2
K. Ozadali et al. / Bioorg. Med. Chem. Lett. xxx (2014) xxx–xxx
diphosphate, as important intermediate for DNA-building blocks18
of M. tuberculosis were selected for the docking studies.
Table 1
Antimycobacterial activity and cytotoxicity of the compounds
The starting compounds, 4-azolylbenzaldehydes 1a–b were
synthesized in accordance with the method described in the liter-
ature.19 1a–b and thiosemicarbazide were refluxed in isopropanol
in the presence of acetic acid to obtain thiosemicarbazones 2a–b.
The target compounds 3–18 were prepared by cyclization of
2a–b with substituted phenacyl bromides (Scheme 1).
H
R
N
N
N
S
X
N
N
The target compounds 3–18 were evaluated for their antimyco-
bacterial activity in vitro against M. tuberculosis H37Rv using the
microplate alamar blue assay method20 in duplicate. The results
of the antimycobacterial activity (MIC values) were reported in Ta-
ble 1. It was observed that, 1-(4-(1H-1,2,4-triazol-1-yl)benzyli-
dene)-2-(4-(4-nitrophenyl)thiazol-2-yl)hydrazine 10 with MIC of
Compound
X
R
MICa
(
l
M)
Cytotoxicityb (% inhibition)
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
CH
CH
CH
CH
CH
CH
CH
CH
N
N
N
N
N
4-H
4-F
4-Cl
4-Br
2,4-DiCl
4-CH3
4-OCH3
4-NO2
4-H
4-F
4-Cl
4-Br
2,4-DiCl
4-CH3
4-OCH3
4-NO2
72.46
4.30
8.22
59.10
15.10
32.98
4.16
1.03
72.25
4.29
8.20
29.48
3.76
21.8
18.7
32.1
36.8
28.8
36.0
26.8
16.4
28.4
35.9
28.4
32.6
8.9
13.6
23.0
12.9
—
—
—
1.03
l
M, was more active than standard compounds, ethambutol
M respectively). Addition-
and ciprofloxacin (MIC = 7.65 and 4.71
l
ally, 4, 9, 12, 15, 16 and 18 were found as active as ethambutol and
ciprofloxacin. Non substituted derivatives exhibited weakest activ-
ity in this series (3 and 11 with MIC of 72.46 and 72.25 lM respec-
tively). These findings show that the presence of substituent on the
phenyl ring (R group) is important for the activity. Introduction of
NO2 which could be probably converted to active amino group by
reduction enhanced the antimycobacterial activity (10 and 18 with
N
N
N
4.33
16.62
3.99
0.36
0.12
INH
MIC of 1.03 and 3.99 lM respectively). The gap of activity between
Rifampin
Ethambutol
Ciprofloxacin
nitro substituted and other compounds may be explained by this
reduction. It was also observed that the presence of Cl and F atoms
on phenyl ring increased antimycobacterial activity remarkably (4,
7.65
4.71
—
a
MIC = minimum inhibitory concentration.
Cytotoxicity against THP1 cell line at 50
5, 12 and 13; MIC = 4.29–8.22
to the phenyl ring did not significantly affect the activity (6 and 14;
MIC = 59.10 and 29.48 M respectively). However, no relationship
lM). However the substitution of Br
b
l
g/mL.
l
can be established between other substituents (2,4-diCl, CH3 and
OCH3) and the antimycobacterial activity in the series. When the
activities of 3 and 11 are compared, it can be assumed that the type
of azole structure is not a decisive factor for the activity.
All compounds were also tested for in vitro cytotoxicity against
THP1cell line at 50 lg/mL by MTT assay method. Percentage
docking results (Fig. 2A) showed that 10 did not make any interac-
tion with CYP121 (Table 2) and the orientation of 10 was com-
pletely different from that of fluconazole (Fig. 3A).
Then, docking studies were carried out with InhA and TMPK.
According to docking results with InhA, it was observed that 10
interacts with Pro156 and Tyr158 (Fig. 2B). For InhA, it is known
that Tyr158 is a key residue for activity22 and it forms a direct
hydrogen bond with inhibitors by its hydroxyl group.23 Differently
growth of cells was reported in Table 1. None of the compounds
showed significant cytotoxicity (8.9–36.8%).
In order to further rationalize the biological results, molecular
docking studies were performed on compound 10, the most active
derivative, with selected enzymes, CYP121, InhA and TMPK of M.
tuberculosis. To check the docking procedure, the ligands which
were taken from crystal structure of the enzyme-ligand complexes,
were rebuilt and redocked. RMSD values of the docking pose from
the original orientation of the ligands were found between 0.33–
0.51 Å. The crucial residues of enzymes that interact with ligands
were given in Table 2.
from mentioned interaction,
p–H interaction was observed be-
tween phenyl ring of 10 and methylene of Tyr158. Docking studies
with TMPK revealed that 10 interacted with Tyr103, Asp163 and
Glu166 (Fig. 2C). It was reported that Tyr103 is a residue which
is responsible for the affinity of TMPK,24 whereas Asp163 and
Glu166 are the residues of lid region25 of TMPK. By regarding the
interactions (Table 2) and orientations (Fig. 3B and C respectively),
it can be proposed that the docking results of InhA and TMPK are
more reasonable than that of CYP121.
Firstly, CYP121, which is known to bind azole drugs (ex.
On the other hand, the order of the energies of the conformers
of 10 in enzymes was found as 10InhA > 10Cyp121 > 10TMPK. Having
fluconazole),21 were selected for docking studies. Unfortunately,
O
O
S
ii
X
i
H
H2N
H
NH
N
NH2
N
H
X
F
N
1a-b
N
O
H
N
H
N
R
Br
iii
N
NH2
N
N
R
S
S
X
N
X
N
N
2a-b
3-18
N
X: CH (a), N (b); R: H, 4-F, 4-Cl, 4-Br, 2,4-diCl, 4-CH3, 4-OCH3, 4-NO2
Scheme 1. Synthesis of the compounds. Reagents and conditions: (i) K2CO3, DMSO, ultrasonic irradiation; (ii) CH3COOH, isopropanol, reflux; (iii) THF, rt.
Ozadali, Keriman
