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Active screening and monitoring of human African trypanosomiasis have brought the disease close to eradication. However, ineffective screening of especially the rural and politically unstable regions of Africa result in underreporting of the disease. New and easily available drugs can have a major effect on the process toward complete eradication of this fatal disease. Over the last decades, several biological targets have gained interest for small molecule drug discovery, of which one is the trypanosome PDEB1/2. Tetrahydrophthalazinone-based TbrPDEB1 inhibitors show great potential as new treatment for human African trypanosomiasis. However, the enzyme selectivity profile for this class of compounds is still undesirably in favor for hPDE4, instead of TbrPDEB1. The aim of this thesis was the structure-based drug development of TbrPDEB1 selective compounds using tetrahydrophthalazinones as core scaffold and exploiting the small parasite specific pocket in TbrPDEB1 as handle to obtain selectivity over hPDE4. Chapter 2 describes the development of the first selective TbrPDEB1 inhibitors by introducing a rigid phenyl linker to activity probe the parasitic P-pocket. Several crystal structures of selective inhibitors in TbrPDEB1 prove the successful targeting of the P-pocket and form the basis of further exploration of selective TbrPDEB1 inhibitors (Chapter 3 to 5). Chapter 3 concentrates on development of diaryl tetrahydrophthalazinones to restore the lost cellular activity of the selective biphenyl class. The typical catechol character of the phthalazinones was reestablished and new linkers were designed based on the crystal structures of selective TbrPDEB1 inhibitors. New linker analogues of the biphenyl tetrahydrophthalazinones are discussed in Chapter 4 to determine the best vector for P-pocket targeting and find alternatives for the lipophilic phenyl moiety. Various linkers with different sizes, aromaticity and vector angles were tested. The combination of good cellular activity and a promising vector for P-pocket targeting in a TbrPDEB1 crystal structure pushed the alkynamide class forward for further development (Chapter 5 and 6). A structure-guided development of new alkynamides is described in Chapter 5. Computational techniques were used to predict new alkynamide modifications to probe the P-pocket to obtain a better TbrPDEB1 selectivity while retaining the cellular activity and cytotoxicity profile. Here we prove that selectivity for TbrPDEB1 over hPDE4 can be obtained for alkynamide phthalazinones, despite their lack of interaction with the P-pocket in the crystal structure. Chapter 6 extends the applicability of the alkynamide tetrahydrophthalazinones as possible new treatments for taxonomically related parasites Trypanosoma cruzi, Leishmania major and Plasmodium falciparum. Furthermore, the compound set is increased by several alkynamide isosteres to investigate the Michael accepting properties of the alkynamide functionality, which is indicated as one of the potential pitfalls. Finally, in Chapter 7 the obtained results will be discussed and will end with some concluding remarks.

Several members of the 3′,5′-cyclic nucleotide phosphodiesterase (PDE) family play an essential role in cellular processes, which has labeled them as interesting targets for various diseases. The parasitic protozoan Trypanosoma brucei, causative agent of human African trypanosomiasis, contains several cyclic AMP specific PDEs from which TbrPDEB1 is validated as a drug target. The recent discovery of selective TbrPDEB1 inhibitors has increased their potential for a novel treatment for this disease. Compounds characterized by a rigid biphenyl tetrahydrophthalazinone core structure were used as starting point for the exploration of novel TbrPDEB1 inhibitors. Using a virtual screening campaign and structure-guided design, diaryl ether substituted phthalazinones were identified as novel TbrPDEB1 inhibitors with IC50 values around 1 μM against T. brucei. This study provides important structure-activity relationship (SAR) information for the future design of effective parasite-specific PDE inhibitors.