Improved rationally designed lead drug structures against African trypanosomiasis, Chagas disease, and leishmaniasis were obtained against trypanothione reductase from Trypanosoma cruzi. Substituted-benzyl [3-(2-chloro-4a,10a-dihydrophenothiazin-10-yl)propyl] dimethyl ammonium salts, synthesized by Menschutkin quaternization of the tertiary alkylamine omega-nitrogen atom of chlorpromazine, were linear, competitive inhibitors of recombinant trypanothione reductase from T. cruzi, with either trypanothione disulfide or N-benzyloxycarbonyl-L-cysteinylglycyl 3-dimethylaminopropylamide disulfide as substrate. The permanent positive charge on the distal nitrogen atom of the tricyclic side chain contribution to binding was estimated as greater than or equal to 5.6 kcal.mol(-1) by comparison with the analogue with the cationic nitrogen atom of the quaternary replaced by an ether oxygen atom. A further major contribution to improving K-i values and inhibition strength was the hydrophobic natures and structures of the N-benzyl substituents. The strongest inhibitor, the (3-(2-chloro-4a,10a-dihydrophenothiazin-10-yl)propyl]-(3,4-dichlorobenzyl)dimethylammonium derivative (K-i 0.12 mu M), was similar to 2 orders of magnitude more inhibitory than the parent chlorpromazine. Several of these quaternary phenothiazines completely inhibited T. brucei parasite growth in vitro at < 1 mu M Antiparasite activity was not solely determined by inhibition strength against trypanothione reductase, there being a strong contribution from hydrophobicity (for example, benzhydryl-quaternized chlorpromazime had ED50 < 1 mu M). Although active against Leishmania donovani, none of the analogues showed major improvement in this activity relative to chlorpromazine or other nonquaternized phenothiazines. The p-tert-butylbenzyl-quaternized analogue very strongly inhibited (ED50 < 1 mu M) growth of the amastigote stage of T. cruzi.
