N-(4-(quinazolin-2-yl)phenyl)benzamide derivatives with potent anti-angiogenesis activities: Synthesis and evaluation

Article abstract of DOI:10.1007/s13738-015-0788-4

Expanding our studies on the anti-angiogenesis activities of 2,4-disubstituted quinazoline derivatives [8], a series of novel N-(2-(quinazolin-2-yl)phenyl)benzamide (SZ) derivatives were designed and synthesized. Cytotoxicity assays indicated that most of these compounds displayed similar cytotoxicity against tumor cells in comparison with our previously reported, but showed a higher cytotoxicity against HUVECs. The SZ derivatives showed a remarkable inhibitive effect against the migration and adhesion of HUVECs, in addition to demonstrating significant in vivo anti-angiogenesis activities in the chick embryo chorioallantoic membrane (CAM) assay. The results proved that the introduction of an aryl group with a basic amide side chain on the 4′ position linked to the amide of the C-2 substituted quinazoline scaffold is an effective approach to improve the anti-angiogenic activity of quinazoline derivatives.

Full text of DOI:10.1007/s13738-015-0788-4

J IRAN CHEM SOC (2016) 13:753–761
DOI 10.1007/s13738-015-0788-4
ORIGINAL PAPER
N‑(4‑(quinazolin‑2‑yl)phenyl)benzamide derivatives with potent
anti‑angiogenesis activities: synthesis and evaluation
Ming Sun1 · Na Lv1 · Zeng Li2 · Qiru Xiong3 · Liang Xu4 · Zongsheng Yin1
Received: 23 August 2015 / Accepted: 20 November 2015 / Published online: 7 December 2015
© Iranian Chemical Society 2015
Abstract Expanding our studies on the anti-angiogenesis
activities of 2,4-disubstituted quinazoline derivatives [8],
a series of novel N-(2-(quinazolin-2-yl)phenyl)benzamide
(SZ) derivatives were designed and synthesized. Cytotoxic-
ity assays indicated that most of these compounds displayed
similar cytotoxicity against tumor cells in comparison with
our previously reported, but showed a higher cytotoxicity
against HUVECs. The SZ derivatives showed a remark-
able inhibitive effect against the migration and adhesion of
HUVECs, in addition to demonstrating significant in vivo
anti-angiogenesis activities in the chick embryo chorioal-
lantoic membrane (CAM) assay. The results proved that the
introduction of an aryl group with a basic amide side chain
on the 4′ position linked to the amide of the C-2 substituted
quinazoline scaffold is an effective approach to improve the
anti-angiogenic activity of quinazoline derivatives.
Introduction
Angiogenesis is the normal process in the growth and
development of new blood vessels from the pre-existing
vasculature of a parasitifer, and participates in many physi-
ological processes, such as wound healing, the menstrual
cycle, pregnancy and organ development [1]. Normally,
there is a balance between molecules that either stimulate
or inhibit angiogenesis so that blood vessels form only
when and where they are needed. Recently, research has
found that the balance is broken in the course of many dis-
eases, especially in various tumor types including solid and
hematopoietic neoplasms. Aberrant angiogenesis would
lead to the formation of abnormal vessels around the tumor,
an essential prerequisite for tumor cell proliferation, inva-
sion and metastasis [2–4]. Therefore, the search for phar-
maceuticals targeting angiogenesis in tumors is a promising
strategy for the development of cancer therapy [5].
ZD6474 (vandetanib, AstraZeneca Company), a het-
eroaromatic-substituted anilinoquinazoline (Fig. 1) was
found to act as a potent and reversible inhibitor of VEGF
receptor-2 (VEGFR-2), EGFR and RET tyrosine kinase,
and showed broad spectrum anti-tumor activity that
was dependent on the inhibition of angiogenesis [6, 7].
Recently, we reported on the synthesis and angiogenesis
inhibiting activity of a new 2,4-disubstituted quinazoline
derivative 11d, which shares the quinazoline scaffold of
ZD6474 [8]. As a result of our continued efforts to screen
synthetic compounds exhibiting anti-tumor properties, a
novel series of quinazoline-based analogs based on 11d
were designed, synthesized and evaluated for their anti-
proliferative and anti-angiogenic activities. In the current
research, we modified the C-2 substituted quinazoline-
based scaffold of 11d by linking a new para-substituted
Keywords 2,4-Disubstituted quinazoline derivatives ·
N-(2-(Quinazolin-2-yl)phenyl)benzamide (SZ) derivatives ·
Synthesized · Anti-angiogenesis
M. Sun and Na Lv contributed equally to the work and should be
regarded as co-first authors.
* Zongsheng Yin
zongshengyin1314@sina.com
1
Department of Stomatology, First Affiliated Hospital
of Anhui Medical University, Hefei 230032, China
2
Department of Chemistry, Anhui Medical University,
Hefei 230032, China
3
Department of Chirurgery, First Affiliated Hospital of Anhui
Medical University, Hefei 230032, China
4
Enantiotech Corporation LTD, Zhongshan 528400, China
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J IRAN CHEM SOC (2016) 13:753–761
N
N
r
B
F
NH
NH
N
HN
N
O
O
N
N
N
HN
N
HN
O
N
N
O
O
R
n
N
H
N
A
B
C
Fig. 1 Structures of a ZD6474, b 11d, and c N-(4-(quinazolin-2-yl)phenyl)benzamide (SZ) derivatives
aryl moiety to the carbonyl of the amide, while still retain-
ing the C-4 substituted amino side chain of 11d. Therefore,
a series of N-(4-(quinazolin-2-yl)phenyl)benzamide (SZ)
derivatives were synthesized (Fig. 1, SZ10a‑d, SZ11a‑d)
and herein we report on the cytotoxicity and anti-angio-
genic effects of these novel quinazoline analogs.
Compound 6: N‑(2‑(4‑(3‑(dimethylamino)propylamino)
quinazolin‑2‑yl)phenyl)‑4‑nitrobenzamide
4-Nitrobenzoyl chloride (50 mmol) was added dropwise
to a solution of 5 (100 mmol) in chloroform (100 mL) at
room temperature and was stirred for 10 h. The gener-
ated solid was filtered, recrystallized with ethanol, and
dried, affording product 6 as a white solid. Yield: 71 %;
1H NMR (400 MHz, CDCl3): δ = 1.88–1.98 (m, 2H),
2.41 (s, 6H), 2.68 (t, J = 5.8 Hz, 2H), 3.90 (dd, J = 7.9,
5.1 Hz, 2H), 7.37–7.58 (m, 3H), 7.60 (d, J = 8.1 Hz, 1H),
7.64 (d, J = 5.9 Hz, 1H), 7.82 (t, J = 8.0 Hz, 1H), 8.34 (d,
J = 7.6 Hz, 2H), 8.44 (d, 2H), 8.79 (d, J = 7.5 Hz, 1H),
8.86 (d, J = 7.6 Hz, 1H), 9.14 (s, 1H), 14.73(s, 1H). ESI–
MS m/z 471 [M + H]+.
Experimental
Synthesis and characterization
1H NMR spectra were recorded on a Bruker 400 NMR with
tetramethylsilane (TMS) as an internal standard. NMR data
are shown only for novel compounds. MS spectra were
obtained using a Shimadzu LC–MS-2010A spectrometer.
Elemental analysis was carried out on an Elementar Vario
EL CHNS Elemental Analyzer. Melting points (m.p.) were
determined using an SRS-OptiMelt automated melting
point instrument without correction.
Compound 7: 4‑amino‑N‑(2‑(4‑(3‑(dimethylamino)
propylamino)quinazolin‑2‑yl)phenyl)benzamide
We adopted the same synthetic method as that of compound
1
5, generating 7 as a white powder. Yield 83 %; H NMR
Compound 2: 2‑(2‑Nitrophenyl)quinazolin‑4(3H)‑one
(400 MHz, CDCl3): δ = 1.90–1.98 (m, 2H), 2.44 (s, 6H),
2.70 (t, J = 5.8 Hz, 2H), 3.91 (dd, J = 7.8, 5.2 Hz, 2H), 4.1
(m, 2H), 6.70 (d, J = 8.1 Hz, 2H), 7.18 (d, J = 7.5 Hz, 1H),
7.41–7.54 (m, 2H), 7.63–7.75 (m, 2H), 7.82 (t, J = 8.3 Hz,
1H), 8.32 (d, J = 7.9 Hz, 2H), 8.79 (d, J = 7.5 Hz, 1H),
8.90 (s, 1H), 9.02 (d, J = 8.4 Hz, 1H), 14.32 (s, 1H). ESI–
MS m/z: 441 [M + H]+.
ZnI2 (10 mol%), DMSO (2 mL), benzyl alcohol (1 mmol),
2-aminobenzamide (1 mmol), TBHP (70 % in H2O,
4 eq.) and a stirring bar were added to a 50 mL pressure
tube. The mixture was kept at 110°Cfor 14 h and the sol-
vent was then removed under vacuum. The pure prod-
uct was isolated by recrystallization from MeOH. Yield
57 %; 1H NMR (400 MHz, DMSO-d6): δ = 7.53–7.61 (m,
1H), 7.69 (t, J = 7.64 Hz, 1H), 7.81–7.95 (m, 4H), 8.33
(dt, J = 8.62 Hz, 2H), 12.75 (s, 1H). ESI–MS m/z: 268
[M + H]+.
General acylation procedure A
A solution of the acid halide (9.6 mmol) in chloroform
(10 mL) was added to a well-stirred mixture of the amino-
substituted compound 7 (8 mmol), followed by the addition
of Na2CO3 (1.6 g) in chloroform (50 mL) at 0 °C, and the
Preparation of 3–5. Compounds 3–5 were synthesized
according to the previously established procedure [8].
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mixture was then allowed to stir overnight. The crude prod-
uct formed as a precipitate was filtered off and was purified
by flash column chromatography with EtOAc/petroleum
ether (1:10) elution to give compounds 8–9.
with petroleum ether/EtOAc (15:1) elution yielded 8
as a white solid. Yield 69 %; mp 164–166 °C. H NMR
1
(400 MHz, CDCl3): δ = 1.12 (t, 6H), 1.81–1.89 (m, 2H),
2.41 (s, 6H), 2.59 (m, 6H), 3.14 (s, 2H), 3.76 (dd, J = 10.3,
5.2 Hz, 2H), 7.07 (t, J = 7.9 Hz, 1H), 7.38 (t, J = 7.4 Hz,
1H), 7.41 (t, 1H), 7.59 (d, J = 8.1 Hz, 1H), 7.65 (d,
J = 7.6 Hz, 1H), 7.69–7.74 (m, 3H), 8.10 (d, J = 8.5 Hz,
2H), 8.72 (d, J = 8.0 Hz, 2H), 8.90 (d, J = 8.3 Hz, 1H),
9.64 (s, 1H), 14.41 (s, 1H). ESI–MS m/z: 554 [M + H]+.
Anal. Cacld for C32H39N7O2.H2O: C, 69.41; H, 7.10; N,
17.71; Found: C, 69.72; H, 7.06; N, 17.69.
Compound 8: 4‑(2‑chloroacetamido)‑N‑(2‑(4‑(3‑(dimethyl
amino)propylamino)quinazolin‑2‑yl)phenyl)benzamide
Compound 7 was treated with chloroacetyl chloride
according to the general acylation procedure, forming com-
pound 8 as a white solid. Yield 74 %; 1H NMR (400 MHz,
CDCl3): δ = 1.88–1.95 (m, 2H), 2.43 (s, 6H), 2.61–2.65
(m, 2H), 3.16 (s, 2H), 3.91 (dd, J = 10.3, 5.5 Hz, 2H),
7.24 (m, 1H), 7.42–7.55 (m, 2H), 7.61 (d, J = 8.0 Hz,
1H), 7.71–7.84 (m, 4H), 8.23 (d, J = 7.9 Hz, 2H), 8.75 (d,
J = 7.9 Hz, 1H), 8.83 (s, 1H), 8.94 (d, J = 8.2 Hz, 1H),
9.52 (s, 1H), 14.39 (s, 1H). ESI–MS m/z: 518 [M + H]+.
Compound SZ10b: N‑(2‑(4‑(3‑(dimethylamino)
propylamino)quinazolin‑2‑yl)phenyl)‑4‑(2‑(piperidin‑1‑yl)
acetamido)benzamide
Compound 8 was treated with excess piperidine accord-
ing to the general aminolysis procedure to afford SZ10b;
purification by column chromatography with petroleum
ether/EtOAc (15:1) elution yielded 8 as a white solid. Yield
Compound 9: 4‑(3‑chloropropanamido)‑N‑(2‑(4‑(3‑(dimet
hylamino)propylamino)quinazolin‑2‑yl)phenyl)benzamide
1
59 %; mp 177–1789 °C. H NMR (400 MHz, CDCl3):
Compound 7 was treated with 3-chloropropanoyl chloride
according to the general acylation procedure to form com-
pound 9 as a white solid. Yield: 71 %; 1H NMR (400 MHz,
CDCl3): δ = 1.89-1.95 (m, 2H), 2.40 (s, 6H), 2.61-2.65 (m,
2H), 3.07 (t, 2H), 3.86 (dd, J = 10.1, 5.4 Hz, 2H), 3.96 (t,
J = 5.6 Hz, 2H), 7.21 (m, 1H), 7.43-7.51 (m, 2H), 7.62 (d,
J = 7.8 Hz, 1H), 7.71-7.86 (m, 4H), 8.21 (d, J = 7.9 Hz,
2H), 8.76 (d, J = 7.9 Hz, 1H), 8.88 (s, 1H), 8.96 (d,
J = 8.4 Hz,1H), 10.34 (s, 1H), 14.41(s, 1H). ESI–MS m/z:
532 [M + H]+.
δ = 1.53–1.62 (m, 4H), 1.76–1.83 (m, 2H), 1.90–1.96
(m, 2H), 2.43 (s, 6H), 2.66 (t, J = 5.8 Hz, 2H), 2.78 (t,
J = 6.3 Hz, 4H), 3.36 (s, 2H), 3.89 (dd, J = 10.1, 5.3 Hz,
2H), 7.20 (t, J = 7.9 Hz, 1H), 7.44 (t, J = 7.2 Hz, 1H), 7.50
(t, J = 7.6 Hz, 1H), 7.68 (d, J = 8.3 Hz, 1H), 7.74–7.85
(m, 4H), 8.18 (d, J = 8.5 Hz, 2H), 8.70 (d, J = 8.0 Hz,
1H), 8.85 (s, 1H), 8.87 (d, J = 8.3 Hz, 1H), 9.14 (s, 1H),
14.43 (s, 1H). ESI–MS m/z: 566 [M + H]+. Anal. Cacld for
C33H39N7O2.2H2O: C, 70.06; H, 6.95; N, 17.33; Found: C,
69.96 H, 7.01; N, 17.25.
General aminolysis procedure B
Compound SZ10c: N‑(2‑(4‑(3‑(dimethylamino)
propylamino)quinazolin‑2‑yl)
To a stirred suspension of compounds 8 or 9 (0.5 mmol)
and KI (0.08 g) in EtOH (20 mL), the appropriate second-
ary amine (2.0 mL) was added dropwise; the mixture was
then stirred under reflux for 6 h. Upon reaction comple-
tion, the mixture was diluted with distilled water, filtered,
and washed with ether. Finally, the crude solid was purified
by chromatography with petroleum ether/EtOAc elution to
afford SZ10a‑10d and SZ11a‑11d.
phenyl)‑4‑(2‑morpholinoacetamido)benzamide
Compound 8 was treated with excess piperidine accord-
ing to the general aminolysis procedure to afford SZ10c;
purification by column chromatography with petroleum
ether/EtOAc (15:1) elution yielded 8 as a white solid.
Yield 68 %; mp 188–189 °C. 1H NMR (400 MHz, CDCl3):
δ = 1.81–1.86 (m, 2H), 2.39 (s, 6H), 2.54–2.63 (m, 6H),
3.21 (s, 2H), 3.78 (t, 4H), 3.93 (dd, J = 10.4, 5.9 Hz, 2H),
7.18 (t, J = 7.9 Hz, 1H), 7.36 (t, J = 8 Hz, 1H), 7.42 (t,
J = 7.5 Hz, 1H), 7.63 (d, J = 8.0 Hz, 1H), 7.71–7.82 (m,
4H), 8.20 (d, J = 8.3 Hz, 2H), 8.70 (d, J = 8.0 Hz, 1H),
8.83 (s, 1H), 8.87 (d, J = 8.3 Hz, 1H), 9.19 (s, 1H), 14.41
(s, 1H). ESI–MS m/z: 568 [M + H]+. Anal. Cacld for
C32H37N7O3.H2O: C, 67.70; H, 6.57; N, 17.27; Found: C,
67.76 H, 6.63; N, 17.04.
Compound SZ10a: 4‑(2‑(diethylamino)acetamido)‑N‑(2‑
(4‑(3‑(dimethylamino)propylamino)quinazolin‑2‑yl)phenyl)
benzamide
Compound 8 was treated with excess diethylamine accord-
ing to the general aminolysis procedure to afford SZ10a;
purification was completed by column chromatography
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Compound SZ10d: N‑(2‑(4‑(3‑(dimethylamino)
propylamino)quinazolin‑2‑yl)
phenyl)‑4‑(2‑(4‑methylpiperazin‑1‑yl)acetamido)
benzamide
J = 8.6 Hz, 2H), 7.74–7.79 (m, 1H), 7.84 (d, J = 8.2 Hz,
1H), 8.17 (d, J = 8.6 Hz, 2H), 8.79 (dd, J = 8.0, 1.5 Hz,
1H), 8.89 (t, J = 4.4 Hz, 1H), 8.96 (dd, J = 8.3, 0.7 Hz,
1H), 11.45 (s, 1H), d 14.42 (s, 1H). ESI–MS m/z: 580
[M + H]+. Anal. Cacld for C34H41N7O2.H2O: C, 70.44; H,
7.13; N, 16.91; Found: C, 70.66; H, 7.25; N, 17.04.
Compound 8 was treated with excess piperidine according
to the general aminolysis procedure to afford SZ10d puri-
fication by column chromatography with petroleum ether/
EtOAc (15:1) elution yielded 8 as a white solid. Yield 44 %;
Compound SZ11c: N‑(2‑(4‑(3‑(dimethylamino)
propylamino)quinazolin‑2‑yl)
phenyl)‑4‑(3‑morpholinopropanamido)benzamide
1
mp 183–184 °C. H NMR (400 MHz, CDCl3): δ = 1.76–
1.88 (m, 2H), 2.11 (s, 3H), 2.37 (s, 6H), 2.43–2.51 (m, 4H),
2.55–2.65 (m, 6H), 3.17 (s, 2H), 3.89 (dd, J = 9.4, 5.3 Hz,
2H), 7.16 (t, J = 7.9 Hz, 1H), 7.38 (t, J = 7.4 Hz, 1H), 7.42
(t, J = 7.8 Hz, 1H), 7.64 (d, J = 8.2 Hz, 1H), 7.73–7.79
(m, 4H), 8.10 (d, J = 8.4 Hz, 2H), 8.68 (d, J = 7.9 Hz,
1H), 8.74 (s, 1H), 8.91 (d, J = 8.3 Hz, 1H), 9.25 (s, 1H),
14.41 (s, 1H). ESI–MS m/z: 581 [M + H]+. Anal. Cacld for
C33H40N8O2.H2O: C, 68.25; H, 6.94; N, 19.30; Found: C,
68.36 H, 6.91; N, 19.15.
Compound 9 was treated with excess piperidine accord-
ing to the general aminolysis procedure to afford SZ11c;
purification by column chromatography with petroleum
ether/EtOAc (15:1) elution yielded 9 as a white solid.
Yield 60 %; mp 174–176 °C. 1H NMR (400 MHz, CDCl3):
δ = 1.78–1.84 (m, 2H), 2.37 (s, 6H), 2.56 (t, J = 5.8 Hz,
2H), 2.59–2.67 (m, 6H), 2.77 (t, J = 5.6 Hz, 2H), 3.81–
3.88 (m, 6H), 7.17 (t, J = 7.3 Hz, 1H), 7.39 (t, J = 7.8 Hz,
1H), 7.46 (t, J = 7.6 Hz, 1H), 7.56 (d, J = 8.3 Hz, 1H),
7.64–7.69 (m, 3H), 7.77 (d, J = 8.2 Hz, 1H), 8.12 (d,
J = 8.5 Hz, 2H), 8.74 (d, J = 8.0 Hz, 1H), 8.82 (s, 1H),
8.93 (d, J = 8.3 Hz, 1H), 11.13 (s, 1H), 14.39 (s, 1H). ESI–
MS m/z: 582 [M + H]+. Anal. Cacld for C33H39N7O3.H2O:
C, 68.14; H, 6.76; N, 16.86; Found: C, 68.09; H, 6.71; N,
16.91.
Compound SZ11a: 4‑(3‑(diethylamino)propanamido)‑
N‑(2‑(4‑(3‑(dimethylamino)propylamino)quinazolin‑2‑yl)
phenyl)benzamide
Compound 9 was treated with excess diethylamine accord-
ing to the general aminolysis procedure to afford SZ11a;
purification by column chromatography with petroleum
ether/EtOAc (15:1) elution yielded 9 as a white solid.
Yield: 68 %; mp 156–157 °C. 1H NMR (400 MHz, CDCl3):
δ = 1.13 (t, 6H), 1.91e1.95 (m, 2H), 2.38 (s, 6H), 2.49 (t,
J = 5.7 Hz, 2H), 2.62–2.69 (m, 6H), 2.77 (t, J = 5.5 Hz,
2H), 3.86 (dd, J = 9.3, 5.8 Hz, 2H), 7.14 (t, J = 7.7 Hz,
1H), 7.41–7.48 (m, 2H), 7.68 (d, J = 8.39 Hz, 4H), 7.74
(d, J = 8.6 Hz, 1H), 8.06 (d, J = 8.2 Hz, 2H), 8.70 (d,
J = 7.2 Hz, 2H), 8.88 (d, J = 8.3 Hz, 1H), 11.37 (s, 1H),
14.39 (s, 1H). ESI–MS m/z: 568 [M + H]+. Anal. Cacld for
C33H41N7O2.2H2O: C, 69.81; H, 7.28; N, 17.27; Found: C,
69.92; H, 7.34; N, 17.26.
Compound SZ11d: N‑(2‑(4‑(3‑(dimethylamino)
propylamino)quinazolin‑2‑yl)
phenyl)‑4‑(3‑(4‑methylpiperazin‑1‑yl)propanamido)
benzamide
Compound 9 was treated with excess piperidine accord-
ing to the general aminolysis procedure to afford SZ11d;
purification by column chromatography with petroleum
ether/EtOAc (15:1) elution yielded 9 as a white solid.
Yield: 47 %; mp 165–166 °C. 1H NMR (400 MHz, CDCl3):
δ = 1.85–1.96 (m, 4H), 2.18 (s, 3H), 2.22 (s, 6H), 2.35–
2.44 (m, 6H), 2.49–0.57 (m, 4H), 2.67 (t, J = 6.6 Hz, 2H),
3.67 (dd, J = 11.4, 6.6 Hz, 2H), 7.26 (t, J = 7.2 Hz, 1H),
7.54 (t, J = 7.8 Hz, 1H), 7.61 (t, J = 7.6 Hz, 1H), 7.76(d,
J = 8.3 Hz, 1H), 7.81–7.87 (m, 3H), 8.04 (d, J = 8.0 Hz,
2H), 8.33 (d, J = 8.0 Hz, 1H), 8.68–8.77 (m, 2H), 8.84
(d, J = 8.1 Hz, 1H), 11.07 (s, 1H), 14.35 (s, 1H). ESI–MS
m/z: 595 [M + H]+. Anal. Cacld for C34H42N8O2.2H2O:
C, 68.66; H, 7.12; N, 18.84; Found: C, 68.60; H, 6.99; N,
18.91.
Compound SZ11b: N‑(2‑(4‑(3‑(dimethylamino)
propylamino)quinazolin‑2‑yl)phenyl)‑4‑(3‑(piperidin‑1‑yl)
propanamido)benzamide
Compound 9 was treated with excess piperidine according
to the general aminolysis procedure to afford SZ11b; puri-
fication by column chromatography with petroleum ether/
EtOAc (15:1) elution yielded 9 as a white solid. Yield 63 %;
1
mp 159–160 °C. H NMR (400 MHz, CDCl3): δ = 1.55–
1.65 (m, 4H), 1.77–1.83 (m, 2H), 1.88–1.94 (m, 2H), 2.45
(s, 6H), 2.56 (t, J = 5.8 Hz, 2H), 2.67 (t, J = 5.5 Hz, 2H),
2.77 (t, J = 6.4 Hz, 4H), 2.90 (t, J = 5.8 Hz, 2H), 3.89 (dd,
J = 10.0, 5.9 Hz, 2H), 7.19–7.24 (m, 1H), 7.42–7.48 (m,
1H), 7.50–7.53 (m, 1H), 7.64 (d, J = 7.9 Hz, 1H), 7.68 (d,
Materials and biological assays
Stock solutions of all compounds (10 mM) were made with
DMSO and stored at 4 °C. HUVEC and tumor cell lines
were obtained from the Animal Laboratory Center of Sun
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Yat-Sen University. The cells were maintained in a DMEM
medium supplemented with 10 % fetal bovine serum,
100 U/mL penicillin, and 100 mg/mL of streptomycin in
25 cm2 culture flasks at 37 °C humidified atmosphere with
5 % CO2. Chick embryos were purchased from Anhui Agri-
cultural University.
wounded area or that protruded from the border of the
wound were visualized, photographed under an inverted
microscope and the area of the migrations was measured
using Photoshop CS3. All measurements were done in
triplicate.
Chicken chorioallantoic membrane (CAM) assay
Methyl thiazolyl tetrazolium (MTT) assay
The anti-angiogenic activity of compounds on Chorioal-
lantoic Membrane (CAM) was assayed as described previ-
ously [15–17]. First, fertilized chicken eggs were incubated
for 8 days. Then, a 1 cm × 1 cm window was punched on
the broad side of the egg to expose the CAM. Sterilized
filter paper disks saturated with 0.9 % NaCl solution or
30 mg/10 mL of compounds were placed upon the CAM.
The eggs were then incubated at 37 °C for a further 48 h.
Finally, an appropriate volume of 4 % paraformaldehyde
was fixed upon the CAMs to observe the density and length
of the vessels toward the CAM face, and the vessel number
from three random fields around the sterilized filter paper
disk was pictured using a microscope.
The HUVEC, CNE-2, PC-3, and SMMC-7721 cells were
seeded in 96-well plates at a concentration of 5000 cells/
well. After overnight incubation, the cells were exposed
to different concentrations of the derivatives for 48 h.
Subsequently,
a 10 mL 3-(4,5-dimethylthiazol-2-yl)-
2,5diphenylte-trazolium bromide (MTT, 5 mg/mL) solu-
tion was added to each well and further incubated for 4 h
at 37 °C. Next, to each well 100 mL of DMSO was added,
and the optical density (OD) was read on the microplate
reader at 490 nm. The IC50 values were determined from
the mean OD values of the triplicate tests versus drug con-
centration curves [9, 10].
Cell adhesion assay
Results and discussion
Chemistry
The cell adhesion assay has been reported previously [11,
12]. Collagen type I (0.02 mg/mL) from the rat trail ten-
don was seeded in 96-well plates at room temperature
overnight. Then, the wells were washed with PBS in tripli-
cate and blocked with 0.2 % bovine serum albumin (BSA)
for 2 h at 37 °C. The wells were washed three times with
PBS (100 mL) again. Subsequently, the HUVECs (50 μL,
104 cells/ml) and related compounds (50 μL, at vari-
ous concentrations) were added to each well. After a 1 or
3 h incubation period, the plates were washed three times
with 100 mL of PBS. Then, the attached cells were fixed
and stained with 0.2 % crystal violet in 20 % methanol
for 10 min, and washed three times with 100 mL of PBS.
Finally, the cell cultures were solubilized by 2 % SDS and
the OD was determined at 570 nm; each experiment was
repeated three times.
The new quinazoline derivatives (SZ derivatives) were
prepared as depicted in Scheme 1, a route that closely fol-
lows the previously reported procedure [8]. To obtain the
quinazolinedione intermediate 2, we employed 2-nitroben-
zaldehyde as the reagent instead of 2-Nitrobenzoyl chlo-
ride [18]. Intermediate 6 was prepared by mixing the C-2
substituted quinazoline analog 5 with 2-nitrobenzoyl chlo-
ride in chloroform at room temperature. The nitro-group
on intermediate 6 was then reduced to the amino group by
Pd/C/H2 in isopropanol, affording intermediate 7, which
was then treated with various acyl chlorides in dichlo-
romethane to generate compounds 8‑9. Finally, intermedi-
ates 8 and 9 were reacted with the appropriate secondary
amines through a nucleophilic substitution reaction to form
the target compounds SZ10a–d and SZ11a–d. The correct
structure of the desired compounds was confirmed through
analytical and spectral data (MS, NMR, etc.).
Reagent: (a) ZnI2 (10 mol %), TBHP (70 % in H2O,
4 eq.), DMSO, 110 °C 14 h. (b) N,N-diethylaniline, POCl3,
toluene, reflux, 6 h; (c) 3-aminopropyldimethyl-amine,
THF, reflux, 5 h; (d) 10 % Pd/C, 80 % N2H.4H2O, isopro-
panol, reflux, 2 h; (e) 4-nitrobenzoyl chloride, K2CO3,
CH3Cl3, rt, 24 h; (f) 10 % Pd/C, 80 % N2H4.H2O, Isopro-
panol, reflux, 2 h; (g) Cl(CH2)nCOCl, K2CO3, CH3Cl3, rt,
24 h; (h) R2NH, KI, ethanol, reflux.
Endothelial cell migration assay
The cell migration assay was used to confirm the effect
of the derivatives on cell invasion activity [13, 14].
HUVEC cell lines were plated in triplicate in pre-treated
48-well culture plates (2 × 104 cells/well) and incubated
for 24 h. The cells were then scraped away with a ster-
ile disposable rubber policeman. The debris was removed
by washing with PBS (phosphate-buffer saline) and
the compounds were added to a DMEM medium. After
24 h incubation, the HUVEC cells that migrated into the
1 3

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J IRAN CHEM SOC (2016) 13:753–761
N
O
NH
N
l
C
O
N
H
NH2
NH2
c
N
O2
N
N
NH
N
b
a
N
N
O
N
O2
O2
O2
4
3
2
N
N
N
NH
NH
NH
d
e
g
f
N
N
N
N
N
N
HN
HN
H2N
5
O
O
N
O2
NH2
7
6
N
N
NH
NH
h
N
N
O
N
c
N
N
a
N
N
N
N
d
HN
b
HN
O
O
O
O
R
n
n=
a-
SZ10 10d
n=1
n=2
8
1
l
C
N
H
N
H
n=
a
-
9
2
SZ11 11d
n
Scheme 1 Synthesis route for the formation of derivatives SZ10a-d and SZ11a-d
Table 1 IC50 cytotoxicity values (µM) of quinazoline derivatives
SZ10a-d and SZ11a-d against tumor cells
Cell growth inhibition
We previously reported on the cytotoxic properties of
11d, but there are fewer studies on the in vitro cytotoxic-
ity of the newly-synthesized SZ derivatives [8]. As was
previously observed with 11d, inhibition of cell prolifera-
tion was observed following treatment of both normal cell
lines (endothelial cell) and tumor cell lines with the SZ
derivatives.
The MTT cytotoxicity assay was carried out to deter-
mine the cytotoxic activities of the SZ derivatives against
Human Umbilical Vein Endothelial Cells (HUVEC) and
human tumor cell lines, including PC-3 (human prostatic
carcinoma), CNE-2 (human nasopharyngeal cancer) and
SMMC-7721 (human liver cancer). Table 1 shows the IC50
values (cytotoxicity potency indexes) of all the derivatives
against four types of cells lines. Comparing quinazoline
derivative 11d with the novel derivatives, SZ10a‑d and
SZ11a‑d which bear an additional aromatic moiety, we find
that the latter (some compounds) have a similar cytotoxic-
ity against the tumor cells and a higher cytotoxicity against
Compound IC50 (µM)
CNE-2
PC-3
SMMC-7721 HUVEC
SZ10a
SZ10b
SZ10c
SZ10d
SZ11a
SZ11b
SZ11c
SZ11d
11d
15.7 0.2 14.2 0.1 16.6 0.1
16.8 0.2 12.4 0.1 22.5 0.3
21.2 0.4 28.1 0.3 19.7 0.5
40.2 0.5
33.6 0.3
41.5 0.2
28.4 0.5
35.3 0.2
29.7 0.4
46.2 0.3
21.8 0.3
45.6 0.2
14.7 0.3 17.2 02
13.6 0.1
13.9 0.2 12.7 0.2 16.4 0.3
17.7 0.2 16.5 0.2 15.4 0.3
24.5 0.5 20.8 0.3 22.4 0.1
15.1 0.2 14.6 0.1 12.9 0.5
11.3 0.2 10.6 0.5 12.4 0.2
Data derived from the mean of three independent assays
the HUVEC cell line. (Table 1). Like in the previous study
with 11d, the IC50 values of all the SZ derivatives tested on
the tumor cell lines also fall in the range of 10–28.1 µM;
however, the IC50 value on the HUVEC line was reduced
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J IRAN CHEM SOC (2016) 13:753–761
759
to 21.8 µM (SZ11d). Among compounds having the same
R-group, those bearing the less basic morpholine group
(SZ10c and SZ11c) had the weakest cytotoxicity to various
cancer cells lines and to the normal cell line. In contrast,
compounds with a more basic amino terminus (N-methyl-
piperazino analogs, SZ10d and SZ11d) showed a higher
activity on all the cell lines. On the other hand, compar-
ing SZ10d and SZ11d, which have the same R group but
vary in the length of the amide side chain (n = 1 and n = 2,
respectively), we observed no significant effect on the
IC50 values. The results demonstrate the importance of the
basicity of the amide side chain to achieve strong cytotox-
icities on the cell lines.
chain and the less basic morpholine group, showed the
weakest inhibition of HUVEC adhesion (34.3
0.2 %
at a dose of 30 μM; 43.2 0.2 % at a dose of 30 μM,
3 h incubation at 37 °C). Similar to the results of the cyto-
toxicity assays, the basicity of the R group was proven to
be an important factor in the potency of the derivative for
inhibition. Comparing the SZ10a‑d and SZ11a‑d deriva-
tives, we can also detect a strengthening of the inhibitory
effect in all cases as the alkyl chain is lengthened from
n = 1 to n = 2. So we can find that all SZ compounds
with a new phenyl group onto the scaffold of quinazoline
derivatives gave an improvement of inhibition effect of
HUVEC adhesion.
Cell adhesion assays
Migration inhibition assays
Cell adhesion plays an important role in maintaining cell
shape, regulation of cell division and movement, as well
as being an important step in the process of angiogenesis.
Therefore, we performed HUVEC cell adhesion assays to
investigate the inhibitory effects of these SZ derivatives on
the attachment of endothelial cells to type I collagen and
thalidomide was selected as positive control.
As shown in Table 2, the inhibition rate of the SZ deriv-
atives for HUVEC adhesion to collagen showed a strong
inhibitory effect at both 1 and 3 h (Inhibition rate range:
31.4 0.3–74.3 0.1 %). SZ11d (n = 2), containing the
more basic piperazino moiety, results in the strongest inhi-
Endothelial cell migration is an essential step in angio-
genesis, hence inhibition of this process could hinder the
formation of new blood vessels. Based on the most sat-
isfactory results of the HUVEC adhesion assay, deriva-
tives SZ10d, SZ11d, thalidomide and 2,4-disubstituted
quinazoline derivative 11d were chosen to evaluate their
inhibition against HUVEC migration in a wound-heal-
ing migration assay. All compounds were tested at con-
centrations of both 15 and 30 μM, and were all able to
inhibit HUVEC migration. Compared with 11d, SZ10d
and SZ11d, with a longer amide side chain in the 4-posi-
tion of the added aryl group, showed a greater inhibitory
potential on HUVEC migration at 15 μM (Fig. 2); in all
cases, the inhibitory effects were more significant when
the concentration was increased to 30 μM. These results
demonstrated that the inhibitory effect on the migration of
HUVECs is significantly enhanced for derivatives bearing
an added aryl group on the C-2 substituted quinazoline
scaffold, which further supports the results of the adhe-
sion studies.
bition among all the derivatives tested (69.2
0.2 % at
a dose of 30 μM, 1 h; 74.3 0.1 % at a dose of 30 μM,
3 h incubation at 37 °C). An analogous result is observed
among the SZ10 derivatives, with SZ10d displaying the
greatest inhibition among the series, albeit lower than that
of SZ11d. Derivative SZ10c, with a shorter amide side
Table 2 Inhibitory effects of SZ derivatives on adhesion of HUVEC
to collagen at a dose of 15 and 30 μM
Derivatives inhibiting angiogenesis in vivo in the CAM
assay
Compound 1 h Inhibition rate (%)
3 h Inhibition rate (%)
Dose
Dose
Dose
Dose 30 μM
15 μM
30 μM
15 μM
The anti-angiogenic activity of the most potent deriva-
tives, SZ10d and SZ11d, was tested using the chick cho-
rioallantoic membrane (CAM) assay which represents an
excellent animal model for the study of angiogenesis, since
the early 1970s when it was developed by Folkmann et al.
[10]. Chorioallantoic membrane was treated with thalido-
mide (a clinical anti-tumor drug targeting tumor angio-
genesis, 30 mg/10 mL), SZ10d (30 mg/10 mL), SZ11d
(30 mg/10 mL) or the vehicle control for 48 h. As shown in
Fig. 3, when SZ derivatives were added on the CAMs, there
was a considerable inhibition of the angiogenic responses.
In the group treated with SZ11d, the formation of ves-
sels in the chick embryos was greatly inhibited, compared
SZ10a
SZ10b
SZ10c
SZ10d
SZ11a
SZ11b
SZ11c
SZ11d
11d
39.7 0.2 46.6 0.1 40.4 0.3 47.7 0.2
36.6 0.1 41.9 0.4 42.3 0.2 49.2 0.1
31.4 0.3 34.3 0.2 35.7 0.5 43.2 0.2
43.4 0.2 51.3 0.1 45.2 0.5 52.6 0.2
41.1 0.4 47.9 0.2 46.2 0.3 55.4 0.5
40.7 0.2 53.6 0.2 46.2 0.2 69.3 0.3
35.4 0.1 51.3 0.4 43.2 0.1 60.8 0.2
51.3 0.5 69.2 0.2 56.4 0.3 74.3 0.1
46.5 0.2 66.3 0.3 52.6 0.2 73.1 0.4
Thalidomide 39.7 0.4 54.6 0.2 42.6 0.3 64.7 0.1
Data derived from the mean of three independent assays
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J IRAN CHEM SOC (2016) 13:753–761
Fig. 2 The inhibitory effects
of quinazoline derivatives on
migration of HUVEC at a dose
of 15 and 30 μM
Fig. 3 The results of the CAM assay. 0.9 % NaCl solution and tha-
lidomide were used as a negative and positive control, respectively.
Representative pictures of independent experiments performed in
duplicate for Thalidomide, derivatives SZ10d, SZ11d and N-(4-
(quinazolin-2-yl)phenyl)benzamide dose and controls. All filter discs
had the same size; the apparent visual size difference is due to the
various distances from which pictures were taken
with those treated with SZ10d and thalidomide, a result
in accord with the aforementioned studies on cytotoxicity,
migration and adhesion.
tumor cells but showed increased cytotoxicity towards
HUVECs. Among these derivatives, compound SZ11d with
an N-methyl piperazino terminus of the amide side chains
(n = 2, with three bonds between basic N terminus and car-
bonyl group) exhibited the most potent inhibitory effect on
the proliferation of HUVECs, CNE-2, PC-3, and SMMC-
7721 cells (IC50 = 21.8 0.3, 15.1 0.2, 14.6 0.1, and
Conclusion
In our efforts towards the discovery of novel 2,4-disubsti-
tuted quinazoline derivatives with enhanced anti-angiogen-
esis activities, a series of N-(2-(quinazolin-2-yl)phenyl)
benzamide (SZ) derivatives were designed and synthe-
sized. Based on previous reports of the use of amide bonds
on ligand design, a new benzene ring was attached to the
scaffold of quinazoline derivatives through amide bond. In
comparison with the previously reported 2,4-disubstituted
quinazoline derivative 11d, cytotoxicity assays indicated
that most of these novel compounds (with the exception of
derivative SZ11c) displayed similar cytotoxicities against
12.9
0.5 μM, respectively). Furthermore, cell adhesion
and migration inhibition assays demonstrated the anti-
angiogenic potential of some derivatives on HUVECs and
their use as possible targets for further optimization studies.
In addition, the chick embryo chorioallantoic membrane
(CAM) assay showed that compounds SZ10d and SZ11d
inhibited the angiogenic responses at 30 mg/10 mL. In con-
clusion, the results proved that introduction an aryl group
linked to the amide of the quinozaline C-2 substituted
scaffold with a basic amide side chain on the 4′ position
is an effective approach to increase angiogenesis inhibition,
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