Journal of Medicinal Chemistry Table 2. SAR Study for Analogues with Substitution on the Piperazine Core compd 31 32 33 34 35 36 15 22 24 27 28 29 31 32 33 34 35 36 52 51.8 51.5 81 99.5 99.5 99.5 80.9 nt 97.1 99.9 99.9 R1 H H Me Me Me Me R2 H O O O H H R3 4-MePh 4-MePh 4-MePh 3Cl,4F-Ph 4-MePh 4-FPh mouse microsome hepatic extraction ratio 0.80 0.70 0.72 0.61 0.77 0.80 0.70 1.26 0.61 3.05 1.2 3.5 8.2 15 nt 3.22 15.56 nt 2.55 2.99 1.3 8.38 0.97 2.37 2.36 14.37 nt 3.35 6.96 nt 0.47 0.48 0.46 0.51 0.4 0.41 0.87 0.66 0.72 0.61 0.87 0.8 nt 0.66 0.69 0.63 0.89 0.79 0.96 0.95 nt 0.68 0.93 0.92 P. falciparum strain EC50 3D7 (nM) 161 ± 13 1079 ± 285 138 ± 10 36 ± 2 13 ± 2 5 ± 1 <0.21 0.49 0.53 0.52 0.55 0.47 0.79 0.44 nt 0.72 0.88 0.92 Article W2 (nM) 178 ± 12 1168 ± 188 136 ± 20 65 ± 3 13 ± 1 5 ± 1 nt 13.4 >30 5.6 21.9 >30 nt nt nt >30 nt nt Table 3. In Vitro ADME of Selected Analogues entry PAMPAa Caco-2b A−B Caco-2b B−A CYP Met CL-Msc (ER) CYP Met CL-rat (ER) CYP Met CL-Hud (ER) hERGe IC50 (μM) aMeasured as percentage absorbed. bunits = (cm/s ×106). cMs = mouse. dHu = human. eQ-patch assay; nt = not tested. Table 4. Complete in Vivo Pharmacokinetic Proﬁle of Selected Analogues in Micea IV PK parameters PO PK parameters entry Vd (L/kg) CL (mL/min/kg) T1/2 (h) AUC(0−24 h) (h·nM) Cmax (nM) Tmax (h) T1/2 (h) AUC(0−24 h) (h·nM) F (%) 15 12.3 188.9 2.7 1072 235 2.0 4.20 965 15 22 10.2 49.2 2.0 4119 1538 1.0 4.35 12155 74 24 7.5 27.5 5.5 7062 1723 3.0 5.21 21058 75 27 23.7 60.0 6.2 3442 1803 1.7 8.95 17894 87 29 7.5 36.1 18.0 7607 1642 1.0 14.54 32409 107 34 2.7 12.5 2.8 16525 3651 1.0 2.71 50619 77 36 1.2 25.9 0.8 9090 6404 1.0 2.95 19447 53 aPO dosing; dose (mg/kg), 20; species/strain, mouse, male balb/c; formulation, 2.5 mg/mL in PEG300/D5W, 3:1, solution; Vd = volume of distribution; CL = clearance; Cmax = maximal concentration; Tmax = time at maximal concentration; AUC = area under the curve; F = oral bioavailability. not evident in rat microsomes. Introduction of the dimethyl group on the 8-position of the piperazine ring does not im- prove overall metabolic stability of the compound (15 vs 28 or 31 vs 35) despite the metabolite identiﬁcation results presented earlier. This may be due to the ethylene bridge in the pipera- zine ring representing a metabolic soft spot and enabling meta- bolic switching. In general, this scaﬀold exhibited poor rat metabolic stability when compared to other species. One inter- esting ﬁnding with the substituted piperazine analogues was the improvement of activity against the hERG channel as deter- mined in the automated patch clamp assay. Compared to the unsubstituted piperazine analogue 27, we were able to decrease inhibition of the hERG channel by at least 2−3-fold, with most analogues (such as 22) suggesting a lower potential for QT interval prolongation in compounds with a substituted piperazine. Pharmacokinetic Properties. Compounds series found to have favorable potency and in vitro ADME properties were tested in snapshot PK, an abbreviated PK study shown to accurately bin compounds into low, moderate, or high oral exposure.9 Compounds predicted to have reasonable oral exposure would be tested in mice using both PO and IV routes of administration (Table 4). The in vivo clearance generally was low to moderate and signiﬁcantly improved when com- pared to the corresponding unsubstituted analogues (15, 22). This resulted in higher maximum concentration as well as 4248 dx.doi.org/10.1021/jm300041e | J. Med. Chem. 2012, 55, 4244−4273