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  • Next our exploration shifted to modifications

    2019-07-11

    Next, our exploration shifted to modifications at the lateral chain of the benzoquinone nucleus (SAR 2). Considering that the CK2 inhibitory activity is favoured by the presence of the -Ph-4-NO2 (SAR 1), the subsequent analogues were investigated with this moiety retained. The influence of the side chain length on the CK2 inhibitory was analysed with the preparation of compounds 23 (R2= (CH2)7CH3), 24 (R2= (CH2)5CH3), 25 (R2= (CH2)3CH3) and 26 (R2 = CH2CH3). These compounds were obtained from the multicomponent reaction of 4-nitrobenzaldehyde, malononitrile and the corresponding benzoquinones IV, V, VI and VII. The benzoquinones (IV-VII) were synthesized following the reactions shown in Scheme 3 [36]. Table 2 shows the results obtained in the evaluation of derivatives (23–26). As we can see the shortening of the side chain produces a drastic loss of activity. In docking model, Medroxyprogesterone acetate what 24 (R2=(CH2)5CH3), for example, showed the same pose and similar interactions as compound 7 into the binding pocket except for the interactions established by the Medroxyprogesterone acetate what alkyl chain. The inactivity of this compound could be explained for the absence of the hydrophobic interactions that the C-7-C-11 fragment of the alkyl chain of compound 7 establish, and reinforces the key role that the C-11 plays driving the orientation of the compounds within the lining of the ATP-binding site. Attending to the SAR 2 region we also evaluated the derivatives 27 and 28 in order to see the effect of the replacement of the alkyl chain and the hydroxyl group by a fused aromatic ring, and also the replacement of the free hydroxyl by a methoxy group. In both cases low percentages of inhibition were obtained (37% (27), 39% (28)), which ratifies the importance of the C-11 alkyl chain and the free hydroxyl group for the activity (Fig. 4). Next, our attention turned to the SAR 3 series bearing the -Ph-4-NO2 (SAR 1), -(CH2)10 CH3 and the free hydroxyl (SAR 2) moieties on the structural template. Since the NH2 group in the docking model establishes a hydrogen bond interaction with Leu 45, the importance of this group for the activity was assumed, and the SAR 3 was focused in the replacement of the CN group with electron-withdrawing groups, which could also act as hydrogen bond acceptors. Because of the CN group is introduced by the malononitrile component in the MCR, we carried out the MCR with other methylene active compound bearing one cyano group (necessary to generate the NH2 functionality) and other electron-withdrawing groups such as methylformyloxy or ethylformyloxy. Replacement of the cyano moiety by these groups (29–30) resulted in loss of activity (see Table 3). Compounds 29 and 30 with a methylformyloxy and an ethylformyloxy groups, respectively, showed a similar pose respect to compound 7. The carbonyl of the methylformyloxy and ethylformyloxy moieties presents the same interaction that the cyano group establishes with Arg 47. The loss of activity of these compounds could be explained considering a plausible intramolecular hydrogen bond between the NH2 group and the corresponding methoxy or ethoxy group, which consequently debilities the interaction between the amino group and Leu 45. The effect of the lead compound 1 (IC50: 0.90 μM) on viability of MCF-7 breast cancer cells was tested using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylte- trazoliumbromide) assay and compared to the effect of compounds 4 (IC50: 1 μM), 7 (0.22 μM), 8 (0.89 μM), 12 (0.57 μM), and 13 (0.84 μM). As it is shown in Fig. 5, all compounds tested showed almost no cytotoxic effect in MCF-7 cells after 48 h incubation at concentration of 10 μM. While using 100 μM (which is far away from any therapeutic dosage) the cytotoxicity was between 50 and 90%. The highest effect was shown by compound 7 with around 90% inhibition, while compound 12 showed the weakest effect with around 50%, and the inhibition effects of other compounds were for 4, 8, and 13 around 60%, 65%, and 55%, respectively.