Reaction of dimethoxycarbene-DMAD zwitterion with 1,2-diones and anhydrides: a novel synthesis of highly substituted dihydrofurans and spirodihydrofurans

The zwitterion formed by the reaction of dimethoxycarbene and DMAD adds efficiently to one of the carbonyl groups of 1,2-dicarbonyl compounds and anhydrides to generate dihydrofurans and spirodihydrofurans in good yields. In many cases, the carbene inserts into the C-C bond of the dione to yield masked vicinal tricarbonyl systems

Structure of limonene synthase, a simple model for terpenoid cyclase catalysis

The crystal structure of (4 S )-limonene synthase from Mentha spic ata , a metal ion-dependent monoterpene cyclase that catalyzes the coupled isomerization and cyclization of geranyl diphosphate, is reported at 2.7-Å; resolution in two forms liganded to the substrate and intermediate analogs, 2-fluorogeranyl diphosphate and 2-fluorolinalyl diphosphate, respectively. The implications of these findings are described for domain interactions in the homodimer and for changes in diphosphate–metal ion coordination and substrate binding conformation in the course of the multistep reaction

Effect of EC13 on mammary gland morphology and uterine weight.

<p>A) Whole mounts of mammary glands of overiectomized C57BL/6 mice treated with E2, EC313 0.1 and 1.0 mg/kg. Magnification- upper panel-10X, lower panel-20X. B) Uterine weight of mice treated for 4 weeks with E2, EC313 0.1 and 1.0 mg/kg. Data represented are average uterine weight in milligrams ±SD (*P<0.001 vs E2-control). C-D) Total duct length and terminal end bud counts in the mammary gland whole mounts after 4 weeks of the treatment with test compounds (*P<0.001 vs E2-control).</p

Effects of Combination of Estradiol with Selective Progesterone Receptor Modulators (SPRMs) on Human Breast Cancer Cells <i>In Vitro</i> and <i>In Vivo</i>

<div><p>Use of estrogen or estrogen / progestin combination was an approved regimen for menopausal hormonal therapy (MHT). However, more recent patient-centered studies revealed an increase in the incidence of breast cancer in women receiving menopausal hormone therapy with estrogen plus progestin rather than estrogen alone. Tissue selective estrogen complex (TSEC) has been proposed to eliminate the progesterone component of MHT with supporting evidences. Based on our previous studies it is evident that SPRMs have a safer profile on endometrium in preventing unopposed estrogenicity. We hypothesized that a combination of estradiol (E2) with selective progesterone receptor modulator (SPRM) to exert a safer profile on endometrium will also reduce mammary gland proliferation and could be used to prevent breast cancer when used in MHT. In order to test our hypothesis, we compared the estradiol alone or in combination with our novel SPRMs, EC312 and EC313. The compounds were effectively controlled E2 mediated cell proliferation and induced apoptosis in T47D breast cancer cells. The observed effects were found comparable that of BZD <i>in vitro</i>. The effects of SPRMs were confirmed by receptor binding studies as well as gene and protein expression studies. Proliferation markers were found downregulated with EC312/313 treatment <i>in vitro</i> and reduced E2 induced mammary gland proliferation, evidenced as reduced ductal branching and terminal end bud growth <i>in vivo</i>. These data supporting our hypothesis that E2+EC312/EC313 blocked the estrogen action may provide basic rationale to further test the clinical efficacy of SPRMs to prevent breast cancer incidence in postmenopausal women undergoing MHT.</p></div

EC312 and EC313 inhibiting DNA synthesis.

<p>A-B) Effect of EC312 and EC313 on cell proliferation as assessed by BrdU incorporation. T47D cells were plated in 96-well plates at a density of 10,000 cells per well. Two days later the culture medium (5% FCS-RPMI) was replaced with phenol red-free RPMI with 5% DCC-FCS. After 24 hours of starvation, the cells were treated with increasing doses of the compounds alone or in combination with E2 (0.1nM). The results are expressed as average OD value of quadruplicate wells (*P<0.01 vs control). C-D) Effect of EC312 and EC313 in inducing apoptosis in the presence or absence of endogenous estrogen in T47D cells. T47D cells were plated in 12 well plated at a density of 80,000 cells/well. Two days later culture media were replaced with phenol red-free RPMI containing 5% FBS or 5% DCC-FBS. After 24 hours of starvation, cells were treated with test compounds at increasing concentrations for 48 hours. Apoptosis was assessed by Caspase-Glo assay kit (Promega). The results were expressed as OD±SEM of 2 wells per treatment (*P<0.01 vs control).</p

Glucocorticoid receptor (GR) antagonistic activity, Progesterone receptor (PR)—agonistic and antagonistic activity is evaluated and IC50 values as well as agonistic/antagonistic ratios were noted.

<p>ND- Not determined.</p

Effect of EC312 and EC313 on apoptosis, gene signatures of proliferation and protein expression levels.

<p>A) Effect of EC312 and EC313 (10nM) on apoptosis in the absence of endogenous estrogen and increasing concentrations of exogenous E2. We have selected the above concentration based on the fact that EC312 and EC313 abrogated E2 induced cell proliferation as comparable to that of BZD at 10nM). T47D cells were plated in 12 well plated at a density of 80,000 cells/well. Two days later culture media were replaced with phenol red-free RPMI containing 5% FBS or 5% DCC-FBS. After 24 hours of starvation, cells were treated with test compounds at increasing concentrations for 48 hours. Apoptosis was assessed by Caspase-Glo assay kit (Promega). The results were expressed as OD±SEM of 2 wells per treatment (*P<0.01 vs control). B) Effect of EC313 on gene signatures of proliferation. Gene expression of T47D cells in response to EC313 for anti-apoptotic genes. T47D cells were grown in phenol red-free RPMI medium containing 5% DCC-FBS for 24 hours and then treated with indicated doses of test compounds alone or in combination with E2 (0.1nM) (*P<0.01 vs E2-control). C) Effect of EC313 on gene signatures of proliferation. Gene expression of T47D cells in response to EC313 for pro-apoptotic genes. T47D cells were grown in phenol red-free RPMI medium containing 5% DCC-FBS for 24 hours and then treated with indicated doses of test compounds alone or in combination with E2 (0.1nM) (*P<0.01 vs E2-control). D) Effect of EC312 and BZD on the expression of ER, Cyclin D1 and phosphorylation of MAPK and AKT. T47D cells grown in 60-mm culture dishes in phenol red-free RPMI medium containing 5%DCC-FBS and treated with E2 (1nM) alone or in combination with EC312 or BZD (100nM) for 24 hours before preparation of cell lysate and analysis by western blot and quantification normalized by beta-actin.</p

Antiestrogenic effect of EC312/313 on cell viability and proliferation in vitro.

<p>A-B) Antiestrogenic effect of EC312 and EC313 on cell growth in the presence and absence of endogenous estrogens. T47D cells were plated at 30,000 cells per well in 5% FBS-RPMI and grown for 2 days before treatment. The cells were then incubated with EC312 and EC313 at indicated concentrations for 5 days and counted for cell number. *P<0.01 vs control. <b>C)</b> The compounds were treated as per the conditions above with and without E2 (0.1nM). Tested compounds were found to inhibit E2 induced cell proliferation as comparable to that of BZD (10nM). EC317 was used as a pure PR antagonist (*P<0.01 vs control).</p

Effect of EC313 on gene expression of proliferation, apoptotic markers and PR regulated genes.

<p>A-B), Gene expression in mammary glands of overiectomized C57BL/6 mice treated with E2, EC313 0.1 and 1.0 mg/kg. Columns are average of relative amount (ΔΔCt) of tested mRNA ± SD (n = 6) (**P<0.01 vs E2-control, *P<0.001 vs E2-control).</p