Investigation of hormone-receptor interactions by means of fluorescence labeling.

Fluorescent-labeled hormones can be used to study hormone-receptor interactions by means of fluorescence polarization, visualization by fluorescence microscopy, or separation methods, e.g., dextran-coated charcoal. Subcellular fragments, single cells, and tissue preparations are amenable to study; in this work rat uterine cytosol was used unless otherwise noted. Estrone labeled with fluorescein at position 17 gives 50% inhibition in the radiometric dextran-coated charcoal assay at 8.3 X 10(-7) M as compared to 3.4 and 3.5 X 10(-8) M for diethylstilbestrol and estradiol, respectively. Scatchard plots from fluorescence polarization are hyperbolic and consistent with two classes of binding sites having association constants 5.6 X 10(10) and 6.4 X 10(7) M-1. Binding by high-affinity sites, which were present at about 3 times the concentraion of "specific" sites (radiometric dextran-coated charcoal assay), was abrogated by estradiol or diethylstilbestrol. Kinetic measurements showed that binding sites that can be blocked by excess estradiol or diethylstilbestrol are those that are both slowly associating and slowly dissociating. Staining of tissues by estrone labeled with fluorescein at position 17 as seen in the fluorescence microscope showed specificity. In normal rat uterus only epithelial cells were stained. In one human infiltrating ductal carcinoma only the malignant ductoid elements stained, while in another there was essentially no staining.

Estrogen receptor cytochemistry by fluorescent estrogen.

A recently synthesized fluorescein-labeled estrogen (17FE, 1-(N)-fluoresceinyl-estrone-thiosemicarbazone) interacts with estrogen-target cells like the native hormone and visualizes the uptake, transport, and distribution of estrogen in intact target cells. Moreover, estrogen binding sites are traced by 17FE in cryostat sections of estrogen target tissues as well. Cell and tissue 17FE binding sites fulfill the accepted criteria for specific estrogen receptors (finite binding capacity, high affinity, steroid and tissue specificity). This fluorescent probe allows estrogen receptors to be studied in a wide variety of cell and tissue preparations under varying conditions of physiologic and pathophysiologic interest.

Transcriptional regulation of estrogen-responsive genes by non-steroidal estrogens: doisynolic and allenolic acids.

Estrogen receptor (ER), a member of the nuclear receptor superfamily, exerts prominent physiological roles in both humans and other species by acting directly as a transcription factor, altering nuclear gene expression. One peculiarity of estrogenic regulation is that it is affected by a wide variety of non-steroidal compounds in addition to the natural hormone, estradiol. Doisynolic and allenolic acid compounds are non-steroidal compounds that act as potent estrogens in animal studies, yet bind to ER extremely poorly in competitive binding assays, raising the possibility of alternative molecular mechanisms for the observed estrogenic effects. In this work we demonstrate that (+/-)-Z-bisdehydrodoisynolic acid, (+/-)-Z-bisdehydrodoisynolic acid 3-methyl ether, and (-) allenolic acid can interact directly with ER. These compounds all serve as ligands for ER in mechanism-specific tissue culture-based reporter gene assays for both positive and negative gene regulation. We have also used a novel assay based on electromobility shift by ER for directly determining relative binding affinities for ER. In addition, we show cell-type-specific activity differences for (+/-)-Z-bisdehydrodoisynolic acid 3-methyl ether, supporting clinical observations indicating a higher potency of this compound in female animals than in humans.

Absolute structure determination of the highly biologically active bisdehydrodoisynolic acids.

In a project designed to relate the unexpected in vivo and in vitro properties exhibited by (+)- and (-)-bisdehydrodoisynolic acid with their absolute stereochemical structure, an X-ray crystal-structure analysis was undertaken of the highly estrogenic, poorly binding (-) enantiomer. (1) and (13)C NMR spectra are also reported for the first time. The crystal structure shows the cis juxtaposition of the carboxyl and ethyl groups, which are separated by a large torsion angle, and that only the carbon atom holding the carboxyl group is out of the plane in which the remainder of the fused three-ring moiety lies. The crystal structure, which unequivocally characterizes the (-) enantiomer as cis-13(S),14(R) and, implicitly, the (+) enantiomer as cis-13(R),14(S), will be useful in continued studies aimed at explaining the selective estrogen receptor modulation (SERM) of these enantiomers which, in some cases, produces significantly different end-organ effects compared to those of estradiol, in both males and females, affording the promise of a variety of therapeutic and pharmacologic applications.

Doisynolic acids: potent estrogens with very low affinity for estrogen receptors.

Doisynolic acids are alkaline degradation products of steroidal estrogens. While these doisynolic acids are potent estrogens, some being more estrogenic than estradiol itself, they bind to cytoplasmic estrogen receptors only feebly compared with estradiol.

Structure of sp-9-hydroxy-9-pivaloylfluorene, product of base-catalyzed autoxidation of ap-9-pivaloylfluorene.

1-(9-Hydroxyfluoren-9-yl)-2,2-dimethyl-1-propanone, C18H18O2, M(r) = 266.34, orthorhombic, Pbcn, a = 18.917 (10), b = 11.843 (8), c = 13.177 (7) A, V = 2952 (5) A3, Z = 8, Dx = 1.198 g cm-3, lambda(Mo K alpha) = 0.71069 A, mu = 0.72 cm-1, F(000) = 1136, T = 296 K, R = 0.044 for 1042 unique observed reflections. Conversion of ap-9-pivaloylfluorene, ap-(I), into lithiated (I)-9-anion followed by the addition of MeOH, then H2O, led to unexpected hydroxylation to provide 20-40% of sp-9-hydroxy-9-pivaloyfluorene, sp-(II), and 60-80% recovery of ap-(I). The singular sp conformation of (II) in solution suggested by NMR was confirmed in the crystalline state by X-ray diffraction which showed the O(1)-C(9)-C(10)-O(2) torsion angle approximately 0 degrees and the C(10) = O(2)...H(9)-O(1) non-bonding distance 1.95 (4) A, suggesting strong intramolecular hydrogen bonding in this conformation.

Unexpected dipivaloylation of 9-lithiated fluorene: formation of 1-(fluoren-9-ylidene)-2,2-dimethylpropyl pivalate.

Treatment of 9-lithiated fluorene with pivaloyl chloride provided ap-9-pivaloylfluorene, (1), the major product, and a minor product ultimately identified as the title compound, C(23)H(26)O(2), (2). The latter was also formed directly, but slowly, from 9-lithiated-(1) treated with pivaloyl chloride. Although (1) exists exclusively as its less sterically restricted ap rotamer, its sp(2)-hybridized anion sterically impedes reaction at the 9-position from either face. While 9-lithiated-(1) is exclusively, but slowly, 9-methylated with methyl iodide, reaction with pivaloyl chloride, also slow, leads only to the O-acylated product, (2). The protons of the tert-butyl-C=C moiety approach a proton on the fluorene ring to well within the sum of their van der Waals radii, resulting in significant molecular compression, strain and distortion. For example, distortion in the moiety C=C(O)(C) is exhibited by the enlargement of C=C-C angle to 130.6 (2) degrees at the expense of the corresponding 'equivalent' C=C-O angle, which is compressed to 116.46 (19) degrees.

9-(1-Acetoxyethylidene)fluorene ("diacetylfluorene"), a by-product from the acetylation of 9-fluorenyllithium.

Treatment of 9-fluorenyllithium with acetyl chloride produces 9-acetylfluorene, (I), and several by-products, among which is "diacetylfluorene", now characterized definitively as 9-(1-acetoxyethylidene)fluorene [IUPAC name: (1-fluoren-9-ylidene)ethyl acetate], (II), C(17)H(14)O(2), derived from acetylation of initially formed (I). Various parameters disclose substantial structural distortion within (II) emanating from A((1,3)) strain associated with the 9-(acetoxyethylidenyl)fluorene system.

1,1-Di(9-fluorenyl)ethanol, a by-product from the acetylation of 9-fluorenyllithium.

Treatment of 9-fluorenyllithium with acetyl chloride produces 9-acetylfluorene, (I), and several by-products. Among them is a compound unequivocally identified for the first time as the addition product of (I) with 9-fluorenyllithium, 1,1-di(9-fluorenyl)ethanol, C(28)H(22)O, (II). The two fluorene-ring planes of (II) are essentially perpendicular [89.90 (9) degrees ]. A number of intermolecular non-bonding distances are well within or close to the sum of their respective van der Waals radii and may be responsible for the rarely observed large bowing of one of the fluorene rings. This bowing apparently arises from two molecules impinging on the convex face of the bowed ring, augmented by hydrogen bonding between the peripheral pi electrons of the concave face and the hydroxyl H atom of another molecule adjacent to that face.

1,1-Di(9-fluorenyl)ethyl acetate, a by-product from the acetylation of 9-fluorenyllithium.

The preparation of sp-9-acetylfluorene from the reaction of 9-fluorenyllithium with acetyl chloride also provided 9-(1-acetoxyethylidene)fluorene ('diacetylfluorene') and 1,1-di(9-fluorenyl)ethanol, (II), as by-products recently characterized by X-ray analysis. A third by-product, 1,1-di(9-fluorenyl)ethyl acetate, (III), C(30)H(24)O(2), has now been unequivocally identified for the first time, and emanates from the acetylation of the oxyanion of (II). In the asymmetric unit, compound (III) exists as two almost identical structures which differ slightly, but significantly, in conformation. Neither possesses the significant fluorene-ring bowing or the perpendicularity of the two ring planes exhibited by (II). The angle between the least-squares planes of the two fluorene rings of (III) is 58.45 (9) and 60.95 (10) degrees, respectively, for the two conformations, and their corresponding bonding parameters also differ slightly in a number of instances.

Comparative effects of the selective estrogen receptor modulators (-)-, (+)- and (+/-)-Z bisdehydrodoisynolic acids on metabolic and reproductive parameters in male and female rats.

Doisynolic acids are non-steroidal estrogenic compounds originally obtained from alkali fusion of estrone and equilenin. Z-bisdehydrodoisynolic acids (Z-BDDA) exhibit a low binding affinity accompanied by a disproportionately high biologic activity. Two experiments were designed to investigate the chronic effects of (+)-, (-)- and (+/-)-Z-BDDA and (+)-17beta-estradiol (E2) in male and female rats. The (+)-, (-)- and (+/-)-forms Z-BDDA were prepared and injected, daily for four to six weeks into male and female rats and changes in body weight, food intake, metabolic parameters, and reproductive parameters were investigated. Results from both experiments demonstrate that in male and female rats, (+)- and (+/-)-Z-BDDA had similar estrogenic effects on reproductive organ weight. Surprisingly, (-)-Z-BDDA did not induce the increase in uterine weight observed with (+)- and (+/-)-Z-BDDA and E2, demonstrating selective estrogen receptor modulation (SERM). Beneficial metabolic effects, although compound- and gender-specific, included a significant weight repression, reduction in cholesterol, reduction in blood glucose, and positive alterations in body fat distribution. Future research defining the optimal dosages of (-)-Z-BDDA that will maximize beneficial effects and minimize undesirable effects on reproductive tissues will lead to more efficacious treatment options for endocrine-responsive conditions in males and females.

Doisynolic-type acids--uterotropically potent estrogens which compete poorly with estradiol for cytosolic estradiol receptors.

Doisynolic acids, a class of seco-steroid acids some of which exhibit greater uterotropic estrogenicity than estradiol-17 beta, are D-ring cleavage products of steroidal estrogens formed by fusion with KOH above 200 degrees C. We have found that electron-transfer reactions between estrone or estradiol and CCl4 or CBrCl3 in KOH-t-BuOH at 25 degrees C rapidly provide 16,16-dichloro- or -dibromodoisynolic acid, respectively, the former approaching estradiol in uterotropic potency. Simple esters from these highly hindered tertiary carboxylic acids, easily prepared via phase-transfer-catalyzed alkylations, also rival estradiol in uterotropic activity. Unlike natural steroidal estrogens or their commonly used artificial equivalents (DES, hexoestrol, ethynylestradiol, etc.) whose uterotropic activity is accompanied by substantial binding affinity for cytosolic estradiol receptors, these highly uterotropic doisynolic-type acids and esters exhibit binding affinities for this receptor of only about 1% that of estradiol-17 beta as determined by the usual competitive binding-inhibition studies with [3H]estradiol. Other highly uterotropic carboxylic acids may exhibit similar characteristics. These unusual results leave open the possibilities that uterotropic seco-steroid and related carboxylic acids undergo some unknown metabolic activation, are exceptionally persistent estrogens, bind to a cytosolic receptor site other than the conventional (type I) estradiol site, or bind directly to type I or type II nuclear receptor sites. At dosages of 1000 times those required for a uterotropic effect, the doisynolic-type acids (24 doses over an 8-week period) were neither toxic nor carcinogenic.

6,7,8,9-Tetrahydro-3-methyl-1H-pyrano-[4,3-b]quinolin-1-one.

The condensation reaction of 4-amino-6-methyl-2-pyrone with 1-cyclohexenecarboxaldehyde and a catalytic amount of (S)-(+)-10-camphorsulfonic acid in toluene at 358 K gave a 1:2.5 ratio of the title compound, (1) (C13H13NO2), and 7,8,9,10-tetrahydro-1H-pyrano[4,3-c]isoquinoline-1-one, (2). The formation of (2) presumably proceeds through an intermediate imine. Both (1) and (2) show inhibitory activities against acetylcholinesterase and human aldose reductase. Of the three linear-fused rings of (1), both ring A and ring B are planar and the angle between these planes is 0.46 (13) degrees. While the two C atoms of cyclohexane ring C attached to its common atoms with ring B are in the plane of the latter, as expected, the remaining two C atoms of ring C are out of this plane, by 0.342 (4) and -0.402 (3) A, respectively.