FIELD OF THE INVENTION

The present invention relates to the treatment of cancer using combination therapy comprising a tyrphostin derivative in combination with another anti-cancer agent, wherein the effect of the tyrphostin derivative and the anti-cancer agent is at least additive, and is in some embodiments synergistic.

BACKGROUND OF THE INVENTION

Tyrphostins are a family of protein tyrosine kinase inhibitors, designed to mimic the tyrosine substrate, the ATP and can inhibit allosterically 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; US Pat. Nos. 5,217,999 and 5,773,476, Posner et al., Mol. Pharmacol. (1994), 45:673-683). The pharmacophores of these tyrphostins, and in particular tyrphostins of the benzylidene malonitril type, are the hydrophilic catechol ring and the more lipophilic substituted cyano- inyl radical. Kinetic studies have shown that some tyrphostin compounds are pure competitive inhibitors vis-a-vis tyrosine substrates whereas for the ATP binding site they act as non-competitive inhibitors (Yaish et al., Science (1988), 242:933-935; Gazit et al., J Med. Chem. (1989), 32:2344-2352). Nonetheless, many tyrphostins have shown competitive inhibition against both the substrate and ATP binding site or mixed competitive (Posner et al., Mol. Pharmacol. (1994), 45:673-683).

In a related group of tyrphostins, the hydrophilic catechol ring was exchanged by lipophilic dichloro- or dimethoxy-phenyl groups, to yield EGFR kinase inhibitors, effective in the low micromolar range (Yoneda et al., Cancer Res. (1991), 51: 4430-4435). These tyrphostins were further administered to tumor-bearing nude mice together with anti-EGFR monoclonal antibodies at a suboptimal dose to afford markedly enhanced inhibition of tumor growth.

WO 2008/0687 1 to some of the inventors of the present invention, discloses novel tyrphostin compounds having increased inhibitory properties of insulin-like growth factor 1 receptor (IGF1R), platelet derived growth factor receptor (PDGFR), epidermal growth factor receptor (EGFR), and IGFlR-related insulin receptor (IR) activation and signaling.

WO 2009/147682 to some of the inventors of the present invention discloses new tyrphostin derivatives acting as protein kinase (PK) and receptor kinase (RK) signaling modulators. Further disclosed in WO 2009/147682 are methods of preparation of the tyrphostin derivative, pharmaceutical compositions including such compounds, and methods of using these compounds and compositions, especially as chemotherapeutic agents for preventions and treatments of PK and RK related disorders such as metabolic, inflammatory, fibrotic, and cell proliferative disorders, in particular cancer.

Cancers treated with conventional radio- or chemo-therapy or other anti-cancer agents frequently develop resistance to these treatments, ultimately leading to recurrent disease that often has a more aggressive phenotype than that observed at the time of the original diagnosis (Li et al., J. Med. Chem. (2009), 52(16): 4981-5004).

In accordance with principles for selecting agents for use in combination chemotherapy regimens, drugs with different mechanisms of action and with additive or synergistic cytotoxic effects on the tumor can be combined (Pazdur et al., Chapter 3: Principles of Oncologic Pharmacotherapy (2005), 9 ^th Edition:23-42). Multi-agent therapy has three important theoretical advantages over single-agent therapy. First, it can maximize cell death while minimizing host toxicities by using agents with non-overlapping dose- limiting toxicities. Second, it may increase the range of drug activity against tumor cells with endogenous resistance to specific types of therapy. Finally, it may also prevent or slow the development of newly resistant tumor cells. Virtually, almost all curative chemotherapy regimens for cancer employ multi-agent drug combinations (Frei and Eder, Cancer medicine (2003), 11:817-837).

Traditional chemotherapeutic agents can be classified by mechanism of action. The alkylating agents impair cell function by forming covalent bonds with the amino, carboxyl, sulfliydryl, and phosphate groups in biologically important molecules. The most important sites of alkylation are DNA, RNA, and proteins. Alkylating agents depend on cell proliferation for activity but are not cell-cycle-phase-specific. Alkylating agents are classified according to their chemical structures and mechanisms of covalent bonding; this drug class includes the nitrogen mustards, nitroso-ureas (BCNU), platinum complexes (cisplatin) and others (dacarbazine). Taxanes are semi-synthetic derivatives of extracted precursors from the needles of yew plants. These drugs have a novel 14-member ring, the taxane. The taxanes (e.g., taxol) promote microtubular assembly and stability, therefore blocking the cell cycle in mitosis. Anti-tumor antibiotics like adriamycin intercalate DNA at guamne-cytosine and guanine-thymine sequences, resulting in spontaneous oxidation and formation of free oxygen radicals that cause strand breakage. Three in vitro models of simulated hypoxia showed that cells under hypoxic conditions are more sensitive than cells under aerobic conditions to agents that inhibit glycolysis, such as 2-deoxy-D-glucose (Hu et al., Biochem. Pharmacol. (2000), 60:1897- 1905; Liu et al., Biochemistry (2001), 40:5542-5547; Liu et al., Biochem. Pharmacol. (2002), 64:1746-1751). Because a slowly proliferating tumor population can be selectively killed with glycolytic inhibitors, combining such agents with chemotherapeutic drugs, which target the rapidly dividing aerobic cells, should raise the overall efficacies of these treatments. Indeed, the combination of 2-deoxy-D-glucose and cisplatin is more effective than either agent alone when applied to various cell lines that are rapidly proliferating in vitro (Yamada et al., Cancer Chemother. Pharmacol. (1999), 44: 59-64). Similar in vitro synergism has been observed with the combination of 2-deoxy-D-glucose and adriamycin (ADR) in MCF7 cells (Kaplan et al., Cancer Res (1990), 50:544-551).

A family of relatively new anti-cancer agents are inhibitors (e.g. antibodies and small molecules) of specific kinases or other signaling enzymes involved in the mitogenic, anti- apoptotic, angiogenic or metastatic pathways in the cancerous cells. Examples of approved drugs included in this family are the EGFR and/or HER2 blockers (e.g. the small molecules gemfrab, erlotinib, lapatinib or antibodies like 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), VEGFR/PDGFR and/or multi kinase inhibitors (e.g. bevacizumab (Avastin®), sorafenib, sunitinib, and pazopanib), and proteasome inhibitors (e.g. bortezomib (Velcade®)) etc. AG1478 (N-(3-Chlorophenyl)-6,7- dimethoxy-4-quinazolinanine hydrochloride) is an example of the family of EGFR inhibitors. Several EGFR inhibitors were approved by the FDA like Tarceva (Erlotinib) in 2004, Iressa (Gefitinib) in 2003, and Lapatinib in 2010, as well as antibodies against EGFR.

The pharmacological activity of tyrphostin compounds makes them attractive candidates as therapeutic agents for the treatment of cancer, alone or in combination with additional anti-cancer agents. There is an unmet need for combinations that are useful for treating cancer, preferably providing at least additive therapeutic effects. Combinations of drugs from different categories are useful to prevent or overcome emergence of drug resistant tumors. SUMMARY OF THE INVENTION

The present invention relates to compositions and methods for treating cancer, by administering a combination comprising at least one tyrphostin derivative of formula I, or any of the tyrphostin derivatives exemplified by this formula, in combination with at least one other anti-cancer agent. In some embodiments, the anti-cancer agent is selected from a protein kinase inhibitor, a proteasome inhibitor, a topoisomerase inhibitor, an alkylating agent and a metabolic inhibitor.

It has been discovered unexpectedly that the combination of a first treatment that includes administration of a tyrphostin derivative of formula (I), as described herein, or any agent covered by such formula, and a second treatment that includes at least one other anticancer agent, preferably a protein kinase inhibitor, a proteasome inhibitor, a topoisomerase inhibitor, an alkylating agent and a metabolic inhibitor, can provide therapeutically beneficial anti-cancer effects. In some embodiments, the effect is synergistic, i.e., the tyrphostin derivative and the at least one other agent together produce a significantly better anti-cancer result (e.g., cell growth arrest, apoptosis, induction of differentiation, cell death, etc.) than the additive effects of the individual constituents. In other embodiments, the effect is additive, i.e., the tyrphostin derivative and the at least one other agent together produce an anti-cancer result (e.g., cell growth arrest, apoptosis, induction of differentiation, cell death, etc.) that is the additive effects of the individual constituents. Each possibility represents a separate embodiment of the present invention.

The combination therapy is particularly advantageous, since the dosage of each agent in a combination therapy can be reduced as compared to mono-therapy with each agent, while still achieving an overall anti-cancer effect. Accordingly, reducing the dosage of each agent may result in decreased side effects. The combination therapy may further reduce the development of resistance to a specific anti-cancer treatment.

The present invention thus provides a pharmaceutical combination comprising a tyrphostin derivative of formula (I), or any agent covered by such formula, in combination with at least one other anti-cancer agent, wherein the tyrphostin derivative and the at least one other anti-cancer agent together provide a therapeutic anti-cancer effect which is at least additive.

In another embodiment, the present invention provides a pharmaceutical combination comprising a tyrphostin derivative of formula (I), or any agent covered by such formula, in combination with at least one other anti-cancer agent selected from a protein kinase inhibitor, a proteasome inhibitor, a topoisomerase inhibitor, an alkylating agent and a metabolic inhibitor, wherein the tyrphostin derivative and the at least one other anti-cancer agent together provide a therapeutic anti-cancer effect which is at least additive.

In another embodiment, the present invention provides a method of treating cancer comprising administering to the subject in need thereof a therapeutically effective amount of a tyrphostin derivative of formula (I), or any agent covered by such formula, in combination with at least one other anti-cancer agent, wherein the tyrphostin derivative and the at least one other anti-cancer agent together provide a therapeutic anti-cancer effect which is at least additive.

In another embodiment, the present invention provides a method of treating cancer comprising administering to the subject in need thereof a therapeutically effective amount of a tyrphostin derivative of formula (I), or any agent covered by such formula, in combination with at least one other anti-cancer agent selected from a protein kinase inhibitor, a proteasome inhibitor, a topoisomerase inhibitor, an alkylating agent and a metabolic inhibitor, wherein the tyrphostin derivative and the at least one other anti-cancer agent together provide a therapeutic anti-cancer effect which is at least additive.

In another embodiment, the present invention provides the use of a tyrphostin derivative of formula (I), or any agent covered by such formula, in combination with at least one other anti-cancer agent, wherein the tyrphostin derivative and the at least one other anticancer agent together provide a therapeutic anti-cancer effect which is at least additive, for treating cancer.

In another embodiment, the present invention provides the use of a tyrphostin derivative of formula (I), or any agent covered by such formula, in combination with at least one other anti-cancer agent selected from a protein kinase inhibitor, a proteasome inhibitor, a topoisomerase inhibitor, an alkylating agent and a metabolic inhibitor, wherein the tyrphostin derivative and the at least one other anti-cancer agent together provide a therapeutic anticancer effect which is at least additive, for treating cancer.

The term "in combination" or "combined treatment" as used herein denotes any form of concurrent or parallel treatment with at least two distinct therapeutic agents. This term is intended to encompass both concomitant administration of the two treatment modalities, i.e., using substantially the same treatment schedule, as well as overlapping administration in sequential or alternating schedules of each treatment. Each possibility represents a separate embodiment of the present invention.

The tyrphostin derivative and the at least one other anti-cancer agent can be administered simultaneously (in the same or in separate dosage forms), or they can be adnii istered sequentially, in any order. The administration can also take place according to alternating dosing schedules, e.g., tyrphostin derivative followed by the at least one other anti-cancer agent, then an additional dose of a tyrphostin derivative, followed by the same or yet another anti-cancer agent and so forth. All administration schedules, including simultaneous, sequential and alternating, are contemplated by the present invention, wherein each possibility represents a separate embodiment of the present invention.

In one embodiment, the tyrphostin derivative and the at least one other anti-cancer agent together provide a therapeutic anti-cancer effect which is synergistic.

According to the principles of the present invention, the at least one other anti-cancer agent is preferably selected from the group consisting of a protein kinase inhibitor, a proteasome inhibitor, a topoisomerase inhibitor, an alkylating agent and a metabolic inhibitor. However, any other anti-cancer agent described herein is suitable for use in the combinations of the present invention. Each possibility represents a separate embodiment of the present invention.

In some embodiments, the protein kinase inhibitor is selected from EGFR and/or HER2 inhibitors (e.g. small molecules such as genifitib, erlotinib, lapatinib and AG 1478 or antibodies such as trastuzumab and cetuximab), B-Raf inhibitors and/or C-Raf inhibitors (e.g. PLX-4032, PLX-4720 and sorafenib), BCR-ABL and/or Src kinase inhibitors (e.g. imatinib, dasatinib and nilotinib), IGF1R inhibitors (e.g., NVP-AEW-541 (7-[cis-3-(l- azetidmyhne1hyl)cyclobutyl]-5-[3-( hen^

amine, dihydrochloride) and VEGFR/PDGFR and/or multi kinase inhibitors (e.g. bevacizumab, sorafenib, sunitinib and pazopanib). Each possibility represents a separate embodiment of the present invention.

In one embodiment, the protein kinase inhibitor is selected from sorafenib, PLX-4720, PLX-4032,AG-1478, NVP-AEW 541 and sunitinib.

In another embodiment, the proteasome inhibitor is bortezomib (PS-341, Velcade®).

In other embodiments, the topoisomerase inhibitor is irinotecan.

In further embodiments, the alkylating agent is dacarbazine or cisplatin.

In further embodiments, the metabolic inhibitor is 2-deoxyglucose (2DG). Any type of combination of the compound of formula (I) and the anti-cancer agent(s) as described herein is contemplated, with non-limiting embodiments including: (i) a tyrphostin derivative of formula A and an anti-cancer agent selected from the group consisting of bortezomib, sorafenib, dacarbazine, irinotecan, cisplatin, PLX-4720, PLX-4032, AG 1478, sunitinib and 2-deoxy glucose; or (ii) a tyrphostin derivative of formula C and cisplatin; or (iii) a tyrphostin derivative of formula D, and an anti-cancer agent selected from AG- 1478 or NVP-AEW-541; or (iv) a tyrphostin derivative of formula E, and cisplatin. Each possibility represents a separate embodiment of the present invention:

The tyrphostin derivative useful in the combinations of the present invention is any compound represented by the structure of formula I:

wherein

R ^l , R ² , R ⁵ and R ⁶ are each independently selected from H, CpC ₄ alkyl, C ₂ -C ₆ alkenyi, C ₂ -C ₆ alkynyl, d-C ₄ alkyl-C ₂ -C ₆ alkenyi, C _r C ₄ alkyl-C ₂ -C ₆ alkynyl, (CH ₂ CH ₂ 0) _n H, C ₃ -C ₇ cycloalkyl, aryl, heterocyclyl, heteroaryl, (Ci-C ₄ )-alkylaryl, (C ₁ -C4)-alkylheterocyclyl, (d- C ₄ )-alkylheteroaryl, haloalkyl, acyl and a functional group that gives rise to hydroxyl upon hydrolysis;

R ³ , R ⁴ , R ⁷ , R ⁸ , R ⁹ , R ¹⁰ , R ^{1 L} , R ¹² , R ¹³ and R ¹⁴ are each independently selected from H, Ci-C alkyl, C ₂ -C ₆ alkenyi, C ₂ -C ₆ alkynyl, Ci-C ₄ alkyl-C ₂ -C ₆ alkenyi, Q-C ₄ alkyl-C ₂ -C ₆ alkynyl, C ₃ -C ₇ cycloalkyl, aryl, heterocyclyl, heteroaryl, (Ci-C4)-alkylaryl, (CrQ)- alkylheterocyclyl, (Ci-C ₄ )-alkylheteroaryl, halogen, haloalkyl, N0 ₂ , CN, N ₃ , S0 ₂ R ^a , COOR ^a , CSNR ^a R , CSOR ^a , OR ⁸ , CONR ^a R ^b , NR ^a R ^b , SR ^a , and CH ₂ SR ^a , wherein R ^a and R ^b are each independently H, d-C ₄ alkyl, C ₂ -C6 alkenyi, C ₂ -C ₆ alkynyl, d-C ₄ alkyl-C ₂ -C ₆ alkenyi, C!-C ₄ alkyl-C ₂ -C ₆ alkynyl, C ₃ -C ₇ cycloalkyl, aryl, heterocyclyl, heteroaryl, (C ₁ -C4)-alkylaryl, (d- C ₄ )-alkylheterocyclyl, (C ₁ -C ₄ )-alkylheteroaryl, haloalkyl, (CH ₂ CH ₂ 0) _n H, acyl or a functional group that gives rise to hydroxyl upon hydrolysis; and

R ¹⁵ is H, Ci-C ₄ alkyl, C ₂ -C ₆ alkenyi, C ₂ -C ₆ alkynyl, d-C ₄ alkyl-C ₂ -C ₆ alkenyi, d-C ₄ alkyl-C ₂ -C6 alkynyl, haloalkyl, or OR ^b wherein R ^b is independently H or Cj-C ₄ alkyl; including salts, hydrates, solvates, polymorphs, optical isomers, geometrical isomers, enantiomers, diastereomers, and mixtures thereof. Each possibility represents a separate embodiment of the invention.

In various embodiments, the tyrphostin derivative is represented by any of the formulae A through E:

B

E

In one embodiment, the tyrphostin derivative is a compound of formula A. In another embodiment, the tyrphostin derivative is a compound of formula B. In another embodiment, the tyrphostin derivative is a compound of formula C. In another embodiment, the tyrphostin derivative is a compound of formula D. In another embodiment, the tyrphostin derivative is a compound of formula E. Each possibility represents a separate embodiment of the invention.

In another embodiment, the tyrphostin compound is represented by the structure of formula II:

(H)

wherein

R ¹ , R ² , R ⁵ and R ⁶ are independently selected from H, C ₁ -C ₄ alkyl, (CH ₂ CH ₂ O),,, wherein n is an integer of 1 to 20, acyl and a functional group that gives rise to hydroxyl upon hydrolysis;

R ³ and R ⁷ are independently selected from H, halogen, Ci-C ₄ alkyl, haloalkyl and OR ¹⁶ wherein R ¹⁶ is H, Q-C4 alkyl, (CH ₂ CH ₂ 0) _n , acyl or a functional group that gives rise to hydroxyl upon hydrolysis; and

R ⁴ is H or CN.

In another embodiment, the tyrphostin compound is represented by the structure of formula III:

(HI) wherein

R ¹ , R ² , R ⁵ and R ⁶ are independently selected from H, C ₁ -C4 alkyl, (CH ₂ CH20) _n , wherein n is an integer of 1 to 20, acyl and a functional group that gives rise to hydroxyl upon hydrolysis;

R ³ , R ⁷ , R ⁸ , R ⁹ , R ¹⁰ , R ¹¹ , R ¹² , R ¹³ and R ^M are independently selected from H, halogen, C _r C ₄ alkyl, haloalkyl and OR ¹⁶ wherein R ¹⁶ is H, Q-C ₄ alkyl,

(CH ₂ CH ₂ 0) _n , acyl or a functional group that gives rise to hydroxyl upon hydrolysis; and

R ⁴ is H or CN.

including salts, hydrates, solvates, polymorphs, optical

isomers, geometrical isomers, enantiomers, diastereomers, and mixtures thereof.

Each possibility represents a separate embodiment of the invention.

In some embodiments, the tyrophotin compounds of the invention (e.g., compounds of formula I, II, A, B, C, D or E or any compound covered by such formulae) show increased inhibitory properties of, but not limited to, insulin-like growth factor 1 receptor (IGF-IR), platelet derived growth factor receptor (PDGFR), epidermal growth factor receptor (EGFR), and IGFlR-related insulin receptor (IR), or proteins affected by or mediated by these PTKs or that are part of the PTK-mediated signal transduction pathway. For example, in some embodiments, the compounds of the present invention are potent inhibitors of insulin-like growth factor 1 receptor (IGF-IR) and or insulin receptor substrate 1 (IRSl) and/or insulin receptor substrate 2 (IRS2) signaling. As such, these compounds are useful in inhibiting, treating or preventing an IGF-IR and/or IRSl and/or IRS2 signaling related disorder, for example cancer. In some embodiments, the tyrphostin compounds trigger any one or more of the following, in any order: (i) serine phosphorylation of the IGF-IR direct substrates IRSl and/or IRS2; (ii) dissociation of IRSl and or IRS2 from the cell membrane; and/or (iii) degradation of IRSl and/or IRS2, thus providing long-lasting effects which enhance the inhibitory activity of these compounds. In some embodiments, the tyrphostin compounds lead to the inhibition of Stat3 phosphorylation in the cancer cells.

The pharmaceutical compositions of the present invention can be provided in any form known in the art, for example in a form suitable for oral administration (e.g., a solution, a suspension, a syrup, an emulsion, a dispersion, a tablet, a pill, a capsule, a pellet, granules and a powder), for parenteral administration (e.g., intravenous, intramuscular, intra-arterial, transdermal, subcutaneous or intra-peritoneal), for topical administration (e.g., an ointment, a gel, a cream), for administration by inhalation or for administration via suppository. Each possibility represents a separate embodiment of the present invention.

The combinations of the present invention are suitable for treating various types of cancer.

In particular, the combinations of the present invention are active against multiple myeloma, ovarian cancer, breast cancer, kidney cancer, stomach cancer, hematopoietic cancers, lymphoma, leukemia, including but not limited to lymphoblastic leukemia, lung carcinoma, melanoma, glioblastoma, hepatocarcinoma, prostate cancer and colon cancer. Each possibility represents a separate embodiment of the present invention.

In some non-Hmiting embodiments, the present invention provides methods and compositions for the treatment of cancer wherein:

(i) the tyrphostin derivative is a compound of formula A, the anticancer agent is bortezomib and the cancer is multiple myeloma; or

(ii) the tyrphostin derivative is a compound of formula A, the anticancer agent is sorafenib and the cancer is melanoma; or

(iii) the tyrphostin derivative is a compound of formula A, the anticancer agent is dacarbazine and the cancer is melanoma; or

(iv) the tyrphostin derivative is a compound of formula A, the anticancer agent is lrinotecan and the cancer is melanoma; or

(v) the tyrphostin derivative is a compound of formula A, the anticancer agent is cisplatin and the cancer is ovarian cancer; or

(vi) the tyrphostin derivative is a compound of formula C, the anticancer agent is cisplatin and the cancer is ovarian cancer; or

(vii) the tyrphostin derivative is a compound of formula E, the anti-cancer agent is cisplatin and the cancer is ovarian cancer; or

(viii) the tyrphostin derivative is a compound of formula A, the anticancer agent is PLX-4720 or PLX-4032, and the cancer is melanoma; or

(ix) the tyrphostin derivative is a compound of formula D, the anticancer agent is AG- 1478, and the cancer is hepatocarcinoma; or

(x) the tyrphostin derivative is a compound of formula A, the anticancer agent is AG- 1478, and the cancer is breast cancer; or (xi) the tyrphostin derivative is a compound of formula A, the anticancer agent is AG- 1478, and the cancer is glioblastoma, or

(xii) the tyrphostin derivative is a compound of formula A, the anticancer agent is sunitinib, and the cancer is ovarian cancer; or

(xiii) the tyrphostin derivative is a compound of formula D, the anticancer agent is NVP-AEW-541, and the cancer is ovarian cancer; or

(xiv) the tyrphostin derivative is a compound of formula A, the anticancer agent is irinotecan and the cancer is colon cancer; or

(xv) the tyrphostin derivative is a compound of formula A, the anticancer agent is 2-deoxyglucose, and the cancer is melanoma.

Further embodiments and the full scope of applicability of the present invention will become apparent from the detailed description given hereinafter. However, it should be understood 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 this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1A. Human multiple myeloma RPMI8226 cell proliferation. Combination of compound A and Velcade®. lAi = compound A (0.3μΜ); lAii = compound A (Ι Μ).

Figure IB. Human multiple myeloma MM IS cell proliferation. Combination of compound A and Velcade®. IBi = compound A (Ο.Ι Μ); IBii compound A (0.3μΜ).

Figure 2. Human melanoma A375 cell proliferation. Combination of compound A and sorafenib.

Figure 3. Human melanoma A375 cell proliferation. Combination of compound A and dacarbazine.

Figure 4. Human melanoma A375 ceil proliferation. Combination of compound A and irinotecan.

Figure 5. Ovarian cancer A2780 cell proliferation. Combination of compounds A, C, and E (5A, 5B and 5C, respectively) with cisplatin.

Figure 6. Combined effects of compound A and BRAF inhibitors PLX4720 (6A&B) or PLX4032 (6C) in human melanoma A375 cell proliferation. BRAF inhibitors were added 5hr (6A&C) or 24hr (6B) following treatment with compound A; 6A&B = compound A (0.6μΜ); 6C = PLX4032 (0.15μΜ).

Figure 7. Combined effect of compound A or D with EGFR inhibitor AG 1478 in human hepatocellular carcinoma HepG2 cells (A), human breast cancer MDA-MB-231 cells (B) and human glioblastoma U87MGwtEGFR overexpressing EGFR (C). 7A = AG 1478 (ΙΟμΜ); 7B = AG1478 (2μΜ); 7C = compound A (ΙμΜ).

Figure 8. Combined effect of Sutent with compound A in human ovary cancer A2780 cell proliferation. Sutent was added 4hr following treatment with 0.15uM compound A.

Figure 9. Combined effect of IGF1R inhibitor NVP-AEW-541 and compound D in human ovary cancer A2780 cell proliferation. NVP-AEW-541 was added 4hr following treatment with 0.05μΜ compound D.

Figure 10. Combined effect of compound A and irinotecan in human colon cancer HCT-15 cell proliferation. Irinotecan was added 5hr following treatment with 0.2μΜ compound A.

Figure 11. Combined effect of compound A and 2-deoxy-glucose (2DG) in human melanoma A375 cell proliferation. 2DG was added 5.5hr following treatment with 0.25uM compound A.

DETAILED DESCRIPTION OF THE PRESENT INVENTION

The present invention relates to compositions and methods for treating cancer, by administering a combination comprising a tyrphostin derivative in combination with at least one other anti-cancer agent. The tyrphostin derivative and the at least one other anti-cancer agent are administered in an amount which provides a therapeutic anti-cancer effect, which is at least additive. In one embodiment, tyrphostin derivative and the at least one other anticancer agent are administered in an amount which provides a therapeutic anti-cancer effect which is synergistic.

The combination therapy can provide a therapeutic advantage in view of the differential toxicity associated with the two individual treatments. For example, treatment with a tyrphostin derivative can lead to a particular toxicity that is not seen with the at least one other anti-cancer agent, and vice versa. As such, this differential toxicity can permit each treatment to be administered at a dose at which said toxicities do not exist or are minimal, such that together the combination therapy provides a therapeutic dose while avoiding the toxicities of each of the constituents of the combination agents. Furthermore, when the therapeutic effects achieved as a result of the combination treatment are enhanced or synergistic, i.e., significantly better than additive therapeutic effects, the doses of each of the agents can be reduced even further, thus lowering the associated toxicities to an even greater extent.

The terms "synergistic", "cooperative" and "super-additive" and their various grammatical variations are used interchangeably herein. An interaction between a tyrphostin derivative and another anti-cancer agent is considered to be synergistic, cooperative or super- additive when the observed effect (e.g., cytotoxicity) in the presence of the drugs together is higher than the sum of the individual effects of each drug administered separately. In one embodiment, the observed combined effect of the drugs is significantly higher than the sum of the individual effects. The term significant means that the observed p<0.05. A non-limiting manner of calculating the effectiveness of the combined treatment comprises the use of the Bliss additivism model (Cardone et al. Science (1998), 282: 1318-1321) using the following formula: Ebliss = EA + EB - EA ^χ EB, where EA and EB are the fractional inhibitions obtained by drug A alone and drug B alone at specific concentrations. When the experimentally measured fractional inhibition is equal to Ebliss, the combination provides an additive therapeutic effect. When the experimentally measured fractional inhibition is greater than Ebliss, the combination provides a synergistic therapeutic effect.

Tyrphostin Derivatives

Any tyrphostin derivative of the general structure of formula I can be used in the compositions of the present invention:

wherein

R ¹ , R ² , R ⁵ and R ⁶ are each independently selected from H, C1-C4 alkyl, C2-C6 alkenyl, C ₂ -C ₆ alkynyl, C _r C ₄ alkyl-C ₂ -C ₆ alkenyl, d-C ₄ alkyl-C ₂ -C ₆ alkynyl, (CH ₂ CH ₂ 0) _n H, C ₃ -C ₇ cycloalkyl, aryl, heterocyclyl, heteroaryl, (C ₁ -C ₄ )-alkylaryl, (Ci-C ₄ )-alkylheterocyclyl, (Ci- C4)-alkylheteroaryl, haloalkyl, acyl and a functional group that gives rise to 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 selected from H, C1-C4 alkyl, C ₂ -C ₆ alkenyl, C ₂ -C ₆ alkynyl, C ₁ -C4 alkyl-C ₂ -C ₆ alkenyl, C,-C ₄ alkyl-C ₂ -C ₆ alkynyl, C ₃ -C ₇ cycloalkyl, aryl, heterocyclyl, heteroaryl, (C ₁ -C ₄ )-alkylaryl ₍ (Ci-C ₄ )- alkylheterocyclyl, (C ₁ -C4)-alkylheteroaryl, halogen, haloalkyl, N0 ₂ , CN, N ₃ , S0 ₂ R ^a , COOR ^a , CSNR ^a R ^b , CSOR ^a , OR ^a , CONR ^a R ^b , NR ^a R ^b , SR ^a , and CH ₂ SR ^a , wherein R ^a and R ^b are each independently H, Q-C4 alkyl, C ₂ -C ₆ alkenyl, C ₂ -C ₆ alkynyl, Cj-C ₄ alkyl-C ₂ -C6 alkenyl, C1-C4 alkyl-C ₂ -C ₆ alkynyl, C3-C7 cycloalkyl, aryl, heterocyclyl, heteroaryl, (Ci-C4)-alkylaryl, (d- C ₄ )-alkylheterocyclyl, (C ₁ -C ₄ )-alkylheteroaryl, haloalkyl, (CH ₂ CH ₂ 0) _n H, acyl or a functional group that gives rise to hydroxyl upon hydrolysis; and

R ¹⁵ is H, C C ₄ alkyl, C ₂ -C ₆ alkenyl, C ₂ -C ₆ alkynyl, C1-C4 alkyl-C ₂ -C ₆ alkenyl, C _r C ₄ alkyl-C ₂ -C6 alkynyl, haloalkyl, or OR ^b wherein R is independently H or Q-C4 alkyl;

including salts, hydrates, solvates, polymorphs, optical isomers, geometrical isomers, enantiomers, diastereomers, and mixtures thereof. Each possibility represents a separate embodiment of the invention.

In an exemplary embodiment, the tyrphostin derivative is a compound represented by formula A:

A

In another exemplary embodiment, the tyrphostin derivative is a compound represented by formula B:

B

In another exemplary embodiment, the tyrphostin derivative is a compound represented by formula C:

In another exemplary embodiment, the tyrphostin derivative is a compound repres

In another exemplary embodiment, the tyrphostin derivative is a compound represented by formula E:

E

In another embodiment, the tyrphostin derivative is a compound of formula I wherein R ¹ , R ² , R ⁴ , R ⁵ , R ⁶ , R ¹⁰ , R ¹² , R ¹³ , R ¹⁴ and R ¹⁵ are each H; R ⁷ is OH; and at least one of R ³ , R ⁸ , R ⁹ and R ¹¹ is halogen.

In another embodiment, the tyrphostin derivative is a compound of formula I wherein R ¹ , R ² , R ⁴ , R ⁵ , R ⁶ , R ^s , R ¹⁰ _} R ¹² , R ¹³ , R ¹⁴ and R ¹⁵ are each H; R ⁷ is OH; and at least one of R ³ , R ⁹ and R ⁿ is halogen.

In another embodiment, the tyrphostin derivative is a compound of formula I wherein R ¹ , R ² , R ⁵ and R ⁶ are each H or a functional group that gives rise to hydroxyl upon hydrolysis.

In another embodiment, the tyrphostin derivative is a compound of formula I wherein R ⁷ is H or OR ^a and R ¹ , R ² , R ⁵ , R ⁶ , and R ^a are each H or a functional group that gives rise to hydroxyl upon hydrolysis. In another embodiment, the tyrphostin derivative is a compound of formula I wherein R ¹³ and R ¹⁴ are each independently H, C1-C4 alkyl, C ₂ -C6 alkenyl, C ₂ -C ₆ alkynyl, C ₁ -C4 alkyl-C ₂ -C6 alkenyl or C!-C ₄ alkyl-C ₂ -C ₆ alkynyl.

In another embodiment, the tyrphostin derivative is a compound of formula I wherein at least one of R ¹³ and R ¹⁴ is H or C C ₄ alkyl.

In another embodiment, the tyrphostin derivative is a compound of formula I wherein 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, N0 ₂ , CN, or CH ₂ SR ^a , wherein R ^a is as defined hereinabove.

In another embodiment, the tyrphostin derivative is a compound of formula I wherein R ⁴ is H.

In another embodiment, the tyrphostin derivative is a compound of formula I wherein R ⁴ is CN.

In another embodiment, the tyrphostin derivative is a compound of formula I wherein R ⁴ , R ¹¹ , R ¹² , R ¹³ , R ¹⁴ and R ¹⁵ are each H.

In another embodiment, the tyrphostin derivative is a compound of formula I wherein R ¹³ , R ¹⁴ and R ¹⁵ are each H.

In another embodiment, the tyrphostin derivative is a compound of formula I wherein R ³ , R ⁷ , R ⁸ , R ⁹ , R ¹⁰ and R ¹¹ are each independently H, halogen, haloalkyl, CH ₂ SR ^a or OH; R ⁴ , R ¹² , R ^!3 and R ¹⁴ are each independently H, C _t -C ₄ alkyl, C ₂ -C ₆ alkenyl, C ₂ -C ₆ alkynyl, d-C ₄ alkyl-C2-C ₆ alkenyl, C C ₄ alkyl-C ₂ -C6 alkynyl, aryl, halogen, haloalkyl, N0 ₂ , or CN; and R ¹⁵ is H.

In another embodiment, the tyrphostin derivative is a compound of formula I wherein R ³ , R ⁷ , R ⁸ , R ⁹ , R ¹⁰ and R ¹¹ are each independently H, halogen, haloalkyl, OH or CH ₂ SR ^a ; and R _; R ¹² , R ¹³ , R ¹⁴ and R ¹⁵ are each H, or a Ci-C ₄ alkyl.

In another embodiment, the tyrphostin derivative is a compound of formula I wherein R ¹ , R ² , R ⁵ and R ⁶ are each H or a functional group that gives rise to hydroxyl upon hydrolysis; R ³ , R ⁸ , and R ⁹ are each independently H, halogen, haloalkyl, or CH ₂ SR ^a ; R ⁷ , R ¹⁰ and R ¹¹ are each independently H, halogen, haloalkyl, OH or a functional group that gives rise to hydroxyl upon hydrolysis; and R ⁴ , R ¹² , R ¹³ , R ¹⁴ and R ¹⁵ are each H, or C ₁ -C4 alkyl.

In another embodiment, the tyrphostin derivative is a compound of formula I wherein the compound is represented by any one of the structures:

I-9b

1-10

1-14

1-15

1-16

Each possibility represents a separate embodiment of the present invention. In other embodiments, the tyrphostin derivative is a compound represented by the structure of formula II:

(Π) wherein

R ¹ , R ² , R ⁵ and R ⁶ are independently selected from H, C ₁ -C ₄ alkyl, acyl and a functional group that gives rise to hydroxyl upon hydrolysis;

R ³ and R ⁷ are independently selected from H, halogen, haloalkyl and OR ⁸ wherein R ⁸ is H, C ₁ -C4 alkyl, acyl or a functional group that gives rise to hydroxyl upon hydrolysis;

R ⁴ is H or CN,

including salts, hydrates, solvates, polymorphs, optical

isomers, geometrical isomers, enantiomers, diastereomers, and mixtures thereof.

In other embodiments, the tyrphostin derivative is a compound represented by the structure of formula II, wherein

R ¹ , R ² , R ⁵ and R ⁶ are independently selected from H, C ₁ -C4 alkyl, (CEkCitC n, wherein n is an integer of 1 to 20, acyl and a functional group that gives rise to hydroxyl upon hydrolysis;

R ³ and R ⁷ are independently selected from H, halogen, C ₁ -C4 alkyl, haloalkyl and OR ¹⁶ wherein R ¹⁶ is H, C ₁ -C4 alkyl, (CH ₂ CH ₂ 0)„, acyl or a functional group that gives rise to hydroxyl upon hydrolysis; and

R ⁴ is H or CN,

including salts, hydrates, solvates, polymorphs, optical isomers, geometrical isomers, enantiomers, diastereomers, and mixtures thereof.

Each possibility represents a separate embodiment of the present invention.

In another embodiment, the tyrphostin derivative is a compound of formula II wherein

R ⁴ is CN.

In other embodiments, the tyrphostin derivative is a compound of formula II wherein R ¹ , R ² , R ⁵ and R ⁶ are each hydrogen. In other embodiments, the tyrphostin derivative is a compound of formula II wherein R ¹ , R ² , R ⁵ and R ⁶ are each CH ₃ .

In other embodiments, the tyrphostin derivative is a compound of formula II wherein R ³ and R ⁷ are each a hydrogen, halogen, halomethyl, OH or OCH3.

In other embodiments, the tyrphostin derivative is a compound of formula II wherein R ¹ , R ² , R ⁵ and R ⁶ are each H, R ³ is halogen and R ⁷ is OH.

In other embodiments, the tyrphostin derivative is a compound of formula II wherein R ¹ , R ² , R ⁵ and R ⁶ are each H, and R ³ and R ⁷ are each halogen.

In other embodiments, the tyrphostin derivative is a compound of formula II wherein R ¹ , R ² , R ⁵ and R ⁶ are each H, R ³ is halomethyl and R ⁷ is OH.

In other embodiments, the tyrphostin derivative is a compound of formula II wherein R ¹ , R ² , R ⁵ and R ⁶ are each H, R ³ is halogen and R ⁷ is H.

In other embodiments, the tyrphostin derivative is a compound of formula II wherein R ¹ , R ² , R ⁵ and R ⁶ are each H, R ³ is OH and R ⁷ is halogen.

In other embodiments, the tyrphostin derivative is a compound of formula II wherein R ¹ , R ² , R ⁵ and R ⁶ are each CH ₃ , R ³ is halogen and R ⁷ is OCH ₃ .

In other embodiments, the tyrphostin derivative is a compound of formula II wherein R ¹ , R ² , R ⁵ and R ⁶ are each CH ₃ , and R ³ and R ⁷ are each halogen.

In other embodiments, the tyrphostin derivative is a compound of formula II wherein R ⁴ is hydrogen.

In other embodiments, the tyrphostin derivative is a compound of formula II wherein R\ R ² , R ⁵ and R ⁶ are each hydrogen.

In other embodiments, the tyrphostin derivative is a compound of formula II wherein R ¹ , R ² , R ⁵ and R ⁶ are each CH ₃ .

In other embodiments, the tyrphostin derivative is a compound of formula II wherein R ³ and R ⁷ are each hydrogen, halogen, halomethyl, OH or OCH3.

In other embodiments, the tyrphostin derivative is a compound of formula II wherein R ¹ , R ² , R ⁵ and R ⁶ are each H, R ³ is halogen and R ⁷ is OH.

In other embodiments, the tyrphostin derivative is a compound of formula II wherein R ¹ , R ² , R ⁵ and R ⁶ are each H, and R ³ and R ⁷ are each halogen.

In other embodiments, the tyrphostin derivative is a compound of formula II wherein R ¹ , R ² , R ⁵ and R ⁶ are each H, R ³ is halomethyl and R ⁷ is OH. In other embodiments, the tyrphostin derivative is a compound of formula II wherein R ¹ , R ² , R ⁵ and R ⁶ are each H, R ³ is halogen and R ⁷ is H.

In other embodiments, the tyrphostin derivative is a compound of formula II wherein R ¹ , R ² , R ⁵ and R ⁶ are each H, R ³ is OH and R ⁷ is halogen.

In other embodiments, the tyrphostin derivative is a compound of formula II wherein R ³ is halogen and R ⁷ is OCH ₃ .

In other embodiments, the tyrphostin derivative is a compound of formula II wherein R ¹ , R ² , R ⁵ and R ⁶ are each CH ₃₎ and R ³ and R ⁷ are each halogen.

In other embodiments, the tyrphostin derivative of formula (II) is represented by any of the following compounds:

II- 2

II- 3

II- 4

II- 5

II- 15

11- 16

II- 18

Each possibility represents a separate embodiment of the present invention. In another embodiment, the tyrphostin compound is represented by the structure of formula III:

wherein

R ¹ , R ² , R ⁵ and R ⁶ are independently selected from H, Cj-C ₄ alkyl,

(CH ₂ CH ₂ 0) _n , wherein n is an integer of 1 to 20, acyl and a functional group that gives rise to hydroxy 1 upon hydrolysis;

R ³ , R ⁷ , R ⁸ , R ⁹ , R ¹⁰ , R ¹¹ , R ¹² , R ¹³ and R ^I4 are independently selected from H, halogen, Ci-C ₄ alkyl, haloalkyl and OR ¹⁶ wherein R ¹⁶ is H, Ci-C ₄ alkyl, (CH ₂ CH ₂ 0) _n , acyl or a functional group that gives rise to hydroxyl upon hydrolysis; and

R ⁴ is H or CN. In other embodiments, the tyrphostin derivative is any of the derivatives described in A) PCT International Patent Application Publication No. WO 2008/068751; or B) PCT International Patent Application Publication No. WO2009/ 147682. The contents of each of the aforementioned references are incorporated by reference herein in their entirety as if fully set forth herein.

It is understood that all conformers, geometrical isomers, stereoisomers, enantiomers and diastereomers of any of the tyrphostin derivatives described herein, are encompassed and may be used in the combinations and methods described by the present application.

Without being bound to any particular theory or mechanism of action, it is contemplated that the compounds of the present invention are inhibitors of PK signaling, such as IGF-1R. It has now been surprisingly been found that these compounds, in addition to being inhibitors of IGF-1R, also lead to the dissociation of the IGF-1R substrates IRS 1/2 from the cell membrane, inhibitory serine phosphorylation and or irreversible degradation of the IRS 1/2 proteins. This activity leads to long lasting inhibition of the IGF-1R pathway, growth inhibition of a wide range of cancer cell types, and potent anti-tumor effects. Thus, in another embodiment, the present invention provides a method of inhibiting, treating or preventing an insulin-like growth factor 1 receptor (IGF-1R) and/or insulin receptor substrate 1 (ERS1) and/or insulin receptor substrate 2 (IRS2) signaling related disorder in a subject comprising the step of administering to said 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 covered by such formula, together with an anti-cancer agent, wherein the compound of formula I and the anti-cancer agent together provide an anti-cancer effect which is at least additive, and is preferably synergistic. In some embodiments, the compound of formula I is an inhibitor of an insulin receptor or an 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 the IGF-1R mediated pathway. In some embodiments, the substrate protein is Insulin Receptor Substrate 1 (IRS1), Insulin Receptor Substrate 2 (ERS2), or a combination thereof. In one particular embodiment, the compound of formula I is an IGF-1 kinase inhibitor that leads to at least one of the dissociation of IRS 1 or IRS2 from the cell membrane, phosphorylation of IRS 1 or IRS2, and or degradation of IRS 1 or IRS2, in any order.

IGF1R and specifically IRS1 are one of the key mechanisms for 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)

Recently, a new drug targeted against mutant B-Raf was approved (PLX4032( K. T. Flaherty et al, N Engl J Med 363, 809 (Aug 26, 2010)), and proved to be remarkably effective against metastatic melanoma. However, responding tumors eventually develop resistance and tumors eventually progress in almost all patients (G. Bollag et al, Nature 467, 596 (Sep 30, 2010)). A worldwide effort commenced to unravel the mechanism of resistance to PLX4032. Meenhard Herlyn et al. showed that IR/IGF1R survival signaling is involved (Cancer Cell. 2010 December 14; 18(6): 683-695). Recent findings show that inhibition of multiple serme/threonine kinases as a treatment against cancer often times leads to unintentional alleviation of the basal feedback loop inhibiting IRS1, thus leading to enhancement of the IRS-PKB axis and consequent resistance to these serine/threonine kinase inhibitors (Buck E. et al. Cancer Res. 2008 Oct 15 ;68(20): 8322-32; Megan Keniry and Ramon Parsons. Cancer Discovery. Aug 2011, 1(3): 203-4). As contemplated herein, treatment with compounds of formula (I), which lead to IRS 1/2 elimination, may enhance the anti-cancer effect of such kinase inhibitors, and prevent resistance development against them. As such, combinations of the compounds of formula (I), together with B-raf inhibitors such as PLX-4032, are particularly preferred.

Chemical Definitions:

An "alkyl" group refers to any saturated aliphatic hydrocarbon, including straight- chain, branched-chain and cyclic alkyl groups, in one embodiment, the alkyl group has 1-12 carbons designated here as Ci-Cn-alkyl. In another embodiment, the alkyl group has 1-6 carbons designated here as Ci-Ce-alkyl. In another embodiment, the alkyl group has 1-4 carbons designated here as Ci-C^alkyl. The alkyl group may be unsubstituted or substituted by one or more groups selected from halogen, hydroxy, alkoxy carbonyl, amido, alkylamido, dialkylamido, nitro, amino, alkylamino, dialkylamino, carboxyl, thio and thioalkyl.

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, the alkenyl group has 2-8 carbon atoms designated here as C2-Cg-alkenyl. In another embodiment, the alkenyl group has 2-6 carbon atoms in the chain designated here as C ₂ -C6-alkenyl. Exemplary alkenyl groups include ethenyl, propenyl, n-butenyl, i-butenyl, 3- methylbut-2-enyl, n-pentenyl, heptenyl, octenyl, cyclohexyl-butenyl and decenyl. The alkenyl group can be unsubstituted or substituted through available carbon atoms with one or more groups defined hereinabove for alkyl.

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

The term "C3-C7 cycloalkyl" 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, cyclohexenyl and the like. The cycloalkyl group can be unsubstituted or substituted with any one or more of the substituents defined above for alkyl. Similarly, the term "cycloalkylene" means a bivalent cycloalkyl, as defined above, where the cycloalkyl radical is bonded at two positions connecting together two separate additional groups.

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

The term "heteroaryl" used herein alone or as part of another group refers to a heteroaromatic system containing at least one heteroatom ring wherein the atom is selected from nitrogen, sulfur and oxygen. The heteroaryl contains 5 or more ring atoms. The heteroaryl group can be monocyclic, bicyclic, tricyclic and the like. Also included in this definition are the benzoheterocyclic rings. If nitrogen is a ring atom, the present invention also contemplates the N-oxides of the nitrogen containing heteroaryls. Non-hraiting examples of heteroaryls include thienyl, benzothienyl, 1-naphthothienyl, thianthrenyl, furyl, benzofuryl, pyrrolyl, imidazolyl, pyrazolyl, pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, indolyl, isoindolyl, indazolyl, purinyl, isoquinolyl, quinolyl, naphthyridinyl, quinoxalinyl, quinazolinyl, cirmolinyl, pteridinyl, carbolinyl, thiazolyl, oxazolyl, isothiazolyl, isoxazolyl and the like. The heteroaryl group can be unsubstituted or substituted through available atoms with one or more groups defined hereinabove for alkyl.

The term "heterocyclic ring" or "heterocyclyl" used herein alone or as part of another group refers to a five-membered to eight-membered rings mat have 1 to 4 heteroatoms, such as oxygen, sulfur and/or nitrogen, in particular nitrogen, either alone or in conjunction with sulfur or oxygen ring atoms. These five-membered to eight-membered rings can be saturated, fully unsaturated or partially unsaturated, with fully saturated rings being preferred. Preferred heterocyclic rings include piperidinyl, pyrroKdinyl pyrrolinyl, pyrazolinyl, pyrazolidinyl, morpholinyl, thiomorpholinyl, pyranyl, thiopyranyl, piperazinyl, indolinyL dihydrofuranyl, tetrahydrofuranyl, dihydrothiophenyl, tetrahydrothiophenyl, dihydropyranyl, tetrahydropyranyl, dihydrothiazolyl, and the like. The heterocyclyl group can be unsubstituted or substituted through available atoms with one or more groups defined hereinabove for alkyl.

The term "acyl" as used herein encompasses groups such as, but not limited to, formyl, acetyl, propionyl, butyryl, pentanoyl, pivaloyl, hexanoyl, heptanoyl, octanoyl, nonanoyL decanoyl, undecanoyl, dodecanoyl, benzoyl and the like. Currently preferred acyl groups are acetyl and benzoyl.

A "hydroxy" group refers to an OH group. An "alkoxy" group refers to an -O-alkyl group wherein R is alkyl as defined above.

A "thio" group refers to an -SH group. An "alkylthio" group refers to an -SR group wherein R is alkyl as defined above.

An "amino" group refers to an NH ₂ group. An alkylamino group refers to an -NHR group wherein R is alkyl is as defined above. A dralkylamino group refers to an -NRR' group wherein R and R' are alkyl as defined above.

An "amido" group refers to a -C(0)N¾ group. An alkylamido group refers to an - C(0)NHR group wherein R is alkyl is as defined above. A dialkylamido group refers to an - C(0) RR' group wherein R and R' are alkyl as defined above.

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

A "polyoxyalkylene" group refers to a (CH ₂ CH ₂ 0) _n H group wherein n=l-20. Currently preferred polyoxyalkylene groups are polyethyleneglycol (PEG) and polypropyleneglycole. The term "halogen" or "halo" as used herein alone or as part of another group refers to chlorine, bromine, fluorine, and iodine. The term "haloalkyl" refers to an alkyl group having some or all of the hydrogens independently replaced by a halogen group including, but not limited to, trichloromethyl, tribromomethyl, trifluoromethyl, triiodomethyl, difluoromethyl, chlorodifluoromethyl, pentafluoroethyl, 1,1-difluoroethyl bromomethyl, chloromethyl, fluoromethyl, iodomethyl, and the like.

Examples of functional groups that give rise to hydroxyl upon hydrolysis include, but are not limited to, esters, anhydrides, carbamates, carbonates and the like. For example, when any of R ¹ , R ² , R ⁵ or R ⁶ is an acyl group (COR), the resulting functional group is an ester (OCOR). When any of R ^l , R ² , R ⁵ or R ⁶ is an amide group (CONHR), the resulting functional group is a carbamate (OCONHR). When any of R ¹ , R ² , R ⁵ or R ⁶ is a carboxylate group (COOR), the resulting functional group is a carbonate (OCOOR).

Within the scope of the present invention are prodrugs of the compounds disclosed herein. The term "prodrug" represents compounds which are rapidly transformed in vivo to any of compounds represented by formula I, for example by hydrolysis in the blood. Thus, the term "prodrug" refers to a precursor of any of the compounds of the present invention that is pharmaceutically acceptable. A prodrug may be inactive when administered to a subject, but is converted in vivo to an active compound. The use of prodrugs is particularly advantageous for facilitating the administration of the compounds. The prodrug compound often offers benefits of solubility, tissue compatibility or delayed release in a mammalian organism. For example the prodrug, according to the principles of the present invention, can be a compound represented by the structure of formula I wherein R ^l , R ² , R ⁵ and R ⁶ are a functional group that gives rise to hydroxyl upon hydrolysis as defined hereinabove.

All stereoisomers of the above tyrphostin derivatives are contemplated, either in admixture or in pure or substantially pure form. The tyrphostin derivatives can have asymmetric centers at any of the atoms. Consequently, the compounds can exist in enantiomeric or diastereomeric forms or in mixtures thereof. The present invention contemplates the use of any racemates {i.e. mixtures containing equal amounts of each enantiomers), enantiomerically enriched mixtures (i.e., mixtures enriched for one enantiomer), pure enantiomers or diastereomers, or any mixtures thereof. The chiral centers can be designated as R or S or R,S or d,D, 1,L or d,l, D,L. Compounds comprising amino acid residues include residues of D-amino acids, L-amino acids, or racemic derivatives of amino acids. Compounds comprising sugar residues include residues of D-sugars, L-sugars, or racemic derivatives of sugars. Residues of D-sugars, which appear in nature, are preferred. In addition, several of the compounds of the invention contain one or more double bonds. The present invention intends to encompass all structural and geometrical isomers including cis, trans, E and Z isomers, independently at each occurrence.

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

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

The present invention also includes solvates of the compounds of the present invention and salts thereof. "Solvate" means a physical association of a compound of the 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 will be capable of isolation. "Solvate" encompasses both solution-phase and isolatable solvates. Non- limiting examples of suitable solvates include ethanolates, methanolates and the like. "Hydrate" is a solvate wherein the solvent molecule is water.

The present invention also includes polymorphs of the compounds of the present invention and salts thereof. The term "polymorph" refers to a particular crystalline or amorphous state of a substance, which can be characterized by particular physical properties such as X-ray diffraction, IR or Raman spectra, melting point, and the like. Anti-Cancer Treatments

The anti-cancer treatments for use in the combinations of the present invention include radiation therapy, chemotherapy, immunotherapy, hormonal therapy and genetic therapy. Each possibility represents a separate embodiment of the present invention.

The additional agent may generally be selected from a chemotherapeutic agent (e.g., a nitroso-urea, a platinum compound, a taxane derivative, a vinca alkaloid, and an anti-tumor antibiotic), a metabolic inhibitor (e.g., 2-deoxy-D-glucose- "2DG"), agents (e.g. antibodies, small molecules) which inhibit kinases or other specific agents which inhibit other enzymes including, but not limited to, EGFR, HER2, B-Raf, PDGFR, VEGFR, cKit, Akt, mTOR, topoisomerase, proteasome, IGFIR, and the like. The tyrphostin derivative and the at least one other agent collectively are administered in an amount which provides a therapeutic effect, which is greater than either one on its own. The combination provides an effect which is at least additive. According to some embodiments, the combination provides an effect which is synergistic. The present invention further relates to the combination of a tyrphostin derivative of the present invention with at least one other anti-cancer treatment (e.g. radiotherapy).

In some embodiments, the at least one anti-cancer agent is selected from an alkylating agent, an antibiotic agent, an anti-metabolic agent, an hormonal agent, a plant-derived agent and their synthetic derivatives, an anti-angiogenic agent, a differentiation inducing agent, a cell growth arrest inducing agent, an apoptosis inducing agent, a cytotoxic agent, an agent which affects cell bioenergetics i.e., which affects cellular ATP levels and molecules/activities regulating these levels, a biologic agent, e.g., a monoclonal antibody, a kinase inhibitor and a growth factor inhibitor and their receptors, a gene therapy agent, a cell therapy agent, e.g., stem cells, or any combination thereof. Each possibility represents a separate embodiment of the present invention.

Suitable anti-cancer agents in accordance with the present invention include, but are not limited to, alkylating agents, antibiotic agents, antimetabolic agents, hormonal agents, plant-derived agents, anti-angiogenic agents, differentiation inducing agents, cell growth arrest inducing agents, apoptosis inducing agents, cytotoxic agents, agents affecting cell bioenergetics, biologic agents, e.g., monoclonal antibodies, kinase inhibitors and inhibitors of growth factors and their receptors, gene therapy agents, cell therapy, e.g., stem cells, or any combination thereof. Each possibility represents a separate embodiment of the present invention.

In one embodiment, the at least one other anti-cancer agent is an alkylating agent Alkylating agents are drugs which impair cell function by forming covalent bonds with amino, carboxyl, suflhydryl and phosphate groups in biologically important molecules. The most important sites of alkylation are DNA, RNA and proteins. Alkylating agents depend on cell proliferation for activity but are not cell-cycle-phase-specific. Non-limiting examples of alkylating agents include bischloroethylamines, nitrogen mustards (e.g. chlorambucil, cyclophosphamide, ifosfamide, mechlorethamine, melphalan, uracil mustard), aziridines (e.g. thiotepa), alkyl alkone sulfonates (e.g. busulfan), nitroso-ureas (e.g. BCNU, carmustine, lomustine, streptozocin), non-classic alkylating agents (e.g., altretamine, dacarbazine, and procarbazine), and inorganic ions including platinum compounds (e.g., carboplatin, oxaloplatin and cisplatin). Each possibility represents a separate embodiment of the present invention. Currently preferred alkylating agents for use in the combinations of the present invention include cisplatin and dacarbazine. Each possibility represents a separate embodiment of the present invention.

In another embodiment, the at least one other anti-cancer agent is a protein kinase inhibitor. Protein kinase inhibitors are small molecules or antibodies which inhibit the phosphorylation of hydroxy groups on tyrosine, serine and threonine residues of proteins, thus affecting cell growth, differentiation and proliferation. Non-limiting examples of protein kinase inhibitors include EGFR and/or HER2 inhibitors (e.g. AG 1478, genifitib, erlotinib, lapatinib, trastuzumab and cetuximab), B-Raf inhibitors (e.g. PLX-4032 and sorafenib), BCR-ABL and/or Src family kinase inhibitors (e.g. imatinib, dasatinib and nilotinib), VEGFR/PDGFR and/or multi kinase inhibitors (e.g. bevacizumab, sorafenib, sunitinib and pazopanib), and IGF1R inhibitors (e.g., NVP-AEW541). Each possibility represents a separate embodiment of the present invention.

In another embodiment, the at least one other anti-cancer agent is a proteasome inhibitor. Proteasome inhibitors have effective anti-tumor activity in cell culture, inducing apoptosis by disrupting the regulated degradation of pro-growth cell cycle proteins. Non- limiting examples of proteasome inhibitors include bortezomib (PS-341, Velcade®) and disulfiram. Each possibility represents a separate embodiment of the present invention.

In yet another embodiment, the at least one other anti-cancer agent is a topoisomerase inhibitor. Topoisomerase inhibitors are agents which interfere with the action of topoisomerase enzymes (topoisomerase I and II) that control the changes in DNA structure by catalyzing the breaking and rejoining of the phosphodiester backbone of DNA strands during the normal cell cycle. Non-limiting examples of topoisomerase inhibitors are irinotecan, topotecan, camptothecin, lamellarin D and etoposide. Each possibility represents a separate embodiment of the present invention. A currently preferred topoisomerase inhibitor is irinotecan.

In further embodiments, the at least one anti-cancer agent is an inhibitor of mTOR, which stands for "mammalian Target Of Rapamycin", has also been called FRAP (FKBP- rapamycin associated protein), RAFT (rapamycin and FKBP target), RAPTl, or SEP. Non- limiting examples of mTOR inhibitors include, but are not limited to rapamycin (Sirolimus), CCI-779, AP23573, RAD-001, Everolimus and Temsirolimus. Each possibility represents a separate embodiment of the present invention.

In further embodiments, the at least one other anti-cancer agent is an anti-tumor antibiotics. Anti-tumor antibiotics like adriamycin intercalate DNA at guanine-cytosine and guanine-thymine sequences, resulting in spontaneous oxidation and formation of free oxygen radicals that cause strand breakage. Non-limiting examples of antibiotic agents include anthracyclines (e. g. doxorubicin, daunorubicin, epirubicin, idarubicin and anthracenedione), mitomycin C, bleomycin, dactinomycin, and plicatomycin. Each possibility represents a separate embodiment of the present invention.

In additional embodiments, the at least one other anti-cancer agent is an anti- metabolic agent. Anti-metabolic agents suitable for use in the present invention include, but are not limited to, 6-mercaptopurine, floxuridine, 5-fiuorouracil, methotrexate, leucovorin, hydroxyurea, thioguanine, mercaptopurine, cytarabine, pentostatin, fludarabine phosphate, cladribme, asparaginase, and gemcitabine. Each possibility represents a separate embodiment of the present invention.

In further embodiments, the at least one other anti-cancer agent is a hormonal agent. Hormonal agents suitable for use in the present invention include, but are not limited to, an estrogen, a progestogen, an antiesterogen, an androgen, an antiandrogen, an LHRH analogue, an aromatase inhibitor, diethylstibestrol, tamoxifen, toremifene, fluoxymesterol, raloxifene, bicalutamide, nilutamide, flutamide, aminoglutethimide, tetrazole, ketoconazole, goserelin acetate, leuprolide, megestrol acetate, and mifepristone. Each possibility represents a separate embodiment of the present invention. In some embodiments, the at least one other anti-cancer agent is a plant derived agent. Plant derived agents include, but are not limited to, taxanes, which are semisynthetic derivatives of extracted precursors from the needles of yew plants. These drugs have a novel 14-member ring, the taxanc. The taxanes (e.g., taxol) promote microtubular assembly and stability, therefore blocking the cell cycle in mitosis. Other plant derived agents include, but are not limited to, vinca alkaloids including vincristine, vinblastine, vindesine, vinzolidine, vinorelbine, teniposide, paclitaxel and docetaxel; podophyllotoxins including etoposide, irinotecan, and topotecan. Each possibility represents a separate embodiment of the present invention. In one embodiment, the plant derived agent is a jasmonate derivative (e.g. methyl jasmonate).

In certain embodiments, the at least one other anti-cancer agent is a biologic agent such as, but not limited to, immuno-modulating proteins, monoclonal antibodies against tumor antigens, tumor suppressor genes, kinase inhibitors and inhibitors of growth factors and their receptors and cancer vaccines. For example, the immuno-modulating protein can be interleukin 2, interleukin 4, interleukin 12, interferon El interferon D, interferon alpha, erythropoietin, granulocyte-CSF, granulocyte, macrophage-CSF, bacillus Calmette- Guerin, levamisole, or octreotide. Each possibility represents a separate embodiment of the present invention. Furthermore, the tumor suppressor gene can be DPC-4,NF-1, NF-2, RB, p53, Tl, BRCA, or BRCA2. Each possibility represents a separate embodiment of the present invention.

Recent developments have introduced, in addition to the traditional cytotoxic and hormonal therapies, additional therapies for the treatment of cancer. For example, many forms of gene therapy are undergoing preclinical or clinical trials. In addition, approaches are currently under development, such approaches are based on the inhibition of tumor vascularization (angiogenesis). The concept of treatment is based on the cut off the tumor from nutrition and oxygen supply provided by a newly built tumor vascular system. In addition, cancer therapy is also being attempted by the induction of terminal differentiation of the neoplastic cells. Suitable differentiation agents include, but are not limited to, hydroxamic acids, derivatives of vitamin D and retinoic acid, steroid hormones, growth factors, tumor promoters, and inhibitors of DNA or RNA synthesis. Each possibility represents a separate embodiment of the present invention. Also, histone deacetylase inhibitors are suitable chemotherapeutic agent to be used in the present invention. Additional anti-cancer agents within the scope of the present invention are glycolytic inhibitors (metabolic inhibitors) such as 2DG oxamate and its derivatives and the like, and other signal transduction inhibitors (small molecules, peptides or antibodies), which block the activation or inhibit the kinase activity of cKit, cRaf, Akt, and/or mTOR. Each possibility represents a separate embodiment of the present invention. In additional embodiments, the present invention provides the combination of the tyrphostin derivatives disclosed herein with at least one other anti-cancer treatment. Anti-cancer treatments include radiation therapy (radiation oncology, radiotherapy) and surgery. Each possibility represents a separate embodiment of the present invention.

Specific compounds for chemotherapeutic treatment in combination with the tyrphostin derivatives of the invention are selected from the group consisting of topoisomerase inhibitors, spindle poison vincas: vinblastine, vincristine, vinorelbine (taxol), paclitaxel, docetaxel; and alkylating agents: mechlorethamine, chlorambucil, cyclophosphamide, melphalan, ifosfamide; methotrexate; 6-mercaptopurine; 5-fluorouracil, cytarabine, gemcitabin; podophyllotoxins: etoposide, irinotecan, topotecan; anticancer chemicals containing a quinone group: carbazilquinone; antibiotics: doxorubicin (adriamycin), daunorubicin, idarubicin, epirubicin, bleomycin, mitomycin; nitrosoureas: carmustine (BCNU), lomustine; inorganic ions: cisplatin, carboplatin; interferon, asparaginase; hormones: tamoxifen, leuprolide, flutamide, and megestrol acetate, and dacarbazine. Each possibility represents a separate embodiment of the present invention. A table of approved anti-cancer drugs for various types of cancers suitable for use in the combinations of the present invention is presented in Table 1 A. A table of FDA approved kinase inhibitors for various types of cancers suitable for use in the combinations of the present invention is presented in Table IB.

Table 1A

Cancer Drug MOA Comments

Melanoma Dacarbazine Alkylating agent

Interferon alfa IFN-a

Aldesleukin

(PROLEUKIN) IL-2

Breast 5-FU Pyrimidine analog

Paclitaxel (TAXOL) Taxane

Docetaxel

(TAXOTERE) Taxane

Gemcitabine

(GEMZAR) Nucleoside analog In combination

Capecitabine 5-FU prodrug (XELODA)

Trastuzumab

(HERCEPTIN) Anti-ErbB2

Epirubicine (Ellence) Anthracycline

In combination with

Lapatinib (TYKERB) TKI capecitabine

Ixabepilone Epothilone B In combination with (IXEMPRA) analog capecitabine

Organoplatinum

Ovary Cisplatin alkylating agent In combination

Organoplatinum

Carboplatin alkylating agent In combination

Paclitaxel (TAXOL) Taxane

Gemcitabine

(GEMZAR) Nucleoside analog In combination

Topotecan Topoisomerase

(HYCAMTIN) inhibitor

Doxorubicin liposomal

(DOXIL) Anthracycline

Pancreas 5-FU Pyrimidine analog

Gemcitabine

(GEMZAR) Nucleoside analog

Rituximab

NHL (RITUXAN) Anti-CD20

Interferon alfa IFN-a

Ibritomumab tiuxetan

(ZEVALIN) Anti-CD20

Tositumomab

(BEXXAR) Anti-CD20

Bendamustine Purine analog and

(TREANDA) alkylator hybrid

Colon 5-FU Pyrimidine analog

Capecitabine

(XELODA) 5-FU prodrug

Irinotecan Topoisomerase

(CAMPTOSAR) inhibitor

Oxaliplatin Organoplatinum In combination with 5- (ELOXATIN) alkylating agent FU/leukovorin

Cetuximab

(ERBITUX) Anti-EGFR

Bevacizumab

(AVASTIN) Anti-VEGF

Panitumumab

(VECTIBIX) Anti-EGFR

Temozolomide

Glioma (TEMODAR)

Busulfan

CML (BUSULFEX) Alkylating agent

Imatinib mesylate TKI (GLEEVEC)

Dasatinib (SPRYCEL) TKI

Nilotinib (TASIGNA) TKI

Arsenic trioxide

APL (TRISENOX)

Gemtuzumab

ozogamicin

AML (MYLOTARG) Anti-CD33

Alemtuzumab

CLL (CAMPATH) Anti-CD52 B-CLL

Bendamustine Purine analog and

(TREANDA) alkylator hybrid

Ofatumumab

(ARZERRA) Anti-CD20

Imatinib mesylate

GIST (GLEEVEC) TKI

Sunitinib (SUTENT) TKI

Bortezomib Proteasome

MM (VELCADE) inhibitor

Organoplatinum

NSCLC Carboplatin alkylating agent

In combination with

Paclitaxel (TAXOL) Taxane cisplatin

Docetaxel

(TAXOTERE) Taxane

Gemcitabine In combination with

(GEMZAR) Nucleoside analog cisplatin

Gefitinib (IRESSA) TKI

Erlotinib (TARCEVA) TKI

In combination with

Mesothelioma Pemetrexed (ALIMTA) Antifolate cisplatin

Sorafenib

Kidney (NEXAVAR) TKI

Sunitinib (SUTENT) TKI

Temsirolimus

(TORISEL) mTOR inhibitor

Pazopanib

(VOTRIENT) TKI (VEGFR)

Bevacizumab

(AVASTIN) Anti-VEGF

Everolimus

(AFINITOR) mTOR inhibitor

Nelarabine Deoxyguanosine

ALL (ARRANON) analogue

Topotecan Topoisomerase

SCLC (HYCAMTIN) inhibitor

Peripheral T- Pralatrexate

cell Lymphoma (FOLOTYN) DHFR inhibitor

Prostate Docetaxel Taxane In combination with (TAXOTERE) prednisone

Cabazitaxel In combination with (JEVTANA) Taxane prednisone

Gastric 5-FU Pyrimidine analog

Docetaxel

(TAXOTERE) Taxane

HNSCC 5-FU Pyrimidine analog

Docetaxel In combination with (TAXOTERE) Taxane cisplatin/5-FU

Organoplatinum

Testis Cisplatin alkylating agent In combination

HCC Nexavar (sorafenib)

Table IB

Name/Year Brand Indication

Company Target

Approved (R) Cancer

Imatinib BCR-ABL,

Gleevec Novartis CML, GIST

2001 PDGFR, cKIT

Gefitinib

Iressa Astra Zeneca EGFR NSCLC

2003

Erlotinib Roche, NSCLC

Tarceva EGFR

2004 Astellas Pancreatic

Sorafenib Bayer, BRaf, VEGFR, HCC

Nexavar

2005 Onyx PDGFR, KIT RCC

Dasatinib BCR-ABL, Src,

Sprycel BMS CML, ALL

2006 Fyn, BRaf

Sunitinib FLT3, PDGFR, RCC, GIST

Sutent Pfizer

2006 VEGFR, KIT Pancreatic NET

Nilotinib BCR-ABL, KIT,

Tasigna Novartis CML 2006 PDGFR

Lapatinib Tykerb Her+ Breast

GSK EGFR, HER2

2010 cancer

Pazopanib VEGFR 1/2/3,

Votrient GSK Kidney cancer 2009 PDGFR, KIT

Everolimus Afinitor/

Novartis mTOR Kidney cancer 2003/2009 Cetican

Temsirolimus Torisel Wyeth mTOR Kidney cancer 2007

Vandetanib Caprelsa

Astra Zeneca VEGFR, EGFR Thyroid

2011 Zactima

Vemurafanib Roche, Daiichi

Zelboraf BRAF Melanoma 2011 Sankyo

Critozinib ALK, HGFR ALK+ve

Xalkori Pfizer

2011 cMet NSCLC

Ruxolitinib Incyte,

Jakafi JAK 1, 2 Myelofibrosis

2011 Novartis

Therapeutic Use

The present invention relates to a method for treating cancer comprising administering to a subject in need thereof a tyrphostin derivative in combination with at least one other anti-cancer agent, wherein the tyrphostin derivative and the at least one other anticancer agent together provide a therapeutic anti-cancer effect. In one embodiment, the therapeutic anti-cancer effect is additive. In another embodiment, the therapeutic anti-cancer effect is synergistic.

The term "cancer" as used herein refers to a disorder in which a population of cells has become, in varying degrees, unresponsive to the control mechanisms that normally govern proliferation and differentiation. Cancer refers to various types of malignant neoplasms and tumors, including primary tumors, and tumor metastasis. Non-limiting examples of cancers which can be treated by the combinations of the present invention are brain, ovarian, colon, prostate, kidney, bladder, breast, lung, oral, and skin cancers. Specific examples of cancers are: carcinomas, sarcomas, myelomas, leukemias, lymphomas and mixed type tumors. Particular categories of tumors include Iymphoproliferative disorders, breast cancer, ovarian cancer, prostate cancer, cervical cancer, endometrial cancer, bone cancer, liver cancer, stomach cancer, colon cancer, pancreatic cancer, cancer of the thyroid, head and neck cancer, cancer of the central nervous system, cancer of the peripheral nervous system, skin cancer, kidney cancer, as well as metastases of all the above. Particular types of tumors include hepatocellular carcinoma, hepatoma, hepatoblastoma, rhabdomyosarcoma, esophageal carcinoma, thyroid carcinoma, ganglioblastoma, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, Ewing's tumor, leimyosarcoma, rhabdotheliosarcoma, invasive ductal carcinoma, papillary adenocarcinoma, melanoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma (well differentiated, moderately differentiated, poorly differentiated or undifferentiated), renal cell carcinoma, hypernephroma, hypernephroid adenocarcinoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, testicular tumor, lung carcinoma including small cell, non-small and large cell lung carcinoma, bladder carcinoma, glioma, astrocyoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, retinoblastoma, neuroblastoma, colon carcinoma, rectal carcinoma, hematopoietic malignancies including all types of leukemia and lymphoma including: acute myelogenous 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 hepatocarcinoma. Each possibility represents a separate embodiment of the present invention.

The term "treatment of cancer" in the context of the present invention includes at least one of the following: a decrease in the rate of growth of the cancer (i.e. the cancer still grows but at a slower rate); cessation of growth of the cancerous growth, i.e., stasis of the tumor growth, and, in preferred cases, the tumor diminishes or is reduced in size. The term also includes reduction in the number of metastases, reduction in the number of new metastases formed, slowing of the progression of cancer from one stage to the other and a decrease in the angiogenesis induced by the cancer. In most preferred cases, the tumor is totally eliminated. Additionally included in this term is lengthening of the survival period of the subject undergoing treatment, lengthening the time of diseases progression, tumor regression, and the like. It is to be understood that the term "treating cancer" also refers to the inhibition of a malignant (cancer) cell proliferation including tumor formation, primary tumors, tumor progression or tumor metastasis. The term "inhibition of proliferation" in relation to cancer cells, may further refer to a decrease in at least one of the following: number of cells (due to cell death which may be necrotic, apoptotic or any other type of cell death or combinations thereof) as compared to control; decrease in growth rates of cells, i.e. the total number of cells may increase but at a lower level or at a lower rate than the increase in control; decrease in the invasiveness of cells (as determined for example by soft agar assay) as compared to control even if their total number has not changed; progression from a less differentiated cell type to a more differentiated cell type; a deceleration in the neoplastic transformation; or alternatively the slowing of the progression of the cancer cells from one stage to the next.

As used herein, the term "administering" refers to bringing in contact with the combination of the present invention. Administration can be accomplished to cells or tissue cultures, or to living organisms, for example humans. In one embodiment, the present invention encompasses administering the combinations of the present invention to a human subject.

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

Pharmaceutical Compositions

Although the components of the combinations of the present invention can be administered alone, it is contemplated that the components are administered in pharmaceutical compositions further containing 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 one pharmaceutical composition.

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, intra-arterial, transdermal and intramuscular), topical, intranasal, or via a suppository. Each possibility represents a separate embodiment of the present invention. Such compositions are prepared in a manner well known in the pharmaceutical art and comprise as an active ingredient at least one compound of the present invention as described hereinabove, and a pharmaceutically acceptable excipient or a carrier. The term "pharmaceutically acceptable" means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals and, more particularly, in humans.

During the preparation of the 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 compositions can be in the form of tablets, pills, capsules, pellets, granules, powders, lozenges, sachets, cachets, elixirs, suspensions, dispersions, emulsions, solutions, syrups, aerosols (as a solid or in a liquid medium), ointments containing, for example, up to 10% by weight of the active compound, soft and hard gelatin capsules, suppositories, sterile injectable solutions, and sterile packaged powders. Each possibility represents a separate embodiment of the present invention.

The carriers may be any of those conventionally used and are limited only by chemical- physical considerations, such as solubility and lack of reactivity with the compound of the invention, and by the route of administration. The choice of carrier will be determined by the particular method used to administer the pharmaceutical composition. Some examples of suitable carriers include lactose, glucose, dextrose, sucrose, sorbitol, mannitol, starches, gum acacia, calcium phosphate, alginates, tragacanth, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water and methylcellulose. Each possibility represents a separate embodiment of the present invention. The formulations can additionally include lubricating agents such as talc, magnesium stearate, and mineral oil; wetting agents, surfactants, emulsifying and suspending agents; preserving agents such as methyl- and propylhydroxybenzoates; sweetening agents; flavoring agents, colorants, buffering agents (e.g., acetates, citrates or phosphates), disintegrating agents, moistening agents, anti-bacterial agents, anti-oxidants (e.g., ascorbic acid or sodium bisulfite), chelating agents (e.g., ethylenediaminetetraacetic acid), and agents for the adjustment of tonicity such as sodium chloride. Other pharmaceutical carriers can 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, sesame oil and the like, polyethylene glycols, glycerin, propylene glycol or other synthetic solvents. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Each possibility represents a separate embodiment of the present invention.

For preparing solid compositions such as tablets, the principal active ingredient(s) is mixed with a pharmaceutical excipient to form a solid preformulation composition containing a homogeneous mixture of a compound of the present invention. When referring to these preformulation compositions as homogeneous, it is meant that the active ingredient is dispersed evenly throughout the composition so that the composition may 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, from about 0.1 mg to about 2000 mg, from about 0.1 mg to about 500 mg, from about 1 mg to about 100 mg, from about 100 mg to about 250 mg, etc. of the active ingredient(s) of the present invention.

Any method can be used to prepare the pharmaceutical compositions. Solid dosage forms can be prepared by wet granulation, dry granulation, direct compression and the like. The solid dosage forms of the present invention may be coated or otherwise compounded to provide a dosage form affording the advantage of prolonged action. For example, the tablet or pill can comprise an inner dosage and an outer dosage component, the latter being in the form of an envelope over the former. The two components can be separated by an enteric layer, which serves to resist disintegration in the stomach and permit 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, such materials including a number of polymeric acids and mixtures of polymeric acids with such materials as shellac, cetyl alcohol, and cellulose acetate. Each possibility represents a separate embodiment of the present invention.

The liquid forms in which the compositions of the present invention may be incorporated, for administration orally or by injection, include aqueous solutions, suitably flavored syrups, aqueous or oil suspensions, and flavored emulsions with edible oils such as cottonseed oil, sesame oil, coconut oil, or peanut oil, as well as elixirs and similar pharmaceutical vehicles. Each possibility represents a separate embodiment of the present invention.

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 masks 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.

Another formulation suitable 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. In yet another embodiment, the composition is prepared for topical administration, e.g. as an ointment, a gel a drop or a cream. For topical administration to body surfaces using, for example, creams, gels, drops, ointments and the like, 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 base forms may include, for example, sodium carboxymethylcellulose, polyacrylates, polyoxyethylene-polyoxypropylene- block polymers, polyethylene glycol and wood wax alcohols. Each possibility represents a separate embodiment of the present invention.

Alternative formulations include nasal sprays, liposomal formulations, slow-release formulations, pumps delivering the drugs into the body (including mechanical or osmotic pumps) controlled-release formulations and the like, as are known in the art.

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

In preparing a formulation, it may be necessary to mill the active ingredient to provide the appropriate particle size prior to combining with the other ingredients. If the active compound is substantially insoluble, it ordinarily is milled to a particle size of less than 200 mesh. If the active ingredient is substantially water soluble, the particle size is normally adjusted by milling to provide a substantially uniform distribution in the formulation, e.g. about 40 mesh.

It may be desirable to administer the pharmaceutical composition of the invention locally to the area in need of treatment; this may be achieved by, for example, and not by way of limitation, local infusion during surgery, infusion to the liver via feeding blood vessels with or without surgery, topical application, e.g., in conjunction with a wound dressing after surgery, by injection, by means of a catheter, by means of a suppository, or by means of an implant, said implant being of a 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 a tumor or neoplastic or pre-neoplastic tissue.

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 localized or it may be systemic. In addition, it may be desirable to introduce the pharmaceutical compositions of the invention into the central nervous system by any suitable route, including intra-ventricular and intra-thecal injection; intra-ventricular injection may be facilitated by an intra-ventricular catheter, for example, attached to a reservoir. Pulmonary administration can also be employed, e.g., by use of an inhaler or nebulizer, and formulation with an aerosolizing agent.

A compound of the present invention can be delivered in an immediate release or in a controlled release system. In one embodiment, an infusion pump may be used to administer a compound of the invention, such as one that is used for delivering 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, a compound of the invention is administered in combination with a biodegradable, biocompatible polymeric implant, which releases the compound over a controlled period of time at a selected site. 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 of the therapeutic target, thus requiring only a fraction of the systemic dose.

Furthermore, at times, the pharmaceutical compositions may be formulated for parenteral administration (subcutaneous, intravenous, intra-arterial, transdermal, intraperitoneal or intramuscular injection) and may include aqueous and non-aqueous, isotonic sterile injection solutions, which can contain anti-oxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions that include suspending agents, solubilizers, thickening agents, 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 detergents may also be used for parenteral administration. The above formulations may also be used for direct intra-tumoral injection. Further, in order to minimize or eliminate irritation at the site of injection, 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 adducts of ethylene oxide with a hydrophobic base, formed by the condensation of propylene oxide with propylene glycol.

The 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 the sterile liquid carrier, for example, water, for injections, immediately prior to use. Extemporaneous injection solutions and suspensions can be prepared from sterile powders, granules, and tablets of the kind previously described and known in the art. Each possibility represents a separate embodiment of the present invention.

Alternatively, the combinations of the present invention can be used in hemodialysis such as leukophoresis and other related methods, e.g., blood is drawn from the patient by a variety of methods such as dialysis through a column/hollow fiber membrane, cartridge etc., is treated with the tyrphostin derivatives and/or at least one other anti-cancer agent ex-vivo, and returned to the patient following treatment Such treatment methods are well known and described in the art. See, e.g., Kolho et al. (J. Med. Virol. 1993, 40(4):318-21); Ting et al. (Transplantation, 1978, 25(l):31-3); the contents of which are hereby incorporated by reference in their entirety.

Doses and Dosing Schedules

The treatment with the tyrphostin derivative and the at least other anti-cancer agent can take place sequentially in any order, simultaneously or a combination thereof. For example, administration of a tyrphostin derivative can take place prior to, after or at the same time as administration of the other anti-cancer agent. For example, a total treatment period can be decided for the tyrphostin derivative. The additional agent(s) can be administered prior to onset of treatment with the tyrphostin derivative or following treatment with the tyrphostin derivative. In addition, the additional agent(s) can be administered during the period of tyrphostin derivative administration but does not need to occur over the entire tyrphostin derivative treatment period. In another embodiment, the treatment regimen includes pre- treatment with one agent, either the tyrophostin derivative or the other anti-cancer agent, followed by the addition of the other agent or agents. Alternating sequences of administration are also contemplated. Alternating administration includes administration of a tyrphostin derivative and another anti-cancer agent in alternating sequences, e.g., tyrphostin derivative, followed by another anti-cancer agent, followed by tyrphostin derivative, etc.

The amount of a compound of the invention (i.e., tyrphostin derivative/anti-cancer agent) that will be effective in the treatment of 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. In addition, in vitro assays may optionally be employed to help identify optimal dosage ranges. The precise dose to be employed in the 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 practitioner and each patient's circumstances. A preferred dosage will be within the range of 0.01-1000 mg/kg of body weight, 0.1 mg kg to 100 mg/kg, 1 mg kg to 1 OOmg kg, 10 mg kg to 75 mg/kg, 0.1-1 mg/kg, etc. Exemplary (non-limiting) amounts of the tyrphostin derivative/another anti-cancer 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 molarity of the administered compound. By way of illustration and not limitation, a tyrphostin derivative (e.g. a compound of any of formulae I, II, A, B, C, D or E) can be administered in a range of 0.1-10 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 illustration and not limitation, a chemotherapeutic agent can be administered in an amount of 1 ng/ml to 100 mg/ml, for example 1-1000 ng/ml, 1-100 ng/ml, 1-1000 g/ml, 1-100 μg/ml, 1-1000 mg/ml, 1-100 mg/ml, etc. Effective doses may be extrapolated from dose- response curves derived from in vitro or animal model test bioassays or systems. When a synergistic effect is observed, the overall dose of each of the components may be lower, thus the side effects experienced by the subject may be significantly lower, while a sufficient anticancer effect is nevertheless achieved.

In one embodiment, the combination therapy reduces the amount of each of its component by a factor of 2, i.e., each component is given at half the dose as compared with single agent therapy, and still achieves the same or similar therapeutic effect. In another embodiment, the combination therapy reduces the amount of each of its component by a factor of 5, 10, 20, 50 or 100. As demonstrated herein, the IC ₅ o of chemotherapeutic agents as anti-proliferative agents in various cancer cells are reduced as compared to the IC50 of the chemotherapeutic agent, when administered alone.

The administration schedule will depend on several factors such as the cancer being treated, the severity and progression, the patient population, age, weight etc. For example, the compositions of the invention can be taken once-daily, twice-daily, thrice daily, once- weekly or once-monthly. In addition, the administration can be continuous, i.e., every day, or intermittently. The terms "intermittent" or "intermittently" as used herein means stopping and starting at either regular or irregular intervals. For example, intermittent administration can be administration one to six days per week or it may mean administration in cycles (e.g. daily administration for two to eight consecutive weeks, then a rest period with no administration for up to one week) or it may mean administration on alternate days. The different components of the combination can. independently of the other, follow different dosing schedules.

The following examples are presented in order to more fully illustrate certain embodiments of the invention. They should in no way, however, be construed as limiting the broad scope of the invention. One skilled in the art can readily devise many variations and modifications of the principles disclosed herein without departing from the scope of the invention.

EXPERIMENTAL DETAILS SECTION

Example 1: Inhibition of Cell Proliferation

Reagents and Antibodies

Velcade® (i.e. bortezomib or PS -341), irinotecan and sorafenib (Nexavar) were obtained from LC Laboratories. Dacarbazine (DTIC), cisplatin and methylene blue were purchased from Sigma. Dulbecco's modified Eagle's medium (DMEM) and fetal calf serum (FCS) were obtained from Biological Industries, Bet-Haemek, Israel. DMSO was obtained from BDH. Inhibition of cell proliferation

Human melanoma A375 cells were plated at a density of 2,500 cells/well, human ovarian cancer A2780 cells at density of 6,000 cells/well and human multiple myeloma cells, MMIS and RPMI8226, were plated at a density of 10,000 cells/well. All cells were plated in 96-well plates in 90 μΐ growth medium containing 10% FCS, 100 units/ml penicillin and 100 μg/ml streptomycin.

Compound A was added a day later in 5 μΐ of 1% DMSO in DDW to the multiple myeloma cell plates to obtain final concentrations of 0, 0.3 and 1 μΜ in RPMI8226 cells and 0, 0.1 and 0.3 μΜ in MMIS cells. The final concentration of DMSO (0.1% DMSO) was kept constant in all samples. Velcade® was added in 5 μΐ of 1% DMSO to obtain final concentrations of 0-0.5 nM in MMIS cells and 0-1 nM in RPMI8226 cells. Cells were exposed to WST-1 reagent for 5 hours following 72 hours treatment with the inhibitors, and optical density values were read at 630nm in ELISA plate reader. The data was analyzed in Microsoft Excel, using the vehicle control as 0% cytotoxicity. The assays were performed in triplicates.

Compound A was added to the cell medium of the A375 plates a day following cell seeding in 5 μΐ aliquots to obtain final concentrations of 0 - 0.9 uM, and 6hr later sorafenib, dacarbazine or irinotecan were added to obtain final concentrations of 0, 2.5, 5 and 10 μΜ sorafenib; 0, 10 30 and 60 uM dacarbazine; and 0.1, 0.3 and 1 μΜ irinotecan. A2780 cells were exposed to a tyrphostin derivative (compound A, compound C or compound E) a day following cell seeding and cisplatin was added at final concentrations of 0.05, 0.2 and 0.8 μΜ. Three days later, cells were fixed in 0.5% gluteraldehyde in medium for 15 min, washed three times with DDW, once with 0.1M sodium borate buffer pH 8.5 and stained with 1% methylene blue dissolved in 0.1M borate buffer solution for 60 min. Excess dye was washed out and cell-bound dye was eluted with 200 μΐ/well of 0.1 M HC1. The optical density values were read at 630nm in ELISA plate reader. The assays were performed in triplicates. Cytotoxicity (%) was calculated as follows: [(absorbance of control cells - absorbance of drug-treated cells) / absorbance of control cells] ^χ 100.

The Bliss additivism model (Cardone et al. Science (1998), 282: 1318-1321) was used to calculate the combined effect using the following formula: Ebliss = EA + EB - EA ^χ EB, where EA and EB are the fractional inhibitions obtained by drug A alone and drug B alone at specific concentrations.

The effect of the combined treatment of compound A and Velacade® was tested on multiple myeloma cells. Figures 1A and IB show strong synergistic effect for the combined treatment of compound A and Velcade® in two multiple myeloma cells, MM IS and RPMI8226. Velcade® was added 5 hr following treatment with compound A. The combined effect of compound A and Velcade® is significantly higher than the calculated additive effect at both 0.5 and 1 nM Velcade® in RPMI8226 cells (Figure 1 A) and at both 0.25 and 0.5 nM Velcade® in MM1S cells (Figure IB). Synergism was found using either 0.3 or 1 μΜ concentrations of compound A in RPMI8226 cells (Figures lAi and lAii, respectively), and 0.1 and 0.3 μΜ compound A in MM1S cells (Figures IBi and IBii, respectively). In advanced metastatic melanoma (AJCC stage IV), the prognosis is still poor, and there is still an unmet need for appropriate systemic treatment for these patients. Sorafenib is an oral multi-kinase inhibitor that targets several kinases which are known to be involved in both tumor proliferation and angiogenesis. These kinases include Raf kinases, platelet-derived growth factor receptor and the vascular endothelial growth factor receptor. Since B-raf gene mutations have been found in 69% of melanoma cell lines, sorafenib was brought into various phase I/II and phase III trials in metastatic melanoma. The combination of sorafenib with compound A in human melanoma A375 cells was tested. A synergic effect was found (Figure 2).

The combination of dacarbazine (an alkylating agent approved for the treatment of melanoma) and compound A was tested in human melanoma A375 cells. Figure 3 shows that the combined effect of compound A and dacarbazine is significantly higher than the calculated additive effect.

Figure 4 and 5 show additive effects of irinotecan and compound A in A375 melanoma cells and of compounds A, C or E with cisplatin in ovarian cancer A2780 cells, respectively.

Example 2: Inhibition of Cell Proliferation

EXPERIMENTAL DETAILS SECTION

Reagents

PLX4720 was purchased from Selleck chemical. Sutent (Sunitinib) and Irinotecan were acquired from LC Laboratories. NVP-AEW-541 was from Cayman chemicals. AG1478 was obtained internally. Compounds A and D were synthesized by and purchased from FineTech IL. 2-deoxy-glucose (2DG) was from Acros Organics.

Inhibition of cell proliferation

Human melanoma A375 cells were plated at a density of 1,500 cells/well, human ovarian cancer A2780 cells at density of 4,500 cells/well, human colon cancer HCT-15 cells at 2500 cells/well, human hepatocellular carcinoma HepG2 cells as well as human breast cancer MDA-MB-231 and U87MGwtEGFR, which are human glioblastoma overexpressing EGFR cells were seeded at a density of 3000 cells/well. All cells were plated in 96-well plates in 90 μΐ growth medium containing 10% FCS, 100 units/ml penicillin and 100 μg ml streptomycin.

Compound A or D was added a day later in 10 μΐ of 2-Hydroxypropyl-p-cyclodextrin (CD) in DDW (up to final concentration of 0.015% CD) to obtain final concentrations indicated at the figure legends, and 4-5.5hr later EGFR inhibitor AG1478 (figure 7), BRAF inhibitors PLX4720 or PLX4032 (figure 6A&C), multi kinase inhibitor Sutent (figure 8), IGF1R inhibitor NVP-AEW-541 (figure 9), topoisomerase inhibitor Irinotecan (figure 10) or metabolic inhibitor 2DG (figure 11) were added to obtain final concentrations indicated at the figures & figure legends. An example in which PLX4720 was added 24hr following compound A is shown as well (figure 6B). Three days following the addition of compound A or D, cells were fixed in 0.5% gluteraldehyde in medium for 15 min, washed three times with DDW, once with 0.1M sodium borate buffer pH 8.5 and stained with 1% methylene blue dissolved in 0.1M borate buffer solution for 60 min. Excess dye was washed out and cell- bound dye was eluted with 200 μΐ/well of 0.1 M HC1. The optical density values were read at 595nm in ELISA plate reader. The assays were performed in triplicates. Cytotoxicity (%) was calculated as follows: [(absorbance of control cells - absorbance of drug-treated cells) / absorbance of control cells] ^χ 100.

The Bliss additivism model (Cardone et al. Science (1998), 282: 1318-1321) was used to calculate the combined effect using the following formula: Ebliss = EA + EB - EA ^χ EB, where EA and EB are the fractional inhibitions obtained by drug A alone and drug B alone at specific concentrations.

The effect of the combined treatment of compound A or D and kinase inhibitors like EGF inhibitor AG 1478 was tested in cells expressing high levels of EGFR, or BRAF inhibitors PLX4720/PLX4032 in cells expressing mutated-BRAF, or IGF1R inhibitor NVP- AE W-541 and multi-kinase inhibitor in ovary cancer cells, representing one of the indications for which such inhibitors are tested for in clinical trials. Exemplified are additive or synergistic effects of the combined treatments. Furthermore, the combined effect of Irinotecan, a drug approved for colon cancer, and compound A were tested in an aggressive colon cancer cell line HCT-15, which is resistant to most drugs, and show preferred effect of the combination versus each treatment alone. An additive effect was shown with the combination of the compounds of formula (I) and metabolic pathway inhibition represented by 2DG.

While certain embodiments of the 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 present invention as described by the claims, which follow.