Synthesis and pharmacology of conformationally restricted raloxifene analogues: highly potent selective estrogen receptor modulators.

The 2-arylbenzothiophene raloxifene, 1, is a selective estrogen receptor modulator (SERM) which is currently under clinical evaluation for the prevention and treatment of postmenopausal osteoporosis. In vivo structure-activity relationships and molecular modeling studies have indicated that the orientation of the basic amine-containing side chain of 1, relative to the stilbene plane, is an important discriminating factor for the maintenance of tissue selectivity. We have constructed a series of analogues of 1 in which this side chain is held in an orientation which is orthogonal to the stilbene plane, similar to the low-energy conformation predicted for raloxifene. Herein, we report on the synthesis of these compounds and on their activity in a series of in vitro and in vivo biological assays reflective of the SERM profile. In particular, we describe their ability to (1) bind the estrogen receptor, (2) antagonize estrogen-stimulated proliferation of MCF-7 cells in vitro, (3) stimulate TGF-beta3 gene expression in cell culture, (4) inhibit the uterine effects of ethynyl estradiol in immature rats, and (5) potently reduce serum cholesterol and protect against osteopenia in ovariectomized (OVX) rats without estrogen-like stimulation of uterine tissue. These data demonstrate that one of these compounds, LY357489,4, is among the most potent SERMs described to date with in vivo efficacy on bone and cholesterol metabolism in OVX rats at doses as low as 0.01 mg/kg/d.

Prenatal detection of de novo duplication of the short arm of chromosome 18 confirmed by fluorescence in situ hybridization (FISH).

We present a patient with developmental delay, minor anomalies, and duplication 18p confirmed by fluorescence in situ hybridization with whole chromosome 18 painting probe (Oncor p5218). Our observation confirms the findings of other investigators that duplication 18p is not associated with major malformations.

Effects of prolonged ethinyl estradiol treatment on calcium channel binding and in vivo calcium-mediated hemodynamic responses in ovariectomized rats.

Ethinyl estradiol (EE2), administered orally to ovariectomized (ovex) rats, has been shown to prevent loss of bone mineral density and to decrease serum cholesterol levels. Radioligand binding studies with the dihydropyridine (DHP) [3H]PN200-110 were undertaken to characterize calcium (Ca++) channels in cardiac and aortic tissues from ovex rats treated for 35 days with EE2 (0.1 mg/kg day p.o.) or vehicle, and from vehicle-treated sham-operated controls (sham). Cardiac tissues from EE2-treated rats displayed significant increases in the density (Bmax) of high-affinity DHP binding sites (505 +/- 46 fmol/mg) compared with vehicle-treated ovex rats (303 +/- 35 fmol/mg); DHP Bmax values from EE2-treated cardiac tissues were not significantly different from vehicle-treated shams (385 +/- 76 fmol/mg). Cardiac Ca++ efflux channels from sarcoplasmic reticulum were assessed with [3H]ryanodine. [3H]Ryanodine Bmax values were not affected by EE2 treatment. However, [3H]ryanodine Kd values in preparations from EE2-treated rats were significantly decreased (10 +/- 2 nM) compared with ovex rats (35 +/- 11 nM) and were similar to values in sham rats (8 +/- 2 nM). Cardiac beta adrenoceptors were not affected by EE2 treatment, which thus confirmed the selective regulation of DHP receptors by EE2. Aortic preparations from EE2-treated rats exhibited significant increases in DHP receptors (125 +/- 37 fmol/mg) compared with both vehicle-treated ovex rats (32 +/- 3 fmol/mg) and vehicle-treated shams (24 +/- 9 fmol/mg). There were no differences in the binding affinity (Kd) of [3H]PN200-110 for cardiac or aortic sites among the three groups. To ascertain if EE2-mediated increases in Ca++ channel density and ryanodine binding affinity affected in vivo responses to agonists that use extracellular and intracellular Ca++ stores, responses to BAY k 8644 and norepinephrine were examined in pithed rats from the same three groups. No significant differences in hemodynamic responses occurred among the three groups to BAY k 8644 or norepinephrine. Thus, in female ovex rats, prolonged treatment with EE2 resulted in increased density of cardiac and aortic calcium channels which did not translate into increased calcium-mediated inotropic, rate or pressor responses. Conversely, EE2 treatment in ovex rats prevented the decrease in cardiac [3H]ryanodine binding affinity evident in vehicle-treated ovex rats. These data suggest that EE2 treatment in normotensive ovex rats resulted in modulation of both the L-type and sarcoplasmic reticulum Ca++ channels, and these alterations maintained cardiovascular homeostasis.

Structure-activity relationships of selective estrogen receptor modulators: modifications to the 2-arylbenzothiophene core of raloxifene.

The 2-arylbenzothiophene raloxifene, 1, is a selective estrogen receptor modulator which is currently under clinical evaluation for the prevention and treatment of postmenopausal osteoporosis. A series of raloxifene analogs which contain modifications to the 2-arylbenzothiophene core have been prepared and evaluated for the ability to bind to the estrogen receptor and inhibit MCF-7 breast cancer cell proliferation in vitro. Their ability to function as tissue-selective estrogen agonists in vivo has been assayed in a short-term, ovariectomized (OVX) rat model with end points of serum cholesterol lowering, uterine weight gain, and uterine eosinophil peroxidase activity. These studies have demonstrated that (1) the 6-hydroxy and, to a lesser extent, the 4'-hydroxy substituents of raloxifene are important for receptor binding and in vitro activity, (2) small, highly electronegative 4'-substituents such as hydroxy, fluoro, and chloro are preferred both in vitro and in vivo, (3) increased steric bulk at the 4'-position leads to increased uterine stimulation in vivo, and (4) additional substitution of the 2-aryl moiety is tolerated while additional substitution at the 4-, 5-, or 7-position of the benzothiophene results in reduced biological activity. In addition, compounds in which the 2-aryl group is replaced by alkyl, cycloalkyl, and naphthyl substituents maintain a profile of in vitro and in vivo biological activity qualitatively similar to that of raloxifene. Several novel structural variants including 2-cyclohexyl, 2-naphthyl, and 6-carbomethoxy analogs also demonstrated efficacy in preventing bone loss in a chronic OVX rat model of postmenopausal osteopenia, at doses of 0.1-10 mg/kg.

Raloxifene (LY139481 HCI) prevents bone loss and reduces serum cholesterol without causing uterine hypertrophy in ovariectomized rats.

There is a medical need for an agent with the positive effects of estrogen on bone and the cardiovascular system, but without the negative effects on reproductive tissue. Raloxifene (LY139481 HCI) is a benzothiophene derivative that binds to the estrogen receptor and inhibits the effects of estrogen on the uterus. In an ovariectomized (OVX) rat model we investigated the effects of raloxifene on bone loss (induced by estrogen deficiency), serum lipids, and uterine tissue. After oral administration of raloxifene for 5 wk (0.1-10 mg/kg per d) to OVX rats, bone mineral density in the distal femur and proximal tibia was significantly greater than that observed in OVX controls (ED50 of 0.03-0.3 mg/kg). Serum cholesterol was lower in the raloxifene-treated animals, which had a minimal effective dose of 0.1 mg/kg and an approximate oral ED50 of 0.2 mg/kg. The effects of raloxifene on bone and serum cholesterol were comparable to those of a 0.1-mg/kg per d oral dose of ethynyl estradiol. Raloxifene diverged dramatically from estrogen in its lack of significant estrogenic effects on uterine tissue. Ethynyl estradiol produced a marked elevation in a number of uterine histologic parameters (e.g., epithelial cell height, stromal eosinophilia). These data suggest that raloxifene has promise as an agent with beneficial bone and cardiovascular effects in the absence of significant uterine effects.