Tuberculosis (TB) is one of the most devastating and second infectious diseases causes of mortality followed by acquired immune deficiency syndrome (AIDS). According to world health organization (WHO) statistics, 33% world’s population has been exposed to TB bacteriuma. In the last year, it was anticipated that there may be around 9.8 million new casesa. A number of anti-TB drugs are ineffective against TB because of the development of resistant strains (1). The limited effectiveness of current chemotherapy stems largely from the lengthy and complicated nature of first-line anti-TB drugs (2,3). The most problematic issue with the current TB regimen is insufficient adherence to the treatment course, attributable to its length, complexity and adverse effects, led to difficult- and expensive-to-treat multidrug-resistant tuberculosis (MDR-TB) (4). The world’s two most populous countries, India and China, account for more than 50% of the world’s MDR-TB cases (5). Treatment for MDR-TB typically requires 18–24 months of combination therapy with second-line drugs those are less efficacious, more toxic and expensive than the first-line drugs (6). In few regions, almost 20% of MDR-TB cases were classified as extensively drug-resistant tuberculosis (XDR-TB) (7). The treatment options for XDR-TB are very limited as XDR-TB bacilli are resistant not only to isoniazid and rifampicin, but also to fluoroquinolones and aminoglycosides (8). More recently, another definition of XDR-TB as MDR-TB resistant to any fluoroquinolone and at least one of the second-line drugs (capreomycin, kanamycin and amikacin) used in TB treatment (9). There are serious adverse effects with most MDR-TB and XDR-TB drugs, such as nephrotoxicity and ototoxicity with aminoglycosides, hepatotoxicity with ethionamide and dysglycaemia with gatifloxacin (10). In few cases, XDR-TB has been shown aggressive form of TB, causing very high mortality (10).
The improvement in TB chemotherapy can be achieved by four primary goals: (i) Shorten and simplify TB treatment; (ii) improve efficacy, safety and reduce long-lasting therapy; (iii) develop drugs for HIV-TB co-infection, which can be readily co-administered with antiretrovirals; and (iv) shorten therapy of latent TB infection (11). Moreover, to effectively treat and control MDR- and XDR-TB patients, physicians and national TB treatment programs require regimens based on safer, tolerable and efficacious drugs having new mechanisms of action (12,13). At present, the global TB development pipeline has nine candidates, but a key issue is how to develop them simultaneously in combination trials to identify the best candidate (14).
In this context, recently, we have explored ubiquitous heterocycles, such as coumarins (15–17), 1,4-dihydropyridines (18,19) and quinolines (20) based scaffolds as promising antituberculars. To understand the structure–activity relationship among the designed conjugates, three-dimensional quantitative structure–activity relationship (3D-QSAR) was also conceived. More recently, we have patented tetrahydropyrimidines as potent anti-TB agents (21–23). The molecules were rationally designed and latter synthesized based on its predicted activities by comparative molecular field analysis and comparative molecular similarity index analysis. Among the naturally occurring pyranocoumarins (calanolide A, calanolide B and its stereoisomer), calanolide A is significantly interesting. The biological activity of calanolide A is not only restricted to anti-TB but also found active for HIV infections (24). Moreover, calanolide B is also claimed to have similar range of activity to calanolide A against mycobacteria. Consequently, we focused on coumarin derivatives such as, 4-styryl coumarins (15), 4-arylamino coumarins (16), coumarin-4-acetic acid benzylidenehydrazides (17) and more recently phenylhydrazono-chroman dione (25) as a potent antituberculars. Furthermore, the coumarin nucleuses have also been found as potent antimycobacterials (26–28) (Figure 1). Interestingly, novel acrylic esters (α,β-unsaturated carbonyls) of versatile (hetero)arenes were also proved as potent antituberculars (29). The 1,5-benzo(thi)diazepine motifs are of particular interest for drug discovery because they have been found active against different families of targets (30). Successful introduction of diltiazem and clentiazem for angina pectoris, hypertension, arrhythmias and other related cardiac disorders have proved the potentials of benzothiazepines skeleton (31,32). At present, in an extensive review, we have highlighted the therapeutic significance of 1,5-benzothiazipine pharmacophore (33). Although the biological evaluation of (benzo)azepines, particularly in the anti-TB research area, is still in its infancy (34–36). Consequently, in a present work, we have envisioned to probe molecular hybrids of coumarins with benzoazepines for their activity against Mycobacterium tuberculosis H37Rv strains (Figure 2).
At the outset, the syntheses of a versatile library of coumarin clubbed with benzoazepines, compounds 5a–o and 7a–k are depicted in Scheme 1. The chalcone derivatives were prepared by Claisen–Schmidt condensations (37). The 3-acetyl-4-hydroxycoumarin 3 was treated with (hetero)aromatic aldehydes using mild base, afforded chalcone derivatives 4. The resulting chalcones were treated with 2-aminothiophenol by employing piperidine as a catalyst at elevated temperature, afforded compounds 5a–o in good to excellent yields. The imine derivatives 6 were prepared by literature described method (38). Latter, the corresponding imines were treated with aldehydes by employing trifluoroacetic acid as a catalyst at higher temperature, afforded easy accessible compounds 7a–k in moderate to good yields (see Appendix S1). Both electron-rich and electron-deficient as well as sterically hindered functional groups were well tolerated under the established reaction conditions. The antimycobacterial activities of synthesized compounds were assessed using the microplate alamar blue assay (MABA) against Mtb H37Rv strains (see Appendix S1). The drug rifampin was used as a positive control. The growth inhibitions of rifampin were observed 97% at minimum inhibitory concentration (MIC) (0.015–0.125 μg/mL). The antimycobacterial activities of synthesized compounds are summarized in Table 1. The molecules bearing electron-rich methoxy group in the ring A, compounds 5c, 5d, 5e and 5l have shown poor activity. The hydrogen bond donor hydrophilic analogue, compound 5j has shown 32% growth inhibition, while 5m has shown very poor potency. The results were quite surprising for the compound 5o bearing hydroxy group at C2 in the ring A, and have exhibited highest growth inhibition and found significantly active in the designed series. Consequently, we may conclude that the potency of the compounds is not only dependent on the presence of particular functional group but regioselectivity may also play a significant role to determine anti-TB activity in this class of compounds. Among three regio-isomers, compounds 5a, 5b and 5k having nitro group in the ring A, the only analogue 5k has shown excellent activity, while the activities of other two isomers were decreased by manyfolds. Next, we replaced the aromatic ring A by heteroarene indole, compound 5n was observed noteworthy. Comparison of the regio-isomers (compounds 5f, 5g and 5h) having moderate electron-poor chloro group in the ring A, the compound 5f was found notable. The incorporation of fluorine group into organic molecules can serve to dramatically alter many of the physical properties of the molecules, for instances, lipophilicity, metabolic stability, conformational behaviour, etc. For these reasons, we have rationally incorporated electro-negative fluoro surrogate, as a result compound 5i has shown promising activity. Having elucidated a few potent inhibitors of Mtb, we next examined the aza-analogues of benzothiazepines. Few benzodiazepines in the designed series have shown moderate activity. The compound 7a having sterically hindered phenoxy group in the ring A have exhibited 35% inhibitions. Despite the lower potency, the ClogP values of the compounds 7b, 7d and 7g having hydrophilic hydroxyl group in the ring A were found <5. The compound 7k was observed least active in the designed series. The compounds demonstrating at least 90% inhibitions in the primary screening were retested at lower concentration against M. tuberculosis H37Rv to determine the actual MIC in the MABA. The MIC is defined as the lowest concentration effecting a reduction in fluorescence of 90% relative to controls such as isoniazid (ATCC 35822) or rifampin (ATCC 35838). Concurrent with the determination of MICs, the compounds were tested for their cytotoxicity (IC50) in Vero cells at concentrations ≤62.5 mg/mL or ten times the MIC for M. tuberculosis H37Rv. After 72 h exposure, viability was assessed on the basis of cellular conversion of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) into a formazan product using the promega cell titre 96 non-radioactive cell proliferation assays. The IC50 values of the compounds 5k and 5o in level-2 screening were observed as >10 and 3.63 μg/mL respectively. Finally, the effect on compound polarity was estimated by calculating the ClogP for each compounds synthesized on ChemDraw Ultra 12. The thumb rule for ClogP values to a drug-like molecule must be <‘5′ to by-pass the cell barrier. The ClogP seems to correlate with some extent in three analogues. Our findings confirmed that electron density, lipophilicity and regioselectivity of the functional groups are enabling to determine the antimycobacterial activity in such compounds.
In summary, a hybrid of coumarins with benzoazepines were synthesized and evaluated for their anti-TB activity against M. tuberculosis H37Rv strain. In a primary screening, compounds 5k, 5n and 5o have shown 93, 80 and 94 percentage growth inhibitions, respectively, at MIC <6.25 μm. The IC50 values of compounds 5k and 5o were found to be >10 μg/mL and 3.63 μg/mL, respectively. The coumarin clubbed with benzodiazepines were not found significant scaffold for anti-TB activity. However, the benzothiazepine is considered as one of the scaffolds for further development of potent anti-TB drugs. The syntheses of more focused library especially having small heterocycles instead of ring A in the core structure and its energy minimization prediction by 3D-QSAR is in progress in our laboratory. The findings will be disseminated in due course.