Dynamic Behavior of Clobazam on High‐Performance Liquid Chromatography Chiral Stationary Phases

Abstract

Clobazam, a 1,5-benzodiazepin-2,4-dione, is a chiral molecule because its ground state conformation features a nonplanar seven-membered ring lacking reflection symmetry elements. The two conformational enantiomers of clobazam interconvert at room temperature by a simple ring-flipping process. Variable temperature HPLC on the Pirkle type (R)-N-(3,5-dinitronenzoyl)phenylglycine and (R,R)-Whelk-O1 chiral stationary phases (CSPs) allowed us to separate for the first time the conformational enantiomers of clobazam and to observe peak coalescence-decoalescence phenomena due to concomitant separation and interconversion processes occurring on the same time scale. Clobazam showed temperature dependent dynamic high-performance liquid chromatography (HPLC) profiles with interconversion plateaus on the two CSPs indicative of on-column enantiomer interconversion. (enantiomerization) in the column temperature range between Tcol = 10°C and Tcol = 30°C, whereas on-column interconversion was absent at temperature close to or lower than Tcol = 5°C. Computer simulation of exchange-deformed HPLC profiles using a program based on the stochastic model yielded the apparent rate constants for the on-column enantiomerization and the corresponding free energy activation barriers. At Tcol = 20°C the averaged enantiomerization barriers, ΔG(‡), for clobazam were found in the range 21.08-21.53 kcal mol(-1) on the two CSPs. The experimental dynamic chromatograms and the corresponding interconversion barriers reported in this article are consistent with the literature data measured by DNMR at higher temperatures and in different solvents.

Recent trends and applications in liquid‐phase chromatography enantioseparation of atropisomers

“…Villani and co‐workers studied the dynamic behavior of 1,4‐benzodiazepin‐2‐ones , a well‐known class of pharmacologically active heterocyclic compounds with sedative, hypnotic, anxiolytic, and anticonvulsant properties (Table ). Medicinal chemistry investigations have shown that conformational chirality of the 1,4‐benzodiazepine core of these molecules plays a key role in determining their bioactivity .…”

Theory and Practice of UHPLC and UHPLC–MS

“…Conformational changes are normally based on monomolecular mechanisms and, as such, do not require the assistance of a secondary molecular partner. Atropoisomerism is an intramolecular event that falls into this class of reactions, featuring pure first-order kinetics (Gasparrini et al, 1997a(Gasparrini et al, , 2000(Gasparrini et al, , 2001(Gasparrini et al, , 2002aDell'Erba et al, 2002;Andreani et al, 2004;Borsato et al, 2004;Dalla Cort et al, 2005;Lunazzi et al, 2010;Levi Mortera et al, 2012;Rizzo et al, 2013Rizzo et al, , 2014Rizzo et al, , 2015Chiarucci et al, 2014;Sabia et al, 2014Sabia et al, , 2016Menta et al, 2015). On the other hand, configurational isomerizations (i.e., processes involving rupture and reformation of chemical bonds) are commonly promoted by species that act as catalysts and that therefore do not modify their concentration during the interconversion (a bimolecular mechanism).…”

Enantiomeric separation of prothioconazole and prothioconazole‐desthio on chiral stationary phases

“…In our work, the chiral resolution of prothioconazole and its chiral metabolite prothioconazole-desthio were performed on commercialized polysaccharide-based columns including Chiralcel OJ-H, Chiralpak IA, Chiralpak IB, Chiralpak IC, Chiralpak AY-H, Chiralpak AZ-H, 17 Lux Cellulose-1, 18 one kind of self-made Chiralcel OD, 17 and another pirkle type column (R,R)-Whelk-O1 19 (as shown in Figure 1B). The influences of mobile phase composition such as n-hexane/IPA or n-hexane/ethanol under normal phase conditions and ACN/water or methanol/water under reverse phase conditions were discussed.…”