Use of an additional hydrophobic binding site, the Z site, in the rationaldrug design of a new class of stronger trypanothione reductase inhibitor, quaternary alkylammonium phenothiazines

Improved rationally designed lead drug structures against African trypanoso miasis, Chagas disease, and leishmaniasis were obtained against trypanothio ne 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 o f chlorpromazine, were linear, competitive inhibitors of recombinant trypan othione reductase from T. cruzi, with either trypanothione disulfide or N-b enzyloxycarbonyl-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 cati onic nitrogen atom of the quaternary replaced by an ether oxygen atom. A fu rther major contribution to improving K-i values and inhibition strength wa s the hydrophobic natures and structures of the N-benzyl substituents. The strongest inhibitor, the (3-(2-chloro-4a,10a-dihydrophenothiazin-10-yl)prop yl]-(3,4-dichlorobenzyl)dimethylammonium derivative (K-i 0.12 mu M), was si milar to 2 orders of magnitude more inhibitory than the parent chlorpromazi ne. Several of these quaternary phenothiazines completely inhibited T. bruc ei parasite growth in vitro at < 1 mu M Antiparasite activity was not solel y determined by inhibition strength against trypanothione reductase, there being a strong contribution from hydrophobicity (for example, benzhydryl-qu aternized chlorpromazime had ED50 < 1 mu M). Although active against Leishm ania donovani, none of the analogues showed major improvement in this activ ity relative to chlorpromazine or other nonquaternized phenothiazines. The p-tert-butylbenzyl-quaternized analogue very strongly inhibited (ED50 < 1 m u M) growth of the amastigote stage of T. cruzi.