Light-Driven Two-Step Preparation of 4-Chromanone Fused to Estrone Derivatives.

A protocol involving the irradiation of some 3-(2-alkenyl)estrone and 3-(2-alkenyl)-17-norestrone derivatives under a nitrogen atmosphere in organic solvents (both hexane and MeOH) followed by base-mediated intramolecular oxa-Michael cyclization reaction was investigated under steady-state conditions. The solvent effect and nature of the acyl group on the preparative photoreaction were studied and the multiplicity of the excited state was also demonstrated. The ortho-regioisomers were obtained in modest to good yields. Intramolecular based-mediate cyclization reaction of these synthons led to the formation of a set of novel substituted 4-chromanone moieties fused to estrone (and 17-norestrone) in good yields. This two-step sequential procedure involving a photochemical/intramolecular thermal cyclization strategy will be useful for the preparation of wide heterocyclic-fused-steroid compounds.

Direct Cysteine Desulfurase Activity Determination by NMR and the Study of the Functional Role of Key Structural Elements of Human NFS1.

The mitochondrial cysteine desulfurase NFS1 is an essential PLP-dependent enzyme involved in iron-sulfur cluster assembly. The enzyme catalyzes the desulfurization of the l-Cys substrate, producing a persulfide and l-Ala as products. In this study, we set the measurement of the product l-Ala by NMR in vitro by means of 1H NMR spectra acquisition. This methodology provided us with the possibility of monitoring the reaction in both fixed-time and real-time experiments, with high sensitivity and accuracy. By studying I452A, W454A, Q456A, and H457A NFS1 variants, we found that the C-terminal stretch (CTS) of the enzyme is critical for function. Specifically, mutation of the extremely conserved position W454 resulted in highly decreased activity. Additionally, we worked on two singular variants: "GGG" and C158A. In the former, the catalytic Cys-loop was altered by including two Gly residues to increase the flexibility of this loop. This variant had significantly impaired activity, indicating that the Cys-loop motions are fine-tuned in the wild-type enzyme. In turn, for C158A, we found an unanticipated increase in l-Cys desulfurase activity. Furthermore, we carried out molecular dynamics simulations of the supercomplex dedicated to iron-sulfur cluster biosynthesis, which includes NFS1, ACP, ISD11, ISCU2, and FXN subunits. We identified CTS as a key element that established interactions with ISCU2 and FXN concurrently; we found specific interactions that are established when FXN is present, reinforcing the idea that FXN not only forms part of the iron-sulfur cluster assembly site but also modulates the internal motions of ISCU2.

Beyond Pseudo-natural Products: Sequential Ugi/Pictet-Spengler Reactions Leading to Steroidal Pyrazinoisoquinolines That Trigger Caspase-Independent Death in HepG2 Cells.

In this work, we describe how stereochemically complex polycyclic compounds can be generated by applying a synthetic sequence comprising an intramolecular Ugi reaction followed by a Pictet-Spengler cyclization on steroid-derived scaffolds. The resulting compounds, which combine a fragment derived from a natural product and a scaffold not found in nature. are both structurally distinct and globally similar to natural products at the same time, and interrogate an alternative region of the chemical space. One of the new compounds showed significant antiproliferative activity on HepG2 cells through a caspase-independent cell-death mechanism, an appealing feature when new antitumor compounds are searched.

Tracing molecular properties throughout evolution: A chemoinformatic approach.

Evolution of metabolism is a longstanding yet unresolved question, and several hypotheses were proposed to address this complex process from a Darwinian point of view. Modern statistical bioinformatic approaches targeted to the comparative analysis of genomes are being used to detect signatures of natural selection at the gene and population level, as an attempt to understand the origin of primordial metabolism and its expansion. These studies, however, are still mainly centered on genes and the proteins they encode, somehow neglecting the small organic chemicals that support life processes. In this work, we selected steroids as an ancient family of metabolites widely distributed in all eukaryotes and applied unsupervised machine learning techniques to reveal the traits that natural selection has imprinted on molecular properties throughout the evolutionary process. Our results clearly show that sterols, the primal steroids that first appeared, have more conserved properties and that, from then on, more complex compounds with increasingly diverse properties have emerged, suggesting that chemical diversification parallels the expansion of biological complexity. In a wider context, these findings highlight the worth of chemoinformatic approaches to a better understanding the evolution of metabolism.

Photochemical Behavior of Some Estrone Aryl and Methyl Sulfonates in Solution: Preparative and Mechanistic Studies.

Direct irradiation of estrone aryl and methyl sulfonates in different organic solvents under nitrogen atmosphere was investigated under steady-state conditions. The estrone derivatives reacted efficiently through the photo-Fries rearrangement reaction involving [1;3]-sulfonyl migration providing the ortho-sulfonyl estrone derivatives and estrone as the photoproducts. In addition, estrone and 2-arylsulfonyl estrone derivatives were epimerized involving a Norrish Type-I reaction. Chemical quenching and photosensitization experiments on the photoreaction have been also carried out to establish the photoreactive excited state. Likewise, the solvent effect and the nature of the sulfonyl group on the photoreactions have been also studied.

Development of an Improved Guanidine-Based Rac1 Inhibitor with in†vivo Activity against Non-Small Cell Lung Cancer.

The Rho GTPase Rac1 is involved in the control of cytoskeleton reorganization and other fundamental cellular functions. Aberrant activity of Rac1 and its regulators is common in human cancer. In particular, deregulated expression/activity of Rac GEFs, responsible for Rac1 activation, has been associated to a metastatic phenotype and drug resistance. Thus, the development of novel Rac1-GEF interaction inhibitors is a promising strategy for finding new preclinical candidates. Here, we studied structure-activity relationships within a new family of N,N'-disubstituted guanidine as Rac1 inhibitors. We found that compound 1D-142, presents superior antiproliferative activity in human cancer cell lines and higher potency as Rac1-GEF interaction inhibitor in vitro than parental compounds. In addition, 1D-142 reduces Rac1-mediated TNFα-induced NF-κB nuclear translocation during cell proliferation and migration in NSCLC. Notably, 1D-142 allowed us to show for the first time the application of a Rac1 inhibitor in a lung cancer animal model.

Validation and development of models using clinical, biochemical and ultrasound markers for predicting pre-eclampsia: an individual participant data meta-analysis.

BACKGROUND: Pre-eclampsia is a leading cause of maternal and perinatal mortality and morbidity. Early identification of women at risk is needed to plan management. OBJECTIVES: To assess the performance of existing pre-eclampsia prediction models and to develop and validate models for pre-eclampsia using individual participant data meta-analysis. We also estimated the prognostic value of individual markers. DESIGN: This was an individual participant data meta-analysis of cohort studies. SETTING: Source data from secondary and tertiary care. PREDICTORS: We identified predictors from systematic reviews, and prioritised for importance in an international survey. PRIMARY OUTCOMES: Early-onset (delivery at < 34 weeks' gestation), late-onset (delivery at ≥ 34 weeks' gestation) and any-onset pre-eclampsia. ANALYSIS: We externally validated existing prediction models in UK cohorts and reported their performance in terms of discrimination and calibration. We developed and validated 12 new models based on clinical characteristics, clinical characteristics and biochemical markers, and clinical characteristics and ultrasound markers in the first and second trimesters. We summarised the data set-specific performance of each model using a random-effects meta-analysis. Discrimination was considered promising for C-statistics of ≥ 0.7, and calibration was considered good if the slope was near 1 and calibration-in-the-large was near 0. Heterogeneity was quantified using I2 and τ2. A decision curve analysis was undertaken to determine the clinical utility (net benefit) of the models. We reported the unadjusted prognostic value of individual predictors for pre-eclampsia as odds ratios with 95% confidence and prediction intervals. RESULTS: The International Prediction of Pregnancy Complications network comprised 78 studies (3,570,993 singleton pregnancies) identified from systematic reviews of tests to predict pre-eclampsia. Twenty-four of the 131 published prediction models could be validated in 11 UK cohorts. Summary C-statistics were between 0.6 and 0.7 for most models, and calibration was generally poor owing to large between-study heterogeneity, suggesting model overfitting. The clinical utility of the models varied between showing net harm to showing minimal or no net benefit. The average discrimination for IPPIC models ranged between 0.68 and 0.83. This was highest for the second-trimester clinical characteristics and biochemical markers model to predict early-onset pre-eclampsia, and lowest for the first-trimester clinical characteristics models to predict any pre-eclampsia. Calibration performance was heterogeneous across studies. Net benefit was observed for International Prediction of Pregnancy Complications first and second-trimester clinical characteristics and clinical characteristics and biochemical markers models predicting any pre-eclampsia, when validated in singleton nulliparous women managed in the UK NHS. History of hypertension, parity, smoking, mode of conception, placental growth factor and uterine artery pulsatility index had the strongest unadjusted associations with pre-eclampsia. LIMITATIONS: Variations in study population characteristics, type of predictors reported, too few events in some validation cohorts and the type of measurements contributed to heterogeneity in performance of the International Prediction of Pregnancy Complications models. Some published models were not validated because model predictors were unavailable in the individual participant data. CONCLUSION: For models that could be validated, predictive performance was generally poor across data sets. Although the International Prediction of Pregnancy Complications models show good predictive performance on average, and in the singleton nulliparous population, heterogeneity in calibration performance is likely across settings. FUTURE WORK: Recalibration of model parameters within populations may improve calibration performance. Additional strong predictors need to be identified to improve model performance and consistency. Validation, including examination of calibration heterogeneity, is required for the models we could not validate. STUDY REGISTRATION: This study is registered as PROSPERO CRD42015029349. FUNDING: This project was funded by the National Institute for Health Research (NIHR) Health Technology Assessment programme and will be published in full in Health Technology Assessment; Vol. 24, No. 72. See the NIHR Journals Library website for further project information.

WHAT IS THE PROBLEM?: Pre-eclampsia, a condition in pregnancy that results in raised blood pressure and protein in the urine, is a major cause of complications for the mother and baby. WHAT IS NEEDED?: A way of accurately identifying women at high risk of pre-eclampsia to allow clinicians to start preventative interventions such as administering aspirin or frequently monitoring women during pregnancy. WHERE ARE THE RESEARCH GAPS?: Although over 100 tools (models) have been reported worldwide to predict pre-eclampsia, to date their performance in women managed in the UK NHS is unknown. WHAT DID WE PLAN TO DO?: We planned to comprehensively identify all published models that predict the risk of pre-eclampsia occurring at any time during pregnancy and to assess if this prediction is accurate in the UK population. If the existing models did not perform satisfactorily, we aimed to develop new prediction models. WHAT DID WE FIND?: We formed the International Prediction of Pregnancy Complications network, which provided data from a large number of studies (78 studies, 25 countries, 125 researchers, 3,570,993 singleton pregnancies). We were able to assess the performance of 24 out of the 131 models published to predict pre-eclampsia in 11 UK data sets. The models did not accurately predict the risk of pre-eclampsia across all UK data sets, and their performance varied within individual data sets. We developed new prediction models that showed promising performance on average across all data sets, but their ability to correctly identify women who develop pre-eclampsia varied between populations. The models were more clinically useful when used in the care of first-time mothers pregnant with one child, compared to a strategy of treating them all as if they were at high-risk of pre-eclampsia. WHAT DOES THIS MEAN?: Before using the International Prediction of Pregnancy Complications models in various populations, they need to be adjusted for characteristics of the particular population and the setting of application.

Innovative Multipodal Ligands Derived from Tröger's Bases for the Sensitization of Lanthanide(III) Luminescence.

Herein, the synthesis and characterization of the first family of multipodal ligands with a Tröger's base framework designed for the preparation of luminescent lanthanide(III) complexes are reported. Eight ligands were designed and synthesized using different strategies, including alkylation reactions, amide couplings, and Ugi multicomponent reactions. All the ligands bear carboxylate groups for the coordination of the lanthanide(III) ions, with the lanthanide(III)-sensitizing units consisting of the Tröger's base framework itself or attached benzamides. Upon irradiation of the chromophoric ligands, green terbium(III) emission was efficiently generated, whereas europium(III) emission was negligible. The geometry and substitution pattern of the ligands allow control of the stoichiometry of the species formed and the TbIII luminescence sensitization efficiency, showing that para-substitution patterns are more efficient than meta substitution for the formation of coordination compounds with lower TbIII /ligand ratio. We propose that the species formed are self-assembled 2:2 or 2:4 metallosupramolecular structures.

A Six-Armed Phenhomazine Ligand with a Potential "Turn-Off" Copper(II) Sensing Capability through Terbium(III) Luminescence Quenching.

Herein, the design, synthesis, and characterization of a phenhomazine ligand are described. The ligand has six pendant acetate arms designed for the combined coordination of copper(II) and lanthanide(III) ions, with the perspective of developing a "turn-off" copper sensor. The key step for the ligand preparation was the one-step endomethylene bridge fission of a diamino Tröger's base with a concomitant alkylation. Fluorescence and absorption spectroscopies as well as nuclear magnetic resonance (NMR) experiments were performed to analyze and understand the coordination properties of the ligand. Transition metal coordination was driven by the synergistic effect of the free nitrogen atoms of the diazocinic core and the two central acetate arms attached to those nitrogen atoms, whereas lanthanide coordination is performed by the external acetate arms, presumably forming a self-assembled 2:2 metallosupramolecular structure. The terbium complex shows the typical green emission with narrow bands and long luminescence lifetimes. The luminescence quenching produced by the presence of copper(II) ions was analyzed. This work sets, therefore, a starting point for the development of a phenhomazine-based "turn-off" copper(II) sensor.

Steroid diversification by multicomponent reactions.

Reports on structural diversification of steroids by means of multicomponent reactions (MCRs) have significantly increased over the last decade. This review covers the most relevant strategies dealing with the use of steroidal substrates in MCRs, including the synthesis of steroidal heterocycles and macrocycles as well as the conjugation of steroids to amino acids, peptides and carbohydrates. We demonstrate that steroids are available with almost all types of MCR reactive functionalities, e.g., carbonyl, carboxylic acid, alkyne, amine, isocyanide, boronic acid, etc., and that steroids are suitable starting materials for relevant MCRs such as those based on imine and isocyanide. The focus is mainly posed on proving the amenability of MCRs for the diversity-oriented derivatization of naturally occurring steroids and the construction of complex steroid-based platforms for drug discovery, chemical biology and supramolecular chemistry applications.

Photo-Fries Rearrangement of Some 3-Acylestrones in Homogeneous Media: Preparative and Mechanistic Studies.

Irradiation of a series of 3-acylestrones under a nitrogen atmosphere in cyclohexane, acetonitrile (MeCN), and methanol (MeOH) was investigated under steady-state conditions. The molecules underwent the photo-Fries rearrangement, with concomitant homolytic fragmentation of the ester group and [1;3]-acyl migration. This pathway afforded the ortho-acyl estrone derivatives, the main photoproducts, together with estrone. During the irradiation of 3-benzoyl estrone, epimerization of estrone through the Norrish type I reaction occurred, providing lumiestrone as the photoproduct. This photoreaction involves the fragmentation of the C-α at the carbonyl group (C-17) of the steroid. On the other hand, epimerization of ortho-regioisomer 2-acetyl estrone occurred during the irradiation of 3-acetyl estrone. Photosensitization with acetone and chemical quenching with N, N, N, N-tetramethyldiazetinedioxide of the photo-Fries reaction confirmed that the photoreaction took place from the singlet excited state while the Norrish type I reaction proceeds efficiently from the triplet excited state. Solvent effects, as well as the nature of the acyl group on the photoreactions, were also studied.

One-step synthesis of N,N'-substituted 4-imidazolidinones by an isocyanide-based pseudo-five-multicomponent reaction.

A pseudo-five-multicomponent reaction involving an isocyanide, a primary amine, two molecules of formaldehyde and water is reported, which gives N,N'-substituted 4-imidazolidinones when trifluoroethanol is used as the solvent. The reaction proceeds with good yields and with a wide variety of amines and isocyanides, providing an efficient new entry to these heterocycles. A preliminary study of the reaction mechanism suggests that trifluoroethanol, although acting as the solvent, is directly involved as a reagent in the reaction pathway.

A Minimalist Approach to the Design of Complexity-Enriched Bioactive Small Molecules: Discovery of Phenanthrenoid Mimics as Antiproliferative Agents.

Over the last decades, much effort has been devoted to the design of the "ideal" library for screening, the most promising strategies being those which draw inspiration from biogenic compounds, as the aim is to add biological relevance to such libraries. On the other hand, there is a growing understanding of the role that molecular complexity plays in the discovery of new bioactive small molecules. Nevertheless, the introduction of molecular complexity must be balanced with synthetic accessibility. In this work, we show that both concepts can be efficiently merged-in a minimalist way-by using very simple guidelines during the design process along with the application of multicomponent reactions as key steps in the synthetic process. Natural phenanthrenoids, a class of plant aromatic metabolites, served as inspiration for the synthesis of a library in which complexity-enhancing features were introduced in few steps using multicomponent reactions. These resulting chemical entities were not only more complex than the parent natural products, but also interrogated an alternative region of the chemical space, which led to an outstanding hit rate in an antiproliferative assay: four out of twenty-six compounds showed in vitro activity, one of them being more potent than the clinically useful drug 5-fluorouracil.

Synthetic stigmastanes with dual antiherpetic and immunomodulating activities inhibit ERK and Akt signaling pathways without binding to glucocorticoid receptors.

BACKGROUND: We have previously shown that some synthetic hydroxylated stigmastanes derived from plant sterols inhibit in vitro HSV-1 replication in ocular cell lines and decrease cytokine production in stimulated macrophages, suggesting that these steroids might combine antiviral and immunomodulating properties. In this paper we report the synthesis of some analogs fluorinated at C-6 in order to study the effect of this modification on bioactivity. METHODS: The following methods were used: organic synthesis of fluorinated analogs, cytotoxicity determination with MTT assays, cytokine production quantification with ELISAs, glucocorticoid activity determination by displacement assays, immunofluorescence and transcriptional activity assays, studies of the activation of signaling pathways by Western blot, antiviral activity evaluation through virus yield reduction assays. RESULTS: We report the chemical synthesis of new fluorinated stigmastanes and show that this family of steroidal compounds exerts its immunomodulating activity by inhibiting ERK and Akt signaling pathways, but do not act as glucocorticoids. We also demonstrate that fluorination enhances the antiviral activity. CONCLUSIONS: Fluorination on C-6 did not enhance the anti-inflammatory effect, however, an increase in the in vitro antiviral activity was observed. Thus, our results suggest that it is possible to introduce chemical modifications on the parent steroids in order to selectively modulate one of the effects. GENERAL SIGNIFICANCE: This family of steroids could allow the development of an alternative treatment for ocular immunopathologies triggered by HSV-1, without the undesirable side effects of the currently used drugs.

Synthetic pregnenolone derivatives as antiviral agents against acyclovir-resistant isolates of Herpes Simplex Virus Type 1.

The conventional therapy for the management of Herpes Simplex Virus Type 1 (HSV-1) infections mainly comprises acyclovir (ACV) and other nucleoside analogues. A common outcome of this treatment is the emergence of resistant viral strains, principally when immunosuppressed patients are involved. Thus, the development of new antiherpetic compounds remains as a central challenge. In this work we describe the synthesis and the in vitro antiherpetic activity of a new family of steroidal compounds derived from the endogenous hormone pregnenolone. Some of these derivatives showed a remarkable inhibitory effect on HSV-1 spread both on wild type and ACV-resistant strains. The results also show that these compounds seem to interfere with the late steps of the viral cycle.

Small Molecules as Anti-TNF Drugs.

Tumor necrosis factor (TNF, TNF-α, cachectin) is a pleiotropic, proinflammatory cytokine with multiple biological effects, many of which are not yet fully understood. Although TNF was initially described as an anti-tumor agent more than three decades ago, current knowledge places it central to immune system homeostasis. TNF plays a critical role in host defense against infection, as well as an inhibitory role in autoimmune disease. However, TNF overproduction generates deleterious effects by inducing the transcription of genes involved in acute and chronic inflammatory responses including asthma, rheumatoid arthritis, Crohn's disease, and psoriasis. Direct inhibition of TNF by biologics, such as monoclonal antibodies and circulating TNF receptor constructs, has produced effective treatments for these disorders and validated the inhibition of this proinflammatory cytokine as an effective therapy. Unfortunately, these biological therapies suffer from several drawbacks, including high cost and the induction of autoantibody production. Thus, the development of small molecules able to modulate TNF production or signaling pathways remains a central challenge in Medicinal Chemistry. Considerable efforts have been made over the past two decades to develop such inhibitors, which could potentially be administered orally and would presumably be cheaper. This review is focused on the recent development of compounds that modulate the activity of this cytokine by acting at different levels, such as TNF expression, processing, binding to its receptors and direct inhibition. These approaches will be compared and discussed.

A bioassay for brassinosteroid activity based on the in vitro fluorimetric detection of nitric oxide production.

Recent studies have shown that low concentrations of brassinolide induce a rapid generation of nitric oxide in mesophyll cells of maize leaves, which can be easily detected by fluorimetric methods. In this work we describe a series of natural and synthetic brassinosteroids that are able to trigger in vitro NO production in tomato cells that exhibits dose-response behavior. We propose that this effect can be used to develop a new rapid and very sensitive bioassay for brassinosteroid activity that offers several advantages when compared to the current methodologies.

Polynomial law for controlling the generation of n-scroll chaotic attractors in an optoelectronic delayed oscillator.

Controlled transitions between a hierarchy of n-scroll attractors are investigated in a nonlinear optoelectronic oscillator. Using the system's feedback strength as a control parameter, it is shown experimentally the transition from Van der Pol-like attractors to 6-scroll, but in general, this scheme can produce an arbitrary number of scrolls. The complexity of every state is characterized by Lyapunov exponents and autocorrelation coefficients.

Multicomponent synthesis of 4,4-dimethyl sterol analogues and their effect on eukaryotic cells.

Most sterols, such as cholesterol and ergosterol, become functional only after the removal of the two methyl groups at C-4 from their biosynthetic precursors. Nevertheless, some findings suggest that 4,4-dimethyl sterols might be involved in specific physiological processes. In this paper we present the synthesis of a collection of analogues of 4,4-dimethyl sterols with a diamide side chain and a preliminary analysis of their in vitro activity on selected biological systems. The key step for the synthesis involves an Ugi condensation, a versatile multicomponent reaction. Some of the new compounds showed antifungal and cytotoxic activity.

Sterol analogues with diamide side chains interfere with the intracellular localization of viral glycoproteins.

The need to develop novel antiviral agents encouraged us to assess the antiviral activity of synthetic sterol analogues with a diamide side chains. Cytotoxicity and antiviral activity of a family of azasterol previously synthesized was evaluated against herpes simplex virus 1 (HSV-1) (KOS and B2006) and vesicular stomatitis virus (VSV). This family of compounds was extended by the synthesis of novel analogs using an Ugi multicomponent reaction and their ability to inhibit viral multiplication was also evaluated. The results show that some of the compounds tested exert an antiviral activity. Besides, the effect of the azasterols on the intracellular localization of viral glycoproteins was examined. Strikingly, alteration on the glycoprotein D (gD) of HSV-1 fluorescence pattern was observed with both the antiherpetic compounds and the inactive azasterols.