Journal of Medicinal Chemistry ring has moved outward by 1.0 Å toward the mouth of the active site. To accommodate this outward movement, the phenol side chain of Tyr110 rotates through 30 . The combination of this side chain rotation and inhibitor translation brings the phenyl group of 29a closer to Tyr110 by 0.5 Å when compared to compounds 1a, 6a, 11e, and 13e (Figure 5), allowing stronger hydrophobic stacking interactions to occur. Therefore, the 4- fold improvement in potency can be attributed to stronger hydrophobic interactions from occupying more of the subpocket and being closer to Tyr110. A more detailed kinetic analysis of 29a revealed that its mode of inhibition is linear mixed with respect to trypanothione, compared to linear competitive for 1a. However, comparison of the Ki and Ki0 values for 29a (Table 5) shows that 29a has a greater aﬃnity to the free enzyme than to the enzyme T[S]2 complex and so is behaving in a similar manner to 1a. To further explore the subpocket, a small collection of analogues containing a C4-aryl (29b e, 32a g, 35, and 36), a C4-cyclohexyl (29f), or a C4-benzyl (34) substituent were prepared and tested (Table 5). All of the analogues in this collection contained an N-ethylpiperidine substitution at N3 in order to make them directly comparable to the majority of the other dihydroquinazolines prepared in this study. The ortho- substituted analogue 1d was known to be inactive (Table 1), probably due to the additional steric bulk of the ortho-chlorine atom, preventing the C4 substituent from occupying the in- duced-ﬁt subpocket. Therefore, all of the phenyl derivatives were substituted at the meta and/or para positions. However, none of the aryl analogues (29b e, 32a c, 32e g, 35, and 36) showed any improvement in potency compared to the para-tolyl ana- logue 32d (Table 5). Increasing the size of the para-substituent to a tert-butyl group (29b) gives an analogue which retains activity, albeit less active than the para-hydrogen analogue (9a). This suggests that either the induced-ﬁt pocket can accommo- date larger hydrophobic groups or that the ligand can bind further away from the top surface of the induced subpocket, although either hypothesis must result in suboptimal ligand binding. This is supported by the observation that the para- methoxy and para-chloro analogues (32c and 32a) are equipo- tent to 9a. The meta-methoxy analogue 29d is 15-fold less active than 9a, suggesting that the protein cannot readily accommodate additional steric bulk in that region of the subpocket. There is no clear relationship between the electronic character of the aryl substituent and TryR inhibitory activity, with the meta-ﬂuoro (29c) and para-ﬂuoro (32b) displaying a 5-fold diﬀerence in potency, which cannot be attributed to sterics. The 4-cyclohexyl analogue 29f is 15-fold less active than the 4-Ph analogue (9a), consistent with the observation that a 4-aryl substituent con- tributes to binding aﬃnity by forming an edge face interaction with Tyr110. Similarly, the 4-benzyl analogue 34 is less potent than 9a. Three of the 4-OH intermediates on the route to the 3,4- dihydroquinazolines (31b d, Scheme 5) were also assayed against TryR (Table 5) and found to retain activity, albeit at a slightly reduced level compared to their reduced counterparts (32b d, respectively). This is consistent with the observed binding mode of the ligands, which suggests that the additional hydroxyl group would point out into the solvent exposed active site and as a result neither enhance nor impede ligand binding. The SAR (Figure S3 of the Supporting Information) and TryR ligand complexes suggest that the N3-substituent makes no signiﬁcant contribution to inhibitor binding. Therefore, quinazolines 6k and 21, which have truncated N3-substituents, ARTICLE were assayed against TryR and found to be equipotent with analogues containing large R3-groups (6a j, 9a f, Table 2). The reduction in substitution size results in compounds with improved ligand eﬃciencies (6k = 0.41 kcal mol 1 per non-H atom) and reduced polar surface area. Cell Screening. Selected dihydroquinazoline analogues were screened against both bloodstream form T. brucei parasites and MRC5 cells in whole cell assays (Tables 2 5). Analogues were selected such that representative examples of each array were screened. For the larger arrays, the most potent TryR inhibitors were preferentially triaged to the cell assays. All of the dihydroquinazoline analogues that were screened demonstrated antitrypanosomal activity greater than that for the hit compound (1a), consistent with them all being more potent TryR inhibitors. However, there is a poor correlation between the TryR IC50 and the T. brucei EC50, with some analogues (9a, 15c, 16a) having T. brucei EC50 values lower than their corre- sponding IC50 values. This suggests that some analogues are not exerting all of their antitrypanosomal activity through inhibition of TryR. The selectivity within the series (as judged by MRC5 EC50/T. brucei EC50) varies from ∼1 to ∼20, the poor selectivity of some analogues being consistent with oﬀ-target activities. It is hoped that, as this series is further optimized against TryR there will be a concomitant reduction in the oﬀ-target eﬀects and an increase in selectivity. Alternatively, the detailed understanding of the ligand binding mode may facilitate the identiﬁcation of alternative inhibitor scaﬀolds with greater selectivity. It is possible to identify some trends from analysis of the cell screening data. The nature of the N3-substituent has a large eﬀect upon both the selectivity and T. brucei EC50:TryR IC50 ratio. Most analogues which contain a basic N3-functionality (9c,e, 11e,h, and 13e,h) have poor selectivity (0.92 2.3). In addition, the amide-containing analogues (11e,h and 13e,h) display the largest (>15-fold) drop oﬀ between their enzyme and cellular inhibition values. However, 9a, which contains an ethylpiper- idine N3-substituent, does not follow this trend, instead being 11-fold selective. Analogues lacking a basic substitution at N3 (1a, 6a,d,g) show reduced cell activity in both assays, suggesting that overall the amine substituent is increasing oﬀ-target activity or is important for cellular uptake. However, although the N3 moiety has an eﬀect upon selectivity and IC50:EC50 ratio within the series, it does not make any signiﬁcant contributions to TryR binding (Figure 5). Compounds 9a, 15c, and 15d, which vary only in their C6- halogen substituent, all display similar activities in all assays (Table 4). However, analogue 15b (R6 = H) is ∼10-fold less potent against T. brucei and as a result signiﬁcantly less selective. This suggests that the nonspeciﬁc antitrypanosomal activity is in part related to the hydrophobicity of the inhibitors (calculated LogD pH 7.4; 15b = 2.1, 15d = 3.2). Compound 16a, which contained a C6 phenyl substituent, is 10-fold more potent against T. brucei cells than it is against TryR (Table 4), identifying this inhibitor as acting primarily oﬀ-target. In addition, 16a is also one of the most potent analogues in the MRC5 assay. Dihydroquinazoline 29a, the most potent analogue against TryR (Ki = 0.19 μM, Ki0 = 1.46 μM), is also one of the most potent against T. brucei (EC50 = 0.73 μM), demonstrating that improved TryR potency can drive antitrypanosomal activity. All C4-substituted analogues (29a,c, 31d, and 32d) retain their antitrypanosomal activity (Table 5 cf. 9a), in contrast with analo- gues containing large N3-substitutions (11e,h and 13e,h, Table 3), 6523 dx.doi.org/10.1021/jm200312v |J. Med. Chem. 2011, 54, 6514–6530