Journal of Medicinal Chemistry and CH3), 1.72 1.68 (m, 4H, 2 CH2), 1.54 1.48 (m, 2H, CH2). 13C NMR (125 MHz, CDCl3): δ 153.5 (C), 145.4 (C), 138.2 (C), 133.9 (C), 129.10 (C), 129.08 (C), 128.7 (CH), 128.5 (CH), 128.1 (CH), 127.4 (CH), 125.4 (CH), 85.2 (C), 58.9 (CH2), 54.6 (CH2), 43.9 (CH2), 25.2 (CH2), 23.6 (CH2), 23.2 (CH3). LRMS (ES+): m/z (%) 418 (100) [35Cl2 M + H]+, 420 (64) [35Cl 37Cl M + H]+. 6-Chloro-4-(4-chlorophenyl)-2-methyl-3-(2-(piperidin-1-yl)ethyl)- 3,4-dihydroquinazoline (32a). Prepared according to general method I from dihydroquinazolinol (31a) (42 mg, 0.1 mmol). The final product was purified from the crude reaction mixture by flash column chroma- tography over silica (MeOH/CH2Cl2 0:100 f 10:90) to give a clear glass (31 mg, 77%). 1H NMR (500 MHz, CDCl3): δ 7.35 7.33 (m, 2H, AA0BB0, 2 ArH), 7.25 7.22 (m, 2H, AA0BB0, 2 ArH), 7.15 7.11 (m, 2H, H-7 and H-8), 6.78 (d, 1H, J = 1.5 Hz, H-5), 5.76 (s, 1H, H-4), 3.52 (ddd, 1H, J = 15.0, 6.5, 6.5 Hz, CHH), 3.29 3.24 (m, 1H, CHH), 2.59 2.53 (m, 1H, CHH), 2.48 2.26 (m, 8H, CHH, CH3 and 2 CH2), 1.57 1.53 (m, 4H, 2 CH2), 1.44 1.39 (m, 2H, CH2). 13C NMR (125 MHz, CDCl3): δ 157.5 (C), 140.7 (C), 135.0 (C), 130.6 (C), 129.70 (CH), 129.2 (CH), 128.2 (CH), 126.2 (CH), 124.4 (C), 122.9 (CH), 62.9 (CH), 57.2 (CH2), 55.1 (CH2), 47.3 (CH2), 26.0 (CH2), 24.0 (CH2), 21.0 (CH3). Note: HMBC analysis suggests that the quaternary carbon peak at 135.0 results from two quaternary carbon atoms. LRMS (ES+): m/z (%) 402 (100) [35Cl2 M + H]+, 404 (71) [35Cl 37Cl M + H]+. HRMS (ES+): calcd for C22H2635Cl2N3 [M + H]+ 402.1498, found 402.1501 ( 0.62 ppm). Biology. TryR Enzyme Assay. Compounds were assayed against recombinant TryR from T. brucei brucei strain S427, which was expressed and purified as previously described.35 Potency was determined as independent duplicates for all compounds tested as previously.9,12 A serial titration of clomipramine was used as a positive control in each assay plate: BTCP was used as an additional control in some screening plates. ActivityBase from IDBS was used for all data processing and analysis. Some inhibitors were screened in a 96-well format essentially as above. The assay was assessed for robustness, yielding the following typical performance statistics: Z0 = 0.84 ( 0.001; %CV (plate) = 3.65 ( 0.4; S:B = 10 ( 0.25; IC50 (clomipramine) = 12.4 ( 0.14 μm. All reported IC50 values are the mean of at least two replicates, with the standard deviations typically being less than 20% of the mean. Cell Assays. Compounds were assayed against bloodstream form T. brucei brucei and MRC5 cells using resazurin fluorescence-based viability assays as previously described. All reported EC50 values are the mean of at least two replicates, with the standard deviations typically being less than 50% of the mean. The HAT drug pentamidine was present in all T. brucei plates as a positive control. Pentamidine has an EC50 of 5 nM (n > 1000) in this assay, which is in close agreement with published EC50 values.37 Mode of Inhibition Studies. These were determined as previously described using a DTNB-coupled assay.12 Briefly, the assay mixture contained 150 μM NADPH, 50 μM DTNB, and 20 mU mL 1 TryR and was initiated by the addition of T[S]2 ranging from 0.58 to 50 μM. The linear rate of increase in absorbance at 412 nm was determined using a Molecular Devices Thermomax plate reader. Each data set was fitted by nonlinear regression to the Michaelis Menten equation using GraFit 5.0 (Erithacus software). The resulting individual fits were examined as Lineweaver Burke transformations and the graphs inspected for diag- nostic inhibition patterns. The entire data set was then globally fitted to the appropriate equation (competitive, mixed, or uncompetitive in- hibition) and goodness-of-fit between models compared using the F-test (see Figure S2 of the Supporting Information for an example). Cloning, Expression, and Purification of His-Tagged TryR. The gene corresponding to T. brucei trypanothione reductase was cloned into a pET15b vector (Novagen) for overexpression with a 6-histidine tag. The plasmid was transformed into Escherichia coli strain BL21 (DE3) codon plus RIL. Cells were cultured in autoinduction media for 3 days and ARTICLE harvested by centrifugation. The cell pellet was resuspended in 25 mM Tris-HCl, pH 8.0, 500 mM NaBr, and 5 mM imidazole and lysed using a French Press at 16000 psi. Insoluble debris was removed by centrifuga- tion at 40000g for 20 min and the soluble lysate clarified by filtration through a 0.2 μm syringe filter. Ni2+ affinity chromatography, thrombin removal of the 6-histidine tag, and Q Sepharose anion exchange chromatography on an Akta purifier (GE Healthcare) yielded 80 mg L 1 of purified protein as assessed by SDS-PAGE. The protein was dialyzed into 50 mM HEPES, pH 7.5, 50 mM NaBr, and concentrated to 15 mg mL 1 to be used for crystallography. Crystallization. Sparse matrix screens (Jena Biosciences) were used to identify initial crystallization conditions, which were optimized and refined using the hanging drop vapor diffusion method to 26% MPD, 10% polyethylene glycol 3350, and 40 mM imidazole, pH 8.0. Thin, plate-type crystals grew in 3 days with dimensions 0.3 0.2 0.05 mm3. The crystallization mother liquor acted as a suitable cryoprotectant during data collection in which the crystals were cooled to 100 K in a stream of nitrogen gas. For the inhibitor complex structures, crystals were soaked for between 2 and 24 h with solid inhibitor compound introduced into the hanging crystallization drops. Data were collected both in-house on a Rigaku 007 MicroMax rotating anode X-ray generator coupled to an R-AXIS IV++ image plate detector and at the European Synchrotron Radiation Facility on beamlines ID14 2 and ID14 4. The crystals were space group P21 with cell dimensions of a = 101.8, b = 63.6, c = 169.8 Å, β = 97.9 . Data were processed and scaled using the XDS package,53,54 and statistics are shown in Table S1 of the Supporting Information. Structure Solution, Model Building, And Refinement. The structure of nonliganded T. brucei TryR was solved by molecular replacement using MOLREP (CCP4) and the T. cruzi TryR model 1aog20 [82% sequence identity]. Matthews coefficient calculations implied that there were two dimers in the asymmetric unit with a solvent content of 51%, which were found by MOLREP. COOT55 was used to correct the nonidentical residues in the TryR model chain A, which was then superposed on chain B, C, and D to generate the two dimers in the asymmetric unit. Further model building and water placement with COOT was interspersed with refinement with REFMAC5. The non- liganded protein was used as a template to solve the complex structures of the inhibitor series and other ligands in the study. Coordinates and libraries for the different inhibitors were generated with PRODRG.56,57 Statistics for each of the complex structures are shown in Table S1 of the Supporting Information. ’ ASSOCIATED CONTENT bS Supporting Information. Additional synthetic proce- dures and analytical information for screening compounds and selected intermediates. Further discussion of the protein X-ray crystal structures. All scheme, ﬁgure, table and section numbers preﬁxed with an ‘S’ are present in the Supporting Information. This material is available free of charge via the Internet at http:// pubs.acs.org. Accession Codes Coordinates of T. brucei TryR and TryR-ligand complexes have been deposited at the RSCB Protein Data Bank, (PDB ID codes 2woi, 2wov, 2wow, 2wp5, 2wp6, 2wpc, 2wpe, 2wpf). ’ AUTHOR INFORMATION Corresponding Author *Phone: (44)1382-385155. Fax: (44)1382-385542. E-mail: a.h.fairlamb@dundee.ac.uk. 6528 dx.doi.org/10.1021/jm200312v |J. Med. Chem. 2011, 54, 6514–6530