Journal of Medicinal Chemistry ARTICLE Table 3. Substituted Amide 3,4-Dihydroquinazoline Analogues and Their Inhibitory Activities against T. brucei TryR and in Cell- Based Assays n R3 R6 R8 TryR T. brucei MRC5 9a 1 N-piperidine Cl H 15b 1 N-piperidine H H 15c 1 N-piperidine Br H 15d 1 N-piperidine I H 16a 1 N-piperidine Ph H 16b 1 N-piperidine 4-pyridine H 16c 1 N-piperidine 2-furan H 16d 1 N-piperidine 3-thiophene H 16e 1 N-piperidine 1-naphthyl H 16f 1 N-piperidine 2-benzo(b)furan H 18 2 NMe2 Cl Br 19 2 NMe2 Cl Ph 1.8 0.78 0.79 0.35 7.3 4.4 1.3 0.79 8 8.5 53 24 a TryR Ki = 0.44 ( 0.03 μM, Ki0 = 2.27 ( 0.3 μM. b TryR Ki = 0.27 ( 0.02 μM, TryR Ki0 = 1.78 ( 0.3 μM. Table 4. C6-Substituted and C8-Substituted 3,4-Dihydro- quinazoline Analogues and Their Inhibitory Activities against T. brucei TryR and in Cell-Based Assays IC50 (μM) EC50 (μM) 1.3 12 0.44 0.55 0.75 14 23 8.4 6.8 3.4 to the positively charged pocket in GR. Analogue 1a was assayed against bloodstream form T. brucei and found to have an EC50 of 40 μM representing a >5-fold reduction in potency between the biochemical and cell assay. In a counter-screen against a mammalian cell line (MRC5 cells), 1a was found to be inactive (EC50 >50 μM). This is consistent with 1a being inactive against GR. Hit Expansion. Screening of commercial analogues demon- strated that the C6-bromine could be replaced with a chlorine atom without a significant loss of activity (1a vs 1c, Table 1). This beneficial reduction in molecular weight together with the availability of starting material 2a resulted in all further com- pound arrays consisting of analogues with a C6-chlorine sub- stitution (except where C6 was the point of variation). Initial synthesis aimed to identify a replacement for the potentially labile ester functionality of 1c. Therefore, benzyl analogues 6a j, amine analogues 9a f, and alcohol-containing analogue 21 were prepared (Table 2, Scheme 1, and Schemes S1 and S2 of the Supporting Information). Analogues 6a j, 9a f, and 21 all demonstrated an improved potency with respect to ester 1c in the T. brucei TryR assay, including examples showing a 10-fold improvement (6a, 6d, 6g, and 9c). However, there is only a 4-fold difference in potency between the most and least potent analogues in the two arrays (6g vs 6e/9d), resulting in a flat SAR. To improve potency, a chemistry-driven, systematic SAR analysis was performed to investigate the N3-, C4-, C6-, and C8-positions. Additional functionality at the C2-position had been shown to result in a large reduction of potency (1i 1l, Table 1, Figure 3), so was not further investigated. To probe the N3-position, amide derivatives of analogues 9f and 9c were prepared (11a h and 13a h, Scheme 2 and Scheme S3 of the Supporting Information). These amides retained their TryR inhibitory activity (Table 3), but there was a narrow range of potencies (IC50 0.37 2.6 μM), giving no identiﬁable SAR. Compounds 11e and 13h were found to have a mixed mode of inhibition, although comparison of their Ki and Ki0 values show that they bind to the free enzyme more readily than to the enzyme T[S]2 complex, similar to the mode of inhi- bition of 1a. Although more potent, the ligand eﬃciencies39 of 13e and 13h are poorer than that of 9c (0.27 and 0.28 vs 0.31 kcal mol 1 per non-H atom, respectively). 6519 dx.doi.org/10.1021/jm200312v |J. Med. Chem. 2011, 54, 6514–6530