Antimycobacterial assessment of salicylanilide benzoates including multidrug-resistant tuberculosis strains

Article abstract of DOI:10.3390/molecules171112812

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.

Full text of DOI:10.3390/molecules171112812

Molecules 2012, 17, 12812-12820; doi:10.3390/molecules171112812
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Antimycobacterial Assessment of Salicylanilide Benzoates
including Multidrug-Resistant Tuberculosis Strains
Martin Krátký 1, Jarmila Vinšová 1,* and Jiřina Stolaříková 2
1
Department of Inorganic and Organic Chemistry, Faculty of Pharmacy, Charles University,
Heyrovského 1203, Hradec Králové 50005, Czech Republic; E-Mail: martin.kratky@faf.cuni.cz
2
Laboratory for Mycobacterial Diagnostics and Tuberculosis, Regional Institute of Public Health in
Ostrava, Partyzánské náměstí 7, Ostrava 70200, Czech Republic; E-Mail: Jirina.Stolarikova@zu.cz
* Author to whom correspondence should be addressed; E-Mail: vinsova@faf.cuni.cz;
Tel.: +420-49506-7343; Fax: +420-49506-7166.
Received: 19 September 2012; in revised form: 24 October 2012 / Accepted: 26 October 2012 /
Published: 31 October 2012
Abstract: 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.
Keywords: antimycobacterial activity; benzoic acid ester; cytotoxicity; in vitro activity;
multidrug-resistant tuberculosis; salicylanilide; salicylanilide ester
1. 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

Molecules 2012, 17
12813
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 [1,3–5]. 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 [7–12].
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.
2. Results and Discussion
2.1. Chemistry
The synthesis and characterization (m.p., IR and NMR spectra, elemental analyses) of salicylanilide
benzoates [2-(phenylcarbamoyl)phenyl benzoates; 1] were published recently [17]; their synthetic
route is depicted in Scheme 1. Yield of esters 1 ranged from 44 up to 88% [17].

Molecules 2012, 17
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Scheme 1. Synthesis of salicylanilides S and corresponding benzoates 1 (R1 for esters
1 = 4-Cl, 5-Cl, 4-Br; R2 = 3-Cl, 4-Cl, 3,4-diCl, 3-Br, 4-Br, 3-F, 4-F, 3-CF3, 4-CF3).
Abbreviations: MW: microwave irradiation, Ph-Cl: chlorobenzene, DCC: N,N'-dicyclohexyl-
carbodiimide, DMF: N,N-dimethylformamide).
2.2. 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.
Table 1. Antimycobacterial activity of salicylanilide benzoates 1.
MIC [mol/L]
M. tuberculosis
M. avium
M. kansasii
M. kansasii
R1
R2
331/88
330/88
235/80
6509/96
14 d
2
4
21 d
2
4
14 d
4
8
21 d
8
8
7 d
2
2
14 d
4
4
21 d
8
4
7 d
4
4
14 d
4
4
21 d
8
8
1a
1b
1c
1d
1e
1f
1g
1h
1i
1j
4-Cl
5-Cl
4-Cl
5-Cl
4-Cl
5-Cl
4-Cl
5-Cl
4-Cl
5-Cl
4-Cl
5-Cl
3-Cl
3-Cl
4-Cl
4-Cl
3-Br
3-Br
4-Br
4-Br
3-F
4
2
2
4
2
2
4
4
4
4
2
4
2
2
4
8
8
4
8
4
4
4
8
8
16
4
16
8
8
4
16
16
16
8
4
2
2
2
2
2
2
2
8
2
4
4
4
4
4
8
8
4
8
4
4
4
8
8
4
2
2
2
2
2
2
2
8
2
4
4
4
4
4
4
8
4
8
4
4
4
4
8
3-F
4-F
4-F
1k
1l
4
8
4
8
8
4
2
2
8
4
8
8
2
4
4
8
4
8
1m
1n
1o
1p
1q
1r
INH
PAS
BA
4-Cl 3,4-diCl
5-Cl 3,4-diCl
1
2
0.5
2
2
1
2
1
2
2
8
8
4
8
8
4
8
8
4
8
2
1
1
1
2
2
4
2
1
2
4
4
4
1
4
4
1
1
2
1
2
2
2
32
250
2
2
2
2
4
2
4
125
1000
2
4
2
4
4
2
4-8
500
1000
4-Cl
5-Cl
4-Cl
4-Br
-
4-CF3
4-CF3
3-CF3
4-CF3
16
4
250
125
1
1
2
2
-
-
-
0.5-1
62.5
0.5-1
62.5
250
32
250
125
250
1000
250
>1000
-
-
>1000 >1000 >1000 >1000 1000 >1000 >1000
One or two best MIC values for each strain are given in bold; INH: isoniazid; PAS: para-aminosalicylic acid;
BA: benzoic acid. MICs of 1m, 1n, 1o and 1r against M. tuberculosis 331/88 were taken from reference [16].

Molecules 2012, 17
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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):
4-CF3 ≥ 3,4-dichloro > 3-CF3 (limited data) ≈ 4-Br > 3-Cl = 3-Br > 4-Cl > 3-F > 4-F. 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.
Table 2. Antimycobacterial activity of parent salicylanilides S [18].
MIC [mol/L]
M. tuberculosis
M. avium
M. kansasii
235/80
R1
R2
331/88
330/88
14 d
4
21 d
8
14 d
21 d
14 d 21 d
S-a
S-b
S-c
S-d
S-e
S-f
5-Cl
4-Cl
5-Cl
4-Cl
5-Cl
4-Cl
5-Cl
4-Cl
5-Cl
4-Cl
5-Cl
4-Cl
5-Cl
4-Cl
5-Cl
4-Cl
5-Cl
4-Br
3-Cl
3-Cl
8
16
8
16
16
8
4
4
8
8
4
4
4-Cl
4
4
8
8
4-Cl
4
4
8
8
4
8
3-Br
NT
NT
8
NT
NT
16
4
NT
NT
8
NT
NT
8
NT
NT
4
NT
NT
4
3-Br
S-g
S-h
S-i
4-Br
4-Br
4
16
31
32
16
32
16
16
8
16
31
32
16
32
16
16
8
4
4
3-F
8
8
8
8
S-j
3-F
8
16
16
16
8
32
4
32
4
S-k
S-l
4-F
16
16
4
4-F
16
4
32
4
S-m
S-n
S-o
S-p
S-q
S-r
3,4-diCl
3,4-diCl
4-CF3
4-CF3
3-CF3
4-CF3
4
4
8
8
2
2
1
1
4
4
8
8
4
4
NT
1
NT
1
NT
1
NT
1
NT
2
NT
4
NT: not tested. MICs of salicylanilides S were taken from reference [18].
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 2012, 17
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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-CF3, 3-CF3, 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 (or was even diminished for 1k vs. S-k), whereas masking of the phenolic
group of 4-chloro-2-hydroxy-N-phenylbenzamides resulted mostly in improved in vitro activity (up to
four times); only 1h retained the same MIC values. Under our experimental conditions, benzoic acid
itself revealed no activity against M. tuberculosis and M. avium and only a very weak inhibition
potency for M. kansasii (MICs ≥ 250 μmol/L).
The most active derivatives proved a similar efficacy when compared to INH against drug-sensitive
M. tuberculosis, and all derivatives exhibited substantially higher activity against M. avium and
M. kansasii 235/80; the activities of INH and benzoates 1 against M. kansasii 6509/96 are almost
identical. Second-line oral drug PAS seems to be significantly less active than the newly synthesized
derivatives against all tested strains.
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.
2.3. 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.

Molecules 2012, 17
Table 3. Activity of selected benzoates against multidrug-resistant strains.
12817
MIC [mol/L]
M.
M.
M.
M.
M.
M.
tuberculosis
234/2005
tuberculosis
53/2009
tuberculosis
tuberculosis
Praha 131
tuberculosis
7357/1998
tuberculosis
Praha 1
9449/2006
14 d
0.25
0.25
0.25
0.5
21 d
0.5
14 d
0.5
1
21 d
14 d
0.5
1
21 d
0.5
1
14 d
0.5
21 d
1
14 d
0.5
21 d
0.5
1
14 d
0.5
2
21 d
1
1m
1n
1o
1r
1
2
0.5
0.5
1
0.5
2
0.25
0.5
0.5
1
1
0.5
1
0.5
1
0.25
0.5
0.5
1
0.25
0.25
14.6
0.5
0.5
14.6
0.5
1
0.5
1
2
INH 14.6
14.6
14.6
14.6
14.6
14.6
14.6
14.6
58.3
58.3
The best MIC value for each strain is given in bold; INH: isoniazid. MDR-TB strains: 357/2005 and 7357/1998
(both resistant to INH, RIF, rifabutine, streptomycin, ethambutol and ofloxacin); 53/2009 (resistant to INH, RIF,
rifabutine, streptomycin, ethambutol); Praha 1 (resistant to INH, RIF, rifabutine, streptomycin, ethambutol and
clofazimine) and 9449/2006 (resistant to INH, RIF, rifabutine and streptomycin); XDR-TB strain: Praha 131
(resistant to INH, RIF, rifabutine, streptomycin, ethambutol, ofloxacin, gentamicin and amikacin).
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].
2.4. Cytotoxicity Evaluation
Salicylanilides and their esters were referred to share some toxic effects on eukaryotic
cells [7–10,16,19]. Therefore we examined the in vitro cytotoxicity of three most anti-MDR-TB active
benzoates (compounds 1m, 1o, 1r) and their parent salicylanilides (S-m, S-o, S-r) in the liver Hep G2
model. The cytotoxicity values, which are expressed as IC50, i.e., concentration which decreases the
viability of the cells to 50% from the maximal viability, was taken over from reference [16] (Table 4).
Table 4. Cytotoxicity and selectivity indexes of selected salicylanilides S and their benzoates 1.
SI for M. tuberculosis 331/88
SI for MDR-TB strains SI for XDR-TB strain
IC50 [µmol/L]
Hep G2
2.54
14 d
2.54
4.80
2.34
0.21
0.18
2.71
21 d
2.54
2.40
2.34
0.10
0.18
2.71
14 d
21 d
14 d
5.08
9.60
4.68
-
14 d
2.54
4.80
2.34
-
2m
2o
5.08–10.16 2.54–5.08
2.40
4.80–9.60
2.40–9.60
2r
2.34
2.34–9.36
1.17–4.68
S-m
S-o
S-r
0.84
-
-
-
-
-
-
0.36
-
-
2.71
-
-
IC50 and MIC values of salicylanilides against M. tuberculosis were taken from reference [16]. SI = IC50/MIC100
.

Molecules 2012, 17
12818
The esterification of 5-chloro-N-(3,4-dichlorophenyl)-2-hydroxybenzamide (S-m) and 5-chloro-2-
hydroxy-N-[4-(trifluoromethyl)phenyl]benzamide (S-o) by benzoic acid led to the derivatives with
significantly decreased toxicity (approximately three and seven times, respectively); contrarily, the
benzoylation of S-r has resulted in a slightly higher cytotoxicity (2.71 vs. 2.34 µmol/L).
Based on the comparison of MIC and IC50, 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 IC50 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 (IC50 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.
3. Experimental
3.1. Chemistry
Synthesis and characterization of the presented 2-(phenylcarbamoyl)phenyl benzoates 1a–r was
published by Krátký et al. [17].
3.2. In Vitro Antimycobacterial Susceptibility Testing
All compounds were tested against Mycobacterium tuberculosis 331/88 (H37Rv) (dilution of the strain
was 10−3) and three nontuberculous strains: Mycobacterium avium 330/88 (resistant to INH, RIF, ofloxacin
and ethambutol; dilution 10−5) and two strains of Mycobacterium kansasii—235/80 (dilution 10−4) and
clinically isolated strain 6509/96 (dilution 10−5). The description of the used method can be found in [12].
The following concentrations of esters were used: 1,000, 500, 250, 125, 62.5, 32, 16, 8, 4, 2, 1, 0.5,
0.25 and 0.125 µmol/L. MIC (µmol/L) is the lowest concentration at which the complete inhibition of
mycobacterial growth was occurred. Isoniazid (INH) and structurally similar para-aminosalicylic acid
(PAS) were chosen as reference drugs for the comparison. The most active compounds were evaluated
in the similar conditions and concentrations against six MDR-TB strains (dilution 10−3) with different
resistance patterns: 7357/1998, 234/2005, 53/2009, 9449/2006, Praha 1 and Praha 131 (XDR-TB
strain). All these strains were resistant to INH, rifamycines, and streptomycin; in some cases additional
resistance was present.

Molecules 2012, 17
12819
4. 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.
Acknowledgments
This work was financially supported by GAUK 27610/2010 and IGA NT 13346 (2012). This
publication is a result of the project implementation: “Support of establishment, development, and
mobility of quality research teams at the Charles University”, project number CZ.1.07/2.3.00/30.0022,
supported by The Education for Competitiveness Operational Programme (ECOP) and co-financed by
the European Social Fund and the state budget of the Czech Republic. We thank J. Urbanová, M.A.,
for language assistance.
Conflict of Interest
The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds S-a–S-r and 1a-r are available from the authors.
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