published or unpublished United States patent applications cited herein are incorporated by reference. All published foreign patents and patent applications cited herein are hereby incorporated by

reference. Genbank and NCBI submissions indicated by accession number cited herein are hereby incorporated by reference. All other published references, documents, manuscripts and scientific literature cited herein are hereby incorporated by reference.

While this invention has been particularly shown and described with references to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims.

Claims

We claim:

1 A composition comprising a selective inhibitor of MAPK/ERK Kinase (MEK), wherein said inhibitor reduces activity of MEK1 and/or MEK2.

2. The compound of claim 1, wherein said compound comprises the structure of :

or a pharmaceutically acceptable salt thereof.

3. The composition of claim 1, wherein said inhibitor of MEK does not substantially reduce activity ofMEK3 and/or MEK5.

4. A compound of Formula I:

or a pharmaceutically acceptable salt thereof,

wherein

m is 0, 1, or 2;

n is 0, 1, or 2;

p is 0 or 1;

X is H or Ci-C₆ alkyl;

Y is F, Cl, Br, I, N0₂, CH , or CF₃; and

R in each instance is independently F, Cl, Br, I, N0₂, CFE, or CF_3.

5. The compound of claim 4, wherein X is H or CH_3.

6. The compound of claim 4, wherein R in each instance comprises F, Cl, Br, or I.

7. A pharmaceutical composition comprising the compound of claim 4, and a pharmaceutically acceptable excipient.

8. The pharmaceutical composition of claim 7, further comprising an additional

therapeutic agent.

9. The pharmaceutical composition of claim 8, wherein the additional therapeutic agent is an anti cancer agent.

10. The pharmaceutical composition of claim 9, wherein the anticancer agent is a

platinum analogue.

11. A method of treating a cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a compound of claim 4, or a pharmaceutically acceptable salt thereof.

12. The method of claim 11, wherein the cancer comprises ovarian cancer, pancreatic cancer, melanoma, medulloblastoma, or neuroblastoma.

13. The method of claim 12, wherein the cancer is clear cell ovarian cancer.

14. The method of claim 12, further comprising administering an additional anticancer agent.

15. The method of claim 14, wherein the additional anticancer agent is a platinum

analogue.

16. The method of claim 15, wherein the compound comprises URML-3881 and the platinum analogue comprises cisplatin.

17. A method of treating a cancer in a subject, comprising administering to said subject an inhibitor of MAPK/ERK Kinase (MEK), wherein said inhibitor reduces activity of MEK1 and/or MEK2.

18. The method of claim 17, wherein said inhibitor does not substantially reduce activity of MEK3 and/or MEK5.

19. The method of claim 17, wherein said inhibitor does not comprise trametinib or cobinetinib.

20. The method of claim 17, wherein said cancer comprises ovarian cancer.

21. The method of claim 17, wherein said cancer comprises an epithelial ovarian cancer.

22. The method of claim 20, wherein said ovarian cancer comprises clear cell ovarian cancer.

23. The method of claim 17, wherein said cancer comprises ovarian cancer, pancreatic cancer, melanoma, medulloblastoma, or neuroblastoma.

24. The method of claim 17, wherein the inhibitor of MAPK/ERK Kinase is selective for MEK1 and/or MEK2.

25. The method of claim 17, wherein said inhibitor does not bind to MEK3 or MEK5.

26. The method of claim 17, further comprising administering to said subject platinum chemotherapy or PI3K/mTOR inhibitory therapy.