3, 9-di-O-substituted coumestrols incorporating basic amine side chains act as novel apoptosis inducers with improved pharmacological selectivity.

There is much interest in the use of phytoestrogens such as coumestrol in breast cancer intervention due to their antiestrogenic activity and multiple modes of tumor cell death. However, the clear beneficial effects of naturally occurring estrogen mimetic coumestrol remain controversial due to experimental evidence that it has been shown to stimulate MCF-7 cell proliferation via agonist effect on estrogen receptor at low concentration. Herein, to disconnect the ER interaction and apoptosis-specific mechanism of coumestrol, various 3, 9-di-O-substituted coumestrols (7a-7e) and their furan ring-opened analogs (5a-5e) were synthesized and assessed for antiproliferative properties. Attachment of a dimethylamine-containing side chain to 3-O of coumestrol led to the most promising compound 7e with improved antiproliferative activity (1.7-fold increase) against MCF-7 cells, decreased estrogen activity (>20 times weaker ERα binder) and a novel action to induce apoptosis. Mechanistic studies revealed that 7e is a tubulin polymerization inhibitor, which could arrest cell cycle at G2/M phase and induce apoptosis along with the decrease of mitochondrial membrane potential. In summary, such subtle modifications to the 3, 9-di-hydroxyl groups of coumestrol allow the generation of a novel apoptosis inducer with distinct pharmacological properties, providing an excellent starting point to future development of novel tumor-vascular disrupting agents targeting tubulin.

Aerobic iron-based cross-dehydrogenative coupling enables efficient diversity-oriented synthesis of coumestrol-based selective estrogen receptor modulators.

An iron-based cross-dehydrogenative coupling (CDC) approach was applied for the diversity-oriented synthesis of coumestrol-based selective estrogen receptor modulators (SERMs), representing the first application of CDC chemistry in natural product synthesis. The first stage of the two-step synthesis of coumestrol involved a modified aerobic oxidative cross-coupling between ethyl 2-(2,4-dimethoxybenzoyl)acetate and 3-methoxyphenol, with FeCl3 (10 mol%) as the catalyst. The benzofuran coupling product was then subjected to sequential deprotection and lactonization steps, affording the natural product in 59% overall yield. Based on this new methodology other coumestrol analogues were prepared, and their effects on the proliferation of the estrogen receptor (ER)-dependent MCF-7 and of the ER-independent MDA-MB-231 breast cancer cells were tested. As a result, new types of estrogen receptor ligands having an acetamide group instead of the 9-hydroxyl group of coumestrol were discovered. Both 9-acetamido-coumestrol and 8-acetamidocoumestrol were found more active than the natural product against estrogen-dependent MCF-7 breast cancer cells, with IC50 values of 30 and 9 nM, respectively.

[Simultaneous determination of four kinds of furocoumarins in cosmetics by high performance liquid chromatography].

OBJECTIVE: A high performance liquid chromatographic (HPLC) method was developed for the simultaneous determination of 4 furocoumarins, including 8-methoxycoumarin, 5-methoxycoumarin, trioxsalen and imperatorin in cosmetics. METHODS: The separation was carried on Diamonsil C18 column (4.6 mm x 250 mm, 5 microm) by gradient elution with methanol-water as mobile phase at a flow rate of 1.0 ml/min and column temperature of 30 degrees C. The analysis of 4 furocoumarins was performed by HPLC with diodearray detector at the wavelength of 248 nm. RESULTS: Identification of 4 furocoumarins was achieved by retention time, and quantification analysis was performed by the external standard method. The limits of quantitation (LOQ) for 4 furocoumarins were 0.54, 0.52, 0.58 and 0.50 microg/g, respectively. The method showed a good linearity in the range of 0.05-10 microg/ml with correlation coefficients more than 0.999. The mean recoveries at spiked levels were in the range of 89.9%-105.2% with RSDs of 0.20%-1.08%. CONCLUSION: The method was accurate and simple, and was suitable for the determination of 4 furocoumarins in cosmetics.

Phenolic compounds from Pueraria lobata protect PC12 cells against Aß-induced toxicity.

Bioassay-guided fractionation of the EtOAc-soluble extract of Pueraria lobata based on the inhibition of Aß-induced toxicity in PC12 cells resulted in the isolation of four known active compounds, genistein (8), biochanin A (9), sissotrin (10), and puerol B (11). Of these, genistein (8) and biochanin A (9) exhibited potent neuroprotective effects with ED(50) values of 33.7 and 27.8 µM, respectively. In addition, a new coumestan, 2-(α,α-dimethylallyl)coumestrol (1) was isolated and characterized, but proved to be inactive, as were additional seven known compounds. The structure of new compound 1 was determined using spectroscopic techniques.

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.

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.

Physicochemical characterization of a new crystal form and improvements in the pharmaceutical properties of the poorly water-soluble antiosteoporosis drug 3,9-bis(N,N-dimethylcarbamoy-loxy)-5H-benzofuro[3,2-c]quinoline-6-one (KCA-098) by solid dispersion with hydroxypropylcellulose.

The present study was undertaken to improve the oral absorption of KCA-098, an antiosteoporosis drug. In this study, the form 2 of KCA-098 was used as a desirable crystal form for pharmaceutical formation among three kinds of crystal forms, 1, 2, and 3. Solid dispersions of KCA-098 with hydroxypropylcellulose (HPC) or poly(vinylpyrrolidone) (PVP) were prepared by the solvent method. The physicopharmaceutical properties of the solid dispersions were characterized by powder x-ray diffraction, FTIR spectroscopy, and differential scanning calorimetry (DSC). The powder x-ray diffractograms suggest that KCA-098 in the HPC-SL solid dispersion existed in a partial crystalline state as a new crystal form that could be produced by recrystallization from the solvent. Dissolution from the solid dispersions was markedly enhanced in comparison with that of the drug alone. The dissolution enhancement was observed to be greater for the solid dispersion with HPC-SL than for that with PVP. The KCA-098/HPC-SL (1:2) solid dispersion capsule showed a 3.5-fold increase in the initial concentration and 2.5-fold increase in initial concentration of dissolved drug after 60 min, compared with the values for a physical mixture of KCA-098 (form 2)/lactose (1:2). The in vivo absorption of the drug was investigated after oral administration of KCA-098 or its solid dispersion. The area under the plasma concentration curve of KCA-098 after oral administration of the KCA-098/HPC-SL (1:2) solid dispersion capsule was three-fold greater than that for the drug itself.

Inclusion complex of 3,9-bis(N,N-dimethylcarbamoyloxy)-5H-benzofuro[3,2-c]quinoline-6-one (KCA-098) with heptakis(2,6-di-O-methyl)-beta-cyclodextrin: interaction and dissolution properties.

Interactions of KCA-098 with heptakis(2,6-di-O-methyl)-beta-cyclodextrin (DM-beta-CyD) in solution and in the solid state were studied by the solubility method, UV and fluorescence spectroscopy, powder X-ray diffractometry, and thermal analysis. The KCA-098/DM-beta-CyD system showed an A(L) type solubility diagram with stability constants of 5870 and 2220 M(-1) in aqueous and 10% methanol solutions, respectively. Following the addition of DM-beta-CyD, the maximum UV wavelength of KCA-098 was shifted to a longer wavelength and the fluorescence intensity was decreased. A similar spectral change was observed when KCA-098 was dissolved in less polar solvents, especially in proton-acceptor solvents, such as acetone and dimethylsulfoxide, suggesting that KCA-098 interacts with DM-beta-CyD through not only a hydrophobic interaction but also hydrogen bonding. The solid complex of KCA-098 with DM-beta-CyD in a molar ratio of 1:1 was prepared by the kneading method and the solvent evaporation method, using organic solvents. Powder X-ray diffractometric and differential scanning calorimetric studies indicated that KCA-098 was dispersed as microparticles on the DM-beta-CyD complex in the solid state prepared by the solvent evaporation method although it dispersed as crystals in the sample prepared by the kneading method. The dissolution of KCA-098 from the solid complex prepared by the former method was markedly faster than that prepared by the latter method, although it slowed down with the passage of time. The reduced dissolution of KCA-098 was explained by crystallization to the hydrate form in the medium. These data indicate that poorly water-soluble KCA-098 interacts with DM-beta-CyD in water and in the solid state and that a fast-dissolving form of KCA-098 can be obtained by evaporating with DM-beta-CyD using organic solvents.

Effect of grinding with hydroxypropyl cellulose on the dissolution and particle size of a poorly water-soluble drug.

A new benzofuroquinoline derivative, 3,9-bis(N,N-dimethylcarbamoyloxy)-5H-benzofuro[3,2-c]quinoli ne-6-one (KCA-098), shows poor oral absorption due to practical insolubility in water. In this study, a co-grinding technique employing a water-soluble polymer was used for improvement of the dissolution rate of KCA-098. Powder X-ray diffraction patterns and IR spectra of KCA-098 showed the conversion of the drug from a crystal state to an amorphous state by grinding with a polymer such as hydroxypropyl cellulose (HPC-SL) or polyvinylpyrrolidone (PVP K30). The particle size of KCA-098 was remarkably reduced to a submicron size by grinding with HPC-SL. The co-ground mixture with HPC-SL showed a rapid dissolution rate and maintained supersaturation for more than 1 h. On the other hand, the co-ground mixture with PVP K30 showed rapid dissolution and supersaturation for a shorter period. These data suggest that the rapid dissolution rate was obtained by the conversion of the drug particles from a crystal to amorphous state by grinding with water-soluble polymers and that a reduction in particle size to the submicron level led to the maintenance of supersaturation due to good dispersion.

Characterization of urinary metabolites of a new benzofuroquinoline derivative, 3,9-bis(N,N-dimethylcarbamoyloxy)-5 H-benzofuro[3,2-c]-quinoline-6-one (KCA-098), in dogs.

The metabolism of 3,9-bis(N,N-dimethylcarbamoyloxy)-5 H-benzofuro[3,2-c]-quinoline-6-one (KCA-098), a new inhibitor of bone resorption and stimulator of bone formation, was examined after oral administration to dogs. Nine metabolites and the unchanged KCA-098 were isolated by extraction and HPLC from dog urine. The structures of these metabolites were characterized by LC/MS or LC/MS/MS, and/or were confirmed by comparison with corresponding authentic standards. The presumed main metabolic pathways were hydrolysis, hydroxylation, and N-demethylation of the N,N-dimethyl-carbamate ester group.

Effect of KCA-098 on the function of osteoblast-like cells and the formation of TRAP-positive multinucleated cells in a mouse bone marrow cell population.

KCA-098 (3,9-bis(N,N-dimethylcarbamoyloxy)-5H-benzofuro[3,2-c]quinol ine-6-one), an analogue of coumestrol (a naturally occurring weak phytoestrogen), dose-dependently increased alkaline phosphatase activity of osteoblastic ROS 17/2.8 cells and freshly-isolated osteoblasts from neonatal mouse calvaria, and reduced cell proliferation. In addition, KCA-098 increased the synthesis of collagenese-digestible protein (CDP) of ROS 17/2.8 cells. On the other hand, KCA-098 had no effect on the basal synthesis of osteocalcin but reduced the 1 alpha,25-dihydroxyvitamin D3 (1 alpha,25(OH)(2)D3)-induced increase in osteocalcin synthesized by ROS 17/2.8 cells. Therefore, KCA-098 had a bidirectional effect on the differentiation of osteoblasts (i.e., stimulating both alkaline phosphatase activity and synthesis of CDP and inhibiting osteocalcin synthesis). However, as KCA-098 stimulated the mineralization of chick embryonic bone in organ culture and recovered the bone density reduced by ovariectomy of rats, it would serve overall to stimulate the differentiation of osteoblasts. On the other hand, KCA-098 inhibited the formation of tartrate-resistant, acid phosphate-positive multinucleated cells (TRAP(+)MNC) induced by 1 alpha,25(OH)(2)D(3), parathyroid hormone (PTH), and prostaglandin E2 (PGE2) in cultures of mouse bone marrow cells, showing that it inhibits the formation of osteoclast-like cells. Coumestrol and 17beta-estradiol had no effect on the proliferation and alkaline phosphatase activity of ROS 17/2.8 cells. They did, however, dose-dependently inhibit osteoclast-like cell formation as well as KCA-098 did, indicating that the main action of coumestrol and 17beta-estradiol on bone tissue is the inhibition of bone resorption.

[Polymorphism of 3,9-bis-(N,N-dimethylcarbamoyloxy)-5H-benzofuro[3,2- c]quinoline-6-one(KCA-098), a new benzofuroquinoline derivative].

Physicochemical properties of polymorphism of 3,9-bis-(N,N-dimethylcarbamoyloxy)-5H-benzofuro[3,2-c]quinol ine-6-one (KCA-098) have been investigated. The existence of four crystalline forms (designated as hydrate, I, II and III) was confirmed by X-ray powder diffraction, IR spectroscopy and thermal analysis (DSC and TG). The hydrate was found to be a monohydrate by elemental analysis and water content measurement. DSC measurement found that the hydrate was transformed fo form III at about 93 degrees C, and then to form II at about 152 degrees C, and finally to form I at about 260 degrees C. On the other hand, when suspended in water the forms I, II and III were transformed into hydrate. The transition rate from form III to hydrate was higher than those from form II to hydrate and from form I to hydrate. Form III as a metastable form showed higher solubility than any of form I, II and hydrate.

Effects of KCA-098 on bone metabolism: comparison with those of ipriflavone.

We previously found that 3,9-bis(N,N-dimethylcarbamoyloxy)-5H- benzofuro[3,2-c]quinoline-6-one (KCA-098) inhibited bone resorption in organ culture. In this study, to determine if KCA-098 is therapeutically applicable for the treatment of osteoporosis, we compared the effect of KCA-098 on bone tissues with that of ipriflavone, a drug that is clinically used for the treatment of osteoporosis. Both KCA-098 and ipriflavone inhibited parathyroid hormone-, prostaglandin E2-, 1 alpha,25-dihydroxyvitamin D3- and interleukin 1 beta-induced bone resorption of fetal rat bones, but the inhibitory activity of KCA-098 was more potent than that of ipriflavone. In fact, the effective concentrations of KCA-098 were 10 to 100 times lower than those of ipriflavone. Oral administration of KCA-098 (1 and 3 mg/kg) or ipriflavone (100 mg/kg) to ovariectomized rats on a low-calcium diet increased the breaking force and bone density of the femora, indicating that KCA-098 is an effective on the whole animal as ipriflavone. Furthermore, KCA-098 increased the length and calcium content of 9-day chick embryonic femora cultured in vitro, whereas ipriflavone did not, suggesting that KCA-098 had a direct stimulatory effect on bone mineralization. Therefore, KCA-098 seems to be more potent than ipriflavone in stimulating bone tissue formation and may thus be expected to become a useful agent for the treatment of osteoporosis.

Induction of osteopenia in confined rats.

We have developed a simple model of osteopenia in rats which is induced by confinement without requiring surgical operation. Each rat was maintained for 8 weeks in a compartment of a commercially-available wire netting cage subdivided into 10 areas (compartment size, 9 x 16 x 14 cm) to restrict exercise. The femora isolated from the confined rats showed significant decreases in mineral (calcium and phosphorus) content, compared with the level in normal rats, 2 weeks after the start of their confinement. Confined rats showed significantly lower values for the physical properties of bones such as breaking energy and breaking force and also density composed with normal rats 4 weeks after the start of confinement. KCA-098 (1 mg/kg), a new benzofuroquinoline derivative that inhibits bone resorption and at the same time stimulates bone mineralization in organ culture, protected against these decreases when given orally for 8 weeks. All these results show that confinement of rats offers a simple and useful animal model of osteopenia.

Effect of KCA-098, a new benzofuroquinoline derivative, on bone mineral metabolism.

The effect of 3,9-bis(N,N-dimethylcarbamoyloxy)-5H-benzofuro[3,2-c]quinoli ne-6-one designated as KCA-098) on the bone mineral metabolism of chick embryonic bone was examined. KCA-098 dose-dependently inhibited bone resorption of cultured chick embryonic femora and calvariae. It increased the length, dry weight, and calcium and phosphorus contents of 9-d-old chick embryonic femurs cultivated for 6 d, indicating that it stimulated bone formation. These results show that KCA-098 has the unique effects of inhibiting bone resorption and stimulating bone formation of chick embryo. In addition, in an in vivo experiment, oral administration of KCA-098 (3.0 mg/kg/d) for 16 weeks led to an increase in calcium and phosphorus content as well as an increase in the amount of force required to break the femur from ovariectomized rats, suggesting that it may be useful for the treatment of bone diseases.

In vitro effect of KCA-098, a derivative of coumestrol, on bone resorption of fetal rat femurs.

The effects of 3,9-bis(N,N-dimethylcarbamoyloxy)-5H-benzofuro[3,2-c]quinoli ne-6-one (KCA-098), a derivative of coumestrol, on bone resorption was studied in organ cultures of 20-day fetal rat femora. KCA-098 increased the length, dry weight, and calcium and phosphorus contents of parathyroid hormone (PTH)-treated fetal rat femur. As PTH significantly reduced the calcium and phosphorus contents of the femora, probably by stimulating bone resorption, KCA-098 seems to inhibit bone resorption. In fact, KCA-098 inhibited the PTH-induced release of 45Ca from pre-labeled fetal rat femora into the medium in organ culture. Coumestrol also inhibited the release of 45Ca from bone into the medium. However, KCA-098 did not increase the uterine weight of ovariectomized rats, whereas coumestrol did so. Thus KCA-098 is a unique, new inhibitor of bone resorption that has no estrogenic activity.