SYNTHESIS AND EVALUATION OF FOSMIDOMYCIN ANALOGUES AS ANTIMALARIAL AGENTS

This Ph.D. study constitutes part of an extensive project dealing with the design and development of new agents with antimalarial activity. The enzyme 1-deoxy-D-xylulose-5-phosphate reductoisomerase (DXR), which is involved in the DOXP pathway for isoprenoid synthesis, was chosen as drug target. This non-mevalonate pathway is not operating in humans and inhibitors of DXR can therefore be considered as selective for parasites. Fosmidomycin and its methyl homologue FR900098, well-known inhibitors of this enzyme that show antimalarial activity in vivo, were used as lead compounds for inhibitor design. The structure of fosmidomycin is characterized by a retrohydroxamic acid group, which acts as a ligand for the metal ion in DXR, and a phosphonate group, which mimics the phosphate group in the substrate structure. It was our objective to further expand the structure-activity relationship (SAR) of fosmidomycin as DXR inhibitor and to discover analogues showing superior activity against the enzyme and/or better antimalarial activity. Modifications were applied to the retrohydroxamic acid group and the propyl chain of the parent structures. Synthetic pathways to these modified fosmidomycines were elaborated. As initial screening method all new compounds were tested for their ability to inhibit DXR of Escherichia coli. The most promising inhibitors were further tested in an in vitro assay on two different Plasmodium falciparum strains. Our investigations are discussed in 4 chapters. 1. To explore the impact of structural modifications of the hydroxyl group of the retrohydroxamic moiety, analogues were synthesized with methyl, ethyl or hydroxyl ethyl substituents on the nitrogen (2.1-2.9). These modifications did not produce any acceptable inhibitors, which demonstrates the importance of the hydroxyl group in ligating the divalent metal. 2. A synthetic procedure for the preparation of α-aryl-substituted fosmidomycin analogues was developed starting from the corresponding benzylphosphonates or the appropriate cinnamaldehydes. Seven analogues were synthesized, allowing to perform an initial SAR study of this α-aryl series (3.1c,e and 3.2a-e). Although the α-aryl analogues were generally weaker DXR inhibitors than fosmidomycin, these analogues unambiguously surpassed the activity of fosmidomycin in inhibiting P. falciparum growth. Remarkably, the formyl analogues 3.1c and 3.1e consistently outperformed the acetyl derivatives 3.2c and 3.2e, both in the enzyme and the parasite growth inhibition assays. Compound 3.1e emerged as the most promising analogue with an IC50 value of 0.059 μM. Due to their straightforward synthesis these compounds are suitable for upscaling in further drug development programmes. The deoxygenated sideproducts 3.17a,c demonstrated the contribution of the hydroxyl group to the overall affinity. The Michael addition procedure, used to prepare the α-substituted analogues from phenyl-substituted cinnamaldehydes, proved also valuable to prepare the fosmidomycin cyclopentyl analogues 3.3 and 3.4. These compounds, featuring a restricted mobility, showed that a trans orientation of the retrohydroxamic acid moiety and the phosphonate group is optimal for binding the enzyme. 3. An efficient and reliable synthesis was established for β- and γ-oxa fosmidomycin analogues. Analogue 4.4, which combines a β-oxa modification with a hydroxamate moiety was almost as potent in inhibiting DXR as FR900098. In an in vitro assay it proved almost twice as active as FR900098 in inhibiting the growth of a P.falciparum 3D7 strain. 4. Several synthetic pathways towards the transition state analogue 5.1 were explored. A 10-step sequence, involving an asymmetric dihydroxylation, afforded compound 5.1 in a reasonable yield. Unfortunately, this compound did not show any worth mentioning inhibitory activity. In conclusion, this Ph.D. thesis highlights some important structural requirements for DXR inhibitors. This study also provides valuable information for modifications on fosmidomycin toward new and more efficient inhibitors: introducing aryl modifications on the α-position of the phosphonate moiety significantly improves the antiplasmodial activity in vitro and one oxa analogue with a hydroxamic acid proved to be a more potent inhibitor and to show superior antiplasmodial activity than the parent compound, fosmidomycin. Further investigation on analogues 3.1e and 4.4 is desirable since these compounds are significantly more potent than fosmidomycin on P. falciparum strains. Especially the latter one is very interesting, since it can be easily synthesized. Before further development the toxicological properties of these analogues need to be investigated and their pharmacokinetic profile needs to be established. In vivo studies, employing murine and monkeys models, are required. Further drug design may involve other sterically hindered analogues as well as prodrugs of the most promising analogues. VULGARISERENDE SAMENVATTING: Elke 40 seconden sterft er op de wereld een kind aan de gevolgen van malaria. De bestaande medicatie om malaria te behandelen verliest zijn werkzaamheid ten gevolge van resistentie van de parasiet voor de bestaande medicatie. Er is dan ook een urgente nood aan nieuwe, efficiënte, goedkope en veilige geneesmiddelen. In Gent aan het laboratorium voor medicinale chemie van de faculteit farmaceutische wetenschappen wordt onderzoek gedaan naar nieuwe geneesmiddelen. Recent zijn daar twee nieuwe fosmidomycine analogen ontdekt die zeer beloftevol bleken in het remmen van de malariaparasiet. Deze nieuwe antibiotica zouden een doorbraak kunnen betekenen in de behandeling van malaria.