Journal of Medicinal Chemistry improvement in AUC values in the oral arms as well. Lactam compound 34 exhibited highest oral AUC than all other ana- logues, and it also had lower intrinsic clearance with moderate volume of distribution. To test whether red cell partitioning was occurring to explain the high volume of distribution for the basic compounds, we carried out an in vitro experiment in which compound 22 was incubated in nai v̈e rat whole blood as well as plasma for 30 min. After centrifuging the blood and quantifying the amount of compound in the red blood cells, we determined that the concentration of the compound was 3.47 times higher in whole blood than in plasma alone, indicating that the compound is either adhering to the RBC’s or getting distributed inside the cells. In Vivo Eﬃcacy. Because we had examples of compounds with diﬀerent modiﬁcations with acceptable bioavailability, we decided to carry on the eﬃcacy experiment in mice. The in vivo anti- malarial activities of the optimized compounds were evaluated using a Plasmodium berghei mouse survival model.8 In this test, groups of three P. berghei infected mice were treated orally with compound one day after infection. The key readouts were the percentage of parasitemia reduction on day 3 postinfection compared to the untreated mice and the prolongation of survival of the infected mice. Table 5 summarizes the in vivo eﬃcacy test results of com- pound in comparison with standard antimalarials chloroquine Table 5. In Vivo Eﬃcacy of Compounds on Plasmodium berghei Mouse Survival Model Following Diﬀerent Dose Levels in Comparison with Known Antimalarialsa 1 × 30 mg/kg 1 × 100 mg/kg activity (%) survival (day) activity (%) survival (day) 3 × 30 mg/kg activity (%) survival (day) entry 22 24 29 34 36 CQb ASb 99.5 99.0 99.0 47.0 90.0 99.5 95.6 16.3 15.0 15.3 6.3 7.0 9.0 5.8 99.4 99.1 99.0 97.0 99.3 99.6 98.0 14.0 16.7 16.7 6.7 9.7 12.5 7.0 99.8 99.7 99.7 66.0 99.7 99.8 99.0 17.7 17.0 18.0 7.0 12.0 13.6 12.2 aActivity = average parasitemia reduction; survival = average lifespan after infection (6−7 days for untreated control mice); compounds were formulated in 75% PEG300/25% D5W (5% dextrose in water); CQ, chloroquine; AS, artesunate. b10% ethanol, 30% PEG400, 6% vitamin ETPGS. (CQ) and artesunate (AS). It was observed that compounds 22, 24, and 29 exhibited >99% parasitemia reduction at a low dose of 30 mg/kg with a modest survival of 15 days or higher. Increasing the dose to 100 mg/kg or dosing the compound 3 × 30 mg/kg did not lead to increase in survival. Currently, we are investigating the possibility of recrudescence of parasites or development of resistance for the compound in vivo to better explain the lack of curative activity in this model. We decided to determine the ED99 representative examples for the substituted and nonsubstituted piperazine cores and found the substituted piperazine core was ∼10-fold more eﬃcacious (compound 22 ED99 = 2.2 mg/kg) than the unsubstituted core (compound 1 ED99 = 26.5 mg/kg). Compounds 34 and 36 clearly proved inferior both in terms of parasitemia reduction as well as survival in mice, indicating the need for the amino acid side chain in providing better mouse eﬃcacy. On the basis of the eﬃcacy results, we decided to study the oral exposure of several compounds in rats. Rat was chosen as Article the second animal species for oral exposure evaluation as it may serve as the in vivo preclinical toxicity species. Taking into account the nonoptimal metabolic stability data for rats for this scaﬀold, we decided to study the oral exposure of the com- pound in rat for a period of 5 h in the oral arm only to get an estimation of the exposure (Table 6).9 The results show that Table 6. Rat Snapshot PK Results for Interesting Compoundsa PK parameters entry AUC(0−5 h) (h·nM) Cmax (nM) AUC(0−5 h)/dose [(min·μg/mL)/(mg/kg)] 22 487 126 1.2 24 367 96.66 0.95 29 BLQ 34 2097 504.44 5.07 36 423 202 0.9 aPO dosing; dose (mg/kg), 20; species/strain, rat, Wistar; formulation, 2.5 mg/mL in PEG300/D5W, 3:1, solution; BLQ, below level of quantiﬁcation; = could not be determined. the 8-substituted piperazine compounds had inferior exposure. α-Methylalanine subclass of compounds like 29 did not exhibit any in vivo exposure and were not further pursued. In line with the better rat metabolic stability (ER = 0.68), nonbasic lactam compound 34 demonstrated the best rat exposure despite lower mouse eﬃcacy. As the oral exposure of compounds 22 was low in rats but demonstrated superior mouse eﬃcacy, we decided to dose compound 22 at higher doses to make sure that suﬃcient multiples of exposure of the mouse eﬃcacious dose could be achieved (Table 7). We observe that when the dose was increased to 30 mg/kg from 10 mg/kg, there was over- proportional 7-fold increase in AUC as well as 2-fold increase in bioavailability. Increasing the dose further to 100 mg/kg dose with suspension formulation led to 55-fold increase in AUC with a 3-fold increase in oral bioavail- ability. The AUC at the mouse eﬃcacious dose (2 mg/kg) was 780 h·nM, therefore a ∼45× exposure multiple is achi- eved at 100 mg/kg in the rat. The multiple of exposure provides a suﬃcient window to use the rat as a rodent toxicology species. It is unclear at this time if this over- proportional exposure at higher doses is due to saturating metabolism or an eﬄux transporter, but given the partial eﬄux observed in Caco-2, this may explain this multiple dose exposure results. In Vitro Toxicology. Compound 22 was subjected to a series of assays to evaluate the toxicity proﬁle. This drug candidate was inactive on a panel of human cytochromes P450 (IC50s are greater than 6 μM for CYP1A2, CYP2C19, CYP2C9, CYP2D6, and CYP3A4), and the observed cytotoxicity CC50 were all greater than 12 μM on a panel of mammalian cell lines (293T, Ba/F3, CHO, HEp2, HeLa, Huh7), which translates to an adequate selectivity index (SI > 100). Compound 20 was found negative in the mini- Ames and micronucleus tests, which indicated a low mutagenic potential. Finally, considering the eﬃcacious plasma Cmax in the mouse at 2 mg/kg is low (∼75 nM), the risk of cardiotoxicity is considered to be minimal where the hERG channel inhibitory activity IC50 = 13.4 μM, providing a greater than 170-fold window. 4249 dx.doi.org/10.1021/jm300041e | J. Med. Chem. 2012, 55, 4244−4273