Publications by authors named "Andrea Carotti"

72 Publications

Fragment based drug design and diversity-oriented synthesis of carboxylic acid isosteres.

Bioorg Med Chem 2020 Nov 28;28(22):115731. Epub 2020 Aug 28.

TES Pharma, via P. Togliatti 22bis, 06073 Terrioli, Corciano, Italy.

The medicinal chemist toolbox is plenty of (bio)isosteres when looking for a carboxylic acid replacement. However, systematic assessment of acid surrogates is often time consuming and expensive, while prediction of both physicochemical properties (logP and logD) as well as acidity would be desirable at early discovery stages for a better analog design. Herein in this work, to enable decision making on a project, we have synthesized by employing a Diversity-Oriented Synthetic (DOS) methodology, a small library of molecular fragments endowed with acidic properties. By combining in-silico and experimental methodologies these compounds were chemically characterized and, particularly, with the aim to know their physicochemical properties, the aqueous ionization constants (pKa), partition coefficients logD and logP of each fragment was firstly estimated by using molecular modeling studies and then validated by experimental determinations. A face to face comparison between data and the corresponding carboxylic acid might help medicinal chemists in finding the best replacement to be used. Finally, in the framework of Fragment Based Drug Design (FBDD) the small library of fragments obtained with our approach showed good versatility both in synthetic and physico-chemical properties.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.bmc.2020.115731DOI Listing
November 2020

Integrating experimental and computational techniques to study chromatographic enantioresolutions of chiral tetrahydroindazole derivatives.

J Chromatogr A 2020 Aug 8;1625:461310. Epub 2020 Jun 8.

Department of Pharmaceutical Sciences, University of Perugia, Via Fabretti 48, 06123 - Perugia, Italy. Electronic address:

With the selection of partially saturated 2H-indazoles as model compounds, we demonstrate the possibility to use Whelk-O1 chiral stationary phases (CSPs) to succeed in efficient small-scale preparative enantioseparations. Runs of three consecutive liquid chromatography injections (about 300 μg of racemate repeatedly injected in a 100 μL loop) produced groups of peaks without band contamination (α = 1.2 and R = 2.57). With this procedure approximately 3.0 mg of each enantiomer, with enantiomeric excess ≥ 97% were obtained. Very profitably, the high volatility of n-hexane used as the sole eluent facilitated the solvent evaporation after the enantiomer recovery. High resolution mass spectrometry analysis confirmed that the chemical identity of the two enantiomers was preserved along the entire process. The ability of Whelk-O1 phases in enantioseparating structurally similar compounds was confirmed with the analysis of other two racemates. Moreover, the relevant chemoselectivity exhibited by the CSP towards the three racemates should allow to simultaneously optimizing the enantioselectivity of different analytes and perform small-scale enantioresolutions of different compounds during the same run. In this study, the integration of experimental off-line electronic circular dichroism analysis with ab initio time-dependent density-functional theory simulations facilitated the assignment of the absolute configuration of the single enantiomers, while a molecular dynamics protocol can be useful to make a priori predictions of the enantioseparation ability of CSP towards selected compounds.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.chroma.2020.461310DOI Listing
August 2020

Fragment-based Design of Zwitterionic, Strong Cation- and Weak Anion-Exchange Type Mixed-mode Liquid Chromatography Ligands and their Chromatographic Exploration.

J Chromatogr A 2020 Jun 20;1621:461075. Epub 2020 Apr 20.

Institute of Pharmaceutical Sciences, Pharmaceutical (Bio-)Analysis, University of Tübingen, Auf der Morgenstelle 8, 72076 Tübingen, Germany. Electronic address:

The role of individual functional groups has been assessed with regard to surface charge and chromatographic retention. Coatings were prepared from various fragments of the chiral zwitterionic materials Chiralpak ZWIX(+) and ZWIX(-). The different chromatographic ligands allowed fine tuning of the surface charge. Chiralpak ZWIX phases showed strongly negative ζ-potentials over the entire pH-range. Zwitterionic congeners with quinuclidine and sulfonic acid moieties but lacking the quinolone ring in the ligand structure exhibited shifted ζ-potentials of around + 5 to 20 mV depending on the surrounding residues. Capillary electrophoretic mobilitiy measurements with the chromatographic ligands and molecular dynamics simulations were carried out to offer some explanation of these surface charge differences of the distinct zwitterionic stationary phases. The new mixed-mode phases were also chromatographically characterized by simple RP and HILIC tests. The results allowed their positioning within a large variety of different commercially available RP, HILIC and mixed-mode phases, which were evaluated as well, by multivariate data processing using principal component analysis. The new mixed-mode phases overall exhibit reasonable hydrophilicity-lipophilicity balance and enable retention of ionic compounds by additional ionic interactions through weak anion-exchange (WAX-type), strong cation-exchange (SCX-type) or both (RP/ZWIX-type). Hence, the new RP/ZWIX phases can be flexible tools for selectivity tuning in RP and HILIC separations.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.chroma.2020.461075DOI Listing
June 2020

Profiling calcium-dependent interactions between Sorcin and intrinsically disordered regions of human proteome.

Biochim Biophys Acta Gen Subj 2020 08 17;1864(8):129618. Epub 2020 Apr 17.

Department of Chemistry - BMC, Uppsala University, Husargatan 3, 751 23 Uppsala, Sweden.

Background: Sorcin is a calcium sensor that exerts many calcium-related functions in the cells, e.g. it regulates calcium concentration in the cytoplasm, endoplasmic reticulum (ER) and mitochondria, by interacting with calcium pumps, exchangers and channels. Albeit Sorcin is an interesting potential cancer target, little is known about its interactors upon calcium-mediated activation. Our previous study suggested that Sorcin may recognize short linear binding motifs as the crystal structure revealed a self-interaction with a GYYPGG stretch in its N-terminus, and combinatorial peptide-phage display provided support for peptide-mediated interactions.

Methods: In this study we screened for motif-based interactions between Sorcin and intrinsically disordered regions of the human proteome using proteomic peptide phage display (ProP-PD). We identified a peptide belonging to protein phosphatase 1 regulatory subunit 3G (PPP1R3G) as a potential novel interactor and confirm the interaction through biophysical and cell-based approaches, and provide structural information through molecular dynamics simulations.

Results: Altogether, we identify a preferred motif in the enriched pool of binders and a peptide belonging to protein phosphatase 1 regulatory subunit 3G (PPP1R3G) as a preferred ligand.

Conclusion: Through this study we gain information on a new Sorcin binding partner and profile Sorcin's motif-based interaction.

General Significance: The interaction between Sorcin and PPP1R3G may suggest a close dependence between glucose homeostasis and calcium concentration in the different cell compartments, opening a completely new and interesting scenery yet to be fully disclosed.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.bbagen.2020.129618DOI Listing
August 2020

Chromatograpic resolution of phenylethanolic-azole racemic compounds highlighted stereoselective inhibition of heme oxygenase-1 by (R)-enantiomers.

Bioorg Chem 2020 06 19;99:103777. Epub 2020 Mar 19.

Department of Drug Sciences, University of Catania, V.le A. Doria 6, 95125 Catania, Italy. Electronic address:

Heme oxygenase-1 (HO-1) has been recognized as extensively involved in the development and aggravation of cancer, cell propagation and at in the mechanism of chemoresistance development. Low micromolar HO-1 inhibitors selective towards HO-2 has been recently reported, wherein the azole core and the hydrophobic residues are linked through a phenylethanolic spacer bearing a chiral center. Since less information are known about the stereoselective requirements for HO-1 inhibition, here we report the enantiomeric resolution of 1-(biphenyl-3-yl)-2-(1H-imidazol-1-yl)ethanol (1) and 1-[4-[(4-bromobenzyl)oxy]phenyl]-2-(1H-imidazol-1-yl)ethanol (2), two among the most potent and selective HO-1 inhibitors known thus far when tested as racemates. The absolute configuration was established for 1 by a combination of experimental and in silico derived electronic circular dichroism spectra, while docking approaches were useful in the case of compound 2. Biological evaluation of pure enantiomers highlighted higher HO-1 inhibitory activity of (R)-enantiomers. Docking studies demonstrated the importance of hydrogen bond interaction, more pronounced for the (R)-enantiomers, with a consensus water molecule within the binding pocket. The present study demonstrates that differences in three-dimensional structure amongst compounds 1 and 2 enantiomers affect significantly the selectivity of these HO-1 inhibitors.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.bioorg.2020.103777DOI Listing
June 2020

New Insights from Crystallographic Data: Diversity of Structural Motifs and Molecular Recognition Properties between Groups of IDO1 Structures.

ChemMedChem 2020 05 14;15(10):891-899. Epub 2020 Apr 14.

Department of Pharmaceutical Sciences, University of Perugia, via del liceo n.1, 06123, Perugia, Italy.

A large number of crystallographic structures of IDO1 in different ligand-bound and -unbound states have been disclosed over the last decade. Yet, only a few of them have been exploited for structure-based drug design (SBDD) campaigns. In this study, we analyzed the structural motifs and molecular-recognition properties of three groups of IDO1 structures: 1) structures containing the heme group and inhibitors in the catalytic site; 2) heme-free structures of IDO1; 3) substrate-bound structures of IDO1. The results suggest that unrelated conformations of the enzyme have been solved with different ligand-induced changes of secondary motifs that localize even in regions remote from the catalytic site. Moreover, the study identified an uncharted region of molecular-recognition space covered by IDO1 binding sites that could guide the selection of diverse structures for additional SBDD studies aimed at the identification of novel lead compounds with differentiated chemical scaffolds.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/cmdc.202000116DOI Listing
May 2020

Binding modes identification through molecular dynamic simulations: A case study with carnosine enantiomers and the Teicoplanin A2-2-based chiral stationary phase.

J Sep Sci 2020 May 6;43(9-10):1728-1736. Epub 2020 Apr 6.

Department of Pharmaceutical Sciences, University of Perugia, Via Fabretti 48, Perugia, 06123, Italy.

In the present study, an in silico methodology able to define the binding modes adopted by carnosine enantiomers in the setting of the chiral recognition process is described. The inter- and intramolecular forces involved in the enantioseparation process with the Teicoplanin A2-2 chiral selector and carnosine as model compound are successfully identified. This approach fully rationalizes, at a molecular level, the (S) < (R) enantiomeric elution order obtained under reversed-phase conditions. Consistent explanations were achieved by managing molecular dynamics results with advanced techniques of data analysis. As a result, the time-dependent identification of all the interactions simultaneously occurring in the chiral selector-enantiomeric analyte binding process was obtained. Accordingly, it was found that only (R)-carnosine is able to engage a stabilizing charge-charge interaction through its ionized imidazole ring with the carboxylate counter-part on the chiral selector. Instead, (S)-carnosine establishes intramolecular contacts between its ionized functional groups, that limit its conformational freedom and impair the association with the chiral selector unit.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/jssc.202000092DOI Listing
May 2020

Discovery of Novel 5-Lipoxygenase-Activating Protein (FLAP) Inhibitors by Exploiting a Multistep Virtual Screening Protocol.

J Chem Inf Model 2020 03 24;60(3):1737-1748. Epub 2020 Feb 24.

Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Gazi University, Etiler, 06560 Yenimahalle, Ankara, Turkey.

Leukotrienes (LTs) are proinflammatory mediators derived from arachidonic acid (AA), which play significant roles in inflammatory diseases. The 5-lipoxygenase-activating protein (FLAP) is an integral membrane protein, which is essential for the initial step in LT biosynthesis. The aim of this study was to discover novel and chemically diverse FLAP inhibitors for treatment of inflammatory diseases requiring anti-LT therapy. Both ligand- and structure-based approaches were applied to explain the activities of known FLAP inhibitors in relation to their predicted binding modes. We gained valuable insights into the binding modes of the inhibitors by molecular modeling and generated a multistep virtual screening (VS) workflow in which 6.2 million compounds were virtually screened, and the molecular hypotheses were validated by testing VS-hit compounds biologically. The most potent hit compounds showed significant inhibition of FLAP-dependent cellular LT biosynthesis with IC values in the range from 0.13 to 0.87 μM. Collectively, this study provided novel bioactive chemotypes with potential for further development as effective anti-inflammatory drugs.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/acs.jcim.9b00941DOI Listing
March 2020

Enantioselective HPLC Analysis to Assist the Chemical Exploration of Chiral Imidazolines.

Molecules 2020 Feb 2;25(3). Epub 2020 Feb 2.

Department of Pharmaceutical Sciences, University of Perugia, Via Fabretti 48, 06123 Perugia, Italy.

In the present work, we illustrate the ability of high-performance liquid chromatography (HPLC) analysis to assist the synthesis of chiral imidazolines within our medicinal chemistry programs. In particular, a Chiralpak IB column containing cellulose tris(3,5-dimethylphenylcarbamate) immobilized onto a 5 μm silica gel was used for the enantioselective HPLC analysis of chiral imidazolines synthesized in the frame of hit-to-lead explorations and designed for exploring the effect of diverse amide substitutions. Very profitably, reversed-phase (RP) conditions succeeded in resolving the enantiomers in nine out of the 10 investigated enantiomeric pairs, with α values always higher than 1.10 and R values up to 2.31. All compounds were analysed with 50% (v) water while varying the content of the two organic modifiers acetonitrile and methanol. All the employed eluent systems were buffered with 40 mM ammonium acetate while the apparent pH was fixed at 7.5. Based on the experimental results, the prominent role of π-π stacking interactions between the substituted electron-rich phenyl groups outside of the polymeric selector and the complementary aromatic region in defining analyte retention and stereodiscrimination was identified. The importance of compound polarity in explaining the retention behaviour with the employed RP system was readily evident when a quantitative structure-property relationship study was performed on the retention factor values (k) of the 10 compounds, as computed with a 30% (v) methanol containing mobile phase. Indeed, good Pearson correlation coefficients of retention factors (r - log k = -0.93; r - log k = -0.94) were obtained with a water solubility descriptor (Ali-logS). Interestingly, a -hexane/chloroform/ethanol (88:10:2, //)-based non-standard mobile phase allowed the almost base-line enantioseparation (α = 1.06; R = 1.26) of the unique compound undiscriminated under RP conditions.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/molecules25030640DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7036806PMC
February 2020

Optimized one-pot derivatization and enantioseparation of cysteine: Application to the study of a dietary supplement.

J Pharm Biomed Anal 2020 Feb 23;180:113066. Epub 2019 Dec 23.

University of Perugia, Department of Pharmaceutical Sciences, Via Fabretti, 48, 06123 Perugia, Italy. Electronic address:

Cysteine is a sulfur-containing amino acid which plays an outstanding role in many biological pathways in mammals. The analysis and quantification of native cysteine remains a critical issue due to its highly reactive thiol group evolving to the disulfide cystine derivative through oxidation reaction. Aimed at improving the derivative stability, cysteine was labelled with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F), which reacts with both amino and thiol groups. The derivatization was optimized and the chemical identity of the reaction product was assessed via high-resolution mass spectrometry. The NBD-cysteine derivative resulted stable for 10 days. This derivative was enantioresolved (α and R equal to 1.25 and 2.70, respectively) thanks to a (R,R)-Whelk-O1 phase with the following chromatographic setting: eluent, MeOH/water-90/10 (v/v) with 15 mM ammonium formate (pwsH 6.0); column temperature, 35 °C; flow rate, 1.0 mL/min. The developed method was validated following the ICH guidelines and applied for the quality control of a L-cysteine containing dietary supplement.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.jpba.2019.113066DOI Listing
February 2020

Mixed-mode chromatography characteristics of chiralpak ZWIX(+) and ZWIX(-) and elucidation of their chromatographic orthogonality for LC × LC application.

Anal Chim Acta 2020 Jan 27;1093:168-179. Epub 2019 Sep 27.

Institute of Pharmaceutical Sciences, Pharmaceutical (Bio-)Analysis, University of Tübingen, Auf der Morgenstelle 8, 72076, Tübingen, Germany. Electronic address:

Two-dimensional liquid chromatography requires orthogonal columns and/or separation principles in the first and second separation dimension. It is sometimes not straightforward to achieve. Chiral columns could expand the toolbox for 2D-LC, but are rarely exploited for this purpose, not least due to missing understanding of retention principles under non-chiral application conditions. To gain more insight, in this study Chiralpak ZWIX(+) and ZWIX(-), based on zwitterionic quinine and quinidine carbamate selectors, were carefully characterized by molecular dynamics simulations, lipophilicity/hydrophilicity measurements of selectors, pH-dependent ζ-potential determinations, and chromatographic characterization in RPLC and HILIC modes combined with unsupervised principal component analysis to extract classification of these columns in comparison to a number of commercial benchmarks (RP, HILIC and mixed-mode columns). The results showed that these chiral columns can be classified as mixed-mode chromatography phases with balanced lipophilic-hydrophilic surface character, excess of negative net charge due to sulfonic acid groups (in spite of weakly basic quinuclidine and quinoline rings), and multimodal applicability (RP, HILIC and polar organic elution modes). Orthogonality mapping in comparison to a number of modern HILIC and mixed-mode columns revealed that Poroshell HILIC-Z (with a zwitterionic ligand on 2.7 μm core-shell particles) can be beneficially combined as second dimension with the ZWIX column for comprehensive LC × LC. The online hyphenation of this 2D-LC system with complementary detection modalities including UV (DAD for chromophoric substances), charged aerosol detection (for universal detection and calibration of non-volatile analytes) and high-resolution mass spectrometry (ESI-QTOF-MS/MS for identification) provided an advanced method for comprehensive impurity profiling, applicable for instance for amino acid pharmaceutical products.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.aca.2019.09.068DOI Listing
January 2020

Tracking Hidden Binding Pockets Along the Molecular Recognition Path of l-Trp to Indoleamine 2,3-Dioxygenase 1.

ChemMedChem 2019 12 14;14(24):2084-2092. Epub 2019 Nov 14.

Department of Pharmaceutical Sciences, University of Perugia, via del liceo n.1, 06123, Perugia, Italy.

Indoleamine 2,3-dioxygenase 1 (IDO1) catalyzes the oxidative cleavage of l-Tryptophan (l-Trp) to yield N-formyl-kynurenine in the first and rate limiting step of the kynurenine pathway. Bioactive metabolites, involved in the regulation of important immunological responses and neurological processes, are then produced by downstream enzymes along the pathway. Inhibitors of IDO1 are being designed and developed as therapeutic agents for immuno-oncology. In this work, we investigated the molecular recognition path of l-Trp to IDO1, integrating biophysical methods with supervised molecular dynamics (suMD) and mutagenesis experiments. Results allowed disclosing for the first time high and low dissociation constants of l-Trp to IDO1, and the presence of a metastable interaction site located at the upper part of a channel whose borders are defined by the EF-loop and the C-terminal part of the JK-loop. Collectively, our results provide new clues for the design of next-generation IDO1 ligands.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/cmdc.201900529DOI Listing
December 2019

Dissecting the allosteric FXR modulation: a chemical biology approach using guggulsterone as a chemical tool.

Medchemcomm 2019 Aug 24;10(8):1412-1419. Epub 2019 Jun 24.

Department of Pharmaceutical Sciences , University of Perugia , Perugia , Italy . Email:

Guggulsterone is a promiscuous ligand for endocrine and metabolic lipid receptors traditionally used to treat a number of diseases including diabesity, hyperlipidemia, atherosclerosis, and osteoarthritis. Although relatively weak, its activity at the farnesoid X receptor (FXR) is particularly intriguing as guggulsterone acts as an antagonist with a peculiar ability of gene selective modulation. We report here a chemical biology study with the aim to further characterize the biological action of guggulsterone at the FXR and to obtain further insights into the functional role played by noncanonical FXR binding pockets S2 and S3. Our results suggest that the FXR accessory pockets might act as potential targets for small molecules able to modulate the metabolic activation of the receptor without affecting the anti-inflammatory activity thus revealing a new approach for disclosing selective FXR modulators that might bypass potential side-effects from chronic treatments.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1039/c9md00264bDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6786047PMC
August 2019

Application of the "inverted chirality columns approach" for the monitoring of asymmetric synthesis protocols.

Talanta 2019 Oct 14;203:147-152. Epub 2019 May 14.

University of Perugia, Department of Pharmaceutical Sciences, Via Fabretti 48, 06123, Perugia, Italy.

In the present study, the "Inverted Chirality Columns Approach (ICCA)" was applied to follow an asymmetric synthetic reaction, namely, the addition of butan-2-one to trans-β-nitrostyrene, catalysed by (S)-proline, leading to the formation of 3-methyl-4-phenyl-5-nitro-2-pentan-2-one. The ICCA method was applied to overcome the lack of pure enantiomeric standards. The two widely employed (R,R)- and (S,S)-Whelk-O1 chiral stationary phases (CSPs), incorporating fully synthesized enantiomeric chiral selectors, were profitably used for this purpose. The enantioselective analysis with the two CSPs was performed under optimized reversed-phase conditions with a water/acetonitrile (60/40, v/v) eluent. In the probe reaction under investigation, a diastereomeric excess >90% was found according to a well-established reaction mechanism, thus affording the enantiomer couple (3S,4R)-3-methyl-4-phenyl-5-nitropentan-2-one and (3R,4S)-3-methyl-4-phenyl-5-nitropentan-2-one as the main product. Therefore, the attention was exclusively focused on this enantiomers pair. Rather similar retention and separation factor [1.12 with (R,R)-Whelk-O1 and 1.13 with (S,S)-Whelk-O1] values as well as resolutions [2.06 with (R,R)-Whelk-O1 and 2.30 with (S,S)-Whelk-O1] were produced by the two enantiomeric CSPs. Applying the ICCA concept allowed to identify the two enantiomers-related peaks in the chromatograms, ultimately indicating a 65-to-35 enantiomeric per cent ratio. Electronic circular dichroism (ECD) and high-resolution mass spectrometry analyses of the two peaks collected during the enantioselective analyses further confirmed the enantiomeric nature of the identified compounds. The (3S,4R) < (3R,4S) enantiomer elution order with the (R,R)-Whelk-O1 was fully disclosed thanks to ECD studies coupled with in silico quantum mechanical simulations. As expected, reversed elution order turned out with (S,S)-Whelk-O1.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.talanta.2019.05.045DOI Listing
October 2019

Enantioseparations by High-Performance Liquid Chromatography Based on Chiral Ligand Exchange.

Methods Mol Biol 2019 ;1985:279-302

Department of Pharmaceutical Sciences, University of Perugia, Perugia, Italy.

Although the first application of chiral ligand-exchange chromatography (CLEC) in HPLC dates back to late 1960s, this enantioselective strategy still represents the elective choice for the direct analysis of compounds endowed with chelating moieties. As a specific feature of the CLEC mechanism, the interaction between the chiral selector and the enantiomer does not take place in direct contact. Indeed, it is mediated by a central metal ion that, acting as a Lewis acid, simultaneously coordinates the two species, selector and analyte, through the activation of dative bonds. As a consequence, two diastereomeric mixed ternary complexes are generated in the column, ultimately leading to the stereoisomeric discrimination. CLEC applications can be carried out both with the chiral selector included in the mobile phase (chiral mobile phase, CMP), or as a part of the stationary phase. In the latter case, the chiral selector can be either covalently immobilized onto a solid support (bonded CSP, B-CSP) or physically adsorbed onto a conventional packing material, coated chiral stationary phase (C-CSP).In this chapter, a selection of CLEC applications with CMP- and C-CSP-based chiral systems is presented.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/978-1-4939-9438-0_15DOI Listing
January 2020

Exploiting Chemical Toolboxes for the Expedited Generation of Tetracyclic Quinolines as a Novel Class of PXR Agonists.

ACS Med Chem Lett 2019 Apr 27;10(4):677-681. Epub 2018 Dec 27.

Department of Pharmaceutical Sciences, University of Perugia, Via del Liceo 1, I-06123 Perugia, Italy.

The discovery of lead compounds relies on the iterative generation of structure-activity relationship data resulting from the synthesis and biological evaluation of hit analogues. Using traditional approaches, a significant time delay may occur from compound design to results, leading to slow and expensive hit-to-lead explorations. Herein, we have exploited the use of chemical toolboxes to expedite lead discovery and optimization. In particular, the integration of flow synthesizers, automation, process analytical technologies, and computational chemistry has provided a prototype system enabling the multicomponent flow synthesis, in-line analysis, and characterization of chiral tetracyclic quinolines as a novel class of PXR agonists. Within 29 compounds, a novel template (3a,11,11a) was identified with an EC of 1.2 μM (efficacy 119%) at the PXR receptor.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/acsmedchemlett.8b00459DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6466813PMC
April 2019

Hydrophilic interaction liquid chromatography of aminoglycoside antibiotics with a diol-type stationary phase.

Anal Chim Acta 2018 Dec 6;1044:174-180. Epub 2018 Aug 6.

University of Perugia, Department of Pharmaceutical Sciences, Section of Chemistry and Technology of Drugs, Via del Liceo 1, 06123, Perugia, Italy.

Owing to their pronounced polarity, hydrophilic interaction liquid chromatography (HILIC) can be considered as the elective choice for the LC analysis of aminoglycoside (AG) antibiotics. In the present work, a gradient program was optimized for the first time with a diol-type stationary phase and an evaporative light scattering detector (ELSD), thus allowing the almost complete separation of the nine analysed AGs: spectinomycin, dihydrostreptomycin, streptomycin A, gentamicin C1, amikacin, kanamycin A, paromomycin, apramycin and neomycin. In the optimized analysis conditions, analyte retention was found to be governed by a multimodal mechanism encompassing electrostatic, partitioning and hydrophilic interactions. However, the gradient mode of elution complicated a deep understanding of the influence of each contribution on the retention behaviour. The developed HILIC-ELSD method was applied for the analysis of commercial tablets containing neomycin co-formulated with the polypeptide antibiotic bacitracin. The method was fully validated according to the guidelines enshrined in the International Conference on Harmonization (ICH). The use of the diol-type stationary phase was well suited for implementing a successful 2D-HPLC system. Indeed, in order to cope with the absence of chemoselectivity for the couples amikacin/kanamycin and paromomycin/apramycin, a successful 2D-HPLC method was implemented with the "heart-cut" approach and the use of either heptafluorobutyric (for the former) or perfluorooctanoic acid (for the latter) as the ion-pair reagent in the second RP-LC dimension.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.aca.2018.08.008DOI Listing
December 2018

Last ten years (2008-2018) of chiral ligand-exchange chromatography in HPLC: An updated review.

J Sep Sci 2019 Jan 12;42(1):21-37. Epub 2018 Sep 12.

Department of Pharmaceutical Sciences, Section of Chemistry and Technology of Drugs, University of Perugia, Perugia, Italy.

Chiral ligand-exchange chromatography is one of the elective strategies for the direct enantioresolution of small chelating compounds: amino acids, diamines, amino alcohols, diols, small peptides, etc. Unlike other methods, the interaction between chiral selector and analyte enantiomers is mediated by a cation, thus producing diastereomeric ternary complexes. Two main approaches are conventionally applied in chiral ligand-exchange chromatography. The first relies upon chiral stationary phases where the chiral selector is either covalently immobilized or physically adsorbed onto suitable packing materials (coated phases). In the second approach, chiral molecules are added to the eluent, thus generating chiral eluent systems. Among the advantages of chiral ligand-exchange chromatography, the generation of UV/vis-active metal complexes, and the use of commercially available or easy-to-synthesize chiral selectors, in combination to rather inexpensive achiral columns for coated phases and chiral eluents, are noteworthy. Besides amino acids and amino alcohols, other species have proven suitable for chiral ligand-exchange chromatography applications. Recently, the use of either chiral ionic liquids or micellar liquid chromatography systems as well as the successful off-column formation of diastereomeric complexes have expanded the selectivity profiles and application fields. All of these issues are touched in the review, shedding light to the contributions appeared in the last decade.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/jssc.201800724DOI Listing
January 2019

Improved chromatographic diastereoresolution of cyclopropyl dafachronic acid derivatives using chiral anion exchangers.

J Chromatogr A 2018 Jul 27;1557:20-27. Epub 2018 Apr 27.

University of Perugia, Department of Pharmaceutical Sciences, Section of Chemistry and Technology of Drugs, Via del Liceo 1, 06123 Perugia, Italy.

In the present paper we describe the optimization and the application of a chromatographic method suitable to get all four diastereoisomers of C24-C25 cyclopropyl dafachronic acid derivatives in sufficient amount for their biological appraisal towards the nuclear hormone receptor transcription factor DAF-12. A preliminary column screening of six anion exchange type Cinchona alkaloid-based chiral stationary phases (CSPs) allowed to identify the one with a quinine scaffold and carrying a O-9-(3,5-bis(trifluoromethyl)phenyl) moiety at C9 position as the best CSP. Few modifications of the experimental conditions revealed that a content of 18mM acetic acid used as counterion and displacer in acetonitrile and a column temperature fixed at 35°C were optimal for the simultaneous discrimination of all four diastereoisomers with a 1.0mL/min flow rate. With such conditions, transoid (S,S) and (R,R) diastereoisomers were resolved with R>1.4. With non chiral reversed-phase columns, neither the cisoid nor the transoid diastereoisomers could be resolved. This way, ca. 1.0mg of each stereoisomer was isolated with a diastereomeric purity >98%, suitable for the following biological tests. The indirect stereochemical assignments of the four diastereoisomers, and hence the corresponding chromatographic elution order (24R,25R)<(24S,25S)<(24R,25S)<(24S,25R) were made in an analogy manner on the basis of the resolution of fully assigned and structurally very similar ursodeoxycholic acid derivatives. As support of this indirect way of assigning the absolute configuration of the C24 and C25 chiral centre a molecular modeling procedure based on dynamic simulation was successfully applied.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.chroma.2018.04.062DOI Listing
July 2018

α-Amino-β-carboxymuconate-ε-semialdehyde Decarboxylase (ACMSD) Inhibitors as Novel Modulators of De Novo Nicotinamide Adenine Dinucleotide (NAD) Biosynthesis.

J Med Chem 2018 02 26;61(3):745-759. Epub 2018 Jan 26.

Laboratory of Integrative and Systems Physiology, Interfaculty Institute of Bioengineering, École Polytechnique Fédérale de Lausanne CH-1015 Lausanne, Switzerland.

NAD has a central function in linking cellular metabolism to major cell-signaling and gene-regulation pathways. Defects in NAD homeostasis underpin a wide range of diseases, including cancer, metabolic disorders, and aging. Although the beneficial effects of boosting NAD on mitochondrial fitness, metabolism, and lifespan are well established, to date, no therapeutic enhancers of de novo NAD biosynthesis have been reported. Herein we report the discovery of 3-[[[5-cyano-1,6-dihydro-6-oxo-4-(2-thienyl)-2-pyrimidinyl]thio]methyl]phenylacetic acid (TES-1025, 22), the first potent and selective inhibitor of human ACMSD (IC = 0.013 μM) that increases NAD levels in cellular systems. The results of physicochemical-property, ADME, and safety profiling, coupled with in vivo target-engagement studies, support the hypothesis that ACMSD inhibition increases de novo NAD biosynthesis and position 22 as a first-class molecule for the evaluation of the therapeutic potential of ACMSD inhibition in treating disorders with perturbed NAD supply or homeostasis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/acs.jmedchem.7b01254DOI Listing
February 2018

Synthesis, physicochemical properties, and biological activity of bile acids 3-glucuronides: Novel insights into bile acid signalling and detoxification.

Eur J Med Chem 2018 Jan 11;144:349-358. Epub 2017 Dec 11.

Department of Pharmaceutical Sciences, University of Perugia, Via del Liceo, 1, 06123 Perugia, Italy. Electronic address:

Glucuronidation is considered an important detoxification pathway of bile acids especially in cholestatic conditions. Glucuronides are less toxic than the parent free forms and are more easily excreted in urine. However, the pathophysiological significance of bile acid glucuronidation is still controversial and debated among the scientific community. Progress in this field has been strongly limited by the lack of appropriate methods for the preparation of pure glucuronides in the amount needed for biological and pharmacological studies. In this work, we have developed a new synthesis of bile acid C3-glucuronides enabling the convenient preparation of gram-scale quantities. The synthesized compounds have been characterized in terms of physicochemical properties and abilities to modulate key nuclear receptors including the farnesoid X receptor (FXR). In particular, we found that C3-glucuronides of chenodeoxycholic acid and lithocholic acid, respectively the most abundant and potentially cytotoxic species formed in patients affected by cholestasis, behave as FXR agonists and positively regulate the gene expression of transporter proteins, the function of which is critical in human conditions related to imbalances of bile acid homeostasis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.ejmech.2017.12.034DOI Listing
January 2018

Exploring the enantiorecognition mechanism of Cinchona alkaloid-based zwitterionic chiral stationary phases and the basic trans-paroxetine enantiomers.

J Sep Sci 2018 Mar 27;41(6):1199-1207. Epub 2017 Dec 27.

Department of Inorganic and Analytical Chemistry, University of Szeged, Szeged, Hungary.

The enantiomers of trans-paroxetine (the selectand) were separated on four chiral stationary phases incorporating either quinine [ZWIX(+), ZWIX(+A)] or quinidine [ZWIX(-), ZWIX(-A)] and (R,R)-aminocyclohexanesulfonic acid [in ZWIX(-), and ZWIX(+A)] or (S,S)-aminocyclohexanesulfonic acid [in ZWIX(+), and ZWIX(-A)] chiral selectors. The zwitterion nature of the phases is due to the presence of either (R,R)- or (S,S)-aminocyclohexanesulfonic acid in the selector structure bearing the quinuclidine moiety. ZWIX(+) and ZWIX(-) phases are available on the market with the commercial names CHIRALPAK ZWIX(+) and CHIRALPAK ZWIX(-), respectively. With the aim of rationalizing the enantiomer elution order with the above chiral stationary phases, a molecular dynamic protocol was applied and two energetic parameters were initially measured: selectand conformational energy and selectand interaction energy. In the search for other descriptors allowing a better fitting with the experimental evidences, in the present work we consider an energetic parameter, defined as the selector conformational energy, which resulted to be relevant in the explanation of the experimental elution order in most of the cases. Very importantly, the computational data produced by the present study strongly support the outstanding role of the conformational energy of the chiral selector as it interacts with the analytes.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/jssc.201701068DOI Listing
March 2018

Targeting Wnt-driven cancers: Discovery of novel tankyrase inhibitors.

Eur J Med Chem 2017 Dec 7;142:506-522. Epub 2017 Oct 7.

Department of Pharmaceutical Sciences, University of Perugia, Via del Liceo 1, 06123 Perugia, Italy. Electronic address:

Recent years have seen substantially heightened interest in the discovery of tankyrase inhibitors (TNKSi) as new promising anticancer agents. In this framework, the aim of this review article is focused on the description of potent TNKSi also endowed with disruptor activity toward the Wnt/β-catenin signaling pathway. Beginning with an overview of the most characterized TNKSi deriving from several drug design approaches and classifying them on the basis of the molecular interactions with the target, we discuss only those ones acting against Wnt cancer cell lines. In addition, comprehensive structure property relationships (SPR) emerging from the hit evolution processes and preclinical results are provided. We then review the most promising TNKSi hitherto reported in literature, acting in vivo models of Wnt-driven cancers. Some outlooks on current issues and future directions in this field are also discussed.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.ejmech.2017.09.030DOI Listing
December 2017

Binding properties of different categories of IDO1 inhibitors: a microscale thermophoresis study.

Future Med Chem 2017 08 3;9(12):1327-1338. Epub 2017 Aug 3.

Dipartimento di Scienze Farmaceutiche, University of Perugia, Via del Liceo 1, 06123 Perugia, Italy.

Aim: Inhibition of IDO1 is a strategy pursued in the immune-oncology pipeline for the development of novel anticancer therapies. At odds with an ever-increasing number of inhibitors being disclosed in the literature and patent applications, only very few compounds have hitherto advanced in clinical settings.

Materials & Methods: We have used MicroScale Thermophoresis analysis and docking calculations to assess on a quantitative basis the binding properties of distinct categories of inhibitors to IDO1.

Results: Results shed further light on hidden molecular aspects governing the recognition by the enzyme of compounds with different mechanism of inhibition.

Conclusion: Results pinpoint specific binding features of distinct inhibitors to IDO1 that offer clues for the design of next-generation inhibitors of the enzyme.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.4155/fmc-2017-0022DOI Listing
August 2017

Enantioresolution and stereochemical characterization of two chiral sulfoxides endowed with COX-2 inhibitory activity.

Chirality 2017 Sep 5;29(9):536-540. Epub 2017 Jul 5.

Department of Pharmaceutical Sciences, University of Perugia, Perugia, Italy.

The capacity of nonsteroidal antiinflammatory drugs (NSAIDs) to prevent prostanoids biosynthesis through the inhibition of COX-2 enzyme is related to their structural backbone, based on the fusion of a cis-stilbene unit with a variety of heterocyclic and carbocyclic rings. By this route, a series of new selective COX-2 inhibitors was developed, by maintaining the 4-methylsulfone or 4-methylsulfonamide substituent on the phenyl moiety, essential for their activity. In this frame, two novel propyl sulfoxide derivatives were synthesized, which proved selective and sufficiently potent COX-2 inhibition activity when tested as racemates. In the present study, the use of a cellulose tris(3,5-dichlorophenylcarbamate)-based chiral stationary phase, in a polar-organic mode of elution, enabled the successful enantioseparation of the investigated compounds. The developed chromatography method reveals a useful tool of monitoring in view of a proper forthcoming enantioselective synthetic protocol. Moreover, the optimized chromatographic conditions allowed the isolation of appropriate amounts of single enantiomers for the electronic circular dichroism studies that, coupled with in silico simulations, allowed assessing the absolute configuration of each species.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/chir.22724DOI Listing
September 2017

Reshaping antibiotics through hydrophobic drug-bile acid ionic complexation enhances activity against Staphylococcus aureus biofilms.

Int J Pharm 2017 Aug 6;528(1-2):144-162. Epub 2017 Jun 6.

Department of Pharmaceutical Sciences, via del Liceo 1, 06123 Perugia, Italy.

The antibiotic era is on the verge of a profound change and facing a ground shaking crisis. The frequent failures of antibiotic treatments are often associated with biofilm formation, which is responsible for chronic infections, exacerbation as well as reinfection. So far, albeit the large number of valuable strategies employed to combat biofilm formation, little success has been recorded. In this work, we propose a simple approach, based on hydrophobic ionic complexation with the bile acids, deoxycholic acid (DCA) and ursodeoxycholic acid (UDCA), to enhance anti-biofilm activity of well-known antibiotics, namely kanamycin (K), amikacin (A) and vancomycin (V). Activity was evaluated against Staphylococcus aureus ATCC 29213 and six methicillin-resistant clinical isolates. The formation of a 1:4 ADCA and KDCA and 1:1 VUDCA complexes was confirmed by HNMR, in silico molecular dynamics simulations, as well as thermal, spectrophotometric and HPLC analyses. The complexes showed higher inhibition of S. aureus growth compared to parent drugs and a concentration-independent biofilm inhibition and dispersion capacity in the order KDCA > ADCA >VUDCA, even at concentrations ten-fold below the MIC. S. aureus growth inhibition evaluated upon treatment with bile acid-drug sequential addition and the complexes as well as the measured complex stability in solution suggest a bile acid carrier role. The complexes showed in vivo toxicity only at 10×MIC concentration on the chicken embryo chorioallantoic membrane model in the order KDCA < ADCA < VUDCA. KDCA was safe at all concentrations. Although several aspects to be addressed, this approach is promising due to its simplicity, the proved in vitro anti-biofilm activity enhancement and tolerability. A potential pulmonary drug delivery application is envisaged.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.ijpharm.2017.06.008DOI Listing
August 2017

N-Decyl-S-trityl-(R)-cysteine, a new chiral selector for "green" ligand-exchange chromatography applications.

J Pharm Biomed Anal 2017 Sep 5;144:31-40. Epub 2017 Feb 5.

Department of Pharmaceutical Sciences, University of Perugia, Via Fabretti 48, 06123 Perugia, Italy.

In search for new enantioselectivity profiles, the N-decyl-S-trityl-(R)-cysteine [C-(R)-STC] was synthesized through a one-step procedure and then hydrophobically adsorbed onto an octadecylsilica surface to generate a stable chiral stationary phase for ligand-exchange chromatography (CLEC-CSP) applications. The CLEC analysis was carried out on underivatized amino acids, by using a Cu(II) sulphate (1.0mM) containing aqueous eluent system. Most of the analysed compounds (34 out of 45) were enantiodiscriminated by the C-(R)-STC-based CSP, with resolution factor (R) values up to 8.86. Conformationally rigid and hydrophobic ligands often experienced the largest enantioselectivity effects. A high loadability emerged from the analysis of rac-NorVal (selected as prototype test compound): up to 20mg/mL were efficiently enantioseparated with the CLEC-CSP. Two in-line hand-made cartridges filled with a strong cation-exchange resin allowed the effective catching of Cu(II) ions after the semi-preparative enantioseparation. The quantitative recovery of the rac-NorVal enantiomers was made possible by flowing through the cartridge a 5% (v) ammonia solution. The CLEC phase proved successful in the enantioselective analysis of a commercially available (S)-Leu containing tablet. Furthermore, in order to understand the molecular basis for a successful use of the C-(R)-STC-based CLEC system, a descriptive structure-separation relationship study was performed. As a result, all compounds with a MEAN-QPlogS (a hydrophilicity descriptor) value lower than 0.373 can be most likely enantioseparated with the CLEC system under investigation. In the work, the numerous aspects complying with the principles of green chromatography are highlighted and discussed.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.jpba.2017.02.009DOI Listing
September 2017

A rational approach to elucidate human monoamine oxidase molecular selectivity.

Eur J Pharm Sci 2017 Apr 7;101:90-99. Epub 2017 Feb 7.

Dipartimento di Farmacia-Scienze del Farmaco, Università di Bari 'Aldo Moro', Via Orabona, 4, 70126 Bari, Italy. Electronic address:

Designing highly selective human monoamine oxidase (hMAO) inhibitors is a challenging goal on the road to a more effective treatment of depression and anxiety (inhibition of hMAO-A isoform) as well as neurodegenerative diseases (inhibition of hMAO-B isoform). To uncover the molecular rationale of hMAOs selectivity, two recently prepared 2H-chromene-2-ones, namely compounds 1 and 2, were herein chosen as molecular probes being highly selective toward hMAO-A and hMAO-B, respectively. We performed molecular dynamics (MD) studies on four different complexes, cross-simulating one at a time the two hMAO-isoforms (dimer embedded in a lipid bilayer) with the two considered probes. Our comparative analysis on the obtained 100ns trajectories discloses a stable H-bond interaction between 1 and Gln215 as crucial for ligand selectivity toward hMAO-A whereas a water-mediated interaction might explain the observed hMAO-B selectivity of compound 2. Such hypotheses are further supported by binding free energy calculations carried out applying the molecular mechanics generalized Born surface area (MM-GBSA) method and allowing us to evaluate the contribution of each residue to the observed isoform selectivity. Taken as whole, this study represents the first attempt to explain at molecular level hMAO isoform selectivity and a valuable yardstick for better addressing the design of new and highly selective MAO inhibitors.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.ejps.2017.02.008DOI Listing
April 2017

Elucidation of the Chromatographic Enantiomer Elution Order Through Computational Studies.

Mini Rev Med Chem 2018 ;18(2):88-97

Department of Pharmaceutical Sciences, University of Perugia, Via Fabretti 48, 06123-Perugia, Italy.

During the last twenty years, the interest towards the development of chiral compound has exponentially been increased. Indeed, the set-up of suitable asymmetric enantioselective synthesis protocols is currently one of the focuses of many pharmaceutical research projects. In this scenario, chiral HPLC separations have gained great importance as well, both for analytical- and preparative-scale applications, the latter devoted to the quantitative isolation of enantiopure compounds. Molecular modelling and quantum chemistry methods can be fruitfully applied to solve chirality related problems especially when enantiomerically pure reference standards are missing. In this framework, with the aim to explain the molecular basis of the enantioselective retention, we performed computational studies to rationalize the enantiomer elution order with both low- and high-molecular weight chiral selectors. Semi-empirical and quantum mechanical computational procedures were successfully applied in the domains of chiral ligand-exchange and chiral ion-exchange chromatography, as well as in studies dealing with the use of polysaccharide-based enantioresolving materials.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.2174/1389557516666161018143629DOI Listing
April 2018

Discovery of 3α,7α,11β-Trihydroxy-6α-ethyl-5β-cholan-24-oic Acid (TC-100), a Novel Bile Acid as Potent and Highly Selective FXR Agonist for Enterohepatic Disorders.

J Med Chem 2016 Oct 4;59(19):9201-9214. Epub 2016 Oct 4.

Department of Pharmaceutical Sciences, University of Perugia , Via del Liceo 1, 06123 Perugia, Italy.

As a continuation of previous efforts in mapping functional hot spots on the bile acid scaffold, we here demonstrate that the introduction of a hydroxy group at the C11β position affords high selectivity for FXR. In particular, the synthesis and FXR/TGR5 activity of novel bile acids bearing different hydroxylation patterns at the C ring are reported and discussed from a structure-activity standpoint. The results obtained led us to discover the first bile acid derivative endowed with high potency and selectivity at the FXR receptor, 3α,7α,11β-trihydroxy-6α-ethyl-5β-cholan-24-oic acid (TC-100, 7) which also shows a remarkable physicochemical and pharmacological profile. Compound 7 combines the excellent physicochemical properties of hydrophilic bile acids such as ursodeoxycholic acid, with the distinct ability to specifically bind and regulate FXR activity in vivo, thus providing a bona fide novel therapeutic agent to treat enterohepatic disorders such as cholestasis, NASH, and inflammatory bowel disease.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/acs.jmedchem.6b01126DOI Listing
October 2016