Synthesis and in vitro antimycobacterial and isocitrate lyase inhibition properties of novel 2-methoxy-2′-hydroxybenzanilides, their thioxo analogues and benzoxazoles

Article abstract of DOI:10.1016/j.ejmech.2012.08.016

A new series of 2-methoxy-2′-hydroxybenzanilide derivatives and their thioxo analogues have been synthesised and characterised by IR, NMR and elemental analysis. These compounds were investigated for their in vitro antimycobacterial activities against Mycobacterium tuberculosis 331/88, Mycobacterium avium 330/88, Mycobacterium kansasii 235/80, clinically isolated M. kansasii 6509/96 and the ability to act as in vitro isocitrate lyase inhibitors. The best ICL inhibitors were two compounds from the thiobenzanilide group (8f, 8m), which exhibited an inhibition potential that was equal to the standard compound, 3-nitropropionic acid. In addition, the best antimycobacterial properties were exhibited by benzanilide derivatives 6h, 6k and 6l with 5-Cl and 4′ or 5′ Cl/Br substitution. For all the thiobenzanilide derivatives tested, two conformers were observed in the NMR spectra, which is most likely due to the hindered rotation of the C-N bond.

Full text of DOI:10.1016/j.ejmech.2012.08.016

European Journal of Medicinal Chemistry 56 (2012) 108e119
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and in vitro antimycobacterial and isocitrate lyase inhibition properties
of novel 2-methoxy-20-hydroxybenzanilides, their thioxo analogues and
benzoxazoles
a
b
c
d
c
b
ꢀ
ꢀ
ꢀ
Ján Kozic , Eva Novotná , Marie Volková , Jirina Stolaríková , Frantisek Trejtnar , Vladimír Wsól ,
a,
*
ꢀ
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
A new series of 2-methoxy-20-hydroxybenzanilide derivatives and their thioxo analogues have been
synthesised and characterised by IR, NMR and elemental analysis. These compounds were investigated
for their in vitro antimycobacterial activities against Mycobacterium tuberculosis 331/88, Mycobacterium
avium 330/88, Mycobacterium kansasii 235/80, clinically isolated M. kansasii 6509/96 and the ability to act
as in vitro isocitrate lyase inhibitors. The best ICL inhibitors were two compounds from the thio-
benzanilide group (8f, 8m), which exhibited an inhibition potential that was equal to the standard
compound, 3-nitropropionic acid. In addition, the best antimycobacterial properties were exhibited by
benzanilide derivatives 6h, 6k and 6l with 5-Cl and 40 or 50 Cl/Br substitution. For all the thiobenzanilide
derivatives tested, two conformers were observed in the NMR spectra, which is most likely due to the
hindered rotation of the CeN bond.
Article history:
Received 10 June 2012
Received in revised form
8 August 2012
Accepted 10 August 2012
Available online 16 August 2012
Keywords:
Tuberculosis
Benzanilide
Thiobenzanilide
Ó 2012 Elsevier Masson SAS. All rights reserved.
Antimycobacterial activity
Isocitrate lyase
E, Z conformation of thiobenzanilide
1. Introduction
estimated 8.8 million new cases of TB with 1.45 million being fatal
and more than one third of the global population being infected
Benzanilide (N-phenylbenzamide) derivatives possess a broad
spectrum of biological activities. They have been found to exhibit
antimalarial [1] and antibacterial properties [2], and the derivatives
of N-(2-hydroxyphenyl)benzamide have been synthesised and
studied for the last few years as the possible metabolites of the
antibacterial active benzo[d]oxazole derivatives [3e5]. Benzanilides
are also well known for their ability to act as potassium channel
openers [6,7] and spasmolytic agents [8]. They have shown anti-
epileptic activity by affecting the central voltage-dependent sodium
channels [9]. More recently, some benzanilide derivatives have been
reported to inhibit the c-Met tyrosine kinase receptor, which is
a potentially important target for the treatment of cancer [10].
Tuberculosis (TB) is one of the most dangerous and the most
widespread infectious diseases. This fact is supported by the new
WHO report released in 2011, which stated that there were an
with Mycobacterium tuberculosis, the causative agent of TB [11].
Although most of the cases are located in developing countries, TB
has become a serious threat to the modern world due to population
migration and its increasing resistance to commonly used anti-
tuberculotics. Currently, M. tuberculosis strains have been found to
be resistant to rifampicin and INH (isoniazid), which are first line
treatments and the most powerful antituberculotics (MDReTB,
multidrug-resistant TB). In addition, strains resistant to fluo-
roquinolones and to at least one of the injectable drugs including
kanamycin, capreomycin and amikacin have been already
described (XDReTB, extensively drug-resistant TB) [12]. A further
health care problem is the coincidence of HIV and TB infection,
which is highlighted by the fact that 1.1 million of all TB cases and
0.35 million of the fatal cases involve HIV positive patients [11].
Therefore, there is still an urgent need for new antituberculotics,
particularly those with a new mode of action, that would be less toxic
and more effective than the current first and second line anti-
tuberculotic drugs that could shorten the total duration of the
* 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.2012.08.016

J. Kozic et al. / European Journal of Medicinal Chemistry 56 (2012) 108e119
109
treatment, improve the treatment of MDReTB and XDReTB, be
effective for co-administration with antiviral agents used to treat
AIDS and ultimately improve the treatment of latent TB infections,
which is crucial for elimination of TB [13,14]. The molecular target for
the last mentioned purpose appears to be the isocitrate lyase (ICL)
enzyme in the glyoxylate pathway that is well characterised in
plants, fungi, most prokaryotes and lower eukaryotes but has not
been observed in vertebrates. The glyoxylate shunt converts iso-
citrate to succinate and glyoxylate, catalysed by ICL, followed by the
addition of Acetyl-CoA to glyoxylate to form malate by malate syn-
thase. The glyoxylate shunt allows above mentioned organisms to
avoid the carbon dioxide generating steps of the Krebs cycle and
enabling them to use carbons from fats to carbohydrate synthesis.
This pathway is also upregulated during the latent phase of
M. tuberculosis where the metabolism differs from the metabolism
observed during the growing phase and the bacteria’s carbon source
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.
Isoniazid (INH), which is a first line antituberculosis drug, was used
as a standard. The compound activity against M. tuberculosis and
M. avium was evaluated after 14 and 21 days, and the compound
activity against M. kansasii was evaluated after 7, 14 and 21 days.
The MIC (minimal inhibition concentration) values represent the
lowest concentration of the tested compounds at which the inhi-
bition of mycobacterium growth is observed.
3.2. In vitro enzymatic assay
All compounds were tested for their ability to act as in vitro
inhibitor of mycobacterial enzyme isocitrate lyase. This evaluation
was performed at the Department of Biochemical Sciences,
Faculty of Pharmacy in Hradec Králové. The results are presented
is shifted to C2 substrates generated by b-oxidation of fattyacids [15].
Herein, we present the results from our study of the anti-
mycobacterial properties of 2-methoxybenzanilides and their thi-
oxo analogues.
as
a percentage of inhibition and compared to standard
3-nitropropionic acid. Ethionamide and isoniazid were employed
as negative controls (inhibition 0%).
2. Chemistry
3.3. In vitro cytotoxicity assay of selected compounds
A new series of 2-methoxy-20-hydroxybenzanilide derivatives (6)
were prepared by the condensation reaction of an appropriate
2-methoxybenzoyl chloride (2) and a substituted 2-aminophenol (5)
(Scheme 1). The acid chloride was used due to the failure of the direct
condensation in the presence of PCl3 [16] and was prepared by the
treatment of acid (1) with excess of SOCl2. Most of the aminophenols
were not commercially available and were prepared from the cor-
responding phenols (3) by mild nitration followed by reduction by
iron in acetic acid [17]. Next, the prepared anilides (6) were converted
to their thioxo analogues (8). The conversion was performed by
reaction with P4S10 in pyridine [18]. Due to the presence of the 20-OH
group in the starting anilides (6), the synthesis was complicated by
formation of a cyclic salt with P4S10, which was hydrolysed by HCl in
chloroform. The substituents on both aromatic parts of the molecule
were selected according to their effect on the antimycobacterial
activity in a previously studied group of salicylanilides [16].
The most active compounds were evaluated for their in vitro
cytotoxicity. This evaluation was performed at the Department of
Pharmacology and Toxicology, Faculty of Pharmacy in Hradec
Králové. For this purpose, the human liver cell line Hep G2 and
a standard colorimetric test based on determining the reductive
metabolic activity of the cells was used. The results are presented as
an inhibitory concentration (IC50), which is the concentration of the
tested compound required for the reduction of the cell viability
value to 50% of the maximum (control) cell viability value. To
characterize relation between toxicity and antimycobacterial
activity, selectivity index (SI) was calculated. SI value indicates the
ratio of IC50 and MIC after 14 days of incubation of M. tuberculosis
331/88.
4. Results
4.1. Discussion and conclusion
3. Pharmacology
Forty-two antimycobacterial active compounds were syn-
thesised and characterised by 1H and 13C NMR spectrometry, IR
spectrometry, elemental analysis and melting point. The best yields
(51e72%) for the 2-methoxy-20-hydroxybenzanilides (6) were ob-
tained when the condensation was performed at room temperature.
3.1. In vitro antimycobacterial assay
All 42 newly prepared compounds were tested in the Laboratory
for Mycobacterial Diagnostics and TB at the Institute of Public
Scheme 1. Preparation of 2-methoxy-20-hydroxybenzanilides (6) and their thioxo analogues (8) (R1 ¼ 4-Cl, 5-Cl, R2 ¼ 3-Cl, 4-Cl 3,4-di-Cl, 3-Br, 4-Br, 3-CF3, 4-CF3 e 2-amino-4/5-
chlorophenols are available). Reagents and conditions: (a) SOCl2, 110 ꢀC, (b) 65% HNO3, 99% CH3COOH, 40 ꢀC, (c) Fe, H2O, CH3COOH, 135 ꢀC, (d) TEA, ether, r.t., (e) 1. P4S10, pyridine,
155 ꢀC, 2. HCl, CHCl3, 110 ꢀC.

110
J. Kozic et al. / European Journal of Medicinal Chemistry 56 (2012) 108e119
The chloride (2) was added drop wise in an ether solutionwith avery
low concentration containing an equivalent of triethylamine to
a more concentrated ether solution of substituted 2-aminophenol
(5). Under these conditions, it was possible to reduce the forma-
tion of esterified amides (7) and the oxidation by-products of the
starting aminophenols (5). Despite this, four esterified amides were
obtained in sufficient amount and purity so could be biologically
evaluated. During the preparation of the thioxo analogues (8), all of
the compounds cyclised to derivatives of 2-(2-methoxyphenyl)
benzo[d]oxazole (9) with yields that were equimolar to the yields of
thioamides (22e49%). This cyclisation was also observed on silica
gel. Most of these benzoxazoles were obtained in sufficient amount
and purity and could also be biologically tested.
and 8f and 8m exhibited an activity equivalent to the standard
compound, 3-nitropropionic acid (Table 4). Benzoxazole derivatives
9 were found to be inactive, but esterified amides 7 were active
(Table 4). Therefore, we can assume that the C]O/S group is
essential for inhibitory activity.
The results of the cytotoxicity assay are presented in Table 5.
Unfortunately, the great drawback of most of the amides and thi-
oamides is their cytotoxicity. Only two compounds (6h and 6k)
were more toxic for M. tuberculosis than for human hepatic cell, 6h
with SI after 14 days of incubation 12.8 and 6k with SI after 14 days
of incubation 2.0. Also the best inhibition of ICL (compounds 8f and
8m) was observed at a concentration, which was higher than IC50
values of these compounds.
The in vitro antimycobacterial evaluation of the prepared
compounds was used to characterise their ability to act against
actively growing mycobacteria under nutrient and oxygen rich
conditions. It was revealed that in the group of benzanilides were
more active derivatives with 5-Cl than with 4-Cl substitution in the
acid portion of the molecule. Further, the substitution of the anilide
moiety in position 4 or 5 by Cl or Br was more effective than the
substitution with CF3 and 4,5-di-Cl (Table 1). The activities in
the thiobenzanilides group were very similar, and surprisingly, the
thioxo group had a negative effect on the activities of the most
active anilides (Table 2). However, thioxo group improved the
activities of compounds with 4,5-di-Cl and CF3 substitution in the
aniline moiety. All of the thioamides exhibited activities against
INH resistant M. avium and M. kansasii 235/80. The cyclisation to
For all of the prepared thiobenzanilides (8), an interesting result
was observed in the proton NMR spectrum, which was measured in
DMSO under ambient conditions. Two sets of hydrogen resonance
signals were observed with a relative integral intensity ratio of
approximately 1:0.15. Contamination of the thioamides (8) by the
starting benzanilides (6) was excluded by comparison of their
hydrogen shifts with the hydrogen shifts of the starting benzani-
lides, whose NMR spectrum was also recorded in DMSO. Further
confirmation of the purity was provided by IR spectra and
elemental analyses. Based on the coupling constant values, the
shape of the peaks and the chemical shift values, the interpretation
of both sets of hydrogen signals revealed that each more intense
hydrogen signal corresponded to the less intense one. Therefore,
there were two OH singlets, two NH singlets, four aromatic
doublets of doublets, eight aromatic doublets and two OCH3
singlets for compound 8i (Fig. 1). A similar situation was observed
in the carbon NMR spectra. However, two complete sets of reso-
nance signals were not always observed due to the low intensity of
the less intense signals. Finally, when the proton NMR spectrum of
compound 8i was recorded in DMSO at different temperatures
ranging from 25 to 100 ꢀC, each pair of hydrogen resonance signals
merged into one signal at 90 ꢀC.
the benzoxazole derivatives
9 negatively affected the anti-
mycobacterial properties (Table 3). Therefore, one possible expla-
nation of low activities of benzoxazole derivatives could be that the
mycobacteria cannot cleave the benzoxazole ring to the amide,
which is most likely the active form. Table 3 also shows the MIC
values of some esterified amides 7.
The enzymatic assay of prepared compounds was used to
characterised their ability to act as ICL inhibitor and thus potentially
against mycobacteria in the latent phase of infection. According to
this assay, thioamides 8 were better inhibitors of ICL than amides 6,
The most likely explanation for this phenomenon is based on
the ability of compounds 8 to adopt relatively stable E or Z
Table 1
Antimycobacterial activities of the prepared 2-methoxy-20-hydroxybenzanilide derivatives 6aen.M
R1
R2
R3
R4
MIC [m ]
mol Lꢁ1
M. tuberculosis My 331/88
M. avium My 330/88
M. kansasii My 235/80
M. kansasii My 6509/96
Days
14
21
14
21
7
14
21
7
14
21
6a
6b
6c
6d
6e
6f
6g
6h
6i
6j
6k
6l
6m
6n
INH
Cl
Cl
Cl
Cl
Cl
Cl
Cl
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
H
Cl
Br
H
CF3
H
Cl
H
Cl
Br
H
CF3
H
H
8
8
62.5
125
>1000
125
62.5
62.5
250
62.5
125
125
500
62.5
62.5
62.5
>250
125
125
62.5
125
500
125
62.5
ea
62.5
125
500
125
62.5
250
250
32
125
125
500
125
62.5
250
250
62.5
16
125
500
8
250
62.5
>250
16
16
62.5
62.5
250
125
62.5
250
250
16
62.5
62.5
250
125
62.5
250
250
16
Cl
Cl
H
Br
H
CF3
H
Cl
Cl
H
125
500
125
125
250
250
4
16
125
4
125
500
125
125
250
250
4
32
125
8
>1000
125
62.5
62.5
250
250
125
125
500
250
125
62.5
250
250
8
250
16
4
16
8
16
16
125
500
4
125
500
8
250
62.5
>250
125
250
4
250
62.5
4
125
250
8
250
125
4
125
250
8
250
125
4
Br
H
CF3
8
8
62.5
62.5
125
500
250
0.5
500
250
0.5
ea
32
>250
>250
INH e isoniazid.
a
MIC value was impossible to determine.

J. Kozic et al. / European Journal of Medicinal Chemistry 56 (2012) 108e119
111
Table 2
Antimycobacterial activities of the prepared 2-methoxy-20-hydroxythiobenzanilide derivatives 8aen.
R1
R2
R3
R4
MIC [m ]
mol Lꢁ1
M. tuberculosis My 331/88
M. avium My 330/88
M. kansasii My 235/80
M. kansasii My 6509/96
Days
14
21
14
21
7
14
21
7
14
21
8a
8b
8c
8d
8e
8f
8g
8h
8i
8j
8k
8l
8m
8n
INH
Cl
Cl
Cl
Cl
Cl
Cl
Cl
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
H
Cl
Br
H
CF3
H
Cl
H
Cl
Br
H
CF3
H
H
16
62.5
32
16
32
32
32
16
16
16
32
16
62.5
32
32
62.5
32
32
32
62.5
62.5
32
32
32
32
32
62.5
32
0.5
32
32
62.5
32
32
62.5
32
32
32
32
62.5
32
62.5
62.5
62.5
62.5
32
62.5
125
32
16
16
16
16
16
ea
32
16
8
32
62.5
32
32
32
62.5
62.5
32
8
62.5
32
62.5
125
62.5
62.5
62.5
62.5
125
62.5
16
62.5
62.5
16
62.5
62.5
>250
16
32
16
16
32
32
32
32
16
16
16
16
32
16
4
32
62.5
32
32
32
62.5
62.5
32
32
32
32
16
62.5
32
4
32
Cl
Cl
H
Br
H
CF3
H
Cl
Cl
H
125
62.5
32
62.5
62.5
125
32
32
62.5
32
32
62.5
62.5
32
32
62.5
>250
32
16
8
Br
H
CF3
16
32
62.5
32
62.5
62.5
>250
ea
32
>250
62.5
62.5
>250
0.5
4
INH e isoniazid.
a
MIC value was impossible to determine.
conformation about the CSeNH bond (Fig. 2). These conformers
manifested themselves spectroscopically in NMR spectra due to
slow rotation on the NMR timescale, but could not be isolated due
to fast rotation on the laboratory timescale.
The literature provides much more information on E, Z confor-
mation of nitrogen containing compounds in the group of
benzanilides than in thiobenzanilide group. But to some extent
these two groups are similar to each other. Benzanilides are well
known for their ability to prefer due to steric reasons either E or Z
conformation about the COeNH bond [19]. The free rotation about
this bond is restricted due to the existence of the rotational barrier,
what can make the conformers observable on the NMR timescale.
Table 3
Antimycobacterial activities of isolated esterified amides 7aed and benzoxazole by-products 9aej.
R1
R2
R3
R4
MIC [m ]
mol Lꢁ1
M. tuberculosis My 331/88
M. avium My 330/88
M. kansasii My 235/80
M. kansasii My 6509/96
Days
14
21
14
21
7
14
21
7
14
21
7a
7b
7c
7d
9a
9b
9c
9d
9e
9f
9g
9h
9i
9j
INH
Cl
Cl
H
H
Cl
Cl
Cl
Cl
Cl
H
H
H
H
H
H
H
Cl
Cl
H
H
H
H
H
Cl
Cl
Cl
Cl
Cl
H
H
Cl
H
H
Cl
Br
H
Cl
CF3
Cl
CF3
Cl
Cl
4
250
8
8
250
8
62.5
62.5
62.5
62.5
62.5
250
250
125
125
250
125
250
125
250
>250
62.5
62.5
62.5
62.5
62.5
500
250
125
125
250
125
250
125
250
>250
16
32
250
8
125
250
8
8
250
4
16
250
8
62.5
250
8
ea
4
250
125
250
250
125
250
125
125
250
125
62.5
0.5
250
125
250
250
125
250
125
125
250
125
62.5
0.5
ea
8
16
8
8
8
62.5
250
250
125
ea
62.5
250
250
125
250
250
125
250
125
250
>250
62.5
250
250
125
250
250
125
250
125
250
>250
62.5
250
250
125
250
250
125
250
125
62.5
4
62.5
250
250
125
250
250
125
250
125
62.5
4
62.5
250
250
125
250
250
125
250
125
62.5
4
H
Br
CF3
H
Cl
H
H
Cl
Cl
Br
H
250
125
250
125
250
>250
Br
CF3
H
INH e isoniazid.
a
MIC value was impossible to determine.

112
J. Kozic et al. / European Journal of Medicinal Chemistry 56 (2012) 108e119
Table 4
Inhibition activity of prepared compounds 6e9 against isocitrate lyase. Concentra-
tion of all the tested and control compounds was 10
m
mol Lꢁ1
.
% Of inhibition
Standard
deviation
% Of inhibition
Standard
deviation
6a
6b
6c
6d
6e
6f
4
0
0
0
0
7
0
ꢂ3.01
6h
6i
6j
6k
6l
6m
6n
10
0
7
0
0
ꢂ0.17
0
0
0
0
0
ꢂ0.50
0
0
ꢂ2.33
2
0
ꢂ0.66
6g
0
0
7a
7b
0
8
0
7c
7d
3
9
ꢂ1.44
ꢂ2.22
ꢂ2.41
8a
8b
8c
8d
8e
8f
10
9
0
10
0
23
0
ꢂ2.74
ꢂ2.28
0
8h
8i
8j
8k
8l
8m
8n
0
0
9
7
8
21
8
0
0
ꢂ3.30
ꢂ1.94
ꢂ1.37
ꢂ2.14
ꢂ4.8
Fig. 1. 1H NMR spectrum of compound 8i in DMSO at ambient temperature. The
relative integral intensity ratio of both conformers 1:0.15. For interpretation of signals
of more intense conformer see Experimental protocols.
ꢂ1.51
0
ꢂ1.36
0
8g
9a
9b
9c
9d
0
0
0
0
0
25
0
0
0
0
0
0
0
4.13
0
9f
9g
9h
9i
0
4
0
0
0
25
0
0
is involved in both H-bonded five-membered ring and H-bond six-
membered ring, resulting in a rigid and planar arrangement of
molecule. In addition, the three-centre hydrogen bond acts cooper-
atively, 5- and 6-membered hydrogen bonding systems enforce each
other. The extraordinary stability of this H-bonds system has been
demonstrated by the formation of helices based on the m-oligomers
of the 2,20-dialkoxybenzanilides [23]. Thus, we can assume that
three-centre hydrogen bond in Z-conformation of thioamides (8)
presented further contribution to the rotational barrier (Fig. 2). This
can also explainwhy we have not observed this phenomenon for the
previously studied 2-methoxythiobenzanilide derivatives without
2-OH group in the anilide part of the molecule.
Finally, some contribution to the rotational barrier of
compounds 8 could be as well caused by the steric effect of the
substituents at the ortho positions of the aromatic rings and thus by
preference of conformations with minimal steric interactions [19].
Steric effect possesses also sulphur atom itself, because of its
greater size and longer C]S bond than C]O bond [24].
ꢂ3.27
0
0
9e
9j
0
4.13
0
3-NPA
ETA
INH
3-NPA
ETA
INH
0
0
0
3-NPA e 3-nitropropionic acid, ETA e ethionamide, INH e isoniazid.
For the substantial contribution to the rotational barrier is
responsible the conjugation of the lone electron pair of nitrogen
atom over the carbonyl group and thus, partial double bond char-
acter of amide bond [20]. The existence of rotational barrier has
been also shown in thioamide group. In thioformamide, it has been
calculated that the CSeNH bond is in greater abundance partial
double than COeNH bond of formamide, because of the greater
conjugation of the lone electron pair of nitrogen over thiocarbonyl
group of thioformamide than over carbonyl group of formamide
[21]. This can be the reason that we have been able to observe the
two conformers of 8 but not of 6.
The conformational interconversion of compounds (8) could
be also slowed down by intramolecular hydrogen bonds. In
Z-conformer it is possible the existence of three-centre hydrogen
bond (Fig. 2), which has been described in similar compound e 2,20-
dimethoxybenzanilide [22]. In this amide, the CONH group hydrogen
Based on this information, we have concluded that this
phenomenon and conformation behaviour of compounds
8
requires more in-depth study, and the results of this study will be
presented in the near future.
5. Experimental protocols
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.
Table 5
Cytotoxicity of 2-methoxy-20-hydroxybenzanilide derivatives 6aen and 2-methoxy-
20-hydroxythiobenzanilide derivatives 8aen.
5.1. Chemistry
IC50
mol Lꢁ1
7.34
87.02
11.52
46.62
5.63
SI for
My 331/88, 14 d
IC50
mol.Lꢁ1
SI for
My 331/88, 14 d
[
m
]
[m
]
5.1.1. General methods
6a
6b
6c
6d
6e
6f
0.92
0.70
0.02
0.37
0.05
0.02
0.10
6h
6i
6j
6k
6l
6m
6n
51.30
ea
12.83
e
The reactions were monitored, and the purity of products was
verified by thin layer chromatography in which the plates were
coated with 0.2 mm of silica gel 60 F254 (Merck KGaA, Darmstadt,
Germany) and visualised using UV irradiation (254 and 366 nm).
2.98
7.98
4.09
4.91
15.43
0.02
2.00
0.51
0.01
0.06
3.90
25.37
6g
8a
8b
8c
8d
8e
8f
5.11
8.49
3.16
3.88
6.53
3.14
5.74
0.32
0.14
0.10
0.24
0.20
0.10
0.18
8h
8i
8j
8k
8l
8m
8n
4.45
8.60
3.72
7.60
8.74
3.69
5.86
0.28
0.54
0.23
0.24
0.55
0.06
0.18
8g
a
IC50 value was impossible to determine.
Fig. 2. The possible conformers of thiobenzanilide derivative 8i.

J. Kozic et al. / European Journal of Medicinal Chemistry 56 (2012) 108e119
113
Column chromatography was performed using silica gel 60 with
1544 (amide II), 1497, 1487, 1467 (
n
CC aromatic) cmꢁ1
;
1H NMR
a particle size of 0.063e0.2 mm (Fluka, Prague, Czech Republic).
(DMSO-d6, 500 MHz): d 11.09 (1H, s, OH), 10.48 (1H, s, NH), 8.56
The melting points were determined on a Melting Point M-560
(1H, s, H60), 8.03 (1H, d, J ¼ 8.5 Hz, H6), 7.35 (1H, d, J ¼ 2.0 Hz, H3),
7.19 (1H, dd, J ¼ 8.5 Hz, J ¼ 2.0 Hz, H5), 7.03 (1H, s, H30), 4.05 (3H, s,
}
apparatus (Buchi Labortechnik AG, Flawil, Switzerland) in open
capillaries and are uncorrected. The IR spectra were recorded on
a Nicolet 6700 FT-IR spectrometer (Thermo Fisher Scientific, Wal-
tham, MA, USA) over the range of 400e4000 cmꢁ1 using the ATR
technique. The NMR spectra were measured in DMSO-d6 or CDCl3
solutions at ambient temperature on a Varian Mercury Vxbb 300
(300 MHz for 1H and 75.5 MHz for 13C, Varian Comp. Palo Alto, CA,
USA) and Varian Mercury (500 MHz for 1H and 125 MHz for 13C,
OCH3); 13C NMR (DMSO-d6, 125 MHz):
d 161.64, 157.95, 146.34,
138.36, 133.00, 127.47, 124.71, 121.56, 120.59, 120.28, 119.76, 115.60,
113.33, 57.33; Anal. Calcd. for C14H10Cl3NO3 (346.59): C 48.52, H
2.91, N 4.04, Found: C 48.65, H 2.97, N 4.00.
5.1.3.4. N-(4-Bromo-2-hydroxyphenyl)-4-chloro-2-methoxybenzam-
ide (6d). Yield 51.5%, grey solid; m. p. 233e235 ꢀC; IR (ATR): 3316
Varian Comp. Palo Alto, CA, USA). The chemical shifts (
d
) are given
(
n
NH), 3178 (b,
1541 (amide II), 1503, 1482, 1458 (
(DMSO-d6, 300 MHz):
n
OH), 1635 (amide I), 1608, 1589 (
CC aromatic) cmꢁ1
10.78 (1H, s, OH), 10.42 (1H, s, NH), 8.30
n
CC aromatic),
in ppm, and tetramethylsilane was employed as the internal stan-
dard. Elemental analysis (C, H, N, S) was performed on an automatic
microanalyser CHNS-O CE instrument, FISONS EA 1110 (Thermo
Fisher Scientific, Waltham, MA, USA).
n
;
1H NMR
d
(1H, d, J ¼ 8.6 Hz, H60), 8.04 (1H, d, J ¼ 8.4 Hz, H6), 7.35 (1H, d,
J ¼ 1.9 Hz, H3), 7.19 (1H, dd, J ¼ 8.4 Hz, J ¼ 1.9 Hz, H5), 7.05 (1H, d,
J ¼ 2.2 Hz, H30), 6.99 (1H, dd, J ¼ 8.6 Hz, J ¼ 2.2 Hz, H50), 4.05 (3H,
5.1.2. General procedure for the preparation of acid chlorides (2)
2.7 mmol of 4/5-chloro-2-methoxybenzoic acid (1) was dis-
solved in 10 mL of SOCl2, and the solution was stirred and refluxed
under a CaCl2 drying tube for 3 h. Then, the excess SOCl2 was
removed under reduced pressure, and the liquid residue was used
without further purification in the next step.
s, OCH3); 13C NMR (DMSO-d6, 75 MHz):
d 161.40, 157.90, 147.72,
138.04, 132.99, 126.73, 122.04, 121.48, 121.20, 120.26, 117.24, 115.07,
113.28, 57.27; Anal. Calcd. for C14H11BrClNO3 (356.60): C 47.15, H
3.11, N 3.93, Found: C 47.26, H 3.46, N 3.93.
5.1.3.5. N-(5-Bromo-2-hydroxyphenyl)-4-chloro-2-methoxybenzam-
ide (6e). Yield 55.8%, grey solid; m. p. 226e228 ꢀC; IR (ATR): 3295 (
n
5.1.3. Generalprocedureforthepreparationofsubstituted2-methoxy-
20-hydroxybenzanilides (6)
NH), 3150 (b,
(amide II), 1503, 1482, 1457 (
d6, 500 MHz):
n
OH), 1644 (amide I), 1608, 1592 (
CC aromatic) cmꢁ1; 1H NMR (DMSO-
10.63 (1H, s, OH), 10.49 (1H, s, NH), 8.55 (1H, d,
n CC aromatic), 1548
n
First, 100 mL of an ether solution containing 2.7 mmol of
substituted 2-methoxybenzoyl chloride (2) and an equimolar
amount of TEA was added drop wise at r.t. (1.5 h) into 40 mL of
a stirred ether solution containing 2.7 mmol of substituted
2-aminophenol (5). After the addition, the mixture was stirred for
1 h at r.t. and then extracted with a 5% solution of HCl (50 mL), with
a 5% solution of NaHCO3 (50 mL) and with water (50 mL). The
organic phase was dried with Na2SO4 and purified using column
chromatography and crystallisation.
d
J ¼ 2.5 Hz, H60), 8.05 (1H, d, J ¼ 8.5 Hz, H6), 7.37 (1H, d, J ¼ 2.0 Hz,
H3), 7.20 (1H, dd, J ¼ 8.5 Hz, J ¼ 2.0 Hz, H5), 7.09 (1H, dd, J ¼ 8.6 Hz,
J ¼ 2.5 HZ, H40), 6.86 (1H, d, J ¼ 8.6 Hz, H30), 4.06 (3H, s, OCH3); 13C
NMR (DMSO-d6, 125 MHz):
d 161.58, 157.94, 145.78, 138.20, 133.01,
128.67, 128.38, 126.26, 121.95, 121.54, 120.06, 116.27, 113.34, 57.31;
Anal. Calcd. for C14H11BrClNO3 (356.60): C 47.15, H 3.11, N 3.93,
Found: C 47.35, H 3.40, N 3.70.
5.1.3.6. 4-Chloro-N-(2-hydroxy-4-(trifluoromethyl)phenyl)-2-metho-
xybenzamide (6f). Yield 51.1%, white solid; m. p. 219e221 ꢀC; IR
5.1.3.1. 4-Chloro-N-(4-chloro-2-hydroxyphenyl)-2-methoxybenza-
mide (6a). Yield 59.0%, grey solid; m. p. 223e225 ꢀC; IR (ATR): 3315
(ATR): 3228 (b,
(amide II), 1483, 1466 (
500 MHz):
n
OH),1646 (amide I),1605,1592 (
n CC aromatic),1549
(
n
NH), 3185 (b,
1548 (amide II), 1508, 1482, 1460 (
(DMSO-d6, 300 MHz):
n
OH), 1638 (amide I), 1608, 1592 (
CC aromatic) cmꢁ1
10.82 (1H, bs, OH), 10.43 (1H, s, NH), 8.35
n
CC aromatic),
n
CC aromatic) cmꢁ1 1H NMR (DMSO-d6,
;
n
;
1H NMR
d
11.01 (1H, s, OH),10.62 (1H, s, NH), 8.55 (1H, d, J ¼ 8.3 Hz,
d
H60), 8.06 (1H, d, J ¼ 8.5 Hz, H6), 7.38 (1H, d, J ¼ 1.9 Hz, H3), 7.20 (1H,
(1H, d, J ¼ 8.7 Hz, H60), 8.05 (1H, d, J ¼ 8.5 Hz, H6), 7.37 (1H, d,
J ¼ 1.9 Hz, H3), 7.20 (1H, dd, J ¼ 8.5 Hz, J ¼ 1.9 Hz, H5), 6.92 (1H, d,
J ¼ 2.3 Hz, H30), 6.87 (1H, dd, J ¼ 8.7 Hz, J ¼ 2.3 Hz, H50), 4.06 (3H, s,
dd, J ¼ 8.5 Hz, J ¼ 1.9 Hz, H5), 7.19e7.12 (2H, m, H30, H50), 4.07 (3H, s,
OCH3);13C NMR(DMSO-d6,125MHz):
d161.79,157.97,146.58,138.32,
133.08, 130.75, 123.80 (1C, q, J ¼ 31.6 Hz, C40), 123.05 (1C, q,
J ¼ 269.8 Hz, CF3),121.55,120.01,119.59,116.55 (1C, q, J ¼ 3.7 Hz, C30),
113.37, 110.65 (1C, m, C50), 57.34; Anal. Calcd. for C15H11ClF3NO3
(345.70): C 52.11, H 3.21, N 4.05, Found: C 52.17, H 3.30, N 4.25.
OCH3); 13C NMR (DMSO-d6, 75 MHz):
d 161.41, 157.92, 147.60,
138.03, 132.99, 127.17, 126.33, 121.50, 120.81, 120.27, 119.09, 114.47,
113.32, 57.29; Anal. Calcd. for C14H11Cl2NO3 (312.15): C 53.87, H
3.55, N 4.49, Found: C 53.45, H 3.94, N 4.23.
5.1.3.7. 4-Chloro-N-(2-hydroxy-5-(trifluoromethyl)phenyl)-2-metho-
xybenzamide (6g). Yield 56.5%, white solid; m. p. 238e240 ꢀC; IR
5.1.3.2. 4-Chloro-N-(5-chloro-2-hydroxyphenyl)-2-methoxybenza-
mide (6b). Yield 62.3%, orange solid; m. p. 240e242 ꢀC; IR (ATR):
(ATR): 3153 (b,
aromatic), 1567 (amide II), 1513, 1484, 1463, 1449 (
cmꢁ1; 1H NMR (DMSO-d6, 500 MHz):
n
OH), 1646 (amide I), 1620, 1605, 1596 (
CC aromatic)
11.25 (1H, s, OH), 10.58 (1H,
n CC
3294 (
aromatic), 1546 (amide II), 1506, 1496, 1480, 1458 (
cmꢁ1; 1H NMR (DMSO-d6, 300 MHz):
n
NH), 3142 (b,
n
OH), 1642 (amide I), 1610, 1593 (
CC aromatic)
10.61 (1H, bs, OH),10.50 (1H,
n
CC
n
n
d
d
s, NH), 8.74 (1H, d, J ¼ 2.2 Hz, H60), 8.06 (1H, d, J ¼ 8.5 Hz, H6), 7.37
(1H, d, J ¼ 2.0 Hz, H3), 7.30 (1H, dd, J ¼ 8.4 Hz, J ¼ 2.2 Hz, H40), 7.21
(1H, dd, J ¼ 8.5 Hz, J ¼ 2.0 Hz, H5), 7.06 (1H, d, J ¼ 8.4 Hz, H30), 4.07
s, NH), 8.42 (1H, d, J ¼ 2.6 Hz, H60), 8.05 (1H, d, J ¼ 8.4 Hz, H6), 7.37
(1H, d, J ¼ 2.0 Hz, H3), 7.21 (1H, dd, J ¼ 8.4 Hz, J ¼ 2.0 Hz, H5), 6.97
(1H, dd, J ¼ 8.6 Hz, J ¼ 2.6 Hz, H40), 6.90 (1H, d, J ¼ 8.6 Hz, H30), 4.06
(3H, s, OCH3); 13C NMR (DMSO-d6, 125 MHz):
d 161.82, 157.97,
(3H, s, OCH3); 13C NMR (DMSO-d6, 75 MHz):
d
161.62,157.96,145.37,
149.65, 138.27, 133.01, 127.53, 125.74 (1C, q, J ¼ 269.7 Hz, CF3),
121.57, 121.18 (1C, q, J ¼ 4.0 Hz, C60), 120.00, 119.86 (1C, q,
J ¼ 31.6 Hz, C50), 116.22 (1C, q, J ¼ 4.0 Hz, C40), 114.67, 113.36, 57.34;
Anal. Calcd. for C15H11ClF3NO3 (345.70): C 52.11, H 3.21, N 4.05,
Found: C 52.31, H 3.37, N 4.04.
138.23, 133.05, 128.29, 123.35, 122.73, 121.57, 120.07, 119.21, 115.71,
113.37, 57.34; Anal. Calcd. for C14H11Cl2NO3 (312.15): C 53.87, H 3.55,
N 4.49, Found: C 53.82, H 3.86, N 4.32.
5.1.3.3. 4-Chloro-N-(4,5-dichloro-2-hydroxyphenyl)-2-methoxyben-
zamide (6c). Yield 54.4%, white solid; m. p. 250e252 ꢀC; IR (ATR):
5.1.3.8. 5-Chloro-N-(4-chloro-2-hydroxyphenyl)-2-methoxybenz
amide (6h). Yield 69.0%, grey solid; m. p. 228e230 ꢀC; IR (ATR):
3302 (
n
NH), 3153 (b,
n
OH), 1633 (amide I), 1589 (
n
CC aromatic),

114
J. Kozic et al. / European Journal of Medicinal Chemistry 56 (2012) 108e119
3600e2849 (b,
n
OH), 1651 (amide I), 1614, 1593 (
CC aromatic) cmꢁ1; 1H NMR (DMSO-
10.83 (1H, bs, OH), 10.52 (1H, s, NH), 8.34 (1H, d,
n
CC aromatic),
aromatic), 1545 (amide II), 1482 (
n
CC aromatic) cmꢁ1
;
1H NMR
1558 (amide II), 1508, 1479 (
d6, 300 MHz):
n
(DMSO-d6, 500 MHz):
d 11.00 (1H, s, OH),10.70 (1H, s, NH), 8.53 (1H,
d
d, J ¼ 8.3 Hz, H60), 7.99 (1H, d, J ¼ 2.9 Hz, H6), 7.61 (1H, dd, J ¼ 8.9 Hz,
J ¼ 2.9 Hz, H4), 7.30 (1H, d, J ¼ 8.9 Hz, H3), 7.17 (1H, dd, J ¼ 8.3 Hz,
J ¼ 2.1 Hz, H50), 7.15 (1H, d, J ¼ 2.1 Hz, H30), 4.04 (3H, s, OCH3); 13C
J ¼ 8.6 Hz, H60), 7.98 (1H, d, J ¼ 2.8 Hz, H6), 7.62 (1H, dd, J ¼ 8.9 Hz,
J ¼ 2.8 Hz, H4), 7.30 (1H, d, J ¼ 8.9 Hz, H3), 6.92 (1H, d, J ¼ 2.3 Hz,
H30), 6.87 (1H, dd, J ¼ 8.6 Hz, J ¼ 2.3 Hz, H50), 4.03 (3H, s, OCH3); 13C
NMR (DMSO-d6, 125 MHz):
d 161.32, 156.21, 146.65, 133.39, 130.62,
NMR (DMSO-d6, 75 MHz):
d
160.96, 156.16, 147.69, 133.19, 130.56,
125.42,124.27 (1C, q, J ¼ 270.0 Hz, CF3),123.95 (1C, q, J ¼ 31.6 Hz, C40),
122.58, 119.66, 116.52 (1C, q, J ¼ 4.0 Hz, C30), 115.11, 110.69 (1C, q,
J ¼ 3.8 Hz, C50), 57.19; Anal. Calcd. for C15H11ClF3NO3 (345.70): C
52.11, H 3.21, N 4.05, Found: C 52.35, H 3.38, N 4.23.
127.35, 126.21, 125.38, 122.88, 120.89, 119.10, 115.11, 114.50, 57.18;
Anal. Calcd. for C14H11Cl2NO3 (312.15): C 53.87, H 3.55, N 4.49,
Found: C 53.46, H 3.74, N 4.40.
5.1.3.9. 5-Chloro-N-(5-chloro-2-hydroxyphenyl)-2-methoxybenza
mide (6i) [6]. Yield 71.8%, grey solid; m. p. 232e234 ꢀC; IR (ATR):
5.1.3.14. 5-Chloro-N-(2-hydroxy-5-(trifluoromethyl)phenyl)-2-
methoxybenzamide (6n). Yield 57.9%, white solid; m. p.190e192 ꢀC;
3296 (b,
(amide II), 1498, 1483 (
500 MHz):
n
OH), 1648 (amide I), 1612, 1595 (
n
CC aromatic), 1551
IR (ATR): 3182 (b,
1565 (amide II), 1513, 1484, 1449 (
(DMSO-d6, 500 MHz):
n
OH), 1648 (amide I), 1620, 1605 (
CC aromatic) cmꢁ1
11.26 (1H, s, OH), 10.66 (1H, s, NH), 8.72
n
CC aromatic),
n
CC aromatic) cmꢁ1
;
1H NMR (DMSO-d6,
n
;
1H NMR
d
10.60 (1H, s, OH), 10.58 (1H, s, NH), 8.40 (1H, d,
d
J ¼ 2.7 Hz, H60), 7.98 (1H, d, J ¼ 2.8 Hz, H6), 7.62 (1H, dd, J ¼ 8.9 Hz,
J ¼ 2.8 Hz, H4), 7.30 (1H, d, J ¼ 8.9 Hz, H3), 6.98 (1H, dd, J ¼ 8.5 Hz,
J ¼ 2.7 Hz, H40), 6.90 (1H, d, J ¼ 8.5 Hz, H30), 4.03 (3H, s, OCH3); 13C
(1H, d, J ¼ 2.3 Hz, H60), 7.99 (1H, d, J ¼ 2.9 Hz, H6), 7.63 (1H, dd,
J ¼ 8.9 Hz, J ¼ 2.9 Hz, H4), 7.31 (1H, d, J ¼ 8.9 Hz, H3), 7.31 (1H, m,
H40), 7.07 (1H, d, J ¼ 8.4 Hz, H30), 4.04 (3H, s, OCH3); 13C NMR
NMR (DMSO-d6, 125 MHz):
d
161.15, 156.19, 145.42, 133.31, 130.57,
(DMSO-d6,125 MHz): d 161.38,156.21,149.73,133.37,130.59,127.39,
128.13, 125.40, 123.50, 122.71, 122.66, 119.27, 115.73, 115.14, 57.19;
Anal. Calcd. for C14H11Cl2NO3 (312.15): C 53.87, H 3.55, N 4.49,
Found: C 54.31, H 3.91, N 4.31.
125.42, 125.33 (1C, q, J ¼ 269.4 Hz, CF3), 122.61, 121.35 (1C, q,
J ¼ 4.0 Hz, C60), 119.78 (1C, q, J ¼ 31.7 Hz, C50), 116.31 (1C, q,
J ¼ 4.0 Hz, C40), 115.15, 114.72, 57.22; Anal. Calcd. for C15H11ClF3NO3
(345.70): C 52.11, H 3.21, N 4.05, Found: C 52.28, H 2.83, N 4.13.
5.1.3.10. 5-Chloro-N-(4,5-dichloro-2-hydroxyphenyl)-2-
methoxybenzamide (6j). Yield 68.4%, white solid; m. p. 248e250 ꢀC;
5.1.3.15. 5-Chloro-2-(4-chloro-2-methoxybenzamido)phenyl
IR (ATR): 3294 (b,
(amide II), 1498, 1479 (
300 MHz):
11.07 (1H, s, OH), 10.55 (1H, s, NH), 8.53 (1H, s, H60),
n
OH), 1649 (amide I), 1593 (
n
CC aromatic), 1541
4-chloro-2-methoxybenzoate (7a). By-product, yield 8.2%, white
n
CC aromatic) cmꢁ1
;
1H NMR (DMSO-d6,
solid; m. p.153e155 ꢀC; IR (ATR): 3341 (
n
NH),1754 (
CC aromatic), 1534 (amide II), 1485, 1462,
9.74 (1H, s,
n CO, ester),1665
d
(amide I), 1594, 1565 (
n
7.95 (1H, d, J ¼ 2.8 Hz, H6), 7.60 (1H, dd, J ¼ 8.8 Hz, J ¼ 2.8 Hz, H4),
1455 ( d
n
CC aromatic) cmꢁ1; 1H NMR (CDCl3, 300 MHz):
7.27 (1H, d, J ¼ 8.8 Hz, H3), 7.02 (1H, s, H30), 4.01 (3H, s, OCH3); 13C
NH), 8.48 (1H, d, J ¼ 9.5 Hz, H30), 8.17 (1H, d, J ¼ 8.4 Hz), 8.01 (1H, d,
J ¼ 8.0 Hz), 7.31e7.23 (2H, m), 7.12e7.03 (3H, m), 6.88 (1H, d,
J ¼ 1.8 Hz), 3.86 (3H, s, OCH3), 3.52 (3H, s, OCH3); 13C NMR (CDCl3,
NMR (DMSO-d6, 75 MHz):
d 161.14, 156.17, 146.38, 133.39, 130.55,
127.32, 125.42, 124.85, 122.32, 120.58, 120.33, 115.60, 115.04, 57.16;
Anal. Calcd. for C14H10Cl3NO3 (346.59): C 48.52, H 2.91, N 4.04,
Found: C 48.58, H 3.21, N 4.13.
75 MHz):
d 162.47, 162.34, 160.66, 157.50, 141.36, 140.83, 139.15,
133.67, 133.55, 129.60, 128.89, 126.63, 123.45, 122.45, 121.92, 120.81,
120.09, 116.30, 113.03, 112.11, 56.34, 55.95; Anal. Calcd. for
C22H16Cl3NO5 (480.73): C 54.97, H 3.35, N 2.91, Found: C 55.26, H
3.68, N 2.85.
5.1.3.11. N-(4-Bromo-2-hydroxyphenyl)-5-chloro-2-methoxybenza
mide (6k). Yield 55.4%, white solid; m. p. 239e241 ꢀC; IR (ATR):
3320 (
aromatic), 1541 (amide II), 1502, 1481, 1462 (
1H NMR (DMSO-d6, 300 MHz):
n
NH), 3254 (b,
n
OH), 1645 (amide I), 1613, 1595 (
CC aromatic) cmꢁ1
10.80 (1H, s, OH), 10.52 (1H, s, NH),
n CC
n
;
5.1.3.16. 2-(4-Chloro-2-methoxybenzamido)-5-(trifluoromethyl)
phenyl 4-chloro-2-methoxybenzoate (7b). By-product, yield 9.5%,
d
8.29 (1H, d, J ¼ 8.6 Hz, H60), 7.98 (1H, d, J ¼ 2.8 Hz, H6), 7.62 (1H, dd,
J ¼ 8.9 Hz, J ¼ 2.8 Hz, H4), 7.30 (1H, d, J ¼ 8.9 Hz, H3), 7.05 (1H, d,
J ¼ 2.2 Hz, H30), 7.00 (1H, dd, J ¼ 8.6 Hz, J ¼ 2.2 Hz, H50), 4.03 (3H, s,
white solid; m. p. 162e164 ꢀC; IR (ATR): 3334 (
n
NH), 1758 (
n
CO,
ester), 1670 (amide I), 1621, 1592, 1569 (
II), 1506, 1488, 1479, 1463 (n ;
n CC aromatic), 1537 (amide
CC aromatic) cmꢁ1 1H NMR (CDCl3,
OCH3); 13C NMR (DMSO-d6, 75 MHz):
d
160.96, 156.15, 147.79,
300 MHz):
d
9.94 (1H, s, NH), 8.75 (1H, d, J ¼ 8.6 Hz, H30), 8.18 (1H, d,
133.18, 130.55, 126.59, 125.37, 122.86, 122.06, 121.28, 117.27, 115.25,
115.10, 57.17; Anal. Calcd. for C14H11BrClNO3 (356.60): C 47.15, H
3.11, N 3.93, Found: C 46.92, H 3.31, N 4.02.
J ¼ 8.3 Hz), 8.05 (1H, d, J ¼ 8.2 Hz), 7.60e7.51 (2H, m), 7.16e7.04 (3H,
m), 6.90 (1H, d, J ¼ 1.8 Hz), 3.88 (3H, s, OCH3), 3.51 (3H, s, OCH3); 13
C
NMR (CDCl3, 75 MHz):
d 162.57, 162.42, 160.75, 157.52, 141.57,
139.80,139.46,134.08,133.75,133.62,125.94 (1C, q, J ¼ 33.3 Hz, C50),
123.68 (1C, q, J ¼ 3.8 Hz, C60),122.32 (1C, q, J ¼ 270.3 Hz, CF3),122.21,
122.03, 120.87, 119.95, 119.50 (1C, q, J ¼ 3.9 Hz, C40), 116.10, 113.07,
112.19, 56.38, 56.01; Anal. Calcd. for C23H16Cl2F3NO5 (514.28): C
53.72, H 3.14, N 2.72, Found: C 53.81, H 3.22, N 2.83.
5.1.3.12. N-(5-Bromo-2-hydroxyphenyl)-5-chloro-2-methoxybenza
mide (6l). Yield 64.7%, grey solid; m. p. 223e224 ꢀC; IR (ATR): 3301
(b,
n OH), 1652 (amide I), 1610, 1595 (n CC aromatic), 1542 (amide II),
1481, 1458 (
n
CC aromatic) cmꢁ1 1H NMR (DMSO-d6, 300 MHz):
;
d
10.64 (1H, s, OH), 10.57 (1H, s, NH), 8.52 (1H, d, J ¼ 2.5 Hz, H60),
7.97 (1H, d, J ¼ 2.8 Hz, H6), 7.62 (1H, dd, J ¼ 8.9 Hz, J ¼ 2.8 Hz, H4),
7.29 (1H, d, J ¼ 8.9 Hz, H3), 7.09 (1H, dd, J ¼ 8.6 Hz, J ¼ 2.5 Hz, H40),
6.85 (1H, d, J ¼ 8.6 Hz, H30), 4.02 (3H, s, OCH3); 13C NMR (DMSO-d6,
5.1.3.17. 4,5-Dichloro-2-(5-chloro-2-methoxybenzamido)phenyl
5-chloro-2-methoxybenzoate (7c). By-product, yield 10.7%, orange
solid; m. p.184e185 ꢀC; IR (ATR): 3324 (
n
NH),1766 (
CC aromatic),1523 (amide II),1488,1462 (
9.88 (1H, s, NH), 8.78
n
CO, ester),1668
75 MHz):
d
161.15, 156.20, 145.88, 133.35, 130.58, 128.54, 126.45,
(amide I),1592,1572 (
n
n
CC
125.41, 122.66, 122.04, 116.33, 115.16, 110.30, 57.21; Anal. Calcd. for
C14H11BrClNO3 (356.60): C 47.15, H 3.11, N 3.93, Found: C 47.03, H
3.43, N 4.04.
aromatic) cmꢁ1; 1H NMR (CDCl3, 300 MHz):
d
(1H, s, H60), 8.18 (1H, d, J ¼ 2.8 Hz), 8.02 (1H, d, J ¼ 2.8 Hz), 7.57
(1H, dd, J ¼ 9.0 Hz, J ¼ 2.8 Hz), 7.40 (1H, dd, J ¼ 8.8 Hz, J ¼ 2.8 Hz), 7.41
(1H, s, H30), 7.02 (1H, d, J ¼ 9.0 Hz), 6.85 (1H, d, J ¼ 8.8 Hz), 3.86 (3H, s,
5.1.3.13. 5-Chloro-N-(2-hydroxy-4-(trifluoromethyl)phenyl)-2-
OCH3), 3.54 (3H, s, OCH3); 13C NMR (CDCl3, 75 MHz):
d 161.97,161.79,
methoxybenzamide (6m). Yield 60.5%, white solid; m. p. 207e
158.70, 155.71, 138.80, 134.97, 133.25, 132.15, 131.86, 130.27, 129.01,
128.20, 127.13, 126.94, 125.55, 123.62, 123.34, 122.53, 113.79, 112.99,
209 ꢀC; IR (ATR): 3253 (b,
n OH), 1651 (amide I), 1610, 1598 (n CC

J. Kozic et al. / European Journal of Medicinal Chemistry 56 (2012) 108e119
115
56.40, 56.09; Anal. Calcd. for C22H15Cl4NO5 (515.17): C 51.29, H 2.93,
N 2.72, Found: C 51.66, H 3.32, N 2.63.
C14H10Cl3NO2S (362.66): C 46.37, H 2.78, N 3.86, S 8.84, Found: C
46.20, H 2.58, N 3.83, S 8.86.
5.1.3.18. 2-(5-Chloro-2-methoxybenzamido)-5-(trifluoromethyl)
phenyl 5-chloro-2-methoxybenzoate (7d). By-product, yield 7.8%,
5.1.4.4. N-(4-Bromo-2-hydroxyphenyl)-4-chloro-2-methoxybenzo-
thioamide (8d). Yield 25.2%, yellow solid; m. p. 167e169 ꢀC,
white solid; m. p. 142e143 ꢀC; IR (ATR): 3333 (
n
NH), 1760 (
n
CO,
decomposition; IR (ATR): 3197 (bp,
1476, 1461, 1454 (
CC aromatic) cmꢁ1
300 MHz): d 11.34 (1H, s, OH), 10.52 (1H, s, NH), 8.05 (1H, d,
n
OH), 1613, 1591, 1557, 1497,
ester), 1681 (amide I), 1619, 1599 (
n
CC aromatic), 1541 (amide II),
n
;
1H NMR (DMSO-d6,
1489, 1482, 1463 (
n
CC aromatic) cmꢁ1; 1H NMR (CDCl3, 300 MHz):
d
10.02 (1H, s, NH), 8.76 (1H, d, J ¼ 8.5 Hz, H30), 8.21 (1H, d,
J ¼ 8.6 Hz, H60), 7.78 (1H, d, J ¼ 8.3 Hz, H6), 7.22 (1H, d, J ¼ 1.9 Hz, H3),
J ¼ 2.8 Hz), 8.06 (1H, d, J ¼ 2.7 Hz), 7.59 (1H, d, J ¼ 2.5 Hz), 7.58e7.53
(2H, m), 7.42 (1H, dd, J ¼ 8.8 Hz, J ¼ 2.8 Hz), 7.03 (1H, d, J ¼ 8.9 Hz),
6.86 (1H, d, J ¼ 8.8 Hz), 3.87 (3H, s, OCH3), 3.55 (3H, s, OCH3); 13C
7.13e7.06 (2H, m, H5, H30), 7.04 (1H, dd, J ¼ 8.6 Hz, J ¼ 2.2 Hz, H50),
3.90 (3H, s, OCH3); 13C NMR (DMSO-d6, 75 MHz):
d 193.45, 155.31,
151.85, 135.75, 133.02, 130.07, 127.18, 126.85, 121.46, 120.43, 119.08,
118.55, 112.53, 56.71; Anal. Calcd. for C14H11BrClNO2S (372.66): C
45.12, H 2.98, N 3.76, S 8.60, Found: C 45.42, H 3.24, N 3.88, S 8.72.
NMR (CDCl3, 75 MHz):
d 162.21, 161.96, 158.71, 155.73, 139.73,
134.91, 133.91, 133.26, 132.23, 131.91, 127.15, 125.73 (1C, q,
J ¼ 33.4 Hz, C50), 125.55, 123.77 (1C, q, J ¼ 3.8 Hz, C60), 123.26 (1C, q,
J ¼ 270.1 Hz, CF3), 122.74, 122.25, 119.50 (1C, q, J ¼ 3.8 Hz, C40),
118.95, 113.77, 113.00, 56.41, 56.10; Anal. Calcd. for C23H16Cl2F3NO5
(514.28): C 53.72, H 3.14, N 2.72, Found: C 53.90, H 3.29, N 2.95.
5.1.4.5. N-(5-Bromo-2-hydroxyphenyl)-4-chloro-2-methoxybenzo-
thioamide (8e). Yield 28.2%, yellow solid; m. p. 155e158 ꢀC,
decomposition; IR (ATR): 3189 (bp,
n OH), 1614, 1587, 1556, 1489,
1475, 1455 (n ;
CC aromatic) cmꢁ1 1H NMR (DMSO-d6, 300 MHz):
5.1.4. General procedure for the preparation of substituted 2-
methoxy-20-hydroxybenzothioanilides (8)
d
11.43 (1H, s, OH), 10.41 (1H, s, NH), 8.39 (1H, d, J ¼ 2.6 Hz, H60),
7.81 (1H, d, J ¼ 8.4 Hz, H6), 7.28 (1H, dd, J ¼ 8.6 Hz, J ¼ 2.6 Hz, H40),
7.23 (1H, d, J ¼ 1.8 Hz, H3), 7.10 (1H, dd, J ¼ 8.4 Hz, J ¼ 1.8 Hz, H5),
6.91 (1H, d, J ¼ 8.6 Hz, H30), 3.91 (3H, s, OCH3); 13C NMR (DMSO-d6,
To a solution containing 1.3 mmol of the benzanilide derivative
(6) in 10 mL of pyridine, 2.8 mmol of P4S10 was added, and the
reaction mixture was stirred and refluxed for 4 h. Then, 40 mL of
CHCl3, 40 mL of a 5% solution of HCl and 10 mL of concentrated HCl
were added, and the mixture was refluxed under vigorous stirring
for 1 h. The organic phase was removed, and the aqueous phase was
extracted three times with CHCl3 (50 mL). Then, the organic phases
were collected, dried with Na2SO4 and evaporated. The oil residue
was purified by column chromatography and crystallisation [18].
75 MHz):
d 193.42, 155.32, 149.93, 135.94, 133.19, 130.00, 129.83,
128.71, 127.33, 120.50, 117.73, 112.57, 108.95, 56.75; Anal. Calcd. for
C14H11BrClNO2S (372.66): C 45.12, H 2.98, N 3.76, S 8.60, Found: C
45.38, H 2.90, N 3.69, S 8.79.
5.1.4.6. 4-Chloro-N-(2-hydroxy-4-(trifluoromethyl)phenyl)-2-metho-
xybenzothioamide (8f). Yield 36.0%, yellow solid; m. p. 132e134 ꢀC,
decomposition; IR (ATR): 3246 (bp,
n
OH), 1618, 1592, 1559, 1477,
5.1.4.1. 4-Chloro-N-(4-chloro-2-hydroxyphenyl)-2-methoxybenzoth-
ioamide (8a). Yield 35.9%, yellow solid; m. p. 168e169 ꢀC, decom-
1462 (
n
CC aromatic) cmꢁ1
;
1H NMR (DMSO-d6, 500 MHz):
d
11.51
(1H, s, OH), 10.79 (1H, bs, NH), 8.38 (1H, d, J ¼ 8.8 Hz, H60), 7.82 (1H,
d, J ¼ 8.5 Hz, H6), 7.24 (1H, d, J ¼ 2.0 Hz, H3), 7.23e7.18 (2H, m, H30,
H50), 7.11 (1H, dd, J ¼ 8.5 Hz, J ¼ 2.0 Hz, H5), 3.92 (3H, s, OCH3); 13C
position; IR (ATR): 3132 (bp,
n
OH), 1620, 1590, 1557, 1499, 1476,
11.34
1456 ( d
n
CC aromatic) cmꢁ1 1H NMR (DMSO-d6, 300 MHz):
;
(1H, s, OH), 10.52 (1H, s, NH), 8.09 (1H, d, J ¼ 8.5 Hz, H60), 7.78 (1H,
d, J ¼ 8.4 Hz, H6), 7.22 (1H, d, J ¼ 1.9 Hz, H3), 7.09 (1H, dd, J ¼ 8.4 Hz,
J ¼ 1.9 Hz, H5), 6.96 (1H, d, J ¼ 2.3 Hz, H30), 6.91 (1H, dd, J ¼ 8.5 Hz,
J ¼ 2.3 Hz, H50), 3.90 (3H, s, OCH3); 13C NMR (DMSO-d6, 75 MHz):
NMR (DMSO-d6, 125 MHz): d 194.03, 155.32, 150.87, 136.00, 133.19,
130.83, 129.91, 127.32 (1C, q, J ¼ 31.5 Hz, C40), 125.98, 124.11 (1C, q,
J ¼ 270.5 Hz, CF3), 120.51, 115.47 (1C, q, J ¼ 3.8 Hz, C30), 112.58,
112.18 (1C, q, J ¼ 3.8 Hz, C50), 56.76; Anal. Calcd. for C15H11ClF3NO2S
(361.77): C 49.80, H 3.06, N 3.87, S 8.86, Found: C 49.95, H 3.15, N
3.99, S 8.75.
d
193.49, 155.28, 151.71, 135.70, 132.96, 130.88, 130.05, 126.88,
126.43, 120.40, 118.51, 115.66, 112.49, 56.68; Anal. Calcd. for
C14H11Cl2NO2S (328.21): C 51.23, H 3.38, N 4.27, S 9.77, Found: C
51.14, H 3.36, N 4.24, S 10.02.
5.1.4.7. 4-Chloro-N-(2-hydroxy-5-(trifluoromethyl)phenyl)-2-metho-
xybenzothioamide (8g). Yield 22.2%, yellow solid; m. p. 153e155 ꢀC,
5.1.4.2. 4-Chloro-N-(5-chloro-2-hydroxyphenyl)-2-methoxybenzoth-
ioamide (8b). Yield 27.8%, yellow solid; m. p. decomposition; IR
decomposition; IR (ATR): 3174 (bp,
1476, 1461, 1449 (
CC aromatic) cmꢁ1
300 MHz):
n
OH), 1626, 1591, 1573, 1557,
n
;
1H NMR (DMSO-d6,
(ATR): 3198 (bp,
n
OH), 1613, 1589, 1555, 1493, 1475, 1457 (
n
CC
d
11.50 (1H, s, OH),11.04 (1H, s, NH), 8.55 (1H, d, J ¼ 2.3 Hz,
aromatic) cmꢁ1; 1H NMR (DMSO-d6, 300 MHz):
d
11.45 (1H, s, OH),
H60), 7.82 (1H, d, J ¼ 8.4 Hz, H6), 7.49 (1H, dd, J ¼ 8.5 Hz, J ¼ 2.3 Hz,
H40), 7.24 (1H, d, J ¼ 2.0 Hz, H3), 7.12 (1H, d, J ¼ 8.5 Hz, H30), 7.11 (1H,
dd, J ¼ 8.4 Hz, J ¼ 2.0 Hz, H5), 3.92 (3H, s, OCH3); 13C NMR (DMSO-d6,
10.40 (1H, s, NH), 8.32 (1H, d, J ¼ 2.6 Hz, H60), 7.83 (1H, d, J ¼ 8.5 Hz,
H6), 7.23 (1H, d, J ¼ 1.9 Hz, H3), 7.17 (1H, dd, J ¼ 8.7 Hz, J ¼ 2.6 Hz, H40),
7.10 (1H, dd, J ¼ 8.5 Hz, J ¼ 1.9 Hz, H5), 6.96 (1H, d, J ¼ 8.7 Hz, H30),
75 MHz):
d 193.92, 155.36, 153.92, 135.98, 133.16, 129.86, 127.48,
3.92 (3H, s, OCH3); 13C NMR (DMSO-d6, 75 MHz):
d
193.33, 155.32,
124.76 (1C, q, J ¼ 4.2 Hz, C60),124.70 (1C, q, J ¼ 269.3 Hz, CF3),122.41
(1C, q, J ¼ 4.2 Hz, C40), 120.51, 119.00 (1C, q, J ¼ 32.2 Hz, C50), 116.39,
112.58, 56.77; Anal. Calcd. for C15H11ClF3NO2S (361.77): C 49.80, H
3.06, N 3.87, S 8.86, Found: C 49.62, H 2.93, N 3.67, S 8.64.
149.40, 135.98, 133.25, 129.78, 128.30, 127.08, 124.42, 121.61, 120.51,
117.15,112.57, 56.76; Anal. Calcd. for C14H11Cl2NO2S(328.21): C 51.23,
H 3.38, N 4.27, S 9.77, Found: C 51.16, H 3.22, N 4.21, S 9.82.
5.1.4.3. 4-Chloro-N-(4,5-dichloro-2-hydroxyphenyl)-2-methoxyben-
zothioamide (8c). Yield 28.9%, yellow solid; m. p. 176e177 ꢀC,
5.1.4.8. 5-Chloro-N-(4-chloro-2-hydroxyphenyl)-2-methoxybenzo-
thioamide (8h). Yield 26.4%, yellow solid; m. p. 144e145 ꢀC,
decomposition; IR (ATR): 3130 (bp,
n
OH), 1589, 1558, 1541, 1491,
decomposition; IR (ATR): 3197 (bp, n OH), 1611, 1591, 1556, 1502,
1477, 1458 (
n
CC aromatic) cmꢁ1 1H NMR (DMSO-d6, 300 MHz):
;
1475, 1450 (n ;
CC aromatic) cmꢁ1 1H NMR (DMSO-d6, 500 MHz):
d
11.48 (1H, s, OH), 10.88 (1H, s, NH), 8.45 (1H, s, H60), 7.81 (1H, d,
d
11.42 (1H, s, OH), 10.50 (1H, s, NH), 8.03 (1H, d, J ¼ 8.6 Hz, H60),
J ¼ 8.4 Hz, H6), 7.22 (1H, d, J ¼ 2.0 Hz, H3), 7.12 (1H, s, H30), 7.09 (1H,
7.71 (1H, d, J ¼ 2.8 Hz, H6), 7.47 (1H, dd, J ¼ 8.8 Hz, J ¼ 2.8 Hz, H4),
7.16 (1H, d, J ¼ 8.8 Hz, H3), 6.97 (1H, d, J ¼ 2.5 Hz, H30), 6.91 (1H, dd,
J ¼ 8.6 Hz, J ¼ 2.5 Hz, H50), 3.87 (3H, s, OCH3); 13C NMR (DMSO-d6,
dd, J ¼ 8.4 Hz, J ¼ 2.0 Hz, H5), 3.90 (3H, s, OCH3); 13C NMR (DMSO-
d6, 75 MHz): d 193.86, 155.37, 150.49, 136.12, 133.26, 129.62, 128.62,
127.55, 126.16, 120.54, 119.72, 117.03, 112.59, 56.78; Anal. Calcd. for
125 MHz): d 193.10, 153.46, 151.88, 132.71, 131.36, 129.99, 126.48,

116
J. Kozic et al. / European Journal of Medicinal Chemistry 56 (2012) 108e119
124.20, 119.29, 117.93, 115.28, 114.91, 113.62, 56.04; Anal. Calcd. for
C14H11Cl2NO2S (328.21): C 51.23, H 3.38, N 4.27, S 9.77, Found: C
50.82, H 3.13, N 4.23, S 9.81.
C30), 114.34, 112. 34 (1C, q, J ¼ 3.7 Hz, C50), 56.68; Anal. Calcd. for
C15H11ClF3NO2S (361.77): C 49.80, H 3.06, N 3.87, S 8.86, Found: C
49.71, H 2.85, N 3.69, S 8.92.
5.1.4.9. 5-Chloro-N-(5-chloro-2-hydroxyphenyl)-2-methoxybenzoth-
5.1.4.14. 5-Chloro-N-(2-hydroxy-5-(trifluoromethyl)phenyl)-2-
ioamide (8i). Yield 48.3%, yellow solid; m. p. 147e149 ꢀC, decom-
methoxybenzothioamide (8n). Yield 26.3%, yellow solid; m. p. 132e
position; IR (ATR): 3315 (bp,
n
OH), 1618, 1594, 1562, 1497, 1477,
11.52
133 ꢀC, decomposition; IR (ATR): 3252 (bp,
n OH), 1624, 1574, 1478,
1454 (
n
CC aromatic) cmꢁ1 1H NMR (DMSO-d6, 300 MHz):
;
d
1460, 1447 (n ;
CC aromatic) cmꢁ1 1H NMR (DMSO-d6, 300 MHz):
(1H, s, OH), 10.36 (1H, s, NH), 8.23 (1H, d, J ¼ 2.6 Hz, H60), 7.76 (1H,
d, J ¼ 2.8 Hz, H6), 7.48 (1H, dd, J ¼ 8.8 Hz, J ¼ 2.8 Hz, H4), 7.18 (1H,
dd, J ¼ 8.7 Hz, J ¼ 2.6 Hz, H40), 7.16 (1H, d, J ¼ 8.8 Hz, H3), 6.96 (1H, d,
J ¼ 8.7 Hz, H30), 3.88 (3H, s, OCH3); 13C NMR (DMSO-d6, 75 MHz):
d
11.57 (1H, s, OH), 11.00 (1H, s, NH), 8.48 (1H, d, J ¼ 2.3 Hz, H60),
7.75 (1H, d, J ¼ 2.7 Hz, H6), 7.55e7.45 (2H, m, H4, H40), 7.17 (1H, d,
J ¼ 8.9 Hz, H3), 7.12 (1H, d, J ¼ 8.5 Hz, H30), 3.89 (3H, s, OCH3); 13C
NMR (DMSO-d6, 75 MHz):
d 193.52, 154.10, 153.50, 132.53, 130.85,
d
192.95, 153.49, 149.62, 132.46, 130.85, 130.67, 128.15, 127.30,
130.58, 127.32, 124.93 (1C, q, J ¼ 3.9 Hz, C60), 124.21, 124.19 (1C, q,
124.73, 124.22, 121.64, 117.29, 114.31, 56.66; Anal. Calcd. for
C14H11Cl2NO2S (328.21): C 51.23, H 3.38, N 4.27, S 9.77, Found: C
51.47, H 3.35, N 4.04, S 9.31.
J ¼ 269.4 Hz, CF3), 122.70 (1C, q, J ¼ 3.8 Hz, C40), 119.05 (1C, q,
J
¼
32.2 Hz, C50), 116.50, 114.30, 56.66; Anal. Calcd. for
C15H11ClF3NO2S (361.77): C 49.80, H 3.06, N 3.87, S 8.86, Found: C
49.66, H 3.15, N 4.02, S 9.05.
5.1.4.10. 5-Chloro-N-(4,5-dichloro-2-hydroxyphenyl)-2-methoxyben-
zothioamide (8j). Yield 33.4%, yellow solid; m. p. decomposition; IR
5.1.4.15. 6-Chloro-2-(4-chloro-2-methoxyphenyl)benzo[d]oxazole
(ATR): 3240 (bp,
n
OH), 1611, 1591, 1555, 1489, 1477, 1456 (
n
CC
(9a). By-product, yield 20.7%, white solid; m. p. 146e147 ꢀC; IR
aromatic) cmꢁ1; 1H NMR (DMSO-d6, 500 MHz):
d
11.55 (1H, bs, OH),
(ATR): 1614, 1592, 1566, 1550, 1487, 1471, 1458, 1446 (n CC aromatic)
10.85 (1H, bs, NH), 8.39 (1H, s, H60), 7.75 (1H, d, J ¼ 2.8 Hz, H6), 7.48
(1H, dd, J ¼ 8.9 Hz, J ¼ 2.8 Hz, H4), 7.17 (1H, d, J ¼ 8.9 Hz, H3), 7.13
(1H, s, H30), 3.88 (3H, s, OCH3); 13C NMR (DMSO-d6, 125 MHz):
cmꢁ1; 1H NMR (CDCl3, 300 MHz):
d
8.01 (1H, d, J ¼ 8.5 Hz, H60), 7.97
(1H, d, J ¼ 1.8 Hz), 7.82 (1H, d, J ¼ 8.5 Hz), 7.45 (1H, dd, J ¼ 8.5 Hz,
J ¼ 1.8 Hz), 7.38 (1H, d, J ¼ 1.8 Hz), 7.21 (1H, dd, J ¼ 8.5 Hz,
d
193.43, 153.51, 150.64, 132.26, 130.94, 130.66, 128.81, 127.39,
J ¼ 1.8 Hz), 3.95 (3H, s, OCH3); 13C NMR (CDCl3, 75 MHz):
d 161.41,
126.39, 124.24, 119.74, 117.13, 114.31, 56.66; Anal. Calcd. for
C14H10Cl3NO2S (362.66): C 46.37, H 2.78, N 3.86, S 8.84, Found: C
46.25, H 2.99, N 3.96, S 8.92.
158.97, 150.50, 140.46, 138.17, 132.45, 129.83, 125.38, 121.05, 120.96,
114.05, 113.53, 111.60, 56.81; Anal. Calcd. for C14H9Cl2NO2 (294.13):
C 57.17, H 3.08, N 4.76, Found: C 56.95, H 3.26, N 4.97.
5.1.4.11. N-(4-Bromo-2-hydroxyphenyl)-5-chloro-2-methoxybenzo-
5.1.4.16. 5,6-Dichloro-2-(4-chloro-2-methoxyphenyl)benzo[d]oxazole
thioamide (8k). Yield 38.9%, yellow solid; m. p. 148e149 ꢀC,
(9b). By-product, yield 42.8%, white solid; m. p. 184e185 ꢀC; IR
decomposition; IR (ATR): 3233 (bp,
n
OH), 1613, 1594, 1556, 1497,
(ATR): 1605, 1592, 1573, 1536, 1479, 1465 (n
CC aromatic) cmꢁ1; 1H
1476, 1451 (
n
CC aromatic) cmꢁ1
;
1H NMR (DMSO-d6, 300 MHz):
NMR (CDCl3, 500 MHz):
d
8.03 (1H, d, J ¼ 8.3 Hz, H60), 7.86 (1H, s),
d
11.42 (1H, s, OH), 10.49 (1H, s, NH), 7.99 (1H, d, J ¼ 8.5 Hz, H60),
7.68 (1H, s), 7.09 (1H, dd, J ¼ 8.3 Hz, J ¼ 1.9 Hz, H50), 7.07 (1H, d,
7.72 (1H, d, J ¼ 2.8 Hz, H6), 7.47 (1H, dd, J ¼ 8.9 Hz, J ¼ 2.8 Hz, H4),
7.16 (1H, d, J ¼ 8.9 Hz, H3), 7.11 (1H, d, J ¼ 2.2 Hz, H30), 7.04 (1H, dd,
J ¼ 8.5 Hz, J ¼ 2.2 Hz, H50), 3.87 (3H, s, OCH3); 13C NMR (DMSO-d6,
J ¼ 1.9 Hz, H30), 4.01 (3H, s, OCH3); 13C NMR (CDCl3, 125 MHz):
d
162.45,159.03,148.84,141.69,139.33,132.10,129.01,128.65,121.16,
121.08, 113.82, 112.88, 112.19, 56.49; Anal. Calcd. for C14H8Cl3NO2
(328.58): C 51.18, H 2.45, N 4.26, Found: C 51.55, H 2.63, N 4.56.
75 MHz):
d 193.01, 153.45, 151.99, 132.70, 130.68, 130.51, 127.37,
126.71, 124.15, 121.50, 119.28, 118.65, 114.26, 56.62; Anal. Calcd. for
C14H11BrClNO2S (372.66): C 45.12, H 2.98, N 3.76, S 8.60, Found: C
45.34, H 3.20, N 3.97, S 8.76.
5.1.4.17. 5-Bromo-2-(4-chloro-2-methoxyphenyl)benzo[d]oxazole
(9c). By-product, yield 24.8%, white solid; m. p. 138e139 ꢀC; IR
(ATR): 1612, 1597, 1572, 1536, 1492, 1479, 1467, 1445 (n CC aromatic)
5.1.4.12. N-(5-Bromo-2-hydroxyphenyl)-5-chloro-2-methoxybenzo-
cmꢁ1; 1H NMR (CDCl3, 300 MHz):
d
8.05 (1H, d, J ¼ 8.3 Hz, H60), 7.93
thioamide (8l). Yield 38.7%, yellow solid; m. p. 148e149 ꢀC,
(1H, s), 7.51e7.40 (2H, s), 7.14e7.02 (2H, m0, H30, H50), 4.02 (3H, s,
decomposition; IR (ATR): 3299 (bp,
n
OH), 1616, 1593, 1562, 1493,
OCH3); 13C NMR (CDCl3, 75 MHz):
d 161.85, 158.97, 149.23, 143.55,
139.00, 132.10, 128.08, 123.10, 121.09, 117.13, 114.20, 112.85, 111.67,
56.50; Anal. Calcd. for C14H9BrClNO2 (338.58): C 49.66, H 2.68, N
4.14, Found: C 49.45, H 2.90, N 4.18.
1477, 1453 (
d
n
CC aromatic) cmꢁ1
;
1H NMR (DMSO-d6, 300 MHz):
11.51 (1H, s, OH), 10.38 (1H, s, NH), 8.32 (1H, d, J ¼ 2.5 Hz, H60),
7.75 (1H, d, J ¼ 2.8 Hz, H6), 7.48 (1H, dd, J ¼ 8.8 Hz, J ¼ 2.8 Hz, H4),
7.29 (1H, dd, J ¼ 8.6 Hz, J ¼ 2.5 Hz, H40), 7.16 (1H, d, J ¼ 8.8 Hz, H3),
6.92 (1H, d, J ¼ 8.6 Hz, H30), 3.88 (3H, s, OCH3); 13C NMR (DMSO-d6,
5.1.4.18. 6-Bromo-2-(4-chloro-2-methoxyphenyl)benzo[d]oxazole
75 MHz):
d
193.03, 153.48, 150.13, 132.50, 130.83, 130.62, 130.20,
(9d). By-product, yield 38.1%, white solid; m. p. 157e158 ꢀC; IR
128.56, 127.61, 124.21, 117.85, 114.30, 108.97, 56.65; Anal. Calcd. for
C14H11BrClNO2S (372.66): C 45.12, H 2.98, N 3.76, S 8.60, Found: C
45.12, H 2.89, N 3.77, S 8.54.
(ATR): 1608,1596,1576,1538,1481,1471,1458 (n ;
CC aromatic) cmꢁ1
1H NMR (CDCl3, 300 MHz):
d
8.04 (1H, d, J ¼ 8.8 Hz, H60), 7.74 (1H, d,
J ¼ 1.8 Hz, H7), 7.66 (1H, d, J ¼ 8.5 Hz, H4), 7.46 (1H, dd, J ¼ 8.5 Hz,
J ¼ 1.8 Hz, H5), 7.11e7.05 (2H, m, H30, H50), 4.02 (3H, s, OCH3); 13C
5.1.4.13. 5-Chloro-N-(2-hydroxy-4-(trifluoromethyl)phenyl)-2-
NMR (CDCl3, 75 MHz): d 161.16,158.95,150.67,141.21,138.90,132.01,
methoxybenzothioamide (8m). Yield 49.0%, yellow solid; m. p. 127e
127.86, 121.15, 121.10, 118.02, 114.23, 113.96, 112.84, 56.49; Anal.
Calcd. for C14H9BrClNO2 (338.58): C 49.66, H 2.68, N 4.14, Found: C
49.68, H 2.83, N 4.23.
129 ꢀC, decomposition; IR (ATR): 3204 (bp,
n OH), 1622, 1595, 1568,
1476, 1458 (n ;
CC aromatic) cmꢁ1 1H NMR (DMSO-d6, 300 MHz):
d
11.58 (1H, bs, OH), 10.77 (1H, bs, NH), 8.31 (1H, d, J ¼ 8.9 Hz, H60),
7.75 (1H, d, J ¼ 2.7 Hz, H6), 7.49 (1H, dd, J ¼ 8.9 Hz, J ¼ 2.7 Hz, H4),
5.1.4.19. 2-(4-Chloro-2-methoxyphenyl)-6-(trifluoromethyl)benzo[d]
oxazole (9e). By-product, yield 27.6%, white solid; m. p.103e105 ꢀC;
7.25e7.19 (2H, m, H30, H50), 7.17 (1H, d, J ¼ 8.9 Hz, H3), 3.89 (3H, s,
OCH3); 13C NMR (DMSO-d6, 75 MHz):
d
193.61, 153.49, 151.06,
IR (ATR): 1617,1595,1569,1555,1491,1466 (n
CC aromatic) cmꢁ1; 1H
132.57, 130.90, 130.69, 130.62, 127.66 (1C, q, J ¼ 31.7 Hz, C40), 126.28,
125.37 (1C, q, J ¼ 270.4 Hz, CF3), 124.25, 115.53 (1C, q, J ¼ 3.9 Hz,
NMR (CDCl3, 500 MHz):
J ¼ 8.4 Hz, H4), 7.86 (1H, d, J ¼ 1.6 Hz, H7), 7.62 (1H, dd, J ¼ 8.4 Hz,
d
8.09 (1H, d, J ¼ 8.4 Hz, H60), 7.88 (1H, d,

J. Kozic et al. / European Journal of Medicinal Chemistry 56 (2012) 108e119
117
J ¼ 1.6 Hz, H5), 7.11 (1H, dd, J ¼ 8.4 Hz, J ¼ 2.0 Hz, H50), 7.09 (1H, d,
J ¼ 2.0 Hz, H30), 4.03 (3H, s, OCH3); 13C NMR (CDCl3, 125 MHz):
glacial acetic acid was added drop wise over 15 min. The mixture
was stirred at r.t. for 45 min and poured into ice-water (400 mL).
The aqueous mixture was extracted four times with CHCl3
(100 mL). Next, the organic phases were collected, dried with
Na2SO4, evaporated and purified by column chromatography (4a,
4c, 4e) or crystallisation from ethanol (4b). Compound 4d was used
for the next step without further purification [17].
d
163.12, 159.16, 149.67, 144.69, 139.40, 132.25, 127.31 (1C, q,
J ¼ 32.9 Hz, C6),124.14 (1C, q, J ¼ 270.3 Hz, CF3),121.70,121.18,120.50,
114.03,112.93,108.28, 56.50; Anal. Calcd. for C15H9ClF3NO2 (327.69):
C 54.98, H 2.77, N 4.27, Found: C 55.15, H 2.98, N 4.35.
5.1.4.20. 5-Chloro-2-(5-chloro-2-methoxyphenyl)benzo[d]oxazole
(9f). By-product, yield 17.9%, white solid; m. p. 136e137 ꢀC; IR
5.1.5.1. 4,5-Dichloro-2-nitrophenol (4a) [17]. Yield 38.0%, yellow
(ATR): 1613,1599,1578,1532,1478, 1464,1449,1434 (
cmꢁ1 1H NMR (CDCl3, 500 MHz): 8.10 (1H, s, H60), 7.78 (1H, s),
7.55e7.39 (2H, m), 7.33 (1H, d, J ¼ 8.5 Hz), 7.01 (1H, d, J ¼ 9.1 Hz,
H30), 4.00 (3H, s, OCH3); 13C NMR (CDCl3,125 MHz):
161.51,157.09,
n
CC aromatic)
solid; m. p. 66e68 ꢀC; IR (ATR): 3275 (b,
n
OH), 1614, 1564 (
CC aromatic), 1344 (ns NO2) cmꢁ1
d 10.47 (1H, s, OH), 8.23 (1H, s, H3), 7.33
n CC
;
d
aromatic), 1513 (nas NO2), 1457 (
n
;
1H NMR (CDCl3, 300 MHz):
d
(1H, s, H6); 13C NMR (CDCl3, 75 MHz):
125.83, 124.34, 121.48.
d 153.44, 142.26, 132.28,
148.87, 143.01, 132.69, 130.78, 129.94, 125.84, 125.59, 120.22, 116.90,
113.49, 111.26, 56.55; Anal. Calcd. for C14H9Cl2NO2 (294.13): C 57.17,
H 3.08, N 4.76, Found: C 57.39, H 3.26, N 5.06.
5.1.5.2. 4-Bromo-2-nitrophenol (4b) [25]. Yield 54.0%, yellow solid;
m. p. 90e92 ꢀC; IR (ATR): 3274 (b,
OH), 1612, 1601, 1571 ( CC
aromatic), 1530 (nas NO2), 1470, 1450 ( CC aromatic), 1323 (ns NO2)
cmꢁ1 1H NMR (CDCl3, 300 MHz):
10.49 (1H, s, OH), 8.25 (1H, d,
n
n
5.1.4.21. 5,6-Dichloro-2-(5-chloro-2-methoxyphenyl)benzo[d]oxa-
n
zole (9g). By-product, yield 33.2%, white solid; m. p. 201e202 ꢀC; IR
;
d
(ATR): 1597, 1575, 1558, 1529, 1481, 1437 (
NMR (DMSO-d6, 500 MHz): 7.86 (1H, s, H7), 7.66 (1H, s, H4), 7.54
(1H, s), 7.27 (1H, m), 6.86 (1H, m), 3.80 (3H, s, OCH3); 13C NMR
(DMSO-d6, 125 MHz): 161.55, 156.70,148.45, 141.07, 132.57, 130.15,
n
CC aromatic) cmꢁ1; 1H
J ¼ 2.4 Hz, H3), 7.66 (1H, dd, J ¼ 9.0 Hz, J ¼ 2.4 Hz, H5), 7.08 (1H, d,
d
J ¼ 9.0 Hz, H6); 13C NMR (CDCl3, 75 MHz):
d 154.09, 140.35, 127.30,
126.72, 121.72, 111.69.
d
128.54, 128.07, 125.18, 120.56, 115.86, 113.27, 111.89, 56.07; Anal.
Calcd. for C14H8Cl3NO2 (328.58): C 51.18, H 2.45, N 4.26, Found: C
51.31, H 2.64, N 4.52.
5.1.5.3. 5-Bromo-2-nitrophenol (4c) [26]. Yield 26.0%, yellow oil; IR
(ATR): 3182 (b, OH), 1608, 1573 ( CC aromatic), 1530 (nas NO2),
1466, 1448 (
CC aromatic), 1325 (ns NO2) cmꢁ1 1H NMR (CDCl3,
500 MHz):
n
n
n
;
d
10.62 (1H, s, OH), 7.97 (1H, d, J ¼ 9.1 Hz, H3), 7.37 (1H,
5.1.4.22. 5-Bromo-2-(5-chloro-2-methoxyphenyl)benzo[d]oxazoꢀ le
d, J ¼ 2.1 Hz, H6), 7.13 (1H, dd, J ¼ 9.1 Hz, J ¼ 2.1 Hz, H4); 13C NMR
(9h). By-product, yield 21.8%, white solid; m. p. 121e122 C; IR
(CDCl3, 125 MHz): d 155.26, 132.69, 132.34, 126.05, 123.88, 122.98.
(ATR): 1598, 1577, 1531, 1481, 1460, 1444 (n ;
CC aromatic) cmꢁ1 1H
NMR (CDCl3, 500 MHz):
d
8.10 (1H, s, H60), 7.94 (1H, s, H7), 7.53e
5.1.5.4. 2-Nitro-5-(trifluoromethyl)phenol (4e) [27]. Yield 26.1%,
yellow oil; IR (ATR): 3273 (b, OH), 1635,1594 ( CC aromatic), 1542
nas NO2), 1487, 1448 (
CC aromatic), 1335 (ns NO2) cmꢁ1; 1H NMR
(CDCl3, 300 MHz):
10.57 (1H, s, OH), 8.23 (1H, d, J ¼ 8.7 Hz, H3),
7.44 (1H, d, J ¼ 1.9 Hz, H6), 7.23 (1H, dd, J ¼ 8.7 Hz, J ¼ 1.9 Hz, H4);
13C NMR (CDCl3, 75 MHz):
7.37 (3H, s, H4, H5, H40), 7.02 (1H, d, J ¼ 9.1 Hz, H30), 4.00 (3H, s,
n
n
OCH3); 13C NMR (CDCl3, 125 MHz):
d
161.33, 157.10, 149.29, 143.48,
(
n
132.71, 130.80, 128.31, 125.84, 123.24, 117.23, 116.85, 113.49, 111.76,
56.56; Anal. Calcd. for C14H9BrClNO2 (338.58): C 49.66, H 2.68, N
4.14, Found: C 49.34, H 2.82, N 4.28.
d
d
154.81, 138.47 (1C, q, J ¼ 33.4 Hz, C5),
135.22, 126.08, 121.50 (1C, q, J ¼ 271.7 Hz, CF3), 117.87 (1C, q,
5.1.4.23. 6-Bromo-2-(5-chloro-2-methoxyphenyl)benzo[d]oxazole
(9i). By-product, yield 28.5%, white solid; m. p.113e115 ꢀC; IR (ATR):
J ¼ 3.9 Hz, C6), 116.64 (1C, q, J ¼ 3.5 Hz, C4).
1608, 1598, 1578, 1534, 1482, 1455, 1435 (
n
CC aromatic) cmꢁ1
;
1H
5.1.6. General procedure for the preparation of substituted 2-
aminophenols (5)
NMR (CDCl3, 300 MHz):
d
8.09 (1H, d, J ¼ 2.7 Hz, H60), 7.75 (1H, d,
J ¼ 1.8 Hz, H7), 7.67 (1H, d, J ¼ 8.5 Hz, H4), 7.47 (1H, dd, J ¼ 8.5 Hz,
J ¼ 1.8 Hz, H5), 7.45 (1H, dd, J ¼ 8.9 Hz, J ¼ 2.7 Hz, H40), 7.01 (1H, d,
J ¼ 8.9 Hz, H30), 4.00 (3H, s, OCH3); 13C NMR (CDCl3, 75 MHz):
An appropriate 2-nitrophenol (4) (40 mmol) was dissolved in
100 mL of glacial acetic acid and 30 mL of water. The reaction
mixture was stirred and heated to reflux. Iron powder (800 mmol)
was added in portions over 15 min. After the addition of the iron
powder, the mixture was refluxed for 30 min and then poured into
ice-water (400 mL). The mixture was extracted four times with
ethyl acetate (200 mL). Then, the organic phases were collected,
extracted ten times with a 5% solution of NaHCO3 (150 mL) and
once with a saturated solution of NaCl (200 mL) before being dried
with Na2SO4 and evaporated. The residue was purified by column
chromatography [17].
d
160.66,157.07,150.73,141.12,132.62,130.70,127.96,125.85,121.27,
118.25, 116.86, 114.04, 113.48, 56.53; Anal. Calcd. for C14H9BrClNO2
(338.58): C 49.66, H 2.68, N 4.14, Found: C 49.63, H 2.91, N 4.16.
5.1.4.24. 2-(5-Chloro-2-methoxyphenyl)-6-(trifluoromethyl)benzo[d]
oxazole (9j). By-product, yield 13.4%, white solid; m. p. 82e84 ꢀC; IR
(ATR): 1618, 1599, 1578, 1551, 1533, 1497, 1479, 1464 (
n
CC aromatic)
cmꢁ1; 1H NMR (CDCl3, 500 MHz):
d
8.14 (1H, m, H60), 7.94e7.85 (2H,
m, H4, H7), 7.63 (1H, dd, J ¼ 8.4 Hz, J ¼ 1.6 Hz, H5), 7.48 (1H, m, H40),
7.04 (1H, d, J ¼ 8.9 Hz, H30), 4.02 (3H, s, OCH3); 13C NMR (CDCl3,
5.1.6.1. 2-Amino-4,5-dichlorophenol (5a) [17]. Yield 66.3%, dark red
125 MHz):
d
162.63,157.29,149.74,144.58,133.05,130.94,127.51 (1C,
solid; m. p. decomposition; IR (ATR): 3467, 3376 (
n NH2), 3298 (b, n
q, J ¼ 32.7 Hz, C6),125.93,123.39 (1C, q, J ¼ 270.7 Hz, CF3),121.76 (1C,
q, J ¼ 3.7 Hz, C7), 120.65, 116.66, 113.57, 108.37 (1C, q, J ¼ 4.2 Hz, C5),
56.54; Anal. Calcd. for C15H9ClF3NO2 (327.69): C 54.98, H 2.77, N 4.27,
Found: C 55.21, H 2.94, N 4.41.
OH), 1610, 1581, 1502 (
n
CC aromatic) cmꢁ1 1H NMR (DMSO-d6,
;
300 MHz): d 9.73 (1H, bs, OH), 6.74 (1H, s, H6), 6.71 (1H, s, H3), 4.95
(2H, bs, NH2); 13C NMR (DMSO-d6, 75 MHz):
d 143.96, 137.98,
120.68, 115.88, 115.14, 114.09.
5.1.5. General procedure for the preparation of substituted 2-
nitrophenols (4)
An appropriate phenol (3) (150 mmol) was dissolved in glacial
acetic acid (50 mL), and the solution was stirred and maintained at
40 ꢀC. Then, a solution containing 11 mL of 65% HNO3 and 30 mL of
5.1.6.2. 2-Amino-4-bromophenol (5b) [25]. Yield 41.7%, dark red
solid; m. p. decomposition; IR (ATR): 3457, 3386 ( NH2), 3278 (b,
OH), 1601, 1582, 1505 (
CC aromatic) cmꢁ1 1H NMR (DMSO-d6,
500 MHz):
9.26 (1H, bs, OH), 6.70 (1H, d, J ¼ 2.5 Hz, H3), 6.54
(1H, d, J ¼ 8.2 Hz, H6), 6.48 (1H, dd, J ¼ 8.2 Hz, J ¼ 2.5 Hz, H5), 4.79
n
n
n
;
d

118
J. Kozic et al. / European Journal of Medicinal Chemistry 56 (2012) 108e119
(2H, bs, NH2); 13C NMR (DMSO-d6, 125 MHz):
118.25, 116.17, 115.82, 110.84.
d
143.46, 139.08,
The resulting pellet was resuspended in BugBuster Protein
Extraction Reagent Novagen (Merck KGaA, Darmstadt, Germany).
The cell debris was removed by centrifugation, and the histidine-
tagged protein was purified using an Äkta purifier (Amersham
Biosciences, Valley Stream, NY, USA). The purity of the protein was
confirmed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE)
followed by Coomassie staining of the gel. The protein concentra-
tion was determined by the Bradford method [30].
5.1.6.3. 2-Amino-5-bromophenol (5c) [26]. Yield 60.5%, dark red
solid; m. p. decomposition; IR (ATR): 3370, 3296 ( NH2), 3100e
2300 (b, OH), 1600, 1501 (
CC aromatic) cmꢁ1 1H NMR (DMSO-
d6, 500 MHz):
9.46 (1H, bs, OH), 6.75 (1H, d, J ¼ 2.5 Hz, H6), 6.67
n
n
n
;
d
(1H, dd, J ¼ 8.3 Hz, J ¼ 2.5 Hz, H4), 6.51 (1H, d, J ¼ 8.3 Hz, H3), 4.66
(2H, bs, NH2); 13C NMR (DMSO-d6, 125 MHz):
d
145.33, 136.52,
The isocitrate lyase activity was assayed according to the
protocol reported by Dixon and Kornberg [31]. The enzyme assay
122.04, 116.84, 115.53, 106.24.
was optimised in the final volume of 100 mL using 96-well plates
5.1.6.4. 2-Amino-4-(trifluoromethyl)phenol (5d) [28]. Yield 65.6%,
orange solid; m. p. 115e118 ꢀC; IR (ATR): 3391, 3318 (
NH2), 3061e
2342 (b, OH), 1618, 1528, 1460 (
CC aromatic) cmꢁ1 1H NMR
(DMSO-d6, 500 MHz): 9.85 (1H, bs, OH), 6.85 (1H, m, H5), 6.80e
6.64 (2H, m, H3, H6), 4.92 (2H, bs, NH2); 13C NMR (DMSO-d6,
125 MHz): 147.28 (1C, s, C1), 137.57 (1C, s, C2), 125.28 (1C, q,
(NUNC, Schoeller, Prague, Czech Republic) and the final conceꢁn1-
n
tration of tested compound in the reaction mixture 10 mmol L .
n
n
;
The reaction buffer contained 50 mmol Lꢁ1 of KH2PO4, 4 mmol Lꢁ1
of MgCl2$6H2O, 4 mmol Lꢁ1 of phenylhydrazine hydrochloride,
12 mmol Lꢁ1 of cysteine, H2O and KOH to pH 7.0. The isocitrate
cleavage was measured by the change in the absorbance at 324 nm,
which is associated with the formation of glyoxylate phenyl
hydrazone. Each tested compound was dissolved in DMSO to
d
d
J ¼ 269.3 Hz, CF3), 120.23 (1C, q, J ¼ 31.0 Hz, C4), 113.91 (1C, s, C6),
113.32 (1C, q, J ¼ 4.3 Hz, C5), 110.11 (1C, q, J ¼ 3.7 Hz, C3).
prepare a 1 mmol Lꢁ1 solution, and 1
to 93.9 L of reaction buffer. Then, it was added 0.1124 m
m
L of this solution was added
L of the
5.1.6.5. 2-Amino-5-(trifluoromethyl)phenol (5e) [29]. Yield 50.5%,
m
orange solid; m. p. 112e115 ꢀC; IR (ATR): 3397, 3316 (
n
NH2), 3500e
;
enzyme in phosphate buffer and glycerol solution with concen-
2300 (b,
n
OH), 1611, 1527, 1444 (
n
CC aromatic) cmꢁ1 1H NMR
tration 0.58 mg mLꢁ1 (Bradford). Finally the reaction was started by
(DMSO-d6, 300 MHz):
d
9.60 (1H, bs, OH), 6.92e6.82 (2H, m, H4,
the addition of 0.2
m
mol of (þ)-potassium Ds-threo-isocitrate in
H6), 6.67 (1H, d, J ¼ 8.7 Hz, H3), 5.18 (2H, bs, NH2); 13C NMR (DMSO-
solution. Ethionamide and isoniazid were employed as negative
controls (inhibition 0%), and 3-nitropropionic acid served as the
positive control. All of the control compounds were added to the
reaction mixture the same way like tested compounds and also
their concentration in the reaction mixture was 10
inhibitory activity of DMSO alone (1 L) was subtracted from the
d6, 75 MHz):
d
143.63 (C1), 141.03 (C2), 125.54 (1C, q, J ¼ 268.3 Hz,
CF3), 117.16 (1C, q, J ¼ 4.2 Hz, C6), 115.87 (1C, q, J ¼ 31.4 Hz, C5),
113.10 (C3), 110.42 (1C, q, J ¼ 3.7 Hz, C4).
m
mol Lꢁ1. The
5.2. In vitro antimycobacterial assay
m
activities of the evaluated compounds.
The in vitro antimycobacterial activity of the prepared
compounds was determined against M. tuberculosis My 331/88
(dilution of strain 10ꢁ3), M. avium My 330/88 (dilution of strain
5.4. In vitro cytotoxicity assay
10ꢁ5), M. kansasii My 235/80 (dilution of strain 10ꢁ4
) and
The measurement of the in vitro cytotoxicity of the most active
prepared compounds was performed in the human hepatic cell line
Hep G2 (passage 12) (ECACC, Salisbury, UK) using the CellTiter 96
AQueous One Solution Assay method (Promega G3580, East Port,
Prague, Czech Republic). In the beginning, 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
consisted of 24 h and was performed in an incubation device (Shel
Lab, Cornelius, OR, USA) at 37 ꢀC with a 5% atmosphere of CO2.
Before the experiment with the tested compounds, the cells were
microscopically examined. Each of the tested compounds was
dissolved in DMSO and evaluated in quadruplicate for each of the
eight increasing concentrations. The samples of the tested
compounds and control samples were incubated for 24 h at 37 ꢀC.
After the incubation period, the reagent from the kit was added to
the wells according to the recommendation of the manufacturer,
and the mixture was maintained at 37 ꢀC for 1.5 h. Then, the
absorbance at 490 nm was measured with a plate analyser Infinite
M200 (Tecan Group, Männedorf, Switzerland). The results were
statistically evaluated in Microsoft Excel 2010 (Microsoft Corpora-
tion, Redmond, WA, USA), and the IC50 value for each tested
compound was determined using GraphPad Prism 5.02 (GraphPad
Software, San Diego, CA, USA).
M. kansasii 6509/96 (dilution of strain 10ꢁ4). 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
ꢀ
was determined in a Sula’s semisynthetic medium (SEVAC, Prague,
Czech Republic) via the micromethod for the determination of the
minimum inhibitory concentration (MIC) at 37 ꢀC after 14 and 21
days and after 7, 14 and 21 days for M. kansasii. The tested
compounds were added to the medium in DMSO solutions, and INH
was used as a standard in a sterile water solution. The concentra-
tions of the tested compounds were used as follows: 1000, 500,
250, 125, 62.5, 32, 16, 8, 4, 2, 1 and 0.5
concentrations within the range of 0.5e250
for INH.
m
m
mol Lꢁ1. The same
mol Lꢁ1 were used
5.3. In vitro enzymatic assay
The mycobacterium H37Rv genomic DNA was used as
a template. The Icl gene (Rv0467) of 1.28 kb was amplified using
PCR. The amplified DNA was cloned into the pET-28b(þ) plasmid
vector Novagen (Merck KGaA, Darmstadt, Germany) using NdeI and
HindIII restriction sites. The recombinant plasmid was transferred
into Escherichia coli HB101. DNA sequencing was employed to
confirm that the inserted coding sequence had no mutations. For
bacterial expression, 25 mL culture volumes were inoculated with
BL21(DE3) cells containing the recombinant plasmid and allowed
to grow until an optical density of OD595 ¼ 0.6 was achieved. Then,
Acknowledgements
the culture was induced with 1 mmol Lꢁ1 isopropyl-
b-D-thio-
galactopyranoside solution and incubated at 30 ꢀC for an additional
This work was financially supported by IGA NT 13346 (2012),
4 h. The cells were harvested by centrifugation at 6000g for 10 min.
GAUK 27610/2010, SVV 2012-265-001, UNCE (No. 17/2012) and

J. Kozic et al. / European Journal of Medicinal Chemistry 56 (2012) 108e119
119
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