Combining IRS / STAT3 dual modulators with anticancer drugs to treat cancer

Abstract

To provide a pharmaceutical composition for treating a tumor that has developed resistance to an Epidermal Growth Factor Receptor (EGFR) inhibitor and / or EGFR antibody.SOLUTION: A pharmaceutical composition contains a compound represented by the following formula or a salt thereof, as well as an EGFR inhibitor selected from the group consisting of AZD9291, afatinib, neratinib, dacomitinib, poziotinib, CO-1686, HM61713 and AP26113 and / or an EGFR antibody selected from the group consisting of cetuximab, panitumumab, and necitumumab.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to the treatment of cancer using a combination therapy comprising: (i) a protein kinase (PK) modulator selected from an epidermal growth factor inhibitor (EGFR inhibitor) and an EGFR antibody; (ii) an inhibitor of mammalian target of rapamycin (mTOR); (iii) a mitogen-activated protein kinase (MEK) inhibitor; (iv) a mutant B-Raf inhibitor; (v) a chemotherapeutic agent such as gemcitabine, 5-FU, irinotecan, and oxaliplatin; and (vi) a dual modulator of insulin receptor substrate (IRS) and signal transducer and activator of transcription 3 (Stat3) in combination with specific combinations thereof. The combination can be used to treat tumors that have developed resistance to EGFR inhibitors, EGFR antibodies, mTOR inhibitors, MEK inhibitors, mutant B-Raf inhibitors, chemotherapeutic agents, and specific combinations thereof, or to prevent acquired tumor resistance to any of the inhibitors or agents, or to prevent tumor recurrence after cessation of treatment with any of the inhibitors or agents, or combinations thereof. The combination provides at least additive, and preferably synergistic, therapeutic effects. The present invention further relates to the treatment of cancer using a combination therapy comprising a dual modulator of IRS and Stat3 in combination with an immunotherapeutic agent, which combination can be used to sensitize tumors to immunotherapy. [Background technology]

[0002] Tyrphostins are a family of protein tyrosine kinase inhibitors designed to mimic the tyrosine substrate, ATP, and are able to allosterically inhibit the enzyme (Levitzki et al., Science (1995), 267:1782-88; Levitzki et al., Biochem. Pharm. (1990), 40:913-920; Levitzki et al., FASEB J. (1992), 6:3275-3282; U.S. Patent Nos. 5,217,999 and 5,773,476; Posner et al., Mol. Pharmacol. (1994), 45:673-683). The pharmacophore of these tyrphostins, and in particular benzylidenemalononitrile-type tyrphostins, is a hydrophilic catechol ring and a more lipophilic substituted cyano-vinyl group. Kinetic studies have shown that some tyrphostin compounds are purely competitive inhibitors of the tyrosine substrate but act as noncompetitive inhibitors of the ATP-binding site (Yaish et al., Science (1988), 242:933-935; Gazit et al., J. Med. Chem. (1989), 32:2344-2352). Nevertheless, many tyrphostins exhibit competitive or mixed competitive inhibition of both the substrate and ATP-binding sites (Posner et al., Mol. Pharmacol. (1994), 45:673-683).

[0003] In a related group of tyrphostins, replacement of the hydrophilic catechol ring with a lipophilic dichloro- or dimethoxy-phenyl group resulted in EGFR kinase inhibitors effective in the low micromolar range (Yoneda et al., Cancer Res. (1991), 51:4430-4435). Furthermore, administration of these tyrphostins to tumor-bearing nude mice at suboptimal doses with anti-EGFR monoclonal antibodies resulted in significantly enhanced inhibition of tumor growth.

[0004] WO 2008 / 068751 by some of the inventors of the present invention discloses compounds with increased inhibitory properties of the activation and signaling of the insulin-like growth factor 1 receptor (IGF1R), the platelet-derived growth factor receptor (PDGFR), the epidermal growth factor receptor (EGFR), and the IGF1R-related insulin receptor (IR).

[0005] WO 2009 / 147682, by some of the inventors of the present invention, discloses compounds that act as signal transduction modulators of protein kinases (PKs) and receptor kinases (RKs). WO 2009 / 147682 further discloses methods for preparing such compounds, pharmaceutical compositions containing such compounds, and methods of using these compounds and compositions, particularly as chemotherapeutic agents for the prevention and treatment of PK- and RK-related disorders, such as metabolic disorders, inflammatory disorders, fibrosis, and cell proliferative disorders, especially cancer.

[0006] WO 2012 / 117396 by some of the inventors of the present invention describes the combination of a compound of WO 2008 / 068751 or WO 2009 / 147682 with an anti-cancer agent for the treatment of cancer.

[0007] Cancers treated with conventional radiation or chemotherapy or other anticancer drugs frequently develop resistance to these treatments, ultimately resulting in recurrent disease that often has a more aggressive phenotype than that observed at the time of original diagnosis (Li et al., J. Med. Chem. (2009), 52(16):4981-5004).

[0008] According to the principles for selecting drugs for use in combination chemotherapy regimens, drugs with different mechanisms of action and additive or synergistic cytotoxic effects on tumors can be combined (Pazdur et al., Chapter 3: Principles of Oncologic Pharmacotherapy (2005), 9th Edition: 23-42). Multidrug therapy has three important theoretical advantages over monotherapy. First, multidrug therapy can maximize cell death while minimizing host toxicity by using drugs with non-overlapping dose-limiting toxicities. Second, multidrug therapy can increase the range of drug activity against tumor cells that have intrinsic resistance to a particular type of treatment. Finally, multidrug therapy can also prevent or reduce the development of new resistant tumor cells. Virtually all curative chemotherapy regimens for cancer use multidrug combinations (Frei and Eder, Cancer Medicine (2003), 11:817-837).

[0009] A relatively new family of anti-cancer drugs are inhibitors (e.g., antibodies and small molecules) of specific kinases or other signaling enzymes involved in mitogenic, anti-apoptotic, angiogenic, or metastatic pathways in cancer cells. Examples of approved drugs within this family include EGFR and / or HER2 blockers (e.g., small molecules gefitinib, erlotinib, lapatinib, or antibodies such as trastuzumab (Herceptin®) and cetuximab (Erbitux®)), B-Raf inhibitors (e.g., PLX-4032, sorafenib), BCR-ABL and / or Src family kinase inhibitors (e.g., imatinib, dasatinib, nilotinib), VEGF inhibitors (e.g., cytochrome P450 / 450), cytochrome P450 / 450, ... These include R / PDGFR and / or multikinase inhibitors (e.g., bevacizumab (Avastin®), sorafenib, sunitinib, and pazopanib), and proteasome inhibitors (e.g., bortezomib (Velcade®)). Several EGFR inhibitors have been approved by the FDA, such as Tarceva (erlotinib) in 2004, Iressa (gefitinib) in 2003, and lapatinib in 2010, as well as antibodies against EGFR.

[0010] Another family of anticancer drugs is inhibitors of the mammalian target of rapamycin (mTOR). mTOR (also known as FRAP (FKBP-rapamycin-related protein), RAFT (rapamycin and FKBP target), RAPT1, or SEP) is a serine / threonine kinase belonging to the phosphatidylinositol-3 kinase (PI3K)-related kinase (PIKK) family. mTOR functions as a central controller of growth, proliferation, metabolism, and angiogenesis, but its signaling is deregulated in various human diseases, particularly certain cancers such as renal cell carcinoma and breast cancer. In cancer, mTOR is frequently overactivated, promoting cancer development and progression. Recent developments have shifted cancer treatment from traditional cytotoxic drugs to drugs that target specific proteins, such as mTOR, called mTOR inhibitors. Rapamycin (sirolimus), a common mTOR inhibitor, is a bacterial product that inhibits mTOR by binding to its intracellular receptor. Two mTOR inhibitors, temsirolimus (CCI-779) and everolimus (Afinitor, RAD-001), which are derivatives of rapamycin, are approved for the treatment of patients with advanced renal cell carcinoma (RCC) and mantle cell lymphoma. Other examples of mTOR inhibitors include ridaforolimus (deforolimus, AP23573), a dual inhibitor of PI3K and mTOR, and NVP-BEZ235.

[0011] First-generation mTOR inhibitors, such as rapamycin, show certain limitations by only blocking the C1 isoform, inducing feedback activation of AKT, and showing resistance to the second isoform, mTORC2. A panel of second-generation drugs has been developed that can inhibit both mTORC1 and mTORC2 by targeting the kinase domain with high selectivity. Examples of second-generation mTOR inhibitors include OSI-027 (OSI Pharmaceuticals), XL765 (Exelixis), INK128, MLN0128, AZD2014, DS-3078a, and Palomid 529.

[0012] Over the past few decades, immunotherapy has become an important part of the treatment of several types of cancer. The goal of cancer immunotherapy is to enable the patient's immune system to specifically recognize and kill cancer cells. Signal transducer and activator of transcription 3 (Stat3) is frequently activated in cancers and is directly involved in establishing and maintaining the immunosuppressive microenvironment of cancer, playing a central role in tumor immune evasion. Summary of the Invention [Problem to be solved by the invention]

[0013] There is an unmet need for combinations that are useful in treating cancer and preferably provide at least an additive therapeutic effect. Combinations of drugs from different categories are useful in preventing or overcoming the emergence of drug-resistant tumors. [Means for solving the problem]

[0014] The present invention relates to compositions and methods for treating cancer by administering a combination comprising at least one compound that is a dual modulator of insulin receptor substrate (IRS) and signal transducer and activator of transcription 3 (Stat3), e.g., a compound of formula (III) or (IV), or any of the compounds included in these formulas, in combination with (i) a modulator of protein kinase (PK) selected from epidermal growth factor inhibitors (EGFR inhibitors) and EGFR antibodies; (ii) an inhibitor of mammalian target of rapamycin (mTOR); (iii) a mitogen-activated protein kinase (MEK) inhibitor; (iv) a mutant B-Raf inhibitor; (v) a chemotherapeutic agent, such as gemcitabine, 5-FU, irinotecan, and oxaliplatin; and (vi) certain combinations thereof. Furthermore, the present invention relates to the treatment of cancer using a combination therapy comprising a dual modulator of IRS and Stat3, e.g., a compound of formula (III) or (IV), or any of the compounds included in these formulas, in combination with an immunotherapeutic agent. [Effects of the Invention]

[0015] The compounds described herein are modulators of insulin receptor substrate 1 (IRS1) and / or insulin receptor substrate 2 (IRS2) signaling. Accordingly, these compounds are referred to herein as "IRS modulators." In some embodiments, the compounds are inhibitors of IRS1 and / or IRS2. In further embodiments, the compounds of the present invention are inhibitors of insulin-like growth factor 1 receptor (IGF-1R). As such, these compounds are useful for inhibiting, treating, or preventing IGF-1R and / or IRS1 and / or IRS2 signaling-related disorders, such as cancer. In some embodiments, the compounds induce any one or more of the following, in any order: (i) dissociation of IRS1 and / or IRS2 from the cell membrane; (ii) serine phosphorylation of IGF-1R's direct substrates, IRS1 and / or IRS2; and / or (iii) degradation of IRS1 and / or IRS2, thus providing a long-term effect that enhances the inhibitory activity of these compounds. In other embodiments, the compounds are also inhibitors of the IGF1R-related insulin receptor (IR), or proteins that are affected or mediated by these PTKs, or proteins that are part of a PTK-mediated signaling pathway.

[0016] The IRS is negatively regulated by downstream elements of EGFR and mTOR / S6K. Therefore, treating patients with drugs that inhibit these targets can, as a side effect, upregulate the IRS and activate the central survival pathway to AKT. As demonstrated herein, in accordance with the principles of the present invention, this feedback mechanism leading to drug resistance can be overcome by combining an IRS disruptor with the treatment.

[0017] The compounds described herein are also modulators of signal transducer and activator of transcription 3 (Stat3). Accordingly, these compounds are also referred to herein as "Stat3 modulators." In some embodiments, the compounds result in the inhibition of Stat3 phosphorylation in cancer cells. Increased levels of Stat3 phosphorylation are detected in various cancers and drug-resistant cancers, and enhance cancer survival. Furthermore, as demonstrated herein, treatment of cancer with PK inhibitors surprisingly results in the induction of Stat3 phosphorylation. Without being bound by any particular theory or mechanism of action, it is contemplated that inhibition of Stat3 activity by the compounds of the present invention may synergize with such PK inhibitors, upregulating Stat3 as a side effect, preventing acquired resistance to such drugs, and may be effective against drug-resistant cancers.

[0018] Due to their dual effects on IRS and Stat3, the compounds are further described herein as "dual IRS / Stat3 modulators."

[0019] Thus, in one embodiment, the present invention relates to a method of treating a tumor that has developed resistance to an epidermal growth factor receptor (EGFR) inhibitor and / or an EGFR antibody, comprising contacting the tumor with an EGFR inhibitor and / or an EGFR antibody in combination with a compound represented by the structure of formula (III) or (IV).

[0020] In another embodiment, the present invention relates to a method for preventing acquired resistance of a tumor to an epidermal growth factor receptor (EGFR) inhibitor and / or an EGFR antibody, comprising contacting the tumor with an EGFR inhibitor and / or an EGFR antibody in combination with a compound represented by the structure of formula (III) or (IV).

[0021] In another embodiment, the present invention relates to a method for preventing or delaying tumor recurrence after cessation of treatment with an EGFR inhibitor and / or an EGFR antibody, comprising contacting the tumor with an EGFR inhibitor and / or an EGFR antibody in combination with a compound represented by the structure of formula (III) or (IV).

[0022] In another embodiment, the invention relates to a pharmaceutical combination comprising a compound represented by the structure of Formula (III) or (IV) in combination with an epidermal growth factor (EGFR) inhibitor and / or an EGFR antibody.

[0023] In another embodiment, the present invention relates to a pharmaceutical combination comprising a compound represented by the structure of Formula (III) in combination with an epidermal growth factor (EGFR) inhibitor and / or an EGFR antibody, wherein the EGFR inhibitor is selected from the group consisting of erlotinib, gefitinib, lapatinib, vandetanib, neratinib, icotinib, afatinib, dacomitinib, poziotinib, AZD9291, CO-1686, HM61713, and AP26113, and the EGFR antibody is selected from the group consisting of trastuzumab, necitumumab, cetuximab, and panitumumab, preferably, the EGFR inhibitor is erlotinib or afatinib, and / or the EGFR antibody is cetuximab.

[0024] In another embodiment, the present invention relates to a pharmaceutical combination comprising a compound represented by the structure of Formula (IV) in combination with an epidermal growth factor (EGFR) inhibitor and / or an EGFR antibody.

[0025] In other embodiments, the present invention further relates to the above pharmaceutical combinations for use in treating tumors that are resistant to EGFR inhibitors and / or EGFR antibodies, or for preventing acquired resistance to EGFR inhibitors and / or EGFR antibodies, or for preventing or delaying tumor recurrence after cessation of treatment with EGFR inhibitors and / or EGFR antibodies.

[0026] In other embodiments, the present invention further relates to the use of the above combination for the preparation of a medicament for the treatment of tumors that are resistant to EGFR inhibitors and / or EGFR antibodies, or for preventing acquired resistance to EGFR inhibitors and / or EGFR antibodies, or for preventing or delaying tumor recurrence after cessation of treatment with EGFR inhibitors and / or EGFR antibodies.

[0027] In one embodiment, the compound is represented by the structure of Formula (III): In another embodiment, the compound is represented by the structure of Formula (IV): Each possibility represents a separate embodiment of the present invention.

[0028] In some embodiments, the tumor is present in a cancer patient whose tumor has acquired resistance to EGFR inhibitor and / or EGFR antibody treatment. In other embodiments, the tumor is present in a cancer patient who is undergoing treatment with an EGFR inhibitor and / or EGFR antibody or who is a candidate for such treatment. In other embodiments, the treatment results in attenuation of growth or regression of the resistant tumor.

[0029] In some embodiments, the EGFR inhibitor is selected from the group consisting of erlotinib, gefitinib, lapatinib, vandetanib, neratinib, icotinib, afatinib, dacomitinib, poziotinib, AZD9291, CO-1686, HM61713, and AP26113. In one currently preferred embodiment, the EGFR inhibitor is erlotinib. In another currently preferred embodiment, the EGFR inhibitor is afatinib. Each possibility represents a separate embodiment of the present invention.

[0030] In some embodiments, the EGFR antibody is selected from the group consisting of trastuzumab, necitumumab, cetuximab, and panitumumab. In one currently preferred embodiment, the EGFR antibody is cetuximab.

[0031] In some embodiments, the compound is a compound of formula (III), represented by the structure of Formula D, in combination with erlotinib or afatinib.

[0032] In other embodiments, the compound is a compound of formula (III) represented by the structure of Formula D in combination with cetuximab.

[0033] In other embodiments, the compound is a compound of formula (III) represented by the structure of Formula D in combination with afatinib and cetuximab.

[0034] In some embodiments, the combination treatment comprises a compound of Formula (III) or (IV) and either an EGFR antibody or an EGFR inhibitor. In other embodiments, the combination treatment comprises a compound of Formula (III) or (IV) and both an EGFR antibody and an EGFR inhibitor. In some currently preferred embodiments, the EGFR inhibitor is erlotinib or afatinib, and the EGFR antibody is cetuximab.

[0035] In one particular embodiment, the present invention relates to a pharmaceutical combination comprising a compound represented by the structure of Formula D in combination with erlotinib. In one particular embodiment, the present invention relates to a pharmaceutical combination comprising a compound represented by the structure of Formula D in combination with afatinib. In another particular embodiment, the present invention relates to a pharmaceutical combination comprising a compound represented by the structure of Formula D in combination with cetuximab. In another particular embodiment, the present invention relates to a pharmaceutical combination comprising a compound represented by the structure of Formula D in combination with afatinib and cetuximab.

[0036] In another aspect, it has now been unexpectedly discovered that a combination of a dual modulator of insulin receptor substrate (IRS) and signal transducer and activator of transcription 3 (Stat3) described herein with an inhibitor of mammalian target of rapamycin (mTOR) provides at least an additive, and preferably a synergistic, therapeutic effect compared to the therapeutic effect of each agent alone. Furthermore, the combination can be used to treat tumors that have developed resistance to mTOR inhibitors, and / or to prevent acquired resistance of tumors to mTOR inhibitors, and / or to prevent or delay tumor recurrence after cessation of treatment with an inhibitor of mammalian target of rapamycin (mTOR).

[0037] Thus, in one embodiment, the present invention relates to a pharmaceutical combination comprising a compound represented by the structure of Formula (III) or (IV) and at least one inhibitor of mammalian target of rapamycin (mTOR), wherein the compound and the at least one mTOR inhibitor together provide a synergistic anti-cancer therapeutic effect.

[0038] In another embodiment, the present invention relates to a method of treating cancer, comprising administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical combination comprising a compound represented by the structure of Formula (III) or (IV) and at least one inhibitor of mammalian target of rapamycin (mTOR), wherein the compound and the at least one mTOR inhibitor together provide a synergistic therapeutic effect.

[0039] In another embodiment, the present invention relates to a method of treating a tumor that has developed resistance to an inhibitor of mammalian target of rapamycin (mTOR), comprising contacting the tumor with an mTOR inhibitor in combination with a compound represented by the structure of Formula (III) or (IV).

[0040] In another embodiment, the present invention relates to a method for preventing acquired resistance of a tumor to an inhibitor of mammalian target of rapamycin (mTOR), comprising contacting the tumor with an mTOR inhibitor in combination with a compound represented by the structure of Formula (III) or (IV).

[0041] In another embodiment, the present invention relates to a method of preventing or delaying tumor recurrence after cessation of treatment with an inhibitor of mammalian target of rapamycin (mTOR), comprising contacting the tumor with an mTOR inhibitor in combination with a compound represented by the structure of Formula (III) or (IV).

[0042] In other embodiments, the present invention further relates to the above pharmaceutical combinations for use in treating tumors that are resistant to mTOR inhibitors, or for preventing acquired resistance to mTOR inhibitors, or for preventing or delaying tumor recurrence after cessation of treatment with an inhibitor of mTOR.

[0043] In other embodiments, the present invention further relates to the use of the above combination for the preparation of a medicament for the treatment of tumors that are resistant to mTOR inhibitors, or for preventing acquired resistance to mTOR inhibitors, or for preventing or delaying tumor recurrence after cessation of treatment with an inhibitor of mTOR.

[0044] In one embodiment, the compound is represented by the structure of Formula (III): In another embodiment, the compound is represented by the structure of Formula (IV): Each possibility represents a separate embodiment of the present invention.

[0045] In some embodiments, the tumor is present in a cancer patient with a tumor that has acquired resistance to mTOR inhibitor treatment. In other embodiments, the tumor is present in a cancer patient who is undergoing treatment with an mTOR inhibitor or is a candidate for such treatment. In other embodiments, the treatment results in attenuation or regression of the growth of the resistant tumor.

[0046] Any mTOR inhibitor known to those skilled in the art can be used in the combination of the present invention. In some embodiments, the mTOR inhibitor is selected from the group consisting of rapamycin (sirolimus), ridaforolimus (deforolimus, AP23573), NVP-BEZ235, everolimus (afinitor, RAD-001), temsirolimus (CCI-779), OSI-027, XL765, INK128, MLN0128, AZD2014, DS-3078a, and Palomid 529. In a currently preferred embodiment, the mTOR inhibitor is everolimus.

[0047] In one particular embodiment, the compound is represented by the structure of Formula D, and the mTOR inhibitor is everolimus (Afinitor).

[0048] In some embodiments, the subject or cancer patient is a human.

[0049] In another embodiment, it has been unexpectedly discovered that dual modulators of IRS and Stat3 can be used to sensitize tumors to immunotherapy. Stat3 is often activated in cancers and is known to be directly involved in establishing and maintaining a tumor immunosuppressive microenvironment, playing a central role in tumor immune evasion. Without being bound by any particular theory or mechanism of action, it is contemplated that inhibition of Stat3 phosphorylation by the compounds of the present invention exposes tumors to the local immune system and sensitizes them to immunotherapy, such as antibodies against PDL, PD1, CTLA4, or any other immunotherapeutic agent.

[0050] Thus, in one embodiment, the present invention relates to a method of sensitizing a tumor to immunotherapy, comprising contacting the tumor with a compound represented by the structure of Formula (III) or (IV) in combination with an immunotherapeutic agent.

[0051] In another embodiment, the present invention further relates to a pharmaceutical combination comprising a compound represented by the structure of Formula (III) or (IV) in combination with an immunotherapeutic agent.

[0052] In another embodiment, the present invention further relates to a combination comprising a compound of formula (III) or (IV) together with an immunotherapeutic agent for use in sensitizing tumors to immunotherapy.

[0053] In another embodiment, the present invention further relates to the use of a combination comprising a compound of formula (III) or (IV) together with an immunotherapeutic agent for the preparation of a medicament for the treatment of a tumor by sensitizing the tumor to immunotherapy.

[0054] In one embodiment, the compound is represented by the structure of Formula (III): In another embodiment, the compound is represented by the structure of Formula (IV): Each possibility represents a separate embodiment of the present invention.

[0055] In some embodiments, the immunotherapeutic agent used in combination with the compound is an antibody against a target selected from the group consisting of PDL, PD1, CTLA4, CD20, CD30, CD33, CD52, VEGF, CD30, EGFR, and ErbB2. In some aspects, the antibody is selected from the group consisting of alemtuzumab, bevacizumab, brentuximab vedotin, cetuximab, gemtuzumab ozogamicin, ibritumomab tiuxetan, ipilimumab, ofatumumab, panitumumab, rituximab, tositumomab, and trastuzumab. Each possibility represents a separate embodiment of the present invention.

[0056] In some embodiments, the tumor is present in a cancer patient who is undergoing or is a candidate for receiving immunotherapy.

[0057] In another aspect, it has now been unexpectedly discovered that a combination of a dual modulator of insulin receptor substrate (IRS) and signal transducer and activator of transcription 3 (Stat3) described herein with an inhibitor of mitogen-activated protein kinase (MEK) and / or a mutant B-Raf inhibitor provides at least an additive, and preferably a synergistic, therapeutic effect compared to the therapeutic effect of each agent alone. Furthermore, the combination can be used to treat tumors that have developed resistance to MEK inhibitors and / or mutant B-Raf inhibitors, and / or to prevent acquired resistance of tumors to MEK inhibitors and / or mutant B-Raf inhibitors, and / or to prevent or delay tumor recurrence after cessation of treatment with the MEK inhibitor and / or mutant B-Raf inhibitor.

[0058] Thus, in some embodiments, the present invention relates to a method of treating a tumor that has developed resistance to a mitogen-activated protein kinase (MEK) inhibitor and / or a mutant B-Raf inhibitor, comprising contacting the tumor with a MEK inhibitor and / or a mutant B-Raf inhibitor in combination with a compound represented by the structure of Formula (III) or (IV).

[0059] In another embodiment, the present invention relates to a method for preventing acquired resistance of a tumor to a MEK inhibitor and / or a mutant B-Raf inhibitor, comprising contacting the tumor with a MEK inhibitor and / or a mutant B-Raf inhibitor in combination with a compound represented by the structure of Formula (III) or (IV).

[0060] In another embodiment, the present invention relates to a method of preventing or delaying tumor recurrence after cessation of treatment with a MEK inhibitor and / or a mutant B-Raf inhibitor, comprising contacting the tumor with a MEK inhibitor and / or a mutant B-Raf inhibitor in combination with a compound represented by the structure of Formula (III) or (IV).

[0061] In other embodiments, the present invention relates to a pharmaceutical combination comprising a compound represented by the structure of Formula (III) in combination with a mitogen-activated protein kinase (MEK) inhibitor, and optionally, a mutant B-Raf inhibitor. In some embodiments, the combination comprises a compound of Formula (III), a MEK inhibitor, and a mutant B-Raf inhibitor, preferably, the MEK inhibitor is trametinib and the mutant B-Raf inhibitor is vemurafenib.

[0062] In another embodiment, the present invention relates to a compound represented by the structure of Formula (IV) in combination with a mitogen-activated protein kinase (MEK) inhibitor, and / or a mutant B-Raf inhibitor.

[0063] In some embodiments, the tumor is present in a cancer patient whose tumor has acquired resistance to MEK inhibitor and / or mutant B-Raf inhibitor treatment. In other embodiments, the treatment results in attenuation of growth or regression of the resistant tumor. In other embodiments, the tumor is present in a cancer patient who is undergoing treatment with a MEK inhibitor and / or mutant B-Raf inhibitor or is a candidate for such treatment.

[0064] Any MEK inhibitor known to those skilled in the art may be used in the combination of the present invention. In some embodiments, the MEK inhibitor is selected from the group consisting of trametinib (GSK1120212), selumetinib, binimetinib (MEK162), PD-325901, cobimetinib, CI-1040, and PD035901, and preferably, the MEK inhibitor is trametin.

[0065] Any mutant B-Raf inhibitor known to those skilled in the art can be used in the combination of the present invention. In some embodiments, the mutant B-Raf inhibitor is selected from the group consisting of vemurafenib (PLX-4032), PLX4720, sorafenib (BAY43-9006), and dabrafenib, and preferably, the mutant B-Raf inhibitor is vemurafenib.

[0066] In one embodiment, the compound is represented by the structure of Formula (III): In another embodiment, the compound is represented by the structure of Formula (IV): Each possibility represents a separate embodiment of the present invention.

[0067] In some embodiments, the compound is represented by the structure of Formula D, and the MEK inhibitor is trametinib.

[0068] In other embodiments, the compound is represented by the structure of Formula D, and the mutant B-Raf inhibitor is vemurafenib.

[0069] In some embodiments, the combination treatment comprises a compound of Formula (III) or (IV) and either a MEK inhibitor or a mutant B-Raf inhibitor, hi other embodiments, the combination treatment comprises a compound of Formula (III) or (IV) and both a MEK inhibitor and a mutant B-Raf inhibitor.

[0070] In some embodiments, the subject or cancer patient is a human.

[0071] In some embodiments, the present invention relates to a pharmaceutical combination comprising a compound represented by the structure of Formula D in combination with trametinib.

[0072] In another embodiment, the present invention relates to a pharmaceutical combination comprising a compound represented by the structure of Formula D in combination with trametinib and vemurafenib.

[0073] In other embodiments, the invention relates to the above combinations for use in treating tumors that are resistant to MEK inhibitors and / or mutant B-Raf inhibitors, or for preventing acquired resistance to MEK inhibitors and / or mutant B-Raf inhibitors, or for preventing or delaying tumor recurrence after cessation of treatment with a MEK inhibitor and / or mutant B-Raf inhibitor.

[0074] In another embodiment, the invention relates to the use of the above combination for the preparation of a medicament for the treatment of tumors that are resistant to MEK inhibitors and / or mutant B-Raf inhibitors, or for preventing acquired resistance to MEK inhibitors and / or mutant B-Raf inhibitors, or for preventing or delaying tumor recurrence after cessation of treatment with MEK inhibitors and / or mutant B-Raf inhibitors.

[0075] In another aspect, it has now been unexpectedly discovered that combinations of dual modulators of insulin receptor substrate (IRS) and signal transducer and activator of transcription 3 (Stat3) described herein with chemotherapeutic agents such as gemcitabine, 5-FU, irinotecan, oxaliplatin, and any combination thereof (e.g., Forfiri or Forfox for combination therapy) provide at least additive, and preferably synergistic, therapeutic effects compared to the therapeutic effects of each agent alone. Furthermore, the combinations can be used to treat tumors that have developed resistance to any of these chemotherapeutic agents or combinations thereof, and / or to prevent acquired resistance of tumors to any of these chemotherapeutic agents or combinations thereof, and / or to prevent or delay tumor recurrence after cessation of treatment with any of these chemotherapeutic agents or combinations thereof.

[0076] Forfili is a combination cancer treatment containing leucovorin (folinic acid), 5-FU, and irinotecan. Forfox is a combination cancer treatment containing leucovorin calcium (folinic acid), 5-FU, and oxaliplatin.

[0077] Thus, according to some embodiments, the present invention relates to a pharmaceutical combination comprising a compound represented by the structure of Formula (III) or (IV) and at least one chemotherapeutic agent selected from gemcitabine, 5-FU, irinotecan, oxaliplatin, and any combination thereof, wherein the compound and the chemotherapeutic agent(s) together provide a synergistic anti-cancer therapeutic effect.

[0078] In some embodiments, the present invention relates to a method of treating cancer, comprising administering to a subject in need thereof a therapeutically effective amount of a combination comprising a compound represented by the structure of Formula (III) or (IV) and at least one chemotherapeutic agent selected from gemcitabine, 5-FU, irinotecan, oxaliplatin, and any combination thereof, wherein the compound and the chemotherapeutic agent(s) together provide a synergistic anti-cancer therapeutic effect.

[0079] In other embodiments, the present invention provides a method of treating a tumor that has developed resistance to at least one chemotherapeutic agent, such as gemcitabine, 5-FU, irinotecan, oxaliplatin, and any combination thereof, comprising contacting the tumor with at least one of the chemotherapeutic agent(s) in combination with a compound represented by the structure of Formula (III) or (IV).

[0080] In other embodiments, the present invention provides a method of preventing acquired resistance of a tumor to at least one chemotherapeutic agent, such as gemcitabine, 5-FU, irinotecan, oxaliplatin, and any combination thereof, comprising contacting the tumor with at least one of the chemotherapeutic agent(s) in combination with a compound represented by the structure of Formula (III) or (IV).

[0081] In other embodiments, the present invention provides a method of preventing or delaying tumor recurrence after cessation of treatment with at least one chemotherapeutic agent, such as gemcitabine, 5-FU, irinotecan, oxaliplatin, and any combination thereof, comprising contacting the tumor with at least one of the chemotherapeutic agent(s) in combination with a compound represented by the structure of Formula (III) or (IV).

[0082] In some embodiments, the tumor is present in a cancer patient whose tumor has acquired resistance to any one or more of the chemotherapeutic agent(s). In other embodiments, the treatment results in attenuation of growth or regression of the resistant tumor. In other embodiments, the tumor is present in a cancer patient who is undergoing treatment with the chemotherapeutic agent(s) or who is a candidate for such treatment.

[0083] In other embodiments, the present invention provides a pharmaceutical combination comprising a compound represented by the structure of Formula (III) or (IV) and at least one chemotherapeutic agent selected from gemcitabine, 5-FU, irinotecan, oxaliplatin, and any combination thereof, for use in treating tumors that are resistant to any one or more of said chemotherapeutic agent(s), or for preventing acquired resistance to said chemotherapeutic agent(s), or for delaying tumor recurrence after cessation of treatment with any one or more of such chemotherapeutic agent(s).

[0084] In another embodiment, the present invention relates to the use of a pharmaceutical combination comprising a compound represented by the structure of Formula (III) or (IV) and at least one chemotherapeutic agent selected from gemcitabine, 5-FU, irinotecan, oxaliplatin, and any combination thereof, for the preparation of a medicament for the treatment of tumors that are resistant to said chemotherapeutic agent(s), or for preventing acquired resistance to said chemotherapeutic agent(s), or for preventing or delaying tumor recurrence after cessation of treatment with such chemotherapeutic agent(s).

[0085] As contemplated herein, the present invention provides several examples in which tumors with activated K-RAS that did not respond to anticancer drugs showed impressive tumor regression when the anticancer drug was combined with Compound D. For example, the combination of Compound D and an anticancer drug converted tumors from "non-responsive" to anticancer drugs to "responders." In Example 1 (FIG. 1), genomic analysis of the erlotinib-treated group, i.e., erlotinib-resistant clones, at endpoint revealed several changes compared to controls. Among these novel genomic alterations, KRAS is known to cause resistance to EGFR inhibitors. KRAS-related genomic alterations include KRAS amplification and NF-1 deletion, which leads to KRAS activation. In contrast to tumors treated with erlotinib, tumors treated with both erlotinib and Compound D did not have KRAS alterations. These tumors (treated with both erlotinib and Compound D) did not acquire resistance to erlotinib, and the tumors did not grow during treatment. Additionally, treatment of erlotinib-resistant tumors with Compound D plus erlotinib resensitized these tumors to erlotinib and induced tumor regression. This suggests that the inclusion of Compound D antagonizes resistance imposed by KRAS amplification and / or activation. In another example, gemcitabine-resistant pancreatic tumors with mutant KRAS (Figures 13A and 13B) were resensitized to gemcitabine treatment in combination with Compound D. Furthermore, supporting data from the literature indicates that "oncogene-addicted" cancer cells treated with many drugs engage in a positive feedback loop that leads to STAT3 activation, ultimately promoting cell survival and limiting overall drug response. This has been observed in cancer cells driven by a variety of activated kinases, including EGFR, HER2, ALK, and MET, as well as mutant KRAS.

[0086] Thus, in some embodiments, the present invention relates to a method of treating a tumor, comprising contacting a tumor that has developed resistance due to mutation and / or amplification of KRAS with a combination of an anti-cancer drug and a compound of Formula (III) or (IV). Any of the anti-cancer agents described herein (e.g., EGFR inhibitors / EGFR antibodies / mTOR inhibitors / immunotherapeutic agents / MEK inhibitors / mutant B-Raf inhibitors / chemotherapeutic agents / combinations of the foregoing) can be used in such methods by treating tumors that have developed resistance to such agents due to mutation and / or amplification of KRAS.

[0087] The compound of formula (III) has the structure [ka] (In the formula, R 1 , R 2 , R 5 and R 6 are independently H, C1-C4 alkyl, (CH2CH2O) n H (wherein n is an integer from 1 to 20), acyl, and a functional group that generates hydroxyl upon hydrolysis; R 3 , R 7 , R 8 , R 9 , R 10 , R 11 , R 12 , R 13 and R 14 are independently H, halogen, C1-C4 alkyl, C1-C4 haloalkyl, CH2SR a (In the formula, R a is selected from H, C1-C4 alkyl, aryl, heterocyclyl, heteroaryl, C1-C4 alkylaryl, C1-C4 alkylheterocyclyl, and C1-C4 alkylheteroaryl, and OR 16 (In the formula, R 16 is H, C1-C4 alkyl, (CH2CH2O) n H, acyl, or a functional group that generates hydroxyl upon hydrolysis; and R4 is H or CN) and includes salts, hydrates, solvates, polymorphs, optical isomers, geometric isomers, enantiomers, diastereomers, and mixtures thereof.

[0088] In some embodiments, the compound of Formula (III) is selected from compounds A, B, C, D, E, F, G, H, I, and J: [ka] TIFF0007624700000003.tif243165.

[0089] In one embodiment, the compound is a compound of formula A. In another embodiment, the compound is a compound of formula B. In another embodiment, the compound is a compound of formula C. In another embodiment, the compound is a compound of formula D. In another embodiment, the compound is a compound of formula E. In another embodiment, the compound is a compound of formula F. In another embodiment, the compound is a compound of formula G. In another embodiment, the compound is a compound of formula H. In another embodiment, the compound is a compound of formula I. In another embodiment, the compound is a compound of formula J. In a currently preferred embodiment, the compound is represented by the structure of formula D.

[0090] The compound of formula (IV) has the structure [ka] (In the formula, A is H or CN; Z is S, SO or SO; X 1 , X 2 , X 3 , X 4 , X 5 , Y 1 and Y 2 are each independently H, halogen, alkyl, haloalkyl, and OR 1 selected from; and Y3 and Y 4 are OR 1 (In the formula, each R 1 are independently H, C1-C4 alkyl, -(CH2CH2O) n H (wherein n is an integer of 1 to 20), acyl, or a functional group that generates hydroxyl upon hydrolysis). and includes salts, hydrates, solvates, polymorphs, optical isomers, geometric isomers, enantiomers, diastereomers, and mixtures thereof.

[0091] In some embodiments, the compound of formula (IV) has the formula (IV-4): [ka] It is represented by the structure:

[0092] It will further be apparent to one skilled in the art that any other compound of formula (I) or (IV) described herein can be used for any of the combination treatments described by this invention.

[0093] The combinations of the present invention are suitable for the treatment of various types of cancer. In particular, the combinations of the present invention are active against head and neck (H&N) cancer, sarcoma, multiple myeloma, ovarian cancer, breast cancer, renal cancer, gastric cancer, hematopoietic cancer, lymphoma, leukemia including lymphoblastic leukemia, lung cancer, melanoma, glioblastoma, liver cancer, prostate cancer and colon cancer. Each possibility represents a separate embodiment of the present invention.

[0094] As used herein, the term "combination" or "combination therapy" refers to any form of simultaneous or parallel treatment with at least two separate therapeutic agents. The term is intended to encompass both simultaneous administration of two treatment modalities, using substantially the same treatment schedule, and overlapping administration of each treatment on a sequential or alternating schedule. Each possibility represents a separate embodiment of the present invention.

[0095] Combination therapy is particularly advantageous because the dosage of each drug in the combination therapy can be reduced while still achieving an overall anti-cancer effect compared to monotherapy with each drug. Thus, reducing the dosage of each drug can result in reduced side effects. As demonstrated herein, combination therapy can reduce the development of resistance to a particular anti-cancer treatment and / or result in tumor regression after resistance has been acquired.

[0096] The compound of formula (III) or (IV) and the EGFR inhibitor / EGFR antibody / mTOR inhibitor / immunotherapeutic agent / MEK inhibitor / mutant B-Raf inhibitor / chemotherapeutic agent / combination thereof can be administered simultaneously (in the same dosage form or in separate dosage forms), or can be administered sequentially in any order. Administration can also be according to an alternating administration schedule, such as a compound of formula (III) or (IV) followed by additional agent(s), then an additional dose of the compound of formula (III) or (IV) followed by the same agent(s) or yet another agent(s). All administration schedules, including simultaneous, sequential, and alternating, are contemplated by the present invention, and each possibility represents a separate embodiment of the present invention.

[0097] The pharmaceutical compositions of the present invention can be provided in any form known in the art, for example, a form suitable for oral administration (e.g., solutions, suspensions, syrups, emulsions, dispersions, tablets, pills, capsules, pellets, granules, and powders), parenteral administration (e.g., intravenous, intramuscular, intraarterial, transdermal, subcutaneous, or intraperitoneal), topical administration (e.g., ointments, gels, creams), administration by inhalation, or administration via suppositories, each possibility representing a separate embodiment of the present invention.

[0098] Further embodiments and the full scope of applicability of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from the detailed description. [Brief explanation of the drawings]

[0099] [Figure 1] Compound D prevents acquired resistance to erlotinib in mice bearing tumors from head and neck (H&N) cancer patients. Mice were treated with (a) vehicle (◇); (b) erlotinib (□); (c) Compound D (Δ); or (d) erlotinib + Compound D (◯). Treatment began when the average tumor size reached approximately 80 mm3. Treatment with erlotinib induced significant tumor regression; however, during treatment, all erlotinib-treated mice developed resistance to erlotinib, and their tumors regrew aggressively. Combined treatment with erlotinib and Compound D induced tumor regression; none of them regrew during treatment. P value (vs. erlotinib) = 0.0001. [Figure 2] Compound D prevents acquired resistance to erlotinib and induces regression of erlotinib-resistant tumors in mice bearing tumors from head and neck (H&N) cancer patients. Mice were treated with (a) vehicle (◇); (b) erlotinib (□); (c) Compound D (Δ); or (d) erlotinib + Compound D (◯). Treatment with erlotinib (group b) initially resulted in tumor regression. During treatment, the tumors developed resistance to erlotinib and regrew. Combined treatment with erlotinib and Compound D induced tumor regression, and none of the tumors regrew (group d), consistent with the results shown in Figure 1. After acquiring resistance to erlotinib, mice in group b, whose tumors were approximately 130 mm3 on day 10, were divided into two groups: the first group received erlotinib alone (□), and the second group received a combination treatment of erlotinib plus compound D (treatment started on day 10) (●). Tumors grew significantly under treatment with erlotinib alone (□), but the combination treatment of compound D and erlotinib induced tumor regression (●). [Figure 3] Compound D prevents acquired resistance to erlotinib in large tumors (700 mm). Mice were treated with vehicle (⋄), erlotinib (□), Compound D (Δ), or erlotinib + Compound D (◯). Treatment began when the mean tumor size was approximately 700 mm. [Figure 4] Combination treatment with Afinitor and Compound D induces tumor regression of aggressive uterine adenosarcoma in mice. Mice bearing xenografts derived from patients with aggressive uterine adenosarcoma were treated with either vehicle (◇), Afinitor (□), Compound D (Δ), or Afinitor plus Compound D (◯) when the mean tumor size reached approximately 130 mm3. The mean tumor volume in the Afinitor-treated group (□) indicated tumor growth inhibition. Compared to controls, Compound D alone (Δ) had no significant effect on tumor growth, whereas combination treatment with everolimus plus Compound D (◯) resulted in tumor regression. Separate analysis of the results (demonstrated in Figure 5) showed that none of the mice responded to Compound D alone (Δ); in the Afinitor-treated group (□), half of the group responded and the other half did not; combination treatment with Afinitor plus Compound D (◯) resulted in a positive response in all mice in this group. [Figure 5]Compound D prevents acquired resistance to Afinitor (A) and leads to regression of Afinitor-resistant tumors (B). Mice bearing patient-derived xenografts of high-grade uterine adenosarcoma were initially treated as described in Figure 4 when tumors reached approximately 130 mm3. Mice were treated with either vehicle (diamonds); Afinitor (squares); Compound D (triangles); or Afinitor plus Compound D (circles). Figure 5A. Afinitor-treated groups were divided into responders (□, Group A, n = 8) and non-responders (gray squares, Group B, n = 7). Afinitor treatment in Group A initially induced tumor regression, but during treatment, all tumors developed resistance to Afinitor and grew aggressively. Combined treatment with Afinitor and Compound D from day 0 (treatment initiation) induced tumor regression, although their mean tumor volume remained low until the end of the experiment (circles). Figure 5B. After acquiring resistance to Afinitor, mice from Group A were divided into two groups: one group received Afinitor alone (□), and the second group received Afinitor plus Compound D combination treatment (treatment starting on day 6) (●). Tumors grew significantly under treatment with Afinitor alone (□), but combination treatment with Compound D and Afinitor induced tumor regression (●). The graph in Figure 5B represents the growth rate in percent; for each tumor, 100% was defined as its volume on day 6. Figure 5C. Combination treatment of Afinitor plus Compound D in high-grade uterine adenosarcoma cancer for which no medical treatment is available delayed acquired resistance to Afinitor, achieving complete remission in 40% of this group. [Figure 6]IRS / Stat3 dual modulators potently inhibit Stat3 phosphorylation in intact cells in a dose-dependent manner, and their inhibitory effect on Stat3 persists long after the modulators are washed out of the cells. A. Human melanoma A375 cells were treated with the indicated concentrations of Compound A or D for 1.3 and 4.5 hours. Cells were lysed and immunoblotted with antibodies against phosphorylated Y705 Stat3 (pSTAT3) and Stat3. Dose-response inhibition of pStat3 with submicromolar IC50 values ​​was demonstrated, and the effect increased over time. B. Cells were treated with 2 μM Compound A and lysed after the indicated times. C. A375 cells were treated with Compound D for 4 hours, then washed several times with medium and lysed after 4, 24, and 48 hours of incubation without inhibitor. D. Dose-dependent inhibition of pStat3 with IC50 values ​​of <1 μM for Compounds A, C, and D; 1 μM for Compound B; and 2 μM for Compound F. E. Complete inhibition of Stat3 Y705-phosphorylation in melanoma A375 cells at 24 hours after treatment with 3 μM of compounds IV-1, IV-2, IV-3 and IV-4. [Figure 7]Acquired resistance to BRAF inhibitors (BRAFi) in melanoma is associated with enhanced Stat3 phosphorylation levels, and treatment of human melanoma cells with BRAFi surprisingly induces a dramatic increase in pStat3. Figure 7A. Highly increased levels of pStat3 in BRAFi-resistant melanoma clones (R) compared with parental melanoma cells (P). Human metastatic melanoma 451-Lu cells (P) and PLX4032-resistant clones, both of which harbor mutant BRAF, were grown in serum-free medium and immunoblotted with anti-pStat3 Ab followed by anti-Stat3 Ab. Figure 7B. Highly increased levels of pStat3 in melanoma cells with mutant BRAF from a patient who had acquired resistance to BRAFi PLX4032 (R) compared with melanoma cells with mutant BRAF from a naive patient (N) who was not treated with PLX4032. Cells from the patient were grown in complete medium and immunoblotted as described above. Figure 7C-E. Treatment of BRAF-mutant human melanoma cells with PLX4032 surprisingly induces a dramatic increase in pStat3. Compounds A / D block both basal and PLX4032-induced pStat3. Treatment of PLX4032-sensitive human melanoma A375 (C), 451-Lu (D), or Mel1617 (E) cells with 1 μM PLX4032 for 18–24 h induces a dramatic increase in pStat3. Compounds A (Figure 7C, D) or D (Figure 7E) block both basal and PLX4032-induced pStat3. OSI-906, an ATP-competitive inhibitor of IGF1R, does not affect pStat3. [Figure 8]Dual IRS / Stat3 modulators efficiently inhibit pStat3 in BRAFi-resistant melanoma cells that have acquired resistance in culture or in patients. The ability to inhibit pStat3 was demonstrated in various cancer types. Figure 8A-B. Compound A, in contrast to the IGF1R inhibitor OSI-906, potently inhibits pStat3 in BRAFi-resistant melanoma clones, 451-BR (Figure 8A) and ME11617-BR (Figure 8B). Figure 8C. Compound A and the more potent compound D inhibit pStat3 in melanoma cells from a patient that acquired resistance to PLX4032 after treatment (i and ii). Figure 8D. Cells of various cancer types were treated with compound D for 4 hours in serum-free medium and for 20 hours in medium with or without 10% serum. The asterisk indicates the 10 μM concentration in RPMI8226 and HepG2 cells. [Figure 9] Treatment of melanoma A375 cells with Compound A induces PBMC chemotaxis. A375 cells were treated with the indicated concentrations of Compound A and washed twice with medium at 4 hours post-treatment (wash). At 30 hours post-treatment, the cell medium was transferred to the lower plate of the chemotaxis apparatus. 10,000 PBMCs per well were added to the upper plate. Additionally, PBMCs were added to the lower plate as a positive control. Figure 9A. PBMC chemotaxis toward Compound A was examined 24 hours later by cell titerglo analysis of the lower plate. Figure 9B. Standard curve of PBMCs at 10-10,000 cells per well. [Figure 10]Combination treatment with Compound D and cetuximab plus afatinib dramatically delayed tumor recurrence compared to cetuximab plus afatinib alone in mice bearing tumors from HNSCC patients. Mice were treated for 9 days with (a) vehicle (◇); (b) Compound D (Δ); (c) cetuximab (□); (d) cetuximab plus afatinib (▪); (e) cetuximab plus Compound D (◯); or (f) cetuximab + afatinib + Compound D (●). Treatment began when the average tumor size reached approximately 1103 mm. Combination treatment with either cetuximab or cetuximab plus afatinib with Compound D for only 9 days significantly delayed the recurrence of regressed tumors and extended the response to cetuximab or cetuximab plus afatinib. [Figure 11] Compound D synergizes with a combination of a mutant B-Raf inhibitor (BRAFi) and a MEK inhibitor (MEKi) to induce dramatic tumor regression in mice implanted with tumor cells from a melanoma patient who had acquired resistance to mutant B-RAF inhibitor treatment. Mice were treated with (a) vehicle (◇); (b) trametinib (MEKi) + vemurafenib (BRAFi) (□); (c) Compound D (Δ); or (d) trametinib + vemurafenib + Compound D (◯). Treatment began when the average tumor size reached approximately 60 mm3. Tumors grew aggressively in all mice treated with trametinib + vemurafenib, but combined treatment with trametinib + vemurafenib and Compound D induced tumor regression in all mice in this group, and none of them regrown during treatment. P value for trametinib + vemurafenib + compound D versus trametinib + vemurafenib = 0.0001. [Figure 12]Compound D synergizes with the MEK inhibitor trametinib to induce tumor regression in mice bearing tumors from patients with adenoid cystic carcinoma harboring a BRAF mutation. Mice were treated with (a) vehicle (◇); (b) trametinib (□); (c) Compound D (Δ); or (d) trametinib plus Compound D (◯). Treatment began when the average tumor size reached approximately 65 mm3. Treatment with trametinib induced tumor regression, but tumors continued to grow during treatment. Combination treatment with trametinib and Compound D induced tumor regression, and none of these tumors regrew during treatment. This study included two treatment phases: days 0-13 and 24-31. [Figure 13] Compound D resensitizes gemcitabine-resistant tumors to gemcitabine in mice transplanted with tumors from liver metastases of pancreatic cancer patients. A. Mice were treated with gemcitabine for 35 days, and one week later, the regressed tumors acquired resistance to gemcitabine and continued to grow. At this point, the average tumor size was already approximately 110 mm3, and the gemcitabine-treated mice were divided into two groups: (a) gemcitabine (□); (b) gemcitabine + Compound D (◯). While all tumors treated with gemcitabine grew, combined treatment with Compound D + gemcitabine resulted in tumor regression in half of this group, resulting in significant tumor growth inhibition in terms of average tumor size in this group compared to the gemcitabine-treated group (P value = 7.35 × 10-5). B. At the end of the experiment, tumor pieces of similar size were cultured in plates (3 tumors per group) and their viability and proliferative activity were tested. After 9 days, the plates were fixed and stained, showing extensive growth in the gemcitabine-treated tumors, in contrast to very low to negligible proliferative activity in tumors from mice treated with gemcitabine plus Compound D. [Figure 14]Compound D prevents acquired resistance to cetuximab in mice bearing tumors from patients with metastatic adnexal adenocarcinoma. Mice were treated for 32 days with (a) vehicle (◇); (b) Compound D (Δ); (c) cetuximab (□); or (d) cetuximab + Compound D (◯). Treatment began when the mean tumor size reached approximately 90 mm. Treatment with cetuximab resulted in a transient attenuation of tumor growth followed by acquired resistance to cetuximab, whereas combined treatment with cetuximab + Compound D induced tumor regression and prevented acquired resistance to cetuximab. [Figure 15] Compound D prevents acquired resistance to the combination of cetuximab and forfili (a treatment approved for colon cancer patients) in mice bearing tumors from colon cancer patients. Mice were treated for 14 days with (a) vehicle (◇); (b) Compound D (Δ); (c) cetuximab + forfili (□); (d) cetuximab + forfili + Compound D (◯); (e) forfili (▪); and (f) cetuximab (▪, dashed line). Treatment began when the average tumor size reached approximately 110 mm. Combination treatment of cetuximab + forfili, with or without Compound D, induced tumor regression during the first week of treatment. All tumors in mice treated with cetuximab + forfili developed resistance to this treatment and grew aggressively during the second week of treatment, but combination treatment with Compound D prevented acquired resistance to cetuximab + forfili. DETAILED DESCRIPTION OF THE INVENTION

[0100] The present invention relates to the treatment of cancer using a combination therapy comprising: (i) a protein kinase (PK) modulator selected from an epidermal growth factor inhibitor (EGFR inhibitor) and an EGFR antibody; (ii) an inhibitor of mammalian target of rapamycin (mTOR); (iii) a mitogen-activated protein kinase (MEK) inhibitor; (iv) a mutant B-Raf inhibitor; (v) a chemotherapeutic agent such as gemcitabine, 5-FU, irinotecan, and oxaliplatin; and (vi) a dual modulator of insulin receptor substrate (IRS) and signal transducer and activator of transcription 3 (Stat3) in combination with specific combinations thereof. The combination can be used to treat tumors that have developed resistance to EGFR inhibitors, EGFR antibodies, mTOR inhibitors, MEK inhibitors, mutant B-Raf inhibitors, chemotherapeutic agents, and specific combinations thereof, or to prevent acquired tumor resistance to any of the inhibitors or agents, or to prevent tumor recurrence after cessation of treatment with any of the inhibitors or agents, or combinations thereof. The combination provides at least additive, and preferably synergistic, therapeutic effects. The present invention further relates to the treatment of cancer using a combination therapy comprising a dual modulator of IRS and Stat3 in combination with an immunotherapeutic agent, which combination can be used to sensitize tumors to immunotherapy.

[0101] Insulin receptor substrate (IRS) / signal transducer and activator of transcription 3 (Stat3) dual modulator Formula (I) [ka] (In the formula, R 1 , R 2 , R 5 and R 6 are each independently H, C1-C4 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C4 alkyl-C2-C6 alkenyl, C1-C4 alkyl-C2-C6 alkynyl, (CH2CH2O) nselected from H, C3-C7 cycloalkyl, aryl, heterocyclyl, heteroaryl, (C1-C4)-alkylaryl, (C1-C4)-alkylheterocyclyl, (C1-C4)-alkylheteroaryl, haloalkyl, acyl and functional groups that generate hydroxyl upon hydrolysis; R 3 , R 4 , R 7 , R 8 , R 9 , R 10 , R 11 , R 12 , R 13 and R 14 are each independently H, C1-C4 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C4 alkyl-C2-C6 alkenyl, C1-C4 alkyl-C2-C6 alkynyl, C3-C7 cycloalkyl, aryl, heterocyclyl, heteroaryl, (C1-C4)-alkylaryl, (C1-C4)-alkylheterocyclyl, (C1-C4)-alkylheteroaryl, halogen, haloalkyl, NO2, CN, N3, SO2R a , COOR a , CSNR a R b , CSOR a , OR a ,CONR a R b , N.R. a R b , S.R. a , and CH2SR a wherein R a and R b are each independently H, C1-C4 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C4 alkyl-C2-C6 alkenyl, C1-C4 alkyl-C2-C6 alkynyl, C3-C7 cycloalkyl, aryl, heterocyclyl, heteroaryl, (C1-C4)-alkylaryl, (C1-C4)-alkylheterocyclyl, (C1-C4)-alkylheteroaryl, haloalkyl, (CH2CHO) n H, acyl, or a functional group that generates a hydroxyl group upon hydrolysis; R15 is H, C1-C4 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C4 alkyl-C2-C6 alkenyl, C1-C4 alkyl-C2-C6 alkynyl, haloalkyl, or OR b (In the formula, R b are independently H or C1-C4 alkyl Any compound of the general structure may be used in the compositions of the present invention, including its salts, hydrates, solvates, polymorphs, optical isomers, geometric isomers, enantiomers, diastereomers, and mixtures thereof, with each possibility representing a separate embodiment of the present invention.

[0102] In an exemplary embodiment, the compound has Formula A: [ka] It is a compound represented by:

[0103] In another exemplary embodiment, the compound has Formula B: [ka] It is a compound represented by:

[0104] In another exemplary embodiment, the compound has formula C: [ka] It is a compound represented by:

[0105] In another exemplary embodiment, the compound has formula D: [ka] It is a compound represented by:

[0106] In another exemplary embodiment, the compound has formula E: [ka] It is a compound represented by:

[0107] In another exemplary embodiment, the compound has formula F: [ka] It is a compound represented by:

[0108] In another exemplary embodiment, the compound has formula G: [ka] It is a compound represented by:

[0109] In another exemplary embodiment, the compound has formula H: [ka] It is a compound represented by:

[0110] In another exemplary embodiment, the compound has Formula I: [ka] It is a compound represented by:

[0111] In another exemplary embodiment, the compound has formula J: [ka] It is a compound represented by:

[0112] In another embodiment, the compound has formula I, where R 1 , R 2 , R 4 , R 5 , R 6 , R 10 , R 12 , R 13 , R 14 and R 15 are H and R, respectively. 7 is OH; and R3 , R 8 , R 9 and R 11 at least one of which is a halogen.

[0113] In another embodiment, the compound has formula I, where R 1 , R 2 , R 4 , R 5 , R 6 , R 8 , R 10 , R 12 , R 13 , R 14 and R 15 are H and R, respectively. 7 is OH; and R 3 , R 9 and R 11 at least one of which is a halogen.

[0114] In another embodiment, the compound has formula I, where R 1 , R 2 , R 5 and R 6 are functional groups that yield H or hydroxyl upon hydrolysis, respectively).

[0115] In another embodiment, the compound has formula I, where R 7 is H or OR a and R 1 , R 2 , R 5 , R 6 , and R a are functional groups that yield H or hydroxyl upon hydrolysis, respectively).

[0116] In another embodiment, the compound has formula I, where R 13 and R 14 are each independently H, C1-C4 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C4 alkyl-C2-C6 alkenyl, or C1-C4 alkyl-C2-C6 alkynyl).

[0117] In another embodiment, the compound has formula I, where R 13 and R 14 at least one of which is H or C1-C4 alkyl.

[0118] In another embodiment, the compound has formula I, where R 3 , R 4 , R 7 , R 8 , R 9 , R 10 , R 11 , R 12 , R 13 and R 14 are each independently H, halogen, haloalkyl, OH, NO, CN, or CHSR a (In the formula, R a is a compound of formula (I) where

[0119] In another embodiment, the compound has formula I, where R 4 is H).

[0120] In another embodiment, the compound has formula I, where R 4 is CN).

[0121] In another embodiment, the compound has formula I, where R 4 , R 11 , R 12 , R 13 , R 14 and R 15 is a compound of

[0122] In another embodiment, the compound has formula I, where R 13 , R 14 and R 15 is a compound of

[0123] In another embodiment, the compound has formula I, where R 3 , R 7 , R8 , R 9 , R 10 and R 11 are each independently H, halogen, haloalkyl, or CHSR a or OH; R 4 , R 12 , R 13 and R 14 are each independently H, C1-C4 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C4 alkyl-C2-C6 alkenyl, C1-C4 alkyl-C2-C6 alkynyl, aryl, halogen, haloalkyl, NO, or CN; and R 15 is H).

[0124] In another embodiment, the compound has formula I, where R 3 , R 7 , R 8 , R 9 , R 10 and R 11 are each independently H, halogen, haloalkyl, OH, or CHSR a and R 4 , R 12 , R 13 , R 14 and R 15 are each H or C1-C4 alkyl).

[0125] In another embodiment, the compound has formula I, where R 1 , R 2 , R 5 and R 6 are H or a functional group that generates a hydroxyl group upon hydrolysis; R 3 , R 8 , and R 9 are each independently H, halogen, haloalkyl, or CHSR a and;R 7 , R 10 and R 11 are each independently H, halogen, haloalkyl, OH, or a functional group that generates hydroxyl upon hydrolysis; and R 4 , R12 , R 13 , R 14 and R 15 are each H or C1-C4 alkyl).

[0126] In another embodiment, the compound is a compound of formula I, wherein the compound is represented by any one of the following structures: [ka] TIFF0007624700000018.tif251163TIFF0007624700000019.tif154162

[0127] Each possibility represents a separate embodiment of the present invention.

[0128] In other embodiments, the compound has Formula II: [ka] (In the formula, R 1 , R 2 , R 5 and R 6 are independently selected from H, C1-C4 alkyl, acyl, and a functional group that generates hydroxyl upon hydrolysis; R 3 and R 7 are independently H, halogen, haloalkyl, and OR 8 (In the formula, R 8 is selected from H, C1-C4 alkyl, acyl, or a functional group that generates hydroxyl upon hydrolysis; and R 4 is H or CN) and includes salts, hydrates, solvates, polymorphs, optical isomers, geometric isomers, enantiomers, diastereomers, and mixtures thereof.

[0129] In other embodiments, the compound has formula II, where R 1 , R 2 , R5 and R 6 are independently H, C1-C4 alkyl, (CH2CH2O) n H (wherein n is an integer from 1 to 20), acyl, and a functional group that generates hydroxyl upon hydrolysis; R 3 and R 7 are independently H, halogen, C1-C4 alkyl, haloalkyl, and OR 16 (In the formula, R 16 is H, C1-C4 alkyl, (CH2CH2O) n H, acyl, or a functional group that generates hydroxyl upon hydrolysis; and R 4 is H or CN) and includes salts, hydrates, solvates, polymorphs, optical isomers, geometric isomers, enantiomers, diastereomers, and mixtures thereof.

[0130] Each possibility represents a separate embodiment of the present invention.

[0131] In another embodiment, the compound has formula II, wherein R 4 is CN).

[0132] In other embodiments, the compound has formula II, where R 1 , R 2 , R 5 and R 6 are hydrogen atoms.

[0133] In other embodiments, the compound has formula II, where R 1 , R 2 , R 5 and R 6 are compounds of the formula (CH3, respectively).

[0134] In other embodiments, the compound has formula II, where R 3 and R 7 are hydrogen, halogen, halomethyl, OH, or OCH3, respectively.

[0135] In other embodiments, the compound has formula II, where R 1 , R 2 , R 5 and R 6 are H and R, respectively. 3 is a halogen and R 7 is OH).

[0136] In other embodiments, the compound has formula II, where R 1 , R 2 , R 5 and R 6 are H and R, respectively. 3 and R 7 are each halogens).

[0137] In other embodiments, the compound has formula II, where R 1 , R 2 , R 5 and R 6 are H and R, respectively. 3 is halomethyl and R 7 is OH).

[0138] In other embodiments, the compound has formula II, where R 1 , R 2 , R 5 and R 6 are H and R, respectively. 3 is a halogen and R 7 is H).

[0139] In other embodiments, the compound has formula II, where R 1 , R 2 , R 5 and R 6 are H and R, respectively. 3 is OH and R 7 is a halogen).

[0140] In other embodiments, the compound has formula II, where R1 , R 2 , R 5 and R 6 are CH3 and R 3 is a halogen and R 7 is OCH3).

[0141] In other embodiments, the compound has formula II, where R 1 , R 2 , R 5 and R 6 are CH3 and R 3 and R 7 are halogens).

[0142] In other embodiments, the compound has formula II, where R 4 is hydrogen).

[0143] In other embodiments, the compound has formula II, where R 1 , R 2 , R 5 and R 6 are hydrogen atoms.

[0144] In other embodiments, the compound has formula II, where R 1 , R 2 , R 5 and R 6 are compounds of the formula (CH3, respectively).

[0145] In other embodiments, the compound has formula II, where R 3 and R 7 are hydrogen, halogen, halomethyl, OH, or OCH3, respectively.

[0146] In other embodiments, the compound has formula II, where R 1 , R 2 , R 5 and R 6 are H and R, respectively. 3 is a halogen and R 7is OH).

[0147] In other embodiments, the compound has formula II, where R 1 , R 2 , R 5 and R 6 are H and R, respectively. 3 and R 7 are each halogens).

[0148] In other embodiments, the compound has formula II, where R 1 , R 2 , R 5 and R 6 are H and R, respectively. 3 is halomethyl, R 7 is OH).

[0149] In other embodiments, the compound has formula II, where R 1 , R 2 , R 5 and R 6 are H and R, respectively. 3 is a halogen and R 7 is H).

[0150] In other embodiments, the compound has formula II, where R 1 , R 2 , R 5 and R 6 are H and R, respectively. 3 is OH and R 7 is a halogen).

[0151] In other embodiments, the compound has formula II, where R 3 is a halogen and R 7 is OCH3).

[0152] In other embodiments, the compound has formula II, where R 1 , R 2 , R 5 and R 6are CH3 and R 3 and R 7 are each halogens).

[0153] In other embodiments, the compound of formula (II) is represented by any of the following compounds: [ka] TIFF0007624700000022.tif233163 TIFF0007624700000023.tif52159

[0154] Each possibility represents a separate embodiment of the present invention. In another embodiment, the compound has formula III: [ka] (In the formula, R 1 , R 2 , R 5 and R 6 are independently H, C1-C4 alkyl, (CH2CH2O) n H (wherein n is an integer from 1 to 20), acyl, and a functional group that generates hydroxyl upon hydrolysis; R 3 , R 7 , R 8 , R 9 , R 10 , R 11 , R 12 , R 13 and R 14 are independently H, halogen, C1-C4 alkyl, haloalkyl, and OR 16 (In the formula, R 16 is H, C1-C4 alkyl, (CH2CH2O) n H, acyl, or a functional group that generates hydroxyl upon hydrolysis; and R 4 is H or CN) It is represented by the structure:

[0155] In other embodiments, the compound has Formula IV: [ka] (In the formula, A is H or CN; Z is S, SO or SO; X 1 , X 2 , X 3 , X 4 , X 5 , Y 1 and Y 2 are each independently H, halogen, alkyl, haloalkyl, and OR 1 selected from; and Y 3 and Y 4 are OR 1 where each R 1 are independently H, C1-C4 alkyl, -(CH2CH2O) n H (wherein n is an integer from 1 to 20), acyl, or a functional group that generates hydroxyl upon hydrolysis) and includes salts, hydrates, solvates, polymorphs, optical isomers, geometric isomers, enantiomers, diastereomers, and mixtures thereof.

[0156] In some embodiments, the compound is of formula IV, wherein A is H.

[0157] In other embodiments, the compound is of formula IV where A is CN.

[0158] In other embodiments, the compound is of formula IV where Z is S.

[0159] In other embodiments, the compound is of formula IV where Z is SO2.

[0160] In other embodiments, the compound is X 1 , X 2 , X 3 , X 4 , Y1 and Y 2 is a compound of formula IV, wherein at least one of

[0161] In other embodiments, the compound is X 1 , X 2 , X 3 , X 4 , Y 1 and Y 2 is a compound of formula IV, wherein at least one of

[0162] In other embodiments, the compound is X 1 , X 2 , X 3 , X 4 , Y 1 and Y 2 wherein at least one of

[0163] In other embodiments, the compound is X 1 , X 2 , X 3 , and X 4 are each selected from H or a halogen, the halogen preferably being Br or I.

[0164] In other embodiments, the compound is X 2 is a compound of formula IV where

[0165] In other embodiments, the compound is X 5 is a compound of formula IV where

[0166] In other embodiments, the compound is X 5 is an alkyl, preferably methyl.

[0167] In other embodiments, the compound is Y 3 and Y 4 are each OH.

[0168] In other embodiments, the compound is Y 1 and Y 2 are each OH.

[0169] In other embodiments, the compound is 3 and Y 4 are OH and X 1 is a halogen selected from Br and I.

[0170] Each possibility represents a separate embodiment of the present invention.

[0171] In other embodiments, the compound of formula (IV) is represented by any of the following compounds: [ka] TIFF0007624700000027.tif201161

[0172] A currently preferred compound of formula IV is the compound of formula IV-4.

[0173] In other embodiments, the compound is any of the derivatives described in A) PCT International Patent Application Publication No. WO2008 / 068751; B) PCT International Patent Application Publication No. WO2009 / 147682; or C) PCT International Patent Application No. WO2012 / 090204. The contents of each of the foregoing references are incorporated herein by reference in their entirety as if fully set forth herein.

[0174] It is understood that all conformational isomers, geometric isomers, stereoisomers, enantiomers and diastereomers of any of the compounds described herein are encompassed and may be used in the combinations and methods described by the present application.

[0175] Without being bound by any particular theory or mechanism of action, it is contemplated that the compounds of the present invention are inhibitors of PK signal transduction, such as IGF-1R.Surprisingly, it has now been found that these compounds, in addition to being inhibitors of IGF-1R, also cause the dissociation of IGF-1R substrate IRS1 / 2 from the cell membrane, the inhibitory serine phosphorylation and / or degradation of IRS1 / 2 proteins.This activity leads to long-term inhibition of the IGF-1R and IR pathways, growth inhibition of a wide range of cancer cell types, and potent anti-tumor effects.These compounds are therefore referred to as "IRS modulators." Thus, in another embodiment, the present invention provides a method for suppressing, treating, or preventing a disorder associated with insulin-like growth factor 1 receptor (IGF-1R) and / or insulin receptor substrate 1 (IRS1) and / or insulin receptor substrate 2 (IRS2) signaling in a subject, comprising administering to the subject a pharmaceutical composition comprising a therapeutically effective amount of at least one compound represented by the structure of Formula I or any of the compounds encompassed by such formula, together with an anti-cancer agent selected from an EGFR inhibitor, an EGFR antibody, an mTOR inhibitor, and / or an immunotherapeutic agent, wherein the compound of Formula I and the anti-cancer agent together provide at least an additive, preferably a synergistic, anti-cancer effect. In some embodiments, the compound of Formula I is an inhibitor of insulin receptor or insulin-like growth factor 1 receptor (IGF-1R) signaling and / or the compound of Formula I interacts with, affects, or inhibits a substrate protein in an IGF-1R-mediated pathway. In some embodiments, the substrate protein is insulin receptor substrate 1 (IRS1), insulin receptor substrate 2 (IRS2), or a combination thereof. In one particular embodiment, the compound of formula I is an IGF-1R kinase inhibitor that leads to at least one of, in any order, dissociation of IRS1 or IRS2 from the cell membrane, phosphorylation of IRS1 or IRS2, and / or degradation of IRS1 or IRS2.

[0176] IGF1R and specifically IRS1 are one of the key mechanisms of resistance to EGFR inhibition (Buck E. et al., Feedback mechanisms promote cooperativity for small molecule inhibitors of epidermal and insulin-like growth factor receptors, Cancer Res. 2008 Oct 15;68(20):8322-32).

[0177] The compounds described herein are also modulators of signal transducer and activator of transcription 3 (Stat3). In some embodiments, the compounds inhibit Stat3 phosphorylation in cancer cells. Increased levels of Stat3 phosphorylation have been detected in various cancers and drug-resistant cancers, resulting in enhanced cancer survival. Without being bound by any particular theory or mechanism of action, it is contemplated that inhibition of Stat3 activity may synergize with PK inhibitors that upregulate Stat3 as a side effect, preventing acquired resistance to such drugs and being effective against drug-resistant cancers. Furthermore, Stat3 is often activated in cancers and is directly involved in establishing and maintaining a cancer immunosuppressive microenvironment, playing a central role in tumor immune evasion. Without being bound by any particular theory or mechanism of action, it is contemplated that inhibition of Stat3 phosphorylation exposes tumors from the local immune system and sensitizes them to immunotherapy, such as antibodies against PDL, PD1, CTLA4, or any other immunotherapeutic agent.

[0178] chemical definition : An "alkyl" group refers to any saturated aliphatic hydrocarbon, including straight-chain and branched-chain alkyl groups. In one embodiment, the alkyl group is a C1-C 12-having 1 to 12 carbons, designated herein as alkyl. In another embodiment, an alkyl group has 1 to 6 carbons, designated herein as C1-C6-alkyl. In another embodiment, an alkyl group has 1 to 4 carbons, designated herein as C1-C4-alkyl. Alkyl groups can be unsubstituted or substituted with one or more groups selected from halogen, hydroxy, alkoxycarbonyl, amido, alkylamido, dialkylamido, nitro, amino, alkylamino, dialkylamino, carboxyl, thio, and thioalkyl.

[0179] An "alkenyl" group refers to an aliphatic hydrocarbon group containing at least one carbon-carbon double bond, including straight-chain, branched-chain, and cyclic alkenyl groups. In one embodiment, an alkenyl group has 2 to 8 carbon atoms, designated herein as C2-C8-alkenyl. In another embodiment, an alkenyl group has 2 to 6 carbon atoms in the chain, designated herein as C2-C6-alkenyl. Exemplary alkenyl groups include ethenyl, propenyl, n-butenyl, i-butenyl, 3-methylbut-2-enyl, n-pentenyl, heptenyl, octenyl, cyclohexyl-butenyl, and decenyl. Alkenyl groups can be unsubstituted or substituted through available carbon atoms with one or more groups as defined above for alkyl.

[0180] An "alkynyl" group refers to an aliphatic hydrocarbon group containing at least one carbon-carbon triple bond, including straight and branched chains. In one embodiment, an alkynyl group has 2 to 8 carbon atoms in the chain, designated herein as C2-C8-alkynyl. In another embodiment, an alkynyl group has 2 to 6 carbon atoms in the chain, designated herein as C2-C6-alkynyl. Exemplary alkynyl groups include ethynyl, propynyl, n-butynyl, 2-butynyl, 3-methylbutynyl, n-pentynyl, heptynyl, octynyl, and decynyl. Alkynyl groups can be unsubstituted or substituted through available carbon atoms with one or more groups as defined above for alkyl.

[0181] The term "C3-C7 cycloalkyl," as used herein alone or as part of another group, refers to any saturated or unsaturated (e.g., cycloalkenyl, cycloalkynyl) monocyclic or polycyclic group. Non-limiting examples of cycloalkyl groups are cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, or cycloheptyl. Non-limiting examples of cycloalkenyl groups include cyclopentenyl and cyclohexenyl. Cycloalkyl groups can be unsubstituted or substituted with any one or more of the substituents defined above for alkyl. Similarly, the term "cycloalkylene" refers to a divalent cycloalkyl, as defined above, in which the cycloalkyl group is bonded at two positions, linking two separate additional groups together.

[0182] The term "aryl," as used herein alone or as part of another group, refers to an aromatic ring system containing 6 to 14 ring carbon atoms. Aryl rings can be monocyclic, bicyclic, tricyclic, and the like. Non-limiting examples of aryl groups include phenyl and naphthyl, including 1-naphthyl and 2-naphthyl. Aryl groups can be unsubstituted or substituted through available carbon atoms with one or more groups as defined above for alkyl.

[0183] The term "heteroaryl," as used herein alone or as part of another group, refers to a heterocyclic aromatic system containing at least one heteroatom ring atom selected from nitrogen, sulfur, and oxygen. Heteroaryls contain five or more ring atoms. Heteroaryl groups can be monocyclic, bicyclic, tricyclic, and the like. Benzoheterocycles are also included in this definition. When nitrogen is a ring atom, the present invention also contemplates the N-oxide of the nitrogen-containing heteroaryl. Non-limiting examples of heteroaryl include thienyl, benzothienyl, 1-naphthothienyl, thianthrenyl, furyl, benzofuryl, pyrrolyl, imidazolyl, pyrazolyl, pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, indolyl, isoindolyl, indazolyl, purinyl, isoquinolyl, quinolyl, naphthyridinyl, quinoxalinyl, quinazolinyl, cinnolinyl, pteridinyl, carbolinyl, thiazolyl, oxazolyl, isothiazolyl, and isoxazolyl, etc. Heteroaryl groups can be unsubstituted or substituted through available atoms with one or more groups as defined above for alkyl.

[0184] The terms "heterocycle" or "heterocyclyl," as used herein alone or as part of another group, refer to a 5- to 8-membered ring having 1 to 4 heteroatoms, such as oxygen, sulfur, and / or nitrogen, especially nitrogen, either alone or linked to sulfur or oxygen ring atoms. These 5- to 8-membered rings can be saturated, fully unsaturated, or partially unsaturated, with fully saturated rings being preferred. Preferred heterocycles include piperidinyl, pyrrolidinyl, pyrrolinyl, pyrazolinyl, pyrazolidinyl, morpholinyl, thiomorpholinyl, pyranyl, thiopyranyl, piperazinyl, indolinyl, dihydrofuranyl, tetrahydrofuranyl, dihydrothiophenyl, tetrahydrothiophenyl, dihydropyranyl, tetrahydropyranyl, and dihydrothiazolyl. Heterocyclyl groups can be unsubstituted or substituted through available atoms with one or more groups as defined above for alkyl.

[0185] As used herein, the term "acyl" includes, but is not limited to, groups such as formyl, acetyl, propionyl, butyryl, pentanoyl, pivaloyl, hexanoyl, heptanoyl, octanoyl, nonanoyl, decanoyl, undecanoyl, dodecanoyl, and benzoyl. Presently preferred acyl groups are acetyl and benzoyl.

[0186] A "hydroxy" group refers to an OH group. An "alkoxy" group refers to an -O-alkyl group where R is alkyl, as defined above.

[0187] A "thio" group refers to an -SH group. An "alkylthio" group refers to an -SR group where R is alkyl, as defined above.

[0188] An "amino" group refers to an NH group. An alkylamino group refers to an -NHR group where R is alkyl, as defined above. A dialkylamino group refers to an -NRR' group where R and R' are alkyl, as defined above.

[0189] An "amido" group refers to a -C(O)NH group. An alkylamido group refers to a -C(O)NHR group where R is alkyl, as defined above. A dialkylamido group refers to a -C(O)NRR group where R and R are alkyl, as defined above.

[0190] A "thioamide" group refers to a -C(S)NHR group, where R is either alkyl, aryl, alkylaryl, or H.

[0191] A "polyoxyalkylene" group is (CH2CH2O) n H group, where n=1 to 20. Presently preferred polyoxyalkylene groups are polyethylene glycol (PEG) and polypropylene glycol.

[0192] The terms "halogen" or "halo," as used herein alone or as part of another group, refer to chlorine, bromine, fluorine, and iodine. The term "haloalkyl" refers to an alkyl group having some or all of its hydrogen atoms independently replaced with halogen groups, such as, but not limited to, trichloromethyl, tribromomethyl, trifluoromethyl, triiodomethyl, difluoromethyl, chlorodifluoromethyl, pentafluoroethyl, 1,1-difluoroethylbromomethyl, chloromethyl, fluoromethyl, and iodomethyl.

[0193] Examples of functional groups that yield hydroxyl upon hydrolysis include, but are not limited to, esters, anhydrides, carbamates, and carbonates. For example, R 1 , R 2 , R 5 or R 6 If either of R is an acyl group (COR), the resulting functional group is an ester (OCOR). 1 , R 2 , R 5 or R 6 If either of R is an amide group (CONHR), the resulting functional group is a carbamate (OCONHR). 1 , R 2 , R 5 or R 6 is a carboxylate group (COOR), the resulting functional group is a carbonate (OCOOR).

[0194] Prodrugs of the compounds disclosed herein are within the scope of the present invention. The term "prodrug" refers to a compound that is rapidly converted in vivo to any of the compounds represented by Formula I, for example, by hydrolysis in blood. Thus, the term "prodrug" refers to any pharmaceutically acceptable precursor of the compounds of the present invention. A prodrug may be inactive when administered to a subject, but is converted in vivo to an active compound. The use of a prodrug is particularly advantageous for facilitating administration of a compound. Prodrug compounds often offer advantages of solubility, tissue compatibility, or delayed release in mammalian organisms. For example, such prodrugs, in accordance with the principles of the present invention, can be prepared by administering a compound of Formula I (wherein R 1 , R 2 , R 5 and R 6 is a functional group that yields a hydroxyl upon hydrolysis, as defined above).

[0195] All stereoisomers of the compounds are contemplated, either in mixture or in pure or substantially pure form. The compounds may have asymmetric centers at any of the atoms. Consequently, the compounds can exist in enantiomeric or diastereomeric forms, or mixtures thereof. The present invention contemplates the use of any racemate (i.e., a mixture containing equal amounts of each enantiomer), an enantiomerically enriched mixture (i.e., a mixture enriched in one enantiomer), a pure enantiomer or diastereomer, or any mixture thereof. Chiral centers can be designated as R or S or R,S or d,D,l,L or d,l,D,L. Compounds containing amino acid residues include residues of D-amino acids, L-amino acids, or racemic derivatives of amino acids. Compounds containing sugar residues include residues of D-sugars, L-sugars, or racemic derivatives of sugars. Residues of naturally occurring D-sugars are preferred. Additionally, some of the compounds of the present invention contain one or more double bonds. The present invention is intended to encompass all structural and geometric isomers, including cis, trans, E and Z isomers, independently at each occurrence.

[0196] One or more of the compounds of the present invention may exist as a salt. The term "salt" encompasses both base and acid addition salts, including, but not limited to, salts with carboxylate or amine nitrogens, and includes salts formed with the organic and inorganic anions and cations described below. Furthermore, the term includes salts formed by standard acid-base reactions of basic groups (such as amino groups) with organic or inorganic acids. Such acids include hydrochloric acid, hydrofluoric acid, trifluoroacetic acid, sulfuric acid, phosphoric acid, acetic acid, succinic acid, citric acid, lactic acid, maleic acid, fumaric acid, palmitic acid, cholic acid, pamoic acid, mucic acid, D-glutamic acid, D-camphoric acid, glutaric acid, phthalic acid, tartaric acid, lauric acid, stearic acid, salicylic acid, methanesulfonic acid, benzenesulfonic acid, sorbic acid, picric acid, benzoic acid, and cinnamic acid, among others. Each possibility represents a separate embodiment of the present invention.

[0197] The term "organic or inorganic cation" refers to a counterion of the anion of a salt. Counterions include, but are not limited to, alkali metals and alkaline earth metals (such as lithium, sodium, potassium, barium, aluminum, and calcium), ammonium, and organic cations such as monoalkylamines, dialkylamines, and trialkylamines such as trimethylamine, cyclohexylamine; and cations such as dibenzylammonium, benzylammonium, 2-hydroxyethylammonium, bis(2-hydroxyethyl)ammonium, phenylethylbenzylammonium, and dibenzylethylenediammonium. See, for example, Berge et al., J. Pharm. Sci. (1977), 66:1-19, which is incorporated herein by reference.

[0198] The present invention also includes solvates of the compounds of the present invention and their salts. "Solvate" refers to a physical association of a compound of the present invention with one or more solvent molecules. This physical association involves varying degrees of ionic and covalent bonding, including hydrogen bonding. In certain instances, the solvate can be isolated. "Solvate" encompasses both solution-phase and isolatable solvates. Non-limiting examples of suitable solvates include ethanolates and methanolates. "Hydrate" is a solvate in which the solvent molecule is water.

[0199] The present invention also includes polymorphs of the compounds of the present invention and their salts. The term "polymorph" refers to a particular crystalline or amorphous state of a substance that can be characterized by particular physical properties such as X-ray diffraction, IR or Raman spectra, and melting point.

[0200] Dual IRS / Stat3 modulators combined with EGFR inhibitors / antibodies In one embodiment, the present invention relates to a method of treating a tumor that has developed resistance to an epidermal growth factor receptor (EGFR) inhibitor and / or an EGFR antibody, comprising the step of contacting the tumor with an EGFR inhibitor and / or an EGFR antibody in combination with a compound of any of Formulas (I), (II), (III), or (IV), or any of the individual compounds within such formulas.

[0201] In another embodiment, the present invention relates to a method for preventing acquired resistance of a tumor to an epidermal growth factor receptor (EGFR) inhibitor and / or an EGFR antibody, comprising the step of contacting the tumor with an EGFR inhibitor and / or an EGFR antibody in combination with a compound of any of formulas (I), (II), (III), or (IV), or any of the individual compounds included in such formulas.

[0202] In another embodiment, the present invention relates to a pharmaceutical combination comprising a compound represented by the structure of any of Formulas (I), (II), (III), (IV), or any of the individual compounds included in such formulas, in combination with an epidermal growth factor (EGFR) inhibitor and / or an EGFR antibody.

[0203] In another embodiment, the present invention further relates to the above combination for use in treating tumors that are resistant to EGFR inhibitors and / or EGFR antibodies or for preventing acquired resistance to EGFR inhibitors and / or EGFR antibodies.

[0204] In another embodiment, the present invention further relates to the use of the above combination for the preparation of a medicament for the treatment of tumors that are resistant to EGFR inhibitors and / or EGFR antibodies or for preventing acquired resistance to EGFR inhibitors and / or EGFR antibodies.

[0205] In some embodiments, the tumor is present in a cancer patient whose tumor has acquired resistance to EGFR inhibitor and / or EGFR antibody treatment. In other embodiments, the treatment results in attenuation of growth or regression of the resistant tumor. In other embodiments, the tumor is present in a cancer patient who is undergoing treatment with an EGFR inhibitor and / or EGFR antibody or is a candidate for such treatment.

[0206] Any EGFR inhibitor or antibody known to those skilled in the art may be used in the combinations of the present invention. In some embodiments, the EGFR inhibitor is selected from the group consisting of erlotinib, gefitinib, lapatinib, vandetanib, neratinib, icotinib, afatinib, dacomitinib, poziotinib, AZD9291, CO-1686, HM61713, and AP26113. In a currently preferred embodiment, the EGFR inhibitor is erlotinib. In a specific embodiment, the compound is represented by the structure of Formula D, and the EGFR inhibitor is erlotinib. Each possibility represents a separate embodiment of the present invention.

[0207] In some embodiments, the EGFR antibody is selected from the group consisting of trastuzumab, necitumumab, cetuximab, and panitumumab. Each possibility represents a separate embodiment of the present invention.

[0208] In one embodiment, the compound is a compound of formula A. In another embodiment, the compound is a compound of formula B. In another embodiment, the compound is a compound of formula C. In another embodiment, the compound is a compound of formula D. In another embodiment, the compound is a compound of formula E. In another embodiment, the compound is a compound of formula F. In another embodiment, the compound is a compound of formula G. In another embodiment, the compound is a compound of formula H. In another embodiment, the compound is a compound of formula I. In another embodiment, the compound is a compound of formula J. In another embodiment, the compound is a compound of formula IV-4. In a currently preferred embodiment, the compound is represented by the structure of formula D. However, it will be apparent to one of skill in the art that any of the compounds described herein may be used in the combinations of the present invention.

[0209] Combining a dual IRS / Stat3 modulator with an mTOR inhibitor In a further aspect of the present invention, it has now been unexpectedly discovered that a combination of an IRS / Stat3 dual modulator having any of formulas (I), (II), (III), or (IV) described herein, or any individual compound within such formulae, with an inhibitor of mammalian target of rapamycin (mTOR) provides at least an additive, and preferably a synergistic, therapeutic effect compared to the therapeutic effect of each agent alone. Furthermore, the combination can be used to treat tumors that have developed resistance to mTOR inhibitors and / or to prevent acquired tumor resistance to mTOR inhibitors.

[0210] Thus, in one embodiment, the present invention relates to a pharmaceutical combination comprising a compound represented by the structure of any of Formulas (I), (II), (III), (IV), or any individual compound within such formulae, and at least one inhibitor of mammalian target of rapamycin (mTOR), wherein the compound and the at least one mTOR inhibitor together provide a synergistic anti-cancer therapeutic effect.

[0211] In another embodiment, the present invention relates to a method of treating cancer, comprising administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical combination comprising a compound represented by the structure of any of Formulas (I), (II), (III), or (IV) and at least one inhibitor of mammalian target of rapamycin (mTOR), wherein the compound and the at least one mTOR inhibitor together provide a synergistic therapeutic effect.

[0212] In another embodiment, the present invention relates to a method of treating a tumor that has developed resistance to an inhibitor of mammalian target of rapamycin (mTOR), comprising the step of contacting the tumor with an mTOR inhibitor in combination with a compound of any of Formulas (I), (II), (III), or (IV), or any of the individual compounds within such formulas.

[0213] In another embodiment, the present invention relates to a method for preventing acquired resistance of a tumor to an inhibitor of mammalian target of rapamycin (mTOR), comprising the step of contacting the tumor with an mTOR inhibitor in combination with a compound of any of formulas (I), (II), (III), (IV), or any of the individual compounds within such formulas.

[0214] In another embodiment, the invention further relates to the above combination for use in treating tumors that are resistant to mTOR inhibitors or for preventing acquired resistance to mTOR inhibitors.

[0215] In another embodiment, the present invention further relates to the use of the above combination for the preparation of a medicament for the treatment of tumors resistant to mTOR inhibitors or for preventing acquired resistance to mTOR inhibitors.

[0216] In another embodiment, the invention further relates to the above combination for use in treating tumors that are resistant to mTOR inhibitors or for preventing acquired resistance to mTOR inhibitors.

[0217] In some embodiments, the tumor is present in a cancer patient with a tumor that has acquired resistance to mTOR inhibitor treatment. In other embodiments, the treatment results in attenuation of growth or regression of the resistant tumor. In other embodiments, the tumor is present in a cancer patient who is undergoing treatment with an mTOR inhibitor or is a candidate for such treatment.

[0218] Any mTOR inhibitor known to those skilled in the art may be used in the combination of the present invention. In some embodiments, the mTOR inhibitor is a first-generation inhibitor selected from the group consisting of rapamycin (sirolimus); ridaforolimus (deforolimus, AP23573, MK-8669); NVP-BEZ235 (2-methyl-2-{4-[3-methyl-2-oxo-8-(quinolin-3-yl)-2,3-dihydro-1H-imidazo[4,5-c]quinolin-1-yl]phenyl}propanenitrile); everolimus (Afinitor, RAD-001, 40-O-(2-hydroxyethyl) derivative of sirolimus); and temsirolimus (CCI-779). In a currently preferred embodiment, the mTOR inhibitor is everolimus.

[0219] In other embodiments, the mTOR inhibitor is OSI-027 (trans-4-[4-amino-5-(7-methoxy-1H-indol-2-yl)imidazo[5,1-f[1,2,4]triazin-7-yl]cyclohexanecarboxylic acid); XL765 (SAR245409); INK128 (3-(2-amino-5-benzoxazolyl)-1-(1-methylethyl)-1H-pyrazolo[3,4-d]pyrimidin-4-yl]cyclohexanecarboxylic acid); amine); MLN0128, AZD2014 (3-(2,4-bis((S)-3-methylmorpholino)pyrido[2,3-d]pyrimidin-7-yl)-N-methylbenzamide); second-generation compounds (inhibitors of mTORC1 and mTORC2) such as DS-3078a and Palomid 529 (3-(4-methoxybenzyloxy)-8-(1-hydroxyethyl)-2-methoxy-6H-benzo[c]chromen-6-one).

[0220] In one specific embodiment, the compound is represented by the structure of Formula D, and the mTOR inhibitor is everolimus (Afinitor).

[0221] In one embodiment, the compound is a compound of formula A. In another embodiment, the compound is a compound of formula B. In another embodiment, the compound is a compound of formula C. In another embodiment, the compound is a compound of formula D. In another embodiment, the compound is a compound of formula E. In another embodiment, the compound is a compound of formula F. In another embodiment, the compound is a compound of formula G. In another embodiment, the compound is a compound of formula H. In another embodiment, the compound is a compound of formula I. In another embodiment, the compound is a compound of formula J. In another embodiment, the compound is a compound of formula IV-4. In a currently preferred embodiment, the compound is represented by the structure of formula D. However, it will be apparent to one of skill in the art that any of the compounds described herein may be used in the combinations of the present invention.

[0222] Combining IRS / Stat3 dual modulators with immunotherapeutic agents In one embodiment, the present invention relates to a method of sensitizing a tumor to immunotherapy, comprising contacting the tumor with a compound of any of formulas (I), (II), (III), (IV), or any individual compound within such formulae, in combination with an immunotherapeutic agent.

[0223] In another embodiment, the present invention relates to a pharmaceutical combination comprising a compound represented by the structure of any of Formulas (I), (II), (III), (IV), or any of the individual compounds included in such formulas, in combination with an immunotherapeutic agent.

[0224] In another embodiment, the present invention further relates to the above combination for use in treating tumors by sensitizing tumors to immunotherapy.

[0225] In another embodiment, the present invention further relates to the use of said combination for the preparation of a medicament for the treatment of tumors by sensitizing the tumors to immunotherapy.

[0226] In some embodiments, the tumor is present in a cancer patient who is undergoing or is a candidate for receiving immunotherapy.

[0227] Any immunotherapeutic agent known to one of skill in the art can be used in the combinations of the present invention, hi some embodiments, the immunotherapeutic agent is an antibody directed against a target selected from the group consisting of PDL, PD1, CTLA4, CD20, CD30, CD33, CD52, VEGF, CD30, EGFR, and ErbB2. In some embodiments, the antibody is selected from the group consisting of alemtuzumab (Campath®), bevacizumab (Avastin®), brentuximab vedotin (Adcetris®), cetuximab (Erbitux®), gemtuzumab ozogamicin (Mylotarg®), ibritumomab tiuxetan (Zevalin®), ipilimumab (Yervoy®), ofatumumab (Arzerra®), panitumumab (Vectibix®), rituximab (Rituxan®), tositumomab (Bexxar®), and trastuzumab (Herceptin®). Each possibility represents a separate embodiment of the present invention.

[0228] In one embodiment, the compound is a compound of formula A. In another embodiment, the compound is a compound of formula B. In another embodiment, the compound is a compound of formula C. In another embodiment, the compound is a compound of formula D. In another embodiment, the compound is a compound of formula E. In another embodiment, the compound is a compound of formula F. In another embodiment, the compound is a compound of formula Ga. In another embodiment, the compound is a compound of formula H. In another embodiment, the compound is a compound of formula I. In another embodiment, the compound is a compound of formula J. In another embodiment, the compound is a compound of formula IV-4. In a currently preferred embodiment, the compound is represented by the structure of formula D. However, it will be apparent to one of skill in the art that any of the compounds described herein may be used in the combinations of the present invention.

[0229] Combination of IRS / Stat3 dual modulators with mitogen-activated protein kinase (MEK) inhibitors and / or mutant B-Raf inhibitors In another aspect, it has now been unexpectedly discovered that a combination of a dual modulator of insulin receptor substrate (IRS) and signal transducer and activator of transcription 3 (Stat3) described herein with an inhibitor of mitogen-activated protein kinase (MEK) and / or a mutant B-Raf inhibitor provides at least an additive, and preferably a synergistic, therapeutic effect compared to the therapeutic effect of each agent alone. Furthermore, the combination can be used to treat tumors that have developed resistance to MEK inhibitors and / or mutant B-Raf inhibitors, and / or to prevent acquired resistance of tumors to MEK inhibitors and / or mutant B-Raf inhibitors, and / or to prevent or delay tumor recurrence after cessation of treatment with the MEK inhibitor and / or mutant B-Raf inhibitor.

[0230] Thus, in some embodiments, the present invention relates to a method of treating a tumor that has developed resistance to a mitogen-activated protein kinase (MEK) inhibitor and / or a mutant B-Raf inhibitor, comprising contacting the tumor with a MEK inhibitor and / or a mutant B-Raf inhibitor in combination with a compound represented by the structure of Formula (III) or (IV).

[0231] In another embodiment, the present invention relates to a method for preventing acquired resistance of a tumor to a MEK inhibitor and / or a mutant B-Raf inhibitor, comprising contacting the tumor with a MEK inhibitor and / or a mutant B-Raf inhibitor in combination with a compound represented by the structure of Formula (III) or (IV).

[0232] In another embodiment, the present invention relates to a method of preventing or delaying tumor recurrence after cessation of treatment with a MEK inhibitor and / or a mutant B-Raf inhibitor, comprising contacting the tumor with a MEK inhibitor and / or a mutant B-Raf inhibitor in combination with a compound represented by the structure of Formula (III) or (IV).

[0233] In other embodiments, the present invention relates to a pharmaceutical combination comprising a compound represented by the structure of Formula (III) in combination with a mitogen-activated protein kinase (MEK) inhibitor, and optionally, a mutant B-Raf inhibitor. In some embodiments, the combination comprises a compound of Formula (III), a MEK inhibitor, and a mutant B-Raf inhibitor, preferably, the MEK inhibitor is trametinib and the mutant B-Raf inhibitor is vemurafenib.

[0234] In another embodiment, the present invention relates to a compound represented by the structure of Formula (IV) in combination with a mitogen-activated protein kinase (MEK) inhibitor, and / or a mutant B-Raf inhibitor.

[0235] In some embodiments, the tumor is present in a cancer patient whose tumor has acquired resistance to MEK inhibitor and / or mutant B-Raf inhibitor treatment. In other embodiments, the treatment results in attenuation of growth or regression of the resistant tumor. In other embodiments, the tumor is present in a cancer patient who is undergoing treatment with a MEK inhibitor and / or mutant B-Raf inhibitor or is a candidate for such treatment.

[0236] Any MEK inhibitor known to one of skill in the art may be used in the combinations of the present invention. In some embodiments, the MEK inhibitor is selected from the group consisting of trametinib (GSK1120212), selumetinib, binimetinib (MEK162), PD-325901, cobimetinib, CI-1040, and PD035901, and preferably, the MEK inhibitor is trametinib.

[0237] Any mutant B-Raf inhibitor known to those skilled in the art may be used in the combinations of the present invention. In some embodiments, the mutant B-Raf inhibitor is selected from the group consisting of vemurafenib (PLX-4032), PLX4720, sorafenib (BAY43-9006), and dabrafenib, and preferably, the mutant B-Raf inhibitor is vemurafenib.

[0238] In one embodiment, the compound is represented by the structure of Formula (III): In another embodiment, the compound is represented by the structure of Formula (IV): Each possibility represents a separate embodiment of the present invention.

[0239] In some embodiments, the compound is represented by the structure of Formula D, and the MEK inhibitor is trametinib.

[0240] In other embodiments, the compound is represented by the structure of Formula D, and the mutant B-Raf inhibitor is vemurafenib.

[0241] In some embodiments, the combination treatment comprises a compound of Formula (III) or (IV) and either a MEK inhibitor or a mutant B-Raf inhibitor, hi other embodiments, the combination treatment comprises a compound of Formula (III) or (IV) and both a MEK inhibitor and a mutant B-Raf inhibitor.

[0242] In some embodiments, the present invention relates to a pharmaceutical combination comprising a compound represented by the structure of Formula D in combination with trametinib.

[0243] In another embodiment, the present invention relates to a pharmaceutical combination comprising a compound represented by the structure of Formula D in combination with trametinib and vemurafenib.

[0244] In another embodiment, the invention relates to the above combination for use in treating tumors that are resistant to MEK inhibitors and / or mutant B-Raf inhibitors or for preventing acquired resistance to MEK inhibitors and / or mutant B-Raf inhibitors.

[0245] In another embodiment, the present invention relates to the use of the above combination for the preparation of a medicament for the treatment of tumors resistant to MEK inhibitors and / or mutant B-Raf inhibitors or for preventing acquired resistance to MEK inhibitors and / or mutant B-Raf inhibitors.

[0246] In one embodiment, the compound is a compound of formula A. In another embodiment, the compound is a compound of formula B. In another embodiment, the compound is a compound of formula C. In another embodiment, the compound is a compound of formula D. In another embodiment, the compound is a compound of formula E. In another embodiment, the compound is a compound of formula F. In another embodiment, the compound is a compound of formula Ga. In another embodiment, the compound is a compound of formula H. In another embodiment, the compound is a compound of formula I. In another embodiment, the compound is a compound of formula J. In another embodiment, the compound is a compound of formula IV-4. In a currently preferred embodiment, the compound is represented by the structure of formula D. However, it will be apparent to one of skill in the art that any of the compounds described herein may be used in the combinations of the present invention.

[0247] Combination of IRS / Stat3 dual modulators and chemotherapeutic agents In another aspect, it has now been unexpectedly discovered that combinations of dual modulators of insulin receptor substrate (IRS) and signal transducer and activator of transcription 3 (STAT3) described herein with chemotherapeutic agents such as gemcitabine, 5-FU, irinotecan, oxaliplatin, and any combination thereof (e.g., Forfiri or Forfox in combination treatment) provide at least additive, and preferably synergistic, therapeutic effects compared to the therapeutic effects of each agent alone. Furthermore, the combinations can be used to treat tumors that have developed resistance to any of these chemotherapeutic agents or combinations thereof, and / or to prevent acquired resistance of tumors to any of these chemotherapeutic agents or combinations thereof, and / or to prevent or delay tumor recurrence after cessation of treatment with any of these chemotherapeutic agents or combinations thereof.

[0248] Forfili is a combination cancer treatment containing leucovorin (folinic acid), 5-FU, and irinotecan. Forfox is a combination cancer treatment containing leucovorin calcium (folinic acid), 5-FU, and oxaliplatin.

[0249] Thus, according to some embodiments, the present invention relates to a pharmaceutical combination comprising a compound represented by the structure of Formula (III) or (IV) and at least one chemotherapeutic agent selected from gemcitabine, 5-FU, irinotecan, oxaliplatin, and any combination thereof, wherein the compound and the chemotherapeutic agent(s) together provide a synergistic anti-cancer therapeutic effect.

[0250] In some embodiments, the present invention relates to a method of treating cancer, comprising administering to a subject in need thereof a therapeutically effective amount of a combination comprising a compound represented by the structure of Formula (III) or (IV) and at least one chemotherapeutic agent selected from gemcitabine, 5-FU, irinotecan, oxaliplatin, and any combination thereof, wherein the compound and the chemotherapeutic agent(s) together provide a synergistic anti-cancer therapeutic effect.

[0251] In other embodiments, the present invention provides a method of treating a tumor that has developed resistance to at least one chemotherapeutic agent, such as gemcitabine, 5-FU, irinotecan, oxaliplatin, and any combination thereof, comprising contacting the tumor with at least one of the chemotherapeutic agent(s) in combination with a compound represented by the structure of Formula (III) or (IV).

[0252] In other embodiments, the present invention provides a method of preventing acquired resistance of a tumor to at least one chemotherapeutic agent, such as gemcitabine, 5-FU, irinotecan, oxaliplatin, and any combination thereof, comprising contacting the tumor with at least one of the chemotherapeutic agent(s) in combination with a compound represented by the structure of Formula (III) or (IV).

[0253] In other embodiments, the present invention provides a method of preventing or delaying tumor recurrence after cessation of treatment with at least one chemotherapeutic agent, such as gemcitabine, 5-FU, irinotecan, oxaliplatin, and any combination thereof, comprising contacting the tumor with at least one of the chemotherapeutic agent(s) in combination with a compound represented by the structure of Formula (III) or (IV).

[0254] In some embodiments, the tumor is present in a cancer patient whose tumor has acquired resistance to the chemotherapeutic agent(s). In other embodiments, the treatment results in attenuation of growth or regression of the resistant tumor. In other embodiments, the tumor is present in a cancer patient who is undergoing treatment with the chemotherapeutic agent(s) or who is a candidate for such treatment.

[0255] In other embodiments, the present invention provides pharmaceutical combinations comprising a compound represented by the structure of Formula (III) or (IV) and at least one chemotherapeutic agent, such as gemcitabine, 5-FU, irinotecan, oxaliplatin, and any combination thereof, for use in treating tumors that are resistant to said chemotherapeutic agent(s), or for preventing acquired resistance to said chemotherapeutic agent(s), or for delaying tumor recurrence after cessation of treatment with such chemotherapeutic agent(s).

[0256] In other embodiments, the present invention relates to the use of a pharmaceutical combination comprising a compound represented by the structure of Formula (III) or (IV) and at least one chemotherapeutic agent, such as gemcitabine, 5-FU, irinotecan, oxaliplatin, and any combination thereof, for the preparation of a medicament for the treatment of tumors that are resistant to said chemotherapeutic agent(s), or for preventing acquired resistance to said chemotherapeutic agent(s), or for preventing or delaying tumor recurrence after cessation of treatment with such chemotherapeutic agent(s).

[0257] It will be apparent to those skilled in the art that other chemotherapeutic agents related to the above non-limiting examples can be used in the combinations of the present invention. For example, the present invention contemplates the use of other platinum compounds (e.g., carboplatin and cisplatin), SN-38 (a metabolite of irinotecan), and other fluoropyrimidines (analogs of 5-FU).

[0258] In one embodiment, the compound is a compound of formula A. In another embodiment, the compound is a compound of formula B. In another embodiment, the compound is a compound of formula C. In another embodiment, the compound is a compound of formula D. In another embodiment, the compound is a compound of formula E. In another embodiment, the compound is a compound of formula F. In another embodiment, the compound is a compound of formula Ga. In another embodiment, the compound is a compound of formula H. In another embodiment, the compound is a compound of formula I. In another embodiment, the compound is a compound of formula J. In another embodiment, the compound is a compound of formula IV-4. In a currently preferred embodiment, the compound is represented by the structure of formula D. However, it will be apparent to one of skill in the art that any of the compounds described herein may be used in the combinations of the present invention.

[0259] Cancer treatment The term "cancer," as used herein, refers to disorders in which populations of cells, to varying degrees, become unresponsive to the control mechanisms that normally govern proliferation and differentiation. Cancer refers to tumors, including various types of malignant neoplasms and primary tumors, as well as tumor metastases. Non-limiting examples of cancers that can be treated with the combinations of the present invention include cancers of the brain, ovary, colon, prostate, kidney, bladder, breast, lung, oral cavity, and skin. Specific examples of cancers include carcinomas, sarcomas, myelomas, leukemias, lymphomas, and mixed tumors. Specific categories of tumors include lymphoproliferative disorders, breast cancer, ovarian cancer, prostate cancer, cervical cancer, endometrial cancer, bone cancer, liver cancer, stomach cancer, colon cancer, pancreatic cancer, thyroid cancer, head and neck cancer, cancers of the central nervous system, cancers of the peripheral nervous system, skin cancer, kidney cancer, and metastases of all of the above. Specific types of tumors include hepatocellular carcinoma, hepatoma, hepatoblastoma, rhabdomyosarcoma, esophageal carcinoma, thyroid carcinoma, ganglioblastoma, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, chordoma, angiosarcoma, endotheliosarcoma, Ewing's tumor, leiomyosarcoma, rhabdotheliosarcoma, invasive ductal carcinoma, papillary adenocarcinoma, melanoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma (well, moderately, poorly, or undifferentiated), renal cell carcinoma, adrenal gland tumor, adrenal adenocarcinoma, cholangiocarcinoma, choriocarcinoma, and seminoma. These include embryonal carcinoma, Wilms' tumor, testicular tumor, lung cancer including small cell, non-small cell, and large cell lung cancer, bladder cancer, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, retinoblastoma, neuroblastoma, colon cancer, rectal cancer, hematopoietic malignancies including all types of leukemia and lymphoma, including acute myeloid leukemia, acute myelocytic leukemia, acute lymphocytic leukemia, chronic myelogenous leukemia, chronic lymphocytic leukemia, mast cell leukemia, multiple myeloma, myeloid lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, and liver cancer. Each possibility represents a separate embodiment of the present invention.

[0260] In some exemplary embodiments, the cancer is selected from the group consisting of head and neck (H&N) cancer, sarcoma, multiple myeloma, ovarian cancer, breast cancer, renal cancer, gastric cancer, hematopoietic cancer, lymphoma, leukemia including lymphoblastic leukemia, lung cancer, melanoma, glioblastoma, liver cancer, prostate cancer, and colon cancer, with each possibility representing a separate embodiment of the present invention.

[0261] The term "treating cancer" in the context of the present invention includes at least one of the following: a reduction in the rate of cancer proliferation (i.e., the cancer is still growing, but at a slower rate); growth arrest of cancer growth, i.e., a stasis of tumor growth, and in preferred cases, the tumor is reduced or reduced in size. The term also includes a reduction in the number of metastases, a reduction in the number of new metastases formed, a delay in the progression of cancer from one stage to another, and a reduction in cancer-induced angiogenesis. In the most preferred cases, the tumor is completely eliminated. The term also includes an extension of the survival time of the subject receiving treatment, an extension of the time to disease progression, tumor regression, etc. It should be understood that the term "treating cancer" also refers to the inhibition of malignant (cancer) cell proliferation, including tumor formation, primary tumor, tumor growth, or tumor metastasis. In relation to cancer cells, the term "inhibition of proliferation" can further refer to a reduction in at least one of the following: a decrease in the number of cells compared to a control (due to cell death, which may be necrotic, apoptotic or any other type of cell death, or a combination thereof); a decrease in the rate of proliferation of the cells, i.e., an increase in the total number of cells, but at a lower level or rate than in the control; a decrease in the invasiveness of the cells compared to a control, even if their total number is unchanged (e.g., as determined by a soft agar assay); a progression from a less differentiated cell type to a more differentiated cell type; a slowdown in neoplastic transformation; or a delay in the progression of cancer cells from one stage to the next.

[0262] As used herein, the term "administration" refers to contacting with a combination of the present invention. Administration can be to a cell or tissue culture, or to an organism, e.g., a human. In one embodiment, the present invention encompasses administration of a combination of the present invention to a human subject.

[0263] A "therapeutic" treatment is a treatment administered to a subject who exhibits symptoms of a pathology for the purpose of reducing or eliminating those symptoms. A "therapeutically effective amount" is the amount of a compound or composition that is sufficient to provide a beneficial effect to the subject to which the compound or composition is administered.

[0264] As used herein, the term "after cessation of treatment" means after treatment with the selected drug has been stopped. For example, according to certain embodiments of the present invention, an IRS / Stat3 dual modulator (e.g., a compound of Formula (III) or (IV)) is administered together (sequentially or simultaneously) with any of the combination treatments described herein for a desired period of time. Treatment (with all compounds) is then stopped, and tumors are monitored for a desired period of time. As contemplated herein, the IRS / Stat3 dual modulators of the present invention can prevent or delay tumor recurrence after cessation of treatment with any of the combination drugs described herein to a greater extent than if either of these drugs were administered alone.

[0265] The term "treating a tumor that has developed resistance" to a particular anticancer drug or the term "preventing tumor acquired resistance" to a particular anticancer drug means any one or more of the following: (i) the tumor acquires or develops resistance to that anticancer drug as a result of treatment; (ii) the tumor acquires or develops resistance as a result of treatment with another anticancer drug; or (iii) the tumor has primary resistance to that anticancer drug.

[0266] Combination therapy can provide therapeutic advantages by taking into account the differential toxicity associated with the two individual therapies. For example, treatment with one compound may result in a particular toxicity not seen with the other compound, or vice versa. Thus, this differential toxicity can allow each treatment to be administered at a dose where toxicity is absent or minimal, so that the combined therapy together provides a therapeutic dose while avoiding the toxicity of each of the combination components. Furthermore, if the therapeutic effect achieved as a result of the combination therapy is enhanced or synergistic, i.e., significantly better than additive therapeutic effect, the dose of each of the agents can be further reduced, thus reducing the associated toxicity to an even greater extent.

[0267] The terms "synergistic," "cooperative," and "superadditive," as well as their various grammatical variations, are used interchangeably herein. An interaction between an IRS / Stat3 dual modulator and another anticancer agent (e.g., an mTOR inhibitor, an EGFR inhibitor, an EGFR antibody, and / or an immunotherapeutic agent) is considered synergistic, cooperative, or superadditive if the observed effect (e.g., cytotoxicity) in the presence of the drugs together is greater than the sum of the individual effects when each drug is administered separately. In one embodiment, the observed combined effect of the drugs is significantly greater than the sum of the individual effects. The term "significant" means that the observed p-value is less than 0.05. A non-limiting method for calculating the efficacy of a combination treatment includes using the Bliss additivity model (Cardone et al., Science (1998), 282:1318-1321), which uses the following formula: Ebliss = EA + EB - EA × EB, where EA and EB are the percentage of inhibition obtained by drug A alone and drug B alone at a particular concentration. If the experimentally determined percentage of inhibition is equal to Ebliss, the combination provides an additive therapeutic effect. If the experimentally determined percentage of inhibition is greater than Ebliss, the combination provides a synergistic therapeutic effect.

[0268] Pharmaceutical Composition While the components of the combination of the present invention can be administered alone, it is contemplated that the components will be administered in a pharmaceutical composition further comprising at least one pharmaceutically acceptable carrier or excipient. Each of the components can be administered in a separate pharmaceutical composition, or the combination can be administered in a single pharmaceutical composition.

[0269] The pharmaceutical compositions of the present invention can be formulated for administration by a variety of routes, including oral, rectal, transdermal, parenteral (subcutaneous, intraperitoneal, intravenous, intraarterial, transdermal and intramuscular), topical, intranasal, or via suppository. Each possibility represents a separate embodiment of the present invention. Such compositions are prepared by methods well known in the pharmaceutical art and comprise, as an active ingredient, at least one compound of the present invention as described above and a pharmaceutically acceptable excipient or carrier. The term "pharmaceutically acceptable" means approved by a federal or state regulatory agency or listed in the United States Pharmacopeia or other generally recognized pharmacopeia for use in animals, more particularly in humans.

[0270] During the preparation of pharmaceutical compositions according to the present invention, the active ingredient is usually mixed with a carrier or excipient, which may be a solid, semi-solid, or liquid material.The composition may be in the form of, for example, tablets, pills, capsules, pellets, granules, powders, lozenges, sachets, cachets, elixirs, suspensions, dispersions, emulsions, solutions, syrups, aerosols (as solids or in liquid media), ointments, soft and hard gelatin capsules, suppositories, sterile injection solutions, and sterile packaged powders, each of which contains up to 10% by weight of the active compound.Each possibility represents a separate embodiment of the present invention.

[0271] The carrier can be any of those conventionally used, and is limited only by chemical and physical considerations such as solubility and lack of reactivity with the compound of the present invention, and by the route of administration.The selection of carrier is determined by the specific method used to administer pharmaceutical composition.Some examples of suitable carrier include lactose, glucose, dextrose, sucrose, sorbitol, mannitol, starch, gum acacia, calcium phosphate, alginate, tragacanth, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water and methylcellulose.Each possibility represents a separate embodiment of the present invention. The formulations may further include lubricants such as talc, magnesium stearate, and mineral oil; wetting agents, surfactants, emulsifying and suspending agents; preservatives such as methylhydroxybenzoate and propylhydroxybenzoate; sweeteners; flavorings, coloring agents, buffers (e.g., acetates, citrates, or phosphates), disintegrating agents, wetting agents, antibacterial agents, antioxidants (e.g., ascorbic acid or sodium bisulfite), chelating agents (e.g., ethylenediaminetetraacetic acid), and agents for adjusting tonicity such as sodium chloride. Other pharmaceutical carriers may be sterile liquids such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, and sesame oil, polyethylene glycol, glycerin, propylene glycol, or other synthetic solvents. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Physiological saline and aqueous dextrose and glycerol solutions can also be used as liquid carriers, particularly for injectable solutions. Each possibility represents a separate embodiment of the invention.

[0272] To prepare solid compositions such as tablets, the primary active ingredient(s) are mixed with pharmaceutical excipients to form a solid preformulation composition containing a homogeneous mixture of the compounds of the present invention. When these preformulation compositions are referred to as homogeneous, it is meant that the active ingredient(s) are dispersed evenly throughout the composition such that the composition can be readily subdivided into equally effective unit dosage forms such as tablets, pills, and capsules. This solid preformulation is then subdivided into unit dosage forms of the type described above containing, for example, about 0.1 mg to about 2000 mg, about 0.1 mg to about 500 mg, about 1 mg to about 100 mg, or about 100 mg to about 250 mg of the active ingredient(s) of the present invention.

[0273] Any method can be used to prepare the pharmaceutical composition. Solid dosage forms can be prepared by wet granulation, dry granulation, direct compression, and the like. The solid dosage forms of the present invention can be coated or otherwise formulated to provide a dosage form that offers the advantage of prolonged action. For example, a tablet or pill can contain an inner dosage component and an outer dosage component, the latter in the form of an envelope over the former. These two components can be separated by an enteric layer that functions to resist disintegration in the stomach and allow the inner component to pass intact into the duodenum or to be delayed in release. A variety of materials can be used for such enteric layers or coatings, including several polymeric acids and mixtures of polymeric acids with materials such as shellac, cetyl alcohol, and cellulose acetate. Each possibility represents a separate embodiment of the present invention.

[0274] Liquid forms into which the compositions of the present invention may be incorporated for oral or injectable administration include aqueous solutions, suitably flavored syrups, aqueous or oily suspensions, and flavored emulsions, along with edible oils such as cottonseed oil, sesame oil, coconut oil, or peanut oil, as well as elixirs and similar pharmaceutical vehicles, each possibility representing a separate embodiment of the present invention.

[0275] Compositions for inhalation or insulation include solutions and suspensions in pharmaceutically acceptable aqueous or organic solvents, or mixtures thereof, and powders. The liquid or solid compositions may contain suitable pharmaceutically acceptable excipients as described above. In one embodiment, the compositions are administered by the oral or nasal respiratory route for local or systemic effect. Compositions in pharmaceutically acceptable solvents may be nebulized by use of inert gases. Nebulized solutions may be breathed directly from the nebulizing device, or the nebulizing device may be attached to a face mask tent or intermittent positive pressure breathing machine. Solution, suspension, or powder compositions may be administered orally or nasally from devices that deliver the formulation in an appropriate manner.

[0276] Another suitable formulation for the compositions and methods of the present invention employs transdermal delivery devices ("patches"). Such transdermal patches may be used to provide continuous or discontinuous infusion of the compounds of the present invention in controlled amounts. The construction and use of transdermal patches for the delivery of pharmaceutical agents is well known in the art.

[0277] In yet another embodiment, the composition is prepared for topical administration, for example, as an ointment, gel, drop, or cream. For topical administration to body surfaces using, for example, creams, gels, drops, and ointments, the compounds of the present invention can be prepared and applied in a physiologically acceptable diluent with or without a pharmaceutical carrier. The present invention may be used topically or transdermally to treat cancer, for example, melanoma. Adjuvants for topical or gel-based forms may include, for example, sodium carboxymethylcellulose, polyacrylate, polyoxyethylene-polyoxypropylene block polymer, polyethylene glycol, and wood wax alcohol. Each possibility represents a separate embodiment of the present invention.

[0278] Alternative formulations include nasal sprays, liposomal formulations, sustained release formulations, pumps that deliver drugs into the body (including mechanical or osmotic pumps), and controlled release formulations, as are known in the art.

[0279] The compositions are preferably formulated in unit dosage form. The term "unit dosage form" refers to physically discrete units suitable as unitary dosages for human subjects and other mammals, each unit containing a predetermined quantity of active substance(s) calculated to produce the desired therapeutic effect, together with suitable pharmaceutical excipients.

[0280] To prepare formulation, active ingredient may need to be pulverized to obtain suitable particle size before being combined with other components.If active compound is substantially insoluble, it is usually pulverized to the particle size of less than 200 mesh.If active ingredient is substantially water-soluble, its particle size is usually adjusted by pulverizing to about 40 mesh, for example, to obtain substantially uniform distribution in formulation.

[0281] It may be desirable to administer the pharmaceutical compositions of the present invention locally to the area in need of treatment. This can be achieved, for example, but not limited to, by local infusion during surgery, injection into the liver via its vascular supply with or without surgery, by topical application in conjunction with a wound dressing after surgery, by injection, by catheter, by suppository, or by implants of porous, non-porous, or gelatinous material. According to some embodiments, administration can be by direct injection, e.g., via a syringe, at the site of the tumor or neoplastic or pre-neoplastic tissue.

[0282] The compounds may also be administered by any convenient route, for example, by infusion or bolus injection, by absorption through epithelial linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.), and may be administered together with other therapeutically active agents. The administration may be local or systemic. In addition, it may be desirable to introduce the pharmaceutical compositions of the present invention into the central nervous system by any suitable route, including intraventricular and intrathecal injection. Intraventricular injection can be facilitated, for example, by an intraventricular catheter attached to a reservoir. Pulmonary administration can also be employed, for example, by use of an inhaler or nebulizer, and by formulation with an aerosolizing agent.

[0283] The compounds of the present invention can be delivered in immediate-release or controlled-release systems. In one embodiment, the compounds of the present invention can be administered using an infusion pump, such as those used to deliver chemotherapy to specific organs or tumors (see Buchwald et al., 1980, Surgery 88:507; Saudek et al., 1989, N. Engl. J. Med. 321:574). In one embodiment, the compounds of the present invention are administered in combination with a biodegradable, biocompatible polymer implant that releases the compound at a selected site over a controlled period of time. Examples of polymeric materials include, but are not limited to, polyanhydrides, polyorthoesters, polyglycolic acid, polylactic acid, polyethylene vinyl acetate, copolymers and blends thereof. In yet another embodiment, a controlled-release system can be placed in proximity to the therapeutic target, thereby requiring only a fraction of the systemic dose.

[0284] Furthermore, sometimes the pharmaceutical compositions may be formulated for parenteral administration (subcutaneous, intravenous, intraarterial, transdermal, intraperitoneal, or intramuscular injection), including aqueous and non-aqueous isotonic sterile injection solutions, which may contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, as well as aqueous and non-aqueous sterile suspensions containing suspending agents, solubilizers, thickeners, stabilizers, and preservatives. Oils, such as petroleum, animal, vegetable, or synthetic oils, and soaps, such as fatty alkali metal, ammonium, and triethanolamine salts, and suitable surfactants, can also be used for parenteral administration. The above formulations can also be used for direct intratumoral injection. Furthermore, to minimize or eliminate irritation at the injection site, the compositions may contain one or more nonionic surfactants. Suitable surfactants include polyethylene sorbitan fatty acid esters, such as sorbitan monooleate, and the high molecular weight adduct of ethylene oxide with a hydrophobic base formed by the condensation of propylene oxide with propylene glycol.

[0285] Parenteral formulations can be presented in unit-dose or multi-dose sealed containers, such as ampoules and vials, and can be stored in a freeze-dried (lyophilized) condition requiring only the addition of a sterile liquid carrier for injection, e.g., water, immediately prior to use. Extemporaneous injection solutions and suspensions can be prepared from sterile powders, granules, and tablets of the kind described above and known in the art. Each possibility represents a separate embodiment of the invention.

[0286] Alternatively, the combinations of the present invention can be used in hemodialysis, such as leukapheresis and other related methods, where blood is withdrawn from a patient by various methods, such as dialysis through a column / hollow fiber membrane, cartridge, etc., treated ex vivo with an IRS / Stat3 dual modulator and / or additional anticancer agent, and returned to the patient after treatment. Such treatment methods are well known and described in the art. See, for example, Kolho et al. (J. Med. Virol. 1993, 40(4):318-21); Ting et al. (Transplantation, 1978, 25(1):31-3), the contents of which are incorporated herein by reference in their entireties.

[0287] Dosage and Administration Schedule Treatment with an IRS / Stat3 dual modulator and another anti-cancer agent (i.e., an EGFR inhibitor / EGFR antibody / mTOR inhibitor / immunotherapeutic agent / MEK inhibitor / mutant B-Raf inhibitor / chemotherapeutic agent, or a combination thereof) can be performed sequentially in any order, simultaneously, or in any combination thereof. For example, administration of an IRS / Stat3 dual modulator can be performed before, after, or simultaneously with administration of the other anti-cancer agent or combination thereof. For example, a total treatment period can be determined for the IRS / Stat3 dual modulator. The other anti-cancer agent can be administered before the start of treatment with the IRS / Stat3 dual modulator or after treatment with the IRS / Stat3 dual modulator. In addition, the other anti-cancer agent can be administered during the administration period of the IRS / Stat3 dual modulator, but need not be administered for the entire treatment period. In another embodiment, the treatment regimen includes prior treatment with one of the following agents: an IRS / Stat3 dual modulator or an EGFR inhibitor / EGFR antibody / mTOR inhibitor / immunotherapeutic agent / MEK inhibitor / mutant B-Raf inhibitor / chemotherapeutic agent, or a combination thereof, followed by the addition of another agent or agents. Alternating administration sequences are also contemplated. Alternating administration includes administration of an IRS / Stat3 dual modulator and another anti-cancer agent in an alternating order, such as an IRS / Stat3 dual modulator followed by the other anti-cancer agent, followed by the IRS / Stat3 dual modulator.

[0288] The amount of compound that will be effective in treating a particular disorder or condition, including cancer, will depend on the nature of the disorder or condition and can be determined by standard clinical techniques. Additionally, in vitro assays can be used, if necessary, to help identify optimal dosage ranges. The precise dose to be used in a formulation will also depend on the route of administration and the progression of the disease or disorder, and should be decided according to the judgment of the physician and each patient's circumstances. Preferred dosages are within the ranges of 0.01 to 1000 mg / kg body weight, 0.1 mg / kg to 100 mg / kg, 1 mg / kg to 100 mg / kg, 10 mg / kg to 75 mg / kg, 0.1 to 1 mg / kg, etc. Exemplary (non-limiting) amounts of IRS / Stat3 dual modulator, EGFR inhibitor / EGFR antibody / mTOR inhibitor / immunotherapeutic / MEK inhibitor / mutant B-Raf inhibitor / chemotherapeutic agent include 0.1 mg / kg, 0.2 mg / kg, 0.5 mg / kg, 1 mg / kg, 5 mg / kg, 10 mg / kg, 20 mg / kg, 50 mg / kg, 60 mg / kg, 75 mg / kg, and 100 mg / kg. Alternatively, the amount administered can be measured and expressed as the molar concentration of the administered compound. By way of example, and not limitation, an IRS / Stat3 dual modulator (e.g., a compound of Formula I, II, II, or IV) can be administered in the range of 0.1 to 10 mM, e.g., 0.1, 0.25, 0.5, 1, and 2 mM. Alternatively, the amount administered can be measured and expressed as mg / ml, μg / ml, or ng / ml. By way of example, and not limitation, an EGFR inhibitor / EGFR antibody / mTOR inhibitor / immunotherapeutic agent / MEK inhibitor / mutant B-Raf inhibitor / chemotherapeutic agent can be administered in an amount of 1 ng / ml to 100 mg / ml, e.g., 1 to 1000 ng / ml, 1 to 100 ng / ml, 1 to 1000 μg / ml, 1 to 100 μg / ml, 1 to 1000 mg / ml, 1 to 100 mg / ml, etc. Effective doses may be estimated from dose-response curves derived from in vitro or animal model test bioassays or systems. If a synergistic effect is observed, the overall dose of each component may be lowered, so that the subject may experience significantly fewer side effects, while still achieving a sufficient anti-cancer effect.

[0289] In one embodiment, the combination therapy reduces the amount of each of the components by 2-fold, i.e., each component is administered at half the dose compared to monotherapy, while still achieving the same or similar therapeutic effect. In another embodiment, the combination therapy reduces the amount of each of the components by 5, 10, 20, 50, or 100-fold. As demonstrated herein, the IC of chemotherapeutic agents as antiproliferative agents in various cancer cells. 50 is the IC of the chemotherapeutic agent when administered alone. 50 Decreased compared to

[0290] The administration schedule depends on several factors, such as the cancer being treated, its severity and progression, the patient population, age, and body weight. For example, the compositions of the present invention can be taken once daily, twice daily, three times daily, weekly, or monthly. In addition, administration can be continuous, i.e., daily, or intermittent. As used herein, the terms "intermittent" or "intermittently" refer to stopping and starting at either regular or irregular intervals. For example, intermittent administration can be administration 1 to 6 days per week, or can refer to administration in cycles (e.g., daily administration for 2 to 8 consecutive weeks followed by a rest period of up to 1 week without administration), or can refer to administration on alternating days. Different components of the combination can follow different administration schedules independently of each other.

[0291] The following examples are presented to more fully illustrate certain embodiments of the present invention. However, they should in no way be construed as limiting the broad scope of the invention. Those skilled in the art can readily devise numerous variations and modifications of the principles disclosed herein without departing from the scope of the invention.

[0292] Experiment Details Section Example 1: Prevention of acquired resistance to erlotinib with Compound D Experimental System : Patient-derived xenografts (PDX) of squamous cell carcinoma of the head and neck (SCCHN) tumor biopsies are implanted subcutaneously into NodScid mice. I. Animals and biopsies Biopsy: Fresh human primary SCCHN tumor biopsy Tumor type: salivary gland mucoepidermoid carcinoma Genomic analysis revealed amplification and mutation (activation) of EGFR. Implantation of tumor biopsy grafts (P0): Fresh human primary SCCHN tumor biopsy grafts were implanted into five female NOD.CB17-Prkdc mice aged 5–6 weeks after 14 days of acclimation. scid The tumor cells were implanted subcutaneously (SC) into / J (NodScid mice) (Harlan, IL). · Implantation of tumor biopsy xenografts (P1) into NodScid mice for efficacy studies : The tumor is approximately 1,200 mm 3 At 3.5 weeks after implantation of the biopsies (P0), when the tumors had reached an average size of 1.5 mm, mice were sacrificed by cervical dislocation and the tumors were excised. The tumors were measured, cut into 2-4 mm pieces, and transferred to gentleMACS tubes containing sterile saline. The tumor volume was adjusted with saline to 1.5 mm per 100 μl of saline. 3 The tumor volume was adjusted to 1.5 mm. The sample was dissociated using a gentleMACS Octo Dissociator. The dissociated tumor tissue was collected with an 18G syringe and injected directly under the skin. 100 μl of the resulting cell solution (approximately 1.5 mm in 100 μl saline per mouse) was injected into the nuchal region of 35 4- to 5-week-old female NodScid mice (Harlan, IL). 3 Tumor volumes of 1000-120 ... The onset of tumor growth (palpable tumor masses) was detected 10 days after cell injection. After 8 days, 32 of 35 injected mice had tumors of approximately 80 mm 3 These mice developed tumors with an average size of 1.5 mm. These mice were randomly divided into four treatment groups with 8 animals per group.

[0293] II. Treatments and Procedures The tumor size is approximately 80 mm 3 On day 0, the following treatments were initiated: 1. Control (vehicle): 100 μl water PO (5 times / week, daily) 2. Compound D 70 mg / kg IV in 20% 2-hydroxypropyl-β-cyclodextrin (HPbCD) (3 times a week, every other day) 3. Erlotinib 100mg / kg PO (5 times / week, daily) 4. Erlotinib 100 mg / kg PO 5 times / week + Compound D 70 mg / kg IV 3 times / week. If administered on the same day, erlotinib was administered approximately 4 hours after Compound D administration. All treatments for each of treatment groups 1-4 were initiated simultaneously.

[0294] The length (l) and width (w) of the tumor were measured four times a week, and the tumor volume was calculated as follows: v = lw 2 / 2. Graphs represent mean tumor volume with standard error (standard deviation / square root of group size). Mice were monitored for weight and behavior at least once a week. After two weeks of treatment, mice were sacrificed and tumors were excised 14 hours after the last drug / inhibitor administration for biochemical and genomic analysis. Three mice in the combination treatment group were not sacrificed at the end of treatment and were kept without further treatment.

[0295] result As shown in Figure 1, treatment with erlotinib, an EGFR TK inhibitor, initially resulted in significant tumor regression in all treated mice (Figure 1, open squares). However, after one week of treatment, all tumors developed resistance to erlotinib and grew aggressively. Combination treatment with erlotinib and Compound D resulted in significant tumor regression in all treated mice, and none of the tumors regrew during the combined treatment period (Figure 1, open circles).

[0296] The two mice that achieved a complete response continued to survive without further treatment and remained disease-free after 3 months without further treatment.

[0297] Although early-stage tumors did not respond to Compound D alone, acquired resistance to erlotinib was completely abolished by Compound D. Evidence from the literature suggests that treatment with erlotinib induces upregulation of IRS, leading to resistance through activation of the IGF1R / IRS to AKT survival pathway. Other reports claim that Stat3 phosphorylation is induced by erlotinib in H&N cancers, and that inhibition of Stat3 and EGFR has a synergistic inhibitory effect on H&N tumors. Without being bound by any particular theory or mechanism of action, Compound D and the other compounds of Formulas (I-IV) described herein are dual inhibitors of IRS1 / 2 and Stat3, and therefore may antagonize these erlotinib-induced mechanisms and prevent resistance.

[0298] Example 2: Regression of erlotinib-resistant tumors by combined treatment with erlotinib and Compound D Experimental System : Patient-derived xenografts (PDX) of squamous cell carcinoma of the head and neck (SCCHN) tumor biopsies are implanted subcutaneously into NodScid mice. I. Animals and biopsies Implantation of SCCHN tumor biopsy xenografts (P8) into NodScid mice for efficacy studies: Using the same procedure as described for P1 xenografts, tumor cells (P8) were injected into autologous 9.5-week-old NodScid mice 5 months after implantation of the SCCHN tumor biopsy xenografts (P1) described above. The original biopsy was the same as above, and P indicates progression (number of xenografts in the mouse). The onset of tumor growth (palpable tumor mass) was detected 7 days after cell injection. After 12 days, the mice had tumors measuring approximately 70 mm 3 These mice were randomly divided into four treatment groups, with four animals treated with vehicle, Compound D, or Compound D plus erlotinib, and the remaining animals treated with erlotinib. Treatments were initiated simultaneously (day 0).

[0299] II. Treatments and Procedures Treatment groups included: 1. Vehicle-Control: 20% 2-hydroxypropyl-β-cyclodextrin (HPbCD) 50 μl / injection, IV (3 times / week, every other day) 2.Compound D 70mg / kg in HPbCD, IV (3 times / week, every other day) 3. Erlotinib 100 mg / kg PO (5 times / week) in HPbCD 4. Erlotinib 100 mg / kg PO 5 times / week + Compound D 70 mg / kg IV 3 times / week. If administered on the same day, erlotinib was administered approximately 4 hours after Compound D administration. All these treatments were started simultaneously.

[0300] Treatment with erlotinib (group 3) resulted in dramatic tumor regression (Figure 2, open squares). During treatment, the tumor developed resistance to erlotinib after 1 week of treatment and grew aggressively. By day 10, the tumor had grown to approximately 130 mm. 3 Erlotinib-treated mice (n=7) that developed tumors were divided into two groups: 5. Group 1 (n=3) continued to receive erlotinib (100 mg / kg PO, 5 times / week), and 6. Group 2 (n=4) began combination treatment with erlotinib (100 mg / kg PO, 5 times per week) plus Compound D (70 mg / kg IV, 3 times per week, every other day) on day 10 of treatment. When administered on the same day, erlotinib was administered approximately 4 hours after Compound D administration.

[0301] The length (l) and width (w) of the tumor were measured four times a week, and the tumor volume was calculated as follows: v = lw 2 / 2. Graphs represent mean tumor volumes with standard error (standard deviation / square root of group size). Mice were monitored for weight and behavior at least once a week. Mice were sacrificed and tumors were excised for biochemical and genomic analysis.

[0302] result As shown in Figure 2, treatment with erlotinib (open squares) resulted in tumor regression in 78% of treated mice (14 of 18 mice responded). However, during treatment, tumors developed resistance to erlotinib after 1 week and grew aggressively. By day 10, tumors had grown to approximately 130 mm. 3 The erlotinib-treated mice (n=7) that had developed ERLOTINIB-like tumors were divided into two groups: the first group (n=3) continued to receive ERLOTINIB (open squares), and the second group (n=4) began combination treatment with ERLOTINIB and Compound D (filled circles) on day 10 of treatment. Dramatic tumor regression was observed after the initiation of combination treatment (Figure 2, late treatment, filled circles), but only tumors in mice treated with ERLOTINIB grew aggressively (Figure 2, open squares). Combination treatment with ERLOTINIB and Compound D, initiated on day 0 (Figure 2, early treatment, open circles), resulted in significant tumor regression in all treated mice, and none of the tumors regrew, consistent with the results of Example 1.

[0303] conclusion In conclusion, the combination of Compound D and erlotinib is highly effective, resulting in dramatic tumor regression after resistance to erlotinib has already developed. Furthermore, in the early treatment of established tumors, Compound D prevents acquired resistance to erlotinib.

[0304] Example 3: Compound D induces very high initial tumor size (700 mm 3 ) to prevent acquired resistance to erlotinib Experimental System : Patient-derived xenografts (PDX) of SCCHN squamous cell carcinoma tumor biopsies were subcutaneously implanted into NRG mice. I. Animals and biopsies Implantation of SCCHN tumor biopsy grafts (P11) into NRG mice for efficacy studies: Eight months after implantation of the above-mentioned SCCHN tumor biopsy grafts (P1), tumor cells (P11) were injected into 20 male, autologous NOD.Cg-Ragltm1Mom I12rgtmlWjl / SzJ mice (generic name: NRG) using the same procedure as described for implantation of P1. The original biopsy was the same as above, and P indicates the progression (number of grafts in the mouse). The onset of tumor growth (palpable tumor masses) was detected 6 days after cell injection. After 13 days, 19 of 20 injected mice had tumors measuring 700-720 mm 3 Tumors with an average size of 1000 mg / kg / day developed (day 0). The mice were randomly divided into four treatment groups, with four mice in the vehicle-treated group and five mice per group in the erlotinib, Compound D, or Compound D + erlotinib-treated groups. All treatments began simultaneously on day 0.

[0305] II. Treatments and Procedures Treatment groups included: 1. Vehicle control: 20% HPbCD 50 μl / injection, IV (3 times / week, every other day), 4 mice 2. Compound D 70 mg / kg in HPbCD, IV (3 times / week, every other day), 5 mice 3. Erlotinib in HPbCD 100 mg / kg, PO (5 times / week), 5 mice 4. Erlotinib 100 mg / kg PO (5 times / week) + Compound D 70 mg / kg IV (3 times / week), 5 mice. If administered on the same day, erlotinib was administered approximately 4 hours after Compound D administration.

[0306] The length (l) and width (w) of the tumor were measured four times a week, and the tumor volume was calculated as follows: v = lw 2 / 2. Graphs represent mean tumor volumes with standard error (standard deviation / square root of group size). Mice were routinely monitored for weight and behavior. Mice were sacrificed and tumors were excised for biochemical and genomic analysis.

[0307] result As shown in Figure 3, treatment with erlotinib resulted in a significant tumor response, with tumor growth halting and regression. However, during treatment, tumors developed resistance to erlotinib one week after treatment initiation and grew aggressively (Figure 3, open squares). Combination treatment with erlotinib plus Compound D (Figure 3, open squares) initiated on day 0 showed the same response as to erlotinib in the first week, but combination treatment with Compound D prevented acquired resistance to erlotinib, prevented tumor regrowth, and induced tumor regression, consistent with the results of Example 1.

[0308] conclusion In conclusion, when treatment was initiated, the initial tumor size was very high (700 mm 3 ), the combination treatment of Compound D plus erlotinib is highly effective and prevents acquired resistance to erlotinib.

[0309] Example 4: Combination treatment of Compound D and Afinitor effectively inhibits the growth of sarcoma patient-derived xenografts in mice Experimental system: Patient-derived xenografts (PDX) of uterine adenosarcoma biopsies are implanted subcutaneously into NodScid mice. I. Animals and biopsies Biopsy: Frozen human primary uterine adenosarcoma (Sample ID: OT_001) Tumor biopsy transplantation (P0): Female NOD.CB17-Prkdc scid / J (NodScid mice, Harlan, IL) were implanted subcutaneously (SC) with frozen human primary uterine adenosarcoma biopsy xenografts (P0). Three months later, tumors were excised, cut into small pieces, and implanted into 38 NodScid mice (P1) for efficacy testing. On day 8 after biopsy (P1) implantation, tumor development was detected in 37 mice. After one week (day 0), tumors in 33 mice were 130 mm 3 Once the mice reached an average size of 100 mg / kg, they were randomly divided into four treatment groups.

[0310] II. Treatments and Procedures Treatment groups included: 1. Control: 20% HPbCD 50 μl IP every other day (6 mice) 2. Compound D 70 mg / kg IV in 20% HPbCD every other day (6 mice) 3. Afinitor 5 mg / kg PO every other day (15 mice) 4. Afinitor 5 mg / kg PO (alternate day) + Compound D 70 mg / kg IV (alternate day), 6 mice. Afinitor was administered approximately 4 hours after Compound D administration. All treatments were initiated simultaneously on day 0.

[0311] The length (l) and width (w) of the tumor were measured every other day, and the tumor volume was calculated as follows: v = lw 2 / 2. The graph shows the mean tumor volume with standard error (standard deviation / square root of group size). On the fourth day after the start of treatment, tumors in the control and Compound D groups had already reached the endpoint, and the mice were sacrificed.

[0312] result As shown in Figure 4, treatment with Afinitor (open squares), an mTOR / S6K inhibitor, resulted in tumor growth inhibition: the mean tumor size in the control group increased 16-fold, whereas the mean tumor size in the Afinitor group increased 5.5-fold.

[0313] Surprisingly, Compound D alone (open triangles) had no significant effect on tumor growth, whereas the combination treatment of Afinitor + Compound D (open circles) resulted in tumor regression. The average tumor size of the combination treatment was 130 mm after only two treatments over four days. 3 From 70mm 3 It regressed to.

[0314] In terms of responder vs. non-responder mice, no response to Compound D alone was detected; only half of the mice in the Afinitor-treated group responded (Group A, n=8) and half did not respond (Group B, n=7); all mice in the combination treatment responded, and most tumors also significantly regressed.

[0315] Example 5: Compound D prevents acquired resistance to Afinitor (A) and causes regression of Afinitor-resistant tumors (B). Experimental system: Patient-derived xenografts (PDX) of uterine adenosarcoma biopsies are implanted subcutaneously into NodScid mice as described in Example 4.

[0316] The experiment described in Example 4 (Phase I) was extended into Phase II (FIG. 5A) and Phase III (FIG. 5B) of the experiment. After treatment as described in Phase I (Example 4), mice whose tumors reached the endpoint were sacrificed and continued treatment as follows: Phase II : 1. The Afinitor responder group (Group A, open squares) was orally administered Afinitor 5 mg / kg once daily (8 mice). 2. The combination treatment group (open circles) continued treatment with Afinitor 5 mg / kg orally every other day plus Compound D 70 mg / kg intravenously every other day (6 mice). Afinitor was administered approximately 4 hours after Compound D administration. Phase III : Tumors in the Afinitor responder group (Group A) regressed but developed resistance to Afinitor during treatment, reaching a mass of 590 mm on day 6. 3 The tumors grew aggressively to an average size of 1000 mg / kg. This group was divided into two groups of four mice each and, from day 6 onwards, received the following treatments: - Group 1 continued to receive oral Afinitor 5 mg / kg (once daily) (4 mice); A second group (4 mice) received a combination treatment of Afinitor 5 mg / kg orally (every other day) plus Compound D 70 mg / kg intravenously (every other day). Afinitor was administered approximately 4 hours after Compound D administration.

[0317] The length (l) and width (w) of the tumor were measured every other day, and the tumor volume was calculated as follows: v = lw 2 / 2. The graph in Figure 5A shows the mean tumor volume with standard error (standard deviation / square root of group size). The graph in Figure 5B shows the growth rate in %, with 100% for each tumor defined as the volume on day 6.

[0318] result Afinitor-treated groups were divided into responders (open squares, Group A, n=8) versus non-responders (gray squares, Group B, n=7). Afinitor treatment in Group A initially induced tumor regression (mean tumor size of 125 mm on day 0). 3 37mm on the 5th day 3 During treatment, all tumors developed resistance to Afinitor (590 mm on day 6). 3 The tumors grew aggressively (to an average tumor size of 1000).

[0319] Combination treatment with Afinitor and Compound D from day 0 (treatment initiation) induced tumor regression, and their mean tumor volumes remained low until the end of the experiment (Figure 5A, circles). Although early tumors did not respond to Compound D alone, acquired resistance to Afinitor was completely abolished by Compound D. Evidence from the literature suggests that treatment with Afinitor induces upregulation of IRS, leading to resistance through activation of the IGF1R / IRS-AKT survival pathway. mTOR / S6K is a negative regulator of IRS proteins. It phosphorylates IRS at serine residues, thereby downregulating IRS levels and reducing its affinity for the receptor tyrosine kinases (RTKs) IGF1R and IR. Inhibition of mTOR / S6K stabilizes IRS1 / 2, increasing their levels and enhancing complex formation between IGF1R and IR, leading to activation of the AKT survival pathway and acquired resistance to mTOR inhibitors. This feedback loop has been described in the literature (Crose LES and Linardic CM Sarcoma 2011, Keniry M. and Parsons R. Cancer Discovery 2011;1:203-204), where it has been shown that phosphorylation of AKT is a clinically observable phenomenon following treatment with the mTOR inhibitors Afinitor / Everolimus in women with breast cancer. Without being bound by any particular theory or mechanism of action, it is believed that removal of IRS1 / 2 from cancer cells by IRS / Stat3 dual modulators, such as Compound D and other compounds of Formulas (I-IV) described herein, may prevent acquired resistance to Afinitor or any other mTOR inhibitor and may synergize with these inhibitors to induce tumor regression after resistance has already been acquired.

[0320] After acquiring resistance to Afinitor, mice from group A were divided into two groups: the first group received Afinitor alone (□), and the second group received a combination treatment of Afinitor and Compound D (●) starting on day 6 of treatment. Tumors grew significantly under treatment with Afinitor alone (□), whereas the combination treatment of Compound D and Afinitor induced tumor regression (●). The graph in Figure 5B represents the growth rate in %, with 100% for each tumor defined as the volume on day 6.

[0321] Example 5A: Combination treatment with Afinitor plus Compound D in an aggressive cancer with no available medical treatment delayed acquired resistance to Afinitor and achieved complete remission in 40% of the group. Experimental system: Patient-derived xenografts (PDX) of uterine adenosarcoma biopsies are implanted subcutaneously into NodScid mice as described in Example 4. The experiment described in Example 4 was repeated with mice purchased from Harlan. treatment : 1. Control: 20% HPbCD 50 μl IP every other day (3 mice) 2. Compound D 70 mg / kg in 20% HPbCD, IV, 3 times a week, every other day (3 mice) 3. Afinitor 5 mg / kg PO, 4 times per week (17 mice) 4. Afinitor 5 mg / kg PO (every other day) + Compound D 70 mg / kg IV (every other day), 3 times per week (5 mice). Afinitor was administered approximately 4 hours after administration of Compound D. Treatment was discontinued on day 17.

[0322] The length (l) and width (w) of the tumor were measured every other day, and the tumor volume was calculated as follows: v = lw 2 / 2. The graph shows the mean tumor volume with standard error (standard deviation / square root of group size). Five days after the start of treatment, tumors in the control and Compound D groups had already reached the endpoint, and the mice were sacrificed.

[0323] result As shown in previous experiments, Compound D alone (open triangles) had no effect on tumor growth, whereas combined treatment with Afinitor and Compound D (filled circles) resulted in tumor regression. Tumors in the Afinitor responder group (14 of 17 treated mice) regressed but acquired resistance to Afinitor after one week of treatment and grew aggressively during treatment (open squares). Combined treatment with Afinitor and Compound D significantly delayed acquired resistance to Afinitor in 60% of the group (3 of 5 treated mice, open circles, dashed line) and completely eliminated tumors in 40% of this group (2 of 5 treated mice, open circles, solid line). These two mice remained alive without further treatment and remained disease-free for more than 3 months without further treatment (Figure 5C).

[0324] Example 6: I. cell line A375 (human melanoma), HCT15 (colon carcinoma), SK-ES.1 (Ewing's sarcoma), NCI-H460 (lung carcinoma), and PC3 (prostate carcinoma) cells were cultured in RPMI containing 10% fetal calf serum (FCS). HePG2 (hepatocellular carcinoma) cells were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% FCS and F12 (1:1). DU145 (prostate cancer) cells were cultured in RPMI containing 5% FCS and 5 mg / L insulin.

[0325] All cell lines were obtained from the American Type Culture Collection. YUMAC, YURIF, and YUSIK (all human melanomas kindly provided by Professor Ruth Halaban, Yale University, New Haven, Connecticut) were cultured in OptiMEM containing 5% FCS. M571, M2068, and M560n (all human melanomas kindly provided by Dr. Michal Rotham, Hadassah Hospital, Jerusalem, Israel) were maintained in RPMI, DMEM, and F12 (1:3:1) containing 10% FCS. 451-Lu (human melanoma) and 451-Lu-BR (PLX4032-resistant melanoma; ref. 32) were maintained in RPMI containing 5% FCS (medium for resistant cells containing 1 mmol / L PLX4032). All media were supplemented with 100 U / mL penicillin and 100 mg / mL streptomycin, and all cells were grown at 37° C. / 5% CO 2 .

[0326] All melanoma cells used and described in Figures 6-9 and Table 1 were of human origin and harbored mutant BRAF 600K / E It has the following characteristics.

[0327] II. Cell proliferation Cells were grown in complete medium and treated with inhibitors on day 1 after plating. After 72 hours, viable cells were quantified by methylene blue staining or WST-1 staining (Roche) for non-adherent cells.

[0328] III Immunoblotting Cells were treated after overnight serum starvation (unless otherwise indicated) as indicated in Figures 6-9 and the corresponding figure legends. When cells were treated with both PLX4032 and Compound A or D, PLX4032 was added 3-4 hours after compound addition. Cells were lysed in boiling sample buffer (10% glycerol, 50 mmol / L Tris-HCl, pH 6.8, 3% SDS, and 5% 2-mercaptoethanol). Western blot analysis was performed on 8% SDS-PAGE using the following antibodies: Aliquots of cell extracts containing equal amounts of protein were separated by 8% SDS-PAGE and electroblotted onto nitrocellulose filters. The membranes were blocked for 0.5 h with low-fat milk diluted 1:20 in TBST (NaCl / Tris containing 0.2% Tween-20) and incubated overnight at 4°C with rabbit anti-phosphorylated 705-Stat3 antibody (Cell Signaling catalog no. 9131), mouse anti-ERK-diphosphorylated YT (Sigma-Aldrich catalog no. M8159), or anti-PARP antibody in 5% BSA in TBST containing 0.05% azide. After extensive washing with TBST, the membranes were then incubated for 45 min at room temperature with horseradish peroxidase-conjugated secondary antibodies in 5% BSA in TBST. Immunoreactive bands were visualized using enhanced chemiluminescence. Membranes were reblotted with mouse anti-Stat3 antibody (Transduction Labs Cat. No. 21320) or rabbit anti-AKT1 / 2 (Santa Cruz Cat. No. sc-8312), or anti-actin as described above.

[0329] IV. Peripheral blood mononuclear cell (PBMC) chemotaxis A375 cells were seeded in 96-well plates (6,000 cells / well) and grown overnight. Cells were treated with Compound A and washed twice with medium at the indicated 4-hour post-treatment time points (wash). At 30 hours post-treatment, 150 μl of medium was transferred to the lower plate of the chemotaxis apparatus. 10,000 PBMCs / 75 μl of medium / well were added to the upper plate. Additionally, PBMCs were added to the lower plate as a positive control (Figure 9B - Cell Titer Glo calibration graph, 10-10,000 cells / well). Chemotaxis was examined 24 hours later by Cell Titer Glo analysis of the lower plate. Additionally, A375 cell viability was analyzed by methylene blue at 30 hours post-treatment with Compound A.

[0330] [Table 1]

[0331] Cells were seeded in 96-well plates in medium containing 5-10% FCS and exposed to various concentrations of Compound A, Compound D, or OSI-906 the next day. After 3 days, cells were stained with methylene blue and the relative cell numbers were quantified.

[0332] result Compounds A and D, previously shown to induce IRS1 / 2 serine phosphorylation, were found to efficiently induce a decrease in Stat3 Y705 phosphorylation levels in cancer cells. These dual IRS / Stat3 modulators potently inhibited Stat3 phosphorylation (pStat3) in a dose-dependent manner (Figure 6A) without affecting Stat3 protein levels. The inhibitory effect demonstrated by compounds A and D increased over time: IC50 values ​​for both compounds were approximately 2 μM at 1.3 h posttreatment and decreased to less than 1 μM after 3 h. The observed inhibitory effect on Stat3 phosphorylation levels was long-lasting (Figure 6B), as the modulators could be detected long after washing from the cells (Figure 6C). Figure 6C also shows that brief exposure of A375 melanoma cells to compound D was sufficient to induce cell apoptosis after 24 and 48 h. Blockade of Stat3 Y705-phosphorylation is exemplified for compounds A, B, C, D, F, IV-1, IV-2, IV-3 and IV-4.

[0333] Because Stat3 has been reported to be involved in both survival and drug resistance and immune evasion in various cancer types, we tested the ability of Stat3 / IRS dual modulators to sensitize tumors to specific PK inhibitors and immunotherapies, respectively.

[0334] Stat3 phosphorylation levels in melanomas that have acquired resistance to BRAF inhibitors (BRAFi), such as PLX4032 (also known as vemurafenib or Zelboraf), are significantly higher than in parental melanoma cells / tumors (Figures 7A and 7B). This is demonstrated in the metastatic melanoma clone 451-LU-BR (R) isolated after 6 months of treatment with BRAFi compared with the parental metastatic melanoma 451-LU cell line (P) [Villanueva et al., Cancer Cell 2010;18:683-95]. Higher levels of Stat3 phosphorylation are further demonstrated in cells obtained from patients (M2068, M560n, M571) who had been treated with PLX4032 and developed resistance to it (R) compared with melanoma cells from naive patients (N) who harbor mutant BRAF (YUMAC, YURIF, YUSIK) but have not yet been treated with BRAFi (Figure 7B).

[0335] Although Stat3 protein levels are similar in all samples, only phosphorylation levels are dramatically enhanced in PLX4032-resistant cells.

[0336] Surprisingly, we found that treatment with 1 μM PLX4032 for 18–24 hours significantly induced Stat3 Y705-phosphorylation (pStat3) in PLX4032-sensitive melanoma cells. This was tested and demonstrated in three different human metastatic melanoma cell lines (Figures 7C–E). The results in Figure 7 suggest that increased pStat3 may play a role in acquired resistance to BRAFi, with resistant cells adapting high and constant pStat3 levels as a survival factor. We therefore speculated that the combination of an IRS / Stat3 dual modulator with BRAFi might prevent acquired resistance to BRAFi and other drugs that induce upregulation of pStat3 and / or IRS1 and / or IRS2. The ability of an IRS / Stat3 dual modulator to prevent acquired resistance to such drugs is demonstrated in Example 1, where we show that compound D prevented acquired resistance to erlotinib in patient-derived HNSCC cells transplanted into mice.

[0337] Furthermore, pStat3 plays a key role in tumor immune evasion, masking tumors from the local immune system by upregulating the expression and secretion of immunosuppressive factors and downregulating pro-inflammatory mediators. In addition to cancer cells, diverse immune subsets in the tumor microenvironment also exhibit constitutively activated Stat3. Blocking Stat3 in immune cells can also induce potent antitumor immune responses, such as increased NK cell and neutrophil cytotoxicity, increased T cell activation, and tumor infiltration. Therefore, we hypothesized that the combination of our IRS / Stat3 dual modulator with immunotherapy could downregulate pStat3 and sensitize tumors to various immunotherapeutic agents.

[0338] Herein, we demonstrate that IRS / Stat3 dual modulators, represented by compounds A and D, blocked both basal and PLX4032-induced levels of pStat3, whereas the IGF1R / IR TK inhibitor OSI-906 had no effect on pStat3 levels (Figures 7E and 8A). To test the significance of this finding in terms of anticancer activity, we compared the antiproliferative activity of IRS / Stat3 dual modulators versus the IGF1R / IR TK inhibitor OSI-906 in various cancer types. Table 1 shows that compounds A and D were significantly more effective than OSI-906 in various melanoma cells (both PLX4032-resistant and PLX4032-sensitive); in colon cancer cells resistant to various chemotherapy regimens and EGFRi; in prostate cancer cells (resistant to several chemotherapy regimens); and in hepatocellular carcinoma cells (resistant to EGFRi). These differences between dual modulators and IGF1R / IR tyrosine kinase inhibitors suggest that inhibition of both Stat3 and IRS, central binding proteins critically involved in survival and drug resistance, may contribute to the ability of dual modulators to sensitize resistant cancer cells to various therapies.

[0339] As previously discussed in Figures 6A and 6B, pStat3 levels are increased in melanoma cells that have developed resistance to BRAFi. Figures 8A and 8B show that compounds A and D completely block Stat3 phosphorylation in these PLX4032-resistant clones of melanoma cell lines (451-LU-BR and Mel1617-BR [Villanueva et al., Cancer Cell 2010;18:683-95]) and in melanoma cells from two patients [M2068(i) and M571(ii)] who had acquired resistance to PLX4032 treatment (Figure 8C). Figure 8C also demonstrates the superior activity of compound D compared to compound A. These results suggest that IRS / Stat3 dual modulators can resensitize melanoma cells that have acquired resistance to BRAFi and that combined therapy with BRAFi and IRS / Stat3 dual modulators can induce tumor regression in resistant tumors. The ability of IRS / Stat3 dual modulators to resensitize drug-resistant tumors to drugs is demonstrated in Examples 2 and 3, demonstrating that the combination of Compound D with erlotinib induces regression of erlotinib-resistant HNSCC tumors in mice.

[0340] As previously demonstrated for their effects on IRS1 / 2 serine phosphorylation and removal, the ability of IRS / Stat3 dual modulators to inhibit pStat3 was demonstrated in various cancer types. Figure 8D illustrates the inhibitory activity of dual modulators on Stat3 Y705 phosphorylation (pStat3) in prostate cancer, multiple myeloma, Ewing's sarcoma, hepatocellular carcinoma, and NSCL.

[0341] The immune system is a powerful and largely untapped force in the fight against tumors. Tumors have developed sophisticated mechanisms to evade the immune system. Stat3 plays a key role in mediating crosstalk between tumor cells and tumor-interacting immune cells. Targeting Stat3 in tumors is associated with bystander tumor cell killing, associated with the infiltration of various immune effector cells. Therefore, we tested whether treatment of tumor cells with IRS / Stat3 dual modulators could induce the recruitment of peripheral blood mononuclear cells (PBMCs) to cancer cells. Human melanoma A375 cells were treated with increasing concentrations of Compound A and washed twice with medium (wash) at the indicated 4-h post-treatment time points. At 30 h post-treatment, cell medium was transferred to the lower plate of the chemotaxis apparatus, and 10,000 human PBMCs per well were added to the upper plate. Chemotaxis of PBMCs toward the A375 medium samples was examined 24 h later by Cell Titer Glo analysis of the lower plate. As shown in Figure 9A, dose-dependent chemotaxis was detected, suggesting that cytokine expression / secretion regulated by compound A induces PBMC recruitment toward the treated tumor. Thus, the combination of immunotherapy with our dual modulators may result in enhanced antitumor effects. These IRS / Stat3 dual modulators may sensitize tumors to other immunotherapies or PK inhibitors (e.g., EGFRi, mTORi) by directly and indirectly affecting tumor cells and the tumor microenvironment, including immune cells interacting with the tumor.

[0342] Example 7: Combination treatment with the EGFR antibody cetuximab and Compound D dramatically delayed tumor recurrence compared to cetuximab alone in mice bearing tumors from patients with head and neck squamous cell carcinoma (HNSCC). This was also the case when cetuximab plus afatinib was used instead of cetuximab.

[0343] Experimental System : Patient-derived xenografts (PDX) of HNSCC tumor biopsies are implanted subcutaneously into NodScid mice. I. Animals and biopsies Implantation of HNSCC tumor biopsy explants (P6) into NodScid mice for efficacy studies: Several months after implantation of frozen HNSCC tumor biopsy explants (P1) into mice, tumor cells (P6) were injected into NodScid mice (generated by autologous breeding) using the same procedure as described for implantation of P1. The original biopsy was the same as above, and P indicates the time course (number of implants in the mouse). The onset of tumor growth (palpable tumor mass) was detected 4 days after cell injection. Five days later, the tumor size reached approximately 113 mm 3 Treatment was initiated in mice that had reached the target of serotonin-positive (SNP) status. Mice were randomly divided into six treatment groups, with four animals per group treated with cetuximab, cetuximab + afatinib, cetuximab + Compound D, or cetuximab + afatinib + Compound D, and three mice from each group were treated with vehicle or Compound D. Treatments were initiated simultaneously (day 0) and were administered for 9 days.

[0344] II. Treatments and Procedures Treatment groups included: 1. Vehicle control: Vehicle (0.5% hydroxymethyl-cellulose, 0.4% Tween-80) 200 μl PO (5 times / week, once daily) 2.Compound D 70mg / kg in HPbCD, IV (3 times / week, every other day) 3. Cetuximab 1mg / mouse IP (twice a week) 4. Cetuximab 1 mg / mouse IP (2 times / week) + Compound D 70 mg / kg IV (3 times / week). If administered on the same day, cetuximab was administered approximately 4 hours after Compound D administration. 5. Cetuximab 1 mg / mouse IP in vehicle (twice weekly) + afatinib 25 mg / kg PO (5 times weekly) 6. Cetuximab 1 mg / mouse IP (2 times / week) + afatinib 25 mg / kg PO (5 times / week) + Compound D 70 mg / kg IV (3 times / week). If administered on the same day, cetuximab and / or afatinib were administered approximately 4 hours after Compound D administration.

[0345] The length (l) and width (w) of the tumor were measured 2–4 times per week, and tumor volume was calculated as follows: v = lw 2 / 2. Graphs represent mean tumor volumes with standard error (standard deviation / square root of group size). Mice were monitored for weight and behavior at least once a week. Mice were sacrificed and tumors were excised for biochemical and genomic analysis.

[0346] result As shown in Figure 10, during the first 4 days of treatment, all tumors grew, but then treatment with either cetuximab, cetuximab + Compound D, cetuximab + afatinib, or cetuximab + afatinib + Compound D resulted in dramatic tumor regression in all mice, whereas all tumors in vehicle-treated and Compound D-treated mice (as stand-alone treatments) grew aggressively. Treatment was applied for only 9 days.

[0347] Eight days after treatment was stopped, tumors in the cetuximab group began to regrow and grew aggressively, and one week later tumors in the cetuximab + afatinib group grew, but the combination with Compound D extended the positive response beyond four weeks after treatment ended.

[0348] Afatinib is a second-generation irreversible EGFR tyrosine kinase inhibitor developed to overcome acquired resistance to EGFR inhibitors, which arises from the EGFR T790M mutation, the most frequent mechanism of acquired resistance to EGFR tyrosine kinase inhibitors.

[0349] conclusion Combination treatment of Compound D with either cetuximab for only 9 days or additionally cetuximab plus afatinib significantly delayed recurrence of regressed tumors and prolonged the response to cetuximab or cetuximab plus afatinib.

[0350] Example 8: Compound D synergizes with a drug combination including inhibitors of mutant BRAF and MEK to induce dramatic tumor regression in mice implanted with tumor cells from a melanoma patient who had acquired resistance to vemurafenib. Experimental System : Patient-derived melanoma xenografts (PDXs) are injected subcutaneously into NodScid mice. I. Animals and biopsies · biopsy Biopsy revealed a mutant BRaF that showed a short-term response to vemurafenib V600E Early passage cells (million cells / mouse) were resected from melanoma patients bearing NOD.CB17-Prkdc mice and plated. scid Female NodScid / J mice were injected subcutaneously (SC). Tumor development was detected 5 days later, and treatment was initiated when the mean tumor volume reached approximately 60 mm. 3 The treatment was started 7 days after cell injection when the

[0351] II. Treatments and Procedures The tumor size is approximately 60 mm 3 On day 0, the following treatments were initiated: 5. Control (vehicle): Vehicle for vemurafenib + trametinib PO - 5% propylene glycol, 0.5% Tween-80, 30% PEG 400 in sterile DDW (5 times / week, once daily), 6 mice 6. Compound D 70 mg / kg in 20% 2-hydroxypropyl-β-cyclodextrin (HPbCD), IV (3 times / week, every other day), 6 mice 7. Vemurafenib 75mg / kg + trametinib 1mg / kg PO (5 times / week, once daily), 20 mice 8. Vemurafenib 75 mg / kg + trametinib 1 mg / kg PO (5 times / week, once daily) + Compound D 70 mg / kg IV (3 times / week), 7 mice. If administered on the same day, vemurafenib + trametinib were administered approximately 4 hours after Compound D. All treatments for each of treatment groups 1-4 were initiated simultaneously.

[0352] The length (l) and width (w) of the tumor were measured four times a week, and the tumor volume was calculated as follows: v = lw 2 / 2. Graphs represent mean tumor volumes with standard error (standard deviation / square root of group size). Mice were monitored for weight and behavior at least twice weekly.

[0353] result As shown in Figure 11, during treatment with vemurafenib + trametinib, tumors grew aggressively on the sixth day of treatment (Figure 11, open squares), but all tumors regressed upon combination treatment with Compound D (vemurafenib + trametinib + Compound D) (Figure 11, open circles).

[0354] Vemurafenib is a BRaf V600 This was the first mutant B-Raf inhibitor approved by the FDA for the treatment of melanoma patients harboring a mutation in BRaf. Unfortunately, several months after initiating treatment, the patient developed resistance to vemurafenib, and the regressed tumors relapsed more aggressively. Consequently, the combination of a mutant B-Raf inhibitor and a MEK inhibitor has not been shown to be effective in treating melanoma patients harboring a mutation in BRaf. V600 Although approved for the treatment of melanoma patients harboring mutations in BRAF, resistance still develops. The inventors show that two feedback pathways are induced by treatment of cells in culture with vemurafenib (a mutant BRaf inhibitor) or trametinib (a MEK inhibitor), resulting in increased levels of both IRS1 / 2 and phosphorylated STAT3. Both pathways are important for cell survival, proliferation, metastasis, and angiogenesis. Without being bound by any particular theory or mechanism of action, Compound D and the compounds of Formulae (I-IV) described herein are dual inhibitors of IRS1 / 2 and Stat3, and therefore may antagonize these mechanisms induced by MAPK pathway inhibitors (such as mutant BRaf inhibitors and MEK inhibitors) and synergize with these inhibitors (either alone or in combination), preventing resistance to these inhibitors.

[0355] Example 9: Compound D synergizes with the MEK inhibitor trametinib to induce tumor regression in mice bearing tumors from a patient with adenoid cystic carcinoma harboring a mutation in BRAF. Experimental System : Patient-derived xenografts (PDX) of adenoid cystic carcinoma tumor biopsies are implanted subcutaneously into NodScid mice. I. Animals and biopsies Biopsy: Fresh human primary adenoid cystic carcinoma tumor biopsy. Genomic analysis revealed a BRaf mutation. Implantation of adenoid cystic carcinoma RA_148 tumor biopsy explants into NodScid mice for efficacy studies. Implantation of tumor biopsy grafts (P0): Fresh human primary adenoid cystic carcinoma tumor biopsy grafts were implanted subcutaneously (SC) into NOD.CB17-Prkdcscid / J (NodScid mice). Implantation of tumor biopsy explants (P5) into NodScid female mice (generated by in-house breeding) for efficacy studies was performed using the same procedure as described above for implantation of HNSCC. The onset of tumor growth (palpable tumor masses) was detected in all mice 5 days after cell injection. After a further 3 days, the mice had tumors measuring approximately 65 mm. 3 The mice developed tumors with an average size of 1.5 mm. These mice were randomly divided into treatment groups containing 5 animals per group. The vehicle treatment group contained 4 mice.

[0356] II. Treatments and Procedures Treatment groups included: 1. Vehicle-Control: Trametinib vehicle (5% propylene glycol, 0.5% Tween-80, 30% PEG 400 in sterile DDW) 200 μl PO (5 times / week, once daily) 2.Compound D 70mg / kg in HPbCD, IV (3 times / week, every other day) 3. Trametinib 1 mg / kg PO (5 times a week, once daily) 4. Trametinib 1 mg / kg PO (5 times / week, once daily) + Compound D 70 mg / kg IV (3 times / week). If administered on the same day, trametinib was administered approximately 4 hours after Compound D.

[0357] All treatments for each of treatment groups 1-4 began simultaneously on day 0, and the study included two treatment phases: days 0-13 and 24-31.

[0358] The length (l) and width (w) of the tumor were measured 2–4 times per week, and tumor volume was calculated as follows: v = lw 2 / 2. Graphs represent mean tumor volumes with standard error (standard deviation / square root of group size). Mice were monitored for weight and behavior at least twice weekly.

[0359] result As shown in Figure 12, treatment with trametinib induced tumor regression, but tumors grew after 10 days of treatment. Combination treatment with trametinib and Compound D induced tumor regression, and none of these tumors regrew during treatment. Phase 2 treatment on days 24-31 induced dramatic tumor regression in all mice treated with trametinib, but the response was transient; after 4 days of treatment, tumors acquired resistance to this treatment and grew aggressively during treatment. Phase 2 treatment with the combination of trametinib and Compound D induced tumor regression, and none of these tumors regrew during treatment.

[0360] conclusion Treatment of early-stage tumors with Compound D alone resulted in modest tumor growth inhibition, whereas combination treatment with trametinib and Compound D resulted in dramatic tumor regression, and acquired resistance to trametinib was abolished by Compound D. Evidence from the literature suggests that treatment with trametinib induces upregulation of IRS, leading to resistance through activation of the IGF1R / IRS to AKT survival pathway. Other reports claim that trametinib induces Stat3 phosphorylation in cancer cells, leading to survival and acquired resistance to trametinib. Without being bound by any particular theory or mechanism of action, Compound D and other compounds of Formulas (I-IV) described herein are dual inhibitors of IRS1 / 2 and Stat3, and therefore may antagonize these trametinib-induced feedback mechanisms and prevent resistance.

[0361] Example 10: Compound D resensitizes gemcitabine-resistant tumors to gemcitabine in mice implanted with tumors from liver metastases of pancreatic cancer patients. Experimental System : Patient-derived xenografts (PDX) of biopsies of pancreatic cancer metastases from the liver are subcutaneously implanted into NodScid mice. I. Animals and biopsies Implantation of pancreatic cancer metastases from liver RA_160 tumor biopsy explants (P5) into NodScid mice for efficacy studies: Several weeks after implantation of pancreatic cancer liver metastasis biopsy explants into mice, tumor cells (P5) were injected into NodScid mice (generated by in-house breeding) using the same procedure as above. The onset of tumor growth (palpable tumor mass) was detected 10 days after cell injection. After 1 week (day 0), tumors grew to 90 mm. 3 Nineteen mice with an average tumor size of 1400 mm were treated with gemcitabine 25 mg / kg IP twice weekly for 35 days. By day 11, all tumors in the gemcitabine-treated mice had regressed, while tumors in all five control mice had grown. By day 21, the average tumor size in the gemcitabine-treated group was 1400 mm compared with 1400 mm in the control group. 3 Compared to about 5 mm 3 It was. Resistance developed 42 days after the start of gemcitabine treatment, and the tumors regressed and grew. After 4 days, the average tumor volume was approximately 110 mm 3 The 16 mice from the gemcitabine-treated group were divided into two groups as follows:

[0362] II. treatment Treatment groups after acquired resistance to gemcitabine included: 1. Gemcitabine 25mg / kg IP twice weekly 2. Gemcitabine 25mg / kg IP + Compound D 70mg / kg IV twice weekly.

[0363] The length (l) and width (w) of the tumor were measured 2–4 times per week, and tumor volume was calculated as follows: v = lw 2 / 2. Graphs represent mean tumor volumes with standard error (standard deviation / square root of group size). Mice were monitored for weight and behavior at least twice weekly.

[0364] result Mice were treated with gemcitabine for over a month until the regressed tumors acquired resistance to gemcitabine and grew. At this point, the gemcitabine-treated mice were divided into two groups (Figure 13): (a) gemcitabine (□); (b) gemcitabine + Compound D (◯). The average tumor size was approximately 110 mm. 3 Treatment was initiated when tumors reached a maximum of 100 mg / kg / day. All tumors treated with gemcitabine grew, but combination treatment with Compound D plus gemcitabine resulted in tumor regression in half of this group and significant tumor growth inhibition in terms of mean tumor size in this group compared to the gemcitabine-treated group (Figure 13A, p-value = 7.35 x 10 -5 ).

[0365] At the end of the experiment, similarly sized tumor pieces, three per group, were cultured in separate plates to test their viability and proliferation activity. After 9 days, plates were fixed and stained, demonstrating extensive proliferation in gemcitabine-treated tumors, in contrast to the very low to negligible proliferation activity in tumors from mice treated with gemcitabine plus Compound D (Figure 13B).

[0366] conclusion Compound D unexpectedly synergized with the chemotherapy drug gemcitabine to counteract the resistance that developed to gemcitabine in pancreatic cancer.

[0367] Example 11: Compound D prevents acquired resistance to cetuximab in mice bearing tumors from patients with metastatic adnexal adenocarcinoma Experimental System : Patient-derived xenografts (PDX) of human primary adnexal adenocarcinoma biopsies are implanted subcutaneously into NodScid mice.

[0368] 1. Animals and biopsies · biopsy : Fresh human adnexal adenocarcinoma metastasis (skin) biopsy (Sample ID: RA-162) · Implantation of tumor biopsy xenografts (P3) into NodScid mice for efficacy studies : The tumor (P2) is approximately 1500 mm 3 When the tumor tissues reached an average size of 1000 mg / kg, they were injected into 50 male NodScid mice, which were generated by in-house breeding in the animal facility of Bar-Ilan University, using the same procedure as described in Example 1. The tumor is approximately 90 mm 3 Nineteen mice that reached an average size of 100 were included in this study.

[0369] These mice were divided into four groups and the following treatments were initiated (day 0): Control: NT219 vehicle (20% HPbCD) 50 μl IV twice weekly - 5 mice Compound D 70 mg / kg IV twice weekly - 6 mice Cetuximab 1mg / mouse IP twice weekly - 5 mice Cetuximab (1 mg / mouse IP) + Compound D (70 mg / kg IV), twice weekly - 3 mice In the combination group, cetuximab was administered approximately 4 hours after Compound D administration.

[0370] The length (l) and width (w) of the tumor were measured four times a week, and the tumor volume was calculated as follows: v = lw 2 / 2. Graphs represent mean tumor volumes with standard error (standard deviation / square root of group size). Mice were monitored for weight and behavior at least once a week.

[0371] result After 5 days of treatment, mice in the control group were examined at the endpoint (1.5 cm 3 (defined as a tumor size greater than 100 μg / kg) (FIG. 14) and the mice were sacrificed.

[0372] Treatment with cetuximab resulted in a transient tumor growth attenuation, after which resistance to cetuximab was acquired and tumors grew during treatment (after day 26 of treatment).

[0373] Combination treatment with cetuximab plus Compound D resulted in significant tumor regression, but the cetuximab-treated group had a tumor regression rate of 500 mm 3 The mean tumor volume for the combination treatment (cetuximab + Compound D) was only 60 mm at the end of the experiment. 3 (Day 34).

[0374] Example 12. Compound D prevents acquired resistance to combined treatment with cetuximab and Forfiri (a treatment approved for colon cancer patients) in mice bearing tumors from colon cancer patients. Forfiri contains the following regimen: FOL - folinic acid (leucovorin), a vitamin B derivative used as a "rescue" drug for high doses of the drug methotrexate, but which increases the cytotoxicity of 5-fluorouracil; F-fluorouracil (5-FU), a pyrimidine analogue and antimetabolite that is incorporated into DNA molecules and stops their synthesis; and · IRI - Irinotecan (Camptosar), a topoisomerase inhibitor that unwinds DNA and prevents it from replicating.

[0375] Experimental System : Patient-derived xenografts (PDX) of human primary colon metastatic cancer biopsies are implanted subcutaneously into NodScid mice. 1. Animals and biopsies Biopsy: Fresh human colon cancer biopsy (Sample ID: RA-149) · Implantation of tumor biopsy xenografts (P4) into NodScid mice for efficacy studies : The tumor (P3) is approximately 1500 mm 3 When the tumor tissues reached an average size of 1000 mg / kg, they were injected into male NodScid mice, which were generated by in-house breeding in the animal facility of Bar-Ilan University, using the same procedure as described in Example 1. The tumor is approximately 110 mm 3 Thirty-six mice that reached an average size of 100 were included in this study.

[0376] Mice were divided into seven groups and the following treatments were initiated (day 0): Control: NT219 vehicle (20% HPbCD) 50 μl IV twice weekly - 5 mice Compound D 70 mg / kg IV twice weekly - 5 mice Cetuximab 1mg / mouse IP twice weekly - 5 mice FOLFIRI IP 5 times per week - 5 mice Cetuximab 1mg / mouse IP twice weekly + FOLFIRI IP 5 times weekly - 5 mice Cetuximab 1mg / mouse IP twice weekly + FOLFIRI IP 5 times weekly + Compound D 70mg / kg IV 2 times weekly - 6 mice In the combination group, cetuximab was administered approximately 4 hours after Compound D administration.

[0377] In colon cancer, cetuximab is not effective as a monotherapy and is therefore approved for patients in combination with chemotherapy such as forfiri, which includes folinic acid (Leucoberin), 5-FU, and irinotecan.

[0378] The length (l) and width (w) of the tumor were measured four times a week, and the tumor volume was calculated as follows: v = lw 2 / 2. Graphs represent mean tumor volumes with standard error (standard deviation / square root of group size). Mice were monitored for weight and behavior at least once a week.

[0379] result No effect on tumor growth was detected in groups treated with either cetuximab, Compound D, or Forfiri alone. However, the combination of Forfiri and cetuximab, with or without Compound D, resulted in significant tumor regression (Figure 15).

[0380] After 12 days of treatment, tumors in the cetuximab + forfili group developed resistance to the treatment and grew, while tumors in the cetuximab + forfili + Compound D group regressed and did not acquire resistance to the treatment (Figure 15).

[0381] conclusion The FDA approved cetuximab (Erbitux) in combination with the Forfili regimen as first-line treatment for patients with metastatic colorectal cancer who have tested negative for KRAS mutations. Cetuximab as monotherapy in colorectal cancer is generally ineffective. The RA_149 biopsy colon cancer PDX model, which follows clinical symptoms, shows that cetuximab as monotherapy is ineffective, but in combination with chemotherapy such as Forfili, it induces dramatic tumor regression. Additionally, as unfortunately observed in patients, acquired resistance occurs and tumors grow. The inventors demonstrate that combining this therapy with Compound D prevents acquired resistance to cetuximab plus Forfili and prolongs positive responses.

[0382] While particular embodiments of the present invention have been illustrated and described, it will be clear that the invention is not limited to the embodiments described herein. Numerous modifications, changes, variations, substitutions and equivalents will be apparent to those skilled in the art without departing from the spirit and scope of the invention, as defined by the following claims.

Claims

1. A pharmaceutical composition comprising an epidermal growth factor receptor (EGFR) inhibitor and / or an EGFR antibody together with a compound represented by the structure of formula (D) or a salt thereof for use in treating tumors that have developed resistance to EGFR inhibitors and / or EGFR antibodies, or for preventing acquired resistance of tumors to EGFR inhibitors and / or EGFR antibodies, or for preventing or delaying tumor recurrence after cessation of treatment with an EGFR inhibitor and / or EGFR antibody, wherein the structure of compound D is represented as follows: 【Chemistry 1】 the EGFR inhibitor is selected from the group consisting of AZD9291, afatinib, neratinib, dacomitinib, poziotinib, CO-1686, HM61713 and AP26113; The EGFR antibody is selected from the group consisting of cetuximab, panitumumab, and necitumumab. Pharmaceutical compositions.

2. The composition of claim 1 , wherein the EGFR inhibitor is AZD9291 or afatinib.

3. The composition of claim 1 , wherein the EGFR inhibitor is AZD9291.

4. 2. The composition of claim 1, wherein the EGFR antibody is cetuximab or panitumumab.

5. The composition of claim 1 , wherein the EGFR antibody is cetuximab.

6. The composition of claim 1 comprising compound D, cetuximab and afatinib.

7. 7. The composition of any one of claims 1 to 6, wherein the tumor is present in a cancer patient having a tumor that has acquired resistance to EGFR inhibitor and / or EGFR antibody treatment, and wherein the treatment results in attenuation of growth or regression of the resistant tumor.

8. The composition of any one of claims 1 to 7, wherein the tumor is present in a cancer patient who is undergoing or is a candidate for undergoing treatment with an EGFR inhibitor and / or an EGFR antibody.

9. 9. The composition of any one of claims 1 to 8, wherein the tumor is present in a cancer patient having a cancer selected from the group consisting of head and neck (H&N) cancer, sarcoma, pancreatic cancer, multiple myeloma, ovarian cancer, breast cancer, renal cancer, hematopoietic cancer, lymphoma, leukemia (including lymphoblastic leukemia), lung cancer, melanoma, glioblastoma, liver cancer, prostate cancer, thyroid cancer, esophageal cancer, gastric cancer, and colon cancer.

10. comprising a compound of formula D and cetuximab, the tumor is present in a patient with head and neck (H&N) cancer, thyroid cancer, esophageal cancer, gastric cancer, or colon cancer; The composition according to any one of claims 1 to 9.

11. Formula D: 【Chemistry 2】 or a salt thereof, for use in treating a tumor resistant to an EGFR inhibitor and / or an EGFR antibody, or for preventing acquired resistance to an EGFR inhibitor and / or an EGFR antibody, or for preventing or delaying tumor recurrence after cessation of treatment with an EGFR inhibitor and / or an EGFR antibody, comprising a compound represented by the structure: the pharmaceutical composition is for administration in combination with an epidermal growth factor receptor (EGFR) inhibitor and / or an EGFR antibody; the EGFR inhibitor is selected from the group consisting of AZD9291, afatinib, neratinib, dacomitinib, poziotinib, CO-1686, HM61713 and AP26113; The EGFR antibody is selected from the group consisting of cetuximab, panitumumab, and necitumumab. Pharmaceutical compositions.

12. The composition of claim 11, wherein the EGFR inhibitor is AZD9291 or afatinib.

13. The composition of claim 11, wherein the EGFR inhibitor is AZD9291.

14. 12. The composition of claim 11, wherein the EGFR antibody is cetuximab or panitumumab.

15. The composition of claim 11 , wherein the EGFR antibody is cetuximab.

16. 12. The composition of claim 11, wherein the EGFR antibody is cetuximab and the EGFR inhibitor is afatinib.

17. The composition of claim 11 , wherein the EGFR inhibitor and / or EGFR antibody and the compound represented by the structure of Formula D are administered in the same pharmaceutical composition.

18. The composition of any one of claims 11 to 16, wherein the EGFR inhibitor and / or EGFR antibody and the compound represented by the structure of Formula D are administered in separate pharmaceutical compositions, simultaneously or sequentially in any order.

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