Article F (%) 20 40 57 Journal of Medicinal Chemistry Table 7. In Vivo Rat Dose Escalation PK Tests for Compound 22 IV PK parameters entry 22 Vd (L/kg) 13.7 CL (mL/min/kg) 67.5 T1/2 (h) 4.6 AUC (h·nM) 1831 ■ SUMMARY Imidazolopiperazine compounds had been proven to be eﬀective against multidrug resistant parasite strains with mini- mum toxicity to host-cell lines. However, early lead compound such as compound 15 and 27 still suﬀer from moderate potencies, relatively poor rat PK, and potent hERG inhibitory activity. Directed by the identiﬁcation of in vitro metabolites formed by compound 1, the piperazine core of 15 was sys- tematically modiﬁed by a gem-dimethyl functionality at all three possible places. The 8,8-dimethyl analogues demonstrated excellent potency as well as improved oral exposure in the mouse. This also allowed us to use the glycine amide that re- duced our hERG risk when compared to 27. The ﬁrst genera- tion of compounds exhibited eﬃcacy that was comparable to the known antimalarials in the P. berghei mouse model, how- ever, compound 22 was more eﬃcacious than chloroquine and artesunate especially when parasitemia reduction is compared in terms of ED99 values. Although compound 22 demonstrated poor rat PK at low doses, with higher doses it was able to achieve the necessary exposure multiples calculated based on the plasma concentration found to be eﬃcacious in the mouse model. Compound 22 is thus currently under pre- clinical safety assessment in rats as a potential candidate for human trials. ■ EXPERIMENTAL SECTION Experimental Protocols (Biology). Maintenance of Plasmo- dium falciparum Cultures. Plasmodium falciparum cultures were grown in O+ RBCs using culturing media: RPMI 1640 media (no phenol red) with L-glutamine and containing 0.05 mg/mL gentamicin, 0.014 mg/mL hypoxanthine, 38.4 mM Hepes, 0.20% sodium bicarbonate, 0.20% glucose, 3.4 nM NaOH, 5% human serum, and 1.25% albumax. Using traditional P. falciparum protocols published previously, 25 mL cultures were maintained in 75 cm2 ﬂasks (Fisher cat. no. 10-126-41) at 5% hematocrit and parasitemia ranging between 1% and 10%.10 Once parasitemia reached 8−10%, cultures are diluted down to 1% or higher depending on needs. Maintenance required daily media changes and fresh blood every two weeks. Cultures were gassed for approximately 30 s to 1 min using a blood gas mixture to maintain a gas composition of 96% nitrogen, 3% carbon dioxide, and 1% oxygen and incubated at 37 °C. On day 1 of the assay, percent parasitemia was determined by obtain- ing a 1 μL blood smear. The smear was ﬁxed onto the slide by placing in methanol for 30 s, stained in 10% Geimsa stain, and the percent of parasitized erythrocytes vs uninfected erythrocytes was determined by microscopy with a light microscope. Antiplasmodial Proliferation Inhibition Assay: 384-Well Plate Format. P. falciparum strains to be used for screening were prepared with screening media (culturing media without human serum but supplemented with 0.5% Albumax II) and fresh erythrocytes. Twenty μL of screening media were dispensed via the MicroFlo (BioTek) into 384-well, black clear-bottom assay plates (μClear GNF custom plates by Griener Bio-One). Then 50 nL of compounds were transferred using a PlateMate Plus (Matrix) or the GNF Systems PinTool into the assay plates containing screening media to a ﬁnal maximum concentration of 10 μM in a 12 point dose−response (1/2 log serial PO PK parameters dose mg/kg Cmax (nM) Tmax (h) T1/2 (h) AUC(all) (h·nM) 10 30 100 91 580 34885 1.5 4.3 8.0 4.7 6.3 8.4 974 7337 34885 dilution). On the basis of the measured parasitemia levels, each para- site strain was diluted to yield 0.5% parasitemia, and 50% hematocrit blood (uninfected erythrocytes) was added to a concentration of 4.17%. This diluted culture was used in dispensing the remaining 60% of the assay volume (30 μL) into the prewarmed assay plates via the MicroFlo liquid dispenser. The ﬁnal parasitemia was 0.3% and ﬁnal hematocrit was 2.5% in a total volume of 50 μL. Assay plates were incubated at 37 °C for 72 h in a gas chamber ﬁlled with low oxygen blood gas. After the 72 h incubation (equivalent to 1−2 cycles during the blood stage), a prepared mixture of the lysis buﬀer (5 mM EDTA, 1.6% Triton X-100, 20 mM Tris-HCl, 0.16% saponin) in water and SYBR Green detection (0.1%) reagent was dispensed at 10 μL per well using the MicroFlo. Cultures were incubated for an additional 24 h at 25 °C before measuring ﬂuorescence intensity using the Envi- sion plate reader (Perkin-Elmer) with a 505 dichroic mirror. Excitation and emission ﬁlters are 485 and 530 nm, respectively. Data were normalized based on maximum ﬂuorescence signal values for DMSO treated wells (no inhibition by compound) and the minimum ﬂuo- rescence signal values for wells containing the highest concentration of inhibitor control compounds, for example, pyrimethamine at a ﬁnal concentration of 10 μM. Data were analyzed on a plate-by-plate basis and compared to reference compounds that were always included on every plate, typically artemisinin, meﬂoquine, and pyrimethamine. EC50 values were obtained using a custom curve ﬁtting model, and a standard logistic regression model was applied for curve ﬁtting. The quality of the assay run was assessed by the performance of the reference compounds where the EC50 must be within 3-fold of the standard reference values for the assay plate to pass requisite data quality control needs. Additionally, all compounds were typically assayed at a minimum in duplicate (independent assay plates) and EC50 values ideally must not vary by more than 2-fold between plates. Metabolic Stability. The metabolic stability of drug candidates was determined in human, mouse, and rat liver microsomes using the compound depletion approach, quantiﬁed by LC/MS/MS. The assay measured the rate and extent of metabolism of chemical compounds by measuring the disappearance of the parent compound. The assay determined the compound’s in vitro half-life (T1/2) and hepatic extraction ratios (ER) and predicted metabolic clearance in human, rat, and mouse species.11 PAMPA Assay. Permeation experiments were carried out in 96- well microtiter ﬁlter plates. The compound concentration was deter- mined by UV absorption measured with a SpectraMax190 microplate spectrophotometer (Molecular Devices Corporation, Sunnyvale, CA, USA) at absorption wavelengths between 260 and 290 nm.12 Caco-2 Assay. The Caco-2 assay was carried out in a 96-well format, and compound concentrations in each chamber were mea- sured by LC/MS. The assay oﬀers apparent permeability data of test compounds from the apical to the basolateral chambers [Papp (A−B)] and from the basolateral to the apical chambers [Papp (B−A)]. The results can be used to predict the in vivo oral absorption (using Papp (A−B)) and the transport mechanism of the test compounds across GI membrane (ratio of Papp (B−A):Papp (A−B) and log P lipophilicity data).13 hERG (Q-Patch) Assay. The assay utilizes electrophysiology measurement on the electric current passing through hERG channel that is heterologously expressed in a stable CHO cell line. Channels were open by a hERG-speciﬁc voltage protocol, and the compound eﬀects were directly characterized by their block of the hERG current. The assay was performed on an automated platform, Q-Patch. All 4250 dx.doi.org/10.1021/jm300041e | J. Med. Chem. 2012, 55, 4244−4273