Journal of Medicinal Chemistry position. The C2-methyl of 1a also stacks with Trp21Cδ1 at a distance of 3 Å, helping to stabilize the inhibitor in this position. The binding mode of 1a is consistent with 1i 1l showing weak or no inhibitory activity (IC50 89 to >100 μM) against TryR (Table 1, Figure 3). These analogues contain bulky substitu- tions at C2 that would prevent ligand binding due to occlusion of this position by the protein. Similarly, compounds 18 and 19 (Table 4) display reduced potency (IC50 53 and 24 μM, respectively) due to a steric clash between their C8 substituents and the surface of the active site cleft (Figure 4C). Residues Glu18, Trp21, and Met113, which form key inter- actions with 1a, are not conserved in human GR (see Figure S4 of the Supporting Information), which is consistent with the fact that 1a is not an inhibitor of the human enzyme (see above). Interestingly, the irreversible TryR inhibitor quinacrine mustard (Figure S7 of the Supporting Information) has also been shown to bind to the same hydrophobic area of the protein.26 Despite binding in the same region as 1a, the binding of quinacrine mustard does not induce a conformational change in the active site of TryR. A more detailed comparison of the TryR dihydroquinazoline complex with the published TryR quina- crine structures is discussed in section 3.4 of the Supporting Information. To help rationalize the SAR derived from the dihydroquinazo- line analogues, additional protein ligand complexes were struc- turally characterized. To investigate the N3 substitution TryR complexes with analogues 6a, 11e, and 13e were solved. Un- fortunately, no complexes with analogues containing C6-aryl substitutions could be determined. Comparison of the complexes for compounds 6a, 11e, and 13e with that for 1a show that the 3,4-dihydroquinazoline core superimposes in identical positions and that the C6-halogen substitutions overlay with one another (Figure 5). Therefore, the N3 substitution does not have any signiﬁcant eﬀect on the position of ligand binding. In analogue 6a, the N3 acetyl ester group has been replaced with a benzyl group, resulting in a 10- fold increase in potency (6a IC50 = 0.93 μM vs 1c IC50 = 9.4 μM). Interestingly, the benzyl group does not stack against Trp21 but is angled such that it is pointing away from the side chain of Trp21 and into the active site cleft in a similar fashion to the methyl acetate of 1a and so does not appear to contribute to inhibitor binding. In compounds 11e and 13e, the ester moiety has been replaced with substituted amides, giving 11- and 22-fold improvements in potency (IC50 0.86 and 0.42 μM, respectively). In 11e and 13e, the N3-substituent runs along the surface of the protein, forming hydrophobic interactions with Trp21 and other residues. The displacement of water molecules from the protein surface and the resultant hydrophobic interactions are likely to be the major eﬀect contributing to the improvement in potency observed with these analogues, as there are no other speciﬁc molecular interactions between the protein and the additional N3-amide functionality. The lack of speciﬁc molecular interac- tions between TryR and the N3-substituent of 11e/13e is consistent with the relatively ﬂat SAR observed from screening 6a j, 9a f, 11a h, and 13a h (Tables 2 and 3). Structure-Based Inhibitor Design. Although numerous modeling studies15,16,21 26,42 52 have predicted binding modes for various classes of TryR inhibitors, the accuracy of these models has not been corroborated by structure-based experi- ments and do not consider the ability of Met113 to undergo a conformational change as observed upon binding of 1a. There- fore, the current study represents a unique opportunity to ARTICLE 1.9 10 5.7 5.0 Table 5. C4-Substituted 3,4-Dihydroquinazoline Analogues and Their Inhibitory Activities against T. brucei TryR and in Cell-Based Assays perform a validated structure-assisted inhibitor design project for TryR. Examination of the TryR ligand structures suggested three primary areas for further investigation: (1) the eﬀect of altering the halogen substitution at C6, (2) challenging the hydrophobic pocket created by the movement of Met113 by altering the C4 phenyl moiety, and (3) reducing the size of the N3 substitution to improve ligand eﬃciency. Comparison of the halogens and hydrogen at the C6-positon (9a, 15b d) (Table 4) indicated that they were approximately equipotent. Therefore, 3,4-dihydroquinazoline-based inhibitors do not need to occupy the Ser14, Leu17, Gly49, and Val63 subpocket (Figure 4A) to eﬀectively inhibit TryR, providing that 15b binds in an analogous fashion to 1a. To investigate the eﬀect of challenging the induced subpocket with additional steric bulk, compound 29a, containing a para- toluene substituent at C4, was prepared as outlined in Scheme 4 and assayed against TryR (Table 5). The introduction of the additional methyl group resulted in a 4-fold improvement in potency (cf. 9e, IC50 = 1.0 μM, Table 2), suggesting that additional hydrophobic interactions in the subpocket are beneﬁcial for ligand binding. Structural determination of the TryR 29a complex revealed that the overall eﬀect of introdu- cing the methyl group is to push the inhibitor further out into the active site by 0.5 Å (Figure 5). The C6-chlorine and N1 atoms act as anchor points, having moved only 0.5 Å, whereas the C4-aryl 6522 dx.doi.org/10.1021/jm200312v |J. Med. Chem. 2011, 54, 6514–6530 IC50 (μM) EC50 (μM) n R3 R4 X TryR T. brucei MRC5 29a 2 NMe2 4-Me-Ph H 0.23a 29b 1 N-piperidine 4-tBu-Ph H 7.9 29c 1 N-piperidine 3-F-Ph H 0.9 29d 1 N-piperidine 3-OMe-Ph H 28 29e 1 N-piperidine 3,4-Cl2-Ph H 1.3 29f 1 N-piperidine cyclohexyl H 27 31b 1 N-piperidine 4-F-Ph OH 8.3 31c 1 N-piperidine 4-OMe-Ph OH 4.9 31d 1 N-piperidine 4-Me-Ph OH 0.71 32a 1 N-piperidine 4-Cl-Ph H 0.73 32b 1 N-piperidine 4-F-Ph H 4.9 32c 1 N-piperidine 4-OMe-Ph H 1.5 32d 1 N-piperidine 4-Me-Ph H 0.59 32e 1 N-piperidine 3-Cl-Ph H 1.2 32f 1 N-piperidine 3-Me-Ph H 4.6 32g 1 N-piperidine 3,4-Me2-Ph H 6.8 34 1 N-piperidine Bn H 50 35 1 N-piperidine 4-OH-Ph H 20 36 1 N-piperidine 4-OH-Ph H 1.6 a TryR Ki = 0.19 ( 0.01 μM, Ki0 = 1.46 ( 0.32 μM. 0.73 2 1.5 1.4