Synthesis and preliminary pharmacological evaluation of coumestans with different patterns of oxygenation

Article abstract of DOI:10.1016/S0960-894X(00)00621-1

Five coumestans with different patterns of oxygenation in rings A and D were synthesized from resorcinol and aromatic aldehydes, and screened for their antimyotoxic activity. The most potent compound (2b, IC₅₀ = 1 μM) was selected for study of

Full text of DOI:10.1016/S0960-894X(00)00621-1

Bioorganic & Medicinal Chemistry Letters 11 (2001) 283±286
Synthesis and Preliminary Pharmacological Evaluation of
Coumestans with Di€erent Patterns of Oxygenation
Alcides J. M. da Silva,a Paulo A. Melo,b Noelson M. V. Silva,b Flavia V. Brito,a
Camilla D. Buarque,a Daniele V. de Souza,b Veronica P. Rodrigues,b Elisa S. C. Pocas,b
Francois Noel,b Edson X. Albuquerqueb,c and Paulo R. R. Costaa,Ã
a
Â
Â
Ã
Â
Laboratorio de Quõmica Bioorganica (LQB), Nucleo de Pesquisas de Produtos Naturais,
Universidade Federal do Rio de Janeiro, Rio de Janeiro, 21941-590, Brazil
b
Â
Departamento de Farmacologia Basica e Clõnica, Centro de Ciencias da Saude, Blocos H e J,
Â
Universidade Federal do Rio de Janeiro, Rio de Janeiro, 21941-590, Brazil
Ã
Â
cDepartment of Pharmacology and Experimental Therapeutics, University of Maryland School of Medicine,
Baltimore, MD 21201, USA
Received 14 August 2000; accepted 30 October 2000
AbstractÐFive coumestans with di€erent patterns of oxygenation in rings A and D were synthesized from resorcinol and aromatic
aldehydes, and screened for their antimyotoxic activity. The most potent compound (2b, IC50=1 mM) was selected for study of its
pharmacological pro®le. # 2001 Elsevier Science Ltd. All rights reserved.
Naturally occurring coumestans, used in folk medicine
as snake antivenom, mainly isolated from plants of the
family Fabaceae, have interesting pharmacological
properties.1 Mors et al.2 and Melo et al.3 reported that
1a, a constituent of the plant Eclipta prostrata L.
(Asteraceae), antagonized the muscle damage, the hae-
morrhagic e€ect, and the proteolytic and phospholipase
activity of di€erent Brazilian and North American
snake venoms and their isolated toxins, in vitro and in
vivo. Wagner and Fessler4 found a potent and selective
5-lipoxygenase-inhibitor action for wedelolactone (1a)
(IC50=2.5 mM) while coumestrol (1b) was 40 times less
potent. Wagner et al.5 also reported an antihepatotoxic
activity for 1a and synthetic derivatives such as 2e in
assays employing CCL4, GaIN, and phalloidin-cyto-
toxicity in rat hepatocytes. As part of a program aimed
at synthesizing biologically active ¯avonoids,6 some
coumestans bearing di€erent patterns of oxygenation at
rings A and D (2a±e) were chosen as target molecules.
Coupound 2a is natural and 2e was previously prepared
by Wagner et al.5 On the other hand, 2b±d are described
for the ®rst time in this letter.
The pharmacological pro®le of a selected coumestan
(2b) was also further examined.
Chemistry
Pterocarpans 7a±e (Scheme 1) were considered as inter-
esting precursors to prepare the target coumestans
through oxidation followed by debenzylation.7 These
compounds could be obtained by a Heck type reaction
between appropriately substituted chromenes (5a,b) and
organomercurials (6a±d). The di€erent patterns of oxy-
genation present in these intermediates was assured by the
use of easily available benzaldehyde derivatives (3a±c),
resorcinol derivatives (4e), and sesamol (4d), as starting
The action of 2a±e as antimyotoxic was evaluated and
compared with that observed for wedelolactone (1a).
ÃCorresponding author. Tel.: +55-21-270-2683; fax: +55-21-523-
5938; e-mail: prrcosta@nppn.ufrj.br
0960-894X/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(00)00621-1

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A. J. M. da Silva et al. / Bioorg. Med. Chem. Lett. 11 (2001) 283±286
Figure 1. E€ect of some coumestan derivatives on CK release induced
by Bothrops jararacussu venom. (A) Mouse EDL muscles were exposed
during 60 min to physiological saline solution (PSS) or B. jararacussu
venom (25 mg/mL) either alone or in the presence of 30 mM of 1a
(wedelolacetone) or one of its analogues (compounds 2a±e). The basal
1
1
rate of CK release in control conditions was 0.35Æ0.07 U g
h
(n=29). (B) Concentration±e€ect curve for compounds 1a and 2b.
Each point represents the meanÆSE (n=3±6).
tion with 3-iodo propanal dimethylacetal followed by
cyclization in acid medium led to the obtention of
chromenes 5a,b. As expected, the coupling reaction
between 5a±e and 6a±d furnished the corresponding
pterocarpans 7a±e. Coumestans 2a±e were prepared by
oxidation of pterocarpans 7a±e with DDQ in THF at
room temperature,9 followed by hydrogenolysis of the
protecting benzil groups.
Biological Results and Discussion
At 30 mM, all ®ve wedelolactone analogues antagonized
the creatine kinase (CK) release10 induced by Bothrops
jararacussu venom (Fig. 1A).
Scheme 1.
materials. The syntheses of the necessary chromenes and
organomercurials were accomplished as shown in
Scheme 1.
Compound 2b inhibited the venom myotoxic activity in
a concentration-dependent manner, with an IC50 (1 mM)
similar to the one of compound 1a (wedelolactone,
Fig. 1B).
Baeyer±Villiger oxidation of aldehydes 3a±c furnished
the corresponding phenols 4a±c,8 while 4d±e (resorcinol
and sesamol, respectively) are commercially available.
Reaction of some of these phenols with (AcO)2Hg led to
the corresponding organomercurials 6a±d, while alkyla-
Similarly to compound 1a, 2b inhibited the proteolytic
and phospholipase activities of Bothrops venom (data
not shown).11 Compound 2b was further submitted to

A. J. M. da Silva et al. / Bioorg. Med. Chem. Lett. 11 (2001) 283±286
285
radioreceptor and enzymatic assays in order to screen for
Acknowledgements
di€erent potential molecular targets. As shown in Figure
2A, both 1a and 2b were relatively potent (IC50=0.7 and
0.5 mM, respectively) for inhibiting rat kidney Na+,
K+-ATPase,12 the enzymatic target for the therapeutic
and toxic e€ects of cardiac glycosides.13 At about the
same concentration (IC50=2 mM), 1a also inhibited
[3H]-¯unitrazepam binding to rat brain synaptosomes,14
indicating a potential modulatory e€ect on the GABAA/
chloride ion channel involved in inhibitory transmission
in the central nervous system.15 On the other hand, 2b
was practically without e€ect in this assay since its IC50
was higher than 100 mM (Fig. 2B).
This work has been done in honour of the 80th birthday
of Professor Walter B. Mors, who stimulated and initi-
ated the study of wedelolactone as a snake antivenom. Our
research was supported by grants from PRONEX (No.
41.96.0888.00), FAPERJ, FUJB-UFRJ and CAPES.
A.J.M.S is a postdoctoral fellow of FAPERJ (No. 26/
151.081/97). F.N. and P.R.R.C. are fellows of CNPq.
F.V.B., C.D.B., D.V.S. and V.P.R. are recipients of
CNPq (PIBIC) fellowships. E.S.C.P. is a recipient of the
FAPERJ fellowship. We thank ROCHE S/A for the
supply of ¯unitrazepam.
The newly synthesized 2b is equipotent to wedelolactone
for its antimyotoxic action and inhibition of Na+, K+-
ATPase. In addition, compound 2b is less potent for
binding to benzodiazepine receptors, a ®nding which
makes compound 2b less susceptible to produce adverse
e€ects in the central nervous system. Furthermore, we
can hypothesize that some of the other derivatives syn-
thesized could di€er in their selectivity for the three
main e€ects described here, due to probable di€erences
in the structural requirements for binding to the respec-
tive molecular targets. Further structure±activity studies
are needed for selecting pro®les of interest.
References and Notes
1. (a) Harbone, J. B.; Mabry, T. J.; Mabry, H. The Flavo-
noids; Chapman and Hall: London, 1975; pp 780±784. (b)
Bicko€, E. M.; Spencer, R. R.; Witt, S. C.; Knuckles, B. E.
Agricultural Research ServiceÐUnited States Department of
Agriculture: Tech. Bull. No. 1408, p. 1.
2. Mors, W. B.; Nascimento, M. C.; Parente, J. P.; Silva,
M. H.; Melo, P. A.; Suarez-Kurtz, G. Toxicon 1989, 27, 1003.
3. (a) Melo, P. A.; do Nascimento, M. C.; Mors, W. B.;
Suarez-Kurtz, G. Toxicon 1994, 32, 595. (b) Melo, P. A.;
Ownby, C. L. Toxicon 1999, 37, 199.
4. Wagner, H.; Fessler, B. Planta Medica 1986, 52, 374.
5. Wagner, H.; Geyer, B.; Kiso, Y.; Hikino, H.; Rao, G. S.
Planta Medica 1986, 52, 370.
6. (a) Costa, P. R. R.; Coelho, A. L.; Simas, A. B. C. Syn-
thesis 1999, 6, 1017. (b) Lichtenfels, R. A.; Coelho, A. L.;
Costa, P. R. R. J. Chem. Soc., Perkin Trans. 1 1995, 949. (c)
Coelho, A. L.; Vasconcellos, M. A. A.; Simas, A. B. C.; Rabi,
J. A.; Costa, P. R. R. Synthesis 1992, 914.
7. The oxidation of one pterocarpan to the corresponding
coumestan was previously reported; Narkhede, D. D.; Iyer,
P. R.; Iyer, C. S. R. J. Nat. Prod. 1989, 3, 502.
8. Barrero, A. F.; Alvarez-Manzaneda, E. J.; Chahboun, R.
Tetrahedron 1998, 54, 5635.
9. To a solution of 7a±e (1 mM) in THF (38 mL) was added
DDQ (2 mM). The resulting mixture was stirred at room tem-
perature for 2 h. The intermediate O-benzylated coumestan
precipitated out of solution and it was collected by ®ltration
and washed with cold hexane. The crude product was allowed
to react with hydrogen (2 atm in acetone for 3 h). The resulting
debenzylated coumestanes 2a±e were puri®ed by ¯ash chro-
matography (hexane/AcOEt, 1/1, v/v).
10. In vitro myotoxicity was evaluated at room temperature
as previously described.3a Brie¯y, extensor digitonum longos
muscles were blotted, weighed rapidly and then transferred to
sample collecting units of 2.5 mL capacity, where they were
superfused continuously at a rate of 3 mL/min with physi-
ological saline solution (PSS) equilibrated with 95% O2/5%
CO2. At 40 min intervals, the solutions perfusing the muscles
were collected and replaced with fresh media. The collected
samples were stored at 4 ꢀC until their CK activity was deter-
mined using a diagnostic kit purchased from Sigma Chemical
Co. The rate of CK release from the isolated muscles is
expressed as international enzyme units released into the
medium per gram of muscle per hour of collection (U g 1 h 1).
The basal release rate refers to the enzyme loss from the mus-
cles into the PSS at the beginning of the experiment, after the
preparations had been mounted in the sample collecting units
for at least 1 h.
The composition of the PSS was (mM) NaCl, 135; KCl, 5
CaCl2, 2; MgCl2, 1; NaHCO3, 15; NaH2PO4, 1; glucose 11.
The pH of this solution after equilibration with 95% O2/5%
Figure 2. (A) E€ect of compounds 1a (wedelolactone) and 2b on rat
kidney Na+, K+-ATPase and (B) [3H]-¯unitrazepam binding to rat
synaptosomes.

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CO2 was 7.4Æ0.02. Bothrops jararacussu venom alone or
associated to the di€erent coumestans was added to the nutri-
ent solution which superfused the isolated muscles.
The experiments with (CK) release were performed in mul-
tiple (at least four) independent experiments.
ATPase inhibition (`Fiske and Subbarrow assay' but with
1.2 mM, instead of 3 mM, ATP) were previously described in:
Noel, F.; Godfraind, T. Biochem. Pharmacol. 1984, 33, 47.
13. Allen, D. G.; Eisner, D. A.; Wray, S. C. Nature 1985, 316,
674.
11. Phospholipase A2 activity of B. jararacussu venom (1±30mg/
mL) was evaluated by the indirect hemolysis of washed rabbit
red blood cell with egg yolk as a substrate, as described earlier.3
12. Procedures for preparation of partially puri®ed micro-
somes from rat kidney and determination of Na+, K+-
14. Crude synaptosomes (200 mg protein) prepared from rat
brain (without cerebellum and brainstem) were incubated at
4 ꢀC for 90 min in a bu€ered Krebs solution containing 0.2 nM
[3H]-¯unitrazepam.
15. Olsen, R. W. Ann. Rev. Pharmacol. Toxicol 1982, 22, 245.