Design, synthesis and biological evaluation of novel quinazoline derivatives as potential anti-cancer agents

Article abstract of DOI:10.3109/14756366.2011.601302

Twenty-two quinazoline derivatives have been synthesised and examined for their anti-tumour activity against three tumour cell lines, namely human breast cancer cell line (MCF-7), human cervical cancer cell line (HeLa) and human hepatoma cell line (HepG2). Twelve of the tested compounds have shown promising anti-tumour activity with an IC50 range of 5.09.7 g/mL. Regarding the spectrum of activity, five compounds exhibited interesting anti-proliferative properties against the three tested cell lines comparable to the reference drug (dasatinib).

Full text of DOI:10.3109/14756366.2011.601302

Journal of Enzyme Inhibition and Medicinal Chemistry, 2012; 27(4): 541–545
© 2012 Informa UK, Ltd.
ISSN 1475-6366 print/ISSN 1475-6374 online
DOI: 10.3109/14756366.2011.601302
research artIcle
Design, synthesis and biological evaluation of novel
quinazoline derivatives as potential anti-cancer agents
Ahmed M. Alafeefy1 and Abdelkader E. Ashour2
2
1Department of Pharmaceutical Chemistry, College of Pharmacy, AlKharj University, Saudi Arabia and Department of
Pharmacology and Toxicology, College of Pharmacy, King Saud University, Riyadh, Saudi Arabia
abstract
Twenty-two quinazoline derivatives have been synthesised and examined for their anti-tumour activity against three
tumour cell lines, namely human breast cancer cell line (MCF-7), human cervical cancer cell line (HeLa) and human
hepatoma cell line (HepG2). Twelve of the tested compounds have shown promising anti-tumour activity with an IC50
range of 5.0–9.7 µg/mL. Regarding the spectrum of activity, five compounds exhibited interesting anti-proliferative
properties against the three tested cell lines comparable to the reference drug (dasatinib).
Keywords: Anti-tumour, dasatinib, quinazoline, synthesis
Introduction
tyrosine kinases8–10. Dasatinib, the standard used in this
study, is a multi-targeted kinase inhibitor11.
With the aging of the world’s population, the westerni-
sation of diet and the widespread environmental pollu-
tion, cancer has emerged as the top threat to human life
worldwide1. For the majority of cancers, chemotherapy,
either alone or as an adjunct to radiotherapy or surgery,
remains the treatment of choice. Most anti-cancer agents
are broadly acting cytotoxic drugs. e heterogeneity
of tumours, the lack of selectivity between normal and
cancerous cells and the development of drug resistance
are some of the major obstacles hindering the reach
of an ultimate cure for such a deadly disease2. In spite
of the substantial progress in many aspects of cancer
research, the current approaches have many disadvan-
tages, including low efficacy and high degree of toxicity.
Notably, many cancer chemotherapeutic agents are
themselves carcinogenic3. erefore, there is an urgent
need to rationally design novel, molecularly targeted
anti-cancer therapies, which are more selective, ideally
less toxic and ultimately more effective than traditional
treatments.
Recently, quinazolines have shown inhibitory activ-
ity against NF-κB, a transcription factor that has been
reported to be involved in cancer development and pro-
gression, as well as in resistance to chemotherapy and
radiotherapy12. Tobe et al.13 have conducted a reporter
gene-based screening, with the reporter DNA having the
binding sequence for NF-κB and the luciferase gene, to
find new structural class of NF-κB activation inhibitors.
is effort led to the identification of quinazoline 1 as a
lead compound (IC50 = 2.3 µM), Figure 1.
Huntington’s disease (HD), a member of the trinu-
cleotide-repeat neurological disorders, is caused by a
heritable, polyglutamine expansion mutation in the
NH3-terminus of the Huntingtin protein (Htt). To search
for small-molecule activators of Htt protein clearance,
Rinderspacher et al.14 used a unique two-tiered functional
genetic screen and found that quinazoline derivatives 2–5
(Figure 1) suppressed tet-regulatable gene expression
with IC50 of 1.3, 2.21, 1.1 and 0.71 µM, respectively.
In this work, a new series of quinazoline compounds
of the general formula represented in Scheme 1 were
designed in such a way to accommodate phenyl group
at position 2 to modify the electronic effects (increase
Numerous quinazoline derivatives have been reported
to have anti-cancer activity4–6. In that context, these
agents have shown inhibitory activity against thymidylate
synthase5–7, dihydrofolate reductase and various receptor
Address for Correspondence: Ahmed M. Alafeefya, Department of Pharmaceutical Chemistry, College of Pharmacy, AlKharj University,
Saudi Arabia. E-mail: ahmed.alafeefy@yahoo.com
(Received 21 April 2011; revised 17 June 2011; accepted 20 June 2011)
541

542 A. M. Alafeefya and A. E. Ashourb
Figure 1.
Scheme 1. Synthesis of the proposed anti-tumour quinazoline derivatives (4–22).
lipophilicity) to study its effect on activity because most of
was alkylated with some selected phenethyl chloride
derivatives in dry dimethylformamide containing
anhydrous potassium carbonate. It was reported that
3H-quinazolin-4-one derivatives react readily with
alkyl halides at the 3-nitrogen and occasionally at the
4-oxygen atom16. e variation of the product ratio has
remained unclear until Bogentoft et al.17 presumed
that a steric hindrance effect of the 2-substituent of the
quinazoline ring facilitated the 4-O alkylation. However,
Hori et al.18 reported that the ratio of O/N alkylation was
not explicable by the steric hindrance alone, but other
factors such as electronic effects should be considered.
In this work, a mixture of two products was obtained
and separated by column chromatography. e first
one had the alkyl group at the 3-nitrogen atom to afford
3-alkylquinazolin-4-one derivatives 4–7. e other
one was identified to be the 4-alkoxy derivatives 8–11,
where alkylation occurred at the 4-oxygen atom. e
structures of these compounds were confirmed by infra
red (IR) and nuclear magnetic resonance (NMR) spectra.
Generally, compounds 4–7 showed the characteristic
thereportedactivecompoundsweredevoidofthismoiety.
Some substituted amines and/or their oxo-isosteres at
position 4, and/or piperidines were synthesised to impart
structural similarity to compounds 1–5. We designed
these compounds with one and two carbon spacer
between the 4-quinazolin-amine backbone and the side
chain at position 4 aiming to identify new candidates that
may be of value as potent, selective and less toxic anti-
tumour agents.
results and discussion
Chemistry
e synthesis of the quinazoline derivatives 1–22 was
carried out following Scheme 1. It outlines the syn-
thetic pathway used to obtain compounds 4–11. e
starting material 2-phenyl-4H-[3,1]benzoxazin-4-one
1, 2-phenyl-3H-quinazolin-4-one
2 and 2-phenyl-
4-chloroquinazoline 3 were prepared in our labora-
tory following a reported procedure15. Compound 2
Journal of Enzyme Inhibition and Medicinal Chemistry

Novel quinazoline derivatives as potential anti-cancer agents 543
carbonyl absorption band around 1695–1680 cm−1 and no
absorption band for the NH group. While the IR spectra
of compounds 8–11 revealed the absence of both the NH
and the endocyclic carbonyl C=O groups of the parent
compounds, and no signal for the NH groups was seen
in the H NMR spectra. On the other hand, the H NMR
spectra of the latter compounds showed triplet at around
δ 4.3, which corresponds to O-CH2, while the N-alkyl
derivatives 8–11 showed their triplet at around δ 3.5,
which corresponds to N-CH2. 13H NMR spectra of O-alkyl
derivatives, 4–7 showed their CH2 carbons at around δ
65 ppm. However, the N-alkyl derivatives 8–11 showed
their CH2 carbons at around δ 44 ppm.
e appropriate piperidine derivative was allowed to
react with the 4-chloro derivative in acetone containing
anhydrous potassium carbonate to afford the target
4-substituted derivatives 12–22. e structures of these
compounds were confirmed by IR, NMR and mass
spectra.
curves. e concentration causing 50% cell growth
inhibition (IC50) was calculated. Concerning sensitiv-
ity of cell lines to the synthesised compounds, MCF-7
cell line was shown to be the most sensitive toward the
tested compounds followed by HeLa and HepG2 cell
lines. As shown in Table 1, five compounds (14, 16–18,
22) showed broad spectrum inhibition of the three cell
lines. In this respect, the potency of these compounds
can be arranged in the following descending order:
22 > 14 > 18 > 16 > 17. Indeed, compounds 22, 14, 18
and 16 exhibited potent anti-tumour activity better
than that of the standard drug dasatinib, whereas the
potency of compound 17 was comparable to that of
dasatinib. On the other hand, compounds 10 and 12
were moderately active against both human breast
cancer cell line MCF-7 (IC50 = 9.7, 9.3 µg/mL, respec-
tively) and human hepatoma cell line HepG2 (IC50 = 9.2,
7.8 µg/mL, respectively). With regard to selectivity of
tested compounds against individual cell lines, most
of the tested compounds showed a distinctive pattern
of selectivity. Compounds 8, 9 and 12 showed high
degree of selectivity against the human breast cancer
cell line MCF-7 (IC50 = 9.3, 5.4, 9.3 µg/mL, respectively),
while compounds 5, 13 and 15 selectively inhibited the
human cervical cancer cell line HeLa with moderate
activity (IC50 = 9.7, 9.7, 7.8 µg/mL). Except for the above-
mentioned results, none of the compounds under test
selectively inhibited HepG2. Regarding the structure-
activity-relationship of the compounds presented in
this study, it is clear that, generally, the 4-substituted
1
1
Pharmacology
All the new compounds were screened for their anti-
tumour activity against HepG2 (human liver cancer
cell line), MCF-7 (human breast cancer cell line)
and HeLa (human cervical cancer cell line) using
sulphorhodamine-B assay as described previously19
and dasatinib as a reference drug. Each cell line was
incubated with five concentrations (0–100 µg/mL) of
each compound and the results were used to create
compound concentration versus survival fraction
Table 1. IC50 of synthesised compounds against cancer cell lines.
IC50 (µg/mL)
Cpd No.
MCF-7
20.7
20.7
23.6
16.0
9.3
HeLa
35.0
9.7
HepG2
44.1
24.5
24.1
34.1
36.0
12.1
9.2
4
5
6
30.3
36.9
43.1
16.0
22.2
38.9
16.4
9.7
7
8
9
5.4
10
9.7
11
37.4
9.3
32.2
7.8
12
13
18.8
5.0
29.3
7.8
14
5.4
15
19.8
5.5
7.8
14.5
7.2
16
7.5
17
9.3
7.7
7.4
18
5.0
9.7
5.9
19
11.3
14.7
13.5
5.0
15.5
16.4
12.0
5.4
13.2
19.1
20.1
5.9
20
21
22
Dasatinib
6.44
8.34
8.14
e cytotoxic activity of synthesised compounds against MCF-7, HeLa and HepG2 cells was examined using sulphorhodamine-B assay.
e dose-response relationships of the compounds were measured for each cell line using concentrations of 10, 25, 50 and 100 µg/
mL, and the concentration causing 50% cell growth inhibition (IC50), compared with control DMSO-treated cells, was calculated. e
clinically used anti-cancer drug dasatinib was used as standard.
© 2012 Informa UK, Ltd.

544 A. M. Alafeefya and A. E. Ashourb
quinazoline derivatives were generally more active
than the 3-substituted ones. Within the substituted pip-
eridine derivatives, the mono-substituted compounds
proved to be far more promising than the di-substituted
ones. It seems that electron releasing groups attached
to the piperidine ring has better impact on activity
than electron withdrawing ones; compounds 18 and
22 with 4-hydroxy and 4-piperidyl-piperidine moieties,
respectively, showed the highest activity, followed by
compounds 16 and 17 with 3-hydroxycarbonyl and
3-ethoxycarbonyl, respectively. While in compounds
20 and 21, the 4,4-di-substituted piperidine derivatives
were inactive or very weakly active. Amongst the flexible
4-substituted benzylamine derivatives, compound 14
with 4-chloro group was the most active one followed
by compound 12 (the unsubstituted), while the 4-fluo-
robenzyl derivative was the least active one.
Synthesis
Compounds 1–3 These compounds were prepared in our
laboratory according to reported procedures.
General procedures for the synthesis of compounds 4–11
mixture of 2-phenyl-4-chloroquinazoline (0.666 g,
A
3
0.003 mol) and the appropriate alkyl halide (0.003 mol)
in dry dimethylformamide (10 mL) in presence of
anhydrous potassium carbonate (0.3 g) was stirred for
24 h at room temperature. e solvent was then diluted
with water (30 mL) and the separated solid was filtered,
washed with water, dried and crystallised from ethanol
to afford compounds 4–1l.
4: Yield, 41%; m.p. 135–137°C; 1H NMR (CDCl3): δ
2.90–2.92 (t, 2H, C6H5CH2, J= 7.0 Hz), 4.34–4.36 (t, 2H,
OCH2, J= 7.0 Hz), 7.60–8.22 (m, 14H, Ar-H). 13C NMR: δ
34.40 (C6H5CH2), 64.98 (OCH2), 118.11, 121.95, 126.50,
127.28, 128.16, 129.23, 130.05, 131.15, 133.80, 140.06,
151.34, 163.28, 181.02 (Ar-C). MS (EI): m/z 326 [M+, 32%].
Anal. (C22H18N2O) C, H, N.
Conclusion
is work led to the development of novel anti-tumour
molecules containing 4-substituted aminoquinazoline
pharmacophore. ree cell lines, including MCF-7,
HeLa and HepG2, were used to measure cytotoxic
activity of the proposed quinazoline derivatives. Five
compounds exhibited promising and more potent anti-
tumour activity than the standard drug dasatinib and one
compound showed activity similar to that of dasatinib.
Most of the tested compounds showed varying degrees
of activity against the three selected cell lines and few
compounds were ineffective. Our preliminary results in
this study where we considered three cancer cell lines
will be followed by further studies to explore the exact
mechanism of action of the most active compounds in
addition to lead modifications.
General procedures for the synthesis of 2-phenyl 4-substi-
tuted amino quinazoline derivatives 12–22 A mixture of
compound 3 (0.36 g, 0.0015 mol) and the appropriate
piperidine derivative (0.0015 mol) in acetone (12 mL) in
presence of anhydrous potassium carbonate (0.5 g) was
heated under reflux for 5 h. e solvent was then evapo-
rated under vacuum and the separated solid was filtered,
washed with water, dried and crystallised from ethanol to
afford compounds l2–22.
12: Yield, 87%; m.p. 159–161°C; 1H NMR (CDCl3): δ4.33
(s, 2H, CH2), 6.70 (s, 1H, NH, exchangeable), 7.48–8.25 (m,
14H, Ar-H). 13C NMR: δ 45.84 (CH2), 117.0, 121.05, 126.42,
127.0, 127.41, 128.76, 129.83, 130.25, 130.64, 134.0, 142.16,
150.18, 157.33, 160.15. MS (EI): m/z 311 [M+, 65%]. Anal.
(C21H17N3) C, H, N.
experimental protocol
Chemistry
General
Pharmacology
Materials
Dasatinib was supplied by LC Laboratories® (Woburn,
MA, USA). All other chemicals were obtained from
Sigma Chemical Company (St. Louis, MO, USA). e cell
lines MCF-7, HeLa and HepG2 were obtained from the
American Type Culture Collection (Rockville, MD, USA).
Tissue culture plates and flasks were purchased from
Costar (Milan, Italy).
All melting points (°C, uncorrected) were determined
on a Stuart melting point apparatus (Stuart Scientific,
Redhill, UK). Elemental analyses (C, H, N) were
performed on Perkin-Elmer 2400 analyser (Perkin-Elmer,
Norwalk, CT, USA) and were in full agreement with the
proposed structures within ≥0.4% of the theoretical
values. IR spectra (KBr) were recorded using Pye Unicam
SP 1000 IR spectrometer (ermoelectron, Egelbach,
Germany) and expressed in wave number υ (cm−1). NMR
spectra were obtained on a Bruker AC 300 Ultra Shield
NMR spectrometer (Bruker, Munich, Germany) at 300
Evaluation of cellular cytotoxicity
e cytotoxic activity of compounds 4–22 against
HepG2 cells, MCF-7 cells and HeLa cells was deter-
mined using sulphorhodamine-B assay as described
by Skehan et al.19. Briefly, stock solutions of the tested
compounds were prepared in dimethyl sulphoxide
(DMSO) and were used for serial dilutions in culture
medium. e final concentration of DMSO in all wells
was adjusted to 0.3%. e tested cell lines were grown
in RPMI-1640 medium supplemented with 10% calf
serum. Exponentially growing cells were suspended in
1
MHz for H and 75 MHz for 13C; the chemical shifts are
expressed in δ (ppm) downfield from tetramethylsilane.
Splitting patterns were designated as follows: s: singlet;
d: doublet; t: triplet; m: multiplet. Electron impact mass
spectra were recorded on a Varian Mat 311-A70eV instru-
ment (Varian, Fort Collins, USA). Chemicals used were
supplied from Sigma-Aldrich (Sigma-Aldrich Chemie
GmbH, Steinheim, Germany).
Journal of Enzyme Inhibition and Medicinal Chemistry

Novel quinazoline derivatives as potential anti-cancer agents 545
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the above-mentioned medium, seeded in sextuplicates
onto 96-well plates at a density of 5 × 104 cells/well and
incubated at 37°C in a humidified 5% CO2 atmosphere
for 24 h. e cell medium in test wells was then changed
to new culture medium containing the required concen-
trations (10, 25, 50, 100 µg/mL) of the tested compounds,
while the cell medium in control wells was changed to
new culture medium containing an equivalent volume
of solvent (DMSO), and all cells were then incubated for
48 h. Following the 48-h exposure to the compounds,
cells were fixed with 50% cold trichloroacetic acid for
1 h, stained for 30 min with 0.4% sulphorhodamine-B
and then washed with 1% acetic acid. e plates were
then air-dried and the optical density of each well was
measured spectrophotometrically at 564 nm using
the enzyme-linked immunosorbent assay microplate
reader (Meter tech.*sigma;∑ 960, USA). Surviving frac-
tion for each cell type was determined, and then IC50
was calculated for each compound under investiga-
tion. It is worth to mention that the cytotoxic activity
of anti-cancer drug dasatinib, a multi-targeted kinase
inhibitor of BCR-ABL and Src family kinases9, against
the three cell lines was examined at the same concen-
trations of tested compounds and used as standard for
comparative purposes.
Declaration of interest
is work was supported by a grant from King Abdulaziz
city for science and technology (KACST; Grant No.: ARP-
29–265) and the National Plan of Science, Technology
and Innovation (Grant No. 10-MED1188-02), and a
funding from the College of Pharmacy Research Center,
King Saud University, Riyadh, Saudi Arabia.
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© 2012 Informa UK, Ltd.