17,20 beta-P and cortisol are the main in vitro metabolites of 17-hydroxy-progesterone produced by spermiating testes of Micropogonias furnieri (Desmarest, 1823) (Perciformes Sciaenidae)

The aim was to investigate the major C21 steroids produced by spermiating white croaker Micropogonias furnieri (Sciaenidae) in order to establish the potential mediator of gamete maturation in males of this species. The testes steroid production at the spawning season was identified incubating the 3H-17-hydroxy-4-pregnene-3,20-dione precursor through thin layer chromatography, high pressure liquid chromatography, enzymatic oxydation, acetylation and immunochemistry analyses. 17,20β-Dihydroxy-4-pregnen-3-one (17,20β-P) and 11β,17,21-Trihydroxy-4-pregnene-3,20-dione (cortisol) were the main metabolites produced. Contrary to what we expected, 17,20β,21-Trihydroxy-4-pregnen-3-one was not detected. Circulating levels of 17,20β-P were undetectable in immature testes and in those at the first spermatogenesis stages, while a clear increase was observed during the whole spermatogenesis and spermiation phases (from undetectable to 1047 pg mL-1). In vitro studies together with plasma detection suggest that 17,20β-P is a good steroid candidate involved in M. furnieri testes maturation. The role of cortisol during late phases of testes development needs further studies.

El objetivo fue investigar cuales eran los esteroides C21 más importantes producidos por los testículos en espermiación de la corvina blanca Micropogonias furnieri (Sciaenidae) para poder identificar los potenciales mediadores de la maduración gamética de los machos de esta especie. Los esteroides producidos por los testículos en espermiación fueron identificados después de su incubación con el precursor 3H-17-hidroxi-4-pregnene-3,20-diona a través de cromatografía de capa fina y cromatografía líquida de alta presión y posteriormente por oxidación enzimática, acetilación e inmunoquímica. Los principales metabolitos producidos por los testículos en espermiación fueron la 17,20β-Dihidroxi-4-pregnen-3-ona (17,20β-P) y la 11β,17,21-Trihidroxi-4-pregnene-3,20-diona (cortisol). Contrariamente a lo esperado, no se encontró el derivado tri-hidroxilado de la progesterona llamado 17,20β,21-Trihidroxi-4-pregnen-3-ona. Los niveles circulantes de 17,20β-P fueron no detectable en los testículos inmaduros y en aquellos en inicios de espermatogénesis, mientras que un aumento claro en las concentraciones circulantes fue detectada en corvinas en plena espermatogénesis y en espermiación (desde no detectable a 1047 pg mL-1). Los resultados obtenidos in vitrojunto a los cambios a nivel plasmático sugieren que la 17,20β-P es un buen candidato a proponer como esteroide involucrado en la regulación del proceso de maduración testicular de la corvina. La función del cortisol a nivel testicular debería ser mejor estudiada en las etapas finales del desarrollo testicular de esta especie.

Recent advances in structure of progestins and their binding to progesterone receptors.

The role of progesterone in women's cancers as well as the knowledge of the progesterone receptor (PR) structure has prompted the design of different therapies. The aim of this review is to describe the basic structure of PR agonists and antagonists as well as the recent treatments for illness associated with the progesterone receptor. The rational design for potent and effective drugs for the treatment of female cancer must consider the structural changes of the androgen and progestogen skeleton which are an indicator of their activity as progestins or antiprogestins. The presence of a hydroxyl group at C-17 in the progesterone skeleton brings about a loss of progestational activity whereas acetylation induces a progestational effect. The incorporation of an ethynyl functional group to the testosterone framework results in a loss of androgenic activity with a concomitant enhancement of the progestational effect. On the other hand, an ester function at C-3 of dehydroepiandrosterone skeleton induces partial antagonism to the PR.

Synthesis and biological evaluation of partially fluorinated antiprogestins and mesoprogestins.

A series of antiprogestins have been synthesized by partially fluorinating the steroid molecule in positions relevant for receptor binding. By introducing fluorine at the exo-methylene of the 17 spirofuran ring, we obtained partial agonists (mesoprogestins) with significant applications for antiproliferative and antiovulatory treatment strategies in gynecological therapy such as uterine fibroids, endometriosis and heavy menstrual bleeding. Compared to the standard drug RU486, our synthesized compounds exhibited considerable dissociation between antiprogestational and antiglucocorticoid PR receptors. Furthermore, our studies have shown that pure antiprogestins can be generated by partially fluorinating the 17 propenyl and propynl group or by substituting the 4' acetyl phenyl group in the 11 position using trifluromethyl group.

Progestógenos: farmacologia e uso clínico / Progestagens: pharmacology and clinical use

Os progestógenos são esteroides que podem ser sintéticos ou naturais. A progesterona é o único progestágeno natural. Os progestógenos sintéticos tentam mimetizar o efeito da progesterona, e são chamados de progestinas. Cada progestina apresenta diferentes propriedades farmacológicas, dependendo da molécula da qual foi originada, usualmente testosterona e progesterona. Pequenas mudanças estruturais nas moléculas originais levam a diferenças consideráveis na atividade de cada uma das progestinas. O objetivo deste trabalho é revisar a origem dos progestógenos, as peculiaridades de cada grupo e seu uso clínico mais comum. As informações já levantadas sobre o efeito das progestinas em patologias importantes e prevalentes, como o câncer de mama e eventos tromboembólicos, também será abordado.

Progestagens are natural or synthetic steroids, and progesterone is the only natural one. Synthetic progestagens, called progestins, were created to mimic the effects of natural progesterone. The progestins have different pharmacological properties depending on the parent molecule, usually testosterone or progesterone, from which they are derived. Very small structural changes in the original molecule may induce considerable differences in the activity of the derivative. The aim of this paper is to review the origin of each progestin, the peculiarities of each group and its most common clinical use. The current knowledge about the effect of progestins on important and prevalent diseases, such as breast cancer and thromboembolic events, will also be addressed.

Bromine- and iodine-substituted 16alpha,17alpha-dioxolane progestins for breast tumor imaging and radiotherapy: synthesis and receptor binding affinity.

Progesterone receptors (PRs) are present in many breast tumors, and their levels are increased by certain endocrine therapies. We describe the synthesis and PR binding affinities of a series of bromine- and iodine-substituted 16alpha,17alpha-dioxolane progestins, some of which, when appropriately radiolabeled, are potential agents for diagnostic imaging of PR-positive breast tumors using positron emission tomography (PET) and for radiotherapy. These compounds were synthesized from halogenated furanyl, phenyl, and thiophenyl aldehydes and a progestin 16alpha,17alpha,21-triol (5) in the presence of HClO4 or Sc(OTf)3 in high yields under optimized conditions. A new reagent, perfluoro-1-butanesulfonyl fluoride (PBSF), was used to convert the C-21 OH to F in high yields. The relative binding affinities (RBAs) of the most promising compounds for the PR (RBA of R5020 = 100) were 16alpha,17alpha-[(R)-1'-alpha-(5-bromofurylmethylidene)dioxyl]-21-hydroxy-19-norpregn-4-ene-3,20-dione (endo-6; RBA = 65 and moderate lipophilicity), 21-fluoro-16alpha,17alpha-[(R)-1'-alpha-(5-iodofurylmethylidene)dioxyl]-19-norpregn-4-ene-3,20-dione (endo-14; RBA = 40) and 21-fluoro-16alpha,17alpha-[(S)-1'-beta-(4-iodophenylmethylidene)dioxyl]-19-norpregn-4-ene-3,20-dione (exo-16; RBA = 34).

El papel de los gestágenos en los anticonceptivos hormonales orales / The role of gestagens in oral hormonal contraceptives

El papel de los gestágenos en los anticonceptivos hormonales orales está en relación directa con los efectos secundarios y el impacto sobre el metabolismo. En la última década se han desarrollado cinco nuevos gestágenos con alta potencia progestogénica y sin actividad androgénica. Es necesario desarrollar y utilizar gestágenos más selectivos para que la píldora sea usada con seguridad y confianza, para aumentar el cumplimiento y así disminuir el número de embarazos no deseados

The role of gestagens in oral hormonal contraceptives was directly related to secondary effects and metabolic impact. In the last decade five new gestagens have been developed, each presenting high progestogenic potency and absent androgenic activity. It is necesary to develop more selective gestagens to reduce secondary effects in order to get a safe and confident use of contraceptive pill, to increase compliance and to decrease the number of unwanted pregnancies

Synthesis and biological activity of 11,19-bridged progestins.

A synthetic approach to 11,19-bridged progestins is described. The key step in the synthesis is a 6-endo-trig radical cyclisation. The new progestins were tested for their biological activities in vitro and in vivo and compared to those of known progestins.

Effects of estrogens and progestogens on the renin-aldosterone system and blood pressure.

Endogenous 17 beta-estradiol (E2) and low parenteral doses of exogenous E2 are vasodilators. High dose estrogens, especially ethinylestradiol (EE) and mestranol, stimulate the synthesis of hepatic proteins including coagulation factors, sex hormone binding globulin, and angiotensinogen (Aogen). In the steady state, high plasma levels of Aogen produce only a very small increase of angiotensin II (AII) and plasma renin activity, because AII inhibits the secretion of renin and lowers plasma renin concentration. However, the increase in AII is sufficient for a slight reduction in renal blood flow and a slight increase in exchangeable sodium and blood pressure; in susceptible women, blood pressure may rise considerably. Effects of estrogens on the brain may also be involved in blood pressure changes. Endogenous progesterone is a mineralocorticoid receptor antagonist. Endogenous or exogenous progesterone leads to sodium loss and a compensatory increase in renin secretion, plasma renin activity, AII, and plasma aldosterone, e.g. in the second half of the menstrual cycle. Synthetic progestogens are commonly devoid of the mineralocorticoid receptor antagonistic effect of progesterone, and some are weak estrogen receptor agonists. Combined use of EE and synthetic progestogens may therefore enhance estrogen effects on body sodium and blood pressure. A new progestogen (Drospirenone) with an antimineralocorticoid effect like that of progesterone is described that slightly lowers body weight and blood pressure in a contraceptive formulation together with EE. An almost ideal oral contraceptive would be progestogen like Drospirenone together with a low dose natural estrogen that does not stimulate Aogen synthesis. Since most oral formulations for postmenopausal estrogen replacement also stimulate hepatic protein synthesis (including Aogen) to some extent, the transdermal route of E2 application for contraceptive purposes should also be investigated, since it has reduced potential for undesirable side effects.

Fluorine-18-labeled progestin 16 alpha, 17 alpha-dioxolanes: development of high-affinity ligands for the progesterone receptor with high in vivo target site selectivity.

We describe the synthesis and tissue biodistribution of two 21-[fluoro-18F]progestin 16 alpha, 17 alpha-furanyl ketals, potential agents for imaging progesterone receptor (PR)-positive breast tumors in humans, using positron emission tomography. 21-Fluro-16 alpha, 17 alpha-[(R)-(1'-alpha-furylmethylidene)dioxy]-19- norpregn-4-ene-3,20-dione (endo-10a) and 21-fluoro-16 alpha, 17 alpha-[(R)-(1'-alpha-furylethylidene)dioxy]-19- norpregn-4-ene-3,20-dione (endo-10b) were chosen for radiochemical synthesis from a series of seven novel progestin 16 alpha, 17 alpha-(furanyldioxolanes) on the basis of their high relative binding affinity to PR (190% and 173%, respectively, relative to R5020 = 100%), their low nonspecific binding (NSB) (log P o/w = 3.87 and 4.13, respectively), and their resulting high binding selectivity indices (BSI; i.e., the ratio of their PR binding affinity to nonspecific binding). Radiochemical synthesis of these two species in high radiochemical purity and at high effective specific activity was accomplished by treatment of the corresponding diastereomerically pure 21-trifluoromethanesulfonates with fluorine-18 anion. In tissue biodistribution studies in estrogen-primed immature female Sprague-Dawley rats, both [18F]-endo-10a and [18F]endo-10b demonstrated high PR-selective uptake in the principal target tissues, the uterus and the ovaries, and relatively low uptake in fat and bone. The metabolism at the 21-position in these progestins (as monitored by in vivo defluorination) appears to be less than that in other 21-fluoroprogestins; this may reflect steric inhibition of metabolism at this site due to the bulk of the furan-substituted dioxolane ring at the 16 alpha, 17 alpha-position. Comparison with other fluorine-18-labeled progestins shows that the PR-specific uptake in uterine tissue correlates with the BSI of the ligand and that the fat uptake correlates with the NSB of the ligand at high levels of statistical significance. These two dioxolanes may prove to be useful as breast tumor-imaging agents in humans.

Effects of D-ring substituents on antiprogestational (antagonist) and progestational (agonist) activity of 11 beta-aryl steroids.

The discovery of antiprogestational steroids by the Roussel-Uclaf group not only was a major scientific advance but also opened the way to new methods of fertility control and new therapies for such conditions as cancer. RU486, the prototype of the series, is distinguished by a p-(N,N-dimethylaminophenyl) substituent at the 11 beta- position of the steroid framework, a 4,9-dien-3-one system and 17 beta-hydroxy-17 alpha-propynyl substituents. We examined the effect of varying the 17 alpha- substituent in 17 beta-hydroxy compounds analogous to RU486, the effect of introducing a progesterone side chain at C-17, and the effects of further substitution at C-17 alpha and C-16 alpha on the activity of these latter compounds. These studies indicate an important role for D-ring substituents in determining the balance of agonist/antagonist activity in this series. For example, 17 alpha-acetoxy-17 beta-acetyl substitution gave a potent antagonist, whereas 16 alpha-ethyl-17 beta-acetyl substitution resulted in a compound with potent progestational (agonist) activity. The compounds present opportunities for further interesting and useful biological investigations.

Progestin-induced growth hormone excess in the dog originates in the mammary gland.

In the dog endogenous progesterone and synthetic progestins may incite overproduction of GH, resulting in acromegaly and insulin resistance. This progrestin-induced excessive GH secretion is characterized by disappearance of the pulsatile secretion pattern and insensitivity to both stimulation with GHRH and inhibition with a somatostatin analog. This progestin-induced GH hypersecretion is not associated with neoplastic transformation at the pituitary level. These observations were the impetus for a search of a possible extrapituitary site of GH production. In four ovariohysterectomized dogs elevated plasma GH levels (46.5 +/- 7.7 micrograms/liter; mean +/- SEM) were induced by administration of synthetic progestins. In these dogs hypophysectomy did not led to a significant decrease in plasma GH levels. Analysis of the GH content of various tissue homogenates revealed that the highest GH immunoreactivity was found in extracts of the mammary gland. Ectopic production of GH in the mammary gland was confirmed by lowering of plasma GH concentration to values within the reference range within 2 h after complete mammectomy in two dogs with progestin-induced elevations of plasma GH levels. In one of these dogs the arterial and elevations of plasma GH levels. In one of these dogs the arterial and venous GH concentrations across the mammary gland were measured and an arterio-venous GH gradient was demonstrated. Displacement studies in the RIA and analysis by reversed-phase HPLC revealed that mammary-derived GH is highly similar to pituitary-derived GH. Immuno-histochemical staining revealed that GH immunoreactivity was localized in focal areas of hyperplastic ductular epithelium. In mammary tissue of healthy untreated female dogs no GH immunoreactivity was found. It is concluded that treatment of dogs with synthetic progestins can induce the overproduction of GH in the mammary gland. This GH is biologically active, highly similar to pituitary derived GH, and originates from foci of hyperplastic ductular epithelium of the mammary gland.

Progestin 16 alpha, 17 alpha-dioxolane ketals as molecular probes for the progesterone receptor: synthesis, binding affinity, and photochemical evaluation.

Chemical probes for steroid receptors have proven useful in providing molecular details about important hormone-receptor interactions. A series of progestin 16 alpha, 17 alpha-dioxolane ketals of acetophenone or substituted acetophenones that bind to the progesterone receptor (PgR) with comparable or higher affinities than the natural ligand, progesterone, have been prepared and evaluated as potential in vitro and in vivo probes for the progesterone receptor. p-Azidoacetophenone ketal 6, the tetrafluoro analog 8, and the p-(benzoyl)acetophenone ketal 9 demonstrate the required combination of high relative binding affinity (RBA) (6 = 15%, 8 = 14%, 9 = 6.6%, progesterone = 13%, R5020 = 100%) and photoinactivation efficiency (6 = 80%, 8 = 77%, 9 = 29% at 30 min) required for potential photoaffinity labeling reagents for the PgR. The synthesis of azide 6 has been modified to accommodate a palladium-catalyzed tritium gas hydrogenolysis of an iodoaryl precursor in the final stage of the synthetic sequence; this procedure has been verified by hydrogenation. In addition, the progestin p-fluoroacetophenone ketal 10 was selected for preparation in fluorine-18-labeled form, on the basis of its high affinity for the PgR (RBA = 53%). Fluorine-18-labeled progestins may be evaluated as potential diagnostic imaging agents for PgR-positive breast tumors. The radiochemical syntheses and further biochemical results with the fluorine-18-labeled ketal 10 and the tritium-labeled aryl azide 6 will be presented in an accompanying paper and elsewhere.

Steroid hormones and the Luftwaffe. A venture into fundamental strategic research and some of its consequences: the Birch reduction becomes a birth reduction.

I recount my involvement in the development of biologically active, totally synthetic steroid hormones since 1941. The reasons for my approach to the first total synthesis of a potent androgen are given. I touch on the even more important general synthetic outcomes from ideas generated by the search for useful amounts of a cortically active hormone, such as novel angular methylations, partial hydrogenations of aromatic systems (Birch reductions), and novel usages in synthesis based on the unique structures so generated. The Birch process was critical for synthesis of the first oral contraceptives. A need to explain the structures of Birch reduction products and the experimental requirements resulted in further elucidations of the mechanism of reduction processes, notably for aromatic compounds. The first theoretical and practical distinction between structures of products as determined by a reaction rate or an equilibrium position was first exploited experimentally in the deconjugation of cholest-4-en-3-one in the final step of the first facile total synthesis of cholesterol. A knowledge gained of the biosynthesis of steroids and its specific enzymes helped to initiate my general polyketide theory of biosynthesis and also my idea of outdoing enzyme achievements with organometallic complexes ("inorganic enzyme chemistry"). I assert the high historic importance of steroids in promoting the advance of general organic chemistry within many fields.

PIP: Arthur J. Birch, chemistry professor at Australian National University, reviewed his role in developing the biologically active, totally synthetic steroid hormones beginning in 1941 after graduating from Oxford University in the UK. His supervisor asked him to serve at the Dyson Perrins Laboratory at Oxford, affiliated with ICI which was affiliated with the UK Government, to produce cortically active hormones for RAF pilots, since it was rumored that German Luftwaffe pilots used them. His research and ideas led him to develop new angular methylations, partial hydrogenations of aromatic systems (Birch reductions), and new synthesis practices based on the unique structures so generated. The Birch reductions were a key element to synthesis of the first oral contraceptives. Specifically, Gregory Pincus orally administered the synthetic 19-nor ethinyl derivatives obtained from Birch reduction and found them to be active progestagens, leading to the development of oral contraceptives. His research into revealing the structures of chemicals produced by Birch reductions and the experimental requirements brought about more clarity of the mechanism of reduction processes, especially for aromatic compounds. His first attempt to differentiate between structures of products as determined by a reaction rate or an equilibrium position was deconjugation of cholest-4-en-3-one in the last step of the first elementary total synthesis of cholesterol. The knowledge acquired from biosynthesis of steroids and of specific enzymes contributed to his general polyketide theory of biosynthesis and to his theory of outdoing enzyme achievements with organometallic complexes, which he called inorganic enzyme chemistry. Professor Birch's research has maintained the very historic significance of steroids in furthering the progress of general organic chemistry within many fields.

Fluorinated androgens and progestins: molecular probes for androgen and progesterone receptors with potential use in positron emission tomography.

In order to develop imaging agents for receptor-positive tumors of the breast and prostate, we have investigated the binding affinity of several fluorine-substituted steroids in the testosterone and nortestosterone series for the androgen receptor and the progesterone receptor. The 6 alpha- and 11 beta-fluoro-, and 16 alpha-fluoroalkyl-substituted steroids were prepared by an olefin bromofluorination reaction followed by dehydrobromination or reductive debromination. The 17 alpha-fluoromethyl derivatives were prepared by fluoride ion attack on the 17-spiroepoxide or 17-spiro sulfate and the 17 alpha-fluoropropynyl derivative, by reaction of a propargyl alcohol precursor with diethylaminosulfur trifluoride. Of the compounds synthesized, 17 alpha-(3-fluoro-I-propynyl)nortestosterone was found to possess the highest binding affinity and selectivity for the progesterone receptor, and 11 beta-fluoronordihydrotestosterone had the greatest affinity for the androgen receptor. Both receptor systems seem to tolerate reasonably well the substitution of fluorine for hydrogen.

7 alpha-Methylnorethindrone enanthate 10 beta-hydroperoxide: isolation and characterization.

10 beta-Hydroperoxy-7 alpha-methylnorethindrone 17-heptanoate (II), a product of allylic autoxidation of 7 alpha-methylnorethindrone enanthate (I), has been isolated and characterized. The synthesis of the hydroperoxide (II) from the 3-ethylene ketal of 7 alpha-methylnorethynodrel (III) was achieved. Esterification of alcohol (III), subsequent deketalization, and photochemical oxygenation resulted in the hydroperoxide (II). Reduction of the hydroperoxide (II) to the 10 beta-alcohol (VI) and acetylation of (II) to the 10 beta-acetoxyperoxide (VII) are described. A single subcutaneous injection of the compounds (II), (VI), and (VII) to rats failed to produce long term inhibition of fertility in contrast to the parent compound (I) which is at least five times more effective than norethindrone enanthate as measured by suppression of vaginal cornification and estrous cycles.

Pregna-D'-pentaranes - a new class of active gestagenes.

A new class of modified progesterones with an additional ring in the 16 alpha , 17 alpha-position (pregna-D'-pentaranes) are described. Compounds containing 4- and 6-membered D'-ring (D'4- and D'6-pentaranes) were synthesized by the cycloaddition of acetylene or 1,3-butadiene, respectively, to the conjugated 16-double bond of 16-dehydro-20-keto steroids. The D'3-pentarane was prepared by the addition of diazomethane to the steroidal olefin followed by decomposition of the intermediate 16 alpha , 17 alpha-pyrazoline. The D'5-pentarane was obtained by conventional contraction of cyclohexane D'-ring of the corresponding D'6-pentarane. Progestational and contraceptive activity has been investigated for these compounds. They were found to exhibit a high progestational activity in the McPhail assay and also to be active in the pregnancy maintenance test in ovariectomized rabbits. Some of the D'-pentaranes displayed a remarkable contraceptive effect in combination with mestranol.

Substituted pyrazolo corticoids as topical antiinflammatory agents.

The synthesis of a series of substituted pyrazolo corticoids is described. Of these 11beta,17alpha,21-trihydroxy-6,16alpha-dimethyl-4,6-pregnadieno[3,2-c]-2'-(4-pyridly)pyrazole (21) shows an excellent separation of systemic to local activity in the model animal test. Compound 21 exhibits high vasoconstriction activity in human volunteers and is clinically effective in the treatment of psoriasis.