WO2014176475A2 - Egfr inhibitors and uses thereof - Google Patents

Abstract

Compounds that inhibit the interaction between HSP90 and proteins having an M-5 loop, such as EGFR, are disclosed, as are their use in treating EGFR-related diseases, including cancers and solid tumors.

Description

EGFR INHIBITORS AND USES THEREOF

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] The benefit is claimed of U.S. provisional application serial no. 61/816476, filed April 26, 2013, the disclosure of which is incorporated by reference in its entirety.

STATEMENT OF U.S. GOVERNMENT INTEREST

[0002] This invention was made with government support under Grant Number

R01CA131290 and Grant Number P50CADE97248. The government has certain rights in the invention.

BACKGROUND

[0003] About 1 out of 4 patients with non- small cell lung cancer (NSCLC) harbors activating kinase mutations in the epidermal growth factor receptor (EGFR). These patients typically respond to existing tyrosine kinase inhibitors (TKI), but their tumors become resistant to TKI's due to the emergence of mutation(s) (such as T790M). In addition to the T790M-EGFR mutation, some patients may acquire amplification in cMet. Few options for treatment exist for such TKI-resistant patients. Besides a gain of function in EGFR such as overexpression or somatic mutation(s) in the kinase domain, tumors can also acquire a mechanism that can enhance EGFR stability, which could lead to resistance. One such mechanism involves enhanced interaction with HSP90. This interaction can protect EGFR from both ligand and drug induced degradation, causing chemoresistance . These studies also show that not just inhibition of EGFR tyrosine kinase activity but EGFR itself is an important therapeutic target in cancer therapy.

[0004] EGFR degradation increases the tumor cell-specific cytotoxicity of chemo and radiotherapy beyond that obtained by EGFR inhibition alone. EGFR degradation can be enhanced by inhibition of HSP90 ATPase activity through the use of ansamycin analogues such as geldanamycin or 17-AAG, which leads to disruption of HSP90 interaction with its client proteins and significant enhancement in both chemo and radiosensitivity. Inhibition of HSP90 can reverse TKI resistance by induction of EGFR degradation. However, this approach is not specific to tumors, as HSP90 has multiple clients that support critical functions in normal tissues as well. Thus, HSP90 inhibition has, so far, produced

unacceptable liver or ocular toxicity in clinical studies. Certain other potential limitations may also exist with the global inhibition of a protein with such a wide range of functions. For example, the HSP90 inhibitor 17-AAG enhances osteoclast formation and actually potentiates bone metastasis in a human breast cancer cell line in vitro and in vivo. Therefore, a need exists for inhibitors that do not inhibit HSP90 activity but only affects its interaction with oncogenic EGFR, which would be more effective and less toxic to normal cells.

[0005] The stability of EGFR is regulated by direct interaction with HSP90. ErbB2 interacts with HSP90 via the M5-loop, which exists between the ccC helix and β4 strand of the kinase domain. Although the M5 loop sequence is different in EGFR, the M5-loop not only provides the contact surface for HSP90 but is also required for EGFR dimerization. EGF-bound EGFR prefers to form homodimers, whereas the non-ligand bound monomer of EGFR preferentially interacts with HSP90. These mechanisms are mutually exclusive ways of enhancing EGFR stability. Therefore, an M5-loop mimetic will be effective in blocking both mechanisms of enhanced EGFR stability and thus lead to EGFR degradation.

SUMMARY

[0006] Provided herein are compounds and methods of using the same, for example, as inhibitors of M-5 loop containing proteins, such as epithelial growth factor receptor (EGFR). Thus, provided herein is a method of increasing degradation of epithelial growth factor receptor (EGFR) or inhibiting EGFR dimerization comprising contacting EGFR with a compound having a structure of formula (I) in an amount sufficient to inhibit EGFR

dimerization or induce EGFR degradation:

, wherein ring A is a 5- membered heteroaryl; Ar is an aryl or heteroaryl; and Ar₂ is an aryl, heteroaryl, cycloalkyl, or heterocycloalkyl, or a pharmaceutically acceptable salt or solvate thereof. In some cases, ring A is selected from the group consisting of diazolyl, triazolyl, tetrazolyl, thiophenyl, and

furanyl. In some embodiments, ring A is diazolyl, for example, . In

various cases is triazolyl, for example, . In some cases, ring A is tetrazolyl,

for example,

. In various embodiments, ring A is further substituted. Ring A can be substituted with an alkyl or amino substituent. [0007] In various cases, the compound has a structure of formula (II):

are each independently a Ci_4 alkyl or together form a spiro heterocyclyl. In some embodiments, each of R 1 and R 2 is a Ci_4 alkyl. In various embodiments, each of R 1 and R2 is methyl. In some cases, R 1 and R2 together form a spiro heterocyclyl. In some cases, the sprio heterocyclyl can be piperdine, pyrrolidine, tetrahydrofuran, tetrahydropyran, dihydrofuran, morpholine, or thiophene. In various embodiments, the spiro heterocyclyl is substituted. The substitution can be, for example, alkyl, alkoxy, or halo. In some cases, the spiro heterocyclyl is N-methyl-piperdine. In some cases, the spiro heterocyclyl is N-methyl-pyrrolidine.

[0008] In various embodiments, Α is aryl. The aryl can be, for example, phenyl or naphthyl. In some cases, Α is substituted aryl. In various embodiments, the phenyl is substituted at the 3 or 4 position. The substitution can be, for example, one or more of halo, alkyl, and alkoxy. In some embodiments, Αη is heteroaryl. The heteroaryl can be thienyl, furyl, pyridyl, oxazolyl, quinolyl, thiophenyl, isoquinolyl, indolyl, triazinyl, triazolyl, isothiazolyl, isoxazolyl, imidazolyl, benzothiazolyl, pyrazinyl, pyrimidinyl, thiazolyl, or thiadiazolyl. In various cases, the heteroaryl can be a substituted heteroaryl. The substitution can be, for example, one or more of halo, alkyl, and alkoxy.

[0009] In various embodiments, Ar₂ is aryl. In some cases, the aryl is phenyl or naphthyl. In various cases, Ar₂ is substituted aryl. The aryl can be substituted phenyl, and in some embodiments, the phenyl is substituted at at least one of the 3 and 4 position. In some cases, the phenyl is substituted at both the 3 and 4 position. The substitution can be, for example, one or more of halo, alkyl, and alkoxy. In various embodiments, Ar₂ is heteroaryl. In some cases, the heteroaryl is thienyl, furyl, pyridyl, oxazolyl, quinolyl, thiophenyl, isoquinolyl, indolyl, triazinyl, triazolyl, isothiazolyl, isoxazolyl, imidazolyl, benzothiazolyl, pyrazinyl, pyrimidinyl, thiazolyl, or thiadiazolyl. In various cases, Ar₂ is substituted heteroaryl. The substitution can be one or more of halo, alkyl, and alkoxy. In some cases, Ar₂ is cycloalkyl. The cycloalkyl can be selected from cyclopropyl, cyclobutyl, cyclopentyl, cylcohexyl, cycloheptyl, or cylcooctyl. In some specific cases, cycloaklyl is cyclpentyl or cyclohexyl. The Ar₂ can be a substituted cycloalkyl, and, in some cases, the substitution can be one or more of halo, alkyl, and alkoxy. [0010] In various embodiments, the compound inhibits EGFR interaction with HSP90. In some cases, the compound inhibits HSP90 activity less than 20%. In some cases, the compound does not inhibit HSP90 activity.

[0011] In various embodiments, the contacting comprises administering to a subject in need thereof. In various embodiments, the subject is diagnosed with cancer. The cancer can be characterized by overexpression of EGFR or expression of a mutant EGFR. In some cases, the cancer is lung cancer, pancreatic cancer, head and neck cancer, or colorectal cancer. In various cases, the cancer is a lung cancer, head and neck cancer, cervical cancer, glioblastoma, colorectal cancer, or breast cancer. In various cases, the compound inhibits one or more of DMPK, Erb2, and cMet. In some cases, the contacting further results in inhibition of one or more of DMPK, Erb2, and cMet.

[0012] In some embodiments, the methods disclosed herein further comprise contacting with a second therapeutic. The second therapeutic can be a chemotherapeutic or radiation therapy. In some embodiments, the chemotherapeutic is one or more of cisplatin and gemcitabine. 0013] In various embodiments, the compound is selected from the group consisting of

, or a salt or solvate thereof. 0014] Further provided herein are compounds having a structure of formula (III):

wherein R is alkyl, C(0)alkyl, C(0)NHalkyl, or C(0)Oalkyl;

Aii is an aryl or heteroaryl; and Ar₂ is an aryl, heteroaryl, cycloalkyl, or heterocycloalkyl, or a pharmaceutically acceptable salt or solvate thereof. In some cases, R¹ is alkyl, for example, methyl or ethyl. In some cases, R¹ is C(0)alkyl, and in some embodiments, the alkyl is methyl or ethyl. In some cases, R¹ is C(0)NHalkyl, and in some embodiments, the alkyl is methyl or ethyl. In some cases, R¹ is C(0)Oalkyl, and in some embodiments, the alkyl is methyl or ethyl. In various embodiments, Ari is aryl. In some cases, the aryl is phenyl or naphthyl. In some cases, Ari is substituted aryl. In some cases, Ar₂ is phenyl, and the phenyl is substituted at the 3 or 4 position. In some cases, the substitution is one or more of halo, alkyl, and alkoxy. In various embodiments, Ari is heteroaryl. The heteroaryl can be thienyl, furyl, pyridyl, oxazolyl, quinolyl, thiophenyl, isoquinolyl, indolyl, triazinyl, triazolyl, isothiazolyl, isoxazolyl, imidazolyl, benzothiazolyl, pyrazinyl, pyrimidinyl, thiazolyl, or thiadiazolyl. In various cases, Ari is substituted heteroaryl. The substitution can be one or more of halo, alkyl, and alkoxy. In some cases, Ar₂ is aryl. The aryl can be, for example, phenyl or naphthyl. Ar₂ can be substituted aryl. The Ar₂ phenyl can be substituted at at least one of the 3 and 4 position. In some cases, the phenyl is substituted at both the 3 and 4 position. In various cases, the substitution is one or more of halo, alkyl, and alkoxy. In various embodiments, Ar₂ is heteroaryl. The heteroaryl can be thienyl, furyl, pyridyl, oxazolyl, quinolyl, thiophenyl, isoquinolyl, indolyl, triazinyl, triazolyl, isothiazolyl, isoxazolyl, imidazolyl, benzothiazolyl, pyrazinyl, pyrimidinyl, thiazolyl, or thiadiazolyl. In some cases, Ar₂ is substituted heteroaryl. The substitution can be one or more of halo, alkyl, and alkoxy. In various embodiments, Ar₂ is cycloalkyl. The cycloalkyl can be cyclopropyl, cyclobutyl, cyclopentyl, cylcohexyl, cycloheptyl, or cylcooctyl. In various cases, the cycloaklyl is cyclpentyl or cyclohexyl. In some embodiments, Ar₂ is a substituted cycloalkyl. The substitution can be one or more of halo, alkyl, and alkoxy. In various embodiments, Ar₂ is heterocyclo alkyl. The heterocyloalkyl can be, for example, piperdine, pyrrolidine, tetrahydrofuran, tetrahydropyran, dihydrofuran, morpholine, or thiophene. The

heterocyclo alkyl can be substituted. The substitution can be, for example, one or more of halo, alkyl, and alkoxy. Also provided herein are methods of inhibiting EGFR or an M-5 loop containing protein by contacting the protein with a compound of formula (III).

BRIEF DESCRIPTION OF THE FIGURES

[0015] FIG 1 shows screening and validation of M5-loop analogues. A, (left panel), NCI- HI 975 cells stably expressing EGFR-reporter were treated with a total of 51 compounds identified in the virtual screen along with the positive controls. After initiation of treatment, change in EGFR activity was recorded at multiple time-points by measurement of

bioluminescence signal. Four compounds showed a concentration dependent increase in the bioluminescence. Fold-change in the bioluminescence is plotted in the right panel. These four compounds showed similar activity in UMSCCl-EGFR reporter cells (not shown). B, Effect on lead compounds (10 μΜ for 2-hrs) on interaction between HSP90 and EGFR as well as total EGFR level (24-hrs) was confirmed by immunoprecipitation and direct

immunoblotting. C, Efficacy and selectivity of lead compound was determined by colony formation assay in several cancer and normal cells (MRC5- lung fibroblasts) and resulting IC₅₀ is shown.

[0016] FIG. 2 shows the results of an in vivo study of the treatment of mice having NCI- HI 975 xenografts with an EGFR inhibitor as disclosed herein.

DETAILED DESCRIPTION

[0017] Somatic mutations in the EGFR kinase domain confer sensitivity to tyrosine kinase inhibitors (TKI), but a majority of patients become resistant to TKI's due to de novo mutations (such as T790M). Preclinical studies have indicated that inhibition of HSP90 can degrade EGFR and reverse TKI resistance. While HSP90 inhibitors have displayed efficacy, their clinical use has been limited by ocular and liver toxicities. The stability of EGFR is regulated by 1) its direct interaction with HSP90 and 2) by EGFR- dimerization. These interactions occur via the M5-loop of the kinase domain of EGFR. An M5-loop mimetic can induce EGFR degradation by blocking its stabilizing interactions independent of TKI- resistant EGFR. A substrate- site directed, protein-protein interaction (PPI) inhibitor peptide, Disruptin, was developed which is effective against TKI-resistant cells and xenografts. Based on the Structural Activity Relationship (SAR) of Disruptin, a class of small molecule PPI inhibitors was identified that also inhibit EGFR interaction with HSP90 and block EGFR dimerization, induce EGFR degradation, and kill TKI resistant lung cancer cells. Provided herein are small molecules that can prevent the protein-protein interaction that occurs between EGFR and HSP90 and also between monomers of EGFR.

[0018] Disruptin is a substrate- site specific, protein-protein interaction (PPI) inhibitor peptide. Treatment with Disruptin causes inhibition of EGFR interaction with HSP90 and blocks EGFR dimerization, which results in degradation of EGFR and inhibition of tumor growth of T790M-EGFR driven lung tumor cell-lines. A small molecule structural analog of the Disruptin (or the M5-loop) can similarly cause degradation of EGFR, by blocking receptor dimerization, as well as by inhibition of EGFR-HSP90 binding. A class of small molecule PPI inhibitors that inhibits the EGFR dimerization and its interaction with HSP90 and selectively kills T790M-EGFR driven lung cancer cells has been identified.

EGFR-HSP90 Interactions

[0019] Heat shock protein 90 (HSP90) is a molecular chaperone that maintains the stability of several oncogenic kinases. EGFR directly interacts with HSP90, which can enhance receptor stability. This finding is in agreement with reports that show that blocking HSP90 activity by geldanamycin (GA) has a profound effect on EGFR stability and chemo and radio-sensitization. Inhibition of HSP90 activity is effective in inducing EGFR degradation which can sensitize drug-resistant tumors in pre-clinical models. Therefore, a number of clinical candidates that directly target the ATP-binding site of HSP90 are in development, but, to date, no agent has been approved due in part to the toxicity associated with its global inhibition. Therefore, a novel approach that would interfere with the EGFR-HSP90 interaction but would not affect HSP90 activity is attractive.

[0020] EGFR plays an important role in the survival of tumor cells. Therefore, promoting EGFR degradation has important implications in targeting tumor cells. EGFR signaling has been inhibited by small molecule tyrosine kinase inhibitors (e.g. erlotinib, gefitinib) or by monoclonal antibodies (cetuximab, panitumumab, etc.). Targeting the receptor for degradation is an important mechanism that regulates gemcitabine and cisplatin cytotoxicity in head and neck, lung, breast and colorectal cancer cells. Knockdown of EGFR with small interfering RNA can induce cell death independent of its tyrosine kinase activity. Together these findings provide compelling evidence that targeting the receptor for degradation is important for cytotoxic effects. Thus, a therapeutic agent that can selectively induce EGFR degradation will be more effective than agents that inhibit receptor activity only. HSP90 inhibitors represent this class of agents, as they are known to induce EGFR-degradation, but HSP90 inhibitors non- selectively degrade other clients of HSP90. EGFR is an important client of HSP90, and there is direct interaction between EGFR and HSP90 by GST pull-down assay. Blocking of HSP90 interaction with EGFR would induce degradation of EGFR and would not affect other clients. HSP90 recognizes a common hydrophobic surface on client proteins, and the interaction between ErbB2 and HSP90 occurs via the M5 loop of the kinase domain, which lies between the aC helix and the β4 strand. Therefore, EGFR, which contains a similar M5 loop, may interact with HSP90 via this loop.

[0021] If EGFR interacts with HSP90 via the M5 loop of EGFR, mutation in this loop should reduce the protein-protein interaction, which would reduce EGFR stability. Therefore, mutants of EGFR, expressed in EGFR-null CHO cells, were assessed for their interactions with HSP90 Mutations in the M5 loop (stretch of 6 amino acid from 768-773) decreased EGFR interaction with HSP90. CHO cells were transfected with WT or 768-773 mutant EGFR constructs, followed by CHX (50 μg/ml) treatment to block the new protein synthesis. The level of EGFR was assessed at multiple time points using immunoblot analysis, and half- life was calculated. The half-life of the mutant EGFR (768-773) was reduced from 6 to 2 h.

[0022] Several mutants of EGFR spanning 20 amino acids around M5 loop were expressed in EGFR null CHO cells. For each mutant, the interaction with HSP90 and steady state level of EGFR was assessed. The interaction between EGFR and HSP90 depends not on any single amino acid but on the 6 amino acid stretch from 768 to 773. There is also a correlation between EGFR and HSP90 interaction and steady state level of EGFR. The M5 loop mutation of EGFR is vulnerable to degradation due to its decreased binding with HSP90. The WT and scrambled 768-773 EGFR in CHO cells were treated with cycloheximide to block protein synthesis. EGFR levels were reduced by 30% in WT-EGFR expressing cells at 3 h and by 50% at 6h. However, levels of scrambled 768-773 EGFR were reduced by about 90% within 3h, showing that scrambled 768-773 EGFR (M5 domain) is a significantly less stable protein than WT-EGFR.

[0023] There is a role of Serine744 (also referred as S768) within the M5 loop in EGFR stability and its dimer formation, as such in addition to HSP90 interaction, the M5 loop is also involved in the formation of EGFR dimers. The WT- kinase domain and mutated the M5 loop were cloned by scrambling 6 amino acids (768-SVDNPH-773 to 768-NHVPSD- 773). A V5 tag was fused with EGFR for molecular studies. To see the effect of M5 loop mutation on EGFR homo-dimerization, EGFR-negative CHO cells were transfected with either WT or M5 mutated KD of EGFR. One day after transfection, cells were treated with Disuccinimidyl suberate (DSS) for 30 min (150 μΜ) to cross-link proteins. EGFR was immunoprecipitated using V5 antibody, and covalently cross-linked EGFR dimers were separated from monomer by reducing SDS-PAGE. Reduction in the 76 KDa dimer in the M5 mutant EGFR relative to WT EGFR confirms the role of M5 loop in EGFR dimerization.

[0024] Oligopeptides that mimic this region were designed. Over a dozen peptides spanning the M5-loop and found that one of the peptides, called "Disruptin", which is analogous to the WT 768-773 EGFR, caused a decrease in cell survival and reduced total EGFR level confirming the importance of this domain in the EGFR protein stability. For further control studies, the amino acids of Disruptin were scrambled to generate a scrambled peptide (control peptide). These two peptides were synthesized along with 11 amino acids selected from the HIV-TAT gene to enable cellular uptake, and a biotin moiety was attached for molecular studies.

[0025] It was first determined whether Disruptin could block HSP90 interaction with EGFR in a cell free assay. Disruptin reduced HSP90 binding to EGFR by 90% compared to scrambled peptide. This effect of Disruptin on blockade of protein-protein interaction may be a result of Disruptin binding with both HSP90 and EGFR. To determine whether Disruptin directly interacts with HSP90 or EGFR, biotin-conjugated peptides were incubated with the whole cell extracts from NCTH1975 (and several other cell lines) for 2 h, and Disruptin (biotin)-bound HSP90 or EGFR was pulled down by streptavidin beads and resolved by immunoblotting. A large amount of HSP90 and EGFR was affinity purified using Disruptin (5 to 10 fold over vehicle), and that this binding depended on the concentration of Disruptin. The scrambled peptide had some affinity towards HSP90 but not towards EGFR, which may be due to partial similarity with Disruptin). In contrast to a typical HSP90 ATPase inhibitor, Disruptin does not affect HSP90 ATPase activity. Disruptin had no effect on HSP90's ability to bind with ATP, as expected, geldanamycin treatment reduced HSP90 binding with ATP. As Disruptin did not block HSP90 activity, it is more selective compared to geldanamycin.

[0026] After a 24-h exposure to Disruptin, a significant reduction in EGFR-HSP90 interaction is observed in two EGFR-driven cancer cell lines (UMSCC1 (WT-EGFR and cMet amplification) and NCI-H1975 (erlotinib resistant T790M-EGFR)), which was comparable to geldanamycin treatment, whereas the control peptide had no effect.

Importantly, Disruptin treatment did not induce HSP90 levels, which were elevated by the HSP90 ATPase inhibitor geldanamycin, indicative of a compensatory response to HSP90 inhibition. A loss of EGFR protein was observed and a decrease in clonogenic survival in both cell lines. These findings demonstrate that Disruptin disrupts EGFR-HSP90 interaction, decreases EGFR stability of both wild type and erlotinib resistant T790M-EGFR, and decreases clonogenic survival, recapitulating the effect of theM5 residues 768-773.

[0027] The short term toxicity of Disruptin relative to geldanamycin in immune-competent C57BL/6 mice was assessed. Mice were dosed with 10 and 30 mg/kg of Disruptin (i.p.), and several organs including lung, liver, heart, kidneys, spleen, stomach, small intestine, mesenteric lymph nodes, cecum, colon, pancreas, ovaries, bone marrow, and eyes were evaluated 3-days post-injection by a board-certified veterinary pathologist. As expected there were no histological alterations in the any of the organs analyzed after treatment with Disruptin. In contrast, mice treated with geldanamycin showed previously known ocular and liver toxicities. Complete blood counts and liver cytosolic enzymes (AST, ALT) were also not different from controls in the Disruptin or scrambled peptide treated mice at either dose.

[0028] These results suggest that Disruptin, at an effective dose, was well-tolerated and lacked the adverse effects seen in case of a typical HSP90 inhibitor. Disruptin was assessed for effectiveness in reducing EGFR expression and growth of NCTH1975 (T790M-EGFR expressing erlotinib resistant cells) and UMSCC1 xenografts. The administration of two injections of Disruptin (8 mg/kg, i.p., 3 days apart) increased median tumor doubling time significantly (P<0.0001) in both the tumor models compared to the scrambled peptide or erlotinib (100 mg/kg, oral, Monday- Friday) treatment. Tumor growth delay upon Disruptin treatment correlated well with decreased EGFR in tumors cells as assessed by

immunoblotting one week into the treatment. Analysis of tumor histology revealed profound fibrotic response upon Disruptin treatment in both the tumor types.

Small Molecule EGFR-HSP90 Interaction Inhibitors

[0029] While Disruptin provides proof of concept that some functions of HSP90 can be inhibited without inhibiting all client functions, an oligopeptide will likely have limited utility as a cancer therapeutic against an intracellular target. Therefore, EGFR-M5-loop (similar to Disruptin) was used as a probe for a ligand-based virtual screen to identify small molecule inhibitors with a similar biological profile. [0030] An EGFR activity reporter assay was used to assess biological activity of potential small molecule inhibitors (see Khan et al., Anal Biochem. 201 l;417(l):57-64). The reporter is based on the reverse complementation of luciferase components where inhibition of EGFR activity causes a proportional increase in the bioluminescence. In this assay a loss of EGFR correlates with inactivation of EGFR, which is reflected in the activation of bioluminescence. Several tumor and normal cells including NCTH1975, UMSCCl and MRC5 were transfected to stably express this bioluminescence reporter to screen effective compounds using a high- throughput platform. EGFR-reporter cells were treated with increasing concentrations of various M5-loop analogues; erlotinib, geldanamycin and AT13386 (another HSP90 inhibitor from CTEP-NCI and Astex Pharmaceutical) were used as positive controls. This assessment

provided an inhibitor having a structure:

(C250-0395, also referred to herein as Compound 95) (Figure 1).

[0031] A set of 27 closely related structural analogs of C250-0395 were tested in the screening funnel to establish structure-activity relationships with EGFR degradation.

Molecules were considered active if there was a 1.5 fold induction of EGFR-reporter activity in the high throughput assay. Active molecules in the assay were selected for follow-up within EGFR dependent cell lines for causing EGFR degradation, and decreasing clonogenic survival while not inducing HSP70 (induction would suggest HSP90 ATPase inhibition). Interestingly, the ICso's of all the active compounds were in the range of 5 to 20μΜ for cancer cells and >50 μΜ for normal fibroblasts, indicating potential selectivity for cancer cells. Based upon this assessment, the structure tolerates substitution at the para position of the left aryl, spiro or dialkyl substitution on the diazoyl ring, and substitution at the 3 or 4 position on the right aryl. The effect of lead molecule (C250-0395) was compared with Disruptin to assess its ability to block EGF induced EGFR dimer formation. Based on the importance of M5-loop in T790M-EGFR homo-dimer contact surface mutation in the M5- loop of EGFR kinase domain is required for EGFR dimerization, that similar to Disruptin, it was hypothesized lead compound C250-0395 would also block EGF-induced dimer formation of EGFR. NCI-H1975 cells were treated with either Disruptin or compound C250- 0395 for 2-hrs; cells were then stimulated with EGF treatment 30-mins. After treatment, cells were washed; proteins were covalently cross-linked by treatment with DSS (150 μΜ, 30 min). Whole cell lysates were prepared, and EGFR-dimers were resolved. Result suggests that compound C250-0395 reduced EGF induced dimerization of EGFR similar to Disruptin. Erlotinib had no effect on EGF-induced EGFR-dimer formation. These findings suggest that the mechanism of action of lead compound is similar to Disruptin.

[0032] Thus, provided herein are compounds having a structure of formula (I):

ring A is a 5-membered heteroaryl;

Ari is an aryl or heteroaryl; and

Ar₂ is an aryl, heteroaryl, cycloalkyl, or heterocycloalkyl,

or a pharmaceutically acceptable salt or solvate thereof. In some cases, the compound of formula (I) has a structure of formula (II):

wherein

R 1 and R 2" are each independently a C_1-4 alkyl or together form a spiro heterocyclyl; and Ar and Ar₂ are as defined for formula (I), or a pharmaceutically acceptable salt or solvate thereof. Further provided are com ounds having a structure of formula (III):

wherein R¹ is alkyl, C(0)alkyl, C(0)NHalkyl, or C(0)Oalkyl, and An and Ar₂ are as defined for formula (I), or a pharmaceutically acceptable salt or solvate thereof.

[0033] In various embodiments, the A ring is selected from the group consisting of diazolyl, triazolyl, tetrazolyl, thiophenyl, and furanyl. The A ring can be a diazolyl, triazolyl,

or tetrazolyl ring. The A ring can be . The A ring can be further substituted. In some cases, the further substitution on the A ring is one or more of alkyl or amino. [0034] When the compound has a structure of formula (II), in some cases, each of R¹ and

R 2 can be a Ci_₄ alkyl, for example, methyl or ethyl. In some cases, both R 1 and R2 are each methyl. In some cases, R 1 and R 2 together form a spiro heterocyclyl, for example piperdine, pyrrolidine, tetrahydrofuran, tetrahydropyran, dihydrofuran, morpholine, or thiophene. The spiro heterocyclyl can be further substituted. The substitution can be one or more of alkyl, alkoxy, and halo. In some specific cases, the spiro heterocyclyl is N-methyl-piperdine or N- methyl-pyrrolidine.

[0035] Ari can be aryl, such as phenyl or naphthyl. The aryl can be substituted, for example, with one or more of halo, alkyl, and alkoxy. Alternatively, Ari can be heteroaryl, such as thienyl, furyl, pyridyl, oxazolyl, quinolyl, thiophenyl, isoquinolyl, indolyl, triazinyl, triazolyl, isothiazolyl, isoxazolyl, imidazolyl, benzothiazolyl, pyrazinyl, pyrimidinyl, thiazolyl, or thiadiazolyl. The heteroaryl can be substituted, for example, with one or more of halo, alkyl, and alkoxy.

[0036] Ar₂ can be aryl, such as phenyl or naphthyl. The aryl can be substituted, for example, with one or more of halo, alkyl, and alkoxy. In some cases, the aryl is substituted at at least one of the 3 and 4 positions, and in various cases, is substituted at both the 3 and 4 positions. Alternatively, Ar₂ can be heteroaryl, such as thienyl, furyl, pyridyl, oxazolyl, quinolyl, thiophenyl, isoquinolyl, indolyl, triazinyl, triazolyl, isothiazolyl, isoxazolyl, imidazolyl, benzothiazolyl, pyrazinyl, pyrimidinyl, thiazolyl, or thiadiazolyl. The heteroaryl can be substituted, for example, with one or more of halo, alkyl, and alkoxy. In some cases, Ar₂ can be cycloalkyl, such as cyclopropyl, cyclobutyl, cyclopentyl, cylcohexyl, cycloheptyl, or cylcooctyl. The cycloalkyl can be substituted, for example with one or more of halo, alkyl, and alkoxy. In some specific cases, the cycloalkyl is cyclopenyl or cyclohexyl, and optionally substituted. In various cases, Ar₂ is heterocycloalkyl, such as piperdine, pyrrolidine, tetrahydrofuran, tetrahydropyran, dihydrofuran, morpholine, or thiophene. The heterocycloalkyl can be further substituted, such as with one or more of alkyl, alkoxyl and halo.

[0037] Further provided herein are com ounds having a structure of formula (III):

wherein R¹ is alkyl, C(0)alkyl, C(0)NHalkyl, or C(0)Oalkyl; Ari is an aryl or heteroaryl; and Ar₂ is an aryl, heteroaryl, cycloalkyl, or heterocycloalkyl, or a pharmaceutically acceptable salt or solvate thereof. R¹ can be alkyl, and in specific cases, can be methyl or ethyl. R¹ can be C(0)alkyl, and in specific cases can be C(0)Me or C(0)Et. R¹ can be C(0)NHalkyl, and in specific cases can be C(0)NHMe or C(0)NHEt. R¹ can be

C(0)Oalkyl, and in specific cases can be C(0)OMe or C(0)OEt.

[0038] In various cases, Ari can be aryl. For example, Ari can be phenyl or naphthyl. The aryl can be substituted. The aryl can be substituted, for example, at the 3 and/or 4 position. The substitution can be one or more of halo, alkyl, and alkoxy. In various cases, Ari can be heteroaryl. For example, Ari can be thienyl, furyl, pyridyl, oxazolyl, quinolyl, thiophenyl, isoquinolyl, indolyl, triazinyl, triazolyl, isothiazolyl, isoxazolyl, imidazolyl, benzothiazolyl, pyrazinyl, pyrimidinyl, thiazolyl, or thiadiazolyl. The heteroaryl can be substituted. For example, the heteroaryl can be substituted with one or more of halo, alkyl, and alkoxy.

[0039] In various cases, Ar₂ can be aryl. For example, Ar₂ can be phenyl or naphthyl. The aryl can be substituted. The aryl can be substituted, for example, at the 3 and/or 4 position. The substitution can be one or more of halo, alkyl, and alkoxy. In various cases, Ar₂ can be heteroaryl. For example, Ar₂ can be thienyl, furyl, pyridyl, oxazolyl, quinolyl, thiophenyl, isoquinolyl, indolyl, triazinyl, triazolyl, isothiazolyl, isoxazolyl, imidazolyl, benzothiazolyl, pyrazinyl, pyrimidinyl, thiazolyl, or thiadiazolyl. The heteroaryl can be substituted. For example, the heteroaryl can be substituted with one or more of halo, alkyl, and alkoxy. In some cases, Ar₂ can be cycloalkyl, such as cyclopropyl, cyclobutyl, cyclopentyl, cylcohexyl, cycloheptyl, or cylcooctyl. The cycloalkyl can be substituted, for example with one or more of halo, alkyl, and alkoxy. In some specific cases, the cycloalkyl is cyclopenyl or cyclohexyl, and optionally substituted. In various cases, Ar₂ is heterocycloalkyl, such as piperdine, pyrrolidine, tetrahydrofuran, tetrahydropyran, dihydrofuran, morpholine, or thiophene. The heterocycloalkyl can be further substituted, such as with one or more of alkyl, alkoxyl and halo. 0040] Specific compounds contemplated in the disclosure include

, or a salt or solvate thereof.

[0041] The compounds disclosed herein can inhibit interaction HSP90 and a protein having an M5 loop. Examples of proteins having an M5 loop include EGFR, DMPK, Erb2, and cMet.

[0042] The term "alkyl" used herein refers to a saturated or unsaturated straight or branched chain hydrocarbon group of one to ten carbon atoms, including, but not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-hexyl, and the like. Alkyls of one to four carbon atoms are also contemplated. The term "alkyl" includes "bridged alkyl," i.e., a bicyclic or polycyclic hydrocarbon group, for example, norbornyl, adamantyl, bicyclo[2.2.2]octyl, bicyclo[2.2.1]heptyl, bicyclo[3.2.1]octyl, or decahydronaphthyl. Alkyl groups optionally can be substituted, for example, with hydroxy (OH), halide, thiol (SH), aryl, heteroaryl, cycloalkyl, heterocycloalkyl, and amino.

[0043] As used herein, the term "cycloalkyl" refers to a cyclic hydrocarbon group, e.g., cyclopropyl, cyclobutyl, cyclohexyl, and cyclopentyl. "Heterocycloalkyl" is defined similarly as cycloalkyl, except the ring contains one to three heteroatoms independently selected from the group consisting of oxygen, nitrogen, and sulfur. Nonlimiting examples of heterocycloalkyl groups include piperdine, tetrahydrofuran, tetrahydropyran, dihydrofuran, morpholine, thiophene, and the like. In some cases, cycloalkyl and heterocycloalkyl groups can be saturated or partially unsaturated ring systems optionally substituted with, for example, one to three groups, independently selected from the group consisting of alkyl, alkyleneOH, C(0)NH₂, NH₂, oxo (=0), aryl, haloalkyl, halo, and OH. Heterocycloalkyl groups containing a N heteroatom optionally can be further N-substituted with alkyl, hydroxyalkyl, alkylenearyl, or alkyleneheteroaryl.

[0044] As used herein, the term "aryl" refers to a monocyclic or polycyclic aromatic group, preferably a monocyclic or bicyclic aromatic group, e.g., phenyl or naphthyl. Unless otherwise indicated, an aryl group can be unsubstituted or substituted with one or more, and in particular one to four groups independently selected from, for example, halo, alkyl, alkenyl, OCF₃, N0₂, CN, NC, OH, alkoxy, amino, C0₂H, C0₂alkyl, aryl, and heteroaryl. Exemplary aryl groups include, but are not limited to, phenyl, naphthyl, tetrahydronaphthyl, chlorophenyl, methylphenyl, methoxyphenyl, trifluoromethylphenyl, nitrophenyl, 2,4- methoxychlorophenyl, and the like. In some specific cases, the aryl can be substituted with one or more of halo, alkyl and alkoxy.

[0045] As used herein, the term "heteroaryl" refers to a monocyclic or bicyclic ring system containing one or two aromatic rings and containing at least one nitrogen, oxygen, or sulfur atom in an aromatic ring. Unless otherwise indicated, a heteroaryl group can be unsubstituted or substituted with one or more, and in particular one to four, substituents selected from, for example, halo, alkyl, alkenyl, OCF₃, N0₂, CN, NC, OH, alkoxy, amino, C0₂H, C0₂alkyl, aryl, and heteroaryl. In some cases, the heteroaryl group is substituted with one or more of alkyl and alkoxy groups. Examples of heteroaryl groups include, but are not limited to, thienyl, furyl, pyridyl, oxazolyl, quinolyl, thiophenyl, isoquinolyl, indolyl, triazinyl, triazolyl, isothiazolyl, isoxazolyl, imidazolyl, benzothiazolyl, pyrazinyl, pyrimidinyl, thiazolyl, and thiadiazolyl. In some cases, the heteroaryl group is selected from the group consisting of

[0046] These compounds can be synthesized in a variety of ways. One such scheme is shown below.

Scheme

[0047] The synthesis of a specific compound is outlined in the below scheme. It will be appreciated that similar such compounds can be prepared using the same synthetic steps and varying the starting reagents used.

Scheme

Methods of Inhibiting a Binding Interaction between EGFR and HSP90

[0048] The compounds disclosed herein inhibit the interaction of HSP90 and a protein having an M-5 loop. Proteins having an M-5 loop include EGFR, cMet, Erb2, and DMPK. The compounds disclosed herein inhibit the interaction between EGFR and HSP90. The compounds can inhibit the interaction between HSP90 and the protein with an M-5 loop at least 20%, at least 30%, at least 40%, at least 50%, or at least 60%, or up to 20%, up to 30%, up to 40%, up to 50%, up to 60%, or up to 70%.

[0049] The compound inhibits the interaction between HSP90 and the protein with an M-5 loop, but does not inhibit activity of HSP90 itself. In various cases, the inhibition of HSP90 activity is less than 20%, less than 15%, less than 10%, or less than 5%, and in some cases, HSP90 activity is not substantively inhibited. In various cases, the protein with the M-5 loop is EGFR, cMet, Erb2, or DMPK. In some cases, the protein with the M-5 loop is EGFR.

[0050] Given the importance of the biological roles of EGFR and HSP90, individually, and as shown herein, in combination with one another, the compounds of the present disclosure are useful for a number of applications in a variety of settings. For example and most simplistically, the compounds of the present disclosure are useful for inhibiting a binding interaction between EGFR and HSP90 in a cell. In this regard, the present disclosure provides a method of inhibiting a binding interaction between EGFR and HSP90 in a cell. The method comprises contacting the cell with a compound of the present disclosure or a pharmaceutically acceptable salt or solvate thereof, in an amount effective to inhibit the binding interaction. In some aspects, the cell is part of an in vitro or ex vivo cell culture or in vitro or ex vivo tissue sample. In some aspects, the cell is an in vivo cell. In certain embodiments, the method is intended for research purposes, and, in other embodiments, the method is intended for therapeutic purposes.

Methods of Increasing EGFR Degradation

[0051] As shown herein for the first time, inhibition of the binding interaction between EGFR and HSP90 leads to an increase in EGFR degradation, or an increase in degradation of a protein having an M-5 loop. Accordingly, the present disclosures further provides a method of increasing EGFR degradation in a cell, or degradation of a protein having an M-5 loop in a cell. The method comprises contacting the cell with a compound of the present disclosure or a pharmaceutically acceptable salt or solvate thereof, in an amount effective to increase the degradation. In some aspects, the cell is part of an in vitro or ex vivo cell culture or in vitro or ex vivo tissue sample. In some aspects, the cell is an in vivo cell. In certain embodiments, the method is intended for research purposes, and, in other embodiments, the method is intended for therapeutic purposes.

Methods of Treating Cancer

[0052] As shown herein, a compound that inhibits a binding interaction between EGFR and HSP90 increases tumor cell death. Thus, the present disclosures provides a method of increasing tumor cell death in a subject. The method comprises administering to the subject a compound of the present disclosures, a pharmaceutically acceptable salt or solvate thereof in an amount effective to increase tumor cell death.

[0053] In accordance with the foregoing, the present disclosure further provides a method of treating a cancer in a subject. The method comprises administering to the subject a compound of the present disclosures or a pharmaceutically acceptable salt or solvate thereof, in an amount effective to treat the cancer in the subject.

[0054] As used herein, the term "treat," as well as words related thereto, do not necessarily imply 100% or complete treatment. Rather, there are varying degrees of treatment of which one of ordinary skill in the art recognizes as having a potential benefit or therapeutic effect. In this respect, the methods of treating cancer of the present disclosure can provide any amount or any level of treatment of cancer. Furthermore, the treatment provided by the method of the present disclosures may include treatment of one or more conditions or symptoms of the cancer, being treated. Also, the treatment provided by the methods of the present disclosure may encompass slowing the progression of the cancer. For example, the methods can treat cancer by virtue of reducing tumor or cancer growth, reducing metastasis of tumor cells, increasing cell death of tumor or cancer cells, and the like.

Methods of Sensitizing Tumors

[0055] The present disclosure furthermore provides a method of sensitizing a tumor to chemotherapy, radiation therapy, or both chemotherapy and radiation therapy, in a subject. The method comprises administering to the subject a compound of the present disclosure or a pharmaceutically acceptable salt or solvate thereof, in an amount effective to sensitize the tumor to the therapy. As used herein, the term "sensitize" refers to rendering the tumor more treatable by the therapy, such that the therapy achieves a greater therapeutic index or efficacy. In some aspects, the chemotherapy comprises any of the chemotherapeutics described herein, including, but not limited to, a platinum coordination compound (e.g., cisplatin),

topoisomerase inhibitor (e.g., camptothecin), antibiotic compound (e.g., doxorubicin, mitomycin, bleomycin, daunorubicin, strep tozocin), an antimitotic alkaloid (e.g., vinblastine, vincristine, videsine, Taxol, vinorelbine), or an anti-viral (e.g., gemcitabine). In some aspects, the radiation therapy is any of those described herein.

[0056] In some embodiments, the compound of the present disclosures, a pharmaceutically acceptable salt thereof, a conjugate comprising the compound, or a multimer or dimer comprising the compound, is administered to the subject simultaneously with the

chemotherapy and/or radiation therapy. In some embodiments, the compound of the present disclosures, a pharmaceutically acceptable salt thereof, a conjugate comprising the

compound, or a multimer or dimer comprising the compound, is administered to the subject before the chemotherapy and/or radiation therapy. In particular aspects, the time of administration of the compound of the present disclosures, a pharmaceutically acceptable salt thereof, a conjugate comprising the compound, or a multimer or dimer comprising the compound, and the time of administration of the chemotherapy and/or radiation therapy are about 1 week or less apart, e.g., about 6 days or less apart, about 5 days or less apart, about 4 days or less apart, about 3 days or less apart, about 48 hours or less apart, about 24 hours or less apart, about 12 hours or less apart, about 8 hours or less apart, about 6 hours or less apart, about 4 hours or less apart, about 3 hours or less apart, about 2 hours or less apart, about 1 hour or less apart, about 45 minutes or less apart, about 30 minutes or less apart, about 15 minutes or less apart.

Cancer

[0057] The cancer treatable by the methods disclosed herein may be any cancer, e.g., any malignant growth or tumor caused by abnormal and uncontrolled cell division that may spread to other parts of the body through the lymphatic system or the blood stream.. In some embodiments, the cancer is a cancer in which an EGFR and an HSP90 are expressed by the cells of the cancer. In some aspects, the cancer is a cancer in which an EGFR protein is over- expressed, the gene encoding EGFR is amplified, and/or an EGFR mutant protein (e.g., truncated EGFR, point-mutated EGFR) is expressed. In some aspects, the cancer is a cancer in which a k-Ras protein is over-expressed, a gene encoding the k-Ras protein is amplified, and/or a k-Ras mutant protein (truncated k-Ras, point-mutated k-Ras) is expressed.

[0058] The cancer in some aspects is one selected from the group consisting of acute lymphocytic cancer, acute myeloid leukemia, alveolar rhabdomyosarcoma, bone cancer, brain cancer, breast cancer, cancer of the anus, anal canal, or anorectum, cancer of the eye, cancer of the intrahepatic bile duct, cancer of the joints, cancer of the neck, gallbladder, or pleura, cancer of the nose, nasal cavity, or middle ear, cancer of the oral cavity, cancer of the vulva, chronic lymphocytic leukemia, chronic myeloid cancer, colon cancer, esophageal cancer, cervical cancer, gastrointestinal carcinoid tumor, Hodgkin lymphoma, hypopharynx cancer, kidney cancer, larynx cancer, liver cancer, lung cancer, malignant mesothelioma, melanoma, multiple myeloma, nasopharynx cancer, non-Hodgkin lymphoma, ovarian cancer, pancreatic cancer, peritoneum, omentum, and mesentery cancer, pharynx cancer, prostate cancer, rectal cancer, renal cancer (e.g., renal cell carcinoma (RCC)), small intestine cancer, soft tissue cancer, stomach cancer, testicular cancer, thyroid cancer, ureter cancer, and urinary bladder cancer. In particular aspects, the cancer is selected from the group consisting of: head and neck, ovarian, cervical, bladder and oesophageal cancers, pancreatic, gastrointestinal cancer, gastric, breast, endometrial and colorectal cancers, hepatocellular carcinoma, glioblastoma, bladder, lung cancer, e.g., non-small cell lung cancer (NSCLC), bronchioloalveolar carcinoma.

Subjects

[0059] In some embodiments of the present disclosures, the subject is a mammal, including, but not limited to, mammals of the order Rodentia, such as mice and hamsters, and mammals of the order Logomorpha, such as rabbits, mammals from the order Carnivora, including Felines (cats) and Canines (dogs), mammals from the order Artiodactyla, including Bovines (cows) and Swines (pigs) or of the order Perssodactyla, including Equines (horses). In some aspects, the mammals are of the order Primates, Ceboids, or Simoids (monkeys) or of the order Anthropoids (humans and apes). In some aspects, the mammal is a human. In some aspects, the human is an adult aged 18 years or older. In some aspects, the human is a child aged 17 years or less.

Kits

[0060] In some embodiments, the composition comprising a compound of the present disclosures, a pharmaceutically acceptable salt thereof, a conjugate comprising the compound, or a multimer or dimer comprising the compound, is provided as a kit or package or unit dose. "Unit dose" is a discrete amount of a therapeutic composition dispersed in a suitable carrier. Accordingly, provided herein are kits comprising a compound of the present disclosures, a pharmaceutically acceptable salt thereof, a conjugate comprising the compound, or a multimer or dimer comprising the compound.

[0061] In some embodiments, the components of the kit/unit dose are packaged with instructions for administration to a patient. In some embodiments, the kit comprises one or more devices for administration to a patient, e.g., a needle and syringe, a dropper, a measuring spoon or cup or like device, an inhaler, and the like. In some aspects, the compound of the present disclosures, a pharmaceutically acceptable salt thereof, a conjugate comprising the compound, or a multimer or dimer comprising the compound, is pre-packaged in a ready to use form, e.g., a syringe, an intravenous bag, an inhaler, a tablet, capsule, etc. In some aspects, the kit further comprises other therapeutic or diagnostic agents or

pharmaceutically acceptable carriers (e.g., solvents, buffers, diluents, etc.), including any of those described herein. In particular aspects, the kit comprises a compound of the present disclosures, a pharmaceutically acceptable salt thereof, a conjugate comprising the compound, or a multimer or dimer comprising the compound, along with an agent, e.g., a therapeutic agent, used in chemotherapy or radiation therapy.

Routes of Administration

[0062] With regard to the present disclosures, the active agent, pharmaceutical

composition comprising the same, may be administered to the subject via any suitable route of administration. The following discussion on routes of administration is merely provided to illustrate exemplary embodiments and should not be construed as limiting the scope in any way. [0063] Formulations suitable for oral administration can consist of (a) liquid solutions, such as an effective amount of the active agent of the present disclosure dissolved in diluents, such as water, saline, or orange juice; (b) capsules, sachets, tablets, lozenges, and troches, each containing a predetermined amount of the active ingredient, as solids or granules; (c) powders; (d) suspensions in an appropriate liquid; and (e) suitable emulsions. Liquid formulations may include diluents, such as water and alcohols, for example, ethanol, benzyl alcohol, and the polyethylene alcohols, either with or without the addition of a

pharmaceutically acceptable surfactant. Capsule forms can be of the ordinary hard- or soft- shelled gelatin type containing, for example, surfactants, lubricants, and inert fillers, such as lactose, sucrose, calcium phosphate, and corn starch. Tablet forms can include one or more of lactose, sucrose, mannitol, corn starch, potato starch, alginic acid, microcrystalline cellulose, acacia, gelatin, guar gum, colloidal silicon dioxide, croscarmellose sodium, talc, magnesium stearate, calcium stearate, zinc stearate, stearic acid, and other excipients, colorants, diluents, buffering agents, disintegrating agents, moistening agents, preservatives, flavoring agents, and other pharmacologically compatible excipients. Lozenge forms can comprise the active agent of the present disclosure in a flavor, usually sucrose and acacia or tragacanth, as well as pastilles comprising the active agent of the present disclosure in an inert base, such as gelatin and glycerin, or sucrose and acacia, emulsions, gels, and the like containing, in addition to, such excipients as are known in the art.

[0064] The active agents of the present disclosure, alone or in combination with other suitable components, can be delivered via pulmonary administration and can be made into aerosol formulations to be administered via inhalation. These aerosol formulations can be placed into pressurized acceptable propellants, such as dichlorodifluoromethane, propane, nitrogen, and the like. They also may be formulated as pharmaceuticals for non-pressured preparations, such as in a nebulizer or an atomizer. Such spray formulations also may be used to spray mucosa. In some embodiments, the active agent is formulated into a powder blend or into microparticles or nanoparticles. Suitable pulmonary formulations are known in the art. See, e.g., Qian et al., Int J Pharm 366: 218-220 (2009); Adjei and Garren,

Pharmaceutical Research, 7(6): 565-569 (1990); Kawashima et al., J Controlled Release 62(1-2): 279-287 (1999); Liu et al., Pharm Res 10(2): 228-232 (1993); International Patent Application Publication Nos. WO 2007/133747 and WO 2007/141411.

[0065] Formulations suitable for parenteral administration include aqueous and nonaqueous, 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 can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives. The term, "parenteral" means not through the alimentary canal but by some other route such as subcutaneous, intramuscular, intraspinal, or intravenous. The active agent of the present disclosure can be administered with a physiologically acceptable diluent in a pharmaceutical carrier, such as a sterile liquid or mixture of liquids, including water, saline, aqueous dextrose and related sugar solutions, an alcohol, such as ethanol or hexadecyl alcohol, a glycol, such as propylene glycol or polyethylene glycol, dimethylsulfoxide, glycerol, ketals such as 2,2- dimethyl-153- dioxolane-4-methanol, ethers, poly(ethyleneglycol) 400, oils, fatty acids, fatty acid esters or glycerides, or acetylated fatty acid glycerides with or without the addition of a

pharmaceutically acceptable surfactant, such as a soap or a detergent, suspending agent, such as pectin, carbomers, methylcellulose, hydroxypropylmethylcellulose, or

carboxymethylcellulose, or emulsifying agents and other pharmaceutical adjuvants.

[0066] Oils, which can be used in parenteral formulations include petroleum, animal, vegetable, or synthetic oils. Specific examples of oils include peanut, soybean, sesame, cottonseed, corn, olive, petrolatum, and mineral. Suitable fatty acids for use in parenteral formulations include oleic acid, stearic acid, and isostearic acid. Ethyl oleate and isopropyl myristate are examples of suitable fatty acid esters.

[0067] Suitable soaps for use in parenteral formulations include fatty alkali metal, ammonium, and triethanolamine salts, and suitable detergents include (a) cationic detergents such as, for example, dimethyl dialkyl ammonium halides, and alkyl pyridinium halides, (b) anionic detergents such as, for example, alkyl, aryl, and olefin sulfonates, alkyl, olefin, ether, and monoglyceride sulfates, and sulfosuccinates, (c) nonionic detergents such as, for example, fatty amine oxides, fatty acid alkanolamides, and polyoxyethylenepolypropylene copolymers, (d) amphoteric detergents such as, for example, alkyl-P-aminopropionates, and 2-alkyl -imidazoline quaternary ammonium salts, and (e) mixtures thereof.

[0068] The parenteral formulations in some embodiments contain from about 0.5% to about 25% by weight of the active agent of the present disclosure in solution. Preservatives and buffers may be used. In order to minimize or eliminate irritation at the site of injection, such compositions may contain one or more nonionic surfactants having a hydrophile- lipophile balance (HLB) of from about 12 to about 17. The quantity of surfactant in such formulations will typically range from about 5% to about 15% by weight. Suitable surfactants include polyethylene glycol 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 in some aspects are 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 excipient, for example, water, for injections, immediately prior to use. Extemporaneous injection solutions and suspensions in some aspects are prepared from sterile powders, granules, and tablets of the kind previously described.

[0069] Injectable formulations are in accordance with the invention. The requirements for effective pharmaceutical carriers for injectable compositions are well-known to those of ordinary skill in the art (see, e.g., Pharmaceutics and Pharmacy Practice, J. B. Lippincott Company, Philadelphia, PA, Banker and Chalmers, eds., pages 238-250 (1982), and ASHP Handbook on Injectable Drugs, Toissel, 4th ed., pages 622-630 (1986)).

[0070] Additionally, the active agent of the present disclosures can be made into suppositories for rectal administration by mixing with a variety of bases, such as emulsifying bases or water-soluble bases. Formulations suitable for vaginal administration can be presented as pessaries, tampons, creams, gels, pastes, foams, or spray formulas containing, in addition to the active ingredient, such carriers as are known in the art to be appropriate.

[0071] It will be appreciated by one of skill in the art that, in addition to the above- described pharmaceutical compositions, the active agent of the disclosure can be formulated as inclusion complexes, such as cyclodextrin inclusion complexes, or liposomes.

Dosages

[0072] The active agents of the disclosure are believed to be useful in methods of inhibiting a binding interaction between EGFR and HSP90, methods of increasing EGFR degradation, methods of treating cancer in a subject, and methods of sensitizing tumors to treatment, as further described herein. For purposes of the disclosure, the amount or dose of the active agent administered should be sufficient to effect, e.g., a therapeutic or prophylactic response, in the subject or animal over a reasonable time frame. For example, the dose of the active agent of the present disclosure should be sufficient to treat cancer as described herein in a period of from about 1 to 4 minutes, 1 to 4 hours or 1 to 4 weeks or longer, e.g., 5 to 20 or more weeks, from the time of administration. In certain embodiments, the time period could be even longer. The dose will be determined by the efficacy of the particular active agent and the condition of the animal (e.g., human), as well as the body weight of the animal (e.g., human) to be treated. [0073] Many assays for determining an administered dose are known in the art. For purposes herein, an assay, which comprises comparing the extent to which cancer is treated upon administration of a given dose of the active agent of the present disclosure to a mammal among a set of mammals, each set of which is given a different dose of the active agent, could be used to determine a starting dose to be administered to a mammal. The extent to which cancer is treated upon administration of a certain dose can be represented by, for example, the cytotoxicity of the active agent or the extent of tumor regression achieved with the active agent in a mouse xenograft model. Methods of measuring cytotoxicity of compounds and methods of assaying tumor regression are known in the art, including, for instance, the methods described in the EXAMPLES set forth below.

[0074] The dose of the active agent of the present disclosure also will be determined by the existence, nature and extent of any adverse side effects that might accompany the

administration of a particular active agent of the present disclosure. Typically, the attending physician will decide the dosage of the active agent of the present disclosure with which to treat each individual patient, taking into consideration a variety of factors, such as age, body weight, general health, diet, sex, active agent of the present disclosure to be administered, route of administration, and the severity of the condition being treated. By way of example and not intending to limit the invention, the dose of the active agent of the present disclosure can be about 0.0001 to about 1 g/kg body weight of the subject being treated/day, from about 0.0001 to about 0.001 g/kg body weight/day, or about 0.01 mg to about 1 g/kg body weight/day.

Controlled Release Formulations

[0075] In some embodiments, the active agents described herein can be modified into a depot form, such that the manner in which the active agent of the present disclosures is released into the body to which it is administered is controlled with respect to time and location within the body (see, for example, U.S. Patent No. 4,450,150). Depot forms of active agents of the present disclosures can be, for example, an implantable composition comprising the active agents and a porous or non-porous material, such as a polymer, wherein the active agent is encapsulated by or diffused throughout the material and/or degradation of the non-porous material. The depot is then implanted into the desired location within the body of the subject and the active agent is released from the implant at a predetermined rate.

[0076] The pharmaceutical composition comprising the active agent in certain aspects is modified to have any type of in vivo release profile. In some aspects, the pharmaceutical composition is an immediate release, controlled release, sustained release, extended release, delayed release, or bi-phasic release formulation. Methods of formulating peptides for controlled release are known in the art. See, for example, Qian et al., J Pharm 374: 46-52 (2009) and International Patent Application Publication Nos. WO 2008/130158,

WO2004/033036; WO2000/032218; and WO 1999/040942.

[0077] The instant compositions may further comprise, for example, micelles or liposomes, or some other encapsulated form, or may be administered in an extended release form to provide a prolonged storage and/or delivery effect.

Timing of Administration

[0078] The disclosed pharmaceutical compositions and formulations may be administered according to any regimen including, for example, daily (1 time per day, 2 times per day, 3 times per day, 4 times per day, 5 times per day, 6 times per day), every two days, every three days, every four days, every five days, every six days, weekly, bi-weekly, every three weeks, monthly, or bi-monthly. Timing, like dosing can be fine-tuned based on dose-response studies, efficacy, and toxicity data, and initially guaged based on timing used for other antibody therapeutics.

Combinations

[0079] In some embodiments, the active agents described herein are administered alone, and in alternative embodiments, the active agents described herein are administered in combination with another therapeutic agent, e.g., another active agent of the present disclosures of different type (e.g., structure), or another therapeutic which does not inhibit a binding interaction between EGFR and HSP90. In some aspects, the other therapeutic aims to treat or prevent cancer. In some embodiments, the other therapeutic is a chemotherapeutic agent. In some aspects, the chemotherapeutic agent is a DNA crosslinker or an agent that targets DNA synthesis (e.g., cisplatin). In some aspects, the chemotherapeutic agent comprises any of a platinum coordination compound (e.g., cisplatin), topoisomerase inhibitor (e.g., camptothecin), antibiotic compound (e.g., doxorubicin, mitomycin, bleomycin, daunorubicin, streptozocin), an antimitotic alkaloid (e.g., vinblastine, vincristine, videsine, Taxol, vinorelbine), or an anti-viral (e.g., gemcitabine). In some embodiments, the other therapeutic is an agent used in radiation therapy for the treatment of cancer. In exemplary aspects, the radiation therapy comprises photon beams (e.g., X rays, gamma rays), electron beams and/or charged particle beams. In certain aspects, the radiation therapy comprises external beam radiation therapy (e.g., 3-dimensional conformal radiation therapy (3D-CRT), intensity-modulated radiation therapy (IMRT), image-guided radiation therapy (IGRT), tomotherapy, stereostatic radiosurgery, stereostatic body radiation therapy, proton therapy, and the like). In alternative aspects, the radiation therapy comprises internal radiation therapy (a.k.a., brachytherapy), such as, interstitial brachytherapy. In some aspects, the radiation therapy comprises systemic radiation therapy, e.g., ibritumomab tiuxetan

(Zevalin®), tositumomab and iodine 1 131 tositumomab (Bexxar®), samarium-153- lexidronam (Quadramet®) and strontium-89 chloride (Metastron®).

[0080] In exemplary embodiments, the active agent is administered simultaneously as the other therapeutic. In alternative embodiments, the active agent is administered either before or after the other therapeutic.

EXAMPLES

[0081] Four TKI resistant cell lines representing a range of EGFR status seen in lung cancer patients such as WT, de novo T790M EGFR or deletion mutant E746-A750 have been selected as has a TKI sensitive tumor cell line (due to L858R-EGFR mutation). Normal lung fibroblasts and EGFR null CHO cells are also included to assess selectivity. For initial screening cells are treated with a range of concentrations (0.1-30 μg/ml), erlotinib and AT13387, as positive controls. Effect of treatment on EGFR is assessed by an increase in bioluminescence over vehicle at multiple time points post treatment (ranging from 30-min to 3-days). The effect on HSP90-EGFR interaction and EGFR steady state level is confirmed using immunoprecipitation of HSP90 followed by immunoblotting for EGFR or by direct immunoblotting .

[0082] Analogs are rationally designed based upon the SAR studies to date and to establish structure- activity relationships and improve potency to sub 100 nM in primary assays. The chemical structure of 2-3 most effective (as determined by selective degradation of EGFR) lead molecules is modified to improve the potency while maintaining the efficacy.

[0083] The overlay of C250-395 with the M5 loop provides a hypothesis generator to guide molecules for synthesis to recapitulate the activity observed by Disruptin. The underlying assumption is that the mode of action of the small molecule C250-395 is similar to Disruptin.

[0084] The SAR of the initial lead compound C250-395 is assessed. At each iteration, hypothesis driven design is used to optimize physicochemical properties such as solubility, potency, absorption, distribution, cross-reactivity and toxicity. [0085] Molecules from this SAR study are assessed for efficacy and selectivity using biochemical and cell-based functional assays. Efficacy of each synthesized analog is assessed for activity using the EGFR reporter assay. Hits within that in vitro test are followed-up by assessing the effect on target using following the parameters: 1) EGFR steady-state level, 2) Interaction between EGFR with HSP90, 3) EGFR homo-dimer formation, 4) EGFR hetero-dimer formation, and 5) effects on EGFR down-stream signal molecules (such as cMet, Erk, AKT, ERK). The disruption of receptor dimerization and blockade of HSP90 binding result in EGFR and cMet degradation independent of TKI- resistant mutation. This results in cell-death of EGFR, or cMet driven TKI resistant cells. Molecules that have a dose dependent effect on EGFR are tested in the clonogenic survival assay. Molecules found active are further profiled to assess the physicochemical properties of a variety of molecules in the series.

[0086] Compounds are tested for CYP (cytochrome P450 monooxygenase) inhibition, dofetilide binding (to determine K+ channel binding) and normal cell toxicity using known technologies. In brief, compounds are evaluated for cellular toxicity by measuring the percentage of viable cells with the CellTiter-Glo Luminescent Cell Viability Assay

(Promega) and visualized with the En- Vision Xcite Multilabel Reader (PerkinElmer). The potential for compounds to exhibit drug-drug interactions is assessed by measurement of the inhibition of the CYPs, in particular, CYP 3A4 and CYP 2D6 by means of Invitrogen's Vivid® or BD's Gentest P450 screening kits which use a 96-well plate, high-throughput format. The potential for compounds to exhibit a hERG liability which can induce a prolongation of the QT interval of cardiac rhythm is assessed via the moderate-throughput [³H] dofetilide competition binding assay as a surrogate indicator of the hERG potassium ion channel activity.

[0087] At each iteration of the closed-loop design, standard quantitative structure-activity relationship (QSAR) analysis and molecule modeling is used to analyze result and correlate with structure, using, e.g., tools readily available in software packages such Molecular Operating Environment by Chemical Computing Group and SYBYL by TRIPOS. The library is designed using the tools available in PIPELINE PILOT. Trends are plotted and documented with SPOTFIRE.

[0088] The safety profile (e.g., effects on various organs) of the compounds is assessed in immunocompetent C57BL/6 mice. Mice are injected with three different doses (5 mice/dose, reducing or increasing dose, as necessary). Additionally, the PK/PD (Pharmacokinetics/ Pharmacodynamics) of the lead compounds is assessed. The concentration of the compounds in blood and plasma are measured using LC/MS/MS. These data are used in the prediction of clearance. Results obtained from these studies help determine the safe dose of the compounds for further efficacy studies.

[0089] Efficacy of compounds is tested in engineered as well as lung explants

(tumorgrafts) wherein EGFR drives the tumor resistance to tyrosine kinase inhibitors. The effect of small molecules on EGFR is correlated with tumor response. This approach produces regression of tumors driven by EGFR without affecting HSP90 activity. Based on the mechanism of action of this class of molecules, they are selectively active against TKI resistant EGFR or cMet driven tumor cells. Treatment with lead compound induces EGFR degradation. The amount of EGFR degradation upon treatment correlates with tumor growth delay. Four to five different cell lines are implanted (3 to 4 TKI resistant and one TKI sensitive), chosen due to either EGFR mutation or cMet amplification (Table 1) in the flanks of nude mice. A bioluminescence reporter is used to monitor EGFR activity in real-time, non- invasively in live mice. This reporter is expressed in all the cell lines and selected clones that are used for efficacy studies. This allows for monitoring not only the effect of lead compound on EGFR during the treatment, but also for assessment of the effect of treatment on potential tumor spread.

[0090] The effectiveness of the most selective molecules is assessed against a TKI resistant flank xenograft tumor model. Lung cancer cells are implanted on both the flanks to induce two tumors/mouse. Once the tumor has reached the size of about 50 mm , mice are randomized into 5 groups containing at least 15 animals per group. At this point 4 tumors from 2 animals are removed for control studies. The remaining mice receive the following treatments: 1) vehicle, 2) compound 1, i.p.); 3) compound 2; 4) compound 3; 5) erlotinib (100 mg/kg Mon-Fri x3, orally), or 6) AT13378 (5 mg/kg, i.p., Mon-Fri x 3). Then 2 mice (4 tumors) are sacrificed on days 1, 7 and 21 post treatment to assess the effect on EGFR- HSP90 interaction, EGFR-dimerization, Ki67 (proliferation marker), and TUNEL (apoptotic marker). In each group EGFR activity is monitored weekly and just before mice are sacrificed, using non-invasive bioluminescence imaging (58). The remaining (7 mice) animals are monitored for growth for 90 days or until tumors have reached a maximum of 1.5 cm x 1.5 cm. The tumor volume doubling time is about a week. The tumor doubling time is correlated with the degree of induction in EGFR-reported activity and EGFR degradation, as assessed by immunoblotting of the tumor specimens collected during the treatment. To assess systemic toxicity, the animals' weight is measured bi-weekly. At the time of sacrifice, vital organs are collected for histology. Correlation between EGFR and cMet levels and tumor growth is assessed in at least two ways. First, for tumors removed on day 21, measures of EGFR level and tumor growth (over 0-21 days) is available for the same tumor. Pearson correlation coefficients with 95% CIs is estimated for each cell line across tumors and treatments. In addition, for a given cell line and treatment, the median doubling time or growth rate (for tumors not removed early) and median EGFR level (for tumors removed early) is estimated. The correlation between these 2 quantities across treatments for a given cell line is assessed. The former approach has the advantage of making 'within tumor' comparisons and assesses the correlation between EGFR levels at day 21 and prior growth. The latter comparison assesses the correlation between average EGFR levels for a given treatment and cell line at day 21 and future tumor growth. The null hypothesis of no correlation is tested for each cell line.

[0091] The most effective molecule from these assessments is tested against 4 low passage human tumorgrafts, and its efficacy will be compared to EGFR and HSP90 inhibitors. The responses of these tumorgrafts combined with the knowledge of their molecular profiles allows for the rational design of future clinical trials. EGFR and cMet and induction of other compensatory pathway (such as ERK, AKT, Src, etc.), combined with genomic signatures of the low passage human tumorgrafts is correlated with therapeutic outcome and acquired resistance to therapy.

[0092] A panel of 4 lung cancer xenografts is subcutaneously implanted. All of the patient- derived tumorgraft models are comprehensively profiled for genomic markers and phosphoproteomic signatures. Treatment is initiated when tumors reach a volume of 50 mg/mm . Building upon the information gained on dosing, mice are randomized into four treatment arms. Animals are treated with either: 1) vehicle, 2) erlotinib, or 3) one of two lead compounds. Treatment is administered as described above. Tumor burden is assessed by calipers. Data obtained with NCI-H1975 and UMSCCl xenograft support the notion that the degradation of EGFR elicits a therapeutically meaningful advantage over erlotinib and AT13387.

[0093] CCSP-rtTA;Tet-op-EGFR L858R-T790M transgenic mice are used. Mice are fed on Doxycycline containing diet to induce transgene expression: detection of the CCSP-rtTA and Tetop- hEGFR-L858R-T790M alleles are performed. Similar to the above tumor xenograft studies, the therapeutic potential of the 1-2 lead compounds is tested on the tumor onset and growth. For this purpose, mice are treated with doxycycline to induce the expression of the T790M+L858R-EGFR transgene in the lung. Six weeks after doxycycline treatment, lung is imaged using MRI scan. If tumors are detected, treatment starts (using optimum dose and schedule as determined in previous studies). Mice continue to be imaged using MRI every week for 4-6 weeks. Mice are sacrificed when tumor triples in volume or when mice start to lose body weight. The entire lung is inflated with formalin and processed for histopathologic analysis to assess the number of tumor lesions. H&E slides of lungs from lead compound, vehicle or positive control (AT13387 or gefitinib) treated tumors are scored blindly, and the effect of lead compound is compared with AT13378 and gefitinib.

[0094] Translational findings (e.g. activation of compensatory pathways) are integrated to advance the most promising agent into a rationally designed human clinical trial. A patient enrichment strategy is designed that incorporates molecular determinants of response defined by sensitive models in the heterogeneous tumorgraft panel. An imaging-based biomarker strategy is incorporated as an early indicator of response to maximize the ultimate impact of the proposed therapy in patients.

[0095] Correlative analysis is carried out to identify common molecular signatures associated with therapeutic outcome. The data is initially pooled from cell-line based xenograft and lung cancer tumorgrafts to simulate one patient population. The degree of tumor growth delay (T-C) forms the basis of judging overall responsiveness to treatment. Net cell kill values are determined from each experiment defined as the net change (logs) in tumor burden during treatment. Use of net log kill allow for normalize data across tumor types and experiments and provide a clinically relevant quantitative assessment of activity. Models are classified as highly responsive (regressions/positive net log kill), moderately responsive (stasis/zero net log kill), or insensitive (progression/negative log kill). Within each response class, investigations into any shared genomic aberrations present in these tumors selected on the basis of their well-documented role in lung tumor progression (e.g.

EGFR/cMet) are made. Since compensatory pathway activation can occur in the absence of accompanying somatic mutations, therapeutic response is correlated with expression levels of phosphorylated Src, ERK and AKT. This provides a rationally designed human clinical trial with an EGFR-HSP90 interaction inhibitor development candidate. While the above analysis informs a patient enrichment strategy, there also exists a need to assess patient response as early as possible.

[0096] The questions posed above specifically address the hypothesis that EGFR driven tumors, irrespective of activation mechanism, prove responsive to therapeutic intervention by blocking EGFR and HSP90 interaction. There are subpopulations of patients harboring EGFR somatic mutations, identifiable from genomic and proteomic profiling, that are especially sensitive to this therapeutic approach. For example, activated cMet signaling that is observed in TKI resistant lung cancer patients, is this class of agents. The information gained from these studies builds a compelling case for pursuing a lung cancer indication for this approach. The data generated here provide an experimental basis for testing the same hypotheses posed above in the clinic.

[0097] The experiments described here deliver mimetics of EGFR-M5-loop capable of blocking the specific interaction between EGFR and HSP90 and also inhibit EGFR dimerization independent of TKI resistant T790M-EGFR mutation status. This results in at least one lead series that is ready for final pre-clinical optimization and validation. This compound possesses good pharmaceutical properties and activity against HSP90 for EGFR in a cell-based assay with an EC₅₀ of 50 nM or better. It further possesses an appropriate level of off-target selectivity and demonstrate excellent drug-like properties, including ADME parameters (absorption, distribution, metabolism, and elimination pharmacokinetic). The compound does not cause non-specific cell death at a concentration of 20 μΜ nor

significantly inhibit P450 or hERG channel binding at a concentration of 10 μΜ. Finally, the effective compound from the mouse studies can be used for subsequent IND enabling preclinical development and exploratory toxicology.

Screening Results

[0098] Compounds were screened in an EGFR induction Assay and the EGFR fold induction was measured.

Reporter

Assay (Fold

Induction)

CH H H CI H H 2.0

3

CH H H OCH H H 1.2

3 3

CH H H Br H H 1.3

3

CH H CI CI H H 1.6

3

CH H CI OCH H H 2.1

3 3

CH H CI OCH H OCH 1.7

3 3 3

CH H CI F H H 1.7

3

CH H CI H CI H 1.6

3

CI CI H CI H H 1.4

H H H CI H H 1.1

CI H H CI H H 0.9

[0099] Mice bearing NCI-H1975 xenografts were treated with compound 95 (20mg/kg, daily for one week).

Bioluminescence was monitored twice a day for one week, the results of which are shown in Fig. 2A. Tumor growth was recorded and relative growth profile is plotted, as shown in Fig. 2B. Relative tumor volume on day 8 is plotted, as shown in Fig. 2C. After one week of treatment mice were euthanized and tumors were harvested, as shown in Fig. 2D. The effect of treatment on EGFR in the tumor cells (n=3 for control and compound 95 group) was assessed by immunoblotting tumor cells lysates, as shown in Fig. 2E.

[0100] All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein. [0101] The use of the terms "a" and "an" and "the" and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The terms "comprising," "having," "including," and "containing" are to be construed as open-ended terms (i.e., meaning "including, but not limited to,") unless otherwise noted.

[0102] Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range and each endpoint, unless otherwise indicated herein, and each separate value and endpoint is incorporated into the specification as if it were individually recited herein.

[0103] All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., "such as") provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[00100] Preferred embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those preferred embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

Claims

What is Claimed:

1. A method of increasing degradation of epithelial growth factor receptor (EGFR) or inhibiting EGFR dimerization comprising

contacting EGFR with a compound having a structure of formula (I) in an amount sufficient to inhibit EGFR dimerization or induce EGFR degradation:

wherein

ring A is a 5-membered heteroaryl;

Ari is an aryl or heteroaryl; and

Ar₂ is an aryl, heteroaryl, cycloalkyl, or heterocycloalkyl,

or a pharmaceutically acceptable salt or solvate thereof.

2. The method of claim 1, wherein ring A is selected from the group consisting of diazolyl, triazolyl, tetrazolyl, thiophenyl, and furanyl.

3. The method of claim 1, wherein ring A is diazolyl.

4. The method of claim 3, wherein the diazolyl is

or

5. The method of claim 1, wherein ring A is triazolyl.

6. The method of claim 5, wherein the triazoyl is

.

7. The method of claim 1, wherein ring A is tetrazolyl.

8. The method of claim 7, wherein the tetrazolyl is

.

9. The method of any one of claims 1-6, wherein ring A is further substituted.

10. The method of claim 9, where ring A is further substituted with an alkyl or amino substituent.

11. The method of claim 1, wherein the compound has a structure of formula (II):

and R 1 and R 2 are each independently a C_1-4 alkyl or together form a spiro heterocyclyl.

12. The method of claim 11, wherein each of R 1 and R 2 is a C_1-4 alkyl.

13. The method of claim 12, wherein each of R 1 and R 2 is methyl.

14. The method of claim 11, wherein R 1 and R 2 together form a spiro

heterocyclyl.

15. The method of claim 14, wherein the sprio heterocyclyl is piperdine, pyrrolidine, tetrahydrofuran, tetrahydropyran, dihydrofuran, morpholine, or thiophene.

16. The method of

17. The method of

18. The method of

19. The method of

pyrrolidine.

20. The method of

21. The method of

22. The method of

23. The method of

position.

24. The method of claim 22 or 23, wherein the substitution is one or more of halo, alkyl, and alkoxy.

25. The method of any one of claims 1-19, wherein is heteroaryl.

26. The method of claim 25, wherein heteroaryl is thienyl, furyl, pyridyl, oxazolyl, quinolyl, thiophenyl, isoquinolyl, indolyl, triazinyl, triazolyl, isothiazolyl, isoxazolyl, imidazolyl, benzothiazolyl, pyrazinyl, pyrimidinyl, thiazolyl, or thiadiazolyl.

27. The method of claim 25 or 26, wherein Α is substituted heteroaryl.

28. The method of claim 27, wherein the substitution is one or more of halo, alkyl, and alkoxy.

29. The method of any one of claims 1-28, wherein Ar₂ is aryl.

30. The method of claim 29, wherein aryl is phenyl or naphthyl.

31. The method of claim 29 or 30, wherein Ar₂ is substituted aryl.

32. The method of claim 31, wherein the phenyl is substituted at at least one of the 3 and 4 position.

33. The method of claim 32, wherein the phenyl is substituted at both the 3 and 4 position.

34. The method of any one of claims 31-33, wherein the substitution is one or more of halo, alkyl, and alkoxy.

35. The method of any one of claims 1-28, wherein Ar₂ is heteroaryl.

36. The method of claim 35, wherein heteroaryl is thienyl, furyl, pyridyl, oxazolyl, quinolyl, thiophenyl, isoquinolyl, indolyl, triazinyl, triazolyl, isothiazolyl, isoxazolyl, imidazolyl, benzothiazolyl, pyrazinyl, pyrimidinyl, thiazolyl, or thiadiazolyl.

37. The method of claim 35 or 36, wherein Ar₂ is substituted heteroaryl.

38. The method of claim 37, wherein the substitution is one or more of halo, alkyl, and alkoxy.

39. The method of any one of claims 1-28, wherein Ar₂ is cycloalkyl.

40. The method of claim 39, wherein cycloalkyl is selected from cyclopropyl, cyclobutyl, cyclopentyl, cylcohexyl, cycloheptyl, or cylcooctyl.

41. The method of claim 39, wherein cycloaklyl is cyclpentyl or cyclohexyl.

42. The method of any one of claims 39-41, wherein Ar₂ is a substituted cycloalkyl.

43. The method of claim 42, wherein the substitution is one or more of halo, alkyl, and alkoxy.

44. The method of any one of claims 1-44, wherein the compound inhibits EGFR interaction with HSP90.

45. The method of claim 44, wherein the compound inhibits HSP90 activity less than 20%.

46. The method of any one of claims 1-45, wherein the compound does not inhibit HSP90 activity.

47. The method of any one of claims 1-46, wherein the contacting comprises administering to a subject in need thereof.

48. The method of claim 47, wherein the subject is diagnosed with cancer.

49. The method of claim 48, wherein the cancer is characterized by

overexpression of EGFR or expression of a mutant EGFR.

50. The method of claim 47 or 48, wherein the cancer is lung cancer, pancreatic cancer, head and neck cancer, or colorectal cancer.

51. The method of claim 47 or 48, wherein the cancer is a lung cancer, head and neck cancer, cervical cancer, glioblastoma, colorectal cancer, or breast cancer.

52. The method of any one of claims 1-51, further comprising contacting with a second therapeutic.

53. The method of claim 52, wherein the second therapeutic is a chemotherapeutic or radiation therapy.

54. The method of claim 53, wherein the chemotherapeutic is one or more of cisplatin and gemcitabine.

55. The method of any one of claims 1-54, wherein the compound inhibits one or more of DMPK, Erb2, and cMet.

56. The method of claim 55, wherein the contacting further results in inhibition of one or more of DMPK, Erb2, and cMet.

57. The method of any one of claims 1 and 44-56, wherein the compound is selected from the roup consisting of

salt or solvate thereof.

A compound havin a structure of formula (III)

wherein

R¹ is alkyl, C(0)alkyl, C(0)NHalkyl, or C(0)Oalkyl;

Ari is an aryl or heteroaryl; and

Ar₂ is an aryl, heteroaryl, cycloalkyl, or heterocycloalkyl, or a pharmaceutically acceptable salt or solvate thereof.

59. The compound of claim 58, wherein R¹ is alkyl.

60. The compound of claim 59, wherein alkyl is methyl

61. The compound of claim 58, wherein R¹ is C(0)alkyl.

62. The compound of claim 61, wherein alkyl is methyl or ethyl.

63. The compound of claim 58, wherein R¹ is C(0)NHalkyl.

64. The compound of claim 63, wherein alkyl is methyl or ethyl.

65. The compound of claim 58, wherein R¹ is C(0)Oalkyl.

66. The compound of claim 65, wherein alkyl is methyl or ethyl.

67. The compound of any one of claims 58-66, wherein Α is aryl.

68. The compound of claim 67, wherein aryl is phenyl or naphthyl.

69. The compound of claim 67 or 68, wherein Ar is substituted aryl.

70. The compound of claim 69, wherein the phenyl is substituted at the 3 or 4 position.

71. The compound of claim 69 or 70, wherein the substitution is one or more of halo, alkyl, and alkoxy.

72. The compound of any one of claims 58-66, wherein Ar is heteroaryl.

73. The compound of claim 72, wherein heteroaryl is thienyl, furyl, pyridyl, oxazolyl, quinolyl, thiophenyl, isoquinolyl, indolyl, triazinyl, triazolyl, isothiazolyl, isoxazolyl, imidazolyl, benzothiazolyl, pyrazinyl, pyrimidinyl, thiazolyl, or thiadiazolyl.

74. The compound of claim 72 or 73, wherein A i is substituted heteroaryl.

75. The compound of claim 74, wherein the substitution is one or more of halo, alkyl, and alkoxy.

76. The compound of any one of claims 58-75, wherein Ar₂ is aryl.

77. The compound of claim 76, wherein aryl is phenyl or naphthyl.

78. The compound of claim 76 or 77, wherein Ar₂ is substituted aryl.

79. The compound of claim 78, wherein the phenyl is substituted at at least one of the 3 and 4 position.

80. The compound of claim 79, wherein the phenyl is substituted at both the 3 and 4 position.

81. The compound of any one of claims 78-80, wherein the substitution is one or more of halo, alkyl, and alkoxy.

82. The compound of any one of claims 58-75, wherein Ar₂ is heteroaryl.

83. The compound of claim 82, wherein heteroaryl is thienyl, furyl, pyridyl, oxazolyl, quinolyl, thiophenyl, isoquinolyl, indolyl, triazinyl, triazolyl, isothiazolyl, isoxazolyl, imidazolyl, benzothiazolyl, pyrazinyl, pyrimidinyl, thiazolyl, or thiadiazolyl.

84. The compound of claim 82 or 83, wherein Ar₂ is substituted heteroaryl.

85. The compound of claim 84, wherein the substitution is one or more of halo, alkyl, and alkoxy.

86. The compound of any one of claims 58-75, wherein Ar₂ is cycloalkyl.

87. The compound of claim 86, wherein the cycloalkyl is cyclopropyl, cyclobutyl, cyclopentyl, cylcohexyl, cycloheptyl, or cylcooctyl.

88. The compound of claim 87, wherein cycloaklyl is cyclpentyl or cyclohexyl.

89. The compound of any one of claims 86-88, wherein Ar₂ is a substituted cycloalkyl.

90. The compound of claim 89, wherein the substitution is one or more of halo, alkyl, and alkoxy.

91. The compound of any one of claims 58-75, wherein Ar₂ is heterocycloalkyl.

92. The compound of claim 91, wherein the heterocyloalkyl is piperdine, pyrrolidine, tetrahydrofuran, tetrahydropyran, dihydrofuran, morpholine, or thiophene.

93. The compound of claim 91 or 92, wherein the heterocycloalkyl is substituted.

94. The compound of claim 94, wherein the substitution is one or more of halo, alkyl, and alkoxy.

95. A method of increasing degradation of epithelial growth factor receptor (EGFR) or inhibiting EGFR dimerization comprising

contacting EGFR with the compound of any one of claims 58-94 in an amount sufficient to inhibit EGFR dimerization or induce EGFR degradation.