In Silico Pharmacokinetic Profile and Antimalarial Efficacy of 3-Chloro-4-(4-Chlorophenoxy)Aniline

Despite the urgent need for new therapies to treat multidrug-resistant virulent Plasmodium falciparum, many promising antimalarial candidates fail to progress to clinical use because their pharmacokinetic (PK) profile is poorly characterized. This study predicted the PK profile of a new compound, 3-chloro-4-(4-chlorophenoxy)aniline (ANI), in silico using the pkCSM online platform. In vitro cytotoxicity in Vero E6 cells and antiplasmodial activity against chloroquine-sensitive (P. falciparum 3D7) and chloroquine-resistant (P. falciparum W2) strains were screened using the MTT colorimetric and the [³H]-hypoxanthine incorporation assays, respectively. In vivo antimalarial efficacy was evaluated in Plasmodium berghei ANKA and piperaquine-resistant (PQ^R) P. berghei parasites in mouse models employing early and established infection tests. In silico predictions show that ANI is a substrate for CYP 3A4 and CYP 2D6 and is unlikely to inhibit the hERG potassium ion channel. ANI exhibited low cytotoxicity, with a CC₅₀ value of 7.90 × 10² ± 86.40 μM, and potent antiplasmodial activity, with IC₅₀ values of 1.72 ± 0.09 μM and 1.84 ± 0.21 μM against the 3D7 and W2 strains, respectively. The selectivity index of greater than 400 indicates that ANI has a broad safety margin. In vivo, ANI demonstrated antimalarial efficacy with ED₅₀ values of 3.07 mg/kg/day against P. berghei ANKA and 2.81 mg/kg/day against PQ^R P. berghei parasites. Notably, mice treated with ANI in the established infection model survived for up to 30 days without observable adverse effects. These findings highlight ANI as a potential antimalarial compound and support further drug evaluation and development.