WO2018145217A1 - Human ets-related gene (erg) compounds as therapeutics and methods for their use - Google Patents

Abstract

This invention provides compound having a structure of Formula (I): (I) Uses of such compounds for treatment of ERG, FLI1, ETV4, or ETV1 -mediated indications, including cancer. Also provided are pharmaceutical composition and methods of treating ERG, FLI1, ETV4, or ETV1-mediated indications, including cancer. The cancer may be selected from the group consisting of: prostate cancer, Ewing's sarcoma, breast cancer or pancreatic cancer.

Description

HUMAN ETS-RELATED GENE (ERG) COMPOUNDS AS THERAPEUTICS

AND METHODS FOR THEIR USE

TECHNICAL FIELD

This invention relates to therapeutic compounds and compositions, and methods for their use in the treatment of various cancers. The therapeutic compounds and compositions may be used to treat prostate cancer, Ewing's sarcoma, breast cancer or pancreatic cancer.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Patent Application Serial No. 62/458,085 filed on 13 February 2017, entitled "HUMAN ETS-RELATED GENE (ERG) COMPOUNDS AS THERAPEUTICS AND METHODS FOR THEIR USE".

BACKGROUND

The E26 transformation-specific or E-twenty-six (ETS) family of transcription factors consists of approximately 30 genes that share a conserved DNA binding domain (ETS domain) (Oikawa, 2004). ETS genes play a vital role during embryonic development and also affect cellular mechanisms such as proliferation, differentiation, and apoptosis. Certain ETS genes are commonly deregulated in human diseases, including prostate cancer, Ewing sarcoma and leukemia (Gutierrez-Hartmann, Duval, & Bradford, 2007). This deregulation of ETS genes, which occurs as the result of genetic rearrangements or aberrant expression, impacts several significant downstream pathways and results in malignant transformation and tumour progression (Sharrocks, 2001).

Among the many cancers that ETS factors have been implicated in (Gutierrez- Hartmann, 2007), prostate cancer (PCa) is the most commonly diagnosed non-skin cancer and one of the leading causes of cancer-related death in Canadian men (Canadian Cancer Society). When diagnosed early, PCa is treated by surgery or radiation (Denmeade & Isaacs, 2002). However, in about 30% of cases when PCa recurs and/or metastasizes, the disease is managed primarily by the androgen deprivation therapy (ADT) that reduces the level of androgens (male hormones) or blocks their binding to the androgen receptor (AR) (Lonergan & Tindall, 2011). Unfortunately, the effectiveness of this therapy is temporary due to emerging resistance mechanisms related to AR.

The majority of clinically used anti-AR drugs, such as Bicalutamide and Enzalutamide (Tran et al., 2009), inhibit the AR by competing with androgens at the androgen binding site (ABS) in the ligand binding domain (LBD) of the protein. However, resistance mutations at the ABS in PCa patients can convert anti-AR drugs from antagonists (i.e. AR inhibition) to agonists (i.e. AR activation) (Balbas etal, 2013; Bohl, Gao, Miller, Bell, & Dalton, 2005; Bohl, Miller, Chen, Bell, & Dalton, 2005). Furthermore, studies indicated that PCa cells can express constitutively active, ligand-independent AR splice isoforms lacking the entire LBD, thus rendering most of the antiandrogens ineffective (Hu et al., 2012; Li et al., 2011). In addition to the AR-related resistance mechanisms, ADT comes with significant side effects on the sexual functions and characteristics of male bodies (Isbarn et al., 2009; Valenca, Sweeney, & Pomerantz, 2015). Moreover, recent studies demonstrated that treating PCa with antiandrogens, such as Enzalutamide, can promote the development of neuroendocrine prostate cancer (NEPC) (Dang et al., 2015), likely due to the inhibition of AR function for maintaining the prostate phenotype and suppressing the neuroendocrine trans-differentiation process (Wright, Tsai, & Aebersold, 2003). Thus, due to the limited effectiveness and significant side effects of current treatments, new therapeutics for the treatment of aggressive and resistant prostate cancer are needed.

Normal AR functions are altered in prostate cancer cells by mechanisms such as mutations and aberrant gene expressions (Berger etal., 2011; Hu etal., 2012; Yu etal., 2010). A prime example is the fusion of the AR DNA response element of transmembrane protease serine 2 (TMPRSS2) and the ETS-related gene (ERG), adjacently coded on chromosome 21 (Tomlins et al., 2005). The TMPRSS2-ERG fusion is the most common genomic rearrangement in prostate cancer to date, occurring in about 50% of PCa patients (Rahim & Uren, 2013; Robinson et al., 2015; Rubin, Maher, & Chinnaiyan, 2011; Tomlins et al., 2005). Normally ERG is not expressed in prostate epithelial cells, but its fusion with the TMPRSS2 promoter causes AR to drive ERG expression. Thus, ERG is one of the most commonly overexpressed genes in PCa, which is confirmed by gene expression analyses of three independent patient datasets (Taylor et al., 2010; The Cancer Genome Atlas; Wyatt et al., 2014). Being an oncogenic hub that activates multiple cancer-inducing pathways, ERG can effectively promote epithelial-mesenchymal transition (EMT) and transform normal prostate cells into cancerous and invasive forms (Adamo & Ladomery, 2015; Dobi, Sreenath, & Srivastava, 2013). Previous studies have associated the ERG-positive status with high Gleason scores, aggressive disease, and poor prognosis in PCa patients (Rahim & Uren, 2013; St John, Powell, Conley-Lacomb, & Chinni, 2012). Moreover, the tumorigenic potential of ERG (especially in combination with loss of tumour suppressors such as PTEN), has been extensively demonstrated in both xenograft and transgenic mouse models (Becker-Santos et al., 2012; Chen etal., 2013).

Researchers have previously targeted the expression of ERG by siRNA (Shao et al., 2012; Sun et al., 2008) and shRNA (J. Wang et al., 2008), which inhibited tumour growth in ERG-positive VCaP xenografts in mice. The DNA site recognized by the ERG protein has been targeted by the compound DB1255, which binds to the minor groove of the DNA (Nhili et al., 2013). A 7-residue peptidomimetic, RI-EIP, is under development to target the ERG protein (OncoFusion Therapeutics Inc., unpublished). In addition, ERG has been targeted indirectly through inhibition of ERG binding proteins including PARPi (Brenner et al., 2011) and USP9X (S. Wang et al., 2014), as well as ERG downstream target genes such as YAPi (Nguyen et al., 2015). Among the various anti-ERG attempts, the only published small molecule that demonstrated direct binding to the ERG protein is an experimental compound, YK-4-279 (CAS #1037184-44-3), which was initially developed to target FLIi in Ewing sarcoma, and was later shown to inhibit ERG-mediated cell invasion in PCa cells (Erkizan etal., 2009; Rahim et al., 2011). Recent reports on YK-4-279 have disclosed toxicity, oral bioavailability and pharmacokinetics concerns (Lamhamedi-Cherradi etal., 2015; Rahim et al., 2014).

Although ERG has been established as a critical factor that drives prostate cancer development and progression (Adamo & Ladomery, 2015; Dobi et al., 2013; St John et al., 2012) and despite the above attempts to target this protein, there is not yet any approved therapy directly targeting the ERG protein (Knox et al., 2011). In fact, despite the involvement of multiple ETS factors in many different cancers (Gutierrez-Hartmann et al., 2007), there are currently no approved drugs directly targeting any members of the ETS family. Unlike the AR or estrogen receptor, which possess a ligand binding site targetable by small molecules, ERG and other ETS factors do not require ligand binding for their activation. Furthermore, due to the complexity of protein-DNA interactions and lack of well-defined pockets that can be easily targeted by small molecules, drug development against transcription factors, such as ETS factors, is an immense challenge (Neher et al., 2011).

SUMMARY OF THE INVENTION

This invention is based in part on the fortuitous discovery that compounds described herein modulate ETS factor activity. Specifically, compounds identified herein, show inhibition of human ETS-related gene (ERG) activity. Compounds that inhibit ERG in cancer, may also provide insights into the identification of similar compounds that target other oncogenic ETS factors. One non-limiting example is FLIi, shares 98% sequence identity at the ETS domain with ERG.

In one aspect, there is provided a compound having the structure of Formula I

I, wherein, R¹ may be selected from H, CH₃, OH, F, CI and Br; R² may be selected from H, CH₃, F, CI and Br; R3 may be selected from

alternatively R³ may be selected from

when R¹ may be H, R² may be H, R4 may be H, R3 may be H and R⁶ may be H; R4 may be selected from H, CH₃, OH, F, CI and Br; R5 may be selected from H, CH₃, F, CI and Br; R⁶ may be H; or alternatively R⁶ is CH₃, when R3 is -OCH₃; provided that the

compound does not have the following structure:

R4 may be selected from H, CH₃, F, CI or Br. R4 may be selected from H, F, CI or Br. R4 may be selected from H or CH₃. R4 may be selected from H or Br. R4 may be selected from H or F. R4 may be selected from H or CI. R4 may be H. R¹ may be H, CH₃, OH, F or CI. R¹ may be H,

CH₃, F or CI. R² may be H, CH₃, F or CI. R5 may be H. R3 may be selected from

The compound may be for use in the treatment of at least one indication selected from the group consisting of: prostate cancer, Ewing's sarcoma, breast cancer and pancreatic cancer.

In a further aspect, there is provided a method for modulating E26 transformation- specific (ETS) activity, the method comprising administering to a mammalian cell in need thereof a compound or pharmaceutically acceptable salt thereof, wherein the compound has

the structure of Formula I wherein, R¹ may be selected from

H, CH₃, OH, F, Cl and Br; R² may be selected from H, CH₃, F, Cl and Br; R3 may be selected

alternatively R³ may be selected from

when R¹ may be H, R² may be may be H, R4 is H, R5 may be H and R⁶ is H; R4 may be selected from H, CH₃, OH, F, CI and Br; R5 may be selected from H, CH₃, F, CI and Br; R⁶ may be H; or alternatively R⁶ is CH₃, when R3 maybe -OCH₃.

R4 may be selected from H, CH₃, F, CI or Br. R4 may be selected from H, F, CI or Br. R4 maybe H. R¹ may be H, CH₃, OH, F or CI. R¹ may be H, CH F or CI. R² may be H, CH₃, F

_; and

ETS activity may be for treatment of at least one indication selected from the group consisting of: prostate cancer, Ewing's sarcoma, breast cancer and pancreatic cancer. The modulating ETS activity may be for the treatment of prostate cancer. The modulating ETS activity may be for the treatment of aggressive prostate cancer. The modulating ETS activity may be for the treatment of resistant prostate cancer. The mammalian cell is a human cell. The cell may be a prostate cell. The cell may be a prostate cancer cell.

In a further aspect, there is provided a compound having the structure of Formula I

I, wherein, R¹ maybe selected from H, CH₃, OH, F, CI and Br;

R² may be selected from H, CH₃, F, CI and Br; R3 may be selected from

alternatively R³ may be selected from

, when R¹ may be H, R² may be H, R4 may be H, R5 may be H and R⁶ may be H; R4 may be selected from H, CH₃, OH, F, CI and Br; R5 may be selected from H, CH₃, F, CI and Br; R⁶ may be H; or alternatively R⁶ may be CH₃, when R3 is -OCH₃; for use in the treatment of at least one indication selected from the group consisting of: prostate cancer, Ewing's sarcoma, breast cancer and pancreatic cancer.

R⁴ may be selected from H, CH₃, F, CI or Br. R4 may be selected from H, F, CI or Br. R4 maybe H. R¹ may be H, CH₃, OH, F or CI. R¹ may be H, CH₃, F or CI. R² may be H, CH₃, F or CI. R⁵maybe H. R3 may be selected from -

In a further aspect, there is provided a use of a compound for modulating ETS activity,

wherein the compound has the structure of Formula I,

wherein, R¹ may be selected from H, CH₃, OH, F, CI and Br; R² may be selected from H, CH₃,

F, CI and Br; R3 may be selected from

alternatively R3 may be selected from

when R¹ is H, R² is H, R4 is H, R5 is H and R⁶ is H; R4 may be selected from H, CH₃, OH, F, CI and Br; R5 may be selected from H, CH₃, F, CI and Br; R⁶ may be H; or alternatively R⁶ may be CH₃, when R3 is -OCH₃.

In a further aspect, there is provided a use of a compound for the manufacture of a medicament for modulating ETS activity, wherein the compound has the structure of Formula

I, wherein, R¹ may be selected from H, CH₃, OH, F, CI and Br;

R² may be selected from H, CH₃, F, CI and Br; R3 may be selected from

a

when R¹ is H, R² is H, R4 is H, R5 is H and R⁶ is H; R4 may be selected from H, CH₃, OH, F, CI and Br; R5 may be selected from H, CH₃, F, CI and Br; R⁶ may be H; or alternatively R⁶ may be CH₃, when R3 is -OCH₃.

R4 may be selected from H, CH₃, F, CI or Br. R4 may be selected from H, F, CI or Br. R4 maybe H. R¹ may be H, CH₃, OH, F or CI. R¹ may be H, CH₃, F or CI. R² may be H, CH₃, F

g y y treatment of at least one indication selected from the group consisting of: prostate cancer, Ewing's sarcoma, breast cancer and pancreatic cancer. The modulating ETS activity is for treatment of prostate cancer.

In a further aspect, there is provided a pharmaceutical composition comprising a compound or pharmaceutically acceptable salt thereof, wherein the compound has the

structure of Formula I

I, wherein, R¹ may be selected from H

CH₃, OH, F, CI and Br; R² may be selected from H, CH₃, F, CI and Br; R3 may be selected from

when R¹ is H, R² is H, R4 is H, R5 is H and R⁶ is H; R4 may be selected from H, CH₃, OH, F, CI and Br; R5 may be selected from H, CH₃, F, CI and Br; R⁶ is H; or alternatively R⁶ may be CH₃, when R3 is -OCH₃. The compound may be selected from the

there is provided a commercial package comprising (a) a compound of any one of claims 35- 48; and (b) instructions for the use thereof for modulating ETS activity.

In a further aspect, there is provided a commercial package comprising (a) a pharmaceutical composition comprising a compound described herein and a pharmaceutically acceptable carrier; and (b) instructions for the use thereof for modulating ETS activity.

In a further aspect, there is provided a compound having the structure of one or more of the following:

for use in the treatment of at least one indication selected from the group consisting of: prostate cancer, Ewing's sarcoma, breast cancer and pancreatic cancer.

The compound may be selected from one or more of the structures described herein, including their analogs, isomers, stereoisomers, or related derivatives, for use in modulating ETS activity. Furthermore, the compounds described herein may be useful in the treatment of various indications where the activity of one or more of ERG, FLIi, ETV4, or ETVi would benefit from modulation. Furthermore, the modulating ETS activity may be for use in treatment of at least one indication selected from the group including prostate cancer, Ewing's sarcoma, breast cancer or pancreatic cancer.

The modulating ETS activity may be for the treatment of prostate cancer. The mammalian cell may be a human cell. The cell may be a prostate cell. The cell may be a prostate cancer cell. The modulating ETS activity may be for the treatment of Ewing's sarcoma. The mammalian cell may be a human cell. The cell may be a bone cell. The cell may be a sarcoma cancer cell. The modulating ETS activity may be for the treatment of breast cancer. The mammalian cell may be a human cell. The cell may be a breast cell. The cell may be a breast cancer cell. The modulating ETS activity may be for the treatment of pancreatic cancer. The mammalian cell may be a human cell. The cell may be a pancreatic cell. The cell may be a pancreatic cancer cell.

DESCRIPTION OF THE DRAWINGS FIGURE 1. ERG as a drug target and discovery of VPC-18005: (A) The pocket within the ERG-ETS domain that was identified by virtual atomic probes and used to screen 3 million molecules from the ZINC database. The DNA backbone is shown for illustration purposes, but was not included in virtual screening. (B) Dose response effect of VPC-18005 (media concentration) in PNTiB-ERG and VCaP cells on ERG-mediated luciferase activity. (C) Dose response effect of YK-4-279 in PNTiB-ERG and VCaP cells on ERG-mediated luciferase activity. The toxicity of YK-4-729 is shown in red filled dots. (D) After treatment with up to 25 μΜ VPC-18005, no effect on cell viability (MTS) was observed with ERG-expressing cells (PNTiB-ERG and VCaP) and non-ERG expressing cells (PC3). In contrast, the published inhibitor YK-4-279 demonstrated cytotoxic effects starting at 1 μΜ in both cell types.

FIGURE 2. Characterization of VPC-18005 binding to the ERG-ETS domain: (A)

Chemical structure of VPC-18005, in the isomeric form used for docking. The R-isomer is calculated to have the most favorable binding energy. (Molecular weight = 318 g/mol at pH 7). (B) A surface representation of predicted VPC-18005 binding pose within the pocket on the ERG-ETS protein domain. (C) Plot of amide chemical shift perturbations of the ERG-ETS domain due to the addition of a 10 fold molar excess of VPC-18005 (derived from FIGURE 8 and data not shown). Grey bars (light and dark) denote the largest changes (dark grey≥ mean + s.d.; light grey≥ mean). (D) Amino acid residues exhibiting the largest chemical shift perturbations were mapped to their corresponding locations on the ERG-ETS domain (solid light grey and solid dark grey (i.e. not grey mesh) as in E). (E) -NMR1 mHonitored titration of VPC-18005 (sharp signals) with increasing concentrations (grey-scale) of the ERG-ETS domain (broad signals). Signals from 1H nuclei directly bonded to the indicated chemical moieties shift and broaden upon binding the protein.

FIGURE 3. VPC-18005 inhibits migration and invasion of prostate cell lines in vitro: (A) PNTiB-Mock cells and (B) PNTiB-ERG cells were seeded in the upper chamber of a real-time cell analysis system (xCelligence) and treated with 5 μΜ compounds or DMSO (control) at 24 hrs. The normalized cell index is a measure of the migration of the cells through the pores of the upper chamber and was used as the migration index. Dotted lines represent standard deviations (N=3). The horizontal dotted line indicates the level of migration the - MOCK cells reached at 48 h in comparison to -ERG cells. (C) Rates of migration were determined by the slopes of the curves between 24 - 48 h. Migration was inhibited in the presence of VPC-18005. YK-4-279 is cytotoxic. (D) Pretreatment of VPC-18005 (10 μΜ) inhibits the subsequent invasion of PNTiB-ERG spheroids into the surrounding matrix. Quantitative analysis was performed on the day of invasion matrix addition to determine the area of the spheroids. After 6 days of growth, those cells treated with VPC-18005 had significantly reduced invasion compared to vehicle control. YK-4-279 (5 μΜ) was cytotoxic and resulted in no invasion from day o. (* p<0.05). Furthermore, the order of the lines in the legends correspond to the order of the lines on the plots and bar graphs for (A)-(D).

FIGURE 4. VPC-18005 inhibits prostate cell line dissemination in vivo: (A) Pre- stained PNTiB-Mock and PNTiB-ERG cells were microinjected into the yolk sac (grey arrows) of the zebrafish, and the metastasis capability of the cells (white arrows) were detected using confocal microscope at day 2 and day 5. Five days following injection, only ERG expressing cells had invaded and metastasized into the head and tail region of the fish. (B) Evaluation of compound toxicity to zebrafish embryos. Zebrafish embryos were treated with increasing concentration of VPC-18005 and YK-4-279 in their water. After 4 days, surviving embryos were counted. YK-4-279 demonstrated toxicity in zebrafish at levels above 10 μΜ. VPC-18005 was non-toxic until concentrations above 75 μΜ. (C) Following 5 days of daily treatment, VPC- 18005 reduced occurrence of metastasis in zebrafish grafted with PNTiB-ERG and VCaP cells. DMSO versus 1 μΜ (p=o.03) and 10 μΜ (p=0.002) VPC-18005.

FIGURE 5. ERG is overexpressed in prostate cancer: (A) A Venn diagram that shows the number of upregulated genes from each of the three gene expression datasets: Vancouver Prostate Centre (VPC) (Wyatt etal. 2014), Memorial Sloan-Kettering Cancer Center (MSKCC) (Taylor et al. 2010), and The Cancer Genome Atlas (TCGA) (2015), based on a bioinformatics protocol. ERG is the only overexpressed gene common to the three datasets. (B) The fold changes of ERG gene expression in PCa tumour samples, compared to normal samples, range from 2.66 to 3.29.

FIGURE 6. In vitro assessment of YK-4-279 and VPC-18005: (A) Incucyte was used to monitor proliferation of PNTiB-ERG cells in the presence of VPC-18005 or (B) YK-4-279. VPC-18005 did not affect the rate of cell proliferation. In comparison, YK-4-279 inhibited cell proliferation at high concentrations. The order of the lines in the legends correspond to the order of the lines on the plots of (A) and (B).

FIGURE 7. VPC-18005 disrupts binding of purified ERG-ETS complex to DNA:

(A) EMSA analysis of the binding of the purified ERG-ETS domain to 1 nM fluorescently- labeled dsDNA. Fitting of these data to a 1:1 binding isotherm yielded a KD value of ~ 5 nM.

(B) Titration of VPC-18005 disrupts the binding of 4 nM ERG-ETS domain with 1 nM fluorescently-labeled dsDNA. Fitting of the data to simple competition isotherm yielded a Ki value of ~ 250 μΜ for the interaction of VPC-18005 with the ERG-ETS domain.

FIGURE 8. VPC-18005 binds the ERG-ETS domain: Fitting of the VPC-18005-induced chemical shift perturbations of the amide ^Ν-1«HΝ signals of residues 319, 323, 334, 371, and 379 (shown) to a simple 1:1 binding isotherm yielded an average KD ~ 3 mM.

FIGURE 9. General scheme of chemical synthesis for VPC-18005.

FIGURE 10. Preliminary SAR studies using derivatives of VPC-18005:

Modifications of the isopropyl moiety (VPC-18005) into tert-butyl (VPC-18065) and cyclobutyl (VPC-18098) improved the IC₅₀ values in the luciferase reporter assays in PNTiB- ERG cells. Removal of the carboxyl moiety (VPC-18100) resulted in the loss of activity. Progressive differences between the derivatives are highlighted.

DETAILED DESCRIPTION

Any terms not directly defined shall be understood to have the meanings commonly associated with them as understood within the art of the invention.

It will be understood by a person of skill that a chemical structure may represent multiple isomeric forms. Unless otherwise a compound specifically identifies an isomeric form or excludes an isomeric form the structure is meant to encompass all sterioisomeric forms of the structure. The structures shown in TABLE l and TABLE 2 are presented as representative 2D images, and includes all stereoisomers, ionic forms and related derivatives.

It will be understood by a person of skill that the functional groups -C(=0)OH and - NH₂ may include the corresponding ionic forms (for example, -C(=0)0^" as shown for VPC- 18005, in TABLE 1 and FIGURE 9 and VPC-18018 in TABLE 2), whereby the carboxylate ions and ammonium ions may be shown, respectively. Alternatively, where the ions are shown, a person of skill in the art will appreciate that the counter ion may also be present. Furthermore, it will be appreciated by a person of skill that other moieties may include the corresponding ions, and where the ions are shown, a person of skill in the art will appreciate that the counter ion may also be present.

In the absence of any approved ETS inhibitor, targeting this family of proteins represents an important step towards creating new therapeutics. This project has utilized a combined approach of in silico, in vitro, and in vivo models to identify candidate drug scaffolds that have been verified to directly bind to the DNA interaction site of the ERG-ETS domain. In a previous study targeting ERG action, only one small molecule, YK-4-279, was been shown to target the ERG protein directly, but the targeted site occurs at a protein -protein interaction interface (Erkizan et al., 2009), and has demonstrated non-specific toxicity. Whereas, DB1255, targets the ETS binding site of the DNA, not the ERG protein directly. Identification of small molecules directly targeting the ERG-DNA interaction would be useful in targeting of ERG and other ETS factors and may be used alternatively or synergistically with current therapies to benefit patients with the PCa.

TABLE 1 and TABLE 2 shows the compounds tested by structure and the associated identifiers. Where the % inhibition or the IC₅₀ has no value given, this may be because no measurement was taken or the value was not calculated. Accordingly, no value given in TABLE 1 or TABLE 2 does not mean that there was no activity. In TABLE 1, Estimated % inhibition in a luciferase reporter assay was performed at 10 μΜ in at least one of prostate cancer, breast cancer or Ewing's sarcoma cell lines. Cell lines used in luciferase reporter assays: prostate cancer (PNTiB-ERG, VCaP, PC3, LNCaP), breast cancer (MDA-MB-231), Ewing's sarcoma (RD-ES).

TABLE 2 - Additional Tested Com ounds

In some embodiments, compounds of TABLE 1 and TABLE 2 maybe selected for use in the systemic treatment of cancer. The cancer may be selected from the group consisting of: prostate cancer, Ewing's sarcoma, breast cancer or pancreatic cancer. In some embodiments, compounds of TABLE 1 and TABLE 2 may be used in the preparation of a medicament or a composition for systemic treatment of an indication described herein. In some embodiments, methods of systemically treating any of the indications described herein are also provided.

Those skilled in the art will appreciate that the point of covalent attachment of the moiety to the compounds as described herein may be, for example, and without limitation, cleaved under specified conditions. Specified conditions may include, for example, and without limitation, in vivo enzymatic or non-enzymatic means. Cleavage of the moiety may occur, for example, and without limitation, spontaneously, or it may be catalyzed, induced by another agent, or a change in a physical parameter or environmental parameter, for example, an enzyme, light, acid, temperature or pH. The moiety may be, for example, and without limitation, a protecting group that acts to mask a functional group, a group that acts as a substrate for one or more active or passive transport mechanisms, or a group that acts to impart or enhance a property of the compound, for example, solubility, bioavailability or localization.

Compounds as described herein may be in the free form or in the form of a salt thereof. In some embodiment, compounds as described herein may be in the form of a pharmaceutically acceptable salt, which are known in the art (Berge S. M. etal, J. Pharm. Sci. (1977) 66(i):i-19). Pharmaceutically acceptable salt as used herein includes, for example, salts that have the desired pharmacological activity of the parent compound (salts which retain the biological effectiveness and/or properties of the parent compound and which are not biologically and/or otherwise undesirable). Compounds as described herein having one or more functional groups capable of forming a salt may be, for example, formed as a pharmaceutically acceptable salt. Compounds containing one or more basic functional groups may be capable of forming a pharmaceutically acceptable salt with, for example, a pharmaceutically acceptable organic or inorganic acid. Pharmaceutically acceptable salts may be derived from, for example, and without limitation, acetic acid, adipic acid, alginic acid, aspartic acid, ascorbic acid, benzoic acid, benzenesulfonic acid, butyric acid, cinnamic acid, citric acid, camphoric acid, camphorsulfonic acid, cyclopentanepropionic acid, diethylacetic acid, digluconic acid, dodecylsulfonic acid, ethanesulfonic acid, formic acid, fumaric acid, glucoheptanoic acid, gluconic acid, glycerophosphoric acid, glycolic acid, hemisulfonic acid, heptanoic acid, hexanoic acid, hydrochloric acid, hydrobromic acid, hydriodic acid, 2- hydroxyethanesulfonic acid, isonicotinic acid, lactic acid, malic acid, maleic acid, malonic acid, mandelic acid, methanesulfonic acid, 2-napthalenesulfonic acid, naphthalenedisulphonic acid, p-toluenesulfonic acid, nicotinic acid, nitric acid, oxalic acid, pamoic acid, pectinic acid, 3-phenylpropionic acid, phosphoric acid, picric acid, pimelic acid, pivalic acid, propionic acid, pyruvic acid, salicylic acid, succinic acid, sulfuric acid, sulfamic acid, tartaric acid, thiocyanic acid or undecanoic acid. Compounds containing one or more acidic functional groups may be capable of forming pharmaceutically acceptable salts with a pharmaceutically acceptable base, for example, and without limitation, inorganic bases based on alkaline metals or alkaline earth metals or organic bases such as primary amine compounds, secondary amine compounds, tertiary amine compounds, quaternary amine compounds, substituted amines, naturally occurring substituted amines, cyclic amines or basic ion-exchange resins. Pharmaceutically acceptable salts may be derived from, for example, and without limitation, a hydroxide, carbonate, or bicarbonate of a pharmaceutically acceptable metal cation such as ammonium, sodium, potassium, lithium, calcium, magnesium, iron, zinc, copper, manganese or aluminum, ammonia, benzathine, meglumine, methylamine, dimethylamine, trimethylamine, ethylamine, diethylamine, triethylamine, isopropylamine, tripropylamine, tributylamine, ethanolamine, diethanolamine, 2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, hydrabamine, choline, betaine, ethylenediamine, glucosamine, glucamine, methylglucamine, theobromine, purines, piperazine, piperidine, procaine, N-ethylpiperidine, theobromine, tetramethylammonium compounds, tetraethylammonium compounds, pyridine, Ν,Ν-dimethylaniline, N- methylpiperidine, morpholine, N-methylmorpholine, N-ethylmorpholine, dicyclohexylamine, dibenzylamine, Ν,Ν-dibenzylphenethylamine, l-ephenamine, N,N'-dibenzylethylenediamine or polyamine resins. In some embodiments, compounds as described herein may contain both acidic and basic groups and may be in the form of inner salts or zwitterions, for example, and without limitation, betaines. Salts as described herein may be prepared by conventional processes known to a person skilled in the art, for example, and without limitation, by reacting the free form with an organic acid or inorganic acid or base, or by anion exchange or cation exchange from other salts. Those skilled in the art will appreciate that preparation of salts may occur in situ during isolation and purification of the compounds or preparation of salts may occur by separately reacting an isolated and purified compound.

In some embodiments, compounds and all different forms thereof (e.g. free forms, salts, polymorphs, isomeric forms) as described herein may be in the solvent addition form, for example, solvates. Solvates contain either stoichiometric or non-stoichiometric amounts of a solvent in physical association the compound or salt thereof. The solvent may be, for example, and without limitation, a pharmaceutically acceptable solvent. For example, hydrates are formed when the solvent is water or alcoholates are formed when the solvent is an alcohol.

In some embodiments, compounds and all different forms thereof (e.g. free forms, salts, solvates, isomeric forms) as described herein may include crystalline and amorphous forms, for example, polymorphs, pseudopolymorphs, conformational polymorphs, amorphous forms, or a combination thereof. Polymorphs include different crystal packing arrangements of the same elemental composition of a compound. Polymorphs usually have different X-ray diffraction patterns, infrared spectra, melting points, density, hardness, crystal shape, optical and electrical properties, stability and/ or solubility. Those skilled in the art will appreciate that various factors including recrystallization solvent, rate of crystallization and storage temperature may cause a single crystal form to dominate.

In some embodiments, compounds and all different forms thereof (e.g. free forms, salts, solvates, polymorphs) as described herein include isomers such as geometrical isomers, optical isomers based on asymmetric carbon, stereoisomers, tautomers, individual enantiomers, individual diastereomers, racemates, diastereomeric mixtures and combinations thereof, and are not limited by the description of the formula illustrated for the sake of convenience.

In some embodiments, compounds may include analogs, isomers, stereoisomers, or related derivatives. Compounds of the present invention may include compounds related to the compounds of TABLE l and TABLE 2 by substitution or replacement of certain substituents with closely related substituents, for instance replacement of a halogen substituent with a related halogen (i.e. bromine instead of chlorine, etc.) or replacement of an alkyl chain with a related alkyl chain of a different length, and the like. In other embodiments, compounds may include compounds within a generic or Markush structure, as determined from structure-activity relationships identified from the data presented in TABLE l and TABLE 2. Different structures that have been demonstrated to have good efficacy may be combined with other efficacious structures. In this way, many different combinations of structures may be expected to also be efficacious. The determination of such structure-activity relationships for the development of generic Markush structures is within the skill of one in the art.

In some embodiments, pharmaceutical compositions as described herein may comprise a salt of such a compound, preferably a pharmaceutically or physiologically acceptable salt. Pharmaceutical preparations will typically comprise one or more carriers, excipients or diluents acceptable for the mode of administration of the preparation, be it by injection, inhalation, topical administration, lavage, or other modes suitable for the selected treatment. Suitable carriers, excipients or diluents (used interchangeably herein) are those known in the art for use in such modes of administration.

Suitable pharmaceutical compositions may be formulated by means known in the art and their mode of administration and dose determined by the skilled practitioner. For parenteral administration, a compound may be dissolved in sterile water or saline or a pharmaceutically acceptable vehicle used for administration of non-water soluble compounds such as those used for vitamin K. For enteral administration, the compound may be administered in a tablet, capsule or dissolved in liquid form. The tablet or capsule may be enteric coated, or in a formulation for sustained release. Many suitable formulations are known, including, polymeric or protein microparticles encapsulating a compound to be released, ointments, pastes, gels, hydrogels, or solutions which can be used topically or locally to administer a compound. A sustained release patch or implant may be employed to provide release over a prolonged period of time. Many techniques known to one of skill in the art are described in Remington: the Science & Practice of Pharmacy by Alfonso Gennaro, 20^th ed., Lippencott Williams & Wilkins, (2000). Formulations for parenteral administration may, for example, contain excipients, polyalkylene glycols such as polyethylene glycol, oils of vegetable origin, or hydrogenated naphthalenes. Biocompatible, biodegradable lactide polymer, lactide/glycolide copolymer, or polyoxyethylene-polyoxypropylene copolymers may be used to control the release of the compounds. Other potentially useful parenteral delivery systems for modulatory compounds include ethylene-vinyl acetate copolymer particles, osmotic pumps, implantable infusion systems, and liposomes. Formulations for inhalation may contain excipients, for example, lactose, or may be aqueous solutions containing, for example, polyoxyethylene-9-lauryl ether, glycocholate and deoxycholate, or may be oily solutions for administration in the form of nasal drops, or as a gel.

Compounds or pharmaceutical compositions as described herein or for use as described herein may be administered by means of a medical device or appliance such as an implant, graft, prosthesis, stent, etc. Also, implants may be devised which are intended to contain and release such compounds or compositions. An example would be an implant made of a polymeric material adapted to release the compound over a period of time.

An "effective amount" of a pharmaceutical composition as described herein includes a therapeutically effective amount or a prophylactically effective amount. A "therapeutically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result, such as reduced tumor size, increased life span or increased life expectancy. A therapeutically effective amount of a compound may vary according to factors such as the disease state, age, sex, and weight of the subject, and the ability of the compound to elicit a desired response in the subject. Dosage regimens may be adjusted to provide the optimum therapeutic response. A therapeutically effective amount is also one in which any toxic or detrimental effects of the compound are outweighed by the therapeutically beneficial effects. A "prophylactically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result, such as smaller tumors, increased life span, increased life expectancy or prevention of the progression of prostate cancer to an androgen-independent form. Typically, a prophylactic dose is used in subjects prior to or at an earlier stage of disease, so that a prophylactically effective amount may be less than a therapeutically effective amount.

It is to be noted that dosage values may vary with the severity of the condition to be alleviated. For any particular subject, specific dosage regimens may be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions. Dosage ranges set forth herein are exemplary only and do not limit the dosage ranges that may be selected by medical practitioners. The amount of active compound(s) in the composition may vary according to factors such as the disease state, age, sex, and weight of the subject. Dosage regimens may be adjusted to provide the optimum therapeutic response. For example, a single bolus may be administered, several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It may be advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. In some embodiments, compounds and all different forms thereof as described herein may be used, for example, and without limitation, in combination with other treatment methods for at least one indication selected from the group consisting of: prostate cancer, Ewing's sarcoma, breast cancer or pancreatic cancer. For example, compounds and all their different forms as described herein may be used as neoadjuvant (prior), adjunctive (during), and/or adjuvant (after) therapy with surgery, radiation (brachytherapy or external beam), or other therapies (for example, HIFU).

In general, compounds as described herein should be used without causing substantial toxicity. Toxicity of the compounds as described herein can be determined using standard techniques, for example, by testing in cell cultures or experimental animals and determining the therapeutic index, i.e., the ratio between the LD50 (the dose lethal to 50% of the population) and the LD100 (the dose lethal to 100% of the population) . In some circumstances however, such as in severe disease conditions, it may be appropriate to administer substantial excesses of the compositions. Some compounds as described herein may be toxic at some concentrations. Titration studies may be used to determine toxic and non-toxic concentrations. Toxicity may be evaluated using animal studies may be used to provide an indication if the compound has any effects on other tissues.

Compounds as described herein may be administered to a subject. As used herein, a "subject" may be a human, non-human primate, rat, mouse, beaver, cow, horse, pig, sheep, goat, dog, cat, etc. The subject may be suspected of having or at risk for having a cancer, such as prostate cancer, Ewing's sarcoma, breast cancer or pancreatic cancer. Diagnostic methods for various cancers, such as prostate cancer, Ewing's sarcoma, breast cancer or pancreatic cancer, and the clinical delineation of cancer, such as prostate cancer, Ewing's sarcoma, breast cancer or pancreatic cancer are known to those of ordinary skill in the art.

Various alternative embodiments and examples of the invention are described herein. These embodiments and examples are illustrative and should not be construed as limiting the scope of the invention.

Discovery of small molecules that target the ETS domain of ERG protein:

ERG overexpression, driven by the TMPRSS2-ERG gene fusion in prostate cancer cells, has been reported by a number of previous studies. To confirm ERG overexpression in PCa, we compared tumor-specific upregulated genes from three published datasets (Taylor etal. 2010, Wyatt etal. 2014, 2015) based on a 2-fold differential expression threshold (FIGURE 5). While there were a number of genes dysregulated in each pair-wise dataset comparison (data not shown), the only upregulated gene common in all three datasets was ERG, which highlights ERG as a potential major influencer of prostate cancer. There are numerous TMPRSS2-ERG fusions that encode for ERG transcripts. Whereas, the majority produce amino terminal-truncated ERG proteins, all retain the C- terminal DNA-binding ETS domain (St John et al. 2012). This DNA-binding ETS domain is essential for ERG to function as a direct transcriptional regulator, and structural data is available for its complex with DNA. Thus, we reasoned that we could use in silico approaches to identify small molecules targeting the ETS domain. Such molecules should therefore inhibit transcriptional activity of all functional ERG mutant proteins by antagonizing their ability to interact with DNA. This in turn might also disrupt ERG-mediated transcriptional events involved in disease development and progression.

A structure-based virtual screening approach, previously established for targeting protein-DNA and protein-protein interaction interfaces (Dalai et al. 2014), was applied to the 1.7A resolution ERG-ETS domain crystal structure [PDB ID: 4IRG] (Regan et al. 2013). The DNA binding interface was identified from a 2.8 A resolution crystal structure of the corresponding ERG-ETS domain/ DNA complex [PDB ID: 4IRI] (structures not shown) was produced. The ERG-ETS domain contained a winged helix-turn-helix motif, with helix 03 positioned within the major groove of the DNA containing a cognate GGAA sequence (Regan et al.,). A top-ranked druggable surface pocket was identified by virtual atomic probes to partially overlap this ERG-DNA interface (FIGURE lA). The identified pocket is adjacent to the DNA recognition helix (03), and thus it was predicted that a small molecule bound at this site will competitively block DNA binding. Three million chemical structures derived from the ZINC database (Irwin et al. 2012) were individually docked into this pocket. Combining the docking scores, binding poses, consensus voting and drug-like properties (detail in supplementary methods), an initial set of 48 compounds, representing 45 different chemical classes, were selected for in vitro analysis.

To evaluate the biological anti-ERG activity of the compounds identified above, we first confirmed ERG expression in a panel of prostate cancer cell lines (in PC3; VCaP; PNTiB; PNTiB-Mock; and PNTiB-ERG cells - data not shown) by Western blot analysis as compared to tubilin. We confirmed expression of the ERG protein in VCaP (endogenous expression) and PNTiB-ERG cells [stable ERG overexpression (Becker-Santos et al. )]. In contrast, PC-3, PNTiB and PNTiB-Mock cells were negative for ERG expression. Each of the compounds was first evaluated in PNTiB-ERG cells at concentrations of 10 μΜ and 25 μΜ for its ability to inhibit ERG transcriptional activation of a transiently transfected Endoglin E3 promoter- derived ETS-responsive Firefly luciferase reporter (pETS-luc) construct containing 3 conserved ETS recognition (GGAA) motif (Pimanda et al. 2006). Compound VPC-18005 was identified as the most potent inhibitor of luciferase activity from this initial set. Before proceeding with in depth analysis, the media solubility of VPC-18005 was assessed. VPC- 18005 exhibited excellent solubility after 3 days as compared to that of the published inhibitor YK-4-279 (93 vs 60%, respectively).

A more thorough dose response analysis was performed using both VCaP and PNTiB- ERG cells to evaluate the potency of VPC-18005. VPC-18005 was found to inhibit pETS-luc reporter activity in PNTiB-ERG and VCaP cells with IC₅₀ values of 3 and 6 μΜ, respectively (FIGURE lB). This compared favorably relative to, YK-4-279 (Rahim et al. 2011), that exhibited IC₅₀ values of 5 μΜ and 16 μΜ in parallel PNTiB-ERG and VCaP cell-based ETS-Luc reporter assays, respectively (FIGURE lC).

In order to assess whether the suppressed pETS-luc reporter activity was due to cytotoxicity, an MTS assay was performed over 72 h to measure the impact of the compounds on cell viability (FIGURE lD). VPC-18005 treatment (0.2 - 25 μΜ) did not decrease viability of either ERG-expressing cells (PNTiB-ERG and VCaP) or non-ERG expressing (PC-3) prostatic cells. Previous reports suggest that the IC₅₀ for YK-4-279 cytotoxicity is 10 μΜ in VCaP cells and > 100 μΜ in PC-3 cells (Rahim et al. 2011). However, in this study we observed YK-4-279-mediated inhibition of cell viability in both ERG expressing and non-ERG expressing cell lines at doses≤ 5 μΜ. This was also observed in the luminescence reporter assays, particularly in VCaP cells where concentrations above 10 μΜ had low Renilla readings, suggestive of altered cell viability (FIGURE lC). This result was further validated using real time cell in vitro analysis (i.e. Incucyte was used to monitor proliferation of PNTiB-ERG cells in the presence of VPC-18005 or YK-4-279 - see FIGURES 6A and 6B), which demonstrated a dose-dependent effect of YK-4-279 on cell growth. Most notably the cells were observed to have apoptotic morphology, indicative of toxicity. In contrast, no suppression of proliferation or induction of apoptosis was observed for VPC-18005. To confirm that VPC-18005 does not have a non-specific cellular effect, this compound was tested against an androgen receptor luciferase reporter (ARR₃tk-luc), and no significant effect on the reporter expression was observed (data not shown). These results indicated that a compound such as VPC-18005 identified by virtual screening could suppress ERG reporter activity without exhibiting overt cytotoxicity. VPC-18005 or YK-4-279 were also compared for their stability in media (% remaining after 3 days 93% VPC-18005 or 60% YK-4-279) and for solubility in media (μΜ) were both >50.

As there was no obvious effect of VPC-18005 on general cytotoxicity, we next assessed whether the impact of VPC-18005 treatment on ETS reporter activity was due to decreased ERG protein stability. After protein production was halted with cyclohexamide, VCaP cells were treated with VPC-18005 at up to 50 μΜ for 4 h. VPC-18005 did not induce ERG protein degradation after compound treatment (data not shown). At extended time points of 24 and 48 h, there was also no observable degradation (data not shown). Since ERG protein levels were stable in cells treated with VPC-18005, we assessed whether VPC-18005 could disrupt ERG-DNA binding. We performed an electrophoretic mobility shift assay (EMSA) using nuclear extracts of VPC-18005-treated VCaP cells and a biotin tagged CXCR4 promoter oligonucleotide (bases -912 to -932), previously identified as an ERG binding site (Singareddy et al. 2013). No probe mobility shift was observed in the absence of VCaP nuclear lysate. Nuclear lysate from untreated VCaP cells resulted in decreased electrophoretic mobility of probe. This band is deduced to be an ERG-CXCR4 oligo complex because the band was eliminated when the nuclear lysate was incubated with ιοχ excess unlabeled probe. Addition of VPC-18005 at 1 μΜ and 5 μΜ to nuclear lysates resulted in no detectable ERG-CXCR4 complex formation. EMSA assays were also performed using purified ERG-ETS domain and an independent DNA oligonucleotide containing the GGAA ETS recognition motif (data not shown). These experiments demonstrated that the recombinant ERG-ETS domain binds this cognate DNA with a KD ~ 5 nM and that VPC-18005 exhibits dose-dependent disruption of recombinant ERG-ETS/DNA complex formation with a Ki value of ~ 250 μΜ (FIGURE 7A & 7B). Collectively, these results indicate that VPC-18005 can disrupt binding of ERG to ETS response elements.

Direct binding of VPC-18005 to the ERG-ETS domain: The chemical structure of VPC-18005 is depicted in FIGURE 2A. Using computational modeling methods, the predicted binding pose of VPC-18005 was visualized in more detail inside the target pocket on the ERG-ETS domain (FIGURE 2B). In this pose, the molecular docking score of VPC-18005 was ranked in the top 0.01% of all 3 million molecules evaluated in the virtual screening discussed earlier. VPC-18005 is composed of a hydrophobic isopropyl benzyl group at one end and a negatively charged 5' carboxyl 4-thiazolidanone group on the other end, linked by an azo moiety with conjugated double bonds. Within the binding pocket on the ERG-ETS domain, VPC-18005 is predicted to form a salt bridge with Lys357, hydrogen bonds with Leu3i3, Trp35i and Tyr372, and hydrophobic interactions with a number of surrounding amino acid residues, including Gln3i2, Trp3i4, Tyr37i, Tyr372, Lys375, Ile377, Ile395, Ala398, and Leu399 (residue numbering based on ERG isoform 5, UniProt ID: P11308-4).

We utilized NMR spectroscopy to directly assess the binding of VPC-18005 with the ERG-ETS domain. The ¹⁵N-HSQC spectrum of ¹⁵N -labelled protein (100 μΜ) was assessed in the presence of increasing concentrations of DMSO-solubilized VPC-18005 (FIGURE 8), as well as with a DMSO control (data not shown). The spectra demonstrated small dose- dependent chemical shifts changes for a number of amide1H Ν g1rHoups that occurred upon addition of VPC-18005, but not DMSO. A chemical shift perturbation plot with VPC-18005 at 1:10 molar ratio (i.e. 1 mM) showed that protein residues with changes greater than the mean (0.01 ppm) were mostly located along helix α1, helix 03 and strand 3 (FIGURE 2C). These amides cluster around the predicted binding pocket of VPC-18005 (FIGURE 2D), supportive of its binding pose with the ERG protein. Of note, residues with perturbed amide chemical shifts, including Leu3i3 on helix cti and TV1371, Try372, Lys375 on helix 03, modeled to interact with VPC-18005 through hydrogen bonds and hydrophobic interactions, have also been previously shown to be involved in ERG-DNA interactions (Regan et al. 2013). Fitting of the ¹⁵N-HSQC titration curves to a simple 1:1 binding isotherm yielded a KD value of ~ 3 mM for the interaction of VPC-18005 with recombinant ERG-ETS domain (FIGURE 8). While this is indicative of relatively weak binding, it is in agreement with the Ki value determined by EMSA analysis for the competitive disruption of the ERG-DNA complex by VPC-18005. To further localize the binding interactions between VPC-18005 and the ERG-ETS domain, the reverse titration was performed. In this case, the 1-HNMR spectrum of VPC-18005 was monitored vs. increasing concentrations of recombinant ERG-ETS domain. Several nuclei 1H of VPC-18005 exhibited ERG-dependent chemical shift perturbations. These include the hydrogens on the aromatic ring (Ή 7.78 and 7.45 ppm), the methyl's on the isopropyl group (Ή 1.25 ppm) and the conjugated double bond (Ή 8.4 ppm) (FIGURE 2E). Due to the spectral overlap with signals from DMSO, perturbations from the CH₂ group near the carboxyl group of VPC-18005 could not be determined. Overall, these two complimentary direct binding assay results are consistent with the proposed model for how VPC-18005 binds to the ERG-ETS domain and disrupts DNA binding. There were no perturbations due to the control titration with DMSO.

As a further experimental test of the in silico model, additional analogs based on the VPC-18005 scaffold were developed through both chemical similarity searches and medicinal chemistry modifications (TABLE 1). Of these candidates VPC-18065 and 18098, with terminal moieties that are more hydrophobic, demonstrated slightly better IC₅₀ values (2 μΜ and ΐμΜ respectively in luciferase assays) compared to VPC-18005 (FIGURE 10). The removal of the carboxyl group in VPC-18100 resulted in the loss of inhibition in the luciferase reporter assays. Although the modifications tested to date do not result in significant sub- micro molar activity, the derivatives do provide a working structure-activity relationship (SAR) that will guide future medicinal chemistry.

VPC-18005 inhibits migration and invasion of ERG-overexpressing cells in vitro: ERG promotes EMT, which enables cells to acquire migratory and invasive characteristics (Adamo and Ladomery 2015). PNTiB cells were previously shown to acquire invasive characteristics when ERG was stably overexpressed (Becker-Santos et al. 2012). Therefore, we aimed to determine if VPC-18005 was able to affect migration of these cells. PNTiB-MOCK and -ERG cells were plated into the upper chamber of a double chamber realtime cell analysis system and treated with VPC-18005 after 24 h. As expected, in the absence of VPC-18005, PNTiB-ERG exhibited an increased rate of migration toward the serum containing bottom chamber compared to the PNTiB-MOCK control (FIGURES 3A & 3B). After 24 h exposure and in comparison to a DMSO control, VPC-18005 (5 μΜ) significantly reduced the rate of migration of the PNTiB-ERG cells relative to vehicle-treated cells, and the resulting migration rate was indistinguishable from that observed for vehicle treated PNTiB- MOCK cells (p = 0.031; FIGURE 3C). In contrast, but consistent with the cytotoxicity results described earlier, treatment with YK-4-279 resulted in cytotoxicity in both cell lines. In addition, a titration was performed which demonstrated a dose dependent effect of VPC- 18005 on cell migration 48-72 h post-exposure (data not shown). To further explore this inhibitory effect of VPC-18005, PNTiB-ERG and PNTiB-MOCK spheroids, pretreated for 24h with vehicle control or VPC-18005, were submerged in matrix in the presence or absence of treatments, and monitored for 6 days. Analysis of images captured every 2 days revealed that VPC-18005 significantly reduced the invasion of ERG-expressing PNTiB cells into the surrounding matrix (p = 0.02; FIGURE 3D).

VPC-18005 inhibits migration of ERG-overexpressing cells in vivo: To determine whether VPC-18005 could affect cell migratory behavior in an animal model, we utilized the zebrafish xenotransplantation model as a tool to investigate cell extravasation (Teng et al. 2013). We first investigated whether PNTiB-MOCK and PNTiB-ERG could disseminate through the zebrafish body (FIGURE 4A). Fluorescently tagged cells were injected into the yolk sac and after 5 days PNTiB-ERG could be seen throughout the body of the fish. In contrast, PNTiB-MOCK cells were not observed outside of the yolk sac. When cultured in the presence of VPC-18005 for 72 h, the embryos remained viable up to a concentration of 75 μΜ. In contrast, YK-4-279-treated embryos exhibited toxicity at concentrations > 10 μΜ (FIGURE 4B). Yolk sac-inoculated PNTiB-ERG and VCaP cells were found to become disseminated toward the head and tail of 65 to 70% of embryos, respectively. When cultured in the presence of VPC-18005 at 1 and 10 μΜ, this percentage of fish with PNTiB-ERG or VCaP dissemination was reduced to 20-30% of inoculated animals (FIGURE 4C). Culturing embryos in YK-4-279 at 1 and 10 μΜ resulted in yolk sac dissemination in 30- 45% of inoculated animals (data not shown). These assays provide first principle evidence that targeted small molecules can antagonize the metastatic potential of ERG-expressing prostate cells.

MATERIALS AND METHODS

In silico modeling and virtual screening: The published ERG-ETS domain X- ray crystal structure (PDB: 4IRG) (Regan et al. 2013) was subjected to the Site Finder™ algorithm, implemented in the Molecular Operating Environment™ (MOE) (Chemical Computing Group), which used virtual atomic probes to search the protein surface for suitable small molecule binding pockets. The crystal structure of an ERG/DNA complex (PDB: 4IRI) (Regan et al. 2013) was used to define the ERG-DNA interface. The top-ranked pocket was identified and used for the subsequent virtual screening. Before molecular docking, the ERG- ETS domain structural model was prepared by using the Protein Preparation Wizard™ module of the Maestro V9.3™ program from the Schrodinger™ 2012 software suite. The docking grid was centered at the pocket composed of the following amino acids: Pro3o6, Gly307, GI11310, Ile311 , GI11312, Leu3i3, Trp3i4, Trp35i, Lys355, Met360, Lys364, Leu365, Ala368, Tyr37i, Tyr372, Lys375, Ile377, Ile395, Ala398, Leu399 (residue numbering based on ERG isoform 5, UniProt ID: P11308-4). A total of 19,607,722 (~20 million) small molecule structures were downloaded from the ZINC database version 12 (Irwin et al. 2012). Among the 20 million set, a total of 2,990,102 (~ 3 million) molecules that possess the following leadlike and drug-like properties were extracted for molecular docking: molecular weight between 250 and 350 Da, logP <= 5, hydrogen-bond donors <= 5, hydrogen -bond acceptors <= 10, number of rotatable bonds <= 10, and number of rings <= 4. Each molecule was given its expected protonation state at pH 7 and energy-minimized under the MMFF94X (solvation: Born) force field using MOE. All of the 3 million molecules were compiled into a single SDF file as the input ligand database for the subsequent molecular docking step. Each molecule was docked into the previously defined docking grid on the ERG-ETS domain protein model, using the Glide program (Small-Molecule Drug Discovery Suite™, version 5.8, Schrodinger™, LLC, New York, NY, 2012). Standard Precision™ with all other parameters set to default. The top 1% (~30,ooo molecules), as ranked by the docking scores calculated based on interaction forces including hydrogen bonds and hydrophobic interactions, were selected to advance into the next stage of virtual screening. Within this set, a predicted p¾ was calculated for each molecule using a custom MOE SVL™ script, and ligand efficiency was calculated using Glide™. To avoid any 'frequent hitters', each chemical structure was computationally screened for any presence of Pan Assay Interference Compounds™ (PAINS), as implemented in FAFDrugs™ (Lagorce et al. 2015). A consensus scoring method was used: 1) each compound within the top 20% p¾ values received one vote; 2) each compound within the top 20% ligand efficiency values also received one vote; and 3) two votes were deducted if a compound was predicted to have PAINS™. The top 3,000 molecules, as ranked by the number of votes, were selected for the final stage of selection. During this step, the chemical structure of each molecule within the predicted ERG-ETS binding pocket was manually examined using the 3D visual environment in MOE. Preference was given to compounds with favorable binding poses and interactions with the surrounding amino acid residues. Molecules were removed from the selection if they contain any problematic or promiscuous moiety. In addition to manual examination, the FAFDrugs™ program was used to assist identification of such problematic groups. A total of 48 compounds were selected for testing. MOE and MarvinSketch™ were used to visualize and represent the protein models and chemical structures. Chemical similarity searches based on the Tanimoto coefficient was performed on the hit compound VPC-18005 in the ZINC™ database, with additional medchem designs done in MOE.

Solubility: Stock solutions of compounds at 50 mM in dimethyl sulfoxide (DMSO) were diluted ιοοοχ into methanol (MeOH), RPMI +5% CSS (media), and phosphate buffered saline (PBS) and vortex mixed for 1 hr, 800 rpm at RT. The resulting solutions were centrifuged at 20000 g for 5 min (RT) and saturated supernatants transferred to fresh Eppendorfs. Saturated PBS samples were further diluted with an equal volume of PBS. Aliquots of these solutions were analysed and the remainder stored at RT in the dark. Aliquots taken at later time points were vortex mixed for 1 h prior to sampling. MeOH and diluted PBS samples required no further processing; media samples were extracted with two volumes acetonitrile (ACN) and centrifuged at 20000g for 5 min. These MeOH, and diluted media and PBS samples were analysed using an Acquity™ UPLC coupled in series with an eLambda PDA™ and a Quattro Premier™ (Waters). A 100 mm BEH C18, 1.7 μ column (Waters) was used for separations with a 10-95% acetonitrile (ACN) gradient from 0.2-7 min followed by a 1 min 95% ACN flush and 2 min re-equilibration for a 10 min run length (0.1% formic acid present throughout). Wavelengths from 210-800 nm at 1.2 nm resolution and 2 points/sec were collected with the PDA. The sampler was maintained at RT and all MS data was collected in ES+ scan or single ion recording (SIR) mode at unit resolution with the following instrument parameters: capillary, 3.0 kV; extractor and RF lens, 3 V and 0.1 V; cone, 40 V; source and desolvation temperatures, 120 °C and 350 °C; desolvation and cone (N2) flow, 900 L/hr and 50 L/hr. The m/z for SIR functions were selected from MeOH scan datasets.

Quanlynx™ (Waters) was used for analysis of data, using extracted wavelength chromatograms selected for best signal to noise for PDA data and SIR for MS data. All compounds dissolved well in MeOH and these were used for calibration purposes with slopes forced through the origin. OD data was used in most cases with MS data mainly for PBS samples; SIR data was calibrated by applying the SIR/OD ratio from corresponding media samples where less saturation of MS data is expected. This rudimentary method is useful to 5θμΜ, performs well for solubility and relative stability at higher concentrations (μΜ) and gives reasonable estimates when the use of MS endpoints is needed.

Bioinformatics and statistical analyses on gene expression datasets from PCa patients: The gene expression datasets included 26 PCa and 5 normal patient samples from Vancouver Prostate Centre (VPC) (Wyatt et al. 2014), 150 PCa and 29 normal patient samples from Memorial Sloan-Kettering Cancer Center (MSKCC) (Taylor etal. 2010), and 498 PCa and 52 normal patient samples from The Cancer Genome Atlas (TCGAX2015). A list of upregulated genes were identified from each dataset by the following steps: 1) log2 transformation; 2) two sample t-test between tumor and normal samples; 3) multiple testing correction on p-values; 4) selection of genes with corrected (adjusted) p-values < 0.05; and 5) among those with significant p-values, selection of genes with fold-change≥ 2 (tumor vs. normal).

Cell Culture: VCaP (CRL-2876) and PC-3 (CRL-1435) human prostate carcinoma cells were obtained from the American Type Culture Collection (ATCC, August 2014). The PNTiB-Mock and PNTiB-ERG lines were generated at the VPC (Becker-Santos et al. 2012). Cells were grown in a humidified, 5% C0₂ incubator at 37 °C. PC-3 cells were maintained in RPMI 1640 medium (Life Technologies™) supplemented with 5 % (v/v) fetal bovine serum (FBS). The VCaP cell line was maintained in DMEM (ATCC) supplemented with 10 % FBS. PNTiB-Mock and -ERG cells were maintained in DMEM (Life Technologies™) supplemented with 10 % FBS and under selection with blasticidin.

Dual reporter luciferase assay: All of the compounds selected from the virtual screening were tested in a luciferase-based ERG-responsive reporter assay, using two ERG- overexpressing cell lines: 1) VCaP cells that harbor an endogenous TMPRSS2-ERG gene fusion; and 2) PNTiB-ERG cells previously developed at VPC (Becker-Santos et al. 2012). Cells (3000) in 150 μL per well of a 96 well plate were seeded and after a 24 h incubation were transfected with 50 ng of an Endoglin E3 promoter-derived ETS-responsive Firefly luciferase reporter (-507/-280 of (E3) promoter (Pimanda etal. 2006) inserted into luciferase reporter vector (Signosis™) and 5 ng of the Renilla luciferase reporter (pRL-tk, Promega™) using TransIT 20/20 transfection reagent (Mirus™, USA). After 16 h incubation, cells were treated with compound for a further 48 h. Luciferase and Renilla activity was measured using a TECAN M200Pro plate reader. Comparison of empty vector versus ETS responsive reporter demonstrates activation only in the presence of the ETS responsive sequence (data not shown). Data were normalized first to Renilla and then to the DMSO-media control on each plate. Initial hit compounds were identified as those with an average normalized luciferase reading (luciferase reading/Renilla reading) that is 60 % or less of the average normalized luciferase reading of the DMSO-media control (i.e. 40 % or more reduction of luciferase activity) at 10 μΜ. The luciferase assays were repeated for each lead compound under multiple concentrations (0.1 to 100 μΜ) to establish a dose-dependent response and an IC₅₀ value. AR reporter assay was performed as previously described (Dalai et al. 2014).

Proliferation and cell viability assays. MTS: Cells were seeded at a density of 3000 cells per well (except VCaP at 20,000/well) in 100 μL of appropriate media in 96 well culture dishes. Twenty four hours later, 100 μL of medium containing vehicle control or compounds. Each treatment was prepared in triplicate. After a 72 hr treatment, cellular viability was assessed using CellTiter 96™ Aqueous One Solution Cell Proliferation Assay™ reagent (Promega™) according to the manufacturer's instructions. Values were normalized to the DMSO control. Incucyte generated growth curves: VCaP Cells (20,000 cells/well) were plated in a 96 well plate. After 24 h, plates were treated with vehicle control, VPC-18005 or YK-4-279. Growth curves were constructed by imaging plates using the Incucyte™ system (Essen Instruments™), where the growth curves were built from real-time confluence measurements acquired during round-the-clock kinetic imaging for 7 days.

NMR spectroscopy: ERG-ETS domain expression and purification: The plasmids encoding residues 307-400 of the ERG-ETS domain were expressed in E. coli BL21 (λDE3). Cultures of 1 L were grown at 37 °C in M9 media supplied with 3 gm/L ¹³C6-glucose and/or 1 gm/L ¹⁵NH₄Cl. Cells were allowed to grow to O.D = 0.6 and protein expression was induced by adding 1 mM IPTG. After an induction time of 4 hrs, cells were harvested by centrifugation and stored at -80 °C for at least 1 round of freeze/thaw. Cells were resuspended in 40 mL of lysis buffer for every 1 L of culture. Cells were lysed by passing through 5 rounds of homogenization and 10 mins of sonication. The cell lysate centrifuged at 15k rpm for 1 hr, and the supernatant subjected to nickel column purification. The column was washed using 25 mM imidazole (50 mM phosphate, 1 M NaCl, pH 7.4) and proteins were eluted with 1 M imidazole. Fractions containing the ETS domain were confirmed by SDS-PAGE and pooled. The His6-tag was cleaved by thrombin and the tag-free sample was concentrated to 2 mL and subjected to S75 size exclusion chromatography. Fractions were checked by SDS-PAGE and those containing the pure sample were pooled and concentrated. The protein ample was dialyzedto NMR buffer (20 mM sodium phosphate, 150 mM NaCl, 2 mM DTT, 0.1 mM EDTA, pH 6.5) for all NMR experiments. NMR spectral assignments: NMR data were recorded at 25 or 28 °C on cryoprobe-equipped 850 MHz Bruker Avance III™ spectrometer. Data were processed and analyzed using NMRpipe™ (Delaglio et al. 1995) and Sparky (T. D. Goddard and D. G. Kneller and 1999). Signals from backbone and sidechain1H, ¹³C, and ¹⁵N nuclei were assigned by standard multidimensional heteronuclear correlation experiments. NMR- monitored titrations: Interactions of compounds with the ERG-ETS domain were monitored via sensitivity-enhanced ¹⁵N-HSQC spectra. Experiments involved titrating unlabeled DMSO-solubilized compound or control DMSO into ¹⁵N-labeled ERG-ETS domain. Chemical shift perturbations were calculated from the combined amide1H ^Ν and ¹⁵N shift changes as Δδ = [(0.2 ΔδΝ)² + (ΔδΗ)²]^ι/2. Reciprocal titrations were carried out using1H - NMR to monitor the effects of progressively adding unlabeled protein to a sample of VPC- 18005 (ι8θμΜ) in 20 mM phosphate, 150 mM NaCl, 2 mM DTT, 0.1 mM EDTA, pH 6.5 . The signal from water was suppressed by pre-saturation. Real time cell analysis (xCELLigence): Cell migration was monitored using CIM- 16 migration plates via the xCELLigence™ platform (ACEA). FBS-supplemented media (160 μL) was added to the lower chamber of the plate and incubated at RT for 30 min. The upper chamber was then mounted and 30 μL of serum free media (SFM) was added to each well and left to equilibrate in the incubator for 1 h at 37 °C. After the incubation, a background reading was taken for each well. PNTiB-ERG or -MOCK cells (24 h starved) were prepared in SFM, and 30,000 cells in 70 μL were seeded to each well of the upper chamber in addition of 100 μL of desired treatment (vehicle control, VPC-18005, and YK-4-279). Real time readings of cell index values were recorded initially every 5 min until the end of the experiment (48 hr).

Spheroid invasion assay: 3D Spheroid BME Cell Invasion Assay™ (Trevigen™) was performed as per manufacturer's instructions. Briefly, 5,000 PNTiB-ERG cells and 5 μL of ECM were prepared in growth media to a total volume of 50 μL and seeded in 3D culture qualified 96 well spheroid formation plate and incubated at 37 °C for 72 hr. Spheroids were pre-treated with VPC-18005 or DMSO for 24 h after which 50 μL gel invasion matrix was added. Spheroids were then incubated at 37 °C for 3 to 7 days, and photographed using Zeiss AxioObserver Zi™ microscope in each well on the day of invasion mix addition and every two days following. Spheroids were retreated with 50 μL of vehicle control or compound after 72 hr.

Western Blot: Cells were lysed on ice with RIPA buffer containing a protease inhibitor cocktail (Pierce™). Primary antibodies: ERG (1:1,000, EPR3864(2), Abeam), ct- Tubulin (1:20,000, Millipore™), Vinculin (1:1,000, Abeam™). Immunoreactivity was detected with the use of the goat anti-rabbit or rabbit anti-mouse horseradish peroxidase (HPR)-conjugated secondary antibody (1:10,000) (Santa Cruz™), and visualization was achieved by chemiluminescence (Pierce™).

Electrophoretic mobility shift assay (EMSA). Using nuclear lysate: EMSA was performed as per manufacturer's protocol (Panomics™). Briefly, 10 μg nuclear extracts (CelLytic NuCLEAR Extraction Kit™, Sigma™) from VCaP cells, 1 μL polyd(I-C), 2 μL 5X binding buffer, and nuclease-free water up to 7 μL were mixed together and incubated for 5 min at RT. Biotin-labeled DNA probe (5' AAT GCG GGC CTT GTC TGG TTC 3'(Singareddy et al. 2013)) was added (0.25 ng) and the resulting samples were incubated for 30 min at 15 °C in a thermal cycler. Samples were resolved on a 6 % native gel (run at 120 V at 4 °C, transferred to a nylon membrane (Pall™), and fixed using UV cross-linker for 3 min. Membrane was incubated with Streptavidin-HRP for 15 min, followed by Detection buffer for 5 min. The membrane was finally incubated in kit solutions I, II and III and exposed to X-ray film. EMSA competitor was conducted using ιοχ excess unlabeled oligo nucleotide in the reaction mix. Using purified ERG-ETS domain: Purified ERG-ETS domain (see NMR spectroscopy) was stored in buffer (20 mM sodium phosphate, 200 mM NaCl, 2 mM DTT, 0.1 mM EDTA, pH 6.5). To prepare the probe for the gel shift assay, equal amounts (200 nM) of Alexa-488™ fluorophore-labeled DNA (5'-CGG CCA AGC CGG AAG TGA GTG-3') and it reverse complement strand were mixed, heated to 95 °C for 30 minutes, and then slowly cooled to 25 °C in several hours. An initial gel shift assay was performed by titrating constant 1 nM labeled dsDNA with ERG (0.5 μΜ to 0.3 pM). Glycerol (3 %) and 0.2 mg/mL BSA was added into the reaction mixture and incubated at room temperature for 1 hr before loading on to 10 % acrylamide native gel, and running at 10 °C. The gel was scanned with Typhoon 9200 Imager™ equipped with blue laser to excite at 490 nm and measure at 520 nm. The scanned image was analyzed with Image J™ (Rasband 1997-2015). Fitting the titration data to a 1:1 binding isotherm yielded the equilibrium dissociation constant (KD value) for the ERG-ETS domain interaction with DNA. This was used to set the molar ratio of ERG-ETS domain:DNA in subsequent competition assays with VPC-18005. For these assay, 4 nM of ERG was mixed with 1 nM of fluorophore-labeled dsDNA, and subsequently titrated with VPC-18005 (diluted from a DMSO stock) and analyzed by the same EMSA protocol. A control experiment was carried out by titrating with equivalent quantities of DMSO.

Zebrafish: Husbandry. Research was carried in accordance with protocols compliant to the Canadian Council on Animal Care and with the approval of the Animal Care Committee at the University of British Columbia. The wildtype zebrafish strain was maintained in aquaria according to standard protocols (Westerfield 2000). Embryos were generated by natural pair-wise matings and raised at 28.5 °C on a 14 h light/ 10 h dark cycle in a 100 mm² petri dish containing aquarium water. Phenylthiourea (0.2 mM PTU, Sigma™) was added to the embryos at 10 hr post-fertilization (hpf) to prevent pigment formation. Dissemination assay. PCa cell lines were fluorescently labelled the day before microinjection with 1.5 μΜ of CellTracker CM-Dil™ dye (Life Technologies™) as per manufacturer's instructions. Wild-type embryos were dechorionated at 2 dpf. Following anaesthetization with tricane, approximately 50-70 cancer cells were microinjected into the yolk sac. Embryos were then transferred to 100 mm² plates that contained aquaria water with added PTU and VPC-18005, YK-4-279 or DMSO control. Embryos were visually assessed for presence of xenograph. Those embryos that did not contain cells were removed from the experiment. Embryos were kept at 35 °C for the duration of the experiment. Approximately, 50 fish were injected per cell line and metastasis was determined on Day 4 and 5 by observation using the Zeiss Axio Observer™ microscope (5X objective) controlled with Zen 2012 software. Fixed (dead) cells were used as a control to ensure that the dissemination observed was not due to yolk sac absorption. Statistics. Data are presented as mean ± SEM unless indicated otherwise. Statistical analyses were performed with the use of the IBM SPSS 22 Statistics (IBM™ Corp.) or GraphPad Prism 6™ (GraphPad Software™, Inc.). Statistical significance for all comparisons was set at p < 0.05.

SYNTHESIS INFORMATION

Synthetic compounds: Compounds were synthesized and purchased from a number of chemical vendors. Compounds described herein may be synthesized as by methods known to a person of skill in the art.

[VPC-18061] The compound was prepared according to: Derivatives of 5- Carboxymethylthiazolidine-2,4-dione, a New Group of Antiviral Compounds. A. Krbavcic, M. Plut, A. Pollak, M. Tisler, M. Likar, P. Schauer. J. Med. Chem., 1966, 9 (3), pp 430-431.

Chemical synthesis of [VPC-18005] (FIGURE 9). General Experimental Procedures: All reagents and solvents were purchased from commercial suppliers and used without further purification unless otherwise stated. The reactions were monitored by thin layer chromatography (TLC) on pre-coated silica gel F254 plates (Sigma-Aldrich™) with a UV indicator using ethylacetate/hexane (1:2 v/v). Yields were of purified product were not optimized. The purities of the newly synthesized compounds were determined by LC-MS analysis using an Agilent 1100 LC system. The compound solution was injected into the ionization source operating positive and negative modes with a mobile phase acetonitrile/water/formic acid (50:50:0.1 % v/v) at 1.0 mL/min. The instrument was externally calibrated for the mass range m/z 100 to 650. The -NMR1 sHpectra were measured on a Varian GEMINI 2000™ NMR spectrometer system with working frequency of 400 MHz. Chemical shifts δ are given in ppm, and the following abbreviations are used: singlet (s), doublet (d), triplet (t), quartet (q), multiplet (m), and broad singlet (br s).

[VPC-i8oo5]:2-((Z)-2-(((Z)-4-isopropylbenzylidene)hydrazono)-4- oxothiazolidin-5-yl)acetic acid (4d). To a stirred solution of 4-isopropylbenzaldehyde (id) (563 mg, 3.8 mmol) in PhMe (2 mL) and DMF (2 mL) were added thiosemicarbazide (2) (290 mg, 3.2 mmol) and p-TsOH acid (5 mg, 0.03 mmol). The reaction mixture was heated in stirred microwave vial for 10 min at 90 °C. After formation of thiosemicarbazone derivate and testing by TLC, maleic anhydride (3) (343 mg, 3.5 mmol) was added, and the reaction mixture was heated for 40 min at 110 °C in the microwave. Recrystallized from AcOH yielded (4d, VPC- 18005) (300 mg, 29 % yield) as a white solid with 99 % purity by LC/MS. -NMR1H (DMSO- de, 400 MHz): 1.20-1.22 (6H, d), 2.69-2.76 (lH, m), 2.90-2.94 (2H, m), 4.25-4.28 (lH, d), 7.31- 7.33 (2H, d), 7.66-7.68 (2H, d), and 8.34 (1H,s). MS (ESI) m/z (M + H)⁺ calculated for Ci₅H₁₇N₃0₃S: 319.4, found: 320.2. The final product is racemic and has several possible isomeric forms that have not been experimentally defined.

General Experimental Procedures for [VPC-18098], [VPC-180104], [VPC18106]:

Scheme of compound synthesis:

Ar = 4-c-BuPh (a); 4-t-Bu-2-OHPh (b); 4-t-Bu-2-FPh

[VPC-i8o98]:2-((Z)-2-(((Z)-4-Cyclobutylbenzylidene)hydrazono)-4- oxothiazolidin-5-yl)acetic acid (4a). To a stirred solution of 4-cyclobutylbenzaldehyde (la) (224 mg, 1.4 mmol) in PhMe (1 mL) and DMF (1 mL) were added thiosemicarbazide (2) (102 mg, 1.1 mmol) and p-TsOH acid (2 mg, 0.01 mmol). The reaction mixture was heated in stirred microwave vial for 10 min at 90 C. After formation of thiosemicarbazone derivate, (followed by TLC) maleic anhydride (3) (88 mg, 0.9 mmol) was added, and the reaction mixture was heated 15 min at 110 C in the microwave. The residue was finally recrystallized from AcOH to give (4a) (16 mg, 4% yield) as a white solid. Purity 100% by LCMS. MS (ESI) calculated for Ci6H₁₇N₃0₃S m/z (M + H)⁺ : 331.4, found:332.i.

[VPC-18104]: 2-((Z)-2-(((Z)-4-(tert-Butyl)-2-hydroxybenzylidene)hydrazono)-4- oxothiazolidin-5-yl)acetic acid (4b). To a stirred solution of 4-(tert-butyl)-2- hydroxybenzaldehyde (lb) (250 mg, 1.4 mmol) in PhMe (2 mL) and DMF (2 mL) were added thiosemicarbazide (2) (102 mg, 1.1 mmol) and p-TsOH acid (2 mg, 0.01 mmol). The reaction mixture was heated in stirred microwave vial for 10 min at 90 C. After formation of thiosemicarbazone derivate, (followed by TLC) maleic anhydride (3) (117 mg, 1.2 mmol) was added, and the reaction mixture was heated 15 min at 110 C in the microwave. The residue was finally recrystallized from AcOH to give (4b) (250 mg, 65% yield) as a white solid. Purity 100% by LCMS1.H NMR (DMSO-d₆, 400MHz): 1.27 (9H, s), 2.90-3.07 (2H, m), 4.43-4.46 (lH, d), 6.92 (lH, d), 6.98-7.00 (lH, d), 7.48-7.50 (lH, d), 10.80 (1H,s), 12.1 (0.5H, bs), 12.7 (0.5H, bs). MS (ESI) calculated for C_l6H₁₉N₃0₄S m/z (M + H)⁺ : 349.4, found:350.i. [VPC-18106]: 2-((Z)-2-(((Z)-4-(tert-Butyl)-3-fluorobenzylidene)hydrazono)-4- oxothiazolidin-5-yl)acetic acid (4c). To a stirred solution of 4-(tert-butyl)-3- fluorobenzaldehyde (lc) (700 mg, 3.5 mmol) in PhMe (2 mL) and DMF (2 mL) were added thiosemicarbazide (2) (260 mg, 2.9 mmol) and p-TsOH acid (5 mg, 0.03 mmol). The reaction mixture was heated in stirred microwave vial for 10 min at 90 C. After formation of thiosemicarbazone derivate, (followed by TLC) maleic anhydride (3) (314 mg, 3.2 mmol) was added, and the reaction mixture was heated 40 min at 110 C in the microwave. The residue was finally recrystallized from AcOH to give (4c) (712 mg, 70% yield) as a white solid. Purity 99% by LCMS. 1H NMR (DMSO-d₆, 400MHz): 1.36 (9H, s), 2.86-3.04 (2H, m), 4.36-4.38 (lH, d), 743-7.53 (3H, m), 8.37 (1H,s), 12.4 (1H, bs). MS (ESI) calculated for C16H18FN3O3S m/z (M + H)⁺ : 351.4, found:352.i.

General Experimental Procedures [VPC-18107 (MHoi-01)], [VPC-18100 (MH01- 02)]: General Scheme of compound synthesis:

Step A: To a solution of compound 1 (37.0 g, 216 mmol) and catalytic quantity of hydroquinone in dry ether (100 mL) a solution of (Et0)₂P0Na (38.0 g, 273 mmol) in dry ether

(150 mL) was added dropwise under argon atmosphere maintaining temperature below 25°C.

The mixture was stirred overnight at room temperature and then filtered. The filtrate was evaporated and the residue was purified by vacuum distillation (1 Torr) to obtain 7.7 g of ~70% pure compound 2 which was use as is in the next step. Step B: A mixture of compound 3

(20.6 g, 127 mmol) and thiosemicarbazide (11.5 g, 126 mmol) in EtOH (60 mL) was stirred at

6o°C for 72 hours. After cooling, the suspension was filtered. The filter cake was washed with

EtOH and dried to yield 18.0 g (76.5 mmol, 61%) of compound 4. Step C: A mixture of compound 4 (1.50 g, 6.37 mmol), methyl 2-clorobutyrate (0.850 g, 6.22 mmol), and melted sodium acetate (0.630 g, 7.68 mmol) in EtOH (15 mL) was refluxed for 2.5 days and then evaporated to dryness. The residue was purified chromatographically to yield 0.670 g (2.21 mmol, 35%) of target compound VPC-18100 (MH01-02). Step D: A mixture of compound 2 (3.70 g, 13.6 mmol), compound 4 (3.40 g, 14.4 mmol), and catalytic amount of KI in dry BuOH (290 mL) was stirred at ioo°C for a 48 hours under argon atmosphere and then evaporated. The residue was purified chromatographically to obtain 0.400 g (0.940 mmol, 7%) of 5. Step E: To suspension of compound 5 (0.260 g, 0.611 mmol) in dry MeCN (30 mL) Me₃SiBr (0.940 g, 6.14 mmol) was added dropwise under argon atmosphere maintaining temperature below o°C. The mixture was stirred for 72 hours at 25°C and then evaporated to dryness at 30-35°C. The residue was dissolved in methanol (10 mL) and mixture was stirred half an hour at room temperature. Then distilled water (0.5 mL) was added and the mixture was stirred for another half an hour at room temperature. The solution was evaporated, and the residue was dried under high vacuum at 40°C to obtain 0.052 g (0.141 mmol, 23%) of target compound VPC-18107 (MHoi-01).

Scheme 1

Scheme 2

Scheme 3.

F2167-9387. To a precooled ( 2 °C) solution of HCOOH (74-7g, 1.38 mol), Et₃N (78 ml) in DMF (250 ml) compounds F2190-0632 (66g, 0445 mol) and 2,2-dimethyl-i,3-dioxane- 4,6-dione (67.4 g, 0.47 mol), were added in one portion (temperature increased to 16 C). The reaction mixture was stirred at room temperature for ihr and at 80 °C for 12 hrs. The cooled down to ambient temperature reaction mixture was poured into stirred water (7 L). The product was precipitated by addition of concentrated aqueous HCl, the precipitate was filtered off and dried on air. Yield 85 %.

F9995-1919. SOCL (39-35g, o.33mol) was added in one portion to a suspension of F2167-9387 (53g, o.276mol) in benzene (80ml). The reaction mixture was stirred under reflux for 2hrs, the solvent was removed in vacuo , the residue (F2167-9387a) was vacuum dried and was used for the next step without additional purification. Yield 99 %. F2167-9387a (61.7g, 029mol) was added in portions at -5°C To a solution of aqueous ammonia (25 %, 3L) . The reaction mixture was stirred for 12 hrs at rt, the precipitate was filtered off washed with water and dried on air to give crude F2167-9387b. Yield 89 %. To a precooled (-5 °C) solution of F2167-9387b (50g, 0.26 mol), MeONa (6i.8g, 1.1 mol) in MeOH (lL) Br2 (50. lg, 16.1 ml, 0.31 mol) was added dropwise. The reaction mixture was stirred at o C for 30 min , ihr at 25 C and 2 hrs at 60 C. After cooling down to 20 °C the reaction mixture was quenched with 80ml of AcOH, the volatiles were removed in vacuo and the residue was triturated with water (stirring for ihr), the precipitate formed was filtered off washed with water and dried on air to give crude F9995-1918. Yield 95%.

F9995-1919- A solution of F9995-1918 (50g, 0.226 mol) in aqueous HC1 (35%, 121 ml) and AcOH ( 200 ml) was stirred under reflux for 24 hrs, the solvents were removed in vacuo, 200ml of toluene was added and evaporated in vacuo ( 2 times). The residue was triturated with MeCN (200 ml) and the solvent was evaporated in vacuo (2 times). The residue was triturated with MeCN (200 ml) the precipitate was filtered off, washed with DEE and dried in vacuo at 80 °C for shrs. Yield 89 %.

F9995-1919a · To a suspension of F9995-1919 (7-3g, 36.6 mmol) in H₂0 (20ml) and DCM (20ml) at o°C CSC1₂ (5g, 3.34 ml, 43.8 mmol) was added in one portion followed by portionwise addition of NaOH . The reaction mixture was stirred at rt for 20 hrs, the organic layer was separated and tried over MgS04, filtered and evaporated in vacuo to give 7g of oil which was distilled in vacuo T = 110-115 °C (1 torr) to give F9995-1919a (yield 80%)

F2167-9486. Compound F9995-1919a (2.6g, 12.6 mmol) was added dropwise to a saturated aqueous solution of ammonia (30%, 20ml) at -10 °C. The reaction mixture was stirred for 2 days at room temperature. The precipitate was filtered off and washed with methanol (ml) and dried in vacuo (shrs, 1 torr, 40 °C). Yield 88 %.

F2190-o632a. A solution of Znl₂ in DEE (2M, 0.5ml ) was added to F2190-0632 (66.3g, 044 mol) at rt. The solution was cooled down to o C and TMSCN (59g, o.59mol) was added drop wise at 0-5 °C. The reaction mixture was stirred at rt for i2hrs, the solvents were removed in vacuo to give after destilation (Tb = 101-110 °C (ltorr)) product in 82 % yield. F2189-0853. Compound F2190-o632a (89g, 0.36 mol) was added dropvise to a suspension of LAH (25.9g, o.68mmol) in DEE (2L) at rt followed by solution of KOH (8.ig) in 90ml H₂0. The reaction mixture was stirred for 3hrs at rt, the precipitate was filtered off, washed with diethyl ether. The filtrate was evaporated in vacuo and the residue was dried (3hrs, l torr, 40 °C). Yield 55 %.

F2189-o853a. To a solition of F2189-0853 (i2g, 66.7 mmol) in CHC1₃ (200 ml) S0C1₂ (23.9g, 14.4 ml, 200 mmol) B was added drop wise at oC. The reaction mixture was stirred for 4ohrs at rt, the volatiles were removed in vacuo and the oily residue was treated with diethyl ether (200ml). After 12 hrs of standing at rt the precipitate was filtered off and washed with EtOEt ( 100 ml) and MeCN ( 100 ml). The product was dried in vacuo (shrs, l torr, 40 °C). Yield 57%. F2189-o853b. Thiophosgene (4-8g, 4.17 mmol) was added in one portion followed by portionwise addition of NaOH(4.87g, 0.122 mol) to a suspension of F2189-o853a in H₂0 (50 ml) and DCM (80 ml) at o °C. The reaction mixture was stirred at rt for 2ohrs. The organic layer was separated dried over MgS04, the solvent was removed in vacuo to give oily residue . Yield 95 %.

F2189-0853C. A solution of F2189-o853b (7-9g, o.033mol) and DIPEA (8.5ig, o.659mol) in toluene (80 ml) was stirred under reflux for 4ohrs (TLC control of conversion EtOAc/hex = 1/ 20). After cooling down the reaction mixture was diluted with water (100ml), the organic layer was separated, dried over MgS04, the volatiles were removed in vacuo, the oily residue (7.8g) was purified by flash column chromatography (250g Si0₂, n- hexane/EtOAc= 50/1). Fraction 1: o.95g ( yield 14%) trans isomer

Fraction 2: i.56g - mixture of cis and trans isomers

F2167-9485. Compound F2189-0853C (o.9g, 4.43 mmol) was added in portions to a saturated aqueous solution of ammonia (30%, 20ml) at o °C . The reaction mixture was stirred for 24 hrs days at rt . The volatiles were evaporated in vacuo and the residue was triturated with mixture DEE/n-hexane (2:1) , the precipitate formed was filtered off and dried in vacuo (1 torr, 3hrs, 40 °C). Yield 92%.

F2190-o632b was synthesized according to published procedure (TMEDAO2 Facilitates Atom Economical/Open Atmosphere Ley-Griffith (TPAP) Tandem Oxidation-Wittig Reactions Supplementary Information Christopher D. G. Read, Peter W. Moore and Craig M. Williams Electronic Supplementary Material (ESI) for Green Chemistry. The Royal Society of Chemistry (2015) pages S1-S47).

F2190-0632C. Compound F2190-o632b ( log, 0.0665 mol) was added to a precooled (o °C) suspension of paraform (2g, o.o699mol) in TMSC1 (84-37g, 0.466 mol) and THF 20 ml. The reaction mixture was stirred at rt for 4 days the volatiles were removed in vacuo, the residue was distilled in vacuo (Tb = 66-8i°C , ltorr). Yield 72%.

F2190-0632d. A suspension of KCN (7.79 g, 0.119 mol) in solution of F2190-0632C (9.i4g, 0.046 mol), 18-C-6 (3i.6ig., 0.12) in MeCN (300 ml) was stirred under reflux for 20 hrs. The reaction mixture was concentrated to 1/3 of its volume and was dilluted with EtOAc (200 ml). The solution was washed with half sturated aqueous solution of KC1 (2x 200 ml) to remove 18-C-6) , and dried over Na₂S0₄ . The folution was filtered, eveporated in vacuo. The residue was distilled in vacuo T = 80-100 °C (1 torr). Additional purification by preparative column chromatography (EtOAc/Hex = 1/20 , 6og Si0₂) gave 4-3g of 92% pure target compound (GCMS). Impurity - ArCH₂CN.

F2190-o632e. Compound F2190-0632d (o.9g, 4.43 mmol) was added in portions to aqueous solution of ammonia (30%, 20ml) at o °C. The reaction mixture was stirred for 24 hrs at rt . The volatiles were evaporated in vacuo and the residue was triturated with mixture DEE/n-hexane (2:1) , the precipitate formed was filtered off and dried in vacuo (1 torr) for 3hrs at 40 C. Yield 92 %.

Scheme 5

Scheme 6

F9995-4278

Scheme 7

F0001-0167a. A solution of F0001-0167 (0.5 g, 4.6mmol) in CH₃S0₃H (2 ml) was warmed to 40 °C. 2-Methyl-butan-2-ol (0.88 g, 10 mmol) was added slowly for 3.5 h in order to keep the temperature between 50-55 °C. After addition the temperature was maintained at 50 C for 2 hrs. The reaction mixture was poured onto ice (1 kg) followed by extraction with diethyl ether (3 x too ml). The combined organic phases were washed with water (3 x too ml), saturated NaHC0₃ (2 x 500 ml), water (2 x 50 ml), dried (MgS0₄) and concentrated using a rotary evaporator giving a yellow oil 4 that was used without further purification in the next step.

F0001-0167b. F0001-0167a (0.66 g, 3.7 mmol) was dissolved DCM (30 mL), and at 3 °C. TfOTF (1.35 g, 4.8 mmol) and pyridine (0.45 g, 5.6 mmol) were added, followed by stirring at the same temperature for 30 min. Water was added to the reaction solution, followed by extraction with methylene chloride (2x30ml). After the extract was concentrated under reduced pressure,

F0001-0167C. A solution of F0001-0167b (3.5 g, 11.3 mmol), (Ph₃P)₄Pd (lg), and Zn(CN)₂ (0.352 g, 3 mmol) in 10 mL of DMF was flushed with nitrogen three times and then stirred at 80 °C. After 24 hrs, the mixture was cooled down to rt, diluted with EtOAc (10 ml), and filtered through a cake of Celite. The solid was washed with EtOAc, and the filtrates were combined and concentrated.

F0001-0167d. To a solution of F0001-0167C ( 5 mmol) in THF (5 ml) was added dropwise a solution of DIBAL-H (1.01 M, 5 mmol) at o°C. The reaction mixture was stirred at room temperature (r.t.) for 1 h. Concentrated aqueous HC1 (0.5ml) was added and the reaction mixture was stirred for ihr at rt. aq)/THF (1:9, 21 mL) and stirred for 1 h at rt. The mixture was diluted with EtOAc (15 mL), and the organic phase was washed with brine (10 mL). The solvents of the dried solution (MgS0₄) were concentrated under reduced pressure to give crude F0001-0167d.

F0001-0167e . Thiosemicarbazide (91.1 mg, 1 mmol), F0001-0167d. (190 mg, 1 mmol) were dissolved in ethanol (10ml) and acetic acid (few drops) was added to the above solution. The reaction mixture was stirred under reflux for 5-6I1 and then cooled down to room temperature. The precipitate was filtered off washed with DEE and dried in vacuo. Yield 83 %. Schem

F0001-0879'. n-BuLi (2.5 M, 44.0 mL) was added dropwise into a cold (-78º C) solution of F0001-0879 (20.34 g, 109.4 mmol) in THF (150 mL). The reaction mixture was stirred at -78º C for 3 hrs, and then tBuCHO ( 9.34 g, 109.4 mmol) in THF (10 mL) was added. The mixture was allowed to warm up to o° C. stirred for 10 min, and then quenched with aqueous ammonium chloride. The mixture was poured into water, acidified with HC1 (2N) and extracted with EtOAc. The organic extracts were dried over MgS0₄. Evaporation and purification by flash chromatography (hexanes/EtOAc 3/ 1) gave F0001-0879' as a yellowish oil. The product was dissolved in acetone (150 mL), cooled to io° C. and Jones Reagent (79.7 mL) was added dropwise. The reaction stirred for poured into water and extracted with ethyl ether. The organic extracts were dried over MgS0₄. Evaporation and purification by flash chromatography (hexanes/EtOAc, 3:1) gave F0001-0879' in 78% yield.

F0001-0879" . To a solution of crude F0001-0879' (3.0 mmol) in dry DCM (18 mL), HSiEt₃ (1.2 eq), and TFA (1.2 eq) was added. Then, the mixture was stirred I2hrs and concentrated under reduced pressure. The residue was purified by flash-column

chromatography on a silica gel using a mixture of Hexane and EtOAc as the eluent to afford F0001-0879" as a clear oil.

F0001-o879a. To a solution of F0001-0879" (3.22 g, 18.1 mmol) in DCM (100 ml) stirring in -78 °C bath was added dropwise 2.14 mL (5.68 g) of BBr₃. The mixture was stirred while warming to room temperature. After 3 hrs, ice was added, and the organic layer separated, dried over Na₂S0₄, filtered and evaporated affording F0001-o879a as an oil in 96 % yield.

Compounds F0001-0879b, F0001-0879C, F0001-o879d and F0001-o879e were prepared as similar compounds of F0001-0167 series.

Scheme 9

F2190-0576a. t-BuLi (1.7 M in pentane) (51.5 ml, 87.6 mmol) was added slowly to a solution of F2190-0576 (7.7 g, 39-8 mmol) in dry THF (100 ml) at -78 °C, under an inert atmosphere. The resulting mixture was stirred overnight, allowing the temperature too gradually warm from -78 °C to room temperature, and then quenched with water. The aqueous layer was extracted three times with ethyl acetate. The combined organic solution was dried over MgS0₄, filtered, and evaporated. The residue was purified by flash chromatography . After removal of the solvent purification was achieved by recrystallization from AcOEt/hexane. Yield 70%. . Compound F2190-0576b was prepared as F0001-0879" from F2190-0576a

Compound F2190-0576C was prepared as F0001-o879a from F2190-0576b

F2190-0576d. A stirred, cooled (ice bath) solution of F2190-0576C (i.5g, lommol) in anhydrous dichloromethane (15ml) was treated with titanium tetrachloride (i.86mL, i7mmol) followed by α,α-dichloromethyl ether (o.9mL, 20mmol). The reaction was allowed to warm to ambient temperature over lh, quenched cautiously with ice and water and extracted with dichloromethane. The organic extract was washed with water and brine, dried over sodium sulfate, filtered and evaporated in vacuo to a residue that was subjected to flash column chromatography using 2-2.5% ethyl acetate in hexane as the eluent to afford the title compound (i-3g, 75%). F2190-0576e was obtained as prepared as F0001-0167e from F2190-0576e and thiosemicarbazide.

Scheme 10.

Compound F0001-i682a was prepared according to published procedure (Journal of Organic Chemistry, 1987, 52(17), 3847-3850)

F0001-i682b. A solution of 2-(dimethylamino)ethanol (o.59g, 6.6 mmol, 3.0 equiv) in dry hexanes (5 mL) was cooled to o°C, and treated dropwise with n-butyllithium (3.6 ml of 3.3M,

11.9 mmol, 5.4 equiv) under an argon atmosphere. After 30mm at o °C, the mixture was cooled to - 20 °C and F0001-i682a (0,3 g, 2.2 mmol, 1.0 equiv.) was added dropwise. After 1 h of stirring, a green-brown solution was observed. The mixture was cooled to - 78 °C and treated dropwise with the solution of dibromotetrafluoro ethane (2.3 g, 8.8 mmol, 4.0 equiv.) in hexanes(6 ml). After 1 h at - 78 °C, the cold mixture was quenched with a saturated solution of sodium bicarbonate (80 mL) and immediately extracted with Et20 (2x300mL). The combined organic layers were dried over potassium carbonate. After evaporation of solvents, the crude product was obtained which was used for next step without further purification.

F0001-1682C. nBuLi (6.25 mL of 1.6m solution in hexanes, 10 mmol, 1 equiv) was added dropwise to a solution of F0001-i682b (2.14 g, 10 mmol) in dry Et₂0 (20 mL), cooled to -

78 °C. The reaction mixture was allowed to warm to 40 C for 15 min, then cooled back to -

78°C again. DMA (1.023 mL, 11 mmol, 1.1 equiv) was added dropwise and the mixture was stirred at -78 °C for 2h. Saturated aqueous ammonium chloride (10 mL) was added and the organic layer was separated. The aqueous layer was extracted with Et₂0 (3 x 10 mL) and the combined organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo to give an oily residue that was subjected to flash column chromatography by using (5% methanol/methylene chloride) to give F0001-1682C.

F0001-i682d was prepared as F0001-0167e from F0001-0167C and

thiosemicarbazide.

Scheme 11

Scheme 12

Schem 13

Compound 701-23-5 was prepared according to known procedure (J. Gen. Chem. USSR (Engl. Transl.), 1964 , vol. 34, p. 3063 - 3066/3099 - 3101)

706751-57-7. To a solution of 536-60-7 (i-5g, 10 mmol), N-hydroxyphthalimide (i-96g, 12 mmol) and PPh₃ (3.14 g, 12 mmol) in 20 mL THF was added diisopropyl azodicarboxylate (2.4 mL, 12 mmol) dropwise at o °C. The reaction was then allowed to warm to room temperature and stirred overnight. After the reaction is finished, hexanes were added and the precipitate was collected and directly used in the next step without further purification. Hydrazine hydrate (12 mmol) was added to a solution of the crude product in

DCM/MeOH (1:1, 10ml). The reaction mixture was stirred under reflux for 20min. The volatiles were removed in vacuo the residue was recrystallized from AcOEt/ Hexane 4:1 to give pure 706751-57-7 (68% yield).

1142201-30-6. A solution of 701-23-5 (i.9g, lommol) Mel (2.13g, 15 mmol) and

DIPEA ( i.95g, 15 mmol) in EtOH (20 ml) was stirred at room temperature 24 hrs. The volatiles were removed in vacuo, the residue was triturated with HC1 (2N in water, 10ml) and extracted with DCM (2x20 ml). The extract was washed with water (2x50 ml) the organic layer was dried over Na₂S0₄, filtered and evaporated in vacuo. The residue was triturated with DEE (40 ml), the precipitate was filtered off washed with DEE and dried in vacuo. Yield 82%.

F1923-0798 . A solution of 1142201-30-6 ( o.2g, l mmol), 706751-57-7 (0.165g, 1 mmol) and DIPEA (o.26g, 2 mmol) in EtOH (5ml) was stirred under reflux for 12 hrs The volatiles were removed in vacuo, the residue was triturated with HCl (2N in water, 5 ml) and extracted with DCM (2x5 ml). The extract was washed with water (2x10 ml) the organic layer was dried over Na₂C0₃, filtered and evaporated in vacuo. The residue was triturated with DEE/Hexane (1:1) the precipitate was filtered off washed with DEE/Hexane and dried in vacuo. Yield 65 %.

5i86o-03-8a. BOC-anhydride (5.0 g, 23 mmol) was dissolved in MeOH (50 mL) and added dropwise to a solution of 51860-03-8 (4.1 g, 25 mmol) in MeOH (50 mL) over a period of 30 min at room temperature. Stirring was continued for 40 min and the mixture was concentrated in vacuo. The oily residue was taken up in EtOAc (100 mL) and washed with sat. NaHC0₃ (2 x 50 mL) and brine (1 x 30 mL). After drying (Na₂S0₄) the solvent was removed under reduced pressure to give 51860-03-88 as a colorless oil (70 % yield) which was used without further purification.

VPC-18190 (F1923-0800). A solution of 1142201-30-6 ( o.2g, l mmol), 51860-03-88

( 0.264 g, i mmol) and DIPEA (o.26g, 0.2 mmol) in EtOH (5ml) was stirred under reflux for 12 hrs. The volatiles were removed in vacuo. The residue was dissolved in HCl/Dioxane (4M, ml) and the solution was stirred at rt for 24 hrs, then evaporated in vacuo. The residue was dissolved in DCM (10 ml) and the solution was kept under Na₂C0₃ for 3 hrs, filtered and evaporated in vacuo. The product was purified by preparative column chromatography (Si0₂, MeCN /H₂0). Yield 43 %.

Although various embodiments of the invention are disclosed herein, many adaptations and modifications may be made within the scope of the invention in accordance with the common general knowledge of those skilled in this art. Such modifications include the substitution of known equivalents for any aspect of the invention in order to achieve the same result in substantially the same way. Numeric ranges are inclusive of the numbers defining the range. The word "comprising" is used herein as an open-ended term, substantially equivalent to the phrase "including, but not limited to", and the word "comprises" has a corresponding meaning. As used herein, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a thing" includes more than one such thing. Citation of references herein is not an admission that such references are prior art to an embodiment of the present invention. The invention includes all embodiments and variations substantially as hereinbefore described and with reference to the examples and drawings.

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Claims

1. A compound having the structure of Formula I

wherein,

R¹ is selected from H, CH₃, OH, F, CI and Br;

R² is selected from H, CH₃, F, CI and Br;

R4 is selected from H, CH₃, OH, F, CI and Br;

R5 is selected from H, CH₃, F, CI and Br;

R⁶ is H; or

alternatively R⁶ is CH₃, when R3 is -OCH₃;

provided that the compound does not have the following structure:

2. The compound of claim l, wherein R4 is selected from H, CH₃, F, CI or Br.

3. The compound of claim l or 2, wherein R4 is selected from H, F, CI or Br.

4. The compound of claim 1, 2 or 3, wherein R4 is H.

5. The compound of any one of claims 1-4, wherein R¹ is H, CH₃, OH, F or CI.

6. The compound of any one of claims 1-5, wherein R¹ is H, CH₃, F or CI.

7. The compound of any one of claims 1-6, wherein R² is H, CH₃, F or CI.

8. The compound of any one of claims 1-7, wherein R5 is H.

9. The compound of any one of claims 1-8, wherein R3 is selected from

10. The compound of any one of claims 1-8, wherein R3 is selected from

12. The compound of any one of claims 1-8, wherein R3 is selected

13. The compound of any one of claims 1-12, wherein R² is H or CH₃.

14. The compound of any one of claims 1-8, wherein the compound is selected from the following:

15. The compound of any one of claims 1-14, for use in the treatment of at least one indication selected from the group consisting of: prostate cancer, Ewing's sarcoma, breast cancer and pancreatic cancer.

16. A method for modulating E26 transformation-specific (ETS) activity, the method comprising administering to a mammalian cell in need thereof a compound or pharmaceutically acceptable salt thereof, wherein the compound has the structure of Formula I

I

wherein,

R¹ is selected from H, CH₃, OH, F, CI and Br;

R² is selected from H, CH₃, F, CI and Br;

alternatively R3 is selected from

and

, when R¹ is H, R² is H, R4 is H, R5 is H and R⁶ is H;

R is selected from H, CH₃, OH, F, CI and Br;

R5 is selected from H, CH₃, F, CI and Br;

R⁶ is H; or

alternatively R⁶ is CH₃, when R3 is -OCH₃.

17. The method of claim 16, wherein R4 is selected from H, CH₃, F, CI or Br.

18. The method of claim 16 or 17, wherein R4 is selected from H, F, CI or Br.

19. The method of claim 16, 17 or 18, wherein R4 is H.

20. The method of any one of claims 16-19, wherein R¹ is H, CH₃, OH, F or CI.

21. The method of any one of claims 16-20, wherein R¹ is H, CH₃, F or CI.

22. The method of any one of claims 16-21, wherein R² is H, CH₃, F or CI.

23. The method of any one of claims 16-22, wherein R5 is H.

24. The method of any one of claims 16-23, wherein R3 is selected from

28. The method of any one of claims 16-27, wherein R² is H or CH₃.

29. The method of any one of claims 16-23, wherein the compound is selected from the following:

84

30. The method of any one of claims 16-29, wherein the modulating ETS activity is for treatment of at least one indication selected from the group consisting of: prostate cancer, Ewing's sarcoma, breast cancer and pancreatic cancer.

31. The method of claim 30, wherein the modulating ETS activity is for the treatment of prostate cancer.

32. The method of any one of claims 16-31, wherein the mammalian cell is a human cell.

33. The method of any one of claims 16-32, wherein the cell is a prostate cell.

34. The method of any one of claims 16-33, wherein the cell is a prostate cancer cell.

35. A compound having the structure of Formula I

wherein,

R¹ is selected from H, CH₃, OH, F, CI and Br;

R² is selected from H, CH₃, F, CI and Br;

R4 is selected from H, CH₃, OH, F, CI and Br;

R5 is selected from H, CH₃, F, CI and Br;

R⁶ is H; or alternatively R⁶ is CH₃, when R3 is -OCH₃;

for use in the treatment of at least one indication selected from the group consisting prostate cancer, Ewing's sarcoma, breast cancer and pancreatic cancer.

36. The compound of claim 35, wherein R4 is selected from H, CH₃, F, CI or Br.

37. The compound of claim 35 or 36, wherein R4 is selected from H, F, CI or Br.

38. The compound of claim 35, 36 or 37, wherein R4 is H.

39. The compound of any one of claims 35-38 wherein R¹ is H, CH₃, OH, F or CI.

40. The compound of any one of claims 35-39, wherein R¹ is H, CH₃, F or CI.

41. The compound of any one of claims 35-40, wherein R² is H, CH₃, F or CI.

42. The compound of any one of claims 35-41, wherein R5 is H.

43. The compound of any one of claims 35-42, wherein R3 is selected from

47. The compound of any one of claims 35-46, wherein R² is H or CH₃.

48. The compound of any one of claims 35-42, wherein the compound is selected from the following:

wherein,

R¹ is selected from H, CH₃, OH, F, CI and Br;

R² is selected from H, CH₃, F, CI and Br;

R4 is selected from H, CH₃, OH, F, CI and Br;

R5 is selected from H, CH₃, F, CI and Br;

R⁶ is H; or

alternatively R⁶ is CH₃, when R3 is -OCH₃.

50. Use of a compound for the manufacture of a medicament for modulating ETS activity, wherein the compound has the structure of Formula I

wherein,

R¹ is selected from H, CH₃, OH, F, CI and Br;

R² is selected from H, CH₃, F, CI and Br;

R4 is selected from H, CH₃, OH, F, CI and Br;

R5 is selected from H, CH₃, F, CI and Br;

R⁶ is H; or

alternatively R⁶ is CH₃, when R3 is -OCH₃.

51· The use of claim 49 or 50, wherein R4 is selected from H, CH₃, F, CI or Br.

52 The use of claim 49, 50 or 51, wherein R4 is selected from H, F, CI or Br.

53 The use of any one of claims 49-52, wherein R4 is H.

54 The use of any one of claims 49-53 wherein R¹ is H, CH₃, OH, F or CI.

55 The use of any one of claims 49-54, wherein RHs H, CH₃, F or CI.

56 The use of any one of claims 49-55, wherein R² is H, CH₃, F or CI.

57· The use of any one of claims 49-56, wherein R5 is H.

58 The use of any one of claims 49-57, wherein R3 is selected from

59. The use of any one of claims 49-57, wherein R3 is selected from

60. The use of any one of claims 49-57, wherein R3 is selected from

62. The use of any one of claims 49-61, wherein R² is H or CH₃.

63. The use of any one of claims 49-57, wherein the compound is selected from the following

64. The use of any one of claims 49-63, wherein the modulating ETS activity is for treatment of at least one indication selected from the group consisting of: prostate cancer, Ewing's sarcoma, breast cancer and pancreatic cancer.

65. The use of any one of claims 49-64, wherein the modulating ETS activity is for treatment of prostate cancer.

66. A pharmaceutical composition comprising a compound or pharmaceutically acceptable salt thereof, wherein the compound has the structure of Formula I

wherein,

R¹ is selected from H, CH₃, OH, F, CI and Br;

R² is selected from H, CH₃, F, CI and Br;

R4 is selected from H, CH₃, OH, F, CI and Br;

R5 is selected from H, CH₃, F, CI and Br;

R⁶ is H; or

alternatively R⁶ is CH₃, when R3 is -OCH₃.

67. The pharmaceutical composition of claim 66, wherein the compound is selected from the following:

68. A commercial package comprising (a) a compound of any one of claims 35-48; and (b) instructions for the use thereof for modulating ETS activity.

69. A commercial package comprising (a) a pharmaceutical composition comprising a compound of any one of claims 35-48 and a pharmaceutically acceptable carrier; and (b) instructions for the use thereof for modulating ETS activity.