Synthesis and evaluation of graveoline and graveolinine derivatives with potent anti-angiogenesis activities

Article abstract of DOI:10.1016/j.ejmech.2010.05.043

A series of graveoline and graveolinine derivatives were synthesized. The biological results showed that most of graveoline derivatives possessed higher cytotoxicity and better inhibitive effect against the adhesion and migration of human umbilical vein e

Full text of DOI:10.1016/j.ejmech.2010.05.043

European Journal of Medicinal Chemistry 45 (2010) 3895e3903
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and evaluation of graveoline and graveolinine derivatives
with potent anti-angiogenesis activities
1
1
**
Zeng-Yun An , Yi-Yong Yan , Dan Peng, Tian-Miao Ou, Jia-Heng Tan , Shi-Liang Huang, Lin-Kun An,
*
Lian-Quan Gu, Zhi-Shu Huang
School of Pharmaceutical Sciences, Sun Yat-sen University, Waihuan East Road 132, Guangzhou 510080, China
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of graveoline and graveolinine derivatives were synthesized. The biological results showed that
most of graveoline derivatives possessed higher cytotoxicity and better inhibitive effect against the
adhesion and migration of human umbilical vein endothelial cell (HUVEC) than graveolinine derivatives.
Among these compounds, 8d was the most potent agents that also showed significant anti-angiogenesis
activities in chick embryo chorioallantoic membrane (CAM) assay.
Received 3 June 2009
Received in revised form
18 May 2010
Accepted 20 May 2010
Available online 26 May 2010
Ó 2010 Elsevier Masson SAS. All rights reserved.
Keywords:
Graveoline derivatives
Graveolinine derivatives
Synthesis
Anti-angiogenesis
1. Introduction
showed comprehensive pharmacological activities such as anti-
bacteria, spasmolysis, and anti-tumor [8e12].
Angiogenesis, the formation of new blood vessels from the pre-
existing vascular bed of the guest, is related to many human
diseases such as cancer, obesity, psoriasis, diabetic retinopathy, and
arthritis [1]. In tumors, this process is extremely necessary because
angiogenesis supply necessary oxygen and nutrient for the growth
of tumor cells [2]. It has been shown that, without angiogenesis,
a solid tumor can only reach the size of 1e2 mm3, which is small
enough to be treated with conventional chemotherapeutic agents
[2]. Tumor angiogenesis is a promising target to inhibit tumor
growth and ease the harm of cancer [3]. Many angiogenesis
inhibitors such as fumagillin derivative TNP-470 [4], SU-5416 [5],
angiostatin [6], and endostatin [7] exhibit anti-tumor or anti-
metastatic activities. Since targeting tumor angiogenesis is expec-
ted to be less toxic than killing cancer cells directly, compounds that
significantly inhibit tumor angiogenesis but possess low cytotox-
icity against normal cells are likely to be found.
In this paper, we synthesized a series of graveoline and grave-
olinine derivatives (Table 1). Their cytotoxicity against normal cell
lines (HUVEC, human umbilical vein endothelial cell line) and
tumor cell lines (NCI-H460, GLC-82, and MCF-7 cell lines) were
evaluated. Furthermore, their anti-angiogenesis activities were
tested by cell adhesion assay, migration assay, and chorioallantoic
membrane of the chick embryo assay (CAM).
2. Results and discussion
2.1. Chemistry
The synthetic routes for the graveoline and graveolinine deriv-
atives were outlined in Schemes 1e4. The purchased starting
material 1-(2-aminophenyl)ethanone was reacted with three
benzoyl chlorides respectively in THF at room temperature, fol-
lowed by strong base t-BuOK to obtain three ring closure inter-
mediates (1, 2 and 3) as shown in Scheme 1. Different from
compound 1 and 2, when compound 3 was synthesized, with the
same treatment of t-BuOK, the halogen of 4-(chloromethyl)benzoyl
group was attacked and then replaced by the basic tert-butoxy
group, which was proved by 1H, 13C NMR and LC-MS.
Graveoline and graveolinine (Fig. 1) were alkaloids isolated from
Ruta graveolens L. (Common Rue Herb) in the south of China, which
* Corresponding author. Tel./fax: þ86 20 3994 3056.
** Corresponding author. Tel.: þ86 20 3994 3068.
E-mail addresses: tanjiah@mail.sysu.edu.cn (J.-H. Tan), ceshzs@mail.sysu.edu.cn
(Z.-S. Huang).
Modifications to the three intermediates (1, 2 and 3) were
mainly focused on the carbonyl group and the benzyl group. In
1
Equally contributed as the first author.
0223-5234/$ e see front matter Ó 2010 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2010.05.043

3896
Z.-Y. An et al. / European Journal of Medicinal Chemistry 45 (2010) 3895e3903
different amines respectively afforded five graveoline derivatives
O
N
O
N
(8ae8e). In Scheme 3, when using intermediate 2 as a starting
materials, two graveoline derivatives (10a, 10b) were obtained by
the same steps showed in Scheme 2. All modifications of 3 were
described in Scheme 4. Graveoline derivatives 12 and 13 were
obtained by subsequently treatment with 3-morpholinopropan-1-
amine to intermediate 11, which was generated from 3 in POCl3.
Methylation of 3 with methane iodide and sodium hydride in DMF
afforded O-methylated product 14. In this step, the tert-butoxy
group which attached to the benzyl group of 3 was changed to
a hydroxyl group, and little N-methylated product was detected,
which was different from the methylation of 1. Then further
treatment of 14 in dichloromethane with thiophene-2-carbonyl
chloride provided graveolinine derivative 15 using reported
method [22].
O
O
O
O
A
B
Fig. 1. Chemical structures of graveoline (A) and graveolinine (B).
Scheme 2, intermediate 1 was reacted with methane iodide and
sodium hydride in N, N-dimethylformamide (DMF) by a reported
method [13,14], yielded 1-methyl-2-(4-methylphenyl)quinolin-4
(1H)-one (5) as the main product (N-methylated) and 4-methoxy-
2-(4-methylphenyl)quinoline (4) as the minor product (O-methyl-
ated). The major product 5 was refluxed in dichloromethane with
N, N-dimethylpropyldiamine to yield graveolinine derivative 6 after
substitution of bromide by N-bromosuccinimide (NBS) [15,16].
Another treatment of intermediate 1 with POCl3 generated 4-
chloro-2-(4-methylphenyl)quinoline (7) by replacing the carbonyl
group with chloride [17e21]. Subsequent reaction of 7 with five
2.2. Cytotoxicity against tumor cells and HUVEC
Folkman firstly proposed anti-angiogenesis as a potential target
in cancer biology [2]. Over the past 30 years most researches on
tumor angiogenesis has been aimed at inhibiting the process of
Table 1
Structures of graveoline derivatives (A) and graveolinine derivatives (B).
11R1
O
N
6
10
9
1
17
12
16
N
3
7
R2
R2
14
B
A
Compd.
4
Struct.
A
R1
R2
Compd.
10a
Struct.
A
R1
R2
OH
OH
OCH3
CH3
CH3
CH3
OCH3
N
H
Cl
OCH3
N
H
7
A
A
10b
12
A
A
OH
Cl
O
O
N
H
H
N
N
N
8a
O
CH3
CH3
H
N
N
OH
H
N
N
H
8b
8c
A
A
13
14
A
A
O
OCH3
OCH3
H
N
OH
S
N
H
N
O
CH3
CH3
N
8d
A
15
A
O
H
N
CH3
8e
9
A
A
5
6
B
B
e
e
O
Cl
OCH3
H
N
N

Z.-Y. An et al. / European Journal of Medicinal Chemistry 45 (2010) 3895e3903
3897
O
N
H
O
O
R2
1
: R2=CH3
: R2=OCH3
Cl
a
b
2
NH2
R1
OH
R1=CH3, OCH3, CH2Cl
N
R2
R2=CH2OC(CH3)3
3:
Scheme 1. Reagents: a) THF, rt; b) t-BuOH/t-BuOK.
tumor induced vessel formation. Endothelial cells from the inner
lining of all blood vessels constitute an essential part of new as well
as pre-existing blood vessels. The proliferation, migration, and
differentiation of endothelial cells are integral parts of angiogen-
esis. Most angiogenesis inhibitors discovered to date target endo-
thelial cells. We thus employed a cell proliferation assay to evaluate
the cytotoxicity of graveoline and graveolinine derivatives against
both normal (HUVEC) and tumor cell lines (NCI-H460, GLC-82, and
MCF-7 cell lines). So that we could screen some compounds that
had moderate cytotoxicity against tumor cells but low cytotoxicity
against HUVEC. Meanwhile, a proper range of concentrations of
these compounds could be determined to study their inhibitive
effect against the proliferation, migration, and differentiation of
HUVEC.
The growth inhibition effect was assayed using MTT method.
The concentration of graveoline derivatives with 50% inhibition
(IC50) on the NCI-H460, GLC-82, MCF-7 and HUVEC cell line was
determined and the results were summarized in Table 2. The results
showed that most of graveoline derivatives (8ae8e, 10a, 10b, 12 and
13) possessed higher cytotoxicity than all of graveolinine deriva-
tives (5, 6) which had no cytotoxicity against all tested cell lines.
The results indicated that introducing an appropriate R group could
increase the cytotoxicity of the parent structure A, but it did not
work for structure B (Table 2).
explained as all of them had both short R1 and R2 groups. While
graveoline derivatives possessed at least one long side chain
(8ae8e, 10a, 10b, 12 and 13), they showed much higher cytotoxicity.
In addition, among these compounds, relative lower cytotoxicity
was found with 8a, 8b, 10a and 10b, all of which had a hydroxyl
group at the end of the R1 group. On the other hand, there were no
significant differences between graveoline derivatives with either
methoxyl group or methyl group as the R2 groups, such as 8a/10a,
and 8b/10b. Comparing with graveoline derivatives 8c and 13,
which had the same R1 group but different R2 groups, it was found
compound 13 was much less cytotoxic against HUVEC than 8c
although they had comparative cytotoxicity against tumor cells.
All the above results showed that the cytotoxicity of these
graveoline and graveolinine derivatives was closely related to their
parent structure and the length of the side chains. However, the
groups at the end of the side chains had relatively weak effects on
the cytotoxicity.
2.3. Inhibition of HUVEC adhesion
An ideal inhibitor of angiogenesis should be low toxic to HUVEC
while highly inhibitive to the proliferation, migration, and differ-
entiation of HUVEC. To study the inhibitory effects of graveoline
and graveolinine derivatives, three subsequently assays: HUVEC
adhesion assay, HUVEC migration assay and CAM assay were
adopted. Firstly, to test the ability of the attachment of endothelial
Among all graveoline derivatives, 4, 7, 9 and 14 showed poor
cytotoxicity against both tumor cells and HUVEC, this could be
O
O
O
b
N
N
N
H
N
N
4 (minor)
6
5 (major)
R
a
Cl
HN
N
c
d
1
N
7
8a-8e
Scheme 2. Reagents: a) CH3I/NaH, DMF; b) NBS/benzoyl peroxide, CCl4; RNH2, CH2Cl2; c) POCl3; d) RNH2, SnCl4. (‘R-’ are shown in Table 1).

3898
Z.-Y. An et al. / European Journal of Medicinal Chemistry 45 (2010) 3895e3903
2.5. Graveoline derivatives inhibits angiogenesis in vivo in the CAM
assay
R
Cl
HN
N
a
b
2
According to the results of cytotoxicity, migration, and adhesion
N
assay of HUVEC, eight graveoline derivatives (8ae8e, 10a, 10b and
12) which possessed moderate cytotoxicity and potent inhibitive
effect against the adhesion and migration of HUVEC were selected
to evaluate their anti-angiogenesis activities by in vivo CAM assay.
CAMs treated with 0.9% NaCl solution (negative control) was
surrounded by allantoic vessels as newly-formed capillaries
converging radially toward the sponge in a “spoked wheel” pattern
(number of vessels ¼ 26 ꢁ 3, Fig. 3). As shown in the Fig. 3, when
graveoline derivatives were added to the CAMs, significant reduc-
tion of the angiogenic responses were found compared to negative
control. Among all CAMs treated with graveoline derivatives,
8ae8e excellently inhibited angiogenesis of chick embryos, which
was in consistent with the results of cytotoxicity, migration, and
adhesion. And all the compounds tested in CAM assay showed
better inhibition of angiogenesis than thalidomide which is a clin-
ical anti-tumor drug targeting tumor angiogenesis [23].
O
O
9
10a 10b
,
Scheme 3. Reagents: a) POCl3; b) RNH2, SnCl4. (‘R-’ are shown in Table 1).
cells to type I collagen, cell adhesion assays were performed. The
inhibitory effects of derivatives on the attachment of HUVEC cells to
collagen type were determined by both 1 and 3 h exposures to
different concentration of graveoline derivatives (15 or 30 mM).
As shown in Table 3, most compounds inhibited HUVEC adhe-
sion to the collagen. 10a, 10b, 8d, 8e and 12 significantly inhibited
HUVEC adhesion at both 1 and 3 h 12 showed the most potent
inhibition of HUVEC adhesion, the inhibitory rate was as much as
69.4% at a dose of 15 mM and 72.3% at a dose of 30 mM when 3 h
incubation at 37 ꢀC. All of graveoline derivatives showed better
inhibitive effect against HUVEC adhesion than graveolinine
derivatives.
3. Conclusion
2.4. Inhibition of HUVEC migration
Tumor angiogenesis is a promising target of cancer therapy.
Based on this target, a series of graveoline and graveolinine deriv-
atives were synthesized and tested their anti-angiogenesis activi-
ties. Cytotoxicity assay indicated that most of graveoline derivatives
showed potent cytotoxicity against both tumor cells and HUVEC
compared to graveolinine derivatives. And their cytotoxicity was
closely related to the parent structures and the length of side
chains. Among all the compounds, 8d comprehensively showed
strong inhibitive effect against the migration, adhesion of HUVEC
and significant anti-angiogenesis activities. In conclusion, the
preliminary in vitro activities and in vivo activities of these
compounds possess potential for design of better future molecules
targeting tumor angiogenesis. Further studies on their mechanism
of anti-angiogenesis activity are in progress.
Based on the result of HUVEC adhesion assay in which most of
graveoline derivatives showed better inhibitory effects against
HUVEC adhesion, graveoline derivatives 7, 9, 8ae8e, 10a and 10b
were further tested in HUVEC wounding assay to evaluate their
inhibition against HUVEC migration, an important step in tube
formation and vessel sprouting. Compounds were tested at both
concentrations of 15 mM and 30 mM, all nine compounds tested in
this assay could inhibit HUVEC migration. Five derivatives, 8aee,
with a longer side chain in the 10-position of parent structure A,
showed a greatly inhibitory potential on HUVEC migration at 15
(Fig. 2), and inhibitory effects was more significant when the
concentration of these compounds was increased to 30 M. Among
mM
m
all these compounds, 8d showed the best inhibitory activity.
Cl
Cl
a
b
3
N
N
O
H
N
d
Cl
N
11
12
O
c
N
HN
N
OH
O
14
N
O
H
N
e
N
O
13
N
S
O
15
O
Scheme 4. Reagents: a) POCl3; b) RNH2/CH2Cl2; c) RNH2, SnCl4; d) CH3I/NaH, DMF; TFA/CH2Cl2 e) thiophene-2-carbonyl chloride/Et3N, CH2Cl2.

Z.-Y. An et al. / European Journal of Medicinal Chemistry 45 (2010) 3895e3903
3899
Table 2
IC50 cytotoxicity values (mM) of graveoline and graveolinine derivatives against tumor cells and HUVEC.
Compound
GLC
NCI
MCF-7
HUVEC
Compound
GLC
56.2
40.9
30.6
34.4
NCI
38.3
24.0
21.1
42.3
MCF-7
HUVEC
4
7
>100
>100
61.1
60.3
49.5
20.6
34.2
>100
>100
53.7
48.1
11.5
6.1
>100
>100
73.7
63.5
45.2
30.3
14.3
>100
>100
94.6
40.4
34.5
23.7
>100
29.3
>100
10a
10b
12
13
14
15
5
74.1
47.6
22.4
39.7
23.8
31.5
8a
8b
8c
8d
8e
9
56.5
>100
>100
>100
>100
>100
>100
99.7
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
24.2
>100
>100
6
4. Experimental
DMSO): d 172.40, 152.83, 141.75, 140.18, 133.36, 129.85, 129.77 (2C),
128.04 (2C), 125.72, 123.82, 121.61, 119.66, 105.15, 20.95; APCI-MS
4.1. Synthesis and characterzation
m/z: 236.1 [M þ 1] þ
.
All chemicals and solvents were obtained from commercial
sources unless otherwise specified. 1H and 13C NMR spectra were
recorded on a Bruker 400 NMR with tetramethylsilane (TMS) as an
internal standard. Only new compounds’ NMR data were shown.
MS spectra were obtained using Shimadzu LC-MS-2010A spec-
trometry. Melting points were determined on an X-6 microscope
melting point instrument and were uncorrected.
4.1.1.2. 2-(4-Methoxyphenyl)quinolin-4(1H)-one (2). A light yellow
solid; 1H NMR (400 MHz, DMSO):
3.90 (s, 3H), 4.98 (brs, 1H, D2O
d
exchangeable), 7.24 (d, 2H, J ¼ 8.8 Hz), 7.34 (s, 1H), 7.70 (t, 1H,
J ¼ 7.6 Hz), 7.97 (s, 1H), 8.01 (d, 2H, J ¼ 8.8 Hz), 8.29 (t, 2H,
J ¼ 8.5 Hz); 13C NMR (100 MHz, DMSO):
d 170.58, 162.34, 153.57,
140.00, 133.84, 130.25 (2C), 126.53, 124.04, 123.47, 120.23, 119.93,
114.79 (2C), 104.06, 55.64; ESI-MS m/z: 252.1 [M þ 1] þ
.
4.1.1. General procedure for the synthesis of the three intermediates
(1, 2, 3)
4.1.1.3. 2-(4-(tert-butoxymethyl)phenyl)quinolin-4-ol (3). A light
yellow solid; 1H NMR (400 MHz, DMSO):
1.26 (s, 9H), 4.50 (d, 2H,
d
1-(2-aminophenyl)ethanone (10 mL, 10 mmol) and 10 mL of
triethylamine were dissolved in 200 mL of THF in an ice bath. Then,
benzoyl chloride (11 mL, 11 mmol) was added dropwise. After
30 min at 0 ꢀC, the mixture was stirred at room temperature
overnight and poured into 50 mL ice water. The precipitate was
collected and washed with water and then with methanol. The
solid was dried under vacuum and suspended in 200 mL of tert-
butyl alcohol. Potassium tert-butoxide (15 g, 134 mmol) was added,
and the mixture was heated at 75 ꢀC for 16 h under N2 atmosphere.
The mixture was cooled and poured into 30 mL of ice water. 10%
aqueous HCl was added until pH ¼ 6. The solid was collected and
washed several times with water. The crude product was recrys-
tallized from CH2Cl2 and methanol to afford the intermediates
(80e90%). All these intermediates were routinely checked by TLC
with Merck Silica Gel 60F-254 glass plates, analyzed by APCI/ESI-
MS, 1H and 13C NMR, and subjected to the subsequent synthesis
without further purification.
J ¼ 11.8 Hz), 6.34 (d, 1H, J ¼ 1.7 Hz), 7.34 (t, 1H, J ¼ 7.5 Hz), 7.52 (d,
2H, J ¼ 8.2 Hz), 7.70e7.64 (m, 1H), 7.76 (s, 1H), 7.82e7.78 (m, 2H),
8.10 (d, 1H, J ¼ 7.9 Hz), 11.68 (m, 1H, D2O exchangeable); 13C NMR
(100 MHz, DMSO):
d 176.89, 149.83, 142.72, 140.46, 132.67, 131.73,
127.50 (2C), 127.13 (2C), 124.83, 124.68, 123.17, 118.64, 107.11, 73.11,
62.82, 27.43; ESI-MS m/z: 308.1 [M þ 1]þ.
4.1.2. 4-Methoxy-2-(4-methylphenyl)quinoline (4) and 1-methyl-
2-(4-methylphenyl) quinolin-4(1H)-one (5)
To a solution of 1 (4.50 g, 20 mmol) in DMF (80 mL) was added
NaH (0.45 g) and methyl iodide (2.8 mL). The mixture was stirred at
50 ꢀC for 4 h under N2 atomsphere. The solvent was evaporated and
the residue was diluted with CH2Cl2, extracted by water, the
combined organic layer was dried over anhydrous MgSO4, filtered
and the solution was evaporated under reduced pressure. The
crude residue was purified by silica gel column chromatography
(eluent CH2Cl2/MeOH ¼ 50:1) to give 4 (0.38 g, 7.5%) and 5 (1.42 g,
28%).
4.1.1.1. 2-(4-Methylphenyl)quinolin-4(1H)-one (1). A light yellow
solid; 1H NMR (400 MHz, DMSO):
d
2.44 (s, 3H), 5.03 (brs, 1H, D2O
4.1.2.1. 4-Methoxy-2-(4-methylphenyl)quinoline (4). A light yellow
solid; m.p. 96e97 ꢀC (lit. [24] m.p. 94e95 ꢀC); 1H NMR (CDCl3,
exchangeable), 7.05 (s, 1H), 7.48 (d, 2H, J ¼ 8.1 Hz), 7.61 (t, 1H,
J ¼ 7.5 Hz), 7.86 (d, 2H, J ¼ 8.1 Hz), 7.91 (dd,1H, J1 ¼13.7, J2 ¼ 6.5 Hz),
8.14 (d, 1H, J ¼ 8.5 Hz), 8.25 (d, 1H, J ¼ 8.1 Hz); 13C NMR (100 MHz,
400 MHz):
d 2.46 (s, 3 H), 4.10 (s, 3H), 7.16 (s, 1H), 7.33 (d, 2H,
J ¼ 7.8 Hz), 7.46e7.47 (m, 1H), 7.70e7.72 (m, 1H), 8.03 (d, 2H,
Table 3
The inhibitory effects of compounds on adhesion of HUVEC on collagen at a dose of 15 and 30 mM.
Compd.
1 h Inhibition rate %
15 30
3 h Inhibition rate %
15 30
Compd.
1 h Inhibition rate %
15 30
3 h Inhibition rate %
15 30 mM
m
M
m
M
m
M
m
M
m
M
m
M
m
M
4
7
11.0
30.6
36.7
31.9
27.4
39.7
34.9
41.7
35.4
39.0
38.3
44.2
47.2
52.2
42.4
52.6
18.1
20.8
28.5
26.0
26.0
35.4
34.8
29.0
20.3
35.2
40.0
33.8
49.2
48.2
48.1
48.2
10a
10b
12
13
14
15
5
52.9
43.1
58.9
15.8
26.6
25.6
6.8
54.1
45.3
67.8
46.2
27.5
37.4
30.3
31.9
39.9
35.9
69.4
nd
nd
1.3
54.9
56.4
72.3
49.2
19.4
27.4
8.1
8a
8b
8c
8d
8e
9
nd
24.1
6
23.8
42.0
data derived from the mean of three independent assays.
nd means no results due to no inhibition.

3900
Z.-Y. An et al. / European Journal of Medicinal Chemistry 45 (2010) 3895e3903
residue 0.5 g was dissolved in CH2Cl2 (2 mL) and amine (1.0 mL)
1.2
1.0
0.8
0.6
0.4
0.2
0.0
15 µM
30 µM
was added. The mixture was stirred at room temperature overnight
and then was treated with 20 mL of distillated water. The mixture
was extracted with CH2Cl2 (3 ꢂ 20 mL), the combined organic layer
was dried over anhydrous MgSO4 and concentrated under vacuum.
The crude residue was purified by silica gel column chromatog-
raphy (eluent CH2Cl2/MeOH ¼ 20:1) to afford 6 (65 mg, 10.3%). A
light yellow solid; m.p. 180e184 ꢀC; 1H NMR (CDCl3, 400 MHz):
d
1.24e1.26 (m, 2H), 1.96e1.99 (m, 2H), 2.32 (s, 6H), 2.42 (s, 3H),
2.72e2.73 (m, 2H), 3.37e3.41 (m, 2H), 3.71 (brs, 1H, D2O
exchangeable), 6.82 (s, 1H), 7.29 (d, 2H, J ¼ 7.6 Hz), 7.40 (t, 1H,
J ¼ 7.6 Hz), 7.63 (t, 1H, J ¼ 7.6 Hz), 7.80 (d, 1H, J ¼ 8.4 Hz), 7.98 (d, 2H,
J ¼ 7.6 Hz), 8.06 (d, 1H, J ¼ 8.4 Hz); ESI-MS m/z: 350.2 [M þ 1]þ.
4.1.4. 4-Chloro-2-(4-methylphenyl)quinoline (7)
7
9
8a
8b
8c
8d
8e 10a 10b Control
Compound 1 (15.0 g) and POCl3 (35 mL) were stirred in ice bath
for 30 min, and refluxed for further 3.5 h. After cooling, the mixture
was poured into 200 mL of ice water. The solution of NaOH (5 M)
was added until pH ¼ 7. The mixture was extracted with CH2Cl2
(3 ꢂ 100 mL), and the combined organic layer was dried with
MgSO4 and evaporated. The crude residue was purified by silica gel
column chromatography (eluent CH2Cl2) to afford 7 (7.10 g, 44%). A
light yellow solid; m.p. 143e145 ꢀC; 1H NMR (CDCl3, 400 MHz):
Compounds
Fig. 2. The inhibitory effects of graveoline derivatives on migration of HUVEC at a dose
of 15 and 30 M.
m
J ¼ 8.1 Hz), 8.11 (dd, 1H, J1 ¼ 8.4 Hz, J2 ¼ 0.6 Hz), 8.18 (dd, 1H,
J1 ¼8.7 Hz, J2 ¼ 1.5 Hz); 13C NMR (CDCl3,100 MHz):
d 21.3, 55.6, 97.8,
d
2.45 (s, 3H), 7.43 (d, 2H, J ¼ 8.0 Hz), 7.82 (t, 1H, J ¼ 8.0 Hz), 8.00 (t,
120.4, 121.6, 125.2, 127.4 (2C), 129.1, 129.5 (2C), 129.9, 137.6, 139.3,
149.2, 158.8, 162.8; APCI-MS m/z: 250.1 [M þ 1]þ.
1H, J ¼ 8.0 Hz), 8.07 (s, 1H), 8.21 (d, 2H, J ¼ 8.0 Hz), 8.30 (d, 1H,
J ¼ 8.0 Hz), 9.31 (d, 1H, J ¼ 8.0 Hz); 13C NMR (CD3OD, 100 MHz):
d
21.5, 121.4, 125.5, 126.6, 127.3, 129.3 (2C), 129.8, 131.1 (2C), 133.8,
4.1.2.2. 1-Methyl-2-(4-methylphenyl)quinolin-4(1H)-one (5). A light
yellow solid; m.p. 187e191 ꢀC; (lit. [25] m.p. 170e174 ꢀC); 1H NMR
(CDCl3, 400 MHz): d 2.45 (s, 3H), 3.63 (s, 3H), 6.32 (s, 1H), 7.29e7.34
133.9, 143.4, 146.4, 148.1, 158.1; APCI-MS m/z: 254.1 [M þ 1]þ.
4.1.5. General procedure for the synthesis of quinoline-4-amines
(8ae8e)
(m, 4H), 7.43 (t, 1H, J ¼ 8.0 Hz), 7.55 (d, 1H, J ¼ 8.0 Hz), 7.69e7.74 (m,
1H), 8.51 (dd, 1H, J1 ¼ 8.0 Hz, J2 ¼ 1.2 Hz); 13C NMR (CDCl3,
A mixture of 7 (1 mmol), amine (1.0 mL), and anhydrous SnCl4 (2
drops) were stirred at 135 ꢀC under N2 atmosphere for 4 h. After
cooling, 10 mL of distillated water was added; the precipitate was
filtered and the residue was purified by silica gel column chroma-
tography (eluent CH2Cl2/MeOH ¼ 10:1), yield 65%e90%.
100 MHz):
d 21.4, 37.3, 112.7, 115.9, 123.0, 123.6, 126.8, 128.5 (2C),
129.4 (2C), 132.3, 133.0, 139.8, 142.0, 155.0, 178.7; APCI-MS m/z:
250.1 [M þ 1]þ.
4.1.3. 2-(4-((3-(Dimethylamino)propylamino)methyl)phenyl)-1-
methylquinolin-4(1H)-one (6)
4.1.5.1. (2-(4-Methylphenyl)quinolin-4-ylamino)ethanol
light yellow solid; m.p. 174e178 ꢀC; 1H NMR (CD3OD, 400 MHz):
2.40 (s, 3H), 3.55 (t, 2H, J ¼ 5.8 Hz), 3.87 (t, 2H, J ¼ 5.8 Hz), 6.85 (s,
1H), 7.29 (d, 2H, J ¼ 8.0 Hz), 7.38e7.42 (m, 1H), 7.60e7.64 (m, 1H),
(8a). A
Compound 5 (1.2 g, 4.8 mmol), NBS (1.04 g, 5.7 mmol), benzoyl
peroxide (0.05 g) in CCl4 (40 mL) were refluxed at 50 ꢀC for 4 h
under N2 atomsphere. After cooling to room temperature, the
precipitate was filtered off and the solvent was removed. The dried
d
Fig. 3. The results of CAM assay. 0.9% NaCl solution and thalidomide were used as a negative control and a positive control, respectively. Representative pictures of 8e10 inde-
pendent experiments performed in duplicate for the graveoline derivatives dose and controls. All filter discs had the same size, visual size difference due to various distances from
which pictures were taken.

Z.-Y. An et al. / European Journal of Medicinal Chemistry 45 (2010) 3895e3903
3901
7.82 (d, 2H, J ¼ 8.0 Hz), 7.91 (d, 1H, J ¼ 8.4 Hz), 8.05 (d, 1H,
400 MHz): d 1.23e1.26 (m, 2H), 1.90e1.98 (m, 2H), 3.39 (q, 2H), 3.83
J ¼ 8.4 Hz); 13C NMR (CD3OD, 100 MHz):
d
21.3, 46.3, 61.0, 97.6,
(s, 3H), 3.85 (brs, 1H, D2O exchangeable), 5.74 (brs, 1H, D2O
exchangeable), 6.63 (s, 1H), 6.97 (d, 2H, J ¼ 8.5 Hz), 7.26e7.32 (m,
1H), 7.55e7.62 (m, 2H), 7.96 (d, 2H, J ¼ 8.5 Hz), 8.03 (d, 1H,
119.4, 121.9, 125.4, 128.8 (2C), 129.0, 130.3 (2C), 130.7, 139.1, 140.4,
149.2, 153.1, 160.3; ESI-MS m/z: 279.2 [M þ 1]þ.
J ¼ 8.4 Hz); 13C NMR (CDCl3, 100 MHz):
d 30.8, 41.8, 55.4, 61.5, 95.9,
113.9 (2C), 117.8, 119.5, 124.1, 128.9 (2C), 129.2, 129.4, 133.4, 148.3,
4.1.5.2. 3-(2-(4-Methylphenyl)quinolin-4-ylamino)propan-1-ol
(8b). A light yellow solid; m.p. 118e120 ꢀC; 1H NMR (DMSO,
150.4, 158.0, 160.5; ESI-MS m/z: 309.2 [M þ 1]þ.
400 MHz): d 1.86e1.93 (m, 2H), 2.38 (s, 3H), 3.43e3.52 (m, 2H), 3.59
(t, 2H, J ¼ 6.0 Hz), 4.66 (brs, 1H, D2O exchangeable), 6.97 (s, 1H), 7.16
(brs, 1H, D2O exchangeable), 7.31 (d, 2H, J ¼ 8.0 Hz), 7.35e7.43 (m,
1H), 7.58e7.65 (m, 1H), 7.84 (d, 1H, J ¼ 8.2 Hz), 8.10 (d, 2H,
J ¼ 8.0 Hz), 8.20 (d, 1H, J ¼ 8.3 Hz); 13C NMR (DMSO, 100 MHz):
4.1.8. 4-Chloro-2-(4-(chloromethyl)phenyl)quinoline (11)
The same procedure as the preparation of 7 afforded 11 (3.67 g,
37%). A light yellow solid; m.p. 110e112 ꢀC; 1H NMR (CDCl3,
400 MHz):
d
4.67 (s, 2H), 7.54 (d, 2H, J ¼ 8.1 Hz), 7.59e7.64 (m, 1H),
d
20.8, 31.2, 39.6, 58.6, 94.7, 118.0, 121.4, 123.5, 126.9 (2C), 128.9,
7.75e7.80 (m, 1H), 7.94 (s, 1H), 8.12e8.18 (m, 3H), 8.21 (d, 1H,
129.0(2C), 129.2, 137.4, 138.3, 148.2, 150.7, 156.5; ESI-MS m/z: 293.2
J ¼ 8.4 Hz); 13C NMR (CDCl3, 100 MHz):
d 45.8, 118.9, 124.0, 125.4,
[M þ 1]þ.
127.4, 127.9 (2C), 129.1 (2C), 130.1, 130.6, 138.7, 139.0, 143.2, 149.1,
156.5; ESI-MS m/z: 288.1 [M þ 1]þ.
4.1.5.3. N-(3-Morpholinopropyl)-2-(4-methylphenyl)quinolin-4-
amine (8c). A light yellow solid; m.p. 144e145 ꢀC (lit. [26] m.p.
146e148 ꢀC); ESI-MS m/z: 362.3 [M þ 1]þ.
4.1.9. N-(4-(4-chloroquinolin-2-yl)benzyl)-3-morpholinopropan-1-
amine (12)
11 (0.8 g) was dissolved in CH2Cl2 (4 mL) and 3-morpholino-
propan-1-amine (1.5 mL) was added. The mixture was stirred at
room temperature overnight and then was treated with 40 mL of
distillated water. The mixture was extracted with CH2Cl2
(3 ꢂ 30 mL), and the combined organic layer was dried over
anhydrous MgSO4, filtered and the solution was evaporated under
vacuum. The crude residue was purified by silica gel column
chromatography (eluente CH2Cl2/MeOH ¼ 20:1) to afford 12 as
4.1.5.4. N1,N1-Dimethyl-N2-(2-(4-methylphenyl)quinolin-4-yl)
ethane-1,2-diamine (8d). A light yellow solid; m.p.þ106e107 ꢀC (lit.
[27] m.p. 108e110 ꢀC); ESI-MS m/z: 306.2 [M þ 1] .
4.1.5.5. N-(4-Methoxyphenyl)-2-(4-methylphenyl)quinolin-4-amine
(8e). A light yellow solid; m.p. 245e248 ꢀC; 1H NMR (CDCl3,
400 MHz):
d 2.27 (s, 3H), 3.77 (s, 3H), 6.67 (s, 1H), 6.82 (d, 2H,
J ¼ 9.0 Hz), 7.15 (d, 2H, J ¼ 8.1 Hz), 7.37e7.33 (m, 3H), 7.48 (t, 1H,
J ¼ 7.5 Hz), 7.72 (d, 2H, J ¼ 8.1 Hz), 8.65 (d, 1H, J ¼ 8.4 Hz), 8.92 (d,
1H, J ¼ 8.4 Hz) 10.71 (brs, 1H, D2O exchangeable); 13C NMR (CDCl3,
a yellow oil (0.75 g, 95%). 1H NMR (400 MHz, CDCl3):
d 1.76e1.83 (m,
2H), 2.42e2.46 (m, 6H), 2.80 (t, 2H, J ¼ 6.8 Hz), 3.67e3.70 (m, 5H,
1H D2O exchangeable), 3.93 (s, 2H), 7.52 (d, 2H, J ¼ 8.0 Hz),
7.59e7.63 (m, 1H), 7.75e7.79 (m, 1H), 7.96 (s, 1H), 8.13 (d, 2H,
J ¼8.0 Hz), 8.17 (d, 1H, J ¼ 8.0 Hz), 8.22 (d, 1H, J ¼ 8.0 Hz); 13C NMR
100 MHz):
d 21.4, 55.5, 97.6, 114.9 (2C), 116.7, 121.0, 124.2, 126.3,
126.7 (2C), 128.3 (2C), 128.9, 129.9 (2C), 130.0, 132.7, 136.0, 142.6,
152.7, 155.3, 158.5; ESI-MS m/z: 341.2 [M þ 1]þ.
(CDCl3, 100 MHz): d 24.5, 47.0, 52.0, 52.7 (2C), 56.6, 65.9 (2C), 118.0,
123.0, 124.3, 126.3, 126.7 (2C), 127.9 (2C), 128.3, 129.0, 129.6, 136.8,
4.1.6. 4-Chloro-2-(4-methoxyphenyl)quinoline (9)
142.2, 148.1, 155.8; ESI-MS m/z: 396.2 [M þ 1]þ.
Compound 2 (2.00 g), POCl3 (10 mL) were stirred in ice bath for
30 min, and refluxed for 3.5 h. After cooling, the mixture was
poured into 30 mL of ice water. The solution of NaOH (5 M) was
added dropwise until pH ¼ 7. The mixture was extracted with
CH2Cl2 (3 ꢂ 30 mL), and the combined organic layer was dried with
MgSO4 and evaporated. The crude residue was purified by silica gel
column chromatography (eluent CH2Cl2) to afford 9 (1.07 g, yield
50%). A light yellow solid; m.p. 75e77 ꢀC; (lit. [28] m.p. 77e78 ꢀC);
ESI-MS m/z: 270.1 [M þ 1]þ.
4.1.10. N-(3-Morpholinopropyl)-2-(4-((3-morpholinopropylamino)
methyl)phenyl)- quinolin-4-amine (13)
A mixture of 12 (0.20 g, 0.5 mmol), 3-morpholinopropan-1-
amine (1.0 mL), and anhydrous SnCl4 (2 drops) were stirred under N2
atmosphere and heated at 135 ꢀC for 4 h. After cooling, 10 mL of
distillated water was added; the precipitatewas filtered and purified
by Al2O3 column chromatography (eluent CH2Cl2/MeOH ¼ 20:1) to
afford 13 as a yellow oil. 1H NMR (CDCl3, 400 MHz):
d 1.64e1.76 (m,
2H), 1.88e1.98 (m, 2H), 2.32e2.42 (m, 6H), 2.52e2.58 (m, 6H), 2.66
(t, 2H, J ¼ 6.8 Hz), 3.42e3.46 (m, 2H), 3.63 (t, 4H, J ¼ 4.8 Hz),
3.77e3.83 (m, 7H, 1H D2O exchangeable), 6.74 (s, 1H), 6.98 (brs, 1H,
D2O exchangeable), 7.32e7.41 (m, 3H), 7.54e7.61 (m,1H), 7.82 (d,1H,
J ¼ 8.4 Hz), 7.96e8.03 (m, 3H); APCI-MS m/z: 504.4 [M þ 1]þ.
4.1.7. General procedure for the synthesis of 4-chloro-2-(4-
methoxyphenyl)quinoline derivatives (10a, 10b)
A mixture of 9 (1 mmol), amine 1.0 mL, and anhydrous SnCl4 (2
drops) were stirred under N2 atmosphere and heated at 135 ꢀC for
4 h. After cooling, 10 mL of distillated water was added; the
precipitate was filtered and purified by silica gel column chroma-
tography (eluent CH2Cl2/MeOH ¼ 10:1), yield 65%e90%.
4.1.11. (4-(4-Methoxyquinolin-2-yl)phenyl)methanol (14)
3 (1.0 g, 3.2 mmol) was dissolved in DMF (10 mL), the mixture
was added to 60% NaH in oil (92 mg, 3.8 mol) at ice bath and stirred
at for 30 min, then was added to CH3I (475 mg, 3.2 mol) and stirred
for 3 h. The reaction mixture was poured into water (20 mL),
extracted with CH2Cl2, the combined organic layer was concen-
trated under reduced pressure, the residue was dissolved in 15 mL
of TFA, the mixture was refluxed at 90 ꢀC overnight. After cooling,
the solvent was evaporated and the residue was purified by silica
gel column chromatography (eluent pet/AcOEt ¼ 1:1) to afford 14
4.1.7.1. 2-(2-(4-Methoxyphenyl)quinolin-4-ylamino)ethanol (10a).
A light yellow solid; m.p. 188e189 ꢀC; 1H NMR (DMSO, 400 MHz):
d
3.49 (d, 2H, J ¼ 5.3 Hz), 3.73 (t, 2H, J ¼ 5.8 Hz), 3.83 (s, 3H), 4.94
(brs,1H, D2O exchangeable), 6.96 (s,1H), 7.05 (d, 2H, J ¼ 8.4 Hz), 7.10
(brs, 1H, D2O exchangeable), 7.38 (t, 1H, J ¼ 7.5 Hz), 7.60 (t, 1H,
J ¼ 7.5 Hz), 7.82 (d,1H, J ¼ 8.3 Hz), 8.16 (d, 2H, J ¼ 8.4 Hz), 8.21 (d,1H,
J ¼ 8.3); 13C NMR (DMSO, 100 MHz):
d 45.1, 55.2, 59.0, 94.5, 113.7
(2C), 117.9, 121.5, 123.3, 128.4 (2C), 129.0, 129.1, 132.5, 148.2, 150.8,
(0.52 g, 61%). A light yellow solid; 1H NMR (400 MHz, CDCl3):
d 4.23
156.2, 160.0; ESI-MS m/z: 295.2 [M þ 1]þ.
(s, 3H), 4.60 (s, 2H), 5.33 (brs, 1H, D2O exchangeable), 7.19 (s, 1H),
7.35 (d, 2H, J ¼ 7.9 Hz), 7.62e7.55 (m, 1H), 7.84 (dd, 1H, J1 ¼11.4 Hz,
J2 ¼ 4.2 Hz), 7.89 (d, 2H, J ¼ 8.0 Hz), 8.10 (dd, 1H, J1 ¼ 8.4 Hz,
J2 ¼ 1.0 Hz), 8.44 (d, 1H, J ¼ 8.6 Hz); APCI-MS m/z: 266.1 [M þ 1]þ.
4.1.7.2. 3-(2-(4-Methoxyphenyl)quinolin-4-ylamino)propan-1-ol
(10b). A light yellow solid; m.p. 144e145 ꢀC; 1H NMR (CDCl3,

3902
Z.-Y. An et al. / European Journal of Medicinal Chemistry 45 (2010) 3895e3903
4.1.12. 4-(4-Methoxyquinolin-2-yl)benzyl thiophene-2-carboxylate
(15)
was added to each well. The wells were then tested after incubation
for 24 h with or without graveoline derivatives at various concen-
trations. 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.
14 (0.13 g, about 0.5 mmol), EtN3 (0.2 mL) was dissolved in
CH2Cl2 (20 mL) in an ice bath. Thiophene-2-carbonyl chloride
(0.1 mL) was added dropwise. After 30 min at 0 ꢀC, the mixture was
stirred at room temperature overnight and poured into 50 mL of ice
water. The mixture was extracted with AcOEt (3 ꢂ 20 mL), and the
organic layer was dried with MgSO4 and evaporated under vacuum.
The crude residue was purified by silica gel column chromatog-
raphy (eluente pet/AcOEt ¼ 2:1) to afford 15 (73 mg, 38.9%). A light
4.2.4. CAM assay
The in vivo CAM angiogenesis model used was initially described
by Folkman [2] and then modified by Maragoudakis [33e37]. CAM
assay in this paper was carried out by a reported method with slight
modification.
Fertilized chicken eggs were used after incubated for 8 days.
A 1 cm ꢂ 1 cm window was made in the shell to create
a pocket to expose the CAM. A filter disc with 0.9% NaCl solution
yellow solid; m.p. 125e127 ꢀC; 1H NMR (CDCl3, 400 MHz):
d 4.14 (s,
3H), 5.42 (s, 2H), 7.12 (m, 1H), 7.18 (s, 1H), 7.51 (m, 1H), 7.59 (m, 3H),
7.71e7.79 (m, 1H), 7.85e7.86 (m, 1H), 8.15 (d, 2H, J ¼ 8.4 Hz),
8.18e8.21 (m, 2H); ESI-MS m/z: 376.1 [M þ 1]þ.
4.2. Materials and biological assays
or 30 mg/10 mL 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.
Stock solutions of all graveoline and graveolinine 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). The cell culture was maintained in a RPMI-
1640 or DMEM medium supplemented with 10% fetal bovine serum,
Acknowledgements
100 U/mL penicillin, and 100 m
g/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).
We thank the National Nature Science Foundation of China
(20772159, 90813011, U0832005), the NSFC/RGC joint Research
Scheme (30731160006) for financial support of this study.
4.2.1. Methyl thiazolyl tetrazolium (MTT) assay
HUVEC, GLC-82, NCI-H460 and MCF-7 cell lines were seeded on
96-well plates at a concentration of 5000 cells per well and exposed
to various concentrations of graveoline and graveolinine deriva-
tives. After 48 h of treatment at 37 ꢀC in a humidified atmosphere of
References
[1] J. Folkman, Nat. Med. 1 (1995) 27e30.
[2] J. Folkman, Adv. Cancer Res. 43 (1985) 175e203.
5% CO2, 10
mL of 5 mg/mL MTT solution was added to each well and
[3] K. Tahtis, R. Backwell, M. Knowles, P. Delby (Eds.), Tumor Angiogenesis.
Introduction to the Cellular and Molecular Biology of Cancer, Oxford Univer-
sity Press, 2005, pp. 289e305.
[4] D. Ingber, T. Fujita, S. Kishimoto, K. Sudo, T. Kanamaru, H. Brem, J. Folkman,
Nature 348 (1990) 555e557.
[5] T.A. Fong, L.K. Shawver, L. Sun, C. Tang, H. App, T.J. Powell, Y.H. Kim,
R. Schreck, X. Wang, W. Risau, A. Ullrich, K.P. Hirth, G. McMahon, Cancer Res.
59 (1999) 99e106.
further incubated for 4 h. Each well was then added 100
m
L of
dimethyl sulfoxide (DMSO), 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.
[6] M.S. O’Reilly, L. Holmgren, Y. Shing, C. Chen, R.A. Rosenthal, M. Moses, W.
S. Lane, Y. Cao, E.H. Sage, J. Folkman, Cell 79 (1994) 315e328.
[7] M.S. O’Reilly, T. Boehm, Y. Shing, N. Fukai, G. Vasios, W.S. Lane, E. Flynn, J.
R. Birkhead, B.R. Olsen, J. Folkman, Cell 88 (1997) 277e285.
[8] A. Ivanovaa, B. Mikhovaa, H. Najdenskib, I. Tsvetkovab, I. Kostovaa, Fitoterapia
76 (2005) 344e347.
[9] T.S. Wu, L.S. Shi, J.J. Wang, S.C. Iou, H.C. Chang, Y.P. Chen, Y.H. Kuo, Y.L. Chang,
C.M. Teng, J. Chin. Chem. Soc. 50 (2003) 171e178.
[10] E.E. Stashenko, R. Acosta, J.R. ’Mart’ınez, J. Biochem. Biophys. Methods 43
(2000) 379e390.
[11] A. Olive, K.M. Meepagala, D.E. Wedge, D. Harries, A.L. Hale, G. Aliotta, S.
O. Duke, J. Agric. Food Chem. 51 (2000) 890e896.
[12] A.L. Hale, K.M. Meepagala, A. Olive, G. Aliotta, S.O. Duke, J. Agric. Food Chem.
52 (2004) 3345e3349.
[13] T.I. Savchenko, O.V. Silin, S.M. Kovalenko, V.I. Musatov, V.M. Nikitchenko, A.
V. Ivachtchenko, Synth. Commun. 37 (2007) 1321e1330.
[14] Y.H. Ahn, J.S. Lee, Y.T. Chang, J. Am. Chem. Soc. 129 (2007) 4510e4511.
[15] R.G. Fan, D.I. Kwok, G.D. Daves Jr., J. Org. Chem. 57 (1992) 2093e2100.
[16] Y.H. Kim, H. Lee, Y.J. Kim, B.T. Kim, J.N. Heo, J. Org. Chem. 73 (2008) 495e501.
[17] Y.L. Chen, C.J. Huang, Z.Y. Huang, C.H.A. Tseng, F.S. Chang, S.H. Yang, S.R. Lin, C.
C. Tzeng, Bioorg. Med. Chem. 14 (2006) 3098e3105.
[18] I. Sánchez, R. Reches, D.H. Caignard, P. Renard, M.D. Pujol, Eur. J. Med. Chem.
41 (2006) 340e352.
[19] B. Lal, N.B. Bhise, R.M. Gidwani, A.D. Lakdawala, K. Joshi, S. Patvardhan,
Arkivoc (2005) 77e97.
[20] J. Lim, N. Stock, R. Pracitto, J.K. Boueres, B.o Munoz, A. Chaudhary, A.M. Santini,
K. Orr, H. Schaffhauser, R.E. Bezverkov, J. Aiyar, S. Venkatraman, Bioorg. Med.
Chem. Lett. 14 (2004) 1913e1916.
[21] K.E. Andersen, B.F. Lundt, A.S. Jgrgensen, C. Braestrup, Eur. J. Med. Chem. 31
(1996) 417e425.
[22] Q.S. Shen, W. Huang, J.L. Wang, X.G. Zhou, Org. Lett. 9 (2007) 4491e4494.
[23] R.J. D’Amato, M.S. Loughnan, E. Flynn, J. Folkman, Proc. Natl. Acad. Sci. U S A.
91 (1994) 4082e4085.
[24] O. Singh, M. Muthukrishnan, M. Sundaravedivelu, Synth. Commun. 36 (2006)
943e950.
4.2.2. Cell adhesion assay
Cell adhesion assay was carried out by a reported method
[29,30] with slight modification. Wells of a 96-well plate were
coated at room temperature overnight with 2
tendon collagen type I in PBS in a final volume of 100
were washed three times with 100 L of PBS and then blocked
with 0.2% bovine serum albumin (BSA) in 100 L PBS for 2 h at
37 ꢀC cell culture incubator. The wells were then washed three
times with 100 L of PBS. HUEVC were detached from the dish,
resuspended in DMEM, and added (104 cells/ml) 50
L to each well
and added 50 L related compounds at various concentrations.
The plates were incubated for 1 h or 3 h at 37 ꢀC and then washed
three times with 100 L of PBS. The attached cells were fixed and
stained with 0.2% crystal violet in 20% methanol for 10 min, then
washed three times with 100 L of PBS. The cells were solubilized
m
g of Rat trail
mL. The wells
m
m
m
m
m
m
m
by 2% SDS and the OD was measured at 570 nm. Attachment was
quantitated after 1 h or 3 h as described above. Each compound
was tested in triplicate and each experiment was repeated three
times.
4.2.3. Cell migration assay
For detection of cell migration, cells were grown in a pre-treated
48-well culture plate at a concentration of 20 000 cells per well in
a volume of 200 mL, and a portion of the cell monolayer was scraped
away with a sterile disposable rubber policeman [30e32]. After the
DMEM medium was removed, the cell monolayer was washed
three times with phosphate-buffer saline (PBS), and fresh medium
[25] H. Singh, S.K. Aggarwal, N. Malhotra, Tetrahedron 42 (1986) 1139e1144.

Z.-Y. An et al. / European Journal of Medicinal Chemistry 45 (2010) 3895e3903
3903
[26] L. Strekowski, M. Say, M. Henary, P. Ruiz, L. Manzel, D.E. MacFarlane, A.
J. Bojarski, J. Med. Chem. 46 (2003) 1242e1249.
[32] M. Weis, C. Heeschen, A.J. Glassford, J.P. Cooke, Circulation 105 (2002)
739e745.
[27] L. Strekowski, J.L. Mokrosz, V.A. Honkan, A. Czarny, M.T. Cegla, R.L. Wydra, S.
E. Patterson, R.F. Schinazi, J. Med. Chem. 34 (1991) 1739e1746.
[28] K.H. Kumar, P.T. Perumal, J. Heterocycl. Chem. 45 (2008) 597e599.
[29] J. Vukanovic, A. Passaniti, T. Hirata, R.J. Traystman, B.H. Asp, J.T. Isaacs, Cancer
Res. 53 (1993) 1833e1837.
[30] K.M. Malinda, M. Nomizu, M. Chung, M. Delgado, Y. Kuratomi, Y. Yamada, H.
K. Kleinman, M.L. Ponce, FASEB J. 13 (1999) 54e62.
[31] S. Dimmeler, E. Dernbach, A.M. Zeiher, FEBS Lett 477 (2000) 258e262.
[33] M.E. Maragoudakis, M. Panoutsakopoulou, M. Sarmonica, Cell 20 (1988)
531e539.
[34] J.K. Jackson, H.M. Burt, A.M. Oktaba, W. Hunter, M.P. Scheid, F. Mouhajir, R.
w. Lauener, Cancer Chemother. Pharmacol. 42 (1998) 326e332.
[35] H.H. Chen, L.L. You, S.B. Li, Cancer Lett. 211 (2004) 163e173.
[36] S.U. Dani, R. Espindola, Genet. Mol. Res. 2 (2002) 167e175.
[37] M. Richardson, D. Wong, S. Lacroix, J. Stanisz, G. Singh, Cancer Chemother.
Pharmacol. 56 (2005) 1e9.