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

Article abstract of DOI:10.1016/j.ejmech.2012.07.044

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

Full text of DOI:10.1016/j.ejmech.2012.07.044

European Journal of Medicinal Chemistry 56 (2012) 387e395
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Short communication
Synthesis and in vitro antimycobacterial activity of 2-methoxybenzanilides
and their thioxo analogues
a
b
c
d
c
a,
*
ꢀ
ꢀ
ꢀ
ꢀ
Ján Kozic , Eva Novotná , Marie Volková , Jirina Stolaríková , Frantisek Trejtnar , Jarmila Vinsová
a Charles University in Prague, Faculty of Pharmacy in Hradec Králové, Department of Inorganic and Organic Chemistry, Heyrovského 1203, 500 05 Hradec Králové, Czech Republic
b Charles University in Prague, Faculty of Pharmacy in Hradec Králové, Department of Biochemical Sciences, Heyrovského 1203, 500 05 Hradec Králové, Czech Republic
c Charles University in Prague, Faculty of Pharmacy in Hradec Králové, Department of Pharmacology and Toxicology, Heyrovského 1203, 500 05 Hradec Králové, Czech Republic
d Laboratory for Mycobacterial Diagnostics and TB, Institute of Public Health in Ostrava, Partyzánské nám. 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:
A new series of N-(3/4-substituted phenyl) 4/5-chloro-2-methoxybenzamides and their thioxo analogues
have been synthesised and evaluated for in vitro antimycobacterial activity against Mycobacterium
tuberculosis H37Rv, as well as the two atypical strains Mycobacterium kansasii and Mycobacterium avium.
Five of the most active compounds were evaluated for cytotoxicity and their ability to inhibit myco-
bacterial isocitrate lyase, which is responsible for latent survival of Mycobacterium. The results showed
that benzthioanilides were more active than the corresponding benzanilides. The most active compound,
4-chloro-2-methoxy-N-(3,4-dichlorophenyl)benzothioamide (4e), had a minimal inhibition concentra-
Received 4 January 2012
Received in revised form
23 July 2012
Accepted 26 July 2012
Available online 4 August 2012
Keywords:
Tuberculosis
Benzanilide
Benzthioanilides
Antimycobacterial activity
Isocitrate lyase
tion (MIC) against M. tuberculosis of 2
m
mol Lꢀ1, which was better than the activity of the previously
published corresponding salicylanilide.
Ó 2012 Elsevier Masson SAS. All rights reserved.
1. Introduction
[1]. Although the absolute number of TB cases per year is slightly
decreasing globally, the number of MDReTB cases is increasing [1].
Recently, several pathways have been characterised as new
possible drug targets, including cell wall metabolism, cellular
respiration and protein processing enzymes [4]. One promising
enzyme target in persistent and latent mycobacterial infection is
isocitrate lyase (ICL), which is a key enzyme in the glyoxylate cycle
essential for growth in macrophages. During steady state growth,
ICL converts isocitrate to succinate and glyoxylate, followed by
condensation of glyoxylate with acetyl-CoA to form malate by
malate synthase [5]. The carbon conserving glyoxylate pathway has
not been observed in the human body, therefore it has been
selected as a potential drug target for new antituberculotic agents
[6].
The need for new antituberculotics, especially ones with a new
mode of action, demands intensive search for new compounds.
Salicylanilides could fit this description, as they act on the bacterial
two component system (TCS), which is not found in animal cells
and is not a target for any current antituberculotic drugs [7e9].
They are currently well-known antibacterial [7] and anti-
mycobacterial agents [10,11], but are too toxic for animal cells
because they decouple oxidative phosphorylation in mitochondria.
The phenolic OH group is responsible for this action, which seems
to also be essential for antibacterial activity [7].
In the last few years, tuberculosis (TB), caused by Mycobacterium
tuberculosis, has again become one of the most dangerous and
lethal infectious diseases. According to the new WHO report in
2010, there were an estimated 8.8 million new cases of TB, 1.45
million TB deaths and more than two billion people infected with
M. tuberculosis, of which approximately 10% will most likely
become ill during their life [1]. This situation is due to HIV infection
(1.1 million of all TB causes and 0.35 million of TB deaths are in
people who are HIV positive) [1], poor compliance of patients,
population migration and increased drug resistance [2]. A partic-
ularly dangerous form of drug-resistant TB is multidrug-resistant
tuberculosis (MDReTB), caused by a strain of M. tuberculosis
resistant to at least rifampicin and INH (isoniazid), the first line and
most powerful antituberculotics, and extensively drug-resistant
tuberculosis (XDReTB), caused by the strains of M. tuberculosis
also resistant to any fluoroquinolone and to at least one of the three
injectable drugs kanamycin, capreomycin and amikacin [3]. For
example, in 2010, there was an estimated 650,000 cases of
MDReTB, and 150,000 died from infection with this form in 2008
* Corresponding author.
E-mail address: vinsova@faf.cuni.cz (J. Vinsová).
ꢀ
0223-5234/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2012.07.044

388
J. Kozic et al. / European Journal of Medicinal Chemistry 56 (2012) 387e395
On the other hand there is little information on the anti-
Department of Pharmacology and Toxicology, Faculty of Pharmacy
in Hradec Králové, Charles University in Prague. For this purpose,
the human liver cell line Hep G2 and a standard colorimetric test
based on the reductive metabolic activity of the cells was used. The
results are presented as the inhibitory concentration (IC50), which
is the concentration of the tested compound required for reduction
of cell viability by 50% of the maximum (control) cell viability. To
characterise the relationship between toxicity and anti-
mycobacterial activity, the selectivity index (SI) was calculated. The
SI is the ratio of IC50 to MIC after 14 days of incubation with
M. tuberculosis 331/88.
mycobacterial properties of benzanilides, the possible metabolites
of benzoxazoles [12]. According to Weidner-Wells et al. [13], who
studied TCS inhibition by benzimidazoles and their isosteres ben-
zoxazoles, neither the benzimidazole hydrogen bond donating
ability nor the basicity of benzimidazole was necessary for their
activity. In addition, they showed that benzoxazoles are also active
even though the oxygen can only accept a hydrogen bond. Thus, it is
possible that a hydrogen bond acceptor at the right position is
sufficient for activity.
We prepared a series of 2-methoxybenzanilides and their thioxo
analogues as the opened reverse form of 2-phenylbenzo[d]oxa-
zoles, expecting that these compounds would show good activity
even if they lacked a 2-OH group (Scheme 1).
4. Discussion and conclusion
Thirty-six novel compounds were synthesised and charac-
terised by 1H and 13C NMR spectroscopy, IR spectroscopy, elemental
analysis and melting point. The yields of thiobenzanilides were
significantly lower (4.9e54.0%) than the yields of benzanilides
(40.7e75.3%). This discrepancy was most likely due to the large
excess of P4S10 and long reaction time which were needed for
complete conversion of amides to thioamides to simplify their
isolation. It was possible that the P4S10 formed some complex with
the carbonyl/thiocarbonyl group of amides/thioamides in the
similar way as Lewis acids with carbonyl groups of products of the
FriedeleCrafts acylation.
2. Chemistry
Scheme 2 shows the preparation of substituted 2-metho-
xybenzanilides and their thioxo analogues. The substituents on
both aromatic parts of the molecule were selected according to
a previous study on salicylanilides [10]. For both synthetic steps, well
known preparations were used: the reaction of substituted 2-
methoxybenzoic acid with an appropriate aniline in the presence
of PCl3 and chlorobenzene as solvent [10], and thionation with P4S10
in pyridine as solvent [14].
All compounds showed in vitro activity against all tested strains,
m
mol Lꢀ1 (Table 1) and thio-
3. Pharmacology
with amides in the range of 4e500
amides in the range of 2e250
m
mol Lꢀ1 (Table 2). In general, thio-
3.1. In vitro antimycobacterial assay
amides were more efficient than benzamides, but all prepared
compounds were less active than their corresponding salicylani-
lides, except 3c, 3g and 4e. For a comparison of the anti-
mycobacterial activities of prepared compounds 3 and 4 and
salicylanilides, see Tables 1e3 [10].
In the thiobenzanilides, slightly higher activity against atypical
strains of Mycobacterium was found for compounds having Cl, Br or
CF3 groups on the aniline and a 5-Cl substitution on the acyl aro-
mate (Table 2). Conversely, the activity of the thioamides against
M. tuberculosis was better if the Cl was in position 4 in the acyl part
of the molecule (Table 2). Most of the thioamides exhibited activ-
ities against M. avium and M. kansasii 235/80 which are resistant to
INH (Table 2).
The newly prepared compounds were tested in the Laboratory
for Mycobacterial Diagnostics and TB at the Institute of Public
Health in Ostrava for their in vitro antimycobacterial activity against
M. tuberculosis 331/88, Mycobacterium avium 330/88, Mycobacte-
rium kansasii 235/80 and clinically isolated M. kansasii 6509/96. The
first line antituberculotic drug INH was used as a standard. The
compound activity against M. tuberculosis and M. avium was eval-
uated after 14 and 21 days, and the compound activity against
M. kansasii was evaluated after 7, 14 and 21 days. The minimal
inhibition concentration (MIC) values represent the lowest
concentration of the tested compounds at which the inhibition of
mycobacterium growth was observed.
The results of in vitro enzymatic assays of the most active
compounds showed that the tested derivatives were not effective
isocitrate lyase inhibitors. The best inhibition activity was found for
4d with 39 ꢁ 5% inhibition. Inhibition by other compounds was
within the range 17e27% (Table 4).
3.2. In vitro enzymatic assay of selected compounds
Five of the most active compounds (4c, 4d, 4e, 4g, 4h) against
M. tuberculosis 331/88 after 14 days of incubation were tested for
their ability to act as in vitro inhibitors of mycobacterial isocitrate
lyase. This evaluation was performed at the Department of
Biochemical Sciences, Faculty of Pharmacy in Hradec Králové,
Charles University in Prague. The results are presented as % of
inhibition and compared to the standard 3-nitropropionic acid.
The best compounds in the cytotoxicity assay were 4c, with an
IC50 of 11.68 m
mol Lꢀ1 and an SI for M. tuberculosis after 14 days of
incubation of 2.92, and 4e, with an IC50 of 6.50 m
mol Lꢀ1 and an SI
for My 331/88 after 14 days of 3.25 (Table 5).
Despite the prepared compounds showing antimycobacterial
activity, use of the 2-methoxy group instead of the 2-hydroxy group
decreased activity and in vitro cytotoxicity did not improve. The
best compound in the cytotoxicity assay was 4e, which was also
more active against M. tuberculosis after 14 days than the corre-
sponding salicylanilide. Compounds 3c and 3g were more active
against M. avium than the corresponding salicylanilides.
3.3. In vitro cytotoxicity assay of selected compounds
Compounds 4c, 4d, 4e, 4g and 4h were also tested for their
in vitro cytotoxicity. This evaluation was performed at the
Scheme 1. 2-Methoxybenzanilides as the opened reverse form of 2-phenylbenzo[d]oxazoles.

J. Kozic et al. / European Journal of Medicinal Chemistry 56 (2012) 387e395
389
Scheme 2. Synthesis of 2-methoxybenzanilides 3 and their thioxo analogues 4.
5. Experimental protocols
for 13C, Varian Co., Palo Alto, CA, USA) and Varian Mercury (500 MHz
for 1H and 125 MHz for 13C, Varian Co., Palo Alto, CA, USA) instru-
All of the chemicals and solvents used in this study were
purchased from SigmaeAldrich, Prague, Czech Republic and Penta,
Prague, Czech Republic, and were used without further purification.
ments. The chemical shifts (d) are given in ppm and are indirectly
related to tetramethylsilane as an internal standard. Elemental
analysis (C, H, N, S) was performed on an automatic microanalyser
CHNSeO CE instrument, FISONS EA 1110 (Thermo Fisher Scientific,
Waltham, MA, USA). MS measurement was performed on an Agilent
500 Ion Trap LC/MS mass spectrometer using APCI ionization tech-
nique (Agilent Technologies, Santa Clara, CA, USA). For MS sample
preparation was used methanol.
5.1. Chemistry
5.1.1. General methods
The reactions were monitored and the purity of products was
verified by thin layer chromatography using plates coated with
0.2 mm silica gel 60 F254 (Merck KGaA, Darmstadt, Germany) and
visualised using UV irradiation (254 and 366 nm). Column chro-
matography was performed using silica gel 60 with a particle size of
0.063e0.2 mm (Fluka, Prague, Czech Republic).
5.1.2. General procedure for the preparation of substituted 2-
methoxybenzamides
Equimolar amounts of 4/5-chloro-2-methoxybenzoic acid
(5.0 mmol), substituted aniline (5.0 mmol) and PCl3 (5.0 mmol)
were dissolved in 20.0 mL of chlorobenzene and stirred at 165 ꢂC
for 3 h. Then, the mixture was immediately filtered, evaporated and
the residue was recrystallised from 96% EtOH/H2O.
Melting points were determined on a Melting Point B-540 appa-
}
ratus (Buchi Labortechnik AG, Flawil, Switzerland) in open capillaries
andare uncorrected. IR spectrawere recordedona Nicolet6700 FT-IR
spectrometer (Thermo Fisher Scientific, Waltham, MA, USA) over the
range 400e4000 cmꢀ1 using the ATR technique. The NMR spectra
were measured in DMSO-d6 or CDCl3 solutions at ambient temper-
ature on a Varian Mercury Vxbb 300 (300 MHz for 1H and 75.5 MHz
5.1.2.1. 4-Chloro-2-methoxy-N-(3-nitrophenyl)benzamide (3a). Yield:
64.5%, yellow solid; m. p. 185e186 ꢂC; IR (ATR): 3331 (
n
NH), 3071
(n
CH aromatic), 1670 (
n
CO þ
d
NH, amide I), 1590 (n CC aromatic),
Table 1
Antimycobacterial activities of 2-methoxybenzanilide derivatives 3.
R1
R2
MIC [
M. tuberculosis My 331/88
m ]
mol Lꢀ1
M. avium My 330/88
M. kansasii My 235/80
M. kansasii My 6509/96
14d
21d
14d
21d
7d
14d
21d
7d
14d
21d
3a
3b
3c
3d
3e
3f
3g
3h
3i
3j
3k
3l
3m
3n
3o
3p
3q
3r
4-Cl
4-Cl
4-Cl
4-Cl
4-Cl
4-Cl
4-Cl
4-Cl
4-Cl
5-Cl
5-Cl
5-Cl
5-Cl
5-Cl
5-Cl
5-Cl
5-Cl
5-Cl
e
30-NO2
40-NO2
30-Cl
125
250
125
125
125
250
62.5
250
125
125
125
250
62.5
125
125
16
125
250
125
125
125
250
125
250
125
125
125
250
62.5
125
125
16
125
250
8
125
250
32
125
125
8
250
250
32
250
250
32
62.5
125
125
32
250
125
125
125
250
250
250
62.5
62.5
16
125
125
62.5
8
125
125
4
125
125
62.5
16
125
125
8
125
125
125
125
250
125
125
62.5
62.5
4
125
125
125
62.5
125
125
32
125
125
125
125
250
125
125
62.5
62.5
16
40-Cl
8
32
8
16
30,40-diCl
30-Br
40-Br
125
125
8
125
125
32
125
125
4
125
125
8
30-CF3
40-CF3
30-NO2
40-NO2
30-Cl
125
62.5
250
125
250
62.5
250
62.5
16
125
125
250
125
250
62.5
250
62.5
32
125
125
125
125
62.5
62.5
62.5
62.5
62.5
4
250
125
125
125
250
250
250
62.5
62.5
4
125
32
125
125
125
62.5
62.5
62.5
32
4
62.5
4
40-Cl
30,40-diCl
30-Br
40-Br
30-CF3
40-CF3
e
4
250
0.5
16
250
0.5
125
250
>250
125
250
>250
125
>250
500
>250
500
>250
250
4
250
4
INH
INH e isoniazid.

390
J. Kozic et al. / European Journal of Medicinal Chemistry 56 (2012) 387e395
Table 2
Antimycobacterial activities of 2-methoxybenzthioanilide derivatives 4.
R1
R2
MIC [
M. tuberculosis My 331/88
m ]
mol Lꢀ1
M. avium My 330/88
M. kansasii My 235/80
M. kansasii My 6509/96
14 d
21 d
14 d
21 d
7 d
14 d
21 d
7 d
14 d
21 d
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
4k
4l
4m
4n
4o
4p
4q
4r
4-Cl
4-Cl
4-Cl
4-Cl
4-Cl
4-Cl
4-Cl
4-Cl
4-Cl
5-Cl
5-Cl
5-Cl
5-Cl
5-Cl
5-Cl
5-Cl
5-Cl
5-Cl
e
30-NO2
40-NO2
30-Cl
8
8
4
4
2
32
4
4
8
250
32
16
16
8
32
16
16
16
0.5
16
32
16
16
8
32
16
8
16
250
32
16
16
8
32
16
16
16
0.5
16
16
16
32
32
125
16
32
32
250
250
32
32
32
32
16
32
16
16
16
16
32
32
16
16
32
16
125
32
16
16
62.5
125
8
32
32
32
62.5
32
125
62.5
32
62.5
32
32
8
16
16
32
32
32
125
32
32
32
250
250
16
16
125
32
32
16
16
4
32
32
32
62.5
32
125
62.5
32
16
16
16
16
125
16
16
8
62.5
125
16
16
62.5
32
16
8
40-Cl
62.5
62.5
125
62.5
32
62.5
250
250
16
62.5
62.5
32
250
16
30,40-diCl
30-Br
40-Br
32
125
62.5
32
30-CF3
40-CF3
30-NO2
40-NO2
30-Cl
32
32
32
250
250
32
32
62.5
32
16
32
16
>250
250
250
16
32
62.5
32
250
16
16
250
250
32
32
125
32
32
16
16
4
40-Cl
8
30,40-diCl
30-Br
32
16
62.5
8
40-Br
30-CF3
40-CF3
e
8
32
>250
8
4
INH
>250
>250
>250
INH e isoniazid.
1544 (
d
NH þ
n
CN, amide II), 1520 (nas NO2), 1477 (
n
CC aromatic),
Calcd. for C14H11ClN2O4 (306.70): C 54.83, H 3.62, N 9.13, Found: C
54.74, H 3.93, N 9.11.
1354 (ns NO2) cmꢀ1 1H NMR (DMSO-d6, 300 MHz):
; d 10.57 (1H, s,
NH), 8.76 (1H, m, H20), 8.04 (1H, m, H40), 7.95 (1H, m, H60), 7.64 (1H,
t, J ¼ 8.1 Hz, H50), 7.63 (1H, d, J ¼ 8.2 Hz, H6), 7.29 (1H, d, J ¼ 1.8 Hz,
5.1.2.2. 4-Chloro-2-methoxy-N-(4-nitrophenyl)benzamide (3b). Yield:
H3), 7.14 (1H, dd, J ¼ 8.2 Hz, J ¼ 1.8 Hz, H5), 3.91 (3H, s, OCH3); 13
C
60.3%, yellow solid; m. p. 223e226 ꢂC; IR (ATR): 3327 (
n
NH), 3115
CH aromatic),1677 ( CCaromatic),1551
NH þ CN, amide II), 1506 (nas NO2), 1479 ( CC aromatic), 1328
1H NMR (DMSO-d6, 300 MHz):
d 10.70 (1H, s, NH),
NMR (DMSO-d6, 75 MHz):
d
164.60 (1C, s, CONH), 157.55, 148.16,
(
(
(
n
n
CO þ
d NH, amide I),1593 (n
140.18, 136.80, 131.19, 130.33, 125.96, 123.77, 120.68, 118.39, 113.99,
d
n
n
112.71, 56.68 (1C, s, OCH3); LRMS-APCIþ: m/z 307 [M þ H]þ; Anal.
ns NO2) cmꢀ1
;
Table 3
Antimycobacterial activities of corresponding salicylanilide derivatives [10] for comparison to prepared compounds 3 and 4.
R1
R2
MIC[
M. tuberculosis My 331/88
m ]
mol Lꢀ1
M. avium My 330/88
M. kansasii My 235/80
14 d
21 d
14 d
21 d
14 d
21 d
4-Cl
4-Cl
4-Cl
4-Cl
4-Cl
4-Cl
5-Cl
5-Cl
5-Cl
5-Cl
5-Cl
5-Cl
5-Cl
INH
30-NO2
30-Cl
8
4
4
4
4
4
8
4
4
4
4
8
2
4
8
4
4
4
4
4
8
8
8
4
8
16
2
4
16
16
8
16
16
8
16
8
8
8
16
8
8
500
16
16
8
16
16
8
16
8
16
8
16
4
4
8
4
4
8
4
4
8
4
4
1
16
8
8
8
4
4
8
4
8
8
4
4
1
40-Cl
30,40-diCl
40-Br
40-CF3
30-NO2
40-NO2
30-Cl
40-Cl
30,40-diCl
40-Br
16
8
8
40-CF3
500
500
500
INHeisoniazid.

J. Kozic et al. / European Journal of Medicinal Chemistry 56 (2012) 387e395
391
Table 4
Inhibition of isocitrate lyase by selected thioamides. Concentration of all of the tested compounds and 3-nitropropionic acid was 100
m
mol Lꢀ1
.
R1
R2
30-Cl
40-Cl
% Inhibition
ꢁ Standard deviation
4c
4d
4e
4g
4-Cl
4-Cl
4-Cl
4-Cl
4-Cl
20
39
17
27
23
67
ꢁ5
ꢁ5
30,40-diCl
40-Br
ꢁ1
ꢁ5
4h
30-CF3
ꢁ6
3-Nitropropionic acid
ꢁ2.68
8.29e8.21 (2H, m, H30, H50), 8.00e7.91 (2H, m, H20, H60), 7.62 (1H, d,
J ¼ 8.2 Hz, H6), 7.29 (1H, d, J ¼ 1.8 Hz, H3), 7.14 (1H, dd, J ¼ 8.2 Hz,
J ¼ 1.8 Hz, H5), 3.90 (3H, s, OCH3); 13C NMR (DMSO-d6, 75 MHz):
(1C, s, OCH3); LRMS-APCIþ: m/z 330 [M þ H]þ; Anal. Calcd. for
C14H10Cl3NO2 (330.59): C 50.86, H 3.05, N 4.24, Found: C 50.95, H
3.37, N 4.23.
d
164.82 (1C, s,CONH),157.55,145.21,142.66,136.88,131.16,125.14 (2C,
s, C30, C50), 123.83, 120.69, 119.56 (2C, s, C20, C60), 112.73, 56.69 (1C, s,
OCH3); LRMS-APCIþ: m/z 307 [M þ H]þ; Anal. Calcd. for C14H11ClN2O4
(306.70): C 54.83, H 3.62, N 9.13, Found: C 54.90, H 3.81, N 9.21.
5.1.2.6. 4-Chloro-2-methoxy-N-(3-bromophenyl)benzamide
Yield: 73.9%, white solid; m. p. 136e137 ꢂC; IR (ATR): 3355 (
(3f).
n
NH),
3081 (
aromatic), 1536 (
cmꢀ1; 1H NMR (DMSO-d6, 500 MHz):
d
n
CH aromatic), 1670 (
NH þ CN, amide II), 1484, 1461 (
10.25 (1H, s, NH), 8.05 (1H,
n
CO þ
d
NH, amide I), 1588 (
n CC
d
n
n
CC aromatic)
5.1.2.3. 4-Chloro-2-methoxy-N-(3-chlorophenyl)benzamide
(3c).
NH),
NH þ
Yield: 71.3%, white solid; m. p. 116e118 ꢂC; IR (ATR): 3355 (
n
m, H20), 7.65 (1H, m, H40), 7.61 (1H, d, J ¼ 8.2 Hz, H6), 7.32e7.25 (3H,
1668 (
n
CO þ
d
NH, amide I), 1587 (
n
CC aromatic), 1534 (
d
n
m, H3, H50, H60), 7.12 (1H, dd, J ¼ 8.2 Hz, J ¼ 1.9 Hz, H5), 3.90 (3H, s,
CN, amide II), 1482, 1460 (
300 MHz):
n
CC aromatic) cmꢀ1; 1H NMR (DMSO-d6,
OCH3); 13C NMR (DMSO-d6, 125 MHz):
d 164.16 (1C, s, CONH),
d
10.28 (1H, s, NH), 7.91 (1H, t, J ¼ 2.1 Hz, H20), 7.65e7.57
157.50, 140.64, 136.61, 131.18, 130.90, 126.44, 123.92, 122.17, 121.73,
120.65, 118.70, 112.67, 56.66 (1C, s, OCH3); LRMS-APCIþ: m/z 340
[M þ H]þ; Anal. Calcd. for C14H11BrClNO2 (340.60): C 49.37, H 3.26,
N 4.11, Found: C 49.33, H 3.60, N 4.17.
(2H, m, H6, H40), 7.36 (1H, t, J ¼ 8.1 Hz, H50), 7.27 (1H, d, J ¼ 1.9 Hz,
H3), 7.15 (1H, m, H60), 7.13 (1H, dd, J ¼ 8.2 Hz, J ¼ 1.9 Hz, H5), 3.90
(3H, s, OCH3); 13C NMR (DMSO-d6, 75 MHz):
d 164.22 (1C, s, CONH),
157.50, 140.52, 136.61, 133.27, 131.18, 130.63, 123.96, 123.55, 120.66,
119.31, 118.32, 112.68, 56.67 (1C, s, OCH3); LRMS-APCIþ: m/z 296
[M þ H]þ; Anal. Calcd. for C14H11Cl2NO2 (296.15): C 56.78, H 3.74, N
4.73, Found: C 56.90, H 3.61, N 4.85.
5.1.2.7. 4-Chloro-2-methoxy-N-(4-bromophenyl)benzamide
(3g).
NH),
3104 ( CC
aromatic), 1539 ( CC aromatic)
cmꢀ1; 1H NMR (DMSO-d6, 300 MHz):
d 10.23 (1H, s, NH), 7.74e7.65
Yield: 66.8%, white solid; m. p. 124e126 ꢂC; IR (ATR): 3350 (
n
n
CH aromatic), 1668 (
n
CO þ
d NH, amide I), 1589 (n
d
NH þ CN, amide II), 1488, 1460 (
n
n
5.1.2.4. 4-Chloro-2-methoxy-N-(4-chlorophenyl)benzamide
(3d).
NH),
CC
CC aromatic)
10.23 (1H, s, NH), 7.79e7.70
Yield: 61.7%, white solid; m. p. 127e128 ꢂC; IR (ATR): 3378 (
n
(2H, m, H30, H50), 7.60 (1H, d, J ¼ 8.2 Hz, H6), 7.56e7.48 (2H, m, H20,
3073 (
aromatic), 1540 (
cmꢀ1; 1H NMR (DMSO-d6, 300 MHz):
n
CH aromatic), 1667 (
n
CO þ
d NH, amide I), 1596 (n
H60), 7.27 (1H, d, J ¼ 1.9 Hz, H3), 7.12 (1H, dd, J ¼ 8.2 Hz, J ¼ 1.9 Hz,
d
NH þ CN, amide II), 1492, 1463 (
n
n
H5), 3.90 (3H, s, OCH3); 13C NMR (DMSO-d6, 75 MHz):
d 164.02 (1C,
d
s, CONH),157.48, 138.47,136.50,131.74 (2C, s, C30, C50),131.16,124.11,
121.81 (2C, s, C20, C60), 120.64, 115.44, 112.66, 56.65 (1C, s, OCH3);
LRMS-APCIþ: m/z 340 [M þ H]þ; Anal. Calcd. for C14H11BrClNO2
(340.60): C 49.37, H 3.26, N 4.11, Found: C 49.61, H 3.58, N 4.15.
(2H, m, H30, H50), 7.61 (1H, d, J ¼ 8.2 Hz, H6), 7.42e7.34 (2H, m, H20,
H60), 7.27 (1H, d, J ¼ 1.9 Hz, H3), 7.12 (1H, dd, J ¼ 8.2 Hz, J ¼ 1.9 Hz,
H5), 3.90 (3H, s, OCH3); 13C NMR (DMSO-d6, 75 MHz):
d 164.00 (1C,
s, CONH), 157.48, 138.05, 136.49, 131.16, 128.82 (2C, s, C30, C50),
127.39, 124.10, 121.44 (2C, s, C20, C60), 120.64, 112.65, 56.64 (1C, s,
5.1.2.8. 4-Chloro-2-methoxy-N-(3-trifluoromethylphenyl)benzamide
(3h). Yield: 73.2%, white solid; m. p. 117e119 ꢂC; IR (ATR): 3355 (
n
OCH3); LRMS-APCIþ: m/z 296 [M
þ
H]þ; Anal. Calcd. for
C14H11Cl2NO2 (296.15): C 56.78, H 3.74, N 4.73, Found: C 56.98, H
4.01, N 4.76.
NH), 1670 (
NH þ CN, amide II), 1493 (
d6, 300 MHz):
n
CO þ
d
NH, amide I), 1592 (
n CC aromatic), 1558 (d
n
n
CC aromatic) cmꢀ1; 1H NMR (DMSO-
d
10.43 (1H, s, NH), 8.22 (1H, m, H20), 7.91 (1H, m,
5.1.2.5. 4-Chloro-2-methoxy-N-(3,4-dichlorophenyl)benzamide (3e).
H40), 7.63 (1H, d, J ¼ 8.1 Hz, H6), 7.56 (1H, t, J ¼ 8.1 Hz, H50), 7.44 (1H,
Yield: 51.5%, white solid; m. p. 165e167 ꢂC; IR (ATR): 3343 (
n NH),
m, H60), 7.28 (1H, d, J ¼ 1.8 Hz, H3), 7.13 (1H, dd, J ¼ 8.1 Hz, J ¼ 1.8 Hz,
3106 (
aromatic), 1525 (
aromatic) cmꢀ1; 1H NMR (DMSO-d6, 300 MHz):
n
CH aromatic), 1665 (
n
CO þ
d
NH, amide I), 1584 (
n
CC
CC
H5), 3.91 (3H, s, OCH3); 13C NMR (DMSO-d6, 75 MHz):
d 164.44 (1C,
d
NH þ CN, amide II), 1476, 1464, 1456 (
n
n
s, CONH), 157.53, 139.84, 136.67, 131.17, 130.16, 129.87 (1C, q,
J ¼ 31.2 Hz, C30), 124.51 (1C, q, J ¼ 270.6 Hz, CF3), 123.91, 123.51,
120.65, 120.19 (1C, q, J ¼ 4.0 Hz, C20), 115.91 (1C, q, J ¼ 4.1 Hz, C40),
112.69, 56.67 (1C, s, OCH3); LRMS-APCIþ: m/z 330 [M þ H]þ; Anal.
Calcd. for C15H11ClF3NO2 (329.70): C 54.64, H 3.36, N 4.25, Found: C
54.30, H 3.48, N 4.47.
d
10.37 (1H, s, NH),
8.09 (1H, d, J ¼ 2.4 Hz, H20), 7.66 (1H dd, J ¼ 8.8 Hz, J ¼ 2.4 Hz, H60),
7.64e7.56 (2H, m, H6, H50), 7.28 (1H, d, J ¼ 1.8 Hz, H3), 7.13 (1H, dd,
J ¼ 8.2 Hz, J ¼ 1.8 Hz, H5), 3.90 (3H, s, OCH3); 13C NMR (DMSO-d6,
75 MHz):
d 164.30 (1C, s, CONH), 157.51, 139.15, 136.77, 131.21,
131.19, 130.86, 125.27, 123.71, 121.07, 120.69, 119.97, 112.70, 56.69
Table 5
Cytotoxicity and selectivity index for selected compounds.
R1
R2
IC50
[m
mol Lꢀ1
]
SI for My 331/88, 14 d
4c
4d
4e
4g
4h
4-Cl
4-Cl
4-Cl
4-Cl
4-Cl
30-Cl
11.68
2.93
6.50
3.55
1.81
2.92
0.73
3.25
0.89
0.45
40-Cl
30,40-diCl
40-Br
30-CF3
SI (selectivity index) ¼ IC50/MIC.

392
J. Kozic et al. / European Journal of Medicinal Chemistry 56 (2012) 387e395
5.1.2.9. 4-Chloro-2-methoxy-N-(4-trifluoromethylphenyl)benzamide
(3i). Yield: 67.8%, white solid; m. p. 141e142 ꢂC; IR (ATR): 3368 (
NH), 1673 ( NH, amide I), 1594 ( CC aromatic), 1541 (
CO þ
NH þ CN, amide II), 1483, 1464 (
CC aromatic) cmꢀ1 1H NMR
(DMSO-d6, 300 MHz):
127.48, 126.95, 124.42, 121.45 (2C, s, C20, C60), 114.19, 56.50 (1C, s,
OCH3); LRMS-APCIþ: m/z 296 [M H]þ; Anal. Calcd. for
C14H11Cl2NO2 (296.15): C 56.78, H 3.74, N 4.73, Found: C 56.40, H
4.16, N 4.66.
n
þ
n
d
n
d
n
n
;
d
10.46 (1H, s, NH), 7.97e7.88 (2H, m, H30,
H50), 7.75e7.66 (2H, m, H20, H60), 7.62 (1H, d, J ¼ 8.2 Hz, H6), 7.28
(1H, d, J ¼ 1.9 Hz, H3), 7.13 (1H, dd, J ¼ 8.2 Hz, J ¼ 1.9 Hz, H5), 3.90
5.1.2.14. 5-Chloro-2-methoxy-N-(3,4-dichlorophenyl)benzamide(3n).
Yield: 50.2%, white solid; m. p. 175e177 ꢂC; IR (ATR): 3343 (
n
NH),
CC
CC aromatic)
10.45 (1H, s, NH), 8.09 (1H, d,
(3H, s, OCH3); 13C NMR (DMSO-d6, 75 MHz):
d
164.51 (1C, s, CONH),
3099 (
aromatic), 1529 (
cmꢀ1; 1H NMR (DMSO-d6, 300 MHz):
n
CH aromatic), 1674 (
n
CO þ
d NH, amide I), 1587 (n
157.53, 142.66, 136.67, 131.16, 126.26 (2C, q, J ¼ 3.9 Hz, C30, C50),
125.25 (1C, q, J ¼ 269.5 Hz, CF3), 124.02, 123.80 (1C, q, J ¼ 31.8 Hz,
C40), 120.66, 119.77 (2C, s, C20, C60), 112.70, 56.67 (1C, s, OCH3);
LRMS-APCIþ: m/z 330 [M þ H]þ; Anal. Calcd. for C15H11ClF3NO2
(329.70): C 54.64, H 3.36, N 4.25, Found: C 54.43, H 3.25, N 4.44.
d
NH þ CN, amide II), 1476, 1469 (
n
n
d
J ¼ 2.3 Hz, H20), 7.65 (1H, dd, J ¼ 9.0 Hz, J ¼ 2.3 Hz, H60), 7.62e7.57
(2H, m, H6, H50), 7.54 (1H, dd, J ¼ 8.9 Hz, J ¼ 2.7 Hz, H4), 7.20 (1H,
d, J ¼ 8.9 Hz, H3), 3.87 (3H, s, OCH3); 13C NMR (DMSO-d6, 75 MHz):
d
163.83 (1C, s, CONH), 155.48, 139.09, 131.80, 131.19, 130.87, 128.99,
5.1.2.10. 5-Chloro-2-methoxy-N-(3-nitrophenyl)benzamide
(3j).
NH),
NH þ
126.55, 125.36, 124.43, 121.07, 119.96, 114.23, 56.52 (1C, s, OCH3);
LRMS-APCIþ: m/z 330 [M þ H]þ; Anal. Calcd. for C14H10Cl3NO2
(330.59): C 50.86, H 3.05, N 4.24, Found: C 50.38, H 3.26, N 4.17.
Yield: 47.5%, white solid; m. p. 190e192 ꢂC; IR (ATR): 3318 (
n
1670 (
CN, amide II), 1530 (nas NO2), 1474, 1458, 1433 (
ns NO2) cmꢀ1 1H NMR (DMSO-d6, 300 MHz):
n
CO þ
d
NH, amide I), 1596 (
n
CC aromatic), 1552 (
CC aromatic), 1338
10.66 (1H, s, NH),
d
n
n
(
;
d
5.1.2.15. 5-Chloro-2-methoxy-N-(3-bromophenyl)benzamide (3o).
8.76 (1H, t, J ¼ 2.2 Hz, H20), 8.03 (1H, m, H40), 7.96 (1H, m, H60), 7.63
(1H, t, J ¼ 8.2 Hz, H50), 7.62 (1H, d, J ¼ 2.8 Hz, H6), 7.56 (1H, dd,
J ¼ 8.8 Hz, J ¼ 2.8 Hz, H4), 7.22 (1H, d, J ¼ 8.8 Hz, H3), 3.87 (3H, s,
Yield: 68.3%, white solid; m. p. 133e134 ꢂC; IR (ATR): 3350 (
n
NH),
NH þ
;
1H NMR (CDCl3,
1661 (
CN, amide II), 1479, 1459 (
300 MHz):
n
CO þ
d
NH, amide I), 1592 (
n
CC aromatic), 1534 (
d
n
n
CC aromatic) cmꢀ1
OCH3); 13C NMR (DMSO-d6, 75 MHz):
d
164.17 (1C, s, CONH), 155.51,
d
9.72 (1H, s, NH), 8.20 (1H, d, J ¼ 2.8 Hz, H6), 7.88 (1H, t,
148.15, 140.14, 131.81, 130.38, 128.97, 126.65, 125.94, 124.42, 118.48,
114.24, 113.99, 56.53 (1C, s, OCH3); LRMS-APCIþ: m/z 307 [M þ H]þ;
Anal. Calcd. for C14H11ClN2O4 (306.70): C 54.83, H 3.62, N 9.13,
Found: C 54.56, H 3.91, N 8.88.
J ¼ 1.9 Hz, H20), 7.55 (1H, m, H40), 7.42 (1H, dd, J ¼ 8.8 Hz, J ¼ 2.8 Hz,
H4), 7.25 (1H, m, H60), 7.21 (1H, t, J ¼ 7.9 Hz, H50), 6.96 (1H, d,
J ¼ 8.8 Hz, H3), 4.05 (3H, s, OCH3); 13C NMR (CDCl3, 75 MHz):
d
161.86 (1C, s, CONH), 155.63, 139.29, 133.03, 132.13, 130.25, 127.28,
127.15, 123.19, 122.67, 122.57, 118.85, 113.05, 56.68 (1C, s, OCH3);
LRMS-APCIþ: m/z 340 [M þ H]þ; Anal. Calcd. for C14H11BrClNO2
(340.60): C 49.37, H 3.26, N 4.11, Found: C 49.04, H 3.56, N 4.04.
5.1.2.11. 5-Chloro-2-methoxy-N-(4-nitrophenyl)benzamide
(3k).
NH),
NH þ
CC aromatic), 1330 (ns
1H NMR (DMSO-d6, 300 MHz):
10.79 (1H, s, NH),
Yield: 49.3%, yellow solid; m. p. 209e211 ꢂC; IR (ATR): 3318 (
n
1677 (
CN, amide II), 1513 (nas NO2), 1478, 1454 (
NO2) cmꢀ1
n
CO þ
d
NH, amide I), 1596 (
n
CC aromatic), 1553 (
d
n
n
5.1.2.16. 5-Chloro-2-methoxy-N-(4-bromophenyl)benzamide (3p).
;
d
Yield: 40.7%, white solid; m. p. 139e141 ꢂC; IR (ATR): 3337 (
n
NH),
NH, amide I), 1594, 1586 (CC
CN, amide II), 1488, 1477 (CC aromatic)
10.31 (1H, s, NH), 7.74e7.66
8.29e8.21 (2H, m, H30, H50), 8.00e7.92 (2H, m, H20, H60), 7.61 (1H, d,
J ¼ 2.8 Hz, H6), 7.57 (1H, dd, J ¼ 8.8 Hz, J ¼ 2.8 Hz, H4), 7.22 (1H, d,
J ¼ 8.8 Hz, H3), 3.87 (3H, s, OCH3); 13C NMR (DMSO-d6, 75 MHz):
3084 (
aromatic), 1535 (
cmꢀ1; 1H NMR (DMSO-d6, 300 MHz):
n
CH aromatic), 1666 (
n
CO þ
d
d
NH þ
n
d
d
164.38 (1C, s, CONH), 155.50, 145.16, 142.72, 131.89, 128.94, 126.69,
(2H, m, H30, H50), 7.59 (1H, d, J ¼ 2.8 Hz, H6), 7.57e7.48 (3H, m, H4,
125.17 (2C, s, C30, C50), 124.43, 119.59 (2C, s, C20, C60), 114.25, 56.54
(1C, s, OCH3); LRMS-APCIþ: m/z 307 [M þ H]þ; Anal. Calcd. for
C14H11ClN2O4 (306.70): C 54.83, H 3.62, N 9.13, Found: C 54.38, H
3.21, N 8.95.
H20, H60), 7.19 (1H, d, J ¼ 8.9 Hz, H3), 3.86 (3H, s, OCH3); 13C NMR
(DMSO-d6, 75 MHz):
d 163.54 (1C, s, CONH), 155.44, 138.42, 131.74
(2C, s, C30, C50), 131.56, 128.95, 126.94, 124.41, 121.81 (2C, s, C20, C60),
115.53, 114.18, 56.49 (1C, s, OCH3); LRMS-APCIþ: m/z 340 [M þ H]þ;
Anal. Calcd. for C14H11BrClNO2 (340.60): C 49.37, H 3.26, N 4.11,
Found: C 49.12, H 3.59, N 4.00.
5.1.2.12. 5-Chloro-2-methoxy-N-(3-chlorophenyl)benzamide (3l).
Yield: 71.7%, white solid; m. p. 133e135 ꢂC; IR (ATR): 3326 (
n
NH),
CC
CC aromatic)
10.35 (1H, s, NH), 7.91 (1H, t,
3080 (
aromatic), 1525 (
cmꢀ1; 1H NMR (DMSO-d6, 300 MHz):
n
CH aromatic), 1667 (
n
CO þ
d
NH, amide I), 1588 (
n
5.1.2.17. 5-Chloro-2-methoxy-N-(3-trifluoromethylphenyl)benzamide
(3q). Yield: 53.2%, white solid; m. p. 107e108 ꢂC; IR (ATR): 3342 (
n
d
NH þ CN, amide II), 1483, 1463 (
n
n
d
NH), 1665 (
NH þ CN, amide II), 1479 (
300 MHz):
n
CO þ
d
NH, amide I), 1597 (
n CC aromatic), 1559 (d
J ¼ 2.1 Hz, H20), 7.65e7.58 (2H, m, H6, H40), 7.54 (1H, dd, J ¼ 8.8 Hz,
J ¼ 2.8 Hz, H4), 7.36 (1H, t, J ¼ 8.1 Hz, H50), 7.20 (1H, d, J ¼ 8.8 Hz,
H3), 7.15 (1H, m, H60), 3.87 (3H, s, OCH3); 13C NMR (DMSO-d6,
n
n
CC aromatic) cmꢀ1; 1H NMR (DMSO-d6,
d
10.51 (1H, s, NH), 8.21 (1H, m, H20), 7.90 (1H, m, H40),
7.68e7.50 (3H, m, H4, H6, H50), 7.44 (1H, m, H60), 7.21 (1H, d,
J ¼ 8.9 Hz, H3), 3.87 (3H, s, OCH3); 13C NMR (DMSO-d6, 75 MHz):
75 MHz):
d 163.71 (1C, s, CONH), 155.47, 140.44, 133.26, 131.66,
130.61, 128.97, 126.76, 124.42, 123.62, 119.33, 118.32, 114.21, 56.50
(1C, s, OCH3); LRMS-APCIþ: m/z 296 [M þ H]þ; Anal. Calcd. for
C14H11Cl2NO2 (296.15): C 56.78, H 3.74, N 4.73, Found: C 56.41, H
3.64, N 4.66.
d 164.01 (1C, s, CONH), 155.50, 139.80, 131.71, 130.19, 129.83 (1C, q,
J ¼ 31.4 Hz, C30), 128.96, 126.78, 124.80 (1C, q, J ¼ 270.6 Hz, CF3),
124.41,123.50,120.27 (1C, q, J ¼ 4.0 Hz, C20),115.95 (1C, q, J ¼ 4.3 Hz,
C40), 114.23, 56.52 (1C, s, OCH3); LRMS-APCIþ: m/z 330 [M þ H]þ;
Anal. Calcd. for C15H11ClF3NO2 (329.70): C 54.64, H 3.36, N 4.25,
Found: C 54.81, H 2.98, N 4.16.
5.1.2.13. 5-Chloro-2-methoxy-N-(4-chlorophenyl)benzamide (3m).
Yield: 70.0%, white solid; m. p. 135e137 ꢂC; IR (ATR): 3335 (
n
NH),
CC
CC aromatic)
10.32 (1H, s, NH), 7.79e7.70
3085 (
aromatic), 1535 (
cmꢀ1; 1H NMR (DMSO-d6, 300 MHz):
n
CH aromatic), 1665 (
n
CO þ
d
NH, amide I), 1591 (
n
5.1.2.18. 5-Chloro-2-methoxy-N-(4-trifluoromethylphenyl)benzamide
(3r). Yield: 75.3%, white solid; m. p. 131e133 ꢂC; IR (ATR): 3336 (
n
d
NH þ CN, amide II), 1491, 1477 (
n
n
d
NH), 1675 (
NH þ CN, amide II),1481 (
300 MHz):
n
CO þ
d
NH, amide I), 1605 (
n CC aromatic), 1545 (d
(2H, m, H30, H50), 7.59 (1H, d, J ¼ 2.8 Hz, H6), 7.54 (1H, dd, J ¼ 8.9 Hz,
n
n
CC aromatic) cmꢀ1; 1H NMR (DMSO-d6,
J ¼ 2.8 Hz, H4), 7.43e7.35 (2H, m, H20, H60), 7.19 (1H, d, J ¼ 8.9 Hz,
d
10.54 (1H, s, NH), 7.97e7.89 (2H, m, H30, H50), 7.75e7.66
H3), 3.86 (3H, s, OCH3); 13C NMR (DMSO-d6, 75 MHz):
d
163.55 (1C,
(2H, m, H20, H60), 7.60 (1H, d, J ¼ 2.7 Hz, H6), 7.55 (1H, dd, J ¼ 8.8 Hz,
s, CONH), 155.45, 138.02, 131.57, 128.97, 128.85 (2C, s, C30, C50),
J ¼ 2.7 Hz, H4), 7.21 (1H, d, J ¼ 8.8 Hz, H3), 3.87 (3H, s, OCH3); 13C

J. Kozic et al. / European Journal of Medicinal Chemistry 56 (2012) 387e395
393
NMR (DMSO-d6, 75 MHz):
d
164.05 (1C, s, CONH), 155.48, 142.59,
75 MHz): d 193.64 (1C, s, CSNH), 155.02,138.53, 137.61,136.35,132.01,
131.71, 128.94, 126.86, 126.25 (2C, q, J ¼ 3.9 Hz, C30, C50), 125.17 (1C,
q, J ¼ 268.9 Hz, CF3), 124.42, 124.06 (1C, q, J ¼ 31.8 Hz, C40), 119.77
(2C, s, C20, C60), 114.22, 56.51 (1C, s, OCH3); LRMS-APCIþ: m/z 330
[M þ H]þ; Anal. Calcd. for C15H11ClF3NO2 (329.70): C 54.64, H 3.36, N
4.25, Found: C 54.26, H 3.11, N 4.21.
128.96 (2C, s, C30, C50), 126.79, 125.52 (2C, s, C20, C60), 121.76, 112.07,
56.68 (1C, s, OCH3); LRMS-APCIþ: m/z 312 [M þ H]þ; Anal. Calcd. for
C14H11Cl2NOS (312.21): C 53.86, H 3.55, N 4.49, S 10.27, Found: C
53.65, H 3.45, N 4.56, S 10.15.
5.1.3.5. 4-Chloro-2-methoxy-N-(3,4-chlorophenyl)benzothioamide
5.1.3. General procedure for the preparation of substituted 2-meth-
oxybenzthioamides
(4e). Yield: 39.0%, yellow solid; m. p. 122e123 ꢂC; IR (ATR): 3300 (
n
NH), 1583, 1542, 1477, 1459 (CC aromatic) cmꢀ1
300 MHz):
;
1H NMR (CDCl3,
A suspension of 2.0 mmol of the starting benzamide (3ae3r)
and an equimolar amount of P4S10 (2.0 mmol) in 25.0 mL of pyri-
dine was stirred and heated at reflux temperature (155 ꢂC) until the
starting benzamide disappeared by TLC (3e8 h). More P4S10 was
added if necessary. The unreacted P4S10 was removed by the
addition of ice water. The mixture was then extracted three times
with 50 mL of chloroform and the organic phase was collected,
dried with Na2SO4 and evaporated. The oil residue was purified by
column chromatography on silica gel with toluene, toluene/hexane
(9/1) for 4h or toluene/hexane (4/1) for 4o as a mobile phase.
d
10.56 (1H, s, NH), 8.40 (1H, d, J ¼ 8.6 Hz, H6), 7.97 (1H,
d, J ¼ 2.5 Hz, H20), 7.59 (1H, dd, J ¼ 8.7 Hz, J ¼ 2.5 Hz, H60), 7.46 (1H,
d, J ¼ 8.7 Hz, H50), 7.06 (1H, dd, J ¼ 8.6 Hz, J ¼ 1.9 Hz, H5), 6.98 (1H,
d, J ¼ 1.9 Hz, H3), 4.01 (3H, s, OCH3); 13C NMR (CDCl3, 75 MHz):
d
193.87 (1C, s, CSNH),155.00,138.80,138.36,136.42,132.58, 130.33,
130.15, 126.52, 125.77, 123.56, 121.84, 112.09, 56.75 (1C, s, OCH3);
LRMS-APCIþ: m/z 346 [M þ H]þ; Anal. Calcd. for C14H10Cl3NOS
(346.66): C 48.51, H 2.91, N 4.04, S 9.25, Found: C 48.27, H 3.05, N
4.15, S 9.02.
5.1.3.6. 4-Chloro-2-methoxy-N-(3-bromophenyl)benzothioamide(4f).
5.1.3.1. 4-Chloro-2-methoxy-N-(3-nitrophenyl)benzothioamide (4a).
Yield: 53.4%, yellow solid; m. p. 129e130 ꢂC; IR (ATR): 3303 (
n
NH),
Yield: 23.0%, yellow solid; m. p. 160e162 ꢂC; IR (ATR): 3304 (
n
1588, 1560,1547, 1477 (CC aromatic) cmꢀ1
300 MHz):
;
1H NMR (CDCl3,
NH),1589, 1561 (CC aromatic), 1524 (nas NO2), 1477, 1462 (CC
d
10.52 (1H, s, NH), 8.41 (1H, d, J ¼ 8.6 Hz, H6), 7.97 (1H, m,
aromatic), 1345 (ns NO2) cmꢀ1
;
1H NMR (DMSO-d6, 300 MHz):
H20), 7.69 (1H, m, H40), 7.41 (1H, m, H60), 7.28 (1H, t, J ¼ 8.1 Hz, H50),
7.06 (1H, m, H5), 6,98 (1H, m, H3), 4.00 (3H, s, OCH3); 13C NMR
(CDCl3, 75 MHz): 193.81 (1C, s, CSNH), 155.00, 140.29, 138.59, 136.35,
130.07, 129.79, 127.04, 126.82, 122.91, 122.21, 121.78, 112.09, 56.72
(1C, s, OCH3); LRMS-APCIþ: m/z 356 [M þ H]þ; Anal. Calcd. for
C14H11BrClNOS (356.67): C 47.14, H 3.11, N 3.93, S 8.99, Found: C
46.96, H 3.34, N 3.79, S 8.94.
d
12.16 (1H, s, NH), 9.13 (1H, t, J ¼ 2.2 Hz, H20), 8.30 (1H, m, H40), 8.11
(1H, m, H60), 7.72 (1H, t, J ¼ 8.2 Hz, H50), 7.51 (1H, d, J ¼ 8.2 Hz, H6),
7.23 (1H, d, J ¼ 1.9 Hz, H3), 7.09 (1H, dd, J ¼ 8.2 Hz, J ¼ 1.9 Hz, H5),
3.85 (3H, s, OCH3); 13C NMR (DMSO-d6, 75 MHz):
d 195.25 (1C, s,
CSNH), 154.98, 147.70, 140.69, 135.34, 132.30, 131.26, 130.33, 128.94,
120.83, 120.34, 116.84, 112.38, 56.58 (1C, s, OCH3); LRMS-APCIþ: m/z
323 [M þ H]þ; Anal. Calcd. for C14H11ClN2O3S (322.77): C 52.10, H
3.44, N 8.68, S 9.93, Found: C 52.31, H 3.53, N 8.94, S 10.07.
5.1.3.7. 4-Chloro-2-methoxy-N-(4-bromophenyl)benzothioamide (4g).
Yield: 29.0%, yellow solid; m. p. 123e124 ꢂC; IR (ATR): 1592, 1566,
5.1.3.2. 4-Chloro-2-methoxy-N-(4-nitrophenyl)benzothioamide (4b).
1486, 1462 (CC aromatic) cmꢀ1 1H NMR (CDCl3, 300 MHz):
; d 10.52
Yield: 20.0%, yellow solid; m. p. 153e154 ꢂC; IR (ATR): 3364 (
n
NH),
(1H, s, NH), 8.42 (1H, d, J ¼ 8.6 Hz, H6), 7.69e7.62 (2H, m, H30, H50),
7.58e7.54 (2H, m, H20, H60), 7.07 (1H, dd, J ¼ 8.6 Hz, J ¼ 2.0 Hz, H5),
6.98 (1H, d, J ¼ 2.0 Hz, H3), 4.00 (3H, s, OCH3); 13C NMR (CDCl3,
1589, 1561 (CC aromatic), 1510 (nas NO2), 1491, 1473 (CC aromatic),
1318 (ns NO2) cmꢀ1 1H NMR (DMSO-d6, 300 MHz):
; d 12.20 (1H, s,
NH), 8.39e8.26 (4H, m. H20, H30, H50, H60), 7.49 (1H, d, J ¼ 8.2 Hz,
H6), 7.23 (1H, d, J ¼ 1.9 Hz, H3), 7.09 (1H, dd, J ¼ 8.2 Hz, J ¼ 1.9 Hz,
75 MHz): d 193.57 (1C, s, CSNH), 155.00, 138.55, 138.10, 136.36, 131.93
(2C, s, C30, C50), 126.81, 125.77 (2C, s, C20, C60), 121.78, 119.88, 112.08,
56.70 (1C, s, OCH3); LRMS-APCIþ: m/z 356 [M þ H]þ; Anal. Calcd. for
C14H11BrClNOS (356.67): C 47.14, H 3.11, N 3.93, S 8.99, Found: C 47.20,
H 3.31, N 3.96, S 9.37.
H5), 3.84 (3H, s, OCH3); 13C NMR (DMSO-d6, 75 MHz):
d 195.83 (1C,
s, CSNH), 154.96, 145.41, 144.17, 135.43, 132.58, 131.31, 124.71 (2C, s,
C30, C50), 122.47 (2C, s, C20, C60), 120.36, 112.38, 56.57 (1C, s, OCH3);
LRMS-APCIþ: m/z 323 [M þ H]þ; Anal. Calcd. for C14H11ClN2O3S
(322.77): C 52.10, H 3.44, N 8.68, S 9.93, Found: C 52.38, H 3.62, N
8.51, S 9.79.
5.1.3.8. 4-Chloro-2-methoxy-N-(3-trifluoromethylphenyl)benzothio-
amide (4h). Yield: 37.0%, yellow solid; m. p. 109e110 ꢂC; IR (ATR):
3289 (
(CDCl3, 300 MHz):
n ;
NH), 1591, 1566, 1483, 1450 (CC aromatic) cmꢀ1 1H NMR
5.1.3.3. 4-Chloro-2-methoxy-N-(3-chlorophenyl)benzothioamide (4c).
d
10.64 (1H, s, NH), 8.44 (1H, d, J ¼ 8.6 Hz, H6),
Yield: 50.2%, yellow solid; m. p. 124e125 ꢂC; IR (ATR): 3290 (
n
NH),
8.05e7.95 (2H, m, H20, H40), 7.60e7.50 (2H, m, H50, H60), 7.08 (1H,
dd, J ¼ 8.6 Hz, J ¼ 1.9 Hz, H5), 7.00 (1H, d, J ¼ 1.9 Hz, H3), 4.03 (3H, s,
1588, 1565, 1479, 1460 (CC aromatic) cmꢀ1
300 MHz):
;
1H NMR (CDCl3,
d
10.53 (1H, s, NH), 8.41 (1H, d, J ¼ 8.6 Hz, H6), 7.85 (1H,
OCH3); 13C NMR (CDCl3, 75 MHz):
d 194.17 (1C, s, CSNH), 155.07,
t, J ¼ 2.0 Hz, H20), 7.62 (1H, m, H40), 7.35 (1H, t, J ¼ 8.0 Hz, H50), 7.26
(1H, m, H60), 7.06 (1H, dd, J ¼ 8.6 Hz, J ¼ 1.9 Hz, H5), 6.98 (1H, d,
J ¼ 1.9 Hz, H3), 4.00 (3H, s, OCH3); 13C NMR (CDCl3, 75 MHz):
139.56, 138.75, 136.46, 131.28 (1C, q, J ¼ 32.5 Hz, C30), 129.38, 127.67,
126.67, 123.66 (1C, q, J ¼ 270.8 Hz, CF3), 123.41 (1C, q, J ¼ 3.8 Hz,
C20), 121.86, 121.09 (1C, q, J ¼ 3.8 Hz, C40), 112.12, 56.75 (1C, s,
d
193.78 (1C, s, CSNH), 154.99, 140.16, 138.57, 136.34, 134.36, 129.81,
OCH3); LRMS-APCIþ: m/z 346 [M
þ
H]þ; Anal. Calcd. for
126.88, 126.84, 124.19, 122.38, 121.77, 112.09, 56.70 (1C, s, OCH3);
LRMS-APCIþ: m/z 312 [M þ H]þ; Anal. Calcd. for C14H11Cl2NOS
(312.21): C 53.86, H 3.55, N 4.49, S 10.27, Found: C 54.11, H 3.71, N
4.72, S 10.02.
C15H11ClF3NOS (345.77): C 52.10, H 3.21, N 4.05, S 9.27, Found: C
52.31, H 3.02, N 4.37, S 9.16.
5.1.3.9. 4-Chloro-2-methoxy-N-(4-trifluoromethylphenyl)benzothio-
amide (4i). Yield: 22.0%, yellow solid; m. p. 95e96 ꢂC; IR (ATR):
5.1.3.4. 4-Chloro-2-methoxy-N-(4-chlorophenyl)benzothioamide (4d).
Yield: 54.0%, yellow solid; m. p. 128e129 ꢂC; IR (ATR): 1590, 1565,
1589, 1565, 1482, 1464 (CC aromatic) cmꢀ1 1H NMR (CDCl3,
;
300 MHz):
d
10.66 (1H, s, NH), 8.43 (1H, d, J ¼ 8.6 Hz, H6),
1527, 1488, 1460 (CC aromatic) cmꢀ1
;
1H NMR (CDCl3, 300 MHz):
8.00e7.89 (2H, m, H30, H50), 7.73e7.64 (2H, m, H20, H60), 7.08 (1H,
dd, J ¼ 8.6 Hz, J ¼ 1.9 Hz, H5), 7.00 (1H, d, J ¼ 1.9 Hz, H3), 4.02 (3H,
d
10.52 (1H, s, NH), 8.42 (1H, d, J ¼ 8.6 Hz, H6), 7.75e7.66 (2H, m, H30,
H50), 7.43e7.34 (2H, m, H20, H60), 7.07 (1H, dd, J ¼ 8.6 Hz, J ¼ 1.9 Hz,
H5), 6.98 (1H, d, J ¼ 1.9 Hz, H3), 4.00 (3H, s, OCH3); 13C NMR (CDCl3,
s, OCH3); 13C NMR (CDCl3, 75 MHz):
d 193.95 (1C, s, CSNH), 154.97,
142.03, 138.76, 136.40, 126.91, 126.06 (2C, q, J ¼ 3.8 Hz, C30, C50),

394
J. Kozic et al. / European Journal of Medicinal Chemistry 56 (2012) 387e395
123.93 (2C, s, C20, C60), 121.89, 112.15, 56.75 (1C, s, OCH3); LRMS-
APCIþ: m/z 346 [M þ H]þ; Anal. Calcd. for C15H11ClF3NOS (345.77):
C 52.10, H 3.21, N 4.05, S 9.27, Found: C 52.33, H 3.39, N 4.26, S
9.39.
(346.66): C 48.51, H 2.91, N 4.04, S 9.25, Found: C 48.41, H 2.75, N
4.25, S 9.02.
5.1.3.15. 5-Chloro-2-methoxy-N-(3-bromophenyl)benzothioamide
(4o). Yield: 50.2%, yellow solid; m. p. 103e105 ꢂC; IR (ATR): 3238 (
n
5.1.3.10. 5-Chloro-2-methoxy-N-(3-nitrophenyl)benzothioamide (4j).
NH), 1590, 1532 (CC aromatic), 1478, 1463 (CC aromatic) cmꢀ1 1H
;
Yield: 5.3%, yellow solid; m. p. 158e159 ꢂC; IR (ATR): 3291 (
n
NH),
NMR (CDCl3, 300 MHz):
d
10.54 (1H, s, NH), 8.41 (1H, d, J ¼ 2.7 Hz,
1560 (
(
n
CC aromatic), 1531 (nas NO2), 1479, 1459 (
n
CC aromatic),1344
12.23 (1H, s, NH),
H6), 7.98 (1H, t, J ¼ 1.9 Hz, H20), 7.69 (1H, m, H40), 7.45e7.35 (2H, m,
ns NO2) cmꢀ1
;
1H NMR (DMSO-d6, 500 MHz):
d
H4, H60), 7.29 (1H, t, J ¼ 8.1 Hz, H50), 6.93 (1H, d, J ¼ 8.8 Hz, H3), 3.96
9.13 (1H, t, J ¼ 2.2 Hz, H20), 8.29 (1H, m, H40), 8.12 (1H, m, H60), 7.73
(1H, t, J ¼ 8.2 Hz, H50), 7.51e7.45 (2H, m, H4, H6), 7.17 (1H, d,
J ¼ 8.5 Hz, H3), 3.82 (3H, s, OCH3); 13C NMR (DMSO-d6, 125 MHz):
(3H, s, OCH3); 13C NMR (DMSO-d6, 75 MHz):
d 193.33 (1C, s, CSNH),
153.20, 140.23, 134.48, 132.12, 130.11, 129.85, 129.61, 126.88, 126.83,
122.77, 122.24, 113.09, 56.78 (1C, s, OCH3); LRMS-APCIþ: m/z 356
[M þ H]þ; Anal. Calcd. for C14H11BrClNOS (356.67): C 47.14, H 3.11, N
3.93, S 8.99, Found: C 47.38, H 3.34, N 3.96, S 9.36.
d
194.55 (1C, s, CSNH), 153.04, 147.70, 140.56, 134.78, 130.36, 128.98,
128.94, 127.85, 124.10, 120.93, 116.90, 113.98, 56.47 (1C, s, OCH3);
LRMS-APCIþ: m/z 323 [M þ H]þ; Anal. Calcd. for C14H11ClN2O3S
(322.77): C 52.10, H 3.44, N 8.68, S 9.93, Found: C 51.92, H 3.28, N
8.37, S 9.78.
5.1.3.16. 5-Chloro-2-methoxy-N-(4-bromophenyl)benzothioamide
(4p). Yield: 50.3%, yellow solid; m. p. 116e117 ꢂC; IR (ATR): 3299 (
n
NH), 1594, 1551, 1479, 1458 (CC aromatic) cmꢀ1 1H NMR (CDCl3,
;
5.1.3.11. 5-Chloro-2-methoxy-N-(4-nitrophenyl)benzothioamide (4k).
300 MHz):
d
10.53 (1H, s, NH), 8.43 (1H, d, J ¼ 2.8 Hz, H6), 7.72e7.62
Yield: 17.0%, yellow solid; m. p. 171e172 ꢂC; IR (ATR): 3276 (
n
NH),
(2H, m, H30, H50), 7.58e7.50 (2H, m, H20, H60), 7.38 (1H, dd,
J ¼ 8.8 Hz, J ¼ 2.8 Hz, H4), 6.93 (1H, d, J ¼ 8.8 Hz, H3), 3.99 (3H, s,
1594 (CC aromatic),1565 (nas NO2),1493, 1477 (CC aromatic),1322 (ns
NO2) cmꢀ1
;
1H NMR (DMSO-d6, 300 MHz):
d
12.28 (1H, s, NH),
OCH3); 13C NMR (CDCl3, 75 MHz):
d 193.09 (1C, s, CSNH), 153.20,
8.38e8.27 (4H, m, H20, H30, H50, H60), 7.52e7.44 (2H, m, H4, H6), 7.16
(1H, d, J ¼ 8.8 Hz, H3), 3.81 (3H, s, OCH3); 13C NMR (DMSO-d6,
138.06,134.50, 132.09, 131.96 (2C, s, C30, C50), 129.63,129.00,1þ26.84,
125.63 (2C, s, C20, C60), 113.08, 56.77 (1C, s, OCH3); LRMS-APCI : m/z
356 [M þ H]þ; Anal. Calcd. for C14H11BrClNOS (356.67): C 47.14, H
3.11, N 3.93, S 8.99, Found: C 47.19, H 3.38, N 4.15, S 9.21.
75 MHz):
d 195.09 (1C, s, CSNH), 153.02, 145.32, 144.25, 135.06,
130.42, 128.98, 124.72 (2C, s, C30, C50), 124.12, 122.53 (2C, s, C20, C60),
113.98, 56.46 (1C, s, OCH3); LRMS-APCIþ: m/z 323 [M þ H]þ; Anal.
Calcd. for C14H11ClN2O3S (322.77): C 52.10, H 3.44, N 8.68, S 9.93,
Found: C 52.37, H 3.35, N 8.85, S 10.05.
5.1.3.17. 5-Chloro-2-methoxy-N-(3-trifluoromethylphenyl)benzothi-
oamide (4q). Yield: 31.3%, yellow solid; m. p. 93e94 ꢂC; IR (ATR):
3303 (
(CDCl3, 300 MHz):
n ;
NH), 1573, 1540, 1484, 1453 (CC aromatic) cmꢀ1 1H NMR
5.1.3.12. 5-Chloro-2-methoxy-N-(3-chlorophenyl)benzothioamide
(4l). Yield: 11.2%, yellow solid; m. p. 103e104 ꢂC; IR (ATR): 3243 (
d
10.66 (1H, s, NH), 8.43 (1H, d, J ¼ 2.8 Hz, H6),
n
8.01, 7.55 (4H, m, H20, H40, H50, H60), 7.39 (1H, dd, J ¼ 8.8 Hz,
NH), 1592, 1480, 1463 (CC aromatic) cmꢀ1
300 MHz):
;
1H NMR (CDCl3,
J ¼ 2.8 Hz, H4), 6.94 (1H, d, J ¼ 8.8 Hz, H3), 4.01 (3H, s, OCH3); 13
C
d
10.55 (1H, s, NH), 8.42 (1H, d, J ¼ 2.7 Hz, H6), 7.86 (1H,
NMR (CDCl3, 75 MHz): d 193.66 (1C, s, CSNH), 153.25, 139.49,
t, J ¼ 2.0 Hz, H20), 7.64 (1H, m, H40), 7.38 (1H, dd, J ¼ 8.9 Hz,
J ¼ 2.7 Hz, H4), 7.36 (1H, t, J ¼ 8.0 Hz, H50), 7.27 (1H, m, H60), 6.93
(1H, d, J ¼ 8.9 Hz, H3), 4.00 (3H, s, OCH3); 13C NMR (CDCl3, 75 MHz):
134.52, 132.24, 131.28 (1C, q, J ¼ 32.5 Hz, C30), 129.41, 128.18, 127.48,
126.85, 124.79 (1C, q, J ¼ 270.9 Hz, CF3), 123.43 (1C, q, J ¼ 3.7 Hz,
C20), 120.90 (1C, q, J ¼ 3.9 Hz, C40), 113.10, 56.78 (1C, s, OCH3);
LRMS-APCIþ: m/z 346 [M þ H]þ; Anal. Calcd. for C15H11ClF3NOS
(345.77): C 52.10, H 3.21, N 4.05, S 9.27, Found: C 52.02, H 3.37, N
3.89, S 8.98.
d
193.31 (1C, s, CSNH), 153.19, 140.10, 134.51, 134.41, 132.13, 129.86,
129.60, 126.95, 126.84, 124.06, 122.27, 113.08, 56.77 (1C, s, OCH3);
LRMS-APCIþ: m/z 312 [M þ H]þ; Anal. Calcd. for C14H11Cl2NOS
(312.21): C 53.86, H 3.55, N 4.49, S 10.27, Found: C 53.54, H 3.27, N
4.65, S 10.35.
5.1.3.18. 5-Chloro-2-methoxy-N-(4-trifluoromethylphenyl)benzothi-
oamide (4r). Yield: 8.1%, yellow solid; m. p. 102e103 ꢂC; IR (ATR):
5.1.3.13. 5-Chloro-2-methoxy-N-(4-chlorophenyl)benzothioamide
3260 (
(CDCl3, 300 MHz):
n ;
NH), 1593, 1550, 1476, 1463 (CC aromatic) cmꢀ1 1H NMR
(4m). Yield: 44.0%, yellow solid; m. p.106e107 ꢂC; IR (ATR): 3292 (
n
d
10.68 (1H, s, NH), 8.42 (1H, d, J ¼ 2.7 Hz, H6),
NH), 1592, 1552, 1477 and 1460 (CC aromatic) cmꢀ1
;
1H NMR
8.00e7.90 (2H, m, H30, H50), 7.74e7.64 (2H, m, H20, H60), 7.40 (1H,
dd, J ¼ 8.8 Hz, J ¼ 2.7 Hz, H4), 6.94 (1H, d, J ¼ 8.8 Hz, H3), 4.01 (3H, s,
(CDCl3, 300 MHz):
d
10.54 (1H, s, NH), 8.43 (1H, d, J ¼ 2.5 Hz, H6),
7.77e7.66 (2H, m, H30, H50), 7.44e7.33 (3H, m, H4, H20, H60), 6.93
(1H, d, J ¼ 8.8 Hz, H3), 3.99 (3H, s, OCH3); 13C NMR (CDCl3, 75 MHz):
OCH3); 13C NMR (CDCl3, 75 MHz):
d 193.50 (1C, s, CSNH), 153.18,
141.97, 134.54, 132.27, 129.69, 128.43 (1C, q, J ¼ 32.9 Hz, C40), 126.94,
126.08 (2C, q, J ¼ 3.7 Hz, C30, C50), 123.80 (2C, s, C20, C60), 123.78 (1C,
q, J ¼ 270.2 Hz, CF3), 113.13, 56.81 (1C, s, OCH3); LRMS-APCIþ: m/z
346 [M þ H]þ; Anal. Calcd. for C15H11ClF3NOS (345.77): C 52.10, H
3.21, N 4.05, S 9.27, Found: C 51.85, H 2.98, N 3.79, S 9.17.
d
193.17 (1C, s, CSNH), 153.22, 137.56, 134.51, 132.08, 129.62, 129.01
(2C, s, C30, C50), 126.84, 125.41 (2C, s, C20, C60), 113.08, 56.76 (1C, s,
OCH3); LRMS-APCIþ: m/z 312 [M H]þ; Anal. Calcd. for
þ
C14H11Cl2NOS (312.21): C 53.86, H 3.55, N 4.49, S 10.27, Found: C
53.74, H 3.65, N 4.52, S 10.39.
5.2. In vitro antimycobacterial assay
5.1.3.14. 5-Chloro-2-methoxy-N-(3,4-chlorophenyl)benzothioamide
(4n). Yield: 4.9%, yellow solid; m. p. 122e123 ꢂC; IR (ATR): 3301 (
n
The in vitro antimycobacterial activity of the prepared
compounds was determined against M. tuberculosis My 331/88
(10ꢀ3 dilution of strain), M. avium My 330/88 (10ꢀ5 dilution of
strain), M. kansasii My 235/80 (10ꢀ4 dilution of strain) and
M. kansasii 6509/96 (10ꢀ4 dilution of strain). All of the strains were
obtained from the Czech National Collection of Type Cultures
(CNCTC) with the exception of M. kansasii 6509/96, which was
clinically isolated. The antimycobacterial activity of the compounds
NH), 1584, 1536, 1475, 1463 (CC aromatic) cmꢀ1
300 MHz):
;
1H NMR (CDCl3,
d
10.57 (1H, s, NH), 8.44 (1H, d, J ¼ 2.7 Hz, H6), 7.98 (1H,
d, J ¼ 2.5 Hz, H20), 7.63 (1H, dd, J ¼ 8.7 Hz, J ¼ 2.5 Hz, H60), 7.49 (1H,
d, J ¼ 8.7 Hz, H50), 7.40 (1H, dd, J ¼ 8.8 Hz, J ¼ 2.7 Hz, H4), 6.94 (1H,
d, J ¼ 8.8 Hz, H3), 4.00 (3H, s, OCH3); 13C NMR (CDCl3, 75 MHz):
d
193.47 (1C, s, CSNH),153.23, 138.35, 134.66, 132.70, 132.33,130.43,
130.28, 129.35, 127.00, 125.71, 123.50, 113.11, 56.84 (1C, s, OCH3);
LRMS-APCIþ: m/z 346 [M þ H]þ; Anal. Calcd. for C14H10Cl3NOS
was determined in a Sula’s semisynthetic medium (SEVAC, Prague,
ꢀ

J. Kozic et al. / European Journal of Medicinal Chemistry 56 (2012) 387e395
395
Czech Republic) via the micromethod for the determination of the
minimum inhibitory concentration (MIC) at 37 ꢂC after 14 and 21
days, and after 7, 14 and 21 days for M. kansasii. The tested
compounds were added to the medium as DMSO solutions and INH
was used as a standard in sterile water solution. The following
concentrations of the tested compounds were used: 1000, 500, 250,
DMSO, a control for determination of 0% viability (incubation with
10% DMSO), a control for determination of possible interaction of
the tested compound with the reagent, and a control for the
background of the incubation medium were also prepared. The
tested compounds and control samples were incubated with the
cells for 24 h at 37 ꢂC. After the incubation period, the reagent from
the kit was added to the wells and the mixture was maintained at
37 ꢂC for 1.5 h. Then, the absorbance at 490 nm was measured with
125, 62.5, 32, 16, 8, 4, 2, 1 and 0.5
m
mol Lꢀ1. INH was used in the
mol Lꢀ1
concentration range 0.5e250
m
.
a
plate analyser Infinite M200 (Tecan Group, Männedorf,
5.3. In vitro enzymatic assay
Switzerland). The results were statistically evaluated in Microsoft
Excel 2010 (Microsoft Corporation, Redmond, WA, USA) and the
IC50 value for each tested compound was determined using
GraphPad Prism 5.02 (GraphPad Software, San Diego, CA, USA).
The gene encoding isocitrate lyase was amplified by PCR from
mycobacterium H37Rv genomic DNA. The amplified DNA was
digested using NdeI and HindIII, cloned into the pET-28b(þ)
plasmid vector Novagen (Merck KGaA, Darmstadt, Germany) and
the recombinant plasmid was transformed into E. coli HB101. DNA
sequencing was used 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 OD595 ¼ 0.6 was achieved. The
Acknowledgments
This work was financially supported by IGA NT 13346 (2012),
ꢀ
GAUK 27610/2010, FRVS 665/2012 and SVV 2012-265-001. The
publication is co-financed by the European Social Fund and the
state budget of the Czech Republic. TEAB, project no. CZ.1.07/2.3.00/
20.0235.
culture was then induced with 1 mmol Lꢀ1 isopropyl-
b-D-thio-
galactopyranoside solution and incubated at 30 ꢂC for an additional
4 h. The cells were harvested by centrifugation at 4 ꢂC and resus-
pended in BugBuster Protein Extraction Reagent Novagen (Merck
KGaA, Darmstadt, Germany). Histidine-tagged protein was purified
using an Äkta purifier (Amersham Biosciences, Valley Stream, NY,
USA) and confirmed by SDS-polyacrylamide gel electrophoresis
(SDS-PAGE) followed by Coomassie staining.
References
[1] WHO report, Global Tuberculosis Control (2011).http://www.who.int/tb/
publications/global report/2011/gtbr11 full.pdf.
[2] S.T. Cole, G. Riccardi, New tuberculosis drugs on the horizon, Curr. Opin.
Microbiol. 14 (2011) 570e576.
[3] P.E.A. Da Silva, J.C. Palomino, Molecular basis and mechanisms of drug resis-
tance in Mycobacterium tuberculosis: classical and new drugs, J. Antimicrob.
Chemother. 66 (2011) 1417e1430.
[4] G. Lamichhane, Novel targets in M. tuberculosis: search for new drugs, Trends
Mol. Med. 17 (2011) 25e33.
[5] E.J. Munoz-Elias, J.D. McKinney, Mycobacterium tuberculosis isocitrate lyases 1
and 2 are jointly required for in vivo growth and virulence, Nat. Med. 11
(2005) 638e644.
[6] J.D. McKinney, K.H. zu Bentrup, E.J. Munoz-Elias, A. Miczak, B. Chen, W.T. Chan,
D. Swenson, J.C. Sacchettini, W.R. Jacobs, D.G. Russell, Persistence of Myco-
bacterium tuberculosis in macrophages and mice requires the glyoxylate shunt
enzyme isocitrate lyase, Nature 406 (2000) 735e738.
Isocitrate lyase activity was assayed according to the protocol
reported by Dixon and Kornberg [15]. The enzyme assay was
optimised in the final volume of 1 mL. The reaction buffer contained
50 mmol Lꢀ1 KH2PO4, 4 mmol Lꢀ1 MgCl2$6H2O, 4 mmol Lꢀ1 phe-
nylhydrazine hydrochloride, 12 mmol Lꢀ1 cysteine, H2O and KOH to
pH 7. The isocitrate cleavage was measured by the change in
absorbance at 324 nm associated with the formation of glyoxylate
phenyl hydrazone. Each tested compound was dissolved in DMSO
to prepare a 10 mmol Lꢀ1 solution, and 10
added to 939 L reaction buffer and 1 L of recombinant isocitrate
mL of this solution was
m
m
[7] M. Kratky, J. Vinsova, Salicylanilide ester prodrugs as potential antimicrobial
agents e a review, Curr. Pharm. Des. 17 (2011) 3494e3505.
lyase. ICL was used as phosphate buffer and glycerol solution with
concentration 0.58 mg mLꢀ1. Finally the reaction was started by the
[8] D.J. Hlasta, J.P. Demers, B.D. Foleno, S.A. Fraga-Spano, J.H. 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.
[9] 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.
addition of 0.2
tion. 3-Nitropropionic acid served as the positive control. The
inhibitory activity of DMSO alone (10 L) was subtracted from the
m
mol of (þ)-potassium Ds-threo-isocitrate in solu-
m
activities of the evaluated compounds.
5.4. In vitro cytotoxicity assay
Measurement of the in vitro cytotoxicity of the most active
compounds was carried out in the human hepatic cell line Hep G2
(passage 12) (Health Protection Agency Culture Collections -
ECACC, Salisbury, UK) using the CellTiter 96 AQueous One Solution
Assay method (Promega G3580, East Port, Prague, Czech Republic).
To start, 10, 000 cells were placed in each well of a 96-well plate
(NUNC, Schoeller, Prague, Czech Republic). The incubation medium
was composed of Minimum Essentials Eagle Medium
(SigmaeAldrich, Prague, Czech Republic), 1% glutamine, 10% foetal
bovine serum (PAA, Biotech, Prague, Czech Republic) and 1%
nonessential amino acids. The incubation period was 24 h and
carried out in an incubation device (Shel Lab, Cornelius, OR, USA) at
37 ꢂC with a 5% atmosphere of CO2. The cells were then inspected
microscopically. Each of the tested compounds was dissolved in
DMSO and evaluated at eight concentrations in quadruplicate. A
control for determination of 100% viability, a control for action of 1%
[10] K. Waisser, O. Bures, P. Holy, J. Kunes, R. Oswald, L. Jiraskova, M. Pour,
V. Klimesova, L. Kubicova, J. Kaustova, Relationship between the structure and
antimycobacterial activity of substituted salicylanilides, Arch. Pharm. 336
(2003) 53e71.
[11] K. Waisser, J. Matyk, H. Divisova, P. Husakova, J. Kunes, V. Klimesova,
J. Kaustova, U. Mollmann, H.M. Dahse, M. Miko, The oriented development of
antituberculotics: salicylanilides, Arch. Pharm. 339 (2006) 616e620.
[12] I.Y. Oren, E. Aki-Sener, C. Ertas, O.T. Arpaci, I. Yalcin, N. Altanlar, Synthesis and
microbiological activity of some substituted N-(2-hydroxy-4-nitrophenyl)
benzamides and phenylacetamides as possible metabolites of antimicrobial
active benzoxazoles, Turk. J. Chem. 28 (2004) 441e449.
[13] M.A. Weidner-Wells, K.A. Ohemeng, V.N. Nguyen, S. Fraga-Spano,
M.J. Macielag, H.M. Werblood, B.D. Foleno, G.C. Webb, J.F. Barrett, D.J. Hlasta,
Amidino benzimidazole inhibitors of bacterial two-component systems, Bio-
org. Med. Chem. Lett. 11 (2001) 1545e1548.
[14] J. Kunes, V. Balsanek, M. Pour, K. Waisser, J. Kaustova, On the relationship
between the substitution pattern of thiobenzanilides and their anti-
mycobacterial activity, Farmaco 57 (2002) 777e782.
[15] G.H. Dixon, H.L. Kornberg, Assay methods for key enzymes of the glyoxylate
cycle, Proceedings of the Biochemical Society, Biochem. J. 72 (1959). 3p.