Synthesis of potential antiprogestogens.

Acylated derivatives of 17 alpha-hydroxyprogesterone were prepared in order to test the hypothesis that dialkylamino alkyl moieties have the effect of transforming progestogens into antiprogestogens. This approach has been successful with certain estrogens. Compounds with other functional groups were synthesized to determine whether these might exert binding influence outside the area occupied by progesterone itself. The compounds were tested for competitive affinity against tritiated progesterone and receptor from rabbit uterus cytosol. The low affinity of all derivatives makes it unlikely that they would be active as antiprogestational agents.     

3- and 19-oximes of 16alpha,17alpha-cyclohexanoprogesterone derivatives: synthesis and interactions with progesterone receptor and other proteins

Series of 3- and 19-oximes of 16alpha,17alpha-cyclohexanoprogesterone derivatives (pregna-d'-pentaranes) have been synthesized with the aim of probing the surfaces of progesterone receptor's and two other protein ligand binding pockets neighboring to 3- and 19-positions of steroid core. The same derivatives were also studied as possible intermediates for attachment to matrixes. The data on affinity constants suggest the presence of hydrophobic cavities with hydrophilic necks in the progesterone receptor and serum pentaranophylin near C19 of bound ligand and the lack of such a cavity in uterine pentaranophylin. Any of 3-oxime substitutions were found to significantly diminish the ligand affinity for the progesterone receptor. It was also found that some of these modifications, in the Z-configuration particularly, might increase the affinity for serum and uterine pentaranophylins. The latter finding suggests the presence of large cavities near C3 of bound ligand in these proteins and interchangeability between 3-keto and 3-oxime groups in ligand-protein interactions.     

Species and tissue distribution specificity of proteins binding 16alpha,17alpha-cycloalkane derivatives of progesterone

The binding of [3H]progesterone and [3H] 16 alpha,17 alpha-cycloalkanoprogesterones to proteins from rat, rabbit, and human uteri and other organs was studied. We found that 16 alpha,17 alpha-cycloalkanoprogesterone derivatives display affinities for the uterine progesterone receptors comparable with that of the natural hormone and no substantial species differences in the affinity. Rabbit uterus was found to have no proteins distinct from the progesterone receptor that specifically bind [3H] 16 alpha,17 alpha-cycloalkanoprogesterones. At the same time, in the human uterus, we found another protein that binds some of these progesterone derivatives; it turned out to be similar to the protein from rat uterus. A similar protein with the same selectivity and affinity for steroids was also found in rat and human kidneys. Blood serum, liver, lung, and a number of other tissues were found to contain a protein of the third type that binds the same 16 alpha,17 alpha-cycloalkanoprogesterones and exhibits submicromolar Kd values for these steroids and a very low affinity for progesterone. We speculated that the introduction of a bulky substituent adjacently to the 17 beta-side chain of progesterone could result in a change in the general biodynamics of the derivative including its transport, uptake, and accumulation in tissues, which may determine the selectivity of its effect.     

The size and/or configuration of the cycloalkane D' ring in pentacyclic progesterone derivatives are crucial for their high-affinity binding to a protein in addition to progesterone receptor in rat uterine cytosol

[(3)H]labeled progesterone and a number of its 16alpha, 17alpha-cycloalkano derivatives with an additional three to six-membered D' ring were investigated for mutual competition and equilibrium binding to proteins from rat uterine cytosol. The interaction of all studied [(3)H]ligands with proteins was characterized by comparable affinity (K(d) in nM region) and apparent homogeneity in terms of affinity. At the same time, the concentrations of binding sites for ligands bearing 16alpha,17alpha cyclopentano, cyclohexano, or cyclohexeno substituents were several-fold higher than those for progesterone or 16alpha, 17alpha-cyclopropanoprogesterone. In mutual competition experiments, when [(3)H]progesterone or [(3)H]16alpha, 17alpha-cyclopropanoprogesterone were used, the curves of 'bound radioactivity-log of competitor concentration' for all compounds studied were parallel and corresponded to a model of 'one protein-two ligands.' However, when [(3)H]ligands with bulky 16alpha, 17alpha-substituents (with the possible exception of cyclohexene derivative) were used, competitive curves for various ligands had different appearances and fell into two groups. Parallel curves for derivatives with 5 or 6 carbons in D' ring described by a model of 'one protein-two ligands' formed the 1st group. The 2nd group comprised curves for progesterone or 16alpha, 17alpha-cyclopropanoprogesterone that had lower slopes and could be described by a model of 'two proteins-two ligands.' Taken together, the results suggest the presence in rat uterine cytosol, of a protein in addition to progesterone receptor capable of discriminating between ligands with no or small 16alpha, 17alpha-cycloalkano substituents and ligands with more bulky substituents.     

Nitrile hydratase from Rhodococcus erythropolis: metabolization of steroidal compounds with a nitrile group.

The progestin dienogest (17alpha-cyanomethyl-17beta-hydroxy-estra-4,9-dien-3-one) was metabolized by the nitrile hydratase-containing microorganism Rhodococcus erythropolis. An enzymatic hydrolysis of the nitrile group at the 17alpha-side chain was intended to obtain novel derivatives and to test them for progesterone receptor affinity. In contrast to the rapid enzymatic hydrolysis of nonsteroidal nitriles, the nitrile group of dienogest was cleaved very slowly. The dominant reaction was an aromatization of ring A. After prolonged fermentation, the 17alpha-acetamido derivatives of estradiol and of 9(11)-dehydroestradiol were formed. Three of the metabolites were also prepared synthetically. They were tested for hormonal activity by assessing their binding to progesterone and estrogen receptors in vitro. Neither the aromatized 17alpha-acetamido derivatives nor the dienogest derivative 17alpha-acetamido-17beta-hydroxy-estra-4,9-dien-3-one, which was prepared synthetically only, exhibited affinity for the progesterone receptor.     

Nonsteroidal progesterone receptor ligands (II); synthesis and SAR of new tetrahydrobenzindolone derivatives

The human progesterone receptor (PR) binding affinity and the PR agonistic or antagonistic potency of tetrahydronaphthofuranone derivatives were shown previously to be markedly influenced by substituents at the 6- and 7-positions. Here, we synthesized tetrahydrobenzindolones possessing a lactam ring, which enabled us to modify the 6- and 7-positions more freely, since tetrahydrobenzindolones are chemically more stable than tetrahydronaphthofuranones. The tetrahydrobenzindolone derivatives generally showed higher PR binding affinity than the corresponding tetrahydronaphthofuranones. We also succeeded in separating the agonistic and antagonistic activities by choosing suitable substituent groups at the 6- and/or 7-position(s) of the tetrahydrobenzindolone. The effects of representative agonists, 12c (CP8668), and 14a (CP8816), and a representative antagonist, 15f (CP8661), were confirmed in in vivo tests. In this report, we mainly describe the synthesis and structure-activity relationships (SAR) of tetrahydrobenzindolone derivatives, as new nonsteroidal PR ligands.     

Synthesis of novel progestin-rhenium conjugates as potential ligands for the progesterone receptor.

To assist in the development of technetium-based radiopharmaceuticals that are useful for the diagnostic imaging of steroid receptor-positive breast tumors, we have synthesized a series of small-sized metal chelates according to 'n + 1' mixed-ligand, thioether-carbonyl and organometallic designs. In these preliminary investigations, rhenium was used as a model for the radioactive technetium. The metal chelates contain the rhenium metal in several oxidation states, being + 5, + 3, and + 1, and they were attached to 21-substituted progesterone derivatives. A competitive receptor-binding assay (rat uterine cytosol, 0 degrees C) was used to determine the binding affinity of these conjugates for the progesterone receptor. The highest affinity of 9% (RU5020 = 100%) was obtained with a '3 + 1' mixed-ligand complex, containing a NMe group as the central donor atom in the tridentate ligand part. This value reflects a relative binding affinity of 75% compared with the parent steroid progesterone.     

Preparation of dehydroepiandrosterone,testosterone and progesterone antigens through 7-carboxymethyl derivatives: Characteristics of the antisera to testosterone and progesterone

Dehydroepiandrosterone, testosterone and progesterone 7-carboxymethyl derivatives were prepared : 7α and 7β epimers were separated and coupled to bovine serum albumin. Preliminary studies of the antisera induced by these antigens showed that they have high affinity and good specificity.     

Steroid metabolism as a mechanism of escape from progesterone-mediated growth inhibition in Trichophyton mentagrophytes

It has been shown by us and others that progesterone inhibits the growth of Trichophyton mentagrophytes and that the organism escapes from this inhibition over time. We report here studies which show that escape from growth inhibition is related to the enzymatic transformation of progesterone to polar metabolites. Isolation and identification of the progesterone metabolites confirm the production of 15 alpha-hydroxyprogesterone. In addition, three other metabolites were isolated. Two of these were determined to be 1-dehydroprogesterone and 11 alpha-hydroxyprogesterone. The third metabolite was a 1-dehydro-hydroxyprogesterone, but the location of the hydroxyl group could not be determined unequivocally. Studies using authentic 15 alpha-hydroxyprogesterone, 1-dehydroprogesterone, and 11 alpha-hydroxyprogesterone reveal that these derivatives are significantly less inhibitory to the growth of T. mentagrophytes than progesterone. Pretreatment of organisms with progesterone augments the rate of metabolism and enhances escape. We have described previously a progesterone-binding protein (PBP) in cytoplasmic extracts of T. mentagrophytes and hypothesized that progesterone mediates growth inhibition by binding to the PBP of this organism. The relative binding affinity that progesterone and its metabolites display for PBP correlates with the relative growth inhibitory potency of these compounds. These results suggest that metabolism of progesterone to more polar and less inhibitory compounds, which exhibit lower affinity for PBP, is the mechanism of escape from progesterone-mediated inhibition of growth in this organism.     

3-O-methoxyimino group inhibits interactions between progestins and blood transcortin

The interactions between E- and Z-isomers of 3-O-methoxyimino-pregn-4-ene-20-one and its 17α-hydroxy derivative and transcortin from human blood were investigated. The substitution of the progesterone 3-oxo group for a 3-O-methoxyimino group was shown to diminish the affinity of the steroid for transcortin by approximately one order of magnitude irrespective of the substituent’s orientation. The data suggests that progesterone derivatives substituted thereby must have higher bioavailability compared to progesterone and must not significantly affect the biodynamics of glucocorticoid in vivo.     

Approaches to the design of selective ligands for membrane progesterone receptor alpha

A number of progesterone derivatives were assayed in terms of their affinity for recombinant human membrane progesterone receptor alpha (mPRα) in comparison with nuclear progesterone receptor (nPR). The 16α,17α-cycloalkane group diminished an affinity of steroids for mPRα without significant influence on affinity for nPR, thus rendering a prominent selectivity of ligands for nPR. On the contrary, substitution of methyl at C10 for ethyl or methoxy group moderately increased the affinity for mPRα and significantly lowered the affinity for nPR. A similar but even more prominent effect was observed upon substitution of the 3-oxo group for the 3-O-methoxyimino group. A significant preference towards mPRα was also rendered by the 17α-hydroxy group and additional C6–C7-double bond. The data suggest that the modes of lig- and interaction with mPRα and nPR in the C3 region of the steroid molecule are different. One can speculate that combination of the above substitutions at C17, C10, C6, and C3 may give ligand(s) with high specificity towards mPRα over nPR.     

Luteal luteinizing hormone receptors during the postovulatory period in the mare

Changes in serum luteinizing hormone (LH) and progesterone concentrations, number of luteal unoccupied LH receptors, receptor affinity constants, luteal weights and luteal progesterone concentrations were determined during the postovulatory period in the mare. The number of unoccupied LH receptors and receptor affinity was less during the early (Days 1-4) and late [Day 15 through 3rd day after start of corpus luteum (CL) regression] luteal phases than during the mid-luteal (Days 9-14) phase of the postovulatory period (P less than 0.01). The number of LH receptors per CL increased 21-fold (P less than 0.001) from Day 1 to Day 14. Receptor affinity increased 5-fold (P less than 0.001) from Day 1 to Day 13. Receptor number was highly correlated with receptor affinity (P less than 0.01) and both were highly correlated with serum and luteal progesterone (P less than 0.01). During regression of the CL, the number of LH receptors and receptor affinity decreased concomitantly with serum and luteal progesterone. Morphologically, luteal cell development and degeneration correlated with the change in receptor numbers, affinity constants and luteal and serum progesterone concentrations. Receptor number and affinity, luteal weight and serum and luteal progesterone concentrations did not differ between the CL from multiple ovulations. Random variations in the data observed between CL from multiple and single ovulations suggested that CL from the two groups were not different in structure and function. In summary, the above results suggest that major factors in regulation of progesterone secretion and maintenance of the equine CL are changes in the number of LH receptors and the affinity constants throughout the postovulatory period.     

Analogies and differences in the modulation of progesterone receptor induction and cell proliferation by estrogens and antiestrogens in MCF-7 human breast cancer cells: study with 24 triphenylacrylonitrile derivatives

Structure-activity relationships in a homogeneous series of 24 triphenylacrylonitrile derivatives were examined with respect to the stimulation of progesterone receptor induction and cell proliferation in MCF-7 cells. In general, triphenylacrylonitrile derivatives were found to be full or partial agonists for both responses; the partial agonists were also able to antagonize the stimulatory action of estradiol. The agonistic activities of these molecules decreased as the size of the lateral side chain increased, but the side-chains correlated with partial agonism of progesterone receptor induction were bulkier than those correlated with partial agonism of cell proliferation. Agonistic and antagonistic effects on both responses were correlated with affinity for the estrogen receptor. Half maximal effects on the two responses occurred at different concentrations (4-fold) of the compounds. It can be concluded that in MCF-7 cells, triphenylacrylonitrile modulation of progesterone receptor induction and cell proliferation are mediated by the estrogen receptor; the two effects, which occur at different concentrations and with slightly different substituents of the compounds, are differentially modulated.     

Structural features of pregna-D'-pentaranes determining their interaction with three rat proteins

Competition of a number of progesterone 16alpha,17alpha-cycloalkane derivatives with 3H-labeled ligands for the binding sites of the rat uterine progesterone receptor, uterine pentaranophilin, and blood serum pentaranophilin was studied. We found that the selective ligands for the progesterone receptor are progesterone, 16alpha,17alpha-cyclopropanoprogesterone, and 16alpha,17alpha-cyclopent-3'-enoprogesterone and the selective ligands for serum pentaranophilin are 6alpha-methyl-16alpha,17alpha-cyclohexanopregna-1,4-diene-3,20-dione and 3beta-hydroxy-16alpha,17alpha-cyclohexanopregn-5-en-20-one. No selective ligands for the uterine pentaranophilin were found. The majority of substituents in rings A, B, and D' we studied decreased the affinity of ligands for all the three proteins. The substitution of the delta5-3beta-hydroxy grouping for the delta4-3-keto grouping exerted the strongest negative effect in the case of the progesterone receptor and the uterine pentaranophilin, whereas the introduction of the 3',4'-dimethyl grouping strongly inhibited the ligand affinity for the uterine pentaranophilin. The extent and even the direction of the effect of a substituent on the affinity of ligands for the proteins substantially depended on the presence of other substituents in the steroid molecules. We hypothesized that a certain similarity exists between three proteins studied in respect to the structures of their ligand-binding pockets. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2004, vol. 30, no. 3; see also http://www.maik.ru.     

Affinities of progestogen and estrogen receptors in rabbit uterus for synthetic progestogens

The affinities of progestogen and estrogen receptors of rabbit uterus for a number of synthetic progestogens in clinical use and some analogues have been measured. Progesterone, 17-hydroxyprogesterone caproate, the chlormadinone, megestrol and cyproterone acetates had similar affinities for the progestogen receptor. Medroxyprogesterone acetate and particularly DU-41164 possessed much higher affinities. 17-hydroxyprogesterone or other agents with a free 17-hydroxyl had much lower receptor affinities than the corresponding 17-esters.Of the nortestosterone derivatives tested, norethisterone equalled progesterone in affinity for the progestogen receptor while d-norgestrel and Wy-4355 were more active. Norethynodrel and ethynodiol diacetate had much lower receptor affinities than progesterone. These results are discussed in relation to possible metabolic bioactivation.Only norethynodrel and ethynodiol (free alcohol) showed marked affinity for the estrogen receptor.     

The significance of the 20-carbonyl group of progesterone in steroid receptor binding: a molecular dynamics and structure-based ligand design study

Polar functional groups in the A- and D-ring (positions 3 and 17beta or 20) are common to all natural and synthetic steroid hormones. It was assumed that these pharmacophoric groups are involved in strong hydrogen bonding interactions with the respective steroid receptors. High resolution X-ray structures of the estrogen and androgen receptors have confirmed these assumptions. Also site-directed mutagenesis studies of the human progesterone receptor (hPR) suggest an important role for Cys891 in the recognition of the progesterone 20-carbonyl group. Surprisingly, the crystal structure of the hPR ligand binding domain (LBD) in complex with progesterone suggests that the carbonyl oxygen in position 20 (O20) is not involved in hydrogen bond contacts. To investigate these surprising and contradicting results further, we performed a molecular dynamics simulation of the hPR-progesterone complex in an aqueous environment. The simulation revealed hPR-Cys891 as the sole but weak hydrogen bonding partner of progesterone in the D-ring. In contrast to the site-directed mutagenesis data a major role of hPR-Cys891 in progesterone recognition could not be confirmed. Isolated hydrogen bond acceptors, such as the prosterone O20 group, in a relatively lipophilic environment of the receptor led to a decrease in affinity of the ligand. Based on this consideration and the structure of the PR, we designed compounds lacking such an acceptor function. If the X-ray structure and the calculations were right, these compounds should bind with comparable or higher affinity versus that of progesterone. E-17-Halomethylene steroids were synthesized and pharmacologically characterized in vitro and in vivo. Although the compounds are unable to form hydrogen bonds with the hPR in the D-ring region, they bind with superior affinity and exert stronger in vivo progestational effects than progesterone itself. Our investigations have confirmed the results of the X-ray structure and disproved the old pharmacophore model for progestogenic activity, comprising two essential polar functional groups on both ends of the steroid core. The 20-carbonyl group of progesterone is likely to play a role beyond PR-binding, e.g. in the context of other functions via the androgen and mineralocorticoid receptors and as a site of metabolic inactivation.     

Affinity labelling of human transcortin

The binding site of transcortin has been studied by using bromoacetyltestosterone and bromoacetylated derivatives of progesterone which were monohydroxylated at different positions of the steroid nucleus. Specificity of affinity labelling was demonstrated by the displad cortisol analog was added to a [3H]cortisol-transcortin complex solution. The binding site crevice was found to be very narrow in the vicinity of the A and B rings of steroid since 2alpha-hydroxyprogesterone, 6alpha- or 6beta-bromoacetoxyprogesterone and dexamethasone could not displace bound cortisol. A specific affinity labelling was obtained with 11alpha-bromoacetoxyprogesterone, 16alpha-bromoacetoxyprogesterone and 17beta-bromoacetyltestosterone. The results of the affinity labelling by these hormone analogs suggested that one methionine and one histidine residues were located within the active site:methionine might interact with the 11beta-hydroxyl group and histidine with the 20 keto group of cortisol.     

A progesterone receptor affinity chromatography reagent: 17α-Hexynyl nortestosterone sepharose

Several affinity chromatography reagents have been proposed for purification of progesterone receptor (PgR), and significant results have been achieved with some of these. None, however, have approached the results achieved in affinity chromatography of estrogen receptor. We have therefore synthesized a number of new 19-nortestosterone derivatives capable of chemically stable linkage with Sepharose beads, and have identified one with very high PgR affinity for further study. We first synthesized the epoxides of 17α-allyl nortestosterone, by analogy with the estradiol derivatization of Greene and Jensen. The relative affinity of these epoxides for PgR from T47D human breast cancer cells, however, was only around 5% that of R5020, and affinity beads prepared from them bound very little PgR. We then reacted appropriately protected 17α-ethynyl-nortestosterone with a series of diiodo alkanes, and found that 17α-(6'-iodohex-1'-ynyl)nortestosterone had an affinity of 22% relative to R5020, equal to the affinity of progesterone itself. Reaction with Thiopropyl-Sepharose 6B yielded hexynyl-nortestosterone-Sepharose beads with a ligand density of about 7 micromoles/ml beads. One-hundred μl of these beads adsorbed 71% of the PgR present in 1 ml ofcytosol from T47D cells. This adsorption was inhibited by 10 μM progesterone but not Cortisol, indicating the specificity of the binding. Comparisions with NADAC and Sterogel, other affinity beads used for PgR purification, show that the former takes up much less receptor, while the latter takes up and releases similar amounts of receptor but more extraneous protein, and is less stable. We therefore believe that hexynyl-nortestosterone-Sepharose, having a high density of a high affinity ligand, and having chemically and biochemically stable covalent bonds, should be a good reagent for affinity purification of PgR.     

Binding of a contraceptive progestogen ORG 2969 and its metabolites to receptor proteins and human sex hormone binding globulin

Org 2969 is an orally active progestogen which can be used in oral contraceptives because of its strong ovulation-inhibiting activity and acceptability. The present study examines tha binding of Org 2969, its metabolites and reference coupounds to the progesterone and estrogen receptors in human and rabbit myometrium, the rat prostate androgen receptor and human SHBG (sex hormone binding globulin). At 4 degrees Centigrade, the affinite of the major metabolite 3-keto-Org 2969 was similar to that of levonorgestrel and 6 and 2 times higher than that of progesterone and norethisterone, respectively. At 30 degrees, the binding affinity of 3-keto-Org 2969 was twice that of levonorgestrel and 25 times that of progesterone. Other metabolites and derivatives tested displayed low but measurable affinities under equilibrium and non-equilibrium conditions. The binding characteristics of Org 2969 and its metabolites differ from those of other progestogens. The major metabolite 3-keto-Org 2969 binds strongly to the progesterone receptor and relatively weakly to the androgen receptor and human SHBG and shows little or no affinity for the estrogen receptor. Org 2969 is a strong and specific progestogen and its use in oral contraceptives will induce a low incidence of androgenic side effects.     

Estrogen receptor binding tolerance of 16 alpha-substituted estradiol derivatives

In order to examine the tolerance of the estrogen receptor for 16 alpha-substituents in estradiol, we have synthesized various 16 alpha-substituted estrogens and determined their binding affinity for receptor by a competitive radiometric binding assay. The substituents ranged from small, single-atom substituents (halogens), two-atom substituents (halomethyl groups), to larger alkyl groups and ultimately alkyl groups bearing various functionality, including fluorescent (nitrobenzoxadiazole, NBD) and photoreactive (nitroazidophenyl, NAP) groups. The estrogen receptor seems to have a moderate tolerance for bulky substituents: All of the halogen and halomethyl substituents bind with an affinity at least 50% that of estradiol; in the three atom alkyl series, the affinity declined markedly from propargyl (44%) and allyl (38%) to propyl (5%), suggestive of detailed steric constraints or a preference for unsaturation. The larger, more highly functionalized derivatives ranged in affinity from 0.1-7%, with the highest affinity binders being benzyl (5%) and 4-phenoxy-2(E)-butenyl (7%); most of the lowest affinity ones were the bulky fluorescent and photoreactive derivatives. Thus, the estrogen receptor has good tolerance for estradiol derivatives substituted at the 16 alpha-position with nonpolar groups of moderate bulk; however, with groups of larger bulk, affinity is much lower and becomes highly dependent upon the polarity and detailed structure of the substituents.