Antimycobacterial Assessment of Salicylanilide Benzoates including Multidrug-Resistant Tuberculosis Strains

The increasing emergence especially of drug-resistant tuberculosis has led to a strong demand for new anti-tuberculosis drugs. Eighteen salicylanilide benzoates were evaluated for their inhibition potential against Mycobacterium tuberculosis, Mycobacterium avium and two strains of Mycobacterium kansasii; minimum inhibitory concentration values ranged from 0.5 to 16 μmol/L. The most active esters underwent additional biological assays. Four benzoates inhibited effectively the growth of five multidrug-resistant strains and one extensively drug-resistant strain of M. tuberculosis at low concentrations (0.25–2 μmol/L) regardless of the resistance patterns. The highest rate of multidrug-resistant mycobacteria inhibition expressed 4-chloro-2-[4-(trifluoromethyl)-phenylcarbamoyl]phenyl benzoate (0.25–1 μmol/L). Unfortunately, the most potent esters were still considerably cytotoxic, although mostly less than their parent salicylanilides.


Introduction
Tuberculosis (TB) represents a contagious infectious disease caused by Mycobacterium tuberculosis complex. It is still a harsh global public health problem, partly due to increasing emergence of OPEN ACCESS multidrug-resistant tuberculosis [MDR-TB, which was defined as the infection that is resistant to at least isoniazid (INH) and rifampicin (RIF), the most effective first-line oral agents], and most recently the extensively drug-resistant tuberculosis (XDR-TB). XDR-TB consists in MDR-TB in combination with both resistance to any fluoroquinolone and at least one second-line injectable drug (kanamycin, amikacin, capreomycin). Every year almost 500,000 people are infected with MDR-TB and about 40,000 new XDR-TB cases are appraised annually, with an increasing trend expected in the future. While the standard therapeutic regimen for drug-sensitive TB lasts six months, the treatment of MDR-TB usually takes at least 18 months, and XDR-TB is often untreatable; the coincidence with HIV infection brings other serious problem [1,2]. Therefore the development of novel antimycobacterial agents is still challenging, and new structures with innovative mechanisms of action are especially needed [1,[3][4][5]. Moreover, infections caused by nontuberculous (atypical) mycobacteria bring some challenges including those in the area of new drug discovery. Compounds with collateral anti-TB and anti-nontuberculosis mycobacteria activity may bring a satisfactory progress [6].
Salicylanilide (2-hydroxy-N-phenylbenzamide) derivatives may be such a promising group with a complex mechanism of action [7]. Their various esters have exhibited a significant antimycobacterial activity in micromolar or lower concentrations, including MDR-TB and atypical mycobacteria; they do not share any resistance with established antimycobacterial drugs. Esterification of salicylanilides may bring some both pharmacodynamic and pharmacokinetic advantages [7][8][9][10][11][12].
Some substituted esters of benzoic acid (BA) with substituted phenols have displayed antimycobacterial properties against typical and atypical species [13,14]. It was reported that M. tuberculosis is uniquely susceptible to weak acids compared to other mycobacteria. Some ester prodrugs of benzoic acid expressed a significant activity, especially at slightly acidic environment [15]. Recently, four salicylanilide benzoates were reported to block the growth of drug-sensitive M. tuberculosis strain with minimum inhibitory concentrations (MICs) ranging between 0.5-2 µmol/L and, moreover, it was found that they act as mild inhibitors of isocitrate lyase and methionine aminopeptidase, two enzymes essential for the maintenance of mycobacterial infection. These targets are different from those affected by clinically used drugs [16]. Salicylanilide benzoates [2-(phenylcarbamoyl)phenyl benzoates; Table 1] were synthesized and their MICs against eight bacterial and fungal strains were reported [17].
This study brings a complex characteristic of antimycobacterial properties (including against atypical, MDR-and XDR-TB strains) of known salicylanilide benzoates. It is a part of our research effort concerned with a group of salicylanilide derivatives with improved activity and/or reduced toxicity in comparison to their parent molecules.

In Vitro Antimycobacterial Evaluation
Eighteen salicylanilide benzoates were evaluated against four mycobacterial strains-one tuberculous and three atypical ones (Mycobacterium avium and two strains of Mycobacterium kansasii). Results are summarized in Table 1. All tested compounds exhibited a significant activity against drug-sensitive M. tuberculosis at micromolar concentrations (0.5-8 μmol/L) with 1m, 1o and 1r showing superiority (MICs ≤ 1 μmol/L). When we evaluated the isomers on the salicylic ring, 4-chloroderivatives were more beneficial than 5-chloro ones-only with 1c vs. 1d being an exception and with no difference in the 1g vs. 1h pair. The order of the moieties on the aniline ring is as follows (according to decreased potency): The benzoylation of salicylanilides provided esters with noticeably improved activity when compared to the parent phenolic molecules S (Table 2) [18]-e.g., even eight-fold for 1m; no ester exhibited inferior activity than its "parent" salicylanilide, and just 1b, 1c, and 1r have identical MIC values. M. avium showed the lowest level of susceptibility among the investigated mycobacterial strains (MICs 4-16 μmol/L). Compounds 1d, 1h, 1o and 1r are the most active esters. In general, derivatives substituted in the aniline part by 3- chloro (1a, 1b), 4-bromo (1g, 1h), and 4-trifluoromethyl (1o, 1p, 1r) moieties exhibited better activity; on the other hand, 3-fluoroderivatives (1i, 1j) offered minimal benefit. With two exceptions, molecules derived from 5-chlorosalicylic acid showed a higher or equal activity than their 4-chloro isomers. The introduction of a benzoyl fragment into salicylanilide molecules resulted in an increased activity against M. avium-only three MIC values are higher than those of the parent salicylanilides (Table 2) [18], while others are equal or mostly lower in the case of benzoates, even four times in some cases.
Both clinically isolated and collection strains of M. kansasii were inhibited by salicylanilide benzoates 1 with MICs  8 μmol/L with clear 1o superiority. 4-CF 3 , 3-CF 3 , 3,4-diCl and 4-Br represent the more suitable aniline substitution patterns; no substituent of the aniline part was evaluated as being significantly less beneficial than others. The influence of the halogen position on the salicylic ring is ambiguous. When concentrated on MICs towards the strain 235/80, there is a surprising fact-when 5-chloro-2-hydroxy-N-phenylbenzamides are esterified, the activity against M. kansasii did not change Salicylanilide benzoates expressed predominantly lower or equal MIC values in comparison to corresponding acetates [8] and benzenesulfonates [12]. Carbamates possessed a slightly higher in vitro inhibitory activity for M. tuberculosis, whereas MIC levels against atypical strains are approximately similar [9]. Salicylanilide N-acetyl-L-phenylalanine esters demonstrated a superior activity against M. tuberculosis and somewhat worse against M. avium [10]. Benzoates surpassed the antimycobacterial activity of salicylanilides esters with different N-benzyloxycarbonyl α-amino acids [11].
In conclusion, the benzoylation of salicylanilides S led to derivatives with predominantly higher in vitro activity against all four mycobacterial strains. The aim of improving the antimycobacterial potency was successfully achieved. The reason may lay in the increased lipophilicity of synthesized esters, which facilitates passage through biomembranes. With respect to the weak intrinsic activity of benzoic acid against M. kansasii, the possibility of synergistic action of released salicylanilides and benzoic acid could be included, as it has been previously observed.

In Vitro Activity against Drug-Resistant Tuberculosis Strains
Four esters with the lowest MICs (≤1 μmol/L against any mycobacterial strain) were selected for advanced biological tests . Benzoates 1m, 1n, 1o and 1r were evaluated for their in vitro activity against five MDR-TB strains and one XDR-TB strain ( Table 3). All four derivatives exhibited very low MICs (0.25-2 μmol/L). Interestingly, in most cases MDR strains are even more sensitive than drug-sensitive M. tuberculosis. This susceptibility is independent on the resistance patterns indicating no cross-resistance with the conventionally used drugs. 4-Trifluoromethyl derivative 1o was assayed as the most active compound. Based on the pair 1m vs. 1n, the preferable location of the chlorine is the position 4 of the salicylic ring (compound 1m). Compound 1o, the derivative of 5-chlorosalicylic acid, exhibited a better in vitro activity than 1r, which was synthesized from 5-bromosalicylic acid. The salicylanilide benzoates 1m, 1n, 1o, and 1r exhibited a higher activity against drug-resistant strains (expressed as MICs) than salicylanilide esters with N-acetyl-L-phenylalanine [10] and similar or slightly better MIC values when compared to salicylanilide carbamates [9].
Based on the comparison of MIC and IC 50 , it is possible to predict that the antimycobacterial activity of salicylanilide benzoates is not only the result of a general cytotoxic impact, but that they probably should have additional specific effect(s) against M. tuberculosis-e.g., recently reported inhibition of isocitrate lyase and methionine aminopeptidase [16].
However, benzoates still exhibited IC 50 values in micromolar range similar to the activities against atypical mycobacteria. Selectivity indexes (SI) for M. tuberculosis ranges from 2.34 to 4.80. The situation for MDR-TB and XDR-TB strains is quite advantageous with SI values of 1.17-10.16; the ratios are more favourable for 1o. Generally, only SI values about the break point of 10 could be considered to be still border sufficient, others are poor.
Although benzoic acid possesses only a very mild cytotoxicity (IC 50 of 2,881 µmol/L in our assay), unfortunately the benzoylation did not fill up our expectation about the significant toxicity reduction of parent salicylanilides, in contrast to improved antimycobacterial efficacy. Otherwise, esterification of salicylanilides may be a perspective way to reduce undesired cytotoxicity; it is necessary to search new acids, because benzoic acid brings a certain, but insufficient benefit.

Conclusions
In summary, salicylanilide benzoates revealed a significant antimycobacterial activity; their mechanism of action is still not fully elucidated and seems to be multiple. The masking of salicylanilide phenolic group by lipophilic aromatic acid resulted in the derivatives with improved antimycobacterial potency in the micromolar range (0.25-16 μmol/L). Additionally, the most active esters stopped the growth of MDR-TB strains with MIC values from 0.25 μmol/L. Salicylanilide benzoates represent a group with a promising in vitro antimycobacterial activity. Nevertheless, the expectancy of the reduced cytotoxicity was accomplished only partly-two esters of three tested ones exhibited a significantly lower toxicity when compared to parent salicylanilides, but these molecules are unfortunately still relatively toxic. Thus, the next search for new highly active and less cytotoxic derivatives still remains a topic of interest.