A series of N-, S-, and COOH-blocked glutathione derivatives were evaluated against the pathogenic parasites Trypanosoma brucei, Trypanosoma cruzi, and Leishmania donovani in vitro, to identify the determinants necessary for activity and for further development into an active lead structure. The results show that N,S-blocked glutathione diesters are the most effective inhibitors of T. brucei with structures 14−16 being the most active, 14 having an IC50 ∼ 1.9 μM. The toxicity effects observed for glutathione derivatives 12, 14, and 16 have been correlated to the K562 antileukemic activity of these compounds and their inhibitory effects on the glyoxalase system of the host. Diester compounds based on S-2,4-dinitrophenylglutathione (17 − 22) were found to be significantly better inhibitors of T. brucei with ED50's in the range 16−0.19 μM. Compounds 19 and 20 were the two best inhibitors, with an ED50 of ∼1.07 and 0.19 μM, respectively; however 20 displayed toxicity in parasitic assays. Monoesters, monoamides, and diamides tested generally exhibited low in vitro activity. The compounds did not inhibit glutathionylspermidine synthetase and trypanothione reductase enzyme targets in the unique trypanothione pathway of these parasites. Diester compounds per se were considered to be ineffective inhibitors of trypanothione metabolism suggesting that these compounds might act as prodrugs, being hydrolyzed in situ into a variety of glutathione derivatives which include combinations of monoesters, free acids, and amines, some of which are inhibitors of trypanothione metabolism.

A series of N-S-blocked glutathione monoester and diester derivatives based on N-benzyloxycarbonyl-S-(2,4-dinitrophenyl)glutathione were evaluated for activity against the pathogenic parasites Trypanosoma brucei brucei, Trypanosoma cruzi, and Leishmania donovani in vitro.Only monoesters 7-9 with a log P value of >2.7 were active inhibitors of T.b. brucei bloodstream form trypomastigotes. Diester compounds 10-15 and 17-27 in most cases were better inhibitors of T.b. brucei than monoester compounds, and some displayed high activity against T. cruzi 14 and L. donovani 17, 19, 29. Compounds 14, 24, and 25 were the most active compounds identified against T.b. brucei having ED(50) values of <0.4 microM. Analysis of the inhibition data (ED(50)) vs calculated log P and E(s) values provided evidence to support membrane penetration and steric factors as the key component in the activity of these compounds. The optimum values for log P and E(s) determined were 5.8 and -0.70, respectively. A QSAR equation relating log(1/ED(50)) vs log P and E(s) was determined and interpreted within the proposed mechanism of activity for these compounds.

A new strategy for the skin delivery of bioactive compounds has been developed, using enzymes involved in the maintenance of the epidermal barrier function and the enzymatic transformation of corresponding precursors. This new strategy has been tested with regard to two enzymatic activities of the skin barrier: extracellular glucosidase and esterase/lipase. An analysis of the requirements for the glycosidic bond hydrolysis of any glycoconjugate by beta-glucocerebrosidase indicates that the release of the moiety linked to the glucose unit is obtained as long as the glycosidic bond being broken is not hindered, and as long as the leaving group property of the released moiety is good enough. This strategy was first applied to the release of the antioxidant delta-tocopherol. It was then extended to retinoic acid by introducing a spacer between the glucose unit and the bioactive moiety. This spacer was either a good leaving group such as hydroquinone, or a structure akin to a ceramide, namely glycerol. In these conditions, beta-glucocerebrosidase releases the complex spacer-active compound that is cleaved by an esterase. One of the advantages of this strategy lies in the slow release of the bioactive compound, extending in time its effect and most likely its tolerance, as is the case for retinoic acid.

Diesters based on N-benzyloxycarbonyl-S-(2,4-dinitrophenyl) GSH (CBzGSDNP) containing linear alcohols 3 to 9, branched alcohols 10 to 20, or heteroatom linear alcohols 21 to 25, were investigated as in vitro inhibitors of pathogenic parasites. Diesters 3 to 25 were better inhibitors of Trypanosoma brucei rhodesiense than of T. brucei brucei and had low cytotoxicities. The most active compound had a 50% effective dose (ED 50 ) of 0.2 M. A quantitative structure activity regression equation relating the log (1/ED 50 ) versus the hydrophobicity parameter (log P), Taft's steric parameter (E s ), molecular weight (MW), and the WienI descriptor (W) was determined, and the species difference was found to be related to membrane penetration and steric effects.Trypanosomiasis and leishmaniasis are parasitic diseases that cause severe infections in humans and domestic animals in the tropics. These infections pose a serious health problem to the countries in tropical regions, in terms of the suffering they inflict and the effects on their economies (22). Chemotherapy for treatment of these diseases is inadequate, because many treatments have poor clinical efficacy, produce side effects, or are toxic, especially in the late chronic stages, which inevitably leads to death. Parasites display a variety of unique metabolic reactions not present in other microorganisms and eukaryotes. One general approach to the development of novel antiparasitic drugs is to identify key differences in metabolism between the host and pathogen and use them in the design of selective toxic agents. Thiol metabolism in the trypanosomatids (15) is characterized by dependence on the hexapeptide, trypano- Fig. 1), an antioxidant that replaces glutathione (GSH), which is the major antioxidant of eukaryotic cells. The importance of trypanothione as an antiprotozoal drug target is highlighted by the fact that existing trypanocidal drugs, notably the arsenicals (e.g., melarsoprol) (3) and difluoromethylornithine (23), may work by interfering with the metabolism or synthesis of this hexapeptide. The central roles of trypanothione in trypanosomiasis and leishmaniasis make thiol-dependent enzymes potential targets for the development of chemotherapeutic drugs (15). Enzymes investigated to date include glutathionylspermidine synthetase, by using substrate analogues (6, 9), and trypanothione reductase, by using substrate analogues (12), subversive substrates (19), irreversible inhibitors (5), and phenothiazine derivatives (1). However, many of these compounds have proven to be inactive against trypanosomes in vitro (6, 9). By using a lead-directed approach to identify potential antiprotozoal compounds, we recently reported the identification of several GSH derivatives active in vitro against trypanosomiasis and leishmaniasis (10). A structure-function study of S-bromobenzylglutathione derivatives identified the antiparasitic activity to be exclusively associated with N, S-blocked GSH diester derivatives (10), with the nature of the N and S groups contributing to...

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The ability of drugs to show therapeutic activity in vitro can be affected by their hydrophobicity (log P). We recently reported the presence of antiparasitic activity within a group of glutathione diesters [I], against Trypanosoma brucei, Leishmania donovanz and Trypanosoma muzi. In this study we show the dependence of antiparasitic activity (ED50) vs log P and indicate an optimum value for the effectiveness of peptidyl drugs against strains of Tb. brucei. Whilst hydrophobicity is a principal component for activity, steric factors also contribute to the observed pattern with these prodrugs. The mechanism of anti-parasitic action of these glutathione prodrugs will be presented and the protein targets qualitatively identified by sensors studies [2].