Synthesis of Coumestrol and Aureol.

A total synthesis of coumestrol (1) and aureol (2) is described. The Perkin condensation of 2-bromo-4-hydroxylphenylacetic acid (6) and o-hydroxybenzaldehydes (7) gave the corresponding 2'-bromo-3-arylcoumarins (9). A copper-catalyzed consecutive hydroxylation and aerobic oxidative coupling of 9 under microwave conditions facilitated the total synthesis of 1 and 2, respectively, with spectroscopic data highly similar to those of natural products.

Ability of a synthetic coumestan to antagonize Bothrops snake venom activities.

We investigated a synthetic coumestan named LQB93 and similar compounds abilities to antagonize activities of Bothrops jararacussu and Bothrops jararaca crude venoms in different protocols. The antimyotoxic activity was evaluated in vitro by the rate of release of creatine kinase (CK) from isolated mouse extensor digitorum longus muscle (EDL) induced by B. jararacussu (25 g/ml). For in vivo studies, B. jararacussu venom (1.0 mg/kg) was preincubated with LQB93 (0.1-30 mg/kg), during 30 min, for later injection in mouse tight and evaluation of the antimyotoxic and anti-edematogenic effects. LQB93 antagonized in vitro, the increase of CK release from the EDL muscle (IC(50)=0.0291 M). It also showed in vivo, antimyotoxic and anti-edematogenic effects that were dose-dependent with ID50 of 0.17 mg/kg and 0.14 mg/kg, respectively. The hemorrhage induced by B. jararaca (1.0 mg/kg) venom in the mouse skin, was abolished by LQB93 (10.0 mg/kg) preincubated with venom. Like wedelolactone, LQB93 protected rat isolated heart on a Langendorff preparation, from the cardiotoxicity of B. jararacussu venom. LQB93 inhibit the effects of Bothrops venoms like wedelolactone, a natural compound isolated from the plant Eclipta prostrata.

Inhibition of the alpha1beta1 isoform of the Na, K-ATPase by 8-methoxycoumestrol without positive inotropic effect in human myocardium--novel aspects of cardiac glycoside pharmacology.

BACKGROUND: The Na,K-ATPase (NKA) is necessary for maintaining the resting membrane potential by transporting Na and K ions across the cell membrane. Although its 3 isoforms expressed in human heart (alpha1beta1, alpha2beta1, and alpha3beta1) possess similar biochemical properties, their specific functions in human tissues remain unknown. In our search for an isoform-specific agent, which can serve to identify isoform-specific functions, we examined 8-methoxycoumestrol in its ability to inhibit the NKA and to produce inotropism in connection with the possibility to identify the NKA isoform-specific functions. METHODS AND RESULTS: In radioligand binding experiments (membrane preparations of yeast expressing isoforms alpha1beta1, alpha2beta1, and alpha3beta1; backdoor phosphorylation; and [H]-ouabain, n = 3), 8-methoxycoumestrol (1-10 microM) produced no or only little inhibition of specific ouabain binding. However, when NKA activity of the alpha1beta1 isoform was measured in membrane preparations from human kidney (reduced form of nicotinamide adenine dinucleotide-coupled assay, n = 3), a concentration-dependent full inhibition of the activity was induced by 8-methoxycoumestrol (IC50: 90 +/- 97 nM), similar to that observed for classical cardiac glycosides digitoxin, digoxin, methyldigoxin, and beta-acetyldigoxin (IC50 = 287 +/- 190 nM, 409 +/- 171 nM, 282 +/- 482 nM, 587 +/- 135 nM, P > 0.05). However, unlike the classical cardiac glycosides, 8-methoxycoumestrol did not increase cardiac contractility of electrically stimulated human right atrial trabeculae. CONCLUSIONS: These results indicate that 8-methoxycoumestrol inhibits the human alpha1beta1 NKA by a mechanism different to that of cardiac glycosides. In addition, the inhibition of the alpha1beta1 NKA activity seems not sufficient to evoke positive inotropy in human trabeculae, indicating that either the positive inotropic effect of cardiac glycosides is not mediated via the alpha1beta1 isoform or the specific glycoside binding to alpha1beta1 is needed for positive inotropy.

An efficient synthesis of coumestrol and coumestans by iodocyclization and Pd-catalyzed intramolecular lactonization.

[reaction: see text] The iodocyclization of acetoxy-containing 2-(1-alkynyl)anisoles and subsequent direct palladium-catalyzed carbonylation/lactonization provide an efficient route to naturally occurring coumestan and coumestrol, and their related analogues.

Synthesis of hapten and conjugates of coumestrol and development of immunoassay.

3-O-Carboxymethylcoumestrol was prepared as the hapten for immunoassay by a partial alkylation of coumestrol with ethyl chloroacetate in acetone alkalized with potassium carbonate. 3-O-Ethoxycarbonylmethylcoumestrol was separated by column chromatography and finally was hydrolyzed with formic acid. 1H and 13C NMR data (APT, COSY, HMQC, and HMBC) revealed that the reaction was regioselective, as 3-O-ethoxycarboxymethylcoumestrol was the only monosubstituted derivative. The hapten was then conjugated to bovine serum albumin and used for immunization of rabbits. A radioimmunoassay (RIA) system was established based on the polyclonal antiserum and a 125I-labeled hapten-tyrosine methyl ester conjugate as the radioligand. Parameters of the RIA: sensitivity: 12 pg per tube, 50% intercept: 140 pg per tube, working range: 20-4000 pg per tube. The cross-reactivity of a panel isoflavonoid and lignan phytoestrogens was either negligible (e.g. formononetin 0.07%; biochanin A 0.06%) or not detectable at all. The major immunoreactive peak in HPLC fractions from an alfalfa extract had the same retention time as coumestrol standard and represented 94.8% of the signal. The remaining 5.2% of immunoreactivity was distributed between five minor peaks. We conclude that after the validation for particular matrices, the method will be a useful tool for analysis of coumestrol, especially in low volume and low concentration samples.