Multiparameter Optimization of Trypanocidal Cruzain Inhibitors With In Vivo Activity and Favorable Pharmacokinetics

Cruzain, the main cysteine protease of Trypanosoma cruzi, plays key roles in all stages of the parasite’s life cycle, including nutrition acquisition, differentiation, evasion of the host immune system, and invasion of host cells. Thus, inhibition of this validated target may lead to the development of novel drugs for the treatment of Chagas disease. In this study, a multiparameter optimization (MPO) approach, molecular modeling, and structure-activity relationships (SARs) were employed for the identification of new benzimidazole derivatives as potent competitive inhibitors of cruzain with trypanocidal activity and suitable pharmacokinetics. Extensive pharmacokinetic studies enabled the identification of metabolically stable and permeable compounds with high selectivity indices. CYP3A4 was found to be involved in the main metabolic pathway, and the identification of metabolic soft spots provided insights into molecular optimization. Compound 28, which showed a promising trade-off between pharmacodynamics and pharmacokinetics, caused no acute toxicity and reduced parasite burden both in vitro and in vivo.


Supporting Figures
. Clearance of compounds evaluated after incubation with human hepatocytes, represented as clearance values. Blue bars correspond to total clearance. Residual clearance after incubation with azamulin is shown as red bars. Residual clearance after incubation with 1-ABT is represented as green bars.          87.9 ± 0.7 82.6 ± 0.3 82.9 ± 3.5 a The % of inhibition values correspond to the mean of three measures.   CLint, intrinsic clearance after incubation with human and mouse microsomes; fu, fraction unbound; CLint_u, corrected clearance (CLint/fu); eLogD, experimentally determined distribution coefficient; PAMPA, parallel artificial membrane permeability assay.

Organic Synthesis Methodology Method A
To a solution of corresponding phenol (1 equiv.) in anhydrous DMF (in a minimum concentration of 0.3 mol. L -1 ) was added potassium carbonate (2 equiv.) and ethyl 2bromoacetate (1.2 equiv.). The reaction mixture was stirred at room temperature for 4-6 hours and monitored by TLC. After completion, the reaction mixture was quenched with water and extracted with diethyl ether (2 times). The organic layers were combined, washed with brine, dried over MgSO4 and concentrated under vacuum.
To a solution of the crude material in methanol (at a concentration of 0.5 mol. L -1 ) was added NaOH (6 mol. L -1 , in an equal volume of methanol) at 0 °C. The reaction mixture was stirred at 0 °C for 30 minutes. Hydrochloric acid (6 mol. L -1 ) was added at 0 °C until pH 2 was reached.
The mixture was then stirred at 0 °C for 10 minutes. The solid was filtered and washed with water to yield the corresponding carboxylic acid.

Method B
To a solution of corresponding phenol (1 equiv.) in anhydrous DMF (in a minimum concentration of 0.3 mol. L -1 ) was added potassium carbonate (2 equiv.) and benzyl 2bromoacetate (1.2 equiv.). The reaction mixture was stirred at room temperature for 4-6 hours and monitored by TLC. After completion, the reaction mixture was quenched with water and extracted with diethyl ether (2 times). The organic layers were combined, washed with brine, dried with MgSO4 and concentrated under vacuum.
To a solution of crude ethyl acetate/methanol (1:1) (in a concentration of 0.2 mol. L -1 ) was added 20% Pd/C (10 mol% equiv.) at room temperature. The reaction mixture was stirred at room temperature and under a hydrogen atmosphere for 1-2 hours. After completion, the mixture was filtered with a celite pad and evaporated under vacuum to yield the corresponding carboxylic acid.

Method C
Phenoxyacetic acid (5 mmol) was suspended in absolute dichloromethane (15 mL) at room temperature. A catalytic amount of N,N-dimethylformamide (0.10 mL) was added, and then neat oxalyl chloride (0.63 g, 5 mmol) was combined portionwise within 10 min. Stirring was continued until gas evolution ceased. The resulting clear solution was cooled down to 0 ºC, solid N-hydroxysuccinimide (6 mmol) was combined and then neat dry triethylamine (1.52 g, 15 mmol) was added portionwise within 10 min. After 15 min., the ice bath was removed, and stirring was continued for 2 h. The solvent was evaporated in vacuum, and the solid evaporation residue was partitioned between ethyl acetate and ice-cold water. The organic layer was washed with cold diluted NaHCO3 solution, brine, dried over Na2SO4, and concentrated in vacuum. The crude Nhydroxysuccinimide ester thus obtained was suspended in diethyl ether (3 mL), and the mixture was stirred at room temperature (15 min). Hexane (3 mL) was added, and the mixture was kept in a freezer overnight. The solid was filtered off, rinsed with cold diethyl ether, collected, dried in vacuum, and stored. In a separate reaction flask, commercial 2-(2-aminoethyl)benzimidazole dihydrochloride (1.07 mmol) was suspended in ordinary dichloromethane (30 mL). Neat, ordinary triethylamine (3.52 mmol) was added, and to the resulting clear solution was added the matching solid N-hydroxysuccinimide ester. The mixture was stirred at room temperature for 15 min. The solvent was evaporated in vacuum, and the evaporation residue was partitioned between ethyl acetate and water. The organic layer was washed with diluted NaOH solution, brine, dried over Na2SO4, and concentrated to dryness in vacuum. The crude solid product was suspended in diethyl ether, and the resulting slurry was stirred for 1 h at room temperature. The precipitate was filtered off, rinsed with diethyl ether, collected, and dried in vacuum. Unless otherwise stated, no further purification was required.

Method D
To a solution of 2-(1H-benzo[d]imidazol-2-yl)ethanamine dihydrochloride (II) (1 equiv.) in anhydrous DMF was added triethylamine (4 equiv.), followed by the corresponding carboxylic acid (1 equiv.), EDC (1.2 equiv.) and HOBt (1 equiv.) at room temperature. The reaction mixture was stirred at room temperature for 8-15 hours. After completion, the mixture was quenched with water (ten times the volume of DMF). The solid was filtered and washed with water to yield the corresponding amide.