Cytotoxic 2',5'-dihydroxychalcones with unexpected antiangiogenic activity.

Article abstract of DOI:10.1016/S0223-5234(02)01443-5

A series of 2',5'-dihydroxychalcones were synthesized and evaluated for cytotoxicity against tumor cell lines and human umbilical venous endothelial cells (HUVEC). It was found that chalcones with electron-withdrawing substituents on the B ring exhibited potent cytotoxicity against a variety of tumor cell lines while compounds with electron-releasing groups were less potent in general. Those compounds with B ring replaced by extended or heteroaromatic rings exhibited significant bioactivity. Several compounds were shown to have marked cytotoxic selectivity towards HUVECs. Especially, among the synthesized compounds, 2-chloro-2',5'-dihydroxychalcone (2-3) showed the highest selectivity index up to 66 in comparison to HCT116 cells. This compound also exhibited strong inhibitory effects on the HUVEC tube formation in an in vitro model. When administered into BDF1 mice bearing Lewis lung carcinoma cells at 50 mg kg(-1) day(-1), 2-3 was found to inhibit the growth of tumor mass by 60.5%.

Full text of DOI:10.1016/S0223-5234(02)01443-5

European Journal of Medicinal Chemistry 38 (2003) 179ꢁ187
/
www.elsevier.com/locate/ejmech
Original article
Cytotoxic 2?,5?-dihydroxychalcones with unexpected antiangiogenic
activity
Nguyen-Hai Nam a, Yong Kim a, Young-Jae You a, Dong-Ho Hong a,
Hwan-Mook Kim b, Byung-Zun Ahn a,*
a College of Pharmacy, Chungnam National University, Taejon 305-764, South Korea
b Korea Research Institute of Bioscience and Biotechnology, Taejon 305-600, South Korea
Received 29 April 2002; received in revised form 4 October 2002; accepted 20 November 2002
Abstract
A series of 2?,5?-dihydroxychalcones were synthesized and evaluated for cytotoxicity against tumor cell lines and human umbilical
venous endothelial cells (HUVEC). It was found that chalcones with electron-withdrawing substituents on the B ring exhibited
potent cytotoxicity against a variety of tumor cell lines while compounds with electron-releasing groups were less potent in general.
Those compounds with B ring replaced by extended or heteroaromatic rings exhibited significant bioactivity. Several compounds
were shown to have marked cytotoxic selectivity towards HUVECs. Especially, among the synthesized compounds, 2-chloro-2?,5?-
dihydroxychalcone (2-3) showed the highest selectivity index up to 66 in comparison to HCT116 cells. This compound also exhibited
strong inhibitory effects on the HUVEC tube formation in an in vitro model. When administered into BDF1 mice bearing Lewis
lung carcinoma cells at 50 mg kgꢀ1 dayꢀ1, 2-3 was found to inhibit the growth of tumor mass by 60.5%.
´
# 2003 Editions scientifiques et me´dicales Elsevier SAS. All rights reserved.
Keywords: 2?,5?-Dihydroxychalcone; Cytotoxicity; Antiangiogenic activity; Antitumor activity
1. Introduction
acceptor, an active moiety often employed in the design
of anticancer drugs [4]. A number of a,b-unsaturated
ketones have demonstrated preferential activity toward
thiol [5]. Alkylation with a cellular thiol such as
glutathione (GSH) may also occur with chalcones like
1, leading to adducts at b-position. Hence, a,b-unsatu-
rated carbonyl-containing compounds may be free from
problems of mutagenicity and carcinogenicity that are
associated with many alkylating agents used in cancer
chemotherapy [6].
Chemically, the alkylation of a,b-unsaturated carbon-
yl compounds like 1 with cellular thiols and other
nucleophiles is greatly dependent on the electrophilicity
of the b-carbon. In this view, we reason that the
introduction of electron-withdrawing groups on B ring
may increase the electrophilicity of the b-carbon, thus
improve the bioactivity of the resulting compounds.
However, our initial results showed that such com-
pounds suffered unexpected chemical instability. In
addition, it is difficult to synthesize such compounds
with diverse substituted patterns in B ring. Therefore,
we sought another solution by reallocation of the 2,5-
In a previous paper we have reported a series of 2,5-
dihydroxychalcones (1) (Fig. 1) with potent cytotoxic
activities in various tumor cell lines [1]. We also noted
that some of them showed modest selectivity towards
human umbilical venous endothelial cells (HUVEC).
Those compounds with selective cytotoxicity against
HUVECs may be potential for development as selective
angiogenesis inhibitors. Indeed, antiangiogenesis has
been widely accepted as a novel and interesting
approach for anticancer drug development recently [2].
Structurally, 1 possesses two distinguished features; a
hydroquinone and an a,b-unsaturated carbonyl. The
hydroquinone has been known to be easily oxidised in
vivo to quinone which elicits cytotoxicity per se or
through a cascade of redox cycling [3]. The a,b-
unsaturated carbonyl can be considered as a Michael
* Corresponding author.
E-mail address: ahnbj@cnu.ac.kr (B.-Z. Ahn).
´
0223-5234/03/$ - see front matter # 2003 Editions scientifiques et me´dicales Elsevier SAS. All rights reserved.
doi:10.1016/S0223-5234(02)01443-5

180
N.-H. Nam et al. / European Journal of Medicinal Chemistry 38 (2003) 179ꢁ187
/
Fig. 1. Structures of 2,5-dihydroxychalcones (1) and 2?,5?-dihydroxychalcones (2).
dihydroxy functionality into ring A, and introduction of
various electron-withdrawing groups into ring B. Thus,
at the same time we could both conserve the important
hydroquinone moiety and make the b-carbon more
electron-deficient. The resulting compounds 2 are ex-
pected to be more bioactive than 1. In this work we have
synthesized and evaluated a series of compounds 2 for
cytotoxicity in both tumor cell lines and HUVEC. And
interestingly, we found compound 2-3 (2-chloro-2,?-5?-
dihydroxychalcone) with unexpectedly large cytotoxic
selectivity against HUVECs. In this paper, the synthesis
of the chalcones in series 2, their cytotoxicity and
detailed biological evaluation of compound 2-3 as an
antiangiogenic antitumor agent are presented and dis-
cussed.
tively, this compound was obtained by condensation
under acidic conditions, thus involving bubbling of
hydrochloride gas into a solution of the two reactants
in anhydrous ethanol. 2-25 in particular was synthesized
by condensation of 3 with cinnamaldehyde followed by
deprotection as described for 2-1. 1H-NMR spectra
revealed that the olefinic bond in these chalcones
adopted the E configuration (the coupling constants of
the two olefinic protons were around 16 Hz).
3. Biological results and discussion
3.1. Cytotoxicity
The synthesized chalcones were evaluated for cyto-
toxic activities in three cancer cell lines including B16
(murine melanoma), HCT116 (human colon cancer
cells), and A431 (human epidermoid carcinoma). As
already widely known, the formation of new blood
vessels from endothelial cells, so-called angiogenesis, is
prerequisite for the growth of solid tumors [7]. Inhibi-
tion of angiogenesis has been postulated to be an
attractive approach for cancer treatment [2] and a
compound showing a selective cytotoxicity against
endothelial cells may be potential for further develop-
ment as an angiogenesis inhibitor. For that reason,
2. Chemistry
Most of chalcones were synthesized by a Claisenꢁ
/
Schmidt condensation of appropriate benzaldehydes,
protected as tetrahydropyranyl ether wherever a phenol
group(s) was present, with 2?,5?-dihydroxyacetophe-
none, also protected as bistetrahydropyranyl ether (3)
(Fig. 2), using barium hydroxide octahydrate as base.
However, these conditions failed to give chalcone 2-7
which possesses a nitro group on the A ring. Alterna-
Fig. 2. (a) Pyridinium p-toluene sulfonate, 3,4-dihydro-2H-pyran, methylene chloride, r.t., 5 h. (b) ArCHO, Ba(OH)2×
(c) p-Toluene sulfonic acid, MeOH, 5 h. (d) HCl gas, ethanol, 4-NO2 ꢁC6H4CHO.
/
8H2O, methanol, 40 8C, 12 h.
/

N.-H. Nam et al. / European Journal of Medicinal Chemistry 38 (2003) 179ꢁ
/187
181
HUVEC, a representative line of endothelial cells, was
included in an assay panel in an attempt to unveil a
selective cytotoxicity against this cell line of the com-
pounds tested. The results expressed as IC50 values are
summarized in Table 1. For comparison of the potency
between compounds within the series, the average
potency (average IC50 values) of each compound in
four cell lines were calculated (see Section 4). The data
are included in Table 1.
Furthermore, to compare the potency of compounds
in series 2 with series 1, the average potencies of the
series 2 in each cell line and in all four cell lines are also
calculated. The results are presented in Table 2. The
data for series 1 shown in this table are adopted from
Ref. [1].
Table 2
Average potency (AP) of series 1 and 2 and of each category a in series
2
Series Category
Average Potency (AP)
B16 HCT116
A431 HUVEC
AP
2
1
2-EWG
2-MIX
2-ERG
2-HET
AP
1.87 2.42
1.33 1.39
5.23 9.15
1.68 3.00
2.52 3.99
4.47 5.62
1.84
1.49
7.81
2.13
3.31
5.13
1.49
1.27
3.49
1.68
1.98
4.52
1.90
1.36
6.12
2.12
2.87
4.90
b
AP
a
Category is classified as described in the text.
Data adopted from Ref. [1].
b
one ERG (2-10ꢀ12) were pooled in a 2-MIX group.
/
To discern the electronic effects of the substituents on
the bioactivity, we divided compounds in series 2 into
four groups. Group 2-EWG comprises of compounds
The last group (2-HET) houses compounds where the B
rings were replaced with extended or heteroaromatic
ones. The AP values of each group were calculated and
included in Table 2.
As shown in Table 1, most of the synthesized
compounds in series 2 exhibited significant cytotoxici-
(2-1ꢀ
(EWG), meanwhile group 2-ERG accommodates com-
pounds (2-13ꢀ19) possessing electron-releasing substi-
/
9) bearing electron-withdrawing substituents
/
tuents (ERG). Three compounds with one EWG and
Table 1
Cytotoxicity of synthesized chalcones in tumor cell lines and HUVECs
a
Cpd
R or A ring for
Cytotoxicity (IC50 b, mg mLꢀ1
)
Cell line
B16
AP
HCT116
A431
HUVEC
2-1
2-2
H
2-F
2.25
1.78
1.34
1.39
2.18
1.84
1.99
2.15
1.99
1.10
1.01
1.87
3.59
11.25
1.00
2.90
10.16
3.77
3.84
1.20
1.36
2.21
2.00
1.97
1.39
0.10
3.29
1.99
1.83
1.81
3.62
2.36
2.38
2.72
1.84
1.18
1.25
1.75
9.09
2.44
1.11
0.09
1.97
2.16
2.54
2.56
1.81
1.84
1.29
1.73
1.49
3.75
13.33
2.58
6.27
26.65
8.06
7.81
2.30
1.97
1.71
2.18
2.37
2.27
1.01
1.89
1.02
0.03
1.71
2.01
1.52
2.01
1.84
1.42
1.24
1.24
1.27
5.48
2.46
1.47
0.82
1.72
2.49
2.06
2.23
2.13
1.77
1.20
1.30
1.59
5.47
2-3
2-4
2-Cl
3-Cl
2-5
2-6
4-Cl
4-Br
2-7
2-8
4-NO2
2,3,4,5,6-(F)5
2,5-(F)2
2-9
2-10
2-11
2-12
2-13
2-14
2-15
2-16
2-17
2-18
2-19
2-20
2-21
2-22
2-23
2-24
2-25
ADR
2-Brꢁ
2-OCH3 ꢁ
2-Brꢁ5-OH
/
5-OCH3
/5-Br
/
4-OCH3
4-N(CH3)2
2,3-(OH)2
3,4-(OH)2
2,5-(OH)2
2,3,4-(OCH3)3
3,4,5-(OCH3)3
4-phenyl
ꢁ/
30
ꢁ/
30
ꢁ/
21.14
2.58
6.47
14.01
6.93
6.12
2.14
2.04
2.16
2.16
2.33
1.92
0.60
5.26
1.48
13.73
14.46
12.17
9.35
3.93
3.57
2.69
2.57
2.49
2.79
1.11
3.00
4.78
3.75
3.49
1.15
1.28
2.04
1.92
2.49
1.24
0.21
2-naphtyl
pyridin-3-yl
indol-3-yl
quinolin-2-yl
CHꢀCHPh-(t)
/
c
a
Cancer cell lines: B16, murine melanoma; HCT116, colon cancer; A431, human epidermoid carcinoma.
A sample’s concentration produces a 50% reduction in cell growth. The values shown were the averages from a triplicate experiment with the
b
individual value varied less than 10%.
c
Adriamycin, used as a positive control.

182
N.-H. Nam et al. / European Journal of Medicinal Chemistry 38 (2003) 179ꢁ187
/
ties. Noteworthy, a review of the AP values of the two
series 1 and 2 shows that compounds in series 2 are in
average twofold more potent than those in 1 (the AP
2-3 might inhibit A431 and HUVEC growth by antag-
onizing EGFs. Whatever an exact mechanism would be,
as stressed earlier, compounds displaying selective
cytotoxicity towards HUVEC, like 2-3, may be potential
for further development as angiogenesis inhibitor.
Therefore, 2-3 was chosen as a candidate for evaluation
in an in vitro angiogenesis assay using HUVEC. Because
a 24 h incubation was used in this assay model, an IC50
value for a 24 h incubation of 2-3 was determined (0.45
mg mLꢀ1) using SRB assay. Accordingly, 2-3 was
assayed at four concentrations of 0.3, 0.1, 0.03 and
0.01 mg mLꢀ1. As a positive inhibitory control, suramin,
an established angiogenesis inhibitor, was also added at
non-growth inhibitory concentrations of 100, 30, 10 mM.
For suramin, an IC50 value for a 24 h incubation using
values of 2 and 1 are 2.87 and 4.90 mg mLꢀ1
,
respectively. For example, 2-3 showed average IC50
value of 0.82 versus 1.32 mg mLꢀ1 of the corresponding
compound 1-3 which bears a 2-Cl substituent on the A
ring [1]. Thus, though we have not yet confirmed by
calculation of the atomic charges of the b-carbons of the
two series, these results strongly support our earlier
hypothesis that by reallocation of the 2,5-dihydroxy into
A ring and introduction of the EWGs into B ring, the
series 2 would be expected to be more active than 1.
Perusal of the data summarized in Table 2 indicates
that compounds with EWGs (2-1ꢀ9, group 2-EWG)
/
showed most potent cytotoxicities while compounds
with ERGs (2-13, -19) were much less active (the AP
values of 2-EWG and 2-ERG are 1.90 and 6.12 mg
mLꢀ1, respectively). These results clearly prove the
favorable effects of the EWGs for the bioactivity.
However, it is unclear that compounds possess mixed
SRB assays was ꢁ100 mM. The results from these
/
assays are summarized in Table 3.
As shown in Table 3, the addition of 2-3 at the
concentrations of 0.1 and 0.3 mg mLꢀ1 resulted in the
complete inhibition of the HUVEC tube formation.
Though signs of cytotoxicity were observed at 0.3 mg
mLꢀ1, the lower concentration of 0.1 mg mLꢀ1
manifested no cytotoxicity while the complete inhibition
was still observed. This concentration was 4.5-fold lower
than the IC50 value of the compound against HUVECs
measured under the same conditions, e.g. cell concen-
substituents (2-10ꢀ
where the B ring has been replaced by extended
aromatic or heteroaromatic rings (2-20ꢀ25, 2-HET
/12, 2-MIX group) and compounds
/
group) also exhibited good activities. For the case of 2-
HET group, it is possible that the extended aromatic
rings or the heteroatoms may increase the binding of
those compounds to appropriate receptors through a
van der Waals force or hydrogen bonding.
tration (8ꢂ
104 cells/mL), and time course (24 h).
/
Compared to the IC50 values of the compound in tumor
cell lines, it was revealed that this concentration was
more than tenfold lower than the average cytotoxicity
(AVR-C) in three tumor cell lines (B16, HCT116, A431).
Especially, at 0.03 mg mLꢀ1, the concentration 36-fold
lower than the AVR-C value, 2-3 considerably inhibited
the tube formation induced by HUVEC. The inhibition
of HUVEC tube formation at this concentration was
comparable to that of 10 mM, or 15 mg mLꢀ1 of
suramin, one established angiogenesis inhibitor cur-
rently in some clinical uses. Collectively, these results
suggest that 2-3 is a cytotoxic agent (IC50 values in
tumor cell lines in the range of 1 mg mLꢀ1) as well as a
3.2. Antiangiogenic activity of 2-3
Interestingly, the results of the screening of chalcones
synthesized in this work in HUVECs revealed several
compounds with significant preferential cytotoxicity
toward HUVEC, in reference to tumor cells. In order
to get a quick view of the compound’s cytotoxic
selectivity, a selectivity index (SI), which refers to the
cytotoxic selectivity of the compound towards HUVEC
in comparison to three tumor cell lines, was calculated
(see Section 4). As a result we found nine out of 25
compounds synthesized, including 2-2, -3, -15, -16, -17, -
18, -19, -20, -25 displayed SI values larger than 2 (data
not shown). Strikingly, compound 2-3 in this series was
revealed to have distinguished SI value of 36, respec-
tively. Noteworthy, 2-3 also showed significant selectiv-
ity in human epidermoid carcinoma cells (A431) with
the IC50 value in this cell line being 15- and 20-fold
lower than that in B16 and HCT116, respectively.
Biologically, the two cell lines A431 and HUVEC share
a common property. The growth of A431 could be
stimulated by three to fourfold in the presence of
epidermal growth factors (EGF) meanwhile HUVEC’s
growth is completely dependent on growth factors like
EGF, PDGF (platelet-derived growth factors), and
FGF (fibroblast growth factors). Thus, it is likely that
Table 3
Summary of the inhibitory effects of 2-3 on in vitro HUVEC assay
2-3
Suramin
a
a
Concentration
(mg mLꢀ1
Activity
Concentration (mM)
Activity
)
b
0.3 (1.10)
0.1 (0.37)
ꢃ
/
ꢃ
/
ꢃ
/
/
100
30
10
3
ꢃ
/
ꢃ
/
ꢃ
/
/
ꢃ
/
ꢃ
/ꢃ
ꢃ
/
ꢃ
/ꢃ
0.03 (0.11)
0.01 (0.04)
ꢃ
/
ꢃ
/
ꢃ
/
ꢃ
/
ꢃ/
ꢀ/
a
The activity was evaluated in a double-blind manner and designed
, inactive; ꢃ, slight; ꢃ , moderate; ꢃ , strong to
complete inhibition.
as follows: ꢀ
/
/
/
ꢃ
/
/
ꢃ
/
ꢃ
/
b
Numbers in parenthesis show the corresponding concentration in
mM.

N.-H. Nam et al. / European Journal of Medicinal Chemistry 38 (2003) 179ꢁ
/187
183
selective angiogenesis inhibitor with large selectivity
index (ꢁ36). In the in vitro model, 2-3 was shown to
be more potent than suramin.
at 50 mg kgꢀ1 dayꢀ1 with the same injection schedule.
It was interesting to observe that, on day 15, 2-3 was
found to be less potent than 2-3; the IR value of 2-4 was
47.3%, lower than that (60.5%) of 2-3. In vitro, the two
compounds were almost equipotent towards tumor cell
lines (IC50 values of 1.34 and 1.39 mg mLꢀ1 in B16 cells,
for instance), however, 2-4 did not show any selective
angiogenesis inhibitory activity meanwhile 2-3 was
shown to be a strong angiogenesis inhibitor. Thus, the
antiangiogenic activity of 2-3 was likely one mechanism
of its in vivo antitumor activity. It was noted that the
inhibition rate of 2-3 on the growth of Lewis lung
carcinoma cells in BDF1 mice was highest on day 11
(72.6%), the last day of the compound injection. After
the interruption of the compound injection, the tumors
continued to growth. On day 16, the IR value decreased
to just 45.9%. Similar phenomena have been documen-
ted for other angiogenesis inhibitors like TNP-470,
endostatin, and thalidomide [8]. And for angiogenesis
inhibitors, continual administration of drugs has been
reported to be required to effect regression of tumors.
From these perspectives, the tumor masses in mice
treated with 2-3 were challenged with a second round
of treatment with three additional injections on days 16,
17, and 18. On day 19, a significant regression of tumors
in this group was achieved with the inhibition rate
recorded as 57.4%. Meanwhile, a similar supplement of
2-4 did not cause any shrinkage of tumor masses in the
group treated with 2-4 (Fig. 3), indicating that tumor
cells in this group had become refractory to this agent.
In summary, we have synthesized a series of 2?,5?-
dihydroxychalcones (2) and found that most of com-
pounds in this series showed potent cytotoxicity against
tumor cell lines. In overall, the series 2 was found to be
twofold more potent than series 1. The electron-with-
/
3.3. Antitumor activity of 2-3
The chalcones prepared in this work showed con-
siderable cytotoxicity in tumor cell lines, and compound
2-3 was found to selectively inhibit angiogenesis in the in
vitro model. To examine whether these chalcones exert
in vivo antitumor activity, one representative com-
pound, 2-3, was chosen for in vivo experiments in
BDF1 mice bearing Lewis lung carcinoma cells. The
compound was evaluated at the maximum tolerated
dose of 50 mg kgꢀ1 dayꢀ1. The administration of the
compound was scheduled to include 6 injections, from
day 1 after tumor cell transplantation and continued
every two days until day 11. The body weights were
recorded and body weight changes were calculated. The
results are included in Table 4. Tumor volumes were
tracked from day 11 and the inhibition rates, calculated
as described in the Section 4, are shown in Table 4.
Etoposide, one clinical anticancer agent, was dosed to
one group of mice at 36 mg kgꢀ1 dayꢀ1 on days 1, 5
and 9, and used as a positive control. The results shown
in Table 4 demonstrate that 2-3 showed efficacy in this
model. On day 15, the tumor masses in the 2-3 treated
group were inhibited by 60.5% compared to the vehicle
treated control. However, this inhibition rate was still
lower than that of etoposide (73.6% on day 15). To
ascertain whether the antiangiogenic activity of this
compound contributed to its in vivo antitumor activity,
an indirect approach was used. Compound 2-4, structu-
rally similar to 2-3, thus their pharmacokinetics would
be expected to be similar, was experimented in parallel
Table 4
Antitumor activity of 2-3 and 2-4
Cpd
R
Cytotoxicity (IC50 a, mg mLꢀ1
)
Dose (mg kgꢀ1 dayꢀ1
)
Antitumor activity
b
c
d
B16
1.34
AVR-C
1.09
HUVEC
0.03
BWC (%)
IR
2-3
2-Cl
50
72.6
60.5
45.9
57.4
51.2
47.3
39.7
37.5
73.6
(day 11)
(day 15)
(day 16)
(day 19)
(day 11)
(day 15)
(day 16)
(day 19)
(day 15)
ꢃ/10.8
2-4
3-Cl
1.39
0.10
1.72
1.11
1.71
0.21
50
ꢃ
/
5.9
e
ETP
36
0
a
b
c
A sample’s concentration produces a 50% reduction in cell growth. The values shown were the averages from a triplicate experiment.
Average IC50 values in three cell lines B16, A431 and HCT116.
Body weight change.
d
Inhibition rate of tumor growth in BDF1 mice bearing Lewis lung carcinoma cells (see Section 4 for calculation method); 2-3 and 2-4 were
injected every 2 days from day 1 after tumor cell transplantation until day 11; a second round of three doses were supplemented every day from day
16.
e
Etoposide, a positive control, was dosed on days 1, 5 and 9.

184
N.-H. Nam et al. / European Journal of Medicinal Chemistry 38 (2003) 179ꢁ187
/
Fig. 3. Tumor volumes in 2-3- and 2-4-treated mice. The data plotted as averages9S.D. The tumor volumes of the treated groups were significantly
/
(p B0.05) lower than the negative control (vehicle) group. ", vehicle group; m, 2-4 treated group; ', 2-3 treated group. Each group of ten mice was
/
used. Samples were injected at 50 mg kgꢀ1 dayꢀ1 every other day from day 1 after tumor cell transplantation until day 11; a second round of three
doses were supplemented every day from day 16.
drawing groups proved to be favorable for bioactivity.
A number of compounds showed significant cytotoxic
selectivity towards HUVE cells. Especially, compound
2-3 (2-chloro-2?,5?-dihydroxychalcone) was revealed to
be a strong angiogenesis inhibitor with marked anti-
tumor activity. Through the experimental results, the
antiangiogenic property of 2-3 was suggested to play a
role in its antitumor activity.
noted. Analytical thin layer chromatography was per-
formed on a plastic sheet (0.2 mm) precoated with silica
gel 60 F254 (E. Merck). Silica gel 60 (70-230 mesh, E.
Merck) was used for column chromatography.
4.1.1. (E)-1-(2,5-Dihydroxyphenyl)-3-phenyl-2-
propene-1-one (2-1)
2?,5?-Dihydroxyacetophenone (3.8 g, 25 mmol) and
pyridinium p-toluene sulfonate (0.15 g, 0.6 mmol) were
stirred at room temperature (r.t.) for 0.5 h in methylene
chloride (100 mL), and then 3,4-dihydro-2H-pyran
(13 mL) in methylene chloride (20 mL) was added
dropwise. The reaction mixture was stirred at r.t) for
4 h, then washed twice with water, dried and evaporated
in vacuo. The residue yielded crude 2?,5?-bis(tetrahydro-
pyran-2-yloxy)acetophenone (3).
Crude 3, benzaldehyde (2.6 g, 25 mmol) and barium
hydroxide octahydrate (8.15 g, 25 mmol) were dissolved
in methanol (100 mL). The reaction mixture was stirred
for 12 h at 40 8C and then evaporated in vacuo. Water
(100 mL) was added and the mixture was neutralized
with HCl (1M, 35 mL) and extracted with methylene
chloride. The organic layer was separated, washed with
water, dried and evaporated in vacuo. The residue
yielded crude (E)-1-[2,5-bis(tetrahydropyran-2-yloxy)-
phenyl]-3-phenyl-2-propene-1-one (4).
4. Experimental protocols
4.1. Chemistry
Chemicals and solvents were reagent grade and used
without further purification. Tumor cell lines were
obtained from a cancer cell bank at the Korea Research
Institute of Bioscience and Biotechnology (KRIBB).
Media, Sera and other reagents used for cell culture were
purchased from GIBCO Co. Ltd. Melting points were
determined on an electrothermal melting point appara-
tus and are uncorrected. IR spectra were recorded on a
Jasco Report-100 IR spectrometer. 1H NMR spectra
were recorded on Varian-Gemini 300 or 90 MHz
spectrometers using tetramethylsilane as internal stan-
dard. Mass spectra were determined with a JEOL JMS-
D-100 mass spectrometer. Elemental analyses were
Crude 4 and p-toluenesulfonic acid (0.2 g, 1.05 mmol)
were dissolved in methanol (100 mL). The reaction
within90.4% of the theoretical values, unless otherwise
/

N.-H. Nam et al. / European Journal of Medicinal Chemistry 38 (2003) 179ꢁ
/187
185
mixture was stirred for 4 h at r.t., and then evaporated
in vacuo. Water (100 mL) was added and the resulting
mixture was neutralized with 5% NaHCO3 (50 mL), and
extracted with ethyl acetate. The organic layer was
separated, washed with water, dried and concentrated in
vacuo. The residue was purified on a silica gel column
4.1.7. (E)-1-(2,5-dihydroxyphenyl)-3-(4-nitrophenyl)-
2-propene-1-one (2-7)
A 100 mL two-neck flask was charged with 1.52 g of
2?,5?-dihydroxyacetophenone (10 mmol), 1.51 g of 4-
nitrobenzaldehyde (10 mmol) dissolved in 30 mL of
ethanol. Dry HCl gas was bubbled into the reaction
mixture for 3 h. The mixture was kept stirring for a
further 9 h and ethanol was removed. The residue was
dissolved in water and extracted with methylene chloride
twice (50 mL each). The methylene chlororide layers
were combined and concentrated in vacuo. The crude
product was purified on a silica gel column eluting with
gradient ethyl acetate in hexane to give a reddish solid;
eluted with hexaneꢁ
65%) as pale yellow solids; IR (KBr) 3400, 1670 cmꢀ1
1H-NMR (DMSO-d6, 90 MHz) d 11.89 (1H, s), 9.58
(1H, s), 7.98 (1H, d, Jꢄ15.12 Hz), 7.71ꢁ7.32 (5H, m),
7.15 (1H, d, Jꢄ2.12 Hz), 7.07ꢁ6.91 (2H, m), 6.79 (1H,
d, Jꢄ15.10 Hz).
/ethyl acetate (4:1) to give 2-1 (4.2 g,
;
/
/
/
/
/
The following chalcones were synthesized by the same
procedure described above unless otherwise noted.
yield 70%; IR (KBr) 3350, 1650 cmꢀ1
(DMSO-d6, 300 MHz) d 12.10 (1H, s), 10.12 (1H, s),
8.02 (H-b, d, Jꢄ 16.00 Hz), 7.91 (H-a, d, Jꢄ16.00 Hz),
7.65ꢁ7.84 (H-2, H-6, m), 7.27ꢁ7.45 (H-6?, H-3, H-5, m),
7.07 (H-4?, dd, Jꢄ2.50, 8.41 Hz), 6.85 (H-3?, d, Jꢄ7.89
Hz).
;
1H-NMR
/
/
4.1.2. (E)-1-(2,5-Dihydroxyphenyl)-3-(2-
fluorophenyl)-2-propene-1-one (2-2)
Red crystals; yield 81%; 1H-NMR (DMSO-d6, 90
/
/
/
/
MHz) d 12.00 (1H, s), 10.18 (1H, s), 8.11 (1H, s, Jꢄ
15.21 Hz), 7.42 (1H, s, Jꢄ15.21 Hz), 7.32ꢁ6.91 (4H, m),
6.88ꢁ6.73 (3H, m).
/
/
/
4.1.8. (E)-1-(2,5-Dihydroxyphenyl)-3-(2,3,4,5,6-
pentafluorophenyl)-2-propene-1-one (2-8)
Red crystals; yield 78%; IR (KBr) 3350, 1670 cmꢀ1
/
;
4.1.3. (E)-3-(2-Chlorophenyl)-1-(2,5-
dihydroxyphenyl)-2-propene-1-one (2-3)
Red crystals; yield 76%; 1H-NMR (DMSO-d6, 90
1H-NMR (DMSO-d6, 90 MHz) d 11.03 (1H, s), 9.22
(1H, s), 7.88 (1H, d, Jꢄ15.93 Hz), 7.50 (1H, d, Jꢄ
15.93 Hz), 7.18 (1H, d, Jꢄ
2.70, 8.82 Hz), 6.82 (1H, d, Jꢄ
/
/
/
2.70 Hz), 7.02 (1H, dd, Jꢄ
/
MHz) d 12.27 (1H, s), 9.98 (1H, s), 8.08 (1H, s, Jꢄ
14.39 Hz), 7.45 (1H, s, Jꢄ14.42 Hz), 7.27ꢁ7.11 (4H, m),
6.85ꢁ6.73 (2H, m), 6.70 (1H, d, Jꢄ8.21 Hz).
/
/8.82 Hz).
/
/
/
/
4.1.9. (E)-3-(2,5-Difluorophenyl)-1-(2,5-
dihydroxyphenyl)-2-propene-1-one (2-9)
Red crystals; yield 57%; IR (KBr) 3520, 1680 cmꢀ1
;
4.1.4. (E)-3-(3-Chlorophenyl)-1-(2,5-
dihydroxyphenyl)-2-propene-1-one (2-4)
Red crystals; yield 59%; 1H-NMR (DMSO-d6, 90
1H-NMR (DMSO-d6, 90 MHz) d 12.17 (1H, s), 10.27
(1H, s), 8.12 (H, d, Jꢄ
Hz), 7.87 (H, d, Jꢄ 15.38 Hz), 7.55ꢁ
6.99 (3H, m).
/
15.38 Hz), 7.96 (H, d, Jꢄ 2.24
/
MHz) d 11.79 (1H, s), 9.37 (1H, s), 8.01 (1H, s, Jꢄ
15.87 Hz), 7.41 (1H, s, Jꢄ15.87 Hz), 7.31 (1H, d, Jꢄ
7.89 Hz), 7.29ꢁ7.02 (2H, m), 6.98 (1H, d, Jꢄ8.12 Hz),
6.86ꢁ6.71 (2H, m).
/
/
/
7.41 (2H, m), 7.46ꢁ
/
/
/
/
/
/
4.1.10. (E)-3-[(2-Bromo-5-methoxy)phenyl]-1-(2,5-
dihydroxyphenyl)-2-propene-1-one (2-10)
4.1.5. (E)-3-(4-Chlorophenyl)-1-(2,5-
dihydroxyphenyl)-2-propene-1-one (2-5)
Red crystals; yield 81%; IR (KBr) 3420, 1670 cmꢀ1
;
1H-NMR (DMSO-d6, 90 MHz) d 12.41 (1H, s), 10.06
Red crystals; yield 65%; IR (KBr) 3360, 1670 cmꢀ1
;
(1H, s), 8.01 (H, d, Jꢄ
/
16.15 Hz), 7.75ꢁ7.56 (3H, m),
/
1H-NMR (DMSO-d6, 300 MHz) d 11.98 (1H, s), 9.89
(1H, s), 7.85 (1H-b, d, Jꢄ 16.3 Hz), 7.78 (1H-a, d, Jꢄ
15.3 Hz), 7.75ꢁ7.71 (H-2, H-6, m), 7.48ꢁ7.42 (H-6?, H-
3, H-5, m), 7.05 (H-4?, dd, Jꢄ8.12 Hz), 6.84 (H-3?, d,
Jꢄ8.12 Hz).
7.27ꢁ6.85 (5H, m), 3.91 (3H, s).
/
/
/
/
/
4.1.11. (E)-3-[(5-Bromo-2-methoxy)phenyl]-1-(2,5-
dihydroxyphenyl)-2-propene-1-one (2-11)
/
Red crystals; yield 69%; IR (KBr) 3400, 1680 cmꢀ1
;
/
1H-NMR (DMSO-d6, 90 MHz) d 12.07 (1H, s), 10.06
(1H, s), 8.02 (H, d, Jꢄ
Hz), 7.83 (H, d, Jꢄ 15.75 Hz), 7.71ꢁ
6.85 (4H, m), 3.98 (3H, s).
/
15.75 Hz), 7.91 (H, d, Jꢄ 2.43
/
4.1.6. (E)-3-(4-Bromophenyl)-1-(2,5-
dihydroxyphenyl)-2-propene-1-one (2-6)
Red crystals; yield 43%; IR (KBr) 3400, 1680 cmꢀ1
/
/
7.59 (1H, m), 7.35ꢁ
/
;
1H-NMR (DMSO-d6, 300 MHz) d 12.41 (1H, s), 10.27
(1H, s), 8.10 (1H-b, d, Jꢄ
16.10 Hz), 7.91ꢁ7.73 (H-2, H-6, m), 7.42ꢁ
3, H-5, m), 7.12 (H-4?, dd, Jꢄ2.50, 8.41 Hz), 6.83 (H-3?,
d, Jꢄ8.41 Hz).
/
16.10 Hz), 7.93 (1H-a, d, Jꢄ
/
4.1.12. (E)-3-[(5-Bromo-2-hydroxy)phenyl]-1-(2,5-
dihydroxyphenyl)-2-propene-1-one (2-12)
/
/
7.31 (H-6?, H-
/
Red crystals; yield 48%; IR (KBr) 3340, 1640 cmꢀ1
;
/
1H-NMR (DMSO-d6, 90 MHz) d 12.31 (1H, s), 10.12

186
N.-H. Nam et al. / European Journal of Medicinal Chemistry 38 (2003) 179ꢁ187
/
(1H, s), 9.21 (1H, s), 8.07 (1H, d, Jꢄ
/
14.57 Hz), 7.63ꢁ
/
4.1.19. (E)-1-(2,5-Dihydroxyphenyl)-3-(3,4,5-
trimethoxyphenyl)-2-propene-1-one (2-19)
7.25 (3H, m), 7.05ꢁ6.75 (4H, m).
/
Yellow crystals; yield 65%; m.p. 188ꢁ
(KBr) 3360, 1680 cmꢀ1 1H-NMR (DMSO-d6, 90
MHz) d 11.93 (1H, s), 9.23 (1H, s), 8.00ꢁ7.54 (3H,
m), 7.33 (2H, s), 7.04 (1H, d, Jꢄ2.75 Hz), 6.88 (1H, d,
Jꢄ8.84 Hz), 3.86 (6H, s), 3.81 (3H, s).
/
191 8C; IR
;
4.1.13. (E)-1-(2,5-Dihydroxyphenyl)-3-(4-
methoxyphenyl)-2-propene-1-one (2-13)
/
/
Yellow crystals; yield 83%; IR (KBr) 3380, 1670
1
/
cmꢀ1; H-NMR (DMSO-d6, 90 MHz) d 12.06 (1H, s),
9.28 (1H, s), 8.02ꢁ
Hz), 7.18ꢁ7.09 (3H, m), 6.93 (1H, d, Jꢄ
(3H, s).
/
7.90 (4H, m), 7.63 (1H, d, Jꢄ
/
2.70
8.82 Hz), 3.88
4.1.20. (E)-1-(2,5-Dihydroxyphenyl)-3-(4-
phenylphenyl)-2-propene-1-one (2-20)
/
/
Yellow crystals; yield 68%; m.p. 147ꢁ
(KBr) 3400, 1680 cmꢀ1 1H-NMR (DMSO-d6, 90
MHz) d 11.97 (1H, s), 10.01 (1H, s), 7.91 (1H, d, Jꢄ
15.24 Hz), 7.51ꢁ22 (10H, m), 7.13ꢁ6.84 (3H, m).
/
150 8C; IR
;
4.1.14. (E)-1-(2,5-Dihydroxyphenyl)-3-(4-
/
dimethylaminophenyl)-2-propene-1-one (2-14)
/
/
Yellow crystals; yield 61%; m.p. 147ꢁ
(KBr) 3350, 1670 cmꢀ1 1H-NMR (DMSO-d6, 90
MHz) d 12.35 (1H, s), 9.16 (1H, s), 7.82 (1H, d, Jꢄ
14.49 Hz), 7.72 (2H, d, Jꢄ8.45 Hz), 7.53 (1H, d, Jꢄ
2.75 Hz), 7.00 (1H, dd, Jꢄ2.75, 8.81 Hz), 6.86 (1H, dd,
Jꢄ15.00 Hz), 6.78 (2H, dd, Jꢄ8.45 Hz), 6.69 (1H, dd,
Jꢄ8.81 Hz), 3.00 (6H, s).
/
149 8C; IR
;
4.1.21. (E)-1-(2,5-Dihydroxyphenyl)-3-(2-naphthyl)-2-
propene-1-one (2-21)
/
/
/
Yellow crystals; yield 45%; m.p. 235ꢁ
(KBr) 3380, 1660 cmꢀ1 1H-NMR (DMSO-d6, 90
MHz) d 8.26 (1H, s), 8.11 (1H, d, Jꢄ15.51 Hz),
7.65ꢁ8.01 (4H, m), 7.57 (1H, d, Jꢄ16.00 Hz), 7.52ꢁ
7.13 (2H, m), 6.77ꢁ6.89 (3H, m).
/
237 8C; IR
/
;
/
/
/
/
/
/
/
/
4.1.15. (E)-1-(2,5-Dihydroxyphenyl)-3-(2,3-
dihydroxyphenyl)-2-propene-1-one (2-15)
4.1.22. (E)-1-(2,5-Dihydroxyphenyl)-3-(pyridin-3-yl)-
2-propene-1-one (2-22)
Yellow crystals; yield 55%; m.p. 145ꢁ
(KBr) 3380, 1670 cmꢀ1 1H-NMR (DMSO-d6, 90
MHz) d 12.21 (1H, s), 9.32 (1H, s), 8.87 (2H, br), 7.89
(1H, d, Jꢄ14.51 Hz), 7.32ꢁ7.05 (3H, m), 7.00ꢁ6.81
(3H, m), 6.56 (1H, d, Jꢄ14.51 Hz).
/
148 8C; IR
;
Yellow crystals; yield 35%; m.p. 119ꢁ
(KBr) 3400, 1680 cmꢀ1 1H-NMR (DMSO-d6, 90
MHz) d 12.35 (1H, s), 10.12 (1H, s), 7.91 (1H, d, Jꢄ
14.57 Hz), 7.75 (1H, s), 7.71ꢁ7.31 (4H, m), 7.15ꢁ6.85
(3H, m).
/
122 8C; IR
;
/
/
/
/
/
/
/
4.1.16. (E)-1-(2,5-Dihydroxyphenyl)-3-(2,5-
dihydroxyphenyl)-2-propene-1-one (2-16)
4.1.23. (E)-1-(2,5-Dihydroxyphenyl)-3-(indol-3-yl)-2-
propene-1-one (2-23)
Yellowish crystals; yield 35%; m.p. 144ꢁ
(KBr) 3345, 1680, 1610, 1590 cmꢀ1; 1H-NMR (DMSO-
d6, 90 MHz) d 7.99 (1H, d, Jꢄ16.11 Hz), 7.41 (1H, d,
Jꢄ16.11 Hz), 6.88ꢁ7.02 (3H, m), 6.44ꢁ6.75 (3H, m).
/
145 8C; IR
Yellow crystals; yield 38%; m.p. 117ꢁ
(KBr) 3380, 1680 cmꢀ1 1H-NMR (DMSO-d6, 90
MHz) d 11.99 (1H, s), 9.48 (1H, s), 8.21 (1H, s), 7.97
(1H, d, Jꢄ15.21 Hz), 7.48 (1H, d, Jꢄ15.21 Hz), 7.32ꢁ
6.99 (5H, m), 6.75ꢁ6.88 (2H, m).
/
121 8C; IR
;
/
/
/
/
/
/
/
/
4.1.17. (E)-1-(2,5-Dihydroxyphenyl)-3-(3,4-
dihydroxyphenyl)-2-propene-1-one (2-17)
Yellowish crystals; yield 28%; IR (KBr) 3350, 1680,
1615, 1585 cmꢀ1; 1H-NMR (DMSO-d6, 90 MHz) d 8.10
4.1.24. (E)-1-(2,5-Dihydroxyphenyl)-3-(quinolin-2-yl)-
2-propene-1-one (2-24)
Yellow crystals; yield 45%; m.p. 141ꢁ
(KBr) 3350, 1690 cmꢀ1 1H-NMR (DMSO-d6, 90
MHz) d 12.00 (1H, s), 9.78 (1H, s), 8.21 (1H, d, Jꢄ
14.38 Hz), 8.07 (1H, s), 7.71ꢁ7.56 (5H, m), 7.48 (1H, d,
Jꢄ14.40 Hz), 7.15ꢁ6.88 (3H, m).
/
145 8C; IR
(1H, d, Jꢄ
/
16.23 Hz), 7.45 (1H, d, Jꢄ
/
16.22 Hz), 6.88ꢁ
/
;
7.11 (3H, m), 6.51ꢁ/6.87 (3H, m).
/
/
/
/
4.1.18. (E)-1-(2,5-Dihydroxyphenyl)-3-(2,3,4-
trimethoxyphenyl)-2-propene-1-one (2-18)
Yellowish crystals; yield 59%; m.p. 128ꢁ
(KBr) 3340, 2910, 1670, 1620, 1580 cmꢀ1
(DMSO-d6, 300 MHz) d 7.95 (1H, d, Jꢄ15.59 Hz), 7.58
(1H, d, Jꢄ15.59 Hz), 7.29 (1H, d, Jꢄ8.6 Hz), 6.66 (1H,
dd, Jꢄ7.82, 2.40 Hz), 6.51 (1H, d, Jꢄ8.61 Hz), 6.44
(1H, d, Jꢄ7.82 Hz), 6.41 (1H, d, Jꢄ 2.40 Hz), 3.95
(3H, s), 3.89 (3H, s), 3.88 (3H, s).
/
130 8C; IR
4.1.25. all-trans-1-(2,5-Dihydroxyphenyl)-5-phenyl-2,4-
dienepentane-1-one (2-25)
;
1H-NMR
/
Yellow crystals; yield 87%; m.p. 142ꢁ
(KBr) 3360, 1680 cmꢀ1 1H-NMR (DMSO-d6, 90
MHz) d 11.75 (1H, s), 9.25 (1H, s), 7.88ꢁ7.26 (8H,
m), 7.13 (1H, dd, Jꢄ 8.81, 2.22 Hz), 6.91 (1H, s), 6.85
(1H, d, Jꢄ8.81 Hz).
/
144 8C; IR
/
/
;
/
/
/
/
/
/
/

N.-H. Nam et al. / European Journal of Medicinal Chemistry 38 (2003) 179ꢁ
/187
187
4.2. Biological assays
per well were photographed. The inhibition was esti-
mated in a double-blind manner and designed as ꢃ
(strong, complete), ꢃ (moderate), ꢃ (slight), and ꢀ
(inactive).
/
ꢃ
/
ꢃ
/
4.2.1. Cells and cell cultures
/
ꢃ
/
/
/
Tumor cell lines were obtained from a cancer cell
bank at Korea Research Institute of Bioscience and
Biotechnology (KRIBB) and cultured in DMEM sup-
plemented with 10% FBS. Human umbilical venous
endothelial cells (HUVEC) were purchased from Sanko
Junyaku Co., Ltd., Tokyo, Japan, and were grown in
M-199 supplemented with heparin (5 U mLꢀ1), en-
4.2.4. Antitumor experiments
Antitumor experiments were carried as described in
our previous reports [10,12]. Briefly, Lewis lung carci-
noma cells were inoculated s.c. into BDF1 mice on day 0
dothelial cell growth supplement (ECGS, 200 mg mLꢀ1
)
and 20% FBS. HUVECs were preserved frozen between
passages 3 and 4. Biological experiments were carried
(1ꢂ
107 cells/mouse/0.2 mL PBS). Test compounds were
/
dissolved in a medium comprising of 5% DMSO and
20% Cremophor†. Each compound was dosed with six
injections from day 1 and every 2 days after. The second
round of three injections was administered everyday
from day 16 until day 18. Body weights were tracked
every day and tumor sizes were measured with calipers
from day 11. Tumor volumes were calculated by the
out with cells between passages 5ꢁ10.
/
4.2.2. Cytotoxicity assays
On day 0, 180 mL of a cell suspension (in culture
medium, 3ꢂ
104 cells mLꢀ1 for tumor cells, 5ꢂ104 cells
/
/
mLꢀ1 for HUVECs) were seeded in each well of 96 well
plates. The plates were incubated in a 5% CO2 incubator
at 37 8C for 24 h, then samples in 20 mL culture medium
were added at various concentrations. The plates were
incubated for another 48 h for tumor cell lines and 72 h
for HUVECs. Cytotoxicity was measured by SRB’s
method as described in literature [9,10]. Compounds
were examined in three independent assays, and the
values, expressed IC50 (a concentration produces 50%
reduction in cell growth), were the averages of three
following equation: tumor volume (mm3)ꢄ
(mm)ꢂ
width (mm)2]/2. The inhibition rate was evalu-
ated as (1ꢀT/C)ꢂ100% (where T is the mean tumor
/[length
/
/
/
volume of the treated group, C is the mean tumor
volume of the control group). Each group consisted of
ten mice. Etoposide was administered at days 1, 5 and 9
at 36 mg kgꢀ1 dayꢀ1. No death was recorded within the
experimented period. Student’s t-test was used for all
statistical analyses.
determinations (S.D.510%). The average potency of
/
each compound in all cell lines was the calculated as the
average of IC50 values (average cytotoxicity). When the
IC50 values were greater than 30 mg mLꢀ1, this figure
References
[1] N.H. Nam, Y.J. You, Y. Kim, D.H. Hong, H.M. Kim, B.Z. Ahn,
Arch. Pharm. Res. 25 (2002) (2002) 590.
was used. The SI was calculated by an equation: SIꢄ
/
[average of IC50 value of the compound in tumor cell
lines (B16, HCT116, A431)]/IC50 value of the compound
in HUVEC.
[2] G. Gastl, T. Hermann, M. Steurer, J. Zmijia, E. Gunsilius, C.
Unger, A. Kraft, Oncology 54 (1997) 177.
[3] K. Ollinger, J. Llopis, E. Cadenas, Arch. Biochem. Biophys. 275
(1989) 514.
[4] B.Z. Ahn, D.E. Sok, Curr. Pharm. Des. 2 (1996) 247.
[5] K.H. Lee, E.S. Huang, C. Piantadosi, J. Pagano, T.A. Geissman,
Cancer Res. 31 (1971) 1649.
4.2.3. In vitro angiogenesis assay
In vitro angiogenesis assays were performed using a
method reported previously [11] with slight modifica-
tions. Briefly, matrigel was thawed on ice to prevent
premature polymerization. An aliquot of 200 mL ma-
trigel was seeded into each well of a 24-well tissue
culture plate and allowed to polymerize at 37 8C for 30
min. Into each well of the matrigel-seeded plate were
added 500 mL of M-199, with or without a test sample.
[6] K.H. Lee, I.H. Hall, C.D. Starness, S.A. Elgebaly, T.G. Waddell,
R.T. Hadgraft, C.G. Ruffler, I. Weidner, Science 196 (1977) 533.
[7] J. Folkman, N. Engl. J. Med. 285 (1971) 1182.
[8] T.G. Zogakis, S.K. Libutti, Exp. Opin. Biol. Ther. 1 (2001) 253.
[9] P. Skehan, R. Storeng, D. Scudiero, A. Monk, J. MacMahon, D.
Vistica, J. Warren, H. Bokesch, S. Kenny, M.R. Boyd, J. Nat.
Cancer Inst. 82 (1990) 1107.
[10] N.H. Nam, Y.J. You, Y. Kim, D.H. Hong, H.M. Kim, B.Z. Ahn,
Phytother. Res. (2002) in press.
Finally, 500 mL of a cell suspension (1.6ꢂ
/
105 cells
mLꢀ1 in a complete HUVEC culture medium) were
added. After incubation for 18 h at 37 8C in a 5% CO2
humidified atmosphere, the formation of HUVEC
induced network was observed under microscope and
[11] H.W. Schnaper, D.S. Grant, W.G. Stterler-Stevenson, R. Frid-
man, G. D’orazi, A.N. Murphy, R.E. Bird, M. Hoythya, T.R.
Fuerst, D. French, J.P. Qigley, H.K. Kleiman, J. Cell. Physiol.
156 (1993) 235.
[12] Y. Kim, S.B. Kim, Y.J. You, B.Z. Ahn, Planta Med. 68 (2002)
271.
four different phase-contrast microscopic fields (ꢂ100)
/