Journal of Medicinal Chemistry Table 1. SAR Study for Analogues with Substitution on the Piperazine Corea Article P. falciparum strain EC50 compd R1 R2 R3 R4 R5 R6 3D7 (nM) W2 (nM) 15 H H gly H H 4-FPh 24 ± 2 23 ± 8 16 H H gly H H 3Cl,4F-Ph 4 ± 3 4 ± 2 17 (S)-Me H gly H H 3Cl,4F-Ph 29 ± 2 24 ± 1 18 (R)-Me H gly H H 3Cl,4F-Ph 31 ± 4 29 ± 3 19 (S)-i-Pr H gly H H 3Cl,4F-Ph 343 ± 59 470 ± 130 20 (R)-i-Pr H gly H H 3Cl,4F-Ph 168 ± 15 202 ± 38 21 Me Me gly H H 3Cl,4F-Ph 27 ± 1 24 ± 1 22 Me Me gly H H 4-FPh 10 ± 3 6 ± 1 23 Me Me gly H H 4-MePh 8 ± 2 6 ± 1 24 Me Me gly H H 3,4-diFPh 4 ± 1 5 ± 1 25 H H gly Me H 4-FPh 82 ± 7 111 ± 17 26 H H gly H Me 4-FPh 80 ± 5 76 ± 15 27 H H α-Me-Ala H H 4-FPh 20 ± 5 25 ± 2 28 Me Me α-Me-Ala H H 4-FPh 10 ± 1 9 ± 1 29 Me Me α-Me-Ala H H 4-MePh 2 ± 1 4 ± 1 30 Me Me α-Me-Ala H H 3,4-diFPh 13 ± 3 7 ± 1 aValues are means of two experiments. Each assay plate has meﬂoquine, sulfadoxine, and artimesinin as internal standards. The EC50 values for standard compounds match the literature values. Scheme 5. Structures of Key Compounds of the piperazine nitrogen could be modulated without complete loss in antimalarial activity. ADME and Pharmacokinetic Properties. In parallel to the SAR studies to optimize the potency of the compounds series, we evaluated the in vitro ADME and in vivo phar- macokinetic properties of key analogues. In general, this series of compounds had very good Lipinski's rule-of-ﬁve compliance and most of the analogues were highly soluble (>175 μM at pH 6.8), with the neutral lactams being least soluble among all the compounds. Table 3 summarizes the in vitro ADME proﬁles of the important compounds. Typically, a range of values in the permeability assays was observed. In the Caco-2 assays, the core piperazine compounds (both lactams as well as nonamide piperazine compounds) exhibited high apical to basal values (31, 32, 34, and 35). The permeability of the compounds was reduced once the side chain amino acid was installed, but metabolic stability is improved (28 vs 35). There was no clear correlation between Caco-2 and PAMPA assay. However, the % absorbed was lower in the PAMPA assay for compounds with poor permeability in the Caco-2 assay. Upon comparing the calculated log P values with PAMPA as well as Caco-2 values, we discovered no correlation with any of the permeability param- eters. Unsurprisingly, log P of 4.5 or higher was needed for having excellent percentage absorbed values in PAMPA assay (see Supporting Information). Removing the basicity of the piperazine ring by introduction of carbonyl group improved the mouse and human microsome stability of the compound, presumably by blocking the ethylene bridge (31 vs 32) but is 4247 dx.doi.org/10.1021/jm300041e | J. Med. Chem. 2012, 55, 4244−4273