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  • br We used epoxy EPA as

    2020-08-12


    We used ω-3-17,18-epoxy-EPA as a lead compound to develop novel anti-cancer agents. The double bonds in ω-3-17,18-epoxy-EPA are sites for oxidative metabolism that leads to rapid degradation in vivo. We therefore prepared the fully saturated analogue of ω-3-17,18-epoxy-EPA - ω-3-17,18-epoxyeicosanoic 6-NBDG (ω-3-EEA, Fig. 1) – which impaired the via-bility of MDA-MB-231 triple-negative breast cancer cells in part by de-creasing energy production and cell cycle progression (Dyari et al., 2014). Although ω-3-EEA decreased the growth of tumours in mice carrying MDA-MB-231 xenografts, coadministration of a soluble epoxide hydrolase (sEH) inhibitor was required to protect the labile epoxide group from sEH-mediated hydrolysis (Morisseau and Hammock, 2013; Dyari et al., 2017). Falck and coworkers have developed urea bioisosteres that retain the biological actions of epoxy fatty acids but have improved stability (Falck et al., 2011; Falck et al., 2009; Falck et al., 2014). We adopted this ap-proach and developed CTU (Fig. 1), an aryl-urea that was derived from ω-3-EEA. CTU effectively decreased the viability of MDA-MB-231 breast cancer cells in vitro and in vivo in a mouse xenograft model without the need for coadministration of a sEH inhibitor (Rawling et al., 2017).
    CTU represents a new lead compound with potential anti-cancer activity, and a well-defined structure-activity relationship (SAR) is now required for its optimization and development. From our initial series of analogues it appeared that substitution of the ω-terminal aryl system with strong electron-withdrawing groups was required because analo-gues with weak electron withdrawing groups or electron donating groups, such as 1 (Table 1) were inactive (Rawling et al., 2017). In the present study we prepared an expanded library of analogues to further probe the relationship between the electronic properties of aryl ring substituents and the capacity of CTU analogues to decrease the viability of human MDA-MB-231 cells.
    2. Results
    2.1. Aryl-urea library design and synthesis
    To further elucidate the SAR of the aryl-urea fatty acid scaffold we designed a series of aryl-urea analogues (Table 1) bearing electron 
    withdrawing and donating groups of varying strengths, determined
    from the Hammett substituent constants (σtotal (Hansch et al., 1991)). Mono-, di-, and tri-substituted analogues were prepared with sub- stituents in the ortho-, meta- and para-positions.
    Aryl-ureas were prepared by two synthetic routes, depending on the commercial availability of the appropriately substituted aryl iso-cyanates required to form the urea moieties (Scheme 1). Aryl-ureas 7–9 were prepared in 2 steps, commencing with reactions of the corre-sponding aryl isocyanates with amine 13a in anhydrous THF for 4 h. Column chromatography of the crude products afforded ester-protected intermediates 7a–9a in good yields. In the final step the ester groups in 7a–9a were hydrolyzed with NaOH at room temperature, and acid-ification of the reaction mixtures precipitated aryl-ureas 7–9, which were isolated by filtration.
    Further aryl-ureas were prepared using an alternate 3-step proce-dure. The syntheses commenced from commercially available and ap-propriately substituted anilines, which were reacted with N,N‑carbonyldiimidazole (CDI) to generate N-carbamoylimidazoles (Scheme 1). N-Carbamoylimidazoles serve as masked isocyanates that are stable in the solid state but dissociate in solution to liberate an aryl isocyanate and imidazole (Rawling et al., 2012). Apart from their synthetic accessibility and stability, N-carbamoylimidazoles have the added advantage that the liberated imidazole catalyzes the rate de-termining proton transfer step in the urea-forming reaction to increase the overall reaction rate (Rawling et al., 2012). Thus, 13a or 13b was reacted with N-carbamoylimidazoles for 1 h, yielding intermediates 2a–6a and 10a–12a after chromatographic purification. As previously described, hydrolysis under basic conditions was used to remove the ester protecting groups to give the desired carboxylic acids.
    2.2. Novel CTU analogues decrease the viability of MDA-MB-231 breast cancer cells
    Table 1
    Chemical structures and electron withdrawing/donating strengths of ring sub-stituents.
    O
    HO
    N N
    O
    H H
    Compound R σtotala
    Cl
    CTU
    Cl
    Cl
    Cl
    Br
    F
    Cl
    Cl
    Cl
    Cl
    Cl
    aHammett substituent constants were taken from published values (Hansch et al., 1991). σp was used for substituents in ortho positions. 
    The analogues 8, 9 and 10 produced minimal decreases in ATP pro-duction, even after treatment of cells for 48 h at 40 μM concentration (Fig. 2A).
    To corroborate the effects on ATP production, the impact of a number of CTU analogues on cell cycle kinetics was investigated by flow cytometry. After 24 h of treatment 2, 4, 6, 7 and CTU increased the proportion of cells in sub-G1 phase to ~7.5–14-fold of control (Fig. 2B). Several analogues also altered the distribution of cells in S phase. The cell population in S-phase was significantly increased by 4 and 7 (17.7 ± 0.5% to 16.9 ± 1.1% of cells, respectively, compared with 13.2 ± 0.3% in DMSO-treated control, P < 0.01; Fig. 2B). 2 and 3 also increased the proportion of cells in S-phase, but to a lesser extent than 4 and 7, while 1, 5, 6, 8 and CTU did not markedly alter the proportion of cells in S-phase after 24 h of treatment (Fig. 2B). Together these findings indicate that several of the new analogues, especially 2, 4, and 7, impaired the viability of MDA-MB-231 cells by decreasing energy production and cell cycle progression. To confirm the selectivity of the decrease in breast cancer cell viability CTU and several other aryl-ureas were tested in the well-differentiated MCF10A control breast cell line and were found to minimally alter cell viability (not shown).