A boronic acid chalcone analog of combretastatin A-4 as a potent anti-proliferation agent

Article abstract of DOI:10.1016/j.bmc.2009.11.003

Chalcones represent a class of natural products that inhibits tubulin assembly. In this study we designed and synthesized boronic acid analogs of chalcones in an effort to compare biological activities with combretastatin A-4, a potent inhibitor of tubuli

Full text of DOI:10.1016/j.bmc.2009.11.003

Bioorganic & Medicinal Chemistry 18 (2010) 971–977
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
A boronic acid chalcone analog of combretastatin A-4 as a potent
anti-proliferation agent
Yali Kong a, Kan Wang a, Michael C. Edler b, Ernest Hamel b, Susan L. Mooberry c, Mikell A. Paige a,
Milton L. Brown a,
*
a Department of Oncology, 3970 Reservoir Road, Drug Discovery Program, Georgetown University Medical Center, Washington DC 20057, United States
b Toxicology and Pharmacology Branch, Developmental Therapeutics Program, Division of Cancer Treatment and Diagnosis,
National Cancer Institute at Frederick, National Institute of Health, Frederick, MD 21702, United States
c Department of Physiology and Medicine, Southwest Foundation for Biomedical Research, San Antonio, TX 78245, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Chalcones represent a class of natural products that inhibits tubulin assembly. In this study we designed
and synthesized boronic acid analogs of chalcones in an effort to compare biological activities with com-
bretastatin A-4, a potent inhibitor of tubulin polymerization. Systematic evaluation of the positional
effects of the carbonyl moiety towards inhibition of tubulin polymerization, cancer cell proliferation
and angiogenesis revealed that placement of the carbonyl adjacent to the trimethoxybenzene A-ring
resulted in more active compounds than when the carbonyl group was placed adjacent to the C-ring.
Our study identified a boronic acid chalcone with inhibition towards 16 human cancer cell lines in the
10–200 nM range, and another three cell lines with GI50-values below 10 nM. Furthermore, this drug
has significant anti-angiogenesis effects demonstrated by HUVEC tube formation and aortic ring assay.
Ó 2009 Elsevier Ltd. All rights reserved.
Received 7 October 2009
Revised 30 October 2009
Accepted 2 November 2009
Available online 10 November 2009
Keywords:
Chalcone
Boronic acid
Combretastatin A-4
Tubulin polymerization
Anti-tumor agent
1. Introduction
has led to the development of a number of diverse ligands designed
to mimic CA-4. Ikeda et al.7 and Edwards et al.8 reported that chal-
Compounds with a chalcone-based structure have promise as
agents for the treatment of human cancers. Moreover, they serve
as precursors in the biosynthesis of flavonoids. The chalcone fla-
vokawain A from kava extracts has strong anti-proliferative and
apoptotic effects against human bladder cancer cells.1 Moreover
there are several reports demonstrating antiinflammatory,2 anti-
bacterial3 and anticancer4 activities of chalcones. Due to their
abundance in plants and ease of synthesis, the chalcone class of
compounds has provoked great interest for possible therapeutic
uses.
The microtubule system of eukaryotic cells is an important tar-
get for the development of antitumor agents. Antimitotic agents
cause mitotic arrest in eukaryotic cells by interfering with the nor-
mal microtubule polymerization/depolymerization process. Fur-
thermore, these agents provide new perspectives on the
inhibition of tumor cell growth and the suppression of cancer.
Combretastatin A-4 (CA-4) is a structurally very simple natural
product with potent cytotoxic activity (Fig. 1).5 The mechanism
of action of CA-4 involves reversible, high affinity binding in the
colchicine site of tubulin.6 The ease of synthesis of CA-4 analogs
cone analogs of CA-4 are potent inhibitory agents toward human
cancer cells. Boronic acid-chalcone analogs have previously been
reported in the literature as fluorescent probes9 and antitumor
agents.10 Boronic acid derivatives are of considerable interest in
drug development because of their moderate pH of 9–10 and air-
stability. VELCADEÒ, a modified dipeptidyl boronic acid was ap-
proved by the FDA in 2003 for the treatment of multiple myeloma
disease. Previously we designed boronic acid CA-4 analog 1 (Fig. 1),
which was found to have potent anti-tubulin, anti-cancer activity,
and have improved solubility compared to CA-4.11 This led us to
explore chalcone analogs incorporating the phenylboronic acid C-
ring of analog 1.
We designed chalcone analogs 4, 6,4,7 11, and 14 as carbonyl-
expanded analogs of CA-4 (Fig. 1). Analogs 11 and 14 are car-
bonyl-inverse analogs of chalcones 4 and 6. Analogs 4 and 11 are
analogs of our previously reported boronic acid analog 1, whereas
analogs 6 and 14 are directly derived from CA-4. We synthesized
and evaluated these analogs for effects on tubulin polymerization,
inhibition of [3H]colchicine binding to tubulin, disruption of the
microtubule network of A-10 cells, and inhibition of the growth
of human MCF-7 breast cancer cells. Boronic acid analog 4 was also
evaluated by the National Cancer Institute against a panel of 54 hu-
man cancer cell lines in an effort to survey activity against a broad
range of human cancers.
* Corresponding author. Tel.: +1 202 960 8769; fax: +1 202 687 7659.
E-mail address: mb544@georgetown.edu (M.L. Brown).
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.11.003

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Y. Kong et al. / Bioorg. Med. Chem. 18 (2010) 971–977
H3CO
A
H3CO
H3CO
A
H3CO
C
C
OH
OCH3
OCH3
B
OH
OH
OCH3
OCH3
1
Combretastatin A-4 (CA-4)
O
O
H3CO
A
H3CO
H3CO
C
C
A
R
H3CO
R
OCH3
OCH3
OCH3
OCH3
11
4
, R = -B(OH)2
, R = -B(OH)2
14, R = -OH
6, R = -OH
Figure 1. Chalcone analogs of CA-4.
Figure 2 demonstrates the effects of compounds 4 and 6 on the
microtubule networks of A-10 cells (Fig. 2). In the control (Fig. 2A),
both the microtubule network and nuclei of the cells are clearly
visible. Following the treatment with boronic acid 4, extensive
thinning of the network of microtubules occurred. In contrast,
2. Results and discussion
2.1. Chemistry
The Claisen–Schmidt condensation was used as a general proce-
dure to synthesize the chalcone analogs (Scheme 1). Base-pro-
moted condensation was found to give the best results for
chalcone synthesis.12,13
20 lM of 6 caused complete disappearance of the microtubule net-
work, with the disassembled tubulin, following its reaction with
the antibodies used in the indirect immunofluorescence assay. This
result is consistent with the relative activities of 4 and 6 as inhib-
itors of tubulin polymerization (Table 1).
Chalcone analogs 4 and 6 were obtained under base-pro-
moted condensation from ketone 2 and the appropriately substi-
tuted aldehyde (Scheme 2A and B). Chalcone 11 was prepared
from 1-bromo-2-methoxybenzene (7) by Friedel–Crafts acyla-
tion,14 followed by ketone protection, lithiation, treatment with
trimethylborate, and acidic work-up to afford 10 in 77% yield.
Standard Claisen–Schmidt condensation gave the substituted
chalcone 11 in 57% yield (Scheme 2C). Analog 14 was synthe-
sized from 3,4-dimethoxyphenyl ethanone 12. Selective demeth-
ylation of 12 by acid-promoted hydrolysis afforded substituted
phenol 13 as a white solid in 43% yield.15 Standard Claisen–
Schmidt condensation gave the substituted chalcone 14 in 68%
yield (Scheme 2D).
To further survey the potential activity spectrum of boronic acid
chalcone 4, the compound was sent to the National Cancer Insti-
tute for evaluation in the human cancer cell screen.16 Data was ob-
tained for 54 human cancer cell lines (Table 2). Boronic acid
chalcone 4 yielded GI50’s between 10 and 200 nM in 16 of the 54
cell lines, and GI50’s below 10 nM in three lines (colon cancer
HCT-15, CNS cancer SNB-19, and ovarian cancer SK-OV-3.
The data shown in Table 1 above demonstrates that boronic
acid chalcone analog 4 is not a potent tubulin inhibitor. However,
the highly cytotoxic activity toward the NCI panel of human cancer
cells suggests it may have a different mechanism for cytotoxicity
and further mechanistic studies are warranted.
We also tested the boronic acid compound 4 on the effect of
MCF-7 cell cycle (Fig. 3). Based on this experiment, compound 4
did not cause significant G2/M phase arrest at the concentration
2.2. Biological data
The biological activities of the chalcone analogs of CA-4 are
summarized in Table 1. The data showed that the known chalcone
6 was the most potent inhibitor, with activity comparable with
that of CA-4 in the tubulin assays. However, compound 6 was 3–
4 fold less active than CA-4 as an inhibitor of MCF-7 cell growth
and much less active in disrupting the microtubules of A-10 cells.
Boronic acid chalcone analog 4 was weakly active as an inhibitor of
colchicine binding and of tubulin polymerization, but nevertheless
had significant cytotoxic activity against the MCF-7 cancer cells
as high as 5 lM, far above the IC50 (Table 1). The results further
support the inhibitory effect on cancer cell proliferation was not
realized through interfering tubulin assembly, which distinguished
this compound from the regular anti-tubulin drugs.
We also tested the anti-angiogenesis property of compound 4 in
HUVEC cells. Interestingly, this compound significantly inhibited
the tube formation at the concentration of 1
assay also showed the compound 4 dramatically inhibited func-
tional angiogenesis at a concentration of 5 M (Fig. 5). The cancer
lM (Fig. 4). Aortic ring
l
(IC50, 0.9 lM). SAR analysis shows that placement of the carbonyl
vascular cells do not generate drug resistance as often as the cancer
epithelium cells, and thus the discovery on drugs targeting angio-
genesis is a very promising approach.
adjacent to the A-ring results in more active analogs than place-
ment adjacent to the C-ring. In all assays, 4 was more active than
11 and 6 was more active than 14.
O
O
O
NaOH
EtOH
H
R2
+
R2
R1
R1
Scheme 1. General synthesis of chalcone analogs.

Y. Kong et al. / Bioorg. Med. Chem. 18 (2010) 971–977
973
A
O
O
H3CO
OHC
B(OH)2
H3CO
a
B(OH)2
OCH3
+
H3CO
OCH3
H3CO
OCH3
OCH3
3
4
2
B
O
O
H3CO
OHC
OH
H3CO
H3CO
OH
a
+
H3CO
OCH3
OCH3
OCH3
2
OCH3
6
5
C
O
O
O
Br
Br
OCH3
Br
b
a
OCH3
OCH3
8
7
9
O
O
H3CO
H3CO
B(OH)2
OCH3
B(OH)2
OCH3
c
d
OCH3
10
11
D
O
O
O
OH
H3CO
H3CO
OH
OCH3
OCH3
b
a
OCH3
OCH3
OCH3
13
12
14
Scheme 2. Synthesis of chalcone analogs.
Table 1
identified as a potent inhibitor of human cancer in cell prolifera-
tion and angiogenesis. Positional addition of boronic acid elimi-
nated tubulin activity, but produced nanomolar cytotoxicity,
warranting further studies on the mechanism of action and
in vivo anti-tumor effects of this new boronic acid.
Inhibition of tubulin polymerization, growth of MCF-7 human breast carcinoma cells,
and the binding of [3H]colchicine to tubulin, and EC50’s for loss of microtubules in A-
10 cells
Compound Tubulina
IC50
MCF-7b IC50
M) SD
[3H]Colchicine
binding inhibitionc
(% SD)
A-10 EC50
(lM)
d
(
l
(lM) SD
4. Experimental
5
l
M
50
lM
4
6
11
14
CA-4
31 3.4
2.6 0.2
>40
>40
2.0 0.1
0.9 0.18
0.11 0.024
>2.5
4.4 8.6 56 8.6
16.5
101 5.5 0.98
4.1. Chemistry general methods
88 6.3
N/D
N/D
N/D
N/D
N/D
>15
18
<0.025
NMR spectra were recorded using a Varian-300 spectrometer
for 1H (300 MHz) and 13C (75 MHz). Chemical shifts (d) are given
in ppm downfield from tetramethylsilane, as internal standard,
and coupling constants (J-values) are in hertz (Hz). Mass spectra
data were collected on a Finnigan MAT LC-Q mass spectrometer
system using electrospray ionization (ESI). Elemental analyses
were performed by Atlantic Microlabs. Melting points were ob-
tained using an electrothermal digital melting point apparatus
and are uncorrected. All purifications by flash chromatography
>2.5
0.032 0.021 99 2.0
a
b
c
Inhibition of tubulin polymerization. Tubulin was at 10
Inhibition of growth of MCF-7 human breast carcinoma cells.
Inhibition of colchicine binding. Compounds were tested at 5 and 50
M, and the [3H]colchicine concentration was 5
All experiments were performed in triplicate.
lM.
l
M. The
tubulin concentration was 1
l
lM.
d
The percentage of cellular microtubule loss was estimated visually over a range
of concentrations. Dose response curves were generated and EC50 values calculated.
The values represent the averages from two independent experiments.
were performed using Geduran silica gel 60, 35–75 lm (VWR).
All solvents used were purified by pressure filtration through acti-
vated alumina. Reactions were run at ambient temperature and
under a nitrogen atmosphere, unless otherwise noted, and were
monitored by TLC using Merck 60 F254 silica gel aluminium sheets.
3. Conclusion
We synthesized novel boronic acid analogs and the correspond-
ing hydroxy chalcone analogs of CA-4. Boronic acid analog 4 was

974
Y. Kong et al. / Bioorg. Med. Chem. 18 (2010) 971–977
Figure 2. Effects of compounds 4 and 6 on interphase microtubules in A-10 cells. (A) Vehicle control; (B) 20 lM compound 4; (C) 20 lM compound 6.
4.2. General procedure a for the preparation of chalcones
lid was washed with petroleum ether to afford 1-(3-bromo-4-
methoxyphenyl)ethanone 8 as a white solid (5.5 g, 96%): mp 86–
88 °C (lit.13 mp 86.5–87.5 °C). 1H NMR (CDCl3, 300 MHz) d 8.10
(d, 1H, J = 2.1 Hz), 7.85 (dd, 1H, J = 8.7, 2.1 Hz), 6.88 (d, 1H,
J = 8.7 Hz), 3.91 (s, 3H), 2.50 (s, 3H); 13C NMR (75 MHz) d 195.68,
159.61, 133.82, 131.24, 129.61, 111.90, 111.16, 56.57, 26.41.
Ketone 8 (0.5 g, 2.18 mmol) was treated with ethylene glycol
(0.73 mL, 13.1 mmol) and p-TsOHÁH2O (0.07 g, 0.37 mmol) in ben-
zene (50 mL) under reflux. A Dean–Stark trap was used to remove
the water generated during reaction. The resulting mixture was
cooled to room temperature, washed with saturated NaHCO3
(20 mL), H2O (20 mL), brine (20 mL), and dried over Na2SO4. The
solution was concentrated and purified by flash chromatography
using 1:19 EtOAc–hexanes to afford 2-(3-bromo-4-methoxy-
phenyl)-2-methyl-1,3-dioxolane 9 as a yellow oil (0.57 g, 96%):
1H NMR (CDCl3, 300 MHz) d 7.65 (d, 1H, J = 2.4 Hz), 7.37 (dd,
1H, J = 8.4, 2.1 Hz), 6.85 (d, 1H, J = 8.7 Hz), 4.02 (m, 2H), 3.88 (s,
3H), 3.76 (m, 2H), 1.62 (s, 3H); 13C NMR (75 MHz) d 155.23,
137.06, 130.24, 125.43, 111.37, 111.16, 107.87, 64.32, 56.06,
27.48.
Compound 9 (0.44 g, 1.61 mmol) was dissolved in dry THF
(25 mL), cooled to À78 °C, and BuLi (0.97 mL, 2.43 mmol, 2.5 M
in hexane) was added dropwise. The resulting mixture was stirred
at À78 °C for an additional 1.5 h, followed by the dropwise addi-
tion of trimethyl borate (0.25 g, 2.43 mmol). The mixture was
warmed to room temperature and stirred overnight. The reaction
was quenched with 3 N HCl (10 mL) and stirred at room tempera-
ture for 30 min, followed by extraction with ether (3 Â 30 mL). The
combined ether layers were extracted with 1 N NaOH (2 Â 10 mL),
and the combined aqueous phase was re-acidified with 1 N HCl to
pH 4. After re-acidification, the mixture was extracted with ether
(2 Â 30 mL). The ether layers were combined and dried over
Na2SO4. The solution was concentrated and purified by flash chro-
matography using 1:1 EtOAc–hexanes to afford 5-acetyl-2-meth-
oxyboronic acid 10 as a white solid (0.24 g, 77%): 1H NMR
(CDCl3, 300 MHz) d 8.46 (d, 1H, J = 2.4 Hz), 8.10 (dd, 1H, J = 8.7,
2.2 Hz), 6.97 (d, 1H, J = 8.7 Hz), 6.45 (broad, 2H), 3.98 (s, 3H),
2.59 (s, 3H); 13C NMR (75 MHz) d 197.54, 168.10, 138.32, 133.61,
130.76, 110.27, 110.11, 56.13, 26.66.
To a stirred solution of an equivalent amount of the appropri-
ately substituted acetophenone and benzaldehyde derivatives in
ethanol was added aqueous NaOH solution (10% w/v, 4.0 equiv).
The resulting solution was stirred at room temperature overnight,
poured into water, and acidified to pH 4 with 1 N HCl. The resultant
precipitate was filtered off, washed with water, and purified by
recrystallization or flash chromatography.
4.2.1. 2-Methoxy-5-[(1E)-3-oxo-3-(3,4,5-trimethoxyphenyl)prop-
1-en-1-yl]boronic acid (4)
General Procedure A was used for the reaction of 1-(3,4,5-tri-
methoxyphenyl)ethanone 2 (0.5 g, 2.38 mmol) and 5-formyl-2-
methoxyboronic acid 3 (0.43 g, 2.38 mmol). The crude solid was
recrystallized from EtOH–H2O to afford product 4 (0.56 g, 63%) as
yellow needles: mp 134–135 °C; 1H NMR (CDCl3, 300 MHz) d
8.18 (d, 1H, J = 2.1 Hz), 7.80 (d, 1H, J = 15.6 Hz), 7.70 (dd, 1H,
J = 8.7, 2.1 Hz), 7.41 (d, 1H, J = 15.6 Hz). 7.24 (s, 2H), 6.96 (d, 1H,
J = 8.7 Hz), 5.74 (s, 2H), 3.98 (s, 3H), 3.95 (s, 6H), 3.93 (s, 3H); 13C
NMR (75 MHz) d 189.60, 166.32, 153.25, 144.60, 142.46, 137.16,
134.01, 133.87, 128.19, 120.24, 110.66, 106.24, 61.13, 56.58,
56.04; ESI m/z 373 (M+H)+. Anal. Calcd for C19H21BO7Á0.9H2O: C,
58.71; H, 5.87. Found: C, 58.98, H, 5.97.
4.2.2. (2E)-3-(3-Hydroxy-4-methoxyphenyl)-1-(3,4,5-trimethoxy-
phenyl)prop-2-en-1-one (6)
General procedure A was used for the reaction of 1-(3,4,5-tri-
methoxyphenyl)ethanone 2 (0.5 g, 2.38 mmol) and 3-hydroxy-4-
methoxybenzaldehyde 5 (0.36 g, 2.38 mmol). The crude solid was
purified by flash chromatography using 1:3 EtOAc–hexanes to af-
ford product 6 (0.37 g, 45%) as a bright yellow solid: mp 127–
129 °C; 1H NMR (CDCl3, 300 MHz) d 7.75 (d, 1H, J = 15.6 Hz), 7.35
(d, 1H, J = 15.6 Hz), 7.31 (d, 1H, J = 1.8 Hz), 7.27 (s, 2H), 7.14 (dd,
1H, J = 8.4, 2.0 Hz), 6.88 (d, 1H, J = 9.0 Hz), 5.71 (s, 1H), 3.95 (s,
9H), 3.94 (s, 3H); 13C NMR (75 MHz) d 189.28, 153.24, 149.10,
146.07, 144.90, 142.39, 133.86, 128.59, 123.15, 119.87, 112.97,
110.74, 106.04, 61.11, 56.48, 56.15; ESI m/z 345 (M+H)+. Anal.
Calcd for C19H20O6: C, 66.27; H, 5.85. Found: C, 66.47, H, 6.04.
General procedure A was used for the reaction of 3,4,5-trimeth-
oxybenzaldehyde (0.24 g, 1.23 mmol) and 5-acetyl-2-methoxybo-
ronic acid 10 (0.24 g, 1.23 mmol). The crude solid was
recrystallized from EtOAc–hexanes to afford product 11 (0.26 g,
57%) as a pale yellow solid: mp 148–150 °C; 1H NMR (CDCl3,
300 MHz) d 8.55 (d, 1H, J = 2.4 Hz), 8.20 (dd, 1H, J = 8.7, 2.4 Hz),
7.73 (d, 1H, J = 15.3 Hz), 7.46 (d, 1H, J = 15.6 Hz). 7.03 (d, 1H,
J = 9.0 Hz), 6.86 (s, 2H), 6.23 (broad, 2H), 4.00 (s, 3H), 3.91 (s,
6H), 3.89 (s, 3H); 13C NMR (75 MHz) d 189.66, 168.20, 153.61,
144.77, 137.96, 134.29, 131.72, 130.72, 121.30, 110.50, 105,87,
61.19, 56.42, 56.18; ESI m/z 373 (M+H)+. Anal. Calcd for
C19H21BO7: C, 61.32; H, 5.69. Found: C, 61.29, H, 5.95.
4.2.3. 2-Methoxy-5-[(2E)-3-(3,4,5-trimethoxyphenyl)prop-2-
enoyl]boronic acid (11)
To a stirred solution of 1-bromo-2-methoxybenzene 7 (4.68 g,
25.0 mmol) in CH2Cl2 (20 mL) was added AlCl3 (8.03 g, 60.2 mmol).
Acetic anhydride was added dropwise to the mixture. The resulting
mixture was stirred at room temperature for 30 min, and heated to
40 °C for an additional 30 min. The solution was poured into con-
centrated HCl (20 mL) and ice (50 g), extracted with CH2Cl2
(3 Â 30 mL), and the combined organic layers were washed with
1 N NaOH (30 mL), H2O (30 mL), brine (30 mL), and dried over
Na2SO4. The solution was concentrated, and the resulting crude so-

Y. Kong et al. / Bioorg. Med. Chem. 18 (2010) 971–977
975
Table 2
mixture was cooled to room temperature, poured into ice (65 g), and
extracted with CH2Cl2 (4 Â 25 mL). The combined CH2Cl2 phase was
extracted into 1 N NaOH (2 Â 20 mL). The aqueous phase was acidi-
fied with concentrated HCl to pH 3, extracted with CH2Cl2
(3 Â 30 mL), and the combined CH2Cl2 extracts were dried over
Na2SO4. The solution was concentrated and purified by flash column
chromatographyusing3:7EtOAc–hexanestoafford13asa whiteso-
lid (0.8 g, 43%): mp 86–88 °C (lit.14 mp 92–93 °C); 1H NMR (CDCl3,
300 MHz) d 7.55 (m, 2H), 6.89 (d, 1H, J = 9.0 Hz), 3.96 (s, 3H), 2.54
(s, 3H); 13C NMR (75 MHz) d 197.50, 151.08, 145.58, 130.80,
122.03, 114.57, 110.02, 56.09, 26.39. ESI m/z 167 (M+H)+.
NCI Cancer Cell line GI50 values
Cell line
GI50 (lM)
Boronic acid chalcone 4
Leukemia
CCRF-CEM
K-562
MOLT-4
RPMI-8226
SR
2.39
3.46
3.60
1.12
2.18
Non-small cell lung cancer
A549/ATCC
EKVX
HOP-62
HOP-92
NCI-H226
NCI-H23
NCI-H322M
NCI-H460
NCI-H522
0.45
1.31
0.098
0.38
4.14
0.14
8.44
0.028
0.20
General procedure A was used for the reaction of 3,4,5-trimeth-
oxybenzaldehyde (0.35 g, 1.81 mmol) and 1-(3-hydroxy-4-
methoxyphenyl)ethanone 13 (0.30 g, 1.81 mmol). The crude solid
was purified by flash column chromatography using 1:4 EtOAc–
hexanes to afford product 14 (0.43 g, 68%) as a pale yellow solid:
mp 130–132 °C; 1H NMR (CDCl3, 300 MHz)
d 7.72 (d, 1H,
J = 15.6 Hz), 7.65 (m, 2H), 7.40 (d, 1H, J = 15.6 Hz), 7.82 (d, 1H,
J = 9.0 Hz), 6.86 (s, 2H), 5.73 (broad, 1H), 3.99 (s, 3H), 3.93 (s,
6H), 3.90 (s, 3H); 13C NMR (75 MHz) d 188.89, 153.56, 150.86,
145.69, 144.41, 140.33, 131.89, 130.65, 122.20, 121.14, 114.90,
110.22, 105.67, 61.11, 56.33, 56.20; ESI m/z 345 (M+H)+. Anal.
Calcd for C19H20O6: C, 66.27; H, 5.85. Found: C, 66.43, H, 5.99.
Colon cancer
COLO 205
HCC-2998
HCT-116
HCT-15
2.20
0.41
0.37
<0.01
15.4
0.037
KM12
SW-620
5. Biological testing
CNS Cancer
SF-268
SF-295
SF-539
SNB-19
U251
0.80
2.73
0.035
<0.01
0.025
5.1. Tubulin polymerization
Tubulin polymerization was followed turbidimetrically at
350 nm in Beckman model DU-7400 and DU-7500 spectrophotom-
eters equipped with electronic temperature controllers, as previ-
Melanoma
LOX IMVI
MALME-3M
M14
SK-MEL-2
SK-MEL-5
UACC-257
UACC-62
0.060
5.49
1.19
3.33
0.31
4.41
0.034
ously described in detail.17 The tubulin concentration was 10
(1.0 mg/mL).
lM
5.2. MCF-7 Cell proliferation assay
IC50 values for inhibition of cell growthwere obtained by measur-
ing the amount of total cell protein with the sulforhodamine B (SRB)
assay.18 The MCF-7 cells were grown for 24 h in the presence and ab-
sence of the drug. IC50 values were determined after an additional
48 h.
Ovarian cancer
IGROV1
0.088
2.08
2.06
1.81
0.30
<0.01
OVCAR-3
OVCAR-4
OVCAR-5
OVCAR-8
SK-OV-3
5.3. [3H]Colchicine binding assay
Renal cancer
786-0
A498
0.39
4.76
0.52
6.39
0.39
0.46
20.90
0.32
The binding of [3H]colchicine to tubulin was measured by the
DEAE-cellulose filter method, as previously described in detail.19
ACHN
CAKI-1
RXF 393
SN12C
TK-10
The tubulin concentration was 1.0
lM (0.1 mg/mL), and the
M.
[3H]colchicine concentration was 5.0
l
5.4. NCI Human cancer cell proliferation assay20
UO-31
Prostate cancer
PC-3
DU-145
0.25
0.050
Compound 4 was tested against human tumor cell lines at five
concentrations in a 10-fold serial dilution. After a 48 h incubation
time period, an SRB protein assay was used to determine inhibition
of cell growth. GI50 values were calculated from log dose response
curves.
Breast cancer
MCF7
NCI/ADR-RES
MDA-MB-231/ATCC
HS 578T
0.034
0.050
0.11
0.046
1.95
MDA-MB-435
BT-549
5.5. Indirect immunofluorescence
0.021
Drug effects on the microtubule network of A-10 cells were
evaluated by indirect immunofluorescent techniques as previously
described.21 Cells were plated onto glass cover slips and treated
with compounds at varying concentrations for 18 h. The cells were
fixed and microtubules visualized using a b-tubulin antibody, and
the nuclei was stained with 4,6-diamidino-2-phenylindole. Cells
were examined with a Nikon ES800 fluorescence microscope,
4.2.4. (2E)-1-(3-Hydroxy-4-methoxyphenyl)-3-(3,4,5-
trimethoxyphenyl)prop-2-en-1-one (14)
1-(3,4-Dimethoxyphenyl)ethanone 12 (2.0 g, 11.1 mmol) was
dissolvedinconcentratedsulfuricacid(10 mL)atroomtemperature.
The resulting solution was heated to 65 °C and stirred overnight. The

976
Y. Kong et al. / Bioorg. Med. Chem. 18 (2010) 971–977
0 µM,
G2/M (5.9 %)
0.1 µM,
G2/M (6.4%)
1 µM,
G2/M (7.1%)
0
50
100
150
200
250
0
50
100
150
200
250
0
50
100
150
200
250
Channels (FL2-A)
Channels (FL2-A)
Channels (FL2-A)
5 µM,
G2/M (7.4%)
10 µM,
G2/M (14.0%)
0
50
100
150
200
250
0
50
100
150
200
250
Channels (FL2-A)
Channels (FL2-A)
Figure 3. The effect of compound 4 on the cell cycle of MCF-7 cells after 48 h treatment.
Figure 4. Anti-angiogenesis affects of compound 4 in MCF-7 cell line. (A) Control; (B) 1
lM of compound 4; (C) 10
lM of compound 4.
Figure 5. The aortic ring assay on the inhibitory effects of compound 4. (A) Control; (B) 1
of compound 4; (F) 100 M of compound 4.
lM of compound 4; (C) 5 lM of compound 4; (D) 10 lM of compound 4; (E) 5 0 lM
l
images were captured with a Photometrics Cool Snap FX3 camera,
and the data was compiled using MetamorphÒ software.
at37 °Covernight, allowthecellsto attachtothebottomof thewells.
Different concentration of compound 4 was added to each well. Cell
cycle was measured by flow cytometry after 48 h treatment.
5.6. Cell cycle assay
5.7. Angiogenesis assay in vitro
Breast cancer cell line MCF-7 (2 Â 105 cells/ml) were seeded into
6-well plates in 2 mL DMEM culture media (GIBCO BRL Company;
Grand Island, USA) with 5% fetal calf serum for each well. Incubation
HUVEC (human umbilical vein endothelial cells, 1 Â 105 cells)
were seeded on the surface of Matrigel in 96 well plates in EBM-

Y. Kong et al. / Bioorg. Med. Chem. 18 (2010) 971–977
977
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well. The tube formation was observed after 16 h treatment.
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tas were excised. The aorta was immediately transferred into a
100 mm petri dish in ice cooled PBS buffer. The fibroadipose tissue
was removed under dissection microscope. The arteries were cut
into 1–1.5 mm long cross sections and washed five times with
PBS. To the cover slip in 6-well plates was added 80 lL of Matrigel
(Chemicon International), and the aortic sections submerged into
the gel and incubate for 15 min (37 °C, 5% CO2). EBM2 (1.5 mL)
with different concentration of compound 4 was added to each
well. The formation of sprouts on the rings were examined on
day 6, and compared with the control group.
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Acknowledgments
We thank the National Cancer Institute’s Developmental Thera-
peutics Program for evaluating compound 4 in the human cancer
cell screen, the Georgetown Medical Center Drug Discovery Pro-
gram and Lombardi Comprehensive Cancer Center for financial
support.
17. Hamel, E. Cell Biochem. Biophys. 2003, 38, 1.
18. Skehan, P.; Storeng, R.; Scudiero, D.; Monks, A.; McMahon, J.; Vistica, D.;
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20. For additional details concerning this screen and the NCI’s drug discovery and
development program, visit http://dtp.nci.nih.gov/branches/btb/ivclsp.html.
21. Tinley, T. L.; Randall-Hlubek, D. A.; Leal, R. M.; Jackson, E. M.; Cessac, J. W.;
Quada, J. C., Jr.; Hemscheidt, T. K.; Mooberry, S. L. Cancer Res. 2003, 63, 3211.
References and notes
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