Imine/amide-imidazole conjugates derived from 5-amino-4-cyano-N1-substituted benzyl imidazole: Microwave-assisted synthesis and anticancer activity via selective topoisomerase-II-α inhibition

Article abstract of DOI:10.1016/j.bmc.2015.07.020

Microwave-accelerated synthesis and anticancer activity of novel imine/amide-imidazole conjugates derived from 5-amino-4-cyano-N1-substituted benzyl imidazole against a panel of seven cancer cell lines are reported for the first time. Compounds ARK-4, 10

Full text of DOI:10.1016/j.bmc.2015.07.020

Bioorganic & Medicinal Chemistry xxx (2015) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Imine/amide–imidazole conjugates derived from 5-amino-4-cyano-
N1-substituted benzyl imidazole: Microwave-assisted synthesis and
anticancer activity via selective topoisomerase-II-
a inhibition
Arvind Negi a, Jimi Marin Alex a, Suyog M. Amrutkar c, Ashish T. Baviskar c, Gaurav Joshi a, Sandeep Singh b,
Uttam C. Banerjee c, Raj Kumar a,
⇑
,
a Laboratory for Drug Design and Synthesis, Centre for Chemical and Pharmaceutical Sciences, Central University of Punjab, 151 001 Bathinda, India
b Centre for Genetic Diseases and Molecular Medicine, Central University of Punjab, 151 001 Bathinda, India
c Department of Pharmaceutical Technology (Biotechnology), National Institute of Pharmaceutical Education and Research (NIPER), Mohali, S.A.S. Nagar, Sec 67, 160 062 Punjab, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Microwave-accelerated synthesis and anticancer activity of novel imine/amide–imidazole conjugates
derived from 5-amino-4-cyano-N1-substituted benzyl imidazole against a panel of seven cancer cell lines
are reported for the first time. Compounds ARK-4, 10 and 12 in the series show promising in vitro anti
proliferative activity with low micromolar IC50 values against A-459 (lung), Hep-G2 (liver) and H-460
(liver) cancer cell lines. Compounds caused the increase in ROS levels as well as mitochondrial membrane
depolarization, which might induce apoptosis. Further, mechanistic interventions on biological and
molecular modeling data supported that compounds inhibited topoisomerase-II selectively.
Ó 2015 Elsevier Ltd. All rights reserved.
Received 26 May 2015
Revised 9 July 2015
Accepted 10 July 2015
Available online xxxx
Keywords:
Imine/amide–imidazole
Anticancer activity
Topoisomerase inhibition
Antioxidant activity
Molecular modeling
1. Introduction
discovery4–9 and development,10 we thought of designing hybrid
pharmacophores derived from imine–imidazoles and imine–
Cancer represents a group of more than hundred diseases char-
acterized by uncontrolled proliferation of the cells.1 According to
WHO, cancer deaths are predicted to rise with an approximation
of 13.1 million deaths in the year 2030 worldwide. Despite avail-
ability of various anticancer drugs, the problems like less therapeu-
tic efficacy with prevailing agents due to multi-drug resistance,
adverse effects and/or poor bioavailability warrant anticancer
screening of new chemical entities. Persistent failure in anticancer
drug development prompted the researchers to use the strategies
of combining pharmacophores as targeted therapeutic agents.2
These drugs have the potential to overcome the fast development
of resistance, enhance patient compliance, and reduce both the
cost and the risk of drug–drug interactions.3 The scope of molecu-
lar hybridization can be clearly exemplified through the US-FDA
approved estramustine and successful ongoing of other hybrid
molecules that are currently in different phases of clinical trials
such as CUDC-101, CBLC-137, PLX3397, E-3810, and CUDC-907.2
In continuation of our previous efforts towards anticancer drug
amides (Fig. 1) in which one of the aromatic rings of stilbenes11,12
/
imines (1) or benzamides (2) was substituted with imidazole scaf-
fold, that is, 5-amino-4-cynao-N1-substituted benzyl imidazole
(3). This is anticipated to solve the issue of limited bioavailability
due to glucuronidation and sulfation as observed with most of stil-
benes and offer an alternative strategy other than acetylation13 and
methylation.14 Also the designed compounds may be expected to
exhibit better anticancer activity of benzamides which are
reported to show their anticancer effect either via inhibition of
protein tyrosine kinases,15 PI3K,16 histone deacetylase,17,18 tubu-
lin19 or DNA methylation.20
2. Results and discussion
2.1. Synthesis
Compound 3 as an intermediate for the synthesis of the target
compounds ARK-4–13 was prepared by using synthetic proce-
dures shown in Scheme 1. Briefly 2,3-diaminomaleonitrile was
treated with CH(OEt)3 in 1,4-dioxane to afford ethyl (Z)-N-(2-
amino-1,2-dicyanovinyl)formimidate (4) which on reaction with
⇑
Corresponding author. Tel.: +91 164 2430586; fax: +91 164 2240555.
E-mail addresses: raj.khunger@gmail.com, rajcps@cup.ac.in (R. Kumar).
CUPB Library Communication Number P007/15.
http://dx.doi.org/10.1016/j.bmc.2015.07.020
0968-0896/Ó 2015 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Negi, A.; et al. Bioorg. Med. Chem. (2015), http://dx.doi.org/10.1016/j.bmc.2015.07.020

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A. Negi et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
CN
N
N
NH2
CN
CN
N
N
N
N
O
R1
Y
X
Y
Cl
R1
X
R1
R1
O
R1
= NH
Y
X = N
3
R1
1
2
Cl
Cl
Imine-imidazole Conjugate
Amide-imidazole Conjugate
Figure 1. Design of target compounds.
Cl
CN
Table 1
N
Standardization of the reaction conditions for the syntheses of (E)-1-(2-chloroben-
zyl)-5-((4-hydroxybenzylidene)amino)-1H-imidazole-4-carbonitrile (ARK-8)
EtO
HN
NH2
Cl
N
NC
NC
NH2
NH2
(iii)
(i)
(ii)
NC
NC
N
NC
NC
N
CN
CN
N
N
N
N
OHC
NH2
+
MW
Cl
NH2
Cl
4
3
5
N
NH2
Reaction
conditions
OH
3
ARK-8
OH
Yielda,b
Scheme 1. Reagents and conditions: (i) CH(OEt)3, dry 1,4-dioxane, reflux, 8–10 h;
(ii) o-Cl-PhCH2NH2, EtOH, aniline hydrochloride (1 mol %), rt, 5–6 h; (iii) 1 M KOH,
rt, 7–8 h.
Entry
Solvent
Temp (°C)
Time (h)
1
2
3
4
5
6
7
8
—
120
80
120
80
120
80
120
80
1
1.5
1.5
1.5
12c
85
89
82
84
85
88
63
72
77
79
Toluened
Toluened
NMPd
o-chlorobenzylamine under the catalytic influence of aniline
hydrochloride in ethanol furnished N0-((Z)-2-amino-1,2-di-
cyanovinyl)-N-(2-aminophenyl)formimidamide (5). Cyclization of
4 under the influence of base resulted in the formation of 1-(2-
chlorobenzyl)-5-amino-1H-imidazole-4-carbonitrile (3), which
acted as the precursor for the formation of target compounds.21,22
NMPd
1.5
MeOHd
MeOHd
H2Od
20 min
15 min
3
3.5
1.7
9
10
11
H2Od
120
80
120
DMFd
DMFd
1.9
a
2.2. Optimization of the reaction conditions for the synthesis of
imine–imidazole and amide–imidazole hybrids
A stirred mixture of 3 (1 mmol) and 4-hydroxybenzaldehyde (1 equiv) in a
sealed vial was heated under microwave irradiations using Biotage Initiator.
b
Isolated yield.
c
Trace amount of product was obtained in carrying out the reaction under
Microwave (MW) assisted organic synthesis23–26 is swiftly
advancing and is now accepted as a valuable tool to overcome
some of the bottlenecks in the drug discovery process and has
enhanced the rapid generation of libraries of compounds in lesser
time, high yields, proficient and speedy dielectric heating of the
reaction mixture in a conserved vessel to temperatures even higher
than the boiling point of the solvent.6,27
In order to determine the optimum reaction conditions for the
synthesis of imine–imidazole hybrids (ARK-4–11), a representa-
tive reaction of 3 (1 equiv) was carried out with 4-hydroxyben-
zaldehyde (1 equiv) under various MW reaction conditions. The
results are summarized in Table 1. The best results were obtained
in carrying out reaction in methanol for 15 min TLC at 120 °C under
MW irradiation. The very low yield and higher time required under
conventional heating could be due to poor nucleophilicity of amino
group because of delocalization of its lone pair in conjugation with
adjacent nitrile group.
conventional heating at 80 °C for 12 h.
d
0.5 mL of solvent was used.
methanol and purified (72–93%) through recrystallization (metha-
nol). The amide–imidazoles obtained after aqueous work-up were
dried, and recrystallized (methanol). All the target compounds
were found to be new and fully characterized by 1H and 13C
NMR, IR, and HRMS data. The appearance of IR absorption frequen-
cies in range of 1642–1688 cm–1 (C@N str.), singlet at range of 8.4–
9.7 d in 1H NMR spectra due to –N@CH– and peak in the range of
160–165 d in case of 13C NMR and mass spectra confirmed the for-
mation of imine–imidazole hybrids (ARK-4–11). The melting
points for these compounds were observed to be in the range of
167–204 °C. The melting points for ARK-12 and ARK-13 were
found to be in the range of 192–194 °C and 231–235 °C,
respectively.
Since imines can have two configurational isomers, that is, E
and Z, and we obtained single isomer (TLC, NMR), we were inter-
ested to know which isomer we are dealing with. We utilized the
structure minimization tool of ChemBio3D Ultra licensed version
(Licenced@ Cambridge-soft) for finding out the stability of iso-
mers. ARK-4 was selected as it has more substituents which
could be profoundly effective in determining the configurations
based on quantum theories. The MMFF94X force-field calcula-
tions supported that E-isomer was found to be more stable
(Fig. 2a) in comparison to the Z (Fig. 2b) based on parameters col-
lected in Table 2. Further, Z-isomer suffers steric clashes which
could be easily determined by observing the torsional and total
energy level.
2.3. Synthesis of imine–imidazole and amide–imidazole hybrids
Utilizing this standardized reaction condition, 3 (1 equiv) was
condensed under MW irradiations with various aryl aldehydes
(1 equiv) in methanol to furnish the imine–imidazole conjugates
compounds (ARK-4–11; Scheme 2). Furthermore, two amide–imi-
dazole conjugates (ARK-12–13) were synthesized (Scheme 3) by
treating 3 (1 equiv) with acetic or benzoic anhydrides (1 equiv)
under MW heating.
The reactions were found to be compatible with aromatic alde-
hydes bearing electron withdrawing and donating substituents.
Imine–imidazole conjugates precipitated and were removed from
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A. Negi et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
3
CN
N
2.4.2. Reactive oxygen species (ROS) and mitochondrial
membrane integrity assays
CN
N
N
N
N
OHC
R4
R3
+
MW
MeOH, 120 oC
Cl
Cl
R4
R3
Anticancer activity of most of the compounds is mediated
through changes in the ROS status in the cell. To evaluate mode
of action of our investigational compounds the ROS study using
DHE based fluorescent detection system has been performed. In
this study ARK-4 was selected as representative compound. The
results showed that there is concentration dependent increase in
ROS levels in the treated cells (Fig. 3A). Increase in ROS levels is
generally associated with changes in mitochondrial membrane
potential,5 which triggers its depolarization and lead to release of
cytochrome-c into cytoplasm, thus inducing apoptosis. JC-1 dye
was used to measure membrane potential of the mitochondria
(Fig. 3B), the results showed that there is steady decrease in
OD590/OD527 ratio indicating increased mitochondrial membrane
depolarization.
NH2
R1
R1
3
R2
R2
CN
CN
N
N
N
Cl
Cl
N
N
Cl
N
HO
OCH3
H3CO
Cl
ARK-4, 10 min, 82%
ARK-5, 20 min, 77%
CN
N
CN
N
Cl
Cl
N
OCH3
N
N
N
H3CO
Cl
ARK-7, 16min, 83%
ARK-6, 20min, 72%
CN
N
2.4.3. TopoII-mediated DNA decatenation assay
CN
N
Since many of the imidazole, its fused derivatives29,30 and imi-
Cl
Cl
N
N
nes4 are known to present anticancer activity via obstructing the
activity of topoisomerases (Topo) I and or IIa, we planned to eval-
N
N
NH
O2N
uate some of the selected compounds of ARK series (ARK-4, 6–10,
12 and 13) for their ability to inhibit them.
ARK-9, 10 min, 91%
ARK-10, 25 min, 76%
CN
N
N
N
To assess the activity against topoisomerase (hTopo) IIa, the
Cl
synthetics were subjected to ATP-dependent decatenation assay
in agarose gel electrophoresis (Fig. 4), a specific assay for the topoi-
somerase II inhibition activity. In this assay kinetoplast DNA
(kDNA) was used as a substrate and etoposide, a known topoiso-
merase II-inhibiting anticancer drug, was used as standard.
Two decatenated products: nicked kDNA (Nck) and supercoiled
(SC)/relaxed (Rel) circular DNA were obtained on incubation of
OH
H3CO
ARK-11, 25 min, 93%
Scheme 2. Synthesis of target compounds (ARK-4–11).
kDNA with hTopoIIa. Quantification of decatenation products
formed (Nck, Rel, and SC) were carried out by densitometric data
obtained using Quantity One (BioRad), and the results were com-
pared with etoposide. As expected, compounds ARK-4, 10 and 12
2.4. Biological studies
2.4.1. Antiproliferative activity
were observed to be more active against hTopoII
and 12 exhibited greater inhibition of hTopoII
a
a
. ARK-4, 10
activity as
In order to determine the antiproliferative potential of the tar-
get compounds ARK-4–13 were screened in vitro against a panel
of seven cancer cell lines, comprising of A-459 (lung), Hep-G2
(liver), H-460 (liver), HeLa (cervix), MCF7 (breast), PC3 (prostate)
and IGROV-1 (ovary). Overall all the tested compounds exhibited
the antiproliferative effects in only three cell lines (results in other
cancer cell lines not shown), A-459 (lung), Hep-G2 (liver) and H-
460 (liver); the results are summarized in Table 3. The compounds
ARK-4, ARK-10 and ARK-12 showed significant in vitro anticancer
activity in A-459, Hep-G2 and H-460 cancer lines. Etoposide
(Etop)28 was used as the positive control. The compounds did not
show significant cytotoxicity towards human peripheral blood
cells (hPBMCs) that served as normal control.
CN
N
CN
N
N
N
Ar
H
N
H
N
Ar
Cl
Cl
Z-isomer
E-isomer
Ar = 3,5-Cl-2-OH-Ph
CN
O
CN
N
N
N
O
O
R'(Ar)
N
H
Cl
MW
120 oC
N
+
(Ar)
NH2
R'
R'
(Ar)
O
a
3
Cl
CN
CN
O
O
N
N
CH3
N
N
H
N
N
H
b
Cl
ARK-12,15 min, 80%
Cl
ARK-13, 13 min, 91%
Figure
dene)amino)-1H-imidazole-4-carbonitrile,
dichloro-2-hydroxybenzylidene)amino)-1H-imidazole-4-carbonitrile.
2. (a)
(E)-1-(2-Chlorobenzyl)-5-((3,5-dichloro-2-hydroxybenzyli-
(b) (Z)-1-(2-chlorobenzyl)-5-((3,5-
Scheme 3. Synthesis of target compounds (ARK-12–13).
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4
A. Negi et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
Table 2
Walker A motif in the ATP binding site of TopoII
a includes the resi-
Comparison of stabilities of E and Z isomers of ARK-4
dues 161–166 and these are considered crucial for the binding of
ATP and other catalytic inhibitors acting on ATPase domain.4
Further studies revealed that ARK-4 might bind at the ATP site of
E-Configuration
Z-Configuration
Stretch
1.4071
1.4532
TopoII
a. The superimposition of the co-crystallized ANP and
Bend
Stretch–Bend
Torsion
Non-1,4 VDW
1,4 VDW
Dipole/dipole
Total energy
23.6455
27.4343
0.0594
1.9418
ꢀ5.2978
17.8784
ꢀ0.0291
43.4402 kcal/mol
ꢀ0.0180
ARK-4 indicated that the 2-chlorobenzyl ring (ring-C) overlapped
with the phosphate group of ANP in the ATP binding site
(Fig. 7a). The interaction model of the most active compound
ARK-4 at the ATPase domain (Fig. 7b) showed that the hydroxyl
group of 3,5-dichloro-2-hydroxybenzyl moiety (ring-A) formed a
H-bond with the water molecule present at the binding site
(d = 2.13 Å). Further, ring-A could show polar interactions with
THR 215, ASN 120 and ASN 95. The 2-chlorobenzyl ring (ring-C)
was involved in the arene–cation interaction with the Mg2+ ion
ꢀ13.3402
ꢀ3.2550
18.4778
ꢀ0.2708
26.6465 kcal/mol
compared to etoposide (Fig. 4A and B). This study indicated that
the inhibition of hTopoII was the putative mode of action of
ARK series. The calculated IC50 value (Fig. and also see
Supplementary information) for one of the representatives of
ARK series, ARK-4 was observed to be 34.93 M which was better
than etoposide (IC50 = 78.4 M).
a
(d = 2.63 Å). Similar kind of
reported in the binding of imine–pyrazolopyrimidinones to the
ATP binding site in topo-IIa 4 The interaction with Mg2+ ion has
p–cation interactions have been
5
.
l
been considered essential for the catalytic activity at the ATPase
binding domain.32 Additionally, this interaction led to the forma-
tion of distorted tetrahedral conformation involving Mg2+ ion, 2-
chlorobenzyl ring (ring-C), ASN 91, and two water molecules
(HOH 927 and HOH 928). These formed a stable complex which
may prevent any further catalytic activity due to the Mg2+ ion.
Thus, the molecular modeling studies suggest that ARK-4 may
act as a catalytic inhibitor by occupying the ATP binding pocket
l
2.4.4. Redefining selectivity of ARK-4 as hTopoIIa inhibitor
In order to assess the potential of the compounds to inhibit
TopoI, we tested the ARK-4 in TopoI relaxation assay which
showed no significant inhibition of hTopoI as compared to camp-
tothecin (C), a known TopoI inhibitory anticancer agent (Fig. 6).
This indicated that the compounds represent a class of selective
of TopoII
acid residues.
a and showing favorable interactions with the key amino
TopoIIa inhibitors.
2.5. Molecular modeling
3. Conclusion
In order to investigate the binding mode of the tested com-
pounds with the active site of TopoII
selected ARK-4, which emerged to be a potent catalytic inhibitor
of TopoII . Molecular docking study of ARK-4 was done using
Maestro 9.6 module in Schrodinger. Molecular docking protocol
was validated by docking ANP (bound ligand) into the crystal
a
(PDB entry: 1ZXM),31 we
The current study began with the development of efficient and
convenient method for the synthesis of imine/amide–imidazole
conjugates by condensation of 1-(2-chlorobenzyl)-5-amino-1H-
imidazole-4-carbonitrile with aryl aldehydes or anhydrides under
microwave heating in sealed tubes. The optimization of the syn-
thetic procedure was carried out and the effect of various solvents
on the condensation reaction was studied. Methanol emerged as
the best suitable solvent for carrying out the reactions. Following
the optimized procedure novel imine/amide–imidazole conjugates
were synthesized. The synthesized compounds showed encourag-
ing anticancer activity in vitro with low micromolar IC50 values
compared to etoposide against lung and liver cancer cell lines
tested and with almost no toxicity to normal cells. The biological
experiments on representative compounds suggested that they
a
structure of ATPase domain of TopoII
a. The docked ANP showed
similar binding pose as the co-crystallized ligand with a root mean
square deviation (rmsd) of about 0.024 Å. The binding model of
ANP with ATPase domain of TopoIIa revealed that the amino group
of adenine moiety showed hydrogen bonding with ASN120 and the
two hydroxyl groups of ribose unit showed hydrogen bond interac-
tion with Ser149. The oxygen atoms in the three phosphate groups
displayed hydrogen bond interactions with the nitrogen atom of
the Walker A motif residues; ARG 162, ASN 163, TYR 165 and
GLY 166. In addition, these phosphate groups showed hydrogen
bonding with ASN 91, LYS 168, and GLN 376. The highly conserved
selectively and catalytically inhibitor hTopoIIa which was further
supported by molecular docking studies. In addition, compounds
were found to increase ROS levels as well as mitochondrial mem-
brane depolarization, which might induce apoptosis, independent
of their Topo-II specific activity. Further detailed investigations of
SARs as well as explorations of antitumor activity are presently
in progress.
Table 3
Antiproliferativeactivity of the compounds under investigations (ARK-4–13)
Compound
A-549
Lung
Hep-G2
Liver
H-460
Liver
a
4. Experimental section
4.1. Chemistry
IC50
(lM)
ARK-4
ARK-5
ARK-6
ARK-7
ARK-8
ARK-9
ARK-10
ARK-11
ARK-12
ARK-13
Etop
7.5
>50
18
30.8
35.1
37
10.9
39
12
11.2
—
b
>50
20
31
38.2
40
28.3
28.1
>40
The reagents were purchased from Sigma–Aldrich, Loba Chemie
Pvt. Ltd, S.D. Fine Chemicals and Sisco Research Laboratory and
used without further purification. The progress of the reaction
was determined by thin layer chromatography (TLC; for imine;
EtOAc/n-hexane::1:1; for amides; EtOAc/n-hexane::3:2) carried
out on pre-coated silica plates. Melting points were recorded on
Stuart SMP—30 melting point apparatus with open glass capillary
tube and are uncorrected. Infrared spectra of compounds were
recorded on Bruker IR spectrophotometer at CUPB. The 1H and
b
—
15
14.6
b
>40
17.1
28
—
16.5
20.3
18.2
13.4
33.9
>30
20.9
a
Values are derived from averaging three independent experiments and each
experiment was done in triplicate.
b
Not tested.
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A. Negi et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
5
Figure 3. (A) DHE based assay to measure intra-cellular reactive oxygen species (ROS) induced by ARK-4. (B) JC-1 dye based assay to measure mitochondrial membrane
potential altered by ARK-4.
A
B
1
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
E
4
6
7
8
9
10
12
13
Investigated compounds
Figure 4. (A) hTopoIIa-inhibitory effect of selected ARK series compounds in kDNA decatenation assay. (B) Quantification of product formed in kDNA decatenation assay.
13C NMR of the compounds were recorded on Bruker Advance II
instrument at 400 MHz and 100 MHz frequencies, respectively, in
CDCl3/DMSO-d6 and TMS (d = 0) as internal standard and HRMS
at SAIF, Punjab University, Chandigarh. The solvents were evapo-
rated on Ilmvac rotary evaporator with Julabo chiller under
reduced pressure.
4.1.1. Ethyl-N-((Z)-2-amino-1,2-dicyanovinyl)formimidate (4)
A mixture of 2,3-diaminomaleonitrile (6 g, 55.5 mmol, 1 equiv)
was refluxed with triethylorthoformate (9.24 mL, 55.5 mmol,
1 equiv) in dry 1,4-dioxane (25 mL) for 8–10 h after completion
of the reaction (TLC), the mixture was allowed to cool overnight.
The dark brown mass obtained after distilling-off 1,4-dioxane
was further extracted with diethyl ether (3 ꢁ 20 mL), dried to
afford 421 as light brown needles (Yield: 78%). Mp 195–197 °C. IR
(KBr cmꢀ1): 3309 (N–H str.), 2247 (CN str.), 2207 (CN str.), 1636
(C@N str.), 1256 (C–O str.). 1H NMR (400 MHz, CDCl3): d = 7.95
(1H, s), 4.64 (D2O exchangeable NH2, 2H, br s), 4.23 (q, 2H,
J = 7.3 Hz), 1.33 (3H, t, J = 7.3 Hz).
Figure 5. Determination of IC50 value of ARK-4 by the inhibition of hTopoII
catalyzed decatenation of kDNA. (Different concentrations of ARK-4 (10–100 lM)
were used.)
a
4.1.2. Ethyl-N-((Z)-2-amino-1,2-dicyanovinyl)formimidate (5)
To a suspension of 4 (1.00 g, 6.09 mmol) and o-chlorobenzy-
lamine (1.01 g, 6.07 mmol) in ethanol was added catalytic amount
of anilinium chloride (1 mol %; 7.8 mg). The mixture was stirred at
room temperature for 5–6 h (TLC). The precipitate obtained from
reaction mixture was washed with diethyl ether, dried and recrys-
tallized in absolute ethanol to afford the light brown crystals 5,21
mp 108–110 °C (decomp.). IR (KBr cmꢀ1): 3417 (N–H str.), 2212
(CN str.), 2191 (CN str.) 1651 (C@N, str.), 1578 (N–H bend.), 741
(C–Cl str.). 1H NMR (400 MHz, d6-DMSO): d = 8.16 (1H, d,
J = 4.6 Hz), 7.74 (1H, d, J = 3.7 Hz), 7.42–7.44 (2H, m), 7.29–7.31
(2H, m), 4.57 (2H, d, J = 5.9 Hz), 6.10 (D2O exchangeable NH2, 2H,
br s).
Figure 6. Topoisomerase
treated with TopoI in the presence of 100
separately.
I relaxation assay: Negatively supercoiled DNA was
lM camptothecin (C) or ARK-4,
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A. Negi et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
Figure 7. (a) Docking pose of ARK-4 (red) at the binding site of AMPPNP (magenta) in ATPase domain of TopoII
important amino acid residues in ATPase domain of TopoII
a; (b) docking pose of ARK-4 showing the interactions with
a.
4.1.3. 5-Amino-1-(2-chlorobenzyl)-1H-imidazole-4-carbonitrile
(3)
4.1.7. (E)-1-(2-Chlorobenzyl)-5-((2,5-dimethoxybenzylidene)-
amino)-1H-imidazole-4-carbonitrile (ARK-6)
A suspension of 5 (1.00 g) in potassium hydroxide solution
(1 M, 10 mL) was stirred at room temperature for approximately
7–8 h until TLC showed complete consumption of the starting
material. The precipitated product was filtered-off, washed with
water (5 mL), air-dried to afford the product 3. IR (KBr cmꢀ1):
3357 (N–H str.), 2212 (CN str.), 1651 (C@N, str.), 1578 (N–H bend.),
741 (C–Cl str.). 1H NMR (400 MHz, d6-DMSO): d = 7.50–7.52 (1H,
m), 7.31–7.35 (2H, m), 7.19 (1H, s), 6.72–6.73 (1H, m), 6.32 (D2O
exchangeable NH2, 2H, br s), 5.16 (2H, s).
Yield: 72%; white solid. Mp = 169–171 °C. 1H NMR (400 MHz,
CDCl3, TMS = 0) d: 9.49 (D2O exchangeable NH, 1H, br s), 7.57
(1H, d, J = 2.8 Hz), 7.52 (1H, s), 7.41–7.43 (1H, m), 7.33 (1H, d,
J = 3.2 Hz), 7.28 (1H, d, J = 1.72 Hz), 7.07–7.15 (3H, m), 5.36 (2H,
s), 3.80 (3H, s), 3.88 (3H, s). HRMS calcd for C20H17ClN4O2 (M+),
380.1040; found, 380.1049.
4.1.8. (E)-5-((4-Chlorobenzylidene)amino)-1-(2-chlorobenzyl)-
1H-imidazole-4-carbonitrile (ARK-7)
Yield: 83%; yellow solid; mp = 167–169 °C IR (KBr cmꢀ1): 2250
(CN str), 1608 (C@N str), 1581 (C@C aromatic, str.), 1206 (C–N str),
731 (C–Cl str). 1H NMR (400 MHz, CDCl3, TMS = 0) d: 9.06 (1H, s),
7.87 (2H, dd, J = 1.8 Hz), 7.54 (1H, s), 7.48 (2H, dd, J = 2.24 Hz),
7.42–7.44 (1H, m), 7.27–7.31 (1H, m), 7.21–7.25 (1H, m), 7.13
(1H, dd, J = 1.96 Hz). 13C NMR (100 MHz, CDCl3, TMS = 0) d:
45.23, 115.61, 127.20, 129.01, 129.39, 129.53, 130.50, 132.37,
133.10, 133.32, 138.05, 138.12, 146.14, 162.17. MS (ESI):
m/z = 355.2 [M+1]+; HRMS calcd for C18H12Cl2N4O (M+Na)+,
377.0331; found, 377.0410.
4.1.4. A representative synthetic procedure of Compounds ARK-
4–11
To a reaction vial, a suspension of 3 (100 mg, 0.47 mmol,
1 equiv) in methanol (0.5 mL) was added aromatic aldehyde
(1 equiv) and the mixture was heated under microwave irradiation
using Biotage initiator for 10–25 min at 120 °C. After the comple-
tion of reaction (TLC), methanol was removed purified through
recrystallization to afford the product.
4.1.5. (E)-1-(2-Chlorobenzyl)-5-((3,5-dichloro-2-hydroxybenz-
ylidene)amino)1H-imidazole-4-carbonitrile (ARK-4)
4.1.9. (E)-1-(2-Chlorobenzyl)-5-((4-hydroxybenzylidene)-
amino)-1H-imidazole-4-carbonitrile (ARK-8)
Yield: 82%; yellow solid; mp = 183–185 °C. IR (KBr cmꢀ1): 2250
(CN str.), 1608 (C@N str.), 1581 (C@C aromatic, str.), 1206 (C–N
str.), 731 (C–Cl str.). 1H NMR (400 MHz, CDCl3, TMS = 0) d: 11.35
(D2O exchangeable OH, 1H, br s), 9.24 (1H, s), 7.9 (1H, s), 7.81
(1H, d, J= 2.6 Hz), 7.62 (1H, d, J = 2.56 Hz), 7.47 (1H, dd,
J1,2 = 1.3 Hz, J1,3 = 7.8 Hz), 7.27–7.36 (2H, m), 7.11 (1H, dd,
J1,2 = 1.6 Hz, J1,3 = 7.4 Hz), 5.45 (2H, s). 13C NMR (100 MHz, CDCl3,
TMS = 0) d: 161.73, 153.95, 145.19, 138.63, 133.26, 132.91,
132.21, 129.70, 129.51, 129.21, 128.19, 127.38, 123.79, 122.55,
122.49, 115.30, 99.85, 45.54. MS (ESI): m/z = 406 [M+1]+. HRMS
calcd for C18H11Cl3N4O (M+Na)+, 426.9891; found, 426.9912.
Yield: 88%; Light yellow solid; mp = 201–203 °C; IR (KBr cmꢀ1):
2241 (CN str.), 1576 (C@N str.), 1571 (C@C aromatic str.), 1261 (C–
N str.), 714 (C–Cl str.). 1H NMR (400 MHz, CDCl3, TMS = 0) d: 9.78
(1H, s), 8.80 (D2O exchangeable OH, 1H, br s), 7.72–7.74 (4H, m),
7.28–7.34 (4H, m), 5.38 (2H, s). 13C NMR (100 MHz, CDCl3,
TMS = 0) d: 45.02, 97.66, 115.96, 116.14, 125.96, 127.31, 129.39,
129.51, 129.57, 131.56, 132.20, 133.46, 137.57, 147.71, 162.43,
163.44; MS (ESI): m/z = 337.2 [M+1]+. HRMS calcd for
C
18H13ClN4O4 (M+Na)+, 359.0670; found, 359.0710.
4.1.10. (E)-1-(2-Chlorobenzyl)-5-((2-nitrobenzylidene)amino)-
1H-imidazole-4-carbonitrile (ARK-9)
4.1.6. (E)-1-(2-Chlorobenzyl)-5-((3,4-dimethoxybenzyl idene)-
amino)-1H-imidazole-4-carbonitrile (ARK-5)
Yield: 91%; Pale Yellow solid; mp = 184–186 °C; IR (KBr cmꢀ1):
2198 (CN str.), 1577 (C@N str.), 1448 (C@C aromatic str.), 1256 (C–
N str.), 801 (C–Cl str.); 1H NMR (400 MHz, CDCl3, TMS = 0) d: 9.54
(1H, s), 8.13 (1H, dd, J1,2 = 1.6 Hz, J1,3 = 7.7 Hz), 8.08 (1H, dd,
J1,2 = 1.3 Hz, J1,3 = 7.9 Hz), 7.68–7.76 (2H, m), 7.56 (1H, s), 7.43–
7.47 (2H, m), 7.12 (1H, dd, J1,2 = 1.6 Hz, J1,3 = 7.5 Hz), 6.96 (1H, dd,
J1,2 = 1.6 Hz, J1,3 = 7.4 Hz), 5.36 (2H, s). MS (ESI): m/z = 366.2
[M+1]+. HRMS calcd for C18H12ClN5O2 (M+Na)+, 388.0572; found,
388.0613.
Yield: 77%; mp = 192–194 °C; IR (KBr cmꢀ1): 2241 (CN str.), 1576
(C@N str.), 1571 (C@C aromatic, str.), 1261 (C–N str.), 731 (C–Cl str.).
1H NMR (400 MHz, CDCl3, TMS = 0) d: 8.97 (1H, s), 7.52 (1H, s), 7.50
(1H, d, J = 1.84 Hz), 7.41–7.45 (2H, m), 7.28–7.30 (1H, m), 7.20–7.24
(1H, m), 7.11–7.14 (1H, dd, J1,2 = 1.6 Hz, J1,3 = 7.6 Hz), 6.95 (1H, d,
J = 8.36 Hz), 5.37 (2H, s), 3.97 (3H, s), 3.95 (3H, s). HRMS calcd for
C
20H17ClN4O2 (M+), 380.1040; found, 380.1051.
Please cite this article in press as: Negi, A.; et al. Bioorg. Med. Chem. (2015), http://dx.doi.org/10.1016/j.bmc.2015.07.020

A. Negi et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
7
4.1.11. (E)-5-(((1H-Indol-2-yl)methylene)amino)-1-(2-
media. The total amount of media per well (100
lL per well of
chlorobenzyl)-1H-imidazole-4-carbonitrile (ARK-10)
96 well plate) was kept constant and all the treatment volumes
were accommodated within these ranges only.
Yield: 76%; Brownish yellow solid mp = 167–169 °C; IR (KBr
cmꢀ1): 2260 (CN str.), 1579 (C@N str.), 1559 (C@C aromatic, str.),
1198 (C–N str.), 771 (C–Cl str.). 1H NMR (400 MHz, CDCl3,
TMS = 0) d: 12.08 (1H, s), 9.05 (1H, s), 8.22 (1H, s), 8.17 (1H, d,
J = 3.04 Hz), 8.0 (1H, d, J = 7.8 Hz), 7.83 (1H, s), 7.46–7.51 (2H, m),
7.22–7.33 (2H, m), 7.08–7.12 (1H, m), 7.01 (1H, dd, J1,2 = 1.6 Hz,
J1,3 = 7.5 Hz), 5.43 (2H, s), MS (ESI): m/z = 360.2 [M+1]+. HRMS calcd
for C20H14ClN5 (M+Na)+, 382.0830; found, 382.0910.
4.3.1. 3-(4,5-Dimethythiazol-2-yl)-2,5-diphenyl tetrazolium
bromide (MTT) assay
MTT assay was carried out using 96-wellplate; each well was
filled by 100
washed with 1% PBS and were mixed with 100
l
L media to which cell were treated and thereafter
L/mL well of
l
MTT (5 mg in 10 mL of 1% PBS) and incubated at room temperature
in dark for 4 h to allow formation of formazan crystals. Each well
4.1.12. (E)-1-(2-Chlorobenzyl)-5-((4-hydroxy-3-methoxybenzyli-
dene)amino)-1H-imidazole-4-carbonitrile (ARK-11)
Yield: 93%; Dark yellow solid; mp = 198–200 °C; IR (KBr cmꢀ1):
2277 (CN str.), 1589 (C@N str.), 1481 (C@C aromatic, str.), 1301 (C–
N str.), 778 (C–Cl str.). 1H NMR (400 MHz, CDCl3, TMS = 0) d: 9.83
(1H, s), 8.95 (1H, s), 7.51 (1H, s), 7.41–7.45 (5H, m), 7.04 (2H, d,
J = 8.48 Hz), 5.36 (2H, s), 3.97 (3H, s). MS (ESI): m/z = 367.2
[M+1]+. HRMS calcd for C19H15ClN4O2 (M+), 366.0884; found,
366.0781.
was then mixed with 100 lL of DMSO to dissolve the crystals fol-
lowed by ELISA readings at 570 nm. The results were then repre-
sented as mean SD obtained from three independent
experiments.
4.3.2. Reactive oxygen species (ROS) and mitochondrial
membrane integrity assays
Cells were seeded in 96-well plate and appropriate treatments
were given. 24 h post treatments, cells were processed for JC-1 or
DHE staining. For DHE staining, cells were washed by 1ꢁ PBS fol-
lowed by staining with DHE at 37 °C for 30 min. Afterwards cells
4.2. A representative synthetic procedure of compounds ARK-
12–13
were washed again with ice-cold 1ꢁ PBS and finally 100 l of 1ꢁ
l
PBS was added to each well followed by measuring OD using
To a reaction vial, a suspension of 3 (100 mg, 0.47 mmol,
1 equiv) was added anhydride (1 equiv) and the mixture was
heated under microwave irradiation using Biotage initiator for
13–15 min at 120 °C. After the completion of reaction (TLC), water
was added; the precipitate was filtered off, dried and recrystallized
to afford the product.
microplate reader.
For JC-1 assay, dye was directly added to the media and cells
were incubated at 37 °C for 30 min followed by washing with 1ꢁ
PBS to remove extra dye. OD was measured using microplate
reader (emission = 527 and 590).
4.3.3. Topoisomerase inhibitory activity evaluation
4.2.1. (N-(1-(2-Chlorobenzyl)-4-cyano-1H-imidazol-5-yl)-
acetamide) (ARK-12)
All the reagents required for the testing of investigational com-
pounds were purchased from TopoGEN, Inc. (Columbus, OH). The
testing of the compounds was performed using a commercially
Yield: 80%; white solid; mp = 192–194 °C; IR (KBr cmꢀ1): 2290
(CN str.), 1609 (C@N str.), 1341 (C@C aromatic, str.), 1213 (C–N
str.), 691 (C–Cl str.). 1H NMR (400 MHz, CDCl3, TMS = 0) d: 10.25
(D2O exchangeable NH, 1H, br s), 7.76 (1H, s), 7.46 (2H, dd,
J = 1.36 Hz), 7.29–7.37 (2H, m), 7.02 (1H, d, J = 7 Hz), 5.22 (2H, s),
2.04 (3H, s). 13C NMR (100 MHz, CDCl3, TMS = 0) d: 169.18,
137.47, 135.06, 132.71, 132.30, 129.75, 129.47, 129.25, 127.39,
114.43, 107.12, 45.92, 22.36; MS (ESI): m/z = 275 [M+1]+. HRMS
calcd for C13H11ClN4O (M+Na)+, 297.0514; found, 297.0612.
available hTopo-IIa drug screening kit. All the synthesized com-
pounds and etoposide were dissolved in DMSO at a concentration
of 1 mM as a stock solution and stored at ꢀ20 °C.
4.3.4. TopoII-mediated DNA decatenation assay
In agarose gel decatenation assay, kinetoplast DNA (kDNA) was
used as a substrate. All the reactions were assembled on ice in
micro centrifuge tubes. Briefly, 2–4 units of purified human topoi-
somerase II, 150 ng catenated DNA (kDNA), and 100 lM drug dis-
4.2.2. N-(1-(2-Chlorobenzyl)-4-cyano-1H-imidazol-5-yl)-
benzamide (ARK-13)
solved in DMSO were combined in freshly prepared 5ꢁ complete
reaction buffer. The reaction mixture was brought to a final volume
Yield: 91%; light Yellow solid; mp = 231–233 °C; IR (KBr cmꢀ1):
2244 (CN str.), 1609 (C@O str.), 1510 (C@C aromatic str.), 1293 (C–
N str.), 817 (C–Cl str.). 1H NMR (400 MHz, CDCl3, TMS = 0) d: 8.30
(2H, d, J = 7.4 Hz), 7.82 (1H, s), 7.58 (1H, d, J = 7.28 Hz), 7.53 (2H,
d, J = 7.84 HZ), 7.46 (2H, d, J = 4.32 Hz), 5.49 (2H, s). HRMS calcd
for C18H12ClN4O (M+Na)+, 359.0670; found, 359.0820.
of 20 lL with deionized, distilled H2O. For the standard assay
method, the order of addition to the assay was H2O, 5ꢁ assay buf-
fer (buffer A: 0.5 M Tris–HCl (pH 8), 1.50 M sodium chloride,
100 mM magnesium chloride, 5 mM dithiothreitol, 300 lg of
bovine serum albumin/mL; buffer B: 20 mM ATP in water),
kDNA, followed by either test compound or standard drug and
finally topoisomerase II. The reaction mixture was incubated at
37 °C for 30 min. The reaction was then stopped with 10% SDS, fol-
lowed by digestion with proteinase K and further incubated at
37 °C for 15 min. Topological forms of kDNA were resolved by run-
ning 1% agarose gel electrophoresis in Tris–acetate–EDTA (TAE)
4.3. Cell culture and treatment
All the cell lines were procured from National cell repository,
NCCS, Pune. Cell lines representing different human cancers were
grown in DMEM media supplemented with 10% fetal bovine serum
(FBS) and antibiotic solution (1% Penstrip, all the reagents from
Invitrogen). Cells were cultured in DMEM media with 10% FBS,
50 U/mL penicillin G, 50 Ig/mL streptomycin sulfate and
1.25 Ig/mL amphotericin B (fungizone). The cells were incubated
at 37 °C with 5% CO2 and 95% humidity conditions. For experi-
ments, cells were seeded in equal numbers after trypan blue cell
counting (8000 cells per well of 96-well plate). The compounds
were dissolved in cell culture grade DMSO up to concentration of
100 mM and further dilutions were done in serum free DMEM
buffer containing 0.5 lg/mL ethidium bromide and further
destained with water for 20 min. The bands of kDNA and decate-
nated products were visualized by UV trans-illumination, and the
products were quantified with Image Lab (BioRad).
4.3.5. Topo I mediated DNA relaxation assay
Synthesized compound was screened for hTopo I mediated DNA
relaxation assay as follows. Reaction mixture containing freshly
prepared 10ꢁ reaction assay buffer (100 mM Tris–HCl (pH-7.9),
10 mM EDTA, 1.5 M sodium chloride, 1% BSA, 1 mM spermidine,
Please cite this article in press as: Negi, A.; et al. Bioorg. Med. Chem. (2015), http://dx.doi.org/10.1016/j.bmc.2015.07.020

8
A. Negi et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
50% glycerol) 250 ng supercoiled DNA (substrate), 100 lM camp-
References and notes
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by addition of purified hTopo-I were incubated at 37 °C for 30 min.
The reaction was terminated with the addition of 10% SDS, fol-
lowed by digestion with proteinase K. Further the reaction mixture
was again incubated at 70 °C for 15 min. each sample was then
subjected to 1% agarose gel electrophoresis in Tris–acetate–EDTA
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Acknowledgments
R.K. and S.S. thank DST and UGC, New Delhi, India for the finan-
cial assistance (F. No. SR/FT/CS-71/2011) and F.30-13/2013(BSR),
respectively. S.M.A., A.T.B. and U.C.B. gratefully acknowledge the
financial aid from DBT Government of India, New Delhi to carry
out hTopoI and hTopoII activity evaluation studies. J.M.A. and
R.K. also thank Maulana Azad National Fellowship (F1-17.1/2013-
14/MANF-2013-14-CHR-DEL-27187)
for
the
same.
Encouragement and support from Prof. P. Ramarao, Dean, and
Prof. R.K. Kohli, Vice Chancellor Central University of Punjab, is
gratefully acknowledged.
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Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2015.07.020.
Please cite this article in press as: Negi, A.; et al. Bioorg. Med. Chem. (2015), http://dx.doi.org/10.1016/j.bmc.2015.07.020