Combined experimental and quantum mechanical elucidation of the synthetically accessible stereoisomers of Hydroxyestradienone (HED), the starting material for vilaprisan synthesis.

Selective progesterone receptor modulators are promising therapeutic options for the treatment of uterine fibroids. Vilaprisan, a new chemical entity that was discovered at Bayer is currently in clinical development. In this study we provide a combined experimental and quantum chemical approach providing the data that allowed to present hydroxyestradienone as an acceptable starting material for drug substance synthesis. Hydroxyestradienone has four stereogenic centers leading to 8 diastereomers and 16 enantiomers of which only six diastereomers were synthetically accessible but two not. A computational multistep protocol resulting in density functional P2PLYP-D3(BJ)/dev2-TZVPP Gibbs free energies and SMD solvation free energies led to a clear separation between the existing and the synthetically not accessible enantiomers, whereas multiple geometry-based and cheminformatic descriptors were not able to explain experimental findings.

X-ray structures of progesterone receptor ligand binding domain in its agonist state reveal differing mechanisms for mixed profiles of 11ß-substituted steroids.

We present here the x-ray structures of the progesterone receptor (PR) in complex with two mixed profile PR modulators whose functional activity results from two differing molecular mechanisms. The structure of Asoprisnil bound to the agonist state of PR demonstrates the contribution of the ligand to increasing stability of the agonist conformation of helix-12 via a specific hydrogen-bond network including Glu(723). This interaction is absent when the full antagonist, RU486, binds to PR. Combined with a previously reported structure of Asoprisnil bound to the antagonist state of the receptor, this structure extends our understanding of the complex molecular interactions underlying the mixed agonist/antagonist profile of the compound. In addition, we present the structure of PR in its agonist conformation bound to the mixed profile compound Org3H whose reduced antagonistic activity and increased agonistic activity compared with reference antagonists is due to an induced fit around Trp(755), resulting in a decreased steric clash with Met(909) but inducing a new internal clash with Val(912) in helix-12. This structure also explains the previously published observation that 16α attachments to RU486 analogs induce mixed profiles by altering the binding of 11ß substituents. Together these structures further our understanding of the steric and electrostatic factors that contribute to the function of steroid receptor modulators, providing valuable insight for future compound design.

Inhibition of 5-lipoxygenase by U73122 is due to covalent binding to cysteine 416.

U73122 which was originally identified as a phospholipase C inhibitor represents a potent direct inhibitor of purified 5-lipoxygenase (5-LO) with an IC50 value of 30 nM. 5-LO catalyzes the conversion of arachidonic acid (AA) into leukotrienes which represent mediators involved in inflammatory and allergic reactions and in host defense reactions against microorganisms. Since the efficient inhibition of the human 5-LO enzyme depended on the thiol reactivity of the maleinimide group of U73122, we used this property to identify cysteine residues in the 5-LO protein that are important for 5-LO inhibition by U73122. We found by MALDI-MS that U73122 covalently binds to cysteine residues 99, 159, 248, 264, 416 and 449. Mutation of Cys416 to serine strongly reduces inhibition of 5-LO by U73122 and the additional mutation of three cysteines close to Cys416 further impairs 5-LO inhibition by the compound. Wash out experiments with U73122 and 5-LO indicated an irreversible binding of U73122. Together, our data suggest that the area around Cys416 which is close to the proposed AA entry channel to the active site is an interesting target for the development of new 5-LO inhibitors.

Direct activation of human phospholipase C by its well known inhibitor u73122.

Phospholipase C (PLC) enzymes are an important family of regulatory proteins involved in numerous cellular functions, primarily through hydrolysis of the polar head group from inositol-containing membrane phospholipids. U73122 (1-(6-((17ß-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione), one of only a few small molecules reported to inhibit the activity of these enzymes, has been broadly applied as a pharmacological tool to implicate PLCs in diverse experimental phenotypes. The purpose of this study was to develop a better understanding of molecular interactions between U73122 and PLCs. Hence, the effects of U73122 on human PLCß3 (hPLCß3) were evaluated in a cell-free micellar system. Surprisingly, U73122 increased the activity of hPLCß3 in a concentration- and time-dependent manner; up to an 8-fold increase in enzyme activity was observed with an EC50=13.6±5 µm. Activation of hPLCß3 by U73122 required covalent modification of cysteines as evidenced by the observation that enzyme activation was attenuated by thiol-containing nucleophiles, l-cysteine and glutathione. Mass spectrometric analysis confirmed covalent reaction with U73122 at eight cysteines, although maximum activation was achieved without complete alkylation; the modified residues were identified by LC/MS/MS peptide sequencing. Interestingly, U73122 (10 µm) also activated hPLCγ1 (>10-fold) and hPLCß2 (∼2-fold); PLCδ1 was neither activated nor inhibited. Therefore, in contrast to its reported inhibitory potential, U73122 failed to inhibit several purified PLCs. Most of these PLCs were directly activated by U73122, and a simple mechanism for the activation is proposed. These results strongly suggest a need to re-evaluate the use of U73122 as a general inhibitor of PLC isozymes.

2-Methoxyestradiol and its derivatives inhibit store-operated Ca2+ entry in T cells: Identification of a new and potent inhibitor.

T cell activation starts with formation of second messengers that release Ca2+ from the endoplasmic reticulum (ER) and thereby activate store-operated Ca2+ entry (SOCE), one of the essential signals for T cell activation. Recently, the steroidal 2-methoxyestradiol was shown to inhibit nuclear translocation of the nuclear factor of activated T cells (NFAT). We therefore investigated 2-methoxyestradiol for inhibition of Ca2+ entry in T cells, screened a library of 2-methoxyestradiol analogues, and characterized the derivative 2-ethyl-3-sulfamoyloxy-17ß-cyanomethylestra-1,3,5(10)-triene (STX564) as a novel, potent and specific SOCE inhibitor. STX564 inhibits Ca2+ entry via SOCE without affecting other ion channels and pumps involved in Ca2+ signaling in T cells. Downstream effects such as cytokine expression and cell proliferation were also inhibited by both 2-methoxyestradiol and STX564, which has potential as a new chemical biology tool.

3-O-phosphate ester conjugates of 17-ß-O-{1-[2-carboxy-(2-hydroxy-4-methoxy-3-carboxamido)anilido]ethyl}1,3,5(10)-estratriene as novel bone-targeting agents.

A series of 3-O-phosphorylated analogs (4-10) of a novel bone-targeting estradiol analog (3) were synthesized after a thorough study of the reaction of 3 with a selection of phosphoryl chlorides under a variety of reaction conditions. Evaluation of these novel phosphate analogs for affinity for hydroxyapatite revealed that they bind with equal or higher affinity when compared to the bone tissue accumulator, tetracycline.

In vitro effects of 2-methoxyestradiol-bis-sulphamate on the non-tumorigenic MCF-12A cell line.

A priority in recent anti-cancer drug development has been attaining better side-effect profiles for potential compounds. To produce highly specific cancer therapies it is necessary to understand both the effects of the proposed compound on cancer and on normal cells comprising the rest of the human body. Thus in vitro evaluation of these compounds against non-carcinogenic cell lines is of critical importance. One of the most recent developments in experimental anti-cancer agents is 2-methoxyestradiol-bis-sulphamate (2ME-BM), a sulphamoylated derivative of 2-methoxyestradiol. The aim of this study was to evaluate the in vitro effects of 2ME-BM on cell proliferation, morphology and mechanisms of cell death in the non-carcinogenic MCF-12A breast epithelial cell line. The study revealed changes in proliferative capacity, morphology and cell death induction in response to 2ME-BM exposure (24 h at 0.4 microM). Microscopy showed decreased cell density and cell death-associated morphology (increased apoptotic characteristics), a slight increase in acidic intracellular vesicles and insignificant ultra-structural aberrations. Mitotic indices revealed a G(2)M-phase cell cycle block. This was confirmed by flow cytometry, where an increased fraction of abnormal cells and a decrease in cyclin B1 levels were observed. These results evidently demonstrate that the non-carcinogenic MCF-12A cell line is less susceptible when compared to 2ME-BM-exposed cancer cell lines previously tested. Further in vitro research into the mechanism of this potentially useful compound is warranted.

Synthesis, antiproliferative, and pharmacokinetic properties of 3- and 17-double-modified analogs of 2-methoxyestradiol.

The syntheses of 21 analogs of 2-methoxyestradiol are presented, including ENMD-1198 which was selected for advancement into Phase 1 clinical trials in oncology. These analogs were evaluated for antiproliferative activity using breast tumor MDA-MB-231 cells, for antiangiogenic activity in HUVEC proliferation assays, and for estrogenic activity in MCF-7 cell proliferation. The most active analogs were evaluated for iv and oral pharmacokinetic properties via cassette dosing in rat and in mice pharmacokinetic models.

Significant antitumor activity in vivo following treatment with the microtubule agent ENMD-1198.

Clinical studies using the microtubule-targeting agent 2-methoxyestradiol (2ME2; Panzem) in cancer patients show that treatment is associated with clinical benefit, including prolonged stable disease, complete and partial responses, and an excellent safety profile. Studies have shown that 2ME2 is metabolized by conjugation at positions 3 and 17 and oxidation at position 17. To define structure-activity relationships for these positions of 2ME2 and to generate metabolically stable analogues with improved anti-tubulin properties, a series of analogues was generated and three lead analogues were selected, ENMD-1198, ENMD-1200, and ENMD-1237. These molecules showed improved metabolic stability with >65% remaining after 2-h incubation with hepatocytes. Pharmacokinetic studies showed that oral administration of the compounds resulted in increased plasma levels compared with 2ME2. All three analogues bind the colchicine binding site of tubulin, induce G(2)-M cell cycle arrest and apoptosis, and reduce hypoxia-inducible factor-1alpha levels. ENMD-1198 and ENMD-1200 showed improved in vitro antiproliferative activities. Significant reductions in tumor volumes compared with vehicle-treated mice were observed in an orthotopic breast carcinoma (MDA-MB-231) xenograft model following daily oral treatment with all compounds (ANOVA, P < 0.05). Significantly improved median survival time was observed with ENMD-1198 and ENMD-1237 (200 mg/kg/d) in a Lewis lung carcinoma metastatic model (P < 0.05). In both tumor models, the high-dose group of ENMD-1198 showed antitumor activity equivalent to that of cyclophosphamide. ENMD-1198 was selected as the lead molecule in this analogue series and is currently in a phase I clinical trial in patients with refractory solid tumors.

Sorption and transport of trenbolone and altrenogest photoproducts in soil-water systems.

This study evaluated the sorption and transport potential of seven phototransformation products of 17α-trenbolone, 17ß-trenbolone, trendione, and altrenogest, along with the parent trienone steroids in batch and column soil-water systems. In batch systems, the target solutes exhibited linear isotherms, with values for sorption coefficients (log Koc) of parent steroids (2.46-2.76) higher than those for photoproducts (1.92-2.57). In column systems, the estimated retardation factors (Rsol) for parents (2.7-5.1) were ∼2-5 times higher than those for photoproducts (0.84-1.7). The log Koc (R2 = 0.75) and Rsol (R2 = 0.89-0.98) were well correlated with measured log Kow values, indicating that hydrophobic partitioning governed the soil-solute interaction of these biologically potent compounds in soil-water systems. These data indicated that photoproducts exhibited reduced sorption affinity and increased transport potential relative to more hydrophobic parent structures. In agroecosystems, traditional runoff management practices would be expected to exhibit reduced treatment effectiveness for photoproducts relative to the parent compounds of commonly used trienone steroids.

Inhibitory mechanism of 17ß-aminoestrogens in the formation of Aß aggregates.

Alzheimer's disease (AD) is a complex neurodegenerative disorder associated with the aggregation of the amyloid-beta peptide (Aß) into large oligomers and fibrils that damage healthy brain cells. The predominant peptide fragments in the plaques are mainly formed by the Aß1-40 and Aß1-42 peptides, albeit the eleven-residue Aß25-35 segment is largely used in biological studies because it retains the neurotoxic properties of the longer Aß peptides. Recent studies indicate that treatment with therapeutic steroid hormones reduces the progress of the disease in AD models. Particularly, treatment with 17ß-aminoestrogens (AEs) has shown a significant alleviation of the AD development by inhibiting oxidative stress and neuronal death. Yet, the mechanism by which the AE molecules exhibit their beneficial effects remains speculative. To shed light into the molecular mechanism of inhibition of the AD development by AEs, we investigated the possibility of direct interaction with the Aß25-35 peptide. First, we calculate various interacting electronic properties of three AE derivatives as follows: prolame, butolame, and pentolame by performing DFT calculations. To account for the polymorphic nature of the Aß aggregates, we considered four different Aß25-35 systems extracted from AD relevant fibril structures. From the calculation of different electron density properties, specific interacting loci were identified that guided the construction and optimization of various complexes. Interestingly, the results suggest a similar inhibitory mechanism based on the direct interaction between the AEs and the M35 residue that seems to be general and independent of the polymorphic properties of the Aß aggregates. Our analysis of the complex formation provides a structural framework for understanding the AE therapeutic properties in the molecular inhibitory mechanism of Aß aggregation.

3,17ß-Bis-sulfamoyloxy-2-methoxyestra-1,3,5(10)-triene and Nonsteroidal Sulfamate Derivatives Inhibit Carbonic Anhydrase IX: Structure-Activity Optimization for Isoform Selectivity.

3,17ß-Bis-sulfamoyloxy-2-methoxyestra-1,3,5(10)-triene (STX140), a bis-sulfamate derivative of the endogenous steroid 2-methoxyestradiol, has shown promising anticancer potency both in vitro and in vivo, with excellent bioavailability. Its activity against taxane-resistant xenografts makes it a potential drug candidate against triple-negative breast cancer (TNBC). These properties are linked to the ability of STX140 to act in a multitargeting fashion in vivo as a microtubule disruptor, leading to cell cycle arrest and with both proapoptotic and anti-angiogenic activities. Carbonic anhydrase IX (CA IX) is a well-established biomarker for aggressive cancers, including TNBC. This study reports, for the first time, the inhibitory activities of a series of steroidal and nonsteroidal sulfamate derivatives against CA IX in comparison to the ubiquitous CA II, with some compounds demonstrating 100-200-fold selectivity for CA IX over CA II. X-ray crystallographic studies of four of the most promising compounds reveal that isoform-specific residue interactions are responsible for the high specificity.

Structure-activity relationships of C-17 cyano-substituted estratrienes as anticancer agents.

The synthesis, SAR, and preclinical evaluation of 17-cyanated 2-substituted estra-1,3,5(10)-trienes as anticancer agents are discussed. 2-Methoxy-17beta-cyanomethylestra-1,3,5(10)-trien-3-ol ( 14), but not the related 2-ethyl derivative 7, and the related 3- O-sulfamates 8 and 15 display potent antiproliferative effects (MCF-7 GI 50 300, 60 and 70 nM, respectively) against human cancer cells in vitro. Investigation of the SAR reveals that a sterically unhindered hydrogen bond acceptor attached to C-17 is most likely key to the enhanced activity. Compound 8 displayed significant in vitro antiangiogenic activity, and its ability to act as a microtubule disruptor was confirmed. Inhibitory activity of the sulfamate derivatives against steroid sulfatase and carbonic anhydrase II (hCAII) was also observed, and the interaction between 15 and hCAII was investigated by protein crystallography. The potential of these multimechanism anticancer agents was confirmed in vivo, with promising activity observed for both 14 and 15 in an athymic nude mouse MDA-MB-231 human breast cancer xenograft model.

A new generation of progesterone receptor modulators.

Progesterone receptor (PR) modulators have evolved both structurally and mechanistically over the past half-century. Classical steroidal PR agonists continue to play an important role in women's health such as in oral contraception and post-menopausal hormone therapy whereas steroid-based PR antagonists and selective PR modulators are being evaluated clinically in a wide range of gynecologic conditions. This review will focus primarily on the newer generation of PR modulators derived from structurally unique chemical scaffolds. For example, tanaproget (TNPR) is described as a non-steroidal PR agonist with high affinity and selectivity for the PR that is significantly more potent than many of its steroidal counterparts in a variety of pre-clinical efficacy models. Similarly, we present numerous examples of unique non-steroidal PR antagonists in various stages of characterization and development. A basic understanding of the structural determinants for high affinity binding of these new PR modulators to the PR ligand-binding domain (LBD) is also discussed. Finally, as the biology of the PR becomes further defined, we speculate on the future development of novel PR modulators.

A structural and in vitro characterization of asoprisnil: a selective progesterone receptor modulator.

Selective progesterone receptor modulators (SPRMs) have been suggested as therapeutic agents for treatment of gynecological disorders. One such SPRM, asoprisnil, was recently in clinical trials for treatment of uterine fibroids and endometriosis. We present the crystal structures of progesterone receptor (PR) ligand binding domain complexed with asoprisnil and the corepressors nuclear receptor corepressor (NCoR) and SMRT. This is the first report of steroid nuclear receptor crystal structures with ligand and corepressors. These structures show PR in a different conformation than PR complexed with progesterone (P4). We profiled asoprisnil in PR-dependent assays to understand further the PR-mediated mechanism of action. We confirmed previous findings that asoprisnil demonstrated antagonism, but not agonism, in a PR-B transfection assay and the T47D breast cancer cell alkaline phosphatase activity assay. Asoprisnil, but not RU486, weakly recruited the coactivators SRC-1 and AIB1. However, asoprisnil strongly recruited the corepressor NCoR in a manner similar to RU486. Unlike RU486, NCoR binding to asoprisnil-bound PR could be displaced with equal affinity by NCoR or TIF2 peptides. We further showed that it weakly activated T47D cell gene expression of Sgk-1 and PPL and antagonized P4-induced expression of both genes. In rat leiomyoma ELT3 cells, asoprisnil demonstrated partial P4-like inhibition of cyclooxygenase (COX) enzymatic activity and COX-2 gene expression. In the rat uterotrophic assay, asoprisnil demonstrated no P4-like ability to oppose estrogen. Our data suggest that asoprisnil differentially recruits coactivators and corepressors compared to RU486 or P4, and this specific cofactor interaction profile is apparently insufficient to oppose estrogenic activity in rat uterus.

Combined Biophysical Chemistry Reveals a New Covalent Inhibitor with a Low-Reactivity Alkyl Halide.

17ß-Hydroxysteroid dehydrogenase type 1 (17ß-HSD1) plays a pivotal role in the progression of estrogen-related diseases because of its involvement in the biosynthesis of estradiol (E2), constituting a valuable therapeutic target for endocrine treatment. In the present study, we successfully cocrystallized the enzyme with the reversible inhibitor 2-methoxy-16ß-( m-carbamoylbenzyl)-E2 (2-MeO-CC-156) as well as the enzyme with the irreversible inhibitor 3-(2-bromoethyl)-16ß-( m-carbamoylbenzyl)-17ß-hydroxy-1,3,5(10)-estratriene (PBRM). The structures of ternary complexes of 17ß-HSD1-2-MeO-CC-156-NADP+ and 17ß-HSD1-PBRM-NADP+ comparatively show the formation of a covalent bond between His221 and the bromoethyl side chain of the inhibitor in the PBRM structure. A dynamic process including beneficial molecular interactions that favor the specific binding of a low-reactivity inhibitor and subsequent N-alkylation event through the participation of His221 in the enzyme catalytic site clearly demonstrates the covalent bond formation. This finding opens the door to a new design of alkyl halide-based specific covalent inhibitors as potential therapeutic agents for different enzymes, contributing to the development of highly efficient inhibitors.

[Synthesis, structure, and biological properties of some 8alpha-analogues of steroid estrogens with fluorine in position 2].

A total synthesis of 8alpha analogues of steroid estrogens with fluorine in position 2 was achieved. Structural features of these compounds were studied by the example of 17beta-acetoxy-2-fluoro-3-methoxy-8alpha-estra-1,3,5(10)-triene. It was shown that the 8alpha analogues of 2-fluorosubstituted steroid estrogens have a low uterotropic activity and retain the osteoprotective and hypocholesterolemic activities.

Evidence of boldenone, nandrolone, 5(10)-estrene-3ß-17α-diol and 4-estrene-3,17-dione as minor metabolites of testosterone in equine.

The detection of boldenone, nandrolone, 5(10)-estrene-3ß,17α-diol, and 4-estrene-3,17-dione in a urine sample collected from a gelding having been treated with testosterone (500 mg 'Testosterone Suspension 100', single dose, injected intramuscularly) in 2009 led the authors' laboratory to suspect that these 'testicular' steroids could be minor metabolites of testosterone in geldings. Administration trials on six castrated horses with Testosterone Suspension 100 confirmed that low levels of boldenone, nandrolone, 5(10)-estrene-3ß,17α-diol, and 4-estrene-3,17-dione could indeed be detected and confirmed in the early post-administration urine samples from all six geldings. Although boldenone has been reported to be present in urine after testosterone administration, there has been no direct evidence reported that boldenone, nandrolone, 5(10)-estrene-3ß,17α-diol, and 4-estrene-3,17-dione are metabolites of testosterone in geldings. Subsequent in vitro experiments involving the incubation of testosterone with horse liver microsomes, liver, adipose and muscle tissues, and adrenal cortex homogenates failed to provide evidence that these four substances are minor metabolites of testosterone. An administration trial using 'Testosterone Suspension 100' supplemented with 13 C-labelled testosterone (500 mg, 1:1 ratio, injected intramuscularly) was performed. The similarities of the excretion curves of 12 C-testosterone and 13 C-testosterone in urine suggest that there was minimal kinetic isotope effect. 13 C-Labelled boldenone, nandrolone and 4-estrene-3,17-dione were detected but not 5(10)-estrene-3ß,17α-diol and its 13 C-counterpart. This is the first unequivocal evidence of boldenone, nandrolone and 4-estrene-3,17-dione being metabolites of testosterone in geldings. In view of these results, caution should be exercised when interpreting findings of boldenone, nandrolone and/or 4-estrene-3,17-dione together with a relatively high level of testosterone in gelding urine. Copyright © 2017 John Wiley & Sons, Ltd.

Sorption and desorption of 17α-trenbolone and trendione on five soils.

The metabolites 17α-trenbolone and 17α-estradiol are principal metabolites in cattle excreta following the administration of Synovex® ONE, which contains trenbolone acetate and estradiol benzoate. As part of the environmental assessment of the use of Synovex ONE, data were generated to characterize the fate of 17α-trenbolone, and its metabolite trendione in the environment. Predictions of the fate and environmental concentrations of these hormones after land application require accurate estimates of the sorption of these compounds in soils. The sorption and desorption of 17α-trenbolone and trendione were measured at 5 nominal concentrations in 5 soils from different geologic settings using a batch equilibrium technique following guideline 106 of the Organisation for Economic Co-operation and Development. Both the sorption and desorption of 17α-trenbolone and trendione to soils were adequately described by the Freundlich sorption model and by linear partition coefficients. The mean sorption coefficients were 9.04 mL/g and 32.2 mL/g for 17α-trenbolone and trendione, respectively. The corresponding mean Freundlich sorption exponents were 0.88 and 0.98, respectively. Sorption of 17α-trenbolone and trendione was correlated principally with soil organic carbon. Average sorption coefficients normalized to soil organic carbon content (KOC ) were 460 mL/g and 1804 mL/g for 17α-trenbolone and trendione, respectively. The mean desorption coefficients were 22.1 mL/g and 43.8 mL/g for 17α-trenbolone and trendione, respectively. Calculated hysteresis coefficients based on the difference in the area between sorption and desorption isotherms indicated that sorption equilibrium was not fully reversible and hysteresis of desorption isotherms occurred for both 17α-trenbolone and trendione. Environ Toxicol Chem 2017;36:613-620. © 2016 SETAC.