[show abstract][hide abstract] ABSTRACT: With the discovery of the bile acid (BA)-activated nuclear and membrane receptors, the role of BAs as signalling molecules in important paracrine and endocrine networks has been fully documented in the last decade. Besides regulating their own synthesis and transport, BAs have been demonstrated being involved in triggering the adaptive response to cholestasis and other insults to liver. More to the point, their recognized ability to control the general energy-related metabolism and inflammation processes has contributed to justify the renewed interest towards this class of amphiphilic steroidal compounds. All these evidences feed a continuing interest in the BA research aimed at designing and synthesizing new side chain- and body-modified derivatives endowed with improved biological and physico-chemical profiles, as well as with proper ADMET behaviour. In this context, the micellar aggregation of BAs, and the respective critical micellization concentration (CMC) value (determined on the BA sodium salt, BS), is considered a key parameter that needs to be determined in the preliminary phase of compound characterization, being implicated in cytotoxicity issues. An extraordinary variety of different analytical techniques and methods have been proposed along the years with the aim of better identifying the start of the self-aggregation process of BS monomers. The unicity of the physico-chemical nature of such class of compounds can be invoked to explain this unusual interest. Accordingly, a number of both invasive and non-invasive approaches have been developed along with a limited number of indirect chromatographic-based estimation strategies. Worth to be mentioned among the non-invasive determination methods are those based on potentiometry, freezing point depression, surface tension, nuclear magnetic resonance, viscosimetry, turbidimetry, microcalorimetry, refractometry, conductimetry, spectrophotometry, cholesterol solubilization, and monoglucuronide solubilization. Dye solubilization- and fluorescence-based methods deserve instead credit among the invasive methodological approaches. Indirect chromatographic methods based on capillary electrophoresis and high performance liquid chromatography analysis also demonstrated to be profitably exploited for the CMC estimation, especially when a small amount of sample is available. The collection of literature data reveals that the CMC value of a given BS is markedly related to the method selected for determining it as well as to the experimental conditions applied during the analysis.
Journal of pharmaceutical and biomedical analysis 07/2013; · 2.45 Impact Factor
[show abstract][hide abstract] ABSTRACT: A rapid and eco-friendly synthesis of a sulfonamide library under flow conditions is described. The study illustrates an efficient, safe and easily scalable preparation of sulfonamides by use of a mesoreactor apparatus, thus demonstrating the impact of flow technologies within drug discovery. Waste minimization, employment of green media and non toxic reactants are achieved by the optimization of the flow set up and experimental protocol designed to sequentially synthesize primary, secondary and tertiary sulfonamides. Isolation of the products involves only extraction and precipitation affording pure compounds in good to high yields without further purification for biological evaluation.
[show abstract][hide abstract] ABSTRACT: Bile acids have emerged as versatile signalling compounds of a complex network of nuclear and membrane receptors regulating various endocrine and paracrine functions. The elucidation of the interconnection between the biological pathways under the bile acid control and manifestations of hepatic and metabolic diseases have extended the scope of this class of steroids for in vivo investigations. In this framework, the design and synthesis of novel biliary derivatives able to modulate a specific receptor requires a deep understanding of both structure-activity and structure-property relationships of bile acids. In this paper, we report the preparation and the critical micellization concentration evaluation of a series of hyodeoxycholic acid derivatives characterized by a diverse side chain length and by the presence of a methyl group at the alpha position with respect to the terminal carboxylic acid moiety. The data collected are instrumental to extend on a quantitative basis, the knowledge of the current structure-property relationships of bile acids and will be fruitful, in combination with models of receptor activity, to design and prioritize the synthesis of novel pharmacokinetically suitable ligands useful in the validation of bile acid-responsive receptors as therapeutic targets.
[show abstract][hide abstract] ABSTRACT: Recent years have seen an increasing awareness that drugs often bind to more than one molecular target, exhibiting polypharmacology. Although this aspect has commonly been considered as undesirable, promiscuity being responsible for unwanted side effects, in many cases it is a key component of the therapeutic efficacy of drugs. Nuclear receptors (NRs) are ligand-dependent transcription factors that offer important druggable targets for therapeutic interventions in multiple disease areas. Many NRs are promiscuous with respect to the wide range of ligands that act as modulators, and many NR modulators are not specific with respect to the number of NRs they bind. In this article, we aim to investigate aspects of ligand polypharmacology in the superfamily of human NRs. To this end, the construction of a target-centric and a ligand-centric chemical space is first discussed as instrumental in charting geometrical and physicochemical properties of NR binding sites and cognate ligands that, being characteristic of specific groups of receptors and/or modulators, could underlie their promiscuity. In the second part of the work, generating a graph network, we depict relationships among geometrical and physicochemical properties of binding sites that are used to infer aspects of polypharmacology in human NRs. Working hypotheses of NR ligand polypharmacology are thus generated and discussed in the light of case studies found in the literature.
Medicinal Chemistry Communication 01/2013; 4:216-227. · 2.72 Impact Factor
[show abstract][hide abstract] ABSTRACT: The employment of the flow N-acyl amidation of natural bile acids (BAs) required the in-line connection with suitable analytical tools enabling the determination of reaction yields as well as of the purity grade of the synthesized glyco- and tauro-conjugated derivatives. In this framework, a unique HPLC method was successfully established and validated for ursodeoxycholic (UDCA), chenodeoxycholic (CDCA), deoxycholic (DCA) and cholic (CA) acids, as well as the corresponding glyco- and tauro-conjugated forms. Because of the shared absence of relevant chromophoric moieties in the sample structure, an evaporative light scattering detector (ELSD) was profitably utilized for the analysis of such steroidal species. For each of the investigated compounds, all the runs were contemporarily carried out on the acidic free and the two relative conjugated variants. The different ELSD response of the free and the corresponding conjugated BAs, imposed to build-up separate calibration curves. In all the cases, very good precision (RSD% values ranging from 1.04 to 6.40% in the long-period) and accuracy (Recovery% values ranging from 96.03 to 111.14% in the long-period) values along with appreciably low LOD and LOQ values (the former being within the range 1-27ngmL(-1) and the latter within the range 2-44ngmL(-1)) turned out.
[show abstract][hide abstract] ABSTRACT: Introduction: The G protein-coupled receptor TGR5 is a key player of the bile acid signaling network, and its activation has been proved to increase the glycemic control, to enhance energy expenditure and to exert anti-inflammatory actions. Accordingly, TGR5 ligands have emerged in drug discovery and preclinical appraisals as promising agents for the treatment of liver diseases, metabolic syndrome and related disorders. Areas covered: Recent advances in the field of TGR5 modulators are reviewed, with a particular attention on patent applications and peer-reviewed publications in the past 6 years. Expert opinion: Activation of TGR5 showed to protect mice from diabesity and insulin resistance, to improve liver functions, as well as to attenuate the development of atherosclerosis. However, although the efficacy of TGR5 agonists in mice is encouraging, further studies are needed to determine their potential in humans and to evaluate carefully the balance between the therapeutic benefits and adverse effects associated with the target. The development of new TGR5 selective ligands to support studies in animal models will surely facilitate the understanding of the complexity of TGR5 signaling network.
Expert Opinion on Therapeutic Patents 10/2012; · 3.53 Impact Factor
[show abstract][hide abstract] ABSTRACT: A multi-gram scale protocol for the N-acyl amidation of bile acids with glycine and taurine has been successfully developed under continuous flow processing conditions. Selecting ursodeoxycholic acid (UDCA) as the model compound and N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ) as the condensing agent, a modular mesoreactor assisted flow set-up was employed to significantly speed up the optimization of the reaction conditions and the flow scale-up synthesis. The results in terms of yield, in line purification, analysis, and implemented flow set-up for the reaction optimization and large scale production are reported and discussed.
[show abstract][hide abstract] ABSTRACT: Grounding on our former 3D QSAR studies, a knowledge-based screen of natural bile acids from diverse animal species has led to the identification of avicholic acid as a selective but weak TGR5 agonist. Chemical modifications of this compound resulted in the disclosure of 6α-ethyl-16-epi-avicholic acid that shows enhanced potency at TGR5 and FXR receptors. The synthesis, biological appraisals, and structure–activity relationships of this series of compounds are herein described. Moreover, a thorough physicochemical characterization of 6α-ethyl-16-epi-avicholic acid as compared to naturally occurring bile acids is reported and discussed.
[show abstract][hide abstract] ABSTRACT: The G protein-coupled receptor TGR5 has been identified as an important component of the bile acid signaling network, and its activation has been linked to enhanced energy expenditure and improved glycemic control. Here, we demonstrate that activation of TGR5 in macrophages by 6α-ethyl-23(S)-methylcholic acid (6-EMCA, INT-777), a semisynthetic BA, inhibits proinflammatory cytokine production, an effect mediated by TGR5-induced cAMP signaling and subsequent NF-κB inhibition. TGR5 activation attenuated atherosclerosis in Ldlr(-/-)Tgr5(+/+) mice but not in Ldlr(-/-)Tgr5(-/-) double-knockout mice. The inhibition of lesion formation was associated with decreased intraplaque inflammation and less plaque macrophage content. Furthermore, Ldlr(-/-) animals transplanted with Tgr5(-/-) bone marrow did not show an inhibition of atherosclerosis by INT-777, further establishing an important role of leukocytes in INT-777-mediated inhibition of vascular lesion formation. Taken together, these data attribute a significant immune modulating function to TGR5 activation in the prevention of atherosclerosis, an important facet of the metabolic syndrome.
[show abstract][hide abstract] ABSTRACT: A new stereoselective synthesis of E-guggulsterone is described starting from androsten-3,17-dione. Protection of the ring A enonic system, followed by regioselective Wittig reaction and C-16 oxidation, affords E-guggulsterone in good yields and high stereoselectivity, making this approach easily accessible and scalable. Moreover, an original normal-phase HPLC method enabling the fast quantitation of the guggulsterone isomeric purity, combined with the suitability for sampling procedures, is detailed. The relying upon the cellulose-based Chiralpak IB column and the chloroform as the "non-standard" component of the eluent mixture, allows to get profitably high chromatographic performances.
[show abstract][hide abstract] ABSTRACT: Ethyl diazo(3-hydroxy-2-oxo-2,3-dihydro-1H-indol-3-yl)acetate was prepared by aldol-type condensation of ethyl diazoacetate with isatin. A systematic and mechanistic study on the Lewis acid induced decomposition reaction of this valuable diazo precursor was carried out with the aim to gain new insights into the mechanistic aspects of the reaction as well as to further understand the factors and experimental conditions which affect the relative product distribution. The reaction, which may proceed via cationic and noncationic mechanisms, was found to be significantly influenced by the reaction environment determined by the characteristics of the Lewis acid employed, by the ability of the Lewis acid to form a complex with the alcohol functionality of the α-diazo-β-hydroxy ester, and by the polarity and nucleophilicity of the solvent used.
The Journal of Organic Chemistry 08/2011; 76(18):7431-7. · 4.56 Impact Factor
[show abstract][hide abstract] ABSTRACT: In a line of research focused on the design, synthesis and development of new bile acid-based compounds, the physico-chemical profile of the molecules must be thoroughly explored and analyzed. In this scenario, a fast and reliable information on the critical micellar concentration (CMC) of specific compounds through a profitable chromatographic parameter can be of aid to rationally direct the synthesis of new molecular entities, mainly during the early stages of the drug-discovery process. The derived 'chromatographic hydrophobicity index' (CHI), usually employed for a fast access to the log P/log D value of physico-chemically diverse compounds and obtained via RP-gradient elution, was for the first time engaged in the bile acid field. Accordingly, 14 unconjugated bile acids harboured with a different number, position and orientation of hydroxy groups, as well as other substituents onto the steroidal backbone and side chain, were selected to build up a calibration curve. Such a collection of compounds was rationally assembled in order to manage an almost continuous range of CMC values (spanning the spectrophotometrically obtained CMCs between 5 and 25 mM). A high degree of correlation between CMC and CHI values was obtained (R(2) and cross-validated R(xv)(2) of the pCMC vs CHI plot equal to 0.975 and 0.966, respectively). A selected new subset of five confidential research bile acids with experimental CMCs in the range 6-19 mM was finally recruited to validate the proposed method. The high statistical quality of the established mathematical model turned out into a very appreciable predictive power.
Analytical and Bioanalytical Chemistry 05/2011; 401(1):267-74. · 3.66 Impact Factor
[show abstract][hide abstract] ABSTRACT: Within our efforts in the discovery of novel potent and selective ligands for the FXR receptor, 23-N-(carbocinnamyloxy)-3α,7α-dihydroxy-6α-ethyl-24-nor-5β-cholan-23-amine was synthesized and evaluated for its ability to activate and modulate the biological response of the receptor. Alphascreen and RT-PCR revealed that the 6α-ethyl-24-norcholanyl-23-amine derivate behaves as full FXR agonist endowed with high binding affinity and efficacy, representing a promising lead candidate for further optimization. In addition, docking studies provide new insights into the molecular basis governing the partial and full agonist activity at FXR.
[show abstract][hide abstract] ABSTRACT: Activation of poly(ADP-ribose) polymerase (PARP) is an important factor in controlling cell survival or death. As a consequence, therapeutic interventions with PARP-1 inhibitors are sought in different pathological conditions such as cancer, cardiovascular and inflammatory diseases, as well as brain ischemia. In the first part of this work, as a continuation of our efforts in the field, we report the design, synthesis and biological appraisal of novel potent PARP-1 inhibitors. A crystallization experiment is carried out to ascertain the mode of binding to PARP-1 of the most potent compound, namely 2-((dimethylamino)methyl)-9-hydroxythieno[2,3-c]isoquinolin-5(4H)-one (HYDAMTIQ), whilst molecular modeling studies are performed to infer the role of water molecules in ligand binding. In the second part of the work, we discuss the results of HYDAMTIQ in models of brain ischemia as well as its preliminary physicochemical and pharmacokinetic characterization. Collectively, the data obtained qualify HYDAMTIQ a
Medicinal Chemistry Communication 01/2011; 2:559-565. · 2.72 Impact Factor
[show abstract][hide abstract] ABSTRACT: A new divergent synthesis of DAF-12 ligands, namely Δ4- and Δ7-dafachronic acid, is described starting from the natural bile acid hyodeoxycholic acid. Homologation of the side chain followed by stereoselective reduction of the Δ24 olefinic linkage, 6α-dehydroxylation and C3-oxidation affords dafachronic acids in good yields and high diastereoselectivity making this approach easily accessible and scalable.
[show abstract][hide abstract] ABSTRACT: New stable [6,6]-methano cycloadducts and fulleropyrazolines containing electron-withdrawing groups have been obtained by the reaction of ethyl diazopyruvate with fullerene. The results obtained by a systematic study conducted both in thermal and catalytic conditions have provided crucial indications concerning the mechanism of this important cluster opening process in fullerene chemistry.Graphical abstract
[show abstract][hide abstract] ABSTRACT: In the framework of the design and development of TGR5 agonists, we reported that the introduction of a C(23)(S)-methyl group in the side chain of bile acids such as chenodeoxycholic acid (CDCA) and 6-ethylchenodeoxycholic acid (6-ECDCA, INT-747) affords selectivity for TGR5. Herein we report further lead optimization efforts that have led to the discovery of 6alpha-ethyl-23(S)-methylcholic acid (S-EMCA, INT-777) as a novel potent and selective TGR5 agonist with remarkable in vivo activity.
Journal of Medicinal Chemistry 12/2009; 52(24):7958-61. · 5.61 Impact Factor
[show abstract][hide abstract] ABSTRACT: A facile one-pot procedure for the synthesis of pyrazole-5-carboxylates by 1,3-dipolar cycloaddition of ethyl diazoacetate is described. Cycloadditions with α-methylene carbonyl compounds utilizing 1,8-diazabicyclo[5.4.0]undec-7-ene as base and acetonitrile as solvent provide pyrazoles with excellent regioselectivity and good yields. The reaction was found to proceed by a domino 1,3-dipolar cycloaddition-water elimination.
[show abstract][hide abstract] ABSTRACT: TGR5 is a G protein-coupled receptor expressed in brown adipose tissue and muscle, where its activation by bile acids triggers an increase in energy expenditure and attenuates diet-induced obesity. Using a combination of pharmacological and genetic gain- and loss-of-function studies in vivo, we show here that TGR5 signaling induces intestinal glucagon-like peptide-1 (GLP-1) release, leading to improved liver and pancreatic function and enhanced glucose tolerance in obese mice. In addition, we show that the induction of GLP-1 release in enteroendocrine cells by 6alpha-ethyl-23(S)-methyl-cholic acid (EMCA, INT-777), a specific TGR5 agonist, is linked to an increase of the intracellular ATP/ADP ratio and a subsequent rise in intracellular calcium mobilization. Altogether, these data show that the TGR5 signaling pathway is critical in regulating intestinal GLP-1 secretion in vivo, and suggest that pharmacological targeting of TGR5 may constitute a promising incretin-based strategy for the treatment of diabesity and associated metabolic disorders.
[show abstract][hide abstract] ABSTRACT: Owing to the re-flourished interest towards the bile acids (BAs) as versatile signalling hormones endowed with diverse endocrine functions, the development of reliable analytical protocols monitoring the synthesis of new BA-based receptor modulators, still represents a cogent concern. On this basis, for the first time, a HPLC study has been engaged with the aim to set up suitable chromatographic conditions for the analysis of three different epimeric couples of 23-methyl-substituted unconjugated BAs. Three different methods (one for each couple) have been successfully established and then validated. Good precision and accuracy (evaluated both in the short and long period) as well as appreciably low LOD and LOQ values have turned out. Moreover, the engagement of an evaporative light scattering detector (ELSD) has proven its high effectiveness for the analysis of such steroidal species.
Journal of Separation Science 07/2009; 32(12):2022-33. · 2.59 Impact Factor