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Schistosomiasis is a neglected tropical disease and one of the most important parasitic diseases in terms of mortality and morbidity. The disease is caused through infection with blood flukes of the genus schistosoma. More than 700 million people in over 78 countries live in endemic areas and more than 200,000 deaths a year are due to this disease. Currently there are only two drugs available to combat schistosomiasis, Praziquantel and Oxamniquine, with Oxamniquine being only partially effective. The intensive use of these drugs for several decades led to an increased probability of resistance. Especially for Praziquantel, decreased sensitivity has been reported. Therefore, targeted research and development of new drugs is essential.
In the Grevelding lab, the schistosomal aldehyde-dehydrogenase was identified as a potential drug target. First experiments with Disulfiram, which is a known inhibitor of the human aldehyde-dehydrogenase and used for treatment of alcohol dependence, were performed. At a concentration of 100 µM, good antischistosomal activity, including morphological effects (i.e. tegumental disruption) was observed for disulfiram (dimer of diethyldithiocarbamate). At the same time, cytotoxic effects occurred at concentrations of 25 µM leading to exclusion of disulfiram itself as drug candidate. Nevertheless, it was used as a starting point in the Schlitzer lab (P. Mäder) for structural modification. Thereby, the class of dithiocarbamates was chosen and further synthesis of several hundred compounds followed. The most active candidates were those with the N-atom of the dithiocarbamate substituted with a sulfonylpiperazine moiety (i.e. small alkyl and phenyl) and the single bonded S-atom carrying a para-nitrobenzyle substituent.
In this work, further development of the sulfonylpiperazine derivatives was performed, by substituting the phenyl-moiety with electron-withdrawing and with electron-donating groups, who possess distinct steric requirements, additionally to which structure-activity relationships were established. The phenyl-moiety was also replaced by several heteroaromatics or fused ring systems. Furthermore, the substitution of the alkyl-group with small polar groups and synthesis of cycloalkyl substituents was carried out. In addition to sulfonamide derivatives, synthesis of asymmetrical substituted sulfuric acid diamides was pursued, since introduction of an additional nitrogen atom should be accompanied by better water solubility. For this purpose, a synthesis route starting from N,N'-sulfurylbisimidazole was established, which was used for the preparation of both tri- and tertra-substituted derivatives. As acylpiperazine derivatives showed good antischistosomal effects previously, piperazine-urea derivatives were considered as a secondary line for development in this work. To verify the necessity of the dithiocarbamate-skeleton, the heteroatoms therein were exchanged. Because of formation of highly reactive intermediates during biotransformation of the nitro-group, which in turn interact negatively with biomolecules, the nitro-group at the S-benzyl residue had to be placed by physiologically better tolerated residues.
For this, several strategies were pursued: (1) monosubstitution with electron-withdrawing groups, (2) multisubstitution to produce a dipole moment comparable to that of the nitrophenyl substituent, (3) substitution with groups of similar geometry and ability to form comparable interactions, (4) substitution with groups of identical molecular shape and volume and approximately equal distribution of electrons, (5) substitution with π-electron-poor heteroaromatics. The best optimizations of all parts of the molecule were combined with each other, to check whether there is an additive relationship with respect to the antischistosomal activity when the optimized structural elements are combined.
Taken together, approximately 280 novel compounds were synthesized and tested in vitro against schistosome pairs of the species S. mansoni. While ninety compounds were initially active at 10 µM, twenty-one compounds were still effective up to a concentration of 5 µM, and ten of these even at concentrations < 5 µM. Of a total of seventy-six derivatives for which cytotoxicity could be determined, thirty-three were non-cytotoxic at 100 µM. The most effective compounds are thus roughly comparable with Praziquantel in vitro but show different phenotypes. To determine the spectrum of action of the dithiocarbamtes, selected compounds were also tested against the juvenile stage of the parasite, other species such as S. japonicum and other helminths (Fasciola hepatica or Echinococcus multilocularis), and were particularly active. This was followed by further biological investigations (long-term tests, out-wash of inhibitors and confocal microscopic examinations of the internal schistosomal morphology). Finally, binding studies of dithiocarbamates using molecular docking on an established homology model of schistosomal aldehyde dehydrogenase and an in-silico based prediction of ADME-Tox parameters and a first up-scaling of the synthesis were performed.