WO2016029002A2 - Growth factor receptor inhibitors - Google Patents

Abstract

Compounds of formula (I) or salts thereof as IGF-1R/INSR inhibitors are provided. The compounds may be used in method of treating cancer. Pharmaceutical compositions containing a compound of formula (I) or a salt thereof and a pharmaceutical acceptable excipient are also provided, as are kits containing a compound of formula (I) or a salt thereof and instructions for use, e.g., in a method of treating cancer.

Description

Growth Factor Receptor Inhibitors

RELATED APPLICATIONS

[0001] This application claims priority to U.S. provisional patent application serial no. 62/040,625, filed August 22, 2014 and U.S. provisional patent application serial no. 62/081,300, filed November 18, 2014, both of which are herein incorporated by referenced in their entirety.

FIELD

[0002] The present disclosure relates to compounds that are capable of inhibiting, modulating and/or regulating growth factor receptors, more particularly insulin-like growth factor I receptor (IGF-1R), insulin receptor (INSR or IR), focal adhesion kinase (FAK), fer (fps/^'fes related) tyrosine kinase (FER), and/or feline sarcoma oncogene (FES).

BACKGROUND

[0003] Protein kinases (PKs) are enzymes that catalyze the phosphorylation of hydroxy groups on tyrosine, serine and threonine residues of proteins. The consequences of this seemingly simple activity are staggering; cell growth, differentiation and proliferation, i.e., virtually all aspects of cell life, in one way or another depend on PK activity. Furthermore, abnormal PK activity has been related to a host of disorders, ranging from relatively non life-threatening diseases such as psoriasis to extremely virulent diseases such as glioblastoma (brain cancer). PKs can be broken into two classes, the protein tyrosine kinases (PTKs) and the serine- threonine kinases (STKs).

[0004] Certain growth factor receptors exhibiting PK activity are known as receptor tyrosine kinases (RTKs). They comprise a large family of transmembrane receptors with diverse biological activity. At present, at least nineteen distinct subfamilies of RTKs have been identified. One RTK subfamily contains the insulin receptor (INSR) and insulin-like growth factor 1 receptor (IGF-IR).

[0005] IGF-IR is a tyrosine kinase membrane receptor having a structure very similar to that of the insulin receptor. The structure of IGF-IR consists of two extracellular a-chains that form the ligand-binding domain and two β-chains that make up the transmembrane and intracellular domains. IGF-IR is the primary receptor for insulin-like growth factor IGF- 1 , although IGF-2 and insulin can also bind with less affinity. There are also at least six IGF-binding proteins, IGFBP1-6, that bind and modulate the availability and other biological effects of IGF-IR. Upon ligand binding, IGF-IR is activated, resulting in autophosphorylation of tyrosines on the intracellular B-subunit. IGF-IR then phosphorylates intracellular proteins such as the insulin receptor substrates 1 to 4 (IRS1 - IRS4) and She. These substrates, in turn, initiate phosphorylation cascades that activate the phosphatidylinositol 3 -kinase (PI- 3K)/protein kinase B (Akt) or mitogen- activated protein kinase (MAPK) pathways (Samani et al. Endocr. Rev. 28:20-47 (2007)).

[0006] Through activation of these signaling cascades, IGF-IR has been implicated in cancer. Extensive studies have implicated IGF-IR, IGF-1, and IGF-2 signaling in cancer development, maintenance, and progression, however the exact role of IGF-IR in cancerremains uncertain and appears to vary according to tumor or cell type. For example, some tumors may depend on IGF-IR signaling for survival, whereas others rely on IGF-IR for proliferation. Yet other tumors may employ IGF- IR overexpression as a mechanism of resistance against cytotoxic agents such as anticancer drugs (Rodon et al. Mol. Cancer Ther. 7:2575-2588 (2008)). Accordingly, inhibition of IGF-IR is an attractive drug strategy for cancer treatment.

[0007] IGF-IR pathway is found to establish resistance to epidermal growth factor receptor (EGFR) inhibitors. Many cancers, particularly those carrying EGFR amplifications, are amenable to treatment with epidermal growth factor receptor (EGFR) inhibitors. However, in many cases these cancers acquire resistance and ultimately become refractory to treatment. Several studies have demonstrated or disclosed that some EGFR kinase inhibitors can improve tumor cell or neoplasia killing when used in combination with IGF-IR inhibitors. BRIEF SUMMARY

[0008] Essentially pure or isolated compounds of formula (I) or salts thereof as inhibitors, modulators and/or regulators of growth factor receptors, more particularly insulin-like growth factor I receptor (IGF-IR), insulin receptor (INSR or IR), focal adhesion kinase (FAK). fer (fps/fes related) tyrosine kinase (FER), and/or feline sarcoma oncogene (FES), are provided.

[0009] Further provided is a pharmaceutical composition comprising a compound of formula (I) or a salt thereof, and a pharmaceutically acceptable excipient.

[0010] Further provided is a method of treating cancer comprising

administering to an individual in need thereof a therapeutically effective amount of a compound of Formula (I) or a pharmaceutically acceptable salt thereof.

wherein R_j = H, R₂ = H; or

wherein R, = -COCH₃, R₂ = H or -COCH₃.

[0011] Further provided is use of a compound of Formula (I) or a salt thereof, in the manufacturing of a medicament for the treatment of cancer.

[0012] Also provided is a kit comprising a compound of Formula (I) or a pharmaceutically acceptable salt thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Fig. 1 shows metabolites structure and the biotransformation pathways leading to generation from CO-1686. [0014] Figs. 2A-C show inhibition of CO-1686 resistance by CO-1686 metabolite M502.

[0015] Figs. 3A-B show evaluation of CO-1686 HBr in an Oral Glucose

Tolerance Test in female Sprague Dawley Rats.

[0016] Fig. 4 shows evaluation of M460 and M502 in an Oral Glucose

Tolerance Test in female Sprague Dawley Rats.

[0017] Fig. 5 shows impact of CO-1686 metabolite M502 on cell morphology

(lOx magnification).

[0018] Fig. 6 shows the effect of CO-1686 and the IGF-1R and INSR inhibitors OSI-906 and M502 in PC-9 cells.

DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS

Definition

[0019] For use herein, unless clearly indicated otherwise, use of the terms "a",

"an" and the like refers to one or more.

[0020] As used herein, "treatment" or "treating" is an approach for obtaining beneficial or desired results including clinical results. For purposes of this invention, beneficial or desired clinical results include, but are not limited to, one or more of the following: decreasing one or more symptoms resulting from the disease, diminishing the extent of the disease, stabilizing the disease (e.g., preventing or delaying the worsening of the disease), preventing or delaying the spread (e.g., metastasis) of the disease, preventing or delaying the occurrence or recurrence of the disease, delay or slowing the progression of the disease, ameliorating the disease state, providing a remission (whether partial or total) of the disease, decreasing the dose of one or more other medications required to treat the disease, enhancing effect of another medication, delaying the progression of the disease, increasing the quality of life, and/or prolonging survival. Also encompassed by "treatment" is a reduction of pathological consequence of cancer. The methods of the invention contemplate any one or more of these aspects of treatment. [0021] As used herein, "delaying" the development of cancer means to defer, hinder, slow, retard, stabilize, and/or postpone development of the disease. This delay can be of varying lengths of time, depending on the history of the disease and/or individual being treated. As is evident to one skilled in the art, a sufficient or significant delay can, in effect, encompass prevention, in that the individual does not develop the disease. A method that "delays" development of cancer is a method that reduces probability of disease development in a given time frame and/or reduces the extent of the disease in a given time frame, when compared to not using the method. Such comparisons are typically based on clinical studies, using a statistically significant number of subjects. Cancer development can be detectable using standard methods, such as routine physical exams, mammography, imaging, or biopsy.

Development may also refer to disease progression that may be initially undetectable and includes occurrence, recurrence, and onset.

[0022] As used herein, "combination therapy" means a therapy that includes two or more different compounds. Thus, in one aspect, a combination therapy comprising a compound detailed herein and another compound is provided. In some variations, the combination therapy optionally includes one or more pharmaceutically acceptable carriers or excipients, non-pharmaceutically active compounds, and/or inert substances.

[0023] As used herein, the term "effective amount" intends such amount of a compound of the invention which in combination with its parameters of efficacy and toxicity, should be effective in a given therapeutic form. As is understood in the art, an effective amount may be in one or more doses, i.e., a single dose or multiple doses may be required to achieve the desired treatment endpoint. An effective amount may be considered in the context of administering one or more therapeutic agents, and a single agent may be considered to be given in an effective amount if, in conjunction with one or more other agents, a desirable or beneficial result may be or is achieved. Suitable doses of any of the co-administered compounds may optionally be lowered due to the combined action (e.g., additive or synergistic effects) of the compounds. In various embodiments, an effective amount of the composition or therapy may (i) reduce the number of cancer cells; (ii) reduce tumor size; (iii) inhibit, retard, slow to some extent, and preferably stop cancer cell infiltration into peripheral organs; (iv) inhibit (e.g., slow to some extent and preferably stop) tumor metastasis; (v) inhibit tumor growth; (vi) prevent or delay occurrence and/or recurrence of a tumor; and/or (vii) relieve to some extent one or more of the symptoms associated with the cancer. In various embodiments, the amount is sufficient to ameliorate, palliate, lessen, and/or delay one or more symptoms of cancer. As is understood in the art, an "effective amount" may be in one or more doses, i.e., a single dose or multiple doses may be required to achieve the desired treatment endpoint.

[0024] A "therapeutically effective amount" refers to an amount of a compound or salt thereof sufficient to produce a desired therapeutic outcome (e.g., reducing the severity or duration of, stabilizing the severity of, or eliminating one or more symptoms of cancer). For therapeutic use, beneficial or desired results include, e.g., decreasing one or more symptoms resulting from the disease (biochemical, histologic and/or behavioral), including its complications and intermediate pathological phenotypes presenting during development of the disease, increasing the quality of life of those suffering from the disease, decreasing the dose of other medications required to treat the disease, enhancing effect of another medication, delaying the progression of the disease, and/or prolonging survival of patients.

[0025] A "prophylactically effective amount" refers to an amount of a compound, or pharmaceutically acceptable salt thereof, sufficient to prevent or reduce the severity of one or more future symptoms of cancer when administered to an individual who is susceptible and/or who may develop cancer. For prophylactic use, beneficial or desired results include, e.g., results such as eliminating or reducing the risk, lessening the severity of future disease, or delaying the onset of the disease (e.g., delaying biochemical, histologic and/or behavioral symptoms of the disease, its complications, and intermediate pathological phenotypes presenting during future development of the disease).

[0026] It is understood that an effective amount of a compound or pharmaceutically acceptable salt thereof, including a prophylactically effective amount, may be given to an individual in the adjuvant setting, which refers to a clinical setting in which an individual has had a history of cancer, and generally (but not necessarily) has been responsive to therapy, which includes, but is not limited to, surgery (e.g., surgical resection), radiotherapy, and chemotherapy. However, because of their history of the cancer, these individuals are considered at risk of developing cancer. Treatment or administration in the "adjuvant setting" refers to a subsequent mode of treatment.

[0027] As used herein, "pharmaceutically acceptable" or "pharmacologically acceptable" means a material that is not biologically or otherwise undesirable, e.g., the material may be incorporated into a pharmaceutical composition administered to a patient without causing any significant undesirable biological effects or interacting in a deleterious manner with any of the other components of the composition in which it is contained. Pharmaceutically acceptable carriers or excipients have preferably met the required standards of toxicological and manufacturing testing and/or are included on the Inactive Ingredient Guide prepared by the U.S. Food and Drug administration.

[0028] "Pharmaceutically acceptable salts" are those salts which retain at least some of the biological activity of the free (non-salt) compound and which can be administered as drugs or pharmaceuticals to an individual. Such salts, for example, include: acid addition salts, formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like; or formed with organic acids such as acetic acid, oxalic acid, propionic acid, succinic acid, maleic acid, tartaric acid and the like. Pharmaceutically acceptable salts can be prepared in situ in the manufacturing process, or by separately reacting a purified compound of the invention in its free base form with a suitable organic or inorganic acid, respectively, and isolating the salt thus formed during subsequent purification.

[0029] The term "excipient" as used herein means an inert or inactive substance that may be used in the production of a drug or pharmaceutical, such as a tablet containing a compound of the invention as an active ingredient. Various substances may be embraced by the term excipient, including without limitation any substance used as a binder, disintegrant, coating, compression/encapsulation aid, cream or lotion, lubricant, solutions for parenteral administration, materials for chewable tablets, sweetener or flavoring, suspending/gelling agent, or wet granulation agent. Binders include, e.g., carbomers, povidone, xanthan gum, etc.; coatings include, e.g., cellulose acetate phthalate, ethylcellulose, gellan gum, maltodextrin, enteric coatings, etc.; compression/encapsulation aids include, e.g., calcium carbonate, dextrose, fructose dc (dc = "directly compressible"), honey dc, lactose (anhydrate or monohydrate; optionally in combination with aspartame, cellulose, or microcrystalline cellulose), starch dc, sucrose, etc.; disintegrants include, e.g., croscarmellose sodium, gellan gum, sodium starch glycolate, etc.; creams or lotions include, e.g., maltodextrin, carrageenan, etc.; lubricants include, e.g., magnesium stearate, stearic acid, sodium stearyl fumarate, etc.; materials for chewable tablets include, e.g., dextrose, fructose dc, lactose (monohydrate, optionally in combination with aspartame or cellulose), etc.; suspending/gelling agents include, e.g., carrageenan, sodium starch glycolate, xanthan gum, etc.; sweeteners include, e.g., aspartame, dextrose, fructose dc, sorbitol, sucrose dc, etc. ; and wet granulation agents include, e.g., calcium carbonate, maltodextrin, microcrystalline cellulose, etc.

[0030] Unless clearly indicated otherwise, the term "individual" as used herein refers to a mammal, including but not limited to, bovine, horse, feline, rabbit, canine, rodent, or primate (e.g., human). In some embodiments, an individual is a human. In some embodiments, an individual is a non-human primate such as chimpanzees and other apes and monkey species. In some embodiments, an individual is a farm animal such as cattle, horses, sheep, goats and swine; pets such as rabbits, dogs and cats; laboratory animals including rodents, such as rats, mice, and guinea pigs; and the like. The invention may find use in both human medicine and in the veterinary context.

[0031] Focal Adhesion Kinase (FAK; aliases: Protein Tyrosine Kinase 2

(PTK2)) is a cytoplasmic, non-receptor protein-tyrosine kinase that plays an essential role in regulating cell migration, adhesion, spreading, reorganization of the actin cytoskeleton, formation and disassembly of focal adhesions and cell protrusions, cell cycle progression, cell proliferation and apoptosis. FAK is the first intracellular step in the signal transduction cascade initiated by the attachment of an integrin to the extracellular matrix at points known as focal adhesions. FAK has 3 functional domains: a focal adhesion targeting domain (FAT), a catalytic domain, and a FERM domain, which mediates interactions with the cytoplasmic domains of integrins and growth factor receptors. (Sulzmaier et al. Nat Rev Cancer. 2014, 14(9), 598-610.) Examples of FAK include UniProtKB/Swiss-Prot: FAK1_HUMAN, Q05397, and Entrez Gene ID: 5747 PTK2 protein tyrosine kinase 2 [ Homo sapiens (human) ].

[0032] Fer (Fps/Fes Related) Tyrosine Kinase (FER; aliases: Feline

Encephalitis Virus-Related Kinase FER2) is a cytoplasmic, non-receptor tyrosine- protein kinase that acts downstream of cell surface receptors for growth factors (for example EGFR, KIT, PDGFRA, and PDGFRB) and plays a role in the regulation of the actin cytoskeleton, microtubule assembly, lamellipodia formation, cell adhesion, cell migration and chemotaxis. FER acts downstream of EGFR to promote activation of NF-kappa-B and cell proliferation. (Craig AW. Front Biosci (Landmark Ed). 2012, 17, 861-75.) Examples of FER include UniProtKB/Swiss-Prot: FER_HUMAN, PI 6591 , and Entrez Gene ID: 2241 FER fer (fps/fes related) tyrosine kinase [ Homo sapiens (human) ].

[0033] Feline Sarcoma Oncogene (FES; aliases: Feline Sarcoma (Snyder-

Theilen) Viral (V-Fes)/Fujinami Avian Sarcoma (PRCII) Viral (V-Fps) Oncogene Homolog) is a cytoplasmic, non-receptor tyrosine-protein kinase that acts downstream of cell surface receptors and plays a role in the regulation of the actin cytoskeleton, microtubule assembly, cell attachment and cell spreading. FES acts down-stream of the activated FCER1 receptor and the mast/stem cell growth factor receptor KIT, plays a role in the regulation of mast cell degranulation, and cell differentiation, and promotes neurite outgrowth in response to NGF signaling and in cell scattering and cell migration in response to HGF-induced activation of EZR. (Hellwig et al. Front Biosci (Landmark Ed). 2011 , 6, 3146-55.) Examples of FES include

UniProtKB/Swiss-Prot: FES_HUMAN, P07332, and Entrez Gene ID: 2242.

[0034] As used herein, "N-dealkylation" can also be considered "amide hydrolysis".

Compounds

[0035] The invention includes the use of all of the compounds described herein, including pharmaceutically acceptable salts and solvates of the compounds described herein, as well as methods of making and/or separating such compounds.

[0036] In one embodiment, provided is a compound of the formula (I) {see also table 1) or a salt thereof:

wherein R_j = H, R₂ = H; or

wherein R_j = -COCH₃, R₂ = H or -COCH₃.

[0037] The compounds of formula (I) are metabolites of CO- 1686 (Scheme

1). CO- 1686 is an oral and targeted covalent (irreversible) inhibitor of mutant forms of EGFR for the treatment of non- small cell lung cancer (NSCLC) in patients with mutation including but not restricted to initial activating EGFR mutations as well as the dominant resistance mutation T790M. Promising results have been observed in phase I studies of CO-1686.

[0038] Also provided are articles of manufacture comprising a compound of formula (I) or a salt thereof, compositions, and unit dosages described herein in suitable packaging for use in the methods described herein. Suitable packaging is known in the art and includes, for example, vials, vessels, ampules, bottles, jars, flexible packaging and the like. An article of manufacture may further be sterilized and/or sealed.

[0039] In one aspect, the compounds of formula (I) are orally bioavailable.

However, the compounds may also be formulated for parenteral (e.g., intravenous) administration.

[0040] Compounds described herein can be used in the preparation of a medicament by combining a compound of formula (I) as an active ingredient with a pharmacologically acceptable carrier. Depending on the therapeutic form of the medication, the carrier may be in various forms. In one variation, the manufacture of a medicament is for use in any of the methods disclosed herein, e.g., for the treatment of cancer.

M460

Pharmaceutical Compositions and Formulations

[0041] One embodiment of the invention includes pharmaceutical compositions comprising a compound described herein or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier or excipient. In one aspect, the pharmaceutically acceptable salt is an acid addition salt, such as a salt formed with an inorganic or organic acid. Pharmaceutical compositions may take a form suitable for oral, buccal, parenteral, nasal, topical or rectal administration or a form suitable for administration by inhalation.

[0042] A compound as detailed herein may in one aspect be in a purified form. Compositions comprising a compound of formula (I) or a salt thereof are provided, such as compositions of substantially pure compounds. In some embodiments, a composition containing a compound as detailed herein or a salt thereof is in substantially pure form. In one variation, "substantially pure" intends a composition that contains no more than 35% impurity. Taking compound M502 as an example, a composition of substantially pure compound M502 intends a composition that contains no more than 35% impurity, wherein the impurity denotes a compound other than compound M502 or a salt thereof. In one variation, a composition of substantially pure compound or a salt thereof is provided wherein the composition contains no more than 25% impurity. In another variation, a composition of substantially pure compound or a salt thereof is provided wherein the composition contains no more than 20% impurity. In still another variation, a composition of substantially pure compound or a salt thereof is provided wherein the composition contains no more than 10% impurity. In a further variation, a composition of substantially pure compound or a salt thereof is provided wherein the composition contains no more than 5% impurity. In another variation, a composition of substantially pure compound or a salt thereof is provided wherein the composition contains no more than 3% impurity. In still another variation, a composition of substantially pure compound or a salt thereof is provided wherein the composition contains no more than 1 % impurity. In a further variation, a composition of substantially pure compound or a salt thereof is provided wherein the composition contains no more than 0.5% impurity. In yet other variations, a composition of substantially pure compound means that the composition contains no more than 15% or preferably no more than 10% or more preferably no more than 5% or even more preferably no more than 3% and most preferably no more than 1 % impurity.

[0043] In one variation, the compounds herein are synthetic compounds prepared for administration to an individual. In another variation, compositions are provided containing a compound in substantially pure form. In another variation, the invention embraces pharmaceutical compositions comprising a compound detailed herein and a pharmaceutically acceptable carrier. In another variation, methods of administering a compound are provided. The purified forms, pharmaceutical compositions and methods of administering the compounds are suitable for any compound or form thereof detailed herein.

[0044] The compound may be formulated for any available delivery route, including an oral, mucosal (e.g., nasal, sublingual, vaginal, buccal or rectal), parenteral (e.g., intramuscular, subcutaneous or intravenous), topical or transdermal delivery form. A compound may be formulated with suitable carriers to provide delivery forms that include, but are not limited to, tablets, caplets, capsules (such as hard gelatin capsules or soft elastic gelatin capsules), cachets, troches, lozenges, gums, dispersions, suppositories, ointments, cataplasms (poultices), pastes, powders, dressings, creams, solutions, patches, aerosols (e.g., nasal spray or inhalers), gels, suspensions (e.g., aqueous or non-aqueous liquid suspensions, oil-in- water emulsions or water-in-oil liquid emulsions), solutions and elixirs. [0045] One or several compounds described herein can be used in the preparation of a formulation, such as a pharmaceutical formulation, by combining the compound or compounds as an active ingredient with a pharmaceutically acceptable carrier, such as those mentioned above. Depending on the therapeutic form of the system (e.g., transdermal patch vs. oral tablet), the carrier may be in various forms. In addition, pharmaceutical formulations may contain preservatives, solubilizers, stabilizers, re-wetting agents, emulgators, sweeteners, dyes, adjusters, and salts for the adjustment of osmotic pressure, buffers, coating agents or antioxidants. Formulations comprising the compound may also contain other substances which have valuable therapeutic properties.

[0046] Compounds as described herein may be administered to individuals in a form of generally accepted oral compositions, such as tablets, coated tablets, and gel capsules in a hard or in soft shell, emulsions or suspensions. Examples of carriers, which may be used for the preparation of such compositions, are lactose, corn starch or its derivatives, talc, stearate or its salts, etc. Acceptable carriers for gel capsules with soft shell are, for instance, plant oils, wax, fats, semisolid and liquid polyols, and so on. In addition, pharmaceutical formulations may contain preservatives, solubilizers, stabilizers, re-wetting agents, emulgators, sweeteners, dyes, adjusters, and salts for the adjustment of osmotic pressure, buffers, coating agents or antioxidants.

Methods of Use

[0047] It has been found that compounds of formula (I) can act as inhibitors of specific signal enzymes which are involved in controlling cell proliferation or resistance. Thus, the compounds described herein may be used for example for the treatment of diseases connected with the activity of these signal enzymes and characterized by excessive or abnormal cell proliferation. In one aspect, compounds of formula (I) are effective kinase inhibitors, exhibiting, in particular, a tyrosine kinase-inhibiting action and particularly an IGF-1R and/or INSR inhibiting action.

[0048] In one aspect, a compound of formula (I) binds to or inhibits binding of a ligand to the IGF-1R/INSR and/or reduces or eliminates an activity of the IGF- 1R/INSR in a reversible or irreversible manner. In some aspects, a compound of formula (I) inhibits binding of a ligand to IFG-1R/INSR by at least about or by about any one of 10%, 20%, 30%,40%, 50%, 60%,70%, 80%, 90%, 95% or 100% as determined by an assay described herein. In some aspects, the receptor inhibitor reduces an activity of the receptor by at least about or about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 100% as compared to the corresponding activity in the same subject prior to treatment with the compound of formula (I) or compared to the corresponding activity in other subjects not receiving the compound of formula (I).

[0049] Compounds and compositions of the invention, such as a

pharmaceutical composition containing a compound of formula (I) or a salt thereof and a pharmaceutically acceptable carrier or excipient, may be used in methods of administration and treatment as provided herein. The compounds and compositions may also be used in in vitro methods, such as in vitro methods of administering a compound or composition to cells for screening purposes and/or for conducting quality control assays.

[0050] In one aspect, the invention provides a method of inhibiting IGF-IR and/or INSR comprising administering to an individual an effective amount of one or more compounds of the invention, or a salt thereof (e.g., a pharmaceutically acceptable salt). In one aspect of the method, a compound of the invention or salt thereof inhibits the IGF-IR and/or INSR and/or reduces or eliminates an activity of the IGF-IR and/or INSR in a reversible or irreversible manner. In some aspects, a compound of the invention inhibits binding of a ligand to the IGF-IR and/or INSR by at least about or by about any one of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 100% as determined by an assay described herein. In some aspects, a compound of the invention reduces an activity of the IGF-IR and/or INSR by at least about or about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 100% as compared to the corresponding activity in the same subject prior to treatment with the receptor inhibitor or compared to the corresponding activity in other subjects not receiving the compound. In one aspect, the individual has or is believed to have a disorder in which IGF-IR and/or INSR is implicated. In some embodiments, the compound or salt thereof inhibits IGF-IR. In some embodiments, the compound or salt thereof inhibits INSR. In certain embodiments, the compound or salt thereof inhibits both IGF-IR and INSR. In certain variations, a compound or composition of the invention is used to treat or prevent an IGF-IR and/or INSR related disorder, such as cancer. In one aspect, the method comprises administering to the individual a compound provided herein, or a pharmaceutically acceptable salt thereof. In one aspect, the individual is a human in need of cancer treatment.

[0051] The invention also provides methods for inhibiting the activity of a protein kinase comprising administering an effective amount of one or more compounds of the invention, or a salt thereof, to an individual. In one aspect, a method of inhibiting IGF-1R/INSR is provided.

[0052] The invention additionally provides methods for treating cancer in an individual (e.g., human) comprising administering to the individual an effective amount of a compound of the invention or a pharmaceutically acceptable salt thereof or a composition comprising the compound or salt thereof. In one aspect, the method comprises administering to the individual a compound provided herein, or a pharmaceutically acceptable salt thereof. The invention also provides methods for preventing, and/or delaying the onset and/or development of cancer in an individual (e.g., human) comprising administering to the individual an effective amount of a compound of the invention or a pharmaceutically acceptable salt thereof or a composition comprising the compound or salt thereof.

[0053] In one variation, a method of treating cancer in an individual (e.g., human) is provided comprising administering to the individual an effective amount of a compound of the invention or a pharmaceutically acceptable salt thereof, wherein the cancer is dependent on a kinase signaling pathway. In one variation, a method of treating cancer in an individual (e.g., human) is provided comprising administering to the individual an effective amount of a compound of the invention or a

pharmaceutically acceptable salt thereof, wherein the cancer is dependent on the IGF- 1R signaling pathway. In another variation, a method of treating cancer in an individual (e.g., human) is provided comprising administering to the individual an effective amount of a compound of the invention or a pharmaceutically acceptable salt thereof, wherein the cancer is characterized by depending on IGF-1R signaling for survival and/or proliferation. In yet another variation, a method of treating cancer in an individual (e.g., human) is provided comprising administering to the individual an effective amount of a compound of the invention or a pharmaceutically acceptable salt thereof, wherein the cancer cells overexpress IGF-1R as compared to non-cancerous cells, e.g., as compared to non-cancerous cells of the same cell type. [0054] In some embodiments, the amount of the compound or

pharmaceutically acceptable salt thereof that is administered to an individual is an amount sufficient to decrease the size of a tumor, decrease the number of cancer cells, or decrease the growth rate of a tumor by at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 100% compared to the corresponding tumor size, number of cancer cells, or tumor growth rate in the same subject prior to treatment or compared to the corresponding activity in other subjects not receiving the treatment. Standard methods can be used to measure the magnitude of this effect, such as in vitro assays with purified enzyme, cell-based assays, animal models, or human testing.

[0055] In some embodiments, the cancer that may be treated is a solid tumor such as sarcomas and carcinomas. In some embodiments, the cancer that may be treated is a liquid tumor such as leukemia. Examples of cancers that may be treated by methods of the invention include, but are not limited to, breast cancer, prostate cancer, ovarian cancer, lung cancer, colon cancer, brain tumors, gastric cancer, liver cancer, thyroid cancer, endometrial cancer, gallbladder cancer, kidney cancer, adrenocortical cancer, sarcoma, skin cancer, head and neck cancer, leukemia, bladder cancer, colorectal cancer, hematopoietic cancer and pancreatic cancer. In some embodiments, the breast cancer is breast carcinoma (ER negative or ER positive), primary breast ductal carcinoma, mammary adenocarcinoma, mammary ductal carcinoma (ER positive, ER negative or HER2 positive), HER2 positive breast cancer, luminal breast cancer or triple negative breast cancer (TNBC). In some embodiments, the breast cancer is unclassified. In some embodiments, the triple negative breast cancer is a basal-like TNBC, a mesenchymal TNBC (mesenchymal or mesenchymal stem-like), an immunomodulatory TNBC, or a luminal androgen receptor TNBC. In some embodiments, the prostate cancer is prostate adenocarcinoma. In some embodiments, the ovarian cancer is ovary adenocarcinoma. In some embodiments, the lung cancer is lung carcinoma, non-small lung carcinoma, adenocarcinoma, mucoepidermoid, anaplastic, large cell, or unclassified. In some embodiments, the colon cancer is colon adenocarcinoma, colon adenocarcinoma from a metastatic site lymph node, metastatic colorectal cancer, or colon carcinoma. In some embodiments, a brain tumor is glioblastoma, astrocytoma, medulloblastoma, meningioma or neuroblastoma. In some embodiments, gastric cancer is stomach cancer. In some embodiments, liver cancer is hepatocellular carcinoma, hepatoblastoma or cholangiocarcinoma. In some embodiments, liver cancer is hepatitis B virus derived. In some embodiments, liver cancer is virus negative. In some embodiments, thyroid cancer is papillary thyroid carcinoma, follicular thyroid cancer or medullary thyroid cancer. In some

embodiments, endometrial cancer is high grade endometrioid cancer, uterine papillary serous carcinoma or uterine clear cell carcinoma. In some embodiments, gallbladder cancer is gallbladder adenocarcinoma or squamous cell gallbladder carcinoma. In some embodiments, kidney cancer is renal cell carcinoma or urothelial cell carcinoma. In some embodiments, adrenocortical cancer is adrenal cortical carcinoma. In some embodiments, sarcoma is synovial sarcoma, osteosarcoma,

rhabdomyosarcoma, fibrosarcoma or Ewing's sarcoma. In some embodiments, skin cancer is basal cell carcinoma, squamous carcinoma or melanoma. In some embodiments, head and neck cancer is oropharyngeal cancer, nasopharyngeal cancer, laryngeal cancer and cancer of the trachea. In some embodiments, the leukemia is acute promyelocytic leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, chronic myelogenous leukemia, mantle cell lymphoma or multiple myeloma.

[0056] The invention additionally provides a method for treating a tumor comprising contacting the tumor with an effective amount of one or more compounds of the invention, or a salt thereof. In one aspect of the method, a compound or salt thereof is administered to an individual in need of tumor treatment. Exemplary tumors are derived from carcinomas of the breast, prostate, ovary, lung, or colon. In one aspect, the treatment results in a reduction of the tumor size. In another aspect, the treatment slows or prevents tumor growth and/or metastasis.

[0057] The invention further provides methods for treating a hematopoietic malignancy comprising administering an effective amount of one or more compounds of the invention to an individual in need thereof. In some embodiments, the hematopoietic malignancy is acute promyelocytic leukemia.

[0058] Any of the methods of treatment provided herein may be used to treat a primary tumor. Any of the methods of treatment provided herein may also be used to treat a metastatic cancer (that is, cancer that has metastasized from the primary tumor). Any of the methods of treatment provided herein may be used to treat cancer at an advanced stage. Any of the methods of treatment provided herein may be used to treat cancer at a locally advanced stage. Any of the methods of treatment provided herein may be used to treat early stage cancer. Any of the methods of treatment provided herein may be used to treat cancer in remission. In some of the embodiments of any of the methods of treatment provided herein, the cancer has reoccurred after remission. In some embodiments of any of the methods of treatment provided herein, the cancer is progressive cancer.

[0059] Any of the methods of treatment provided herein may be used to treat an individual (e.g., human) who has been diagnosed with or is suspected of having cancer. In some embodiments, the individual may be a human who exhibits one or more symptoms associated with cancer. In some embodiments, the individual may have advanced disease or a lesser extent of disease, such as low tumor burden. In some embodiments, the individual is at an early stage of a cancer. In some embodiments, the individual is at an advanced stage of cancer. In some of the embodiments of any of the methods of treatment provided herein, the individual may be a human who is genetically or otherwise predisposed (e.g., has one or more so- called risk factors) to developing cancer who has or has not been diagnosed with cancer. In some embodiments, these risk factors include, but are not limited to, age, sex, race, diet, history of previous disease, presence of precursor disease, genetic (e.g., hereditary) considerations, and environmental exposure. In some embodiments, the individuals at risk for cancer include, e.g., those having relatives who have experienced this disease, and those whose risk is determined by analysis of genetic or biochemical markers.

[0060] Any of the methods of treatment provided herein may be practiced in an adjuvant setting. In some embodiments, any of the methods of treatment provided herein may be used to treat an individual who has previously been treated for cancer, e.g., with one or more other therapies such as radiation, surgery or chemotherapy. Any of the methods of treatment provided herein may be used to treat an individual who has not previously been treated for cancer. Any of the methods of treatment provided herein may be used to treat an individual at risk for developing cancer, but who has not been diagnosed with cancer. Any of the methods of treatment provided herein may be used as a first line therapy. Any of the methods of treatment provided herein may be used as a second line therapy.

[0061] Any of the methods of treatment provided herein in one aspect reduce the severity of one or more symptoms associated with cancer by at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 100% compared to the corresponding symptom in the same subject prior to treatment or compared to the corresponding symptom in other subjects not receiving a compound or composition of the invention.

[0062] Any of the methods of treatment provided herein may be used to treat, stabilize, prevent, and/or delay any type or stage of cancer. In some embodiments, the individual is at least about any of 40, 45, 50, 55, 60, 65, 70, 75, 80, or 85 years old. In some embodiments, one or more symptoms of the cancer are ameliorated or eliminated. In some embodiments, the size of a tumor, the number of cancer cells, or the growth rate of a tumor decreases by at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 100%. In some embodiments, the cancer is delayed or prevented.

[0063] In some embodiments, a compound or composition of the invention may be used to treat or prevent cancer in conjunction with a second therapy useful to reduce one or more side effects associated with administering the compound or composition of the invention. In some embodiments, the second compound for such combination therapy is selected from agents used for the treatment of glucose-related disorders such as Type 2 diabetes mellitus, impaired glucose tolerance, Insulin Resistance Syndrome and hyperglycemia. Examples of such agents include oral antidiabetic compounds from the classes of sulfonylureas, biguanides,

thiazolidinediones, alpha-glucosidase inhibitors, meglitinides, other insulin- sensitizing compounds and/or other antidiabetic agents. Particular examples comprise Metformin (Ν,Ν-dimethylimidodicarbonimidic diamide), sulfonylureas and the like, or a salt of the forgoing. Testing of glucose concentration levels in an individual receiving a compound of the present invention may be followed by the coadministration of such a second agent (e.g., Metformin) where appropriate (e.g., where the results of a glucose concentration level test in an individual indicate that such combination therapy will be or is expected to be beneficial for the individual).

[0064] In some embodiments, the compounds and compositions of the invention may be used to treat or prevent cancer in conjunction with a second therapy useful for cancer treatment. The second therapy includes, but is not limited to, surgery, radiation, and/or chemotherapy. [0065] One aspect of the invention is directed to a method of treating cancer which method comprises administering to a patient a therapeutically effective amount of a compound of Formula (I) or a salt thereof, in combination with treatments selected from surgery, radiation, monoclonal antibody, one marrow or peripheral blood stem cell transplantation, and chemotherapeutic agents.

[0066] In another embodiment, the chemotherapeutic agent is selected from taxane, a topoisomerase inhibitor, a signal transduction inhibitor, a cell cycle inhibitor, an IGF/IGF- 1R system modulator, a farnesyl protein transferase (FPT) inhibitor, an epidermal growth factor receptor (EGFR) inhibitor, a HER2 inhibitor, a vascular epidermal growth factor (VEGF) receptor inhibitor, a mitogen activated protein (MAP) kinase inhibitor, a MEK inhibitor, an AKT inhibitor, a mTOR inhibitor, a pi 3 kinase inhibitor, a Raf inhibitor, a cyclin dependent kinase (CDK) inhibitor, a microtubule stabilizer, a microtubule inhibitor, a SERM/Antiestrogen, an aromatase inhibitor, an anthracycline, a proteasome inhibitor, PD-Ll , an agent which inhibits insulin- like growth factor (IGF) production, an anti-sense inhibitor of IGF- 1R, IGF-1 or IGF-2, and an alkylating agent.

[0067] Non-limiting examples of taxane include paclitaxel and docetaxel.

Non-limiting examples of the microtubule inhibitor include vincristine, vinblastine, and a podophyllotoxin, epothilone B. Non-limiting examples of the epidermal growth factor receptor (EGFR) inhibitor include CO- 1686, AZD9291, gefitinib, erlotinib, cetuximab, lapatanib, and canertinib. Non-limiting examples of the farnesyl protein transferase inhibitor include lonafarnib and tipifamib. Non-limiting examples of the selective estrogen receptor modulator (SERM)/antiestrogen include tamoxifen, raloxifene, fulvestrant, acolbifene, pipendoxifene, arzoxifene, toremifene, lasofoxifene, bazedoxifene, and idoxifene. Non-limiting examples of the

anthracycline include doxorubicin, daunorubicin and epirubicin. Non-limiting examples of HER2 inhibitors include trastuzumab. Non- limiting examples of the topoisomerase inhibitor include etoposide, topotecan, camptothecin and irinotecan. Non-limiting examples of the alkylating agent include mitomycin C, adozelesin, cis- platinum, nitrogen mustard, 5-fluorouracil (5FU), etoposide (VP-16), camptothecin, actinomycin-D, and cisplatin (CDDP).

[0068] In another embodiment, the cancer is acute myelogenous leukemia

(AML), and the treatments are selected from bone marrow or peripheral blood stem cell transplantation, radiation, monoclonal antibody, and chemotherapeutic agents. In another embodiment, the monoclonal antibody is Gemtuzumab ozogamicin

(Mylotarg); and the chemotherapeutic agents are selected from daunorubicin, doxorubicin, cytarabine (ara-C), an anthracycline drug such as daunorubicin or idarubicin (Daunomycin, Idamycin), 6-thioguanine, and a granulocyte colony- stimulating factor such as Neupogen or Leukine.

[0069] In another embodiment, the cancer is acute lymphocytic leukemia

(ALL) or Philadelphia Chromosome- Associated Acute Lymphoblastic Leukemia (Ph+ALL) and the treatments are selected from bone marrow or peripheral blood stem cell transplantation, monoclonal antibody, and chemotherapeutic agents. In another embodiment, the monoclonal antibody is rituximab (Rituxan). In another

embodiment, the chemotherapeutic agents are selected from vincristine, prednisone, dexamethasone, anthracycline, L-asparaginase, Gleevec®, cyclophosphamide, doxorubicin (Adriamycin), daunorubicin, methotrexate, cytarabine (ara-C), etoposide, and 6-mercaptopurine (6-MP).

[0070] In another embodiment, the cancer is chronic lymphocytic leukemia

(CLL), and the treatments are selected from bone marrow or peripheral blood stem cell transplantation, monoclonal antibody, radiation, and chemotherapeutic agents. In another embodiment, the monoclonal antibody is Alemtuzumab (Campath) or Rituximab (Rituxan); and the chemotherapeutic agents are selected from

cyclophosphamide, chlorambucil, a corticosteroid such as prednisone, fludarabine, doxorubicin, vincristine, pentostatin, and cladribine (2-CdA).

[0071] In another embodiment, the cancer is chronic myelogenous leukemia

(CML) and the treatments are selected from bone marrow or peripheral blood stem cell transplantation, radiation, and chemotherapeutic agents. Non-limiting examples of the chemotherapeutic agents include interferon therapy such as interferon-a, Gleevec®, hydroxyurea (Hydrea), cytosine, cytosine arabinoside, dasatinib, and cytarabine (ara-C).

[0072] In another embodiment, the cancer is gastrointestinal stromal cancer and the treatments are selected from surgery, radiation, and chemotherapeutic agents. In another embodiment, the surgical procedure is selected from cryosurgery, embolization, and ethanol ablation; and the chemotherapeutic agent is Gleevec®. [0073] In another embodiment, the cancer is small-cell lung cancer, and the treatments are selected from surgery, radiation, and chemotherapeutic agents selected from a platin such as cisplatin or carboplatin, vinorelbine, docetaxel, paclitaxel, etoposide, ifosfamide, cyclophosphamide, doxorubicin, vincristine, gemcitabine, paclitaxel, vinorelbine, topotecan, irinotecan, methotrexate, and docetaxel.

[0074] In another embodiment, the cancer is non-small cell lung cancer, and the treatments are selected from surgery, radiation, and chemotherapeutic agents selected from gefitinib (Iressa) and erlotinib (Tarceva).

[0075] In another embodiment, the cancer is prostate cancer, and the treatments are selected from surgery (including cryosurgery), radiation, and chemotherapeutic agents. Non-limiting examples of the chemotherapeutic agents include hormone therapy (also called androgen deprivation therapy or androgen suppression therapy), mitoxantrone, prednisone, docetaxel (Taxotere), doxorubicin, etoposide, vinblastine, paclitaxel, and carboplatin.

[0076] In another embodiment, the cancer is breast cancer and the treatments include surgery, radiation, a monoclonal antibody, and chemotherapeutic agents. Non- limiting examples of the monoclonal antibody include Trastuzumab (Herceptin®). Non-limiting examples of the chemotherapeutic agents include hormone therapy such as Tamoxifen, Raloxifene (Evista); Toremifene (Fareston), and Fulvestrant

(Faslodex); luteinizing hormone-releasing hormone (LHRH) analogs including goserelin and leuprolide; Megestrol acetate (Megace); Aromatase inhibitors including etrozole (Femara), anastrozole (Arimidex), and exemestane (Aromasin); pamidronate or zoledronic acid (to treat bone weakness); CMF (cyclophosphamide, fluoruracil, and methotrexate); AC (adriamycin and Cyclophosphamide); axane (paclitaxel or Docetaxel); adriamyclin; and cyclophosphamide.

[0077] One aspect of the invention is directed to a method of treating T790M

EGFR mediated cancer which method comprises administering to a patient a therapeutically effective amount of a compound of formula (I) or a salt thereof, in combination with a wild type sparing EGFR inhibitor.

[0078] One aspect of the invention comprises a pharmaceutical combination comprising a compound of formula (I) or a pharmaceutically acceptable salt thereof and an EGFR inhibitor for use in the treatment of cancer in a subject in need thereof. The combination of the present invention can be used to treat subjects suffering from, for example, cancers having EGFR amplification, EGFR activating mutations, and IGF-1R activating signature (e.g., overexpression of IGF-1R, high circulating levels of IGF-1 , or high levels of one or more IGF binding proteins). Suitable cancers include, without limitation, lung cancer, e.g., non-small cell lung cancer.

[0079] In a further embodiment, the present invention comprises a pharmaceutical combination comprising a compound of formula (I) or a

pharmaceutically acceptable salt thereof and an EGFR inhibitor for use in the treatment of cancers that are resistant or refractive to currently-available therapies, e.g., EGFR amplified cancers that are resistant or refractive to EGFR inhibitors or IGF-1R inhibitors, in a subject in need thereof.

[0080] In one embodiment, the EGFR inhibitor is an irreversible wild type sparing T790M EGFR TKI. In another embodiment, the EGFR inhibitor is CO- 1686.

[0081] In a further embodiment, the invention comprises the combination of a compound of formula (I) or a pharmaceutically acceptable salt thereof and an EGFR inhibitor displaying a synergistic effect.

[0082] In another embodiment, the invention provides a pharmaceutical composition comprising a first irreversible EGFR TKI compound, wherein upon administration to a patient the first compound is metabolized to a second compound that is an IGF-1R inhibitor, an INSR inhibitor, or a dual target IGF-1R and INSR inhibitor. In one aspect, the second compound has a t of greater than 13 hours. In another aspect, the first compound is CO- 1686. In another aspect, the second compound is a compound of formula (I).

[0083] In another embodiment, the invention provides a pharmaceutical composition comprising a dual targeting compound, wherein the compound is a wild type sparing irreversible T790M EGFR TKI. The compound is metabolized to an IGF-1R or INSR inhibitor upon administration.

[0084] In another embodiment, the invention provides a method for prolonging the effectiveness or delaying the development of resistance of the treatment of cancer in a mammal. The method include the steps of administering to a patient a first compound that is an irreversible wild type sparing T790M EGFR TKI, coadministering to the patient a second compound that is an IGF-1R inhibitor or INSR inhibitor with a t of greater than 13 hours, wherein the coadministration of the second compound prolongs the effectiveness of the first compound in treating cancer.

[0085] The present invention provides a method of treating cancer in a subject

(e.g., human) by administering to the subject in need of such treatment a

therapeutically effective amount or dose of a combination of a compound or formula (I) or a pharmaceutically acceptable salt thereof and an EGFR inhibitor.

[0086] In one embodiment, the present invention provides a method of treating cancer by administering to a subject in need of such treatment a quantity of a compound of formula (I) or pharmaceutically acceptable salt thereof and an EGFR inhibitor which is jointly therapeutically effective for said treatment.

[0087] In a further embodiment, a compound of formula (I) and an EGFR inhibitor are in a single formulation or unit dosage form. In a further embodiment, a compound of formula (I) and an EGFR inhibitor are in separate formulations or unit dosage forms.

[0088] In a further embodiment, a compound of formula (I) and/or an EGFR inhibitor are administered at substantially the same time. In a further embodiment, a compound of formula (I) and/or an EGFR inhibitor are administered at different times. In a further embodiment, a compound of formula (I) is administered to the subject prior to administration of an EGFR inhibitor. In a further embodiment, an EGFR inhibitor is administered to the subject prior to administration of a compound of formula I.

[0089] One aspect of the invention provides a method for treating a cancer that is resistant or refractive to prior treatment with an EGFR modulator or IGF-1R inhibitor, comprising administering a therapeutically effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof and an EGFR inhibitor to a subject in need thereof.

[0090] One aspect of the invention further provides a method for the treatment of cancer that is resistant or refractive to treatment with an EGFR inhibitor by administering a therapeutically effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof.

[0091] One aspect of the invention provides a use of the pharmaceutical combination comprising a compound of formula (I) or a pharmaceutically acceptable salt thereof and an EGFR inhibitor for the manufacture of a pharmaceutical preparation or medicament for the treatment of cancer.

[0092] One aspect of the invention further provides the use of a

pharmaceutical combination comprising a compound of formula (I) or a

pharmaceutically acceptable salt thereof and an EGFR inhibitor for the manufacture of a pharmaceutical preparation or medicament for the treatment of cancer that is resistant or refractive to treatment with an EGFR modulator or IGF-1R inhibitor.

[0093] One aspect of the invention further provides the use of a compound of formula (I) or a pharmaceutically acceptable salt thereof for the treatment of cancer that is resistant or refractive to treatment with an EGFR inhibitor.

[0094] In one embodiment, the present invention relates to a pharmaceutical composition or pharmaceutical formulation comprising (a) a compound of formula (I) or a pharmaceutically acceptable salt thereof, and (b) an EGFR inhibitor, and optionally one or more pharmaceutically acceptable carriers.

[0095] In a further embodiment, the present invention further relates to a pharmaceutical composition or pharmaceutical formulation comprising (a) a compound of formula (I) or a pharmaceutically acceptable salt thereof, and (b) an EGFR inhibitor, and optionally one or more pharmaceutically acceptable carriers, for use in the treatment of cancer.

[0096] In a further embodiment, the present invention relates to (a) a pharmaceutical combination comprising a compound of formula (I) or a

pharmaceutically acceptable salt thereof, and (b) a pharmaceutical composition comprising an EGFR inhibitor administered in separate pharmaceutical compositions to a subject in need thereof.

[0097] One aspect of the invention also provides compositions (including pharmaceutical compositions) as described herein for the use in treating, preventing, and/or delaying the onset and/or development of cancer and other methods described herein.

Dosing and Method of Administration

[0098] The dose of a compound administered to an individual (such as a human) may vary with the particular compound or salt thereof, the method of administration, and the particular stage of cancer being treated. The amount should be sufficient to produce a desirable response, such as a therapeutic or prophylactic response against cancer. In some embodiments, the amount of the compound or salt thereof is a therapeutically effective amount. In some embodiments, the amount of the compound or salt thereof is a prophylactically effective amount. In some

embodiments, the amount of compound or salt thereof is below the level that induces a toxicological effect (e.g., an effect above a clinically acceptable level of toxicity) or is at a level where a potential side effect can be controlled or tolerated when the composition is administered to the individual.

[0099] In some embodiments, the amount of compound or salt thereof is an amount sufficient to inhibit IGR-1R and/or INSR, inhibit the phosphorylation of AKT, inhibit cancer cell growth and/or proliferation or increase apoptosis of cancer cells.

[00100] The effective amount of the compound may in one aspect be a dose of between about 0.01 and about 100 mg/kg.

[00101] Any of the methods provided herein may in one aspect comprise administering to an individual a pharmaceutical composition that contains an effective amount of a compound provided herein or a salt thereof and a pharmaceutically acceptable excipient.

[00102] A compound or composition of the invention may be administered to an individual in accordance with an effective dosing regimen for a desired period of time or duration, such as at least about one month, at least about 2 months, at least about 3 months, at least about 6 months, or at least about 12 months or longer, which in some variations may be for the duration of the individual's life. In one variation, the compound is administered on a daily or intermittent schedule. The compound can be administered to an individual continuously (for example, at least once daily) over a period of time. The dosing frequency can also be less than once daily, e.g., about once weekly dosing. The dosing frequency can be more than once daily, e.g., twice or three times daily. The dosing frequency can also be intermittent, including a 'drug holiday' (e.g., once daily dosing for 7 days followed by no doses for 7 days, repeated for any 14 day time period, such as about 2 months, about 4 months, about 6 months or more). Any of the dosing frequencies can employ any of the compounds described herein together with any of the dosages described herein.

[00103] The compounds provided herein or a salt thereof may be administered to an individual via various routes, including, e.g., intravenous, intramuscular, subcutaneous, oral and transdermal. A compound provided herein can be administered frequently at low doses, known as 'metronomic therapy,' or as part of a maintenance therapy using compound alone or in combination with one or more additional drugs. Metronomic therapy or maintenance therapy can comprise administration of a compound provided herein in cycles. Metronomic therapy or maintenance therapy can comprise intra-tumoral administration of a compound provided herein.

[00104] In one aspect, the invention provides a method of treating cancer in an individual by parenterally administering to the individual (e.g., a human) an effective amount of a compound or salt thereof. In some embodiments, the route of administration is intravenous, intra-arterial, intramuscular, or subcutaneous. In some embodiments, the route of administration is oral. In still other embodiments, the route of administration is transdermal.

Kits

[00105] The invention further provides kits for carrying out the methods of the invention, which comprises one or more compounds described herein or a pharmacological composition comprising a compound described herein. The kits may employ any of the compounds disclosed herein. In one variation, the kit employs a compound described herein or a pharmaceutically acceptable salt thereof. The kits may be used for any one or more of the uses described herein, and, accordingly, may contain instructions for the treatment of cancer.

[00106] Kits generally comprise suitable packaging. The kits may comprise one or more containers comprising any compound described herein. Each component (if there is more than one component) can be packaged in separate containers or some components can be combined in one container where cross-reactivity and shelf life permit.

[00107] The kits may be in unit dosage forms, bulk packages (e.g., multi-dose packages) or sub-unit doses. For example, kits may be provided that contain sufficient dosages of a compound as disclosed herein and/or a second pharmaceutically active compound useful for a disease detailed herein (e.g., hypertension) to provide effective treatment of an individual for an extended period, such as any of a week, 2 weeks, 3 weeks, 4 weeks, 6 weeks, 8 weeks, 3 months, 4 months, 5 months, 7 months, 8 months, 9 months, or more. Kits may also include multiple unit doses of the compounds and instructions for use and be packaged in quantities sufficient for storage and use in pharmacies (e.g., hospital pharmacies and compounding pharmacies).

[00108] The kits may optionally include a set of instructions, generally written instructions, although electronic storage media (e.g., magnetic diskette or optical disk) containing instructions are also acceptable, relating to the use of component(s) of the methods of the present invention. The instructions included with the kit generally include information as to the components and their administration to an individual.

[00109] Examples of compounds of formula (I) were evaluated in several standard pharmacological test procedures that showed that the compounds of this invention inhibit IGF-1R and/or INSR activity. Based on the activity shown in the standard pharmacological test procedures, the compounds of this invention are therefore useful as anti-cancer agents. Associated cancers are selected from the group consisting of breast, colon, lung, prostate, melanoma, epidermal, leukemia, kidney, bladder, mouth, larynx, esophagus, stomach, ovary, pancreas, liver, skin and brain. The test procedures used and results obtained are shown below. The invention is further illustrated by the following non-limiting examples.

Examples

1. Identification of CO- 1686 Metabolites

[00110] Analysis of plasma samples from healthy subjects following dosing of unlabeled CO- 1686, and from in vitro hepatocyte profiling identified one primary and two secondary metabolites of CO- 1686 referenced according to their molecular ion as M502 (primary metabolites) and M460 and M544 (secondary metabolites).

Metabolite structure and the biotransformation pathways leading to generation from CO- 1686 are outlined in Figure 1.

[00111] In brief, cryopreserved hepatocytes from mice, rats, dogs, monkeys, and human were purchased from In Vitro Technologies (Baltimore, MD). Vials of hepatocytes were thawed in a 37 °C warm bath. For metabolite profiling, a final concentration of 20 μΜ CO-1686 was incubated with 3 x 105 hepatocytes per mL in duplicate for three hours in a 37 °C incubator while shaking at 600 rpm. The final concentration of dimethyl sulfoxide (DMSO) was 0.2%. At time zero and 3 hours, acetonitrile was added to cells to stop the reaction. Duplicate samples were pooled and the samples were centrifuged for 20 minutes at 5000 rpm at room temperature. The resulting supernatant was dried under a gentle stream of nitrogen at 37 °C using Turbo Vap LV evaporator. The samples were re-constituted in 250 μL· of 50/50 mixture of 0.1% formic acid in acetonitrile and 0.1% formic acid in 5 mM ammonium acetate and analyzed by LC- MS/MS/UV analysis.

[00112] For human plasma analysis, plasma samples from six healthy subjects receiving 500 mg CO-1686 hydrobromide (HBr) tablets twice daily for four consecutive days were used for profiling and identification of CO-1686 metabolites using LC-MS/MS. Plasma samples from two subjects at all PK time points on dosing day 1 and day 4 were pooled using the method proposed by Hamilton (Hamilton et al. Clin Pharmacol Ther 1981, 29(3):408-413) to generate one sample for each subject on each day. Pooled plasma samples were extracted by protein precipitation, and the extracted samples were then analyzed by LC-MS/MS to generate product ion spectra of tentative metabolites. Relative abundance of the metabolites and parent compound was obtained by analyzing individual samples from subjects at 2 and 12 hr post dose on day 1 and day 4 using Multiple Reaction Monitoring (MRM) detection. Five metabolites, designated as M460, M502, M544, M664, and M677, were detected and identified. Their chemical structures were proposed based on the product ion spectra and rationale of biotransformation. From the identification of these metabolites, the biotransformation pathways for CO-1686 have been characterized as N-dealkylation, acetylation, glucuronidation, and addition of cysteine. Based on the mass spectrometric responses, M502, M544, and M664 are tentatively identified as the major metabolites, while M460 and M677 are the minor ones. Metabolite M677 was not characterized further.

2. Preparation/Purification of Compounds

A. Synthesis of M460 (Scheme 2)

[00113] CO- 1686 (18 g, 32 mmol) were dissolved in a solution of EtOH (200 proof, 250 mL) and concentrated HC1 solution (250 mL). The reaction mixture was heated to reflux for 6 hrs. After cooling down, half of the solvent was removed. The precipitate was collected by filtration and rinsed with cold ethanol (50 mL). After drying in vacuum oven, M460 (15 g) was obtained in 84% yield. The product may be further slurried in EtOH (50 mL) for 1 hr, and then filtered. The precipitate is collected and dried.

B. Synthesis of M544 (Scheme 3)

Scheme 3

M460 M544

[00114] M460 hydrochloride salt (20 g, 35 mmol) was dissolved in water (150 mL) and THF (150 mL) mixed solvent and Na₂C03 was added to adjust the solution pH value about 10. Acetic anhydride (10 g, 98 mmol) was then added and stirred overnight. THF was then removed and the aqueous solution was then extracted with ethyl acetate (100 mL x 3). The combined solution was dried over anhydrous Na₂S04, and then the solvent was removed under vacuum. The residue was purified by flash chromatography (Eluent: Ethyl acetate : Methanol, from 100 : 0 to 96 : 4) to give pure M544 (15 g, 27.6 mmol) in 77% yield. The product may be further slurried in ethyl acetate (25 mL) at 50°C. After cooling down, the precipitate is collected by filtration, and dried in vacuum oven.

D. Synthesis of M502 (Scheme 4)

[00115] Step 1 : Trifluoromethyl-dichloro-pyrimidine C-024450 is coupled with N-Boc-m-phenylenediamine C-024451 in ethanol at reduced temperature (-50°C to - 30°C) in the presence of triethylamine. Upon completion of the reaction, the desired product is precipitated by the addition of methylcyclohexane and collected by filtration. Residual triethylamine hydrochloride is removed by dissolution in ethyl acetate and washing with water. Following a solvent exchange to heptanes, the intermediate C -024452 is collected via filtration.

[00116] Step 2: Intermediate C-024452 is coupled with C-024646 in 1-butanol at elevated temperature in the presence of Ν,Ν-diisopropylethylamine (DIPEA). The coupling product, C-024705, is isolated initially in dichloromethane (DCM) followed by solvent exchange to ethyl acetate and filtration of solid intermediate C-024705.

[00117] Step 3: Intermediate C-024705 is deprotected by treatment with trifluoroacetic acid (TFA) in DCM at room temperature. The reaction is quenched with aqueous sodium carbonate to yield M502.

Scheme 4

3. Biochemical and Cellular Profiling of CO-1686 metabolites

[00118] The potency and selectivity of CO-1686 and related metabolites M460, M502, and M544 was assessed by apparent Kd profiling against the WT EGFR, EGFR T790M, IGF-1R, and INSR.

[00119] In vitro profiling of selected kinases was performed at Reaction Biology Corporation using the "HotSpot" assay platform (see Anastassiadis et al. Nat Biotechnol. 2011 ; 29(11): 1039-45). Briefly, specific kinase/substrate pairs along with required cofactors were prepared in reaction buffer; 20 mM Hepes pH 7.5, 10 mM MgCl₂, 1 mM EGTA, 0.02% Brij 35, 0.02 mg/ml BSA, 0.1 mM Na₃V0₄, 2 mM DTT, and 1 % DMSO. Compounds were delivered into the reaction, followed -20 min later by addition of a mixture of ATP and ³³P ATP to a final concentration of 10 μΜ. CO- 1686 and the control compound staurosporine (Enzo Life Sciences) were tested in 10- dose IC50 mode with 3-fold serial dilutions starting at 10 μΜ. Reactions were carried out at 25 °C for 120 min, followed by spotting of the reactions onto P81 ion exchange filter paper. Unbound phosphate was removed by extensive washing of filters in 0.75% phosphoric acid. After subtraction of background derived from control reactions containing inactive enzyme, kinase activity data were expressed as the % remaining kinase activity in test samples compared to vehicle (DMSO) reactions. GraphPad Prism was used to obtain curve fits and IC50 values.

[00120] The results from biochemical profiling are summarized in Table 2. Metabolites M460, M502, and M544 displayed limited (IC50 > 1 μΜ) potency against WT EGFR and EGFR T790M. In vitro kinase profiling revealed increased potency against IGF-IR and INSR for several of the metabolites, with M460 and M502 having the greatest potency. M460 and M502 were 8-9 fold more potent against INSR as compared to CO- 1686. Similarly, M460 and M502 were 6-7 fold more potent against IGF-IR as compared to CO-1686.

[00121] Cell viability was measured following three days of treatment using CO-1686 and metabolites of CO-1686 in cell lines harboring the EGFR

L858R/T790M resistance mutation (NCI-H1975), or wild-type EGFR (A431).

[00122] Cells were seeded at 3,000 cells/well in a 96-well white, clear-bottom plate and incubated overnight at 37°C with 5% CO2. Viability and cell density of trypsinized cell lines was determined using a Vi-Cell™ XR instrument (Beckman Coulter; Indianapolis, IN) prior to plating. CO-1686 and related metabolites were serially diluted in 100% dimethyl sulfoxide (DMSO) and then further diluted in growth media before being added to cells (0.5% final DMSO concentration once applied to cells). Depending on the experimental replicate, final concentration of each compound ranged from 20 μΜ to 0.34 nM, diluted in 3-fold steps or 10 μΜ to 0.01 nM, diluted in 4-fold steps. Treatments were performed in duplicate on the same plate. The cells were incubated with compound for three days in 5% FBS containing RPMI at 37°C with 5% CO2. Cell Titer-Glo® Luminescent Cell Viability Assay (Promega Corp.; Madison, WI) was then added to each well and luminescence was measured using a VICTOR™ X4 2030 Multilabel Reader (PerkinElmer; Waltham, MA) according to the manufacturer's instructions. The relative luminescence units (RLU) were plotted as a function of the concentration of the test compounds. Wells containing only growth media were used to control for background signal, which was subtracted from each value on the plate. Each background- subtracted value was then normalized to wells containing cells treated with 0.5% DMSO, yielding experimental values that represent relative cell growth versus DMSO control. The background- subtracted, normalized values were used in conjunction with GraphPad Prism 6 (GraphPad Software; La Jolla, CA) to calculate GI50 values. The curve was fit using nonlinear regression with the software-incorporated "log(agonist) vs. response - Variable slope (four parameters)". All other software options were default.

[00123] Cell viability data is summarized in Table 2. CO- 1686 metabolites M460, M502, and M544 are poor inhibitors of the mutant EGFR L858R/T790M NCI- H1975 cell line. Importantly, metabolites of CO-1686 have a negligible effect on the growth of EGFR wild-type A431 cells, indicating retention of the wild-type sparing characteristics of CO-1686.

4. Further biochemical profiling of CO-1686 metabolites

[00124] The potency and selectivity of CO-1686 and related metabolites M460, M502, and M544 was assessed in vitro by functional biochemical profiling against 350 wild-type (WT) kinases by at Reaction Biology Corporation (Malvern, PA) using the Kinase HotSpotSM radiometric assay platform assay platform (see Anastassiadis et al. Nat Biotechnol. 2011 ; 29(11): 1039-45). Briefly, specific kinase/substrate pairs along with required cofactors were prepared in reaction buffer; 20 mM HEPES pH 7.5, 10 mM MgC12, 1 mM EGTA, 0.02% Brij35, 0.02 mg/mL BSA, 0.1 mM

Na3V04, 2 mM DTT, and 1% DMSO. Compounds were delivered into the reaction, followed -20 min later by addition of a mixture of ATP (Sigma) and 33P ATP (PerkinElmer) to a final concentration of 10 μΜ. CO-1686, M460, M502, or M544 were tested in duplicate at concentrations of 1 μΜ and 0.1 μΜ. Staurosporine (Enzo Life Sciences) was used as a control compound, and was tested in 10-dose IC50 mode with 3 -fold serial dilutions starting at 20 μΜ. Reactions were carried out at 25 °C for 120 min, followed by spotting of the reactions onto P81 ion exchange filter paper (Whatman). Unbound phosphate was removed by extensive washing of filters in 0.75% phosphoric acid. After subtraction of background derived from control reactions containing inactive enzyme, kinase activity data were expressed as the % remaining kinase activity in test samples compared to vehicle (DMSO) reactions. [00125] The results from biochemical profiling of CO- 1686 and its metabolites on the WT kinome are summarized in Table 3. Results for kinases inhibited > 50% are displayed.

[00126] In comparison to CO- 1686, a number of WT kinases were inhibited more potently by the metabolites M460, M520, and M544. Examples included LRRK2, ALK, FER, FES/FPS, IRR/INSRR, INSR, FAK/PTK2, STK22D/TSSK1 , FLT3, IGF1R, EGFR, ERBB4/HER2, JAK3 and ERBB2/HER2.

5. Biochemical Kd determination of CO- 1686 metabolites against EGFR, IR,

IGF1R, FAK, FER and FES

[00127] The specific biochemical Kd of CO- 1686 and its metabolites was determined against a subset of WT kinases identified from the screening of the kinome. Work was panel at Reaction Biology Corporation Specific kinase/substrate pairs along with required cofactors were prepared in reaction buffer; 20 mM Hepes pH 7.5, 10 mM MgCl₂, 1 mM EGTA, 0.02% Brij35, 0.02 mg/ml BSA, 0.1 mM

Na3V0₄, 2 mM DTT, and 1% DMSO. Compounds were delivered into the reaction, followed -20 min later by addition of a mixture of ATP and 33P ATP to a final concentration of Km [ATP]. CO- 1686 was tested in 10-dose IC50 mode with 3 -fold serial dilution starting at 10 μΜ. Reactions were carried out at 25 °C for 120 min, followed by spotting of the reactions onto P81 ion exchange filter paper. Unbound phosphate was removed by extensive washing of filters in 0.75% phosphoric acid.

After subtraction of background derived from control reactions containing inactive enzyme, kinase activity data were expressed as the % remaining kinase activity in test samples compared to vehicle (DMSO) reactions. GraphPad Prism was used to obtain curve fits and IC50 values.

[00128] The results from biochemical profiling are summarized in Table 4. Metabolites M460, M502, and M544 displayed limited (IC50 > 1 μΜ) potency against WT EGFR and EGFR T790M. In comparison, the biochemical Kd determination for IGF-1R, INSR, FAK, FER and FES for all of the metabolites examined revealed a higher potency compared to CO- 1686 with M460 and M502 having the greatest potency. M460 and M502 were 6-9 fold more potent against INSR and IGF-1R as compared to CO- 1686. Similarly, M460 and M502 were 2-23 fold more potent against FAK, FER and FES as compared to CO- 1686. 6. Plasma CO- 1686 metabolite levels in NSCLC patients dosed with CO- 1686

[00129] Quantification of plasma levels of CO- 1686 metabolites in NSCLC patients following CO- 1686 dosing was determined by LC/MS/MS using standard methodologies.

[00130] Human exposure (table 5) is presented as Day 1 mean C_max (nM) for CO- 1686 and steady state mean concentration (nM) for metabolites at CO- 1686 therapeutic doses of 900 mg FB BID (n=16), or HBr BID at 500 (n=l 1), 625 (n=l 1), 750 (n=9), and 1000 mg (n=6).

7. Evaluation of CO-1686 and metabolites potency in IGFIR and INSR

reporter assays

[00131] Mouse Ba/F3 cells were engineered to depend upon the INSR and IGFIR pathways for survival such that inhibition of kinase activity decreases cellular viability. Cell lines were maintained in RPMI-1640 culture media containing 10% fetal calf serum and antibiotics. Cells in logarithmic -phase growth were harvested and 5,000 cells were distributed into each well of a 384-well plate in 50 μL· of growth media. Fifty nL of diluted compound were added to appropriate wells, in duplicate, and the cells were cultured for 48 hr at 37°C in a humidified 5% C02 atmosphere. Viability was determined by adding \5 μL· CellTiter-Glo and measuring

luminescence, which is reported as relative light units (RLU) measured in counts per second. The data (expressed in relative light units) for each compound were normalized to the average maximal response obtained in the presence of vehicle (DMSO) alone. These data were used to derive the percent inhibition (100 - % maximal response) and the average of two data points/concentration was used to calculate the IC50 values (concentration causing a half-maximal inhibition of cell survival) using non-linear regression analysis.

[00132] In vitro kinase and cellular reporter assay profiling of CO-1686 and these 4 metabolites was performed against IGFIR and INSR (Table 6). Cellular profiling demonstrated that M460 and M502 have 2-3 fold and 3-7 fold greater potency against INSR and IGFIR, respectively, as compared to CO-1686. Metabolite M544 demonstrated comparable or reduced potency towards IGFIR and INSR to that of CO-1686.

8. Evaluation of CO-1686 and metabolites M460, M502, and M544 in wild-type and mutant EGFR lung cancer cell lines

[00133] The effect of CO- 1686 and metabolites M460, M502 and M544 on the cellular viability of four EGFR mutant NSCLC adenocarcinoma cell lines (NCI- H1975 [L858R/T790M EGFR], PC-9 [dell 9 EGFR], HCC827 [dell 9 EGFR], HCC4006 [dell 9 EGFR]) and five WT EGFR cell lines (A431, A549, NCI-H661 , NCI-H1048, NCI-H1299) was determined using the Cell Titer-Glo® Luminescent Cell Viability Assay according to the manufacturer's recommendations (Promega; Madison, WI).

[00134] The EGFR WT A431 epidermoid carcinoma cell line express high levels of the WT EGFR protein on the cell surface due to amplification of the EGFR gene, and the growth of A431 cells is known to be dependent on WT EGFR signaling. The A549 (adenocarcinoma), NCI-H661 (large cell lung cancer), NCI-H1048 (small cell lung cancer) and NCI-H1299 (adenocarcinoma) cell lines have a WT EGFR gene and represent non-mutated, lung cancer cell line controls. NCI-H1975, HCC827, HCC4006, A431, A549, NCI-H661 , NCI-H1048 and NCI-H1299 cells were obtained from the American Type Culture Collection (Manassas, VA). PC-9 cells were obtained from the National Cancer Center Research Institute and Shien-Lab (Tokyo, Japan). Cells were cultured in RPMI 1640 (NCI-H1975, HCC827, HCC4006, PC-9, NCI-H661 , and NCI-H1299), F12K (A549) or DMEM (A431 and NCI-H1048) supplemented with 10% FBS, IX penicillin/streptomycin, and IX GlutaMAX. Cells were seeded in 96-well plates at 3,000 cells/well and allowed to adhere overnight. The following day, cells were treated with increasing concentrations of CO-1686 or metabolites for 72 hours and the number of viable cells in culture was determined based on quantitation of the adenosine triphosphate (ATP) present, which signals the presence of metabolically active cells. Depending on the experimental replicate, final concentration of each compound ranged from 20 μΜ to 0.008 μΜ, diluted in 3-, 4-, or 5-fold steps. GI50 values were calculated using GraphPad Prism 6.0 software

(GraphPad Software; La Jolla, CA) by setting 0.5% DMSO-treated control cells as 100% of viable cells. Curve-fits were performed using nonlinear regression with the software-incorporated "log (agonist) vs. response - Variable slope (four parameters)" option. All other software options were default. Each reported GI50 value represents > 3 independent experiments.

[00135] Cell viability data is summarized in table 7. Consistent with Walter et al. (Cancer Discov. 2013, 3(12), 1404-15), CO-1686 was preferentially potent on cell lines that were EGFR mutant, with IC50 values ranging from 23-106 nM in mutant NSCLC lines, compared to EGFR WT cells which had IC50 values of 952 to >5000 nM. Relative to CO-1686, metabolites M460, M502 and M544 demonstrated a lower potency in cell lines with EGFR mutations, with IC50 values ranging from 697 to >5000 nM. Metabolites M460, M502 and M544 did not retain the mutant selective cell potency of the parental CO-1686 molecule. EGFR WT IC50 values for M460, M502 and M544 were comparable to EGFR mutant cell lines.

9. Glucose Tolerance Test of CO-1686 in Rats

[00136] To evaluate the role of CO-1686 on glucose homeostasis, an oral glucose tolerance test was performed in female Sprague-Dawley rats. The insulin-like growth factor receptor-1 (IGF-1R) and insulin receptor (INSR) inhibitor, BMS- 754807, was used as a positive control. Single dose and 4-day repeat BID dosing with CO-1686 was performed prior to the OGTT challenge. Glucose was administered to rats by oral gavage 15 minutes after compound administration, and blood was collected and used for glucose, insulin, and PK analysis.

[00137] Sprague Dawley rats which were approximately 9 weeks of age were selected out of a cohort of 20 cannulated at the jugular vein and used in this study. On Study Day -3, animals were randomized into treatment groups based on body weight measurement.

[00138] All treatments were administered by oral gavage. The dose volume for each animal (10 mL/kg in 0.5% methylcellulose) was based on the most recent body weight measurement taken just prior to dosing. Observations were recorded at each dose. Body weights were recorded prior to study start (Day -3) and prior to each subsequent dose. Standard rodent chow (Purina Lab Diet 5001) and drinking water was provided ad libitum.

[00139] On Study Day 5, an oral glucose tolerance test was conducted following an overnight fast (food removed -1700 hrs on Day 4). Rats were given their respective compound dose via oral gavage and 15 minutes later were given 1 g/kg (10 mL/kg) of glucose also via gavage at a pace of 2 minutes per rat. Blood glucose was checked via glucometer at the following times relative to glucose dose: baseline (same time as overnight fasted bleed -0900 hrs, prior to compound dose), 15, 30, 60, 90, and 120 min. Blood collection for insulin and PK analysis (details below) was collected at the following times relative to glucose dose: baseline (same time as overnight fasted bleed -0900 hrs, prior to compound dose), 15, 30, 60, 90, and 120 min. Food was returned to all animals following the 120 min timepoint.

[00140] Samples for insulin during OGTT were collected as follows: 40 μL· of blood were pipetted into K2EDTA tubes and were centrifuged at 2200 x g for 10 minutes at 5°C ± 3°C. 15 plasma was aliquoted into small volume 96 well plates and stored at -70°C for insulin analysis. Samples for PK during OGTT were collected as follows: -200 μL· of blood was pipetted into K₂EDTA tubes and was centrifuged at 2200 x g for 10 minutes at 5°C + 3°C. 100 μL· plasma was aliquoted into small volume 96 well plates and stored at -70°C until shipped (on dry ice) to the client for PK analysis.

[00141] Statistical analyses were performed on all data using Graph Pad Prism statistical software. For OGTT serial sampling, a two-way RM ANOVA (with a Bonferonni post-hoc test comparing all groups to the vehicle group) was conducted. For baseline values a one-way ANOVA (with a Bonferonni post-hoc test comparing all groups to the vehicle group) was conducted.

[00142] The insulin-like growth factor receptor- 1 (IGF-1R) and insulin receptor (INSR) inhibitor, BMS-754807, elicited a significant elevation in post-prandial glucose excursion as expected whereas single dose and 4-day repeat-dose BID dosing with CO- 1686 (HK7) prior to the OGTT challenge did not significantly elevate plasma glucose or insulin levels over non-treated animals (Figure 3).

10. Effects of M460 and M502 on Blood Glucose

[00143] To evaluate the role of M460 and M502 on glucose homeostasis, an oral glucose tolerance test was performed in female Sprague-Dawley rats. The IGF- 1R and INSR inhibitor, BMS-754807, was used as a positive control. A single dose of M460, M502, or BMS-754807 was performed prior to the OGTT challenge. Glucose was administered to rats by oral gavage 15 minutes after compound administration, and blood was collected and used for glucose, insulin, and PK analysis.

[00144] Sprague Dawley rats approximately 9 weeks of age were used in this study. Standard rodent chow (Purina Lab Diet 5001) and drinking water were provided ad libitum. Animals were randomized into treatment groups based on body weight measurements.

[00145] On the day of dosing, an oral glucose tolerance test was conducted following an overnight fast (food removed -1700 hrs). Vehicle, M460, and M502 were administered IP at dose volumes of 10 mL/kg in 5% DMSO/5 Solutol/90 PBS. The positive control, BMS-754807 was given by oral gavage at a dose volume of 10 mL/kg in 0.5% Methylcellulose. Doses were based on the most recently recorded body weight.

[00146] Observations were recorded at each dose. Rats were dosed with M460 (10 mg/kg), M502 (1000 mg/kg), BMS-754807 (25 mg/kg) 15 minutes later were given 1 g/kg (10 mL/kg) of glucose via gavage at a pace of 2 minutes for each rat. Blood glucose was measured via a glucometer at the following times relative to glucose dose: baseline (same time as overnight fasted bleed -0900 hrs, prior to compound dose), 15, 30, 60, 90, and 120 min. Blood collection for insulin and PK analysis (details below) was collected at the following times relative to glucose dose: baseline (same time as overnight fasted bleed -0900 hrs, prior to compound dose), 15, 30, 60, 90, and 120 min. Food was returned to all animals following the 120 min timepoint.

[00147] Samples for insulin during the oral glucose tolerance test were collected as follows: 40 μL· of blood were pipetted into K₂EDTA tubes and were centrifuged at 2200 x g for 10 minutes at 5°C + 3°C. 15 plasma was aliquoted into small volume 96 well plates and stored at -70°C for insulin analysis. Samples for PK during OGTT were collected as follows: -200 μL· of blood was pipetted into

K₂EDTA tubes and was centrifuged at 2200 x g for 10 minutes at 5°C ± 3°C. 100 plasma was aliquoted into small volume 96 well plates and stored at -70°C and shipped on dry ice to the client for drug level analysis.

[00148] Statistical analyses were performed on all data using Graph Pad Prism statistical software. For OGTT serial sampling, a two-way RM ANOVA (with a Bonferonni post-hoc test comparing all groups to the vehicle group) was conducted. For baseline values a one-way ANOVA (with a Bonferonni post-hoc test comparing all groups to the vehicle group) was conducted.

[00149] The IGF-IR and INSR inhibitor, BMS-754807, elicited a significant elevation in post-prandial glucose excursion as expected. Additionally, M460 and M502 showed elevations in post-prandial glucose excursion, consistent with peripheral insulin resistance resulting from IGF-IR and INSR inhibition by M460 and M502 (Figure 4).

11. Evaluation of CO- 1686 and metabolite M502 in EGFR NSCLC cell line

[00150] The impact of M502 on the development of CO- 1686 resistance was examined in 3 EGFR mutant cell lines: PC-9, HCC827 and NCI-H1975. A specific IR/IGF-1R inhibitor, OSI-906 (Mulvihill et al. Future Med Chem. 2009, 1(6), 1153- 71) was used as a control to determine what component of M502 activity, if any, could be explained by IGF-1R/IR activity. Cells were seeded at 100,000 cells/well in a 6/well plate and incubated overnight at 37 °C with 5% C0₂. Viability and cell density of trypsinized cell lines was determined using a Vi-Cell™ XR instrument (Beckman Coulter; Indianapolis, IN) prior to plating. CO- 1686, OSI-906 and M502 stock solutions (in 100% DMSO) were diluted in growth media before being added to cells (0.5% final DMSO concentration once applied to cells). The final concentration of each compound was 1 μΜ. Cells were incubated with compound for up to 86 days in 10% FBS containing RPMI at 37°C with 5% C0₂. Media was replaced twice weekly and wells were examined microscopically each day for growth. Following the development of cell confluence cell density was determined on the Vi-Cell™ XR instrument. Confluent wells were re-seeded at 100,000 cells/well. If the well reached confluence and was split, total cell number was determined by multiplying the cell count by the split ratio used to seed the plate. GraphPad Prism 6 (GraphPad Software; La Jolla, CA) was used to plot cell number.

Cell count data is summarized in Figure 2. CO- 1686 as a single agent reduced cell number compared to the DMSO control in all 3 EGFR mutant NSCLC cell lines examined, however, after 20-40 days a CO- 1686 resistant cell population was observed to rapidly expand and grow out. OSI-906 as a single agent reduced cell number in all 3 EGFR mutant cell lines, although the impact compared to OSI-906 was modest. The impact of M502 monotherapy was more variable on cell number across cell lines. In the HCC827 cell line, M502 as a single agent was superior to CO- 1686 in reducing total cell number over time. In the NCI-H1975 cell line, M502 and CO- 1686 activity was comparable and in PC-9, CO- 1686 was superior to M502. In all EGFR mutant cell lines examined, M502 reduced cell number to a greater extent than OSI-906, indicating that the impact of M502 cannot be explained by IR/IGF-1R inhibition alone and additional kinase targets of M502 such as FAK, FER and FES play a role. Consistent with the inhibition of FAK, FER and FES (Ivanova et al. Oncogene. 2013, 32(50), 5582-92; Hellwig et al. Chem Biol. 2012, 19(4), 529-40). M502 addition was noted to alter cell morphology following 10 days of incubation, inducing a "flattened" cellular morphology (Figure 5). The use of M502 in combination with CO- 1686 further reduced cell number over time compared to either compound used as a monotherapy in all 3 EGFR mutant cell lines examined. The addition of OSI-906 to CO- 1686 was also observed to be more effective than either agent alone, however, the effect was not as pronounced as for M502 + CO- 1686, consistent with additional non-IR/IGF-lR activities of M502 being responsible for the increased efficacy observed.

12. Effects of CO-1686 in combination with IGF1R and INSR inhibitors

on acquired resistance in PC-9 cells

[00151] Combinations of the mutant selective EGFR inhibitor CO-1686 and the IGF1R and INSR inhibitors OSI-906 and M502 were evaluated in the mutant EGFR cell line PC-9 to assess the potential of this combination to prevent the emergence of acquired resistance.

[00152] PC-9 cells were seeded at lxlO⁵ cells/well in a 6-well tissue culture dish. PC-9 cells have a deletion 19 activating mutation in EGFR commonly observed in NSCLC patients. The following day CO-1686, OSI-906, and M502 were serially diluted in DMSO and then added to cells at a final concentration of 1 μΜ. The cells were incubated with compound for four days in 10% FBS containing RPMI at 37°C with 5% C0₂. Representative photographs of wells were taken at 5x magnification. [00153] As anticipated, cellular viability was clearly reduced in PC-9 cells treated with CO- 1686 as compared to DMSO alone. Limited or no impact on cellular viability was observed with treatment of PC-9 cells with OSI-906 and M502. The combination of CO- 1686 and the IGFIR and INSR inhibitors OSI-906 or M502 resulted in a significant decrease in cellular viability as compared to PC-9 cells dosed with CO- 1686 alone. The combination of an EGFR inhibitor with an IGFIR and INSR inhibitor thus has a greater impact in cellular viability and will delay the emergence of resistant cells as compared to either an EGFR inhibitor or an IGFIR and INSR inhibitor dosed alone (Figure 6).

Table 1

Table 2. Biochemical and cellular profiling of CO- 1686 metabolites

Table 3

In this table IR refers to the insulin receptor, whereas the approved gene name of INSR is used throughout the rest of this document.

Table 4. Biochemical profiling of CO- 1686 metabolites

Table 5: CO-1686 and metabolite levels in NSCLC patients treated with CO-1686

Table 6. Cellular profiling of CO-1686 metabolites against IGFIR and INSR

Table 7: Activity of CO-1686 and related metabolites in EGFR mutant and EGFR wild-type (WT) cells

GI₅₀ = the concentration of compound required for 50% inhibition of cell growth

GI₅₀ values represent the average GI₅₀ ± SEM of >3 independent experiments

ND = not determined

Claims

WHAT IS CLAIMED IS: Claims

1. An essentially pure or isolated compound of formula (I) or a salt thereof,

(T)

wherein Ri = H, R₂ = H; or

wherein Ri = -COCH3, R₂ = H or -COCH3.

2. A pharmaceutical composition comprising a compound of formula (I) or a salt thereof, and a pharmaceutically acceptable excipient.

3. The pharmaceutical composition according to claim 2, further comprising an EGFR inhibitor.

4. The pharmaceutical composition according to claim 3, wherein the EGFR inhibitor is a TKI.

5. A method of treating a mammalian disease condition mediated by epidermal growth factor receptor (EGFR), insulin-like growth factor- 1 receptor (IGF- 1R), insulin receptor (INSR), focal adhesion kinase (FAK). fer (fps/fes related) tyrosine kinase (FER), or feline sarcoma oncogene (FES), the method comprising administering to a mammal in need thereof a therapeutically effective amount of the composition of claim 2.

6. The method according to claim 5, wherein the mammal has a cancer.

7. The method according to claim 6, wherein the cancer comprises lung cancer, colon cancer, breast cancer, prostate cancer, liver cancer, pancreas cancer, brain cancer, kidney cancer, ovarian cancer, stomach cancer, skin cancer, bone cancer, gastric cancer, breast cancer, pancreatic cancer, glioma, gliobastoma, hepatocellular carcinoma, papillary renal carcinoma, head and neck squamous cell carcinoma, leukemia, lymphomas, myeloma, or a solid tumor.

8. The method according to claim 5, wherein the composition further comprises an EGFR inhibitor.

9. The method according to claim 5, wherein the mammal is identified as being in need of IGF-IR inhibition, INSR inhibition, FAK inhibition, FER inhibition or FES inhibition for the treatment of cancer.

10. The method according to claim 5, wherein the mammal is a human.

11. The method according to claim 5, wherein the compound of formula (I) directly or indirectly interacts with, affects, or inhibits IGF-IR mediated pathway, insulin receptor (INSR) pathway, focal adhesion kinase (FAK) pathway, fer (fps/fes related) tyrosine kinase (FER) pathway, or feline sarcoma oncogene (FES) pathway.

12. The method according to claim 5, wherein the compound of formula (I) is an inhibitor of IGF-IR, or wherein the compound of formula (I) directly or indirectly interacts with, affects, or inhibits IGF-IR mediated pathway.

13. The method according to claim 5, wherein the compound of formula (I) is an inhibitor of INSR, or wherein the compound of formula (I) directly or indirectly interacts with, affects, or inhibits INSR mediated pathway.

14. The method according to claim 5, wherein the compound of formula (I) is an inhibitor of FAK, or wherein the compound of formula (I) directly or indirectly interacts with, affects, or inhibits FAK mediated pathway.

15. The method according to claim 5, wherein the compound of formula (I) is an inhibitor of FER, or wherein the compound of formula (I) directly or indirectly interacts with, affects, or inhibits FER mediated pathway.

16. The method according to claim 5, wherein the compound of formula (I) is an inhibitor of FES, or wherein the compound of formula (I) directly or indirectly interacts with, affects, or inhibits FES mediated pathway.

17. A method for preventing resistance to an EGFR inhibitor in a subject in need thereof, comprising administering to the subject an effective amount of a composition of claim 2.

18. The method according to claim 17, wherein the subject has a cancer.

19. The method according to claim 18, wherein the cancer comprises lung cancer, colon cancer, breast cancer, prostate cancer, liver cancer, pancreas cancer, brain cancer, kidney cancer, ovarian cancer, stomach cancer, skin cancer, bone cancer, gastric cancer, breast cancer, pancreatic cancer, glioma, gliobastoma, hepatocellular carcinoma, papillary renal carcinoma, head and neck squamous cell carcinoma, leukemia, lymphomas, myeloma, or a solid tumor.

20. The method according to claim 17, wherein the subject is a human.

21. A kit comprising the composition of claim 2 and instructions for use of the composition in treating a disease or disorder in a subject in need thereof.

22. A pharmaceutical composition comprising a first irreversible EGFR TKI compound, wherein upon administration to a patient the first compound is metabolized to a second compound that is an IGF-IR inhibitor, an INSR inhibitor, or a dual target IGF-IR and INSR inhibitor, wherein second compound has a t of greater than 12 hours.

23. The pharmaceutical composition according to claim 22, wherein the first compound is CO- 1686.

24. The pharmaceutical composition according to claim 22, wherein the second compound is as in claim 1.

25. A method of treating cancer in a mammal comprising administering to said mammal the pharmaceutical composition of claim 22.

26. The method according to claim 25, wherein the first compound is CO-

1686.

27. The method according to claim 25, wherein the second compound is as in claim 1.

28. A pharmaceutical composition comprising a dual targeting compound, wherein said compound is a wild type sparing irreversible T790M EGFR TKI, and upon administration an IGF-IR inhibitor, INSR inhibitor, FER inhibitor, FAK inhibitor, or FER inhibitor.

29. A method of treating cancer in a mammal comprising administering said mammal the pharmaceutical composition of claim 28.

30. A method of treating cancer in a mammal, the method comprising administering to the mammal a therapeutically effective amount of the composition of claim 2.

31. The method of claim 30 wherein the cancer is mediated by epidermal growth factor receptor (EGFR), insulin-like growth factor-1 receptor (IGF-IR), insulin receptor (INSR), focal adhesion kinase (FAK), f r (fps/fes related) tyrosine kinase (FER), or feline sarcoma oncogene (FES).