Structure-activity relationship of wedelolactone analogues: Structural requirements for inhibition of Na+,K+-ATPase and binding to the central benzodiazepine receptor

Article abstract of DOI:10.1016/j.bmc.2006.07.053

Coumestans 2a-i, bearing different patterns of substitution in A- and D-rings, were synthesized and evaluated as inhibitors of kidney Na⁺,K⁺-ATPase and ligands for the central benzodiazepine (BZP) receptor. The presence of a hydroxyl group in position 2 favours the effect on Na⁺,K⁺-ATPase but decreases the affinity for the BZP receptor, allowing the design of more selective molecules than the natural wedelolactone. On the other hand, the presence of a catechol in ring D is important for the effect on both molecular targets.

Full text of DOI:10.1016/j.bmc.2006.07.053

Bioorganic & Medicinal Chemistry 14 (2006) 7962–7966
Structure–activity relationship of wedelolactone analogues:
Structural requirements for inhibition of Na+,K+-ATPase
and binding to the central benzodiazepine receptor
Elisa S. C. Poˆc¸as,a Daniele V. S. Lopes,a Alcides J. M. da Silva,b,c Paulo H. C. Pimenta,a
a
b
b
b
´
Fernanda B. Leitao, Chaquip D. Netto, Camilla D. Buarque, Flavia V. Brito,
˜
Paulo R. R. Costab,* and Franc¸ois Noe¨la,*
a
ˆ
Departamento de Farmacologia Ba´sica e Clı´nica, Instituto de Ciencias Biomedicas,
´
Universidade Federal do Rio de Janeiro, RJ 21941-590, Brazil
ˆ
b
Laborato´rio de Quı´mica Bioorganica, Nucleo de Pesquisas de Produtos Naturais,
Universidade Federal do Rio de Janeiro, RJ 21941-590, Brazil
´
cUniversidade Severino Sombra, Curso de Farma´cia, Vassouras 27700-000, Brazil
Received 28 April 2006; revised 4 July 2006; accepted 26 July 2006
Available online 30 August 2006
Abstract—Coumestans 2a–i, bearing different patterns of substitution in A- and D-rings, were synthesized and evaluated as inhibitors of
kidney Na+,K+-ATPase and ligands for the central benzodiazepine (BZP) receptor. The presence of a hydroxyl group in position 2
favours the effect on Na+,K+-ATPase but decreases the affinity for the BZP receptor, allowing the design of more selective molecules than
the natural wedelolactone. On the other hand, the presence of a catechol in ring D is important for the effect on both molecular targets.
ꢀ 2006 Elsevier Ltd. All rights reserved.
R1
1. Introduction
MeO
O
O
4
R2O
O
O
A
3
A
1
Wedelolactone (1, Fig. 1) is a naturally occurring
coumestan from Eclipta prostrata, a Leguminosae used
in folk medicine against snake poison.1 Some pharmaco-
logical effects of wedelolactone are probably important
for this use, such as its antihaemorrhagic, antiproteolyt-
ic and antiphospholipase activities.1,2 On the other
hand, other effects of wedelolactone are probably unre-
lated to its antimyotoxic action, such as the inhibition of
Na+,K+-ATPase and [3H]flunitrazepam binding.3a Both
effects are potentially relevant and could be useful for
the development of new molecules acting on the
Na+,K+-ATPase or the central benzodiazepine receptor,
two molecular targets of clinical importance, as briefly
discussed below. In such case, however, it should be
important to design molecules with selectivity for only
one of these two targets, since there is no rationale for
a dual action. We previously described in details the
R3
2
8
OH
OH
O
OH
D
OH
O
2a-c
D
1
9
OH
Wedelolactone
a, R1=H; R2=Me, R3=OH
b, R1=R2=H; R3=OMe
c, R1=R2=R3= H
R1
R2O
R3
O
O
R1
R2O
R3
O
O
O
O
2d-f
O
O
OMe
OH
2g,h
d, R1=R2=R3=H
e, R1=OH; R2=R3=H
f, R1=H; R2=H, R3=OH
g, R1=H; R2=Me; R3=OH
h, R1=R2=R3=H
HO
O
O
O
OH
2i
Keywords: Wedelolactone; ATPase,Na-K; Benzodiazepine; Coume-
stan; Isoflavonoid; SAR.
OMe
*
Figure 1. Wedelolactone (1) and analogues bearing different patterns
of substitution in rings A and D.
Corresponding authors. Tel.: +55 021 25626732; fax: +55 021
25626659; e-mail: fnoel@pharma.ufrj.br
0968-0896/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2006.07.053

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E. S. C. Poc¸as et al. / Bioorg. Med. Chem. 14 (2006) 7962–7966
7963
molecular mechanism of action of 2-methoxy-3,8,9-tri-
2. Chemistry
hydroxy coumestan (2b), a non-steroidal original syn-
thetic derivative from wedelolactone (1) (Fig. 1), on
both Na+,K+-ATPase and (central) benzodiazepine
(BZP) site present on the GABAA receptor complex.4,5
Chromenes 6a–c were used as intermediates to prepare
coumestans 2b, 2f and 2h. Baeyer–Villiger oxidation of
easily available aldehydes 3a–c followed by O-alkylation
of the resulting phenols 4a–c with 3-iodo-1,1-dimeth-
oxypropane led to intermediates 5a–c (Scheme 1). Cycli-
zation of these intermediates took place under acid
conditions, furnishing chromenes 6a–c. Phenols 4a–c
were also used as precursors of organomercurials 7a–c.
Oxa-Heck coupling between 6a–c and 7a–c led to the
corresponding protected pterocarpans 8a–c. Oxidation
of the 8a–c with DDQ in THF furnished the coumestans
9a–c. The benzyl protecting groups in 9a–c were re-
moved by hydrogenolysis (H2, Pd–C) to give the desired
coumestans 2b, 2f and 2h.
Our first molecular target, the Na+,K+-ATPase, is a
plasma membrane protein responsible for the mainte-
nance of low Na+, high K+ cellular concentrations using
the energy derived from the hydrolysis of ATP. Besides
that, this protein is considered as the molecular target of
cardiac glycosides,6 drugs used in the treatment of heart
failure due to their positive inotropic effect and benefi-
cial effects on hemodynamics.7 The use of cardiac glyco-
sides such as digoxin in the treatment of moderate and
severe congestive heart failure, mainly in combined ther-
apy, has been recently reinforced by two multicentric
studies8,9 that confirmed the clinical efficacy of digoxin
and its positive effects on morbidity. In addition, a clin-
ical trial sponsored by the NIH10 demonstrated that the
use of digoxin reduces the number of hospitalizations
for these patients. However, the adverse effects and nar-
row therapeutic index of digoxin render its use difficult
and support the interest for searching new inotropic
compounds acting through the inhibition of Na+,K+-
ATPase. The interest of non-steroidal or altered ste-
roid-like inhibitors of Na+,K+-ATPase has recently
emerged in the literature,11–13 since failure to improve
the therapeutic index of digoxin has been attributed to
the preservation of the C/D-cis junction in the steroid
backbone, unique to the cardiac glycosides.14
3. Pharmacology
All the 10 coumestans tested inhibited the Na+,K+-
ATPase activity, but with significant differences in
potency (Table 1). Comparing the coumestans 2a, 2b
and 2c, all bearing a catechol group in the D-ring,
with wedelolactone (1) we can observe that the natu-
rally occurring coumestan 1 and the synthetic isomer
2a were the most potent molecules (IC50 ꢀ 0.7 lM,
Table 1). This result indicates that in A-ring the
change of the hydroxyl group from position 1, as in
1, to position 2, as in 2a, did not affect the capacity
to inhibit the Na+,K+-ATPase. On the other hand,
when the positions of the hydroxyl and methoxy
groups present in 2a are inverted, as in 2b, a five
times loss of affinity is observed, suggesting that the
hydroxyl group in position 2 is important for the
Our second molecular target, the central benzodiazepine
receptor, is an allosteric modulatory site present on the
GABAA receptor complex responsible for inhibitory
pathways in the central nervous system. Classical benzo-
diazepines (agonists) potentiate the GABA neuropsy-
chological effects by increasing GABA affinity for its
binding sites15 leading to anxiolytic, sedative/hypnotic,
anticonvulsant and myorelaxant effects and amnesia.
In spite of being among the most frequently prescribed
drugs, benzodiazepines have limitations, particularly in
relation to long-term use. As a consequence of an im-
proved definition of the appropriate target (specific
sub-type of receptor) the development of anxiolytics
without the undesirable effects of classical benzodiaze-
pines is currently a hot topic in medicinal chemistry.
On the other hand, BZP receptor inverse agonists are
capable of producing convulsions and anxiety but if this
molecule bound at a particular sub-type of receptor (a5)
their effects in cognitive function could be clinically use-
ful in Alzheimer’s disease and related dementia.16 The
aim of the present work was to perform a structure–ac-
tivity relationship (SAR) study with nine coumestans
(2a–i) in order to obtain information on the structural
requirements for each of the two molecular targets de-
scribed above, namely Na+,K+-ATPase and BZP recep-
tor. Wedelolactone was used as a reference compound.
The syntheses of new, non-natural coumestans 2b and
2f, as well as 8-methoxycoumestrol (2h), a natural prod-
uct isolated from Medicago sativa,17 are described for
the first time, while coumestans 2a, 2c, 2d, 2e, 2g and
2i were synthesized as previously described.3
interaction with this enzyme. The absence of
a
O
R3O
R2
O
R3O
R2
OH
ii
R3O
R2
iii
i
OMe
47-53%
70-85%
90-98%
4
3
5
OMe
ClHg
HO
OR8
R3O
O
R3O
R2
O
H
OR9
R2
7
iv
H
(+/-)-8
OR8
OR9
O
6
45-55%
6a + 7a = 8a
6b + 7b = 8b
6c + 7c = 8c
7
3-6
8
a, R8=Bn, R9=Bn
b, R8+R9=CH2
c, R8=Me, R9=Bn
a, R3=Bn, R2=OMe
b, R3=Bn, R2=OBn
c, R3=Bn, R2=H
a, R3=Bn; R2=OMe, R8=R9=Bn
b, R3=Bn; R2=OBn, R8+R9=CH2
c, R3=Bn; R2=H, R8=Me, R9=Bn
R3O
v
O
O
vi
2b, 2f, 2h
R2
40-55%
for two steps
OR8
O
OR9
9
a, R3=Bn; R2=OMe, R8=R9=Bn
b, R3=Bn; R2=OBn, R8+R9=CH2
c, R3=Bn; R2=H, R8=Me, R9=Bn
Scheme 1. Synthesis of coumestans 2b, 2f and 2h. Reagents and
conditions: (i) MCPBA, CH2Cl2; (ii) 3-iodo-1,1-dimethoxypropane,
KOH, THF; (iii) HCl (20%), THF; (iv) PdCl2, LiCl, acetone; (v) DDQ,
THF; (vi) H2 (3.5 atm), Pd–C.

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E. S. C. Poc¸as et al. / Bioorg. Med. Chem. 14 (2006) 7962–7966
7964
Table 1. IC50 values for inhibition of rat kidney Na+,K+-ATPase activity (NaK) and [3H]flunitrazepam binding to rat cerebral synaptosomes (BZD)
1
2a
2b
2c
2d
20
ꢁ30
2e
2f
2g
2h
2i
NaK
BZP
0.7
2
0.7
ꢁ100
3
16
10
2
25
ꢁ100
3
6
30
20
25
50
ꢁ30
ꢁ30
IC50 values, expressed in lM, were obtained from at least three experiments performed in triplicate. When sufficient data were available, these IC50
were calculated by non-linear regression. In the other cases, the values were estimated graphically (see detailed results available in supplementary
material). Symbol ꢁ indicates that much less than 50% inhibition was obtained at the highest concentration tested (indicated in lM).
hydroxyl group at position 2, as in 2c, leads to an
even higher loss of affinity. The same pattern was
observed comparing the potency of 2d, 2e and 2f, all
bearing a less polar methylenedioxy group at the
D-ring but a different pattern of oxygenation at the
A-ring. Coumestan 2f, bearing a hydroxyl group at
the 2-position, is 7–8 times more potent than 2d and
2e. When the phenol group at C8 (D-ring) in 2a
was methylated, as in 2g, the potency also decreased
ten times. Finally, the comparison of 2c with 2h and
2i, which do not present a hydroxyl group at C2,
shows that the catechol group at the D-ring is the best
substituent for Na+,K+-ATPase inhibition.
5. Experimental
5.1. Chemistry
1H NMR spectra were recorded on a Varian Gemini
(200 MHz) instrument using tetramethylsilane (TMS)
as standard and CDCl3 as solvent. J values are given
in Hz.
5.1.1. Baeyer-Villiger oxidation of aldehydes. Synthesis of
4a–c. m-Chloroperbenzoic acid (4.5 mmol) was added to a
solution of the aldehyde 3a–c (3.0 mmol) in dichlorometh-
ane (15 mL), and the mixture was stirred at room temper-
ature for 24 h and then diluted with ethyl acetate. The
organic solution was successively washed with saturated
aqueous Na2CO3 solution and brine. The solvent was
evaporated in vacuo to give corresponding formiate.
NaOH (6 N) was added to a stirred solution of crude form-
iate in MeOH (15 mL). After stirring at room temperature
for 5 min, was added 10% aq HCl solution. The reaction
mixture was diluted with ethyl acetate (50 mL), washed
with brine and dried over anhydrous Na2SO4. The flash
chromatography (7:3 hexane/ethyl acetate) furnished the
compounds 4a–c as solids (70–85% yield).
The BZP binding affinity of coumestans was evaluated
by their ability to displace [3H]flunitrazepam from its
specific binding in rat brain synaptosomes (Table 1).
Wedelolactone (1) inhibited this binding with a tenuous
difference of potency when compared to its inhibitory ef-
fect on Na+,K+-ATPase (IC50 = 2 and 0.7 lM, respec-
tively). However, the structural requirement here seems
to be very different from that for Na+,K+-ATPase since
the presence of a hydroxyl in position 1 or 2 is here
unnecessary or even unfavourable, as illustrated by the
equipotency of 2c (lacking a hydroxyl in C1 and C2)
and 1 (with a hydroxyl in C1) and by the loss of activity
of 2a as compared to 2b and 1.
1
Compound 4a: H NMR d 7.37 (5H, m); 6.75( 1H, d,
J 8.61 Hz); 6.45 (1H, d, J 2.84 Hz); 6.34 (1H, dd,
J 8.61, 2.84 Hz); 5.09 (2H, s); 3.82 (3H, s).
Besides that, the loss of the catechol group in the D-ring
resulted in a decrease of affinity for the benzodiazepine
receptor, as observed when comparing 2c with 2d, 2h
and 2i.
1
Compound 4b: H NMR d 7.36 (10H, m); 6.79 (1H, d,
J 8.61 Hz); 6.49 (1H, d, J 2.84 Hz); 6.29 (1H, dd,
J 8.61, 2.84 Hz); 5.11(2H, s); 5.06 (2H, s).
1
Compound 4c: H NMR d 7.37 (5H, m); 7.09 (1H, t,
J 8.0 Hz); 6.48 (3H, m); 5.10 (2H, s).
4. Conclusion
Our present results indicate that it is chemically possi-
ble to separate the inhibition of Na+,K+-ATPase from
the benzodiazepine-like effect and thus to design more
selective compounds against the two molecular targets
studied here, namely Na+,K+-ATPase and central ben-
zodiazepine receptor. Both the hydroxyl group in po-
sition 2 and the catechol in the D-ring are important
to modulate the inhibition of rat Na+,K+-ATPase. On
the other hand, binding to the central BZP receptor is
decreased if a hydroxyl is present in C2. As a conse-
quence, comparing to the natural wedelolactone (1)
which is nearly equipotent for these two molecular
targets, it was possible to construct a molecule, like
2a, with a high selectivity towards the Na+,K+-ATP-
ase and another, like 2c, with a relative selectivity
for the benzodiazepine receptor. In both cases this
could be interesting for clinical purposes.
5.1.2. O-Alkylation of phenols. Synthesis of 5a–c. A mix-
ture of KOH (224 mg, 2 mmol) and phenols 4a–c
(2.15 mmol) in THF (25 mL) was stirred for 10 min at
room temperature. Then 3-iodopropionaldehyde
dimethyl acetal (1 mmol) was added slowly and the
resulting mixture refluxed for 5 h. The reaction mixture
was cooled and the 3-iodopropionaldehyde dimethyl
acetal (1 mmol) was added slowly and the resulting mix-
ture refluxed for 12 h. The reaction mixture was cooled,
extracted with ethyl acetate, washed with brine, dried
over sodium sulfate and concentrated. The flash chro-
matography (7:3 hexane/ethyl acetate) furnished a yel-
low oil (90–98% yield).
1
Compound 5a: H NMR d 7.37 (5H, m); 6.81 (1H, d, J
8.80 Hz); 6.60 (1H, d, J 2.60 Hz); 6.43 (1H, dd, J 8.80,
2.60 Hz); 5.13 (2H, s); 4.61 (1H, t, J 5.8 Hz); 3.95 (2H,

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7965
t, J 6.40 Hz); 3.84 (3H, s); 3.36 (6H, s); 2.04 (1H, q,
J 6.00 Hz).
J 5.49 Hz); 5.89 (1H, d, J 5.49 Hz); 5.41 (1H, d,
J6.87 Hz); 5.13 (2H, s); 5.11 (2H, s); 4.18 (1H, dd, J
10.8, 4.63 Hz); 3.59 (1H, t, J 10.75 Hz); 3.46 (1H, m).
13C NMR d 153.90 (C); 150.53 (C); 150.23 (C); 147.84
(C); 143.71 (C); 141.82 (C); 137.15 (C); 136.56 (C);
128.31–126.97 (10 CH); 117.67 (C); 116.60 (CH);
111.31 (C); 104.51 (CH); 103.18 (CH); 101.06 (CH2);
93.53 (CH); 78.30 (CH); 72.01 (CH2); 70.59 (CH2);
66.31 (CH2); 40.00 (CH).
1
Compound 5b: H NMR d 7.38 (10H, m); 6.84 (1H, d,
J 8.79 Hz); 6.57 (1H, d, J 2.80 Hz); 6.29 (1H, dd,
J 8.80, 2.80 Hz); 5.12 (2H, s); 5.07 (2H, s); 4.60 (1H, t,
J 5.74 Hz); 3.94 (2H, t, J 6.35 Hz); 3.35 (6H, s); 2.03
(2H, q, J 6.35 Hz).
Compound 5c: 1H NMR d 7.4 (5H, m); 7.2 (1H, t,
J 8.00 Hz); 6.6 (3H, m); 5.00 (2H, s); 4.60 (1H, t,
J 5.50 Hz); 4.00 (2H, t, J 5.50 Hz); 3.4 (6H, s); 2.1
(2H, q, J 5.50 Hz). 13C NMR d 160.00 (C); 159.90 (C);
136.80 (C); 129.80 (CH); 128.40 (CH); 127.80 (CH);
127.30 (CH); 101.9 (CH2); 101.80 (CH); 101.70 (CH);
69.80 (CH2); 63.80 (CH2); 53.10 (CH3); 32.6 (CH2).
1
Compound 8c: H NMR d 7.40 (10H, m); 6.85 (1H, s),
6.70 (1H, dd, J 8.60, 2.37 Hz); 6.53 (3H, m); 5.46 (1H, d,
J 6.23 Hz); 5.11 (2H, s); 5.05 (2H, s); 4.26 (1H, dd,
J 10.07, 4.21 Hz); 3.86 (3H, s); 3.65 (1H, t,
J 10.62 Hz); 3.53 (1H, m).
5.1.5. Synthesis of coumestans 2b, 2f and 2h. To a solu-
tion of 8a–c (0.1 mmol) in THF (3.5 mL) was added
DDQ (41.3 mg, 0.2 mmol). The resulting mixture was
stirred at room temperature for 12 h. The coumestans
9a–c precipitated out of solution and they were col-
lected by filtration and washed with cold hexane.
The crude products were allowed to react with hydro-
gen (3 atm) in acetone for 6 h. After this time, the cat-
alyst was filtered (Celite)ꢂ and concentrated in
vacuum to furnish the compounds 2b, 2f and 2h
(40–55% overall yield). Homogeneous spectra were ob-
tained in the three cases. In the case of 2b and 2h, the
purity was also checked by HPLC (CH3CN/H2O;
55:45) and was around 91–96%.
5.1.3. Synthesis of chromens 6a–c. To a solution of 5a–c
(0.98 mmol) in THF (5 mL) was added 10% aq HCl at
0 ꢁC. The reaction mixture was stirred at 0 ꢁC for 1 h.
Then the ice bath was removed and the reaction mixture
was stirred for 12 h at room temperature. After this time
saturated aqueous NaHCO3 solution was added and the
mixture was extracted with ethyl acetate, washed with
brine, dried over sodium sulfate, concentrated. The flash
chromatography (8:2 hexane/ethyl acetate) furnished a
yellow oil (47–53%).
1
Compound 6a: H NMR d 7.40 (5H, m); 6.59 (1H, s);
6.42 (1H, s); 6.30 (1H, dt, J 13.00, 3.20 Hz); 5.61 (1H,
dt, J 13.00, 4.90); 5.10 (2H,s); 4.78 (2H, dd, J 4.90,
3.20); 3.82 (3H, s).
1
Compound 2b: H NMR d 10.39 (1H, s); 9.48 (1H, s);
9.42 (1H, s); 7.37 (1H, s); 7.23 (1H, s); 7.17 (1H, s);
6.95 (1H, s); 3.91 (3H, s). Anal. Calcd for C16H10O7:
C, 61.15; H, 3.21. Found: C, 62.00; H, 3.11.
1
Compound 6c: H NMR d 7.40 (5H, m); 6.90 (1H, d, J
9.80 Hz); 6.50 (2H, dd, J 9.80, 3.00 Hz); 6.50 (1H, d, J
3.00 Hz); 6.40 (1H, dt, J 13.00, 3.2 Hz); 5.60 (1H, dt, J
13.00, 4.90 Hz); 5.00 (2H, s); 4.80 (2 H, dd, J 4.90,
3.20). 13C NMR d 159.60 (C); 155.10 (C); 136.70 (C);
128.40 (CH); 127.30 (CH); 127.10 (CH); 131.10 (C);
124.00 (CH); 118.80 (CH); 115.80 (C); 107.70 (CH);
102.50 (CH); 69.90 (CH2); 65. 40 (CH2).
Compound 2f: 1H NMR d 7.38 (1H, s); 7.36 (1H, s); 7.33
(1H, s); 6.99 (1H, s); 6.16 (2H, s). Anal. Calcd for
C16H8O7: C, 61.55; H, 2.58. Found: C, 61.00; H, 2.48.
1
Compound 2h: H NMR d 10.66 (1H, s); 9.62 (1H, s);
7.85 (1H, d, J 9.16 Hz); 7.32 (1H, s); 7.25 (1H, s); 6.94
5.1.4. Oxa-Heck coupling for the synthesis of 8a–c. To a
mixture of PdCl2 (87 mg, 0.49 mmol) and LiCl (42 mg,
1.0 mmol) in acetone (5 mL) were added chromens 6a–
c (0.46 mmol) in acetone (10 mL). This mixture was stir-
red for 15 min at 0 ꢁC and then 2-chloromercurio–phe-
nols 7a–c (0.42 mmol) in acetone (10 mL) were added.
The suspension obtained was stirred for 12 h at 25 ꢁC.
After this time, brine (150 mL) was added to it and
the mixture was extracted with ethyl acetate (3·
50 mL), the organic extract dried (Na2SO4) and submit-
ted to column chromatography (8:2 hexane/ethyl ace-
tate) to give the compounds 8a–c as solids (45–55%
yield).
(2H, m).17
5.2. Pharmacology
A 30 mM stock solution in DMSO was made for all
compounds. Further dilutions were done immediately
before use in water or Tris–HCl-buffered Krebs solution
for Na+,K+-ATPase inhibition and [3H]flunitrazepam
binding, respectively.
5.3. Tissue preparation
5.3.1. Na+,K+-ATPase. Adult male Wistar rats were
killed by decapitation and their kidneys were rapidly ex-
cised and stored at À80 ꢁC. Preparations enriched in
Na+,K+-ATPase were obtained by chaotropic treatment
with 2 M KI for 1 h and 0.1% DOC (sodium deoxicho-
late) overnight, followed by differential centrifugation,
as earlier described.18 The protein concentration was
measured according to the method of Lowry et al.19
using bovine serum albumin as the standard.
1
Compound 8a: H NMR d 7.40 (5H, m); 6.96 (1H, s);
6.85 (1H, s); 6.54 (1H, s); 6.49 (1H, s); 5.42 (1H, d,
J 6.41 Hz); 5.13 (2H, s); 5.10 (2H, s); 5.06 (2H, s); 4.14
(1H, dd, J 9.61, 3.75 Hz); 3.89 (3H, s); 3.50 (2H, m).
1
Compound 8b: H NMR d 7.37 (10H, m); 7.07 (1H, s);
6.70 (1H, s); 6.53 (1H, s); 6.43 (1H, s); 5.90 (1H, d,

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7966
5.3.2. Benzodiazepine receptor. Brain hemispheres were
obtained from male Wistar rats sacrificed by decapita-
tion. Briefly, tissues were homogenized in a Potter appa-
ratus with a motor-driven Teflon pestle at 4 ꢁC in 15
volumes of ice-cold 0.32 M buffered sucrose (pH 7.4)
per gram of organ. After centrifuging at 1000gmax for
10 min, the supernatant was centrifuged at 48,000gav
for 20 min to obtain the crude synaptosomes that were
resuspended in a buffered Krebs solution and stored at
À80 ꢁC until use.
5.3.3. Inhibition of Na+,K+-ATPase activity. The
Na+,K+-ATPase activity was determined by the Fiske
and Subbarow method with slight modifications, as de-
scribed earlier.20 The specific activity of the enzyme corre-
sponds to the difference between the total ATPase activity
and the activity measured in the presence of 1 mM oua-
bain. The reaction was started by addition of the prepara-
tion, incubated at 37 ꢁC for 2 h, in a total volume of
0.5 mL. The incubation was performed in the presence
of 84 mM NaCl, 3 mM KCl, 3 mM MgCl2, 1.2 mM ATP-
Na2, 2.5 mM EGTA, 10 mM sodium azide and 20 mM
maleic acid buffered to pH 7.4 with Tris, in the presence
or absence of increasing concentrations of the
coumestans.
Supplementary data
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/
j.bmc.2006.07.053.
References and notes
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5.3.4. [3H]Flunitrazepam binding assay. Synaptosomes
(200 lg protein) were incubated at 4 ꢁC for 90 min in a
buffered Krebs solution (Tris–HCl, pH 7.4, at 4 ꢁC),
containing 0.2 nM [3H]flunitrazepam (85 Ci/mmol,
New England Nuclear Life Science Products, USA).
After incubation, samples were rapidly diluted with
3 mL of ice-cold Krebs buffer and immediately filtered
on glass fibre filters (GMF3, Filtrak, Germany) under
vacuum. Filters were then washed once more with
3 mL buffer, dried and immersed in a scintillation cock-
tail (0.1 g/L POPOP and 4.0 g/L PPO in toluene) and the
radioactivity retained in the filters was counted in a
Packard Tri-Carb 1600 TR liquid scintillation analyzer.
Non-specific binding was estimated in the presence of
5 lM unlabelled flunitrazepam. Competition experi-
ments were performed adding increasing concentrations
of the coumestans.
Acknowledgments
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Our research was supported by grants from PRONEX
(No. E-26/171.162/2003), FAPERJ, FUJB-UFRJ and
CAPES, in Brazil. E.S.C.P., D.V.S.L., F.B.L.,
P.H.C.P., C.D.N., C.D.B. and F.V.B. were supported
in part by CAPES, FAPERJ and CNPq.
20. Noe¨l, F.; Pardon, S. Life Sci. 1989, 44, 1677.