Design, Synthesis and Biological Evaluation of Novel Inhibitors of Trypanosoma brucei Pteridine Reductase1

Human African trypanosomiasis (HAT) is a serious health problem in sub-Saharan Africa, with an estimated 50000 new infections each year, and over 60 million people in 36 countries are at risk of infection. HAT is a progressive and ultimately fatal disease. The causative agents of HAT are the protozoan parasites Trypanosoma brucei gambiense and T. b. rhodesiense, which are transmitted by the bite of a tsetse fly. In the initial stage of the infection, parasites multiply in the blood and lymphatic systems of the host, causing fever, headaches and joint pains. Eventually they cross the blood–brain barrier (BBB) to invade the central nervous system (CNS). Once the infection penetrates the CNS, it is very difficult to treat, causing the classical symptoms of mental deterioration leading to coma and death, which give the disease its more commonly recognisable name of ‘sleeping sickness’. The current drugs to treat HAT are inadequate, due to poor efficacy, side effects and the requirement for parenteral administration, which is not appropriate for a rural African setting. Folate metabolism has been successfully used as a drug target in a number of diseases such as cancer, bacterial infections and malaria. In particular, the enzyme dihydrofolate reductase (DHFR) is a clinically validated drug target in some diseases. 4] Figure 1 shows examples of known DHFR inhibitors. Although folate metabolism is a potential drug target in Trypanosoma and Leishmania, known DHFR inhibitors are not potent inhibitors of parasite growth. One possible explanation involves pteridine reductase 1 (PTR1), an NADPH-dependent enzyme that not only carries out the reduction of biopterin to dihydrobiopterin and dihydrobiopterin to tetrahydrobiopterin, but also the reduction of dihydrofolate to tetrahydrofolate. 7] Thus, PTR1 serves as a possible by-pass/resistance mechanism in Leishmania to DHFR inhibitors. Data from our laboratory, however, indicates that PTR1 may be a drug target in its own right in T. brucei, as PTR1 knockdown by RNA interference in the bloodstream form of T. brucei results in loss of viability in culture and loss of virulence in animal models of infection. Genetic studies indicate that the enzyme pteridine reductase 1 (PTR1) is essential for the survival of the protozoan parasite Trypanosoma brucei. Herein, we describe the development and optimisation of a novel series of PTR1 inhibitors, based on benzo[d]imidazol-2-amine derivatives. Data are reported on 33 compounds. This series was initially discovered by a virtual screening campaign (J. Med. Chem. , 2009, 52, 4454). The inhibitors adopted an alternative binding mode to those of the natural ligands, biopterin and dihydrobiopterin, and classical inhibitors, such as methotrexate. Using both rational medicinal chemistry and structure-based approaches, we were able to derive compounds with potent activity against T. brucei PTR1 (K i =7 nm), which had high selectivity over both human and T. brucei dihydrofolate reductase. Unfortunately, these compounds displayed weak activity against the parasites. Kinetic studies and analysis indicate that the main reason for the lack of cell potency is due to the compounds having insufficient potency against the enzyme, which can be seen from the low Km to Ki ratio (Km=25 nm and Ki=2.3 nm, respectively).