New amino acid esters of salicylanilides active against MDR-TB and other microbes

Article abstract of DOI:10.1016/j.ejmech.2010.09.040

Eleven halogenated (S)-2-(phenylcarbamoyl)phenyl 2-acetamido-3- phenylpropanoates (3a-3k) were designed and synthesized as potential antimicrobial agents. They were evaluated in vitro against some mycobacterial, bacterial and fungal strains. These compounds were active against drug-sensitive and atypical mycobacterial strains with general MIC values from 0.25 to 16 μmol/L. The most active compounds were (S)-4-chloro-2-(4-(trifluoromethyl) phenylcarbamoyl)phenyl 2-acetamido-3-phenylpropanoate (3i) and (S)-4-bromo-2-(4-(trifluoromethyl)phenylcarbamoyl)phenyl 2-acetamido-3- phenylpropanoate (3k) which exhibited activity against MDR and XDR-TB strains with MICs from 1 to 2 μmol/L. 3k was shown to be less cytotoxic with higher IC₅₀. Some compounds exhibited low MICs on Gram-positive bacteria (MICs ≥ 0.98 μmol/L) and on fungi (MICs ≥ 3.9 μmol/L).

Full text of DOI:10.1016/j.ejmech.2010.09.040

European Journal of Medicinal Chemistry 45 (2010) 6106e6113
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Short communication
New amino acid esters of salicylanilides active against MDR-TB and other
microbes
a
a,
b,
c
d
d
e
*
ꢀ
ꢀ
ꢀ
Martin Krátký , Jarmila Vinsová , Vladimír Buchta , Kata Horvati , Szilvia Bösze , Jirina Stolaríková
a Department of Inorganic and Organic Chemistry, Faculty of Pharmacy, Charles University, Heyrovského 1203, 500 05 Hradec Králové, Czech Republic
b Department of Clinical Microbiology, Faculty of Medicine and University Hospital, Charles University, Sokolská 581, 500 12 Hradec Králové, Czech Republic
c Department of Biological and Medical Sciences, Faculty of Pharmacy, Charles University, Heyrovského 1203, 500 05 Hradec Králové, Czech Republic
d Research Group of Peptide Chemistry, Eötvös Lórand University, Hungarian Academy of Science, Pázmány Péter Sétány 1/A, Budapest, H-1117, Hungary
e
ꢀ
Laboratory for TBC, Regional Institute of Public Health in Ostrava, Partyzánské námestí 7, 702 00 Ostrava, Czech Republic
a r t i c l e i n f o
a b s t r a c t
Article history:
Eleven halogenated (S)-2-(phenylcarbamoyl)phenyl 2-acetamido-3-phenylpropanoates (3ae3k) were
designed and synthesized as potential antimicrobial agents. They were evaluated in vitro against some
mycobacterial, bacterial and fungal strains. These compounds were active against drug-sensitive and
Received 30 June 2010
Received in revised form
13 September 2010
atypical mycobacterial strains with general MIC values from 0.25 to 16
mmol/L. The most active
Accepted 16 September 2010
compounds were (S)-4-chloro-2-(4-(trifluoromethyl)phenylcarbamoyl)phenyl 2-acetamido-3-phenyl-
propanoate (3i) and (S)-4-bromo-2-(4-(trifluoromethyl)phenylcarbamoyl)phenyl 2-acetamido-3-phe-
nylpropanoate (3k) which exhibited activity against MDR and XDR-TB strains with MICs from 1 to
Keywords:
Tuberculosis
Multidrug-resistant tuberculosis
Salicylanilide
2
m
mol/L. 3k was shown to be less cytotoxic with higher IC50. Some compounds exhibited low MICs on
Gram-positive bacteria (MICs ꢀ 0.98 mol/L) and on fungi (MICs ꢀ 3.9 mol/L).
Ó 2010 Elsevier Masson SAS. All rights reserved.
m
m
Amino acid ester
Antibacterial activity
Antifungal activity
1. Introduction
Multidrug-resistant M. tuberculosis was defined as resistant to
isoniazid (INH) and rifampicin (RIF), but it may include resistance
to more antituberculotics [23]. XDR-TB was defined as a resistance
to any fluoroquinolone and to at least one of three injectable drugs
capreomycin, kanamycin or amikacin, in addition to MDR-TB. XDR-
TB is very often untreatable. HIV co-infection with MDR-TB in
immunocompromised patients is a serious challenge for the
research since it is demanded a new type of drugs or prodrugs by
a new mechanism of action [20].
Waisser et al. [24,25] described a good antimycobacterial
activity of substituted salicylanilides against drug-sensitive
M. tuberculosis and some atypical mycobacterial strains (Mycobac-
terium avium, Mycobacterium kansasii) with MICs for di- and tri-
Salicylanilides (2-hydroxy-N-phenylbenzamides) have been the
subject of interest in medicinal chemistry for many years [1],
mainly due to their antibacterial [1,2], antiprotozoal [1,3] and
antifungal activity [1,4]. They showed a wide-range of biological
activities including potential anticancer efficacy [5e8], influence on
interleukins production [9e12], anti-inflammatory activity [13],
activity against different viruses [14e18], or effects on ion channels
[19].
Increasing emergence of drug-resistant tuberculosis is alarming
[20,21]. It is estimated that one third of the world’s population is
currently infected with Mycobacterium tuberculosis, and each year
8e9 million new cases develop. Every year almost 500,000 people
are infected with multidrug-resistant TB (MDR-TB) and there are
estimated 40,000 new cases of extensively drug-resistant TB (XDR-
TB) annually [22].
halogenated salicylanilides in the range of 1e32
mmol/L for all the
tested strains.
Designing of new salicylanilide derivatives based on esterifica-
tion is a known trend. First recent article dealing with antimicrobial
esters of salicylanilides was published in 2006 [26]. Hydrophobicity
is one of the factors influencing biological activity of salicylanilide.
While the presence of phenolic hydroxyls seems to be necessary for
the activity, it could have irritative properties. Temporary masking
of this group by esterification and resulting changes in physico-
chemical properties could be advantageous in a high activity, an
* Corresponding author. Heyrovského 1203, 500 05 Hradec Králové, Czech
Republic. Tel.: þ420 495067343; fax: þ420 495067166.
ꢀ
E-mail address: jarmila.vinsova@faf.cuni.cz (J. Vinsová).
0223-5234/$ e see front matter Ó 2010 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2010.09.040

M. Krátký et al. / European Journal of Medicinal Chemistry 45 (2010) 6106e6113
6107
improved solubility, a better bioavailability, passing through the
mycobacterial cell wall and a lower toxicity. When amino acids are
used for esterification, they could facilitate the possibility of tar-
geting drugs as a drug delivery system [4,26,27].
Esters of salicylanilides (3) with lipophilic amino acid N-acetyl-
-phenylalanine (2) were obtained by the activation with N,N0-
L
dicyclohexylcarbodiimide (DCC) in dry N,N-dimethylformamide
(DMF) (Scheme 1). This method, which was taken from peptide
chemistry, has been recently published by our group [30].
All newly prepared compounds were characterised by the
melting point, IR and NMR spectroscopy, elementary analysis and
optical activity.
The worldwide epidemic of antibiotic resistance is touching all
people, because antibiotic-resistant bacteria are increasingly seen
to be just as virulent as their sensitive counterparts. For example,
the global emergence of methicillin-resistant Staphylococcus aureus
(MRSA) has arguably been the biggest setback in the history of
antimicrobial therapy. MRSA has caused serious problems in the
empirical use of the major commonly applied antibiotics. The
situation in Gram-negative bacteria is no less serious, too [28].
The conditions in the fungal kingdom are quite similar. Fungi
can become resistant to each of clinically used antifungal drugs by
specific mechanisms. The phenomenon of multidrug-resistance
was described analogously to bacteria [29].
3. Pharmacology
3.1. Minimum inhibitory concentration assays
3.1.1. In vitro antimycobacterial susceptibility testing
All the prepared compounds were tested in vitro for their anti-
mycobacterial activity in the Laboratory for Tuberculosis in Ostrava
against M. tuberculosis 331/88 (H37Rv) (dilution of the strain was
10ꢁ3) and moreover for some non-tuberculous INH-resistant
strains e M. avium (330/88, dilution 10ꢁ5) and M. kansasii (235/80,
dilution 10ꢁ4). One strain was clinically isolated (M. kansasii 6509/
96 in the dilution 10ꢁ5), other strains were obtained from the Czech
National Collection of Type Cultures. The micromethod for the
determination of the minimum inhibitory concentration (MIC) was
used.
Salicylanilide esters showed antimycobacterial activity in the
range from 0.5 to 62.5
mmol/L [4,27,30,31]. Salicylanilides, their
esters and some structures containing them have shown and
confirmed recently their good antibacterial activity, especially on
Gram-positive strains in the concentration of 0.25
mg/mL and
higher [2,32e38]. About the mechanism of action e salicylanilides
were identified as inhibitors of the two-component regulatory
systems of bacteria by a mechanism related to the effect on
uncoupling oxidative phosphorylation, but other injurious effects
on prokaryote cells were described, too [2,31,32,36]. Recently, it
was also found in the studies that salicylanilides are selective
inhibitors of interleukin-12p40 production playing a specific role in
the initiation, expansion and control of the cellular response to TB
infection [10,12]. The antifungal activity of salicylanilide esters is
The most active compounds 3g, 3i and 3k were evaluated against
MDR-TB in the similar conditions using six M. tuberculosis strains
(dilution 10ꢁ3) with different resistance patterns: 357/2005, 362/
1998, 53/2009, Praha 1, Praha 131 (XDR-TB strain), and 9449/2007.
3.1.2. In vitro antibacterial susceptibility testing
significant for acetates and
and ꢀ1.95 mol/L, respectively [4,30].
Therefore we designed and synthesized a new ester series
of halogenated salicylanilides and N-acetyl- -phenylalanine as
a
-amino acids esters with MICs ꢀ 0.49
The broth microdilution test M27-A [40] was used for the inves-
tigation of invitroantibacterialactivityofesters(3)againstfourGram-
positive and four Gram-negative strains: S. aureus, methicillin-resis-
tant S.aureus, Staphylococcusepidermidis, Enterococcussp.;Escherichia
coli, Klebsiella pneumoniae, EBSL-positive K. pneumoniae, and Pseu-
domonas aeruginosa. The method used was microdilution panel
bouillon method with twofold dilution of the compounds.
m
L
potential antibacterial and antifungal agents, primarily against
M. tuberculosis including MDR-TB strains and atypical mycobacteria.
2. Chemistry
3.1.3. In vitro antifungal susceptibility testing
The broth microdilution test M27-A was used for the investi-
gation of in vitro antifungal activity of esters (3) against four yeast
strains: Candida albicans, Candida tropicalis, Candida krusei, Candida
glabrata, and four moulds: Trichosporon beigelii, Aspergillus fumi-
gatus, Absidia corymbifera, and Trichophyton mentagrophytes. The
method used was microdilution panel bouillon method with
twofold dilution of the compounds.
The starting salicylanilides were selected according to their
previously described high in vitro antimycobacterial activity [24] and
the N-protected amino acid N-acetyl-
inary publications about good influence of N-Cbz-
antimycobacterial and antifungal activity of salicylanilides [4,27].
The syntheses of salicylanilide N-acetyl- -phenylalanine esters
L-phenylalanine due to prelim-
L-phenylalanine on
L
consist of two steps. At first, starting halogenated salicylanilides (1)
were prepared routinely by the reaction of substituted salicylic
acids and appropriate anilines in the presence of PCl3 in chloro-
benzene. The reaction was carried out in a microwave reactor
which led to an increase of the yield and shortening the reaction
time [39].
3.2. In vitro cytotoxicity assay
Cytotoxicity was determined on human peripheral blood
mononuclear cells (PBMC). After incubation the cell viability was
Scheme 1. Esterification of salicylanilides by N-acetyl-L-phenylalanine (R1 of esters ¼ 4-Cl, 5-Cl, 4-Br; R2 ¼ 3-Cl, 4-Cl, 3,4-diCl, 4-Br, 4-CF3). Reagents and conditions: (a) DMF, DCC,
ꢁ20 ꢂC.

6108
M. Krátký et al. / European Journal of Medicinal Chemistry 45 (2010) 6106e6113
determined by MMT-assay [41]. Cytotoxicity was calculated after
determination of optical density and IC50 values were determined
from the dose-response curves.
Values of SI indicate rate between IC50 and MIC after 14 days of
incubation of M. tuberculosis 331/88 and all MDR-TB strains.
4.4. Cytotoxicity evaluation
Cytotoxicity was determined only for three most anti-
mycobacterial effective esters 3g, 3i and 3k (Table 5). All three
compounds presented very good SI values for drug-sensitive
M. tuberculosis and with a partly exception of 3i good SI for MDR-TB.
If SI value ꢀ10, then it is considered to be promising for a further
screening [42]. The lowest SI for resistant strains could be caused by
4. Results
the lowest investigated concentration of 1 mmol/L.
4.1. Chemistry
5. Discussion
Eleven new salicylanilide esters were prepared in two steps
from salicylic acids and anilines to form salicylanilides, which were
esterified by the using of DCC. This method was described recently
[4,30]. The yields of esters (3) ranged from 43 to 82%.
The prepared esters are showing the high antituberculous
activity (the lowest MICs 0.25 mol/L). Generally, this series is more
m
active then starting salicylanilides [24]. Except 3j which is less
active and 3c, 3d, where the MIC values are analogous, esters are
more active on M. tuberculosis than salicylanilides, in some cases
MIC was decreased eight-fold (e.g. 3e, 3g, 3i). The effect of esteri-
fication on the activity against M. avium is ambivalent e in six cases
it resulted in increasing and in five cases in decreasing activity e
and toward M. kansasii 235/80 there were five esters more active
and two less active than salicylanilides.
Structureeactivity relationships against M. tuberculosis. On the
salicylic ring, relatively independent on the aniline ring substitution,
4-bromo/chloro substitutions are the most convenient for the anti-
mycobacterial activity; the lowest is the contribution of 5-Cl (but
these derivatives are still comparable or better than non-substituted
ones). The optimal moieties on the positions 3 and 4 of the aniline
ring are 4-CF3 (in combinationwith 4-substituted salicylic part of the
molecule)and3,4-diCl. Themonosubstitutionbychlorineonposition
3 or 4 provides a minimal benefit when compared with other ones.
The lipophility influences the activity, but the relationship is not
direct. In general, the most lipophilic derivatives 3g (logP 5.12), 3i
(4.92), 3k (5.19) and partly 3e (4.83) showed the highest anti-
mycobacterial activity, but the effects of substituents and their
positions are very important, too. The LogP values, that are the
logarithm of the partition coefficient for octan-1-ol/water, calcu-
lated using the program CS ChemOffice Ultra version 11.0 (Cam-
bridgeSoft, USA), range from 4.56 to 5.19.
4.2. Antimycobacterial activity
Esters 3 were evaluated against four mycobacterial strains
(Table 1). All compounds inhibited tested strains with MICs in the
range from 0.25 to 32
M. tuberculosis, least M. avium. Compounds 3g, 3i and 3k exhibited
an excellent activity against M. tuberculosis (MICs ꢃ 1 mol/L) and
3i and 3k towards two strains of M. kansasii with MICs up to
mol/L. All compounds have shown a very good MIC value. Three
mmol/L. The most susceptible strain was
m
2
m
compounds presented the same or a better activity than INH and all
esters were more efficient than para-aminosalicylic acid (PAS). N-
Acetyl-
L-phenylalanine alone is quite inactive in all assays (data not
shown).
Based on anti-TB activities, three esters were selected for next
assessment against MDR-TB strains. All compounds were effective
in the lowest used concentration of 1
mmol/L and the maximal level
was in one case 4 mol/L and therefore they exhibited a very good
m
efficacy (Table 2). 3i was evaluated as the most active compound,
closely followed by 3k. All these esters inhibited the growth of
XDR-TB strain Praha 131 with MICs of 1e2 mmol/L.
4.3. Antibacterial and antifungal assays
Prepared esters (3) were evaluated for their in vitro antibacterial
and antifungal activity.
The strategyto esterifysalicylanilides by N-acetyl-L-phenylalanine
to develop new antituberculotics is with the respect to MICs more
Tables 3 and 4 bring the results. Three compounds (3a, 3e, and
3g) showed no inhibition of growth in antibacterial assessment in
convenient than by acetic acid, especially against M. tuberculosis and
M. kansasii [30], lipophilic N-Cbz-
with a slight superiority in some cases with carbamates [31].
The MDR-TB results (MICs 1e2 mol/L) of 3g, 3k and 3i postulate
a-amino acids [27] and comparable
the concentration of ꢃ125
mmol/L. Tested compounds expressed
only a weak or no effect against Gram-negative bacteria, when
m
P. aeruginosa and EBSL-positive K. pneumoniae were completely
them to be promising candidates for other testing for MDR-TB
treatment and they are comparable, although the lowest tested
unaffected by these derivatives in the concentration of 125
and lower; E. coli was affected only by 3d and 3f (MIC 31.25 and
15.62 mol/L, respectively) and K. pneumoniae by 3d (62.5 mol/L).
Some of derivatives had a very good MIC value against Gram-
positive cocci with MIC of 0.98 mol/L, including MRSA and
S. epidermidis strains (3f, 3i; MIC 0.98 mol/L), or Enteroccocus (3d;
3.9 mol/L). The most susceptible strain was S. aureus.
In general, these esters have shown a significant antifungal
activity. Table 4 includes only compounds with at least one MIC
lower than125 mol/L (3a and 3g displayed no inhibition activity in
the MIC value of 125 mol/L). The higher activity against one or
mmol/L
concentrationwas 1
were published recently on the website LeadDiscovery [43]. SI of
esters 3 is favourable than N-Cbz- -amino acid ester [27] and for
mmol/L, to salicylanilide carbamates [31], which
m
m
a
m
M. tuberculosis 331/88 than carbamates [31]. When we compared 3i
and 3k, the second compound has almost the same antitubercular
efficacy as 3i, and SI is higher e these results make 3k optimal for
next investigation as a promising antimycobacterial agent.
m
m
For the activity against bacteria, in general, it seems that the most
convenient substituents are 40-CF3 (3i, 3k) and 40-Br (3f). When
compared substituents on salicylic ring, favourable are derivatives
with5-Clthan4-Clwith3i being anexception. Therefore some of these
esters could be potential agents against Gram-positive infections.
The antifungal activity, generally worse than antibacterial and
antimycobacterial activity when compared on the base of MICs, is
positively modulated by the presence of 4-chloro substitution of the
aniline moiety(3b, 3d) and 4-bromo substitutionof salicylic ring(3k).
However, the antifungal potential of esters 3 (expressed as MICs) is
not such hopeful when compared with the antimycobacterial and
m
m
more fungi presented in summary 3c, 3d, 3f and 3k. Candida strains
were less susceptible than moulds e the lowest MICs were 15.62
and 3.9 mmol/L, respectively. The major resistance demonstrated
C. glabrata, on the other hand, the most susceptible are A. cor-
ymbifera and T. mentagrophytes. Eight compounds exhibited activ-
ities against fluconazole-resistant A. fumigatus and A. corymbifera in
the range from 3.9 to 125
mmol/L and 3k showed only a slightly
poorer activity for T. mentagrophytes (3.91/7.81
mmol/L).

M. Krátký et al. / European Journal of Medicinal Chemistry 45 (2010) 6106e6113
6109
Table 1
Antimycobacterial activity of esters 3.
MIC [
R1
m
mol/L]
R2
M. tuberculosis
M. avium
330/88
14 d
M. kansasii
235/80
7 d
M. kansasii
6509/96
7 d
331/88
14 d
21 d
21 d
32
8
16
16
8
8
8
16
4
32
4
14 d
21 d
14 d
21 d
3a
3b
3c
4-Cl
5-Cl
4-Cl
5-Cl
4-Cl
5-Cl
4-Cl
5-Cl
4-Cl
5-Cl
4-Br
e
3-Cl
3-Cl
4-Cl
4-Cl
4-Br
4-Br
3,4-diCl
3,4-diCl
4-CF3
4-CF3
4-CF3
e
2
2
4
4
2
2
0.5
2
0.25
4
4
4
4
2
4
1
2
8
4
8
8
4
4
8
8
4
8
4
4
4
4
2
1
4
0.5
4
8
4
4
4
4
4
2
4
1
8
8
4
4
4
4
4
4
2
4
2
2
2
2
4
4
4
1
1
1
4
1
2
32
8
4
4
8
8
8
3d
3e
3f
8
4
4
16
8
8
3g
3h
3i
2
2
2
4
4
2
0.5
3j
8
16
16
4
>250
32
4
2
8
8
3k
INH
PAS
0.25
0.5
0.5
0.5e1
0.5
>250
125
2
4
125
2
4e8
500
>250
125
>250
1000
>250
>1000
e
e
62.5
62.5
Bold values represent lowest values of minimal inhibition concentration (MIC).
M. avium strain 330/88 resistant to INH, RIF, ofloxacin and ethambutol; INH e isoniazid; PAS e para-aminosalicylic acid.
antifungal activity. The introduction of N-acetyl-
is slightly less advantageous than esterification of salicylanilides with
N-Cbz- -amino acids [4] and significantly less advantageous than
L
-phenylalanine acyl
melting points were determined on a Melting Point machine B-540
(Büchi) apparatus using open capillaries and they are uncorrected.
Optical activities were measured on polarimeter ADP 220 BS (Bel-
lingham Stanley Ltd.). Infrared spectra (KBr pellets) were recorded
on FT-IR spectrometer Nicolet 6700 FT-IR in the range of
400e4000cmꢁ1. TheNMRspectraweremeasuredinCDCl3 orDMSO-
d6 solutions at ambient temperature on a Varian Mercury e Vxbb 300
(300 MHz for 1H and 75.5 MHz for 13C; Varian Comp. Palo Alto, USA).
a
with acetic acid [30].
In conclusion, salicylanilides N-acetyl-L-phenylalanine esters
were found to be a new highly active group promising a wide-range
antimicrobial activity.
The chemical shifts
d are given in ppm, related to tetramethylsilane
6. Experimental protocols
as an internal standard. The coupling constants (J) are reported in Hz.
Elemental analysis (C, H, N) were performed on an automatic
microanalyser CHNS-O CE instrument (FISONS EA 1110, Italy).
6.1. Chemistry
6.1.1. General methods
6.1.2. Synthetic procedure for (S)-2-(phenylcarbamoyl)phenyl
2-acetamido-3-phenylpropanoates
The halogenated (S)-2-(phenylcarbamoyl)phenyl 2-acetamido-
3-phenylpropanoates were prepared by a two-step synthesis
described previously by our group [30].
All used reagents and solvents were purchased from commercial
sources (SigmaeAldrich). Commercial grade reagents were used
without a further purification. Reactions were monitored by thin
layer chromatography plates coated with 0.2 mm silica gel 60 F254
(Merck), which were visualized by UV irradiation (254 nm). All
Table 2
Activity of selected esters against MDR strains.
MIC [mmol/L]
M. tuberculosis
M. tuberculosis
M. tuberculosis
M. tuberculosis
M. tuberculosis
M. tuberculosis
357/2005
53/2009
Praha 1
Praha 131
362/1998
9449/2007
14 d
21 d
14 d
21 d
14 d
21 d
14 d
21 d
14 d
21 d
14 d
21 d
3g
3i
2
1
2
2
1
1
2
1
1
1
2
2
2
1
2
2
1
1
2
1
2
2
4
2
3k
INH
2
14.6
2
14.6
1
14.6
2
14.6
1
14.6
2
14.6
2
14.6
2
14.6
1
14.6
1
14.6
1
58.3
2
58.3
Bold values represent lowest values of minimal inhibition concentration (MIC).
MDR-TB M. tuberculosis strains: 357/2005 and 362/1998 (both resistant to INH, RIF, rifabutine, streptomycin, ethambutol, and ofloxacine); 53/2009 (resistant to INH, RIF,
rifabutine, streptomycin, ethambutol); Praha 1 (resistant to INH, RIF, rifabutine, streptomycin, ethambutol and clofazimine); Praha 131 (resistant to INH, RIF, rifabutine,
streptomycin, ethambutol, ofloxacine, gentamicin and amikacin; XDR-TB strain); 9449/2007 resistant to INH, RIF, rifabutine, and streptomycin.

6110
M. Krátký et al. / European Journal of Medicinal Chemistry 45 (2010) 6106e6113
Table 3
Antibacterial activity of prepared esters.
MIC/IC80
SA
[m
mol/L]
MRSA
24 h
SE
EF
EC
KP
24 h
48 h
48 h
24 h
48 h
24 h
48 h
24 h
48 h
24 h
48 h
3b
3c
3d
3f
3h
3i
31.25
62.5
1.95
0.98
125
0.98
125
3.9
125
125
125
1.95
0.98
125
0.98
125
7.81
125
62.5
125
1.95
0.98
125
0.98
125
15.62
125
125
125
3.9
7.81
125
31.25
125
15.62
>125
>125
>125
125
>125
>125
>125
125
>125
>125
31.25
15.62
125
>125
>125
250
>125
>125
>125
125
>125
>125
>125
500
>125
>125
62.5
>125
>125
>125
>125
>500
>125
>125
125
>125
>125
>125
>125
>500
>125
15.62
1.95
>125
1.95
>125
31.25
7.81
>125
7.81
>125
31.25
1.95
>125
3.9
3j
3k
>125
31.25
>125
125
>125
62.5
Bold values represent lowest values of minimal inhibition concentration (MIC).
SA: Staphylococcus aureus CCM 4516/08; MRSA: methicillin-resistant Staphylococcus aureus H 5996/08; SE: Staphylococcus epidermidis H 6966/08; EF: Enterococcus sp. J 14365/
08; EC: E. coli CCM4517; KP: Klebsiella pneumoniae D 11750/08.
At first, starting halogenated salicylanilides (1) were prepared
routinely by the modified reaction of substituted salicylic acids and
appropriate anilines (in equivalent amounts; Scheme 1) in the
presence of PCl3 (0.5 equiv.) in chlorobenzene. The reaction was
carried out with vigorously stirring in a microwave reactor (530 W,
600 rpm, MicroSYNTH Milestone) for 20 min to reflux. This
procedure led to an increase of the yield and shortening the reac-
tion time from several hours to minutes. The reaction mixture was
filtered while hot, let stand at 20 ꢂC and then at 4 ꢂC for 24 h. The
crude product was filtered off and once or more times recrystallized
from aqueous ethanol to obtain the pure product.
CH2), 2.89 (1H, dd, J ¼ 14.0 Hz, J ¼ 10.0 Hz, CH2), 1.76 (3H, s, CH3). 13
C
NMR (75 MHz, DMSO): d 170.0, 169.8, 163.4, 148.4, 139.9, 137.6, 135.5,
133.2, 131.2, 130.6, 129.5, 129.1, 128.3, 126.8, 126.6, 124.0, 120.3, 119.4,
117.8, 53.9, 36.2, 22.3. Anal. Calcd. for C24H20Cl2N2O4 (471.33): C, 61.16;
H, 4.28; N, 5.94. Found: C, 61.34; H, 4.39; N, 6.01.
6.1.2.3. (S)-4-Chloro-2-(4-chlorophenylcarbamoyl)phenyl
2-acet-
amido-3-phenylpropanoate (3c). White solid; yield 52%; mp
164e166 ꢂC; ½a 2D3
ꢁ26.8 (c 0.77; ethyl acetate). IR (KBr pellet): 3305,
ꢄ
3066, 3031, 2927, 1751 (CO ester), 1651, 1595, 1526, 1493, 1403, 1371,
1313, 1197, 1107, 1015, 823, 697, 504. 1H NMR (300 MHz, DMSO):
Salicylanilide esters (3) with N-acetyl-
L
-phenylalanine (2) were
d
10.64 (1H, s, NH), 8.49 (1H, bs, NH) 7.90 (1H, d, J ¼ 2.6 Hz, H3),
obtained by the activation with N,N0-dicyclohexylcarbodiimide
(DCC) in N,N-dimethylformamide (DMF) (Scheme 1). The N-pro-
tected amino acid (2) and substituted salicylanilide (1) (both
0.001 mol) were dissolved in dry DMF (15 mL). This solution was
cooled to ꢁ15 ꢂC and DCC in a mild excess (0.0011 mol) was added
in three portions during 1 h. Next the mixture was stirred for 3 h at
the same temperature and stored at þ4 ꢂC for 48 h. The precipitate
of by-product N,N0-dicyclohexylurea was filtered off and the solvent
was evaporated in vacuo. The remnant was dissolved in a small
amount of ethyl acetate and the insoluble portion (another N,N0-
dicyclohexylurea) was filtered off. The filtrate was again evaporated
in vacuo. The crude product 3 was purified by the crystallization
(commonly once or twice) from ethyl acetateehexane.
7.78e7.73 (2H, m, H20, H60), 7.64 (1H, dd, J ¼ 8.6 Hz, J ¼ 2.6 Hz, H5),
7.47e7.37 (2H, m, H30, H50), 7.29e7.20 (6H, m, H6, H200, H300, H400,
H500, H600), 4.60 (1H, m, CH), 3.16 (1H, dd, J ¼ 14.0 Hz, J ¼ 4.6 Hz,
CH2), 2.85 (1H, dd, J ¼ 9.3 Hz, J ¼ 4.5 Hz, CH2), 1.76 (3H, s, CH3). 13
C
NMR (75 MHz, DMSO):
d 170.1, 169.8, 162.7, 146.5, 138.0, 137.3,
133.2, 131.5, 130.4, 129.5, 129.2, 129.1, 128.3, 126.7, 125.2, 122.4,
121.5, 53.9, 36.2, 22.3. Anal. Calcd. for C24H20Cl2N2O4 (471.33): C,
61.16; H, 4.28; N, 5.94. Found: C, 61.02; H, 4.50; N, 5.71.
6.1.2.4. (S)-5-Chloro-2-(4-chlorophenylcarbamoyl)phenyl
2-acet-
amido-3-phenylpropanoate (3d). White solid; yield 43%; mp
147.5e150 ꢂC; ½a D23
ꢁ16.3 (c 0.4; ethyl acetate). IR (KBr pellet): 3406,
ꢄ
3030, 2930, 1765 (CO ester), 1657, 1629, 1602, 1536, 1400, 1384, 1314,
1200, 1093, 829, 745, 700, 510. 1H NMR (300 MHz, DMSO):
d 10.59
6.1.2.1. (S)-4-Chloro-2-(3-chlorophenylcarbamoyl)phenyl
2-acet-
(1H, s, NH), 8.50 (1H, bs, NH), 7.89 (1H, d, J ¼ 8.6 Hz, H3), 7.76e7.72
(2H, m, H20, H60), 7.52 (1H, dd, J ¼ 8.3 Hz, J ¼ 2.1 Hz, H4), 7.44e7.40
(2H, m, H30, H50), 7.38 (1H, d, J ¼ 2.0 Hz, H6), 7.31e7.18 (5H, m, H200,
H300, H400, H500, H600), 4.54 (1H, m, CH), 3.17 (1H, dd, J ¼ 14.0 Hz,
J ¼ 4.7 Hz, CH2), 2.85 (1H, dd, J ¼ 9.8 Hz, J ¼ 3.4 Hz, CH2), 1.76 (3H, s,
amido-3-phenylpropanoate (3a). White solid; yield 63%; mp
143e145 ꢂC; ½a 2D3
ꢁ27.8 (c 0.72; ethyl acetate). IR (KBr pellet): 3297,
ꢄ
3062, 2933,1775 (CO ester), 1651, 1594,1544, 1481,1427, 1372,1320,
1200, 1105, 874, 779, 698, 681. 1H NMR (300 MHz, DMSO):
d 10.76
(1H, s, NH), 8.50 (1H, bs, NH), 7.92 (1H, t, J ¼ 1.8 Hz, H20), 7.78 (1H, d,
J ¼ 2.6 Hz, H3), 7.67 (1H, dd, J ¼ 8.7 Hz, J ¼ 2.6 Hz, H5), 7.59 (1H, d,
J ¼ 8.2 Hz, H6), 7.37 (1H, t, J ¼ 8.0 Hz, H50), 7.28e7.14 (7H, m, H60, H40,
H200, H300, H400, H500, H600), 4.61 (1H, m, CH), 3.17 (1H, dd, J ¼ 14.1 Hz,
J ¼ 4.6 Hz, CH2), 2.88 (1H, dd, J ¼ 14.0 Hz, J ¼ 10.0 Hz, CH2),1.76 (3H, s,
CH3). 13C NMR (75 MHz, DMSO):
d 170.0, 169.8, 163.3, 148.4, 138.0,
137.3, 136.4, 135.4, 131.1, 129.5, 129.2, 129.1, 128.3, 126.9, 126.8, 122.5,
121.5, 53.9, 36.2, 22.3. Anal. Calcd. for C24H20Cl2N2O4 (471.33): C,
61.16; H, 4.28; N, 5.94. Found: C, 61.44; H, 4.22; N, 6.17.
CH3). 13C NMR (75 MHz, DMSO):
d
170.1, 169.8, 162.9, 146.5, 140.4,
6.1.2.5. (S)-2-(4-Bromophenylcarbamoyl)-4-chlorophenyl2-acetamido-
137.3,133.2,131.6,131.4,130.6,130.4,129.5,129.1,128.3,126.8,125.2,
123.8, 119.5, 118.4, 53.9, 36.2, 22.3. Anal. Calcd. for C24H20Cl2N2O4
(471.33): C, 61.16; H, 4.28; N, 5.94. Found: C, 60.95; H, 4.41; N, 5.85.
3-phenylpropanoate (3e). White solid; yield 82%; mp 170.5e171.5 ꢂC;
½
a 2D3
ꢄ
ꢁ22.4 (c 0.67; ethyl acetate). IR (KBr pellet): 3303, 3064, 2928,
1767 (CO ester), 1654, 1592, 1529, 1489, 1394, 1314, 1197, 1105, 1072,
1011, 824, 748, 698, 506. 1H NMR (300 MHz, DMSO):
10.63 (1H, s,
d
6.1.2.2. (S)-5-Chloro-2-(3-chlorophenylcarbamoyl)phenyl
2-acet-
NH), 8.49 (1H, bs, NH) 7.90 (1H, d, J ¼ 2.7 Hz, H3), 7.71e7.66 (2H, m,
H20, H60), 7.56 (1H, dd, J ¼ 8.6 Hz, J ¼ 2.6 Hz, H5), 7.29e7.18 (8H, m, H6,
H30, H50, H200, H300, H400, H500, H600), 4.63 (1H, m, CH), 3.16 (1H, dd,
J ¼ 14.3 Hz, J ¼ 4.5 Hz, CH2), 2.85 (1H, dd, J ¼ 9.0 Hz, J ¼ 4.9 Hz, CH2),
amido-3-phenylpropanoate (3b). White solid; yield 43%; mp
177.5e179 ꢂC; ½a D23
ꢁ27.1 (c 0.7; ethyl acetate). IR (KBr pellet): 3300,
ꢄ
3065, 2930, 1777 (CO ester), 1651, 1594, 1542, 1493, 1426, 1372, 1198,
1109, 827, 780, 700, 503.1H NMR (300 MHz, DMSO):
d
10.63(1H, s, NH),
1.76 (3H, s, CH3). 13C NMR (75 MHz, DMSO):
d 170.1, 169.8, 162.8, 146.5,
8.52 (1H, bs, NH), 7.91 (1H, t, J ¼ 1.9 Hz, H20), 7.88 (1H, d, J ¼ 8.2 Hz, H3),
7.41e7.35 (3H, m, H4, H6, H50), 7.28e7.15 (7H, m, H40, H60, H200, H300,
H400, H500, H600), 4.63 (1H, m, CH), 3.17 (1H, dd, J ¼ 14.2 Hz, J ¼ 4.8 Hz,
138.4, 137.3, 133.2, 131.5, 130.4, 129.5, 129.2, 129.1, 128.3, 126.8, 125.2,
122.8, 121.9, 53.9, 36.2, 22.3. Anal. Calcd. for C24H20BrClN2O4 (515.78):
C, 55.89; H, 3.91; N, 5.43. Found: C, 55.59; H, 3.69; N, 5.62.

M. Krátký et al. / European Journal of Medicinal Chemistry 45 (2010) 6106e6113
6111
6.1.2.6. (S)-2-(4-Bromophenylcarbamoyl)-5-chlorophenyl 2-acetami-
do-3-phenylpropanoate (3f). White solid; yield 61%; mp
167e168.5 ꢂC; ½a D23
ꢁ11.7 (c 0.9; dimethyl sulfoxide). IR (KBr
ꢄ
pellet): 3292, 3241, 3083, 1750 (CO ester), 1653, 1602, 1521, 1488,
1391, 1370, 1313, 1197, 1143, 1074, 1013, 895, 815, 749, 697. 1H NMR
(300 MHz, DMSO): d 10.57 (1H, s, NH), 8.49 (1H, bs, NH), 7.73e7.66
(3H, m, H3, H20, H60), 7.55e7.49 (3H, m, H4, H30, H50), 7.30 (1H, d,
J ¼ 2.1 Hz, H6), 7.24e7.17 (5H, m, H200, H300, H400, H500, H600), 4.61 (1H,
m, CH), 3.16 (1H, dd, J ¼ 14.0 Hz, J ¼ 4.7 Hz, CH2), 2.88 (1H, dd,
J ¼ 13.6 Hz, J ¼ 10.3 Hz, CH2), 1.76 (3H, s, CH3). 13C NMR (75 MHz,
DMSO):
d 170.0, 169.8, 163.3, 148.4, 138.4, 137.3, 136.4, 131.8, 131.1,
129.5, 129.2, 128.9, 128.3, 126.8, 123.3, 122.8, 121.9, 53.9, 36.2, 22.3.
Anal. Calcd. for C24H20BrClN2O4 (515.78): C, 55.89; H, 3.91; N, 5.43.
Found: C, 56.08; H, 3.80; N, 5.51.
6.1.2.7. (S)-4-Chloro-2-(3,4-dichlorophenylcarbamoyl)phenyl 2-aceta-
mido-3-phenylpropanoate (3g). White solid; yield 69%; mp
160e162 ꢂC; ½a 2D3
ꢁ14.8 (c 0.73; ethyl acetate). IR (KBr pellet): 3308,
ꢄ
3251, 3083, 1748 (CO ester), 1647, 1578, 1543, 1513, 1471, 1379, 1304,
1196, 1149, 1104, 1030, 907, 788, 694, 652. 1H NMR (300 MHz,
DMSO):
d
10.77 (1H, s, NH), 8.50 (1H, bs, NH), 8.10 (1H, d, J ¼ 1.3 Hz,
H20), 7.85 (1H, d, J ¼ 2.7 Hz, H3), 7.80 (1H, d, J ¼ 2.7 Hz, H50),
7.71e7.60 (3H, m, H5, H6, H60), 7.28e7.17 (5H, m, H200, H300, H400, H500,
H600), 4.64 (1H, m, CH), 3.17 (1H, dd, J ¼ 14.0 Hz, J ¼ 4.8 Hz, CH2), 2.84
(1H, dd, J ¼ 9.5 Hz, J ¼ 4.3 Hz, CH2), 1.76 (3H, s, CH3). 13C NMR
(75 MHz, DMSO): d 170.1, 169.8, 162.9, 146.5, 138.5, 137.3, 136.4, 131.7,
130.8, 130.4, 129.5, 129.2, 128.9, 128.3, 126.9, 126.8, 125.6, 122.9, 121.2,
53.9, 36.2, 22.3. Anal. Calcd. for C24H19Cl3N2O4 (505.78): C, 56.99; H,
3.79; N, 5.54. Found: C, 56.77; H, 3.94; N, 5.76.
6.1.2.8. (S)-5-Chloro-2-(3,4-dichlorophenylcarbamoyl)phenyl 2-aceta-
mido-3-phenylpropanoate (3h). White solid; yield 43%; mp
145e146.5 ꢂC; ½a D23
ꢁ32.9 (c 0.73; ethyl acetate). IR (KBr pellet):
ꢄ
3373, 3302, 3082, 1954, 1760 (CO ester), 1657, 1591, 1514, 1474, 1375,
1305, 1199, 1156, 1082, 1053, 906, 818, 741, 668. 1H NMR (300 MHz,
DMSO):
d
10.72 (1H, s, NH), 8.50 (1H, bs, NH), 8.11 (1H, d, J ¼ 2.2 Hz,
H20), 7.85 (1H, d, J ¼ 8.3 Hz, H3), 7.52 (1H, dd, J ¼ 8.3 Hz, J ¼ 2.1 Hz,
H4), 7.43e7.37 (3H, m, H6, H50, H60), 7.28e7.17 (5H, m, H200, H300, H400,
H500, H600), 4.64 (1H, m, CH), 3.17 (1H, dd, J ¼ 14.0 Hz, J ¼ 4.8 Hz, CH2),
2.84 (1H, dd, J ¼ 14.3 Hz, J ¼ 4.8 Hz, CH2), 1.76 (3H, s, CH3). 13C NMR
(75 MHz, DMSO): d 170.0,169.8,163.5,148.4,138.6,137.3,135.7,131.2,
130.8, 129.5, 129.2, 128.9, 128.3, 126.9, 126.8, 125.8, 123.3, 121.9, 119.3,
53.9, 36.2, 22.3. Anal. Calcd. for C24H19Cl3N2O4 (505.78): C, 56.99; H,
3.79; N, 5.54. Found: C, 56.68; H, 3.83; N, 5.42.
6.1.2.9. (S)-4-Chloro-2-(4-(trifluoromethyl)phenylcarbamoyl)phenyl
2-acetamido-3-phenylpropanoate (3i). White solid; yield 59%; mp
132e134 ꢂC; ½a 2D3
ꢁ17.9 (c 0.7; ethyl acetate). IR (KBr pellet): 3289,
ꢄ
3030, 2925, 1764 (CO ester), 1650, 1603, 1537, 1413, 1374, 1317, 1102,
1067, 1017, 881, 823, 739, 696. 1H NMR (300 MHz, DMSO):
d 10.87
(1H, s, NH), 8.50 (1H, bs, NH), 7.95e7.90 (2H, m, H30, H50), 7.88 (1H,
d, J ¼ 2.7 Hz, H3), 7.80 (1H, d, J ¼ 2.6 Hz, H6), 7.75e7.65 (3H, m, H5,
H20, H60), 7.26e7.17 (5H, m, H200, H300, H400, H500, H600), 4.60 (1H, m,
CH), 3.16 (1H, dd, J ¼ 14.0 Hz, J ¼ 4.7 Hz, CH2), 2.84 (1H, dd,
J ¼ 13.3 Hz, J ¼ 4.0 Hz, CH2), 1.75 (3H, s, CH3). 13C NMR (75 MHz,
DMSO):
d 170.1, 169.8, 163.2, 146.5, 142.6, 137.3, 133.2, 131.7, 130.4,
129.5, 129.0, 128.3, 126.8, 126.6, 126.2, 125.2, 124.3, 119.9, 53.9, 36.2,
22.3. Anal. Calcd. for C25H20ClF3N2O4 (504.89): C, 59.47; H, 3.99; N,
5.55. Found: C, 59.73; H, 4.14; N, 5.65.
6.1.2.10. (S)-5-Chloro-2-(4-(trifluoromethyl)phenylcarbamoyl)phenyl
2-acetamido-3-phenylpropanoate (3j). White solid; yield 57%; mp
155e156.5 ꢂC; ½a D23
ꢁ29.9 (c 0.67; ethyl acetate). IR (KBr pellet):
ꢄ
3365, 3289, 2929, 1753 (CO ester), 1680, 1650, 1602, 1534, 1408,
1373, 1320, 1114, 1067, 1018, 909, 839, 741, 701. 1H NMR (300 MHz,

6112
M. Krátký et al. / European Journal of Medicinal Chemistry 45 (2010) 6106e6113
Table 5
Cytotoxicity and SI for selected esters.
R1
R2
PBMC IC50
(
mmol/L)
SI for M. tuberculosis 331/88
SI for M. tuberculosis (MDR-TB strains)
3g
3i
3k
4-Cl
4-Cl
4-Br
3,4-diCl
4-CF3
4-CF3
44
12.2
48.7
88
48.8
194.8
22e44
6.1e12.2
24.1e48.7
SI (selectivity index) ¼ IC50/MIC; MICs were chosen after 14 d of M. tuberculosis 331/88 and all MDR-TB incubation .
DMSO):
d: 10.82 (1H, s, NH), 8.49 (bs, 1H, NH), 7.95e7.91 (2H, m,
6.2.2. In vitro antibacterial susceptibility testing
H30, H50), 7.90 (1H, d, J ¼ 2.9 Hz, H3), 7.74e7.68 (2H, m, H20, H60),
7.53 (1H, dd, J ¼ 8.3 Hz, J ¼ 2.0 Hz, H4), 7.32 (1H, dd, J ¼ 2.0, H6),
7.28e7.17 (5H, m, H200, H300, H400, H500, H600), 4.63 (1H, m, CH), 3.16
(1H, dd, J ¼ 14.2 Hz, J ¼ 4.8 Hz, CH2), 2.85 (1H, dd, J ¼ 9.8 Hz,
The broth microdilution test M27-A [40] was used for the
investigation of in vitro antibacterial activity of esters (3) against
four Gram-positive and four Gram-negative strains: S. aureus,
methicillin-resistant S. aureus, S. epidermidis, Enterococcus sp.; E.
coli, K. pneumoniae, EBSL-positive K. pneumoniae, and P. aeruginosa.
These assays were performed in the Department of Medical and
Biological Sciences at the Faculty of Pharmacy, Charles University,
Hradec Králové. The method used was microdilution panel bouillon
method with twofold dilution of the compounds in Mueller-Hinton
bouillon (HiMedia Laboratories, India). The esters were dissolved in
DMSO and the final concentrations of the compounds ranged from
J ¼ 2.4 Hz, CH2),1.75 (3H, s, CH3). 13C NMR (75 MHz, DMSO):
d 170.0,
169.8, 163.7, 148.4, 142.2, 137.6, 135.6, 131.2, 129.5, 129.2, 129.1,
128.3, 126.7, 126.6, 126.5, 126.2, 124.0, 119.3, 53.9, 36.2, 22.3. Anal.
Calcd. for C25H20ClF3N2O4 (504.89): C, 59.47; H, 3.99; N, 5.55.
Found: C, 59.51; H, 3.86; N, 5.59.
6.1.2.11. (S)-4-Bromo-2-(4-(trifluoromethyl)phenylcarbamoyl)phenyl
2-acetamido-3-phenylpropanoate (3k). White solid; yield 55%; mp
500 to 0.49
mmol/L. Drug-free controls were included. The
155e157 ꢂC; ½a 2D3
ꢄ
ꢁ20.1 (c 0.67; ethyl acetate). IR (KBr pellet): 3349,
minimum inhibitory concentrations (MICs) were defined as 80%
(IC80) or higher reduction of growth in comparison with the control.
The determination of results was performed visually and photo-
metrically. The values of MICs were determined after 24 and 48 h of
incubation in the darkness at 35 ꢂC in a humid atmosphere.
3261, 3023, 2941,1747 (CO ester),1665,1644,1604,1530,1408,1372,
1322, 1204, 1163, 1066, 1018, 973, 833, 747, 699. 1H NMR (300 MHz,
DMSO):
d
10.86 (1H, s, NH), 8.49 (1H, bs, NH), 7.95e7.90 (3H, 0m, H03,
H30, H50), 7.82 (1H, d, J ¼ 2.5 Hz, H6), 7.75e7.69 (2H, m, H2 , H6 ),
7.58 (1H, dd, J ¼ 8.8 Hz, J ¼ 2.6 Hz, H5), 7.27e7.16 (5H, m, H200, H300,
H400, H500, H600), 4.60 (1H, m, CH), 3.16 (1H, dd, J ¼ 13.9 Hz, J ¼ 4.6 Hz,
6.2.3. In vitro antifungal susceptibility testing
CH2), 2.85 (1H, dd, J ¼ 13.8 Hz, J ¼ 10.7 Hz, CH2),1.75 (3H, s, CH3). 13
C
The broth microdilution test M27-Awas used for the investigation
of in vitro antifungal activity of esters (3) against four yeast strains: C.
albicans, C. tropicalis, C. krusei, C. glabrata, and four moulds: T. beigelii,
A. fumigatus, A. corymbifera, and T. mentagrophytes. Fluconazole was
used as a reference drug. The method used was microdilution panel
bouillon method with twofold dilution of the compounds in RPMI-
1640 with glutamine (Sevapharma, Czech Republic) buffered to pH
7.0 with 0.165 M of 3-morpholinopropane-1-sulphonic acid (Sigma,
Germany). Other conditions and testing workplace were the same as
for antibacterial assay, only for T. mentagrophytes the final MICs were
determined after 72 and 120 h of incubation.
NMR (75 MHz, DMSO):
d 170.0, 169.8, 163.1, 147.0, 142.6, 137.3,
134.6, 131.8, 129.5, 129.1, 128.3, 126.7, 126.6, 126.4, 126.2, 125.5,
124.5, 119.6, 53.9, 36.2, 22.3. Anal. Calcd. for C25H20BrF3N2O4
(504.89): C, 54.66; H, 3.67; N, 5.10. Found: C, 54.83; H, 3.88; N, 5.40.
6.2. Pharmacology
6.2.1. In vitro antimycobacterial susceptibility testing
All the prepared compounds were tested in vitro for their anti-
mycobacterial activity in the Laboratory for Tuberculosis in Ostrava
against M. tuberculosis 331/88 (H37Rv) (dilution of the strain was
10ꢁ3) and moreover for some non-tuberculous INH-resistant strains
e M. avium (330/88, resistant to INH, RIF, ofloxacin and ethambutol;
dilution 10ꢁ5) and M. kansasii (235/80, dilution 10ꢁ4). One strainwas
clinically isolated (M. kansasii 6509/96 in the dilution 10ꢁ5); other
strains were obtained from the Czech National Collection of Type
Cultures. The micromethod for the determination of the minimum
inhibitory concentration (MIC) was used. Antimycobacterial activi-
6.2.4. In vitro cytotoxicity assay
Cytotoxicity was determined on human peripheral blood
mononuclear cells. The cells were cultured in RPMI-1640 medium
with 10% fetal calf serum, 2 mM L-glutamine and 160 mg/mL of
gentamicin. Cell cultures were maintained at 37 ꢂC, 5% CO2 in
water-saturated atmosphere. Cells were plated into 96-well plate
with initial cell number of 1.5e2.0 ꢅ 105 cells per well. After 24 h
ꢀ
ties were determined in the Sula semisynthetic medium (SEVAC,
incubation cells were treated with compounds in 100 mL serum free
Czech Republic). The tested compounds were added to the medium
as solutions in dimethylsulfoxide (DMSO). The following concen-
trations were used: 1000, 500, 250, 125, 62, 32, 16, 8, 4, 2, 1, 0.5, and
medium overnight. Controls were included. Four parallel
measurements were performed.
After incubation the cell viability was determined by MMT-assay
[41]. 45 mL MTT-solution (2 mg/mL) was added to each well [44]. After
0.25
m
mol/L. The MICs were determined after incubation at 37 ꢂC for
14 and 21 days, for M. kansasii additionally for 7 days. MIC (
m
mol/L)
4 h of incubation cells were centrifuged for 5 min (2000 rpm) and
supernatant was removed. The obtained formazan crystals were
was the lowest concentration at which the inhibition of mycobac-
terial growth occurred. As reference compounds were chosen
isoniazid and PAS.
The most active compounds 3g, 3i and 3k were evaluated
against MDR-TB in the similar conditions using six M. tuberculosis
strains (dilution 10ꢁ3) with different resistance patterns: 357/2005,
362/1998, 53/2009, Praha 1, Praha 131 (XDR-TB strain), and 9449/
2007. All strains are resistant to INH, RIF, rifabutine and strepto-
mycin, in some cases is present other resistance. The following
concentrations were used: 1000, 500, 250, 125, 62, 32, 16, 8, 4, 2,
dissolved in 50 or 100
mL DMSO and optical density (OD) was
measured at 540 and 620 nm using ELISA Reader (iEMS Reader,
Labsystems, Finland). OD620 values were subtracted from OD540
values. Cytotoxicity was calculated using the following equation:
cytotoxicity (%) ¼ [1 ꢁ (ODtreated/ODcontrol)] ꢅ 100; where ODtreated
and ODcontrol correspondtotheopticaldensities of the treated and the
control cells, respectively. Two independent experiments were
carried out with parallel measurements. IC50 values were determined
from the dose-response curves (defined using MicrocalTM Origin1
software, version 6.0).
and 1 mmol/L.

M. Krátký et al. / European Journal of Medicinal Chemistry 45 (2010) 6106e6113
6113
[20] A. Wright, M. Zignol, Anti-tuberculosis drug resistance in the world (fourth
global report) [online] Available from: http://www.who.int/tb/publications/
2008/drs_report4_26feb08.pdf (2008) (cited 06.04.10.).
Values of SI indicate rate between IC50 and MIC after 14 days of
incubation of M. tuberculosis 331/88 and all MDR-TB strains.
[21] A. Jain, P. Dixit, Multidrug resistant to extensively drug resistant tuberculosis:
what is next? J. Biosci. 33 (2008) 605e616.
ꢀ
Acknowledgments
[22] J. Vinsová, M. Krátký, Tuberculosis e the Development of New MDR-TB Drugs.
in: S. Ngụy, Z. K’ung (Eds.), Drug-resistant Tuberculosis: Causes, Diagnosis and
Treatment. Nova Science Publishers, New York, 2009 (Chapter 3).
[23] B.E. Laughon, New tuberculosis drugs in development, Curr. Top. Med. Chem.
7 (2007) 463e473.
This work was financially supported by GAUK 27610/2010, the
Research project MSM 0021620822, IGA NS 10367-3 and SVV-2010-
261-001.
ꢀ
ꢀ
[24] K. Waisser, O. Bures, P. Holý, J. Kunes, R. Oswald, L. Jirásková, M. Pour,
ꢀ
V. Klimesová, L. Kubicová, J. Kaustová, Relationship between the structure and
antimycobacterial activity of substituted salicylanilides, Arch. Pharm. Pharm.
Med. Chem. 1 (2003) 53e71.
References
ꢀ
ꢀ
ꢀ
[25] K. Waisser, J. Matyk, H. Divisová, P. Husáková, J. Kunes, V. Klimesová,
J. Kaustová, U. Mollman, H.M. Dahse, M. Miko, The oriented development of
antituberculotics: salicylanilides, Arch. Pharm. Chem. Life Sci. 339 (2006)
616e620.
ꢀ
[1] J. Vinsová, A. Imramovský, Salicylanilidy e stále aktuální skupina s potenciální
antibakteriální aktivitou, Cesk. Slov. Farm 53 (2004) 294e299.
[2] M.J. Macielag, J.P. Demers, S.A. Fraga-Spano, D.J. Hlasta, S.G. Johnson,
R.M. Kanojia, R.K. Russel, Z.H. Sui, M.A. Weidner-Wells, H. Werblood,
B.D. Foleno, R.M. Goldschmidt, M.J. Loeloff, G.C. Webb, J.F. Barrett, Substituted
salicylanilides as inhibitors of two-component regulatory systems in bacteria,
J. Med. Chem. 41 (1998) 2939e2945.
[3] G.S. Hassan, G.H. Hegazy, H.M. Safwat, Synthesis of furo-salicylanilides and
their heterocyclic derivatives with anticipated molluscicidal activity, Arch.
Pharm. Chem. Life Sci. 339 (2006) 448e455.
ꢀ
ꢀ
ꢀ
[26] A. Imramovský, J. Vinsová, J.M. Férriz, J. Kunes, M. Pour, M. Dolezal, Salicyla-
nilide esterification: unexpected formation of novel seven-membered rings,
Tetrahedron Lett. 29 (2006) 5007e5011.
ꢀ
ꢀ
[27] A. Imramovský, J. Vinsová, J.M. Férriz, R. Dolezal, J. Jampílek, J. Kaustová,
F. Kunc, New Antituberculotics originated from salicylanilides with prom-
ising in vitro activity against atypical Mycobacterial strains, Bioorg. Med.
Chem. 17 (2009) 3572e3579.
[28] I.M. Gould, Antibiotic resistance: the perfect storm, Int. J. Antimicrob. Agents
34 (2009) S2eS5.
ꢀ
[4] A. Imramovský, J. Vinsová, J.M. Férriz, V. Buchta, J. Jampílek, Salicylanilide
[29] J. Morschhäuser, Regulation of multidrug resistance in pathogenic fungi,
Fungal Genet. Biol. 47 (2010) 94e106.
esters of N-protected amino acids as novel antimicrobial agents, Bioorg. Med.
Chem. Lett. 19 (2009) 348e351.
[30] J. Vinsova, A. Imramovsky, V. Buchta, M. Ceckova, M. Dolezal, F. Staud,
J. Jampilek, J. Kaustova, Salicylanilide acetates: synthesis and antibacterial
evaluation, Molecules 12 (2007) 1e12.
[5] C. Liechti, U. Séquin, G. Bold, P. Furet, T. Meyer, P. Traxler, Salicylanilides as
inhibitors of the protein tyrosine kinase epidermal growth factor receptor,
Eur. J. Med. Chem. 39 (2004) 11e26.
[6] W. Deng, Z. Guo, Y. Guo, Z. Feng, Y. Juany, F. Chu, Acryloylamino-salicylanilides
as EGFR PTK inhibitors, Bioorg. Med. Chem. Lett. 16 (2006) 469e472.
[7] X.F. Zhu, J.S. Wang, L.L. Cai, Y.X. Zeng, D. Yang, SUCI02 inhibits the erbB-2
tyrosine kinase receptor signaling pathway and arrests the cell cycle in G1
phase in breast cancer cells, Cancer Sci. 97 (2006) 84e89.
ꢀ
ꢀ
[31] J.M. Férriz, K. Vávrová, F. Kunc, A. Imramovský, J. Stolaríková, E. Vavríková,
ꢀ
J. Vinsová, Salicylanilide carbamates: antitubercular agents active against
multidrug-resistant Mycobacterium tuberculosis strains, Bioorg. Med. Chem.
18 (2010) 1054e1061.
[32] J.J. Hilliard, R.M. Goldschmidt, L. Licata, E.Z. Baum, K. Bush, Multiple mecha-
nisms of action for inhibitors of histidine protein kinases from bacterial two-
component systems, Antimicrob. Agents Chemother. 43 (1999) 1693e1699.
[8] S. Kamath, J.K. Buolamwini, Targeting EGFR and HER-2 receptor tyrosine
kinases for cancer drug discovery and development, Med. Res. Rev. 26 (2006)
569e594.
ꢀ
ꢀ
[33] K. Waisser, M. Machácek, H. Dostál, J. Gregor, L. Kubicová, V. Klimesová,
ꢀ
ꢁ
J. Kunes, K. Palát, J. Hladuvková, J. Kaustová, U. Mollmann, Relationships
between the chemical structure of substances and their antimycobacterial
activity against atypical strains. Part 18. 3-Phenyl-2H-1,3-benzoxazine-2,4
(3H)-diones and isosteric 3-phenylquinazoline-2,4-(1H,3H)-diones, Collect.
Czech. Chem. Commun. 24 (1999) 1902e1924.
[9] M.R. Edwards, N.W. Bartlett, D. Clarke, M. Birrell, M. Belvisi, S.L. Johnston,
Targeting the NF-
kB pathway in asthma and chronic obstructive pulmonary
disease, Pharmacol. Ther. 121 (2009) 1e13.
[10] M.E. Brown, J.N. Fitzner, T. Stevens, W. Chin, C.D. Wright, J.P. Boyce, Salicy-
lanilides: selective inhibitors of interleukin-12p40 production, Bioorg. Med.
Chem. 16 (2008) 8760e8764.
[11] A. Sugita, H. Ogawa, M. Azuma, S. Muto, A. Honjo, H. Yanagawa, Y. Nishioka,
K. Tani, A. Itai, S. Sone, Antiallergic and anti-inflammatory effects of a novel
IkB kinase b inhibitor, IMD-0354, in a mouse model of allergic inflammation,
Int. Arch. Allergy Immunol. 148 (2009) 186e198.
[12] J.P. Boyce, M.E. Brown, W. Chin, J.N. Fitzner, R.J. Paxton, M. Shen, T. Stevens,
[34] Y. Cui, Y. Dang, Y. Yang, R. Ji, Synthesis of novel oxazolidinone derivatives for
antibacterial investigation, Curr. Sci. 89 (2005) 531e534.
[35] M.K. Dahlgren, A.M. Kauppi, I.M. Olsson, A. Linusson, M. Elofsson, Design,
synthesis, and multivariate quantitative structureeactivity relationship of
salicylanilides e potent inhibitors of type III secretion in Yersinia, J. Med.
Chem. 50 (2007) 6177e6188.
[36] D.J. Hlasta, J.P. Demers, B.D. Foleno, S.A. Fraga-Spano, J. Guan, J.J. Hilliard,
M.J. Macielag, K.A. Ohemeng, C.M. Sheppard, Z.H. Sui, G.C. Webb,
M.A. Weidner-Wells, H. Werblood, J.F. Barrett, Novel inhibitors of bacterial
two-component systems with Gram positive antibacterial activity: pharma-
cophore identification based on the screening hit closantel, Bioorg. Med.
Chem. Lett. 8 (1998) 1923e1928.
M.F. Wolfson, C.D. Wright, Identification of 14-3-3z by chemical affinity with
salicylanilide inhibitors of interleukin-12p40 production, Bioconjugate Chem.
19 (2008) 1775e1784.
[13] G.X. Zhong, L.L. Chen, J. Li, W.X. Hu, M.Y. Qi, Synthesis and anti-inflammatory-
analgesic activity of 20,40-difluoro-3-(carbamoyl)biphenyl-4-yl benzoates,
Chin. Chem. Lett. 19 (2008) 1419e1422.
[37] R. De La Fuente, N.D. Sonawane, D. Arumainayagam, A.S. Verkman, Small
molecules with antimicrobial activity against E. coli and P. aeruginosa iden-
tified by high-throughput screening, Br. J. Pharmacol. 149 (2006) 551e559.
[38] I.M.C. Ienacu, A.X. Lupea, D. Hadaruga, N. Hadaruga, I.M. Popescu, The anti-
microbial activity and quantitative structure e biological activity relation-
ships evaluation of some novel 2-hydroxybenzamide derivatives, Rev. Chim.
59 (2008) 247e250.
[14] K. Sudo, Y. Matsumoto, M. Matsushima, K. Konno, K. Shimotohno, S. Shigeta,
T. Yokota, Novel hepatitis C virus protease inhibitors: 2,4,6-trihydroxy, 3-nitro-
benzamide derivatives, Antiviral Chem. Chemother. 8 (1997) 541e544.
[15] C.J. Wu, J.T. Jan, C.M. Chen, H.P. Hsieh, D.R. Hwang, H.W. Liu, C.Y. Liu,
H.W. Huang, S.C. Chen, C.F. Hong, R.K. Lin, Y.S. Chao, J.T.A. Hsu, Inhibition of
severe acute respiratory syndrome coronavirus replication by niclosamide,
Antimicrob. Agents Chemother. 48 (2004) 2693e2696.
[16] X.W. Zhang, Y.L. Yap, Old drugs as lead compounds for a new disease? Binding
analysis of SARS coronavirus main proteinase with HIV, psychotic and parasite
drugs, Bioorg. Med. Chem. 12 (2004) 2517e2521.
ꢀ
[39] A. Imramovský, J.M. Férriz, K. Pauk, M. Krátký, J. Vinsová, A synthetic route for
the preparation of 2-hydroxy-N-(1-oxo-(phenylamino)-alkan-2-yl)benza-
mides, J. Comb. Chem. 12 (2010) 414e416.
[40] National Committee for Clinical Laboratory Standards, Reference Method for
Broth Dilution Antifungal Susceptibility Testing of Yeast, Approved Standard
NCCLS document, M27-A, Villanova, NCCLS (1997).
[41] T. Mosmann, Rapid colorimetric assay for cellular growth and survival:
application to proliferation and cytotoxicity assays, J. Immunol. Methods
65 (1983) 55e63.
[17] Y. Liu, P.L. Donner, J.K. Pratt, W.W. Jiang, T. Ng, V. Gracias, S. Baumeister,
P.E. Wiedeman, L. Traphagen, U. Warrior, C. Maring, W.M. Kati, S.W. Djuric,
A. Molla, Identification of halosalicylamide derivatives as a novel class of
allosteric inhibitors of HCV NS5B polymerase, Bioorg. Med. Chem. Lett. 18
(2008) 3173e3177.
[18] H.C. Li, C. Wang, T. Sanchez, Y.M. Tan, C.Y. Jiang, N. Neamati, G.S. Zhao, Amide-
containing diketoacids as HIV-1 integrase inhibitors: synthesis, structur-
eeactivity relationship analysis, and biological activity, Bioorg. Med. Chem. 17
(2009) 2913e2919.
[42] http://www.taacf.org/Process-text.htm (cit. 21.04.10.).
[43] http://www.leaddiscovery.co.uk/search/?search¼tuberculosis&;
FilterType¼Articles&Submit¼Search&type¼2&page¼5 (cit. 21.04.10.).
[44] F. Denizot, R. Lang, Rapid colorimetric assay for cell-growth and survival e
modifications to the tetrazolium dye procedure giving improved sensitivity
and reliability, J. Immunol. Methods 89 (1986) 271e277.
[19] V. Calderone, A. Coi, F.L. Fiamingo, I. Giorgi, M. Leonardi, O. Livi, A. Martelli,
E. Martinotti, Structural modifications of benzanilide derivatives, effective
potassium channel openers. X, Eur. J. Med. Chem. 41 (2006) 1421e1429.