Design, Synthesis and Biological Evaluation of Novel Inhibitors of Trypanosoma brucei Pteridine Reductase 1

Genetic studies indicate that the enzyme pteridine reductase 1 (PTR1) is essential for the survival of the protozoan parasite Trypanosoma brucei. Herein, we describe the development and optimisation of a novel series of PTR1 inhibitors, based on benzo[d]imidazol-2-amine derivatives. Data are reported on 33 compounds. This series was initially discovered by a virtual screening campaign (J. Med. Chem., 2009, 52, 4454). The inhibitors adopted an alternative binding mode to those of the natural ligands, biopterin and dihydrobiopterin, and classical inhibitors, such as methotrexate. Using both rational medicinal chemistry and structure-based approaches, we were able to derive compounds with potent activity against T. brucei PTR1 (=7 nm), which had high selectivity over both human and T. brucei dihydrofolate reductase. Unfortunately, these compounds displayed weak activity against the parasites. Kinetic studies and analysis indicate that the main reason for the lack of cell potency is due to the compounds having insufficient potency against the enzyme, which can be seen from the low Km to Ki ratio (Km=25 nm and Ki=2.3 nm, respectively).

ethanol. The reaction was stirred at 20°C for 18 h and concentrated, washed, then concentrated again to afford a cream coloured solid. The crude product was purified by flash column chromatography (6% methanol / dichloromethane eluant), to yield compound 11 as a white solid (512 mg, 47%). 1

H NMR and LCMS
confirmed purity at >98%.
Absolute regiochemistry of compound 26 was confirmed by NOESY NMR: no interaction between CH 2 -dichlorobenzyl protons at 5.27 ppm (2H, s, CH 2 ) and the 7-OCH 2 -benzyl signal at 5.25 ppm (2H, s, CH 2 ) was observed, indicating groups are not in close proximity. Also, the relative 1 H NMR signal for the CH 2 -dichlorobenzyl protons at 5.27 ppm, versus 5.40 ppm for compound 31, agrees with the expected shift for a 4-alkoxy substituted N1-benzylated benzimidazole versus a 7-substituted benzimidazole. This observed relative shift is supported by the literature..
Absolute regiochemistry of compound 28 was confirmed by the relative 1

H NMR and LCMS
confirmed purity at >98%.
Absolute regiochemistry of compound 31 was confirmed by NOESY NMR: a strong interaction between CH 2 -dichlorobenzyl protons at 5.40 ppm (2H, s, CH 2 ) and the 7-OCH 2 -benzyl signal at 5.08 ppm (2H, s, CH 2 ) was observed, indicating close proximity of these substituents. Also, the relative 1 H NMR signal for the CH 2 -dichlorobenzyl protons at 5.40 ppm, versus 5.27 ppm for compound 31, agrees with the expected shift for a 7-substituted N1-benzylated benzimidazole versus a 4-substituted benzimidazole. This observed relative shift is supported by the literature. [8] Compound 26 (along with a second batch of compound 31) was obtained from the filtrate by flash column chromatography. 1
Absolute regiochemistry of compound 32 was confirmed by NOESY NMR: a strong interaction between CH 2 -dichlorobenzyl protons at 4.88 ppm (2H, s, CH 2 ) and the ortho proton signals of the pendant phenyl ring at 7.12 ppm (2H, d, CH) was observed, indicating close proximity of these substituents. Also an x-ray crystal structure of compound 32 in the PTR1 enzyme was obtained, confirming the absolute regiochemistry of compound 32 as 7-phenyl-1-(3,4-dichloro)-1Hbenzo(d)imidazol-2-amine.
Absolute regiochemistry of compound 33 was confirmed by the relative 1

H NMR and LCMS
confirmed purity at >98%.

3-Methoxybenzene-1,2-diamine (47)
To a solution of 2-methoxy-6-nitroaniline, compound 48 (500 mg, 2.98 mmol) in ethanol (15 ml) was added tin(II) chloride (2.83g, 14.9 mmol). The reaction was heated in a microwave reactor at 140°C for 10 min. The reaction mixture was poured into NaHCO 3 solution (30 ml) and the product was extracted into ethyl acetate (2x25 ml). The organic layers were combined and washed with sodium bicarbonate solution (1x30 ml), water (1x30 ml) and brine (2x40 ml), before being dried with MgSO4 and concentrated to remove ethyl acetate. Afforded compound 47 as a pale yellow oil in quantitative yield. 1 H NMR and LCMS confirmed purity at >95%.

Enzyme Assays
Inhibitor sensitivity against PTR1 and DHFR were determined using the cytochrome-c coupled assay method. [9] . An HPLC method was used to determine the sensitivity of PTR1 to compound 32 in clarified cell lysates. Assays were carried out in 20 mM sodium citrate (pH 6.0) containing 1 mM EDTA, 1% (v/v) DMSO, 0.1% (v/v) Triton X-100 and 100 μM NADPH cofactor. TbPTR1 (1.1 nM) was preincubated with varying concentrations of compound 32 (0-1 µM) for 5 min, before reactions were initiated using 25 nM dihydrobiopterin. Aliquots (100 μl) of enzymatic reactions were removed after 2 min and oxidized with iodine under alkaline conditions and analysed by HPLC as previously described. [9] The IC 50 of 32 against native PTR1 in T. brucei clarified lysates (20,000 g, 30 min, 4 °C; final assay concentration 100 µg ml -1 ) was determined in a similar fashion. Lysates were prepared [10] using T. brucei harvested from rats, [11] as previously described. Trypanothione reductase activity was measured in clarified lysates as previously described [12] to ensure adequate extraction of parasites.

Trypanosome Growth Assays
These were performed as previously described. [13]