Design, synthesis and discovery of 5-hydroxyaurone derivatives as growth inhibitors against HUVEC and some cancer cell lines

Article abstract of DOI:10.1016/j.ejmech.2010.09.061

A series of 4′-substituted 5-hydroxyaurone derivatives were synthesized and their inhibitory activities against the proliferation of endothelial cells and two cancer cell lines were studied. Some of these compounds functioned as potent inhibitors against the proliferation of endothelial cells and cancer cells but possessed much weaker cytotoxic activities against non-cancer cell line of CCC-HPF-1. It was demonstrated that two most active compounds 16 and 27 effectively inhibited in vitro endothelial cell motility and tube formation, which are basic properties of endothelial cells for angiogenesis. Moreover, 16 and 27 also showed significant activities against in vitro cancer cell invasion, indicating that they have potential to inhibit cancer metastasis. These composite results suggest that 4′-substituted 5-hydroxyaurone is indeed a candidate structural scaffold for anticancer agent targeting activated endothelial cells and fast-proliferating cancer cells.

Full text of DOI:10.1016/j.ejmech.2010.09.061

European Journal of Medicinal Chemistry 45 (2010) 5950e5957
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Design, synthesis and discovery of 5-hydroxyaurone derivatives as growth
inhibitors against HUVEC and some cancer cell lines
,
,
Huimin Cheng a d, Lianwen Zhang a e, Yingxue Liu a, Shaopeng Chen a, Hao Cheng a, Xin Lu a,
Zhuxia Zheng a e, Guo-Chun Zhou a
,
,b,c,
*
a Guangzhou Institute of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, China
b College of Biotechnology and Pharmaceutical Engineering, Nanjing University of Technology, Nanjing 210009, China
c College of Pharmaceutical Sciences, Nanjing University of Technology, Nanjing 210009, China
d Graduate School of Chinese Academy of Sciences, Beijing 100039, China
e School of Life Science, University of Science and Technology of China, Hefei 230027, China
a r t i c l e i n f o
a b s t r a c t
A series of 40-substituted 5-hydroxyaurone derivatives were synthesized and their inhibitory activities
against the proliferation of endothelial cells and two cancer cell lines were studied. Some of these
compounds functioned as potent inhibitors against the proliferation of endothelial cells and cancer cells
but possessed much weaker cytotoxic activities against non-cancer cell line of CCC-HPF-1. It was
demonstrated that two most active compounds 16 and 27 effectively inhibited in vitro endothelial cell
motility and tube formation, which are basic properties of endothelial cells for angiogenesis. Moreover,
16 and 27 also showed significant activities against in vitro cancer cell invasion, indicating that they have
potential to inhibit cancer metastasis. These composite results suggest that 40-substituted 5-hydrox-
yaurone is indeed a candidate structural scaffold for anticancer agent targeting activated endothelial cells
and fast-proliferating cancer cells.
Article history:
Received 24 June 2010
Received in revised form
27 September 2010
Accepted 28 September 2010
Available online 7 October 2010
Keywords:
5-Hydroxyaurone
Anti-angiogenesis agent
Conventional anticancer agent
Cell migration
Ó 2010 Elsevier Masson SAS. All rights reserved.
Tube formation
Cell invasion
1. Introduction
death of the tumor are two major cancer chemotherapies [7,8].
However, independent application of anti-angiogenesis agents will
Cancer cells in a dormant tumor are supplied with a limited
quantity of oxygen and nutrients by diffusion, therefore, the size of
the tumor is arrested at 1e2 mm3 [1e3]. After tumor angiogenesis
has been established, cancer cells have ability to acquire enough
oxygen and nutrients for their unlimited proliferation, in turn, the
tumor becomes active and grows very fast [4,5]. Unlike angiogenic
activity in the adult normal organism, which is generally low and
adjustable, the regulatory mechanisms of angiogenesis in an active
tumor are destroyed by overgrowing cancer cells [1,6]. Unchecked
growth of cancer cells is the fundamental characteristics of a tumor
[4] and excessive activation of angiogenesis is the basal patholog-
ical process for tumor progression and metastasis [5,6]; as a result,
blockade of angiogenesis by targeting activated endothelial cells
and regression of rapidly growing tumor through inducing cell
not be enough to cure cancer and the tumor can also recur after
drug administration; conventional anticancer treatment alone
often has toxic side effects on normal cells and easily produces drug
resistance. Many conventional anticancer agents and some angio-
genesis inhibitors have been used in the clinic and they have been
demonstrated in recent years to be beneficial when used in
combination [8e10]. In addition to the combination therapy, it is
possible in theory that one agent possesses the antitumor activities
by targeting activated endothelial cells and fast-proliferating
cancer cells, like sorafenib [11] (receptor tyrosine kinase inhibitor),
combretastatin A-4 [12] (CA-4, vascular targeting agent), benga-
mides [13] (methionine aminiopeptidase inhibitors), etc.
Aurones, (Z)-2-benzylidenebenzofuran-3(2H)-ones, as one class
of flavonoids, are abound in many plants, especially in flowers and
fruits [14,15], and also exists in marine organisms [16]. Natural and
synthetic aurones possess a strong and broad variety of biological
activities [17], some activities in connection with anti-proliferative
properties against cancer cells including binding P-glycoprotein
[18,19], inhibiting cyclin-dependent kinases (CDKs) [20], blocking
sphingosine kinase [21], interfering microtubule assembly [22e24],
* Corresponding author. Guangzhou Institute of Biomedicine and Health, Chinese
Academy of Sciences, Guangzhou 510530, China. Tel.: þ86 013433968219; fax: þ86
20 32015351.
E-mail address: allspringzhou@yahoo.com (G.-C. Zhou).
0223-5234/$ e see front matter Ó 2010 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2010.09.061

H. Cheng et al. / European Journal of Medicinal Chemistry 45 (2010) 5950e5957
5951
subsequent demethylation of 1 using BBr3 [31]. 1 and 2 were at last
cyclized by sodium acetate in ethanol into 3 and 4 [27,33],
respectively.
O
O
4
A
7
3
HO
5
6
2
C
1'
2'
4'
The assignment of the olefinic geometrical configuration of the
title aurones is mainly based on the chemical shift of the olefinic
proton singlet signal being around 6.80 ppm for the (Z)-isomer as
reported in the literature [35]. Generally, H-4 in a 5-hydroxyaurone
derivative is weakly coupled by meta-aromatic proton of H-6 with
J & 3.0 Hz, while no coupled signal was observed between H-4 and
H-6 in 5-methoxyaurones or 5-acetoxyaurones. In our studies,
most of the reactions only generated Z-form products, whereas, it
seemed that Z-form aurones were occasionally accompanied by the
trace amount of E-form aurones observed by TLC and HPLC in some
reactions (data not shown).
O
O
1
3'
B
6'
4'-substituted
5-hydroxyaurone
5'
Aurone
R
Fig. 1. General structures of aurones and 40-substituted 5-hydroxyaurones.
modulating ABCG2 (breast cancer resistance protein)-mediated
multi-drug resistance [25], exhibiting antioxidative activity [26,27],
DNA strandescission activity [28], etc. Among hydroxylated
derivatives of aurones, 4-hydroxyaurones and 6-hydroxyaurones
are more commonly studied but reports for 5-hydroxyaurones and
7-hydroxyaurones are currently limited.
In our aims to search for small molecular entities with anti-
angiogenesis activity and conventional anticancer property for
cancer treatment, we investigated 5-hydroxyl derivatives of aurones
(Fig. 1). To our knowledge, aurones have never been reported to be
anti-angiogenesis agents even there are many reports that aurones
are active against cancer cell growth. In this study, we have found for
the first time that synthetic 40-substituted 5-hydroxyaurone deriv-
atives exhibited potent inhibitory activities against the proliferation
of endothelial cells [29]. In addition, some of these compounds were
also active against the proliferation of cancer cells but showed much
weakercytotoxic activities against non-cancercell line of CCC-HPF-1
[30]. Based on the biological evaluation data of anti-proliferation
assay, we studied their preliminary structureeactivity relationship
(SAR). Furthermore, it was demonstrated that two most active
compounds 16 and 27 effectively inhibited in vitro endothelial cell
motility and tube formation, which are basic properties of endo-
thelial cells for angiogenesis [29]. Moreover, 16 and 27 showed
significant activities against in vitro cancer cell invasion, indicating
that they have potential to inhibit cancer metastasis. These
composite results suggest that 40-substituted 5-hydroxyaurone is
indeed a candidate structural scaffold for anticancer agent targeting
activated endothelial cells and fast-proliferating cancer cells.
3. Results and discussion
The strategy in this study was to submit these synthesized
compounds to a preliminary screening test aimed to evaluate their
in vitro inhibitory effects on the proliferation of endothelial cells
and cancer cells prior to measure their in vitro anti-angiogenesis
activities along with their activities against in vitro cancer cell
invasion. HUVECs (human umbilical vein endothelial cells) were
used as target endothelial cells, while A549 (non-small cell lung
cancer cell line) and MCF-7 (breast cancer cell line) were used as
target cancer cells. The anti-proliferative IC50 values for HUVEC and
cancer cell lines A549 and MCF-7 are summarized in Table 1.
In our study streamline, unsubstituted 5-hydroxyaurone (5) was
firstly prepared and tested but it was not active against the cell
growth. Then we functionated 40-position of aurone core by intro-
duction of hydroxyl group to afford 40-hydroxy-5-hydroxyaurone
(6), which exhibited the inhibitory activity against HUVEC prolifer-
ation. Meanwhile, 40-methoxy-5-hydroxyaurone (7) preserved
anti-HUVEC proliferation activity of 6 and it also exhibited weak
inhibitoryactivityagainst the cell growth of A549. Next, we explored
the syntheses and inhibitory activities of 40-halide substituted
5-hydroxyaurones. Fluoride
8 exhibited almost no inhibitory
activity as parent 5-hydroxyaurone; however, chloride 9 and
bromide 10 showed close anti-HUVEC proliferation activities to 40-
hydroxy- and 40-methoxy-5-hydroxyaurones 6 and 7. Both 9 and 10
emerged very weak anti-proliferative activities to cancer cells. It
seems here that the size not the electronegativity of fluorine
contributes to the inhibitory activity of fluoride 8.
Considering the effects of the substitute group’s electronega-
tivity on the activity, some groups with different electronegativity
were introduced at 40-position. Electron-withdrawing tri-
fluoromethyl group slightly increased anti-HUVEC proliferation
activity and showed weak inhibitory activity against cancer cell
proliferation; however, introduction of conjugated 40-nitrile and
40-formamide groups dramatically decreased the inhibitory activi-
ties. We further explored the introduction of amine group and found
that40-amino-5-hydroxyaurone (14) exhibited the similaractivity to
2. Chemistry
There are many reported synthetic methodologies to aurones.
5-Methoxyaurones or 5-hydroxyaurones in this study were
synthesized by means of an aldol condensation of 5-methoxy-
benzofuranone (3) [31] or 5-hydroxy-benzofuranone (4) [32] with
the appropriate benzaldehyde in glacial acetic acid, which was
catalyzed by concentrated hydrochloric acid (Scheme 1) [33]. 5-
Substituted benzofuranones 3 and 4 were prepared starting from
1,4-dimethoxybenzene (Scheme 1). Briefly, the FriedeleCrafts
reaction of 1,4-dimethoxybenzene with chloroacetylchloride under
the catalyst of anhydrous AlCl3 afforded
methoxyacetophenone (1) [34] with the medium yield of 76%.
Chloro-2,5-dihydroxy-acetophenone (2) [32] was generated by
a-chloro-2-hydroxy-5-
a
-
O
OH
O
OMe
O
R1O
d
R1O
Cl
a
c
O
O
R1 = H, 5-22
OR1
R1 = Me, 1
R1 = H, 2
OMe
e
R1 = Me, 3
R1 = H, 4
R1 = Ac, 26-28
R1 = Me, 23-25
R2
b
Scheme 1. Synthesis of 40-substituted 5-hydroxyaurones. Reagents and conditions: a) AlCl3, chloroacetyl chloride, 76%; b) BBr3, ꢀ78 ꢁC to room temperature, 82%; c) sodium acetate,
92% for 3 and 88% for 4; d) benzaldehydes, glacial acetic acid, concentrated hydrochloric acid (cat.), room temperature; e) acetic anhydride, triethylamine, room temperature.

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H. Cheng et al. / European Journal of Medicinal Chemistry 45 (2010) 5950e5957
40-hydroxy- and 40-methoxy-5-hydroxyaurones. Taken together, it
is proposed that the electronegativity of the substitute group is not
a key for the anti-proliferative activity but conjugated electron-
withdrawing groups sharply decreased the inhibitory activity.
Even 40-O-methylation (compound 7) did not affect the inhibi-
tory activity compared with 40-hydroxy-5-hydroxyaurone (6), N-
methylation and N-ethylation of 40-amino group significantly
enhanced the activities against the proliferation of HUVEC and two
cancer cell lines of A549 and MCF-7. Importantly, N-ethylated
compound 16 were 5-fold more active than N-methylated
compound 15 against the proliferation of these cells. However, it is
unexpected that the introduction of cyclic amino groups of 1-
piperidinyl and 1-pyrrolidinyl at 40-position made the inhibitory
activities of 17 and 18 completely lost, suggesting that the negative
effects of these nitrogen-containing rings on the inhibitory activi-
ties may be due to the reduction of the flexibility of 40-substitution.
With regards to the increase in potency from 40-amino-5-
hydroxyaurone (14) to 40-(N,N-dialkyl)amino-5-hydroxyaurones (15
and 16) and the inactivity of 17 and 18, we wondered if the size and
the shape of a substitute are more important than its electronega-
tivityinexecutingtheinhibitoryactivity. Sincethesizesandshapes of
i-Pr and t-Bu groups are close to those of dimethylamino group, 40-
alkyl products of 20 and 21 along with 19 were synthesized and
assayed. Interestingly, they possessed the similar activities against
HUVECproliferationtocompounds 6, 7, 9,10,11 and14. Moreover, the
results showed that more bulky alkyl substitution at C-40 led to more
potent activityinterms ofanti-HUVECproliferationevencompounds
20 and 21 were not as active as 40-dimethylamino product 15 against
HUVEC proliferation and inactive against the proliferation of cancer
cell lines. The combined data support the hypothesis that thesize and
the shape of a substitute are more important than its electronega-
tivity at least against the proliferation of HUVEC.
Table 1
The activities of 40-substituted 5-hydroxyaurones 5e28.
O
R1O
O
R2
Cmpd.a
R1
R2
IC50
(m
M)b
CC50 (m
M)b
HUVEC
MCF-7
A549
CCC-HPF-1
5
6
7
8
H
H
/
/
/
/
ND
ND
ND
ND
/
ND
48.6
ND
ND
ND
25.08
7.92
ND
ND
ND
/
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
OH
OMe
F
Cl
Br
CF3
CN
CONH2
NH2
NMe2
NEt2
1-piperidinyl
1-pyrrolidinyl
Me
9.63
9.86
39.45
7.93
8.97
5.35
22.74
35.58
7.33
1.35
0.25
/
/
/
/
/
29.02
/
35.71
44.7
29.98
/
/
44.6
18.66
1.25
/
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
Fumagillin
Taxol
40.9
34.39
/
/
/
10.12
1.81
/
/
/
/
/
/
/
/
9.35
6.38
3.49
/
/
/
48.75
/
/
/
/
/
/
6.5
1.29
/
/
i-Pr
t-Bu
/
H
NHAc
NMe2
NEt2
t-Bu
NMe2
NEt2
t-Bu
ND
ND
ND
ND
/
7.77
ND
ND
ND
Me
Me
Me
Ac
Ac
Ac
/
/
/
/
3.18
0.23
46.4
0.0089
ND
8.39
2.95
/
In an effort to study the function of 5-hydroxy group, we were
interested in modification of 5-hydroxyl group by acetylation and
methylation. Methylation of 5-hydroxy group resulted in blocking
the role of phenol; whereas, acetylation of 5-hydroxy group tempo-
rarily blockedthe roleof phenol in that phenol group can often invivo
and sometimes in vitro be free by hydrolysis of acetyl group, which is
/
0.021
0.0049
a
Notes: The purity of these compounds was analyzed by HPLC and listed in
“Experimental”.
b
“/” means IC50 or CC50 ꢂ 50
mM and “ND” means “Not Detected”.
Fig. 2. Compounds 16 and 27 inhibited HUVEC migration (AeC) and tubular structure formation (DeF). A) Carrier-treated for 24 h after wounding; B) 0.1
m
M of compound
16-treated for 24 h after wounding; C) 0.1
compound 27-treated for 6 h.
m
M of compound 27-treated for 24 h after wounding; D) Carrier-treated for 6 h; E) 2.0
m
M of compound 16-treated for 6 h; F) 2.0 M of
m

H. Cheng et al. / European Journal of Medicinal Chemistry 45 (2010) 5950e5957
5953
100%
80%
60%
40%
20%
0%
activities against A549 and MCF-7 cancer cell lines. However, these
compounds possessed much weaker cytotoxicities against non-
cancer cell line of CCC-PPF-1. Our current observations make these
chemical entities attractive for pursuits of anti-angiogenesis and
conventional anticancer activities. Further investigations are
underway in our laboratory to discover more potent and drug-like
anti-angiogenesis compounds with conventional anticancer
activity and to clarify their possible mechanism/s of action.
5. Experimental
ctr
cmpd 16
cmpd 27
ctr
cmpd 16
cmpd 27
MCF-7
A549
5.1. Chemistry
Fig. 3. 1.0 mM Compounds 16 and 27 inhibited cancer cell invasion of MCF-7 and A549
cell lines assayed by Matrigel-coated transwell (drug-treated for 24 h).
1H and 13C NMR spectra were recorded on a Bruker AV-400
spectrometer at 400 and 100 MHz, respectively, in CDCl3 unless
otherwise mentioned. Coupling constants (J) are expressed in hertz
generally used as a prodrug strategy. In addition, we also studied the
acetylation blocking of 40-amino group of 14. As anticipated, 5-O-
methylation of 15, 16 and 21 damaged significantly the inhibitory
activities; meanwhile, 5-O-acetylation of 21 and 40-N-acetylation of
14 led to complete loss of inhibitory activities in the cell-based assay.
Acetylated product of 26 increased its inhibitory activity to A549
cancer cell line and only slightly decreased its activity against HUVEC
proliferation compared with 15; however, the prodrug 27 showed
close inhibitory activities to its parent compound 16.
In order to study the selectivity of these relatively active
compounds, growth inhibition by them against human embryonic
lung diploid cell line (CCC-HPF-1) [30], a non-cancer cell line, was
measured through the MTT assay. Results listed in Table 1 showed
that the cytotoxicity (CC50) values of these compounds to CCC-HPF-
1 were much higher (w20 times) than their IC50 values to HUVEC,
and higher than those of MCF-7 and A549. On the basis of these
data, it is concluded that these compounds are selective against the
proliferation of HUVEC and one or both of two cancer cell lines.
Although angiogenesis is a very complex process, cell migration
and tube formation are critical for endothelial cells to form blood
vessels in tumor angiogenesis and thus is necessary for tumor
growth and metastasis to establish newtumor focus [29]. The effects
of twomostactive compounds 16 and27 on thechemotactic motility
ofHUVECswere measured bywound-healing migration assayand as
shown in Fig. 2AeC, both of 16 and 27 significantly inhibited HUVEC
(Hz). Chemical shifts (d) of NMR are reported in parts per million
(ppm)unitsrelativetothesolvent. TheloworhighresolutionofESIMS
was recorded on an Agilent 1200 HPLC-MSD mass spectrometer or
Applied Biosystems Q-STAR Elite ESI-LC-MS/MS mass spectrometer,
respectively. The purity of compounds was determined by reverse-
phase HPLC analysis. HPLC instrument: DIONEX SUMMIT HPLC;
Column: Diamonsil C18, 5.0
detection: wavelength
¼ 280 nm, PDA-100 Photodiode Array
Detector; Elution: MeOH in water; Flow rate: 1.0 ml/min.
mm, 4.6 ꢃ 250 mm (Dikma Technologies);
l
5.1.1.
a-Chloro-2-hydroxy-5-methoxyacetophenone (1) [34]
To the cooled mixture (ꢀ20 ꢁC) of 1,4-dimethoxybenzene
(10.0 g, 72.4 mmol) and anhydrous AlCl3 (20.3 g, 152.0 mmol) in
200 ml dry dichloromethane, chloroacetyl chloride (7.0 ml,
86.9 mmol) was added dropwise in 1 h. The resulting solution was
raised to room temperature gradually and was stirred overnight.
The reaction solution was poured into a mixture of crushed ice and
10 ml con. HCl and stirred for 20 min. The organic phase was
separated and the aqueous phase was extracted with dichloro-
methane. The combined organic phase was washed with saturated
NaHCO3 solution and brine and dried over anhydrous Na2SO4. After
filtered, volatile solvents were evaporated to yield pale yellowish
solid 11.1 g (76% yield). The crude product was crystallized from
petroleum ether-ethyl acetate to afford pure 1. 1H NMR (400 MHz,
migration at 0.1 mM compared with the control group. The potential
DMSO-d6):
d
10.73 (s, 1H, OH), 7.24 (d, J ¼ 3.2 Hz, 1H, H-6), 7.13 (dd,
inhibitory activities of 16 and 27 against the tubular structure
formation of endothelial cells were examined by two-dimensioned
Matrigel assay. Elongated and robust tube-like structures were well
established by HUVECs after incubation for 6 h in the control group;
J ¼ 3.2, 9.2 Hz, 1H, H-4), 6.95 (d, J ¼ 8.8 Hz, 1H, H-3), 5.10 (s, 2H,
CH2Cl), 3.73 (s, 3H, 4-OCH3) ppm.
5.1.2.
a-Chloro-2,5-dihydroxyacetophenone (2) [32]
however, treatment of HUVECs with 2.0 mM of 16 or 27 completely
A solution of 1 (3.55 g, 17.7 mmol) in 50 ml dry dichloromethane
was cooled to ꢀ78 ꢁC, BBr3 (2.4 ml, 25.8 mmol) was added drop-
wise. The resulting solution was stirred at this temperature for
30 min and then was warmed to room temperature and continued
to stir for additional 1 h. The reaction was treated carefully with
iced water and extracted with dichloromethane. The combined
organic phase was washed with saturated NaHCO3 solution and
brine and dried over anhydrous Na2SO4. After filtered and
concentrated, the crude product 2 was purified by silica gel column
chromatography (petroleum ether/ethyl acetate 6/1) to give pale
yellowish solid 2.70 g (82% yield). 1H NMR (400 MHz, DMSO-d6):
inhibited the formation of tubular structures (Fig. 2DeF). These
observations showed that 16 and 27 could block invitroangiogenesis
by inhibiting endothelial cell migration and tube formation.
In addition to their inhibitory effects on HUVEC migration and
tube formation, we also investigated the effects of 16 and 27 on
cancer invasion by Matrigel-coated transwell cell invasion assay
since the invasive ability of cancer cells is one of fundamental
properties for cancer metastasis and establishment of new tumor
focus. The results are showed in Fig. 3 that 16 and 27 inhibited more
than 50% invasion of A549 and MCF-7 at 1.0 mM by Matrigel-coated
transwell assays, suggesting that they are potential inhibitors
against cancer invasion and metastasis.
d
10.48 (s, 1H, OH), 9.17 (s, 1H, OH), 7.12 (d, J ¼ 3.2 Hz, 1H, H-6), 6.96
(dd, J ¼ 2.8, 8.8 Hz, 1H, H-4), 6.84 (d, J ¼ 8.7 Hz, 1H, H-3), 5.05 (s, 2H,
CH2Cl) ppm.
4. Conclusions
5.1.3. 5-Methoxy-2,3-dihydrobenzofuran-3-one (3) [31] and 5-hydr
oxy-2,3-dihydrobenzofuran-3-one (4) [32]
A mixture of 1.0 equiv of 1 or 2 and 2.1 equiv of sodium acetate
was dissolved in ethanol and heated under reflux for 2 h [27,33].
The reaction mixture was filtered and the filter cake was washed
In summary, we disclosed for the first time the inhibition of in
vitro angiogenesis of HUVEC proliferation, motility and tube
formation by 40-substituted 5-hydroxyaurone derivatives. Some of
these molecules also exhibited anti-proliferative and anti-invasive

5954
H. Cheng et al. / European Journal of Medicinal Chemistry 45 (2010) 5950e5957
with ethyl acetate. The filtrate was evaporated to about one third,
and then diluted with water and extracted with ethyl acetate. The
combined organic phase was washed with saturated NaHCO3
solution and brine and dried over anhydrous Na2SO4. The filtered
organic solution was evaporated to dryness to yield crude orange-
red product, which was purified by silica gel column chromatog-
raphy (petroleum ether/ethyl acetate 6/1) to afford 3 or 4. 3 (92%
CH) ppm; 13C NMR (100 MHz, DMSO-d6): 184.4, 164.41 and 161.93
d
(d, J ¼ 248.0 Hz, 1C, C-40), 159.8, 154.4, 147.31 and 147.28 (d,
J ¼ 3.0 Hz, 1C, ]CH), 134.16 and 134.07 (d, J ¼ 9.0 Hz, 2C, C-20, C-60),
129.24 and 129.21 (d, J ¼ 3.0 Hz, 1C, C-10), 126.3, 121.7, 116.74 and
116.53 (d, J ¼ 21.0 Hz, 2C, C-30, C-50), 114.4, 111.1, 108.3 ppm; HRMS
(ESI): m/z 257.0613 [M þ H]þ (100), calcd for C15H10O3F, 257.0614,
D
¼ ꢀ0.1 mmu; HPLC analysis: 76:24 methanolewater, 11.09 min,
yield); 1H NMR (400 MHz, CDCl3):
d
7.23 (dd, J ¼ 2.8, 9.2 Hz, 1H, H-
98.6% in purity.
6), 7.06 (d, J ¼ 8.4 Hz, 1H, H-7), 7.04 (s, 1H, H-4), 4.63 (s, 2H, 2-CH2),
3.80 (s, 3H, 5-OCH3) ppm. 4 (88% yield); 1H NMR (400 MHz, DMSO-
5.1.4.5. (Z)-5-Hydroxy-2-(4-chlorobenzylidene)benzofuran-3(2H)-
one (9). Starting from 4-chlorobenzaldehyde and 4; yield: 70%;
yellow solid; M.p. 238e239 ꢁC; 1H NMR (400 MHz, DMSO-d6):
d6):
d
9.57 (s, 1H, 5-OH), 7.18 (dd, J ¼ 2.8, 8.8 Hz, 1H, H-6), 7.12 (d,
J ¼ 8.8 Hz, 1H, H-7), 6.86 (d, J ¼ 2.6 Hz, 1H, H-4), 4.73 (s, 2H, 2-CH2)
ppm; MS (ESI): m/z 151 [M þ H]þ (100), 149 [M ꢀ H]ꢀ (100).
d
9.82 (s, 1H, 5-OH), 7.99 (d, J ¼ 8.4 Hz, 2H, H-20, H-60), 7.57 (d,
J ¼ 8.4 Hz, 2H, H-30, H-50), 7.39 (d, J ¼ 9.2 Hz, 1H, H-7), 7.23 (dd,
5.1.4. General procedure of condensation of benzaldehyde
derivatives with benzofuran-3-ones (3 or 4) [33]
J ¼ 2.4, 8.8 Hz, 1H, H-6), 7.03 (d, 1H, J ¼ 2.4 Hz, H-4), 6.90 (s, 1H, ]
CH) ppm; 13C NMR (100 MHz, DMSO-d6):
d 184.4,159.8,154.5, 147.8,
To the solution of 1.0 mmol of 3 or 4 and 1.2 mmol of appropriate
benzaldehyde derivatives in 10 ml of glacial acetic acid, 4 drops of
concentrated hydrochloric acid was added at the room temperature
and then the reaction was stirred at the same temperature for 3 h.
The reaction mixture was poured into ice-cooled water and filtered.
The filter cake was washed with water and then was purified by
column chromatography using methanol-dichloromethane
(2:100e5:100).
135.0, 133.3,131.5,129.6, 126.4, 121.6, 114.4, 110.8, 108.3 ppm; HRMS
(ESI): m/z 273.0315 [M þ H]þ (100), calcd for C15H10O3Cl, 273.0318,
D
¼ ꢀ0.3 mmu; HPLC analysis: 76:24 methanolewater, 18.20 min,
99.7% in purity.
5.1.4.6. (Z)-5-Hydroxy-2-(4-bromobenzylidene)benzofuran-3(2H)-
one (10). Starting from 4-bromobenzaldehyde and 4; yield: 77%;
yellow solid; M.p. 243e244 ꢁC; 1H NMR (400 MHz, DMSO-d6):
d
9.83 (s, 1H, 5-OH), 7.91 (d, J ¼ 8.4 Hz, 2H, H-20, H-60), 7.71 (d,
5.1.4.1. (Z)-5-Hydroxy-2-benzylidene-benzofuran-3(2H)-one
(5)
J ¼ 8.4 Hz, 2H, H-30, H-50), 7.40 (d, J ¼ 8.8 Hz, 1H, H-7), 7.23 (dd,
[36]. Starting from benzaldehyde and 4; yield: 95%; yellow
J ¼ 2.4, 8.8 Hz, 1H, H-6), 7.03 (d, J ¼ 2.4 Hz, 1H, H-4), 6.88 (s, 1H, ]
powder; M.p. 224e225 ꢁC; 1H NMR (400 MHz, DMSO-d6):
d
9.83 (s,
CH) ppm; 13C NMR (100 MHz, DMSO-d6):
d 184.4,159.8,154.5, 147.9,
1H, 5-OH), 7.97 (d, J ¼ 7.2 Hz, 2H, H-20, H-60), 7.41e7.52 (m, 3H, H-30,
H-40, H-50), 7.40 (d, J ¼ 8.8 Hz, 1H, H-7), 7.22 (dd, J ¼ 2.8, 8.8 Hz, 1H,
H-6), 7.03 (d, J ¼ 2.8 Hz, 1H, H-4), 6.88 (s, 1H,]CH) ppm; 13C NMR
133.5, 132.5, 131.8, 126.4, 123.9, 121.6, 114.4,110.8, 108.3 ppm; HRMS
(ESI): m/z 316.9813 [M þ H]þ (100), calcd for C15H10O3Br, 316.9813,
D
¼ 0.0 mmu; HPLC analysis: 76:24 methanolewater, 19.73 min,
(100 MHz, DMSO-d6):
d
184.4, 159.9, 154.4, 147.7, 132.6, 131.7, 130.4,
98.9% in purity.
129.4, 126.3, 121.7, 114.3, 112.1, 108.3 ppm; MS (ESI): m/z 239
[M þ H]þ (100), 237 [M ꢀ H]- (100), 475 [2M ꢀ H]ꢀ (85); HPLC
analysis: 76:24 methanolewater, 11.15 min, 98.8% in purity.
5.1.4.7. (Z)-5-Hydroxy-2-(4-trifluoromethylbenzylidene)benzofuran-
3(2H)-one (11). Starting from 4-trifluoromethylbenzaldehyde and
4; yield: 70%; yellow solid; M.p. 210e211 ꢁC; 1H NMR (400 MHz,
5.1.4.2. (Z)-5-Hydroxy-2-(4-hydroxybenzylidene)benzofuran-3(2H)-
one (6) [36]. Starting from 4-hydroxybenzaldehyde and 4; yield:
68%; yellow solid; M.p. 319e320 ꢁC; 1H NMR (400 MHz, DMSO-d6):
DMSO-d6):
d
9.84 (s, 1H, 5-OH), 8.15 (d, J ¼ 8.0 Hz, 2H, H-20, H-60),
7.83 (d, J ¼ 8.4 Hz, 2H, H-30, H-50), 7.39 (d, J ¼ 8.8 Hz, 1H, H-7), 7.24
(dd, J ¼ 2.4, 8.8 Hz, 1H, H-6), 7.04 (d, J ¼ 2.4 Hz, 1H, H-4), 6.94 (s, 1H,
d
10.18 (brs, 1H, 40-OH), 9.81 (brs, 1H, 5-OH), 7.84 (d, J ¼ 8.4 Hz, 2H,
]CH) ppm; 13C NMR (100 MHz, DMSO-d6):
d 184.5, 160.0, 154.6,
H-20, H-60), 7.37 (d, J ¼ 8.8 Hz, 1H, H-7), 7.20 (dd, J ¼ 2.4, 8.8 Hz, 1H,
148.7, 136.6, 132.1, 130.0e123.1 (m, 6C, peaks of C-20, C-30, C-40, C-50,
C-60 and 40-CF3 were split by fluorides), 121.4, 114.4, 109.9, 108.4;
HRMS (ESI): m/z 307.0581 [M þ H]þ (100), calcd for C16H10O3F3,
H-6), 7.01 (d, J ¼ 2.8 Hz, 1H, H-4), 6.89 (d, J ¼ 8.8 Hz, 2H, H-30, H-50),
6.83 (s, 1H, ]CH) ppm; 13C NMR (100 MHz, DMSO-d6):
d 183.9,
160.1, 159.4, 154.2, 146.1, 134.0, 125.8, 123.6, 122.1, 116.6, 114.2, 113.2,
108.1 ppm; MS (ESI): m/z 255 [M þ H]þ (100), 253 [M ꢀ H]ꢀ (100).
HPLC analysis: 76:24 methanolewater, 5.49 min, 99.0% in purity.
307.0582,
17.48 min, 99.0% in purity.
D
¼ ꢀ0.1 mmu; HPLC analysis: 76:24 methanolewater,
5.1.4.8. (Z)-5-Hydroxy-2-(4-cyanobenzylidene)benzofuran-3(2H)-
one (12). Starting from 4-cyanobenzaldehyde and 4; yield: 64%;
yellow solid; M.p. 285e286 ꢁC; 1H NMR (400 MHz, DMSO-d6):
5.1.4.3. (Z)-5-Hydroxy-2-(4-methoxybenzylidene)benzofuran-3(2H)-
one (7). Starting from 4-methoxybenzaldehyde and 4; yield: 71%;
yellow solid; M.p. 251e252 ꢁC; 1H NMR (400 MHz, DMSO-d6):
d
9.88 (s, 1H, 5-OH), 8.09 (d, J ¼ 8.4 Hz, 2H, H-30, H-50), 7.92 (d,
d
9.77 (s, 1H, 5-OH), 7.95 (d, J ¼ 8.8 Hz, 2H, H-20, H-60), 7.38 (d,
J ¼ 8.4 Hz, 2H, H-20, H-60), 7.39 (d, J ¼ 8.8 Hz, 1H, H-7), 7.23 (dd,
J ¼ 8.8 Hz, 1H, H-7), 7.21 (dd, J ¼ 2.4, 8.8 Hz, 1H, H-6), 7.08 (d,
J ¼ 8.8 Hz, 2H, H-30, H-50), 7.02 (d, J ¼ 2.4 Hz, 1H, H-4), 6.87 (s, 1H, ]
CH), 3.83 (s, 3H, 40-OCH3) ppm; 13C NMR (100 MHz, DMSO-d6):
J ¼ 2.4, 8.8Hz, 1H, H-6), 7.02 (d, J ¼ 2.4 Hz, 1H, H-4), 6.91 (s, 1H, ]
CH) ppm; 13C NMR (100 MHz, DMSO-d6):
d 184.5, 159.9, 154.6,
148.9, 137.2, 133.1, 132.0, 126.6, 121.3, 119.1, 114.4, 111.9, 109.7,
d
184.0, 161.3, 159.6, 154.3, 146.6, 133.7, 126.0, 125.1, 122.0, 115.2,
108.4 ppm; HRMS (ESI): m/z 264.0658 [M þ H]þ (100), calcd for
114.2, 112.6, 108.2, 55.9 ppm; HRMS (ESI): m/z 269.0812 [M þ H]þ
C16H10NO3, 264.0661,
anolewater, 7.19 min, 99.4% in purity.
D
¼ ꢀ0.3 mmu; HPLC analysis: 76:24 meth-
(100), calcd for C16H13O4, 268.0814,
76:24 methanolewater, 11.33 min, 99.3% in purity.
D
¼ ꢀ0.2 mmu. HPLC analysis:
5.1.4.9. (Z)-5-Hydroxy-2-(4-carbamoylbenzylidene)benzofuran-3
(2H)-one (13). Starting from 4-carbamoylbenzaldehyde and 4;
yield: 67%; yellow solid; M.p. 294e295 ꢁC; 1H NMR (400 MHz,
5.1.4.4. (Z)-5-Hydroxy-2-(4-fluorobenzylidene)benzofuran-3(2H)-
one (8). Starting from 4-fluorobenzaldehyde and 4; yield: 93%;
yellow solid; M.p. 226e227 ꢁC; 1H NMR (400 MHz, DMSO-d6):
DMSO-d6):
d
9.84 (s, 1H, 5-OH), 8.07 (br s, 1H, 40-CONH2), 8.03 (d,
d
9.80 (s,1H, 5-OH), 8.03 (dd, J ¼ 5.6, 8.8 Hz, 2H, H-20, H-60), 7.36 (dd,
J ¼ 8.4 Hz, 2H, H-30, H-50), 7.96 (d, J ¼ 8.4 Hz, 2H, H-20, H-60), 7.48 (s,
1H, 40-CONH2), 7.43 (d, J ¼ 8.8 Hz, 1H, H-7), 7.24 (dd, J ¼ 2.8, 8.8 Hz,
1H, H-6), 7.04 (d, J ¼ 2.8 Hz, 1H, H-4), 6.92 (s, 1H, ]CH) ppm; 13C
J ¼ 8.8, 12.6 Hz, 2H, H-30, H-50), 7.32 (d, J ¼ 8.8 Hz, 1H, H-7), 7.21 (dd,
J ¼ 2.8, 8.8 Hz, 1H, H-6), 7.01 (d, J ¼ 2.8 Hz, 1H, H-4), 6.92 (s, 1H, ]

H. Cheng et al. / European Journal of Medicinal Chemistry 45 (2010) 5950e5957
5955
NMR (100 MHz, DMSO-d6):
d
184.5, 167.7, 160.0, 154.5, 148.2, 135.4,
149.2, 145.1, 134.0, 125.1, 122.6, 119.1, 115.0, 114.1, 112.5, 107.8, 47.7,
135.2, 131.4, 128.4, 126.4, 121.6, 114.4, 110.9, 108.4 ppm; HRMS (ESI):
25.4 ppm; HRMS (ESI): m/z 308.1288 [M þ H]þ (100), calcd for
m/z 282.0764 [M þ H]þ (100), calcd for C16H12NO4, 282.0766,
C19H18NO3, 308.1287,
D
¼ 0.1 mmu; HPLC analysis: 76:24 meth-
D
¼ ꢀ0.2 mmu; HPLC analysis: 76:24 methanolewater, 4.12 min,
anolewater, 24.49 min, 96.1% in purity.
99.5% in purity.
5.1.4.15. (Z)-5-Hydroxy-2-(4-methylbenzylidene)benzofuran-3(2H)-
one (19). Starting from 4-methylbenzaldehyde and 4; yield: 91%;
pale yellow solid; M.p. 227e228 ꢁC; 1H NMR (400 MHz, DMSO-d6):
5.1.4.10. (Z)-5-Hydroxy-2-(4-aminobenzylidene)benzofuran-3(2H)-
one (14). Starting from 4-aminobenzaldehyde and 4; yield: 52%;
red brown solid; decomposede220 ꢁC; 1H NMR (400 MHz, DMSO-
d
9.79 (s, 1H, 5-OH), 7.88 (d, J ¼ 8.0 Hz, 2H, H-20, H-60), 7.40 (d,
d6):
d
9.69 ppm (s, 1H, 5-OH), 7.70 (d, J ¼ 8.4 Hz, 2H, H-20, H-60), 7.35
J ¼ 8.8 Hz, 1H, H-7), 7.32 (d, J ¼ 8.0 Hz, 2H, H-30, H-50), 7.22 (dd,
J ¼ 2.8, 8.8 Hz, 1H, H-6), 7.02 (d, J ¼ 2.4 Hz, 1H, H-4), 6.86 (s, 1H, ]
CH), 2.36 (s, 3H, 40-CH3) ppm; 13C NMR (100 MHz, DMSO-d6):
(d, J ¼ 8.8 Hz, 1H, H-7), 7.16 (dd, J ¼ 2.8, 8.8 Hz, 1H, H-6), 6.98 (d,
J ¼ 2.8 Hz, 1H, H-4), 6.76 (s, 1H, ]CH), 6.64 (d, J ¼ 8.4 Hz, 2H, H-30,
H-50), 6.05 (s, 2H, 40-NH2) ppm; 13C NMR (100 MHz, DMSO-d6):
d
184.3, 159.7, 154.3, 147.2, 140.6, 131.8, 130.2, 129.8, 126.2, 121.8,
d
183.2, 158.9, 154.0, 151.9, 145.1, 134.2, 125.2, 122.6, 119.6, 115.0,
114.4, 112.4, 108.2, 21.6 ppm; HRMS (ESI): m/z 253.0864 [M þ H]þ
114.4, 114.0, 107.9 ppm; HRMS (ESI): m/z 254 [M þ H]þ (100); HPLC
analysis: 76:24 methanolewater, 4.46 min, 88.8% in purity. Purifi-
cation of 14 is very difficult and we did not get more pure 14. In
HPLC analysis, the E-isomer was suspected as a major impurity.
(100), calcd for C16H13O3, 253.0865,
85:15 methanolewater, 7.98 min, 98.6% in purity.
D
¼ ꢀ0.1 mmu; HPLC analysis:
5.1.4.16. (Z)-5-Hydroxy-2-(4-isopropylbenzylidene)benzofuran-3
(2H)-one (20). Starting from 4-isopropylbenzaldehyde and 4;
yield: 72%; yellow solid; M.p. 168e169 ꢁC; 1H NMR (400 MHz,
5.1.4.11. (Z)-5-Hydroxy-2-(4-dimethylaminobenzylidene)benzo-
furan-3(2H)-one (15). Starting from 4-dimethylaminobenzalde-
hyde and 4; yield: 69%; red brown solid; M.p. 263e264 ꢁC; 1H NMR
DMSO-d6):
d
9.78 (s, 1H, 5-OH), 7.89 (d, J ¼ 8.0 Hz, 2H, H-20, H-60),
7.39 (d, J ¼ 8.8 Hz, 1H, H-7), 7.37 (d, J ¼ 8.8 Hz, 2H, H-30, H-50), 7.22
(dd, J ¼ 2.4, 8.8 Hz, 1H, H-6), 7.03 (d, J ¼ 2.8 Hz, 1H, H-4), 6.85 (s, 1H,
]CH), 2.93 (m, 1H, 40-CH(CH3)2), 1.22 (d, J ¼ 6.8 Hz, 6H, 40-CH
(400 MHz, DMSO-d6):
d
9.68 (s, 1H, 5-OH), 7.82 (d, J ¼ 8.8 Hz, 2H, H-
20, H-60), 7.35 (d, J ¼ 8.8 Hz, 1H, H-7), 7.17 (dd, J ¼ 2.8, 8.8 Hz, 1H, H-
6), 6.99 (d, J ¼ 2.8 Hz, 1H, H-4), 6.82 (s, 1H, ]CH), 6.80 (d, J ¼ 8.8 Hz,
2H, H-30, H-50), 3.02 (s, 6H, 40-N(CH3)2) ppm; 13C NMR (100 MHz,
(CH3)2) ppm; 13C NMR (100 MHz, DMSO-d6):
d 184.3, 159.7, 154.3,
151.3, 147.3, 131.9, 130.1, 127.5, 126.2, 121.8, 114.3, 112.4, 108.2, 33.9,
DMSO-d6):
d
183.2, 158.9, 154.0, 151.7, 145.4, 133.8, 125.2, 122.5,
24.0 ppm; HRMS (ESI): m/z 281.1182 [M þ H]þ (100), calcd for
119.6, 114.6, 114.1, 112.4, 107.8, 40.1 ppm; HRMS (ESI): m/z 282.1129
C18H17O3, 281.1178,
anolewater, 27.92 min, 98.2% in purity.
D
¼ 0.4 mmu; HPLC analysis: 76:24 meth-
[M þ H]þ (100), calcd for C17H16NO3, 282.1130,
D
¼ ꢀ0.1 mmu;
HPLC analysis: 76:24 methanolewater, 13.63 min, 98.2% in purity.
5.1.4.17. (Z)-5-Hydroxy-2-(4-tert-butylbenzylidene)benzofuran-3
(2H)-one (21). Starting from 4-tert-butylbenzaldehyde and 4;
yield: 82%; pale yellow solid; M.p. 201e202 ꢁC; 1H NMR (400 MHz,
5.1.4.12. (Z)-5-Hydroxy-2-(4-diethylaminobenzylidene)benzofuran-3
(2H)-one (16). Starting from 4-diethylaminobenzaldehyde and 4;
yield: 76%; red brown solid; M.p. 198e199 ꢁC; 1H NMR (400 MHz,
DMSO-d6):
d
9.80 (s, 1H, 5-OH), 7.89 (d, J ¼ 8.4 Hz, 2H, H-20, H-60),
DMSO-d6):
d
9.67 (s, 1H, 5-OH), 7.81 (d, J ¼ 8.8 Hz, 2H, H-20, H-60),
7.51 (d, J ¼ 8.4 Hz, 2H, H-30, H-50), 7.38 (d, J ¼ 8.8 Hz, 1H, H-7), 7.22
(dd, 1H, J ¼ 2.8, 8.8 Hz, H-6), 7.03 (d, J ¼ 2.4 Hz, 1H, H-4), 6.85 (s, 1H,
]CH), 1.29 (s, 9H, 40-C(CH3)3) ppm; 13C NMR (100 MHz, DMSO-d6):
7.34 (d, J ¼ 8.8 Hz,1H, H-7), 7.16 (dd, J ¼ 2.4, 8.8 Hz,1H, H-6), 6.99 (d,
J ¼ 2.4 Hz, 1H, H-4), 6.80 (s, 1H, ]CH), 6.77 (d, J ¼ 8.8 Hz, 2H, H-30,
H-50), 3.43 (q, J ¼ 6.8 Hz, 4H, 40-N(CH2CH3)2), 1.13 (t, J ¼ 6.8 Hz, 6H,
d
184.3, 159.7, 154.3, 153.4, 147.4, 131.7, 129.8, 126.3, 126.2, 121.8,
40-N(CH2CH3)2) ppm; 13C NMR (100 MHz, DMSO-d6):
d
183.0, 158.8,
114.3, 112.3, 108.2, 35.1, 31.3 ppm; HRMS (ESI): m/z 295.1337
154.0,149.3,145.2, 134.2, 125.1, 122.6, 118.8,114.8,114.0,112.0, 107.8,
[M þ H]þ (100), calcd for C19H19O3, 295.1334,
D
¼ 0.3 mmu; HPLC
44.3, 12.9 ppm; HRMS (ESI): m/z 310.1441 [M þ H]þ (100), calcd for
analysis: 79:21 methanolewater, 30.26 min, 98.8% in purity.
C19H20NO3, 310.1443,
anolewater, 21.30 min, 99.9% in purity.
D
¼ ꢀ0.2 mmu; HPLC analysis: 76:24 meth-
5.1.4.18. (Z)-5-Hydroxy-2-(4-acetamidobenzylidene)benzofuran-3
(2H)-one (22). Starting from 4-acetamidobenzaldehyde and 4; yield:
58%; yellow solid; M.p. 309e310 ꢁC; 1H NMR (400 MHz, DMSO-d6):
5.1.4.13. (Z)-5-Hydroxy-2-(4-(1-piperidinyl)benzylidene)benzofuran-
3(2H)-one (17). Starting from 4-(1-piperidinyl)benzaldehyde and
4; yield: 88%; brown solid; M.p. 200e201 ꢁC; 1H NMR (400 MHz,
d
10.23 (s, 1H, 5-OH), 7.92 (d, J ¼ 8.8 Hz, 2H, H-20, H-60), 7.72 (d,
J ¼ 8.8 Hz, 2H, H-30, H-50), 7.38 (d, J ¼ 8.8 Hz,1H, H-7), 7.21 (dd, J ¼ 2.8,
8.8 Hz,1H, H-6), 7.02 (d, J ¼ 2.4 Hz,1H, H-4), 6.82 (s,1H, ]CH), 3.16 (s,
1H, 40-NHCOCH3), 2.08 (s, 3H, 40-NHCOCH3) ppm; 13C NMR (100 MHz,
DMSO-d6):
d
9.70 (s, 1H, 5-OH), 7.81 (d, J ¼ 8.8 Hz, 2H, H-20, H-60),
7.36 (d, J ¼ 8.8 Hz, 1H, H-7), 7.18 (dd, J ¼ 2.8, 8.8 Hz, 1H, H-6),
6.99e7.01 (m, 3H, H-30, H-50, H-4), 6.81 (s, 1H, ]CH), 3.35 (m, 4H,
40-N(CH2)2), 1.59 (m, 6H, CH2eCH2eCH2) ppm; 13C NMR (100 MHz,
DMSO-d6): d 184.1, 169.2, 159.6, 154.4, 146.9, 141.4, 132.7, 127.1, 126.1,
121.9, 119.6, 114.2, 112.3, 108.2, 24.6 ppm; HRMS (ESI): m/z 296.0925
DMSO-d6):
d
183.4, 159.1, 154.1, 152.3, 145.8, 133.7, 125.4, 122.4,
[M þ H]þ (100), calcd for C17H14NO4, 296.0923,
D
¼ 0.2 mmu; HPLC
121.0, 114.7, 114.0, 113.9, 108.0, 48.4, 25.4, 24.4 ppm; HRMS (ESI): m/
analysis: 76:24 methanolewater, 5.28 min, 94.6% in purity.
z 322.1447 [M þ H]þ (100), calcd for C20H20NO3, 322.1443,
D
¼ 0.4 mmu; HPLC analysis: 76:24 methanolewater, 27.94 min,
5.1.4.19. (Z)-5-Methoxy-2-(4-dimethylaminobenzylidene)benzo-
furan-3(2H)-one (23). Starting from 4-dimethylaminobenzalde-
hyde and 3; yield: 69%; red brown solid; M.p. 153e154 ꢁC; 1H NMR
99.8% in purity.
5.1.4.14. (Z)-5-Hydroxy-2-(4-(1-pyrrolidinyl)benzylidene)benzo-
furan-3(2H)-one (18). Starting from 4-(1-pyrrolidinyl)benzalde-
hyde and 4; yield: 86%; brown solid; decomposede300 ꢁC; 1H NMR
(400 MHz, DMSO-d6):
d
7.83 (d, J ¼ 8.8 Hz, 2H, H-20, H-60), 7.46 (d,
J ¼ 8.8 Hz, 1H, H-7), 7.33 (dd, J ¼ 2.8, 8.8 Hz, 1H, H-6), 7.21 (d,
J ¼ 2.8 Hz, 1H, H-4), 6.87 (s, 1H, ]CH), 6.80 (d, J ¼ 8.8 Hz, 2H, H-30,
H-50), 3.81 (s, 3H, 5-OCH3), 3.02 (s, 6H, 40-N(CH3)2) ppm; 13C NMR
(400 MHz, DMSO-d6):
d
9.70 (s, 1H, 5-OH), 7.81 (d, J ¼ 8.4 Hz, 2H, H-
20, H-60), 7.35 (d, J ¼ 8.8 Hz, 1H, H-7), 7.16 (dd, J ¼ 2.8, 8.8 Hz, 1H, H-
6), 6.99 (d, J ¼ 2.8 Hz, 1H, H-4), 6.81 (s, 1H, ]CH), 6.63 (d, J ¼ 8.8 Hz,
2H, H-30, H-50), 3.32 (t, J ¼ 6.4 Hz, 4H, 40-N(CH2)2), 1.97 (m, 4H,
(100 MHz, DMSO-d6): d 182.9, 160.0, 156.1, 151.9, 145.4, 133.9, 125.4,
122.5, 119.5, 115.0, 114.3, 112.5, 105.8, 56.4, 40.0 ppm; MS (ESI): m/z
296 [M þ H]þ (100). HPLC analysis: 76:24 methanolewater,
29.47 min, 98.5% in purity.
CH2CH2) ppm; 13C NMR (100 MHz, DMSO-d6):
d 183.0, 158.8, 153.9,

5956
H. Cheng et al. / European Journal of Medicinal Chemistry 45 (2010) 5950e5957
5.1.4.20. (Z)-5-Methoxy-2-(4-diethylaminobenzylidene)benzofuran-
3(2H)-one (24). Starting from 4-diethylaminobenzaldehyde and 3;
yield: 52%; red brown solid; M.p. 120e121 ꢁC; 1H NMR (400 MHz,
(CH3)3) ppm; 13C NMR (100 MHz, DMSO-d6):
d 183.5, 169.7, 163.2,
153.9, 147.2, 147.0, 131.9, 131.7, 129.5, 126.4, 122.0, 117.4, 114.5, 113.4,
35.2, 31.3, 21.2 ppm; HRMS (ESI): m/z 337.1439 [M þ H]þ (100),
DMSO-d6):
d
7.81 (d, J ¼ 8.8 Hz, 2H, H-20, H-60), 7.44 (d, J ¼ 8.8 Hz,
calcd for C21H21O4, 337.1440,
methanolewater, 15.40 min, 95.2% in purity.
D
¼ ꢀ0.1 mmu; HPLC analysis: 85:15
1H, H-7), 7.32 (dd, J ¼ 2.4, 8.8Hz, 1H, H-6), 7.20 (d, J ¼ 2.0 Hz, 1H, H-
4), 6.85 (s, 1H, ]CH), 6.76 (d, J ¼ 8.8 Hz, 2H, H-30, H-50), 3.80 (s, 3H,
5-OCH3), 3.42 (q, J ¼ 7.2 Hz, 4H, 40-N(CH2CH3)2), 1.12 (t, J ¼ 7.2 Hz,
5.2. Biology
6H, 40-N(CH2CH3)2) ppm; 13C NMR (100 MHz, DMSO-d6):
d 182.7,
159.8, 156.1, 149.5, 145.2, 134.2, 125.3, 122.5, 118.8, 115.2, 114.3, 112.1,
Human umbilical vein endothelial cells (HUVECs) were isolated
from freshly delivered umbilical cords and HUVECs of 3e8 passages
were used for assays; CCC-HPF-1 cell line was bought from Cell
Center of Institute of Basic Medical Sciences, Chinese Academy of
Medical Sciences; all cancer cell lines were from ATCC; FBS (Fetal
Bovine Serum) for HUVEC and CCC-HPF-1 was obtained from
Hyclone; NBCS (Newborn Calf Serum) for A549 and MCF-7 was
purchased from GIBCO; EGM-2 for HUVEC was from LONZA; RPMI
1640 for A549 and DMEM (Dulbecco’s Modified Eagle’s Medium) for
MCF-7 andCCC-HPF-1 were from Hyclone. Allcells were incubated at
37 ꢁC and 5% CO2. The positive inhibitor for individual cell prolifer-
ation was fumagillin for HUVEC and taxol for A549 and MCF-7. IC50
values for HUVEC and cancer cell lines A549 and MCF-7, or CC50
values for CCC-HPF-1 are summarized in Table 1. Tested compound
was dissolved in DMSO and diluted with PBS into a stock solution
containing 2% DMSO before use. The stock solution of carrier is 2%
DMSO in PBS. The final concentration of DMSO is 0.1% for all wells.
105.7, 56.4, 44.3, 12.9 ppm; HRMS (ESI): m/z 324.1604 [M þ H]þ
(100), calcd for C20H22NO3, 324.1600,
85:15 methanolewater, 17.26 min, 99.6% in purity.
D
¼ 0.4 mmu; HPLC analysis:
5.1.4.21. (Z)-5-Methoxy-2-(4-tert-butylbenzylidene)benzofuran-3
(2H)-one (25). Starting from 4-tert-butylbenzaldehyde and 3;
yield: 48%; pale yellow solid; M.p. 200e201 ꢁC; 1H NMR (400 MHz,
DMSO-d6):
d
7.90 (d, J ¼ 8.4 Hz, 2H, H-20, H-60), 7.52 (d, J ¼ 8.4 Hz,
2H, H-30, H-50), 7.48 (d, J ¼ 8.8 Hz, 1H, H-7), 7.38 (dd, J ¼ 2.8, 8.8 Hz,
1H, H-6), 7.25 (d, J ¼ 2.8 Hz, 1H, H-4), 6.90 (s, 1H, ]CH), 3.81 (s, 3H,
5-OCH3), 1.30 (s, 9H, 40-C(CH3)3) ppm; 13C NMR (100 MHz, DMSO-
d6): d 184.1, 160.9, 156.4, 153.6, 147.4, 131.7, 129.7, 126.4, 126.3, 121.8,
114.6, 112.7, 106.2, 56.5, 35.1, 31.3 ppm; HRMS (ESI): m/z 309.1487
[M þ H]þ (100), calcd for C20H21O3, 309.1491,
D
¼ ꢀ0.4 mmu;
HPLC analysis: 85:15 methanolewater, 27.48 min, 98.7% in purity.
5.1.5. General procedure for acetylation of 15, 16 and 19
1.0 mmol of (Z)-5-hydroxyaurone was dissolved in 20 ml dry
dichloromethane and then 1.2 mmol of acetic anhydride and 2.0 mmol
of triethylamine were added to the solution at the room temperature.
After the reaction mixture was stirred at room temperature for 30 min,
it was poured into water and extracted with ethyl acetate. The
combined organic phase was washed with saturated NaHCO3 solution
and brine and then dried over anhydrous Na2SO4. The organic phase
was evaporated and the residue was purified by silica gel column
chromatography (petroleum ether: ethyl acetate ¼ 10:1).
5.2.1. Anti-proliferation assay
The activities of target compounds against the proliferation of
cancer cells and HUVEC were assayed by the MTT method. Briefly,
1000 cells/well of HUVEC or 1500 cells/well of MCF-7 and CCC-HPF-
1 or 2000 cells/well of A549 in 190
seeded in 96-well plates and allowed to attach or stabilize for 20 h
before being treated with 10 l stock solutions of different
concentration of tested compound or carrier. Every concentration
of the compound or carrier was in triplicate. After incubation for
96 h, cells were treated with MTT reagents and the relative cell
viability of each well was calculated. IC50 or CC50 value of tested
compound was calculated based on the relationship of cell viability
vs the corresponding concentration.
ml corresponding medium were
m
5.1.5.1. (Z)-5-Acetoxy-2-(4-dimethylaminobenzylidene)benzofuran-3
(2H)-one (26). Starting from 15; yield: 89%; red brown solid; M.p.
174e175 ꢁC;1H NMR (400 MHz, DMSO-d6):
d
7.86(d, J ¼ 9.2 Hz,2H, H-
20, H-60), 7.54 (AB, J ¼ 10.0 Hz, 2H, H-6, H-7), 7.52 (s,1H, H-4), 6.93 (s,
1H, ]CH), 6.81 (d, J ¼ 9.2 Hz, 2H, H-30, H-50), 3.03 (s, 6H, 40-N(CH3)2),
5.2.2. HUVEC wound-healing assay
2.29 (s, 3H, 5-COCH3) ppm; 13C NMR (100 MHz, DMSO-d6):
d
182.2,
HUVECs (5 ꢃ 104/well) were allowed to grow into full conflu-
ence in 24-well plates. After cells were wounded by pipette tips, the
media and dislodged cells were aspirated and the plates were
rinsed with PBS. Fresh media with tested compound or carrier were
added to the wells and the cells were incubated for 24 h. The width
of the wound was visualized with Olympus CKX41 inverted
microscope at the magnification of 40.
169.7, 162.3, 152.1, 146.7, 145.2, 134.1, 130.6, 122.7, 119.3, 116.8, 115.8,
114.3, 112.5, 40.0, 21.2 ppm; HRMS (ESI): m/z 324 [M þ H]þ (100);
HPLC analysis: 76:24 methanolewater, 15.86 min, 91.1% in purity.
Purification of 26 is very difficult and we did not get more pure 26. In
HPLC analysis, the E-isomer was suspected as a major impurity.
5.1.5.2. (Z)-5-Acetoxy-2-(4-diethylaminobenzylidene)benzofuran-3
(2H)-one (27). Starting from 16; yield: 92%; red brown solid; M.p.
5.2.3. HUVEC tube formation assay
134e135 ꢁC; 1H NMR (400 MHz, DMSO-d6):
d
7.85 (d, J ¼ 8.8 Hz, 2H,
HUVECs (1 ꢃ 105/well) with tested compound or carrier were
H-20, H-60), 7.54 (AB, J ¼ 9.6 Hz, 2H, H-6, H-7), 7.51 (s, 1H, H-4), 6.91
(s, 1H, ]CH), 6.79 (d, J ¼ 8.8 Hz, 2H, H-30, H-50), 3.44 (q, J ¼ 6.8 Hz,
4H, 40-N(CH2CH3)2), 2.29 (s, 3H, 5-COCH3), 1.13 (t, J ¼ 6.8 Hz, 6H, 40-
cultured in 12-well plates coated with 150 ml Matrigel basement
(the polymerized mixture of 50 ml Matrigel and 100 ml serum-free
DMEM) for 6 h. Endothelial cell tubular structure was observed by
Olympus CKX41 inverted microscope at the magnification of 100.
N(CH2CH3)2) ppm; 13C NMR (100 MHz, DMSO-d6):
d
182.0, 169.8,
162.1, 149.6, 146.5, 144.9, 134.5, 130.5, 122.7, 118.6, 116.9, 116.1, 114.2,
112.0, 44.3, 21.2, 12.9 ppm; HRMS (ESI): m/z 352.1543 [M þ H]þ
5.2.4. Cancer cell in vitro invasion assay
(100), calcd for C21H22NO4, 352.1549,
D
¼ ꢀ0.6 mmu; HPLC anal-
Cancer cell invasion was measured by examining cell numbers
ysis: 76:24 methanolewater, 26.87 min, 98.9% in purity.
through Matrigel (60
m
l ꢃ 250
mg/ml)-coated transwell poly-
carbonate filters. Cells were serum-starved for 24 h and detached
from tissue culture plate by trypsin, washed twice with PBS and
then re-suspended in the medium containing 0.1% FBS. 20000 cells
5.1.5.3. (Z)-5-Acetoxy-2-(4-tert-butylbenzylidene)benzofuran-3(2H)-
one (28). Starting from 21; yield: 90%; pale yellow solid; M.p.
157e158 ꢁC; 1H NMR (400 MHz, DMSO-d6):
d
7.94 (d, J ¼ 8.4 Hz, 2H,
per well (200
(Corning Costar 3422) and the transwell was inserted into a 24-well
plate containing 600 l of the medium with 10% FBS or NBCS. Cells
ml) were seeded in the transwell with 8 mm pores
H-20, H-60), 7.56e7.63 (m, 3H, H-4, H-6, H-7), 7.55 (d, J ¼ 8.4 Hz, 2H,
H-30, H-50), 6.97 (s, 1H, ]CH), 2,29 (s, 3H, 5-COCH3), 1.31 (s, 9H, 40-C
m

H. Cheng et al. / European Journal of Medicinal Chemistry 45 (2010) 5950e5957
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Acknowledgements
This work was supported by Knowledge Innovation Program of
Chinese Academy of Sciences (KSCX-2-YW-R-22), National Basic
Research Program of China (2007CB914504) and Natural Science
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