Journal of Medicinal Chemistry ARTICLE Figure 1. The TryR-catalyzed reduction of trypanothione disulﬁde (T[S]2) to dihydrotrypanothione (T[SH]2, N1,N8-bis(glutathionyl)spermidine). Figure 2. 3,4-Dihydroquinazolines 1a and 1b, small molecule inhibitors of T. cruzi and T. brucei TryR (see Table 1). the large, solvent-exposed trypanothione-binding site of the enzyme.20 Although it has been possible to readily identify TryR inhibitor series by both high-throughput9,13 and virtual screening,21,22 the development of these hits into submicromolar inhibitors has been limited by the lack of crystallographic information required to guide structure-based drug design. None of the proposed TryR inhibitor binding modes derived from docking experiments11,21,23 25 have been veriﬁed by structural techni- ques and the only high-resolution TryR inhibitor complex that has been reported is that from a covalent adduct between quinacrine mustard and T. cruzi TryR.26 In this current article, we report the synthesis and evaluation of a series of novel T. brucei TryR inhibitors based on the 3,4- dihydroquinazoline core structure (Figure 2). This series was identiﬁed from a high-throughput screen against Trypanosoma cruzi TryR13 and subsequently conﬁrmed to also inhibit T. brucei TryR. Inhibition data are reported for all analogues against T. brucei TryR and for selected compounds against the parasite in a whole cell assay. The structure activity relationship (SAR) derived from the in vitro data is discussed. Additionally, the key molecular interactions formed between the 3,4-dihydroquinazo- lines and TryR have been identiﬁed by determining the X-ray crystal structure of inhibitors in complex with T. brucei TryR. The liganded structures were used to guide further synthesis, resulting in the preparation of analogues with increased potency against the target enzyme. ’ CHEMISTRY The general synthetic route used to access the 3,4-dihydro- quinazoline analogues is outlined in Scheme 1. This route was based on a modiﬁcation of a literature route for the preparation of 2-triﬂuoromethyl-3,4-dihydroquinazolines.27 Brieﬂy, substituted 2-aminobenzophenones (2a d) were converted to amides (3a d) by treatment with functionalized acid chlorides. The ketone functionality of 3a d was subsequently reacted with primary amines under microwave irradiation to aﬀord substi- tuted imines (4a k, 7a d, 10, 12, 14b d, 20). Reduction of the imines with NaBH4 gave the corresponding secondary amines, from which the 3,4-dihydroquinazolines could be accessed either by reaction with POCl3 or by temperature-assisted cyclization. Alternatively, both the imine reduction and cyclization reactions could be achieved in a single pot by treating the imines with NaBH4 in reﬂuxing EtOH (Scheme 1). This synthetic route proved to be tolerant of a variety of substituents and was used to prepare a number of small compound arrays and individual compounds. The full range of compounds prepared is shown in Tables 2 5. To prepare two small collections of substituted amide analo- gues (Scheme 2) the t-Boc-protected analogues 11a and 13a were ﬁrst prepared by following the chemical transformations outlined above (Scheme 1). Deprotection of 11a and 13a by treatment with excess HCl, followed by reaction of the resultant amines with a collection of acid chloride building blocks, gave amides 11b h and 13b h, respectively (Scheme 2). Variation at the R6 position required the synthesis of inter- mediate 5-substituted-benzophenones. Where R6 was halogen (15c and 15d), this was obtained by regiospeciﬁc halogenation of 2-aminobenzophenone28 (Scheme S4 of the Supporting In- formation). Using the 6-bromo derivative (15c), it was possible to introduce aromatic rings at the 6-position using Suzuki chemistry29 (Scheme 3). Similarly, reaction of the 8-bromo-de- rivative 18 under Suzuki reaction conditions29 gave the 8-phenyl analogue (19) (Scheme 3). To further investigate the series, it was necessary to prepare analogues where the substituent R4 was varied. This required substituted 2-aminobenzophenone starting materials, which were synthesized by three diﬀerent routes (Scheme 4). Following a literature sequence, anthranilate 23 was converted to benzox- azinone 2430 and subsequently reacted with p-tolylmagnesium bromide to give benzophenone 27a in an 18% yield.31 Benzo- phenone 27a was then converted to quinazoline analogue 29a using the general reaction sequence described above. Given the poor yield of 27a, an improved two-step synthesis was employed for the preparation of 27b e (Scheme 4). Brieﬂy, anthranilate 23 was treated with substituted aryl lithiums followed by TMSCl to give the substituted 2-aminobenzophenones 26b e.32 Sub- sequent reaction with AcCl gave amides 27b e, which were converted to 29b e as outlined in Scheme 4. Alternatively, benzophenone analogues could be accessed by reacting Grignard reagents with nitrile 25, followed by hydrolysis of the resultant imine (Scheme 4). This was useful for the preparation of analogues that were not accessible by other routes (Scheme S6 of the Supporting Information). The route used to prepare each of 29b f required four dedicated steps from 23, or 25 making this route unsuitable for the rapid generation of analogues. Therefore, a shorter, more suitable route (Scheme 5) was developed for the preparation of a 6515 dx.doi.org/10.1021/jm200312v |J. Med. Chem. 2011, 54, 6514–6530