br myricetin and the morphinan alkaloid di O
myricetin and the morphinan alkaloid 6,7-di-O-acetyl-sinococuline in-duced anti-proliferative cell cycle arrest in T24 CFTRinh-172 carcinoma cells, and MDA-MB-231 and MCF-7, respectively, in part by down-regulation of cyclin B1 (Sun et al., 2012; Li et al., 2015). The lignin erythro-aus-trobailignan-6 down-regulated the cyclin D1/CDK4 functional complex in MCF-7 cells (Han et al., 2017) and the glycoalkaloid solanine in-hibited prostate cancer cell proliferation by targeting several cyclins and CDKs (Pan et al., 2016). Similarly, epigallocatechin gallate elicited cell cycle arrest in G1 phase in oral squamous cell carcinoma cells by down-regulating cyclins D and E (Lee et al., 2015). We found previously that ω-3-17,18-epoxy-EPA selectively decreased the expression of the cyclin D1/CDK4 complex in endothelial cells and prevented cell cycle
progression (Cui et al., 2011). In contrast, however, cyclin E1, cyclin B1 and CDK6 were relatively refractory. The present findings that CTU and other active aryl-ureas down-regulated multiple cyclins and CDKs may offer an opportunity to efficiently decrease cancer cell viability by targeting cell cycle progression at multiple points.
Whereas proliferative signals activate the ERK MAP kinases to promote the accumulation of cyclin D1 and accelerate cell cycle pro-gression (Mebratu and Tesfaigzi, 2009), activation of the p38 MAP ki-nase decreases cyclin D1 and cell proliferation (Conrad et al., 1999). Consistent with these findings, a number of PUFA epoxides have been found to modulate MAP kinase pathways. Thus, ω-6 AA-derived epox-yeicosatrienoic acids activate ERK MAP kinase signalling and cyclin D1
expression (Potente et al., 2002; Chen et al., 2001). In contrast, the anti-proliferative ω-3-17,18-epoxy-EPA has been shown to activate the p38 MAP kinase and down-regulate cyclin D1 expression in endothelial cells (Cui et al., 2011). The natural products solanine, erythro-aus-trobailignan-6, epigallocatechin-3-gallate, 6,7-di-O-acetyl-sinococuline and myricetin, that have been reported to decrease the expression of cyclins and CDKs in multiple tumour cell types, have also been shown to activate the p38 MAP kinase anti-proliferative pathway (Sun et al., 2012; Li et al., 2015; Han et al., 2017; Pan et al., 2016; Lee et al., 2015). Together these findings suggest that a viable strategy for the develop-ment of novel anticancer agents could be the activation of the p38 MAP kinase to target the expression of multiple cell cycle regulatory proteins. The present study has identified a number of aryl-urea fatty acid ep-oxide bioisosteres based on ω-3-17,18-epoxy-EPA and the lead aryl-urea CTU that strongly activated the p38 MAP kinase in MDA-MB-231 cells and decreased the expression of important cyclins and CDKs to impair cancer cell viability.
We have evaluated the role of the electronic properties of the aro-matic substituent in the actions of aryl-ureas to decrease the viability of breast cancer cells. The SAR that emerged indicated that strong electron
withdrawing substituents (σtotal ≥ 0.66) in the meta- and para-positions were required for activity, and that ortho-substitutions were not toler- ated. Effective analogues were found to down-regulate cyclins D1, E1 and B1 as well as CDKs 4 and 6, that was related to activation of the p38 MAP kinase signalling pathway. Effective analogues also decreased MDA-MB-231 cell proliferation and activated apoptosis to varying ex-tents. Agents such as these may offer a new approach to target the cell cycle at multiple stages in order to efficiently prevent cancer cell ex-pansion.
5. Experimental section
Compounds CTU, 1, 10 and 13a were prepared as previously re-ported (Rawling et al., 2017). All other reagents and anhydrous solvents were purchased from Sigma-Aldrich (Castle Hill, NSW, Australia) and Fluorochem (Hadfield, Derbyshire, UK). Dry Column Vacuum Chro-matography was used to purify reaction products on silica gel with gradient elution. TLC was performed on silica gel 60 F254 plates. Melting points were measured on a Stuart SMP10 melting point appa-ratus. 1H and 13C NMR spectra were recorded on an Agilent 500 MHz
There was one less resonance than expected in the 13C NMR spectra of 3, 4 and 8, which results from signal overlap with the solvent peak (DMSO‑d6). The missing resonance arises from the carbon proximal to the urea group, which resonates at ~40.65 ppm in the spectra of their ester intermediates 3a, 4a and 8a. 3, 4 and 8 were not sufficiently soluble to obtain 13C NMR spectra in an alternate solvent. 12a and 12 were not sufficiently soluble in any solvent to record 13C NMR spectra. High resolution mass spectrometry (HRMS) was performed on an Agi-lent Technologies 6510 Q-TOF LCMS.