Synthesis, mechanistic and synergy studies of diarylidenecyclohexanone derivatives as new antiplasmodial pharmacophores

Diarylidenecyclohexanone (DAC) derivatives (Ia-i, IIa-c and IIIa-b) were synthesized, characterized and screened for their in vitro antiplasmodial activities against erythrocytic stages of chloroquine (CQ) sensitive and resistant strains of P. falciparum by using SYBR green I fluorescence assay. SAR studies of DAC derivatives showed antiplasmodial activity in the order of 3-NO2 (Ib, IC50 0.95 µM) > 3-chloro (Id, IC50 3 µM) > 4-chloro (Ie, IC50 8.5 µM) > 2-chloro (Ic, IC50 13 µM). Further Ib and Id exhibited nearly equal potencies against CQ-resistant strains P. falciparum Dd2, {IC50 1 µM (Ib) and 2.7 µM (Id)} and PfINDO {IC50 1.1 µM (Ib) and 2.5 µM (Id)}. Drug exposure followed by drug withdrawal-based stage-specific kill kinetic studies showed that Ib is shizonticidal within 3 h while the earliest killing actions against Trophozoites and Rings were seen at >3 h and >6 h, respectively. Combination studies of the most potent leads viz. Ib and Id showed strong to moderate synergistic effects with Artemisinin (ƩFIC50: 0.34 to 0.63) whereas no interaction (ƩFIC50: 0.65 to 2.36) was observed with Chloroquine. The DACs showed significant in silico binding affinity with β-haematin and P. falciparum lactate dehydrogenase (PfLDH) suggesting these to be the targets of their antiplasmodial action. High compliance with Lipinski rule of 5 and high selectivity index of Ib (105.3) and Id (8.3) against HeLa cell line indicated that Diarylidenecyclohexanones could serve as structural templates towards lead optimization of compounds for discovery of novel, potent, safe and affordable drugs against malaria.