Pharmacophore based 3D-QSAR study of biphenyl derivatives as nonsteroidal aromatase inhibitors in JEG-3 cell lines.

Breast cancer is one of the most high-profile malignant diseases in modern society. Among postmenopausal women affected by the disease, a substantial portion has breast tumors that are estrogen-receptor positive. A common therapeutic intervention for this type of cancer is through endocrine therapy. Endocrine agents can act by either diminishing the availability or inhibiting the binding of estrogens to ER. Aromatase catalyzes the conversion of androgens to estrogens in the final step of the biosynthesis of estrogens and is therefore an attractive therapeutic target for inhibition. 3DQSAR pharmacophore modeling studies were undertaken for biphenyl derivatives as aromatase inhibitors in JEG-3 cell lines. A four-point pharmacophore with two H-bond acceptors and two aromatic rings as pharmacophoric features was developed. The pharmacophore hypothesis yielded a statistically significant 3D-QSAR model, with a correlation coefficient of R² = 0.977 for training set molecules. The generated model showed excellent predictive power, with a correlation coefficient of Q² = 0.946 for an external test set. The 3D-QSAR plots illustrated insights into the structure activity relationship of these compounds which may help in the design and development of potent biphenyl derivatives as new aromatase inhibitors.

Hydroxybenzothiazoles as new nonsteroidal inhibitors of 17ß-hydroxysteroid dehydrogenase type 1 (17ß-HSD1).

17ß-estradiol (E2), the most potent estrogen in humans, known to be involved in the development and progession of estrogen-dependent diseases (EDD) like breast cancer and endometriosis. 17ß-HSD1, which catalyses the reduction of the weak estrogen estrone (E1) to E2, is often overexpressed in breast cancer and endometriotic tissues. An inhibition of 17ß-HSD1 could selectively reduce the local E2-level thus allowing for a novel, targeted approach in the treatment of EDD. Continuing our search for new nonsteroidal 17ß-HSD1 inhibitors, a novel pharmacophore model was derived from crystallographic data and used for the virtual screening of a small library of compounds. Subsequent experimental verification of the virtual hits led to the identification of the moderately active compound 5. Rigidification and further structure modifications resulted in the discovery of a novel class of 17ß-HSD1 inhibitors bearing a benzothiazole-scaffold linked to a phenyl ring via keto- or amide-bridge. Their putative binding modes were investigated by correlating their biological data with features of the pharmacophore model. The most active keto-derivative 6 shows IC50-values in the nanomolar range for the transformation of E1 to E2 by 17ß-HSD1, reasonable selectivity against 17ß-HSD2 but pronounced affinity to the estrogen receptors (ERs). On the other hand, the best amide-derivative 21 shows only medium 17ß-HSD1 inhibitory activity at the target enzyme as well as fair selectivity against 17ß-HSD2 and ERs. The compounds 6 and 21 can be regarded as first benzothiazole-type 17ß-HSD1 inhibitors for the development of potential therapeutics.

Formation of bisphenol A by thermal degradation of poly(bisphenol A carbonate).

The thermal decomposition of poly(bisphenol A carbonate) (PoC) results in the formation of the endocrine disruptor bisphenol A (BPA). In the present work, we investigated the kinetics of the thermal decomposition of PoC, and the subsequent decomposition of BPA, under pyrolysis conditions and in the presence of oxygen by using infrared image furnace-ion attachment mass spectrometry. The decomposition of PoC obeyed Arrhenius kinetics, which allowed us to determine the activation energy (E(a)) for thermal decomposition to BPA from Arrhenius plots. From the selected ion monitoring curves for BPA, E(a) for thermal decomposition in a nitrogen atmosphere was calculated to be 133.2 kcal mol(-1), whereas E(a) for oxidative thermal decomposition was calculated to be approximately 35% lower (86.5 kcal mol(-1)).

Structure-function relationships of estrogenic triphenylethylenes related to endoxifen and 4-hydroxytamoxifen.

Estrogens can potentially be classified into planar (class I) or nonplanar (class II) categories, which might have biological consequences. 1,1,2-Triphenylethylene (TPE) derivatives were synthesized and evaluated against 17beta-estradiol (E2) for their estrogenic activity in MCF-7 human breast cancer cells. All TPEs were estrogenic and, unlike 4-hydroxytamoxifen (4OHTAM) and Endoxifen, induced cell growth to a level comparable to that of E2. All the TPEs increased ERE activity in MCF-7:WS8 cells with the order of potency as followed: E2 > 1,1-bis(4,4'-hydroxyphenyl)-2-phenylbut-1-ene (15) > 1,1,2-tris(4-hydroxyphenyl)but-1-ene (3) > Z 4-(1-(4-hydroxyphenyl)-1-phenylbut-1-en-2-yl)phenol (7) > E 4-(1-(4-hydroxyphenyl)-1-phenylbut-1-en-2-yl)phenol (6) > Z(4-(1-(4-ethoxyphenyl)-1-(4-hydroxyphenyl)but-1-en-2-yl)phenol (12) > 4-OHTAM. Transient transfection of the ER-negative breast cancer cell line T47D:C4:2 with wild-type ER or D351G ER mutant revealed that all of the TPEs increased ERE activity in the cells expressing the wild-type ER but not the mutant, thus confirming the importance of Asp351 for ER activation by the TPEs. The findings confirm E2 as a class I estrogen and the TPEs as class II estrogens. Using available conformations of the ER liganded with 4OHTAM or diethylstilbestrol, the TPEs optimally occupy the 4OHTAM ER conformation that expresses Asp351.

Elemental isomerism: a boron-nitrogen surrogate for a carbon-carbon double bond increases the chemical diversity of estrogen receptor ligands.

To increase the chemical diversity of bioactive molecules by incorporating unusual elements, we have examined the replacement of a C=C double bond with the isoelectronic, isostructural B-N bond in the context of nonsteroidal estrogen receptor (ER) ligands. While the B-N bond was hydrolytically labile in the unhindered cyclofenil system, the more hindered anilino dimesitylboranes, analogs of triarylethylene estrogens, were easily prepared, hydrolytically stable, and demonstrated substantial affinity for ERs. X-ray analysis of one ERalpha-ligand complex revealed steric clashes with the para methyl groups distorting the receptor; removal of these groups resulted in an increase in affinity, potency, and transcriptional efficacy. These studies define the structural determinants of stability and cellular bioactivity of a B-N for C=C substitution in nonsteroidal estrogens and provide a framework for further exploration of "elemental isomerism" for diversification of drug-like molecules.

Structure-based drug design: synthesis, crystal structure, biological evaluation and docking studies of mono- and bis-benzo[b]oxepines as non-steroidal estrogens.

Mono- and bis-benzo[b]oxepine derivatives have been rationally synthesized to meet the molecular requirement for interaction with estrogen receptor. Bis-benzo[b]oxepines (7 and 9) and mono-benzo[b]oxepine (10) acquire geometry with phenolic groups disposed in a fashion to stimulate estrogen receptor. Structure-based investigation, in vivo activity and docking studies have been described and correlated to demonstrate a practical approach for suitable ligand design.

Convenient synthesis of 5,6,11,12,17,18- hexahydrocyclononal[1,2-b:4,5-b':7,8-b'']triindole, a novel phytoestrogen.

An efficient one-pot synthesis is described of 5,6,11,12,17,18-hexahydrocyclononal[1,2-b:4,5-b':7,8-b'']triindole (CTr), a potent estrogen agonist from food plants. For the procedure, gramine is treated with dimethyl sulfate and sodium in ethanol at room temperature. Quenching of the reaction with water and workup of the product provides CTr in approximately 75% yield.

Solid phase synthesis of complex natural products and libraries thereof.

Natural products have served as an important source of medicinal compounds and pharmaceutical leads over the last century. Within the last 10 years, significant interest has developed in applying combinatorial chemistry techniques to the study of natural products and their biological activities. In this review, we examine several representative efforts wherein natural product skeletons have been constructed or immobilized on solid support and subsequently derivatized, giving rise to analog libraries useful in understanding the structure-activity relationships of the parent natural product. Issues such as target selection, library design, linker development, automation, and library characterization are addressed.

Total syntheses of (S)-(-)-zearalenone and lasiodiplodin reveal superior metathesis activity of ruthenium carbene complexes with imidazol-2-ylidene ligands.

Total syntheses of the bioactive orsellinic acid derivatives zearalenone 3 and lasiodiplodin 1 are reported based on a ring-closing metathesis (RCM) reaction of styrene precursors as the key steps. These and closely related macrocyclizations are catalyzed with high efficiency by the "second generation" ruthenium carbene catalyst 5 bearing a N-heterocyclic carbene ligand, whereas the standard Grubbs carbene 4 fails to afford any cyclized product. Only the (E)-isomer of the macrocyclic cycloalkene is formed in all cases. The substrates for RCM can be obtained either via a Stille cross-coupling reaction of tributylvinylstannane or, even more efficiently, by Heck reactions of the aryl triflate precursors with pressurized ethene. Furthermore, the synthesis of 1 via RCM is compared with an alternative approach employing a low-valent titanium-induced McMurry coupling of dialdehyde 47 for the formation of the large ring. This direct comparison clearly ends in favor of metathesis which turned out to be superior in all preparatively relevant respects.

Effects of the ethyl and benzyl ethers of indenestrols A and B on cytotoxicity in Chinese hamster V79 cells.

We recently investigated the relationship between the structures of various indenestrols and their cytotoxicity, and reported that indenestrol A (IA), a metabolite of the synthetic nonsteroidal estrogen diethylstilbestrol, and indenestrol B (IB), an analog of IA, disrupt the microtubule architecture of Chinese hamster V79 cells in vitro. We then synthesized 16 optically active indenestrol derivatives by substituting monoethyl, monobenzyl and diethyl ether groups at the 6- and/or 4'-hydroxyl positions, and examined their cytotoxic activities in Chinese hamster V79 cells. The results indicated that the monoethyl ethers had cytotoxic activities similar to monomethyl ethers. However, the (+)- and (-)-monobenzyl ethers were less cytotoxic than the corresponding monomethyl and monoethyl derivatives.

Acid and base-catalyzed hydrolysis of bisphenol A-related compounds.

In order to ascertain whether an estrogenic bisphenol A is produced from bisphenol A-related monomers by chemical-induced hydrolysis and to clarify their hydrolytic mechanisms, bisphenol A dimethacrylate (Bis-DMA) and bisphenol A bis(glycidyl methacrylate) (Bis-GMA) were reacted with phosphoric acid, hydrochloric acid, and sodium hydroxide in methanol or methanol/water mixed media at 37 degrees C. Amounts of monomethacrylate intermediates as well as bisphenol A (BPA) were determined by the use of high-performance liquid chromatography (HPLC), and time-conversion curves of hydrolytic products were prepared. BPA and bisphenol A monomethacrylate were produced by acid-catalyzed hydrolysis of Bis-DMA. Bis-GMA was partly converted into monomethacrylate by phosphoric acid and into monomethacrylate and 2,2-bis[4-(2,3-dihydroxypropoxy) phenyl]propane (BHP) by hydrochloric acid. Hydrolytic reactions by sodium hydroxide were completed almost within 1 day, resulting in the production of BPA from Bis-DMA, and BHP from Bis-GMA. No BPA was formed from Bis-GMA by chemical-induced hydrolysis. The hydrolytic behaviors of these monomers were discussed.

[Hormone dysregulators. Pseudo-estrogens in dental composite resins and sealanta?]. / Hormoonontregelaars. Pseudo-oestrogenen in tandheelkundige composieten en sealants?

A number of polluting chemicals in the ecosystems must be characterized as hormone disruptors. Among others, male animals appear to become feminized by the action of the so-called pseudo-estrogens and under their influence mens' fertility is said to decrease. Composites and sealants based on Bis-GMA resin may contain bisphenol-A as an impurity and Bis-DMA, from which in saliva bisphenol-A will be formed by hydrolytic degradation. Therefore, in extreme circumstances a weak estrogenic effect is not impossible on the short-term. However, the amounts of these probably not very potent estrogenic compounds are small, thereby resulting in a tolerable risk on the short term. Long-term-effects and synergism with pseudo-estrogens from other sources prompt further studies in order to verify the safety of the Bis-GMA containing products.

A urinary profile study of dietary phytoestrogens. The identification and mode of metabolism of new isoflavonoids.

The metabolic fate of the dietary isoflavones daidzein and genistein was investigated in human volunteers challenged with soya. Urinary diphenols, isolated by partition chromatography on Sephadex LH-20, were characterized and identified by profile capillary gas chromatography (GC) and electron ionization mass spectrometry (GC-EIMS) analysis of the trimethylsilyl ether (TMS) derivatives. Novel isoflavonic phytoestrogens found in the urine of volunteers were those of tetrahydrodaidzein, dihydrogenistein, 6'-hydroxy-O-demethylangolesin and 2-dehydro-O-demethylangolensin. Other known diphenols identified were those of equal, dehydrodaidzein, O-demethylangolensin, daidzein, genistein, glycitein, and the lignan enterolactone. Two other urinary isomers with a fragmentation pattern closely resembling that of the persilylated TMS ethers of cis/trans-isomers of tetrahydrodaidzein, were characterized based on the elucidation of fragments associated with the loss of a non-phenolic-OTMS functional group in ring-C. These are fragments presented in the persilylated mass spectra of isoflavan-4-ols and isoflav-3-ene-4-ols, demonstrated here by a combination of simple and tandem mass spectrometry study of the deuterated persilylated TMS ethers of dihydrodaidzein. In a similar study we also present the data on the structural identification and fragment elucidation of the keto/enol tautomers of the TMS ether derivatives of the dihydro derivatives of daidzein and genistein, observed in the urine of volunteers and considered probable products of the derivatization process. Finally, the GC and GC-MS data of two unknown isoflavonoids and that of a lignan-like compound are presented together with those of dihydrodaidzein, dihydrogenistein, tetrahydrodaidzein and 2-dehydro-O-demethylangolensin. The latter four were obtained here as products of small scale chemical synthesis in a preliminary study on the tentative identification of urinary isoflavonoids in human volunteers challenged with soya.

Synthesis and labeling of isoflavone phytoestrogens, including daidzein and genistein.

The synthesis of the important diphenolic isoflavone type phytoestrogens starting from the corresponding unprotected phenols and arylacetic acids is discussed. The aryl rings may carry additional alkyl, methoxy, and/or halogeno groups. Intermediate polyhydroxy deoxybenzoins can also be isolated in good yield. Isotopically labeled isoflavone phytoestrogens were prepared for use as internal standards in ion exchange chromatography and GC-MS selected ion monitoring (SIM technique). Traditional methods rely on total synthesis using deuterated starting materials for the preparation of labeled isoflavonoid structures. We have used successfully an application where the H/D exchange is performed within the finished molecular framework, based on the exchange of aromatic protons that are ortho or para to a phenolic OH group. By this method the deuterated products are available in an isotopic purity of 90% or higher.

Influence of alkyl-chain fluorination on the action of mammary tumor inhibiting 2,3-bis(hydroxyphenyl)butanes and 2,3-bis(hydroxyphenyl)but-2-enes.

trans-1,2-Bis(trifluoromethyl)-1,2-bis(4- and 3-hydroxyphenyl)ethenes 2 and 4 were prepared by reductive coupling (TiCl4/Zn/pyridine) of the methoxy-substituted alpha, alpha, alpha-trifluoroacetophenones, separation of the resulting cis- and trans-stilbene derivatives, and ether cleavage with BBr3. The cis-stilbenes were catalytically hydrogenated to give meso-1,1,1,4,4,4-hexafluoro-2,3-bis(4- and 3-hydroxyphenyl)butanes 6 and 8. Compounds 2, 4, 6, and 8 showed 2- to 10-fold increased binding affinities for the estradiol receptor (E2R) and enhanced estrogenicity in the uterine weight test of the immature mouse compared to their unfluorinated analogues. Compound 8 exhibited a 46% inhibition of the estrone-stimulated uterine growth. Antitumor activity was evaluated with use of the transplantable, hormone-dependent MXT mammary tumor of the BD2F1 mouse. All compounds showed tumor growth inhibitory activity corresponding to their RBA values. The most interesting compound 8 led to a significant inhibition of the tumor growth on the DMBA-induced hormone-dependent mammary carcinoma of the Sprague-Dawley rat.

Benzo[a]carbazole derivatives. Synthesis, estrogen receptor binding affinities, and mammary tumor inhibiting activity.

A number of 11-alkylbenzo[a]carbazoles and their 5,6-dihydro derivatives with one or two hydroxy groups in the aromatic rings were synthesized and studied for their binding affinities for the estrogen receptor. Best conditions for the receptor binding are provided by one hydroxy group at C-3 and a second one at position 8 or 9. The binding affinities of the benzo[a]carbazoles are somewhat lower than those of the dihydro derivatives but still high regarding the planar structure of these molecules. The highest relative binding affinity (RBA) values (e.g., 30 for 13b, 13 for 16b, 20 for 25a; estradiol = 100) are close to those of the corresponding 2-phenylindole derivatives. Depending on the positions of the oxygen functions, the benzo[a]carbazoles behaved as strong estrogens (13c, 25a) or impeded estrogens (16c, 28a) in the immature mouse. Derivative 16c inhibited the growth of dimethylbenzanthracene-induced hormone-dependent mammary tumors of the rat at a dose of 6 X 1 mg/kg per week. In vitro, 16b and 28b showed inhibitory activity on estrogen receptor positive MCF-7 breast cancer cells. A mode of action involving the estrogen receptor system is assumed.

The synthesis of non-steroidal estrogen receptor binding compounds labeled with 80mBr.

Estrogen receptor (ER) binding radiopharmaceuticals have potential for use in the diagnosis and treatment of cancers of the female reproductive system. Two triphenylethylene derivatives based on the structure of hydroxytamoxifen 4, a high ER binding metabolite of tamoxifen 5, have been prepared: 1-(4-dimethylaminoethoxy)phenyl]-1-(4-hydroxy)phenyl-2-bromo-2-phenyl ethylene 2 and 1,1-bis (p-hydroxyphenyl)-2-bromo-2-phenylethylene 3. Both 2 and 3 bind strongly to the ER. Compound 3 has been labeled in modest yield by direct bromination with 80mBr, which was produced by the 83Kr (d,n alpha) reaction. Radiolabeled 22, a dimethoxy precursor of 3, has been prepared in yields ranging between 40 and 60% by a bromination destannylation reaction.

Estrogen receptor based imaging agents. 1. Synthesis and receptor binding affinity of some aromatic and D-ring halogenated estrogens.

Steroidal and nonsteroidal estrogens substituted with halogens ortho to the phenolic hydroxyl group in the D ring at C-16 have been prepared as potential estrogen receptor-based imaging agents for human breast tumors. Estrogens bearing an aromatic fluorine ortho to a phenolic hydroxyl group were prepared by the Schiemann reaction on the corresponding methyl esters; other ortho-halogenated estrogens were prepared by direct halogenation. Steroidal estrogens substituted at the 16 alpha position were prepared by halogenation of estrone 3-acetate (17-enol acetate) followed by hydride reduction, and those substituted at the 16 beta position were prepared by epimerization prior to reduction. The binding affinity of these halogenated estrogens to the uterine estrogen receptor was measured relative to that of [3H]estradiol by a competitive binding assay. All of the monosubstituted ortho-fluorinated estrogens show very high binding affinity for the receptor (64--250% that of estradiol). The monosubstituted and symmetrically disubstituted bromo- and iodohexestrols and 2- and 4-substituted estradiols have binding affinities considerably lower than those of the fluoro compounds, the 4-substituted estradiols have affinities greater than the corresponding 2-substituted isomers. Introduction of a halogen (Cl, Br, I) at the 16 alpha position of 17 beta-estradiol results in compounds with receptor affinities comparable to that of 17 beta-estradiol itself; the 16 beta-epimers and the estrone derivatives are bound less well. Thus, provided that they can be labeled with suitable gamma-emitting radioisotopes at sufficiently high specific activity, it appears that the A-ring fluoroestrogens and 16 alpha-bromo- and 16 alpha-iodoestradiol-17 beta are excellent candidates for receptor-based imaging of human breast tumors.

alpha-(2-Pyridine)benzyl aryl ketones as potential hypocholesteremic agents.

A series of alpha-(2-pyridine)benzyl aryl ketones were prepared as potential hypocholesteremic agents. The synthesis of these compounds was by conversion of 2-benzylpyridine to its anion with n-butyllithium and condensation of the anion with selected aromatic esters. The ketones were tested for their hypocholesteremic activity in rats, and those compounds showing activity were further tested for estrogenicity. Only those aryl ketones with substituents in the ortho position showed a statistically significant reduction in serum cholesterol. Of these compounds the tert-butyl derivative had the most favorable hypocholesteremic to estrogenic ratio.