S S symmetry Article Low Temperature Dynamic Chromatography for the Separation of the Interconverting Conformational Enantiomers of the Benzodiazepines Clonazolam, Flubromazolam, Diclazepam and Flurazepam Roberta Franzini , Alessia Rosetti and Claudio Villani * Dipartimento di Chimica e Tecnologie del Farmaco, Sapienza Università di Roma, p.le Aldo Moro, 5, 00185 Rome, Italy; [email protected] (R.F.); [email protected] (A.R.) * Correspondence: [email protected] Abstract: Benzodiazepines (BZDs) are an important class of psychoactive drugs with hypnotic- sedative, myorelaxant, anxiolytic and anticonvulsant properties due to interaction with the GABAa receptor in the central nervous system of mammals. BZDs are interesting both in clinical and forensic toxicology for their pharmacological characteristics and potential of abuse. The presence of a non-planar diazepine ring generates chiral conformational stereoisomers, even in the absence of stereogenic centers. A conformational enrichment of BZD at the binding sites has been reported in the literature, thus making interesting a stereodynamic screening of a wide range of BZDs. Herein, we report the investigation of three stereolabile 1,4-benzodiazepine included in the class of “designer benzodiazepines” (e.g., diclazepam, a chloro-derivative of diazepam, and two triazolo- Citation: Franzini, R.; Rosetti, A.; benzodiazepines, flubromazolam and clonazolam) and a commercially available BZD known as Villani, C. Low Temperature Dynamic flurazepam, in order to study the kinetic of the “ring-flip” process that allows two conformational Chromatography for the Separation enantiomers to interconvert at high rate at room temperature. A combination of low temperature of the Interconverting enantioselective dynamic chromatography on chiral stationary phase and computer simulations of Conformational Enantiomers of the the experimental chromatograms allowed us to measure activation energies of enantiomerization Benzodiazepines Clonazolam, (DG‡) lower than 18.5 kcal/mol. The differences between compounds have been correlated to the Flubromazolam, Diclazepam and pattern of substitutions on the 1,4-benzodiazepinic core. Flurazepam. Symmetry 2021, 13, 1012. https://doi.org/10.3390/sym13061012 Keywords: conformational enantiomers; chirality; chiral stationary phases; benzodiazepines; low Academic Editor: Angela Patti temperature dynamic chromatography; enantiomerization Received: 10 May 2021 Accepted: 3 June 2021 Published: 4 June 2021 1. Introduction Chirality is a common property in pharmaceutically active compounds [1], and the Publisher’s Note: MDPI stays neutral investigation of the stereoselectivity in the interaction of drugs with their biological target is with regard to jurisdictional claims in a fundamental step in drug design. The constant increase in the request for enantiomerically published maps and institutional affil- pure active compounds in the pharmaceutical market [2] urges for the improvement iations. of stereoselective analytical and preparative methodologies and stereochemical stability studies [3,4]. Benzodiazepines are one of the most prescribed drugs in the world for their sedative, anxiolytic and anticonvulsant properties [5,6] but are also known for their many adverse effects and potential of abuse [7–9]. Copyright: © 2021 by the authors. The seven membered ring of the 1,4-benzodiazepine core lacks planarity and adopts Licensee MDPI, Basel, Switzerland. a boat conformation; the chirality that derives from it has been the focus of many recent This article is an open access article investigations in the last few decades [10–13]. The two possible boat-shaped conformations distributed under the terms and are non-superimposable mirror images, so the compound is chiral despite the fact that it conditions of the Creative Commons lacks a chiral center. Attribution (CC BY) license (https:// Conformational enantiomers of 1,4 benzodiazepines interconvert via a ring inversion creativecommons.org/licenses/by/ mechanism with an energetic barrier calculated for diazepam of about 17.6 kcal/mol [12]. 4.0/). Symmetry 2021, 13, 1012. https://doi.org/10.3390/sym13061012 https://www.mdpi.com/journal/symmetry Symmetry 2021, 13, x FOR PEER REVIEW 2 of 10 Symmetry 2021, 13, 1012 2 of 9 Conformational enantiomers of 1,4 benzodiazepines interconvert via a ring inversion mechanism with an energetic barrier calculated for diazepam of about 17.6 kcal/mol [12]. The interestThe interest in investigating in investigating the chirality the chirality of BZD of BZD results results from from the theevidence evidence of a of stereose- a stereoselec- lectivetive association association with with both both human human serum serum albumin albumin HSA HSA and and GABAa GABAa receptors receptors with with the the (M)- (M)-chiralchiral conformation conformation as as the the preferential one in thethe interaction,interaction, asas revealedrevealed byby has-inducedhas-in- ducedcircular circular dichroism dichroism measured measured for for fast fast interconverting interconverting species species such such as as diazepam diazepam [14 ,15] [14,15]and and by by the the direct direct measurements measurements ofof GABAa receptors receptors’’ affinity affinity of of single single enantiomers enantiomers of Symmetry 2021, 13, x FOR PEER REVIEW 3 of 10 of slowlyslowly interconverting interconverting derivatives derivatives [16,17]. [16,17 Recently,]. Recently, many many reviews reviews pointed pointed out out the the role role of of conformationalconformational chirality chirality in inmodern modern medicina medicinall chemistry chemistry [18 [18–25–25]. Basically,]. Basically, a classifica- a classification tion of conformationally chiral compounds,compounds, alsoalso knownknown asas atropisomers, atropisomers, can can be be made made based basedon on the the energetic energetic barriersbarriers of interconversion ( ( ΔGǂ ) between ) between the t he stereoisomers: stereoisomers: the the first first group comprises those species whose single enantiomers group comprises those species whose single enantiomers featurefeature half-lives half-lives of racemization of racemization covering the range from days to months or years at room covering the range from days to months or years at room temperaturetemperature with ΔGⱡ values larger than 27 kcal/mol. Drugs or drug-like molecules of the firstvalues group larger are stereochem- than 27 kcal/mol. Drugs or drug-like molecules of the first group are ically stable enough to be developed as single enantiomers.stereochemically A second group stable includes enough to be developed as single enantiomers. A second group stereochemically less stable compounds, characterized by energeticincludes barriers stereochemically smaller than less stable compounds, characterized by energetic barriers 20 kcal/mol, featuring half-lives of racemization for the individualsmaller enantiomers than 20 kcal/mol, spanning featuring half-lives of racemization for the individual enantio- from minutes to seconds (or fractions of seconds) at room temperature.mers spanning Thus, from atropiso- minutes to seconds (or fractions of seconds) at room temperature. mers that equilibrate with short half-lives, if selected as candidatesThus, atropisomers in drug design, that equilibrate will with short half-lives, if selected as candidates in drug be developed to be administered as an equilibrating mixture.design, In this will context, be developed to achieve to be administered as an equilibrating mixture. In this context, information on the rate of racemization or the equilibrium ratioto achieve for diastereoisomers information on is the es- rate of racemization or the equilibrium ratio for diastereoi- sential for understanding the results of spectroscopic and chromatographicsomers is essential analysis for understanding of the the results of spectroscopic and chromatographic investigated drug. Specifically for benzodiazepines, HPLC separationanalysis of thethe enantiomersinvestigated is drug. Specifically for benzodiazepines, HPLC separation of achievable at or near room temperature, only for those derivativesthe enantiomers that present is a achievable pattern of at or near room temperature, only for those derivatives that substitution that increases the energy barrier, for example, bulkypresent groups a pattern on N1 (ofiPr substitution or tBu). that increases the energy barrier, for example, bulky Dynamic NMR and dynamic HPLC (coupled with computationalgroups on N1 techniques) (iPr or tBu).are two of the most common methods to measure the free energy of interconversionDynamic NMR betweenand dynamic HPLC (coupled with computational techniques) are stereoisomers [26–33]. Dynamic HPLC with chiral stationarytwo phase of the (CSP) most is common successfully methods to measure the free energy of interconversion between employed in the determination of the enantiomerization barriersstereoisomers of fast interconverting [26–33]. Dynamic HPLC with chiral stationary phase (CSP) is successfully enantiomers that exchange on a time-scale comparable to thatemployed of the separationin the determination process. of the enantiomerization barriers of fast interconverting In a typical dynamic HPLC experiment, the racemic mixtureenantiomers of the studied that exchange compound on a time-scale comparable to that of the separation process. is analyzed while the temperature of the column is increasedIn a ortypical decreased dynamic by HPLC defined experiment, the racemic mixture of the studied compound