2-aminobenzimidazoles for leishmaniasis: From initial hit discovery to in vivo profiling

Leishmaniasis is a major infectious disease with hundreds of thousands of new cases and over 20,000 deaths each year. The current drugs to treat this life-threatening infection have several drawbacks such as toxicity and long treatment regimens. A library of 1.8 million compounds, from which the hits reported here are publicly available, was screened against Leishmania infantum as part of an optimization program; a compound was found with a 2-aminobenzimidazole functionality presenting moderate potency, low metabolic stability and high lipophilicity. Several rounds of synthesis were performed to incorporate chemical groups capable of reducing lipophilicity and clearance, leading to the identification of compounds that are active against different parasite strains and have improved in vitro properties. As a result of this optimization program, a group of compounds was further tested in anticipation of in vivo evaluation. In vivo tests were carried out with compounds 29 (L. infantum IC50: 4.1 μM) and 39 (L. infantum IC50: 0.5 μM) in an acute L. infantum VL mouse model, which showed problems of poor exposure and lack of efficacy, despite the good in vitro potency.


Method A: Nucleophilic substitution reactions with amines
To a stirring solution of 2-fluoronitrobenzene derivatives (i) in DMF (concentration of 0.5 mol.L -1 ) was added K2CO3 (1.0 equiv.), KF (1.0 equiv.) and corresponding primary amines (x equiv.) at 0°C. The reaction was stirred at room temperature until the consumption of the 2-fluoronitrobenzene. Water (15-fold DMF amount) was added at room temperature and the formed solid was filtered and washed with water. The solid was dried in the high vacuum, generating the desired aniline. When a solid was not formed after water addition, the solution was extracted with diethyl ether. The organic layer was dried with magnesium sulfate, filtered e evaporated, giving the aniline product. 1

Method B: Reduction reaction of nitro groups
To a stirring solution of nitrobenzene derivatives in mixture of EtOAc and MeOH (1:1, concentration of 0.2 mol.L -1 ) was added 10% Pd/C (10 mol %) at room temperature. 4 Hydrogen gas (1 bar) was added until the consumption of the nitrobenzene derivative.
The solution was filtered through celite and evaporated under reduced pressure, generating the phenylenediamine derivatives (ii).

Method C: Construction of aminobenzimidazole fragment
To a stirring solution of phenylenediamine derivatives (ii) in methanol (concentration of 1.0 mol.L -1 ) in a sealed tube, was added a 1.0 mol.L -1 fresh solution of cyanogen bromine (1.5 equiv.) at room temperature. The reaction was stirred at 60°C until the consumption of phenylenediamine derivative. The reaction was quenched at room temperature with a solution of NaOH (2.0 mol.L -1 ). The organic layer was separated and washed with brine.
The DCM layer was dried with magnesium sulfate, filtered and evaporated under reduced pressure, giving the desired aminobenzimidazole fragment (iii). 2

Method D: Amidation reaction
To a stirring solution of aminobenzimidazole (iii) in a minimal possible amount of DMF was added EDC (1.2 equiv.), HOBt (1.0 equiv.) and the corresponding carboxylic acid (1.1 equiv.) at room temperature. The reaction was stirred until the consumption of aminobenzimidazole (iii). Excess of water was added and the mixture was stirred for 15 minutes at room temperature. The solid was filtered and washed with water and dried in 5 the high vacuum, generating the desired acyl aminobenzimidazole 2-48. A flesh chromatography column was performed for some final compounds.

Method E: Nucleophilic substitution reactions with ammonium chloride salts.
To a stirring solution of 2-fluoronitrobenzene derivatives in DMSO (0.1 mol.L -1 ) was added the ammonium chloride salt (2.0 equiv.) and DIPEA (4.0 equiv.) in a sealed tube.
The reaction was stirred at 80°C until the consumption of the 2-fluoronitrobenzene. Water (15-fold DMSO amount) was added at room temperature and the formed solid was filtered and washed with water. The solid was dried in the high vacuum, generating the desired aniline. giving the aniline product.
The solution of cyanogen bromine was prepared as reported in the literature. 3 The compound ii was prepared in 73% yield using the Method D. To a solution of ii (80 mg; 0.28 mmol) in DMF (2 mL) was added potassium carbonate (58 mg; 0.42 mmol) and n-propyl iodide (41 L; 0.42 mmol) at room temperature. After 19 h at room temperature was added water (6 mL). The mixture was extracted with EtOAc (3 x 5 mL), the organic 6 layer was dried with magnesium sulfate, filtered and evaporated under reduced pressure.
Then, the reaction was stirred 36 h at rt, quenched with water (2 mL) and diluted with DCM (2 mL). The product was extracted with DCM, washed with brine and dried over MgSO4 and concentrated. The residue was purified by flash column chromatography (Hexanes:AcOEt 20% to 40%) to yield 110 mg (77%) as a sum of separable regioisomers XVII and XVIII.
The mixture was evaporated under reduced pressure and purified by flash chromatographic column (9 DCM / 1 MeOH), giving the thiourea derivative in 75% yield.
To a solution of this thiourea derivative (1.06 g; 4.53 mmol) in acetic acid (20 mL) was added HBr (48%, 1.03 mL; 6.11 mmol) and bromine (0.84 mL; 16.31 mmol) at 0°C. After 15 h, a solution of NaOH (2.5 mol.L -1 ) was added at 0° C until pH 5. The mixture was extracted with EtOAc (2 x 50 mL). The organic layer was deried with magnesium sulfate, filtered and evaporated under reduced pressure. The crude was purified by flash cromatographic column (1 Hex/ 1 EtOAc), giving the bromine xxiv in 28% yield. The bromine xxiv (62 mg; 0.22 mmol) was solubilized in an ammonia solution (7 mol.L -1 in metanol; 3 mL) and heated for 2 h at 120°C in a microwave reactor. The reaction solution was evaporated under reduced pressure, generating the aminobenzimidazole analogue xxv in a quantitative yield. 55 was prepared in 61% yield, using the Method D from aminobenzimidazole xxii.