Antimalarial benzimidazoles and related structures incorporating an intramolecular hydrogen bonding motif: medicinal chemistry and mechanistic studies

Doctoral Thesis


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Malaria, an infectious disease caused by Plasmodium parasites, continues to take an enormous toll on human health, particularly in tropical regions. According to the World Health Organization (WHO), progress against malaria eradication has stalled, specifically in the African region. Global efforts to curb the disease are being undermined by the gaps in access to vital tools. In 2019, about 229 million cases were recorded compared to the 228 million cases recorded in 2018. This is an annual estimate that has not changed significantly over the last four (4) years. Also, children under the age of five (5) years account for most malaria deaths worldwide. Chemotherapy represents one of the most effective control measures to mitigate the malaria burden, with the WHO presently recommending the use of artemisinin combination therapies (ACTs) to treat uncomplicated malaria. However, there is compelling evidence from Southeast Asia and recently in Rwanda (Africa) describing the emergence and spread of ACT resistance, characterized by reduced clearance rates of P. falciparum parasites. In some countries, resistance to partner drugs such as amodiaquine has been observed. These developments highlight the need to expand the antimalarial drug arsenal by exploring and developing new compound classes, preferably with a combination of novel modes of action, multistage activity, good safety profile, efficacy at low doses and reduced tendency to the development of resistance. In this study, two classes of compounds, benzimidazoles and imidazopyridines, incorporating an intramolecular hydrogen bonding (IMHB) motif, were explored for their antimalarial potential. These two chemotypes were selected on account of their privileged nature due to their capacity to interact with various biological systems, leading to a wide variety of biological activities, including antimalarial activity. Structural modifications around the benzimidazole scaffold resulted in the classification of these analogues into 1H-benzimidazoles and N-benzyl benzimidazoles (astemizole-based). In this regard, 33 benzimidazole analogues were synthesized, fully characterized and evaluated in vitro for their antiplasmodium activity against both the drug-sensitive NF54 and the multidrug-resistant K1 strains of the Plasmodium parasite. As a result, the 1H-benzimidazole analogues manifested sub-micromolar potencies against the chloroquine-sensitive NF54 strain of P. falciparum, with IC50 values between 0.079 µM and 0.968 µM. The most potent analogue within this series was compound 1.3 (Figure 1) with an IC50 of 0.079 µM against the chloroquine-sensitive strain and 0.335 µM against the multidrug-resistant (K1) strain. The resistance index of compound 1.3 (RI = 4) suggests the possibility of cross-resistance with drugs like chloroquine. The N-benzyl benzimidazole (astemizole-based) also displayed sub-micromolar activity against the chloroquine-sensitive strain of the parasite, with compound 2.3 (Figure 1) displaying the highest potency (IC50 PfNF54 = 0.029 µM and IC50 PfK1 = 0.117 µM) within the series. Generally, the benzimidazole analogues exhibited poor activity against the sexual gametocyte stage of the Plasmodium parasite in comparison to the asexual blood stage. However, compound 1.3 displayed sub-micromolar potency (IC50 = 0.382 µM) against earlystage gametocytes. Furthermore, selected potent analogues showed low cytotoxicity (SI = 39- 1500) when tested in vitro against the Chinese Hamster Ovary cells. The N-benzyl benzimidazole analogues, designed based on the known antihistamine drug astemizole, were tested against the hERG (human ether-a-go-go-related)-encoded potassium ion channel. These analogues expressed >40% inhibition against the hERG ion channel at the highest test concentration with potencies between 0.96 and 13.24 µM. Regardless, these compounds showed an improved cardiotoxicity risk relative to verapamil, a potent hERG channel inhibitor (IC50 = 0.58 µM), and the control drug used in the experiment. In addition, the five (5) selected potent analogues displayed low microsomal metabolic stability in mouse, rat and human liver microsomes. This impeded the advancement of these potent analogues to in vivo efficacy studies. Meanwhile, metabolite identification studies provided insight into the metabolic hotspots, which can be addressed in future optimization campaigns to address this liability. On the other hand, the imidazopyridine analogues were designed using the 1H-benzimidazole frontrunner analogue 1.3 as a guide. A structure-activity relationship (SAR) plan was pursued to produce diverse analogues due to modifications around the core scaffold. The SAR was explored with aromatic and aliphatic groups. As a result, 19 structural variants were synthesized and evaluated in vitro for their antiplasmodium activity against both the drug sensitive NF54 and the multidrug-resistant K1 strains of the Plasmodium parasite. 13 of these analogues showed potencies of <1 µM with compound 3.14 (IC50 = 0.08 µM) displaying the highest potency within the series. Subsequently, most of the active analogues showed a favourable cytotoxicity profile against CHO cells, with compound 3.14 being the least cytotoxic (SI = 466). Like the benzimidazoles, selected potent imidazopyridine analogues exhibited low microsomal metabolic stability in mouse, rat and human liver microsomes, posing a hurdle to the progression of these compounds to in vivo proof of concept studies. Aqueous solubility studies and physicochemical profiling of all the target compounds were carried out. The solubility results obtained were correlated with physicochemical parameters such as cLogP, melting points, TLC retardation factors and HPLC retention times to establish a solubility-property relationship across both classes of compounds. The correlation assessment revealed that different factors simultaneously affect the solubility of compounds across a series; hence, it may be crucial to assess these factors based on individual cases rather than an entire class of compounds. Also, the physicochemical assessment showed that both the benzimidazoles and the imidazopyridines complied with Lipinski's RO5 and Veber's rule. Single crystal X-ray structure analysis, IR spectroscopy, and DFT calculations were used to ascertain the presence of IMHB in the target compounds. Representative analogues 1.2 and 2.2 were used for these studies. In an effort to elucidate the mechanism of action, novel fluorescent analogues [1.3-NBD (IC50 PfNF54 = 0.044 µM) and 3.14-NBD (IC50 PfNF54 = 0.049 µM)] of the frontrunner compounds 1.3 and 3.14 were synthesized and pharmacologically validated as suitable probes for fluorescence live-cell imaging. The extrinsic fluorophore 7-nitrobenz-2-oxa-1,3-diazole (NBD) was employed due to the absence of intrinsic fluorescence properties in both compounds. Live-cell microscopy showed localization of both fluorescent analogues in all the studied organelles except the nucleus. While this suggests that the nucleus may not be a site of action for antiplasmodium activity, incorrect localization due to the NBD tag cannot be excluded. Based on the results from the live-cell imaging where both fluorescent probes accumulated in acidic organelles like the digestive vacuole and the neutral lipid bodies that have been implicated in hemozoin formation, it was hypothesized that the parent compounds 1.3 and 3.14 could be inhibiting the formation of hemozoin. Docking studies employed to investigate this hypothesis predicted intermolecular interactions between the parent compounds and the heme/hemozoin surfaces to inhibit hemozoin formation. The heme fractionation studies of compound 1.3 showed a dose-dependent increase in heme levels with a subsequent decrease in hemozoin levels at increasing compound concentrations. In essence, these observations support hemozoin inhibition as a mechanism of action of compound 1.3 while pointing to other targets within the parasite based on widespread association with other organelles. However, compound 3.14 showed no significant change in heme levels, but a decrease in hemozoin levels with increasing compound concentration was observed. This indicates that compound 3.14 is not a hemozoin inhibitor but could be targeting different digestive vacuole processes.