RBA Values Research Articles

Bioorthogonal “Click Chemistry” synthesis of fluorescent derivative of Ethinyl estradiol for probing estrogen receptor on Poly(dimethylsiloxane)-Based microarrays

To date, binding affinities of estrogen receptors (ERs) have been studied but with a struggle of achieving good biocompatibility, thermal stability, optical transparency, low manufacturing costs and robust results. Herein, we reported bioorthogonal “click chemistry” synthetic route to yield a fluorescent (Cy3) derivative of ethinyl estradiol (EE2) in order to demonstrate its importance as a probe for affinity assays of estrogen receptor (ER) on poly(dimethylsiloxane) (PDMS)-based microarrays. Furthermore, a novel three-step Mitsunobu route has been used to synthesize a 17α-alkynyl amine derivative of E2 (E2-NH2, 3) followed by its labeling with Cy3 (through EDC/NHS chemistry) in order to explore and compare its receptor binding affinity on the aforementioned PDMS platform. An indirect method was also investigated, in which ER was firstly exposed to EE2 and followed by applying of optimized bioorthogonal “click chemistry” reaction conditions against the cognate EE2 and Cy3 azide. Eventually, we found that indirect method is more specific than direct method and thus established the importance of bioorthogonal “click chemistry”. The optimized direct and indirect methods were further explored for real samples of MCF-7 and MBD-MB-231 cell lines and yielded excellent results. Conclusively, an exhibition of minimum nonspecific binding was yielded in this case which suggested that PDMS is a robust and affordable material in a common lab for developing fluorescence binding assays. Comparative docking studies have also been performed in a supporting role to reveal the interaction of Cy3-EE2 and Cy3-3 molecules with the same target of ER, present in breast cancer and which. We found a better correlation between the computed experimentally determined binding affinities as we have achieved almost equivalent or better results (RBA values) than those of earlier reports.

Potential antioxidative components from Syringa oblata Lindl stems revealed by affinity ultrafiltration with multiple drug targets

Traditional Chinese medicine is the main source of natural products due to its remarkable clinical efficacy. Syringa oblata Lindl (S. oblata) was widely used because of its extensive biological activities. However, to explore the antioxidant components of S. oblata against tyrosinase, the experiments of antioxidation in vitro were employed. At the same time, the determination of TPC was also use to assess the antioxidant ability of CE, MC, EA and WA fractions and the liver protective activity of the EA fraction was evaluated by mice in vivo. Next, UF-LC-MS technology was performed to screen and identify the efficient tyrosinase inhibitors in S. oblata. The results showed that alashinol (G), dihydrocubebin, syripinin E and secoisolariciresinol were characterized as potential tyrosinase ligands and their RBA values were 2.35, 1.97, 1.91 and 1.61, respectively. Moreover, these four ligands can effectively dock with tyrosinase molecules, with binding energies (BEs) ranging from 0.74 to −0.73 kcal/mol. In addition, tyrosinase inhibition experiment was employed to evaluate the tyrosinase inhibition activities of four potential ligands, the result showed that compound 12 (alashinol G, IC50 = 0.91 ± 0.20 mM) showed the strongest activity to tyrosinase, followed by secoisolariciresinol (IC50 = 0.99 ± 0.07 mM), dihydrocubebin (IC50 = 1.04 ± 0.30 mM) and syripinin E (IC50 = 1.28 ± 0.23 mM), respectively. The results demonstrate that S. oblata might have excellent antioxidant activity, and UF-LC-MS technique is a effective means to filter out tyrosinase inhibitors from natural products.

Long-Term Dynamics of Forest Fires in Southern Siberia

The research focus. Wildfire is a critical environmental disturbance affecting forest dynamics, succession, and the carbon cycle in Siberian forests. Forests of southern Siberia experienced an increase of fire rate. We analyzed spatial and temporal dynamics of burned area in the southern Central Siberian Mountains. We considered burning rate within different forest types: dark needle conifers (DNC) composed by Siberian pine, spruce and fir, light-needle composed by Scotch pine and larch, and mix wood (composed by birch, aspen and conifers). Methods. We used long-term (1982–2021) climatic variables from the ERA5-Land database. Satellite observations included MODIS wildfire data (2002–2021) and gravimetric (GRACE) data (2002–2021) for estimation soil moisture content. We analyzed the spatial distribution of burned areas considering dominant forest stands and terrain (elevation, slope aspect and steepness). Results. We found a significant decreasing trend in burned area. On average, about 978.7 ± 447.5 thousand ha including 257.1 ± 154.3 thousand ha of forests are disturbed by fire annually. Light coniferous forests are characterized by the highest degree of the fire disturbance (relative burned area, RBA, was ca. 0.83% per year); whereas DNC stands have the lowest RBA (ca. 0.15% per year). The spatial distribution of fires in light coniferous forests showed predominance of fires on the southern slopes comparing to northern slopes (by 15–20%). We found a decreasing logarithmic trend in burned area between plains and low mountains and high mountains due to lower forest fuel loads and increased amount of precipitation on higher elevations. We also observed lower RBA values (by 300–400%) on steep slopes for light coniferous forests and mixed forests comparing to gentle slopes due to lower available forest fuels. The long-term dynamics of burned area in the region was related to the variation of climatic variables, such as soil moisture (r2 = 0.70, p < 0.01), moisture content (r2 = 0.63, p < 0.01) and drought index (r2 = 0.3, p < 0.05).

Effect of Polishing on Lead and Cadmium Bioavailability in Rice and Its Health Implications.

Rice polishing is an important approach to reducing the concentrations of heavy metals in rice, but knowledge of its effect on the Pb and Cd bioavailability in produced rice and the related health risk remains limited. In this study, the effects of rice polishing on the bioaccessibility (BAC) and bioavailability (RBA) of Pb and Cd in rice are assessed using an in vitro method and an in vivo mouse bioassay. The Pb removal rate in brown rice (40%), lightly processed brown rice (62%), germinated rice (74%), and polished rice (79%) gradually enhanced with an increase in the polishing degree, while Cd was difficult to remove by polishing. The Pb and Cd BAC in germinated rice was the highest, while that in brown rice was the lowest. The polished rice Pb and Cd RBA in the liver and kidneys were significantly higher than those in the brown rice group. The Pb RBA in the livers and kidneys in the polished rice group was 26.6% ± 1.68% and 65.3% ± 0.83%, respectively, which was 1.6- and 2.6-times higher than that in the brown rice group, respectively. The Cd RBA values in both the livers and kidneys of the polished rice group were 1.3-times higher than those in the brown rice group. Although polishing reduced the total Pb in the polished rice, it was not enough to offset the increase in bioavailability, and its consumption risk was not weakened. This study highlighted the value of the oral-bioavailability-corrected health risk assessment for assessing the influence of rice polishing on Pb and Cd exposure via rice consumption.

Rational design, synthesis, antiproliferative activity against MCF-7, MDA-MB-231 cells, estrogen receptors binding affinity, and computational study of indenopyrimidine-2,5-dione analogs for the treatment of breast cancer

Based on the structural architecture of estrogen receptors (ER) agonists/antagonists, we rationally designed and synthesized indenopyrimidine-2,5-dione analogs as a starting point of current research targeting estrogen receptors. These analogs were evaluated for their antiproliferative activities against breast cancer MCF-7 (ER+), MDA-MB-231 (ER-) and non-cancerous HEK-293 cells using MTT assay. Compounds with high antiproliferative activity against MCF-7 breast cancer cells were found devoid of cytotoxicity against HEK-293 cells. Competitive binding assay of estrogen receptors ERα and ERβ showed that diethanolamine derivative of 4-trifluoromethyl phenyl derivative 30 displayed 77.5-fold strong binding affinity towards ERα (IC50 = 0.004 μM) as compared to ERβ (IC50 = 0.31 μM). The calculated RBA value of compound 30 indicated that it has greater affinity with ER than estradiol. By docking studies, we demonstrated that high binding affinity with ERα is due to binding orientation and interaction of CF3 with a number of key amino acid residues present in the active site of ERα.

Effects of Different In Situ Remediation Strategies for an As-Polluted Soil on Human Health Risk, Soil Properties, and Vegetation

The demand for soils for recreational uses, gardening, or others in urban and periurban areas is increasing, and thus the presence of polluted technosols in these areas requires nature-based in situ remediation technologies. In this context, the capacity of three amendments, namely zero valent iron nanoparticles (nZVI), compost and a mixture of compost and biochar, to immobilise As in a polluted technosol simultaneously cultivated with Lolium perenne L. were tested and compared. The characteristics of the soil were comprehensively characterised by chemical and X-ray analysis to determine As contents, distribution, and mineralogy. As mobility was evaluated by the RBA methodology and then potential human health risks, both carcinogenic and non-carcinogenic, were assessed in all treatments. The nZVI treatment reduced risks due to the As immobilisation obtained (41% As decrease, RBA test), whereas the organic amendments did not imply any significant reduction of the RBA values. As to soil properties, the organic treatments applied lowered the pH values, increasing cation exchange capacity, and carbon and nutrient contents. To determine impacts over plant production, fresh biomass, As, Ca, Fe, K, Mg, Na and P were measured in Lolium under the different treatments. Notably, organic amendments improved As extraction by plants (57% increase), as well as fresh biomass (56% increase). On the contrary, nZVI diminished As extraction (65% decrease) and promoted a fresh biomass decrease of 57% due to nutrients immobilisation (61% decrease of P in plants tissues).

Synthesis of benzoylbenzamide derivatives of 17α-E-vinyl estradiol and evaluation as ligands for the estrogen receptor-α ligand binding domain

A series consisting of substituted benzoylbenzamide derivatives of 17α-E-vinyl estradiol 6a-i and 7a-d was prepared in good overall yields from the corresponding novel iodinated benzoylbenzamide precursors using Pd(0)-catalyzed Stille coupling. Biological evaluation using competitive binding assays indicated that all compounds were effective ligands for the ERα- and ERβ-LBD (RBA = 0.5–10.0% of estradiol). Most of the compounds expressed lower stimulatory (agonist) potency (RSA <0.2–0.5%) compared to their binding affinity, however, the meta-substituted isomer 6h demonstrated a level of efficacy (RSA = 5.7%) comparable to its affinity (RBA = 9.5%). Docking studies of 6b, 6h, and 6i with the 2YAT crystal structure suggested that higher affinity and efficacy of 6h are due to an effective set of interactions with exposed receptor sidechains not observed with the ortho- and para- isomers. In this binding model, the terminal ring of the ligand is exposed to the solvent space, which would explain both the small variation in RBA values and the narrow SAR for the diverse structural features.

Design, synthesis, biological evaluation and molecular docking studies of novel 3-aryl-4-anilino-2H-chromen-2-one derivatives targeting ERα as anti-breast cancer agents

The estrogen receptor (ER) has played an important role in breast cancer development and progression and is a central target for anticancer drug discovery. In order to develop novel selective ERα modulators (SERMs), we designed and synthesized 18 novel 3-aryl-4-anilino-2H-chromen-2-one derivatives based on previously reported lead compounds. The biological results indicated that most of the compounds presented potent ERα binding affinity and possessed better anti-proliferative activities against MCF-7 and Ishikawa cell lines than the positive control tamoxifen. The piperidyl substituted compounds such as 16d and 18d demonstrated strong ERα binding affinities and excellent anti-proliferative activities respectively. Compound 18d displayed the most potent ERα binding affinity with RBA value of 2.83%, while 16d exhibited the best anti-proliferative activity against MCF-7 cells with IC50 value of 4.52±2.47μM. Further molecular docking studies were also carried out to investigate binding pattern of the newly synthesized compounds with ERα. All these results together with the structure–activity relationships (SARs) indicated that these 3-aryl-4-anilino-2H-chromen-2-one derivatives with basic side chain could serve as promising leads for further optimization as novel SERMs.

Tracheobronchial Angle Measurements in Children: An Anthropometric Retrospective Study With Multislice Computed Tomography.

ObjectivesThe purpose of this study is to investigate if any change exists in the values of tracheal bifurcation angles (subcarinal angle [SCA] and interbronchial angle [IBA]), right and left bronchial angles (RBA and LBA) in different pediatric age groups.MethodsChest computed tomography (CT) images of children aged 18 years and younger were reviewed retrospectively by two radiologists who were blinded to each other’s measurements. One hundred and eighteen children were involved. RBA, LBA, SCA, and IBA were measured on coronal reformatted images. Subjects were classified into three groups according to their age. Measurement of IBA was done by measuring the angle between the lines drawn along the central axis of right and left main bronchi over their length. RBA and LBA were measured at the intersection points of the lines drawn along the inferior borders of the right and left main bronchi and the line passing through the longitudinal axis of trachea. Sums of RBA and LBA gave SCA. Interobserver agreement was also analyzed.ResultsSCA, IBA, and RBA values were statistically significant between children of ages less than 10 years and over 10 years P<0.01). Interobserver agreement was excellent with an intraclass correlation coefficient score of 0.87 (95% confidence interval) for RBA, SCA, and IBA measurements.Conclusion We concluded that tracheal bifurcation angles are wider in children of age 10 years and younger. As age increases values of SCA, IBA, and RBA decrease.

Natural phenolics and nuclear receptors (ER & PPAR): Isolation, structure determination and biological evaluation

Plants originated mainly from Mediterranean region as well as from other higly deverse hot-spots all over the world have been phytochemically explored in the context of our continuing study towards the discovery of novel, active natural products presenting high affinity to nuclear reseptors. In particular estrogen receptor (ER) and peroxisome proliferator-activated receptor (PPAR) were the target-reseptors under investigation. Our study is mainly focused to the identification of secondary metabolites which present the capability to activate both isoforms of estrogen receptor (ERα& ERβ) [1] as well as the two isoforms of the peroxisome proliferator-activated receptor (PPARγ& PPARδ) [2]. Numerous of secondary metabolites belonging mostly to simple phenolics, to flavonoids and specifically to flavones, flavonols and methylether derivatives thereof and isoflavonoids mainly isoflavones, prenylated isoflavones and 2-arylbenzofurans have been identified using a bioguided approach or LC-MS-based methodologies. The structure elucidation of the isolated compounds was performed via HRMSn and 1 & 2D NMR [3], [4], [5], [6]. The biological evaluation was focused on the determination of the relative binding activity (RBA values) of the purified compounds to ERα and ERβ and/or PPARγ and PPARδ as well as on the exploration of the biological michanism of action using e-screen, reporter gene and analysis of gene expression assays. Determination of their proliferative and/or cytotoxic properties was also performed. A series of novel natural compounds belonging mostly to 2-arylbenzofurans and prenylated isoflavones were identified whereas a numerous thereof found to present significant estrogenic, antiestrogenic, SERM and/or antidiabetic activity [7], [8], [9].

The Presence of a Ferrocenyl Unit on an Estrogenic Molecule is Not Always Sufficient to Generate in vitro Cytotoxicity

We recently reported the dual (antihormonal and cytotoxic) functionality of ferrocifens, which are organometallic complexes derived from hydroxytamoxifen, the standard molecule in the treatment of hormone-dependent breast cancers. To test the hypothesis that the presence of a ferrocenyl substituent on molecules with an affinity for the estrogen receptor is sufficient to give them cytotoxic properties in vitro, we prepared complexes derived from estradiol with a ferrocenyl substituent at positions 7alpha and 17alpha. The complexes thus obtained retain a satisfactory level of affinity for the estrogen receptor (RBA values higher than 12 %). At low concentrations (0.1-1 microM) the complexes show an estrogenic effect in vitro equivalent to that of estradiol on hormone-dependent (MCF-7) breast cancer cells, and no cytotoxic effect on hormone-independent (MDA-MB-231) breast cancer cells. At high concentrations (up to 50 microM) the 17alpha-ethynylferrocenyl estradiol and 7alpha-ferrocenylmethylthio estradiol become cytotoxic (IC(50)=13.2 microM and 18.8 microM, respectively) while the 17alpha-ferrocenylestradiol remains non toxic. The low toxicity of these compounds support our hypothesis that electronic communication between the ferrocenyl and phenol moieties in the hydroxyferrocifens series is a key parameter in the generation of cytotoxic effects at submicromolar concentrations.

Quantitative Structure−Activity Relationship Models for Prediction of the Toxicity of Polybrominated Diphenyl Ether Congeners

Levels of polybrominated diphenyl ethers (PBDEs) are increasing in the environment and may cause long-term health problems in humans. The similarity in the chemical structures of PBDEs and other halogenated aromatic pollutants hints on the possibility that they might share similar toxicological effects. In this work, three-dimensional quantitative structure activity relationships (3-D-QSAR) models, using comparative molecular field analysis (CoMFA) and comparative similarity indices analysis (CoMSIA), were built based on calculated structural indices and a reported experimental toxicology index (aryl hydrocarbon receptor relative binding affinities, RBA) of 18 PBDEs congeners, to determine the factors required for the RBA of these PBDEs. After performing leave-one-out cross-validation, satisfactory results were obtained with cross-validation O2 and R2 values of 0.580 and 0.995 by the CoMFA model and 0.680 and 0.982 by the CoMSIA model, respectively. The results showed clearly that the nonplanar conformations of PBDEs result in the lowest energy level and that the electrostatic index was the main factor reflecting the RBA of PBDEs. The two QSAR models were then used to predict the RBA value of 46 PBDEs for which experimental values are unavailable at present.

2,5-Diphenylfuran-based pure antiestrogens with selectivity for the estrogen receptor α

The estrogen receptor α (ERα) is understood to play an important role in the progression of breast cancer. Therefore, pure antiestrogens with a preference for this receptor form are of interest as new agents for the treatment of this malignancy. Several chemical structures with selective binding affinity for ERα have been identified and might be useful for the synthesis of ERα-selective pure antiestrogens. In this study we applied the 2,5-diphenylfuran system which is closely related to the triphenylfurans described by others. Various side chains with amino and/or sulfur functions were linked to C3 to convert the furans to estrogen antagonists without residual estrogenic activity. The degree of α-selectivity which ranges from 2.5- to 236-fold is strongly influenced by the alkyl group at C4. Antiestrogenic potency was determined in MCF-7/2a breast cancer cells stably transfected with a luciferase gene under the control of an ERE. The 2,5-bis(4-hydroxyphenyl)furan with an ethyl substituent and a 6-[ N-methyl- N-(3-pentylthiopropyl)amino]hexyl side chain exerted the strongest antiestrogenic effect in this series with an IC 50 value of 50 nM in cells stimulated with 1 nM estradiol. The RBA values of this derivative were 18% (ERα) and 3.4% (ERβ) of estradiol, respectively. It inhibited the growth of wild-type MCF-7 cells with an IC 50 value of 22 nM. The data show that the 2,5-diphenylfuran system is appropriate for the development of pure antiestrogens with preference for ERα.

Platinum(II) and technetium(I) complexes anchored to ethynylestradiol: a way to drug targeting and delivery

Starting from ethynylestradiol ( 1), or, more precisely, from its 3,17β-bis-(triethylsilyloxy) derivative 2, two new ligands containing the ethylenediamino motif were synthesised by a Mannich aminomethylation, namely N-methyl- N-(prop-2-ynyl-3-(17α-estradiolyl))- N ′, N ′-dimethylethylenediamine ( 3) and N-(prop-2-ynyl-3-(17α-estradiolyl))- N ′-methylpiperazine ( 4). The corresponding platinum(II)-malonato complexes ( 7 and 8) were prepared through the PtI 2 intermediates (namely 5 and 6) by Dhara’s method. The structures of the two platinum complexes were energy-minimised by molecular mechanics employing the Amber force field. Both ligands were joined to the [ 99mTc(CO) 3Cl] moiety, 99mTc being the chief γ-emitter employed in nuclear medicine. Unfortunately, piperazine ligand 4 afforded complexes that were unstable under physiological conditions. The RBA values for both ligands and complexes derived from 3, measured for the two forms of estrogen receptor, were less than 1%. Such a poor degree of ligand recognition may be due to the partial protonation of the amino groups at physiological pH, making the carrier quite hydrophilic, therefore unsuitable for entering the hydrophobic pocket of estrogen receptors.

Synthesis and evaluation of 17alpha-20E-21-(4-substituted phenyl)-19-norpregna-1,3,5(10),20-tetraene-3,17beta-diols as probes for the estrogen receptor alpha hormone binding domain.

As part of our program to develop probes for the hormone binding domain of the estrogen receptor alpha (ERalpha), we prepared a series of 4-para-substituted phenylvinyl estradiol derivatives using a combination of solution and solid-phase Pd(0)-catalyzed methods. The compounds 5a-j were evaluated for their binding affinity using the ERalpha hormone binding domain (HDB) isolated from transfected BL21 cells. The results indicated that although the new compounds were somewhat lower in relative binding affinity (RBA at 25 degrees C is 1-60%) than estradiol (100%), most had higher affinity than the unsubstituted parent phenylvinyl estradiol (RBA = 9%). Because the substituents did not generate a structure-activity relationship directly based on physicochemical properties, the series was evaluated using molecular modeling and molecular dynamics to determine key interactions between the ligand, especially the para substituent, and the protein. The results suggest that the observed relative binding affinities are directly related to the calculated binding energies and that amino acids juxtaposed to the para position play a significant but not dominant role in binding. In conclusion, we have identified the 17alpha-E-(4-substituted phenyl)vinyl estradiols as a class of ligands that retain significant affinity for the ERalpha-HBD. In particular, 4-substitution tends to increase receptor affinity compared to the unsubstituted analogue, as exemplified by 5e (4-COCH(3)), which had the highest RBA value (60%) of the series. Palladium(0)-catalyzed coupling reactions on solid support or in solution using suitably substituted iodo arenes and 17alpha-E-tributylstannylvinyl estradiols offer a flexible approach to their preparation. Molecular modeling studies of the receptor suggest that there exists additional ligand accessible regions within the ERalpha-HBD to generate interactions that may enhance receptor affinity or modify efficacy in developing new therapeutic agents. Studies to undertake modification in the properties and/or position of the aryl substituents in subsequent series to further define that role are in progress.

Conformational studies on (17alpha,20Z)-21-(X-Phenyl)-19-norpregna-1, 3,5(10),20-tetraene-3,17beta-diols using 1D and 2D NMR spectroscopy and GIAO calculations of (13)C shieldings.

Differences in agonist responses of the novel estrogen receptor ligands (17alpha,20Z)-(p-methoxyphenyl)vinyl estradiol (1), (17alpha, 20Z)-(o-alpha,alpha,alpha-trifluoromethylphenyl)vinyl estradiol (2), and (17alpha,20Z)-(o-hydroxymethylphenyl)vinyl estradiol (3) led us to investigate their solution conformation. In competitive binding assay studies, we observed that several phenyl-substituted (17alpha, 20E/Z)-(X-phenyl)vinyl estradiols exhibited significant estrogen receptor binding, but with variation (RBA (1) = 20; RBA (2) = 23; RBA (3) = 140 where estradiol RBA = 100) depending on the phenyl substitution pattern. Because the 17alpha-phenylvinyl substituent interacts with the key helix-12 of the ligand binding domain, we considered that differences in the preferred conformation of 1-3 could account for their varying binding affinity. 2D NMR experiments at 500 MHz allowed the complete assignment of the (13)C and (1)H spectra of 1-3. The conformations of these compounds in solution were established by 2D and 1D NOESY spectroscopy. A statistical approach of evaluating contributing conformers of 1-3 from predicted (13)C shifts correlated quite well with the NOE data. The 17alpha substituents of 1 and 2 exist in similar conformational equilibria with some differences in relative populations of conformers. In contrast, the 17alpha substituent of 3 exists in a different conformational equilibrium. The similarity in solution conformations of 1 and 2 suggests they occupy a similar receptor volume, consistent with similar RBA values of 20 and 23. Conversely, the different conformational equilibria of 3 may contribute to the significant binding affinity (RBA = 140) of this ligand.

1,3-Diphenylpropane-1,3-diamines, X: Synthesis and biochemical evaluation of highly substituted 1,3-diphenylpropane-1,3-diamines and preparation of their Pt(II) complexes

The ligands of the title complexes1 and2 were prepared from the pertinent chalcone5 and hydrazine hydrate, followed by N-N cleavage. The estrogenic activity of the diamines11 and12 was determined by measuring the RBA values (calf uterine cytosol) and by a luciferase test in MCF 7-2a cells. The compounds are by far less active thanSchonenberger's most active compound ([meso-1,2-bis(2,6-dichloro-4-hydroxyphenyl)ethane-1,2-diamine]dichloro-platinum(II),3).

11 beta-Substituted estradiol derivatives, potential high-affinity carbon-11-labeled probes for the estrogen receptor: a structure-affinity relationship study.

In view of their possible development as carbon-11-labeled receptor-based radiotracers for imaging estrogen-responsive breast tumors, we have synthesized a series of estradiols (1), estriols (2), 11 beta-ethylestradiols (3), 11 beta-ethylestriols (4), 11 beta-methoxyestradiols (5), and 11 beta-methoxyestriols (6), differing in the type of substituent R present at the 17 alpha-position (a, -H; b, -CH3; c, -C identical to CH; d, -C identical to CCH3; e, -Ph; f, -CH = CHMe cis), and measured their binding affinity for the estrogen receptor relative to estradiol (RBA). As expected, all the derivatives having an 11 beta-ethyl substituent have good binding properties (3a-d, 4a-d, RBA (25 degrees C): 109-3000%), and among them there are several promising candidates for carbon-11 labeling. Moxestrol (RBA (25 degrees C) = 185%) and its corresponding estriol derivative (4c, RBA (25 degrees C) = 20%) were the analogs having the highest affinity in the 11 beta-methoxyestradiol (5a-f) and 11 beta-methoxyestriol (6a-e) series, respectively; other analogs (R = Me, C identical to CMe, Ph, or cis-CH = CHMe) had uniformly lower RBA values.

Receptor binding of norgestimate — A new orally active synthetic progestational compound

Binding of the new progestagen, norgestimate (D-(−)-13β-ethyl-17β-acetoxy-17-ethinyl-4-gonen-3-one-oxime), and its metabolites (levonorgestrel-3-oxime, levonorgestrel-17-acetate and levonorgestrel) to the progesterone receptor was investigated by competition experiments using cytosol from human myometrial tissue. Compared to R5020, a highly potent synthetic ligand for progesterone receptor analysis, the L-isomer of norgestimate shows only a weak specific behaviour with regard to binding to the progesterone receptor from uterine cytosol with an RBA value of 0.8%, whereas the D-isomer of this compound is characterized by a lack of binding activity to the progesterone receptor. Levonorgestrel-3-oxime, one of the possible metabolites of norgestimate, binds to the progesterone receptor with an RBA value of 8%, whereas levonorgestrel-17-acetate, the other possible metabolite of norgestimate, binds with a binding affinity of 110% which is in the same order of magnitude as levonorgestrel itself. The competition experiments suggest that norgestimate is a prodrug and that the metabolites, levonorgestrel levonorgestrel-17-acetate, which actively bind to the progesterone receptor, must first be formed from the parent drug via metabolic processes in vivo. These are the actual biologically active compounds which are responsible for the gestagenic potency.

Dihydrospirorenone (ZK30595): A novel synthetic progestagen-characterization of binding to different receptor proteins

Dihydrospirorenone (6β,7β,15β,16β-dimethylen-3-oxo-17a-pregn-4-en-21,17-carbolacton) is a new type of progestin with no other agonistic activities, in particular no estrogenic, androgenic or glucocorticoid activity. Dihydro-spirorenone is a potent aldosterone antagonist 8 times as potent as spirono-lactone and antiandrogenic (0.3 times cyproterone acetate). The aim of the present study is to make a broad characterization of dihydrospirorenone at the receptor level and to discuss the results in comparison to those of established progestins. Kinetic studies of 3H-dihydrospirorenone uptake show a rapid increase in the amount of specific binding during the first three hours. The dissociation of 3H-dihydrospirorenone from the cytoplasmic human uterine progesterone receptor, measured by displacement of labeled steroid with dextran-coated charcoal treatment at 4 °C at various times, showed a monophasic or one-component, first order dissociation curve like progesterone. Sucrose density gradient centrifugation of the 3H-dihydrospirorenone-labeled myometrial cytosol showed that the dihydrospirorenone binding components sedimented in the 4S and 8S region which is typical for the progesterone receptor under low salt conditions. The high binding affinity of dihydrospirorenone to the binding sites of the mineralocorticoid receptor of rat kidney with an RBA value of 230% compared to aldosterone is remarkable. This reflects the strong antimineralocorticoid activity of this compound which was evaluated in adrenalectomized rats.