EYA2 inhibitors and methods for using and producing the same

Abstract

The present disclosure provides a compound of the formula Ar1-L1-Ar2-S-(L2)m-Ar3 (I), wherein m is 0 or 1; Ar1 is aryl or heteroaryl each of which is optionally substituted; L1 is a first linker selected from the group consisting of (II), III and (IV); Ar2 is arylene or heteroarylene each of which is optionally substituted; L2 is alkylene; Ar3 is aryl or heteroaryl each of which is optionally substituted or -NR1 R2; each X1 is O, NR1, or S; each X2 is O, NR2, or S; each of Y is independently H or halide; and each of R1, R2, and Ra is independently H, alkyl, or a nitrogen protecting group, provided when Ar2 is furylene or optionally substituted phenylene, then L1 is not -C(=O)-NH-N=CH- The present disclosure also provides methods for using compound of Formula I in treating a clinical condition associated with EYA2 tyrosine phosphatase activity.

Description

EYA2 INHIBITORS AND METHODS FOR USING AND PRODUCINGTHE SAMEFIELD OF THE INVENTION

[0001] The present disclosure relates generally to the fields of oncology, molecular biology, and medicine. More particularly, the disclosure relates to a compound of the formula, where Ar1, L1, Ar2, L2, m, and Ar3are those defined herein, and methods for using the same in treating a clinical condition associated with EYA2 tyrosine phosphatase activity.BACKGROUND OF THE INVENTION

[0002] SIX1 is a homeoprotein critical for the development of numerous organs during embryogenesis. In development, SIX1 is important for maintaining progenitor cell populations and for enhancing cell growth, survival, migration and invasion. However, its expression postembryogenesis is low and / or undetectable, but it is re-expressed in many types of adult and pediatric cancers. Re-expression of Sixl contributes to both tumor induction and metastasis in transgenic and xenograft mammary / breast cancer models, and in other adult and pediatric cancer models. See Blevins, M.A. et al., Expert opinion on therapeutic targets, 19, 213-225 (2015).

[0003] While SIX1 contains a DNA binding domain, it lacks an intrinsic activation domain and thus requires EYA cofactors to activate transcription. Four EYA family members exist, and they are also critical for organ development by activating transcription in cooperation with SIX family members. The EYA family members all contain a highly conserved C-terminal EYA domain (ED), which interacts with SIX proteins. The ED also contains protein Tyrosine Phosphatase (Tyr Phosphatase) activity, which is implicated in various biological activities, such as motility, DNA damage response, angiogenesis, inhibition of the anti-tumor activity of Estrogen Receptor-0, etc. This Tyr Phosphatase activity has been shown to be important for transcriptional activation of Myc in complex with Sixl in development. Of clinical significance, EYA also has been shown to be aberrantly re-expressed in cancer.

[0004] The N-terminal domain (NTD) of EYA family members contains a proline / serine / threonine (PST) rich region that serves as a transactivation domain (TAD). The NTD also contains a serine / threonine (Ser / Thr) Phosphatase activity which shows no homology to any known Phosphatases. The EYA Ser / Thr Phosphatase has been implicated in innate immunity and in regulating MYC protein stability during embryonic development and in breast cancer metastasis. The Ser / Thr Phosphatase activity of Eya is mediated through an interaction with the B55a regulatory subunit of Protein Phosphatase 2A (PP2A), and that this interaction is important for the ability of EYA to enhance MYC stability. See, for example, Xu J., et al., Dev Cell., 2014 Nov 24; 31(4), 434-47; Okabe, Y. el al., Nature, 2009, 460(7254), 520-4; Zhang, L. et al., Nat. Commun, 2018 Mar 13, 9(1), 1047.

[0005] The MYC family of transcription factors is comprised of C-, N- and L-MYC. MYC, when expressed at physiologic levels, primarily binds to active promoters to regulate a distinct set of genes. However, when C-MYC (referred to as MYC) is overexpressed, it begins to bind at distal sites, such that it has been argued to regulate anywhere from -15% of all human genes, to almost all genes within a cell type that contain active promoters and enhancers. MYC overexpression therefore leads to its “invasion” of active genes, making it a ‘super-controller’ of the genome. These data explain in part why MYC is such a potent oncogene that regulates a myriad of critical cellular processes including (but not limited to) proliferation, survival, metabolism, and immune regulation. MYC is a key driver of many different tumor types, and its deregulation can be observed in 70% of all human cancers. MYC protein stability is controlled by the dynamic phosphorylation / dephosphorylation of its T58 and S62 residues. MAPK and CDK phosphorylate S62, followed by T58 phosphorylation by GSK3. PP2A-B56-mediated dephosphorylation of S62 leads to degradation of MYC through the proteasome pathway. EYA proteins recruit PP2A-B55a to dephosphorylate phospho-threonine 58 (pT58) on MYC, leading to maintenance of phosphorylation on S62 and stabilizing the MYC protein. The intrinsic Tyr Phosphatase activity of EYA has been shown to cooperate with the Ser / Thr Phosphatase activity to regulate pT58 dephosphorylation and MYC stability. Thus, the Tyr Phosphatase activity of Eya is linked to both Myc transcription (Li, X. et al., Nature, 2003, 426, 247-254) and protein stabilization (Li, J. et al., Mol. and Cellular Biology (2016)).

[0006] MYC is considered one of the critical “undruggable” targets that need to bepursued. To date, six studies, three published by the inventors (see, for example, Wolin, A R., et al., Neuro. Oncol, (2023)), performed in breast cancer, prostate cancer, glioblastoma, leukemia, medulloblastoma have shown that EYA2 regulates MYC in cancer. Since the EYA2 Tyr Phosphatase is critical to induce MYC transcription and protein stabilization, targeting it serves as a novel means to inhibit EYA2 / MYC driven cancers. Some direct or indirect inhibitors are now being developed that target MYC. For example, Omomyc, a cell penetrating peptide-based inhibitor which acts as a dominant negative form of MYC, has demonstrated efficacy in animal models and has moved into clinical trials. Less direct MYC inhibitors such as JQ1, a BET inhibitor which downregulates MYC transcription (as well as transcription of numerous other genes), has been shown to result in compensatory mutations that prevent MYC downregulation. However, their efficacy in clinical settings remains uncertain. It is likely that at best the MYC inhibitors currently under development need to be used in combination with other drugs to maximize their effect on MYC inhibition. Indeed, identification of a clinically relevant MYC inhibitor has been compared to the Higgs boson hunt and the goal of identifying viable clinical MYC inhibitors has not yet been met.

[0007] While some studies have examined use of EYA2 tyrosine Phosphatase activity inhibitors in treating cancer, unfortunately various obstacles, such as bioavailability, stability, solubility, and / or side-effects have prevented their use in medicine.

[0008] Therefore, there is a continuing need for EYA2 tyrosine phosphatase inhibitors that can be used for treating a clinical condition associated with EYA2 tyrosine phosphatase activity.SUMMARY OF THE INVENTION

[0009] Some aspects of the invention are based on the discovery that EYA2 Tyr Phosphatase is associated with activation of SIX1 and MYC and that inhibitors of EYA2 Tyr Phosphatase activity can disrupt the activity of these oncogenic transcription factors. In one particular aspect of the disclosure, the present inventors have discovered that compounds disclosed herein are allosteric EYA2 Tyr Phosphatase inhibitors and can be used to treat a clinical condition associated with EYA2 tyrosine phosphatase expression, including but not limited to, cancer.

[0010] Accordingly, some aspects of the disclosure provide a compound that inhibits EYA2 tyrosine phosphatase activity. Other aspects of the disclosure provide a method for treating a clinical condition associated with EYA2 tyrosine phosphatase activity by administering a therapeutically effective amount of EYA2 tyrosine phosphatase inhibitor of the disclosure.

[0011] In one aspect of the disclosure, the EYA2 tyrosine phosphatase inhibitor is of the formula:where m is 0 or 1;Ar1is aryl or heteroaryl each of which is optionally substituted;L1is a first linker selected from the group consisting of:Ar2is arylene or heteroarylene each of which is optionally substituted;L2is alkylene;Ar3is aryl or heteroaryl each of which is optionally substituted or -NR’R2; each X1is O, NR1, or S; each X2is O, NR2, or S; each of Y is independently H or halide; and each of R1, R2, and Rais independently H, alkyl, or a nitrogen protecting group, provided when Ar2is furylene or optionally substituted phenylene, then L1is not -C(=O) NH N~CH .

[0012] In some embodiments, Ar1is phenyl, naphthyl, anthracyl, thiophenyl, or furyl, each of which is optionally substituted. Still in other embodiments, Ar1is optionally substituted phenyl, i.e., a moiety of the formula:where each of Xais independently halide, alkyl, haloalkyl, alkoxide, optionally substituted aryl, optionally substituted aryloxide, - (where Rbis H or alkyl), haloalkoxide, or-NCh;and n is an integer from 0 to 5, typically 0 to 4, often 0 to 3, more often 0 to 2, and most often 0 or 1. In one particular embodiment, n is 1.

[0013] In some embodiments, the EYA2 tyrosine phosphatase inhibitor is of the formula: Ar'-L'-Ar-S-Ar3(Formula II), where Ar1, L1, and Ar2are those defined herein and Ar3is aryl or heteroaryl each of which is optionally substituted.

[0014] Another aspect of the disclosure provides a method for treating a clinical condition associated with EYA2 tyrosine phosphatase activity. The method generally comprises administering a therapeutically effective amount of a compound disclosed herein to a subject in need of such a treatment.

[0015] Still another aspect of the disclosure provides a method for treating cancer. The method includes administering to a subject in need of such a treatment a therapeutically effective amount of a pharmaceutically active agent that inhibits EYA2 tyrosine phosphatase activity. In some embodiments, the pharmaceutically active agent comprises a compound disclosed herein. Cancers that can be treated using a compound of the disclosure include, but are not limited to, medulloblastoma, glioblastoma, lung cancer, breast cancer, leukemia, ovarian cancer, kidney cancers, hepatocellular cancer, colorectal cancer, thyroid cancer, prostate cancer, endometrial cancer, esophageal cancer, osteosarcoma, pancreatic cancer, cervical cancer, meningioma, or a combination thereof. In general, the method includes treating any cancer that overexpresses EYA2. Still in other embodiments, the method includes administering a second cancer therapy, such as radiotherapy, immunotherapy, chemotherapy, hormonal therapy, gene therapy, or a combination thereof.

[0016] Yet another aspect of the disclosure provides a method for modulating EYA2 tyrosine phosphatase activity in a cell. The method includes contacting the cell with a compound disclosed herein.

[0017] Another aspect of the disclosure provides a pharmaceutical composition comprising a compound disclosed herein.BRIEF DESCRIPTION OF THE DRAWINGS

[0018] FIG. 1A is a graph showing EYA2 expression in Group 3 MB (GSE124814) compared to other subgroup tumors or brain tissue from epileptic patients.

[0019] FIG. IB is a graph showing EYA2 expression in SHH-driven (DAOY, ONS-76) and Group 3 (D425, D458, D283) MB models.

[0020] FIG. 1C is Western blot of EYA2 and MYC protein expression in SHH-driven and Group 3 medulloblastoma cell models, representative of four independent experiments.

[0021] FIG. 2A a graph showing EYA2 and MYC mRNA relative expression in D425 shNC and EYA2 KD cells.

[0022] FIG. 2B is a graph showing EYA2 and MYC mRNA relative expression in D458 shNC and EYA2 KD cells.

[0023] FIG. 2C is Western blot showing EYA2, MYC, and phospho-threonine 58 (pT58) MYC levels in shNC and EYA2 KD in D425 and D458 cells.

[0024] FIG. 2D is a graph showing pT58-MYC:MYC ratio in EYA2 KD in D425 and D458 cells.

[0025] FIG. 3A is pathway analysis of differentially expressed genes in shNC / EYA2 KD D425 cells.

[0026] FIG. 3B is a pathway analysis of differentially expressed genes in shNC / EYA2 KD D458 cells.

[0027] FIG. 4 is a graph showing loss of EYA2 in D425 and D458 cells elicits severe growth defects as measured by CellTiterGlo. Statistical analysis: Longitudinal mixed-effects model in R.

[0028] FIG. 5 is a graph showing EYA2 is required for MB growth in vivo. Tumor volume as measured by MRI of shNC and EYA2 KD D458 tumors. Statistical analysis: Longitudinal mixed-effects model in R.

[0029] FIG. 6A is GSEA pre-ranked analysis on genes correlated with EYA2 expression in primary MB tumors (GSE85217).

[0030] FIG. 6 B is GSEA plot for MYC_targets_V2 genes.

[0031] FIG. 6C is a graph showing MYC and EYA2 gene expression across all MB subgroups. Correlation between EYA2 and MYC expression in Group 3 MB only (top line), or all other MB groups (bottom line).

[0032] FIG. 7A is a graph showing EYA2 Correlates with Worsened Prognosis in MB. Patient survival across all MB subtypes stratified by differential EYA2 expression. Statistical analysis: Log-Rank analysis in R.

[0033] FIG. 7B is a graph showing EYA2 Correlates with Worsened Prognosis in MB. Patient survival across all MB subtypes stratified by differential MYC expression. Statistical analysis: Log-Rank analysis in R.

[0034] FIG. 7C is a graph showing EYA2 Correlates with Worsened Prognosis in MB. Patient survival across all MB subtypes stratified by differential EYA2 and MYC expression. Statistical analysis: Log-Rank analysis in R.

[0035] FIG. 7D is a graph showing patient survival within Group 3 MB stratified by high vs low (above or below the mean) EYA2 expression. Statistical analysis: Log-Rank analysis in R.

[0036] FIG. 7E is a graph showing patient survival within Group 3 MB stratified by high vs low (above or below the mean) MYC expression. Statistical analysis: Log-Rank analysis in R.

[0037] FIG. 7F is a graph showing patient survival within Group 3 MB stratified by high vs low (above or below the mean) EYA2 and MYC expression. Statistical analysis: Log-Rank analysis in R.

[0038] FIG. 8 is a graph showing quantitation of total MYC / Actin to pT58-MYC / Actin in cells treated with indicated amounts of 3 different EYA2 Tyr Ptase inhibitors.

[0039] FIG. 9A is a CD analyses showing the EYA3 NTD is disordered.

[0040] FIG. 9B shows the interaction between the EYA2 NTD Q) and B55a (2) as predicted by Alpha fold.

[0041] FIG. 10A shows a representative class of benzohydrazide compounds that selectively inhibit the EYA2 Tyr P’tase, namely, MLS000544460 and an analog NCGC00249987 (numbers in parentheses are IC50s).

[0042] FIG. 10B shows compound 9987 selectively inhibits the phosphatase activity of EYA2 relative to EYA1 and 4.

[0043] FIG. 11 A is a crystal structure of the EYA2-9987 complex. The compound (1) binding to an allosteric pocket opens up the HBM and catalytic domain interface as well as leads to a dramatic conformational change of the 01 -al loop critical for Mg2+ binding.

[0044] FIG. 1 IB is a surface representation of the EYA2-9987 complex structure showing vectors for possible optimization of the compound (green arrow).

[0045] FIG. 12A is a normalized graph showing MYC mRNA expression in D458 cells cultured with 24 pM 9987.

[0046] FIG. 12B is Western blot and graph of D458 cells cultured with 24 pM 9987 showing reduced MYC protein and increased pT58-MYC relative to total MYC.

[0047] FIG. 12C shows growth analysis of D458 cells cultured with indicated doses of 9987.

[0048] FIG. 12D shows pharmacokinetic analysis of 9987 in plasma and brain tissue through 24 hours.

[0049] FIG. 12E is a picture showing representative bioluminescence images (left) of end-point tumors in mice treated with vehicle or 25 mg / kg 9987, and intracranial tumor growth over time of vehicle or 9987 treated mice, measured as photon flux.

[0050] FIG. 12F is Kaplan-Meier curve of mice treated with vehicle or 25 mg / kg 9987.

[0051] FIG. 12G shows IHC images of MYC from tumor bearing mice treated with vehicle or 25 mg / kg 9987. Scale bars represent 100 pm.

[0052] FIG. 12H is a graph showing quantification of MYC in end-point tumors of mice treated with vehicle or 25 mg / kg 9987.

[0053] FIG. 13 A is a graph showing IC50of compounds LG1-34 and LG1-37.

[0054] FIG. 13B shows GI50of some of the compounds of the disclosure in inhibiting growth of D458 cells.

[0055] FIG. 14A shows chemical structures of 9987, LG1-34, and SD3-146, where the arrows indicate changes compared to compound 9987.

[0056] FIG. 14B is a graph showing IC50 of compounds LG1-34 and SD3-146 obtained from in vitro phosphatase assays using purified mammalian EYA2.

[0057] FIG. 14C is a graph showing GI50of compounds LG1-34 and SD3-146 in D458 cells.

[0058] FIG. 14D shows results of Cellular Thermal Shift Assay (CETSAs) of compounds 9987, LG1-34, and SD3-146.

[0059] FIG. 15A is a graph showing effects of EYA2 inhibitors on cell growth.

[0060] FIG. 15B is a graph showing effects of EYA2 inhibitors on cell cycle.

[0061] FIG. 15C is a graph showing effect of EYA2 inhibitors on apoptosis.

[0062] FIG. 16 shows EYA2 Tyr Ptase activity is required for MYC levels in MYC driven MB cells. Statistical analysis was performed using a Kruskal -Wallis test with Dunn’s correction for multiple comparisons. Representative of three biological replicates.

[0063] FIG. 17 is a graph showing GI50 of two representative compounds of the disclosure that are analogs of 9987 in CEM cells.DETAILED DESCRIPTION OF THE INVENTION

[0064] “Alkyl” refers to a saturated linear monovalent hydrocarbon moiety of one to twelve, typically one to six, carbon atoms or a saturated branched monovalent hydrocarbon moiety of three to twelve, preferably three to six, carbon atoms. Exemplary alkyl group include, but are not limited to, methyl, ethyl, n-propyl, 2-propyl, tert-butyl, pentyl, and the like.

[0065] “Alkylene” refers to a saturated linear divalent hydrocarbon moiety of one to twelve, typically one to six, carbon atoms or a branched saturated divalent hydrocarbon moiety of three to twelve, typically three to six, carbon atoms. Exemplary alkylene groups include, but are not limited to, methylene, ethylene, propylene, butylene, pentylene, cyclopropylene, cyclopentylene, cyclohexylene, and the like.

[0066] “Aryl” refers to a monovalent mono-, bi- or tricyclic aromatic hydrocarbon moiety of 6 to 15 ring atoms which is optionally substituted with 0-5, typically 0-4, often 0-3, more often 0-2, still more often 0 or 1 substituents within the ring structure. When two or more substituents are present in an aryl group, each substituent is independently selected. Exemplary substituents for the aryl group include, but are not limited to, halide, alkyl, haloalkyl, alkoxide, optionally substituted aryl, optionally substituted aryloxide, optionally substituted heteroaryl, optionally substituted heteroaryloxide(where Rbis H or alkyl), haloalkoxide, -NO2, amino, mono- or dialkyl amino, -(alkylene)n-COOR (where n is 0 or 1 and R is hydrogen, alkyl, or haloalkyl), or -(alkylene)n-CONRaRb(where n is 0 or 1, and each of Raand Rbis independently hydrogen or alkyl). More specifically the term aryl includes, but is not limited to, phenyl, 1 -naphthyl, and 2-naphthyl, anthracyl, and the like.

[0067] “Arylene” refers to a divalent aryl group, e.g., aryl group having two points of attachments. Thus, the term phenylene refers to a phenyl ring in which two points of attachment is indicated in the formula. For example, Ar2in Formula I.

[0068] “Aralkyl” refers to a moiety of the formula -RbRcwhere Rbis an alkylene group and Rcis an aryl group as defined herein. Exemplary aralkyl groups include, but are not limited to, benzyl, phenylethyl, 3 -(3 -chi orophenyl)-2 -methylpentyl, and the like.

[0069] “Chiral center” (i.e., stereochemical center, stereocenter, or stereogenic center) refers to an asymmetrically substituted atom, e.g., a carbon atom to which four different groups are attached. The ultimate criterion of a chiral center, however, is nonsuperimposability of its mirror image.

[0070] The terms “cycloalkyl” and “cyclyl” are used interchangeably herein and refer to a non-aromatic, typically saturated, monovalent mono- or bicyclic hydrocarbon moiety of three to ten ring carbons. The cycloalkyl can be optionally substituted with one or more, typically one,two, or three, substituents within the ring structure. When two or more substituents are present in a cycloalkyl group, each substituent is independently selected. Exemplary substituents for a cycloalkyl group include, but are not limited to, alkyl, haloalkyl, halo, nitro, cyano, heteroalkyl, optionally substituted phenyl, optionally substituted heteroaralkyl, or -C(O)R (where R is hydrogen, alkyl, haloalkyl, amino, monsubstituted amino, disubstituted amino, hydroxy, alkoxy, or optionally substituted phenyl). Exemplary cycloalkyls include, but are not limited to, cyclopropyl, cyclopentyl, cyclohexyl, and the like, each of which is optionally substituted.

[0071] “Cycloalkylalkyl” refers to a moiety of the formula -RdRewhere Rdis an alkylene group and Reis a cycloalkyl group as defined herein. Exemplary cycloalkylalkyl groups include, but are not limited to, cyclopropylmethyl, cyclohexylpropyl, 3-cyclohexyl-2-methylpropyl, etc.

[0072] The terms “halo,” “halogen” and “halide” are used interchangeably herein and refer to fluoride, chloride, bromide, or iodide. In some embodiments, halide is fluoride or chloride. In one particular embodiment, halide is fluoride.

[0073] “Haloalkyl” refers to an alkyl group as defined herein in which one or more hydrogen atom is replaced by same or different halide. The term “haloalkyl” also includes perhalogenated alkyl groups in which all alkyl hydrogen atoms are replaced by halides. Exemplary haloalkyl groups include, but are not limited to, -CH2CI, -CF3, -CH2CF3, -CH2CCI3, and the like.

[0074] The term “heteroaryl” means a monovalent monocyclic or bicyclic aromatic moiety of 5 to 12 ring atoms containing one, two, or three ring heteroatoms selected from N, O, or S, the remaining ring atoms being C. The heteroaryl ring is optionally substituted independently with one or more substituents. When substituted, heteroaryl typically contains one to four, often one to three, and more often one or two substituents, each of which is independently selected. Exemplary substituents for heteroaryl includes those disclosed for aryl group. Exemplary heteroaryls include, but are not limited to, pyridyl, furyl, thiophenyl, thiazolyl, isothiazolyl, triazolyl, triazinyl, imidazolyl, oxazolyl, isoxazolyl, pyrrolyl, pyrazolyl, pyrazinyl, pyrimidinyl, benzofuranyl, isobenzofuranyl, benzothiazolyl, benzoisothiazolyl, benzotri azolyl, indolyl, isoindolyl, benzoxazolyl, thiazolyl, isothiazolyl, quinolyl, isoquinolyl, benzimidazolyl, benzisoxazolyl, benzothiophenyl, dibenzofuran, benzodiazepin-2-one-5-yl, and the like.

[0075] “Heteroarylene” refers to a divalent heteroaryl. For example, furan ring with two points of attachment is called furylene, and thiophene with two points of attachment is called thiophenylene, etc.

[0076] ‘Enantiomeric excess” refers to the difference between the amount of enantiomers. The percentage of enantiomeric excess (%ee) can be calculated by subtracting the percentage of one enantiomer from the percentage of the other enantiomer. For example, if the percent of (R)-enantiomer is 99% and the percent of (S)-enantiomer is 1%, the %ee of (R)- isomer is 99%-l% or 98%.

[0077] “Leaving group” has the meaning conventionally associated with it in synthetic organic chemistry, i.e., an atom or a group capable of being displaced by a nucleophile and includes halo (such as chloro, bromo, and iodo), alkanesulfonyloxy, arenesulfonyloxy, alkylcarbonyloxy (e.g., acetoxy), arylcarbonyloxy, mesyloxy, tosyloxy, trifluoromethanesulfonyloxy, aryloxy (e.g., 2,4-dinitrophenoxy), methoxy, N,O- dimethylhydroxylamino, and the like.

[0078] “Pharmaceutically acceptable excipient” refers to an excipient that is useful in preparing a pharmaceutical composition that is generally safe, non-toxic and neither biologically nor otherwise undesirable, and includes excipient that is acceptable for veterinary use as well as human pharmaceutical use.

[0079] “Pharmaceutically acceptable salt” of a compound means a salt that is pharmaceutically acceptable and that possesses the desired pharmacological activity of the parent compound. Such salts include: (1) acid addition salts, formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like; or formed with organic acids such as acetic acid, propionic acid, hexanoic acid, cyclopentanepropionic acid, glycolic acid, pyruvic acid, lactic acid, malonic acid, succinic acid, malic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, 3-(4- hydroxybenzoyl)benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, 1,2-ethane-disulfonic acid, 2-hydroxy ethanesulfonic acid, benzenesulfonic acid, 4-chlorobenzenesulfonic acid, 2-naphthalenesulfonic acid, 4-toluenesulfonic acid, camphorsulfonic acid, 4-methylbicyclo[2.2.2]-oct-2-ene-l carboxylic acid, glucoheptonic acid, 3- phenylpropionic acid, trimethylacetic acid, tertiary butylacetic acid, lauryl sulfuric acid, gluconicacid, glutamic acid, hydroxynaphthoic acid, salicylic acid, stearic acid, muconic acid, and the like; or (2) salts formed when an acidic proton present in the parent compound either is replaced by a metal ion, e.g., an alkali metal ion, an alkaline earth ion, or an aluminum ion; or coordinates with an organic base such as ethanolamine, diethanolamine, triethanolamine, tromethamine, N- methylglucamine, and the like.

[0080] The terms “pro-drug” and “prodrug” are used interchangeably herein and refer to a pharmacologically substantially inactive derivative of a parent drug molecule that requires biotransformation, either spontaneous or enzymatic, within the organism to release the active drug. Prodrugs are variations or derivatives of the compounds of this invention which have groups cleavable under metabolic conditions. Prodrugs become the compounds of the invention which are pharmaceutically active in vivo when they undergo solvolysis under physiological conditions or undergo enzymatic degradation. Prodrug compounds of this invention may be called single, double, triple etc., depending on the number of biotransformation steps required to release the active drug within the organism, and indicating the number of functionalities present in a precursor-type form. Prodrug forms often offer advantages of solubility, tissue compatibility, or delayed release in the mammalian organism (see, Bundgard, Design of Prodrugs, pp. 7-9, 21-24, Elsevier, Amsterdam 1985 and Silverman, The Organic Chemistry of Drug Design and Drug Action, pp. 352-401, Academic Press, San Diego, Calif, 1992).Prodrugs commonly known in the art include acid derivatives that are well known to one skilled in the art, such as, but not limited to, esters prepared by reaction of the parent acids with a suitable alcohol, or amides prepared by reaction of the parent acid compound with an amine, or basic groups reacted to form an acylated base derivative. Moreover, the prodrug derivatives of this invention may be combined with other features herein taught to enhance bioavailability. For example, a compound of the invention having free amino, amido, hydroxy or carboxylic groups can be converted into prodrugs. Prodrugs include compounds wherein an amino acid residue, or a polypeptide chain of two or more (e.g., two, three or four) amino acid residues which are covalently joined through peptide bonds to free amino, hydroxy or carboxylic acid groups of compounds of the invention. The amino acid residues include the 20 naturally occurring amino acids commonly designated by three letter symbols and also include, 4-hydroxyproline, hydroxylysine, demosine, isodemosine, 3-methylhistidine, norvalin, beta-alanine, gamma- aminobutyric acid, citrulline homocysteine, homoserine, ornithine and methionine sulfone.Prodrugs also include compounds wherein carbonates, carbamates, amides and alkyl esters which are covalently bonded to the above substituents of a compound of the invention through the carbonyl carbon prodrug sidechain.

[0081] ‘Protecting group” refers to a moiety, except alkyl groups, that when attached to a reactive group in a molecule masks, reduces or prevents that reactivity. Examples of protecting groups can be found in T.W. Greene and P.G.M. Wuts, Protective Groups in Organic Synthesis, 3rdedition, John Wiley & Sons, New York, 1999, and Harrison and Harrison et al., Compendium of Synthetic Organic Methods, Vols. 1-8 (John Wiley and Sons, 1971-1996), which are incorporated herein by reference in their entirety. Representative hydroxy protecting groups include acyl groups, benzyl and trityl ethers, tetrahydropyranyl ethers, trialkylsilyl ethers and allyl ethers. Representative amino protecting groups include, formyl, acetyl, trifluoroacetyl, benzyl, benzyloxycarbonyl (CBZ), ter / -butoxycarbonyl (Boc), trimethyl silyl (TMS), 2- trimethylsilyl-ethanesulfonyl (SES), trityl and substituted trityl groups, allyloxycarbonyl, 9- fluorenylmethyloxycarbonyl (FMOC), nitro-veratryloxycarbonyl (NVOC), and the like.

[0082] “Corresponding protecting group” means an appropriate protecting group corresponding to the heteroatom (i.e., N, O, P or S) to which it is attached.

[0083] “A therapeutically effective amount” means the amount of a compound that, when administered to a mammal for treating a disease, is sufficient to affect such treatment for the disease. The “therapeutically effective amount” will vary depending on the compound, the disease and its severity and the age, weight, etc., of the mammal to be treated.Compounds o f the Disclosure

[0084] For the sake of brevity and clarity, the present disclosure will now be described with regard to methods and compounds for treating cancer. However, it should be appreciated that the scope of the disclosure is not limited to treating cancer. In general, methods and compounds of the disclosure can be used in treating any clinical condition associated with EYA2 tyrosine phosphatase activity. Discussion on treating cancer is provided solely for the purpose of illustrating the practice of the disclosure and do not constitute limitations on the scope thereof.

[0085] Some aspects of the disclosure provide a compound of the formula:where m is 0 or 1;Ar1is aryl or heteroaryl each of which is optionally substituted;L1is a first linker selected from the group consisting ofAr2is arylene or heteroarylene each of which is optionally substituted;L2is alkylene;Ar3is aryl or heteroaryl each of which is optionally substituted or -NR’R2; each X1is O, NR1, or S; each X2is O, NR2, or S; each of Y is independently H or halide; and each of R1, R2, and Rais independently H, alkyl, or a nitrogen protecting group, provided when Ar2is furylene or optionally substituted phenylene, then L1is not -C(=O)-NH-N=CH-

[0086] In some embodiments, Ar1is phenyl, naphthyl, anthracyl, thiophenyl, or furyl, each of which is optionally substituted. In one particular embodiments, Ar1is of the formula:where each of Xais independently halide, alkyl, haloalkyl, alkoxide, optionally substituted aryl, optionally substituted aryloxide, (where Rbis H or alkyl), haloalkoxide, or -NCh;and n is an integer from 0 to 5.

[0087] In one particular embodiment, n is i . Yet in another particular embodiment, Ar1is of the formula:where Xais as defined herein.

[0088] Still in other embodiments, L1is of the formula:wherein X1is as defined herein. In some particular embodiments, X1is O.

[0089] Yet in other embodiments, Li is of the formula:where X1and X2are as defined herein. In some particular embodiments, X1and X2are O.

[0090] In further embodiments, L1is of the formula:where X1, X2, and Y are as defined herein. It should be appreciated it that while the cyclopropyl ring is depicted herein as a particular stereochemistry, the scope of the disclosure includes its enantiomer, racemic mixture, diastereomer, as well as enantiomeric excess of a particular isomer. In some embodiments, X1and X2are O. Still in other embodiments, each of Y is independently H, F, or Cl.

[0091] Still yet in other embodiments, Ar2is an optionally substituted heteroaryl of the formula:where Z1is NRC, O, or S; Z2is CH or N; and Rcis H or alkyl. In some particular embodiments, Z1is S. In other embodiments, Z1is O. Yet in other particular embodiments, Z2is CH. In other embodiments, Z2is N. In other embodiments, Ar2is phenylene or a pyridylene, each of which is optionally substituted, e.g., a moiety of the formula:respectively, where Xais as defined herein. In another embodiment, Ar2is optionally substituted phenylene.

[0092] Yet in other embodiments, Ar3is a five-membered heteroaryl, a six-membered heteroaryl, or a 9-membered bicycloheteroaryl, each of which is optionally substituted. In one particular embodiment, Ar3is a moiety of the formula:each of which is optionally substituted, where each of W1, W2, and W3are independently N or CH, provided at least one of W1, W2, and W3is N; each of W4and W6is independently CH or N; each of W5and W7is independently NRd, O, or S, wherein Rdis H, alkyl, or a nitrogen protecting group.

[0093] In one particular embodiment, Ar3is optionally substituted moiety of the formula:where W1, W2, and W3are as defined herein. In certain embodiments, W1is N and one or both of W2and W3is CH.

[0094] Still in another particular embodiment, Ar3is optionally substituted moiety of the formula:where W4and W5are as defined herein. In one embodiment, W4is N.

[0095] Yet in other embodiments, W5is O, NRdor S, wherein Rdis as defined herein.

[0096] Still further, combinations of various embodiments described herein form other embodiments. For example, in one particularly embodiment Ar1is halogenated phenyl (e.g., 3- fluorophenyl, 3 -chlorophenyl, or 3 -bromophenyl), L1is a moiety of the formula:, X1and X2are O, Ar2is 2,5-thiophenylene, i.e., a moiety of the formula:, Ar3is 2-pyridyl, i.e., a moiety of the formula:. In this manner, a variety of compounds are embodied within the present disclosure.

[0097] In other embodiments, m is 0.

[0098] Still in other embodiments m is 1. Within these embodiments, in some cases L2is methylene or ethylene. Still in other cases Ar3is -NR'R2. When Ar3is -NRjR2, in some instances, each of R1and R2is independently H or alkyl. Still in other instances, each of R1and R2is independently alkyl. In one particular embodiment, R1and R2is methyl.

[0099] In some embodiments, the EYA2 tyrosine phosphatase inhibitor is of the formula: Ar'-I.'-Ar-S-Ar3(Formula II), or a pharmaceutically acceptable salt thereof, or a prodrug thereof, whereAr1is aryl or heteroaryl each of which is optionally substituted;L1is a first linker selected from the group consisting of:each of Ar2and Ar3is independently aryl or heteroaryl each of which is optionally substituted; each X1is O, NR1, or S; each X2is O, NR2, or S; each of Y is independently H or halide; and each of R1, R2, and Rais independently H, alkyl, or a nitrogen protecting group,provided that when Ar2is furylene or optionally substituted phenylene, then L1is not -C(=O)-NH-N=CH-.

[0100] Compounds disclosed herein can be readily prepared by one skilled in the art having read the present disclosure, including the Examples section. In general, the synthetic strategy shown in the Examples section of this disclosure can be utilized by one skilled in the art of organic chemistry to prepare various compounds embodied within the present disclosure simply by utilizing different starting materials for each of Ar1, L1, Ar2, m, L2, and Ar3. For example, some representatives Ar1, L1, Ar2, m, L2, and Ar3moieties that can be used to prepare a wide variety of compounds embodied within this disclosure is schematically illustrated in Scheme I below:It should be noted that Ar2 in Scheme I can also include arylene or heteorarylene moieties, each of which is optionally substituted, such as for example, optionally substituted phenylene and optionally substituted pyridylene. In this manner, a wide variety of compounds of the disclosure have been prepared. Some of the representative compounds of the disclosure include, but are not limited to, the following list of compounds:Utility

[0101] Another aspect of the disclosure provides a method for treating a clinical condition associated with EYA2 tyrosine phosphatase activity. The method comprises administering a therapeutically effective amount of a compound disclosed herein, or a pharmaceutically acceptable salt thereof, or a prodrug thereof to a subject in need of such a treatment. In some embodiments, the clinical condition associated with EYA2 tyrosine phosphatase activity comprises cancer. Exemplary cancers that can be treated using methods and compounds of the disclosure include, but are not limited to, medulloblastoma, glioblastoma, lung cancer, breast cancer, leukemia, ovarian cancer, kidney cancers, hepatocellular cancer, colorectal cancer, thyroid cancer, prostate cancer, endometrial cancer, esophageal cancer, osteosarcoma, pancreatic cancer, cervical cancer, and meningioma.

[0102] Still another aspect of the disclosure provides a method for treating cancer in a subject in need of such a treatment. The method comprises administering a therapeutically effective amount of a pharmaceutically active agent that inhibits EYA2 tyrosine phosphatase activity. Typically, the pharmaceutically active agent comprises a compound disclosed herein. In some embodiments, the method further comprises administering to said subject a second cancer therapy. Exemplary second therapies include, but are not limited to, radiotherapy, immunotherapy, chemotherapy, hormonal therapy, gene therapy, or a combination thereof.

[0103] Yet in another aspect of the disclosure provides a method for modulating EYA2 tyrosine phosphatase activity in a cell. The method comprises contacting the cell with a compound disclosed herein.

[0104] In yet further aspect of the disclosure provides a pharmaceutical composition comprising a compound disclosed herein and a pharmaceutically acceptable excipient.

[0105] In some embodiments, the invention provides compositions and methods for the treatment of cancer. In one embodiment, the invention provides a method of treating cancer comprising administering to a patient an effective amount of an EYA2 tyrosine phosphatase inhibitor disclosed herein. This treatment can be combined with additional cancer treatments. One of skill in the art is aware of many treatments that can be combined with the methods of the present disclosure, some but not all of which are described below solely for the purpose of illustrating the practice of the disclosure and does not constitute limitations on the scope of the present disclosure.

[0106] In general, the cancers that can be treated using compounds of the disclosure are characterized by overexpression of Sixl and / or EYA2. Exemplary cancers that can be treated using compounds of the disclosure include, but are not limited to, cancers of the brain, lung, liver, spleen, kidney, lymph node, pancreas, small intestine, blood cells, colon, stomach, breast, endometrium, prostate, testicle, ovary, skin, head and neck, esophagus, bone marrow, blood or other tissue. In many contexts, it is not necessary that the tumor cell be killed or induced to undergo normal cell death or “apoptosis.” Rather, to accomplish a meaningful treatment, all that is required is that the tumor growth be slowed to some degree. It may be that the tumor growth is completely blocked, however, or that some tumor regression is achieved. Clinical terminology such as “remission” and “reduction of tumor” burden also are contemplated given their normal usage.Formulations and Routes for Administration to Patients

[0107] In some embodiments, the invention provides a method of treating cancer comprising providing to a patient an effective amount of a compound as provided herein. Where clinical applications are contemplated, it will be necessary to prepare pharmaceutical compositions in a form appropriate for the intended application. Generally, this will entail preparing compositions that are essentially free of pyrogens, as well as other impurities that could be harmful to humans or animals. One will generally desire to employ appropriate salts and buffers to render delivery stable and allow for uptake by target cells.

[0108] Aqueous compositions of the present disclosure comprise an effective amount of the compound of the disclosure, dissolved or dispersed in a pharmaceutically acceptable carrier or aqueous medium. The phrase “pharmaceutically or pharmacologically acceptable” refer to molecular entities and compositions that do not produce adverse, allergic, or other undesired reactions when administered to an animal or a human. As used herein, “pharmaceutically acceptable carrier” includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the compound of the disclosure, its use in therapeutic compositions is contemplated. Supplementary active ingredients also can be incorporated into the compositions.

[0109] The active compositions of the disclosure can include classic pharmaceutical preparations. Administration of these compositions according to the present disclosure will be via any common route so long as the target tissue is available via that route. This includes oral, nasal, buccal, rectal, vaginal or topical. Alternatively, administration can be by intradermal, subcutaneous, intramuscular, intraperitoneal or intravenous injection. Such compositions would normally be administered as pharmaceutically acceptable compositions. Of particular interest is direct intratumoral administration, perfusion of a tumor, or administration local or regional to a tumor, for example, in the local or regional vasculature or lymphatic system, or in a resected tumor bed (e.g., post-operative catheter). For practically any tumor, systemic delivery also is contemplated. This will prove especially important for attacking microscopic or metastatic cancer.

[0110] The active compounds can also be administered as free base or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.[0U1] The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases the form should be sterile and should be fluid tothe extent that easy syringability exists. It should be stable under the conditions of manufacture and storage and should be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it is desirable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

[0112] Sterile injectable solutions can be prepared by incorporating the active compounds in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, optionally followed by fdtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, typical methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-fdtered solution thereof.

[0113] The compositions of the present disclosure can be formulated in a neutral or salt form. Pharmaceutically-acceptable salts include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like.

[0114] Upon formulation, solutions can be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The actual dosage amountof a composition of the present disclosure administered to a patient or subject can be readily determined by physical and physiological factors such as body weight, severity of condition, the type of disease being treated, previous or concurrent therapeutic interventions, idiopathy of the patient, and on the route of administration. The practitioner responsible for administration can, in any event, readily determine the concentration of active ingredient(s) in a composition and appropriate dose(s) for the individual subject.

[0115] In additional embodiments of the disclosure, methods can include identifying a patient in need of treatment. A patient may be identified, for example, based on taking a patient history or based on findings on clinical examination, or screening a patient for EYA2 level.Cancer Combination Treatments

[0116] Some aspects of the disclosure include a method for treating cancer in a patient using a compound of the present disclosure in combination with a conventional cancer treatment. In the context of the present disclosure, it is contemplated that this combined treatment can include, but is not limited to, chemotherapeutic, radiation, a polypeptide inducer of apoptosis, or other therapeutic intervention. The scope of the present disclosure also includes using more than one administration of the treatment.

[0117] Chemotherapy

[0118] In addition to using a compound of the present disclosure, one embodiment includes utilizing a wide variety of other conventional chemotherapeutic agents. The term “chemotherapy” refers to the use of drugs to treat cancer. A “chemotherapeutic agent” is used to connote a compound or composition that is administered in the treatment of cancer. These agents or drugs are categorized by their mode of activity within a cell, for example, whether and at what stage they affect the cell cycle. Alternatively, an agent may be characterized based on its ability to directly cross-link DNA, to intercalate into DNA, or to induce chromosomal and mitotic aberrations by affecting nucleic acid synthesis. Most chemotherapeutic agents fall into the following categories: alkylating agents, antimetabolites, antitumor antibiotics, mitotic inhibitors, and nitrosoureas. Examples of chemotherapeutic agents include, but are not limited to, alkylating agents such as thiotepa and cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine,trietylenephosphoramide, triethiylenethiophosphoramide and trimethylolomelamine; acetogenins (especially bullatacin and bullatacinone); a camptothecin (including the synthetic analogue topotecan); bryostatin; cally statin; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogues); cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189 and CB1-TM1); eleutherobin; pancrati statin; a sarcodictyln; spongistatin; nitrogen mustards such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; antibiotics such as the enediyne antibiotics (e.g., calicheamicin, especially calicheamicin gammall and calicheamicin omegall; dynemicin, including dynemicin A; bisphosphonates, such as clodronate; an esperamicin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antiobiotic chromophores, aclacinomysins, actinomycin, authrarnycin, azaserine, bleomycins, cactinomycin, carabicin, carminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo- 5-oxo-L-norleucine, doxorubicin (including morpholino-doxorubicin, cyanomorpholinodoxorubicin, 2-pyrrolino-doxorubicin and deoxy doxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalarnycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexate and 5 -fluorouracil (5-FU); folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, di deoxy uridine, doxifluridine, enocitabine, floxuridine; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; anti-adrenals such as aminoglutethimide, mitotane, trilostane; folic acid replenisher such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elformithine; elliptinium acetate; an epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSKpolysaccharide complex); razoxane; rhizoxin; sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2"-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A and anguidine); urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside (“Ara-C”); cyclophosphamide; thiotepa; taxoids, e.g., paclitaxel and doxetaxel; chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum coordination complexes such as cisplatin, oxaliplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; vinorelbine; novantrone; teniposide; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; irinotecan (e.g., CPT-11); topoisomerase inhibitor RFS 2000; difluorometlhylornithine (DMFO); retinoids such as retinoic acid; capecitabine; cisplatin (CDDP), carboplatin, procarbazine, mechlorethamine, cyclophosphamide, camptothecin, ifosfamide, melphalan, chlorambucil, busulfan, nitrosurea, dactinomycin, daunorubicin, doxorubicin, bleomycin, plicomycin, mitomycin, etoposide (VP 16), tamoxifen, raloxifene, estrogen receptor binding agents, taxol, paclitaxel, docetaxel, gemcitabien, navelbine, farnesyl-protein tansferase inhibitors, transplatinum, 5-fluorouracil, vincristin, vinblastin and methotrexate and pharmaceutically acceptable salts, acids or derivatives of any of the above.

[0119] Radiotherapy

[0120] Radiotherapy, also called radiation therapy, is the treatment of cancer and other diseases with ionizing radiation. Ionizing radiation deposits energy that injures or destroys cells in the area being treated by damaging their genetic material, making it impossible for these cells to continue to grow. Although radiation damages both cancer cells and normal cells, the latter are able to repair themselves and function properly. Radiation therapy used according to the present disclosure can include, but is not limited to, the use of y-rays, X-rays, positrons, and / or the directed delivery of radioisotopes to tumor cells. Other forms of DNA damaging factors are also contemplated such as microwaves and UV-irradiation. It is most likely that all of these factors effect a broad range of damage on DNA, on the precursors of DNA, on the replication and repair of DNA, and on the assembly and maintenance of chromosomes. Dosage ranges for X-rays range from daily doses of 50 to 200 roentgens for prolonged periods of time (3 to 4 wk), to single doses of 2000 to 6000 roentgens. Dosage ranges for radioisotopes vary widely, and depend on the half-life of the isotope, the strength and type of radiation emitted, and the uptake by the neoplastic cells.

[0121] Radiotherapy can include the use of radiolabeled antibodies to deliver doses of radiation directly to the cancer site (radioimmunotherapy). Antibodies are highly specific proteins that are made by the body in response to the presence of antigens (substances recognized as foreign by the immune system). Some tumor cells contain specific antigens that trigger the production of tumor-specific antibodies. Large quantities of these antibodies can be made in the laboratory and attached to radioactive substances (a process known as radiolabeling). Once injected into the body, the antibodies actively seek out the cancer cells, which are destroyed by the cell-killing (cytotoxic) action of the radiation. This approach can minimize the risk of radiation damage to healthy cells.

[0122] Conformal radiotherapy uses the same radiotherapy machine, a linear accelerator, as the normal radiotherapy treatment but metal blocks are placed in the path of the x-ray beam to alter its shape to match that of the cancer. This ensures that a higher radiation dose is given to the tumor. Healthy surrounding cells and nearby structures receive a lower dose of radiation, so the possibility of side effects is reduced. A device called a multi-leaf collimator has been developed and can be used as an alternative to the metal blocks. The multi-leaf collimator consists of a number of metal sheets which are fixed to the linear accelerator. Each layer can be adjusted so that the radiotherapy beams can be shaped to the treatment area without the need for metal blocks. Precise positioning of the radiotherapy machine is very important for conformal radiotherapy treatment and a special scanning machine may be used to check the position of your internal organs at the beginning of each treatment.

[0123] High-resolution intensity modulated radiotherapy also uses a multi-leaf collimator. During this treatment the layers of the multi-leaf collimator are moved while the treatment is being given. This method is likely to achieve even more precise shaping of the treatment beams and allows the dose of radiotherapy to be constant over the whole treatment area. Although research studies have shown that conformal radiotherapy and intensity modulated radiotherapy may reduce the side effects of radiotherapy treatment, it is possible that shaping the treatment area so precisely could stop microscopic cancer cells just outside the treatment area being destroyed.

[0124] Still in other embodiments, methods of the disclosure can include using a radiosensitizer to make the tumor cells more likely to be damaged, or radioprotectors to protect normal tissues from the effects of radiation. Yet in other embodiments, methods of the disclosure can include using hyperthermia in sensitizing tissue to radiation.

[0125] Immunotherapy

[0126] In cancer treatment, immunotherapeutics generally rely on the use of immune effector cells and molecules to target and destroy cancer cells. Antibodies, such as trastuzumab (Herceptin™) is such an example. The immune effector may be, for example, an antibody specific for some marker on the surface of a tumor cell. The antibody alone may serve as an effector of therapy or it may recruit other cells to actually affect cell killing. The antibody also may be conjugated to a drug or toxin (chemotherapeutic, radionuclide, ricin A chain, cholera toxin, pertussis toxin, etc.) and serve merely as a targeting agent. Alternatively, the effector may be a lymphocyte carrying a surface molecule that interacts, either directly or indirectly, with a tumor cell target. Various effector cells include cytotoxic T cells and NK cells. The combination of therapeutic modalities, i .e., direct cytotoxic activity and inhibition or reduction of ErbB2 would provide therapeutic benefit in the treatment of ErbB2 overexpressing cancers.

[0127] Other Secondary Therapies

[0128] In yet other embodiments, administration of a compound of the disclosure can be combined with other secondary therapies for treating cancer. Exemplary secondary cancer treatments or therapies include those discussed above, as well as surgery, gene therapy, miRNAs, as well as other agents such as immunomodulatory agents, agents that affect the upregulation of cell surface receptors and GAP junctions, cytostatic and differentiation agents, inhibitors of cell adhesion, agents that increase the sensitivity of the hyperproliferative cells to apoptotic inducers, or other biological agents.

[0129] Another type of therapy used in treating glioblastoma patients is Tumor Treating Fields (TTF). TTF is administered in the home or outpatient setting and involves wearing scalp transducers on the shaved head to deliver alternating electrical fields, with frequent reshaving and reapplying transducers. The administration of the compounds of the disclosure can be combined with TTF.

[0130] Additional objects, advantages, and novel features of this invention will become apparent to those skilled in the art upon examination of the following examples thereof, which are not intended to be limiting. In the Examples, procedures that are constructively reduced to practice are described in the present tense, and procedures that have been carried out in the laboratory are set forth in the past tense.EXAMPLES

[0131] Synthesis and Characterization Data

[0132] Synthesis of 1. To a solution of pyrimidine-2-thione (1.0 eqv.) in dry DMF(0.2M) were added 5-bromofuran-2-carbaldehyde (1.1 eqv.), potassium carbonate (3.0 eqv.) under argon atmosphere. The reaction mixture was heated to 75°C and stirred for 2 hours. After the completion of the reaction, it was diluted with water and ethyl acetate (EtOAc), and extracted. The organic layer was washed with brine three times. The organic layer was dried over anhydrous sodium sulfate and reduced under a vacuum. The crude product was purified by flash chromatography by EtOAc and Hexane to get the pure product, 1, 82% yield.

[0133] Synthesis of 2. To a solution of pyrimidine-2-thione (1.0 eqv.) in dry DMF (0.2M) were added 5-bromothiophen-2-carbaldehyde (1.1 eqv.), potassium carbonate (3.0 eqv.) under argon atmosphere. The reaction mixture was heated to 75 °C and stirred for 2 hours. After the completion of the reaction, it was diluted with water and ethyl acetate (EA), and extracted. The organic layer was washed with brine three times. The organic layer was dried overanhydrous sodium sulfate and reduced under vacuum. The crude product was purified by flash chromatography by EtOAc and Hexane to get the pure product, 1A, 80% yield.

[0134] Synthesis of acylhydrazones. To a solution of 1A (1.0 eqv.) in ethanol (0.1M) was added desired hydrazides (1.0 eqv.) at room temperature and heated to 95 °C for 12 hours. After the completion of the reaction, it was diluted with water and extracted with chloroform. The organic layer was dried over anhydrous sodium sulfate and concentrated under a vacuum. The crude was purified by column chromatography by EtOAc and hexane. In this manner, the following acylhydrazone compounds were prepared.

[0135] 1H), 8.67 (d, 7= 4.9 Hz, 2H), 8.33 (s, 1H), 7.75 (d, 7= 7.8 Hz, 1H), 7.70 (d, 7= 9.8 Hz, 1H),7.64 - 7.55 (m, 1H), 7.47 (t, 7= 8.3 Hz, 1H), 7.34 (t, 7= 4.9 Hz, 1H), 7.18 - 7.08 (m, 2H).

[0136] (d, J= 4.9 Hz, 2H), 8.26 (s, 1H), 8.08 (s, 1H), 7.92 (d, J= 7.8 Hz, 1H), 7.73 (d, J= 8.2 Hz, 1H), 7.45 (t, J = 7.8 Hz, 1H), 7.32 (t, J = 4.9 Hz, 1H), 7.11 (d, 7= 3.4 Hz, 1H), 7.02 (d, 7 = 3.5 Hz, 1H).

[0137] 24:1H NMR (300 MHz, DMSO) 812.12 (s, 1H), 8.68 (d, J= 4.9 Hz, 2H), 8.35 (s, 1H), 8.22 (d, J= 8.6 Hz, 2H), 8.00 (d, J= 7.9 Hz, 1H), 7.80 (t, J= 7.8 Hz, 1H), 7.35 (t, J= 4.9 Hz, 1H), 7.15 (q, 7= 3.5 Hz, 2H).

[0138] 25: 'H NMR (300 MHz, DMSO) 512.02 (s, 1H), 8.68 (d, J= 4.8 Hz, 2H), 8.33 (s, 1H), 7.95 (s, 1H), 7.87 (d, J= 7.7 Hz, 1H), 7.69 (d, J=8.0Hz, 1H), 7.58 (t, J= 7.9 Hz, 1H), 7.35 (t, J= 4.9 Hz, 1H), 7.15 (d, J= 3.5 Hz, 1H), 7.12 (d, .7= 3.5 Hz, 1H).

[0139] 30: 'H NMR (300 MHz, DMSO) 812.01 (s, 1H), 8.70 (d, J= 4.9 Hz, 2H), 8.65 (s, 1H), 7.77 (d, J= 7.7 Hz, 1H), 7.72 (d, J= 12.8 Hz, 1H), 7.61 (d, J=6.0Hz, 1H), 7.54 (d,J=3.8Hz, 1H), 7.49 (d,J=9.4Hz, 1H), 7.43 (d, J = 3.8 Hz, 1H), 7.34 (t, J= 4.8 Hz, 1H).

[0140] 33:1H NMR (300 MHz, DMSO) 812.05 (s, 1H), 8.68 (d,J=4.8Hz, 2H), 8.34 (s, 1H), 7.96 (d,J=7.5Hz, 1H), 7.85 (s, 1H), 7.70 (t,J=7.8Hz, 1H), 7.64 (d, .7= 8.3 Hz, 1H), 7.35 (t, J= 4.9 Hz, 1H), 7.16 (d, J= 3.5 Hz, 1H), 7.13 (d, J=3.5Hz, 1H).

[0141] 34: 'H NMR (300 MHz, DMSO) 812.01 (s, 1H), 8.65 (s, 1H), 8.45 (d, J= 4.8 Hz, 1H), 7.78 - 7.67 (m, 3H), 7.64 - 7.58 (m, 1H), 7.57 (d, J=4.0Hz, 1H), 7.46 (d, J=3.7Hz, 2H), 7.28-7.17 (m, 1H), 7.12 (d, J= 8.1 Hz, 1H). HRMS (ESI+) : calcd [M-H]+358.0405; found 358.0478.

[0142] 37: 'HNMR (300 MHz, DMSO) 812.04(s, 1H), 8.64 (s, 1H), 8.47 - 8.43 (m, 1H), 7.94 (d, J = 2.0 Hz, 1H), 7.86 (d, J= 7.7 Hz, 1H), 7.74 (td, .7 = 7.8, 1.9 Hz, 1H), 7.68 (d,J=8.7Hz, 1H), 7.59 (d, J=7.5Hz, 1H), 7.56 (d, J= 4.7 Hz, 1H), 7.46 (d, J=3.8Hz, 1H), 7.22 (ddd, J= 7.5, 4.8, 1.0 Hz, 1H), 7.12 (d, J= 8.1 Hz, 1H).HRMS (ESI+) : calcd [M-H]+374.0110; found 374.0194.

[0143] 38: ’H NMR (300 MHz, DMSO) 812.03(s, 1H), 8.70 (d, J= 4.9 Hz, 2H), 8.64 (s, 1H), 7.95 (d, J= 1.9 Hz, 1H), 7.87 (d, J= 7.8 Hz, 1H), 7.69 (d, J=8.1 Hz, 1H), 7.60 (d, .7 = 7.8 Hz, 1H), 7.54 (d, J=3.8Hz, 1H), 7.43 (d, J=3.8Hz, 1H), 7.34 (t, J =4.9 Hz, 1H).

[0144] 41:XH NMR (300 MHz, DMSO) 811.99(s, 1H), 8.43 (ddd, J= 4.8, 1.9, 0.9 Hz, 1H), 8.34 (s, 1H), 7.77 - 7.70 (m, 2H), 7.68 (s, 1H), 7.64 - 7.55 (m, 1H), 7.53 - 7.41 (m, 1H), 7.23 (ddd, J= 7.5, 4.9, 1.0 Hz, 1H), 7.19 (d, J= 3.5 Hz, 1H), 7.15 (d, J= 3.5 Hz, 1H), 6.99 (dt,J=8.1, 1.0 Hz, 1H).

[0145] 42: 'H NMR (300 MHz, DMSO) 812.03(s, 1H), 8.44 (ddd, J= 4.9, 1.9, 0.9 Hz, 1H), 8.34 (s, 1H), 7.94 (d, J= 2.1 Hz, 1H), 7.86 (d, J =7.7 Hz, 1H), 7.78 - 7.71 (m, 1H), 7.68 (d, J= 2.1 Hz, 1H), 7.58 (t, J= 7.8 Hz, 1H), 7.24 (ddd, J= 7.5, 4.9, 1.0 Hz, 1H), 7.20 (d,J=3.5Hz, 1H), 7.16 (d, J= 3.5 Hz, 1H), 7.00 (dt, J=8.1, 1.0 Hz, 1H).

[0146] ' H NMR (300 MHz, DMSO) 812.03 (s, 1H), 8.64 (s, 1H), 8.45 (ddd, J =4.8, 2.0, 0.9 Hz, 1H), 8.07 (d,J = 1.9 Hz, 1H), 7.90 (dd, J=8.0, 1.5 Hz, 1H), 7.81 (ddd, J= 8.1, 2.0, 1.0 Hz, 1H), 7.74 (td, J= 7.8, 1.9 Hz, 1H), 7.57 (d, J = 3.8Hz, 1H), 7.52 (d,J=7.9Hz, 1H), 7.47 (t, J = 4.1 Hz, 1H), 7.22 (ddd, J= 7.5, 4.8, 1.0 Hz, 1H), 7.11 (dt, J= 8.1, 1.0 Hz, 1H). HRMS (ESI+) : calcd [M-H]+417.9605; found 417.9722.:TH NMR (300 MHz, DMSO) 812.14 (s, 1H), 8.66 (s, 1H), 8.46 (ddd, J= 4.9, 1.9, 0.9 Hz, 1H), 8.21 (d,J=9.4Hz, 2H), 7.99 (d, J=7.8Hz, 1H), 7.81 (d, J=7.8Hz, 1H), 7.77-7.70 (m, 1H), 7.58 (d, J= 3.8 Hz, 1H), 7.47 (d, J=3.8Hz, 1H), 7.22 (ddd, J= 7.3, 4.9, 1.1 Hz, 1H), 7.12 (dt, J= 8.1, 1.0 Hz, 1H). 'H NMR (300 MHz, DMSO)811.97 (s, 1H), 8.64 (s, 1H), 8.45 (d,J=4.8Hz, 1H), 7.73 (td, J= 7.8, 1.9 Hz, 1H), 7.68 (d,J = 8.1 Hz, 1H), 7.60 - 7.49 (m, 3H), 7.49 - 7.38 (m, 3H), 7.25 - 7.19 (m, 3H), 7.09 (t, J= 9.0 Hz, 3H).

[0149] 52: ’H NMR (300 MHz, DMSO) 811.91 (s, 1H), 8.67 (s, 1H), 8.51-8.41 (m, 1H), 7.74 (td, J= 7.8, 1.9 Hz, 1H), 7.55 (d, J=3.8 Hz, 1H), 7.51-7.39 (m, 4H), 7.22 (ddd, J= 7.5, 4.8, 1.0 Hz, 1H), 7.19-7.14(m, 1H), 7.11 (d, J = 8.1 Hz, 1H), 3.83 (s, 3H).

[0150] 55: 'HNMR (300 MHz, DMSO) 812.07(s, 1H), 8.70 (d, J= 4.8 Hz, 1H), 8.66 (s, 1H), 8.23 (s, 1H), 7.90 (td, J = 7.7, 1.8 Hz, 1H), 7.74 (t, J=7.6Hz, 1H), 7.68 (s, 1H), 7.62-7.55 (m, 1H), 7.48 (d, J= 10.1 Hz, 1H), 7.44 (dd, J =13, 5.0 Hz, 2H).

[0151] 56:1H NMR (300 MHz, DMSO) 811.99(s, 1H), 8.58 (ddd, J= 4.9, 1.9, 1.0 Hz, 1H), 8.52 (s, 1H), 8.26 (s, 1H), 7.89-7.82 (m, 1H), 7.77 (d, J=7.8Hz, 1H), 7.72 (d,J=9.9Hz, 1H), 7.65 - 7.58 (m, 1H), 7.54 (dt, J= 8.1, 1.0 Hz, 1H), 7.49 (d,J=7.9Hz, 1H), 131 (ddd, J= 7.5, 4.9, 1.1 Hz, 1H).

[0152] 58: ’H NMR (300 MHz, DMSO) 812.06(s, 1H), 8.97 (d, J= 1.3 Hz, 1H), 8.67 (s, 1H), 8.58 (d, J= 5.5 Hz, 1H), 7.76 (d, J= 7.8 Hz, 1H), 7.70 (d, J= 9.9 Hz, 1H), 7.62 (d, J= 4.0 Hz, 1H), 7.58 (d, J= 1.1 Hz, 1H), 7.54 (d, J= 3.8 Hz, 1H), 7.47 (t, J= 8.5 Hz, 1H), 7.19 (dd, J= 5.5, 1.3 Hz, 1H). HRMS (ESI+): calcd [M-H]+359.0358; found 359.0440

[0153] 59: 'H NMR (300 MHz, DMSO) 812.02 (s, 1H), 8.64 (s, 1H), 8.45 (d, J= 5.0 Hz, 1H), 7.92 (d, J= 8.3 Hz, 2H), 7.73 (t, J= 7.9 Hz, 1H), 7.62 (d, J= 8.3 Hz, 2H), 7.56 (d, J= 3.8 Hz, 1H), 7.46 (d, J= 3.7 Hz, 1H), 7.25 - 7.19 (m, 1H), 7.11 (d, J = 8.0 Hz, 1H).

[0154] 60:1H NMR (300 MHz, DMSO) 811.91 (s, 1H), 8.65 (s, 1H), 8.49 - 8.41 (m, 1H), 7.79 - 7.66 (m, 3H), 7.54 (d, J= 3.8 Hz, 1H), 7.45 (d, .7= 3.8 Hz, 1H), 7.41 (d, .7 = 4.5 Hz, 2H), 7.22 (ddd, J = 1.5, 4.8, 1.0 Hz, 1H), 7.11 (d, J = 8.1 Hz, 1H), 3.33 (s, 3H).

[0155] 63: ’H NMR (300 MHz, DMSO) 8 12.04(s, 1H), 8.64 (s, 1H), 7.75 (d, ,7 = 7.8 Hz, 1H), 7.70 (d, J= 9.6 Hz, 1H), 7.64 - 7.58 (m, 1H), 7.56 (s, 2H), 7.46 (t, J= 8.5 Hz, 1H), 7.27 (d, J = 1.2 Hz, 1H), 2.33 (d, J= 1.0 Hz, 3H).

[0156] 66:1H NMR (300 MHz, DMSO) 812.08 (s, 1H), 8.64 (s, 1H), 7.94 (s, 1H), 7.86 (d, J = 7.8 Hz, 1H), 7.79 (d, J = 3.4 Hz, 1H), 7.75 (d, J= 3.4 Hz, 1H), 7.69 (d, J= 8.2 Hz, 1H), 7.64 - 7.53 (m, 3H).

[0157] 12.02 (s, 1H), 8.59 (ddd, J= 4.9, 1.9, 0.9 Hz, 1H), 8.53 (s, 1H), 8.27 (s, 1H), 7.96 (s, 1H), 7.87 (ddd, J= 9.7, 6.2, 2.2 Hz, 2H), 7.70 (d, J= 8.0 Hz, 1H), 7.60 (d, J= 7.8 Hz, 1H), 7.55 (dt, J = 8.0, 1.0 Hz, 1H), 7.38 (ddd, J = 7.5, 4.9, 1.1 Hz, 1H).

[0158] 'H NMR (300 MHz, DMSO) 812.07 (s, 1H), 8.98 (d, J= 1.3 Hz, 1H), 8.68 (s, 1H), 8.59 (d, J= 5.5 Hz, 1H), 7.94 (d, J= 8.3 Hz, 2H), 7.64 (d, J= 3.0 Hz, 1H), 7.63 - 7.61 (m, 2H), 7.54 (d, J= 3.8 Hz, 1H), 7.20 (dd, J = 5.5, 1.3 Hz, 1H).

[0159] TH NMR (300 MHz, DMSO) 812.12 (s, 1H), 8.75 (s, 1H), 8.50 - 8.41 (m, 2H), 7.74 (ddd, J= 8.1, 7.5, 1.9 Hz, 1H), 7.51 (d, J =3.8 Hz, 1H), 7.45 (d, J= 3.8 Hz, 1H), 7.23 (ddd, J= 7.4, 4.8, 1.0 Hz, 1H), 7.12 (dt, J= 8.1, 1.0 Hz, 1H).

[0160] 1H NMR (300 MHz, DMSO) 811.83 (s, 1H), 8.62 (s, 1H), 8.46 (d, J= 4.9 Hz, 1H), 8.18 (s, 1H), 7.74 (td, J= 7.8, 1.9 Hz, 1H), 7.57 (d, .7= 2.6 Hz, 2H), 7.46 (d, .7= 3.8 Hz, 1H), 7.22 (dd, J = 1.3, 5.0 Hz, 1H), 7.11 (d, J= 8.1 Hz, 1H). HRMS (ESI+) : calcd [M-H]+379.9674; found 379.9758.

[0161] 'H NMR (300 MHz, DMSO) 812.07 (s, 1H), 8.69 (s, 1H), 8.16 (t, J = 1.9 Hz, 1H), 7.94 - 7.85 (m, 2H), 7.80 (d, J= 3.4 Hz, 1H), 7.78 (d, J= 1.6 Hz, 1H), 7.76 - 7.74 (m, 2H), 7.65 (d, J= 7.8 Hz, 1H), 7.62 - 7.54 (m, 2H), 7.52 (d, J= 7.7 Hz, 1H), 7.43 (t, J= 7.3 Hz, 1H).

[0162] : ' H NMR (300 MHz, DMSO) 8 12.06 (s,1H), 8.34 (s, 1H), 8.08 (t, J= 1.8 Hz, 1H), 7.94 - 7.87 (m, 1H), 7.82 (dt, J= 8.0, 1.3 Hz, 1H),7.78 (d, .7= 3.3 Hz, 1H), 7.74 (d, .7 = 3.4 Hz, 1H), 7.52 (t, J= 7.9 Hz, 1H), 7.31 (d, J= 3.5 Hz, 1H), 7.16 (d, J= 3.5 Hz, 1H).LG1 88:JH NMR (300 MHz,DMSO) 8 11.99 (s, 1H), 8.35 (s, 1H), 7.77 (d, J= 3.4 Hz, 1H), 7.74 (d, J= 3.3 Hz, 1H), 7.68 (d, .7= 7.7 Hz, 1H), 7.57 (d, J= 8.0 Hz, 1H), 7.53 (s, 1H), 7.44 (t, J = 7.8 Hz, 2H), 7.30 (d, J = 3.5 Hz, 1H), 7.27 - 7.16 (m, 2H), 7.14 (d, J= 3.5 Hz, 1H), 7.10 - 7.06 (m, 2H).

[0164] ’H NMR (300 MHz, DMSO) 812.09 (s, 1H), 8.64 (s, 1H), 8.22 (s, 1H), 7.74 (d, J= 7.8 Hz, 1H), 7.69 (d, J= 10.0 Hz, 1H), 7.64 (d, J= 1.2 Hz, 1H), 7.62 - 7.56 (m, 1H), 7.47 (t, J= 8.6 Hz, 1H), 2.46 (d, J= 1.0 Hz, 3H).HRMS (ESI+) : calcd [M-H]+379.0079; found 379.0163.

[0165] 22: 'H NMR (300 MHz, DMSO) 8 12.04(s, 1H), 8.98 (d, J= 1.3 Hz, 1H), 8.61 (d, J= 5.5 Hz, 1H), 8.37 (s, 1H), 7.76 (d, J= 7.8 Hz, 1H), 7.71 (d, J= 10.1 Hz, 1H), 7.61 (q, J= 7.5 Hz, 1H), 7.48 (t, J= 8.4 Hz, 1H), 7.29 (d, J= 3.5 Hz, 1H), 7.20 (d, .7= 3.5 Hz, 1H), 7.15 (dd, J= 5.5, 1.4 Hz, 1H).

[0166] 23:1H NMR (300 MHz, DMSO) 812.04 (s, 1H), 8.36 (s, 1H), 7.78 (d, J = 3.3 Hz, 1H), 7.74 (d, ,7 = 3.3 Hz, 1H), 7.73 - 7.67 (m, 2H), 7.61 (td, J = 8.0, 5.8 Hz, 1H), 7.48 (t, J= 8.9 Hz, 1H), 7.31 (d, .7= 3.5 Hz, 1H), 7.16 (d, J = 3.5 Hz, 1H). HRMS (ESI+) : calcd [M-H]+348.0198; found 348.0283.

[0167] 24: ’H NMR (300 MHz, DMSO) 8 11.94(s, 1H), 8.98 (d, J= 1.4 Hz, 1H), 8.61 (d, J= 5.5 Hz, 1H), 8.37 (s, 1H), 7.71 (s, 2H), 7.43 (d, J = 4.6 Hz, 2H), 7.28 (d, J= 3.5 Hz, 1H), 7.18 - 7.13 (m, 2H), 2.40 (s, 3H).

[0168] 25:1H NMR (300 MHz, DMSO) 8 11.95(s, 1H), 8.36 (s, 1H), 7.78 (s, 1H), 7.75 (s, 1H), 7.71 (s, 2H), 7.42 (d, J= 4.4 Hz, 2H), 7.31 (s, 1H), 7.13 (s, 1H), 2.40 (s, 3H).

[0169] 26: 'H NMR (300 MHz, DMSO) 812.06 (s, 1H), 8.98 (d, J= 1.3 Hz, 1H), 8.61 (d, J= 5.6 Hz, 1H), 8.36 (s, 1H), 8.08 (s, 1H), 7.91 (d, J= 7.8 Hz, 1H), 7.83 (d, J= 8.0 Hz, 1H), 7.52 (t, J= 7.9 Hz, 1H), 7.29 (d, J= 3.5 Hz, 1H), 7.19 (d, .7= 3.5 Hz, 1H), 7.15 (dd, J= 5.5, 1.4 Hz, 1H).

[0170] Compounds of the disclosure having a cyclopropyl ring moiety in L1can be prepared using the following exemplary reaction scheme.

[0171] Intermediate 3. A solution of triethyl phosphonoacetate (1.2 eqv.) was added dropwise to a solution of sodium-tert-butoxide (1.0 eqv.) in dry THF (0.9M) at -5 °C over a period of 10 mins and stirred at 0 °C for 30 mins. Then a solution of 2 (1.0 eqv.) in dry THF wasadded dropwise at 0 °C over a period of 15 mins and stirred at RT for 12 h. After completion, the reaction mixture was poured into ice water and extracted with EtOAc (2 x). The combined extracts were washed with sat NaHCCh solution, water, and brine, dried over anhydrous Na2SO4, fdtered, and evaporated to afford crude 3. It was directly utilized for the next step without purification.

[0172] Intermediate 4. Trimethyl sulfoxonium iodide (5.0 eqv) was added portion wise to a suspension of NaH (5.0 eqv.) in dry DMSO (0.7 M) at RT over a period of 20 min. and stirred for 1 h t the formation of a clear solution. A solution of 3 (1.0 eqv.) in dry DMSO (IM) was added dropwise and stirred at RT for 12 hrs. After completion, the reaction mixture was poured into ice water, extracted with EtOAc. Combined organic extracts were washed with ice water, and brine, dried over anhydrous Na2SO4 , filtered, and evaporated to afford crude 4. It was directly utilized for the next step without purification.

[0173] Intermediate 5. An aqueous solution of NaOH (2.0 eqv) was added to a solution of 4 (1.0 eqv.) in Methanol at 0 °C and stirred at RT for 16 h. After completion, the solvent was evaporated, the residue was diluted with water, neutralized with Acetic acid and extracted with EtOAc (4 x). The combined extracts were washed with water, and brine, dried over anhydrous Na2SO4 , filtered, and evaporated to afford 5. It was directly utilized for the next step without purification.

[0174] Intermediate 6. Ethyl chloroformate (1.3 eqv.) was added to a solution of 5 (1.0 eqv) and triethyl amine (1.5 eqv.) in acetone (0.7 M) at -20 °C and stirred for 1 h, then a solution ofNaN.3 (1.5 eqv.) in water was added and stirred at RT for 16 hours. After completion, the solvent was evaporated, the residue was dissolved in EA, washed with water (2 x ), and brine, dried over anhydrous Na2SC>4, filtered, and evaporated to afford 6. It was directly utilized for the next step without purification.

[0175] Intermediate 7. A solution of 6 (8.4 g, 44.66 mmol) in tert-butanol was heated at 90 °C for 16 h. After completion, the solvent was evaporated and crude residue was purified by column chromatography (SiCE) using EtOAc: Petroleum ether (25: 75) to afford colorless liquid.

[0176] Hydrochloride HC1 in Dioxane was added to a solution of the previous product in 1,4-di oxane at 0 °C and stirred at RT for 12 h. After completion, the solvent was evaporated andthe residue was triturated with diethyl ether) followed by hexane to get hydrochloride salt. It was basified with sat. NaHCOs to get the free base, 7. It was directly utilized for the next step without purification.

[0177] Compounds SD_3_139 and SD_3_142. To a dry DCM (0.2M) solution of 7, were added EDCI (1.4 eqv.), the desired benzoic acids (1.0 eqv.), DIEA( 1.4 eqv.) at RT. The mixture was stirred overnight. Ater completion of the reaction, it was washed with IN HC1, sat. NaHCCh and brine and DCM. The combined organic layer was dried over anhydrous Na2SC>4 and evaporated under reduced pressure. The crude was purified by column chromatography to afford the desired products.

[0178] Using this general synthetic scheme, the following compounds were prepared:

[0179] 139, yellow solid, 20% yield, ‘HNMR (300MHz, CDCI3) 8 8.43 (ddd, J = 4.8, 1.9, 0.9 Hz, 1H), 7.78 (t, J = 2.0 Hz, 1H), 7.67 (dt, J = 7.6, 1.4 Hz, 1H), 7.56 - 7.48 (m, 2H), 7.44 - 7.38 (m, 1H), 7.22 (d, J = 3.6 Hz, 1H), 7.03 (ddd, J = 7.5, 4.9, 1.0 Hz, 1H), 6.97 - 6.92 (m, 2H), 6.40 (s, 1H), 3.16-3.19 (m, 1H), 2.41- 2.34 (m, 1H), 1.49 - 1.38 (m, 2H) ppm. HRMS (ESI+): cacld [M+H]+387.0314, found 387.0346. yellow solid, 28% yield, 'H NMR (300MHz, CDC13) 8 8.42 (ddd, J = 4.9, 1.9, 0.9 Hz, 1H), 7.59 - 7.48 (m, 3H), 7.47 - 7.38 (m, 1H), 7.24 (ddd, J = 8.3, 2.6, 1.1 Hz, 1H), 7.20 (d, J = 3.6 Hz, 1H), 7.02 (ddd, J = 7.5, 4.9, 1.1 Hz, 1H), 6.97 - 6.90 (m, 2H), 6.56 (s, 1H), 3.16 (ddd, J = 8.1, 6.4, 3.1 Hz, 1H), 2.42 - 2.32 (m, 1H), 1.45- 1.40 (m, 2H) ppm. HRMS (EST): cacld [M-H]’ 369.0610; found 369.0514.

[0181] Compounds of the disclosure where L1is a moiety of the formula:can be prepared using the following general synthetic strategy.

[0182] SD_3_120 and SD_3_149. Intermediate 8 and compounds SD_3_120 andSD_3_149 were synthesized following the procedures of intermediate 5 and SD_3_122synthesis.

[0183] SD 3 120, yellow soild, 65% yield, 'H NMR (300MHz, CDCh) 8 8.43 (ddd, J = 4.9, 1.9, 0.9 Hz, 1H), 8.33 (s, 1H), 7.83 (s, H), 7.78 (s, 1H), 7.59 (ddd, J = 8.1, 7.5, 1.9 Hz, 2H), 7.47 (ddd, J = 8.2, 2.1, 1.0 Hz, 1H), 7.28 - 7.25 (m, 2H), 7.24 - 7.17 (m, 1H), 7.13 - 7.06 (m, 3H), 6.38 (dd, J = 15.1, 0.5 Hz, 1H) ppm.

[0184] yellow solid, 55% yield, 'H NMR (300MHz, CDCI3) 8 8.45 (ddd, J = 4.9, 1.9, 0.9 Hz, 1H), 7.96 (s, IH), 7.83 (dd, J = 15.1, 0.7 Hz, 1H), 7.64 (s, 1H), 7.58 (ddd, J = 8.1, 7.5, 1.9 Hz, 2H), 7.30 (d, J = 2.6 Hz, 1H), 7.27 - 7.21 (m, 2H), 7.13 - 7.05 (m, 2H), 6.88 - 6.80 (m, 1H), 6.36 (d, J = 15.2 Hz, 1H) ppm. HRMS (ESI ): calcd [M-H]- 355.0453, found 355.0387.

[0185] Compounds of the disclosure where L1is a moiety of the formula:can be prepared using the following general synthetic strategy.

[0186] Intermediate 9. To a solution of CrCh (15.0 eq.) in THF was added CHI3 (5.0 eq.) at 0°C under argon, and the mixture was stirred the same temperature for 15 min. When the color of the reaction mixture turned to dark red, substrate 3 (1.0 eqv.) in THF at 0°C, was added at the 0°C, and stirred at the same temperature for Ih. Then the mixture was stirred at room temperature overnight. The reaction was diluted by water and quenched by sat. NaHCCh and extracted with DCM (4 x). The combined organic layer was dried over anhydrous Na2SO4 and evaporated under reduced pressure. The crude was purified by column chromatography to afford pure 9, 85% yield.TH NMR (300 MHz, CDCI3) 8 8.42 (ddd, J = 4.9, 1.9, 0.9 Hz, IH), 7.55 -7.49 (tn, 1H), 7.45 (d, J= 15.2 Hz, 1H), 7.22 (d, J = 3.6 Hz, 1H), 7.03 (ddd, J = 7.5, 4.9, 1.0 Hz, 1H), 6.99 - 6.92 (m, 2H), 6.73 (dd, J = 14.8, 0.5 Hz, 1H) ppm.

[0187] Compounds SD 3 146 and SD 3 150. To a dry DMS solution of 9, were added desired benzamides (2.0 eqv.), Cui (1.5 eqv.), A7, TV ’-dimethyl diaminoethane (DMDA) (3.0 eqv.), and K2CO3 (6.0 eqv.) at room temperature and stirred for overnight. After the completion of the reaction, it was diluted with EtOAc and filtered through a pad of cellite. The filtrate was washed with EtOAc water. The organic layer was dried over anhydrous Na?SO4 before being concentrated under vaccuo. The crude was purified by column chromatography to get desired enamides.

[0188] SD 3 146, yellow solid, 65% yield, 'HNMR(300 MHz, CDC13) 5 9.20 (d, J = 10.4 Hz, 1H), 8.40 (ddd, J = 4.9, 1.9, 0.9 Hz, 1H), 8.02 (s, 1H), 7.87 (t, J = 1.9 Hz, 1H), 7.76 (dt, J = 7.7, 1.4 Hz, 1H), 7.63 - 7.54 (m, 1H), 7.54 - 7.45 (m, 2H), 7.36 (t, J = 7.9 Hz, 1H), 7.15 (d, J = 3.7 Hz, 1H), 7.08 - 6.96 (m, 2H), 6.87 (d, J = 3.8 Hz, 1H), 6.48 (dd, J = 14.5, 0.6 Hz, 1H) ppm. HRMS(ESE): calcd. [M+H]+373.0158; found 373.0227.

[0189] 150, yellow solid, 67% yield, 'H NMR(300 MHz, CDCI3) 5 9.43 (d, J = 10.3 Hz, 1H), 8.41 (ddd, J = 4.9, 1.9, 0.9 Hz, 1H), 7.72 (ddd, J= 7.7, 1.6, 1.0 Hz, 1H), 7.69 - 7.55 (m, 2H), 7.51 (ddd, J = 8.1, 7.5, 1.9 Hz, 1H), 7.47 - 7.37 (m, 1H), 7.23 (ddd, J = 8.3, 2.6, 1.0 Hz, 1H), 7.20 (s, 1H), 7.02 (ddd, J = 7.5, 4.9, 1.0 Hz, 1H), 6.97 (dt, J = 8.1, 1.0 Hz, 1H), 6.91 (dt, J = 3.7, 0.5 Hz, 1H), 6.53 (dd, J = 14.5, 0.7 Hz, 1H). HRMS (EST): calcd. [M-H]’ 355.0453; found 355.0387.

[0190] (pyridin-2-yl)sulfanyl]thiophen-2-yl}ethenyl]benzamide (3c), SD_4_236. Method B, yield 48%. Rf = 0.5, yellow solid, 'HNMR(300 MHz, CDCI3) <5 8.43 (ddd, J= 4.8, 1.9, 0.9 Hz, 1H), 8.23 (d, J= 10.7 Hz, 1H), 7.90 - 7.81 (m, 2H), 7.69 - 7.61 (m, 1H), 7.61 - 7.53 (m, 2H), 7.52 - 7.45 (m, 2H), 7.20 (d, J= 3.7 Hz, 1H), 7.08 - 7.03 (m, 1H), 7.02 - 6.99 (m,lH), 6.92 (dd, J= 3.8, 0.6 Hz, 1H), 6.44 (dd, J= 14.5, 0.7 Hz, 1H) ppm.yl)sulfanyl]thiophen-2-yl}ethenyl]benzamide (3d), SD_4_18. Method A, 28%, Rf = 0.4, yellow solid,JH NMR (300 MHz, CDC13) 3 8.98 (s, 1H), 8.46 (s, 1H), 8.39 (d, J = 11.3 Hz, 1H), 7.94 - 7.87 (m, 1H), 7.72 (ddd, J= 7.7, 1.8, 1.1 Hz, 1H), 7.57 (ddd, J= 8.0, 2.1, 1.1 Hz, 1H), 7.51 - 7.42 (tn, 1H), 7.38 (d, J= 3.8 Hz, 1H), 7.23 (dd, J= 11.2, 9.5 Hz, 1H), 7.10 (dt, J= 3.8, 0.6 Hz, 1H), 6.99 (dd, J= 5.5, 1.2 Hz, 1H), 6.08 (dt, J= 9.4, 0.7 Hz, 1H) ppm.yl)sulfanyl]thiophen-2-yl}ethenyl]-3-(trifluoromethyl)benzamide(3e), SD_3_199. Method A, yield 37%, Rf = 0.5, red oil, 'H NMR (300 MHz, CDCI3) 3 9.45 (d, J = 10.3 Hz, 1H), 8.42 (ddd, J= 4.9, 1.9, 0.9 Hz, 1H), 8.20 (dq, J= 1.8, 0.9 Hz, 1H), 8.18 - 8.12 (m, 1H), 7.80 (dddt, J= 7.8, 1.9, 1.3, 0.6 Hz, 1H), 7.70 - 7.58 (m, 2H), 7.53 (ddd, J = 8.1, 7.4, 1.9 Hz, 1H), 7.20 (d, J = 3.6 Hz, 1H), 7.07 - 7.02 (m, 1H), 7.02 - 6.96 (m, 1H), 6.93 (dt, J = 3.7, 0.5 Hz, 1H), 6.55 (dd, J = 14.5, 0.7 Hz, 1H) ppm.

[0193] (dimethylamino)ethyl]sulfanyl}thiophen-2-yl)ethenyl]benzamide (3f), SD_4_56 / AL_1_78.Method A, yield 64%, Rf = 0.3, red oil, ’HNMR (300 MHz, CDCI3) d 7.91 (d, J= 10.9 Hz, 1H), 7.85 (t, J= 1.9 Hz, 1H), 7.73 (dt, J = 1.6, 1.4 Hz, 1H), 7.60 - 7.50 (m, 2H), 7.49 - 7.38 (m, 1H), 7.01 (d, .7= 3.7 Hz, 1H), 6.80 (d, J= 3.7 Hz, 1H), 6.47 - 6.31 (m, 1H), 3.02 - 2.94 (m, 2H), 2.75 - 2.66 (m, 2H), 2.38 (s, 6H) ppm.

[0194] yl)sulfanyl]thiophen-2-yl}ethenyl]benzamide (3g), SD_3_232. Method A, yield 41%, Rf = 0.6, yellow solid, 'H NMR (300 MHz, CDCI3) 3 8.14 (d, J= 10.6 Hz, 1H), 7.86 (t, J= 1.9 Hz, 1H), 7.74 (dt, J= 7.8, 1.4 Hz, 1H), 7.68 (d, J= 3.4 Hz, 1H), 7.67 - 7.60 (m, 1H), 7.56 (ddd, J= 8.0, 2.1, 1.1 Hz, 1H), 7.45 (t, J = 7.8 Hz, 1H), 7.30 (d, J= 3.7 Hz, 1H), 7.24 (d, J= 3.4 Hz, 1H), 6.94 (d, J= 3.8 Hz, 1H), 6.45 (dd, J= 14.5, 0.7 Hz, 1H) ppm.

[0195] (dimethylamino)ethyl] sulfanyl }thiophen-2-yl)ethenyl]-3 -fluorobenzamide (3h), AL_1_75.Method A, yield 32%. Rf = 0.3, yellow solid, 'H NMR (300 MHz, CDCh) 3 7.98 (d, J= 10.6 Hz, 1H), 7.86 (t, J= 1.9 Hz, 1H), 7.74 (d, J= 7.6 Hz, 1H), 7.60 - 7.50 (m, 2H), 7.50 - 7.39 (m, 1H), 7.00 (d, J= 3.7 Hz, 1H), 6.79 (d, J= 3.7 Hz, 1H), 6.42 (d, J= 14.4 Hz, 1H), 3.05 - 2.88 (m, 2H), 2.69 (t, J= 7.4 Hz, 2H), 2.37 (s, 6H) ppm.

[0196] yl)methyl]sulfanyl }furan-2-yl)ethenyl]benzamide (3i), AL_l_101. Method A, yield 38%, Rf = 0.3, yellow solid, 'H NMR (300 MHz, CDCh) 3 7.86 (t, J= 1.9 Hz, 1H), 7.77 - 7.70 (m, 1H), 7.65 (dd, J= 11.8, 2.6 Hz, 1H), 7.56 (ddd, J= 8.0, 2.1, 1.1 Hz, 1H), 7.49 - 7.42 (m, 1H), 6.51 (d, J= 3.2 Hz, 1H), 6.19 (d, J= 3.3 Hz, 1H), 6.15 (d, J= 14.4 Hz, 1H), 3.02 - 2.91 (m, mH), 2.67 (t, J = 7.2 Hz, 2H), 2.36 (s, 6H) ppm.

[0197] yl)methyl] sulfanyl }thiophen-2-yl)ethenyl]benzamide (3j), AL_1_98. Method A, yield 54%, Rf = 0.5, yellow oil, 'H NMR (300 MHz, CDC13) b 8.62 - 8.53 (m, 1H), 7.89 - 7.79 (m, 2H), 7.71 (ddd, J= 7.6, 1.7, 1.1 Hz, 1H), 7.64 (td, J = 1.1, 1.8 Hz, 1H), 7.59 - 7.52 (m, 1H), 7.51 - 7.39 (m, 2H), 7.19 (ddd, J= 7.5, 3.7, 1.4 Hz, 2H), 6.83 (d, J= 3.7 Hz, 1H), 6.73 (dt, J= 3.7, 0.5 Hz, 1H), 6.35 (dd, J= 14.4, 0.7 Hz, 1H), 4.12 (s, 2H) ppm.

[0198] yl)sulfanyl]furan-2-yl}ethenyl]benzamide (3k), AL_1_94,. Method A, yield 39%, Rf = 0.5, yellow oil,rH NMR (300 MHz, CDC13) <5 8.45 (ddd, J= 4.9, 1.9, 0.9 Hz, 1H), 7.91 (d, J= 10.9 Hz, 1H), 7.85 - 7.82 (m, 1H), 7.76 - 7.68 (m, 2H), 7.61 - 7.49 (m, 2H), 7.45 (ddd, J= 8.1, 7.7, 0.5 Hz, 1H), 7.07 (ddd, J= 7.5, 4.9, 1.0 Hz, 1H), 6.95 (dt, J = 8.1, 1.0 Hz, 1H), 6.83 (d, J= 3.3 Hz, 1H), 6.36 (dt, J= 3.3, 0.5 Hz, 1H), 6.20 (d, J= 14.4 Hz, 1H) ppm.

[0199] yl)sulfanyl]furan-2-yl}ethenyl]benzamide (31), AL_l_105, Method A, yield 47%, Rf = 0.6, yellow solid, 'H NMR (300 MHz, CDC13) <57.94 (d, J= 11.0 Hz, 1H), 7.88 - 7.81 (m, 1H), 7.78 - 7.72 (m, 1H), 7.72 - 7.67 (m, 2H), 7.56 (ddd, J= 8.0, 2.1, 1.1 Hz, 1H), 7.45 (ddd, J= 8.1, 7.7, 0.5 Hz, 1H), 7.26 (d, J= 3.4 Hz, 1H), 6.88 (d, J= 3.3 Hz, 1H), 6.38 - 6.30 (m, 1H), 6.19 (d, J = 14.5 Hz, 1H) ppm.DISCUSSION

[0200] EYA2 Tyr Phosphatase plays important roles in Medulloblastoma progression'.Medulloblastoma (MB) is a heterogeneous disease that was traditionally divided into four distinct molecular groups (WNT, SHH, Groups 3 and 4). Group 3 MB demonstrate the highestrisk features, and patients with these subtypes exhibit the poorest overall 5-year survival (50%).Group 3 tumors express high levels of MYC.

[0201] The present inventors have discovered that EYA2 is highly expressed in Group 3 Medulloblastoma (MB). A differential gene expression dataset was generated containing SHH and Group 3 MB as well as control, non-cancerous brain tissue obtained from epileptic patients were analyzed. This analysis identified EYA2 as significantly increased in high MYC expressing Group 3 MB. The expression of all four EYAs was then analyzed in a publicly available batch- normalized gene expression dataset (GSE124814) from 1350 MB patient tumors and 291 control brain tissue samples from epileptic patients. The present inventors have discovered that in this larger dataset, EYA2 was significantly overexpressed in Group 3 MB samples compared to control brain tissue and other MB subtypes (FIG. 1A). EYA2 mA MYC expression were also analyzed in multiple MB human cell lines derived from Group 3 (D425, D458, D283) and SHH tumors (DAOY, ONS-76) to determine whether commonly used cell line models would mimic what was observed in human MB. Group 3 cell lines known to exhibit MYC amplification (D425, D458) were chosen, as well as a non-MYC amplified, Group 3 / 4 cell line (D283) and confirmed that in these models, EYA2 and MYC were both elevated (at the mRNA and protein levels, shown only for EYA2 for mRNA, but for both at protein level) in the Group 3 and Group 3 / 4 MB cell lines compared to the SHH lines (FIGS. 1B-C).

[0202] Control (shNC) and EYA2 knockdown cell lines (KD1, KD2) in both D425 and D458 cell lines were generated (FIGS. 2A-2C). EYA2 KD in D425 and D458 cells consistently led to decreased MYC mRNA expression (FIGS. 2A and 2B), indicating that EYA2 transcriptionally regulates MYC in Group 3 MB, similar to its function in development. To determine if EYA2 post-translationally regulates MYC in Group 3 MB via its ability to dephosphorylate pT58, likely through its association with PP2A, levels of EYA2, MYC, and MYC-pT58 were assessed by Western blot analysis. Results showed that EYA2 KD leads to a decrease in MYC protein levels, and that a greater proportion of the remaining MYC protein is phosphorylated on T58 in EYA2 KD cells when compared to shNC cells (FIGS. 2C and 2D). Because pT58-MYC marks the protein for degradation, this alteration of MYC phosphorylation is likely to be partially responsible for the decreased levels of total MYC protein. These datasupport the hypothesis that in addition to regulating MYC mRNA, EYA2 regulates MYC stability in Group 3 MB by promoting pT58 dephosphorylation.

[0203] MYC-associated gene expression is globally altered by EYA2 KD. Briefly, RNA-sequencing was performed on shNC and EYA2 KD (KD1 and KD2, data combined) D425 and D458 cells in biological triplicate and differential gene expression analysis and pathway enrichment using gene set enrichment analysis (GSEA) was performed. In both cell lines, MYC target gene sets were among the most significantly enriched gene sets in shNC vs. EYA2 KD lines (FIG. 3). Additional pathways altered with EYA2 KD include E2F target and G2 / M checkpoint pathways, both associated with MYC driven proliferation. Thus, data showed that EYA2 regulates MYC levels and transcriptional output specifically in Group 3 MB, suggesting that it has a significant role for regulating survival and / growth for this MB subtype, given its dependence on MYC.

[0204] The inventors then showed that KD of EYA2 in D425 and D458 cells dramatically reduced growth, as determined by a CellTiterGlo assay (FIG. 4). For CeilTiter-Glo assays, 1500 cells incubated with various compounds were plated in white 96-well tissue culture plates. Cells were treated for 72 hours with concentrations of 128,64,32, I6,8,4,2,l,0.5uM and DMSO only. 100 pl of CellTiter-Glo 2.0® Reagent (Promega Corporation, G9243) was added to each well and incubated at room temperature for 10 mins and luminescence was measured. IC50 calculated in Graphpad Prism vlO by Nonlinear regression analysis. The growth defects of EYA2 KD were so profound in vitro that it was not possible to maintain stable KD lines, and thus KD lines were continually re-established for assays.

[0205] Control (shNC) or EYA2 KD D458 were intracranially transplanted into brains of NSG mice. Mice that received D458 EYA2 KD cells exhibited reduced tumor growth (FIG. 5) and had a significant survival advantage (data not shown) compared to mice that received D458 shNC tumors. However, all tumors eventually grew out, and the mice receiving D458 EYA2 KD cells ultimately succumbed to the disease (data not shown). IHC analysis of the D458 end-point tumors for EYA2 and MYC protein revealed no difference in EYA2 or MYC protein levels between shNC and EYA2 KD tumors, indicating that the cells capable of growing out had escaped EYA2 KD and the associated decrease in MYC levels (data not shown). Taken together, these data show that an EYA2 / MYC axis is critical for the growth and survival of Group 3 MB.

[0206] A Pearson correlation was then performed between EYA2 expression and all other genes in 763 MB patient samples (GSE85217) and used the ranked gene list as an input for GSEA (FIG. 6A). Data showed that MYC target gene-sets are highly positively correlated with EYA2 expression in patient MB (FIGS. 6A and 6B). To determine how EYA2 and MYC expression correlate at the individual tumor level, MYC and EYA2 gene expression were plotted as z-scores and stratified Group 3 tumors compared to other MB subgroups. This analysis demonstrates that EYA2 and MYC are more highly correlated in Group 3 MB than in the other subtypes of MB (FIG. 6C). The available patient survival data was also used to stratify survival according to EYA2 and MYC expression among all MB subtypes. It was found that patients with higher EYA2 and MYC expression had worse survival outcomes than patients with lower EYA2 and MYC expression (FIG. 7A-C). In Group 3 MB specifically, it was found that patients with high EYA2 levels had worse survival outcomes than those with lower EYA2 levels (FIGS. 7D- F). When stratified by MYC expression, there was no significant difference in patient outcome, presumably due to the high dependence and overall high expression of MYC in these tumors (FIG. 7E). Patients with both low EYA2 / low MYC had a modestly improved survival outcome, which was not significant, likely due to an underpowered patient number (FIG. 7F). Overall, these patient data corroborate data obtained from cell-based findings, demonstrating that EYA2 strongly correlates with MYC in Group 3 MB, that it is associated with MYC gene expression signatures, and that the presence of EYA2 and MYC together correlate with worsened survival in MB. Thus, confirming that targeting EYA2 in Group 3 MBs is a sound therapeutic strategy to decrease MYC levels and activity, inhibiting the progression of these devastating tumors.

[0207] Studies have shown that EYA2 Tyr Phosphatase inhibitors are also effective in vivo in treating other tumors, including, but not limited to, glioblastoma (Zhang, G. J. Exp. Med. 2021 Vol. 218 No. 11 e20202669, doi.org / 10.1084 / jem.20202669) and breast cancer.Compounds disclosed herein can be used in treating numerous tumor types. Due to the paucity of expression of EYA2 post-developmentally in normal differentiated tissues, inhibiting expression of EYA2 selectively inhibits the tumor while sparing normal tissue. Such inhibitors are sorely needed in all cancer types but are particularly relevant in pediatric tumors where children treated with standard therapies experience devastating, life-long, morbidities.

[0208] Inhibition ofEYA2 Tyr Phosphatase in Medulloblastoma. In collaboration with the NIH Chemical Genomics Center (NCGC), a high throughput screen of -300,000 compounds using purified EYA2 ED was carried out. The screen identified a class of benzohydrazide compounds represented by MLS000544460 (abbreviated as 4460, see V Krueger, A.B., et al., J Biomol Screen, 2013, 18, 85-96. Krueger, A.B., et al. Allosteric inhibitors of the Eya2 phosphatase are selective and inhibit Eya2- mediated cell migration. J Biol Chem 289, 16349- 16361 (2014). (FIG. 10A, note that the IC50 numbers in this figure are different from those reported in our published papers because the assay condition was modified to be more sensitive to the potency of the analogs to facilitate compound optimization). These screening hits were also confirmed in a malachite green-based secondary Phosphatase assay using the phosphorylated histone H2AX (p-H2AX) peptide, a known target of EYA2 Tyr Phosphatase activity, as the enzyme substrate. An analog, NCGC00249987 (abbreviated as 9987) has slightly better potency and higher aqueous solubility than 4460 (51 vs 8.9 pg / mL) and is used for subsequent cell culture and animal experiments. This class of compounds also inhibits full-length EYA2.

[0209] The specificity of the screening hits was also tested against EYAs 1,3,4 and several other cellular Phosphatases including PTP1B (a Tyr Phosphatase), PPM1 A (a Mg2+- dependent Ser / Thr Phosphatase), and Scpl (a HAD family Ser / Thr Phosphatase), in OMFP- based Phosphatase assays. These compounds showed surprising selectivity. These compounds not only do not inhibit other cellular Phosphatases, and also do not inhibit the highly homologous EYA family members (FIG. 10B).

[0210] Enzyme kinetics studies indicated that these compounds are non-competitive inhibitors, consistent with mutagenesis studies demonstrating that mutations of multiple residues around the active site have no effect on compound inhibition. Co-crystal structure of EYA2 in complex with 9987 was determined, revealing that it binds to a compound-induced pocket distant from the active site. Compound 9987 binding to this allosteric pocket leads to a conformational change of the active site that is unfavorable for Mg2+binding, thereby inhibiting the Tyr Phosphatase activity of EYA2 (FIGS. HA and 11B).

[0211] A variety of compounds of the class disclosed herein were synthesized and evaluated in the EYA2 phosphatase assays in order to understand its SARs. Co-crystal structuresof EYA2 ED and several potent inhibitors showed that three aromatic fragments can tolerate modifications, and several key interactions between the small molecules and EYA2 ED (e.g., H- bondings, π / π stacking interactions, and hydrophobic interactions) were identified. These provided valuable insight for further optimization.

[0212] Compound 9987 was shown to inhibit Group 3 MB both in vitro and in vivo. Treatment of D458 cells with 9987 led to reduced MYC mRNA (FIG. 12A) and protein (FIG. 12B) and to increased pT58-MYC relative to total MYC (FIG. 12B). In addition, D458 cell growth in vitro was significantly inhibited after treatment with 9987 (Figure 12C). Inhibition of growth of additionally tested Group 3 MB lines (D341) was also observed, but not SHH MB lines (DAOY) (data not shown). To determine whether 9987 could inhibit growth of Group 3 MB lines in vivo, first PK analysis was conducted to assess whether this compound could have utility in the clinically relevant setting (the brain). Briefly, pharmacokinetic (PK) analysis was conducted using mass spectrometry (MS) of plasma and brain tissue from mice treated orally with 25 mg / kg 9987. It was found that up to 24 hours, brain tissue contained 9987 at levels comparable to that seen in plasma, demonstrating that 9987 is able to cross the blood brain barrier (BBB) (FIG. 12D). Another experiment was conducted to test whether EYA2 Tyr Phosphatase inhibition would reduce Group 3 MB growth when cells were implanted intracranially. Data in FIG. 12 show that 9987, when delivered via oral gavage once daily at 25 mg / kg, attenuates intracranial Group 3 MB growth, prolonging survival (FIGS. 12E-F).Furthermore, end-point tumors demonstrate a significant reduction in MYC protein, as measured by IHC, when mice are treated with 9987 (FIGS. 12G-H). This finding is the first to demonstrate that EYA2 tyrosine phosphatase inhibitors disclosed herein can cross the BBB, decrease MYC expression, and inhibit the growth of Group 3 MB.

[0213] The above results showed that 9987 and its analogs provide an excellent starting point as a lead compound. Iterative structure-based design, parallel synthesis, and comprehensive in vitro characterizations are carried out to synthesize and identify other EYA2 tyrosine phosphatase inhibitors. Many series of 9987 analogs are designed (see Scheme I above). Cheminformatic software Schrodinger is used to compute each analog’s molecular descriptors, such as molecular weight (MW), clogP, polar surface area (PSA), and H-bond donors and acceptors. The analogs with attributes of potential successful CNS drugs (e.g., MW <450, clogP<5, and PSA <70 A2) are prioritized for synthetic efforts. Once synthesized, these analogs are evaluated for their activity in EYA2 Phosphatase inhibitory assay using established protocol. Krueger, A.B., et al., J Biol Chem 289, 16349-16361 (2014). Compounds showing improved activity are tested against additional Phosphatases, such as PPM1A, PTP1B, and Scpl, to determine their selectivity. Compounds showing selectivity of >100 fold against other Phosphatases are then tested in a panel of Group 3 MB and SHH cell lines with varying levels of EYA2, (SHH MB lines expressing low EYA2 vs Group 3 MB lines- see FIG. 1). Compounds with improved cellular activity in Group 3 MB cells with low general toxicity (thus not inhibiting low EYA2 / MYC lines such as DAOY and ONS-76) are analyzed for their in vitro drug-like properties including aqueous solubility, membrane permeability, and metabolic stability. Compounds with improved properties are identified as the new leads for further optimization.

[0214] New analogs were synthesized using Scheme I above, and some have shown similar or slightly better activity in in vitro phosphatase assays and significant improvement (4-7 fold) in cellular activity over 9987. Some representative results are shown in Table 2. For example, compared to 9987 (IC50 = 0.39 pM), LG1-34 and 37 inhibit EYA2 with IC50S of 0.31 and 0.16 pM, respectively (FIG. 13A), and are >100 fold more selective against other phosphatases tested, such as PPM1A, PTP1B, and Scpl (not shown). Most importantly, their half growth inhibition concentrationsare 8.9 and 5.0 pM, respectively, in the D458 Group 3 MB cell line, whereas the 9987 GI50 is 36 mM (FIG. 13B).Table 2: ResultsTable 2: Results

[0215] 9987 is further optimized by designing, synthesizing, and evaluating the activity and selectivity of structurally closely related scaffolds (e.g., thiophene, benzene, or pyridine instead of furan, and enamide or cyclopropyl amide instead of benzohydrazide, Scheme I above) to identify the optimal structural motifs, and to further improve the potency, selectivity, mammalian toxicity, and DMPK properties by exploring the substituents of the best core structures. To maximize the effectiveness and efficiency of chemistry efforts, activity, selectivity, toxicity, and drug-like properties of lead compounds are simultaneously optimized.

[0216] The designed compounds are synthesized using the state-of-the-art parallel synthesis and purification techniques such as utilization of a microwave reactor and HPLCpurification systems, which have been well established. All new compounds are characterized via HPLC-MS and 'H NMR and produced with purity of 95% or higher by both methods and in an average amount greater than 30 mg, which is sufficient for profiling in all biochemical and cell-based assays, as well as initial in vitro DMPK studies. Compounds demonstrating low pM or nM range activity, selectivity, and drug-like properties are re-synthesized in larger scale (100 mg) for PK studies in mice.

[0217] Synthetic analogues are evaluated for their inhibitory activity against EYA2 as described previously. To evaluate their selectivity, whether they inhibit other phosphatases, such as EYA1, EYA3, EYA4, PPM1A, PTP1B, and Sept are also tested using established protocols. Highly active compounds that show IC50 < 1 pM against EYA2 with good selectivity against other Phosphatases (i.e., IC50S >50 pM for other Phosphatases) are further analyzed for their cellular activity. The crystal structure of selected analogs are also determined to facilitate compound optimization.

[0218] Screening compounds of the invention for effect on Eya2 Tyr Phosphatase

[0219] The ED of Eya2 was used to screen compounds for phosphatase inhibition in a fluorescence-based Phosphatase Assay

[0220] Regular Screening Conditions. Eya2 Eya domain (ED) phosphatase activity was measured in black, half-volume 96-well plates (Greiner Bio-One) using the substrate 3-O- methylfluorescein Phosphate (OMFP, Sigma). 100 pL reactions were carried out in triplicate using 25 mM MES, pH 6.5, 50 mM NaCl, 5 mM MgCh, and 5 mM DTT. Eya2 ED was added to buffer, followed by the addition of small molecule compounds (serially diluted in DMSO to eight concentrations) or DMSO as control, in 50 pL total. Reactions were started by the addition of 50 pL OMFP, yielding 150 nM Eya2 ED and 25 pM OMFP at final reaction conditions. Reactions were allowed to proceed at room temperature for 1 hour, then were quenched by the addition of 30 pL of 0.45 M EDTA, pH 8.0. Fluorescence intensity was measured using 485 / 515 nm excitation / emission using a BioTek Synergy Hl plate reader (Agilent). Data were normalized to blank and DMSO controls, and IC50 values were calculated using Prism software (GraphPad).

[0221] More Sensitive Screening Conditions'. Eya2 Eya domain (ED) phosphatase activity was measured in black, half-volume 96-well plates (Greiner Bio-One) using the substrate3 -0-m ethylfluorescein Phosphate (OMFP, Sigma). 50 pL reactions were carried out in triplicate using 50 mM MES, pH 6.5, 50 mM NaCl, 5 pM MgCh, and 1 mM DTT. Eya2 ED was added to buffer, followed by the addition of small molecule compounds (serially diluted in DMSO to eleven concentrations) or DMSO as control, in 46.67 pL total. Reactions were started by the addition of 3.33 pL OMFP, yielding 800 nM Eya2 ED and 100 pM OMFP at final reaction conditions. Reactions were allowed to proceed at room temperature for 1 hour, then were quenched by the addition of 10 pL of 0.45 M EDTA, pH 8.0. Fluorescence intensity was measured using 485 / 515 nm excitation / emission using a BioTek Synergy Hl plate reader (Agilent). Data were normalized to blank and DMSO controls, and IC50 values were calculated using Prism software (GraphPad).

[0222] Selected potent and selective analogs are tested in cell lines with varying levels of EYA2, such as DAOY, ONS-76 (two SHH MB lines expressing low EYA2) and D425, D458, D283, and D341 using the mammalian CellTiter-Glo® luminescent cell viability assay. Briefly, 1500 cells incubated with various compounds are plated in white 96-well tissue culture plates. Cells are treated for 72 hours with concentrations of 128, 64, 32, 16, 8, 4, 2, 1,0.5uM and DMSO only. 100 pL of CellTiter-Glo 2.0® Reagent (Promega Corporation, G9243) is added to each well and incubated at room temperature for 10 mins and luminescence was measured. IC50 calculated in Graphpad Prism vlO by Nonlinear regression analysis. Compounds with Ghos <5 pM in EYA2 / MYC high MB lines are also tested against human immortalized liver (FA2N-4)80and kidney lines (RPTEC / TERT1) using CellTiter-Glo assay for general toxicity. Compounds showing much lower GI50S (>20 fold) in these cells are prioritized to demonstrate direct on target effects in Group 3 MB cells. To this end, one can examine whether the compounds inhibit the dephosphorylation of a known target, pY142 on H2AX, particularly in response to DNA damage (9987 is able to do this in other contexts, data not shown). If additional direct targets of the EYA2 Tyr Phosphatase are identified, phosphorylation changes in these targets are also examined in response to these compounds.

[0223] For compounds that show high potency and selectivity against EYA2 in biochemical and cell-based assays with low general toxicity, their in vitro DMPK properties were evaluated at the Pharmacology D3SR Shared Resource. These studies include a Caco-2 permeability test (CacoReadyTM, ADMEcell), metabolic stability tests using pooled mixed-gender human and rat liver microsome, plasma protein binding test using the Rapid Equilibrium Dialysis (RED) plate system (Thermo Scientific), and a solubility test using the 96-well MultiScreen® HTS-PCF system (Millipore). Potential liabilities of lead compounds, such as hERG K+channel inhibition (Predictor TM, Life Technologies) and CYP450 inhibition, are also assessed. Some of the properties of drug candidates are compounds having the following properties: activity GLo(Group 3 MB lines) <1 pM), low toxicityor HepG2) >50 pM), and sufficient CNS drug-like properties (aqueous solubility>50 pM, permeability >lxl0’6cm / s, and low hERG IC50 >50 pM).

[0224] Compounds with sufficient aqueous solubility, membrane permeability, and in vitro DMPK properties are evaluated for their in vivo mouse PK properties via intravenous (i.v.), intraperitoneal (i.p.) and oral (p.o.) administrations, respectively. The maximum tolerated dose (MTD) up to 100 mg / kg for each compound is determined. For PK studies, groups of three CD-I mice are treated with compound at a single dose of 25 mg / kg or MTD. Plasma samples are collected at 0.25, 0.5, 1, 3, 6, 9, 12 and 24 h and stored at -70°C until analysis of compound concentration by LC / MS / MS. Brain samples are also collected for the determination of brain exposure of the lead compounds. The assay lower limit of quantitation and the range of the coefficient of variation are calculated. A noncompartmental model are used for PK analyses. The concentration of drug in plasma over time is plotted and the maximum serum concentration (Cmax) and the time at which the Cmax is observed is calculated. PK parameters, including elimination half-life (T1 / 2) and concentration at time zero (Co), are calculated via nonlinear leastsquares techniques. The area under the concentration-time curve (AUC) are calculated by the trapezoidal rule, yielding bioavailability and enabling the establishment of a dosage regimen based on decay and clearance in mice. Compounds with low toxicity to mammalian cells and sufficient in vivo PK properties (e.g., half-life (T1 / 2) >4 h, Cmax(brain) >5xGho(Group 3 MB lines)) are evaluated for their efficacy in in vivo MB models. In some instances, the in vivo PK properties of lead compounds are further optimized while maintaining their high activity and selectivity, as well as low toxicity.

[0225] Based on the MTD and PK results lead compounds are chosen to determine their dose, frequency, and route of administration. The lead compounds are then tested in intracranial MB xenograft and patient derived xenograft (PDX) models to study the effects on MB growth.Similar to FIG. 12, D458 or PDX411 are intracranially implanted into at least 10 female and 10 male mice per group to address sex as a variable as MB occurs in both males and females. Five days after implantation of tumor cells (once the tumors are growing and easily imaged by IVIS in the brain, for established cell lines, and by MRI for PDX models), vehicle or compound treatment is commenced. The mice are randomized, and half of each group are administered test compounds via the route and MTD determined in PK studies, whereas the other half are administered vehicle for the remaining 2 weeks of the study. The tumors are tracked by IVIS imaging following IP injection of luciferin (for D458). Because PDX tumors are not luciferase tagged, PDX411 are tracked using MRI during the course of the experiment, and then examined at the endpoint. The results are repeated on the two additional Group 3 PDX lines. In all settings, 3-6 weeks post-tumor cell transplantation, or once mice develop symptoms of morbidity, the mice are sacrificed and tumors excised and processed to analyze various markers using RT-qPCR and WB: SIX1, EYA2, c-MYC, pT58 c-MYC. Immunohistochemistry (IHC) are used to monitor the markers as well, and to monitor proliferation (BrdU / Ki67) and apoptosis (TUNEL, PARP cleavage, or activated caspase 3). All IHC analyses are done in a blinded manner. For statistical analyses, longitudinal mixed effects models are used for tumor growth over time and log-rank Mantel-Cox test are used for survival curves. For IHC analyses, multiple regression analysis is used to assess Ki67 / activated caspase 3 / MYC as a function of pair-wise and three-way interactions with EYA2.

[0226] To determine a possibility of hydrolysis of compounds disclosed herein, and thereby leading to toxicity and limiting use in vivo, EYA2 inhibitory activity and Group 3 MB toxicity of the potential hydrolysis products of LG1-34 and LG1-37 (i.e., aldehydes LG1-35, LG1-46 and benzohydrazides 449-55-8 and 1673-47-8) were evaluated. The results show that these fragments do not show significant inhibition against the EYA2 Tyr Phosphatase or Group 3 MB cell line growth (FIG. 13B). Other EYA2 tyrosine phosphatase inhibitors are identified using Core Hopping (Schrodinger) based on the benzohydrazide structure to serve as an alternative chemotype. See, for example, Table 2 above.

[0227] EYA2 inhibitors are more potent Tyr Ptase inhibitors than 9987. FIG. 14A shows chemical compound structures of 9987 and two representative compounds of the present disclosure, namely, LG1-34, and SD3-146. Differences in the chemical structure betweencompound 9987 and those of LG1-34 and SD3-146 are indicated by arrows. Compounds LG1- 34 and SD3-146 had a better potency of in vitro phosphatase activity compared to compound 9987 using purified mammalian EYA2, as can be seen in FIG. 14B (IC50 of compounds LG1-34 and SD3-146 was 0.31 pM and 0.36 pM, respectively).

[0228] The half-growth inhibition concentration of compounds LG1-34 and SD3-146 are shown in FIG. 14C, which was determined by culturing using about 1500 D458 cells per well in technical sextuplet for 72 hours with 0 -128 pM of respective compounds. CellTiterGlo was used to determine viability. Cellular thermal shift assay (CETSA) was conducted by culturing cells with 20 pM of each compound for 24 hours. Whole cell protein lysates were extracted and exposed to 42 °C for 3 minutes, and the soluble fraction of thermally stable proteins was probed for EYA2 and GAPDH via western blot. The results are shown in FIG. 14D.

[0229] A summary of results and properties of compounds LG1-34 and SD3-146 compared to compound 9987 is shown in table below:As can be seen, both compounds of the disclosure, LG1-34 and SD3-146, have better potency and pharmacological properties compared to compound 9987.

[0230] EYA2 Tyr Ptase inhibitors affect growth, cell cycle, and apoptotic phenotypes. Ability of LG1-34 and SD3-146 to affect growth, cell cycle, and apoptosis of D458 cells were evaluated. Representative Incucyte growth curves of D458 cells cultured with LG1- 34 and SD3-146 for 72 hours is shown in FIG. 15A. As can be seen, EYA2 inhibitors of the disclosure significantly inhibit cell growth. Bromodeoxyuridine (BrdU) cell cycle analysis ofD458 cells cultured in the presence of SD3-146 and LG1-34 for 72 hours at various concentrations are shown in FIG. 15B. As can be seen, EYA2 inhibitors of the disclosure also effect cell cycle. Furthermore, Incucyte apoptosis analysis using CyQuant Caspase 3 / 7 reagent for D458 cells cultured with SD3-146 and LG1-34 shows EYA2 inhibitors of the disclosure also promote apoptosis. FIG. 15C.

[0231] EYA2 Tyr Ptase activity is required for MYC levels in MYC driven MB cells.FIG. 16 shows western blot analysis of D458 cells cultured with EYA2 inhibitors compound 9987, LG1-34, and SD3-146. The right panel shows quantification of relative MYC protein produced by D458 cells in the presence of these EYA2 inhibitors. As can be seen in FIGS. 16, EYA2 inhibitors of the disclosure, e.g., compounds LG1-34 and SD3-146, significantly reduce MYC protein synthesis.

[0232] Half growth inhibition concentrations (Ghos) curves of two EYA2 inhibitors of the disclosure, namely, compound LG1-113 and compound LG1-34, two analogs of 9987, in CEM cells (an acute lymphoblastic leukemia line) are shown in FIG. 17. Compound LG1-113 had GI50 of 4.15 pM while compound LG1-34 has GI50 is 1.20 pM.

[0233] The foregoing Discussion of the invention has been presented for purposes of illustration and description. The foregoing is not intended to limit the invention to the form or forms disclosed herein. Although the description of the invention has included description of one or more embodiments and certain variations and modifications, other variations and modifications are within the scope of the invention, e.g., as may be within the skill and knowledge of those in the art, after understanding the present disclosure. It is intended to obtain rights which include alternative embodiments to the extent permitted, including alternate, interchangeable and / or equivalent structures, functions, ranges or steps to those claimed, whether or not such alternate, interchangeable and / or equivalent structures, functions, ranges or steps are disclosed herein, and without intending to publicly dedicate any patentable subject matter. All references cited herein are incorporated by reference in their entirety.

Claims

What is Claimed is:

1. A compound of the formula:or a pharmaceutically acceptable salt thereof, or a prodrug thereof, wherein m is 0 or 1;Ar1is aryl or heteroaryl each of which is optionally substituted;L1is a first linker selected from the group consisting of:Ar2is arylene or heteroarylene each of which is optionally substituted;L2is alkylene;Ar3is aryl or heteroaryl each of which is optionally substituted or -NR1R2; each X1is O, NR1, or S; each X2is O, NR2, or S; each of Y is independently H or halide; and each of R1, R2, and Rais independently H, alkyl, or a nitrogen protecting group, provided when Ar2is furylene or optionally substituted phenylene, then L1is not-C(=O)-NH-N=CH-.

2. The compound of claim 1, wherein m is 0.

3. The compound of any one of claims 1-2, wherein Ar3is aryl or heteroaryl each of which is optionally substituted.

4. The compound of any one of claims 1-3, wherein Ar1is phenyl, naphthyl, anthracyl, thiophenyl, or furyl, each of which is optionally substituted.

5. The compound of any one of claims 1-4, wherein Ar1is of the formula:wherein each of Xais independently halide, alkyl, haloalkyl, alkoxide, optionally substituted aryl, optionally substituted aryloxide, -CN, -C02Rb, (where Rbis H or alkyl), haloalkoxide, or -NCh; and n is an integer from 0 to 5.

6. The compound of any one of claims 1-5, wherein n is 1.

7. The compound of any one of claims 1-6, wherein Ar1is of the formula:wherein Xais as defined in claim 3.

8. The compound of any one of claims 1-7, wherein L1is of the formula:wherein X1is as defined in claim 1.

9. The compound of any one of claims 1-8, wherein X1is O.

10. The compound of any one of claims 1-7, wherein L1is of the formula:, wherein X1and X2are as defined in claim 1.

11. The compound of any one of claims 1-7 or 10, wherein X1and X2are O.

10. The compound of any one of claims 1-7, wherein L1is of the formula:Y, wherein X1, X2, and Y are as defined in claim 1.

12. The compound of any one of claims 1-7 and 11, wherein X1and X2are O.

13. The compound of any one of claim 1-7 and 11-12, wherein each of Y is independently H, F, or Cl.

14. The compound of any one of claims 1 -13, wherein Ar2is an optionally substituted heteroaryl of the formula:whereinZ1is NRC, O, or S;Z2is CH or N; andRcis H or alkyl.

15. The compound of any one of claims 1-14, wherein Z1is S.

16. The compound of any one of claims 1-15, wherein Z2is CH.

17. The compound of any one of claim 1-13, wherein Ar2is phenylene or pyridylene each of which is optionally substituted.

18. The compound of any one of claims 1-17, wherein Ar3is a five-membered heteroaryl, a six-membered heteroaryl, or a 9-membered bicycloheteroaryl, each of which is optionally substituted.

19. The compound of any one of claims 1-18, wherein Ar3is a moiety of the formula:, each of which is optionally substituted,wherein each of W1, W2, and W3are independently N or CH, provided at least one of W1, W2, and W3is N; each of W4and W6is independently CH or N; each of W5and W7is independently NRd, O, or S, wherein Rdis H, alkyl, or a nitrogen protecting group.

20. The compound of claim 19, wherein Ar3is optionally substituted moiety of the formula:, wherein W1, W2, and W3are as defined in claim 19.

21. The compound of any one of claims 19-20, wherein W1is N and one or both of W2and W3is CH.

22. The compound of claim 19, wherein Ar3is optionally substituted moiety of the formula:, wherein W4and W5are as defined in claim 19.

23. The compound of claim 19 or 22, wherein W4is N.

24. The compound of any one of claims 19 and 22-23, wherein W5is O or S.

25. The compound of claim 1, wherein m is 1.

26. The compound of any one of claims 1 and 25, wherein L2is methylene or ethylene.

27. The compound of any one of claims 1 and 25-26, wherein Ar3is -NRJR2.

28. The compound of any one of claims 1 and 25-27, wherein each of R1and R2is independently H or alkyl.

29. The compound of any one of claims 1 and 25-28, wherein each of R1and R2is independently alkyl.

30. A method for treating a clinical condition associated with EYA2 tyrosine phosphatase activity, said method comprising administering a therapeutically effective amount of a compound of any one of claims 1-29 to a subject in need of such a treatment.

31. The method of claim 30, wherein said clinical condition associated with EYA2 tyrosine phosphatase activity comprises cancer.

32. The method of any one of claims 30-31, wherein said clinical condition associated with EYA2 phosphatase activity comprises medulloblastoma, glioblastoma, lung cancer, breastcancer, leukemia, ovarian cancer, kidney cancers, hepatocellular cancer, colorectal cancer, thyroid cancer, prostate cancer, endometrial cancer, esophageal cancer, osteosarcoma, pancreatic cancer, cervical cancer, or meningioma.

33. The method of any one of claims 30-32, wherein said clinical condition associated with EYA2 tyrosine phosphatase activity comprises medulloblastoma.

34. The method of any one of claims 30-33, wherein said clinical condition associated with EYA2 tyrosine phosphatase activity comprises Group 3 medulloblastoma.

35. A method for treating cancer in a subject in need of such a treatment, said method comprising administering a therapeutically effective amount of a pharmaceutically active agent that inhibits EYA2 tyrosine phosphatase activity, wherein said pharmaceutically active agent comprises a compound of any one of claims 1-29.

36. The method of claim 35, wherein said cancer comprises medulloblastoma, glioblastoma, lung cancer, breast cancer, leukemia, ovarian cancer, kidney cancers, hepatocellular cancer, colorectal cancer, thyroid cancer, prostate cancer, endometrial cancer, esophageal cancer, osteosarcoma, pancreatic cancer, cervical cancer, meningioma, or a combination thereof.

37. The method of any one of claims 35-36 further comprising administering to said subject a second cancer therapy.

38. The method of claim 37, wherein said second cancer therapy comprises radiotherapy, immunotherapy, chemotherapy, hormonal therapy, gene therapy, or a combination thereof.

39. The method of any one of claims 35-38, wherein cells of said cancer overexpress EYA2.

40. A method for modulating EYA2 tyrosine phosphatase activity in a cell, said method comprising contacting said cell with a compound of any one of claims 1-29.

41. A pharmaceutical composition comprising a compound of any one of claims 1-29 and a pharmaceutically acceptable excipient.

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