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]dimethylammonium 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 >/=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 microM), was approximately 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 microM. 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 ED(50) < 1 microM). 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 (ED(50) < 1 microM) growth of the amastigote stage of T. cruzi.

Quaternization of the nitrogen atom of 2-amino-4-chlorophenyl phenyl sulfide analogues of chlorpromazine improved inhibition approximately 40-fold (3',4'-dichlorobenzyl-[5-chloro-2-phenylsulfanyl-phenylamino)-propyl]-dimethylammonium chloride inhibited trypanothione reductase from Trypanosoma cruzi with a linear competitive Ki value of 1.7 +/- 0.2 microM). Molecular modelling explained docking orientations and energies by: (i) involvement of the Z-site hydrophobic pocket (roughly bounded by F396', P398', and L399'), (ii) ionic interactions for the cationic nitrogen with Glu-466' or -467'. A series of N-acyl-2-amino-4-chlorophenyl sulfides showed mixed inhibition (Ki, Ki' = 11.3-42.8 microM). The quaternized analogues of the 2-chlorophenyl phenyl sulfides had strong antitrypanosomal and antileishmanial activity in vitro against T. brucei rhodesiense STIB900, T. cruzi Tulahuan, and Leishmania donovani HU3. The N-acyl-2-amino-4-chlorophenyl sulfides were active against Plasmodium falciparum. The phenothiazine and diaryl sulfide quaternary compounds were also powerful antimalarials, providing a new structural framework for antimalarial design.