Design and development of bioresponsive pseudoglucosinolates, non-cytotoxic coumarins with antibiofilm properties and studies towards the synthesis of [13]cytochalasans

Abstract

The main aim of this thesis was the development of multifunctional pseudoglucosinolates (psGSLs) and artificial multivalent glucosinolates (mv-GSLs) as ITC releasing prodrugs. They were utilized as imaging, enzyme labelling and controlled drug delivery tools. Beyond that it also dealt with the development of 6,7-dihydroxycoumarins as non-cytotoxic antibiofilm agents. Lastly, studies towards the total synthesis of [13]cytochalasans (chaetoglobosin B and D) were conducted. The first chapter focused on the exploration and expansion of the utility of glucosinolate (GSL) inspired compounds for diverse biochemical and therapeutic applications. The first project within this chapter involved the development of enzyme-responsive pseudoglucosinolates (psGSLs) as prodrugs for the controlled release of isothiocyanates (ITCs). Incorporating nitroreductase (NTR) and azoreductase (AzoR)-responsive masking groups and a polyethylene glycol (PEG) chain enhanced solubility and functionality of the probes. Further attachment of fluorophores enabled imaging of enzymatic activation. Synthesized via multistep routes, psGSLs demonstrated successful ITC release upon enzymatic cleavage, confirmed by LC-MS and covalent binding of the ITCs were confirmed by SDS-PAGE analysis. In vivo validation in C. elegans further highlighted the covalent binding of ITCs. These results showcase the potential of psGSLs as flexible tools for imaging, and enzyme dependent labelling of biomolecules. The second project within this chapter investigated the design and synthesis of psGSL-based antimicrobial prodrugs for controlled drug release upon enzymatic activation. Antibiotics were attached to the psGSL thiohydroximate unit via carbamates, replacing the O-sulfonate group. LC-MS analysis showed release of antibiotics upon activation of the antimicrobial prodrugs by nitroreductase (NTR). Although the conversions were slow and incomplete, proof-of-principle for the activity of these probes was provided. Photo-cleavable psGSLs were also developed to bypass enzymatic incompatibility by incorporating o-nitrobenzyl moiety for UV-activated drug release at 365 nm. Additionally, azide groups in the psGSL structure allowed for fluorophore attachment or immobilization on surfaces, enabling potential applications as imaging agents or antimicrobial coatings. The third project within this chapter is introducing the first artificial multivalent glucosinolates (mv-GSLs). Compounds which bear more than one glycosidic thiohydroximate-O-sulfonate moiety, potentially amplifying the bioactivities observed for natural monovalent antetypes. Multistep synthesis and biochemical evaluation of mv-GSLs, featuring aliphatic and aromatic cores, were conducted. Myrosinase from Sinapis alba efficiently released multivalent isothiocyanates (mv-ITCs) for selected substrates, while specifier protein AtNSP3 facilitated only partial conversion to nitriles. Further optimization of the structures and developing a bigger panel of compounds could facilitate better conversion efficiency with myrosinase. The second chapter focused on the synthesis and bio evaluation of novel 6,7-dihydroxycoumarin-5-carboxylates (DHCou and 4-MeDHCou) as non-cytotoxic antibiofilm agents. These compounds were synthesized through multistep route in 11% and 8% overall yield respectively. Biological evaluation revealed significant retention of biofilm inhibition activity, with 4-MeDHCou demonstrating efficacy against Staphylococcus aureus and Candida albicans, while DHCou selectively inhibited C. albicans biofilms. Importantly, both derivatives exhibited no cytotoxicity against mammalian cell lines, unlike their parent compounds, esculetin (II-2) and 4-methylesculetin (II-29). Therefore, incorporation of a carboxylate moiety at the C5 position successfully produced compounds with reduced cytotoxicity and retained biofilm activity. These results establish a basis for further exploration of dihydroxycoumarin derivatives as antibiofilm agents, with potential applications in developing artificial siderophores or antimicrobial drug conjugates. The third chapter focused on the studies towards the total synthesis of [13]cytochalasans chaetoglobosin B and D. Efforts toward the total synthesis of chaetoglobosin D were centered on the construction of its isoindolone core (III-148) and macrocyclic fragment (III-149). Initial strategies faced significant challenges, including unsuccessful attempts to form the Wittig salt (III-145) during the construction of the macrocyclic fragment as well as decomposition of intermediates (III-122, III-128a, III-128b) during Diels-Alder reactions for construction of the isoindolone core. These drawbacks necessitated a shift to the synthesis of chaetoglobosin B, employing a revised strategy. Key precursors, diene III-163 and dienophile III-171, were synthesized via multistep routes, though the processes were hindered by low yields and issues with product selectivity, such as inseparable E/Z-isomer mixtures and material loss during intermediate preparation. While the assembly of the isoindolone core (III-173) and macrocyclic fragment (III-180) was not completed, a new synthetic pathway was proposed, incorporating intramolecular Diels-Alder reactions and optimized strategies for coupling and protecting intermediates. Future efforts could refine these routes, improve yields, and enable the total synthesis of chaetoglobosin B and related compounds. This work lays a foundation for advancing synthetic methodologies toward complex natural products.