Simple synthesis and biological evaluation of flocoumafen and its structural isomers

Article abstract of DOI:10.1007/s12039-010-0071-2

Simple synthesis and biological properties of flocoumafen 1 and its structural isomers are described. The key synthetic strategies involve Knoevenagel condensation, Grignard reaction, intramolecular ring cyclization and coupling reaction. Flocoumafen 1 was easily separated into cis and trans forms using flash column chromatography. They were then evaluated for suppression of LPS-induced NO generation and anti-excitotoxicity in vitro. It was found that the trans-flocoumafen was potent suppressor of NO generation with the concentration of 10 μM in vitro, while no significant effect for neurotoxicity in cultured cortical neurons. Indian Academy of Sciences.

Full text of DOI:10.1007/s12039-010-0071-2

J. Chem. Sci., Vol. 122, No. 6, November 2010, pp. 833–838. © Indian Academy of Sciences.
Simple synthesis and biological evaluation of flocoumafen and its
structural isomers
1
JAE-CHUL JUNG , SOYONG JANG , SEIKWAN OH and OEE-SOOK PARK *
1
1
2,
1
Department of Neuroscience, Ewha Global Challenge, BK21 and Medical Research Institute,
School of Medicine, Ewha Womans University, Seoul 158-710, South Korea
2
Department of Chemistry, Institute for Basic Sciences, College of Natural Sciences,
Chungbuk National University, Cheongju 361–763, Chungbuk, Korea
e-mail: ospark@cbnu.ac.kr; jcjung10@yahoo.co.kr
MS received 13 January 2010; revised 22 March 2010; accepted 22 June 2010
Abstract. Simple synthesis and biological properties of flocoumafen 1 and its structural isomers are
described. The key synthetic strategies involve Knoevenagel condensation, Grignard reaction, intramolecu-
lar ring cyclization and coupling reaction. Flocoumafen 1 was easily separated into cis and trans forms
using flash column chromatography. They were then evaluated for suppression of LPS-induced NO gen-
eration and anti-excitotoxicity in vitro. It was found that the trans-flocoumafen was potent suppressor of
NO generation with the concentration of 10 µM in vitro, while no significant effect for neurotoxicity in
cultured cortical neurons.
Keywords. Flocoumafen; intramolecular ring cyclization; coupling reaction, NO-generation; neuro-
toxicity.
1. Introduction
cially, the hydroxy group attached to coumarin
nucleus at 4-position is important in biosynthesis
pathway. Currently, 4-hydroxycoumarins are regis-
tered for the control of rats and mice in and around
farm structures, households, and domestic dwell-
ings, inside transport vehicles, commercial transpor-
Many 4-hydroxycoumarins have been naturally
obtained from several plants such as tonka bean,
lavender, sweet clover grass, strawberries, and cin-
namon. The 4-hydroxycoumarin and its derivatives
have been effectively used as anticoagulants such as
warfarin, brodifacoum, difethialone, bromadiolone,
coumatetralone, and flocoumafen for the treatment
of disorders in which there is excessive or undesir-
able clotting, such as thrombophlebitis, pulmonary
10
tation facilities, and industrial areas. Recent report
described a simple synthesis of 4-hydroxycoumarin
derivatives and their screening assay for their bio-
logical properties. Actually, most of the work to
date, regarding the synthetic approaches of warfarin
1
embolism, and certain cardiac conditions. Recently,
11
type anticoagulants were reported by the Ferreira
the 4-hydroxycoumarins have evoked a great deal of
interest due to their characteristic structural features
and biological activities. The coumarin have been
studied as cell growth stimulation, bacteriostatic
activity as well as various pharmacological effective-
group through the formation of the carbon backbone
using organocopper methodology, the ring cycliza-
tion, and the coupling reaction with the 4-hydroxy-
coumarin moiety.
The scheme 1 reveals representative retrosyn-
thetic approach for syntheses of 1 and 2. The major
synthetic strategies involve intramolecular ring
cyclization, O-alkylation for preparation of key
fragments tetrahydronaphthalen-1-ol 3 or tetrahy-
dronaphthalen-1-bromide 4 and coupling reaction of
3, 4 with 4-hydroxycoumarin 5 or 4-hydroxy-
thiocoumarin 6 to generate the target flocoumafen 1
or thioflocoumafen 2. The skeletons of 2H-1-
benzopyran-2-ones and tetrahydronaphthalens are
2
ness such as analgesic, arthritis, anti-inflammatory,
3
4
5
6
7
anti-pyretic, anti-bacterial, anti-viral, and anti-
8
cancer effects. Especially, the flocoumafen 1 and
thioflocoumafen 2 are very effective anticoagulant
9
agents among the 4-hydroxycoumarin derivatives.
The coumarin nucleus is derived from cinnamic acid
as phenylacrylic skeleton in the biosynthesis. Espe-
*For correspondence
833

834
Jae-Chul Jung et al
Scheme 1. Retrosynthetic analysis of flocoumafen 1 and thioflocoumafen 2.
essential structural features for the 4-hydroxy- use. Thin layer chromatography (TLC) was per-
coumarin derivatives of second generation rodenti- formed on precoated silica gel G and GP uniplates
cides 1–2. They have traditionally been coupled from Analtech and visualized with 254 nm UV light.
with acidic media. Even though improved condensa- Flash chromatography was carried out on silica gel
tion reactions of 4-hydroxycoumarins 5–6 with com- 60 [Scientific Adsorbents Incorporated (SAI), parti-
1
pounds 3 or 4 using Bronsted–Lowry acids (HCl, cle size 32–63 μm, pore size 60 Å]. H NMR and
11–12
13
H2SO4) have been reported,
to preferential dehyrdohalogenation to give low yield. 500 at 500 MHz and 125 MHz, respectively. The
Thus, an efficient coupling condition was required chemical shifts are reported in parts per million
to obtain high yield. (ppm) downfield from tetramethylsilane, and J val-
these reactions led
C NMR spectra were recorded on a Bruker DPX
In the context of our medicinal research program ues are in Hz. Infrared (IR) spectra were obtained on
dealing with the synthesis of biologically active 4- an ATI Mattson FT/IR spectrometer. Mass spectra
hydroxycoumarin derivatives, we hope to report a were recorded with a Waters Micromass ZQ LC–
simple synthesis of flocoumafen from intramolecu- Mass system and high resolution mass spectra
lar ring cyclization, O-alkylation, and coupling reac- (HRMS) were measured with a Bruker BioApex
tion. Flocoumafen 1 was firstly separated into cis FTMS system by direct injection using an electros-
form and trans form using flash silica-column pray interface (ESI). When necessary, chemicals
13
chromatography. Biological activities of flocou- were purified according to the reported procedures.
mafen and its structural isomers for suppression of
LPS-induced NO generation in vitro suggests that 2.1a Synthesis of 3-[1,2,3,4-Tetrahydro-3-[4-(4-
they can be possible lead compounds for anti- trifluoromethylbenzyloxy)phenyl]-1-naphthalen-1-ol
inflammatory agents.
(3): To a stirred solution of ketone (13, 0⋅8 g,
2⋅0 mmol) in EtOH (6 mL) was added portion-wise
sodium borohydride (91 mg, 2⋅4 mmol) and then the
mixture was stirred at room temperature for 2 h. The
reaction mixture was diluted with water (5 mL) and
acidified with 1 N HCl aqueous solution. The result-
ing mixture was extracted with dichloromethane
(10 mL × 3). The combined organic layer was washed
with saturated aqueous NH4Cl solution (15 mL) and
organic phase was separated, dried over anhydrous
2. Experimental
2.1 General
Reactions requiring anhydrous conditions were per-
formed with the usual precautions for rigorous
exclusion of air and moisture. Tetrahydrofuran was
distilled from sodium benzophenone ketyl prior to

Simple synthesis and biological evaluation of flocoumafen and its structural isomers
835
MgSO4, filtered, and concentrated under reduced (m, 5/2H, CH, CH2), 2⋅51–2⋅40 (m, 1/2H, CH2),
pressure. The residue was purified by flash column 2⋅38–2⋅24 (m, 1/2H, CH2), 1⋅96–1⋅82 (m, 1/2H,
13
chromatography (silica gel, ethyl acetate/hexanes = CH2). C NMR (CDCl3, 125⋅76 MHz) δ 163⋅5
1 : 3, v/v) to afford alcohol 3 (0⋅56 g, 70%) as a (C=O), 160⋅8 (C), 157⋅2 (C), 157⋅1 (C), 152⋅7 (C),
white solid. Rf = 0⋅3 (30% ethyl acetate/hexanes); 152⋅6 (C), 141⋅3 (C), 138⋅1 (C), 137⋅9 (C), 137⋅7
m.p. 111⋅6°C. IR (neat, NaCl) ν 3388 (OH), 2922 (C), 134⋅3 (C), 132⋅1 (CH), 132⋅0 (CH), 130⋅8 (C),
(C–H), 1611 (C=C), 1584, 1512, 1454, 1244, 1066 130⋅7 (C), 130⋅6 (CH), 129⋅3 (CH), 128⋅7 (CH),
–1
1
(C–O), 824 cm . H NMR (CDCl3, 500.14 MHz) δ

128⋅1 (CH), 128⋅0 (C), 127⋅9 (C), 127⋅8 (C), 127⋅5
7⋅63 (dd, 3H, J = 8⋅5, 8⋅5 Hz, aromatic-H), 7⋅56 (d, (C), 127⋅4 (C), 125⋅7 (C), 125⋅6 (C), 124⋅1 (CH),
2H, J = 8⋅0 Hz, aromatic-H), 7⋅29–7⋅20 (m, 4H, aro- 124⋅0 (CH), 123⋅2 (CH), 123⋅1 (CH), 116⋅6 (CH),
matic-H), 7⋅10 (d, 1H, J = 7⋅5 Hz, aromatic-H), 6⋅95 116⋅5 (CH), 116⋅3 (C), 115⋅1 (C), 115⋅0 (C), 109⋅4
(d, 2H, J = 8⋅0 Hz, aromatic-H), 5⋅13 (s, 2H, benzyl- (C), 108⋅8 (C), 69⋅3 (benzyl-C), 39⋅8 (CH), 38⋅6
H), 5⋅02–4⋅96 (m, 1H, CH), 3⋅09–2⋅88 (m, 2H, CH2), (CH2), 38⋅1 (CH2), 37⋅5 (CH), 37⋅0 (CH), 36⋅5
2⋅51–2⋅45 (m, 1H, CH), 1⋅92 (q, 1H, J = 12⋅5 Hz, (CH2), 35⋅9 (CH2). HRMS: m/z = 543⋅1772 (calcd.
13
+
CH2), 1⋅78 (d, 1H, J = 8⋅0 Hz, CH2). C NMR 543⋅1783 for C33H26F3O4: [M + H] ); cis-Flocouma-
1
(CDCl3, 125⋅76 MHz) δ 157⋅2, 141⋅4, 139⋅3, 138⋅4, fen: m.p. 180⋅1°C. H NMR (CDCl3, 500⋅14 MHz) δ

136⋅4, 128⋅7, 127⋅9 (2C), 127⋅6, 127⋅5 (2C), 126⋅9, 7⋅72 (d, 1H, J = 7⋅5 Hz, aromatic-H), 7⋅62 (d, 2H,
126⋅7, 125⋅7 (2C), 125⋅6 (2C), 115⋅1 (2C), 70⋅3, J = 8⋅0 Hz, aromatic-H), 7⋅53 (dd, 3H, J = 7⋅5,
69⋅4, 41⋅0, 38⋅7, 38⋅6. HRMS: m/z = 399⋅1585 2⋅0 Hz, aromatic-H), 7⋅34 (d, 1H, J = 8⋅0 Hz, aro-
+
(calcd. 399⋅1572 for C24H22F3O2: [M + H] ).
matic-H), 7⋅32–7⋅23 (m, 5H, aromatic-H), 7⋅21 (d,
2H, J = 9⋅0 Hz, aromatic-H), 6⋅92 (d, 2H, J = 9⋅0,
8⋅5 Hz, aromatic-H), 5⋅64 (brs, 1H, OH), 5⋅12 (s,
2H, benzyl-H), 4⋅87 (q, 1H, J=5⋅5 Hz, CH), 3⋅13–
3⋅02 (m, 3H, CH, CH2), 2⋅52–2⋅42 (m, 1H, CH2),
2.1b Synthesis of flocoumafen; 4-hydroxy-3-[1,2,3,4-
tetrahydro-3-[4-(4-trifluoromethylbenzyloxy)phenyl]-
1-naphthyl]coumarin (1): To a stirred solution of
secondary alcohol (3, 0⋅2 g, 0⋅5 mmol) in dichloro-
methane (4 mL) was added p-toluenesulfonic acid
(5 mg, cat) and 4-hydroxycoumarin (0⋅1 g, 0⋅6 mmol)
and then the mixture was refluxed for 6 h. The reac-
tion mixture was cooled to room temperature and
washed with water (5 mL). The organic layer was
separated and the aqueous layer was extracted with
dichloromethane (10 mL × 3). The combined organic
layer was washed with saturated aqueous NH4Cl
solution (15 mL) and organic phase was separated,
dried over anhydrous MgSO4, filtered, and concen-
trated under reduced pressure. The residue was puri-
fied by flash column chromatography (silica gel,
ethyl acetate/hexanes = 1 : 4, v/v) to give flocou-
mafen 1 (0⋅18 g, 64%) as a white solid. Rf = 0⋅2
(25% ethyl acetate/hexanes); m.p. 137⋅5°C. IR (neat,
NaCl) ν 3396 (OH), 1668 (C=O), 1610 (C=C),
1570, 1510, 1452, 1419, 1326, 1240, 1066 (C–O),
+
1⋅95–1⋅80 (m, 1H, CH2); LC–MS (ESI ) m/z 565⋅1 –
1
[M + Na]; trans-Flocoumafen: m.p. 107⋅3°C. H
NMR (CDCl3, 500⋅14 MHz): δ = 7⋅66 (dd, 1H,
J = 1⋅5, 1⋅5 Hz, aromatic-H), 7⋅63 (d, 2H, J = 8⋅0 Hz,
aromatic-H), 7⋅57–7⋅52 (m, 3H, aromatic-H), 7⋅39–
7⋅34 (m, 2H, aromatic-H), 7⋅33–7⋅29 (m, 2H,
aromatic-H), 7⋅27–7⋅20 (m, 2H, aromatic-H), 7⋅16
(d, 2H, J = 8⋅5 Hz, aromatic-H), 6⋅90 (d, 2H,
J = 8⋅5 Hz, aromatic-H), 5⋅12 (s, 2H, benzyl-H),
4⋅72 (t, 1H, J=4⋅0 Hz, CH), 3⋅23 (d, 1H, J =
12⋅0 Hz, CH2), 3⋅12–2⋅99 (m, 2H, CH, CH2), 2⋅36–
+
2⋅32 (m, 2H, CH2); LC–MS (ESI ) m/z 565⋅3 –
[M + Na].
2.2 Biological activity test
2.2a Cell culture: Cerebral cortices were removed
from the brains of 15⋅5-day-old fetal mice. The neo-
cortices were triturated and plated on 24-well plates
–1
1
825 cm . H NMR (CDCl3, 500⋅14 MHz) δ 7⋅72 (d,
1/2H, J = 8⋅0 Hz, aromatic-H), 7⋅68–7⋅61 (m, 5/2H,
aromatic-H), 7⋅57–7⋅50 (m, 3H, aromatic-H), 7⋅38–
7⋅29 (m, 4H, aromatic-H), 7⋅28–7⋅23 (m, 2H, aro-
matic-H), 7⋅19 (dd, 2H, J = 8⋅5, 8⋅5 Hz, aromatic-
H), 6⋅91 (dd, 2H, J = 8⋅5, 8⋅5 Hz, aromatic-H), 5⋅12
(s, 1H, benzyl-H), 5⋅10 (s, 1H, benzyl-H), 4⋅86 (q,
1/2H, J = 6⋅0 Hz, CH), 4⋅72 (t, 1/2H, J = 4⋅5 Hz,
CH), 3⋅23 (d, 1/2H, J = 12⋅5 Hz, CH2), 3⋅13–2⋅98
6
(with approximately 10 cells/mL) precoated with
100 μg/mL poly-D-lysine and 4 μg/mL laminine, in
Eagle’s minimal essential media (Earle’s salts, sup-
plied glutamine-free), and supplemented with horse
serum (5%), fetal bovine serum (5%), 2 mM gluta-
mine, and 20 mM glucose. Cultures were maintained
at 37°C in a humidified atmosphere of 5% CO2.
After 6 days in vitro (DIV), the cultures were shifted

836
Jae-Chul Jung et al
value that is found after a 24 h exposure to 300 μM
NMDA (defined as 0) or a sham control (defined as
100).
to the plating media containing 10 μM cytosine
arabinoside without fetal serum. Cultures were then
fed twice per week. Mixed cortical cell cultures con-
taining neurons and glia (DIV 16-14) were exposed
to excitatory amino acid, glutamate, in Eagle’s
minimal essential media without serum. The mor-
phology of the degenerating neurons was observed
under a phase contrast microscope over the next
24 h. The murine BV2 cell line (a generous gift from
Dr. W. Kim, Korea Research Institute of Bioscience
and Biotechnology, Korea), which is immortalized
after infection with a v-raf/v-myc recombinant ret-
rovirus, exhibits the phenotypic and functional
3. Results and discussion
3.1 Chemistry
The linear synthesis of flocoumafen 1 is summarized
in scheme 2. Commercially available 4-methoxy-
bezaldehyde 7 was condensed with dimethyl malo-
nate as a Knoevenagel condensation in the presence
of AcOH and pyrrolidine to afford diketo ester 8,
which was treated with freshly prepared benzylmag-
nesium bromide in the presence of copper (I) chlo-
ride to give compound 9. Subsequent hydrolysis of 9
was accomplished with aqueous potassium hydroxide.
The resulting diacid 10 was decarboxylated under
acidic condition and then cyclized intramolecularly
using polyphosphoric acid to yield tetralone 11 in
14
properties of reactive microglial cells BV2 cells
were maintained at 37°C at 5% CO2 in DMEM sup-
plemented with 10% FBS, 100 μg/mL streptomycin,
and 100 U/mL penicillin. BV2 cells were grown in
5
24-well plates at a concentration of 1 × 10 cells/
well followed by proper treatment.
16
two steps. Demethylation of tetralone 11 was per-
2.2b Nitrite assay: NO production from activated
microglial cells was determined by measuring the
amount of nitrite, a relatively stable oxidation product
formed by hydrobromic acid in acetic acid to give
phenol 12. It was O-alkylated with freshly prepared
3-(trifluoromethyl)benzyl bromide in sodium hydride/
15
of NO, as described previously. Cells were incu-
17
THF to generate ether 13. Reduction of ketone
bated with or without LPS (1 μg/mL) in the presence
or absence of various concentrations of compounds
for 24 h. The nitrite accumulation in the supernatant
was assessed by Griess reaction. In brief, an aliquot
of the conditioned medium was mixed with an equal
volume of 1% sulfanilamide in water and 0⋅1% N-1-
naphthylethylenediamine dihydrochloride in 5%
phosphoric acid. The absorbance was determined at
540 nm in an automated microplate reader.
group of compound 13 with sodium borohydride in
methanol gave secondary alcohol 3, which was
treated with p-toluenesulfonic acid to give flocou-
18
mafen 1 in 64% yield as 1 : 1 structural isomeric
mixture.
On the other hand, the coupling reaction of bro-
mide 4 with 4-hydroxycoumarin 5 was not effective
for preparation of flocoumafen 1 due to generation
of undesired dehydroxyhalogenated product and
almost recovered starting materials. The obtained
flocoumarin 1 was a 1 : 1 mixture of cis-flocoumafen
(cis-FCF) and trans-flocoumafen (trans-FCF). For
the biological activity test, we separated it into cis-
FCF and trans-FCF using flash silica-column chro-
matography. Also, we could recrystallize flocou-
marin 1 using the co-solvent (ethyl acetate/hexanes)
system in order to separately yield cis and trans
forms.
2.2c Cell viability: Cortical neuronal cell number
and viability were assessed by using the reagent
WST-1 (Roche, Indianapolis, IN). This colorimetric
assay measures the metabolic activity of viable cells
based on cleavage of the tetrazolium salt WST-1
substrate 4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-
5-tetrazolio-1,3-benzene disulphonate into formazan
by mitochondrial dehydrogenase in live cells. This
was followed by incubation with WST-1 reagent at a
dilution of 1 : 10 in the original conditioned media at
37°C for 2 h. After thorough shaking, the formazan
produced by the metabolically active cells in each
sample was measured at a wavelength of 450 nm
and a reference wavelength 650 nm. Absorbance
readings were normalized against control wells with
untreated cells. Neuronal death was analysed 24 h
later, and the percentage of neurons undergoing
actual neuronal death was normalized to the mean
3.2 Suppression of NO-generation and anti-
neurotoxicity
NO production from activated microglial cells was
determined by measuring the amount of nitrite after
incubation with or without LPS (1 μg/mL) in the
presence or absence of various concentrations of

Simple synthesis and biological evaluation of flocoumafen and its structural isomers
837
Scheme 2. Reagents and conditions: (a) Dimethyl malonate, AcOH, pyr-
rolidine, toluene, reflux, 3 h; (b) benzylmagnesium bromide, Cu Cl , THF, 0°C,
2
2
1 h; (c) KOH/H O, reflux, 3 h; (d) H SO /H O, reflux, 8 h; and then PPA, 80°C,
2 2 4 2
1 h; (e) HBr, AcOH, reflux, 6 h; (f) 3-(trifluoromethyl)benzyl bromide, sodium
hydride, THF, 0°C, 1 h; (g) NaBH , MeOH, r.t., 2 h; (h) 4-hydroxycoumarin, p-
4
TsOH, dichloromethane, 80°C, 3 h.
Figure 1. Suppression of NO production in LPS-treated microglia. The cells
were treated with 1 µg/mL of LPS only or LPS plus different concentrations (1,
5, and 10 µM) of compounds at 37°C for 24 h. At the end of incubation, 50 µL
of the medium was removed to measure nitrite production. All values represent
mean SE of three independent experiments performed in triplicate.
compounds for 24 h. All compounds (10 μM) showed agonist) resulted in a rapid swelling of the neuronal
considerable suppression of LPS-induced NO gen- cell body within 2 h, and caused 90 to 100% neu-
eration (figure 1). The trans form of FCF has more ronal death over the next day. The 60 μM of gluta-
potent activity than that of cis-FCF in anti- mate induced the 60% of neurotoxicity after
inflammatory action. However, cis-FCF is slightly 24 h exposure in cultured neurons. These excitotoxic
more active than trans-FCF in the inhibition of glu- neuronal deaths were weakly prevented by all of
tamate-induced neurotoxicity in cultured cortical compounds (1 μM) (figure 2). The results revealed
neurons. Exposure of cortical cell cultures to that suppression of NO production in LPS-treated
300 μM NMDA (prototype of glutamate receptor microglia cell of the trans-flocoumafen is increased

838
Jae-Chul Jung et al
Figure 2. Inhibition of glutamate-induced neurotoxicity in cultured cortical
neurons. Glutamate (60 µM) and compounds (1 µM) were applied for 24 h at
37°C. After incubation of neurons with water soluble tetrazolium salts WST-1
for 2 h, the compounds were quantified spectrophotometrically. All values rep-
resent the mean S.E. of three independent experiments performed in triplicate.
4. Kontogiorgis C A and Hadjipavlou-Litina D J 2005
J. Med. Chem. 48 6400
much more than that of the cis-flocoumafen due to
the favourable electronic conjugation system. We
have found that the trans-FCF exhibited signifi-
cantly high efficacy comparable to mix-FCF or cis-
FCF in vitro LPS-induced NO generation, while all
of them did not show significant neurotoxicity in
cultured cortical neurons.
5. Kurokawa M, Kumeda C A, Yamamura J I,
Kamiyama T and Shiraki K 1998 Eur. J. Pharmacol.
348 45
6. Nawrot-Modranka J, Nawrot E, and Graczyk J, 2006
Eur. J. Med. Chem. 41 1301
7. Al-Soud Y A, Al-Sa’doni H H, Amajaour H A S,
Salih K S M, Mubarak M S, Al-Masoudi N A and
Jaber I H 2008 Zeit. Fuer Naturforschung, B. Chem.
Sci. 63 83
4. Conclusion
8. Nolan K A, Zhao H, Faulder P F, Frenkel A D,
Timson D J, Siegel D, Ross D, Jr. Burke T R,
Stratford I J and Bryce R A 2007 J. Med. Chem. 50
6316
We synthesized flocoumafen 1 from inexpensive
and readily available materials and then separated it
into cis-flocoumafen (cis-FCF) and trans-flocou-
mafen (trans-FCF). Their biological activities have
been evaluated for the NO generation and anti-
excitotoxicity in vitro. We expect that this simple
synthesis of flocoumafen 1 and key fragments are
useful for the synthesis of flocoumafen analogues.
9. Ito Y 2003 Med. Entomol. Zool. 54 337
10. Stanchev S, Momekov G, Jensen F and Manolov I
2008 Eur. J. Med. Chem. 43 694
11. Van Heerden P S, Bezuidenhoudt B C B and Ferreira
D 1997 J. Chem. Soc. Perkin Trans. 1 8 1141
12. Chen D U, Kuo P Y and Yang D Y 2005 Bioorg. &
Med. Chem. Lett. 15 2665
Acknowledgements
13. Armarego W L F, Perrin D D and Butterworth-
Heinemann 1997 Purification of Laboratory Chemi-
cals (Oxford: Pergamon Press) 4th edn
This work was supported by the research grant of
the Chungbuk National University in 2009.
14. Bocchini V, Mazzolla R, Barluzzi R, Blasi E, Sick P
and Kettenmann H J 1992 J. Neurosci. Res. 31 616
15. Green L C, Wagner D A, Glogowski J, Skipper P L,
Wishnock J S and Tannenbaum PSR 1982 Anal. Bio-
chem. 126 131
References
1. Au N and Rettie A E 2008 Drug Metabolism Rev. 40
355
16. Selvaraj S, Rajendran A S and Arumugam N 1987
Indian J. Chem. B26 1047
2. Ghate M, Kusanur R A and Kulkarni M V 2005 Eur.
J. Med. Chem. 40 882
17. Jung J C, Kim J C and Park O S 1999 Synth.
Commun. 29 3587
3. Cuzzocrea S, Mazzon E, Bevilaqua C, Costantino G,
Britti D, Mazzullo G, De Sarro A and Caputi AP 18. Park O S and Jang B S 1995 Arch. Pharm. Res. 18
2000 Brit. J. Pharmacol. 131 1399 277