L. E. Kulikova

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.

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

Competition of a number of progesterone 16α,17α-cycloalkane derivatives with 3H-labeled ligands for the binding sites of rat uterine progesterone receptor, uterine pentaranophilin, and blood serum pentaranophilin was studied. We found that the selective ligands for the progesterone receptor are progesterone, 16α,17α-cyclopropanoprogesterone, and 16α,17α-cyclopent-3′-enoprogesterone and the selective ligands for serum pentaranophilin are 6α-methyl-16α,17α-cyclohexanopregna-1,4-diene-3,20-dione and 3β-hydroxy-16α,17α-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 Δ5-3β-hydroxy grouping for the Δ4-3-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.

Species and Tissue Distribution of Proteins Binding 16α,17α-Cycloalkanoprogesterone Derivatives

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 binding of [3H]progesterone and [3H]16α,17α-cycloalkanoprogesterones to proteins from rat, rabbit, and human uteri and other organs was studied. We found that 16α,17α-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]16α,17α-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 16α,17α-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 17β-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.

Synthesis and structure of 4′,4′-dimethyl[16α,17α]spiropentanopregn-4-ene-3,20-dione

Abstract4′,4′-Dimethyl[16α,17α]spiropentanopregn-4-ene-3,20-dione was synthesized. The addition of diazo-2,2-dimethylcyclopropane generated from N-(2,2-dimethylcyclopropyl)-N-nitrosourea to 16,17-didehydropregnenolone acetate occurs regio-and stereospecifically to give 3β-acetoxy-1′,1′-dimethyl-20-oxopregn-5-ene-[16α,17α;7′,6′]-4′, 5′-diazaspiro[2.4]-hept-4′-ene in high yield. Its thermolysis affords a spiropentane-containing steroid, which is transformed into the target diketone. The anti position of the gem-dimethyl group in the fused spiropentane fragment is evident from the X-ray diffraction study of the final product.

Synthesis of 19-substituted steroids of the 16α,17α-cyclohexanopregnane series and study of their interactions with rat uterine cytosol and blood serum proteins

New 16α,17α-cyclohexanopregnanes (pregna-D′6-pentaranes) containing alkyl or heteroatomic substituents in position 19, namely, 19-methylidene-, 19-methyl-, 19-methoxyimino-, and 19-(3-methoxycarbonylpropoxyimino)-16α,17α-cyclohexanopregn-4-ene-3,20-diones were synthesized, and their interactions with the progesterone receptor and pentarane-binding proteins of rat uterus cytosol and rat blood serum was studied. The same substituent in position 19 of the steroid molecule can affect in opposite direction its affinity to these proteins. These results suggest differences in the structures of the ligand-binding pockets of the three proteins in the region surrounding the C(10) atom of the steroid nucleus.

Synthesis of 6(E)- and 6(Z)-(3-ethoxycarbonylpropyl)oximes of 16α,17α-cyclohexanopregn-4-ene-3,6,20-trione and study of their interaction with proteins of the rat uterine cytosol and blood serum

Abstract6(E)- and 6(Z)-(3-Ethoxycarbonylpropyl)- and -(3-carboxypropyl)oximes of 16α,17α-cyclohexanopregn-4-ene-3,6,20-trione were synthesized. The reactions of these ester ligands with pentarane-binding proteins of the uterine cytosol and blood serum were studied; the latter exhibits a higher affinity. The preferred binding of the oxime (E)-isomer relative to the (Z)-isomer was noted.

Some derivatives of pregna-D6′-pentaranes as possible antagonists of progesterone

Some derivatives of progestins of the pregna-D6′-pentarane series have been obtained and examined for their possible hormonal and antihormonal activity in the Clauberg-McPhail assay as well as in the pregnancy maintenance test in ovariectomized rabbits. 16α, 17α-Cyclohexanoprogesterones of the 19-methyl and 19-nor series (1–4) saturated in ring A, which are inactive as progestins, exhibited a remarkable antiprogesterone effect. They decreased the McPhail index under combined administration with progesterone in a dose-dependable manner and completely inhibited the action of the latter in the pregnancy maintenance test.

Selection of progesterone derivatives specific to membrane progesterone receptors

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.

The synthesis of O-substituted 3-oximes of 6α -methyl-16α,17α-cyclohexanopregn-4-ene-3,20-diones tritium-labeled in the 1,2-position

Abstract1,2-Tritium-labeled 3-(O-carboxypropyl)- and 3-(O-carbomethoxypropyl)-oximes of 6α-methyl-16α,17α-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.

Catalysis by lewis acids at high pressure as a method of involving hindered steroid dienophiles in the diels—Alder reaction

The joint action of Lewis acids and high pressure permits the Diels—Alder reaction to be conducted with high yields with greatly sterically hindered steroid dienophiles, which are virtually unreactive under normal conditions. The [2 + 4]-cycloadditions of dienes to Δ16-20-, Δ1-3-, Δ1,4-3-, and Δ4,6-3-ketones proceed stereospecifically with the formation of one stereoisomer for each cycloadduct. The reactions of the steroid dienes studied give mixtures of two monocycloadducts at both double bonds of the steroid, with predominance of the adduct at the sterically less-hindered double bond (Δ1- and Δ6-). The method developed is a new method of producing various modified steroids with possible hormonal or antihormonal activity.