Journal of Medicinal Chemistry ARTICLE Figure 4. The X-ray protein crystal structure of 1a in complex with T. brucei TryR. (A) and (B) show two orientations, rotated about 90 , of the TryR compound 1a complex active site showing experimental density (magenta) for the inhibitor contoured at 2.5 σ. Compound 1a is shown in blue and FAD in turquoise. Carbon atoms are shown in brown, oxygens in red, nitrogens in dark blue, and sulfurs in yellow. Important residues have been highlighted in black including Trp21 and Met113, forming the hydrophobic wall and electron donor Glu18. Those residues involved in binding the halogen component are labeled in green, and those involved in the novel hydrophobic pocket are in blue. (C) Shows the same complex with the solvent accessible surface shown in beige and the ligand in green to demonstrate where the inhibitor is occupying subpockets within the active site cleft. (D) Surface representation of TryR active site cleft with bound compound 1a. The structure of T[SH]2 (yellow) bound to TryR from PDB entry 2wow has been overlaid. When investigating the C6-position (Scheme 3, Table 4), the N3 ester was replaced with a more potent basic moiety (e.g., 9a vs 1c). The potency of analogues 1a and 15c demonstrated that a bromine atom was tolerated at C6 (Tables 1 and 4). Therefore, the C6-position was explored by introducing aryl substituents, prepared as outlined in Scheme 3. The C6-aryl analogues all demonstrated low micromolar TryR IC50 values (Table 4), giving no signiﬁcant improvement in potency. It was observed that the furan and thiophene analogues (16c and 16d) were 5- to 10-fold more potent than the other C6-aryls (16a,b,e,f). The fact that analogues containing large bicyclic substitutions at C6 retain some activity (16e,f) suggests that the protein does not entirely occlude the C6 position of the inhibitor upon binding. In addition, the observation that the C6-bromine analogue (15c) is 10-fold more potent than the similarly sized C6- phenyl analogue (16a) implies that the beneﬁcial eﬀect of a bromine substitution is not entirely due to its steric bulk and hydrophobic character. The C8-substituted analogues (18 and 19) were signiﬁcantly less active when assayed against TryR (Table 4), suggesting that the introduction of a large hydrophobic group at C8 is detri- mental to the inhibitor's ability to bind to TryR. As the chemistry-driven approach failed to identify inhibitors with improved ligand eﬃciency, protein crystallographic inves- tigations were initiated in order to facilitate an alternative rational inhibitor design program. The SAR expanded to include the analogues in Tables 2 4 above is summarized in Figure S3 of the Supporting Information. X-ray Crystallography, Analysis of Ligand Binding, and Rationalization of SAR. The X-ray crystal structure for the hit compound 1a in complex with T. brucei TryR was solved and refined at a resolution of 2.8 Å with an R factor and Rfree of 16.4% and 21.9%, respectively (Figure 4A,B, Table S1 of the Supporting Information). 6520 dx.doi.org/10.1021/jm200312v |J. Med. Chem. 2011, 54, 6514–6530