New estrogen receptor antagonists. 3,20-Dihydroxy-19-norpregna-1,3,5(10)-trienes: Synthesis, molecular modeling, and biological evaluation.

New estrogen receptor α (ERα) antagonists - 3,20-dihydroxy-19-norpregna-1,3,5(10)-trienes containing an additional carbocyclic ring D' at the 16α,17α positions - were synthesized. The effects of the new compounds on the MCF-7 breast cancer cells and ERα activation were investigated. All the steroids studied were synthesized starting from estrone methyl ether. The scaffold of compounds containing the six-membered ring D' was constructed via the Diels-Alder reaction of butadiene with 3-methoxy-19-norpregna-1,3,5(10),16-tetraen-20-one 5. The hydrogenation of primary 16α,17α-cyclohexeno-adduct 7 followed by 3-demethylation (by HBr-AcOH) and reduction of 20-oxo group (by LiAlH4) or in one step by DIBAH gave target mono- and dihydroxy steroids 9-11. The Corey-Chaykovsky reaction of the same 3-methoxy-19-norpregna-1,3,5(10),16-tetraen-20-one 5 gave 16α,17α-methylene-substituted compound. The reaction of the latter with DIBAH immediately yielded 3,20-dihydroxy-16α,17α-methyleno-19-norpregna-1,3,5(10)-triene 13. The same procedures using 3-methoxy-19-norpregna-1,3,5(10),16-tetraen-20-one 5 produced corresponding 3,20-dihydroxy-16,17-19-norpregna-1,3,5(10)-triene 16 and 3,20-dihydroxy-19-norpregna-1,3,5(10),16-tetraene 17. All compounds were fully characterized by 1D and 2D NMR, HRMS, and X-ray diffraction. The molecular docking showed that the target compounds can bind to ER, their binding mode being similar to that of natural estradiol. 16α,17α-Methylene- or unsubstituted compounds exhibit the highest cytotoxicity against MCF-7 cells, being simultaneously relatively weak ERα inhibitors. 3,20-Dihydroxy steroids containing the six-membered ring D' proved to be the most effective ERα inhibitors. These compounds displayed moderate cytotoxicity comparable of that of tamoxifen and showed no toxic effect on MCF-10A normal, nontumorigenic epithelial cells. The new ER antagonists were found to be good candidates for further testing as agents for the treatment and prevention of ERα-positive breast cancers.

Synthesis and evaluation of isomeric (17alpha,20E)-11beta-methoxy-21-(trifluoromethylphenyl)-19-norpregna-1,3,5(10),20-tetraene-3,17beta-diols as ERalpha-hormone binding domain ligands: effect of the methoxy group on receptor binding and uterotrophic growth.

In this study we have introduced the 11beta-methoxy group, a substituent known to increase in vivo potency in other steroidal estrogens, into the (17alpha,20E)-21-(trifluoromethylphenyl)-19-norpregna-1,3,5(10),20-tetraene-3,17beta-diols: (trifluoromethylphenyl)vinyl estradiols. Receptor binding, using the ERalpha-HBD, indicated that the 11beta-methoxy group had little effect on the relative binding affinity of the target compounds compared to the corresponding 11beta-unsubstituted analogs, however, the 11beta-methoxy derivatives were significantly more potent in stimulating uterotrophic growth in immature female rats. Molecular modeling studies suggest that while the 11beta-methoxy group does not contribute significantly to the overall binding energy of the ligand-ERalpha-HBD complex, it stabilizes residues associated with the coregulator protein binding site. Such effects would influence the dynamics of subsequent events, such as transcription and biological responses.

Chromatographic analysis and decomposition product characterization of compound SR16157 (NSC 732011).

SR16157 (21-(2-N,N-diethylaminoethyl)oxy-7alpha-methyl-19-norpregna-1,3,5(10)-triene-3-O-sulfamate) is a novel, dual-acting estrone sulfatase inhibitor currently in preclinical development for use in breast cancer therapy. The compound has a dual mechanism of action: the sulfamate-containing parent compound SR16157 inhibits estrogen biosynthesis by irreversibly inhibiting the enzyme estrone sulfatase. The phenolic metabolite, SR16137, generated by the sulfatase enzyme is a potent antiestrogen in breast tissues and has beneficial effects in bone and the cardiovascular system. As part of the ongoing preclinical studies, an HPLC assay method has been developed and validated for SR16157. The assay method is specific, accurate (recovery=99.4-101.1), linear (r(2)> or =0.9999), precise (intraday R.S.D.< or =1.1%, intermediate R.S.D.< or =0.8%), and sensitive (limit of detection=1.0 microg/ml). It separates SR16157 from its impurities and forced decomposition products, which have been characterized by LC coupled with mass and UV spectral data. Major decomposition pathways are hydrolysis, hydroxylation, and oxidation.

Synthesis and evaluation of (17alpha,20Z)-21-(4-substituted-phenyl)-19-norpregna-1,3,5(10),20-tetraene-3,17beta-diols as ligands for the estrogen receptor-alpha hormone binding domain: comparison with 20E-isomers.

As part of our ongoing program to develop probes for the hormone binding domain of the estrogen receptor-alpha (ERalpha), we prepared and evaluated a series of 17alpha,Z-(4-substituted-phenyl)vinyl estradiol derivatives. The results indicated that the relative binding affinities (RBAs) at 25 degrees C for the new compounds were significant (RBA = 9-57) although less than that of estradiol (RBA = 100) or of the parent unsubstituted phenylvinyl estradiol (RBA = 66). All of the Z-compounds were full agonists in the uterotrophic assay, indicating that the ligands formed estrogen-like complexes with the estrogen receptor-alpha hormone binding domain (ERalpha-HBD). Comparison of corresponding Z- and E-4-substituted phenylvinyl ligands complexed with the ERalpha-HBD indicated small but significant differences in binding modes that may account for the differing trends seen in the structure-activity relationships for the two series.

Fundamental differences in the action of estrogens and antiestrogens on the uterus: comparison between compounds with similar duration of action.

In order to understand differences in the mechanism of action of estrogens and antiestrogens, it is essential to make comparisons between compounds with similar duration of action. Hence, in these studies, we compare the action of a long-acting estrogen (17alpha-ethinyl estriol-3-cyclopentyl ether, EE3CPE) and a long-acting antiestrogen (U-11,100AUA) on the immature rat uterus and analyze different dosage regimens (single and multiple injections) in studying the effects of these compounds on the uterine estrogen receptor and on uterine growth and sensitivity to estradiol. During the first 24 h, UA (50 microng) and EE3CPE (5 microng) evoke remarkably similar receptor distribution patterns and uterine wet weight increase; however, pronounced differences are seen with long-term, multiple injection regimens (every 12 or 24 h for 72 h). Such treatment with UA results in maintenance of high nuclear receptor levels and very low cytoplasmic receptor levels (ca. 10% of total), but no further increase in uterine weight, DNA or protein content, or total receptor content beyond 24-48 h. In contrast, multiple injections of EE3CPE produce not only a prolonged nuclear retention of receptor, but a progressive increase in total receptor content in the tissue and 35-50% of total receptor is cytoplasmic; uterine weight and DNA and protein content also continue to increase markedly above the 24 h level, and responsiveness to estradiol is maintained. However, regardless of whether the uterus continues to grow (as the EE3CPE) or stops growing after 24-48 h (as with UA), the receptor content on a cell basis is similar. Hence, uterine responsiveness to estradiol and continued uterine growth appear not to be related to the total content of receptor per cell, but rather are correlated with the cytoplasmic receptor level within the cell. As there is a continuous translocation of cytoplasmic receptor to the necleus in the growing uterus, the antagonistic action of antiestrogens appears to derive from their ability to effect a marked perturbation in the subcellular distribution of receptor, whereby very little of receptor (ca. 10%) is cytoplasmic, and further estrogen receptor accumulation (most likely synthesis) is blocked.

Synthesis and evaluation of 17alpha-20E-21-(4-substituted phenyl)-19-norpregna-1,3,5(10),20-tetraene-3,17beta-diols as probes for the estrogen receptor alpha hormone binding domain.

As part of our program to develop probes for the hormone binding domain of the estrogen receptor alpha (ERalpha), we prepared a series of 4-para-substituted phenylvinyl estradiol derivatives using a combination of solution and solid-phase Pd(0)-catalyzed methods. The compounds 5a-j were evaluated for their binding affinity using the ERalpha hormone binding domain (HDB) isolated from transfected BL21 cells. The results indicated that although the new compounds were somewhat lower in relative binding affinity (RBA at 25 degrees C is 1-60%) than estradiol (100%), most had higher affinity than the unsubstituted parent phenylvinyl estradiol (RBA = 9%). Because the substituents did not generate a structure-activity relationship directly based on physicochemical properties, the series was evaluated using molecular modeling and molecular dynamics to determine key interactions between the ligand, especially the para substituent, and the protein. The results suggest that the observed relative binding affinities are directly related to the calculated binding energies and that amino acids juxtaposed to the para position play a significant but not dominant role in binding. In conclusion, we have identified the 17alpha-E-(4-substituted phenyl)vinyl estradiols as a class of ligands that retain significant affinity for the ERalpha-HBD. In particular, 4-substitution tends to increase receptor affinity compared to the unsubstituted analogue, as exemplified by 5e (4-COCH(3)), which had the highest RBA value (60%) of the series. Palladium(0)-catalyzed coupling reactions on solid support or in solution using suitably substituted iodo arenes and 17alpha-E-tributylstannylvinyl estradiols offer a flexible approach to their preparation. Molecular modeling studies of the receptor suggest that there exists additional ligand accessible regions within the ERalpha-HBD to generate interactions that may enhance receptor affinity or modify efficacy in developing new therapeutic agents. Studies to undertake modification in the properties and/or position of the aryl substituents in subsequent series to further define that role are in progress.

3,17 beta-Dihydroxy-20,21-epoxy-19-norpregna-1,3,5(10)-trienes: synthesis, rearrangement, cytotoxicity, and estrogen-receptor binding.

Diastereoisomers of 3,17 beta-dihydroxy-20,21-epoxy-19-norpregna-1,3,5(10)-triene have been prepared as potential antitumor agents. Both isomers undergo the base-catalyzed Payne rearrangement. The isomers were cytotoxic to mammalian cells in culture and were able to displace [3H]estradiol from binding sites in rat uterine cytosols with 1/7 and 1/70 the potency of estradiol. The reasons for this difference are discussed.

Comparison of quinestrol and Tace for relief of postpartum breast discomfort.

A single 2-mg dose of quinestrol was demonstrated safe and effective for controlling postpartum lactation and for alleviating breast discomfort. A double-blind comparison to Tace 72 mg every 12 hours for 2 days, and to placebo, was made in 134 patients. The single oral dose of quinestrol showed efficacy equal to the 2-day regimen of Tace. Both were superior to placebo.

Replacement estrogen therapy for menopausal vasomotor flushes. Comparison of quinestrol and conjugated estrogens.

Quinestrol, conjugated estrogens, or placebo was used to treat 156 patients with pernicious vasomotor instability in a prospective, double-blind, randomized, multiinvestigator trial. Vasomotor flushes were severe in approximately 80% of the cases and moderate in 20%, relatively equally distributed among the various drug groups. Both qinestrol and conjugated estrogens were significantly more effective than placebo in relieving vasomotor symptoms (by chi2 analysis, P less than or equal to 0.05). Greatest improvement was seen in the group receiving the higher once weekly quinestrol dosage of 0.2 mg followed by the group on the lower quinestrol dosage of 0.1 mg once weekly and the group on conjugated estrogens, 1.25 mg daily for 21 days on and 7 days off. No significant difference in relief of vasomotor flushes was shown between the active drug groups. No drug-related complications or side reactions of significance occurred. The results indicate that once weekly quinestrol is effective in relieving the vasomotor symptoms of the menopause. Either of two once weekly quinestrol regimens is an effective as conjugated estrogens given daily in a cyclic manner and therefore offers an alternative form of exogenous estrogen therapy.

The effects of a once-a-week steroid contraceptive (R2323) on lipid and carbohydrate metabolism in women during three months of use.

Forty-four women were prospectively evaluated with a 3-hour oral glucose tolerance test while they used a new weekly oral contraceptive drug, R2323. The blood tests were carried out before the drug was administered and again 3 months after its use. Both blood glucose and plasma insulin levels were measured. In the 40 women with a "normal" control glucose tolerance test, it was found that only the 1-hour blood glucose value was significantly elevated after 3 months of drug use and the plasma insulin values were unchanged. The tests of three of the four women with borderline abnormal to abnormal control test curves converted to normal with 3 months of treatment and that of one remained unchanged. The fasting plasma cholesterol and triglyceride levels were unchanged over the 3 months' time. These results suggest that this steroid contraceptive has no effect on lipid metabolism. It also has minimal adverse effects on normal carbohydrate metabolism and may possibly improve abnormal metabolism.

The effect of a synthetic progestogen, ethylnorgestrienone, on hypothalamic-pituitary-ovarian function, cervical mucus, vaginal cytology, and endometrial morphology.

The antifertility mechanism of a new progestin preparation, ethylnorgestrienone (13 beta-ethyl-17 alpha-ethynyl-17-hydroxy-gona-4,9,11-triene-3-one) (R2323), was evaluated. The compound was administered orally in doses of 5 mg weekly to seven subjects for a total of nine treatment cycles. Each woman served as her own control and was studied during a normal menstrual cycle followed by a cycle in which she received R2323. Serum concentrations of luteinizing hormone (LH), follicle-stimulating hormone (FSH), estradiol, and progesterone, as well as cervical mucus properties, were studied serially during the control and treatment cycles. Results indicated that all control cycles were ovulatory. Ovulatory gonadotropin patterns were observed in four treated cycles, but preovulatory FSH and LH peaks and progesterone production during the luteal phase were suppressed significantly. Estradiol levels showed an early increase during the follicular phase and a significantly decreased preovulatory peak. Cervical mucus properties were altered and sperm penetration in cervical mucus was inhibited in all treatment cycles. These findings suggest that at least two different factors might be involved in the contraceptive mechanism of R2323: (1) alteration of the ovulatory process and progesterone production, and (2) cervical mucus changes leading to inhibition of sperm migration.

PIP: The antifertility mechanism of a new progestin preparation, ethylnorgestrienone (13 beta-ethyl-17alpha-ethynyl-17-hydroxy-gona-4,9,11-triene-3-one) (R2323) was evaluated. The compound was administered orally in doses of 5 mg weekly to 7 subjects for a total of 9 treatment cycles. Each woman served as her own control and was studied during a mormal menstrual cycle followed by a cycle in which she received R2323. Serum concentrations of (LH) luteinizing hormone, (FSH) follicle stimulating hormone, estradiol, and progesterone, as well as cervical mucus properties, were studied serially during the control and treatment cycles. Results indicated that all control cycles were ovulatory. Ovulatory gonadotropin patterns were observed in 4 treated cycles, but prevulatory FSH and LH peaks and the progesterone production during the luteal phase were suppressed significantly. Estradiol levels showed an early increase during the follicular phase and a significantly decreased preovulatory peak. Cervical mucus properties were altered and sperm penetration in cervical mucus was inhibited in all treatment cycles. These findings suggest that at least 2 different factors might be involved in the contraceptive mechanism of R2323: 1) alteration of the ovulatory process and progesterone production, and 2) cervical mucus changes leading to the inhibition of sperm migration.

Studies on the mechanism of action of danazol and gestrinone (R2323) in the rat: evidence for a masked estrogen component.

The effects of androgen and estrogen receptor antagonists on the action of danazol and gestrinone (R2323) were investigated. The tropic effect of danazol and gestrinone on sex accessory tissues of castrated, immature male rats was inhibited by the antiandrogen flutamide, whereas the uterotropic action of these steroids in immature female and adult ovariectomized rats was not inhibited by flutamide. In contrast, the uterotropic effect of danazol was reduced by the antiestrogen, LY156758. The estrogen-sensitive endpoints, vaginal keratinization and uterine progesterone receptor concentration, were enhanced by treatment with a combination of flutamide and either danazol or gestrinone. These data indicate that danazol and gestrinone have estrogenic activity that is masked by the androgenic component of these drugs.

The inhibitory effects of danazol, danazol metabolites, gestrinone, and testosterone on the growth of human endometrial cells in vitro.

Danazol and gestrinone are effective drugs in the treatment of endometriosis. Their mechanism of action remains uncertain, but may be related to their androgenic activity. The authors examined the effect of danazol on human endometrial cells cultured in vitro, its two major metabolites, ethisterone and 2 hydroxymethyl ethisterone, gestrinone, and testosterone (T) at 1X and 10X expected plasma concentrations. Danazol and T suppressed growth by 20.8 and 25.0% (P less than 0.01), respectively, at the lower dose, and by 26.9 and 35.5% (P less than 0.01), respectively, at the 10-fold higher dose. No significant suppression of growth occurred with gestrinone, ethisterone, or 2 hydroxymethyl ethisterone. The results provide further evidence that danazol and T (but not gestrinone) may act by a direct effect on endometrial tissue.

Effects of quinestrol on hepatic function in the rat.

Hepatic function in female rats given 200 microgram of quinestrol every 15 days for up to 210 days was studied. Prolonged treatment with quinestrol affected liver weight, serum cholestrol levels, biliar flux, and bromosulphthalein test, but these parameters tended toward control levels after 30 days of treatment. Quinestrol failed to affect alkaline phosphatase, oxalacetic and pyruvic transaminases and the concentration of bilirubin in serum and bile. The effects of quinestrol on hepatic function are similar to the effects of other estrogen derivatives.

Experimental study of cervical blockage induced by continuous low-dose oral progestogens.

An investigation correlating scanning electron microscopic observations with sperm penetration tests carried out on cervical mucus under the influence of low-dose continuous progestogen (Norgestrienone) is presented. The results demonstrate that such type of contraceptive is involved in drastic alterations of mid-cycle cervical mucus at the macromolecular level. The meshwork which constitutes the infrastructure of the cervical secretion appears to be greatly tightened as a result of the treatment, thus giving the woof a general appearance typical of cervical mucus in the late luteal phase. The immobilizing effect of such modified mucus on spermatozoa is demonstrated and the duration of effectiveness after the administration of a last pill on the morning of day 13 is determined.

PIP: The antifertility and obstructive effects toward spermatozoa of a continuous low-dose progestogen contraceptive are examined in this study. Scanning electron microscopy (SEM) observations were correlated with sperm penetration tests carried out on cervical mucus. 11 healthy women aged 19 to 36 who were using no oral contraceptives were studied for 2 consecutive cycles. During the 2nd cycle, the women took a single, daily low-dose progestogen pill (350 ug of Norgestrienone) from the 1st till the 13th day. Blood samples were collected for determination of plasma estrogen levels, which were found to be normal during the control cycle. The progestogen was found to dramatically alter midcycle cervical mucus at the macromolecular level. The meshwork constituting the infrastructure of cervical secretion appeared to be greatly tightened by the treatment, giving the woof a general appearance typical of cervical mucus in the late luteal phase. The mucus framework revealed a tangle of micelles, and sperms leaving the seminal pool in the vagina were faced with such a succession of exhausting physical obstacles that they were unable to penetrate the uterine cavity. The SEM data shows that rendering the midcycle cervical mucus hostile interferes with sperm transport and this probably accounts for the effectiveness of the continuous low-dose progestogen therapy.

Biochemical studies with once-a-month contraceptive pill containing quinestrol-quingestanol acetate.

PIP: Biochemical studies with a once-a-month contraceptive pill containing 2 mg quinestrol and 2.5 mg quingestanol acetate were undertaken during a period of 24 months. The 1st pill was given on the 2nd day of the menstrual cycle and the 2nd on the 22nd day of the same cycle; thereafter the pills were given every 28th day from the last pill. Blood samples were obtained before therapy and after 6, 12, 18, and 24 pills. Hemoglobin, packed cell volume, liver function tests, and serum proteins were not markedly altered. There was a significant (p less than .001) elevation in serum triglycerides, free fatty acids, phospholipids, and cholesterol after the ingestion of 6-12 pills in 17 women who were continuously followed for 2 years; however, serum triglycerides and phospholipids were less after the ingestion of 12 pills than after 6 pills. It appeared that these changes in serum lipids were similar to those reported in women taking daily combination pills. In no case did the serum lipids exceed the upper limit of normal. The drop-out rate was high after 12 pills. Th nonacceptability was of greater significance, therefore, than the metabolic effects in these trials.^ieng

Contraception with long acting subdermal implants: I. An effective and acceptable modality in international clinical trials.

This paper presents results of a double blind, multi-centered and multi-national study of two progestin only subdermal implants used for contraception. A regimen of six capsules of levonorgestrel (Ng) used by 492 women had a net cumulative 12-month pregnancy rate of 0.6 percent and a continuation rate of 74.6 percent. 498 women used six capsules of norgestrienone (R2010) and experienced a net cumulative 12-month pregnancy rate of 3.5 percent and a continuation rate of 79.4 percent. The difference in the pregnancy rate was significant at P less than 0.01, while there was no significant difference in the continuation rates. Menstrual problems were the principal reason for termination of the levonorgestrel regimen, accounting for approximately half of all terminations. There were significantly fewer menstrual problems among users of the norgestrienone (R2010) capsules; the net cumulative 12-month termination rate for this reason was 4.3 percent. Results are compared with continuation and termination rates for acceptors of the Copper T 200 at the same clinics. The low pregnancy rate and reasonably high continuation rate of the norgestrel implants coupled with the fact that the expected effective lifetime of a set of capsules is of the order of 3-5 years appears to warrant further development of this contraceptive regimen.

Long acting contraceptive implants. An analysis of menstrual bleeding patterns.

The menstrual patterns of women using subdermal implants of levonorgestrel and norgestrienone included in a double-blind clinical trial was evaluated by the method developed by the International Committee on Contraception Research. Women using Copper T200 randomly selected in the same clinics were used as controls. Both implant regimens were associated with a high frequency of reduced bleeding and norgestrel patients had also a high proportion of increased and irregular bleeding. Termination of use was associated to increased bleeding but not so much to reduced bleeding.

Fertility following termination of the once-a-month oral contraceptive.

PIP: The rate of fertility return as evidenced by conception after discontinuance, endometrial biopsies and return to menstruation was evaluated in 290 women who used the oral contraceptive quinestrol and quingestanol acetate once a month. 73% had their first menstrual flow 25-45 days after the last drug induced bleeding with a significant direct relationship of minimal influence between the duration of therapy and the time interval to menstruation. 104 of 111 women who desired a pregnancy conceived in an average of 7 months after their last treatment. There was no significant relationship between the duration of treatment, time to conceive or the autrome of the therapy. 72% of the 152 biopsies taken 14 days or less before the first regular menstruation showed a normal secretory endothelium with no correlation to duration of therapy.^ieng

The effect of progestin R 2323 released from vaginal rings on ovarian function.

PIP: The effect of progestin R 2323 released from intravaginal rings (IVR) on ovarian function was studied in 20 young healthy volunteers. The silastic IVRs containing 10, 20, 50, or 200 mg of progestin R 2323 were inserted on the 1st day after menstrual bleeding and removed 21 days after insertion. The IVRs were tolerated well without irritation or alteration of the vaginal mucosa. Control plasma estradiol and progesterone levels revealed 18 of 20 cycles were ovulatory. No ovulation was observed during treatment, as revealed by consistently suppressed progesterone levels. Normal ovulation resumed after removal of the rings. 3 women experienced withdrawal bleeding; 9 had breakthrough bleeding and 13 had a normal postovulatory bleeding 15-33 days after ring removal. It was concluded that the bleeding control was unsatisfactory with every dosage of R 2323 used in this study.^ieng