Journal of Medicinal Chemistry Table 1. Inhibitory Activities of Commercially Available 3,4-Dihydroquinazoline Analogues against T. brucei TryR R2 R3 R4 R6 TryR IC50 (μM) 6.8a 1a CH3 CO2CH3 Ph Br 67b 1b CH3 CO2CH3 Ph CH3 1c CH3 CO2CH3 Ph Cl 9.4 1d CH3 CO2CH3 Br >100 1e CH3 CO2CH2CH3 Ph Br 12 1f CH3 CO2H Ph Br 37 1g CH3 CONHNH2 Ph Br 15 1h CH2CH3 CO2CH3 Ph Br 23 1i CH2-N-morpholine CO2CH3 Ph Br 89 1j CH2CH2-N-morpholine CO2CH3 Ph Br 93 1k Ph CO2CH3 Ph CH3 >100 1l 3,4,5-OCH3 phenyl CO2CH3 Ph CH3 >100 1m CH2NHCO Ph Br 37 1n CH2CH2NHCO Ph Br 39 a T. cruzi TryR IC50 = 19 μM. b T. cruzi TryR IC50 = 38 μM. (IC50 19 and 38 μM, respectively). The 3,4-dihydroquinazolines were considered promising screening hits for further investiga- tion due to their low molecular weights (373 and 308), reason- able ligand efficiencies (0.28 and 0.27 kcal mol 1 per non-H atom, respectively), and low polar surface area, an important property due to the requirement for blood brain barrier perme- ability to treat stage 2 HAT. (As defined here, ligand efficiency = free energy of binding (ΔG)/number of non-hydrogen (“heavy”) atoms, where ΔG = RT 3 lnKd with R = 1.987 cal K 1 mol 1; T = 300 K; and assuming Kd ∼ IC50 (M).) To determine the validity of this series as a starting point for the development of a lead compound for the treatment of HAT, 1a, 1b, and a small collection of commercially available analogues (1c n) were assayed against T. brucei TryR (Table 1). The hit compounds 1a and 1b were found to have IC50 values of 6.8 and 67 μM, respectively, conﬁrming their activity against the T. brucei enzyme. The similar levels of activity observed for 1a and 1b against T. cruzi and T. brucei TryR (<3-fold diﬀerence) is as expected given the high degree of sequence identity between TryR in the two species (83% at the amino acid level overall, 100% for active site residues). The remainder of the commercial analogues (1c n) display a range of TryR inhibitory activities (IC50 9.4 to >100 μM, Table 1). Although no analogues with improved potency were identiﬁed, it was possible to develop a preliminary SAR analysis (Figure 3). Therefore, the 3,4-dihy- droquinazolines represent a validated TryR inhibitor series and a hit expansion program was initiated. Biological Characterization of the Hit Series. A more detailed kinetic analysis (Figure S2 of the Supporting In- formation) established that 1a is a linear competitive inhibitor of TryR (with respect to trypanothione disulfide), with a Ki value of 0.92 ( 0.06 μM, in reasonable agreement with the IC50 value determined in the high-throughput assay (Table 1). Therefore, it is possible that the 3,4-dihydroquinazolines are binding to TryR in an orientation that partially occludes the substrate binding ARTICLE Figure 3. SAR derived from screening analogues 1a n (Table 1). Table 2. N3-Substituted 3,4-Dihydroquinazoline Analogues and Their Inhibitory Activities against T. brucei TryR and in Cell-Based Assays R3 1c CO2Me 6a Ph 6b Ph 6c 2-OMe-Ph 6d 2-F-Ph 6e 3-Cl-Ph 6f 3-CF3-Ph 6g 3-NMe2-Ph 6h 4-Me-Ph 6i 4-Cl-Ph 6j 4-CF3-Ph 6k Me 9a N-piperidine 9b N-morpholine 9c N-4-Me-piperazine 9d NMe2 9e NMe2 9f NHMe 21 OH a TryR Ki = 0.32 ( 0.01 μM. n 1 1 2 1 1 1 1 1 1 1 1 1 2 2 2 2 3 2 2 IC50 (μM) TryR EC50 (μM) T. brucei MRC5 9.4 0.93 2.0 1.1 0.94 3.5 1.5 0.81 1.3 2.2 2.2 1.2 1.8 2.8 0.93a 3.5 1.0 2.0 1.7 13 14 6.4 1.3 4.4 2.7 46 36 27 14 4.6 2.7 pocket. Compound 1a is a selective TryR inhibitor because it was inactive against human GR (IC50 >100 μM), the nearest human homologue of TryR. This is consistent with 1a binding to the negatively charged substrate-binding pocket of TryR, in contrast 6518 dx.doi.org/10.1021/jm200312v |J. Med. Chem. 2011, 54, 6514–6530