Sigfrid Schwarz

Sulfamates of various estrogens are prodrugs with increased systemic and reduced hepatic estrogenicity at oral application

Oral therapy with natural or synthetic estrogens, like ethinylestradiol, suffers from low, suboptimally defined bioavailability and excess hepatic estrogen actions. N,N-alkylated and non-alkylated sulfamates of ethinylestradiol, estradiol and estrone overcome these deficiencies. Ovariectomized Wistar rats (n = 6-7/group) were orally treated for 7 days, and killed on day 8, plasma was gained on days 0, 4, and 8. Systemic estrogenicity was quantified by assessment of uterine weight, vaginal cornification, and measurement of gonadotropins by homologous RIA. Estrogenicity in the liver was analysed. Angiotensinogen was estimated by RIA of angiotensin-1 after incubation of EDTA-plasma with porcine renin. Total and high-density cholesterol were measured by enzymatic methods. Preliminary biotransformation studies were performed after oral administration of 10 micrograms, 5 microCi [2,4,6,7-3H]estradiol sulfamate. Ethinylestradiol led to distinct elevation of angiotensin-1 and dramatic depression of cholesterol fractions, reflecting hepatic estrogen effects, already at doses with marginal systemic effects. Estradiol and estrone had systemic and hepatic estrogenic activity at much higher doses only. Estrogen sulfamates had systemic estrogen activity 10-90-fold above that of their parent estrogen. Non-alkylated sulfamates of given estrogens were more active than N-alkylated ones. Elevation of systemic estrogen activity was always combined with a dramatic reduction of hepatic estrogenicity. Estradiol sulfamate had a 90-fold elevated systemic estrogen activity vs estradiol, but lacked hepatic activity including the 30-fold dose inducing vaginal response. Three hours after administration no unchanged estradiol sulfamate was detectable in plasma. Rather peaks, probably representing estradiol and estrone, were found. Estrogen sulfamates are considered prodrugs of their parent estrogen, which do not interact with any liver function during the first-pass. They represent a new strategy of oral hormone administration. Their main potential seems to be the systemic generation of natural estrogens when used in oral contraceptives.

Novel “scavestrogens” and their radical scavenging effects, iron-chelating, and total antioxidative activities: Δ8,9-dehydro derivatives of 17α-estradiol and 17β-estradiol

Abstract Antioxidant effects of Δ8,9-dehydro derivatives of 17α-estradiol and 17β-estradiol were investigated using four different in vitro models: rat synaptosomal lipid peroxidation induced by Fentons reagent, Fe(II)-chelating activities, the formation of superoxide anion radicals, and total antioxidative activities. The parent molecules, 17α-estradiol and 17β-estradiol as well as estrone and estriol inhibit iron-dependent lipid peroxidation and stimulate total antioxidant activity. In contrast, Δ8,9-dehydro estrogens such as J811, J861, J835, or J851 not only exhibit the antioxidative activities as the parent molecules do but also directly alter the iron redox chemistry and drastically inhibit the formation of superoxide anion radicals generated by a xanthine/xanthine oxidase-dependent luminescence reactions. These in vitro findings indicate that 17α-estradiol as well as 17β-estradiol, modified with an additional double bond in the basic structure, trigger more potent antioxidant properties. These results suggest that relatively minor modifications in the chemical structure of estrogenic compounds can enhance antioxidative actions.

Novel estrogens and their radical scavenging effects, iron-chelating, and total antioxidative activities: 17α-substituted analogs of Δ9(11)-dehydro-17β-estradiol

Abstract Antioxidant effects of N,N-dimethyl-p-toluidine, p-cresol, and p-(hydroxy)thioanisol 17α-substituted analogs of 17β-estradiol and their Δ9(11)-dehydro homologs were investigated using four different in vitro models: rat synaptosomal lipid peroxidation induced by Fentons reagent, Fe(II)-chelating activities, the formation of superoxide anion radicals, and total antioxidative activity. Whereas the classical estrogen 17β-estradiol as well as selected phenolic compounds was only moderately inhibiting iron-dependent lipid peroxidation and stimulating total antioxidative activity, besides Δ9(11)-dehydro-17β-estradiol (J 1213), novel estrogens such as C-17-oriented side chain analogs of 17β-estradiol (J 843, J 872, and J 897) and Δ9(11)-dehydro homologs (J 844, J 864, and J 898) directly altered the iron redox chemistry and diminished the formation of superoxide anion radicals generated by a xanthine/xanthine oxidase-dependent luminescence reaction to a great extent. These results suggest that definite modifications in the chemical structure of 17β-estradiol, e.g., the introduction of a Δ9(11)-double bond and/or p-cresol as well as p-(hydroxy)thioanisol C-17 substitution, may result in substantial changes in their antioxidant behavior. These compounds may be drug candidates for treating pathologies related to free radical formation.

Synthesis of estrogen sulfamates: Compounds with a novel endocrinological profile

Estrogen sulfamates are promising hormones by oral administration. Therefore, generally applicable and convenient methods for the multigram synthesis of these derivatives are desirable. Numerous estra-1,3,5(10)-trienes derived from estrone, estradiol. 14 alpha,15 alpha-methylenestradiol, ethinylestradiol, and estriol have been esterified with sulfamoyl chloride and N-methylsulfamoyl chloride by a novel approach involving the use of 2,6-di-tert-butylpyridines as bases and chemoselective hydroxy group protections. These pathways circumvent the nonselective formation of esters and side reactions by in situ generated azasulfenes. For toxicological and clinical studies a new synthesis of estrone sulfamate on a 100-g scale was developed using dimethylformamide as the solvent and base.

NOVEL OESTROGEN SULFAMATES : A NEW APPROACH TO ORAL HORMONE THERAPY

Sulfamate substitution at the C-3 position of the steroid skeleton leads to orally active prodrugs of ethinyloestradiol, oestradiol, oestrone, oestriol and probably many other natural or synthetic steroidal oestrogens. These compounds have higher systemic, but reduced hepatic, oestrogenic activity than their parent steroids. The release of the parent oestrogen results from the passage of the sulfamates in the erythrocyte compartment through the liver and the systemic hydrolysis of the sulfamate ester. Sulfamates may be used to generate natural oestrogens, oestradiol and other oestrogens for different types of oral oestrogen therapy, e.g., as oestriol for hindered oestrogen or as 17-alpha oestradiol for non-feminising oestrogen. Oestradiol sulfamate J995 is currently in the initial stages of clinical testing. It has per se no oestrogen receptor affinity or oestrogenic activity in vitro, i.e., is inactive without hydrolysis. Its oral uterotropic activity in rats is approximately 100-times higher than that of oestradiol, its hepatotropic activity, however, is only 2- to 3-fold higher than oestradiol. Oral oestradiol sulfamate treatment should, therefore, reduce the hepatic side-effects seen with conventional oral hormone therapy, while at the same time lead to full stimulation of the uterus and the vagina, and suppression of gonadotropin. In the rat, orally administered oestradiol is effectively extracted from the portal vein and excreted via the bile. In contrast, oestradiol sulfamate is present in the circulation at high concentrations, up to 30% of the dose. Initially, 98% of this is found in the erythrocyte compartment and only 2% in the plasma. Significant oestradiol levels in the blood are recorded during the depletion of this pool. Pharmacokinetic and toxicological studies reveal complete excretion of the compound and its metabolites. No toxic effects have been seen in rats and cynomolgus monkeys at any dose, in spite of the compounds distinct oestrogenicity. The evaluation of genotoxicity also yielded negative results.

Effects of estradiol and estradiol sulfamate on the uterus of ovariectomized or ovariectomized and hypophysectomized rats

Estradiol sulfamate (J995), estradiol-17beta with a substituted sulfamate group in position 3, has much higher systemic estrogenic activity after oral administration than 17beta-estradiol (E2) due to reduced hepatic metabolism of the drug. The lower dose necessary for achievement of adequate systemic estrogenic effects results in a substantial reduction of otherwise commonly observed hepatic side-effects. This makes J995 a strong candidate as an estrogen suitable for oral administration. The present study was performed to examine and compare the effects of J995 and E2 on the uterus after oral or subcutaneous administration to ovariectomized or ovariectomized+hypophysectomized female rats, in particular on the levels of the estrogen receptor (ER) (alpha+beta), ERalpha mRNA and insulin-like growth factor-I (IGF-I) mRNA. The ER levels were determined using a ligand binding assay and the mRNA levels using solution hybridization. The doses of J995 or E2 were chosen to achieve comparable uterotrophic activity. The rats were treated with hormones for 7 days and the treatment was initiated 14 days after surgery. We conclude that there are no major differences in the uterine response to treatment with J995 or E2 with respect to the effects on ER and ERalpha mRNA levels. The IGF-I mRNA level though, is more affected by J995 than by E2 after 7 days of treatment, indicating a prolonged effect of J995.

Influence of subchronic administration of oestradiol, ethinyloestradiol and oestradiol sulphamate on bile flow, bile acid excretion, and liver and biliary glutathione status in rats.

Abstract The cholestatic effect of the newly developed oestradiol sulphamate (J995) was compared with that of the natural oestrogen oestradiol (E) and of the cholestatic standard oestrogen ethinyloestradiol (EE). Female ovariectomized rats were orally treated for 7 days with oestrogen doses molar equivalent to 0.01, 0.1, 1, and 10 mg E/kg body wt. Bile flow, biliary bile acid and glutathione excretion as well as liver glutathione status were determined after bile duct cannulation under the influence of ketamine anaesthesia. For systemic oestrogen activity, the increase of uterine weight was measured. J995 showed the highest oestrogenic activity followed by EE and E. Impairment of bile flow and biliary glutathione excretion (GSH, GSSG) were found to be in the order Eu2009<u2009J995u2009<u2009EE. As all three oestrogens did not influence bile acid excretion, we postulate that mainly the bile acid-independent fraction of bile flow was inhibited, caused at least partly by impairment of canalicular glutathione transport. Based on the results of these studies, we conclude that a tenfold higher dose of J995 exhibited the same cholestatic effect as EE. Together with higher systemic oestrogenic activity, J995 may be used as a prodrug with reduced hepatic side-effects to replace EE in contraception strategies.

17β-Hydroxy-11α-(3'-sulfanylpropyl)oxy-estra-1,3,5(10)-trien-3-yl sulfamate : A novel hapten structure : Toward the development of a specific enzyme immunoassay (EIA) for estra-1,3,5(10)-triene-3-yl sulfamates

The title compound 17 has been synthesized for the use as hapten in the development of a competitive enzyme immunoassay for estrogen sulfamates. The synthesis started from estradiol diacetate 2. Oxyfunctionalization at C-11 to give 11alpha-hydroxy steroid 8 was accomplished by hydroboration/alkaline hydrogen peroxide oxidation of the 9(11)-dehydro derivative 7, which was obtained from compound 2 via 9-hydroxylation with dimethyldioxirane. After transformation of compound 8 into the allyl ether 9, the side chain was thio-functionalized at the omega-position affording the thioate 11 in two steps. Selective silylether deprotection at position 3 followed by sulfamoylation gave the sulfamate 19, which in turn was demasked at position 17 and treated with sodium borohydride/aluminum chloride to liberate the side chain thiol. Alternatively, title compound 17 was synthesized via the disulfides 13-16. For the preparation of the immunogen the title compound 17 was coupled to bovine gamma globulin in a two-step procedure using an amine and thiol specific bifunctional crosslinker. The immunization of rabbits resulted in the formation of antibodies which clearly discriminated the sulfamoylated estrogens from the non-esterified estrogens. The use of a biotinylated hapten derivative as a tracer in combination with a streptavidin-peroxidase-tetramethylbenzidine based detection system allowed the measurement of estradiol 3-sulfamate (1) in the range of about 1 to 1000 pg/well.

Synthesis and conformation of four 16,17-diols in the 3-methoxy-13α-estra-1,3,5(10)-triene series

All four diasteromeric 16,17-diols in the 3-methoxy-13alpha-estra-1,3,5(10)-triene series have been synthesized. The trans-diols 1 and 2 can be obtained by hydroborating the 17-enol acetate 6 (61%, ratio 27:73, preferred alpha attack). OsO(4) dihydroxylation of the olefin 7 yielded the cis-diols 3 and 4 (ratio 13:87). The dihydroxylation proceeds with preference for beta attack caused by a C-ring twist-boat form of 7. The conformations of the diols 2 and 4, the 17-benzyl-17-hydroxy compounds 9 and 10 (obtained by Grignard reaction), and the 16alpha-bromo-17beta-hydroxy compound 8 were determined by X-ray analysis and by 1H NMR spectroscopy in solution. Some compounds, in spite of a 17beta-hydroxy group, had a conformation with a ring C chair form (4, 8, 9) caused by intermolecular interaction in the solid state. The rest of the compounds studied here (2, 10) possessed a conformation with a ring C twist-boat form, which has been also found for all 17beta-substituted compounds in solution. The preferred conformation of the D-ring with 17beta-substituents seems to be the 16beta-envelope form or near this form, but the existence of the 16alpha-envelope form (inversion of the ring D) for some compounds showed great variance in the conformation of ring D, which is substituent dependent.

The remarkable influence of steroid A/B-ring junction on the Wittig olefination reaction of the 11-oxo group : Towards the synthesis of 5α- and 5β-oriented Δ3-isomers of desogestrel

Abstract The 5α- and 5β-oriented Δ 3 -double bond isomers 8, 9 of the widely used progestin desogestrel (7) were synthesized. Wittig olefination reaction of the 5α-intermediate 12 showed a dramatically reduced reaction rate compared with the olefination of the 5β-intermediate 13 . Computational studies suggest that different energies of the intermediary 1,2-oxaphosphetanes may, at least partially, have been the reason for this phenomenon.