Synthesis and biological activities of fluorinated chalcone derivatives

Article abstract of DOI:10.1016/S0968-0896(01)00319-4

We have designed and synthesized new 5-lipoxygenase inhibitors, fluorinated 3,4-dihydroxychalcones, and evaluated their biological activities with respect to antiperoxidation activity and in vitro antitumor activities. All fluorinated chalcones tested showed 5-lipoxygenase inhibition on rat basophilic leukemia-1 (RBL-1) cells and inhibitory action on Fe³⁺-ADP induced NADPH-dependent lipid peroxidation in rat liver microsomes. The potencies were comparable or better to that of the lead 3,4-dihydroxychalcone. 6-Fluoro-3,4-dihydroxy-2′,4′-dimethoxy chalcone (7) was the most effective compound in the in vitro assay using a human cancer cell line panel (HCC panel) consisting of 39 systems.

Full text of DOI:10.1016/S0968-0896(01)00319-4

Bioorganic & Medicinal Chemistry 10 (2002) 699–706
Synthesis and Biological Activities of Fluorinated Chalcone
Derivatives
Chika Nakamura,a Nobuhide Kawasaki,a Hideki Miyataka,a,*
Ezhuthachan Jayachandran,b In Ho Kim,b Kenneth L. Kirk,b Takeo Taguchi,c
Yoshio Takeuchi,d Hitoshi Horie and Toshio Satoha
aFaculty of Pharmaceutical Sciences, Tokushima Bunri University, Tokushima 770-8514, Japan
bLaboratory of Bioorganic Chemistry, National Institute of Diabetes and Digestive and Kidney Diseases,
National Institutes of Health, Bethesda, MD 20892, USA
cSchool of Pharmacy, Tokyo University of Pharmacy and Life Science, Hachioji, Tokyo 192-0392, Japan
dFaculty of Pharmaceutical Sciences Toyama Medical & Pharmaceutical University, Toyama 930-0194, Japan
eDepartment of Biological Science and Technology, Faculty of Engineering, University of Tokushima,
Tokushima 770-8506, Japan
Received 2 July 2001; accepted 8 September 2001
Abstract—We have designed and synthesized new 5-lipoxygenase inhibitors, fluorinated 3,4-dihydroxychalcones, and evaluated
their biological activities with respect to antiperoxidation activity and in vitro antitumor activities. All fluorinated chalcones tested
showed 5-lipoxygenase inhibition on rat basophilic leukemia-1 (RBL-1) cells and inhibitory action on Fe3+-ADP induced
NADPH-dependent lipid peroxidation in rat liver microsomes. The potencies were comparable or better to that of the lead 3,4-
dihydroxychalcone. 6-Fluoro-3,4-dihydroxy-20,40-dimethoxy chalcone (7) was the most effective compound in the in vitro assay
usinga human cancer cell line panel (HCC panel) consistingof 39 systems. # 2002 Elsevier Science Ltd. All rights reserved.
Introduction
further indication of the potential utility of these
agents.6 Inhibition of 5-hydroxy-6,8,11,14-eicosate-
traenoic acid (5-HETE) biosynthesis from arachidonic
acid has been implicated in this response.
The leukotrienes are mediators of such important func-
tions as smooth muscle contraction, increased vascular
permeability, leukocyte chemotaxis, hypersensitivity
reactions and inflammation.1,2 These eicosanoides are
formed from the unstable epoxide, leukotriene A4
(LTA4), which is transformed either by LTB4 synthase
to give LTB4 or by LTC4 synthase to produce the
cysteinyl leukotrienes. The synthesis of LTA4 from ara-
chidonic acid is mediated by the enzyme, 5-lipoxygenase
(5-LO). Because of the potent activities of the leuko-
trienes, development of 5-LO inhibitors has become the
focus of much research.3ꢀ5 Chemotherapeutic goals of
this research include treatment of diseases associated
with inflammatory responses, includingasthma and
inflammatory skin diseases such as psoriasis and contact
dermatitis.1 The ability of a 5-LO inhibitor to trigger
massive apoptosis in human prostate cancer cells is
It was previously shown that a series of 3,4-dihy-
droxychalcones had potent inhibitory effects on 5-LO
with antioxidative effects, and some inhibited cyclo-
oxygenase.7
The C–F bond frequently is used as a replacement of a
C–H or C–OH bond in biologically active compounds
because these substitutions result in altered physico-
chemical properties of the molecule, but introduce no
major steric changes.8 Because of this, useful alterations
in biological activities often result from these substitu-
tions.9 This report deals with the synthesis of fluori-
nated chalcone derivatives and their 5-LO inhibiting
action on rat basophilic leukemia-1 (RBL-1) cells, pre-
ventive action on Fe3+-ADP induced NADPH-depen-
dent lipid peroxidation in rat liver microsomes, and
action against a human cancer cell line panel (HCC
panel) consistingof 39 systems.
*Correspondingauthor. Fax: +81-88-655-3051; e-mail: hideki@ph.
bunri-u.ac.jp
0968-0896/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(01)00319-4

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C. Nakamura et al. / Bioorg. Med. Chem. 10 (2002) 699–706
Scheme 1.
Table 1. Effect of chalcone derivatives on Fe3+-ADP induced NADPH dependent lipid peroxidation in rat liver microsomes and on RBL-1 cell 5-
lipoxygenase activity
Compd
IC50 (M)
logP (C)c
5-Lipoxygenasea
Lipid peroxidationb
4.0ꢁ10ꢀ8
4.5ꢁ10ꢀ6
3.07
1
2
3
4
5
6
7
8
8.3ꢁ10ꢀ8
1.2ꢁ10ꢀ7
1.2ꢁ10ꢀ7
7.0ꢁ10ꢀ8
1.2ꢁ10ꢀ7
8.7ꢁ10ꢀ8
4.3ꢁ10ꢀ8
2.3ꢁ10ꢀ6
3.7ꢁ10ꢀ6
7.4ꢁ10ꢀ6
1.0ꢁ10ꢀ6
1.8ꢁ10ꢀ6
3.5ꢁ10ꢀ6
3.5ꢁ10ꢀ6
3.12
3.33
3.39
3.17
3.38
3.38
3.43
aTest compound concentration: 1ꢁ10ꢀ6 – 1ꢁ10ꢀ8 M.
bTest compound concentration: 1ꢁ10ꢀ5 M – 3ꢁ10ꢀ7 M.
clogP(C) was calculated usingPrologP 5.1 (PALLAS for Windows, CompuDrugInternational Inc., South San Francisco, CA, USA).

Figure 1. Dose–response curves of compound 7 on HCC panel. Test compound concentration; 10ꢀ4–10ꢀ8 M.

702
C. Nakamura et al. / Bioorg. Med. Chem. 10 (2002) 699–706
Chemistry
plate 24 h after cancer cells were enclosed into it. The
percentage of cell proliferation was evaluated against
12
Claisen–Schmidt condensations of acetophenones with
bis-THP-protected fluorinated dihydroxybenzaldehydes
were carried out as previously reported (Scheme 1).7
Without purification, the protectinggroups were removed
by acid treatment, and the catecholic chalcones (2–8) were
purified by chromatography and/or recrystallization.
the control by determiningcolorimetrically
the
amount of the cells after 48 h incubation. First, pre-
screeningwas performed usingthree cell lines, MCF-7
(breast), NCI-H460 (lung), and SF-268 (CNS), at a
compound concentration of 10ꢀ4 M (Table 2). The
results on the nonfluorinated chalcone (1) and the
fluorinated chalcone derivatives (3, 5, 7, 8) showed that
all of these compounds have a cell proliferation sup-
pressingaction on the three cell lines used. Amongthese
Results and Discussion
Preventive action on Fe3+-ADP induced NADPH-
dependent lipid peroxydation in rat liver microsomes
and 5-LO inhibitory action on RBL-1 cells were inves-
tigated for the fluorinated chalcone derivatives synthe-
sized (Table 1). The lipid peroxidation preventingaction
was induced with Fe3+-ADP in rat liver microsomes
and the amount formed of peroxidized lipids was deter-
mined as the amount of malondialdehyde produced in
their reaction with 2-thiobarbituric acid (TBA). The
percentage of prevention was calculated against the
control on the basis of the experimental data obtained.
All of the chalcone derivatives exhibited a concentra-
tion-dependent lipid peroxidation preventingaction at
concentrations from 1ꢁ10ꢀ5 to 3ꢁ10ꢀ7 M and the value
of IC50 was in a range of 1.0ꢁ10ꢀ6 to 7.4ꢁ10ꢀ6M. The
IC50 value for nonfluorinated 3,4-dihydroxy-20,40-
dimethoxychalcone (1) was 4.5ꢁ10ꢀ6 M.
Table 3. Effect of compound 7 on HCC panel
Cell
GI50 (mM)
TGI (mM)
LC50 (mM)
Breast
HBC-4
BSY-1
HBC-5
1.9
0.48
1.9
46
13
9.5
>100
64
>100
>100
>100
MCF7
MDA-MB-231
0.55
38
54
>100
CNS
U251
10
16
19
14
4.0
7.4
26
63
>100
38
29
63
>100
>100
99
>100
>100
SF-268
SF-295
SF-539
SNB-75
SNB-78
72
Colon
HCC2998
KM-12
HT-29
HCT-15
HCT-116
10
5.4
22
3.9
14
27
29
53
44
35
67
>100
>100
>100
80
5-LO inhibition was evaluated from the amount formed
of 5-HETE determined by HPLC after addingarachi-
donic acid as the substrate to cell homogenate. The
percentage of inhibition was calculated against the con-
trol usingthe analytical data obtained. All fluorinated
chalcone derivatives showed a concentration-dependent
5-LO inhibitingaction in their concentration raneg
from 1ꢁ10ꢀ6 to 1ꢁ10ꢀ8 M. The IC50 value for the
fluorinated compounds ranged from 1.2ꢁ10ꢀ7 to
4.3ꢁ10ꢀ8 M while that for the nonfluorinated com-
pound (1) was 4.0ꢁ10ꢀ8 M.
Lung
NCI-H23
NCH-H226
NCI-H522
NCI-H460
A549
14
11
2.3
8.2
22
47
39
24
32
93
33
27
>100
>100
>100
>100
>100
>100
>100
DMS273
DMS114
10
2.2
Melanoma
LOX-IMVI
21
47
>100
Amongthe fluorinated chalcone derivatives synthesized,
20,40-dimethoxy type compounds (5–8) showed stronger
antioxydative and 5-LO inhibitory actions than 20,50-
dimethoxy type compounds (2–4). Fluorination was
found to cause no significant change in these activities.
Ovarian
OVCAR-3
OVCAR-4
OVCAR-5
OVCAR-8
SK-OV-3
0.53
13
18
17
15
26
41
46
84
49
83
>100
>100
>100
>100
HCC-panel10,11 was employed to examine the anti-
carcinogenic activity of the fluorinated compounds.
Each of the test compounds was added to a microtiter
Renal
RXF-631L
ACHN
16
11
36
47
81
>100
Stomach
St-4
Table 2. Effect of chalcone derivatives on human cancer cell lines
24
17
9.8
1.8
9.0
3.9
61
45
45
28
26
24
>100
>100
>100
>100
71
MKN1
MKN7
MKN128
MKN45
MKN74
Compd
Growth (%)
MCF-7
NCH-H460
SF-268
1
3
5
7
8
ꢀ73
ꢀ59
ꢀ51
ꢀ84
ꢀ30
ꢀ65
ꢀ50
ꢀ79
ꢀ100
10
ꢀ30
ꢀ65
ꢀ26
ꢀ100
19
>100
Prostate
DU-145
PC-3
14
12
51
44
>100
>100
Test compound concentration: 10ꢀ4 M.
Test compound concentration: 10ꢀ4ꢀ10ꢀ8 M.

C. Nakamura et al. / Bioorg. Med. Chem. 10 (2002) 699–706
703

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C. Nakamura et al. / Bioorg. Med. Chem. 10 (2002) 699–706
compounds, 7 was most anticarcinogenic and killed the
cells of MCF-7 and SF-268 almost completely.
mixture of fluorinated 3,4-dihydroxybenzaldehyde (20
mmol) and pyridinium p-toluenesulfonate (0.12 g, 0.48
mmol) in 80 mL of methylene chloride. The reaction
mixture was stirred at room temperature until all the
components dissolved. The reaction mixture was
washed twice with water, dried, and evaporated in
vacuo. The residual 3,4-bis-(tetrahydro-pyran-2-yloxy)-
fluorobenzaldehydes were used in the condensation step
without further purification.
Screeningwas then conducted for 7 makinguse of an
HCC panel consistingof 39 cell lines. The compound
exhibited a concentration-dependent inhibitory action
on cell proliferation for all cell lines (Fig. 1). Evalua-
tions were made on the values of GI50 (compound con-
centration necessary to suppress cell proliferation to
50% of the control), TGI (compound concentration
necessary to suppress cell proliferation to the cell num-
ber at time zero), and LC50 (compound concentration
necessary to reduce cell number to 50% of that at time
zero) for these cell lines (Table 3). The values obtained
for each of the three indexes were averaged respectively
over the 39 cell lines and the mean values thus calcu-
lated were used to denote the relative sensitivity of each
cell line to the test compound (Fig. 2). This sort of figure
is called mean graph and allows to grasp the specificity
of cancer cells to drug. Many 7-sensitive cell strains
were found in breast cancer cell lines. Based on these
mean values, an analysis was carried out usingthe
compare program13,14 to make a comparison between
the compound and the conventional anticancer agents.
As a result, the most similar agent was found to be vin-
cristine15,16 with a tubulin inhibitingaction but the cor-
relation coefficient was small (0.57). Examination of the
tubulin inhibitingaction of 7 revealed that the com-
pound has no such action (data not shown). This sug-
gests an action mechanism of 7 differingfrom that of
the conventional anticancer agents to make the
compound a unique anticarcinogenic agent.
General condensation procedure
A solution of the above crude 3,4-bis-(tetrahydro-
pyran-2-yloxy)-fluorobenzaldehyde and 20,40- or 20,50-
dimethoxyacetophenone (20 mmol) and barium
hydroxide octahydrate (6.52 g, 20 mmol) in 100 mL of
o
MeOH was stirred at 40 C for 12 h. The mixture was
concentrated in vacuo to give the crude protected chal-
cone. This was dissolved in 80 mL of MeOH and p-
toluenesulfonic acid monohydrate (0.091 g, 0.48 mmol)
was added to it. The mixture was stirred at room tem-
perature for 3 h (monitored by TLC for completion of
the reaction) and then concentrated in vacuo. Water
(100 mL) was added to the mixture, the aqueous solu-
tion was neutralized with 5% NaHCO3 and extracted
with EtOAc. The organic layer was separated, washed
with water and brine, dried over MgSO4, filtered and
evaporated. The residue was chromatographed on silica
gel to afford the fluorinated chalcones, data for which
are given below.
20,50-Dimethoxy-2-fluoro-3,4-dihydroxychalcone
(2).
Yield 35%; dark yellow solid, mp 185–186 ꢂC; 1H
NMR (300 MHz, CD3OD) d 7.62 (d, J=15.9 Hz, 1H),
7.34 (d, J=15.9 Hz, 1H), 7.09 (brs, 3H), 7.04 (d, J=8.1
Hz, 1H), 6.65 (dd, J=8.7 and 1.8 Hz, 1H), 3.86 (s, 3H),
3.79 (s, 3H); 13C NMR (75 MHz, DMSO-d6) d 191.5,
153.1, 151.8, 151.4 (d, J=244.7 Hz), 150.5 (d, J=6.2
Hz), 136.2, 133.5 (d, J=13.7 Hz), 129.4, 125.5 (d, J=6.3
Hz), 119.4 (d, J=4.5 Hz), 118.4, 114.2, 114.1, 113.9 (d,
J=1.1Hz), 111.8, 56.3, 55.6; HRMS (FAB+) m/z calcd
for C17H16O5F (M+1) 319.0982, obsd 319.0972.
In summary, we have synthesized fluorinated derivatives
of 3,4-dihydroxy-dimethoxychalcone and shown that
they have antioxidative and 5-LO preventingactions
which are almost equivalent to those of the non-
fluorinated compound. We have also shown that the
compounds synthesized exhibit an anticarcinogenic
action and suggested that 7 may act on cancer cells in a
way quite different from that of the conventional antic-
ancer agents. The compound may thus be an extremely
useful anticancer agent.
20,50-Dimethoxy-6-fluoro-3,4-dihydroxychalcone
(3).
Yield 30%; yellow solid, mp 180–183 ꢂC; 1H NMR
(300 MHz, DMSO-d6) d 10.19 (brs, 1H), 9.24 (brs, 1H),
7.48 (d, J=15.9 Hz, 1H), 7.18 (d, J=15.9 Hz, 1H),
7.12–7.09 (m, 3H), 7.03 (brd, J=1.8 Hz, 1H), 6.63 (d,
J=11.7 Hz, 1H), 3.82 (s, 3H), 3.74 (s, 3H); 13C NMR
(75 MHz, DMSO-d6) d 191.3, 155.4 (d, J=242.5 Hz),
153.1, 151.9, 149.9 (d, J=11.9 Hz), 142.5, 135.3 (d,
J=2.9 Hz), 129.4, 125.0 (d, J=5.1 Hz), 118.5, 114.02,
113.96, 113.3 (d, J=4.0 Hz), 112.2 (d, J=12.5 Hz),
103.3 (d, J=25.6 Hz), 56.4, 55.6; HRMS (FAB+) m/z
calcd for C17H16O5F (M+1) 319.0982, obsd 319.0975.
Experimental
Chemistry
Meltingpoints are uncorrected. 1H and 13C NMR
spectra were recorded at 300 and 75 MHz, respectively,
with CD3OD and DMSO-d6 as solvents. Commercial
reagents and solvents were used without further pur-
ification. All reactions were performed under a dry N2
atmosphere. Analytical TLC was performed with Kisegel
60 GF254(Merck) and flash column chromatography was
performed with silica gel 60 (230–400 mesh, Merck).
20,50-Dimethoxy-5,6-difluoro-3, 4-dihydroxy chalcone (4).
Yield 35%, dark yellow solid, mp 129–130 ꢂC; 1H NMR
(300 MHz, CD3OD) d 7.60 (d, J=15.9 Hz, 1H), 7.33 (d,
J=15.9 Hz, 1H), 7.11 (brs, 3H), 6.88 (dd, J=6.6 and
2.1 Hz, 1H), 3.88 (s, 3H), 3.79 (s, 3H); 13C NMR
(75 MHz, CD3OD) d 194.3, 155.2, 154.2, 146.4 (dd,
J=243.9 and 11.4 Hz), 144.6 (dd, J=4.6 and 2.3 Hz),
General procedure for formation of THP-protected
fluorinated catechualdehydes
A solution of 3,4-dihydro-2H-pyran in 120 mL of
methylene chloride was added dropwise to a stirred

C. Nakamura et al. / Bioorg. Med. Chem. 10 (2002) 699–706
705
142.3 (dd, J=238.4 and 15.4 Hz), 139.4 (dd, J=11.4
and 2.9 Hz), 136.7 (brt, J=3.4 Hz), 130.5, 127.9 (d,
J=5.7 Hz), 120.3, 115.7, 114.8, 114.7, 108.6, 56.9, 56.3;
HRMS (FAB+) m/z calcd for C17H15O5F2 (M+1)
337.0888, obsd 337.0877.
Pharmaceutical Co., Ltd. 5-HETE was obtained from
Sigma Chemical Co., Ltd. ADP and b-NADPH were
obtained from Wako Pure Chemical Industries, Ltd.
Arachidonic acid was obtained from Nacalai Tesque,
Inc. HCC panel screeningwere performed by National
Cancer Institute, National Institutes of Health, USA
and Cancer Chemotherapy Center, ScreeningCommit-
tee of New Anticancer Agents supported by Grant-in-
Aid for Scientific Research on Priority Area ‘Cancer’,
from The Ministry of Education, Science, Sports and
Culture, Japan.
20,40-Dimethoxy-2-fluoro-3,4-dihydroxy chalcone (5).
Yield 32%; yellow solid, mp 185–186 ꢂC; 1H NMR
(300 MHz, DMSO-d6) d 10.12 (brs, 1H), 9.29 (brs, 1H),
7.60 (d, J=8.4 Hz, 1H), 7.53 (d, J=16.2 Hz, 1H), 7.43
(d, J=15.9 Hz, 1H), 7.11 (t, J=8.4 Hz, 1H), 6.68–6.62
(m, 3H), 3.89 (s, 3H), 3.85 (s, 3H); 13C NMR (75 MHz,
DMSO-d6) d 189.2, 163.9, 160.1, 151.4 (d, J=244.1 Hz),
150.2 (d, J=6.2 Hz), 134.7, 133.5 (d, J=13.7 Hz),
132.0, 125.8 (d, J=6.3 Hz), 121.5, 119.2 (d, J=4.0 Hz),
114.5 (d, J=9.7 Hz), 111.7, 106.0, 98.6, 55.9, 55.6;
HRMS (FAB+) m/z calcd for C17H16O5F (M+1)
319.0982, obsd 319.0974.
Measurement of Fe3+–ADP induced NADPH dependent
lipid peroxidation in rat liver microsome.17 The modified
method of Kiso et al.18 was used. Microsomes were
prepared from male Wistar rats weighing about 200 g.
The rat liver was homogenized in cold 0.25 M sucrose.
The homogenate was centrifuged at 4 ꢂC (8000ꢁg, 10
min). The supernatant fraction was then collected and
ultracentrifuged at 4 ꢂC (105,000ꢁg, 30 min). The pellet
obtained was resuspended in 83.5 mM KCl–37.2 mM
Tris–HCl buffer (pH 7.4) and stocked at ꢀ20 ꢂC until
20,40-Dimethoxy-5-fluoro-3,4-dihydroxy chalcone (6).
Yield 29%; yellow solid, mp 194–195 ꢂC; 1H NMR
(300 MHz, CD3OD) d 7.65 (d, J=8.7 Hz, 1H), 7.44 (d,
J=15.9 Hz, 1H), 7.36 (d, J=15.9 Hz, 1H), 6.93–6.89
(m, 2H), 6.62 (d, J=9.0 Hz, 1H), 6.62 (d, J=11.1 Hz,
1H), 3.93 (s, 3H), 3.88 (s, 3H); 13C NMR (75 MHz,
CD3OD) d 193.0, 166.4, 162.3, 153.6 d, J=236.8 Hz),
149.0 (d, J=6.3 Hz), 143.7 (d, J=3.1 Hz), 137.5 (d,
J=15.4 Hz), 133.7, 127.7 (d, J=8.6 Hz), 126.5, 123.0,
112.1 (d, J=1.7 Hz), 108.7 (d, J=19.9 Hz), 107.0, 99.7,
56.5, 56.3; HRMS (FAB+) m/z calcd for C17H16O5F
(M+1) 319.0982, obsd 319.0983.
use. Protein concentration was determined by the
19
method of Lowry et al.
The assay system (1 mL)
consisted of 83.5 mM KCl–37.2 mM Tris–HCl buffer
(pH 7.4), the test compound in 1% DMSO, 0.2 mM
NADPH, 1 mM ADP, 1 mgprotein/mL rat liver
microsomes and 10 mM FeCl3. The reaction mixture
was incubated at 37 ꢂC for 20 min, and then cooled on
ice to terminate the reaction. Lipid peroxide was mea-
20
sured by the method of Ohkawa et al.
Thus, 8.1%
sodium dodecyl sulfate (0.2 mL), 20% AcOH contain-
ing0.27 M HCl adjusted to pH 3.5 with NaOH (1.5 mL)
and 0.8% TBA (1.5 mL) were added to the reaction
mixture. The mixture was then boiled at 100 ꢂC for 20
min and the reaction was stopped by coolingon ice.
Thereafter, n-BuOH–pyridine (15:1, 4 mL) was added,
and vigorous mixing was performed. After centrifuga-
tion (780ꢁg, 10 min), the organic layer was separated,
and the absorbance was measured at 532 nm. The
amount of TBA-positive material was expressed as a
correspondingamount of malondialdehyde.
20,40-Dimethoxy-6-fluoro-3,4-dihydroxy chalcone (7).
Yield 41%; yellow crystals, mp 179–180 ꢂC; H NMR
1
(300 MHz, DMSO-d6) d 10.11 (brs, 1H), 9.23 (brs, 1H),
7.60 (d, J=8.4 Hz, 1H), 7.52 (d, J=15.6 Hz, 1H), 7.34
(d, J=15.6 Hz, 1H), 7.09 (d, J=7.5 Hz, 1H), 6.69–6.62
(m, 3H), 3.90 (s, 3H), 3.85 (s, 3H); 13C NMR (75 MHz,
DMSO-d6) d 188.9, 163.9, 160.2, 155.3 (d, J=241.9 Hz),
149.5 (d, J=12.0 Hz), 142.4, 133.8, 132.1, 125.4 (d,
J=5.2 Hz), 121.5, 113.3 (d, J=4.0 Hz), 112.6 (d,
J=12.5 Hz), 106.1, 103.3 (d, J=26.2 Hz), 98.7, 56.0,
55.6; HRMS (FAB+) m/z calcd for C17H16O5F (M+1)
319.0982, obsd 319.0974.
Measurement of RBL-1 cell 5-lipoxygenase activity.7
21
The modified method of Blackham et al.
was used.
20,40-Dimethoxy-5, 6-difluoro-3,4-dihydroxychalcone (8).
Yield 30%; yellow solid, mp 218–219 ꢂC; 1H NMR
(300 MHz, DMSO-d6) d 10.31 (brs, 1H), 9.80 (brs, 1H),
7.63 (d, J=8.7 Hz, 1H), 7.50 (d, J=16.2 Hz, 1H), 7.42
(d, J=15.9 Hz, 1H), 6.94 (dd, J=6.9 and 1.8 Hz, 1H),
6.69 (d, J=2.1 Hz, 1H), 6.65 (dd, J=8.7 and 2.4 Hz,
1H), 3.91 (s, 3H), 3.86 (s, 3H); 13C NMR (75 MHz,
DMSO-d6) d 188.6, 164.2, 160.3, 144.0 (dd, J=242.5 and
11.4 Hz), 143.4 (brd, J=3.4 Hz), 140.6 (dd, J=237.9 and
15.4 Hz), 137.6 (dd, J=10.9 and 2.3 Hz), 132.8, 132.2,
127.0 (d, J=5.7 Hz), 121.2, 112.7 (d, J=9.1 Hz), 107.7,
106.2, 98.7, 56.0, 55.6; HRMS (FAB+) m/z calcd for
C17H15O5F2 (M+1) 337.0888, obsd 337.0884.
RBL-1 cells were grown in Roswell Park Memorial
Institute (RPMI)-1640 medium containing10% heat-
inactivated newborn calf serum, 100 units/mL penicillin
and 0.1 mg/mL streptomycin. Cells were cultured at
37 ꢂC in 5% CO2/air. Cells in the growth phase (5ꢁ105
to 106 cells/mL) were collected by centrifugation
(100ꢁg, 5 min) and suspended at 3ꢁ107 cells/mL in 50
mM phosphate buffer (0.25 M sucrose, 1 mM EDTA, 2
mM glutathione, pH 7.4). Cells were stored at ꢀ80 ꢂC
until use. The assay system (0.5 mL) consisted of 50 mM
phosphate buffer (0.25 M sucrose, 1mM EDTA, 2 mM
glutathione, pH 7.4), the test compound in 1% DMSO,
2 mM CaCl2, 0.2 mg/mL arachidonic acid (10 mg/mL
MeOH soln; 10 mL) and 107 cells/mL RBL-1 cells
homogenate. Reaction mixture was incubated at 37 ꢂC
for 3 min, and then 0.5 mL of MeOH was added to
terminate the reaction. The mixture was centrifuged
(2000ꢁg, 15 min), 5-HETE in the supernatant was
Pharmacology
Materials. Male Wistar rats were obtained from Japan
SLC Inc. RBL-1 cell was obtained from Dainippon

706
C. Nakamura et al. / Bioorg. Med. Chem. 10 (2002) 699–706
analyzed by HPLC (column; COSMOSIL 5C18-MS
Waters 4.6ꢁ150 mm (Nacalai Tesque, Inc.), mobile
phase; CH3CN–0.1% AcOH aq (6:4), flow rate; 1 mL/
min, temperature; rt, absorbance; 235 nm).
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Evaluation of anticarcinogenic action using HCC.10,11
Each of the test compounds was added to a microtiter
plate 24 h after cancer cells were enclosed into it. After
48 h incubation, the amount of the cells was color-
imetrically determined usingsulforhodamine B as the
reagent12 and the percentage of cell proliferation was
calculated against the control. An HCC panel consisting
of 39 cell lines was employed for screeningtests, in
which GI50, TGI, and LC50 were evaluated at 10ꢀ4–10ꢀ8
M compound concentrations for each cell line, the mean
value for each index over the cell lines was calculated, the
relative sensitivity of each cell line to each test compound
was estimated usingthe mean index values obtained, and
comparisons were made between each test compound and
the conventional anticancer agents through analyses mak-
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13, 14
inguse of the compare program.
Acknowledgements
We wish to express our thanks to ScreeningCommittee
of New Anticancer Agents supported by Grant-in-Aid
for Scientific Research on Priority Area ‘Cancer’, from
The Ministry of Education, Science, Sports and Culture,
Japan, and Dr. Edward Sausville of National Cancer
Institute for their examinations on the anticarcinogenic
activities of the compounds we synthesized.
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G. R.; Hamel, E. J. Biol. Chem. 1991, 266, 15882.
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