Journal of Medicinal Chemistry Scheme 4. Synthetic Routes for the Preparation of 3,4-Dihydroquinazoline Analogues Containing Diﬀerent Substitutions at R4 (29a f) (Also See Scheme S7 of the Supporting Information; For Details of Individual Substituents, See Table 5)a ARTICLE a Reagents and conditions: (i) Ac2O, 138 C, 2 h; (ii) (1) R4Li, THF, 0 C, 2 h; (2) TMSCl, 0 25 C, 15 min; (iii) R4 MgX, Et2O, 0 25 C, 16 h; (iv) AcCl, DMAP, pyridine, CH2Cl2, 25 C, 16 h; (v) R4 MgX, THF, 25 C, 30 min, 67 C, 2 h; (vi) H2N(CH2)nCH2R3, EtOH, 160 C (MW), 2 h; (vii) NaBH4, EtOH, 78 C, 16 h. Scheme 5. Shortened Synthetic Strategy for the Preparation of 3,4-Dihydroquinazoline Analogues Substituted at R4 (For Details of Individual Substituents, See Table 5)a a Reagents and conditions: (i) (1) 1-(2-aminoethyl)piperidine, CH2Cl2, 25 C, 1 h; (2) formamide, 130 C (MW), 10 min; (ii) R4 MgCl, THF, 0 25 C, 2 4 h; (iii) BF3 3 OEt, HSiEt3, CH2Cl2, 25 C, 16 h. The two C4 methoxyphenyl analogues (32c and 29d) were converted into the corresponding phenol analogues (35 and 36) by treatment with BBr3 (Scheme S8 of the Supporting In- formation). This gave a total of 15 analogues containing diﬀerent C4 substitutions (Table 5). ’ BIOLOGICAL ASSAYS Compounds were assayed against recombinant T. brucei TryR using a spectrophotometric nonenzymatically coupled assay.35,36 In this assay, the activity of TyrR is coupled to the reduction of DTNB (5,50-dithiobis-(2-nitrobenzoic acid)) to 2TNB , which can be measured as an increase in absorbance at 412 nm (Figure S1 of the Supporting Information). The same assay was used to conduct a more detailed kinetic analysis in order to determine the mode of inhibition of selected inhibitors (see section 2.2 of the Supporting Information for more details). Selected compounds were also assayed against bloodstream form T. brucei using the resazurin ﬂuorescence-based cell viability assay37 modiﬁed from a previous method.38 In addition, the compounds were as- sayed using a similar protocol against MRC-5 cells as an indica- tor of mammalian toxicity. Compound 1a was also assayed against human GR using a protocol conceptually similar to the TryR assay. ’ RESULTS AND DISCUSSION Identification and Validation of the 3,4-Dihydroquinazo- lines as a Hit Series. A high-throughput screen of 100000 com- pounds against T. cruzi TryR13 identified the 3,4-dihydroquina- zolines 1a and 1b (Figure 2, Table 1) as low potency inhibitors 6517 dx.doi.org/10.1021/jm200312v |J. Med. Chem. 2011, 54, 6514–6530