Design and synthesis of antitubercular agents

Tuberculosis (TB) is a life-threatening disease which has plagued humans for centuries. Despite significant advances in modern medicine for tackling diseases, treatment of TB infections remains a challenging task: 6-12 months of treatment are required with daily dosing of up to 6 different antibiotics. The World Health Organisation (WHO) has placed the causative agent Mycobacterium tuberculosis (M. tb) on a priority list for novel drug development which led to an explosion of research in this field.

The electron transport chain (ETC) within M. tb has become a desirable drug target, with the thought that inhibition of this process would result in rapid bacterial killing and shorter treatment time. The oxidoreductase enzymes, cytochrome (cyt) bc and bd, are redundant. Therefore, simultaneous inhibition of these enzymes is required to induce a bactericidal effect. There are efficacious and safe cyt bc inhibitors, however, a synergistic cyt bd inhibitor is yet to be discovered. This thesis focuses on the development of cyt bd inhibitors, and methods to release a payload capable of simultaneous cyt bc and cyt bd inhibition.

In Chapter 2, the development of a structure activity relationship (SAR) between the reference natural product for cyt bd inhibition, aurachin D (AuD), and cyt bd is discussed. A series of analogues exhibiting aromatic substitutions, alkyl chain modifications, or quinolone modifications were synthesised. The half-maximal inhibitory concentration (IC₅₀) for each analogue was assessed against the M. tb cyt bd, which established preference and nanomolar inhibition for fluoro, methoxy, and hydroxy substituents in the C-5, C-6, and C-7 positions. Inhibition was also related to the LogP of the alkyl chain, with preference for LogP = >5. Methylation of the quinolone nitrogen or changing it to an oxygen (chromone system) reduced inhibitory activity. A comparison of the experimental IC₅₀ results to in silico docking studies showed consistency, and this drove the development of a series of second generation analogues. The second generation analogue library consisted of an indole or oxindole core structure, however, these analogues were not active against the M. tb cyt bd in IC₅₀ analysis.

AuD is a cytotoxic chemical scaffold, so Chapter 3 focused on prodrug development of AuD and an additional 6-hydroxy analogue 20e. Self-immolative linkers (SILs) were synthesised and conjugated to the analogues, and the in vitro release was measured by high performance liquid chromatography (HPLC). Disulphide triggered cyclisation SILs were toxic to mammalian THP-1 cells, and the release of active inhibitor was <40% over 72 hours incubation at 37 °C with glutathione (GSH). 1,4- and 1,6-releasing systems were generally non-toxic to THP-1 cells, which included nitro, azide, or boron pinacol ester triggers.

Chapter 4 developed synthetic methodology towards a prodrug system capable of releasing both a cyt bc and cyt bd inhibitor (referred to as a ‘codrug’). Initial synthesis utilised the commercially available cyt bc inhibitor lansoprazole sulphide (LPZS). The presence of a nucleophilic benzimidazole in LPZS capable of conjugating to a linker via a carbamate was thought to simplify the synthesis, however, the cyt bd inhibitors (AuD and 20e) were preferentially substituted and a codrug could not be conclusively characterised. An alternative codrug was synthesised, utilising Q203 as the cyt bc inhibitor and inclusion of an aminomethyl spacer to facilitate release of an amide moiety.

Finally, with desire to move away from AuD as a cyt bd inhibitor due to toxicity and solubility challenges, synthetic methodology towards novel chemical scaffolds was discussed in Chapter 5. The retrosynthetic analysis of lead compounds 218, 219, and 220 was performed, and the synthetic procedures towards developing analogues was optimised for future SAR analysis with the M. tb cyt bd. A total of 6 analogues of 218 were synthesised and analysed for activity, however, these were less potent than 218. Future work will explore the SAR, with optimised inhibitors utilised in the developed codrug system (Chapter 4).