Synthesis and biological evaluation of hydroxylated 3-phenylcoumarins as antioxidants and antiproliferative agents

Article abstract of DOI:10.1016/j.bmcl.2011.08.090

Based on the observed biological activities of coumarins and resveratrol, we synthesized fourteen hydroxylated 3-phenylcoumarins (stilbene-coumarin hybrids) including six novel ortho-hydroxy-methoxy substituted derivatives, 1-14, by Perkin reaction. We ch

Full text of DOI:10.1016/j.bmcl.2011.08.090

Bioorganic & Medicinal Chemistry Letters 21 (2011) 6420–6425
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and biological evaluation of hydroxylated 3-phenylcoumarins as
antioxidants and antiproliferative agents
Jie Yang, Guo-Yun Liu, Fang Dai, Xiao-Yan Cao, Yan-fei Kang, Li-Mei Hu, Jiang-Jiang Tang, Xiu-Zhuang Li,
⇑
⇑
Yan Li, Xiao-Ling Jin , Bo Zhou
State Key Laboratory of Applied Organic Chemistry, Lanzhou University, Lanzhou, Gansu 730000, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Based on the observed biological activities of coumarins and resveratrol, we synthesized fourteen
hydroxylated 3-phenylcoumarins (stilbene-coumarin hybrids) including six novel ortho-hydroxy-meth-
oxy substituted derivatives, 1–14, by Perkin reaction. We characterized these compounds concerning
their antioxidant activity against 2,20-azobis(2-amidinopropane hydrochloride) (AAPH)-induced
pBR322 DNA strand breakage, and their antiproliferative effects on human promyelocytic leukemia
HL-60 and human lung adenocarcinoma epithelial A549 cells. Structure–activity relationship information
suggests that the introduction of ortho-hydroxy-methoxy groups and ortho-dihydroxy groups on the aro-
matic A ring could efficiently improve antiproliferative activity. Interestingly, a new derivative, 6-meth-
oxy-7-hydroxy-3-(40-hydroxyphenyl)coumarin, 9, behaved as a poor antioxidant but appeared to be the
most potent antiproliferative agent among the compounds examined, and this activity was mediated by
deregulation in cell cycle and induction of apoptosis.
Received 20 April 2011
Revised 10 June 2011
Accepted 19 August 2011
Available online 25 August 2011
Keywords:
Resveratrol
Coumarin
Hybrid
Antioxidant
Antiproliferative effect
Apoptosis
Ó 2011 Elsevier Ltd. All rights reserved.
Coumarins (1,2-benzopyrones) are an important family of natu-
rally occurring compounds, and are being extensively investigated
because of their wide rage of biological activities, such as
anticancer,1 enzyme inhibition,2,3 vasorelaxant,4 antimicrobial,5,6
antioxidant,7 and anti-inflammatory.4,7 and anti-HIV.8 Most of
coumarins are C7-hydroxylated in nature.9,10 Especially, scopoletin
(6-methoxy-7-hydroxycoumarin) and esculetin (6,7-dihydroxy-
coumarin) were reported to exhibit an antiproliferative effect on
leukemic cells by inducing apoptosis.11,12
Stilbenes are also a class of natural products derived from the
phenylpropanoid pathway, and act as phytoalexins to defend plants
against pathogen attack or environmental stress.13 Over the course
of the past decade, one of the most extensively studied stilbenes is
the 3,5,40-trihydroxy-trans-stilbene, known as resveratrol which is
found in grapes and other food products. This molecule is endowed
with several remarkable biological properties including antioxidant
and cancer chemoprevention,14–16 and its simplicity in structure has
resulted in extensive effort to find more active antioxidants and can-
cer chemoprevention agents by synthesizing its analogs.16,17 We re-
cently synthesized resveratrol analogs by the introduction of
electron-donating (ED) groups in the ortho- or para-position of 4-
OH or 40-OH18,19 and elongation of the conjugated links,20 and found
that the two structural modifications are the important strategies to
improve the antioxidant and antiproliferative activities of the
resveratrol. For instance, a resveratrol analogue, 4,40-dihydroxy-
trans-stilbene (4,40-DHS), which contains two OH in 4 and 40 posi-
tions, exhibited stronger antioxidant activity and antiproliferative
activity against human promyelocytic leukemia HL-60 cells than
resveratrol.19 The two activities were further strengthened by elon-
gation of the conjugated links as examplified in a triene compound
(trans,trans,trans-1,6-bis(4-hydroxyphenyl)-1,3,5-hexatriene) be-
aring the same 4,40-dihydroxy groups.20 Additionally, incorporating
or linking the skeletons of two or more kinds of natural products
with similar biological properties by the hybrid approach, has re-
cently attracted much attention and opened new avenues in drug
discovery.21 We have also constructed resveratrol analogs by incor-
porating a chroman moiety of vitamin E, leading to the remarkable
increase in radical-scavenging activity.22
In view of these similar properties of resveratrol and coumarins,
it is of interest to design and synthesize stilbene-coumarin hybrids
as examplified in 3-phenylcoumarins, in which 3,4-double bond of
the coumarin nucleus fixes the trans configuration of the stilbene
double bond.2,23–25 It has been reported that 3-phenylcoumarins
can be characterized as potent and selective monoamine oxidase-
B inhibitors,23,24 vasorelaxants and platelet antiaggregants25 as
well as horseradish peroxidase catalytic activity inhibitors.2
Encouraged by the aforementioned information, in the present
work we synthesized fourteen hydroxylated 3-phenylcoumarins,
1–14, including six novel ortho-hydroxy-methoxy substituted
derivatives (9–14) with the aim of developing some compounds
with antioxidant and antiproliferative activities superior to that
⇑
Corresponding authors. Fax: +86 931 8915557.
E-mail addresses: jinxling@lzu.edu.cn (X.-L. Jin), bozhou@lzu.edu.cn (B. Zhou).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.08.090

J. Yang et al. / Bioorg. Med. Chem. Lett. 21 (2011) 6420–6425
6421
of resveratrol, and understanding the structure–activity relation-
ship. In the compounds, different numbers and positions of hydro-
AAPH-induced DNA strand breakage depended on the specific
compound used, and the majority of the compounds showed better
activity than resveratrol. On the basis of the density of intact
supercoiled DNA and damaged open circular and linear DNA, the
xy group were introduced to aromatic
A and/or B ring(s).
Especially, most of them contained ortho-hydroxy-methoxy groups
and ortho-dihydroxy groups because the introduction of ED groups
such as methoxy and hydroxy in the ortho position of OH, helps in-
crease antioxidant activity.26
activity followed the order:
6
(6,7,40-trihydroxy groups) ꢀ 7
(6,7-dihydroxy groups) ꢀ 8 (6,7,30,40-tetrahydroxy groups) > 12
(6,7,40-trihydroxy-3-methoxy groups) ꢀ 13 (7,40-dihydroxy-6,8-
dimethoxy groups) ꢀ 14 (7-hydroxy-6,8,30,40,50-pentamethoxy
groups) > 10 (7,40-dihydroxy-30-methoxy groups) ꢀ 11 (7,40-dihy-
droxy-6,30-dimethoxy groups) ꢀ 5 (7,30,40-trihydroxy groups) > 3
(7,40-dihydroxy groups) ꢀ 4 (7,30,50-trihydroxy groups) ꢀ resvera-
The overall strategy for the synthesis of hydroxylated 3-phen-
ylcoumarins is outlined in Scheme 1. Methoxy and/or acetoxyl
substituted 3-phenylcoumarins were prepared from the corre-
sponding phenyl acetic acid sodium and salicylaldehyde in dry ace-
tic anhydride by Perkin reaction.2,27 Hydrolysis of acetoxyl group
was performed with excess NH3 solution, giving the methoxy
and/or hydroxy substituted 3-phenylcoumarins, 1–14. For syn-
thetic details and characterization of all the hydroxylated 3-phen-
ylcoumarins, as well as experimental details of the subsequent
biological evaluation, please see the Supplementary data.
Free radical-mediated oxidative damage of DNA has been sug-
gested to be a major factor in the development of cancer, thus,
we first evaluated their antioxidant activity against 2,20-azobis(2-
amidinopropane hydrochloride) (AAPH)-induced plasmid pBR322
DNA strand breakage using agarose gel electrophoresis analysis
( Fig. 1).28 DNA samples were incubated with 10 mM AAPH at
37 °C for 1 h and subjected to agarose gel electrophoresis. The na-
tive DNA was mostly in its supercoiled form associated with small
amount of open circular form depending on the batch (Fig. 1A and
B, native lane). In the presence of AAPH, the supercoiled DNA was
transformed completely into its open circular and linear forms,
trol > 9
(7,40-dihydroxy-6-methoxy groups) > 1
(7-hydroxy
group) > 2 (40-hydroxy group). Among them, the compounds (6,
7, and 8) bearing ortho-dihydroxy groups on aromatic A ring were
the most active, and completely protected plasmid DNA from
strand breakage induced by AAPH. The higher activity of the com-
pounds bearing ortho-dihydroxy groups is usually explained by
stabilization of ortho-hydroxyphenoxyl radical through formation
of an intramolecular hydrogen bond.26 A comparison of the activity
for 6 and 5, indicates clearly that ortho-dihydroxy groups on the
aromatic A ring are more active than that on the aromatic B ring.
The same tendency was also observed between monohydroxy
derivatives (1 and 2), in which hydroxy group on the A ring is more
effective than that on the B ring. Additionally, compound 3 was less
potent than the compounds 10, 11, 13, and 14, suggesting that the
introduction of methoxy group in the ortho-position of hydroxy
group increases significantly the activity. However, compound 9
bearing ortho-hydroxy-methoxy groups was an exception, and it
appeared to be less active.
indicating
a single and double strand breakage, respectively
(Fig. 1A and B, control lane). It can be seen from Figure 1 that
antioxidative effect of hydroxylated 3-phenylcoumarins against
We next investigated their antiproliferative effects
against human promyelocytic leukemia HL-60 and human lung
Scheme 1. Synthesis of hydroxylated 3-phenylcoumarins, 1–14. Reagents and conditions: (a) dry acetic anhydride, reflux, 8 h; (b) NH3 solution (15%), ethanol, rt, 12 h.

6422
J. Yang et al. / Bioorg. Med. Chem. Lett. 21 (2011) 6420–6425
Figure 1. Protection against AAPH-induced pBR322 DNA strand breakage by hydroxylated 3-phenylcoumarins. DNA (100 ng/25
ll) was incubated with 10 mM AAPH and
20 M hydroxylated 3-phenylcoumarins at 37 °C for 1 h in 10 mM PBS (pH 7.4). Lanes: native, only DNA; control, DNA and AAPH; others, DNA, AAPH and compounds 1–14 or
l
resveratrol (res), respectively.
Figure 2. Effect of 9 on morphology of A549 cells. A549 cells were treated with culture medium, 5, 10, and 15 lM of 9, respectively (A–D) for 24 h prior to analysis of cell
morphology using Hoechst 33258/propidium iodide nuclear staining and fluorescence microscopy (original magnification, 200ꢁ). Living cells was blue-stained with smooth
appearance; early apoptotic cells was blue-stained with bright specks of condensed chromatin. Data were representative of three separate experiments.
adenocarcinoma epithelial A549 cells by MTT assay29 and the IC50
values are summarized in Table 1. Compounds 6, 7, 8, 9, and 11
exhibited higher antiproliferative activity than resveratrol on HL-
60 and A549 cells. A comparison of the antiproliferative activity
for compounds 5–8 or 9–11, suggests clearly that ortho-dihydroxy
or ortho-hydroxy–methoxy groups on the aromatic A ring contrib-
utes critically the activity. It can also be seen from Table 1 that the
compounds (i.e., 6–8) with high antioxidant activity exhibited spe-
cific antiproliferative activity, whereas the compounds (i.e., 1–3)
with weak antioxidant activity were inactive in the antiprolifera-
tive effects. However, interestingly, a new derivative 9 bearing
ortho-hydroxy-methoxy groups on the aromatic A ring, behaved
as a poor antioxidant but displayed the highest potency among
Therefore, there is not a very direct relationship between the anti-
proliferative activity and antioxidant ability.
Further, to determine whether the excellent antiproliferative
activity of 9 is due to apoptotic cell death, A549 cells were treated
with the compound (5–15 lM) for 24 h, and change in nuclear
morphology was observed by fluorescence microscopy using
hoechst 33258/propidium iodide staining according to the
reported procedure with some modifications.30–32 As shown in
Figure 2, 9 altered significantly cell morphology with respect to
those of control cultures, and caused a significant reduction in
the level of living cell and a clear early apoptosis characteristic
by blue-stained cells with bright specks of condensed chromatin,
in a concentration-dependent manner. Finally, to explore whether
9 has a cell cycle arrest effect in A549 cells, the cells treated with
different concentrations of the compound for 24 h were subjected
to flow cytometric analysis after propidium iodide staining.32 It
all compounds tested with IC50 values of 5.2 and 7.5 lM in HL-60
and A549 cells. The compound was about 7 and 16 times more
active than resveratrol in HL-60 and A549 cells, respectively.

J. Yang et al. / Bioorg. Med. Chem. Lett. 21 (2011) 6420–6425
6423
Figure 3. A549 cell cycle changes in response to treatment of 9. After A549 cells were incubated with culture medium, 10, 15, 20, 30, and 40
l
M 9 for 24 h, cell cycle
distribution was determined using flow cytometry with propidium iodide staining expressed as percent of G0/G1, S, and G2/M cycle phases. Data were representative of three
separate experiments.
Table 1
Antiproliferative activity of hydroxylated 3-phenylcoumarins on HL-60 and A549 cellsa
Compounds
IC50
(lM)
Compounds
IC50
(lM)
HL-60
>200
A549
HL-60
A549
1
>200
>200
>200
9
5.2 0.6
7.5 0.6
2
3
>200
>200
10
11
91.7 1.8
24.3 0.6
>200
21.4 1.8
(continued on next page)

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J. Yang et al. / Bioorg. Med. Chem. Lett. 21 (2011) 6420–6425
Table 1 (continued)
Compounds
IC50
(lM)
Compounds
IC50
(lM)
HL-60
A549
HL-60
A549
b
4
5
127.0 0.1
>200
12
—
43.4 3.9
42.1 4.0
85.0 1.6
13
14
25.1 1.5
45.8 3.1
6
7
25.0 0.1
42.5 2.7
72.2 4.5
>200
14.0 1.2
22.1 2.0
27.0 1.3
48.8 6.3
36.3 1.8c
119.6 6.9
8
a
HL-60 and A549 cells were treated with hydroxylated 3-phenylcoumarins for 48 and 72 h, respectively. Antiproliferative activity is expressed as IC50 values, the
concentration for the compound to cause 50% inhibition of cell proliferation. Data are expressed as the mean SD for three determinations.
b
The accurate IC50 values could not be measured because of the formation of other color compounds in the experiments.
Cited from Ref. 19.
c
can be seen from Figure 3 that 9-treated cells were remarkably
blocked in the G2/M phase depending on its doses, and this
change in G2/M phase was accompanied by a decrease in the
proportion of cells in G0/G1 phase. Specifically, 9 increased the
percentage of cells in the G2/M phase to 16.6, 40.65, 70.08,
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2011.08.090.
80.95 and 84.03% at 10, 15, 20, 30, and 40 lM, respectively. The
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This work was supported by the National Natural Science Foun-
dation of China (Grant Nos. 20972063), the 111 Project, and the
Fundamental Research Funds for the Central Universities
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