Synthesis and biological evaluation of imidazo[1,5-a]pyridine-benzimidazole hybrids as inhibitors of both tubulin polymerization and PI3K/Akt pathway

Article abstract of DOI:10.1039/c4ob01930j

A series of imidazo[1,5-a]pyridine-benzimidazole hybrids (5a-aa) were prepared and evaluated for their cytotoxic activity against a panel of sixty human tumor cell lines. Among them compounds 5d and 5l showed significant cytotoxic activity with GI_50<

Full text of DOI:10.1039/c4ob01930j

Organic &
Biomolecular Chemistry
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Synthesis and biological evaluation of imidazo-
1,5-a]pyridine-benzimidazole hybrids as inhibitors
[
Cite this: DOI: 10.1039/c4ob01930j
of both tubulin polymerization and PI3K/Akt
pathway†
a,b
a
a
a
Ahmed Kamal,* A. V. Subba Rao, V. Lakshma Nayak, N. V. Subba Reddy,
b
a
b
Konderu Swapna, G. Ramakrishna and Mallika Alvala
A series of imidazo[1,5-a]pyridine-benzimidazole hybrids (5a–aa) were prepared and evaluated for their
cytotoxic activity against a panel of sixty human tumor cell lines. Among them compounds 5d and 5l
showed significant cytotoxic activity with GI50 values ranging from 1.06 to 14.9 µM and 0.43 to 7.73 µM,
respectively. Flow cytometric analysis revealed that these compounds arrest the cell cycle at G2/M phase
and induced cell death by apoptosis. The tubulin polymerization assay (IC50 of 5d is 3.25 µM and 5l is
1
.71 µM) and immunofluorescence analysis showed that these compounds effectively inhibited the micro-
tubule assembly in human breast cancer cells (MCF-7). Further, the apoptotic effects of compounds were
confirmed by Hoechst staining, mitochondrial membrane potential, cytochrome c release, ROS gene-
ration, caspase 9 activation and DNA fragmentation analysis. After treatment with these compounds for
48 h, p-PTEN and p-AKT levels were markedly decreased. Moreover, these compounds did not signifi-
cantly inhibit the normal human embryonic kidney cells, HEK-293. The molecular docking simulations
predicted the binding interactions of 5d and 5l with colchicine binding site of the tubulin, which is in
compliance with the antiproliferative activity data.
Received 11th September 2014,
Accepted 13th October 2014
DOI: 10.1039/c4ob01930j
www.rsc.org/obc
motility, cell signaling, formation and maintenance of cell
shape, secretion and intracellular transport. Therefore, micro-
Introduction
5
Cancer has been one of the major causes of death across the tubules have become an important target, and there has been
world for decades. The effectiveness of many existing anti- considerable interest in the discovery and development of
cancer drugs is limited because of their toxicity to normal small molecules that affect tubulin polymerization.
rapidly growing cells and that these cells may develop resist-
In recent years, different heterocyclic compounds have been
ance to these drugs. Another drawback is that a majority of the identified through molecular biology, empirical screening, and
drugs currently in the market are not specific. The microtubule rational drug design for the development of new anticancer
6
system of eukaryotic cells is essential for cell division because agents. Nitrogen-bridgehead fused heterocycles containing an
1
microtubules are the key components of the mitotic spindle.
imidazole ring are common structural components in several
Microtubules are in dynamic equilibrium with tubulin dimers pharmacologically important molecules that display a wide
as tubulin is assembled into microtubules, which in turn are range of activities for diverse number of targets. One of the
2
–4
disassembled to tubulin.
This dynamicity is characterized most widely used heterocyclic systems from this group is the
7
by the continuous turnover of α,β-tubulin heterodimers in the imidazopyridines, which show a broad spectrum of biological
polymeric microtubules. Thus, microtubules play an important activities such as inhibitors of aromatase, estrogen production
8
9
role in fundamental cellular processes, such as regulation of suppressors, positive inotropic agents, platelet aggregation
10
inhibitors, and thromboxane synthetase inhibitors. They
also exhibit antiviral
1
1,12
13
and antibacterial
properties.
a
Imidazopyridines exhibit anticancer activity through different
Medicinal Chemistry & Pharmacology, CSIR-Indian Institute of Chemical
Technology, Hyderabad 500 007, India. E-mail: ahmedkamal@iict.res.in;
Fax: (+)91-40-27193189; Tel: (+)91-40-27193157
molecular mechanisms such as vascular endothelial growth
factor (VEGF)-receptor KDR inhibition and the induction of
b
Department of Medicinal Chemistry, National Institute of Pharmaceutical Education
14,15
apoptosis.
Recently, we have reported that the synthesis of
and Research (NIPER), Hyderabad-500 037, India
imidazopyridine-oxindole derivatives (4) as apoptosis inducing
†
Electronic supplementary information (ESI) available. See DOI: 10.1039/
1
6
c4ob01930j
agents.
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Similarly, benzimidazoles are of great importance because of improved pharmacological profiles. The promising biological
their biological properties and have been reported to possess activity exhibited by these hybrids prompted us to develop
1
7–25
potential anticancer activity and anti-HIV activity,
apart some new hybrid molecules by linking the imidazopyridine
antifungal, antiviral and antioxidant pharmacophore with a benzimidiazole scaffold with a view to
Compounds such as nocodazole (NSC-238189) enhance their anticancer activity. In this context we have
26,27
from antibacterial,
2
7–31
activities.
(
3
2
33
34
1), FB642 (2), and Hoechst-33258 (3) are some biologically designed and synthesized imidazopyridine-benzimdiazole
active antitumor agents that possess a benzimidazole moiety. hybrids (5a–aa), which were evaluated for their anticancer
Some of the potent hybrid molecules, which have been potential in the Development Therapeutic Program (DTP) of
recently developed as new anticancer agents were obtained by the National Cancer Institute (NCI), USA, against a panel
3
5,36
the combination of different pharmacophores.
Benzimid- of sixty cell lines, apart from their effect on the inhibition of
azole attached to other heterocyclic moieties including fused tubulin polymerization and apoptotic cell death (Table 1)
rings resulted in compounds (hybrid molecules) with (Fig. 1).
Fig. 1 Lead structures of biologically active antitumor agents having benzimidazole scaffold: nocodazole (1), FB642 (2), Hoechst-33258 (3) and syn-
thesized in our laboratory imidiazopyridine derivative (4) as well as proposed library of imidazopyridine-benzimidazole hybrids (5a–aa).
Compound
R
1
R
2
R
3
Compound
R
1
R
2
R
3
Compound
R
1
R
2
R
3
5
5
5
5
5
5
5
5
5
a
b
c
d
e
f
g
h
i
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Cl
Cl
Me
5j
5k
5l
5m
5n
5o
5p
5q
5r
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
H
H
H
H
H
H
H
Cl
Cl
Me
5s
5t
CF
3
CF
3
CF
3
CF
3
CF
3
CF
3
CF
3
CF
3
CF
3
H
H
H
H
H
H
H
Cl
Cl
Me
OMe
Me
F
Cl
CF
F
OMe
Me
F
Cl
CF
F
OMe
Me
F
Cl
CF
F
5u
5v
5w
5x
5y
5z
5aa
3
3
3
Cl
Me
Cl
Me
Cl
Me
Org. Biomol. Chem.
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Paper
a
Table 1 In vitro anticancer activity (GI50 µM) of compounds 5d, 5f, 5k–p, and 5z on 60 human cancer cell lines
a
Growth inhibition (GI50 µM)
d
e
f
g
h
i
j
k
l
m
c
Cancer panel/cell line
5d
5f
5k
5l
5m
5n
5o
5p
5q
5z
Leukaemia
CCRF-CEM
HL-60(TB)
K-562
MOLT-4
RPMI-8226
SR
c
5.28
14.9
3.47
3.60
2.60
4.45
5.64
27.8
11.0
28.3
3.84
3.65
2.53
2.54
3.60
3.37
1.72
3.21
1.97
2.58
2.92
2.17
1.47
2.77
4.39
39.8
14.0
3.83
2.86
6.63
27.3
49.8
5.40
7.40
3.91
4.97
4.84
4.21
3.02
3.87
NAc
NA
NAc
NA
c
NA
b
c
35.3
2.81
NTc
NAc
c
NA
NA
NAc
b
3.55
12.6
NT
3.74
6.26
NA
NA
c
32.3
Non-small lung
A549/ATCC
HOP-62
b
2.86
1.94
1.73
1.80
3.54
3.18
2.07
2.43
3.12
2.46
3.66
10.9
3.21
3.35
2.93
2.99
3.17
5.07
2.31
6.57
10.26
3.22
3.23
2.89
2.67
3.72
0.416
3.88
3.85
2.38
2.28
1.95
3.96
7.41
3.67
2.72
1.87
9.53
6.83
4.49
1.82
1.96
2.19
1.94
7.52
5.63
2.23
4.24
28.5
38.0
4.04
19.8
3.24
14.5
29.1
6.87
5.43
4.39
2.35
1.81
1.82
2.19
NT
3.04
2.97
2.16
6.57
9.45
c
HOP-92
NA
b
NCI-H226
NCI-H23
NCI-H322M
NCI-H460
NCI-H522
Colon
COLO 205
HCC-2998
HCT-116
HCT-15
HT29
KM12
SW-620
CNS
SF-268
SF-295
SF-539
SNB-19
NTc
b
NAc
NA
NT
NT
b
b
9.73
3.01
3.37
NT
c
NA
10.6
c
b
c
3.42
4.11
2.00
4.29
3.25
3.31
3.97
4.88
5.98
1.85
3.50
3.47
5.11
3.17
7.19
3.96
3.63
3.76
3.75
3.88
4.26
3.23
2.93
3.26
2.83
3.36
2.38
3.12
7.81
6.60
3.61
3.36
7.13
7.03
2.24
3.61
18.6
2.41
3.89
3.66
7.68
4.07
NA
7.10
4.03
6.09
5.72
4.26
4.56
5.38
NTc
NAc
51.8
26.1
NA
NA
2.65
30.1
4.42
2.92
30.5
c
NA
b
58.2
36.4
NT
c
b
NA
NTc
c
NA
8.36
2.59
NA
c
3.28
5.63
3.85
1.69
6.44
1.68
1.98
4.53
2.80
7.20
2.93
NAc
9.11
4.19
3.90
2.08
6.88
1.45
3.13
b
b
b
b
b
b
b
NT
NT
NT
NT
NT
NA
NT
NT
c
2.32
2.37
2.09
2.22
9.71
5.26
24.0
4.42
3.32
2.97
5.49
3.27
1.04
1.12
3.40
1.36
1.76
3.60
1.87
3.16
NAc
39.8
8.35
5.31
6.67
1.86
2.64
1.73
3.05
NA
SNB-75
U251
48.1
c
NA
Melanoma
LOX IMVI
MALME-3M
M14
MDA-MB-435
SK-MEL-2
SK-MEL-28
SK-MEL-5
UACC-257
UACC-62
Ovarian
IGROV1
OVCAR-3
OVCAR-4
OVCAR-5
OVCAR-8
NCI/ADR-RES
SK-OV-3
Renal
b
4.86
2.07
3.83
2.59
2.85
2.13
1.55
2.16
1.96
1.89
2.71
17.7
5.47
1.49
9.22
1.90
13.1
3.19
4.80
13.3
3.74
3.14
4.07
1.85
1.60
4.39
4.51
3.03
2.83
3.15
2.30
2.51
4.61
1.39
2.41
2.14
10.8
5.32
12.9
8.05
10.1
22.8
2.20
9.15
5.41
2.85
2.35
4.24
2.30
2.71
2.14
4.27
2.63
4.89
31.3
NA
5.36
20.0
51.5
3.80
6.86
3.60
2.51
4.92
5.77
NT
c
3.56
b
83.1
47.0
NT
NT
b
c
b
NAc
NT
NT
9.49
c
b
b
NA
NA
NT
47.0
2.22
3.15
6.05
2.91
5.77
5.59
4.20
c
c
NA
NA
51.1
15.6
c
1.95
4.29
1.93
3.07
1.87
2.23
2.40
4.03
4.08
1.81
6.77
2.70
3.71
1.86
2.70
4.97
4.53
11.4
2.53
3.86
36.1
2.18
2.99
3.81
4.69
2.05
2.71
6.95
6.63
6.14
6.92
4.52
3.76
6.50
10.3
4.95
2.61
1.89
7.33
2.40
2.78
2.24
NAc
4.91
8.75
7.93
3.73
3.18
2.00
16.1
3.74
1.87
NA
b
NTc
c
c
c
NA
NA
NA
NA
22.7
85.3
3.45
5.50
2.53
3.90
2.65
6.61
6.10
2.74
c
c
NA
NA
c
786-0
2.23
2.08
2.25
3.38
4.50
3.25
1.75
2.99
1.92
1.69
2.23
5.07
2.59
2.86
4.36
2.06
8.47
4.28
3.57
3.81
3.91
2.65
2.47
24.2
3.67
2.47
2.76
2.83
3.08
3.39
1.67
7.73
8.82
3.83
4.28
12.1
13.3
16.1
6.99
6.05
1.60
5.61
1.86
2.09
3.75
2.01
1.74
1.94
NAc
8.94
2.31
7.34
5.35
11.2
5.25
2.62
4.65
1.79
4.20
2.61
3.32
2.19
4.58
3.90
A498
ACHN
CAKI-1
SN12C
TK-10
UO-31
RXF 393
Prostate
PC-3
DU-145
Breast
NA
63.5
c
b
NAc
NTc
c
NAc
NAc
NA
NA
NA
2.36
1.87
1.76
8.15
b
NT
30.5
2.26
b
1.82
5.56
4.11
7.50
3.01
4.11
1.88
2.85
6.14
10.7
2.13
3.27
4.73
4.58
6.58
2.58
NT
NT
c
b
b
NA
NT
b
MCF7
1.37
2.45
3.56
2.76
1.06
1.49
2.40
2.04
3.65
4.23
1.71
2.02
3.40
5.65
2.57
3.20
2.70
4.08
2.70
3.09
2.17
2.55
2.27
2.32
6.15
4.34
8.54
4.84
3.55
4.12
3.15
2.77
2.76
2.74
3.66
1.99
26.3
50.6
4.57
5.82
12.2
4.91
3.53
2.93
7.20
2.79
2.68
3.08
4.06
5.04
NT
MDA-MB231/ATCC
HS 578T
BT-549
T-47D
MDA-MB-468
2.49
3.99
8.69
4.05
3.05
c
NAc
NA
5.33
13.4
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Table 1 (Contd.)
a
Growth inhibition (GI50 µM)
d
e
f
g
h
i
j
k
l
m
Cancer panel/cell line
5d
5f
5k
5l
5m
5n
5o
5p
5q
5z
Non-small lung
A549/ATCC
HOP-62
b
2.86
1.94
1.73
1.80
3.54
3.18
2.07
2.43
3.12
2.46
3.66
10.9
3.21
3.35
2.93
2.99
3.17
5.07
2.31
6.57
10.26
3.22
3.23
2.89
2.67
3.72
0.416
3.88
3.85
2.38
2.28
1.95
3.96
7.41
3.67
2.72
1.87
9.53
6.83
4.49
1.82
1.96
2.19
1.94
7.52
5.63
2.23
4.24
28.5
38.0
4.04
19.8
3.24
14.5
29.1
6.87
5.43
4.39
2.35
1.81
1.82
2.19
NT
3.04
2.97
2.16
6.57
9.45
c
HOP-92
NA
b
NCI-H226
NCI-H23
NCI-H322M
NCI-H460
NCI-H522
Colon
COLO 205
HCC-2998
HCT-116
HCT-15
HT29
KM12
SW-620
CNS
SF-268
SF-295
SF-539
SNB-19
NTc
b
NAc
NT
NT
b
NA
b
9.73
3.01
3.37
NT
c
NA
10.6
c
b
c
3.42
4.11
2.00
4.29
3.25
3.31
3.97
4.88
5.98
1.85
3.50
3.47
5.11
3.17
7.19
3.96
3.63
3.76
3.75
3.88
4.26
3.23
2.93
3.26
2.83
3.36
2.38
3.12
7.81
6.60
3.61
3.36
7.13
7.03
2.24
3.61
18.6
2.41
3.89
3.66
7.68
4.07
NA
7.10
4.03
6.09
5.72
4.26
4.56
5.38
NTc
NAc
51.8
26.1
NA
NA
2.65
30.1
4.42
2.92
30.5
c
NA
b
58.2
36.4
NT
c
b
NA
NTc
c
NA
8.36
2.59
NA
c
3.28
5.63
3.85
1.69
6.44
1.68
1.98
4.53
2.80
7.20
2.93
NAc
9.11
4.19
3.90
2.08
6.88
1.45
3.13
b
b
b
b
b
b
b
NT
NT
NT
NT
NT
NA
NT
NT
c
2.32
2.37
2.09
2.22
9.71
5.26
24.0
4.42
3.32
2.97
5.49
3.27
1.04
1.12
3.40
1.36
1.76
3.60
1.87
3.16
NAc
39.8
8.35
5.31
6.67
1.86
2.64
1.73
3.05
NA
SNB-75
U251
48.1
c
NA
Melanoma
LOX IMVI
MALME-3M
M14
MDA-MB-435
SK-MEL-2
SK-MEL-28
SK-MEL-5
UACC-257
UACC-62
b
4.86
2.07
3.83
2.59
2.85
2.13
1.55
2.16
1.96
1.89
2.71
17.7
5.47
1.49
9.22
1.90
13.1
3.19
4.80
13.3
3.74
3.14
4.07
1.85
1.60
4.39
4.51
3.03
2.83
3.15
2.30
2.51
4.61
1.39
2.41
2.14
10.8
5.32
12.9
8.05
10.1
22.8
2.20
9.15
5.41
2.85
2.35
4.24
2.30
2.71
2.14
4.27
2.63
4.89
31.3
NA
5.36
20.0
51.5
3.80
6.86
3.60
2.51
4.92
5.77
NT
c
3.56
b
83.1
47.0
NT
NT
b
c
b
NAc
NT
NT
9.49
c
b
b
NA
NA
NT
47.0
2.22
3.15
6.05
2.91
5.77
5.59
4.20
c
c
NA
NA
51.1
15.6
a
b
c
Values are reported as GI50, the µM concentration of the compound required to cause 50% inhibition of cell growth. NT: Not Tested. NA: Not
d
e
f
g
h
i
j
k
Active. 5d (NSC 766340). 5f (NSC 774952). 5k (NSC 766330). 5l (NSC 766338). 5m (NSC 766325). 5n (NSC 766332). 5o (NSC 766331). 5p
l
m
(NSC 766333). 5q (NSC 766337). 5z (NSC 774957).
Institute (NCI), for their anticancer activity at a single concen-
Chemistry
tration of 10− M against a panel of sixty cancer cell lines.
The synthesis of the imidazo[1,5-a]pyridine-benzimidazole hybrids The human tumor cell lines were derived from nine different
5a–aa) was accomplished as described in Scheme 1. Differently cancer types: leukemia, melanoma, lung, colon, CNS, ovarian,
5
37–39
(
substituted benzoylchlorides (7a–c) were reacted with 2-pyridyl- renal, prostate and breast cancers. The compounds were
methanamine (6) in presence of the base triethylamine to give N1- added at a single concentration and the cell cultures were
(
2-pyridylmethyl)-substituted benzamides (8a–c), which were incubated for 48 h and the end point determinations were
cyclized to corresponding imidazo[1,5-a]pyridines (9a–c) by reflux- made with a protein binding dye, sulforhodamine B (SRB).
ing in POCl . The formylation of these imidazo[1,5-a]pyridine Among the 17 compounds tested, ten compounds exhibited
3
derivatives (9a–c) under Vilsmeier–Haack conditions with POCl in significant growth inhibition at a single dose of 10 µM concen-
3
dry DMF yielded the intermediates 3-(substituted phenyl)imidazo- tration, which were further evaluated against the sixty cell line
[1,5-a]pyridine-1-carbaldehydes (10a–c). The reaction of (10a–c) panel at a five dose concentration, and the results are summar-
with substituted o-phenylenediamines (11a–i) in ethanol using ized in Table 1. From the five dose assay results, it is observed
sodium metabisulphate afforded the final benzimidiazole-derived that most of the tested compounds exhibited significant anti-
imidazo[1,5-a]pyridines (5a-aa) in excellent yields.
proliferative activity with distinct selectivity on different cell
lines. Two compounds 5d and 5l emerged as hit compounds,
which show remarkable growth inhibition on all the tested cell
line with GI50 values in the range of 1.06–14.9 µM and
Biological evaluation
Cytotoxic activity
0.46–7.73 µM, respectively.
All the newly synthesized imidazo[1,5-a]pyridine-benzimid-
The in vitro cytotoxicity values of these ten selected com-
azole hybrids (5a–aa) were evaluated by the National Cancer pounds allow a rudimentary picture of the structure–activity
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Scheme 1 Reagents and conditions: (a) TEA, dry THF, 0 °C to rt, 3 h; (b) POCl
reflux, 8 h.
3 3 2 2 4
, reflux, 3 h; (c) DMF, POCl , 0 °C to 100 °C, 3 h; (d) Na S O , EtOH,
Fig. 2 SAR of selected compounds 5f, 5l and 5o.
relationship (SAR) to be drawn. Four compounds (5d, 5f, 5l pattern for individual compounds with the same binding site,
and 5n) exhibited better cytotoxicity profiles when compared a correlation of the cytotoxicity to substituents (R , R and R )
1
2
3
to the other congeners. It is interesting to note that the benz- on the compounds was not attempted at this stage.
imidazole half of these four compounds carriesa single substi-
Specific killing of cancer cells without affecting normal cell
tuent (R ≠ H, R = H), which is the benzenoid subunit. A growth is a key safety feature of cancer chemotherapy. There-
2
3
comparison of the most inactive member of the series 5o (R
1
=
fore, these compounds (5d and 5l) were evaluated for possible
OMe, R2 = CF , R = H) with the significantly active com- cytotoxicity towards normal human embryonic kidney cells,
3
3
pounds 5f (R = H, R = CF , R = H) and 5l (R = OMe, R = HEK-293 by MTT assay. The assay results suggested that these
1
2
3
3
1
2
3
Me, R = H) reveals certain interesting observations (Fig. 2). 5o compounds did not significantly (compound 5d and 5l
and 5f differ only in the nature of the R substituent (OMe and showed 75.85 and 97.72 µM IC50 values respectively) affect the
1
H, respectively). 5l has the same R
but a different R substituent (Me instead of CF
near-complete loss of activity for 5o cannot be attributed to
the individual substituents R or R , but it may rather be the The data from MTT assay showed that the test compounds 5d
1
substituent (OMe) as 5o growth of normal human embryonic kidney cells, HEK-293.
2
3
). Thus, the
Cell cycle analysis
1
2
result of a different binding pattern for individual compounds and 5l induced the significant inhibition of breast cancer cells
to their cellular targets. Some evidence in support of this infer- with IC50 values 1.79 and 2.85 µM, respectively. It was of inter-
ence has been obtained in the docking experiments, in which est to examine cell-cycle alterations caused by these conjugates
4
0
two active compounds 5d and 5l showed very different binding in MCF-7 cancer cells to understand the phase distribution.
poses with the colchicine binding site of tubulin (vide infra). Thus, cells were treated with these conjugates at concen-
In view of the possibility of such an opportunistic binding trations of 1 and 3 µM for 48 h, and the results indicated that
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Fig. 3 Flow cytometric analysis in MCF-7 breast cancer cell line; MCF-7 cells were treated with the 5d and 5l at concentrations of 1 and 3 µM for
8 h.
4
these compounds showed cell cycle arrest in G2/M phase at 1 (Fig. 4). Tubulin polymerization inhibition was also observed
µM concentration (Fig. 3). Interestingly, when the concen- in case of standard nocodazole (66.98%). Furthermore, these
tration was increased from 1 µM to 3 µM, it was observed that conjugates (5d, 5l and nocodazole) were evaluated for their
the percentage of accumulation of the cells in sub G1 phase inhibition of tubulin polymerization assays at different con-
4
1
increased, which indicates the onset of apoptosis.
centrations. It was observed that compound 5l showed potent
inhibition of tubulin polymerization with an IC50 value
Inhibition of tubulin polymerization
1.71 µM, which is similar to that of nocodazole (1.75 µM),
Compounds that alter cell-cycle parameters with preference to whereas compound 5d inhibited with an IC50 value of 3.25 µM
the G2/M blockade are known to exhibit effects on tubulin as shown in Table 2.
assembly. Moreover, the inhibition of tubulin polymerization
4
2
Immunohistochemistry of tubulin
is strongly associated with G2/M cell-cycle arrest. These com-
pounds arrest the cell cycle at G2/M phase at 1 µM concen- In addition to in vitro tubulin polymerization studies, we
tration, hence, it was of considerable interest to investigate the investigated alterations in the microtubule network induced by
tubulin polymerization aspect. As tubulin subunits hetero- the compounds 5d and 5l in MCF-7 cells by the immunofluore-
dimerize and self-assemble to form microtubules in a time sence microscopy of immunohistochemistry studies, as most
4
4
dependent manner, we have investigated the progression of antimitotic agents affect microtubules. Therefore, MCF-7
4
3
tubulin polymerization by monitoring the increase in fluo- cells were treated with compounds 5d and 5l at 1 µM concen-
rescence emission at 420 nm (excitation wavelength is 360 nm) tration for 48 h. Nocodazole was used as a reference com-
in 384 well plate for 1 h at 37 °C with and without the com- pound in this study. Results revealed that untreated human
pounds at 3 µM concentration.
breast cancer cells displayed the normal distribution of micro-
Compounds, 5d and 5l inhibited tubulin polymerization by tubules (Fig. 5). However, cells treated with these compounds
7.92% and 71.27%, respectively, compared to the control (5d and 5l), as well as nocodazole showed disrupted micro-
4
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Fig. 5 IHC analysis of compounds on the microtubule network: MCF-7
cells were treated with compounds 5d, 5l and nocodazole at 1 µM con-
centration for 48 h.
Fig. 4 Effect of compounds on tubulin polymerization: tubulin
polymerization was monitored by the increase in fluorescence at
360 nm (excitation) and 420 nm (emission) for 1 h at 37 °C. All the com-
pounds were included at a final concentration of 3 µM. Nocodazole was
used as a positive controls. Values indicated are the mean ± SD of two
different experiments performed in triplicates.
Table 2 Inhibition of tubulin polymerization (IC50) of compounds 5d
and 5l
a
Compound
IC50 ± SD (µM)
5
5
d
l
3.25 ± 0.42
1.71 ± 0.08
1.75 ± 0.08
Nocodazole
Fig. 6 Hoechst staining in MCF-7 breast cancer cell line.
a
Concentration of drug to inhibit 50% of tubulin assembly.
mitochondrial membrane potential (ΔΨm), are key events that
take place during drug-induced apoptosis. To determine the
changes in mitochondrial membrane potential (ΔΨm), we
examined the lipophilic dye JC-1 specific for mitochondria.
Mitochondria that maintain a normal mitochondrial mem-
brane potential (ΔΨm) concentrate JC-1 into aggregates, indi-
cated by red fluorescence.
However, JC-1 forms monomers (green fluorescence) in
depolarized mitochondria. MCF-7 cells were treated with com-
pounds 5d and 5l at 1 µM concentration and incubated for
tubule organization, as shown in Fig. 5, thus, demonstrating
the inhibition of tubulin polymerization.
Hoechst staining for apoptosis
Apoptosis is one of the major pathways that lead to cell death,
wherein chromatin condensation and DNA fragmentation are
known as the classic characteristics of apoptosis. It was of con-
siderable interest to investigate the apoptotic inducing effects of
these compounds (5d and 5l) by Hoechst staining (H 33258) in
MCF-7 cancer cells. Therefore, cells were treated with these com-
pounds at 1 µM concentration for 48 h. On the basis of chromatin
condensation, fragmented nuclei, the presence of apoptotic
bodies, and relative fluorescence of the test compounds (5d and
48 h. After 48 h of treatment with these compounds, a
decrease in the mitochondrial membrane potential (ΔΨm)
when compared to untreated control (Fig. 7) was observed.
5l) revealed a significant increase in the percentage of apoptotic
cells relative to that of untreated control cells (Fig. 6).
Cytochrome c release
An important consequence of apoptotic pathway is the mito-
Measurement of mitochondrial membrane potential (ΔΨm)
46,47
chondrial dysfunction and cytochrome c release.
In this
The maintenance of mitochondrial membrane potential context, human breast cancer cells, MCF-7, were treated with
ΔΨm) is significant for mitochondrial integrity and bio ener- test compounds (5d and 5l) at 1 µM concentration for 48 h
(
4
5
getic function. Mitochondrial changes, including the loss of and a Western blot analysis was performed to examine the
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Fig. 9 Effect of compounds 5d and 5l on caspase-9 activity; MCF-7
breast cancer cells were treated with these compounds at 2 µM concen-
tration for 48 h. Values indicated are the mean ± SD of two different
experiments performed in triplicates.
Fig. 7 Figure represents the loss of mitochondrial membrane potential
in human breast cancer cells, MCF-7 after 48 h of drug treatment with
compounds 5d and 5l at 1 µM concentration.
effect on cytochrome c levels. After treatment with these
compounds (5d and 5l), it was observed that there was an
increase in the cytochrome c protein levels (Fig. 11), suggesting
pared to the untreated control (Fig. 9), which suggests that they
have the ability to induce cell death by apoptosis in MCF-7 cells.
that the cytosolic cytochrome c release from mitochondria DNA fragmentation assay
might be responsible for the induction of apoptosis by these
compounds in the human breast cancer cells (MCF-7).
DNA laddering was carried out in order to elucidate the mode
of action of these compounds especially for their ability to
induce oligonucleosomal DNA fragmentation (DNA ladder),
which is a characteristic feature of the programmed cell death
Effect on intracellular ROS generation
5
1,52
Many anticancer agents have demonstrated to exert their cyto-
or apoptosis.
During apoptosis, DNA is cleaved into
toxic effects by the generation of reactive oxygen species
smaller fragments by endonucleases that can be observed by
gel electrophoresis as ladders. MCF-7 cells were treated with
these compounds (5d and 5l) at 1 µM concentration for 48 h
and DNA was isolated from these cells. The DNA was run on
48,49
(ROS),
which is considered as one of the key mediators of
apoptotic signaling. Therefore, we decided to investigate the
role of these compounds (5d and 5l) in inducing the production
of ROS that could potentially lead to the cytotoxic effect in the
MCF-7 cells. This production of ROS was examined by using an
oxidant-sensitive fluorescent probe, DCFDA (2′,7′dichlorofluor-
escin diacetate). After treatment with these compounds at a 1
µM concentration, it was observed that 5d and 5l significantly
increased the production of ROS in MCF-7 cells (Fig. 8).
2% agarose gel electrophoresis after staining with ethidium
bromide under UV illumination. It was observed that these
produced significant DNA fragmentation (Fig. 10), which is
indicative of apoptosis.
Western blot analysis
The PI3K pathway plays a key role in the cell signal transduc-
tion. AKT is the downstream target of PI3K, which controls
The activation of caspases plays an important role in the angiogenesis and tumor growth. The PTEN genesis is fre-
process of programmed cell death or apoptosis. Caspases, or quently mutated or inactivated in a high proportion of human
cysteine-aspartic protease, are a family of cysteine proteases,
Activation of caspase 9
5
3
which are crucial mediators of apoptosis. The MCF-7 cells lack
endogenous caspase-3, whereas caspase-9 plays an important
5
0
role in mediating drug-induced apoptosis.
MCF-7 cells were treated with these compounds (5d and 5l)
at 2 µM concentration for 48 h. The results demonstrate that
there was a 2–3 fold induction in caspase-9 activity when com-
Fig. 8 The effect of 5d and 5l on the ROS production in human breast
cancer cells (MCF-7).
Fig. 10 DNA fragmentation assay: Lane-1: Marker (100 bp), Lane-2:
Control DNA, Lane-3: 5d-1 µM and Lane-4: 5l-1 µM.
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these compounds’ inhibition of cancer-cell growth may be via
the inhibition of PI3K/AKT signaling pathway.
Molecular modelling studies
The crystal co-ordinates of αβ-tubulin subunits were taken
5
7
from the protein data bank (PDB ID: 3E22). As the colchicine
binds at the interface of αβ-tubulin subunits, both the chains
were considered for molecular docking studies. The multi-step
Schrödinger’s (Schrödinger L.L.C., USA) protein preparation
tool (PPrep) has been used for the final preparation of receptor
model. PPrep neutralizes side chains and residues, which are
not involved in salt bridges. Then, this step is followed by
restrained minimization using the OPLS 2005 force field to
RMSD of 0.3 Å. The synthesized compounds 5d and 5l were
sketched by using 2D sketcher and prepared for docking using
5
8
Fig. 11 Effect of compounds on p-Akt, p-PTEN level and Cytochrome Ligprep, a module of Schrödinger, and a total of 10 confor-
c release. MCF-7 cells were treated with compounds 5d and 5l at 1 µM mations were generated for 5d and 5l. An active site pocket of
concentration for 48 h. The cell lysates were collected and the
expression levels of p-Akt, p-PTEN and Cytochrome c release were
of X: −187.5857; Y: −54.3614; Z: −39.887, and the GLIDE (3)-XP
determined by Western blot analysis.β-actin was used as a loading
colchicine was considered for grid generation with coordinates
59
(
Extra Precision) flexible program was used for docking.
control.
Molecular docking studies revealed that these compounds
(
5d and 5l) bind to the colchicine binding site of the tubulin
with a binding affinity of −6.58 and −7.00, respectively, com-
cancers, including up to 30% of breast cancers, resulting in pared to colchicine (−5.76). The NH group of the benzimid-
5
4
hyper-activation of the PI3K/Akt signaling pathway. Increas- azole ring and nitrogen of imidazopyridine group in 5d
ing PTEN expression and down-regulating PTEN phosphoryl- showed a hydrogen bond interaction with Asn: A: 101 at dis-
5
5
ation are important steps in retarding tumor growth.
tance of 1.99 Å and 2.14 Å, respectively (Fig. 12). In case of 5l,
Therefore, controlling the PTEN or AKT signal pathway may be the NH group of the benzimidazole ring showed hydrogen
5
6
an important anticancer mechanism. In order to understand bond interactions with aminonitrogen of Asn: A: 101 at dis-
the effect of these compounds on phosphorylated levels of tance of 2.04 Å and substitution with a methoxy phenyl group
p-PTEN and p-Akt, we performed a western blot analysis. In contributed to the hydrogen bond interactions with the
this study, MCF-7 cells were treated with these compounds (5d hydroxyl group of Ser: A: 140 at a distance of 2.09 Å, which is
and 5l) at 1 µM concentration for 48 h. After treatment with responsible for the flipping of the structure wherein the orien-
these compounds, phospho-PTEN and phospho-Akt levels tation of benzimidazole group is on opposite side of the
were significantly reduced (Fig. 11). This result suggests that imidazopyridine core. This orientation is fruitful for extensive
Fig. 12 Docking pose and interacting aminoacids of compound 5d, 5l. Green dotted lines: hydrophobic interaction, pink dotted lines: hydrogen
bond interaction.
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interactions, such as hydrophobic interactions with Tyr: A: Coupling constants are reported in hertz (Hz). Melting points
24, Leu: B: 248, Ala: B: 316 and Leu: B: 255, within 3 Å dis- were determined in a capillary tube using an Electrothermal
2
tance of the active site pocket with the amino acid residues apparatus (Model IA9200) and are uncorrected. The IR spectra
present in the protein cavity. Therefore, substitution with were recorded by employing a Nicolet FTIR model MX-1
methoxyphenyl in 5l resulted in improved docking score, spectrophotometer. Analytical thin layer chromatography
which contributed for the enhanced antiproliferative activity. (TLC) was performed on MERCK precoated silica gel 60-F254
Hence, from the molecular modelling studies it is clear that (0.5 mm) glass plates. The visualization of the spots on TLC
compound 5l exhibits significant binding with colchicine plates was achieved either by exposure to iodine vapour or UV
binding site of the tubulin and this correlates well with the light or by dipping the plates into methanolic sulphuric acid-
observed antiproliferative activity.
β-naphthol, ethanolic anisaldehyde-sulphuric acid–acetic acid,
or ethanolic ninhydrin solution and heating the plates to
120 °C. Column chromatography was performed using silica
gel 60–120 and 100–200 mesh. Moisture sensitive reactions
were carried out using standard syringe septum techniques
Conclusion
In conclusion, a series of imidazo[1,5-a]pyridine-benzimid- and under an inert atmosphere of nitrogen. All the solvents
azole hybrids (5a–5aa) were synthesized and evaluated for and reagents were purified by standard techniques. O-Phenyl-
their anticancer activity against a panel of sixty human tumor ene di-amines (11a–i) and substituted benzoyl chlorides (7a–c)
cell lines derived from nine different cancer types: leukemia, are commercially available and were used as received. All the
lung, colon, CNS, melanoma, ovarian, renal, prostate and solvents were evaporated under reduced pressure on Laborota-
breast. Among them, ten compounds (5d, 5f, 5k–p, and 5z) 4000 rotary evaporator below 45 °C. The names of all the com-
with promising activity were taken up for a five dose concen- pounds given in the Experimental section were taken from
tration assay by the NCI. Interestingly some of these com- Chem Ultra, Version 11.0.
pounds exhibited promising anticancer activity, and two of
them (5d and 5l) significantly showed GI50 values ranging
from 1.06 to 14.9 µM and 0.43 to 7.73 µM, respectively, against
General procedure for the synthesis of N1-(2-pyridylmethyl)
substituted benzamides (8a–c)
the cell lines tested. FACS analysis revealed that these com-
Method A. To a stirred solution of 2-pyridylmethanamine 6
pounds arrest the cell cycle at G2/M phase and induced cell (1 mmol) in dry THF was added triethylamine (3.0 mmol) fol-
death by apoptosis. These potential compounds (5d and 5l) lowed by substituted benzoylchlorides 7a–c (1.1 mmol) at 0 °C.
showed significant inhibition of tubulin polymerization with The reaction mixture was stirred for 3 h and the reaction was
IC50 values 3.25 and 1.75 µM, respectively. Immunohistochem- monitored by TLC. After the completion of the reaction, THF
istry study results also suggest that these compounds disrupt was removed under vacuum to get the crude products. This
the microtubule assembly in MCF-7 cells. The induction of was further purified by column chromatography (EtOAc–
apoptosis was further confirmed by Hoechst staining, loss of Hexane) to obtain the pure compounds (8a–c).
mitochondrial membrane potential, cytochrome c release, ROS
N1-(2-Pyridylmethyl) benzamide (8a). This compound was
generation, activation of caspase 9 and DNA fragmentation prepared according to the method A, employing benzoylchlor-
1
analysis. Moreover, these compounds significantly suppressed ide 7a (1 mmol) to obtain the pure product 8a. Yield: 76%; H
3
the expression levels of p-Akt and p-PTEN. Overall, the current NMR (300 MHz, CDCl ): δ 8.53 (d, J = 4.7 Hz, 1H), 7.88 (d, J =
study demonstrates that the synthesis of imidazo[1,5-a]pyri- 6.4 Hz, 2H), 7.67 (t, J = 7.7 Hz, 2H), 7.47–7.39 (m, 3H), 7.32 (d,
dine-benzimidazole hybrids as promising cytotoxic agents J = 7.9 Hz, 1H), 7.20 (t, J = 5.0 Hz, 1H), 4.73 (d, J = 4.7 Hz, 2H);
+
with apoptotic-inducing ability by inhibiting both tubulin MS (ESI): m/z 213 [M + H] .
polymerization, as well as PI3K/Akt pathway. Moreover, these
N1-(2-Pyridylmethyl)-4-methoxybenzamide (8b). This com-
compounds did not affect the normal human embryonic pound was prepared according to the method A, employing
kidney cells (HEK-293), suggesting that these molecules selec- 4-methoxy benzoylchloride 7b (1 mmol) to obtain the pure
1
tively inhibits the growth of cancer cells.
product 8b. Yield: 80%; H NMR (300 MHz, CDCl ): δ 8.56 (d,
3
J = 4.8 Hz, 1H), 7.83 (d, J = 8.7 Hz, 2H), 7.68 (t, J = 7.8 Hz, 1H),
7
6
.47 (brs, 1H), 7.32 (d, J = 7.8 Hz, 1H), 7.21 (t, J = 5.8 Hz, 1H),
.93 (d, J = 8.7 Hz, 2H), 4.75 (d, J = 4.8 Hz, 2H), 3.85 (s, 3H);
Experimental
Chemistry
+
MS (ESI): m/z 243 [M + H] .
N-(Pyridin-2-ylmethyl)-4-(trifluoromethyl)benzamide
1
Materials and methods. H NMR spectra were recorded on (8c). This compound was prepared according to the method A,
Avance 300, Inova 400, Avance 500, and Bruker 600 MHz employing 4-trifluoromethyl benzoylchloride 7c (1 mmol) to
spectrometers using tetramethyl silane (TMS) as the internal obtain the pure product 8c. Yield: 77%; H NMR (300 MHz,
1
standard. Chemical shifts are reported in parts per million CDCl ): δ 8.51 (d, J = 5.2 Hz, 1H), 8.15 (brs, 1H), 7.96 (d, J = 7.5
3
(
ppm) downfield from tetramethyl silane. Spin multiplicities Hz, 2H), 7.63 (d, J = 8.3 Hz, 2H), 7.30 (d, J = 7.5 Hz, 1H), 7.19
are described as s (singlet), brs (broad singlet), d (doublet), dd (q, J = 5.2, 1.5 Hz, 1H), 4.71 (d, J = 4.5 Hz, 2H); MS (ESI): m/z
+
(
double doublet), t (triplet), q (quartet), and or m (multiplet). 281 [M + H] .
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General procedure for the synthesis of substituted
3-(4-(Trifluoromethyl)phenyl)imidazo[1,5-a]pyridine-1-carb-
phenylimidazo[1,5-a]pyridine (9a–c)
aldehyde (10c). This compound was prepared according to
1
the method C. Yield: 80%; H NMR (300 MHz, CDCl
3
): δ 10.16
Method B. To N1-(2-pyridylmethyl)-substituted benzamides
(
s, 1H), 8.42–8.38 (m, 2H), 7.95 (d, J = 8.3 Hz, 2H), 7.83 (d, J =
(
8a–c) (1 mmol), 4 mL of POCl was added and refluxed for
3
8
7
.3 Hz, 2H), 7.34–7.29 (q, J = 6.0, 3.0 Hz, 1H), 6.97 (t, J =
.5 Hz, 1H); MS (ESI): m/z 291 [M + H] .
3
h. This was poured into cold water and neutralized with
NaHCO solution. This water layer was extracted three times
with ethylacetate. The combined organic phases were dried
over anhydrous Na SO and evaporated under vacuum. Thus,
+
3
General procedure for the synthesis of compounds (5a–aa)
2
4
Method D. To a solution appropriate aldehyde (10a–c) in
ethanol (10 mL), appropriate o-phenylene diamines (11a–c)
the residue obtained was purified by column chromatography
using ethylacetate and hexane as solvent system to afford the
pure compounds (9a–c).
(
H
1 mmol) and sodium meta bisulphate (2 mmol) dissolved in
O (5 mL) were sequentially added, and then the reaction
2
3
-Phenylimidazo[1,5-a]pyridine (9a). This compound was
1
mixture was refluxed for 4 h. After the completion of reaction,
solvent was removed by vacuo to obtain crude product, which
was extracted with ethyl acetate. The combined organic
prepared according to the method B. Yield: 78%; H NMR
300 MHz, CDCl ): δ 8.24 (d, J = 7.5 Hz, 1H), 7.76 (d, J = 6.7 Hz,
H), 7.50–7.36 (m, 5H), 6.67 (dd, J = 9.0, 6.7 Hz, 1H), 6.51 (t,
(
3
2
+
2 4
extracts were dried over anhydrous Na SO and concentrated
J = 6.7 Hz, 1H); MS (ESI): m/z 195 [M + H] .
-(4-Methoxyphenyl)imidazo[1,5-a]pyridine (9b). This com-
to obtain crude products, which were further purified by
column chromatography using ethylacetate and hexane as
solvent system to afford pure compounds (5a–aa).
3
pound was prepared according to the method B. Yield: 80%;
1
H NMR (300 MHz, CDCl ): δ 8.34 (d, J = 8.7 Hz, 1H), 8.32 (d,
3
2-(3-Phenylimidazo[1,5-a]pyridin-1-yl)-1H-benzo[d]imidazole
J = 6.8 Hz, 1H), 7.71 (d, J = 8.7 Hz, 2H), 7.32–7.17 (m, 1H), 7.07
(
5a). This compound was prepared according to the method
(
3
d, J = 8.7 Hz, 2H), 6.98 (s, 1H), 6.88 (t, J = 6.8 Hz, 1H), 3.90 (s,
1
+
D. Yield (166 mg, 78%); Yellow solid, mp: 205–210 °C; H NMR
300 MHz, CDCl ): δ 10.29 (br s, 1H), 8.62 (d, J = 9.2 Hz, 1H),
H); MS (ESI): m/z 225 [M + H] .
-(4-(Trifluoromethyl)phenyl)imidazo[1,5-a]pyridine
(
3
3
(9c).
8.34 (d, J = 7.1 Hz, 1H), 7.79 (d, J = 6.9 Hz, 3H), 7.6–7.47 (m,
This compound was prepared according to the method
1
4H), 7.23 (dd, J = 5.6, 2.6 Hz, 2H), 7.05 (dd, J = 9.6, 6.6 Hz, 1H),
B. Yield: 78%; H NMR (300 MHz, CDCl ): δ 8.28 (d, J = 7.5 Hz,
3
1
3
6
.75 (td, J = 7.3, 1.3 Hz, 1H); C NMR (75 MHz, DMSO): δ
1
1
H), 7.91 (d, J = 8.3 Hz, 2H), 7.73 (d, J = 8.3 Hz, 2H), 7.54 (s,
H), 7.47 (d, J = 9.0 Hz, 1H), 6.73 (q, J = 6.7, 2.2 Hz, 1H), 6.59
148.44, 143.50, 138.34, 129.27, 129.11, 129.13, 129.01, 128.92,
+
128.14, 127.99, 122.27, 122.07, 121.78, 120.80, 114.56; IR (KBr,
(t, J = 6.0 Hz, 1H); MS (ESI): m/z 263 [M + H] .
−
1
cm ): 3310 (NH), 2952, 2352, 2327, 1634, 1519, 1251, 1031;
+
+
MS (ESI): m/z 311 [M + H] ; HRMS calcd for C20
11.12912, found 311.12877.
-Methoxy-2-(3-phenylimidazo[1,5-a]pyridin-1-yl)-1H-benzo-
15 4
H N [M + H]
General procedure for the synthesis of 3-(substituted phenyl)-
imidazo[1,5-a]pyridine-1-carbaldehydes (10a–c)
3
6
Method C. To an ice-water cooled solution of substituted [d]imidazole (5b). This compound was prepared according to
phenyl imidazo[1,5-a]pyridines (9a–c) (1 mmol) in DMF the method D. Yield (172 mg, 75%); Yellow solid, mp:
1
(
1.4 mmol) was added drop wise POCl (1.4 mmol)with stir- 195–200 °C; H NMR (300 MHz, CDCl ): δ 10.19 (br s, 1H), 8.63
3
3
ring, and then reaction mixture was heated to 100 °C for 2 h. (d, J = 8.2 Hz, 1H), 8.28 (d, J = 7.3 Hz, 1H), 7.81 (d, J = 7.3 Hz,
After the completion of the reaction, the reaction mixture was 2H), 7.50 (td, J = 15.6, 7.3 Hz, 5H), 7.03 (t, J = 6.4 Hz, 1H), 6.87
1
3
poured into ice water and quenched with ammonium hydrox- (d, J = 8.2 Hz, 1H), 6.73 (t, J = 7.3 Hz, 1H), 3.87 (s, 3H);
C
ide solution. The aqueous solution was extracted with ethyl NMR (75 MHz, DMSO): δ 155.38, 148.11, 137.65, 129.90,
acetate and the resultant organic layer was dried over anhy- 129.18, 129.04, 128.42, 127.75, 127.41, 122.67, 122.38, 122.32,
drous sodium sulphate and evaporated under vacuum. Thus, 119.66, 114.66, 110.64, 55.36; IR (KBr, ¯ν ): 3416 (NH), 2352,
−
1
the residue obtained was purified by column chromatography 2318, 1642, 1581, 1453, 1344, 1154 cm ; MS (ESI): m/z 341
+
+
using ethylacetate and hexane as solvent system to afford pure [M + H] ; HRMS calcd for C21
compounds (10a–c). found 341.13905.
-Phenylimidazo[1,5-a]pyridine-1-carbaldehyde (10a). This 6-Methyl-2-(3-phenylimidazo[1,5-a]pyridin-1-yl)-1H-benzo[d]-
compound was prepared according to the method C. Yield: imidazole (5c). This compound was prepared according to the
17 4
H N O [M + H] 341.13969,
3
1
8
0%; H NMR (300 MHz, CDCl ): δ 10.10 (s, 1H), 8.39 (d, J = method D. Yield (169 mg, 77%); Yellow solid, mp: 182–184 °C;
3
1
8
.3 Hz, 2H), 7.77 (d, J = 6.6 Hz, 2H), 7.58–7.46 (m, 3H), 7.22
3
H NMR (300 MHz, CDCl ): δ 8.65–8.62 (m, 1H), 8.28 (t, J =
(
2
dd, J = 9.4, 6.7 Hz, 1H), 6.86 (t, J = 6.7 Hz, 1H); MS (ESI): m/z 6.5 Hz, 1H), 7.82–7.80 (m, 3H), 7.55 (td, J = 7.3, 1.8 Hz, 2H),
+
23 [M + H] .
-(4-Methoxyphenyl)imidazo[1,5-a]pyridine-1-carbaldehyde 2H), 6.75–6.71 (m, 1H), 2.47 (s, 3H); C NMR (75 MHz,
10b). This compound was prepared according to the method DMSO): δ 148.08, 137.68, 130.41, 130.08, 129.16, 128.98,
7.50–7.46 (m, 1H), 7.22 (dd, J = 5.9, 3.0 Hz, 1H), 7.06–7.01 (m,
1
3
3
(
1
C. Yield: 76%; H NMR (300 MHz, CDCl ): δ 10.16 (s, 1H), 8.34 128.20, 122.76, 122.70, 122.48, 122.23, 119.67, 114.66, 21.30;
(
8
3
3
d, J = 8.7 Hz, 2H), 8.31 (d, J = 6.8 Hz, 1H), 7.26–7.23 (dd, J = IR (KBr, cm− ): 3444 (NH), 2352, 2327, 1633, 1593, 1445, 1317,
1
+
.7 Hz, 2H), 7.07 (d, J = 8.7 Hz, 2H), 6.88 (t, J = 7.8 Hz, 1H), 1275, 1031; MS (ESI): m/z 325 [M + H] ; HRMS calcd for
+
+
.90 (s, 3H); MS (ESI): m/z 253 [M + H] .
C H N [M + H] 325.14360, found 325.14371.
21 17 4
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6
-Fluoro-2-(3-phenylimidazo[1,5-a]pyridin-1-yl)-1H-benzo[d]- 1H), 7.76–7.65 (m, 4H), 7.35 (dd, J = 9.0, 6.6 Hz, 1H), 7.06 (t,
1
3
imidazole (5d). This compound was prepared according to the J = 6.7 Hz, 1H); C NMR (75 MHz, CDCl + DMSO): δ 149.57,
3
method D. Yield (163 mg, 73%); Yellow solid, mp: 234–235 °C; 137.02, 129.76, 127.86, 127.58, 126.94, 122.91, 121.29, 120.86,
1
−1
H NMR (300 MHz, CDCl
3
): δ 10.60 (br s, 1H), 8.63 (d, J = 120.26, 118.65, 113.27; IR (KBr, cm ): 3410 (NH), 2954, 2355,
+
9
7
9
.0 Hz, 1H), 8.29 (d, J = 7.3 Hz, 1H), 7.82 (d, J = 8.3 Hz, 2H), 2321, 1633, 1565, 1381, 1135; MS (ESI): m/z 379 [M + H] ;
.56–7.43 (m, 4H), 7.06 (dd, J = 8.6, 6.6 Hz, 2H), 6.94 (td, J = HRMS calcd for C20H N Cl [M + H] 379.05118, found
13 4 2
.8, 1.8 Hz, 1H), 6.73 (t, J = 6.4 Hz, 1H); C NMR (75 MHz, 379.05130.
5,6-Dimethyl-2-(3-phenylimidazo[1,5-a]pyridin-1-yl)-1H-benzo[d]-
27.65, 127.34, 127.21, 126.52, 120.57, 120.53, 118.30, 112.85, imidazole (5i). This compound was prepared according to the
+
1
3
CDCl3 + DMSO): δ 158.43, 155.32, 148.18, 136.29, 128.81,
1
1
1
−
1
07.55, 107.22; IR (KBr, cm ): 3416 (NH), 3010, 2353, 2327, method D. Yield (166 mg, 72%); Yellow solid, mp: 218–220 °C;
+
1
632, 1510, 1391, 1284, 1126; MS (ESI): m/z 329 [M + H] ;
H NMR (300 MHz, DMSO): δ 10.11 (br s, 1H), 8.67 (d, J = 9.0
F [M + H] 329.11970, found Hz, 1H), 8.29 (d, J = 7.1 Hz, 1H), 7.82 (d, J = 7.1 Hz, 2H),
7.59–7.47 (m, 4H), 7.07–7.00 (m, 2H), 6.73 (t, J = 6.4 Hz, 1H),
+
HRMS calcd for C20
29.11907.
H
14
N
4
3
1
3
6
-Chloro-2-(3-phenylimidazo[1,5-a]pyridin-1-yl)-1H-benzo[d]- 2.4 (s, 6H); C NMR (125 MHz, DMSO): δ 137.58, 130.09,
imidazole (5e). This compound was prepared according to the 129.21, 128.97, 128.21, 123.58, 122.61, 122.48, 122.32, 119.84,
method D. Yield (159 mg, 68%); Yellow solid, mp: 236–238 °C; 114.66, 19.03; IR (KBr, cm− ): 3443 (NH), 2920, 2352, 2327,
1
1
+
H NMR (300 MHz, CDCl ): δ 10.70 (br s, 1H), 8.62 (d, J = 9.0 1578, 1504, 1373, 1251, 1031; MS (ESI): m/z 339 [M + H] ;
3
+
Hz, 1H), 8.34 (d, J = 7.3 Hz, 1H), 7.79 (d, J = 7.1 Hz, 3H), HRMS calcd for
.55–7.43 (m, 4H), 7.15 (d, J = 7.7 Hz, 1H), 7.08 (dd, J = 8.4, 6.4 339.16011.
C
22
H
19
N
4
[M + H] 339.16042, found
7
1
3
Hz, 1H), 6.80 (t, J = 6.6 Hz, 1H); C NMR (75 MHz, CDCl + 2-(3-(4-Methoxyphenyl)imidazo[1,5-a]pyridin-1-yl)-1H-benzo-
DMSO): δ 148.38, 137.00, 129.60, 127.96, 127.88, 127.62, [d]imidazole (5j). This compound was prepared according to
3
1
1
1
26.96, 125.20, 121.17, 120.84, 120.44, 118.74, 114.02, 113.98, the method D. Yield (175 mg, 86%); Yellow solid, mp:
−
1
1
13.28, 112.92; IR (KBr, cm ): 3421 (NH), 3062, 2354, 2327, 165–167 °C; H NMR (300 MHz, CDCl ): δ 8.63 (d, J = 9.1 Hz,
3
+
639, 1504, 1381, 1273, 1039; MS (ESI): m/z 345 [M + H] ; 1H), 8.20 (d, J = 7.3 Hz, 1H), 7.77 (d, J = 8.6 Hz, 3H), 7.20 (dd,
+
HRMS calcd for C20
H
14
N
4
Cl [M + H] 345.09015, found J = 5.7, 2.8 Hz, 2H), 7.03 (d, J = 8.6 Hz, 3H), 7.01–6.99 (m, 1H),
1
3
3
45.08968.
-(3-Phenylimidazo[1,5-a]pyridin-1-yl)-6-(trifluoromethyl)- CDCl
H-benzo[d]imidazole (5f). This compound was prepared 120.54, 120.11, 119.99, 119.70, 119.39, 117.96, 112.48, 53.36;
6.71 (dt, J = 7.4, 1.2 Hz, 1H), 3.85 (s, 3H); C NMR (75 MHz,
2
3
+ DMSO): δ 157.97, 146.69, 136.07, 128.22, 127.86,
1
according to the method D. Yield (166 mg, 65%), Yellow solid, IR (KBr, cm− ): 3418 (NH), 2952, 2358, 1594, 1457, 1345, 1251,
1
1
+
mp: 222–225 °C; H NMR (300 MHz, CDCl
3
): δ 10.90 (br s, 1H), 1030; MS (ESI): m/z 341 [M + H] ; HRMS calcd for C21
H
17
N
4
O
+
8
8
9
.64 (d, J = 9.0 Hz, 1H), 8.32 (d, J = 7.1 Hz, 1H), 7.81 (dd, J = [M + H] 341.13969, found 341.13924.
.3, 1.3 Hz, 2H), 7.54–7.41 (m, 5H), 7.26 (s, 1H), 7.11 (dd, J =
.0, 6.6 Hz, 1H), 6.80 (t, J = 7.1 Hz, 1H); C NMR (75 MHz, yl)-1H-benzo[d]imidazole (5k). This compound was prepared
+ DMSO): δ 150.10, 137.35, 130.12, 128.27, 128.19, according to the method D. Yield (176 mg, 80%); Yellow solid,
6-Methoxy-2-(3-(4-methoxyphenyl) imidazo[1,5-a]pyridin-1-
1
3
CDCl
3
1
1
1
27.89, 127.28, 125.89, 122.29, 122.17, 121.76, 121.42, 120.99, mp: 153–154 °C; H NMR (300 MHz, CDCl ): δ 8.62 (d, J =
3
20.91, 119.17, 117.13, 117.09, 113.52; IR (KBr): cm−1: 3428 9.0 Hz, 1H), 8.22 (d, J = 7.3 Hz, 1H), 7.74 (d, J = 8.6 Hz, 2H),
(
3
NH), 3072, 2356, 2328, 1595, 1330, 1248, 1112; MS (ESI): m/z 7.06 – 6.98 (m, 5H), 6.84 (dd, J = 8.6, 2.2 Hz, 1H), 6.70 (t, J = 6.4
+
+
13
79 [M + H] ; HRMS calcd for C H N F [M + H] 379.11651, Hz, 1H), 3.85 (d, J = 6.4 Hz, 6H); C NMR (75 MHz, CDCl ): δ
2
1
14
4
3
3
found 379.11632.
160.38, 156.14, 148.33, 138.41, 130.23, 129.73, 122.15, 121.74,
5
-Chloro-6-fluoro-2-(3-phenylimidazo[1,5-a]pyridin-1-yl)-1H- 121.57, 121.52, 120.86, 114.64, 114.44, 111.43, 55.81, 55.43;
benzo[d]imidazole (5g). This compound was prepared accord- IR (KBr, cm− ): 3462 (NH), 2935, 2360, 1617, 1590, 1327, 1129,
1
+
ing to the method D. Yield (164 mg, 67%); Yellow solid, mp: 1068; MS (ESI): m/z 371 [M + H] ; HRMS calcd for C22
H
19
N
4
O
1
+
2
(
3
56–257 °C; H NMR (300 MHz, CDCl
d, J = 9.2 Hz, 1H), 8.32 (d, J = 7.1 Hz, 1H), 7.78 (d, J = 6.9 Hz,
H), 7.55–7.43 (m, 4H), 7.09 (dd, J = 8.8, 6.6 Hz, 1H), 6.80 (t, 1H-benzo[d]imidazole (5l). This compound was prepared
J = 6.7 Hz, 1H); C NMR (75 MHz, DMSO): δ 155.22, 151.96, according to the method D. Yield (178 mg, 84%); Yellow solid,
50.98, 143.98, 141.72, 138.68, 133.17, 130.79, 129.01, 128.25, mp: 199–201 °C; H NMR (500 MHz, CDCl ): δ 10.23 (br s, 1H),
23.28, 122.94, 121.25, 119.33, 118.41, 114.81, 113.09, 111.35, 8.64 (d, J = 9.2 Hz, 1H), 8.19 (d, J = 7.3 Hz, 1H), 7.70 (d, J =
05.06, 98.87; IR (KBr, cm ): 3416 (NH), 3060, 2354, 2327, 8.8 Hz, 3H), 7.06–7.98 (m, 5H), 7.02 (dt, J = 7.3, 1.1 Hz, 1H),
633, 1462, 1351, 1146; MS (ESI): m/z 363 [M + H] ; HRMS 6.70 (t, J = 7.1 Hz, 1H), 3.87 (s, 3H), 2.46 (s, 3H); C NMR
3
): δ 10.83 (br s, 1H), 8.58 [M + H] 371.15025, found 371.15007.
2-(3-(4-Methoxyphenyl) imidazo[1,5-a]pyridin-1-yl)-6-methyl-
1
3
1
1
1
1
1
3
−1
+
13
+
calcd for C21
,6-Dichloro-2-(3-phenylimidazo[1,5-a]pyridin-1-yl)-1H-benzo[d]- 129.93, 129.01, 128.53, 121.92, 120.72, 120.54, 120.42, 118.94,
imidazole (5h). This compound was prepared according to the 113.32, 113.09, 113.01, 112.87, 112.63, 54.04, 20.34; IR (KBr,
H
14
N
4
F
3
[M + H] 363.08073, found 363.08047.
3
(75 MHz, CDCl + DMSO): δ 158.85, 146.98, 136.98, 136.38,
5
−
1
method D. Yield (161 mg, 63%); Yellow solid, mp: 266–268 °C; cm ): 3340 (NH), 2952, 2361, 1578, 1520, 1332, 1237, 1132,
H NMR (300 MHz, DMSO): δ 8.65 (d, J = 6.9 Hz, 1H), 8.56 (d, 928; MS (ESI): m/z 355 [M + H] ; HRMS calcd for C22
J = 9.0 Hz, 1H), 8.43 (s, 1H), 8.50 (d, J = 6.9 Hz, 1H), 7.98 (s, [M + H] 355.15534, found 355.15507.
1
+
H
19
N
4
O
2
+
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-Fluoro-2-(3-(4-methoxyphenyl)imidazo[1,5-a]pyridin-1-yl)- 9.0 Hz, 1H), 8.29 (d, J = 7.1 Hz, 1H), 7.77–7.68 (m, 4H),
1
3
1
H-benzo[d]imidazole (5m). This compound was prepared 7.11–7.05 (m, 3H), 6.81 (t, J = 6.3 Hz, 1H), 3.91 (s, 1H);
C
according to the method D. Yield (173 mg, 81%); Yellow solid, NMR (75 MHz, CDCl
3
+ DMSO): δ 158.70, 149.63, 137.04,
1
mp: 210–215 °C; H NMR (300 MHz, CDCl
3
): δ 8.61 (d, J = 129.43, 128.38, 122.74, 120.95, 120.79, 120.07, 119.83, 118.53,
.2 Hz, 1H), 8.26 (d, J = 7.3 Hz, 1H), 7.69 (d, J = 8.8 Hz, 3H), 112.94, 53.85; IR (KBr, cm− ): 3427 (NH), 3129, 2357, 2339,
1
9
7
7
+
.06–7.00 (m, 4H), 6.91 (dt, J = 9.4, 1.7 Hz, 1H), 6.73 (dt, J = 1592, 1518, 1444, 1307, 1290; MS (ESI): m/z 409 [M + H] ;
1
3
+
3 15 4 2
.1, 1.7 Hz, 1H), 3.85 (s, 3H); C NMR (75 MHz, CDCl ): HRMS calcd for C21H N OCl [M + H] 409.06174, found
δ 160.80, 160.39, 157.66, 138.63, 130.58, 129.66, 122.02, 409.06079.
21.82, 121.34, 120.73, 114.64, 114.49, 110.02, 109.69, 55.38; 2-(3-(4-Methoxyphenyl)imidazo[1,5-a]pyridin-1-yl)-5,6-dimethyl-
IR (KBr, cm ): 3432 (NH), 2930, 2352, 1609, 1583, 1504, 1411, 1H-benzo[d]imidazole (5r). This compound was prepared
1
−
1
+
1
C
248, 1131, 1043; MS (ESI): m/z 359 [M + H] ; HRMS calcd for according to the method D. Yield (157 mg, 71%); Yellow solid,
+
1
21
H
16
N
4
OF [M + H] 359.13027, found 359.12957.
mp: 250–252 °C; H NMR (300 MHz, DMSO): δ 8.58 (d, J =
6
-Chloro-2-(3-(4-methoxyphenyl)imidazo[1,5-a]pyridin-1-yl)- 9.0 Hz, 1H), 8.30 (d, J = 7.1 Hz, 1H), 7.77 (d, J = 8.6 Hz, 2H),
1
H-benzo[d]imidazole (5n). This compound was prepared 7.33 (d, J = 8.1 Hz, 1H), 7.09 (d, J = 8.6 Hz, 2H), 7.07–6.97
according to the method D. Yield (169 mg, 76%); Yellow solid, (m, 2H), 6.79 (t, J = 6.9 Hz, 1H), 3.91 (s, 3H), 2.58 (s, 3H),
1
−1
mp: 224–226 °C; H NMR (300 MHz, CDCl
3
): δ 11.54 (br s, 1H), 2.39 (s, 3H); IR (KBr, cm ): 3361 (NH), 2919, 2353, 2319,
+
8
8
3
.58 (d, J = 9.2 Hz, 1H), 8.27 (d, J = 7.1 Hz, 1H), 7.67 (d, J = 1588, 1313, 1236, 1121; MS (ESI): m/z 369 [M + H] ;
.6 Hz, 3H), 7.07 (dd, J = 9.2, 6.4 Hz, 2H), 6.93 (d, J = 8.6 Hz, HRMS calcd for C23H N O [M + H] 369.17099, found
21 4
+
13
H), 6.79 (td, J = 7.3, 1.1 Hz, 1H), 3.81 (s, 3H); C NMR 369.17051.
2-(3-(4-(Trifluoromethyl)phenyl)imidazo[1,5-a]pyridin-1-yl)-
30.87, 129.69, 122.51, 121.94, 121.42, 120.96, 120.72, 115.07, 1H-benzo[d]imidazole (5s). This compound was prepared
(
75 MHz, CDCl ): δ 160.52, 157.88, 155.76, 152.58, 138.87,
3
1
1
2
3
3
−1
14.70, 114.59, 55.41; IR (KBr, cm ): 3444 (NH), 3121, 2359, according to the method D. Yield (156 mg, 79%); Yellow solid,
341, 1611, 1575, 1409, 1349, 1251, 1141, 1039; MS (ESI): m/z mp: 176–180 °C; H NMR (300 MHz, CDCl ): δ 10.21 (br s, 1H),
75 [M + H] ; HRMS calcd for C21
75.10072, found 375.10135.
1
3
+
+
H
16
N
4
OCl [M + H]
8.67 (d, J = 9.0 Hz, 1H), 8.31 (d, J = 7.1 Hz, 1H), 7.98 (d, J =
8.1 Hz, 2H), 7.82 (d, J = 8.1 Hz, 3H), 7.26 (s, 3H), 7.10 (dd, J =
1
3
2
-(3-(4-Methoxyphenyl)imidazo[1,5-a]pyridin-1-yl)-6-(trifluoro- 8.8, 6.4 Hz, 1H), 6.82 (t, J = 7.1 Hz, 1H); C NMR (75 MHz,
methyl)-1H-benzo[d]imidazole (5o). This compound was pre- DMSO): δ 159.90, 156.79, 149.82, 136.27, 132.99, 131.01,
pared according to the method D. Yield (164 mg, 67%); Yellow 130.77, 128.93, 128.59, 125.80, 125.76, 123.29, 122.98, 122.36,
1
−1
solid, mp: 224–226 °C; H NMR (300 MHz, CDCl ): δ 8.65 (d, 119.52, 115.12; IR (KBr, cm ): 3381 (NH), 2952, 2353, 2327,
3
J = 9.0 Hz, 1H), 8.27 (d, J = 7.1 Hz, 1H), 7.68 (d, J = 8.7 Hz, 3H), 1642, 1580, 1411, 1326, 1165, 1110, 1068; MS (ESI): m/z 379
+
+
7
21 4
.39 (d, J = 7.6 Hz, 1H), 7.09 (dd, J = 8.8, 6.4 Hz, 1H), 6.95 (d, [M + H] ; HRMS calcd for C23H N O [M + H] 379.11651,
1
3
J = 8.5 Hz, 3H), 6.78 (t, J = 7.1 Hz, 1H), 3.81 (s, 3H); C NMR found 379.11652.
75 MHz, CDCl ): δ 159.77, 150.68, 138.07, 130.49, 129.33, 6-Methoxy-2-(3-(4-(trifluoromethyl)phenyl)imidazo[1,5-a]-
26.44, 123.01, 122.82, 122.59, 121.71, 121.40, 121.12, 121.06, pyridin-1-yl)-1H-benzo[d]imidazole (5t). This compound was
19.86, 117.82, 113.88, 54.83; IR (KBr, cm ): 3435 (NH), 3083, prepared according to the method D. Yield (154 mg, 73%);
358, 2340, 1612, 1523, 1331, 1287, 1113, 1040; MS (ESI): m/z Yellow solid, mp: 180–187 °C; H NMR (300 MHz, CDCl
(
3
1
1
2
4
4
−
1
1
3
):
+
+
09 [M + H] ; HRMS calcd for C22
H
16
N
4
F
3
O [M + H]
δ 8.64 (d, J = 9.0 Hz, 1H), 8.30 (d, J = 7.1 Hz, 1H), 7.98 (d, J =
8.1 Hz, 3H), 7.82 (d, J = 8.1 Hz, 2H), 7.52 (br s, 1H), 7.08 (dd,
09.12707, found 409.12652.
5
-Chloro-6-fluoro-2-(3-(4-methoxyphenyl)imidazo[1,5-a]pyridin- J = 6.4, 2.6 Hz, 2H), 6.89 (dd, J = 8.8, 2.2 Hz, 1H), 6.80 (t, J =
1
3
1
-yl)-1H-benzo[d]imidazole (5p). This compound was prepared 6.9 Hz, 1H), 3.87 (s, 3H); C NMR (125 MHz, DMSO): δ 155.39,
according to the method D. Yield (168 mg, 72%); Yellow solid, 133.11, 130.46, 128.75, 128.59, 128.57, 128.50, 125.82, 125.79,
1
3
mp: 210–213 °C; H NMR (300 MHz, CDCl ): δ 10.97 (br s, 1H), 125.16, 123.00, 122.96, 122.88, 122.81, 119.78, 119.63, 115.08,
1
8
8
.58 (d, J = 9.2 Hz, 1H), 8.26 (d, J = 7.1 Hz, 1H), 7.77 (d, J = 110.70, 55.33; IR (KBr, cm− ): 3417 (NH), 3018, 2355, 2328,
+
.8 Hz, 3H), 7.10 (d, J = 7.5 Hz, 1H), 7.07–7.04 (m, 1H), 7.02 (d, 1633, 1583, 1410, 1325, 1128, 1066;MS (ESI): m/z 409 [M + H] ;
J = 8.6 Hz, 2H) 6.74 (dt, J = 7.5, 1.3 Hz, 1H), 3.84 (s, 3H); HRMS calcd for C22H N F O [M + H] 409.12707, found
16 4 3
C NMR (75 MHz, DMSO): δ 159.79, 150.04, 149.61, 145.40, 409.12646.
43.25, 138.11, 134.93, 132.97, 130.33, 130.23, 129.78, 125.58, 6-Methyl-2-(3-(4-(trifluoromethyl)phenyl)imidazo[1,5-a]pyridin-
25.49, 122.76, 121.31, 121.07, 119.38, 118.98, 117.20, 114.49, 1-yl)-1H-benzo[d]imidazole (5u). This compound was prepared
+
1
3
1
1
1
2
3
3
−
1
14.37, 112.05, 110.57, 55.24; IR (KBr, cm ): 3444 (NH), 3121, according to the method D. Yield (151 mg, 74%); Yellow solid,
356, 2339, 1611, 1591, 1454, 1317, 1275, 1038; MS (ESI): m/z mp: 124–128 °C; H NMR (300 MHz, CDCl ): 8.66 (d, J = 9.2
1
3
+
+
93 [M + H] ; HRMS calcd for C21
93.09129, found 393.09085.
H
15
N
4
ClF [M + H]
Hz, 1H), 8.29 (d, J = 7.1 Hz, 1H), 7.98 (d, J = 8.1 Hz, 3H), 7.83
(d, J = 8.3 Hz, 3H), 7.05 (d, J = 8.8 Hz, 2H), 6.81 (t, J = 6.2 Hz,
1H), 2.49 (s, 3H); C NMR (75 MHz, DMSO): δ 143.39, 136.09,
1
3
5
,6-Dichloro-2-(3-(4-methoxyphenyl)imidazo[1,5-a]pyridin-1-
yl)-1H-benzo[d]imidazole (5q). This compound was prepared 134.57, 133.93, 133.51, 133.12, 132.56, 130.86, 130.67, 129.05,
according to the method D. Yield (154 mg, 63%); Yellow solid, 128.86, 128.63, 128.50, 127.81, 125.83, 122.95, 120.40, 119.77,
1
−1
mp: 220–222 °C; H NMR (300 MHz, CDCl ): δ 8.54 (d, J = 117.71, 115.13, 113.92, 111.00, 110.70, 21.29; IR (KBr, cm ):
3
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+
+
3
1
13 4 6
436 (NH), 3123, 2354, 2328, 1616, 1519, 1325, 1284, 1125, m/z 431 [M + H] ; HRMS calcd for C22H N F [M + H]
+
067; MS (ESI): m/z 393 [M + H] ; HRMS calcd for C H N F
431.06811, found 431.06707.
5,6-Dichloro-2-(3-(4-(trifluoromethyl)phenyl)imidazo[1,5-a]-
-Fluoro-2-(3-(4-(trifluoromethyl)phenyl)imidazo[1,5-a]pyridin- pyridin-1-yl)-1H-benzo[d]imidazole (5z). This compound was
-yl)-1H-benzo[d]imidazole (5v). This compound was prepared prepared according to the method D. Yield (142 mg, 61%);
2
2
16 4 3
+
[M + H] 393.13216, found 393.13193.
6
1
1
according to the method D. Yield (156 mg, 76%); yellow solid, Yellow solid, mp: 300–302 °C; H NMR (300 MHz, CDCl
3
):
1
mp: 230–232 °C; H NMR (300 MHz, CDCl
3
+ DMSO): δ 8.63 δ 8.54 (dd, J = 9.0, 7.1 Hz, 2H), 8.11 (d, J = 8.1 Hz, 2H), 7.87 (d,
(
2
d, J = 9.2 Hz, 1H), 8.52 (d, J = 6.9 Hz, 1H), 8.12 (d, J = 8.1 Hz, J = 8.1 Hz, 2H), 7.71 (s, 2H), 7.20 (dd, J = 8.6, 6.6 Hz, 1H), 6.95
H), 7.89 (t, J = 6.4 Hz, 2H), 7.54 (dd, J = 8.6, 4.9 Hz, 1H), 7.30 (t, J = 6.9 Hz, 1H); IR (KBr, cm− ): 3457 (NH), 2923, 2358, 1617,
1
+
(dd, J = 9.2, 1.8 Hz, 1H), 7.16 (dd, J = 8.6, 6.6 Hz, 1H), 6.93 (dt, 1566, 1444, 1329, 1287, 1109, 1068; MS (ESI): m/z 447 [M + H] ;
1
3
+
J = 8.3, 2.2 Hz, 2H); C NMR (75 MHz, DMSO): δ 159.90, HRMS calcd for C H N F Cl [M + H] 447.03856, found
2
1
12
4
3
2
1
1
1
56.79, 149.82, 136.27, 132.99, 131.01, 130.77, 128.93, 128.59, 447.03875.
25.80, 125.76, 123.29, 122.98, 122.36, 119.52, 115.12, 111.38, 5,6-Dimethyl-2-(3-(4-(trifluoromethyl)phenyl)imidazo[1,5-a]-
−
1
09.17; IR (KBr, cm ): 3388 (NH), 3021, 2356, 2338, 1619, pyridin-1-yl)-1H-benzo[d]imidazole (5aa). This compound was
+
1
582, 1410, 1325, 1130, 1068; MS (ESI): m/z 397 [M + H] ; prepared according to the method D. Yield (140 mg, 66%);
+
1
HRMS calcd for C22
H
16
N
4
F
3
[M + H] 397.10709, found Yellow solid, mp: 245–147 °C; H NMR (300 MHz, CDCl
δ 10.15 (br s, 1H), 8.70 (d, J = 8.8 Hz, 1H), 8.30 (d, J = 7.1 Hz,
-Chloro-2-(3-(4-(trifluoromethyl)phenyl)imidazo[1,5-a]pyridin- 1H), 7.98 (d, J = 8.1 Hz, 2H), 7.82 (d, J = 8.3 Hz, 2H), 7.07(dd,
-yl)-1H-benzo[d]imidazole (5w). This compound was prepared J = 8.4, 6.0 Hz, 2H), 6.79 (dd, J = 7.1, 0.9 Hz, 1H), 2.41 (s, 6H);
3
):
3
97.10688.
6
1
1
3
according to the method D. Yield (145 mg, 68%); Yellow solid,
C NMR (75 MHz, CDCl ): δ 147.46, 136.49, 132.94, 131.03,
3
1
mp: 229–230 °C; H NMR (300 MHz, CDCl
3
): δ 10.34 (br s, 1H), 128.24, 126.21, 126.16, 124.56, 122.28, 121.38, 121.27, 115.24,
.67 (d, J = 9.2 Hz, 1H), 8.35 (d, J = 7.3 Hz, 1H), 7.98 (d, J = 8.3 19.38; IR (KBr, cm− ): 3443 (NH), 2919, 2357, 2317, 1675, 1562,
1
8
+
Hz, 3H), 7.80 (d, J = 8.1 Hz, 3H), 7.20 (d, J = 8.0 Hz, 1H), 7.12 1478, 1334, 1239, 1101; MS (ESI): m/z 407 [M + H] ; HRMS
13
+
(
(
1
1
3
dd, J = 9.0, 6.6 Hz, 1H), 6.84 (t, J = 6.6 Hz, 1H); C NMR calcd for C23
75 MHz, CDCl + DMSO): δ 148.37, 135.28, 131.89, 130.12,
28.99, 128.56, 127.19, 125.30, 124.62, 124.57, 121.65, 121.47,
20.76, 120.52, 119.14, 113.90; IR (KBr, cm ): 3400 (NH),
029, 2357, 1617, 1574, 1409, 1326, 1131, 1068; MS (ESI): was determined using MTT assay.
18 4 3
H N F [M + H] 407.14781, found 407.14713.
3
Biology
Anticancer activity. The cytotoxic activity of the compounds
−
1
6
0,61
4
1 × 10 cells per well
+
m/z 413 [M
+
H] ; HRMS calcd for C H N F Cl were seeded in 200 µl DMEM, supplemented with 10% FBS in
2
1
13 4 3
+
[
M + H] 413.07754, found 413.07716.
-(Trifluoromethyl)-2-(3-(4-(trifluoromethyl)phenyl)imidazo- at 37 °C in a CO
1,5-a]pyridin-1-yl)-1H-benzo[d]imidazole (5x). This com- desired concentrations in culture medium, were added to the
each well of 96-well microculture plates and incubated for 24 h
6
2
incubator. Compounds, diluted to the
[
pound was prepared according to the method D. Yield wells with respective vehicle control. After 48 h of incubation,
1
(
(
146 mg, 63%); Yellow solid, mp: 200–202 °C; H NMR 10 µl MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetr-
−
1
300 MHz, CDCl ): δ 10.49 (br s, 1H), 8.67 (d, J = 8.6 Hz, 1H), azolium bromide) (5 mg mL ) was added to each well and the
3
8
5
6
1
1
1
1
.36 (d, J = 7.1 Hz, 1H), 7.95 (d, J = 8.1 Hz, 2H), 7.81 (d, J = plates were further incubated for 4 h. Then, the supernatant
.4 Hz, 3H), 7.63–7.46 (m, 2H), 7.16 (dd, J = 8.8, 6.4 Hz, 1H), from each well was carefully removed, formazon crystals were
1
3
.87 (t, J = 6.9 Hz, 1H); C NMR (75 MHz, DMSO): δ 150.69, dissolved in 100 µL of DMSO and absorbance at 570 nm wave-
36.69, 132.92, 131.52, 129.13, 128.77, 128.29, 125.89, 125.84, length was recorded.
23.93, 123.21, 122.30, 121.91, 121.75, 119.44, 118.27, 118.23,
−
1
Cell cycle analysis
15.33; IR (KBr, cm ): 3444 (NH), 2903, 2352, 1620, 1596,
+
410, 1327, 1249, 1112, 1038; MS (ESI): m/z 447 [M + H] ; Flow cytometric analysis (FACS) was performed to evaluate the
+
HRMS calcd for C22
47.10339.
H
13
N
4
F
6
[M + H] 447.10389, found distribution of the cells through the cell cycle phases. MCF-7,
4
breast cancer cells were incubated with compounds (5d and
5
-Chloro-6-fluoro-2-(3-(4-(trifluoromethyl)phenyl)imidazo[1,5- 5l) at 1 and 3 µM concentrations for 48 h. Untreated and
a]pyridin-1-yl)-1H-benzo[d]imidazole (5y). This compound was treated cells were harvested, washed with PBS, fixed in ice-cold
prepared according to the method D. Yield (152 mg, 68%); 70% ethanol and stained with propidium iodide (Sigma
1
Yellow solid, mp: 260–262 °C; H NMR (300 MHz, CDCl
3
+
Aldrich). Cell cycle was performed by flow cytometry (Becton
6
2
DMSO): δ 10.82 (br s, 1H), 8.60 (d, J = 9.0 Hz, 1H), 8.46 (d, J = Dickinson FACS Caliber) as described earlier.
7
7
6
.3 Hz, 1H), 8.07 (d, J = 8.1 Hz, 2H), 7.88 (d, J = 8.3 Hz, 2H),
.60 (d, J = 6.6 Hz, 1H), 7.40 (d, J = 9.4 Hz, 1H), 7.16 (dd, J =
.4, 2.6 Hz, 1H), 6.91 (t, J = 7.1 Hz, 1H); C NMR (75 MHz, A fluorescence based in vitro tubulin polymerization assay was
Inhibition of tubulin polymerization
1
3
CDCl3 + DMSO): δ 154.69, 151.54, 150.33, 136.45, 132.89, performed according to the manufacturer’s protocol (BK011,
1
1
31.16, 128.99, 128.63, 125.81, 125.76, 123.64, 123.09, 122.25, Cytoskeleton, Inc.). In brief, the reaction mixture in a total
21.82, 119.37, 115.20, 113.21, 112.94; IR (KBr, cm−1): 3438 volume of 10 µL contained PEM buffer, GTP (1 µM) in the pres-
(NH), 3049, 2358, 1618, 1581, 1407, 1324, 1173, 1067; MS (ESI): ence or absence of test compounds (final concentration of 3
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µM). Tubulin polymerization was followed by a time depen- measuring mitochondrial membrane potential (ΔΨm), accord-
dent increase in fluorescence because of the incorporation of a ing to the manufacturer’s instructions.
fluorescence reporter into microtubules as polymerization pro-
ROS generation
ceeds. Fluorescence emission at 420 nm (excitation wavelength
is 360 nm) was measured by using a Varioscan multimode To evaluate intracellular reactive oxygen species (ROS) levels,
plate reader (Thermo scientific Inc.). Nocodazole was used as 2′,7′-dichlorofluorescein diacetate (DCFH-DA, Molecular
positive control in each assay. The IC50 value was defined as Probes) fluorescent dye was used to clarify this issue. The non-
the drug concentration required for inhibiting 50% of tubulin polar DCFH-DA is converted to the polar derivative DCFH by
assembly compared to control. The reaction mixture for these esterases when it is taken up by the cell. DCFH is nonfluores-
−
1
experiments include: tubulin (3 mg ml ) in PEM buffer, GTP cent but is rapidly oxidized to the highly fluorescent DCF by
(
1 µM), in the presence or absence of test compounds at 2.5, 5, intracellular H O or nitric oxide. MCF-7 cells were treated
2 2
1
0, and 15 µM concentrations. Polymerization was monitored with compound 5d and 5l at 1 µM concentration for 48 h. To
by increase in the fluorescence as mentioned above at 37 °C.
determine the production of ROS, cells were treated with 1 µM
of DCFH2-DA at 37 °C for 30 min, and the fluorescence of DCF
was measured at 530 nm after excitation at 485 nm (DCFH2-
Immunohistochemistry
MCF-7 cells were seeded on glass cover slips and incubated for DA, after deacetylation to DCFH2, is oxidized intracellular to
6
4
4
8 h in the presence or absence of test compounds 5d and 5l its fluorescent derivative, DCF).
at 1 µM concentration. Following the termination of incu-
bation, cells were fixed with 3% paraformaldehyde, 0.02% glu-
Activation of caspase 9
taraldehyde in PBS and permeabilized by dipping the cells in Caspase-9 assay was conducted for the detection of apoptosis
00% methanol followed by overnight incubation at 4 °C. in breast cancer cell line (MCF-7). The commercially available
1
Later, cover slips were blocked with 1% BSA in phosphate apoptosis detection kit (Sigma-Caspase 9 Assay kit, Colori-
buffered saline for 1 h followed by incubation with a primary metric) was used. MCF-7 cells were treated with compounds
anti tubulin (mouse monoclonal) antibody and FITC conju- 5d and 5l at 1 µM concentrations for 48 h. After 48 h of treat-
gated secondary mouse anti IgG antibody. Images were ment, cells were collected by centrifugation, washed once with
obtained using the fluorescence microscope, equipped with PBS, and cell pellets were collected. Suspended the cell pellet
FITC settings and the pictures were analyzed for the integrity in lysis buffer and incubated for 15 min. After incubation, cells
of microtubule network. In parallel experiments, nocodazole were centrifuge at 20 000 rpm for 15 min and collected the
(1 µM) was used as positive control for analyzing microtubule supernatant. Supernatants were used for measuring caspase 9
integrity.
activity using an ELISA-based assay, according to the manufac-
turer’s instructions.
Hoechst staining
DNA fragmentation analysis
Cells were seeded at a density of 10 000 cells over 18 mm cover
6
slips and incubated for 24 h. After incubation, cells were Cells were seeded (1 × 10 ) in six well plates and incubated for
treated with the compounds 5d and 5l at 1 µM concentration 24 h. After incubation, cells were treated with compounds 5d
for 48 h. Hoechst 33258 (Sigma Aldrich) was added to the cells and 5l at 1 µM concentration for 48 h. After 48 h of drug treat-
−
1
at a concentration of 0.5 mg mL and incubated for 30 min at ment cells were collected and centrifuged at 2500 rpm for
7 °C. Later, the cells were washed with phosphate buffered 5 min at 4 °C. Pellet was collected and washed with phosphate
3
saline (PBS). Cells from each cover slip were captured from ran- buffered saline (PBS), added 100 µl of lysis buffer, centrifuged
domly selected fields under fluorescent microscope (Leica, at 3000 rpm for 5 min at 4 °C and collected supernatant and
Germany) to qualitatively determine the proportion of viable add 10 µl of 10% SDS and 10 µl of (50 mg mL− ) RNase-A and
and apoptotic cells based on their relative fluorescence and incubated for 2 h at 56 °C. After that 10 µl of Proteinase K
1
6
3
−1
nuclear fragmentation.
(25 mg mL ) was added and incubated at 37 °C for 2 h. After
incubation, 65 µl of 10 M Ammonium acetate and 500 µl of ice
cold ethanol was added and mixed well. And this sample was
Measurement of mitochondrial membrane potential
Mitochondrial membrane potential (ΔΨm) was determined by incubated at 80 °C for 1 h. After that samples were centrifuged
using JC-1 dye. Cell-permeable cationic carbocyanine dye JC-1 at 12 000 rpm for 20 min at 4 °C, washed with 80% ethanol fol-
(Sigma Aldrich India, Catlog CS0390), also known as 5,5′,6,6′- lowed by air dried for 10 min at room temperature. The pellet
tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine was dissolved in 50 µl TE buffer, and DNA laddering was deter-
6
5
iodine, emits green fluorescence (525 nm) in its monomeric mined by using 2% agarose gel electrophoresis in TE buffer.
form. However, upon transfer to the membrane environment
of a functionally active mitochondrion, it exhibits an aggrega-
Protein extraction and western blot analysis
tion dependent orange-red fluorescence (emission at 590 nm). MCF-7 cells were treated with compounds 5d and 5l at 1 µM
In brief, cultures were treated with the test drugs for 48 h. concentration for 48 h. The cell lysates were obtained by lysing
After drug treatment the cells were incubated with JC-1 dye for the cells in ice-cold radio immune precipitation assay (RIPA)
2
0 min at 37 °C. After incubation cultures were used for the buffer (1 × PBS, 1% NP-40 detergent, 0.5% sodium deoxycho-
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−
1
late, and 0.1% SDS) containing 100 mg mL phenylmethane- 12 S. Mavel, J. L. Renou, C. Galtier, R. Snoeck, G. Andrei,
−
1
−1
sulfonyl fluoride (PMSF), 5 mg mL aprotinin, 5 mg mL leu-
peptin, 5 mg mL− pepstatin, and 100 mg mL−1 NaF. After
J. Balzarini, E. D. C. A. Gueiffier and A. Forsch, Drug Res.,
2001, 51, 304.
1
centrifugation at 12 000 rpm for 10 min, the protein in the 13 J. C. Teulade, G. Grassy, J. P. Girard, J. P. Chapat and
supernatant was quantified by the Bradford method (BIO-RAD) M. S. DeBuochberg, Eur. J. Med. Chem., 1978, 13, 271.
using a Multimode Varioskan instrument (Thermo Fischer 14 (a) T. Veikkola, M. Karkkainen, L. Claesson-Welsh and
Scientifics Ltd). Protein (50 mg per lane) was applied in 12%
SDS polyacrylamide gel. After electrophoresis, the protein was
K. Alitalo, Cancer Res., 2000, 60, 203; (b) K. A. Thomas,
J. Biol. Chem., 1996, 271, 603.
transferred to a polyvinylidenedifluoride (PVDF) membrane 15 Z. Wu, M. E. Fraley, M. T. Bilodeau, M. S. Kaufman,
(
Thermo Scientific Inc.). The membrane was blocked at room
E. S. Tasber, A. E. Balitza, G. D. Hartman, K. E. Coll,
K. Rickert, J. Shipman, B. Shi, L. Sepp-Lorenzino and
K. A. Thomas, Bioorg. Med. Chem. Lett., 2004, 14, 909.
temperature for 2 h in Tris-buffered saline (TBS) with 0.1%
Tween 20 (TBST) containing 5% blocking powder (Santa Cruz).
The membrane was washed with TBST for 5 min, and then 16 A. Kamal, G. Ramakrishna, P. Raju, A. V. Subba Rao,
primary antibody was added and the membrane was incubated
A. Viswanath, V. Lakshma Nayak and S. Ramakrishna,
Eur. J. Med. Chem., 2011, 46, 2427.
(
ser473)
(ser380)
at 48 °C overnight. Cytochrome c, p-Akt,
p-PTEN
and β-actin antibodies were purchased from Cell Signaling 17 T. D. Penning, G. D. Zhu, V. B. Gandhi, J. Gong, X. Liu,
Technology (CST). The membrane was incubated with the
corresponding horseradish peroxidase labeled secondary anti-
body (1 : 2000; CST) at room temperature for 1 h. Membranes
were washed with TBST three times for 15 min, and the blots
Y. Shi, V. Klinghofer, E. F. Johnson, C. K. Donawho,
D. J. Frost, V. B. Diaz, J. J. Bouska, D. J. Osterling,
A. M. Olson, K. C. Marsh, Y. Luo and V. L. Giranda, J. Med.
Chem., 2009, 52, 514.
were visualized with chemiluminescence reagent (Thermo 18 D. Hao, J. D. Rizzo, S. Stringer, R. V. Moore, J. Marty,
Fischer Scientifics Ltd). Images were captured by using the
UVP ChemiDoc imager (BIO-RAD).
D. L. Dexter, G. L. Mangold, J. B. Camden, D. D. Von-Hoff
and S. D. Weitman, Invest. New Drugs, 2002, 20, 261.
9 H. M. Refaat, Eur. J. Med. Chem., 2010, 45, 2949.
0 S. C. Lio, J. Johnson, A. Chatterjee, J. W. Ludwig, D. Millis,
H. Banie, J. C. Sircar, A. Sinha and M. L. Richards, Cancer
Chemother. Pharmacol., 2008, 61, 1045.
6
6
1
2
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