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.

Preclinical pharmacokinetic, toxicological and biomarker evaluation of SR16157, a novel dual-acting steroid sulfatase inhibitor and selective estrogen receptor modulator.

PURPOSE: SR16157 is a novel dual-acting inhibitor of estrogen action that irreversibly inhibits the estrogen biosynthetic enzyme steroid sulfatase (STS) and releases the selective estrogen receptor modulator SR16137, which blocks the estrogen receptor. SR16157 is a promising agent for the endocrine therapy of breast cancer. We conducted preclinical in vivo toxicity evaluations to determine the maximum-tolerated dose (MTD), target organ(s) of toxicity, reversibility, dose-limiting toxicity, no observable adverse effect level (NOAEL), and toxicokinetics (TK) and to investigate a potential biomarker for use in SR16157 clinical trials. METHODS: SR16157 was administered to female Fischer 344 rats or beagle dogs by oral gavage (po) or capsule. Intravenous (iv) groups were included for the determination of bioavailability. Endpoints evaluated included clinical observations, body weights, hematology, serum chemistry, pharmacokinetics, TK, pathology of tissues, and STS activity in liver, or peripheral blood mononuclear cells (PBMCs). RESULTS: For rats, the MTD (i.e., the highest dose that did not cause lethality but produced toxicity) was 33 mg/kg/day (198 mg/m(2)/day), and the NOAEL was <10 mg/kg/day (60 mg/m(2)/day). For dogs, the MTD was estimated to exceed 10 mg/kg/day (200 mg/m(2)/day), and the NOAEL was estimated to be at or above 2.5 mg/kg/day (50 mg/m(2)/day). CONCLUSIONS: Our studies demonstrate that SR16157 has excellent pharmacokinetic properties and an acceptable toxicological profile. Modulation of STS activity in PBMCs appeared to be a possible biomarker for use in future clinical trials of SR16157.

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.

A novel dual-target steroid sulfatase inhibitor and antiestrogen: SR 16157, a promising agent for the therapy of breast cancer.

Endocrine therapy is the ideal treatment choice for estrogen receptor alpha (ERalpha)-positive breast cancer patients. Principal used therapies target either the ERalpha e.g. by selective ERalpha modulators (SERMs) such as tamoxifen or target estrogen biosynthesis with aromatase inhibitors. Steroid sulfatase (STS) plays a crucial role in formation of compounds with estrogenic properties, converting inactive sulfate-conjugated steroids to active non-conjugated forms. Steroid sulfates are considered as a reservoir for active steroids due to their prolonged half-life and increased concentration in plasma. STS is present in several tissues including the breast, and the STS the mRNA level and enzyme activity is significantly increased in ERalpha-positive breast tumors. Inhibition of STS is therefore a new approach for decreasing estrogenic steroids that stimulate breast cancer. The novel dual-acting compound SR 16157 is designed as a sulfamate-containing STS inhibitor that releases a tissue-selective SERM SR 16137. Use of a dual-target STS inhibitor and SERM represents a new strategy in the treatment of hormone-dependent breast cancer. In this study, we tested the potential of SR 16157 and SR 16137 on STS activity, cell growth and ERalpha function in MCF-7 breast cancer cells. We confirmed that the dual-target compound SR 16157 exerts STS inhibition and antiestrogenic effects. SR 16157 was a highly effective growth inhibitor, being 10 times more potent than the antiestrogens SR 16137 and tamoxifen. Relative to tamoxifen, SR 16137 displays profoundly improved ERalpha binding affinity and antiestrogenic effects on expression of estrogen-regulated genes. Thus, the dual-target SR 16157 is possibly a promising new treatment alternative, superior to tamoxifen.

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.

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.

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.

Structure-activity relationships of estrogenic ligands: synthesis and evaluation of (17 alpha, 20E)- and (17 alpha, 20Z)-21-halo-19-norpregna-1,3,5(10),20-tetraene-3,17 beta-diols.

As part our program to probe the molecular requirement for estrogen-receptor binding we undertook the synthesis and evaluation of the 17 alpha,E and 17 alpha,Z halovinyl estradiols. By use of an improved variation of the existing synthetic strategy, the targeted compounds were prepared stereospecifically and in 92-98% yields from the corresponding 17 alpha,E or 17 alpha,Z [(tri-n-butylstannyl)vinyl]estradiol 3-acetates. The novel estradiol derivatives were evaluated for their relative binding affinity (RBA) for the estrogen receptor with use of a rat uterine preparation. The results demonstrated a marked difference between the E and Z isomers and among the halogen employed. The Z isomers possessed significantly higher RBA values and the larger halogens (I, Br) were more effective than the smaller Cl substituent. These results modify the previous interpretations of estrogen-receptor binding for steroidal ligands. As a result, our design of (radio)halogenated ligands will incorporate this concern for Z vs E stereochemistry.

Gestrinone in the treatment of menorrhagia.

The role of gestrinone, 2.5 mg twice weekly, in treating proven menorrhagia (greater than 80 ml) was examined in 19 women. They were treated for five cycles (2 placebo, 3 active), taking one capsule twice weekly. Placebo had no effect on menstrual blood loss (MBL). On gestrinone 10 women became amenorrhoeic, in five MBL was markedly reduced (5-74 ml) and four did not respond. In three of the non-responders submucous leiomyomas were found at subsequent hysterectomy. Follow-up periods showed a persistent reduction in MBL for nine women in the first post-treatment menstruation.

[Treatment of uterine fibroma using LH-RH analogs and gestrinone. Limits and indications]. / Traitement des fibromes utérins par les analogues du LH-RH et la gestrinone. Limites et indications.

Medical treatment of fibroma has changed radically in recent years with the introduction of analogs of LH-RH (luteinizing hormone releasing hormone). These agents, which are active only via parenteral administration, have proved remarkably effective, and are devoid of metabolic effects. They do engender some disagreeable adverse reactions, and unfortunately their efficacy is transitory and their cost high. Compared with these analogs, gestrinone, a progestogen as yet unused in treatment of luteal insufficiency, seems very interesting in the treatment of fibromas, due to its prolonged antigonadotropic effect and its antiprogesterone effect. Further studies are required to confirm the first results published by Coutinho.

A randomized double-blind prospective trial of two doses of gestrinone in the treatment of endometriosis.

The purpose of this randomized double blind prospective trial was to study the efficacy and safety of two doses of oral gestrinone in the treatment of endometriosis. Six patients received gestrinone 1.25 mg twice weekly (group I) and six patients received gestrinone 2.5 mg twice weekly (group II). Patients underwent pretreatment and post-treatment laparoscopies and their endometriosis scores were recorded. The mean total endometriosis scores declined significantly from 20.0 +/- 5.2 (mean +/- standard error of the mean) pretreatment to 9.5 +/- 3.9 post-treatment in group I and from 19.1 +/- 4.8 pretreatment to 7.1 +/- 2.1 post-treatment in group II. A total of 67% of patients reported side effects. This study suggests that oral gestrinone 1.25 mg or 2.5 mg twice weekly is efficacious and safe in the treatment of endometriosis.

Evaluation of histological and ultrastructural aspects of endometrium during treatment with gestrinone in women with amenorrhea or spotting.

Nineteen patients with a laparoscopic diagnosis of endometriosis were treated with gestrinone at a dosage of 2.5 mg twice a week for 6 months. In 7 who reported spotting in the first 3 months, the dose was increased to 2.5 mg three times a week during the second trimester. An endometrial sample was obtained from each patient at the time of laparoscopy (basal) and at 3 and 6 months of treatment. Endometrial structure and ultrastructure were studied. Areas of hemorrhage and of loss of surface epithelium and a lesser degree of involution of the surface epithelium were observed in the 3-month samples of 7 patients with spotting, compared with 12 with amenorrhea. It is hypothesized that incomplete endometrial involution could be due to differences in gestrinone pharmacokinetics in individual patients, in the quality and/or quantity of endometrial cytosolic receptors for sex steroids, or in endocrine compensation to administration of the drug.

Endocrine, metabolic, and clinical effects of gestrinone in women with endometriosis.

The effect of oral gestrinone, 2.5 mg twice weekly for 6 months, was studied in 11 women with mild or moderate endometriosis laparoscopically confirmed. The mean laparoscopic score decreased from 17.18 to 9.09 (P greater than 0.005). Painful symptoms were relieved in all patients within 2 months from start of therapy. Gonadotropins, prolactin (PRL) 17 beta-estradiol (17 beta-E2), estrone (E1), progesterone (P), androstenedione (A), and dehydroepiandrosterone sulfate (DHEA-S) remained in the follicular phase range. Total testosterone (TT) and sex hormone-binding globulin (SHBG) decreased, whereas free testosterone (FT) slightly increased. Metabolic studies showed a decrease of total triglycerides, very low-density lipoprotein (VLDL) triglycerides, and high-density lipoprotein (HDL) and VLDL cholesterol, parallel to the decrease of associated apoproteins. Low-density lipoprotein cholesterol and apoprotein B increased during therapy. The results suggest that gestrinone possesses antiestrogenic, androgenic, and progestigenic effects at therapeutic dosages both by acting on central and peripheral steroid receptors. For its efficacy and good tolerance, gestrinone may be considered an option for treating endometriosis.

Structure of (20R)-20,21-epoxy-19-norpregna-1,3,5(10)-triene-3,17 beta-diol.

C20H26O3, Mr = 314.4, orthorhombic, P2(1)2(1)2(1), a = 10.25 (1), b = 25.36 (2), c = 13.11 (1) A, V = 3410.14 A3, Z = 8, Dx = 1.225 g cm-3, lambda(Mo K alpha) = 0.71069 A, mu = 0.45 cm-1, F(000) = 1352, T = 293 K. The structure was solved by direct methods and refined to R = 0.051 for 2384 reflections with F/signa(F) greater than 5. Both the crystallographically independent molecules are in one 20R configuration and confirm predictions based on mechanistic and stereochemical arguments. In the crystal lattice the molecules form a two-dimensional network of hydrogen bonds approximately perpendicular to c.

Long-term treatment of leiomyomas with gestrinone.

A prospective randomized study was conducted in 100 women with leiomyomas in order to evaluate the effect of gestrinone, a synthetic derivative of ethynil-nor-testosterone. Patients in group A received capsules containing 2.5 mg of gestrinone three times weekly orally. Those in group B received capsules containing 5.0 mg twice weekly, also orally. In group C, patients used by vaginal route tablets containing 5 mg of gestrinone three times weekly. Reduction in uterine volume occurred in all three groups of patients. Of patients who discontinued treatment at 6 months, uterine volume remained lower than pretreatment values in 89%, 18 months after discontinuation. Of those patients who discontinued at 1 year, uterine volume remained below pretreatment levels in 76% 1 year after discontinuation. In patients treated continuously for 24 months, mean uterine volume decreased from a mean 339 cm3 to 273 cm3, a statistically significant difference. The vaginal route showed statistically more significant volume decreases than the oral route for all treatment intervals.

Gestrinone versus danazol in the treatment of endometriosis.

Thirty-nine infertile patients with laparoscopic diagnosis of endometriosis were allocated randomly to treatment with gestrinone 2.5 mg twice weekly (20 patients) or danazol 600 mg/day (19 patients) for 6 months. If amenorrhea was not obtained after 1 month of treatment, the gestrinone dose was increased to 2.5 mg three times a week (7 patients) and the danazol dose to 800 mg/day (2 patients). One month after the end of the treatment, a repeat laparoscopy was performed only in the women who agreed (7 of the gestrinone treated group, 9 of the danazol group). All of the patients were followed for at least 12 months after the end of the treatment, during which time they attempted to conceive. There was a marked improvement of pain symptoms during the treatment in the patients of both groups. The repeat laparoscopy did not reveal significant differences between the two groups in the reduction of the disease extent. Eighteen months after treatment suspension, the cumulative pregnancy rate was 33% in the patients treated with gestrinone and 40% in those treated with danazol. Pain symptoms recurred during the follow-up in 57% of the gestrinone and 53% of the danazol group. The side effects were more frequent and severe with the danazol treatment, whereas those caused by gestrinone were mostly weight gain and acne. The results of this study suggest that gestrinone is as effective as danazol in the treatment of infertility associated with endometriosis and is better tolerated.

[Effects of gestrinone on serum lipid and lipoprotein levels in women with endometriosis].

Gestrinone (G) was given to 12 females with endometriosis in weekly doses of 5 or 10mg for 4 to 6 months, and the change in serum lipids and lipoproteins was analysed. G decreased total cholesterol by 20% (p less than 0.05), triglycerides by 36% (p less than 0.05), phospholipids by 28% (p less than 0.01) and lipid peroxides by 34% (p less than 0.05), among which reductions in them were statistically significant when compared with the pretreatment levels. Levels of high density lipoproteins (HDL) also fell: HDL-cholesterol by 41% (p less than 0.01), HDL-triglycerides by 49% (p less than 0.05) and HDL-phospholipids by 38% (p less than 0.01) which were significant. Concurrently apolipoproteins (Apo) and lecithin-cholesterol acyltransferase activity (LCAT) decreased: Apo A-I by 31% (p less than 0.01), Apo A-II by 13% (p less than 0.05) and LCAT by 53% (p less than 0.05), which were significant. In contrast, there were few changes in the levels of low density lipoproteins (LDL) and Apo B. There was also little effect on very low density lipoproteins (VLDL) except VLDL-triglycerides which decreased by 52% (p less than 0.05). Meanwhile free fatty acids increased by 61% (p less than 0.05). Therefore, the atherogenic index defined as the ratio of LDL-cholesterol to HDL-cholesterol rose as much as 92% (p less than 0.01) of the initial value in 24 weeks of medication. When these results were examined with respect to the 5 and 10mg administration group, dose-dependent effects were observed, but these were not marked.(ABSTRACT TRUNCATED AT 250 WORDS)

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 rationale for endocrine therapy.

There are three major types of peritoneal endometriotic implant as distinguished by their laparoscopic and morphological characteristics; the microscopic or epithelial-type plaque, the vesicular and papular type and the fibrotic, nodular type. Fluctuations in hormone levels during the menstrual cycle and throughout hormonal therapy have differential effects on the three types of implant. After only a short period of treatment with gestrinone the active types of peritoneal endometriotic implant exhibit inactivity and involutionary changes. Regressive changes in the implants may explain the abolition of the symptoms.