title: Evaluation and comparison of three different separation techniques for analysis of retroamide enantiomers and their biological evaluation against h-P2X7 receptor.
abstract: The P2X receptors are seven-transmembrane domain G protein-coupled receptors and the 7 subtypes of P2X receptors identified in humans, and named P2X1 to P2X7, are channel receptors whose endogenous ligand is ATP. New antagonists of the P2X7 receptor were developed, since this purinergic receptor was highlighted to be involved in many diseases such as different types of pain, cancer, ischemia, neurodegenerative diseases (including Parkinson's and Alzheimer's diseases) characterized by inflammatory processes. With the aim of evaluate the impact of chirality on the pharmacological activity of a new P2X7R antagonist, a semi-preparative method was developed in supercritical fluid chromatography (SFC). Among four polysaccharide based chiral stationary phases: Chiralcel OD-H and OJ-H and Chiralpak AS-H and AD-H, the last one namely amylose tris (3,5-dimethylphenylcarbamate) with a mobile phase consisted of carbon dioxide-ethanol (80:20, v/v), led to the successful separation of the enantiomers in short run time and with good resolution. Limits of detection and quantification were calculated and were found equal for compound 1, to 1.37 μM and 4.57 μM respectively, for peak 1 and were equal to 1.60 μM and 5.30 μM respectively, for peak 2 at λ=210 nm. Before carrying out the pharmacological evaluation of each enantiomer, two complementary methodologies, e.g. liquid chromatography and capillary electrophoresis were performed in parallel to improve the limits of detection and quantification to assess the enantiomeric purity. HPLC using a Chiralpak AD stationary phase led to four times lower limits of detection and quantification with regard to SFC. In the same time, capillary electrophoresis involving dual cyclodextrins system constituted of a SBE-β-CD and a MM-β-CD mixture enhanced the signal-to-noise ratio and led to similar limits of detection and quantification with regard to SFC. No trace of the other enantiomer was found in the isolated one. Biological activities of individual enantiomers were then evaluated and revealed no cytotoxicity against cell lines and a significant difference in terms of their IC50 values with respect to the investigated racemate (6.43 μM): 3.49 μM for the (R)-enantiomer and >10(-4)μM for the (S)-enantiomer, for compound 1, showing that, this antagonist activity is stereospecific.
authors.affiliation: Inserm U995, Université de Lille, Laboratoire de Chimie Analytique, UFR Pharmacie, F-59000 Lille, France; Inserm U995, Université de Lille, Hautes Etudes Ingénieur, F-59000 Lille, France.
authors.forename: Davy
authors.initials: D
authors.lastname: Baudelet
authors.affiliation: Université de Lille, UDSL, Plateforme de binding, ICPAL, F-59000 Lille, France.
authors.forename: Christophe
authors.initials: C
authors.lastname: Furman
authors.affiliation: Inserm U995, Université de Lille, Hautes Etudes Ingénieur, F-59000 Lille, France.
authors.forename: Alina
authors.initials: A
authors.lastname: Ghinet
authors.affiliation: Université de Lille, UDSL, Plateforme de binding, ICPAL, F-59000 Lille, France.
authors.forename: Xavier
authors.initials: X
authors.lastname: Dezitter
authors.affiliation: Inserm 905, Université de Rouen, Département de Médecine, F-76983 Rouen, France.
authors.forename: Sahil
authors.initials: S
authors.lastname: Adriouch
authors.affiliation: Université de Lille, USTL, UMR 8181, Unité de Catalyse et de Chimie du Solide, F-59000 Lille, France.
authors.forename: Frédéric
authors.initials: F
authors.lastname: Capet
authors.affiliation: Université de Lille, EA GRITA, Laboratoire de Chimie Analytique, UFR Pharmacie, F-59000 Lille, France.
authors.forename: Tiphaine
authors.initials: T
authors.lastname: Rogez-Florent
authors.affiliation: Inserm U995, Université de Lille, Hautes Etudes Ingénieur, F-59000 Lille, France.
authors.forename: Philippe
authors.initials: P
authors.lastname: Gautret
authors.affiliation: Inserm U995, Université de Lille, Laboratoire de Chimie Analytique, UFR Pharmacie, F-59000 Lille, France.
authors.forename: Benoit
authors.initials: B
authors.lastname: Rigo
authors.affiliation: Inserm U995, Université de Lille, Laboratoire de Chimie Analytique, UFR Pharmacie, F-59000 Lille, France.
authors.forename: Régis
authors.initials: R
authors.lastname: Millet
authors.affiliation: Université de Lille, EA GRITA, Laboratoire de Chimie Analytique, UFR Pharmacie, F-59000 Lille, France.
authors.forename: Claude
authors.initials: C
authors.lastname: Vaccher
authors.affiliation: Inserm U995, Université de Lille, Laboratoire de Chimie Analytique, UFR Pharmacie, F-59000 Lille, France. Electronic address: emmanuelle.lipka@univ-lille2.fr.
authors.forename: Emmanuelle
authors.initials: E
authors.lastname: Lipka
mesh_terms: D000688:Amylose / Q000031:analogs & derivatives* / Q000737:chemistry; D002482:Cellulose / Q000031:analogs & derivatives* / Q000737:chemistry; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D016014:Linear Models; D048448:Phenylcarbamates / Q000737:chemistry*; D058920:Purinergic P2X Receptor Antagonists / Q000032:analysis / Q000737:chemistry* / Q000302:isolation & purification*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013237:Stereoisomerism
chemical_list: D048448:Phenylcarbamates; D058920:Purinergic P2X Receptor Antagonists; C065943:cellulose tris-3,5-dimethylphenyl-carbamate; C080033:Chiralpak AD; D002482:Cellulose; D000688:Amylose
keywords: Amylose tris (3,5-dimethylphenylcarbamate); Capillary electrophoresis; Dual cyclodextrins system; High performance liquid chromatography; N-[1-(2,4-Dichlorobenzyl)-5-oxopyrrolidin-2-yl]-2-(2,4-dichlorophenyl) acetamide; Supercritical fluid chromatography