Synthesis and anticancer activity of N -substituted 2-arylquinazolinones bearing trans -stilbene scaffold

Article abstract of DOI:10.1016/j.ejmech.2015.03.057

A novel series of 2-arylquinazolinones 7a-o bearing trans-stilbene moiety were designed, synthesized, and evaluated against human breast cancer cell lines including human breast adenocarcinoma (MCF-7 and MDA-MB-231) and human ductal breast epithelial tumor (T-47D). Among the tested compounds, the sec-butyl derivative 7h showed the best profile of activity (IC₅₀ < 5 μM) against all cell lines, being 2-fold more potent than standard drug, etoposide. Our investigation revealed that the cytotoxic activity was significantly affected by N3-alkyl substituents. Furthermore, the morphological analysis by acridine orange/ethidium bromide double staining test and flow cytometry analysis indicated that the prototype compound 7h can induce apoptosis in MCF-7 and MDA-MB-231 cells.

Full text of DOI:10.1016/j.ejmech.2015.03.057

European Journal of Medicinal Chemistry 95 (2015) 492e499
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and anticancer activity of N-substituted 2-
arylquinazolinones bearing trans-stilbene scaffold
,
e
Mohammad Mahdavi a, Keyvan Pedrood b, Maliheh Safavi c, Mina Saeedi d
,
Mahboobeh Pordeli f, Sussan Kabudanian Ardestani f, Saeed Emami g, Mehdi Adib b,
,
*
Alireza Foroumadi a, Abbas Shafiee a
a Department of Medicinal Chemistry, Faculty of Pharmacy and Pharmaceutical Sciences Research Center, Tehran University of Medical Sciences, Tehran
14176, Iran
b School of Chemistry, College of Science, University of Tehran, PO Box 14155-6455, Tehran, Iran
c Department of Biotechnology, Iranian Research Organization for Science and Technology, Tehran, Iran
d Medicinal Plants Research Center, Tehran University of Medical Sciences, Tehran, Iran
e Persian Medicine and Pharmacy Research Center, Tehran University of Medical Sciences, Tehran, Iran
f Department of Biochemistry, Institute of Biochemistry and Biophysics, University of Tehran, Tehran, Iran
g Department of Medicinal Chemistry and Pharmaceutical Sciences Research Center, Faculty of Pharmacy, Mazandaran University of Medical Sciences, Sari,
Iran
a r t i c l e i n f o
a b s t r a c t
Article history:
A novel series of 2-arylquinazolinones 7a-o bearing trans-stilbene moiety were designed, synthesized,
and evaluated against human breast cancer cell lines including human breast adenocarcinoma (MCF-7
and MDA-MB-231) and human ductal breast epithelial tumor (T-47D). Among the tested compounds, the
Received 7 February 2015
Received in revised form
7 March 2015
Accepted 25 March 2015
Available online 26 March 2015
sec-butyl derivative 7h showed the best profile of activity (IC50 < 5 mM) against all cell lines, being 2-fold
more potent than standard drug, etoposide. Our investigation revealed that the cytotoxic activity was
significantly affected by N3-alkyl substituents. Furthermore, the morphological analysis by acridine or-
ange/ethidium bromide double staining test and flow cytometry analysis indicated that the prototype
compound 7h can induce apoptosis in MCF-7 and MDA-MB-231 cells.
Keywords:
Anti-cancer
Cytotoxic agents
3H-quinazolin-4-one
trans-stilbene
© 2015 Elsevier Masson SAS. All rights reserved.
1. Introduction
antimicrobial [6e8], anti-virus [9], antimalarial [10], anti-
tuberculosis [11], antioxidant [12], anti-hypertensive [13,14], anti-
Cancer has been emerged as a major health problem and an
important cause of mortality and morbidity worldwide [1]. Several
main factors such as incorrect diet, genetic predisposition, and
environmental contaminants contribute to the outbreak of cancers
[2]. Although major advances in cellular and molecular biology
have led to the improvement of chemotherapeutic management of
cancer, the continued efforts to discover new anticancer agents in
order to overcome resistance and toxicity concerns, is necessary [3].
To develop novel lead compounds as efficient anticancer agents,
quinazoline derivatives attracted our attention. Quinazolines are
considered as important bioactive scaffold due to their diverse
convulsant [15,16], anti-psychotic [17], anti-diabetes [18], and
antihyperlipidemic [19] activities. In particular, many quinazoline
derivatives have been reported as anti-cancer agents [20e26].
Furthermore, some quinazolinone analogs demonstrated potent
anticancer activity via inhibition of dihydrofolate reductase enzyme
[27]. Xia et al. reported a series of 2-aryl quinazolinones (Fig. 1) as
antitumor agents and inhibitor of tubulin polymerization [28].
Accordingly the quinazoline scaffold is considered as an attractive
heterocycle in the field of anti-cancer drug design and
development.
In continuation of our efforts to develop anti-cancer agents
[29e31], herein, we decided to investigate novel series of 3H-qui-
nazolin-4-one derivatives bearing trans-stilbene moiety at 2-
position. For this purpose, various 2-aryl quinazolinones 7a-o
were designed, synthesized and evaluated for their cytotoxic ac-
tivity. Also, the structure-activity relationship of the corresponding
biological
activities
including
anti-inflammatory
[4,5],
* Corresponding author.
E-mail address: ashafiee@ams.ac.ir (A. Shafiee).
http://dx.doi.org/10.1016/j.ejmech.2015.03.057
0223-5234/© 2015 Elsevier Masson SAS. All rights reserved.

M. Mahdavi et al. / European Journal of Medicinal Chemistry 95 (2015) 492e499
493
tested compounds were listed in Table 1, compared with the
standard drug etoposide.
Structurally, tested compounds can be classified as N-alkyl, N-
aryl, and N-benzyl derivatives (7a-i, 7j-l, and 7m-o respectively). In
general, N-alkyl derivatives 7a and 7c-h showed significant cyto-
toxic activity against all cell lines. Among the N-aryl (7j-o) and N-
benzyl derivatives (7m-o), only compound 7n (R
methylbenzyl) was active against all cell lines and also, N-phenyl
analog 7j showed inhibitory activity against MCF-7 cells.
¼
4-
The obtained IC50 values related to MCF-7 and T-47D cells
revealed that compound 7h had the highest activity against these
cell lines. In the case of MDA-MB-231 cells, compounds 7e and 7f
were the most potent compounds (IC50s ¼ 4.1 and 4.0
mM,
Fig. 1. Structures of 2-aryl quinazolines and designed copounds 7a-o as cytotoxic
agents.
respectively). The activities of compound 7c, 7d, and 7h against
MCF-7 were superior to that of standard drug, etoposide. Com-
pounds 7a and 7c-h with IC50 values of 4.0e8.4 mM were more
compounds was studied (Fig. 1).
potent than etoposide against MDA-MB-231 cells. Against T-47D,
compounds 7c-f and 7h showed higher activities when compared
to etoposide. In particular, compounds 7h displayed IC50 values of
2. Results and discussion
3.8, 4.9 and 3.6
lines, being 2-fold less than those of etoposide (IC50s ¼ 7.6, 10.3 and
8.9 M, respectively).
As mentioned above, N-alkyl substituent was more biologically
favored than N-aryl and N-benzyl groups. However, N-cyclopentyl
analog 7i showed no activity towards all cell lines and N-propyl
derivative 7b was active only against MCF-7. Interestingly, the
susceptibility of all tested cell lines to the most of N-alkyl quina-
zolinones was similar and the observed IC50s showed no significant
differences. However, in the case of compound 7g, the suscepti-
bility of MCF-7 was significantly less than MDA-MB-231 and T-47D.
In contrast, MCF-7 cells were susceptible to compounds 7b at
mM against MCF-7, MDA-MB-231, and T-47D cell
2.1. Chemistry
m
The synthetic route to target compounds 7 was illustrated in
Scheme 1. First, 2-bromobenzaldehyde (1) reacted with styrene (2)
in the presence of palladium (II) acetate, and sodium acetate to give
trans-stilbene 3 [32]. Also, the anthranilamide derivatives 5 were
prepared from the reaction of isatoic anhydride (4) and appropriate
primary amine in water at room temperature [33]. The reaction of
compound 5 with aldehyde 3 in the presence of potassium car-
bonate in refluxing methanol afforded the corresponding 2,3-
dihydroquinazolinone 6. Finally, compounds 6a-o were converted
to quinazolinone derivatives 7a-o using tetrabutylammonium
bromide (TBAB) and potassium tert-butoxide in dry THF.
concentrations less than 30
mM but MDA-MB-231 and T-47D were
resistant to this compound (IC50 > 100
mM).
The IC50 values of compounds 7j-o demonstrated that the aryl or
benzyl substituents were not favorable from anti-cancer activity
point of view; however, 4-methyl substitution on benzyl moiety in
compound 7n, showed mild activity against all cells (IC50
2.2. Cytotoxic activity
The cytotoxic activity of final compounds 7a-o was evaluated
against three human cancer cell lines including MCF-7, MDA-MB-
231, and T-47D using MTT assay. The obtained IC50 values for the
values ¼ 23.8e28.2
mM). It is speculated that the substituent at the
Table 1
Cytotoxic activities (IC50s,
mM) of compounds 7a-o against human cancer cell lines.
O
R
N
N
Ph
Compound
R
MCF-7
MDA-MB-231
T-47D
7a
7b
7c
7d
7e
7f
7g
7h
Ethyl-
Propyl-
Isopropyl-
Cyclopropyl-
Allyl-
Butyl-
Isobutyl-
sec-Butyl-
Cyclopentyl-
Phenyl-
8.7 1.4
29.4 2.8
6.7 2.8
5.3 1.3
10.3 2.8
9.8 1.8
23.3 1.3
3.8 2.1
>100
8.4 2.1
>100
10.2 2.3
>100
8.4 0.1
5.5 0.8
4.1 2.2
4.0 0.9
6.9 0.4
4.9 1.6
>100
6.2 2.1
6.8 2.4
5.8 1.1
7.9 1.8
9.3 2.6
3.6 1.2
>100
7i
7j
7k
44.9 0.9
>100
>100
>100
>100
>100
p-Tolyl-
7l
7m
4-Methoxyphenyl-
Benzyl-
>100
>100
>100
>100
>100
>100
7n
7o
4-Methylbenzyl-
4-Methoxybenzyl-
24.7 1.1
>100
23.8 0.2
>100
28.2 1.5
>100
Etoposide
7.6 2.1
10.3 1.0
8.9 2.1
Scheme 1. Synthesis of compounds 7.

494
M. Mahdavi et al. / European Journal of Medicinal Chemistry 95 (2015) 492e499
N-3 position of quinazolinone ring interacts with a hydrophobic
pocket in the target binding site by means of van der Waals in-
teractions. Varying the length and bulk of the substituent (e.g. alkyl,
aryl or benzyl groups) allows us to probe the depth and width of the
pocket. It seems the depth and width of the pocket is favorable for
N-alkyl groups such as ethyl, isopropyl, cyclopropyl, butyl, isobutyl,
sec-butyl or allyl, but more bulky substituents (e.g., cyclopentyl,
aryl or benzyl groups) may prevent the molecule from binding to
the target. Furthermore, a great variation between isobutyl and sec-
butyl derivatives (compounds 7g and 7h, respectively) was
observed in biological activity against MCF-7. This could be due to
the different bond connectivity of isobutyl and sec-butyl groups to
the N-3 position which resulted in different steric effect on 2-aryl
moiety.
2.4. Flow cytometry analysis
Flow cytometry analyses were performed to confirm apoptosis
induced by representative compounds 7d and 7h in comparison to
standard drug etoposide. Annexin V-FITC/PI double staining fol-
lowed by flow cytometric analysis detects the externalization of
phosphatidylserine in apoptotic cells using recombinant annexin V
conjugated to green-fluorescent FITC dye and dead cells using
propidium iodide (PI). Flow cytometric analysis revealed that cells
undergo apoptosis after treatment with the test compounds. As
shown in Figs. 4 and 5, the apoptotic index of the compounds 7d
and 7h were compared with negative control and etoposide in
MDA-MB-231 and MCF-7 cell lines. Although the tested com-
pounds have caused necrosis in treated cells to some extent the
results showed that the exposure of MDA-MB-231 cells to IC50
concentrations of 7h and 7d induced apoptosis in 16.55% and
22.41% of the cells after 12 h, respectively. The apoptosis inducing
effect of compound 7d in MDA-MB-231 cells is comparable with
etoposide apoptosis inducing ability. The results also revealed that
the percentage of MCF-7 cells undergoing apoptosis after 12 h of
exposure to compounds 7h and 7d was higher than MDA-MB-231
(26.46% and 25.88% apoptosis in the 7h and 7d-treated cells,
respectively). According to these data the cytotoxic activity of
compounds 7h and 7d in MDA-MB-231 and MCF-7 cell lines occurs
through apoptosis.
2.3. Morphological analysis
Based on the molecular classification of breast carcinoma, MCF-
7 and T-47D are luminal A cell lines and MDA-MB-231 is a triple
negative breast cancer cell line. In this study, MCF-7 and MDA-MB-
231 cells were selected as a representative for each class. Also, two
potent compounds 7d and 7h which showed very good activity
against all three cancer cell lines were nominated for more detailed
investigations. Accordingly, compounds 7d and 7h were investi-
gated in comparison with etoposide for identification of apoptosis
induced by MCF-7 and MDA-MB-231 cells.
Morphological analysis of 7d and 7h-treated cells was accom-
plished using fluorescence microscopy after double staining with
acridine orange/ethidium bromide. As shown in Figs. 2 and 3,
morphological findings indicated that the compounds 7d and 7h
reduced cell viability and induced apoptosis in MCF-7 and MDA-
MB-231 cells.
3. Conclusion
We described the synthesis of N3-substituted quinazolinones
7a-o bearing trans-stilbene moiety as anti-proliferative agents
against cancer cell lines. The results of cytotoxic assay against MCF-
7, MDA-MB-231 and T-47D cells revealed that N3-alkyl derivatives
had significant inhibitory activity against all tested cell lines. In
contrast, the most of N3-aryl and N3-benzyl analogs were inactive.
Among the tested compounds, the sec-butyl derivative 7h showed
Fig. 2. Morphological analysis of MCF-7 cells by double staining method. a) Control condition, b) cells treated with IC50 value of etoposid for 24 h, c) cells treated with IC50 value of
7h for 24 h. d) cells treated with IC50 value of 7d for 24 h. White arrow indicates live cells, dashed arrow shows apoptosis.

M. Mahdavi et al. / European Journal of Medicinal Chemistry 95 (2015) 492e499
495
Fig. 3. Morphological analysis of MDA-MB-231 cells by double staining method. a) Control condition, b) cells treated with IC50 value of etoposid for 24 h, c) cells treated with IC50
value of 7h for 24 h. d) cells treated with IC50 value of 7d for 24 h. White arrow indicates live cells, dashed arrow shows apoptosis.
the best profile of activity as its IC50 was less than 5
m
M for all tested
4.1.1. General procedure for the preparation of (E)-2-
styrylbenzaldehyde (3)
cell lines. It was two times more potent than standard drug, eto-
poside. In addition to compound 7h, the activities of isopropyl and
cyclopropyl derivatives (compounds 7c and 7d, respectively) were
higher than that of etoposide against all cell lines. The results of our
study revealed that the N3-substituted quinazolinones having
trans-stilbene moeity could be considered as a new bioactive
structure and the optimum cytotoxic potency of the molecule
would be achieved by introduction of an appropriate alkyl group
such as cyclopropyl, isopropyl or sec-butyl on the 3-position of
quinazoline ring. It seems that the substituent at the N-3 position of
quinazolinone ring interacts with a hydrophobic pocket in the
target binding site by means of van der Waals interactions. Thus,
the length and bulk of the optimum substituents such as cyclo-
propyl, isopropyl or sec-butyl allows to favorable fitting to the
pocket. However, more bulky substituents such as cyclopentyl, aryl
or benzyl groups may prevent the prototype molecule from binding
to the target and results in diminishing the activity.
A mixture of 2-bromobenzaldehyde (1, 1 mmol), styrene (2,
2 mmol), palladium(II) acetate (0.01 mmol), and sodium acetate
(4 mmol) in polyethylene glycol (3 mL) was heated at 100 ꢀC. After
3 h, the reaction mixture was poured into water (20 mL), extracted
with diethyl ether (3 ꢁ 20 mL) and purified by column chroma-
tography on silica gel (petroleum ether/ethyl acetate, 15:1) (yield
90%).
4.1.2. General procedure for the preparation of 2-aminobenzamides
5a-o
A mixture of isatoic anhydride (4, 10 mmol) and appropriate
amine (10 mmol) in water (20 mL) was stirred for 3e5 h at room
temperature. After completion of the reaction (checked by TLC), the
resulting off-white benzamides 5a-o were filtered off and used for
the next reaction without further purifications (yields 85e95%).
4.1.3. General procedure for the preparation of 2,3-
dihydroquinazolin-4(1H)-one derivatives 6a-o
A
mixture of 2-aminobenzamide 5a-o (1 mmol), (E)-2-
4. Experimental
styrylbenzaldehyde (3, mmol), and potassium carbonate
1
(1.2 mmol) in MeOH (5 mL) was heated under reflux for 2e4 h.
After completion of the reaction (checked by TLC), potassium car-
bonate was filtered off from the hot solution and pure products 6a-
o were obtained as white crystals after the solution was cooled
down to room temperature (yields 90e95%).
4.1. Chemistry
Melting points were taken on a Kofler hot stage apparatus and
are uncorrected. 1H and 13C NMR spectra were recorded on Bruker
FT-400 and 500, using TMS as an internal standard. The IR spectra
were obtained on a Nicolet Magna FT-IR 550 spectrophotometer
(potassium bromide disks). Mass spectra were recorded on an
Agilent Technology (HP) mass spectrometer operating at an ioni-
zation potential of 70 eV. The elemental analysis was performed
with an Elemetar Analysen system GmbH VarioEL CHNS mode.
Purification of all product were conducted by column chromatog-
raphy on silica gel using petroleum ether and ethyl acetate as
eluent.
4.1.4. General procedure for the preparation of quinazolin-4(3H)-
one derivatives 7a-o
A mixture of 2,3-dihydroquinazolin-4(1H)-one 6a-o (1 mmol),
tetrabutylammonium bromide (TBAB, 1 mmol), potassium tert-
butoxide (1 mmol, 0.11 g) in dry THF (3 mL) was stirred at room
temperature for 4e6 h. Upon completion of reaction, the solvent
was evaporated under vacuum. After addition of water to the

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M. Mahdavi et al. / European Journal of Medicinal Chemistry 95 (2015) 492e499
Fig. 4. Flow cytometric analysis of MDA-MB231 cells treated with synthetic compounds 7h and 7d. Cells were stained with Annexin V-FITC/PI and quantitated by flow cytometry.
The cells treated with A) DMSO 1% (negative control); B) IC50 values of etoposide as positive control; C) IC50 values of compound 7h; D) IC50 values of compound 7d for 12 h.
residue, the mixture was extracted with diethyl ether (3 ꢁ 20 mL)
and purified by column chromatography eluting with petroleum
ether/ethyl acetate (10:1).
100 MHz): 11.2, 21.9, 47.2, 121.0, 124.3, 125.7, 126.7, 126.8, 127.1,
127.6, 127.7, 128.2, 128.6, 128.7, 130.0, 132.1, 133.9, 134.4, 135.4,
136.6, 147.3, 155.5, 162.0. Anal. Calcd for C25H22N2O: C, 81.94; H,
6.05; N, 7.64. Found: C, 82.14; H, 5.86; N, 7.80.
4.1.4.1. (E)-3-Ethyl-2-(2-styrylphenyl)quinazolin-4(3H)-one
(7a).
Yield: 78%; white crystals; mp 154e156 ꢀC. IR (KBr): 3055, 2974,
2867, 1671, 1597 cm-1. 1H NMR (CDCl3, 400 MHz): 1.17 (t, J ¼ 6.8 Hz,
3H, CH3), 3.72 (dq, J ¼ 20.8, 6.8 Hz, 1H, CH2), 4.20 (dq, J ¼ 20.8,
6.8 Hz, 1H, CH2), 6.90 (d, J ¼ 16.4 Hz, 1H, CH), 7.16 (d, J ¼ 16.4 Hz, 1H,
CH), 7.22e7.29 (m, 3H, Ph), 7.32e7.35 (m, 2H, H6, H50), 7.44e7.47
(m, 2H, H8, H30), 7.54e7.60 (m, 2H, H7, H40), 7.79e7.82 (m, 2H, Ph),
7.85 (d, J ¼ 8.0 Hz,1H, H60), 8.41 (dd, J ¼ 7.6, 0.8 Hz,1H, H5). 13C NMR
(CDCl3, 100 MHz): 13.8, 40.9, 121.1, 124.3, 125.7, 126.7, 126.8, 127.1,
127.6, 127.8, 128.2, 128.5, 128.7, 130.1, 132.2, 133.9, 134.4, 135.4,
136.6, 147.3, 155.4, 161.8. MS: m/z (%) ¼ 352 (100) [M]þ, 323 (22),
275 (31), 247 (63). Anal. Calcd for C24H20N2O: C, 81.79; H, 5.72; N,
7.95. Found: C, 81.57; H, 5.58; N, 8.18.
4.1.4.3. (E)-3-Isopropyl-2-(2-styrylphenyl)quinazolin-4(3H)-one
(7c). Yield: 75%; white crystals; mp 147e149 ꢀC. IR (KBr): 3056,
2926, 2868, 1676, 1590 cm-1. 1H NMR (CDCl3, 400 MHz): 1.51 (d,
J ¼ 6.8 Hz, 3H, CH3), 1.56 (d, J ¼ 6.8 Hz, 3H, CH3), 4.17 (septet,
J ¼ 6.8 Hz, 1H, CH), 6.95 (d, J ¼ 16.0 Hz, 1H, CH), 7.15 (d, J ¼ 16.0 Hz,
1H, CH), 7.22e7.32 (m, 3H, Ph), 7.33e7.36 (m, 2H, H8, H30), 7.40 (td,
J ¼ 8.0 Hz, 1H, H50), 7.43 (td, J ¼ 8.0 Hz, 1H, H6), 7.52e7.58 (m, 2H,
H7, H40), 7.75e7.82 (m, 2H, Ph), 7.85 (d, J ¼ 8.0 Hz,1H, H60), 8.40 (dd,
J ¼ 8.0, 0.8 Hz, 1H, H5). 13C NMR (CDCl3, 100 MHz): 19.3, 19.4, 54.1,
122.3, 124.6, 125.7, 126.5, 126.7, 127.0, 127.4, 127.8, 128.0, 128.2,
128.7, 129.8, 132.0, 134.2, 134.9, 135.3, 136.6, 146.9, 155.8, 162.4.
Anal. Calcd for C25H22N2O: C, 81.94; H, 6.05; N, 7.64. Found: C,
81.84; H, 6.22; N, 7.51.
4.1.4.2. (E)-3-Propyl-2-(2-styrylphenyl)quinazolin-4(3H)-one (7b).
Yield: 80%; white crystals; mp 158e160 ꢀC. IR (KBr): 3059, 3030,
2960, 2874, 1671, 1601, 1583 cm-1. 1H NMR (CDCl3, 400 MHz): 0.75
(t, J ¼ 7.2 Hz, 3H, CH3), 1.49e1.59 (m, 1H, CH2), 1.60e1.72 (m, 1H,
CH2), 3.58 (ddd, J ¼ 14.4, 9.2, 5.6 Hz, 1H, NCH2), 4.10 (ddd, J ¼ 14.4,
9.2, 5.6 Hz, 1H, NCH2), 6.90 (d, J ¼ 16.0 Hz, 1H, CH), 7.16 (d,
J ¼ 16.0 Hz, 1H, CH), 7.25e7.35 (m, 5H, Ph, H6, H50), 7.43e7.45 (m,
2H, H8, H30), 7.53e7.59 (m, 2H, H7, H40), 7.80e7.82 (m, 2H, Ph), 7.85
(d, J ¼ 8.0 Hz, 1H, H60), 8.40 (d, J ¼ 7.6 Hz, 1H, H5). 13C NMR (CDCl3,
4.1.4.4. (E)-3-Cyclopropyl-2-(2-styrylphenyl)quinazolin-4(3H)-one
(7d). Yield: 75%; white crystals; mp 177e179 ꢀC. IR (KBr): 3062,
3029, 2923, 2852, 1685, 1587 cm-1. 1H NMR (CDCl3, 400 MHz):
0.56e0.62 (m, 2H, CH2), 0.80e0.90 (m, 2H, CH2), 2.92e2.96 (m, 1H,
NCH), 7.15e7.32 (m, 7H, Ph, CH, H30, H50), 7.43 (dd, J ¼ 8.0, 1.2 Hz,
1H, H8), 7.49 (m, 2H, H6, H40), 7.56 (td, J ¼ 8.0, 0.8 Hz, 1H, H7),
7.79e7.80 (m, 2H, Ph), 7.83 (d, J ¼ 8.0 Hz,1H, H60), 8.37 (d, J ¼ 8.0 Hz,
1H, H5). 13C NMR (CDCl3, 100 MHz): 9.9, 10.7, 29.2, 121.2, 125.0,

M. Mahdavi et al. / European Journal of Medicinal Chemistry 95 (2015) 492e499
497
Fig. 5. Flow cytometric analysis of MCF-7 cells treated with synthetic compounds 7h and 7d. Cells were stained with Annexin V-FITC/PI and quantitated by flow cytometry. The cells
treated with A) DMSO 1% (negative control); B) IC50 values of etoposide as positive control; C) IC50 values of compound 7h; D) IC50 values of compound 7d for 12 h.
125.8,126.7,126.8,127.1,127.5,127.6,128.2,128.7,129.0,129.8,131.8,
134.4, 134.9, 135.6, 136.7, 147.0, 156.9, 163.4. Anal. Calcd for
134.4, 135.4, 136.6, 147.3, 155.5, 162.0. Anal. Calcd for C26H24N2O: C,
82.07; H, 6.36; N, 7.36. Found: C, 82.28; H, 6.51; N, 7.19.
C
25H20N2O: C, 82.39; H, 5.53; N, 7.69. Found: C, 82.50; H, 5.68; N,
7.80.
4.1.4.7. (E)-3-Isobutyl-2-(2-styrylphenyl)quinazolin-4(3H)-one (7g).
Yield: 75%; white crystals; mp 131e133 ꢀC. IR (KBr): 3058, 3028,
2961, 2869,1675,1595,1588 cm-1.1H NMR (CDCl3, 400 MHz): 0.70 (d,
J ¼ 7.0 Hz, 3H, CH3), 0.79 (d, J ¼ 7.0 Hz, 3H, CH3),1.98 (m,1H, CH), 3.54
(dd, J ¼ 13.5, 7.5 Hz, 1H, NCH2), 4.11 (dd, J ¼ 13.5, 7.5 Hz, 1H, NCH2),
6.90 (d, J ¼ 16.0 Hz,1H, CH), 7.15 (d, J ¼ 16.0 Hz,1H, CH), 7.22e7.35 (m,
5H, Ph, H6, H50), 7.41e7.48 (m, 2H, H8, H30), 7.52e7.59 (m, 2H, H7,
H40), 7.81e7.84 (m, 3H, Ph, H60), 8.40 (d, J ¼ 8.0 Hz,1H, H5). 13C NMR
(CDCl3, 100 MHz): 20.0, 20.1, 27.7, 51.9, 120.9, 124.3, 125.6, 126.7,
127.0, 127.1,127.6, 127.7, 128.2,128.7, 129.4,130.0,132.2,133.9,134.4,
135.5, 136.6, 147.2, 155.8, 162.4. Anal. Calcd for C26H24N2O: C, 82.07;
H, 6.36; N, 7.36. Found: C, 82.31; H, 6.19; N, 7.51.
4.1.4.5. (E)-3-Allyl-2-(2-styrylphenyl)quinazolin-4(3H)-one
(7e).
Yield: 78%; white crystals; mp 140e142 ꢀC. IR (KBr): 3058, 3028,
2926, 2850, 1675, 1606 cm-1. 1H NMR (CDCl3, 500 MHz): 4.22 (dd,
J ¼ 15.3, 6.0 Hz, 1H, NCH2), 4.80 (dd, J ¼ 15.3, 5.3 Hz, 1H, NCH2), 4.85
(d, J ¼ 17.0 Hz,1H, ¼CH2), 5.07 (d, J ¼ 10.1 Hz,1H, ¼CH2), 5.77 (dddd,
J ¼ 17.0,10.2, 6.0, 5.3 Hz,1H, CH), 6.89 (d, J ¼ 16.1 Hz,1H, CH), 7.13 (d,
J ¼ 16.1 Hz, 1H, CH), 7.23e7.33 (m, 5H, Ph, H6, H50), 7.40e7.41 (m,
2H, H8, H30), 7.54e7.58 (m, 2H, H7, H40), 7.82e7.83 (m, 3H, Ph, H60),
8.40 (d, J ¼ 8.1 Hz, 1H, H5). 13C NMR (CDCl3, 125 MHz): 47.7, 118.2,
120.9, 124.5, 125.7, 126.7, 126.9, 127.2, 127.5, 128.2, 128.3, 128.7,
128.9,130.1,131.4,132.2,133.5, 134.5,135.4, 136.6,147.2, 155.4,161.7.
Anal. Calcd for C25H20N2O: C, 82.39; H, 5.53; N, 7.69. Found: C,
82.54; H, 5.31; N, 7.82.
4.1.4.8. (E)-3-(sec-Butyl)-2-(2-styrylphenyl)quinazolin-4(3H)-one
(7h). Yield: 75%; white crystals; mp 146e148 ꢀC. IR (KBr): 3059,
2925, 2927, 1673, 1603, 1563 cm-1. 1H NMR (CDCl3, 400 MHz): 0.69
(t, J ¼ 7.6 Hz, 3H, CH3), 1.52 (d, J ¼ 1.2 Hz, 3H, CH3), 1.87e1.84 (m, 1H,
CH2), 2.16e2.28 (m, 1H, CH2), 3.83 (sextet, J ¼ 6.8 Hz, 1H, NCH), 6.92
(d, J ¼ 16.4 Hz, 1H, CH), 7.03 (d, J ¼ 16.4 Hz, 1H, CH), 7.23e7.33 (m,
5H, Ph, H6, H5), 7.35e7.45 (m, 2H, H8, H30), 7.51e7.57 (m, 2H, H7,
H40), 7.76e7.81 (m, 2H, Ph), 7.84 (d, J ¼ 8.0 Hz, 1H, H60), 8.36e8.39
(m, 1H, H5). 13C NMR (CDCl3, 100 MHz): 11.5, 17.5, 26.3, 59.9, 122.1,
124.7, 125.6,126.5,126.7,127.0, 127.3, 127.7,127.9, 128.2,128.7, 129.7,
131.6, 134.2, 134.8, 135.2, 136.5, 146.8, 156.1, 162.3. MS: m/z
(%) ¼ 380 (28) [M]þ, 323 (12), 247 (100), 233 (81). Anal. Calcd for
4.1.4.6. (E)-3-Butyl-2-(2-styrylphenyl)quinazolin-4(3H)-one
(7f).
Yield: 80%; white crystals; mp 132e134 ꢀC. IR (KBr): 3069, 3015,
2957, 2925, 2863, 1679, 1590 cm-1.1H NMR (CDCl3, 400 MHz): 0.72
(t, J ¼ 7.5 Hz, 3H, CH3), 1.10e1.21 (m, 2H, CH2), 1.38e1.53 (m, 1H,
CH2), 1.59e1.70 (m, 1H, CH2), 3.64 (ddd, J ¼ 14.5, 9.2, 5.2 Hz, 1H,
NCH2), 4.13 (ddd, J ¼ 14.5, 9.2, 5.2 Hz, 1H, NCH2), 6.91 (d, J ¼ 16.2 Hz,
1H, CH), 7.16 (d, J ¼ 16.2 Hz, 1H, CH), 7.22e7.35 (m, 5H, Ph, H6, H50),
7.44e7.47 (m, 2H, H8, H30), 7.53e7.59 (m, 2H, H7, H40), 7.79e7.86
(m, 2H, Ph), 7.85 (d, J ¼ 8.0 Hz, 1H, H60), 8.40 (d, J ¼ 8.0 Hz, 1H, H5).
13C NMR (CDCl3, 100 MHz): 13.3, 19.9, 30.4, 45.4, 121.0, 124.4, 125.7,
126.7, 126.8, 127.1, 127.6, 127.7, 128.2, 128.7, 128.8, 130.1, 132.2, 133.9,
C26H24N2O: C, 82.07; H, 6.36; N, 7.36. Found: C, 81.89; H, 6.44; N,
7.50.

498
M. Mahdavi et al. / European Journal of Medicinal Chemistry 95 (2015) 492e499
4.1.4.9. (E)-3-Cyclopentyl-2-(2-styrylphenyl)quinazolin-4(3H)-one
(7i). Yield: 78%; white crystals; mp 210e212 ꢀC. IR (KBr): 3032,
2964, 2866, 1674, 1585 cm-1. 1H NMR (CDCl3, 400 MHz): 1.39e1.51
(m, 2H, CH2), 1.66e1.78 (m, 2H, CH2), 1.99e2.09 (m, 2H, CH2),
2.32e2.43 (m, 2H, CH2), 4.32 (pentet, J ¼ 8.0 Hz, 1H, NCH), 6.96 (d,
J ¼ 16.2 Hz, 1H, CH), 7.15 (d, J ¼ 16.2 Hz, 1H, CH), 7.22e7.35 (m, 5H,
Ph, H6, H50), 7.40e7.46 (m, 2H, H8, H30), 7.52e7.58 (m, 2H, H7, H40),
7.63e7.82 (m, 2H, Ph), 7.86 (d, J ¼ 8.0 Hz,1H, H60), 8.37 (d, J ¼ 8.0 Hz,
1H, H5). 13C NMR (CDCl3, 100 MHz): 26.1, 26.2, 29.0, 29.1, 61.8, 122.1,
124.6, 125.6, 126.4, 126.7, 127.1, 127.4, 128.0, 128.1, 128.2, 128.7,
129.8, 131.8, 134.2, 135.0, 135.2, 136.6, 146.9, 156.3, 161.9. Anal. Calcd
for C27H24N2O: C, 82.62; H, 6.16; N, 7.14. Found: C, 82.74; H, 6.31; N,
7.31.
136.3, 136.6, 147.3, 155.6, 162.4. Anal. Calcd for C29H22N2O: C, 84.03;
H, 5.35; N, 6.76. Found: C, 83.90; H, 5.48; N, 6.87.
4.1.4.14. (E)-3-(4-Methylbenzyl)-2-(2-styrylphenyl)quinazolin-
4(3H)-one (7n). Yield: 85%; white crystals; mp 130e132 ꢀC. IR
(KBr): 3054, 3025, 2957, 2921, 1662, 1595 cm-1. 1H NMR (CDCl3,
400 MHz): 2.19 (s, 3H, CH3), 5.03 (d, J ¼ 14.8 Hz, 1H, NCH2), 5.29 (d,
J ¼ 14.8 Hz, 1H, NCH2), 6.71 (d, J ¼ 16.0 Hz, 1H, CH), 6.62 (d,
J ¼ 7.6 Hz, 2H, H300, H500), 6.79 (d, J ¼ 7.6 Hz, 2H, H200, H600), 7.00 (d,
J ¼ 16.0 Hz, 1H, CH), 7.21e7.30 (m, 7H, Ph, H8, H6, H50, H30), 7.51 (t,
J ¼ 8.0 Hz, 1H, H40), 7.59 (td, J ¼ 8.0, 2.4 Hz, 1H, H7), 7.77 (d,
J ¼ 8.0 Hz, 1H, H60), 7.80e7.85 (m, 2H, Ph), 8.46 (d, J ¼ 8.0 Hz, 1H,
H5). 13C NMR (CDCl3, 100 MHz): 21.0, 48.1, 121.1, 124.2, 125.6, 126.8,
127.2, 127.3, 127.5, 127.6, 127.7, 127.8, 128.1, 128.6, 129.0, 130.0, 131.8,
133.3, 133.8, 134.6, 135.7, 136.6, 137.3, 147.3, 155.7, 162.4. MS: m/z
(%) ¼ 428 (76) [M]þ, 323 (100), 247 (37), 105 (55). Anal. Calcd for
4.1.4.10. (E)-3-Phenyl-2-(2-styrylphenyl)quinazolin-4(3H)-one (7j).
Yield: 80%; off-white crystals; mp 153e155 ꢀC. IR (KBr): 3059, 3025,
2925, 1688, 1601, 1582 cm-1. 1H NMR (CDCl3, 500 MHz): 6.95 (d,
J ¼ 16.2 Hz, 1H, CH), 7.06e7.09 (m, 3H, Ph, CH), (t, J ¼ 7.7 Hz, 1H,
H50), 7.20e7.21 (m, 3H, Ph), 7.25e7.28 (m, 3H, Ph), 7.31e7.34 (m, 2H,
H6, H40), 7.37e7.39 (m, 2H, H8, H30), 7.52 (d, J ¼ 7.7 Hz,1H, H60), 7.60
(td, J ¼ 7.5, 2.0 Hz, 1H, H7), 7.85e7.88 (m, 2H, Ph), 8.42 (d, J ¼ 7.5 Hz,
1H, H5). 13C NMR (CDCl3, 125 MHz): 121.2, 125.3, 125.5, 125.6, 126.6,
126.9, 127.2, 127.4, 127.8, 128.1, 128.5, 128.6, 128.7, 129.4, 129.5,
129.6, 131.7, 134.0, 134.8, 135.3, 136.8, 147.2, 154.9, 162.1. MS: m/z
(%) ¼ 400 (97) [M]þ, 323 (100), 278 (13), 178 (17), 77 (50). Anal.
Calcd for C28H20N2O: C, 83.98; H, 5.03; N, 7.00. Found: C, 84.17; H,
5.24; N, 6.87.
C30H24N2O: C, 84.08; H, 5.65; N, 6.54. Found: C, 84.21; H, 5.80; N,
6.37.
4.1.4.15. (E)-3-(4-Methoxybenzyl)-2-(2-styrylphenyl)quinazolin-
4(3H)-one (7o). Yield: 85%; white crystals; mp 145e147 ꢀC. IR
(KBr): 3055, 2924, 2833, 1674, 1605, 1585 cm-1. 1H NMR (CDCl3,
400 MHz): 3.62 (s, 3H, OCH3), 5.04 (d, J ¼ 14.8 Hz, 1H, NCH2), 5.23
(d, J ¼ 14.8 Hz, 1H, NCH2), 6.62 (d, J ¼ 8.5 Hz, 2H, H300, H500), 6.71 (d,
J ¼ 16.0 Hz, 1H, CH), 6.79 (d, J ¼ 8.5 Hz, 2H, H200, H600), 6.98 (d,
J ¼ 16.0 Hz, 1H, CH), 7.21e7.30 (m, 6H, Ph, H8, H50, H30), 7.33 (t,
J ¼ 7.5 Hz,1H, H6), 7.52 (t, J ¼ 7.5 Hz,1H, H40), 7.61 (td, J ¼ 7.5, 2.4 Hz,
1H, H7), 7.80 (d, J ¼ 7.5 Hz, 1H, H60), 7.80e7.83 (m, 2H, Ph), 8.47 (d,
J ¼ 7.5 Hz, 1H, H5). 13C NMR (CDCl3,100 MHz): 47.7, 55.1, 113.6, 121.1,
124.1, 125.7, 126.8, 127.2, 127.3, 127.5, 127.7, 128.1, 128.4, 128.6, 129.0,
129.2,130.1,131.7,133.8,134.6,135.7,136.6,147.3,155.6,159.0,162.5.
Anal. Calcd for C30H24N2O2: C, 81.06; H, 5.44; N, 6.30. Found: C,
80.91; H, 5.23; N, 6.54.
4.1.4.11. (E)-2-(2-Styrylphenyl)-3-(p-tolyl)quinazolin-4(3H)-one
(7k). Yield: 80%; white crystals; mp 186e188 ꢀC. IR (KBr): 3061,
2922, 2855, 1683, 1582 cm-1. 1H NMR (CDCl3, 400 MHz): 2.26 (s, 3H,
CH3), 6.96 (m, 5H, CH, H200, H300, H500, H600), 7.10 (d, J ¼ 16.4 Hz, 1H,
CH), 7.19 (t, J ¼ 7.6 Hz, 1H, H50), 7.26e7.41 (m, 7H, Ph, H6, H8, H30,
H40), 7.55 (d, J ¼ 7.6 Hz, 1H, H60), 7.60 (t, J ¼ 7.6 Hz, 1H, H7),
7.87e7.89 (m, 2H, Ph), 8.43 (d, J ¼ 7.6 Hz, 1H, H5). 13C NMR (CDCl3,
100 MHz): 21.5, 121.2, 125.4, 125.5, 126.7, 127.0, 127.3, 127.4, 127.8,
128.1, 128.2, 128.4, 128.5, 128.8, 129.4, 129.5, 131.6, 134.2, 134.8,
135.3, 136.9, 138.4, 147.3, 155.2, 162.3. MS: m/z (%) ¼ 414 (100) [M]þ,
337 (77), 308 (19), 278 (10). Anal. Calcd for C29H22N2O: C, 84.03; H,
5.35; N, 6.76. Found: C, 83.87; H, 5.19; N, 6.85.
4.2. Cell lines and cell culture
The human cancer cells MCF-7, MDA-MB-231 and T-47D were
purchased from Pastor Institute of Iran. The cells were maintained
in RPMI 1640 medium supplemented with 10% heat-inactivated
fetal calf serum and streptomycin (100
mg/mL) and penicillin
(100 U/ml) at 37 ꢀC in a humidified atmosphere with 5% CO2 in air.
4.1.4.12. (E)-3-(4-Methoxyphenyl)-2-(2-styrylphenyl)quinazolin-
4(3H)-one (7l). Yield: 80%; off-white crystals; mp 153e155 ꢀC. IR
(KBr): 3046, 2922, 2839, 1680, 1634, 1609 cm-1. 1H NMR (CDCl3,
400 MHz): 3.74 (s, 3H, OCH3), 6.72 (d, J ¼ 6.8 Hz, 2H, H300, H500),
6.98e7.02 (m, 3H, CH, H200, H600), 7.06 (d, J ¼ 16.4 Hz, 1H, CH), 7.18 (t,
J ¼ 7.5 Hz, 1H, H50), 7.28e7.40 (m, 7H, Ph, H6, H8, H30, H40), 7.55 (dd,
J ¼ 7.5, 1.6 Hz, 1H, H60), 7.60 (td, J ¼ 8.0, 2.5 Hz, 1H, H7), 7.86e7.88
(m, 2H, Ph), 8.43 (dd, J ¼ 8.0, 2.5 Hz, 1H, H5). 13C NMR (CDCl3,
100 MHz): 55.3, 113.9, 121.2, 125.4, 125.6, 126.6, 127.1, 127.3, 127.4,
127.8, 128.1, 128.8, 129.3, 129.4, 129.5, 129.7, 131.6, 134.3, 134.8,
135.3,136.9,147.3, 155.4,159.2,162.4. Anal. Calcd for C29H22N2O2: C,
80.91; H, 5.15; N, 6.51. Found: C, 81.11; H, 5.28; N, 6.40.
4.3. MTT assay
The cytotoxic activities of compounds 7a-o were evaluated by
using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium
bromide) colorimetric assay as reported method [34,35]. The
absorbance was read at 492 nm with an ELISA plate reader (Biotek
Instruments, Winooski, Vt.). The inhibition percentage of com-
pounds was calculated as: ODwells treated with DMSO1% ꢂ ODwells treated
compounds/OD
DMSO1%*100 (OD ¼ absorbance).
with
wells treated with
Then, IC50 values were calculated by nonlinear regression analysis
and expressed in mean SD.
4.1.4.13. (E)-3-Benzyl-2-(2-styrylphenyl)quinazolin-4(3H)-one (7m).
Yield: 90%; white crystals; mp 135e137 ꢀC. IR (KBr): 3029, 2953,
2923, 2855, 1679, 1601, 1582 cm-1. 1H NMR (CDCl3, 400 MHz): 5.01
(d, J ¼ 15.0 Hz, 1H, NCH2), 5.42 (d, J ¼ 15.0 Hz, 1H, NCH2), 6.77 (d,
J ¼ 16.0 Hz, 1H, CH), 6.88e6.90 (m, 2H, Ph), 7.04 (d, J ¼ 16.0 Hz, 1H,
CH), 7.12e7.14 (m, 3H, Ph), 7.17 (dd, J ¼ 7.6, 0.8 Hz, 1H, H30),
7.23e7.32 (m, 6H, Ph, H8, H6, H50), 7.51 (td, J ¼ 7.6, 0.8 Hz, 1H, H40),
7.61 (td, J ¼ 7.6, 2.0 Hz, 1H, H7), 7.86 (d, J ¼ 7.6 Hz, 1H, H60),
7.82e7.85 (m, 2H, Ph), 8.47 (d, J ¼ 7.6 Hz, 1H, H5). 13C NMR (CDCl3,
100 MHz): 48.2, 121.1, 124.3, 125.7, 126.8, 127.2, 127.3, 127.5, 127.6,
127.8, 127.7, 128.1, 128.3, 128.6,129.0, 130.1, 132.0, 133.7, 134.6, 135.7,
4.4. Acridine orange/ethidium bromide double staining test
The acridine orange/ethidium bromide test was used to detect
apoptosis induced by selected compounds 7d and 7h according to
the previously described method [35,36].
4.5. Annexin V-FITC/PI double staining and flow cytometric analysis
The double staining test was performed using Annexin V-FITC
Apoptosis Detection Kit (Bio Vision) as described in protocol. Using
this technique, living cells (annexin V-FITCꢂ/PIꢂ), early apoptotic

M. Mahdavi et al. / European Journal of Medicinal Chemistry 95 (2015) 492e499
499
cells (Annexin V-FITCþ/PIꢂ), late apoptotic or secondary apoptotic
cells (Annexin V-FITCþ/PIþ), and necrotic cells (Annexin V-FITCꢂ/
PIþ) were distinguished. In brief, the cancer cell lines were treated
with IC50 of compounds 7d and 7h, and etoposide as reference
drug. After 12 h incubation, cells were collected by centrifugation
ꢁ
~
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and resuspended in 500
were double stained with Annexin V-FITC and 5
m
l of 1ꢁ Binding Buffer. Finally, the cells
m
l of PI and the
samples were gently mixed and incubated for 5 min at room
temperature in the dark before flow cytometry. Annexin V-FITC
binding was analyzed by flow cytometry (Ex
¼
488 nm;
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exhibits high potent antitumor activity against human gynecologic malig-
nancies, Invest. New. Drugs 28 (2010) 132e138.
Em ¼ 530 nm) using FITC signal detector (FL1) and PI staining by
the phycoerythrin emission signal detector (FL2).
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activities of novel 4-anilinoquinazoline derivatives, Eur. J. Med. Chem. 44
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Acknowledgments
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M.C. Brady, S. Green, S.P. Heaton, S. Kearney, N.J. Keen, R. Odedra, S.R. Wedge,
R.W. Wilkinson, Synthesis and SAR of 1-acetanilide-4-aminopyrazole-
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H.F. VanBrocklin, Synthesis of 6-acrylamido-4-(2-[18F]fluoroanilino)quinazo-
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in vitro antitumor activity of 4(3H)-quinazolinone derivatives with dithio-
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This research has been supported by a grant from the Research
Council of Tehran University of Medical Sciences and Iran National
Science Foundation (INSF).
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2015.03.057.
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