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Identification of progesterone selective agonists and antagonists that act through one of the nuclear progesterone receptor isoforms is of particular importance for the development of tissue-specific drugs in gynecology and anticancer therapy. Fourteen pregna-D'6- and pregna-D'3-pentarane progesterone derivatives with 16α,17α-cycloalkane groups and two progesterone 3-deoxyderivatives were examined for their ability to regulate transcriptional activity of human nuclear progesterone receptor isoform B (nPR-B) expressed in Saccharomyces cerevisiae yeast. Transcriptional activity of nPR-B was measured from the expression of the ß-galactosidase reporter gene with a hormone-responsible element in the promoter. Among the compounds tested, two were full progesterone agonists, four were partial agonists, one compound possessed both agonistic and antagonistic activity, one compound displayed only partial antagonistic activity, and eight compounds did not show any activity. Modifications of the pentarane structure, precisely, introduction of an additional double bound in the A or B rings and/or modification at the 6th position of progesterone, lead to a switch from the complete agonistic activity to partial agonistic or mixed activities. These modifications enable progestins to act as selective modulators of progesterone receptor. Steroids with reduced A-ring and 3-ketogroups lose their ability to regulate PR-B activity. Both 3-deoxycompounds, being selective ligands of progesterone membrane receptors, do not affect PR-B activity.

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The search of selective agonists and antagonists of membrane progesterone receptors (mPRs) is a starting point for the study of progesterone signal transduction mechanisms mediated by mPRs, distinct from nuclear receptors. According to preliminary data, the ligand affinity for mPRs differs significantly from that for classical nuclear progesterone receptors (nPRs), which might indicate structural differences in the ligand-binding pocket of these proteins. In the present work, we analyzed the affinity of several progesterone derivatives for mPRs of human pancreatic adenocarcinoma BxPC3 cell line that is characterized by a high level of mPR mRNA expression and by the absence of expression of nPR mRNA. The values were compared with the affinity of these compounds for nPRs. All tested compounds showed almost no affinity for nPRs, whereas their selectivity towards mPRs was different. Derivatives with an additional 19-hydroxyl group and removed 3-keto group had the highest selectivity for mPRs. These results suggest these compounds as the most selective progesterone analogs for studying the mechanisms of progestin action via mPRs.

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The cytotoxic activity of synthetic progestins (pregna-D'-pentaranes) II-V full agonists of the progesterone receptor (PR) for PR-positive and PR-negative cells of human breast carcinoma was studied. These compounds were more active in the PR-positive MCF-7 cells than in the PR-negative MDA-MB-453 cells. Cytotoxic effects of tested compounds against normal epithelial MDCK cells were not found. Molecular modeling of studied steroids with PR showed that all progestins with close energy values can bind to the ligand binding domain (LBD) of PR and the magnitude of the energy exceeds the value estimated for the progesterone molecule. Thus, the studied progestins are active against different molecular subtypes of breast cancer and represent a promising class of chemical compounds for oncology.

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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 ligand 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.

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1,2-Tritium-labeled 3-(O-carboxypropyl)- and 3-(O-carbomethoxypropyl)-oximes of 6alpha-methyl-16alpha,17alpha-cyclohexanopregn-4-ene-3,20-diones were obtained by the homogeneous catalytic hydrogenation of 1,2-dehydroprecursors with gaseous tritium and the subsequent separation of the resulting mixtures by HPLC. The specific radioactivities of 50-55 Ci/mmol were prepared using tris-(triphenylphosphine)-rhodium chloride.

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(19R)- and (19S)-tert-Butyldimethylsilyl (TBS) ethers of 19-hydroxy-19-vinyl-16alpha,17alpha-cyclohexanopregn-5-en-20-ones were synthesized. These compounds containing the 1,5-oxydienoic motif were subjected to the anionic oxy-Cope rearrangement to obtain 3beta-TBS ether of 6beta-(3-oxopropyl)-16alpha,17alpha-cyclohexano-19-nor-pregn-5(10)-en-20-one. The structures of the compounds synthesized were confirmed by the analysis of their H and 13C NMR spectra.

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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.

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Changes in the uterus morphology of mature female rats were studied on the model of pseudopregnancy after treatment with the progestin 16 alpha,17 alpha-cyclohexanoprogesterone (PR) and the antirpogestins 5 alpha(H)-16 alpha,17 alpha-cyclohexano-4,5-dihydroprogesterone (APR1) and 5 beta(H)-16 alpha,17 alpha-cyclohexano-4,5-dihydroprogesterone (APR2). The rats were preliminarily estrogenized with 17 beta-estradiol at a dose of 1 microgram/(animal day) for 4 days and then treated with PR at a dose of 0.2 mg/(animal day) for 14 days. The first group was then left without any treatment, whereas APR1 and APR2 were injected at the dose of 0.2 mg/(animal day) for 4 days to the animals of the second and the third groups, respectively. Light and electron microscopy of the uterus preparations demonstrated that the PR action provoked a complete pseudopregnancy picture characterized by the endometrium functionalization and the myometrium hypertrophy. Subsequent treatment with APR1 and APR2 caused the hypertrophy to cease, which had a more pronounced effect in the case of APR1. At the same time, some indications of the endometrium functionalization remained observable after treatment with APR1 and APR2. The specific binding sites for 3H-labeled APR1 and APR2 were absent from the uterus cytosol for the rats gestagenized with PR.

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6-O-(3-Methoxycarbonylpropyl)- and 6-O-(3-carboxypropyl)oximes of 16 alpha,17 alpha-cyclohexanopregn-4-ene-3,6,20-trione labeled by tritium in position 1,2 were synthesized. When using homogenous catalysts, the molar radioactivity of the resulting preparations was 1.5-1.7 PBq/mol.

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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.

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Antiprogestin activity of 5H-progesterone metabolites and their analogues 16alpha,17alpha-cyclohexan-5H-pregnan-3,20-diones was tested on rats. Modified pregnancy interruption test showed that 5alpha (H)-isomers of natural and synthetic hormones exhibit maximum activity.

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The interaction of 6alpha-methyl-[1,2-3H]16alpha,17alpha-cyclohexanoprogesterone with rat serum proteins has been studied. Specific binding of this ligand characterized by Kd = 0.36 +/- 0.10 microM and concentration of binding sites (Bmax) of about 1 microM (27.8 +/- 12.5 pmol/mg total protein) was found. According to competitive analysis, the affinity of the studied progestins to a protein that differs from transcortin was to some extent correlated with their hydrophobicity. The dissociation kinetics of 3H-ligand-protein complexes were biphasic, the binding sites forming stable and labile complexes with 3H-ligand being eluted in the same region during ion-exchange chromatography. In overall properties, the serum protein differs from the progesterone receptor and the pregna-D'-pentarane-specific protein from rat uterus. It is suggested that the revealed protein may provide high progestagenic activity of 6alpha-methyl-16alpha,17alpha-cyclohexanoprogesterone by prolonging its retention in the bloodstream.

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We studied competitive activities of 16 alpha,17 alpha-cyclohexano-5 alpha- and 5 beta-dihydroprogesterone in replacing(3)H-progesterone and(3)H-16 alpha,17 alpha-cyclohexano-6 alpha-methylprogesterone from protein complexes. Direct binding of(3)H-5-reduced derivatives with proteins of soluble fractions from rat and rabbit uteri was also assayed. C(d) values for 5-reduced derivatives were in the micro- or submicromolar range. The data suggest that biological effects of these analogues are not mediated via soluble uterine receptors.

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[(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.

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A comparative study of the progestational activity of modified pentarans depending on the dose and the route of administration was carried out. It was shown that modification of the molecule of 16 alpha, 17 alpha-cyclohexanoprogesterones by introducing and additional double bond in positions 6, 7 and methyl group in position 6 provided a high gestational effect, being particularly pronounced at intramuscular administration.

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The synthesis of 2' beta-methyl-16 alpha,17 alpha-cyclohexanoprogesterone and its MM2 conformational analysis have been performed. The acetyl side chain was shown to have an unusual conformation with the torsion angle C13-C17-C20-O20 being -32.1 degrees. This conformation is by 5.4 kJ.mol-1 more stable than the usual one with the torsion angle 130.3 degrees. 2' beta-Methyl-16 alpha,17 alpha-cyclohexanoprogesterone proved to be inactive as a progestogen (pregnancy maintenance and McPhail tests). The lack of the activity may be due to the additional methyl group in D'-ring causing a change of the conformation of the 17 beta-acetyl side chain, thus hindering the formation of the conformation necessary for binding to the progesterone receptor.

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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.

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It has been shown in experiments on rats, mice and rabbits that 6 alpha-methyl-16 alpha, 17 alpha-cyclohexano-pregna-4-en-3, 20-dion and 6-methyl-16 alpha, 17 alpha-cyclohexano-pregna-4,6-dien-3,20-dion belonging to the series of D'6-pentaranes exhibit high contraceptive activity when combined with mestranol. The contraceptive activity of D'6-pentaranes combined with mestranol arises primarily from their inhibitory effect on luteinizing function of the hypophysis.

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