N-benzylsalicylthioamides: Highly active potential antituberculotics

Article abstract of DOI:10.1002/ardp.200800032

A gseries of 29 new derivatives of N-benzylsalicylthioamides was synthesized and the compounds were tested for in-vitro antimycobacterial activity against Mycobacterium tuberculosis, Mycobacterium kansasii, and Mycobacterium avium. The activity was analyz

Full text of DOI:10.1002/ardp.200800032

ˇ
Arch. Pharm. Chem. Life Sci. 2009, 342, 113–119
R. Dolezal et al.
113
Full Paper
N-Benzylsalicylthioamides: Highly Active Potential
Antituberculotics
1
1
1
1
1
1
ˇ
Rafael Dolezal , Karel Waisser , Eva Petrlꢀkovꢁ , Jirꢀ KuneÐ , Lenka Kubicovꢁ , MiloÐ Machꢁcek ,
Jarmila Kaustovꢁ2, and Hans Martin Dahse3
1 Department of Inorganic and Organic Chemistry, Faculty of Pharmacy in Hradec Krꢀlovꢁ, Charles University
in Prague, Hradec Krꢀlovꢁ, Czech Republic
2 Department for Diagnostic of Mycobacteria, Regional Institute of Public Health in Ostrava, Ostrava, Czech
Republic
3 Leibniz Institute for Natural Product Research and Infection Biology – Hans Knꢂll Institute, Jena, Germany
A gseries of 29 new derivatives of N-benzylsalicylthioamides was synthesized and the compounds
were tested for in-vitro antimycobacterial activity against Mycobacterium tuberculosis, Mycobacte-
rium kansasii, and Mycobacterium avium. The activity was analyzed by quantitative structure-activ-
ity relationship (QSAR). Activity increased with increasing lipophilicity and electron donating
effect of the substituents in the acyl moiety and decreased with the electrophilic superdelocaliz-
ability of the molecules. The most active compounds are more active than isoniazid (INH) and
are active against INH-resistant potential pathogenic strains of mycobacterium.
Keywords: Antimycobacterial activity / Antituberculotic activity / N-Benzylsalicylamides / QSAR / Salicylthioamides /
Received: January 29, 2008; accepted: June 19, 2008
DOI 10.1002/ardp.200800032
diseases. The development of new antituberculotic
agents with high activity is the principal goal of our
research. Our group is especially interested in the devel-
opment of drugs against M. tuberculosis and potentially
pathogenic strains like M. avium and M. kansasii. We have
recently studied a number of structurally different com-
pounds, such as salicylanilides [1], benzoxazinediones [2],
alkoxyphenylcarbamic acids [3], tetrazoles [4, 5], dihy-
droindolethiones [6], derivatives of benzoxazine with thi-
oxo group [7], and other heterocycles [8]. The aim of this
paper is the synthesis, antimycobacterial evaluation, and
quantitative structure-activity relationship (QSAR) study
of N-benzylsalicylthioamides.
Introduction
Since 1985, the world is confronted with the return of
tuberculosis to Europe and North America. In the devel-
oping countries, due to insufficient medical care,
hygienic standards, and compliance of the population
with the treatment, a number of mycobacterial strains
became resistant to modern chemical drugs (i.e., the
development of multidrug resistant strains); infection
has been often transferred to Europe and North America,
since migration towards developed countries is a contem-
porary feature. The multidrug resistant strains of M.
tuberculosis coming to Central Europe are mainly from
the countries of the former Soviet Union. In addition, the
unfavorable state is also being influenced by an increase
in AIDS, which is often accompanied by mycobacterial
Results
Chemistry
Correspondence: Karel Waisser, Charles University in Prague, Faculty
of Pharmacy in Hradec Krꢀlovꢁ, Heyrovskꢁho 1203, 500 05 Hradec
Krꢀlovꢁ, Czech Republic.
E-mail: waisser@faf.cuni.cz
Fax: +42 0495 067166
The synthesis of the title compounds is illustrated in
Scheme 1. The synthesis of most of the starting N-benzyl-
salicylamides was described in our previous paper [9]. We
completed a group of starting N-benzylsalicylamides
with fourteen new compounds (see Table 1 and Fig. 1).
Abbreviation: multiple linear regression (MLR)
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114
R. Dolezal et al.
Arch. Pharm. Chem. Life Sci. 2009, 342, 113–119
zylsalicylamides and P4S10 produce heterocyclic com-
pounds that hydrolyze to N-benzylsalicylthioamides (see
Fig. 1). The structures are summarized in Table 3 and
Fig. 2. The yields, melting points, and carbonyl frequen-
cies are summarized in Table 4. The application of the
Willgerodt–Kindler reaction [11] in case of N-benzylsali-
cylamides was unsuccessful. The structure of the com-
1
pounds was established by IR, H- and 13C-NMR spectro-
scopy and verified by elemental analysis.
For substituents R1 and R2 see Tables 1 and 3.
Biology
Scheme 1. Synthetic pathway to N-Benzylsalicylthioamides.
In-vitro antimycobacterial activity of the compounds was
evaluated against Mycobacterium tuberculosis CNCTC My
331/88, Mycobacterium kansasii CNCTC My 235/80, Myco-
bacterium avium CNCTC My 330/88, and Mycobacterium
kansasii 6509/96 using the micromethod for the determi-
nation of the minimum inhibitory concentration (MIC).
All strains were obtained from the Czech National Collec-
tion of Type Cultures (CNCTC), National Institute of Pub-
Yields, melting points, and carbonyl frequencies are sum-
marized in Table 2. The synthesis of N-benzylsalicylthioa-
mides was performed by the method elaborated in our
group and described in a patent [10].
The synthesis consists of the microwave-promoted
thionation of the starting N-benzylsalicylamides. N-Ben-
Table 1. Antimycobacterial activity (minimum inhibition concentrations, MICs) of N-benzylsalicylamides 1–15.
Compound R1
R2
MIC (lmol/L)
M. tuberculosis My 331/88 M. avium My 330/88 M. kansasii My 235/80 M. kansasii 6509/96
14 d
16
62.5
32
21 d
32
62.5
32
32
14 d
21 d
62.5
16
32
14 d
62.5
16
32
21 d
62.5
32
62.5
32
14 d
32
62.5
32
21 d
62.5
62.5
32
1
2
3
4
H
H
5-Br
5-Br
4-tert-but
3-CF3
3-Br
32
16
32
16
4-Br
16
32
32
32
32
5
6
3,5-Cl2
4-Cl
4-tert-but
4-Br
32
16
32
32
62.5
32
62.5
32
62.5
32
62.5
32
62.5
32
62.5
32
7
8
9
10
4-CH3
4-CH3
4-CH3
4-CH3
4-CH3
4-OCH3
3-CH3
3-CH3
3,5-Br2
H
4-CH3
4-Cl
4-tert-but
3-NO2
3-Cl
H
4-Cl
4-CF3
62.5
125
62.5
32
62.5
125
62.5
16
62.5
1
125
500
125
62.5
62.5
125
125
32
62.5
62.5
62.5
62.5
16
125
125
125
125
250
250
125
500
250
250
125
16
250
125
32
500
250
32
125
125
125
62.5
62.5
A250
62.5
62.5
11
32
62.5
62.5
62.5
62.5
62.5
62.5
62.5
4
125
125
125
62.5
62.5
4
12
62.5
62.5
32
62.5
A250
62.5
62.5
62.5
62.5
62.5
62.5
62.5
62.5
13a)
14
15
Isoniazid
1
A250
A250
a)
Synthesis in ref. [18].
Table 2. Yield, melting point, and carbonyl frequency of N-benzylsalicylamides 1–15.
Compound
Yield
(%)
M.p.
(8C)
mC=O
(cm– 1
Compound
Yield
(%)
M. p.
(8C)
mC=O
(cm– 1
)
)
1
2
3
4
5
6
7
73
91
51
49
76
75
76
130–131
116–118
168–170
154–156
91–92
1645
1636
1641
1622
1641
1621
1644
8
9
81
89
94
84
77
92
85
141–143
162–163
118–120
162–163
105–107
97–99
1642
1641
1650
1640
1636
1643
1630
10
11
12
14
15
153–156
105–107
129–130
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Arch. Pharm. Chem. Life Sci. 2009, 342, 113–119
N-Benzylsalicylthioamides as Potential Antituberculotics
115
Table 3. Antimycobacterial activity (minimum inhibition concentrations, MICs) of N-benzylsalicylthioamides 16–45.
Compound R1
R2
MIC (lmol/L)
M. tuberculosis My 331/88 M. avium My 330/88 M. kansasii My 235/80 M. kansasii 6509/96
14 d
0.5
0.25
1
21 d
14 d
21 d
14 d
21 d
14 d
2
0.5
0.5
4
2
2
2
2
21 d
16a)
17
18
19
20
21
22
23
24
H
H
H
H
H
H
H
H
H
4-CH3
4-Cl
4-OCH3
3,4-Cl2
4-F
3-CH3
4-tert-but
3-Cl
1
0.5
2
4
0.98
1
1
2
1
2
1
0.5
1
4
2
4
2
2
1
2
1
1
4
2
4
4
4
1
2
1
1
4
4
2
2
2
1
0.25
0.5
0.98
0.98
1
0.5
0.25
1
0.5
0.5
0.98
0.98
2
1
0.25
1
4
0.49
0.5
0.5
1
H
1
1
1
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
H
3-CF3
3,4-Cl2
3-Br
4-Br
H
3,4-Cl2
4-F
3,4-Cl2
4-tert-but 32
4-Br
H
4-CH3
4-Cl
4-tert-but
3-NO2
H
H
3-Cl
4-CH3
4-Cl
4-CF3
0.5
4
1
1
2
2
1
16
1
4
2
2
2
4
2
0.5
16
8
4
8
16
8
62.5
62.5
2
1
0.25
0.5
1
1
4
4
1
1
16
8
4
8
32
8
62.5
62.5
4
2
16
8
8
8
32
8
32
62.5
8
2
0.25
0.5
1
2
16
8
2
125
2
62.5
A250
2
32
8
16
8
32
16
62.5
62.5
16
2
0.5
1
1
4
32
8
2
125
2
62.5
A250
2
16
8
4
8
16
8
32
62.5
4
1
0.5
1
1
4
16
8
1
125
2
62.5
4
2
16
8
4
8
16
8
32
62.5
8
2
5-Br
5-Br
5-Br
5-Cl
5-Cl
5-Cl
3,5-Cl2
3,5-Cl2
4-Cl
4-CH3
4-CH3
4-CH3
4-CH3
4-CH3
5-OCH3
4-OCH3
4-OCH3
5-NO2
3-CH3
3,5-Br2
32
32
2
1
0.25
0.5
0.5
2
8
4
1
62.5
4
32
1
1
0.5
0.125
0.5
0.5
2
4
2
0.5
32
2
0.5
0.5
2
2
4
1
1
1
4
16
8
1
125
4
62.5
41
42
43
44
4
2
A125
1
32
A125
1
32
2
32
1
45
Isoniazid
A250
A250
4
a)
Synthesis in ref. [19].
at 378C for 14 and 21 days. For the sake of comparison,
we also included the values of MICs of the standard iso-
niazid (INH). The method is described in our previous
papers [1–9]. The antiproliferative activity and cytotoxic-
ity of some compounds were investigated at the Leibniz
Institute for Natural Product Research and Infection Biol-
ogy, Hans Knꢀll Institute in Jena. The results are summar-
ized in Table 5. The methods were described in the pre-
vious papers [12, 13] and in the experimental part (Sec-
tion 4) herein.
Figure 1. A survey of structures of N-benzylsalicylamides. For
substituents R1 and R2 see Table 1.
Calculations
The program HyperChem, Release 7.52, was used to cal-
culate log P (octanol / water system), volume, surface, and
polarizability of the compounds under study [14]. The
Figure 2. A survey of structures of N-benzylthiosalicylamides.
For substituents R1 and R2 see Table 3.
lic Health, Prague, with the exception of M. kansasii 6509/ program Gaussian 03, Revision C.02, was used for the
96, a clinical isolate. Tables 1 and 3 summarize the MICs quantum chemical calculations [15]. All molecular mod-
of the new starting compounds and the title products, els were computed by the B3LYP/6-31G* method. The
respectively. The MICs were determined after incubation obtained wave-function data were processed in a self-
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R. Dolezal et al.
Arch. Pharm. Chem. Life Sci. 2009, 342, 113–119
Table 4. Yield, melting point, and frequency of hydroxyl vibration of N-benzylsalicylthioamides 17–45.
Compound
Yield
(%)
M.p.
(8C)
mC=O
(cm-1)
Compound
Yield
(%)
M. p.
(8C)
mC=O
(cm-1)
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
34
17
43
41
34
42
80
51
40
37
32
38
55
34
40
73–74
101–102
92–93
96–97
64–65
85–86
89–90
96–97
29–30
117–118
136–137
131–132
109–110
133–134
112–113
3311
3324
3332
3301
3316
3302
3312
3305
3311
3365
3366
3309
3311
3291
3363
32
33
34
35
36
37
38
39
40
41
42
43
44
45
19
31
49
45
81
45
52
10
37
29
49
25
42
5
91–93
120–122
106–108
91–93
99–100
131–133
121–122
99–100
94–95
74–75
73–74
125–127
94–95
130–132
3355
3338
3320
3308
3313
3312
3327
3315
3273
3377
3376
3324
3376
3347
developed program to compute quantum chemical
descriptors. The complete set of 177 molecular descrip-
tors consisted of 166 different quantum chemical
descriptors (e.g., superdelocalizabilities, indices of fron-
tier electron densities, Mulliken charges, etc.), Hammett
constants, Hansch constants, and several chemical shifts
from 13C-NMR spectra. A detailed review of quantum
chemical descriptors is available in the literature [16].
The structure-activity relationships were searched for by
the program STATOO (R. Doleˇzal). The program STATOO
is based on multiple linear regression (MLR). Its selection
algorithm searches the best parameters in correlation
equations. Statistical evaluation is given under every
equation. The symbols are usual, q2 is the cross-validated
correlation coefficient determined by leave-one-out pro-
cedure.
Figure 3. Comparison of antimycobacterial activity against M.
tuberculosis (log MIC after 14 d incubation) of N-benzylsalicylth-
ioamides 17–45 with that of N-benzylsalicylamides. The values
of the activity of corresponding N-benzylsalicylamides not pre-
sented in this paper were culled from Ref. [9].
bonyl oxygen for sulfur is demonstrated in Fig. 3. Inter-
estingly, the antimycobacterial activities of N-benzylsali-
cylamides do not correlate significantly with those of the
corresponding N-benzylsalicylthioamides. The newly syn-
thesized compounds form a new promising group of
antimycobacterials with a broad spectrum of antimyco-
bacterial activity. Two most active compounds, 17 and
36, were chosen for preclinical testing. Antiproliferative
activity and cytotoxicity of compounds 16, 17, 18, 19, 20,
23, and 36 were investigated (Table 5). Unfortunately, the
compounds under study are cytotoxic.
The set of 177 descriptors was used for QSAR study. The
best three-parameter QSAR models were searched for
using MLR technique in the STATOO program. The most
important parameters were: log P – partition coefficient
for system octanol / water; rR1 – Hammett constants of
substituents R1 in the acyl moiety; SumSew – sum of
Discussion
In general, the synthesized compounds possess in-vitro
activities against all tested mycobacterial strains, which
are better than or comparable to that of INH (Tables 1
and 3). The values of MICs are generally within the range
from 0.125 to 125 lmol/L, most often between 0.5 and
4 lmol/L. The most active compounds, 17 and 36, were
more active against M. tuberculosis 331/88 than INH. All
new compounds, again, were more effective against M.
kansasii 235/80 and M. avium 330/88 than INH. The
replacement of an oxo group in the starting N-benzylsali-
cylamides for a thioxo group increases the antimycobac-
terial activity of the title compounds against all mycobac-
terial strains. The increase in antimycobacterial activity
against M. tuberculosis (incubation 14 d) by replacing car-
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Arch. Pharm. Chem. Life Sci. 2009, 342, 113–119
N-Benzylsalicylthioamides as Potential Antituberculotics
117
Table 5. Antiproliferative activity and cytotoxicity.
of N-benzylsalicylthioamides for their antimycobacterial
activities.
Compound
Antiproliferative activity Cytotoxicity
14d
M tuber:
log MIC
= – 0.478 (l 0.107)log P + 1.551 (l 0.206)rR1
Huvec GI50
K-562 GI50
HeLa CC50 / CC10
(lg/mL)
(lg/mL)
(lg/mL)
+ 0.048 (l 0.009)SumSew – 2.46 (l 0.646)
Eq. (1)
Eq. (2)
Eq. (3)
Eq. (4)
Eq. (5)
Eq. (6)
16
17
18
19
20
23
36
INH
ETH
9.2
10.0
5.9
19.2
5.9
8.7
9.9
5.2
14.8
5.2
3.5
13.0
A50.0
50.0
11.1 / 3.9
12.5 / 5.7
5.5 / 2.7
21.6 / 9.2
5.6 / 2.1
r2 = 0.811 q2 = 0.758 s = 0.282 F = 37.407 n = 30
21d
log MIC = – 0.491 (l 0.097)log P + 1.581 (l 0.187)rR1
M tuber:
+ 0.043 (l 0.008)SumSew – 1.725 (l 0.586)
4.6
3.7 / 1.7
12.5
A50.0
37.2
12.4 / 4.5
A50.0 / 50.0
40.8 / 40.2
r2 = 0.828 q2 = 0.776 s = 0.256 F = 41.595 n = 30
14d
M avi:
log MIC
= 1.819 (l 0.163)rR1 – 0.013 (l 0.002)Sn39
+ 0.043 (l 0.007)SumSew + 2.118 (l 0.765)
weighted electrophilic superdelocalizabilities for all
atoms in the molecule (calculated from the output of the
Gaussian); Sn39 – nucleophilic superdelocalizability on
carbon 3 on the benzyl (calculated from output of the
Gaussian); charge1 – Mulliken charge on carbon 1 in the
acyl moiety (calculated from output of the Gaussian); SeH
– electrophilic superdelocalizability of hydrogen atoms
in the thioamide group; Sumfe – sum of indices of elec-
trophilic frontier electron density of all atoms in the mol-
r2 = 0.913 q2 = 0.88 s = 0.219 F = 86.974 n = 29
21d
M avi:
log MIC
= 1.611 (l 0.19)rR1 – 0.012 (l 0.002)Sn39
+ 0.041 (l 0.008)SumSew + 2.101 (l 0.887)
r2 = 0.864 q2 = 0.819 s = 0.254 F = 52.98 n = 29
14d
M kan:
log MIC
= – 0.472 (l 0.106)log P + 1.948 (l 0.204)rR1
+ 0.04 (l 0.009)SumSew – 1.27 (l 0.64)
ecules (calculated from output of the Gaussian); pR2
–
r2 = 0.846 q2 = 0.796 s = 0.28 F = 47.615 n = 30
Hansch constant of substituents R2 on the benzyl; char-
geH – Mulliken charge on the hydrogen atom in the thio-
amide group. The program STATOO determined the stat-
istically most significant correlation equations (see Eqs.
1–8). In accordance with the correlations found, the anti-
mycobacterial activities are always enhanced by electron
donating substituents R1 in the acyl moiety (i.e., when sR1
is negative). Lipophilicity (log P or pR2) mostly increases
the activity. Other significant descriptors correspond
with the local properties of certain atoms (i.e., Sn39, char-
geH, charge1 and SeH) or express the overall molecular
properties (i.e., SumSew and Sumfe). A higher nucleophilic
superdelocalizability on carbon 39 (Sn39) increases the
activity against M. avium Eq. (3) and Eq. (4). Electrophilic
superdelocalizability (SeH) and Mulliken charge (char-
geH) on the hydrogen atom of the thioamide group are
both statistically associated with Hammett constant (rR1),
and therefore, when they are lower, they also enhance
the activity in the same way Eqs. (6 and 7). A lower Mul-
liken charge on carbon 1 (charge1) enhances the activity
Eq. (6). The sum of weighted electrophilic superdelocaliz-
abilities (SumSew), present in most equations, should be
lower in order to increase the activity. The sum of indices
of electrophilic electron densities (Sumfe) increases the
activity when it is higher Eq. (6). All presented QSAR mod-
els show acceptable statistical significance suggesting a
high importance of lipophilic and electronic properties
21d
M kan:
log MIC
= 39.247 (l 10.225)charge1 + 98.36 (l 9.011)SeH
– 3.271 (l 0.43)Sumfe + 13.978 (l 2.456)
r2 = 0.886 q2 = 0.856 s = 0.236 F = 67.402 n = 30
14d
M kan: cl:
log MIC
= – 0.486 (l 0.103)pR2 + 177.82 (l 18.757) chargeH
+ 0.046 (l 0.007)SumSew – 64.828 (l 6.336)
Eq. (7)
Eq. (8)
r2 = 0.869 q2 = 0.811 s = 0.249 F = 57.257 n = 30
21d
M kan: cl:
log MIC
= – 0.401 (l 0.092)log P + 1.731 (l 0.177)rR1
+ 0.034 (l 0.008)SumSew –1.006 (l 0.556)
r2 = 0.852 q2 = 0.791 s = 0.243 F = 50.012 n = 30
The most active compound can be obtained by a syn-
thesis of four compounds if the Topliss approach [17] is
used.
The eight above-mentioned MLR QSAR models repre-
sent the best three-parameter linear relationships found
in 177 investigated descriptors. Generally, the antimyco-
bacterial activities of N-benzylsalicylthioamides against
M. tuberculosis, M. avium, M. kansasii, and M. kansasii cl. are
enhanced by electron-donating as well as lipophilic sub-
stituents at the acyl moiety of N-benzylsalicylthioamides.
It seems that electronic properties of the substituents in
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R. Dolezal et al.
Arch. Pharm. Chem. Life Sci. 2009, 342, 113–119
under reflux for 45 min in a microwave reactor. Without isola-
tion of the intermediary product, the reaction mixture was
added to a mixture of toluene (150 mL) and dilute HCl (150 mL,
pH 1), and refluxed under vigorous stirring for 3 h. The toluene
layer was separated, toluene was evaporated, and the residue
was chromatographed (silica gel, toluene). The product was
recrystallized from ethanol / water (yields were in the range of
17–81%).
the acyl moiety of N-benzylsalicylthioamides induce
more distinctive changes in the activity than the sub-
stituents on the benzyl. For further development of N-
benzylsalicylthioamides, we suggest to introduce potent
electron-donating substituents (e.g., –N(CH3)2) into the
acyl moiety and to keep lipophilic substituents in the
amine moiety. Contrary to isosteres of salicylanilides, the
antimycobacterial activity of which has been recently
found to be enhanced by electron-withdrawing substitu-
ents (paper to be published), the N-benzylsalicylthioa-
mides may act with a different mechanism. We suppose
that increasing the electron density on the sulfur of the
CSNH group is the key factor for increasing the antimyco-
bacterial activity.
Tests for cytotoxicity and antiproliferative activity
Cells and culture conditions
Cells /cell-culture medium: A: Huvec (ATCC CRL-1730) / DMEM
(Cambrex 12-614F; Cambrex Bio Science at Biotech A.S., Prague,
Czech Republic); B: K-562 (DSM ACC 10) / RPMI 1640 (Cambrex
12-167F); C: HeLa (DSM ACC 57) / RPMI 1640 (Cambrex 12-167F).
Cells were grown in the appropriate cell-culture medium sup-
plemented with 10 mL/L ultraglutamine I (Cambrex 17-605E/
U1), 500 lL/L gentamicin sulfate (Cambrex 17-518Z), and 10%
heat-inactivated fetal bovine serum (PAA A15-144) at 378C in
high-density polyethylene flasks (NUNC 156340; Fisher Scien-
tific spol. s.r.o., Pradubice, Czech Republic).
This study is a part of the research project number
MSM0021620822 of the Ministry of Education of the Czech
Republic.
The authors have declared no conflict of interest.
Antiproliferative assay
The test substances were dissolved in DMSO before being diluted
in DMEM / Dulbecco's modified Eagle medium. The adherent
cells were harvested at the logarithmic growth phase after soft
trypsinization, using 0.25% trypsin in PBS containing 0.02%
EDTA (Biochrom KG L2163; Biochrom, Berlin, Germany). For
each experiment approximately 10000 cells were seeded with
0.1 mL culture medium per well of the 96-well microplates
(NUNC 167008).
Experimental
Melting points were determined on a Kofler block (C. Reichert,
Vienna, Austria) and are uncorrected. The IR spectra were meas-
ured in KBr pellets or in CHCl3 solutions on a Nicolet Impact 400
apparatus (Nicolet, Madison, WI, USA); the wave numbers are
given in cm1. The NMR spectra were recorded on a Varian Mer-
cury–Vx BB 300 spectrometer (Varian Inc., Palo Alto, CA, USA)
operating at 300 MHz for H and 75 MHz for 13C in d6-DMSO.
1
Cytotoxic assay
For the cytotoxic assay, HeLa cells were 48 hours pre-incubated
without the test substances. The dilutions of the compounds
were carried out carefully on the subconfluent monolayers of
HeLa cells after the pre-incubation time.
Chemical shifts were recorded as d values in ppm, and were indi-
rectly referenced to tetramethylsilane via the solvent signal
(7.26 for 1H and 77.0 for 13C). The coupling constants J are given
in Hz. Elemental analyses were done on a CHNS-O CE FISONS
EA1110 elemental analyzer (Fisons Instruments, Italy). Analyses
of the C, H, N, and S contents were within l 0.4% of the theoret-
ical values. To check the purity of the products, TLC was perform-
ed on silica gel plates precoated with a fluorescent indicator,
Silufol UV 254 + 366 (Kavalier, Votice, The Czech Republic), in
cyclohexane / acetone 3 : 1. Interpretation of NMR spectra and
values of elemental analyses are in the attachment to the jour-
nal.
Condition of incubation
The cells were incubated with dilutions of the test substances for
72 hours at 378C in a humidified atmosphere and 5% CO2.
Method of evaluation
To estimate the influence of chemical compounds on cell prolif-
eration of K-562, we determinate the numbers of viable cells
present in multi-well plates via CellTiter-Blue1 assay. It uses the
indicator dye resazurin to measure the metabolic capacity of
cells as the indicator of cell viability. Viable cells of untreated
control retain the ability to reduce resazurin into resorufin,
which is highly fluorescent. Non-viable cells rapidly lose meta-
bolic capacity, do not reduce the indicator dye, and thus do not
generate a fluorescent signal. Under our experimental condi-
tions, the signal from the CellTiter-Blue1 reagent is proportional
to the number of viable cells.
General procedure for preparation of
N-benzylsalicylamides 1–15
A suspension of substituted salicylic acid (0.02 mol) and substi-
tuted benzylamine (0.02 mol) in chlorobenzene (100 mL) was
heated under reflux in the presence of PCl3 (0.01 mol) for 3 h.
The reaction mixture was filtered while hot, and the solvent was
evaporated under reduced pressure. The product was recrystal-
lized from ethanol / water (yields were in the range of 49–95%).
The adherent Huvec and HeLa cells were fixed by glutaralde-
hyde and stained with a 0.05% solution of methylene blue for
15 min. After gently washing, the stain was eluted with 0.2 mL
of 0.33 N HCl in the wells. The optical densities were measured
at 660 nm in SUNRISE microplate reader (TECAN, Switzerland).
General procedure for the preparation of
N-benzylsalicylthioamides 16–45
A suspension of starting N-benzylsalicylamide (0.005 mol) and
an equivalent amount of P4S10 in pyridine (10 mL) was heated
i 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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Arch. Pharm. Chem. Life Sci. 2009, 342, 113–119
N-Benzylsalicylthioamides as Potential Antituberculotics
119
The GI50 and CC50 values were defined as the value at the inter-
section of the dose response curve with the 50% line, compared
to untreated control. These comparisons of the different values
were performed with the software Magellan (TECAN).
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