Synthesis, antitubercular activity and docking study of novel cyclic azole substituted diphenyl ether derivatives

Abstract

The re-emergence of tuberculosis (TB) as a global health problem over the past few decades, accompanied by the rise of drug-resistant strains of Mycobacterium tuberculosis, emphasizes the need for discovery of new therapeutic drugs against this disease. The emerging serious problem both in terms of TB control and clinical management prompted us to synthesize a novel series of heterocyclic o/m/p substituted diphenyl ether derivatives and determine their activity against H37Rv strain of Mycobacterium. All 10 compounds inhibited the growth of the H37Rv strain of mycobacterium at concentrations as low as 1 μg/mL. This level of activity was found comparable to the reference drugs rifampicin and isoniazid at the same concentration. Molecular modeling of the binding of the diphenyl ether derivatives on enoyl-ACP reductase, the molecular target site of triclosan, indicated that these compounds fit within the binding domain occupied by triclosan. Hence the diphenyl ether derivatives tested in this study were docked to ENR and the binding of the diphenyl ether derivatives was also estimated using a variety of scoring functions that have been compiled into the single consensus score. As the scores ranged from 47.27% to 65.81%, these bioactive compounds appear to have a novel mechanism of action against M. tuberculosis, and their structural features should be studied further for their potential use as new antitubercular drugs.

Introduction

Tuberculosis (TB) is a chronic infectious disease caused by mycobacteria of the “tuberculosis complex”, including primarily Mycobacterium tuberculosis, but also Mycobacterium bovis and Mycobacterium africanum [1], [2]. In the last decade, TB has re-emerged as one of the leading causes of death worldwide (nearly 3 million deaths annually) [3]. The estimated 8.8 million new cases every year correspond to 52,000 deaths per week or more than 7,000 each day [4], [5]. These numbers however, are only a partial depiction of the global TB threat. More than 80% of TB patients are in the economically productive age of 15–49 years, which results in tremendous economic and social problems.

It was estimated that nearly 1 billion more people will be infected with TB in the next 20 years. About 15% of that group (150 million) will exhibit symptoms of the disease, and about 3.6% (36 million) will die from TB if new disease prevention and treatment measures are not developed [6]. In 2005, the TB incidence rate was stable or in decline in all six WHO regions, and had reached a peak worldwide. However, the total number of new TB cases is still rising slowly, because the case-load continues to grow in the African, Eastern Mediterranean and South-East Asia regions [6]. The dramatic increase in TB cases observed in the recent years is a result of two major factors. First is the increased susceptibility of people infected with Acquired Immunodeficiency Syndrome (AIDS) to TB, which augments the risk of developing the disease 100-fold [7]. Second is the increase in resistant strains of the disease [8] with some showing cross-resistance to as many as nine drugs [7].

Although one possible long term solution to the problem is a better vaccine, in the short term, the major reliance will be on chemotherapy [9] requiring the development of novel, effective and non-toxic antitubercular agents [9], [10], [11]. The identification of novel target sites will also be needed to circumvent the problems associated with the increasing occurrence of multi-drug resistant strains. To do this, biochemical pathways specific to the mycobacteria and related organisms' disease cycle must be better understood. Many unique metabolic processes occur during the biosynthesis of mycobacterial cell wall components [12]. One of these attractive targets for the rational design of new antitubercular agents are the mycolic acids, the major components of the cell wall of M. tuberculosis [13].

Mycolic acids are high molecular weight C74–C90 α-alkyl, β-hydroxy fatty acids covalently linked to arabino-galactan [13], [14], [15]. They represent the major lipid components of the mycobacterial cell walls and are unique to mycobacteria and related species [16]. Enzymes that comprise the fatty acid synthetase (FAS) complex responsible for fatty acid biosynthesis are considered ideal targets for designing new antibacterial agents. Enoyl-ACP reductase (ENR) is a key regulatory enzyme in fatty acid elongation, and it catalyses the NADH-dependent stereospecific reduction of α,β-unsaturated fatty acids bound to the acyl carrier protein [17], [18], [19]. Two studies have shown that 5-chloro-pyrazinamide (5-Cl-PZA) [20], [21] and pyrazinamide (PZA) [21] inhibit M. tuberculosis FAS I, indicating that FAS I is also a good drug target.

The diphenyl ether, triclosan 5-chloro-2-(2,4-dichloro-phenoxy) phenol ether, is a broad-spectrum biocide that has been used for over 30 years mainly as a component of antimicrobial wash in consumer products such as toothpastes, mouthwashes, deodarant soaps, lotions, children toys and cutting boards [22].

Until recently, it was thought that triclosan, being a relatively small hydrophobic molecule, was absorbed via diffusion into the bacterial cell wall and that its antibacterial activity was the result of a non-specific disruption of the organism's cell wall [23], [24]. However, the first evidence that this diphenyl ether inhibits fatty acid biosynthesis came when a genetic analysis of an Escherichia coli strain resistant to triclosan linked the resistance to the FabI gene which encodes for ENR [25]. Subsequently extensive biochemical and structural studies have been performed to confirm that triclosan is a specific inhibitor of E. coli ENR [18], [26], [27], [28]. Triclosan also directly inhibits ENR from Staphylococcus aureus [29], Haemophilus influenzae [30], M. tuberculosis and Mycobacterium smegmatis (encoded by InhA) [31], [32], [33] and Plasmodium falciparum, the malarial parasite [34], [35], [36]. The common theme in the inhibition of ENRs by triclosan is the requirement of the NAD⁺ cofactor. The interaction of triclosan with ENR is stabilized by the π–π stacking interaction between the hydroxyl chloro phenyl ring (ring A in Fig. 1) and the hydroxyl group of a tyrosine from hydrogen bonding interactions with the hydroxyl group of triclosan. Ring B of triclosan makes several hydrophobic contacts with ENR. The ether oxygen of triclosan may also be critical in the formation of the stable ENR–triclosan–NAD⁺ complex, since the replacement of the group by a sulfur atom abolishes the inhibitory activity [26].