Chavalala, Hlamulo Edward. (2025). Synthesis and biological evaluation of Plasmodium falciparum calcium-dependent protein kinase inhibitors for malaria treatment and transmission-blocking. [PhD thesis, University of the Witwatersrand, Johannesburg]. WIReDSpace.

Abstract

Malaria is a mosquito-borne disease that was responsible for an estimated 249 million cases and 608 000 mortalities within 85 malaria endemic countries and areas in 2022, with children under the age of 5-years most burdened. Only five of the Plasmodium parasites are known to infect humans, with P. falciparum causing the most severe form of malaria. Several drugs have been used in the treatment of malaria, however, due to the emergence of parasite resistance, the efficacy of most available drugs has been reduced, prompting a need for the development of novel antimalarial agents to prevent an increase in malaria incidences and mortalities. Ideal novel antimalarial agents are those that target both the asexual and sexual forms of the Plasmodium parasite in the human host, to both treat and prevent the transmission of malaria. Two P. falciparum calcium-dependent protein kinases (PfCDPKs), PfCDPK1 and PfCDPK4, have emerged as attractive targets for such antimalarial compounds owing to their involvement in both the asexual and sexual blood stages of the parasite, and their absence in the human kinome. In this PhD thesis, we explore the pyrrolo[2,3-d]pyrimidine derivatives and their ring open analogues, the 4,6 diaminopyrimidine derivatives, as potential inhibitors of PfCDPK1 and PfCDPK4 towards the treatment of malaria and blocking of parasite transmission from human host to the mosquito. The first part of the thesis involves the design and in silico study of the targeted compounds, the pyrrolo[2,3-d]pyrimidine and the 4,6-diaminopyrimidine derivatives. We assessed the binding affinities of these compounds in silico against the PfCDPK4 model prepared from the crystal structure 4QOX from the RCSB Protein Data Bank, and the PfCDPK1 model prepared through homology modelling. The compounds were docked into the active site of the enzyme and interacted in the binding site in a similar way as the adenine ring of the natural substrate, adenosine triphosphate (ATP). Substituents important for selectivity and potency were chosen, and shown to occupy additional binding pockets, suggesting that our target compounds are potential inhibitors of PfCDPK1 and PfCDPK4. A representative docking pose from each series of compounds was chosen to evaluate the compound-enzyme stability using molecular dynamics and we found that the afforded poses are relatively stable with the hinge region interaction between the enzymes and the ligands playing a central role. Furthermore, we evaluated the compounds’ pharmacokinetic properties and toxicity potential, and results indicated that the molecules have good oral bioavailability and poor potential to cause neurological side effects but could cause cardiac-based complications. The second part of the research focuses on the synthesis and biological evaluation of the targeted pyrrolo[2,3-d]pyrimidine and 4,6-diaminopyrimidine-based compounds. The synthesis of the pyrrolo[2,3-d]pyrimidine compounds was achieved using a synthetic route consisting of nine synthetic steps which includes the formation of the pyrrolo[2,3-d]pyrimidine ring and the addition of side-groups that confer potency and selectivity to the compounds. We modified some of the synthetic steps using microwave irradiation, including the Sonogashira coupling reaction. Three compounds were found to be exclusively active against recombinant PfCDPK4 in the micromolar range, while one was active against PfCDPK1, also in the micromolar range. The 4,6-diaminopyrimidine derivatives bearing a two-atom linker to a bulky naphthalene substituent were divided into three classes the ethyne-, ethene-, and ethane-linked compounds. The ethyne- and ethene-linked compounds were afforded through at least four synthetic steps starting from 4,6-dichloropyrimidine. Three of these synthetic steps were modified using microwave irradiation. We could not prepare the target ethane-linked compounds by our planned hydrogenation of the corresponding alkyne precursors, as this reaction afforded only alkene products. This could be due to steric hindrance, although alternative reduction methods were not attempted. Unfortunately, the compounds from these series are yet to be tested for biological activity against PfCDPK1 and PfCDPK4. A second series of 4,6-diaminopyrimidine derivatives bearing a one-atom linker to a bulky naphthalene substituent were divided into two classes: those bearing a carbon-linker, and heteroatom-linked compounds. We developed the synthetic protocol for the synthesis of carbon linked 4,6-diaminopyrimidine compounds on a model study as far as the formation of 5-benzyl-6 chloropyrimidin-4-amine through five synthetic steps; with only one synthetic step remaining to afford an analogue of the target compounds. We have yet to apply the developed protocol in the synthesis of the targeted carbon-linked 4,6-diaminopyrimidine compounds and test them for biological activity. The development of a synthetic route to afford heteroatom-linked 4,6-diaminopyrimidine derivatives went as far as four synthetic steps of the planned six synthetic steps. Microwave irradiation played a central role in affording three of the intermediates, significantly reducing the synthesis time and solvent quantities. We have yet to complete and apply the synthetic route to our targeted compounds which will be tested for biological activity against PfCDPK1 and PfCDPK4. Molecular modelling has proved to be a viable tool in the design of our ligands and prediction of their binding potential in the active site of the targeted enzymes. To the scope of our research, the models we developed have shown good accuracy in predicting the binding affinity of the ligands in vivo as confirmed by experimental data. It gives us confidence that the biological results from the targeted 4,6-diaminopyrimidine derivative will agree with in silico data to a great degree. Also, it justifies the further use of molecular modelling in the design of new ligands and improvement of current ligands towards finding molecules that are potent inhibitors of the targeted enzymes.