Antibacterial activity of salicylanilide 4-(trifluoromethyl)benzoates

Article abstract of DOI:10.3390/molecules18043674

The development of novel antimicrobial agents represents a timely research topic. Eighteen salicylanilide 4-(trifluoromethyl)benzoates were evaluated against Mycobacterium tuberculosis, M. avium and M. kansasii, eight bacterial strains including methicillin-resistant Staphylococcus aureus (MRSA) and for the inhibition of mycobacterial isocitrate lyase. Some compounds were further screened against drug-resistant M. tuberculosis and for their cytotoxicity. Minimum inhibitory concentrations (MICs) for all mycobacterial strains were within 0.5-32 μmol/L, with 4-chloro-2-[4- (trifluoromethyl)phenylcarbamoyl] phenyl 4-(trifluoromethyl)benzoate superiority. Grampositive bacteria including MRSA were inhibited with MICs ≥ 0.49 μmol/L, while Gramnegative ones were much less susceptible. Salicylanilide 4-(trifluoromethyl)benzoates showed significant antibacterial properties, for many strains being comparable to standard drugs (isoniazid, benzylpenicillin) with no cross-resistance. All esters showed mild inhibition of mycobacterial isocitrate lyase and four compounds were comparable to 3-nitropropionic acid without a direct correlation between in vitro MICs and enzyme inhibition.

Full text of DOI:10.3390/molecules18043674

Molecules 2013, 18, 3674-3688; doi:10.3390/molecules18043674
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Antibacterial Activity of Salicylanilide
4-(Trifluoromethyl)benzoates
Martin Krátký 1, Jarmila Vinšová 1,*, Eva Novotná 2, Jana Mandíková 3, František Trejtnar 3
and Jiřina Stolaříková 4
1
Department of Inorganic and Organic Chemistry, Faculty of Pharmacy, Charles University,
Heyrovského 1203, 500 05 Hradec Králové, Czech Republic; E-Mail: martin.kratky@faf.cuni.cz
2
Department of Biochemical Sciences, Faculty of Pharmacy, Charles University, Heyrovského 1203,
500 05 Hradec Králové, Czech Republic; E-Mail: eva.novotna@faf.cuni.cz
3
Department of Pharmacology and Toxicology, Faculty of Pharmacy, Charles University,
Heyrovského 1203, 500 05 Hradec Králové, Czech Republic;
E-Mails: jana.mandikova@faf.cuni.cz (J.M.); frantisek.trejtnar@faf.cuni.cz (F.T.)
4
Laboratory for Mycobacterial Diagnostics and Tuberculosis, Regional Institute of Public Health in
Ostrava, Partyzánské náměstí 7, 702 00 Ostrava, Czech Republic; E-Mail: Jirina.Stolarikova@zu.cz
* Author to whom correspondence should be addressed; E-Mail: jarmila.vinsova@faf.cuni.cz;
Tel.: +420-495-067-343; Fax: +420-495-067-166.
Received: 1 February 2013; in revised form: 18 March 2013 / Accepted: 18 March 2013 /
Published: 25 March 2013
Abstract: The development of novel antimicrobial agents represents a timely research
topic. Eighteen salicylanilide 4-(trifluoromethyl)benzoates were evaluated against
Mycobacterium tuberculosis, M. avium and M. kansasii, eight bacterial strains including
methicillin-resistant Staphylococcus aureus (MRSA) and for the inhibition of mycobacterial
isocitrate lyase. Some compounds were further screened against drug-resistant
M. tuberculosis and for their cytotoxicity. Minimum inhibitory concentrations (MICs)
for all mycobacterial strains were within 0.5–32 μmol/L, with 4-chloro-2-[4-
(trifluoromethyl)phenylcarbamoyl]phenyl 4-(trifluoromethyl)benzoate superiority. Gram-
positive bacteria including MRSA were inhibited with MICs  0.49 μmol/L, while Gram-
negative ones were much less susceptible. Salicylanilide 4-(trifluoromethyl)benzoates
showed significant antibacterial properties, for many strains being comparable to standard
drugs (isoniazid, benzylpenicillin) with no cross-resistance. All esters showed mild inhibition
of mycobacterial isocitrate lyase and four compounds were comparable to 3-nitropropionic
acid without a direct correlation between in vitro MICs and enzyme inhibition.

Molecules 2013, 18
Keywords: antibacterial activity; antimycobacterial activity; cytotoxicity; isocitrate lyase
3675
inhibitor; multidrug-resistant tuberculosis; salicylanilide ester; 4-(trifluoromethyl)benzoic
acid ester
1. Introduction
The inappropriate use or misuse of established antimicrobial drugs has led to the increasing
emergence of microbial resistance. The possible loss of the global control of infectious diseases
represents a serious threat for public health, since resistance has been described for many important
human pathogens.
Tuberculosis (TB) is responsible for millions of human deaths and about 1.4 million people died
from TB in 2010 [1]. The drug-resistant TB forms, especially multidrug-resistant and extensively drug-
resistant tuberculosis (MDR- and XDR-TB), bring grave problems. MDR-TB consists in a resistance
to at least isoniazid (INH) and rifampicin (RIF), the most effective first-line oral agents, while XDR-
TB is MDR-TB in combination with both resistance to any fluoroquinolone and at least one second-
line injectable drug (kanamycin, amikacin, capreomycin) [2]. Latent TB and also coincidence of TB
with HIV-infection signify other principal impetuses for novel antimycobacterial drugs.
Contemporary research trends are focused, i.e., on the identification of unique mycobacterial
pathways. Isocitrate lyase (ICL), an essential enzyme for the metabolism of fatty acids which splits
isocitrate to succinate and glyoxylate, is one of two glyoxylate shunt enzymes. This pathway
replenishes tricarboxylic acids cycle intermediates during growth on C2 substrates and is lacking in
vertebrates. Disruption of icl gene attenuated bacterial persistence and virulence without affecting the
bacterial growth during the acute phase of infection. ICL might contribute to mycobacterial adaptation
to hypoxia. Conventional anti-tuberculosis drugs exhibit mostly an insufficient activity against slow-
or non-growing (latent) mycobacteria. This is thought to be an important reason for the length of
anti-tuberculosis therapy [3]. Novel ICL inhibitors may bring the shortening of TB treatment course
and targeting of ICL represents one of the up-to-date research topics [4,5].
Nosocomial infections are a major challenge because of the high rates of morbidity and mortality.
Worldwide, the state of resistance increased considerably in both Gram-positive and Gram-negative
pathogens. Methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant Enterococcus
spp., Klebsiella pneumoniae, Escherichia coli, Pseudomonas spp. or Acinetobacter baumannii
producing extended spectrum β-lactamases (ESBL) represent such a most common and problematic
drug-resistant bacteria, which have been rapidly spread throughout the world [6].
Consequently, novel drugs and strategies to control microbial spread are of critical importance,
especially molecules with unique mechanism of action and no structural similarity. They can replace or
combine with conventional antibiotics to combat problematic strains. The question if a new drug
should be broad-spectral rather than have a narrow-spectrum of antimicrobial activity is controversial [7].
Salicylanilides (2-hydroxy-N-phenylbenzamides) and their derivatives have been reported to share a
significant antibacterial activity, especially against atypical mycobacteria, Mycobacterium tuberculosis
(Mtb.) including drug-resistant strains, Gram-positive cocci including MRSA with minimum inhibitory

Molecules 2013, 18
3676
concentrations (MICs) in micromolar range [8–14]. Additionally, salicylanilides inhibit moderately
mycobacterial isocitrate lyase and methionine aminopeptidase [4], but they affect multiple targets
within the cells of pathogens [4,8,14,15].
Salicylanilide scaffold represents still an investigated group of compounds with many interesting
pharmacological properties. Recently, they have been reported, e.g., as potential agents with a
significant activity against Onchocerca volvulus [15], Toxoplasma gondii parasite [16] or with
anticancer features [17–20].
Salicylanilides derived from 4-(trifluoromethyl)aniline have exhibited predominantly a high
antimycobacterial activity; in general, the introduction of CF3 moiety into the salicylanilide scaffold
enhanced antimicrobial properties [4,9–13]. Salicylanilide benzoates revealed a notable activity
especially toward MDR-TB [13] as well as antibacterial one [11]. Aromatic esters of
4-(trifluoromethyl)benzoic acid have been reported as a mild antibacterial agents against Pseudomonas
fluorescens and Bacillus subtilis, since inactive for S. aureus and E. coli [21], and some salicylanilide
4-(trifluoromethyl)benzoates exhibited antifungal activity, especially against moulds with
MICs  0.49 μmol/L [22]. Additionally, an increased lipophilicity may ameliorate passing through cell
walls and biomembranes, that is why we selected highly lipophilic salicylanilide 4-(trifluoromethyl)benzoates
for the searching of a potential antibacterial and antimycobacterial agents with expected lower MIC.
2. Results and Discussion
Salicylanilide 4-(trifluoromethyl)benzoates 1 were evaluated for their in vitro antimycobacterial and
antibacterial activity, cytotoxicity and inhibition of mycobacterial isocitrate lyase.
2.1. Chemistry
The synthesis and characterization of the salicylanilide 4-(trifluoromethyl)benzoates (Scheme 1)
were published recently [22]. Yield of esters synthesized via N,N'-dicyclohexylcarbodiimide coupling
ranged from 49% up to 86%.
Scheme 1. Synthesis of salicylanilides SAL and corresponding 4-(trifluoromethyl)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 (530 W, 600 rpm, 20 min); Ph-Cl: chlorobenzene;
DCC: N,N'-dicyclohexylcarbodiimide; DMF: N,N-dimethylformamide.

Molecules 2013, 18
3677
2.2. Antimycobacterial Evaluation
Table 1 summarizes the antimycobacterial activity of esters 1, isoniazid and para-aminosalicylic
acid, two comparative drugs. All of the 4-(trifluoromethyl)benzoates displayed a significant
antimycobacterial activity, with MICs of 0.5–32 μmol/L against all strains, with 1o and 1r showing
superiority (1–4 μmol/L). Mycobacterium avium showed the highest MIC values, up to 32 μmol/L. All
esters displayed a better in vitro activity against four mycobacterial strains than PAS, a second-line
oral drug sharing a structural similarity. Additionally, salicylanilide derivatives exhibited lower MICs
than INH for M. kansasii 235/80 and M. avium; MIC values against M. kansasii 6509/96 were
comparable or a slightly better in the case of esters. Only three esters (1e, 1o and 1r) showed equal
activity like INH against M. tuberculosis (1 μmol/L). 4-(Trifluoromethyl)benzoic acid exhibited only a
very mild intrinsic activity towards M. kansasii (MICs  250 μmol/L), whereas other mycobacteria
were completely insusceptible at 1,000 µmol/L.
Table 1. Antimycobacterial activity of salicylanilide 4-(trifluoromethyl)benzoates 1.
MIC [mol/L]
Mtb.
M. avium
M. kansasii
M. kansasii
6509/96
R1
R2
331/88
330/88
235/80
14 d
21 d
4
4
2
4
1
2
2
8
2
4
8
4
8
8
1
2
14 d
8
8
4
4
4
2
2
8
8
8
16
8
16
4
1
2
4
2
21 d
16
8
8
8
4
8
8
16
8
7 d
0.5
1
1
1
1
1
2
4
1
1
8
4
4
2
1
2
14 d
2
4
2
4
2
2
4
8
4
4
16
8
16
8
2
2
4
2
21 d
4
4
2
4
4
4
4
8
8
4
16
8
16
8
2
4
8
4
7 d
2
2
1
1
1
1
1
2
2
2
8
8
8
4
1
1
14 d
8
8
4
4
2
2
4
8
8
4
16
8
16
8
2
4
4
2
21 d
8
8
4
4
4
4
4
8
8
4
16
16
16
8
2
4
8
4
1a
1b
1c
1d
1e
1f
4-Cl
5-Cl
4-Cl
5-Cl
4-Cl 3,4-diCl
5-Cl 3,4-diCl
3-Cl
3-Cl
4-Cl
4-Cl
2
4
2
4
1
1
1
4
2
2
8
4
8
4
1
2
2
1
1g
1h
1i
1j
1k
1l
4-Cl
5-Cl
4-Cl
5-Cl
4-Cl
5-Cl
3-Br
3-Br
4-Br
4-Br
3-F
8
32
16
32
4
4
8
3-F
1m 4-Cl
4-F
1n
1o
1p
1q
1r
5-Cl
4-Cl
5-Cl
4-Cl
4-Br
4-F
4-CF3
4-CF3
3-CF3
4-CF3
2
1
16
4
2
1
1
1
INH
PAS
0.5–1 0.5–1
62.5 62.5
2–4
4–8
125
8–16
500
250 250 250 250
32 125 125
250
1000 >1000 32
CF3-BA
>1,000 >1,000 >1,000 >1,000 1,000 >1,000 >1,000 250 1,000 1,000
INH = isoniazid; PAS = p-aminosalicylic acid; CF3-BA = 4-(trifluoromethyl)benzoic acid; Two best MICs
for each strain are given in bold.

Molecules 2013, 18
3678
In general, the most active derivatives share a trifluoromethyl moiety (compounds 1o–r) or two
chlorines (compounds 1e, 1f) on the aniline ring; on the other side, fluorine atom provided only a
minimal benefit. Esters of 5-chlorosalicylanilides showed a higher antimycobacterial activity than
those derived from 4-chlorosalicylanilides, with fluorinated molecules 1k–n being an exception.
Importantly, salicylanilide 4-(trifluoromethyl)benzoates 1 were assayed being predominantly more
active than parent salicylanilides [23] – e.g., up to eight times for 1f, 1n and 1o against M. avium or 1e
for M. tuberculosis. In some cases, esters and parent phenolic molecules share the same MIC values,
e.g., 1b, 1k or 1d for M. tuberculosis. Sporadically, the esterification led to the decreased potency;
namely 1i, 1k, 1m and 1o against M. kansasii, 1m and 1r for M. avium. 5-Bromo-2-hydroxy-N-[4-
(trifluoromethyl)phenyl]benzamide (SAL-7) represents only one salicylanilide, which esterification
did not improve activity against any mycobacterial strain. Additionally, the most active compounds
(i.e., with at least one MIC against any strain ≤ 1 μmol/L) were evaluated against five MDR-TB strains
and one XDR-TB strain with different resistance patterns (Table 2).
Table 2. MICs of salicylanilide 4-(trifluoromethyl)benzoates 1 towards MDR- and XDR-TB.
MIC [mol/L]
Mtb.
Mtb.
Mtb.
Mtb.
Mtb.
Mtb. Praha 131
R1
R2
7357/1988 9449/2006
53/2009
234/2005
Praha 1
(XDR-TB)
14 d 21 d 14 d 21 d 14 d 21 d 14 d 21 d 14 d 21 d
14 d
4
21 d
4
1a
1b
1c
1d
1e
1f
4-Cl
5-Cl
4-Cl
5-Cl
4-Cl
5-Cl
4-Cl
4-Cl
5-Cl
4-Cl
4-Br
3-Cl
3-Cl
4
8
4
8
2
4
2
2
2
4
2
1
1
4
4
2
4
2
2
1
2
2
1
1
4
4
2
4
2
2
2
4
2
1
1
4
8
4
4
2
2
2
4
2
1
1
4
8
4
4
4
2
2
4
2
1
2
4
8
4
8
4
4
2
2
2
2
2
1
1
4
4
4
4
8
8
4-Cl
2
4
2
2
2
2
4-Cl
4
4
2
4
2
4
3,4-diCl
3,4-diCl
3-Br
1
1
1
2
2
2
2
1
1
2
1
2
1g
1i
2
1
1
2
1
2
4-Br
2
2
2
2
2
2
1j
4-Br
2
2
2
2
1
2
1o
1r
4-CF3
4-CF3
0.5
1
0.5
0.5
0.5
0.5
0.5
1
0.5
0.5
1
1
MIC values of 1 μmol/L and lower are given in bold.
Importantly, all evaluated esters showed a significant activity towards MDR- and XDR-TB strains
independently of the particular resistances. Interestingly, not only esters with the best activity towards
M. tuberculosis (MICs ≤ 1 μmol/L) were assayed, as in previous studies [9,13,24], as even additional
derivatives with MICs in the range of 2–4 μmol/L, were also powerful. All of these derivatives
affected the growth of drug-resistant M. tuberculosis at equal or almost equal concentrations required
for the inhibition of drug-sensitive one; drug-resistant strains were more susceptible to esters
1j, 1o and 1r than M. tuberculosis H37Rv. These findings indicate that salicylanilide
4-(trifluoromethyl)benzoates do not share any cross-resistance with conventionally used drugs (INH,
rifamycines, EMB, STM, OFX, clofazimine, aminoglycosides) and they may be prospective and
perspective agents for combating drug-resistant TB.

Molecules 2013, 18
3679
4-(Trifluoromethyl)benzoates 1 possess potent in vitro antimycobacterial properties; although
sharing a high lipophilicity, their efficacy did not surpassed esters with N-acetyl-L-phenylalanine [9]
with MDR- and XDR-TB being an exception, but esters 1 exhibited lower MICs than,
e.g., salicylanilide benzenesulfonates [10] or esters with N-benzyloxycarbonyl amino acids [25].
Carbamates showed better in vitro activity against M. tuberculosis including drug-resistant strains,
while atypical mycobacteria were inhibited at similar concentrations [24]. When compared to closely
related salicylanilide benzoates [13], the introduction of trifluoromethyl moiety into a benzoic acid
ring did not improved activity, although the presence of this substituent in parent salicylanilides
resulted unambiguously in the excellent antimycobacterial properties.
2.3. Isocitrate Lyase Inhibition Assay
Salicylanilide 4-(trifluoromethyl)benzoates were evaluated for their inhibition of mycobacterial ICL
to elucidate their impact on one of the known targets for latent TB infection (Table 3). Compounds
with dual effect on both the actively growing and latent mycobacterial populations may bring an
appreciable benefit.
Table 3. ICL inhibition activity of salicylanilide 4-(trifluoromethyl)benzoates 1.
R1
1a 4-Cl
1b 5-Cl
1c 4-Cl
1d 5-Cl
R2
% ICL inhibition at 10 μmol/L (± standard deviation)
3-Cl
3-Cl
4-Cl
4-Cl
18 ± 1.9
13 ± 1.7
12 ± 2.7
12 ± 2.3
18 ± 1.5
17 ± 1.6
27 ± 0.2
21 ± 1.8
18 ± 1.2
14 ± 4.0
25 ± 0.8
20 ± 0.5
23 ± 1.4
17 ± 1.3
13 ± 1.2
17 ± 2.3
19 ± 0.5
23 ± 0.1
25 ± 4.1
0
1e 4-Cl 3,4-diCl
1f 5-Cl 3,4-diCl
1g 4-Cl
1h 5-Cl
3-Br
3-Br
4-Br
4-Br
3-F
1i
1j
4-Cl
5-Cl
1k 4-Cl
1l 5-Cl
3-F
1m 4-Cl
1n 5-Cl
4-F
4-F
1o 4-Cl 4-CF3
1p 5-Cl 4-CF3
1q 4-Cl 3-CF3
1r 4-Br 4-CF3
3-NP
INH
INH = isoniazid; 3-NP = 3-nitropropionic acid; Inhibition rate values comparable to 3-NP or higher are given in bold.
Salicylanilide 4-(trifluoromethyl)benzoates 1 showed 12%–27% inhibition of isocitrate lyase at
10 µmol/L concentration. Six esters demonstrated  20% enzyme inhibition rate (1g, 1h, 1k, 1l, 1m, 1r)
with superiority of the 3-bromoaniline derivative 1g; this molecule also possessed higher potency than
3-NP, a well-known standard. Derivatives of 5-chlorosalicylic acid mostly exhibited higher potency

Molecules 2013, 18
3680
than those related to 4-chlorosalicylic acid (1a vs. 1b, 1g vs. 1h, 1k vs. 1l, etc.). The substitution of
aniline ring at the position 3 is linked with enhanced enzyme inhibition when compared to
4-substituted anilines (e.g., 1a vs. 1c, 1g vs. 1i, 1o vs. 1q).
Salicylanilide 4-(trifluoromethyl)benzoates 1 produced significantly and consistently higher ICL-1
inhibition than previously reported salicylanilide derivatives – salicylanilides, their pyrazinoates,
benzoates, benzenesulfonates or esters with N-acetyl-L-phenylalanine [4].
The additional ICL inhibition assay was performed with the derivatives at the concentration of
100 µmol/L; nevertheless, some of the compounds precipitated in the medium after a very short
period during the investigation. This effect at the higher concentration may be render due to a
high lipophilicity.
Obviously, even though salicylanilide 4-(trifluoromethyl)benzoates 1 exhibited a significant ICL-1
inhibition, their concentration values are higher (IC50 > 10 µmol/L) in comparison with MICs
necessary for the mycobacterial growth arrest. Importantly, the inhibition of isocitrate lyase does not
facilitate the elimination of mycobacteria during the acute phase of infection – in contrast to the
chronic phase [3]. Therefore there is not a direct relationship of in vitro MICs of salicylanilides
towards growing mycobacteria and the ICL inhibition, especially when the suppression of ICL may
affect the slowly- or non-growing mycobacterial subpopulations existing on the nutrition poor
substrates. Thus, the salicylanilide derivatives share a complex and still not fully elucidated
mechanism of action for actively growing mycobacteria, since more cellular targets have been
identified for this chemical group (e.g., [4,5,15]). Further experiments dealing with the mechanism(s)
of action are appropriate.
2.4. Antibacterial Evaluation
Table 4 overviews the antibacterial activity. For the remaining ten esters (1a, 1c, 1d, 1f, 1g, 1i, 1l,
1n, 1q, 1r), whose MICs are not reported, it was not possible to determine them. Similarly as in
previously reported antifungal assay [22], they could not be dissolved in the testing medium well or
they precipitated rapidly in them due to an escalated lipophilicity. 4-(Trifluoromethyl)benzoic acid
showed no antibacterial action under our conditions (MIC > 500 µmol/L).
All soluble derivatives showed a very good activity against Gram-positive cocci, in some cases (1e,
1j, 1p, particularly 1b, 1h and 1k) even excellent MICs lower than 1 μmol/L, despite the methicillin-
resistance. Based on MIC values, these esters act likely mainly as bactericidal agents. MICs against
S. aureus were comparable to benzylpenicillin and, moreover, 4-(trifluoromethyl)benzoates 1 exceeded
its activity for MRSA, S. epidermidis and partly for Enterococcus (1e, 1j, 1p). Among Gram-negative
bacteria, only E. coli possessed a partial susceptibility from 31.25 μmol/L (1p), while the growth of
P. aeruginosa and both strains of K. pneumoniae was not reduced up to the concentration of 500 μmol/L.
Due to a limited number of assayed esters, it is difficult to discuss the structure-activity relationship.
In the pair of 1o and 1p, the 5-chloro derivative (1p) showed lower MICs and when compared 1k vs.
1m, 3'-F moiety resulted in a higher activity.
Based on limited data, 4-(trifluoromethyl)benzoates displayed more significant activity against
Gram-positive bacteria than salicylanilide benzoates [11], pyrazinoates [12] or esters with N-acetyl-L-
phenylalanine [9].

Molecules 2013, 18
Table 4. MICs of salicylanilide 4-(trifluoromethyl)benzoates 1 towards selected bacteria.
3681
MIC/IC90 [μmol/L]
S. aureus
MRSA
S. epidermidis
H 6966/08
Enterococcus sp.
E. coli
CCM 4517
R1
R2
CCM 4516/08
H 5996/08
J 14365/08
24 h
0.98
0.49
0.98
0.49
3.9
48 h
1.95
0.98
0.98
0.98
3.9
24 h
48 h
1.95
0.98
0.98
0.98
1.95
15.62
7.81
0.98
125
24 h
48 h
1.95
0.98
0.98
0.98
3.9
24 h
7.81
0.49
31.25
1.95
31.25
500
48 h
15.62
3.9
24 h
>250
>500
>500
250
48 h
1b
1e
5-Cl
4-Cl
5-Cl
5-Cl
4-Cl
4-Cl
4-Cl
5-Cl
3-Cl
3,4-diCl
3-Br
0.98
0.49
0.49
0.49
0.98
15.62
3.9
0.98
0.49
0.49
0.49
3.9
>250
>500
>500
>250
>250
>500
>125
250
1h
1j
250
4-Br
7.81
>250
>500
62.5
3.9
1k
1m
1o
1p
3-F
>250
>500
>125
31.25
>500
4-F
7.81
3.9
7.81
7.81
0.98
0.98
31.25
3.9
>500
7.81
0.98
250
4-CF3
4-CF3
7.81
0.49
7.81
0.49
0.98
0.49
62.5
0.49
250
PNC
15.62
>500
MRSA: methicillin-resistant Staphylococcus aureus; PNC: benzylpenicillin; One or two best MIC(s) for each strain are
given in bold.
2.5. Cytotoxicity Evaluation
Since salicylanilide derivatives have displayed cytotoxicity for human cells (e.g., [4,9,20,24]), we
examined in vitro cytotoxicity of six 4-(trifluoromethyl)benzoates, which shared the highest rate of
MDR-TB growth suppression (1e, 1f, 1g, 1j, 1o, 1r), as well as esters with the lowest MICs for
Staphylococcus sp. (1e, 1j, 1p). For comparison, seven parent salicylanilides (SAL-1 – SAL-7) and
4-(trifluoromethyl)benzoic acid were also investigated in the liver HepG2 cells model (Table 5). The
parameter IC50 was used as a measure of toxicity, which allowed the quantitative comparison of the
toxicity among the tested compounds.
IC50 of 4-(trifluoromethyl)benzoates 1 were within the range 1.33–4.82 μmol/L and IC50 of parent
salicylanilides of 0.36–1.98 μmol/L. For all pairs salicylanilide and its 4-(trifluoromethyl)benzoate, the
esterification alleviated mildly the strong cytotoxicity of the parent salicylanilides containing free
phenolic group, which is probably mainly responsible for the cytotoxicity. Its temporary masking leads
to the derivatives with somewhat higher IC50 values – from 1.2-fold (1r) up to 3.7-fold for 1e and 1p,
but still in similar concentration range. 4-(Trifluoromethyl)benzoic acid was many time less toxic with
IC50 of 563.7 µmol/L.
Selectivity indexes (SI) ranges from 0.1 up to 1.67 for M. tuberculosis for parent salicylanilides,
while esters with a significant activity against MDR-TB showed SI of 0.89–4.46 for drug-sensitive
M. tuberculosis and of 0.78–4.46 for drug-resistant strains. SI values for S. aureus were within
0.17–9.84; SI of 1j almost reached the value of 10, which is generally considered being borderline.
With respect to the purposed cytotoxicity reduction of the parent salicylanilides, the results are
not satisfactory.

Molecules 2013, 18
Table 5. IC50 and SI for selected salicylanilide 4-(trifluoromethyl)benzoates 1.
3682
IC50 [µmol/L]
HepG2
24 h
SI for Mtb.
SI for
SI for
SI for
R1
R2
H37Rv 331/88
MDR-TB strains
XDR-TB strain Staphylococcus sp.
14 d
3.13
4.46
2.63
2.41
1.33
0.89
2.03
0.21
0.45
NT
21 d
3.13
2.23
1.32
1.21
1.33
0.89
2.03
0.10
0.45
NT
14 d
21 d
14 d
14 d
24 h
48 h
1e
1f
4-Cl
5-Cl
4-Cl
5-Cl
4-Cl
5-Cl
4-Br
5-Cl
4-Cl
5-Cl
4-Cl
5-Cl
4-Cl
5-Br
3,4-diCl
3,4-diCl
3-Br
3.13
1.57–3.13 0.78–1.57
1.57
1.57
6.39
3.19
4.46
2.23–4.46
1.32–2.63
2.24
2.23
1.32
2.24
4.46
2.23
-
-
1g
2.63
2.63
1.32
-
-
1j
4-Br
4.82
4.82
2.41
9.84
4.92
1o
4-CF3
4-CF3
4-CF3
3,4-diCl
3,4-diCl
3-Br
1.33
1.33–2.66 1.33–2.66
2.66
1.33
0.34
0.17
1p
1.77
-
-
-
-
3.61
1.81
1r
2.03
2.03–4.06 1.02–2.03
4.06
2.03
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
SAL-1
SAL-2
SAL-3
SAL-4
SAL-5
SAL-6
SAL-7
0.84
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
1.80
1.54
4-Br
1.98
0.5
0.5
4-CF3
4-CF3
4-CF3
0.36
0.18
0.17
1.67
0.18
0.17
1.67
0.69
1.67
CF3-BA
563.70
-
-
-
-
-
-
-
-
CF3-BA = 4-(trifluoromethyl)benzoic acid. SI = IC50/MIC100; MIC values of salicylanilides against M. tuberculosis were
taken from reference [23].
When compared to other salicylanilide esters, 4-(trifluoromethyl)benzoates showed about ten times
higher cytotoxicity than salicylanilide carbamates [24], esters with N-acetyl-L-phenylalanine [9] or
even more than esters with N-benzyloxycarbonyl amino acids [25], while it was approximately
comparable with salicylanilide benzenesulfonates and benzoates [4].
However, salicylanilide esterification may represent one successful way to reduce their cytotoxicity
for human cells as it was demonstrated especially for esters with N-protected -amino acids [9,25] and
carbamates [24]; it is necessary to search new type(s) of acid(s), because 4-(trifluoromethyl)benzoic
acid brought a very mild and still insufficient benefit.
From other point of view, salicylanilides and their 4-(trifluoromethyl)benzoates may be considered
as potential anticancer agents. One study [20] reported some salicylanilides being proposed and
synthesized as potential cytotoxic agents against hepatocellular carcinoma. All of our salicylanilide
derivatives showed IC50 values similar to the most active salicylanilides reported by Zhu et al. [20]
(1.3 and 1.7 μmol/L); even three salicylanilides (SAL-1, SAL-5, SAL-6) were more active with
5-chloro-2-hydroxy-N-[4-(trifluoromethyl)phenyl]benzamide (SAL-5) being superior with IC50 of
0.36 μmol/L. Especially salicylanilide 4-(trifluoromethyl)benzoates 1 could be more convenient
because previously it was demonstrated that some 3- and 4-(trifluoromethyl)benzoate esters of
scaffolds with an intrinsic activity showed superiority among other esters [26,27].
The mechanism of cytotoxicity seems to be multiple – besides non-specific damage of cellular
structures and function by the in vivo released phenolic group [8], it has been described a mild
inhibition of human methionine aminopeptidase [4], inhibition of EGFR protein tyrosine
kinases [17,19,20,28,29] or other human enzymes [18,19].

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3. Experimental
3.1. Chemistry
The synthesis and characterization (m.p., IR and NMR spectra, elemental analyses) of the presented
salicylanilide 4-(trifluoromethyl)benzoates [2-(phenylcarbamoyl)phenyl 4-(trifluoromethyl)benzoates;
Scheme 1] 1a–r was published recently by Krátký et al. [22].
3.2. Antimycobacterial Evaluation
All compounds were tested for their in vitro against Mycobacterium tuberculosis 331/88 (H37Rv;
dilution of the strain was 10−3), nontuberculous mycobacteria: Mycobacterium avium 330/88 [resistant
to INH, RIF, ofloxacin (OFX), and ethambutol (EMB); dilution 10−5], one strain of Mycobacterium
kansasii 235/80 from the Czech National Collection of Type Cultures (CNCTC) (dilution 10−4) and a
clinically isolated strain of M. kansasii 6509/96 (dilution 10−5). The micromethod for the determination
of MICs was used. Antimycobacterial activities were determined in the Šula´s semisynthetic medium
(SEVAC, Prague, Czech Republic). The tested compounds were added to the medium as solutions in
dimethyl sulfoxide (DMSO). The following concentrations were used: 1000, 500, 250, 125, 62.5, 32,
16, 8, 4, 2, 1, 0.5, 0.25, and 0.125 μmol/L. The MICs were determined after incubation at 37 °C for 14
and 21 days, for M. kansasii additionally for 7 days. MIC (in μmol/L) was the lowest concentration at
which the complete inhibition of mycobacterial growth occurred. The first-line anti-tuberculosis drug
isoniazid (INH) and structurally similar para-aminosalicylic acid (PAS), a second-line oral agent, were
chosen as the reference drugs.
The most active esters were evaluated at similar conditions and concentrations against six MDR-TB
strains (dilution 10−3) with different resistance patterns. All strains are resistant to INH, RIF,
rifabutine, and streptomycin (STM); an additional resistance was present in some cases: 7357/1998
and 234/2005 both resistant to INH, rifamycines, STM, EMB, and OFX; 9449/2006 resistant to INH,
rifamycines, and STM; 53/2009 resistant to INH, rifamycines, STM, and EMB; Praha 1 resistant to
INH, rifamycines, STM, EMB, and clofazimine; and Praha 131 resistant to INH, rifamycines, STM,
EMB, OFX, gentamicin and amikacin (i.e., XDR-TB strain). The following concentrations were used:
1000, 500, 250, 125, 62.5, 32, 16, 8, 4, 2, 1, 0.5, 0.25, and 0.125 μmol/L.
3.3. Isocitrate Lyase Inhibition Assay (ICL1)
The Mycobacterium tubeculosis H37Rv genomic DNA was used as a template. The icl gene
(Rv0467) of 1.28 kb was amplified using PCR. The amplified DNA was cloned into the pET-28b(+)
plasmid vector Novagen (Merck KGaA, Darmstadt, Germany) using NdeI and HindIII restriction sites.
The recombinant plasmid was transferred into E. coli HB101. DNA sequencing was employed to
confirm that the inserted coding sequence had no mutations. For bacterial expression, 25 mL culture
volumes were inoculated with BL21(DE3) cells containing the recombinant plasmid and allowed to
grow until an optical density of OD595 = 0.6 was achieved. Then, the culture was induced with
1 mmol/L isopropyl-β-D-thiogalactopyranoside solution and incubated at 30 °C for additional 4 h. The
cells were harvested at 4 °C by centrifugation at 6,000 g for 10 min. The resulting pellet was

Molecules 2013, 18
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resuspended in BugBuster Protein Extraction Reagent Novagen (Merck KGaA, Darmstadt, Germany).
The cell debris was removed by centrifugation, and the histidine-tagged protein was purified using an
Äkta purifier (Amersham Biosciences, Valley Stream, NY, USA). The purity of the protein was
confirmed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) followed by Coomassie blue
staining of the gel. The protein concentration was determined by the Bradford method [30].
The isocitrate lyase activity was assayed according to the protocol reported by Dixon and Kornberg [31].
The enzyme assay was optimised in the final volume of 100 μL using 96-well plates (NUNC,
Schoeller, Prague, Czech Republic) and the final concentration of tested compound in the reaction
mixture 10 μmol/L. The reaction buffer contained 50 mmol/L of KH2PO4, 4 mmol/L of MgCl2·6H2O,
4 mmol/L of phenylhydrazine hydrochloride, 12 mmol/L of L-cysteine, H2O and KOH to pH 7.0. The
isocitrate cleavage was measured by the change in the absorbance at 324 nm, which is associated with
the formation of glyoxylate phenyl hydrazone. Each tested compound was dissolved in DMSO to
prepare a 1 mmol/L solution, and 1 μL of this solution was added to 93.9 μL of reaction buffer. Then,
it was added 0.1124 μL of the recombinant enzyme in phosphate buffer and glycerol solution with
concentration of 0.58 mg/mL (Bradford). Finally the reaction was started by the addition of 0.2 μmol
of (+)-potassium Ds-threo-isocitrate in solution.
Isoniazid was employed as a negative control (inhibition 0%), and 3-nitropropionic acid (3-NP)
served as the positive control. All of the control compounds were added to the reaction mixture
the same way like tested compounds and also their concentration in the reaction mixture was
10 μmol/L. The inhibitory activity of DMSO alone (1 μL) was subtracted from the activities of the
evaluated compounds.
3.4. Antibacterial Evaluation
The in vitro antibacterial activity was assayed against eight Gram-positive and Gram-negative
strains: Staphylococcus aureus CCM 4516/08, methicillin-resistant Staphylococcus aureus H 5996/08
(MRSA), Staphylococcus epidermidis H 6966/08, Enterococcus sp. J 14365/08; Escherichia coli CCM
4517, Klebsiella pneumoniae D 11750/08, ESBL-positive Klebsiella pneumoniae J 14368/08, and
Pseudomonas aeruginosa CCM 1961.
The microdilution broth method modified according to standard M07-A07 in Mueller-Hinton broth
(HiMedia Laboratories, Mumbai, India) adjusted to pH 7.4 (±0.2) was used. The tested compounds
were dissolved in DMSO to the final concentrations ranging from 500 to 0.49 mmol/L.
Benzylpenicillin (penicillin G) was used as a comparative drug. Bacterial inoculum in sterile water was
prepared to match 0.5 McFarland scale (1.5 ± 108 CFU/mL). The minimum inhibitory concentrations
were assayed as 90% (IC90) or higher reduction of growth when compared to the control.
The determination of results was performed visually and spectrophotometrically (at 540 nm). The
values of MICs were determined after 24 and 48 h of incubation in the darkness at 35 °C (±0.1) in a
humid atmosphere.
3.5. Cytotoxicity Evaluation (HepG2 Cells)
Seven esters and their parent salicylanilides with the most robust antimicrobial activity were tested
for cytotoxicity in the human hepatocellular liver carcinoma cell line HepG2 (passages 21–24;

Molecules 2013, 18
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ECACC, Salisbury, UK) using a standard colorimetric method measuring a tetrazolium salt reduction
(CellTiter(R) 96 AQueous One Solution Assay, Promega G3580, Madison, WI, USA).
The cells were routinely cultured in Minimum Essentials Eagle Medium (Sigma-Aldrich,
Darmstadt, Germany) supplemented with 10% foetal bovine serum (PAA; Biotech, Prague, Czech
Republic), 1% of L-glutamine solution (Sigma-Aldrich), and non-essential amino acids solution
(Sigma-Aldrich) in a humidified atmosphere containing 5% CO2 at 37 °C. For subculturing, the cells
were harvested after trypsin/EDTA (Sigma-Aldrich) treatment at 37 °C. The cells treated with the
tested substances were used as experimental groups. Untreated HepG2 cells were used as control groups.
The cells were seeded in density 1 × 104 cells per well in a 96-well plate with microscopic control.
Next day, the cells were treated with each of the tested substances. Because of the low solubility of
some investigated compounds in water they were dissolved in a very small amount of DMSO and in a
small volume added to cell culture; a final solution contained less than 1% DMSO. The most of the
tested compounds were prepared in incubation concentrations of 0.01–20 µM in triplicates.
4-(Trifluoromethyl)benzoic acid was applied in the concentrations of 10–4,000 µM in triplicates. The
following types of controls were included: determination of 100% viability and 0% viability (the cells
treated by 10% DMSO), no cell control and vehiculum controls. These checking samples were
prepared in triplicates.
The treated cells were incubated together with controls at 37 °C for 24 h in 5% CO2 atmosphere.
After this time reagent from the kit CellTiter 96 AQueous One Solution Cell Proliferation Assay was
added. The CellTiter 96 assay is based on the reduction of tetrazolium salt MTS [3-(4,5-
dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] to water-soluble
formazan dye by metabolically active cells. The reduction of the reagent is attributed to availability of
NADH or NADPH. The decline in levels of these metabolically important compounds in the cell
causes that the production of formazan is reduced. The tested plate was incubated for 2 h at 37 °C and
5% CO2 and after this time, the absorbance was recorded at 490 nm using a 96-well plate reader
(TECAN, Infinite M200, Grödig, Austria).
The results were expressed as inhibitory concentration which is necessary to inhibit cell viability to
50% from the maximal (control) viability (IC50). A standard toxicological parameter IC50 was
calculated in each of the tested substances using GraphPad Prism software (version 5.02; GraphPad
Software Inc., San Diego, CA, USA).
4. Conclusions
Eighteen salicylanilide 4-(trifluoromethyl)benzoates with a known antifungal activity underwent
additional biological screening for the antimycobacterial and antibacterial activity, cytotoxicity and
inhibition of mycobacterial isocitrate lyase. All compounds showed a significant antimycobacterial
activity including against drug-resistant strains; in some cases comparable or even better than
isoniazid. 3,4-Dichloro- and trifluoromethyl moieties represent the optimal substitution of aniline ring.
Esters caused consistently a mild inhibition of isocitrate lyase; moreover, some derivatives are
comparable to 3-nitropropionic acid. The substitution of aniline ring at the position 3 enhances this
action. Although salicylanilide 4-(trifluoromethyl)benzoates failed with respect to our intention of

Molecules 2013, 18
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finding the most antimycobacterial active salicylanilide esters, they offer very low MICs against
Gram-positive strains and IC50 values for HepG2 cells.
In contrast to the aniline ring, the introduction of a 4-trifluoromethyl moiety into an acyl part of the
salicylanilide esters did not provide sharply improved antimycobacterial activity, when compared to
salicylanilide benzoates. The most potent esters are still considerably cytotoxic in micromolar values,
although less than parent salicylanilides. Their cytotoxicity was mostly a slightly higher than for
corresponding benzoates. In summary, salicylanilides and their 4-(trifluoromethyl)benzoates may be
promising initial step compounds for anticancer research with advantageous broad-spectrum
antimicrobial properties.
Acknowledgments
This work was financially supported by 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 Ida Dufková for the excellent performance of antibiotic susceptibility tests (under the
supervision of Vladimír Buchta), Vladimír Wsól for the supervision of enzyme inhibition assay and
Jaroslava Urbanová, M.A., for the language help.
Conflict of Interest
The authors declare no conflict of interest.
References
1. WHO. 2011/2012 Tuberculosis Global Facts. Available online: http://www.who.int/tb/
publications/2011/factsheet_tb_2011.pdf (accessed on 12 December 2012).
2. Caminero, J.A. Extensively drug-resistant tuberculosis: Is its definition correct? Eur. Respir. J.
2008, 32, 1413–1415.
3. McKinney, J.D.; Bentrup, K.H.; Munoz-Elias, E.J.; Miczak, A.; Chen, B.; Chan, W.T.; Swenson, D.;
Sacchettini, J.C.; Jacobs, W.R.; Russell, D.G. Persistence of Mycobacterium tuberculosis in
macrophages and mice requires the glyoxylate shunt enzyme isocitrate lyase. Nature 2000, 406,
735–738.
4. Krátký, M.; Vinšová, J.; Novotná, E.; Mandíková, J.; Wsól, V.; Trejtnar, F.; Ulmann, V.;
Stolaříková, J.; Fernandes, S.; Bhat, S.; Liu, J.O. Salicylanilide derivatives block Mycobacterium
tuberculosis through inhibition of isocitrate lyase and methionine aminopeptidase. Tuberculosis
2012, 92, 434–439.
5. Krátký, M.; Vinšová, J. Advances in mycobacterial isocitrate lyase targeting and inhibitors.
Curr. Med. Chem. 2012, 19, 6126–6137.
6. Cabrera, C.E.; Gómez, R.F.; Zuñiga, A.E.; Corral, R.H.; López, B.; Chávez, M. Epidemiology of
nosocomial bacteria resistant to antimicrobials. Colomb. Med. 2011, 42, 117–125.

Molecules 2013, 18
3687
7. Scientific Blueprint for Tuberculosis Drug Development. Available online: http://www.tballiance.org/
downloads/publications/TBA_Scientific_Blueprint.pdf (accessed on 12 December 2012).
8. Krátký, M.; Vinšová, J. Salicylanilide ester prodrugs as potential antimicrobial agents—A review.
Curr. Pharm. Des. 2011, 17, 3494–3505.
9. Krátký, M.; Vinšová, J.; Buchta, V.; Horvati, K.; Bösze, S.; Stolaříková, J. New amino acid esters
of salicylanilides active against MDR-TB and other microbes. Eur. J. Med. Chem. 2010, 45,
6106–6113.
10. Krátký, M.; Vinšová, J.; Rodriguez, N.G.; Stolaříková, J. Antimycobacterial activity of
salicylanilide benzenesulfonates. Molecules 2012, 17, 492–503.
11. Krátký, M.; Vinšová, J.; Buchta, V. In vitro antibacterial and antifungal activity of salicylanilide
benzoates. ScientificWorldJournal 2012, 12, doi:10.1100/2012/290628.
12. Krátký, M.; Vinšová, J.; Buchta, V. In vitro antibacterial and antifungal activity of salicylanilide
pyrazine-2-carboxylates. Med. Chem. 2012, 8, 732–741.
13. Krátký, M.; Vinšová, J.; Stolaříková, J. Antimycobacterial assessment of salicylanilide benzoates
including multidrug-resistant tuberculosis strains. Molecules 2012, 17, 12812–12820.
14. Cheng, T.J.R.; Wu, Y.T.; Yang, S.T.; Lo, K.H.; Chen, S.K.; Chen, Y.H.; Huang, W.I.; Yuan, C.H.;
Guo, C.W.; Huang, L.Y.; et al. High-throughput identification of antibacterials against
methicillin-resistant Staphylococcus aureus (MRSA) and the transglycosylase. Bioorg. Med. Chem.
2010, 18, 8512–8529.
15. Garner, A.L.; Gloeckner, C.; Tricoche, N.; Zakhari, J.S.; Samje, M.; Cho-Ngwa, F.; Lustigman, S.;
Janda, K.D. Design, synthesis, and biological activities of closantel analogues: Structural
promiscuity and its impact on Onchocerca volvulus. J. Med. Chem. 2011, 54, 3963–3972.
16. Fomovska, A.; Wood, R.D.; Mui, E.; Dubey, J.P.; Ferreira, L.R.; Hickman, M.R.; Lee, P.J.;
Leed, S.E.; Auschwitz, J.M.; Welsh, W.J.; et al. Salicylanilide inhibitors of Toxoplasma gondii.
J. Med. Chem. 2012, 55, 8375−8391.
17. Ding, N.; Zhang, W.; Xiao, H.L.; Wang, P.; Li, Y.X. Synthesis and biological evaluation of a
series of novel salicylanilides as inhibitors of EGFR protein tyrosine kinases. Chin. Chem. Lett.
2012, 23, 529–532.
18. Steffen, J.D.; Coyle, D.L.; Damodaran, K.; Beroza, P.; Jacobson, M.K. Discovery and structure-
activity relationships of modified salicylanilides as cell permeable inhibitors of poly(ADP-ribose)
glycohydrolase (PARG). J. Med. Chem. 2011, 54, 5403–5413.
19. Zuo, M.; Zheng, Y.W.; Lu, S.M.; Li, Y.; Zhang, S.Q. Synthesis and biological evaluation of
N-aryl salicylamides with a hydroxamic acid moiety at 5-position as novel HDAC–EGFR dual
inhibitors. Bioorg. Med. Chem. 2012, 20, 4405–4412.
20. Zhu, Z.W.; Shi, L.; Ruan, X.M.; Yang, Y.; Li, H.Q.; Xu, S.P.; Zhu, H.L. Synthesis and
antiproliferative activities against Hep-G2 of salicylanide derivatives: potent inhibitors of the
epidermal growth factor receptor (EGFR) tyrosine kinase. J. Enzym. Inhib. Med. Chem. 2011, 26,
37–45.
21. Liu, X.H.; Lv, P.C.; Li, B.; Zhu, H.L.; Song, B.A. Synthesis, structure, and antibacterial activity
of novel 5-arylpyrazole derivatives. Aust. J. Chem. 2008, 61, 223–230.
22. Krátký, M.; Vinšová, J. Antifungal activity of salicylanilides and their esters with
4-(trifluoromethyl)benzoic acid. Molecules 2012, 17, 9426–9442.

Molecules 2013, 18
3688
23. Waisser, K.; Bureš, O.; Holý, P.; Kuneš, J.; Oswald, R.; Jirásková, L.; Pour, M.; Klimešová, V.;
Kubicová, L.; Kaustová, J. Relationship between the structure and antimycobacterial activity of
substituted salicylanilides. Arch. Pharm. Pharm. Med. Chem. 2003, 336, 53–71.
24. Imramovský, A.; Vinšová, J.; Férriz, J.M.; Doležal, R.; Jampílek, J.; Kaustová, J.; Kunc, F. New
antituberculotics originated from salicylanilides with promising in vitro activity against atypical
mycobacterial strains. Bioorg. Med. Chem. 2009, 17, 3572–3579.
25. Férriz, J.M.; Vávrová, K.; Kunc, F.; Imramovský, A.; Stolaříková, J.; Vavříková, E.; Vinšová, J.
Salicylanilide carbamates: Antitubercular agents active against multidrug-resistant Mycobacterium
tuberculosis strains. Bioorg. Med. Chem. 2010, 18, 1054–1061.
26. Liu, X.H.; Song, B.A.; Bhadury, P.S.; Zhu, H.L.; Cui, P.; Hou, K.K.; Xu, H.L. Novel 5-(3-
(substituted)-4,5-dihydroisoxazol-5-yl)-2-methoxyphenyl derivatives: Synthesis and anticancer
activity. Aust. J. Chem. 2008, 61, 864–869.
27. Wada, K.; Ohkoshi, E.; Morris-Natschke, S.L.; Bastow, K.F.; Lee, K.H. Cytotoxic esterified
diterpenoid alkaloid derivatives with increased selectivity against a drug-resistant cancer cell line.
Bioorg. Med. Chem. Lett. 2012, 22, 249–252.
28. Deng, W.; Guo, Z.; Guo, Y.; Feng, Z.; Jiang, Y.; Chu, F. Acryloylamino-salicylanilides as EGFR
PTK inhibitors. Bioorg. Med. Chem. Lett. 2006, 16, 469–472.
29. Liechti, C.; Séquin, U.; Bold, G.; Furet, P.; Meyer, T.; Traxler P. Salicylanilides as inhibitors of
the protein tyrosine kinase epidermal growth factor receptor. Eur. J. Med. Chem. 2004, 39, 11–26.
30. Bradford, M.M. Rapid and sensitive method for quantitation of microgram quantities of protein
utilizing principle of protein-dye binding. Anal. Biochem. 1976, 72, 248–254.
31. Dixon, G.H.; Kornberg, H.L. Assay methods for key enzymes of the glyoxylate cycle. Biochem. J.
1959, 72, P3.
Sample Availability: Samples of the compounds 1a–r and SAL-1 – SAL-7 are available from the authors.
© 2013 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article
distributed under the terms and conditions of the Creative Commons Attribution license
(http://creativecommons.org/licenses/by/3.0/).