In the current study, the efficacy of three major by-products of ISM synthesis was compared to purified ISM, as well as the commercial products, Veridium® and Samorin®.

For the first time, the red and blue isomers and the disubstituted compound were synthesised, purified by HPLC and tested individually for trypanocidal activity against T. congolense and T. b. brucei in vitro, in addition to analysis of trypanocidal and prophylactic activity against T. congolense in vivo. Analysis of trypanocidal activity in Tanespimycin supplier vitro necessitated the development of a 96-well sensitivity test to determine IC50 values of the individual compounds. T. congolense IL1180, T. congolense IL3000 (kindly provided by the International Livestock Research Institute, Nairobi, Kenya)

and T. b. brucei Antat 1.1 strains were used for this study. T. congolense IL3000 and T. b. brucei Antat 1.1 bloodstream forms were used since the cultures of these strains are standardised ( Baltz et al., 1985 and Coustou et al., 2010). T. congolense Selleckchem EGFR inhibitor IL1180 was used for in vivo sensitivity tests since it causes a chronic infection in mice ( Coustou et al., 2010) and was previously used as a reference drug-sensitive strain for Samorin® uptake studies ( Peregrine et al., 1988) since it is ISM sensitive ( Hirumi, 1993). Isometamidium (M&B 4180A), the blue isomer (M&B almost 4250), red isomer (M&B 38897) and disubstituted compound (M&B 4596) were synthesised and purified by Provence Technologies SAS (Hôtel Technologique-BP100, Technopôle de Château-Gombert, 13382 Marseille Cedex 13-France). The compounds were analysed, and structures confirmed by LC/MS (M+) and NMR (1H and 13C). Respective purities were 99.7%, 97.6%, 95.4% and 97.9% for ISM, the blue isomer, red isomer and disubstituted compound. Veridium®, Samorin® and the compounds were dissolved in distilled water (10 mg/ml)

and stored at −20 °C. For in vitro drug sensitivity tests, drugs were subsequently diluted in the trypanosome culture medium. Prior to the drug sensitivity tests, observation of the general growth patterns of T. congolense IL3000 and T. b. brucei Antat 1.1 in 96-well plates for 72 h established that an inoculum of 4000 cells was optimal for exponential growth for 48 h. Parasites, counted using a haemocytometer (4000/well, 100 μl), were added to 96-well plates and incubated at 34 °C (T. congolense) or 37 °C (T. b. brucei) with 4% CO2 for ∼4 h before addition of drugs. The drugs were initially dissolved in water (10 mg/ml) and serial dilutions made in culture media (1:10). Each dilution of drug (100 μl) was added to 100 μl of culture in wells (duplicates were made for each dilution from 1 mg/ml until 1.10−19 mg/ml). Subsequently, parasites were cultivated under the aforementioned conditions for 24 and 48 h.