Metronidazole hydrazone conjugates: Design, synthesis, antiamoebic and molecular docking studies

Article abstract of DOI:10.1016/j.bmcl.2015.06.091

Abstract Metronidazole hydrazone conjugates (2-13) were synthesized and screened in vitro for antiamoebic activity against HM1: IMSS strain of Entamoeba histolytica. Six compounds were found to be better inhibitors of E. histolytica than the reference dru

Full text of DOI:10.1016/j.bmcl.2015.06.091

Accepted Manuscript
Metronidazole hydrazone conjugates: design, synthesis, antiamoebic and mo-
lecular docking studies
Mohammad Fawad Ansari, Shadab Miyan Siddiqui, Subhash M. Agarwal,
Kunwar Somesh Vikramdeo, Neelima Mondal, Amir Azam
PII:
S0960-894X(15)00696-4
DOI:
http://dx.doi.org/10.1016/j.bmcl.2015.06.091
BMCL 22881
Reference:
To appear in:
Bioorganic & Medicinal Chemistry Letters
Received Date:
Revised Date:
Accepted Date:
11 February 2015
31 May 2015
24 June 2015
Please cite this article as: Ansari, M.F., Siddiqui, S.M., Agarwal, S.M., Vikramdeo, K.S., Mondal, N., Azam, A.,
Metronidazole hydrazone conjugates: design, synthesis, antiamoebic and molecular docking studies, Bioorganic &
Medicinal Chemistry Letters (2015), doi: http://dx.doi.org/10.1016/j.bmcl.2015.06.091
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Graphical Abstract
Metronidazole hydrazone conjugates: design,
synthesis, antiamoebic and molecular
docking studies
a
a
b
Mohammad Fawad Ansari , Shadab Miyan Siddiqui , Subhash M. Agarwal , Kunwar Somesh
c
c
a
Vikramdeo , Neelima Mondal and Amir Azam *
a
Department of Chemistry, Jamia Millia Islamia, Jamia Nagar, New Delhi 110025 India.
Bioinformatics Division, Institute of Cytology and Preventive Oncology (ICPO) I-7, Sector - 39, Noida – 201301, Uttar Pradesh,
b
India.
c
School of Life Sciences, Jawaharlal Nehru University, New Delhi 110062, India.

Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com
Metronidazole hydrazone conjugates: design, synthesis, antiamoebic and molecular
docking studies
a
a
b
c
Mohammad Fawad Ansari , Shadab Miyan Siddiqui , Subhash M. Agarwal , Kunwar Somesh Vikramdeo ,
c
a
Neelima Mondal and Amir Azam *
a
Department of Chemistry, Jamia Millia Islamia, Jamia Nagar, New Delhi 110025 India.
b
Bioinformatics Division, Institute of Cytology and Preventive Oncology (ICPO) I-7, Sector - 39, Noida – 201301, Uttar Pradesh, India.
c
School of Life Sciences, Jawaharlal Nehru University, New Delhi 110062, India.

Corresponding author. Tel.: +91-11-26981717/3254; fax: 91-11-26980229; e-mail: amir_sumbul@yahoo.co.in (A. Azam)
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
Metronidazole hydrazone conjugates (2-13) were synthesized and screened in vitro for
antiamoebic activity against HM1: IMSS strain of Entamoeba histolytica. Six
compounds were found to be better inhibitors of E. histolytica than the reference drug
metronidazole. These compounds showed greater than 50-60% viability against HeLa
cervical cancer cell line after 72hr treatment. Also, molecular docking study was
undertaken on E. histolytica thioredoxin reductase (EhTHRase) protein which showed
significant binding affinity in the active site. Out of the six actives, some of the
compounds showed lipophilic characteristics.
Available online
Keywords:
Metronidazole
Amoebiasis
Entamoeba histolytica
MTT-Assay
2
009 Elsevier Ltd. All rights reserved.
Thioredoxin reductase
Amoebiasis, a contagious disease of the human
novel metronidazole hydrazone conjugates. In this paper, we
herein report the synthesis, antiamoebic activity, molecular
docking and lipophilic studies of metronidazole hydrazone
conjugates (Figure 4).
gastrointestinal tract caused by parasitic protozoa Entamoeba
1
-2
histolytica (E. histolytica).
The parasite causes invasive
infections and induces tissue destruction, producing amoebic
colitis, dysentery and liver abscesses that affects 50 million
3
-4
people and causes 100,000 death per annum world wide.
N
Moreover, relapses of intestinal and hepatic amoebiasis have
N
5
been reported. Metronidazole (MNZ), tinidazole (TZ) and
N
O N
2
N
ornidazole (OZ) (Figure 1) are the widely used medicament for
the treatment of protozoal infections, in which MNZ is the drug
of choice for the treatment of amoebiasis, but long term use
O N
2
N
O N
2
N
O
OH
6
S
causes several side effects. Although, it is mutagenic in bacteria,
O
7
carcinogenic to rodents and genotoxic to human cells. However,
OH
MNZ
Cl
OZ
due to inadequate epidemiological evidence, it is not considered
as a risk factor of cancer in humans.
TZ
The side chains attached to MNZ provide an
opportunity to carry out modifications to derive novel molecules
which might exhibit better antiamoebic activity and lesser
toxicity for the host. In our previous studies some metronidazole
conjugates have been found to exhibit promising antiamoebic
Figure 1: Antiamoebic drugs having imidazole ring
The synthetic pathway leading to target compounds (2-
3) is depicted in Scheme 1. The key intermediate 4-[2-(2-
1
methyl-5-nitro-1H-imidazole-1-yl) ethoxy]benzaldehyde (1), was
8
-10
activities
(Figure 2). Also, hydrazone derivatives are one of
14
synthesized by a reported method. Further, the condensation
the widely studied pharmacophores showing a vast range of
reaction of intermediate (1) with various aryl hydrazides
furnished the final compounds (2-13). The structures of all the
1
1
12
biological activities. Hydrazone bearing pyridyl or quinoline
1
3
moiety showing promising antiamoebic activity have been
reported by us (Figure 3). Considering this perspective, it was
envisaged to modify the metronidazole framework to synthesize
1
13
compounds were elucidated on the basis of FT-IR, H NMR,
C
NMR and ESI-MS. The purity of the compounds was confirmed
by the elemental analyses.

In order to explore the possible antiamoebic potential of
newly synthesized metronidazole-hydrazone conjugates (2-13),
all the compounds were screened against HM1: IMSS strain of E.
NO2
N
N
1
5
N
histolytica by microdilution method and the results were
compared with the most widely used antiamoebic drug MNZ that
had 50% inhibitory concentration (IC ) 1.81 μM in our
N
5
0
N N
experiments. All the title compounds (2-13) exhibited better IC50
values (0.20-7.12 μM) than the compound 1 (IC50 = 11.48 μM).
Compound 3 having chloro group at para position of phenyl ring
exhibited most promising antiamoebic activity (IC50 = 0.20 μM)
followed by compound 5 (IC50 = 0.36 μM) with nitro group at
para position which can be attributed to electron withdrawing
effect. Incorporation of hydroxy (4, IC50 = 0.38 μM), amino (8,
IC50 = 0.43 μM) and methyl (7, IC50 = 0.49 μM) group at para
position, exerted significant inhibitory effect whereas the
introduction of methoxy (6, IC50 = 7.12 μM) and tertiary butyl
group (9, IC50 = 1.98 μM) at the same position did not affect the
antiamoebic activity. Compounds 4 and 12 had mono-hydroxy
and di-hydroxy substitution on the phenyl group respectively but
their antiamoebic activities were almost same. However
compounds having mono-methoxy (6), di-methoxy (11) and tri-
methoxy (13) group vary in their antiamoebic activities with the
increase in number of methoxy groups. Therefore, it can be
concluded that the antiamoebic activity varied with the nature as
well as the position of the substituents. However, a comparison
between precursor (1) and final compounds revealed that the
better antiamoebic activities of metronidazole-hydrzone
conjugates are due to presence of hydrazone moiety.
(IC = 0.008 µM)
5
0
Figure 2: Metronidazole based compounds having antiamoebic
activity
NO2
O
N
N
H
N
(
IC50 = 0.370 µM)
O
O
N
N
H
N
(IC50 = 0.390 µM)
Therefore, it can be concluded that the combination of
5
-nitroimidazole, hydrazone and the nature as well as the position
of substitution on the phenyl group was responsible for
antiamoebic activity.
N
O
Br
N
N
N
NO2
NO2
N
N
N
O
H
O
O
O
O
(
IC = 0.040 µM)
50
S
S
O
Figure 3: Hydrazones with antiamoebic activity
(
IC = 0.028 µM)
(IC50 = 0.015 µM)
5
0
R
O
N
NO2
N
N
N
N
O
N
O
O
H
O
NO2
S
O
Figure 4: General structure of metronidazole hydrazone
conjugates (blue and red colour depicts imidazole ring and
hydrazone linkage respectively)
(
IC = 0.021 µM)
50
Further to assess the effect of the compounds (3, 4, 5, 7,
8 and 12) on cervical cancer cell line, HeLa, cells
N
H
H
N
N
H
O N
2
C C
H H
C N
N
(4000cells/well) were plated in 96 well plate in triplicate. Cells
were treated with compounds (3, 4, 5, 7, 8 and 12) as indicated in
the Figure 5, the value correspond to the IC50 value observed for
these compounds for E. histolytica (Table 2). In case of 7 and 4
these values resulted in reduction of viability to 50.8 and 54.4
percent respectively, whereas 60 % or more of the cells were
viable in response to compounds 12, 8, 5 and 3.
S
OH
(
IC50 = 0.560 µM)

N
12
OH
0
.37
±0.004
N
O N
2
N
O N
2
N
(a)
OH
O
1
3
OCH3
OCH3
O
2.60
±0.004
±0.029
N
OCH3
O
HN
MNZ
N
1
.81
O
O N
2
R
N
(1)
(2-13)
OH
Scheme 1: Synthesis of Metronidazole hydrazone conjugates
2—13): Reagents and condition: (a) Different aryl hydrazides,
ethanol, reflux.
a
The values obtained in at least three separate assays done in
(
triplicate
Standard Deviation.
b
Table 1: In vitro antiamoebic activity of metronidazole –
hydrazone conjugates (2-13) against HM1: IMSS strain of E.
histolytica
DMSO
7
4
8
2
5
3
b
Compound No.
R
Antiamoebic
Activity
SD
a
1
IC (µM)
5
0
1
2
-
1
2
1.48
.02
±0.003
±0.017
MNZ
3
4
5
0
0
0
.20
.38
.36
±0.007
±0.004
±0.002
Figure 5: Assessment of viability of HeLa cells in response to
compound 7, 4, 8, 12, 5 and 3. Cells were plated in triplicates for
24h, 48h and 72h and treated with the compounds. Cells treated
with DMSO are used as the control. MTT was added after
completion of stipulated time intervals and processed.
Absorbance was taken at 570 nm. Results were plotted taking
control (DMSO) as 100%.
Cl
OH
NO2
Studies have indicated that E. histolytica thioredoxin
reductase (EhTHRase) is an important drug target for
6
7
7
0
.12
.49
±0.005
±0.007
1
6-17
nitroimidazole based drugs including metronidazole
making
it a suitable candidate for the search of novel leads against
amoebiasis. In order to understand the possible mechanism of
action of the compounds docking has been extensively exploited.
OCH3
Thus, using the previously published homology model of
8
CH3
EhTHRase by our group we docked the most active analogue so
8
9
as to identify the key interactions between the inhibitor–target
complex. It was observed that the compound 3 fits tightly into the
active binding site (Figure 6 and 7) and exhibits GOLD score of
0
1
.43
.98
±0.005
±0.004
NH2
5
4.44, much higher than the metronidazole complex (score:
39.33). Protein-ligand interactions identified that nitrogen atom
of His 182 and Arg 183 of the receptor active site forms
hydrogen bonds with oxygen atoms of compound 3 (Figure 6 and
7
). The two H-bonds could be responsible for enhanced binding
affinity and thereby antiamoebic activity. Also, the docking
studies demonstrates the possible role of the nitro group present
in these compounds which is known to get reduced to highly
reactive intermediate which then further reduces the enzyme like
1
1
0
1
CH3
5.96
±0.005
±0.003
1
6
thioredxin reductase. Moreover, the residues Lys 122, Val 158,
Gly 159, Gly 160, Gly 161, Ala 163, Glu 210, Ala 245, Ile 246,
Gly 247, His 248, Ser 249 and Asp 264 contribute towards the
hydrophobic interactions (Figure 6B). Additionally, we observed
Pi-interaction (represented in orange) with Arg 183 which further
stabilizes the complex (Figure 7). The molecular docking thereby
supports the previous observations that 5-nitroimidazole
analogues with hydrophobic groups show better antiamoebic
CH3
OCH3
4
.39
OCH3
8
-9, 18
activity
and thus paves the way for identifying new leads

1
9-
which could be useful for developing novel antiamoebic drugs.
To evaluate the lipophilicity of the compounds (3, 4, 5,
2
0
7
, 8, 12) having IC50 values better than MNZ, relative
2
1
fluorescence intensity was analyzed using the Nile red stain.
The compounds
5 and 12 showed significantly higher
fluorescence (Figure 8), which was due to the binding of Nile red
stain to the hydrophobic groups. Thus, it can be construed that
the active compounds 3, 4, 7 and 8 are highly less lipophilic than
5
and 12.
Figure 6: (A) Molecular surface representation of EhTHRase
protein with compound 3 (shown in stick) docked in the active
site (B) Ligplot showing hydrogen bonding interaction with
green dashed lines, and hydrophobic contacts by red arcs with
radiating lines.
Figure 8: Relative fluorescence intensity for compound 3, 4, 5, 7,
and 12.
8
In summary, six metronidazole-hydrazone conjugates
showed better antiamoebic activity than metronidazole and the
activity was substituent dependent. Molecular docking study of
the most promising compound i.e. compound 3 demonstrated that
the compound 3-ETHRase complex had significant binding
affinity. Compound 5 was most lipophilic compound followed
by compound 12. These promising results suggests the possibility
of developing metronidazole hydrazone conjugates as potential
drug candidates therefore, it may be hoped that the present study
will stimulate efforts towards the development of novel effective
antiamoebic agents.
Figure7: 2-D diagrams depicting protein-ligand interactions.
Table 2: Effect on cell viability of HeLa cells in response to
compounds 7, 4, 8, 12, 5 and 3 as assessed by MTT assay
Acknowledgements
This work was supported by University Grants Commission
Compo Conc.
24h
48h
72h
(grant # 41-275/2012(SR), New Delhi, India). MFA is thankful
und
No.
in nM
for JRF.
7
4
8
490
66.25± 2.7
83.85±8.5
87.10± 5.7
80.10± 8.6
92.10± 17.4
88.90± 8.2
93.70±3.3
50.80± 1.1
54.40± 3.2
60.30±5.3
59.80±13.0
68.30± 10.7
65.90± 4.3
Supplementary data
Supplementary data (synthetic procedures, spectral data,
antiamoebic assay, MTT assay and molecular docking)
associated with this article can be found, in the online version, at
doi:
387 81.90± 13.8
References:
435
370
79.80± 8.9
93.40±19.0
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