Isolation and characterization of African marine natural products and repositioning of the natural product antibiotic fusidic acid and privileged benzimidazole scaffold for tuberculosis and malaria

Dziwornu, Godwin Akpeko

Isolation and characterization of African marine natural products and repositioning of the natural product antibiotic fusidic acid and privileged benzimidazole scaffold for tuberculosis and malaria

Doctoral Thesis

2019

Abstract

Many bioactive compounds isolated from nature (microbes, plants and animals) are successfully being used as drugs to prevent and/or cure diseases. A greater number of these natural products have not progressed to the latter stages of development as drugs largely due to the drawbacks of toxicity, limited supply, growing concerns to conserve terrestrial and marine ecosystems and their biodiversity, which impedes sample collections in adequate quantities for research, among others. However, in spite of these drawbacks, the medicinal chemistry explorations of bioactive natural products, which hitherto may have been abandoned in the repositories of research laboratories, have afforded an even greater number of drugs in clinical use. The collaboration of natural products chemistry and medicinal chemistry to discover, optimize and develop bioactive compounds as drugs remains an important endeavor today. A chemistry-guided natural products investigation of the newly identified marine red alga Laurencia alfredensis, which is an endemic species of South Africa, afforded ten (10) newly reported compounds from the genus. Isolation and purification of the compounds from the DCM/MeOH 1:1 v/v crude extract was achieved through a combination of normal phase benchtop column chromatography, preparative TLC, and semi-preparative HPLC. Elucidation of the chemical structures was accomplished by 1D- and 2D-NMR, HR-ESI-MS, UV and IR spectroscopic techniques, and X-ray diffraction analysis. The isolated compounds belong to the Laurencia secondary metabolite classes of labdane diterpenoids, polyether triterpenoids and sterols, and a glycolipid, which is reported herein for the first time from the genus. The compounds displayed low micromolar to poor in vitro antiproliferative activity towards the human breast (MDA-MB-231) and cervical (HeLa) cancer cell lines with IC50 values in the 8.8 – 133.8 µM range. A further natural products research effort involving the chemical investigation of two marine sponge species, Halichondria sp. and Hymeniacidon sp., is also reported herein. The specimens were collected from the Prince Edwards Islands in the Southern Ocean in the vicinity of Antarctica. With the aim to investigate the chemical and biological properties of the secondary metabolites from the cold continent, the organic crude extracts (DCM/MeOH 1:1 v/v) of the specimens were purified by DIOL flash chromatography and reverse phase semi-preparative HPLC. Two previously unreported compounds were isolated from the Halichondria sp., comprising of a bis-zooanemonin betaine metabolite and a trimer of proline betaine. The two compounds were inactive (IC50 >10 µg/mL) towards the drug-sensitive strain of Plasmodium falciparum (NF54), in vitro. Meanwhile, the Hymeniacidon sp. afforded the known nucleoside, thymidine, the amino acids, tyrosine, leucine, isoleucine and valine, and the small alkaloids, choline and agmatine. The natural product antibiotic, fusidic acid, has been in clinical use as, largely, a bacteriostatic agent against staphylococcal pathogens since 1962. Having exhibited a similar mode of action via inhibition of Mycobacteria tuberculosis and P. falciparum elongation factor G (EF-G), the current work expanded the structure-activity relationship (SAR) explorations in the medicinal chemistry approach of repositioning for tuberculosis and malaria. Twenty-nine (29) fusidic acid C-21 ethanamides, anilides and benzyl amides were synthesized and evaluated in vitro against the M. tuberculosisstrain H37Rv and asexual blood stage and gametocytes parasites of the human malaria parasite Plasmodium falciparum. Good antimycobacterial activity (MIC90 10 µM) was observed in the analogues, which possessed bulky and/or polar substituents in close proximity to the amide bond. Moreover, hydrophilic electron-withdrawing substituents seem to enhance antimycobacterial activity. A fusidic acid anilide formed from the Mannich base, 3-((N,Ndiethylamino)methyl)-4-hydroxy aniline recorded the most potent antimycobacterial activity with MIC90 values of 0.40 µM and 10.35 µM in the 7H9/CAS and 7H9/ADC media, respectively. Meanwhile, multi-stage antiplasmodium activity was more favored in the fusidic acid ethanamides. While the activity was not affected by diastereoisomerism, potency was enhanced by para-substituted lipophilic electron-withdrawing/donating groups. The (R)-4-fluoro ethanamide analogue of fusidic acid displayed the most potent asexual erythrocytic blood stage antiplasmodium activity with IC50 values of 0.57 and 0.47 µM against the NF54 and K1 strains of P. falciparum, respectively. The synthesized compounds, however, exhibited moderate to no inhibition of gametocyte growth at the highest concentration tested of 5 µM. The privileged benzimidazole scaffold has afforded antiviral, antiulcer, antimicrobial, antitumor, antihypertensive, anti-inflammatory and antihistaminic clinical chemotherapeutic agents. A limited number of studies describing the utility of benzimidazoles as potential antimalarial and antimycobacterial agents have been reported. In this context, novel benzimidazole analogues were rationally designed by incorporation of phenolic Mannich bases and synthesized in this thesis work. The synthesized compounds exhibited good in vitro potency towards Mtb in the 7H9/CAS media and sub-micromolar activity against the asexual blood stage P. falciparum parasites. All the compounds were relatively non-cytotoxic (IC50 >10 µM) to the Chinese hamster ovarian (CHO) cell line, recording acceptable selectivity indices, S1>10. No correlation was observed between the antiplasmodium activity of the synthesized analogues and their likely mode of action as inhibitors of hemozoin formation. The 1-((4-trifluoromethyl)benzyl) benzimidazole analogue based on the 3-((N,N-diethylamino)methyl)-2-hydroxy-5-methyl aniline Mannich base was the most potent analogue in both assays. It recorded a MIC90 value of 2.00 µM in the 7H9/CAS media. Moreover, IC50 values of 0.19 µM and 0.06 µM were observed against the asexual blood stage NF54 and K1 strains of P. falciparum, respectively, while it inhibited 91% of growth of early stage gametocytes at 1 µM. Although it showed poor aqueous kinetic solubility as a free base at pH 7.4, it conformed to Lipinski and Veber rules for oral absorption and bioavailability, while it shared a similar chemical space with the clinically used 4-aminoquinoline drug naphthoquine. The results presented herein substantiates the fact that the complimentary efforts of natural products research and medicinal chemistry exploration of natural products and privileged scaffolds continue to remain a viable approach to discovering potent bioactive drug candidates.

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