Exploration of imidazole and imidazopyridine dimers as anticancer agents: Design, synthesis, and structure–activity relationship study

Article abstract of DOI:10.1002/ardp.201900011

Dimerization of proteins/receptors plays a critical role in various cellular processes, including cell proliferation and differentiation. Therefore, targeting such dimeric proteins/receptors by dimeric small molecules could be a potential therapeutic appr

Full text of DOI:10.1002/ardp.201900011

Received: 10 January 2019
Revised: 2 September 2019
Accepted: 7 September 2019
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DOI: 10.1002/ardp.201900011
F U L L P A P E R
Exploration of imidazole and imidazopyridine dimers as
anticancer agents: Design, synthesis, and structure–activity
relationship study
Sangeetha Meenakshisundaram1
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Manoj Manickam2
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Thanigaimalai Pillaiyar3
1Department of Chemistry, Sri Krishna
Abstract
College of Engineering and Technology,
Coimbatore, Tamil Nadu, India
Dimerization of proteins/receptors plays a critical role in various cellular processes,
including cell proliferation and differentiation. Therefore, targeting such dimeric
proteins/receptors by dimeric small molecules could be a potential therapeutic approach
to treating various diseases, including inflammation‐associated diseases like cancer. A
novel series of bis‐imidazoles (13–18) and bis‐imidazo[1,2‐a]pyridines (19–28) were
designed and synthesized from Schiff base dimers (1–12) for their anticancer activities.
All the synthesized compounds were screened for anticancer activities against three
cancer cell lines, including cervical (HeLa), breast (MDA‐MB‐231), and renal cancer
(ACHN). From structure–activity relationship studies, imidazo[1,2‐a]pyridines (19–28)
showed remarkable cytotoxic activities, with compounds 19 and 24 showing the best
inhibitory activities against all three cell lines. Especially, both 19 and 24 were very
effective against the breast cancer cell line (19, GI50 = 0.43 µM; 24, GI50 = 0.3 µM),
exceeding the activity of the control adriamycin (GI50 = 0.51 µM). The in vivo anticancer
activity results of compounds 19 and 24 were comparable with those of the animals
treated with the standard drug tamoxifen. Therefore, the dimeric imidazo[1,2‐a]pyridine
scaffold could serve as a potential lead for the development of novel anticancer agents.
2Polymer Engineering Laboratory, PSG
Institute of Technology and Applied Research,
Coimbatore, Tamil Nadu, India
3Department of Pharmaceutical and Medicinal
Chemistry, PharmaCenter Bonn,
Pharmaceutical Institute, University of Bonn,
Bonn, Germany
Correspondence
Thanigaimalai Pillaiyar, Department of
Pharmaceutical and Medicinal Chemistry,
PharmaCenter Bonn, Pharmaceutical Institute,
University of Bonn, An der Immenburg 4,
D‐53121 Bonn, Germany.
Email: thanigai@uni-bonn.de
K E Y W O R D S
anticancer activity, bis‐imidazoles, bis‐imidazopyridines, cytotoxicity studies, Schiff base dimers
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1
INTRODUCTION
such as human growth hormone and erythropoietin are bivalent, and
one ligand binds simultaneously to two receptors and creates a 1:2
Dimerization of proteins/receptors plays critical roles in various
cellular processes, including cell proliferation and differentiation.
Dimers exist as homodimers, physical association of two identical
proteins or heterodimers, or interaction of two different proteins. In
some cases, dimerization is thought to be important for receptor
function. For example, the cellular growth factors vascular endothelial
growth factor and platelet‐derived growth factor are monomers in the
cell membrane, but ligand binding induces dimerization of these
receptors.[1,2] Structural studies have shown that certain cytokines,
ligand–receptor complex.[3,4] Upon ligand binding, the estrogen
receptor (ER) isoforms ERα and ERβ form homo‐ or heterodimers.[5–7]
Heat shock protein 90, a chaperone protein which is responsible for
the maturation of numerous signaling proteins including the proteins
required for tumor growth, exists as a homodimer.[8] The presence of
such dimeric proteins/receptors as therapeutic targets suggests that
an interaction/disruption of these dimers by dimeric small molecules
could be
diseases.
a potential therapeutic approach for treating various
- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -
This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium,
provided the original work is properly cited.
© 2019 The Authors. Archiv der Pharmazie published by Wiley‐VCH Verlag GmbH & Co. KGaA on behalf of Deutsche Pharmazeutische Gesellschaft
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Arch Pharm Chem Life Sci. 2019;e1900011.
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FIGURE 1 Representative examples of biologically important dimers and their monomers
There are many natural and synthetic dimers with potential
biological activities such as anticancer,[9] anti‐HIV,[10] antimalarial,[11]
antibacterial,[12] as well as opioid antagonist.[13,14] For example,
indol‐3‐carbinol (I3C, Figure 1), which is found in cruciferous
vegetables,[15] is a naturally occurring modulator of carcinogenesis
that is dependent on the conversion of its dimer, diindolylmethane
(DIM)[16,17] in the stomach during digestion. The structure–activity
relationship (SAR) of DIM has been extensively studied and many
analogs have been developed with the immune stimulatory G
protein‐coupled receptor 84 (GPR84) agonistic activities, especially
the fluoro substituted DIMs (e.g., PSB‐15160 and PSB‐16671, see
Figure 1).[18,19] GPR84 has been proposed as a drug target for acute
myeloid leukemia and might have potential for treating other cancers
as well. Similarly, numerous synthetic bis‐indoles possess cytotoxic
activities compared with its monomer.[20] Aaptamine (Figure 1), a
naphthyridine alkaloid isolated from marine sponge Aaptos aaptos,
was found to possess cancer cell growth inhibitory activity,[21] while
its dimer bis‐aaptamine (Figure 1), isolated from the marine sponge
Aaptos suberitoides showed potent cytotoxicity against P388 cell
lines.[22] Research has been focused on the replacement of
chloroquine by its dimeric piperaquine for the treatment of malarial
disease caused by Plasmodium falciparum.[23]
FIGURE 2 Rational design of bis‐
imidazoles and bis‐imidazopyridines from
Schiff base dimers

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_
1 5
3 hr
_
7 11
3 hr
SCHEME 1 Preparation of Schiff bases. Reagents and conditions: ethanol, reflux, 3 hr. For the synthesis of 6 and 12, cyclohexylamine was
used
Schiff bases have been shown to exhibit a broad range of biological
activities[24,25] and serve as a building block for the synthesis of
biologically important heterocycles, such as imidazoles[26] and imidazo-
pyridines.[27] The clinically useful anticancer drug, dacarbazine[28] holds
imidazole moiety. Similarly, imidazopyridine drugs such as zolpidem[29]
(used in the treatment of insomnia) and alpidem (as an anxiolytic
agent),[30] are available in the market. Although a large number of
imidazole and imidazopyridine analogs have been studied for various
biological activities, their dimeric analogs are not well explored either
synthetically and biologically.
with the parent compound 1 (unsubstituted one). To study the
hydrophobic effect, the benzene ring was replaced by the
cyclohexyl group as shown in compound 6. The next series of
Schiff bases (7–12) were derived from isophthalaldehyde to
visualize the effect of biological activity on 1,3‐positional isomers
(Scheme 1). The substitution pattern was the same as that of the
earlier series. Further, to explore the effect of 1,2‐positional
isomers of the corresponding Schiff bases, we intended to react
various amines with phthalaldehyde. However, the reaction did not
yield the desired product, probably due to the steric factor.
Compound 30 was synthesized from the reaction of aniline and
benzaldehyde (equimolar ratio) using the reaction condition of
Scheme 1. The chemical structures of the synthesized compounds
are summarized in Table 1.
Given the biological importance of Schiff bases, an attempt
has been made in the present work to synthesize their 1,4‐
dimeric analogs (Step I, Figure 2). The utility of Van Leusen
synthesis was explored to prepare imidazole dimers from the
corresponding Schiff base dimers (Step II, Figure 2). In addition to
that, the hydrogen bonding property in the imidazole scaffold
encouraged us to design the bis‐imidazopyridine analogs as a
fusion between the phenyl ring and the imidazole ring (Step III,
Figure 2). All the prepared dimers of Schiff base, imidazole, and
imidazopyridine were biologically evaluated for their anticancer
activities. For selected compounds, anticancer activities demon-
strated an in vivo model.
Next, imidazole analogs were synthesized from the corresponding
dimeric Schiff base by Van Leusen imidazole synthesis[31] using p‐
toluenesulfonylmethyl isocyanide (TOSMIC) in the presence of K2CO3.
The reaction yielded both the dimeric imidazoles (13–16) as well as
imidazole–Schiff base hybrid analogs (17–18, Scheme 2). The imidazo-
pyridine derivatives (19–28, Scheme 3) were synthesized by multi-
component reactions (A3‐coupling and 5‐exo‐dig cyclo isomerization)[27]
in which terephthalaldehyde was reacted with 2‐aminopyridines and
phenylacetylenes in the presence of CuSO4 and D‐glucose in ethanol. On
the other hand, isophthalaldehyde was used for the preparation of
corresponding 1,3‐positional isomers of imidazopyridines in the same
condition (Scheme 3). However, it was not possible to synthesize
1,2‐positional isomers of imidazopyridines (e.g., imidazo[1,2‐a]pyridine)
using phthalaldehyde. For the comparison, the monomer 29 (Scheme 3)
was synthesized from the reaction of 2‐aminopyridine and benzalde-
hyde under the same above condition.
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2
RESULTS AND DISCUSSION
Chemistry
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2.1
The first series of 1,4‐isomers (1–6) of dimeric Schiff bases were
prepared from terephthalaldehyde and a variety of amines that
diverge the electronic effects of the aromatic ring. The substituents
on the phenyl ring of the amine were electron‐donating methyl
groups (ortho‐ and para‐substitution) as shown in compounds 2, 3
and para‐methoxy group as indicated in 4, electron‐withdrawing
chloro group at para‐position as demonstrated in compound 5 along
The structures of the synthesized compounds were characterized
by 1H, 13C NMR, HRMS (for selected compounds), and elementary
analysis (C, H, and N). Besides, IR spectra were recorded for
functional group assignment.

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TABLE 1 Structure of synthesized compounds, melting points, and yields
Melting
Melting
Compound Structure
1
point (°C) Yield (%) Compound
Structure
point (°C)
Yield (%)
150–152 85
16
255–257
35
2
171–173 85
17
203–205
18
3
4
183–185 82
192–194 78
18
19
198–200
184–186
15
70
5
155–157 73
20
190–192
72
6
7
8
130–132 81
112–114 75
127–129 80
21
22
23
204–206
260–262
198–200
73
73
69
9
150–152 65
24
25
170–172
185–187
73
71
10
137–139 75
11
106–108 78
26
196–198
72
(Continues)

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TABLE 1 (Continued)
Melting
Melting
Compound Structure
12
point (°C) Yield (%) Compound
Structure
point (°C)
Yield (%)
115–117 78
27
240–242
78
13
14
262–264 32
28
182–184
75
78
290–292 38
29[28]
150–152
(144–146)a
15
234–236 33
30
Viscous oil 74
aLiterature melting point.
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2.2
Pharmacology
Table 2. The GI50 values are listed in parentheses below along with
this sequence of these cell lines written here. The LC50 and TGI were
also calculated and are mentioned in Table 2.
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2.2.1
In vitro anticancer activity and SARs
All the synthesized compounds 1–30 were evaluated for their in vitro
anticancer activities against three human cancer cell lines (cervical
HeLa, breast MDA‐MB‐231, and renal ACHN) using SRB assay. The
concentrations required for 50% growth inhibition (GI50) are listed in
At first, Schiff base dimers 1–12 were tested in all three cell
lines (Table 2). Unfortunately, none of them showed any anticancer
activity. Next, the corresponding imidazoles were screened.
Compound 13 without any substitution on phenyl rings showed
hr
hr
SCHEME 2 Preparation of imidazole dimers and imidazole–Schiff base hybrids. Reagents and conditions: p‐Toluenesulfonylmethyl
isocyanide, K2CO3, DMF, 36 hr. DMF, dimethylformamide

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O4 H2
hr
_
19 23
O4 5H2
hr
_
24 28
.
O4 5H2
hr
SCHEME 3 Preparation of imidazopyridine dimers. Reagents and conditions: (i) Ethanol, 10 min, (ii) CuSO4·5H2O, D‐glucose, reflux, 10 hr
better cell growth inhibition compared with the Schiff base dimers.
Methyl substitution at the para‐position on the phenyl ring (14)
increased the cell growth inhibitory activity against all three cancer
cell lines (HeLa; GI50 = 1.86 µM, MDA‐MB‐231; GI50 = 1.16 µM,
ACHN; GI50 = 3.78 µM), which was comparable to the standard
compound adriamycin. The 1,3‐positional isomers 15 and 16 had
low cytotoxic activities. The imidazole–Schiff base hybrid molecules
17 and 18 showed significant inhibitory activities on cancer cell
growth (17: GI50 = 1.92 µM, GI50 = 1.20 µM, GI50 = 2.24 µM and 18:
GI50 = 3.10 µM, GI50 = 1.90 µM, GI50 = 3.86 µM, respectively) at all
cell lines. In spite of the lower LC50 and TGI values for the
compounds 14, 17, and 18, it was clearly shown that the drastic
improvement of the cytotoxicity of imidazole derivatives was due to
the introduction of additional nitrogen in the imidazole ring.
Encouraged by this result, imidazo[1,2‐a]pyridines were screened
and all of them showed good to excellent activities. The unsub-
the electron‐withdrawing chloro group resulted in a poor inhibitory
activity (22: GI50 = 5.24 µM, GI50 = 4.50 µM, GI50 = 7.72 µM) imply-
ing that the electron‐donating or electron‐withdrawing groups in
the imidazopyridine ring exercise no positive influence on the
anticancer activity. Therefore we did not go for substituent with
more electron donating effect such as the methoxy group in the
imidazopyridine moiety toward anticancer activity.
Besides, the 1,3‐positional isomers also showed good to
excellent anticancer activities. The unsubstituted compound 24
(GI50 = 0.36 µM, GI50 = 0.30 µM, GI50 = 0.38 µM) showed the best
anticancer activity, and in fact compound 24 was the most potent
anticancer agent of the present series. The 5‐methyl (25:
GI50 = 0.84 µM, GI50 = 0.65 µM, GI50 = 0.98 µM) and 6‐methyl
(26: GI50 = 0.97 µM, GI50 = 0.58 µM, GI50 = 0.85 µM) derivatives
showed equipotent cell growth inhibitory activity to the com-
pound 24 and the standard adriamycin. The electron‐withdrawing
chloro group was not suitable on the imidazopyridine ring as it
demonstrated a weak activity (27: GI50 = 4.00 µM, GI50 = 1.60 µM,
GI50 = 1.82 µM). The 4‐methyl substitution on the benzylic ring 28
(GI50 = 0.73 µM, GI50 = 0.59 µM, GI50 = 0.62 µM) was also effec-
tive for the anticancer activity against all three cell lines. The
monomers of the imidazopyridine scaffold 29 and the Schiff base
30 did not prove versatile.
stituted parent analog 19 showed a remarkable activity (GI50
=
0.55 µM, GI50 = 0.43 µM, GI50 = 0.55 µM), which exceeds the activity
of the standard (ariamycin). This result suggests the importance of
the bis‐imidazopyridine ring. Next, we focused on the effect of
various substituents in the ring on the anticancer activity. The
introduction of the electron‐donating methyl group at the position 5
resulted in a comparable inhibitory activity (20: GI50 = 1.20 µM,
GI50 = 0.88 µM, GI50 = 1.16 µM) whereas the 6‐methyl derivative
showed a weak inhibitory activity (21: GI50 = 2.25 µM, GI50 = 2.05
µM, GI50 = 1.90 µM) when compared with 19. The introduction of
Figure 3 summarizes the SARs of the investigated imidazo-
pyridine dimers for their anticancer activities in all three cancer
cell lines. All the above facts clearly emphasize that the dimeric

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TABLE 2 Anticancer activity of the synthesized compounds against three cell linesa
HeLa
LC50 (µM)
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
MDA‐MB‐231
LC50 (µM)
>10
ACHN
LC50 (µM)
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
TGI (µM)
>10
>10
9.47
>10
>10
>10
9.79
9.67
>10
>10
>10
>10
9.76
9.76
>10
9.76
>10
>10
>10
>10
>10
3.73
9.76
>10
>10
>10
9.74
9.76
>10
>10
>10
GI50 (µM)
>10
TGI (µM)
>10
>10
>10
>10
>10
>10
>10
>10
8.45
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
GI50 (µM)
>10
TGI (µM)
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
GI50 (µM)
>10
Compound
1
2
>10
>10
>10
>10
3
>10
>10
>10
>10
4
>10
>10
>10
>10
5
>10
>10
>10
>10
6
>10
>10
>10
>10
7
8.23
>10
>10
>10
>10
8
>10
8.90
>10
>10
9
9.12
8.23
>10
>10
7.50
>10
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
>10
>10
>10
>10
>10
>10
>10
>10
>10
4.23
1.86
>10
>10
5.14
1.16
6.88
4.26
1.20
1.90
0.43
0.88
2.05
4.50
1.30
0.30
0.65
0.58
1.60
0.59
>10
8.24
3.78
9.88
7.15
2.24
3.86
0.55
1.16
1.90
7.72
1.32
0.38
0.98
0.85
1.82
0.62
8.24
>10
>10
>10
6.85
1.92
3.10
0.55
1.20
2.25
5.24
0.96
0.36
0.84
0.97
4.00
0.73
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
Adriamy-
cinb
0.52
>10
0.51
0.58
Abbreviations: GI50, concentration of drug causing 50% inhibition of cell growth; LC50, concentration of drug causing 50% cell kill; TGI, concentration of
drug causing total inhibition of cell growth.
Bold values indicate the activity best compounds.
aInhibitory activity was expressed in micromolar.
bPositive control.
imidazopyridines were more important for the anticancer activity
when compared with the monomer as well as to the Schiff base
dimers and imidazole dimers. In the dimer series of imidazopyr-
idines, the 1,3‐positional isomer was found to be more effective
than the 1,4‐positional isomer. In both 1,4 and 1,3 isomers, the
unsubstituted parent compounds 19 (1,4 isomer) and 24 (1,3
isomer) were the two best compounds among all compounds
tested in the present study. Especially, both 19 and 24 were very
effective against the breast cancer cell line (MDA‐MB‐231)
among all three cell lines selected (see Figure 4).
ERs (ERα, ERβ) are prominent in breast cancer cells and therefore
current breast cancer drugs like tamoxifene, rolaxifene, toremifene act
as either agonist or antagonist by binding to this receptor.[32,33]
As mentioned earlier, the ERs form homo‐or heterodimers upon ligand
binding. Based on these facts and to account for the mechanistic
pathway of the imidazopyridines dimers, particularly toward breast
cancer cell line, we believe that the two effective positional isomers 19
and 24 bind to the ERs and upon binding, the receptors could be
dimerized. However, the mechanistic studies are under process and
the results will be explored elsewhere.

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FIGURE 3 Structure–activity
relationships of imidazopyridine dimers as
anticancer agents (R1 = R1′, and R2 = R2′ as
these are symmetrical dimers)
|
2.2.2
In vivo anticancer activity
are found to be elevated in the breast cancer‐induced rats (Group II)
compared with the control rats (Group I). When compound 19
(Group III) and 24 (Group IV) were treated, the contents of
hemoglobin, PCV, WBC, RBC, and platelets were found to be
comparable to the standard drug, tamoxifen (Group V).
Selected compounds such as 19 and 24 were tested for in vivo anticancer
activities. In the experiment design, the animals were divided into five
groups each consisting of six rats. Group I: Control rats received normal
pelleted diet. Group II: Mammary carcinoma was induced in overnight
fasted rats by a single dose of 7,12‐dimethylbenz[a]anthracene (DMBA)
in olive oil (25 mg/kg body weight) by gastric intubation.[34] Group III:
Mammary carcinoma rats treated with compound 19 (50 mg/kg body
weight) for 14 continuous days by gastric intubation. Group IV: Mammary
carcinoma rats treated with compound 24 (50 mg/kg body weight) for 14
continuous days by gastric intubation. Group V: Mammary carcinoma rats
treated with (standard drug) tamoxifen (20 mg/kg body weight) for 14
continuous days by gastric intubation.
Serum cancer markers carcinoembryonic antigen (CEA) and
cancer antigen 15‐3 (CA 15‐3) are used to monitor response to
cancer treatment, particularly breast cancer treatment and disease
recurrence.[35] In Table 4, CEA and CA are elevated in cancer‐
induced rats (Group II) whereas the antigens were reduced due to
the effect of compound 19 (Group III) and compound 24 (Group IV),
which was also comparable to the drug tamoxifen (Group V).
Liver marker enzymes act as significant indicators of malignant
disorders.[36] The concentrations of liver marker enzymes in the
serum, such as aspartate aminotransferase (AST), alanine amino-
transferase (ALT), alkaline phosphatase (ALP), and acid phosphatase
(ACP) in the experimental groups are shown in Table 5. A marked
upsurge in the levels of serum marker enzymes was found in cancer‐
induced rats (Group II) compared with the control group (Group I)
The values outside the normal range of the hematological
parameters are diagnostic for cancer.[34] In Table 3, the results of
the hematological assay of the whole blood sample of the rats of five
groups are denoted. The contents of hemoglobin, packed cell volume
(PCV), white blood cells (WBC), red blood cells (RBC), and platelets
FIGURE 4 Cytotoxic activity of the compounds 19 and 24 against MDA‐MB‐231 cell line (adriamycin used as a positive control)
TABLE 3 Hematological parameters of breast cancer‐induced rats
Groups
Hb (g/dl)
PCV (%)
43 0.96
WBC (thousands/mm3)
RBC (millions/mm3)
Platelets (lakhs/mm3)
Group I
Group II
Group III
Group IV
Group V
14.33 0.32
8.86 0.25
13.51 0.20
13.61 0.21
13.91 0.28
7.16 0.10
6.08 0.09
8.30 0.10
26.60 0.75
40.55 0.61
40.85 0.64
41.75 0.85
10.18 0.19
9.25 0.10
5.11 0.11
4.48 0.24
5.25 0.16
6.18 0.09
8.85 0.21
5.40 0.08
6.58 0.25
8.08 0.21
6.38 0.14
7.13 0.12
Note: Values are expressed as mean SD for six animals.
Abbreviations: Hb, hemoglobin; PCV, packed cell volume; RBC, red blood cells; SD, standard deviation; WBC, white blood cells.

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TABLE 4 Analysis of serum cancer markers of the animals of five
groups
To study the SARs of each class, all the synthesized compounds were
screened for anticancer activities against HeLa, MDA‐MB‐231, and
ACHN cell lines. The bis‐imidazoles 14, imidazole–Schiff base hybrid
analog 17 and imidazopyridines (19–28) showed good to excellent
anticancer activities against three cell lines. Especially, imidazopyridines
were more promising as anticancer agents. The substituents in
the imidazopyridine ring, namely, 1,4‐bis(3‐benzyl‐6‐methylimidazo
[1,2‐a]‐pyridin‐2‐yl)benzene (20), 1,4‐bis(3‐benzyl‐7‐methylimidazo
[1,2‐a]pyridin‐2‐yl)benzene (21), 1,4‐bis(3‐benzyl‐6‐chloroimidazo[1,2‐
a]pyridin‐2‐yl)benzene (22), 1,4‐bis(3‐(4‐methylbenzyl)imidazo[1,2‐a]
pyridin‐2‐yl)‐benzene (23), 1,3‐bis(3‐benzyl‐6‐methylimidazo[1,2‐a]pyr-
idin‐2‐yl)benzene (25), 1,3‐bis(3‐benzyl‐7‐methylimidazo[1,2‐a]pyridin‐
2‐yl)benzene (26), 1,3‐bis(3‐benzyl‐6‐chloroimidazo[1,2‐a]pyridin‐2‐yl)
benzene (27), and 1,3‐bis(3‐(4‐methylbenzyl)imidazo[1,2‐a]pyridin‐2‐yl)
benzene (28), demonstrated good activity; however, the unsubstituted
analogs, 1,4‐bis‐(3‐benzylimidazo[1,2‐a]pyridin‐2‐yl)benzene (19) and
1,3‐bis(3‐benzylimidazo[1,2‐a]pyridin‐2‐yl)benzene (24), showed the
best result and, in fact, they were the two best compounds among all
series in the present study. In vivo studies were proved that these two
compounds (19 and 24) were effective in treating the cancers. Although
the molecular targets for these new class of compounds as anticancer
agents were not possible to explore in the present study, we believe
that this novel class of compounds could target ERs, as they are
potential target in treating the breast cancer. However, the mechanistic
studies for identifying molecular target for these new inhibitors are
under progress and it will be explored in the future. In summary, 1,4‐
and 1,3‐positional isomers of dimeric imidazo[1,2‐a]pyridine could serve
as potent leads for the development of anticancer agents.
Groups
CEA (ng/ml)
0.42 0.04
4.2 0.08
CA 15‐3 (ng/ml)
0.56 0.05
8.1 0.20
Group I
Group II
Group III
Group IV
Group V
2.45 0.15
2.21 0.07
1.7 0.12
3.75 0.22
3.08 0.21
2.75 0.13
Note: Values are expressed as mean SD for six animals.
Abbreviations: CA 15‐3, cancer antigen 15‐3; CEA, carcinoembryonic
antigen; SD, standard deviation.
rats. However, these levels were substantially decreased in the
animals treated with compound 19 (Group III) and compound 24
(Group IV). The results were comparable with that of the animals
treated with the standard drug tamoxifen (Group V).
In Figure 5, no histopathological alterations were observed in the
mammary gland of the control group (Group I). The histopathological
features of the tumor developed in rats (Group II) injected by DMBA
were shown to possess anaplastic activity. However, these levels
were substantially decreased and the histopathological structures
were restored in the animals treated with compound 19 (Group III)
and compound 24 (Group IV). The in vivo anticancer activity results
of compounds 19 and 24 results were comparable with that of the
animals treated with the standard drug tamoxifen (Group V).
The histopathology of the liver is shown in Figure 6. No
histopathological alterations were observed in the liver of control
group (Group I). The alterations in the histopathological features of
the liver of rats injected by DMBA are shown in Group II. However,
the histopathological structures were restored in the animals treated
with compound 19 (Group III) and compound 24 (Group IV).
The in vivo anticancer activity results of compounds 19 and 24
results were comparable with that of the animals treated with the
standard drug tamoxifen (Group V).
|
4
EXPERIMENTAL
Chemistry
|
4.1
|
4.1.1
General
Melting points were determined on Mettler FP 51 apparatus (Mettler
Instruments, Switzerland) and are uncorrected. They are expressed in
degree centigrade (°C). The IR spectra (in KBr pellets) were recorded
|
3
CONCLUSION
on a Shimadzu FT‐IR 157 spectrophotometer (Shimadzu). 1H and 13
C
A novel series of dimers of imidazole (13–18) and imidazo[1,2‐a]pyridine
(19–28) were designed and synthesized from Schiff base dimers (1–12).
NMR spectra (see the Supporting Information) were recorded using
CDCl3 as solvent and TMS as an internal standard on a Bruker
TABLE 5 Analysis of serum liver enzymes of the animals of five groups
AST (µmoles of
ALT (µmoles of
ALP (µmoles of
ACP (µmoles of
Groups
pyruvate liberated/l)
pyruvate liberated/l)
phenol liberated/l)
phenol liberated/l)
Group I
Group II
Group III
Group IV
Group V
45.63 1.98
96.30 2.28
59.21 1.84
56.08 1.16
51.60 0.95
50.63 1.54
104.56 1.78
67.26 1.85
65.2 0.70
144.78 1.38
196.13 1.80
163.05 2.79
156.83 1.36
152.35 0.83
78.18 1.91
56.05 1.37
65.53 0.79
66.56 1.13
68.13 0.63
61.88 0.94
Note: Values are expressed as mean SD for six animals.
Abbreviations: ACP, acid phosphatase; ALP, alkaline phosphatase; ALT, alanine aminotransferase; AST, aspartate aminotransferase; SD, standard deviation.

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MEENAKSHISUNDARAM ET AL.
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FIGURE 5 Representative photomicrographs of breast tissues of Group I to Group V
300 MHz/400 MHz NMR spectrometer (Bruker). Chemical shift
values are given in δ (ppm scale). Mass spectra were recorded on a
Bruker Maxis HRMS (Bruker) by ESI techniques and Auto Spec EI
Shimadzu QP 2010 PLUS GC‐MS mass spectrometer (Shimadzu).
Microanalyses were performed on a Vario EL III model CHNS
analyzer (Vario, Germany) at the Department of Chemistry,
Bharathiar University. The purity of the compounds was checked
by thin‐layer chromatography (TLC) on silica gel plates using
petroleum ether and ethyl acetate.
The InChI codes of the investigated compounds together with
some biological activity data are provided as Supporting Information.
|
4.1.2
General procedure for the preparation of
Schiff bases 1–12 and 30
To a solution of corresponding dialdehyde (10.0 mmol) in ethanol
(10 ml), corresponding amines (20.0 mmol) were added and refluxed
for 6 hr. The obtained precipitate was collected by filtration and
FIGURE 6 Representative photomicrographs of liver tissues of Group I to Group V

MEENAKSHISUNDARAM ET AL.
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recrystallized from ethanol. The compound 30 was synthesized from
3.16–3.25 (m, 1H), 7.75 (s, 2H), 8.32 (s, 1H, CH═N). 13C NMR (CDCl3)
δ (ppm): 24.80, 25.63, 34.33, 70.14, 128.23, 138.28, 158.17. Anal.
calcd. for C20H28N2 (296): C, 81.03, H, 9.52, N, 9.45. Found: C, 81.10,
H, 9.49, N, 9.41%.
the reaction of aniline and benzaldehyde (equimolar ratio).
(N,N′E,N,N′E)‐N,N′‐(1,4‐Phenylenebis(methan‐1‐yl‐1‐ylidene))diani-
line (1)
White solid. IR (KBr) νmax (cm−1): 3,019, 2,872, 1,611, 1,042, 838. 1
H
(N,N′E,N,N′E)‐N,N′‐(1,3‐Phenylenebis(methan‐1‐yl‐1‐ylidene))diani-
line) (7)
NMR (CDCl3) δ (ppm): 7.27–7.29 (m, 3H), 7.42 (t, 2 H, J = 6.80 Hz),
8.02 (s, 2H), 8.52 (s, 1H, CH═N). 13C NMR (CDCl3) δ (ppm): 120.94,
129.18, 129.23, 130.04, 138.85, 151.68, 159.44. EI‐MS, m/z (%): 285
(10), 282 (100), 207 (38), 181 (35), 130 (20), 94 (42), 72 (55), 54 (45).
Anal. calcd. for C20H16N2 (284): C, 84.48, H, 5.67, N, 9.85. Found: C,
84.69, H, 5.07, N, 10.24%.
Yellow liquid. IR (KBr) νmax (cm−1): 3,019, 2,850, 1,618, 1,067, 842.
1H NMR (CDCl3) δ (ppm): 7.25–7.28 (m, 3H), 7.38–7.43 (m, 2H), 7.55
(m, 1H), 8.00 (d, 1H, Hz, J = 7.52 Hz), 8.38 (s, 1H), 8.53 (s, 1H, CH═N).
13C NMR (CDCl3) δ (ppm): 120.85, 129.24, 129.35, 129.80, 130.85,
136.25, 137.10, 149.12, 158.72. Anal. calcd. for C20H16N2 (284): C,
84.48, H, 5.67, N, 9.85. Found: C, 84.40, H, 5.69, N, 9.91%.
(N,N′E,N,N′E)‐N,N′‐(1,4‐Phenylenebis(methan‐1‐yl‐1‐ylidene))bis(2‐
methylaniline) (2)
White solid. IR (KBr) νmax (cm−1): 3,020, 2,874, 1,609, 1,041, 836. 1
H
(N,N′E,N,N′E)‐N,N′‐(1,3‐Phenylenebis(methan‐1‐yl‐1‐ylidene))bis(4‐
methylaniline) (8)
White solid. IR (KBr) νmax (cm−1): 3,017, 2,880, 1,615, 1,081, 837. 1
NMR (CDCl3) δ (ppm): 2.39 (s, 3H), 6.98 (d, J = 7.80 Hz, 1H),
7.13–7.25 (m, 3H), 8.03 (s, 2H), 8.43 (s, 1H, CH═N). 13C NMR
(CDCl3) δ (ppm): 17.87, 117.49, 126.03, 126.75, 129.04, 130.36,
132.20, 138.80, 150.81, 158.53. Anal. calcd. for C20H20N2 (312): C,
84.58, H, 6.45, N, 8.97. Found: C, 84.47, H, 6.58, N, 8.95%.
H
NMR (CDCl3) δ (ppm): 2.38 (s, 3H), 7.17 (d, 2H, J = 8.50 Hz), 7.21 (d,
2H, J = 8.00 Hz), 7.57 (m, 1H), 8.03 (d, J = 7.50 Hz, 1H), 8.39 (s, 1H),
8.55 (s, 1H, CH═N). 13C NMR (CDCl3) δ (ppm): 21.03, 120.86, 129.2,
129.84, 131.02, 134.12, 136.16, 136.93, 149.09, 158.73. Anal. calcd.
for C22H20N2 (312): C, 84.58, H, 6.45, N, 8.97. Found: C, 84.59, H,
6.46, N, 8.95%.
(N,N′E,N,N′E)‐N,N′‐(1,4‐Phenylenebis(methan‐1‐yl‐1‐ylidene))bis(4‐
methylaniline) (3)
Yellowish solid. IR (KBr) νmax (cm−1): 3,015, 2,875, 1,607, 1,075, 829.
1H NMR (CDCl3) δ (ppm): 2.39 (s, 3H), 7.18 (d, 2H, J = 6.80 Hz), 7.22
(d, 2H, J = 8.00 Hz), 8.00 (s, 2H), 8.53 (s, 1H, CH═N). 13C NMR
(CDCl3) δ (ppm): 21.05, 120.91, 129.04, 129.84, 136.30, 138.85,
149.29, 158.62. Anal. calcd. for C22H20N2 (312): C, 84.58, H, 6.45, N,
8.97. Found: C, 84.50, H, 6.46, N, 9.04%.
(N,N′E,N,N′E)‐N,N′‐(1,3‐Phenylenebis(methan‐1‐yl‐1‐ylidene))bis(2‐
methylaniline) (9)
Yellow liquid. IR (KBr) νmax (cm−1): 3,019, 2,884, 1,614, 1,079, 835.
1H NMR (CDCl3) δ (ppm): 2.39 (s, 3H), 6.98 (d, J = 6.80 Hz, 1H),
7.08–7.26 (m, 3H), 7.60 (m, 1H), 8.08 (d, J = 7.20 Hz, 1H), 8.41 (s, 1H),
8.46 (s, 1H, CH═N). 13C NMR (CDCl3) δ (ppm): 17.87, 117.60, 125.90,
126.76, 129.51, 130.34, 131.02, 131.66, 134.11, 137.07, 150.84,
158.69. Anal. calcd. for C20H20N2 (312): C, 84.58, H, 6.45, N, 8.97.
Found: C, 84.53, H, 6.43, N, 9.04%.
(N,N′E,N,N′E)‐N,N′‐(1,4‐Phenylenebis(methan‐1‐yl‐1‐ylidene))bis(4‐
methoxyaniline) (4)
Brown solid. IR (KBr) νmax (cm−1): 3,016, 2,871, 1,600, 1,035, 830. 1
H
NMR (CDCl3) δ (ppm): 3.85 (s, 3H), 6.95 (d, J = 7.80 Hz, 2H), 7.30 (d,
J = 7.40 Hz, 2H), 8.00 (s, 2H), 8.54 (s, 1H, CH═N). 13C NMR (CDCl3) δ
(ppm): 55.52, 114.46, 122.29, 129.20, 130.78, 137.00, 157.53,
158.58. Anal. calcd. for C22H20N2O2 (344): C, 76.72, H, 5.85, N,
8.13. Found: C, 76.76, H, 5.83, N, 8.09%.
(N,N′E,N,N′E)‐N,N′‐(1,3‐Phenylenebis(methan‐1‐yl‐1‐ylidene))bis(4‐
methoxyaniline) (10)
Brown solid. IR (KBr) νmax (cm−1): 3,018, 2,875, 1,618, 1,076, 841. 1
H
NMR (CDCl3) δ (ppm): 3.87 (s, 3H), 6.99 (d, 2H, J = 6.80 Hz), 7.33 (d,
2H, J = 8.80 Hz), 7.59 (m, 1H), 8.03 (d, J = 7.00 Hz, 1H), 8.40 (s, 1H),
8.59 (s, 1H, CH═N). Anal. calcd. for C22H20N2O2 (344): C, 76.72, H,
5.85, N, 8.13. Found: C, 76.10, H, 5.49, N, 8.41%.
(N,N′E,N,N′E)‐N,N′‐(1,4‐Phenylenebis(methan‐1‐yl‐1‐ylidene))bis(4‐
chloroaniline) (5)
Yellow solid. IR (KBr) νmax (cm−1): 3,018, 2,875, 1,604, 1,035, 829. 1
H
NMR (CDCl3) (ppm): 7.19 (d, 2H, J = 8.80 Hz), 7.38 (d, 2H,
δ
J = 8.50 Hz), 8.01 (s, 2H), 8.49 (s, 1H, CH═N). 13C NMR (CDCl3) δ
(ppm): 122.28, 129.24, 129.35, 130.05, 138.58, 150.11, 159.82. Anal.
calcd. for C20H14Cl2N2 (353): C, 68.00, H, 3.99, N, 7.93. Found: C,
68.04, H, 3.94, N, 7.97%.
(N,N′E,N,N′E)‐N,N′‐(1,3‐Phenylenebis(methan‐1‐yl‐1‐ylidene))bis(4‐
chloroaniline) (11)
Yellow solid. IR (KBr) νmax (cm−1): 3,024, 2,879, 1,621, 1,033, 851. 1
H
NMR (CDCl3) (ppm): 7.18 (d, 2H, J = 9.00 Hz), 7.37 (d, 2H,
δ
J = 9.00 Hz), 7.60 (m, 1H), 8.04 (d, 1H, J = 7.50 Hz, 2H), 8.41 (s, 1H),
8.52 (s, 1H, CH═N). 13C NMR (CDCl3) δ (ppm): 122.23, 129.35,
130.21, 131.58, 131.87, 134.19, 136.63, 150.09, 159.71. Anal. calcd.
for C20H14Cl2N2 (353): C, 68.00, H, 3.99, N, 7.93. Found: C, 68.10, H,
3.49, N, 7.91%.
(N,N′E,N,N′E)‐N,N′‐(1,4‐Phenylenebis(methan‐1‐yl‐1‐ylidene))dicyclo-
hexanamine (6)
White solid. IR (KBr) νmax (cm−1): 3,021, 2,855, 1,621, 1,065, 920,
840. 1H NMR (CDCl3) δ (ppm): 1.24–1.45 (m, 3H), 1.53–1.87 (m, 7H),

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(N,N′E,N,N′E)‐N,N′‐(1,3‐Phenylenebis(methan‐1‐yl‐1‐ylidene))dicyclo-
hexanamine (12)
7.91 (s, 1H). 13C NMR (CDCl3) δ (ppm): 125.40, 127.41, 127.87,
128.11, 128.38, 128.94, 130.06, 130.86, 132.19, 138.30, 139.24.
Anal. calcd. for C24H18N4 (362): C, 79.54, H, 5.0, N, 15.46. Found:
C,79.57, H, 5.05, N, 15.38%.
White solid. IR (KBr) νmax (cm−1): 3,022, 2,875, 1,619, 1,032, 848. 1
H
NMR (CDCl3) δ (ppm): 1.24–1.87 (m, 10H), 3.18–3.25 (m, 1H), 7.43
(m, 1H), 7.81 (d, J = 8.40 Hz, 1H), 8.06 (s, 1H), 8.35 (s, 1H, CH═N). 13C
NMR (CDCl3) δ (ppm): 24.81, 25.64, 34.36, 69.98, 127.90, 128.75,
129.71, 137.00, 158.20. Anal. calcd. for C20H28N2 (296): C, 81.03, H,
9.52, N, 9.45. Found: C, 81.09, H, 9.50, N, 9.41%.
1,3‐Bis(1‐p‐tolyl‐1H‐imidazol‐5‐yl)benzene (16)
White solid. IR (KBr) νmax (cm−1): 2,989, 2,944, 1,642, 1,465, 1,189,
1,076. 1H NMR (CDCl3) δ (ppm): 2.38 (s, 3H), 7.07 (d, J = 7.40 Hz, 2H),
7.20 (s, 1H), 7.21 (d, J = 7.80 Hz, 4H), 7.28 (m, 1H), 7.65 (d,
J = 7.20 Hz, 1H), 7.69 (s, 1H), 7.90 (s, 1H). 13C NMR (CDCl3) δ
(ppm): 21.09, 125.40, 127.45, 127.92, 128.03, 128.50, 129.31,
130.90, 132.49, 134.10, 138.29, 139.20. Anal. calcd. for C26H22N4
(390): C, 79.97, H, 5.68, N, 14.35. Found: C, 80.03, H, 5.65, N, 14.32%.
(E)‐N‐Benzylideneaniline (30)
Viscous oil. 1H NMR (CDCl3) δ (ppm): 7.18–7.21 (m, 3H), 7.28–7.53
(m, 5H), 7.82 (d, J = 7.40 Hz, 2H), 8.42 (s, 1H, CH═N). Anal. calcd. for
C13H11N (181.23): C, 86.15, H, 6.12, N, 7.73. Found: C, 86.22, H, 6.08,
N, 7.70%.
(E)‐4‐Methyl‐N‐(4‐(1‐p‐tolyl‐1H‐imidazol‐5‐yl)benzylidene)aniline
(17)
|
4.1.3
General procedure for the synthesis of
Light brown solid. IR (KBr) νmax (cm−1): 2,933, 1,641, 1,615, 1,435,
1,194, 1,075, 956, 821. 1H NMR (CDCl3) δ (ppm): 2.37 (s, 3H), 2.40 (s,
3H), 7.09–7.13 (m, 4H), 7.18–7.26 (m, 6H), 7.36 (s, 1H), 7.70 (s, 1H),
7.78 (d, J = 7.8 Hz, 2H), 8.43 (s, 1H, CH═N). Anal. calcd. for C24H21N3
(351): C, 82.02, H, 6.02, N, 11.96. Found: C, 82.04, H, 6.05, N, 11.90%.
imidazoles 13–16 and the imidazole–Schiff base
hybrids 17 and 18
To
a solution of the corresponding Schiff base (2.0 mmol) in
dimethylformamide, TOSMIC (4.0 mmol) and K2CO3 (6.0 mmol) were
added. The solution was stirred at ambient temperature for 36 hr.
The formation of products was monitored by TLC. Water was added
to the reaction mixture and extracted using ethyl acetate. The
organic layer was dried over anhydrous sodium sulfate and
concentrated. The obtained crude product was purified by column
chromatography to get the imidazole dimers 13–16 and imidazo-
le–Schiff base hybrid compounds 17 and 18. The yields of the
imidazole dimers 13–16 were 30–40%. The yields of the imidazo-
le–Schiff base hybrid compounds 17 and 18 were 10–20%. The
reaction was not completed even after 36 hr and the unreacted
starting material was recovered.
(E)‐N‐(3‐(1‐Phenyl‐1H‐imidazol‐5‐yl)benzylidene)aniline (18)
Light brown solid. IR (KBr) νmax (cm−1): 2,932, 1,639, 1,620, 1,437,
1,191, 1,076, 958, 826. 1H NMR (CDCl3) δ (ppm): 7.17–7.20 (m, 3H),
7.28–7.34 (m, 7H), 7.34 (s, 1H), 7.38 (m, 1H), 7.68 (s, 1H), 7.79 (d,
J = 8.00 Hz, 2H), 8.18 (s, 1H), 8.42 (s, 1H, CH═N). Anal. calcd. for
C22H17N3 (323): C, 82.02, H, 6.02, N, 11.96. Found: C, 82.04, H, 6.0 4,
N, 11.92%.
|
4.1.4
General procedure for the synthesis of
imidazopyridines 19–29
1,4‐Bis(1‐phenyl‐1H‐imidazol‐5‐yl)benzene (13)
A mixture of 2‐aminopyridine (1 mmol) and the corresponding
White solid. IR (KBr) νmax (cm−1): 2,994, 2,943, 1,639, 1,461, 1,186,
1,074. 1H NMR (CDCl3) δ (ppm): 7.03 (s, 1H), 7.20–7.25 (m, 3H), 7.27
(s, 2H), 7.28–7.30 (m, 2H), 7.68 (s, 1H). Anal. calcd. for C24H18N4
(362): C, 79.54, H, 5.01, N, 15.46. Found: C, 79.59, H, 5.05, N, 14.32%.
dialdehyde (1 mmol) in absolute ethanol (5 ml) was stirred for
15 min. To this reaction mixture phenylacetylene (1.5 mmol) was
added followed by the addition of CuSO4·5H2O (15 mol%) and D‐
glucose (30 mol%). Again ethanol (5 ml) was added to the reaction
mixture and refluxed at 100°C for 10 hr. After the completion of
reaction as indicated by TLC, the resultant mixture was directly
adsorbed on neutral alumina (without workup) and the product was
purified by column chromatography. The monomer 29 was synthe-
sized from the reaction of 2‐aminopyridine and benzaldehyde under
the same above condition.
1,4‐Bis(1‐p‐tolyl‐1H‐imidazol‐5‐yl)benzene (14)
White solid. IR (KBr) νmax (cm−1): 2,995, 2,946, 1,645, 1,462, 1,182,
1,076. 1H NMR (CDCl3) δ (ppm): 2.40 (s, 3H), 7.02 (s, 1H), 7.05 (d,
J = 8.00 Hz, 2H), 7.18 (d, J = 8.00 Hz, 2H), 7.26 (s, 2H), 7.66 (s, 1H).
13C NMR (CDCl3) δ (ppm): 21.11, 125.44, 127.49, 127.90, 128.47,
130.92, 132.47, 134.04, 138.28, 139.19. HRMS (ESI): m/z [M+H]+
calcd. for C26H22N4 391.1843, found: 391.1922. Anal. calcd. for
C26H22N4 (390): C, 79.97, H, 5.68, N, 14.35. Found: C, 80.07, H, 5.55,
N, 14.38%.
1,4‐Bis(3‐benzylimidazo[1,2‐a]pyridin‐2‐yl)benzene (19)
White solid. IR (KBr) νmax (cm−1): 3,027, 2,926, 1,602, 1,499, 1,342,
1,049, 800. 1H NMR (CDCl3) δ (ppm): 4.54 (s, 2H), 6.74 (t, J = 7.40 Hz,
1H), 7.15–7.27 (m, 5H), 7.30–7.33 (m, 2H), 7.73 (d, J = 7.40 Hz, 1H),
7.89 (s, 2H). 13C NMR (CDCl3) δ (ppm): 29.82, 112.59, 117.39,
118.05, 123.49, 124.78, 126.71, 127.04, 127.75, 128.54, 129.14,
130.37, 136.56, 144.67. Anal. calcd. for C34H26N4 (490): C, 83.24, H,
5.34, N, 11.42. Found: C, 83.27, H, 5.32, N, 11.41%.
1,3‐Bis(1‐phenyl‐1H‐imidazol‐5‐yl)benzene (15)
White solid. IR (KBr) νmax (cm−1): 2,993, 2,942, 1,647, 1,460, 1,186,
1,074. 1H NMR (CDCl3) δ (ppm): 7.19–7.23 (m, 3H), 7.21 (s, 1H),
7.28–7.31 (m, 2H), 7.29 (m, 1H), 7.67 (d, J = 7.40 Hz, 1H), 7.70 (s, 1H),

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1,4‐Bis(3‐benzyl‐6‐methylimidazo[1,2‐a]pyridin‐2‐yl)benzene (20)
White solid. IR (KBr) νmax (cm−1): 3,025, 2,924, 1,609, 1,497, 1,345,
1,047, 802. 1H NMR (CDCl3) δ (ppm): 2.26 (s, 3H), 4.49 (s, 2H), 7.10
(d, J = 7.20 Hz, 1H), 7.14–7.16 (m, 2H), 7.24–7.34 (m, 3H), 7.51 (s,
1H), 7.68 (d, J = 8.20 Hz, 1H), 7.85 (s, 2H). 13C NMR (CDCl3) δ (ppm):
18.35, 29.77, 116.61, 117.80, 121.07, 127.01, 127.74, 127.75,
128.41, 128.42, 129.13, 129.14, 136.67, 145.31, 149.88. HRMS
(ESI): m/z [M+H]+ calcd. for C36H30N4: 519.2468, found: 519.2548.
Anal. calcd. for C36H30N4 (518): C, 83.37, H, 5.83, N, 10.80. Found: C,
83.34, H, 5.89, N, 10.77%.
1,3‐Bis(3‐benzyl‐6‐methylimidazo[1,2‐a]pyridin‐2‐yl)benzene (25)
White solid. IR (KBr) νmax (cm−1): 3,027, 2,920, 1,596, 1,498, 1,339,
1,041, 855. 1H NMR (CDCl3) δ (ppm): 2.32 (s, 3H), 4.43 (s, 2H),
7.05–7.08 (m, 2H), 7.14 (d, J = 7.40 Hz, 1H), 7.19–7.25 (m, 3H), 7.53
(t, J = 8.00 Hz, 1H), 7.58 (s, 1H), 7.61–7.65 (m, 2H), 8.17 (s, 1H). 13C
NMR (101 MHz, CDCl3) δ 143.81, 143.63, 136.85, 134.88, 128.96,
128.84, 127.71, 127.64, 127.53, 127.23, 126.67, 121.67, 120.94,
117.49, 116.75, 77.33, 29.67, 18.32. Anal. calcd. for C36H30N4 (518):
C, 83.37, H, 5.83, N, 10.80. Found: C, 83.34, H, 5.86, N, 10.80%.
1,3‐Bis(3‐benzyl‐7‐methylimidazo[1,2‐a]pyridin‐2‐yl)benzene (26)
White solid. IR (KBr) νmax (cm−1): 3,026, 2,924, 1,595, 1,495, 1,333,
1,040, 852. 1H NMR (DMSO‐d6) δ (ppm): 2.35 (s, 3H), 4.45 (s, 2H),
6.59 (d, J = 7.40 Hz, 1H), 7.08–7.10 (m, 2H), 7.20–7.29 (m, 3H), 7.41
(t, J = 7.80 Hz, 1H), 7.57 (s, 1H), 7.63 (d, J = 6.80 Hz, 1H), 7.67 (d,
J = 7.80 Hz, 1H), 8.03 (s, 1H). 13C NMR (DMSO‐d6) δ (ppm): 13C NMR
(151 MHz, DMSO) δ 160.00, 147.18, 144.44, 141.98, 137.58, 135.23,
135.06, 128.98, 128.89, 127.71, 126.97, 126.68, 126.68, 126.44,
123.65, 118.01, 115.25, 114.78, 20.86. Anal. calcd. for C36H30N4
(518): C, 83.37, H, 5.83, N, 10.80. Found: C, 83.33, H, 5.91, N, 10.76%.
1,4‐Bis(3‐benzyl‐7‐methylimidazo[1,2‐a]pyridin‐2‐yl)benzene (21)
White solid. IR (KBr) νmax (cm−1): 3,022, 2,927, 1,615, 1,498, 1,342,
1,044, 807. 1H NMR (DMSO‐d6) δ (ppm): 2.41 (s, 3H), 4.50 (s, 2H),
6.60 (d, J = 7.20 Hz, 1H), 7.14–7.16 (m, 2H), 7.25–7.32 (m, 3H), 7.53
(s, 1H), 7.61 (d, J = 8.00 Hz, 1H), 7.87 (s, 2H). 13C NMR (DMSO‐d6) δ
(ppm): 13C NMR (151 MHz, DMSO) δ 192.84, 141.73, 140.82, 140.58,
137.77, 137.49, 135.82, 134.98, 133.87, 132.61, 130.10, 128.99,
126.83, 123.89, 119.78, 118.11, 115.29, 114.83, 28.93, 20.93, 15.36.
Anal. calcd. for C36H30N4 (518): C, 83.37, H, 5.83, N, 10.80. Found: C,
83.35, H, 5.87, N, 10.78%.
1,3‐Bis(3‐benzyl‐6‐chloroimidazo[1,2‐a]pyridin‐2‐yl)benzene (27)
White solid. IR (KBr) νmax (cm−1): 3,027, 2,931, 1,594, 1,492, 1,335,
1,042, 851. 1H NMR (DMSO‐d6) δ (ppm): 4.40 (s, 2H), 7.07–7.09 (m,
2H), 7.17 (d, J = 7.80 Hz, 1H), 7.24–7.32 (m, 3H), 7.55 (t, J = 7.40 Hz,
1H), 7.67 (d, J = 7.80 Hz, 1H), 7.72 (s, 1H), 7.84 (d, J = 7.8 Hz, 1H), 8.17
(s, 1H). 13C NMR (151 MHz, DMSO‐d6) δ 144.08, 142.33, 137.44,
135.14, 128.94, 127.75, 126.75, 124.65, 124.43, 118.68, 117.04,
112.35, 40.11, 39.97, 39.84, 39.70, 39.56, 39.42, 39.28, 28.85. HRMS
(ESI): m/z [M+H]+ calcd for C34H24Cl2N4: 559.1375, found: 559.1451.
Anal. calcd. for C34H24Cl2N4 (559): C, 72.99, H, 4.32, N, 10.01. Found:
C, 72.86, H, 4.35, N, 10.06%.
1,4‐Bis(3‐benzyl‐6‐chloroimidazo[1,2‐a]pyridin‐2‐yl)benzene (22)
White solid. IR (KBr) νmax (cm−1): 3,025, 2,922, 1,617, 1,494, 1,339,
1,042, 801. 1H NMR (CDCl3) δ (ppm): 4.51 (s, 4H), 7.13–7.15 (m, 4H),
7.28–7.33 (m, 3H), 7.41–7.44 (m, 5H), 7.61 (d, J = 8.0 Hz, 2H), 7.74 (d,
J = 8.4 Hz, 2H), 8.01 (s, 2H). 13C NMR (CDCl3) δ (ppm): 29.83, 114.13,
119.21, 122.27, 123.50, 124.75, 126.64, 127.12, 127.98, 128.54,
129.15, 130.37, 136.57. Anal. calcd. for C34H24Cl2N4 (559): C, 72.99,
H, 4.32, N, 10.01. Found: C, 72.95, H, 4.30, N, 10.08%.
1,4‐Bis(3‐(4‐methylbenzyl)imidazo[1,2‐a]pyridin‐2‐yl)benzene (23)
White solid. IR (KBr) νmax (cm−1): 3,020, 2,919, 1,615, 1,484, 1,340,
1,045, 803. 1H NMR (CDCl3) δ (ppm): 2.33 (s, 3H), 4.49 (s, 2H), 6.86
(t, J = 7.40 Hz, 1H), 7.02 (d, J = 7.80 Hz, 2H), 7.13 (d, J = 8.00 Hz, 2H),
7.28–7.35 (m, 2H), 7.79 (d, J = 7.20 Hz, 1H), 7.90 (s, 2H). 13C NMR
(CDCl3) δ (ppm): 21.10, 29.44, 112.69, 117.44, 123.84, 124.93,
127.93, 128.07, 128.35, 129.29, 129.87, 129.95, 133.48, 136.65,
144.59. Anal. calcd. for C36H30N4 (518): C, 83.37, H, 5.83, N, 10.80.
Found: C, 83.35. H, 5.87, N, 10.78%.
1,3‐Bis(3‐(4‐methylbenzyl)imidazo[1,2‐a]pyridin‐2‐yl)benzene (28)
White solid. IR (KBr) νmax (cm−1): 3,024, 2,932, 1,590, 1,491, 1,337,
1,045, 853. 1H NMR (DMSO‐d6) δ (ppm): 2.32 (s, 3H), 4.49 (s, 2H),
6.90 (t, J = 7.20 Hz, 1H), 6.99 (d, J = 7.80 Hz, 2H), 7.08 (d, J = 8.00 Hz,
2H), 7.37–7.43 (m, 2H), 7.63 (t, J = 7.80 Hz, 1H), 7.79 (d, J = 7.20 Hz,
1H), 7.94 (d, J = 7.20 Hz, 1H), 8.25 (s, 1H). 13C NMR (DMSO‐d6) δ
(ppm): 21.35, 29.66, 112.70, 117.51, 118.42, 123.85, 127.14, 128.01,
128.21, 128.22, 129.15, 129.35, 132.03, 135.13, 136.22, 144.05,
145.11. Anal. calcd. for C36H30N4 (518): C, 83.37, H, 5.83, N, 10.80.
Found: C, 83.32, H, 5.85, N, 10.83%.
1,3‐Bis(3‐benzylimidazo[1,2‐a]pyridin‐2‐yl)benzene (24)
White solid. IR (KBr) νmax (cm−1): 3,028, 2,919, 1,599, 1,494, 1,343,
1,036, 852. 1H NMR (CDCl3) δ (ppm): 4.42 (s, 2H), 6.68 (t, J = 8.20 Hz,
1H), 7.07–7.09 (m, 2H), 7.16 (d, J = 7.80 Hz, 1H), 7.20–7.28 (m, 3H),
7.55 (t, J = 8.00 Hz, 1H), 7.65–7.67 (m, 2H), 7.85 (d, J = 7.80 Hz, 1H),
8.18 (s, 1H). 13C NMR (CDCl3) δ (ppm): 29.88, 112.33, 117.72,
118.09, 123.81, 124.36, 127.02, 127.99, 128.18, 128.19, 129.15,
129.39, 134.99, 136.89, 144.10, 144.96. Anal. calcd. for C34H26N4
(490): C, 83.24, H, 5.34, N, 11.42. Found: C, 83.25, H, 5.37, N, 11.38%.
3‐Benzyl‐7‐methyl‐2‐phenylimidazo[1,2‐a]pyridine (29)
White solid. 1H NMR (CDCl3) δ (ppm): 2.26 (s, 3H), 4.48 (s, 2H), 7.11
(dd, J = 1.80, 8.40 Hz, 1H), 7.16 (d, J = 7.4 Hz, 2H), 7.27–7.38 (m, 4H),
7.43–7.45 (m, 2H), 7.51 (s, 1H), 7.69 (d, J = 8.80 Hz, 1H), 7.79 (dd,
J = 2.0, 7.40 Hz, 2H). Anal. calcd. for C21H18N2 (298.38): C, 84.53, H,
6.08, N, 9.39. Found: C, 84.52, H, 6.10, N, 9.38%.

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Anticancer activity (in vivo)
4.2
Pharmacology
4.2.2
|
4.2.1
Anticancer activity (in vitro)
Collection of samples
In the experiment design, the animals were divided into five groups
each consisting of six rats. After the experimental regimen (16
weeks), the animals were killed by cervical dislocation under mild
chloroform anesthesia. Blood was collected in EDTA and centrifuge
tubes by an incision made in the jugular veins and serum was
separated by centrifugation at 2,000 rpm for 20 min and utilized for
various biochemical assays. The liver and mammary tissue were
excised immediately and thoroughly washed with ice‐cold physiolo-
gical saline and blotted dry. A part of the tissues such as liver and
breast were removed and fixed in 10% formalin for histopathological
study.
The anticancer screening was performed by SRB assay.[34,37–39] The
cell lines were grown in RPMI 1640 medium containing 10% fetal
bovine serum and
2 mM L‐glutamine. For present screening
experiment, cells were inoculated into 96‐well microtiter plates in
90 µl at 5,000 cells per well. After cell inoculation, the microtiter
plates were incubated at 37°C, 5% CO2, 95% air, and 100% relative
humidity for 24 hr before addition of experimental drugs. Experi-
mental drugs were solubilized in appropriate solvent to prepare stock
of 10−2 concentration. At the time of experiment, four 10‐fold serial
dilutions were made using complete medium. Aliquots of 10 µl of
these different drug dilutions were added to the appropriate
microtiter wells already containing 90 µl of medium, resulting in
the required final drug concentrations.
Hematological parameters
The hematological parameters such as hemoglobin, PCV, WBC, RBC,
and platelets, were assayed. The whole blood sample was analyzed
for the changes in the blood cells using SYSMEX Xs—800 i automatic
hematology analyzer (Bio‐Rad Laboratories, Inc.).
After compound addition, plates were incubated at standard
conditions for 48 hr and assay was terminated by the addition of
cold TCA. Cells were fixed in situ by the gentle addition of 50 µl of
cold 30% (w/v) TCA (final concentration, 10% TCA) and incubated
for 60 min at 4°C. The supernatant was discarded; the plates
were washed five times with tap water and air dried. Sulforho-
damine B (SRB) solution (50 µl) at 0.4% (w/v) in 1% acetic acid
was added to each of the wells, and plates were incubated for
20 min at room temperature. After staining, unbound dye was
recovered and the residual dye was removed by washing five
times with 1% acetic acid. The plates were air dried. Bound stain
was subsequently eluted with 10 mM Trizma base, and the
absorbance was read on an enzyme‐linked immunosorbent assay
(ELISA) plate reader at a wavelength of 540 nm, with 690 nm
reference wavelength.
|
4.2.3
Estimation of aspartate transaminase[40]
Principle
The enzyme catalysis the following reaction
AST
L‐Aspartate + α‐oxoglutarate
→
oxaloacetate + L‐glutamate.
The oxaloacetate is measured by the reaction with 2,4‐dinitro-
phenyl hydrazine giving brown‐colored hydrazone after the
a
Percentage of growth was calculated on a plate‐by‐plate basis
for test wells relative to control wells. Percentage of growth was
expressed as the ratio of average absorbance of the test well to the
average absorbance of the control wells × 100. Using the six
absorbance measurements (time zero [Tz], control growth [C], and
test growth in the presence of drug at the four concentration levels
[Ti]), the percentage growth was calculated at each of the drug
concentration levels. The dose response parameters were calcu-
lated for each test article. Growth inhibition of 50% (GI50) was
calculated from [(Ti − Tz)/(C − Tz)] × 100 = 50, which is the drug
concentration resulting in a 50% reduction in the net protein
increase (as measured by SRB staining) in control cells during the
drug incubation. The drug concentration resulting in total growth
inhibition (TGI) was calculated from Ti = Tz. The LC50 (concentration
of drug resulting in a 50% reduction in the measured protein at the
end of the drug treatment as compared with that at the beginning)
indicating a net loss of cells following treatment is calculated from
[(Ti − Tz)/Tz] × 100 = −50. Values were calculated for each of these
three parameters if the level of activity was reached; however, if the
effect was not reached or was exceeded, the values for that
parameter were expressed as greater or less than the maximum or
minimum concentration tested.
addition of sodium hydroxide (NaOH). The color developed is read
at 520 nm.
Reagents
(a) Phosphate buffer, 0.1 M, pH 7.5, (b) substrate: 146 mg ketoglu-
tarate and 13.3 g aspartic acid in 1 N NaOH. Adjusted the pH to 7.4
and made up to 1,000 ml with phosphate buffer, (c) standard
pyruvate, 2 mML: 22 mg sodium pyruvate/100 ml phosphate buffer,
(d) dinitrophenyl hydrazine reagent, 1 mmol/l in 1 M HCl, and (e)
0.4 N NaOH.
Procedure
To 0.2 ml of sample, added 1.0 ml of the buffered substrate and
incubated for 60 min at 37°C. To the control tubes, enzyme was added
after arresting the reaction with 1.0 ml of DNPH and the tubes were
kept at room temperature for 20 min. Then 10 ml of 0.4 N NaOH was
added. A set of standard pyruvate in the concentration 0.4–2.0 µM
was also treated in a similar manner. The color developed was read at
520 nm. The enzyme activity was expressed as µmol of pyruvate
liberated/l in serum and µmol of pyruvate liberated/min/mg protein in
tissues.

MEENAKSHISUNDARAM ET AL.
15 of 16
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4.2.4
Estimation of alanine transaminase[40]
added immediately. Mixed well and centrifuged. To 4.0 ml of the
supernatant added 2.0 ml of sodium carbonate. Standards were also
run. Incubated all the tubes at 37°C for 15 min, the color developed
were read at 700 nm. The enzyme activity was expressed as µmol of
phenol liberated/l in serum and µmol of phenol liberated/min/mg
protein.
Principle
The enzyme catalyses the following reaction
ALT
L‐Alanine + α‐oxoglutarate
→
pyruvate + L‐glutamate.
The oxaloacetate is measured by the reaction with 2,4‐dinitro-
phenyl hydrazine giving brown‐colored hydrazone after the
Estimation of ACP[42]
a
|
4.2.6
addition of NaOH. The color developed is read at 520 nm.
Principle
The method used was that of King and Armstrong in which disodium
phenyl phosphate is hydrolyzed with the liberation of phenol and
inorganic phosphate. The liberated phenol is measured at 700 nm
with Folin‐Ciocalteu reagent.
Reagents
(a) Phosphate buffer 0.1 M, pH 7.5, (b) substrate: 146 mg ketoglu-
tarate and 17.8 g L‐alanine in 1 N NaOH. Adjusted the pH to 7.4 and
made up to 1,000 ml with phosphate buffer, (c) standard pyruvate,
2 mM: 22 mg sodium pyruvate/100 ml phosphate buffer, (d) dinitro-
phenyl hydrazine reagent, 1 mmol/l in 1 M HCl, and (e) 0.4 N NaOH.
Reagents
(i) Citrate buffer: 0.1 M, pH 5. A: citric acid (21.01 g in 1,000 ml), B:
sodium citrate (29.41 g in 1,000 ml), 20.5 ml of A and 29.5 ml of B,
diluted to a total of 100 ml. (ii) Disodium phenyl phosphate, 100 mM:
dissolved 2.18 g in water, heated to boil, cooled and made to a liter.
Added 1.0 ml of chloroform and stored in the refrigerator. (iii) Buffer‐
substrate: prepared by mixing equal volume of the above two solutions.
This has a pH of 5.0. (iv) Folin‐Ciocalteu reagent: mixed 1.0 ml of
reagent with 2.0 ml of water. (v) Sodium carbonate solution, 15%:
dissolved 15 g of anhydrous sodium carbonate in 100 ml of water. (vi)
Standard phenol solution, 1 g/l: dissolved 19 g pure crystalline phenol in
100 mM HCl and made to a liter with the acid. (vii) Working standard
solution: diluted 10 ml of stock standard to 100 ml with water. This
contains 100 μg phenol/ml.
Procedure
To 0.2 ml of sample, added 1.0 ml of the buffered substrate and
incubated for 30 min at 37°C. To the control tubes, enzyme was
added after arresting the reaction with 1.0 ml of DNPH and the tube
was kept at room temperature for 20 min. Then 10 ml of 0.4 N NaOH
was added.
A set of standard pyruvate in the concentration
0.4–2.0 µM was also treated in a similar manner. The color developed
was read at 520 nm. The enzyme activity was expressed as µmol of
pyruvate liberated/l in serum and µmoles of pyruvate liberated/min/
mg protein in tissues.
Estimation of alkaline phosphatase[41]
|
4.2.5
Procedure
Principle
Pipetted out 4.0 ml of the buffered substrate into a test tube and
incubated at 37°C for 5 min. Added 0.2 ml of sample and incubated
further for exact 60 min, removed and immediately added 1.8 ml of
diluted phenol reagent. At the same time a control was set up
containing 4.0 ml buffered substrate and 0.2 ml of sample to which
1.8 ml of phenol reagent was added immediately. Mixed well and
centrifuged. To 4.0 ml of the supernatant added 2.0 ml of sodium
carbonate. Took 4.0 ml of working standard solution and for blank
taken 3.2 ml water and 0.8 ml of phenol reagent. Then added 2.0 ml
of sodium carbonate. Incubated all the tubes at 37°C for 15 min.
Read the color developed at 700 nm.
The method used was that of King and Armstrong in which disodium
phenyl phosphate is hydrolyzed with the liberation of phenol and
inorganic phosphate. The liberated phenol is measured at 700 nm
with Folin‐Ciocalteu reagent.
Reagents
(a) Sodium carbonate/sodium bicarbonate buffer, 100 mM, (b)
disodium phenyl phosphate, 100 mM, (c) buffered substrate: mix
equal volume of above two solutions (pH 10), (d) Folin‐Ciocalteu
reagent: mixed 1.0 ml of reagent with 2.0 ml of water, (e) sodium
carbonate solution, 15%, (f) standard: 1.0 g crystalline phenol/l in
0.1 M HCl, and (g) working standard: added 100 ml dilute phenol
reagent to 5.0 ml of stock standard and diluted to 500 ml with water.
This contained 10 µg phenol/ml.
The activity of serum ACP was expressed in μmol of phenol
liberated/l. The activity in tissue homogenate was expressed as μmol
of phenol liberated/min/mg protein.
ELISA of CEA and CA 15‐3[43]
|
4.2.7
Procedure
Pipetted out 4.0 ml of the buffered substrate into a test tube and
incubated at 37°C for 5 min. Added 0.2 ml of serum and incubated for
15 min. Removed and immediately added 1.8 ml of diluted phenol
reagent. A control was run simultaneously with 4.0 ml buffered
substrate and 0.2 ml sample to which 1.8 ml phenol reagent was
Quantitative estimation of tumor markers namely, CEA and CA 15‐3
was carried out by solid phase ELISA using the UBI MAGIWELL (USA)
enzyme immunoassay kit. The desired numbers of coated wells were
secured in the holder. Ten microliters of standards, controls, or serum
samples were then dispensed into appropriate wells. One well was

16 of 16
MEENAKSHISUNDARAM ET AL.
|
saved for the blank, to which standards or enzyme conjugate should
not be added. Fifty microliters of antibody solution was dispensed into
each well expect the blank well. The wells were then incubated for 1 hr
at room temperature. After incubation mixture the wells were rinsed
with working washing buffer (50 ml) five times. Then, 100 µl of
solution A (a phosphate buffer solution containing hydrogen peroxide)
and 100 µl solution B (tetramethylbenzidine solution) was added into
each well including the blank well. This was then incubated for 30 min
at room temperature, after which, the enzyme reaction was stopped
by the addition of 50 µl of stop reagent and the intensity of the color
was measured with microreader at 450 nm.
[19] T. Pillaiyar, M. Köse, K. Sylvester, H. Weighardt, D. Thimm, G.
Borges, I. Förster, I. von Kügelgen, C. E. Müller, J. Med. Chem. 2017,
60, 3636.
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1149.
[21] K. Shaari, K. C. Ling, Z. Mat Rashid, T. P. Jean, F. Abas, S. M. Raof, Z.
Zainal, N. H. Lajis, H. Mohamad, A. M. Ali, Mar. Drugs 2009, 7, 1.
[22] C. Liu, X. Tang, P. Li, G. Li, Org. Lett. 2012, 14, 1994.
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[24] M. S. Karthikeyan, D. J. Prasad, B. Poojary, K. Subrahmanya Bhat, B.
S. Holla, N. S. Kumari, Bioorg. Med. Chem. 2006, 14, 7482.
[25] B. M. Sahoo, S. C. Dinda, B. V. V. Kumar, J. Panda, P. S.
Brahmkshatriya, Lett. Drug Des. Discov. 2014, 11, 82.
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ACKNOWLEDGMENTS
[27] S. K. Guchhait, A. L. Chandgude, G. Priyadarshani, J. Org. Chem. 2012,
The authors thank IISc, Bangalore Hyderabad Central University for
NMR and mass analysis. Sangeetha Meenakshisundaram thank
Professor K.J. Rajendra Prasad, Department of Chemistry, Bharathiar
University for his invaluable support throughout this study.
77, 4438.
[28] Y. Özkay, I. Işıkdağ, Z. Incesu, G. Akalın, Eur. J. Med. Chem. 2010, 45,
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CONFLICT OF INTERESTS
The authors declare that there are no conflict of interests.
[32] C. Palmieri, G. J. Cheng, S. Saji, M. Zelada‐Hedman, A. Warri, Z.
Weihua, S. Van Noorden, T. Wahlstrom, R. C. Coombes, M. Warner,
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[36] Z. Wang, X. Zhang, Front. Pharmacol. 2017, 8, 1.
[37] B. K. Banin Hirata, J. M. M. Oda, R. LosiGuembarovski, C. B. Ariza, C.
E. C. Oliveira, M. A. E. Watanabe, Dis. Markers 2014, 2014, 1.
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[39] M. R. Boyd, The NCI In Vitro Anticancer Drug Discovery Screen,
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p. 23.
ORCID
Thanigaimalai Pillaiyar
http://orcid.org/0000-0001-5575-8896
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