Synthesis and evaluation of 2,4-disubstituted quinazoline derivatives with potent anti- Angiogenesis activities

Article abstract of DOI:10.3390/molecules19078916

A series of 2,4-disubstituted quinazoline derivatives were designed and synthesized. The biological results showed that most of quinazoline derivatives exhibited potent antiproliferative activities against a panel of three tumor cell lines and a good inhibitory effect against the adhesion and migration of human umbilical vein endothelial cells (HUVECs). Among these compounds, 11d was the most potent agent, that also exhibited the highest anti- Angiogenesis activities in the chick embryo chorioallantoic membrane (CAM) assay.

Full text of DOI:10.3390/molecules19078916

Molecules 2014, 19, 8916-8932; doi:10.3390/molecules19078916
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Synthesis and Evaluation of 2,4-Disubstituted Quinazoline
Derivatives with Potent Anti-Angiogenesis Activities
Guangjin Yu 1,†, Zeng Li 2,†, Liang Tang 1 and Qiru Xiong 1,*
1
Department of Chirurgery, First Affiliated Hospital of Anhui Medical University, Hefei 230032,
China; E-Mails: yusysu@163.com (G.Y.); tang@163.com (L.T.)
2
Department of Chemistry, Anhui Medical University, Hefei 230032, China;
E-Mail: stronglz110732@163.com
†
These authors contributed equally to this work.
* Author to whom correspondence should be addressed; E-Mail: xiongqiru@gmail.com;
Tel.: +86-551-6512-4322; Fax: +86-551-5516-1136.
Received: 23 April 2014; in revised form: 15 June 2014 / Accepted: 18 June 2014 /
Published: 26 June 2014
Abstract: A series of 2,4-disubstituted quinazoline derivatives were designed and
synthesized. The biological results showed that most of quinazoline derivatives exhibited
potent antiproliferative activities against a panel of three tumor cell lines and a good
inhibitory effect against the adhesion and migration of human umbilical vein endothelial
cells (HUVECs). Among these compounds, 11d was the most potent agent, that also
exhibited the highest anti-angiogenesis activities in the chick embryo chorioallantoic
membrane (CAM) assay.
Keywords: 2,4-disubstituted quinazoline derivatives; synthesis; anti-angiogenesis
1. Introduction
Angiogenesis, the physiological process through which new blood vessels form from pre-existing
vessels, often provides nutrients and oxygen to energetically proliferating tumor cells to support solid
tumor growth [1]. Thus, it plays a key role in tumor progression and metastasis. Recent reports have
been shown that without angiogenesis, a solid tumor can only reach the size of 1–2 mm3, which is
small enough to be treated with conventional cytotoxic chemotherapeutic drugs [2]. In the past two

Molecules 2014, 19
8917
decades, a number of angiogenesis inhibitors have been advanced into clinical development in cancer
therapy, such as the fumagillin derivative TNP-470 [3], SU-5416 [4], angiostatin [5], and endostatin [6].
Therefore, the search for anti-cancer drugs targeting angiogenesis is a promising strategy for
cancer therapy.
Quinazolines, which are heterocyclic compounds consisting of two closed six-membered aromatic
rings, represent an important class of scaffolds are found in a number of naturally occurring and
synthetic molecules possessing a broad spectrum of biological activities. Astra Zeneca’s ZD6474
(vandetanib, Figure 1) is a novel heteroaromatic-substituted anilinoquinazoline that was discovered to be
a potent and reversible inhibitor of VEGF receptor-2 (VEGFR-2), EGFR and RET tyrosine kinase (y) [7].
In preclinical trials, ZD6474 was shown to have broad spectrum antitumor activity in mouse models
and was found to prevent the growth of tumors by inhibition of angiogenesis [8].
Figure 1. Structure of ZD6474 and 2,4-disubstituted quinazoline derivatives.
According to the structural features of ZD6474, we synthesized a series of 2,4-disubstituted
quinazoline derivatives (Figure 1), hoping to screen for new and better inhibitors of angiogenesis. The
approach was divided into two steps: (i) replacement of the C-4 anilino moiety of ZD6474 with a long
side chain with a terminal amino group; (ii) introduction of another side chain such as an
alkylaminoanilino side chain on the 2-position of the substituted aromatic group. This can significantly
increase the ability of these compounds to form hydrogen bonds with the relevant desired receptors
such as VEGFR-2, EGFR or RET tyrosine kinase. Their cytotoxicity against normal cell lines (human
umbilical vein endothelial cell line, HUVEC) and tumor cell lines (CNE-2, PC-3, and SMMC-7721 cell
lines) were evaluated. Furthermore, their anti-angiogenesis activities were examined by biophysical
and biochemical assays, including cell adhesion, migration and chorioallantoic membrane of the chick
(CAM) embryo assays.
2. Results and Discussion
2.1. Chemistry
The synthetic route to the quinazoline derivatives is outlined in Scheme 1. The uncyclized amide
intermediate 2 was obtained by amidating 2-nitrobenzoyl chloride with anthranilamide in
trichloromethane. The heterocyclic skeleton of quinazolinone 3 was formed through one step reaction
of substrate 2, with an oxidative ring closure reaction under basic conditions [9]. Then, treatment of the
quinazolinone 3 with excess phosphorus oxychloride in refluxing toluene in the presence of

Molecules 2014, 19
8918
N,N-diethylaniline gave compound 4. Coupling of 3-(dimethylamino)-1-propylamine with quinazoline
intermediate 4 in THF at 66 °C, afforded compound 5. In order to obtain the amine functionality,
compound 6 was prepared by treatment of nitro-substituted compound 5 with 80% hydrazine hydrate
and 10% Pd/C in isopropanol. Treatment of the amine-substituted compound 6 with acyl chlorides and
potassium carbonate in dichloromethane gave compounds 7–9 at 69%–85% yield. Finally, the target
compounds 10a–i, 11a–e, 12a–e were obtained through aminolysis of the compounds 7, 8, and 9 under
reflux through the treatment with the appropriate secondary amines.
Scheme 1. Synthesis of quinazoline derivatives.
Reagents and Conditions: (i) 2 equiv. of TEA, CHCl3, rt, 5 h; (ii) 10% aqueous KOH, EtOH, reflux,
2 h; (iii) N,N-diethylaniline, POCl3, toluene, reflux, 6 h; (iv) 3-aminopropyldimethyl- amine, THF,
.
reflux, 5 h; (v) 10% Pd/C, 80% N2H4 H2O, isopropanol, reflux, 2 h; (vi) Cl(CH2)nCOCl, K2CO3,
CH2Cl2, rt, 24 h; (vii) R2NH, KI, ethanol, reflux.
2.2. Cytotoxicity Against Tumor Cells and HUVEC
In the early 80s, scientists proposed anti-angiogenesis as a potential target in cancer therapy [10]. Much
research in the past 30 years has targeted tumor angiogenesis as a means of inhibiting the process of
tumor-caused blood vessel formation. Endothelial cells, which come from the inner lining of blood
vessels, makes up a fundamental part of new blood vessels as well as pre-existing ones. A complete
angiogenesis process includes the proliferation, migration, and differentiation of endothelial cells, so a
great deal of angiogenesis inhibitors discovered to date target endothelial cells. We thus employed a
cell proliferation assay to evaluate the cytotoxicity of quinazoline derivatives against both normal
(HUVEC) and tumor cell lines (CNE-2, PC-3, and SMMC-7721 cell lines) so we can screen some
molecules that have cytotoxicity against tumor cells but low cytotoxicity against HUVECs. Meanwhile,
an appropriate concentration range of these compounds could be determined to further study their
inhibitive effects against the proliferation, migration, and differentiation of HUVECs. The cytotoxic
activities of quinazoline derivatives against human tumor cell lines CNE-2 (human nasopharyngeal

Molecules 2014, 19
8919
cancer), PC-3 (human prostatic carcinoma), SMMC-7721 (human liver cancer) and Human Umbilical
Vein Endothelial Cells (HUVECs) were determined by using the MTT cytotoxicity assay. The results
showed that most of the 2,4-disubstituted quinazoline derivatives possessed high cytotoxicity against
human tumor cell lines and moderate cytotoxicity against HUVECs. Table 1 showed the IC50 values
(cytotoxicity potency indexes) of all the derivatives against four types of cells lines.
Table 1. IC50 cytotoxicity values (µM) of the quinazoline derivatives against tumor cells and HUVEC.
IC50 (μM)
IC50 (μM)
PC-3 7721
9.8 ± 0.3 10.9 ± 0.2 47.2 ± 0.2
ompd
10a
10b
10c
10d
10e
11a
11b
11c
CNE-2
PC-3
7721
HUVEC Compd
CNE-2
HUVEC
13.6 ± 0.2 16.8 ± 0.1 18.1 ± 0.2 45.7 ± 0.2
14.9± 0.2 16.7 ± 0.2 17.9 ± 0.4 39.3 ± 0.3
17.1 ± 0.4 17.6 ± 0.1 19.4 ± 0.1 56.2 ± 0.2
10.5 ± 0.3 11.3 ± 0.5 10.4 ± 0.1 38.7 ± 0.1
19.7 ± 0.2 20.5 ± 0.3 22.6 ± 0.2 79.8 ± 0.1
14.2 ± 0.1 18.1 ± 0.2 19.9 ± 0.4 57.4 ± 0.5
14.3 ± 0.1 17.4 ± 0.1 15.1 ± 0.2 46.3 ± 0.3
16.8 ± 0.4 18.7 ± 0.3 13.8 ± 0.1 66.8 ± 0.3
11d
11e
12a
12b
12c
12d
12e
9.3 ± 0.2
20.1 ± 0.2 19.6 ± 0.2 20.9 ± 0.3 84.9 ± 0.3
17.3 ± 0.3 17.7 ± 0.2 16.5 ± 0.1 41.7 ± 0.3
16.4 ± 0.3 18.9 ± 0.4 15.3 ± 0.2 39.4 ± 0.1
19.9 ± 0.2 17.1 ± 0.3 17.4 ± 0.1 44.5 ± 0.2
11.4 ± 0.1 10.9 ± 0.2 10.3 ± 0.5 40.2 ± 0.2
20.6 ± 0.2 21.9 ± 0.2 20.8 ± 0.3 76.3 ± 0.2
Data derived from the mean of three independent assays.
The results indicate that the IC50 values on the tested tumor cells are in the range of 10–40 µM.
Among all the derivatives, 11d was the most potent in inhibiting the tumor cell proliferation with the
lowest IC50 values of 9.3 ± 0.2 μM (CNE-2), 9.8 ± 0.3 μM (PC-3) and 10.9 ± 0.2 μM (SMMC-7721),
respectively. Compound 10e exhibited the lowest cytotoxicity to the various cancer cells lines.
Comparing with quinazoline derivatives 11d, 12d and 10d, which had the same R group, it was found
compound 10d was much less cytotoxic against HUVECs than 11d and 12d, although they had
comparative cytotoxicity against tumor cells. As for the structure-activity relationships, the n = 2
amide side chain analogues of compounds (11 series) show the highest cytotoxicity against human
tumor cell lines, while no significant effect on IC50 values was observed with the extension of the
amide side chain (n > 2). On the other hand, alterations of the basic terminus of the amide side chains
show that the N-methylpiperazino analogues (10d, 11d and 12d) had the most enhanced cytotoxicity
for human tumor cell lines compared to other haloalkanamido-substituted compounds. All the above
results showed that the cytotoxicity of these quinazoline derivatives was closely related to their R
group and the length of the side chains.
2.3. Inhibition of HUVEC Adhesion
A perfect angiogenesis inhibitor must be show lower toxicity toward HUVECs, while showing high
inhibitive capability towards the proliferation, migration, and differentiation of HUVECs. To assess the
inhibitory effects of 2,4-disubstituted quinazoline derivatives, three assays were used: HUVEC
adhesion assay, HUVEC migration assay and CAM assay. Firstly, to test the inhibitory effects of
quinazoline derivatives on the attachment of endothelial cells to type I collagen, cell adhesion assays
were carried out.
As shown in Table 2, most compounds inhibited HUVEC adhesion to collagen. Most of the
quinazoline derivatives significantly inhibited HUVEC adhesion at both 1 h and 3 h. Compound 11d

Molecules 2014, 19
8920
showed the most potent inhibition of HUVEC adhesion, with an inhibitory rate of as much as
65.8% ± 0.2% at a dose of 15 μM and 71.2% ± 0.1% at a dose of 30 μM after 3 h incubation at 37 °C.
Compound 12d showed similar results with 11d for the experiment, while 10d, with the shortest amide
side chains and the same basic terminus as 11d showed an average inhibition of HUVEC adhesion,
with a inhibitory rate of 63.7% ± 0.2% at a dose of 15 µM and 63.4% ± 0.3% at a dose of 30 µM when
3 h incubation at 37 °C. Alterations of the basic terminus of the amide side chains show that the
N-methylpiperazino analogues had strongly enhanced inhibition of HUVEC adhesion compared to the
piperidino, pyrrolidino and diethylamino analogues, with the less basic morpholino analogues proving
favorable to HUVEC adhesion. These results demonstrated the importance of then amide side chain
length and basicity for potent inhibition of HUVEC adhesion.
Table 2. The inhibitory effects of compounds on adhesion of HUVEC on collagen at a
dose of 15 μM and 30 μM.
Compd.
1 h Inhibition Rate %
Dose 15 μM Dose 30 μM
26.7 ± 0.1 31.6 ± 0.3
3 h Inhibition Rate %
Dose 15 μM
29.2 ± 0.2
27.8 ± 0.2
16.4 ± 0.4
32.5 ± 0.4
7.9 ± 0.3
Dose 30 μM
37.6 ± 0.1
34.8 ± 0.2
29.5 ± 0.1
47.2 ± 0.1
17.6 ± 0.3
59.3 ± 0.1
51.7 ± 0.2
36.3 ± 0.4
71.2 ± 0.1
36.4 ± 0.3
61.2 ± 0.3
60.1 ± 0.2
49.5 ± 0.2
63.4 ± 0.3
39.4 ± 0.2
10a
10b
10c
10d
10e
11a
11b
11c
11d
11e
12a
12b
12c
12d
12e
28.3±0.1
18.9 ± 0.3
34.8 ± 0.2
nd
33.0 ± 0.2
27.8 ± 0.2
44.6 ± 0.4
14.3 ± 0.1
49.6 ± 0.5
54.3 ± 0.3
38.7 ± 0.2
65.8 ± 0.2
32.3 ± 0.2
57.9 ± 0.3
49.3 ± 0.1
44.3 ± 0.2
63.7 ± 0.2
34.7 ± 0.2
37.4 ± 0.3
35.4 ± 0.3
29.9 ± 0.2
47.4 ± 0.1
27.4 ± 0.3
33.6 ± 0.2
29.8 ± 0.1
24.2 ± 0.3
46.8 ± 0.2
26.5 ± 0.2
38.2 ± 0.1
35.2 ± 0.3
29.4 ± 0.2
53.3 ± 0.3
25.6 ± 0.3
40.6 ± 0.3
38.4 ± 0.2
27.9 ± 0.2
47.5 ± 0.3
17.2 ± 0.4
Data derived from the mean of three independent assays; nd means no results due to no inhibition.
2.4. Inhibition of HUVEC Migration
According to the HUVEC adhesion assay results in which most of the 2,4-disubstituted quinazoline
derivatives showed better inhibitory effects against HUVEC adhesion, compounds 10d, 11a–e, 12d
were chosen to evaluate their inhibition against HUVEC migration in a HUVEC wounding assay
which is an important procedure in tube formation and vessel sprouting. Compounds were tested at
concentrations of both 15 μM and 30 μM, and all seven compounds tested in this assay could inhibit
HUVEC migration. Five derivatives, 11a–e, with a longer side chain in the 2-position of the parent
structure C, showed a greatly inhibitory potential on HUVEC migration at 15 μM (Figure 2), and the
inhibitory effects were more significant when the concentration of these compounds was increased to
30 μM. Among all these compounds, 11d showed the best inhibitory activity. As for the structure-
activity relationship, the results from these migration experiments suggest that quinazoline derivatives

Molecules 2014, 19
8921
bearing the longer amide chains (n ≥ 2) should have potent inhibitory activity against HUVEC
migration, which further supported the results of the adhesion studies indicating the importance of
amide side chain length.
Figure 2. The inhibitory effects of quinazoline derivatives on HUVEC migration at doses
of 15 μM and 30 μM.
2.5. 2,4-Disubstituted Quinazoline Derivatives Inhibits Angiogenesis in Vivo in the CAM Assay
Based on the HUVEC cytotoxicity, migration, and adhesion assay results, some
2,4-disubstituted quinazoline derivatives (compounds 10d, 11a–d, 12d) possessing moderate
cytotoxicity and potent inhibitive effect against the adhesion and migration of HUVEC, were chosen to
assess their anti-angiogenesis activities by an in vivo CAM assay. CAMs treated with 0.9% NaCl
solution (negative control) were surrounded by all antoic vessels as newly-formed capillaries
converging radially toward the sponge in a “spoked wheel” pattern (number of vessels 24 ± 3). As
shown in Figure 3, there was a significant reduction of the angiogenic response when quinazoline
derivatives were added to the CAMs. Among all CAMs treated with 2,4-disubstituted quinazoline
derivatives, compounds 11d, 12d show excellent inhibition of angiogenesis in chick embryos, which
further supports the cytotoxicity, adhesion and migration results. A comparison of the CAM assay
results with the reference thalidomide (a clinical anti-tumor drug targeting tumor angiogenesis) and all
the compounds tested revealed that these quinazoline derivatives show better inhibition of
angiogenesis than thalidomide [11].

Molecules 2014, 19
Figure 3. The results of CAM assay. NaCl solution (0.9%) and thalidomide were used as a
8922
negative control and a positive control, respectively. Representative pictures of
independent experiments performed in duplicate for the quinazoline derivatives 10d, 11a–d
and 12d doses and controls. All filter discs had the same size, and any visual size
differences are due to the various distances from which pictures were taken.
3. Experimental
3.1. General Information
1H-NMR (400 MHz) and 13C-NMR (101 MHz) spectra were recorded on a Bruker 400 NMR
instrument with tetramethylsilane (TMS) as an internal standard. Only new compounds’ NMR data is
given. MS spectra were obtained using a Shimadzu LC-MS-2010A spectrometer. Melting points were
determined on an X-6 microscope melting point instrument and are uncorrected.
N-[2-(Aminocarbonyl)phenyl]-2-nitrobenzamide (2). 2-Nitrobenzoic acid (11.04 g, 54.9 mmol) and
thionyl chloride (50 mL) were mixed and the reaction mixture was refluxed for 1.5 h at 80 °C. The
solution was allowed to cool at room temperature, followed by the evaporation of excess thionyl
chloride in vacuo. The resulting wine red solution was taken and added dropwise to a solution of
anthranilamide (1; 9.36 g, 76.6 mmol) and triethylamine (27.9 mL, 153.2 mmol) in chloroform (200 mL)
and stirred at room temperature for 5 h. The precipitated solid was collected by filtration, washed with
ethanol, and dried. Recrystallization from ethanol afforded the product 2 (15.1 g, 86%) as a white
solid; mp 219–221 °C. 1H-NMR (DMSO-d6) δ = 12.56 (s, 1H), 8.44 (d, J = 8.2 Hz, 1H), 8.39 (s, 1H),
8.26 (d, J = 1.6 Hz, 1H), 8.04–7.96 (m, 1H), 7.93–7.85 (m, 2H), 7.81 (s, 1H), 7.59 (t, J = 7.8 Hz, 1H),
13
7.24 (t, J = 7.6 Hz, 1H). C-NMR (DMSO-d6) δ = 170.63, 162.27, 148.00, 138.95, 135.72, 133.61,
132.35, 130.45, 129.92, 128.68, 124.56, 123.52, 120.63, 120.38. ESI-MS m/z: 320 [M+H]+.
2-(2-Nitrophenyl)-3H-quinazolin-4-one (3). A mixture of benzamide 2 (12 g, 37.6 mmol) in 10%
aqueous KOH (100 mL) and EtOH (100 mL) was heated to reflux for 2 h. Ethanol was removed in vacuo,
and the aqueous layer was acidified with hydrochloric acid to pH 3 to give a solid residue. The solid
was filtered and washed with water, which was purified by column chromatography, eluting with
petroleum ether/EtOAc (3:1) to furnish pure 3 (11.2 g, 92%) as a white solid; mp 263–264 °C.

Molecules 2014, 19
8923
1H-NMR (DMSO-d6) δ = 12.87 (s, 1H), 8.19 (d, J = 2.0 Hz, 1H), 8.05 (d, J = 8.0 Hz, 1H), 7.89 (dd, J = 8.4,
2.0 Hz, 1H), 7.78 (d, J = 8.4 Hz, 1H), 7.71 (t, J = 7.8 Hz, 1H), 7.51 (d, J = 8.2 Hz, 1H), 7.44 (t, J = 7.7 Hz,
1H). 13C-NMR (DMSO-d6) δ = 161.46, 150.60, 148.30, 148.18, 135.66, 134.55, 133.53, 132.99,
127.70, 127.26, 127.15, 125.86, 124.41, 121.19. ESI-MS m/z: 302 [M+H]+.
2-(4-Chloro-2-nitrophenyl)quinazoline (4). To a mechanically stirred suspension of 3 (10 g, 33.2 mmol) in
toluene (100 mL) was added N,N-diethylaniline (8,2 g, 55.2 mmol), followed by phosphorus
oxychloride (6.1 g, 39.8 mmol). The reaction was then heated to 80 °C for 6 h, allowed to cool then
washed with iced water (200 mL). The organic layer was then washed sequentially with 20% NaOH
(2 × 100 mL), iced water (200 mL) and brine (200 mL) and dried over anhydrous sodium sulfate. The
organic solvent was removed in vacuo and the residue was purified by column chromatography with
CHCl3/CH3OH (15:1) to give 4 (5.1 g, 48%) as a colorless powder. The aqueous layers were combined
and extracted with dichloromethane. The organic layer was reduced in vacuo to yield a yellow solid,
which was purified by column chromatography as above, providing further product to give a total yield
of 6.5 g (61%). mp 189–191 °C. 1H-NMR (DMSO-d6) δ = 8.36–8.31 (m, 1H), 8.26 (d, J = 2.1 Hz, 1H),
13
8.23–8.17 (m, 2H), 8.10 (d, J = 8.1 Hz, 1H), 7.96 (ddd, J = 9.1, 5.9, 2.2 Hz, 2H). C-NMR (CDCl3)
δ = 162.69, 157.00, 151.29, 150.32, 136.63, 135.33, 132.94, 132.06, 130.16, 129.47, 129.00, 125.82,
124.35, 122.50. ESI-MS m/z: 321 [M+H]+.
N1-(2-(2-Nitrophenyl)quinazolin-4-yl)-N3,N3-dimethylpropane-1,3-diamine (5). To a suspension of 4
(5 g, 15.7 mmol) in dried THF (100 mL) was slowly added dried 3-(dimethylamino)-1-propylamine
(5.9 mL, 47.1 mmol) under a N2 atmosphere. The mixture was refluxed for 5 h, at which point TLC
analysis indicated reaction completion. After concentration in vacuo, the resulting residue was
dissolved in CHCl3, washed with water and brine, dried over anhydrous MgSO4 and evaporated. The
residue was chromatographed on silica gel eluting with CHCl3/CH3OH (30:1) to give 5 (3.1 g, 51%) as
1
a white solid. mp 179–181 °C. H-NMR (CDCl3) δ = 8.95 (s, 1H), 8.21 (d, J = 8.4 Hz, 1H), 7.84 (d,
J = 8.3 Hz, 1H), 7.75–7.66 (m, 2H), 7.62–7.56 (m, 2H), 7.45 (t, J = 7.3 Hz, 1H), 3.66 (dd, J = 10.4, 5.6 Hz,
2H), 2.63–2.55 (m, 2H), 2.38 (s, 6H), 1.88–1.82 (m, 2H). 13C-NMR (CDCl3) δ = 159.58, 158.07,
150.86, 149.87, 135.18, 132.62, 132.51, 132.45, 131.33, 128.50, 126.17, 123.53, 121.14, 114.15, 59.46,
45.36, 42.16, 24.64. ESI-MS m/z: 386 [M+H]+.
N1-(2-(2-Aminophenyl)quinazolin-4-yl)-N3,N3-dimethylpropane-1,3-diamine (6).
A
mixture of
compound 5 (10 g, 26 mmol) in isopropanol (100 mL) and 80% hydrazine hydrate (40 mL) was added
to a suspension of 10% Pd/C (1 g) in chilled isopropanol (30 mL), and the suspension was refluxed for 2 h.
After the reaction was complete, the mixture was filtered through a pad of Celite, washed with
isopropanol and then concentrated in vacuo. The residue was then suspended in 5% NH3(aq) solution
(100 mL). The solution was then extracted with EtOAc (3 × 100 mL), the organics dried over
anhydrous Na2SO4 and then removed in vacuo. The solid obtained was dried under vacuum to give 6
(8.7 g, 96%) as a white powder. mp 182–183 °C. 1H-NMR (CDCl3) δ = 8.57 (s, 1H), 8.51 (d, J = 8.7 Hz,
1H), 7.78–7.73 (m, 1H), 7.70–7.61 (m, 2H), 7.41–7.35 (m, 1H), 6.77 (s, 2H), 6.71 (dt, J = 8.3, 1.9 Hz,
2H), 3.84 (dd, J = 10.5, 5.8 Hz, 2H), 2.69–2.62 (m, 2H), 2.42 (s, 6H), 1.96 (dd, J = 6.7, 4.9 Hz, 2H).

Molecules 2014, 19
8924
13C-NMR (CDCl3) δ = 161.56, 158.98, 149.96, 149.33, 136.28, 132.59, 132.12, 127.96, 125.05,
120.95, 118.78, 116.51, 115.96, 113.71, 59.72, 45.48, 42.44, 24.76. ESI-MS m/z: 356 [M+H]+.
General acylation procedure. A solution of the appropriate acid halide (9.6 mmol) in CH2Cl2 (10 mL)
was added during to a well-stirred mixture of the amino-substituted compound 6 (2.6 g, 8 mmol) and
K2CO3 (1.6 g) in CH2Cl2 (50 mL) at 0 °C in a ice bath. The reaction was then allowed to warm up to
RT, then stirred overnight. After cooling to 0 °C, the precipitate formed was filtered off and was
further purified by flash column chromatography with petroleum ether/EtOAc (10:1) elution to give the
compounds 7–9.
2-Chloro-N-(2-(4-(3-(dimethylamino)propylamino)quinazolin-2-yl)phenyl)acetamide (7). Compound 6
was treated with chloroacetyl chloride according to general acylation procedure to afford 7 as a white
solid in 85% yield; mp 227–228 °C. 1H-NMR (CDCl3) δ = 14.29 (s, 1H), 8.84 (s, 1H), 8.81 (d, J = 2.1 Hz,
1H), 8.62 (d, J = 8.6 Hz, 1H), 7.95 (d, J = 8.7 Hz, 1H), 7.84 (d, J = 8.3 Hz, 1H), 7.73 (ddd, J = 8.3, 7.0,
1.3 Hz, 1H), 7.50–7.44 (m, 1H), 7.17 (dd, J = 8.6, 2.2 Hz, 1H), 4.29 (s, 2H), 3.92 (dd, J = 11.0, 5.4 Hz,
2H), 2.86–2.76 (m, 2H), 2.55 (s, 6H), 2.09–2.02 (m, 2H). 13C-NMR (CDCl3) δ = 165.43, 160.27,
159.32, 148.24, 139.93, 136.52, 132.56, 131.94, 127.54, 125.95, 123.37, 121.10, 120.39, 113.92, 59.82,
45.44, 43.79, 42.62, 24.47. ESI-MS m/z: 432 [M+H]+.
3-Chloro-N-(2-(4-(3-(dimethylamino)propylamino)quinazolin-2-yl)phenyl)propanamide (8). Using the
general acylation procedure compound 6 was treated with 3-chloropropanoyl chloride to afford 8 as a white
solid in 73% yield; mp 181–183 °C. 1H-NMR (CDCl3) δ 14.20 (s, 1H), 8.95 (s, 1H), 8.73 (d, J = 1.9 Hz,
1H), 7.73 (d, J = 3.9 Hz, 2H), 7.62 (d, J = 8.1 Hz, 1H), 7.47–7.41 (m, 2H), 7.17 (d, J = 8.3 Hz, 1H),
3.97 (t, J = 6.9 Hz, 2H), 3.86 (dd, J = 10.3, 5.5 Hz, 2H), 3.01 (t, J = 6.9 Hz, 2H), 2.65–2.61 (m, 2H),
2.40 (s, 6H), 1.95–1.88 (m, 2H). 13C-NMR (DMSO-d6) δ 169.96, 160.30, 159.12, 147.56, 139.90,
133.13, 130.85, 130.41, 126.81, 126.00, 122.97, 122.73, 122.17, 119.57, 113.31, 56.93, 45.00, 44.80,
34.76, 28.14, 26.12. ESI-MS m/z: 446 [M+H]+.
4-Chloro-N-(2-(4-(3-(dimethylamino)propylamino)quinazolin-2-yl)phenyl)butanamide (9). Compound
6 was treated with 4-chlorobutyryl chloride according to the general acylation procedure to afford 9 as
a white solid in 69% yield; mp 156–157 °C. 1H-NMR (CDCl3) δ = 14.20 (s, 1H), 8.94 (s, 1H), 8.85 (d,
J = 2.2 Hz, 1H), 8.67 (d, J = 8.6 Hz, 1H), 7.79–7.69 (m, 3H), 7.50–7.44 (m, 1H), 7.11 (dd,
J = 8.6, 2.2 Hz, 1H), 3.90 (dd, J = 10.5, 5.7 Hz, 2H), 3.72 (t, J = 6.2 Hz, 2H), 2.77 (t, J = 5.0 Hz, 2H),
13
2.74 (d, J = 6.9 Hz, 2H), 2.49 (s, 6H), 2.30 (dt, J = 13.4, 6.6 Hz, 2H), 2.04–1.98 (m, 2H). C-NMR
(CDCl3) δ = 170.56, 160.34, 159.00, 147.86, 141.09, 136.62, 132.59, 131.77, 127.30, 125.93, 122.34,
121.68, 121.20, 119.78, 113.83, 59.77, 44.66, 42.70, 35.29, 28.29, 24.45. ESI-MS m/z: 460 [M+H]+
General aminolysis procedure. To a stirred refluxing suspension of the chloride compounds 7, 8 or 9
(0.5 mmol) and KI (0.08 g) in EtOH (15 mL) was added dropwise the appropriate secondary amine
(2.0 mL) in EtOH (5 mL). The mixture was stirred at reflux for 5 h, cooled to 0 °C, then diluted with
distilled water, filtered, and washed with ether and water, then evaporated under vacuum. The crude
solid was purified by chromatography with petroleum ether/ EtOAc elution to afford 10a–e, 11a–e and
12a, 12d, 12e.

Molecules 2014, 19
8925
N-(2-(4-(3-(Dimethylamino)propylamino)quinazolin-2-yl)phenyl)-2-(piperidin-1-yl)acetamide (10a).
Compound 7 was treated with excess piperidine according to the general aminolysis procedure to
afford 10a as a white solid in 78% yield after column chromatography with petroleum ether/EtOAc
(20:1) elution; mp 180–182 °C. 1H-NMR (CDCl3) δ = 13.51 (s, 1H), 9.00 (s, 1H), 8.89 (d, J = 2.1 Hz,
1H), 8.51 (d, J = 8.6 Hz, 1H), 7.99 (d, J = 8.2 Hz, 1H), 7.72 (t, J = 7.6 Hz, 1H), 7.60 (d, J = 8.0 Hz,
1H), 7.45 (t, J = 7.4 Hz, 1H), 7.12 (dd, J = 8.6, 2.1 Hz, 1H), 3.86 (dd, J = 10.2, 5.6 Hz, 2H), 3.26 (s,
2H), 2.67–2.61 (m, 2H), 2.57–2.44 (m, 4H), 2.41 (s, 6H), 1.94–1.87 (m, 2H), 1.46 (dt, J = 10.9, 5.6 Hz,
4H), 1.37–1.29 (m, 2H). 13C-NMR (CDCl3) δ = 170.12, 160.56, 159.41, 148.66, 140.14, 136.35,
132.17, 131.77, 128.41, 125.85, 123.76, 122.69, 121.02, 120.66, 113.84, 65.22, 59.93, 54.81, 45.50,
42.73, 25.28, 24.45, 23.84. HRMS (ESI): Calcd for (M−H)− (C26H33ClN6O) requires m/z 479.2326,
found 479.2320. Anal. Calcd for C26H33ClN6O.H2O: C, 62.57; H, 7.07; N, 16.84. Found: C, 62.46; H,
7.03; N, 16.84.
N-(2-(4-(3-(dimethylamino)propylamino)quinazolin-2-yl)phenyl)-2-(pyrrolidin-1-yl)acetamide (10b).
Compound 7 was treated with excess pyrrolidine according to the general aminolysis procedure to
afford 10b as a white solid in 74% yield after column chromatography with petroleum ether/EtOAc
(20:1) elution; mp 174–175 °C. 1H-NMR (CDCl3) δ = 13.84 (s, 1H), 8.93 (s, 2H), 8.57 (d, J = 8.1 Hz,
1H), 7.92 (d, J = 7.8 Hz, 1H), 7.69 (d, J = 6.6 Hz, 1H), 7.60 (d, J = 7.5 Hz, 1H), 7.43 (t, J = 6.9 Hz, 1H),
7.12 (d, J = 8.1 Hz, 1H), 3.86 (dd, J = 10.0, 5.8 Hz, 2H), 3.43 (s, 2H), 2.68 (t, J = 7.0 Hz, 4H), 2.65–2.52
(m, 2H), 2.40 (s, 6H), 1.97–1.84 (m, 2H), 1.71 (t, J = 7.2 Hz, 4H). 13C-NMR (CDCl3) δ = 170.55, 160.50,
159.38, 148.72, 140.36, 136.42, 131.99, 131.76, 128.01, 125.79, 123.54, 122.66, 121.01, 120.69,
113.80, 62.22, 59.78, 54.52, 45.44, 42.53, 24.51, 23.93. HRMS (ESI): Calcd for (M−H)−
(C25H31ClN6O) requires m/z 465.2170, found 465.2162. Anal. Calcd for C25H31ClN6O.H2O: C, 61.91;
H, 6.86; N, 17.33. Found: C, 61.88; H, 6.53; N, 17.38.
N-(2-(4-(3-(Dimethylamino)propylamino)quinazolin-2-yl)phenyl)-2-(diethylamino)acetamide
(10c).
Compound 7 was treated with excess diethylamine according to the general aminolysis procedure to
afford 10c as a white solid in 81% yield after column chromatography with petroleum ether/EtOAc
(20:1) elution; mp 10–172 °C 1H-NMR (CDCl3) δ = 13.60 (s, 1H), 8.92 (d, J = 2.1 Hz, 2H), 8.53 (d,
J = 8.6 Hz, 1H), 7.90 (d, J = 8.3 Hz, 1H), 7.71 (t, J = 7.3 Hz, 1H), 7.60 (d, J = 8.0 Hz, 1H), 7.43 (t,
J = 7.5 Hz, 1H), 7.13 (dd, J = 8.6, 2.1 Hz, 1H), 3.86 (dd, J = 10.3, 5.5 Hz, 2H), 3.30 (s, 2H), 2.68 (q,
J = 7.2 Hz, 4H), 2.65–2.55 (m, 2H), 2.40 (s, 6H), 1.90 (dd, J = 16.7, 5.9 Hz, 2H), 0.99 (t, J = 7.1 Hz,
6H). 13C-NMR (CDCl3) δ = 172.03, 160.53, 159.44, 148.88, 140.01, 136.26, 132.04, 131.93, 128.02,
125.71, 124.22, 122.73, 121.02, 120.86, 113.85, 59.92, 58.81, 49.09, 45.51, 42.65, 24.53, 11.64.
HRMS (ESI): Calcd for (M−H)− (C25H33ClN6O) requires m/z 467.2326, found 467.2319. Anal. Calcd
for C25H33ClN6O.H2O: C, 61.65; H, 7.24; N, 17.26. Found: C, 61.58; H, 7.21; N, 17.04.
N-(2-(4-(3-(Dimethylamino)propylamino)quinazolin-2-yl)phenyl)-2-(4-methylpiperazin-1-yl)acetamide
(10d). Compound 7 was treated with excess N-methylpiperazine according to the general aminolysis
procedure to afford 10d as a white solid in 66% yield after column chromatography with petroleum
ether/EtOAc (15:1) elution; mp 174–175 °C. 1H-NMR (DCl3) δ = 13.51 (s, 1H), 8.98 (s, 1H), 8.86 (d,
J = 2.1 Hz, 1H), 8.49 (d, J = 8.6 Hz, 1H), 7.95 (d, J = 8.2 Hz, 1H), 7.77–7.67 (m, 2H), 7.47 (t, J = 7.5 Hz,

Molecules 2014, 19
8926
1H), 7.13 (dd, J = 8.6, 2.1 Hz, 1H), 3.87 (dd, J = 10.4, 5.5 Hz, 2H), 3.31 (s, 2H), 2.72–2.67 (m, 2H),
2.67–2.51 (m, 4H), 2.46 (s, 6H), 2.40–2.24 (m, 4H), 2.12 (s, 3H), 2.01–1.91 (m, 2H). 13C-NMR (CDCl3)
δ = 169.36, 160.52, 159.45, 148.55, 139.93, 136.37, 132.42, 131.75, 128.46, 126.01, 123.79, 122.85,
121.29, 120.67, 113.86, 64.20, 59.29, 54.15, 53.35, 45.80, 45.20, 42.04, 24.31. HRMS (ESI): Calcd for
(M−H)− (C26H34ClN7O) requires m/z 494.2435, found 494.2421. Anal. Calcd for C26H34ClN7O.H2O: C,
60.75; H, 7.06; N, 19.07. Found: C, 60.66; H, 6.97; N, 18.89.
N-(2-(4-(3-(Dimethylamino)propylamino)quinazolin-2-yl)phenyl)-2-morpholinoacetamide
(10e).
Compound 7 was treated with excess morpholine according to the general aminolysis procedure to
afford 10e as a white solid in 78% yield after column chromatography with petroleum ether/EtOAc
(20:1) elution; mp 179–181 °C. 1H-NMR (CDCl3) δ = 13.67 (s, 1H), 9.09 (s, 1H), 8.88 (d, J = 2.1 Hz,
1H), 8.53 (d, J = 8.6 Hz, 1H), 7.92 (d, J = 8.2 Hz, 1H), 7.77–7.69 (m, 1H), 7.62 (d, J = 8.1 Hz, 1H),
7.46 (t, J = 7.6 Hz, 1H), 7.13 (dd, J = 8.6, 2.2 Hz, 1H), 3.86 (dd, J = 10.2, 5.6 Hz, 2H), 3.64–3.53 (m,
13
4H), 3.32 (s, 2H), 2.68–2.62 (m, 2H), 2.63–2.53 (m, 4H), 2.42 (s, 6H), 1.94–1.85 (m, 2H). C-NMR
(CDCl3) δ = 168.97, 160.56, 159.40, 148.43, 140.04, 136.45, 132.35, 131.83, 128.02, 126.02, 123.51,
122.86, 121.22, 120.55, 113.89, 66.34, 64.81, 59.95, 53.78, 45.51, 42.80, 24.41. HRMS (ESI): Calcd
for (M−H)− (C25H31ClN6O2) requires m/z 481.2119, found 481.2107. Anal. Calcd for C25H31ClN6O2: C,
62.17; H, 6.47; N, 17.40. Found: C, 62.24; H, 6.36; N, 17.55.
N-(2-(4-(3-(Dimethylamino)propylamino)quinazolin-2-yl)phenyl)-3-(piperidin-1-yl)propanamide (11a).
Compound 8 was treated with excess piperidine according to the general aminolysis procedure to
afford 11a as a white solid in 69% yield after column chromatography with petroleum ether/EtOAc
(15:1) elution; mp 178–179 °C. 1H-NMR (CDCl3) δ = 14.06 (s, 1H), 9.06 (s, 1H), 8.85 (d, J = 2.0 Hz,
1H), 8.66 (d, J = 8.6 Hz, 1H), 7.79–7.68 (m, 2H), 7.59 (d, J = 8.1 Hz, 1H), 7.48–7.40 (m, 1H), 7.08 (dd,
J = 8.6, 2.1 Hz, 1H), 3.84 (dd, J = 10.2, 5.5 Hz, 2H), 2.92–2.82 (m, 2H), 2.80–2.71 (m, 2H), 2.67–2.58
(m, 2H), 2.54–2.42 (m, 4H), 2.40 (s, 6H), 1.96–1.85 (m, 2H), 1.61–1.53 (m, 4H), 1.46–1.37 (m, 2H).
13C-NMR (CDCl3) δ = 170.93, 160.60, 159.11, 148.02, 141.16, 136.75, 132.58, 131.74, 127.45,
125.93, 122.33, 121.79, 121.21, 120.01, 113.86, 59.94, 54.99, 54.42, 45.51, 42.83, 36.52, 25.97, 24.39,
24.30. HRMS (ESI): Calcd for (M−H)− (C27H35ClN6O) requires m/z 493.2483, found 493.2472. Anal.
Calcd for C27H35ClN6O: C, 65.51; H, 7.13; N, 16.98. Found: C, 65.70; H, 7.24; N, 16.86.
N-(2-(4-(3-(Dimethylamino)propylamino)quinazolin-2-yl)phenyl)-3-(pyrrolidin-1-yl)propanamide (11b).
Compound 8 was treated with excess pyrrolidine according to the general aminolysis procedure to
afford 11b as a white solid in 71% yield after column chromatography with petroleum ether/EtOAc (15:1)
elution; mp 163–165 °C 1H-NMR (CDCl3) δ = 14.12 (s, 1H), 9.07 (s, 1H), 8.86 (d, J = 2.0 Hz, 1H), 8.67 (d,
J = 8.6 Hz, 1H), 7.74 (dt, J = 14.9, 7.6 Hz, 2H), 7.60 (d, J = 8.1 Hz, 1H), 7.45 (dd, J = 11.0, 3.9 Hz, 1H),
7.10 (dd, J = 8.6, 2.1 Hz, 1H), 3.87 (dd, J = 10.2, 5.5 Hz, 2H), 3.01 (t, J = 7.5 Hz, 2H), 2.82 (t, J = 7.5 Hz,
13
2H), 2.74–2.55 (m, 6H), 2.41 (s, 6H), 1.97–1.88 (m, 2H), 1.86–1.74 (m, 4H). C-NMR (CDCl3)
δ = 170.62, 160.68, 159.16, 148.07, 141.15, 136.84, 132.64, 131.73, 127.50, 125.94, 122.39, 121.81,
121.19, 120.09, 113.88, 59.96, 54.18, 52.08, 45.51, 42.84, 38.41, 24.40, 23.52. HRMS (ESI): Calcd for
(M−H)− (C26H33ClN6O) requires m/z 479.2326, found 479.2318. Anal. Calcd for C26H33ClN6O: C,
64.92; H, 6.91; N, 17.47. Found: C, 65.02; H, 6.68; N, 17.55.

Molecules 2014, 19
8927
N-(2-(4-(3-(Dimethylamino)propylamino)quinazolin-2-yl)phenyl)-3-(diethylamino)propanamide (11c).
Compound 8 was treated with excess diethylamine according to the general aminolysis procedure to
afford 11c as a white solid in 60% yield after column chromatography with petroleum ether/EtOAc
(15:1) elution; mp 158–160 °C. 1H-NMR (CDCl3) δ = 14.08 (s, 1H), 9.07 (s, 1H), 8.86 (d, J = 2.1 Hz,
1H), 8.67 (d, J = 8.6 Hz, 1H), 7.77–7.68 (m, 2H), 7.59 (d, J = 8.1 Hz, 1H), 7.44 (ddd, J = 8.1, 6.1, 2.0
Hz, 1H), 7.09 (dd, J = 8.6, 2.2 Hz, 1H), 3.86 (dd, J = 10.2, 5.6 Hz, 2H), 3.07–2.95 (m, 2H), 2.76–2.68
(m, 2H), 2.64 (t, J = 4.1 Hz, 2H), 2.63–2.56 (m, 4H), 2.41 (s, 6H), 1.97–1.85 (m, 2H), 1.05 (t, J = 7.1 Hz,
6H). 13C-NMR (CDCl3) δ = 171.05, 160.60, 159.10, 147.99, 141.19, 136.74, 132.56, 131.74, 127.35,
125.93, 122.31, 121.74, 121.24, 119.95, 113.87, 59.90, 48.93, 47.01, 45.49, 42.73, 36.64, 24.41, 11.90.
HRMS (ESI): Calcd for (M−H)− (C26H35ClN6O) requires m/z 481.2483, found 481.2472. Anal. Calcd for
C26H35ClN6O.H2O: C, 62.32; H, 7.44; N, 16.77. Found: C, 62.15; H, 7.57; N, 16.63.
N-(2-(4-(3-(Dimethylamino)propylamino)quinazolin-2-yl)phenyl)-3-(4-methylpiperazin-1-yl)propane-
mide (11d). Compound 8 was treated with excess N-methylpiperazine according to the general
aminolysis procedure to afford 11d as a white solid in 44% yield after column chromatography with
1
petroleum ether/EtOAc (10:1) elution; mp 167–168 °C. H-NMR (CDCl3) δ = 14.07 (s, 1H), 9.07 (s,
1H), 8.85 (d, J = 1.5 Hz, 1H), 8.66 (d, J = 8.6 Hz, 1H), 7.72 (d, J = 3.9 Hz, 2H), 7.60 (d,
J = 8.1 Hz, 1H), 7.45 (dt, J = 8.1, 4.0 Hz, 1H), 7.09 (dd, J = 8.6, 1.8 Hz, 1H), 3.86 (dd, J = 10.2, 5.3 Hz,
2H), 2.92 (t, J = 7.4 Hz, 2H), 2.75 (t, J = 7.4 Hz, 2H), 2.67–2.63 (m, 2H), 2.59 (dd, J = 19.2, 11.0 Hz,
4H), 2.49–2.41 (m, 4H), 2.41 (s, 6H), 2.26 (s, 3H), 1.95–1.87 (m, 2H). 13C-NMR (CDCl3)
δ = 170.62, 160.71, 159.19, 148.08, 141.12, 136.87, 132.69, 131.77, 127.40, 125.98, 122.46, 121.85,
121.28, 120.12, 113.92, 59.92, 55.08, 54.20, 52.99, 46.01, 45.51, 42.80, 36.39, 24.42. HRMS (ESI):
Calcd for (M−H)− (C27H36ClN7O) requires m/z 508.2592, found 508.2588. Anal. Calcd for
C27H36ClN7O: C, 63.58; H, 7.11; N, 19.22. Found: C, 63.49; H, 7.26; N, 19.36.
N-(2-(4-(3-(Dimethylamino)propylamino)quinazolin-2-yl)phenyl)-3-morpholinopropanamide
(11e).
Compound 8 was treated with excess morpholine according to the general aminolysis procedure to
afford (11e) as a white solid in 67% yield after column chromatography with petroleum ether/EtOAc
(15:1) elution; mp 165–167 °C. 1H-NMR (CDCl3) δ = 14.10 (s, 1H), 9.08 (s, 1H), 8.84 (d, J = 2.0 Hz,
1H), 8.67 (d, J = 8.6 Hz, 1H), 7.75–7.69 (m, 2H), 7.62 (d, J = 8.1 Hz, 1H), 7.45 (ddd, J = 8.2, 5.2, 2.9 Hz,
1H), 7.10 (dd, J = 8.6, 2.1 Hz, 1H), 3.86 (dd, J = 10.2, 5.4 Hz, 2H), 3.69–3.61 (m, 4H), 2.89 (t, J = 7.1 Hz,
2H), 2.75 (t, J = 7.2 Hz, 2H), 2.68–2.62 (m, 2H), 2.58–2.48 (m, 4H), 2.42 (s, 6H), 1.95–1.89 (m, 2H).
13C-NMR (CDCl3) δ = 170.52, 160.59, 159.12, 147.94, 141.04, 136.77, 132.63, 131.77, 127.20,
125.99, 122.46, 121.80, 121.34, 120.02, 113.88, 66.88, 59.72, 54.61, 53.53, 45.46, 42.61, 36.28, 24.44.
HRMS (ESI): Calcd for (M−H)− (C26H33ClN6O2) requires m/z 495.2275, found 495.2271. Anal. Calcd
.
for C26H33Cl N6O2 H2O: C, 60.63; H, 6.85; N, 16.32. Found: C, 60.51; H, 6.92; N, 16.36.
N-(2-(4-(3-(Dimethylamino)propylamino)quinazolin-2-yl)phenyl)-4-(piperidin-1-yl)butanamide (12a).
Compound 9 was treated with excess piperidine according to the general aminolysis procedure to
afford 12a as a white solid in 73% yield after column chromatography with petroleum ether/EtOAc
(15:1) elution; mp 159–160 °C. 1H-NMR (CDCl3) δ = 14.04 (s, 1H), 9.03 (s, 1H), 8.86 (d, J = 2.2 Hz,
1H), 8.68 (d, J = 8.6 Hz, 1H), 7.80–7.70 (m, 2H), 7.61 (d, J = 8.0 Hz, 1H), 7.45 (ddd, J = 8.2, 6.2, 2.0 Hz,

Molecules 2014, 19
8928
1H), 7.10 (dd, J = 8.6, 2.2 Hz, 1H), 3.87 (dd, J = 10.2, 5.7 Hz, 2H), 2.69–2.62 (m, 2H), 2.59 (t, J = 7.3 Hz,
2H), 2.53–2.46 (m, 2H), 2.46–2.41 (m, 4H), 2.41 (s, 6H), 2.04 (dt, J = 14.8, 7.4 Hz, 2H), 1.92 (dt,
13
J = 11.3, 5.8 Hz, 2H), 1.56 (dt, J = 11.0, 5.5 Hz, 4H), 1.45–1.36 (m, 2H). C-NMR (CDCl3)
δ = 171.94, 160.74, 159.20, 148.17, 141.29, 136.90, 132.64, 131.77, 127.54, 125.94, 122.33, 121.81,
121.23, 120.05, 113.92, 60.00, 58.70, 54.56, 45.53, 42.86, 36.90, 25.95, 24.45, 22.94. HRMS (ESI):
Calcd for (M−H)− (C28H37ClN6O) requires m/z 507.2639, found 507.2629. Anal. Calcd for
C28H37ClN6O: C, 66.06; H, 7.33; N, 16.51. Found: C, 66.10; H, 7.49; N, 16.32.
N-(2-(4-(3-(Dimethylamino)propylamino)quinazolin-2-yl)phenyl)-4-(pyrrolidin-1-yl)propanamide (12b).
Compound 9 was treated with excess pyrrolidine according to the general aminolysis procedure to
afford 12b as a white solid in 66% yield after column chromatography with petroleum ether/EtOAc
1
(15:1) elution; mp 162–164 °C. H-NMR (CDCl3) δ 13.90 (s, 1H), 8.97 (s, 1H), 8.81–8.65 (m, 2H),
7.78–7.69 (m, 2H), 7.61 (d, J = 8.0 Hz, 1H), 7.47–7.40 (m, 2H), 7.17–7.12 (m, 1H), 3.88 (dd,
J = 10.2, 5.7 Hz, 2H), 2.62 (dd, J = 13.8, 6.6 Hz, 3H), 2.54 (s, 2H), 2.40 (s, 3H), 2.11–2.02 (m, 2H),
13
1.95–1.88 (m, 2H), 1.80–1.71 (m, 2H). C-NMR (CDCl3) δ 171.76, 161.30, 159.18, 148.24, 140.35,
132.45, 130.95, 130.69, 127.42, 125.75, 123.68, 122.26, 121.25, 120.26, 113.77, 59.59, 55.83, 54.07,
45.45, 42.40, 36.92, 25.19, 24.68, 23.45. HRMS (ESI): Calcd for (M−H)− (C28H37ClN6O) requires m/z
507.2639, found 507.2629. Anal. Calcd for C28H37ClN6O: C, 66.06; H, 7.33; N, 16.51. Found: C,
66.10; H, 7.49; N, 16.32.
N-(2-(4-(3-(Dimethylamino)propylamino)quinazolin-2-yl)phenyl)-4-(diethylamino)propanamide (12c).
Compound 9 was treated with excess diethylamine according to the general aminolysis procedure to
afford 12c as a white solid in 63% yield after column chromatography with petroleum ether/EtOAc
(15:1) elution; mp 159–161 °C. 1H-NMR (CDCl3) δ 13.84 (s, 1H), 9.06 (s, 1H), 8.65 (dd, J = 12.9, 4.9 Hz,
2H), 7.71–7.61 (m, 3H), 7.36 (dd, J = 11.1, 4.2 Hz, 2H), 7.12–7.03 (m, 1H), 3.81 (dd, J = 9.9, 5.4 Hz,
2H), 2.99–2.90 (m, 2H), 2.69–2.59 (m, 8H), 2.54 (q, J = 7.1 Hz, 4H), 1.90–1.81 (m, 2H), 1.07 (t,
J = 7.1 Hz, 6H), 0.98 (t, J = 7.1 Hz, 6H). 13C-NMR (101 MHz, CDCl3) δ 170.79, 161.43, 159.14,
148.27, 140.27, 132.51, 131.02, 130.64, 127.49, 125.55, 123.73, 122.34, 121.59, 120.34, 113.78 ,
53.54, 48.96, 46.98, 42.97, 36.53, 24.23, 11.43. HRMS (ESI): Cacld for (M-H)− (C28H38N6O) requires
m/z 475.3185, found 475.3186.
N-(2-(4-(3-(Dimethylamino)propylamino)quinazolin-2-yl)phenyl)-4-(4-methylpiperazin-1-yl)butanami-de
12d. The compound 9 was treated with excess N-methyl piperazine according to general aminolysis
procedure to afford 12d as a white solid in 58% yield after column chromatography with petroleum
ether/EtOAc (10:1) elution; mp 155–157 °C. 1H-NMR (CDCl3) δ = 14.03 (s, 1H), 9.02 (s, 1H), 8.85 (d,
J = 2.1 Hz, 1H), 8.66 (d, J = 8.7 Hz, 1H), 7.72 (dd, J = 3.9, 1.6 Hz, 2H), 7.60 (d, J = 8.2 Hz, 1H), 7.43
(ddd, J = 8.2, 5.4, 2.8 Hz, 1H), 7.08 (dd, J = 8.7, 2.2 Hz, 1H), 3.89 (dd, J = 10.2, 5.5 Hz, 2H),
2.75–2.69 (m, 2H), 2.66 (t, J = 7.3 Hz, 2H), 2.59 (t, J = 7.3 Hz, 2H), 2.55–2.46 (m, 4H), 2.43 (s, 6H),
2.36–2.28 (m, 4H), 2.23 (s, 3H), 2.02 (dt, J = 14.5, 7.3 Hz, 2H), 1.97–1.90 (m, 2H). 13C-NMR (CDCl3)
δ = 171.86, 160.64, 159.12, 148.05, 141.26, 136.80, 132.60, 131.75, 127.42, 125.91, 122.26, 121.71,
121.26, 119.95, 113.89, 59.89, 57.81, 55.10, 53.11, 45.99, 45.49, 42.76, 36.75, 24.41, 22.93. HRMS

Molecules 2014, 19
8929
(ESI): Calcd for (M−H)− (C28H38ClN7O) requires m/z 522.2748, found 522.2747. Anal. Calcd for
C28H38ClN7O: C, 64.17; H, 7.31; N, 18.71. Found: C, 64.03; H, 7.26; N, 18.59.
N-(2-(4-(3-(Dimethylamino)propylamino)quinazolin-2-yl)phenyl)-4-morpholinobutanamide
(12e).
Compound 9 was treated with excess morpholine according to the general aminolysis procedure to
afford 12e as a white solid in 65% yield after column chromatography with petroleum ether/EtOAc
(15:1) elution; mp 152–154 °C. 1H-NMR (CDCl3) δ = 14.07 (s, 1H), 9.03 (s, 1H), 8.87 (d, J = 2.2 Hz,
1H), 8.68 (d, J = 8.6 Hz, 1H), 7.78–7.69 (m, 2H), 7.65 (d, J = 8.2 Hz, 1H), 7.46 (ddd, J = 8.2, 5.0, 3.2 Hz,
1H), 7.10 (dd, J = 8.6, 2.2 Hz, 1H), 3.88 (dd, J = 10.3, 5.6 Hz, 2H), 3.70–3.56 (m, 4H), 2.73–2.64 (m,
2H), 2.61 (t, J = 7.2 Hz, 2H), 2.47 (t, J = 6.3 Hz, 2H), 2.44 (s, 6H), 2.08–1.99 (m, 2H), 1.98–1.91 (m,
13
2H), 1.86–1.72 (m, 4H). C-NMR (CDCl3) δ = 171.82, 160.65, 159.12, 148.02, 141.24, 136.83,
132.60, 131.77, 127.30, 125.96, 122.32, 121.68, 121.32, 119.93, 113.89, 66.97, 59.81, 58.27, 53.67,
45.48, 42.68, 36.62, 24.43, 22.52. HRMS (ESI): Calcd for (M−H)− (C27H35ClN6O2) requires m/z
509.2432, found 509.2429. Anal. Calcd for C27H35ClN6O2: C, 63.45; H, 6.90; N, 16.44. Found: C,
63.31; H, 7.05; N, 16.51.
3.2. Materials and Biological Assays
Stock solutions of all quinazoline derivatives (10 mM) were made with DMSO and stored at 4 °C.
All tumor cell lines were obtained from the American Type Culture Collection (ATCC, Rockville,
MD, USA). The cell culture was maintained in a RPMI-1640 or DMEM medium supplemented with
10% fetal bovine serum, 100 U/mL penicillin, and 100 mg/mL streptomycin in 25 cm2 culture flasks at
37 °C humidified atmosphere with 5% CO2. Chick embryos were purchased from South China
Agricultural University (SCAU).
3.2.1. Methyl Thiazolyl Tetrazolium (MTT) Assay
HUVEC, CNE-2, PC-3, and 7721 cell lines were seeded on 96-well plates at a concentration of
5,000 cells per well and exposed to various concentrations of quinazoline derivatives. After 48 h of
treatment at 37 °C in a humidified atmosphere of 5% CO2, 5 mg/mL MTT solution (10 μL) was added
to each well and further incubated for 4 h. Each well was then added dimethyl sulfoxide (DMSO, 100 μL),
and the optical density (OD) was recorded at 490 nm. All measurements were done in triplicate, and
the IC50 values were derived from the mean OD values of the triplicate tests versus drug concentration
curves [12,13].
3.2.2. Cell Adhesion Assay
Cell adhesion assay was carried out by a reported method [14,15] with slight modification. Wells of
a 96-well plate were coated at room temperature overnight with rat trail tendon collagen type I (2 μg)
in PBS in a final volume of 100 μL. The wells were washed three times with PBS (100 mL) and then
blocked with 0.2% bovine serum albumin (BSA) in PBS (100 mL) for 2 h at 37 °C in a cell culture
incubator. The wells were then washed three times with PBS (100 mL). HUEVCs were detached from
the dish, resuspended in DMEM, and added (50 μL, 104 cells/mL) to each well followed by the related
compounds (50 μL) at various concentrations. The plates were incubated for 1 h or 3 h at 37 °C and

Molecules 2014, 19
8930
then washed three times with PBS (100 μL). The attached cells were fixed and stained with 0.2 %
crystal violet in 20% methanol for 10 min, then washed three times with PBS (100 mL). The cells were
solubilized by 2% SDS and the OD was measured at 570 nm. Attachment was determined by both
1 and 3 h as described above. Each compound was tested in triplicate and each experiment was
repeated three times.
3.2.3. Cell Migration Assay
The HUVEC cell line was seeded on a pre-treated 48-well culture plate at a concentration of
2 × 104 cells per well in a volume of 200 μL, and a portion of the cell monolayer was scraped off with
a sterile disposable rubber policeman [15–17]. After the DMEM medium was removed, the cells in
each well were then washed three times with PBS (phosphate-buffer saline), and fresh medium was
added to each well. The wells were then tested after incubation for 24 h with or without quinazoline
derivatives at various concentrations. Two random pictures were taken for each wound using a
microscope. The area of the migrations was measured using Photoshop CS3. All measurements were
done in triplicate, and the experiments were done at least three times.
3.2.4. CAM Assay
The in vivo CAM angiogenesis model as initially described by Folkman [10] and then modified by
Maragoudakis [18–21] was used. Fertilized chicken eggs were used after incubating them for 8 days. A
1 cm × 1 cm window was made in the shell to create a pocket to expose the CAM. A filter disc with
0.9% NaCl solution or 30 μg/10 μL of compounds was then placed upon the CAM from 10 eggs per
treatment and 48 h later, the CAMs were fixed with 4% paraformaldehyde and the vessel number from
three random fields around the filter disc were pictured using a microscope.
4. Conclusions
Tumor angiogenesis is a promising target of cancer therapy. Based on this target, a structure-based
drug design plan led us to synthesize a series of 2,4-disubstituted quinazoline derivatives that were
then subjected to pharmacological evaluation as angiogenesis inhibitors. Cytotoxicity assays indicated
that most of 2,4-disubstituted quinazoline derivatives showed potent cytotoxicity against both tumor
cells and HUVECs,and that their cytotoxicity toward tumor cells was closely related to the length of
side chains and basicity. Among them, compound 11d with a three carbon chain length and the N-
methylpiperazino group showed a remarkable inhibitive effect against the migration, adhesion of
HUVECs and significant anti-angiogenesis activities in the chick embryo chorioallantoic membrane
(CAM) assay, which are consistent with those previously reported for effective angiogenesis inhibitors.
Finally, this preliminary research on these quinazoline derivatives’ in vitro activities and in vivo
activities provides us with another new entry into angiogenesis inhibitors and conformation studies for
targeting tumor angiogenesis. Additional studies and tests on their mechanism of anti-angiogenesis
activity are underway.

Molecules 2014, 19
8931
Acknowledgments
We thank the National Nature Science Foundation of China (81302701), the Natural Science
Foundation of Anhui Province (1308085QC53) for financial support of this study.
Author Contributions
The listed authors contributed to this work as described in the following. Qiru Xiong gave the
concepts of work, interpreted the results and prepared the manuscript. Guangjin Yu interpreted the
results and cooperated in the preparation of the manuscript, Zeng Li and Liang Tang carried out of the
synthetic work and cooperated in the preparation of the manuscript. All authors read and approved the
final manuscript.
Conflicts of Interest
The authors declare no conflict of interest.
References
1. Grothey, A.; Galanis, E. Targeting angiogenesis: Progress with anti-VEGF treatment with large
molecules. Nat. Rev. Clin. Oncol. 2009, 6, 507–518.
2. Folkman, J. Tumor angiogenesis. Adv. Cancer Res. 1985, 43, 175–203.
3. Ingber, D.; Fujita, T.; Kishimoto, S.; Sudo, K.; Kanamaru, T.; Brem, H.; Folkman, J. Synthetic
analogues of fumagillin that inhibit angiogenesis and suppress tumour growth. Nature 1990, 348,
555–557.
4. Fong, T.A.; Shawver, L.K.; Sun, L.; Tang, C.; App, H.; Powell, T.J.; Kim, Y.H.; Schreck, R.;
Wang, X.; Risau, W.; et al. SU5416 is a potent and selective inhibitor of the vascular endothelial
growth factor receptor (Flk-1/KDR) that inhibits tyrosine kinase catalysis, tumor vascularization,
and growth of multiple tumor types. Cancer Res. 1999, 59, 99–106.
5. O’Reilly, M.S.; Holmgren, L.; Shing, Y.; Chen, C.; Rosenthal, R.A.; Moses, M.; Lane, W.S.;
Cao, Y.; Sage, E.H.; Folkman, J. Angiostatin: A novel angiogenesis inhibitor that mediates the
suppression of metastases by a Lewis lung carcinoma. Cell 1994, 79, 315–328.
6. O’Reilly, M.S.; Boehm, T.; Shing, Y.; Fukai, N.; Vasios, G.; Lane, W.S.; Flynn, E.; Birkhead, J.R.;
Olsen, B.R.; Folkman, J. Endostatin: An endogenous inhibitor of angiogenesis and tumor growth.
Cell 1997, 88, 277–285.
7. Morabito, A.; Piccirillo, M.C.; Falasconi, F.; de Feo, G.; del Giudice, A.; Bryce, J.; di Maio, M.;
De Maio, E.; Normanno, N.; Perrone, F. Vandetanib (ZD6474), a dual inhibitor of vascular
endothelial growth factor receptor (VEGFR) and epidermal growth factor receptor (EGFR)
tyrosine kinases: Current status and future directions. Oncologist 2009, 14, 378–390.
8. Meyerhardt, J.A.; Ancukiewicz, M.; Abrams, T.A.; Schrag, D.; Enzinger, P.C.; Chan, J.A.;
Kulke, M.H.; Wolpin, B.M.; Goldstein, M.; Blaszkowsky, L.; et al. Phase I study of cetuximab,
irinotecan, and vandetanib (ZD6474) as therapy for patients with previously treated metastastic
colorectal cancer. PLoS One 2012, 7, e38231.

Molecules 2014, 19
8932
9. Roy, A.D.; Subramanian, A.; Roy, R. Auto-redox reaction: tin(II) chloride-mediated one-step
reductive cyclization leading to the synthesis of novel biheterocyclic 5,6-dihydro-quinazolino[4,3-
b]quinazolin-8-ones with three-point diversity. J. Org. Chem. 2006, 71, 382–385.
10 Bellou, S.; Pentheroudakis, G.; Murphy, C.; Fotsis, T. Anti-angiogenesis in cancer therapy:
Hercules and hydra. Cancer Lett. 2013, 338, 219–228.
11. D’Amato, R.J.; Loughnan, M.S.; Flynn, E.; Folkman, J. Thalidomide is an inhibitor of
angiogenesis. Proc. Natl. Acad. Sci. USA 1994, 91, 4082–4085.
12. Woo, H.J.; Jun do, Y.; Lee, J.Y.; Woo, M.H.; Yang, C.H.; Kim, Y.H. Apoptogenic activity of
2α,3α-dihydroxyurs-12-ene-28-oic acid from Prunella vulgaris var. lilacina is mediated via
mitochondria-dependent activation of caspase cascade regulated by Bcl-2 in human acute
leukemia Jurkat T cells. J. Ethnopharmacol. 2011, 135, 626–635.
13. Park, C.; Park. S.; Chung, Y.H.; Kim, G.Y.; Choi, Y.W.; Kim, B.W.; Choi, Y.H. Induction of
apoptosis by a hexane extract of aged black garlic in the human leukemic U937cells. Nutr. Res.
Pract. 2014, 8, 132–137.
14. Vukanovic, J.; Passaniti, A.; Hirata, T.; Traystman, R.J.; Hartley-Asp, B.; Isaacs, J.T.
Antiangiogenic effects of the quinoline-3-carboxamide linomide. Cancer Res. 1993, 53, 1833–1837.
15. Malinda, K.M.; Nomizu, M.; Chung, M.; Delgado, M.; Kuratomi, Y.; Yamada, Y.; Kleinman, H.K.;
Ponce, M.L. Identification of laminin alpha1 and beta1 chain peptides active for endothelial cell
adhesion, tube formation, and aortic sprouting. FASEB J. 1999, 13, 53–62.
16. Song, Y.; Jin, J.; Li, J.; He, R.; Zhang, M.; Chang, Y.; Chen, K.; Wang, Y.; Sun, B.; Xing, G.
Gd@C82(OH)22 nanoparticles constrain macrophages migration into tumor tissue to prevent
metastasis. J. Nanosci. Nanotechnol. 2014, 14, 4022–4028.
17. He, Z.; Chen, H.; Li, G.; Zhu, H.; Gao, Y.; Zhang, L.; Sun, J. Diosgenin inhibits the migration of
human breast cancer MDA-MB-231 cells by suppressing Vav2 activity. Phytomedicine 2014,
21, 871–876.
18. Ferreira, J.F.; Luthria, D.L.; Sasaki, T.; Heyerick, A. Flavonoids from Artemisia annua L. as
antioxidants and their potential synergism with artemisinin against malaria and cancer. Molecules
2010, 15, 3135–3170.
19. Huan-huan, C.; Li-Li, Y.; Shang-Bin, L. Artesunate reduces chicken chorioallantoic membrane
neovascularisation and exhibits antiangiogenic and apoptotic activity on human microvascular
dermal endothelial cell. Cancer Lett. 2004, 211, 163–173.
20. Liu, K.; Yao, S.; Lou, Y.; Xu, Y. Synthesis and anti-angiogenetic activity evaluation of N-(3-aryl
acryloyl)aminosaccharide derivatives. Carbohydr. Res. 2013, 381, 83–92.
21. Krusche, B.; Arend, J.; Efferth, T. Synergistic inhibition of angiogenesis by artesunate and captopril
in vitro and in vivo. Evid. Based Complement. Alternat. Med. 2013, doi:10.1155/2013/454783.
Sample Availability: Samples of the compounds are available from the authors.
© 2014 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article
distributed under the terms and conditions of the Creative Commons Attribution license
(http://creativecommons.org/licenses/by/3.0/).