Journal of Chromatography A, 1363 (2014) 162–168 Contents lists available at ScienceDirect Journal of Chromatography A jo ur nal ho me pag e: www.elsevier.com/locate/chroma Enantioresolution, stereochemical characterization and biological activity of a chiral large-conductance calcium-activated potassium channel opener Roccaldo Sardella a , Andrea Carotti a , Giuseppe Manfroni a , Daniele Tedesco b , Alma Martelli c , Carlo Bertucci b , Violetta Cecchetti a , Benedetto Natalini a,∗ a Department of Pharmaceutical Sciences, University of Perugia, via Fabretti 48, 06123 Perugia, Italy b Department of Pharmacy and Biotechnology, University of Bologna, via Belmeloro 6, 40126 Bologna, Italy c Department of Pharmacy, University of Pisa, Via Bonanno 6, 56126 Pisa, Italy a r t i c l e i n f o Article history: Received 28 February 2014 Received in revised form 4 June 2014 Accepted 4 June 2014 Available online 12 June 2014 Keywords: Polysaccharide-based stationary phases Preparative enantioresolution BK channel opener Electronic circular dichroism Absolute configuration Vasorelaxing potency a b s t r a c t A number of large-conductance calcium-activated potassium (BK) channel openers based on the 2-aryl-1,4-benzothiazine scaffold were previously synthesized, and 2-(5-bromo-2-methoxyphenyl)-6- trifluoromethyl-2H-1,4-benzothiazin-3(4H)-one (1) was identified as the most active compound. Since a stereoselective activation of BK channels was demonstrated for arylindolone derivatives, the effect of the absolute configuration at the C-2 position on the vasorelaxing potency of 2-aryl-1,4-benzothiazines is investigated in this article. Compound 1 was initially evaluated as a racemate: subsequently, the “racemic approach” was used to isolate its enantiomers. The excellent enantioresolution obtained using the Sepapak-4 column (CSP 4, cellulose tris(4-chloro-3-metylphenylcarbamate); R S = 8.36; ˛ = 2.03) allowed to collect highly pure enantiomeric fractions, with enantiomeric excess (e.e.) values higher than 97% and 98% for the first- and second-eluted enantiomer, respectively. Electronic circular dichroism (ECD) stud- ies on the two isolated enantiomers, combined with time-dependent density functional theory (TD-DFT) calculations allowed to characterize the configuration of the enantiomers and determine a (R), (S) elu- tion order. Results from biological assays indicated that the racemate and the isolated enantiomers are endowed with comparable vasorelaxing potency. © 2014 Elsevier B.V. All rights reserved. 1. Introduction Recent efforts toward the identification of potent and selective activators of potassium channels led to the development of a struc- turally novel class of large-conductance calcium-activated potas- sium (BK) channel openers based on the 2-aryl-1,4-benzothiazine scaffold. As a result, the racemic 2-(5-bromo-2-methoxyphenyl)- 6-trifluoromethyl-2H-1,4-benzothiazin-3(4H)-one (1, Fig. 1) was found to be the most active compound [1]. BK channels are present in virtually every cell type where they play a pivotal and specific role in a wide range of physiological processes, spanning from mediating fast after-hyperpolarizations following action poten- tials, to inhibition of neurotransmitter release, and relaxation of smooth muscle cells in bladder, arterioles and airways [2]. ∗ Corresponding author. Tel.: +39 075 5855131; fax: +39 075 5855161. E-mail address: benedetto.natalini@unipg.it (B. Natalini). The functional versatility of BK channel proteins is conferred by a variety of means, including extensive alternative splicing [3] of the pore-forming -subunit encoded by the single gene slo1 [4] and co-assembly with auxiliary -subunits [5]. Thus, a consider- able diversity is generated within the BK family, which may be tissue and organ-specific [2,6]. Due to these properties and their central role in regulating cell activity, BK channels are particularly appealing as a therapeutic drug target [7,8]. In particular, BK chan- nel openers, decreasing cell excitability and causing smooth muscle relaxation, could offer a novel therapeutic approach to several dis- eases associated with both the central nervous system and smooth muscles, such as stroke, epilepsy, bladder overactivity, asthma, and hypertension [9,10]. Several agents have been reported to activate BK channels [8,11]. Among the prototypical BK openers, the class of arylindolone derivatives [12,13], represented by the eutomer BMS- 204352 (2, Fig. 1) [14], were studied in detail providing evidence for chiral discrimination by the BK protein. This indication prompted the present study on the influence of the absolute configuration at the C-2 stereogenic center on http://dx.doi.org/10.1016/j.chroma.2014.06.020 0021-9673/© 2014 Elsevier B.V. All rights reserved.