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WO2018145217A1 - Composés de gènes (erg) associés à l'ets humain utilisés en tant qu'agents thérapeutiques et leurs procédés d'utilisation - Google Patents

Composés de gènes (erg) associés à l'ets humain utilisés en tant qu'agents thérapeutiques et leurs procédés d'utilisation Download PDF

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Publication number
WO2018145217A1
WO2018145217A1 PCT/CA2018/050162 CA2018050162W WO2018145217A1 WO 2018145217 A1 WO2018145217 A1 WO 2018145217A1 CA 2018050162 W CA2018050162 W CA 2018050162W WO 2018145217 A1 WO2018145217 A1 WO 2018145217A1
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Prior art keywords
compound
erg
vpc
cancer
ets
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English (en)
Inventor
Artem Tcherkassov
Paul. S. RENNIE
Michael Hsing
Miriam S. BUTLER
Mani ROSHAN-MONIRI
Michael E. Cox
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University of British Columbia
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University of British Columbia
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D277/00—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings
    • C07D277/02—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings not condensed with other rings
    • C07D277/20—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members
    • C07D277/32—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D277/54—Nitrogen and either oxygen or sulfur atoms

Definitions

  • 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.
  • ETS E26 transformation-specific or E-twenty-six
  • ETS domain conserved DNA binding domain
  • 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).
  • PCa prostate cancer
  • ADT androgen deprivation therapy
  • 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.
  • ABS androgen binding site
  • 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).
  • 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).
  • ADT comes with significant side effects on the sexual functions and characteristics of male bodies (Isbarn et al., 2009; Valenca, Sweeney, & Pomerantz, 2015).
  • TMPRSS2 transmembrane protease serine 2
  • ETS-related gene ETS-related gene
  • 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).
  • ERG is not expressed in prostate epithelial cells, but its fusion with the TMPRSS2 promoter causes AR to drive ERG expression.
  • 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).
  • ERG epithelial-mesenchymal transition
  • 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).
  • PARPi Binding Protein
  • USP9X S. Wang et al., 2014
  • ERG downstream target genes such as YAPi (Nguyen et al., 2015).
  • YK-4-279 CAS #1037184-44-3
  • Recent reports on YK-4-279 have disclosed toxicity, oral bioavailability and pharmacokinetics concerns (Lamhamedi-Cherradi etal., 2015; Rahim et al., 2014).
  • 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).
  • 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.
  • ERG ETS-related gene
  • FLIi shares 98% sequence identity at the ETS domain with ERG.
  • R 1 may be selected from H, CH 3 , OH, F, CI and Br
  • R 2 may be selected from H, CH 3 , F, CI and Br
  • R3 may be selected from
  • R 3 may be selected from
  • R 1 when R 1 may be H, R 2 may be H, R4 may be H, R3 may be H and R 6 may be H; R4 may be selected from H, CH 3 , OH, F, CI and Br; R5 may be selected from H, CH 3 , F, CI and Br; R 6 may be H; or alternatively R 6 is CH 3 , when R3 is -OCH 3 ; provided that the
  • R4 may be selected from H, CH 3 , F, CI or Br.
  • R4 may be selected from H, F, CI or Br.
  • R4 may be selected from H or CH 3 .
  • 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.
  • R 1 may be H, CH 3 , OH, F or CI.
  • R 1 may be H,
  • R 2 may be H, CH 3 , 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.
  • ETS E26 transformation-specific
  • R 1 may be selected from
  • R 1 when R 1 may be H, R 2 may be may be H, R4 is H, R5 may be H and R 6 is H; R4 may be selected from H, CH 3 , OH, F, CI and Br; R5 may be selected from H, CH 3 , F, CI and Br; R 6 may be H; or alternatively R 6 is CH 3 , when R3 maybe -OCH 3 .
  • R4 may be selected from H, CH 3 , F, CI or Br.
  • R4 may be selected from H, F, CI or Br.
  • R4 maybe H.
  • R 1 may be H, CH 3 , OH, F or CI.
  • R 1 may be H, CH F or CI.
  • R 2 may be H, CH 3 , F
  • 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.
  • R 1 maybe selected from H, CH 3 , OH, F, CI and Br;
  • R 2 may be selected from H, CH 3 , F, CI and Br; R3 may be selected from alternatively R 3 may be selected from
  • R 1 when R 1 may be H, R 2 may be H, R4 may be H, R5 may be H and R 6 may be H; R4 may be selected from H, CH 3 , OH, F, CI and Br; R5 may be selected from H, CH 3 , F, CI and Br; R 6 may be H; or alternatively R 6 may be CH 3 , when R3 is -OCH 3 ; 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.
  • R 4 may be selected from H, CH 3 , F, CI or Br.
  • R4 may be selected from H, F, CI or Br.
  • R4 maybe H.
  • R 1 may be H, CH 3 , OH, F or CI.
  • R 1 may be H, CH 3 , F or CI.
  • R 2 may be H, CH 3 , F or CI.
  • R 5 maybe H.
  • R3 may be selected from -
  • a use of a compound for modulating ETS activity
  • R 1 may be selected from H, CH 3 , OH, F, CI and Br;
  • R 2 may be selected from H, CH 3 ,
  • R3 may be selected from
  • R3 may be selected from when R 1 is H, R 2 is H, R4 is H, R5 is H and R 6 is H; R4 may be selected from H, CH 3 , OH, F, CI and Br; R5 may be selected from H, CH 3 , F, CI and Br; R 6 may be H; or alternatively R 6 may be CH 3 , when R3 is -OCH 3 .
  • R 1 may be selected from H, CH 3 , OH, F, CI and Br;
  • R 4 when R 1 is H, R 2 is H, R4 is H, R5 is H and R 6 is H; R4 may be selected from H, CH 3 , OH, F, CI and Br; R5 may be selected from H, CH 3 , F, CI and Br; R 6 may be H; or alternatively R 6 may be CH 3 , when R3 is -OCH 3 .
  • R4 may be selected from H, CH 3 , F, CI or Br.
  • R4 may be selected from H, F, CI or Br.
  • R4 maybe H.
  • R 1 may be H, CH 3 , OH, F or CI.
  • R 1 may be H, CH 3 , F or CI.
  • R 2 may be H, CH 3 , F
  • the modulating ETS activity is for treatment of prostate cancer.
  • composition comprising a compound or pharmaceutically acceptable salt thereof, wherein the compound has the
  • R 1 may be selected from H
  • R 4 when R 1 is H, R 2 is H, R4 is H, R5 is H and R 6 is H; R4 may be selected from H, CH 3 , OH, F, CI and Br; R5 may be selected from H, CH 3 , F, CI and Br; R 6 is H; or alternatively R 6 may be CH 3 , when R3 is -OCH 3 .
  • the compound may be selected from the
  • 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.
  • prostate cancer 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.
  • 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.
  • 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.
  • 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.
  • FIGURE 2 Characterization of VPC-18005 binding to the ERG-ETS domain: (A)
  • VPC-18005 YK-4-279 is cytotoxic.
  • D Pretreatment of VPC-18005 (10 ⁇ ) 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 ⁇ ) 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.
  • VPC-18005 was non-toxic until concentrations above 75 ⁇ .
  • C Following 5 days of daily treatment, VPC- 18005 reduced occurrence of metastasis in zebrafish grafted with PNTiB-ERG and VCaP cells.
  • 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.
  • VPC Vancouver Prostate Centre
  • MSKCC Memorial Sloan-Kettering Cancer Center
  • TCGA Cancer Genome Atlas (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:
  • FIGURE 8 VPC-18005 binds the ERG-ETS domain: Fitting of the VPC-18005-induced chemical shift perturbations of the amide ⁇ -1 «H ⁇ 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:
  • VPC-18005 isopropyl moiety
  • VPC-18065 tert-butyl
  • VPC-18098 cyclobutyl
  • the ions are shown, a person of skill in the art will appreciate that the counter ion may also be present.
  • 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.
  • TABLE 1 and TABLE 2 shows the compounds tested by structure and the associated identifiers. Where the % inhibition or the IC 50 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.
  • Estimated % inhibition in a luciferase reporter assay was performed at 10 ⁇ 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
  • 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.
  • 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.
  • methods of systemically treating any of the indications described herein are also provided.
  • 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.
  • 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
  • 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.
  • 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, theo
  • 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.
  • compounds and all different forms thereof 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.
  • hydrates are formed when the solvent is water or alcoholates are formed when the solvent is an alcohol.
  • compounds and all different forms thereof 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.
  • compounds and all different forms thereof 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • a medical device or appliance such as an implant, graft, prosthesis, stent, etc.
  • 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.
  • 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.
  • 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.
  • dosage values may vary with the severity of the condition to be alleviated.
  • 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.
  • parenteral compositions in dosage unit form for ease of administration and uniformity of dosage.
  • 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.
  • 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).
  • 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.
  • 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.
  • 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.
  • 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.
  • Each of the compounds was first evaluated in PNTiB-ERG cells at concentrations of 10 ⁇ and 25 ⁇ 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).
  • VPC-18005 was found to inhibit pETS-luc reporter activity in PNTiB-ERG and VCaP cells with IC 50 values of 3 and 6 ⁇ , respectively (FIGURE lB). This compared favorably relative to, YK-4-279 (Rahim et al. 2011), that exhibited IC 50 values of 5 ⁇ and 16 ⁇ in parallel PNTiB-ERG and VCaP cell-based ETS-Luc reporter assays, respectively (FIGURE lC).
  • VPC-18005 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 (ARR 3 tk-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 ( ⁇ ) were both >50.
  • VPC-18005 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 ⁇ 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.
  • VPC-18005 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.
  • 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).
  • VPC-18005 exhibited ERG-dependent chemical shift perturbations. These include the hydrogens on the aromatic ring ( ⁇ 7.78 and 7.45 ppm), the methyl's on the isopropyl group ( ⁇ 1.25 ppm) and the conjugated double bond ( ⁇ 8.4 ppm) (FIGURE 2E). Due to the spectral overlap with signals from DMSO, perturbations from the CH 2 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.
  • VPC-18005 scaffold 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 IC 50 values (2 ⁇ and ⁇ 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.
  • SAR working structure-activity relationship
  • 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.
  • VPC- 18005 a dose dependent effect of VPC- 18005 on cell migration 48-72 h post-exposure (data not shown).
  • 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.
  • the ERG- ETS domain structural model was prepared by using the Protein Preparation WizardTM module of the Maestro V9.3TM program from the SchrodingerTM 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 predicted p3 ⁇ 4 was calculated for each molecule using a custom MOE SVLTM script, and ligand efficiency was calculated using GlideTM.
  • PAINS Pan Assay Interference CompoundsTM
  • a consensus scoring method was used: 1) each compound within the top 20% p3 ⁇ 4 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 PAINSTM.
  • Solubility Stock solutions of compounds at 50 mM in dimethyl sulfoxide (DMSO) were diluted ⁇ 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.
  • DMSO dimethyl sulfoxide
  • 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.
  • ACN acetonitrile
  • These MeOH, and diluted media and PBS samples were analysed using an AcquityTM UPLC coupled in series with an eLambda PDATM and a Quattro PremierTM (Waters).
  • a 100 mm BEH C18, 1.7 ⁇ 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.
  • SIR single ion recording
  • QuanlynxTM 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 5 ⁇ , performs well for solubility and relative stability at higher concentrations ( ⁇ ) 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).
  • VPC Vancouver Prostate Centre
  • MSKCC Memorial Sloan-Kettering Cancer Center
  • TCGAX2015 The Cancer Genome Atlas
  • 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).
  • VCaP (CRL-2876) and PC-3 (CRL-1435) human prostate carcinoma cells were obtained from the American Type Culture Collection (ATCC, August 2014).
  • PC-3 cells were maintained in RPMI 1640 medium (Life TechnologiesTM) 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 TechnologiesTM) 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 ⁇ L 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.
  • 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 ⁇ .
  • the luciferase assays were repeated for each lead compound under multiple concentrations (0.1 to 100 ⁇ ) to establish a dose-dependent response and an IC 50 value.
  • AR reporter assay was performed as previously described (Dalai et al. 2014).
  • MTS 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 ⁇ L of appropriate media in 96 well culture dishes. Twenty four hours later, 100 ⁇ L of medium containing vehicle control or compounds. Each treatment was prepared in triplicate. After a 72 hr treatment, cellular viability was assessed using CellTiter 96TM Aqueous One Solution Cell Proliferation AssayTM reagent (PromegaTM) 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.
  • 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 IIITM spectrometer. Data were processed and analyzed using NMRpipeTM (Delaglio et al.
  • Reciprocal titrations were carried out using1H - NMR to monitor the effects of progressively adding unlabeled protein to a sample of VPC- 18005 ( ⁇ 8 ⁇ ) 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 xCELLigenceTM platform (ACEA).
  • FBS-supplemented media 160 ⁇ L was added to the lower chamber of the plate and incubated at RT for 30 min.
  • the upper chamber was then mounted and 30 ⁇ L 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.
  • SFM serum free media
  • PNTiB-ERG or -MOCK cells 24 h starved were prepared in SFM, and 30,000 cells in 70 ⁇ L were seeded to each well of the upper chamber in addition of 100 ⁇ L 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 AssayTM (TrevigenTM) was performed as per manufacturer's instructions. Briefly, 5,000 PNTiB-ERG cells and 5 ⁇ L of ECM were prepared in growth media to a total volume of 50 ⁇ L 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 ⁇ L gel invasion matrix was added. Spheroids were then incubated at 37 °C for 3 to 7 days, and photographed using Zeiss AxioObserver ZiTM microscope in each well on the day of invasion mix addition and every two days following. Spheroids were retreated with 50 ⁇ L of vehicle control or compound after 72 hr.
  • Electrophoretic mobility shift assay Using nuclear lysate: EMSA was performed as per manufacturer's protocol (PanomicsTM). Briefly, 10 ⁇ g nuclear extracts (CelLytic NuCLEAR Extraction KitTM, SigmaTM) from VCaP cells, 1 ⁇ L polyd(I-C), 2 ⁇ L 5X binding buffer, and nuclease-free water up to 7 ⁇ L 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.
  • 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-488TM 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.
  • Alexa-488TM fluorophore-labeled DNA 5'-CGG CCA AGC CGG AAG TGA GTG-3'
  • An initial gel shift assay was performed by titrating constant 1 nM labeled dsDNA with ERG (0.5 ⁇ 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 ImagerTM equipped with blue laser to excite at 490 nm and measure at 520 nm. The scanned image was analyzed with Image JTM (Rasband 1997-2015).
  • 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 mm 2 petri dish containing aquarium water. Phenylthiourea (0.2 mM PTU, SigmaTM) 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 ⁇ of CellTracker CM-DilTM dye (Life TechnologiesTM) 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 mm 2 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.
  • 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.
  • 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 2000TM NMR spectrometer system with working frequency of 400 MHz. Chemical shifts ⁇ 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).
  • Ar 4-c-BuPh (a); 4-t-Bu-2-OHPh (b); 4-t-Bu-2-FPh
  • 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) 2 P0Na (38.0 g, 273 mmol) in dry ether
  • Step B A mixture of compound 3
  • 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) Me 3 SiBr (0.940 g, 6.14 mmol) was added dropwise under argon atmosphere maintaining temperature below o°C.
  • 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 %.
  • F2189-o853a To a solition of F2189-0853 (i2g, 66.7 mmol) in CHC1 3 (200 ml) S0C1 2 (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 H 2 0 (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 %.
  • 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).
  • 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).
  • F2190-0632d 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 Na 2 S0 4 . The folution was filtered, eveporated in vacuo.
  • 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 %.
  • 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), (Ph 3 P) 4 Pd (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 réelle 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 (MgS0 4 ) 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 %.
  • 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 MgS0 4 .
  • 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 BBr 3 . The mixture was stirred while warming to room temperature. After 3 hrs, ice was added, and the organic layer separated, dried over Na 2 S0 4 , filtered and evaporated affording F0001-o879a as an oil in 96 % yield.
  • 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 MgS0 4 , 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 ⁇ , ⁇ -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.
  • F2190-0576e was obtained as prepared as F0001-0167e from F2190-0576e and thiosemicarbazide.
  • F0001-i682d was prepared as F0001-0167e from F0001-0167C and
  • Compound 701-23-5 was prepared according to known procedure (J. Gen. Chem. USSR (Engl. Transl.), 1964 , vol. 34, p. 3063 - 3066/3099 - 3101)
  • VPC-18190 F1923-0800.
  • NMRPipe a multidimensional spectral processing system based on UNIX pipes. J Biomol NMR 6(3): 277-293.
  • TMPRSS2-ERG fusion a common genomic alteration in prostate cancer activates C-MYC and abrogates prostate epithelial differentiation. Oncogene 27(40): 5348-5353.

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  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • General Chemical & Material Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Health & Medical Sciences (AREA)
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  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

La présente invention concerne un composé ayant une structure de formule (I) : (I) des utilisations de tels composés pour le traitement d'indications induites par l'ERG, FLI1, ETV4 ou l'ETV1, y compris le cancer. L'invention concerne également une composition pharmaceutique et des procédés de traitement d'indications induites par l'ERG, FLI1, ETV4 ou l'ETV1, y compris le cancer. Le cancer peut être choisi dans le groupe constitué par : le cancer de la prostate, le sarcome d'Ewing, le cancer du sein ou le cancer du pancréas.
PCT/CA2018/050162 2017-02-13 2018-02-13 Composés de gènes (erg) associés à l'ets humain utilisés en tant qu'agents thérapeutiques et leurs procédés d'utilisation Ceased WO2018145217A1 (fr)

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Publication number Priority date Publication date Assignee Title
CN112279804A (zh) * 2020-11-20 2021-01-29 上海凌凯医药科技有限公司 一种制备2-氯-4-新戊基吡啶的方法
WO2025085030A1 (fr) * 2023-10-18 2025-04-24 Erciyes Universitesi Synthèse de dérivés semi-synthétiques de pénicilline contenant un cycle imidazole et thiazolidine dérivé d'hydrazone

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BUTLER, M.S ET AL.: "Discovery and characterization of small molecules targeting the DNA-binding ETS domain of ERG in prostate cancer", ONCOTARGET, vol. 8, no. 26, 2017, pages 42438 - 42454, XP055533633 *
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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112279804A (zh) * 2020-11-20 2021-01-29 上海凌凯医药科技有限公司 一种制备2-氯-4-新戊基吡啶的方法
CN113896678A (zh) * 2020-11-20 2022-01-07 上海凌凯医药科技有限公司 一种制备2-氯-4-新戊基吡啶的方法
WO2025085030A1 (fr) * 2023-10-18 2025-04-24 Erciyes Universitesi Synthèse de dérivés semi-synthétiques de pénicilline contenant un cycle imidazole et thiazolidine dérivé d'hydrazone

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