Large numbers of structurally related congeners of pentamidine were synthesized by Tidwell RR et al [24] by introducing substitutions on the cationic groups, changing the position of dications from 4,4' to 3,3' position, changing the length of the aliphatic chain between the two aromatic rings, adding substituent on the aromatic rings at 2,2' and 3,3' position and replacing oxygen atoms in the alkyl linker with isosteric sulfur or nitrogen atoms (Figure 2a-g) . On comparing the activities of the synthesized compounds with pentamidine and melarsoprol, it was found that few compounds showed activity in the range or better than that of pentamidine and/or melarsoprol. The unsubstituted diamidine compound 64 ( Figure 3a ) with secondary amino group in place of oxygen atom exhibited subnanomolar activity (<0.001 µM) against T. b. rhodesiense and was nearly twice as selective against the pathogen as pentamidine. At the same time the diimidazoline compound 66 (Figure 3b ) exhibited the second highest anti-trypanosomal activity in the series with the IC 50 value of 0.001 µM and also had the highest parasite selectivity (SI T = 34500), being 63 times more selective against T. b. rhodesiense than pentamidine. The replacement of oxygen atom with sulphur atom as in compound 62 ( Figure 3c ) also generated active congeners having IC 50 (0.005 µM) value better than that of pentamidine (0.007 µM). The compounds 20-31 ( Figure 2c ) in which the cationic groups were present in the 3,3′-position of the aromatic rings and the length of the carbon linker was varied from 3-6 exhibited lower anti-trypanosomal activity compared to pentamidine whereas among the compounds 12-19 ( Figure 2b ) of the series, in which the amidine groups were present at 4,4' position and the length of aliphatic chain was varied from three carbon atoms to six carbon atoms, compound 12 ( Figure 3d , IC 50 =0.007 µM) with three methylene linker between the aromatic rings had same in vitro activity as that of pentamidine (0.007 µM). Further introduction of 2,2′-dichloro substituent in compound 12 improved the antitrypanosomal properties as evident from the in vitro activity of compound 32 (Figure 3e , IC 50 =0.004 µM).