Novel benzophenone-, acetophenone-, and benzohydrazide-containing triazolopyrimidines as inhibitors of Plasmodium falciparum dihydroorotate dehydrogenase (PfDHODH): Design utilizing combined in silico approaches, synthesis, in vitro enzyme inhibition, and in vivo antimalarial activity

PfDHODH is an essential enzyme in malaria parasites for the production of pyrimidines. Unlike humans, the parasite is unable to recycle pyrimidines, making PfDHODH a promising target for antimalarial drug discovery. This research focuses on the design, synthesis, and biological evaluation of novel benzophenone, acetophenone, and benzohydrazide derivatives bearing triazolopyrimidines as PfDHODH inhibitors and antimalarial agents. A dataset of 99 triazolopyrimidine derivatives was used to develop CoMFA (r2ncv = 0.986, q2 = 0.626) and CoMSIA (r2ncv = 0.940, q2 = 0.616) models. Based on a 3D QSAR study, a series of 176 benzophenone, acetophenone, and benzohydrazide derivatives containing a triazolopyrimidine scaffold was designed and predicted for PfDHODH inhibitory activity. The designed compounds, which were predicted to have good inhibitory activity (pIC50 values of >9.5) were subjected to prediction of in silico ADMET parameters. The docking study was then conducted to select the best compounds for synthesis. Some of the designed compounds, such as 35d, 95d, 100d, 127d, 128d, and 144d, exhibited docking scores in the range of -10.4 to -9.0 kcal/mol. Molecular dynamics (MD) simulations confirmed the stability of the inhibitor within the PfDHODH binding site, and binding free energy calculations correspond to a calculated inhibition constant (Ki) of 58.57 nM, indicating the strong binding of the designed compound 128d to PfDHODH. A total of 9 out of the 176 designed compounds were selected for synthesis based on their predicted activity, in silico ADMET profile, and docking score, and characterized using NMR, mass spectrometry, and FTIR analysis, then screened in an in vitro PfDHODH enzyme inhibition assay. Furthermore, 4 compounds with good enzyme inhibition activity were selected for in vivo antimalarial assay in mice model of Plasmodium berghei. Compounds 13d and 13e were able to reduce parasitaemia levels by 30 - 35 %, respectively, proving the goodness of the approach.