Antibiotics are the primary treatment for bacterial infections, with their discovery and development leading to cures for these once untreatable diseases. However, the overuse, misuse and limited options of antibiotics have led to the emergence of multi-drug-resistant bacteria, causing a global resistance problem [1]. Among the bacteria causing concern is Mycobacterium tuberculosis (Mtb), the causative agent of Tuberculosis (TB), which was the leading source of global fatalities from a single agent before the COVID-19 pandemic [2]. Notwithstanding this, in recent years, TB treatment progress has halted and, in some aspects, has reversed, resulting in an estimated increase in deaths associated with TB [2].

Current treatment regimens for TB infections require patients to take drug combinations for at least six months, which is costly, prone to differential response rates, presents significant challenges with patient compliance and adherence, and most notably an increased prevalence of resistance to these therapies, leading to a reduction in treatment efficacy and a worsening prognosis [3,4,5,6,7,8]. Consequently, the identification of novel therapeutics and treatment strategies for the management of TB, especially drug-resistant disease, is becoming increasingly urgent.

In recent years, several new therapeutic approaches have entered development and/or trial, such as bedaquiline, delamanid, clofazimine, and combination therapy bedaquiline-protomanid-linezolid [9, 10]. However, resistance to several of these new regimens and monotherapies has already been reported. The reason is that the vast majority of these new treatments are either derivatives of current drugs or target the same biochemical pathways, making them prone to the same acquired drug resistance mechanisms [11,12,13,14,15,16]. For example, acquired resistance to bedaquiline has been observed due to a mutation in an efflux pump-related gene associated with clofazimine resistance [17]. Therefore, the development of successful and improved drug therapies for TB, particularly drug-resistant disease, requires the discovery of new drug targets and novel drug structures, alongside innovative pharmacological approaches to circumvent resistance mechanisms and offer a new dimension to treatment [18, 19].

Benzoxa-[2,1,3]-diazole, benzothia-[2,1,3]-diazole and benzotriazoles are a class of heterocycle that exhibit widespread therapeutic utility, including use as antifungals, anti-cancer therapeutics and, to some extent, antibiotics [20]. Their application to the treatment of TB is limited to date, albeit with promising studies supporting their potential. For instance, the screening of simple substituted nitrobenzoxadiazoles against the mycobacterial ATP phosphoribosyl transferase (HisG) (IC50 = 12 µM) confirmed 1 as a capable inhibitor of Mycobacterium smegmatis in whole-cell assays (12 µg/ml) (Fig. 1) [21]. Further support is provided with the development of benzoxadiazoles substituted with heavily decorated heterocycles 2 and N-oxide containing species 3, identified to target the adenosine 5’-phosphosulfate reductase which catalyses the first step in bacterial sulfate reduction (Fig. 1) [22]. Although it remains to be determined whether these benzoxadiazoles would translate as putative drugs for Mtb, it is noted that they would likely evade the problematic acquired drug-resistant phenotype, due to the exploitation of different molecular pathways and divergence from structural pharmacophores of the current drugs, providing good support to their therapeutic utility in this context.

Benzoxadiazole frameworks as potential antitubercular agents

In our previous studies, we synthesised a small library of benzoxa-[2,1,3]-diazoles and their analogues, identifying benzoxa-[2,1,3]-diazole-substituted amino acid hydrazides 4 as promising antibacterials against Mtb, with an acceptable therapeutic index based on Mtb selectivity, potency, and efficacy [23]. This foundational work not only established the significance of the benzoxa-[2,1,3]-diazole core in anti-Mtb activity but also revealed key opportunities for structural modifications—specifically, variations in the amino acid (AA) and substituted aryl hydrazine (R1) moieties—to enhance potency [23].

Building on these insights, the present study aims to refine the structure-activity relationship (SAR) of the molecular architecture 4 and further assess the potential of these compounds to overcome Mtb drug resistance. We have focused on elucidating the precise role of the amino acid (AA) and aryl hydrazine substitution (R1) functionality while maintaining the essential benzoxa-[2,1,3]-diazole framework. By advancing the analysis of these critical modifications, this study seeks to optimise the antibacterial efficacy of these compounds and provide a deeper understanding of their therapeutic potential (Fig. 2).

Inhibitors of Mycobacterium tuberculosis based on the benzoxa-[2,1,3]-diazole framework, highlighting the key modification sites for the work presented herein