Design, synthesis of novel quinazolin-4-one derivatives and biological evaluation against human MCF-7 breast cancer cell line

Article abstract of DOI:10.1007/s11164-015-2117-z

A new series of 6,8-dibromo-2-(4-chlorophenyl)quinazolin-4(3H)-one derivatives VI-XIII were synthesized. Their chemical structures were confirmed by spectral and elemental analysis. The cytotoxic effect of the newly synthesized compounds was tested in vitro against human breast cancer cell line (MCF-7). Most of the tested compounds have shown promising cytotoxic activity. Compounds X and XIIIb exerted a powerful cytotoxic effect against MCF-7 with a very low IC50 (0.0015 and 0.0047 μmol/ml), while compounds VI, VII, VIII, XIIb, XI, XIIIc and IX exerted a moderate cytotoxic effect (IC50 0.01523, 0.0213, 0.031, 0.0478, 0.049, 0.068 and 0.079 μmol/ml respectively), compared to doxorubicin (0.0025 μmol/ml). Exploring their apoptotic effect; interestingly,all compounds activated apoptotic cascade in MCF-7. Compounds VI, XIIIb, XIIb, XI, XIIa, VII, V and VIII showed potent effect even much more than doxorubicin by 12.87-5.91 folds, while compounds XIIIc, IX, XIIIa, XIIc and X showed moderate increase in CASP3 activity by 4.96-3.22 folds relative to untreated cells more or less similar to doxorubicin (5.57 folds).

Full text of DOI:10.1007/s11164-015-2117-z

Res Chem Intermed
DOI 10.1007/s11164-015-2117-z
Design, synthesis of novel quinazolin-4-one derivatives
and biological evaluation against human MCF-7 breast
cancer cell line
Marwa F. Ahmed1 Amel AA. Hashim2
•
Received: 30 March 2015 / Accepted: 26 May 2015
Ó Springer Science+Business Media Dordrecht 2015
Abstract A new series of 6,8-dibromo-2-(4-chlorophenyl)quinazolin-4(3H)-one
derivatives VI–XIII were synthesized. Their chemical structures were confirmed by
spectral and elemental analysis. The cytotoxic effect of the newly synthesized
compounds was tested in vitro against human breast cancer cell line (MCF-7). Most
of the tested compounds have shown promising cytotoxic activity. Compounds X
and XIIIb exerted a powerful cytotoxic effect against MCF-7 with a very low IC50
(0.0015 and 0.0047 lmol/ml), while compounds VI, VII, VIII, XIIb, XI, XIIIc and
IX exerted a moderate cytotoxic effect (IC50 0.01523, 0.0213, 0.031, 0.0478, 0.049,
0.068 and 0.079 lmol/ml respectively), compared to doxorubicin (0.0025 lmol/ml).
Exploring their apoptotic effect; interestingly,all compounds activated apoptotic
cascade in MCF-7. Compounds VI, XIIIb, XIIb, XI, XIIa, VII, V and VIII showed
potent effect even much more than doxorubicin by 12.87–5.91 folds, while com-
pounds XIIIc, IX, XIIIa, XIIc and X showed moderate increase in CASP3 activity
by 4.96–3.22 folds relative to untreated cells more or less similar to doxorubicin
(5.57 folds).
Keywords Synthesis Á Quinazolines Á Breast cancer Á IC50 Á Caspase-3
&
Marwa F. Ahmed
marwafarag80@yahoo.com
Amel AA. Hashim
weiss_rosa@yahoo.com; dr.hashim@helwan.edu.eg
1
2
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Helwan University, Cairo,
Egypt
Department of Biochemistry and Molecular Biology, Faculty of Pharmacy, Helwan University,
Cairo, Egypt
123

M. F. Ahmed, A. AA. Hashim
Introduction
Cancer is considered one of the major health problems, with increasing incidence
worldwide. Malignant tumors are ranked the third in developing countries after
infectious-parasitic and cardiovascular diseases [1]. The worldwide burden of
cancer rose to an estimated 14 million new cases per year. Cancer deaths are
predicted to rise from an estimated 8.2 million annually to 13 million per year.
Breast cancer incidence was (1.7 million, 11.9 % of the total) [2]. In cancer as well
as many other serious diseases, the body loses control over apoptosis and
angiogenesis where apoptosis is hindered and excessive angiogenesis occurs [1].
Programmed cell death ‘‘apoptosis’’ is one of the various cell death modalities
regulating cell proliferation, as well as eliminating potentially harmful ones
resulting from pathophysiological conditions. It was described as a major defense
strategy that prevents cells from acquiring tumorigenic potential. This suicide
mechanism is controlled by multiple and interrelated pathways ensuring that
proteolytic initiators and executioners ‘‘Caspases’’ are triggered only in cells
requiring termination [3]. Caspase-3 ‘‘CASP3’’ is a member of the cysteine protease
family, which plays a crucial role in apoptotic pathways by cleaving a variety of key
cellular proteins. Previous results suggest that lack of CASP3 can attenuate
apoptosis in response to certain stimuli. CASP3 can be activated by diverse death-
inducing signals, including chemotherapeutic agents [4].
Discovering new chemotherapeutic agents has become one of the most
challenging tasks in medicinal chemistry and remains critically important,
especially the design and synthesis of new ones that can trigger apoptosis. One
of the most important scaffolds in medicinal chemistry is quinazoline. It is well
known that quinazoline derivatives have a wide range of biological activities
such as anticancer [5–8], antibacterial [9], antiviral [10], antifungal [9],
antihypertensive [11], anti-inflammatory [12], analgesic and COX-II inhibitors
[13–16]. Several quinazoline derivatives have been approved by FDA as
anticancer drugs such as Erlotinib, Lapatinib, Gefitinib and Caneratinib (Fig. 1).
Literature survey revealed that 2,3-disubstituted quinazoline-4-one derivatives
have good anticancer activity against MCF-7 tumor cell line [17–19]. Moreover,
quinazoline derivatives bearing phenyl moiety at position 3 show potent activity
against breast cancer [19, 20]. On the other hand many 6,8-dibromoquinazolin-4-
one derivatives showed greater activity than doxorubicin against MCF-7 [5, 6].
In view of previous rationale and in continuation of our drug research program
concerning synthesis of new safer and more biologically active quinazolin-4-one
derivatives [5–7, 9, 13, 15, 16], our interest was to synthesize new series of 6,8
dibromoquinazolin-4-one compounds having various substituents at position 3
and p-chorophenyl moiety at position 2 aiming to obtain new quinazolin-4-one
derivatives with high activity against breast cancer.
123

Design, synthesis of novel quinazolin-4-one derivatives…
O
large fragment
large fragment
F
Cl
NH
N
NH
N
O
O
N
N
O
O
O
O
S
(tarceva)
O
Erlotinib
Lapatinib
large fragment
large fragment
F
F
Cl
NH
O
Cl
NH
O
O
O
N
O
N
N
N
N
N
NH
O
Gefitinib (Iressa)
Caneratinib (CI 1033)
Fragment Replacement
O
N
X
R
Br
N
Small lipophilic group insertion
Br
Cl
Lipophilic aromatic insertion
Fig. 1 Reported and new designed antitumor quinazoline derivatives
Results and discussion
Chemistry
The reaction of 3,5 dibromoanthranilic acid I and p-chlorobenzoylchloride II
afforded the amide analog III which was refluxed in acetic anhydride to obtain 6,8-
dibromo-2-(4-chlorophenyl)-4H-benzo[d] [1, 3] oxazin-4-one IV [21] (Scheme 1).
Compound IV reacted with formamide to afford compound V [22] (Scheme 1). The
reaction of urea, thiourea, aniline, p-toluidine, p-aminoacetophenone with benzox-
azine derivative IV by fusion at high temperature afforded the corresponding
quinazoline-4-one derivatives VI–X (Scheme 2). The benzoxazine derivative IV
123

M. F. Ahmed, A. AA. Hashim
O
Cl
O
O
Br
pyridine
Br
OH
NH2
OH
NH
Br
O
Cl
(II)
Br
(I)
Cl
(III)
AC2O
O
O
N
Br
O
Br
HCONH2
NH
N
Cl
Cl
Br
Br
(V)
(IV)
Scheme 1 Synthesis of compounds III–V
O
N
S
O
N
Br
N
Br
N
NH2
Cl
thiourea
Br
Cl
Br
Br
VIII
VII
O
O
O
N
O
N
Br
O
Br
N
NH2
p-toulidine
Cl
N
N
Br
Cl
Cl
Br
Br
IV
VI
IX
O
N
Br
OH
N
O
COCH3
Cl
Br
N
Cl
Br
N
Br
XI
X
Scheme 2 Synthesis of the target compounds VI–XI
was also reacted with hydroxylamine hydrochloride in dry pyridine to afford
compound XI (Scheme 2).
6,8-dibromo-2-(4-chlorophenyl)quinazolin-4(3H)-one V reacted with primary
amines like aniline, p-toluidine, 2-amino pyridine in the presence of formamide and
glacial acetic acid to give Mannich bases XII a–c (Scheme 3). On the other hand
123

Design, synthesis of novel quinazolin-4-one derivatives…
O
C H
O
N
O
Br
H
Br
NH
R
N
NH
N
primary amine
Cl
Cl
Br
Br
XII a-c
V
XIIa,
R =
R =
Paraformaldehyde
secondary amine
XIIb,
XIIc,
N
R =
O
R
Br
N
N
Cl
Br
XIIIa,
N
R =
R =
R =
XIIIb,
XIIIc,
N
N
O
Scheme 3 Synthesis of the target compounds XII a–c and XIII a–c
compound V react with secondary amine like piperidine, 2-methyl piperidine,
morpholine in presence of paraformaldehyde to give the corresponding Mannich
bases XIII a–c (Scheme 3).
Biological activity
In vitro cytotoxic activity
In this study a novel series of 6,8-dibromo-2-(4-chlorophenyl)quinazolin-4(3H)-one
derivatives were designed and synthesized (Schemes 1, 2, 3). All synthesized
compounds were screened for their in vitro cytotoxic and growth inhibitory
activities against MCF-7 cell line, in comparison with doxorubicin as reference
drug. The cytotoxic activities of our tested compounds were expressed as IC50
values (the dose that reduces survival to 50 %) in lmol/ml. Growth curves showed
that our tested compounds exhibit significant efficacy against MCF-7 with IC50
values range 0.0015–0.317 lmol/ml, compared to doxorubicin (0.0025 lmol/ml).
As shown in Table 1: Compounds X and XIIIb exerted a powerful cytotoxic effect
against MCF-7 with a very low IC50 (0.0015 and 0.0047 lmol/ml). Compounds VI,
123

M. F. Ahmed, A. AA. Hashim
IC50 %
Table 1 Cytotoxity of tested
compounds against human
breast cancer cell line (MCF-7)
Compounds
V
0.12823 0.00022
0.01523 0.00008
0.0213 0.00004
0.031 0.00001
0.079 0.00028
0.0015 0.00001
0.049 0.00027
0.0956 0.00022
0.0478 0.00016
0.208 0.00018
0.317 0.00032
0.0047 0.00024
0.068 0.00005
0.0025 0.000028
VI
VII
VIII
IX
X
XI
XIIa
XIIb
XIIc
XIIIa
XIIIb
XIIIc
Doxo
Data is expressed as
M
SD (lmol/ml)
Doxo doxorubicin (reference
drug), IC50 in vitro
concentration of a drug that is
required for 50 % inhibition
VII, VIII, XIIb, XI, XIIIc and IX exerted a moderate cytotoxic effect (IC50 0.01523,
0.0213, 0.031, 0.0478, 0.049, 0.068 and 0.079 lmol/ml respectively), while
compounds XIIa, V, XIIc, and XIIIa exerted a week cytotoxic effect against
MCF-7 (IC50 0.0956, 0.12823, 0.208 and 0.317 lmol/ml respectively), as
compared to doxorubicin.
In vitro apoptotic effect
It is well established that the induction of apoptotic cascade is one of the main
mechanisms of chemotherapy-induced cell death [6]. CASP3 can be activated by
diverse death-inducing signals, including chemotherapeutic agents [4]. Previous
study has shown that approximately 75 % of tumors as well as morphologically
normal peritumoral tissue samples lacked CASP3 transcript and protein expression
[4]. In addition, CASP3 mRNA levels in commercially available total RNA samples
from breast (MCF-7) and cervical studied tumors were undetectable [4].
In order to determine whether the chemo sensitizing effect of our newly
synthesized 6,8-dibromo-2-(4-chlorophenyl)quinazolin-4(3H)-one derivatives is
secondary to its ability to activate an apoptotic cascade, MCF-7 were treated with
test compounds at their IC50 concentrations, for 6 h. The activity of CASP3 was
measured using in vitro human CASP3 assay. Figure 2 represents enzyme activity
expressed in folds relative to untreated cells.
All our tested compounds caused significant increase of CASP3 activity against
MCF-7 compared to doxorubicin. Compounds VI, XIIIb, XIIb, XI, XIIa, VII, V and
VIII increased CASP3 activity even more than doxorubicin by 12.87–5.91 folds,
while compounds XIIIc, IX, XIIIa, XIIc and X showed moderate increase in CASP3
activity by 4.96–3.22 folds relative to untreated cells more or less similar to
doxorubicin (5.57 folds).
123

Design, synthesis of novel quinazolin-4-one derivatives…
Fig. 2 Apoptotic effect ‘‘in terms of caspase-3 activity’’ of tested compounds after 6 h treatment of
human breast cancer cell line (MCF-7). Enzymatic activity of caspase-3 expressed in folds relative to
untreated cells. Control untreated cells (-ve control), Doxo doxorubicin (?ve control)
The sensitivity of CASP3- deficient breast cancer (MCF-7) cells to undergo
apoptosis in response to doxorubicin and other apoptotic stimuli could be
augmented by reconstituting CASP3 expression. These results suggest that loss of
CASP3 expression may represent an important cell survival mechanism in breast
cancer patients [5].
SAR
Substitution of 6,8-dibromo-2-(4-chlorophenyl)quinazolin-4(3H)-one moiety with
4-acetylphenyl group at position 3 gave the most potent anticancer activity against
MCF-7 exhibited by compounds X.
It is interesting to note that a minor alteration in the molecular configuration of
investigated compounds may have a pronounced effect on anticancer activity, As
demonstrated hereby: compound XIIIb with ‘‘(2-methylpiperidin-1-yl) methyl’’
substitution at position 3 led to a potent cytotoxic effect against MCF-7, while
substitution with ‘‘morpholinomethyl’’ give compound XIIIc with moderate
cytotoxic activity, on the other hand substitution with ‘‘piperidin-1-ylmethyl’’ at
the same position give compound XIIIa with low cytotoxic activity.
Introduction of ‘‘carboxamide, carbothioamide, phenyl, p-tolyl, hydroxyl and/or
(p-tolylamino)methyl)’’ group at position 3 give compounds VI, VII, VIII, IX, XI
and XIIb respectively which exerted a moderate cytotoxic effect against MCF-7.
On the other hand substitution with ‘‘(phenylamino) methyl’’ and/or ‘‘(pyridin-2-
ylamino) methyl’’ at position 3 gave compounds XIIa and XIIc respectively with
weak cytotoxic effect.
On discussing the SAR of apoptotic activity of our newly synthesized derivatives;
substitution of 6,8-dibromo-2-(4-chlorophenyl)quinazolin-4(3H)-one moiety at po-
sition 3 with ‘‘carboxamide, (2-methylpiperidin-1-yl) methyl, (p-tolylamino)methyl),
123

M. F. Ahmed, A. AA. Hashim
hydroxyl, (phenylamino) methyl, carbothioamide, phenyl’’ give compounds VI, XIIIb,
XIIb, XI, XIIa, VII and VIII respectively which increase CASP3 activity even more
than doxorubicin by 12.87 to 5.91 folds.
Conclusion
A new series 6,8-dibromo-2-(4-chlorophenyl)quinazolin-4(3H)-one derivatives VI–
XIII were synthesized. All synthesized compounds were tested in vitro against
human breast cancer cell line (MCF-7). Most of the tested compounds have shown
promising cytotoxic effect. Substitution of 6,8-dibromo-2-(4-chlorophenyl)quina-
zolin-4(3H)-one moiety with ‘‘4-acetylphenyl’’ and/or ‘‘(2-methylpiperidin-1-yl)
methyl’’ at position 3 gave compounds X and XIIIb respectively which exert the
most potent anticancer activity against MCF-7 with a very low IC50 (0.0015 and
0.0047 lmol/ml),compared to doxorubicin (0.0025 lmol/ml).
All compounds activated apoptotic cascade in MCF-7. Substitution of 6,8-
dibromo-2-(4-chlorophenyl)quinazolin-4(3H)-one moiety at position 3 with ‘‘car-
boxamide, (2-methylpiperidin-1-yl) methyl, (p-tolylamino)methyl), hydroxyl,
(phenylamino) methyl, carbothioamide, phenyl’’ give compounds VI, XIIIb, XIIb,
XI, XIIa, VII and VIII respectively which increase CASP3 activity by 12.87 to 5.91
folds relative to untreated cells compared to doxorubicin (5.57 folds).
Experimental
Chemistry
All melting points are uncorrected, elemental analyses were carried out in the micro
analytical unit of National Research Centre and Cairo University, Egypt. IR spectra
were recorded on FT-IR spectrophotometer- Nexus 670-Nicolet (USA) and Perkin
1
Elmer-9712 spectrophotometer. H NMR spectra were determined on a Varian-
Gemini 300 MHz and Joel-Ex 270 MHz NMR spectrometer using TMS as an
internal standard. 13C NMR (DMSO-d6) spectra were recorded at 100.62 MHz at
the aforementioned research center in Cairo University. Mass spectra were
determined on Finnigan Mat SSQ 7000, mode EI 70 eV (Thermo Inst. Sys. Inc.
USA). Thin layer chromatography was carried out on silica gel 60 F254 (Merck)
plates using chloroform/methanol (9:1) as an eluent system.
General procedure for the synthesis of 3(4H)-substituted 6,8-dibromo-2-(4-
chlorophenyl)-4-oxoquinazoline (VI–X)
Equimolar amount of benzoxazine IV and different primary amino containing
moieties like urea, thiourea, aniline, p-toluidine, p-aminoacetophenone were fused
together at 200o C in an oil bath for 1 h. The mixture was cooled and methanol was
added to the mixture. The separated solid was collected by filtration, washed with
123

Design, synthesis of novel quinazolin-4-one derivatives…
methanol, dried and crystallized from proper solvent to give the compounds VI–X
respectively.
6,8-dibromo-2-(4-chlorophenyl)-4-oxoquinazoline-3(4H)-carboxamide (VI) Crys-
tallized from ethanol to give yellow crystals of compound VI, m.p. 230 °C, 85 %
yield. Analysis calculated for C15H8Br2ClN3O2; Calcd.: %C, 39.38; H, 1.76; N,
9.18, Found: %C, 39.29; H, 1.8; N, 9.2. IR: tmax./cm-1 3300 (NH2),3050 (C–H
aromatic), 1690 (C=O of quinazolinone), 1650 (C=O of amide), 1630 (C=N) and at
1
1585 (C=C). H-NMR (DMSO-d6, ppm): d 7.2–8.1 (m, 6H, Ar–H) and at 10.5 (s,
2H, NH2, exchangeable with D2O). 13C NMR (DMSO-d6):169, 160, 155.9, 148,
136.3, 136, 133, 126.5, 129, 128.5, 124, 121.9. MS: m/z = 461
6,8-dibromo-2-(4-chlorophenyl)-4-oxoquinazoline-3(4H)-carbothioamide (VII) Crys-
tallized from ethanol to give yellow crystals of compound VII, m.p. 270 °C, 80 %
yield. Analysis calculated for C15H8Br2ClN3OS; Calcd.: %C, 38.04; H, 1.70; N,
8.87, Found: %C, 38.12; H, 1.76; N, 8.94. IR: tmax./cm-1 3600 (NH2), 3100 (C–H
aromatic), 1690 (C=O of quinazolinone), 1633 (C=N), 1590 (C=C) and at 1560 and
1320 (C=S). 1H-NMR (DMSO-d6, ppm): d 7.5–8.3 (m, 6 H, Ar–H) and at 9.4 (s, 2H,
NH2, exchangeable with D2O). 13C NMR (DMSO-d6):183, 171, 156, 155, 140, 136,
132, 130, 129. 127, 122, 114. MS: m/z = 477
6,8-dibromo-2-(4-chlorophenyl)-3-phenylquinazolin-4(3H)-one (VIII) Crystallized
from ethanol to give brown crystals of compound VIII, m.p. 140 °C, 80 % yield.
Analysis calculated for C20H11Br2ClN2O; Calcd.: %C, 48.97; H, 2.26; N, 5.70,
Found: %C, 48.95; H, 2.20; N,5.62. IR: tmax./cm-1 3110 (C–H aromatic), 1680
1
(C=O of quinazolinone), 1630 (C=N) and at 1600 (C=C). H-NMR (DMSO-d6,
ppm): d 7.1–8.2 (m, 11 H, Ar–H). 13C NMR (DMSO-d6):164, 160.5, 155, 140, 135,
131, 129, 128.9, 128.5, 128, 126.7, 122, 113. MS: m/z = 494
6,8-dibromo-2-(4-chlorophenyl)-3-p-tolylquinazolin-4(3H)-one (IX) Crystallized
from ethanol to give yellow crystals of compound IX, m.p. 110 °C, 75 % yield.
Analysis calculated for C21H13Br2ClN2O; Calcd.: %C, 49.98; H, 2.60; N, 5.55,
Found: %C, 49.90; H, 2.50; N,5.47. IR: tmax./cm-1 3100 (C–H aromatic), 1685
1
(C=O of quinazolinone), 1625 (C=N) and at 1590 (C=C). H-NMR (DMSO-d6,
ppm): d 2.30 (s, 3H, CH3), and at 7.2–8.1 (m, 10 H, Ar–H). 13C NMR (DMSO-
d6):165, 161, 154.4, 139.4, 136.9, 135.7, 129.8, 129.5, 129.2, 128.9, 128.4, 122, 113,
21.4. MS: m/z = 508
3-(4-acetylphenyl)-6,8-dibromo-2-(4-chlorophenyl)quinazolin-4(3H)-one (X) Crys-
tallized from ethanol to give brown crystals of compound X, m.p. 180 °C, 70 %
yield. Analysis calculated for C22H13Br2ClN2O2; Calcd.: %C, 40.07; H, 1.99; N,
4.25, Found: %C 40.03; H, 1.90; N, 4.20. IR: tmax./cm-1 3200 (C–H aromatic),
1710 (C=O of acetyl), 1690 (C=O of quinazolinone), 1635 (C=N) and at 1600
1
(C=C). H-NMR (DMSO-d6, ppm): d 2.60 (3H, s, CH3) and at 7.5–8.3 (m, 10 H,
123

M. F. Ahmed, A. AA. Hashim
Ar–H). 13C NMR (DMSO-d6):197, 164, 160.6, 154.3, 139.4, 137.1, 136.9, 136, 132,
129.5, 129, 128.9, 126.5, 125, 124, 123, 113, 26.7. MS: m/z = 536
6,8-dibromo-2-(4-chlorophenyl)-3-hydroxyquinazolin-4(3H)-one (XI)
A mixture of benzoxazine IV (0.01 mol) and hydroxylamine hydrochloride
(0.01 mol) in dry pyridine (30 ml) was heated under reflux for 8 h and the reaction
mixture was then concentrated to half its volume poured into crushed ice containing
few drops HCl. The separated solid was filtered, washed with water and crystallized
with ethanol m.p. 245 °C, 60 % yield. Analysis calculated for C14H7Br2ClN2O2;
Calcd.: %C, 39.06; H, 1.64; N, 6.51, Found: %C 39.12; H, 1.70; N, 6.53. IR: tmax.
/
cm-1 3400 (OH), 3050 (C–H aromatic), 1690 (C=O of quinazolinone), 1630 (C=N)
1
and at 1600 (C=C). H-NMR (DMSO-d6, ppm): d 7.4–8.2 (m, 6 H, Ar–H) and at
10.60 (s, 1H, OH). 13C NMR (DMSO-d6):165, 162, 154.3, 139, 135, 132, 129,
128.5, 126.9, 125, 122, 115. MS: m/z = 434
General procedure for the synthesis of 6,8-dibromo-2-(4-chlorophenyl)-3-((substituted)
methyl)quinazolin-4(3H)-one (XIIa–c)
A mixture of V (0.01 mol) and respective primary amines like aniline, p-toluidine,
2-amino pyridine (0.01 mol) in 30 mL of glacial acetic acid, formaldehyde solution
30 % (0.05 mol) was added. The solution was refluxed for 8 h, the reaction mixture
was cooled and immediately poured into crushed ice. The product obtained was
filtered, washed with methanol and crystallized from proper solvent to give the
compounds XIIa–c respectively.
6,8-dibromo-2-(4-chlorophenyl)-3-((phenylamino)methyl)quinazolin-4(3H)-one
(XIIa) Crystallized from ethanol to give white crystals of compound XIIa, m.p.
260 °C, 65 % yield. Analysis calculated for C21H14Br2ClN3O; Calcd.: %C, 48.54;
H, 2.72; N, 8.09, Found: %C 48.43; H, 2.65; N, 8.03. IR: tmax./cm-1 3420 (N–H),
3100 (C–H aromatic), 1670 (C=O of quinazolinone), 1620 (C=N) and at 1590
1
(C=C). H-NMR (DMSO-d6, ppm): d 4.70 (s, 2H, CH2), 7.5–8.3 (m, 11 H, Ar–H)
and at 9.5 (s,1H, NH, exchangeable with D2O). 13C NMR (DMSO-d6):169, 162,155,
148, 139.4, 135, 131.9, 129.5, 129.2, 12809, 126.7, 125.2, 120.7, 122, 113, 60.5.
MS: m/z = 523
6,8-dibromo-2-(4-chlorophenyl)-3-((p-tolylamino)methyl)quinazolin-4(3H)-one
(XIIb) Crystallized from ethanol to give white crystals of compound XIIb, m.p.
290 °C, 70 % yield. Analysis calculated for C22H16Br2ClN3O; Calcd.: %C, 49.52;
H, 3.02; N, 7.87, Found: %C 49.59; H, 3.14; N, 7.9. IR: tmax./cm-1 3400 (N–H),
3100 (C–H aromatic), 1665 (C=O of quinazolinone), 1620 (C=N) and at 1595
(C=C). 1H-NMR (DMSO-d6, ppm): d 2.30 (s, 3H, CH3), 4.60 (s, 2H, CH2), 7.3–8.1
(m, 10 H, Ar–H) and at 9.4 (s, 1H, NH, exchangeable with D2O). 13C NMR
(DMSO-d6):162, 160, 153, 144, 139.7, 135.5, 131, 129.7, 129.4, 129, 128, 126.7,
125.1, 122, 113, 60.3. MS: m/z = 537
123

Design, synthesis of novel quinazolin-4-one derivatives…
6,8-dibromo-2-(4-chlorophenyl)-3-((pyridin-2-ylamino)methyl)quinazolin-4(3H)-one
(XIIc) Crystallized from ethanol to give white crystals of compound XIIc, m.p.
255 °C, 65 % yield. Analysis calculated for C20H13Br2ClN4O; Calcd.: %C, 46.14;
H, 2.52; N, 10.76, Found: %C 46.25; H,2.61; N,10.80. IR: tmax./cm-1 3450 (N–H),
3150 (C–H aromatic), 1675 (C=O of quinazolinone), 1630 (C=N) and at 1600
1
(C=C). H-NMR (DMSO-d6, ppm): d 4.70 (s, 2H, CH2), 6.9–8.1 (m, 10 H, Ar–H)
and at 9.9 (s, 1H, NH, exchangeable with D2O). 13C NMR (DMSO-d6):165, 162,
158, 155, 148, 139.5, 138.6, 135.7, 131.3, 129.2, 128.9, 125, 126, 122, 118, 115,
107.4, 60.7. MS: m/z = 524
General procedure for the synthesis of 6,8-dibromo-2-(4-chlorophenyl)-3-(substituted-
1-ylmethyl)quinazolin-4(3H)-one (XIIIa–c)
A mixture of compound V (0.01 mol), paraformaldehyde (0.01 mol) and the
appropriate secondary amine (0.01 mol) namely, piperidine, 2-methyl piperidine
and/or morpholine in DMF (20 mL) was refluxed for 8 h. The excess solvent was
evaporated under vacuum and the obtained residue was poured into crushed ice. The
solid product was filtered off and washed with water to obtain the desired products
XIIIa–c respectively.
6,8-dibromo-2-(4-chlorophenyl)-3-(piperidin-1-ylmethyl)quinazolin-4(3H)-one
(XIIIa) Crystallized from ethanol to give brown crystals of compound XIIIa,
m.p. 285 °C, 60 % yield. Analysis calculated for C20H18Br2ClN3O; Calcd.: %C,
46.95; H, 3.55; N, 8.21, Found: %C 46.90; H, 3.46; N,8.10. IR: tmax./cm-1 3110
(C–H aromatic), 1670 (C=O of quinazolinone), 1630 (C=N) and at 1600 (C=C).
1H-NMR (DMSO-d6, ppm): d 1.55 (m, 6H, 3CH2 of piperdine), 2.43 (m, 4H,
CH2NCH2 of piperdine), 4.20 (s, 2H, CH2) and at 7.2–8.13 (m, 6H, aromatic-H).
13C NMR (DMSO-d6):161.6, 155, 139, 135.5, 131.3, 129.1, 128.9, 126, 125.2, 122,
113, 57.4, 51.7, 25.7, 24.5. MS: m/z = 515
6,8-dibromo-2-(4-chlorophenyl)-3-((2-methylpiperidin-1-yl)methyl)quinazolin-4(3H)-
one (XIIIb) Crystallized from ethanol to give yellow crystals of compound XIIIb,
m.p. 170 °C, 60 % yield. Analysis calculated for C21H20Br2ClN3O; Calcd.: %C,
47.98; H, 3.83 N, 7.99, Found: %C 47.92; H, 3.76; N,7.91. IR: tmax./cm-1 3100
(C–H aromatic), 1660 (C=O of quinazolinone), 1610 (C=N) and at 1595 (C=C).
1H-NMR (DMSO-d6, ppm): d 1.12 (s, 3H, CH3), 1.4 (m, 6H, 3CH2 of piperdine),
2.40 (m, 3H, CHNCH2 of piperdine), 3.90 (s, 2H, CH2) and at 7.4–8.3 (m, 6H,
aromatic-H). 13C NMR (DMSO-d6):164, 161.9, 155, 139.6, 136, 132, 129.4, 128.8,
126, 125,122, 113 54.8, 54.2, 49.5, 33.9, 25.9, 23.3. MS: m/z = 529
6,8-dibromo-2-(4-chlorophenyl)-3-(morpholinomethyl)quinazolin-4(3H)-one (XIIIc) Crys-
tallized from ethanol to give yellow crystals of compound XIIIc, m.p. 235 °C, 60 %
yield. Analysis calculated for C19H16Br2ClN3O2; Calcd.: %C, 44.43; H, 3.14 N,
8.18, Found: %C 44.32; H, 3.10; N,8.10. IR: tmax./cm-1 3150 (C–H aromatic), 1690
1
(C=O of quinazolinone), 1640 (C=N) and at 1600 (C=C). H-NMR (DMSO-d6,
123

M. F. Ahmed, A. AA. Hashim
ppm): d 2.5 (t, 4H, 2CH2 of morpholine), 3.6 (t, 4H, 2CH2 of morpholine), 4.25 (s,
2H, CH2) and at 7.4–8.2 (m, 6H, aromatic-H). 13C NMR (DMSO-d6): 167, 163, 156,
139.6, 136.4, 131.5, 129.4, 128.9, 126.7, 125.1,123,113, 66.4, 57.4, 50.7. MS:
m/z = 517
Biological studies
Cytotoxicity
Cell line culture Human breast cancer cell line (MCF-7) was grown in DMEM
medium with: 10 % fetal bovine serum, penicillin100 units/ml and strepto-
mycin100 lg/mL. Cell line was maintained at 37 °C in humidified 5 % CO2
atmosphere. Tested compounds and doxorubicin solutions were prepared in DMSO
and stored at–20 °C. Control (untreated cells) was treated with DMSO alone
(DMSO concentration was always less than 0.1 %.
Cell viability assay (SulfoRhodamine B assay) To assess cellular proliferation,
Sulforhodamine B assay was used according to the manufacturer’s instructions.
Briefly, cells were grown in tissue culture flasks, and then harvested by treating the
flasks with 0.025 % trypsin and 0.25 mM EDTA for 5 min. Once detached, cells
were washed, counted and an aliquot (5 9 103 cells) was placed in each well of a
96-well cell culture plate in a total volume of 100 ll. Cells were allowed to attach
overnight and then treated with or without increasing concentrations of tested
compounds at serial concentrations of 100–6.25 lmol/ml (each repeated thrice).
After treatment, cell medium was aspirated, and 100 mm3/well of 10 %
trichloroacetic acid was added. After fixation at 4 °C and washing, 50 mm3 of
0.4 % (w/v) sulforhodamine B (SRB; Sigma-Aldrich) was added. Plates were
incubated at room temperature for 30 min. Unbound SRB was removed with 1 %
acetic acid. Bound SRB was solubilized with 100 mm3 of 10 mM Tris-base
solution. Absorbance was measured using a precision microplate reader (StatFax
2100, USA) at 570 and 650 nm (background). Doxorubicin was used as reference
drug in this study; positive control [6, 23]. Cell viability was calculated using
sigmoidal concentration response curve fitting models (Sigmaplot software) and
expressed as percentage of survival relative to untreated cells.
Apoptotic effect (Caspase-3 activity)
Human CASP3 assay is an in vitro enzyme-linked immunosorbent assay for the
quantitative measurement of Caspase-3 activities of cell culture supernatants. Six
hours after (MCF-7) cell lines treatment with tested compounds or doxorubicin,
CASP3 activity was determined according to manufacturer’s protocol (Cat.
KA1868, Ver01, Abnova). Absorbance was measured at 450 nm, using a precision
microplate reader (StatFax 2100, USA). Cellular apoptosis was calculated using
Sigma plot software (best-fit straight line) and expressed in folds relative to
untreated cells (negative control).
123

Design, synthesis of novel quinazolin-4-one derivatives…
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