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Synthesis and in vitro antimycobacterial activity of 2-methoxybenzanilides and their thioxo analogues

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Abstract

A new series of N-(3/4-substituted phenyl) 4/5-chloro-2-methoxybenzamides and their thioxo analogues have been synthesised and evaluated for in vitro antimycobacterial activity against Mycobacterium tuberculosis H37Rv, as well as the two atypical strains Mycobacterium kansasii and Mycobacterium avium. Five of the most active compounds were evaluated for cytotoxicity and their ability to inhibit mycobacterial isocitrate lyase, which is responsible for latent survival of Mycobacterium. The results showed that benzthioanilides were more active than the corresponding benzanilides. The most active compound, 4-chloro-2-methoxy-N-(3,4-dichlorophenyl)benzothioamide (4e), had a minimal inhibition concentration (MIC) against M. tuberculosis of 2 μmol L−1, which was better than the activity of the previously published corresponding salicylanilide.

Highlights

► A new series of substituted-2-methoxybenzamides and their thioxo analogues was prepared. ► Benzthioanilides are more active than corresponding benzanilides against Mycobacterium strains. ► Evaluation for isocitrate lyase inhibition showed medium activity.

Introduction

In the last few years, tuberculosis (TB), caused by Mycobacterium tuberculosis, has again become one of the most dangerous and lethal infectious diseases. According to the new WHO report in 2010, there were an estimated 8.8 million new cases of TB, 1.45 million TB deaths and more than two billion people infected with M. tuberculosis, of which approximately 10% will most likely become ill during their life [1]. This situation is due to HIV infection (1.1 million of all TB causes and 0.35 million of TB deaths are in people who are HIV positive) [1], poor compliance of patients, population migration and increased drug resistance [2]. A particularly dangerous form of drug-resistant TB is multidrug-resistant tuberculosis (MDR–TB), caused by a strain of M. tuberculosis resistant to at least rifampicin and INH (isoniazid), the first line and most powerful antituberculotics, and extensively drug-resistant tuberculosis (XDR–TB), caused by the strains of M. tuberculosis also resistant to any fluoroquinolone and to at least one of the three injectable drugs kanamycin, capreomycin and amikacin [3]. For example, in 2010, there was an estimated 650,000 cases of MDR–TB, and 150,000 died from infection with this form in 2008 [1]. Although the absolute number of TB cases per year is slightly decreasing globally, the number of MDR–TB cases is increasing [1].

Recently, several pathways have been characterised as new possible drug targets, including cell wall metabolism, cellular respiration and protein processing enzymes [4]. One promising enzyme target in persistent and latent mycobacterial infection is isocitrate lyase (ICL), which is a key enzyme in the glyoxylate cycle essential for growth in macrophages. During steady state growth, ICL converts isocitrate to succinate and glyoxylate, followed by condensation of glyoxylate with acetyl-CoA to form malate by malate synthase [5]. The carbon conserving glyoxylate pathway has not been observed in the human body, therefore it has been selected as a potential drug target for new antituberculotic agents [6].

The need for new antituberculotics, especially ones with a new mode of action, demands intensive search for new compounds. Salicylanilides could fit this description, as they act on the bacterial two component system (TCS), which is not found in animal cells and is not a target for any current antituberculotic drugs [7], [8], [9]. They are currently well-known antibacterial [7] and antimycobacterial agents [10], [11], but are too toxic for animal cells because they decouple oxidative phosphorylation in mitochondria. The phenolic OH group is responsible for this action, which seems to also be essential for antibacterial activity [7].

On the other hand there is little information on the antimycobacterial properties of benzanilides, the possible metabolites of benzoxazoles [12]. According to Weidner-Wells et al. [13], who studied TCS inhibition by benzimidazoles and their isosteres benzoxazoles, neither the benzimidazole hydrogen bond donating ability nor the basicity of benzimidazole was necessary for their activity. In addition, they showed that benzoxazoles are also active even though the oxygen can only accept a hydrogen bond. Thus, it is possible that a hydrogen bond acceptor at the right position is sufficient for activity.

We prepared a series of 2-methoxybenzanilides and their thioxo analogues as the opened reverse form of 2-phenylbenzo[d]oxazoles, expecting that these compounds would show good activity even if they lacked a 2-OH group (Scheme 1).

Section snippets

Chemistry

Scheme 2 shows the preparation of substituted 2-methoxybenzanilides and their thioxo analogues. The substituents on both aromatic parts of the molecule were selected according to a previous study on salicylanilides [10]. For both synthetic steps, well known preparations were used: the reaction of substituted 2-methoxybenzoic acid with an appropriate aniline in the presence of PCl3 and chlorobenzene as solvent [10], and thionation with P4S10 in pyridine as solvent [14].

In vitro antimycobacterial assay

The newly prepared compounds were tested in the Laboratory for Mycobacterial Diagnostics and TB at the Institute of Public Health in Ostrava for their in vitro antimycobacterial activity against M. tuberculosis 331/88, Mycobacterium avium 330/88, Mycobacterium kansasii 235/80 and clinically isolated M. kansasii 6509/96. The first line antituberculotic drug INH was used as a standard. The compound activity against M. tuberculosis and M. avium was evaluated after 14 and 21 days, and the compound

Discussion and conclusion

Thirty-six novel compounds were synthesised and characterised by 1H and 13C NMR spectroscopy, IR spectroscopy, elemental analysis and melting point. The yields of thiobenzanilides were significantly lower (4.9–54.0%) than the yields of benzanilides (40.7–75.3%). This discrepancy was most likely due to the large excess of P4S10 and long reaction time which were needed for complete conversion of amides to thioamides to simplify their isolation. It was possible that the P4S10 formed some complex

Experimental protocols

All of the chemicals and solvents used in this study were purchased from Sigma–Aldrich, Prague, Czech Republic and Penta, Prague, Czech Republic, and were used without further purification.

Acknowledgments

This work was financially supported by IGA NT 13346 (2012), GAUK 27610/2010, FRVŠ 665/2012 and SVV 2012-265-001. The publication is co-financed by the European Social Fund and the state budget of the Czech Republic. TEAB, project no. CZ.1.07/2.3.00/20.0235.

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