(+)-z-bisdehydrodoisynolic Acid enhances Basal metabolism and Fatty Acid oxidation in female obese zucker rats.

We have previously reported that the synthetic estrogen, (+)-Z-bisdehydrodoisynolic Acid [(+)-Z-BDDA], attenuated weight gain and cardiovascular risk in obese rodents. To determine if these antiobesity effects were attributed to changes in basal metabolism, we assessed indirect calorimetry and metabolic profile in female obese Zucker (OZR) rats provided (+)-Z-BDDA (0.0002% food admixture) for 11 weeks. Similar to our previous findings, (+)-Z-BDDA reduced weight gain and improved lipid and glucose homeostasis in OZR rats. Furthermore, resting energy expenditure was increased by (+)-Z-BDDA, as evident by heat production and oxygen consumption. We also observed a marked reduction in respiratory quotient (RQ) along with a corresponding induction of hepatic AMPK in rodents provided (+)-Z-BDDA. Collectively, these findings indicate that (+)-Z-BDDA partially attenuated obesity and associated pathologies through increased resting energy expenditure and fatty acid utilization. Further investigation is required to fully elucidate the mechanisms involved as well as to determine the potential therapeutic implications for (+)-Z-BDDA on obesity and its related pathologies.

A short, economical synthesis of 2-methoxyestradiol, an anticancer agent in clinical trials.

2-Methoxyestradiol, a natural metabolite of estradiol and potential therapeutic agent for many types of cancers, has been synthesized successfully in three steps, starting from estradiol and cumyl methyl peroxide.

Derivatives of Z-bisdehydrodoisynolic acid provide a new description of the binding-activity paradox and selective estrogen receptor modulator activity.

Z-Bisdehydrodoisynolic acid [(+/-)-Z-BDDA], an estrogenic carboxylic acid, is highly active in vivo yet binds poorly to estrogen receptors (ERs). Studies of Z-BDDA and its enantiomers demonstrate therapeutic potential as selective ER modulators; however, the activity vs. binding paradox has remained. One possible explanation is that the carboxylic acid group of Z-BDDA may be modified in vivo to an ester or amide. Synthesis of these derivatives showed the relative binding affinity (RBA) of the methyl ester for ERalpha and ERbeta was increased approximately 14- and 20-fold, respectively, relative to the parent compound. Yet, this increased affinity did not result in increased reporter gene expression. In contrast, the amide showed an unexpected approximately 4-fold decrease in RBA to both ERs compared with the parent. The relationship among the RBAs of ester, acid, and amide is consistent with their predicted polarity, suggesting the carboxylic acid, and not the carboxylate of BDDA, binds to ERs. Studies at pH 6.5, 7.4, and 8.0 were consistent with a simple acid-base equilibrium model, with BDDA binding as the undissociated acid and with affinity equal to or exceeding that of estradiol, consistent with high in vivo potency. Furthermore, the alcohol BDD-OH also demonstrated high affinity and increased activity in gene expression assays. In addition to suggesting a resolution to the decades-old binding/activity paradox, these studies may provide a direction for definitive in vivo metabolic and pharmacokinetic studies and provide additional insight into the chemical and metabolic determinants of BBDA's unique tissue selectivity and selective ER modulator activities.

(+)-Z-Bisdehydrodoisynolic acid ameliorates obesity and the metabolic syndrome in female ZDF rats.

OBJECTIVE: The putative selective estrogen receptor modulator (+)-Z-bisdehydrodoisynolic acid (Z-BDDA) has been found to improve cardiovascular risk in rodents. The objective of this study was to investigate the effectiveness of (+)-Z-BDDA compared with the antidiabetic drug, rosiglitazone, in treating obesity and risk factors associated with the metabolic syndrome. RESEARCH METHODS AND PROCEDURES: Female Zucker Diabetic Fatty rats were randomly assigned to three treatment groups for 29 weeks: control (C), 1.8 mg (+)-Z-BDDA/kg diet [control diet + (+)-Z-BDDA (CB)], or 100 mg rosiglitazone/kg diet [control diet + rosiglitazone (CR)]. At sacrifice, physiological, biochemical, and molecular parameters were examined. RESULTS: CB animals gained less weight and exhibited a decrease in total body lipids (p < 0.05) as compared with C or CR rats. Body weight and total body lipids were the highest in CR rats (p < 0.05). Liver weights in CB and CR rats were lower (p < 0.05) than in C rats, whereas kidney weights were lower in CB (p < 0.05) than in C and CR animals. Fasting plasma glucose was lower (p < 0.05) in the CB and CR animals when compared with C animals. C rats exhibited the highest concentration of total plasma cholesterol, and CR-treated rats exhibited the lowest concentration. Plasma triglycerides followed the same pattern as plasma cholesterol. Histomorphometry of heart vasculature revealed that CB and CR treatments produced a significant shift from small to large venules and arterioles compared with C (p < 0.05). Liver expression profiles of peroxisome proliferator-activated receptor (PPAR) alpha, PPARgamma, and PPAR-regulated genes revealed encouraging CB-induced effects. DISCUSSION: These results suggest that (+)-Z-BDDA may have applications in treating obesity and complications associated with the metabolic syndrome.

Stereochemistry of reactions involving rotationally restricted, sterically hindered cations, radicals, and anions: 9-fluorenyl systems.

A study of the stereochemical pathways of reactions involving rotationally restricted, sterically hindered cations, radicals, and anions has been undertaken utilizing chiral 9-(o-tert-butylphenyl)fluorenes. Previous reports of studies with these or related achiral compounds contained erroneous or equivocal conclusions. This study shows that (+)-sp-9-(o-tert-butylphenyl)-9-methoxy-2-methylfluorene, treated with Tf(2)O-CHCl(3) to form 100% of the 9-cation, then with NaOMe-MeOH, provided 29% of re-formed substrate (configurational retention) and 71% of the (-)-sp enantiomer (inversion). The same substrate treated with HI-CHCl(3) was converted into the 9-radical, which was rapidly reduced, affording 100% isolation of (-)-sp-9-(o-tert-butylphenyl)-2-methylfluorene (inversion). Treatment of the latter with n-BuLi-THF provided the 9-anion which, on acidification, yielded 100% of the enantiomeric (+)-sp-9-(o-tert-butylphenyl)-2-methylfluorene (inversion). The substrates in these reactions were the thermodynamically favored sp rotamers. Inversion directly produced the higher energy nonenantiomeric ap rotamers, which rapidly rotated into the sp products that were enantiomeric with the substrates. These results are explained by the rotational restriction and partial steric hindrance by the tert-butyl group to the original face of the sp(3) antiaromatic 9-cation (4n pi electrons), and the rotational restriction and extensive blockage to the original face of the sp(2) nonaromatic 9-radical (4n + 1 pi electrons) and aromatic (4n + 2 pi electrons) 9-anion. The barrier to rotation in some of the ortho-substituted 9-arylfluorenes is great enough to allow their sp and ap rotamers to be detected coexisting in solution, although their crystals were composed exclusively of one. Rotational restriction and steric hindrance at the 9-position have a large influence on the pK(a) values of these fluorenes and can offset the classic electronic effects of the substituents.

sp-9-(o-Methylphenyl)fluorene.

While the barriers of rotation of the sp and ap rotamers of 9-(o-methylphenyl)fluorene, C(20)H(16), are sufficiently similar to permit them to equilibrate, both being observed (NMR) in solution, crystallization provides the sp rotamer, (I), exclusively. Although in the sp conformation the intramolecular distance between adjacent C atoms of the phenyl and fluorene rings is small [3.382 (4) A, within 0.02 A of the sum of the van der Waals radii], in the ap conformation the distance between the adjacent o-CH(3) group on the phenyl ring and C atom of the fluorene ring would be much closer, based on that exhibited in the crystalline ap progenitor 9-(o-methylphenyl)-9-fluorenol. The angle between the fluorene and 9-aryl planes of (I) is 75.82 (10) degrees.

ap-9-(o-Methylphenyl)-9-fluorenol, a structure exhibiting several aryl-H...pi(arene) intermolecular interactions.

The title compound, C(20)H(16)O, (I), which crystallized exclusively as its ap rotamer, exhibits several intermolecular aryl-H.pi(arene) interactions, resulting in planar molecular arrays in which each molecule interacts with six adjacent molecules. Surprisingly, there were no O-H.O-H or O-H.pi(arene) interactions within hydrogen-bonding distances. Crystalline (I) melted sharply without molecular decomposition (NMR), but the cooled melt recrystallized only after several hours.

ap-9-(meta-tert-butylphenyl)fluorene.

The title compound, C(23)H(22), (I), crystallizes in an ap conformationThe designations sp (synperiplanar) and ap (antiperiplanar) for these fluorene rotamers are in accordance with Rule E-6.6, IUPAC Tentative Rules, Section E, Fundamental Stereochemistry [J. Org. Chem. (1970), 35, 2861]. and its melt readily recrystallizes on cooling, in contrast to the corresponding 9-fluorenol compound, (II), which is sp and which melts without decomposition and fails to recrystallize over a long period. Both of these differences are ascribed to the intermolecular hydrogen bonding in (II), which is absent in (I) and which leads to distinctly different molecular packing in the two compounds.

Unexpected differences in the alpha-halogenation and related reactivity of sulfones with perhaloalkanes in KOH-t-BuOH.

Most alkyl phenyl sulfones are readily alpha-chlorinated with CCl(4) and alpha-brominated with CBrCl3 in KOH-t-BuOH via radical-anion radical pair (RARP) reactions. While isopropyl mesityl sulfone (4) is easily alpha-chlorinated with CCl(4), it was completely recovered when treated with the more reactive CBrCl3. Subsequent investigations showed the latter result to be due to the poor acidity of 4 together with the rapid depletion of CBrCl3 and KOH by their reaction with each other, and led to a variety of other important results. 4-Hydroxyphenyl isopropyl sulfone (6) is unreactive with either CCl4 or CBrCl3 in KOH-t-BuOH, its phenoxide anion strongly reducing the electronegativity of the sulfonyl group, thereby inhibiting alpha-anion formation. This effect is reversed by the electron-withdrawing influence of two alpha-phenyls, so that benzhydryl 4-hydroxyphenyl sulfone (8) is readily alpha-halogenated in KOH-t-BuOH with CCl4 or CBrCl3. On further contact with KOH-t-BuOH the alpha-halogenated sulfones from 8 are decomposed into benzophenone and phenol. While the alpha-halogenated derivatives of 4-methoxyphenyl benzhydryl sulfone (9) are stable to base, they are decomposed even under mildly acidic conditions into 4-methoxyphenyl 4-methoxybenzenethiolsulfonate (9c), phenol, and benzophenone. Mono-alpha-halogenation of benzyl phenyl sulfone (10) enhances the rate of the subsequent halogenation, so that alpha,alpha-dihalogenation is attained while much substrate is still present and the mono-alpha-halogenated product is not detected. The ease of reductive debromination of alpha-bromo sulfones with Cl3C- was correlated with the stability of the formed alpha-anions, explaining the success with alpha-bromobenzylic sulfones but failure with alpha-bromoalkyl sulfones. In the presence of air and the absence of competing halogenation, formation of the alpha-anions of alkyl aryl sulfones is quickly accompanied by oxidative cleavage by atmospheric O2, leading to the formation of arenesulfonyl alcohols, arenesulfonyl halides, and haloarenes.

Activities of a non-classical estrogen, Z-bis-dehydrodoisynolic acid, with ERalpha and ERbeta.

(+/-)-Z-bis-Dehydrodoisynolic acid [(+/-)-Z-BDDA] is highly estrogenic in vivo, yet binds to estrogen receptor (ER) poorly. This paradox has raised the possibility of alternative ERs and/or molecular mechanisms. To address the possibility of high activities of Z-BDDA with ERbeta, we determined the activities of (+)-Z-BDDA and (-)-Z-BDDA, in cell culture and in vitro, comparing ERbeta to ERalpha. Transfectional analysis in Hela cells showed (-)-Z-BDDA is an agonist for gene activation with both ERalpha (EC(50) congruent with 0.3nM) and ERbeta (EC(50) congruent with 5nM), while little to no activity was observed with (+)-Z-BDDA. Similarly, in gene repression assays, (-)-Z-BDDA was active (EC(50) congruent with 0.2nM), but again minimal activity was exhibited by (+)-Z-BDDA. Binding to ERalpha and ERbeta in vitro used both competition and a direct binding assay. For ERalpha, the relative affinity of (-)-Z-BDDA was approximately 6% by competition and 1.7% by direct binding versus 17beta-estradiol (E2; 100%), while (+)-Z-BDDA also demonstrated binding, but with relative affinities of only 0.08% by competition and 0.3% by the direct assay. For ERbeta, the affinity of (-)-Z-BDDA was approximately 7% by competition and 1.5% by the direct assay relative to E2 (100%), while (+)-Z-BDDA had lower affinity, approximately 0.2% that of E2 by both assays. The paradox of potent in vivo activity but lower activity in receptor binding and in cell culture reporter gene assays, previously seen with ERalpha is now also associated with ERbeta. The failure of ERbeta to explain the activity-binding paradox indicates the need for additional in vivo metabolic and pharmacokinetic studies and continued consideration of alternative mechanisms.

Antioxidant effects of phyto-and synthetic-estrogens on cupric ion-induced oxidation of human low-density lipoproteins in vitro.

Oxidation of low-density lipoproteins (LDL) promotes the formation of atherosclerotic plaques. Estrogenic compounds (EC) from foods and other natural products, and synthetic estrogenic compounds (SECs) may prevent heart disease by inhibiting LDL oxidation. In the present study, we tested the antioxidant capacities of two phytoestrogens, daidzein (DAI) and genistein (GEN), and four SECs, (+)- and (-)-Z-bisdehydrodoisynolic acid (ZBDDA), and (+)- and (-)-hydroxy-allenoic acid (HAA), on isolated human LDL subjected to oxidation by cupric sulfate. The effects of these estrogenic compounds on the kinetics of conjugated diene formation in LDL undergoing oxidation were evaluated with a lag-time assay with continuous monitoring of absorbance at 234 nm. Lag-time data revealed that (+)-HAA, (-)-HAA, (+)-ZBDDA, and (-)-ZBDDA had similarly stronger antioxidant activities than either GEN or DAI. We also found that (+)-HAA, (-)-HAA, (+)-ZBDDA, and (-)-ZBDDA strongly inhibited the formation of Cu+-induced thiobarbituric acid reactive substances (TBARS) in LDL, and that GEN and DAI were less effective for inhibiting LDL lipid peroxidation. Finally, electrophoretic evaluation suggested that (+)-HAA, (-)-HAA, (+)-ZBDDA, and (-)-ZBDDA protected the apolipoprotein B-100 of LDL against oxidation better than did GEN or DAI. In summary, the four SECs, (+)-HAA, (-)-HAA, (+)-ZBDDA, and (-)-ZBDDA, were more potent antioxidants than the phytoestrogens, DAI and GEN.

trans-3-Ethyl-cis-2,6,6-trimethyl-4-oxocyclohexanecarboxylic acid: an intermediate in the synthesis of a highly potent estrogen.

The title compound, C(12)H(20)O(3), (IV), the ethyl ester of which is an intermediate in the synthesis of a compound reported to be highly estrogenic, has been prepared. After the initial steps reported for the synthesis of this ester intermediate were followed, it was converted into the crystalline acid, (IV), for X-ray analysis. It was verified that (IV) was racemic when prepared. X-ray analysis showed that anti-hydrogenation of the double bond had occurred in the synthesis, making the orientation of the carboxyl group cis to the 2-methyl group and trans to the 3-ethyl group. NMR spectroscopy showed that the stereochemistry of (IV) was identical with that of its ester precursor. While the earlier report did not note the stereochemistry of this ester, it pointed out that the estrogenic product derived from it possessed the opposite carboxyl-2-methyl orientation, i.e. trans, although no X-ray analysis was performed. In the light of these results and the importance of correlating biological activity with compound structure, the unequivocal characterization of the highly estrogenic compound is warranted.