Synthesis and anticancer activity of oxindole derived imidazo[1,5-a] pyrazines

Article abstract of DOI:10.1016/j.ejmech.2011.03.027

A series of oxindole derivatives of imidazo[1,5-a]pyrazines were prepared and confirmed by ¹H NMR, mass and HRMS data. These compounds were evaluated for their anticancer activity against a panel of 52 human tumor cell lines derived from nine d

Full text of DOI:10.1016/j.ejmech.2011.03.027

European Journal of Medicinal Chemistry 46 (2011) 2427e2435
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European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and anticancer activity of oxindole derived imidazo[1,5-a]pyrazines
*
Ahmed Kamal , G. Ramakrishna, P. Raju, A.V. Subba Rao, A. Viswanath, V. Lakshma Nayak, Sistla Ramakrishna
Divisions of Organic Chemistry and Pharmacology, Indian Institute of Chemical Technology, Tarnaka, Hyderabad 500 607, India
a r t i c l e i n f o
a b s t r a c t
A series of oxindole derivatives of imidazo[1,5-a]pyrazines were prepared and confirmed by 1H NMR,
mass and HRMS data. These compounds were evaluated for their anticancer activity against a panel of 52
human tumor cell lines derived from nine different cancer types: leukemia, lung, colon, CNS, melanoma,
ovarian, renal, prostate and breast. Among them compound 7l showed significant anticancer activity
Article history:
Received 27 September 2010
Received in revised form
8 March 2011
Accepted 14 March 2011
Available online 23 March 2011
with GI50 values ranging from 1.54 to 13.0
mM. Cell cycle arrest was observed in G0/G1 phase upon
treatment of A549 cells with 6.5 M (IC50) concentration of compound 7l and induced apoptosis. This
m
was confirmed by Annexin V-FITC as well as DNA fragmentation analysis and interestingly this
compound (7l) did not affect the normal cells.
Keywords:
Imidazo[1,5-a]pyrazines
Anticancer activity
Cell cycle arrest
G0/G1
Ó 2011 Elsevier Masson SAS. All rights reserved.
DNA fragmentation
Hoechst staining
Annexin V-FITC
Apoptosis
1. Introduction
KDR (VEGFR-2) [10]. Substituted 2-(N-trifluoroacetylamino)imida-
zopyridines are known to arrest cell cycle at G2/M phase and
One of the major diseases causing death across the world is
cancer. Nitrogen-bridgehead fused heterocycles containing an
imidazole ring are a common structural moiety in many pharma-
cologically important molecules that display a wide range of
activities for diverse number of targets. One of the most widely
used heterocyclic system from this group is imidazopyridine [1].
Imidazopyridines show a spectrum of biological activities like
inhibitors of aromatase estrogen production suppressors [2], posi-
tive inotropic agents [3], platelet aggregation inhibitors, throm-
boxane synthetase inhibitors [4], antiviral [5,6], antibacterial [7],
hypnoselective, and anxioselective activities [8]. Imidazopyridines
exhibit different type of molecular mechanisms in cancer chemo-
therapy. Recently, Wu and coworkers reported 3,7-diarylimidazo-
pyridines as inhibitors of the vascular endothelial growth factor
(VEGF)-receptor KDR [9]. VEGF is a regulator of vascular perme-
ability and an inducer of endothelial cell proliferation, migration,
and survival. Activation of the VEGF pathway is a fundamental
regulation of angiogenesis, the formation of new capillaries from
established blood vessels. In molecular mechanisms, the mitogenic
signal of VEGF is mediated through the receptor tyrosine kinase
induce apoptosis in SK-LU-1 human cancer cell line [11]. Oxindoles
are important pharmacophores that are known to enhance anti-
cancer activity of some core molecules. Similarly, substituted E-3-
(3-Indolylmethylene)-1,3-dihydroindol-2-ones are also reported as
anticancer agents and induce apoptosis [12e17]. Therefore in the
present study oxindole-derived imidazo[1,5-a]pyrazines have been
synthesized and evaluated for their anticancer activity. Further the
mode of action of one of the potent compound (7l) has been
investigated.
2. Results and discussion
2.1. Chemistry
Synthesis of the oxindole-derived imidazo[1,5-a]pyrazine
(7a-r) derivatives was accomplished as described in Scheme 1.
The intermediates N1-(2-pyridylmethyl)-substituted benzamides
(3a-h) were prepared by the reaction of different substituted
benzoylchlorides (2a-h) and 2-pyridylmethanamine (1) in the
presence of triethylamine. The cyclization of N1-(2-pyridylmethyl)-
substituted benzamides (3a-h) in phosphorous oxychloride gave
the intermediates substituted phenyl imidazo[1,5-a]pyridines
(4a-h) in good yield. Formylation of substituted phenyl imidazo
[1,5-a]pyridines (4a-h) in DMF and phosphorous oxychloride
* Corresponding author. Tel.: þ91 40 27193157; fax: þ91 40 27193189.
E-mail address: ahmedkamal@iict.res.in (A. Kamal).
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.03.027

2428
A. Kamal et al. / European Journal of Medicinal Chemistry 46 (2011) 2427e2435
R2
O
R1
R1
O
R3
R4
ii
N
R2
R3
i
H
N
N
Cl
R1
N
N
NH2
R2
R4
R4
4a-h
3a-h
2a-h
1
R3
O
R5
O
N
H
N
N
R6
N
N
iv
iii
O
R1
R2
R1
R2
R6
N
R5
R4
R4
R3
7a-r
R3
5a-h
6a-d
7a = R1 = H, R2 = H, R3 = H, R4 = H, R5 = H, R6 = H
7b
= R1 = H, R2 = H, R3 = H, R4 = H, R5 = CH3, R6 = H
7c = R1 = H, R2 = H, R3 = OCH3, R4 = H, R5 = H, R6 = H
7d
= R1 = H, R2 = H, R3 = OCH3, R4 = H, R5 = CH3, R6 = H
7e = R1 = H, R2 = H, R3 = OCH3 R4 = H, R5 = H, R6 = F
7f = R1 = H, R2 = OCH3, R3 = OCH3, R4 = H, R5 = H, R6 = H
7g
= R1 = H, R2 = OCH3, R3 = OCH3, R4 = H, R5 = CH3, R6 = H
7h = R1 = H, R2 = OCH3, R3 = OCH3, R4 = OCH3, R5 = CH3, R6 = H
7i
= R1 = H, R2 = H, R3 = CH3, R4 = H, R5 = H, R6 = H
7j = R1 = H, R2 = H, R3 = CH3, R4 = H, R5 =H, R6 = F
7k = R1 = H, R2 = H, R3 = CH3, R4 = H, R5 = CH3, R6 = F
7l
= R1 = F, R2 = H, R3 = H, R4 = H, R5 = H, R6 = H
7m = R1 = F, R2 = H, R3 = H, R4 = H, R5 = CH3, R6 = H
7n
= R1 = F, R2 = H, R3 = H, R4 = H, R5 = H, R6 = F
7o = R1 = F, R2 = H, R3 = H, R4 = H, R5 = CH3, R6 = F
7p = R1 = H, R2 = F, R3 = H, R4 = H, R5 = CH3, R6 = H
7q
= R1 = H, R2 = H, R3 = F, R4 = H, R5 = H, R6 = H
7r = R1 = H, R2 = H, R3 = F, R4 = H, R5 = H, R6 = F
Scheme 1. Reagents and conditions: (i) TEA, dry THF, 0 ꢁC to rt, 3 h; (ii) POCl3, reflux, 2 h; (iii) DMF, POCl3; (iv) EtOH, piperidine, reflux, 3 h.
gave 3-(substituted phenyl)imidazo[1,5-a]pyridine-1-carbaldehyde
(5a-h) [18].
The reaction between 3-(substituted phenyl)imidazo[1,5-a]
pyridine-1-carbaldehyde (5a-h) and oxindole (6a-d) in methanol in
the presence of piperidine gave the final oxindole-derived imidazo
[1,5-a]pyrazines (7a-r) in excellent yield. The structures of these
compounds were established on the basis of their spectroscopic
data. The ESIMS (mass spectra) of the compounds showed [M þ H]þ
peaks corresponding to their molecular formula. The purity of new
Table 1
Anticancer screening data in concentration 10-5 M.
Compound Meangrowth Range of
growth %
Most sensitive
cell line
Growth % of the
most sensitive
cell line
%
7a
87.51
ꢀ1.63 to 123.93 MBA-MB-468
(Breast)
ꢀ1.63
I
compounds synthesized was verified by H NMR and high resolu-
tion mass spectra.
7b
7c
7d
7e
7f
85.21
72.54
77.31
101.96
82.66
103.43
52.68 to 129.58 T-47D (Breast)
17.53 to 120.98 ACHN (Renal)
ꢀ7.02 to 119.01 SR (Leukemia)
48.75 to 126.47 MCF-7 (Breast)
21.84 to 117.12 MCF-7 (Breast)
70.51 to 138.54 NCI-H522
(Non-small cell
52.68
17.53
ꢀ7.03
48.75
21.84
70.51
2.2. Anticancer activity
7g
All synthesized new oxindole-derived imidazo[1,5-a]pyrazines
(7a-r) were evaluated by the National Cancer Institute, Bethesda for
their anticancer activity at single concentration of 10ꢀ5 M toward
a panel of approximately sixty cancer cell lines. The human tumor
cell lines were derived from nine different cancer types: leukemia,
melanoma, lung, colon, CNS, ovarian, renal, prostate and breast
cancers. Primary anticancer assays were performed according to the
US NCI protocol [19e21]. The compounds were added at a single
concentration and the cell culture was incubated for 48 h. End point
determinations were made with a protein binding dye, sulforhod-
amine B (SRB). The results for each compound are reported as the
percent growth of treated cells when compared to untreated control
cells (shown in Table 1). Range of growth% shows the lowest and the
highest growth % found among different cancer cell lines.
lung)
7h
7i
7j
98.17
78.55
74.57
99.39
42.32 to 126.81 SR (Leukemia)
42.32
ꢀ16.33 to 133.23 DU-145 (Prostate) ꢀ16.33
ꢀ23.07 to 110.36 DU-145 (Prostate) ꢀ23.07
7k
60.48 to 128.17 NCI-H522
60.48
(Non-small cell
lung)
7l
18.49
ꢀ68.25 to 86.08 SNB-75
ꢀ68.25
(CNS Cancer)
7m
7n
7o
7p
7q
84.72
80.90
99.95
70.98
39.31
27.49 to 115.62 DU-145 (Prostate) 27.49
ꢀ7.63 to 130.24 DU-145 (Prostate) ꢀ7.63
69.77 to 146.09 COLO-205 (Colon) 69.77
IGROV1 (Ovarian)
39.31 to 118.39 MDA-MB-468
(Breast)
7.47 to 101.45
7.47
39.31
7r
97.53
ꢀ13.90 to 131.85 A498 (Renal)
ꢀ13.90

A. Kamal et al. / European Journal of Medicinal Chemistry 46 (2011) 2427e2435
2429
Interestingly, among all the compounds one compound (7l) was
found active and selected for five dose. In five dose the compound
(7l) was dissolved in DMSO and evaluated for anticancer activity
using five concentrations at 10-fold dilutions, the highest being
10ꢀ4 M and others 10ꢀ5, 10ꢀ6, 10ꢀ7 and 10ꢀ8 M. The results of five
dose data have been showed in Table 2. Compound (7l) is extremely
repair, induces apoptosis and/or cell cycle arrest [23e25]. Data from
MTT assay showed that compound (7l) induced significant inhibi-
tion of lung cancer cells. It was of interest to understand whether
this inhibition of cell growth was on account of cell cycle arrest.
Hence, we studied the cell cycle distribution of this compound by
treating the cells with propidium iodide-labeling and followed
fluorescence-activated cell sorting analysis. Results indicated that
potent with GI50 values ranging from 1.54 to 13.0
mM. This
compound is active at <2 M against HCT-116 (colon cancer), SNB-
m
compound 7l (6.5 mM) showed cell cycle arrest at G0/G1 Phase in
75 (CNS cancer), OVACAR-3, OVACAR-4, OVACAR-5 (ovarian cancers)
and MDA-MB- 468 (breast cancer) cell lines. It is interesting to note
that the mean graph midpoint values of compound 7l are log10 GI50
(ꢀ5.34), log10 TGI (ꢀ4.82) and log10 LC50 (ꢀ4.41). These results
specify that compound 7l possess remarkable anticancer activity.
This compound was considered for further studies like cell cycle
analysis and understanding the mechanism of action.
24 h and 48 h as compared with untreated control as shown in Fig.1.
Manual field quantification of percent apoptotic cells based on
cytoplasmic condensation, presence of apoptotic body, nuclear
fragmentation and relative fluorescence in cells treated with test
compounds revealed that compound 7l in A549 cells resulted in
significant increase in percentage of apoptotic cell (Fig. 2).
This potent compound 7l (IC50 values of 6.5 mM, against A549
Specific killing of cancer cells without affecting normal cell
growth is a key safety feature of cancer chemotherapy. Therefore
compound (7l) was evaluated for possible cytotoxicity to normal
cells, for example, HEK-293. Compound (7l), did not significantly
affect the growth of HEK-293, suggesting that the molecule selec-
tively inhibits the growth of cancer cells.
cells) was also investigated for the inhibition of cell growth to
understand whether it is due to physiological apoptosis or non-
specific necrosis. The ability of compound 7l to induce apoptosis
was measured by Annexin V-FITC and propidium iodide (PI)
labeling of A549 cells, after treatment with 6.5 mM for 24 h and 48 h.
It was observed that this compound (7l) showed significant
apoptosis against A549 cells as illustrated in Fig. 3.
2.3. Cell cycle analysis and apoptotic changes
2.4. DNA fragmentation
The tumor suppressor gene p53 is a multifunctional protein
responsible for maintaining genomic integrity and its mutation is
known to cause tumors in humans [22]. In response to DNA
damage, aberrant growth signals, or chemotherapeutic drugs, p53
activates signaling for DNA repair initially, and in failure of DNA
The ability of 7l for the induction of intranucleosomal DNA
fragments at 24 and 48 h of exposure to A549 cells have been
demonstrated by agarose gel electrophoresis technique. DNA lad-
dering is significantly observed in the A549 cells exposed to 6.5 uM
of 7l. Our results show that 7l harbor DNA fragmentation in A549
cells, evident by Fig. 4, which is associated with the last events of
drug induced apoptosis.
Table 2
In vitro cytotoxicity of the compound 7l on 52 human cancer cell lines.
3. Conclusion
Panel/Cell Line
GI50
(m
M)
Panel/Cell Line
GI50 (mM)
Leukemia
Melanoma
MALME-3M
M 14
In conclusion, we synthesized a series of oxindole-derived
imidazo[1,5-a]pyrazines derivatives and evaluated this for anti-
cancer activity. One of the compound 7l showed significant cyto-
toxicity against a panel of 52 human cancer cell lines. The FACS
analysis showed that the compound 7l arrests the cell cycle in G0/
G1 phase in A549 cells and ultimately leading to cell death. This was
further confirmed by Annexin V-FITC, DNA fragmentation analysis
and Hoechst staining. Moreover, compound 7l did not affect the
normal cells (HEK-293). Based on these observations, compound
(7l) could be considered as important lead compound for potential
application in anticancer chemotherapy.
3.07
5.65
3.71
4.15
6.81
2.80
5.20
6.32
3.95
K-562
SR
6.35
11.0
SK-MEL-28
LOX IMV
SK-MEL-2
SK-MEL-5
UACC-257
UACC-62
MDA-MB-435
Ovarian
Non-small cell lung
A549/ATCC
EKVX
HOP-62
NCI-H226
NCI-H23
NCI-H322M
NCI-H460
NCI-H522
3.46
10.6
2.37
4.78
4.55
4.16
2.17
2.18
IGROV1
2.04
1.90
1.81
1.80
2.49
3.36
3.46
OVCAR-3
OVCAR-4
OVCAR-5
OVCAR-8
NCI/ADR-RES
SK-OV-3
Renal Cancer
786-0
A498
ACHN
RXF 393
SN12C
TK-10
4. Experimental
Colon
4.1. Chemistry
COLO 205
HCT-116
HCT-15
HT29
KM12
SW-620
4.08
1.97
13.0
3.50
3.49
3.97
3.44
2.81
3.30
3.41
4.32
4.05
2.12
All chemicals and reagents were obtained from Aldrich (Sig-
maeAldrich, St. Louis, MO, USA), Lancaster (Alfa Aesar, Johnson
Matthew Company, Ward Hill, MA, USA) and were used without
purification. Reactions were monitored by TLC, performed on silica
gel glass plates containing 60 F-254, and visualization on TLC was
achieved by UV light or iodine indicator. Column chromatography
was performed with Merck 60e120 mesh silica gel. 1H NMR spectra
were recorded on Gemini Varian VXR-unity (400 and 500 MHz) or
Bruker UXNMR/XWIN-NMR (300 MHz) instruments. Chemical
CNS Cancer
SF-268
SF-295
SF-539
SNB-19
SNB-75
U251
UO-31
4.65
3.65
3.10
4.70
1.79
2.57
Breast Cancer
MCF7
MDA-MB-231/ATCC
BT-549
T-47D
MDA-MB-468
2.10
2.27
3.36
2.36
1.54
shifts (d) are reported in ppm downfield from internal TMS stan-
dard. ESI spectra were recorded on Micro mass, Quattro LC using
ESIþ software with capillary voltage 3.98 kV and ESI mode positive
ion trap detector. High-resolution mass spectra (HRMS) were
recorded on QSTAR XL Hybrid MS/MS mass spectrometer. Melting
Prostate Cancer
PC-3
DU-145
6.81
3.26

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A. Kamal et al. / European Journal of Medicinal Chemistry 46 (2011) 2427e2435
Fig. 1. Cell cycle analysis of 7l in A549 cells, A: Control cells, B: Cells treated with 7l for 24 h in 6.5 mM concentration, C: Cells treated with 7l for 48 h in 6.5 mM concentration.
points were determined with an Electrothermal melting point
apparatus, and are uncorrected.
4.1.1.3. N1-(2-Pyridylmethyl)-3,4-dimethoxybenzamide (3c). Yield
78%, 1H NMR (300 MHz, CDCl3):
(2H, m), 7.45 (1H, d, J ¼ 2.2 Hz), 7.39e7.31 (2H, m), 7.20 (1H, dd,
J ¼ 6.0, 5.2 Hz), 6.82 (1H, d, J ¼ 8.3 Hz), 4.70 (2H, d, J ¼ 5.2 Hz), 3.92
(3H, s), 3.90 (3H, s); MS (ESI): m/z 273 [M þ H]þ.
d
8.52 (d,1H, J ¼ 4.5 Hz), 7.70e7.62
4.1.1. General procedure for the synthesis of N1-(2-pyridylmethyl)-
substituted benzamides (3a-h)
To a stirred solution of 2-pyridylmethanamine (1) (1 mmol) in
dry THF was added triethylamine (1.1 mmol) followed by
substituted benzoylchlorides (2a-h) at 0 ꢁC. The reaction mixture
was stirred for 3 h and the reaction was monitored by TLC. After
completion of reaction, THF was removed under vacuum to get the
crude products. This was further purified by column chromatog-
raphy (EtOAc-Hexane) to get the pure compounds (3a-h).
4.1.1.4. N1-(2-Pyridylmethyl)-3,4,5-trimethoxybenzamide
(3d). Yield 76%, 1H NMR (300 MHz, CDCl3):
d 8.50 (d,1H,
J ¼ 4.5 Hz), 7.93e7.90 (1H, brs), 7.67 (1H, t, J ¼ 8.3, 7.5 Hz), 7.34
(1H, d, J ¼ 7.5 Hz), 7.19 (1H, dd, J ¼ 6.7, 5.2 Hz), 7.05 (2H, s), 4.68
(2H, d, J ¼ 5.2 Hz), 3.84 (6H, s), 3.82 (3H, s); MS (ESI): m/z 303
[M þ H]þ.
4.1.1.1. N1-(2-Pyridylmethyl)benzamide (3a). Yield 76%, 1H NMR
4.1.1.5. N1-(2-Pyridylmethyl)-4-methylbenzamide (3e). Yield 74%,
1H NMR (300 MHz, CDCl3):
d
8.35 (d,1H, J ¼ 4.5 Hz), 8.33 (1H, s),
(300 MHz, CDCl3):
d
8.53 (d,1H, J ¼ 4.7 Hz), 7.88 (2H, d, J ¼ 6.4 Hz),
7.67 (2H, t, J ¼ 7.7, 6.2 Hz), 7.47e7.39 (3H, m), 7.32 (1H, d, J ¼ 7.9 Hz),
7.20 (1H, t, J ¼ 5.0, 5.0 Hz), 4.73 (2H, d, J ¼ 4.7 Hz); MS (ESI): m/z 213
[M þ H]þ.
7.64 (2H, d, J ¼ 8.3 Hz), 7.32 (2H, d, J ¼ 8.3 Hz), 7.20 (1H, dd, J ¼ 6.7,
6.0 Hz), 6.83 (1H, t, J ¼ 8.3, 6.7 Hz), 4.70 (2H, d, J ¼ 5.0 Hz), 2.46 (3H,
s); MS (ESI): m/z 227 [M þ H]þ.
4.1.1.2. N1-(2-Pyridylmethyl)-4-methoxybenzamide (3b). Yield 80%,
4.1.1.6. N1-(2-Pyridylmethyl)-2-fluorobenzamide (3f). Yield 82%,
1H NMR (300 MHz, CDCl3):
d
8.56 (d,1H, J ¼ 4.8 Hz), 7.83 (2H, d,
1H NMR (400 MHz, CDCl3):
d
8.55 (d,1H, J ¼ 4.8 Hz), 8.12 (1H, t,
J ¼ 8.7 Hz), 7.68 (1H, t, J ¼ 7.8, 5.8 Hz), 7.47 (1H, brs), 7.32 (1H, d,
J ¼ 7.8 Hz), 7.21 (1H, t, J ¼ 5.8, 4.8 Hz), 6.93 (2H, d, J ¼ 8.7 Hz), 4.75
(2H, d, J ¼ 4.8 Hz), 3.85 (3H, s); MS (ESI): m/z 243 [M þ H]þ.
J ¼ 7.2, 5.6 Hz), 8.06e7.98 (1H, brs), 7.66 (1H, t, J ¼ 8.0, 5.6 Hz),
7.47e7.41 (1H, m), 7.32 (1H, d, J ¼ 8.0 Hz), 7.25 (1H, d, J ¼ 8.0 Hz),
7.19 (1H, dd, J ¼ 7.2, 4.8 Hz), 7.11 (1H, dd, J ¼ 8.0, 4.0 Hz), 4.77 (2H, d,
J ¼ 4.8 Hz); MS (ESI): m/z 231 [M þ H]þ.
4.1.1.7. N1-(2-Pyridylmethyl)-3-fluorobenzamide (3g). Yield 71%, 1H
NMR (300 MHz, CDCl3):
d
8.53 (d,1H, J ¼ 4.5 Hz), 7.80 (1H, brs),
7.70e7.57 (3H, m), 7.38 (1H, dd, J ¼ 8.3, 5.2 Hz), 7.32 (1H, d,
J ¼ 7.5 Hz), 7.22e7.14 (1H, m), 4.70 (2H, d, J ¼ 5.2 Hz); MS (ESI): m/z
231 [M þ H]þ.
4.1.1.8. N1-(2-Pyridylmethyl)-4-fluorobenzamide (3h). Yield 75%, 1H
NMR (300 MHz, CDCl3):
d
8.55 (d,1H, J ¼ 4.6 Hz), 8.00 (1H, brs), 7.72
(2H, d, J ¼ 8.1 Hz), 7.53 (1H, t, J ¼ 6.2, 6.0 Hz), 7.40 (2H, d, J ¼ 8.1 Hz),
7.25 (1H, m), 7.2 (1H, dd, J ¼ 6.2,5.6 Hz), 4.71 (2H, d, J ¼ 5.0 Hz); MS
(ESI): m/z 231 [M þ H]þ.
Fig. 2. Hoechst staining of 7l in A549 cells.

A. Kamal et al. / European Journal of Medicinal Chemistry 46 (2011) 2427e2435
2431
Fig. 3. Annexine V-FITC, (A) Control ells of A549 (B) A549 cells treated with 7l for 24 hrs-6.5
m
M(1 ꢂ IC50) (C) A549 cells treated with 7l for 48 hrs-6.5 M(1 ꢂ IC50).
m
4.1.2. General procedure for the synthesis of substituted phenyl
imidazo [1,5-a]pyridine (4a-h)
To N1-(2-pyridylmethyl)-substituted benzamides (3a-h),
added 4 mL of POCl3 and refluxed for 3 h. This was poured into
cold water and neutralized with NaHCO3 solution. This water
layer was extracted three times with ethylacetate. The combined
organic phases was dried over anhydrous Na2SO4 and evaporated
under vacuum. The residue, thus obtained was purified by
column chromatography using ethylacetate and hexane as
solvent system.

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A. Kamal et al. / European Journal of Medicinal Chemistry 46 (2011) 2427e2435
4.1.2.8. 3-(4-Fluorophenyl)imidazo [1,5-a]pyridine (4h). Yield 85%,
1H NMR (300 MHz, CDCl3):
d
8.16 (1H, d, J ¼ 7.5 Hz), 7.75 (2H, dd,
J ¼ 9.0, 5.2 Hz), 7.49 (1H, s), 7.47 (1H, d, J ¼ 9.0 Hz), 7.26e7.17 (2H,
m), 6.69 (1H, dd, J ¼ 9.0, 6.7 Hz), 6.53 (1H, t, J ¼ 6.7, 6.0 Hz); MS
(ESI): m/z 213 [M þ H]þ.
4.1.3. General procedure for the synthesis of 3-(substituted phenyl)
imidazo [1,5-a]pyridine-1-carbaldehyde (5a-h)
To an ice-water cooled solution of substituted phenyl imidazo
[1,5-a]pyridines (4a-h) (1 mmol) in DMF (1.4 mmol) was added
dropwise and with stirring phosphorus oxychloride (1.4 mmol). A
strongly exothermic reaction occurred. After 2 h heating on oil-
bath, the mixture was poured into ice water made basic with
concentrated ammonium hydroxide and extracted trice with eth-
ylacetate. The organic layer was dried over anhydrous sodium
sulfate and evaporated under vacuum. The residue, thus obtained
was purified by column chromatography using ethylacetate and
hexane as solvent system.
4.1.3.1. 3-Phenylimidazo [1,5-a]pyridine-1-carbaldehyde (5a). Yield
80%, 1H NMR (300 MHz, CDCl3):
d 10.10 (1H, s), 8.39 (2H, d,
Fig. 4. DNA laddering of 7l on A549 cells.
J ¼ 8.3 Hz), 7.77 (2H, d, J ¼ 6.6 Hz), 7.58e7.46 (3H, m), 7.22 (1H,
dd, J ¼ 9.4,6.7 Hz), 6.86 (1H, t, J ¼ 6.7 Hz); MS (ESI): m/z 223
[M þ H]þ.
4.1.2.1. 3-Phenylimidazo [1,5-a]pyridine (4a). Yield 78%, 1H NMR
(300 MHz, CDCl3):
d
8.24 (1H, d, J ¼ 7.5 Hz), 7.76 (2H, d, J ¼ 6.7 Hz),
4.1.3.2. 3-(4-Methoxyphenyl)imidazo [1,5-a]pyridine-1-carbaldehyde
7.50e7.36 (5H, m), 6.67 (1H, dd, J ¼ 9.0,6.7 Hz), 6.51 (1H, t, J ¼ 6.7,
(5b). Yield 76%, 1H NMR (300 MHz, CDCl3):
d 10.16 (1H, s), 8.34 (2H,
6.0 Hz); MS (ESI): m/z 195 [M þ H]þ.
d, J ¼ 8.7 Hz), 8.31 (1H, d, J ¼ 6.8 Hz), 7.26e7.23 (2H, dd, J ¼ 8.7 Hz),
7.07 (2H, d, J ¼ 8.7 Hz), 6.88 (1H, t, J ¼ 7.8, 6.8 Hz), 3.90 (3H, s); MS
(ESI): m/z 253 [M þ H]þ.
4.1.2.2. 3-(4-Methoxyphenyl)imidazo[1,5-a]pyridine(4b). Yield 80%,
1H NMR (300 MHz, CDCl3):
d
8.34 (1H, d, J ¼ 8.7 Hz), 8.32 (1H, d,
J ¼ 6.8 Hz), 7.71 (2H, d, J ¼ 8.7 Hz), 7.32e7.17 (1H, m), 7.07 (2H, d,
J ¼ 8.7 Hz), 6.98 (1H, s), 6.88 (1H, t, J ¼ 6.8 Hz), 3.90 (3H, s); MS
(ESI): m/z 225 [M þ H]þ.
4.1.3.3. 3-(3,4-Dimethoxyphenyl)imidazo [1,5-a]pyridine-1-carbalde-
hyde (5c). Yield 82%, 1H NMR (500 MHz, CDCl3):
d 10.12 (1H, s), 8.35
(2H, d, J ¼ 5.6 Hz), 7.30 (1H, s), 7.25 (1H, s), 7.21 (1H, t, J ¼ 7.2, 6.4 Hz),
6.96 (1H, d, J ¼ 8.0 Hz), 6.84 (1H, t, J ¼ 7.2, 6.2 Hz), 3.97 (3H, s), 3.95
(3H, s); MS (ESI): m/z 283 [M þ H]þ.
4.1.2.3. 3-(3,4-Dimethoxyphenyl)imidazo [1,5-a]pyridine (4c). Yield
76%, 1H NMR (500 MHz, CDCl3):
d
8.18 (1H, d, J ¼ 7.2 Hz), 7.47
(1H, s), 7.40 (1H, d, J ¼ 9.3 Hz), 7.30 (1H, s), 7.27 (1H, d,
J ¼ 8.3 Hz), 6.93 (1H, d, J ¼ 7.2 Hz), 6.64 (1H, dd, J ¼ 7.2, 6.2 Hz),
6.49 (1H, t, J ¼ 7.2, 6.2 Hz), 3.90 (3H, s), 3.87 (3H, s); MS (ESI): m/z
255 [M þ H]þ.
4.1.3.4. 3-(3,4,5-Trimethoxyphenyl)imidazo
[1,5-a]pyridine-1-car-
10.09 (1H,
baldehyde (5d). Yield 72%, 1H NMR (300 MHz, CDCl3):
d
s), 8.36 (1H, d, J ¼ 7.5 Hz), 8.31 (1H, d, J ¼ 9.0 Hz), 7.22 (1H, dd,
J ¼ 6.7, 5.2 Hz), 6.95 (2H, s), 6.87 (1H, t, J ¼ 6.7, 6.0 Hz), 3.94 (6H, s),
3.90 (3H, s); MS (ESI): m/z 313 [M þ H]þ.
4.1.2.4. 3-(3,4,5-Trimethoxyphenyl)imidazo [1,5-a]pyridine (4d). Yield
74%, 1H NMR (300 MHz, CDCl3):
d
8.39 (1H, d, J ¼ 7.5 Hz), 8.31 (1H, d,
4.1.3.5. 3-(4-Methylphenyl)imidazo [1,5-a]pyridine-1-carbaldehyde
J ¼ 9.0 Hz), 7.22 (1H, dd, J ¼ 9.0, 5.2 Hz), 7.08 (1H, s), 6.95 (2H, s), 6.87
(1H, t, J ¼ 6.7, 6.0 Hz), 3.94 (6H, s), 3.90 (3H, s); MS (ESI): m/z 285
[M þ H]þ.
(5e). Yield 75%, 1H NMR (300 MHz, CDCl3):
d 10.11 (1H, s), 8.32 (2H,
d, J ¼ 7.5 Hz), 7.64 (2H, d, J ¼ 8.3 Hz), 7.33 (2H, s, d, J ¼ 7.5 Hz), 7.20
(1H, dd, J ¼ 9.0, 6.7 Hz), 6.84 (1H, t, J ¼ 6.7, 6.0 Hz), 2.46 (3H, s); MS
(ESI): m/z 237 [M þ H]þ.
4.1.2.5. 3-(4-Methylphenyl)imidazo [1,5-a]pyridine (4e). Yield 82%,
1H NMR (300 MHz, CDCl3):
d
8.06 (1H, d, J ¼ 7.3 Hz), 7.51 (2H, d,
4.1.3.6. 3-(2-Fluorophenyl)imidazo [1,5-a]pyridine-1-carbaldehyde
J ¼ 8.1 Hz), 7.32 (1H, s), 7.29 (1H, d, J ¼ 9.0 Hz), 7.14 (2H, d,
J ¼ 7.9 Hz), 6.49 (1H, dd, J ¼ 6.4, 6.2 Hz), 6.33 (1H, t, J ¼ 7.3, 7.1 Hz),
2.27 (3H, s); MS (ESI): m/z 209 [M þ H]þ
(5f). Yield 83%, 1H NMR (400 MHz, CDCl3):
d 10.13 (1H, s), 8.37
(1H, d, J ¼ 8.8 Hz), 7.93 (1H, t, J ¼ 5.6, 5.2 Hz), 7.77 (1H, t, J ¼ 7.3,
7.1 Hz), 7.53 (1H, q, J ¼ 7.1, 6.7 Hz), 7.36 (1H, d, J ¼ 7.1 Hz), 7.30e7.24
(2H, m), 6.90 (1H, t, J ¼ 6.7, 6.6 Hz); MS (ESI): m/z 241 [M þ H]þ.
4.1.2.6. 3-(2-Fluorophenyl)imidazo [1,5-a]pyridine (4f). Yield 79%,
1H NMR (400 MHz, CDCl3):
d
7.79e7.72 (2H, m), 7.56 (1H, s),
4.1.3.7. 3-(3-Fluorophenyl)imidazo [1,5-a]pyridine-1-carbaldehyde
7.49e7.41 (2H, m), 7.30 (1H, t, J ¼ 7.2 Hz), 7.21 (1H, dd, J ¼ 9.7,
8.9 Hz), 6.74 (1H, dd, J ¼ 6.4, 5.6 Hz), 6.56 (1H, t, J ¼ 7.2, 6.4 Hz); MS
(ESI): m/z 213 [M þ H]þ.
(5g). Yield 78%, 1H NMR (400 MHz, CDCl3):
d 10.12 (1H, s), 8.37 (2H,
d, J ¼ 8.3 Hz), 7.60e7.50 (3H, m), 7.28e7.18 (2H, m), 6.90 (1H, t,
J ¼ 7.5, 6.0 Hz); MS (ESI): m/z 241 [M þ H]þ.
4.1.2.7. 3-(3-Fluorophenyl)imidazo [1,5-a]pyridine (4g). Yield 72%,
4.1.3.8. 3-(4-Fluorophenyl)imidazo [1,5-a]pyridine-1-carbaldehyde
1H NMR (400 MHz, CDCl3):
d
8.24 (1H, d, J ¼ 6.7 Hz), 7.57 (1H, d,
(5h). Yield 70%, 1H NMR (300 MHz, CDCl3):
d 10.11 (1H, s), 8.39 (1H,
J ¼ 7.5 Hz), 7.51e7.41 (4H, m), 7.07 (1H, t, J ¼ 9.8, 8.3 Hz), 6.70 (1H,
dd, J ¼ 9.0, 6.7 Hz), 6.54 (1H, t, J ¼ 6.7, 6.0 Hz); MS (ESI): m/z 213
[M þ H]þ.
d, J ¼ 9.0 Hz), 8.30 (1H, d, J ¼ 6.7 Hz), 7.78 (2H, t, J ¼ 8.3, 5.2 Hz),
7.29e7.18 (3H, m), 6.87 (1H, t, J ¼ 6.7 Hz); MS (ESI): m/z 241
[M þ H]þ.

A. Kamal et al. / European Journal of Medicinal Chemistry 46 (2011) 2427e2435
2433
4.1.4. General procedure for the synthesis of compounds 7a-r
The appropriate aldehyde (5a-h) (1 mmol) was dissolved in
methanol (50 mL) and treated with appropriate oxindole (6a-d)
(1 mmol) and piperidine (1 mL). the reaction mixture was refluxed
3 h and the precipitate formed on cooling was collected by filtra-
tion. Compounds were purified by column chromatography by
using chloroform and methanol as solvent system.
J ¼ 7.5 Hz), 3.92 (3H, s), 3.90 (3H, s); 13C NMR (150 MHz, DMSO-
d6):
d 170.01, 149.87, 149.07, 141.45, 140.06, 136.02, 128.09, 126.89,
126.16, 124.41, 123.97, 123.18, 122.95, 121.38, 120.68, 120.47,
119.98, 117.57, 115.46, 112.01, 111.46, 108.96, 55.67, 55.50; MS (ESI):
m/z 398 [M þ H]þ; HRMS calcd for C24H20N3O3 [M þ H]þ
398.1504, found 398.1495.
4.1.4.7. 3-(E)-1-[3-(3,4-Dimethoxyphenyl)imidazo [1,5-a]pyridin-1-
4.1.4.1. 3-[(E)-1-(3-Phenylimidazo [1,5-a]pyridin-1-yl)methylidene]-
yl]methylidene-1-methyl-2-indolinone
(7g). Yield
9.59 (1H, d, J ¼ 7.3 Hz), 8.37
74%,
mp:
2-indolinone (7a). Yield 70%, mp: 258e262 ꢁC; 1H NMR (300 MHz,
218e223 ꢁC; 1H NMR (300 MHz, CDCl3):
d
DMSO):
d
10.43 (1H, s), 9.42 (1H, d, J ¼ 7.7 Hz), 8.62 (1H, d,
(1H, d, J ¼ 7.1 Hz), 8.03 (1H, s), 7.97 (1H, d, J ¼ 9.2 Hz), 7.54 (1H, d,
J ¼ 6.2 Hz), 7.44 (1H, dd, J ¼ 8.3, 8.1 Hz), 7.29e7.23 (1H, m), 7.10e7.01
(3H, m), 6.86e6.75 (2H, m), 4.01 (3H, s), 3.99 (3H, s), 3.34 (3H, s); 13C
J ¼ 7.1 Hz), 8.21 (1H, d, J ¼ 9.0 Hz), 7.98 (2H, d, J ¼ 7.1 Hz), 7.85 (1H,
m), 7.74e7.55 (3H, m), 7.30 (1H, dd, J ¼ 6.7,6.6 Hz), 7.17 (1H, t, J ¼ 7.7,
7.5 Hz), 7.05e6.96 (2H, m), 6.84 (1H, d, J ¼ 7.5 Hz); 13C NMR
NMR (150MHz,CDCl3): d 150.08,149.76,149.16,141.36,140.02,136.03,
(150 MHz, DMSO-d6):
d 169.95, 141.46, 132.92, 129.80, 129.54,
128.06, 127.85, 126.48, 124.04, 122.85, 122.73, 122.33, 122.15, 121.63,
120.38, 120.00, 118.20, 114.68, 111.77, 111.09, 107.11, 56.00, 55.68,
26.12; MS (ESI): m/z 412 [M þ H]þ; HRMS calcd for C25H22N3O3
[M þ H]þ 412.1661, found 412.1643.
129.31, 128.17, 128.06, 127.16, 125.96, 125.87, 124.62, 123.78, 123.05,
120.99, 120.81, 120.17, 117.61, 115.55, 108.94; MS (ESI): m/z 338
[M þ H]þ; HRMS calcd for C22H16N3O [M þ H]þ 338.1288, found
338.1288.
4.1.4.8. 3-(E)-1-[3-(3,4,5-Trimethoxyphenyl)imidazo [1,5-a]pyridin-
4.1.4.2. 1-Methyl-3-[(E)-1-(3-phenylimidazo
methylidene]-2-indolinone (7b). Yield 65%, mp: 246e251 ꢁC;
1H NMR (500 MHz, CDCl3):
[1,5-a]pyridin-1-yl)
1-yl]methylidene-2-indolinone (7h). Yield 65%, mp: 223e228 ꢁC; 1H
NMR (300 MHz, CDCl3):
d
9.56 (1H, d, J ¼ 7.1 Hz), 8.42 (1H, d,
d
9.54 (1H, d, J ¼ 7.8 Hz), 8.37 (1H, d,
J ¼ 7.1 Hz), 8.03 (1H, s), 7.99 (1H, d, J ¼ 9.0 Hz), 7.31e7.23 (1H, m),
J ¼ 6.8 Hz), 8.04 (1H, s), 7.97 (1H, d, J ¼ 8.7 Hz), 7.93 (1H, d,
J ¼ 6.8 Hz), 7.61 (2H, t, J ¼ 7.8, 6.8 Hz), 7.54 (1H, d, J ¼ 7.8 Hz),
7.28e7.25 (2H, m), 7.12e7.07 (2H, m), 6.83 (1H, d, J ¼ 7.8 Hz), 6.80
7.15 (2H, s), 7.12e7.00 (2H, m), 6.86e6.80 (2H, m), 3.98 (6H, s), 3.97
(3H, s), 3.34 (3H, s); 13C NMR (150 MHz, CDCl3):
d 158.10, 156.20,
153.72, 143.01, 139.96, 136.03, 128.09, 127.97, 126.49, 124.77, 123.86,
122.91, 122.76, 122.61, 121.57, 120.52, 118.18, 114.79, 107.11, 105.42,
60.94, 56.22, 26.09; MS (ESI): m/z 442 [M þ H]þ; HRMS calcd for
C26H24N3O4 [M þ H]þ 442.1767, found 442.1749.
(1H, t, J ¼ 6.8 Hz), 3.34 (3H, s); 13C NMR (150 MHz, CDCl3):
d 140.19,
136.09, 134.57, 129.48, 129.42, 129.13, 129.07, 128.15, 128.06, 127.94,
126.52, 123.96, 122.99, 122.82, 122.62, 121.86, 120.55, 118.17, 114.71,
107.05, 26.15; MS (ESI): m/z 352 [M þ H]þ; HRMS calcd for
C23H18N3O [M þ H]þ 352.1449, found 352.1434.
4.1.4.9. 3-(E)-1-[3-(4-Methylphenyl)imidazo
methylidene-2-indolinone (7i). Yield 71%, mp: 309e315 ꢁC; 1H NMR
(300 MHz, DMSO):
[1,5-a]pyridin-1-yl]
4.1.4.3. 3-(E)-1-[3-(4-Methoxyphenyl)imidazo [1,5-a]pyridin-1-yl]
d
9.38 (1H, s), 8.39 (1H, d, J ¼ 7.5 Hz), 7.56 (1H, d,
methylidene-2-indolinone (7c). Yield 80%, mp: 286e291 ꢁC; 1H NMR
J ¼ 7.1 Hz), 7.28 (1H, s), 7.16 (1H, d, J ¼ 9.0 Hz), 6.86 (2H, d,
J ¼ 7.9 Hz), 6.47 (2H, d, J ¼ 8.1 Hz), 6.25 (1H, dd, J ¼ 6.6,6.4 Hz), 6.14
(1H, t, J ¼ 7.5, 6.7 Hz), 6.03e5.92 (2H, m), 5.81 (1H, d, J ¼ 7.5 Hz),
(500 MHz, DMSO):
d
10.23 (1H, s), 9.35 (1H, d, J ¼ 7.2 Hz), 8.49 (1H, d,
J ¼ 7.2 Hz), 8.05 (1H, d, J ¼ 9.3 Hz), 7.87 (2H, d, J ¼ 8.2 Hz), 7.80 (1H, s),
7.23e7.05 (4H, m), 6.97e6.86 (2H, m), 6.81 (1H, d, J ¼ 8.2 Hz), 3.90
(3H, s); MS (ESI): m/z 368 [M þ H]þ; HRMS calcd for C23H18N3O2
[M þ H]þ 368.1399, found 368.1383.
1.92 (3H, s); 13C NMR (150 MHz, DMSO-d6):
d 169.91, 141.44, 140.01,
139.25, 135.98, 129.80, 128.07, 127.94, 127.02, 126.15, 126.07, 124.45,
123.74, 123.04, 122.88, 120.72, 120.09, 117.57, 115.40, 108.88, 20.96;
MS (ESI): m/z 352 [M þ H]þ; HRMS calcd for C23H18N3O [M þ H]þ
352.1449, found 352.1442.
4.1.4.4. 3-(E)-1-[3-(4-Methoxyphenyl)imidazo [1,5-a]pyridin-1-yl]
methylidene-1-methyl-2-indolinone
(7d). Yield
76%,
mp:
257e262 ꢁC; 1H NMR (500 MHz, CDCl3):
d
9.53 (1H, d, J ¼ 6.8 Hz),
4.1.4.10. 5-Fluoro-3-(E)-1-[3-(4-methylphenyl)imidazo
idin-1-yl]methylidene-2-indolinone (7j). Yield67%, mp: 330e335 ꢁC;
1H NMR (300 MHz, DMSO):
[1,5-a]pyr-
8.29 (1H, d, J ¼ 7.8 Hz), 8.02 (1H, s), 7.93 (1H, d, J ¼ 8.7 Hz), 7.83 (2H,
d, J ¼ 7.8 Hz), 7.33e7.21 (1H, m), 7.20e7.00 (4H, m), 6.83 (1H, d,
J ¼ 7.8 Hz), 6.76 (1H, t, J ¼ 6.8 Hz), 3.92 (3H, s), 3.34 (3H, s); MS
(ESI): m/z 382 [M þ H]þ; HRMS calcd for C24H20N3O2 [M þ H]þ
382.1555, found 382.1541.
d
9.57 (1H, s), 8.51 (1H, d, J ¼ 7.8 Hz), 7.78
(1H, d, J ¼ 6.9 Hz), 7.38 (1H, d, J ¼ 8.6 Hz), 7.13e6.96 (3H, m), 6.62 (2H,
d, J ¼ 7.9 Hz), 6.46 (1H, t, J ¼ 8.8, 6.9 Hz), 6.27e6.07 (2H, m), 6.02e5.90
(1H, m), 2.03 (3H, s); 13C NMR (150 MHz, CDCl3):
d 149.81, 141.39,
139.92, 139.19, 135.78, 129.60, 127.95, 127.81, 127.00, 126.11, 126.00,
124.32, 123.64, 122.97, 122.82, 120.70, 121.10, 117.32, 115.10, 107.13,
27.12, 20.90; MS (ESI): m/z 370 [M þ H]þ; HRMS calcd for C23H17FN3O
[M þ H]þ 370.1350, found 370.1367.
4.1.4.5. 5-Fluoro-3-(E)-1-[3-(4-methoxyphenyl)imidazo
idin-1-yl]methylidene-2-indolinone (7e). Yield 82%, mp: 317e321 ꢁC;
1H NMR (500 MHz, DMSO):
[1,5-a]pyr-
d
10.03 (1H, s), 8.98 (1H, d, J ¼ 8.8 Hz),
8.20 (1H, d, J ¼ 6.8 Hz), 7.83 (1H, d, J ¼ 9.7 Hz), 7.53 (3H, t, J ¼ 8.8,
7.8 Hz), 6.94 (1H, t, J ¼ 8.8, 6.8 Hz), 6.84 (2H, d, J ¼ 7.8 Hz), 6.67e6.57
(2H, m), 6.44 (1H, dd, J ¼ 8.8, 7.8 Hz), 3.92 (3H, s); 13C NMR (150 MHz,
4.1.4.11. 5-Fluoro-1-methyl-3-(E)-1-[3-(4-methylphenyl)imidazo
[1,5-a]pyridin-1-yl]methylidene-2-indolinone (7k). Yield 83%, mp:
DMSO-d6):
d 169.92, 160.23, 155.84, 140.31, 137.53, 136.41, 129.54,
293e297 ꢁC; 1H NMR (300 MHz, CDCl3):
d
8.56 (1H, d, J ¼ 7.0 Hz),
126.69, 124.91, 124.27, 123.87, 121.05, 117.52, 115.49, 114.65, 113.95,
113.63, 113.05, 112.69, 109.06, 55.36; MS (ESI): m/z 386 [M þ H]þ;
HRMS calcd for C23H17FN3O2 [M þ H]þ 386.1304, found 386.1288.
7.92 (1H, d, J ¼ 7.1 Hz), 7.54 (1H, d, J ¼ 8.3 Hz), 7.33e7.01 (3H, m),
6.82 (2H, d, J ¼ 8.0 Hz), 6.71e6.52 (1H, t, J ¼ 7.8,6.8 Hz), 6.23e6.20
(2H, m), 6.12e6.00 (1H, m), 3.32 (3H, s), 1.98 (3H, s); MS (ESI): m/z
384 [M þ H]þ; HRMS calcd for C24H19FN3O [M þ H]þ 384.1512,
found 384.1499.
4.1.4.6. 3-(E)-1-[3-(3,4-Dimethoxyphenyl)imidazo [1,5-a]pyridin-1-
yl]methylidene-2-indolinone(7f). Yield 68%, mp: 258e263 ꢁC; 1H
NMR (300 MHz, DMSO):
d
10.44 (1H, s), 9.47 (1H, d, J ¼ 7.5 Hz),
8.66 (1H, d, J ¼ 6.9 Hz), 8.18 (1H, d, J ¼ 9.2 Hz), 7.85 (1H, s), 7.57
4.1.4.12. 3-(E)-1-[3-(2-Fluorophenyl)imidazo [1,5-a]pyridin-1-yl]meth-
(2H, m), 7.35e7.16 (3H, m), 7.05e6.95 (2H, m), 6.87 (1H, d,
ylidene-2-indolinone (7l). Yield 78%, mp: 288e293 ꢁC; 1H NMR

2434
A. Kamal et al. / European Journal of Medicinal Chemistry 46 (2011) 2427e2435
(300 MHz, DMSO):
d
9.43 (1H, s), 8.31 (1H, d, J ¼ 7.5 Hz), 7.23 (2H, d,
4.2. Cell culture
J ¼ 9.6 Hz), 6.94e6.86 (2H, m), 6.71 (1H, q, J ¼ 7.3, 6.4 Hz), 6.54 (2H, q,
J ¼ 7.9, 6.7 Hz), 6.32 (1H, dd, J ¼ 6.7,6.4 Hz), 6.15 (1H, t, J ¼ 8.3, 7.7 Hz),
6.03 (1H, t, J ¼ 7.1, 6.9 Hz), 5.92 (1H, t, J ¼ 7.7, 6.9 Hz), 5.84 (1H, d,
The antiproliferative activities of the compounds were deter-
mined using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl
tetrazolium bromide) reduction assay [26]. 1 ꢂ 104 cells/well were
seeded in 100 mL DMEM (Dulbecco’s Modified Eagle’s Medium)
supplemented with 10% FBS in each well of 96-well microculture
plates and incubated for 24 h at 37 ꢁC in a CO2 incubator.
Compounds, diluted to the desired concentrations in culture
medium. After 24 h of incubation, media was removed and to each
J ¼ 7.7 Hz); 13C NMR (150 MHz, DMSO-d6):
d 169.90, 158.30, 143.03,
136.02, 135.68, 132.27, 131.71, 128.26, 126.13, 125.44, 124.68, 124.10,
122.90, 122.73, 120.76, 120.58, 117.44, 116.86, 117.74, 116.48, 115.37,
108.92; MS (ESI): m/z 356 [M þ H]þ; HRMS calcd for C22H15FN3O
[M þ H]þ 356.1199, found 356.1208.
4.1.4.13. 3-(E)-1-[3-(2-Fluorophenyl)imidazo [1,5-a]pyridin-1-yl]meth-
well 10
incubated for 4 h. Supernatant from each well was carefully
removed, formazon crystals were dissolved in 100 L of DMSO and
ml of MTT (5 mg/mL) was added and the plates were further
ylidene-1-methyl-2-indolinone (7m). Yield 72%, mp: 260e265 ꢁC; 1H
NMR (300 MHz, CDCl3):
d
8.37 (1H, d, J ¼ 7.2 Hz), 7.52 (2H, d,
m
J ¼ 8.2 Hz), 7.20e6.90 (2H, m), 6.85 (1H, m), 6.58 (2H, q, J ¼ 6.8,
6.2 Hz), 6.42 (1H, m), 6.21 (1H, t, J ¼ 6.3, 6.0 Hz), 6.09 (1H, t, J ¼ 7.1,
6.0 Hz), 6.01 (1H, t, J ¼ 7.2, 6.0 Hz), 5.89 (1H, d, J ¼ 6.3 Hz), 3.52 (3H, s);
absorbance at 540 nm of wavelength was recorded.
4.3. In vitro growth percentage
13C NMR (150 MHz, CDCl3):
d 161.61, 158.29, 143.03, 136.02, 135.74,
132.16, 131.69, 131.59, 128.03, 126.50, 125.06, 123.89, 123.55, 123.46,
123.21, 122.76, 121.89, 117.79, 116.47, 116.18, 114.48, 107.09, 26.14; MS
(ESI): m/z 370 [M þ H]þ; HRMS calcd for C23H17FN3O [M þ H]þ
370.1355, found 370.1371.
The screening of anticancer compounds in National Cancer
Institute is a two step process. In first step the evaluation of all
compounds against the 60 cell lines at a single concentration of
10ꢀ5 M. Compounds which exhibit significant growth inhibition are
selected for second step. In second step compounds are evaluated
against the 60 cell panel at five concentration levels by the NCI
according to standard procedures (http://dtp.nci.nih.gov/branches/
btb/ivclsp.html).
4.1.4.14. 5-Fluoro-3-(E)-1-[3-(2-fluorophenyl)imidazo [1,5-a]pyridin-
1-yl]methylidene-2-indolinone (7n). Yield 65%, mp: 341e346 ꢁC; 1H
NMR (300 MHz, DMSO):
d
9.62 (1H, s), 8.42 (1H, d, J ¼ 7.9 Hz), 7.44
(2H, d, J ¼ 8.6 Hz), 7.12e7.03 (2H, m), 6.99e6.81 (1H, m), 6.78e6.61
(2H, m), 6.54 (1H, dd, J ¼ 6.9,6.4 Hz), 6.24 (1H, t, J ¼ 7.1, 6.4 Hz), 6.15
(1H, t, J ¼ 9.2, 8.8 Hz), 6.01e5.93 (1H, m); MS (ESI): m/z 374
[M þ H]þ; HRMS calcd for C22H14F2N3O [M þ H]þ 374.1099, found
374.1113.
4.4. Cytotoxicity assay in HEK-293
Cytotoxic effects of the compounds were determined using MTT
(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide)
reduction assay [26].1 ꢂ104 cells/well were seeded in 100
ml DMEM
4.1.4.15. 5-Fluoro-3-(E)-1-[3-(2-fluorophenyl)imidazo [1,5-a]pyridin-
1-yl]methylidene-1-methyl-2-indolinone (7o). Yield 60%, mp:
supplemented with 10% FBS in each well of 96-well microculture
plates and incubated for 24 h at 37 ꢁC in a CO2 incubator. Compound
(7l), diluted to the desired concentrations in culture medium. After
297e302 ꢁC; 1H NMR (300 MHz, CDCl3):
d
8.62 (1H, d, J ¼ 8.0 Hz),
7.52 (2H, d, J ¼ 7.9 Hz), 7.11e7.08 (2H, m), 7.03e6.94 (1H, m),
6.87e6.72 (2H, m), 6.64 (1H, dd, J ¼ 6.8,6.5 Hz), 6.32 (1H, t, J ¼ 7.2,
6.8 Hz), 6.23 (1H, m), 6.11e5.98 (1H, m), 3.54 (3H, s); MS (ESI): m/z
388 [M þ H]þ; HRMS calcd for C23H16F2N3O [M þ H]þ 388.1261,
found 388.1249.
48 h of incubation, media were removed and to each well 10 ml of
MTT (5 mg/mL) was added and the plates were further incubated
for 4 h. Supernatant from each well was carefully removed, for-
mazon crystals were dissolved in 100
540 nm wavelength was recorded.
ml of DMSO and absorbance at
4.1.4.16. 3-(E)-1-[3-(3-Fluorophenyl)imidazo
methylidene-1-methyl-2-indolinone (7p). Yield 72%, mp: 228e232 ꢁC;
1H NMR (300 MHz, CDCl3):
[1,5-a]pyridin-1-yl]
4.5. Cell cycle analysis
d
9.43 (1H, d, J ¼ 7.5 Hz), 8.39 (1H, d,
To determine the effect of compound on the stages of cell cycle,
J ¼ 7.1 Hz), 8.01e7.95 (2H, m), 7.72e7.51 (3H, m), 7.29e7.19 (2H, m),
A549 cells (1 ꢂ106) were seeded in six-well plates and treated with
7.10 (2H, t, J ¼ 8.6, 6.7 Hz), 6.84e6.80 (2H, m), 3.34 (3H, s); 13C NMR
compound 7l at a final concentration of 6.5 mM for 24 h and 48 h.
(150 MHz, CDCl3):
d
161.41, 143.06, 136.19, 130.90, 130.78, 128.49,
After 24 h and 48 h treatments, both floating and trypsinized
adherent cells were collected and fixed with 70% ethanol. After
fixation cells were washed with PBS and stained with 50 mg/mL
propidium iodide in hypotonic lysis buffer (0.1% sodium citrate,
0.1% Triton X-100) containing DNase-free RNase-A for 20 min.
Stained cells were analyzed using fluorescence-activated cell sorter
caliber (Becton Dickinson) [26].
128.17, 126.47, 123.72, 123.59, 123.56, 123.20, 122.70, 122.46, 120.98,
118.29,116.55,116.26,115.31,115.09,115.00,107.19, 26.17; MS (ESI): m/
z 370 [M þ H]þ; HRMScalcd for C23H17FN3O [Mþ H]þ 370.1355, found
370.1366.
4.1.4.17. 3-(E)-1-[3-(4-Fluorophenyl)imidazo
methylidene-2-indolinone (7q). Yield 68%, mp: 290e295 ꢁC; 1H NMR
(300 MHz, DMSO):
[1,5-a]pyridin-1-yl]
d
10.12 (1H, s), 9.30 (1H, d, J ¼ 7.9 Hz), 8.44 (1H, d,
4.6. Morphological analysis for apoptosis with Hoechst staining
J ¼ 6.6 Hz), 8.05e7.77 (4H, m), 7.34 (2H, t, J ¼ 8.3 Hz), 7.13 (2H, q,
J ¼ 8.3, 8.1 Hz), 6.98e6.78 (3H, m); MS (ESI): m/z 356 [M þ H]þ;
HRMS calcd for C22H15FN3O [M þ H]þ 356.1193, found 356.1209.
Cells were seeded at a density of 10,000 cells over 18-mm
coverslips and incubated for 24 h. Then, the medium was
replaced, and cells were treated with 6.5 mM 24 h and 48 h. Cells
4.1.4.18. 5-Fluoro-3-(E)-1-[3-(4-fluorophenyl)imidazo [1,5-a]pyridin-
1-yl]methylidene-2-indolinone (7r). Yield 81%, mp: 335e340 ꢁC; 1H
treated with vehicle (0.001% DMSO) were included as controls
for all experiments. After overnight treatment, Hoechst 33342
(SigmaeAldrich) were added to medium at a concentration of
NMR (300 MHz, DMSO):
d
10.14 (1H, s), 9.47 (1H, d, J ¼ 7.2 Hz), 8.52
(1H, d, J ¼ 6.2 Hz), 8.22e8.02 (3H, m), 7.67 (2H, t, J ¼ 8.1 Hz), 7.32
(2H, q, J ¼ 8.2, 7.9 Hz), 7.02e6.94 (3H, m); MS (ESI): m/z 374
[M þ H]þ; HRMS calcd for C22H14F2N3O [M þ H]þ 374.1099, found
374.1097.
0.5 mg/mL containing 4% paraformaldehyde. After incubation for
30 min 37 ꢁc, cells from each dish were captured from randomly
selected fields under fluorescent microscope (Leica, Germany) to
qualitatively determine the proportion of viable and apoptotic

A. Kamal et al. / European Journal of Medicinal Chemistry 46 (2011) 2427e2435
2435
cells based on their relative fluorescence and nuclear fragmen-
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Acknowledgments
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We thank the National Cancer Institute, Bethesda, for anticancer
assay in human cell lines. The authors (G. R. K, P. R, A. V. S, and A. V)
thank CSIR and UGC, New Delhi for financial assistance.
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